U.S. patent application number 10/223507 was filed with the patent office on 2003-11-06 for non-stochastic generation of genetic vaccines.
Invention is credited to Short, Jay M..
Application Number | 20030207287 10/223507 |
Document ID | / |
Family ID | 27533273 |
Filed Date | 2003-11-06 |
United States Patent
Application |
20030207287 |
Kind Code |
A1 |
Short, Jay M. |
November 6, 2003 |
Non-stochastic generation of genetic vaccines
Abstract
This invention provides methods of obtaining vaccines by use of
non-stochastic methods of directed evolution (DirectEvolution.TM.).
These methods include non-stochastic polynucleotide site-saturation
mutagenesis (Gene Site Saturation Mutagenesis.TM.) and
non-stochastic polynucleotide reassembly (GeneReassembly.TM.).
Through use of the claimed methods, vectors can be obtained which
exhibit increased efficacy for use as genetic vaccines. Vectors
obtained by using the methods can have, for example, enhanced
antigen expression, increased uptake into a cell, increased
stability in a cell, ability to tailor an immune response, and the
like.
Inventors: |
Short, Jay M.; (Rancho Santa
Fe, CA) |
Correspondence
Address: |
HALE AND DORR LLP
300 PARK AVENUE
NEW YORK
NY
10022
US
|
Family ID: |
27533273 |
Appl. No.: |
10/223507 |
Filed: |
August 19, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10223507 |
Aug 19, 2002 |
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09495052 |
Jan 31, 2000 |
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6479258 |
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09495052 |
Jan 31, 2000 |
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09276860 |
Mar 26, 1999 |
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6352842 |
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09276860 |
Mar 26, 1999 |
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09267118 |
Mar 9, 1999 |
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6238884 |
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09267118 |
Mar 9, 1999 |
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09246178 |
Feb 4, 1999 |
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6171820 |
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09246178 |
Feb 4, 1999 |
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09185373 |
Nov 3, 1998 |
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6335179 |
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09185373 |
Nov 3, 1998 |
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08760489 |
Dec 5, 1996 |
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5830696 |
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08962504 |
Oct 31, 1997 |
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08677112 |
Jul 9, 1996 |
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5965408 |
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60008311 |
Dec 7, 1995 |
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60008316 |
Dec 7, 1995 |
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Current U.S.
Class: |
435/6.14 ;
435/320.1; 435/325; 435/69.1; 514/44R; 800/288 |
Current CPC
Class: |
C12Y 301/11002 20130101;
C12N 15/102 20130101; C12N 15/1034 20130101; C07K 14/445 20130101;
C12N 9/00 20130101; C12N 15/1027 20130101; C12N 9/88 20130101; C12N
9/14 20130101; A61K 2039/53 20130101; C12N 9/16 20130101; A61K
39/00 20130101 |
Class at
Publication: |
435/6 ; 435/69.1;
435/320.1; 435/325; 514/44; 800/288 |
International
Class: |
C12Q 001/68; A61K
048/00; A01H 005/00; C12P 021/02; C12N 005/06; A61K 031/70; A01N
043/04; C12P 021/06; A01H 001/00; C12N 015/82; C12N 015/87; C12N
015/00; C12N 015/09; C12N 015/63; C12N 015/70; C12N 015/74; C12N
005/00; C12N 005/02 |
Claims
What is claimed is:
1. A method for obtaining an immunomodulatory polynucleotide that
has an optimized modulatory effect on an immune response, or
encodes a polypeptide that has an optimized modulatory effect on an
immune response, the method comprising: creating a library of
non-stochastically generated progeny polynucleotides from a
parental polynucleotide set; wherein optimization can thus be
achieved using one or more of the directed evolution methods as
described herein in any combination, permutation and iterative
manner; whereby these directed evolution methods include the
introduction of mutations by non-stochastic methods, including by
"gene site saturation mutagenesis" as described herein; and whereby
these directed evolution methods also include the introduction
mutations by non-stochastic polynucleotide reassembly methods as
described herein; including by synthetic ligation polynucleotide
reassembly as described herein.
2. The method of claim 1, wherein said optimized modulatory effect
on an immune response is induced by a genetic vaccine vector.
Screening
3. A method for obtaining an immunomodulatory polynucleotide that
has an optimized modulatory effect on an immune response, or
encodes a polypeptide that has an optimized modulatory effect on an
immune response, the method comprising: screening a library of
non-stochastically generated progeny polynucleotides to identify an
optimized non-stochastically generated progeny polynucleotide that
has, or encodes a polypeptide that has, a modulatory effect on an
immune response; wherein the optimized non-stochastically generated
polynucleotide or the polypeptide encoded by the non-stochastically
generated polynucleotide exhibits an enhanced ability to modulate
an immune response compared to a parental polynucleotide from which
the library was created.
4. The method of claim 3, wherein said optimized modulatory effect
on an immune response is induced by a genetic vaccine vector.
Evolution & Screening
5. A method for obtaining an immunomodulatory polynucleotide that
has an optimized modulatory effect on an immune response, or
encodes a polypeptide that has an optimized modulatory effect on an
immune response, the method comprising: a) creating a library of
non-stochastically generated progeny polynucleotides from a
parental polynucleotide set; and b) screening the library to
identify an optimized non-stochastically generated progeny
polynucleotide that has, or encodes a polypeptide that has, a
modulatory effect on an immune response induced by a genetic
vaccine vector; wherein the optimized non-stochastically generated
polynucleotide or the polypeptide encoded by the non-stochastically
generated polynucleotide exhibits an enhanced ability to modulate
an immune response compared to a parental polynucleotide from which
the library was created; whereby optimization can thus be achieved
using one or more of the directed evolution methods as described
herein in any combination, permutation, and iterative manner;
whereby these directed evolution methods include the introduction
of point mutations by non-stochastic methods, including by "gene
site saturation mutagenesis" as described herein; and whereby these
directed evolution methods also include the introduction mutations
by non-stochastic polynucleotide reassembly methods as described
herein; including by synthetic ligation polynucleotide reassembly
as described herein.
6. The method of claim 5, wherein said optimized modulatory effect
on an immune response is induced by a genetic vaccine vector.
7. The method of any of claims 1-6, wherein the optimized
non-stochastically generated polynucleotide is incorporated into a
genetic vaccine vector.
8. The method of any of claims 1-6, wherein the optimized
non-stochastically generated polynucleotide, or a polypeptide
encoded by the optimized non-stochastically generated
polynucleotide, is administered in conjunction with a genetic
vaccine vector.
9. The method of any of claims 1-6, wherein the library of
non-stochastically generated progeny polynucleotides is created by
a process selected from the group consisting of gene reassembly,
oligonucleotide-directed saturation mutagenesis, and any
combination, permutation and iterative manner.
10. The method of any of claims 1-6, wherein the optimized
non-stochastically generated polynucleotide that has a modulatory
effect on an immune response is obtained by: a) non-stochastically
reassembling at least two parental template polynucleotide, each of
which is, or encodes a molecule that is, involved in modulating an
immune response; wherein the first and second parental templates
differ from each other in two or more nucleotides, to produce a
library of non-stochastically generated polynucleotides; and b)
screening the library to identify at least one optimized
non-stochastically generated polynucleotide that exhibits, either
by itself or through the encoded molecule, an enhanced ability to
modulate an immune response in comparison to a parental
polynucleotide from which the library was created.
11. The method of claim 10, wherein the method further comprises
the steps of: c) subjecting a working optimized non-stochastically
generated polynucleotide to a further round of non-stochastic
reassembly with at least one additional polynucleotide, which is
the same or different from the first and second polynucleotides, to
produce a further working library of recombinant polynucleotides;
d) screening the further working library to identify at least one
further optimized non-stochastically generated polynucleotide that
exhibits an enhanced ability to modulate an immune response in
comparison to a parental polynucleotide from which the library was
created; and e) optionally repeating c) and d) as necessary, until
a desirable further optimized non-stochastically generated
polynucleotide that exhibits an enhanced ability to modulate an
immune response than a form of the nucleic acid from which the
library was created.
12. The method of any of claims 1-6, wherein the optimized
non-stochastically generated polynucleotide encodes a polypeptide
that can interact with a cellular receptor involved in mediating an
immune response; wherein the polypeptide acts as an agonist or
antagonist of the receptor.
13. The method of claim 12, wherein the cellular receptor is a
macrophage scavenger receptor.
14. The method of claim 12, wherein the cellular receptor is
selected from the group consisting of a cytokine receptor and a
chemokine receptor.
15. The method of claim 14, wherein the chemokine receptor is
CCR6.
16. The method of claim 12, wherein the polypeptide mimics the
activity of a natural ligand for the receptor but does not induce
immune reactivity to said natural ligand.
17. The method of claim 12, wherein the library is screened by: i)
expressing the non-stochastically generated progeny polynucleotides
so that the encoded polypeptides are produced as fusions with a
protein displayed on the surface of a replicable genetic package;
ii) contacting the replicable genetic packages with a plurality of
cells that display the receptor; and iii) identifying cells that
exhibit a modulation of an immune response mediated by the
receptor.
18. The method of claim 17, wherein the replicable genetic package
is selected from the group consisting of a bacteriophage, a cell, a
spore, and a virus.
19. The method of claim 18, wherein the replicable genetic package
is an M13 bacteriophage and the protein is encoded by geneIII or
geneVIII.
20. The method of claim 12, which method further comprises
introducing the optimized non-stochastically generated
polynucleotide into a genetic vaccine vector and administering the
vector to a mammal, wherein the peptide or polypeptide is expressed
and acts as an agonist or antagonist of the receptor.
21. The method of claim 12, which method further comprises
producing the polypeptide encoded by the optimized
non-stochastically generated polynucleotide and introducing the
polypeptide into a mammal in conjunction with a genetic vaccine
vector.
22. The method of claim 12, wherein the optimized
non-stochastically generated polynucleotide is inserted into an
antigen-encoding nucleotide sequence of a genetic vaccine
vector.
23. The method of claim 22, wherein the optimized
non-stochastically generated polypeptide is introduced into a
nucleotide sequence that encodes an M-loop of an HBsAg
polypeptide.
24. The method of any of claims 1-6, wherein the optimized
non-stochastically generated polynucleotide comprises a nucleotide
sequence rich in unmethylated CpG.
25. The method of any of claims 1-6, wherein the optimized
non-stochastically generated polynucleotide encodes a polypeptide
that inhibits an allergic reaction.
26. The method of claim 25, wherein the polypeptide is selected
from the group consisting of interferon-.alpha.,
interferon-.gamma., IL-10, IL-12, an antagonist of IL-4, an
antagonist of IL-5, and an antagonist of IL-13.
27. The method of 1, wherein the optimized recombinant
polynucleotide encodes an antagonist of IL-10.
28. The method of claim 27, wherein the antagonist of IL-10 is
soluble or defective IL-10 receptor or IL-20/MDA-7.
29. The method of any of claims 1-6, wherein the optimized
non-stochastically generated polynucleotide encodes a
co-stimulator.
30. The method of claim 29, wherein the co-stimulator is B7-1
(CD80) or B7-2 (CD86) and the screening step involves selecting
variants with altered activity through CD28 or CTLA-4.
31. The method of claim 29, wherein the co-stimulator is CD1, CD40,
CD154 (ligand for CD40) or CD150 (SLAM).
32. The method of claim 29, wherein the co-stimulator is a
cytokine.
33. The method of claim 32, wherein the cytokine is selected from
the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,
IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-16,
IL-17, IL-18, GM-CSF, G-CSF, TNF-.alpha., IFN-.alpha., IFN-.gamma.,
and IL-20 (MDA-7).
34. The method of 33, wherein the library of non-stochastically
generated polynucleotides is screened by testing the ability of
cytokines encoded by the non-stochastically generated
polynucleotides to activate cells which contain a receptor for the
cytokine.
35. The method of claim 34, wherein the cells contain a
heterologous nucleic acid that encodes the receptor for the
cytokine.
36. The method of 33, wherein the cytokine is interleukin-12 and
the screening is performed by: growing mammalian cells which
contain the genetic vaccine vector in a culture medium; and
detecting whether T cell proliferation or T cell differentiation is
induced by contact with the culture medium.
37. The method of 33, wherein the cytokine is interferon-.alpha.
and the screening is performed by: i) expressing the
non-stochastically generated polynucleotides so that the encoded
polypeptides are produced as fusions with a protein displayed on
the surface of a replicable genetic package; ii) contacting the
replicable genetic packages with a plurality of B cells; and iii)
identifying phage library members that are capable of inhibiting
proliferation of the B cells.
38. The method of claim 33, wherein the immune response of interest
is differentiation of T cells to T.sub.H1 cells and the screening
is performed by contacting a population of T cells with the
cytokines encoded by the members of the library of recombinant
polynucleotides and identifying library members that encode a
cytokine that induces the T cells to produce IL-2 and
interferon-.gamma..
39. The method of claim 32, wherein the cytokine encoded by the
optimized non-stochastically generated polynucleotide exhibits
reduced immunogenicity compared to a cytokine encoded by a
non-optimized polynucleotide, and the reduced immunogenicity is
detected by introducing a cytokine encoded by the
non-stochastically generated polynucleotide into a mammal and
determining whether an immune response is induced against the
cytokine.
40. The method of claim 29, wherein the co-stimulator is B7-1
(CD80) or B7-2 (CD86) and the cell is tested for ability to
costimulate an immune response.
41. The method of any of claims 1-6, wherein the optimized
recombinant polynucleotide encodes a cytokine antagonist.
42. The method of claim 41, wherein the cytokine antagonist is
selected from the group consisting of a soluble cytokine receptor
and a transmembrane cytokine receptor having a defective signal
sequence.
43. The method of claim 41, wherein the cytokine antagonist is
selected from the group consisting of .DELTA.IL-1 OR and
.DELTA.IL-4R.
44. The method of any of claims 1-6, wherein the optimized
non-stochastically generated polynucleotide encodes a polypeptide
capable of inducing a predominantly T.sub.H1 immune response.
45. The method of any of claims 1-6, wherein the optimized
non-stochastically generated polynucleotide encodes a polypeptide
capable of inducing a predominantly T.sub.H2 immune response.
Decreased Immune Response
46. The method of any of claims 1-6, wherein said optimized
modulatory effect on an immune response is a decrease in an
unwanted modulatory effect on an immune response; whereby
application of the method can be used to generate a molecule having
a decreased ability to elicit an immune response from a host
recipient of said molecule, where said recipient can be a human or
an animal host; and whereby application of the method can thus be
used to generate a molecule having decreased antigenicity with
respect to at least one host recipient of said molecule. Increased
Immune Response
47. The method of any of claims 1-6, wherein said optimized
modulatory effect on an immune response is an increase in a
desirable modulatory effect on an immune response; whereby
application of the method can be used to generate a molecule having
an increased ability to elicit an immune response from a host
recipient of said molecule, where said recipient can be a human or
an animal host; and whereby application of the method can thus be
used to generate a molecule having increased antigenicity with
respect to at least one host recipient of said molecule. Decreased
and Increased Immune Response
48. The method of any of claims 1-6, wherein said optimized
modulatory effect on an immune response is both a decrease in a
first unwanted modulatory effect on an immune response as well as
an increase in a second desirable modulatory effect on an immune
response; whereby application of the method can be used to generate
a molecule having both a decreased ability to elicit a first immune
response from a first host recipient of said molecule as well as a
an increased ability to elicit a second immune response from a
second host recipient of said molecule; whereby the first and the
second recipient hosts can be the same or different; whereby each
of the first and the second recipient hosts can be a human or an
animal host; and whereby application of the method can thus be used
to generate a molecule having both a first decreased antigenicity
with respect to at least one host recipient of said molecule and a
second decreased antigenicity with respect to at least one host
recipient of said molecule.
49. The method of claim 48, wherein said first and said second
modulatory effect on an immune response are evolved for
respectively a first and a second module on the same multimodule
vaccine vector; whereby a module is exemplified by the following
modules, as well as by a fragment derivative or analog thereof: an
antigen coding sequence, a polyadenylation sequence, a sequence
coding for a co-stimulatory molecule, a sequence coding for an
inducible repressor or transactivator, a eukaryotic origin or
replication, a prokaryotic origin of replication, a sequence coding
for a prokaryotic marker,, and enhancer, a promoter, and operator,
and an intron. Stability
50. The method of any of claims 1-6, wherein the optimized
modulatory effect on an immune response is comprised of an increase
in the stability of the immunomodulatory (IM) polynucleotide or
polypeptide encoded thereby; whereby application of the method can
be used to generate a molecule having an increased stability ex
vivo, thus, for example, increasing shelf-life and/or ease of
storage and/or length of time before expiration of activity upon
storage; and whereby application of the method can also be used to
generate a molecule having an increased stability in vivo upon
administration to a host recipient, thus, for example, increasing
resistance to digestive acids and/or increasing stability in the
circulation and/or any other method of elimination or destruction
by the host recipient. Human Vaccines
50. The method of any of claims 1-6, wherein the immunomodulatory
(IM) polynucleotide or polypeptide encoded thereby; has an
optimized modulatory effect on an immune response in a human host
recipient; whereby application of the method can thus be used to
generate an optimized genetic vaccine for human recipeints. Animal
Vaccines
51. The method of any of claims 1-6, wherein the immunomodulatory
(IM) polynucleotide or polypeptide encoded thereby; has an
optimized modulatory effect on an immune response in an animal host
recipient; whereby application of the method can thus be used to
generate an optimized genetic vaccine for animal recipients,
including animals that are farmed or raised by man, animals that
are not farmed or raised by man, domesticated animals, and
non-domesticated animals. Accessory Molecules
52. A method for obtaining an optimized polynucleotide that encodes
an accessory molecule that improves the transport or presentation
of antigens by a cell, the method comprising: a) creating a library
of non-stochastically generated polynucleotides by subjecting to
optimization by non-stochastic directed evolution a parental
polynucleotide set in which is encoded all or part of the accessory
molecule; and b) screening the library to identify an optimized
non-stochastically generated progeny polynucleotide that encodes a
recombinant molecule that confers upon a cell an increased or
decreased ability to transport or present an antigen on a surface
of the cell compared to an accessory molecule encoded by template
polynucleotides not subjected to the non-stochastic reassembly;
whereby application of the method can thus be used to generate an
optimized molecule for human recipients &/or animal recipients,
including animals that are farmed or raised by man, animals that
are not farmed or raised by man, domesticated animals, and
non-domesticated animals; whereby optimization can thus be achieved
using one or more of the directed evolution methods as described
herein in any combination, permutation, and iterative manner;
whereby these directed evolution methods include the introduction
of point mutations by non-stochastic methods, including by "gene
site saturation mutagenesis" as described herein; and whereby these
directed evolution methods also include the introduction mutations
by non-stochastic polynucleotide reassembly methods as described
herein; including by synthetic ligation polynucleotide reassembly
as described herein.
53. The method of claim 52, wherein the screening involves: i)
introducing the library of non-stochastically generated
polynucleotides into a genetic vaccine vector that encodes an
antigen to form a library of vectors; introducing the library of
vectors into mammalian cells; and ii) identifying mammalian cells
that exhibit increased or decreased immunogenicity to the
antigen.
54. The method of claim 52, wherein the accessory molecule
comprises a proteasome or a TAP polypeptide.
55. The method of claim 52, wherein the accessory molecule
comprises a cytotoxic T-cell inducing sequence.
56. The method of claim 55, wherein the cytotoxic T-cell inducing
sequence is obtained from a hepatitis B surface antigen.
57. The method of claim 52, wherein the accessory molecule
comprises an immunogenic agonist sequence. Plant Expression
58. A method for obtaining an immunomodulatory polynucleotide that
has, an optimized expression in a recombinant expression host, the
method comprising: creating a library of non-stochastically
generated progeny polynucleotides from a parental polynucleotide
set; whereby optimization can thus be achieved using one or more of
the directed evolution methods as described herein in any
combination, permutation and iterative manner; whereby these
directed evolution methods include the introduction of mutations by
non-stochastic methods, including by "gene site saturation
mutagenesis" as described herein; and whereby these directed
evolution methods also include the introduction mutations by
non-stochastic polynucleotide reassembly methods as described
herein; including by synthetic ligation polynucleotide reassembly
as described herein.
59. A method for obtaining an immunomodulatory polynucleotide that
has an optimized expression in a recombinant expression host, the
method comprising: screening a library of non-stochastically
generated progeny polynucleotides to identify an optimized
non-stochastically generated progeny polynucleotide that has an
optimized expression in a recombinant expression host when compared
to the expression of a parental polynucleotide from which the
library was created.
60. A method for obtaining an immunomodulatory polynucleotide that
has an optimized expression in a recombinant expression host, the
method comprising: a) creating a library of non-stochastically
generated progeny polynucleotides from a parental polynucleotide
set; and b) screening a library of non-stochastically generated
progeny polynucleotides to identify an optimized non-stochastically
generated progeny polynucleotide that has an optimized expression
in a recombinant expression host when compared to the expression of
a parental polynucleotide from which the library was created;
whereby optimization can thus be achieved using one or more of the
directed evolution methods as described herein in any combination,
permutation, and iterative manner; whereby these directed evolution
methods include the introduction of point mutations by
non-stochastic methods, including by "gene site saturation
mutagenesis" as described herein; and whereby these directed
evolution methods also include the introduction mutations by
non-stochastic polynucleotide reassembly methods as described
herein; including by synthetic ligation polynucleotide reassembly
as described herein.
61. The method of any of claims 58-60, wherein the recombinant
expression host is a prokaryote.
62. The method of any of claims 58-60, wherein the recombinant
expression host is a eukaryote.
63. The method of claim 62, wherein the recombinant expression host
is a plant.
64. The method of any of claims 63, wherein the recombinant
expression host is a monocot.
65. The method of any of claims 63, wherein the recombinant
expression host is a dicot.
66. The method of any of claims 1-6, 52, or 58-60, wherein creating
a library of non-stochastically generated progeny polynucleotides
from a parental polynucleotide set is comprised of subjecting the
parental polynucleotide set to "gene site saturation mutagenesis"
as described herein.
67. The method of any of claims 1-6, 52, or 58-60, wherein creating
a library of non-stochastically generated progeny polynucleotides
from a parental polynucleotide set is comprised of subjecting the
parental polynucleotide set to "synthetic ligation polynucleotide
reassembly" as described herein.
68. The method of any of claims 1-6, 52, or 58-60, wherein creating
a library of non-stochastically generated progeny polynucleotides
from a parental polynucleotide set is comprised of subjecting the
parental polynucleotide set to both "gene site saturation
mutagenesis" as described herein, and to "synthetic ligation
polynucleotide reassembly" as described herein.
Description
[0001] The present application is a continuation-in-part of U.S.
application Ser. No. 09/276,860, filed on Mar. 26, 1999 (entitled
Exonuclease-Mediated Gene Assembly in Directed Evolution), which is
hereby incorporated by reference, which is a continuation-in-part
of U.S. application Ser. No. 09/267118, filed on Mar. 9, 1999
(entitled End Selection in Directed Evolution), which is hereby
incorporated by reference, which is a continuation-in part of U.S.
application Ser. No. 09/246178, filed Feb. 4, 1999 (entitled
Saturation Mutagenesis in Directed Evolution), which is hereby
incorporated by reference; which is a continuation-in part of U.S.
application Ser. No. 09/185,373 filed on Nov. 3, 1998 (entitled
Directed Evolution of Thermophilic Enzymes), which is hereby
incorporated by reference; which is a continuation of U.S.
application Ser. No. 08/760,489 filed on Dec. 5, 1996 (entitled
Directed Evolution of Thermophilic Enzymes, now U.S. Pat. No.
5,830,696), which is hereby incorporated by reference; which is a
continuation-in-part of U.S. provisional application No. 60/008,311
filed on Dec. 7, 1995, which is hereby incorporated by
reference.
[0002] U.S. application Ser. No. 09/246178, filed Feb. 4, 1999
(entitled Saturation Mutagenesis in Directed Evolution) is also a
continuation-in-part of U.S. application Ser. No. 08/962,504 filed
on Oct. 31, 1997 (entitled Method of DNA Shuffling), which is
hereby incorporated by reference; which is a continuation-in-part
of U.S. application Ser. No. 08/677,112 filed on Jul. 9, 1996
(entitled Method of DNA Shuffling with Polynucleotides Produced by
Blocking or Interrupting A Synthesis or Amplification Process, now
U.S. Pat. No. 5,965,408), which is hereby incorporated by
reference.
[0003] U.S. application Ser. No. 09/246178, filed Feb. 4, 1999
(entitled Saturation Mutagenesis in Directed Evolution) is also a
continuation-in-part of U.S. application Ser. No. 08/651,568 filed
on May 22, 1996 (entitled Combinatorial Enzyme Development, now
U.S. Pat. No. 5,939,250), which is hereby incorporated by
reference; which is a continuation-in-part of U.S. provisional
application serial No. 60/008,316, filed Dec. 7, 1995, which is
hereby incorporated by reference.
CONTENTS
[0004] 1. GENERAL
[0005] 1.1. FIELD OF THE INVENTION
[0006] 1.2. BACKGROUND
[0007] 1.3. SUMMARY OF THE INVENTION
[0008] 1.4. BRIEF DESCRIPTION OF THE DRAWINGS
[0009] 2. DETAILED DESCRIPTION OF THE INVENTION
[0010] 2.1. DEFINITIONS
[0011] 2.2. GENERAL CONSIDERATIONS & FORMATS FOR
RECOMBINATION
[0012] 2.3. VECTORS USED IN GENETIC VACCINATION
[0013] 2.3.1. VIRAL VECTORS
[0014] 2.3.1.1. ADENOVIRUSES
[0015] 2.3.1.2. ADENO-ASSOCIATED VIRUS (AAV)
[0016] 2.3.1.3. PAPILLOMA VIRUS
[0017] 2.3.1.4. RETROVIRUSES
[0018] 2.3.2. NON-VIRAL GENETIC VACCINE VECTORS
[0019] 2.4. MULTICOMPONENT GENETIC VACCINES
[0020] 2.4.1. VECTOR "AR",DESIGNED TO PROVIDE OPTIMAL ANTIGEN
RELEASE
[0021] 2.4.2. VECTOR COMPONENTS "CTL-DC", "CTL-LC" AND "CTL-MM"I
DESIGNED FOR OPTIMAL PRODUCTION OF CTLs
[0022] 2.4.3. VECTORS "M" DESIGNED FOR OPTIMAL RELEASE OF IMMUNE
MODULATORS
[0023] 2.4.4. VECTORS "CK", DESIGNED TO DIRECT RELEASE OF
CHEMOKINES
[0024] 2.4.5. OTHER VECTORS
[0025] 2.5. SCREENING METHODS
[0026] 2.5.1. SCREENING FOR VECTOR LONGEVITY OR TRANSLOCATION TO
DESIRED TISSUE
[0027] 2.5.1.1. SELECTION FOR EXPRESSION OF CELL SURFACE-LOCALIZED
ANTIGEN
[0028] 2.5.1.2. SELECTION FOR EXPRESSION OF SECRETED
ANTIGEN/CYTOKINE/CHEMOKINE
[0029] 2.5.2. FLOW CYTOMETRY
[0030] 2.5.3. ADDITIONAL IN VITRO SCREENING METHODS
[0031] 2.5.4. ANTIGEN LIBRARY IMMUNIZATION
[0032] 2.5.5. SCREENINGFOR OPTIMAL INDUCTION OF PROTECTIVE
IMMUNITY
[0033] 2.5.6. SCREENING OF GENETIC VACCINE VECTORS THAT ACTIVATE
HUMAN ANTIGEN-SPECIFIC LYMPHOCYTE RESPONSES
[0034] 2.5.7. SCID-HUMAN SKIN MODEL FOR VACCINATION STUDIES
[0035] 2.5.8. MOUSE MODEL FOR STUDYING THE EFFICIENCY OFGENETIC
VACCINES IN TRANSFECTING HUMAN MUSCLE CELLS AND INDUCING HUMAN
IMMUNE RESPONSES IN VIVO
[0036] 2.5.9. SCREENINGFOR IMPROVED DELIVERY OF VACCINES
[0037] 2.5.10. ENHANCED ENTRY OF GENETIC VACCINE VECTORS INTO
CELLS
[0038] 2.6. OPTIMIZATION OF GENETIC VACCINE COMPONENTS
[0039] 2.6.1. EPISOMAL VECTOR MAINTENANCE
[0040] 2.6.2. EVOLUTION OF OPTIMIZED PROMOTERS FOR EXPRESSION OF AN
ANTIGEN
[0041] 2.6.2.1. CONSTITUTIVE PROMOTERS
[0042] 2.6.2.2. CELL-SPECIFIC PROMOTERS
[0043] 2.6.2.3. INDUCIBLE PROMOTERS
[0044] 2.6.3. EVOLUTION OF BINDING POLYPEPTIDES THAT ENHANCE
SPECIFICITY AND EFFICIENCY OF GENETIC VACCINES
[0045] 2.6.4. EVOLUTION OF BACTERIOPHAGE VECTORS
[0046] 2.6.4.1. EVOLUTION OF EFFICIENT DELIVERY OF BACTERIGPLIAGE
VEHICLES BY INHALATION OR ORAL DELIVE
[0047] 2.6.4.2. EVOLUTION OF BACTERIOPHAGE VEHICLES FOR EFFICIENT
HOMING TO APCs
[0048] 2.6.4.3. EVOLUTION OF BACTERIOPHAGE FOR INVASION OF APCs
[0049] 2.6.5. EVOLUTION OF IMPROVED IMMUNOMODULATORY SEQUENCES
[0050] 2.6.5.1. IMMUNOSTIMULATORY DNA SEQUENCES
[0051] 2.6.5.2. CYTOKINES, CHEMOKINES, AND ACCESSORY MOLECULES
[0052] 2.6.5.3. AGONISTS OR ANTAGONISTS OF CELLULAR RECEPTORS
[0053] 2.6.5.4. COSTIMULATORY MOLECULES CAPABLE OF INHIBITING OR
ENHANCING ACTIVATION, DIFFERENTIATION, OR ANERGY OF
ANTIGEN-SPECIFIC T CELLS
[0054] 2.6.6. EVOLUTION OF GENETIC VACCINE VECTORS FOR INCREASED
VACCINATION EFFICACY AND EASE OF VACCINATION
[0055] 2.6.6.1. TOPICAL APPLICATION OF GENETIC VACCINE VECTORS
[0056] 2.6.6.2. ENHANCED ABILITY TO ESCAPE HOST IMMUNE SYSTEM
[0057] 2.6.6.3. ENHANCED ANTIVIRAL ACTIVITY
[0058] 2.6.6.4. EVOLUTION OF VECTORS HAVING INCREASED COPY NUMBER
IN PRODUCTION CELLS
[0059] 2.7. OPTIMIZATION OF TRANSPORT AND PRESENTATION
OFANTIGENS
[0060] 2.7.1. PROTEASOMES
[0061] 2.7.2. ANTIGEN TRANSPORT
[0062] 2.7.3. CYTOTOXIC T-CELL INDUCING SEQUENCES AND IMMUNOGENIC
AGONIST SEQUENCES
[0063] 2.8. GENETIC VACCINE PHARMACEUTICAL COMPOSITIONS AND METHODS
OF ADMINISTRATION
[0064] 2.9. USES OF GENETIC VACCINES
[0065] 2.9.1. INFECTIOUS DISEASES
[0066] 2.9.1.1. BACTERIAL PATHOGENS AND TOXINS
[0067] 2.9.1.2. VIRAL PATHOGENS
[0068] 2.9.2. INFLAMMATORY AND AUTOIMMUNE DISEASES
[0069] 2.9.3. ALLERGY AND ASTHMA
[0070] 2.9.4. CANCER
[0071] 2.9.5. PARASITES
[0072] 2.9.6. CONTRACEPTION
[0073] 2.10. MALARIAL ANTIGENS AND VACCINES
[0074] 2.10.1. MALARIAL POLYPEPTIDES
[0075] 2.10.2. MALARIAL NUCLEIC ACIDS AND CELLS CAPABLE OF
EXPRESSING SAME
[0076] 2.10.3. ANTIBODIES
[0077] 2.10.4. METHODS OF USE
[0078] 2.10.4.1. DIAGNOSTIC APPLICATIONS
[0079] 2.10.4.2. SCREENING APPLICATIONS
[0080] 2.10.4.3. THERAPEUTIC AND PROPHYLACTIC APPLICATIONS
[0081] 2.11. DIRECTED EVOLUTION METHODS
[0082] 2.11.1. SATURATION MUTAGENESIS
[0083] 2.11.2. CHIMERIZATIONS
[0084] 2.11.2.1. "SHUFFLING"
[0085] 2.11.2.2. EXONUCLEASE-MEDIATED REASSEMBLY
[0086] 2.11.2.3. NON-STOCHASTIC LIGATION REASSEMBLY
[0087] 2.11.2.4. END-SELECTION
[0088] 2.11.3. ADDITIONAL SCREENING METHODS
[0089] 3. LITERATURE CITED
1. GENERAL
1.1. FIELD OF THE INVENTION
[0090] This invention pertains to the field of genetic vaccines.
Specifically, the invention provides multi-component genetic
vaccines that contain components that are optimized for a
particular vaccination goal. In a particular aspect this invention
provides methods for improving the efficacy of genetic vaccines by
providing materials that facilitate targeting of a genetic vaccine
to a particular tissue or cell type of interest.
[0091] This invention also pertains to the field of modulation of
immune responses such as those induced by genetic vaccines and also
pertains to the field of methods for developing immunogens that can
induce efficient immune responses against a broad range of
antigens.
[0092] Thus, the present invention also relates generally to novel
proteins, and fragments thereof, as well as nucleic acids which
encode these proteins, and methods of making and using these
proteins in diagnostic, prophylactic and therapeutic applications.
In a particular exemplification, the present invention relates to
proteins from the Plasmodium falciparum erythrocyte membrane
protein 1 ("PfEMP 1") gene family and fragments thereof which are
derived from malaria parasitized erythrocytes. In particular, these
proteins are derived from the erythrocyte membrane protein of
Plasmodium falciparum parasitized erythrocytes, also termed
"PfEMP1". The present invention also provides nucleic acids
encoding these proteins, which proteins and nucleic acids are
associated with the pathology of malaria infections, and which may
be used as vaccines or other prophylactic treatments for the
prevention of malaria infections, and/or in diagnosing and treating
the symptoms of patients who suffer from malaria and associated
diseases.
[0093] This invention also relates to the field of protein
engineering. Specifically, this invention relates to a directed
evolution method for preparing a polynucleotide encoding a
polypeptide. More specifically, this invention relates to a method
of using mutagenesis to generate a novel polynucleotide encoding a
novel polypeptide, which novel polypeptide is itself an improved
biological molecule &/or contributes to the generation of
another improved biological molecule. More specifically still, this
invention relates to a method of performing both non-stochastic
polynucleotide chimerization and non-stochastic site-directed point
mutagenesis.
[0094] Thus, in one aspect, this invention relates to a method of
generating a progeny set of chimeric polynucleotide(s) by means
that are synthetic and non-stochastic, and where the design of the
progeny polynucleotide(s) is derived by analysis of a parental set
of polynucleotides &/or of the polypeptides correspondingly
encoded by the parental polynucleotides. In another aspect this
invention relates to a method of performing site-directed
mutagenesis using means that are exhaustive, systematic, and
non-stochastic.
[0095] Furthermore this invention relates to a step of selecting
from among a generated set of progeny molecules a subset comprised
of particularly desirable species, including by a process termed
end-selection, which subset may then be screened further. This
invention also relates to the step of screening a set of
polynucleotides for the production of a polypeptide &/or of
another expressed biological molecule having a useful property.
[0096] Novel biological molecules whose manufacture is taught by
this invention include genes, gene pathways, and any molecules
whose expression is affected thereby, including directly encoded
polypetides &/or any molecules affected by such polypeptides.
Said novel biological molecules include those that contain a
carbohydrate, a lipid, a nucleic acid, &/or a protein
component, and specific but non-limiting examples of these include
antibiotics, antibodies, enzymes, and steroidal and non-steroidal
hormones.
[0097] In a particular non-limiting aspect, the present invention
relates to enzymes, particularly to thermostable enzymes, and to
their generation by directed evolution. More particularly, the
present invention relates to thermostable enzymes which are stable
at high temperatures and which have improved activity at lower
temperatures.
1.2. BACKGROUND
[0098] Providing Protective Immunity Even in Situations When the
Pathogens are Poorly Characterized or Cannot be Isolated or
Cultured in Laboratory Environment
[0099] Genetic immunization represents a novel mechanism of
inducing protective humoral and cellular immunity. Vectors for
genetic vaccinations generally consist of DNA that includes a
promoter/enhancer sequence, the gene of interest and a
polyadenylation/transcriptional terminator sequence. After
intramuscular or intradermal injection, the gene of interest is
expressed, followed by recognition of the resulting protein by the
cells of the immune system. Genetic immunizations provide means to
induce protective immunity even in situations when the pathogens
are poorly characterized or cannot be isolated or cultured in
laboratory environment.
[0100] Small Improvement in the Efficiency of Genetic Vaccine
Vectors can Result in Dramatic Increase if the Level of Immune
Response
[0101] The efficacy of genetic vaccination is often limited by
inefficient uptake of genetic vaccine vectors into cells.
Generally, less than 1% of the muscle or skin cells at the sites of
injections express the gene of interest. Even a small improvement
in the efficiency of genetic vaccine vectors to enter the cells can
result in a dramatic increase in the level of immune response
induced by genetic vaccination. A vector typically has to cross
many barriers which can result in only a very minor fraction of the
DNA ever being expressed.
[0102] Various Limitations to Immunogenicity
[0103] Limitations to immunogenicity include: loss of vector due to
nucleases present in blood and tissues; inefficient entry of DNA
into a cell; inefficient entry of DNA into the nucleus of the cell
and preference of DNA for other compartments; lack of DNA stability
in the nucleus (factor limiting nuclear stability may differ from
those affecting other cellular and extracellular compartments),
and, for vectors that integrate into the chromosome, the efficiency
of integration and the site of integration. Moreover, for many
applications of genetic vaccines, it is preferable for the genetic
vaccine to enter a particular target tissue or cell.
[0104] Thus, a need exists for genetic vaccines that can be
targeted to specific cell and tissue types of interest, and which
exhibit an increased ability to enter the target cells. The present
invention fulfills these and other needs.
[0105] Pathways for Immune Responses Induced by Gentic Vaccines
[0106] Elicitation of a desired in vivo response by a genetic
vaccine generally requires multiple cellular processes in a complex
sequence. Several potential pathways exist along which a genetic
vaccine can exert its effect on the mammalian immune system. In one
pathway, the genetic vaccine vector enters cells that are the
predominant cell type in the tissue that receives vaccine (e.g.,
muscle or epithelial cells). These cells express and release the
antigen encoded by the vector. The vaccine vector can be engineered
to have the antigen released as an intact protein from living
transfected cells (i.e., via a secretion process) or directed to a
membrane-bound form on the surface of these cells. Antigen can also
be released from an intracellular compartment of such cells if
those cells die.
[0107] The Antigen Derived from Vaccine Vector Internalization and
Antigen Expression Within the Predominant Cell Type in the Tissue
Ends Up Within APC, Which Then Process the Antigen Internally to
Prime MHC Class I and or Class II, Essential Steps in Activation of
CD4.sup.+ T-Helper Cells and Development of Potent Specific Immune
Responses
[0108] Extracellular antigen derived from any of these situations
interacts with antigen presenting cells (APC) either by binding to
the cell surface (specifically via IgM or via other
non-immunoglobulin receptors) and subsequent endocytosis of outer
membrane, or by fluid phase micropinocytosis wherein the APC
internalizes extracellular fluid and its contents into an endocytic
compartment. Interaction with APC may occur before or after partial
proteolytic cleavage in the extracellular environment. In any case,
the antigen derived from vaccine vector internalization and antigen
expression within the predominant cell type in the tissue ends up
within APC. The APC then process the antigen internally to prime
MHC Class I and or Class II, essential steps in activation of
CD4.sup.+ T-helper cells (T.sub.H1 and/or T.sub.H2) and development
of potent specific immune responses.
[0109] The Genetic Vaccine Plasmid Enters APC and Antigen is
Proteolytically Cleaved in the Cell Cytoplasm
[0110] In a parallel pathway, the genetic vaccine plasmid enters
APC (or the predominant cell type in the tissue) and, instead of
antigen derived from plasmid expression being directed to
extracellular export, antigen is proteolytically cleaved in the
cell cytoplasm (in a proteasome dependent or independent process).
Often, intracellular processing in such cells occurs via
proteasomal degradation into peptides that are recognized by the
TAP-1 and TAP-2 proteins and transported into the lumen of the
rough endoplasmic reticulum (RER).
[0111] The Peptide Fragments are Transported into the RER Complex,
Expressed on the Cell Surface; in the Presence of Appropriate
Additional Signals, Can Differentiate into Functional CTLs
[0112] The peptide fragments transported into the RER complex with
MHC Class I. Such antigen fragments are then expressed on the cell
surface in association with Class I. CD8.sup.+ cytotoxic T
lymphocytes (CTL) bearing specific T cell receptor then recognize
the complex and can, in the presence of appropriate additional
signals, differentiate into functional CTLs.
[0113] By Virtue of Poorly Characterized Pathways for Trafficking
of Cytoplasmically Generated Peptides into Endosomal Compartments,
a Genetic Vaccine Vector Can Lead to CD4.sup.+ T Cell
Stimulation
[0114] In addition, poorly characterized pathways, which are
generally not dominant, exist in APC for trafficking of
cytoplasmically generated peptides into endosomal compartments
where they can end up complexed with MHC Class II, and thereby act
to present antigen peptides to CD4.sup.+ T.sub.H1 and T.sub.H2
cells. Because activation, proliferation, differentiation and
immunoglobulin isotype switching by B lymphocytes requires help of
CD4.sup.+ T cells, antigen presentation in the context of MHC Class
II molecules is crucial for induction of antigen-specific
antibodies. By virtue of this pathway, a genetic vaccine vector can
lead to CD4.sup.+ T cell stimulation in addition to the dominant
CD8.sup.+ CTL activation process described above. This alternative
pathway is, however, of little consequence in muscle cells where
levels of MHC Class II expression are very low or zero.
[0115] In This Case Cytokines are Derived Not Only from Processes
Intrinsic to the Interaction of DNA with Cells, or Specific Cell
Responses to the Antigen, But Via Synthesis Directed by the Vaccine
Plasmid
[0116] Genetic vaccination can also elicit cytokine release from
cells that bind to or take up DNA. So-called immunostimulatory or
adjuvant properties of DNA are derived from its interaction with
cells that internalize DNA. Cytokines can be released from cells
that bind and/or internalize DNA in the absence of gene
transcription. Separately, interaction of antigen with APC followed
by presentation and specific recognition also stimulates release of
cytokines that have positive feedback effects on these cells and
other immune cells. Chief among these effects are the direction of
CD4.sup.+ T.sub.H cells to differentiate/proliferate preferentially
to T.sub.H1 or T.sub.H2 phenotypes. Furthermore, cytokines released
at the site of DNA vaccination, regardless of the mechanism of
their release, contribute to recruitment of other immune cells from
the immediate local area and more distant sites such as draining
lymph nodes. In recognition of the importance of cytokines in
elicitation of a potent immune response, some investigators have
included the genes for one or more cytokines in the DNA vaccine
plasmid along with the target antigen for immunization. In this
case cytokines are derived not only from processes intrinsic to the
interaction of DNA with cells, or specific cell responses to the
antigen, but via synthesis directed by the vaccine plasmid.
[0117] Movement of Immune Cells From the Blood Stream and Different
Sites to the Site of Immunization and Also From the Site of
Immunization to Other Sites
[0118] Immune cells are recruited to the site of immunization from
distant sites or the bloodstream. Specific and non-specific immune
responses are then greatly amplified. Immune cells, including APC,
bearing antigen fragments complexed to MHC molecules or even
expressing antigen from uptake of plasmid, also move from the
immunization site to other sites (blood, hence to all tissues;
lymph nodes; spleen) where additional immune recruitment and
qualitative and quantitative development of the immune response
ensue.
[0119] Current Genetic Vaccine Vectors Employ Simple Methods for
Expression of the Desired Antigen with Few If Any Design Elements
That Control the Precise Intracellular Fate of the Antigen or the
Immunological Consequences of Antigen Expression
[0120] While these pathways often compete, previously available
genetic vaccines have incorporated all components for influencing
each of the pathways into a single polynucleotide molecule. Because
separate cell types are involved in the complex interactions
required for a potent immune response to a genetic vaccine vector,
mutually incompatible consequences can arise from administration of
a genetic vaccine that is incorporated in a single vector molecule.
Current genetic vaccine vectors employ simple methods for
expression of the desired antigen with few if any design elements
that control the precise intracellular fate of the antigen or the
immunological consequences of antigen expression. Thus, although
genetic vaccines show great promise for vaccine research and
development, the need for major improvements and several severe
limitations of these technologies are apparent.
[0121] Existing Genetic Vaccine Vectors Have Not Been Optimized for
Human Tissue, Providing Low and Short-Lasting Expression of the
Antigen of Interest, With Insufficient Stability, Inducibility, or
Levels of Expression in vivo, Among Other Things
[0122] Largely due to the lack of suitable laboratory models, none
of the existing genetic vaccine vectors have been optimized for
human tissues. The existing genetic vaccine vectors typically
provide low and short-lasting expression of the antigen of
interest, and even large quantities of DNA do not always result in
sufficiently high expression levels to induce protective immune
responses. Because the mechanisms of the vector entry into the
cells and transfer into the nucleus are poorly understood,
virtually no attempts have been made to improve these key
properties. Similarly, little is known about the mechanisms that
regulate the maintenance of vector functions, including gene
expression. Furthermore, although there is increasing amount of
data indicating that specific sequences alter the immunostimulatory
properties of the DNA, rational engineering is a very laborious and
time-consuming approach when using this information to generate
vector backbones with improved immunomodulatory properties.
[0123] Moreover, presently available genetic vaccine vectors do not
provide sufficient stability, inducibility or levels of expression
in vivo to satisfy the desire for vaccines which can deliver
booster immunization without additional vaccine administration.
Booster immunizations are typically required 3-4 weeks after the
primary injection with existing genetic vaccines.
[0124] Therefore a need exists for improved genetic vaccine vectors
and formulations, and methods for development of such vectors. The
present invention fulfills these and other needs.
[0125] The interactions between pathogens and hosts are results of
millions of years of evolution, during which the mammalian immune
system has evolved sophisticated means to counterattack pathogen
invasions. However, bacterial and viral pathogens have
simultaneously gained a number of mechanisms to improve their
virulence and survival in hosts, providing a major challenge for
vaccine research and development despite the powers of modem
techniques of molecular and cellular biology. Similar to the
evolution of pathogen antigens, several cancer antigens are likely
to have gained means to downregulate their immunogenicity as a
mechanism to escape the host immune system.
[0126] Efficient vaccine development is also hampered by the
antigenic heterogeneity of different strains of pathogens, driven
in part by evolutionary forces as means for the pathogens to escape
immune defenses. Pathogens also reduce their immunogenicity by
selecting antigens that are difficult to express, process and/or
transport in host cells, thereby reducing the availability of
immunogenic peptides to the molecules initiating and modulating
immune responses. The mechanisms associated with these challenges
are complex, multivariate and rather poorly characterized.
Accordingly, a need exists for vaccines that can induce a
protective immune response against bacterial and viral pathogens.
The present invention fulfills this and other needs.
[0127] Antigen processing and presentation is only one factor which
determines the effectiveness of vaccination, whether performed with
genetic vaccines or more classical methods. Other molecules
involved in determining vaccine effectiveness include cytokines
(interleukins, interferons, chemokines, hematopoietic growth
factors, tumor necrosis factors and transforming growth factors),
which are small molecular weight proteins that regulate maturation,
activation, proliferation and differentiation of the cells of the
immune system.
[0128] Characteristic features of cytokines are pleiotropy and
redundancy; that is, one cytokine often has several functions and a
given function is often mediated by more than one cytokine. In
addition, several cytokines have additive or synergistic effects
with other cytokines, and a number of cytokines also share receptor
components.
[0129] Due to the complexity of the cytokine networks, studies on
the physiological significance of a given cytokine have been
difficult, although recent studies using cytokine gene-deficient
mice have significantly improved our understanding on the functions
of cytokines in vivo. In addition to soluble proteins, several
membrane-bound costimulatory molecules play a fundamental role in
the regulation of immune responses. These molecules include CD40,
CD40 ligand, CD27, CD80, CD86 and CD150 (SLAM), and they are
typically expressed on lymphoid cells after activation via antigen
recognition or through cell-cell interactions.
[0130] T helper (T.sub.H) cells, key regulators of the immune
system, are capable of producing a large number of different
cytokines, and based on their cytokine synthesis pattern T.sub.H
cells are divided into two subsets (Paul and Seder (1994) Cell 76:
241-251). T.sub.H1 cells produce high levels of IL-2 and IFN- and
no or minimal levels of IL-4, IL-5 and IL-13. In contrast, T.sub.H2
cells produce high levels of IL-4, IL-5 and IL-13, and IL-2 and
IFN-production is minimal or absent. T.sub.H1 cells activate
macrophages, dendritic cells and augment the cytolytic activity of
CD8.sup.+ cytotoxic T lymphocytes and NK cells (Id.), whereas
T.sub.H.sup.2 cells provide efficient help for B cells and they
also mediate allergic responses due to the capacity of T.sub.H2
cells to induce IgE isotype switching and differentiation of B
cells into IgE secreting cell (De Vries and Punnonen (1996) In
Cytokine regulation of humoral immunity: basic and clinical
aspects. Eds. Snapper, C. M., John Wiley & Sons, Ltd., West
Sussex, UK, p. 195- 215). The exact mechanisms that regulate the
differentiation of T helper cells are not fully understood, but
cytokines are believed to play a major role. IL-4 has been shown to
direct T.sub.H2 differentiation, whereas IL-12 induces development
of T.sub.H1 cells (Paul and Seder, supra.). In addition, it has
been suggested that membrane bound costimulatory molecules, such as
CD80, CD86 and CD150, can direct T.sub.H1 and/or T.sub.H2
development, and the same molecules that regulate T.sub.H cell
differentiation also affect activation, proliferation and
differentiation of B cells into Ig-secreting plasma cells (Cocks et
al. (1995) Nature 376: 260-263; Lenschow et al. (1996) Immunity 5:
285-293; Punnonen et al. (1993) Proc. Nat'l. Acad. Sci. USA 90:
3730-3734; Punnonen et al. (1997) J Exp. Med. 185: 993-1004).
[0131] Studies in both man and mice have demonstrated that the
cytokine synthesis profile of T helper (T.sub.H) cells plays a
crucial role in determining the outcome of several viral, bacterial
and parasitic infections. High frequency of T.sub.H1 cells
generally protects from lethal infections, whereas dominant
T.sub.H2 phenotype often results in disseminated, chronic
infections. For example, T.sub.HI phenotype is observed in
tuberculoid (resistant) form of leprosy and T.sub.H2 phenotype in
lepromatous, multibacillary (susceptible) lesions (Yamamura et al.
(199 1) Science 254: 277-279). Similarly, late-stage HIV patients
have T.sub.H2-like cytokine synthesis profiles, and T.sub.H1
phenotype has been proposed to protect from AIDS (Maggi et al.
(1994) J Exp. Med. 180: 489-495). Furthermore, the survival from
meningococcal septicemia is genetically determined based on the
capacity of peripheral blood leukocytes to produce TNF- and IL-10.
Individuals from families with high production of IL-10 have
increased risk of fatal meningococcal disease, whereas members of
families with high TNF-production were more likely to survive the
infection (Westendorp et al. (1997) Lancet 349: 170-173).
[0132] Cytokine treatments can dramatically influence
T.sub.H1/T.sub.H2 cell differentiation and macrophage activation,
and thereby the outcome of infectious diseases. For example, BALB/c
mice infected with Leishmania major generally develop a
disseminated fatal disease with a T.sub.H2 phenotype, but when
treated with anti-IL-4 mAbs or IL-12, the frequency of T.sub.H1
cells in the mice increases and they are able to counteract the
pathogen invasion (Chatelain et al. (1992) J Immunol. 148:
1182-1187). Similarly, IFN-protects mice from lethal Herpes Simplex
Virus (HSV) infection, and MCP-1 prevents lethal infections by
Pseudomonas aeruginosa or Salmonella typhimurium. In addition,
cytokine treatments, such as recombinant IL-2, have shown
beneficial effects in human common variable immunodeficiency
(Cunningham-Rundles et al. (1994) N. Engl. J Med. 331:
918-921).
[0133] The administration of cytokines and other molecules to
modulate immune responses in a manner most appropriate for treating
a particular disease can provide a significant tool for the
treatment of disease. However, presently available immunomodulator
treatments can have several disadvantages, such as insufficient
specific activity, induction of immune responses against, the
immunomodulator that is administered, and other potential problems.
Thus, a need exists for immunomodulators that exhibit improved
properties relative to those currently available. The present
invention fulfills this and other needs.
[0134] Erythrocytes infected with the malaria parasite P.
falciparum disappear from the peripheral circulation as they mature
from the ring stage to trophozoites (Bignami and Bastianeli,
Reforma Medica (1889) 6:1334-1335). This phenomenon, known as
sequestration, results from parasitized erythrocyte ("PE")
adherence to microvascular endothelial cells in diverse organs
(Miller, Am. J Trop. Med. Hyg. (1969) 18:860-865). Sequestration is
associated temporally with expression of knob protrusions (Leech et
al., J. Cell. Biol. (1984) 98:1256-1264), expression of a very
large antigenically variant surface protein, called PfEMP1 (Aley et
al., J. Exp. Med. (1984) 160:1585-1590; Leech et al., J. Exp. Med.
(1984) 159:1567-1575; Howard et al., Molec. Biochem. Parasitol.
(1988) 27:207-223), and expression of new receptor properties which
mediate adherence to endothelial cells (Miller, supra; Udeinya et
al., Science (1981) 213:555-557. Endothelial cell surface proteins
such as CD36, thrombospondin (TSP) and ICAM-1 have been identified
as major host receptors for mature PE. See, e.g., Barnwell et al.,
J. Immunol. (1985) 135:3494-3497; Roberts et al., Nature (1985)
318:64-66; and Berendt et al., Nature (1989) 341:57-59.
[0135] PE sequestration confers unique advantages for P. falciparum
parasites (Howard and Gilladoga, Blood (1989) 74:2603-2618), but
also contributes directly to the acute pathology of P. falciparum
(Miller et al., Science (1994) 264:1878-1883). Of the four human
malarias, only P. falciparum infection is associated with
neurological impairment and cerebral pathology seen increasingly in
severe drug-resistant malaria (Howard and Gilladoga, supra).
[0136] Although the genesis of human cerebral malaria is likely due
to a combination of factors including particular parasite
phenotypes (Berendt et al., Parasitol. Today (1994) 10:412-414),
inappropriate immune responses and the phenotype of endothelial
cell surface molecules in the cerebral microvasculature (Pasloske
and Howard, Ann. Rev. Med. (1994) :283-295), adherence of PE to
cerebral blood vessels and consequent local microvascular occlusion
is a major contributing factor. See, e.g., Berendt et al., supra;
Patnaik et al., Am. J. Trop. Med. Hyg. (1994) 51:642-647.
[0137] The capacity of P. falciparum PE to express variant forms of
PfEMP1 contributes to the special virulence of this parasite.
Variant parasites can evade variant-specific antibodies elicited by
earlier infections. The P. falciparum variant antigens have been
defined in vitro using antiserum prepared in Aotus monkeys infected
with individual parasite strains (Howard et al., Molec. Biochem.
Parasitol. (1988) 27:207-223). Antibodies raised against a
particular parasite will only react by PE agglutination, indirect
immuno-fluorescence or immunoelectronmicroscopy with PE from the
same strain (van Schravendijk et al., Blood (1991) 78:226-236).
[0138] Such studies with PE from malaria patients in diverse
geographic locations and sera from the same or different patients
confirm that PE in natural isolates express variant surface
antigens and that individual patients respond to infection by
production of isolate-specific antibodies (Marsh and Howard,
Science (1986) 231:150-153; Aguiar et al., Am. J. Trop. Med. Hyg.
(1992) 47:621-632; Iqbal et al., Trans. R. Soc. Trop. Med. Hyg.
(1993) 87:583-588. Expression of a variant antigen on PE has also
been demonstrated in several simian, murine and human malaria
species, including P. knowlesi (Brown and Brown, Nature (1965)
208:1286-1288; Barnwell et al., Infect. Immun. (1983) 40:985-994),
P. chabaudi (Gilks et al., Parasite Immunol. (1990) 12:45-64;
Brannan et al., Proc. R. Soc. Lond. Biol. Sci. (1994) 256:71- 75),
P. fragile (Handunnetti et al., J. Exp. Mod. (1987) 165:1269-1283)
and P. vivax (Mendis et al., Am. J. Txop. Med. Hyg. (1988)
38:42-46). Laboratory studies with P. knowlesi (Brown and Brown,
supra; Barnwell et al., supra) or P. falciparum (Hommel et al., J.
Exp. Med. (1983) 157:1137-1148) in monkeys and P. chabaudi in mice
(Gilks et al., supra) confirmed that antigenic variation at the PE
surface is associated with prolonged or chronic infection and the
capacity to repeatedly re-establish blood infection in previously
infected animals. Studies with cloned parasites demonstrated that
antigenic variants can arise with extraordinary frequency, e.g., 2%
per generation with P. falciparum (Roberts et al., Nature (1992)
357:689-692) and 1.6% per generation with P. chabaudi (Brannan et
al., supra).
[0139] PfEMP1 was identified as a .sup.125I-labeled, size diverse
protein (200-350 kD) on PE that is lacking from uninfected
erythrocytes, and that is also labeled by biosynthetic
incorporation of radiolabeled amino acids (Leech et al., J. Exp.
Med. (1984) 159:1567-1575; Howard et al., Molec. Biochem.
Parasitol. (1988) 27:207-223). PfEMP1 is not extracted from PE by
neutral detergents such as Triton X-100 but is extracted by SDS,
suggesting that it is linked to the erythrocyte cytoskeleton (Aley
et al., J. Med. Exp. (1984) 160:1585-1590). After addition of
excess Triton X-100, PfEMP1 is immunoreactive with appropriate
serum antibodies (Howard et al., (1988), supra). Mild
trypsinization of intact PE rapidly cleaves PfEMP1 from the cell
surface (Leech et al., J. Exp. Mod. (1984) 159:1567-1575). PfEMP1
bears antigenically diverse epitopes since it is immunoprecipitated
from particular strains of P. falciparum by antibodies from sera of
Aotus monkeys infected with the same strain, but not by antibodies
from animals infected with heterologous strains (Howard et al.
(1988), supra). Knobless PE derived from parasite passage in
splenectomized Aotus monkeys (Aley et al., supra) do not express
surface PfEMP1 and are not agglutinated with sera from immune
individuals or infected monkeys (Howard et al. (1988), supra;
Howard and Gilladoga, Blood (1989) 74:2603-2618). In general, sera
that react with the PE surface by indirect immunofluorescence and
antibody-mediated PE agglutination are the only sera to
immunoprecipitate .sup.125I-labeled PfEMP1 from any particular
strain (Howard et al., (1988), supra; van Schravendijk et al.,
Blood (1991) 78:226- 236; Biggs et al., J. Immunol. (1992)
149:2047-2054).
[0140] The adherence of parasitized erythrocytes to endothelial
cells is mediated by multiple receptor/counter-receptor
interactions, including CD36, thrombospondin and intracellular
adhesion molecule-1 (ICAM.sub.--1) as the major host cell receptors
(Howard and Gilladoga, Blood (1989) 74:2603- 2618, Pasloske and
Howard, Ann. Rev. Med. (1994) 45:283-295).
[0141] Vascular cell adhesion molecule-1 (VCAM-1) and endothelial
leukocyte adhesion molecule-1 (ELAM-1) have also been implicated as
additional endothelial cell receptors that can mediate adherence of
a minority of P. falciparum PE (Ockenhouse, et al., J. Exp. Med.
(1992) 176:1183-1189, and Howard and Paslaske, supra). The
adherence receptors on the surface of PE has not yet been
conclusively identified, and several molecules, including AG 332
(Udomsangpetch, et al., Nature (1989) 338:763-765), modified band 3
(Crandall, et al., Proc. Nat'l Acad. Sci. USA (1993) 90:4703-4707),
Sequestrin (Ockenhouse, Proc. Nat'l Acad. Sci. USA (1991)
88:3175-3179), and PfEMP1 (Howard and Gilladoga, supra, and
Pasloske and Howard, supra), have been proposed as candidates.
Several pieces of indirect evidence have linked expression of
PfEMP1 with the acquisition of new host protein receptor properties
on the surface of PE (Howard and Gilladoga, supra; Pasloske and
Howard, Ann. Rev. Med. (1994) 45:283-295). PE adherence is
correlated with the expression of PfEMP1 on the surface of mature
stage PE (Leech, et al., J. Exp. Med. (1984) 159:1567-1575).
Alterations in the adherence phenotype of the PE selected for in
vitro are usually associated with the emergence of new forms of
PfEMP1 (Biggs, et al., J. Immunol. (1992) 149:2047-2054; Roberts,
et al., Nature (1992) 357:689-692). Mild trypsinization of intact
mature PE cleaves the extracellular portion of PfEMP1 and at the
same time, reduces or eliminates PE cytoadherence (Leech, et al.,
supra) Previously described antibody mediated blockade or reversal
of cytoadherence is strain specific and is correlated with the
ability of the reacting sera to agglutinate the corresponding PE
and to immunoprecipitate the surface labeled .sup.125I-PfEMP1
(Howard, et al., Molec. Biochem. Parasitol. (1988) 27:207-224).
Pfalhesin (modified band 3) have been shown to bind CD36 under
non-physiological conditions (Crandall, et al., Exp. Parasitol.
(1994) 78:203-209). Sequestrin, which appears to be homologous to
PfEMP1, extracted with TX100 from knobless PE, was shown to bind to
immobilized CD36 (Ockenhouse, Proc. Nat'l Acad. Sci. USA (1991)
88:3175-3179).
[0142] The complex nature and/or mechanism of malarial antigenic
variation, and its particular virulence has created a need for
methods and compositions which may be useful in the treatment
diagnosis and prevention of malaria infections. The present
invention meets these and other needs.
[0143] General Overview of Problems & Considerations in
Directed Evolution
[0144] The approach, termed directed evolution, of experimentally
modifying a biological molecule towards a desirable property, can
be achieved by mutagenizing one or more parental molecular
templates and by idendifying any desirable molecules among the
progeny molecules. Currently available technologies in directed
evolution include methods for achieving stochastic (i.e. random)
mutagenesis and methods for achieving non-stochastic (non-random)
mutagenesis. However, critical shortfalls in both types of methods
are identified in the instant disclosure.
[0145] In prelude, it is noteworthy that it may be argued
philosophically by some that all mutagenesis--if considered from an
objective point of view--is non-stochastic; and furthermore that
the entire universe is undergoing a process that--if considered
from an objective point of view--is non-stochastic. Whether this is
true is outside of the scope of the instant consideration.
Accordingly, as used herein, the terms "randomness", "uncertainty",
and "unpredictability" have subjective meanings, and the knowledge,
particularly the predictive knowledge, of the designer of an
experimental process is a determinant of whether the process is
stochastic or non-stochastic.
[0146] By way of illustration, stochastic or random mutagenesis is
exemplified by a situation in which a progenitor molecular template
is mutated (modified or changed) to yield a set of progeny
molecules having mutation(s) that are not predetermined. Thus, in
an in vitro stochastic mutagenesis reaction, for example, there is
not a particular predetermined product whose production is
intended; rather there is an uncertainty--hence
randomness--regarding the exact nature of the mutations achieved,
and thus also regarding the products generated. In contrast,
non-stochastic or non-random mutagenesis is exemplified by a
situation in which a progenitor molecular template is mutated
(modified or changed) to yield a progeny molecule having one or
more predetermined mutations. It is appreciated that the presence
of background products in some quantity is a reality in many
reactions where molecular processing occurs, and the presence of
these background products does not detract from the non-stochastic
nature of a mutagenesis process having a predetermined product.
[0147] Thus, as used herein, stochastic mutagenesis is manifested
in processes such as error-prone PCR and stochastic shuffling,
where the mutation(s) achieved are random or not predetermined. In
contrast, as used herein, non-stochastic mutagenesis is manifested
in instantly disclosed processes such as gene site-saturation
mutagenesis and synthetic ligation reassembly, where the exact
chemical structure(s) of the intended product(s) are
predetermined.
[0148] In brief, existing mutagenesis methods that are
non-stochastic have been serviceable in generating from one to only
a very small number of predetermined mutations per method
application, and thus produce per method application from one to
only a few progeny molecules that have predetermined molecular
structures. Moreover, the types of mutations currently available by
the application of these non-stochastic methods are also limited,
and thus so are the types of progeny mutant molecules.
[0149] In contrast, existing methods for mutagenesis that are
stochastic in nature have been serviceable for generating somewhat
larger numbers of mutations per method application--though in a
random fashion & usually with a large but unavoidable
contingency of undesirable background products. Thus, these
existing stochastic methods can produce per method application
larger numbers of progeny molecules, but that have undetermined
molecular structures. The types of mutations that can be achieved
by application of these current stochastic methods are also
limited, and thus so are the types of progeny mutant molecules.
[0150] It is instantly appreciated that there is a need for the
development of non-stochastic mutagenesis methods that:
[0151] 1) Can be used to generate large numbers of progeny
molecules that have predetermined molecular structures;
[0152] 2) Can be used to readily generate more types of
mutations;
[0153] 3) Can produce a correspondingly larger variety of progeny
mutant molecules;
[0154] 4) Produce decreased unwanted background products;
[0155] 5) Can be used in a manner that is exhaustive of all
possibilities; and
[0156] 6) Can produce progeny molecules in a systematic &
non-repetitive way.
[0157] The instant invention satisfies all of these needs.
[0158] Directed Evolution Supplements Natural Evolution: Natural
evolution has been a springboard for directed or experimental
evolution, serving both as a reservoir of methods to be mimicked
and of molecular templates to be mutagenized. It is appreciated
that, despite its intrinsic process-related limitations (in the
types of favored &/or allowed mutagenesis processes) and in its
speed, natural evolution has had the advantage of having been in
process for millions of years & and throughout a wide diversity
of environments. Accordingly, natural evolution (molecular
mutagenesis and selection in nature) has resulted in the generation
of a wealth of biological compounds that have shown usefulness in
certain commercial applications.
[0159] However, it is instantly appreciated that many unmet
commercial needs are discordant with any evolutionary pressure
&/or direction that can be found in nature. Moreover, it is
often the case that when commercially useful mutations would
otherwise be favored at the molecular level in nature, natural
evolution often overrides the positive selection of such mutations,
e.g. when there is a concurrent detriment to an organism as a whole
(such as when a favorable mutation is accompanied by a detrimental
mutation). Additionally, natural evolution is often slow, and
favors fidelity in many types of replication. Additionally still,
natural evolution often favors a path paved mainly by consecutive
beneficial mutations while tending to avoid a plurality of
successive negative mutations, even though such negative mutations
may prove beneficial when combined, or may lead--through a
circuitous route--to final state that is beneficial.
[0160] Moreover, natural evolution advances through specific steps
(e.g. specific mutagenesis and selection processes), with avoidance
of less favored steps. For example, many nucleic acids do not reach
close enough proximity to each other in a operative environment to
undergo chimerization or incorporation or other types of transfers
from one species to another. Thus, e.g., when sexual intercourse
between 2 particular species is avoided in nature, the
chimerization of nucleic acids from these 2 species is likewise
unlikely, with parasites common to the two species serving as an
example of a very slow passageway for inter-molecular encounters
and exchanges of DNA. For another example, the generation of a
molecule causing self-toxicity or self-lethality or sexual
sterility is avoided in nature. For yet another example, the
propagation of a molecule having no particular immediate benefit to
an organism is prone to vanish in subsequent generations of the
organism. Furthermore, e.g., there is no selection pressure for
improving the performance of molecule under conditions other than
those to which it is exposed in its endogenous environment; e.g. a
cytoplasmic molecule is not likely to acquire functional features
extending beyond what is required of it in the cytoplasm.
Furthermore still, the propagation of a biological molecule is
susceptible to any global detrimental effects--whether caused by
itself or not--on its ecosystem. These and other characteristics
greatly limit the types of mutations that can be propagated in
nature.
[0161] On the other hand, directed (or experimental)
evolution--particularly as provided herein--can be performed much
more rapidly and can be directed in a more streamlined manner at
evolving a predetermined molecular property that is commercially
desirable where nature does not provide one &/or is not likely
to provide. Moreover, the directed evolution invention provided
herein can provide more wide-ranging possibilities in the types of
steps that can be used in mutagenesis and selection processes.
Accordingly, using templates harvested from nature, the instant
directed evolution invention provides more wide-ranging
possibilities in the types of progeny molecules that can be
generated and in the speed at which they can be generated than
often nature itself might be expected to in the same length of
time.
[0162] In a particular exemplification, the instantly disclosed
directed evolution methods can be applied iteratively to produce a
lineage of progeny molecules (e.g. comprising successive sets of
progeny molecules) that would not likely be propagated (i.e.,
generated &/or selected for) in nature, but that could lead to
the generation of a desirable downstream mutagenesis product that
is not achievable by natural evolution.
[0163] Previous Directed Evolution Methods Are Suboptimal:
[0164] Mutagenesis has been attempted in the past on many
occasions, but by methods that are inadequate for the purpose of
this invention. For example, previously described non-stochastic
methods have been serviceable in the generation of only very small
sets of progeny molecules (comprised often of merely a solitary
progeny molecule). By way of illustration, a chimeric gene has been
made by joining 2 polynucleotide fragments using compatible sticky
ends generated by restriction enzyme(s), where each fragment is
derived from a separate progenitor (or parental) molecule. Another
example might be the mutagenesis of a single codon position (i.e.
to achieve a codon substitution, addition, or deletion) in a
parental polynucleotide to generate a single progeny polynucleotide
encoding for a single site-mutagenized polypeptide.
[0165] Previous non-stochastic approaches have only been
serviceable in the generation of but one to a few mutations per
method application. Thus, these previously described non-stochastic
methods thus fail to address one of the central goals of this
invention, namely the exhaustive and non-stochastic chimerization
of nucleic acids. Accordingly previous non-stochastic methods leave
untapped the vast majority of the possible point mutations,
chimerizations, and combinations thereof, which may lead to the
generation of highly desirable progeny molecules.
[0166] In contrast, stochastic methods have been used to achieve
larger numbers of point mutations and/or chimerizations than
non-stochastic methods; for this reason, stochastic methods have
comprised the predominant approach for generating a set of progeny
molecules that can be subjected to screening, and amongst which a
desirable molecular species might hopefully be found. However, a
major drawback of these approaches is that--because of their
stochastic nature--there is a randomness to the exact components in
each set of progeny molecules that is produced. Accordingly, the
experimentalist typically has little or no idea what exact progeny
molecular species are represented in a particular reaction vessel
prior to their generation. Thus, when a stochastic procedure is
repeated (e.g. in a continuation of a search for a desirable
progeny molecule), the re-generation and re-screening of previously
discarded undesirable molecular species becomes a labor-intensive
obstruction to progress, causing a circuitous--if not
circular--path to be taken. The drawbacks of such a highly
suboptimal path can be addressed by subjecting a stochastically
generated set of progeny molecules to a labor-incurring process,
such as sequencing, in order to identify their molecular
structures, but even this is an incomplete remedy.
[0167] Moreover, current stochastic approaches are highly
unsuitable for comprehensively or exhaustively generating all the
molecular species within a particular grouping of mutations, for
attributing functionality to specific structural groups in a
template molecule (e.g. a specific single amino acid position or a
sequence comprised of two or more amino acids positions), and for
categorizing and comparing specific grouping of mutations.
Accordingly, current stochastic approaches do not inherently enable
the systematic elimination of unwanted mutagenesis results, and
are, in sum, burdened by too many inherently shortcomings to be
optimal for directed evolution.
[0168] In a non-limiting aspect, the instant invention addresses
these problems by providing non-stochastic means for
comprehensively and exhaustively generating all possible point
mutations in a parental template. In another non-limiting aspect,
the instant invention further provides means for exhaustively
generating all possible chimerizations within a group of
chimerizations. Thus, the aforementioned problems are solved by the
instant invention.
[0169] Specific shortfalls in the technological landscape addressed
by this invention include:
[0170] 1) Site-directed mutagenesis technologies, such as sloppy or
low-fidelity PCR, are ineffective for systematically achieving at
each position (site) along a polypeptide sequence the full
(saturated) range of possible mutations (i.e. all possible amino
acid substitutions).
[0171] 2) There is no relatively easy systematic means for rapidly
analyzing the large amount of information that can be contained in
a molecular sequence and in the potentially colossal number or
progeny molecules that could be conceivably obtained by the
directed evolution of one or more molecular templates.
[0172] 3) There is no relatively easy systematic means for
providing comprehensive empirical information relating structure to
function for molecular positions.
[0173] 4) There is no easy systematic means for incorporating
internal controls, such as positive controls, for key steps in
certain mutagenesis (e.g. chimerization) procedures.
[0174] 5) There is no easy systematic means to select for a
specific group of progeny molecules, such as full-length chimeras,
from among smaller partial sequences.
[0175] An exceedingly large number of possibilities exist for the
purposeful and random combination of amino acids within a protein
to produce useful hybrid proteins and their corresponding
biological molecules encoding for these hybrid proteins, i.e., DNA,
RNA. Accordingly, there is a need to produce and screen a wide
variety of such hybrid proteins for a desirable utility,
particularly widely varying random proteins.
[0176] The complexity of an active sequence of a biological
macromolecule (e.g., polynucleotides, polypeptides, and molecules
that are comprised of both polynucleotide and polypeptide
sequences) has been called its information content ("IC"), which
has been defined as the resistance of the active protein to amino
acid sequence variation (calculated from the minimum number of
invariable amino acids (bits) required to describe a family of
related sequences with the same function). Proteins that are more
sensitive to random mutagenesis have a high information
content.
[0177] Molecular biology developments, such as molecular libraries,
have allowed the identification of quite a large number of variable
bases, and even provide ways to select functional sequences from
random libraries. In such libraries, most residues can be varied
(although typically not all at the same time) depending on
compensating changes in the context. Thus, while a 100 amino acid
protein can contain only 2,000 different mutations, 20.sup.100
sequence combinations are possible.
[0178] Information density is the IC per unit length of a sequence.
Active sites of enzymes tend to have a high information density. By
contrast, flexible linkers of information in enzymes have a low
information density.
[0179] Current methods in widespread use for creating alternative
proteins in a library format are error-prone polymerase chain
reactions and cassette mutagenesis, in which the specific region to
be optimized is replaced with a synthetically mutagenized
oligonucleotide. In both cases, a substantial number of mutant
sites are generated around certain sites in the original
sequence.
[0180] Error-prone PCR uses low-fidelity polymerization conditions
to introduce a low level of point mutations randomly over a long
sequence. In a mixture of fragments of unknown sequence,
error-prone PCR can be used to mutagenize the mixture. The
published error-prone PCR protocols suffer from a low processivity
of the polymerase. Therefore, the protocol is unable to result in
the random mutagenesis of an average-sized gene. This inability
limits the practical application of error-prone PCR. Some computer
simulations have suggested that point mutagenesis alone may often
be too gradual to allow the large-scale block changes that are
required for continued and dramatic sequence evolution. Further,
the published error-prone PCR protocols do not allow for
amplification of DNA fragments greater than 0.5 to 1.0 kb, limiting
their practical application. In addition, repeated cycles of
error-prone PCR can lead to an accumulation of neutral mutations
with undesired results, such as affecting a protein's
immunogenicity but not its binding affinity.
[0181] In oligonucleotide-directed mutagenesis, a short sequence is
replaced with a synthetically mutagenized oligonucleotide. This
approach does not generate combinations of distant mutations and is
thus not combinatorial. The limited library size relative to the
vast sequence length means that many rounds of selection are
unavoidable for protein optimization. Mutagenesis with synthetic
oligonucleotides requires sequencing of individual clones after
each selection round followed by grouping them into families,
arbitrarily choosing a single family, and reducing it to a
consensus motif. Such motif is re-synthesized and reinserted into a
single gene followed by additional selection. This step process
constitutes a statistical bottleneck, is labor intensive, and is
not practical for many rounds of mutagenesis.
[0182] Error-prone PCR and oligonucleotide-directed mutagenesis are
thus useful for single cycles of sequence fine tuning, but rapidly
become too limiting when they are applied for multiple cycles.
[0183] Another limitation of error-prone PCR is that the rate of
down-mutations grows with the information content of the sequence.
As the information content, library size, and mutagenesis rate
increase, the balance of down-mutations to up-mutations will
statistically prevent the selection of further improvements
(statistical ceiling).
[0184] In cassette mutagenesis, a sequence block of a single
template is typically replaced by a (partially) randomized
sequence. Therefore, the maximum information content that can be
obtained is statistically limited by the number of random sequences
(i.e., library size). This eliminates other sequence families which
are not currently best, but which may have greater long term
potential.
[0185] Also, mutagenesis with synthetic oligonucleotides requires
sequencing of individual clones after each selection round. Thus,
such an approach is tedious and impractical for many rounds of
mutagenesis.
[0186] Thus, error-prone PCR and cassette mutagenesis are best
suited, and have been widely used, for fine-tuning areas of
comparatively low information content. One apparent exception is
the selection of an RNA ligase ribozyme from a random library using
many rounds of amplification by error-prone PCR and selection.
[0187] In nature, the evolution of most organisms occurs by natural
selection and sexual reproduction. Sexual reproduction ensures
mixing and combining of the genes in the offspring of the selected
individuals. During meiosis, homologous chromosomes from the
parents line up with one another and cross-over part way along
their length, thus randomly swapping genetic material. Such
swapping or shuffling of the DNA allows organisms to evolve more
rapidly.
[0188] In recombination, because the inserted sequences were of
proven utility in a homologous environment, the inserted sequences
are likely to still have substantial information content once they
are inserted into the new sequence.
[0189] Theoretically there are 2,000 different single mutants of a
100 amino acid protein. However, a protein of 100 amino acids has
20.sup.100 possible sequence combinations, a number which is too
large to exhaustively explore by conventional methods. It would be
advantageous to develop a system which would allow generation and
screening of all of these possible combination mutations.
[0190] Some workers in the art have utilized an in vivo site
specific recombination system to generate hybrids of combine light
chain antibody genes with heavy chain antibody genes for expression
in a phage system. However, their system relies on specific sites
of recombination and is limited accordingly. Simultaneous
mutagenesis of antibody CDR regions in single chain antibodies
(scFv) by overlapping extension and PCR have been reported.
[0191] Others have described a method for generating a large
population of multiple hybrids using random in vivo recombination.
This method requires the recombination of two different libraries
of plasmids, each library having a different selectable marker. The
method is limited to a finite number of recombinations equal to the
number of selectable markers existing, and produces a concomitant
linear increase in the number of marker genes linked to the
selected sequence(s).
[0192] In vivo recombination between two homologous, but truncated,
insect-toxin genes on a plasmid has been reported as a method of
producing a hybrid gene. The in vivo recombination of substantially
mismatched DNA sequences in a host cell having defective mismatch
repair enzymes, resulting in hybrid molecule formation has been
reported.
1.3. SUMMARY OF THE INVENTION
[0193] Directing an Immune Response so as to Achieve an Optimal
Response to Vaccination
[0194] The present invention provides multicomponent genetic
vaccines that include at least one, and preferably two or more
genetic vaccine components that confer upon the vaccine the ability
to direct an immune response so as to achieve an optimal response
to vaccination. For example, the genetic vaccines can include a
component that provides optimal antigen release; a component that
provides optimal production of cytotoxic T lymphocytes; a component
that directs release of an immunomodulator; a component that
directs release of a chemokine; and/or a component that facilitates
binding to, or entry into, a desired target cell type. For example,
a component can confer improved improves binding to, and uptake of,
the genetic vaccine to target cells such as antigen-expressing
cells or antigen-presenting cells.
[0195] Additional components include those that direct antigen
peptides derived from uptake of an antigen into a cell to
presentation on either Class I or Class II molecules. For example,
one can include a component that directs antigen peptides to
presentation on Class I molecules and comprises a polynucleotide
that encodes a protein such as tapasin, TAP-1 and TAP-2, and/or a
component that directs antigen peptides to presentation on Class Il
molecules and comprises a polynucleotide that encodes a protein
such as an endosomal or lysosomal protease.
[0196] In a particularly preferred aspect, this invention provides
a method for obtaining an immunomodulatory polynucleotide that has
an optimized modulatory effect on an immune response, or encodes a
polypeptide that has an optimized modulatory effect on an immune
response, the method comprising: creating a library of
non-stochastically generated progeny polynucleotides from a
parental polynucleotide set; wherein optimization can thus be
achieved using one or more of the directed evolution methods as
described herein in any combination, permutation and iterative
manner; whereby these directed evolution methods include the
introduction of mutations by non-stochastic methods, including by
"gene site saturation mutagenesis" as described herein; and whereby
these directed evolution methods also include the introduction
mutations by non-stochastic polynucleotide reassembly methods as
described herein; including by synthetic ligation polynucleotide
reassembly as described herein.
[0197] In another particularly preferred aspect, this invention
provides a method for obtaining an immunomodulatory polynucleotide
that has an optimized modulatory effect on an immune response, or
encodes a polypeptide that has an optimized modulatory effect on an
immune response, the method comprising:
[0198] screening a library of non-stochastically generated progeny
polynucleotides to identify an optimized non-stochastically
generated progeny polynucleotide that has, or encodes a polypeptide
that has, a modulatory effect on an immune response; wherein the
optimized non-stochastically generated polynucleotide or the
polypeptide encoded by the non-stochastically generated
polynucleotide exhibits an enhanced ability to modulate an immune
response compared to a parental polynucleotide from which the
library was created.
[0199] In another particularly preferred aspect, this invention
provides a method for obtaining an immunomodulatory polynucleotide
that has an optimized modulatory effect on an immune response, or
encodes a polypeptide that has an optimized modulatory effect on an
immune response, the method comprising: a) creating a library of
non-stochastically generated progeny polynucleotides from a
parental polynucleotide set; and b) screening the library to
identify an optimized non-stochastically generated progeny
polynucleotide that has, or encodes a polypeptide that has, a
modulatory effect on an immune response induced by a genetic
vaccine vector; wherein the optimized non-stochastically generated
polynucleotide or the polypeptide encoded by the non-stochastically
generated polynucleotide exhibits an enhanced ability to modulate
an immune response compared to a parental polynucleotide from which
the library was created; whereby optimization can thus be achieved
using one or more of the directed evolution methods as described
herein in any combination, permutation, and iterative manner;
whereby these directed evolution methods include the introduction
of point mutations by non-stochastic methods, including by "gene
site saturation mutagenesis" as described herein; and whereby these
directed evolution methods also include the introduction mutations
by non-stochastic polynucleotide reassembly methods as described
herein; including by synthetic ligation polynucleotide reassembly
as described herein.
[0200] In another particularly preferred aspect, this invention
provides a method for obtaining an immunomodulatory polynucleotide
that has, an optimized expression in a recombinant expression host,
the method comprising: creating a library of non-stochastically
generated progeny polynucleotides from a parental polynucleotide
set; whereby optimization can thus be achieved using one or more of
the directed evolution methods as described herein in any
combination, permutation and iterative manner; whereby these
directed evolution methods include the introduction of mutations by
non-stochastic methods, including by "gene site saturation
mutagenesis" as described herein; and whereby these directed
evolution methods also include the introduction mutations by
non-stochastic polynucleotide reassembly methods as described
herein; including by synthetic ligation polynucleotide reassembly
as described herein.
[0201] In another particularly preferred aspect, this invention
provides a method for obtaining an immunomodulatory polynucleotide
that has an optimized expression in a recombinant expression host,
the method comprising: screening a library of non-stochastically
generated progeny polynucleotides to identify an optimized
non-stochastically generated progeny polynucleotide that has an
optimized expression in a recombinant expression host when compared
to the expression of a parental polynucleotide from which the
library was created.
[0202] In another particularly preferred aspect, this invention
provides a method for obtaining an immunomodulatory polynucleotide
that has an optimized expression in a recombinant expression host,
the method comprising: a) creating a library of non-stochastically
generated progeny polynucleotides from a parental polynucleotide
set; and b) screening a library of non-stochastically generated
progeny polynucleotides to identify an optimized non-stochastically
generated progeny polynucleotide that has an optimized expression
in a recombinant expression host when compared to the expression of
a parental polynucleotide from which the library was created;
whereby optimization can thus be achieved using one or more of the
directed evolution methods as described herein in any combination,
permutation, and iterative manner; whereby these directed evolution
methods include the introduction of point mutations by
non-stochastic methods, including by "gene site saturation
mutagenesis" as described herein; and whereby these directed
evolution methods also include the introduction mutations by
non-stochastic polynucleotide reassembly methods as described
herein; including by synthetic ligation polynucleotide reassembly
as described herein.
[0203] In one aspect, this invention provides that the ability to a
vaccine, for example a genetic vaccine, or a component of a
vaccine, for example a component of a genetic vaccine by optimizing
its immunogenicity. Moreover, the present invention provides for
the modification of other properties, including its:
[0204] Catalysed reaction(s)
[0205] Reaction type
[0206] Natural substrate(s)
[0207] Substrate spectrum
[0208] Product spectrum
[0209] Inhibitor(s)
[0210] Cofactor(s)/prostetic group(s)
[0211] Metal compounds/salts that affect it
[0212] Turnover number
[0213] Specific activity
[0214] Km value
[0215] pH optimum
[0216] pH range
[0217] Temperature optimum
[0218] Temperature range
[0219] It is also instantly appreciated that the serviceability of
amolecule with an immunogenic effect can be affected by additional
physical properties, which can likewise be modified by directed
evolution as provided herein, such as how it is affected by
subjection to:
[0220] Isolation/Preparation
[0221] Purification
[0222] Renaturating conditions (reversibility or retention of
activity upon: heating and cooling, urea, salts, detergents, pH
extremes)
[0223] Crystallization
[0224] pH
[0225] Temperature
[0226] Oxidation
[0227] Organic solvent(s)
[0228] Miscellaneous storage conditions
[0229] Moreover, the instant invention provides for the
modification of molecule's immunogenic properties properties such
as
[0230] Exposure to biological compartments (stomach acids, in vivo
degradation)
[0231] Expression (e.g.Transcription &/or Translation)
level
[0232] mRNA stability
[0233] Any in vivo interactions with other cells or biologicals
[0234] Method for Obtaining the Genetic Components
[0235] In some embodiments, one or more of the genetic vaccine
components is obtained by a method that involves: (1) reassembling
(&/or subjecting to one or more directed evolution methods
described herein) at least first and second forms of a nucleic acid
which can confer a desired property upon a genetic vaccine, wherein
the first and second forms differ from each other in two or more
nucleotides, to produce a library of recombinant nucleic acids; and
(2) screening the library to identify at least one optimized
recombinant component that exhibits an enhanced capacity to confer
the desired property upon the genetic vaccine. If further
optimization of the component is desired, the following additional
steps can be conducted: (3) reassembling (&/or subjecting to
one or more directed evolution methods described herein) at least
one optimized recombinant component with a further form of the
nucleic acid, which is the same or different from the first and
second forms, to produce a further library of recombinant nucleic
acids; (4) screening the further library to identify at least one
further optimized recombinant component that exhibits an enhanced
capacity to confer the desired property upon the genetic vaccine;
and (5) repeating (3) and (4), as necessary, until the further
optimized recombinant component exhibits a further enhanced
capacity to confer the desired property upon the genetic
vaccine.
[0236] Members of a Gene Family
[0237] In some embodiments of the invention, the first form of the
nucleic acid is a first member of a gene family and the second form
of the nucleic acid comprises a second member of the gene family.
Additional forms of the module nucleic acid can also be members of
the gene family. As an example, the first member of the gene family
can be obtained from a first species of organism and the second
member of the gene family obtained from a second species of
organism. If desired, the optimized recombinant genetic vaccine
component obtained by the methods of the invention can be
backcrossed by, for example, reassembling (&/or subjecting to
one or more directed evolution methods described herein) the
optimized recombinant genetic vaccine component with a molar excess
of one or both of the first and second forms of the substrate
nucleic acids to produce a further library of recombinant genetic
vaccine components; and screening the further library to identify
at least one optimized recombinant genetic vaccine component that
further enhances the capability of a genetic vaccine vector that
includes the component to modulate the immune response.
[0238] Methods of Obtaining a Genetic Vaccine Component that
Confers upon a Genetic Vaccine Vector an Enhanced Ability to
Replicate in a Host Cell
[0239] Additional embodiments of the invention provide methods of
obtaining a genetic vaccine component that confers upon a genetic
vaccine vector an enhanced ability to replicate in a host cell.
These methods involve creating a library of recombinant nucleic
acids by subjecting to reassembly (&/or one or more additonal
directed evolution methods described herein) at least two forms of
a polynucleotide that can confer episomal replication upon a vector
that contains the polynucleotide; introducing into a population of
host cells a library of vectors, each of which contains a member of
the library of recombinant nucleic acids and a polynucleotide that
encodes a cell surface antigen; propagating the population of host
cells for multiple generations; and identifying cells which display
the cell surface antigen on a surface of the cell, wherein cells
which display the cell surface antigen are likely to harbor a
vector that contains a recombinant vector module which enhances the
ability of the vector to replicate episomally.
[0240] Obtaining Genetic Vaccine Components that Confer Upon a
Vector an Enhanced Ability to Replicate in a Host Cell
[0241] Genetic vaccine components that confer upon a vector an
enhanced ability to replicate in a host cell can also be obtained
by creating a library of recombinant nucleic acids by subjecting to
reassembly (&/or one or more additonal directed evolution
methods described herein) at least two forms of a polynucleotide
derived from a human papillomavirus that can confer episomal
replication upon a vector that contains the polynucleotide;
introducing a library of vectors, each of which contains a member
of the library of recombinant nucleic acids, into a population of
host cells; propagating the host cells for a plurality of
generations; and identifying cells that contain the vector.
[0242] In additional embodiments, the invention provides methods
obtaining a genetic vaccine component that confers upon a vector an
enhanced ability to replicate in a human host cell by creating a
library of recombinant nucleic acids by subjecting to reassembly
(&/or one or more additonal directed evolution methods
described herein) at least two forms of a polynucleotide that can
confer episomal replication upon a vector that contains the
polynucleotide; introducing a library of genetic vaccine vectors,
each of which comprises a member of the library of recombinant
nucleic acids, into a test system that mimics a human immune
response; and determining whether the genetic vaccine vector
replicates or induces an immune response in the test system. A
suitable test system can involve human skin cells present as a
xenotransplant on skin of an immunocompromised non-human host
animal, for example, or a non-human mammal that comprises a
functional human immune system. Replication in these systems can be
detected by determining whether the animal exhibits an immune
response against the antigen.
[0243] The invention also provides methods of obtaining a genetic
vaccine component that confers upon a genetic vaccine an enhanced
ability to enter an antigen-presenting cell. These methods involve
creating a library of recombinant nucleic acids by subjecting to
reassembly (&/or one or more additonal directed evolution
methods described herein) at least two forms of a polynucleotide
that can confer episomal replication upon a vector that contains
the polynucleotide; introducing a library of genetic vaccine
vectors, each of which comprises a member of the library of
recombinant nucleic acids, into a population of antigen-presenting
or antigen-processing cells; and determining the percentage of
cells in the population which contain the nucleic acid vector.
Antigen- presenting or antigen-processing cells of interest
include, for example, B cells, monocytes/macrophages, dendritic
cells, Langerhans cells, keratinocytes, and muscle cells.
[0244] The present invention provides methods of obtaining a
polynucleotide that has a modulatory effect on an immune response
that is induced by a genetic vaccine, either directly (i.e., as an
immunomodulatory polynucleotide) or indirectly (i.e., upon
translation of the polynucleotide to create an immunomodulatory
polypeptide. The methods of the invention involve: creating a
library of experimentally generated (in vitro &/or in vivo)
polynucleotides; and screening the library to identify at least one
optimized experimentally generated (in vitro &/or in vivo)
polynucleotide that exhibits, either by itself or through the
encoded polypeptide, an enhanced ability to modulate an immune
response than a form of the nucleic acid from which the library was
created. Examples include, for example, CpG-rich polynucleotide
sequences, polynucleotide sequences that encode a costimulator
(e.g., B7-1, B7-2, CD1, CD40, CD154 (ligand for CD40), CD150
(SLAM), or a cytokine. The screening step used in these methods can
include, for example, introducing genetic vaccine vectors which
comprise the library of recombinant nucleic acids into a cell, and
identifying cells which exhibit an increased ability to modulate an
immune response of interest or increased ability to express an
immunomodulatory molecule. For example, a library of recombinant
cytokine-encoding nucleic acids can be screened by testing the
ability of cytokines encoded by the nucleic acids to activate cells
which contain a receptor for the cytokine. The receptor for the
cytokine can be native to the cell, or can be expressed from a
heterologous nucleic acid that encodes the cytokine receptor. For
example, the optimized costimulators can be tested to identify
those for which the cells or culture medium are capable of inducing
a predominantly T.sub.H2 immune response, or a predominantly
T.sub.H1 immune response.
[0245] In some embodiments, the polynucleotide that has a
modulatory effect on an immune response is obtained by: (1)
reassembling (&/or subjecting to one or more directed evolution
methods described herein) at least first and second forms of a
nucleic acid that is, or encodes a molecule that is, involved in
modulating an immune response, wherein the first and second forms
differ from each other in two or more nucleotides, to produce a
library of experimentally generated (in vitro &/or in vivo)
polynucleotides; and (2) screening the library to identify at least
one optimized experimentally generated (in vitro &/or in vivo)
polynucleotide that exhibits, either by itself or through the
encoded polypeptide, an enhanced ability to modulate an immune
response than a form of the nucleic acid from which the library was
created. If additional optimization is desired, the method can
further involve: (3) reassembling (&/or subjecting to one or
more directed evolution methods described herein) at least one
optimized experimentally generated (in vitro &/or in vivo)
polynucleotide with a further form of the nucleic acid, which is
the same or different from the first and second forms, to produce a
further library of experimentally generated (in vitro &/or in
vivo) polynucleotides; (4) screening, the further library to
identify at least one further optimized experimentally generated
(in vitro &/or in vivo) polynucleotide that exhibits an
enhanced ability to modulate an immune response than a form of the
nucleic acid from which the library was created.; and (5) repeating
(3) and (4), as necessary, until the further optimized
experimentally generated (in vitro &/or in vivo) polynucleotide
exhibits an further enhanced ability to modulate an immune response
than a form of the nucleic acid from which the library was
created.
[0246] In some embodiments of the invention, the library of
experimentally generated (in vitro &/or in vivo)
polynucleotides is screened by: expressing the experimentally
generated (in vitro &/or in vivo) polynucleotides so that the
encoded peptides or polypeptides are produced as fusions with a
protein displayed on the surface of a replicable genetic package;
contacting the replicable genetic packages with a plurality of
cells that display the receptor; and identifying cells that exhibit
a modulation of an immune response mediated by the receptor.
[0247] The invention also provides methods for obtaining a
polynucleotide that encodes an accessory molecule that improves the
transport or presentation of antigens by a cell. These methods
involve creating a library of experimentally generated (in vitro
&/or in vivo) polynucleotides by subjecting to reassembly
(&/or one or more additonal directed evolution methods
described herein) nucleic acids that encode all or part of the
accessory molecule; and screening the library to identify an
optimized experimentally generated (in vitro &/or in vivo)
polynucleotide that encodes a recombinant accessory molecule that
confers upon a cell an increased or decreased ability to transport
or present an antigen on a surface of the cell compared to an
accessory molecule encoded by the non-recombinant nucleic acids. In
some embodiments, the screening step involves: introducing the
library of experimentally generated (in vitro &/or in vivo)
polynucleotides into a genetic vaccine vector that encodes an
antigen to form a library of vectors; introducing the library of
vectors into mammalian cells; and identifying mammalian cells that
exhibit increased or decreased immunogenicity to the antigen.
[0248] In some embodiments of the invention, the cytokine that is
optimized is interleukin-12 and the screening is performed by
growing mammalian cells which contain the genetic vaccine vector in
a culture medium, and detecting whether T cell proliferation or T
cell differentiation is induced by contact with the culture medium.
In another embodiment, the cytokine is interferon--and the
screening is performed by expressing the recombinant vector module
as a fusion protein which is displayed on the surface of a
bacteriophage to form a phage display library, and identifying
phage library members which are capable of inhibiting proliferation
of a B cell line. Another embodiment utilizes B7-1 (CD80) or B7-2
(CD86) as the costimulator and the cell or culture medium is tested
for ability to modulate an immune response.
[0249] The invention provides methods of using stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly to obtain optimized
recombinant vector modules that encode cytokines and other
costimulators that exhibit reduced immunogenicity compared to a
corresponding polypeptide encoded by a non-optimized vector module.
The reduced immunogenicity can be detected by introducing a
cytokine or costimulator encoded by the recombinant vector module
into a mammal and determining whether an immune response is induced
against the cytokine.
[0250] The invention also provides methods of obtaining optimized
immunomodulatory sequences that encode a cytokine antagonist. For
example, suitable cytokine agonists include a soluble cytokine
receptor and a transmembrane cytokine receptor having, a defective
signal sequence. Examples include sIL-10R and sIL-4R, and the
like.
[0251] The present invention provides methods for obtaining a
cell-specific binding molecule that is useful for increasing uptake
or specificity of a genetic vaccine to a target cell. The methods
involve: creating a library of experimentally generated (in vitro
&/or in vivo) polynucleotides that by reassembling (&/or
subjecting to one or more directed evolution methods described
herein) a nucleic acid that encodes a polypeptide that comprises a
nucleic acid binding domain and a nucleic acid that encodes a
polypeptide that comprises a cell-specific binding domain; and
screening the library to identify a experimentally generated (in
vitro &/or in vivo) polynucleotide that encodes a binding
molecule that can bind to a nucleic acid and to a cell-specific
receptor. Target cells of particular interest include
antigen-presenting and antigen-processing cells, such as muscle
cells, monocytes, dendritic cells, B cells, Langerhans cells,
keratinocytes, and M-cells.
[0252] In some embodiments, the methods of the invention for
obtaining a cell-specific binding moiety useful for increasing
uptake or specificity of a genetic vaccine to a target cell
involve:
[0253] (1) reassembling (&/or subjecting to one or more
directed evolution methods described herein) at least first and
second forms of a nucleic acid which comprises a polynucleotide
that encodes a nucleic acid binding domain and at least first and
second forms of a nucleic acid which comprises a cell-specific
ligand that specifically binds to a protein on the surface of a
cell of interest, wherein the first and second forms differ from
each other in two or more nucleotides, to produce a library of
recombinant binding moiety- encoding nucleic acids;
[0254] (2) transfecting into a population of host cells a library
of vectors, each of which comprises: a) a binding site specific for
the nucleic acid binding domain and b) a member of the library of
recombinant binding moiety-encoding nucleic acids, wherein the
recombinant binding moiety is expressed and binds to the binding
site to form a vector-binding moiety complex;
[0255] (3) lysing the host cells under conditions that do not
disrupt binding of the vector-binding moiety complex;
[0256] (4) contacting the vector-binding moiety complex with a
target cell of interest; and
[0257] (5) identifying target cells that contain a vector and
isolating the optimized recombinant cell-specific binding moiety
nucleic acids from these target cells.
[0258] If further optimization is desired, the methods can further
involve:
[0259] (6) reassembling (&/or subjecting to one or more
directed evolution methods described herein) at least one optimized
recombinant binding moiety-encoding nucleic acid with a further
form of the polynucleotide that encodes a nucleic acid binding
domain and/or a further form of the polynucleotide that encodes a
cell-specific ligand, which are the same or different from the
first and second forms, to produce a further library of recombinant
binding moiety-encoding nucleic acids;
[0260] (7) transfecting into a population of host cells a library
of vectors that comprise: a) a binding site specific for the
nucleic acid binding domain and 2) the recombinant binding
moiety-encoding nucleic acids, wherein the recombinant binding
moiety is expressed and binds to the binding site to form a
vector-binding moiety complex;
[0261] (8) lysing the host cells under conditions that do not
disrupt binding of the vector-binding moiety complex;
[0262] (9) contacting the vector-binding moiety complex with a
target cell of interest and identifying target cells that contain
the vector; and
[0263] (10) isolating the optimized recombinant binding moiety
nucleic acids from the target cells which contain the vector;
and
[0264] (11) repeating (6) through (10), as necessary, to obtain a
further optimized cell-specific binding moiety useful for
increasing uptake or specificity of a genetic vaccine vector to a
target cell.
[0265] The invention also provides cell-specific recombinant
binding moieties produced by expressing in a host cell an optimized
recombinant binding moiety-encoding nucleic acid obtained by the
methods of the invention.
[0266] In another embodiment, the invention provides genetic
vaccines that include: a) an optimized recombinant binding moiety
that comprises a nucleic acid binding domain and a cell-specific
ligand, and b) a polynucleotide sequence that comprises a binding
site, wherein the nucleic acid binding domain is capable of
specifically binding to the binding site.
[0267] A further embodiment of the invention provides methods for
obtaining an optimized cell-specific binding moiety useful for
increasing uptake, efficacy, or specificity of a genetic vaccine
for a target cell by:
[0268] (1) reassembling (&/or subjecting to one or more
directed evolution methods described herein) at least first and
second forms of a nucleic acid that comprises a polynucleotide
which encodes a non-toxic receptor binding moiety-of an enterotoxin
or other toxin, wherein the first and second forms differ from each
other in two or more nucleotides, to produce a library of
recombinant nucleic acids;
[0269] (2) transfecting vectors that contain the library of nucleic
acids into a population of host cells, wherein the nucleic acids
are expressed to form recombinant cell-specific binding moiety
polypeptides;
[0270] (3) contacting the recombinant cell-specific binding moiety
polypeptides with a cell surface receptor of a target cell; and
[0271] (4) determining which recombinant cell-specific binding
moiety polypeptides exhibit enhanced ability to bind to the target
cell. Methods of enhancing uptake of a genetic vaccine vector by a
target cell by coating the genetic vaccine vector with an optimized
recombinant cell-specific binding moiety produced by these methods
are also provided by the invention.
[0272] The present invention also provides methods for evolving a
vaccine delivery vehicle, genetic vaccine vector, or a vector
component to obtain an optimized delivery vehicle or component that
has, or confers upon a vector, enhanced ability to enter a selected
mammalian tissue upon administration to a mammal. These methods
involve:
[0273] (1) reassembling (&/or subjecting to one or more
directed evolution methods described herein) members of a pool of
polynucleotides to produce a library of experimentally generated
(in vitro &/or in vivo) polynucleotides;
[0274] (2) administering to a test animal a library of replicable
genetic packages, each of which comprises a member of the library
of experimentally generated (in vitro &/or in vivo)
polynucleotides operably linked to a polynucleotide that encodes a
display polypeptide, wherein the experimentally generated (in vitro
&/or in vivo) polynucleotide and the display polypeptide are
expressed as a fusion protein which is which is displayed on the
surface of the replicable genetic package; and
[0275] (3) recovering replicable genetic packages that are present
in the selected tissue of the test animal at a suitable time after
administration, wherein recovered replicable genetic packages have
enhanced ability to enter the selected mammalian tissue upon
administration to the mammal.
[0276] If further optimization of the delivery vehicle is desired,
the methods of the invention further involve:
[0277] (4) reassembling (&/or subjecting to one or more
directed evolution methods described herein) a nucleic acid that
comprises at least one experimentally generated (in vitro &/or
in vivo) polynucleotide obtained from a replicable genetic package
recovered from the selected tissue with a further pool of
polynucleotides to produce a further library of experimentally
generated (in vitro &/or in vivo) polynucleotides;
[0278] (5) administering to a test animal a library of replicable
genetic packages, each of which comprises a member of the further
library of experimentally generated (in vitro &/or in vivo)
polynucleotides operably linked to a polynucleotide that encodes a
display polypeptide, wherein the experimentally generated (in vitro
&/or in vivo) polynucleotide and the display polypeptide are
expressed as a fusion protein which is which is displayed on the
surface of the replicable genetic package;
[0279] (6) recovering replicable genetic packages that are present
in the selected tissue of the test animal at a suitable time after
administration; and
[0280] (7) repeating (4) through (6), as necessary, to obtain a
further optimized recombinant delivery vehicle that exhibits
further enhanced ability to enter a selected mammalian tissue upon
administration to a mammal. Methods of administration that are of
particular interest include, for example, oral, topical, and
inhalation. Where the administration is intravenous, mammalian
tissues of interest include, for example, lymph node and
spleen.
[0281] In another embodiment, the invention provides methods for
evolving a vaccine delivery vehicle, genetic vaccine vector, or a
vector component to obtain an optimized delivery vehicle or
component to obtain an optimized delivery vehicle or vector
component that has, or confers upon a vector containing the
component, enhanced specificity for antigen-presenting cells
by:
[0282] (1) reassembling (&/or subjecting to one or more
directed evolution methods described herein) members of a pool of
polynucleotides to produce a library of experimentally generated
(in vitro &/or in vivo) polynucleotides;
[0283] (2) producing a library of replicable genetic packages, each
of which comprises a member of the library of experimentally
generated (in vitro &/or in vivo) polynucleotides operably
linked to a polynucleotide that encodes a display polypeptide,
wherein the experimentally generated (in vitro &/or in vivo)
polynucleotide and the display polypeptide are expressed as a
fusion protein which is which is displayed on the surface of the
replicable genetic package;
[0284] (3) contacting the library of recombinant replicable genetic
packages with a non-APC to remove replicable genetic packages that
display non-APC-specific fusion polypeptides; and
[0285] (4) contacting the recombinant replicable genetic packages
that did not bind to the non-APC with an APC and recovering those
that bind to the APC, wherein the recovered replicable genetic
packages are capable of specifically binding to APCs.
[0286] In an additional embodiment, the invention provides methods
for evolving a vaccine delivery vehicle, genetic vaccine vector, or
a vector component to obtain an optimized delivery vehicle or
component to obtain an optimized delivery vehicle or vector
component that has, or confers upon a vector containing the
component, an enhanced ability to enter a target cell by:
[0287] (1) reassembling (&/or subjecting to one or more
directed evolution methods described herein) at least first and
second forms of a nucleic acid which encodes an invasin
polypeptide, wherein the first and second forms differ from each
other in two or more nucleotides, to produce a library of
recombinant invasin nucleic acids;
[0288] (2) producing a library of recombinant bacteriophage, each
of which displays on the bacteriophage surface a fusion polypeptide
encoded by a chimeric gene that comprises a recombinant invasin
nucleic acid operably linked to a polynucleotide that encodes a
display polypeptide;
[0289] (3) contacting the library of recombinant bacteriophage with
a population of target cells; (4) removing unbound phage and phage
which is bound to the surface of the target cells; and
[0290] (5) recovering phage which are present within the target
cells, wherein the recovered phage are enriched for phage that have
enhanced ability to enter the target cells.
[0291] In some embodiments, the optimized recombinant genetic
vaccine vectors, delivery vehicles, or vector components obtained
using these methods exhibit improved ability to enter an antigen
presenting cell. These methods can involve washing the cells after
the transfection step to remove vectors which did not enter an
antigen presenting cell.; culturing the cells for a predetermined
time after transfection; lysing the antigen presenting cells; and
isolating the optimized recombinant genetic vaccine vector from the
cell lysate.
[0292] Antigen Presenting Cells that Contain an Optimized
Recombinant Genetic Vaccine Vectors can be Identified by, For
Example, Detecting Expression of a Marker Gene that is Included in
the Vectors
[0293] The invention also provides methods of evolving a
bacteriophage-derived vaccine delivery vehicle to obtain a delivery
vehicle having enhanced ability to enter a target cell. These
methods involve the steps of.
[0294] (1) reassembling (&/or subjecting to one or more
directed evolution methods described herein) at least first and
second forms of a nucleic acid which encodes an invasin
polypeptide, wherein the first and second forms differ from each
other in two or more nucleotides, to produce a library of
recombinant invasin nucleic acids;
[0295] (2) producing a library of recombinant bacteriophage, each
of which displays on the bacteriophage surface a fusion polypeptide
encoded by a chimeric gene that comprises a recombinant invasin
nucleic acid operably linked to a polynucleotide that encodes a
display polypeptide;
[0296] (3) contacting the library of recombinant bacteriophage with
a population of target cells;
[0297] (4) removing unbound phage and phage which is bound to the
surface of the target cells; and
[0298] (5) recovering phage which are present within the target
cells, wherein the recovered phage are enriched for phage that have
enhanced ability to enter the target cells. Again, if further
optimization is desired, the methods can include the further steps
of.
[0299] (6) reassembling (&/or subjecting to one or more
directed evolution methods described herein) a nucleic acid which
comprises at least one recombinant invasin nucleic acid obtained
from a bacteriophage which is recovered from a target cell with a
further pool of polynucleotides to produce a further library of
recombinant invasin polynucleotides;
[0300] (7) producing a further library of recombinant
bacteriophage, each of which displays on the bacteriophage surface
a fusion polypeptide encoded by a chimeric gene that comprises a
recombinant invasin nucleic acid operably linked to a
polynucleotide that encodes a display polypeptide;
[0301] (8) contacting the library of recombinant bacteriophage with
a population of target cells;
[0302] (9) removing unbound phage and phage which is bound to the
surface of the target cells; and
[0303] (10) recovering phage which are present within the target
cells; and
[0304] (11) repeating (6) through (10), as necessary, to obtain a
further optimized recombinant delivery vehicle which exhibits
further have enhanced ability to enter the target cells.
[0305] In some embodiments the methods of evolving a
bacteriophage-derived vaccine delivery vehicle to obtain a delivery
vehicle having enhanced ability to enter a target cell can include
the additional steps of.
[0306] (12) inserting into the optimized recombinant delivery
vehicle a polynucleotide which encodes an antigen of interest,
wherein the antigen of interest is expressed as a fusion
polypeptide which comprises a second display polypeptide;
[0307] (13) administering the delivery vehicle to a test animal;
and (14) determining whether the delivery vehicle is capable of
inducing a CTL response in the test animal.
[0308] Alternatively, the following steps can be employed:
[0309] (12) inserting into the optimized recombinant delivery
vehicle a polynucleotide which encodes an antigen of interest,
wherein the antigen of interest is expressed as a fusion
polypeptide which comprises a second display polypeptide;
[0310] (13) administering the delivery vehicle to a test animal;
and
[0311] (14) determining whether the delivery vehicle is capable of
inducing neutralizing antibodies against a pathogen which comprises
the antigen of interest. An example of a target cell of interest
for these methods is an antigen-presenting cell.
[0312] The present invention provides recombinant multivalent
antigenic polypeptides that include a first antigenic determinant
from a first disease-associated polypeptide and at least a second
antigenic determinant from a second disease-associated polypeptide.
The disease-associated polypeptides can be selected from the group
consisting of cancer antigens, antigens associated with
autoimmunity disorders, antigens associated with inflammatory
conditions, antigens associated with allergic reactions, antigens
associated with infectious agents, and other antigens that are
associated with a disease condition.
[0313] In another embodiment, the invention provides a recombinant
antigen library that contains recombinant nucleic acids that encode
antigenic polypeptides. The libraries are typically obtained by
reassembling (&/or subjecting to one or more directed evolution
methods described herein), at least first and second forms of a
nucleic acid which includes a polynucleotide sequence that encodes
a disease-associated antigenic polypeptide, wherein the first and
second forms differ from each other in two or more nucleotides, to
produce a library of recombinant nucleic acids.
[0314] Another embodiment of the invention provides methods of
obtaining a polynucleotide that encodes a recombinant antigen
having improved ability to induce an immune response to a disease
condition. These methods involve:
[0315] (1) reassembling (&/or subjecting to one or more
directed evolution methods described herein) at least first and
second forms of a nucleic acid which comprises a polynucleotide
sequence that encodes an antigenic polypeptide that is associated
with the disease condition, wherein the first and second forms
differ from each other in two or more nucleotides, to produce a
library of recombinant nucleic acids; and
[0316] (2) screening the library to identify at least one optimized
recombinant nucleic acid that encodes an optimized recombinant
antigenic polypeptide that has improved ability to induce an immune
response to the disease condition.
[0317] These methods optionally further involve:
[0318] (3) reassembling (&/or subjecting to one or more
directed evolution methods described herein) at least one optimized
recombinant nucleic acid with a further form of the nucleic acid,
which is the same or different from the first and second forms, to
produce a further library of recombinant nucleic acids;
[0319] (4) screening the further library to identify at least one
further optimized recombinant nucleic acid that encodes a
polypeptide that has improved ability to induce an immune response
to the disease condition; and
[0320] (5) repeating (3) and (4), as necessary, until the further
optimized recombinant nucleic acid encodes a polypeptide that has
improved ability to induce an immune response to the disease
condition.
[0321] In some embodiments, the optimized recombinant nucleic acid
encodes a multivalent antigenic polypeptide and the screening is
accomplished by expressing the library of recombinant nucleic acids
in a phage display expression vector such that the recombinant
antigen is expressed as a fusion protein with a phage polypeptide
that is displayed on a phage particle surface; contacting the phage
with a first antibody that is specific for a first serotype of the
pathogenic agent and selecting those phage that bind to the first
antibody; and contacting those phage that bind to the first
antibody with a second antibody that is specific for a second
serotype of the pathogenic agent and selecting those phage that
bind to the second antibody; wherein those phage that bind to the
first antibody and the second antibody express a multivalent
antigenic polypeptide.
[0322] The Invention also Provides Methods of Obtaining a
Recombinant Viral Vector Which has an Enhanced Ability to Induce an
Antiviral Response in a Cell
[0323] Methods of Obtaining a Recombinant Genetic Vaccine Component
that Confers Upon a Genetic Vaccine an Enhanced Ability to Induce a
Desired Immune Response in a Mammal
[0324] In additional embodiments, the invention provides methods of
obtaining a recombinant genetic vaccine component that confers upon
a genetic vaccine an enhanced ability to induce a desired immune
response in a mammal. These methods involve: (1) reassembling
(&/or subjecting to one or more directed evolution methods
described herein) at least first and second forms of a nucleic acid
which comprise a genetic vaccine vector, wherein the first and
second forms differ from each other in two or more nucleotides, to
produce a library of recombinant genetic vaccine vectors; (2)
transfecting the library of recombinant vaccine vectors into a
population of mammalian cells selected from the group consisting of
peripheral blood T cells, T cell clones, freshly isolated
monocytes/macrophages and dendritic cells; (3) staining the cells
for the presence of one or more cytokines and identifying cells
which exhibit a cytokine staining pattern indicative of the desired
immune response; and (4) obtaining recombinant vaccine vector
nucleic acid sequences from the cells which exhibit the desired
cytokine staining pattern.
[0325] Methods of Improving the Ability of a Genetic Vaccine Vector
to Modulate an Immune Response
[0326] Also provided by the invention are methods of improving the
ability of a genetic vaccine vector to modulate an immune response
by: (1) reassembling (&/or subjecting to one or more directed
evolution methods described herein) at least first and second forms
of a nucleic acid which comprise a genetic vaccine vector, wherein
the first and second forms differ from each other in two or more
nucleotides, to produce a library of recombinant genetic vaccine
vectors; (2) transfecting the library of recombinant genetic
vaccine vectors into a population of antigen presenting cells; and
(3) isolating from the cells optimized recombinant genetic vaccine
vectors which exhibit enhanced ability to modulate a desired immune
response.
[0327] Methods of Obtaining a Recombinant Genetic Vaccine Vector
that has an Enhanced Ability to Induce a Desired Immune Response in
a Mammal Upon Administration to the Skin of the Mammal
[0328] Another embodiment of the invention provides methods of
obtaining a recombinant genetic vaccine vector that has an enhanced
ability to induce a desired immune response in a mammal upon
administration to the skin of the mammal. These methods involve:
(1) reassembling (&/or subjecting to one or more directed
evolution methods described herein) at least first and second forms
of a nucleic acid which comprise a genetic vaccine vector, wherein
the first and second forms differ from each other in two or more
nucleotides, to produce a library of recombinant genetic vaccine
vectors; (2) topically applying the library of recombinant genetic
vaccine vectors to skin of a mammal; (3) identifying vectors that
induce an immune response; and (4) recovering genetic vaccine
vectors from the skin cells which contain vectors that induce an
immune response.
[0329] Methods of Inducing an Immune Response in a Mammal by
Topically Applying to Skin of the Mammal a Genetic Vaccine Vector,
Wherein the Genetic Vaccine Vector is Optimized for Topical
Application Through Use of Stochastic (e.g. Polynucleotide
Shuffling & Interrupted Synthesis) and Non-Stochastic
Polynucleotide Reassembly
[0330] The invention also provides methods of inducing an immune
response in a mammal by topically applying to skin of the mammal a
genetic vaccine vector, wherein the genetic vaccine vector is
optimized for topical application through use of stochastic (e.g.
polynucletide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly. In some embodiments, the
genetic vaccine is administered as a formulation selected from the
group consisting of a transdermal patch, a cream, naked DNA, a
mixture of DNA and a transfection-enhancing agent. Suitable
transfection-enhancing agents include one or more agents selected
from the group consisting of a lipid, a liposome, a protease, and a
lipase.
[0331] Alternatively, or in addition, the genetic vaccine can be
administered after pretreatment of the skin by abrasion or hair
removal.
[0332] Methods of Obtaining an Optimized Genetic Vaccine Component
that Confers Upon a Genetic Vaccine Containing the Component an
Enhanced Ability to Induce or Inhibit Apoptosis of a Cell into
Which the Vaccine is Introduced
[0333] In another embodiment, the invention provides methods of
obtaining an optimized genetic vaccine component that confers upon
a genetic vaccine containing the component an enhanced ability to
induce or inhibit apoptosis of a cell into which the vaccine is
introduced. These methods involve: (1) reassembling (&/or
subjecting to one or more directed evolution methods described
herein) at least first and second forms of a nucleic acid which
comprise a nucleic acid that encodes an apoptosis-modulating
polypeptide, wherein the first and second forms differ from each
other in two or more nucleotides, to produce a library of
recombinant nucleic acids; (2) transfecting the library of
recombinant nucleic acids into a population of mammalian cells; (3)
staining the cells for the presence of a cell membrane change which
is indicative of apoptosis initiation; and (4) obtaining
recombinant apoptosis-modulating genetic vaccine components from
the cells which exhibit the desired apoptotic membrane changes.
[0334] Methods of Obtaining a Genetic Vaccine Component that
Confers Upon a Genetic Vaccine Reduced Susceptibility to a CTL
Immune Response in a Host Mammal
[0335] Other embodiments of the invention provide methods of
obtaining a genetic vaccine component that confers upon a genetic
vaccine reduced susceptibility to a CTL immune response in a host
mammal. These methods can involve: (1) reassembling (&/or
subjecting to one or more directed evolution methods described
herein) at least first and second forms of a nucleic acid which
comprises a gene that encodes an inhibitor of a CTL immune
response, wherein the first and second forms differ from each other
in two or more nucleotides, to produce a library of recombinant CTL
inhibitor nucleic acids; (2) introducing genetic vaccine vectors
which comprise the library of recombinant CTL inhibitor nucleic
acids into a plurality of human cells; (3) selecting cells which
exhibit reduced MHC class I molecule expression; and (4) obtaining
optimized recombinant CTL inhibitor nucleic acids from the selected
cells.
[0336] Methods of Obtaining a Genetic Vaccine Component that
Confers Upon a Genetic Vaccine Reduced Susceptibility to a CTL
Immune Response in a Host Mammal
[0337] The invention also provides methods of obtaining a genetic
vaccine component that confers upon a genetic vaccine reduced
susceptibility to a CTL immune response in a host mammal. These
methods involve: (1) reassembling (&/or subjecting to one or
more directed evolution methods described herein) at least first
and second forms of a nucleic acid which comprises a gene that
encodes an inhibitor of a CTL immune response, wherein the first
and second forms differ from each other in two or more nucleotides,
to produce a library of recombinant CTL inhibitor nucleic acids;
(2) introducing viral vectors which comprise the library of
recombinant CTL inhibitor nucleic acids into mammalian cells; (3)
identifying mammalian cells which express a marker gene included in
the viral vectors a predetermined time after introduction, wherein
the identified cells are resistant to a CTL response; and (4)
recovering as the genetic vaccine component the recombinant CTL
inhibitor nucleic acids from the identified cells.
[0338] It is a general object of the invention to provide proteins
and polypeptides that are derived from PfEMP 1 proteins, nucleic
acids encoding these proteins and antibodies that are specifically
immunoreactive with these proteins. It is a further object to
provide methods of using these various compositions in diagnosis,
treatment or prevention of the onset of symptoms of a malaria
parasite infection. It is a further object to provide methods of
screening compounds to identify further compositions which may be
used in these methods.
[0339] In one embodiment, the present invention provides
substantially pure polypeptides which have amino acid sequences
substantially homologous to the amino acid sequence of a PfEMP1
protein, or biologically active fragments thereof.
[0340] In preferred aspects, the polypeptides of the present
invention are substantially homologous to the amino acid sequence
shown, described &/or referenced herein (including incorporated
by reference), biologically active fragments or analogues thereof.
Also provided are pharmaceutical compositions comprising these
polypeptides.
[0341] In another embodiment, the present invention provides
nucleic acids which encode the above-described polypeptides.
Particularly preferred nucleic acids will be substantially
homologous to a part or whole of the nucleic acid sequence shown,
described &/or referenced herein (including incorporated by
reference) or the nucleic acid encoding for the sequences shown,
described &/or referenced herein (including incorporated by
reference). The present invention also provides expression vectors
comprising these nucleic acid sequences and cells capable of
expressing same.
[0342] In an additional embodiment, the present invention provides
antibodies which recognize and bind PfEMP1 polypeptides or
biologically active fragments thereof. More preferred are those
peptides which recognize and bind PfEMP1 proteins associated with
infection by more than one variant of P. falciparum.
[0343] In a further embodiment, the present invention provides
methods of inhibiting the formation of PfEMP1/ligand complex,
comprising contacting PfEMP1 or its ligands with polypeptides of
the present invention.
[0344] In a related embodiment, the present invention provides
methods of inhibiting sequestration of erythrocytes in a patient
suffering from a malaria infection, comprising administering to
said patient, an effective amount of a polypeptide of the present
invention. such administration may be carried out prior to or
following infection.
[0345] In still another embodiment, the present invention provides
a method of detecting the presence or absence of PfEMP1 in a
sample. The method comprises exposing the sample to an antibody of
the invention, and detecting binding, if any, between the antibody
and a component of the sample.
[0346] In an additional embodiment, the present invention provides
a method of determining whether a test compound is an antagonist of
PfEMP1/ligand complex formation. The method comprises incubating
the test compound with PfEMP1 or a biologically active fragment
thereof, and its ligand, under conditions which permit the
formation of the complex. The amount of complex formed in the
presence of the test compound is determined and compared with the
amount of complex formed in the absence of the test compound. A
decrease in the amount of complex formed in the presence of the
test compound is indicative that the compound is an antagonist of
PfEMP1/ligand complex formation.
[0347] Summary of Directed Evolution Approaches
[0348] This invention also relates generally to the field of
nucleic acid engineering and correspondingly encoded recombinant
protein engineering. More particularly, the invention relates to
the directed evolution of nucleic acids and screening of clones
containing the evolved nucleic acids for resultant activity(ies) of
interest, such nucleic acid activity(ies) &/or specified
protein, particularly enzyme, activity(ies) of interest.
[0349] Mutagenized molecules provided by this invention may have
chimeric molecules and molecules with point mutations, including
biological molecules that contain a carbohydrate, a lipid, a
nucleic acid, &/or a protein component, and specific but
non-limiting examples of these include antibiotics, antibodies,
enzymes, and steroidal and non-steroidal hormones.
[0350] This invention relates generally to a method of: 1)
preparing a progeny generation of molecule(s) (including a molecule
that is comprised of a polynucleotide sequence, a molecule that is
comprised of a polypeptide sequence, and a molecules that is
comprised in part of a polynucleotide sequence and in part of a
polypeptide sequence), that is mutagenized to achieve at least one
point mutation, addition, deletion, &/or chimerization, from
one or more ancestral or parental generation template(s); 2)
screening the progeny generation molecule(s)--preferably using a
high throughput method--for at least one property of interest (such
as an improvement in an enzyme activity or an increase in stability
or a novel chemotherapeutic effect); 3) optionally obtaining
&/or cataloguing structural &/or and functional information
regarding the parental &/or progeny generation molecules; and
4) optionally repeating any of steps 1) to 3).
[0351] In a preferred embodiment, there is generated (e.g. from a
parent polynucleotide template)--in what is termed "codon
site-saturation mutagenesis"--a progeny generation of
polynucleotides, each having at least one set of up to three
contiguous point mutations (i.e. different bases comprising a new
codon), such that every codon (or every family of degenerate codons
encoding the same amino acid) is represented at each codon
position. Corresponding to--and encoded by--this progeny generation
of polynucleotides, there is also generated a set of progeny
polypeptides, each having at least one single amino acid point
mutation. In a preferred aspect, there is generated--in what is
termed "amino acid site-saturation mutagenesis"--one such mutant
polypeptide for each of the 19 naturally encoded
polypeptide-forming alpha-amino acid substitutions at each and
every amino acid position along the polypeptide. This yields--for
each and every amino acid position along the parental
polypeptide--a total of 20 distinct progeny polypeptides including
the original amino acid, or potentially more than 21 distinct
progeny polypeptides if additional amino acids are used either
instead of or in addition to the 20 naturally encoded amino
acids
[0352] Thus, in another aspect, this approach is also serviceable
for generating mutants containing--in addition to &/or in
combination with the 20 naturally encoded polypeptide-forming
alpha-amino acids--other rare &/or not naturally-encoded amino
acids and amino acid derivatives. In yet another aspect, this
approach is also serviceable for generating mutants by the use
of--in addition to &/or in combination with natural or
unaltered codon recognition systems of suitable hosts--altered,
mutagenized, &/or designer codon recognition systems (such as
in a host cell with one or more altered tRNA molecules).
[0353] In yet another aspect, this invention relates to
recombination and more specifically to a method for preparing
polynucleotides encoding a polypeptide by a method of in vivo
re-assortment of polynucleotide sequences containing regions of
partial homology, assembling the polynucleotides to form at least
one polynucleotide and screening the polynucleotides for the
production of polypeptide(s) having a useful property.
[0354] In yet another preferred embodiment, this invention is
serviceable for analyzing and cataloguing--with respect to any
molecular property (e.g. an enzymatic activity) or combination of
properties allowed by current technology--the effects of any
mutational change achieved (including particularly saturation
mutagenesis). Thus, a comprehensive method is provided for
determining the effect of changing each amino acid in a parental
polypeptide into each of at least 19 possible substitutions. This
allows each amino acid in a parental polypeptide to be
characterized and catalogued according to its spectrum of potential
effects on a measurable property of the polypeptide.
[0355] In another aspect, the method of the present invention
utilizes the natural property of cells to recombine molecules
and/or to mediate reductive processes that reduce the complexity of
sequences and extent of repeated or consecutive sequences
possessing regions of homology.
[0356] It is an object of the present invention to provide a method
for generating hybrid polynucleotides encoding biologically active
hybrid polypeptides with enhanced activities. In accomplishing
these and other objects, there has been provided, in accordance
with one aspect of the invention, a method for introducing
polynucleotides into a suitable host cell and growing the host cell
under conditions that produce a hybrid polynucleotide.
[0357] In another aspect of the invention, the invention provides a
method for screening for biologically active hybrid polypeptides
encoded by hybrid polynucleotides. The present method allows for
the identification of biologically active hybrid polypeptides with
enhanced biological activities.
1.4. BRIEF DESCRIPTION OF THE DRAWINGS
[0358] FIG. 1. Exonuclease Activity. FIG. 1 shows the activity of
the enzyme exonuclease III. This is an exemplary enzyme that can be
used to shuffle, assemble, reassemble, recombine, and/or
concatenate polynucleotide building blocks. The asterisk indicates
that the enzyme acts from the 3' direction towards the 5' direction
of the polynucleotide substrate.
[0359] FIG. 2. Generation of A Nucleic Acid Building Block by
Polymerase-Based Amplification. FIG. 2 illustrates a method of
generating a double-stranded nucleic acid building block with two
overhangs using a polymerase-based amplification reaction (e.g.,
PCR). As illustrated, a first polymerase-based amplification
reaction using a first set of primers, F.sub.2 and R.sub.1, is used
to generate a blunt-ended product (labeled Reaction 1, Product 1),
which is essentially identical to Product A. A second
polymerase-based amplification reaction using a second set of
primers, F.sub.1 and R.sub.2, is used to generate a blunt-ended
product (labeled Reaction 2, Product 2), which is essentially
identical to Product B. These two products are then mixed and
allowed to melt and anneal, generating a potentially useful
double-stranded nucleic acid building block with two overhangs. In
the example of FIG. 1, the product with the 3' overhangs (Product
C) is selected for by nuclease-based degradation of the other 3
products using a 3' acting exonuclease, such as exonuclease III.
Alternate primers are shown in parenthesis to illustrate
serviceable primers may overlap, and additionally that serviceable
primers may be of different lengths, as shown.
[0360] FIG. 3. Unique Overhangs And Unique Couplings. FIG. 3
illustrates the point that the number of unique overhangs of each
size (e.g. the total number of unique overhangs composed of 1 or 2
or 3, etc. nucleotides) exceeds the number of unique couplings that
can result from the use of all the unique overhangs of that size.
For example, there are 4 unique 3' overhangs composed of a single
nucleotide, and 4 unique 5' overhangs composed of a single
nucleotide. Yet the total number of unique couplings that can be
made using all the 8 unique single-nucleotide 3' overhangs and
single-nucleotide 5' overhangs is 4.
[0361] FIG. 4. Unique Overall Assembly Order Achieved by
Sequentially Coupling the Building Blocks
[0362] FIG. 4 illustrates the fact that in order to assemble a
total of "n" nucleic acid building blocks, "n-1" couplings are
needed. Yet it is sometimes the case that the number of unique
couplings available for use is fewer that the "n-1" value. Under
these, and other, circumstances a stringent non-stochastic overall
assembly order can still be achieved by performing the assembly
process in sequential steps. In this example, 2 sequential steps
are used to achieve a designed overall assembly order for five
nucleic acid building blocks. In this illustration the designed
overall assembly order for the five nucleic acid building blocks
is: 5'-(#1-#2-#3-#4-#5)-3', where #1 represents building block
number 1, etc.
[0363] FIG. 5. Unique Couplings Available Using a Two-Nucleotide 3'
Overhang. FIG. 5 further illustrates the point that the number of
unique overhangs of each size (here, e.g. the total number of
unique overhangs composed of 2 nucleotides) exceeds the number of
unique couplings that can result from the use of all the unique
overhangs of that size. For example, there are 16 unique 3'
overhangs composed of two nucleotides, and another 16 unique 5'
overhangs composed of two nucleotides, for a total of 32 as shown.
Yet the total number of couplings that are unique and not
self-binding that can be made using all the 32 unique
double-nucleotide 3' overhangs and double-nucleotide 5' overhangs
is 12. Some apparently unique couplings have "identical twins"
(marked in the same shading), which are visually obvious in this
illustration. Still other overhangs contain nucleotide sequences
that can self-bind in a palindromic fashion, as shown and labeled
in this figure; thus they not contribute the high stringency to the
overall assembly order.
[0364] FIG. 6. Generation of an Exhaustive Set of Chimeric
Combinations by Synthetic Ligation Reassembly. FIG. 6 showcases the
power of this invention in its ability to generate exhaustively and
systematically all possible combinations of the nucleic acid
building blocks designed in this example. Particularly large sets
(or libraries) of progeny chimeric molecules can be generated.
Because this method can be performed exhaustively and
systematically, the method application can be repeated by choosing
new demarcation points and with correspondingly newly designed
nucleic acid building blocks, bypassing the burden of re-generating
and re-screening previously examined and rejected molecular
species. It is appreciated that, codon wobble can be used to
advantage to increase the frequency of a demarcation point. In
other words, a particular base can often be substituted into a
nucleic acid building block without altering the amino acid encoded
by progenitor codon (that is now altered codon) because of codon
degeneracy. As illustrated, demarcation points are chosen upon
alignment of 8 progenitor templates. Nucleic acid building blocks
including their overhangs (which are serviceable for the formation
of ordered couplings) are then designed and synthesized. In this
instance, 18 nucleic acid building blocks are generated based on
the sequence of each of the 8 progenitor templates, for a total of
144 nucleic acid building blocks (or double-stranded oligos).
Performing the ligation synthesis procedure will then produce a
library of progeny molecules comprised of yield of 8.sup.18 (or
over 1.8.times.10.sup.16) chimeras.
[0365] FIG. 7. Synthetic genes from oligos:. According to one
embodiment of this invention, double-stranded nucleic acid building
blocks are designed by aligning a plurality of progenitor nucleic
acid templates. Preferably these templates contain some homology
and some heterology. The nucleic acids may encode related proteins,
such as related enzymes, which relationship may be based on
function or structure or both. FIG. 7 shows the alignment of three
polynucleotide progenitor templates and the selection of
demarcation points (boxed) shared by all the progenitor molecules.
In this particular example, the nucleic acid building blocks
derived from each of the progenitor templates were chosen to be
approximately 30 to 50 nucleotides in length.
[0366] FIG. 8. Nucleic acid building blocks for synthetic ligation
gene reassembly. FIG. 8 shows the nucleic acid building blocks from
the example in FIG. 7. The nucleic acid building blocks are shown
here in generic cartoon form, with their compatible overhangs,
including both 5' and 3' overhangs. There are 22 total nucleic acid
building blocks derived from each of the 3 progenitor templates.
Thus, the ligation synthesis procedure can produce a library of
progeny molecules comprised of yield of 3.sup.22 (or over
3.1.times.10.sup.10) chimeras.
[0367] FIG. 9. Addition of Introns by Synthetic Ligation
Reassembly. FIG. 9 shows in generic cartoon form that an intron may
be introduced into a chimeric progeny molecule by way of a nucleic
acid building block. It is appreciated that introns often have
consensus sequences at both termini in order to render them
operational. It is also appreciated that, in addition to enabling
gene splicing, introns may serve an additional purpose by providing
sites of homology to other nucleic acids to enable homologous
recombination. For this purpose, and potentially others, it may be
sometimes desirable to generate a large nucleic acid building block
for introducing an intron. If the size is overly large easily
genrating by direct chemical synthesis of two single stranded
oligos, such a specialized nucleic acid building block may also be
generated by direct chemical synthesis of more than two single
stranded oligos or by using a polymerase-based amplification
reaction as shown, described &/or referenced herein (including
incorporated by reference).
[0368] FIG. 10. Ligation Reassembly Using Fewer Than All The
Nucleotides Of An Overhang. FIG. 10 shows that coupling can occur
in a manner that does not make use of every nucleotide in a
participating overhang. The coupling is particularly lively to
survive (e.g. in a transformed host) if the coupling reinforced by
treatment with a ligase enzyme to form what may be referred to as a
"gap ligation" or a "gapped ligation". It is appreciated that, as
shown, this type of coupling can contribute to generation of
unwanted background product(s), but it can also be used
advantageously increase the diversity of the progeny library
generated by the designed ligation reassembly.
[0369] FIG. 11. Avoidance of unwanted self-ligation in palindromic
couplings. As mentioned before and shown, described &/or
referenced herein (including incorporated by reference), certain
overhangs are able to undergo self-coupling to form a palindromic
coupling. A coupling is strengthened substantially if it is
reinforced by treatment with a ligase enzyme. Accordingly, it is
appreciated that the lack of 5' phosphates on these overhangs, as
shown, can be used advantageously to prevent this type of
palindromic self-ligation. Accordingly, this invention provides
that nucleic acid building blocks can be chemically made (or
ordered) that lack a 5' phosphate group (or alternatively they can
be remove--e.g. by treatment with a phosphatase enzyme such as a
calf intestinal alkaline phosphatase (CIAP)--in order to prevent
palindromic self-ligations in ligation reassembly processes.
[0370] FIG. 12. Site-directed mutagenesis by polymerase-based
extension. Panel A. This figure shows one method of site-directed
mutagenesis, among many methods of site-directed mutagenesis, that
are serviceable for performing site-saturation mutagenesis. Section
(1) shows the first and second mutagenic primer annealed to a
circular closed double-stranded plasmid. The dot and the open-sided
triangle indicate the mutagenic sites in the mutagenic primers. The
arrows indicate the direction of synthesis. Section (2) shows the
newly synthesized (mutagenized) DNA strands annealed to each other.
The parental DNA can be treated with a selection enzyme. The
mutagenized DNA strands are shown as being annealed to form a
double-stranded mutagenized circular DNA intermediate. The dot and
the open-sided triangle indicate the mutagenic sites in the
experimentally generated progeny (mutagenized) DNA strands. Note
that the staggered openings on the mutagenized DNA strands form
"sticky" ends. Section (3) shows the first and second mutagenic
primer annealed to the mutagenized DNA strands of Section (2). The
arrows indicate the direction of synthesis. Note the opening on
each of the mutagenized DNA strands (i.e. they have not been
ligated). Section (4) shows a "Gapped Product", which is composed
of second generation mutagenized DNA strands, synthesized using the
mutagenized DNA strands (shown in Step (2)) as a template. The DNA
strands of the "Gapped Product" are shown as being annealed to form
a double-stranded mutagenized circular DNA intermediate. The dot
and the open-sided triangle indicate the mutagenic sites in the
mutagenized DNA strands. Note the large gap in each of the
mutagenized DNA strands. Section (5) shows the "Gapped Product"
annealed to the parental (non-mutated) plasmid, enabling
polymerase-based synthesis to occur. The arrows indicate the
direction of synthesis. Section (6) shows the newly synthesized DNA
strands, as being annealed to form a double-stranded mutagenized
circular DNA product. The dot and the open-sided triangle indicate
the mutagenic sites in the mutagenized DNA strands. Note the
staggered openings on the mutagenized DNA strands. Also note the
presence of both mutagenic sites on each of the mutagenized DNA
strands. Panel B. This figure shows two possible molecular
structures produced from the amplification steps of FIG. 12A.
Molecule (A) is shown also in Section (2) of FIG. 12A. Molecule (B)
is also shown in Section (6) of FIG. 12A.
[0371] FIG. 13. Site-directed mutagenesis by polymerase-based
extension and ligase-based ligation. Panel A. This figure shows one
method of site-directed mutagenesis, among many methods of
site-directed mutagenesis, that are serviceable for performing
site-saturation mutagenesis. Section (1) shows the first and second
mutagenic primer annealed to a circular closed double-stranded
plasmid. The dot and the open-sided triangle indicate the mutagenic
sites in the mutagenic primers. The arrows indicate the direction
of synthesis. Section (2) shows the newly synthesized (mutagenized)
DNA strands annealed to each other. The parental DNA can be treated
with a selection enzyme. The mutagenized DNA strands are shown as
being annealed to form a double-stranded mutagenized circular DNA
intermediate. The dot and the open-sided triangle indicate the
mutagenic sites in the experimentally generated progeny
(mutagenized) DNA strands. Note that the staggered openings on the
mutagenized DNA strands form "sticky" ends. Section (3) shows the
resultant double-stranded mutagenized circular DNA molecule
produced after the double-stranded mutagenized circular DNA
intermediate of Section (2) is ligated (e.g. with T4 DNA ligase).
Section (4) shows the first and second mutagenic primer annealed to
the mutagenized DNA strands of Section (3). The arrows indicate the
direction of synthesis. Section (5) shows the recently generated
(blue) mutagenized DNA strands as being annealed to form a
double-stranded mutagenized circular DNA intermediate. The dot and
the open-sided triangle indicate the mutagenic sites in the
recently generated mutagenized DNA strands (blue). Note that the
staggered openings on the mutagenized DNA strands form "sticky
ends". Also note the presence of both mutagenic sites on each of
the two recently generated mutagenized DNA strands (blue). Note the
opening on each of the mutagenized DNA strands (i.e. they have not
been ligated). Section (6) shows the resultant double-stranded
mutagenized circular DNA molecule produced after the
double-stranded mutagenized circular DNA intermediate of Section
(5) is ligated (e.g. using T4 DNA ligase). The dot and the
open-sided triangle indicate the mutagenic sites in the mutagenized
DNA molecules. Again, note the presence of both mutagenic sites on
each of the mutagenized DNA strands.
[0372] Panel B. This figure shows two molecular structures produced
from the amplification steps of FIG. 13A. Molecule (A) is also
shown in Section (3) of FIG. 13A. Molecule (B) is produced in
Section (6) of FIG. 13A.
[0373] FIG. 14: Strategy for Obtaining and Using Nucleic Acid
Binding Proteins that Facilitate Entry of Genetic Vaccines.
[0374] Shown here is a strategy for obtaining and using nucleic
acid binding proteins that facilitate entry of genetic vaccines, in
particular, naked DNA, into target cells. Members of a library
obtained by the directed evolution methods described herein are
linked to a coding region of M 13 protein VIII so that a fusion
protein is displayed on the surface of the phage particles. Phage
that efficiently enter the desired target tissue are identified,
and the fusion protein is then used to coat a genetic vaccine
nucleic acid.
[0375] FIG. 15: A schematic representation of a method for
generating a chimeric, multivalent antigen that has immunogenic
regions from multiple antigens.
[0376] Antibodies to each of the non-chimeric parental immunogenic
polypeptides are specific for the respective organisms (A, B, C).
After carrying out [the directed evolution] and selection methods
of the invention, however, a chimeric immunogenic polypeptide is
obtained that is recognized by antibodies raised against each of
the three parental immunogenic polypeptides.
[0377] FIG. 16A and FIG. 16B: Method for Obtaining
Non-Stochastically Generated Polypeptides that can induce a
Broad-Spectrum Immune Response.
[0378] Shown here is a schematic for a method by which one can
obtain non-stochastically generated polypeptides that can induce a
broad-spectrum immune response. In FIG. 16A, wild-type immunogenic
polypeptides from the pathogens A, B, and C provide protection
against the corresponding pathogen from which the polypeptide is
derived, but little or no cross-protection against the other
pathogens (left panel). After evolving, an A/B/C chimeric
polypeptide is obtained that can induce a protective immune
response against all three pathogen types (right panel). In FIG.
16B, directed evolution is used with substrate nucleic acids from
two pathogen strains (A, B), which encode polypeptides that are
protective only against the corresponding pathogen. After directed
evolution, the resulting chimeric polypeptide can induce an immune
response that is effective against not only the two parental
pathogen strains, but also against a third strain of pathogen
(C).
[0379] FIG. 17: Possible factors for determining whether a
particular polynucleotide encodes an immunogenic polypeptide having
a desired property.
[0380] Shown here are some of the possible factors that can
determine whether a particular polynucleotide encodes an
immunogenic polypeptide having a desired property, such as enhanced
immunogenicity and/or cross-reactivity. Those sequence regions that
positively affect a particular property are indicated as plus signs
along the antigen gene, while those sequence regions that have a
negative effect are shown as minus signs. A pool of related antigen
genes are non-stochastically generated using the methods described
herein and screened to obtain those evolved nucleic acids that have
gained positive sequence regions and lost negative regions. No
pre-existing knowledge as to which regions are positive or negative
for a particular trait is required.
[0381] FIG. 18: Screening strategy for antigen library
screening.
[0382] Shown here is a schematic representation of the screening
strategy for antigen library screening.
[0383] FIG. 19: Strategy for pooling and deconvolution as used in
antigen library screening.
[0384] Shown here is a schematic representation of a strategy for
pooling and deconvolution as used in antigen library screening.
[0385] FIG. 20A: Expression of non-stochastically generated
envelope gp120 protein in a genetic vaccine.
[0386] Shown here is a diagram of a method for expressing HIV gp
120 using genetic vaccine vectors and generation of a library of
non-stochastically generated gp120 genes.
[0387] FIG. 20B: PCR primers for genetic vaccine for HIV.
[0388] Shown here are PCR primers that are useful for obtaining gp
120 nucleic acid substrates for directed evolution reactions.
Primers suitable for generating substrates include 6025F (SEQ ID
NO: 4), 7773R (SEQ ID NO: 5), and primers suitable for amplifying
the non-stochastically generated nucleic acids include 6196F (SEQ
ID NO: 6) and 7746R (SEQ ID NO: 7). The primer BssH2-6205F (SEQ ID
NO: 8) can be used to clone the resulting fragment into a genetic
vaccine vector.
[0389] FIG. 21. Schematic representation of a multimodule genetic
vaccine vector.
[0390] Shown here is a schematic representation of a multimodule
genetic vaccine vector. A typical genetic vaccine vector will
include one or more of the components indicated, each of which can
be native or optimized using the directed evolution methods
described herein. These directed evolution methods can include the
introduction of point mutations by stochastic methods &/or by
non-stochastic methods, including "gene site saturation
mutagenesis" as described herein. These directed evolution methods
can also include stochastic polynucleotide reassembly methods, for
example by interrupted synthesis (as described in U.S. Pat. No.
5,965,408). These directed evolution methods can also include
non-stochastic polynucleotide reassembly methods as described
herein, including synthetic ligation polynucleotide reassembly as
described herein. The components can be present on the same vaccine
vector, or can be included in a genetic vaccine as separate
molecules.
[0391] FIG. 22A and FIG. 22B. Generation of vectors with multiple T
cell epitopes. Shown here are two different strategies for
generating vectors that contain multiple T cell epitopes obtained,
for example, by directed evolution. In FIG. 60A, each individual
non-stochastically generated epitope-encoding gene is linked to a
single promoter, and multiple promoter-epitope gene constructs can
be placed in a single vector. The scheme shown, described &/or
referenced herein (including incorporated by reference) involves
linking multiple epitope-encoding genes to a single promoter.
[0392] FIG. 23. Generation of optimized genetic vaccines by
directed evolution. Shown here is a diagram of the application of
directed evolution to the generation of optimized genetic vaccines.
Different forms of polynucleotides having known functional
properties (e.g., regulatory, coding, and the like) are evolved and
screened to identify variants that exhibit improved properties for
use as genetic vaccines.
[0393] FIG. 24. Recursive application of directed evolution and
selection of evolved promoter sequences as an example of flow
cytometry-based screening methods. Shown here is a diagram of flow
cytometry-based screening methods (FACS) for selection of optimized
promoter sequences evolved using recursive applications of the
directed evolution methods as described herein. A cytomegalovirus
(CMV) promoter is used for illustrative purposes.
[0394] FIG. 25. An apparatus for microinjections of skin and
muscle. Shown here is an apparatus that is suitable for
microinjection of genetic vaccines and other reagents into tissue
such as skin and muscle. The apparatus is particularly useful for
screening large numbers of agents in vivo, being based on a 96-well
format. The tips of the apparatus are movable to allow adjustment
so that the tips fit into a microtiter plate. After obtaining a
reagent of interest is obtained from a plate, the tips are adjusted
to a distance of about 2-3 min apart, enabling transfer of 96
different samples to an area of about 1.6 cm by 2.4 cm to about 2.4
cm by 3.6 cm. If desired, the volume of each sample transferred can
be electronically controlled; typically the volumes transferred
range from about 2 ul to about 5 ul. Each reagent can be mixed with
a marker agent or dye to facilitate recognition of the injection
site in the tissue. For example, gold particles of different sizes
and shaped can be mixed with the reagent of interest, and
microscopy and immunohistochemistry used to identify each injection
site and to study the reaction induced by each reagent. When muscle
tissue is injected, the injection site is first revealed by
surgery.
[0395] FIG. 26. Polynucleotide reassembly. Shown in Panel A is an
example of directed evolution. n different strains of a virus are
used in this illustration, but the technique is applicable to any
single nucleic acid as well as to any nucleic acid for which
different strains, species, or gene families have homologous
nucleic acids that have one or more nucleotide changes compared to
other homologous nucleic acids. The different variant nucleic acids
are experimentally generated, preferably non-stochastically, as
described herein, and screened or selected to identify those
variants that exhibit the desired property. The directed evolution
method(s) and screening can be repeated one or more times to obtain
further improvement. Panel B shows that successive rounds of
directed evolution can produce progressively enhanced properties,
and that the combination of individual beneficial mutations can
lead to an enhance improvement compared to the improvement achieved
by an individual beneficial mutation.
[0396] FIG. 27. Vector for promoter evolution. Shown here is an
example of a vector that is useful for screening to identify
improved promoters from a library of promoter nucleic acids evolved
using the directed evolution methods as described herein.
Experimentally generated putative promoters are inserted into the
vector upstream of a reporter gene for which expression is readily
detected. For many applications, it is desirable that the product
of the reporter gene be a cell surface protein so that cells which
express high levels of the reporter gene can be sorted using flow
cytometry-based cell sorting using the reporter gene product.
Examples of suitable reporter genes include, for example, B7-2 and
mAb179 epitopes. A polyadenylation region is typically placed
downstream of the reporter gene (SV40 polyA is illustrated). The
vector can also include a second reporter gene an internal control
(GFP; green fluorescent protein); this gene is linked to a promoter
(SR p). The vector also typically includes a selectable marker
(kanamycin/neomycin resistance is shown), and origins of
replication that are functional in mammalian (SV40 ori) and/or
bacterial (pUC ori) cells.
[0397] FIG. 28. Iterative evolution of inducible promoters using
directed evolution and flow cytometry-based selection. Shown here
is a diagram of a scheme for iterative evolution of inducible
promoters using the directed evolution methods as described herein
and flow cytometry-based selection. A library of experimentally
generated (i.e. produced by one or more directed evolution methods
as descried herein) promoter nucleic acids present in appropriate
vectors is transfected into the cells, and those cells which
exhibit the least expression of marker antigen when grown under
uninduced conditions are selected. The vectors (&/or cells
containing them) are recovered, and the vectors are introduced into
cells (if not contained therein already), and grown under inducing
conditions. Those cells that express the highest level of marker
antigen are selected.
[0398] FIG. 29. Evolving a genetic vaccine vector for Oral,
Intravenous, Intramuscular, Intradermal, Anal, Vaginal, or Topical
Delivery. Illustrated is a strategy for screening of M13 libraries
(e.g. generated experimentally using directed evolution as descried
herein) for desired targeting of various tissues. The particular
example shown here is a schematic diagram of a method for evolving
a genetic vaccine vector for improved oral delivery. This may
comprise selecting for stability under the acidic conditions of the
stomach, and resistance to other degredatory factors of the
digestive tract. The particular example illustrated relates to
screening for improved oral delivery, but the same principle
applies to libraries administered by other routes, including
intravenously, intramuscularly, intradermally, anally, vaginally,
or topically. After delivery to a test animal, the M13 phage (or a
product thereof) is recovered from the tissue of interest. The
procedure can be repeated to obtain further optimization.
[0399] FIG. 30. An alignment of the nucleotide sequences of the
immediate/early gene of two human CMV strains and two monkey
strains. Shown here is an alignment of the nucleotide sequences of
the immediate/early gene of two human cytomegalovirus (CMV) strains
and two monkey strains. This alignment is serviceable for
performing non-stochastic polynucleotide reassembly. Shared
nucleotide sequences of 3 or more bases are underlined to
illustrate preferred but non-limiting examples of reassembly
points.
[0400] FIG. 31. An alignment of Intron A sequences from CMV strains
Towne and AD169. Shown here is an alignment of Intron A nucleotide
sequences from CMV strains Towne and AD169. This alignment is
serviceable for performing non-stochastic polynucleotide
reassembly. Shared nucleotide sequences of 3 or more bases are
underlined to illustrate preferred but non-limiting examples of
reassembly points.
[0401] FIG. 32. Generation of a Library of Evolved CMV Promoters.
Shown here is a schematic presentation of the
promoter/enhancer/intron sequences derived from human (AD 169 and
Towne) and monkey (rhesus and vervet monkey) cytomegaloviruses by
PCR amplification. These amplified fragments are suitable for use
in directed evolution. The ability to align them renders them
serviceable for subjection to non-stochastic polynucleotide
reassembly.
[0402] FIG. 33. Non-stochastic Reassembly of oligo-directed CpG
knock-outs. Shown here is a schematic representation of the use of
the non-stochastic methods described herein to generate promoter
sequences in which unnecessary CpG sequences are deleted,
potentially useful CpG sequences are added, and non-replaceable CpG
sequences are identified. Additionally, other sequences (aside from
the CpG sequences) can be substituted into, added to, &/or
deleted from working polynucleotides.
[0403] FIG. 34. An Example of a CTIS obtained from HbsAg
polypeptide (PreS2 plus S regions). Shown here is an example of a
cytotoxic T-cell inducing sequence (CTIS) obtained from HBsAg
polypeptide (PreS2 plus S regions).
[0404] FIG. 35. A CTIS Having Heterologous Epitopes Attached to the
Cytoplasmic Portion. Shown here is a CTIS having heterologous
epitopes attached to the cytoplasmic portion.
[0405] FIG. 36. Method for preparing immunogenic agonist sequences
(IAS). Shown here is a method for preparing immunogenic agonist
sequences (IAS). Wild-type (WT) and mutated forms of nucleic acids
encoding a polypeptide of interest are assembled and subjected to
non-stochastic reassembly to obtain a nucleic acid encoding a
poly-epitope region that contains potential agonist sequences.
[0406] FIG. 37. Improving Immunostimulatory Sequences (ISS) Using
Directed Evolution. Shown here is a scheme for improving
immunostimulatory sequences by the directed evolution methods
described herein. Oligonucleotide building blocks (e.g.
synthetically generated), oligos with known ISS, CpG containing
hexamers &/or oligos containing CpG containing hexamers, poly
A, C, G, T, etc . . . can be assembled. The resultant molecule(s)
can then by subjected to 1 or more directed evolution methods as
described herein.
[0407] FIG. 38. Screening to identify IL-12 genes that encode
recombinant IL-12 having an increased ability to induce T Cell
proliferation. Shown here is a diagram of a procedure by which
experimentally generated molecules, e.g. non-stochastically
generated libraries of human IL-12 genes can be screened to
identify evolved IL-12 genes that encode evolved forms of IL-12
having increased ability to induce T cell proliferation.
[0408] FIG. 39. Model of induction of T cell activation or anergy
by genetic vaccine vectors encoding different CD80 and/or CD86
variants. Shown here is a model of how T cell activation or anergy
can be induced by genetic vaccine vectors that encode different
B7-1 (CD80) and/or B7-2 (CD86) variants.
[0409] FIG. 40. Screening of CD80/CD86 variants that have improved
capacity to induce T cell activation or anergy. Shown here is a
method for using directed evolution as described herein to obtain
CD80/CD86 variants that have improved capacity to induce T cell
activation or anergy.
[0410] FIG. 41. An alignment of the nucleotide sequences for human
and mouse IL-10 receptor sequences. Shown here is an alignment of
the nucleotide sequences for human and mouse IL-10 receptor
sequences. This alignment is serviceable for performing
non-stochastic polynucleotide reassembly. Shared nucleotide
sequences of 3 or more bases are underlined (with number of
consecutive bases indicated) to illustrate preferred but
non-limiting examples of reassembly points.
[0411] FIG. 42: An alignment of the nucleotide sequences for human,
rhesus monkey, and rabbit B7-1 (CD80) genes. Shown here is an
alignment of the nucleotide sequences of B7-1 (CD80) genes from
human, rhesus monkey, and rabbit. This alignment is serviceable for
performing non-stochastic polynucleotide reassembly. Shared
nucleotide sequences of 3 or more bases are underlined (with number
of consecutive bases indicated) to illustrate preferred but
non-limiting examples of reassembly points.
2. DETAILED DESCRIPTION OF THE INVENTION
2.1. DEFINITIONS OF TERMS
[0412] In order to facilitate understanding of the examples
provided herein, certain frequently occurring methods and/or terms
will be described.
[0413] The term "agent" is used herein to denote a chemical
compound, a mixture of chemical compounds, an array of spatially
localized compounds (e.g., a VLSIPS peptide array, polynucleotide
array, and/or combinatorial small molecule array), biological
macromolecule, a bacteriophage peptide display library, a
bacteriophage antibody (e.g., scFv) display library, a polysome
peptide display library, or an extract made form biological
materials such as bacteria, plants, fungi, or animal (particular
mammalian) cells or tissues. Agents are evaluated for potential
activity as anti-neoplastics, anti-inflammatories or apoptosis
modulators by inclusion in screening assays described hereinbelow.
Agents are evaluated for potential activity as specific protein
interaction inhibitors (i.e., an agent which selectively inhibits a
binding interaction between two predetermined polypeptides but
which doe snot substantially interfere with cell viability) by
inclusion in screening assays described hereinbelow.
[0414] An "ambiguous base requirement" in a restriction site refers
to a nucleotide base requirement that is not specified to the
fullest extent, i.e. that is not a specific base (such as, in a
non-limiting exemplification, a specific base selected from A, C,
G, and T), but rather may be any one of at least two or more bases.
Commonly accepted abbreviations that are used in the art as well as
herein to represent ambiguity in bases include the following: R=G
or A; Y=C or T; M=A or C; K=G or T; S=G or C; W=A or T; H=A or C or
T; B=G or T or C; V=G or C or A; D=G or A or T; N=A or C or G or
T.
[0415] "Alignment" with respect to molecular sequences is a way to
determine similarity between 2 or more sequences. Optimal alignment
of sequences for comparison can be conducted, e.g., by the local
homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482
(1981), by the homology alignment algorithm of Needleman &
Wunsch, J Mol. Biol. 48:443 (1970), by the search for similarity
method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444
(1988), by computerized implementations of these algorithms (GAP,
BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software
Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.),
or by visual inspection (see generally Ausubel et al., infra).
[0416] One example of an algorithm that is suitable for determining
percent sequence identity and sequence similarity is the BLAST
algorithm, which is described in Altschul et al., J Mol. Biol.
215:403-410 (1990). Software for performing BLAST analyses is
publicly available through the National Center for Biotechnology
Information (http://www.ncbl.nlm.nih.go- v/). This algorithm
involves first identifying high scoring sequence pairs (HSPs) by
identifying short words of length W in the query sequence, which
either match or satisfy some positive-valued threshold score T when
aligned with a word of the same length in a database sequence. T is
referred to as the neighborhood word score threshold (Altschul et
al., supra). These initial neighborhood word hits act as seeds for
initiating searches to find longer HSPs containing them. The word
hits are then extended in both directions along each sequence for
as far as the cumulative alignment score can be increased.
Cumulative scores are calculated using, for nucleotide sequences,
the parameters M (reward score for a pair of matching residues;
always >0) and N (penalty score for mismatching residues; always
<0). For amino acid sequences, a scoring matrix is used to
calculate the cumulative score. Extension of the word hits in each
direction are halted when: the cumulative alignment score falls off
by the quantity X from its maximum achieved value; the cumulative
score goes to zero or below, due to the accumulation of one or more
negative-scoring residue alignments; or the end of either sequence
is reached.
[0417] The BLAST algorithm parameters W, T, and X determine the
sensitivity and speed of the alignment. The BLASTN program (for
nucleotide sequences) uses as defaults a wordlength (W) of 11, an
expectation (E) of 10, a cutoff of 100, M=5, N=-4, and a comparison
of both strands. For amino acid sequences, the BLASTP program uses
as defaults a wordlength (W) of 3, an expectation (E) of 10, and
the BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989)
Proc. Natl. Acad. Sci. USA 89:10915).
[0418] In addition to calculating percent sequence identity, the
BLAST algorithm also performs a statistical analysis of the
similarity between two sequences (see, e.g., Karlin & Altschul
(1993) Proc. Nat'l. Acad. Sci. USA 90:5873-5787). One measure of
similarity provided by the BLAST algorithm is the smallest sum
probability (P(N)), which provides an indication of the probability
by which a match between two nucleotide or amino acid sequences
would occur by chance. For example, a nucleic acid is considered
similar to a reference sequence if the smallest sum probability in
a comparison of the test nucleic acid to the reference nucleic acid
is less than about 0.1, more preferably less than about 0.01, and
most preferably less than about 0.001.
[0419] Another indication that two nucleic acid sequences are
substantially identical is that the two molecules hybridize to each
other under stringent conditions. The phrase "hybridizing
specifically to", refers to the binding, duplexing, or hybridizing
of a molecule only to a particular nucleotide sequence under
stringent conditions when that sequence is present in a complex
mixture (e.g., total cellular) DNA or RNA. "Bind(s) substantially"
refers to complementary hybridization between a probe nucleic acid
and a target nucleic acid and embraces minor mismatches that can be
accommodated by reducing the stringency of the hybridization media
to achieve the desired detection of the target polynucleotide
sequence.
[0420] The term "amino acid" as used herein refers to any organic
compound that contains an amino group (--NH.sub.2) and a carboxyl
group (--COOH); preferably either as free groups or alternatively
after condensation as part of peptide bonds. The "twenty naturally
encoded polypeptide-forming alpha-amino acids" are understood in
the art and refer to: alanine (ala or A), arginine (arg or R),
asparagine (asn or N), aspartic acid (asp or D), cysteine (cys or
C), gluatamic acid (glu or E), glutamine (gln or Q), glycine (gly
or G), histidine (his or H), isoleucine (ile or I), leucine (leu or
L), lysine (lys or K), methionine (met or M), phenylalanine (phe or
F), proline (pro or P), serine (ser or S), threonine (thr or T),
tryptophan (trp or W), tyrosine (tyr or Y), and valine (val or
V).
[0421] The term "amplification" means that the number of copies of
a polynucleotide is increased.
[0422] The term "antibody", as used herein, refers to intact
immunoglobulin molecules, as well as fragments of immunoglobulin
molecules, such as Fab, Fab', (Fab').sub.2, Fv, and SCA fragments,
that are capable of binding to an epitope of an antigen. These
antibody fragments, which retain some ability to selectively bind
to an antigen (e.g., a polypeptide antigen) of the antibody from
which they are derived, can be made using well known methods in the
art (see, e.g., Harlow and Lane, supra), and are described further,
as follows.
[0423] (1) An Fab fragment consists of a monovalent antigen-binding
fragment of an antibody molecule, and can be produced by digestion
of a whole antibody molecule with the enzyme papain, to yield a
fragment consisting of an intact light chain and a portion of a
heavy chain.
[0424] (2) An Fab' fragment of an antibody molecule can be obtained
by treating a whole antibody molecule with pepsin, followed by
reduction, to yield a molecule consisting of an intact light chain
and a portion of a heavy chain. Two Fab' fragments are obtained per
antibody molecule treated in this manner.
[0425] (3) An (Fab').sub.2 fragment of an antibody can be obtained
by treating a whole antibody molecule with the enzyme pepsin,
without subsequent reduction. A (Fab').sub.2 fragment is a dimer of
two Fab' fragments, held together by two disulfide bonds.
[0426] (4) An Fv fragment is defined as a genetically engineered
fragment containing the variable region of a light chain and the
variable region of a heavy chain expressed as two chains.
[0427] (5) An single chain antibody ("SCA") is a genetically
engineered single chain molecule containing the variable region of
a light chain and the variable region of a heavy chain, linked by a
suitable, flexible polypeptide linker.
[0428] The term "Applied Molecular Evolution" ("AME") means the
application of an evolutionary design algorithm to a specific,
useful goal. While many different library formats for AME have been
reported for polynucleotides, peptides and proteins (phage, lacI
and polysomes), none of these formats have provided for
recombination by random cross-overs to deliberately create a
combinatorial library.
[0429] A molecule that has a "chimeric property" is a molecule that
is: 1) in part homologous and in part heterologous to a first
reference molecule; while 2) at the same time being in part
homologous and in part heterologous to a second reference molecule;
without 3) precluding the possibility of being at the same time in
part homologous and in part heterologous to still one or more
additional reference molecules. In a non-limiting embodiment, a
chimeric molecule may be prepared by assemblying a reassortment of
partial molecular sequences. In a non-limiting aspect, a chimeric
polynucleotide molecule may be prepared by synthesizing the
chimeric polynucleotide using plurality of molecular templates,
such that the resultant chimeric polynucleotide has properties of a
plurality of templates.
[0430] The term "cognate" as used herein refers to a gene sequence
that is evolutionarily and functionally related between species.
For example, but not limitation, in the human genome the human CD4
gene is the cognate gene to the mouse 3d4 gene, since the sequences
and structures of these two genes indicate that they are highly
homologous and both genes encode a protein which functions in
signaling T cell activation through MHC class II-restricted antigen
recognition.
[0431] A "comparison window," as used herein, refers to a
conceptual segment of at least 20 contiguous nucleotide positions
wherein a polynucleotide sequence may be compared to a reference
sequence of at least 20 contiguous nucleotides and wherein the
portion of the polynucleotide sequence in the comparison window may
comprise additions or deletions (i.e., gaps) of 20 percent or less
as compared to the reference sequence (which does not comprise
additions or deletions) for optimal alignment of the two sequences.
Optimal alignment of sequences for aligning a comparison window may
be conducted by the local homology algorithm of Smith (Smith and
Waterman, Adv Appl Math, 1981; Smith and Waterman, J Teor Biol,
1981; Smith and Waterman, J Mol Biol, 1981; Smith et al, J Mol
Evol, 1981), by the homology alignment algorithm of Needleman
(Needleman and Wuncsch, 1970), by the search of similarity method
of Pearson (Pearson and Lipman, 1988), by computerized
implementations of these algorithms (GAP, BESTFIT, FASTA, and
TFASTA in the Wisconsin Genetics Software Package Release 7.0,
Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by
inspection, and the best alignment (i.e., resulting in the highest
percentage of homology over the comparison window) generated by the
various methods is selected.
[0432] As used herein, the term "complementarity-determining
region" and "CDR" refer to the art-recognized term as exemplified
by the Kabat and Chothia CDR definitions also generally known as
supervariable regions or hypervariable loops (Chothia and Lesk,
1987; Clothia et al, 1989; Kabat et al, 1987; and Tramontano et al,
1990). Variable region domains typically comprise the
amino-terminal approximately 105-115 amino acids of a
naturally-occurring immunoglobulin chain (e.g., amino acids 1-110),
although variable domains somewhat shorter or longer are also
suitable for forming single-chain antibodies.
[0433] "Conservative amino acid substitutions" refer to the
interchangeability of residues having similar side chains. For
example, a group of amino acids having aliphatic side chains is
glycine, alanine, valine, leucine, and isoleucine; a group of amino
acids having aliphatic-hydroxyl side chains is serine and
threonine; a group of amino acids having amide-containing side
chains is asparagine and glutamine; a group of amino acids having
aromatic side chains is phenylalanine, tyrosine, and tryptophan; a
group of amino acids having basic side chains is lysine, arginine,
and histidine; and a group of amino acids having sulfur-containing
side chains is cysteine and methionine. Preferred conservative
amino acids substitution groups are: valine-leucine-isoleuci- ne,
phenylalanine-tyrosine, lysine-arginine, alanine-valine, and
asparagine-glutamine.
[0434] "Conservatively modified variations" of a particular
polynucleotide sequence refers to those polynucleotides that encode
identical or essentially identical amino acid sequences, or where
the polynucleotide does not encode an amino acid sequence, to
essentially identical sequences. Because of the degeneracy of the
genetic code, a large number of functionally identical nucleic
acids encode any given polypeptide. For instance, the codons CGU,
CGC, CGA, CGG, AGA, and AGG all encode the amino acid arginine.
[0435] Thus, at every position where an arginine is specified by a
codon, the codon can be altered to any of the corresponding codons
described without altering the encoded polypeptide. Such nucleic
acid variations are "silent variations," which are one species of
"conservatively modified variations." Every polynucleotide sequence
described herein which encodes a polypeptide also describes every
possible silent variation, except where otherwise noted.
[0436] One of skill will recognize that each codon in a nucleic
acid (except AUG, which is ordinarily the only codon for
methionine) can be modified to yield a functionally identical
molecule by standard techniques. Accordingly, each "silent
variation" of a nucleic acid which encodes a polypeptide is
implicit in each described sequence.
[0437] Furthermore, one of skill will recognize that individual
substitutions, deletions or additions which alter, add or delete a
single amino acid or a small percentage of amino acids (typically
less than 5%, more typically less than 1%) in an encoded sequence
are "conservatively modified variations" where the alterations
result in the substitution of an amino acid with a chemically
similar amino acid. Conservative substitution tables providing
functionally similar amino acids are well known in the art. The
following five groups each contain amino acids that are
conservative substitutions for one another:
[0438] Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine
(L), Isoleucine (1);
[0439] Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan
(W);
[0440] Sulfur-containing: Methionine (M), Cysteine (C);
[0441] Basic: Arginine (R), Lysine (K), Histidine (H);
[0442] Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine
(N), Glutamine (Q).
[0443] See also, Creighton (1984) Proteins, W. H. Freeman and
Company, for additional groupings of amino acids. In addition,
individual substitutions, deletions or additions which alter, add
or delete a single amino acid or a small percentage of amino acids
in an encoded sequence are also "conservatively modified
variations".
[0444] The term "corresponds to" is used herein to mean that a
polynucleotide sequence is homologous (i.e., is identical, not
strictly evolutionarily related) to all or a portion of a reference
polynucleotide sequence, or that a polypeptide sequence is
identical to a reference polypeptide sequence. In
contradistinction, the term "complementary to" is used herein to
mean that the complementary sequence is homologous to all or a
portion of a reference polynucleotide sequence. For illustration,
the nucleotide sequence "TATAC" corresponds to a reference "TATAC"
and is complementary to a reference sequence "GTATA."
[0445] The term "cytokine" includes, for example, interleukins,
interferons, chemokines, hematopoietic growth factors, tumor
necrosis factors and transforming growth factors. In general these
are small molecular weight proteins that regulate maturation,
activation, proliferation and differentiation of the cells of the
immune system.
[0446] The term "degrading effective" amount refers to the amount
of enzyme which is required to process at least 50% of the
substrate, as compared to substrate not contacted with the enzyme.
Preferably, at least 80% of the substrate is degraded.
[0447] As used herein, the term "defined sequence framework" refers
to a set of defined sequences that are selected on a non-random
basis, generally on the basis of experimental data or structural
data; for example, a defined sequence framework may comprise a set
of amino acid sequences that are predicted to form a .beta.-sheet
structure or may comprise a leucine zipper heptad repeat motif, a
zinc-finger domain, among other variations. A "defined sequence
kernal" is a set of sequences which encompass a limited scope of
variability. Whereas (1) a completely random 10-mer sequence of the
20 conventional amino acids can be any of (20).sup.10 sequences,
and (2) a pseudorandom 10-mer sequence of the 20 conventional amino
acids can be any of (20).sup.10 sequences but will exhibit a bias
for certain residues at certain positions and/or overall, (3) a
defined sequence kernal is a subset of sequences if each residue
position was allowed to be any of the allowable 20 conventional
amino acids (and/or allowable unconventional amino/imino acids). A
defined sequence kernal generally comprises variant and invariant
residue positions and/or comprises variant residue positions which
can comprise a residue selected from a defined subset of amino acid
residues), and the like, either segmentally or over the entire
length of the individual selected library member sequence. Defined
sequence kernels can refer to either amino acid sequences or
polynucleotide sequences. Of illustration and not limitation, the
sequences (NNK).sub.10 and (NNM).sub.10, wherein N represents A, T,
G, or C; K represents G or T; and M represents A or C, are defined
sequence kernels.
[0448] "Digestion" of DNA refers to catalytic cleavage of the DNA
with a restriction enzyme that acts only at certain sequences in
the DNA. The various restriction enzymes used herein are
commercially available and their reaction conditions, cofactors and
other requirements were used as would be known to the ordinarily
skilled artisan. For analytical purposes, typically 1 .mu.g of
plasmid or DNA fragment is used with about 2 units of enzyme in
about 20 .mu.l of buffer solution. For the purpose of isolating DNA
fragments for plasmid construction, typically 5 to 50 .mu.g of DNA
are digested with 20 to 250 units of enzyme in a larger volume.
Appropriate buffers and substrate amounts for particular
restriction enzymes are specified by the manufacturer. Incubation
times of about 1 hour at 37.degree. C. are ordinarily used, but may
vary in accordance with the supplier's instructions. After
digestion the reaction is electrophoresed directly on a gel to
isolate the desired fragment.
[0449] "Directional ligation" refers to a ligation in which a 5'
end and a 3' end of a polynuclotide are different enough to specify
a preferred ligation orientation. For example, an otherwise
untreated and undigested PCR product that has two blunt ends will
typically not have a preferred ligation orientation when ligated
into a cloning vector digested to produce blunt ends in its
multiple cloning site; thus, directional ligation will typically
not be displayed under these circumstances. In contrast,
directional ligation will typically displayed when a digested PCR
product having a 5' EcoR I-treated end and a 3' BamH I-is ligated
into a cloning vector that has a multiple cloning site digested
with EcoR I and BamH I.
[0450] The term "DNA shuffling" is used herein to indicate
recombination between substantially homologous but non-identical
sequences, in some embodiments DNA shuffling may involve crossover
via non-homologous recombination, such as via cer/lox and/or
flp/frt systems and the like.
[0451] As used in this invention, the term "epitope" refers to an
antigenic determinant on an antigen, such as a phytase polypeptide,
to which the paratope of an antibody, such as an phytase-specific
antibody, binds. Antigenic determinants usually consist of
chemically active surface groupings of molecules, such as amino
acids or sugar side chains, and can have specific three-dimensional
structural characteristics, as well as specific charge
characteristics. As used herein "epitope" refers to that portion of
an antigen or other macromolecule capable of forming a binding
interaction that interacts with the variable region binding body of
an antibody. Typically, such binding interaction is manifested as
an intermolecular contact with one or more amino acid residues of a
CDR.
[0452] An "exogenous DNA segment", "heterologous sequence" or a
"heterologous nucleic acid", as used herein, is one that originates
from a source foreign to the particular host cell, or, if from the
same source, is modified from its original form. Thus, a
beterologous gene in a host cell includes a gene that is endogenous
to the particular host cell, but has been modified. Modification of
a heterologous sequence in the applications described herein
typically occurs through the use of stochastic (e.g. polynucleotide
shuffling & interrupted synthesis) and non-stochastic
polynucleotide reassembly. Thus, the terms refer to a DNA segment
which is foreign or heterologous to the cell, or homologous to the
cell but in a position within the host cell nucleic acid in which
the element is not ordinarily found.
[0453] "Exogenous" DNA segments are expressed to yield exogenous
polypeptides.
[0454] The term "gene" is used broadly to refer to any segment of
DNA associated with a biological function. Thus, genes include
coding sequences and/or the regulatory sequences required for their
expression. Genes also include nonexpressed DNA segments that, for
example, form recognition sequences for other proteins. Genes can
be obtained from a variety of sources, including cloning from a
source of interest or synthesizing from known or predicted sequence
information, and may include sequences designed to have desired
parameters.
[0455] An "experimentally generated (in vitro &/or in vivo)
polynucleotide" (which term includes a "recombinant
polynucleotide") or an "experimentally (in vitro &/or in vivo)
generated polypeptide" (which term includes a "experimentally
generated polypeptide") is a non-naturally occurring polynucleotide
or polypeptide that includes nucleic acid or amino acid sequences,
respectively, from more than one source nucleic acid or
polypeptide, which source nucleic acid or polypeptide can be a
naturally occurring nucleic acid or polypeptide, or can itself have
been subjected to mutagenesis or other type of modification. The
source polynucleotides or polypeptides from which the different
nucleic acid or amino acid sequences are derived are sometimes
homologous (i.e., have, or encode a polypeptide that encodes, the
same or a similar structure and/or function), and are often from
different isolates, serotypes, strains, species, of organism or
from different disease states, for example.
[0456] The terms "fragment", "derivative" and "analog" when
referring to a reference polypeptide comprise a polypeptide which
retains at least one biological function or activity that is at
least essentially same as that of the reference polypeptide.
Furthermore, the terms "fragment", "derivative" or "analog" are
exemplified by a "pro-form" molecule, such as a low activity
proprotein that can be modified by cleavage to produce a mature
enzyme with significantly higher activity.
[0457] A method is provided herein for producing from a template
polypeptide a set of progeny polypeptides in which a "full range of
single amino acid substitutions" is represented at each amino acid
position. As used herein, "full range of single amino acid
substitutions" is in reference to the naturally encoded 20
naturally encoded polypeptide-forming alpha-amino acids, as
described herein.
[0458] The term "gene" means the segment of DNA involved in
producing a polypeptide chain; it includes regions preceding and
following the coding region (leader and trailer) as well as
intervening sequences (introns) between individual coding segments
(exons).
[0459] "Genetic instability", as used herein, refers to the natural
tendency of highly repetitive sequences to be lost through a
process of reductive events generally involving sequence
simplification through the loss of repeated sequences. Deletions
tend to involve the loss of one copy of a repeat and everything
between the repeats.
[0460] The term "heterologous" means that one single-stranded
nucleic acid sequence is unable to hybridize to another
single-stranded nucleic acid sequence or its complement. Thus areas
of heterology means that areas of polynucleotides or
polynucleotides have areas or regions within their sequence which
are unable to hybridize to another nucleic acid or polynucleotide.
Such regions or areas are for example areas of mutations.
[0461] The term "homologous" or "homeologous" means that one
single-stranded nucleic acid nucleic acid sequence may hybridize to
a complementary single-stranded nucleic acid sequence. The degree
of hybridization may depend on a number of factors including the
amount of identity between the sequences and the hybridization
conditions such as temperature and salt concentrations as discussed
later. Preferably the region of identity is greater than about 5
bp, more preferably the region of identity is greater than 10
bp.
[0462] An immunoglobulin light or heavy chain variable region
consists of a "framework" region interrupted by three hypervariable
regions, also called CDR's. The extent of the framework region and
CDR's have been precisely defined; see "Sequences of Proteins of
Immunological Interest" (Kabat et al, 1987). The sequences of the
framework regions of different light or heavy chains are relatively
conserved within a specie. As used herein, a "human framework
region" is a framework region that is substantially identical
(about 85 or more, usually 90-95 or more) to the framework region
of a naturally occurring human immunoglobulin. the framework region
of an antibody, that is the combined framework regions of the
constituent light and heavy chains, serves to position and align
the CDR's. The CDR's are primarily responsible for binding to an
epitope of an antigen.
[0463] The benefits of this invention extend to "commercial
applications" (or commercial processes), which term is used to
include applications in commercial industry proper (or simply
industry) as well as non-commercial commercial applications (e.g.
biomedical research at a non-profit institution). Relevant
applications include those in areas of diagnosis, medicine,
agriculture, manufacturing, and academia.
[0464] The term "identical" or "identity" means that two nucleic
acid sequences have the same sequence or a complementary sequence.
Thus, "areas of identity" means that regions or areas of a
polynucleotide or the overall polynucleotide are identical or
complementary to areas of another polynucleotide or the
polynucleotide.
[0465] The terms "identical" or percent "identity," in the context
of two or more nucleic acid or polypeptide sequences, refer to two
or more sequences or subsequences that are the same or have a
specified percentage of amino acid residues or nucleotides that are
the same, when compared and aligned for maximum correspondence, as
measured using one of the following sequence comparison algorithms
or by visual inspection.
[0466] For sequence comparison, typically one sequence acts as a
reference sequence to which test sequences are compared. When using
a sequence comparison algorithm, test and reference sequences are
input into a computer, subsequence coordinates are designated, if
necessary, and sequence algorithm program parameters are
designated. The sequence comparison algorithm then calculates the
percent sequence identity for the test sequence(s) relative to the
reference sequence, based on the designated program parameters.
[0467] A further indication that two nucleic acid sequences or
polypeptides are substantially "identical" is that the polypeptide
encoded by the first nucleic acid is immunologically cross reactive
with, or specifically binds to, the polypeptide encoded by the
second nucleic acid. Thus, a polypeptide is typically substantially
identical to a second polypeptide, for example, where the two
peptides differ only by conservative substitutions.
[0468] The term "isolated" means that the material is removed from
its original environment (e.g., the natural environment if it is
naturally occurring). For example, a naturally-occurring
polynucleotide or enzyme present in a living animal is not
isolated, but the same polynucleotide or enzyme, separated from
some or all of the coexisting materials in the natural system, is
isolated. Such polynucleotides could be part of a vector and/or
such polynucleotides or enzymes could be part of a composition, and
still be isolated in that such vector or composition is not part of
its natural environment.
[0469] The term "isolated", when applied to a nucleic acid or
protein, denotes that the nucleic acid or protein is essentially
free of other cellular components with which it is associated in
the natural state. It is preferably in a homogeneous state although
it can be in either a dry or aqueous solution. Purity and
homogeneity are typically determined using analytical chemistry
techniques such as polyacrylamide gel electrophoresis or high
performance liquid chromatography. A protein which is the
predominant species present in a preparation is substantially
purified. In particular, an isolated gene is separated from open
reading frames which flank the gene and encode a protein other than
the gene of interest.
[0470] By "isolated nucleic acid" is meant a nucleic acid, e.g., a
DNA or RNA molecule, that is not immediately contiguous with the 5'
and 3' flanking sequences with which it normally is immediately
contiguous when present in the naturally occurring genome of the
organism from which it is derived. The term thus describes, for
example, a nucleic acid that is incorporated into a vector, such as
a plasmid or viral vector; a nucleic acid that is incorporated into
the genome of a heterologous cell (or the genome of a homologous
cell, but at a site different from that at which it naturally
occurs); and a nucleic acid that exists as a separate molecule,
e.g., a DNA fragment produced by PCR amplification or restriction
enzyme digestion, or an RNA molecule produced by in vitro
transcription. The term also describes a recombinant nucleic acid
that forms part of a hybrid gene encoding additional polypeptide
sequences that can be used, for example, in the production of a
fusion protein.
[0471] As used herein "ligand" refers to a molecule, such as a
random peptide or variable segment sequence, that is recognized by
a particular receptor. As one of skill in the art will recognize, a
molecule (or macromolecular complex) can be both a receptor and a
ligand. In general, the binding partner having a smaller molecular
weight is referred to as the ligand and the binding partner having
a greater molecular weight is referred to as a receptor.
[0472] "Ligation" refers to the process of forming phosphodiester
bonds between two double stranded nucleic acid fragments (Sambrook
et al, 1982, p. 146; Sambrook, 1989). Unless otherwise provided,
ligation may be accomplished using known buffers and conditions
with 10 units of T4 DNA ligase ("ligase") per 0.5 .mu.g of
approximately equimolar amounts of the DNA fragments to be
ligated.
[0473] As used herein, "linker" or "spacer" refers to a molecule or
group of molecules that connects two molecules, such as a DNA
binding protein and a random peptide, and serves to place the two
molecules in a preferred configuration, e.g., so that the random
peptide can bind to a receptor with minimal steric hindrance from
the DNA binding protein.
[0474] As used herein, a "molecular property to be evolved"
includes reference to molecules comprised of a polynucleotide
sequence, molecules comprised of a polypeptide sequence, and
molecules comprised in part of a polynucleotide sequence and in
part of a polypeptide sequence. Particularly relevant--but by no
means limiting--examples of molecular properties to be evolved
include enzymatic activities at specified conditions, such as
related to temperature; salinity; pressure; pH; and concentration
of glycerol, DMSO, detergent, &/or any other molecular species
with which contact is made in a reaction environment. Additional
particularly relevant--but by no means limiting--examples of
molecular properties to be evolved include stabilities--e.g. the
amount of a residual molecular property that is present after a
specified exposure time to a specified environment, such as may be
encountered during storage.
[0475] A "multivalent antigenic polypeptide" or a "recombinant
multivalent antigenic polypeptide" is a non-naturally occurring
polypeptide that includes amino acid sequences from more than one
source polypeptide, which source polypeptide is typically a
naturally occurring polypeptide. At least some of the regions of
different amino acid sequences constitute epitopes that are
recognized by antibodies found in a mammal that has been injected
with the source polypeptide. The source polypeptides from which the
different epitopes are derived are usually homologous (i.e., have
the same or a similar structure and/or function), and are often
from different isolates, serotypes, strains, species, of organism
or from different disease states, for example.
[0476] The term "mutations" includes changes in the sequence of a
wild-type or parental nucleic acid sequence or changes in the
sequence of a peptide. Such mutations may be point mutations such
as transitions or transversions. The mutations may be deletions,
insertions or duplications. A mutation can also be a
"chimerization", which is exemplified in a progeny molecule that is
generated to contain part or all of a sequence of one parental
molecule as well as part or all of a sequence of at least one other
parental molecule. This invention provides for both chimeric
polynucleotides and chimeric polypeptides.
[0477] As used herein, the degenerate "N,N,G/T" nucleotide sequence
represents 32 possible triplets, where "N" can be A, C, G or T.
[0478] The term "naturally-occurring" as used herein as applied to
the object refers to the fact that an object can be found in
nature. For example, a polypeptide or polynucleotide sequence that
is present in an organism (including viruses bacteria, protozoa,
insects, plants or mammalian tissue) that can be isolated from a
source in nature and which has not been intentionally modified by
man in the laboratory is naturally occurring. Generally, the term
naturally occurring refers to an object as present in a
non-pathological (un-diseased) individual, such as would be typical
for the species.
[0479] The term "nucleic acid" refers to deoxyribonucleotides or
ribonucleotides and polymers thereof in either single- or
double-stranded form. Unless specifically limited, the term
encompasses nucleic acids containing known analogues of natural
nucleotides which have similar binding properties as the reference
nucleic acid and are metabolized in a manner similar to naturally
occurring nucleotides. Unless otherwise indicated, a particular
nucleic acid sequence also implicitly encompasses conservatively
modified variants thereof (e.g. degenerate codon substitutions) and
complementary sequences and as well as the sequence explicitly
indicated. Specifically, degenerate codon substitutions may be
achieved by generating sequences in which the third position of one
or more selected (or all) codons is substituted with mixed-base
and/or deoxyinosine residues (Batzer et al. (1991) Nucleic Acid
Res. 19: 5081; Ohtsuka et al. (1985) J Biol. Chem. 260: 2605-2608;
Cassol et al. (1992) Rossolini et al. (1994) Mol. Cell. Probes 8:
91-98). The term nucleic acid is used interchangeably with gene,
cDNA, and mRNA encoded by a gene.
[0480] "Nucleic acid derived from a gene" refers to a nucleic acid
for whose synthesis the gene, or a subsequence thereof, has
ultimately served as a template. Thus, an mRNA, a cDNA reverse
transcribed from an mRNA, an RNA transcribed from that cDNA, a DNA
amplified from the cDNA, an RNA transcribed from the amplified DNA,
etc., are all derived from the gene and detection of such derived
products is indicative of the presence and/or abundance of the
original gene and/or gene transcript in a sample.
[0481] As used herein, a "nucleic acid molecule" is comprised of at
least one base or one base pair, depending on whether it is
single-stranded or double-stranded, respectively. Furthermore, a
nucleic acid molecule may belong exclusively or chimerically to any
group of nucleotide-containing molecules, as exemplified by, but
not limited to, the following groups of nucleic acid molecules:
RNA, DNA, genomic nucleic acids, non-genomic nucleic acids,
naturally occurring and not naturally occurring nucleic acids, and
synthetic nucleic acids. This includes, by way of non-limiting
example, nucleic acids associated with any organelle, such as the
mitochondria, ribosomal RNA, and nucleic acid molecules comprised
chimerically of one or more components that are not naturally
occurring along with naturally occurring components.
[0482] Additionally, a "nucleic acid molecule" may contain in part
one or more non-nucleotide-based components as exemplified by, but
not limited to, amino acids and sugars. Thus, by way of example,
but not limitation, a ribozyme that is in part nucleotide-based and
in part protein-based is considered a "nucleic acid molecule".
[0483] In addition, by way of example, but not limitation, a
nucleic acid molecule that is labeled with a detectable moiety,
such as a radioactive or alternatively a non-radioactive label, is
likewise considered a "nucleic acid molecule".
[0484] The terms "nucleic acid sequence coding for" or a "DNA
coding sequence of" or a "nucleotide sequence encoding" a
particular enzyme--as well as other synonymous terms--refer to a
DNA sequence which is transcribed and translated into an enzyme
when placed under the control of appropriate regulatory sequences.
A "promotor sequence" is a DNA regulatory region capable of binding
RNA polymerase in a cell and initiating transcription of a
downstream (3' direction) coding sequence. The promoter is part of
the DNA sequence. This sequence region has a start codon at its 3'
terminus. The promoter sequence does include the minimum number of
bases where elements necessary to initiate transcription at levels
detectable above background. However, after the RNA polymerase
binds the sequence and transcription is initiated at the start
codon (3' terminus with a promoter), transcription proceeds
downstream in the 3' direction. Within the promotor sequence will
be found a transcription initiation site (conveniently defined by
mapping with nuclease S1) as well as protein binding domains
(consensus sequences) responsible for the binding of RNA
polymerase.
[0485] The terms "nucleic acid encoding an enzyme (protein)" or
"DNA encoding an enzyme (protein)" or "polynucleotide encoding an
enzyme (protein)" and other synonymous terms encompasses a
polynucleotide which includes only coding sequence for the enzyme
as well as a polynucleotide which includes additional coding and/or
non-coding sequence.
[0486] In one preferred embodiment, a "specific nucleic acid
molecule species" is defined by its chemical structure, as
exemplified by, but not limited to, its primary sequence. In
another preferred embodiment, a specific "nucleic acid molecule
species" is defined by a function of the nucleic acid species or by
a function of a product derived from the nucleic acid species.
Thus, by way of non-limiting example, a "specific nucleic acid
molecule species" may be defined by one or more activities or
properties attributable to it, including activities or properties
attributable its expressed product.
[0487] The instant definition of "assembling a working nucleic acid
sample into a nucleic acid library" includes the process of
incorporating a nucleic acid sample into a vector-based collection,
such as by ligation into a vector and transformation of a host. A
description of relevant vectors, hosts, and other reagents as well
as specific non-limiting examples thereof are provided hereinafter.
The instant definition of "assembling a working nucleic acid sample
into a nucleic acid library" also includes the process of
incorporating a nucleic acid sample into a non-vector-based
collection, such as by ligation to adaptors. Preferably the
adaptors can anneal to PCR primers to facilitate amplification by
PCR.
[0488] Accordingly, in a non-limiting embodiment, a "nucleic acid
library" is comprised of a vector-based collection of one or more
nucleic acid molecules. In another preferred embodiment a "nucleic
acid library" is comprised of a non-vector-based collection of
nucleic acid molecules. In yet another preferred embodiment a
"nucleic acid library" is comprised of a combined collection of
nucleic acid molecules that is in part vector-based and in part
non-vector-based. Preferably, the collection of molecules
comprising a library is searchable and separable according to
individual nucleic acid molecule species.
[0489] The present invention provides a "nucleic acid construct" or
alternatively a "nucleotide construct" or alternatively a "DNA
construct". The term "construct" is used herein to describe a
molecule, such as a polynucleotide (e.g., a phytase polynucleotide)
may optionally be chemically bonded to one or more additional
molecular moieties, such as a vector, or parts of a vector. In a
specific--but by no means limiting--aspect, a nucleotide construct
is exemplified by a DNA expression DNA expression constructs
suitable for the transformation of a host cell.
[0490] An "oligonucleotide" (or synonymously an "oligo") refers to
either a single stranded polydeoxynucleotide or two complementary
polydeoxynucleotide strands which may be chemically synthesized.
Such synthetic oligonucleotides may or may not have a 5' phosphate.
Those that do not will not ligate to another oligonucleotide
without adding a phosphate with an ATP in the presence of a kinase.
A synthetic oligonucleotide will ligate to a fragment that has not
been dephosphorylated. To achieve polymerase-based amplification
(such as with PCR), a "32-fold degenerate oligonucleotide that is
comprised of, in series, at least a first homologous sequence, a
degenerate N,N,G/T sequence, and a second homologous sequence" is
mentioned. As used in this context, "homologous" is in reference to
homology between the oligo and the parental polynucleotide that is
subjected to the polymerase-based amplification.
[0491] A nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
instance, a promoter or enhancer is operably linked to a coding
sequence if it increases the transcription of the coding
sequence.
[0492] As used herein, the term "operably linked" refers to a
linkage of polynucleotide elements in a functional relationship. A
nucleic acid is "operably linked" when it is placed into a
functional relationship with another nucleic acid sequence. For
instance, a promoter or enhancer is operably linked to a coding
sequence if it affects the transcription of the coding sequence.
Operably linked means that the DNA sequences being linked are
typically contiguous and, where necessary to join two protein
coding regions, contiguous and in reading frame. However, since
enhancers generally function when separated from the promoter by
several kilobases and intronic sequences may be of variable
lengths, some polynucleotide elements may be operably linked but
not contiguous.
[0493] A coding sequence is "operably linked to" another coding
sequence when RNA polymerase will transcribe the two coding
sequences into a single mRNA, which is then translated into a
single polypeptide having amino acids derived from both coding
sequences. The coding sequences need not be contiguous to one
another so long as the expressed sequences are ultimately processed
to produce the desired protein.
[0494] As used herein the term "parental polynucleotide set" is a
set comprised of one or more distinct polynucleotide species.
Usually this term fis used in reference to a progeny polynucleotide
set which is preferably obtained by mutagenization of the parental
set, in which case the terms "parental", "starting" and "template"
are used interchangeably.
[0495] As used herein the term "physiological conditions" refers to
temperature, pH, ionic strength, viscosity, and like biochemical
parameters which are compatible with a viable organism, and/or
which typically exist intracellularly in a viable cultured yeast
cell or mammalian cell. For example, the intracellular conditions
in a yeast cell grown under typical laboratory culture conditions
are physiological conditions. Suitable in vitro reaction conditions
for in vitro transcription cocktails are generally physiological
conditions. In general, in vitro physiological conditions comprise
50-200 mM NaCl or KCl, pH 6.5-8.5, 20-45.degree. C. and 0.001-10 mM
divalent cation (e.g., Mg.sup.++, Ca.sup.++); preferably about 150
mM NaCl or KCl, pH 7.2-7.6, 5 mM divalent cation, and often include
0.01-1.0 percent nonspecific protein (e.g., BSA). A non-ionic
detergent (Tween, NP-40, Triton X-100) can often be present,
usually at about 0.001 to 2%, typically 0.05-0.2% (v/v). Particular
aqueous conditions may be selected by the practitioner according to
conventional methods. For general guidance, the following buffered
aqueous conditions may be applicable: 10-250 mM NaCl, 5-50 mM Tris
HCl, pH 5-8, with optional addition of divalent cation(s) and/or
metal chelators and/or non-ionic detergents and/or membrane
fractions and/or anti-foam agents and/or scintillants.
[0496] Standard convention (5' to 3') is used herein to describe
the sequence of double standed polynucleotides.
[0497] The term "population" as used herein means a collection of
components such as polynucleotides, portions or polynucleotides or
proteins. A "mixed population: means a collection of components
which belong to the same family of nucleic acids or proteins (i.e.,
are related) but which differ in their sequence (i.e., are not
identical) and hence in their biological activity.
[0498] A molecule having a "pro-form" refers to a molecule that
undergoes any combination of one or more covalent and noncovalent
chemical modifications (e.g. glycosylation, proteolytic cleavage,
dimerization or oligomerization, temperature-induced or pH-induced
conformational change, association with a co-factor, etc.) en route
to attain a more mature molecular form having a property difference
(e.g. an increase in activity) in comparison with the reference
pro-form molecule. When two or more chemical modification (e.g. two
proteolytic cleavages, or a proteolytic cleavage and a
deglycosylation) can be distinguished en route to the production of
a mature molecule, the referemce precursor molecule may be termed a
"pre-pro-form" molecule.
[0499] As used herein, the term "pseudorandom" refers to a set of
sequences that have limited variability, such that, for example,
the degree of residue variability at another position, but any
pseudorandom position is allowed some degree of residue variation,
however circumscribed.
[0500] The term "purified" denotes that a nucleic acid or protein
gives rise to essentially one band in an electrophoretic gel.
Particularly, it means that the nucleic acid or protein is at least
about 50% pure, more preferably at least about 85% pure, and most
preferably at least about 99% pure.
[0501] "Quasi-repeated units", as used herein, refers to the
repeats to be re-assorted and are by definition not identical.
Indeed the method is proposed not only for practically identical
encoding units produced by mutagenesis of the identical starting
sequence, but also the reassortment of similar or related sequences
which may diverge significantly in some regions. Nevertheless, if
the sequences contain sufficient homologies to be reasserted by
this approach, they can be referred to as "quasi-repeated"
units.
[0502] As used herein "random peptide library" refers to a set of
polynucleotide sequences that encodes a set of random peptides, and
to the set of random peptides encoded by those polynucleotide
sequences, as well as the fusion proteins contain those random
peptides.
[0503] As used herein, "random peptide sequence" refers to an amino
acid sequence composed of two or more amino acid monomers and
constructed by a stochastic or random process. A random peptide can
include framework or scaffolding motifs, which may comprise
invariant sequences.
[0504] As used herein, "receptor" refers to a molecule that has an
affinity for a given ligand. Receptors can be naturally occurring
or synthetic molecules. Receptors can be employed in an unaltered
state or as aggregates with other species. Receptors can be
attached, covalently or non-covalently, to a binding member, either
directly or via a specific binding substance. Examples of receptors
include, but are not limited to, antibodies, including monoclonal
antibodies and antisera reactive with specific antigenic
determinants (such as on viruses, cells, or other materials), cell
membrane receptors, complex carbohydrates and glycoproteins,
enzymes, and hormone receptors.
[0505] The term "recombinant" when used with reference to a cell
indicates that the cell replicates a heterologous nucleic acid, or
expresses a peptide or protein encoded by a heterologous nucleic
acid. Recombinant cells can contain genes that are not found within
the native (non-recombinant) form of the cell. Recombinant cells
can also contain genes found in the native form of the cell wherein
the genes are modified and re-introduced into the cell by
artificial means. The term also encompasses cells that contain a
nucleic acid endogenous to the cell that has been modified without
removing the nucleic acid from the cell; such modifications include
those obtained by gene replacement, site-specific mutation, and
related techniques.
[0506] "Recombinant enzymes" refer to enzymes produced by
recombinant DNA techniques, i.e., produced from cells transformed
by an exogenous DNA construct encoding the desired enzyme.
"Synthetic" enzymes are those prepared by chemical synthesis.
[0507] A "recombinant expression cassette" or simply an "expression
cassette" is a nucleic acid construct, generated recombinantly or
synthetically, with nucleic acid elements that are capable of
effecting expression of a structural gene in hosts compatible with
such sequences. Expression cassettes include at least promoters and
optionally, transcription termination signals. Typically, the
recombinant expression cassette includes a nucleic acid to be
transcribed (e.g., a nucleic acid encoding a desired polypeptide),
and a promoter. Additional factors necessary or helpful in
effecting expression may also be used as described herein. For
example, an expression cassette can also include nucleotide
sequences that encode a signal sequence that directs secretion of
an expressed protein from the host cell. Transcription termination
signals, enhancers, and other nucleic acid sequences that influence
gene expression, can also be included in an expression
cassette.
[0508] The term "related polynucleotides" means that regions or
areas of the polynucleotides are identical and regions or areas of
the polynucleotides are heterologous.
[0509] "Reductive reassortment", as used herein, refers to the
increase in molecular diversity that is accrued through deletion
(and/or insertion) events that are mediated by repeated
sequences.
[0510] The following terms are used to describe the sequence
relationships between two or more polynucleotides: "reference
sequence," "comparison window," "sequence identity," "percentage of
sequence identity," and "substantial identity."
[0511] A "reference sequence" is a defined sequence used as a basis
for a sequence comparison; a reference sequence may be a subset of
a larger sequence, for example, as a segment of a full-length cDNA
or gene sequence given in a sequence listing, or may comprise a
complete cDNA or gene sequence. Generally, a reference sequence is
at least 20 nucleotides in length, frequently at least 25
nucleotides in length, and often at least 50 nucleotides in length.
Since two polynucleotides may each (1) comprise a sequence (i.e., a
portion of the complete polynucleotide sequence) that is similar
between the two polynucleotides and (2) may further comprise a
sequence that is divergent between the two polynucleotides,
sequence comparisons between two (or more) polynucleotides are
typically performed by comparing sequences of the two
polynucleotides over a "comparison window" to identify and compare
local regions of sequence similarity.
[0512] "Repetitive Index (RI)", as used herein, is the average
number of copies of the quasi-repeated units contained in the
cloning vector.
[0513] The term "restriction site" refers to a recognition sequence
that is necessary for the manifestation of the action of a
restriction enzyme, and includes a site of catalytic cleavage. It
is appreciated that a site of cleavage may or may not be contained
within a portion of a restriction site that comprises a low
ambiguity sequence (i.e. a sequence containing the principal
determinant of the frequency of occurrence of the restriction
site). Thus, in many cases, relevant restriction sites contain only
a low ambiguity sequence with an internal cleavage site (e.g.
G/AATTC in the EcoR I site) or an immediately adjacent cleavage
site (e.g./CCWGG in the EcoR II site). In other cases, relevant
restriction enzymes [e.g. the Eco57 I site or CTGAAG(16/14)]
contain a low ambiguity sequence (e.g. the CTGAAG sequence in the
Eco57 I site) with an external cleavage site (e.g. in the N.sub.16
portion of the Eco57 I site). When an enzyme (e.g. a restriction
enzyme) is said to "cleave" a polynucleotide, it is understood to
mean that the restriction enzyme catalyzes or facilitates a
cleavage of a polynucleotide.
[0514] The term "screening" describes, in general, a process that
identifies optimal antigens. Several properties of the antigen can
be used in selection and screening including antigen expression,
folding, stability, immunogenicity and presence of epitopes from
several related antigens. Selection is a form of screening in which
identification and physical separation are achieved simultaneously
by expression of a selection marker, which, in some genetic
circumstances, allows cells expressing the marker to survive while
other cells die (or vice versa). Screening markers include, for
example, luciferase, beta-galactosidase and green fluorescent
protein. Selection markers include drug and toxin resistance genes,
and the like. Because of limitations in studying primary immune
responses in vitro, in vivo studies are particularly useful
screening methods. In these studies, the antigens are first
introduced to test animals, and the immune responses are
subsequently studied by analyzing protective immune responses or by
studying the quality or strength of the induced immune response
using lymphoid cells derived from the immunized animal. Although
spontaneous selection can and does occur in the course of natural
evolution, in the present methods selection is performed by
man.
[0515] In a non-limiting aspect, a "selectable polynucleotide" is
comprised of a 5' terminal region (or end region), an intermediate
region (i.e. an internal or central region), and a 3' terminal
region (or end region). As used in this aspect, a 5' terminal
region is a region that is located towards a 5' polynucleotide
terminus (or a 5' polynucleotide end); thus it is either partially
or entirely in a 5' half of a polynucleotide. Likewise, a 3'
terminal region is a region that is located towards a 3'
polynucleotide terminus (or a 3' polynucleotide end); thus it is
either partially or entirely in a 3' half of a polynucleotide. As
used in this non-limiting exemplification, there may be sequence
overlap between any two regions or even among all three
regions.
[0516] The term "sequence identity" means that two polynucleotide
sequences are identical (i.e., on a nucleotide-by-nucleotide basis)
over the window of comparison. The term "percentage of sequence
identity" is calculated by comparing two optimally aligned
sequences over the window of comparison, determining the number of
positions at which the identical nucleic acid base (e.g., A, T, C,
G, U, or I) occurs in both sequences to yield the number of matched
positions, dividing the number of matched positions by the total
number of positions in the window of comparison (i.e., the window
size), and multiplying the result by 100 to yield the percentage of
sequence identity. This "substantial identity", as used herein,
denotes a characteristic of a polynucleotide sequence, wherein the
polynucleotide comprises a sequence having at least 80 percent
sequence identity, preferably at least 85 percent identity, often
90 to 95 percent sequence identity, and most commonly at least 99
percent sequence identity as compared to a reference sequence of a
comparison window of at least 25-50 nucleotides, wherein the
percentage of sequence identity is calculated by comparing the
reference sequence to the polynucleotide sequence which may include
deletions or additions which total 20 percent or less of the
reference sequence over the window of comparison.
[0517] As known in the art "similarity" between two enzymes is
determined by comparing the amino acid sequence and its conserved
amino acid substitutes of one enzyme to the sequence of a second
enzyme. Similarity may be determined by procedures which are
well-known in the art, for example, a BLAST program (Basic Local
Alignment Search Tool at the National Center for Biological
Information).
[0518] As used herein, the term "single-chain antibody" refers to a
polypeptide comprising a V.sub.H domain and a V.sub.L domain in
polypeptide linkage, generally liked via a spacer peptide (e.g.,
[Gly-Gly-Gly-Gly-Ser].sub.x), and which may comprise additional
amino acid sequences at the amino- and/or carboxy-termini. For
example, a single-chain antibody may comprise a tether segment for
linking to the encoding polynucleotide. As an example, a scFv is a
single-chain antibody. Single-chain antibodies are generally
proteins consisting of one or more polypeptide segments of at least
10 contiguous amino substantially encoded by genes of the
immunoglobulin superfamily (e.g., see Williams and Barclay, 1989,
pp. 361-368, which is incorporated herein by reference), most
frequently encoded by a rodent, non-human primate, avian, porcine
bovine, ovine, goat, or human heavy chain or light chain gene
sequence. A functional single-chain antibody generally contains a
sufficient portion of an immunoglobulin superfamily gene product so
as to retain the property of binding to a specific target molecule,
typically a receptor or antigen (epitope).
[0519] The phrase "specifically (or selectively) binds to an
antibody" or "specifically (or selectively) immunoreactive with",
when referring to a protein or peptide, refers to a binding
reaction which is determinative of the presence of the protein, or
an epitope from the protein, in the presence of a heterogeneous
population of proteins and other biologics. Thus, under designated
immunoassay conditions, the specified antibodies bind to a
particular protein and do not bind in a significant amount to other
proteins present in the sample. The antibodies raised against a
multivalent antigenic polypeptide will generally bind to the
proteins from which one or more of the epitopes were obtained.
Specific binding to an antibody under such conditions may require
an antibody that is selected for its specificity for a particular
protein. A variety of immunoassay formats may be used to select
antibodies specifically immunoreactive with a particular protein.
For example, solid-phase ELISA immunoassays, Western blots, or
immunohistochemistry are routinely used to select monoclonal
antibodies specifically immunoreactive with a protein. See Harlow
and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor
Publications, New York "Harlow and Lane"), for a description of
immunoassay formats and conditions that can be used to determine
specific immunoreactivity. Typically a specific or selective
reaction will be at least twice background signal or noise and more
typically more than 10 to 100 times background.
[0520] The members of a pair of molecules (e.g., an
antibody-antigen pair or a nucleic acid pair) are said to
"specifically bind" to each other if they bind to each other with
greater affinity than to other, non-specific molecules. For
example, an antibody raised against an antigen to which it binds
more efficiently than to a non-specific protein can be described as
specifically binding to the antigen. (Similarly, a nucleic acid
probe can be described as specifically binding to a nucleic acid
target if it forms a specific duplex with the target by base
pairing interactions (see above).)
[0521] A "specific binding affinity" between two molecules, for
example, a ligand and a receptor, means a preferential binding of
one molecule for another in a mixture of molecules. The binding of
the molecules can be considered specific if the binding affinity is
about 1.times.10.sup.4 M.sup.-1 to about 1.times.10.sup.6M.sup.-I
or greater.
[0522] "Specific hybridization" is defined herein as the formation
of hybrids between a first polynucleotide and a second
polynucleotide (e.g., a polynucleotide having a distinct but
substantially identical sequence to the first polynucleotide),
wherein substantially unrelated polynucleotide sequences do not
form hybrids in the mixture.
[0523] The term "specific polynucleotide" means a polynucleotide
having certain end points and having a certain nucleic acid
sequence. Two polynucleotides wherein one polynucleotide has the
identical sequence as a portion of the second polynucleotide but
different ends comprises two different specific
polynucleotides.
[0524] The T.sub.m is the temperature (under defined ionic strength
and pH) at which 50% of the target sequence hybridizes to a
perfectly matched probe. Very stringent conditions are selected to
be equal to the T,,, for a particular probe. An example of
stringent hybridization conditions for hybridization of
complementary nucleic acids which have more than 100 complementary
residues on a filter in a Southern or northern blot is 50%
formamide with I mg of heparin at 42'C., with the hybridization
being carried out overnight.
[0525] "Stringent hybridization conditions" means hybridization
will occur only if there is at least 90% identity, preferably at
least 95% identity and most preferably at least 97% identity
between the sequences. See Sambrook et al, 1989, which is hereby
incorporated by reference in its entirety.
[0526] An example of highly "stringent" wash conditions is 0.15M
NaCl at 72'C. for about 15 minutes. An example of stringent wash
conditions is a 0.2.times.SSC wash at 65'C. for 15 minutes (see,
Sambrook, infra., for a description of SSC buffer). Often, a high
stringency wash is preceded by a low stringency wash to remove
background probe signal. An example medium stringency wash for a
duplex of, e.g., more than 100 nucleotides, is 1.times.SSC at
45.degree. C. for 15 minutes. An example low stringency wash for a
duplex of, e.g., more than 100 nucleotides, is .sup.4-6.times.SSC
at 40.degree. C. for 15 minutes. For short probes (e.g., about 10
to 50 nucleotides), stringent conditions typically involve salt
concentrations of less than about 1.0 M Na.sup.+ ion, typically
about 0.01 to 1.0 M Na.sup.+ ion concentration (or other salts) at
pH 7.0 to 8.3, and the temperature is typically at least about
30.degree. C. Stringent conditions can also be achieved with the
addition of destabilizing agents such as formamide. In general, a
signal to noise ratio of 2.times.(or higher) than that observed for
an unrelated probe in the particular hybridization assay indicates
detection of a specific hybridization. Nucleic acids which do not
hybridize to each other under stringent conditions are still
substantially identical if the polypeptides which they encode are
substantially identical. This occurs, e.g., when a copy of a
nucleic acid is created using the maximum codon degeneracy
permitted by the genetic code.
[0527] "Stringent hybridization conditions" and "stringent
hybridization wash conditions" in the context of nucleic acid
hybridization experiments such as Southern and northern
hybridizations are sequence dependent, and are different under
different environmental parameters. Longer sequences hybridize
specifically at higher temperatures.
[0528] An extensive guide to the hybridization of nucleic acids is
found in Tijssen (1993) Laboratory Techniques in Biochemistry and
Molecular Biology--Hybridization with Nucleic Acid Probes part I
chapter 2 "Overview of principles of hybridization and the strategy
of nucleic acid probe assays", Elsevier, N.Y. Generally, highly
stringent hybridization and wash conditions are selected to be
about 5.degree. C. lower than the thermal melting point (T.sub.m)
for the specific sequence at a defined ionic strength and pH.
Typically, under "stringent conditions" a probe will hybridize to
its target subsequence, but to no other sequences.
[0529] Also included in the invention are polypeptides having
sequences that are "substantially identical" to the sequence of a
phytase polypeptide, such as one of SEQ ID 1. A "substantially
identical" amino acid sequence is a sequence that differs from a
reference sequence only by conservative amino acid substitutions,
for example, substitutions of one amino acid for another of the
same class (e.g., substitution of one hydrophobic amino acid, such
as isoleucine, valine, leucine, or methionine, for another, or
substitution of one polar amino acid for another, such as
substitution of arginine for lysine, glutamic acid for aspartic
acid, or glutamine for asparagine).
[0530] The phrase "substantially identical," in the context of two
nucleic acids or polypeptides, refers to two or more sequences or
subsequences that have at least 60%, preferably 80%, most
preferably 90-95% nucleotide or amino acid residue identity, when
compared and aligned for maximum correspondence, as measured using
one of the following sequence comparison algorithms or by visual
inspection. Preferably, the substantial identity exists over a
region of the sequences that is at least about 50 residues in
length, more preferably over a region of at least about 100
residues, and most preferably the sequences are substantially
identical over at least about 150 residues. In some embodiments,
the sequences are substantially identical over the entire length of
the coding regions.
[0531] A "subsequence" refers to a sequence of nucleic acids or
amino acids that comprise a part of a longer sequence of nucleic
acids or amino acids (e.g., polypeptide) respectively.
[0532] Additionally a "substantially identical" amino acid sequence
is a sequence that differs from a reference sequence or by one or
more non-conservative substitutions, deletions, or insertions,
particularly when such a substitution occurs at a site that is not
the active site the molecule, and provided that the polypeptide
essentially retains its behavioural properties. For example, one or
more amino acids can be deleted from a phytase polypeptide,
resulting in modification of the structure of the polypeptide,
without significantly altering its biological activity. For
example, amino- or carboxyl-terminal amino acids that are not
required for phytase biological activity can be removed. Such
modifications can result in the development of smaller active
phytase polypeptides.
[0533] The present invention provides a "substantially pure
enzyme". The term "substantially pure enzyme" is used herein to
describe a molecule, such as a polypeptide (e.g., a phytase
polypeptide, or a fragment thereof) that is substantially free of
other proteins, lipids, carbohydrates, nucleic acids, and other
biological materials with which it is naturally associated. For
example, a substantially pure molecule, such as a polypeptide, can
be at least 60%, by dry weight, the molecule of interest. The
purity of the polypeptides can be determined using standard methods
including, e.g., polyacrylamide gel electrophoresis (e.g.,
SDS-PAGE), column chromatography (e.g., high performance liquid
chromatography (HPLC)), and amino-terminal amino acid sequence
analysis.
[0534] As used herein, "substantially pure" means an object species
is the predominant species present (i.e., on a molar basis it is
more abundant than any other individual macromolecular species in
the composition), and preferably substantially purified fraction is
a composition wherein the object species comprises at least about
50 percent (on a molar basis) of all macromolecular species
present. Generally, a substantially pure composition will comprise
more than about 80 to 90 percent of all macromolecular species
present in the composition. Most preferably, the object species is
purified to essential homogeneity (contaminant species cannot be
detected in the composition by conventional detection methods)
wherein the composition consists essentially of a single
macromolecular species. Solvent species, small molecules (<500
Daltons), and elemental ion species are not considered
macromolecular species.
[0535] As used herein, the term "variable segment" refers to a
portion of a nascent peptide which comprises a random,
pseudorandom, or defined kernal sequence. A variable segment"
refers to a portion of a nascent peptide which comprises a random
pseudorandom, or defined kernal sequence. A variable segment can
comprise both variant and invariant residue positions, and the
degree of residue variation at a variant residue position may be
limited: both options are selected at the discretion of the
practitioner. Typically, variable segments are about 5 to 20 amino
acid residues in length (e.g., 8 to 10), although variable segments
may be longer and may comprise antibody portions or receptor
proteins, such as an antibody fragment, a nucleic acid binding
protein, a receptor protein, and the like.
[0536] The term "wild-type" means that the polynucleotide does not
comprise any mutations. A "wild type" protein means that the
protein will be active at a level of activity found in nature and
will comprise the amino acid sequence found in nature.
[0537] The term "working", as in "working sample", for example, is
simply a sample with which one is working. Likewise, a "working
molecule", for example is a molecule with which one is working.
2.2. GENERAL CONSIDERATIONS & FORMATS FOR RECOMBINATION
[0538] Component Modules Provides Genetic Vaccine with the
Acquisition of or Improvement in a useful Property or
Characteristic
[0539] The present invention provides multicomponent genetic
vaccines that include one or more component modules, each of which
provides the genetic vaccine with the acquisition of or an
improvement in a property or characteristic useful in genetic
vaccination.
[0540] The invention provides significant advantages over
previously used genetic vaccines. Through use of a multicomponent
vaccine, one can obtain an immune response that is particularly
effective for a particular application. A multicomponent genetic
vaccine can, for example, contain a component that is optimized for
optimal antigen expression, as well as a component that confers
improved activation of cytotoxic T lymphocytes (CTLs) by enhancing
the presentation of the antigen on dendritic cell MHC Class I
molecules. Additional examples are described herein.
[0541] The invention provides a new approach to vaccine
development, which is termed "antigen library immunization." No
other technologies are available for generating libraries of
related antigens or optimizing known protective antigens. The most
powerful previously existing methods for identification of vaccine
antigens, such as high throughput sequencing or expression library
immunization, only explore the sequence space provided by the
pathogen genome. These approaches are likely to be insufficient,
because they generally only target single pathogen strains, and
because natural evolution has directed pathogens to downregulate
their own immunogenicity. In contrast, the immunization protocols
of the invention, which use experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis) antigen libraries, provide a means to identify novel
antigen sequences. Those antigens that are most protective can be
selected from these pools by in vivo challenge models. Antigen
library immunization dramatically expands the diversity of
available immunogen sequences, and therefore, these antigen chimera
libraries can also provide means to defend against newly emerging
pathogen variants of the future. The methods of the invention
enable the identification of individual chimeric antigens that
provide efficient protection against a variety of existing
pathogens, providing improved vaccines for troops and civilian
populations.
[0542] The methods of the invention provide an evolution-based
approach, such as stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide reassembly
in particular, that is an optimal approach to improve the
immunogenicity of many types of antigens. For example, the methods
provide means of obtaining optimized cancer antigens useful for
preventing and treating malignant diseases. Furthermore, an
increasing number of self-antigens, causing autoimmune diseases,
and allergens, causing atopy, allergy and asthma, have been
characterized. The immunogenicity and manufacturing of these
antigens can likewise be improved with the methods of this
invention.
[0543] The antigen library immunization methods of the invention
provide a means by which one can obtain a recombinant antigen that
has improved ability to induce an immune response to a pathogenic
agent. A "pathogenic agent" refers to an organism or virus that is
capable of infecting a host cell. Pathogenic agents typically
include and/or encode a molecule, usually a polypeptide, that is
immunogenic in that an immune response is raised against the
immunogenic polypeptide. Often, the immune response raised against
an immunogenic polypeptide from one serotype of the pathogenic
agent is not capable of recognizing, and thus protecting against, a
different serotype of the pathogenic agent, or other related
pathogenic agents. In other situations, the polypeptide produced by
a pathogenic agent is not produced in sufficient amounts, or is not
sufficiently immunogenic, for the infected host to raise an
effective immune response against the pathogenic agent.
[0544] These problems are overcome by the methods of the invention,
which typically involve reassembling (&/or subjecting to one or
more directed evolution methods described herein) two or more forms
of a nucleic acid that encode a polypeptide of the pathogenic
agent, or antigen involved in another disease or condition. These
reassembly methods, including stochastic (e.g. polynucleotide
shuffling & interrupted synthesis) and non-stochastic
polynucleotide reassembly, use as substrates forms of the nucleic
acid that differ from each other in two or more nucleotides, so a
library of recombinant nucleic acids results. The library is then
screened to identify at least one optimized recombinant nucleic
acid that encodes an optimized recombinant antigen that has
improved ability to induce an immune response to the pathogenic
agent or other condition.
[0545] The resulting recombinant antigens often are chimeric in
that they are recognized by antibodies (Abs) reacting against
multiple pathogen strains, and generally can also elicit broad
spectrum immune responses. Specific neutralizing antibodies are
known to mediate protection against several pathogens of interest,
although additional mechanisms, such as cytotoxic T lymphocytes,
are likely to be involved. The concept of chimeric, multivalent
antigens inducing broadly reacting antibody responses is further
illustrated in FIG. 1.
[0546] In preferred embodiments, the different forms of the nucleic
acids that encode antigenic polypeptides are obtained from members
of a family of related pathogenic agents.
[0547] This scheme of performing stochastic (e.g. polynucleotide
shuffling & interrupted synthesis) and non-stochastic
polynucleotide reassembly using nucleic acids from different
organisms is shown schematically in Figure ???. Therefore, these
stochastic (e.g. polynucleotide shuffling & interrupted
synthesis) and non-stochastic polynucleotide reassembly methods
provide an effective approach to generate multivalent,
crossprotective antigens. The methods are useful for obtaining
individual chimeras that effectively protect against most or all
pathogen variants (FIG. 3A).
[0548] Moreover, immunizations using entire libraries or pools of
experimentally evolved (e.g. by polynucleotide reassembly &/or
polynucleotide site-saturation mutagenesis) antigen chimeras can
also result in identification of chimeric antigens that protect
against pathogen variants that were not included in the starting
population of antigens (for example, protection against strain C by
experimentally evolved (e.g. by polynucleotide reassembly &/or
polynucleotide site-saturation mutagenesis) library of
chimeras/mutants of strains A and B in FIG. 313).
[0549] Accordingly, the antigen library immunization approach
enables the development of immunogenic polypeptides that can induce
immune responses against poorly characterized, newly emerging
pathogen variants.
[0550] Sequence reassembly (&/or one or more additonal directed
evolution methods described herein) can be achieved in many
different formats and permutations of formats, as described in
further detail below. These formats share some common principles.
For example, the targets for modification vary in different
applications, as does the property sought to be acquired or
improved. Examples of candidate targets for acquisition of a
property or improvement in a property include genes that encode
proteins which have immunogenic and/or toxigenic activity when
introduced into a host organism.
[0551] The methods use at least two variant forms of a starting
target. The variant forms of candidate substrates can show
substantial sequence or secondary structural similarity with each
other, but they should also differ in at least one and preferably
at least two positions. The initial diversity between forms can be
the result of natural variation, e.g., the different variant forms
(homologs) are obtained from different individuals or strains of an
organism, or constitute related sequences from the same organism
(e.g., allelic variations), or constitute homologs from different
organisms (interspecific variants).
[0552] Alternatively, initial diversity can be induced, e.g., the
variant forms can be generated by error-prone transcription, such
as an error-prone PCR or use of a polymerase which lacks
proof-reading activity (see, Liao (1990) Gene 88:107-111), of the
first variant form, or, by replication of the first form in a
mutator strain (mutator host cells are discussed in further detail
below, and are generally well known). A mutator strain can include
any mutants in any organism impaired in the functions of mismatch
repair. These include mutant gene products of mutS, mutT, mutH,
mutL, ovrD, dcm, vsr, umuC, umuD, sbcB, recJ, etc. The impairment
is achieved by genetic mutation, allelic replacement, selective
inhibition by an added reagent such as a small compound or an
expressed antisense RNA, or other techniques. Impairment can be of
the genes noted, or of homologous genes in any organism. Other
methods of generating initial diversity include methods well known
to those of skill in the art, including, for example, treatment of
a nucleic acid with a chemical or other mutagen, through
spontaneous mutation, and by inducing an error-prone repair system
(e.g., SOS) in a cell that contains the nucleic acid. The initial
diversity between substrates is greatly augmented in subsequent
steps of reassembly (&/or one or more additonal directed
evolution methods described herein) for library generation.
[0553] Properties involved in Immunogenicity
[0554] Polynucleotide sequences that can positively or negatively
affect the immunogenicity of an antigen encoded by the
polynucleotide are often scattered throughout the entire antigen
gene. Several of these factors are shown diagrammatically in FIG.
4. By reassembling (&/or subjecting to one or more directed
evolution methods described herein) different forms of
polynucleotide that encode the antigen using stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly, followed by selection for
those chimeric polynucleotides that encode an antigen that can
induce an improved immune response, one can obtain primarily
sequences that have a positive influence on antigen immunogenicity.
Those sequences that negatively affect antigen immunogenicity are
eliminated (FIG. 4). One need not know the particular sequences
involved.
[0555] The present invention provides methods for obtaining
polynucleotide sequences that, either directly or indirectly (i.e.,
through encoding a polypeptide), can modulate an immune response
when present on a genetic vaccine vector. In another embodiment,
the invention provides methods for optimizing the transport and
presentation of antigens. The optimized immunomodulatory
polynucleotides obtained using the methods of the invention are
particularly suited for use in conjunction with vaccines, including
genetic vaccines. One of the advantages of genetic vaccines is that
one can incorporate genes encoding immunomodulatory molecules, such
as cytokines, costimulatory molecules, and molecules that improve
antigen transport and presentation into the genetic vaccine
vectors. This provides opportunities to modulate immune responses
that are induced against the antigens expressed by the genetic
vaccines.
[0556] Obtaining Components for use in Genetic Vaccines that are
more Effective through the Creation of a Library, the Screening of
the Library, and the use of Recombinant Nucleic Acids that Exhibit
Improved Properties
[0557] In additional embodiments, the present invention provides
methods of obtaining components for use in genetic vaccines,
including the multicomponent vaccines, that are more effective in
conferring a desired immune response property upon a genetic
vaccine. The methods involve creating a library of recombinant
nucleic acids and screening the library to identify those library
members that exhibits an enhanced capacity to confer a desired
property upon a genetic vaccine. Those recombinant nucleic acids
that exhibit improved properties can be used as components in a
genetic vaccine, either directly as a polynucleotide or as a
protein that is obtained by expression of the component nucleic
acid.
[0558] Improvement Goals
[0559] The properties or characteristics that can be sought to be
acquired or improved vary widely, and, of course depend on the
choice of substrate. For genetic vaccines, improvement goals
include higher titer, more stable expression, improved stability,
higher specificity targeting, higher or lower frequency of
integration, reduced immunogenicity of the vector or an expression
product thereof, increased immunogenicity of the antigen, higher
expression of gene products, and the like. Other properties for
which optimization is desired include the tailoring of an immune
response to be most effective for a particular application.
Examples of genetic vaccine components are shown, described
&/or referenced herein (including incorporated by reference).
Two or more components can be included in a single vector molecule,
or each component can be present in a genetic vaccine formulation
as a separate molecule.
[0560] Sequence Reassembly (&/or One or More Additonal Directed
Evolution Methods Described Herein) can be Achieved through
Different Formats which Share some Common Principles
[0561] In the methods of the invention, at least two variant forms
of a nucleic acid are reassembled (&/or subjected to one or
more directed evolution methods described herein) to produce a
library of recombinant nucleic acids, which is then screened to
identify at least one recombinant component that is optimized for
the particular vaccine property. Often, improvements are achieved
after one round of reassembly (&/or one or more additonal
directed evolution methods described herein) and selection.
Sequence reassembly (&/or one or more additonal directed
evolution methods described herein) can be achieved in many
different formats and permutations of formats, as described in
further detail below. These formats share some common principles. A
family of nucleic acid molecules that have some sequence identity
to each other, but differ in the presence of mutations, is
typically used as a substrate for reassembly (&/or one or more
additonal directed evolution methods described herein). In any
given cycle, reassembly (&/or one or more additonal directed
evolution methods described herein) can occur in vivo or in vitro,
intracellularly or extracellularly. Furthermore, diversity
resulting from reassembly (&/or one or more additonal directed
evolution methods described herein) can be augmented in any cycle
by applying prior methods of mutagenesis (e.g., error-prone PCR or
cassette mutagenesis) to either the substrates or products of
reassembly (&/or one or more additonal directed evolution
methods described herein). In some instances, a new or improved
property or characteristic can be achieved after only a single
cycle of in vivo or in vitro reassembly (&/or one or more
additonal directed evolution methods described herein), as when
using different, variant forms of the sequence, as homologs from
different individuals or strains of an organism, or related
sequences from the same organism, as allelic variations. However,
recursive sequence reassembly (&/or one or more additonal
directed evolution methods described herein), which entails
successive cycles of reassembly (&/or one or more additonal
directed evolution methods described herein), can also be employed
to achieve still further improvements in a desired property, or to
bring about new (or "distinct") properties, or to. generate further
molecular diversity.
[0562] In a presently preferred embodiment, polynucleotides that
encode optimized recombinant antigens are subjected to molecular
backcrossing, which provides a means to breed the experimentally
evolved (e.g. by polynucleotide reassembly &/or polynucleotide
site-saturation mutagenesis) chimeras/mutants back to a parental or
wild-type sequence, while retaining the mutations that are critical
to the phenotype that provides the optimized immune responses. In
addition to removing the neutral mutations, molecular backcrossing
can also be used to characterize which of the many mutations in an
improved variant contribute most to the improved phenotype. This
cannot be accomplished in an efficient library fashion by any other
method. Backcrossing is performed by reassembling (optionally in
combination with other directed evolution methods described herein)
the improved sequence with a large molar excess of the parental
sequences.
[0563] Stochastic (e.g. Polynucleotide Shuffling & Interrupted
Synthesis) and Non-stochastic Polynucleotide Reassembly is used to
obtain the Library of Recombinant Nucleic Acids, using a Variety of
Substrates to Acquire or Improve Various Properties for Different
Applications
[0564] Creation of Recombinant Libraries
[0565] The invention involves creating recombinant libraries of
polynucleotides that are then screened to identify those library
members that exhibit a desired property. The recombinant libraries
can be created using any of various methods.
[0566] Initial Diversity Between Substrates
[0567] The substrate nucleic acids used for the reassembly
(&/or one or more additonal directed evolution methods
described herein) can vary depending upon the particular
application. For example, where a polynucleotide that encodes (369-
"a cytokine, chemokine, or other accessory molecule") a nucleic
acid binding domain or a ligand for a cell-specific receptor is to
be optimized, different forms of nucleic acids that encode all or
part of the (369- "cytokine, chemokine, or other accessory
molecule") nucleic acid binding domain or a ligand for a
cell-specific receptor are subjected to reassembly (&/or one or
more additonal directed evolution methods described herein).
[0568] In a presently preferred embodiment, stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly is used to obtain the
library of recombinant nucleic acids. stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly, which is diagrammed in
Figure ???, can result in optimization of a desired property even
in the absence of a detailed understanding of the mechanism by
which the particular property is mediated. The substrates for this
modification, or evolution, vary in different applications, as does
the property sought to be acquired or improved. Examples of
candidate substrates for acquisition of a property or improvement
in a property include viral and nonviral vectors used in genetic
vaccination, as well as nucleic acids that are involved in
mediating a particular aspect of an immune response. The methods
require at least two variant forms of a starting substrate. The
variant forms of candidate components can have substantial sequence
or secondary structural similarity with each other, but they should
also differ in at least two positions. The initial diversity
between forms can be the result of natural variation, e.g., the
different variant forms (homologs) are obtained from different
individuals or strains of an organism (including geographic
variants) or constitute related sequences from the same organism
(e.g., allelic variations). Alternatively, the initial diversity
can be induced, e.g., the second variant form can be generated by
error-prone transcription, such as an error- prone PCR or use of a
polymerase which lacks proof-reading activity (see, Liao (1990)
Gene 88:107-111), of the first variant form, or, by replication of
the first form in a mutator strain (mutator host cells are
discussed in further detail below). The initial diversity between
substrates is greatly augmented in subsequent steps of recursive
sequence reassembly (&/or one or more additonal directed
evolution methods described herein).
[0569] Screening or selection after a reassembly (&/or one or
more additonal directed evolution methods described herein) cycle
(screening after in vitro and in vivo reassembly (&/or one or
more additonal directed evolution methods described herein)
cycles)
[0570] Once one has performed stochastic (e.g. polynucleotide
shuffling & interrupted synthesis) and non-stochastic
polynucleotide reassembly to obtain a library of polynucleotides
that encode recombinant antigens, the library is subjected to
selection and/or screening to identify those library members that
encode antigenic peptides that have improved ability to induce an
immune response to the pathogenic agent. Selection and screening of
experimentally generated polynucleotides that encode polypeptides
having an improved ability to induce an immune response can involve
either in vivo and in vitro methods, but most often involves a
combination of these methods. For example, in a typical embodiment
the members of a library of recombinant nucleic acids are picked,
either individually or as pools. The clones can be subjected to
analysis directly, or can be expressed to produce the corresponding
polypeptides. In a presently preferred embodiment, an in vitro
screen is performed to identify the best candidate sequences for
the in vivo studies. Alternatively, the library can be subjected to
in vivo challenge studies directly. The analyses can employ either
the nucleic acids themselves (e.g., as genetic vaccines), or the
polypeptides encoded by the nucleic acids. A schematic diagram of a
typical strategy shown, described &/or referenced herein
(including incorporated by reference). Both in vitro and in vivo
methods are described in more detail below.
[0571] A cycle of reassembly (&/or one or more additonal
directed evolution methods described herein) is usually followed by
at least one cycle of screening or selection for molecules having a
desired property or characteristic. If a cycle of reassembly
(&/or one or more additonal directed evolution methods
described herein) is performed in vitro, the products of reassembly
(&/or one or more additonal directed evolution methods
described herein), i.e., recombinant segments, are sometimes
introduced into cells before the screening step. Recombinant
segments can also be linked to an appropriate vector or other
regulatory sequences before screening.
[0572] Alternatively, products of reassembly (&/or one or more
additonal directed evolution methods described herein) generated in
vitro are sometimes packaged as viruses (in viruses- e.g.,
bacteriophage) before screening. If reassembly (&/or one or
more additonal directed evolution methods described herein) is
performed in vivo, product of reassembly (&/or one or more
additonal directed evolution methods described herein) can
sometimes be screened in the cells in which reassembly (&/or
one or more additional directed evolution methods described herein)
occurred. In other applications, recombinant segments are extracted
from the cells, and optionally packaged as viruses, before
screening.
[0573] Component Sequences having Different Roles than the Product
of Reassembly (&/or One or More Additional Directed Evolution
Methods Described Herein)
[0574] The nature of screening or selection depends on what
property or characteristic is to be acquired or the property or
characteristic for which improvement is sought, and many examples
are discussed below. It is not usually necessary to understand the
molecular basis by which particular products of reassembly
(&/or one or more additional directed evolution methods
described herein) (recombinant segments) have acquired new or
improved properties or characteristics relative to the starting
substrates. For example, a genetic vaccine vector can have many
component sequences each having a different intended role (e.g.,
coding sequence, regulatory sequences, targeting sequences,
stability-conferring sequences, immunomodulatory sequences,
sequences affecting antigen presentation, and sequences affecting
integration). Each of these component sequences can be varied and
reassembled (&/or subjected to one or more directed evolution
methods described herein) simultaneously. Screening/selection can
then be performed, for example, for recombinant segments that have
increased episomal maintenance in a target cell without the need to
attribute such improvement to any of the individual component
sequences of the vector.
[0575] Initial Screenings in Bacterial Cells vs. Later Screening in
Mammalian Cells
[0576] Depending on the particular screening protocol used for a
desired property, initial round(s) of screening can sometimes be
performed in bacterial cells due to high transfection efficiencies
and ease of culture. However, especially for testing of immunogenic
activity, test animals are used for library expression and
screening. Later rounds, and other types of screening which are not
amenable to screening in bacterial cells, are generally performed
(in cells selected for use in an environment close to that of their
intended use) in mammalian cells to optimize recombinant segments
for use in an environment close to that of their intended use.
Final rounds of screening can be performed in (383- "cells or
organisms that are as close as possible to") the precise cell type
of intended use (e.g., a human antigen-presenting cell). In some
instances, this cell can be obtained from a patient to be treated
with a view, for example, to minimizing problems of immunogenicity
in this patient. In some methods, use of a genetic vaccine vector
in treatment can itself be used as a round of screening. That is,
genetic vaccine vectors that are successively taken up and/or
expressed by the intended target cells in one patient are recovered
from those target cells and used to treat another patient. The
genetic vaccine vectors that are recovered from the intended target
cells in one patient are enriched for vectors that have evolved,
i.e., have been modified by recursive reassembly (&/or one or
more additional directed evolution methods described herein),
toward improved or new properties or characteristics for specific
uptake, immunogenicity, stability, and the like.
[0577] Identifying a Subpopulation of Recombinant Segments
[0578] The screening or selection step identifies a subpopulation
of recombinant segments that have evolved toward acquisition of a
new or improved desired property or properties useful in genetic
vaccination. Depending on the screen, the recombinant segments can
be screened as components of cells, components of viruses or other
vectors, or in free form. More than one round of screening or
selection can be performed after each round of reassembly (&/or
one or more additional directed evolution methods described
herein).
[0579] The Second Round of Reassembly (&/or One or More
Additional Directed Evolution Methods Described Herein)
[0580] If further improvement in a property is desired, at least
one and usually a collection of recombinant segments surviving a
first round of screening/selection are subject to a further round
of reassembly (&/or one or more additional directed evolution
methods described herein). These recombinant segments can be
reassembled (&/or subjected to one or more directed evolution
methods described herein) with each other or with exogenous
segments representing the original substrates or further variants
thereof. Again, reassembly (&/or one or more additional
directed evolution methods described herein) can proceed in vitro
or in vivo. If the previous screening step identifies desired
recombinant segments as components of cells, the components can be
subjected to further reassembly (&/or one or more additional
directed evolution methods described herein) in vivo, or can be
subjected to further reassembly (&/or one or more additional
directed evolution methods described herein) in vitro, or can be
isolated before performing a round of in vitro reassembly (&/or
one or more additional directed evolution methods described
herein). Conversely, if the previous screening step identifies
desired recombinant segments in naked form or as components of
viruses or other vectors, these segments can be introduced into
cells to perform a round of in vivo reassembly (&/or one or
more additional directed evolution methods described herein). The
second round of reassembly (&/or one or more additional
directed evolution methods described herein), irrespective how
performed, generates further recombinant segments which encompass
additional diversity compared to recombinant segments resulting
from previous rounds.
[0581] Additional Rounds of Reassembly (&/or One or More
Additional Directed Evolution Methods Described Herein)/Screening
to Sufficiently Evolve the Recombinant Segments
[0582] The second round of reassembly (&/or one or more
additional directed evolution methods described herein) can be
followed by a further round of screening/selection according to the
principles discussed above for the first round. The stringency of
screening/selection can be increased between rounds. Also, the
nature of the screen and the property being screened for can vary
between rounds if improvement in more than one property is desired
or if acquiring more than one new property is desired.
[0583] Additional rounds of reassembly (&/or one or more
additional directed evolution methods described herein) and
screening can then be performed until the recombinant segments have
sufficiently evolved to acquire the desired new or improved
property or function.
[0584] The practice of this invention involves the construction of
recombinant nucleic acids and the expression of genes in
transfected host cells. Molecular cloning techniques to achieve
these ends are known in the art. A wide variety of cloning and in
vitro amplification methods suitable for the construction of
recombinant nucleic acids such as expression vectors are well-known
to persons of skill. General texts which describe molecular
biological techniques useful herein, including mutagenesis, include
Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods
in Enzymology volume 152 Academic Press, Inc., San Diego, Calif.
(Berger); Sambrook et al., Molecular Cloning--A Laboratory Manual
(2nd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y., 1989 ("Sambrook") and Current Protocols in Molecular
Biology, F. M. Ausubel et al., eds., Current Protocols, a joint
venture between Greene Publishing Associates, Inc. and John Wiley
& Sons, Inc., (supplemented through 1998) ("Ausubel")).
[0585] Examples of techniques sufficient to direct persons of skill
through in vitro amplification methods, including the polymerase
chain reaction (PCR) the ligase chain reaction (LCR), Q--replicase
amplification and other RNA polymerase mediated techniques (e.g.,
NASBA) are found in Berger, Sambrook, and Ausubel, as well as
Mullis et al. (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide
to Methods and Applications (Innis et al. eds) Academic Press Inc.
San Diego, Calif. (1990) (Innis); Antheirn & Levinson (Oct. 1,
1990) C&EN 36-47; The Journal Of NIH Research (1991) 3, 81-94;
(Kwoh et al. (1989) Proc. Natl. Acad Sci. USA 86, 1173; Guatelli el
al. (1990) Proc. Natl. Acad Sci. USA 87, 1874; Lowell et al. (1989)
J Clin. Chem 35, 1826; Landegren et al. (1988) Science 241,
1077-1080; Van Brunt (1990) Biotechnology 8, 291-294; Wu and
Wallace (1989) Gene 4, 560; Barringer et al. (1990) Gene 89, 117,
and Sooknanan and Malek (1995) Biotechnology 13: 563-564.
[0586] Improved methods of cloning in vitro amplified nucleic acids
are described in Wallace et al., U.S. Pat. No. 5,426,039. Improved
methods of amplifying large nucleic acids by PCR are summarized in
Cheng et al. (1994) Nature 369: 684-685 and the references therein,
in which PCR amplicons of up to 40 kb are generated. One of skill
will appreciate that essentially any RNA can be converted into a
double stranded DNA suitable for restriction digestion, PCR
expansion and sequencing using reverse transcriptase and a
polymerase. See, Ausubel, Sambrook and Berger, all supra.
[0587] Oligonucleotides for use as probes, e.g., in in vitro
amplification methods, for use as gene probes, or as reassembly
targets (e.g., synthetic genes or gene segments) are typically
synthesized chemically according to the solid phase phosphoramidite
triester method described by Beaucage and Caruthers (1981)
Tetrahedron Letts., 22(20):1859-1862, e.g., using an automated
synthesizer, as described in Needham-VanDevanter et al. (1984)
Nucleic Acids Res., 12:6159-6168. Oligonucleotides can also be
custom made and ordered from a variety of commercial sources known
to persons of skill.
[0588] Indeed, essentially any nucleic acid with a known sequence
can be custom ordered from any of a variety of commercial sources,
such as The Midland Certified Reagent Company (mcrc@oligos.com),
The Great American Gene Company (http://www.genco.com), ExpressGen
Inc. (www.expressgen.com), Operon Technologies Inc. (Alameda,
Calif.) and many others. Similarly, peptides and antibodies can be
custom ordered from any of a variety of sources, such as
PeptidoGenic (pkim@ccnet.com), HTI Bio-products, Inc.
(http://www.htibio.com), BMA Biomedicals Ltd (U.K.), Bio-Synthesis,
Inc., and many others.
[0589] Different Formats are Available for Performing Reassembly
(&/or Additional Directed Evolution Methods described herein)
and Screening/Selection which allow for Large Numbers of Mutations
in a Minimum Number of Selection Cycles and does not require the
Extensive Analysis and Computation Required by Conventional
Methods
[0590] A number of different formats are available by which one can
create a library of recombinant nucleic acids for screening. In
some embodiments, the methods of the invention entail performing
reassembly (&/or one or more additional directed evolution
methods described herein) and screening or selection to "evolve"
individual genes, whole plasmids or viruses, multigene clusters, or
even whole genomes (Stemmer (1995) Bio/Technology 13:549-553).
Reiterative cycles of reassembly (&/or one or more additional
directed evolution methods described herein) and
screening/selection can be performed to further evolve the nucleic
acids of interest. Such techniques do not require the extensive
analysis and computation required by conventional methods for
polypeptide engineering. Reassembly allows the combination of large
numbers of mutations in a minimum number of selection cycles, in
contrast to traditional, pair wise recombiantion events (e.g., as
occur during sexual replication). Thus, the directed evolution
techniques described herein provide particular advantages in that
they provide reassembly (optionally in combination with one or more
additional directed evolution methods described herein) between any
or all of the mutations, thereby providing a very fast way of
exploring the manner in which different combinations of mutations
can affect a desired result. In some instances, however, structural
and/or functional information is available which, although not
required for sequence reassembly (&/or one or more additional
directed evolution methods described herein), provides
opportunities for modification of the technique.
[0591] Four different Approaches to Improve Immunogenic Activity as
well as Broaden Specificity: Reassembly (Optionally in Combination
with other Directed Evolution Methods Described Herein) on Single
Gene, Sequence Comparison of Homologous Genes, whole Genome
Reassembly, Codon Modification of Polypeptide-encoding Genes
[0592] The stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide reassembly
methods can involve one or more of at least four different
approaches to improve immunogenic activity as well as to broaden
specificity. First, stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide reassembly
can be performed on a single gene. Secondly, several highly
homologous genes can be identified by sequence comparison with
known homologous genes. These genes can be synthesized and
experimentally evolved (e.g. by polynucleotide reassembly &/or
polynucleotide site-saturation mutagenesis) as a family of
homologs, to select recombinants with the desired activity. The
experimentally evolved (e.g. by polynucleotide reassembly &/or
polynucleotide site-saturation mutagenesis) genes can be introduced
into appropriate host cells, which can include E. coli, yeast,
plants, fungi, animal cells, and the like, and those having the
desired properties can be identified by the methods described
herein. Third, whole genome reassembly can be performed to shuffle
genes that can confer a desired property upon a genetic vaccine
(along with other genomic nucleic acids). For whole genome
reassembly approaches, it is not even necessary to identify which
genes are being experimentally evolved (e.g. by polynucleotide
reassembly &/or polynucleotide site-saturation mutagenesis).
Instead, e.g., bacterial cell or viral genomes are combined and
experimentally evolved (e.g. by polynucleotide reassembly &/or
polynucleotide site-saturation mutagenesis) to acquire recombinant
nucleic acids that, either itself or through encoding a
polypeptide, have enhanced ability to induce an immune response, as
measured in any of the assays described herein. Fourth,
polypeptide-encoding genes can be codon modified to access
mutational diversity not present in any naturally occurring
gene.
[0593] References for Formats and Examples for Sequence Reassembly
(&/or one or more Additional Directed Evolution Methods
Described Herein) and for other Methods
[0594] Exemplary formats and examples for polynucleotide
reassembly, gene site saturation mutagenesis, interrupted
synthesis, and additional directed evolution methods described
herein have been described by the present inventors and co-workers
in issued and co-pending applications including U.S. Pat. No.
5,965,408 (issued Oct. 12, 1999), U.S. Pat. No. 5,830,696 (issued
Nov. 03, 1998), and U.S. Pat. No. 5939,250 (issued Aug. 17,
1999).
[0595] Other methods for obtaining libraries of experimentally
generated polynucleotides and/or for obtaining diversity in nucleic
acids used as the substrates for directed evolution including
stochastic (e.g. polynucleotide shuffling & interrupted
synthesis) and non-stochastic polynucleotide reassembly include,
for example, WO98/42727; Smith, Ann. Rev. Genet. 19: 423-462
(1985); Botstein and Shortle, Science 229: 1193-1201 (1985);
Carter, Biochem. J 237: 1-7 (1986); Kunkel, "The efficiency of
oligonucleotide directed mutagenesis" in Nucleic acids &
Molecular Biology, Eckstein and Lilley, eds., Springer Verlag,
Berlin (1987)). Included among these methods are
oligonucleotide-directed mutagenesis (Zoller and Smith, Nucl. Acids
Res. 10: 6487-6500 (1982), Methods in Enzymol. 100: 468-500 (1983),
and Methods in Enzymol. 154: 329-350 (1987))
phosphothioate-modified DNA mutagenesis (Taylor et al., Nucl. Acids
Res. 13: 8749-8764 (1985); Taylor et al., Nucl. Acids Res. 13:
8765-8787 (1985); Nakamaye and Eckstein, Nucl. Acids Res. 14:
9679-9698 (1986); Sayers et al., Nucl. Acids Res. 16: 791-802
(1988); Sayers et al., Nucl. Acids Res. 16: 803-814 (1988)),
mutagenesis using uracil-containing templates (Kunkel, Proc. Nat'l.
Acad. Sci. USA 82: 488-492 (1985) and Kunkel et al., Methods in
Enzymol. 154: 367-382)); mutagenesis using gapped duplex DNA
(Kramer et al., Nucl. Acids Res. 12: 9441-9456 (1984); Kramer and
Fritz, Methods in Enzymol. 154: 350-367 (1987); Kramer et al.,
Nucl. Acids Res. 16: 7207 (1988)); and Fritz et al., Nucl. Acids
Res. 16: 6987-6999 (1988)). Additional suitable methods include
point mismatch repair (Kramer et al., Cell 38: 879-887 (1984)),
mutagenesis using repair-deficient host strains (Carter et al.,
Nucl. Acids Res. 13: 4431-4443 (1985); Carter, Methods in Enzymol.
154: 382-403.(1987)), deletion mutagenesis (Eghtedarzadeh and
Henikoff, Nucl. Acids Res. 14: 5115 (1986)), restriction-selection
and restriction-purification (Wells et al., Phil. Trans. R. Soc.
Lond. A 317: 415-423 (1986)), mutagenesis by total gene synthesis
(Nambiar et al., Science 223: 1299-1301 (1984); Sakamar and
Khorana, Nucl. Acids Res. 14: 6361-6372 (1988); Wells et al., Gene
34: 315-323 (1985); and Grundstr6m et al., Nucl. Acids Res. 13:
3305-3316 (1985). Kits for mutagenesis are commercially available
(e.g., Bio-Rad, Amersharn International, Anglian
Biotechnology).
[0596] For Reassembly (&/or One or More Additional Directed
Evolution Methods Described Herein) to Generate Increased Diversity
Relative to the Starting Materials, the Starting Materials must
Differ from Each Other in at Least Two Nucleotide Positions
[0597] The reassembly procedure starts with at least two substrates
that generally show substantial sequence identity to each other
(i.e., at least about 30%, 50%, 70%, 80% or 90% sequence identity),
but differ from each other at certain positions. The difference can
be any type of mutation, for example, substitutions, insertions and
deletions. Often, different segments differ from each other in
about 5-20 positions. For reassembly (&/or one or more
additional directed evolution methods described herein) to generate
increased diversity relative to the starting materials, the
starting materials must differ from each other in at least two
nucleotide positions. That is, if there are only two substrates,
there should be at least two divergent positions. If there are
three substrates, for example, one substrate can differ from the
second at a single position, and the second can differ from the
third at a different single position. The starting DNA segments can
be natural variants of each other, for example, allelic or species
variants. The segments can also be from nonallelic genes showing
some degree of structural and usually functional relatedness (e.g.,
different genes within a superfamily, such as the family of
Yersinia V-antigens, for example). The starting DNA segments can
also be induced variants of each other. For example, one DNA
segment can be produced by error-prone PCR replication of the
other, the nucleic acid can be treated with a chemical or other
mutagen, or by substitution of a mutagenic cassette. Induced
mutants can also be prepared by propagating one (or both) of the
segments in a mutagenic strain, or by inducing an error-prone
repair system in the cells.
[0598] The Different Segments Forming the Starting Materials are
Related, and Might or Might Not be of Similar Length
[0599] In these situations, strictly speaking, the second DNA
segment is not a single segment but a large family of related
segments. The different segments forming the starting materials are
often the same length or substantially the same length. However,
this need not be the case; for example; one segment can be a
subsequence of another. The segments can be present as part of
larger molecules, such as vectors, or can be in isolated form.
[0600] The Starting DNA Segments are Reassembled (&/or
Subjected to One or More Directed Evolution Methods Described
Herein) to Generate a Library of Recombinant DNA Segments Varying
in Size which will include Full Length Coding Sequences and any
Essential Regulatory
[0601] The starting DNA segments are reassembled (&/or
subjected to one or more directed evolution methods described
herein) by any of the sequence reassembly (&/or one or more
additional directed evolution methods described herein) formats
provided herein to generate a diverse library of recombinant DNA
segments. Such a library can vary widely in size from having fewer
than 10 to more than 10.sup.5, 10.sup.9, 10.sup.12 or more members.
In some embodiments, the starting segments and the recombinant
libraries generated will include full-length coding sequences and
any essential regulatory sequences, such as a promoter and
polyadenylation sequence, required for expression. In other
embodiments, the recombinant DNA segments in the library can be
inserted into a common vector providing sequences necessary for
expression before performing screening/selection.
[0602] Using Reassembly PCR to Assemble Multiple Segments that have
been Separately Evolved into a Full Length Nucleic Acid Template
such as a Gene
[0603] A further technique for recombining mutations in a nucleic
acid sequence utilizes "reassembly PCR". This method can be used to
assemble multiple segments that have been separately evolved into a
full length nucleic acid template such as a gene. This technique is
performed when a pool of advantageous mutants is known from
previous work or has been identified by screening mutants that may
have been created by any mutagenesis technique known in the art,
such as PCR mutagenesis, cassette mutagenesis, doped oligo
mutagenesis, chemical mutagenesis, or propagation of the DNA
template in vivo in mutator strains. Boundaries defining segments
of a nucleic acid sequence of interest preferably lie in intergenic
regions, introns, or areas of a gene not likely to have mutations
of interest.
[0604] Oligos are Synthesized for PCR Amplification of Segments of
the Nucleic Acid Sequence of Interest so that the Oligos Overlap
the Junctions of Two Segments by, Typically, about 10 to 100
Nucleotides
[0605] Preferably, oligonucleotide primers (oligos) are synthesized
for PCR amplification of segments of the nucleic acid sequence of
interest, such that the sequences of the oligonucleotides overlap
the junctions of two segments. The overlap region is typically
about 10 to 100 nucleotides in length. Each of the segments is
amplified with a set of such primers. The PCR products are then
"reassembled" according to assembly protocols such as those
discussed herein to assemble non-stochastically generated nucleic
acid building blocks &/or randomly fragmented genes. In brief,
in an assembly protocol the PCR products are first purified away
from the primers, by, for example, gel electrophoresis or size
exclusion chromatography. Purified products are mixed together and
subjected to about 1-10 cycles of denaturing, reannealing, and
extension in the presence of polymerase and deoxynucleoside
triphosphates (dNTP's) and appropriate buffer salts in the absence
of additional primers ("self-priming"). Subsequent PCR with primers
flanking the gene are used to amplify the yield of the fully
reassembled and experimentally evolved (e.g. by polynucleotide
reassembly &/or polynucleotide site-saturation mutagenesis)
genes.
[0606] PCR Primers are used to Introduce Variation into the Gene of
Interest and the Mutations at Sites of Interest are Screened or
Selected by Sequencing Homologues of the Nucleic Acid Sequence
[0607] In a further embodiment, PCR primers for amplification of
segments of the nucleic acid sequence of interest are used to
introduce variation into the gene of interest as follows. Mutations
at sites of interest in a nucleic acid sequence are identified by
screening or selection, by sequencing homologues of the nucleic
acid sequence, and so on.
[0608] Using Oligonucleotide PCR Primers (Encoding Wild Type or
Mutant Information) in PCR to Generate Libraries of Full Length
Genes Encoding Permutations of said info. where the Alternative
Screening or Selection Process is Expensive, Cumbersome, or
Impractical
[0609] Oligonucleotide PCR primers are then synthesized which
encode wild type or mutant information at sites of interest. These
primers are then used in PCR mutagenesis to generate libraries of
full length genes encoding permutations of wild type and mutant
information at the designated positions. This technique is
typically advantageous in cases where the screening or selection
process is expensive, cumbersome, or impractical relative to the
cost of sequencing the genes of mutants of interest and
synthesizing mutagenic oligonucleotides.
2.3. VECTORS USED IN GENETIC VACCINATION
[0610] Evolution of Genetic Vaccines and Components by Stochastic
(e.g. Polynucleotide Shuffling & Interrupted Synthesis) and
Non-stochastic Polynucleotide Reassembly (FIG. 3)
[0611] The invention provides multicomponent genetic vaccines, and
methods of obtaining genetic vaccine components that improve the
capability of the genetic vaccine for use in nucleic acid-mediated
immunomodulation. A general approach for evolution of genetic
vaccines and components by stochastic (e.g. polynucleotide
shuffling & interrupted synthesis) and non-stochastic
polynucleotide reassembly is shown schematically in FIG. 3.
[0612] Including an Origin of Replication is Useful to Obtain
Sufficient Quantities of the Vector Prior to Administration to a
Patient, but might be Undesirable if the Vector is Designed to
Integrate into host Chromosomal DNA or Bind to Host mRNA or DNA
[0613] Broadly speaking, a genetic vaccine vector is an exogenous
polynucleotide which produces a medically useful phenotypic effect
upon the mammalian cell(s) and organisms into which it is
transferred. A vector may or may not have an origin of replication.
For example, it is useful to include an origin of replication in a
vector to allow for propagation of the vector in order to obtain
sufficient quantities of the vector prior to administration to a
patient. If the vector is designed to integrate into host
chromosomal DNA or bind to host mRNA or DNA, or if replication in
the host is otherwise undesirable, the origin of replication can be
removed before administration, or an origin can be used that
functions in the cells used for vector production but not in the
target cells. However, in certain situations, including some of
those discussed herein, it is desirable that the genetic vaccine
vector be capable of replicating in appropriate host cells.
[0614] Incorporating Nucleic Acids that are Modified by Stochastic
(e.g. Polynucleotide Shuffling & Interrupted Synthesis) and
Non-stochastic Polynucleotide Reassembly into Viral Vectors to be
used in Genetic Vaccination
[0615] Vectors used in genetic vaccination can be viral or
nonviral. Viral vectors are usually introduced into a patient as
components of a virus. Illustrative viral vectors into which one
can incorporate nucleic acids that are modified by the stochastic
(e.g. polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassemblyg methods of the invention
include, for example, adenovirus-based vectors (Cantwell (1996)
Blood 88:4676-4683; Ohashi (1997) Proc. Nat'l. Acad. Sci USA
94:1287-1292), Epstein-Barr virus-based vectors (Mazda (1997) J.
Immunol. Methods 204:143-15 1), adenovirus- associated virus
vectors, Sindbis virus vectors (Strong (1997) Gene Ther. 4:
624-627), herpes simplex virus vectors (Kennedy (1997) Brain 120:
1245-1259) and retroviral vectors (Schubert (1997) Curr. Eye Res.
16:656-662).
[0616] Techniques for Transferring DNA into a Cell useful in vivo
(Naked DNA Delivered using Liposomes Fusing to Cellular Membrane or
Entering through Endocytosis; Permeabilize the Cells and use DNA
Binding Protein to Transport into Cell; and Bombardment of Skin
with Particles Coated with DNA Delivered Mechanically)
[0617] Nonviral vectors, typically dsDNA, can be transferred as
naked DNA or associated with a transfer-enhancing vehicle, such as
a receptor- recognition protein, liposome, lipoamine, or cationic
lipid. This DNA can be transferred into a cell using a variety of
techniques well known in the art. For example, naked DNA can be
delivered by the use of liposomes which fuse with the cellular
membrane or are endocytosed, i.e., by employing ligands attached to
the liposome, or attached directly to the DNA, that bind to surface
membrane protein receptors of the cell resulting in endocytosis.
Alternatively, the cells may be permeabilized to enhance transport
of the DNA into the cell, without injuring the host cells. One can
use a DNA binding protein, e.g., HBGF-1, known to transport DNA
into a cell. Furthermore, DNA can be delivered by bombardment of
the skin by gold or other particles coated with DNA which are
delivered by mechanical means, e.g., pressure. These procedures for
delivering naked DNA to cells are useful in vivo. For example, by
using liposomes, particularly where the liposome surface carries
ligands specific for target cells, or are otherwise preferentially
directed to a specific organ, one may provide for the introduction
of the DNA into the target cells/organs in vivo.
2.3.1. VIRAL VECTORS
[0618] Structure of Viral Vectors often Consist of a Modified Viral
Genome and a Coat Structure Surrounding it, a Structure which can
be Changed in many ways for the Viral Nucleic Acid in a Vector
Designed for Genetic Vaccination
[0619] Various viral vectors, such as retroviruses, adenoviruses,
adenoassociated viruses and herpes viruses, are commonly used in
genetic vaccination. They are often made up of two components, a
modified viral genome and a coat structure surrounding it (see
generally Smith (1995) Annu. Rev. Microbiol. 49, 807-83 8),
although sometimes viral vectors are introduced in naked form or
coated with proteins other than viral proteins. Most current viral
vectors have coat structures similar to a wild type virus. This
structure packages and protects the viral nucleic acid and provides
the means to bind and enter target cells. In contrast, the viral
nucleic acid in a vector designed for genetic vaccination can be
changed in many ways. The goals of these changes can be, for
example, to enhance or, reduce replication of the virus in target
cells while maintaining its ability to grow in vector form in
available packaging or helper cells, to incorporate new sequences
that encode and enable appropriate expression of a gene of interest
(e.g., an antigen-encoding gene), and to alter the immunogenicity
of the viral vector itself Viral vector, nucleic acids generally
comprise two components: essential cis-acting viral sequences for
replication and packaging in a helper line and a transcription unit
for the exogenous gene. Other viral functions can be expressed in
trans in a specific packaging or helper cell line.
2.3.1.1. ADENOVIRUSES
[0620] The Normal Life Cycle and Production Infection Cycle of
Adenoviruses
[0621] Adenoviruses comprise a large class of nonenveloped viruses
that contain linear double-stranded DNA. The normal life cycle of
the virus does not require dividing cells and involves productive
infection in permissive cells during which large amounts of virus
accumulate. The productive infection cycle takes about 32-36 hours
in cell culture and comprises two phases, the early phase, prior to
viral DNA synthesis, and the late phase, during which structural
proteins and viral DNA are synthesized and assembled into
virions.
[0622] In General, Adenovirus Infections are Associated with Mild
Disease in Humans
[0623] E3-deletion Vectors Studied; Replication in Cultured Cells
does not Require E3 Region, allowing Insertion of Exogenous DNA
Sequences to Yield Vectors Capable of Productive Infection and the
Transient Synthesis of Relatively Large Amounts of Encoded
Protein
[0624] Adenovirus vectors are somewhat larger and more complex than
retrovirus or AAV vectors, partly because only a small fraction of
the viral genome is removed from most current vectors. If
additional genes are removed, they are provided in trans to produce
the vector, which so far has proved difficult. Instead, two general
types of adenovirus-based vectors have been studied, E3-deletion
and E1-deletion vectors. Some viruses in laboratory stocks of
wild-type lack the E3 region and can grow in the absence of helper.
This ability does not mean that the E3 gene products are not
necessary in the wild, only that replication in cultured cells does
not require them. Deletion of the E3 region allows insertion of
exogenous DNA sequences to yield vectors capable of productive
infection and the transient synthesis of relatively large amounts
of encoded protein.
[0625] E1 Replacement Vectors Grown in 293 Cells Utilized in most
Gene Therapy Applications Involving Adenoviruses
[0626] Deletion of the E1 region disables the adenovirus, but such
vectors can still be grown because there exists an established
human cell line (called "293") that contains the E1 region of Ad5
and that constitutively expresses the E1 proteins. Most recent
gene-therapy applications involving adenovirus have utilized E1
replacement vectors grown in 293 cells.
[0627] Adenovirus Vectors Capable of Efficient Episomal Gene
Transfer, easy to Grow, can be Topically Applied to Skin for
Antigen Delivery, Induction of Antigen Specific Immune Responses
can be Observed, but Host Response Limits Duration of Expression
and Ability to Repeat Dosing in cases with High Doses of First
Generation Vectors
[0628] The main advantages of adenovirus vectors are that they are
capable of efficient episomal gene transfer in a wide range of
cells and tissues and that they are easy to grow in large amounts.
Adenovirus-based vectors can also be used to deliver antigens after
topical application onto the skin, and induction of
antigen-specific immune responses can be observed following
delivery to the skin (Tang et al. (1997) Nature 388: 729-730). The
main disadvantage is that the host response to the virus appears to
limit the duration of expression and the ability to repeat dosing,
at least with high doses of first-generation vectors.
[0629] This Invention Provides for the First Time a Phagemid System
Capable of Cloning Large DNA Inserts of Over 10 Kilobases and
Generating ssDNA in vitro and in vivo Corresponding to those Large
Inserts
[0630] In one embodiment, the directed evolution methods of the
invention are used to construct a novel adenovirus-phagemid capable
of packaging DNA inserts over 10 kilobases in size. Incorporation
of a phage f1 ??? origin in a plasmid using the methods of the
invention also generates a novel in vivo reassembly or shuffling
format capable of evolving whole genomes of viruses, such as the 36
kb family of human adenoviruses. The widely used human adenovirus
type 5 (Ad5) has a genome size of 36 kb. It is difficult to shuffle
this large genome in vitro without creating an excessive number of
changes which may cause a high percentage of nonviable recombinant
variants. To minimize this problem and achieve whole genome
reassembly of Ad5, an adenovirus-phagemid was constructed. The
Ad-phagemid has been demonstrated to accept inserts as large as 15
and 24 kilobases and to effectively generate ssDNA of that size. In
a further embodiment, larger DNA inserts, as large as 50 to 100 kb
are inserted into the Ad-phagemid of the invention; with generation
of full length ssDNA corresponding to those large inserts.
Generation of such large ssDNA non-stochastically generated nucleic
acid building blocks &/or fragments provides a means to evolve,
i.e. modify by the recursive reassembly methods (&/or one or
more additional recursive directed evolution methods described
herein) of the invention, entire viral genomes. Thus, this
invention provides for the first time a unique phagemid system
capable of cloning large DNA inserts (>10 KB) and generating
ssDNA in vitro and in vivo corresponding to those large
inserts.
[0631] In vivo Reassembly or Shuffling of the Genomes of Related
Serotypes of Human Adenoviruses using System is useful for Creation
of Recombinant Adenovirus Variants with Changes in Multiple
Genes
[0632] The genomes of related serotypes of human adenovirus are
experimentally evolved (e.g. by polynucleotide reassembly &/or
polynucleotide site-saturation mutagenesis) in vivo using this
unique phagemid system, as described in International Application
No. PCT/US97/17302 (Publ. No. WO98/13485). The genomic DNA is first
cloned into a phagemid vector, and the resulting plasmid,
designated an "Admid," can be used to produce single-stranded (ss)
Admid phage by using a helper M13 phage. To achieve in vivo
reassembly (&/or one or more additional directed evolution
methods described herein), ssAdmid phages containing the genome of
homologous human adenoviruses are used to perform high multiplicity
of infection (MOI) on F.sup.+ MutS E. coli cells. The ssDNA is a
better substrate for reassembly (&/or one or more additional
directed evolution methods described herein) enzymes such as RecA.
The high MOI ensures that the probability of having multiple
cross-overs between copies of the infecting ssAdmid DNA is high.
The experimentally evolved (e.g. by polynucleotide reassembly
&/or polynucleotide site-saturation mutagenesis) adenovirus
genome is generated by purification of the double stranded Admid
DNA from the infected cells and is introduction into a permissive
human cell line to produce the adenovirus library. This genomic
reassembly strategy is useful for creation of recombinant
adenovirus variants with changes in multiple genes. This allows
screening or selection of recombinant variant phenotypes resulting
from combinations of variations in multiple genes.
2.3.1.2. ADENO-ASSOCIATED VIRUS (AAV)
[0633] AAV is a small, simple, nonautonomous virus containing
linear single-stranded DNA. See, Muzycka, Current Topics Microbiol.
Immunol. 158, 97-129 (1992). The virus requires co-infection with
adenovirus or certain other viruses in order to replicate. AAV is
widespread in the human population, as evidenced by antibodies to
the virus, but it is not associated with any known disease. AAV
genome organization is straightforward, comprising only two genes:
rep and cap. The termini of the genome comprises terminal repeats
(ITR) sequences of about 145 nucleotides.
[0634] Growth of AAV is Cumbersome and Helper Virus such as
Adenovirus is often Required
[0635] AAV-based vectors typically contain only the ITR sequences
flanking the transcription unit of interest. The length of the
vector DNA cannot greatly exceed the viral genome length of 4680
nucleotides. Currently, growth of AAV vectors is cumbersome and
involves introducing into the host cell not only the vector itself
but also a plasmid encoding rep and cap to provide helper
functions. The helper plasmid lacks ITRs and consequently cannot
replicate and package. In addition, helper virus such as adenovirus
is often required.
[0636] Advantage: Long-term Expression in Nondividing Cells
[0637] The potential advantage of AAV vectors is that they appear
capable of long-term expression in nondividing cells, possibly,
though not necessarily, because the viral DNA integrates. The
vectors are structurally simple, and they may therefore provoke
less of a host-cell response than adenovirus.
2.3.1.3. PAPILLOMA VIRUS
[0638] Papillomaviruses are small, nonenveloped, icosahedral DNA
viruses that replicate in the nucleus of squamous epithelial cells.
Papillomaviruses consist of a single molecule of double-stranded
circular DNA about 8,000 bp in size within a spherical protein coat
of 72 capsomeres. Such papillomaviruses are classified by the
species they infect (e.g., bovine, human, rabbit) and by type
within species. Over 50 distinct human papillomaviruses ("HPV")
have been described. See, e.g., Fields Virology (3rd ed., eds.
Fields et al., Lippincott-Raven, Philadelphia, 1996).
Papillomaviral vectors are described in detail in ???
[0639] Cellular Tropism for Epithelial Cells
[0640] Papillomaviruses display a marked degree of cellular tropism
for epithelial cells. Specific viral types have a preference for
either cutaneous or mucosal epithelial cells.
[0641] Benign, Low-risk, Intermediate-risk, and High-risk HPVs
[0642] All papillomaviruses have the capacity to induce cellular
proliferation. The most common clinical manifestation of
proliferation is the production of benign warts. However, many
papillomaviruses have capacity to be oncogenic in some individuals
and some papillomaviruses are highly oncogenic. Based on the
pathology of the associated lesions, most human papillomaviruses
(HPVs) can be classified in one of four major groups, benign,
low-risk, intermediate-risk and high-risk (Fields Virology, (Fields
et al., eds., Lippincott-Raven, Philadelphia, 3d ed. 1996); DNA
Tumor Viruses: Papilloma in (Encyclopedia of Cancer, Academic
Press) Vol. 1, p 520-531). For example, viruses HPV-1, HPV-2,
HPV-3, HPV-4, and HPV-27 are associated with benign cutaneous
lesions. Viruses HPV-6 and HPV-11 are associated with vulval,
penile, and laryngeal warts and are considered low-risk viruses as
they are rarely associated with invasive carcinomas. Viruses
HPV-16, HPV-18, HPV-31, and HPV-45 are considered high risk virus
as they are associated with a high frequency with adeno- and
squamous carcinoma of the cervix. Viruses HPV-5 and HPV-8 are
associated with benign cutaneous lesion in a multifactorial disease
Epidermodysplasia Verruciformis (EV). Such lesions, however, can
progress into squamous cell carcinomas.
[0643] HPVs Classified for Risk based on Frequency of Cancerous
Lesions Relative to Previously Classified HPVs
[0644] These viruses do not fall under one of the four major risk
groups. Newly discovered HPVs can classified for risk based on the
frequency of cancerous lesions relative to that of HPVs that have
already been classified for risk.
[0645] HPV vectors can be subjected to iterative cycles of
reassembly (&/or one or more additional directed evolution
methods described herein) and screening with a view to obtaining
vectors with improved properties. Improved properties include
increased tissue specificity, altered tissue specificity, increased
expression level, prolonged expression, increased episomal copy
number, increased or decreased capacity for chromosomal
integration, increased uptake capacity, and other properties as
discussed herein. The starting materials for reassembling
(optionally in combination with other directed evolution methods
described herein) are typically vectors of the kind described above
constructed from different strains of human papillomaviruses, or
segments or variants of such generated by e.g., error-prone PCR or
cassette mutagenesis. The human papillomaviruses, or at least the
E1 and E2 coding regions thereof are preferably human cutaneous
papillomaviruses.
2.3.1.4. RETROVIRUSES
[0646] Normal Viral Life Cycle and Viral Genome Organization
[0647] Retroviruses comprise a large class of enveloped viruses
that contain single-stranded RNA as the viral genome. During the
normal viral life cycle, viral RNA is reverse-transcribed to yield
double-stranded DNA that integrates into the host genome and is
expressed over extended periods. As a result, infected cells shed
virus continuously without apparent harm to the host cell. The
viral genome is small (approximately 10 kb), and its prototypical
organization is extremely simple, comprising three genes encoding
gag, the group specific antigens or core proteins; pot, the reverse
transcriptase; and env, the viral envelope protein. The termini of
the RNA genome are called long terminal repeats (LTRs) and include
promoter and enhancer activities and sequences involved in
integration. The genome also includes a sequence required for
packaging viral RNA and splice acceptor and donor sites for
generation of the separate envelope mRNA. Most retroviruses can
integrate only into replicating cells, although human
immunodeficiency virus (HIV) appears to be an exception.
[0648] Providing the Missing Viral Functions to the Retrovirus
Vector and Adding/removing Additional Features to Render the
Vectors more Efficacious or Reduce the Possibility of Contamination
by Helper Virus
[0649] Retrovirus vectors are relatively simple, containing the 5'
and 3' LTRs, a packaging sequence, and a transcription unit
composed of the gene or genes of interest, which is typically an
expression cassette. To grow such a vector, one must provide the
missing viral functions in trans using a so-called packaging cell
line. Such a cell is engineered to contain integrated copies of
gag, pol, and env but to lack a packaging signal so that no helper
virus sequences become encapsidated. Additional features added to
or removed from the vector and packaging cell line reflect attempts
to render the vectors more efficacious or reduce the possibility of
contamination by helper virus.
[0650] Potentially Capable of Long-term Expression, can be Grown in
Large Amounts, but must Ensure the Absence of Helper virus
[0651] For some genetic vaccine applications, retroviral vectors
have the advantage of being able integrate in the chromosome and
therefore potentially capable of long-term expression. They can be
grown in relatively large amounts, but care is needed to ensure the
absence of helper virus.
2.3.2. NON-VIRAL GENETIC VACCINE VECTORS
[0652] Nonviral nucleic acid vectors used in genetic vaccination
include plasmids, RNAs, polyamide nucleic acids, and yeast
artificial chromosomes (YACs), and the like.
[0653] Vector Organization; Insertion of Enhancer Sequence
Increases Transcription
[0654] Such vectors typically include an expression cassette for
expressing a polypeptide against which an immune response is
induced. The promoter in such an expression cassette can be
constitutive, cell type-specific, stage-specific, and/or
modulatable (e.g., by tetracycline ingestion;
tetracycline-responsive promoter). Transcription can be increased
by inserting an enhancer sequence into the vector. Enhancers are
cis-acting sequences, typically between 10 to 300 base pairs in
length, that increase transcription by a promoter. Enhancers can
effectively increase transcription when either 5' or 3' to the
transcription unit. They are also effective if located within an
intron or within the coding sequence itself. Typically, viral
enhancers are used, including SV40 enhancers, cytomegalovirus
enhancers, polyoma enhancers, and adenovirus enhancers. Enhancer
sequences from mammalian systems are also commonly used, such as
the mouse immunoglobulin heavy chain enhancer.
[0655] Methods for Introduction of Nonviral Vectors into an
Animal
[0656] Nonviral vectors encoding products useful in gene therapy
can be introduced into an animal by means such as lipofection,
biolistics, virosomes, liposornes, immunoliposomes,
polycation:nucleic acid conjugates, naked DNA injection, artificial
virions, agent-enhanced uptake of DNA, ex vivo transduction.
Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386,
4,946,787; and 4,897,355) and lipofection reagents are sold
commercially (e.g., Transfectam.TM. and Lipofectin.TM.). Cationic
and neutral lipids that are suitable for efficient
receptor-recognition lipofection of polynucleotides include those
of Felgner, WO 91/17424, WO 91/16024. Naked DNA genetic vaccines
are described in, for example, U.S. Pat. No. 5,589,486.
2.4. MULTICOMPONENT GENETIC VACCINES
[0657] Use of Two or More Separate Genetic Vaccine Components for
Immunization, Providing a means for Eliciting Differentiated
Responses in Different Cell Types
[0658] The invention provides multicomponent genetic vaccines that
are designed to obtain an optimal immune response upon
administration to a mammal. In these vaccines, two or more separate
genetic vaccine components are used for immunization, preferably in
the same formulation. Each component can be optimized for
particular functions that will occur in some cells and not in
others, thus providing a means for eliciting differentiated
responses in different cell types. When mutually incompatible
consequences are derived from use of one plasmid, those activities
are separated into different vectors that will have different fates
and effects in vivo. Genetic vaccines are ideal for the formulation
of several biologically active entities into one preparation. The
vectors are preferably all of the same chemical type so there is no
incompatibility of this nature, and can all be manufactured by the
same chemical and/or biological processes. The vaccine preparation
can consist of a defined molar ratio of the separate vector
components that can be formulated exactly and repeatedly.
[0659] Developing Vector Components without Knowledge of Mechanism
by which a Particular Feature is Controlled or Property to be
Modified
[0660] Several genetic vaccine vector components that can be used
as components of a multicomponent genetic vaccine are described
below. The methods of the invention greatly simplify the
development of such vector components, because the mechanism by
which a particular feature is controlled and the properties of a
molecule that, when modified, will enhance that feature, need not
be known. Even in the absence of such knowledge, by carrying out
the reassembly (&/or one or more additional directed evolution
methods described herein) and screening methods of the invention,
one can obtain vector components that are improved for each of the
properties listed.
2.4. VECTOR "AR",DESIGNED TO PROVIDE OPTIMAL ANTIGEN RELEASE
[0661] Genetic vaccine vector component "AR" is designed to provide
optimal release of antigen in a form that will be recognized by
antigen presenting cells (APC) and taken up by those cells for
efficient intracellular processing and presentation to T helper
(T.sub.H) cells. Cells transfected with AR plasmid can be
considered as an antigen factory for APC. AR plasmids typically
have one or more of the following properties, each of which can be
optimized using the stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide reassembly
methods of the invention.
[0662] Optimal Plasmid Binding to and Uptake by the Chosen Antigen
Expressing Cells (e.g., Myocytes for Intramuscular Immunization or
Epithelial Cells for Mucosal Immunization)
[0663] This is a critical property which differentiates AR from
other vector components in the multicomponent DNA vaccine. Optimal
vector binding to the target cell includes not only the concept of
very avid binding and subsequent internalization into target cells,
but relative inability to bind to and enter other cells.
Optimization of this ratio of desired binding to undesired binding
will significantly increase the number of target cells transfected.
This property can be optimized using stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly according to the present
invention as described herein. For example, variant vector
component sequences obtained by stochastic (e.g. polynucleotide
shuffling & interrupted synthesis) and non-stochastic
polynucleotide reassembly, combinatorial assembly of vector
components, insertion of random oligonucleotide sequences, and the
like, can first be selected for those that bind to target cells,
after which this population of cells is depleted for those that
bind to other cells. Vector components for targeting genetic
vaccine vectors to particular cell types, and methods of obtaining
improved targeting, are described in
[0664] (a) Optimal Trafficking of the Vector DNA to the Nucleus
[0665] Again, the present invention provides methods by which one
can obtain genetic vaccine components that are optimal for such
properties.
[0666] (b) Optimal Transcription of the Antigen Gene(s)
[0667] This can involve, for example, the use of optimized
promoters, enhancers, introns, and the like. In a preferred
embodiment, cell-specific promoters are used that only allow
transcription of the genes when the vector is within the nucleus of
the target cell type. In this case, specificity is derived not only
from selective vector entry into target cells.
[0668] (c) Optimal Trafficking of mRNA to the Cytoplasm and Optimal
Longevity of the mRNA in the Cytoplasm
[0669] To achieve this property, the methods of the invention are
used to obtain optimal 3' and 5' non-translated regions of the
mRNA.
[0670] (d) Optimal Translation of the mRNA
[0671] Again, the stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide reassembly
methods are used to obtain optimized recombinant sequences which
exhibit optimal ribosome binding and assembly of translational
machinery, plus optimal codon preference.
[0672] (e) Optimal Antigen Structure for Efficient Uptake by
APC
[0673] Extracellular antigen is taken up by APC by at least five
non-exclusive mechanisms. One mechanism is sampling of the external
fluid phase by micropinocytosis and internalization of a
vesicle.
[0674] Additional Mechanistic Considerations
[0675] The first mechanism has, as far as is presently known, no
structural requirements for an antigen in the fluid phase and is
therefore not relevant to considerations of designing antigen
structure. A second mechanism involves binding of antigen to
receptors on the APC surface; such binding occurs according to
rules that are only now being studied (these receptors are not
immunoglobulin family members and appear to represent several
families of proteins and glycoproteins capable of binding different
classes of extracellular proteins/glycoproteins). This type of
binding is followed by receptor-mediated internalization, also in a
vesicle. Because this mechanism is poorly understood at present,
elements of antigen design cannot be incorporated in a rational
design process. However, application of stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly methods, an empirical
approach of selection of variant DNA molecules most successful at
entry into APC, can select for variants that are improved
throughout this mechanism.
[0676] The other three mechanisms all relate to specific antibody
recognition of the extracellular antigen. The first mechanism
involves immunoglobulin-mediated recognition of the specific
antigen via IgG that is bound to Fc receptors on the cell surface.
APC such as monocytes, macrophages and dendritic cells can be
decorated with surface membrane IgG of diverse specificities. In a
primary response, this mechanism will not be operative. In
previously immunized animals, IgG on the surface of APC can
specifically bind extracellular antigen and mediate uptake of the
bound antigen into an intracellular endosomal compartment. Another
mechanism involves binding to clonally-derived surface membrane
immunoglobulin which is present on each B cells (IgM in the case of
primary B cells and IgG when the animal has been previously exposed
to the antigen). B cells are efficient APC. Extracellular antigen
can bind specifically to surface Ig and be internalized and
processed in a membrane compartment for presentation on the B cell
surface. Finally, extracellular antigen can be recognized by
specific soluble immunoglobulin (IgM in the case of a primary
immunization and IgG in the previously immunized animals).
Complexing with Ig will elicit binding to the surface of APC (via
Fc receptor recognition in the case of IgG) and
internalization.
[0677] In each of these latter three mechanisms, the extent to
which the conformation of the antigen is the same as the
recognition specificity of the pre-existing antibody is critical to
the efficiency of the process of antigen presentation. Antibodies
can recognize linear protein epitopes as well as conformational
epitopes determined by the three dimensional structure of the
protein antigen. Protective antibodies that will recognize an
extracellular virus or bacterial pathogen and by binding to its
surface prevent infection or mediate its immune destruction
(complement mediated lysis, immune complex formation and
phagocytosis) are almost exclusively generated against
conformational determinants on the proteins with native structure
displayed on the surface of the pathogen. Hence, it is imperative
for generation of host protective humoral immunity, to have those
naive B cells which bear antibody specific for conformational
epitopes present on the pathogen be stimulated by direct contact
with T helper cells after intracellular processing of the antigen
and presentation of degradation peptides in the context of MHC
Class II. This T help will allow selective proliferation of the
relevant B cells with consequent mutation of antibody and antigen
driven selection for antibodies with increased specificity, as well
as antibody class switching.
[0678] To summarize, optimal uptake of antigen by APC to elicit
humoral immunity, as well as specific CD4.sup.+ cytotoxic T cells,
requires that the antigen be in native protein conformation (as
presented subsequently to the immune system upon natural infection)
and recognized by naive B cells bearing the appropriate membrane
antibody. Native protein conformation includes appropriate protein
folding, glycosylation and any other post-translational
modifications necessary for optimal reactivity with the receptors
(immunoglobulin and possibly non-immunoglobulin) on APC. In
addition to the three dimensional structure of the expressed
antigen required for recognition by specific antibody and
elicitation of the required immune responses, the structure (and
sequence) can be optimized for increased protein stability outside
the expressing cell, until the time when it is recognized by immune
cells, including APCs. The reassembly (&/or one or more
additional directed evolution methods described herein) and
screening methods of the invention can be used to optimize the
antigen structure (and sequence) for subsequent processing after
uptake by APC so that intracellular processing results in
derivation of the required peptide fragments for presentation on
Class I or Class II on APC and desired immune responses.
[0679] (f) Optimal Partitioning of the Nascent Antigen into the
Desired Subcellular Compartment or Compartments
[0680] This can be directed by signal and trafficking signals
embodied in the antigen sequence. It may be desirable for all of
the antigen to be secreted from these cells; alternatively, all or
part of the antigen could be directed to be expressed on the cell
surface of these factory cells. Signals to direct vesicles
containing the antigen to other subcellular compartments for
post-translational modifications, including glycosylation, can be
embodied in the antigen sequence.
[0681] (g) Optimal Display of the Antigen on the Cell Surface or
Optimal Release of the Antigen from the Cells
[0682] A variation on items (f) and (g) is to design the expression
of the antigen within the cytoplasm of the factory cell followed by
lysis of that cell to release soluble antigen. Cell death can be
engineered by expression on the same genetic vaccine vector of an
intracellular protein that will elicit apoptosis. In this case, the
timing of cell death is balanced with the need for the cell to
produce antigen, as well as the potential deleterious effect of
killing some cells in a designed process.
[0683] In combination, items (a)-(h) lead to a variety of scenarios
for the optimizing the longevity and extent of antigen expression.
It is not always desirable that the antigen be expressed for the
longest time at the highest level. In certain clinical
applications, it will be important to have antigen expression that
is short time-low expression, short time-high expression, long
time-low expression, long time-high expression or somewhere in
between.
[0684] Plasmid AR can be designed to express one or more variants
of a single antigen gene or several quite different targets for
immunization. Methods for obtaining optimized antigens for use in
genetic vaccines are described in ???. Multiple antigens can be
expressed from a monocistronic or multicistronic form of the
vector.
2.4.2. VECTOR COMPONENTS "CTL-DC", "CTL-LC" AND "CTL-MM", DESIGNED
FOR OPTIMAL PRODUCTION OF CTLs
[0685] Genetic vector components "CTL-DC", "CTL-LC" and "CTL-MM"
are designed to direct optimal production of cytotoxic CD8.sup.+
lymphocytes (CTLs) by dendritic cells (CTL-DC), Langerhan's cells
(CTL-LC), and monocytes and macrophages (CTL-MM) These vector
components direct presentation of optimal antigen fragments in
association with MHC Class I, thereby ensuring maximal cytotoxic T
cell immune responses. Cells transfected with CTL vector components
can be considered as the direct activators of this arm of specific
immunity that is usually critically important for protection
against viral diseases.
[0686] CTL vector components are typically designed to have one or
more of the following properties, each of which can be optimized
using the stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide reassembly
methods of the invention:
[0687] (a) Optimal Vector Binding to, and Uptake by, the Chosen
Antigen Presenting Cells (e.g., Dendritic Cells,
Monocytes/macrophages, Langerhan's Cells)
[0688] This is a critical property to differentiate CTL series
vectors from other vectors in the multicomponent DNA vaccine. CTL
series vectors preferably do not bind to or enter cells that are
chosen to be the extracellular antigen expression host via AR
vectors. This separation of functions is critical, as the
intracellular fate and trafficking of antigen destined for
stimulation of immune cells after release from an antigen
expressing cell is quite different than the fate of antigen
destined to be presented on the cell surface in association with
MHC Class I. In the former case, antigen is directed via a signal
secretion sequence to be delivered intact to the lumen of the rough
endoplasmic reticulum (RER) and then secreted. In the latter case,
antigen is directed to remain in the cytoplasm and there be
degraded into peptide fragments by the proteasomal system followed
by delivery to the lumen of the RER for association with MHC Class
I. These complexes of peptide and MHC Class I are then delivered to
the cell surface for specific interaction with CD8.sup.+ cytotoxic
T cells. Vector components, and methods for obtaining optimized
vector components, that are optimized for targeting to desired cell
types are described in
[0689] Optimizing Transcription of the Antigen Gene(s)
[0690] This can be accomplished by optimizing promoters, enhancers,
introns, and the like, as discussed herein. Cell specific promoters
are valuable in such vectors as an additional level of
selectivity.
[0691] (b) Optimal Longevity of the mRNA
[0692] Optimal 3' and 5' non-translated regions of the mRNA can be
obtained using the methods of the invention.
[0693] (c) Optimal Translation of the mRNA
[0694] Again, the stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide reassembly
and selection methods of the invention can be used to obtain
polynucleotide sequences for optimal ribosome binding and assembly
of translational machinery, as well as optimal codon
preference.
[0695] (d) Optimal Protein Conformation
[0696] In this case, the optimal protein conformation yields
appropriate cytoplasmic proteolysis and production of the correct
peptides for presentation on MHC Class I and elicitation of the
desired specific CTL responses, rather than a conformation that
will interact with specific antibody or other receptors on the
surface of APC.
[0697] (e) Optimal Proteolysis to Generate the Correct Peptides
[0698] The order of specific proteolytic cleavages will depend on
the nature of protein folding and the nature of proteases either in
the cytoplasm or in the proteasome.
[0699] (f) Optimal Transport of the Antigen Peptides Across the
Endoplasmic Reticulum Membrane to be Delivered into the RER
Lumen
[0700] This may be mediated by recognition of the peptides by TAP
proteins or by other membrane transporters.
[0701] (h) Optimal Association of the Peptides with the Class l-2
Microglobulin Complex and Trafficking to the Cell Surface via the
Secretory Pathway
[0702] (i) Optimal Display of the MHC-peptide Complex with
Associated accessory molecules for recognition by Specific CTL
[0703] Vector CTL can be designed to express one or more variants
of a single antigen gene or several different targets for
immunization. Multiple optimized antigens can be expressed from a
monocistronic or multicistronic form of the vector.
2.4.3. VECTORS "M" DESIGNED FOR OPTIMAL RELEASE OF IMMUNE
MODULATORS
[0704] Vectors "M" are designed to direct optimal release of immune
modulators, such as cytokines and other growth factors, from target
cells. Target cells can be either the predominant cell type in the
immunized tissue or immune cells such dendritic cells (M-DC),
Langerhan's cells (M-LC), monocytes & macrophages (M-MM)".
These vectors direct simultaneous expression of optimal levels of
several immune cell "modulators" (cytokines, growth factors, and
the like) such that the immune response is of the desired type, or
combination of types, and of the desired level. Cells transfected
with M vectors can be considered as the directors of the nature of
the vaccine immune response (CTL vs T.sub.H1 vs T.sub.H2 vs NK
cell, etc.) and its magnitude. The properties of these vectors
reflect the nature of the cell in which the vectors are designed to
operate. For example, the vectors are designed to bind to and enter
the desired cell type, and/or can have cell-specific regulated
promoters that drive transcription in the desired cell type. The
vectors can also be engineered to direct maximal synthesis and
release of the cell modulator proteins from the target cells in the
desired ratio.
[0705] "M" genetic vaccine vectors are typically designed to have
one or more of the following properties, each of which can be
optimized using the stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide reassembly
methods of the invention:
[0706] (a) Optimal Vector Binding to and Uptake by the Chosen
Modulator Expressing Cell
[0707] Suitable expressing cells include, for example, muscle
cells, epithelial cells or other dominant (by number) cell types in
the target tissue, antigen presenting cells (e.g. dendritic cells,
monocytes/macrophages, Langerhans cells). This is a critical
property which differentiates M series vectors from those designed
to bind to and enter other cells.
[0708] (b) Optimal Transcription of the Immune Modulator
Gene(s)
[0709] Again, promoters, enhancers, introns, and the like can be
optimized according to the methods of the invention. Cell specific
promoters are very valuable here as an additional level of
selectivity.
[0710] (c) Optimal Longevity of the mRNA
[0711] Optimal 3' and 5' non-translated regions of the mRNA can be
obtained using the methods of the invention.
[0712] (d) Optimal Translation of the mRNA
[0713] Again, the stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide reassembly
and selection methods of the invention can be used to obtain
polynucleotide sequences for optimal ribosome binding and assembly
of translational machinery, as well as optimal codon
preference.
[0714] (e) Optimal Trafficking of the Modulator into the Lumen of
the RER (via a Signal Secretion Sequence)
[0715] An alternative strategy for modulation of the immune
response uses membrane anchored modulators rather than secretion of
soluble modulator. Anchored modulator can be retained on the
surface of the synthesizing cell by, for example, a hydrophobic
tail and phosphoinositol glycan linkage.
[0716] (f) Optimal Protein Conformation for Each Modulator
[0717] In this case, the optimal protein conformation is that which
allows extracellular modulator and/or cell membrane anchored
modulator to interact with the relevant receptor.
[0718] (g) the Ratio of Modulators and their Type can be Determined
Empirically
[0719] One will test sets of modulators that are known to work in
concert to direct the immune response in the direction of a T.sub.H
response (e.g., production of IL-2 and/or IFN) or T.sub.H2 response
(e.g., IL-4, IL-5, IL-13), for example. Vector M can be designed to
express one or more modulators. Optimized immunomodulators, and
methods for obtaining optimized immunomodulators, are described in
copending, commonly assigned U.S. patent application Ser. No.,
filed Feb. 10, 1999 as TTC Attorney Docket No.1 8907-0303US, which
is entitled "Optimization of Immunomodulatory Molecules." These
optimized immunomodulatory sequences are particularly suitable for
use as components of the multicomponent genetic vaccines of the
invention. Multiple modulators can be expressed from a
monocistronic or multicistronic form of the vector.
2.4.4. VECTORS "CK", DESIGNED TO DIRECT RELEASE OF CHEMOKINES
[0720] Genetic vaccine vectors designated "CK" are designed to
direct optimal release of chemokines from target cells. Target
cells can be either the predominant cell type in the immunized
tissue, or can be immune cells such as dendritic cells (CK-DC),
Langerhan's cells (CK-LC), or monocytes and macrophages (CK-MM).
These vectors typically direct simultaneous expression of optimal
levels of several chemokines such that the recruitment of immune
cells to the site of immunization is optimal. Cells transfected
with CK vectors can be considered as the traffic police, regulating
the immune cells critical for the vaccine immune response. The
properties of these vectors reflect the nature of the cell in which
the vectors are designed to operate. For example, the vectors are
designed to bind to and enter the desired cell type, and/or can
have cell-specific regulated promoters that drive transcription in
the desired cell type. The vectors are also engineered to direct
maximal synthesis and release of the chemokines from the target
cells in the desired ratio. Genetic vaccine components, and methods
for obtaining components, that provide optimal release of
chemokines are described in ???
[0721] CK vectors are typically designed to have one or more of the
following properties, each of which can be optimized using the
stochastic (e.g. polynucleotide shuffling & interrupted
synthesis) and non-stochastic polynucleotide reassembly methods of
the invention:
[0722] (a) Optimal Vector Binding to and Uptake by the Chosen
Chemokine Expressing Cell
[0723] Suitable cells include, for example, muscle cells,
epithelial cells, or cell types that are dominant (by number) in
the particular tissue of interest. Also suitable are antigen
presenting cells (e.g. dendritic cells, monocytes and macrophages,
Langerhans cells). This is a critical property which differentiates
CK series vectors from those designed to bind to and enter other
cells.
[0724] (b) Optimal Transcription of the Chemokine Gene(s)
[0725] Again, promoters, enhancers, introns, and the like can be
optimized according to the methods of the invention.
[0726] Cell specific promoters are very valuable here as an
additional level of selectivity.
[0727] (c) Optimal Longevity of the mRNA
[0728] Optimal 3' and 5' non-translated regions of the mRNA can be
obtained using the methods of the invention.
[0729] (d) Optimal Translation of the mRNA
[0730] Again, the stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide reassembly
and selection methods of the invention can be used to obtain
polynucleotide sequences for optimal ribosome binding and assembly
of translational machinery, as well as optimal codon
preference.
[0731] (e) Optimal Trafficking of the Chemokine into the Lumen of
the RER (via a Signal Secretion Sequence)
[0732] An alternative strategy for modulation of the immune
response via recruitment of cells will use membrane anchored
chemokine rather than secretion of soluble chemokine.
[0733] Anchored chemokine will be retained on the surface of the
synthesizing cell by a hydrophobic tail and phosphoinositol glycan
linkage.
[0734] (f) Optimal Protein Conformation for Each Chemokine
[0735] In this case, the optimal protein conformation is that which
allows extracellular chemokine/cell membrane anchored chemokine to
interact with the relevant receptor.
[0736] (g) the Ratio of Diverse Chemokines can be Determined
Empirically
[0737] One can test sets of chemokines that are known to work in
concert to direct recruitment of CTL, T.sub.H cells, B cells,
monocytes/macrophages, eosinophils, and/or neutrophils as
appropriate.
[0738] Vector CK can be designed to express one or more chemokines.
Multiple chemokines can be expressed from a monocistronic or
multicistronic form of the vector.
2.4.5. OTHER VECTORS
[0739] Genetic vaccines which contain one or more additional
component vector moieties are also provided by the invention. For
example, the genetic vaccine can include a vector that is designed
to specifically enter dendritic cells and Langerhans cells, and
will migrate to the draining lymph nodes.
[0740] This Vector is Designed to Provide for Expression of the
Target Antigen(s), as well as a Cocktail of Cytokines and
Chemokines Relevant to Elicitation of the Desired Immune Response
in the Node
[0741] Depending on the clinical goals and nature of the antigen,
the vector can be optimized for relatively long lived expression of
the target antigen so that stimulation of the immune system is
prolonged at the node. Another example is a vector that
specifically modulates MHC expression in B cells. Such vectors are
designed to specifically bind to and enter B cells, cells either
resident in the injection site or attracted into the site. Within
the B cell, this vector directs the association of antigen peptides
derived from specific uptake of antigen into the endocytic
compartment of the cell to either association with Class I or Class
II, hence directing the elicitation of specific immunity via
CD4.sup.+ T helper cells or CD8.sup.+ cytotoxic lymphocytes.
Numerous means exist for this intracellular direction of the fate
of processed peptide that are discussed herein.
[0742] Examples of molecules that direct Class I presentation
include tapasin, TAP-1 and TAP-2 (Koopman et al. (1997) Curr. Opin.
Immunol. 9: 80-88), and those affecting Class II presentation
include, for example, endosomal/lysosomal proteases (Peters (1997)
Curr. Opin. Immunol. 9: 89-96). Genetic vaccine components, and
methods for obtaining components, that provide optimized Class I
presentation are described in commonly assigned, copending U.S.
patent application Ser. No. 5 filed Feb. 10, 1999 as TTC Attorney
Docket No. 18097-0303US, entitled "Optimization of Immunomodulatory
Molecules." An optimal DNA vaccine could, for example, combine an
AR vector (antigen release), a CTL-DC vector (CTL activation via
dendritic cell presentation of antigen peptide on MHC Class I), an
M-MM vector for release of IL-12 and IFNg from resident tissue
macrophages, and a CK vector for recruitment of T.sub.H cells into
the immunization site.
[0743] Directed Evolution Aid the Following DNA Vaccination
Goals
[0744] DNA vaccination can be used for diverse goals that can
include the following, among others:
[0745] stimulation of a CTL response and/or humoral response ready
to react rapidly and aggressively against an invading bacterial or
viral pathogen at some time in the distant future
[0746] a continuous but non-aggressive response to prevent
inappropriate responses to allergens
[0747] a continuous non-aggressive and tolerization of immunity to
an autoantigen in autoimmune disease
[0748] elicitation of an aggressive CTL response as rapidly as
possible against tumor cell antigens
[0749] redirection of the immune response away from a strong but
inappropriate immune response to an on-going chronic infection in
the direction of desired responses to clear the pathogen and/or
prevent pathology.
[0750] These goals cannot always be met by the format of a single
vector DNA vaccine, particularly wherein competing goals are
embodied within one DNA sequence. A multicomponent format allows
the generation of a portfolio of DNA vaccine vectors, some of which
will be reconstructed on each occasion (e.g., those vectors
containing antigen) while others will be used as well characterized
and understood reagents for numerous different clinical
applications (e.g., the same chemokine-expressing vector can be
used in different situations).
2.5. SCREENING METHODS
[0751] Screening Assay Varies Depending of Property for which
Improvement is Sought
[0752] Recombinant nucleic acid libraries that are obtained by the
methods described herein are screened to identify those DNA
segments that have a property which is desirable for genetic
vaccination. The particular screening assay employed will vary, as
described below, depending on the particular property for which
improvement is sought. Typically, the experimentally evolved (e.g.
by polynucleotide reassembly &/or polynucleotide
site-saturation mutagenesis) nucleic acid library is introduced
into cells prior to screening. If the stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly format employed is an in
vivo format, the library of recombinant DNA segments generated
already exists in a cell. If the sequence reassembly (&/or one
or more additional directed evolution methods described herein) is
performed in vitro, the recombinant library is preferably
introduced into the desired cell type before screening/selection.
The members of the recombinant library can be linked to an episome
or virus before introduction or can be introduced directly.
[0753] Cell Types
[0754] A wide variety of cell types can be used as a recipient of
evolved genes. Cells of particular interest include many bacterial
cell types that are used to deliver vaccines or vaccine antigens
(Courvalin et al.(1995) C. R. Acad. Sci. 11118: 1207-12), both
gram-negative and gram-positive, such as salmonella (Attridge et
al. (1997) Vaccine 15: 155-62), clostridium. (Fox et al. (1996)
Gene Ther. 3: 173-8), lactobacillus, shigella (Sizemore et al.
(1995) Science 270: 299-302), E. coli, streptococcus (Oggioni and
Pozzi (1996) Gene 169: 85-90), as well as mammalian cells,
including human cells. In some embodiments of the invention, the
library is amplified in a first host, and is then recovered from
that host and introduced to a second host more amenable to
expression, selection, or screening, or any other desirable
parameter. The manner in which the library is introduced into the
cell type depends on the DNA-uptake characteristics of the cell
type, e.g., having viral receptors, being capable of conjugation,
or being naturally competent. If the cell type is unsusceptible to
natural and chemical-induced competence, but susceptible to
electroporation, one would usually employ electroporation. If the
cell type is unsusceptible to electroporation as well, one can
employ biolistics. The biolistic PDS-1000 Gene Gun (Biorad,
Hercules, Calif.) uses helium pressure to accelerate DNA-coated
gold or tungsten microcarriers toward target cells.
[0755] Competent or Potentially Competent Tissue
[0756] The process is applicable to a wide range of tissues,
including plants, bacteria, fungi, algae, intact animal tissues,
tissue culture cells, and animal embryos. One can employ electronic
pulse delivery, which is essentially a mild electroporation format
for live tissues in animals and patients (Zhao, Advanced Drug
Delivery Reviews 17:257-262 (1995)). Novel methods for making cells
competent are described in International Patent Application
PCT/US97/04494 (Publ. No. WO97/35957). After introduction of the
library of recombinant DNA genes, the cells are optionally
propagated to allow expression of genes to occur.
[0757] Identifying Cells that Contain a Vector Through Inclusion of
a Selectable Marker Gene
[0758] In many assays, a means for identifying cells that contain a
particular vector is necessary. Genetic vaccine vectors of all
kinds can include a selectable marker gene. Under selective
conditions, only those cells that express the selectable marker
will survive.
[0759] Examples of Selectable Marker Genes
[0760] Examples of suitable markers include, the dihydrofolate
reductase gene (DHFR), the thymidine kinase gene (TK), or
prokaryotic genes conferring drug resistance, gpt (xanthine-guanine
phosphoribosyltransfera- se, which can be selected for with
mycophenolic acid; neo (neomycin phosphotransferase), which can be
selected for with G418, hygromycin, or puromycin; and DHFR
(dihydrofolate reductase), which can be selected for with
methotrexate (Mulligan Southern & Berg (1982) J Mol. Appl.
Genet. 1: 327).
[0761] Identifying Cells that Contain a Vector Through Inclusion of
a Screenable Marker Gene
[0762] As an alternative to, or in addition to, a selectable
marker, a genetic vaccine vector can include a screenable marker
which, when expressed, confers upon a cell containing the vector a
readily identifiable phenotype. For example, gene that encodes a
cell surface antigen that is not normally present on the host cell
is suitable. The detection means can be, for example, an antibody
or other ligand which specifically binds to the cell surface
antigen. Examples of suitable cell surface antigens include any CD
(cluster of differentiation) antigen (CD1 to CD163) from a species
other than that of the host cell which is not recognized by
host-specific antibodies. Other examples include green fluorescent
protein (GFP, see, e.g., Chalfie et al. (1994) Science 263:802-805;
Crameri et al. (1996) Nature Biotechnol. 14: 315-319; Chalfie et
al. (1995) Photochem. Photobiol. 62:651-656; Olson et al. (1995) J
Cell. Biol. 130:639-650) and related antigens, several of which are
commercially available.
2.5.1. SCREENING FOR VECTOR LONGEVITY OR TRANSLOCATION TO DESIRED
TISSUE
[0763] For certain applications, it is desirable to identify those
vectors with the greatest longevity as DNA, or to identify vectors
which end up in tissues distant from the injection site. This can
be accomplished by administering to an animal a population of
recombinant genetic vaccine vectors by the chosen route of
administration and, at various times thereafter excise the target
tissue and recover vector from the tissue by standard molecular
biology procedures. The recovered vector molecules can be amplified
in, for example, E. coli and/ or by PCR in vitro. The PCR
amplification can involve further polynucleotide (e.g. gene,
promoter, enhancer, intron, & the like) reassembly (optionally
in combination with other directed evolution methods described
herein), after which the derived selected population used for
readministration to animals and further improvement of the vector.
After several rounds of this procedure, the selected vectors can be
tested for their capacity to express the antigen in the correct
conformation under the same conditions as the vector was selected
in vivo.
[0764] Methods for in vitro Identification of Cells Expressing the
Desired Antigen
[0765] Because antigen expression is not part of the selection or
screening process described above, not all vectors obtained are
capable of expressing the desired antigen. To overcome this
drawback, the invention provides methods for identifying those
vectors in a genetic vaccine population that exhibit not only the
desired tissue localization and longevity of DNA integrity in vivo,
but retention of maximal antigen expression (or expression of other
genes such as cytokines, chemokines, cell surface accessory
molecules, MHC, and the like).
[0766] The methods involve in vitro identification of cells which
express the desired molecule using cells purified from the tissue
of choice, under conditions that allow recovery of very small
numbers of cells and quantitative selection of those with different
levels of antigen expression as desired.
[0767] Two embodiments of the invention are described, each of
which uses a library of genetic vaccine vectors as the starting
point. The goal of each method is to identify those vectors that
exhibit the desired biological properties in vivo. The recombinant
library represents a population of vectors that differ in known
ways (e.g., a combinatorial vector library of different functional
modules), or has randomly generated diversity generated either by
insertion of random nucleotide stretches, or has been
experimentally evolved (e.g. by polynucleotide reassembly &/or
polynucleotide site-saturation mutagenesis) in vitro to introduce
low level mutations across all or part of the vector.
2.5.1.1.SELECTION FOR EXPRESSION OF CELL SURFACE-LOCALIZED
ANTIGEN
[0768] In a first embodiment, the invention method involves
selection for expression of cell surface-localized antigen. The
antigen gene is engineered in the vaccine vector library such that
it has a region of amino acids which is targeted to the cell
membrane. For example, the region can encode a hydrophobic stretch
of C-terminal amino acids which signals the attachment of a
phosphoinositol-glycan (PIG) terminus on the expressed protein and
directs the protein to be expressed on the surface of the
transfected cell. With an antigen that is naturally a soluble
protein, this method will likely not affect the three dimensional
folding of the protein in this engineered fusion with a new
C-terminus. With an antigen that is naturally a transmembrane
protein (e.g., a surface membrane protein on pathogenic viruses,
bacteria, protozoa or tumor cells) there are at least two
possibilities. First, the extracellular domain can be engineered to
be in fusion with the C-terminal sequence for signaling
PIG-linkage. Second, the protein can be expressed in toto relying
on the signaling of the host cell to direct it efficiently to the
cell surface. In a minority of cases, the antigen for expression
will have an endogenous PIG terminal linkage (e.g., some antigens
of pathogenic protozoa).
[0769] Collection, Purification, Identification and Separation of
Target Cells
[0770] The vector library is delivered in vivo and, after a
suitable interval of time tissue and/or cells from diverse target
sites in the animal are collected. Cells can be purified from the
tissue using standard cell biological procedures, including the use
of cell specific surface reactive monoclonal antibodies as affinity
reagents. It is relatively facile to purify isolated epithelial
cells from mucosal sites where epithelium may have been inoculated
or myoblasts from muscle. In some embodiments, minimal physical
purification is performed prior to analysis. It is sometimes
desirable to identify and separate specific cell populations from
various tissues, such as spleen, liver, bone marrow, lymph node,
and blood. Blood cells can be fractionated readily by FACS to
separate B cells, CD4.sup.+ or CD8.sup.+ T cells, dendritic cells,
Langerhans cells, monocytes, and the like, using diverse
fluorescent monoclonal antibody reagents.
[0771] Identification and Purification of Cells Expressing the
Antigen
[0772] Those cells expressing the antigen can be identified with a
fluorescent monoclonal antibody specific for the C-terminal
sequence on PIG-linked forms of the surface antigen. FACS analysis
allows quantitative assessment of the level of expression of the
correct form of the antigen on the cell population. Cells
expressing the maximal level of antigen are sorted and standard
molecular biology methods used to recover the plasmid DNA vaccine
vector that conferred this reactivity. An alternative procedure
that allows purification of all those cells expressing the antigen
(and that may be useful prior to loading onto a cell sorter since
antigen expressing cells may be a very small minority population),
is to rosette or pan-purify the cells expressing surface antigen.
Rosettes can be formed between antigen expressing cells and
erythrocytes bearing covalently coupled antibody to the relevant
antigen. These are readily purified by unit gravity sedimentation.
Panning of the cell population over petri dishes bearing
immobilized monoclonal antibody specific for the relevant antigen
can also be used to remove unwanted cells.
[0773] Cells expressing the required conformational structure of
the target antigen can be identified using specific
conformationally-dependen- t monoclonal antibodies that are known
to react specifically with the same structure as expressed on the
target pathogen.
[0774] Using Several Monoclonal Antibodies in the Selection Process
to Minimize the Possibility of an Antigen which Reacts with High
Affinity to the Diagnostic Antibody but does not Yield the Correct
Conformation
[0775] Because one monoclonal antibody cannot define all aspects of
correct folding of the target antigen, one can minimize the
possibility of an antigen which reacts with high affinity to the
diagnostic antibody but does not yield the correct conformation as
defined by that in which the antigen is found on the surface of the
target pathogen or as secreted from the target pathogen. One way to
minimize this possibility is to use several monoclonal antibodies,
each known to react with different conformational epitopes in the
correctly folded protein, in the selection process. This can be
achieved by secondary FACS sorting for example.
[0776] The enriched plasmid population that successfully expressed
sufficient of the antigen in the correct body site for the desired
time is then used as the starting population for another round of
selection, incorporating gene reassembling (optionally in
combination with other directed evolution methods described herein)
to expand the diversity. In this manner, one recovers the desired
biological activity encoded by plasmid from tissues in DNA
vaccine-immunized animals.
[0777] This method can also provide the best in vivo selected
vectors that express immune accessory molecules that one may wish
to incorporate into DNA vaccine constructs. For example, if it is
desired to express the accessory protein B7.1 or B7.2 in antigen-
presenting-cells (APC) (to promote successful presentation of
antigen to T cells) one can sort APC isolated from different
tissues (at or different to the inoculation site) using
commercially available monoclonal antibodies that recognize
functional B7 proteins.
2.5.1.2.SELECTION FOR EXPRESSION OF SECRETED
ANTIGEN/CYTOKINE/CHEMOKINE
[0778] Select Vectors that are Optimal in Inducing Secretion of
Soluble Proteins that can Affect the Qualitative and Quantitative
Nature of an Elicited Immune Response in vivo
[0779] The invention also provides methods to identify plasmids in
a genetic vaccine vector population that are optimal in secretion
of soluble proteins that can affect the qualitative and
quantitative nature of an elicited immune response. For example,
the methods are useful for selecting vectors that are optimal for
secretion of particular cytokines, growth factors and chemokines.
The goal of the selection is to determine which particular
combinations of cytokines, chemokines and growth factors, in
combination with different promoters, enhancers, polyA tracts,
introns, and the like, elicits the required immune response in
vivo.
[0780] Genes Encoding the Polypeptides are Typically Present in the
Vaccine Vector Library in Combination with Optimal Signal Secretion
Sequences (Proteins are Secreted from the Cells.)
[0781] Combinations of the genes for the soluble proteins of
interest can be present in the vectors; transcription can be either
from a single promoter, or the genes can be placed in
multicistronic arrangements. Typically, the genes encoding the
polypeptides are present in the vaccine vector library in
combination with optimal signal secretion sequences, such that the
expressed proteins are secreted from the cells.
[0782] Generating Vectors Capable of Secreting Different
Combinations of Soluble Factors in vitro and Capable of Expressing
those Factors for Desired Lengths of Time
[0783] The first step in these methods is to generate vectors that
are capable of secreting high (or in some case low) levels of
different combinations of soluble factors in vitro and that will
express those factors for a short or long time as desired. This
method allows one to select for and retain an inventory of plasmids
which can be characterized by known patterns of soluble protein
expression in known tissues for a known time. These vectors can
then be tested individually for in vivo efficacy, after being
placed in combination with the genetic vaccine antigen in an
appropriate expression construct.
[0784] Delivery of Vector Library and Subsequent Collection,
Testing, and Purification using FACS Sorting, Affinity Panning,
Resetting, or Magnetic Bead Separation to Separate Cell Populations
Prior to Identification
[0785] The vector library is delivered to a test animal and, after
a chosen interval of time, tissue and/or cells from diverse sites
on the animal are collected. Cells are purified from the tissue
using standard cell biological procedures, which often include the
use of cell specific surface reactive monoclonal antibodies as
affinity reagents. As is the case for cell surface antigens
described above, physical purification of separate cell populations
can be performed prior to identification of cells which express the
desired protein. For these studies, the target cells for expression
of cytokines will most usually be APC or B cells or T cells rather
than muscle cells or epithelial cells. In such cases FACS sorting
by established methods will be preferred to separate the different
cell types. The different cell types described above may also be
separated into relatively pure fractions using affinity panning,
resetting or magnetic bead separation with panels of existing
monoclonal antibodies known to define the surface membrane
phenotype of murine immune cells.
[0786] Identifying and Selecting Purified Cells through Visual
Inspection or Flow Cytometry for use in Another Round of Selection
Incorporating Gene Reassembling (Optionally in Combination with
other Directed Evolution Methods Described Herein) to Expand the
Diversity
[0787] Purified cells are plated onto agar plates under conditions
that maintain cell viability. Cells expressing the required
conformational structure of the target antigen are identified using
conformationally-dependent monoclonal antibodies that are known to
react specifically with the same structure as expressed on the
target pathogen. Release of the relevant soluble protein from the
cells is detected by incubation with monoclonal antibody, followed
by a secondary reagent that gives a macroscopic signal (gold
deposition, color development, fluorescence, luminescence). Cells
expressing the maximal level of antigen can be identified by visual
inspection, the cell or cell colony picked and standard molecular
biology methods used to recover the plasmid DNA vaccine vector that
conferred this reactivity. Alternatively, flow cytometry can be
used to identify and select cells harboring plasmids that induce
high levels of gene expression. The enriched plasmid population
that successfully expressed sufficient of the soluble factor in the
correct body site for the desired time is then used as the starting
population for another round of selection, incorporating gene
reassembling (optionally in combination with other directed
evolution methods described herein) to expand the diversity, if
further improvement is desired. In this manner, one recovers the
desired biological activity encoded by plasmid from tissues in DNA
vaccine-immunized animals.
[0788] Using Monoclonal Antibody to Confirm that the Initial
Results from Screening still Hold when Several Conformational
Epitopes are Probed
[0789] Several monoclonal antibodies, each known to react with
different conformational epitopes in the correctly folded cytokine,
chemokine or growth factor, can be used to confirm that the initial
results from screening with one monoclonal antibody reagent still
hold when several conformational epitopes are probed. In some cases
the primary probe for functional cytokine released from the
cell/cell colony in agar could be a soluble domain of the cognate
receptor.
2.5.2. FLOW CYTOMETRY
[0790] Most of the Vector Module Libraries can be Assayed by Flow
Cytometry to Select Individual Human Tissue Culture Cells that
Contain the Experimentally Generated Nucleic Acid Sequences that
have the Greatest Improvement in the Desired Property
[0791] Flow cytometry provides a means to efficiently analyze the
functional properties of millions of individual cells. The cells
are passed through an illumination zone, where they are hit by a
laser beam; the scattered light and fluorescence is analyzed by
computer-linked detectors. Flow cytometry provides several
advantages over other methods of analyzing cell populations.
Thousands of cells can be analyzed per second, with a high degree
of accuracy and sensitivity. Gating of cell populations allows
multiparameter analysis of each sample. Cell size, viability, and
morphology can be analyzed without the need for staining. When dyes
and labeled antibodies are used, one can analyze DNA content, cell
surface and intracytoplasmic proteins, and identify cell type,
activation state, cell cycle stage, and detect apoptosis. Up to
four colors (thus, four separate antigens stained with different
fluorescent labels) and light scatter characteristics can be
analyzed simultaneously (four colors requires two-laser instrument;
one-laser instrument can analyze three colors). The expression
levels of several genes can be analyzed simultaneously, and
importantly, flow cytometry-based cell sorting ("FACS sorting")
allows selection of cells with desired phenotypes. Most of the
vector module libraries, including the promoter, enhancer, intron,
episomal origin of replication, expression level aspect of antigen,
bacterial origin and bacterial marker, can be assayed by flow
cytometry to select individual human tissue culture cells that
contain the reassembled (&/or subjected to one or more directed
evolution methods described herein) nucleic acid sequences that
have the greatest improvement in the desired property. Typically
the selection is for high level expression of a surface antigen or
surrogate marker protein, as diagrammed in FIG. 4. The pool of the
best individual sequences is recovered from the cells selected by
flow cytometry-based sorting. An advantage of this approach is that
very large numbers (>10.sup.7) can be evaluated in a single vial
experiment.
2.5.3. ADDITIONAL IN VITRO SCREENING METHODS
[0792] Screening for Improved Vaccination Properties using Various
in vitro Testing Methods such as Screening for Improved Adjuvant
Activity and Immunostimulatory Properties
[0793] Genetic vaccine vectors and vector modules can be screened
for improved vaccination properties using various in vitro testing
methods that are known to those of skill in the art. For example,
the optimized genetic vaccines can be tested for their effect on
induction of proliferation of the particular lymphocyte type of
interest, e.g., B cells, T cells, T cell lines, and T cell clones.
This type of screening for improved adjuvant activity and
immunostimulatory properties can be performed using, for example,
human or mouse cells.
[0794] Screening for Improved Vaccination Properties using Various
in vitro Testing Methods such as Screening for Cytokine Production
(ELISA and/or Cytoplasmic Cytokine Staining and Flow Cytometry) or
for Alterations in the Capacity of the Vectors to Direct T.sub.H1/
T.sub.H2 Differentiation
[0795] A library of genetic vaccine vectors, e.g. obtained either
from polynucleotide reassembly (optionally in combination with
other directed evolution methods described herein), or of vectors
harboring genes encoding cytokines, costimulatory molecules etc.)
can be screened for cytokine production (e.g., IL-2, IL-4, IL-5,
IL-6, IL-10, IL-12, IL-13, IL-15, IFN-, TNF-) by B cells, T cells,
monocytes/macrophages, total human PBMC, or (diluted) whole blood.
Cytokines can be measured by ELISA or and cytoplasmic cytokine
staining and flow cytometry (single-cell analysis). Based on the
cytokine production profile, one can screen for alterations in the
capacity of the vectors to direct T.sub.H1/ T.sub.H2
differentiation (as evidenced, for example, by changes in ratios of
IL-4/IFN-, IL-4/IL-2, IL-5/IFN-, IL-5/IL-2, IL-I 3/ IFN-,
IL-13/IL-2). Induction of APC activation can be detected based on
changes in surface expression levels of activation antigens, such
as B7-1 (CD80), 137-2 (CD86), MHC class I and II, CD14, CD23, and
Fc receptors, and the like.
[0796] Analyzing Genetic Vaccine Vectors for their Capacity to
Induce T Cell Activation Through Isolating Spleen Cell of Infected
Mice and Studying the Capacity of Cytotoxic T Lymphocytes to Lyse
Infected, Autologous Target Cells
[0797] In some embodiments, genetic vaccine vectors are analyzed
for their capacity to induce T cell activation. More specifically,
spleen cells from injected mice can be isolated and the capacity of
cytotoxic T lymphocytes to lyse infected, autologous target cells
is studied. The spleen cells are reactivated with the specific
antigen in vitro. In addition, T helper cell differentiation is
analyzed by measuring proliferation or production of T.sub.H1 (IL-2
and IFN-) and T.sub.H2 (IL-4 and IL-5) cytokines by ELISA and
directly in CD4.sup.+ T cells by cytoplasmic cytokine staining and
flow cytometry.
[0798] Testing for Ability to Induce Humoral Immune Responses with
Assays using, for Example, Peripheral B Lymphocytes from Immunized
Individuals or other Assays Involving Detection of Antigen
Expression by the Target Cells
[0799] Genetic vaccines and vaccine components can also be tested
for ability to induce humoral immune responses, as evidenced, for
example, by induction of B cell production of antibodies specific
for an antigen of interest. These assays can be conducted using,
for example, peripheral B lymphocytes from immunized individuals.
Such assay methods are known to those of skill in the art. Other
assays involve detection of antigen expression by the target cells.
For example, FACS selection provides the most efficient method of
identifying cells which produce a desired antigen on the cell
surface. Another advantage of FACS selection is that one can sort
for different levels of expression; sometimes lower expression may
be desired. Another method involves panning using monoclonal
antibodies on a plate. This method allows large numbers of cells to
be handled in a short time, but the method only selects for highest
expression levels. Capture by magnetic beads coated with monoclonal
antibodies provides another method of identifying cells which
express a particular antigen.
[0800] Screening for Ability to Inhibit Proliferation of Tumor Cell
Lines in vitro
[0801] Genetic vaccines and vaccine components that are directed
against cancer cells can be screened for their ability to inhibit
proliferation of tumor cell lines in vitro. Such assays are known
in the art. An indication of the efficacy of a genetic vaccine
against, for example, cancer or an autoimmune disorder, is the
degree of skin inflammation when the vector is injected into the
skin of a patient or test animal. Strong inflammation is correlated
with strong activation of antigen-specific T cells. Improved
activation of tumor-specific T cells may lead to enhanced killing
of the tumors. In case of autoantigens, one can add
immunomodulators that skew the responses towards T.sub.H2. Skin
biopsies can be taken, enabling detailed studies of the type of
immune response that occurs at the sites of each injection (in mice
large numbers of injections/vectors can be analyzed) Other suitable
screening methods can involve detection of changes in expression of
cytokines, chemokines, accessory molecules, and the like, by cells
upon challenge by a library of genetic vaccine vectors.
[0802] Expressing the Recombinant Peptides or Polypeptides as
Fusions with a Protein Displayed on the Surface of a Replicable
Genetic Package
[0803] Various screening methods for particular applications are
described herein. In several instances, screening involves
expressing the recombinant peptides or polypeptides encoded by the
experimentally generated polynucleotides of the library as fusions
with a protein that is displayed on the surface of a replicable
genetic package. For example, phage display can be used. See, e.g.,
Cwirla et al., Proc. Natl. Acad. Sci. USA 87: 6378-6382 (1990);
Devlin et al., Science 249: 404-406 (1990), Scott Ladner et al.,
U.S. Pat. No. 5,571,698. Other replicable genetic packages include,
for example, bacteria, eukaryotic viruses, yeast, and spores.
[0804] Purification and in vitro Analysis of Recombinant Nucleic
Acids and Polypeptides
[0805] Once stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and/or non-stochastic polynucleotide
reassembly has been performed, the resulting library of
experimentally generated polynucleotides can be subjected to
purification and preliminary analysis in vitro, in order to
identify the most promising candidate recombinant nucleic acids.
Advantageously, the assays can be practiced in a high-throughput
format. For example, to purify individual experimentally evolved
(e.g. by polynucleotide reassembly &/or polynucleotide
site-saturation mutagenesis) recombinant antigens, clones can
robotically picked into 96-well formats, grown, and, if desired,
frozen for storage.
[0806] Whole cell lysates (V-antigen), periplasmic extracts, or
culture supernatants (toxins) can be assayed directly by ELISA as
described below, but high throughput purification is sometimes also
needed. Affinity chromatography using immobilized antibodies or
incorporation of a small nonimmunogenic affinity tag such as a
hexahistidine peptide with immobilized metal affinity
chromatography will allow rapid protein purification. High
binding-capacity reagents with 96-well filter bottom plates provide
a high throughput purification process. The scale of culture and
purification will depend on protein yield, but initial studies will
require less than 50 micrograms of protein. Antigens showing
improved properties can be purified in larger scale by FPLC for
re-assay and animal challenge studies.
[0807] In some embodiments, the experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis) antigen-encoding polynucleotides are assayed as
genetic vaccines. Genetic vaccine vectors containing the
experimentally evolved (e.g. by polynucleotide reassembly &/or
polynucleotide site-saturation mutagenesis) antigen sequences can
be prepared using robotic colony picking and subsequent robotic
plasmid purification. Robotic plasmid purification protocols are
available that allow purification of 600-800 plasmids per day. The
quantity and purity of the DNA can also be analyzed in 96-well
plates, for example. In a presently preferred embodiment, the
amount of DNA in each sample is robotically normalized, which can
significantly reduce the variation between different batches of
vectors.
[0808] Once the proteins and/or nucleic acids are picked and
purified as desired, they can be subjected to any of a number of in
vitro analysis methods. Such screenings include, for example, phage
display, flow cytometry, and ELISA assays to identify antigens that
are efficiently expressed and have multiple epitopes and a proper
folding pattern. In the case of bacterial toxins, the libraries may
also be screened for reduced toxicity in mammalian cells.
[0809] As one example, to identify recombinant antigens that are
cross-reactive, one can use a panel of monoclonal antibodies for
screening. A humoral immune response generally targets multiple
regions of antigenic proteins. Accordingly, monoclonal antibodies
can be raised against various regions of immunogenic proteins
(Alving et al. (1995) Immunol. Rev. 145: 5). In addition, there are
several examples of monoclonal antibodies that only recognize one
strain of a given pathogen, and by definition, different serotypes
of pathogens are recognized by different sets of antibodies. For
example, a panel of monoclonal antibodies have been raised against
VEE envelope proteins, thus providing a means to recognize
different subtypes of the virus (Roehrig and Bolin (1997) J Clin.
Microbiol. 35: 1887). Such antibodies, combined with phage display
and ELISA screening, can be used to enrich recombinant antigens
that have epitopes from multiple pathogen strains. Flow cytometry
based cell sorting will further allow for the selection of variants
that are most efficiently expressed.
[0810] Phage display provides a powerful method for selecting
proteins of interest from large libraries (Bass et al. (1990)
Proteins: Struct. Funct. Genet. 8: 309; Lowman and Wells (1991)
Methods: A Companion to Methods Enz. 3(3);205-216. Lowman and Wells
(1993) J Mol. Biol. 234;564-578). Some recent reviews on the phage
display technique include, for example, McGregor (1996) Mol
Biotechnol. 6(2):15 5-62; Dunn (1996) Curr. Opin. Biotechnol.
7(5):547-53; Hill et al. (1996) Mol Microbiol 20(4):685-92; Phage
Display of Peptides and Proteins: A Laboratory Manual. B K. Kay, J.
Winter, J, McCafferty eds., Academic Press 1996; O'Neil et al.
(1995) Curr. Opin. Struct. Biol. 5(4):443-9; Phizicky et al. (1995)
Microbiol Rev. 59(l):94-123; Clackson et al. (1994) Trends
Biotechnol. 12(5):173-84; Felici et al. (1995) Biotechnol. Annu.
Rev. 1: 149-83; Burton (1995) Immunotechnology 1(2):87-94.) See,
also, Cwirla et al., Proc. Natl. Acad Sci. USA 87: 6378-6382
(1990); Devlin et al., Science 249: 404-406 (1990), Scott &
Smith, Science 249: 386-388 (1990); Ladner et al., U.S. Pat. No.
5,571,698. Each phage particle displays a unique variant protein on
its surface and packages the gene encoding that particular variant.
The experimentally evolved (e.g. by polynucleotide reassembly
&/or polynucleotide site-saturation mutagenesis) genes for the
antigens are fused to a protein that is expressed on the phage
surface, e.g., gene III of phage M 13, and cloned into phagemid
vectors. In a presently preferred embodiment, a suppressible stop
codon (e.g., an amber stop codon) separates the genes so that in a
suppressing strain of E. coli, the antigen-gIIIp fusion is produced
and becomes incorporated into phage particles upon infection with M
13 helper phage. The same vector can direct production of the
unfused antigen alone in a nonsuppressing E. coli for protein
purification.
[0811] Most Frequently Used Genetic Packages for Display
Libraries
[0812] The genetic packages most frequently used for display
libraries are bacteriophage, particularly filamentous phage, and
especially phage M13, Fd and F1. Most work has involved inserting
libraries encoding polypeptides to be displayed into either gIII or
gVIII of these phage forming a fusion protein. See, e.g., Dower, WO
91/19818; Devlin, WO 91/18989; MacCafferty, WO 92/01047 (gene III);
Huse, WO 92/06204; Kang, WO 92/18619 (gene VIII). Such a fusion
protein comprises a signal sequence, usually but not necessarily,
from the phage coat protein, a polypeptide to be displayed and
either the gene III or gene VIII protein or a fragment thereof.
Exogenous coding sequences are often inserted at or near the
N-terminus of gene III or gene VIII although other insertion sites
are possible.
[0813] Use of Eukaryotic Viruses to Display Polypeptides
[0814] Eukaryotic viruses can be used to display polypeptides in an
analogous manner. For example, display of human heregulin fused to
gp70 of Moloney murine leukemia virus has been reported by Han et
al., Proc. Natl. Acad. Sci. USA 92: 9747-9751 (1995). Spores can
also be used as replicable genetic packages. In this case,
polypeptides are displayed from the outer surface of the spore. For
example, spores from B. subtilis have been reported to be suitable.
Sequences of coat proteins of these spores are provided by Donovan
et al., J. Mol. Biol. 196, 1-10 (1987). Cells can also be used as
replicable genetic packages. Polypeptides to be displayed are
inserted into a gene encoding a cell protein that is expressed on
the cells surface. Bacterial cells including Salmonella
typhimurium, Bacillus subtilis, Pseudomonas aeruginosa, Vibrio
cholerae, Klebsiella pneumonia, Neisseria gonorrhoeae, Neisseria
meningitidis, Bacteroides nodosus, Moraxella bovis, and especially
Escherichia coli are preferred. Details of outer surface proteins
are discussed by Ladner et al., U.S. Pat. No. 5,571,698 and
references cited therein. For example, the lamB, protein of E. coli
is suitable.
[0815] Establishment of a Physical Association Between Polypeptides
and Their Genetic Material
[0816] A basic concept of display methods that use phage or other
replicable genetic package is the establishment of a physical
association between DNA encoding a polypeptide to be screened and
the polypeptide. This physical association is provided by the
replicable genetic package, which displays a polypeptide as part of
a capsid enclosing the genome of the phage or other package,
wherein the polypeptide is encoded by the genome. The establishment
of a physical association between polypeptides and their genetic
material allows simultaneous mass screening of very large numbers
of phage bearing different polypeptides. Phage displaying a
polypeptide with affinity to a target, e.g., a receptor, bind to
the target and these phage are enriched by affinity screening to
the target. The identity of polypeptides displayed from these phage
can be determined from their respective genomes.
[0817] Using these methods a polypeptide identified as having a
binding affinity for a desired target can then be synthesized in
bulk by conventional means, or the polynucleotide that encodes the
peptide or polypeptide can be used as part of a genetic
vaccine.
[0818] Variants with specific binding properties, in this case
binding to family-specific antibodies, are easily enriched by
panning with immobilized antibodies. Antibodies specific for a
single family are used in each round of panning to rapidly select
variants that have multiple epitopes from the antigen families. For
example, A-family specific antibodies can be used to select those
experimentally evolved (e.g. by polynucleotide reassembly &/or
polynucleotide site-saturation mutagenesis) clones that display
A-specific epitopes in the first round of panning. A second round
of panning with B-specific antibodies will select from the "A"
clones those that display both A- and B-specific epitopes. A third
round of panning with C-specific antibodies will select for
variants with A, B, and C epitopes. A continual selection exists
during this process for clones that express well in E. coli and
that are stable throughout the selection. Improvements in factors
such as transcription, translation, secretion, folding and
stability are often observed and will enhance the utility of
selected clones for use in vaccine production.
[0819] Phage ELISA methods can be used to rapidly characterize
individual variants. These assays provide a rapid method for
quantitation of variants without requiring purification of each
protein. Individual clones are arrayed into 96-well plates, gown,
and frozen for storage. Cells in duplicate plates are infected with
helper phage, grown overnight and pelleted by centrifugation. The
supernatants containing phage displaying particular variants are
incubated with immobilized antibodies and bound clones are detected
by anti- M13 antibody conjugates. Titration series of phage
particles, immobilized antigen, and/or soluble antigen competition
binding studies are all highly effective means to quantitate
protein binding. Variant antigens displaying multiple epitopes will
be further studied in appropriate animal challenge models.
[0820] Several groups have reported an in vitro ribosome display
system for the screening and selection of mutant proteins with
desired properties from large libraries. This technique can be used
similarly to phage display to select or enrich for variant antigens
with improved properties such as broad cross reactivity to
antibodies and improved folding (see, e.g., Hanes et al. (1997)
Proc. Nat'l. A cad. Sci. USA 94(10):493 7-42; Mattheakis et al.
(1994) Proc. Nat 7. Acad. Sci. USA 91(19):9022-6; He et al. (1997)
Nucl. Acids Res. (24):5132-4; Nemoto et al. (1997) FEBS Lett.
414(2):405-8).
[0821] Other display methods exist to screen antigens for improved
properties such as increased expression levels, broad cross
reactivity, enhanced folding and stability. These include, but are
not limited to display of proteins on intact E. coli or other cells
(e.g., Francisco et al. (1993) Proc. Nat'l. Acad. Sci. USA 90:
1044-10448; Lu et al. (1995) BiolTechnology 13: 366-372). Fusions
of experimentally evolved (e.g. by polynucleotide reassembly
&/or polynucleotide site-saturation mutagenesis) antigens to
DNA-binding proteins can link the antigen protein to its gene in an
expression vector (Schatz et al. (1996) Methods Enzymol. 267:
171-91; Gates et al. (1996) J Mol. Biol. 255: 373-86.) The various
display methods and ELISA assays can be used to screen for
experimentally evolved (e.g. by polynucleotide reassembly &/or
polynucleotide site-saturation mutagenesis) antigens with improved
properties such as presentation of multiple epitopes, improved
immunogenicity, increased expression levels, increased folding
rates and efficiency, increased stability to factors such as
temperature, buffers, solvents, improved purification properties,
etc. Selection of experimentally evolved (e.g. by polynucleotide
reassembly &/or polynucleotide site-saturation mutagenesis)
antigens with improved expression, folding, stability and
purification profile under a variety of chromatographic conditions
can be very important improvements to incorporate for the vaccine
manufacturing process.
[0822] To identify recombinant antigenic polypeptides that exhibit
improved expression in a host cell, flow cytometry is a useful
technique.
[0823] Flow cytometry provides a method to efficiently analyze the
functional properties of millions of individual cells. One can
analyze the expression levels of several genes simultaneously, and
flow cytometry-based cell sorting allows for the selection of cells
that display properly expressed antigen variants on the cell
surface or in the cytoplasm. Very large numbers (>10.sup.7) of
cells can be evaluated in a single vial experiment, and the pool of
the best individual sequences can be recovered from the sorted
cells. These methods are particularly useful in the case of, for
example, Hantaan virus glycoproteins, which are generally very
poorly expressed in mammalian cells. This approach provides a
general solution to improve expression levels of pathogen antigens
in mammalian cells, a phenomenon that is critical for the function
of genetic vaccines.
[0824] To use flow cytometry to analyze polypeptides that are not
expressed on the cell surface, one can engineer the experimentally
generated polynucleotides in the library such that the
polynucleotide is expressed as a fusion protein that has a region
of amino acids which is targeted to the cell membrane. For example,
the region can encode a hydrophobic stretch of C-terminal amino
acids which signals the attachment of a phosphoinositol-glycan
(PIG) terminus on the expressed protein and directs the protein to
be expressed on the surface of the transfected cell (Whitehorn et
al. (1995) Biotechnology (N Y) 13:1215-9). With an antigen that is
naturally a soluble protein, this method will likely not affect the
three dimensional folding of the protein in this engineered fusion
with a new C-terminus. With an antigen that is naturally a
transmembrane protein (e.g., a surface membrane protein on
pathogenic viruses, bacteria, protozoa or tumor cells) there are at
least two possibilities.
[0825] First, the extracellular domain can be engineered to be in
fusion with the C-terminal sequence for signaling PIG-linkage.
Second, the protein can be expressed in toto relying on the
signaling of the host cell to direct it efficiently to the cell
surface. In a minority of cases, the antigen for expression will
have an endogenous PIG terminal linkage (e.g., some antigens of
pathogenic protozoa).
[0826] Those cells expressing the antigen can be identified with a
fluorescent monoclonal antibody specific for the C-terminal
sequence on PIG-linked forms of the surface antigen. FACS analysis
allows quantitative assessment of the level of expression of the
correct form of the antigen on the cell population. Cells
expressing the maximal level of antigen are sorted and standard
molecular biology methods are used to recover the plasmid DNA
vaccine vector that conferred this reactivity. An alternative
procedure that allows purification of all those cells expressing
the antigen (and that may be useful prior to loading onto a cell
sorter since antigen expressing cells may be a very small minority
population), is to rosette or pan-purify the cells expressing
surface antigen. Rosettes can be formed between antigen expressing
cells and erythrocytes bearing covalently coupled antibody to the
relevant antigen. These are readily purified by unit gravity
sedimentation. Panning of the cell population over petri dishes
bearing immobilized monoclonal antibody specific for the relevant
antigen can also be used to remove unwanted cells.
[0827] In the high throughput assays of the invention, it is
possible to screen up to several thousand different experimentally
evolved (e.g. by polynucleotide reassembly &/or polynucleotide
site-saturation mutagenesis) variants in a single day. For example,
each well of a microtiter plate can be used to run a separate
assay, or, if concentration or incubation time effects are to be
observed, every 5-10 wells can test a single variant. Thus, a
single standard microtiter plate can assay about 100 (e.g., 96)
reactions. If 1536 well plates are used, then a single plate can
easily assay from about 100 to about 1500 different reactions. It
is possible to assay several different plates per day; assay
screens for up to about 6,000-20,000 different assays (i.e.,
involving different nucleic acids, encoded proteins,
concentrations, etc.) is possible using the integrated systems of
the invention. More recently, microfluidic approaches to reagent
manipulation have been developed, e.g., by Caliper Technologies
(Palo Alto, Calif.).
[0828] In one aspect, library members, e.g., cells, viral plaques,
or the like, are separated on solid media to produce individual
colonies (or plaques). Using an automated colony picker (e.g., the
Q-bot, Genetix, U.K.), colonies or plaques are identified, picked,
and up to 10,000 different mutants inoculated into 96 well
microtiter dishes, optionally containing glass balls in the wells
to prevent aggregation. The Q-bot does not pick an entire colony
but rather inserts a pin through the center of the colony and exits
with a small sampling of cells (or viruses in plaque applications).
The time the pin is in the colony, the number of dips to inoculate
the culture medium, and the time the pin is in that medium each
effect inoculum size, and each can be controlled and optimized. The
uniform process of the Q-bot decreases human handling error and
increases the rate of establishing cultures (roughly 10,000/4
hours). These cultures are then shaken in a temperature and
humidity controlled incubator. The glass balls in the microtiter
plates act to promote uniform aeration of cells dispersal of cells,
or the like, similar to the blades of a fermentor. Clones from
cultures of interest can be cloned by limiting dilution. Plaques or
cells constituting libraries can also be screened directly for
production of proteins, either by detecting hybridization, protein
activity, protein binding to antibodies, or the like.
[0829] The ability to detect a subtle increase in the performance
of a experimentally evolved (e.g. by polynucleotide reassembly
&/or polynucleotide site-saturation mutagenesis) library member
over that of a parent strain relies on the sensitivity of the
assay. The chance of finding the organisms having an improvement in
ability to induce an immune response is increased by the number of
individual mutants that can be screened by the assay. To increase
the chances of identifying a pool of sufficient size, a prescreen
that increases the number of mutants processed by I 0-fold can be
used. The goal of the prescreen will be to quickly identify mutants
having equal or better product titers than the parent strain(s) and
to move only these mutants forward to liquid cell culture for
subsequent analysis.
[0830] A number of well known robotic systems have also been
developed for solution phase chemistries useful in assay systems.
These systems include automated workstations like the automated
synthesis apparatus developed by Takeda Chemical Industries, LTD.
(Osaka, Japan) and many robotic systems utilizing robotic arms
(Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca,
Hewlett-Packard, Palo Alto, Calif.) which mimic the manual
synthetic operations performed by a scientist. Any of the above
devices are suitable for use with the present invention, e.g., for
high- throughput screening of molecules encoded by codon-altered
nucleic acids. The nature and implementation of modifications to
these devices (if any) so that they can operate as discussed herein
with reference to the integrated system will be apparent to persons
skilled in the relevant art.
[0831] High throughput screening systems are commercially available
(see, e.g., Zymark Corp., Hopkinton, Mass.; Air Technical
Industries, Mentor, Ohio; Beckman Instruments, Inc. Fullerton,
Calif.; Precision Systems, Inc., Natick, Mass., etc.). These
systems typically automate entire procedures including all sample
and reagent pipetting, liquid dispensing, timed incubations, and
final readings of the microplate in detector(s) appropriate for the
assay. These configurable systems provide high throughput and rapid
start up as well as a high degree of flexibility and
customization.
[0832] The manufacturers of such systems provide detailed protocols
the various high throughput. Thus, for example, Zymark Corp.
provides technical bulletins describing screening systems for
detecting the modulation of gene transcription, ligand binding, and
the like. Microfluidic approaches to reagent manipulation have also
been developed, e.g., by Caliper Technologies (Palo Alto,
Calif.).
[0833] Optical images viewed (and, optionally, recorded) by a
camera or other recording device (e.g., a photodiode and data
storage device) are optionally further processed in any of the
embodiments herein, e.g., by digitizing the image and/or storing
and analyzing the image on a computer. As noted above, in some
applications, the signals resulting from assays are florescent,
making optical detection approaches appropriate in these instances.
A variety of commercially available peripheral equipment and
software is available for digitizing, storing and analyzing a
digitized video or digitized optical image, e.g., using PC (Intel
.times.86 or Pentium chip-compatible DOS, OS2 WINDOWS, WINDOWS NT
or VIMOWS95 based machines), MACINTOSH, or LTNIX based (e.g., SLJN
work station) computers.
[0834] One conventional system carries light from the assay device
to a cooled charge-coupled device (CCD) camera, in common use in
the art. A CCD camera includes an array of picture elements
(pixels). The light from the specimen is imaged on the CCD.
Particular pixels corresponding to regions of the specimen (e.g.,
individual hybridization sites on an array of biological polymers)
are sampled to obtain light intensity readings for each position.
Multiple pixels are processed in parallel to increase speed. The
apparatus and methods of the invention are easily used for viewing
any sample, e.g., by fluorescent or dark field microscopic
techniques.
[0835] Integrated systems for analysis in the present invention
typically include a digital computer with high-throughput liquid
control software, image analysis software, data interpretation
software, a robotic liquid control armature for transferring
solutions from a source to a destination operably linked to the
digital computer, an input device (e.g., a computer keyboard) for
entering data to the digital computer to control high throughput
liquid transfer by the robotic liquid control armature and,
optionally, an image scanner for digitizing label signals from
labeled assay component. The image scanner interfaces with the
image analysis software to provide a measurement of optical
intensity. Typically, the intensity measurement is interpreted by
the data interpretation software to show whether the optimized
recombinant antigenic polypeptide products are produced.
2.5.4. ANTIGEN LIBRARY IMMUNIZATION
[0836] In a presently preferred embodiment, antigen library
immunization (ALI) is used to identify optimized recombinant
antigens that have improved immunogenicity. ALI involves
introduction of the library of recombinant antigen-encoding nucleic
acids, or the recombinant antigens encoded by the experimentally
evolved (e.g. by polynucleotide reassembly &/or polynucleotide
site-saturation mutagenesis) nucleic acids, into a test animal. The
animals are then subjected to in vivo challenge using live
pathogens. Neutralizing antibodies and cross-protective immune
responses are studied after immunization with the entire libraries,
pools and/or individual antigen variants.
[0837] Methods of immunizing test animals are well known to those
of skill in the art. In presently preferred embodiments, test
animals are immunized twice or three times at two week intervals.
One week after the last immunization, the animals are challenged
with live pathogens (or mixtures of pathogens), and the survival
and symptoms of the animals is followed. Immunizations using test
animal challenge are described in, for example, Roggenkamp et al.
(1997) Infect. Immun. 65: 446; Woody et al. (1997) Vaccine 2: 133;
Agren et al. (1997) J Immunol. 158: 3936; Konishi et al. (1992)
Virology 190: 454; Kinney et al. (1988) J Virol. 62: 4697;
Iacono-Connors et al. (1996) Virus Res. 43: 125; Kochel et al.
(1997) Vaccine 15: 547; and Chu et al. (1995) J Virol. 69:
6417.
[0838] The immunizations can be performed by injecting either the
experimentally generated polynucleotides themselves, i.e., as a
genetic vaccine, or by immunizing the animals with polypeptides
encoded by the experimentally generated polynucleotides. Bacterial
antigens are typically screened primarily as recombinant proteins,
whereas viral antigens are preferably analyzed using genetic
vaccinations.
[0839] To dramatically reduce the number of experiments required to
identify individual antigens having improved immunogenic
properties, one can use pooling and deconvolution, as diagrammed in
FIG. 6. Pools of recombinant nucleic acids, or polypeptides encoded
by the recombinant nucleic acids, are used to immunize test
animals. Those pools that result in protection against pathogen
challenge are then subdivided and subjected to additional analysis.
The high throughput in vitro approaches described above can be used
to identify the best candidate sequences for the in vivo
studies.
[0840] The challenge models that can be used to screen for
protective antigens include pathogen and toxin models, such as
Yersinia bacteria, bacterial toxins (such as Staphylococcal and
Streptococcal enterotoxins, E. coli/V. cholerae enterotoxins),
Venezuelan equine encephalitis virus (VEE), Flaviviruses (Japanese
encephalitis virus, Tick-borne encephalitis virus, Dengue virus),
Hantaan virus, Herpes simplex, influenza virus (e.g., Influenza A
virus), Vesicular Steatites Virus, Pseudomonas aeruginosa,
Salmonella typhimurium, Escherichia coli, Klebsiella pneumoniae,
Toxoplasma gondii, Plasmodium yoeliii, Herpes simplex, influenza
virus (e.g., Influenza A virus), and Vesicular Steatites Virus.
However, the test animals can also be challenged with tumor cells
to enable screening of antigens that efficiently protect against
malignancies. Individual experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis) antigens or pools of antigens are introduced into the
animals intradermally, intramuscularly, intravenously,
intratracheally, anally, vaginally, orally, or intraperitoneally
and antigens that can prevent the disease are chosen, when desired,
for further rounds of reassembly (optionally in combination with
other directed evolution methods described herein) and selection.
Eventually, the most potent antigens, based on in vivo data in test
animals and comparative in vitro studies in animals and man, are
chosen for human trials, and their capacity to prevent and treat
human diseases is investigated.
[0841] In some embodiments, antigen library immunization and
pooling of individual clones is used to immunize against a pathogen
strain that was not included in the sequences that were used to
generate the library. The level of crossprotection provided by
different strains of a given pathogen can significantly. However,
homologous titer is always higher than heterologous titer. Pooling
and deconvolution is especially efficient in models where minimal
protection is provided by the wild-type antigens used as starting
material for reassembly (optionally in combination with other
directed evolution methods described herein) (for example minimal
protection by antigens A and B against strain C in FIG. 3B). This
approach can be taken, for example, when evolving the V-antigen of
Yersinae or Hantaan virus glycoproteins.
[0842] In some embodiments, the desired screening involves analysis
of the immune response based on immunological assays known to those
skilled in the art. Typically, the test animals are first immunized
and blood or tissue samples are collected for example one to two
weeks after the last immunization. These studies enable one to one
can measure immune parameters that correlate to protective
immunity, such as induction of specific antibodies (particularly
IgG) and induction of specific T lymphocyte responses, in addition
to determining whether an antigen or pools of antigens provides
protective immunity.
[0843] Spleen cells or peripheral blood mononuclear cells can be
isolated from immunized test animals and measured for the presence
of antigen-specific T cells and induction of cytokine synthesis.
ELISA, ELISPOT and cytoplasmic cytokine staining, combined with
flow cytometry, can provide such information on a single-cell
level.
[0844] Common immunological tests that can be used to identify the
efficacy of immunization include antibody measurements,
neutralization assays and analysis of activation levels or
frequencies of antigen presenting cells or lymphocytes that are
specific for the antigen or pathogen. The test animals that can be
used in such studies include, but are not limited to, mice, rats,
guinea pigs, hamsters, rabbits, cats, dogs, pigs and monkeys.
[0845] Monkey is a particularly useful test animal because the MHC
molecules of monkeys and humans are very similar. Virus
neutralization assays are useful for detection of antibodies that
not only specifically bind to the pathogen, but also neutralize the
function of the virus. These assays are typically based on
detection of antibodies in the sera of immunized animal and
analysis of these antibodies for their capacity to inhibit viral
growth in tissue culture cells. Such assays are known to those
skilled in the art. One example of a virus neutralization assay is
described by Dolin R (J. Infect. Dis. 1995, 172:1175-83). Virus
neutralization assays provide means to screen for antigens that
also provide protective immunity.
[0846] In some embodiments, experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis) antigens are screened for their capacity to induce T
cell activation in vivo. More specifically, peripheral blood
mononuclear cells or spleen cells from injected mice can be
isolated and the capacity of cytotoxic T lymphocytes to lyse
infected, autologous target cells is studied. The spleen cells can
be reactivated with the specific antigen in vitro. In addition, T
helper cell activation and differentiation is analyzed by measuring
cell proliferation or production of T.sub.H (IL-2 and IFN-) and
T.sub.H2 (IL-4 and IL-5) cytokines by ELISA and directly in CD4+ T
cells by cytoplasmic cytokine staining and flow cytometry. Based on
the cytokine production profile, one can also screen for
alterations in the capacity of the antigens to direct
T.sub.H1/T.sub.H2 differentiation (as evidenced, for example, by
changes in ratios of IL-4/IFN-, IL-4/IL-2, IL-5/IFN-, IL-5/IL-2,
IL-13/IFN-, IL-I 3/IL-2). The analysis of the T cell activation
induced by the antigen variants is a very useful screening method,
because potent activation of specific T cells in vivo correlates to
induction of protective immunity.
[0847] The frequency of antigen-specific CD8+ T cells in vivo can
also be directly analyzed using tetramers of MHC class I molecules
expressing specific peptides derived from the corresponding
pathogen antigens (Ogg and McMichael, Curr. Opin. Immunol. 1998,
10:393-6; Altman et al., Science 1996, 274:94-6). The binding of
the tetramers can be detected using flow cytometry, and will
provide information about the efficacy of the experimentally
evolved (e.g. by polynucleotide reassembly &/or polynucleotide
site-saturation mutagenesis) antigens to induce activation of
specific T cells. For example, flow cytometry and tetramer
stainings provide an efficient method of identifying T cells that
are specific to a given antigen or peptide. Another method involves
panning using plates coated with tetramers with the specific
peptides. This method allows large numbers of cells to be handled
in a short time, but the method only selects for highest expression
levels. The higher the frequency of antigen-specific T cells in
vivo is, the more efficient the immunization has been, enabling
identification of the antigen variants that have the most potent
capacity to induce protective immune responses. These studies are
particularly useful when conducted in monkeys, or other primates,
because the MHC class I molecules of humans mimic those of other
primates more closely than those of mice.
[0848] Measurement of the activation of antigen presenting cells
(APC) in response to immunization by antigen variants is another
useful screening method. Induction of APC activation can be
detected based on changes in surface expression levels of
activation antigens, such as 137-1 (CD80). 137-2 (CD86), MHC class
I and 11, CD14, CD23, and Fc receptors, and the like.
[0849] Experimentally evolved (e.g. by polynucleotide reassembly
&/or polynucleotide site-saturation mutagenesis) cancer
antigens that induce cytotoxic T cells that have the capacity to
kill cancer cells can be identified by measuring the capacity of T
cells derived from immunized animals to kill cancer cells in vitro.
Typically the cancer cells are first labeled with radioactive
isotopes and the release of radioactivity is an indication of tumor
cell killing after incubation in the presence of T cells from
immunized animals. Such cytotoxicity assays are known in the
art.
[0850] An indication of the efficacy of an antigen to activate T
cells specific for, for example, cancer antigens, allergens or
autoantigens, is also the degree of skin inflammation when the
antigen is injected into the skin of a patient or test animal.
Strong inflammation is correlated with strong activation of
antigen-specific T cells. Improved activation of tumor-specific T
cells may lead to enhanced killing of the tumors. In case of
autoantigens, one can add immunomodulators that skew the responses
towards T.sub.H2, whereas in the case of allergens a T.sub.H1
response is desired. Skin biopsies can be taken, enabling detailed
studies of the type of immune response that occurs at the sites of
each injection (in mice and monkeys large numbers of
injections/antigens can be analyzed). Such studies include
detection of changes in expression of cytokines, chemokines,
accessory molecules, and the like, by cells upon injection of the
antigen into the skin.
[0851] To screen for antigens that have optimal capacity to
activate antigen-specific T cells, peripheral blood mononuclear
cells from previously infected or immunized humans individuals can
be used. This is a particularly useful method, because the MHC
molecules that will present the antigenic peptides are human MHC
molecules. Peripheral blood mononuclear cells or purified
professional antigen-presenting cells (APCs) can be isolated from
previously vaccinated or infected individuals or from patients with
acute infection with the pathogen of interest. Because these
individuals have increased frequencies of pathogen-specific T cells
in circulation, antigens expressed in PBMCs or purified APCs of
these individuals will induce proliferation and cytokine production
by antigen-specific CD4.sup.+ and CD8.sup.+ T cells. Thus, antigens
that simultaneously harbor epitopes from several antigens can be
recognized by their capacity to stimulate T cells from various
patients infected or immunized with different pathogen antigens,
cancer antigens, autoantigens or allergens. One buffy coat derived
from a blood donor contains lymphocytes from 0.5 liters of blood,
and up to 10.sup.4 PBMC can be obtained, enabling very large
screening experiments using T cells from one donor.
[0852] When healthy vaccinated individuals (lab volunteers) are
studied, one can make EBV-transformed B cell lines from these
individuals. These cell lines can be used as antigen presenting
cells in subsequent experiments using blood from the same donor;
this reduces interassay and donor-to-donor variation. In addition,
one can make antigen-specific T cell clones, after which antigen
variants are introduced to EBV transformed B cells. The efficiency
with which the transformed B cells induce proliferation of the
specific T cell clones is then studied. When working with specific
T cell clones, the proliferation and cytokine synthesis responses
are significantly higher than when using total PBMCs, because the
frequency of antigen-specific T cells among PBMC is very low.
[0853] CTL epitopes can be presented by most cells types since the
class I major histocompatibility complex (MHC) surface
glycoproteins are widely expressed. Therefore, transfection of
cells in culture by libraries of experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis) antigen sequences in appropriate expression vectors
can lead to class I epitope presentation. If specific CTLs directed
to a given epitope have been isolated from an individual, then the
co-culture of the transfected presenting cells and the CTLs can
lead to release by the CTLs of cytokines, such as IL-2, IFN-, or
TNF, if the epitope is presented. Higher amounts of released TNF
will correspond to more efficient processing and presentation of
the class I epitope from the experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis), evolved sequence. Experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis) antigens that induce cytotoxic T cells that have the
capacity to kill infected cells can also be identified by measuring
the capacity of T cells derived from immunized animals to kill
infected cells in vitro. Typically the target cells are first
labeled with radioactive isotopes and the release of radioactivity
is an indication of target cell killing after incubation in the
presence of T cells from immunized animals. Such cytotoxicity
assays are known in the art.
[0854] A second method for identifying optimized CTL epitopes does
not require the isolation of CTLs reacting with the epitope. In
this approach, cells expressing class I MHC surface glycoproteins
are transfected with the library of evolved sequences as above.
After suitable incubation to allow for processing and presentation,
a detergent soluble extract is prepared from each cell culture and
after a partial purification of the MHC-epitope complex (perhaps
optional) the products are submitted to mass spectrometry
(Henderson et al. (1993) Proc. Nat'l. Acad. Sci. USA 90:
10275-10279). Since the sequence is known of the epitope whose
presentation to be increased, one can calibrate the mass
spectrogram to identify this peptide. In addition, a cellular
protein can be used for internal calibration to obtain a
quantitative result; the cellular protein used for internal
calibration could be the MHC molecule itself. Thus one can measure
the amount of peptide epitope bound as a proportion of the N4HC
molecules.
2.5.5. SCREENINGFOR OPTIMAL INDUCTION OF PROTECTIVE IMMUNITY
[0855] Vectors that can Provide Efficient, Protective Immunity are
Selected using Lethal Infection Models to Choose Vectors that can
Prevent the Disease for Further Rounds of Reassembly (Optionally in
Combination with other Directed Evolution Methods Described Herein)
and Selection
[0856] To select genetic vaccine vectors that provide efficient
protective immunity, one can screen the vector libraries in a test
mammal using lethal infection models, such as Pseudomonas
aeruginosa, Salmonella typhimurium, Escherichia coli, Klebsiella
pneumoniae, Toxoplasma gondii, Plasmodium yoeliii, Herpes simplex,
influenza virus (e.g., Influenza A virus), and Vesicular Steatites
Virus. Pools of genetic vaccine vectors or individual vectors are
introduced into the animals intradermally, intramuscularly,
intravenously, intratracheally, anally, vaginally, orally, or
intraperitoneally and vectors that can prevent the disease are
chosen for further rounds of reassembly (optionally in combination
with other directed evolution methods described herein) and
selection.
[0857] Examples: Anti-IL-4 mAbs or Recombinant IL-12; Recombinant
IL-12 (Advantage of Latter Model is that Infection Occurs Through
Lung, Common Route of Human Pathogen Invasion)
[0858] As an example, optimal vectors can be screened in mice
infected with Leishmania major parasites. When injected into
footpads of BALB/c mice, these parasites cause a progressive
infection later resulting in a disseminated disease with fatal
outcome, which can be prevented by anti-IL-4 mAbs or recombinant
IL-12 (Chatelain et al. (1992) J. Immunol. 148: 1182-1187). Pools
of plasmids can be injected intravenously, intraperitoneally or
into footpads of these mice, and pools that can prevent the disease
are chosen for further analysis and screened for vectors that can
cure existing infections. The size of the footpad swelling can be
followed visually providing simple yet precise monitoring of the
disease progression. Mice can be infected intratracheally with
Klebsiella pneumoniae resulting in lethal pneumonia, which can be
prevented by recombinant IL-12 (Greenberger et al. (1996) J
Immunol. 157: 3006-3012). The advantage of this model is that the
infection occurs through the lung, which is a common route of human
pathogen invasion. The vectors can be given to the lung together
with the pathogen or they can be administered after symptoms are
evident in order to screen for vectors that can cure established
infections.
[0859] Example: Influenza-provides a way to Screen for Vectors that
Provide Protection at Very Low Quantities of DNA and/or High Virus
Concentrations, and it also Allows One to Analyze the Levels of
Antigen Specific Abs and CTLs induced in vivo
[0860] In another example, the genetic vaccines are a mouse
vaccination model for Influenza A virus. Influenza was one of the
first models in which the efficacy of genetic vaccines was
demonstrated (Ulmer et al. (1993) Science 259: 1745-1749). Several
Influenza strains are lethal in mice providing an easy means to
screen for efficacy of genetic vaccines.
[0861] For example, Influenza virus strain A/PR/8/34, which is
available through the American Type Culture Collection (ATCC
VR-95), causes lethal infection, but 100% survival can be obtained
when the mice are immunized with and influenza hemagglutinin (HA)
genetic vaccine (Deck et al. (1997) Vaccine 15: 71-78). This model
provides a way to screen for vectors that provide protection at
very low quantities of DNA and/or high virus concentrations, and it
also allows one to analyze the levels of antigen specific Abs and
CTLs induced in vivo.
[0862] Example: Mycobacterium Tuberculosis (Partial Protection,
Requires Major Improvements)
[0863] The genetic vaccine vectors can also be analyzed for their
capacity to provide protection against infections by Mycobacterium
tuberculosis. This is an example of a situation where genetic
vaccines have provided partial protection, and where major
improvements are required.
[0864] Identification of Candidate Vectors Followed by More
Testing
[0865] Once a number of candidate vectors has been identified,
these vectors can be subjected to more detailed analysis in
additional models. Testing in other infectious disease models (such
as HSV, Mycoplasma pulmonis, RSV and/or rotavirus) will allow
identification of vectors that are optimal in each infectious
disease.
[0866] Optimal Plasmids from the First Round of Screening are used
as the Starting Material for the Next Round, the Successful Vectors
are Sequenced and the Corresponding Human Genes are Cloned into
Genetic Vaccine Vectors which are Characterized in vitro for their
Capacity to Induce Differentiation of a Desired Trait
[0867] In each case, the optimal plasmids from the first round of
screening can be used as the starting material for the next round
of reassembly (optionally in combination with other directed
evolution methods described herein), assembly and selection.
Vectors that are successful in animal models are sequenced and the
corresponding human genes are cloned into genetic vaccine vectors.
These vectors are then characterized in vitro for their capacity to
induce differentiation of T.sub.H1/T.sub.H2 cells, activation of
T.sub.H cells, cytotoxic T lymphocytes and monocytes/macrophages,
or other desired trait. Eventually, the most potent vectors, based
on in vivo data in mice and comparative in vitro studies in mice
and man, are chosen for human trials, and their capacity to
counteract various human infectious diseases is investigated.
[0868] Methods for Measuring Immune Parameters that Correlate to
Protective Immunity
[0869] In addition to determining whether a vector pool provides
protective immunity, one can measure immune parameters that
correlate to protective immunity, such as induction of specific
antibodies (particularly IgG) and induction of specific CTL
responses. Spleen cells can be isolated from vaccinated mice and
measured for the presence of antigen-specific T cells and induction
of T.sub.H1 cytokine synthesis profiles. ELISA and cytoplasmic
cytokine staining, combined with flow cytometry, can provide such
information on a single-cell level.
2.5.6. SCREENING OF GENETIC VACCINE VECTORS THAT ACTIVATE HUMAN
ANTIGEN-SPECIFIC LYMPHOCYTE RESPONSES
[0870] Isolation of PBMCs or APCs to Screen for Vectors with
Optimal Immunostimulatory Properties for the Human Immune
System
[0871] To screen for vectors with optimal immunostimulatory
properties for the human immune system, peripheral blood
mononuclear cells (PBMCs) or purified professional
antigen-presenting cells (APCs) can be isolated from previously
vaccinated or infected individuals or from patients with acute
infection with the pathogen of interest.
[0872] Genetic Vaccine Vectors Encoding the Antigen for which the
Individuals have Specific T Cells can be Transfected into PBMC and
Induction of T Cell Proliferation and Cytokine Synthesis can be
Measured; also Possible to Screen for Spontaneous Entry of Genetic
Vaccine Vector into APCs
[0873] Because these individuals have increased frequencies of
pathogen-specific T cells in circulation, antigens expressed in
PBMCs or purified APCs of these individuals will induce
proliferation and cytokine production by antigen-specific CD4+ and
CD8+ T cells. Thus, genetic vaccine vectors encoding the antigen
for which the individuals have specific T cells can be transfected
into PBMC of the individuals, after which induction of T cell
proliferation and cytokine synthesis can be measured.
Alternatively, one can screen for spontaneous entry of the genetic
vaccine vector into A-PCs, thus providing a means by which to
screen simultaneously for improved transfection efficiency,
improved expression of antigen and improved induction of activation
of specific T cells. Vectors with the most potent immunostimulatory
properties can be screened based on their capacity to induce B cell
proliferation and immunoglobulin synthesis. One buffy coat derived
from a blood donor contains PBMC lymphocytes from 0.5 liters of
blood, and up to 10.sup.4 PBMC can be obtained, enabling very large
screening experiments using T cells from one donor.
[0874] Making EBV-transformed B Cell Lines from Healthy Vaccinated
Individuals for Subsequent Experiments
[0875] When healthy vaccinated individuals (lab volunteers) are
studied, one can make EBV-transformed B cell lines from these
individuals. These cell lines can be used as antigen presenting
cells in subsequent experiments using blood from the same donor;
this reduces interassay and donor-to-donor variation). In addition,
one can make antigen-specific T cell clones, after which genetic
vaccines are transfected into EBV transformed B cells.
[0876] Efficiency with which the Transformed B Cells Induce
Proliferation of the Specific T Cell Clones
[0877] The efficiency with which the transformed B cells induce
proliferation of the specific T cell clones is then studied. When
working with specific T cell clones, the proliferation and cytokine
synthesis responses are significantly higher than when using total
PBMCs, because the frequency of antigen-specific T cells among PBMC
is very low.
[0878] Transfection of Cells in Culture by Libraries of
Experimentally Evolved (e.g. by Polynucleotide Reassembly &/or
Polynucleotide Site-saturation Mutagenesis) DNA Sequences in
Appropriate Expression Vectors can Lead to Class I Epitope
Presentation
[0879] CTL epitopes can be presented by most cells types since the
class I major histocompatibility complex (MHC) surface
glycoproteins are widely expressed. Therefore, transfection of
cells in culture by libraries of experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis) DNA sequences in appropriate expression vectors can
lead to class I epitope presentation. If specific CTLs directed to
a given epitope have been isolated from an individual, then the
co-culture of the transfected presenting cells and the CTLs can
lead to release by the CTLs of cytokines, such as IL-2, IFN-, or
TNF, if the epitope is presented. Higher amounts of released TNF.
will correspond to more efficient processing and presentation of
the class I epitope from the experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis), evolved sequence.
[0880] Transfecting Cells Expressing Class I MHC Surface
Glycoproteins with Library of Evolved Sequences, Preparing a
Detergent Soluble Extract, Performing a Partial Purification of the
MHC-epitope Complex, and then Submitting the Products to Mass
Spectrometry
[0881] A second method for identifying optimized CTL epitopes does
not require the isolation of CTLs reacting with the epitope. In
this approach, cells expressing class I MHC surface glycoproteins
are transfected with the library of evolved sequences as above.
After suitable incubation to allow for processing and
presentation,. a detergent soluble extract is prepared from each
cell culture and after a partial purification of the MHC-epitope
complex (perhaps optional) the products are submitted to mass
spectrometry (Henderson et al. (1993) Proc. Nat'l. Acad. Sci. USA
90: 10275-10279). Since the sequence is known of the epitope whose
presentation to be increased, one can calibrate the mass
spectrogram to identify this peptide. In addition, a cellular
protein can be used for internal calibration to obtain a
quantitative result; the cellular protein used for internal
calibration could be the MHC molecule itself. Thus one can measure
the amount of peptide epitope bound as a proportion of the MHC
molecules.
2.5.7. SCID-HUMAN SKIN MODEL FOR VACCINATION STUDIES
[0882] Use of Mouse Models in Vaccine Studies Limited in that the
MHC Molecules in Mice and Man are Substantially Different, Meaning
that Proteins and Peptides that Efficiently Induce Protective
Immune Responses in Mice do not Necessarily Function in Humans
[0883] Successful genetic vaccinations require transfection of the
target cells after injection of the vector, expression of the
desired antigen, processing the antigen in antigen presenting
cells, presentation of the antigenic peptides in the context of MHC
molecules, recognition of the peptide/MHC complex by T cell
receptors, interactions of T cells with B cells and professional
APCs and induction of specific T cell and B cell responses. All
these events could be differentially regulated in mouse and man. A
limitation of mouse models in vaccine studies is the fact that the
MHC molecules of mice and man are substantially different.
Therefore, proteins and peptides that effectively induce protective
immune responses in mice do not necessarily function in humans.
[0884] Mouse Models can be used to Study Human Tissues in Mice in
vivo for Studies of Transfection Efficiency, Transfer Sequences,
and Gene Expression Levels
[0885] To overcome these limitations mouse models can be used to
study human tissues in mice in vivo. Live pieces of human skin are
xenotransplant onto the back of immunodeficient mice, such as SCID
mice, allowing screening of the vector libraries for optimal
properties in human cells in vivo. Recursive selection of episomal
vectors provides strong selection pressure for vectors that remain
episomal, yet provide high level of gene expression. These mice
provide an excellent model for studies on transfection efficiency,
transfer sequences and gene expression levels. In addition, antigen
presenting cells (APCs) derived from these mice can also be used to
assess the level of antigens delivered to professional APCs, and to
study the capacity of these cells to present antigens and induce
activation of antigen-specific CD4+ and CD8+ T cells in vitro.
Significantly, although SCID mice have severely deficient T and B
cell components, antigen presenting cells (dendritic cells and
monocytes) are relatively normal in these mice.
[0886] Rendering Immunocompetent Mice Immunodeficient in Order to
Aid Transplantation of Human Tissue, Enabling Vaccine Studies in
Human Skin Xenotransplanted into Mice with Genetically Normal
Immune Systems as well, Due to the Transient Nature of the in vivo
Immunosuppression
[0887] In one embodiment of this model system, immunocompetent mice
are rendered immunodeficient in order to enable transplantation of
human tissue. For example, blocking of CD28 and CD40 pathways
promotes long-term survival of allogeneic skin grafts in mice
(Larsen et al. (1996) Nature 381: 434). Because the in vivo
immunosuppression is transient, this model also enables vaccine
studies in human skin xenotransplanted into mice with genetically
normal immune systems. Several methods of blocking CD28-137
interactions and CD40-CD40 ligand interactions are known to those
of skill in the art, including, for example, administration of
neutralizing anti-B7-1 and 137-2 antibodies, soluble CTLA-4, a
soluble form of the extracellular portion of CTLA-4, a fusion
protein that includes CTLA-4 and an Fc portion of an IgG molecule,
and neutralizing anti-CD40 or anti-CD40 ligand antibodies.
Additional methods by which one can improve transient
immunosuppression include administration of one or more of the
following reagents: cyclosporin A, anti-IL-2 receptor-chain Ab,
soluble IL-2 receptor, IL-10, and combinations thereof.
[0888] A model in which SCID-mice transplanted with human skin are
injected with HLA-matched PBMC can be used to analyze vectors that
provide long lasting expression in vivo. In this model, the vectors
are injected, or topically applied, into the human skin.
[0889] If the HLA-matched PBMC Injected into Mice Contains
Lymphocytes Specific for the Vector the Transfected Cells will be
Recognized, and Eventually Destroyed, by these Vector-specific
Lymphocytes, Providing the Possibility to Screen for Vectors that
Efficiently Escape Destruction
[0890] Thereafter, HLA-matched PBMC are injected into these mice.
If the PBMC contains lymphocytes specific for the vector, the
transfected cells will be recognized, and eventually destroyed, by
these vector-specific lymphocytes. Therefore, this model provides
possibilities to screen for vectors that efficiently escape
destruction by the immune cells. It has been shown that human PBLs
injected into mice with human skin transplants reject the organ,
indicating that the CTLs reach the skin in this model. Obtaining
HLA-matching skin and blood is possible (e.g. blood sample and skin
graft from a patient undergoing skin removal due to malignancy, or
blood and foreskin from the same infant).
[0891] SCIDhu Mouse Model: Additionally, Transplanting Human Skin
Allows Studies on the Efficacy of Genetic Vaccine Vectors Following
Injection to the Skin
[0892] An additional model that is suitable for screening as
described herein is the modified SCIDhu mouse model, in which
pieces of human fetal thymus, liver and bone marrow are
transplanted into SCID mice providing functional human immune
system in mice (Roncarolo et al. (1996) Semin. Immunol. 8: 207).
Functional human B and T cells, and APCs can be observed in these
mice. When additionally human skin is transplanted, it is likely to
allow studies on the efficacy of genetic vaccine vectors following
injection into the skin. Cotransplantation of skin is likely to
improve the model because it will provide an additional source of
professional APCs.
2.5.8. MOUSE MODEL FOR STUDYING THE EFFICIENCY OFGENETIC VACCINES
IN TRANSFECTING HUMAN MUSCLE CELLS AND INDUCING HUMAN IMMUNE
RESPONSES IN VIVO
[0893] There is a Lack of Suitable in vivo Models for Studies of
the Efficiency of Genetic Vaccines and the Vast Majority of Studies
are Performed on the Mouse Model, in which it is Sometimes
Difficult to Predict Whether the Results Obtained Reliably Predict
Similar Vaccinations in Humans because of the Complexity of Events
Occurring after Genetic Vaccination
[0894] A lack of suitable in vivo models has hampered studies of
the efficiency of genetic vaccines in inducing antigen expression
in human muscle cells and in inducing specific human immune
responses. The vast majority of studies on the capacity of genetic
vaccines to transfect muscle cells and to induce specific immune
responses in vivo have employed a mouse model. Because of the
complexity of events occurring after genetic vaccination, however,
it is sometimes difficult to predict whether results obtained in
the mouse model reliably predict the outcome of similar
vaccinations in humans. The events required in successful genetic
vaccination include transfection of the cells after delivery of the
plasmid, expression of the desired antigen, processing the antigen
in antigen presenting cells, presentation of the antigenic peptides
in the context of MHC molecules, recognition of the peptide/MHC
complex by T cell receptors, interactions of T cells with B cells
and professional antigen presenting cells and finally induction of
specific T cell and B cell responses. All these events are likely
to be somewhat differentially regulated in mouse and man.
[0895] The Invention Provides an in vivo Model for Human Muscle
Cell Transfection
[0896] Muscle tissue, obtained for example from cadavers, is
transplanted subcutaneously into immunodeficient mice, which can be
transplanted with tissues from other species without rejection.
This model system is especially valuable because there is no in
vitro culture system available for normal muscle cells. Muscle
tissue, obtained for example from cadavers, is transplanted
subcutaneously into immunodeficient mice. Immunodeficient mice can
be transplanted with tissues from other species without rejection.
Mice suitable for xenotransplantations include, but are not limited
to, SCID mice, nude mice and mice rendered deficient in their genes
encoding RAG1 or RAG2 genes. SCID mice and RAG deficient mice lack
functional T and B cells, and therefore are severely
immunocompromised and are unable to reject transplanted organs.
Previous studies indicate that these mice can be transplanted with
human tissues, such as skin, spleen, liver, thymus or bone, without
rejection (Roncarolo et al. (1996) Semin. Immunol. 8: 207). After
transplantation of human fetal lymphoid tissues into SCID mice,
functional human immune system can be demonstrated in these mice, a
model generally referred to as SCID-hu mice. When human muscle
tissue is transplanted into SCID-hu mice, one can not only study
transfection efficiency and expression of the desired antigen, but
one can also study induction of specific human immune responses
induced by genetic vaccines in vivo. In this case, muscle and
lymphoid organs from the same donor are used. Fetal muscle also has
an advantage in that it contains few mature lymphocytes of donor
origin decreasing likelihood of graft versus host reaction.
[0897] Genetic Vaccine Vectors are Introduced into the Human Muscle
Tissue to Study the Expression of the Antigen of Interest
[0898] Once the human muscle tissue is established in the mouse,
genetic vaccine vectors are introduced into the human muscle tissue
to study the expression of the antigen of interest. When studying
transfection efficiency only, RAG deficient mice are preferred,
because these mice never have mature B or T cells in the
circulation, whereas "leakiness" of SCID phenotype has been
demonstrated which may cause variation in the transplantation
efficiency.
[0899] Model Provides an Efficient Means to Study Gene Expression
in Human Muscle Cells in vivo, despite the Limited Survival of the
Tissue in Mice
[0900] The survival of human muscle tissue in mice is likely to be
limited even in immuno-compromised mice. However, because
expression studies can be performed within one or two days, this
model provides an efficient means to study gene expression in human
muscle cells in vivo. A modified SCID-hu mouse model with human
muscle transplanted into these mice can be used to study human
immune responses in mice in vivo.
2.5.9. SCREENING FOR IMPROVED DELIVERY OF VACCINES
[0901] Identifying Genetic Vaccine Vectors that are Capable of
being Administered in a Particular Manner
[0902] For certain applications, it is desirable to identify
genetic vaccine vectors that are capable of being administered in a
particular manner, for example, orally or through the skin. The
following screening methods provide suitable assays; additional
assays are also described herein in conjunction with particular
genetic vaccine properties for which the assays are especially
suitable.
[0903] Screening for Oral Delivery either in vitro (Based on Caco-2
cells) or in vivo
[0904] Screening for oral delivery can be performed either in vitro
or in vivo. An example of an in vitro method is based on Caco-2
(human colon adenocarcinoma) cells which are grown in tissue
culture. When grown on semipermeable filters, these cells
spontaneously differentiate into cells that resemble human small
intestine epithelium, both structurally and functionally. Genetic
vaccine libraries and/or vectors can be placed on one side of the
Caco-2 cell layer, and vectors that are able to move through the
cell layer are detected on the opposite side of the layer.
[0905] Libraries can also be screened for amenability to oral
delivery in vivo. For example, a library of vectors can be
administered orally, after which target tissues are assayed for
presence of vectors. Intestinal epithelium, liver, and the
bloodstream are examples of tissues that can be tested for presence
of library members. Vectors that are successful in reaching the
target tissue can be recovered and, if further improvement is
desired, used in succeeding rounds of reassembly (optionally in
combination with other directed evolution methods described herein)
and selection.
[0906] Apparatus which Permits Large Numbers of Vectors to be
Screened Efficiently and can be used to Study the Effect of Large
Numbers of Agents in vivo
[0907] For screening a library of genetic vaccine vectors for
ability to transfect cells upon injection into skin or muscle, the
invention provides an apparatus which permits large numbers of
vectors to be screened efficiently. This apparatus (FIG. 5) is
based on 96-well format and is designed to transfer small volumes
(2-5 .mu.l) from a microtiter plate to skin or muscle of laboratory
animals, such as mice and rats. Moreover, human muscle or skin
transplanted into immunodeficient mice can be injected.
[0908] The apparatus is designed in such a way that the tips move
to fit a microtiter plate. After the reagent of interest has been
obtained from the plate, the distance of the tips from each other
is decreased to 2-3 mm, enabling transfer of 96 reagents to an area
of 1.6 cm.times.2.4 cm to 2.4 cm.times.3.6 cm. The volume of each
sample transferred is electronically controlled. Each reagent is
mixed with a marker agent or dye to enable recognition of injection
site in the tissue. For example, gold particles of different sizes
and shapes are mixed with the reagent of interest, and microscopy
and immunohistochemistry can be used to identify each injection
site and to study the reaction induced by each reagent. When muscle
tissue is injected the injection site is first revealed by
surgery.
[0909] This apparatus can be used to study the effects of large
numbers of agents in vivo. For example, this apparatus can be used
to screen efficiency of large numbers of different DNA vaccine
vectors to transfect human skin or muscle cells transplanted into
immunodeficient mice.
2.5.10. ENHANCED ENTRY OF GENETIC VACCINE VECTORS INTO CELLS
[0910] Using Stochastic (e.g. Polynucleotide Shuffling &
Interrupted Synthesis) and Non-stochastic Polynucleotide Reassembly
to Efficiently Improve the Capacity of DNA to Enter the Cytoplasm
and Subsequently the Nucleus of Human Cells
[0911] The methods involve subjecting to stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly polynucleotides which are
involved in cell entry. Such polynucleotides are referred to herein
as "transfer sequences" or "transfer modules." Transfer modules can
be obtained which increase transfer in a cell-specific manner, or
which act in a more general manner. Because the exact sequences
that affect DNA binding and transfer are not often known,
stochastic (e.g. polynucleotide shuffling & interrupted
synthesis) and non-stochastic polynucleotide reassembly may be the
only efficient method to improve the capacity of DNA to enter the
cytoplasm and subsequently the nucleus of human cells.
[0912] The Stochastic (e.g. Polynucleotide Shuffling &
Interrupted Synthesis) and Non-stochastic Polynucleotide Reassembly
Methods of the Invention Provide means for Optimizing DNA Sequences
and the Three-dimensional Structure of the Plasmids for Ability to
Confer upon a Vector the Ability to Enter a Cell even in the
Absence of Detailed Information as to the Mechanism by which this
Effect is Achieved
[0913] The methods involve reassembling (&/or subjecting to one
or more directed evolution methods described herein) at least first
and second forms of a nucleic acid that comprises a transfer
sequence. The first and second forms differ from each other in two
or more nucleotides. Suitable substrates include, for example,
transcription factor binding sites, CpG sequences, poly A, C, G, T
oligonucleotides, non-stochastically generated nucleic acid
building blocks ,and random DNA fragments such as, for example,
genomic DNA, from human or other mammalian species. It has been
suggested that cell surface proteins, such as the macrophage
scavenger receptor, may act as receptors for specific DNA binding
(Pisetsky (1996) Immunity 5:303). It is not known whether these
receptors recognize specific DNA sequences or whether they bind DNA
in a sequence non-specific manner. However, GGGG tetrads have been
shown to enhance DNA binding to cell surfaces (Id.). In addition to
the DNA sequence, the three-dimensional structure of the plasmids
may play a role in the capacity of these plasmids to enter cells.
The stochastic (e.g. polynucleotide shuffling & interrupted
synthesis) and non-stochastic polynucleotide reassembly methods of
the invention provide means for optimizing such sequences for
ability to confer upon a vector the ability to enter a cell even in
the absence of detailed information as to the mechanism by which
this effect is achieved.
[0914] Clonal Isolates of Vectors Bearing Recombinant Segments are
used to Infect Separate Cultures of Cells and the Percentage of
Vectors Which Enter Cells is then Determined by, for Example,
Counting Cells Expressing a Marker Expressed by the Vectors in the
Course of Transfection
[0915] The resulting library of recombinant transfer modules are
screened to identify at least one optimized recombinant transfer
module that enhances the capability of a vector comprising the
transfer module to enter a cell of interest. For example, vectors
that include a recombinant transfer module can be contacted with a
population of cells under conditions conducive to entry of the
vector into the cells, after which the percentage of cells in the
population which contain the nucleic acid vector is determined.
Preferably, the vector will contain a selectable or screenable
marker to facilitate identification of cells which contain the
vector. In a preferred embodiment, clonal isolates of vectors
bearing recombinant segments are used to infect separate cultures
of cells. The percentage of vectors which enter cells can then be
determined by, for example, counting cells expressing a marker
expressed by the vectors in the course of transfection.
[0916] The Reassembly (&/or One or More Additional Directed
Evolution Methods Described Herein) and Rescreening Process can be
Repeated as Necessary, until a Transfer Module that has Sufficient
Ability to Enhance Transfer is Obtained
[0917] Typically, the reassembly (&/or one or more additional
directed evolution methods described herein) process is repeated by
reassembling (&/or subjecting to one or more directed evolution
methods described herein) at least one optimized transfer sequence
with a further form of the transfer sequence to produce a further
library of recombinant transfer modules. The further form can be
the same or different from the first and second forms. The new
library is screened to identify at least one further optimized
recombinant vector module that exhibits an enhancement of the
ability of a genetic vaccine vector that includes the optimized
transfer module to enter a cell of interest.
[0918] The reassembly (&/or one or more additional directed
evolution methods described herein) and rescreening process can be
repeated as necessary, until a transfer module that has sufficient
ability to enhance transfer is obtained. After one or more of
reassembly (&/or one or more additional directed evolution
methods described herein) and screening, vector modules are
obtained which are capable of conferring upon a nucleic acid vector
the ability to enter at least about 50 percent more target cells
than a control vector which does not contain the optimized module,
more preferably at least about 75 percent more, and most preferably
at least about 95 or 99 percent more target cells than a control
vector.
[0919] For Integration by Homologous Recombination, Important
Factors are the Degree and Length of Homology to Chromosomal
Sequences, the Frequency of such Sequences in the Genome, and the
Specific Sequence Mediating Homologous Recombination; for
Nonhomologous, Illegitimate and Site-Specific Recombination,
Recombination is Mediated by Specific Sites on the Therapy Vector
Which Interact with Cell Encoded Recombination Proteins
[0920] Although for vaccine purposes non-integrating vectors are
generally preferred, for some applications it may be desirable to
use an integrating vector; for these applications DNA sequences
that directly or indirectly affect the efficiency of integration
can be included in the genetic vaccine vector. For integration by
homologous recombination, important factors are the degree and
length of homology to chromosomal sequences, as well as the
frequency of such sequences in the genome (e.g., Alu repeats). The
specific sequence mediating homologous recombination is also
important, since integration occurs much more easily in
transcriptionally active DNA. Methods and materials for
constructing homologous targeting constructs are described by e.g.,
Mansour (1988) Nature 336:348; Bradley (1992) Bio/Technology
10:534. For nonhomologous, illegitimate and site-specific
recombination, recombination is mediated by specific sites on the
therapy vector which interact with cell encoded recombination
proteins, e.g., Cre/Lox and FIp/Frt systems. See, e.g., Baubonis
(1993) Nucleic Acids Res. 21:2025-2029, which reports that a vector
including a LoxP site becomes integrated at a LoxP site in
chromosomal DNA in the presence of Cre recombinase enzyme.
2.6. OPTIMIZATION OF GENETIC VACCINE COMPONENTS
[0921] Optimizing Properties that can Influence the Efficacy of a
Genetic Vaccine in Modulating an Immune Response in a Mammalian
System
[0922] Many factors can influence the efficacy of a genetic vaccine
in modulating an immune response. The ability of the vector to
enter a cell, for example, has a significant effect on the ability
of the vector to modulate an immune response. The strength of an
immune response is also mediated by the immunogenicity of an
antigen expressed by a genetic vaccine vector and the level at
which the antigen is expressed. The presence or, absence of
costimulatory molecules produced by the genetic vaccine vector can
affect not only the strength, but also the type of immune response
that arises due to introduction of the vector into a mammal. An
increase in the persistence of a vector in an organism can lengthen
the time of immunomodulation, and also makes feasible self-boosting
vectors which do not require multiple administrations to achieve
long-lasting protection. The present invention provides methods for
optimizing many of these properties, thus resulting in genetic
vaccine vectors that exhibit improved ability to elicit the desired
effect on a mammalian immune system.
[0923] The Selection from Large Libraries using Recursive Cycles of
Reassembly (Optionally in Combination with Other Directed Evolution
Methods Described Herein) to Maximally Access all the Fortuitous
but Complex Mechanisms that Cannot be Approached Rationally
[0924] Genetic vaccines can contain a variety of functional
components, whose preferred sequences are best determined by
stochastic (e.g. polynucleotide shuffling & interrupted
synthesis) and non-stochastic polynucleotide reassembly, the
empirical sequence evolution described in detail herein. The
methods of the invention involve, in general, constructing a
separate library for each of the major vector components by
stochastic (e.g. polynucleotide shuffling & interrupted
synthesis) and non-stochastic polynucleotide reassembly of multiple
homologous starting sequences, or other methods of generating a
population of recombinants, resulting in a complex mixture of
chimeric sequences. The best sequences are selected from these
libraries using the high-throughput assays described below. After
one or more cycles of selection from each of the single module
libraries, the pools of the best sequences of different modules can
be combined by stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide reassembly
as long as the screens are compatible. The screens for promoter,
enhancer, intron, transfer sequences, mammalian ori, bacterial ori
and bacterial marker, and the like, can eventually be combined,
resulting in co-optimization of the context of each sequence. An
important aspect in these experiments is the selection from large
libraries using recursive cycles of reassembly (optionally in
combination with other directed evolution methods described herein)
to maximally access all the fortuitous but complex mechanisms that
cannot be approached rationally, such as DNA transfer into the
cell.
[0925] A Library of Different Vectors can be Generated by
Assembling Vector Modules that Provide Promoters, Cytokines,
Cytokine Antagonists, Chemokines, Immunostimulatory Sequences, and
Costimulatory Molecules using Assembly PCR and Combinatorial
Molecular Biology
[0926] Assembly PCR is a method for assembly of long DNA sequences,
such as genes, non-stochastically generated nucleic acid building
blocks, and fragments of plasmids. In contrast to PCR, there is no
distinction between primers and template, because the
non-stochastically generated nucleic acid building blocks &/or
fragments to be assembled prime each other. The library of vector
modules obtained as described herein can be fused with promoters,
which can themselves be optimized by the stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly methods of the invention.
The resulting genes can be assembled combinatorially into DNA
vaccine vectors, where each gene is expressed under a different
promoter (e.g., a promoter derived from a library of experimentally
evolved (e.g. by polynucleotide reassembly &/or polynucleotide
site-saturation mutagenesis) CMV promoters), and the vector library
is screened as described herein to identify vectors which exhibit
the desired effect on the immune system.
[0927] Properties that Influence the Efficacy or Desirability of
the Vaccine
[0928] The methods of the invention are useful for obtaining
genetic vaccines that are optimized for one or more of many
properties that influence the efficacy or desirability of the
vaccine. These properties include, but are not limited to, the
following.
2.6.1. EPISOMAL VECTOR MAINTENANCE
[0929] Episomally Replicating Vectors are Maintained in a Cell for
a Longer Period of Time and Permit the Development of Self-Boosting
Vaccines
[0930] One property that one can optimize using the sequence
reassembly methods of the invention is the ability of a genetic
vaccine vector to replicate episomally in a mammalian cell.
Episomal replication of a vaccine vector is advantageous in many
situations. For example, episomally replicating vectors are
maintained in a cell for a longer period of time than
non-replicating vectors, thus resulting in an increased length of
immune response modulation or increased delivery of a
therapeutically useful protein. Episomal replication also permits
the development of self-boosting vaccines which, unlike traditional
vaccines, do not require multiple vaccine administrations. For
example, a self-boosting vaccine vector can include an
antigen-encoding gene which is under the control of an inducible
control element which allows induction of antigen expression, and
the corresponding immune response, in response to a specific
stimulus. However, screening for naturally occurring vector modules
which result in enhanced episomal maintenance using traditional
approaches or attempts to rationally design mutants with improved
properties would require many person-years of research. The
invention provides methods for generating and screening orders of
magnitude more diversity in a short time period.
[0931] Using Stochastic (e.g. Polynucleotide Shuffling &
Interrupted Synthesis) and Non-Stochastic Polynucleotide Reassembly
to Recombine at Least Two Forms of a Nucleic Acid which is Capable
of Conferring upon a Genetic Vector the Ability to Replicate
Autonomously in Mammalian Cells
[0932] The ability of a genetic vaccine vector to replicate
episomally can be optimized by using stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly to recombine at least two
forms of a nucleic acid which is capable of conferring upon a
genetic vector the ability to replicate autonomously in mammalian
cells. The two or more forms of the episomal replication vector
module differ from each other in two or more nucleotides. A library
of recombinant episomal replication vector modules is produced, and
the library is screened to identify one or more optimized
replication vector modules which, when placed in a genetic vaccine
vector, confer upon the vector an enhanced ability to replicate
autonomously compared to a vector which contains a non-optimized
episomal replication vector module.
[0933] Repetition of the Stochastic (e.g. Polynucleotide Shuffling
& Interrupted Synthesis) and Non-Stochastic Polynucleotide
Reassembly Process at Least Once to Identify Modules Which Exhibit
Enhanced Ability to Confer Episomal Maintenance Upon a Vector
Containing the Module
[0934] In one embodiment, the stochastic (e.g. polynucleotide
shuffling & interrupted synthesis) and non-stochastic
polynucleotide reassembly process is repeated at least once using
as a substrate an optimized episomal replication vector module
obtained from a previous round of stochastic (e.g. polynucleotide
shuffling & interrupted synthesis) and non-stochastic
polynucleotide reassembly. The optimized vector module obtained in
the earlier round is reassembled (&/or subjected to one or more
directed evolution methods described herein) with a further form of
the vector module, which can be the same as one of the forms used
in the earlier round, or can be a different form of a nucleic acid
that functions as an episomal replication element. Again, a library
of recombinant episomal replication vector modules is produced, and
the screening process is repeated to identify those episomal
replication modules which exhibit enhanced ability to confer
episomal maintenance upon a vector containing the module.
[0935] Ability to Replicate Autonomously in Eukaryotic
Cells-Examples
[0936] Nucleic acids which are useful as substrates for the use of
stochastic (e.g. polynucleotide shuffling & interrupted
synthesis) and non-stochastic polynucleotide reassembly to optimize
episomal replication ability include any nucleic acid that is
involved in conferring upon a vector the ability to replicate
autonomously in eukaryotic cells. For example, papillomavirus
sequences E I and E2, simian virus 40 (SV40) origin of replication,
and the like.
[0937] Genes from Human Papillomaviruses are Exemplary Episomal
Replication Vector Modules
[0938] Exemplary episomal replication vector modules that can be
optimized using the methods of the invention are genes from human
papillomaviruses (HPV) which are involved in episomal replication.
HPV are non-tumorigenic viruses which replicate episomally in skin
and are stably expressed in vivo for years. Bernard and Apt (1994)
Arch. Dermatol. 130:210.
[0939] Increased Episomal Maintenance of the HPV Genes Involved in
Episomal Replication using Directed Evolution
[0940] Despite these in vivo properties, it has not been possible
to maintain HPV episomally in tissue culture due to
underreplication. The invention provides methods by which HPV genes
involved in episomal maintenance can be optimized for use in
genetic vaccine vectors. HPV genes involved in episomal replication
include, for example, the E1 and E2 genes. Thus, according to one
embodiment of the invention, either or both of the HPV E I and E2
genes are subjected to stochastic (e.g. polynucleotide shuffling
& interrupted synthesis) and non-stochastic polynucleotide
reassembly to obtain a recombinant episomal replication module
which, when placed in a nucleic acid vaccine vector, results in
increased maintenance of the vector in mammalian cells. In a
preferred embodiment, the HPV E1 and E2 genes from different, but
closely related, benign HPVs are used in a polynucleotide
reassembly procedure, as shown, described &/or referenced
herein (including incorporated by reference). For example,
polynucletide shuffling of HPV E1 and E2 genes from closely related
strains of HPV (such as, for example, HPV 2, 27, and 57) can be
used to obtain a library of recombinant E1 and E2 genes which are
then subjected to an appropriate screening method to identify those
that exhibit improved episomal maintenance properties.
[0941] Identification, Selection, Enrichment of Recombinant
Episomal Replication Vector Modules that Exhibit Improved Ability
to Mediate Episomal Maintenance
[0942] To identify recombinant episomal replication vector modules
that exhibit improved ability to mediate episomal maintenance,
members of the library of recombinant vector modules are inserted
into vectors which are introduced into mammalian cells. The cells
are propagated for at least several generations, after which cells
that have maintained the vector are identified. Identification can
be accomplished, for example, employing a vector that includes a
selectable marker. Cells containing the library members are
propagated in the absence of selection for the selectable marker
for at least several generations, after which selective pressure is
added. Cells which survive selection are enriched for cells that
harbor vectors which contain a recombinant vector module which
enhances the ability of the vector to replicate episomally. DNA is
recovered from the selected cells and introduced into bacterial
host cells, allowing recovery of episomal, non-integrated
vectors.
[0943] Screening by Introducing to a Vector Containing a
Polynucleotide Encoding an Antigen that is Present on the Surface
of the Cell when Expressed
[0944] In another embodiment of the invention, the screening step
is accomplished by introducing members of the library of
recombinant episomal replication vector modules into a vector that
includes a polynucleotide that encodes an antigen which, when
expressed, is present on the surface of a cell. The library of
vectors is introduced into mammalian cells which are propagated for
at least several generations, after which cells which display the
cell surface antigen on the surface of the cell are identified.
Such cells most likely harbor a genetic vaccine vector which
enhances the ability of the vector to replicate autonomously.
[0945] Use of Optimized Recombinant Episomal Replication Vector
Module to Construct Genetic Vaccine Vectors
[0946] Upon identifying cells which contain an episomally
maintained vector, the optimized recombinant episomal replication
vector module is obtained and used to construct genetic vaccine
vectors. Cell surface antigens which are suitable for use in the
screening methods are described above, and others are known to
those of skill in the art. Preferably, an antigen is used for which
a convenient means of detection is available.
[0947] Preferred Cells for Use in the Screening Methods
[0948] Cells which are suitable for use in the screening methods
include both cultured mammalian cells and cells which are present
in an animal. To screen for recombinant vector modules that are
intended for use in humans, the preferred cells for screening
purposes are human cells. Generally, initial screening is
accomplished in cell culture, where processing of large libraries
of experimentally evolved (e.g. by polynucleotide reassembly
&/or polynucleotide site-saturation mutagenesis) material is
feasible. In a preferred embodiment, cells which display a
vector-encoded cell surface antigen on the cell surface are
identified by flow cytometry based cell sorting methods, such as
fluorescence activated cell sorting. This approach allows very
large numbers (>10.sup.7) cells to be evaluated in a single vial
experiment.
[0949] Further Testing for Durability in vivo in an Animal
Model
[0950] Constructs which replicate autonomously in cell culture and
give rise to strong marker gene expression can be further tested
for durability in vivo in an animal model. For example, mouse
models for studies of human tissues in mice in vivo are described
in ???. Live pieces of human skin are xenotransplanted onto the
back of SCID mice, allowing screening of the vector libraries for
optimal properties in human cells in vivo. Recursive selection of
episomal vectors will provide strong selection pressure for vectors
that remain episomal, yet provide high level of gene
expression.
[0951] Introducing a Genetic Vaccine Vector into a Mammal that has
a Functional Human Immune System and Testing for the Existence of
an Immune Response Against the Antigen
[0952] In another embodiment, the screening step involves
introducing a genetic vaccine vector which includes the recombinant
episomal replication vector module, as well as polynucleotide that
encodes an antigen or pharmaceutically useful protein, into a
mammal that has a functional human immune system. The animal is
then tested for the existence of an immune response against the
antigen. In a preferred embodiment, the mammals used for such
assays are non-human mammals that have a functional human immune
system. For example, a functional human immune system can be
created in an immunodeficient mouse by introducing one or more of a
human fetal tissue selected from the group consisting of liver,
thymus, and bone marrow (Roncarolo et al. (1996) Semin. Immunol.
8:207).
[0953] Episomally Maintained Vectors Result in High Signal-To-Noise
Ratios upon FACS Selection and Significantly Improve the
Possibility to Recover the Plasmids from a Small Number of Selected
Cells
[0954] Stable episomal vectors which are obtained using the methods
of the invention are useful not only as genetic vaccines, but also
are useful tools in other library screening applications. In
contrast to randomly integrating and transient vectors, episomally
maintained vectors result in high signal-to-noise ratios upon FACS
selection, and they also significantly improve the possibility to
recover the plasmids from a small number of selected cells.
2.6.2. EVOLUTION OF OPTIMIZED PROMOTERS FOR EXPRESSION OF AN
ANTIGEN
[0955] Optimizing the Promoter and/or other Control Sequence to
Improve the Efficacy of Genetic Vaccinations, Reduce the Amount of
DNA Required for Protective Immunity and Thereby the Cost of
Vaccination, Control the Type of Cell in Which the Gene is
Expressed, and/or the Timing of the Antigen Expression
[0956] In another embodiment, the invention provides methods of
optimizing vector modules such as promoters and other gene
expression control signals. Usually, a coding sequence for an
antigen that is delivered by a genetic vaccine is operably linked
to an additional sequence, such as a regulatory sequence, to ensure
its expression. These regulatory sequences can include one or more
of the following: an enhancer, a promoter, a signal peptide
sequence, an intron and/or a polyadenylation sequence. A desirable
goal is to increase the level of expression of functional
expression product relative to that achieved with conventional
vectors. The efficacy of a genetic vaccine vector often depends on
the level of expression of an antigen by the vaccine vector. An
optimized promoter and/or other control sequence is likely to
result in improved efficacy of genetic vaccinations, reduce the
amount of DNA required for protective immunity and thereby the cost
of vaccination.
[0957] Moreover, it is sometimes desirable to have control over the
type of cell in which a gene is expressed, and/or the timing of
antigen expression. The methods of the invention provide for
optimization of these and other factors which are influenced by
promoters and other control sequences.
[0958] Improving Expression by Increasing the Rate of Production of
an Expression Product, Decreasing the Rate of Degradation of the
Expression Product, or Improving the Capacity of Expression Product
to Perform its Intended Function using Stochastic (e.g.
Polynucleotide Shuffling & Interrupted Synthesis) and
Non-Stochastic Polynucleotide Reassembly of Polynucleotides
Involved in Control of Gene Expression
[0959] Improved expression of selection markers can be achieved by
performing stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide
reassembly, for example. Expression can effectively be improved by
a variety of means, including increasing the rate of production of
an expression product, decreasing the rate of degradation of the
expression product or improving the capacity of the expression
product to perform its intended function. The methods involve
subjecting to stochastic (e.g. polynucleotide shuffling &
interrupted synthesis),and non-stochastic polynucleotide reassembly
polynucleotides which are involved in control of gene expression.
At least first and second forms of a nucleic acid that comprises a
control sequence, which forms differ from each other in two or more
nucleotides, are reassembled (&/or subjected to one or more
directed evolution methods described herein) as described above.
The resulting library of recombinant transfer modules are screened
to identify at least one optimized recombinant control sequence
that exhibits enhanced strength, inducibility, or specificity.
[0960] Introduction of the Recombinant Segments at the Level of
Fragments (Non-Tochastically Generated &/or Randomly Generated)
and in vitro
[0961] The substrates for reassembly (&/or one or more
additional directed evolution methods described herein) can be the
full-length vectors, or fragments thereof, which include a coding
sequence and/or regulatory sequences to which the coding sequence
is operably linked. The substrates can include variants of any of
the regulatory and/or coding sequence(s) present in the vector. If
reassembly (&/or one or more additional directed evolution
methods described herein) is effected at the level of fragments,
the recombinant segments should be reinserted into vectors before
screening. If reassembly (&/or one or more additional directed
evolution methods described herein) proceeds in vitro, vectors
containing the recombinant segments are usually introduced into
cells before screening. An example of a vector suitable for use in
screening of experimentally evolved (e.g. by polynucleotide
reassembly &/or polynucleotide site-saturation mutagenesis)
promoters and other regulatory regions is shown, described &/or
referenced herein (including incorporated by reference).
[0962] Using an Easily Detected Selection Marker (Green Fluorescent
Protein, Cell Surface Protein) when an Additional or Substitute
Marker is Required
[0963] Cells containing the recombinant segments can be screened by
detecting expression of the gene encoded by the selection marker.
For purposes of selection and/or screening, a gene product
expressed from a vector is sometimes an easily detected marker
rather than a product having an actual therapeutic purpose, e.g., a
green fluorescent protein (see, Crameri (1996) Nature Biotechnol.
14:315-319) or a cell surface protein. For example, if this marker
is green fluorescent protein, cells with the highest expression
levels can be identified by flow cytometry-based cell sorting. If
the marker is a cell surface protein, the cells are stained with a
reagent having affinity for the protein, such as antibody, and
again analyzed by flow cytometry-based cell sorting. However, some
genes having a therapeutic purpose, e.g., drug resistance genes,
themselves provide a selectable marker, and no additional or
substitute marker is required. Alternatively, the gene product can
be a fusion protein comprising any combination of detection and
selection markers. Internal reference marker genes can be included
on the vector to detect and compensate for variations in copy
number or insertion site.
[0964] Further Round of Reassembly (&/or One or More Additional
Directed Evolution Methods Described Herein) and Screening.
[0965] Recombinant segments from the cells showing highest
expression of the marker gene can be used as some or all of the
substrates in a further round of reassembly (&/or one or more
additional directed evolution methods described herein) and
screening, if additional improvement is desired.
2.6.2.1.CONSTITUTIVE PROMOTERS
[0966] Evolving Control Sequences (Promoters, Enhancers, etc.) to
Express a Gene of Interest at a Higher Level than is a Gene
Operably Linked to a Non-Evolved Control Sequences
[0967] The invention provides methods of evolving nucleotide
sequences that are capable of directing constitutive expression of
a gene of interest which is operably linked to the control
sequence. Typically, the control sequences, which can include
promoters, enhancers, and the like, are evolved so that a gene of
interest is expressed at a higher level than is a gene operably
linked to a non-evolved control sequence. To screen for control
sequences which are of increased strength, a recombinant library of
control sequences can be introduced into a population of cells and
the level of expression of a detectable marker operably linked to
the control sequences determined. Preferably, the optimized
promoter is capable of expressing an operably linked gene at a
level that is at least about 30% greater than that of a control
promoter construct, more preferably the optimized promoter is at
least about 50% stronger than a control, and most preferably at
least about 75% or more stronger than a control promoter.
[0968] Using Improved CMV Promoter/Enhancer Elements (SV40 and Sra)
to Express Foreign Genes Both in Animal Models and in Clinical
Applications
[0969] Examples of promoters which can be used as substrates in the
methods include any constitutive promoter that functions in the
intended host cell. The major immediate-early (IE) region
transcriptional regulatory elements, including promoter and
enhancer sequences (the promoter/enhancer region), of
cytomegalovirus (CMV) is widely used for regulating transcription
in vectors used for gene therapy because it is highly active in a
broad range of cell types. Optimized CMV transcriptional regulatory
elements which direct increased levels of antigen expression is
generated by the recursive reassembly (&/or one or more
additional directed evolution methods described herein) methods of
the invention, resulting in improved efficacy of gene therapy. As
the CMV promoter and enhancer is active in human and animal cells,
the improved CMV promoter/enhancer elements are used to express
foreign genes both in animal models and in clinical applications.
Other constitutive promoters that are amenable to use in the
claimed methods include, for example, promoters from SV40 and SR,
and other promoters known to those of skill in the art.
[0970] Creating a Library of Chimeric Transcriptional Regulatory
Elements Through Stochastic (e.g. Polynucleotide Shuffling &
Interrupted Synthesis) and Non-Stochastic Polynucleotide Reassembly
of Wild-Type Sequences from Two or More of the Five Related Strains
of CMV, Obtaining the Promoter, Enhancer and First Intron Sequences
of the IE Region through PCR of the CMV Strains
[0971] In a preferred embodiment, a library of chimeric
transcriptional regulatory elements is created by stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly of wild-type sequences
from two or more of the five related strains of CMV. The promoter,
enhancer and first intron sequences of the IE region are obtained
by PCR from the CMV strains: human VR-53 8 strain AD 169 (Rowe (195
6) Proc. Soc. Exp. Biol. Med. 92:418; human V-977 strain Towne
(Plotkin (1975) Infect. Immunol. 12:521-527); rhesus VR-677 strain
68-1 (Asher (1969) BacterioL Proc. 269:91); vervet VR-706 strain
CSG (Black (1963) Proc. Soc. Exp. Biol. Med. 112:60 1); and,
squirrel monkey VR-1398 strain SqSHV (Rangan (1980) Lab. Animal
Sci. 30:532). The promoter/ enhancer sequences of the human CMV
strains are 95% homologous, and share 70% homology with the
sequences of the monkey isolates, allowing the use of
polynucleotide reassembly (optionally in combination with other
directed evolution methods described herein) to generate a library
great diversity. Following reassembly (optionally in combination
with other directed evolution methods described herein), the
library is cloned into a plasmid backbone and used to direct
transcription of a marker gene in mammalian cells. An internal
marker under the control of a native promoter is typically included
in the plasmid vector, which will allow analysis and sorting of
cells harboring equal numbers of vectors.
[0972] Expression markers, such as green fluorescent protein (GFP)
and CD86 (also known as B7.2, see Freeman (1993) J Exp. Med
178:2185, Chen (1994) J Immunol. 152:4929) can also be used. In
addition, transfection of SV40 T antigen-transformed cells can be
used to amplify a vector which contains an SV40 origin of
replication. The transfected cells are screened by FACS sorting to
identify those which express high levels of the marker gene,
normalized against the internal marker to account for differences
in vector copy numbers per cell. If desired, vectors carrying
optimal, recursively reassembled (&/or subjected to one or more
directed evolution methods described herein) promoter sequences are
recovered and subjected to further cycles of reassembly (optionally
in combination with other directed evolution methods described
herein) and selection.
2.6.2.2. CELL-SPECIFIC PROMOTERS
[0973] Reducing the Risk of Autoimmune Disorder Following
Introduction of Foreign Antigens into Host Cells and Providing for
Efficient Induction of Protective Immunity through the Expression
of Genetic Vaccines in Professional APCs, such as Dendritic Cells
and Macrophages
[0974] One of the safety concerns associated with genetic vaccines
has been the possibility of autoimmune disorders following
introduction, of foreign antigens into host cells. This risk can be
reduced if the pathogen antigen is specifically expressed in
professional APCs that express the proper costimulatory molecules.
Although it is somewhat debatable which cells are the most
important cells expressing the pathogen antigen following genetic
vaccinations, it is likely that professional APCs are involved. It
has been shown that blood monocytes express antigen following
intramuscular injection of genetic vaccine vectors, and dendritic
cells derived from lymph nodes of vaccinated animals efficiently
induced antigen-specific T cell activation (C. Bona, The First
Gordon Conference on Genetic Vaccines, Plymouth, N.H., Jul. 21,
1997). These data, together with previous studies indicating that
small number of dendritic cells expressing antigen or antigenic
peptides is sufficient to induce activation of antigen-specific T
cells (Thomas and Lipsky, Stem Cells 14:196, 1996), support the
conclusion that genetic vaccines specifically expressed in
professional APC, such as dendritic cells and macrophages, are
likely to provide efficient induction of protective immunity with
minimized chance of adverse effects.
[0975] Methods for Obtaining Promoters and Enhancers that Induce
High Expression Levels Specifically in Professional APCs,
Exploiting Natural Diversity as a Source of Substrates for
Stochastic (e.g. Polynucleotide Shuffling & Interrupted
Synthesis) and Non-Stochastic Polynucleotide Reassembly
[0976] The present invention provides methods of obtaining
promoters and enhancers that induce high expression levels
specifically in professional APCs. Previously existing APC-specific
vectors did not provide sufficient expression levels following
genetic vaccinations. The methods involve performing stochastic
(e.g. polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly as described above using
as substrates different forms of a nucleic acid that comprises an
APC-specific promoter or other control signal. Suitable promoters
include, for example, the MHC Class II, and the CD11b, CD11c, and
CD40 promoters. Natural diversity of the promoters can be exploited
as a highly appropriate source of substrates for the stochastic
(e.g. polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly. For example, genomic DNA
from monkeys, pigs, dogs, cows, cats, rabbits, rats and mice, can
be obtained, and the proper sequences obtained by using multiple
PCR primers specific for the most conserved regions based on known
sequence information. The selection of the optimal promoters can be
done in monocytic or B cell lines, such as U937, HL60 or Jijoye,
using FACS-sorting. In addition, SV40.sup.+cell lines, such as
COS-1 and COS-7, can be used to improve the recovery of the
plasmids. Further analysis can be undertaken in human dendritic
cells obtained by culturing peripheral blood monocytes in the
presence of IL-4 and GM-CSF as described (Chapuis et al. (1997)
Eur. J Immunol. 27:43 1).
2.6.2.3. INDUCIBLE PROMOTERS
[0977] Using Stochastic (e.g. Polynucleotide Shuffling &
Interrupted Synthesis) and Non-Stochastic Polynucleotide Reassembly
of Two Substrates, such as Tetracycline and Hormone Inducible
Expression Systems, to Increase the Expression Level and
Inducibility in vivo of the Promoter Controlling Transgene
Expression
[0978] A particularly desirable property of a genetic vaccines
would be an ability to induce the promoter controlling transgene
expression simply by taking an innocuous oral drug, resulting in a
boost of the immune response. Essential requirements for inducible
promoters are low base-line expression and strong inducibility.
Several promoters with exquisite in vitro regulation exist, but the
expression level and inducibility of each is too low to be useable
in vivo. The invention overcome these problems by stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly using as substrates two or
more variants of a nucleic acid that functions as an inducible
control sequence. Suitable substrates include, for example,
tetracycline and hormone inducible expression systems, and the
like. Hormones that have been used to regulate gene expression
include, for example, estrogen, tomoxifen, toremifen and ecdysone
(Ramkumar and Adler (1995) Endocrinology 136:536-542). Libraries of
recombinant inducible promoters are screened as described above in
the presence and absence of the inducer.
[0979] Tetracycline Responsive System Provides Possibilities to
Induce and Turn off Gene Expression (Ecdysone Responsive Element
Another Candidate)
[0980] The most commonly used inducible gene expression protocol is
the tetracycline responsive system, which provides possibilities to
both induce and turn off gene expression (Gossen and Bujard (1992)
Proc. Nat'l. Acad. Sci. USA 89:5547; Gossen et al. (1995) Science
268:1766). A repressor gene is located on the plasmid and binds to
an operator in the promoter. Tetracycline or doxycycline modulates
the binding ability of the repressor. Interestingly, four amino
acid changes convert the repressor into an activator. In addition
to the tetracycline responsive system, other candidates for
inducible promoter evolution include the ecdysone responsive
element (No et al., Proc. Nat'l. Acad Sci. USA 93:3346,1997).
[0981] Inducible Promoters Provide a Means by Which a Vaccine Dose
can be Administered Subsequent to the Initial Administration Simply
by Ingestion of a Reagent that Causes Induction of the Inducible
Promoter
[0982] Inducible promoters such as those obtained using the methods
of the invention are useful in autoboost vaccines. Particularly
when combined with a stably maintained episomal vector obtained as
described above, the inducible promoters provide a means by which a
vaccine dose can be administered subsequent to the initial
administration simply by ingestion of a reagent that causes
induction of the inducible promoter. FIG. 8 demonstrates a flow
cytometry-based screening protocol that is suitable for
optimization of inducible promoters.
[0983] Testing the Functionality of Autoboosting Vaccines in a
Mouse Model
[0984] The functionality of autoboosting vaccines can be tested in
a mouse model such as that described above. Genetic vaccine vectors
are injected into the skin of normal mice and into human skin in
SCID-human skin mice. A gene encoding hepatitis B surface antigen
(HBsAg) or other surface antigen is incorporated into these vectors
enabling direct measurements of the levels of antigen produced,
because HBsAg levels can be measured in cell culture supernates and
in the circulation of the mice. The drug inducing the expression of
the antigen is given after 1, 2, 4 and 6 weeks, and the expression
levels of HBsAg are studied. Moreover, the levels of anti-HBsAg
antibodies are measured. The mice are also injected with a vector
containing a pathogen antigen discovered by ELI, and specific
immune responses are followed.
[0985] In vivo Assessment of Functionality of Autoboosting Genetic
Vaccines in Human Immune System using SCID-Human Skin Model with
SCID-hu Mouse Model
[0986] Combining the SCID-human skin model with traditional SCID-hu
mouse model (Roncarolo et al., Semin. Immunol. 8:207, 1996) allows
the assessment of functionality of autoboosting genetic vaccines in
human immune system in vivo, and also allows measurements of human
Ab responses in vivo. This model can also be used to assess
production of HBsAg after oral boosting of novel genetic vaccine
vectors harboring the gene encoding HBsAg.
2.6.3. EVOLUTION OF BINDING POLYPEPTIDES THAT ENHANCE SPECIFICITY
AND EFFICIENCY OF GENETIC VACCINES
[0987] The present invention also provides methods for obtaining
recombinant nucleic acids that encode polypeptides which can
enhance the ability of genetic vaccines to enter target cells.
Although the mechanisms involved in DNA uptake are not well
understood, the methods of the invention enable one to obtain
genetic vaccines that exhibit enhanced entry to cells, and to
appropriate cellular compartments.
[0988] Enhancing the Efficiency and Specificity of a Genetic
Vaccine Nucleic Acid Uptake by a Given Cell Type by Coating the
Nucleic Acid with an Evolved Protein that Binds to the Genetic
Vaccine Nucleic Acid, and is also capable of Binding to the Target
Cell
[0989] In one embodiment, the invention provides methods of
enhancing the efficiency and specificity of a genetic vaccine
nucleic acid uptake by a given cell type by coating the nucleic
acid with an evolved protein that binds to the genetic vaccine
nucleic acid, and is also capable of binding to the target cell.
The vector can be contacted with the protein in vitro or in vivo.
In the latter situation, the protein is expressed in cells
containing the vector, optionally from a coding sequence within the
vector. The nucleic acid binding proteins to be evolved usually
have nucleic acid binding activity but do not necessarily have any
known capacity to enhance or alter nucleic acid DNA uptake.
[0990] DNA Binding Proteins that can be used in these Methods
[0991] DNA binding proteins which can be used in these methods
include, but are not limited to, transcriptional regulators,
enzymes involved in DNA replication (e.g., recA) and reassembly
(&/or one or more additional directed evolution methods
described herein), and proteins that serve structural functions on
DNA (e.g., histones, protamines). Other DNA binding proteins that
can be used include the phage 434 repressor, the lambda phage cl
and cro repressors, the E. coli CAP protein, myc, proteins with
leucine zippers and DNA binding basic domains such as fos and jun;
proteins with`POU` domains such as the Drosophila paired protein;
proteins with domains whose structures depend on metal ion
chelation such as Cys.sub.2His.sub.2 zinc fingers found in TFIIIA,
Zn.sub.2(Cys).sub.6 clusters such as those found in yeast Gal4, the
Cys.sub.3 His box found in retroviral nucleocapsid proteins, and
the Zn.sub.2(Cys).sub.8 clusters found in nuclear hormone
receptor-type proteins; the phage P22 Arc and Mnt repressors (see
Knight et al. (1989) J Biol. Chem. 264:3639-3642 and Bowie &
Sauerkl 989) J Biol. Chem. 264:7596-7602. RNA binding proteins are
reviewed by Burd & Dreyfuss (1994) Science 265:615-62 1, and
include HIV Tat and Rev.
[0992] Formats for Performing Reassembly (&/or One or More
Additional Directed Evolution Methods Described Herein)
[0993] As in other methods of the invention, evolution of DNA
binding proteins toward acquisition of improved or altered uptake
efficiency is effective by one or more cycles of reassembly
(&/or one or more additional directed evolution methods
described herein) and screening. The starting substrates can be
nucleic acid segments encoding natural or induced variants of one
or nucleic acid binding proteins, such as those mentioned above.
The nucleic acid segments can be present in vectors or in isolated
form for the reassembly (&/or one or more additional directed
evolution methods described herein) step. reassembly (&/or one
or more additional directed evolution methods described herein) can
proceed through any of the formats described herein.
[0994] For screening purposes, the reassembled (&/or subjected
to one or more directed evolution methods described herein) nucleic
acid segments are typically inserted into a vector, if not already
present in such a vector during the reassembly (&/or one or
more additional directed evolution methods described herein)
step.
[0995] Including Binding Site in Vector for DNA Binding Protein
Recognizing a Specific Binding Site
[0996] The vector generally encodes a selective marker capable of
being expressed in the cell type for which uptake is desired. If
the DNA binding protein being evolved recognizes a specific binding
site (e.g., lacl binding protein recognizes lacO), this binding
site can be included in the vector. Optionally, the vector can
contain multiple binding sites in tandem.
[0997] Transforming Vectors Containing Recombinant Segments into
Host Cells and Lysing Cells Under Mild Conditions that do not
Disrupt Binding of Vectors to DNA Binding Proteins
[0998] The vectors containing different recombinant segments are
transformed into host cells, usually E. coli, to allow recombinant
proteins to be expressed and bind to the vector encoding their
genetic material. Most cells take up only a single vector and so
transformation results in a population of cells, most of which
contain a single species of vector. After an appropriate period to
allow for expression and binding, cells are lysed under mild
conditions that do not disrupt binding of vectors to DNA binding
proteins. For example, a lysis buffer of 35 mM HEPES (pH 7.5 with
KOH), 0.1 mM EDTA, 100 mM Na glutamate, 5% glycerol, 0.3 mg/ml BSA,
1 mM DTT, and 0.1 mM PMSF) plus lysozyme (0.3-ml at 10 mg/ml) is
suitable (see Schatz et al., US 5,338,665). The complexes of vector
and nucleic acid binding protein are then contacted with cells of
the type for which improved or altered uptake is desired under
conditions favoring uptake. Suitable recipient cells include the
human cell types that are common targets in DNA vaccination. These
cells include muscle cells, monocytes/macrophages, dendritic cells,
B cells, Langerhans cells, keratinocytes, and the M-cells of the
gut. Cells from mammals including, for example, human, mouse, and
monkey can be used for screening. Both primary cells and cells
obtained from cell lines are suitable.
[0999] Recovery of Cells Expressing Marker and Enriching for
Recombinant Segments for Further Rounds of Selection
[1000] After incubation, cells are plated with selection for
expression of the selective marker present in the vector containing
the recombinant segments. Cells expressing the marker are
recovered. These cells are enriched for recombinant segments
encoding nucleic acid binding proteins that enhance uptake of
vectors encoding the respective recombinant segments. The
recombinant segments from cells expressing the marker can then be
subjected to a further round of selection. Usually, the recombinant
segments are first recovered from cells, e.g., by PCR amplification
or by recovery of the entire vectors. The recombinant segments can
then be reassembled (&/or subjected to one or more directed
evolution methods described herein) with each other or with other
sources of DNA binding protein variants to generate further
recombinant segments. The further recombinant segments are screened
in the same manner as before.
[1001] Using Stochastic (e.g. Polynucleotide Shuffling &
Interrupted Synthesis) and Non-Stochastic Polynucleotide Reassembly
to Evolve, Particularly, the Carboxy- and Amino-Terminal Peptide
Extensions of the Histone Protein, to Increase the Efficiency of
DNA Transfer into the Cells
[1002] One example of a method to evolve an optimized nucleic acid
binding domain involves the reassembly (optionally in combination
with other directed evolution methods described herein) of histone
genes. Histone-condensed DNA can result in increased gene transfer
into cells. See, e.g., Fritz et al. (1996) Human Gene Therapy
7:1395-1404. Thus, stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide reassembly
can be used to evolve the histone protein, particularly the
carboxy- and amino-terminal peptide extensions, to increase the
efficiency of DNA transfer into cells. In this approach, the
histone is encoded by the DNA to which it will be bound.
[1003] Construction of the Histone Library
[1004] The histone library can be constructed by, for example, 1)
reassembly (optionally in combination with other directed evolution
methods described herein) of many related histone genes from
natural diversity, 2) addition of random or partially randomized
peptide sequences at the N- and C-terminal sequences of the
histone, 3) by addition of pre-selected protein-encoding regions to
the N- or C-termini, such as whole cDNA libraries, nuclear protein
ligand libraries, etc. These proteins can be partially randomized
and linked to the histone by a library of linkers.
[1005] Starting Substrates for Evolving Nucleic Acid Binding Sites
Contain Variant Binding Sites and Recombinant forms of these Sites
are Screened as a Component of a Vector that also Encodes a Nucleic
Acid Binding Protein
[1006] In a variation of the above procedure, a binding site
recognized by a nucleic acid binding protein can be evolved instead
of, or as well as, the nucleic acid binding protein. Nucleic acid
binding sites are evolved by an analogous procedure to nucleic acid
binding proteins except that the starting substrates contain
variant binding sites and recombinant forms of these sites are
screened as a component of a vector that also encodes a nucleic
acid binding protein.
[1007] When the Evolved DNA Binding Protein does not have a High
Degree of Sequence Specificity and it is Unknown Precisely Which
Sites of the Vector used in Screening are Bound by the Protein, the
Vector should include all or most of the Screening Vector Sequences
Together with Additional Sequences Required to Effect Vaccination
or Therapy
[1008] Evolved nucleic acid segments encoding DNA binding proteins
and/or evolved DNA binding sites can be included in genetic vaccine
vectors. If the affinity of the DNA binding protein is specific to
a known DNA binding site, it is sufficient to include that binding
site and the sequence encoding the DNA binding protein in the
genetic vaccine vector together with such other coding and
regulatory sequences are required to effect gene therapy. In some
instances, the evolved DNA binding protein may not have a high
degree of sequence specificity and it may be unknown precisely
which sites on the vector used in screening are bound by the
protein. In these circumstances, the vector should include all or
most of the screening vector sequences together with additional
sequences required to effect vaccination or therapy. An exemplary
selection scheme which employs M 13 protein VIII is shown,
described &/or referenced herein (including incorporated by
reference).
[1009] Target Cells of Interest
[1010] Target cells of interest include, for example, muscle cells,
monocytes, dendritic cells, B cells, Langerhans cells,
keratinocytes, M-cells of the gut, and the like. Cell-specific
ligands that are suitable for use with each of the cell types are
known to those of skill in the art. For example, suitable proteins
to direct binding to antigen presenting cells include CD2, CD28,
CTLA-4, CD40 ligand, fibrinogen, factor X, ICAM-1, -glycan
(zymosan), and the Fc portion of immunoglobulin G. (Weir's Handbook
of Experimental Immunology, Eds. L.A. Herzenberg, D.M. Weir, L.A.
Herzenberg, C. Blackwell, 5th edition, volume IV, chapters 156 and
174) because their respective ligands are present on APCs,
including dendritic cells, monocytes/macrophages, B cells, and
Langerhans cells. Bacterial enterotoxins or subunits thereof are
also of interest for targeting purposes.
[1011] LPS Facilitates the Interaction between Vector and Monocytes
and is also likely to Act as an Adjuvant, Further Potentiating the
Immune Responses
[1012] The ability of the vectors to enter and activate APC, such
as monocytes, can also be enhanced by coating the vectors with
small quantities of lipopolysaccharide (LPS). This facilitates the
interaction between vector and monocytes, which have a cell surface
receptor for LPS. Due to its immunostimulatory activities, LPS is
also likely to act as an adjuvant, thereby further potentiating the
immune responses.
[1013] Receptor Binding Components of Enterotoxins can be Evolved
for Improved Attachment to Cell Surface Receptors, Improved Entry
to and Transport Across the Cells of the Intestinal Epithelium, and
Improved Binding to, and Activation of, B Cells or Other APCs
[1014] Enterotoxins produced by certain pathogenic bacteria are
useful as agents that bind cells and thus enhance delivery of
vaccines, antigens, gene therapy vectors and pharmaceutical
proteins. In an exemplary embodiment of the: invention, receptor
binding components of enterotoxins derived from Vibrio cholerae and
enterotoxigenic strains of E. coli are evolved for improved
attachment to cell surface receptors and for improved entry to and
transport across the cells of the intestinal epithelium. In
addition, they can be evolved for improved binding to, and
activation of, B cells or other APCs. An antigen of interest can be
fused to these toxin subunits to illustrate the feasibility of the
approach in oral delivery of proteins and to facilitate the
screening of evolved enterotoxin subunits. Examples of such
antigens include growth hormone, insulin, myelin basic protein,
collagen and viral envelope proteins.
[1015] Vectors that Contain the Library of Recombinant Enterotoxin
Binding Moiety Nucleic Acids are Transfected into a Population of
Host Cells, Wherein the Recombinant Enterotoxin Binding Moiety
Nucleic Acids are Expressed to Form Recombinant Enterotoxin Binding
Moiety Polypeptides
[1016] These methods involve reassembling (&/or subjecting to
one or more directed evolution methods described herein) at least
first and second forms of a nucleic acid which comprises a
polynucleotide that encodes a preferably non-toxic receptor binding
moiety of an enterotoxin. The first and second forms differ from
each other in two or more nucleotides, so the stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly results in production of a
library of recombinant enterotoxin binding moiety nucleic acids.
Suitable enterotoxins include, for example, a V cholerae
enterotoxin, enterotoxins from enterotoxigenic strains of E. coli,
salmonella toxin, shigella toxin and campylobacter toxin. Vectors
that contain the library of recombinant enterotoxin binding moiety
nucleic acids are transfected into a population of host cells,
wherein the recombinant enterotoxin binding moiety nucleic acids
are expressed to form recombinant enterotoxin binding moiety
polypeptides. In a preferred embodiment, the recombinant
enterotoxin binding moiety polypeptides are expressed as fusion
proteins on the surface of bacteriophage particles. The recombinant
enterotoxin binding moiety polypeptides can be screened by
contacting the library with a cell surface receptor of a target
cell and determining which recombinant enterotoxin binding moiety
polypeptides exhibit enhanced ability to bind to the target cell
receptor. The cell surface receptor can be present on the surface
of a target cell itself, or can be attached to a different cell, or
binding can be tested using cell surface receptor that is not
associated with a cell. Examples of suitable cell surface receptors
include, for example, Gm I. Similarly, one can evolve bacterial
superantigens for altered (increased or decreased) binding to T
cell receptor and MHC class H molecules. These superantigens
activate T cells in an antigen nonspecific manner.
[1017] Superantigens binding to T cell receptor/MHC class II
molecules include Staphylococcal enterotoxin B, Urtica dioica
superantigen (Musette et al. (1996) Eur. J Immunol. 26:618-22) and
Staphylococcal enterotoxin A (Bavari et al. (1996) J Infect. Dis.
174:338-45). Phage display has been shown to be effective when
selecting superantigens that bind MHC class H molecules (Wung and
Gascoigne (1997) J Immunol. Methods. 204:33-41).
[1018] Both CT and CT-B have been Shown to have Potent Adjuvant
Activities in vivo and they Enhance Immune Responses after Oral
Delivery of Antigens and Vaccines
[1019] Cholera toxin (CT) is an oligomeric protein of 84,000
daltons which consists of one toxic A subunit (CT-A) covalently
linked to five B subunits (CT-B). CT-B functions as the-receptor
binding component and binds to G.sub.MI, ganglioside receptors on
mammalian cell surfaces. The toxic A-subunit is not necessary for
the function of CT, and in the absence of CT-A, functional CT-B
pentamers can form (Lebens and Holingren (1994) Dev. Biol. Stand.
82:215-227). Both CT and CT-B have been shown to have potent
adjuvant activities in vivo and they enhance immune responses after
oral delivery of antigens and vaccines (Czerkinsky et al. (1996)
Ann. NY Acad. Sci. 778:185-93; Van Cott et al. (1996) Vaccine
14:392-8). Moreover, a single dose of CT-B conjugated to myelin
basic protein prevented onset of auto immune encephalomyelitis
(EAE), a murine model of multiple sclerosis (Czerkinsky et al.,
supra.). Furthermore, feeding animals with myelin basic protein
conjugated to CT-B after the onset of clinical symptoms (7 days)
attenuated the symptoms in these animals. Other bacterial toxins,
such as enterotoxins of E. coli, Salmonella toxin, Shigella toxin
and Campylobacter toxin, have structural similarities with CT.
Enterotoxins of E. coli have the same A-B structure as CT and they
also have sequence homology and share functional similarities.
[1020] Family Stochastic (e.g. Polynucleotide Shuffling &
Interrupted Synthesis) and Non-Stochastic Polynucleotide Reassembly
is Feasible among Enterotoxin-Encoding Nucleic Acids from Different
Bacterial Species
[1021] Bacterial enterotoxins can be evolved for improved affinity
and entry to cells by polynucleotide (e.g. gene, promoter,
enhancer, intron, & the like) reassembly (optionally in
combination with other directed evolution methods described
herein). The similarity of E. coli-derived enterotoxin subunit and
CT-B is 78%, and several completely conserved regions of more than
eight nucleotides can be found. B subunits from two different
strains of E. coli are 98% homologous both at sequence and protein
levels. Thus, family stochastic (e.g. polynucleotide shuffling
& interrupted synthesis) and non-stochastic polynucleotide
reassembly is feasible among enterotoxin-encoding nucleic acids
from different bacterial species.
[1022] Screen the Secretion of Chimeric Proteins by V cholerae by
Culturing the Bacteria in Agar in the Presence of Monoclonal
Antibodies Specific for the Antigen that was Fused to the
Toxins
[1023] The libraries of experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis) toxin subunits can be expressed in a suitable host
cell, such as V cholerae. For safety reasons, strains in which the
toxic CT-A is deleted are preferred. An antigen of interest can be
fused to the receptor-binding subunit. Secretion of chimeric
proteins by V cholerae can be screened by culturing the bacteria in
agar in the presence of monoclonal antibodies specific for the
antigen that was fused to the toxins and the level of secretion is
detected as immunoprecipitation in the agar around the
colonies.
[1024] Evolving for Improved Binding to the G.sub.M1, Ganglioside
Receptor and Other Receptors, Detecting Binding Between Receptor
and Chimeric Fusion Proteins with a Monoclonal Antibody Specific
for the Antigen that was Fused to the Toxin
[1025] One can also add G.sub.M1, ganglioside receptors to the agar
in order to detect colonies secreting functional enterotoxin
subunits. Colonies producing significant levels of the fusion
protein are then cultured in 96-well plates, and the culture medium
is tested for the presence of molecules capable of binding to cells
or receptors in solution. Binding of chimeric fusion proteins to
G.sub.M1, ganglioside receptors on cell surface or in solution can
be detected by a monoclonal antibody specific for the antigen that
was fused to the toxin. The assay using whole cells has the
advantage that one may evolve for improved binding also to
receptors other than the G.sub.M1, ganglioside receptor. When
increasing concentrations of wild-type enterotoxins are added to
these assays, one can detect mutants that bind to receptors with
improved affinities. Affinity and specificity of toxin binding can
also be determined by surface plasmon resonance (Kuziemko et al.
(1996) Biochemistry 35:6375-84).
[1026] Advantage of Large Scale Production and Avoidance of
Problems Associated with Expression on Phage in the Bacterial
Expression System
[1027] The advantage of the bacterial expression system is that the
fusion protein is secreted by bacteria that could potentially be
used in large scale production. Moreover, because the fusion
protein is in solution during selection, possible problems
associated with expression on phage (such as bias towards selection
of mutants that only function on phage) can be avoided.
[1028] In Phage Display, Mutants can be easily Further Selected in
in vivo Assays When Screening to Identify Enterotoxins with
Improved Affinities
[1029] Nevertheless, phage display is useful for screening to
identify enterotoxins with improved affinities. A library of
experimentally evolved (e.g. by polynucleotide reassembly &/or
polynucleotide site-saturation mutagenesis) mutants can be
expressed on phage, such as M 13, and mutants with improved
affinity are selected based on binding to, for example, G.sub.M1
ganglioside receptors in solution or on a cell surface. The
advantage of this approach is that the mutants can be easily
further selected in in vivo assays as discussed below. A screening
approach using fusion to M 13 protein VIII is diagrammed in FIG.
1.
[1030] The Recombinant Binding Moiety is Expressed in the Cells and
Binds to the Nucleic Acid Binding Domain to form a Vector-Binding
Moiety Complex
[1031] Finally, the resulting evolved enterotoxin can be fused with
DNA binding protein, and genetic vaccine vectors are coated with
this fusion protein. The stochastic (e.g. polynucleotide shuffling
& interrupted synthesis) and non-stochastic polynucleotide
reassembly can be done either separately, in which case the two
domains are assembled after reassembly (optionally in combination
with other directed evolution methods described herein), or in a
combined reaction. reassembly (optionally in combination with other
directed evolution methods described herein) results in production
of a library of recombinant binding moiety nucleic acids which can
be screened by transfecting vectors which contain the library, as
well as a binding site specific for the nucleic acid binding
domain, into a population of host cells. The binding moiety is
expressed in the cells and binds to the nucleic acid binding domain
to form a vector-binding moiety complex. Host cells can then be
lysed under conditions that do not disrupt binding of the
vector-binding moiety complex.
[1032] Optimized Recombinant Binding Moiety Nucleic Acids are
Isolated from Cells Containing the Vector
[1033] The vector-binding moiety complex can then be contacted with
a cell of interest, after which cells are identified that contain a
vector and the optimized recombinant binding moiety nucleic acids
are isolated from the cells.
[1034] Increasing the Number of Copies of Target DNA taken into
those Cells that Initially take up the same DNA (Mammalian
Cells)
[1035] Another method for obtaining enhanced uptake of a target DNA
by mammalian cells is also provided by the invention. Specifically,
the method increases the number of copies of target DNA taken into
those cells that initially take up the same DNA.
[1036] Cells that take up the Target Molecule of DNA (Cell Surface
Expression of Membrane-Associated DNA Binding Domains) will Express
the Factor and have Increased Specific Affinity for Target DNA that
Remains Extracellular, while Cells that did not take up DNA will be
at a Competitive Disadvantage as they will not bear the Cell
Surface Target DNA-Specific Binding Domain, Which is Required for
Specifically Mediated DNA Uptake
[1037] The method uses cell surface expression of
membrane-associated DNA binding domains of, for example,
transcription factors, that are encoded in the target DNA sequence,
which also includes the cognate recognition sequence for the
binding domain. Uptake of one molecule of target DNA into a cell
(by any process, passive uptake, electroporation, osmotic shock,
other stress) will lead to transcription of the gene encoding the
polynucleotide binding domain. The gene encoding the binding domain
is engineered so that the binding domain is expressed in a membrane
anchored form. For example, a hydrophobic stretch of amino acids
can be encoded at the carboxyl terminus of the binding domain, thus
leading to phospho-inositol-glycan (PIG) conjugation after partial
cleavage of this terminal sequence. This, in turn, leads to
trafficking and positioning of the binding domain on the cell
surface. The same cells that took up the first molecule of DNA will
express the factor and have increased specific affinity for target
DNA that remains extracellular. Cells that did not take up DNA will
be at a competitive disadvantage as they will not bear the cell
surface target DNA-specific binding domain, which is required for
specifically mediated DNA uptake.
[1038] Enhanced binding of the target DNA to the target cell will
increase the efficiency of DNA internalization and desired
intracellular function. This process represents a positive feedback
for increased DNA uptake into cells that take up DNA first.
[1039] Practical Means for Determining Which Transcription Factor
or Combination of Factors to use with any Particular Target DNA
[1040] The target DNA, whether a circular or linear plasmid,
oligonucleotide, bacterial or mammalian chromosomal fragment, is
engineered to bear one or more copies of a DNA recognition sequence
for a mammalian or bacterial transcription factor. Many target
sequences will already bear one or more such motifs; these can be
identified by sequence analysis. Endogenous motifs recognized by
these factors also can be identified experimentally by
demonstrating that the target DNA binds to one or more of a panel
of transcription factors in an appropriate assay format. This
provides a practical means for determining which factor or
combination of factors to use with any particular target DNA.
[1041] Motif(s) in the Case of a Small Oligonucleotide or a DNA
Plasmid and in the Cases Where more than one DNA Binding Protein
will be Expressed on the Cell Surface
[1042] In the case of a small oligonucleotide or a DNA plasmid
(such as used for a DNA vaccine), appropriate motifs can be
engineered into the sequence. A particular motif can be engineered
in one or more copies, in tandem or dispersed in the target
sequence. Alternatively, a set of different motifs can be
engineered, in tandem or separated, in cases where more than one
DNA binding protein will be expressed on the cell surface.
2.6.4. EVOLUTION OF BACTERIOPHAGE VECTORS
[1043] Using Stochastic (e.g. Polynucleotide Shuffling &
Interrupted Synthesis) and Non-Stochastic Polynucleotide
Reassembly, Phage Genetics and Display Technologies to Rapidly
Evolve Highly Novel, Potent, and Generic Vaccine Vehicles
[1044] The invention provides methods of obtaining bacteriophage
vectors that exhibit desirable properties for use as genetic
vaccine vectors. The principle behind the approach provided by the
invention is to combine the power of stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly with the extraordinary
power of bacteriophage genetics and the wealth of recent advances
in phage display technologies to rapidly evolve highly novel,
potent, and generic vaccine vehicles.
[1045] Methods for Delivery of Antigens from Pathogens to
Professional APCs, Maximizing Efficiency Through Increasing the
Kinetics and Potency of the Immune Response to the Vaccine
[1046] The evolved vaccine vehicles can present antigen either (1)
in native form on the surface of these APCs for the induction of an
antibody response or (2) selectively invade APCs and deliver DNA
vaccine constructs to APCs for intracellular expression, processing
and presentation to CTLs. More efficient methods for delivery of
antigens from pathogens to professional APCs will increase the
kinetics and potency of the immune response to the vaccine.
[1047] Affinity Maturation Process, Essential for the Generation of
Antibodies with Sufficient Affinity to Neutralize Pathogenic
Antigens, Occurs in Germinal Centers (Spleen) Where Follicular
Dendritic Cells Present Protein Antigens to B Cells and Processed
Antigen Fragments to T Cells, Making Efficient Delivery of Antigens
to FDCs Essential in Increasing the Kinetics and Potency of the
Immune Response to the Immunizing Antigen
[1048] Genetic vaccine delivery vehicles that are evolved according
to the methods of the invention are particularly valuable for the
rapid induction of high affinity antibodies which can effectively
neutralize viral epitopes or pathogenic toxins such as
superantigens or cholera toxin. High affinity antibodies are
generated by somatic mutation of low affinity primary response
antibodies. This so-called affinity maturation process is essential
for the generation of antibodies with sufficient affinity to
neutralize pathogenic antigens. Affinity maturation occurs in the
spleen in germinal centers where follicular dendritic cells (FDCs),
professional antigen presenting cells, present protein antigens to
B cells and processed antigen fragments to T cells. Clonally
expanding B cell populations which have undergone somatic mutation
are selected for those mutant B cells expressing antibodies with
improved affinity for antigen. Thus, efficient delivery of antigen
to FDCs will increase the kinetics and potency of the immune
response to the immunizing antigen. Additionally, processed antigen
bound to MHC is required to stimulate antigen specific T cells.
Genetic vaccines are particularly efficient at priming class I MHC
restricted responses due to intracellular expression of antigen,
with a resultant trafficking of antigen fragments to the class I
MHC pathway. Thus, invasive bacteriophage vectors capable of
delivery of genetic vaccine constructs or protein antigens to FDCs
are useful.
[1049] Preferred Bacteriophage for the Purpose of Evolution are
those that have been Genetically Well Characterized and Developed
for the Display of Foreign Protein Epitopes (of Special Note was
M13 Bacteriophage, a Small Filamentous Phage Which is a Versatile,
Highly Evolvable Vehicle for Efficient and Targeted Delivery of
Protein or DNA Vaccine Vehicles to Cellular Targets of Interest
[1050] Any of several bacteriophage can be evolved according to the
methods of the invention. Preferred bacteriophage for these
purposes are those that have been genetically well characterized
and developed for the display of foreign protein epitopes; these
include, for example, lambda, T7, and M13 bacteriophage. The
filamentous phage M13 is a particularly preferred vector for use in
the methods of the invention. M 13 is a small filamentous
bacteriophage that has been used widely to display polypeptide
fragments in functional, folded form on the surface of
bacteriophage particles. Polypeptides have been fused to both the
gene III and gene VIII coat proteins for such display purposes.
Thus, M13 is a versatile, highly evolvable vehicle for efficient
and targeted delivery of protein or DNA vaccine vehicles to
cellular targets of interest.
[1051] Improvements in Methods (Efficient Delivery of Phage, Homing
to APCs, and Invasion of Target Cells using Experimentally Evolved
(e.g. by Polynucleotide Reassembly &/or Polynucleotide
Site-Saturation Mutagenesis) Bacterial Invasion Proteins)
Exemplified for Bacteriophage Vectors and Applicable to Other Types
of Genetic Vaccine Vectors
[1052] The following three properties are examples of the type of
improvements that can be achieved by use of the methods of the
invention to evolve bacteriophage genetic vaccine vectors: (1)
efficient delivery of phage to the bloodstream by inhalation or
oral delivery, (2) efficient homing to APCs, and (3) efficient
invasion of target cells using experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis) bacterial invasion proteins. Where M13 is used,
fusions can be made to both gene 111 and gene VIII coat proteins so
that two evolved properties can be combined into a single phage
particle. These studies can be performed in test animals such as
laboratory mice so that the evolved constructs can be rapidly
characterized with respect to their potency as vaccine vehicles.
Evolved inhalable and/or orally deliverable vehicles and evolved
invasins will translate directly for use in human cells, while the
principles developed in evolving the ability to home to test animal
APCs are readily transferable to human cells by performing
analogous selections on human APCs. While these methods are
exemplified for bacteriophage vectors, the methods are also
applicable to other types of genetic vaccine vectors.
2.6.4.1. EVOLUTION OF EFFICIENT DELIVERY OF BACTERIOPHAGE VEHICLES
BY INHALATION OR ORAL DELIVERY
[1053] Method for the Formulation of Proteins into Inhalable
Colloids that can be Absorbed into the Blood Stream Through the
Lung (Preparation Involved in the Invention)
[1054] The invention provides methods for obtaining genetic vaccine
vectors that are capable of efficient delivery to the bloodstream
upon administration by inhalation or by oral administration.
Methods have been developed for the formulation of proteins into
inhalable colloids that can be absorbed into the blood stream
through the lung. The mechanisms by which proteins are transported
into the blood stream are not clearly understood, and thus
improvements are readily approached by evolutionary methods. Using
M 13 as an example, the invention involves preparation of a library
of, for example, peptide ligands, adhesion molecules, bacterial
enterotoxins, and randomly fragmented cDNA, which are fused to gene
111, for example, of M13. Libraries of>10.sup.10 individual
fusions are readily achievable with this technology.
[1055] M13 Phage Enters the Blood Stream, can be Recovered and
Amplified in E. coli Cells, Pass Through Several Rounds of
Enrichment, and be Further Characterized and Evolved by Sequencing
and Reassembling (Optionally in Combination with Other Directed
Evolution Methods Described Herein) the Entire Phage Genome and
Subjecting the Phage to Reiterated Cycles of Delivery, Recovery,
Amplification, and Reassembly (Optionally in Combination with Other
Directed Evolution Methods Described Herein)
[1056] Screening involves preparation of high titer stocks
(preferably>10.sup.12 phage particles) in standard colloidal
formulations which are delivered intranasally to test animals, such
as mice. Blood samples are taken over the course of the ensuing day
and circulating phage are amplified in E. coli. It has been
established that M13 circulates for long periods in the blood after
injection intravenously, and thus it is reasonable to expect that
phage that successfully enter the blood stream through the lung can
be efficiently recovered and amplified E. coli cells. In a
preferred embodiment, several rounds of enrichment are applied to
the initial libraries in order to enrich for phage that can
efficiently enter the blood stream when delivered intranasally.
Candidate clones are typically tested individually for their
relative efficiency of entry, and the best clones can be further
characterized by sequencing to identify the nature of the fusions
that confer efficient delivery (of particular interest from the
cDNA libraries). Selected clones can be further evolved and for
improved entry by reassembling (optionally in combination with
other directed evolution methods described herein) the entire phage
genome and subjecting the phage to reiterated cycles of delivery,
recovery, amplification, and reassembly (optionally in combination
with other directed evolution methods described herein).
[1057] To Obtain Vaccine Vectors that are Effective When Taken
Orally, Recombinant Vectors Prepared Through Reassembly (Optionally
in Combination with Other Directed Evolution Methods Described
Herein) are Administered, Surviving, Stable Vectors are Recovered
from the Stomach, and Vectors that Efficiently enter the
Bloodstream and/or Lymphatic Tissue can be Recovered from the
Blood/Lymph (FIG. 2).
[1058] An analogous procedure is used to obtain vaccine vectors
that are effective when delivered orally. A genetic vaccine vector
library is prepared by stochastic (e.g. polynucleotide shuffling
& interrupted synthesis) and non-stochastic polynucleotide
reassembly. The recombinant vectors are packaged and administered
to a test animal. Vectors that are stable in the stomach/intestinal
environment are recovered, for example, by recovering surviving
vectors from the stomach. Vectors that efficiently enter the
bloodstream and/or lymphatic tissue can be identified by recovering
vectors that reach the blood/lymph. A schematic of this selection
method is shown, described &/or referenced herein (including
incorporated by reference).
2.6.4.2. EVOLUTION OF BACTERIOPHAGE VEHICLES FOR EFFICIENT HOMING
TO APCs
[1059] Two Selection Formats: the First Consisting of Enriching the
Libraries of Random Peptide Ligands and cDNAs used in (A) Above for
Phage Which Selectively Bind to APCs and Using Either Negative or
Positive Selection; the Second Consists of Injecting Phage
Libraries Intravenously, Collecting Target Organs of Interest,
Liberating the Phage by Sonication, Further Amplifying and
Enriching.
[1060] The invention also provides methods of evolving
bacteriophage vectors, as well as other types of genetic vaccine
vectors, for efficient homing to professional antigen presenting
cells. Libraries of random peptide ligands and cDNAs used in (A)
above are enriched for phage which selectively bind to APCs by
first negatively selecting for binding to non-APC cell types, and
then positively selecting for binding to APCs. The selections is
typically performed by mixing high titer stocks of phage from the
libraries (>10.sup.12 phage particles) with cells
(.about.10.sup.7 cells per selection cycle) and either taking the
nonbinding phage (negative selection) or the binding phage from
cell pellets (positive selection). An alternative selection format
consists of injecting phage libraries intravenously, allowing the
libraries to circulate for several hours, collecting target organs
of interest (lymph node, spleen), and liberating the phage by
sonication. The positively selected phage can be amplified in E.
coli and further rounds of enrichment are performed (3-5 rounds) if
further optimization is desired. After the chosen number of rounds,
individual phage are characterized for their ability to home to
lymphoid organs. The best few candidates can be subjected to
further evolution through iterated rounds of selection,
amplification, and reassembly (optionally in combination with other
directed evolution methods described herein).
2.6.4.3. EVOLUTION OF BACTERIOPHAGE FOR INVASION OF APCs
[1061] The methods of the invention are also useful for evolving
bacteriophage and other genetic vaccine vehicles for invasion of
target cells. This opens up the possibility of targeting the class
I MHC antigen processing pathways with either internalized protein
antigen or antigen expressed by DNA vaccine vehicles carried in by
the evolved vector.
[1062] Efficient Internalization of Pathogenic Bacteria Through
Invasin Interaction with Integrins
[1063] Invasins comprise a large family of bacterial proteins which
interact with integrins and promote the efficient internalization
of pathogenic bacteria such as Salmonella.
[1064] Reassembly (Optionally in Combination with Other Directed
Evolution Methods Described Herein) of Different Forms of
Polynucleotides Encoding Invasins, Cloning as Fusions to the M13
Gene VIII Coat Protein Gene, Preparing Libraries and Mixing these
Libraries with Target APCs
[1065] This embodiment of the invention involves reassembling
(optionally in combination with other directed evolution methods
described herein) different forms of polynucleotides that encode
invasins. For example, two or more genes which encode the invasin
family of proteins can be experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis). The experimentally evolved (e.g. by polynucleotide
reassembly &/or polynucleotide site-saturation mutagenesis)
polynucleotides can be cloned as fusions to the M 13 gene VIII coat
protein gene, for example, and high titer stock of such libraries
will be prepared. These libraries of bacteriophage can be mixed
with target APCs.
[1066] Removing Free Phage and Phage Bound to the Cell Surface
[1067] After incubation, the cells are exhaustively washed to
remove free phage and phage bound to the surface of the cells can
be removed by panning against polyclonal anti-M13 antibodies.
[1068] Obtaining Successful Phage, Amplifying, Reassembling
(Optionally in Combination with Other Directed Evolution Methods
Described Herein), and Selecting, Characterizing for Relative
Invasiveness, Combing with Gene III Fusions (Encoding Pathogenic
Epitopes of Interest) and Testing for Relative Abilities to Induce
a CTL Response to the Pathogenic Antigens
[1069] The cells are then sonicated, thus releasing phage that have
successfully entered the target cells (thus protecting them from
the polyclonal anti-M13 antiserum). These phage can, if desired, be
amplified, experimentally evolved (e.g. by polynucleotide
reassembly &/or polynucleotide site-saturation mutagenesis),
and the selective cycle will be iteratively applied for, e.g.,
3-times. Individual phage from the final cycle can then be
characterized with respect to their relative invasiveness. The best
candidates can then be combined with gene III fusions that encode
pathogenic epitopes of interest. These phage can be injected into
mice and tested for their relative abilities to induce a CTL
response to the pathogenic antigens.
[1070] Bacteriophage vaccine vehicles evolved for activity in mice
according to the above methods will establish the principles for
the evolution of similar vehicles for potent human vaccines. The
ability to induce more rapid and potent CTL and neutralizing
antibody responses with such vehicles is an important new tool for
the evolution of improved countermeasures against pathogens of
interest.
2.6.5. EVOLUTION OF IMPROVED IMMUNOMODULATORY SEQUENCES
[1071] Cytokines can dramatically influence macrophage activation
and T.sub.H1/T.sub.H2 cell differentiation, and thereby the outcome
of infectious diseases. In addition, recent studies strongly
suggest that DNA itself can act as adjuvant by activating the cells
of the immune system. Specifically, unmethylated CpG-rich DNA
sequences were shown to enhance T.sub.H1 cell differentiation,
activate cytokine synthesis by monocytes and induce proliferation
of B lymphocytes. The invention thus provides methods for enhancing
the immunomodulatory properties of genetic vaccines (a) by evolving
the stimulatory properties of DNA itself and (b) by evolving genes
encoding cytokines and related molecules that are involved in
immune system regulation. These genes are then used in genetic
vaccine vectors.
[1072] Of particular interest are IFN- (x and IL-12, which skew
immune responses towards a T helper I (T.sub.H1) cell phenotype
and, thereby, improve the host's capacity to counteract pathogen
invasions. Also provided are methods of obtaining improved
immunomodulatory nucleic acids that are capable of inhibiting or
enhancing activation, differentiation, or anergy of
antigen-specific T cells. Because of the limited information about
the structures and mechanisms that regulate these events, molecular
breeding C71 techniques of the invention provide much faster
solutions than rational design.
[1073] The methods of the invention typically involve the use of
stochastic (e.g. polynucleotide shuffling & interrupted
synthesis) and non-stochastic polynucleotide reassembly or other
methods to create a library of experimentally generated (in vitro
&/or in vivo) polynucleotides. The library is then screened to
identify experimentally generated polynucleotides in the library,
when included in a genetic vaccine vector or administered in
conjunction with a genetic vaccine, are capable of enhancing or
otherwise altering an immune response induced by the vector. The
screening step, in some embodiments, can involve introducing a
genetic vaccine vector that includes the experimentally generated
polynucleotides into mammalian cells and determining whether the
cells, or culture medium obtained by growing the cells, is capable
of modulating an immune response.
[1074] Optimized recombinant vector modules obtained through
polynucleotide reassembly (&/or one or more additional directed
evolution methods described herein) are useful not only as
components of genetic vaccine vectors, but also for production of
polypeptides, e.g., modified cytokines and the like, that can be
administered to a mammal to enhance or shift an immune response.
Polynucleotide sequences obtained using the stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly methods of the inventions
can be used as a component of a genetic vaccine, or can be used for
production of cytokines and other immunomodulatory polypeptides
that are themselves used as therapeutic or prophylactic reagents.
If desired, the sequence of the optimized immunomodulatory
polypeptide-encoding polynucleotides can be determined and the
deduced amino acid sequence used to produce polypeptides using
methods known to those of skill in the art.
2.6.5.1 IMMUNOSTIMULATORY DNA SEQUENCES
[1075] The invention provides methods of obtaining polynucleotides
that are immunostimulatory when introduced into a mammal.
Oligonucleotides that contain hexamers with a central CpG flanked
by two 5' purines (GpA or ApA) and two 3' pyrimidines (TpC or TpT)
efficiently induce cytokine synthesis and B cell proliferation
(Krieg et al. (1995) Nature 374:546; Klinman et al. (1996) Proc.
Nat'l. Acad. Sci. USA 93:2879; Pisetsky (1996) Immunity 5:303-10)
in vitro and act as adjuvants in vivo. Genetic vaccine vectors in
which immunostimulatory sequence- (ISS) containing oligos are
inserted have increased capacity to enhance antigen-specific
antibody responses after DNA vaccination. The minimal length of an
ISS oligonucleotide for functional activity in vitro is eight
(Klinman et al., supra.). Twenty-mers with three CG motifs were
found to be significantly more efficient in inducing cytokine
synthesis than a 15-mer with two CG motifs (1 d.). GGGG tetrads
have been suggested to be involved in binding of DNA to cell
surfaces (macrophages express receptors. for example scavenger
receptors, that bind DNA) (Pisetsky et al., supra.).
[1076] According to the invention, a library is generated by
subjecting to reassembly (&/or one or more additional directed
evolution methods described herein) random DNA (e.g., fragments of
human, murine, or other genomic DNA), oligonucleotides that contain
known ISS, poly A, C, G or T sequences, or combinations thereof.
The DNA, which includes at least first and second forms which
differ from each other in two or more nucleotides, are reassembled
(&/or subjected to one or more directed evolution methods
described herein) to produce a library of experimentally generated
polynucleotides.
[1077] The library is then screened to identify those
experimentally generated polynucleotides that exhibit
immunostimulatory properties, For example, the library can be
screened for induction cytokine production in vitro upon
introduction of the library into an appropriate cell type. A
diagram of this procedure is shown, described &/or referenced
herein (including incorporated by reference). Among the cytokines
that can be used as an indicator of immunostimulatory activity are,
for example, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, IL-15,
and IFN-. One can also test for changes in ratios of IL-4,IFN-y,
IL-4/IL-2, IL-5/IFN-, IL-5/IL-2, IL-I 3/IFN-, IL-13/IL-2. An
alternative screening method is the determination of the ability to
induce proliferation of cells involved in immune responses, such as
B cells, T cells, monocytes/macrophages, total PBL, and the like.
Other screens include detecting induction of APC activation based
on changes in expression levels of surface antigens, such as B7-1
(CD80), B7-2 (CD86), MHC class I and II, and CD 14.
[1078] Other useful screens include identifying, experimentally
generated polynucleotides that induce T cell proliferation. Because
ISS sequences induce B cell activation, and because of several
homologies between surface antigens expressed by T cells and B
cells, polynucleotides can be obtained that have stimulatory
activities on T cells.
[1079] Libraries of experimentally generated polynucleotides can
also be screened for improved CTL and antibody responses in vivo
and for improved protection from infection, cancer, allergy or
autoimmunity. Experimentally generated polynucleotides that exhibit
the desired property can be recovered from the cell and, if further
improvement is desired, the reassembly (optionally in combination
with other directed evolution methods described herein) and
screening, can be repeated. Optimized ISS sequences can used as an
adjuvant separately from an actual vaccine, or the DNA sequence of
interest can be fused to a genetic vaccine vector.
2.6.5.2. CYTOKINES, CHEMOKINES, AND ACCESSORY MOLECULES
[1080] The invention also provides methods for obtaining optimized
cytokines, cytokine antagonists, chemokines, and other accessory
molecules that direct, inhibit, or enhance immune responses. For
example, the methods of the invention can be used to obtain genetic
vaccines and other reagents (e.g., optimized cytokines, and the
like) that, when administered to a mammal, improve or alter an
immune response. These optimized immunomodulators are useful for
treating infectious diseases, as well as other conditions such as
inflammatory disorders, in an antigen non-specific manner.
[1081] For example, the methods of the invention can be used to
develop optimized immunomodulatory molecules for treating
allergies. The optimized immunomodulatory molecules can be used
alone or in conjunction with antigen-specific genetic vaccines to
prevent or treat allergy. Four basic mechanisms are available by
which one can achieve specific immunotherapy of allergy. First, one
can administer a reagent that causes a decrease in
allergen-specific T.sub.H2 cells. Second, a reagent can be
administered that causes an increase in allergen-specific T.sub.H1
cells. Third, one can direct an increase in suppressive CD8.sup.+ T
cells.
[1082] Finally, allergy can be treated by inducing anergy of
allergen-specific T cells. In this Example, cytokines are optimized
using the methods of the invention to obtain reagents that are
effective in achieving one or more of these immunotherapeutic
goals. The methods of the invention are used to obtain
anti-allergic cytokines that have one or more properties such as
improved specific activity, improved secretion after introduction
into target cells, are effective at a lower dose than natural
cytokines, and fewer side effects. Targets of particular interest
include interferon-/, IL-10, IL-12, and antagonists of IL-4 and
IL-13.
[1083] The optimized immunomodulators, or optimized experimentally
generated polynucleotides that encode the immunomodulators, can be
administered alone, or in combination with other accessory
molecules. Inclusion of optimal concentrations of the appropriate
molecules can enhance a desired immune response, and/or direct the
induction or repression of a particular type of immune response.
The polynucleotides that encode the optimized molecules can be
included in a genetic vaccine vector, or the optimized molecules
encoded by the genes can be administered as polypeptides.
[1084] In the methods of the invention, a library of experimentally
generated polynucleotides that encode immunomodulators is created
by subjecting substrate nucleic acids to a reassembly (&/or one
or more additional directed evolution methods described herein)
protocol, such as stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide reassembly
or other method known to those of skill in the art. The substrate
nucleic acids are typically two or more forms of a nucleic acid
that encodes an immunomodulator of interest.
[1085] Cytokines are among the immunomodulators that can be
improved using the 0 methods of the invention. Cytokine synthesis
profiles play a crucial role in the capacity of the host to
counteract viral, bacterial and parasitic infections, and cytokines
can dramatically influence the efficacy of genetic vaccines and the
outcome of infectious diseases. Several cytokines, for example
IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,
IL-12, IL-13, IL-14, IL-1 5, IL-16, IL-17, IL-18, G-CSF, GM-CSF,
IFN-, IFN-, TGF-, TNF-, TNF-, IL-20 (MDA-7), and flt-3 ligand have
been shown stimulate immune responses in vitro or in vivo. Immune
functions that can be enhanced using appropriate cytokines include,
for example, B cell proliferation, Ig synthesis, Ig isotype
switching, T cell proliferation and cytokine synthesis,
differentiation of T.sub.H1 and T.sub.H2 cells, activation and
proliferation of CTLs, activation and cytokine production by
monocytes/macrophages/dendritic cells, and differentiation of
dendritic cells from monocytes/macrophages.
[1086] In some embodiments, the invention provides methods of
obtaining optimized immummomodulators that can direct an immune
response towards a T.sub.H1 or a T.sub.H2 response. The ability to
influence the direction of immune responses in this manner is of
great importance in development of genetic vaccines. Altering the
type of T.sub.H response can fundamentally change the outcome of an
infectious disease. A high frequency of T.sub.H1 cells generally
protects from lethal infections with intracellular pathogens,
whereas a dominant T.sub.H2 phenotype often results in
disseminated, chronic infections. For example, in human, the
T.sub.H1 phenotype is present in the tuberculoid (resistant) form
of leprosy, while the T.sub.H2 phenotype is found in lepromatous,
multibacillary (susceptible) lesions (Yamamura et al. (1991)
Science 254:277). Late-stage AIDS patients have the T.sub.H2
phenotype. Studies in family members indicate that survival from
meningococcal septicemia depends on the cytokine synthesis profile
of PBL, with high IL-10 synthesis being associated with a high risk
of lethal outcome and high TNF- being associated with a low risk.
Similar examples are found in mice. For example, BALB/c mice are
susceptible to Leishmania major infection; these mice develop a
disseminated fatal disease with a T.sub.H2 phenotype. Treatment
with anti-IL-4 monoclonal antibodies or with IL-12 induces a
T.sub.H1 response, resulting in healing. Anti-interferon-monoclonal
antibodies exacerbate the disease. For some applications, it is
preferable to direct an immune response in the direction of a
T.sub.H2 response.
[1087] For example, where increased mucosal immunity is desired,
including protective immunity, enhancing the T.sub.H.sup.2 response
can lead to increased antibody production, particularly IgA. T
helper (T.sub.H) cells are probably the most important regulators
of the immune system. T.sub.H cells are divided into two subsets,
based on their cytokine synthesis pattern (Mosmann and Coffman
(1989) Adv. Immunol. 46:111). T.sub.H1 cells produce high levels of
the cytokines IL-2 and IFN- and no or minimal levels of IL-4, IL-5
and IL- 13. In contrast, T.sub.H2 cells produce high levels of
IL-4, IL-5 and IL-13, and IEL-2 and IFN- production is minimal or
absent. T.sub.H1 cells activate macrophages, dendritic cells and
augment the cytolytic activity of CD8.sup.+ cytotoxic T lymphocytes
and natural killer (NK) cells (Paul and Seder (1994) Cell 76:24 1),
whereas T.sub.H2 cells provide efficient help for B cells and also
mediate allergic responses due to the capacity of T.sub.H2 cells to
induce IgE isotype switching and differentiation of B cells into
IgE secreting cells (Punnonen et al. (1993) Proc. Nat'l. Acad. Sci.
USA 90:3730).
[1088] The screening methods for improved cytokines, chemokines,
and other accessory molecules are generally based on identification
of modified molecules that exhibit improved specific activity on
target cells that are sensitive to the respective cytokine,
chemokine, or other accessory molecules. A library of recombinant
cytokine, chemokine, or accessory molecule nucleic acids can be
expressed on phage or as purified protein and tested using in vitro
cell culture assays, for example. Importantly, when analyzing the
recombinant nucleic acids as components of DNA vaccines, one can
identify the most optimal DNA sequences (in addition to the
functions of the protein products) in terms of their
immunostimulatory properties, transfection efficiency, and their
capacity to improve the stabilities of the vectors. The identified
optimized recombinant nucleic acids can then be subjected to new
rounds of reassembly (optionally in combination with other directed
evolution methods described herein) and selection.
[1089] In one embodiment of the invention, cytokines are evolved
that direct differentiation of T.sub.H1 cells. Because of their
capacities to skew immune responses towards a T.sub.H1 phenotype,
the genes encoding interferon- (IFN-) and interleukin-12 (IL-12)
are preferred substrates for reassembly (&/or one or more
additional directed evolution methods described herein) and
selection in order to obtain maximal specific activity and capacity
to act as adjuvants in genetic vaccinations. IFN- is a particularly
preferred target for optimization using the methods of the
invention because of its effects on the immune system, tumor cells
growth and viral replication. Due to these activities, IFN- was the
first cytokine to be used in clinical practice. Today, IFN- is used
for a wide variety of applications, including several types of
cancers and viral diseases. IFN-also efficiently directs
differentiation of human T cells into T.sub.H1 phenotype (Parronchi
et al. (1992) J Immunol. 149:2977). However, it has not been
thoroughly investigated in vaccination models, because, in contrast
to human systems, it does not affect T.sub.H1 differentiation in
mice.
[1090] The species difference was recently explained by data
indicating that, like IL-12, IFN- induces STAT4 activation in human
cells but not in murine cells, and STAT4 has been shown to be
required in IL-12 mediated T.sub.H1 differentiation (Thierfelder et
al. (1996) Nature 382:171).
[1091] Family stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide reassembly
is a preferred method for optimizing IFN-, using as substrates the
mammalian IFN-. genes, which are 85% - 97% homologous. Greater
10.sup.26 distinct recombinants can be generated from the natural
diversity in these genes. To allow rapid parallel analysis of
recombinant interferons, one can employ high throughput methods for
their expression and biological assay as fusion proteins on
bacteriophage.
[1092] Recombinants with improved potency and selectivity profiles
are being selectively bred for improved activity. Variants which
demonstrate improved binding to IFN- receptors can be selected for
further analysis using a screen for mutants with optimal capacity
to direct T.sub.H1 differentiation. More specifically, the
capacities of IFN- mutants to induce IL-2 and IFN- production in in
vitro human T lymphocyte cultures can be studied by
cytokine-specific ELISA and cytoplasmic cytokine staining and flow
cytometry.
[1093] IL-12 is perhaps the most potent cytokine that directs
T.sub.H1 responses, and it has also been shown to act as an
adjuvant and enhance T.sub.H1 responses following genetic
vaccinations (Kim et al. (1997) J Immunol. 15 8:816). IL-12 is both
structurally and functionally a unique cytokine. It is the only
heterodimeric cytokine known to date, composed of a 35 kD light
chain (p35) and a 40 kD heavy chain (p40) (Kobayashi et al (1989) J
Exp. Med. 170:827; Stem et al. (1990) Proc. Nat'l. Acad. Sci. USA
87:6808).
[1094] Recently Lieschke et al. ((1997) Nature Biotech. 15:3 5)
demonstrated that a fusion between p35 and p40 genes results in a
single gene that has activity comparable to that of the two genes
expressed separately. These data indicate that it is possible to
reassemble IL-12 genes as one entity, which is beneficial in
designing the reassembly protocol (optionally in combination with
other directed evolution methods described herein). Because of its
T cell growth promoting activities, one can use normal human
peripheral blood T cells in the selection of the most active IL-12
genes, enabling direct selection of IL-12 mutants with the most
potent activities on human T cells. IL-12 mutants can be expressed
in CHO cells, for example, and the ability of the supernatants to
induce T cell proliferation determined (FIG. 6). The concentrations
of IL-12 in the supernatants can be normalized based on a specific
ELISA that detects a tag fused to the experimentally evolved (e.g.
by polynucleotide reassembly &/or polynucleotide
site-saturation mutagenesis) IL-12 molecules.
[1095] Incorporation of evolved IFN- and/or IL-12 genes into
genetic vaccine vectors is expected to be safe. The safety of IFN-
has been demonstrated in numerous clinical studies and in everyday
hospital practice. A Phase II trial of IL-12 in the treatment of
patients with renal cell cancer resulted in several unexpected
adverse effects (Tahara et al. (1995) Human Gene Therapy 6:1607).
However, IL-12 gene as a component of genetic vaccines alms at high
local expression levels, whereas the levels observed in circulation
are minimal compared to those observed after systemic bolus
injections. In addition, some of the adverse effects of systemic
IL-12 treatments are likely to be related to its unusually long
half-life (up to 48 hours in monkeys). stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly may allow selection for a
shorter half-life, thereby reducing the toxicity even after high
bolus doses.
[1096] In other cases, genetic vaccines that can induce T.sub.H2
responses are preferred, especially when improved antibody
production is desired. As an example, IL-4 has been shown to direct
differentiation of T.sub.H2 cells (which produce high levels of
IL-4, IL-5 and IL-13, and mediate allergic immune responses).
Immune responses that are skewed towards T.sub.H2 phenotype are
preferred when genetic vaccines are used to immunize against
autoimmune diseases prophylactically. T.sub.H1 responses are also
preferred when the vaccines are used to treat and modulate existing
autoimmune responses, because autoreactive T cells are generally of
T.sub.H1 phenotype (Liblau et al. (1995) Immunol. Today 16:34-38).
IL-4 is also the most potent cytokine in induction of IgE
synthesis; IL-4 deficient mice are unable to produce IgE. Asthma
and allergies are associated with an increased frequency of IL-4
producing cells, and are genetically linked to the locus encoding
IL-4, which is on chromosome 5 (in close proximity to genes
encoding IL-3, IL-5, IL-9, IL-13 and GM-CSF). IL-4, which is
produced by activated T cells, basophils and mast cells, is a
protein that has 153 amino acids and two potential N-glycosylation
sites. Human IL-4 is only approximately 50% identical to mouse
IL-4, and IL-4 activity is species-specific. In human, IL-13 has
activities similar to those of IL-4, but IL-13 is less potent than
IL-4 in inducing IgE synthesis. IL-4 is the only cytokine known to
direct T.sub.H2 differentiation.
[1097] Improved IL-2 agonists are also useful in directing T.sub.H2
cell differentiation, whereas improved IL-4 antagonists can direct
T.sub.H1 cell differentiation. Improved IL-4 agonists and
antagonists can be generated by the reassembly. (optionally in
combination with other directed evolution methods described herein)
of IL-4 or soluble IL-4 receptor. The IL-4 receptor consists of an
IL-4R -chain (140 kD high-affinity binding unit) and an IL-2R-chain
(these cytokine receptors share a common 7-chain). The IL-4R-chain
is shared by IL-4 and IL-13 receptor complexes. Both IL-4 and IL-13
induce phosphorylation of the IL-4R-chain, but expression of
IL-4R-chain alone on transfectants is not sufficient to provide a
functional IL-4R. Soluble IL-4 receptor currently in clinical
trials for the treatment of allergies. Using the stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly methods of the invention,
one can evolve a soluble IL-4 receptor that has improved affinity
for IL-4. Such receptors are useful for the treatment of asthma and
other T.sub.H2 cell mediated diseases, such as severe allergies.
The reassembly (optionally in combination with other directed
evolution methods described herein) reactions can take advantage of
natural diversity present in cDNA libraries from activated T cells
from human and other primates. In a typical embodiment, a
experimentally evolved (e.g. by polynucleotide reassembly &/or
polynucleotide site-saturation mutagenesis) IL-4R-chain library is
expressed on a phage, and mutants that bind to IL-4 with improved
affinity are identified. The biological activity of the selected
mutants is then assayed using cell-based assays.
[1098] IL-2 and IL-1 5 are also of particular interest for use in
genetic vaccines. IL-2 acts as a growth factor for activated B and
T cells, and it also modulates the functions of NK-cells. IL-2 is
predominantly produced by T.sub.H1-like T cell clones, and,
therefore, it is considered mainly to function in delayed type
hypersensitivity reactions. However, IL-2 also has potent, direct
effects on proliferation and Ig-synthesis by B cells. The complex
immunoregulatory properties of IL-2 are reflected in the phenotype
of IL-2 deficient mice, which have high mortality at young age and
multiple defects in their immune functions including spontaneous
development of inflammatory bowel disease. IL-15 is a more recently
identified cytokine produced by multiple cell types. IL-15 shares
several, but not all, activities with IL-2. Both IL-2 and IL-15
induce B cell growth and differentiation. However, assuming that
IL-15 production in IL-2 deficient mice is normal, it is clear that
IL-15 cannot substitute for the function of IL-2 in vivo, since
these mice have multiple immunodeficiencies. IL-2 has been shown to
synergistically enhance IL-10-induced human Ig production in the
presence of anti-CD40 mAbs, but it antagonized the effects of IL-4.
IL- 2 also enhances IL-4-dependent IgE synthesis by purified B
cells. On the other hand, IL-2 was shown to inhibit IL-4-dependent
murine IgG1 and IgE synthesis both in vitro and in vivo. Similarly,
IL-2 inhibited IL-4-dependent human IgE synthesis by unfractionated
human PBMC, but the effects were less significant than those of
IFN- or IFN-. Due to their capacities to activate both B and T
cells, IL-2 and IL- 15 are useful in vaccinations. In fact, IL-2,
as protein and as a component of genetic vaccines, has been shown
to improve the efficacy of the vaccinations. Improving the specific
activity and/or expression levels/kinetics of IL-2 and IL-15
through use of the stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide reassembly
methods of the invention increases the advantageous effects
compared to wild-type IL-2 and IL-1 5.
[1099] Another cytokine of particular interest for optimization and
use in genetic vaccines according to the methods of the invention
is interleukin-6. IL-6 is a monocyte-derived cytokine that was
originally described as a B cell differentiation factor or B cell
stimulatory factor-2 because of its ability to enhance Ig levels
secreted by activated B cells.
[1100] IL-6 has also been shown to enhance IL-4-induced I-E
synthesis. It has also been suggested that IL-6 is an obligatory
factor for human IgE synthesis, because neutralizing anti-IL-6 mAbs
completely blocked IL-4-induced IgE synthesis. IL-6 deficient mice
have impaired capacity to produce IgA. Because of its potent
activities on the differentiation of B cells, IL-6 can enhance the
levels of specific antibodies produced following vaccination. It is
particularly useful as a component of DNA vaccines because high
local concentrations can be achieved, thereby providing the most
potent effects on the cells adjacent to the transfected cells
expressing the immunogenic antigen. IL-6 with improved specific
activity and/or with improved expression levels, obtained by
stochastic (e.g. polynucleotide shuffling & interrupted
synthesis) and non-stochastic polynucleotide reassembly, will have
more beneficial effects than the wild-type IL-6.
[1101] Interleukin-8 is another example of a cytokine that, when
modified according to the methods of the invention, is useful in
genetic vaccines. IL-8 was originally identified as a
monocyte-derived neutrophil chemotactic and activating factor.
Subsequently, IL-8 was also shown to be chemotactic for T cells and
to activate basophils resulting in enhanced histamine and
leukotriene release from these cells. Furthermore, IL-8 inhibits
adhesion of neutrophils to cytokine-activated endothelial cell
monolayers, and it protects these cells from neutrophil-mediated
damage. Therefore, endothelial cell derived IL-8 was suggested to
3.sub.--31 attenuate inflammatory events occurring in the proximity
of blood vessel walls. IL-8 also modulates immunoglobulin
production, and inhibits IL-4-induced IgG4 and IgE synthesis by
both unfractionated human PBMC and purified B cells in vitro. This
inhibitory effect was independent of IFN-, IFN- or prostaglandin
E2. In addition, IL-8 inhibited spontaneous IgE synthesis by PBMC
derived from atopic patients. Due to its capacity to attract
inflammatory cells, IL-8, like other chemotactic agents, is useful
in potentiating the functional properties of vaccines, including
DNA vaccines (acting as an adjuvant). The beneficial effects of
IL-8 can be improved by using the stochastic (e.g. polynucleotide
shuffling & interrupted synthesis) and non-stochastic
polynucleotide reassembly methods of the invention to obtain IL-8
with improved specific activity and/or with improved expression in
target cells.
[1102] Interleukin-5, and antagonists thereof, can also be
optimized using the methods of the invention for use in genetic
vaccines. IL-5 is primarily produced by T.sub.H2-type T cells and
appears to play an important role in the pathogenesis of allergic
disorders because of its ability to induce eosinophilia. IL-5 acts
as an eosinophil differentiation and survival factor in both mouse
and man. Blocking IL-5 activity by use of neutralizing monoclonal
antibodies strongly inhibits pulmonary eosinophilia and
hyperactivity in mouse models, and IL-5 deficient mice do not
develop eosinophilia. These data also suggest that IL-5 antagonists
may have therapeutic potential in the treatment of allergic
eosinophilia.
[1103] IL-5 has also been shown to enhance both proliferation of,
and Ig synthesis by, activated mouse and human B cells. However,
other studies suggested that IL-5 has no effect on proliferation of
human B cells, whereas it activated eosinophils. IL-5 apparently is
not crucial for maturation or differentiation of conventional B
cells, because antibody responses in IL-5 deficient mice are
normal. However, these mice have a developmental defect in their
CD5.sup.+ B cells indicating that IL-5 is required for normal
differentiation of this B cell subset in mice. At suboptimal
concentrations of IL-4, IL-5 was shown to enhance IgE synthesis by
human B cells in vitro. Furthermore, a recent study suggested that
the effects of IL-5 on human B cells depend on the mode of B cell
stimulation. IL-5 significantly enhanced IgM synthesis by B cells
stimulated with Moraxella catarrhalis. In addition, IL-5 synergized
with suboptimal concentrations of IL-2, but had no effect on
I-synthesis by SAC-activated B cells. Activated human B cells also
expressed IL-5 mRNA suggesting that IL-5 may also regulate B cell
function, including I-E synthesis, by autocrine mechanisms.
[1104] The invention provides methods of evolving an IL-5
antagonist that efficiently binds to and neutralizes IL-5 or its
receptor. These antagonists are useful as a component of vaccines
used for prophylaxis and treatment of allergies. Nucleic acids
encoding IL-5, for example, from human and other mammalian species,
are experimentally evolved (e.g. by polynucleotide reassembly
&/or polynucleotide site-saturation mutagenesis) and screened
for binding to immobilized IL-5R for the initial screening.
Polypeptides that exhibit the desired effect in the initial
screening assays can then be screened for the highest biological
activity using assays such as inhibition of growth of IL-5
dependent cells lines cultured in the presence of recombinant
wild-type IL-5. Alternatively, experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis) IL-5R-chains are screened for improved binding to
IL-5.
[1105] Tumor necrosis factors (and) and their receptors are also
suitable targets for modification and use in genetic vaccines.
TNF-, which was originally described as cachectin because of its
ability to cause necrosis of tumors, is a 17 kDa protein that is
produced in low quantities by almost all cells in the human body
following activation. TNF- acts as an endogenous pyrogen and
induces the synthesis of several proinflammatory cytokines,
stimulates the production of acute phase proteins, and induces
proliferation of fibroblasts. TNF- plays a major role in the
pathogenesis of endotoxin shock. A membrane-bound form of TNF-
(mTNF-), which is involved in interactions between B- and T-cells,
is rapidly upregulated within four hours of T cell activation.
mTNF- plays a role in the polyclonal B cell activation observed in
patients infected with HIV. Monoclonal antibodies specific for
mTNF-. or the p55 TNF- receptor strongly inhibit IgE synthesis
induced by activated CD4.sup.+ T cell clones or their membranes.
Mice deficient for p55 TNF-R are resistant to endotoxic shock, and
soluble TNF-R prevents autoimmune diabetes mellitus in NOD mice.
Phase III trials using sTNF-R in the treatment of rheumatoid
arthritis are in progress, after promising results obtained in the
phase II trials.
[1106] The methods of the invention can be used to, for example,
evolve a soluble TNF-R that has improved affinity, and thus is
capable of acting as an antagonist for TNF activity. Nucleic acids
that encode TNF-R and exhibit sequence diversity, such as the
natural diversity observed in cDNA libraries from activated T cells
of human and other primates, are experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis). The experimentally evolved (e.g. by polynucleotide
reassembly &/or polynucleotide site-saturation mutagenesis)
nucleic acids are expressed, e.g., on phage, after which mutants
are selected that bind to TNF- with improved affinity. If desired,
the improved mutants can be subjected to further assays using
biological activity, and the experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis) genes can be subjected to one or more rounds of
reassembly (optionally in combination with other directed evolution
methods described herein) and screening.
[1107] Another target of interest for application of the methods of
the invention is interferon-y, and the evolution of antagonists of
this cytokine. The receptor for IFN- consists of a binding
component glycoprotein of 90 kD, a 228 amino acid extracellular
portion, a transmembrane region, and a 222 amino acid intracellular
region. Glycosylation is not required for functional activity. A
single chain provides high affinity binding (10.sup.-9-10.sup.-10
M), but is not sufficient for signaling. Receptor components
dimerize upon ligand binding.
[1108] The mouse IFN- receptor is 53% identical to that of mouse at
the amino acid level. The human and mouse receptors only bind human
and mouse IFN-, respectively. Vaccinia, cowpox and camelpox viruses
have homologues of sIFN-R, which have relatively low amino acid
sequence similarity (.about.20%), but are capable of efficient
neutralization of IFN- in vitro. These homologues bind human,
bovine, rat (but not mouse) IFN-, and may have in vivo activity as
IFN- antagonists. All eight cysteines are conserved in human,
mouse, myxoma and Shope fibroma virus (6 in vaccinia virus) IFN-R
polypeptides, indicating similar 3-D structures. An extracellular
portion of m IFN-R with a kD of 100-300 pM has been expressed in
insect cells. Treatment of NZB/W mice (a mouse model of human SLE)
with msIFN- receptor (100 mg/three times a week i.p.) inhibits the
onset of glomerulonephritis. All mice treated with sIFN- or
anti-IFN-niAbs were alive 4 weeks after the treatment was
discontinued, compared with 50% in a placebo group, and 78% of
IFN--treated mice died.
[1109] The methods of the invention can be used to evolve soluble
IFN-R receptor polypeptides with improved affinity, and to evolve
IFN- with improved specific activity and improved capacity to
activate cellular immune responses. In each case nucleic acids
encoding the respective polypeptide, and which exhibit sequence
diversity (e.g., that observed in CDNA libraries from activated T
cells from human and other primates), are subjected to reassembly
(&/or one or more additional directed evolution methods
described herein) and screened to identify those recombinant
nucleic acids that encode a polypeptide having improved activity.
In the case of experimentally evolved (e.g. by polynucleotide
reassembly &/or polynucleotide site-saturation mutagenesis)
IFN-R, the library of experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis) nucleic acids can be expressed on phage, which are
screened to identify mutants that bind to IFN- with improved
affinity. In the case of IFN-, the experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis) library is analyzed for improved specific activity and
improved activation of the immune system, for example, by using
activation of monocytes/macrophages as an assay. The evolved IFN-
molecules can improve the efficacy of vaccinations (e.g. when used
as adjuvants). Diseases that can be treated using high-affinity
sIFN-R polypeptides obtained using the methods of the invention
include, for example, multiple sclerosis, systemic lupus
erythematosus (SLE), organ rejection after treatment, and graft
versus host disease. Multiple sclerosis, for example, is
characterized by increased expression of IFN- in the brain of the
patients, and increased production of IFN- by patients' T cells in
vitro. IFN- treatment has been shown to significantly exacerbate
the disease (in contrast to EAE in mice).
[1110] Transforming growth factor (TGF)- is another cytokine that
can be optimized for use in genetic vaccines using the methods of
the invention. TGF- has growth regulatory activities on essentially
all cell types, and it has also been shown to have complex
modulatory effects on the cells of the immune system. TGF- inhibits
proliferation of both B and T cells, and it also suppresses
development of and differentiation of cytotoxic T cells and NK
cells, TGF- has been shown to direct IgA switching in both murine
and human B cells. It was also shown to induce germline a
transcription in murine and human B cells, supporting the
conclusion that TGF- can specifically induce IgA switching.
[1111] Due to its capacity to direct IgA switching, TGF- is useful
as a component of DNA vaccines which aim at inducing potent mucosal
immunity, e.g. vaccines for diarrhea. Also, because of its potent
anti-proliferative effects TGF- is useful as a component of
therapeutical cancer vaccines. TGF- with improved specific activity
and/or with improved expression levels/kinetics will have increased
beneficial effects compared to the wild-type TGF-.
[1112] Cytokines that can be optimized using the methods of the
invention also include granulocyte colony stimulating factor
(G-CSF) and granulocyte/macrophage colony stimulating factor
(GM-CSF). These cytokines induce differentiation of bone marrow
stem cell into granulocytes/macrophages. Administration of G-CSF
and GM-CSF significantly improve recovery from bone marrow (BM)
transplantation and radiotherapy, reducing infections and time the
patients have to spend in hospitals. GM-CSF enhances antibody
production following DNA vaccination. G-CSF is a 175 amino acid
protein, while GM-CSF has 127 amino acids. Human G-CSF is 73%
identical at the amino acid level to murine G-CSF and the two
proteins show species cross-reactivity. G-CSF has a homodimeric
receptor (dimeric with kD of .about.200 pM, monomeric .about.2.4
nM), and the receptor for GM-CSF is a three subunit complex. Cell
lines transfected with cDNA encoding G-CSF R proliferate in
response to G-CSF. Cell lines dependent of GM-CSF available (such
as TF-1). G-CSF is nontoxic and is presently working very well as a
drug. However, the treatment is expensive, and more potent G-CSF
might reduce the cost for patients and to the health care.
Treatments with these cytokines are typically short-lasting and the
patients are likely to never need the same treatment again reducing
likelihood of problems with immunogenicity.
[1113] The methods of the invention are useful for evolving G-CSF
and/or GM-CSF which have improved specific activity, as well as
other polypeptides that have G-CSF and/or GM-CSF activity. G-CSF
and/or GM-CSF nucleic acids having sequence diversity, e.g., those
obtained from cDNA libraries from diverse species, are
experimentally evolved (e.g. by polynucleotide reassembly &/or
polynucleotide site-saturation mutagenesis) to create a library of
experimentally evolved (e.g. by polynucleotide reassembly &/or
polynucleotide site-saturation mutagenesis) G-CSF and/or GM-CSF
genes. These libraries can be screened by, for example, picking
colonies, transfecting the plasmids into a suitable host cell
(e.g., CHO cells), and assaying the supernatants using
receptor-positive cell lines. Alternatively, phage display or
related techniques can be used, again using receptor-positive cell
lines. Yet another screening method involves transfecting the
experimentally evolved (e.g. by polynucleotide reassembly &/or
polynucleotide site-saturation mutagenesis) genes into
G-CSF/GM-CSF-dependent cell lines. The cells are grown one cell per
well and/or at very low density in large flasks, and the cells that
grow fastest are selected. Experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis) genes from these cells are isolated; if desired, these
genes can be used for additional rounds of reassembly (optionally
in combination with other directed evolution methods described
herein) and selection.
[1114] Ciliary neurotrophic factor (CNTF) is another suitable
target for application of the methods of the invention. CNTF has
200 amino acids which exhibit 80% sequence identity between rat and
rabbit CNTF polypeptides. CNTF has IL-6-like inflammatory effects,
and induces synthesis of acute phase proteins. CNTF is a cytosolic
protein which belongs to the IL-6/IL-11I/LIF/oncostatin M -family,
and becomes biologically active only after becoming available
either by cellular lesion or by an unknown release mechanism. CNTF
is expressed by myelinating Schwann cells, astrocytes and sciatic
nerves.
[1115] Structurally, CNTF is a dimeric protein, with a novel
anti-parallel arrangement of the subunits. Each subunit adopts a
double crossover four-helix bundle fold, in which two helices
contribute to the dimer interface. Lys-155 mutants lose activity,
and some Glu-153 mutants have 5-10 higher biological activity. The
receptor for CNTF consists of a specific CNTF receptor chain,
gpl3O, and a LIF- receptor. The CNTFR-chain lacks a transmembrane
domain portion, instead being GPI-anchored. At high concentration,
CNTF can mediate CNTFR-independent responses. Soluble CNTFR binds
CNTF and thereafter can bind to LIFR and induce signaling through
gp 130. CNTF enhances survival of several types of neurons, and
protects neurons in an animal model of Huntington disease (in
contrast to NGF, neurotrophic factor, and neurotrophin-3). CNTF
receptor knockout mice have severe motor neuron deficits at birth,
and CNTF knockout mice exhibit such deficits postnatally. CNTF also
reduces obesity in mouse models. Decreased expression of CNTF is
sometimes observed in psychiatric patients. Phase I studies in
patients with ALS (annual incidence .about.{fraction (1/100 000)},
5% familiar cases, 90% die within 6 years) found significant side
effects after doses higher than 5 mg/kg/day subcutaneously
(including anorexia, weight loss, reactivation of herpes simplex
virus (HSV1), cough, increased oral secretions). Antibodies against
CNTF were detected in almost all patients, thus illustrating the
need for alternative CNTF with different immunological
properties.
[1116] The reassembly (&/or one or more additional directed
evolution methods described herein) and screening methods of the
invention can be used to obtain modified CNTF polypeptides that
exhibit decreased immunogenicity in vivo; higher also obtainable
using the methods. reassembly (optionally in combination with other
directed evolution methods described herein) is conducted using
nucleic acids encoding CNTF. In a preferred embodiment, an
IL-6/LIF/(CNTF) hybrid is obtained by reassembly (optionally in
combination with other directed evolution methods described herein)
using an excess of oliconucleotides that encode to the receptor
binding sites of CNTF. Phage display can then be used to test for
lack of binding to the IL-6/LIF receptor.
[1117] This initial screen is followed by a test for high affinity
binding to the CNTF receptor, and, if desired, functional assays
using CNTF responsive cell lines. The experimentally evolved (e.g.
by polynucleotide reassembly &/or polynucleotide
site-saturation mutagenesis) CNTF polypeptides can be tested to
identify those that exhibit reduced immunogenicity upon
administration to a mammal.
[1118] Another way in which the reassembly (&/or one or more
additional directed evolution methods described herein) and
screening methods of the invention can be used to optimize CNTF is
to improve secretion of the polypeptide. When a CNTF cDNA is
operably linked to a leader sequence of hNGF, only 35-40 percent of
the total CNTF produced is secreted.
[1119] Target diseases for treatment with optimized CNTF, using
either the experimentally evolved (e.g. by polynucleotide
reassembly &/or polynucleotide site-saturation mutagenesis)
gene in an expression vector as in DNA vaccines, or a purified
protein, include obesity, amyotrophic lateral sclerosis (ALS, Lou
Gehrig's disease), diabetic neuropathy, stroke, and brain
surgery.
[1120] Polynucleotides that encode chemokines can also be optimized
using the methods of the invention and included in a genetic
vaccine vector. At least three classes of chemokines are known,
based on structure: C chemokines (such as lymphotactin), C--C
chemokines (such as MCP-1, MCP-2, MCP-3, MCP-4, MIP-1a, MIP-1b,
RANTES), C--X--C chemokines (such as IL-8, SDF-1, ELR, Mig, 1P 10)
(Premack and Schall (1996) Nature Med. 2:1174). Chemokines can
attract other cells that mediate immune and inflammatory functions,
thereby potentiating the immune response. Cells that are attracted
by different types of chemokines include, for example, lymphocytes,
monocytes and neutrophils. Generally, C--X--C chemokines are
chemoattractants for neutrophils but not for monocytes, C--C
chemokines attract monocytes and lymphocytes but not neutrophils, C
chemokine attracts lymphocytes.
[1121] Genetic vaccine vectors can also include optimized
experimentally generated polynucleotides that encode surface-bound
accessory molecules, such as those that are involved in modulation
and potentiation of immune responses. These molecules, which
include, for example, B7-1 (CD80), B7-2 (CD86), CD40, ligand for
CD40, CTLA-4, CD28, and CD 150 (SLAM), can be subjected to
stochastic (e.g. polynucleotide shuffling & interrupted
synthesis) and non-stochastic polynucleotide reassembly to obtain
variants have altered and/or improved activities.
[1122] Optimized experimentally generated polynucleotides that
encode CD1 molecules are also useful in a genetic vaccine vector
for certain applications. CD1 are nonpolymorphic molecules that are
structurally and functionally related to MEC molecules.
Importantly, CD I has MHC-like activities, and it can function as
an antigen presenting molecule (Porcelli (1995) Adv. Immunol.
59:1). CD1 is highly expressed on dendritic cells, which are very
efficient antigen presenting cells. Simultaneous transfection of
target cells with DNA vaccine vectors encoding CD1 and an antigen
of interest is likely to boost the immune response. Because CD1
cells, in contrast to MHC molecules, exhibit limited allelic
diversity in an outbred population (Porcelli, supra.), large
populations of individuals with different genetic backgrounds can
be vaccinated with one CD1 allele. The functional properties of CD1
molecules can be improved by the stochastic (e.g. polynucleotide
shuffling & interrupted synthesis) and non-stochastic
polynucleotide reassembly methods of the invention.
[1123] Optimized recombinant TAP genes and/or gene products can
also be included in a genetic vaccine vector. TAP genes and their
optimization for various purposes are discussed in more detail
below. Moreover, heat shock proteins (HSP), such as HSP70, can also
be evolved for improved presentation and processing of antigens.
HSP70 has been shown to act as adjuvant for induction of CD8+T cell
activation and it enhances immunogenicity of specific antigenic
peptides (Blachere et al. (1997) J Exp. Med. 186:1315-22). When
HSP70 is encoded by a genetic vaccine vector, it is likely to
enhance presentation and processing of antigenic peptides and
thereby improve the efficacy of the genetic vaccines. stochastic
(e.g. polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly can be used to further
improve the properties, including adjuvant activity, of heat shock
proteins, such as HSP70.
[1124] Recombinantly produced cytokine, chemokine, and accessory
molecule polypeptides, as well as antagonists of these molecules,
can be used to influence the type of immune response to a given
stimulus. However, the administration of polypeptides sometimes has
shortcomings, including short half life, high expense, difficult to
store (must be stored at 4.degree. C.), and a requirement for large
volumes. Also, bolus injections can sometimes cause side effects.
Administration of polynucleotides that encode the recombinant
cytokines or other molecules overcomes most or all of these
problems. DNA, for example, can be prepared in high purity, is
stable, temperature resistant, noninfectious, easy to manufacture.
In addition, polynucleotide-mediated administration of cytokines
can provide long-lasting, consistent expression, and administration
of polynucleotides in general is regarded as being safe.
[1125] The functions of cytokines, chemokines and accessory
molecules are redundant and pleiotropic, and therefore can be
difficult to determine which cytokines or cytokine combinations are
the most potent in inducing and enhancing antigen specific immune
responses following vaccination. Furthermore, the most useful
combination of cytokines and accessory molecules is typically
different depending on the type of immune C, response that is
desired following vaccination. As an example, IL-4 has been shown
to direct differentiation of T.sub.H2 cells (which produce high
levels of IL-4, IL-5 and IL-13, and mediate allergic immune
responses), whereas IFN- and IL-12 direct differentiation of
T.sub.H1 cells (which produce high levels of IL-2 and IFN- ), and
mediate delayed type immune responses. Moreover, the most useful
combination of cytokines and accessory molecules is also likely to
depend on the antigen used in the vaccination. The invention
provides a solution to this problem of obtaining an optimized
genetic vaccine cocktail. Different combinations of cytokines,
chemokines and accessory molecules are assembled into vectors using
the methods described herein. These vectors are then screened for
their capacity to induce immune responses in vivo and in vitro.
[1126] Large libraries of vectors, generated by polynucleotide
(e.g. gene, promoter, enhancer, intron, & the like) reassembly
(optionally in combination with other directed evolution methods
described herein) and combinatorial molecular biology, are screened
for maximal capacity to direct immune responses towards, for
example, a T.sub.H1 or T.sub.H2 phenotype, as desired. A library of
different vectors can be generated by assembling different evolved
promoters, (evolved) cytokines, (evolved) cytokine antagonists,
(evolved) chemokines, (evolved) accessory molecules and
immunostimulatory sequences, each of which can be prepared using
methods described herein. DNA sequences and compounds that
facilitate the transfection and expression can be included. If the
pathogen(s) is known, specific DNA sequences encoding immunogenic
antigens from the pathogen can be incorporated into these vectors
providing protective immunity against the pathogen(s) (as in
genetic vaccines).
[1127] Initial screening is preferably carried out in vitro. For
example, the library can be introduced into cells which are tested
for ability to induce differentiation of T cells capable of
producing cytokines that are indicative of the type of immune
response desired. For a T.sub.H1 response, for example, the library
is screened to identify experimentally generated polynucleotides
that are capable of inducing T cells to produce IL-2 and IFN-,
while screening for induction of T cell production of IL-4, IL-5,
and IL-13 is performed to identify experimentally generated
polynucleotides that favor a T.sub.H2 response.
[1128] Screening can also be conducted in vivo, using animal
models. For example, vectors produced using the methods of the
invention can be tested for ability to protect against a lethal
infection. Another screening method involves injection of
Leishmania major parasites into footpads of BALB/c mice
(nonhealer). Pools of plasmids are injected i.v., i.p. or into
footpads of these mice and the size of the footpad swelling is
followed. Yet another in vivo screening method involves detection
of IgE levels after infection with Nippostrongylus brasiliensis.
High levels indicate a T.sub.H2 response, while low levels of IgE
indicate a T.sub.H1 response.
[1129] Successful results in animal models are easy to verify in
humans. In vitro screening can be conducted to test for human
T.sub.H1 or T.sub.H2 phenotype, or for other desired immune
response. Vectors can also be tested for ability to induce
protection against infection in humans. Because the principles of
immune functions are similar in a wide variety of infections,
immunostimulating DNA vaccine vectors may not only be useful in the
treatment of a number of infectious diseases but also in prevention
of the infections, when the vectors are delivered to the sites of
the entry of the pathogen (e.g., the lung or gut).
2.6.5.3. AGONISTS OR ANTAGONISTS OF CELLULAR RECEPTORS
[1130] The invention also provides methods for obtaining optimized
experimentally generated polynucleotides that encode a peptide or
polypeptide that can interact with a cellular receptor that is
involved in mediating an immune response. The optimized
experimentally generated polynucleotides can act as an agonist or
an antagonist of the receptor.
[1131] Cytokine Antagonists can be used as Components of Genetic
Vaccine Cocktails
[1132] Blocking immunosuppressive cytokines, rather than adding
single proinflammatory cytokines, is likely to potentiate the
immune response in a more general manner, because several pathways
are potentiated at the same time. By appropriate choice of
antagonist, one can tailor the immune response induced by a genetic
vaccine in order to obtain the response that is most effective in
achieving the desired effect. Antagonists against any cytokine can
be used as appropriate; particular cytokines of interest for
blocking include, for example, IL-4, IL-13, IL-10, and the
like.
[1133] The invention provides methods of obtaining cytokine
antagonists that exhibit greater effectiveness in blocking the
action of the respective cytokine. Polynucleotides that encode
improved cytokine antagonists can be obtained by using
polynucleotide (e.g. gene, promoter, enhancer, intron, & the
like) reassembly (optionally in combination with other directed
evolution methods described herein) to generate a recombinant
library of polynucleotides which are then screened to identify
those that encode an improved antagonist. As substrates for the
stochastic (e.g. polynucleotide shuffling & interrupted
synthesis) and non-stochastic polynucleotide reassembly, one can
use, for example, polynucleotides that encode receptors for the
respective cytokine. At least two forms of the substrate will be
present in the reassembly (&/or one or more additional directed
evolution methods described herein) reaction, with each form
differing from the other in at least one nucleotide position. In a
preferred embodiment, the different forms of the polynucleotide are
homologous cytokine receptor genes from different organisms. The
resulting library of experimentally generated polynucleotides is
then screened to identify those that encode cytokine antagonists
with the desired affinity and biological activity.
[1134] As one example of the type of effect that one can achieve by
including a cytokine antagonist in a genetic vaccine cocktail, as
well as how the effect can be improved using the stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly methods of the invention,
IL-10 is discussed. The same rationale can be applied to obtaining
and using antagonists of other cytokines. Interleukin-10 (IL-10) is
perhaps the most potent anti-inflammatory cytokine known to date.
IL-10 inhibits a number of pathways that potentiate inflammatory
responses. The biological activities of IL-10 include inhibition of
MHC class II expression on monocytes, inhibition of production of
IL-1, IL-6, IL-12, TNF-. by monocytes/macrophages, and inhibition
of proliferation and IL-2 production by T lymphocytes. The
significance of IL-10 as a regulatory molecule of immune and
inflammatory responses was clearly demonstrated in IL-10 deficient
mice.
[1135] These mice are growth-retarded, anemic and spontaneously
develop an inflammatory bowel disease (Kuhn et al. (1993) Cell
75:263). In addition, both innate and acquired immunity to Listeria
monocytogenes were shown to be elevated in IL-10 deficient mice
(Dai et al. (1997) J Immunol. 158:2259). It has also been suggested
that genetic differences in the levels of IL-10 production may
affect the risk of patients to die from complications
meningococcal. infection. Families with high IL-10 production had
20-fold increased risk of fatal outcome of meningococcal. disease
(Westendorp et al. (1997) Lancet 349:170).
[1136] IL-10 has been shown to activate normal and malignant B
cells in vitro, but it does not appear to be a major growth
promoting cytokine for normal B cells in vivo, because IL-10
deficient mice have normal levels of B lymphocytes and Ig in their
circulation. In fact, there is evidence that IL-10 can indirectly
downregulate B cell function through inhibition of the accessory
cell function of monocytes. However, IL-10 appears to play a role
in the growth and expansion of malignant B cells. Anti-IL-10
monoclonal antibodies and IL-10 antisense oligonucleotides have
been shown to inhibit transformation of B cells by EBV in vitro. In
addition, B cell lymphomas are associated with EBV and most
EBV.sup.+ lymphomas produce high levels of IL-10, which is derived
both from the human gene and the homologue of IL-10 encoded by EBV.
AIDS-related B cell lymphomas also secrete high levels of IL-10.
Furthermore, patients with detectable serum IL-10 at the time of
diagnosis of intermediate/high-grade non-Hodgkin's lymphoma have
short survival, further suggesting IDID In a role for IL-10 in the
pathogenesis of B cell malignancies.
[1137] Antagonizing IL-10 in vivo can be beneficial in several
infectious and malignant diseases, and in vaccination. The effect
of blocking of IL-10 is an enhancement of immune responses that is
independent of the specificity of the response. This is useful in
vaccinations and in the treatment of serious infectious diseases.
Moreover, an IL-10 antagonist is useful in the treatment of B cell
malignancies which exhibit overproduction of IL-10 and viral IL-10,
and it may also be useful in boosting general anti-tumor immune
response in cancer patients. Combining an IL-10 antagonist with
gene therapy vectors may be useful in gene therapy of tumor cells
in order to obtain maximal immune response against the tumor cells.
If the reassembly (optionally in combination with other directed
evolution methods described herein) of IL-10 results in IL-10 with
improved specific activity, this IL-10 molecule would have
potential in the treatment of autoimmune diseases and inflammatory
bowel diseases. IL-10 with improved specific activity may also be
useful as a component of gene therapy vectors in reducing the
immune response against vectors which are recognized by memory
cells and it may also reduce the immunogenicity of these
vectors.
[1138] An antagonist of IL-10 has been made by generating a soluble
form of IL-10 receptor (sIL-10R; Tan et al. (1995) J Biol. Chem.
270:12906). However, sIL-10R binds IL-10 with Kd of 560 pM, whereas
the wild-type, surface-bound receptor has affinity of 35-200 pM.
Consequently, 150-fold molar excess of sIL-10R is required for
half-maximal inhibition of biological function of IL-10. Moreover,
affinity of viral IL-10 (IL-10 homologue encoded by Epstein-Barr
virus) to sIL-10R is more than 1000 fold less than that of hIL-10,
and in some situations, such as when treating EBV-associated B cell
malignancies, it may be beneficial if one can also block the
function of viral IL-10. Taken together, this soluble form of
IL-10R is unlikely to be effective in antagonizing IL-10 in
vivo.
[1139] To obtain an IL-10 antagonist that has sufficient affinity
and antagonistic activity to function in vivo, stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly can be performed using
polynucleotides that encode IL-10 receptor. IL-10 receptor with
higher than normal affinity will function as an IL-10 antagonist,
because it strongly reduces the amount of IL-10 available for
binding to functional, wild-type IL-10R. In a preferred embodiment,
IL-10R is experimentally evolved (e.g. by polynucleotide reassembly
&/or polynucleotide site-saturation mutagenesis) using
homologous cDNAs encoding IL-10R derived from human and other
mammalian species.
[1140] An alignment of human and mouse IL-10 receptor sequences is
shown, described &/or referenced herein (including incorporated
by reference) to illustrate the feasibility of family stochastic
(e.g. polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly when evolving IL-10
receptors with improved affinity. A phage library of IL-10 receptor
recombinants can be screened for improved binding of experimentally
evolved (e.g. by polynucleotide reassembly &/or polynucleotide
site-saturation mutagenesis) IL-10 R to human or viral IL-10.
Wild-type IL-10 and/or viral IL-0 are added at increasing
concentrations to demand for higher affinity. Phage bound to IL-10
can be recovered using anti-IL-10 monoclonal antibodies. If
desired, the shuffling can be repeated one or more times, after
which the evolved soluble IL-10R is analyzed in functional assays
for its capacity to neutralize the biological activities of
IL-10/viral IL-10. More specifically, evolved soluble IL-10R is
studied for its capacity to block the inhibitory effects of IL-10
on cytokine synthesis and MHC class II expression by monocytes,
proliferation by T cells, and for its capacity to inhibit the
enhancing effects of IL-10 on proliferation of B cells activated by
anti-CD40 monoclonal antibodies.
[1141] An IL-10 antagonist can also be generated by evolving IL-10
to obtain variants that bind to IL-10R with higher than wild-type
affinity, but without receptor activation. The advantage of this
approach is that one can evolve an IL-10 molecule with improved
specific activity using the same methods. In a preferred
embodiment, IL-10 is experimentally evolved (e.g. by polynucleotide
reassembly &/or polynucleotide site-saturation mutagenesis)
using homologous cDNAs encoding IL-10 derived from human and other
mammalian species. In addition, a gene encoding viral IL-10 can be
included in the reassembly (optionally in combination with other
directed evolution methods described herein). A library of IL-10
recombinants is screened for improved binding to human IL-10
receptor. Library members bound to IL-10R can be recovered by
anti-IL-10R monoclonal antibodies. This screening protocol is
likely to result in IL-10 molecules with both antagonistic and
agonistic activities. Because initial screen demands for higher
affinity, a proportion of the agonists are likely to have improved
specific activity when compared to wild-type human IL-10. The
functional properties of the mutant IL-10 molecules are determined
in biological assays similar to those described above for
ultrahigh-affinity IL-10 receptors (cytokine synthesis and MHC
class II expression by monocytes, proliferation of B and T cells).
An antagonistic IL-4 mutant has been previously generated
illustrating the general feasibility of the approach (Kruse et al.
(1992) EMBO J. 11:3237-3244). One amino acid mutation in IL-4
resulted in a molecule that efficiently binds to IL-4R a-chain but
has minimal IL-4-like agonistic activity.
[1142] Another example of an IL-1 0 antagonist is IL-20/mda-7,
which is a 206 amino acid secreted protein. This protein was
originally characterized as mda-7, which is a melanoma cell-derived
negative regulator of tumor cell growth (Jiang et al. (1995)
Oncogene 11:2477; (1996) Proc. Nat'l. Acad. Sci. USA 93:9160).
IL-20/mda-7 is structurally related to IL-10, and it antagonizes
several functions of IL-10 (Abstract of the 13.sup.th European
Immunology Meeting, Amsterdam, Jun. 22-25, 1997). In contrast to
IL-10, IL-20/mda-7 enhances expression of CD80 (B7-1) and CD86
(B7-2) on human monocytes and it upregulates production of TNF- and
IL-6. IL-20/mda-7 also enhances production of IFN- by PHA-activated
PBMC. The invention provides methods of improving genetic vaccines
by incorporation of IL-20/mda-7 genes into the genetic vaccine
vectors. The methods of the invention can be used to obtain
IL-20/mda-7 variants that exhibit improved ability to antagonize
IL-10 activity.
[1143] When a cytokine antagonist is used as a component of DNA
vaccine or gene therapy vectors, maximal local effect is desirable.
Therefore, in addition to a soluble form of a cytokine antagonist,
a transmembrane form of the antagonist can be generated. The
soluble form can be given in purified polypeptide form to patients
by, for example, intravenous injection. Alternatively, a
polynucleotide encoding the cytokine antagonist can be used as a
component as a component of a genetic vaccine or a gene therapy
vector. In this case, either or both of the soluble and
transmembrane forms can be used. Where both soluble and
transmembrane forms of the antagonist are encoded by the same
vector, the target cells express both forms, resulting in maximal
inhibition of cytokine function on the target cell surface and in
their immediate vicinity.
[1144] The peptides or polypeptides obtained using these methods
can substitute for the natural ligands of the receptors, such as
cytokines or other costimulatory molecules in their ability to
exert an effect on the immune system via the receptor. A potential
disadvantage of administering cytokines or other costimulatory
molecules themselves is that an autoimmune reaction could be
induced against the natural molecule, either due to breaking
tolerance (if using a natural cytokine or other molecule) or by
inducing cross-reactive immunity (humoral or cellular) when using
related but distinct molecules. Through using the methods of the
invention, one can obtain agonists or antagonists that avoid these
potential drawbacks. For example, one can use relatively small
peptides as agonists that can mimic the activity of the natural
immunomodulator, or antagonize the activity, without inducing
cross-reactive immunity to the natural molecule. In a presently
preferred embodiment, the optimized agonist or antagonist obtained
using the methods of the invention is about 50 amino acids or
length or less, more preferably about 30 amino acids or less, and
most preferably is about 20 amino acids in length, or less. The
agonist or antagonist peptide is preferably at least about 4 amino
acids in length, and more preferably at least about 8 amino acids
in length. Polynucleotides that flank the coding sequence of the
mimetic peptide can also be optimized using the methods of the
invention in order to optimize the expression, conformation, or
activity of the mimetic peptide.
[1145] The optimized agonist or antagonist peptides or polypeptides
are obtained by generating a library of experimentally generated
polynucleotides and screening the library to identify those that
encode a peptide or polypeptide that exhibits an enhanced ability
to modulate an immune response. The library can be produced using
methods such as stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide reassembly
or other methods described herein or otherwise known to those of
skill in the art. Screening is conveniently conducted by expressing
the peptides encoded by the library members on the surface of a
population of replicable genetic packages and identifying those
members that bind to a target of interest, e.g., a receptor.
[1146] The optimized experimentally generated polynucleotides that
are obtained using the methods of the invention can be used in
several ways. For example, the polynucleotide can be placed in a
genetic vaccine vector, under the control of appropriate expression
control sequences, so that the mimetic peptide is expressed upon
introduction of the vector into a mammal. If desired, the
polynucleotide can be placed in the vector embedded in the coding
sequence of the surface protein (e.g., geneIII or geneVIII) in
order to preserve, the conformation of the mimetic. Alternatively,
the mimetic-encoding polynucleotide can be inserted directly into
the antigen-encoding sequence of the genetic vaccine to form a
coding sequence for a "mimotope-on-antigen" structure. The
polynucleotide that encodes the mimotope-on-antigen structure can
be used within a genetic vaccine, or can be used to express a
protein that is itself administered as a vaccine. As one example of
this type of application, a coding sequence of a mimetic peptide is
introduced into a polynucleotide that encodes the "M-loop" of the
hepatitis B surface antigen (HBsAg) protein. The M-loop is a six
amino acid peptide sequence bounded by cysteine residues, which is
found at amino acids 139-147 (numbering within the S protein
sequence). The M-loop in the natural HBsAg protein is recognized by
the monoclonal antibody RFHB7 (Chen et al., Proc. Nat'l. Acad. Sci.
USA, 93:1997-2001 (1996)). According to Chen et al., the M-loop
forms an epitope of the HBsAg that is non-overlapping and separate
from at least four other HBsAg epitopes.
[1147] Because of the probable Cys-Cys disulfide bond in this
hydrophilic part of the protein, amino acids 139-147 are likely in
a cyclic conformation. This structure is therefore similar to that
found in the regions of the filamentous phage proteins pIII and
pVIII where mimotope sequences are placed. Therefore, one can
insert a mimotope obtained using the methods of the invention into
this region of the HBsAg amino acid sequence.
[1148] The chemokine receptor CCR6 is an example of a suitable
target for a peptide mimetic obtained using the methods. The CCR6
receptor is a 7- transmembrane domain protein (Dieu et al.,
Biochem. Biophys. Res. Comm. 236:212-217 (1997) and J. Biol. Chem.
272:14893-14898 (1997)) that is involved in the chemoattraction of
immature dendritic cells, which are found in the blood and migrate
to sites of antigen uptake (Dieu et al., J Exp. Med. 188:373-386
(1998)). CCR6 binds the chemokine MIP-3, so a mimetic peptide that
is capable of activating CCR6 can provide a further chemoattractant
function to a given antigen and thus promote uptake by dendritic
cells after immunization with the antigen antigen-mimetic fusion or
a DNA vector that expresses the antigen.
[1149] Another application of this method of the invention is to
obtain molecules that can act as an agonist for the macrophage
scavenger receptor (MSR; see, Wloch et al., Hum. Gene Ther.
9:1439-1447 (1998)). The MSR is involved in mediating the effects
of various immunomodulators. Among these are bacterial DNA,
including the plasmids used in DNA vaccination, and
oligonucleotides, which are often potent immunostimulators.
[1150] Oligonucleotides of certain chemical structure (e.g.,
phosphothio-oligonucleotides) are particularly potent, while
bacterial or plasmid DNA must be used in relatively large
quantities to produce an effect. Also mediated by the MSR is the
ability of oligonucleotides that contain dG residues to stimulate B
cells and enhance the activity of immunostimulatory CpG motifs, and
of lipopolysaccharides to activate macrophages. Some of these
activities are toxic. Each of these immunomodulators, along with a
variety of polyanionic ligands, binds to the MSR. The methods of
the invention can be used to obtain mimetics of one or more of
these immunomodulators that bind to the MSR with high affinity but
are devoid of toxic properties. Such mimetic peptides are useful as
immunostimulators or adjuvants.
[1151] The MSR is a trimeric integral membrane glycoprotein. The
three extracellular C-terminal cysteine-rich regions are connected
to the transmembrane domain by a fibrous region that is composed of
an (x-helical coil and a collagen-like triple helix (see, Kodama et
al., Nature 343:531-535 (1990)). Therefore, screening of the
library of experimentally generated polynucleotides can be
accomplished by expressing the extracellular receptor structure and
artificially attaching it to plastic surfaces. The libraries can be
expressed, e.g., by phage display, and screened to identify those
that bind to the receptors with high affinity.
[1152] The optimized experimentally generated polynucleotides
identified by this method can be incorporated into antigen-encoding
sequences to evaluate their modulatory effect on the immune
response.
2.6.5.4. COSTIMULATORY MOLECULES CAPABLE OF INHIBITING OR ENHANCING
ACTIVATION, DIFFERENTIATION, OR ANERGY OF ANTIGEN-SPECIFIC T
CELLS
[1153] Also provided are methods of obtaining optimized
experimentally generated polynucleotides that, when expressed, are
capable of inhibiting or enhancing the activation, differentiation,
or anergy of antigen-specific T cells. T cell activation is
initiated when T cells recognize their specific antigenic peptides
in the context of MHC molecules on the plasma membrane of antigen
presenting cells (APC), such as monocytes, dendritic cells (DC),
Langerhans cells or B cells. Activation of CD4.sup.+ T cells
requires recognition by the T cell receptor (TCR) of an antigenic
peptide in the context of MHC class II molecules, whereas CD8.sup.+
T cells recognize peptides in the context of MHC class I
molecules.
[1154] Importantly, however, recognition of the antigenic peptides
is not sufficient for induction of T cell proliferation and
cytokine synthesis. An additional costimulatory signal, "the second
signal", is required. The costimulatory signal is mediated via
CD28, which binds to its ligands B7-1 (CD80) or B7-2 (CD86),
typically expressed on the antigen presenting cells. In the absence
of the costimulatory signal, no T cell activation occurs, or T
cells are rendered anergic. In addition to CD28, CTLA-4 (CD152)
also functions as a ligand for B7-1 and B7- 2. However, in contrast
to CD28, CTLA-4 mediates a negative regulatory signal to T cells
and/or to induce anergy and tolerance (Walunas et al. (1994)
Immunity 1:405; Karandikar et al. (1996) J Exp. Med. 184:783).
[1155] B7-1 and B7-2 have been shown to be able to regulate several
immunological responses, and they have been implicated to be of
importance in the immune regulation in vaccinations, allergy,
autoimmunity and cancer. Gene therapy and genetic vaccine vectors
expressing B7-1 and/or B7-2 have also been shown to have
therapeutic potential in the treatment of the above mentioned
diseases and in improving the efficacy of genetic vaccines.
[1156] FIG. 10 illustrates interaction of APC and CD4.sup.+ T
cells, but the same principle is true with CD8.sup.+ T cells, with
the exception that the T cells recognize the antigenic peptides in
the context of MHC class I molecules. Both B7-1 and B7-2 bind to
CD28 and CTLA-4, even though the sequence similarities between
these four molecules are very limited (20-30%). It is desirable to
obtain mutations in B7-1 and B7-2 that only influence binding to
one ligand but not to the other, or improve activity through one
ligand while decreasing the activity through the other. Moreover,
because the affinities of B7 molecules to their ligands appear to
be relatively low, it would also be desirable to find mutations
that improve/alter the activities of the molecules. However,
rational design does not enable predictions of useful mutations
because of the complexity of the molecules.
[1157] The invention provides methods of overcoming these
difficulties, enabling one to generate and identify functionally
different B7 molecules with altered relative capacities to induce T
cell activation, differentiation, cytokine production, anergy
and/or tolerance. Through use of the methods of the invention, one
can find mutations in B7-1 and B7-2 that only influence binding to
one ligand but not to the other, or that improve activity through
one ligand while decreasing the activity through the other.
stochastic (e.g. polynucleotide shuffling & interrupted
synthesis) and non-stochastic polynucleotide reassembly is likely
to be the most powerful method in discovering new B7 variants with
altered relative binding capacities to CD28 and CTLA-4. B7 variants
which act through CD28 with improved activity (and with decreased
activity through CTLA-4) are expected to have improved capacity to
induce activation of T cells. In contrast, B7 variants which bind
and act through CTLA-4 with improved activity (and with decreased
activity through CD28) are expected to be potent negative
regulators of T cell functions and to induce tolerance and
anergy.
[1158] stochastic (e.g. polynucleotide shuffling & interrupted
synthesis) and non-stochastic polynucleotide reassembly or other
reassembly (&/or one or more additional directed evolution
methods described herein) method is used to generate B7 (e.g.,
B7-1/CD80 and B7-2/CD86) variants which have altered relative
capacity to act through CD28 and CTLA-4 when compared to wild-type
B7 molecules. In a preferred embodiment, the different forms of
substrate used in the reassembly (&/or one or more additional
directed evolution methods described herein) reaction are B7 cDNAs
from various species. Such cDNAs can be obtained by methods known
to those of skill in the art, including RT-PCR- Typically, genes
encoding these variant B7 molecules are incorporated into genetic
vaccine vectors encoding an antigen, so that one the vectors can be
used to modify antigen-specific T cell responses. Vectors that
harbor B7 genes that efficiently act through CD28 are useful in
inducing, for example, protective immune responses, whereas vectors
that harbor genes encoding B7 genes that efficiently act through
CTLA-4 are useful in inducing, for example, tolerance and anergy of
allergen- or autoantigen- specific T cells. In some situations,
such as in tumor cells or cells inducing autoimmune reactions, the
antigen may already be present on the surface of the target cell,
and the variant B7 molecules may be transfected in the absence of
additional exogenous antigen gene. FIG. 11 illustrates a screening
protocol that one can use to identify B7-1 (CD80) and/or B7-2
(CD86) variants that have increased capacity to induce T cell
activation or anergy, and the application of this strategy is
described in more detail in Example 1.
[1159] Several approaches for screening of the variants can be
taken. For example, one can use a flow cytometry-based selection
systems. The library of B7-1 and B7-2 molecules is transfected into
cells that normally do not express these molecules (e.g., COS-7
cells or any cell line from a different species with limited or no
cross-reactivity with man regarding B7 ligand binding). An internal
marker gene can be incorporated in order to analyze the copy number
per cell. Soluble CTLA-4 and CD28 molecules can be generated to for
use in the flow cytometry experiments. Typically, these will be
fused with the Fc portion of IgG molecule to improve the stability
of the molecules and to enable easy staining by labeled anti-IgG
mAbs, as described by van der Merwe et al. (J. Exp. Med: 185:393,
1997). The cells transfected with the library of B7 molecules are
then stained with the soluble CTLA-4 and CD28 molecules. Cells
demonstrating increased or decreased CTLA- 4/CD28 binding ratio
will be sorted. The plasmids are then recovered and the
experimentally evolved (e.g. by polynucleotide reassembly &/or
polynucleotide site-saturation mutagenesis) B7 variant-encoding
sequences identified. These selected B7 variants can then-be
subjected to new rounds of reassembly (optionally in combination
with other directed evolution methods described herein) and
selection, and/or they can be further analyzed using functional
assays as described below.
[1160] The B7 variants can also be directly selected based on their
functional properties. For in vivo studies, the B7 molecules can
also be evolved to function on mouse cells. Bacterial colonies with
plasmids with mutant B7 molecules are picked and the plasmids are
isolated. These plasmids are then transfected into antigen
presenting cells, such as dendritic cells, and the capacities of
these mutants to activate T cells is analyzed. One of the
advantages of this approach is that no assumptions on the binding
affinities or specificities to the known ligands are made, and
possibly new activities through yet to be identified ligands can be
found. In addition to dendritic cells, other cells that are
relatively easy to transfect (e.g., U937 or COS-7) can be used in
the screening, provided that the "first T cell signal" is induced
by, for example, anti-CD3 monoclonal antibodies. T cell activation
can be analyzed by methods known to those of skill in the art,
including, for example, measuring proliferation, cytokine
production, CTL activity or expression of activation antigens such
as IL-2 receptor, CD69 or HLA-DR molecules. Usage of
antigen-specific T, cell clones, such as T cells specific for house
dust mite antigen Der p I, will allow analysis of antigen-specific
T cell activation (Yssel et al. (1992) J Immunol. 148:738-745).
Mutants are identified that can enhance or inhibit T cell
proliferation or enhance or inhibit CTL responses. Similarly
variants that have altered capacity to induce cytokine production
or expression of activation antigens as measured by, for example,
cytokine- specific ELISAs or flow cytometry can be identified.
[1161] The B7 variants are useful in modulating immune responses in
autoimmune diseases, allergy, cancer, infectious disease and
vaccination. B7 variants which act through CD28 with improved
activity (and with decreased activity through CTLA-4) will have
improved capacity to induce activation of T cells. In contrast, B7
variants which bind and act through CTLA-4 with improved activity
(and with decreased activity through CD28) will be potent negative
regulators of T cell functions and to induce tolerance and anergy.
Thus, by incorporating genes encoding these variant B7 molecules
into genetic vaccine vectors encoding an antigen, it is possible to
modify antigen-specific T cell responses. Vectors that harbor B7
genes that efficiently act through CD28 are useful in inducing, for
example, protective immune responses, whereas vectors that harbor
genes encoding B7 genes that efficiently act through CTLA-4 are
useful in inducing, for example, tolerance and anergy of allergen-
or autoantigen-specific T cells. In some situations, such as in
tumor cells or cells inducing autoimmune reactions, the antigen may
already be present on the surface of the target cell, and the
variant B7 molecules may be transfected in the absence of
additional exogenous antigen gene.
[1162] The methods of the invention are also useful for obtaining
B7 variants that have increased effectiveness in directing either
T.sub.H1 or T.sub.H2 cell differentiation. Differential roles have
been observed for B7-1 and B7-2 molecules in the regulation of T
helper (T.sub.H) cell differentiation (Freeman et al. (1995)
Immunity 2:523; Kuchroo et al. (1995) Cell 80:707). T.sub.H cell
differentiation can be measured by analyzing, the cytokine
production profiles induced by each particular variant. High levels
of IL-4, IL-5 and/or IL-13 are an indication of efficient T.sub.H2
cell differentiation whereas high levels of IFN- or IL-2 production
can be used as a marker of T.sub.H1 cell differentiation. B7
variants with altered capacity to induce T.sub.H1 or T.sub.H2 cell
differentiation are useful, for example, in the treatment of
allergic, malignant, autoimmune and infectious diseases and in
vaccination.
[1163] Also provided by the invention are methods of obtaining B7
variants that have enhanced capacity to induce IL-10 production by
antigen-specific T cells. Elevated production of IL-10 is a
characteristic of regulatory T cells, which can suppress
proliferation of antigen-specific CD4.sup.+ T cells (Groux et al.
(1997) Nature 389:737). stochastic (e.g. polynucleotide shuffling
& interrupted synthesis) and non-stochastic polynucleotide
reassembly is performed as described above, after which recombinant
nucleic acids encoding B7 variants having enhanced capability of
inducing IL-10 can be identified by, for example, ELISA or flow
cytometry using intracytoplasmic cytokine staining. The variants
that induce high levels of IL-10 production are useful in the
treatment of allergic and autoimmune diseases.
2.6.6. EVOLUTION OF GENETIC VACCINE VECTORS FOR INCREASED
VACCINATION EFFICACY AND EASE OF VACCINATION
[1164] This section discusses the application of the invention to
some specific goals in genetic vaccination. Many of these goals
relate to improvements in vectors used in vaccine delivery. Unless
otherwise indicated the methods are applicable to both viral and
nonviral vectors.
2.6.6.1. TOPICAL APPLICATION OF GENETIC VACCINE VECTORS
[1165] Low Efficiency of Topical Application; Protective Immune
Responses have not been Demonstrated
[1166] The invention provides methods of improving the ability of
genetic vaccine vectors to induce a desired response after topical
application of the vector. Adenoviral vectors topically applied to
bare skin have been shown to be capable of acting as vaccine
antigen delivery vehicles (Tang et al. (1997) Nature 388:729-730).
An adenoviral vector that encoded carcinoembryonic antigen (CA) was
shown to induce antibodies specific for CA after application to the
skin. However, the efficiency of topical application is generally
quite low, and protective immune responses have not been
demonstrated after topical application.
[1167] Optimizing the Topical Application Efficiency using the
Methods of the Invention
[1168] The invention provides methods of obtaining vectors that
exhibit improved efficiency when topically administered. Several
factors can influence topical application efficiency, each of which
can be optimized using the methods of the invention. For example,
the invention provides methods of improving vector affinity for
skin cells, improved skin cell transfection efficiency, improved
persistence of the vector in skin cells (both through improved
replication or through avoidance of destruction by immune cells),
and improved antigen expression in skin cells, and improved
induction of an immune response.
[1169] Methods of Reassembly (Optionally in Combination with other
Directed Evolution Methods Described Herein), Selection, and
Screening
[1170] These methods involve performing stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly using as substrates
plasmid, naked DNA vectors, or viral vector nucleic acids,
including, for example, adenoviral vectors. Libraries of
experimentally evolved (e.g. by polynucleotide reassembly &/or
polynucleotide site-saturation mutagenesis) nucleic acids are
screened to identify those nucleic acids that confer upon a vector
an enhanced ability to induce an immune response upon topical
administration. Screening can be conducted by, for example,
topically applying a library of experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis) vectors to skin, either mouse skin, monkey skin, or
human skin that has been transplanted to immunodeficient mice, or
to normal human skin in vivo. Vectors that persist and/or provide
efficient and long-lasting expression of marker gene are recovered
from the skin samples. In a preferred embodiment, the desired cells
are first selected by cell sorting, magnetic beads, or panning. For
example, recovery can be effected through expression of a marker
gene (e.g., GFP) and detecting cells that are transfected using
fluorescence microscopy or flow cytometry. Cells that express the
marker gene can be isolated using flow cytometry based cell
sorting. Screening can also involve selection of vectors that
induce the highest specific antibody or CTL responses upon
administration to a test mammal, or the identification of vectors
that provide an enhanced protective immune response to challenge
with a corresponding pathogen. Experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis) polynucleotides are then recovered, e.g., by
polymerase chain reaction, or the entire vectors can be purified
from these selected cells. If desired, further optimization of
topical application efficiency can be obtained by subjecting the
recovered experimentally evolved (e.g. by polynucleotide reassembly
&/or polynucleotide site-saturation mutagenesis)
polynucleotides to new rounds of reassembly (optionally in
combination with other directed evolution methods described herein)
and selection.
[1171] Administration of Genetic Vaccine Vectors Optimized for
Topical Application
[1172] Genetic vaccine vectors that are optimized for topical
application can be applied topically to the skin, or by
intramuscular, intravenous, intradermal, oral, anal, or vaginal
delivery. The vector can be delivered in any of the suitable forms
that are known to those of skill in the art, such as a patch, a
cream, as naked DNA, or as a mixture of DNA and one or more
transfection-enhancing agents such as liposomes and/or lipids. In
preferred embodiments, the genetic vaccine vector is applied after
the skin or other target is rendered more susceptible to uptake of
the vector by, for example, mechanical abrasion, removal of hair
(e.g., by treatment with a commercially available product such as
Nair.TM., Neet.TM., and the like). In one embodiment, the skin is
pretreated with proteases or lipases to make it more susceptible to
DNA delivery. In addition, the DNA can be mixed with the proteases
or lipases to enhance gene transfer. Alternatively, a droplet
containing the vector and other vaccine components, if any, can
simply be administered to the skin.
2.6.6.2. ENHANCED ABILITY TO ESCAPE HOST IMMUNE SYSTEM
[1173] Limitations of Host Immune Responses Directed Against the
Viral Vector Sometimes even before Target Cells are Entered
[1174] Immunogenicity is a particular concern with viral vectors,
since a host immune response can prevent a virus from reaching its
intended target particularly in repeated administrations. The
efficacy of some viral vectors which are used for genetic
vaccination and gene delivery is limited by host immune responses
directed against the viral vector. For example, most individuals
have pre-existing antibodies against adenovirus. Adenoviral vectors
can sometimes induce strong immune responses which can destroy
cells harboring adenoviral vectors or clear adenoviral vectors from
the host even before target cells are entered. Cellular immune
responses can also be induced against nonviral vectors administered
in naked form or shielded with a coat such as liposomes.
[1175] Methods to Create Genetic Vaccine Vectors with Improved
Ability to Avoid the Humoral and Cellular Immune Systems
[1176] The invention provides methods to create genetic vaccine
vectors that can escape immune responses that would otherwise be
detrimental to obtaining the desired effect. These methods are
useful for prolonging expression and secretion of pathogen antigen
or pharmaceutically useful protein by genetic vaccine vectors.
Several strategies are provided by which one can improve a genetic
vaccine vector's ability to avoid the humoral (Ab) and cellular
(CTL) immune systems. These strategies can be used in combination
to obtain optimal avoidance such as may be required for highly
immunogenic vectors such as adenovirus.
[1177] Incorporating into Genetic Vaccines one or more Components
that Inhibit Peptide Transport and/or MHC Class I Expression in
Order to Obtain Viral Vectors that are Capable of Escaping a Host
CTL Immune Response
[1178] In one embodiment, the invention provides methods of
obtaining viral vectors that are capable of escaping a host CTL
immune response. This method can be used in conjunction with
methods for obtaining genetic vaccine vectors that can escape the
humoral response; the combination of approaches is often desirable,
as different viral serotypes often have CTL epitopes in common,
suggesting that virus variants which are not recognized by
antibodies still are likely to be recognized by CTLs. This
embodiment of the invention involves incorporating into genetic
vaccines one or more components that inhibit peptide transport
and/or MHC class I expression. An essential element in the
activation of cytotoxic T lymphocyte (CTL) responses is an
interaction between T cell receptors on CTLs and antigenic
peptide-MHC class I molecule complexes on antigen presenting cells.
Expression of MHC class I molecules on thymocytes and antigen
presenting cells is a requirement for maturation and activation of
antigen-specific CD8.sup.+ T lymphocytes. Thus, genes that encode
inhibitors of MHC class I-mediated antigen presentation can be
experimentally evolved (e.g. by polynucleotide reassembly &/or
polynucleotide site-saturation mutagenesis) as described herein and
placed into viral vectors to obtain vectors that, when present in
target cells, do not induce destruction of the target cells by the
cells of the immune system. This can result in prolonged survival
of cells harboring genetic vaccine vectors, including those that
express a pathogen antigen, as well as vectors that express a
pharmaceutically useful protein. In the case of genetic vaccines,
reduced expression of MHC class I molecules will allow secretion of
the pathogen antigen, which then will be presented by professional
antigen presenting cells elsewhere. In the case of vectors encoding
pharmaceutical proteins, reduced expression of MHC class I
molecules prevents recognition by the immune system prolonging the
survival of the cells expressing the gene.
[1179] Reassembly (Optionally in Combination with other Directed
Evolution Methods Described Herein) Genes that Encode Inhibitors of
TAP Activity to Obtain Genes that Encode Optimized TAP
Inhibitors
[1180] Among the proteins involved in MHC class I molecule
expression and antigen presentation are those encoded by TAP genes
(transporters associated with antigen processing), which are
described above. In one embodiment of the invention, genes that
encode inhibitors of TAP activity are experimentally evolved (e.g.
by polynucleotide reassembly &/or polynucleotide
site-saturation mutagenesis) to obtain genes that encode optimized
TAP inhibitors. The substrates for these methods can include, for
example, one or more of the viral genes that are known to regulate
levels of MHC class I molecule expression. TAP I and TAP2 gene
expression is 5-10-fold and 100-fold reduced, respectively, in
cells transformed by adenovirus 12, which results in reduced class
I expression and thus leads to reduced virus-specific cytotoxic T
lymphocyte responses. Similarly, TAP gene expression is
downregulated in 49% of HPV-16.sup.+cervical carcinomas (Seliger et
al. (1997) Immunol. Today 18:292). Thus, adenovirus and HPV viral
nucleic acids provide examples of suitable substrates for carrying
out(the methods of the invention. Additional examples of suitable
stochastic (e.g. polynucleotide shuffling & interrupted
synthesis) and non-stochastic polynucleotide reassembly substrates
for this embodiment of the invention include the human
cytomegalovirus (CMV) encoded genes US2, US3 and US 11, which can
downregulate MHC class I expression (Wiertz et al. (1996) Nature
384:432 and Cell (1996) 84:769; Ahn et al. (1996) Proc. Nat'l. Acad
Sci. USA 93:10990). Another human CMV gene that encodes an
inhibitor of TAP-dependent peptide translocation is US6 (Lehner et
al. (1997) Proc. Nat'l. Acad Sci. USA 94:6904-9). Cells transfected
with US6 had reduced expression of MHC class I molecules on their
surface and reduced capacity to activate cytotoxic T
lymphocytes.
[1181] Reassembly (Optionally in Combination with other Directed
Evolution Methods Described Herein) this 7kb Cluster of Genes in
Order to find the most Potent Sequence for Inhibiting the
Expression of MHC Class I Molecules, Which can also be used for
Generation of Animal Models
[1182] Thus, in one embodiment, the invention involves stochastic
(e.g. polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly of this cluster of genes
(approximately 7kb), or fragments thereof, in order to identify the
sequences that are most potent in inhibiting the expression of MHC
class I molecules. Such optimized TAP inhibitor polynucleotide
sequences are useftil not only for use in constructing vectors that
can escape CTL immune responses, but also for generation of animal
models for use with human viruses that normally are eliminated in
laboratory animals due to their immunogenicity. The desired
expression levels and functional properties of TAP inhibitors may
vary depending on whether genetic vaccine vector, gene therapy
vector or animal model is evolved.
[1183] Reassembly (Optionally in Combination with Other Directed
Evolution Methods Described Herein) Other Genes Involved in
Downregulating Expression of MHC Class I Molecules and/or Antigen
Presentation
[1184] Alternative embodiments of the invention involve stochastic
(e.g. polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly of other genes that are
involved in downregulating expression of MHC class I molecules
and/or antigen presentation. Examples of other possible target
genes include genes encoding adenoviral E3 protein, herpes simplex
ICP47 protein, and tapasin antagonists (Seliger et al. (1997)
Immunol. Today 18:292-299; Galoncha et al. (1997) J Exp. Med.
185:1565-1572; Li et al. (1997) Proc. Nat'l. Acad. Sci. USA
94:8708-8713; Ortmann et al. (1997) Science 277:1306-1309.
[1185] A Gene that Encodes an MHC-Like Molecule that Inhibits NK
Cell Function but is unable to Present Antigens to T
Lymphocytes
[1186] Because reduced expression of MHC class I molecules on cell
surfaces may act as a stimulus for NK cells, it may be useful to
include in genetic vaccine vectors a gene that encodes an MHC like
molecule that inhibits NK cell function but is unable to present
antigens to T lymphocytes. An example of such molecule is MHC class
I homologue encoded by cytomegalovirus (Farrell et al. (1997)
Nature 3 86:510-514).
[1187] Obtaining Viral Vectors that Exhibit an Enhanced Capability
of Avoiding Attack by CD4.sup.+ T Lymphocytes
[1188] The invention also provides methods of obtaining viral
vectors that exhibit an enhanced capability of avoiding attack by
CD4.sup.+ T lymphocytes. Such vectors are particularly useful in
situations where the target cells are capable of expressing MHC
class II molecules, such as in the case of vaccinations and gene
therapy targeted to the cells of the immune system. Substrates for
stochastic (e.g. polynucleotide shuffling & interrupted
synthesis) and non-stochastic polynucleotide reassembly include
genes that encode inhibitors of MHC class II molecules such as, for
example, IL-10 and antagonists of IFN- (such as soluble IFN-
receptor).
[1189] Improving Sequences that Result in Inhibition of MHC Class I
Expression, MHC Class II Expression, and Additional Sequences that
Encode Homologs of MHC Class I Molecules
[1190] Vectors that have the greatest capability of escaping the
host immune system, will typically include DNA sequences that
result in inhibition of MHC class I expression and MHC class II
expression, and additional sequences that encode homologs of MHC
class I molecules. The properties of all these can be further
improved by stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide reassembly
according to the methods of the invention.
[1191] Methods for Screening the Library to Identify those
Polynucleotides that Exhibit the Desired Effect on the Host Immune
Response
[1192] Once a library of experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis) DNA molecules is obtained, any of several methods are
available for screening the library to identify those
polynucleotides that, when present in a viral vector (or in an
animal model) exhibit the desired effect on the host immune
response. For example, to obtain experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis) polynucleotides that inhibit MHC class I expression
and/or antigen presentation, a library of experimentally evolved
(e.g. by polynucleotide reassembly &/or polynucleotide
site-saturation mutagenesis) genes can be incorporated into genetic
vaccine or gene therapy vectors and transfected into human cell
lines, such as, for example, HeLa, U937 or Jijoye, in a single tube
transfection. Primary human monocytes, or dendritic cells generated
by culturing human cord blood cells or monocytes in the presence of
IL-4 and GM-CSF, are also suitable. Initial screening can be done
using FACS-sorting.
[1193] Cells Expressing the Lowest Levels of MHC Class I Molecules
are Expected to have the Lowest Capacity to Induce CTL
Responses
[1194] Cells expressing the lowest levels of MHC class I molecules
are selected, the polynucleotides that encode the MHC inhibitors,
or whole plasmids containing the sequences, are recovered. If
desired, the selected sequences can be subjected to new rounds of
reassembly (optionally in combination with other directed evolution
methods described herein) and selection. Cells expressing the
lowest levels of MHC class I molecules are expected to have the
lowest capacity to induce CTL responses.
[1195] Screening Method: Injecting Library of Experimentally
Evolved (e.g. by Polynucleotide Reassembly &/or Polynucleotide
Site-Saturation Mutagenesis) Polynucleotides that Encode Inhibitors
of MHC Class I Expression Incorporated into HPV Vectors
[1196] Another screening method involves incorporating libraries of
experimentally evolved (e.g. by polynucleotide reassembly &/or
polynucleotide site-saturation mutagenesis) polynucleotides that
encode inhibitors of MHC class I expression are incorporated into
human papillomavirus (HPV) vectors. This library is injected into
the skin of mice.
[1197] Normally, Murine Cells Expressing HPV are Destroyed by the
Host Immune System. Cells Expressing Potent Inhibitors of Peptide
Transportation and/or MHC Class Expression Will be able to Escape
the Immune Response
[1198] However, cells expressing potent inhibitors of peptide
transportation and/or MHC class expression will be able to escape
the immune response. The cells that express a marker gene present
on the vector, such as GFP, for extended periods of time are
selected, the sequences or whole plasmids are recovered, and, if
further optimization is desired, the selected sequences are
subjected to new rounds of reassembly (optionally in combination
with other directed evolution methods described herein) and
selection. Long-lasting maintenance of HPV in mice will allow drug
screening and vaccine studies, which to date have not been possible
due to high immunogenicity of HPV in mice.
[1199] Evolved Inhibitors Will Block Efficient Presentation of
Immunogenic Peptides, and Hence, Will Strongly Downregulate
Activation of Antigen-Specific CTLs Allowing Long-Lasting Transgene
Expression in vivo
[1200] In another embodiment, the libraries of experimentally
evolved (e.g. by polynucleotide reassembly &/or polynucleotide
site-saturation mutagenesis) polynucleotides encoding inhibitors of
MHC class I expression are incorporated into human adenovirus
vectors. This library is transfected into human cell lines, such as
HeLa cells, and cells expressing the lowest levels of MHC class I
molecules are selected as described above. The sequences that
provide the lowest levels of MHC class I expression are further
tested by analyzing the capacity of antigen-presenting cells
transfected with adenovirus harboring evolved inhibitors of MHC
class I expression to activate specific T cell lines or clones.
These inhibitors will block efficient presentation of immunogenic
peptides, and hence, will strongly downregulate activation of
antigen-specific CTLs allowing long-lasting transgene expression in
vivo.
[1201] Methods to Screen for Inhibitors
[1202] Methods to screen for improved inhibitors of MHC class II
expression include detection of MHC class II molecules on the
surface of the target cells by fluorescent labeled specific
monoclonal antibodies, fluorescence microscopy, and flow cytometry.
In addition, the inhibitors can be analyzed in functional assays by
studying the capacity of the inhibitors to block activation of MHC
class II restricted antigen-specific CD4.sup.+ T lymphocytes. For
example, one can determine the capacity of the inhibitor to inhibit
induction of CD4.sub.+ T cell proliferation induced by autologous
antigen presenting cells, such as monocytes, dendritic cells, B
cells or EBV-transformed B cell lines, that harbor genes encoding
the MHC class II inhibitor or have been treated with supernatant
containing the inhibitor.
2.6.6.3. ENHANCED ANTIVIRAL ACTIVITY
[1203] Obtaining a Recombinant Viral Vector Which has an Enhanced
Ability to Induce an Antiviral Response in a Cell
[1204] The invention also provides methods of obtaining a
recombinant viral vector which has an enhanced ability to induce an
antiviral response in a cell. These methods can include the steps
of:
[1205] (1) reassembling (&/or subjecting to one or more
directed evolution methods described herein) at least first and
second forms of a nucleic acid which comprise a viral vector,
wherein the first and second forms differ from each other in two or
more nucleotides, to produce a library of recombinant viral
vectors;
[1206] (2) transfecting the library of recombinant viral vectors
into a population of mammalian cells;
[1207] (3) staining the cells for the presence of Mx protein;
and
[1208] (4) isolating recombinant viral vectors from cells which
stain positive for Mx protein, wherein recombinant viral vectors
from positive staining cells exhibit enhanced ability to induce an
antiviral response.
[1209] Stochastic (e.g. polynucleotide shuffling & interrupted
synthesis) and non-stochastic polynucleotide reassembly is used to
produce a library of recombinant viral vectors. The library is
transfected into a population of mammalian cells, which are then
tested for ability to induce an antiviral response. One suitable
test involves staining the cells for the presence of Mx protein,
which is produced by cells that are exhibiting an antiviral
response (see, e.g., Hallimen et al. (1997) Pediatric Research
41:647-650; Melen et al. (1994) J Biol. Chem. 269:2009-2015).
[1210] Recombinant viral vectors can be isolated from cells which
stain positive for Mx protein. These recombinant viral vectors from
positive staining cells are enriched for those that exhibit
enhanced ability to induce an antiviral response. Viral vectors for
which this method is useful include, for example, influenza
virus.
2.6.6.4. EVOLUTION OF VECTORS HAVING INCREASED COPY NUMBER IN
PRODUCTION CELLS
[1211] Desirability of Method to Increase the Plasmid Copy Number
after all Elements have been Cloned in the Vector, Especially when
the Plasmid is to be Manufactured on a Large Scale
[1212] The invention provides methods for obtaining vector
components that, when present in a genetic vaccine vector (such as
a plasmid) the ability to replicate to a high copy number in a cell
used to produce the vector. Plasmids can incorporate various
heterologous DNA sequences, however the size or the nature of the
cloned sequences in a given plasmid vector may render that vector
less able to grow to high copy number in the bacteria in which it
is propagated. It is therefore desirable to have a method to
increase the plasmid copy number after all elements have been
cloned into the vector. This is especially important when the
plasmid is to be manufactured on a large scale as will be the case
for genetic vaccines.
[1213] Incorporating into the Plasmid One or more Polynucleotide
Sequences that Bind Proteins Which would Otherwise be Toxic to the
Bacterium
[1214] The methods of the invention involve incorporating into the
plasmid one or more polynucleotide sequences that bind proteins
which would otherwise be toxic to the bacterium. One suitable toxic
moiety and binding site combination is the transcription factor
GATA-1 and its recognition site. It has been shown that expression
of a DNA-binding fragment of GATA-1 is toxic to bacteria; this
toxicity apparently results from inhibition of bacterial DNA
replication. Trudel et al. ((1996) Biotechniques 20:684-693) have
described a plasmid (pGATA) that expresses the Z2B2 region of
GATA-1 as a GST fusion protein. The expression of the fusion
protein in this plasmid is under the control of the IPTG-inducible
lac promoter. The GST-GATA-1 fragment also binds strongly to a
sequence from the mouse-globin gene promoter as well as to the
C-oligonucleotide from the -globin gene 3' enhancer; either or both
of these are suitable for use as binding sites in the methods of
the invention.
[1215] Including Only a Single Form of the Selectable Marker in the
Shiffling Reaction to Achieve Significant Diversity in the
Experimentally Evolved (e.g. by Polynucleotide Reassembly &/or
Polynucleotide Site-Saturation Mutagenesis) Library to Recover a
Plasmid Which is Improved in its Growth Properties While Fully
Retaining the Appropriate Selection Function of the Plasmid
[1216] The plasmids preferably also include a selectable marker
such as, for example, kanamycin resistance (aminoglycoside
3'-phosphotransferase (EC 2.7.1.95)) and the like. The plasmid
backbone polynucleotide sequence is subjected to stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly as described herein to
generate a library of plasmids which have different backbone
sequences and possibly different supercoil densities. In order to
introduce sufficient sequence diversity to search for improved
function, it is preferable to perform family stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly. This can be accomplished
in the context of the present invention by including in the
reassembly (optionally in combination with other directed evolution
methods described herein) reaction(s) only a single form of the
selectable marker. In this way, significant diversity can be
achieved in the experimentally evolved (e.g. by polynucleotide
reassembly &/or polynucleotide site-saturation mutagenesis)
library to recover a plasmid which is improved in its growth
properties while fully retaining the appropriate selection function
of the plasmid.
[1217] Selecting for High Copy Number Plasmids
[1218] The selection for high copy number plasmids is performed by
introducing the library of experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis) recombinant plasmids into the desired host cell. The
host cells also express the toxic moiety, preferably under the
control of a promoter which is inducible. For example, the pGATA
plasmid is suitable for use in E. coli host cells. The
experimentally evolved (e.g. by polynucleotide reassembly &/or
polynucleotide site-saturation mutagenesis) plasmids are introduced
into the cells under non-inducing conditions. Transformed cells are
then placed under conditions which induce expression of the toxic
moiety. For example, E. coli cells that contain pGATA can be placed
on media containing increasing concentrations of IPTG. Those target
plasmids which grow to high copy number in the bacteria will
express correspondingly higher numbers of the binding sequences for
GATA-1. The target plasmids will bind the GST-GATA-1 fusion protein
and thus neutralize the toxic effects on the bacteria.
[1219] Plasmids with the highest copy number are detected as those
which confer the best growth to bacteria on the inducer-containing
growth media. Such plasmids can be recovered and transformed into
bacteria which lack the gene that encodes the toxic moiety; these
plasmids should retain their high copy number characteristics.
Further rounds of reassembly (optionally in combination with other
directed evolution methods described herein) can be used to isolate
high copy number plasmids by the above selection procedure.
Alternatively, manual screening can be done in the bacterial host
of choice, lacking the toxic moiety-encoding plasmid, to avoid any
effects due to the presence of this extraneous plasmid.
2.7. OPTIMIZATION OF TRANSPORT AND PRESENTATION OF ANTIGENS
[1220] The invention also provides methods of obtaining genetic
vaccines and accessory molecules that can improve the transport and
presentation of antigenic peptides. A library of experimentally
generated polynucleotides is created and screened to identify those
that encode molecules that have improved properties compared to the
wild-type counterparts. The polynucleotides themselves can be used
in genetic vaccines, or the gene products of the polynucleotides
can be utilized for therapeutic or prophylactic applications.
2.7.1. PROTEASOMES
[1221] The class I peptides presented on major histocompatibility
complex molecules are generated by cellular proteasomes.
Interferon-gamma can stimulate antigen presentation, and part of
the mechanism of action of interferon may be due to induction of
the proteasome beta-subunits LMP2 and LMP7, which replace the
homologous beta-subunits Y (delta) and X (epsilon). Such a
replacement changes the peptide cleavage specificity of the
proteasome and can enhance class I epitope immunogenicity. The Y
(delta) and X (epsilon) subunits, as well as other recently
discovered proteasome subunits such as the MECL-1 homologue MC14,
are characteristic of cells which are not specialized in antigen
presentation. Thus, the incorporation into cells by DNA transfer of
LMP2, LMP7, MECL-1 and/or other epitope presentation-specific and
potentially interferon-inducible subunits can enhance epitope
presentation. It is likely that the peptides generated by the
proteasome containing the interferon-inducible subunits are
transported to the endoplasmic reticulum by the TAP molecules.
[1222] The invention provides methods of obtaining proteasomes that
exhibit increased or decreased ability to specifically process MHC
class I epitopes. According to the methods, stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly is used to obtain evolved
proteins that can either have new specificities which might enhance
the immunogenicity of some proteins and/or enhance the activity of
the subunits once they are bound to the proteasome. Because the
transition from a non-specific proteasome to a class I
epitope-specific proteasome can pass through several states (in
which some but not all of the interferon-inducible subunits are
associated with the proteasome), many different proteolytic
specificities can potentially be achieved. Evolving the specific
LMP-like subunits can therefore create new proteasome compositions
which have enhanced functionality for the presentation of
epitopes.
[1223] The methods involve performing stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly using as substrates two or
more forms of polynucleotides which encode proteasome components,
where the forms of polynucleotides differ in at least one
nucleotide. reassembly (optionally in combination with other
directed evolution methods described herein) is performed as
described herein, using polynucleotides that encode any one or more
of the various proteasome components, including, for example, LMP2,
LMP7, MECL-1 and other individual proteasome components that are
specifically involved in class I epitope presentation. Examples of
suitable substrates are described in, e.g., Stoliwasser et al.
(1997) Eur. J Immunol. 27:1182-1187 and Gaczynska et al. (1996) J
Biol. Chem. 271:17275-17280. In a preferred embodiment,
polynucleotide reassembly (optionally in combination with other
directed evolution methods described herein) is used, in which the
different substrates are proteasome component-encoding
polynucleotides from different species.
[1224] After the reassembly (&/or one or more additional
directed evolution methods described herein) reaction is completed,
the resulting library of experimentally generated polynucleotides
is screened to identify those which encode proteasome components
having the desired effect on class I epitope production. For
example, the experimentally generated polynucleotides can be
introduced into a genetic vaccine vector which also encodes a
particular antigen of interest. The library of vectors can then be
introduced into mammalian cells which are then screened to identify
cells which exhibit increased antigen-specific immunogenicity.
Methods of analyzing proteasome activity are described in, for
example, Groettrup et al. (1997) Proc. Nat'l. Acad. Sci. USA
94:8970-8975 and Groettrup et al. (1997) Eur. J. Immunol.
26:863-869.
[1225] Alternatively, one can use the methods of the invention to
evolve proteins which bind strongly to the proteasome but have
decreased or no activity, thus antagonizing the proteasome activity
and diminishing a cells ability to present class I molecules. Such
molecules can be applied to gene therapy protocols in which it is
desirable to lower the immunogenicity of exogenous proteins
expressed in the cells as a result of the gene therapy, and which
would otherwise be processed for class I presentation allowing the
cell to be recognized by the immune system. Such high-affinity
low-activity LMP- like subunits will demonstrate immuno suppressive
effects which are also of use in other therapeutic protocols where
cells expressing a non-self protein need to be protected from an
immune response.
[1226] The specificity of the proteasome and the TAP molecules
(discussed below) may have co-evolved naturally. Thus it may be
important that the two pathways of the class I processing system be
functionally matched. A further aspect of the invention involves
performing stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide reassembly
simultaneously on the two gene families followed by random
combinations of the two in order to discover appropriate matched
proteolytic and transport specificities.
2.7.2. ANTIGEN TRANSPORT
[1227] The invention provides methods of improving transport of
antigenic peptides from the cytosolic compartment to the
endoplasmic reticulum. and thereby to the cell surface in the
context of MHC class I molecules. Enhanced expression of antigenic
peptides results in enhanced immune response, particularly in
improved activation of CD8.sup.+ cytotoxic lymphocytes. This is
useful in the development of DNA vaccines and in gene therapy.
[1228] In one embodiment, the invention involves evolving TAP-genes
(transporters associated with antigen processing) to obtain genes
that exhibit improved antigen presentation. TAP genes are members
of ATP-binding cassette family of membrane translocators. These
proteins transport antigenic peptides to MHC class I molecules and
are involved in the expression and stability of MHC class I
molecules on the cell surface. Two TAP genes, TAP1 and TAP2, have
been cloned to date (Powis et al. (1996) Proc. Nat'l. Acad. Sci.
USA 89:1463-1467; Koopman et al. (1997) Curr. Opin. Immunol. 9:
80-88; Monaco (1995) J Leukocyte Biol. 57:543-57). TAP1 and TAP2
form a heterodimer and these genes are required for transport of
peptides into the endoplasmic reticulum, where they bind to MHC
class I molecules. The essential role of TAP gene products in
presentation of antigenic peptides was demonstrated in mice with
disrupted TAP genes. TAP 1-deficient mice have drastically reduced
levels of surface expression of MHC class I, and positive selection
of CD8.sup.+ T cells in the thymus is strongly reduced. Therefore,
the number of CD8.sup.+ T lymphocytes in the periphery of
TAP-deficient mice is extremely low. Transfection of TAP genes back
into these cells restores the level of MHC class I expression.
[1229] TAP genes are a good target for polynucleotide (e.g. gene,
promoter, enhancer, intron, & the like) reassembly (optionally
in combination with other directed evolution methods described
herein) because of natural polymorphism and because these genes of
several mammalian species have been cloned and sequenced, including
human (Beck et al. (1992) J Mol. Biol. 228:433-441; Genbank
Accession No. Y13582; Powis et al., supra.), gorilla TAP1 (Laud et
al. (1996) Human Immunol. 50:91-102), mouse (Reiser et al. (1988)
Proc. Nat'l. Acad. Sci. USA 85:2255-2259; Marusina et al. (1997) J
Immunol, 158:5251-5256, TAP1: Genbank Accession Nos. U60018,
U60019, U60020, U60021, U60022, and L76468-L67470; TAP2: Genbank
Accession Nos. U60087, U60088, U6089, U60090, U60091 and U60092),
hamster (TAP 1, Genbank Accession Nos. AF001154 and AF001157; TAP2,
Genbank Accession Nos. AF001 156 and AF001155). Furthermore, it has
been shown that point mutations in TAP genes may result in altered
peptide specificity and peptide presentation. Also, functional
differences in TAP genes derived from different species have been
observed. For example, human TAP and rat TAP containing the rTA.P2a
allele are rather promiscuous, whereas mouse TAP is restrictive and
select against peptides with C-terminal small polar/hydrophobic or
positively charged amino acids. The basis for this selectivity is
unknown.
[1230] The methods of the invention involve performing stochastic
(e.g. polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly of TAP1 and TAP2 genes
using as substrates at least two forms of TAP1 and/or TAP2
polynucleotide sequences which differ in at least one nucleotide
position. In a preferred embodiment, TAP sequences derived from
several mammalian species are used as the substrates for reassembly
(optionally in combination with other directed evolution methods
described herein).
[1231] Natural polymorphism of the genes can provide additional
diversity of substrate. If desired, optimized TAP genes obtained
from one round of reassembly (optionally in combination with other
directed evolution methods described herein) and screening can be
subjected to additional reassembly (optionally in combination with
other directed evolution methods described herein)/screening rounds
to obtain further optimized TAP-encoding polynucleotides.
[1232] To identify optimized TAP-encoding polynucleotides from a
library of recombinant TAP genes, the genes can be expressed on the
same plasmid as a target antigen of interest. If this step is
limiting the extent of antigen presentation, then enhanced
presentation to CD8.sup.+ CTL will result. Mutants of TAPs may act
selectively to increase expression of a particular antigen peptide
fragment for which levels of expression are otherwise limiting, or
to cause transport of a peptide that would normally never be
transferred into the RER and made available to bind to MHC Class
I.
[1233] When used in the context of gene therapy vectors in cancer
treatment, evolved TAP genes provide a means to enhance expression
of MHC class I molecules on tumor cells and obtain efficient
presentation of antigenic tumor- specific peptides. Thus, vectors
that contain the evolved TAP genes can induce potent immune
responses against the malignant cells. Experimentally evolved (e.g.
by polynucleotide reassembly &/or polynucleotide
site-saturation mutagenesis) TAP genes can be transfected into
malignant cell lines that express low levels of MHC class I
molecules usina retroviral vectors or electroporation.
[1234] Transfection efficiency can be monitored using marker genes,
such as green fluorescent protein, encoded by the same vector as
the TAP genes. Cells expressing equal levels of green fluorescent
protein but the highest levels of MHC class I molecules, as a
marker of efficient TAP genes, are then sorted using flow
cytometry, and the evolved TAP genes are then recovered from these
cells by, for example, PCR or by recovering the entire vectors.
[1235] These sequences can then subjected into new rounds of
reassembly (optionally in combination with other directed evolution
methods described herein), selection and recovery, if further
optimization is desired. Molecular evolution of TAP genes can be
combined with simultaneous evolution of the desired antigen.
Simultaneous evolution of the desired antigen can further improve
the efficacy of presentation of antigenic peptides following DNA
vaccination. The antigen can be evolved, using polynucleotide
reassembly (optionally in combination with other directed evolution
methods described herein), to contain structures that allow optimal
presentation of desired antigenic peptides when optimal TAP genes
are expressed. TAP genes that are optimal for presentation of
antigenic peptides of one given antigen may be different from TAP
genes that are optimal for presentation of antigenic peptide of
another antigen. Polynucleotide (e.g. gene, promoter, enhancer,
intron, & the like) reassembly (optionally in combination with
other directed evolution methods described herein) technique is
ideal, and perhaps the only, method to solve this type of problems.
Efficient presentation of desired antigenic peptides can be
analyzed using specific cytotoxic T lymphocytes, for example, by
measuring the cytokine production or CTL activity of the T
lymphocytes using methods known to those of skill in the art.
2.7.3. CYTOTOXIC T-CELL INDUCING SEQUENCES AND IMMUNOGENIC AGONIST
SEQUENCES
[1236] Certain proteins are better able than others to carry MHC
class I epitopes because they are more readily used by the cellular
machinery involved in the necessary processing for class I epitope
presentation. The invention provides methods of identifying
expressed polypeptides that are particularly efficient in
traversing the various biosynthetic and degradative steps leading
to class I epitope presentation and the use of these polypeptides
to enhance presentation of CTL epitopes from other proteins.
[1237] In one embodiment, the invention provides Cytotoxic T-cell
Inducing Sequences (CTIS), which can be used to carry heterologous
class I epitopes for the purpose of vaccinating against the
pathogen from which the heterologous epitopes are derived. One
example of a CTIS is obtained from the hepatitis B surface antigen
(HBsAg), which has been shown to be an effective carrier for its
own CTL epitopes when delivered as a protein under certain
conditions. DNA immunization with plasmids expressing the HBsAg
also induces high levels of CTL activity. The invention provides a
shorter, truncated fragment of the HBsAg polypeptide which
functions very efficiently in inducing CTL activity, and attains
CTL induction levels that are higher than with the HBsAg protein or
with the plasmids encoding the full-length HBsAg polypeptide.
Synthesis of a CTIS derived from HBsAg is described in Example 3;
and a diagram of a CTIS is shown, described &/or referenced
herein (including incorporated by reference).
[1238] The ER localization of the truncated polypeptide may be
important in achieving suitable proteolytic liberation of the
peptide(s). containing the CTL epitopes (see Cresswell Craiu et
al. (1997) Proc. Nat'l. Acad. Sei. USA 94:10850-10855). The preS2
region and the transmembrane region provide T-helper epitopes which
may be important for the induction of a strong cytotoxic immune
response. Because the truncated CTIS polypeptide has a simple
structure (see FIG. 1), it is possible to attach one or more
heterologous class I epitope sequences to the C-terminal end of the
polypeptide without having to maintain any specific protein
conformation. Such sequences are then available to the class I
epitope processing mechanisms. The size of the polypeptide is not
subject to the normal constraints of the native HBsAg structure.
Therefore the length of the heterologous sequence and thus the
number of included CTL epitopes is flexible. This is shown
schematically in FIG. 2. The ability to include a long sequence
containing either multiple and distinct class I sequences, or
alternatively different variations of a single CTL sequence, allows
stochastic (e.g. polynucleotide shuffling & interrupted
synthesis) and non-stochastic polynucleotide reassembly methodology
to be applied.
[1239] The invention also provides methods of obtaining Immunogenic
Agonist Sequences (IAS) which induce CTLs capable of specific lysis
of cells expressing the natural epitope sequence. In some cases,
the reactivity is greater than if the CTL response is induced by
the natural epitope (see, Example 3 and FIG. 3). Such IAS-induced
CTL may be drawn from a T-cell repertoire different from that
induced by the natural sequence. In this way, poor responsiveness
to a given epitope can be overcome by recruiting T cells from a
larger pool. In order to discover such IAS, the amino acid at each
position of a CTL-inducing peptide (excluding perhaps the positions
of the so-called anchor residues) can be varied over the range of
the 19 amino acids not normally present at the position. stochastic
(e.g. polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly methodology can be used to
scan a large range of sequence possibilities.
[1240] A synthetic gene segment containing multiple copies of the
original epitope sequence can be prepared such that each copy
possesses a small number of nucleotide changes. The gene segment
can be experimentally evolved (e.g. by polynucleotide reassembly
&/or polynucleotide site-saturation mutagenesis) to create a
diverse range of CTL epitope sequences, some of which should
function as IAS. This process is illustrated in FIG. 4.
[1241] In practice, oligonucleotides are typically constructed in
accordance with the above design and polymerized enzymatically to
form the synthetic gene segment of the concatenated epitopes.
Restriction sites can be incorporated into a fraction of the
oligonucleotides to allow for cleavage and selection of given size
ranges of the concatenated epitopes, most of which will have
different sequences and thus will be potential IAS. The
epitope-containing gene segment can be joined by appropriate
cloning methods to a CTIS, such as that of HBsAg. The resulting
plasmid constructions can be used for DNA-based C immunization and
CTL induction.
2.8. GENETIC VACCINE PHARMACEUTICAL COMPOSITIONS AND METHODS OF
ADMINISTRATION
[1242] Using Genetic Vaccines in Prophylaxis and Therapy of
Infectious Diseases, Autoimmune Diseases, Other Inflammatory
Conditions, Allergies, Asthma, and Cancer and the Prevention of
Metastasis
[1243] The vector components and multicomponent genetic vaccines of
the invention are useful for treating and/or preventing various
diseases and other conditions. For example, genetic vaccines that
employ the reagents obtained according to the methods of the
invention are useful in both prophylaxis and therapy of infectious
diseases, including those caused by any bacterial, fungal, viral,
or other pathogens of mammals. The reagents obtained using the
invention can also be used for treatment of autoimmune diseases
including, for example, rheumatoid arthritis, SLE, diabetes
mellitus, myasthenia gravis, reactive arthritis, ankylosing
spondylitis, and multiple sclerosis. These and other inflammatory
conditions, including IBD, psoriasis, pancreatitis, and various
immunodeficiencies, can be treated using genetic vaccines that
include vectors and other components obtained using the methods of
the invention. Genetic vaccine vectors and other reagents obtained
using the methods of the invention can be used to treat allergies
and asthma. Moreover, the use of genetic vaccines have great
promise for the treatment of cancer and prevention of metastasis.
By inducing an immune response against cancerous cells, the body's
immune system can be enlisted to reduce or eliminate cancer.
[1244] Use of Recombinant Multivalent Antigens
[1245] The multivalent antigens of the invention are useful for
treating and/or preventing the various diseases and conditions with
which the respective antigens are associated. For example, the
multivalent antigens can be expressed in a suitable host cell and
are administered in polypeptide form. Suitable formulations and
dosage regimes for vaccine delivery are well known to those of
skill in the art. The improved immunomodulatory polynucleotides and
polypeptides of the invention are useful for treating and/or
preventing the various diseases and conditions with which the
respective antigens are associated.
[1246] An Antigen For a Particular Condition Can Be Optimized Using
Reassembly (&/or One or More Additional Directed Evolution
Methods Described Herein) and Selection Methods Analogous to Those
Described Herein.
[1247] In presently preferred embodiments, the reagents obtained
using the invention (e.g. optimized experimentally generated
polynucleotides that encode improved allergens), are used in
conjunction with a genetic vaccine. The choice of vector and
components can also be optimized for the particular purpose of
treating allergy or other conditions. In presently preferred
embodiments, the optimized genetic vaccine components are used in
conjunction with other optimized genetic vaccine reagents. For
example, an antigen that is useful for a particular condition can
be optimized by methods analogous to the reassembly (&/or one
or more additional directed evolution methods described herein) and
screening methods described herein.
[1248] The polynucleotide that encodes the recombinant antigenic
polypeptide can be placed under the control of a promoter, e.g., a
high activity or tissue-specific promoter. The promoter used to
express the antigenic polypeptide can itself be optimized using
reassembly (&/or one or more additional directed evolution
methods described herein) and selection methods analogous to those
described herein., as described in International Application No.
PCTIUS97/17300 (International Publication No. WO 98/13487).
[1249] The vector can contain immunostimulatory sequences such as
described herein. A vector engineered to direct a T.sub.H1 response
is preferred for many of the immune responses mediated by the
antigens described herein. The reagents obtained using the methods
of the invention can also be used in conjunction with
multicomponent genetic vaccines, which are capable of tailoring an
immune response as is most appropriate to achieve a desired effect.
It is sometimes advantageous to employ a genetic vaccine that is
targeted for a particular target cell type (e.g., an antigen
presenting cell or an antigen processing cell); suitable targeting
methods are described herein.
[1250] Delivery of Genetic Vaccines and Delivery Vehicles to
Mammals in vivo and ex vivo
[1251] Genetic vaccines, (e.g. genetic vaccines that include the
optimized experimentally generated polynucleotides obtained as
described herein, such as genetic vaccines that encode the
multivalent antigens described herein, including the multicomponent
genetic vaccines described herein), can be delivered to a mammal
(including humans) to induce a therapeutic or prophylactic immune
response. Vaccine delivery vehicles can be delivered in vivo by
administration to an individual patient, typically by systemic
administration (e.g., intravenous, intraperitoneal, intramuscular,
subdermal, intracranial, anal, vaginal, oral, buccal route or they
can be inhaled) or they can be administered by topical
application.
[1252] Alternatively, vectors can be delivered to cells ex vivo,
such as cells explanted from an individual patient (e.g.,
lymphocytes, bone marrow aspirates, tissue biopsy) or universal
donor hematopoietic stem cells, followed by reimplantation of the
cells into a patient, usually after selection for cells which have
incorporated the vector.
[1253] Delivery Methods and References
[1254] A large number of delivery methods are well known to those
of skill in the art. Such methods include, for example
liposome-based gene delivery (Debs and Zhu (1993) WO 93/24640;
Mannino and Gould-Fogerite (1988) BioTechniques 6(7): 682-691; Rose
U.S. Pat. No. 5,279,833; Brigham (1991) WO 91/06309; and Felgner et
al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7414), as well as
use of viral vectors (e.g., adenoviral (see, e.g., Berns et al.
(1995) Ann. NY Acad Sci. 772: 95-104; Ali et al. (1994) Gene Ther.
1: 367-3 84; and Haddada et al. (1995) Curr. Top. Microbiol.
Immunol. 199 (Pt 3): 297-306 for review), papillomaviral,
retroviral (see, e.g., Buchscher et al. (1992) J Virol. 66(5)
2731-2739; Johann et al. (1992) J Virol. 66 (5):163 5-1640 (1992);
Sommerfelt et al., (1990) Virol. 176:58-59; Wilson et al. (1989) J
Virol. 63:2374-2378; Miller et al., J Virol. 65:2220-2224 (1991);
Wong-Staal et al., PCT/US94/05700, and Rosenburg and Fauci (1993)
in Fundamental Immunology, Third Edition Paul (ed) Raven Press,
Ltd., New York and the references therein, and Yu et al., Gene
Therapy (1994) supra.), and adeno-associated viral vectors (see,
West et al. (1987) Virology 160:3 8-47; Carter et al. (1989) U.S.
Pat. No. 4,797,3 68; Carter et al. WO 93/24641 (1993); Kotin (1994)
Human Gene Therapy 5:793-801; Muzyczka (1994) J Clin. Invst.
94:1351 and Samulski (supra) for an overview of AAV vectors; see
also, Lebkowski, U.S. Pat. No. 5,173,414; Tratschin et al. (1985)
Mol. Cell. Biol. 5(11):3251-3260; Tratschin, et al. (1984) Mol.
Cell. Biol., 4:2072- 2081; Hermonat and Muzyczka (1984) Proc. Natl.
Acad Sci. USA, 81:6466-6470; McLaughlin et al. (1988) and Samulski
et al. (1989) J Virol., 63:03822-3 828), and the like.
[1255] Introduction of "Naked" DNA and/or RNA That Comprises a
Genetic Vaccine Directly Into a Tissue or Using "Biolistic" or
Particle-Mediated Transformation, Both in vivo and ex vivo
[1256] "Naked" DNA and/or RNA that comprises a genetic vaccine can
be introduced directly into a tissue, such as muscle. See, e.g.,
U.S. Pat. No. 5,580,859. Other methods such as "biolistic" or
particle-mediated transformation (see, e.g., Sanford et al., U.S.
Pat. No. 4,945,050; U.S. Pat. No. 5,036,006) are also suitable for
introduction of genetic vaccines into cells of a mammal according
to the invention. These methods are useful not only for in vivo
introduction of DNA into a mammal, but also for ex vivo
modification of cells for reintroduction into a mammal. As for
other methods of delivering genetic vaccines, if necessary, vaccine
administration is repeated in order to maintain the desired level
of immunomodulation.
[1257] Methods of Administering Packaged Nucleic Acids in Mammals
For Transduction of Cells in vivo
[1258] Genetic vaccine vectors (e.g., adenoviruses, liposomes,
papillomaviruses, retroviruses, etc.) can be administered directly
to the mammal for transduction of cells in vivo. The genetic
vaccines obtained using the methods of the invention can be
formulated as pharmaceutical compositions for administration in any
suitable manner, including parenteral (e.g., subcutaneous,
intramuscular, intradermal, or intravenous), topical, oral, rectal,
intrathecal, buccal (e.g., sublingual), or local administration,
such as by aerosol or transdermally, for prophylactic and/or
therapeutic treatment. Pretreatment of skin, for example, by use of
hair-removing agents, may be useful in transdermal delivery.
Suitable methods of administering such packaged nucleic acids are
available and well known to those of skill in the art, and,
although more than one route can be used to administer a particular
composition, a particular route can often provide a more immediate
and more effective reaction than another route.
[1259] Pharmaceutically acceptable carriers are determined in part
by the particular composition being administered, as well as by the
particular method used to administer the composition. Accordingly,
there is a wide variety of suitable formulations of pharmaceutical
compositions of the present invention. A variety of aqueous
carriers can be used, e.g., buffered saline and the like. These
solutions are sterile and generally free of undesirable matter.
These compositions may be sterilized by conventional, well known
sterilization techniques. The compositions may contain
pharmaceutically acceptable auxiliary substances as required to
approximate physiological conditions such as pH adjusting and
buffering agents, toxicity adjusting agents and the like, for
example, sodium acetate, sodium chloride, potassium chloride,
calcium chloride, sodium lactate and the like. The concentration of
genetic vaccine vector in these formulations can vary widely, and
will be selected primarily based on fluid volumes, viscosities,
body weight and the like in accordance with the particular mode of
administration selected and the patient's needs.
[1260] Formulations suitable for oral administration can consist of
(a) liquid solutions, such as an effective amount of the packaged
nucleic acid suspended in diluents, such as water, saline or PEG
400; (b) capsules, sachets or tablets, each containing a
predetermined amount of the active ingredient, as liquids, solids,
granules or gelatin; (c) suspensions in an appropriate liquid; and
(d) suitable emulsions. Tablet fonns can include one or more of
lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn
starch, potato starch, tragacanth, microcrystalline cellulose,
acacia, gelatin, colloidal silicon dioxide, croscannellose sodium,
talc, magnesium stearate, stearic acid, and other excipients,
colorants, fillers, binders, diluents, buffering agents, moistening
agents, preservatives, flavoring agents, dyes, disintegrating
agents, and pharmaceutically compatible carriers.
[1261] Lozenge forms can comprise the active ingredient in a
flavor, usually sucrose and acacia or tragacanth, as well as
pastilles comprising the active ingredient in an inert base, such
as gelatin and glycerin or sucrose and acacia emulsions, gels, and
the like containing, in addition to the active ingredient, carriers
known in the art. It is recognized that the genetic vaccines, when
administered orally, must be protected from digestion. This is
typically accomplished either by complexing the vaccine vector with
a composition to render it resistant to acidic and enzymatic
hydrolysis or by packaging the vector in an appropriately resistant
carrier such as a liposome. Means of protecting vectors from
digestion are well known in the art. The pharmaceutical
compositions can be encapsulated, e.g., in liposomes, or in a
formulation that provides for slow release of the active
ingredient.
[1262] The packaged nucleic acids, alone or in combination with
other suitable components, can be made into aerosol formulations
(e.g., they can be "nebulized") to be administered via inhalation.
Aerosol formulations can be placed into pressurized acceptable
propellants, such as dichlorodifluoromethane, propane, nitrogen,
and the like. Suitable formulations for rectal administration
include, for example, suppositories, which consist of the packaged
nucleic acid with a suppository base. Suitable suppository bases
include natural or synthetic triglycerides or paraffin
hydrocarbons. In addition, it is also possible to use gelatin
rectal capsules which consist of a combination of the packaged
nucleic acid with a base, including, for example, liquid
triglycerides, polyethylene glycols, and paraffin hydrocarbons.
[1263] Formulations suitable for parenteral, administration, such
as, for example, by intraarticular (in the joints), intravenous,
intramuscular, intradermal, intraperitoneal, and subcutaneous
routes, include aqueous and non-aqueous, isotonic sterile injection
solutions, which can contain antioxidants, buffers, bacteriostats,
and solutes that render the formulation isotonic with the blood of
the intended recipient, and aqueous and non-aqueous sterile
suspensions that can include suspending agents, solubilizers,
thickening agents, stabilizers, and preservatives. In the practice
of this invention, compositions can be administered, for example,
by intravenous infusion, orally, topically, intraperitoneally,
intravesically or intrathecally.
[1264] Parenteral Administration and Intravenous Administration Are
the Preferred Methods of Administration
[1265] The formulations of packaged nucleic acid can be presented
in unit-dose or multi-dose sealed containers, such as ampoules and
vials. Injection solutions and suspensions can be prepared from
sterile powders, granules, and tablets of the kind previously
described. Cells transduced by the packaged nucleic acid can also
be administered intravenously or parenterally.
[1266] Dose Size
[1267] The dose administered to a patient, in the context of the
present invention should be sufficient to effect a beneficial
therapeutic response in the patient over time. The dose will be
determined by the efficacy of the particular vector employed and
the condition of the patient, as well as the body weight or
vascular surface area of the patient to be treated. The size of the
dose also will be determined by the existence, nature, and extent
of any adverse side-effects that accompany the administration of a
particular vector, or transduced cell type in a particular
patient.
[1268] In determining the effective amount of the vector to be
administered in the treatment or prophylaxis of an infection or
other condition, the physician evaluates vector toxicities,
progression of the disease, and the production of anti-vector
antibodies, if any. In general, the dose equivalent of a naked
nucleic acid from a vector is from about 1 .mu.g to 1 mg for a
typical 70 kilogram patient, and doses of vectors used to deliver
the nucleic acid are calculated to yield an equivalent amount of
therapeutic nucleic acid. Administration can be accomplished via
single or divided doses.
[1269] In therapeutic applications, compositions are administered
to a patient suffering from a disease (e.g., an infectious disease
or autoimmune disorder) in an amount sufficient to cure or at least
partially arrest the disease and its complications. An amount
adequate to accomplish this is defined as a "therapeutically
effective dose." Amounts effective for this use will depend upon
the severity of the disease and the general state of the patient's
health. Single or multiple administrations of the compositions may
be administered depending on the dosage and frequency as required
and tolerated by the patient. In any event, the composition should
provide a sufficient quantity of the proteins of this invention to
effectively treat the patient.
[1270] In prophylactic applications, compositions are administered
to a human or other mammal to induce an immune response that can
help protect against the establishment of an infectious disease or
other condition.
[1271] Ability to Determine Toxicity Therapeutic Efficacy
[1272] The toxicity and therapeutic efficacy of the genetic vaccine
vectors provided by the invention are determined using standard
pharmaceutical procedures in cell cultures or experimental animals.
One can determine the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population) using procedures presented herein and
those otherwise known to those of skill in the art.
[1273] More on Dosage
[1274] A typical pharmaceutical composition for intravenous
administration would be about 0.1 to 10 mg per patient per day.
Dosages from 0.1 up to about 100 mg per patient per day may be
used, particularly when the drug is administered to a secluded site
and not into the blood stream, such as into a body cavity or into a
lumen of an organ. Substantially higher dosages are possible in
topical administration. Actual methods for preparing parenterally
administrable compositions will be known or apparent to those
skilled in the art and are described in more detail in such
publications as Remington's Pharmaceutical Science, 15th ed., Mack
Publishing Company, Easton, Pa. (1980).
[1275] Packaging/Dispenser Devices
[1276] The genetic vaccines obtained using the methods of the
invention (383 and 368-"the multivalent antigenic polypeptides of
the invention, and genetic vaccines that express the polypeptides")
can be packaged in packs, dispenser devices, and kits for
administering genetic vaccines to a mammal. For example, packs or
dispenser devices that contain one or more unit dosage forms are
provided. Typically, instructions for administration of the
compounds will be provided with the packaging, along with a
suitable indication on the label that the compound is suitable for
treatment of an indicated condition. For example, the label may
state that the active compound within the packaging is useful for
treating a particular infectious disease, autoimmune disorder,
tumor, or for preventing or treating other diseases or conditions
that are mediated by, or potentially susceptible to, a mammalian
immune response.
2.9. USES OF GENETIC VACCINES
[1277] Genetic vaccines which include optimized vector modules and
other reagents provided by the invention are useful for treating
many diseases and other conditions that are either mediated by a
mammalian immune system or are susceptible to treatment by an
appropriate immune response. Representative examples of these
diseases are listed below; antigens appropriate for each are
described in copending, commonly assigned U.S. patent application
Ser. No. filed Feb. 10, 1999 as TTC Attorney Docket No.
18097-028710US.
[1278] Substrates For Evolution of Optimized Recombinant
Antigens
[1279] The invention provides methods of obtaining experimentally
generated polynucleotides that encode antigens that exhibit
improved ability to induce an immune response to a pathogenic
agent. The methods are applicable to a wide range of pathogenic
agents, including potential biological warfare agents and other
organisms and polypeptides that can cause disease and toxicity in
humans and other animals. The following examples are merely
illustrative, and not limiting.
2.9.1. INFECTIOUS DISEASES
[1280] Genetic vaccine vectors obtained according to the methods of
the invention are useful in both prophylaxis and therapy of
infectious diseases, including those caused by any bacterial,
fungal, viral, or other pathogens of mammals. In some embodiments,
protection is conferred by use of a genetic vaccine vector that
will express an antigen (either or both of a humoral antigen or a T
cell antigen) of the pathogen of interest. In preferred
embodiments, the antigen is evolved using the methods of the
invention in order to obtain optimized antigens as described
herein. The vector induces an immune response against the antigen.
One or several antigens or antigen fragments can be included in one
genetic vaccine delivery vehicle. Examples of pathogens and
corresponding polypeptides from which an antigen can be obtained
include, but are not limited to, HIV (gp120, gp160), hepatitis B,
C, D, E (surface antigen), rabies (glycoprotein), Schistosoma
mansoni (calpain; Jankovic (1996) J Immunol. 157: 806-14). Other
pathogen infections that are treatable using genetic vaccine
vectors include, for example, herpes zoster, herpes simplex -1 and
-2, tuberculosis (including chronic, drug-resistant), lyme disease
(Borrelia burgorferii), syphilis, parvovirus, rabies, human
papillomavirus, and the like.
2.9.1.1 BACTERIAL PATHOGENS AND TOXINS
[1281] In some embodiments, the methods of the invention are
applied to bacterial pathogens, as well as to toxins produced by
bacteria and other organisms. One can use the methods to obtain
experimentally generated polypeptides that can induce an immune
response against the pathogen, as well as recombinant toxins that
are less toxic than native toxin polypeptides. Often, the
polynucleotides of interest encode polypeptides that are present on
the surface of the pathogenic organism. Among the pathogens for
which the methods of the invention are useful for producing
protective immunogenic experimentally generated polypeptides are
the Yersinia species.
[1282] Yersinia pestis, the causative agent of plague, is one of
the most virulent bacteria known with LD.sub.50 values in mouse of
less than 10 bacteria. The pneumonic form of the disease is readily
spread between humans by aerosol or infectious droplets and can be
lethal within days. A particularly preferred target for obtaining a
experimentally generated polypeptide that can protect against
Yersinia infection is the V antigen, which is a 37 kDa virulence
factor, induces protective immune responses and is currently being
evaluated as a subunit vaccine (Brubaker (1991) Current
Investigations of the Microbiology of Yersinae, 12: 127). The
V-antigen alone is not toxic, but Y pestis isolates that lack the
V-antigen are avirulent. The Yersinia V-antigen has been
successfully produced in E. coli by several groups (Leary et al.
(1995) Infect. Immun. 3: 2854). Antibodies that recognize the
V-antigen can provide passive protection against homologous
strains, but not against heterologous strains. Similarly,
immunization with purified V antigen protects against only
homologous strains. To obtain cross-protective recombinant V
antigen, in a preferred embodiment, V antigen genes from various
Yersinia species are subjected to polynucleotide reassembly
(optionally in combination with other directed evolution methods
described herein). The genes encoding the V antigen from Y. pestis,
Y. enterocolitica, and Y. pseudotuberculosis, for example, are
92-99% identical at the DNA level, making them ideal for
optimization using family reassembly (optionally in combination
with other directed evolution methods described herein) according
to the methods of the invention. After reassembly (optionally in
combination with other directed evolution methods described
herein), the library of recombinant nucleic acids is screened
and/or selected for those that encode recombinant V antigen
polypeptides that can induce an improved immune response and/or
have greater cross-protectivity.
[1283] Bacillus anthracis, the causative agent of anthrax, is
another example of a bacterial target against which the methods of
the invention are useful. The anthrax protective antigen (PA)
provides protective immune responses in test animals, and
antibodies against PA also provide some protection. However, the
immunogenicity of PA is relatively poor, so multiple injections are
typically required when wild-type PA is used. Co-vaccination with
lethal factor (LF) can improve the efficacy of wild-type PA
vaccines, but toxicity is a limiting factor. Accordingly the
stochastic (e.g. polynucleotide shuffling & interrupted
synthesis) and non-stochastic polynucleotide reassembly and antigen
library immunization methods of the invention can be used to obtain
nontoxic LF. Polynucleotides that encode LF from various B.
anthracis strains are subjected to family reassembly (optionally in
combination with other directed evolution methods described
herein). The resulting library of recombinant LF nucleic acids can
then be screened to identify those that encode recombinant LF
polypeptides that exhibit reduced toxicity. For example, one can
inoculate tissue culture cells with the recombinant LF polypeptides
in the presence of PA and select those clones for which the cells
survive. If desired, one can then backcross the nontoxic LF
polypeptides to retain the immunogenic epitopes of LF. Those that
are selected through the first screen can then be subjected to a
secondary screen. For example, one can test for the ability of the
recombinant nontoxic LF polypeptides to induce an immune response
(e.g., CTL or antibody response) in a test animal such as mice. In
preferred embodiments, the recombinant nontoxic LF polypeptides are
then tested for ability to induce protective immunity in test
animals against challenge by different strains of B. anthracis.
[1284] The protective antigen (PA) of B. anthracis is also a
suitable target for the methods of the invention. PA-encoding
nucleic acids from various strains of B. anthracis are subjected to
stochastic (e.g. polynucleotide shuffling & interrupted
synthesis) and non-stochastic polynucleotide reassembly. One can
then screen for proper folding in, for example, E. coli, using
polyclonal antibodies. Screening for ability to induce
broad-spectrum antibodies in a test animal is also typically used,
either alone or in addition to a preliminary screening method. In
presently preferred embodiments, those experimentally generated
polynucleotides that exhibit the desired properties can be
backcrossed so that the immunogenic epitopes are maintained.
Finally, the selected recombinants are tested for ability to induce
protective immunity against different strains of B. anthracis in a
test animal.
[1285] The Staphylococcus aureus and Streptococcus toxins are
another example of a target polypeptide that can be altered using
the methods of the invention. Strains of Stapkvlococcus aureus and
group A Streptococci are involved in a range of diseases, including
food poisoning, toxic shock syndrome, scarlet fever and various
autoimmune disorders. They secrete a variety of toxins, which
include at least five cytolytic toxins, a coagulase, and a variety
of enterotoxins. The enterotoxins are classified as superantigens
in that they crosslink MHC class II molecules with T cell receptors
to cause a constitutive T cell activation (Fields et al. (1996)
Nature 384: 188). This results in the accumulation of pathogenic
levels of cytokines that can lead to multiple organ failure and
death. At least thirty related, yet distinct enterotoxins have been
sequenced and can be phylogenetically grouped into families.
Crystal structures have been obtained for several members alone and
in complex with MHC class II molecules. Certain mutations in the
MHC class II binding site of the toxins strongly reduce their
toxicity and can form the basis of attenuated vaccines (Woody et
al. (1997) Vaccine 15: 133). However, a successful immune response
to one type of toxin may provide protection against closely related
family members, whereas little protection against toxins from the
other families is observed. Family reassembly (optionally in
combination with other directed evolution methods described herein)
of enterotoxin genes from various family members can be used to
obtain recombinant toxin molecules that have reduced toxicity and
can induce a cross-protective immune response. Experimentally
evolved (e.g. by polynucleotide reassembly &/or polynucleotide
site-saturation mutagenesis) antigens can also be screened to
identify antigens that elicit neutralizing antibodies in an
appropriate animal model such as mouse or monkey. Examples of such
assays can include ELISA formats in which the elicited antibodies
prevent binding of the enterotoxin to the MHC complex and/or T cell
receptors on cells or purified forms. These assays can also include
formats where the added antibodies would prevent T cells from being
cross-linked to appropriate antigen presenting cells.
[1286] Cholera is an ancient, potentially lethal disease caused by
the bacterium Vibrio cholerae and an effective vaccine for its
prevention is still unavailable. Much of the pathogenesis of this
disease is caused by the cholera enterotoxin. Ingestion of
microgram quantities of cholera toxin can induce severe diarrhea
causing loss of tens of liters of fluid.
[1287] Cholera toxin is a complex of a single catalytic A subunit
with a pentameric ring of identical B subunits. Each subunit is
inactive on its own. The B subunits bind to specific ganglioside
receptors on the surface of intestinal epithelial cells and trigger
the entry of the A subunit into the cell. The A subunit
ADP-ribosylates a regulatory G protein initiating a cascade of
events causing a massive, sustained flow of electrolytes and water
into the intestinal lumen resulting in extreme diarrhea.
[1288] The B subunit of cholera toxin is an attractive vaccine
target for a number of reasons. It is a major target of protective
antibodies generated during cholera infection and contains the
epitopes for antitoxin neutralizing antibodies. It is nontoxic
without the A subunit, is orally effective, and stimulates
production of a strong IgA-dominated gut mucosal immune response,
which is essential in protection against cholera and cholera toxin.
The B subunit is also being investigated for use as an adjuvant in
other vaccine preparations, and therefore, evolved toxins may
provide general improvements for a variety of different vaccines.
The heat-labile enterotoxins (LT) from enterotoxigenic E. coli
strains are structurally related to cholera toxin and are 75%
identical at the DNA sequence level. To obtain optimized
recombinant toxin molecules that exhibit reduced toxicity and
increased ability to induce an immune response that is protective
against V. cholerae and E. coli, the genes that encode the related
toxins are subjected to stochastic (e.g. polynucleotide shuffling
& interrupted synthesis) and non-stochastic polynucleotide
reassembly.
[1289] The recombinant toxins are then tested for one or more of a
several desirable traits. For example, one can screen for improved
cross-reactivity of antibodies raised against the recombinant toxin
polypeptides, for lack of toxicity in a cell culture assay, and for
ability to induce a protective immune response against the
pathogens and/or against the toxins themselves. The experimentally
evolved (e.g. by polynucleotide reassembly &/or polynucleotide
site-saturation mutagenesis) clones can be selected by phage
display and/or screened by phage ELISA and ELISA assays for the
presence of epitopes from the different serotypes. Variant proteins
with multiple epitopes can then be purified and used to immunize
mice or other test animal. The animal serum is then assayed for
antibodies to the different B chain subtypes and variants that
elicit a broad cross-reactive response will be evaluated further in
a virulent challenge model. The E. coli and V. cholerae toxins can
also act as adjuvants that are capable of enhancing mucosal
immunity and oral delivery of vaccines and proteins.
[1290] Accordingly, One Can Test the Library of Recombinant Toxins
For Enhancement of the Adjuvant Activity
[1291] Experimentally evolved (e.g. by polynucleotide reassembly
&/or polynucleotide site-saturation mutagenesis) antigens can
also be screened for improved expression levels and stability of
the B chain pentamer, which may be less stable than when in the
presence of the A chain in the hexameric complex. Addition of a
heat treatment step or denaturing agents such as salts, urea,
and/or guanidine hydrochloride can be included prior to ELISA
assays to measure yields of correctly folded molecules by
appropriate antibodies. It is sometimes desirable to screen for
stable monomeric B chain molecules, in an ELISA format, for
example, using antibodies that bind monomeric, but not pentameric B
chains. Additionally, the ability of experimentally evolved (e.g.
by polynucleotide reassembly &/or polynucleotide
site-saturation mutagenesis) antigens to elicit neutralizing
antibodies in an appropriate animal model such as mouse or monkey
can be screened. For example, antibodies that bind to the B chain
and prevent its binding to its specific ganglioside receptors on
the surface of intestinal epithelial cells may prevent disease.
Similarly antibodies that bind to the B chain and prevent its
pentamerization or block A chain binding may be useful in
preventing disease.
[1292] The bacterial antigens that can be improved by stochastic
(e.g. polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly for use as vaccines also
include, but are not limited to, Helicobacter pylori antigens CagA
and VacA (Blaser (1996) Aliment. Pharmacol. Ther. 1: 73-7; Blaser
and Crabtree (1996) Am. J Clin. Pathol. 106: 565-7; Censini et al.
(1996) Proc. Nat'l. Acad. Sci. USA 93: 14648-14643).
[1293] Other suitable H. pylori antigens include, for example, four
immunoreactive proteins of 45-65 kDa as reported by Chatha et al.
(1997) Indian J Med. Res. 105: 170-175 and the H. pylori GroES
homologue (HspA) (Kansau et al. (1996) Mol. Microbiol. 22:
1013-1023. Other suitable bacterial antigens include, but are not
limited to, the 43-kDa and the fimbrilin (41 kDa) proteins of P.
gingivalis (Boutsl et al. (1996) Oral Microbiol. Immunol. 11: 236-
241); pneumococcal surface protein A (Briles et al. (1996) Ann.
NYAcad. Sci. 797: 118-126); Chlamydia psittaci antigens, 80-90 kDa
protein and 110 kDa protein (Buendia et al. (1997) FEMSMicrobiol.
Lett. 150: 113-9); the chlainydial exoglycolipid antigen (GLXA)
(Whittum-Hudson et al. (1996) Nature Med. 2: 1116-1121); Chlamlydia
pneumoniae species-specific antigens in the molecular weight ranges
92-98, 51-55, 43-46 and 31.5-33 kDa and genus-specific antigens in
the ranges 12, 26 and 65-70 kDa (Halme et al. (1997) Scand. J
Immunol. 45: 378-84); Neisseria gonorrhoeae (GC) or Escherichia
coli phase-variable opacity (Opa) proteins (Chen and Gotschlich
(1996) Proc. Nat'l. Acad. Sci. USA 93: 14851-14856), any of the
twelve immunodominant proteins of Schistosoma mansoni (ranging in
molecular weight from 14 to 208 kDa) as described by Cutts and
Wilson (1997) Parasitolog-v 114: 245-55; the 17-kDa protein antigen
of Brucella abortus (De Mot et al. (1996) Curr. Microbiol. 33:
26-30); a gene homolog of the 17-kDa protein antigen of the
Gram-negative pathogen Brucella abortus identified in the
nocardioform actinomycete Rhodococcus sp. N186/21 (De Mot et al.
(1996) Curr. Microbiol. 33: 26-30); the staphylococcal enterotoxins
(SEs) (Wood et al. (1997) FEMS Immunol. Med. Microbiol. 17: 1-10),
a 42-kDa M. hy,opneunioniae NrdF ribonucleotide reductase R2
protein or 15-kDa subunit protein of M. hyopneumoniae (Fagan et al.
(1997) Infect. Immun. 65: 2502-2507), the meningococcal antigen
PorA protein (Feavers et al. (1997) Clin. Diagn. Lab. Immunol. 3:
444-50); pneumococcal surface protein A (PspA) (McDaniel et al.
(1997) Gene Ther. 4: 375-377); F. tularensis outer membrane protein
FopA (Fulop et al. (1996) FEMSImmunol. Med. Microbiol. 13:
245-247); the major outer membrane protein within strains of the
genus Actinobacillus (Hartmann et al. (1996) Zentralbl. Bakteriol.
284: 255-262); p60 or listeriolysin (Hly) antigen of Listeria
monocytogenes (Hess et al. (1996) Proc. Nat'l. Acad. Sci. USA 93:
1458-1463); flagellar (G) antigens observed on Salmonella
enteritidis and S. pullorum (Holt and Chaubal (1997) J. Clin.
Microbiol. 35: 1016-1020); Bacillus anthracis protective antigen
(PA) (Ivins et al. (1995) Vaccine 13: 1779-1784); Echinococcus
granulosus antigen 5 (Jones et al. (1996) Parasitology 113:
213-222); the rol genes of Shigella dvsenteriae I and Escherichia
coli K-12 (Klee et al. (1997) J. Bacteriol. 179: 2421-2425); cell
surface proteins Rib and alpha of group B streptococcus (Larsson et
al. (1996) Infect. Immun. 64: 3518-3523); the 37 kDa secreted
polypeptide encoded on the 70 kb virulence plasmid of pathogenic
Yersinia spp. (Leary et al. (1995) Contrib. Microbiol. Immunol. 13:
216-217 and Roggenkamp et al. (1997) Infect. Immun. 65: 446-51);
the OspA (outer surface protein A) of the Lyme disease spirochete
Borrelia burgdorferi (Li et al. (1997) Proc. Nat'l. Acad Sci. USA
94: 3584-3589, Padilla et al. (1996) J Infect. Dis. 174: 739-746,
and Wallich et al. (1996) Infection 24: 396-397); the Brucella
melitensis group 3 antigen gene encoding Omp28 (Lindler et al.
(1996) Infect. Immun. 64: 2490-2499); the PAc antigen of
Streptococcus mutans (Murakami et al. (1997) Infect. Immun. 65:
794-797); pneumolysin, Pneumococcal neuraminidases, autolysin,
hyaluronidase, and the 37 kDa pneumococcal surface adhesin A (Paton
et al. (1997) Microb. Drug Resist. 3: 1-10); 29-32, 41-45,
63-71.times.10(3) MW antigens of Salmonella typhi (Perez et al.
(1996) Immunology 89: 262-267); K-antigen as a marker of Klebsiella
pneumoniae (Priamukhina and Morozova (1996) Klin. Lab. Diagn.
47-9); nocardial antigens of molecular mass approximately 60, 40,
and 15-10 kDa (Prokesova et al. (1996) Int. J Immunopharmacol. 18:
661- 668); Staphylococcus aureus antigen ORF-2 (Rieneck et al.
(1997) Biochim Biophys Acta 1350: 128-132); GlpQ antigen of
Borrelia hermsii (Schwan et al. (1996) J Clin. Microbiol. 34:
2483-2492); cholera protective antigen (CPA) (Sciortino (1996) J.
Diarrhoeal Dis. Res. 14: 16-26); a 190-kDa protein antigen of
Streptococcus mutans (Senpuku et al. (1996) Oral Microbiol.
Immunol. 11: 121-128); Anthrax toxin protective antigen (PA)
(Sharma et al. (1996) Protein Expr. Purif. 7: 33-38); Clostridium
perfringens antigens and toxoid (Strom et al. (1995) Br. J.
Rheumatol. 34: 1095-1096); the SEF14 fimbrial antigen of Salmonella
enteritidis (Thorns et al. (1996) Microb. Pathog. 20: 235-246); the
Yersinia pestis capsular antigen (F I antigen) (Titball et al.
(1997) Infect. Immun. 65: 1926-1930); a 35-kilodalton protein of
Mycobacterium leprae (Triccas et al. (1996) Infect. Immun. 64:
5171-5177); the major outer membrane protein, CD, extracted from
Moraxella (Branhamella) catarrhalis (Yang et al. (1997) FEMS
Immunol. Med. Microbiol. 17: 187-199); pH6 antigen (PsaA protein)
of Yersinia pestis (Zav'yalov et al. (1996) FEMS Immunol. Med.
Microbiol. 14: 53-57); a major surface glycoprotein, gp63, of
Leishmania major (Xu and Liew (1994) Vaccine 12:1534-1536; Xu and
Liew (1995) Immunology 84: 173-176); mycobacterial heat shock
protein 65, mycobacterial antigen (Mycobacterium leprae hsp65)
(Lowrie et al. (1994) Vaccine 12: 1537-1540; Ragno et al. (1997)
Arthritis Rheum. 40: 277-283; Silva (1995) Braz. J Med. Biol. Res.
28: 843-851); Mycobacterium tuberculosis antigen 85 (Ag85) (Huygen
et al. (1996) Nat. Med. 2: 893-898); the 45/47 kDa antigen complex
(APA) of Mycobacterium tuberculosis, M. bovis and BCG (Horn et al.
(1996) J Immunol. Methods 197: 151-159); the mycobacterial antigen,
65-kDa heat shock protein, hsp65 (Tascon et al. (1996) Nat. Med. 2:
888-892); the mycobacterial antigens MPB64, MPB70, MPB57 and alpha
antigen (Yamada et al. (1995) Kekkaku 70: 63 9-644); the M.
tuberculosis 38 kDa protein (Vordenneier et al. (1995) Vaccine 13:
1576-1582); the MPT63, MPT64 and MPT-59 antigens from Mycobacterium
tuberculosis (Manca et al. (1997) Infect. Immun. 65: 16-23;
Oettinger et al. (1997) Scand. J Immunol. 45: 499-503; Wilcke et
al. (1996) Tuber. Lung Dis. 77: 250-256); the 35-kilodalton protein
of Mycobacterium leprae (Triccas et al. (1996) Infect. Immun. 64:
5171-5177); the ESAT-6 antigen of virulent mycobacteria (Brandt et
al. (1996) J Immunol. 157: 3527-3533; Pollock and Andersen (1997) J
Infect. Dis. 175: 1251-1254); A.about.vcobacterium tuberculosis
16-kDa antigen (Hspl6.3) (Chang et al. (1996) J Biol. Chem. 271:
7218-7223); and the 18-kilodalton protein of Mycobacterium leprae
(Baumgart et al. (1996) Infect. Immun. 64: 2274-228 1).
2.9.1.2. VIRAL PATHOGENS
[1294] The methods of the invention are also useful for obtaining
recombinant nucleic acids and polypeptides that have enhanced
ability to induce an immune response against viral pathogens. While
the bacterial recombinants described above are typically
administered in polypeptide form, recombinants that confer viral
protection are preferably administered in nucleic acid form, as
genetic vaccines.
[1295] One illustrative example is the Hantaan virus. Glycoproteins
of this virus typically accumulate at the membranes of the Golgi
apparatus of infected cells. This poor expression of the
glycoprotein prevents the development of efficient genetic vaccines
against these viruses. The methods of the invention solve this
problem by performing stochastic (e.g. polynucleotide shuffling
& interrupted synthesis) and non-stochastic polynucleotide
reassembly on nucleic acids that encode the glycoproteins and
identifying those recombinants that exhibit enhanced expression in
a host cell, and/or for improved immunogenicity when administered
as a genetic vaccine. A convenient screening method for these
methods is to express the experimentally generated polynucleotides
as fusion proteins to PIG, which results in display of the
polypeptides on the surface of the host cell (Whitehorn et al.
(1995) Biotechnology (N Y) 13:1215-9). Fluorescence-activated cell
sorting is then used to sort and recover those cells that express
an increased amount of the antigenic polypeptide on the cell
surface. This preliminary screen can be followed by immunogenicity
tests in mammals, such as mice. Finally, in preferred embodiments,
those recombinant nucleic acids are tested as genetic vaccines for
their ability to protect a test animal against challenge by the
virus.
[1296] The flaviviruses are another example of a viral pathogen for
which the methods of the invention are useful for obtaining a
experimentally generated polypeptide or genetic vaccine that is
effective against a viral pathogen. The flaviviruses consist of
three clusters of antigenically related viruses: Dengue 1-4 (62-77%
identity), Japanese, St. Louis and Murray Valley encephalitis
viruses (75-82% identity), and the tick-borne encephalitis viruses
(77-96% identity). Dengue virus can induce protective antibodies
against SLE and Yellow fever (40-50% identity), but few efficient
vaccines are available. To obtain genetic vaccines and
experimentally generated polypeptides that exhibit enhanced
cross-reactivity and immunogenicity, the polynucleotides that
encode envelope proteins of related viruses are subjected to
stochastic (e.g. polynucleotide shuffling & interrupted
synthesis) and non-stochastic polynucleotide reassembly. The
resulting experimentally generated polynucleotides can be tested,
either as genetic vaccines or by using the expressed polypeptides,
for ability to induce a broadly reacting neutralizing antibody
response. Finally, those clones that are favorable in the
preliminary screens can be tested for ability to protect a test
animal against viral challenge.
[1297] Viral antigens that can be evolved by stochastic (e.g.
polynucleotide shuffling & interrupted synthesis) and
non-stochastic polynucleotide reassembly for improved activity as
vaccines include, but are not limited to, influenza A virus N2
neuraminidase (Kilbourne et al. (1995) Vaccine 13: 1799-1803);
Dengue virus envelope (E) and premembrane (prM) antigens (Feighny
et al. (1994) Am. J Trop. Med. Hyg. 50: 322-328; Putnak et al.
(1996) Am. J Trop. Med. Hyg. 5 5: 5 04-10); HIV antigens Gag, Pol,
Vif and Nef (Vogt et al. (1995) Vaccine 13: 202-208); HIV antigens
gp 120 and gp 160 (Achour et al. (1995) Cell. Mol. Biol. 41:
395-400; Hone et al. (1994) Dev. Biol. Stand. 82: 159-162); gp41
epitope of human immunodeficiency virus (Eckhart et al. (1996) J
Gen. Virol. 77: 2001-2008); rotavirus antigen VP4 (Mattion et al.
(1995) J Virol. 69: 5132-5137); the rotavirus protein VP7 or VP7sc
(Emslie et al. (1995) J Virol. 69: 1747-1754; Xu et al. (1995) J
Gen. Virol. 76: 1971-1980); herpes simplex virus (HSV)
glycoproteins gB, gC, gD, gE, gG, gH, and gl (Fleck et al. (1994)
Med. Microbiol. Immunol. (Berl) 183: 87-94 [Mattion, 1995]; Ghiasi
et al. (1995) Invest. Ophthalmol. Vis. Sci. 36: 1352-1360; McLean
et al. (1994) J Infect. Dis. 170: 1100-1109); immediate-early
protein ICP47 of herpes simplex virus--type 1 (HSV-1) (Banks et al.
(1994) Virology 200: 23 6-245); immediate-early (IE) proteins
ICP27, ICPO, and ICP4 of herpes simplex virus (Manickan et al.
(1995) J Virol. 69: 4711-4716); influenza virus nucleoprotein and
hemagglutinin (Deck et al. (1997) Vaccine 15: 71-78; Fu et al.
(1997) J Virol. 71: 2715-272 1); B 19 parvovirus capsid proteins
VP1 (Kawase et al. (1995) Virology 211: 359-366) or VP2 (Brown et
al. (1994) Virology 198: 477-488); Hepatitis B virus core and e
antigen (Schodel et al. (1996) Intervirology 39:104-106); hepatitis
B surface antigen (Shiau and Murray (1997) J. Med. Virol. 51:
159-166); hepatitis B surface antigen fused to the core antigen of
the virus (Id.); Hepatitis B virus core-preS2 particles (Nemeckova
et al. (1996) Acta Virol. 40: 273-279); HBV preS2-S protein
(Kutinova et al. (1996) Vaccine 14: 1045-1052); VZV glycoprotein I
(Kutinova et al. (1996) Vaccine 14: 1045-1052); rabies virus
glycoproteins (Xiang et al. (1994) Virology 199: 132-140; Xuan et
al. (1995) Virus Res. 36: 151-161) or ribonucleocapsid (Hooper
eta/. (1994) Proc. Nat'l. Acad. Sci. USA 91: 10908-10912); human
cytomegalovirus (HCMV) glycoprotein B (LTL55) (Britt et al. (1995)
J Infect. Dis. 171: 18-25); the hepatitis C virus (HCV)
nucleocapsid protein in a secreted or a nonsecreted form, or as a
fusion protein with the middle (pre-S2 and S) or major (S) surface
antigens of hepatitis B virus (HBV) (Inchauspe et al. (1997) DNA
Cell Biol. 16: 185-195; Major et al. (1995) J Virol. 69:
5798-5805); the hepatitis C virus antigens: the core protein (pC);
E1 (pE1) and E2 (pE2) alone or as fusion proteins (Saito et al.
(1997) Gastroenterology 112: 1321-1330); the gene encoding
respiratory syncytial virus fusion protein (PFP-2) (Falsey and
Walsh (1996) Vaccine 14: 1214-1218; Piedra et al. (1996) Pediatr.
Infect. Dis. J. 15: 23-3 1); the VP6 and VP7 genes of rotaviruses
(Choi et al. (1997) Virology 232: 129-13 8; Jin et al. (1996) Arch.
Virol. 141: 2057-2076); the E 1, E2, E3, E4, E5, E6 and E7 proteins
of human papillomavirus (Brown et al. (1994) Virology 201: 46-54;
Dillner et al. (1995) Cancer Detect. Prev. 19: 3 81- 393; Krul et
al. (1996) Cancer Immunol. Immunother. 43: 44-48; Nakagawa et al.
(1997) J Infect. Dis. 175: 927-93 1); a human T-lymphotropic virus
type I gag protein (Porter et al. (1995) J Med Virol. 45: 469-474);
Epstein-Barr virus (EBV) gp340 (Mackett et al. (1996) J Med. Virol.
50: 263-271); the Epstein-Barr virus (EBV) latent membrane protein
LMP2 (Lee et al. (1996) Eur. J Immunol. 26: 1875-1883);
Epstein-Barr virus nuclear antigens 1 and 2 (Chen and Cooper (1996)
J Virol. 70: 4849-4853; Khanna et al. (1995) Virology 214:
633-637); the measles virus nucleoprotein (N) (Fooks et al. (1995)
Virology 210: 456-465); and cytomegalovirus glycoprotein gB
(Marshall et al. (1994) J Med. Virol. 43: 77-83) or glycoprotein gH
(Rasmussen et al. (1994) J Infect. Dis. 170: 673-677).
2.9.2. INFLAMMATORY AND AUTOIMMUNE DISEASES
[1298] Autoimmune diseases are characterized by immune response
that attacks tissues or cells of ones own body, or
pathogen-specific immune responses that also are harmful for ones
own tissues or cells, or non-specific immune activation which is
harmful for ones own tissues or cells. Examples of autoinimune
diseases include, but are not limited to, rheumatoid arthritis,
SLE, diabetes mellitus, myasthenia gravis, reactive arthritis,
ankylosing spondylitis, and multiple sclerosis. These and other
inflammatory conditions, including IBD, psoriasis, pancreatitis,
and various immunodeficiencies, can be treated using genetic
vaccines that include vectors and other components obtained using
the methods of the invention (e.g. using antigens that are
optimized using the methods of the invention).
[1299] These conditions are often characterized by an accumulation
of inflammatory cells, such as lymphocytes, macrophages, and
neutrophils, at the sites of inflammation. Altered cytokine
production levels are often observed, with increased levels of
cytokine production. Several autoimmune diseases, including
diabetes and rheumatoid arthritis, are linked to certain MHC
haplotypes. Other autoimmune-type disorders, such as reactive
arthritis, have been shown to be triggered by bacteria such as
Yersinia and Shigella, and evidence suggests that several other
autoimmune diseases, such as diabetes, multiple sclerosis,
rheumatoid arthritis, may also be initiated by viral or bacterial
infections in genetically susceptible individuals.
[1300] Current strategies of treatment generally include
anti-inflammatory drugs, such as NSAID or cyclosporin, and
antiproliferative drugs, such as methotrexate. These therapies are
non-specific, so a need exists for therapies having greater
specificity, and for means to direct the immune responses towards
the direction that inhibits the autoimmune process.
[1301] The present invention provides several strategies by which
these needs can be fulfilled. First, the invention provides methods
of obtaining vaccines which exhibit improved delivery of
tolerogenic antigens (e.g. methods of obtaining antigens having
greater tolerogenicity and/or have improved antigenicity), antigens
which have improved antigenicity, genetic vaccine-mediated
tolerance, and modulation of the immune response by inclusion of
appropriate accessory molecules. In a preferred embodiment, the
vaccines (e.g. optimized antigens) prepared according to the
invention exhibit improved induction of tolerance by oral
delivery.
[1302] Oral tolerance is characterized by induction of
immunological tolerance after oral administration of large
quantities of antigen (Chen et al. (1995) Science 265: 123 7-1240;
Haq et al. (1995) Science 268: 714-716). In animal models, this
approach has proven to be a very promising approach to treat
autoimmune diseases, and clinical trials are in progress to address
the efficacy of this approach in the treatment of human autoimmune
diseases, such as rheumatoid arthritis and multiple sclerosis (Chen
et al. (1994) Science 265:1237-40; Whitacre et al. (1996) Clin.
Immunol. Immunopathol. 80: S31-9; Hohol et al. (1996) Ann. N.Y Acad
Sci. 778:243-50). It has also been suggested that induction of oral
tolerance against viruses used in gene therapy might reduce the
immunogenicity of gene therapy vectors.
[1303] However, the amounts of antigen required for induction of
oral tolerance are very high and improved methods for oral delivery
of antigenic proteins would significantly improve the efficacy of
induction of oral tolerance. (383-"However, the amounts of antigen
required for induction of oral tolerance are very high and the
methods of the invention provide a means for obtaining antigens
that exhibit a significant improvement in induction of oral
tolerance.")
[1304] Expression library immunization (Barry et al. (1995) Nature
3 77: 632) is a particularly useful method of screening for optimal
antigens for use in genetic vaccines. For example, to identify
autoantigens present in Yersinia, Shigella, and the like, one can
screen for induction of T cell responses in HLA-B27 positive
individuals. Complexes that include epitopes of bacterial antigens
and MHC molecules associated with autoimmune diseases, e.g.,
HLA-B27 in association with Yersinia antigens can be used in the
prevention of reactive arthritis and ankylosing spondylitis in
HLA-B27 positive individuals.
[1305] Treatment of autoimmune and inflammatory conditions can
involve not only administration of tolerogenic antigens, but also
the use of a combination of cytokines, costimulatory molecules, and
the like. Such cocktails are formulated for induction of a
favorable immune response, typically induction of
autoantigen-specific tolerance. Cocktails can also include, for
example, CD1, which is crucially involved in recognition of self
antigens by a subset of T cells (Porcelli (1995) Adv. Immunol. 5 9:
1). Genetic vaccine vectors and cocktails that skew immune
responses towards the T.sub.H2 are often used in treating
autoimmune and inflammatory conditions, both with antigen-specific
and antigen non-specific vectors.
[1306] Screening of genetic vaccines and accessory molecules (and
optimized antigens-383) can be done in animal models which are
known to those of skill in the art. Examples of suitable models for
various conditions include collagen induced arthritis, the NFS/sld
mouse model of human Sjogren's syndrome; a 120 kD organ-specific
autoantigen recently identified as an analog of human cytoskeletal.
protein .alpha.-fodrin (Haneji et al. (1997) Science 276: 604), the
New Zealand Black/White F1 hybrid mouse model of human SLE, NOD
mice, a mouse model of human diabetes mellitus, fas/fas ligand
mutant mice, which spontaneously develop autoimmune and
lymphoproliferative disorders (Watanabe-Fukunaga et al. (1992)
Nature 356: 314), and experimental autoimmune encephalomyelitis
(EAE), in which myelin basic protein induces a disease that
resembles human multiple sclerosis.
[1307] Autoantigens (that can be experimentally evolved (e.g. by
polynucleotide reassembly &/or polynucleotide site-saturation
mutagenesis) according to the methods of the invention) that are
useful in genetic vaccines for treating multiple sclerosis include,
but are not limited to, myelin basic protein (Stinissen et al.
(1996) J Neurosci. Res. 45: 500-511) or a fusion protein of myelin
basic protein and proteolipid protein in multiple sclerosis
(Elliott et al. (1996) J Clin. Invest. 98: 1602-1612), proteolipid
protein (PLP) (Rosener et al. (1997) J Neuroimmunol. 75: 28-34),
2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) (Rosener et
al. (1997) J Neuroimmunol. 75: 28-34), the Epstein Barr virus
nuclear antigen-1 (EBNA-1) in multiple sclerosis (Vaughan et al.
(1996) J Neuroimmunol. 69: 95-102), HSP70 in multiple sclerosis
(Salvetti et al. (1996) J Neuroimmunol. 65: 143-53; Feldmann et al.
(1996) Cell 85: 307).
[1308] Target antigens that, after reassembly (optionally in
combination with other directed evolution methods described herein)
according to the methods of the invention, can be used to treat
scleroderma, systemic sclerosis, and systemic lupus erythematosus
include, for example, (-2-GPI, 50 kDa glycoprotein (Blank et al.
(1994) J Autoimmun. 7: 441-455), Ku (p70/p80) autoantigen, or its
80-kd subunit protein (Hong et al. (1994) Invest. Ophthalmol. Vis.
Sei. 35: 4023-4030; Wang et al. (1994) J Cell Sci. 107: 3223-3233),
the nuclear autoantigens La (SS-B) and Ro (SS-A) (Huang et al.
(1997) J Clin. Immunol. 17: 212-219; lgarashi et al. (1995)
Autoimmunity 22: 33-42; Keech et al. (1996) Clin. Exp. Immunol.
104: 255-263; Manoussakis et al. (1995) J Autoimmun. 8: 959-969;
Topfer et al. (1995) Proc. Nat'l. Acad. Sci. USA 92: 875-879),
proteasome (-type subunit C9 (Feist et al. (1996) J Exp. Med. 184:
1313-1318), Scleroderma antigens Rpp 30, Rpp 38 or Scl-70 (Eder et
al. (1997) Proc. Nat'l. Acad. Sci. USA 94: 1101-1106; Hietarinta et
al. (1994) Br. J Rheumatol. 33: 323-326), the centrosome
autoantigen PCM-1 (Bao et al. (1995) Autoimmunitv 22: 219-228),
polymyositis-scleroderma autoantigen (PM-Scl) (Kho et al. (1997) J
Biol. Chem. 272: 13426-1343 1), scleroderma (and other systemic
autoimmune disease) autoantigen CENP-A (Muro et al. (1996) Clin.
Immunol. Immunopathol. 78: 86-89), U5, a small nuclear
ribonucleoprotein (snRNP) (Okano et al. (1996) Clin. Immunol.
Immunopathol. 81: 41-47), the I 00-kd protein of PM-Scl autoantigen
(Ge et al. (1996) Arthritis Rheum. 39: 1588-1595), the nucleolar
U3- and Th(7-2) ribonucleoproteins (Verheijen et al. (1994) J.
Immunol. Methods 169: 173-182), the ribosomal protein L7 (Neu et
al. (1995) Clin. Exp. Immunol. 100: 198-204), hPop 1 (Lygerou et
al. (1996) EMBO J. 15: 5936-5948), and a 36-kd protein from nuclear
matrix antigen (Deng et al. (1996) Arthritis Rheum. 39:
1300-1307).
[1309] Hepatic autoimmune disorders can also be treated using
improved recombinant antigens that are prepared according to the
methods described herein. Among the antigens that are useful in
such treatments are the cytochromes P450 and
UDP-glucuronosyl-transferases (Obermayer-Straub and Manns (1996)
Baillieres Clin. Gastroenterol. 10: 501-532), the cytochromes P450
2C9 and P450 1A2 (Bourdi et al. (1996) Chem. Res. Toxicol. 9:
1159-1166; Clemente et al. (1997) J Clin. Endocrinol. Metab. 82:
1353-1361), LC-1 antigen (Klein et al. (1996) J Pediatr.
Gastroenterol. Nutr. 23: 461-465), and a 230-kDa Golgi-associated
protein (Funaki et al. (1996) Cell Struct. Funct. 21: 63-72).
[1310] For treatment of autoimmune disorders of the skin, useful
antigens include, but are not limited to, the 450 kD human
epidermal autoantigen (Fujiwara et al. (1996) J Invest. Dermatol.
106: 1125-1130), the 230 kD and 180 kD bullous pemphigoid antigens
(Hashimoto (1995) Keio J Med. 44: 115-123; Murakami et al. (1996) J
Dermatol. Sci. 13: 112-117), pemphigus foliaceus antigen
(desmoglein 1), pemphigus vulgaris antigen (desmoglein 3), BPAg2,
BPAg1, and type VII collagen (Batteux et al. (1997) J Clin.
Immunol. 17: 228-233; Hashimoto et al. (1996) J Dermatol. Sci. 12:
10-17), a 168-kDa mucosal antigen in a subset of patients with
cicatricial pemphigoid (Ghohestani et al. (1996) J Invest.
Dermatol. 107: 136-139), and a 218-kd nuclear protein (218-kd Mi-2)
(Seelig et al. (1995) Arthritis Rheum. 38: 1389-1399).
[1311] The methods of the invention are also useful for obtaining
improved antigens for treating insulin dependent diabetes mellitus,
using one or more of antigens which include, but are not limited
to, insulin, proinsulin, GAD65 and GAD67, heat-shock protein 65
(hsp65), and islet-cell antigen 69 (ICA69) (French et al. (1997)
Diabetes 46: 34-39; Roep (1996) Diabetes 45: 1147-1156; Schloot et
al. (1997) Diabetologia 40: 332-338), viral proteins homologous to
GAD65 (Jones and Crosby (1996) Diabetologia 39: 1318-1324), islet
cell antigen-related protein-tyrosine phosphatase (PTP) (Cui et al.
(1996) J Biol. Chem. 271: 24817-24823), GM2-1 ganglioside (Cavallo
et al. (1996) J Endocrinol. 150: 113-120; Dotta et al. (1996)
Diabetes 45: 1193-1196), glutarnic acid decarboxylase (GAD) (Nepom
(1995) Curr. Opin. Immunol. 7: 825-830; Panina-Bordignon et al.
(1995) J Exp. Med. 181: 1923-1927), an islet cell antigen (ICA69)
(Karges et al. (1997) Biochim. Biophys. Acta 1360: 97-101; Roep et
al. (1996) Eur. J Immunol. 26: 1285-1289), Tep69, the single T cell
epitope recognized by T cells from diabetes patients (Karges et al.
(1997) Biochim. Biopkys. Acta 1360: 97-101), ICA 512, an
autoantigen of type I diabetes (Solimena et al. (1996) EMBOJ. 15:
2102-2114), an islet-cell protein tyrosine phosphatase and the 37-
kDa autoantigen derived from it in type I diabetes (including IA-2,
IA-2) (La Gasse et al. (1997) Mol. Med. 3: 163-173), the 64 kDa
protein from In-111 cells or human thyroid follicular cells that is
immunoprecipitated with sera from patients with islet cell surface
antibodies (ICSA) (Igawa et al. (1996) Endocr. J. 43: 299-306),
phogrin, a homologue of the human transmembrane protein tyrosine
phosphatase, an autoantigen of type I diabetes (Kawasaki et al.
(1996) Biochem. Biophys. Res. Commun. 227: 440-447), the 40 kDa and
37 kDa tryptic fragments and their precursors IA-2 and IA-2 in IDDM
(Lampasona et al. (1996) J Immunol. 157: 2707-2711; Notkins et al.
(1996) J A utoimmun. 9: 677-682), insulin or a cholera
toxoid-insulin conjugate (Bergerot et al. (1997) Proc. Nat'l. Acad.
Sci. USA 94: 4610-4614), carboxypeptidase H, the human homologue of
gp330, which is a renal epithelial glycoprotein involved in
inducing Heymann nephritis in rats, and the 38-kD islet
mitochondrial autoantigen (Arden et al. (1996) J Clin. Invest. 97:
551-561.
[1312] Rheumatoid arthritis is another condition that is treatable
using optimized antigens prepared according to the present
invention. Useful antigens for rheumatoid arthritis treatment
include, but are not limited to, the 45 kDa DEK nuclear antigen, in
particular onset juvenile rheumatoid arthritis and iridocyclitis
(Murray et al. (1997) J Rheumatol. 24: 560-567), human cartilage
glycoprotein-39, an autoantigen in rheumatoid arthritis (Verheijden
et al. (1997) Arthritis Rheum. 40: 1115-1125), a 68k autoantigen in
rheumatoid arthritis (Blass et al. (1997) Ann. Rheum. Dis. 56:
317-322), collagen (Rosloniec et al. (1995) J Immunol. 155:
4504-4511), collagen type II (Cook et al. (1996) Arthritis Rheum.
39: 1720-1727; Trentham (1996) Ann. N. Y. Acad. Sci. 778: 306-314),
cartilage link protein (Guerassimov et al. (1997) J Rheumatol. 24:
95 9-964), ezrin, radixin and moesin, which are auto-immune
antigens in rheumatoid arthritis (Wagatsuma et al. (1996) Mol.
Immunol. 33: 1171-1176), and mycobacterial heat shock protein 65
(Ragno et al. (1997) Arthritis Rheum. 40: 277-283).
[1313] Also among the conditions for which one can obtain an
improved antigen suitable for treatment are autoimmune thyroid
disorders. Antigens that are useful for these applications include,
for example, thyroid peroxidase and the thyroid stimulating hormone
receptor (Tandon and Weetman (1994) J R. Coll. Physicians Lond. 28:
10-18), thyroid peroxidase from human Graves' thyroid tissue
(Gardas et al. (1997) Biochem. Biophys. Res. Commun. 234: 366-370;
Zimmer et al. (1997) Histochem. Cell. Biol. 107: 115-120), a 64-kDa
antigen associated with thyroid-associated ophthalmopathy (Zhang et
al. (1996) Clin. Immunol. Immunopathol. 80: 23 6-244), the human
TSH receptor (Nicholson et al. (1996) J Mol. Endocrinol. 16:
159-170), and the 64 kDa protein from In-111 cells or human thyroid
follicular cells that is immunoprecipitated with sera from patients
with islet cell surface antibodies (ICSA) (Igawa et al. (1996)
Endocr. J. 43: 299-306).
[1314] Other conditions and associated antigens include, but are
not limited to, Sjogren's syndrome (-fodrin; Haneji et al. (1997)
Science 276: 604-607), myastenia gravis (the human M2 acetylcholine
receptor or fragments thereof, specifically the second
extracellular loop of the human M2 acetylcholine receptor; Fu et
al. (1996) Clin. Immunol. Immunopathol. 78: 203-207), vitiligo
(tyrosinase; Fishman et al. (1997) Cancer 79: 1461-1464), a 450 kD
human epidermal autoantigen recognized by serum from individual
with blistering skin disease, and ulcerative colitis (chromosomal
proteins HMG1 and HMG2; Sobajima et al. (1997) Clin. Exp. Immunol.
107: 135-140).
2.9.3. ALLERGY AND ASTHMA
[1315] The invention also provides methods of obtaining reagents
that are useful for treating allergy. In one embodiment, the
methods involve making a library of experimentally generated
polynucleotides that encode an allergen, and screening the library
to identify those experimentally generated polynucleotides that
exhibit improved properties when used as immunotherapeutic reagents
for treating allergy. For example, specific immunotherapy of
allergy using natural antigens carries a risk of inducing
anaphylaxis, which can be initiated by cross-linking of
high-affinity IgE receptors on mast cells. Therefore, allergens
that are not recognized by pre-existing IgE are desirable. The
methods of the invention provide methods by which one can obtain
such allergen variants. Another improved property of interest is
induction of broader immune responses, increased safety and
efficacy.
[1316] Genetic vaccine vectors and other reagents obtained using
the methods of the invention can be used to treat allergies and
asthma. Allergic immune responses are results of complex
interactions between B cells, T cells, professional
antigen-presenting cells (APC), eosinophils and mast cells. These
cells take part in allergic immune responses both as modulators of
the immune responses and are also involved in producing factors
directly involved in initiation and maintenance of allergic
responses.
[1317] Synthesis of Polyclonal and Allergen-Specific IgE Requires
Multiple Interactions Between B Cells, T Cells and Professional
Antigen-Presenting Cells (APC).
[1318] Activation of naive, unprimed B cells is initiated when
specific B cells recognize the allergen by cell surface
immunoglobulin (sIg). However, costimulatory molecules expressed by
activated T cells in both soluble and membrane-bound forms are
necessary for differentiation of B cells into IgE-secreting plasma
cells. Activation of T helper cells requires recognition of an
antigenic peptide in the context of MHC class II molecules on the
plasma membrane of APC, such as monocytes, dendritic cells,
Langerhans cells or primed B cells. Professional APC can
efficiently capture the antigen and the peptide-MHC class II
complexes are formed in a post-Golgi, proteolytic intracellular
compartment and subsequently exported to the plasma membrane, where
they are recognized by T cell receptor (TCR) (Monaco (1995) J Leuk.
Biol. 57: 543-547). In addition, activated B cells express CD80
(B7-1) and CD86 (B7-2, B70), which are the counter receptors for
CD28 and which provide a costimulatory signal for T cell activation
resulting in T cell proliferation and cytokine synthesis (Bluestone
(1995) Immunity 2: 555-559). Since allergen-specific T cells from
atopic individuals generally belong to the T.sub.H2 cell subset,
activation of these cells also leads to production of IL-4 and
IL-13, which, together with membrane-bound costimulatory molecules
expressed by activated T helper cells, direct B cell
differentiation into IgE-secreting plasma cells (de Vries and
Punnonen, In Cytokine Regulation of Humoral Immunity: Basic and
Clinical Aspects, Ed. C M Snapper, John Wiley & Sons Ltd, West
Sussex, UK, p. 195-215, 1996).
[1319] Mast cells and eosinophils are key cells in inducing
allergic symptoms in target organs. Recognition of specific antigen
by IgE bound to high-affinity IgE receptors on mast cells,
basophils or eosinophils results in crosslinking of the receptors
leading to degranulation of the cells and rapid release of mediator
molecules, such as histamine, prostaglandins and leukotrienes,
causing allergic symptoms.
[1320] Immunotherapy of allergic diseases currently includes
hyposensibilization treatments using increasing doses of allergen
injected to the patient. These treatments result skewing of immune
responses towards T.sub.H1 phenotype and increase the ratio of
IgG/IgE antibodies specific for allergens. Because these patients
have circulating IgE antibodies specific for the allergens, these
treatments include significant risk of anaphylactic reactions.
[1321] In these reactions, free circulating allergen is recognized
by IgE molecules bound to high-affinity IgE receptors on mast cells
and eosinophils. Recognition of the allergen results in
crosslinking of the receptors leading to release of mediators, such
as histamine, prostaglandins, and leukotrienes, which cause the
allergic symptoms, and occasionally anaphylactic reactions. Other
problems associated with hyposensibilization include low efficacy
and difficulties in producing allergen extracts reproducibly.
[1322] Genetic vaccines provide a means of circumventing the
problems that have limited the usefulness of previously known
hyposensibilization treatments. For example, by expressing antigens
on the surface of cells, such as muscle cells, the risk of
anaphylactic reactions is significantly reduced. This can be
achieved by using genetic vaccine vectors that encode transmembrane
forms of allergens. The allergens can also be modified in such a
way that they are efficiently expressed in transmembrane forms,
further reducing the risk of anaphylactic reactions. Another
advantage provided by the use of genetic vaccines for
hyposensibilization is that the genetic vaccines can include
cytokines and accessory molecules which further direct the immune
responses towards the T.sub.H1 phenotype, thus reducing the amount
of IgE antibodies produced and increasing the efficacy of the
treatments. Vectors can also be evolved to induce primarily IgG and
IgM responses, with little or no IgE response. (see, e.g., U.S.
patent application Ser. No. 09/021,769, filed Feb. 11, 1998).
[1323] Furthermore, stochastic (e.g. polynucleotide shuffling &
interrupted synthesis) and non-stochastic polynucleotide reassembly
can be used to generate allergens that are not recognized by the
specific IgE antibodies preexisting in vivo, yet are capable of
inducing efficient activation of allergen-specific T cells. For
example, using phage display selection, one can express
experimentally evolved (e.g. by polynucleotide reassembly &/or
polynucleotide site-saturation mutagenesis) allergens on phage, and
only those that are not recognized by specific IgE antibodies are
selected. These are further screened for their capacity to induce
activation of specific T cells. An efficient T cell response is an
indication that the T cell epitopes are functionally intact,
although the B cell epitopes were altered, as indicated by lack of
binding of specific antibodies.
[1324] In these methods, polynucleotides encoding known allergens,
or homologs or fragments thereof (e.g., immunogenic peptides) are
inserted into DNA vaccine vectors and used to immunize allergic and
asthmatic individuals. Alternatively, the experimentally evolved
(e.g. by polynucleotide reassembly &/or polynucleotide site-
saturation mutagenesis) allergens are expressed in manufacturing
cells, such as E. coli or yeast cells, and subsequently purified
and used to treat the patients or prevent allergic disease.
stochastic (e.g. polynucleotide shuffling & interrupted
synthesis) and non-stochastic polynucleotide reassembly can be used
to obtain antigens that activate T cells but cannot induce
anaphylactic reactions. For example, a library of experimentally
generated polynucleotides that encode allergen variants can be
expressed in cells, such as antigen presenting cells, which are
than contacted with PBMC or T cell clones from atopic patients.
Those library members that efficiently activate T.sub.H cells from
the atopic patients can be identified by assaying for T cell
proliferation, or by cytokine synthesis (e.g., synthesis of IL-2,
IL-4, IFN-.gamma.. Those recombinant allergen variants that are
positive in the in vitro tests can then be subjected to in vivo
testing.
[1325] Examples of Allergies That Can Be Treated Include, But Are
Not Limited to, Allergies Against House Dust Mite, Grass Pollen,
Birch Pollen, Ragweed Pollen, Hazel Pollen, Cockroach, Rice, Olive
Tree Pollen, Ftmgi, Mustard, Bee Venom.
[1326] Antigens of interest include those of animals, including the
mite (e.g., Dermatophagoides pteronyssinus,
Dermatophagoidesfarinae, Blomia tropicalis), such as the allergens
der p1 (Scobie et al. (1994) Biochem. Soc. Trans. 22: 448S; Yssel
et al. (1992) J Immunol. 148: 738-745), der p2 (Chua et al. (1996)
Clin. Exp. Allergy 26: 829-83 7), der p3 (Smith and Thomas (1996)
Clin. Exp. Allergy 26: 571-579), der p5, der p V (Lin et al. (1994)
J Allergy Clin. Immunol. 94: 989-996), der p6 (Bennett and Thomas
(1996) Clin. Exp. Allergy 26: 1150- 1154), der p7 (Shen et al.
(1995) Clin. Exp. Allergy 25: 416-422), der f2 (Yuuki et al. (1997)
Int. Arch. Allergy Immunol. 112: 44-48), der f3 (Nishiyama et al.
(1995) FEBSLett. 377: 62-66), der f7 (Shen et al. (1995) Clin. Exp.
Allergy 25: 1000-1006); Mag 3 (Fujikawa et al. (1996) Mol. Immunol.
33: 311-319). Also of interest as antigens are the house dust mite
allergens Tyr p2 (Eriksson et al. (1998) Eur. J Biochem. 251:
443-447), Lep d 1 (Schmidt et al. (1995) FEBS Lett. 3 70: 11-14),
and glutathione S-transferase (O'Neill et al. (1995) Immunol Lett.
48: 103-107); the 25,589 Da, 219 amino acid polypeptide with
homology with glutathione S-transferases (ONeill et al. (1994)
Biochim. Biophys. Acta. 1219: 521-528); Blo t 5 (Arruda et al.
(1995) Int. Arch. Allergy Immunol. 107: 456-45 7); bee venom
phospholipase A2 (Carballido et al. (1994) J Allergy Clin. Immunol.
93: 758-767; Jutel et al. (1995) J Immunol. 154: 4187-4194); bovine
dermal/dander antigens BDA 11 (Rautiainen et al. (1995) J. Invest.
Dermatol. 105: 660-663) and BDA20 (Mantyj arvi et al. (1996) J
Allergy Clin. Immunol. 97: 1297-1303); the major horse allergen Equ
c1 (Gregoire et al. (1996) J Biol. Chem. 271: 32951-32959); Jumper
and M. pilosula allergen Myr p 1 and its homologous allergenic
polypeptides Myr p2 (Donovan et al. (1996) Biochem. Mol. Biol. Int.
39: 877-885); 1-13, 14, 16 kD allergens of the mite Blomia
tropicalis (Caraballo et al. (1996) J Allergy Clin. Immunol. 98:
573-579); the cockroach allergens Bla g Bd90K (Helm et al. (1996) J
Allergy Clin. Immunol. 98: 172-80) and Bla g 2 (Arruda et al.
(1995) J Biol. Chem. 270: 19563-19568); the cockroach Cr-PI
allergens (Wu et al. (1996) J Biol. Chem. 271: 1793 7-17943); fire
ant venom allergen, Sol i 2 (Schmidt et al. (1996) J Allergy Clin.
Immunol. 98: 82-88); the insect Chironomus thumini major allergen
Chi t 1-9 (Kipp et al. (1996) Int. Arch. Allergy Immunol. 110:
348-353); dog allergen Can f 1 or cat allergen Fel d 1 (Ingram et
al. (1995) J Allergy Clin. Immunol. 96: 449-456); albumin, derived,
for example, from horse, dog or cat (Goubran Botros et al. (1996)
Immunology 88: 340-347); deer allergens with the molecular mass of
22 kD, 25 kD or 60 kD (Spitzauer et al. (1997) Clin. Exp. Allergy
27: 196-200); and the 20 kd major allergen of cow (Ylonen et al.
(1994) J Allergy Clin. Immunol. 93: 851-858). Pollen and grass
allergens are also useful in vaccines, particularly after
optimization of the antigen by the methods of the invention. Such
allergens include, for example, Hor v9 (Astwood and Hill (1996)
Gene 182: 53-62, Lig v 1 (Batanero et al. (1996) Clin. Exp. Allergy
26: 1401-1410); Lol p 1 (Muller et al. (1996) Int. Arch. Allergy
Immunol. 109: 352-355), Lol p II (Tamborini et al. (1995) Mol.
Immunol. 32: 505-513), Lol pVA, Lol pVB (Ong et al. (1995) Mol.
Immunol. 32: 295-302), Lol p 9 (Blaher et al. (1996) J Allergy
Clin. Immunol. 98: 124-132); Par J I (Costa et al. (1994) FEBS
Lett. 341: 182-186; Sallusto et al. (1996) J Allergy Clin. Immunol.
97: 627-637), Parj 2.0101 (Duro et al. (1996) FEBS Lett. 399:
295-298); Bet v1 (Faber et al. (1996) J Biol. Chem. 271:
19243-19250), Bet v2 (Rihs et al. (1994) Int. Arch. Allergy
Immunol. 105: 190-194); Dac g3 (Guerin-Marchand et al. (1996) Mol.
Immunol. 33: 797-806); Phl p 1 (Petersen et al. (1995) J Allergy
Clin. Immunol. 95: 987-994), Phl p 5 (Muller et al. (1996) Int.
Arch. Allergy Immunol. 109: 352-355), Phl p 6 (Petersen et al.
(1995) Int. Arch. Allergy Immunol. 108: 55-59); Cry j I (Sone et
al. (1994) Biochem. Biophys. Res. Commun. 199: 619-625), Cry j II
(Namba et al. (1994) FEBS Lett. 353: 124-128); Cor a 1 (Schenk et
al. (1994) Eur. J Biochem. 224: 717-722); cyn d 1 (Smith et al.
(1996) J Allergy Clin. Immunol. 98: 331-343), cyn d 7 (Suphioglu et
al. (1997) FEBS Lett. 402: 167-172); Pha a 1 and isoforms of Pha a
5 (Suphioglu and Singh (1995) Clin. Exp. Allergy 25: 853-865); Cha
o 1 (Suzuki et al. (1996) Mol. Immunol. 33: 451-460); profilin
derived, e.g, from timothy grass or birch pollen (Valenta et al.
(1994) Biochem. Biopkys. Res. Commun. 199:106-118); P0149(Wuet al.
(1996) Plant Mol.Biol. 32: 1037-1042); Ory s1 (Xuet al. (1995) Gene
164:255-259); and Amb a V and Amb t5 (Kim et al. (1996) Mol.
Immunol. 33: 873-880; Zhu et al. (1995) J Immunol. 155: 5064-
5073).
[1327] Vaccines against food allergens can also be developed using
the methods of the invention. Suitable antigens for reassembly
(optionally in combination with other directed evolution methods
described herein) include, for example, profilin (Rihs et al.
(1994) Int. Arch. Allergy Immunol. 105: 190-194); rice allergenic
cDNAs belonging to the alpha-amylase/trypsin inhibitor gene family
(Alvarez et al. (1995) Biochim Biophys Acta 1251: 201-204); the
main olive allergen, Ole e I (Lombardero et al. (1994) Clin Exp
Allergy 24: 765-770); Sin a 1, the major allergen from mustard
(Gonzalez De La Pena et al. (1996) Eur J Biochem. 237: 827-832);
parvalbumin, the major allergen of salmon (Lindstrom et al. (1996)
Scand. J Immunol. 44: 335-344); apple allergens, such as the major
allergen Mal d 1 (Vanek-Krebitz et al. (1995) Biochem. Biophys.
Res. Commun. 214: 538-551); and peanut allergens, such as Ara h I
(Burks et al. (1995) J Clin. Invest. 96: 1715- 1721).
[1328] The methods of the invention can also be used to develop
recombinant antigens that are effective against allergies to fungi.
Fungal allergens useful in these vaccines include, but are not
limited to, the allergen, Cla h III, of Cladosporium herbarum
(Zhang et al. (1995) J Immunol. 154: 710-717); the allergen Psi c
2, a fungal cyclophilin, from the basidiomycete Psilocybe cubensis
(Homer et al. (1995) Int. Arch. Allergy Immunol. 107: 298-300); hsp
70 cloned from a cDNA library of Cladosporium herbarum (Zhang et
al. (1996) Clin Exp Allergy 26: 88-95); the 68 kD allergen of
Penicillium notatum (Shen et al. (1995) Clin. Exp. Allergy 26:
350-356); aldehyde dehydrogenase (ALDH) (Achatz et al. (1995) Mol
Immunol. 32: 213-227); enolase (Achatz et al. (1995) Mol. Immunol.
32: 213-227); YCP4 (Id.); acidic ribosomal protein P2 (Id.).
[1329] Other allergens that can be used in the methods of the
invention include latex allergens, such as a major allergen (Hev b
5) from natural rubber latex (Akasawa et al. (1996) J Biol. Chem.
271: 25389-25393; Slater et al. (1996) J Biol. Chem. 271:
25394-25399).
[1330] The invention also provides a solution to another
shortcoming of genetic vaccination as a treatment for allergy and
asthma. While genetic vaccination primarily induces CD8.sup.+ T
cell responses, induction of allergen-specific IgE responses is
dependent on CD4.sup.+ T cells and their help to B cells.
T.sub.H2-type cells are particularly efficient in inducing IgE
synthesis because they secrete high levels of IL-4, IL-5 and IL-13,
which direct Ig isotype switching to IgE synthesis. IL-5 also
induces eosinophilia. The methods of the invention can be used to
develop genetic vaccines that efficiently induce CD4.sup.+ T cell
responses, and direct differentiation of these cells towards the
T.sub.H1 phenotype.
[1331] The invention also provides methods by which the level of
antigen release by a genetic vaccine vector is regulated.
Regulation of the antigen dose is crucial at the onset of
hyposensibilization for safety reasons. Low antigen levels are
preferably used at first, with the antigen level increasing once
evidence has been obtained that the antigen does not induce adverse
effects in the individual. The stochastic (e.g. polynucleotide
shuffling & interrupted synthesis) and non-stochastic
polynucleotide reassembly methods of the invention allow generation
of genetic vaccine vectors that induce expression of different
(high and low) levels of antigen. For example, two or more
different evolved promoters can be used for antigen expression.
Alternatively, the antigen gene itself can be evolved for different
levels of expression by, for example, altering codon usage. Vectors
that induce different levels of antigen expression can be screened
by use of specific monoclonal antibodies, and cell sorting (e.g,
FACS).
2.9.4. CANCER
[1332] Immunotherapy has great promise for the treatment of cancer
and prevention of metastasis. By inducing an immune response
against cancerous cells, the body's immune system can be enlisted
to reduce or eliminate cancer. (e.g. using the improved antigens
obtained using the methods of the invention). Genetic vaccines
prepared using the methods of the invention, as well as accessory
molecules described herein, provide cancer immunotherapies of
increased effectiveness compared to those that are presently
available.
[1333] One approach to cancer immunotherapy is vaccination using
genetic vaccines that include or encode antigens that are specific
for tumor cells or by injecting the patients with purified
recombinant cancer antigens. The methods of the invention can be
used for (obtaining antigens that exhibit an) enhancement of immune
responses against known tumor-specific antigens, and also to search
for novel protective antigenic sequences. Genetic vaccines that
exhibit optimized antigen expression, processing, and presentation
can be obtained as described herein. The methods of the invention
are also suitable for obtaining optimized cytokines, costimulatory
molecules, and other accessory molecules that are effective in
induction of an antitumor immune response, as well as for obtaining
genetic vaccines and cocktails that include these and other
components present in optimal combinations. The approach used for
each particular cancer can vary. For treatment of hormone-sensitive
cancers (for example, breast cancer and prostate cancer), methods
of the invention can be used to obtain optimized hormone
antagonists. For highly immunogenic tumors, including melanoma, one
can screen for genetic vaccine vectors (recombinant antigens) that
optimally boost the immune response against the tumor.
[1334] Breast Cancer, in Contrast, is of Relatively Low
Immunogenicity and Exhibits Slow Progression, So Individual
Treatments Can Be Designed For Each Patient. Prevention of
Metastasis is Also a Goal in Design of Genetic Vaccines.
[1335] Among the tumor-specific antigens that can be used in the
antigen reassembly (optionally in combination with other directed
evolution methods described herein) methods of the invention are:
bullous pemphigoid antigen 2, prostate mucin antigen (PMA) (Beckett
and Wright (1995) Int. J Cancer 62: 703-710), tumor associated
Thomsen-Friedenreich antigen (Dahlenborg et al. (1997) Int. J
Cancer 70: 63-71), prostate-specific antigen (PSA) (Dannull and
Belldegrun (1997) Br. J Urol. 1: 97-103), luminal epithelial
antigen (LEA. 135) of breast carcinoma and bladder transitional
cell carcinoma (TCC) (Jones et al. (1997) Anticancer Res. 17:
685-687), cancer-associated serum antigen (CASA) and cancer antigen
125 (CA 125) (Kierkegaard et al. (1995) Gynecol. Oncol. 59:
251-254), the epithelial glycoprotein 40 (EGP40) (Kievit et al.
(1997) Int. J Cancer 71: 237-245), squamous cell carcinoma antigen
(SCC) (Lozza et al. (1997) Anticancer Res. 17: 525-529), cathepsin
E (Mota et al. (1997) Ant. J Pathol. 150: 1223-1229), tyrosinase in
melanoma (Fishman et al. (1997) Cancer 79: 1461-1464), cell nuclear
antigen (PCNA) of cerebral cavemomas (Notelet et al. (1997) Surg.
Neurol. 47: 364-370), DF3/MUC1 breast cancer antigen
(Apostolopoulos et al. (1996) Immunol. Cell. Biol. 74: 45 7-464;
Pandey et al. (1995) Cancer Res. 5 5: 4000-4003), carcinoembryonic
antigen (Paone et al. (1996) J Cancer Res. Clin. Oncol. 122:
499-503; Schlom et al. (1996) Breast Cancer Res. Treat. 38: 27-39),
tumor-associated antigen CA 19-9 (Tolliver and O'Brien (1997) South
Med. J. 90: 89-90; Tsuruta et al. (1997) Urol. Int. 5 8: 20-24),
human melanoma antigens MART-I/Melan-A27- and gplOO (Kawakami and
Rosenberg (1997) Int. Rev. Immunol. 14: 173-192; Zajac et al.
(1997) Int. J Cancer 71: 491-496), the T and Tn pancarcinoma (CA)
glycopeptide epitopes (Springer (1995) Crit. Rev. Oncog. 6: 57-85),
a 35 kD tumor-associated autoantigen in papillary thyroid carcinoma
(Lucas et al. (1996) Anticancer Res. 16: 2493-2496), KH-I
adenocarcinoma antigen (Deshpande and Danishefsky (1997) Nature
387: 164-166), the A60 mycobacterial antigen (Maes et al. (1996) J
Cancer Res. Clin. Oncol. 122: 296-300), heat shock proteins (HSPs)
(Blachere and Srivastava (1995) Semin. Cancer Biol. 6: 349-355),
and MAGE, tyrosinase, melan-A and gp75 and mutant oncogene products
(e.g., p53, ras, and HER-2/neu (Bueler and Mulligan (1996) Mol.
Med. 2: 545-555; Lewis and Houghton (1995) Semin. Cancer Biol. 6:
321-327; Theobald et al. (1995) Proc. Nat'l. Acad. Sci. USA 92:
11993-11997).
2.9.5. PARASITES
[1336] Antigens from parasites can also be optimized by the methods
of the invention. These include, but are not limited to, the
schistosome gut-associated antigens CAA (circulating anodic
antigen) and CCA (circulating cathodic antigen) in Schistosoma
mansoni, S. haematobium or S. japonicum (Deelder et al. (1996)
Parasitology 112: 21-35); a multiple antigen peptide (MAP) composed
of two distinct protective antigens derived from the parasite
Schistosoma mansoni (Ferru et al. (1997) Parasite Immunol. 19:
1-11); Leishmania parasite surface molecules (Lezama-Davila (1997)
Arch. Med Res. 28: 47-53); third-stage larval (L3) antigens of L.
loa (Akue et al. (1997) J Infect. Dis. 175: 158-63); the genes,
Tams 1-1 and Tams 1-2, encoding the 30-and 32-kDa major merozoite
surface antigens of Theileria annulata (Ta) (d'Oliveira et al.
(1996) Gene 172: 33-39); Plasmodium falciparum merozoite surface
antigen 1 or 2 (al-Yaman et al. (1995) Trans. R. Soc. Trop. Med.
Hyg. 89: 555-559; Beck et al. (1997) J Infect. Dis. 175: 921-926;
Rzepczyk et al. (1997) Infect. Immun. 65: 1098-1100);
circurnsporozoite (CS) protein-based B-epitopes from Plasmodium
berghei, (PPPPNPND)2 and Plasmodium yoelii, (QGPGAP)3QG, along with
a P. berghei T-helper epitope KQIRDSITEEWS (Reed et al. (1997)
Vaccine 15: 482-488); NYVAC-Pf7 encoded Plasmodium falciparum
antigens derived from the sporozoite (circumsporozoite protein and
sporozoite surface protein 2), liver (liver stage antigen 1), blood
(merozoite surface protein 1, serine repeat antigen, and apical
membrane antigen 1), and sexual (25-kDa sexual-stage antigen)
stages of the parasite life cycle were inserted into a single NYVAC
genome to generate NYVAC-Pf7 (Tine et al. (1996) Infect. Immun. 64:
3833-3844); Plasmodium falciparum antigen Pfs230 (Williamson et al.
(1996) Mol. Biochem. Parasitol. 78: 161-169); Plasmodium falciparum
apical membrane antigen (AMA-1) (Lal et al. (1996) Infect. Immun.
64: 1054-1059); Plasmodium falciparum proteins Pfs28 and Pfs25
(Duffy and Kaslow (1997) Infect. Immun. 65: 1109-1113); Plasmodium
falciparum merozoite surface protein, MSP1 (Hui et al. (1996)
Infect. Immun. 64: 1502- 1509); the malaria antigen Pf332 (Ahlborg
et al. (1996) Immunology 88: 630-635); Plasmodium falciparum
erythrocyte membrane protein I (Baruch et al. (1995) Proc. Nat'l.
Acad. Sci. USA 93: 3497-3502; Baruch et al. (1995) Cell 82: 77-87);
Plasmodium falciparum merozoite surface antigen, PfMSP-1 (Egan et
al. (1996) J Infect. Dis. 173: 765-769); Plasmodium falciparum
antigens SERA, EBA-175, RAP1 and RAP2 (Riley (1997) J Pharm.
Pharmacol. 49: 21-27); Schistosoma japonicum paramyosin (Sj97) or
fragments thereof (Yang et al. (1995) Biochem. Biophys. Res.
Commun. 212: 1029-1039); and Hsp70 in parasites (Maresca and
Kobayashi (1994) Experientia 50: 1067-1074).
2.9.6. CONTRACEPTION
[1337] Genetic vaccines that contain optimized antigens obtained by
the methods of the invention are also useful for contraception. For
example, genetic vaccines can be obtained that encode sperm cell
specific antigens, and thus induce anti-sperm immune responses.
Vaccination can be achieved by, for example, administration of
recombinant bacterial strains, e.g. Salmonella and the like, which
express sperm antigen, as well as by induction of neutralizing
anti-hCG antibodies by vaccination by DNA vaccines encoding human
chorionic gonadotropin (hCG), or a fragment thereof.
[1338] Sperm antigens which can be used in the genetic vaccines
include, for example, lactate dehydrogenase (LDH-C4),
galactosyltransferase (GT), SP-10, rabbit sperm autoantigen (RSA),
guinea pig (g)PH-20, cleavage signal protein (CS-1), HSA-63, human
(h)PH-20, and AgX-1 (Zhu and Naz (1994) Arch. Androl 33: 141-144),
the synthetic spenn peptide, P10G (O'Rand et al. (1993) J Reprod.
Immunol. 25: 89-102), the 135 kD, 95 kD, 65 kD, 47 kD, 41 kD and 23
kD proteins of sperm, and the FA-1 antigen (Naz et al. (1995) Arch.
Androl. 35: 225-23 1), and the 35 kD fragment of cytokeratin 1
(Lucas et al. (1996) Anticancer Res. 16: 2493-2496).
[1339] The methods of the invention can also be used to obtain
genetic vaccines that are expressed specifically in testis. For
example, polynucleotide sequences that direct expression of genes
that are specific to testis can be used (e.g., fertilization
antigen-1 and the like). In addition to sperm antigens, antigens
expressed on oocytes or hormones regulating reproduction may be
useful targets of contraceptive vaccines. For example, genetic
vaccines can be used to generate antibodies against gonadotropin
releasing hormone (GnRH) or zona pellucida proteins (Miller et al.
(1997) Vaccine 15:185 8-1862). Vaccinations using these molecules
have been shown to be efficacious in animal models (Miller et al.
(1997) Vaccine 15:1858-1862). Another example of a useful component
of a genetic contraceptive vaccine is the ovarian zona pellucida
glycoprotein ZP3 (Tung et al. (1994) Reprod Fertil. Dev.
6:349-355).
2.10. MALARIAL ANTIGENS AND VACCINES
[1340] The present invention generally relates to the Plasmodium
falciparum erythrocyte membrane protein 1 ("PfEMP1"), nucleic acids
which encode PfEMP1, and antibodies which specifically recognize
PfEMP1. The polypeptides, antibodies and nucleic acids are useful
in a variety of applications including therapeutic, prophylactic,
including vaccination, diagnostic and screening applications.
[1341] The data described herein, indicates that PfEMP1 is
responsible for both antigenic variation and receptor properties on
PE, both of which are central to the special virulence and
pathology of P. falciparum. The central role of PfEMP1 in P.
falciparum biology, as the malarial adherence receptor for host
proteins on microvascular endothelium, as described herein,
indicates its usefulness in a malaria vaccine, in modelling
prophylactic drugs, and also as a target for therapeutics to
reverse PE adherence in acute cerebral malaria (Howard and
Gilladoga, 1989).
2.10.1. MALARIAL POLYPEPTIDES
[1342] Soluble PfEMP1 has been reported to bind to CD36, TSP and
ICAM-1, and tryptic fragments of PfEMP1 cleaved from the PE surface
have been shown to bind to TSP or CD36 (Baruch, et al., Molecular
Parasitology Meeting at Woods Hole, Sept 18-22, 1994). Accordingly,
in one aspect, the present invention provides substantially pure
PfEMP1 polypeptides, analogs or biologically active fragments
thereof.
[1343] The terms "substantially pure" or "isolated" refer,
interchangeably, to proteins, polypeptides and nucleic acids which
are separated from proteins or other contaminants with which they
are naturally associated. A protein or polypeptide is considered
substantially pure when that protein makes up greater than about
50% of the total protein content of the composition containing that
protein, and typically, greater than about 60% of the total protein
content. More typically, a substantially pure protein will make up
from about 75 to about 90% of the total protein. Preferably, the
protein will make up greater than about 90%, and more preferably,
greater than about 95% of the total protein in the composition.
[1344] The term "biologically active fragment" as used herein,
refers to portions of the proteins or polypeptides, e.g., a PfEMP1
derived polypeptide, which portions possess a particular biological
activity, e.g., one or more activities found in a full length
PfEMP1 polypeptide. For example, such biological activity may
include the ability to bind a particular protein, substrate or
ligand, to elicit antibodies reactive with PE, PfEMP 1, the
recombinant proteins or fragments thereof, to block, reverse or
otherwise inhibit an interaction between two proteins, between an
enzyme and its substrate, between an epitope and an antibody, or
may include a particular catalytic activity. With regard to the
polypeptides of the present invention, particularly preferred
polypeptides or biologically active fragments include, e.g.,
polypeptides that possess one or more of the biological activities
described above, such as the ability to bind a ligand of PfEMP1 or
inhibit the binding of PfEMP1 to one or more of its ligands, e.g.,
CD36, TSP, ICAM-1, VCAM-1, ELAM-1, Chondroitin sulfate or by the
presence within the polypeptide fragment of antigenic determinants
which permit the raising of antibodies to that fragment.
[1345] The polypeptides of the present invention may also be
characterized by their immunoreactivity with antibodies raised
against PfEMP1 proteins or polypeptides. In particularly preferred
aspects, the polypeptides are capable of inhibiting an interaction
between a PfEMP1 protein and an antibody raised against a PfEMP1
protein. Additionally or alternatively, such fragments may be
specifically immunoreactive with an antibody raised against a
PfEMP1 protein. Such fragments are also referred to herein as
"immunologically active fragments." Generally, such biologically
active fragments will be from about 5 to about 500 amino acids in
length.
[1346] Typically, these peptides will be from about 20 to about 250
amino acids in length, and preferably from about 50 to about 200
amino acids in length. Generally, the length of the fragment may
depend, in part, upon the application for which the particular
peptide is to be used. For example, for raising antibodies, the
peptides may be of a shorter length, e.g., from about 5 to about 50
amino acids in length, whereas for binding applications, the
peptides may have a greater length, e.g., from about 50 to about
500 amino acids in length, preferably, from about 100 to about 250
amino acids in length, and more preferably, from about 100 to about
200 amino acids in length.
[1347] The polypeptides of the present invention may generally be
prepared using recombinant or synthetic methods well known in the
art. Recombinant techniques are generally described in Sambrook, et
al., Molecular Cloning: A Laboratory Manual, (2nd ed.) Vols. 1-3,
Cold Spring Harbor Laboratory, (1989). Techniques for the synthesis
of polypeptides are generally described in Merrifield, J. Amer.
Chem. Soc. 85:2149-2456 (1963), Atherton, et al., Solid Phase
Peptide Synthesis: A Practical Approach, IRL Press (1989), and,
Merrifield, Science 232:341-347 (1986).
[1348] In preferred aspects, the polypeptides of the present
invention may be expressed by a suitable host cell that has been
transfected with a nucleic acid of the invention, as described in
greater detail below. Isolation and purification of the
polypeptides of the present invention can be carried out by methods
that are generally well known in the art. For example, the
polypeptides may be purified using readily available
chromatographic methods, e.g., ion exchange, hydrophobic
interaction, HPLC or affinity chromatography, to achieve the
desired purity. Affinity chromatography may be particularly
attractive in allowing the investigator to take advantage of the
specific biological activity of the desired peptide, e.g., ligand
binding, presence of antigenic determinants, or the like.
[1349] Exemplary polypeptides of the present invention will
generally comprise an amino acid sequence that is substantially
homologous to the amino acid sequence of a PfEMP1 protein, or
biologically active fragments thereof, or may include sequences
that may take on a homologous conformation. In particularly
preferred aspects, the polypeptides of the present invention will
comprise an amino acid sequence that is substantially homologous to
the amino is acid sequence shown, described &/or referenced
herein (including incorporated by reference), or a biologically
active fragment thereof.
[1350] By "substantially homologous" is meant an amino acid
sequence which is at least about 50% homologous to the amino acid
sequence of PfEMP1 or a biologically active fragment thereof,
preferably at least about 90% homologous, and wore preferably at
least about 95% homologous. In some aspects, substantially
homologous may include a sequence that is at least 50% homologous,
but that presents a homologous structure in three dimensions, i.e.,
includes a substantially similar surface charge or presentation of
hydrophobic groups.
[1351] Examples of preferred polypeptides include polypeptides
having an amino acid sequence substantially homologous to the MC
PfEMP1 amino acid sequence as shown, described &/or referenced
herein (including incorporated by reference), and PfEMP1 of other
P. falciparum strains as shown, described &/or referenced
herein (including incorporated by reference), as well as
biologically active fragments of these polypeptides. Preferred
peptides include those peptide fragments of PfEMP1 that are
involved in the sequestration of parasitized erythrocytes. Examples
of these preferred peptides include peptides which comprise an
amino acid sequence which is substantially homologous to amino
acids 576 through 755 of the PfEMP1 amino acid sequence shown,
described &/or referenced herein (including incorporated by
reference).
[1352] Also among the particularly preferred peptides of the
present invention are those peptides and peptide fragments of PfEMP
1 which are relatively conserved among the variant strains of P.
falciparum or which contain regions of high homology to PfEMP1
proteins from other strains. The term "relatively conserved"
generally refers to amino acid sequences that are substantially
homologous to portions of the amino acid sequence shown, described
&/or referenced herein (including incorporated by reference).
However, also included within the definition of this term are
peptides which are encoded by a nucleic acid which is a PCR product
of primer probes, and particularly, universal primers, derived from
the PfEMP1 nucleic acid sequence. In particular, primer is probes
derived from the nucleic acid sequence shown, described &/or
referenced herein (including incorporated by reference), may be
used to amplify nucleic acids from other strains of P. falciparum.
Particularly preferred primer sequences include the primer
sequences shown in Table 1, below. Similarly, universal primer
compositions, described in greater detail below and also shown in
Table 1, may be used to amplify sequences that encode the peptides
of the present invention.
[1353] Specific examples of relatively conserved peptides include
those that are contained in a region of PfEMP1 proteins that
corresponds to amino acids 576 through 755 of the amino acid
sequence of MC PfEMP1, as shown, described &/or referenced
herein (including incorporated by reference).
[1354] Similar regions have been specifically elucidated in a
number of P. falciparum strains (See FIGS. 20 and 21). In general,
these corresponding regions may be described as containing amino
acid sequences that are encoded by the universal primer sequences
described below. Generally, these amino acid sequences have one or
more of the following general structures:
TTIDKX.sub.1LX.sub.2HE and/or FFWX.sub.3WVX.sub.4X.sub.5ML
[1355] where X.sub.1 is selected from leucine or isoleucine,
X.sub.2 is selected from glutamine and asparagine, X.sub.3 is
selected from the methionine, lysine and aspartic acid, X.sub.4 is
selected from histidine, threanine and tyrosine and X.sub.5 is
selected from aspartic acid, glutamic acid and histidine. In
particularly preferred aspects, the polypeptides may contain both
of the above general amino acid sequences. Particularly preferred
amino acid sequences will possess the conserved amino acids shown
in the various fragments shown, described &/or referenced
herein (including incorporated by reference). In particular,
conserved amino acid sequences of six amino acids or greater,
shown, described &/or referenced herein (including incorporated
by reference), may be used as epitopes for generation of antibodies
that cross react with multiple P. falciparum strains.
[1356] The peptides of the invention may be free or tethered, or
may include labeled groups for detection of the presence of the
polypeptides. Suitable labels include radioactive, fluorescent and
catalytic labeling groups that are well known in the art and that
are substantially described herein, e.g., signaling enzymes,
chemical reporter groups, polypeptide signals, biotin and the like.
Additionally, the peptides may include modifications to the N and
C-termini of the peptide, e.g., an acylated N-terminus or amidated
C-terminus.
[1357] Also included within the present invention are amino acid
variants of the above described polypeptides. These variants may
include insertions, deletions and substitutions with other amino
acids. For example, in some aspects, amino acids may be substituted
with different amino acids having similar structural
characteristics, e.g., net charge, hydrophobicity, or the like. For
example, phenylalanine may be substituted with tyrosine, as a
similarly hydrophobic residue. Glycosylation modifications, either
changed, increased amounts or decreased amounts, as well as other
sequence modifications are also envisioned.
[1358] In addition to the above polypeptides which consist only of
naturally-occurring amino acids, peptidomimetics of the
polypeptides of the present invention are also provided. Peptide
analogs are commonly used in the pharmaceutical industry as
non-peptide drugs with properties analogous to those of the
template peptide. These types of non-peptide compound are termed
"peptide mimetics" or "peptidomimetics" (Fauchere, J. (1986) Adv.
Drug Res. 15:29; Veber and Freidinger (1985) TINS p.392; and Evans
et al. (1987) J. Med. Chem 30:1229, and are usually developed with
the aid of computerized molecular modeling. Peptide mimetics that
are structurally similar to therapeutically useful peptides may be
used to produce an equivalent therapeutic or prophylactic effect.
Generally, peptidomimetics are structurally similar to a paradigm
polypeptide (i.e., a polypeptide that has a biological or
pharmacological activity), such as naturally-occurring
receptor-binding polypeptide, but have one or more peptide linkages
optionally replaced by a linkage selected from the group consisting
of: --CH.sub.2NH--, --CH.sub.2S--, --CH.sub.2--CH.sub.2--,
--CH.dbd.CH-- (cis and trans), --COCH.sub.2--, --CH(OH)CH.sub.2--,
and --CH.sub.2SO--, by methods known in the art and further
described in the following references: Spatola, A. F. in Chemistry
and Biochemistry of Amino Acids, Peptides, and Proteins, B.
Weinstein, eds., Marcel Dekker, New York, p. 267 (1983); Spatola,
A. F., Vega Data (March 1983), Vol. 1, Issue 3, "Peptide Backbone
Modifications" (general review); Morley, J. S., Trends Pharm Sci
(1980) pp. 463-468 (general review); Hudson, D. et al., Int J Pept
Prot Res (1979) 14:177-185 (--CH.sub.2NH--, CH.sub.2CH.sub.2--) ;
Spatola, A. F. et al., Life Sci (1986) 38:1243-1249
(--CH.sub.2--S); Hann, M. M., J Chem Soc Perkin Trans I (1982)
307-314 (--CH--CH--, cis and trans); Almquist, R. G. et al., J Med
Chem (1980) 23:1392-1398 (--COCH.sub.2--); Jennings-White, C. et
al., Tetrahedron Lett (1982) 23:2533 (--COCH.sub.2--); Szelke, M.
et al., European Appln. EP 45665 (1982) CA: 97:39405 (1982)
(--CH(OH)CH.sub.2--); Holladay, M. W. et al., Tetrahedxon Lett
(1983) 24:4401-4404 (--C(OH)CH.sub.2--); and Hruby, V. J., Life Sci
(1982) 31:189-199 (--CH.sub.2--S--)' Peptide mimetics may have
significant advantages over polypeptide embodiments, including, for
example: more economical production, greater chemical stability,
enhanced pharmacological properties (half-life, absorption,
potency, efficacy, etc.), altered specificity (e.g., a
broad-spectrum of biological activities), reduced antigenicity, and
others.
[1359] Labeling of peptidomimetics usually involves covalent
attachment of one or more labels, directly or through a spacer
(e.g., an amide group), to non-interfering position(s) on the
peptidomimetic that are predicted by quantitative
structure-activity data and/or molecular modeling. Such
non-interfering positions generally are positions that do not form
direct contacts with the molecules to which the peptidomimetic
binds (e.g., CD36) to produce the therapeutic effect.
Derivitization (e.g., labeling) of peptidomimetics should not
substantially interfere with the desired biological or
pharmacological activity of the peptidomimetic. Generally,
peptidomimetics of peptides of the invention bind to their ligands
(e.g., CD36) with high affinity and possess detectable biological
activity (i.e., are agonistic or antagonistic to one or more
ligand-mediated phenotypic changes).
[1360] Systematic substitution of one or more amino acids of a
consensus sequence with a D-amino acid of the same type (e.g.,
D-lysine in place of L-lysine) may be used to generate more stable
peptides. In addition, constrained peptides comprising a consensus
sequence or a substantially identical consensus sequence variation
may be generated by methods known in the art (Rizo and Gierasch
(1992) Ann. Rev. Blochem. 61: 387; for example, by adding internal
cysteine residues capable of forming intramolecular disulfide
bridges which cyclize the peptide.
[1361] Polypeptides of the present invention may also be
characterized by their ability to bind antibodies raised against
PfEMP1, or fragments thereof. Preferably, these antibodies
recognize polypeptide domains that are homologous to the PfEMP1
proteins from a number of variants of P. falciparum. These
homologous domains will generally be present throughout the family
of PfEMP1 proteins. A variety of immunoassay formats may be used to
select antibodies specifically immunoreactive with a particular
protein or domain. For example, solid-phase ELISA immunoassays are
routinely used to select monoclonal antibodies specifically
immunoreactive with a protein. See Harlow and Lane (1988)
Antibodies, A Laboratory Manual, Cold Spring Harbor Publications,
New York, for a description of immunoassay formats and conditions
that can be used to determine specific immunoreactivity. Antibodies
to PfEMP1 and its fragments are discussed in greater detail, below.
As used herein, the terms "polypeptide" or "peptide" are used
interchangeably to refer to peptides, peptidomimetics, analogs, and
the like, as described above.
[1362] The polypeptides of the present invention may be used as
isolated polypeptides, or may exist as fusion proteins. A "fusion
protein" generally refers to a composite protein made up of two or
more separate, heterologous proteins which are normally not fused
together as a single protein.
[1363] Thus, a fusion protein may comprise a fusion of two or more
heterologous or homologous sequences, provided these sequences are
not normally fused together. Fusion proteins will generally be made
by either recombinant nucleic acid methods, i.e., as a result of
transcription and translation of a gene fusion comprising a segment
encoding a polypeptide comprising a PfEMP1 protein and a segment
which encodes one or more heterologous proteins, or by chemical
synthesis methods well known in the art.
2.10.2. MALARIAL NUCLEIC ACIDS AND CELLS CAPABLE OF EXDRESSING
SAME
[1364] Also provided in the present invention are isolated nucleic
acid sequences which encode the above described polypeptides and
biologically active fragments. Typically, such nucleic acid
sequences will comprise a segment that is substantially homologous
to a portion or fragment of the nucleic acid sequence shown,
described &/or referenced herein (including incorporated by
reference). Preferably, the nucleic acids of the present invention
will comprise at least about 15 consecutive nucleotides of the
nucleic acid, more preferably, at least about 20 contiguous
nucleotides, still more preferably, at least about 30 contiguous
nucleotides, and still more preferably, at least about 50
contiguous nucleotides from the nucleotide sequence.
[1365] Substantial homology in the nucleic acid context means that
the segments, or their complementary strands, when compared, are
the same when properly aligned with the appropriate nucleotide
insertions or deletions, in at least about 60% of the nucleotides,
typically, at least about 70%, more typically, at least about 80%,
usually, at least about 90%, and more usually, at least about 95%
to 98% of the nucleotides. Alternatively, substantial homology
exists when the segments will hybridize under selective
hybridization conditions to a strand, or its complement, typically
using a sequence of at least about 15 contiguous nucleotides
derived from the PfEMP1 nucleic acid sequence. However, larger
segments will usually be preferred, e.g., at least about 20 or
contiguous nucleotides, more usually about 40 contiguous
nucleotides, and preferably more than about 50 contiguous
nucleotides. Selective hybridization exists when hybridization
occurs which is more selective than total lack of specificity. See,
Kanchisa, Nucleic Acid Res. 12:203-213 (1984).
[1366] Nucleic acids of the present invention include RNA, cDNA,
genomic DNA, synthetic forms and mixed polymers, both sense and
antisense strands. Furthermore, different alleles of each isoform
are also included. The present invention also provides recombinant
nucleic acids which are not otherwise naturally occurring. The
nucleic acids included in the present invention will typically
comprise RNA or DNA or mixed polymers. The DNA compositions will
generally include a coding region which encodes a polypeptide
comprising an amino acid sequence substantially homologous to the
amino acid sequence of a PfEMP1 protein. More preferred are those
DNA segments comprising a nucleotide sequence which encodes a CD36
binding fragment of the PfEMP1 protein.
[1367] cDNA encoding the polypeptides of the present invention, or
fragments thereof, may be readily employed as a probe useful for
obtaining genes which encode the PfEMP1 polypeptides of the present
invention. Preparation of these probes may be carried out by
generally well known methods. For example, the cDNA probes may be
prepared from the amino acid sequence of the PfEMP1 protein. In
particular, probes may be prepared based upon segments of the amino
acid sequence which possess relatively low levels of degeneracy,
i.e., few or one possible nucleic acid sequences which encode
therefor.
[1368] Suitable synthetic DNA fragments may then be prepared, e.g.,
by the phosphoramidite method described by Beaucage and Carruthers,
Tetra. Letts. 22:1859-1862 (1981). Alternatively, nucleotide
sequences which are relatively conserved among the PfEMP1 coding
sequences for the various P. falciparum strains may be used as
suitable probes. A double stranded probe may then be obtained by
either synthesizing the complementary strand and hybridizing the
strands together under appropriate conditions or by adding the
complementary strand using DNA polymerase with an appropriate
primer sequence. Such cDNA probes may be used in the design of
oligonucleotide probes and primers for screening and cloning such
genes, e.g., using well known PCR techniques, or, alternatively,
may be used to detect the presence or absence of a PfEMP1 gene in a
cell. Such nucleic acids, or fragments may comprise part or all of
the cDNA sequence that encodes the polypeptides of the present
invention. Effective cDNA probes may comprise as few as 15
consecutive nucleotides in the cDNA sequence, but will often
comprise longer segments. Further, these probes may further
comprise an additional nucleotide sequence, such as a
transcriptional primer sequence for cloning, or a detectable group
for easy identification and location of complementary
sequences.
[1369] cDNA or genomic libraries of various types may be screened
for new alleles or related sequences using the above probes. The
choice of cDNA libraries normally corresponds to tissue sources
which are abundant in mRNA for the desired polypeptides. Phage
libraries are normally preferred, e.g., .lambda.gt11, but plasmid
or YAC libraries may also be used. Clones of a library are spread
onto plates, transferred to a substrate for screening, denatured,
and probed for the presence of the desired sequences.
[1370] In a related aspect, the nucleic acids of the present
invention also include the PCR product or RT-PCR product, produced
using the above described primer probes. For example, primer probes
derived from the nucleotide sequence shown, described &/or
referenced herein (including incorporated by reference), may be
used to amplify sequences from different malaria parasites, and in
particular, different strains of P. falciparum. Examples of
particularly preferred nucleic acid sequences include those nucleic
acid sequences which are PCR amplified using the following
oligonucleotide probes:
1 5' 1.sup.a: AAGGAAGACAAAATTATGTCCTAT 25: AATGGAGAGACGAACATGG 53:
TCCAAAAATGGGTTGAACAAAAA 80: ATTGGACTCATGATGATTTTC 96:
TTTTGGAAATTATTCAGGATACT 135: CTAAAGGTTTTGTCGCTGAAA 156:
AAGCCGATAAATGCCTAAAAAC 3' 59: TTTTTGTTCAACCCATTTTTGGA 87:
TGAAGAAAATCATCATGAGTCCA 102: AGTATCCTGAATAATTTCCAA 140:
TTCAGCGACAAAACCTTTAGT 179: GAGCGGGCGACACTTCTATCT 192:
CTTAGGGTCGGCAGGTGGTG 233: ATCCGTCTTTTCCTCCTGGACTT
[1371] a. The number designation indicates the amino acid position
within amino acids 575 through 808 of the amino acid sequence
shown, described &/or referenced herein (including incorporated
by reference), which is encoded by the respective end of the probe
(31 or 51).
[1372] Included among the most preferred of the above described
nucleic acid sequences are the nucleic acids which are PCR
amplified using the following primer probe combinations: 5'-1:
3'59, 3'140, 3'179, 5'53: 3'140, 3'179; and 5'140:3'179.
[1373] Also included among the most preferred oligonucleotides are
those nucleic acid segments which encode the relatively conserved
peptides described above. Examples of these oligonucleotides which
have been identified from the previously described P. falciparum
strains are shown in Table 1, below:
2TABLE 1 Plasmodium falciparum Strain 3' primer MC type ACT ACA ATT
GAT AAA TTA CTA CAA CAC GAA ITG type ACC ACA ATT GAT AAA TTG CTC
AAT CAC GAA HB-3 type ACT ACA ATT GAT AAA ATA CTA CAA CAC GAA
Degenerate ACC ACA ATT GAT AAA TTA CTA CAA CAC GAA Universal T A G
C A T Primers 5'primer MC type TTT TTT TGG ATG TGG GTA CAT GAT ATG
TTA ITG type TTT TTT TGG AAG TGG GTT ACC GAA ATG TTA HB-3 type TTT
TTT TGG GAT TGG GTT TAT CAT ATG TTA Degenerate TTT TTT TGG GAG TGG
GTA TAT GAT ATG TTA Universal ATT T ACC C A Primers C
[1374] In the synthesis of the universal primer sequences, single
sequence lines indicate the primary sequence of the primer. Where
two bases are shown for a single position, e.g., A and T, it refers
to a step in the synthesis of the primer sequence where equal
amounts of each base were added to the synthesis step, resulting in
equal amounts of each base being coupled to growing oligomers in
that position.
[1375] Similarly, where three bases are shown for a given position,
equal amounts of the three bases are added to the synthesis step.
This results in a mixture of oligonucleotide sequences having all
possible combinations of sequences reflecting the multiple bases at
each of the indicated positions. In some cases, expression of the
full length primer required the addition of additional bases to the
5' primer, e.g., a CTT before the TTT, to correct for truncation
problems upon inserting the primer into the vector used.
[1376] Thus, based upon the above sequences, the general structure
of the universal 3' primer sequence can be described as a mixture
of a number of individual primer sequences where each individual
primer has the following general structure:
ACX.sub.6ACA ATT GAT AAA X.sub.7TX.sub.8 CTX.sub.9
X.sub.10AX.sub.11 CAC GAA
[1377] where X.sub.6 is selected from C and T, X.sub.7 is selected
from T and A, X.sub.8 is selected from G and A, X.sub.9 is selected
from C and A, X.sub.10 is selected from C and A and X.sub.11 is
selected from T and A.
[1378] Similarly, each of the individual primer sequences within
the universal 5' primer is represented by the general
structure:
TTT TTT TGG X.sub.12X.sub.13X.sub.14 TGG GTX.sub.15
X.sub.16X.sub.17X.sub.18 X.sub.19AX.sub.20 ATG TTA
[1379] where X.sub.12 is selected from G and A, X.sub.13 'S
selected from A and T, X.sub.14 is selected from G and T, X.sub.15
is selected from A and T, X.sub.16 is selected from of T, A and C,
X.sub.17 is selected from A and C, X.sub.18 is selected from of T
and C, X.sub.19 is selected from G and C and X.sub.20 is selected
from T and A.
[1380] The above-described universal primer sequences are
particularly useful in identifying corresponding gene sequences in
different strains of P. falciparum, as well as in the design of
particularly preferred peptides of the invention.
[1381] The above universal primers may be particularly useful in
generating a "finger print" identification of individual P.
falciparum cells and clones by amplifying a distinct set of PCR
products of varying sizes from the var genes and/or the expressed
var genes of these cells and clones.
[1382] The nucleic acids of the present invention may be present in
whole cells, cell lysates or in partially pure or substantially
pure or isolated form. Such "substantially pure" or "isolated"
forms of these nucleic acids generally refer to the nucleic acid
separated from contaminants with which it is generally associated,
e.g., lipids, proteins and other nucleic acids. The nucleic acids
of the present invention will be greater than about 50% pure.
Typically, the nucleic acids will be more than about 60% pure, more
typically, from about 75% to about 90% pure, and preferably, from
about 95% to about 98% pure.
[1383] The present invention also provides substantially similar
nucleic acid sequences, allelic variations and natural or induced
sequences of the above described nucleic acids, as well as
chemically modified and substituted nucleic acids, e.g., those
which incorporate modified nucleotide bases or which incorporate a
labeling group. In addition to comprising a segment which encodes a
PfEMP1 protein or fragment thereof, the nucleic acids of the
present invention may also comprise a segment encoding a
heterologous protein, such that the gene is expressed to produce
the two proteins as a fusion protein, as substantially described
above.
[1384] In addition to their use as probes, the nucleic acids of the
present invention may also be used in the preparation of the
polypeptides of the present invention, as described above. DNA
encoding the polypeptides of the present invention will typically
be incorporated into DNA constructs capable of introduction to and
expression in an in vitro cell culture. Often, the nucleic acids of
the present invention may be used to produce a suitable recombinant
host cell.
[1385] Specifically, DNA constructs will be suitable for
replication in a unicellular host, such as bacteria, e.g., E. coli,
viruses or yeast, but may also be intended for introduction into a
cultured mammalian, plant, insect, or other eukaryotic cell lines.
DNA constructs prepared for introduction into bacteria or yeast
will typically include a replication system recognized by the host,
the intended DNA segment encoding the desired polypeptide,
transcriptional and translational initiation and termination
regulatory sequences operably linked to the polypeptide encoding
segment. A DNA segment is operably linked when it is placed into a
functional relationship with another DNA segment. For example, a
promoter or enhancer is operably linked to a coding sequence if it
stimulates the transcription of the sequence; DNA for a signal
sequence is operably linked to DNA encoding a polypeptide if it is
expressed as a preprotein that participates in the secretion of the
polypeptide. Generally, DNA sequences that are operably linked are
contiguous, and in the case of a signal sequence both contiguous
and in reading phase. However, enhancers need not be contiguous
with the coding sequences whose transcription they control. Linking
is accomplished by ligation at convenient restriction sites or at
adapters or linkers inserted in lieu thereof. The selection of an
appropriate promoter sequence will generally depend upon the host
cell selected for the expression of the DNA segment.
[1386] Examples of suitable promoter sequences include prokaryotic,
and eukaryotic promoters well known in the art. See, e.g., Sambrook
et al., supra. The transcriptional regulatory sequences will
typically include a heterologous enhancer or promoter which is
recognized by the host. The selection of an appropriate promoter
will depend upon the host, but promoters such as the trp, lac and
phage promoters, tRNA promoters and glycolytic enzyme promoters are
known and available. See Sambrook et al., supra.
[1387] Conveniently available expression vectors which include the
replication system and transcriptional and translational regulatory
sequences together with the insertion site for the PfEMP1
polypeptide encoding segment may be employed. Examples of workable
combinations of cell lines and expression vectors are described in
Sambrook et al., supra, and in Metzger et al., Nature 334:31-36
(1988).
[1388] The vectors containing the DNA segments of interest, e.g.,
those encoding polypeptides comprising a PfEMP1 protein or
fragments thereof, can be transferred into the host cell by well
known methods, which may vary depending upon the type of host used.
For example, calcium chloride transfection is commonly used for
prokaryotic cells, whereas calcium phosphate treatment may be used
for other hosts. See, Sambrook et al., supra. The term "transformed
cell" as used herein, includes the progeny of originally
transformed cells.
[1389] Techniques for manipulation of nucleic acids which encode
the polypeptides of the present invention, i.e., subcloning the
nucleic acids into expression vectors, labeling probes, DNA
hybridization and the like, are generally described in Sambrook, et
al., supra. In recombinant methods, generally the nucleic acid
encoding a peptide of the present invention is first cloned or
isolated in a form suitable for ligation into an expression vector.
After ligation, the vectors containing the nucleic acids fragments
or inserts are introduced into a suitable host cell, for the
expression of the polypeptide of the invention. The polypeptides
may then be purified or isolated from the host cells. Methods for
the synthetic preparation of oligonucleotides are generally
described in Gait, oligonucleotide Synthesis: A Practical Approach,
IRL Press (1990).
[1390] There are various methods of isolating the nucleic acids
which encode the polypeptides of the present invention. Typically,
the DNA is isolated from a genomic or cDNA library using labeled
oligonucleotide probes specific for sequences in the desired DNA.
Restriction endonuclease digestion of genomic DNA or cDNA
containing the appropriate genes can be used to isolate the DNA
encoding the binding domains of these proteins. From the PfEMP1
sequence given in FIG. 12, a panel of restriction endonucleases can
be constructed to give cleavage of the DNA in desired regions,
i.e., to obtain segments which encode biologically active fragments
of the PfEMP1 protein. Following restriction endonuclease
digestion, DNA encoding the polypeptides of the present invention
is identified by its ability to hybridize with a nucleic acid probe
in, for example a Southern blot format. These regions are then
isolated using standard methods. See, e.g., Sambrook, et al.,
supra.
[1391] The polymerase chain reaction, or "PCR" can also be used to
prepare nucleic acids which encode the polypeptides of the present
invention. PCR technology is used to amplify nucleic acid sequences
of the desired nucleic acid, e.g., the DNA which encodes the
polypeptides of the invention, directly from mRNA, cDNA, or genomic
or cDNA libraries.
[1392] Appropriate primers and probes for amplifying the nucleic
acids described herein, may be generated from analysis of the
PfEMP1 oligonucleotide sequence, such as those shown, described
&/or referenced herein (including incorporated by reference)
and Table 1. Briefly, oligonucleotide primers complementary to the
two 31 borders of the DNA region to be amplified are synthesized.
The PCR is then carried out using the two primers. See, e.g., PCR
Protocols: A Guide to Methods and Applications (Innis, M., Gelfand,
D., Sninsky, J. and White, T., eds.) Academic Press (1990). Primers
can be selected to amplify various sized segments from the PfEMP1
oligonucleotide sequence. The primers may also contain a
restriction site and additional bases to permit "in-frame" cloning
of the insert into an appropriate expression vector, using the
restriction sites present on the primers.
2.10.3. ANTIBODIES
[1393] The nucleic acids and polypeptides of the present invention,
or fragments thereof, are also useful in producing antibodies,
either polyclonal or monoclonal. These antibodies are produced by
immunizing an appropriate vertebrate host, e.g., rat, mouse, rabbit
or goat, with a polypeptide of the invention, or its fragment, or
plasmid DNA containing a nucleic acid of the invention, alone or in
conjunction with an adjunct. Usually, two or more immunizations are
involved, and a few days following the last injection, the blood or
spleen of the host will be harvested.
[1394] For production of polyclonal antibodies, an appropriate
target immune system is selected, typically a mouse or rabbit, but
also including goats, sheep, cows, guinea pigs, monkeys and rats.
The substantially purified antigen or plasmid is presented to the
immune system in a fashion determined by methods appropriate for
the animal. These and other parameters are well known to
immunologists. Typically, injections are given in the footpads,
intramuscularly, intradermally or intraperitoneally. The
immunoglobulins produced by the host can be precipitated, isolated
and purified by routine methods, including affinity
purification.
[1395] For monoclonal antibodies, appropriate animals will be
selected and the desired immunization protocol followed. After the
appropriate period of time, the spleens of these animals are
excised and individual spleen cells are fused, typically, to
immortalized myeloma cells under appropriate selection conditions.
Thereafter, the cells are clonally separated and the supernatants
of each clone are tested for the production of an appropriate
antibody specific for the desired region of the antigen. Techniques
for producing antibodies are well known in the art. See, e.g.,
Goding et al., Monoclonal Antibodies: Principles and Practice (2d
ed.) Acad. Press, N.Y., and Harlow and Lane, Antibodies: A
Laboratory Manual, Cold Spring Harbor Laboratory, New York (1988).
Other suitable techniques involve the in vitro exposure of
lymphocytes to the antigenic polypeptides or alternatively, to
selection of libraries of antibodies in phage or similar vectors.
Huse et al., Generation of Large Combinatorial Library of the
Immunoglobulin Repertoire in Phage Lambda, Science 246:1275-1281
(1989). Monoclonal antibodies with affinities of 10.sup.8
liters/mole, preferably 10.sup.9 to 10.sup.10 or stronger, will be
produced by these methods.
[1396] The antibodies generated can be used for a number of
purposes, e.g., as probes in immunoassays, for inhibiting PfEMP1
binding to its ligands, thereby inhibiting or reducing erythrocyte
sequestration, in diagnostics or therapeutics, or in research to
further elucidate the mechanism of various aspects of malarial
infection, and particularly, P. falciparum infection. The
antibodies of the present invention can be used with or without
modification. Frequently, the antibodies will be labeled by
joining, either covalently or non-covalently, a substance which
provides for a detectable signal. Such labels include those that
are well known in the art, such as the labels described previously
for the polypeptides of the invention. Additionally, the antibodies
of the invention may be chimeric, human-like or humanized, in order
to reduce their potential antigenicity, without reducing their
affinity for their target. Chimeric, human-like and humanized
antibodies have generally been described in the art. Generally,
such chimeric, human-like or humanized antibodies comprise variable
regions, e.g., complementarity determining regions (CDR) (for
humanized antibodies), from a mammalian animal, i.e., a mouse, and
a human framework region. By incorporating as little foreign
sequence as possible in the hybrid antibody, the antigenicity is
reduced. Preparation of these hybrid antibodies may be carried out
by methods well known in the art.
[1397] Preferred antibodies are those that are specifically
immunoreactive with the polypeptides of the present invention and
their immunologically active fragments. The phrase "specifically
immunoreactive," when referring to the interaction between an
antibody of the invention and a particular protein, refers to an
antibody that specifically recognizes and binds with relatively
high affinity to the particular protein, such that this binding is
determinative of the presence of the protein in a heterogeneous
population of proteins and other biologics. Thus, under designated
immunoassay conditions, the specified antibodies bind to a
particular protein and do not bind in a significant amount to other
proteins present in the sample. A variety of immunoassay formats
may be used to select antibodies specifically immunoreactive with a
particular protein. For example, solid-phase ELISA immunoassays are
routinely used to select monoclonal antibodies specifically
immunoreactive with a protein. See Harlow and Lane (1988)
Antibodies, A Laboratory Manual, Cold Spring Harbor Publications,
New York, for a description of immunoassay formats and conditions
that can be used to determine specific immunoreactivity.
[1398] The antibodies generated can be used for a number of
purposes, e.g., as probes in immunoassays, for inhibiting
interaction between a PfEMP1 protein and its ligand, e.g., CD-36,
TSP, ICAM-1, VCAM-1, ELAM-1, or Chondroitin sulfate, thereby
inhibiting or reducing the level of PfEMP1-ligand interaction, in
diagnostics or therapeutics, or in research to further elucidate
the mechanism of malarial pathology, e.g., erythrocyte
sequestration. Where the antibodies are used to block or reverse
the interaction between a polypeptide of the invention and an
associating ligand or PE, the antibody will generally be referred
to as a "blocking antibody." Preferred antibodies are those
monoclonal or polyclonal antibodies which specifically recognize
and bind the polypeptides of the invention. Accordingly, these
preferred antibodies will specifically recognize and bind the
polypeptides which have an amino acid sequence that is
substantially homologous to the relevant amino acid sequence shown,
described &/or referenced herein (including incorporated by
reference), or immunologically active fragments thereof. Still more
preferred are antibodies which are capable of forming an
antibody-ligand complex with the relatively conserved polypeptide
fragments of PfEMP1 sequences, and are thereby capable of blocking
an interaction of PfEMP1 from a variety of P. falciparum strains,
and PfEMP1 ligands.
2.10.4. METHODS OF USE
[1399] The polypeptides, antibodies, and nucleic acids of the
present invention have a variety of important uses, including, but
not limited to, diagnostic, screening, prophylactic, including
vaccination, and therapeutic applications.
2.10.4.1. DIAGNOSTIC APPLICATIONS
[1400] In a particularly preferred aspect, the present invention
provides methods and reagents useful in detecting the presence of
PfEMP1 in a sample. These detection methods are particularly useful
in diagnosing malarial infections in a patient. For example, in a
particularly preferred aspect, the antibodies of the present
invention may be used to assay for the presence or absence of
PfEMP1 in a sample. Immunoassay techniques for the detection of the
particular antigen are very well known in the art. For a review of
immunological and immunoassay procedures in general, see Basic and
Clinical Immunology 7th Edition (D. Stites and A. Terr ed.)
1991.
[1401] Moreover, the immunoassays of the present invention can be
performed in any of several configurations, which are reviewed
extensively in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press,
Boca Raton, Fla. (1980); "Practice and Theory of Enzyme
Immunoassays," P. Tijssen, Laboratory Techniques in Biochemistry
and Molecular Biology, Elsevier Science Publishers B. V. Amsterdam
(1985); and, Harlow and Lane, Antibodies, A Laboratory Manual,
supra. Generally, these methods comprise contacting the antibody
with a sample to be tested, and detecting any specific binding
between the antibody and a protein within the sample. Typically,
this will be in a blot format, e.g., western blot, or in an ELISA
format. Methods of performing these assay formats are well known in
the art. See, e.g., Basic and Clinical Immunology, 7th ed. (D.
Stites and A Terr, eds., 1991).
[1402] Typically, these diagnostic methods comprise contacting a
sample with an antibody to PfEMP1, as described herein, and
determining whether the antibody binds to any portion of the
sample. In the case of human diagnostic techniques, the sample may
be a whole blood sample, or some fraction thereof, e.g. an
erythrocyte containing sample. Generally, such diagnostic methods
are well known in the art, and are described in the above described
references. The immunoreactivity of the antibody with the sample,
indicates the presence of PfEMP1 in the sample, and, in the case of
a sample derived from a patient, a possible malarial infection.
[1403] Alternatively, labeled polypeptides of the present invention
may be used as diagnostic reagents in detecting the presence or
absence of antibodies to PfEMP1, in a patient. The presence of
antibodies within a patient would be indicative that the patient
had been exposed to a malaria parasite sufficiently to result in an
antigenic response.
[1404] Similarly, the nucleic acid probes of the invention may be
used in a similar manner, i.e., to identify the presence in a
sample of a DNA segment encoding a PfEMP1 polypeptide, or as PCR or
RT-PCR primers to amplify and then detect PfEMP1 encoding nucleic
acid segments. Such assays typically involve the immobilization of
nucleic acids in the sample, followed by interrogation?? of the
immobilized sequences with a chemically labeled oligonucleotide
probe, as described herein. Hybridization of the probe to the
immobilized sample indicates the presence of a DNA segment encoding
PfEMP1, and thus, a malarial infection. As described above, assays
may be further designed to indicate not only the presence of a
Malarial parasite, but also indicate the strain of parasite
present. Although described in terms of an immobilized sample
probed with a solution based oligonucleotide probe, a wide variety
of assay conformations may be adopted, which conformations are
generally well known in the art.
2.10.4.2. SCREENING APPLICATIONS
[1405] In another particularly preferred aspect, the present
invention provides methods for screening compounds to determine
whether or not the particular compound is an antagonist of a
symptom of a malarial infection. In particular, the screening
methods of the present invention can be used to determine whether a
test compound is an antagonist of the sequestration of erythrocytes
which is associated with P. falciparum malaria. More particularly,
the screening methods can determine whether a compound is an
antagonist of the PfEMP11/ligand interaction. Ligands of PfEMP1
generally include, e.g., CD36, TSP, ELAM-1, ICAM-1, VCAM-1 or
Chondroitin sulfate.
[1406] Generally, the screening methods of the present invention
comprise contacting PfEMP1 protein, or a fragment thereof, and/or
ligand protein, with a compound which is to be screened ("test
compound"). The level of PfEMP1/ligand complex formed may then be
detected and compared to a control, e.g., in the absence of the
test compound. A decrease in the level of PfEMP1/ligand interaction
is indicative that the test compound is an antagonist of that
interaction.
[1407] A test compound may be a chemical compound, a mixture of
chemical compounds, a biological macromolecule, or an extract made
from biological materials, such as bacteria, phage, yeast, plants,
fungi, animal cells or tissues. Test compounds are evaluated for
potential activity as antagonists of PfEMP1/ligand interaction by
inclusion in the screening assays described herein. An "antagonist"
refers to a compound which will diminish the level of PfEMP1/ligand
interaction, over a control.
[1408] It will often be desirable in the screening assays of the
present invention, to provide one of the PfEMP1 or ligand proteins
immobilized on a solid support. Suitable solid supports include,
e.g., agarose, cellulose, dextran, Sephadex, Sepharose,
carboxymethyl cellulose, polystyrene, filter paper, nitrocellulose,
ion exchange resins, plastic films, glass beads,
polyaminemethylvinylether maleic acid copolymer, amino acid
copolymer, ethylene-maleic acid copolymer, nylon, silk, etc. The
support may be in the form of, e.g., a test tube, microtiter plate,
beads, test strips, flat surface, e.g., for blotting formats, or
the like. The reaction of the PfEMP1 polypeptide or its ligand with
the particular solid support may be carried out by methods well
known in the art, e.g., binding to an immobilized anti-PfEMP1
antibody, or binding to prederivatized solid support.
[1409] In addition to the foregoing, it may also be desirable to
provide either the PfEMP1 or its ligand linked to a suitable
detectable group to make detection of binding of one protein to the
other, simpler. Useful detectable groups, or labels, are generally
well known in the art. For example, a detectable group may be a
radiolabel, such as, .sup.125I, .sup.32p or .sup.35S, or a
fluorescent or chemiluminescent group.
[1410] Alternatively, the detectable group may be a substrate,
cofactor, inhibitor, affinity ligand, antibody binding epitope tag,
or an enzyme which is capable of being assayed. Suitable enzymes
include, e.g., horseradish peroxidase, luciferase, or another
readily assayable enzymes. These enzyme groups may be attached to
the PfEMP1 polypeptide, or its ligand by chemical means or maybe
expressed as a fusion protein, as already described.
[1411] Generally, where one of the above proteins, e.g., the PfEMP1
ligand, is immobilized on a solid support, the other protein, e.g.,
PfEMP1 or its fragment, will be labeled with an appropriate
detectable group. Assaying whether a compound is an antagonist of
the interaction of the two proteins is then a matter of contacting
the labeled PfEMP1 polypeptide or fragment with the immobilized
ligand, in the presence of the test compound, under conditions
which allow specific binding of the two proteins. The amount of
label bound to the solid support is compared to a control, where no
test compound was added. Where a test compound results in a
reduction of the amount of label which binds to a solid support,
that compound is an antagonist of the PfEMP1/ligand
interaction.
2.10.4.3. THERAPEUTIC AND PROPHYLACTIC APPLICATIONS
[1412] In addition to the above described uses, the polypeptides of
the present invention may also be used in therapeutic applications,
for the treatment of human and/or non-human mammalian patients. The
therapeutic uses of the polypeptides of the present invention
include the treatment of symptoms of existing disorders, as well as
prophylactic applications. The term "prophylactic" refers to the
prevention of a particular disorder, or symptoms of a particular
disorder. Thus, prophylactic treatments will generally include
drugs which actively participate in the prevention of a particular
disorder such as a malaria infection, or symptoms thereof.
Prophylactic applications will also include treatments which elicit
a preventative response from a patient, including, for example, an
immunological response as in the case of vaccination.
[1413] Typically, both therapeutic and prophylactic applications
will comprise administering an effective amount of the compositions
of the present invention to a patient, to treat or prevent
symptoms, or the onset of a malarial parasite infection. An
"effective amount", as the term is used herein, is defined as the
amount of the composition which is necessary to achieve the desired
goal, i.e. alleviation of symptoms, prevention of symptoms or
infection, or treatment of disease.
[1414] In prophylactic applications, the polypeptides of the
present invention may be used in a variety of treatments. For
example, the polypeptides of the invention are particularly useful
as a vaccine, to elicit an immunological response by a patient,
e.g., production of antibodies specific for PfEMP1. In particular,
such vaccine applications generally involve the administration of
the PfEMP1 protein or biologically active fragments thereof, to the
host or patient.
[1415] In response to this administration, the patient's immune
system will generate antibodies to the particular PfEMP1 protein or
fragment introduced. An amount of the polypeptides sufficient to
produce an immunological response in a patient is termed "an
immunogenically effective amount." Thus, the vaccines of the
present invention will contain an immunogenically effective amount
of the polypeptides of the present invention. The immune response
of the patient may include generation of antibodies, activation of
cytotoxic T-lymphocytes against cells expressing the polypeptides,
e.g., PE, or other mechanisms known to the skilled artisan. See,
e.g., Paul, Fundamental Immunology, 2d Edition, Raven Press. Useful
carriers are well known in the art, and include for example,
thyroglobulin, albumins such as human serum albumin, tetanus
toxoid, polyamino acids such as poly(D-lysine; D-glutamic acid),
influenza, hepatitis B virus core protein, hepatitis B virus
recombinant vaccine. The vaccines can also contain a
physiologically tolerable diluent, such as water, buffered water,
buffered saline, saline and typically may further include an
adjuvant, such as incomplete Freunds adjuvant, aluminum phosphate,
aluminum hydroxide, alum, or other materials well known in the
art.
[1416] Alternatively, the nucleic acids of the present invention
may also be used as vaccines for the prevention of malaria
symptoms, and/or infection by malaria parasites. See Sedegah, et
al. Proc. Nat'l Acad. Sci. (1994) 91:9866-9870.
[1417] For example, plasmid DNA comprising the nucleic acids of the
present invention may be directly administered to a patient.
Expression of this "naked" DNA will have effects similar to the
injection of. the actual polypeptides, as described above.
Specifically, the patient's immune response to the presence of the
proteins expressed from the DNA, will result in the production of
antibodies to that protein. The nucleic acids may also be used to
design antisense probes to interrupt transcription of PfEMP1
peptides in parasitized erythocytes.
[1418] Antisense methods are generally well known in the art. The
polypeptides of the present invention, and analogs thereof, may
also be used as prophylactic treatments to prevent the onset of
symptoms of malarial infection. For example, administration of the
polypeptides can directly inhibit, block or reverse the
sequestration of erythrocytes in patients suffering from P.
falciparuin malaria infections. In particular, the polypeptides of
the invention may be used to compete with or displace PE associated
PfEMP1 in binding CD36.
[1419] The blockage or reversal of sequestration will reduce or
eliminate the microvascular occlusion generally associated with the
pathology of this type of malaria, which, again, can lead to
destruction of the PE by the host. The antibodies of the invention
may also be used in a similar fashion. In particular, the
antibodies, which are capable of binding the polypeptides of the
present invention, may be directly administered to a patient. By
binding PfEMP1, the antibodies of the present invention are
effective in blocking, reducing or reversing PfEMP1 mediated
interactions, e.g., erythrocyte sequestration. Chimeric, human-like
or humanized antibodies are particularly useful for administration
to human patients. Additionally, such antibodies may also be used
as a passive vaccination method to provide a subject with a short
term immunization, much as anti-hepatitis A injections have been
used previously.
[1420] In alternative aspects, the polypeptides, antibodies and
nucleic acids of the invention may be used to treat a patient
already suffering from a malarial infection. In particular, the
compositions of the present invention may be administered to a
patient suffering from a malarial infection to treat symptoms
associated with that infection. More particularly, these
compositions may be administered to the patient to prevent or
reduce erythrocyte sequestration and the resulting microvascular
occlusion associated with malarial, and more specifically, P.
falciparum, infections.
[1421] Although the polypeptides, nucleic acids and antibodies of
the present invention may be administered alone, for therapeutic
and prophylactic applications, these elements will generally be
administered as part of a pharmaceutical composition, e.g., in
combination with a pharmaceutically acceptable carrier. Typically,
a single composition may be used in both therapeutic and
prophylactic applications. Pharmaceutical formulations suitable for
use in the present invention are generally described in Remington's
Pharmaceutical Sciences, Mack Publishing Co., 17th ed. (1985).
[1422] The pharmaceutical compositions of the present invention are
intended for parenteral, topical, oral, or local administration.
Where the pharmaceutical compositions are administered
parenterally, the invention provides pharmaceutical compositions
that comprise a solution of the agents described above, e.g.,
polypeptides of the invention, dissolved or suspended in a
pharmaceutically acceptable carrier, preferably an aqueous carrier.
A variety of aqueous carriers may be used, e.g., water, buffered
water, saline glycine, and the like. These compositions may be
sterilized by conventional, well known methods, e.g., sterile
filtration. The resulting aqueous solutions may be packaged for use
as is, or lyophilized for combination with a sterile solution prior
to administration. The compositions may contain pharmaceutically
acceptable auxiliary substances as required to approximate
physiological conditions, such as pH adjusting and buffering
agents, tonicity adjusting agents, wetting agents, and the like,
for example sodium acetate, sodium lactate, sodium chloride,
potassium chloride, calcium chloride, sorbitan monolaurate,
triethanolamine oleate,.etc.
[1423] For solid compositions, conventional nontoxic solid carriers
may be used which include, for example, pharmaceutical grades of
mannitol, lactose starch, magnesium stearate, sodium saccharin,
talcum, cellulose, glucose, sucrose, magnesium carbonate, and the
like. For oral administration, a pharmaceutically acceptable
nontoxic composition may be formed by incorporating any of the
normally employed excipients, such as the previously listed
carriers, and generally, 10-95% of active ingredient, and more
preferably 25-75% active ingredient. In addition, for oral
administration of peptide based compounds, the pharmaceutical
compositions may include the active ingredient as part of a matrix
to prevent proteolytic degradation of the active ingredient by
digestive process, e.g., by providing the pharmaceutical
composition within a liposomal composition, according to methods
well known in the art. See, e.g., Remington's Pharmaceutical
Sciences, Mack Publishing Co., 17th Ed. (1985).
[1424] For aerosol administration, the polypeptides are generally
supplied in finely divided form along with a surfactant or
propellant. Preferably, the surfactant will be soluble in the
propellant. Representative of such agents are the esters or partial
esters of fatty acids containing from 6 to 22 carbon atoms, such as
caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic,
olesteric and oleic acids, with an aliphatic polyhydric alcohol or
its cyclic anhydride. Mixed esters, such as mixed or natural
glycerides may be employed. A carrier can also be included, as
desired, as with, e.g., lecithin for intranasal delivery. The above
described compositions are suitable for a single administration or
a series of administrations. When given as a series, e.g., as a
vaccine booster, the inoculations subsequent to the initial
administration are given to boost the immune response, and are
typically referred to as booster inoculations.
[1425] The amount of the above compositions to be administered to
the patient will vary depending upon what is to be administered to
the patient, the state of the patient, the manner of
administration, and the particular application, e.g., therapeutic
or prophylactic. In therapeutic applications, the compositions are
administered to the patient already suffering from a malarial
infection, in an amount sufficient to inhibit the spread of the
parasite through the erythrocytes, and thereby cure or at least
partially arrest the symptoms of the disease and its associated
complications.
[1426] An amount adequate to accomplish this is termed "a
therapeutically effective amount." Amounts effective for this use
will depend upon the severity of the disease and the weight and
general state of the patient, but will generally be in the range of
from about 1 mg to about 5 g of active agent per day, preferably
from about 50 mg per day to about 500 mg per day, and more
preferably, from about 50 mg to about 100 mg per day, for a 70 kg
patient.
[1427] For prophylactic applications, immunogenically effective
amounts will also depend upon the composition, the manner of
administration and the weight and general state of the patient, as
well as the judgment of the prescribing physician. For the peptide,
peptide analog and antibody based pharmaceutical compositions, the
general range for the initial immunization (for either prophylactic
or therapeutic applications) will be from about 100 .mu.g to about
1 g of polypeptide for a 70 kg patient, followed by boosting
dosages of from about 1 .mu.g to about 1 gm of polypeptide pursuant
to a boosting regimen over weeks to months, depending upon the
patient's response and condition, e.g., by measuring the level of
parasite or antibodies in the patient's blood. For nucleic acids,
typically from about 30 to about 100 .mu.g of nucleic acid is
injected into a 70 kg patient, more typically, about 50 to 150
.mu.g of nucleic acid is injected, followed by boosting treatments
as appropriate.
[1428] The present invention is further illustrated by the
following examples. These examples are merely to illustrate aspects
of the present invention and are not intended as limitations of
this invention.
2.11. DIRECTED EVOLUTION METHODS
[1429] In one aspect the invention described herein is directed to
the use of repeated cycles of reductive reassortment, recombination
and selection which allow for the directed molecular evolution of
highly complex linear sequences, such as DNA, RNA or proteins
thorough recombination.
[1430] In vivo shuffling of molecules can be performed utilizing
the natural property of cells to recombine multimers. While
recombination in vivo has provided the major natural route to
molecular diversity, genetic recombination remains a relatively
complex process that involves 1) the recognition of homologies; 2)
strand cleavage, strand invasion, and metabolic steps leading to
the production of recombinant chiasma; and finally 3) the
resolution of chiasma into discrete recombined molecules. The
formation of the chiasma requires the recognition of homologous
sequences.
[1431] In a preferred embodiment, the invention relates to a method
for producing a hybrid polynucleotide from at least a first
polynucleotide and a second polynucleotide. The present invention
can be used to produce a hybrid polynucleotide by introducing at
least a first polynucleotide and a second polynucleotide which
share at least one region of partial sequence homology into a
suitable host cell. The regions of partial sequence homology
promote processes which result in sequence reorganization producing
a hybrid polynucleotide. The term "hybrid polynucleotide", as used
herein, is any nucleotide sequence which results from the method of
the present invention and contains sequence from at least two
original polynucleotide sequences. Such hybrid polynucleotides can
result from intermolecular recombination events which promote
sequence integration between DNA molecules. In addition, such
hybrid polynucleotides can result from intramolecular reductive
reassortment processes which utilize repeated sequences to alter a
nucleotide sequence within a DNA molecule.
[1432] The invention provides a means for generating hybrid
polynucleotides which may encode biologically active hybrid
polypeptides. In one aspect, the original polynucleotides encode
biologically active polypeptides. The method of the invention
produces new hybrid polypeptides by utilizing cellular processes
which integrate the sequence of the original polynucleotides such
that the resulting hybrid polynucleotide encodes a polypeptide
demonstrating activities derived from the original biologically
active polypeptides. For example, the original polynucleotides may
encode a particular enzyme from different microorganisms. An enzyme
encoded by a first polynucleotide from one organism may, for
example, function effectively under a particular environmental
condition, e.g. high salinity. An enzyme encoded by a second
polynucleotide from a different organism may function effectively
under a different environmental condition, such as extremely high
temperatures. A hybrid polynucleotide containing sequences from the
first and second original polynucleotides may encode an enzyme
which exhibits characteristics of both enzymes encoded by the
original polynucleotides. Thus, the enzyme encoded by the hybrid
polynucleotide may function effectively under environmental
conditions shared by each of the enzymes encoded by the first and
second polynucleotides, e.g., high salinity and extreme
temperatures.
[1433] Enzymes encoded by the original polynucleotides of the
invention include, but are not limited to; oxidoreductases,
transferases, hydrolases, lyases, isomerases and ligases. A hybrid
polypeptide resulting from the method of the invention may exhibit
specialized enzyme activity not displayed in the original enzymes.
For example, following recombination and/or reductive reassortment
of polynucleotides encoding hydrolase activities, the resulting
hybrid polypeptide encoded by a hybrid polynucleotide can be
screened for specialized hydrolase activities obtained from each of
the original enzymes, i.e. the type of bond on which the hydrolase
acts and the temperature at which the hydrolase functions. Thus,
for example, the hydrolase may be screened to ascertain those
chemical functionalities which distinguish the hybrid hydrolase
from the original hydrolyases, such as: (a) amide (peptide bonds),
i.e. proteases; (b) ester bonds, i.e. esterases and lipases; (c)
acetals, i.e., glycosidases and, for example, the temperature, pH
or salt concentration at which the hybrid polypeptide
functions.
[1434] Sources of the original polynucleotides may be isolated from
individual organisms ("isolates"), collections of organisms that
have been grown in defined media ("enrichment cultures"), or, most
preferably, uncultivated organisms ("environmental samples"). The
use of a culture-independent approach to derive polynucleotides
encoding novel bioactivities from environmental samples is most
preferable since it allows one to access untapped resources of
biodiversity.
[1435] "Environmental libraries" are generated from environmental
samples and represent the collective genomes of naturally occurring
organisms archived in cloning vectors that can be propagated in
suitable prokaryotic hosts. Because the cloned DNA is initially
extracted directly from environmental samples, the libraries are
not limited to the small fraction of prokaryotes that can be grown
in pure culture. Additionally, a normalization of the environmental
DNA present in these samples could allow more equal representation
of the DNA from all of the species present in the original sample.
This can dramatically increase the efficiency of finding
interesting genes from minor constituents of the sample which may
be under-represented by several orders of magnitude compared to the
dominant species.
[1436] For example, gene libraries generated from one or more
uncultivated microorganisms are screened for an activity of
interest. Potential pathways encoding bioactive molecules of
interest are first captured in prokaryotic cells in the form of
gene expression libraries. Polynucleotides encoding activities of
interest are isolated from such libraries and introduced into a
host cell. The host cell is grown under conditions which promote
recombination and/or reductive reassortment creating potentially
active biomolecules with novel or enhanced activities.
[1437] The microorganisms from which the polynucleotide may be
prepared include prokaryotic microorganisms, such as Eubacteria and
Archaebacteria, and lower eukaryotic microorganisms such as fungi,
some algae and protozoa. Polynucleotides may be isolated from
environmental samples in which case the nucleic acid may be
recovered without culturing of an organism or recovered from one or
more cultured organisms. In one aspect, such microorganisms may be
extremophiles, such as hyperthermophiles, psychrophiles,
psychrotrophs, halophiles, barophiles and acidophiles.
Polynucleotides encoding enzymes isolated from extremophilic
microorganisms are particularly preferred. Such enzymes may
function at temperatures above 100.degree. C. in terrestrial hot
springs and deep sea thermal vents, at temperatures below 0.degree.
C. in arctic waters, in the saturated salt environment of the Dead
Sea, at pH values around 0 in coal deposits and geothermal
sulfur-rich springs, or at pH values greater than 11 in sewage
sludge. For example, several esterases and lipases cloned and
expressed from extremophilic organisms show high activity
throughout a wide range of temperatures and pHs.
[1438] Polynucleotides selected and isolated as hereinabove
described are introduced into a suitable host cell. A suitable host
cell is any cell which is capable of promoting recombination and/or
reductive reassortment. The selected polynucleotides are preferably
already in a vector which includes appropriate control sequences.
The host cell can be a higher eukaryotic cell, such as a mammalian
cell, or a lower eukaryotic cell, such as a yeast cell, or
preferably, the host cell can be a prokaryotic cell, such as a
bacterial cell. Introduction of the construct into the host cell
can be effected by calcium phosphate transfection, DEAE-Dextran
mediated transfection, or electroporation (Davis et al, 1986).
[1439] As representative examples of appropriate hosts, there may
be mentioned: bacterial cells, such as E. coli, Streptomyces,
Salmonella typhimurium; fungal cells, such as yeast; insect cells
such as Drosophila S2 and Spodoptera S.function.9; animal cells
such as CHO, COS or Bowes melanoma; adenoviruses; and plant cells.
The selection of an appropriate host is deemed to be within the
scope of those skilled in the art from the teachings herein.
[1440] With particular references to various mammalian cell culture
systems that can be employed to express recombinant protein,
examples of mammalian expression systems include the COS-7 lines of
monkey kidney fibroblasts, described in "SV40-transformed simian
cells support the replication of early SV40 mutants" (Gluzman,
1981), and other cell lines capable of expressing a compatible
vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
Mammalian expression vectors will comprise an origin of
replication, a suitable promoter and enhancer, and also any
necessary ribosome binding sites, polyadenylation site, splice
donor and acceptor sites, transcriptional termination sequences,
and 5' flanking nontranscribed sequences. DNA sequences derived
from the SV40 splice, and polyadenylation sites may be used to
provide the required nontranscribed genetic elements.
[1441] Host cells containing the polynucleotides of interest can be
cultured in conventional nutrient media modified as appropriate for
activating promoters, selecting transformants or amplifying genes.
The culture conditions, such as temperature, pH and the like, are
those previously used with the host cell selected for expression,
and will be apparent to the ordinarily skilled artisan. The clones
which are identified as having the specified enzyme activity may
then be sequenced to identify the polynucleotide sequence encoding
an enzyme having the enhanced activity.
[1442] In another aspect, it is envisioned the method of the
present invention can be used to generate novel polynucleotides
encoding biochemical pathways from one or more operons or gene
clusters or portions thereof. For example, bacteria and many
eukaryotes have a coordinated mechanism for regulating genes whose
products are involved in related processes. The genes are
clustered, in structures referred to as "gene clusters," on a
single chromosome and are transcribed together under the control of
a single regulatory sequence, including a single promoter which
initiates transcription of the entire cluster. Thus, a gene cluster
is a group of adjacent genes that are either identical or related,
usually as to their function. An example of a biochemical pathway
encoded by gene clusters are polyketides. Polyketides are molecules
which are an extremely rich source of bioactivities, including
antibiotics (such as tetracyclines and erythromycin), anti-cancer
agents (daunomycin), immunosuppressants (FK506 and rapamycin), and
veterinary products (monensin). Many polyketides (produced by
polyketide synthases) are valuable as therapeutic agents.
Polyketide synthases are multifunctional enzymes that catalyze the
biosynthesis of an enormous variety of carbon chains differing in
length and patterns of functionality and cyclization. Polyketide
synthase genes fall into gene clusters and at least one type
(designated type I) of polyketide synthases have large size genes
and enzymes, complicating genetic manipulation and in vitro studies
of these genes/proteins.
[1443] The ability to select and combine desired components from a
library of polyketides, or fragments thereof, and postpolyketide
biosynthesis genes for generation of novel polyketides for study is
appealing. The method of the present invention makes it possible to
facilitate the production of novel polyketide synthases through
intermolecular recombination.
[1444] Preferably, gene cluster DNA can be isolated from different
organisms and ligated into vectors, particularly vectors containing
expression regulatory sequences which can control and regulate the
production of a detectable protein or protein-related array
activity from the ligated gene clusters. Use of vectors which have
an exceptionally large capacity for exogenous DNA introduction are
particularly appropriate for use with such gene clusters and are
described by way of example herein to include the f-factor (or
fertility factor) of E. coli. This f-factor of E. coli is a plasmid
which affect high-frequency transfer of itself during conjugation
and is ideal to achieve and stably propagate large DNA fragments,
such as gene clusters from mixed microbial samples. Once ligated
into an appropriate vector, two or more vectors containing
different polyketide synthase gene clusters can be introduced into
a suitable host cell. Regions of partial sequence homology shared
by the gene clusters will promote processes which result in
sequence reorganization resulting in a hybrid gene cluster. The
novel hybrid gene cluster can then be screened for enhanced
activities not found in the original gene clusters.
[1445] Therefore, in a preferred embodiment, the present invention
relates to a method for producing a biologically active hybrid
polypeptide and screening such a polypeptide for enhanced activity
by:
[1446] 1) introducing at least a first polynucleotide in operable
linkage and a second polynucleotide in operable linkage, said at
least first polynucleotide and second polynucleotide sharing at
least one region of partial sequence homology, into a suitable host
cell;
[1447] 2) growing the host cell under conditions which promote
sequence reorganization resulting in a hybrid polynucleotide in
operable linkage;
[1448] 3) expressing a hybrid polypeptide encoded by the hybrid
polynucleotide;
[1449] 4) screening the hybrid polypeptide under conditions which
promote identification of enhanced biological activity; and
[1450] 5) isolating the a polynucleotide encoding the hybrid
polypeptide.
[1451] Methods for screening for various enzyme activities are
known to those of skill in the art and discussed throughout the
present specification. Such methods may be employed when isolating
the polypeptides and polynucleotides of the present invention.
[1452] As representative examples of expression vectors which may
be used there may be mentioned viral particles, baculovirus, phage,
plasmids, phagemids, cosmids, fosmids, bacterial artificial
chromosomes, viral DNA (e.g. vaccinia, adenovirus, foul pox virus,
pseudorabies and derivatives of SV40), P1-based artificial
chromosomes, yeast plasmids, yeast artificial chromosomes, and any
other vectors specific for specific hosts of interest (such as
bacillus, aspergillus and yeast). Thus, for example, the DNA may be
included in any one of a variety of expression vectors for
expressing a polypeptide. Such vectors include chromosomal,
nonchromosomal and synthetic DNA sequences. Large numbers of
suitable vectors are known to those of skill in the art, and are
commercially available. The following vectors are provided by way
of example; Bacterial: pQE vectors (Qiagen), pBluescript plasmids,
pNH vectors, (lambda-ZAP vectors (Stratagene); ptrc99a, pKK223-3,
pDR540, pRIT2T (Pharmacia); Eukaryotic: pXT1, pSG5 (Stratagene),
pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia). However, any other plasmid
or other vector may be used as long as they are replicable and
viable in the host. Low copy number or high copy number vectors may
be employed with the present invention.
[1453] A preferred type of vector for use in the present invention
contains an f-factor origin replication. The f-factor (or fertility
factor) in E. coli is a plasmid which effects high frequency
transfer of itself during conjugation and less frequent transfer of
the bacterial chromosome itself. A particularly preferred
embodiment is to use cloning vectors, referred to as "fosmids" or
bacterial artificial chromosome (BAC) vectors. These are derived
from E. coli f-factor which is able to stably integrate large
segments of genomic DNA. When integrated with DNA from a mixed
uncultured environmental sample, this makes it possible to achieve
large genomic fragments in the form of a stable "environmental DNA
library."
[1454] Another preferred type of vector for use in the present
invention is a cosmid vector. Cosmid vectors were originally
designed to clone and propagate large segments of genomic DNA.
Cloning into cosmid vectors is described in detail in "Molecular
Cloning: A laboratory Manual" (Sambrook et al, 1989).
[1455] The DNA sequence in the expression vector is operatively
linked to an appropriate expression control sequence(s) (promoter)
to direct RNA synthesis. Particular named bacterial promoters
include lac, lacZ, T3, T7, gpt, lambda P.sub.R, P.sub.L and trp.
Eukaryotic promoters include CMV immediate early, HSV thymidine
kinase, early and late SV40, LTRs from retrovirus, and mouse
metallothionein-I. Selection of the appropriate vector and promoter
is well within the level of ordinary skill in the art. The
expression vector also contains a ribosome binding site for
translation initiation and a transcription terminator. The vector
may also include appropriate sequences for amplifying expression.
Promoter regions can be selected from any desired gene using CAT
(chloramphenicol transferase) vectors or other vectors with
selectable markers.
[1456] In addition, the expression vectors preferably contain one
or more selectable marker genes to provide a phenotypic trait for
selection of transformed host cells such as dihydrofolate reductase
or neomycin resistance for eukaryotic cell culture, or such as
tetracycline or ampicillin resistance in E coli.
[1457] Generally, recombinant expression vectors will include
origins of replication and selectable markers permitting
transformation of the host cell, e.g., the ampicillin resistance
gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived
from a highly-expressed gene to direct transcription of a
downstream structural sequence. Such promoters can be derived from
operons encoding glycolytic enzymes such as 3-phosphoglycerate
kinase (PGK), .alpha.-factor, acid phosphatase, or heat shock
proteins, among others. The heterologous structural sequence is
assembled in appropriate phase with translation initiation and
termination sequences, and preferably, a leader sequence capable of
directing secretion of translated protein into the periplasmic
space or extracellular medium.
[1458] The cloning strategy permits expression via both vector
driven and endogenous promoters; vector promotion may be important
with expression of genes whose endogenous promoter will not
function in E coli.
[1459] The DNA isolated or derived from microorganisms can
preferably be inserted into a vector or a plasmid prior to probing
for selected DNA. Such vectors or plasmids are preferably those
containing expression regulatory sequences, including promoters,
enhancers and the like. Such polynucleotides can be part of a
vector and/or a composition and still be isolated, in that such
vector or composition is not part of its natural environment.
Particularly preferred phage or plasmid and methods for
introduction and packaging into them are described in detail in the
protocol set forth herein.
[1460] The selection of the cloning vector depends upon the
approach taken, for example, the vector can be any cloning vector
with an adequate capacity for multiply repeated copies of a
sequence, or multiple sequences that can be successfully
transformed and selected in a host cell. One example of such a
vector is described in "Polycos vectors: a system for packaging
filamentous phage and phagemid vectors using lambda phage packaging
extracts" (Alting-Mecs and Short, 1993). Propagation/maintenance
can be by an antibiotic resistance carried by the cloning vector.
After a period of growth, the naturally abbreviated molecules are
recovered and identified by size fractionation on a gel or column,
or amplified directly. The cloning vector utilized may contain a
selectable gene that is disrupted by the insertion of the lengthy
construct. As reductive reassortment progresses, the number of
repeated units is reduced and the interrupted gene is again
expressed and hence selection for the processed construct can be
applied. The vector may be an expression/selection vector which
will allow for the selection of an expressed product possessing
desirable biologically properties. The insert may be positioned
downstream of a functional promotor and the desirable property
screened by appropriate means.
[1461] In vivo reassortment is focused on "inter-molecular"
processes collectively referred to as "recombination" which in
bacteria, is generally viewed as a "RecA-dependent" phenomenon. The
present invention can rely on recombination processes of a host
cell to recombine and re-assort sequences, or the cells' ability to
mediate reductive processes to decrease the complexity of
quasi-repeated sequences in the cell by deletion. This process of
"reductive reassortment" occurs by an "intra-molecular",
RecA-independent process.
[1462] Therefore, in another aspect of the present invention, novel
polynucleotides can be generated by the process of reductive
reassortment. The method involves the generation of constructs
containing consecutive sequences (original encoding sequences),
their insertion into an appropriate vector, and their subsequent
introduction into an appropriate host cell. The reassortment of the
individual molecular identities occurs by combinatorial processes
between the consecutive sequences in the construct possessing
regions of homology, or between quasi-repeated units. The
reassortment process recombines and/or reduces the complexity and
extent of the repeated sequences, and results in the production of
novel molecular species. Various treatments may be applied to
enhance the rate of reassortment. These could include treatment
with ultra-violet light, or DNA damaging chemicals, and/or the use
of host cell lines displaying enhanced levels of "genetic
instability". Thus the reassortment process may involve homologous
recombination or the natural property of quasi-repeated sequences
to direct their own evolution.
[1463] Repeated or "quasi-repeated" sequences play a role in
genetic instability. In the present invention, "quasi-repeats" are
repeats that are not restricted to their original unit structure.
Quasi-repeated units can be presented as an array of sequences in a
construct; consecutive units of similar sequences. Once ligated,
the junctions between the consecutive sequences become essentially
invisible and the quasi-repetitive nature of the resulting
construct is now continuous at the molecular level. The deletion
process the cell performs to reduce the complexity of the resulting
construct operates between the quasi-repeated sequences. The
quasi-repeated units provide a practically limitless repertoire of
templates upon which slippage events can occur. The constructs
containing the quasi-repeats thus effectively provide sufficient
molecular elasticity that deletion (and potentially insertion)
events can occur virtually anywhere within the quasi-repetitive
units.
[1464] When the quasi-repeated sequences are all ligated in the
same orientation, for instance head to tail or vice versa, the cell
cannot distinguish individual units. Consequently, the reductive
process can occur throughout the sequences. In contrast, when for
example, the units are presented head to head, rather than head to
tail, the inversion delineates the endpoints of the adjacent unit
so that deletion formation will favor the loss of discrete units.
Thus, it is preferable with the present method that the sequences
are in the same orientation. Random orientation of quasi-repeated
sequences will result in the loss of reassortment efficiency, while
consistent orientation of the sequences will offer the highest
efficiency. However, while having fewer of the contiguous sequences
in the same orientation decreases the efficiency, it may still
provide sufficient elasticity for the effective recovery of novel
molecules. Constructs can be made with the quasi-repeated sequences
in the same orientation to allow higher efficiency.
[1465] Sequences can be assembled in a head to tail orientation
using any of a variety of methods, including the following:
[1466] a) Primers that include a poly-A head and poly-T tail which
when made single-stranded would provide orientation can be
utilized. This is accomplished by having the first few bases of the
primers made from RNA and hence easily removed RNAseH.
[1467] b) Primers that include unique restriction cleavage sites
can be utilized. Multiple sites, a battery of unique sequences, and
repeated synthesis and ligation steps would be required.
[1468] c) The inner few bases of the primer could be thiolated and
an exonuclease used to produce properly tailed molecules.
[1469] The recovery of the re-assorted sequences relies on the
identification of cloning vectors with a reduced RI. The
re-assorted encoding sequences can then be recovered by
amplification. The products are re-cloned and expressed. The
recovery of cloning vectors with reduced RI can be effected by:
[1470] 1) The use of vectors only stably maintained when the
construct is reduced-in complexity.
[1471] 2) The physical recovery of shortened vectors by physical
procedures. In this case, the cloning vector would be recovered
using standard plasmid isolation procedures and size fractionated
on either an agarose gel, or column with a low molecular weight cut
off utilizing standard procedures.
[1472] 3) The recovery of vectors containing interrupted genes
which can be selected when insert size decreases.
[1473] 4) The use of direct selection techniques with an expression
vector and the appropriate selection.
[1474] Encoding sequences (for example, genes) from related
organisms may demonstrate a high degree of homology and encode
quite diverse protein products. These types of sequences are
particularly useful in the present invention as quasi-repeats.
However, while the examples illustrated below demonstrate the
reassortment of nearly identical original encoding sequences
(quasi-repeats), this process is not limited to such nearly
identical repeats.
[1475] The following example demonstrates the method of the
invention. Encoding nucleic acid sequences (quasi-repeats) derived
from three (3) unique species are depicted. Each sequence encodes a
protein with a distinct set of properties. Each of the sequences
differs by a single or a few base pairs at a unique position in the
sequence which are designated "A", "B" and "C". The quasi-repeated
sequences are separately or collectively amplified and ligated into
random assemblies such that all possible permutations and
combinations are available in the population of ligated molecules.
The number of quasi-repeat units can be controlled by the assembly
conditions. The average number of quasi-repeated units in a
construct is defined as the repetitive index (RI).
[1476] Once formed, the constructs may, or may not be size
fractionated on an agarose gel according to published protocols,
inserted into a cloning vector, and transfected into an appropriate
host cell. The cells are then propagated and "reductive
reassortment" is effected. The rate of the reductive reassortment
process may be stimulated by the introduction of DNA damage if
desired. Whether the reduction in RI is mediated by deletion
formation between repeated sequences by an "intra-molecular"
mechanism, or mediated by recombination-like events through
"inter-molecular" mechanisms is immaterial. The end result is a
reassortment of the molecules into all possible combinations.
[1477] Optionally, the method comprises the additional step of
screening the library members of the shuffled pool to identify
individual shuffled library members having the ability to bind or
otherwise interact (e.g., such as catalytic antibodies) with a
predetermined macromolecule, such as for example a proteinaceous
receptor, peptide oligosaccharide, viron, or other predetermined
compound or structure.
[1478] The displayed polypeptides, antibodies, peptidomimetic
antibodies, and variable region sequences that are identified from
such libraries can be used for therapeutic, diagnostic, research
and related purposes (e.g., catalysts, solutes for increasing
osmolarity of an aqueous solution, and the like), and/or can be
subjected to one or more additional cycles of shuffling and/or
affinity selection. The method can be modified such that the step
of selecting for a phenotypic characteristic can be other than of
binding affinity for a predetermined molecule (e.g., for catalytic
activity, stability oxidation resistance, drug resistance, or
detectable phenotype conferred upon a host cell).
[1479] The present invention provides a method for generating
libraries of displayed antibodies suitable for affinity
interactions screening. The method comprises (1) obtaining first a
plurality of selected library members comprising a displayed
antibody and an associated polynucleotide encoding said displayed
antibody, and obtaining said associated polynucleotide encoding for
said displayed antibody and obtaining said associated
polynucleotides or copies thereof, wherein said associated
polynucleotides comprise a region of substantially identical
variable region framework sequence, and (2) introducing said
polynucleotides into a suitable host cell and growing the cells
under conditions which promote recombination and reductive
reassortment resulting in shuffled polynucleotides. CDR
combinations comprised by the shuffled pool are not present in the
first plurality of selected library members, said shuffled pool
composing a library of displayed antibodies comprising CDR
permutations and suitable for affinity interaction screening.
Optionally, the shuffled pool is subjected to affinity screening to
select shuffled library members which bind to a predetermined
epitope (antigen) and thereby selecting a plurality of selected
shuffled library members. Further, the plurality of selectively
shuffled library members can be shuffled and screened iteratively,
from 1 to about 1000 cycles or as desired until library members
having a desired binding affinity are obtained.
[1480] In another aspect of the invention, it is envisioned that
prior to or during recombination or reassortment, polynucleotides
generated by the method of the present invention can be subjected
to agents or processes which promote the introduction of mutations
into the original polynucleotides. The introduction of such
mutations would increase the diversity of resulting hybrid
polynucleotides and polypeptides encoded therefrom. The agents or
processes which promote mutagenesis can include, but are not
limited to: (+)-CC-1065, or a synthetic analog such as
(+)-CC-1065-(N3-Adenine, see Sun and Hurley, 1992); an N-acelylated
or deacetylated 4'-fluro-4-aminobiphenyl adduct capable of
inhibiting DNA synthesis (see, for example, van de Poll et al,
1992); or a N-acetylated or deacetylated 4-aminobiphenyl adduct
capable of inhibiting DNA synthesis (see also, van de Poll et al,
1992, pp. 751-758); trivalent chromium, a trivalent chromium salt,
a polycyclic aromatic hydrocarbon ("PAH") DNA adduct capable of
inhibiting DNA replication, such as
7-bromomethyl-benz[.alpha.]anthracene ("BMA"),
tris(2,3-dibromopropyl)pho- sphate ("Tris-BP"),
1,2-dibromo-3-chloropropane ("DBCP"), 2-bromoacrolein (2BA),
benzo[.alpha.]pyrene-7,8-dihydrodiol-9-10-epoxide ("BPDE"), a
platinum(II) halogen salt,
N-hydroxy-2-amino-3-methylimidazo[4,5-.functio- n.]-quinoline
("N-hydroxy-IQ"), and N-hydroxy-2-amino-1-methyl-6-phenylimi-
dazo[4,5-.function.]-pyridine ("N-hydroxy-PhIP"). Especially
preferred "means for slowing or halting PCR amplification consist
of UV light (+)-CC-1065 and (+)-CC-1065-(N3-Adenine). Particularly
encompassed means are DNA adducts or polynucleotides comprising the
DNA adducts from the polynucleotides or polynucleotides pool, which
can be released or removed by a process including heating the
solution comprising the polynucleotides prior to further
processing.
[1481] In another aspect the present invention is directed to a
method of producing recombinant proteins having biological activity
by treating a sample comprising double-stranded template
polynucleotides encoding a wild-type protein under conditions
according to the present invention which provide for the production
of hybrid or re-assorted polynucleotides.
[1482] The invention also provides the use of polynucleotide
shuffling to shuffle a population of viral genes (e.g., capsid
proteins, spike glycoproteins, polymerases, and proteases) or viral
genomes (e.g., paramyxoviridae, orthomyxoviridae, herpesviruses,
retroviruses, reoviruses and rhinoviruses). In an embodiment, the
invention provides a method for shuffling sequences encoding all or
portions of immunogenic viral proteins to generate novel
combinations of epitopes as well as novel epitopes created by
recombination; such shuffled viral proteins may comprise epitopes
or combinations of epitopes as well as novel epitopes created by
recombination; such shuffled viral proteins may comprise epitopes
or combinations of epitopes which are likely to arise in the
natural environment as a consequence of viral evolution; (e.g.,
such as recombination of influenza virus strains).
[1483] The invention also provides a method suitable for shuffling
polynucleotide sequences for generating gene therapy vectors and
replication-defective gene therapy constructs, such as may be used
for human gene therapy, including but not limited to vaccination
vectors for DNA-based vaccination, as well as anti-neoplastic gene
therapy and other general therapy formats.
[1484] In the polypeptide notation used herein, the left-hand
direction is the amino terminal direction and the right-hand
direction is the carboxy-terminal direction, in accordance with
standard usage and convention. Similarly, unless specified
otherwise, the left-hand end of single-stranded polynucleotide
sequences is the 5' end; the left-hand direction of double-stranded
polynucleotide sequences is referred to as the 5' direction. The
direction of 5' to 3' addition of nascent RNA transcripts is
referred to as the transcription direction; sequence regions on the
DNA strand having the same sequence as the RNA and which are 5' to
the 5' end of the RNA transcript are referred to as "upstream
sequences"; sequence regions on the DNA strand having the same
sequence as the RNA and which are 3' to the 3' end of the coding
RNA transcript are referred to as "downstream sequences".
2.11.1. SATURATION MUTAGENESIS
[1485] In one aspect, this invention provides for the use of
proprietary codon primers (containing a degenerate N,N,G/T
sequence) to introduce point mutations into a polynucleotide, so as
to generate a set of progeny polypeptides in which a full range of
single amino acid substitutions is represented at each amino acid
position. The oligos used are comprised contiguously of a first
homologous sequence, a degenerate N,N,G/T sequence, and preferably
but not necessarily a second homologous sequence. The downstream
progeny translational products from the use of such oligos include
all possible amino acid changes at each amino acid site along the
polypeptide, because the degeneracy of the N,N,G/T sequence
includes codons for all 20 amino acids.
[1486] In one aspect, one such degenerate oligo (comprised of one
degenerate N,N,G/T cassette) is used for subjecting each original
codon in a parental polynucleotide template to a full range of
codon substitutions. In another aspect, at least two degenerate
N,N,G/T cassettes are used--either in the same oligo or not, for
subjecting at least two original codons in a parental
polynucleotide template to a full range of codon substitutions.
Thus, more than one N,N,G/T sequence can be contained in one oligo
to introduce amino acid mutations at more than one site. This
plurality of N,N,G/T sequences can be directly contiguous, or
separated by one or more additional nucleotide sequence(s). In
another aspect, oligos serviceable for introducing additions and
deletions can be used either alone or in combination with the
codons containing an N,N,G/T sequence, to introduce any combination
or permutation of amino acid additions, deletions, and/or
substitutions.
[1487] In a particular exemplification, it is possible to
simultaneously mutagenize two or more contiguous amino acid
positions using an oligo that contains contiguous N,N,G/T triplets,
i.e. a degenerate (N,N,G/T)n sequence.
[1488] In another aspect, the present invention provides for the
use of degenerate cassettes having less degeneracy than the N,N,G/T
sequence. For example, it may be desirable in some instances to use
(e.g. in an oligo) a degenerate triplet sequence comprised of only
one N, where said N can be in the first second or third position of
the triplet. Any other bases including any combinations and
permutations thereof can be used in the remaining two positions of
the triplet. Alternatively, it may be desirable in some instances
to use (e.g. in an oligo) a degenerate N,N,N triplet sequence, or
an N,N, A/C triplet sequence.
[1489] It is appreciated, however, that the use of a degenerate
N,N,G/T triplet as disclosed in the instant invention is
advantageous for several reasons. In one aspect, this invention
provides a means to systematically and fairly easily generate the
substitution of the full range of possible amino acids (for a total
of 20 amino acids) into each and every amino acid position in a
polypeptide. Thus, for a 100 amino acid polypeptide, the instant
invention provides a way to systematically and fairly easily
generate 2000 distinct species (i.e. 20 possible amino acids per
position.times.100 amino acid positions). It is appreciated that
there is provided, through the use of an oligo containing a
degenerate N,N,G/T triplet, 32 individual sequences that code for
20 possible amino acids. Thus, in a reaction vessel in which a
parental polynucleotide sequence is subjected to saturation
mutagenesis using one such oligo, there are generated 32 distinct
progeny polynucleotides encoding 20 distinct polypeptides. In
contrast, the use of a non-degenerate oligo in site-directed
mutagenesis leads to only one progeny polypeptide product per
reaction vessel.
[1490] This invention also provides for the use of nondegenerate
oligos, which can optionally be used in combination with degenerate
primers disclosed. It is appreciated that in some situations, it is
advantageous to use nondegenerate oligos to generate specific point
mutations in a working polynucleotide. This provides a means to
generate specific silent point mutations, point mutations leading
to corresponding amino acid changes, and point mutations that cause
the generation of stop codons and the corresponding expression of
polypeptide fragments.
[1491] Thus, in a preferred embodiment of this invention, each
saturation mutagenesis reaction vessel contains polynucleotides
encoding at least 20 progeny polypeptide molecules such that all 20
amino acids are represented at the one specific amino acid position
corresponding to the codon position mutagenized in the parental
polynucleotide. The 32-fold degenerate progeny polypeptides
generated from each saturation mutagenesis reaction vessel can be
subjected to clonal amplification (e.g. cloned into a suitable E.
coli host using an expression vector) and subjected to expression
screening. When an individual progeny polypeptide is identified by
screening to display a favorable change in property (when compared
to the parental polypeptide), it can be sequenced to identify the
correspondingly favorable amino acid substitution contained
therein.
[1492] It is appreciated that upon mutagenizing each and every
amino acid position in a parental polypeptide using saturation
mutagenesis as disclosed herein, favorable amino acid changes may
be identified at more than one amino acid position. One or more new
progeny molecules can be generated that contain a combination of
all or part of these favorable amino acid substitutions. For
example, if 2 specific favorable amino acid changes are identified
in each of 3 amino acid positions in a polypeptide, the
permutations include 3 possibilities at each position (no change
from the original amino acid, and each of two favorable changes)
and 3 positions. Thus, there are 3.times.3.times.3 or 27 total
possibilities, including 7 that were previously examined--6 single
point mutations (i.e. 2 at each of three positions) and no change
at any position.
[1493] In yet another aspect, site-saturation mutagenesis can be
used together with shuffling, chimerization, recombination and
other mutagenizing processes, along with screening. This invention
provides for the use of any mutagenizing process(es), including
saturation mutagenesis, in an iterative manner. In one
exemplification, the iterative use of any mutagenizing process(es)
is used in combination with screening.
[1494] Thus, in a non-limiting exemplification, this invention
provides for the use of saturation mutagenesis in combination with
additional mutagenization processes, such as process where two or
more related polynucleotides are introduced into a suitable host
cell such that a hybrid polynucleotide is generated by
recombination and reductive reassortment.
[1495] In addition to performing mutagenesis along the entire
sequence of a gene, the instant invention provides that mutagenesis
can be use to replace each of any number of bases in a
polynucleotide sequence, wherein the number of bases to be
mutagenized is preferably every integer from 6 to 90,000. Thus,
instead of mutagenizing every position along a molecule, one can
subject every a discrete number of bases (preferably totaling from
6 to 90,000) to mutagenesis. Preferably, a separate nucleotide is
used for mutagenizing each position or group of positions along a
polynucleotide sequence. A group of 3 positions to be mutagenized
may be a codon. The mutations are preferably introduced using a
mutagenic primer, containing a heterologous cassette. Preferred
cassettes can have from 1 to 500 bases. Each nucleotide position in
such heterologous cassettes be N, A, C, G, T, A/C, A/G, A/T, C/G,
C/T, G/T, C/G/T, A/G/T, A/C/T, A/C/G, or E, where E is any base
that is not A, C, G, or T (E can be referred to as a designer
oligo). The tables below show exemplary tri-nucleotide cassettes
(there are over 3000 possibilities in addition to N,N,G/T and N,N,N
and N,N,A/C).
[1496] In a general sense, saturation mutagenesis is comprised of
introducing all mutations in a grouping of mutations into each
location ina defined sequence. Such groupings are exemplified by
deletions, additions, groupings of particular codons, and groupings
of particular nucleotide cassettes. Defined sequences are
preferably a whole gene, an entire open reading frame (ORF), and
intire promoter, enhancer, repressor/transactivator, origin of
replicaion, intron, operator, or any polynucleotide functional
group. Additionally preferred "defined sequences" for this purpose
include at least a 15-base polynucleotide sequence, and
polynucleotide sequences of lengths between 15-bases and 15,000
bases (with every integer in named). Considerations in choosing
groupings of codons include types of amino acids encoded by a
degenerate mutagenic cassette.
3 TABLE # triplet sequence Site 1 Site 2 Site 3 1. N,N,G/T N N G/T
2. N,N,G/C N N G/C 3. N,N,G/A N N G/A 4. N,N,A/C N N A/C 5. N,N,A/T
N N A/T 6. N,N,C/T N N C/T 7. N,N,N N N N 8. N,N,G N N G 9. N,N,A N
N A 10. N,N,C N N C 11. N,N,T N N T 12. N,N,C/G/T N N C/G/T 13.
N,N,A/G/T N N A/G/T 14. N,N,A/C/T N N A/C/T 15. N,N,A/C/G N N A/C/G
16. N,A,A N A A 17. N,A,C N A C 18. N,A,G N A G 19. N,A,T N A T 20.
N,C,A N C A 21. N,C,C N C C 22. N,C,G N C G 23. N,C,T N C T 24.
N,G,A N G A 25. N,G,C N G C 26. N,G,G N G G 27. N,G,T N G T 28.
N,T,A N T A 29. N,T,C N T C 30. N,T,G N T G 31. N,T,T N T T 32.
N,A/C,A N A/C A 33. N,A/G,A N A/G A 34. N,A/T,A N A/T A 35. N,C/G,A
N C/G A 36. N,C/T,A N C/T A 37. N,T/G,A N T/G A 38. N,C/G/T,A N
C/G/T A 39. N,A/G/T,A N A/G/T A 40. N,A/C/T,A N A/C/T A 41.
N,A/C/G,A N A/C/G A 42. A,N,N A N N 43. C,N,N C N N 44. G,N,N G N N
45. T,N,N T N N 46. A/C,N,N A/C N N 47. A/C,N,N A/G N N 48. A/T,N,N
A/T N N 49. C/G,N,N C/G N N 50. C/T,N,N C/T N N 51. G/T,N,N G/T N N
52. N,A,N N A N 53. N,C,N N C N 54. N,C,N N G N 55. N,T,N N T N 56.
N,A/C,N N A/C N 57. N,A/C,N N A/G N 58. N,A/T,N N A/T N 59. N,C/C,N
N C/G N 60. N,C/T,N N C/T N 61. N,G/T,N N G/T N 62. N,A/C/G,N N
A/C/G N 63. N,A/C/T,N N A/C/T N 64. N,A/C/T,N N A/G/T N 65.
N,C/G/T,N N C/G/T N 66. C,C,N C C N 67. G,G,N G C N 68. G,C,N G C N
69. G,T,N G T N 70. C,C,N C G N 71. C,T,N C T N 72. T,C,N T C N 73.
A,C,N A C N 74. G,A,N G A N 75. A,T,N A T N 76. C,A,N C A N 77.
T,T,N T T N 78. A,A,N A A N 79. T,A,N T A N 80. T,G,N T G N 81.
A,G,N A G N 82. G/C,C,N G/C G N 83. G/C,C,N G/C C N 84. G/C,A,N G/C
A N 85. G/C,T,N G/C T N
[1497]
4TABLE 1 N, N, G/T CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGT YES GLYCINE 2 NONPOLAR 15 GGC NO (NPL) GGA
NO GGG YES GCT YES ALANINE 2 GCC NO GCA NO GCG YES GTT YES VALINE 2
GTC NO GTA NO GTG YES TTA NO LEUCINE 3 TTG YES CTT YES CTC NO CTA
NO CTG YES ATT YES ISOLEUCINE 1 ATC NO ATA NO ATG YES METHIONINE 1
TTT YES PHENYLALANINE 1 TTC NO TGG YES TRYPTOPHAN 1 CCT YES PROLINE
2 CCC NO CCA NO CCG YES TCT YES SERINE 3 POLAR 9 TCC NO
NONIONIZABLE TCA NO (POL) TCG YES AGT YES AGC NO TGT YES CYSTEINE 1
TGC NO AAT YES ASPARAGINE 1 AAC NO CAA NO GLUTAMINE 1 CAG YES TAT
YES TYROSINE 1 TAC NO ACT YES THREONINE 2 ACC NO ACA NO ACG YES GAT
YES ASPARTIC ACID 1 IONIZABLE: ACIDIC 2 GAC NO NEGATIVE CHARGE GAA
NO GLUTAMIC ACID 1 (NEG) GAG YES AAA NO LYSINE 1 IONIZABLE: BASIC 5
AAG YES POSITIVE CHARGE CGT YES ARGININE 3 (POS) CGC NO CGA NO CGG
YES AGA NO AGG YES CAT YES HISTIDINE 1 CAC NO TAA NO STOP CODON 1
STOP SIGNAL 1 TAG YES (STP) TGA NO TOTAL 64 32 20 Amino Acids Are
Represented NPL: POL: NEG: POS: STP = 15: 9: 2: 5: 1
[1498]
5TABLE 2 N, N, G/C CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGT NO GLYCINE 2 NONPOLAR 15 GGC YES (NPL) GGA
NO GGG YES GCT NO ALANINE 2 GCC YES GCA NO GCG YES GTT NO VALINE 2
GTC YES GTA NO GTG YES TTA NO LEUCINE 3 TTG YES CTT NO CTC YES CTA
NO CTG YES ATT NO ISOLEUCINE 1 ATC YES ATA NO ATG YES METHIONINE 1
TTT NO PHENYLALANINE 1 TTC YES TGG YES TRYPTOPHAN 1 CCT NO PROLINE
2 CCC YES CCA NO CCG YES TCT NO SERINE 3 POLAR 9 TCC YES
NONIONIZABLE TCA NO (POL) TCG YES AGT NO AGC YES TGT NO CYSTEINE 1
TGC YES AAT NO ASPARAGINE 1 AAC YES CAA NO GLUTAMINE 1 CAG YES TAT
NO TYROSINE 1 TAC YES ACT NO THREONINE 2 ACC YES ACA NO ACG YES GAT
NO ASPARTIC ACID 1 IONIZABLE: ACIDIC 2 GAC YES NEGATIVE CHARGE GAA
NO GLUTAMIC ACID 1 (NEC) GAG YES AAA NO LYSINE 1 IONIZABLE: BASIC 5
AAG YES POSITIVE CHARGE CGT NO ARGININE 3 (POS) CGC YES CGA NO CGG
YES AGA NO AGG YES CAT NO HISTIDINE 1 CAC YES TAA NO STOP CODON 1
STOP SIGNAL 1 TAG YES (STP) TGA NO TOTAL 64 32 20 Amino Acids Are
Represented NPL: POL: NEG: POS: STP = 15: 9: 2: 5: 1
[1499]
6TABLE 3 N, N, G/A CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGT NO GLYCINE 2 NONPOLAR 15 GGC NO (NPL) GGA
YES GGG YES GCT NO ALANINE 2 GCC NO GCA YES GCG YES GTT NO VALINE 2
GTC NO GTA YES GTG YES TTA YES LEUCINE 4 TTG YES CTT NO CTC NO CTA
YES CTG YES ATT NO ISOLEUCINE 1 ATC NO ATA YES ATG YES METHIONINE 1
TTT NO PHENYLALANINE 0 TTC NO TGG YES TRYPTOPHAN 1 CCT NO PROLINE 2
CCC NO CCA YES CCG YES TCT NO SERINE 2 POLAR 6 TCC NO NONIONIZABLE
TCA YES (POL) TCG YES AGT NO AGC NO TGT NO CYSTEINE 0 TGC NO AAT NO
ASPARAGINE 0 AAC NO CAA YES GLUTAMINE 2 CAG YES TAT NO TYROSINE 0
TAC NO ACT NO THREONINE 2 ACC NO ACA YES ACG YES GAT NO ASPARTIC
ACID 0 IONIZABLE: ACIDIC 2 GAC NO NEGATIVE CHARGE GAA YES GLUTAMIC
ACID 2 (NEG) GAG YES AAA YES LYSINE 2 IONIZABLE: BASIC 6 AAG YES
POSITIVE CHARGE CGT NO ARGININE 4 (POS) CGC NO CGA YES CGG YES AGA
YES AGG YES CAT NO HISTIDINE 0 CAC NO TAA YES STOP CODON 3 STOP
SIGNAL 3 TAG YES (STP) TGA YES TOTAL 64 32 14 Amino Acids Are
Represented NPL: POL: NEG: POS: STP = 15: 6: 2: 6: 3
[1500]
7TABLE 4 N, N, A/C CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGT NO GLYCINE 2 NONPOLAR 14 GGC YES (NPL) GGA
YES GGG NO GCT NO ALANINE 2 GCC YES GCA YES GCG NO GTT NO VALINE 2
GTC YES GTA YES GTG NO TTA YES LEUCINE 3 TTG NO CTT NO CTC YES CTA
YES CTG NO ATT NO ISOLEUCINE 2 ATC YES ATA YES ATG NO METHIONINE 0
TTT NO PHENYLALANINE 1 TTC YES TGG NO TRYPTOPHAN 0 CCT NO PROLINE 2
CCC YES CCA YES CCG NO TCT NO SERINE 3 POLAR 9 TCC YES NONIONIZABLE
TCA YES (POL) TCG NO AGT NO AGC YES TGT NO CYSTEINE 1 TGC YES AAT
NO ASPARAGINE 1 AAC YES CAA YES GLUTAMINE 1 CAG NO TAT NO TYROSINE
1 TAC YES ACT NO THREONINE 2 ACC YES ACA YES ACG NO GAT NO ASPARTIC
ACID 1 IONIZABLE: ACIDIC 2 GAC YES NEGATIVE CHARGE GAA YES GLUTAMIC
ACID 1 (NEG) GAG NO AAA YES LYSINE 1 IONIZABLE: BASIC 5 AAG NO
POSITIVE CHARGE CGT NO ARGININE 3 (POS) CGC YES CGA YES CGG NO AGA
YES AGG NO CAT NO HISTIDINE 1 CAC YES TAA YES STOP CODON 2 STOP
SIGNAL 2 TAG NO (STP) TGA YES TOTAL 64 32 18 Amino Acids Are
Represented NPL: POL: NEG: POS: STP = 14: 9: 2: 5: 2
[1501]
8TABLE 5 N, N, A/T CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGT YES GLYCINE 2 NONPOLAR 14 GGC NO (NPL) GGA
YES GGG NO GCT YES ALANINE 2 GCC NO GCA YES GCG NO GTT YES VALINE 2
GTC NO GTA YES GTG NO TTA YES LEUCINE 3 TTG NO CTT YES CTC NO CTA
YES CTG NO ATT YES ISOLEUCINE 2 ATC NO ATA YES ATG NO METHIONINE 0
TTT YES PHENYLALANINE 1 TTC NO TGG NO TRYPTOPHAN 0 CCT YES PROLINE
2 CCC NO CCA YES CCG NO TCT YES SERINE 3 POLAR 9 TCC NO
NONIONIZABLE TCA YES (POL) TCG NO AGT YES AGC NO TGT YES CYSTEINE 1
TGC NO AAT YES ASPARAGINE 1 AAC NO CAA YES GLUTAMINE 1 CAG NO TAT
YES TYROSINE 1 TAG NO ACT YES THREONINE 2 ACC NO ACA YES ACG NO GAT
YES ASPARTIC ACID 1 IONIZABLE: ACIDIC 2 GAC NO NEGATIVE CHARGE GAA
YES GLUTAMIC ACID 1 (NEG) GAG NO AAA YES LYSINE 1 IONIZABLE: BASIC
5 AAG NO POSITIVE CHARGE CGT YES ARGININE 3 (POS) CGC NO CGA YES
CGG NO AGA YES AGG NO CAT YES HISTIDINE 1 CAC NO TAA YES STOP CODON
2 STOP SIGNAL 2 TAG NO (STP) TGA YES TOTAL 64 32 18 Amino Acids Are
Represented NPL: POL: NEG: POS: STP = 14: 9: 2: 5: 2
[1502]
9TABLE 6 N, N, C/T CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGT YES GLYCINE 2 NONPOLAR 14 GGC YES (NPL)
GGA NO GGG NO GCT YES ALANINE 2 GCC YES GCA NO GCG NO GTT YES
VALINE 2 GTC YES GTA NO GTG NO TTA NO LEUCINE 2 TTG NO CTT YES CTC
YES CTA NO CTG NO ATT YES ISOLEUCINE 2 ATC YES ATA NO ATG NO
METHIONINE 0 TTT YES PHENYLALANINE 2 TTC YES TGG NO TRYPTOPHAN 0
CCT YES PROLINE 2 CCC YES CCA NO CCG NO TCT YES SERINE 4 POLAR 12
TCC YES NONIONIZABLE TCA NO (POL) TCG NO AGT YES AGC YES TGT YES
CYSTEINE 2 TGC YES AAT YES ASPARAGINE 2 AAC YES CAA NO GLUTAMINE 0
CAG NO TAT YES TYROSINE 2 TAC YES ACT YES THREONINE 2 ACC YES ACA
NO ACG NO GAT YES ASPARTIC ACID 2 IONIZABLE: ACIDIC 2 GAC YES
NEGATIVE CHARGE GAA NO GLUTAMIC ACID 0 (NEG) GAG NO AAA NO LYSINE 0
IONIZABLE: BASIC 4 AAG NO POSITIVE CHARGE CGT YES ARGININE 2 (POS)
CGC YES CGA NO CGG NO AGA NO AGG NO CAT YES HISTIDINE 2 CAC YES TAA
NO STOP CODON 0 STOP SIGNAL 0 TAG NO (STP) TGA NO TOTAL 64 32 15
Amino Acids Are Represented NPL: POL: NEG: POS: STP = 14: 12: 2: 4:
0
[1503]
10TABLE 7 N, N, N CODON Represented AMINO ACID (Frequency) CATEGORY
(Frequency) GGT YES GLYCINE 4 NONPOLAR 29 GGC YES (NPL) GGA YES GGG
YES GCT YES ALANINE 4 GCC YES GCA YES GCG YES GTT YES VALINE 4 GTC
YES GTA YES GTG YES TTA YES LEUCINE 6 TTG YES CTT YES CTC YES CTA
YES CTG YES ATT YES ISOLEUCINE 3 ATC YES ATA YES ATG YES METHIONINE
1 TTT YES PHENYLALANINE 2 TTC YES TGG YES TRYPTOPHAN 1 CCT YES
PROLINE 4 CCC YES CCA YES CCG YES TCT YES SERINE 6 POLAR 18 TCC YES
NONIONIZABLE TCA YES (POL) TCG YES ACT YES AGC YES TGT YES CYSTEINE
2 TGC YES AAT YES ASPARAGINE 2 AAC YES CAA YES GLUTAMINE 2 CAG YES
TAT YES TYROSINE 2 TAC YES ACT YES THREONINE 4 ACC YES ACA YES ACG
YES GAT YES ASPARTIC ACID 2 IONIZABLE: ACIDIC 4 GAC YES NEGATIVE
CHARGE GAA YES GLUTAMIC ACID 2 (NEG) GAG YES AAA YES LYSINE 2
IONIZABLE: BASIC 10 AAG YES POSITIVE CHARGE CGT YES ARGININE 6
(POS) CGC YES CGA YES CGG YES AGA YES AGG YES CAT YES HISTIDINE 2
CAC YES TAA YES STOP CODON 3 STOP SIGNAL 3 TAG YES (STP) TGA YES
TOTAL 64 64 20 Amino Acids Are Represented NPL: POL: NEG: POS: STP
= 29: 18: 4: 10: 3
[1504]
11TABLE 8 N, N, G CODON Represented AMINO ACID (Frequency) CATEGORY
(Frequency) GGT NO GLYCINE 1 NONPOLAR 8 GGC NO (NPL) GGA NO GGG YES
GCT NO ALANINE 1 GCC NO GCA NO GCG YES GTT NO VALINE 1 GTC NO GTA
NO GTG YES TTA NO LEUCINE 2 TTG YES CTT NO CTC NO CTA NO CTG YES
ATT NO ISOLEUCINE 0 ATC NO ATA NO ATG YES METHIONINE 1 TTT NO
PHENYLALANINE 0 TTC NO TGG YES TRYPTOPHAN 1 CCT NO PROLINE 1 CCC NO
CCA NO CCG YES TCT NO SERINE 1 POLAR 3 TCC NO NONIONIZABLE TCA NO
(POL) TCG YES AGT NO AGC NO TGT NO CYSTEINE 0 TGC NO AAT NO
ASPARAGINE 0 AAC NO CAA NO GLUTAMINE 1 CAG YES TAT NO TYROSINE 0
TAC NO ACT NO THREONINE 1 ACC NO ACA NO ACG YES GAT NO ASPARTIC
ACID 0 IONIZABLE: ACIDIC 1 GAC NO NEGATIVE CHARGE GAA NO GLUTAMIC
ACID 1 (NEG) GAG YES AAA NO LYSINE 1 IONIZABLE: BASIC 3 AAG YES
POSITIVE CHARGE CGT NO ARGININE 2 (POS) CGC NO CGA NO CGG YES AGA
NO AGG YES CAT NO HISTIDINE 0 CAC NO TAA NO STOP CODON 1 STOP
SIGNAL 1 TAG YES (STP) TGA NO TOTAL 64 16 13 Amino Acids Are
Represented NPL: POL: NEG: POS: STP = 8: 3: 1: 3: 1
[1505]
12TABLE 9 N, N, A CODON Represented AMINO ACID (Frequency) CATEGORY
(Frequency) GGT NO GLYCINE 1 NONPOLAR 7 GGC NO (NPL) GGA YES GGG NO
GCT NO ALANINE 1 GCC NO GCA YES GCG NO GTT NO VALINE 1 GTC NO GTA
YES GTG NO TTA YES LEUCINE 2 TTG NO CTT NO CTC NO CTA YES CTG NO
ATT NO ISOLEUCINE 1 ATC NO ATA YES ATG NO METHIONINE 0 TTT NO
PHENYLALANINE 0 TTC NO TGG NO TRYPTOPHAN 0 CCT NO PROLINE 1 CCC NO
CCA YES CCG NO TCT NO SERINE 1 POLAR 3 TCC NO NONIONIZABLE TCA YES
(POL) TCG NO AGT NO AGC NO TGT NO CYSTEINE 0 TGC NO AAT NO
ASPARAGINE 0 AAC NO CAA YES GLUTAMINE 1 CAG NO TAT NO TYROSINE 0
TAC NO ACT NO THREONINE 1 ACC NO ACA YES ACG NO GAT NO ASPARTIC
ACID 0 IONIZABLE: ACIDIC 1 GAC NO NEGATIVE CHARGE GAA YES GLUTAMIC
ACID 1 (NEG) GAG NO AAA YES LYSINE 1 IONIZABLE: BASIC 3 AAG NO
POSITIVE CHARGE CGT NO ARGININE 2 (POS) CGC NO CGA YES CGG NO AGA
YES AGG NO CAT NO HISTIDINE 0 CAC NO TAA YES STOP CODON 2 STOP
SIGNAL 2 TAG NO (STP) TGA YES TOTAL 64 16 12 Amino Acids Are
Represented NPL: POL: NEG: POS: STP = 7: 3: 1: 3: 2
[1506]
13TABLE 10 N, N, C CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGT NO GLYCINE 1 NONPOLAR 7 GGC YES (NPL) GGA
NO GGG NO GCT NO ALANINE 1 GCC YES GCA NO GCG NO GTT NO VALINE 1
GTC YES GTA NO GTG NO TTA NO LEUCINE 1 TTG NO CTT NO CTC YES CTA NO
CTG NO ATT NO ISOLEUCINE 1 ATC YES ATA NO ATG NO METHIONINE 0 TTT
NO PHENYLALANINE 1 TTC YES TGG NO TRYPTOPHAN 0 CCT NO PROLINE 1 CCC
YES CCA NO CCG NO TCT NO SERINE 2 POLAR 6 TCC YES NONIONIZABLE TCA
NO (POL) TCG NO AGT NO AGC YES TGT NO CYSTEINE 1 TGC YES AAT NO
ASPARAGINE 1 AAC YES CAA NO GLUTAMINE 0 CAG NO TAT NO TYROSINE 1
TAC YES ACT NO THREONINE 1 ACC YES ACA NO ACG NO GAT NO ASPARTIC
ACID 1 IONIZABLE: ACIDIC 1 GAC YES NEGATIVE CHARGE GAA NO GLUTAMIC
ACID 0 (NEG) GAG NO AAA NO LYSINE 0 IONIZABLE: BASIC 2 AAG NO
POSITIVE CHARGE CGT NO ARGININE 1 (POS) CGC YES CGA NO CGG NO AGA
NO AGG NO CAT NO HISTIDINE 1 CAC YES TAA NO STOP CODON 0 STOP
SIGNAL 0 TAG NO (STP) TGA NO TOTAL 64 16 15 Amino Acids Are
Represented NPL: POL: NEG: POS: STP = 7: 6: 1: 2: 0
[1507]
14TABLE 11 N, N, T CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGT YES GLYCINE 1 NONPOLAR 7 GGC NO (NPL) GGA
NO GGG NO GCT YES ALANINE 1 GCC NO GCA NO GCG NO GTT YES VALINE 1
GTC NO GTA NO GTG NO TTA NO LEUCINE 1 TTG NO CTT YES CTC NO CTA NO
CTG NO ATT YES ISOLEUCINE 1 ATC NO ATA NO ATG NO METHIONINE 0 TTT
YES PHENYLALANINE 1 TTC NO TGG NO TRYPTOPHAN 0 CCT YES PROLINE 1
CCC NO CCA NO CCG NO TCT YES SERINE 2 POLAR 6 TCC NO NONIONIZABLE
TCA NO (POL) TCG NO AGT YES AGC NO TGT YES CYSTEINE 1 TGC NO AAT
YES ASPARAGINE 1 AAC NO CAA NO GLUTAMINE 0 CAG NO TAT YES TYROSINE
1 TAC NO ACT YES THREONINE 1 ACC NO ACA NO ACG NO GAT YES ASPARTIC
ACID 1 IONIZABLE: ACIDIC 1 GAC NO NEGATIVE CHARGE GAA NO GLUTAMIC
ACID 0 (NEG) GAG NO AAA NO LYSINE 0 IONIZABLE: BASIC 2 AAG NO
POSITIVE CHARGE CGT YES ARGININE 1 (POS) CGC NO CGA NO CGG NO AGA
NO AGG NO CAT YES HISTIDINE 1 CAC NO TAA NO STOP CODON 0 STOP
SIGNAL 0 TAG NO (STP) TGA NO TOTAL 64 16 15 Amino Acids Are
Represented NPL: POL: NEG: POS. STP = 7: 6: 1: 2: 0
[1508]
15TABLE 12 N, N, C/G/T CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGT YES GLYCINE 3 NONPOLAR 22 GGC YES (NPL)
GGA NO GGG YES GCT YES ALANINE 3 GCC YES GCA NO GCG YES GTT YES
VALINE 3 GTC YES GTA NO GTG YES TTA NO LEUCINE 4 TTG YES CTT YES
CTC YES CTA NO CTG YES ATT YES ISOLEUCINE 2 ATC YES ATA NO ATG YES
METHIONINE 1 TTT YES PHENYLALANINE 2 TTC YES TGG YES TRYPTOPHAN 1
CCT YES PROLINE 3 CCC YES CCA NO CCG YES TCT YES SERINE 5 POLAR 15
TCC YES NONIONIZABLE TCA NO (POL) TCG YES AGT YES AGC YES TGT YES
CYSTEINE 2 TGC YES AAT YES ASPARAGINE 2 AAC YES CAA NO GLUTAMINE 1
CAG YES TAT YES TYROSINE 2 TAC YES ACT YES THREONINE 3 ACC YES ACA
NO ACG YES GAT YES ASPARTIC ACID 2 IONIZABLE: ACIDIC 3 GAC YES
NEGATIVE CHARGE GAA NO GLUTAMIC ACID 1 (NEG) GAG YES AAA NO LYSINE
1 IONIZABLE: BASIC 7 AAG YES POSITIVE CHARGE CGT YES ARGININE 4
(POS) CGC YES CGA NO CGG YES AGA NO AGG YES CAT YES HISTIDINE 2 CAC
YES TAA NO STOP CODON 1 STOP SIGNAL 1 TAG YES (STP) TGA NO TOTAL 64
48 20 Amino Acids Are Represented NPL: POL: NEG: POS. STP= 22: 15:
3: 7: 0
[1509]
16TABLE 13 N, N, A/G/T CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGT YES GLYCINE 3 NONPOLAR 22 GGC NO (NPL) GGA
YES GGG YES GCT YES ALANINE 3 GCC NO GCA YES GCG YES GTT YES VALINE
3 GTC NO GTA YES GTG YES TTA YES LEUCINE 5 TTG YES CTT YES CTC NO
CTA YES CTG YES ATT YES ISOLEUCINE 2 ATC NO ATA YES ATG YES
METHIONINE 1 TTT YES PHENYLALANINE 1 TTC NO TGG YES TRYPTOPHAN 1
CCT YES PROLINE 3 CCC NO CCA YES CCG YES TCT YES SERINE 4 POLAR 12
TCC NO NONIONIZABLE TCA YES (POL) TCG YES AGT YES AGC NO TGT YES
CYSTEINE 1 TGC NO AAT YES ASPARAGINE 1 AAC NO CAA YES GLUTAMINE 2
CAG YES TAT YES TYROSINE 1 TAC NO ACT YES THREONINE 3 ACC NO ACA
YES ACG YES GAT YES ASPARTIC ACID 1 IONIZABLE: ACIDIC 3 GAC NO
NEGATIVE CHARGE GAA YES GLUTAMIC ACID 2 (NEG) GAG YES AAA YES
LYSINE 2 IONIZABLE: BASIC 8 AAG YES POSITIVE CHARGE CGT YES
ARGININE 5 (POS) CGC NO CGA YES CGG YES AGA YES AGG YES CAT YES
HISTIDINE 1 CAC NO TAA YES STOP CODON 3 STOP SIGNAL 3 TAG YES (STP)
TGA YES TOTAL 64 48 20 Amino Acids Are Represented NPL: POL: NEG:
POS. STP= 22: 12: 3: 8: 3
[1510]
17TABLE 14 N, N, A/C/T CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGT YES GLYCINE 3 NONPOLAR 21 GGC YES (NPL)
GGA YES GGG NO GCT YES ALANINE 3 GCC YES GCA YES GCG NO GTT YES
VALINE 3 GTC YES GTA YES GTG NO TTA YES LEUCINE 4 TTG NO CTT YES
CTC YES CTA YES CTG NO ATT YES ISOLEUCINE 3 ATC YES ATA YES ATG NO
METHIONINE 0 TTT YES PHENYLALANINE 2 TTC YES TGG NO TRYPTOPHAN 0
CCT YES PROLINE 3 CCC YES CCA YES CCG NO TCT YES SERINE 5 POLAR 15
TCC YES NONIONIZABLE TCA YES (POL) TCG NO AGT YES AGC YES TGT YES
CYSTEINE 2 TGC YES AAT YES ASPARAGINE 2 AAC YES CAA YES GLUTAMINE 1
CAG NO TAT YES TYROSINE 2 TAC YES ACT YES THREONINE 3 ACC YES ACA
YES ACG NO GAT YES ASPARTIC ACID 2 IONIZABLE: ACIDIC 3 GAC YES
NEGATIVE CHARGE GAA YES GLUTAMIC ACID 1 (NEG) GAG NO AAA YES LYSINE
1 IONIZABLE: BASIC 7 AAG NO POSITIVE CHARGE CGT YES ARGININE 4
(POS) CGC YES CGA YES CGG NO AGA YES AGG NO CAT YES HISTIDINE 2 CAC
YES TAA YES STOP CODON 2 STOP SIGNAL 2 TAG NO (STP) TGA YES TOTAL
64 48 18 Amino Acids Are Represented NPL: POL: NEG: POS. STP= 21:
15: 3: 7: 2
[1511]
18TABLE 15 N, N, A/C/G CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGT NO GLYCINE 3 NONPOLAR 22 GGC YES (NPL) GGA
YES GGG YES GCT NO ALANINE 3 GCC YES GCA YES GCG YES GTT NO VALINE
3 GTC YES GTA YES GTG YES TTA YES LEUCINE 5 TTG YES CTT NO CTC YES
CTA YES CTG YES ATT NO ISOLEUCINE 2 ATC YES ATA YES ATG YES
METHIONINE 1 TTT NO PHENYLALANINE 1 TTC YES TGG YES TRYPTOPHAN 1
CCT NO PROLINE 3 CCC YES CCA YES CCG YES TCT NO SERINE 4 POLAR 12
TCC YES NONIONIZABLE TCA YES (POL) TCG YES AGT NO AGC YES TGT NO
CYSTEINE 1 TGC YES AAT NO ASPARAGINE .sup.1 AAC YES CAA YES
GLUTAMINE 2 CAG YES TAT NO TYROSINE 1 TAC YES ACT NO THREONINE 3
ACC YES ACA YES ACG YES GAT NO ASPARTIC ACID 1 IONIZABLE: ACIDIC 3
GAC YES NEGATIVE CHARGE GAA YES GLUTAMIC ACID 2 (NEG) GAG YES AAA
YES LYSINE 2 IONIZABLE: BASIC 8 AAG YES POSITIVE CHARGE CGT NO
ARGININE 5 (POS) CGC YES CGA YES CGG YES AGA YES AGG YES CAT NO
HISTIDINE 1 CAC YES TAA YES STOP CODON 3 STOP SIGNAL 3 TAG YES
(STP) TGA YES TOTAL 64 48 20 Amino Acids Are Represented NPL: POL:
NEG: POS. STP= 22: 12: 3: 8: 3
[1512]
19TABLE 16 N, A, A CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 0 ALANINE 0 (NPL) VALINE 0
LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0
PROLINE 0 SERINE 0 POLAR 1 CYSTEINE 0 NONIONIZABLE ASPARAGINE 0
(POL) CAA YES GLUTAMINE 1 TYROSINE 0 THREONINE 0 ASPARTIC ACID 0
IONIZABLE: ACIDIC 1 GAA YES GLUTAMIC ACID 1 NEGATIVE CHARGE (NEG)
AAA YES LYSINE 1 IONIZABLE: BASIC 1 ARGININE 0 POSITIVE CHARGE
HISTIDINE 0 (POS) TAA YES STOP CODON 1 STOP SIGNAL 1 (STP) TOTAL 4
3 Amino Acids Are Represented NPL: POL: NEG: POS: STP = 0: 1: 1: 1:
1
[1513]
20TABLE 17 N, A, C CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 0 ALANINE 0 (NPL) VALINE 0
LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0
PROLINE 0 SERINE 0 POLAR 2 CYSTEINE 0 NONIONIZABLE AAC YES
ASPARAGINE 1 (POL) GLUTAMINE 0 TAC YES TYROSINE 1 THREONINE 0 GAC
YES ASPARTIC ACID 1 IONIZABLE: ACIDIC 1 GLUTAMIC ACID 0 NEGATIVE
CHARGE (NEG) LYSINE 0 IONIZABLE: BASIC 1 ARGININE 0 POSITIVE CHARGE
CAC YES HISTIDINE 1 (POS) STOP CODON 0 STOP SIGNAL 0 (STP) TOTAL 4
4 Amino Acids Are Represented NPL: POL: NEG: POS: STP = 0: 2: 1: 1:
0
[1514]
21TABLE 18 N, A, G CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 0 ALANINE 0 (NPL) VALINE 0
LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0
PROLINE 0 SERINE 0 POLAR 1 CYSTEINE 0 NONIONIZABLE ASPARAGINE 0
(POL) CAG YES GLUTAMINE 1 TYROSINE 0 THREONINE 0 ASPARTIC ACID 0
IONIZABLE: ACIDIC 1 GAG YES GLUTAMIC ACID 1 NEGATIVE CHARGE (NEG)
AAG YES LYSINE 1 IONIZABLE: BASIC 1 ARGININE 0 POSITIVE CHARGE
HISTIDINE 0 (POS) TAG YES STOP CODON 1 STOP SIGNAL 1 (STP) TOTAL 4
3 Amino Acids Are Represented NPL: POL: NEG: POS: STP = 0: 1: 1: 1:
1
[1515]
22TABLE 19 N, A, T CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 0 ALANINE 0 (NPL) VALINE 0
LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0
PROLINE 0 SERINE 0 POLAR 2 CYSTEINE 0 NONIONIZABLE AAT YES
ASPARAGINE 1 (POL) GLUTAMINE 0 TAT YES TYROSINE 1 THREONINE 0 GAT
YES ASPARTIC ACID 1 IONIZABLE: ACIDIC 1 GLUTAMIC ACID 0 NEGATIVE
CHARGE (NEG) LYSINE 0 IONIZABLE: BASIC 1 ARGININE 0 POSITIVE CHARGE
CAT YES HISTIDINE 1 (POS) STOP CODON 0 STOP SIGNAL 0 (STP) TOTAL 4
4 Amino Acids Are Represented NPL: POL: NEG: POS: STP = 0: 2: 1: 1:
0
[1516]
23TABLE 20 N, C, A CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 2 GCA YES ALANINE 1 (NPL)
VALINE 0 LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0
TRYPTOPHAN 0 CCA YES PROLINE 1 TCA YES SERINE 1 POLAR 2 CYSTEINE 0
NONIONIZABLE ASPARAGINE 0 (POL) GLUTAMINE 0 TYROSINE 0 ACA YES
THREONINE 1 ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0
NEGATIVE CHARGE (NEG) LYSINE 0 IONIZABLE: BASIC 0 ARGININE 0
POSITIVE CHARGE HISTIDINE 0 (POS) STOP CODON 0 STOP SIGNAL 0 (STP)
TOTAL 4 4 Amino Acids Are Represented NPL: POL: NEG: POS: STP = 2:
2: 0: 0: 0
[1517]
24TABLE 21 N, C, C CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 2 GCC YES ALANINE 1 (NPL)
VALINE 0 LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0
TRYPTOPHAN 0 CCC YES PROLINE 1 TCC YES SERINE 1 POLAR 2 CYSTEINE 0
NONIONIZABLE ASPARAGINE 0 (POL) GLUTAMINE 0 TYROSINE 0 ACC YES
THREONINE 1 ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0
NEGATIVE CHARGE (NEG) LYSINE 0 IONIZABLE: BASIC 0 ARGININE 0
POSITIVE CHARGE HISTIDINE 0 (POS) STOP CODON 0 STOP SIGNAL 0 (STP)
TOTAL 4 4 Amino Acids Are Represented NPL: POL: NEG: POS: STP = 2:
2: 0: 0: 0
[1518]
25TABLE 22 N, C, G CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 2 GCG YES ALANINE 1 (NPL)
VALINE 0 LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0
TRYPTOPHAN 0 CCG YES PROLINE 1 TCG YES SERINE 1 POLAR 2 CYSTEINE 0
NONIONIZABLE ASPARAGINE 0 (POL) GLUTAMINE 0 TYROSINE 0 ACG YES
THREONINE 1 ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0
NEGATIVE CHARGE (NEG) LYSINE 0 IONIZABLE: BASIC 0 ARGININE 0
POSITIVE CHARGE HISTIDINE 0 (POS) STOP CODON 0 STOP SIGNAL 0 (STP)
TOTAL 4 4 Amino Acids Are Represented NPL: POL: NEG: POS: STP = 2:
2: 0: 0: 0
[1519]
26TABLE 23 N, C, T CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 2 GCT YES ALANINE 1 (NPL)
VALINE 0 LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0
TRYPTOPHAN 0 CCT YES PROLINE 1 TCT YES SERINE 1 POLAR 2 CYSTEINE 0
NONIONIZABLE ASPARAGINE 0 (POL) GLUTAMINE 0 TYROSINE 0 ACT YES
THREONINE 1 ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0
NEGATIVE CHARGE (NEG) LYSINE 0 IONIZABLE: BASIC 0 ARGININE 0
POSITIVE CHARGE HISTIDINE 0 (POS) STOP CODON 0 STOP SIGNAL 0 (STP)
TOTAL 4 4 Amino Acids Are Represented NPL: POL: NEG: POS: STP = 2:
2: 0: 0: 0
[1520]
27TABLE 24 N, G, A CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGA YES GLYCINE 1 NONPOLAR 1 ALANINE 0 (NPL)
VALINE 0 LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0
TRYPTOPHAN 0 PROLINE 0 SERINE 0 POLAR 0 CYSTEINE 0 NONIONIZABLE
ASPARAGINE 0 (POL) GLUTAMINE 0 TYROSINE 0 THREONINE 0 ASPARTIC ACID
0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE (NEG) LYSINE
0 IONIZABLE: BASIC 2 CGA YES ARGININE 2 POSITIVE CHARGE AGA YES
(POS) HISTIDINE 0 TGA YES STOP CODON 1 STOP SIGNAL 1 (STP) TOTAL 4
2 Amino Acids Are Represented NPL: POL: NEG: POS: STP = 1: 0: 0: 2:
1
[1521]
28TABLE 25 N, G, C CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGC YES GLYCINE 1 NONPOLAR 1 ALANINE 0 (NPL)
VALINE 0 LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0
TRYPTOPHAN 0 PROLINE 0 AGC YES SERINE 1 POLAR 2 TGC YES CYSTEINE 1
NONIONIZABLE ASPARAGINE 0 (POL) GLUTAMINE 0 TYROSINE 0 THREONINE 0
ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE
(NEG) LYSINE 0 IONIZABLE: BASIC 1 CGC YES ARGININE 1 POSITIVE
CHARGE HISTIDINE 0 (POS) STOP CODON 0 STOP SIGNAL 0 (STP) TOTAL 4 4
Amino Acids Are Represented NPL: POL: NEG: POS: STP = 1: 2: 0: 1:
0
[1522]
29TABLE 26 N, G, G CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGG YES GLYCINE 1 NONPOLAR 2 ALANINE 0 (NPL)
VALINE 0 LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0 TGG
YES TRYPTOPHAN 1 PROLINE 0 SERINE 0 POLAR 0 CYSTEINE 0 NONIONIZABLE
ASPARAGINE 0 (POL) GLUTAMINE 0 TYROSINE 0 THREONINE 0 ASPARTIC ACID
0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE (NEG) LYSINE
0 IONIZABLE: BASIC 2 CGG YES ARGININE 2 POSITIVE CHARGE AGG YES
(POS) HISTIDINE 0 STOP CODON 0 STOP SIGNAL 0 (STP) TOTAL 4 3 Amino
Acids Are Represented NPL: POL: NEG: POS: STP = 2: 0: 0: 2: 0
[1523]
30TABLE 27 N, G, T CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGT YES GLYCINE 1 NONPOLAR 1 ALANINE 0 (NPL)
VALINE 0 LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0
TRYPTOPHAN 0 PROLINE 0 ACT YES SERINE 1 POLAR 2 TGT YES CYSTEINE 1
NONIONIZABLE ASPARAGINE 0 (POL) GLUTAMINE 0 TYROSINE 0 THREONINE 0
ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE
(NEG) LYSINE 0 IONIZABLE: BASIC 1 CGT YES ARGININE 1 POSITIVE
CHARGE HISTIDINE 0 (POS) STOP CODON 0 STOP SIGNAL 0 (STP) TOTAL 4 4
Amino Acids Are Represented NPL: POL: NEG: POS: STP = 1: 2: 0: 1:
0
[1524]
31TABLE 28 N, T, A CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 4 ALANINE 0 (NPL) GTA YES
VALINE 1 TTA YES LEUCINE 2 CTA YES ATA YES ISOLEUCINE 1 METHIONINE
0 PHENYLALANINE 0 TRYPTOPHAN 0 PROLINE 0 SERINE 0 POLAR 0 CYSTEINE
0 NONIONIZABLE ASPARAGINE 0 (POL) GLUTAMINE 0 TYROSINE 0 THREONINE
0 ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE
CHARGE (NEG) LYSINE 0 IONIZABLE: BASIC 0 ARGININE 0 POSITIVE CHARGE
HISTIDINE 0 (POS) STOP CODON 0 STOP SIGNAL 0 (STP) TOTAL 4 3 Amino
Acids Are Represented NPL: POL: NEG: POS: STP = 4: 0: 0: 0: 0
[1525]
32TABLE 29 N, T, C CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 4 ALANINE 0 (NPL) GTC YES
VALINE 1 CTC YES LEUCINE 1 ATC YES ISOLEUCINE 1 METHIONINE 0 TTC
YES PHENYLALANINE 1 TRYPTOPHAN 0 PROLINE 0 SERINE 0 POLAR 0
CYSTEINE 0 NONIONIZABLE ASPARAGINE 0 (POL) GLUTAMINE 0 TYROSINE 0
THREONINE 0 ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0
NEGATIVE CHARGE (NEG) LYSINE 0 IONIZABLE: BASIC 0 ARGININE 0
POSITIVE CHARGE HISTIDINE 0 (POS) STOP CODON 0 STOP SIGNAL 0 (STP)
TOTAL 4 4 Amino Acids Are Represented NPL: POL: NEG: POS: STP = 4:
0: 0: 0: 0
[1526]
33TABLE 30 N, T, G CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 4 ALANINE 0 (NPL) GTG YES
VALINE 1 TTG YES LEUCINE 2 CTG YES ISOLEUCINE 0 ATG YES METHIONINE
1 PHENYLALANINE 0 TRYPTOPHAN 0 PROLINE 0 SERINE 0 POLAR 0 CYSTEINE
0 NONIONIZABLE ASPARAGINE 0 (POL) GLUTAMINE 0 TYROSINE 0 THREONINE
0 ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE
CHARGE (NEC) LYSINE 0 IONIZABLE: BASIC 0 ARGININE 0 POSITIVE CHARGE
HISTIDINE 0 (POS) STOP CODON 0 STOP SIGNAL 0 (STP) TOTAL 4 3 Amino
Acids Are Represented NPL: POL: NEG: POS: STP = 4: 0: 0: 0: 0
[1527]
34TABLE 31 N, T, T CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 4 ALANINE 0 (NPL) GTT YES
VALINE 1 CTT YES LEUCINE 1 ATT YES ISOLEUCINE 1 METHIONINE 0 TTT
YES PHENYLALANINE 1 TRYPTOPHAN 0 PROLINE 0 SERINE 0 POLAR 0
CYSTEINE 0 NONIONIZABLE ASPARAGINE 0 (POL) GLUTAMINE 0 TYROSINE 0
THREONINE 0 ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0
NEGATIVE CHARGE (NEG) LYSINE 0 IONIZABLE: BASIC 0 ARGININE 0
POSITIVE CHARGE HISTIDINE 0 (POS) STOP CODON 0 STOP SIGNAL 0 (STP)
TOTAL 4 4 Ammo Acids Are Represented NPL: POL: NEG: POS: STP = 4:
0: 0: 0: 0
[1528]
35TABLE 32 N, A/C, A CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 2 GCA YES ALANINE 1 (NPL)
VALINE 0 LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0
TRYPTOPHAN 0 CCA YES PROLINE 1 TCA YES SERINE 1 POLAR 3 CYSTEINE 0
NONIONIZABLE ASPARAGINE 0 (POL) CAA YES GLUTAMINE 1 TYROSINE 0 ACA
YES THREONINE 1 ASPARTIC ACID 0 IONIZABLE: ACIDIC 1 GAA YES
GLUTAMIC ACID 1 NEGATIVE CHARGE (NEC) AAA YES LYSINE 1 IONIZABLE:
BASIC 1 ARGININE 0 POSITIVE CHARGE HISTIDINE 0 (POS) TAA YES STOP
CODON 1 STOP SIGNAL 1 (STP) TOTAL 8 7 Amino Acids Are Represented
NPL: POL: NEG: POS: STP = 2: 3: 1: 1: 1
[1529]
36TABLE 33 N, A/G, A CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGA YES GLYCINE 1 NONPOLAR 1 ALANINE 0 (NPL)
VALINE 0 LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0
TRYPTOPHAN 0 PROLINE 0 SERINE 0 POLAR 1 CYSTEINE 0 NONIONIZABLE
ASPARAGINE 0 (POL) CAA YES GLUTAMINE 1 TYROSINE 0 THREONINE 0
ASPARTIC ACID 0 IONIZABLE: ACIDIC 1 GAA YES GLUTAMIC ACID 1
NEGATIVE CHARGE (NEC) AAA YES LYSINE 1 IONIZABLE: BASIC 3 CGA YES
ARGININE 2 POSITIVE CHARGE AGA YES (POS) HISTIDINE 0 TAA YES STOP
CODON 2 STOP SIGNAL 2 TGA YES (STP) TOTAL 8 5 Amino Acids Are
Represented NPL: POL: NEG: POS: STP = 1: 1: 1: 3: 2
[1530]
37TABLE 34 N, A/T, A CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 4 ALANINE 0 (NPL) GTA YES
VALINE 1 TTA YES LEUCINE 2 CTA YES ATA YES ISOLEUCINE 1 METHIONINE
0 PHENYLALANINE 0 TRYPTOPHAN 0 PROLINE 0 SERINE 0 POLAR 1 CYSTEINE
0 NONIONIZABLE ASPARAGINE 0 (POL) CAA YES GLUTAMINE 1 TYROSINE 0
THREONINE 0 ASPARTIC ACID 0 IONIZABLE: ACIDIC 1 GAA YES GLUTAMIC
ACID 1 NEGATIVE CHARGE (NEG) AAA YES LYSINE 1 IONIZABLE: BASIC 1
ARGININE 0 POSITIVE CHARGE HISTIDINE 0 (POS) TAA YES STOP CODON 1
STOP SIGNAL 1 (STP) TOTAL 8 6 Amino Acids Are Represented NPL: POL:
NEG: POS: STP = 4: 1: 1: 1: 1
[1531]
38TABLE 35 N, C/G, A CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGA YES GLYCINE 1 NONPOLAR 3 GCA YES ALANINE 1
(NPL) VALINE 0 LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0
TRYPTOPHAN 0 CCA YES PROLINE 1 TCA YES SERINE 1 POLAR 2 CYSTEINE 0
NONIONIZABLE ASPARAGINE 0 (POL) GLUTAMINE 0 TYROSINE 0 ACA YES
THREONINE 1 ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0
NEGATIVE CHARGE (NEG) LYSINE 0 IONIZABLE: BASIC 2 CGA YES ARGININE
2 POSITIVE CHARGE AGA YES (POS) HISTIDINE 0 TGA YES STOP CODON 1
STOP SIGNAL 1 (STP) TOTAL 8 6 Amino Acids Are Represented NPL: POL:
NEG: POS: STP = 3: 2: 0: 2: 1
[1532]
39TABLE 36 N, C/T, A CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 6 GCA YES ALANINE 1 (NPL)
GTA YES VALINE 1 TTA YES LEUCINE 2 CTA YES ATA YES ISOLEUCINE 1
METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0 CCA YES PROLINE 1 TCA YES
SERINE 1 POLAR 2 CYSTEINE 0 NONIONIZABLE ASPARAGINE 0 (POL)
GLUTAMINE 0 TYROSINE 0 ACA YES THREONINE 1 ASPARTIC ACID 0
IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE (NEG) LYSINE 0
IONIZABLE: BASIC 0 ARGININE 0 POSITIVE CHARGE HISTIDINE 0 (POS)
STOP CODON 0 STOP SIGNAL 0 (STP) TOTAL 8 7 Amino Acids Are
Represented NPL: POL: NEG: POS: STP = 6: 2: 0: 0: 0
[1533]
40TABLE 37 N, T/G, A CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGA YES GLYCINE 1 NONPOLAR 5 ALANINE 0 (NPL)
GTA YES VALINE 1 TTA YES LEUCINE 2 CTA YES ATA YES ISOLEUCINE 1
METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0 PROLINE 0 SERINE 0 POLAR
0 CYSTEINE 0 NONIONIZABLE ASPARAGINE 0 (POL) GLUTAMINE 0 TYROSINE 0
THREONINE 0 ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0
NEGATIVE CHARGE (NEG) LYSINE 0 IONIZABLE: BASIC 2 CGA YES ARGININE
2 POSITIVE CHARGE AGA YES (POS) HISTIDINE 0 TGA YES STOP CODON 1
STOP SIGNAL 1 (STP) TOTAL 8 5 Amino Acids Are Represented NPL: POL:
NEG: POS: STP = 5: 0: 0: 2: 1
[1534]
41TABLE 38 N, C/G/T, A CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGA YES GLYCINE 1 NONPOLAR 7 GCA YES ALANINE 1
(NPL) GTA YES VALINE 1 TTA YES LEUCINE 2 CTA YES ATA YES ISOLEUCINE
1 METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0 CCA YES PROLINE 1 TCA
YES SERINE 1 POLAR 2 CYSTEINE 0 NONIONIZABLE ASPARAGINE 0 (POL)
GLUTAMINE 0 TYROSINE 0 ACA YES THREONINE 1 ASPARTIC ACID 0
IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE (NEG) LYSINE 0
IONIZABLE: BASIC 2 CGA YES ARGININE 2 POSITIVE CHARGE AGA YES (POS)
HISTIDINE 0 TGA YES STOP CODON 1 STOP SIGNAL 1 (STP) TOTAL 12 9
Amino Acids Are Represented NPL: POL: NEG: POS: STP = 7: 2: 0: 2:
1
[1535]
42TABLE 39 N, A/G/T, A CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGA YES GLYCINE 1 NONPOLAR 5 ALANINE 0 (NPL)
GTA YES VALINE 1 TTA YES LEUCINE 2 CTA YES ATA YES ISOLEUCINE 1
METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0 PROLINE 0 SERINE 0 POLAR
1 CYSTEINE 0 NONIONIZABLE ASPARAGINE 0 (POL) CAA YES GLUTAMINE 1
TYROSINE 0 THREONINE 0 ASPARTIC ACID 0 IONIZABLE: ACIDIC 1 GAA YES
GLUTAMIC ACID 1 NEGATIVE CHARGE (NEG) AAA YES LYSINE 1 IONIZABLE:
BASIC 3 CGA YES ARGININE 2 POSITIVE CHARGE AGA YES (POS) HISTIDINE
0 TAA YES STOP CODON 2 STOP SIGNAL 2 TGA YES (STP) TOTAL 12 8 Amino
Acids Are Represented NPL: POL: NEG: POS: STP = 5: 1: 1: 3: 2
[1536]
43TABLE 40 N, A/C/T, A CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 6 GCA YES ALANINE 1 (NPL)
GTA YES VALINE 1 TTA YES LEUCINE 2 CTA YES ATA YES ISOLEUCINE 1
METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0 CCA YES PROLINE 1 TCA YES
SERINE 1 POLAR 3 CYSTEINE 0 NONIONIZABLE ASPARAGINE 0 (POL) CAA YES
GLUTAMINE 1 TYROSINE 0 ACA YES THREONINE 1 ASPARTIC ACID 0
IONIZABLE: ACIDIC 1 GAA YES GLUTAMIC ACID 1 NEGATIVE CHARGE (NEG)
AAA YES LYSINE 1 IONIZABLE: BASIC 1 ARGININE 0 POSITIVE CHARGE
HISTIDINE 0 (POS) TAA YES STOP CODON 1 STOP SIGNAL 1 (STP) TOTAL 12
10 Amino Acids Are Represented NPL: POL: NEG: POS: STP = 6: 3: 1:
1: 1
[1537]
44TABLE 41 N, A/C/G, A CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGA YES GLYCINE 1 NONPOLAR 3 GCA YES ALANINE 1
(NPL) VALINE 0 LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0
TRYPTOPHAN 0 CCA YES PROLINE 1 TCA YES SERINE 1 POLAR 3 CYSTEINE 0
NONIONIZABLE ASPARAGINE 0 (POL) CAA YES GLUTAMINE 1 TYROSINE 0 ACA
YES THREONINE 1 ASPARTIC ACID 0 IONIZABLE: ACIDIC 1 GAA YES
GLUTAMIC ACID 1 NEGATIVE CHARGE (NEG) AAA YES LYSINE 1 IONIZABLE:
BASIC 3 CGA YES ARGININE 2 POSITIVE CHARGE AGA YES (POS) HISTIDINE
0 TAA YES STOP CODON 2 STOP SIGNAL 2 TGA YES (STP) TOTAL 12 9 Amino
Acids Are Represented NPL: POL: NEG: POS: STP = 3: 3: 1: 3: 2
[1538]
45TABLE 42 A, N, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 4 ALANINE 0 (NPL) VALINE 0
LEUCINE 0 ATT YES ISOLEUCINE 3 ATC YES ATA YES ATG YES METHIONINE 1
PHENYLALANINE 0 TRYPTOPHAN 0 PROLINE 0 ACT YES SERINE 2 POLAR 8 AGC
YES NONION1ZABLE CYSTEINE 0 (POL) AAT YES ASPARAGINE 2 AAC YES
GLUTAMINE 0 TYROSINE 0 ACT YES THREONINE 4 ACC YES ACA YES ACG YES
ASPARTIC ACID 0 IONIZABLE ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE
(NEG) AAA YES LYSINE 2 IONIZABLE. BASIC 4 AAG YES POSITIVE CHARGE
AGA YES ARGININE 2 (POS) AGG YES HISTIDINE 0 STOP CODON 0 STOP
SIGNAL 0 (STP) TOTAL 16 7 Amino Acids Are Represented NPL: POL:
NEG: POS: STP = 4: 8: 0: 4: 0
[1539]
46TABLE 43 C, N, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 8 ALANINE 0 (NPL) VALINE 0
CTT YES LEUCINE 4 CTC YES CTA YES CTG YES ISOLEUCINE 0 METHIONINE 0
PHENYLALANINE 0 TRYPTOPHAN 0 CCT YES PROLINE 4 CCC YES CCA YES CCG
YES SERINE 0 POLAR 2 CYSTEINE 0 NONIONIZABLE ASPARAGINE 0 (POL) CAA
YES GLUTAMINE 2 CAG YES TYROSINE 0 THREONINE 0 ASPARTIC ACID 0
IONIZABLE ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE (NEG) LYSINE 0
IONIZABLE BASIC 6 CGT YES ARGININE 4 POSITIVE CHARGE CGC YES (POS)
CGA YES CGG YES CAT YES HISTIDINE 2 CAC YES STOP CODON 0 STOP
SIGNAL 0 (STP) TOTAL 16 5 Amino Acids Are Represented NPL: POL:
NEG: POS: STP = 8: 2: 0: 6: 0
[1540]
47TABLE 44 G, N, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGT YES GLYCINE 4 NONPOLAR 12 GGC YES (NPL)
GGA YES GGG YES GCT YES ALANINE 4 GCC YES GCA YES GCG YES GTT YES
VALINE 4 GTC YES GTA YES GTG YES LEUCINE 0 ISOLEUCINE 0 METHIONINE
0 PHENYLALANINE 0 TRYPTOPHAN 0 PROLINE 0 SERINE 0 POLAR 0 CYSTEINE
0 NONIONIZABLE ASPARAGINE 0 (POL) GLUTAMINE 0 TYROSINE 0 THREONINE
0 GAT YES ASPARTIC ACID 2 IONIZABLE ACIDIC 4 GAC YES NEGATIVE
CHARGE GAA YES GLUTAMIC ACID 2 (NEG) GAG YES LYSINE 0 IONIZABLE
BASIC 0 ARGININE 0 POSITIVE CHARGE HISTIDINE 0 (POS) STOP CODON 0
STOP SIGNAL 0 (STP) TOTAL 16 5 Amino Acids Are Represented NPL:
POL: NEG: POS: STP = 12 0: 4: 0: 0
[1541]
48TABLE 45 T, N, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 5 ALANINE 0 (NPL) VALINE 0
TTA YES LEUCINE 2 TTG YES ISOLEUCINE 0 METHIONINE 0 TTT YES
PHENYLALANINE 2 TTC YES TGG YES TRYPTOPHAN 1 PROLINE 0 TCT YES
SERINE 4 POLAR 8 TCC YES NONIONIZABLE TCA YES (POL) TCG YES TGT YES
CYSTEINE 2 TGC YES ASPARAGINE 0 GLUTAMINE 0 TAT YES TYROSINE 2 TAC
YES THREONINE 0 ASPARTIC ACID 0 IONIZABLE ACIDIC 0 GLUTAMIC ACID 0
NEGATIVE CHARGE (NEG) LYSINE 0 IONIZABLE BASIC 0 ARGININE 0
POSITIVE CHARGE HISTIDINE 0 (POS) TAA YES STOP CODON 3 STOP SIGNAL
3 TAG YES (STP) TGA YES TOTAL 16 6 Amino Acids Are Represented NPL:
POL: NEG: POS: STP = 5: 8: 0: 0: 3
[1542]
49TABLE 46 A/C, N, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 12 ALANINE 0 (NPL) VALINE 0
CTT YES LEUCINE 4 CTC YES CTA YES CTG YES ATT YES ISOLEUCINE 3 ATC
YES ATA YES ATG YES METHIONINE 1 PHENYLALANINE 0 TRYPTOPHAN 0 CCT
YES PROLINE 4 CCC YES CCA YES CCG YES AGT YES SERINE 2 POLAR 10 AGC
YES NONIONIZABLE CYSTEINE 0 (POL) AAT YES ASPARAGINE 2 AAC YES CAA
YES GLUTAMINE 2 CAG YES TYROSINE 0 ACT YES THREONINE 4 ACC YES ACA
YES ACG YES ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0
NEGATIVE CHARGE (NEG) AAA YES LYSINE 2 IONIZABLE: BASIC 10 AAG YES
POSITIVE CHARGE CGT YES ARGININE 6 (POS) CGC YES CGA YES CGG YES
AGA YES AGG YES CAT YES HISTIDINE 2 CAC YES STOP CODON 0 STOP
SIGNAL 0 (STP) TOTAL 32 11 Amino Acids Are Represented NPL: POL:
NEG: POS: STP = 12: 10: 0: 10: 0
[1543]
50TABLE 47 A/G, N, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGT YES GLYCINE 4 NONPOLAR 16 GGC YES (NPL)
GGA YES GGG YES GCT YES ALANINE 4 GCC YES GCA YES GCG YES GTT YES
VALINE 4 GTC YES GTA YES GTG YES LEUCINE 0 ATT YES ISOLEUCINE 3 ATC
YES ATA YES ATG YES METHIONINE 1 PHENYLALANINE 0 TRYPTOPHAN 0
PROLINE 0 AGT YES SERINE 2 POLAR 8 AGC YES NONIONIZABLE CYSTEINE 0
(POL) AAT YES ASPARAGINE 2 AAC YES GLUTAMINE 0 TYROSINE 0 ACT YES
THREONINE 4 ACC YES ACA YES ACG YES GAT YES ASPARTIC ACID 2
IONIZABLE: ACIDIC 4 GAC YES NEGATIVE CHARGE GAA YES GLUTAMIC ACID 2
(NEG) GAG YES AAA YES LYSINE 2 IONIZABLE: BASIC 4 AAG YES POSITIVE
CHARGE AGA YES ARGININE 2 (POS) AGG YES HISTIDINE 0 STOP CODON 0
STOP SIGNAL 0 (STP) TOTAL 32 12 Amino Acids Are Represented NPL:
POL: NEG: POS: STP = 16: 8: 4: 4: 0
[1544]
51TABLE 48 A/T, N, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 9 ALANINE 0 (NPL) VALINE 0
TTA YES LEUCINE 2 TTG YES ATT YES ISOLEUCINE 3 ATC YES ATA YES ATG
YES METHIONINE 1 TTT YES PHENYLALANINE 2 TTC YES TGG YES TRYPTOPHAN
1 PROLINE 0 TCT YES SERINE 6 POLAR 16 TCC YES NONIONIZABLE TCA YES
(POL) TCG YES AGT YES AGC YES TGT YES CYSTEINE 2 TGC YES AAT YES
ASPARAGINE 2 AAC YES GLUTAMINE 0 TAT YES TYROSINE 2 TAC YES ACT YES
THREONINE 4 ACC YES ACA YES ACG YES ASPARTIC ACID 0 IONIZABLE:
ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE (NEG) AAA YES LYSINE 2
IONIZABLE: BASIC 4 AAG YES POSITIVE CHARGE AGA YES ARGININE 2 (POS)
AGG YES HISTIDINE 0 TAA YES STOP CODON 3 STOP SIGNAL 3 TAG YES
(STP) TGA YES TOTAL 32 12 Amino Acids Are NPL: POL: NEG: POS: STP =
Represented 9: 16: 0: 4: 3
[1545]
52TABLE 49 C/G, N, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGT YES GLYCINE 4 NONPOLAR 20 GGC YES (NPL)
GGA YES GGG YES GCT YES ALANINE 4 GCC YES GCA YES GCG YES GTT YES
VALINE 4 GTC YES GTA YES GTG YES CTT YES LEUCINE 4 CTC YES CTA YES
CTG YES ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0 CCT
YES PROLINE 4 CCC YES CCA YES CCG YES SERINE 0 POLAR 2 CYSTEINE 0
NONIONIZABLE ASPARAGINE 0 (POL) CAA YES GLUTAMINE 2 CAG YES
TYROSINE 0 THREONINE 0 GAT YES ASPARTIC ACID 2 IONIZABLE: ACIDIC 4
GAC YES NEGATIVE CHARGE GAA YES GLUTAMIC ACID 2 (NEG) GAG YES
LYSINE 0 IONIZABLE: BASIC 6 CGT YES ARGININE 4 POSITIVE CHARGE CGC
YES (POS) CGA YES CGG YES CAT YES HISTIDINE 2 CAC YES STOP CODON 0
STOP SIGNAL 0 (STP) TOTAL 32 10 Amino Acids Are NPL: POL: NEG: POS:
STP = Represented 20: 2: 4: 6: 0
[1546]
53TABLE 50 C/T, N, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 13 ALANINE 0 (NPL) VALINE 0
TTA YES LEUCINE 6 TTG YES CTT YES CTC YES CTA YES CTG YES
ISOLEUCINE 0 METHIONINE 0 TTT YES PHENYLALANINE 2 TTC YES TGG YES
TRYPTOPHAN 1 CCT YES PROLINE 4 CCC YES CCA YES CCG YES TCT YES
SERINE 4 POLAR 10 TCC YES NONIONIZABLE TCA YES (POL) TCG YES TGT
YES CYSTEINE 2 TGC YES ASPARAGINE 0 CAA YES GLUTAMINE 2 CAG YES TAT
YES TYROSINE 2 TAC YES THREONINE 0 ASPARTIC ACID 0 IONIZABLE:
ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE (NEG) LYSINE 0 IONIZABLE:
BASIC 6 CGT YES ARGININE 4 POSITIVE CHARGE CGC YES (POS) CGA YES
CGG YES CAT YES HISTIDINE 2 CAC YES TAA YES STOP CODON 3 STOP
SIGNAL 3 TAG YES (STP) TGA YES TOTAL 32 10 Amino Acids Are NPL:
POL: NEG: POS: STP = Represented 13: 10: 0: 6: 3
[1547]
54TABLE 51 G/T, N, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGT YES GLYCINE 4 NONPOLAR 17 GGC YES (NPL)
GGA YES GGG YES GCT YES ALANINE 4 GCC YES GCA YES GCG YES GTT YES
VALINE 4 GTC YES GTA YES GTG YES TTA YES LEUCINE 2 TTG YES
ISOLEUCINE 0 METHIONINE 0 TTT YES PHENYLALANINE 2 TTC YES TGG YES
TRYPTOPHAN 1 PROLINE 0 TCT YES SERINE 4 POLAR 8 TCC YES
NONIONIZABLE TCA YES (POL) TCG YES TGT YES CYSTEINE 2 TGC YES
ASPARAGINE 0 GLUTAMINE 0 TAT YES TYROSINE 2 TAC YES THREONINE 0 GAT
YES ASPARTIC ACID 2 IONIZABLE: ACIDIC 4 GAC YES NEGATIVE CHARGE GAA
YES GLUTAMIC ACID 2 (NEG) GAG YES LYSINE 0 IONIZABLE: BASIC 0
ARGININE 0 POSITIVE CHARGE HISTIDINE 0 (POS) TAA YES STOP CODON 3
STOP SIGNAL 3 TAG YES (STP) TGA YES TOTAL 32 11 Amino Acids Are
NPL: POL: NEG: POS: STP = Represented 17: 8: 4: 0: 3
[1548]
55TABLE 52 N, A, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 0 ALANINE 0 (NPL) VALINE 0
LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0
PROLINE 0 SERINE 0 POLAR 6 CYSTEINE 0 NONIONIZABLE AAT YES
ASPARAGINE 2 (POL) AAC YES CAA YES GLUTAMINE 2 CAG YES TAT YES
TYROSINE 2 TAC YES THREONINE 0 GAT YES ASPARTIC ACID 2 IONIZABLE
ACIDIC 4 GAC YES NEGATIVE CHARGE GAA YES GLUTAMIC ACID 2 (NEG) GAG
YES AAA YES LYSINE 2 IONIZABLE BASIC 4 AAG YES POSITIVE CHARGE
ARGININE 0 (POS) CAT YES HISTIDINE 2 CAC YES TAA YES STOP CODON 2
STOP SIGNAL 2 TAG YES (STP) TOTAL 16 7 Amino Acids Are Represented
NPL: POL: NEG: POS: STP = 0: 6: 4: 4: 2
[1549]
56TABLE 53 N, C, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 8 GCT YES ALANINE 4 (NPL)
GCC YES GCA YES GCG YES VALINE 0 LEUCINE 0 ISOLEUCINE 0 METHIONINE
0 PHENYLALANINE 0 TRYPTOPHAN 0 CCT YES PROLINE 4 CCC YES CCA YES
CCG YES TCT YES SERINE 4 POLAR 8 TCC YES NONIONIZABLE TCA YES (POL)
TCG YES CYSTEINE 0 ASPARAGINE 0 GLUTAMINE 0 TYROSINE 0 ACT YES
THREONINE 4 ACC YES ACA YES ACG YES ASPARTIC ACID 0 IONIZABLE
ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE (NEG) LYSINE 0 IONIZABLE
BASIC 0 ARGININE 0 POSITIVE CHARGE HISTIDINE 0 (POS) STOP CODON 0
STOP SIGNAL 0 (STP) TOTAL 16 4 Amino Acids Are Represented NPL:
POL: NEG: POS: STP = 8: 8: 0: 0: 0
[1550]
57TABLE 54 N, G, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGT YES GLYCINE 4 NONPOLAR 5 GGC YES (NPL) GGA
YES GGG YES ALANINE 0 VALINE 0 LEUCINE 0 ISOLEUCINE 0 METHIONINE 0
PHENYLALANINE 0 TGG YES TRYPTOPHAN 1 PROLINE 0 ACT YES SERINE 2
POLAR 4 AGC YES NONIONIZABLE TGT YES CYSTEINE 2 (POL) TGC YES
ASPARAGINE 0 GLUTAMINE 0 TYROSINE 0 THREONINE 0 ASPARTIC ACID 0
IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE (NEC) LYSINE 0
IONIZABLE: BASIC 6 CGT YES ARGININE 6 POSITIVE CHARGE CGC YES (POS)
CGA YES CGG YES AGA YES AGG YES HISTIDINE 0 TGA YES STOP CODON 1
STOP SIGNAL 1 (STP) TOTAL 16 5 Amino Acids Are Represented NPL:
POL: NEG: POS: STP = 5: 4: 0: 6: 1
[1551]
58TABLE 55 N, T, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 16 ALANINE 0 (NPL) GTT YES
VALINE 4 GTC YES GTA YES GTG YES TTA YES LEUCINE 6 TTG YES CTT YES
CTC YES CTA YES CTG YES ATT YES ISOLEUCINE 3 ATC YES ATA YES ATG
YES METHIONINE 1 TTT YES PHENYLALANINE 2 TTC YES TRYPTOPHAN 0
PROLINE 0 SERINE 0 POLAR 0 CYSTEINE 0 NONIONIZABLE ASPARAGINE 0
(POL) GLUTAMINE 0 TYROSINE 0 THREONINE 0 ASPARTIC ACID 0 IONIZABLE:
ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE (NEG) LYSINE 0 IONIZABLE:
BASIC 0 ARGININE 0 POSITIVE CHARGE HISTIDINE 0 (POS) STOP CODON 0
STOP SIGNAL 0 (STP) TOTAL 16 5 Amino Acids Are Represented NPL:
POL: NEG: POS: STP = 16: 0: 0: 0: 0
[1552]
59TABLE 56 N, A/C, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 8 GCT YES ALANINE 4 (NPL)
GCC YES GCA YES GCG YES VALINE 0 LEUCINE 0 ISOLEUCINE 0 METHIONINE
0 PHENYLALANINE 0 TRYPTOPHAN 0 CCT YES PROLINE 4 CCC YES CCA YES
CCG YES TCT YES SERINE 4 POLAR 14 TCC YES NONIONIZABLE TCA YES
(POL) TCG YES CYSTEINE 0 AAT YES ASPARAGINE 2 AAC YES CAA YES
GLUTAMINE 2 CAG YES TAT YES TYROSINE 2 TAC YES ACT YES THREONINE 4
ACC YES ACA YES ACG YES GAT YES ASPARTIC ACID 2 IONIZABLE: ACIDIC 4
GAC YES NEGATIVE CHARGE GAA YES GLUTAMIC ACID 2 (NEG) GAG YES AAA
YES LYSINE 2 IONIZABLE: BASIC 4 AAG YES POSITIVE CHARGE ARGININE 0
(POS) CAT YES HISTIDINE 2 CAC YES TAA YES STOP CODON 2 STOP SIGNAL
2 TAG YES (STP) TOTAL 32 11 Amino Acids Are Represented NPL: POL:
NEG: POS: STP = 8: 14: 4: 4: 2
[1553]
60TABLE 57 N, A/G, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGT YES GLYCINE 4 NONPOLAR 5 GGC YES (NPL) GGA
YES GGG YES ALANINE 0 VALINE 0 LEUCINE 0 ISOLEUCINE 0 METHIONINE 0
PHENYLALANINE 0 TGG YES TRYPTOPHAN 1 PROLINE 0 ACT YES SERINE 2
POLAR 10 AGC YES NONIONIZABLE TGT YES CYSTEINE 2 (POL) TGC YES AAT
YES ASPARAGINE 2 AAC YES CAA YES GLUTAMINE 2 CAG YES TAT YES
TYROSINE 2 TAC YES THREONINE 0 GAT YES ASPARTIC ACID 2 IONIZABLE:
ACIDIC 4 GAC YES NEGATIVE CHARGE GAA YES GLUTAMIC ACID 2 (NEG) GAG
YES AAA YES LYSINE 2 IONIZABLE: BASIC 10 AAG YES POSITIVE CHARGE
CGT YES ARGININE 6 (POS) CGC YES CGA YES CGG YES AGA YES AGG YES
CAT YES HISTIDINE 2 CAC YES TAA YES STOP CODON 3 STOP SIGNAL 3 TAG
YES (STP) TGA YES TOTAL 32 12 Amino Acids Are Represented NPL: POL:
NEG: POS: STP = 5: 10: 4: 10: 3
[1554]
61TABLE 58 N, A/T, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 16 ALANINE 0 (NPL) GTT YES
VALINE 4 GTC YES GTA YES GTG YES TTA YES LEUCINE 6 TTG YES CTT YES
CTC YES CTA YES CTG YES ATT YES ISOLEUCINE 3 ATC YES ATA YES ATG
YES METHIONINE 1 TTT YES PHENYLALANINE 2 TTC YES TRYPTOPHAN 0
PROLINE 0 SERINE 0 POLAR 6 CYSTEINE 0 NONIONIZABLE AAT YES
ASPARAGINE 2 (POL) AAC YES CAA YES GLUTAMINE 2 CAG YES TAT YES
TYROSINE 2 TAC YES THREONINE 0 GAT YES ASPARTIC ACID 2 IONIZABLE:
ACIDIC 4 GAC YES NEGATIVE CHARGE GAA YES GLUTAMIC ACID 2 (NEG) GAG
YES AAA YES LYSINE 2 IONIZABLE: BASIC 4 AAG YES POSITIVE CHARGE
ARGININE 0 (POS) CAT YES HISTIDINE 2 CAC YES TAA YES STOP CODON 2
STOP SIGNAL 2 TAG YES (STP) TOTAL 32 12 Amino Acids Are Represented
NPL: POL: NEG: POS: STP = 16: 6: 4: 4: 2
[1555]
62TABLE 59 N, C/G, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGT YES GLYCINE 4 NONPOLAR 13 GGC YES (NPL)
GGA YES GGG YES GCT YES ALANINE 4 GCC YES GCA YES GCG YES VALINE 0
LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0 TGG YES
TRYPTOPHAN 1 CCT YES PROLINE 4 CCC YES CCA YES CCG YES TCT YES
SERINE 6 POLAR 12 TCC YES NONIONIZABLE TCA YES (POL) TCG YES AGT
YES AGC YES TGT YES CYSTEINE 2 TGC YES ASPARAGINE 0 GLUTAMINE 0
TYROSINE 0 ACT YES THREONINE 4 ACC YES ACA YES ACG YES ASPARTIC
ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE (NEG)
LYSINE 0 IONIZABLE: BASIC 6 CGT YES ARGININE 6 POSITIVE CHARGE CGC
YES (POS) CGA YES CGG YES AGA YES AGG YES HISTIDINE 0 TGA YES STOP
CODON 1 STOP SIGNAL 1 (STP) TOTAL 32 8 Amino Acids Are Represented
NPL: POL: NEG: POS: STP = 13: 12: 0: 6: 1
[1556]
63TABLE 60 N, C/T, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 24 GCT YES ALANINE 4 (NPL)
GCC YES GCA YES GCG YES GTT YES VALINE 4 GTC YES GTA YES GTG YES
TTA YES LEUCINE 6 TTG YES CTT YES CTC YES CTA YES CTG YES ATT YES
ISOLEUCINE 3 ATC YES ATA YES ATG YES METHIONINE 1 TTT YES
PHENYLALANINE 2 TTC YES TRYPTOPHAN 0 CCT YES PROLINE 4 CCC YES CCA
YES CCG YES TCT YES SERINE 4 POLAR 8 TCC YES NONIONIZABLE TCA YES
(POL) TCG YES CYSTEINE 0 ASPARAGINE 0 GLUTAMINE 0 TYROSINE 0 ACT
YES THREONINE 4 ACC YES ACA YES ACG YES ASPARTIC ACID 0 IONIZABLE:
ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE (NEG) LYSINE 0 IONIZABLE:
BASIC 0 ARGININE 0 POSITIVE CHARGE HISTIDINE 0 (POS) STOP CODON 0
STOP SIGNAL 0 (STP) TOTAL 32 9 Amino Acids Are Represented NPL:
POL: NEG: POS: STP = 24: 8: 0: 0: 0
[1557]
64TABLE 61 N, G/T, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGT YES GLYCINE 4 NONPOLAR 21 GGC YES (NPL)
GGA YES GGG YES ALANINE 0 GTT YES VALINE 4 GTC YES GTA YES GTG YES
TTA YES LEUCINE 6 TTG YES CTT YES CTC YES CTA YES CTG YES ATT YES
ISOLEUCINE 3 ATC YES ATA YES ATG YES METHIONINE 1 TTT YES
PHENYLALANINE 2 TTC YES TGG YES TRYPTOPHAN 1 PROLINE 0 AGT YES
SERINE 2 POLAR 4 AGC YES NONIONIZABLE TGT YES CYSTEINE 2 (POL) TGC
YES ASPARAGINE 0 GLUTAMINE 0 TYROSINE 0 THREONINE 0 ASPARTIC ACID 0
IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE (NEG) LYSINE 0
IONIZABLE: BASIC 6 CGT YES ARGININE 6 POSITIVE CHARGE CGC YES (POS)
CGA YES CGG YES AGA YES AGG YES HISTIDINE 0 TGA YES STOP CODON 1
STOP SIGNAL 1 (STP) TOTAL 32 10 Amino Acids Are Represented NPL:
POL: NEG: POS: STP 21: 4: 0: 6: 1
[1558]
65TABLE 62 N, A/C/G, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGT YES GLYCINE 4 NONPOLAR 13 GGC YES (NPL)
GGA YES GGG YES GCT YES ALANINE 4 GCC YES GCA YES GCG YES VALINE 0
LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0 TGG YES
TRYPTOPHAN 1 CCT YES PROLINE 4 CCC YES CCA YES CCG YES TCT YES
SERINE 6 POLAR 18 TCC YES NONIONIZABLE TCA YES (POL) TCG YES AGT
YES AGC YES TGT YES CYSTEINE 2 TGC YES AAT YES ASPARAGINE 2 AAC YES
CAA YES GLUTAMINE 2 CAG YES TAT YES TYROSINE 2 TAC YES ACT YES
THREONINE 4 ACC YES ACA YES ACG YES GAT YES ASPARTIC ACID 2
IONIZABLE: ACIDIC 4 GAC YES NEGATIVE CHARGE GAA YES GLUTAMIC ACID 2
(NEG) GAG YES AAA YES LYSINE 2 IONIZABLE: BASIC 10 AAG YES POSITIVE
CHARGE CGT YES ARGININE 6 (POS) CGC YES CGA YES CGG YES AGA YES AGG
YES CAT YES HISTIDINE 2 CAC YES TAA YES STOP CODON 3 STOP SIGNAL 3
TAG YES (STP) TGA YES TOTAL 48 15 Amino Acids Are Represented NPL:
POL: NEG: POS: STP 13: 18: 4: 10: 3
[1559]
66TABLE 63 N, A/C/T, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 24 GCT YES ALANINE 4 (NPL)
GCC YES GCA YES GCG YES GTT YES VALINE 4 GTC YES GTA YES GTG YES
TTA YES LEUCINE 6 TTG YES CTT YES CTC YES CTA YES CTG YES ATT YES
ISOLEUCINE 3 ATC YES ATA YES ATG YES METHIONINE 1 TTT YES
PHENYLALANINE 2 TTC YES TRYPTOPHAN 0 CCT YES PROLINE 4 CCC YES CCA
YES CCG YES TCT YES SERINE 4 POLAR 14 TCC YES NONIONIZABLE TCA YES
(POL) TCG YES CYSTEINE 0 AAT YES ASPARAGINE 2 AAC YES CAA YES
GLUTAMINE 2 CAG YES TAT YES TYROSINE 2 TAC YES ACT YES THREONINE 4
ACC YES ACA YES ACG YES GAT YES ASPARTIC ACID 2 IONIZABLE: ACIDIC 4
GAC YES NEGATIVE CHARGE GAA YES GLUTAMIC ACID 2 (NEG) GAG YES AAA
YES LYSINE 2 IONIZABLE: BASIC 4 AAG YES POSITIVE CHARGE ARGININE 0
(POS) CAT YES HISTIDINE 2 CAC YES TAA YES STOP CODON 2 STOP SIGNAL
2 TAG YES (STP) TOTAL 48 16 Amino Acids Are Represented NPL: POL:
NEG: POS: STP 24: 14: 4: 4: 2
[1560]
67TABLE 64 N, A/G/T, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGT YES GLYCINE 4 NONPOLAR 21 GGC YES (NPL)
GGA YES GGG YES ALANINE 0 GTT YES VALINE 4 GTC YES GTA YES GTG YES
TTA YES LEUCINE 6 TTG YES CTT YES CTC YES CTA YES CTG YES ATT YES
ISOLEUCINE 3 ATC YES ATA YES ATG YES METHIONINE 1 TTT YES
PHENYLALANINE 2 TTC YES TGG YES TRYPTOPHAN 1 PROLINE 0 AGT YES
SERINE 2 POLAR 10 AGC YES NONIONIZABLE TGT YES CYSTEINE 2 (POL) TGC
YES AAT YES ASPARAGINE 2 AAC YES CAA YES GLUTAMINE 2 CAG YES TAT
YES TYROSINE 2 TAG YES THREONINE 0 GAT YES ASPARTIC ACID 2
IONIZABLE: ACIDIC 4 GAC YES NEGATIVE CHARGE GAA YES GLUTAMIC ACID 2
(NEG) GAG YES AAA YES LYSINE 2 IONIZABLE: BASIC 10 AAG YES POSITIVE
CHARGE CGT YES ARGININE 6 (POS) CGC YES CGA YES CGG YES AGA YES AGG
YES CAT YES HISTIDINE 2 CAC YES TAA YES STOP CODON 3 STOP SIGNAL 3
TAG YES (STP) TGA YES TOTAL 48 17 Amino Acids Are Represented NPL:
POL: NEG: POS: STP 21: 10: 4: 10: 3
[1561]
68TABLE 65 N, C/G/T, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGT YES GLYCINE 4 NONPOLAR 29 GGC YES (NPL)
GGA YES GGG YES GCT YES ALANINE 4 GCC YES GCA YES GCG YES GTT YES
VALINE 4 GTC YES GTA YES GTG YES TTA YES LEUCINE 6 TTG YES CTT YES
CTC YES CTA YES CTG YES ATT YES ISOLEUCINE 3 ATC YES ATA YES ATG
YES METHIONINE 1 TTT YES PHENYLALANINE 2 TTC YES TGG YES TRYPTOPHAN
1 CCT YES PROLINE 4 CCC YES CCA YES CCG YES TCT YES SERINE 6 POLAR
12 TCC YES NONIONIZABLE TCA YES (POL) TCG YES ACT YES AGC YES TGT
YES CYSTEINE 2 TGC YES ASPARAGINE 0 GLUTAMINE 0 TYROSINE 0 ACT YES
THREONINE 4 ACC YES ACA YES ACG YES ASPARTIC ACID 0 IONIZABLE:
ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE (NEG) LYSINE 0 IONIZABLE:
BASIC 6 CGT YES ARGININE 6 POSITIVE CHARGE CGC YES (POS) CGA YES
CGG YES AGA YES AGG YES HISTIDINE 0 TGA YES STOP CODON 1 STOP
SIGNAL 1 (STP) TOTAL 48 13 Amino Acids Are Represented NPL: POL:
NEG: POS: STP 29: 12: 0: 6: 1
[1562]
69TABLE 66 C, C, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 4 ALANINE 0 (NPL) VALINE 0
LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0
CCT YES PROLINE 4 CCC YES CCA YES CCG YES SERINE 0 POLAR 0 CYSTEINE
0 NONIONIZABLE ASPARAGINE 0 (POL) GLUTAMINE 0 TYROSINE 0 THREONINE
0 ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE
CHARGE (NEG) LYSINE 0 IONIZABLE: BASIC 0 ARGININE 0 POSITIVE CHARGE
HISTIDINE 0 (POS) STOP CODON 0 STOP SIGNAL 0 (STP) TOTAL 4 1 Amino
Acid Is Represented NPL: POL: NEG: POS: STP 4: 0: 0: 0: 0
[1563]
70TABLE 67 G, G, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGT YES GLYCINE 4 NONPOLAR 4 GGC YES (NPL) GGA
YES GGG YES ALANINE 0 VALINE 0 LEUCINE 0 ISOLEUCINE 0 METHIONINE 0
PHENYLALANINE 0 TRYPTOPHAN 0 PROLINE 0 SERINE 0 POLAR 0 CYSTEINE 0
NONIONIZABLE ASPARAGINE 0 (POL) GLUTAMINE 0 TYROSINE 0 THREONINE 0
ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE
(NEG) LYSINE 0 IONIZABLE: BASIC 0 ARGININE 0 POSITIVE CHARGE
HISTIDINE 0 (POS) STOP CODON 0 STOP SIGNAL 0 (STP) TOTAL 0 4 1
Amino Acid Is Represented NPL: POL: NEG: POS: STP 4: 0: 0: 0: 0
[1564]
71TABLE 68 G, C, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 4 GCT YES ALANINE 4 (NPL)
GCC YES GCA YES GCG YES VALINE 0 LEUCINE 0 ISOLEUCINE 0 METHIONINE
0 PHENYLALANINE 0 TRYPTOPHAN 0 PROLINE 0 SERINE 0 POLAR 0 CYSTEINE
0 NONIONIZABLE ASPARAGINE 0 (POL) GLUTAMINE 0 TYROSINE 0 THREONINE
0 ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE
CHARGE (NEG) LYSINE 0 IONIZABLE: BASIC 0 ARGININE 0 POSITIVE CHARGE
HISTIDINE 0 (POS) STOP CODON 0 STOP SIGNAL 0 (STP) TOTAL 4 1 Amino
Acid Is Represented NPL: POL: NEG: POS: STP 4: 0: 0: 0: 0
[1565]
72TABLE 69 G, T, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 4 ALANINE 0 (NPL) GTT YES
VALINE 4 GTC YES GTA YES GTG YES LEUCINE 0 ISOLEUCINE 0 METHIONINE
0 PHENYLALANINE 0 TRYPTOPHAN 0 PROLINE 0 SERINE 0 POLAR 0 CYSTEINE
0 NONIONIZABLE ASPARAGINE 0 (POL) GLUTAMINE 0 TYROSINE 0 THREONINE
0 ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE
CHARGE (NEG) LYSINE 0 IONIZABLE: BASIC 0 ARGININE 0 POSITIVE CHARGE
HISTIDINE 0 (POS) STOP CODON 0 STOP SIGNAL 0 (STP) TOTAL 4 1 Amino
Acid Is Represented NPL: POL: NEG: POS: STP 4: 0: 0: 0: 0
[1566]
73TABLE 70 C, G, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 0 ALANINE 0 (NPL) VALINE 0
LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0
PROLINE 0 SERINE 0 POLAR 0 CYSTEINE 0 NONIONIZABLE ASPARAGINE 0
(POL) GLUTAMINE 0 TYROSINE 0 THREONINE 0 ASPARTIC ACID 0 IONIZABLE:
ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE (NEG) LYSINE 0 IONIZABLE:
BASIC 4 CGT YES ARGININE 4 POSITIVE CHARGE CGC YES (POS) CGA YES
CGG YES HISTIDINE 0 STOP CODON 0 STOP SIGNAL 0 (STP) TOTAL 4 1
Amino Acid Is Represented NPL: POL: NEG: POS: STP 0: 0: 0: 4: 0
[1567]
74TABLE 71 C, T, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 4 ALANINE 0 (NPL) VALINE 0
CTT YES LEUCINE 4 CTC YES CTA YES CTG YES ISOLEUCINE 0 METHIONINE 0
PHENYLALANINE 0 TRYPTOPHAN 0 PROLINE 0 SERINE 0 POLAR 0 CYSTEINE 0
NONIONIZABLE ASPARAGINE 0 (POL) GLUTAMINE 0 TYROSINE 0 THREONINE 0
ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE
(NEG) LYSINE 0 IONIZABLE: BASIC 0 ARGININE 0 POSITIVE CHARGE
HISTIDINE 0 (POS) STOP CODON 0 STOP SIGNAL 0 (STP) TOTAL 4 1 Amino
Acid Is Represented NPL: POL: NEG: POS: STP 4: 0: 0: 0: 0
[1568]
75TABLE 72 T, C, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 0 ALANINE 0 (NPL) VALINE 0
LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0
PROLINE 0 TCT YES SERINE 4 POLAR 4 TCC YES NONIONIZABLE TCA YES
(POL) TCG YES CYSTEINE 0 ASPARAGINE 0 GLUTAMINE 0 TYROSINE 0
THREONINE 0 ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0
NEGATIVE CHARGE (NEG) LYSINE 0 IONIZABLE: BASIC 0 ARGININE 0
POSITIVE CHARGE HISTIDINE 0 (POS) STOP CODON 0 STOP SIGNAL 0 (STP)
TOTAL 4 1 Amino Acid Is Represented NPL: POL: NEG: POS: STP 0: 4:
0: 0: 0
[1569]
76TABLE 73 A, C, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 0 ALANINE 0 (NPL) VALINE 0
LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0
PROLINE 0 SERINE 0 POLAR 4 CYSTEINE 0 NONIONIZABLE ASPARAGINE 0
(POL) GLUTAMINE 0 TYROSINE 0 ACT YES THREONINE 4 ACC YES ACA YES
ACG YES ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0
NEGATIVE CHARGE (NEG) LYSINE 0 IONIZABLE: BASIC 0 ARGININE 0
POSITIVE CHARGE HISTIDINE 0 (POS) STOP CODON 0 STOP SIGNAL 0 (STP)
TOTAL 4 1 Amino Acid Is Represented NPL: POL: NEG: POS: STP 0: 4:
0: 0: 0
[1570]
77TABLE 74 G, A, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 0 ALANINE 0 (NPL) VALINE 0
LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0
PROLINE 0 SERINE 0 POLAR 0 CYSTEINE 0 NONIONIZABLE ASPARAGINE 0
(POL) GLUTAMINE 0 TYROSINE 0 THREONINE 0 GAT YES ASPARTIC ACID 2
IONIZABLE: ACIDIC 4 GAC YES NEGATIVE CHARGE GAA YES GLUTAMIC ACID 2
(NEG) GAG YES LYSINE 0 IONIZABLE: BASIC 0 ARGININE 0 POSITIVE
CHARGE HISTIDINE 0 (POS) STOP CODON 0 STOP SIGNAL 0 (STP) TOTAL 4 2
Amino Acids Are Represented NPL: POL: NEG: POS: STP 0: 0: 4: 0:
0
[1571]
78TABLE 75 A, T, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 4 ALANINE 0 (NPL) VALINE 0
LEUCINE 0 ATT YES ISOLEUCINE 3 ATC YES ATA YES ATG YES METHIONINE 1
PHENYLALANINE 0 TRYPTOPHAN 0 PROLINE 0 SERINE 0 POLAR 0 CYSTEINE 0
NONIONIZABLE ASPARAGINE 0 (POL) GLUTAMINE 0 TYROSINE 0 THREONINE 0
ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE
(NEG) LYSINE 0 IONIZABLE: BASIC 0 ARGININE 0 POSITIVE CHARGE
HISTIDINE 0 (POS) STOP CODON 0 STOP SIGNAL 0 (STP) TOTAL 4 2 Amino
Acids Are Represented NPL: POL: NEG: POS: STP 4: 0: 0: 0: 0
[1572]
79TABLE 76 C, A, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 0 ALANINE 0 (NPL) VALINE 0
LEUCINE 0 ISOLEUCINE 0 PHENYLALANINE 0 TRYPTOPHAN 0 PROLINE 0
SERINE 0 POLAR 2 CYSTEINE 0 NONIONIZABLE ASPARAGINE 0 (POL) CAA YES
GLUTAMINE 2 CAG YES TYROSINE 0 THREONINE 0 ASPARTIC ACID 0
IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE (NEG) LYSINE 0
IONIZABLE: BASIC 2 ARGININIE 0 POSITIVE CHARGE CAT YES HISTIDINE 2
(POS) CAC YES STOP CODON 0 STOP SIGNAL 0 (STP) TOTAL 4 2 Amino
Acids Are Represented NPL: POL: NEG: POS: STP 0: 2: 0: 2: 0
[1573]
80TABLE 77 T, T, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 4 ALANINE 0 (NPL) VALINE 0
TTA YES LEUCINE 2 TIG YES ISOLEUCINE 0 METHIONINE 0 TTT YES
PHENYLALANINE 2 TTC YES TRYPTOPHAN 0 PROLINE 0 SERINE 0 POLAR 0
CYSTEINE 0 NONIONIZABLE ASPARAGINE 0 (POL) GLUTAMINE 0 TYROSINE 0
THREONINE 0 ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0
NEGATIVE CHARGE (NEG) LYSINE 0 IONIZABLE: BASIC 0 ARGININE 0
POSITIVE CHARGE HISTIDINE 0 (POS) STOP CODON 0 STOP SIGNAL 0 (STP)
TOTAL 4 2 Amino Acids Are Represented NPL: POL: NEG: POS: SYP 4: 0:
0: 0 : 0
[1574]
81TABLE 78 A, A, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 0 ALANINE 0 (NPL) VALINE 0
LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0
PROLINE 0 SERINE 0 POLAR 2 CYSTEINE 0 NONIONIZABLE AAT YES
ASPARAGINE 2 (POL) AAC YES GLUTANINE 0 TYROSINE 0 THREONINE 0
ASPARTIC ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE
(NEG) AAA YES LYSINE 2 IONIZABLE: BASIC 2 AAG YES POSITIVE CHARGE
ARGININE 0 (POS) HISTIDINE 0 STOP CODON 0 STOP SIGNAL 0 (STP) TOTAL
4 2 Amino Acids Are Represented NPL: POL: NEG: POS: STP 0: 2: 0: 2:
0
[1575]
82TABLE 79 T, A, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 0 ALANINE 0 (NPL) VALINE 0
LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANIE 0 TRYPTOPHAN 0
PROLINE 0 SERINE 0 POLAR 2 CYSTEINE 0 NONIONIZABLE ASPARAGINE 0
(POL) GLUTAMINE 0 TAT YES TYROSINE 2 TAC YES THREONINE 0 ASPARTIC
ACID 0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE: CHARGE (NEG)
LYSINE 0 IONIZABLE: BASIC 0 ARGININE 0 POSITIVE CHARGE HISTIDINE 0
(POS) TAA YES STOP CODON 2 STOP SIGNAL 2 TAG YES (SYP) TOTAL 4 1
Amino Acid Is Represented NPL: POL: NEG: POS: STP 0: 2: 0: 0: 2
[1576]
83TABLE 80 T , G, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 1 ALANINE 0 (NPL) VALINE 0
LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0 TGG YES
TRYPTOPHAN 1 PROLINE 0 SERINE 0 POLAR 2 TGT YES CYSTEINE 2
NONIONIZABLE TGC YES (POL) TYROSINE 0 THREONINE 0 ASPARTIC ACID 0
IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE (NEG) LYSINE 0
IONIZABLE: BASI /C 0 ARGININE 0 POSITIVE CHARGE (POS) HISTIDINE 0
TGA YES STOP CODON 1 STOP SIGNAL 1 (STP) TOTAL 4 Amino Acids Are
Represented NPL: POL: NEG: POS: STP 1: 2: 0: 0: 1
[1577]
84TABLE 81 A, G, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 0 ALANINE 0 (NPL) VALINE 0
LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0
PROLINE 0 AGT YES SERINE 2 POLAR 2 AGC YES NONIONIZABLE CYSTEINE 0
(POL) ASPARAGINE 0 GLUTAMINE 0 TYROSINE 0 THREONINE 0 ASPARTIC ACID
0 IONIZABLE: ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE (NEG) LYSINE
0 IONIZABLE: BASIC 2 AGA YES ARGININE 2 POSITIVE CHARGE AGG YES
(POS) HISTIDINE 0 STOP CODON 0 STOP SIGNAL 0 (STP) TOTAL 4 2 Amino
Acids Are Represented NPL: POL. NEG POS STP 0 2 0 2 0
[1578]
85TABLE 82 G/C, G, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GGT YES GLYCINE 4 NONPOLAR 4 GGC YES (NPL) GGA
YES GGG YES ALANINE 0 VALINE 0 LEUCINE 0 ISOLEUCINE 0 METHIONINE 0
PHENYLALANINE 0 TRYPTOPHAN 0 PROLINE 0 SERINE 0 POLAR 0 CYSTEINE 0
NONIONIZABLE ASPARAGINE 0 (POL) GLUTAMINE 0 TYROSINE 0 THREONINE 0
ASPARTIC ACID 0 IONIZABLE ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE
(NEG) LYSINE 0 IONIZABLE BASIC 4 CGT YES ARGININE 4 POSITIVE CHARGE
CGC YES (POS) CGA YES CGG YES HISTIDINE 0 STOP CODON 0 STOP SIGNAL
0 (STP) TOTAL 8 2 Amino Acids Are Represented NPL: POL NEG P0S. STP
4 0 0 4 0
[1579]
86TABLE 83 G/C, C, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 8 GCT YES ALANINE 4 (NPL)
GCC YES GCA YES GCG YES VALINE 0 LEUCINE 0 ISOLEUCINE 0 METHIONINE
0 PHENYLALANINE 0 TRYPTOPHAN 0 CCT YES PROLINE 4 CCC YES CCA YES
CCG YES SERINE 0 POLAR 0 CYSTEINE 0 NONIONIZABLE ASPARAGINE 0 (POL)
GLUTAMINE 0 TYROSINE 0 THREONINE 0 ASPARTIC ACID 0 IONIZABLE -
ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE (NEG) LYSINE 0 IONIZABLE
BASIC 0 ARGININE 0 POSITIVE CHARGE HISTIDINE 0 (POS) STOP CODON 0
STOP SIGNAL 0 (STP) TOTAL 8 2 Amino Acids Are Represented NPL. POL
NEG POS STP 8 0 0. 0 0
[1580]
87TABLE 84 G/C, A, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 0 ALANINE 0 (NPL) VALINE 0
LEUCINE 0 ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0
PROLINE 0 SERINE 0 POLAR 2 CYSTEINE 0 NONIONIZABLE ASPARAGINE 0
(POL) CAA YES GLUTAMINE 2 CAG YES TYROSINE 0 THREONINE 0 GAT YES
ASPARTIC ACID 2 IONIZABLE - ACIDIC 4 GAC YES NEGATIVE CHARGE GAA
YES GLUTAMIC ACID 2 (NEG) GAG YES LYSINE 0 IONIZABLE: BASIC 2
ARGININE 0 POSITIVE CHARGE CAT YES HISTIDINE 2 (POS) CAC YES STOP
CODON 0 STOP SIGNAL 0 (STP) TOTAL 8 4 Amino Acids Are Represented
NPL- POL- NEG POS STP 0 2. 4 2 0
[1581]
88TABLE 85 G/C, T, N CODON Represented AMINO ACID (Frequency)
CATEGORY (Frequency) GLYCINE 0 NONPOLAR 8 ALANINE 0 (NPL) GTT YES
VALINE 4 GTC YES GTA YES GTG YES CTT YES LEUCINE 4 CTC YES CTA YES
CTG YES ISOLEUCINE 0 METHIONINE 0 PHENYLALANINE 0 TRYPTOPHAN 0
PROLINE 0 SERINE 0 POLAR 0 CYSTEINE 0 NONIONIZABLE ASPARAGINE 0
(POL) GLUTAMINE 0 TYROSINE 0 THREONINE 0 ASPARTIC ACID 0 IONIZABLE
ACIDIC 0 GLUTAMIC ACID 0 NEGATIVE CHARGE (NEG) LYSINE 0 IONIZABLE
BASIC 0 ARGININE 0 POSITIVE CHARGE HISTIDINE 0 (POS) STOP CODON 0
STOP SIGNAL 0 (STP) TOTAL 8 2 Amino Acids Are Represented NPL- POL.
NEG POS. STP 8. 0 0 0 0
2.11.2. CHIMERIZATIONS
2.11.2.1 "SHUFFLING"
[1582] Nucleic acid shuffling is a method for in vitro or in vivo
homologous recombination of pools of shorter or smaller
polynucleotides to produce a polynucleotide or polynucleotides.
Mixtures of related nucleic acid sequences or polynucleotides are
subjected to sexual PCR to provide random polynucleotides, and
reassembled to yield a library or mixed population of recombinant
hybrid nucleic acid molecules or polynucleotides.
[1583] In contrast to cassette mutagenesis, only shuffling and
error-prone PCR allow one to mutate a pool of sequences blindly
(without sequence information other than primers).
[1584] The advantage of the mutagenic shuffling of this invention
over error-prone PCR alone for repeated selection can best be
explained with an example from antibody engineering. Consider DNA
shuffling as compared with error-prone PCR (not sexual PCR). The
initial library of selected pooled sequences can consist of related
sequences of diverse origin (i.e. antibodies from naive mRNA) or
can be derived by any type of mutagenesis (including shuffling) of
a single antibody gene. A collection of selected complementarity
determining regions ("CDRs") is obtained after the first round of
affinity selection. In the diagram the thick CDRs confer onto the
antibody molecule increased affinity for the antigen. Shuffling
allows the free combinatorial association of all of the CDR1s with
all of the CDR2s with all of the CDR3s, for example.
[1585] This method differs from error-prone PCR, in that it is an
inverse chain reaction. In error-prone PCR, the number of
polymerase start sites and the number of molecules grows
exponentially. However, the sequence of the polymerase start sites
and the sequence of the molecules remains essentially the same. In
contrast, in nucleic acid reassembly or shuffling of random
polynucleotides the number of start sites and the number (but not
size) of the random polynucleotides decreases over time. For
polynucleotides derived from whole plasmids the theoretical
endpoint is a single, large concatemeric molecule.
[1586] Since cross-overs occur at regions of homology,
recombination will primarily occur between members of the same
sequence family. This discourages combinations of CDRs that are
grossly incompatible (e.g., directed against different epitopes of
the same antigen). It is contemplated that multiple families of
sequences can be shuffled in the same reaction. Further, shuffling
generally conserves the relative order, such that, for example,
CDR1 will not be found in the position of CDR2.
[1587] Rare shufflants will contain a large number of the best (eg.
highest affinity) CDRs and these rare shufflants may be selected
based on their superior affinity.
[1588] CDRs from a pool of 100 different selected antibody
sequences can be permutated in up to 1006 different ways. This
large number of permutations cannot be represented in a single
library of DNA sequences. Accordingly, it is contemplated that
multiple cycles of DNA shuffling and selection may be required
depending on the length of the sequence and the sequence diversity
desired.
[1589] Error-prone PCR, in contrast, keeps all the selected CDRs in
the same relative sequence, generating a much smaller mutant
cloud.
[1590] The template polynucleotide which may be used in the methods
of this invention may be DNA or RNA. It may be of various lengths
depending on the size of the gene or shorter or smaller
polynucleotide to be recombined or reassembled. Preferably, the
template polynucleotide is from 50 bp to 50 kb. It is contemplated
that entire vectors containing the nucleic acid encoding the
protein of interest can be used in the methods of this invention,
and in fact have been successfully used.
[1591] The template polynucleotide may be obtained by amplification
using the PCR reaction (U.S. Pat. No. 4,683,202 and U.S. Pat. No.
4,683,195) or other amplification or cloning methods. However, the
removal of free primers from the PCR products before subjecting
them to pooling of the PCR products and sexual PCR may provide more
efficient results. Failure to adequately remove the primers from
the original pool before sexual PCR can lead to a low frequency of
crossover clones.
[1592] The template polynucleotide often should be double-stranded.
A double-stranded nucleic acid molecule is recommended to ensure
that regions of the resulting single-stranded polynucleotides are
complementary to each other and thus can hybridize to form a
double-stranded molecule.
[1593] It is contemplated that single-stranded or double-stranded
nucleic acid polynucleotides having regions of identity to the
template polynucleotide and regions of heterology to the template
polynucleotide may be added to the template polynucleotide, at this
step. It is also contemplated that two different but related
polynucleotide templates can be mixed at this step.
[1594] The double-stranded polynucleotide template and any added
double-or single-stranded polynucleotides are subjected to sexual
PCR which includes slowing or halting to provide a mixture of from
about 5 bp to 5 kb or more. Preferably the size of the random
polynucleotides is from about 10 bp to 1000 bp, more preferably the
size of the polynucleotides is from about 20 bp to 500 bp.
[1595] Alternatively, it is also contemplated that double-stranded
nucleic acid having multiple nicks may be used in the methods of
this invention. A nick is a break in one strand of the
double-stranded nucleic acid. The distance between such nicks is
preferably 5 bp to 5 kb, more preferably between 10 bp to 1000 bp.
This can provide areas of self-priming to produce shorter or
smaller polynucleotides to be included with the polynucleotides
resulting from random primers, for example.
[1596] The concentration of any one specific polynucleotide will
not be greater than 1% by weight of the total polynucleotides, more
preferably the concentration of any one specific nucleic acid
sequence will not be greater than 0.1% by weight of the total
nucleic acid.
[1597] The number of different specific polynucletides in the
mixture will be at least about 100, preferably at least about 500,
and more preferably at least about 1000.
[1598] At this step single-stranded or double-stranded
polynucleotides, either synthetic or natural, may be added to the
random double-stranded shorter or smaller polynucleotides in order
to increase the heterogeneity of the mixture of
polynucleotides.
[1599] It is also contemplated that populations of double-stranded
randomly broken polynucleotides may be mixed or combined at this
step with the polynucleotides from the sexual PCR process and
optionally subjected to one or more additional sexual PCR
cycles.
[1600] Where insertion of mutations into the template
polynucleotide is desired, single-stranded or double-stranded
polynucleotides having a region of identity to the template
polynucleotide and a region of heterology to the template
polynucleotide may be added in a 20 fold excess by weight as
compared to the total nucleic acid, more preferably the
single-stranded polynucleotides may be added in a 10 fold excess by
weight as compared to the total nucleic acid.
[1601] Where a mixture of different but related template
polynucleotides is desired, populations of polynucleotides from
each of the templates may be combined at a ratio of less than about
1:100, more preferably the ratio is less than about 1:40. For
example, a backcross of the wild-type polynucleotide with a
population of mutated polynucleotide may be desired to eliminate
neutral mutations (e.g., mutations yielding an insubstantial
alteration in the phenotypic property being selected for). In such
an example, the ratio of randomly provided wild-type
polynucleotides which may be added to the randomly provided sexual
PCR cycle hybrid polynucleotides is approximately 1:1 to about
100:1, and more preferably from 1:1 to 40:1.
[1602] The mixed population of random polynucleotides are denatured
to form single-stranded polynucleotides and then re-annealed. Only
those single-stranded polynucleotides having regions of homology
with other single-stranded polynucleotides will re-anneal.
[1603] The random polynucleotides may be denatured by heating. One
skilled in the art could determine the conditions necessary to
completely denature the double-stranded nucleic acid. Preferably
the temperature is from 80.degree. C. to 100.degree. C., more
preferably the temperature is from 90.degree. C. to 96.degree. C.
other methods which may be used to denature the polynucleotides
include pressure (36) and pH.
[1604] The polynucleotides may be re-annealed by cooling.
Preferably the temperature is from 20.degree. C. to 75.degree. C.,
more preferably the temperature is from 40.degree. C. to 65.degree.
C. If a high frequency of crossovers is needed based on an average
of only 4 consecutive bases of homology, recombination can be
forced by using a low annealing temperature, although the process
becomes more difficult. The degree of renaturation which occurs
will depend on the degree of homology between the population of
single-stranded polynucleotides.
[1605] Renaturation can be accelerated by the addition of
polyethylene glycol ("PEG") or salt. The salt concentration is
preferably from 0 mM to 200 mM, more preferably the salt
concentration is from 10 mM to 100 mm. The salt may be KCl or NaCl.
The concentration of PEG is preferably from 0% to 20%, more
preferably from 5% to 10%.
[1606] The annealed polynucleotides are next incubated in the
presence of a nucleic acid polymerase and dNTP's (i.e. dATP, dCTP,
DGTP and dTTP). The nucleic acid polymerase may be the Klenow
fragment, the Taq polymerase or any other DNA polymerase known in
the art.
[1607] The approach to be used for the assembly depends on the
minimum degree of homology that should still yield crossovers. If
the areas of identity are large, Taq polymerase can be used with an
annealing temperature of between 45-65.degree. C. If the areas of
identity are small, Klenow polymerase can be used with an annealing
temperature of between 20-30.degree. C. One skilled in the art
could vary the temperature of annealing to increase the number of
cross-overs achieved.
[1608] The polymerase may be added to the random polynucleotides
prior to annealing, simultaneously with annealing or after
annealing.
[1609] The cycle of denaturation, renaturation and incubation in
the presence of polymerase is referred to herein as shuffling or
reassembly of the nucleic acid. This cycle is repeated for a
desired number of times. Preferably the cycle is repeated from 2 to
50 times, more preferably the sequence is repeated from 10 to 40
times.
[1610] The resulting nucleic acid is a larger double-stranded
polynucleotide of from about 50 bp to about 100 kb, preferably the
larger polynucleotide is from 500 bp to 50 kb.
[1611] This larger polynucleotides may contain a number of copies
of a polynucleotide having the same size as the template
polynucleotide in tandem. This concatemeric polynucleotide is then
denatured into single copies of the template polynucleotide. The
result will be a population of polynucleotides of approximately the
same size as the template polynucleotide. The population will be a
mixed population where single or double-stranded polynucleotides
having an area of identity and an area of heterology have been
added to the template polynucleotide prior to shuffling. These
polynucleotides are then cloned into the appropriate vector and the
ligation mixture used to transform bacteria.
[1612] It is contemplated that the single polynucleotides may be
obtained from the larger concatemeric polynucleotide by
amplification of the single polynucleotide prior to cloning by a
variety of methods including PCR (U.S. Pat. No. 4,683,195 and U.S.
Pat. No. 4,683,202), rather than by digestion of the
concatemer.
[1613] The vector used for cloning is not critical provided that it
will accept a polynucleotide of the desired size. If expression of
the particular polynucleotide is desired, the cloning vehicle
should further comprise transcription and translation signals next
to the site of insertion of the polynucleotide to allow expression
of the polynucleotide in the host cell. Preferred vectors include
the pUC series and the pBR series of plasmids.
[1614] The resulting bacterial population will include a number of
recombinant polynucleotides having random mutations. This mixed
population may be tested to identify the desired recombinant
polynucleotides. The method of selection will depend on the
polynucleotide desired.
[1615] For example, if a polynucleotide which encodes a protein
with increased binding efficiency to a ligand is desired, the
proteins expressed by each of the portions of the polynucleotides
in the population or library may be tested for their ability to
bind to the ligand by methods known in the art (i.e. panning,
affinity chromatography). If a polynucleotide which encodes for a
protein with increased drug resistance is desired, the proteins
expressed by each of the polynucleotides in the population or
library may be tested for their ability to confer drug resistance
to the host organism. One skilled in the art, given knowledge of
the desired protein, could readily test the population to identify
polynucleotides which confer the desired properties onto the
protein.
[1616] It is contemplated that one skilled in the art could use a
phage display system in which fragments of the protein are
expressed as fusion proteins on the phage surface (Pharmacia,
Milwaukee Wis.). The recombinant DNA molecules are cloned into the
phage DNA at a site which results in the transcription of a fusion
protein a portion of which is encoded by the recombinant DNA
molecule. The phage containing the recombinant nucleic acid
molecule undergoes replication and transcription in the cell. The
leader sequence of the fusion protein directs the transport of the
fusion protein to the tip of the phage particle. Thus the fusion
protein which is partially encoded by the recombinant DNA molecule
is displayed on the phage particle for detection and selection by
the methods described above.
[1617] It is further contemplated that a number of cycles of
nucleic acid shuffling may be conducted with polynucleotides from a
sub-population of the first population, which sub-population
contains DNA encoding the desired recombinant protein. In this
manner, proteins with even higher binding affinities or enzymatic
activity could be achieved.
[1618] It is also contemplated that a number of cycles of nucleic
acid shuffling may be conducted with a mixture of wild-type
polynucleotides and a sub-population of nucleic acid from the first
or subsequent rounds of nucleic acid shuffling in order to remove
any silent mutations from the sub-population.
[1619] Any source of nucleic acid, in purified form can be utilized
as the starting nucleic acid. Thus the process may employ DNA or
RNA including messenger RNA, which DNA or RNA may be single or
double stranded. In addition, a DNA-RNA hybrid which contains one
strand of each may be utilized. The nucleic acid sequence may be of
various lengths depending on the size of the nucleic acid sequence
to be mutated. Preferably the specific nucleic acid sequence is
from 50 to 50000 base pairs. It is contemplated that entire vectors
containing the nucleic acid encoding the protein of interest may be
used in the methods of this invention.
[1620] The nucleic acid may be obtained from any source, for
example, from plasmids such a pBR322, from cloned DNA or RNA or
from natural DNA or RNA from any source including bacteria, yeast,
viruses and higher organisms such as plants or animals. DNA or RNA
may be extracted from blood or tissue material. The template
polynucleotide may be obtained by amplification using the
polynucleotide chain reaction (PCR, see U.S. Pat. No. 4,683,202 and
U.S. Pat. No. 4,683,195). Alternatively, the polynucleotide may be
present in a vector present in a cell and sufficient nucleic acid
may be obtained by culturing the cell and extracting the nucleic
acid from the cell by methods known in the art.
[1621] Any specific nucleic acid sequence can be used to produce
the population of hybrids by the present process. It is only
necessary that a small population of hybrid sequences of the
specific nucleic acid sequence exist or be created prior to the
present process.
[1622] The initial small population of the specific nucleic acid
sequences having mutations may be created by a number of different
methods. Mutations may be created by error-prone PCR. Error-prone
PCR uses low-fidelity polymerization conditions to introduce a low
level of point mutations randomly over a long sequence.
Alternatively, mutations can be introduced into the template
polynucleotide by oligonucleotide-directed mutagenesis. In
oligonucleotide-directed mutagenesis, a short sequence of the
polynucleotide is removed from the polynucleotide using restriction
enzyme digestion and is replaced with a synthetic polynucleotide in
which various bases have been altered from the original sequence.
The polynucleotide sequence can also be altered by chemical
mutagenesis. Chemical mutagens include, for example, sodium
bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid.
other agents which are analogues of nucleotide precursors include
nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine.
Generally, these agents are added to the PCR reaction in place of
the nucleotide precursor thereby mutating the sequence.
Intercalating agents such as proflavine, acriflavine, quinacrine
and the like can also be used. Random mutagenesis of the
polynucleotide sequence can also be achieved by irradiation with
X-rays or ultraviolet light. Generally, plasmid polynucleotides so
mutagenized are introduced into E. coli and propagated as a pool or
library of hybrid plasmids.
[1623] Alternatively the small mixed population of specific nucleic
acids may be found in nature in that they may consist of different
alleles of the same gene or the same gene from different related
species (i.e., cognate genes). Alternatively, they may be related
DNA sequences found within one species, for example, the
immunoglobulin genes.
[1624] Once the mixed population of the specific nucleic acid
sequences is generated, the polynucleotides can be used directly or
inserted into an appropriate cloning vector, using techniques
well-known in, the art.
[1625] The choice of vector depends on the size of the
polynucleotide sequence and the host cell to be employed in the
methods of this invention. The templates of this invention may be
plasmids, phages, cosmids, phagemids, viruses (e.g., retroviruses,
parainfluenzavirus, herpesviruses, reoviruses, paramyxoviruses, and
the like), or selected portions thereof (e.g., coat protein, spike
glycoprotein, capsid protein). For example, cosmids and phagemids
are preferred where the specific nucleic acid sequence to be
mutated is larger because these vectors are able to stably
propagate large polynucleotides.
[1626] If the mixed population of the specific nucleic acid
sequence is cloned into a vector it can be clonally amplified by
inserting each vector into a host cell and allowing the host cell
to amplify the vector. This is referred to as clonal amplification
because while the absolute number of nucleic acid sequences
increases, the number of hybrids does not increase. Utility can be
readily determined by screening expressed polypeptides.
[1627] The DNA shuffling method of this invention can be performed
blindly on a pool of unknown sequences. By adding to the reassembly
mixture oligonucleotides (with ends that are homologous to the
sequences being reassembled) any sequence mixture can be
incorporated at any specific position into another sequence
mixture. Thus, it is contemplated that mixtures of synthetic
oligonucleotides, PCR polynucleotides or even whole genes can be
mixed into another sequence library at defined positions. The
insertion of one sequence (mixture) is independent from the
insertion of a sequence in another part of the template. Thus, the
degree of recombination, the homology required, and the diversity
of the library can be independently and simultaneously varied along
the length of the reassembled DNA.
[1628] This approach of mixing two genes may be useful for the
humanization of antibodies from murine hybridomas. The approach of
mixing two genes or inserting alternative sequences into genes may
be useful for any therapeutically used protein, for example,
interleukin I, antibodies, tPA and growth hormone. The approach may
also be useful in any nucleic acid for example, promoters or
introns or 31 untranslated region or 51 untranslated regions of
genes to increase expression or alter specificity of expression of
proteins. The approach may also be used to mutate ribozymes or
aptamers.
[1629] Shuffling requires the presence of homologous regions
separating regions of diversity. Scaffold-like protein structures
may be particularly suitable for shuffling. The conserved scaffold
determines the overall folding by self-association, while
displaying relatively unrestricted loops that mediate the specific
binding. Examples of such scaffolds are the immunoglobulin
beta-barrel, and the four-helix bundle which are well-Known in the
art. This shuffling can be used to create scaffold-like proteins
with various combinations of mutated sequences for binding.
[1630] In vitro Shuffling
[1631] The equivalents of some standard genetic matings may also be
performed by shuffling in vitro. For example, a "molecular
backcross" can be performed by repeatedly mixing the hybrid's
nucleic acid with the wild-type nucleic acid while selecting for
the mutations of interest. As in traditional breeding, this
approach can be used to combine phenotypes from different sources
into a background of choice. It is useful, for example, for the
removal of neutral mutations that affect unselected characteristics
(i.e. immunogenicity). Thus it can be useful to determine which
mutations in a protein are involved in the enhanced biological
activity and which are not, an advantage which cannot be achieved
by error-prone mutagenesis or cassette mutagenesis methods.
[1632] Large, functional genes can be assembled correctly from a
mixture of small random polynucleotides. This reaction may be of
use for the reassembly of genes from the highly fragmented DNA of
fossils. In addition random nucleic acid fragments from fossils may
be combined with polynucleotides from similar genes from related
species.
[1633] It is also contemplated that the method of this invention
can be used for the in vitro amplification of a whole genome from a
single cell as is needed for a variety of research and diagnostic
applications. DNA amplification by PCR is in practice limited to a
length of about 40 kb. Amplification of a whole genome such as that
of E. coli (5, 000 kb) by PCR would require about 250 primers
yielding 125 forty kb polynucleotides. This approach is not
practical due to the unavailability of sufficient sequence data. On
the other hand, random production of polynucleotides of the genome
with sexual PCR cycles, followed by gel purification of small
polynucleotides will provide a multitude of possible primers. Use
of this mix of random small polynucleotides as primers in a PCR
reaction alone or with the whole genome as the template should
result in an inverse chain reaction with the theoretical endpoint
of a single concatamer containing many copies of the genome.
[1634] 100 fold amplification in the copy number and an average
polynucleotide size of greater than 50 kb may be obtained when only
random polynucleotides are used. It is thought that the larger
concatamer is generated by overlap of many smaller polynucleotides.
The quality of specific PCR products obtained using synthetic
primers will be indistinguishable from the product obtained from
unamplified DNA. It is expected that this approach will be useful
for the mapping of genomes.
[1635] The polynucleotide to be shuffled can be produced as random
or non-random polynucleotides, at the discretion of the
practitioner. Moreover, this invention provides a method of
shuffling that is applicable to a wide range of polynucleotide
sizes and types, including the step of generating polynucleotide
monomers to be used as building blocks in the reassembly of a
larger polynucleotide. For example, the building blocks can be
fragments of genes or they can be comprised of entire genes or gene
pathways, or any combination thereof.
[1636] In vivo Shuffling
[1637] In an embodiment of in vivo shuffling, the mixed population
of the specific nucleic acid sequence is introduced into bacterial
or eukaryotic cells under conditions such that at least two
different nucleic acid sequences are present in each host cell. The
polynucleotides can be introduced into the host cells by a variety
of different methods. The host cells can be transformed with the
smaller polynucleotides using methods known in the art, for example
treatment with calcium chloride. If the polynucleotides are
inserted into a phage genome, the host cell can be transfected with
the recombinant phage genome having the specific nucleic acid
sequences. Alternatively, the nucleic acid sequences can be
introduced into the host cell using electroporation, transfection,
lipofection, biolistics, conjugation, and the like.
[1638] In general, in this embodiment, the specific nucleic acids
sequences will be present in vectors which are capable of stably
replicating the sequence in the host cell. In addition, it is
contemplated that the vectors will encode a marker gene such that
host cells having the vector can be selected. This ensures that the
mutated specific nucleic acid sequence can be recovered after
introduction into the host cell. However, it is contemplated that
the entire mixed population of the specific nucleic acid sequences
need not be present on a vector sequence. Rather only a sufficient
number of sequences need be cloned into vectors to ensure that
after introduction of the polynucleotides into the host cells each
host cell contains one vector having at least one specific nucleic
acid sequence present therein. It is also contemplated that rather
than having a subset of the population of the specific nucleic
acids sequences cloned into vectors, this subset may be already
stably integrated into the host cell.
[1639] It has been found that when two polynucleotides which have
regions of identity are inserted into the host cells homologous
recombination occurs between the two polynucleotides. Such
recombination between the two mutated specific nucleic acid
sequences will result in the production of double or triple hybrids
in some situations.
[1640] It has also been found that the frequency of recombination
is increased if some of the mutated specific nucleic acid sequences
are present on linear nucleic acid molecules. Therefore, in a
preferred embodiment, some of the specific nucleic acid sequences
are present on linear polynucleotides.
[1641] After transformation, the host cell transformants are placed
under selection to identify those host cell transformants which
contain mutated specific nucleic acid sequences having the
qualities desired. For example, if increased resistance to a
particular drug is desired then the transformed host cells may be
subjected to increased concentrations of the particular drug and
those transformants producing mutated proteins able to confer
increased drug resistance will be selected. If the enhanced ability
of a particular protein to bind to a receptor is desired, then
expression of the protein can be induced from the transformants and
the resulting protein assayed in a ligand binding assay by methods
known in the art to identify that subset of the mutated population
which shows enhanced binding to the ligand. Alternatively, the
protein can be expressed in another system to ensure proper
processing.
[1642] Once a subset of the first recombined specific nucleic acid
sequences (daughter sequences) having the desired characteristics
are identified, they are then subject to a second round of
recombination.
[1643] In the second cycle of recombination, the recombined
specific nucleic acid sequences may be mixed with the original
mutated specific nucleic acid sequences (parent sequences) and the
cycle repeated as described above. In this way a set of second
recombined specific nucleic acids sequences can be identified which
have enhanced characteristics or encode for proteins having
enhanced properties. This cycle can be repeated a number of times
as desired.
[1644] It is also contemplated that in the second or subsequent
recombination cycle, a backcross can be performed. A molecular
backcross can be performed by mixing the desired specific nucleic
acid sequences with a large number of the wild-type sequence, such
that at least one wild-type nucleic acid sequence and a mutated
nucleic acid sequence are present in the same host cell after
transformation. Recombination with the wild-type specific nucleic
acid sequence will eliminate those neutral mutations that may
affect unselected characteristics such as immunogenicity but not
the selected characteristics.
[1645] In another embodiment of this invention, it is contemplated
that during the first round a subset of the specific nucleic acid
sequences can be generated as smaller polynucleotides by slowing or
halting their PCR amplification prior to introduction into the host
cell. The size of the polynucleotides must be large enough to
contain some regions of identity with the other sequences so as to
homologously recombine with the other sequences. The size of the
polynucleotides will range from 0.03 kb to 100 kb more preferably
from 0.2 kb to 10 kb. It is also contemplated that in subsequent
rounds, all of the specific nucleic acid sequences other than the
sequences selected from the previous round may be utilized to
generate PCR polynucleotides prior to introduction into the host
cells.
[1646] The shorter polynucleotide sequences can be single-stranded
or double-stranded. If the sequences were originally
single-stranded and have become double-stranded they can be
denatured with heat, chemicals or enzymes prior to insertion into
the host cell. The reaction conditions suitable for separating the
strands of nucleic acid are well known in the art.
[1647] The steps of this process can be repeated indefinitely,
being limited only by the number of possible hybrids which can be
achieved. After a certain number of cycles, all possible hybrids
will have been achieved and further cycles are redundant.
[1648] In an embodiment the same mutated template nucleic acid is
repeatedly recombined and the resulting recombinants selected for
the desired characteristic.
[1649] Therefore, the initial pool or population of mutated
template nucleic acid is cloned into a vector capable of
replicating in a bacteria such as E. coli. The particular vector is
not essential, so long as it is capable of autonomous replication
in E. coli. In a preferred embodiment, the vector is designed to
allow the expression and production of any protein encoded by the
mutated specific nucleic acid linked to the vector. It is also
preferred that the vector contain a gene encoding for a selectable
marker.
[1650] The population of vectors containing the pool of mutated
nucleic acid sequences is introduced into the E. coli host cells.
The vector nucleic acid sequences may be introduced by
transformation, transfection or infection in the case of phage. The
concentration of vectors used to transform the bacteria is such
that a number of vectors is introduced into each cell. Once present
in the cell, the efficiency of homologous recombination is such
that homologous recombination occurs between the various vectors.
This results in the generation of hybrids (daughters) having a
combination of mutations which differ from the original parent
mutated sequences.
[1651] The host cells are then clonally replicated and selected for
the marker gene present on the vector. Only those cells having a
plasmid will grow under the selection.
[1652] The host cells which contain a vector are then tested for
the presence of favorable mutations. Such testing may consist of
placing the cells under selective pressure, for example, if the
gene to be selected is an improved drug resistance gene. If the
vector allows expression of the protein encoded by the mutated
nucleic acid sequence, then such selection may include allowing
expression of the protein so encoded, isolation of the protein and
testing of the protein to determine whether, for example, it binds
with increased efficiency to the ligand of interest.
[1653] Once a particular daughter mutated nucleic acid sequence has
been identified which confers the desired characteristics, the
nucleic acid is isolated either already linked to the vector or
separated from the vector. This nucleic acid is then mixed with the
first or parent population of nucleic acids and the cycle is
repeated.
[1654] It has been shown that by this method nucleic acid sequences
having enhanced desired properties can be selected.
[1655] In an alternate embodiment, the first generation of hybrids
are retained in the cells and the parental mutated sequences are
added again to the cells. Accordingly, the first cycle of
Embodiment I is conducted as described above. However, after the
daughter nucleic acid sequences are identified, the host cells
containing these sequences are retained.
[1656] The parent mutated specific nucleic acid population, either
as polynucleotides or cloned into the same vector is introduced
into the host cells already containing the daughter nucleic acids.
Recombination is allowed to occur in the cells and the next
generation of recombinants, or granddaughters are selected by the
methods described above.
[1657] This cycle can be repeated a number of times until the
nucleic acid or peptide having the desired characteristics is
obtained. It is contemplated that in subsequent cycles, the
population of mutated sequences which are added to the preferred
hybrids may come from the parental hybrids or any subsequent
generation.
[1658] In an alternative embodiment, the invention provides a
method of conducting a "molecular" backcross of the obtained
recombinant specific nucleic acid in order to eliminate any neutral
mutations. Neutral mutations are those mutations which do not
confer onto the nucleic acid or peptide the desired properties.
Such mutations may however confer on the nucleic acid or peptide
undesirable characteristics. Accordingly, it is desirable to
eliminate such neutral mutations. The method of this invention
provide a means of doing so.
[1659] In this embodiment, after the hybrid nucleic acid, having
the desired characteristics, is obtained by the methods of the
embodiments, the nucleic acid, the vector having the nucleic acid
or the host cell containing the vector and nucleic acid is
isolated.
[1660] The nucleic acid or vector is then introduced into the host
cell with a large excess of the wild-type nucleic acid. The nucleic
acid of the hybrid and the nucleic acid of the wild-type sequence
are allowed to recombine. The resulting recombinants are placed
under the same selection as the hybrid nucleic acid. Only those
recombinants which retained the desired characteristics will be
selected. Any silent mutations which do not provide the desired
characteristics will be lost through recombination with the
wild-type DNA. This cycle can be repeated a number of times until
all of the silent mutations are eliminated.
[1661] Thus the methods of this invention can be used in a
molecular backcross to eliminate unnecessary or silent
mutations.
2.11.2.3. EXONUCLEASE-MEDIATED REASSEMBLY
[1662] In a particular embodiment, this invention provides for a
method for shuffling, assembling, reassembling, recombining,
&/or concatenating at least two polynucleotides to form a
progeny polynucleotide (e.g. a chimeric progeny polynucleotide that
can be expressed to produce a polypeptide or a gene pathway). In a
particular embodiment, a double stranded polynucleotide end (e.g.
two single stranded sequences hybridized to each other as
hybridization partners) is treated with an exonuclease to liberate
nucleotides from one of the two strands, leaving the remaining
strand free of its original partner so that, if desired, the
remaining strand may be used to achieve hybridization to another
partner.
[1663] In a particular aspect, a double stranded polynucleotide end
(that may be part of--or connected to--a polynucleotide or a
nonpolynucleotide sequence) is subjected to a source of exonuclease
activity. Serviceable sources of exonuclease activity may be an
enzyme with 3' exonuclease activity, an enzyme with 5' exonuclease
activity, an enzyme with both 3' exonuclease activity and 5'
exonuclease activity, and any combination thereof. An exonuclease
can be used to liberate nucleotides from one or both ends of a
linear double stranded polynucleotide, and from one to all ends of
a branched polynucleotide having more than two ends. The mechanism
of action of this liberation is believed to be comprised of an
enzymatically-catalyzed hydrolysis of terminal nucleotides, and can
be allowed to proceed in a time-dependent fashion, allowing
experimental control of the progression of the enzymatic
process.
[1664] By contrast, a non-enzymatic step may be used to shuffle,
assemble, reassemble, recombine, and/or concatenate polynucleotide
building blocks that is comprised of subjecting a working sample to
denaturing (or "melting") conditions (for example, by changing
temperature, pH, and /or salinity conditions) so as to melt a
working set of double stranded polynucleotides into single
polynucleotide strands. For shuffling, it is desirable that the
single polynucleotide strands participate to some extent in
annealment with different hybridization partners (i.e. and not
merely revert to exclusive reannealment between what were former
partners before the denaturation step). The presence of the former
hybridization partners in the reaction vessel, however, does not
preclude, and may sometimes even favor, reannealment of a single
stranded polynucleotide with its former partner, to recreate an
original double stranded polynucleotide.
[1665] In contrast to this non-enzymatic shuffling step comprised
of subjecting double stranded polynucleotide building blocks to
denaturation, followed by annealment, the instant invention further
provides an exonuclease-based approach requiring no
denaturation--rather, the avoidance of denaturing conditions and
the maintenance of double stranded polynucleotide substrates in
annealed (i.e. non-denatured) state are necessary conditions for
the action of exonucleases (e.g., exonuclease III and red alpha
gene product). Additionally in contrast, the generation of single
stranded polynucleotide sequences capable of hybridizing to other
single stranded polynucleotide sequences is the result of covalent
cleavage--and hence sequence destruction--in one of the
hybridization partners. For example, an exonuclease III enzyme may
be used to enzymatically liberate 3' terminal nucleotides in one
hybridization strand (to achieve covalent hydrolysis in that
polynucleotide strand); and this favors hybridization of the
remaining single strand to a new partner (since its former partner
was subjected to covalent cleavage).
[1666] By way of further illustration, a specific exonuclease,
namely exonuclease III is provided herein as an example of a 3'
exonuclease; however, other exonucleases may also be used,
including enzymes with 5' exonuclease activity and enzymes with 3'
exonuclease activity, and including enzymes not yet discovered and
enzymes not yet developed. It is particularly appreciated that
enzymes can be discovered, optimized (e.g. engineered by directed
evolution), or both discovered and optimized specifically for the
instantly disclosed approach that have more optimal rates &/or
more highly specific activities &/or greater lack of unwanted
activities. In fact it is expected that the instant invention may
encourage the discovery &/or development of such designer
enzymes. In sum, this invention may be practiced with a variety of
currently available exonuclease enzymes, as well enzymes not yet
discovered and enzymes not yet developed.
[1667] The exonuclease action of exonuclease III requires a working
double stranded polynucleotide end that is either blunt or has a 5'
overhang, and the exonuclease action is comprised of enzymatically
liberating 3' terminal nucleotides, leaving a single stranded 5'
end that becomes longer and longer as the exonuclease action
proceeds (see FIG. 1). Any 5' overhangs produced by this approach
may be used to hybridize to another single stranded polynucleotide
sequence (which may also be a single stranded polynucleotide or a
terminal overhang of a partially double stranded polynucleotide)
that shares enough homology to allow hybridization. The ability of
these exonuclease III-generated single stranded sequences (e.g. in
5' overhangs) to hybridize to other single stranded sequences
allows two or more polynucleotides to be shuffled, assembled,
reassembled, &/or concatenated.
[1668] Furthermore, it is appreciated that one can protect the end
of a double stranded polynucleotide or render it susceptible to a
desired enzymatic action of a serviceable exonuclease as necessary.
For example, a double stranded polynucleotide end having a 3'
overhang is not susceptible to the exonuclease action of
exonuclease III. However, it may be rendered susceptible to the
exonuclease action of exonuclease III by a variety of means; for
example, it may be blunted by treatment with a polymerase, cleaved
to provide a blunt end or a 5' overhang, joined (ligated or
hybridized) to another double stranded polynucleotide to provide a
blunt end or a 5' overhang, hybridized to a single stranded
polynucleotide to provide a blunt end or a 5' overhang, or modified
by any of a variety of means).
[1669] According to one aspect, an exonuclease may be allowed to
act on one or on both ends of a linear double stranded
polynucleotide and proceed to completion, to near completion, or to
partial completion. When the exonuclease action is allowed to go to
completion, the result will be that the length of each 5' overhang
will be extend far towards the middle region of the polynucleotide
in the direction of what might be considered a "rendezvous point"
(which may be somewhere near the polynucleotide midpoint).
Ultimately, this results in the production of single stranded
polynucleotides (that can become dissociated) that are each about
half the length of the original double stranded polynucleotide (see
FIG. 1). Alternatively, an exonuclease-mediated reaction can be
terminated before proceeding to completion.
[1670] Thus this exonuclease-mediated approach is serviceable for
shuffling, assembling &/or reassembling, recombining, and
concatenating polynucleotide building blocks, which polynucleotide
building blocks can be up to ten bases long or tens of bases long
or hundreds of bases long or thousands of bases long or tens of
thousands of bases long or hundreds of thousands of bases long or
millions of bases long or even longer.
[1671] This exonuclease-mediated approach is based on the action of
double stranded DNA specific exodeoxyribonuclease activity of E.
coli exonuclease III. Substrates for exonuclease III may be
generated by subjecting a double stranded polynucleotide to
fragmentation. Fragmentation may be achieved by mechanical means
(e.g., shearing, sonication, etc.), by enzymatic means (e.g. using
restriction enzymes), and by any combination thereof. Fragments of
a larger polynucleotide may also be generated by
polymerase-mediated synthesis.
[1672] Exonuclease III is a 28K monomeric enzyme, product of the
xthA gene of E. coli with four known activities:
exodeoxyribonuclease (alternatively referred to as exonuclease
herein), RNaseH, DNA-3'-phosphatase, and AP endonuclease. The
exodeoxyribonuclease activity is specific for double stranded DNA.
The mechanism of action is thought to involve enzymatic hydrolysis
of DNA from a 3' end progressively towards a 5' direction, with
formation of nucleoside 5'-phosphates and a residual single strand.
The enzyme does not display efficient hydrolysis of single stranded
DNA, single-stranded RNA, or double-stranded RNA; however it
degrades RNA in an DNA-RNA hybrid releasing nucleoside
5'-phosphates. The enzyme also releases inorganic phosphate
specifically from 3' phosphomonoester groups on DNA, but not from
RNA or short oligonucleotides. Removal of these groups converts the
terminus into a primer for DNA polymerase action.
[1673] Additional examples of enzymes with exonuclease activity
include red-alpha and venom phosphodiesterases. Red alpha
(red.alpha.) gene product (also referred to as lambda exonuclease)
is of bacteriophage .lambda. origin. The red.alpha. gene is
transcribed from the leftward promoter and its product is involved
(24 kD) in recombination. Red alpha gene product acts processively
from 5'-phosphorylated termini to liberate mononucleotides from
duplex DNA (Takahashi & Kobayashi, 1990). Venom
phosphodiesterases (Laskowski, 1980) is capable of rapidly opening
supercoiled DNA.
2.11.2.3. NON-STOCHASTIC LIGATION REASSEMBLY
[1674] In one aspect, the present invention provides a
non-stochastic method termed synthetic ligation reassembly (SLR),
that is somewhat related to stochastic shuffling, save that the
nucleic acid building blocks are not shuffled or concatenated or
chimerized randomly, but rather are assembled
non-stochastically.
[1675] A particularly glaring difference is that the instant SLR
method does not depend on the presence of a high level of homology
between polynucleotides to be shuffled. In contrast, prior methods,
particularly prior stochastic shuffling methods require that
presence of a high level of homology, particularly at coupling
sites, between polynucleotides to be shuffled. Accordingly these
prior methods favor the regeneration of the original progenitor
molecules, and are suboptimal for generating large numbers of novel
progeny chimeras, particularly full-length progenies. The instant
invention, on the other hand, can be used to non-stochastically
generate libraries (or sets) of progeny molecules comprised of over
10.sup.100 different chimeras. Conceivably, SLR can even be used to
generate libraries comprised of over 10.sup.1000 different progeny
chimeras with (no upper limit in sight).
[1676] Thus, in one aspect, the present invention provides a
method, which method is non-stochastic, of producing a set of
finalized chimeric nucleic acid molecules having an overall
assembly order that is chosen by design, which method is comprised
of the steps of generating by design a plurality of specific
nucleic acid building blocks having serviceable mutually compatible
ligatable ends, and assembling these nucleic acid building blocks,
such that a designed overall assembly order is achieved.
[1677] The mutually compatible ligatable ends of the nucleic acid
building blocks to be assembled are considered to be "serviceable"
for this type of ordered assembly if they enable the building
blocks to be coupled in predetermined orders. Thus, in one aspect,
the overall assembly order in which the nucleic acid building
blocks can be coupled is specified by the design of the ligatable
ends and, if more than one assembly step is to be used, then the
overall assembly order in which the nucleic acid building blocks
can be coupled is also specified by the sequential order of the
assembly step(s). FIG. 4, Panel C illustrates an exemplary assembly
process comprised of 2 sequential steps to achieve a designed
(non-stochastic) overall assembly order for five nucleic acid
building blocks. In a preferred embodiment of this invention, the
annealed building pieces are treated with an enzyme, such as a
ligase (e.g. T4 DNA ligase), achieve covalent bonding of the
building pieces.
[1678] In a preferred embodiment, the design of nucleic acid
building blocks is obtained upon analysis of the sequences of a set
of progenitor nucleic acid templates that serve as a basis for
producing a progeny set of finalized chimeric nucleic acid
molecules. These progenitor nucleic acid templates thus serve as a
source of sequence information that aids in the design of the
nucleic acid building blocks that are to be mutagenized, i.e.
chimerized or shuffled.
[1679] In one exemplification, this invention provides for the
chimerization of a family of related genes and their encoded family
of related products. In a particular exemplification, the encoded
products are enzymes. As a representative list of families of
enzymes which may be mutagenized in accordance with the aspects of
the present invention, there may be mentioned, the following
enzymes and their functions:
[1680] 1 Lipase/Esterase
[1681] a. Enantioselective hydrolysis of esters
(lipids)/thioesters
[1682] 1) Resolution of racemic mixtures
[1683] 2) Synthesis of optically active acids or alcohols from
meso-diesters
[1684] b. Selective syntheses
[1685] 1) Regiospecific hydrolysis of carbohydrate esters
[1686] 2) Selective hydrolysis of cyclic secondary alcohols
[1687] c. Synthesis of optically active esters, lactones, acids,
alcohols
[1688] 1) Transesterification of activated/nonactivated esters
[1689] 2) Interesterification
[1690] 3) Optically active lactones from hydroxyesters
[1691] 4) Regio- and enantioselective ring opening of
anhydrides
[1692] d. Detergents
[1693] e. Fat/Oil conversion
[1694] f. Cheese ripening
[1695] 2 Protease
[1696] a. Ester/amide synthesis
[1697] b. Peptide synthesis
[1698] c. Resolution of racemic mixtures of amino acid esters
[1699] d. Synthesis of non-natural amino acids
[1700] e. Detergents/protein hydrolysis
[1701] 3 Glycosidase/Glycosyl Transferase
[1702] a. Sugar/polymer synthesis
[1703] b. Cleavage of glycosidic linkages to form mono, di-and
oligosaccharides
[1704] c. Synthesis of complex oligosaccharides
[1705] d. Glycoside synthesis using UDP-galactosyl transferase
[1706] e. Transglycosylation of disaccharides, glycosyl fluorides,
aryl galactosides
[1707] f. Glycosyl transfer in oligosaccharide synthesis
[1708] g. Diastereoselective cleavage of
.beta.-glucosylsulfoxides
[1709] h. Asymmetric glycosylations
[1710] i. Food processing
[1711] j. Paper processing
[1712] 4 Phosphatase/Kinase
[1713] a. Synthesis/hydrolysis of phosphate esters
[1714] 1) Regio-, enantioselective phosphorylation
[1715] 2) Introduction of phosphate esters
[1716] 3) Synthesize phospholipid precursors
[1717] 4) Controlled polynucleotide synthesis
[1718] b. Activate biological molecule
[1719] c. Selective phosphate bond formation without protecting
groups
[1720] 5 Mono/Dioxygenase
[1721] a. Direct oxyfunctionalization of unactivated organic
substrates
[1722] b. Hydroxylation of alkane, aromatics, steroids
[1723] c. Epoxidation of alkenes
[1724] d. Enantioselective sulphoxidation
[1725] e. Regio- and stereoselective Bayer-Villiger oxidations
[1726] 6 Haloperoxidase
[1727] a. Oxidative addition of halide ion to nucleophilic
sites
[1728] b. Addition of hypohalous acids to olefinic bonds
[1729] c. Ring cleavage of cyclopropanes
[1730] d. Activated aromatic substrates converted to ortho and para
derivatives
[1731] e. 1.3 diketones converted to 2-halo-derivatives
[1732] f. Heteroatom oxidation of sulfur and nitrogen containing
substrates
[1733] g. Oxidation of enol acetates, alkynes and activated
aromatic rings
[1734] 7 Lignin Peroxidase/Diarylpropane Peroxidase
[1735] a. Oxidative cleavage of C-C bonds
[1736] b. Oxidation of benzylic alcohols to aldehydes
[1737] c. Hydroxylation of benzylic carbons
[1738] d. Phenol dimerization
[1739] e. Hydroxylation of double bonds to form diols
[1740] f. Cleavage of lignin aldehydes
[1741] 8 Epoxide Hydrolase
[1742] a. Synthesis of enantiomerically pure bioactive
compounds
[1743] b. Regio- and enantioselective hydrolysis of epoxide
[1744] c. Aromatic and olefinic epoxidation by monooxygenases to
form epoxides
[1745] d. Resolution of racemic epoxides
[1746] e. Hydrolysis of steroid epoxides
[1747] 9 Nitrile hydratase/nitrilase
[1748] a. Hydrolysis of aliphatic nitrites to carboxamides
[1749] b. Hydrolysis of aromatic, heterocyclic, unsaturated
aliphatic nitrites to corresponding acids
[1750] c. Hydrolysis of acrylonitrile
[1751] d. Production of aromatic and carboxamides, carboxylic acids
(nicotinamide, picolinamide, isonicotinamide)
[1752] e. Regioselective hydrolysis of acrylic dinitrile
[1753] f. .alpha.-amino acids from .alpha.-hydroxynitriles
[1754] 10 Transaminase
[1755] a. Transfer of amino groups into oxo-acids
[1756] 11 Amidase/Acylase
[1757] a. Hydrolysis of amides, amidines, and other C-N bonds
[1758] b. Non-natural amino acid resolution and synthesis
[1759] These exemplifications, while illustrating certain specific
aspects of the invention, do not portray the limitations or
circumscribe the scope of the disclosed invention.
[1760] Thus according to one aspect of this invention, the
sequences of a plurality of progenitor nucleic acid templates are
aligned in order to select one or more demarcation points, which
demarcation points can be located at an area of homology, and are
comprised of one or more nucleotides, and which demarcation points
are shared by at least two of the progenitor templates. The
demarcation points can be used to delineate the boundaries of
nucleic acid building blocks to be generated. Thus, the demarcation
points identified and selected in the progenitor molecules serve as
potential chimerization points in the assembly of the progeny
molecules.
[1761] Preferably a serviceable demarcation point is an area of
homology (comprised of at least one homologous nucleotide base)
shared by at least two progenitor templates. More preferably a
serviceable demarcation point is an area of homology that is shared
by at least half of the progenitor templates. More preferably still
a serviceable demarcation point is an area of homology that is
shared by at least two thirds of the progenitor templates. Even
more preferably a serviceable demarcation points is an area of
homology that is shared by at least three fourths of the progenitor
templates. Even more preferably still a serviceable demarcation
points is an area of homology that is shared by at almost all of
the progenitor templates. Even more preferably still a serviceable
demarcation point is an area of homology that is shared by all of
the progenitor templates.
[1762] The process of designing nucleic acid building blocks and of
designing the mutually compatible ligatable ends of the nucleic
acid building blocks to be assembled is illustrated in FIGS. 6 and
7. As shown, the alignment of a set of progenitor templates reveals
several naturally occurring demarcation points, and the
identification of demarcation points shared by these templates
helps to non-stochastically determine the building blocks to be
generated and used for the generation of the progeny chimeric
molecules.
[1763] In a preferred embodiment, this invention provides that the
ligation reassembly process is performed exhaustively in order to
generate an exhaustive library. In other words, all possible
ordered combinations of the nucleic acid building blocks are
represented in the set of finalized chimeric nucleic acid
molecules. At the same time, in a particularly preferred
embodiment, the assembly order (i.e. the order of assembly of each
building block in the 5' to 3 sequence of each finalized chimeric
nucleic acid) in each combination is by design (or non-stochastic).
Because of the non-stochastic nature of this invention, the
possibility of unwanted side products is greatly reduced.
[1764] In another preferred embodiment, this invention provides
that, the ligation reassembly process is performed systematically,
for example in order to generate a systematically compartmentalized
library, with compartments that can be screened systematically,
e.g. one by one. In other words this invention provides that,
through the selective and judicious use of specific nucleic acid
building blocks, coupled with the selective and judicious use of
sequentially stepped assembly reactions, an experimental design can
be achieved where specific sets of progeny products are made in
each of several reaction vessels. This allows a systematic
examination and screening procedure to be performed. Thus, it
allows a potentially very large number of progeny molecules to be
examined systematically in smaller groups.
[1765] Because of its ability to perform chimerizations in a manner
that is highly flexible yet exhaustive and systematic as well,
particularly when there is a low level of homology among the
progenitor molecules, the instant invention provides for the
generation of a library (or set) comprised of a large number of
progeny molecules. Because of the non-stochastic nature of the
instant ligation reassembly invention, the progeny molecules
generated preferably comprise a library of finalized chimeric
nucleic acid molecules having an overall assembly order that is
chosen by design. In a particularly preferred embodiment of this
invention, such a generated library is comprised of preferably
greater than 10.sup.3 different progeny molecular species, more
preferably greater than 10.sup.5 different progeny molecular
species, more preferably still greater than 10.sup.10 different
progeny molecular species, more preferably still greater than
10.sup.15 different progeny molecular species, more preferably
still greater than 10.sup.20 different progeny molecular species,
more preferably still greater than 10.sup.30 different progeny
molecular species, more preferably still greater than 10.sup.40
different progeny molecular species, more preferably still greater
than 10.sup.50 different progeny molecular species, more preferably
still greater than 10.sup.60 different progeny molecular species,
more preferably still greater than 10.sup.70 different progeny
molecular species, more preferably still greater than 10.sup.80
different progeny molecular species, more preferably still greater
than 10.sup.100 different progeny molecular species, more
preferably still greater than 10.sup.110 different progeny
molecular species, more preferably still greater than 10.sup.120
different progeny molecular species, more preferably still greater
than 10.sup.130 different progeny molecular species, more
preferably still greater than 10.sup.140 different progeny
molecular species, more preferably still greater than 10.sup.150
different progeny molecular species, more preferably still greater
than 10.sup.175 different progeny molecular species, more
preferably still greater than 10.sup.200 different progeny
molecular species, more preferably still greater than 10.sup.300
different progeny molecular species, more preferably still greater
than 10.sup.400 different progeny molecular species, more
preferably still greater than 10.sup.500 different progeny
molecular species, and even more preferably still greater than
10.sup.1000 different progeny molecular species.
[1766] In one aspect, a set of finalized chimeric nucleic acid
molecules, produced as described is comprised of a polynucleotide
encoding a polypeptide. According to one preferred embodiment, this
polynucleotide is a gene, which may be a man-made gene. According
to another preferred embodiment, this polynucleotide is a gene
pathway, which may be a man-made gene pathway. This invention
provides that one or more man-made genes generated by this
invention may be incorporated into a man-made gene pathway, such as
pathway operable in a eukaryotic organism (including a plant).
[1767] It is appreciated that the power of this invention is
exceptional, as there is much freedom of choice and control
regarding the selection of demarcation points, the size and number
of the nucleic acid building blocks, and the size and design of the
couplings. It is appreciated, furthermore, that the requirement for
intermolecular homology is highly relaxed for the operability of
this invention. In fact, demarcation points can even be chosen in
areas of little or no intermolecular homology. For example, because
of codon wobble, i.e. the degeneracy of codons, nucleotide
substitutions can be introduced into nucleic acid building blocks
without altering the amino acid originally encoded in the
corresponding progenitor template. Alternatively, a codon can be
altered such that the coding for an originally amino acid is
altered. This inventiop provides that such substitutions can be
introduced into the nucleic acid building block in order to
increase the incidence of intermolecularly homologous demarcation
points and thus to allow an increased number of couplings to be
achieved among the building blocks, which in turn allows a greater
number of progeny chimeric molecules to be generated.
[1768] In another exemplifaction, the synthetic nature of the step
in which the building blocks are generated allows the design and
introduction of nucleotides (e.g. one or more nucleotides, which
may be, for example, codons or introns or regulatory sequences)
that can later be optionally removed in an in vitro process (e.g.
by mutageneis) or in an in vivo process (e.g. by utilizing the gene
splicing ability of a host organism). It is appreciated that in
many instances the introduction of these nucleotides may also be
desirable for many other reasons in addition to the potential
benefit of creating a serviceable demarcation point.
[1769] Thus, according to another embodiment, this invention
provides that a nucleic acid building block can be used to
introduce an intron. Thus, this invention provides that functional
introns may be introduced into a man-made gene of this invention.
This invention also provides that functional introns may be
introduced into a man-made gene pathway of this invention.
Accordingly, this invention provides for the generation of a
chimeric polynucleotide that is a man-made gene containing one (or
more) artificially introduced intron(s).
[1770] Accordingly, this invention also provides for the generation
of a chimeric polynucleotide that is a man-made gene pathway
containing one (or more) artificially introduced intron(s).
Preferably, the artificially introduced intron(s) are functional in
one or more host cells for gene splicing much in the way that
naturally-occurring introns serve functionally in gene splicing.
This invention provides a process of producing man-made
intron-containing polynucleotides to be introduced into host
organisms for recombination and/or splicing.
[1771] The ability to achieve chimerizations, using couplings as
described herein, in areas of little or no homology among the
progenitor molecules, is particularly useful, and in fact critical,
for the assembly of novel gene pathways. This invention thus
provides for the generation of novel man-made gene pathways using
synthetic ligation reassembly. In a particular aspect, this is
achieved by the introduction of regulatory sequences, such as
promoters, that are operable in an intended host, to confer
operability to a novel gene pathway when it is introduced into the
intended host. In a particular exemplification, this invention
provides for the generation of novel man-made gene pathways that is
operable in a plurality of intended hosts (e.g. in a microbial
organism as well as in a plant cell). This can be achieve, for
example, by the introduction of a plurality of regulatory
sequences, comprised of a regulatory sequence that is operable in a
first intended host and a regulatory sequence that is operable in a
second intended host. A similar process can be performed to achieve
operability of a gene pathway in a third intended host species,
etc. The number of intended host species can be each integer from 1
to 10 or alternatively over 10. Alternatively, for example,
operability of a gene pathway in a plurality of intended hosts can
be achieved by the introduction of a regulatory sequence having
intrinsic operability in a plurality of intended hosts.
[1772] Thus, according to a particular embodiment, this invention
provides that a nucleic acid building block can be used to
introduce a regulatory sequence, particularly a regulatory sequence
for gene expression. Preferred regulatory sequences include, but
are not limited to, those that are man-made, and those found in
archeal, bacterial, eukaryotic (including mitochondrial), viral,
and prionic or prion-like organisms. Preferred regulatory sequences
include but are not limited to, promoters, operators, and activator
binding sites. Thus, this invention provides that functional
regulatory sequences may be introduced into a man-made gene of this
invention. This invention also provides that functional regulatory
sequences may be introduced into a man-made gene pathway of this
invention.
[1773] Accordingly, this invention provides for the generation of a
chimeric polynucleotide that is a man-made gene containing one (or
more) artificially introduced regulatory sequence(s). Accordingly,
this invention also provides for the generation of a chimeric
polynucleotide that is a man-made gene pathway containing one (or
more) artificially introduced regulatory sequence(s). Preferably,
an artificially introduced regulatory sequence(s) is operatively
linked to one or more genes in the man-made polynucleotide, and are
functional in one or more host cells.
[1774] Preferred bacterial promoters that are serviceable for this
invention include lacI, lacZ, T3, T7, gpt, lambda P.sub.R, P.sub.L
and trp. Serviceable eukaryotic promoters include CMV immediate
early, HSV thymidine kinase, early and late SV40, LTRs from
retrovirus, and mouse metallothionein-I. Particular plant
regulatory sequences include promoters active in directing
transcription in plants, either constitutively or stage and/or
tissue specific, depending on the use of the plant or parts
thereof. These promoters include, but are not limited to promoters
showing constitutive expression, such as the 35S promoter of
Cauliflower Mosaic Virus (CaMV) (Guilley et al., 1982), those for
leaf-specific expression, such as the promoter of the ribulose
bisphosphate carboxylase small subunit gene (Coruzzi et al., 1984),
those for root-specific expression, such as the promoter from the
glutamin synthase gene (Tingey et al., 1987), those for
seed-specific expression, such as the cruciferin A promoter from
Brassica napus (Ryan et al., 1989), those for tuber-specific
expression, such as the class-I patatin promoter from potato
(Rocha-Sasa et al., 1989; Wenzler et al., 1989) or those for
fruit-specific expression, such as the polygalacturonase (PG)
promoter from tomato (Bird et al., 1988).
[1775] Other regulatory sequences that are preferred for this
invention include terminator sequences and polyadenylation signals
and any such sequence functioning as such in plants, the choice of
which is within the level of the skilled artisan. An example of
such sequences is the 3' flanking region of the nopaline synthase
(nos) gene of Agrobacterium tumefaciens (Bevan, 1984). The
regulatory sequences may also include enhancer sequences, such as
found in the 35S promoter of CaMV, and mRNA stabilizing sequences
such as the leader sequence of Alfalfa Mosaic Cirus (A1MV) RNA4
(Brederode et al., 1980) or any other sequences functioning in a
like manner.
[1776] A man-made genes produced using this invention can also
serve as a substrate for recombination with another nucleic acid.
Likewise, a man-made gene pathway produced using this invention can
also serve as a substrate for recombination with another nucleic
acid. In a preferred instance, the recombination is facilitated by,
or occurs at, areas of homology between the man-made
intron-containing gene and a nucleic acid with serves as a
recombination partner. In a particularly preferred instance, the
recombination partner may also be a nucleic acid generated by this
invention, including a man-made gene or a man-made gene pathway.
Recombination may be facilitated by or may occur at areas of
homology that exist at the one (or more) artificially introduced
intron(s) in the man-made gene.
[1777] The synthetic ligation reassembly method of this invention
utilizes a plurality of nucleic acid building blocks, each of which
preferably has two ligatable ends. The two ligatable ends on each
nucleic acid building block may be two blunt ends (i.e. each having
an overhang of zero nucleotides), or preferably one blunt end and
one overhang, or more preferably still two overhangs.
[1778] A serviceable overhang for this purpose may be a 3' overhang
or a 5' overhang. Thus, a nucleic acid building block may have a 3'
overhang or alternatively a 5' overhang or alternatively two 3'
overhangs or alternatively two 5' overhangs. The overall order in
which the nucleic acid building blocks are assembled to form a
finalized chimeric nucleic acid molecule is determined by
purposeful experimental design and is not random.
[1779] According to one preferred embodiment, a nucleic acid
building block is generated by chemical synthesis of two
single-stranded nucleic acids (also referred to as single-stranded
oligos) and contacting them so as to allow them to anneal to form a
double-stranded nucleic acid building block.
[1780] A double-stranded nucleic acid building block can be of
variable size. The sizes of these building blocks can be small or
large depending on the choice of the experimenter. Preferred sizes
for building block range from 1 base pair (not including any
overhangs) to 100,000 base pairs (not including any overhangs).
Other preferred size ranges are also provided, which have lower
limits of from 1 bp to 10,000 bp (including every integer value in
between), and upper limits of from 2 bp to 100, 000 bp (including
every integer value in between).
[1781] It is appreciated that current methods of polymerase-based
amplification can be used to generate double-stranded nucleic acids
of up to thousands of base pairs, if not tens of thousands of base
pairs, in length with high fidelity. Chemical synthesis (e.g.
phosphoramidite-based) can be used to generate nucleic acids of up
to hundreds of nucleotides in length with high fidelity; however,
these can be assembled, e.g. using overhangs or sticky ends, to
form double-stranded nucleic acids of up to thousands of base
pairs, if not tens of thousands of base pairs, in length if so
desired.
[1782] A combination of methods (e.g. phosphoramidite-based
chemical synthesis and PCR) can also be used according to this
invention. Thus, nucleic acid building block made by different
methods can also be used in combination to generate a progeny
molecule of this invention.
[1783] The use of chemical synthesis to generate nucleic acid
building blocks is particularly preferred in this invention &
is advantageous for other reasons as well, including procedural
safety and ease. No cloning or harvesting or actual handling of any
biological samples is required. The design of the nucleic acid
building blocks can be accomplished on paper. Accordingly, this
invention teaches an advance in procedural safety in recombinant
technologies.
[1784] Nonetheless, according to one preferred embodiment, a
double-stranded nucleic acid building block according to this
invention may also be generated by polymerase-based amplification
of a polynucleotide template. In a non-limiting exemplification, as
illustrated in FIG. 2, a first polymerase-based amplification
reaction using a first set of primers, F.sub.2 and R.sub.1, is used
to generate a blunt-ended product (labeled Reaction 1, Product 1),
which is essentially identical to Product A. A second
polymerase-based amplification reaction using a second set of
primers, F.sub.1 and R.sub.2, is used to generate a blunt-ended
product (labeled Reaction 2, Product 2), which is essentially
identical to Product B. These two products are mixed and allowed to
melt and anneal, generating potentially useful double-stranded
nucleic acid building blocks with two overhangs. In the example of
FIG. 2, the product with the 3' overhangs (Product C) is selected
by nuclease-based degradation of the other 3 products using a 3'
acting exonuclease, such as exonuclease III. It is appreciated that
a 5' acting exonuclease (e.g. red alpha) may be also be used, for
example to select Product D instead. It is also appreciated that
other selection means can also be used, including
hybridization-based means, and that these means can incorporate a
further means, such as a magnetic bead-based means, to facilitate
separation of the desired product.
[1785] Many other methods exist by which a double-stranded nucleic
acid building block can be generated that is serviceable for this
invention; and these are known in the art and can be readily
performed by the skilled artisan.
[1786] According to particularly preferred embodiment, a
double-stranded nucleic acid building block that is serviceable for
this invention is generated by first generating two single stranded
nucleic acids and allowing them to anneal to form a double-stranded
nucleic acid building block. The two strands of a double-stranded
nucleic acid building block may be complementary at every
nucleotide apart from any that form an overhang; thus containing no
mismatches, apart from any overhang(s). According to another
embodiment, the two strands of a double-stranded nucleic acid
building block are complementary at fewer than every nucleotide
apart from any that form an overhang. Thus, according to this
embodiment, a double-stranded nucleic acid building block can be
used to introduce codon degeneracy. Preferably the codon degeneracy
is introduced using the site-saturation mutagenesis described
herein, using one or more N,N,G/T cassettes or alternatively using
one or more N,N,N cassettes.
[1787] Contained within an exemplary experimental design for
achieving an ordered assembly according to this invention are:
[1788] 1) The design of specific nucleic acid building blocks.
[1789] 2) The design of specific ligatable ends on each nucleic
acid building block.
[1790] 3) The design of a particular order of assembly of the
nucleic acid building blocks.
[1791] An overhang may be a 3' overhang or a 5' overhang. An
overhang may also have a terminal phosphate group or alternatively
may be devoid of a terminal phosphate group (having, e.g., a
hydroxyl group instead). An overhang may be comprised of any number
of nucleotides. Preferably an overhang is comprised of 0
nucleotides (as in a blunt end) to 10,000 nucleotides. Thus, a wide
range of overhang sizes may be serviceable. Accordingly, the lower
limit may be each integer from 1-200 and the upper limit may be
each integer from 2-10,000. According to a particular
exemplification, an overhang may consist of anywhere from 1
nucleotide to 200 nucleotides (including every integer value in
between).
[1792] The final chimeric nucleic acid molecule may be generated by
sequentially assembling 2 or more building blocks at a time until
all the designated building blocks have been assembled. A working
sample may optionally be subjected to a process for size selection
or purification or other selection or enrichment process between
the performance of two assembly steps. Alternatively, the final
chimeric nucleic acid molecule may be generated by assembling all
the designated building blocks at once in one step.
[1793] Utility
[1794] The in vivo recombination method of this invention can be
performed blindly on a pool of unknown hybrids or alleles of a
specific polynucleotide or sequence. However, it is not necessary
to know the actual DNA or RNA sequence of the specific
polynucleotide.
[1795] The approach of using recombination within a mixed
population of genes can be useful for the generation of any useful
proteins, for example, interleukin I, antibodies, tPA and growth
hormone. This approach may be used to generate proteins having
altered specificity or activity. The approach may also be useful
for the generation of hybrid nucleic acid sequences, for example,
promoter regions, introns, exons, enhancer sequences, 31
untranslated regions or 51 untranslated regions of genes. Thus this
approach may be used to generate genes having increased rates of
expression. This approach may also be useful in the study of
repetitive DNA sequences. Finally, this approach may be useful to
mutate ribozymes or aptamers.
[1796] Scaffold-like regions separating regions of diversity in
proteins may be particularly suitable for the methods of this
invention. The conserved scaffold determines the overall folding by
self-association, while displaying relatively unrestricted loops
that mediate the specific binding. Examples of such scaffolds are
the immunoglobulin beta barrel, and the four-helix bundle. The
methods of this invention can be used to create scaffold-like
proteins with various combinations of mutated sequences for
binding.
[1797] The equivalents of some standard genetic matings may also be
performed by the methods of this invention. For example, a
"molecular" backcross can be performed by repeated mixing of the
hybrid's nucleic acid with the wild-type nucleic acid while
selecting for the mutations of interest. As in traditional
breeding, this approach can be used to combine phenotypes from
different sources into a background of choice. It is useful, for
example, for the removal of neutral mutations that affect
unselected characteristics (i.e. immunogenicity). Thus it can be
useful to determine which mutations in a protein are involved in
the enhanced biological activity and which are not.
2.11.2.4. END-SELECTION
[1798] This invention provides a method for selecting a subset of
polynucleotides from a starting set of polynucleotides, which
method is based on the ability to discriminate one or more
selectable features (or selection markers) present anywhere in a
working polynucleotide, so as to allow one to perform selection for
(positive selection) &/or against (negative selection) each
selectable polynucleotide. In a preferred aspect, a method is
provided termed end-selection, which method is based on the use of
a selection marker located in part or entirely in a terminal region
of a selectable polynucleotide, and such a selection marker may be
termed an "end-selection marker".
[1799] End-selection may be based on detection of naturally
occurring sequences or on detection of sequences introduced
experimentally (including by any mutagenesis procedure mentioned
herein and not mentioned herein) or on both, even within the same
polynucleotide. An end-selection marker can be a structural
selection marker or a functional selection marker or both a
structural and a functional selection marker. An end-selection
marker may be comprised of a polynucleotide sequence or of a
polypeptide sequence or of any chemical structure or of any
biological or biochemical tag, including markers that can be
selected using methods based on the detection of radioactivity, of
enzymatic activity, of fluorescence, of any optical feature, of a
magnetic property (e.g. using magnetic beads), of immunoreactivity,
and of hybridization.
[1800] End-selection may be applied in combination with any method
serviceable for performing mutagenesis. Such mutagenesis methods
include, but are not limited to, methods described herein (supra
and infra). Such methods include, by way of non-limiting
exemplification, any method that may be referred herein or by
others in the art by any of the following terms: "saturation
mutagenesis", "shuffling", "recombination", "re-assembly",
"error-prone PCR", "assembly PCR", "sexual PCR", "crossover PCR",
"oligonucleotide primer-directed mutagenesis", "recursive (&/or
exponential) ensemble mutagenesis (see Arkin and Youvan, 1992)",
"cassette mutagenesis", "in vivo mutagenesis", and "in vitro
mutagenesis". Moreover, end-selection may be performed on molecules
produced by any mutagenesis &/or amplification method (see,
e.g., Arnold, 1993; Caldwell and Joyce, 1992; Stemmer, 1994;
following which method it is desirable to select for (including to
screen for the presence of) desirable progeny molecules.
[1801] In addition, end-selection may be applied to a
polynucleotide apart from any mutagenesis method. In a preferred
embodiment, end-selection, as provided herein, can be used in order
to facilitate a cloning step, such as a step of ligation to another
polynucleotide (including ligation to a vector). This invention
thus provides for end-selection as a serviceable means to
facilitate library construction, selection &/or enrichment for
desirable polynucleotides, and cloning in general.
[1802] In a particularly preferred embodiment, end-selection can be
based on (positive) selection for a polynucleotide; alternatively
end-selection can be based on (negative) selection against a
polynucleotide; and alternatively still, end-selection can be based
on both (positive) selection for, and on (negative) selection
against, a polynucleotide. End-selection, along with other methods
of selection &/or screening, can be performed in an iterative
fashion, with any combination of like or unlike selection &/or
screening methods and serviceable mutagenesis methods, all of which
can be performed in an iterative fashion and in any order,
combination, and permutation.
[1803] It is also appreciated that, according to one embodiment of
this invention, end-selection may also be used to select a
polynucleotide is at least in part: circular (e.g. a plasmid or any
other circular vector or any other polynucleotide that is partly
circular), &/or branched, &/or modified or substituted with
any chemical group or moiety. In accord with this embodiment, a
polynucleotide may be a circular molecule comprised of an
intermediate or central region, which region is flanked on a 5'
side by a 5' flanking region (which, for the purpose of
end-selection, serves in like manner to a 5' terminal region of a
non-circular polynucleotide) and on a 3' side by a 3' terminal
region (which, for the purpose of end-selection, serves in like
manner to a 3' terminal region of a non-circular polynucleotide).
As used in this non-limiting exemplification, there may be sequence
overlap between any two regions or even among all three
regions.
[1804] In one non-limiting aspect of this invention, end-selection
of a linear polynucleotide is performed using a general approach
based on the presence of at least one end-selection marker located
at or near a polynucleotide end or terminus (that can be either a
5' end or a 3' end). In one particular non-limiting
exemplification, end-selection is based on selection for a specific
sequence at or near a terminus such as, but not limited to, a
sequence recognized by an enzyme that recognizes a polynucleotide
sequence. An enzyme that recognizes and catalyzes a chemical
modification of a polynucleotide is referred to herein as a
polynucleotide-acting enzyme. In a preferred embodiment,
serviceable polynucleotide-acting enzymes are exemplified
non-exclusively by enzymes with polynucleotide-cleaving activity,
enzymes with polynucleotide-methylating activity, enzymes with
polynucleotide-ligating activity, and enzymes with a plurality of
distinguishable enzymatic activities (including non-exclusively,
e.g., both polynucleotide-cleaving activity and
polynucleotide-ligating activity).
[1805] Relevant polynucleotide-acting enzymes thus also include any
commercially available or non-commercially available polynucleotide
endonucleases and their companion methylases including those
catalogued at the website http://www.neb.com/rebase, and those
mentioned in the following cited reference (Roberts and Macelis,
1996). Preferred polynucleotide endonucleases include--but are not
limited to--type II restriction enzymes (including type IIS), and
include enzymes that cleave both strands of a double stranded
polynucleotide (e.g. Not I, which cleaves both strands at 5'. . .
GC/GGCCGC. . .3') and enzymes that cleave only one strand of a
double stranded polynucleotide, i.e. enzymes that have
polynucleotide-nicking activity, (e.g. N. BstNB I, which cleaves
only one strand at 5'. . . GAGTCNNNN/N. . .3'). Relevant
polynucleotide-acting enzymes also include type III restriction
enzymes.
[1806] It is appreciated that relevant polynucleotide-acting
enzymes also include any enzymes that may be developed in the
future, though currently unavailable, that are serviceable for
generating a ligation compatible end, preferably a sticky end, in a
polynucleotide.
[1807] In one preferred exemplification, a serviceable selection
marker is a restriction site in a polynucleotide that allows a
corresponding type II (or type IIS) restriction enzyme to cleave an
end of the polynucleotide so as to provide a ligatable end
(including a blunt end or alternatively a sticky end with at least
a one base overhang) that is serviceable for a desirable ligation
reaction without cleaving the polynucleotide internally in a manner
that destroys a desired internal sequence in the polynucleotide.
Thus it is provided that, among relevant restriction sites, those
sites that do not occur internally (i.e. that do not occur apart
from the termini) in a specific working polynucleotide are
preferred when the use of a corresponding restriction enzyme(s) is
not intended to cut the working polynucleotide internally. This
allows one to perform restriction digestion reactions to completion
or to near completion without incurring unwanted internal cleavage
in a working polynucleotide.
[1808] According to a preferred aspect, it is thus preferable to
use restriction sites that are not contained, or alternatively that
are not expected to be contained, or alternatively that unlikely to
be contained (e.g. when sequence information regarding a working
polynucleotide is incomplete) internally in a polynucleotide to be
subjected to end-selection. In accordance with this aspect, it is
appreciated that restriction sites that occur relatively
infrequently are usually preferred over those that occur more
frequently. On the other hand it is also appreciated that there are
occasions where internal cleavage of a polypeptide is desired, e.g.
to achieve recombination or other mutagenic procedures along with
end-selection.
[1809] In accord with this invention, it is also appreciated that
methods (e.g. mutagenesis methods) can be used to remove unwanted
internal restriction sites. It is also appreciated that a partial
digestion reaction (i.e. a digestion reaction that proceeds to
partial completion) can be used to achieve digestion at a
recognition site in a terminal region while sparing a susceptible
restriction site that occurs internally in a polynucleotide and
that is recognized by the same enzyme. In one aspect, partial
digest are useful because it is appreciated that certain enzymes
show preferential cleavage of the same recognition sequence
depending on the location and environment in which the recognition
sequence occurs. For example, it is appreciated that, while lambda
DNA has 5 EcoR I sites, cleavage of the site nearest to the right
terminus has been reported to occur 10 times faster than the sites
in the middle of the molecule. Also, for example, it has been
reported that, while Sac II has four sites on lambda DNA, the three
clustered centrally in lambda are cleaved 50 times faster than the
remaining site near the terminus (at nucleotide 40,386). Summarily,
site preferences have been reported for various enzymes by many
investigators (e.g., Thomas and Davis, 1975; Forsblum et al, 1976;
Nath and Azzolina, 1981; Brown and Smith, 1977; Gingeras and
Brooks, 1983; Kruger et al, 1988; Conrad and Topal, 1989; Oller et
al, 1991; Topal, 1991; and Pein, 1991; to name but a few). It is
appreciated that any empirical observations as well as any
mechanistic understandings of site preferences by any serviceable
polynucleotide-acting enzymes, whether currently available or to be
procured in the future, may be serviceable in end-selection
according to this invention.
[1810] It is also appreciated that protection methods can be used
to selectively protect specified restriction sites (e.g. internal
sites) against unwanted digestion by enzymes that would otherwise
cut a working polypeptide in response to the presence of those
sites; and that such protection methods include modifications such
as methylations and base substitutions (e.g. U instead of T) that
inhibit an unwanted enzyme activity. It is appreciated that there
are limited numbers of available restriction enzymes that are rare
enough (e.g. having very long recognition sequences) to create
large (e.g. megabase-long) restriction fragments, and that
protection approaches (e.g. by methylation) are serviceable for
increasing the rarity of enzyme cleavage sites. The use of M.Fnu II
(mCGCG) to increase the apparent rarity of Not I approximately
twofold is but one example among many (Qiang et al, 1990; Nelson et
al, 1984; Maxam and Gilbert, 1980; Raleigh and Wilson, 1986).
[1811] According to a preferred aspect of this invention, it is
provided that, in general, the use of rare restriction sites is
preferred. It is appreciated that, in general, the frequency of
occurrence of a restriction site is determined by the number of
nucleotides contained therein, as well as by the ambiguity of the
base requirements contained therein. Thus, in a non-limiting
exemplification, it is appreciated that, in general, a restriction
site composed of, for example, 8 specific nucleotides (e.g. the Not
I site or GC/GGCCGC, with an estimated relative occurrence of 1 in
4.sup.8, i.e. 1 in 65,536, random 8-mers) is relatively more
infrequent than one composed of, for example, 6 nucleotides (e.g.
the Sma I site or CCC/GGG, having an estimated relative occurrence
of 1 in 4.sup.6, i.e. 1 in 4,096, random 6-mers), which in turn is
relatively more infrequent than one composed of, for example, 4
nucleotides (e.g. the Msp I site or C/CGG, having an estimated
relative occurrence of 1 in 4.sup.4, i.e. 1 in 256, random 4-mers).
Moreover, in another non-limiting exemplification, it is
appreciated that, in general, a restriction site having no
ambiguous (but only specific) base requirements (e.g. the Fin I
site or GTCCC, having an estimated relative occurrence of 1 in
4.sup.5, i.e. 1 in 1024, random 5-mers) is relatively more
infrequent than one having an ambiguous W (where W=A or T) base
requirement (e.g. the Ava II site or G/GWCC, having an estimated
relative occurrence of 1 in 4.times.4.times.2.times.4.times.4-i.e.
1 in 512-random 5-mers), which in turn is relatively more
infrequent than one having an ambiguous N (where N=A or C or G or
T) base requirement (e.g. the Asu I site or G/GNCC, having an
estimated relative occurrence of 1 in
4.times.4.times.1.times.4.times.4, i.e. 1 in 256-random 5-mers).
These relative occurrences are considered general estimates for
actual polynucleotides, because it is appreciated that specific
nucleotide bases (not to mention specific nucleotide sequences)
occur with dissimilar frequencies in specific polynucleotides, in
specific species of organisms, and in specific groupings of
organisms. For example, it is appreciated that the % G+C contents
of different species of organisms are often very different and wide
ranging.
[1812] The use of relatively more infrequent restriction sites as a
selection marker include--in a non-limiting fashion--preferably
those sites composed at least a 4 nucleotide sequence, more
preferably those composed at least a 5 nucleotide sequence, more
preferably still those composed at least a 6 nucleotide sequence
(e.g. the BamH I site or G/GATCC, the Bgl II site or A/GATCT, the
Pst I site or CTGCA/G, and the Xba I site or T/CTAGA), more
preferably still those composed at least a 7 nucleotide sequence,
more preferably still those composed of an 8 nucleotide sequence
nucleotide sequence (e.g. the Asc I site or GG/CGCGCC, the Not I
site or GC/GGCCGC, the Pac I site or TTAAT/TAA, the Pme I site or
GTTT/AAAC, the SrfI site or GCCC/GGGC, the Sse838 I site or
CCTGCA/GG, and the Swa I site or ATTT/AAAT), more preferably still
those composed of a 9 nucleotide sequence, and even more preferably
still those composed of at least a 10 nucleotide sequence (e.g. the
BspG I site or CG/CGCTGGAC). It is further appreciated that some
restriction sites (e.g. for class IIS enzymes) are comprised of a
portion of relatively high specificity (i.e. a portion containing a
principal determinant of the frequency of occurrence of the
restriction site) and a portion of relatively low specificity; and
that a site of cleavage may or may not be contained within a
portion of relatively low specificity. For example, in the Eco57 I
site or CTGAAG(16/14), there is a portion of relatively high
specificity (i.e. the CTGAAG portion) and a portion of relatively
low specificity (i.e. the N16 sequence) that contains a site of
cleavage.
[1813] In another preferred embodiment of this invention, a
serviceable end-selection marker is a terminal sequence that is
recognized by a polynucleotide-acting enzyme that recognizes a
specific polynucleotide sequence. In a preferred aspect of this
invention, serviceable polynucleotide-acting enzymes also include
other enzymes in addition to classic type II restriction enzymes.
According to this preferred aspect of this invention, serviceable
polynucleotide-acting enzymes also include gyrases, helicases,
recombinases, relaxases, and any enzymes related thereto.
[1814] Among preferred examples are topoisomerases (which have been
categorized by some as a subset of the gyrases) and any other
enzymes that have polynucleotide-cleaving activity (including
preferably polynucleotide-nicking activity) &/or
polynucleotide-ligating activity. Among preferred topoisomerase
enzymes are topoisomerase I enzymes, which is available from many
commercial sources (Epicentre Technologies, Madison, Wis.;
Invitrogen, Carlsbad, Calif.; Life Technologies, Gathesburg, Md.)
and conceivably even more private sources. It is appreciated that
similar enzymes may be developed in the future that are serviceable
for end-selection as provided herein. A particularly preferred
topoisomerase I enzyme is a topoisomerase I enzyme of vaccinia
virus origin, that has a specific recognition sequence (e.g. 5'. .
. AAGGG. . . 3') and has both polynucleotide-nicking activity and
polynucleotide-ligating activity. Due to the specific
nicking-activity of this enzyme (cleavage of one strand), internal
recognition sites are not prone to polynucleotide destruction
resulting from the nicking activity (but rather remain annealed) at
a temperature that causes denaturation of a terminal site that has
been nicked. Thus for use in end-selection, it is preferable that a
nicking site for topoisomerase-based end-selection be no more than
100 nucleotides from a terminus, more preferably no more than 50
nucleotides from a terminus, more preferably still no more than 25
nucloetides from a terminus, even more preferably still no more
than 20 nucleotides from a terminus, even more preferably still no
more than 15 nucleotides from a terminus, even more preferably
still no more than 10 nucleotides from a terminus, even more
preferably still no more than 8 nucleotides from a terminus, even
more preferably still no more than 6 nucleotides from a terminus,
and even more preferably still no more than 4 nucleotides from a
terminus.
[1815] In a particularly preferred exemplification that is
non-limiting yet clearly illustrative, it is appreciated that when
a nicking site for topoisomerase-based end-selection is 4
nucleotides from a terminus, nicking produces a single stranded
oligo of 4 bases (in a terminal region) that can be denatured from
its complementary strand in an end-selectable polynucleotide; this
provides a sticky end (comprised of 4 bases) in a polynucleotide
that is serviceable for an ensuing ligation reaction. To accomplish
ligation to a cloning vector (preferably an expression vector),
compatible sticky ends can be generated in a cloning vector by any
means including by restriction enzyme-based means. The terminal
nucleotides (comprised of 4 terminal bases in this specific
example) in an end-selectable polynucleotide terminus are thus
wisely chosen to provide compatibility with a sticky end generated
in a cloning vector to which the polynucleotide is to be
ligated.
[1816] On the other hand, internal nicking of an end-selectable
polynucleotide, e.g. 500 bases from a terminus, produces a single
stranded oligo of 500 bases that is not easily denatured from its
complementary strand, but rather is serviceable for repair (e.g. by
the same topoisomerase enzyme that produced the nick).
[1817] This invention thus provides a method--e.g. that is vaccinia
topoisomerase-based &/or type II (or IIS) restriction
endonuclease-based &/or type III restriction endonuclease-based
&/or nicking enzyme-based (e.g. using N. BstNB I)--for
producing a sticky end in a working polynucleotide, which end is
ligation compatible, and which end can be comprised of at least a 1
base overhang. Preferably such a sticky end is comprised of at
least a 2-base overhang, more preferably such a sticky end is
comprised of at least a 3-base overhang, more preferably still such
a sticky end is comprised of at least a 4-base overhang, even more
preferably still such a sticky end is comprised of at least a
5-base overhang, even more preferably still such a sticky end is
comprised of at least a 6-base overhang. Such a sticky end may also
be comprised of at least a 7-base overhang, or at least an 8-base
overhang, or at least a 9-base overhang, or at least a 10-base
overhang, or at least 15-base overhang, or at least a 20-base
overhang, or at least a 25-base overhang, or at least a 30-base
overhang. These overhangs can be comprised of any bases, including
A, C, G, or T.
[1818] It is appreciated that sticky end overhangs introduced using
topoisomerase or a nicking enzyme (e.g. using N. BstNB I) can be
designed to be unique in a ligation environment, so as to prevent
unwanted fragment reassemblies, such as self-dimerizations and
other unwanted concatamerizations.
[1819] According to one aspect of this invention, a plurality of
sequences (which may but do not necessarily overlap) can be
introduced into a terminal region of an end-selectable
polynucleotide by the use of an oligo in a polymerase-based
reaction. In a relevant, but by no means limiting example, such an
oligo can be used to provide a preferred 5' terminal region that is
serviceable for topoisomerase I-based end-selection, which oligo is
comprised of: a 1-10 base sequence that is convertible into a
sticky end (preferably by a vaccinia topoisomerase I), a ribosome
binding site (i.e. and "RBS", that is preferably serviceable for
expression cloning), and optional linker sequence followed by an
ATG start site and a template-specific sequence of 0-100 bases (to
facilitate annealment to the template in the a polymerase-based
reaction). Thus, according to this example, a serviceable oligo
(which may be termed a forward primer) can have the sequence:
5'[terminal sequence=(N).sub.1-10] [topoisomerase I site &
RBS=AAGGGAGGAG][linker=(N).sub.1-100][start codon and
template-specific sequence=ATG(N).sub.0-100]3'.
[1820] Analogously, in a relevant, but by no means limiting
example, an oligo can be used to provide a preferred 3' terminal
region that is serviceable for topoisomerase I-based end-selection,
which oligo is comprised of: a 1-10 base sequence that is
convertible into a sticky end (preferably by a vaccinia
topoisomerase I), and optional linker sequence followed by a
template-specific sequence of 0-100 bases (to facilitate annealment
to the template in the a polymerase-based reaction). Thus,
according to this example, a serviceable oligo (which may be termed
a reverse primer) can have the sequence: 5'[terminal
sequence=(N).sub.1-10[topoisomerase I
site=AAGGG][linker=(N).sub.1-100][t- emplate-specific
sequence=(N).sub.0-100]3'.
[1821] It is appreciated that, end-selection can be used to
distinguish and separate parental template molecules (e.g. to be
subjected to mutagenesis) from progeny molecules (e.g. generated by
mutagenesis). For example, a first set of primers, lacking in a
topoisomerase I recognition site, can be used to modify the
terminal regions of the parental molecules (e.g. in
polymerase-based amplification). A different second set of primers
(e.g. having a topoisomerase I recognition site) can then be used
to generate mutated progeny molecules (e.g. using any
polynucleotide chimerization method, such as interrupted synthesis,
template-switching polymerase-based amplification, or interrupted
synthesis; or using saturation mutagenesis; or using any other
method for introducing a topoisomerase I recognition site into a
mutagenized progeny molecule as disclosed herein) from the
amplified template molecules. The use of topoisomerase I-based
end-selection can then facilitate, not only discernment, but
selective topoisomerase I-based ligation of the desired progeny
molecules.
[1822] Annealment of a second set of primers to thusly amplified
parental molecules can be facilitated by including sequences in a
first set of primers (i.e. primers used for amplifying a set
parental molecules) that are similar to a toposiomerase I
recognition site, yet different enough to prevent functional
toposiomerase I enzyme recognition. For example, sequences that
diverge from the AAGGG site by anywhere from 1 base to all 5 bases
can be incorporated into a first set of primers (to be used for
amplifying the parental templates prior to subjection to
mutagenesis). In a specific, but non-limiting aspect, it is thus
provided that a parental molecule can be amplified using the
following exemplary--but by no means limiting--set of forward and
reverse primers:
89 Forward Primer: 5' CTAGAAGAGAGGAGAAAACCATG(N).sub.10-- 1003',
and Reverse Primer: 5' GATCAAAGGCGCGCCTGCAGG(N).sub.10-1003'
[1823] According to this specific example of a first set of
primers, (N).sub.10-100 represents preferably a 10 to 100
nucleotide-long template-specific sequence, more preferably a 10 to
50 nucleotide-long template-specific sequence, more preferably
still a 10 to 30 nucleotide-long template-specific sequence, and
even more preferably still a 15 to 25 nucleotide-long
template-specific sequence.
[1824] According to a specific, but non-limiting aspect, it is thus
provided that, after this amplification (using a disclosed first
set of primers lacking in a true topoisomerase I recognition site),
amplified parental molecules can then be subjected to mutagenesis
using one or more sets of forward and reverse primers that do have
a true topoisomerase I recognition site. In a specific, but
non-limiting aspect, it is thus provided that a parental molecule
can be used as templates for the generation of a mutagenized
progeny molecule using the following exemplary--but by no means
limiting--second set of forward and reverse primers:
90 Forward Primer: 5' CTAGAAGGGAGGAGAAAACCATG 3' Reverse Primer: 5'
GATCAAAGGCGCGCCTGCAGG 3' (contains Asc I recognition sequence)
[1825] It is appreciated that any number of different primers sets
not specifically mentioned can be used as first, second, or
subsequent sets of primers for end-selection consistent with this
invention. Notice that type II restriction enzyme sites can be
incorporated (e.g. an Asc I site in the above example). It is
provided that, in addition to the other sequences mentioned, the
experimentalist can incorporate one or more N,N,G/T triplets into a
serviceable primer in order to subject a working polynucleotide to
saturation mutagenesis. Summarily, use of a second and/or
subsequent set of primers can achieve dual goals of introducing a
topoisomerase I site and of generating mutations in a progeny
polynucleotide.
[1826] Thus, according to one use provided, a serviceable
end-selection marker is an enzyme recognition site that allows an
enzyme to cleave (including nick) a polynucleotide at a specified
site, to produce a ligation-compatible end upon denaturation of a
generated single stranded oligo. Ligation of the produced
polynucleotide end can then be accomplished by the same enzyme
(e.g. in the case of vaccinia virus topoisomerase I), or
alternatively with the use of a different enzyme. According to one
aspect of this invention, any serviceable end-selection markers,
whether like (e.g. two vaccinia virus topoisomerase I recognition
sites) or unlike (e.g. a class II restriction enzyme recognition
site and a vaccinia virus topoisomerase I recognition site) can be
used in combination to select a polynucleotide. Each selectable
polynucleotide can thus have one or more end-selection markers, and
they can be like or unlike end-selection markers. In a particular
aspect, a plurality of end-selection markers can be located on one
end of a polynucleotide and can have overlapping sequences with
each other.
[1827] It is important to emphasize that any number of enzymes,
whether currently in existence or to be developed, can be
serviceable in end-selection according to this invention. For
example, in a particular aspect of this invention, a nicking enzyme
(e.g. N. BstNB I, which cleaves only one strand at 5'. . .
GAGTCNNNN/IN . . .3') can be used in conjunction with a source of
polynucleotide-ligating activity in order to achieve end-selection.
According to this embodiment, a recognition site for N. BstNB
I--instead of a recognition site for topoisomerase I--should be
incorporated into an end-selectable polynucleotide (whether
end-selection is used for selection of a mutagenized progeny
molecule or whether end-selection is used apart from any
mutagenesis procedure).
[1828] It is appreciated that the instantly disclosed end-selection
approach using topoisomerase-based nicking and ligation has several
advantages over previously available selection methods. In sum,
this approach allows one to achieve direction cloning (including
expression cloning). Specifically, this approach can be used for
the achievement of: direct ligation (i.e. without subjection to a
classic restriction-purification-ligation reaction, that is
susceptible to a multitude of potential problems from an initial
restriction reaction to a ligation reaction dependent on the use of
T4 DNA ligase); separation of progeny molecules from original
template molecules (e.g. original template molecules lack
topoisomerase I sites that not introduced until after mutagenesis),
obviation of the need for size separation steps (e.g. by gel
chromatography or by other electrophoretic means or by the use of
size-exclusion membranes), preservation of internal sequences (even
when topoisomerase I sites are present), obviation of concerns
about unsuccessful ligation reactions (e.g. dependent on the use of
T4 DNA ligase, particularly in the presence of unwanted residual
restriction enzyme activity), and facilitated expression cloning
(including obviation of frame shift concerns). Concerns about
unwanted restriction enzyme-based cleavages--especially at internal
restriction sites (or even at often unpredictable sites of unwanted
star activity) in a working polynucleotide--that are potential
sites of destruction of a working polynucleotide can also be
obviated by the instantly disclosed end-selection approach using
topoisomerase-based nicking and ligation.
2.11.3. ADDITIONAL SCREENING METHODS
[1829] Peptide Display Methods
[1830] The present method can be used to shuffle, by in vitro
and/or in vivo recombination by any of the disclosed methods, and
in any combination, polynucleotide sequences selected by peptide
display methods, wherein an associated polynucleotide encodes a
displayed peptide which is screened for a phenotype (e.g., for
affinity for a predetermined receptor (ligand).
[1831] An increasingly important aspect of bio-pharmaceutical drug
development and molecular biology is the identification of peptide
structures, including the primary amino acid sequences, of peptides
or peptidomimetics that interact with biological macromolecules.
one method of identifying peptides that possess a desired structure
or functional property, such as binding to a predetermined
biological macromolecule (e.g., a receptor), involves the screening
of a large library or peptides for individual library members which
possess the desired structure or functional property conferred by
the amino acid sequence of the peptide.
[1832] In addition to direct chemical synthesis methods for
generating peptide libraries, several recombinant DNA methods also
have been reported. One type involves the display of a peptide
sequence, antibody, or other protein on the surface of a
bacteriophage particle or cell. Generally, in these methods each
bacteriophage particle or cell serves as an individual library
member displaying a single species of displayed peptide in addition
to the natural bacteriophage or cell protein sequences. Each
bacteriophage or cell contains the nucleotide sequence information
encoding the particular displayed peptide sequence; thus, the
displayed peptide sequence can be ascertained by nucleotide
sequence determination of an isolated library member.
[1833] A well-known peptide display method involves the
presentation of a peptide sequence on the surface of a filamentous
bacteriophage, typically as a fusion with a bacteriophage coat
protein. The bacteriophage library can be incubated with an
immobilized, predetermined macromolecule or small molecule (e.g., a
receptor) so that bacteriophage particles which present a peptide
sequence that binds to the immobilized macromolecule can be
differentially partitioned from those that do not present peptide
sequences that bind to the predetermined macromolecule. The
bacteriophage particles (i.e., library members) which are bound to
the immobilized macromolecule are then recovered and replicated to
amplify the selected bacteriophage sub-population for a subsequent
round of affinity enrichment and phage replication. After several
rounds of affinity enrichment and phage replication, the
bacteriophage library members that are thus selected are isolated
and the nucleotide sequence encoding the displayed peptide sequence
is determined, thereby identifying the sequence(s) of peptides that
bind to the predetermined macromolecule (e.g., receptor). Such
methods are further described in PCT patent publications WO
91/17271, WO 91/18980, WO 91/19818 and WO 93/08278.
[1834] The latter PCT publication describes a recombinant DNA
method for the display of peptide ligands that involves the
production of a library of fusion proteins with each fusion protein
composed of a first polypeptide portion, typically comprising a
variable sequence, that is available for potential binding to a
predetermined macromolecule, and a second polypeptide portion that
binds to DNA, such as the DNA vector encoding the individual fusion
protein. When transformed host cells are cultured under conditions
that allow for expression of the fusion protein, the fusion protein
binds to the DNA vector encoding it. Upon lysis of the host cell,
the fusion protein/vector DNA complexes can be screened against a
predetermined macromolecule in much the same way as bacteriophage
particles are screened in the phage-based display system, with the
replication and sequencing of the DNA vectors in the selected
fusion protein/vector DNA complexes serving as the basis for
identification of the selected library peptide sequence(s).
[1835] Other systems for generating libraries of peptides and like
polymers have aspects of both the recombinant and in vitro chemical
synthesis methods. In these hybrid methods, cell-free enzymatic
machinery is employed to accomplish the in vitro synthesis of the
library members (i.e., peptides or polynucleotides). In one type of
method, RNA molecules with the ability to bind a predetermined
protein or a predetermined dye molecule were selected by alternate
rounds of selection and PCR amplification (Tuerk and Gold, 1990;
Ellington and Szostak, 1990). A similar technique was used to
identify DNA sequences which bind a predetermined human
transcription factor (Thiesen and Bach, 1990; Beaudry and Joyce,
1992; PCT patent publications WO 92/05258 and WO 92/14843). In a
similar fashion, the technique of in vitro translation has been
used to synthesize proteins of interest and has been proposed as a
method for generating large libraries of peptides. These methods
which rely upon in vitro translation, generally comprising
stabilized polysome complexes, are described further in PCT patent
publications WO 88/08453, WO 90/05785, WO 90/07003, WO 91/02076, WO
91/05058, and WO 92/02536. Applicants have described methods in
which library members comprise a fusion protein having a first
polypeptide portion with DNA binding activity and a second
polypeptide portion having the library member unique peptide
sequence; such methods are suitable for use in cell-free in vitro
selection formats, among others.
[1836] The displayed peptide sequences can be of varying lengths,
typically from 3-5000 amino acids long or longer, frequently from
5-100 amino acids long, and often from about 8-15 amino acids long.
A library can comprise library members having varying lengths of
displayed peptide sequence, or may comprise library members having
a fixed length of displayed peptide sequence. Portions or all of
the displayed peptide sequence(s) can be random, pseudorandom,
defined set kernal, fixed, or the like. The present display methods
include methods for in vitro and in vivo display of single-chain
antibodies, such as nascent scFv on polysomes or scfv displayed on
phage, which enable large-scale screening of scfv libraries having
broad diversity of variable region sequences and binding
specificities.
[1837] The present invention also provides random, pseudorandom,
and defined sequence framework peptide libraries and methods for
generating and screening those libraries to identify useful
compounds (e.g., peptides, including single-chain antibodies) that
bind to receptor molecules or epitopes of interest or gene products
that modify peptides or RNA in a desired fashion. The random,
pseudorandom, and defined sequence framework peptides are produced
from libraries of peptide library members that comprise displayed
peptides or displayed single-chain antibodies attached to a
polynucleotide template from which the displayed peptide was
synthesized. The mode of attachment may vary according to the
specific embodiment of the invention selected, and can include
encapsulation in a phage particle or incorporation in a cell.
[1838] A method of affinity enrichment allows a very large library
of peptides and single-chain antibodies to be screened and the
polynucleotide sequence encoding the desired peptide(s) or
single-chain antibodies to be selected. The polynucleotide can then
be isolated and shuffled to recombine combinatorially the amino
acid sequence of the selected peptide(s) (or predetermined portions
thereof) or single-chain antibodies (or just VHI, VLI or CDR
portions thereof). Using these methods, one can identify a peptide
or single-chain antibody as having a desired binding affinity for a
molecule and can exploit the process of shuffling to converge
rapidly to a desired high-affinity peptide or scfv. The peptide or
antibody can then be synthesized in bulk by conventional means for
any suitable use (e.g., as a therapeutic or diagnostic agent).
[1839] A significant advantage of the present invention is that no
prior information regarding an expected ligand structure is
required to isolate peptide ligands or antibodies of interest. The
peptide identified can have biological activity, which is meant to
include at least specific binding affinity for a selected receptor
molecule and, in some instances, will further include the ability
to block the binding of other compounds, to stimulate or inhibit
metabolic pathways, to act as a signal or messenger, to stimulate
or inhibit cellular activity, and the like.
[1840] The present invention also provides a method for shuffling a
pool of polynucleotide sequences selected by affinity screening a
library of polysomes displaying nascent peptides (including
single-chain antibodies) for library members which bind to a
predetermined receptor (e.g., a mammalian proteinaceous receptor
such as, for example, a peptidergic hormone receptor, a cell
surface receptor, an intracellular protein which binds to other
protein(s) to form intracellular protein complexes such as
hetero-dimers and the like) or epitope (e.g., an immobilized
protein, glycoprotein, oligosaccharide, and the like).
[1841] Polynucleotide sequences selected in a first selection round
(typically by affinity selection for binding to a receptor (e.g., a
ligand)) by any of these methods are pooled and the pool(s) is/are
shuffled by in vitro and/or in vivo recombination to produce a
shuffled pool comprising a population of recombined selected
polynucleotide sequences. The recombined selected polynucleotide
sequences are subjected to at least one subsequent selection round.
The polynucleotide sequences selected in the subsequent selection
round(s) can be used directly, sequenced, and/or subjected to one
or more additional rounds of shuffling and subsequent selection.
Selected sequences can also be back-crossed with polynucleotide
sequences encoding neutral sequences (i.e., having insubstantial
functional effect on binding), such as for example by back-crossing
with a wild-type or naturally-occurring sequence substantially
identical to a selected sequence to produce native-like functional
peptides, which may be less immunogenic. Generally, during
back-crossing subsequent selection is applied to retain the
property of binding to the predetermined receptor (ligand).
[1842] Prior to or concomitant with the shuffling of selected
sequences, the sequences can be mutagenized. In one embodiment,
selected library members are cloned in a prokaryotic vector (e.g.,
plasmid, phagemid, or bacteriophage) wherein a collection of
individual colonies (or plaques) representing discrete library
members are produced. Individual selected library members can then
be manipulated (e.g., by site-directed mutagenesis, cassette
mutagenesis, chemical mutagenesis, PCR mutagenesis, and the like)
to generate a collection of library members representing a kernal
of sequence diversity based on the sequence of the selected library
member. The sequence of an individual selected library member or
pool can be manipulated to incorporate random mutation,
pseudorandom mutation, defined kernal mutation (i.e., comprising
variant and invariant residue positions and/or comprising variant
residue positions which can comprise a residue selected from a
defined subset of amino acid residues), codon-based mutation, and
the like, either segmentally or over the entire length of the
individual selected library member sequence. The mutagenized
selected library members are then shuffled by in vitro and/or in
vivo recombinatorial shuffling as disclosed herein.
[1843] The invention also provides peptide libraries comprising a
plurality of individual library members of the invention, wherein
(1) each individual library member of said plurality comprises a
sequence produced by shuffling of a pool of selected sequences, and
(2) each individual library member comprises a variable peptide
segment sequence or single-chain antibody segment sequence which is
distinct from the variable peptide segment sequences or
single-chain antibody sequences of other individual library members
in said plurality (although some library members may be present in
more than one copy per library due to uneven amplification,
stochastic probability, or the like).
[1844] The invention also provides a product-by-process, wherein
selected polynucleotide sequences having (or encoding a peptide
having) a predetermined binding specificity are formed by the
process of: (1) screening a displayed peptide or displayed
single-chain antibody library against a predetermined receptor
(e.g., ligand) or epitope (e.g., antigen macromolecule) and
identifying and/or enriching library members which bind to the
predetermined receptor or epitope to produce a pool of selected
library members, (2) shuffling by recombination the selected
library members (or amplified or cloned copies thereof) which binds
the predetermined epitope and has been thereby isolated and/or
enriched from the library to generate a shuffled library, and (3)
screening the shuffled library against the predetermined receptor
(e.g., ligand) or epitope (e.g., antigen macromolecule) and
identifying and/or enriching shuffled library members which bind to
the predetermined receptor or epitope to produce a pool of selected
shuffled library members.
[1845] Antibody Display and Screening Methods
[1846] The present method can be used to shuffle, by in vitro
and/or in vivo recombination by any of the disclosed methods, and
in any combination, polynucleotide sequences selected by antibody
display methods, wherein an associated polynucleotide encodes a
displayed antibody which is screened for a phenotype (e.g., for
affinity for binding a predetermined antigen (ligand).
[1847] Various molecular genetic approaches have been devised to
capture the vast immunological repertoire represented by the
extremely large number of distinct variable regions which can be
present in immunoglobulin chains. The naturally-occurring germ line
immunoglobulin heavy chain locus is composed of separate tandem
arrays of variable segment genes located upstream of a tandem array
of diversity segment genes, which are themselves located upstream
of a tandem array of joining (i) region genes, which are located
upstream of the constant region genes. During B lymphocyte
development, V-D-J rearrangement occurs wherein a heavy chain
variable region gene (VH) is formed by rearrangement to form a
fused D segment followed by rearrangement with a V segment to form
a V-D-J joined product gene which, if productively rearranged,
encodes a functional variable region (VH) of a heavy chain.
Similarly, light chain loci rearrange one of several V segments
with one of several J segments to form a gene encoding the variable
region (VL) of a light chain.
[1848] The vast repertoire of variable regions possible in
immunoglobulins derives in part from the numerous combinatorial
possibilities of joining V and i segments (and, in the case of
heavy chain loci, D segments) during rearrangement in B cell
development. Additional sequence diversity in the heavy chain
variable regions arises from non-uniform rearrangements of the D
segments during V-D-J joining and from N region addition. Further,
antigen-selection of specific B cell clones selects for higher
affinity variants having non-germline mutations in one or both of
the heavy and light chain variable regions; a phenomenon referred
to as "affinity maturation" or "affinity sharpening". Typically,
these "affinity sharpening" mutations cluster in specific areas of
the variable region, most commonly in the
complementarity-determining regions (CDRs).
[1849] In order to overcome many of the limitations in producing
and identifying high-affinity immunoglobulins through
antigen-stimulated .beta. cell development (i.e., immunization),
various prokaryotic expression systems have been developed that can
be manipulated to produce combinatorial antibody libraries which
may be screened for high-affinity antibodies to specific antigens.
Recent advances in the expression of antibodies in Escherichia coli
and bacteriophage systems (see "alternative peptide display
methods", infra) have raised the possibility that virtually any
specificity can be obtained by either cloning antibody genes from
characterized hybridomas or by de novo selection using antibody
gene libraries (e.g., from Ig cDNA).
[1850] Combinatorial libraries of antibodies have been generated in
bacteriophage lambda expression systems which may be screened as
bacteriophage plaques or as colonies of lysogens (Huse et al,
1989); Caton and Koprowski, 1990; Mullinax et al, 1990; Persson et
al, 1991). Various embodiments of bacteriophage antibody display
libraries and lambda phage expression libraries have been described
(Kang et al, 1991; Clackson et al, 1991; McCafferty et al, 1990;
Burton et al, 1991; Hoogenboom et al, 1991; Chang et al, 1991;
Breitling et al, 1991; Marks et al, 1991, p. 581; Barbas et al,
1992; Hawkins and Winter, 1992; Marks et al, 1992, p. 779; Marks et
al, 1992, p. 16007; and Lowman et al, 1991; Lerner et al, 1992; all
incorporated herein by reference). Typically, a bacteriophage
antibody display library is screened with a receptor (e.g.,
polypeptide, carbohydrate, glycoprotein, nucleic acid) that is
immobilized (e.g., by covalent linkage to a chromatography resin to
enrich for reactive phage by affinity chromatography) and/or
labeled (e.g., to screen plaque or colony lifts).
[1851] One particularly advantageous approach has been the use of
so-called single-chain fragment variable (scfv) libraries (Marks et
al, 1992, p. 779; Winter and Milstein, 1991; Clackson et al, 1991;
Marks et al, 1991, p. 581; Chaudhary et al, 1990; Chiswell et al,
1992; McCafferty et al, 1990; and Huston et al, 1988). Various
embodiments of scfv libraries displayed on bacteriophage coat
proteins have been described.
[1852] Beginning in 1988, single-chain analogues of Fv fragments
and their fusion proteins have been reliably generated by antibody
engineering methods. The first step generally involves obtaining
the genes encoding VH and VL domains with desired binding
properties; these V genes may be isolated from a specific hybridoma
cell line, selected from a combinatorial V-gene library, or made by
V gene synthesis. The single-chain Fv is formed by connecting the
component V genes with an oligonucleotide that encodes an
appropriately designed linker peptide, such as
(Gly-Gly-Gly-Gly-Ser)3 or equivalent linker peptide(s). The linker
bridges the C-terminus of the first V region and N-terminus of the
second, ordered as either VH-linker-VL or VL-linker-VH' In
principle, the scfv binding site can faithfully replicate both the
affinity and specificity of its parent antibody combining site.
[1853] Thus, scfv fragments are comprised of VH and VL domains
linked into a single polypeptide chain by a flexible linker
peptide. After the scfv genes are assembled, they are cloned into a
phagemid and expressed at the tip of the M13 phage (or similar
filamentous bacteriophage) as fusion proteins with the
bacteriophage PIII (gene 3) coat protein. Enriching for phage
expressing an antibody of interest is accomplished by panning the
recombinant phage displaying a population scfv for binding to a
predetermined epitope (e.g., target antigen, receptor).
[1854] The linked polynucleotide of a library member provides the
basis for replication of the library member after a screening or
selection procedure, and also provides the basis for the
determination, by nucleotide sequencing, of the identity of the
displayed peptide sequence or VH and VL amino acid sequence. The
displayed peptide (s) or single-chain antibody (e.g., scfv) and/or
its VH and VL domains or their CDRs can be cloned and expressed in
a suitable expression system. Often polynucleotides encoding the
isolated VH and VL domains will be ligated to polynucleotides
encoding constant regions (CH and CL) to form polynucleotides
encoding complete antibodies (e.g., chimeric or fully-human),
antibody fragments, and the like. Often polynucleotides encoding
the isolated CDRs will be grafted into polynucleotides encoding a
suitable variable region framework (and optionally constant
regions) to form polynucleotides encoding complete antibodies
(e.g., humanized or fully-human), antibody fragments, and the like.
Antibodies can be used to isolate preparative quantities of the
antigen by immunoaffinity chromatography. Various other uses of
such antibodies are to diagnose and/or stage disease (e.g.,
neoplasia) and for therapeutic application to treat disease, such
as for example: neoplasia, autoimmune disease, AIDS, cardiovascular
disease, infections, and the like.
[1855] Various methods have been reported for increasing the
combinatorial diversity of a scfv library to broaden the repertoire
of binding species (idiotype spectrum) The use of PCR has permitted
the variable regions to be rapidly cloned either from a specific
hybridoma source or as a gene library from non-immunized cells,
affording combinatorial diversity in the assortment of VH and VL
cassettes which can be combined. Furthermore, the VH and VL
cassettes can themselves be diversified, such as by random,
pseudorandom, or directed mutagenesis. Typically, VH and VL
cassettes are diversified in or near the
complementarity-determining regions (CDRS), often the third CDR,
CDR3. Enzymatic inverse PCR mutagenesis has been shown to be a
simple and reliable method for constructing relatively large
libraries of scfv site-directed hybrids (Stemmer et al, 1993), as
has error-prone PCR and chemical mutagenesis (Deng et al, 1994).
Riechmann (Riechmann et al, 1993) showed semi-rational design of an
antibody scfv fragment using site-directed randomization by
degenerate oligonucleotide PCR and subsequent phage display of the
resultant scfv hybrids. Barbas (Barbas et al, 1992) attempted to
circumvent the problem of limited repertoire sizes resulting from
using biased variable region sequences by randomizing the sequence
in a synthetic CDR region of a human tetanus toxoid-binding
Fab.
[1856] CDR randomization has the potential to create approximately
1.times.10.sup.20 CDRs for the heavy chain CDR3 alone, and a
roughly similar number of variants of the heavy chain CDR1 and
CDR2, and light chain CDR1-3 variants. Taken individually or
together, the combination possibilities of CDR randomization of
heavy and/or light chains requires generating a prohibitive number
of bacteriophage clones to produce a clone library representing all
possible combinations, the vast majority of which will be
non-binding. Generation of such large numbers of primary
transformants is not feasible with current transformation
technology and bacteriophage display systems. For example, Barbas
(Barbas et al, 1992) only generated 5.times.10.sup.7 transformants,
which represents only a tiny fraction of the potential diversity of
a library of thoroughly randomized CDRS.
[1857] Despite these substantial limitations, bacteriophage display
of scfv have already yielded a variety of useful antibodies and
antibody fusion proteins. A bispecific single chain antibody has
been shown to mediate efficient tumor cell lysis (Gruber et al,
1994). Intracellular expression of an anti-Rev scfv has been shown
to inhibit HIV-1 virus replication in vitro (Duan et al, 1994), and
intracellular expression of an anti-p21rar, scfv has been shown to
inhibit meiotic maturation of Xenopus oocytes (Biocca et al, 1993).
Recombinant scfv which can be used to diagnose HIV infection have
also been reported, demonstrating the diagnostic utility of scfv
(Lilley et al, 1994). Fusion proteins wherein an scFv is linked to
a second polypeptide, such as a toxin or fibrinolytic activator
protein, have also been reported (Holvost et al, 1992; Nicholls et
al, 1993).
[1858] If it were possible to generate scfv libraries having
broader antibody diversity and overcoming many of the limitations
of conventional CDR mutagenesis and randomization methods which can
cover only a very tiny fraction of the potential sequence
combinations, the number and quality of scfv antibodies suitable
for therapeutic and diagnostic use could be vastly improved. To
address this, the in vitro and in vivo shuffling methods of the
invention are used to recombine CDRs which have been obtained
(typically via PCR amplification or cloning) from nucleic acids
obtained from selected displayed antibodies. Such displayed
antibodies can be displayed on cells, on bacteriophage particles,
on polysomes, or any suitable antibody display system wherein the
antibody is associated with its encoding nucleic acid(s). In a
variation, the CDRs are initially obtained from mRNA (or cDNA) from
antibody-producing cells (e.g., plasma cells/splenocytes from an
immunized wild-type mouse, a human, or a transgenic mouse capable
of making a human antibody as in WO 92/03918, WO 93/12227, and WO
94/25585), including hybridomas derived therefrom.
[1859] Polynucleotide sequences selected in a first selection round
(typically by affinity selection for displayed antibody binding to
an antigen (e.g., a ligand) by any of these methods are pooled and
the pool(s) is/are shuffled by in vitro and/or in vivo
recombination, especially shuffling of CDRs (typically shuffling
heavy chain CDRs with other heavy chain CDRs and light chain CDRs
with other light chain CDRs) to produce a shuffled pool comprising
a population of recombined selected polynucleotide sequences. The
recombined selected polynucleotide sequences are expressed in a
selection format as a displayed antibody and subjected to at least
one subsequent selection round. The polynucleotide sequences
selected in the subsequent selection round(s) can be used directly,
sequenced, and/or subjected to one or more additional rounds of
shuffling and subsequent selection until an antibody of the desired
binding affinity is obtained. Selected sequences can also be
back-crossed with polynucleotide sequences encoding neutral
antibody framework sequences (i.e., having insubstantial functional
effect on antigen binding), such as for example by back-crossing
with a human variable region framework to produce human-like
sequence antibodies. Generally, during back-crossing subsequent
selection is applied to retain the property of binding to the
predetermined antigen.
[1860] Alternatively, or in combination with the noted variations,
the valency of the target epitope may be varied to control the
average binding affinity of selected scfv library members. The
target epitope can be bound to a surface or substrate at varying
densities, such as by including a competitor epitope, by dilution,
or by other method known to those in the art. A high density
(valency) of predetermined epitope can be used to enrich for scfv
library members which have relatively low affinity, whereas a low
density (valency) can preferentially enrich for higher affinity
scfv library members.
[1861] For generating diverse variable segments, a collection of
synthetic oligonucleotides encoding random, pseudorandom, or a
defined sequence kernal set of peptide sequences can be inserted by
ligation into a predetermined site (e.g., a CDR). Similarly, the
sequence diversity of one or more CDRs of the single-chain antibody
cassette(s) can be expanded by mutating the CDR(s) with
site-directed mutagenesis, CDR-replacement, and the like. The
resultant DNA molecules can be propagated in a host for cloning and
amplification prior to shuffling, or can be used directly (i.e.,
may avoid loss of diversity which may occur upon propagation in a
host cell) and the selected library members subsequently
shuffled.
[1862] Displayed peptide/polynucleotide complexes (library members)
which encode a variable segment peptide sequence of interest or a
single-chain antibody of interest are selected from the library by
an affinity enrichment technique. This is accomplished by means of
a immobilized macromolecule or epitope specific for the peptide
sequence of interest, such as a receptor, other macromolecule, or
other epitope species. Repeating the affinity selection procedure
provides an enrichment of library members encoding the desired
sequences, which may then be isolated for pooling and shuffling,
for sequencing, and/or for further propagation and affinity
enrichment.
[1863] The library members without the desired specificity are
removed by washing. The degree and stringency of washing required
will be determined for each peptide sequence or single-chain
antibody of interest and the immobilized predetermined
macromolecule or epitope. A certain degree of control can be
exerted over the binding characteristics of the nascent peptide/DNA
complexes recovered by adjusting the conditions of the binding
incubation and the subsequent washing. The temperature, pH, ionic
strength, divalent cations concentration, and the volume and
duration of the washing will select for nascent peptide/DNA
complexes within particular ranges of affinity for the immobilized
macromolecule. Selection based on slow dissociation rate, which is
usually predictive of high affinity, is often the most practical
route. This may be done either by continued incubation in the
presence of a saturating amount of free predetermined
macromolecule, or by increasing the volume, number, and length of
the washes. In each case, the rebinding of dissociated nascent
peptide/DNA or peptide/RNA complex is prevented, and with
increasing time, nascent peptide/DNA or peptide/RNA complexes of
higher and higher affinity are recovered.
[1864] Additional modifications of the binding and washing
procedures may be applied to find peptides with special
characteristics. The affinities of some peptides are dependent on
ionic strength or cation concentration. This is a useful
characteristic for peptides that will be used in affinity
purification of various proteins when gentle conditions for
removing the protein from the peptides are required.
[1865] One variation involves the use of multiple binding targets
(multiple epitope species, multiple receptor species), such that a
scfv library can be simultaneously screened for a multiplicity of
scfv which have different binding specificities. Given that the
size of a scfv library often limits the diversity of potential scfv
sequences, it is typically desirable to us scfv libraries of as
large a size as possible. The time and economic considerations of
generating a number of very large polysome scFv-display libraries
can become prohibitive. To avoid this substantial problem, multiple
predetermined epitope species (receptor species) can be
concomitantly screened in a single library, or sequential screening
against a number of epitope species can be used. In one variation,
multiple target epitope species, each encoded on a separate bead
(or subset of beads), can be mixed and incubated with a
polysome-display scfv library under suitable binding conditions.
The collection of beads, comprising multiple epitope species, can
then be used to isolate, by affinity selection, scfv library
members. Generally, subsequent affinity screening rounds can
include the same mixture of beads, subsets thereof, or beads
containing only one or two individual epitope species. This
approach affords efficient screening, and is compatible with
laboratory automation, batch processing, and high throughput
screening methods.
[1866] A variety of techniques can be used in the present invention
to diversify a peptide library or single-chain antibody library, or
to diversify, prior to or concomitant with shuffling, around
variable segment peptides found in early rounds of panning to have
sufficient binding activity to the predetermined macromolecule or
epitope. In one approach, the positive selected
peptide/polynucleotide complexes (those identified in an early
round of affinity enrichment) are sequenced to determine the
identity of the active peptides. Oligonucleotides are then
synthesized based on these active peptide sequences, employing a
low level of all bases incorporated at each step to produce slight
variations of the primary oligonucleotide sequences. This mixture
of (slightly) degenerate oligonucleotides is then cloned into the
variable segment sequences at the appropriate locations. This
method produces systematic, controlled variations of the starting
peptide sequences, which can then be shuffled. It requires,
however, that individual positive nascent peptide/polynucleotide
complexes be sequenced before mutagenesis, and thus is useful for
expanding the diversity of small numbers of recovered complexes and
selecting variants having higher binding affinity and/or higher
binding specificity. In a variation, mutagenic PCR amplification of
positive selected peptide/polynucleotide complexes (especially of
the variable region sequences, the amplification products of which
are shuffled in vitro and/or in vivo and one or more additional
rounds of screening is done prior to sequencing. The same general
approach can be employed with single-chain antibodies in order to
expand the diversity and enhance the binding affinity/specificity,
typically by diversifying CDRs or adjacent framework regions prior
to or concomitant with shuffling. If desired, shuffling reactions
can be spiked with mutagenic oligonucleotides capable of in vitro
recombination with the selected library members can be included.
Thus, mixtures of synthetic oligonucleotides and PCR produced
polynucleotides (synthesized by error-prone or high-fidelity
methods) can be added to the in vitro shuffling mix and be
incorporated into resulting shuffled library members
(shufflants).
[1867] The present invention of shuffling enables the generation of
a vast library of CDR-variant single-chain antibodies. One way to
generate such antibodies is to insert synthetic CDRs into the
single-chain antibody and/or CDR randomization prior to or
concomitant with shuffling. The sequences of the synthetic CDR
cassettes are selected by referring to known sequence data of human
CDR and are selected in the discretion of the practitioner
according to the following guidelines: synthetic CDRs will have at
least 40 percent positional sequence identity to known CDR
sequences, and preferably will have at least 50 to 70 percent
positional sequence identity to known CDR sequences. For example, a
collection of synthetic CDR sequences can be generated by
synthesizing a collection of oligonucleotide sequences on the basis
of naturally-occurring human CDR sequences listed in Kabat (Kabat
et al, 1991); the pool (s) of synthetic CDR sequences are
calculated to encode CDR peptide sequences having at least 40
percent sequence identity to at least one known naturally-occurring
human CDR sequence. Alternatively, a collection of
naturally-occurring CDR sequences may be compared to generate
consensus sequences so that amino acids used at a residue position
frequently (i.e., in at least 5 percent of known CDR sequences) are
incorporated into the synthetic CDRs at the corresponding
position(s). Typically, several (e.g., 3 to about 50) known CDR
sequences are compared and observed natural sequence variations
between the known CDRs are tabulated, and a collection of
oligonucleotides encoding CDR peptide sequences encompassing all or
most permutations of the observed natural sequence variations is
synthesized. For example but not for limitation, if a collection of
human VH CDR sequences have carboxy-terminal amino acids which are
either Tyr, Val, Phe, or Asp, then the pool(s) of synthetic CDR
oligonucleotide sequences are designed to allow the
carboxy-terminal CDR residue to be any of these amino acids. In
some embodiments, residues other than those which naturally-occur
at a residue position in the collection of CDR sequences are
incorporated: conservative amino acid substitutions are frequently
incorporated and up to 5 residue positions may be varied to
incorporate non-conservative amino acid substitutions as compared
to known naturally-occurring CDR sequences. Such CDR sequences can
be used in primary library members (prior to first round screening)
and/or can be used to spike in vitro shuffling reactions of
selected library member sequences. Construction of such pools of
defined and/or degenerate sequences will be readily accomplished by
those of ordinary skill in the art.
[1868] The collection of synthetic CDR sequences comprises at least
one member that is not known to be a naturally-occurring CDR
sequence. It is within the discretion of the practitioner to
include or not include a portion of random or pseudorandom sequence
corresponding to N region addition in the heavy chain CDR; the N
region sequence ranges from 1 nucleotide to about 4 nucleotides
occurring at V-D and D-J junctions. A collection of synthetic heavy
chain CDR sequences comprises at least about 100 unique CDR
sequences, typically at least about 1,000 unique CDR sequences,
preferably at least about 10,000 unique CDR sequences, frequently
more than 50,000 unique CDR sequences; however, usually not more
than about 1.times.106 unique CDR sequences are included in the
collection, although occasionally 1.times.107 to 1.times.108 unique
CDR sequences are present, especially if conservative amino acid
substitutions are permitted at positions where the conservative
amino acid substituent is not present or is rare (i.e., less than
0.1 percent) in that position in naturally-occurring human CDRS. In
general, the number of unique CDR sequences included in a library
should not exceed the expected number of primary transformants in
the library by more than a factor of 10. Such single-chain
antibodies generally bind of about at least 1.times.10 m-,
preferably with an affinity of about at least 5.times.10.sup.7M-1,
more preferably with an affinity of at least 1.times.10.sup.8 M-1
to 1.times.10.sup.9M-1 or more, sometimes up to 1.times.10.sup.10
M-1 or more. Frequently, the predetermined antigen is a human
protein, such as for example a human cell surface antigen (e.g.,
CD4, CD8, IL-2 receptor, EGF receptor, PDGF receptor), other human
biological macromolecule (e.g., thrombomodulin, protein C,
carbohydrate antigen, sialyl Lewis antigen, Lselectin), or nonhuman
disease associated macromolecule (e.g., bacterial LPS, virion
capsid protein or envelope glycoprotein) and the like.
[1869] High affinity single-chain antibodies of the desired
specificity can be engineered and expressed in a variety of
systems. For example, scfv have been produced in plants (Firek et
al, 1993) and can be readily made in prokaryotic systems (Owens and
Young, 1994; Johnson and Bird, 1991). Furthermore, the single-chain
antibodies can be used as a basis for constructing whole antibodies
or various fragments thereof (Kettleborough et al, 1994). The
variable region encoding sequence may be isolated (e.g., by PCR
amplification or subcloning) and spliced to a sequence encoding a
desired human constant region to encode a human sequence antibody
more suitable for human therapeutic uses where immunogenicity is
preferably minimized. The polynucleotide(s) having the resultant
fully human encoding sequence(s) can be expressed in a host cell
(e.g., from an expression vector in a mammalian cell) and purified
for pharmaceutical formulation.
[1870] The DNA expression constructs will typically include an
expression control DNA sequence operably linked to the coding
sequences, including naturally-associated or heterologous promoter
regions. Preferably, the expression control sequences will be
eukaryotic promoter systems in vectors capable of transforming or
transfecting eukaryotic host cells. Once the vector has been
incorporated into the appropriate host, the host is maintained
under conditions suitable for high level expression of the
nucleotide sequences, and the collection and purification of the
mutant "engineered" antibodies.
[1871] As stated previously, the DNA sequences will be expressed in
hosts after the sequences have been operably linked to an
expression control sequence (i.e., positioned to ensure the
transcription and translation of the structural gene). These
expression vectors are typically replicable in the host organisms
either as episomes or as an integral part of the host chromosomal
DNA. Commonly, expression vectors will contain selection markers,
e.g., tetracycline or neomycin, to permit detection of those cells
transformed with the desired DNA sequences (see, e.g., U.S. Pat.
No. 4,704,362, which is incorporated herein by reference).
[1872] In addition to eukaryotic microorganisms such as yeast,
mammalian tissue cell culture may also be used to produce the
polypeptides of the present invention (see Winnacker, 1987), which
is incorporated herein by reference). Eukaryotic cells are actually
preferred, because a number of suitable host cell lines capable of
secreting intact immunoglobulins have been developed in the art,
and include the CHO cell lines, various COS cell lines, HeLa cells,
and myeloma cell lines, but preferably transformed Bcells or
hybridomas. Expression vectors for these cells can include
expression control sequences, such as an origin of replication, a
promoter, an enhancer (Queen et al, 1986), and necessary processing
information sites, such as ribosome binding sites, RNA splice
sites, polyadenylation sites, and transcriptional terminator
sequences. Preferred expression control sequences are promoters
derived from immunoglobulin genes, cytomegalovirus, SV40,
Adenovirus, Bovine Papilloma Virus, and the like.
[1873] Eukaryotic DNA transcription can be increased by inserting
an enhancer sequence into the vector. Enhancers are cis-acting
sequences of between 10 to 300 bp that increase transcription by a
promoter. Enhancers can effectively increase transcription when
either 51 or 31 to the transcription unit. They are also effective
if located within an intron or within the coding sequence itself.
Typically, viral enhancers are used, including SV40 enhancers,
cytomegalovirus enhancers, polyoma enhancers, and adenovirus
enhancers. Enhancer sequences from mammalian systems are also
commonly used, such as the mouse immunoglobulin heavy chain
enhancer.
[1874] Mammalian expression vector systems will also typically
include a selectable marker gene. Examples of suitable markers
include, the dihydrofolate reductase gene (DHFR), the thymidine
kinase gene (TK), or prokaryotic genes conferring drug resistance.
The first two marker genes prefer the use of mutant cell lines that
lack the ability to grow without the addition of thymidine to the
growth medium. Transformed cells can then be identified by their
ability to grow on non-supplemented media. Examples of prokaryotic
drug resistance genes useful as markers include genes conferring
resistance to G418, mycophenolic acid and hygromycin.
[1875] The vectors containing the DNA segments of interest can be
transferred into the host cell by well-known methods, depending on
the type of cellular host. For example, calcium chloride
transfection is commonly utilized for prokaryotic cells, whereas
calcium phosphate treatment. lipofection, or electroporation may be
used for other cellular hosts. Other methods used to transform
mammalian cells include the use of Polybrene, protoplast fusion,
liposomes, electroporation, and micro-injection (see, generally,
Sambrook et al, 1982 and 1989).
[1876] Once expressed, the antibodies, individual mutated
immunoglobulin chains, mutated antibody fragments, and other
immunoglobulin polypeptides of the invention can be purified
according to standard procedures of the art, including ammonium
sulfate precipitation, fraction column chromatography, gel
electrophoresis and the like (see, generally, Scopes, 1982). Once
purified, partially or to homogeneity as desired, the polypeptides
may then be used therapeutically or in developing and performing
assay procedures, immunofluorescent stainings, and the like (see,
generally, Lefkovits and Pernis, 1979 and 1981; Lefkovits,
1997).
[1877] The antibodies generated by the method of the present
invention can be used for diagnosis and therapy. By way of
illustration and not limitation, they can be used to treat cancer,
autoimmune diseases, or viral infections. For treatment of cancer,
the antibodies will typically bind to an antigen expressed
preferentially on cancer cells, such as erbB-2, CEA, CD33, and many
other antigens and binding members well known to those skilled in
the art.
[1878] Two-Hybrid Based Screening Assays
[1879] Shuffling can also be used to recombinatorially diversify a
pool of selected library members obtained by screening a two-hybrid
screening system to identify library members which bind a
predetermined polypeptide sequence. The selected library members
are pooled and shuffled by in vitro and/or in vivo recombination.
The shuffled pool can then be screened in a yeast two hybrid system
to select library members which bind said predetermined polypeptide
sequence (e.g., and SH2 domain) or which bind an alternate
predetermined polypeptide sequence (e.g., an SH2 domain from
another protein species).
[1880] An approach to identifying polypeptide sequences which bind
to a predetermined polypeptide sequence has been to use a so-called
"two-hybrid" system wherein the predetermined polypeptide sequence
is present in a fusion protein (Chien et al, 1991). This approach
identifies protein-protein interactions in vivo through
reconstitution of a transcriptional activator (Fields and Song,
1989), the yeast Gal4 transcription protein. Typically, the method
is based on the properties of the yeast Gal4 protein, which
consists of separable domains responsible for DNA-binding and
transcriptional activation. Polynucleotides encoding two hybrid
proteins, one consisting of the yeast Gal4 DNA-binding domain fused
to a polypeptide sequence of a known protein and the other
consisting of the Gal4 activation domain fused to a polypeptide
sequence of a second protein, are constructed and introduced into a
yeast host cell. Intermolecular binding between the two fusion
proteins reconstitutes the Gal4 DNA-binding domain with the Gal4
activation domain, which leads to the transcriptional activation of
a reporter gene (e.g., lacz, HIS3) which is operably linked to a
Gal4 binding site. Typically, the two-hybrid method is used to
identify novel polypeptide sequences which interact with a known
protein (Silver and Hunt, 1993; Durfee et al, 1993; Yang et al,
1992; Luban et al, 1993; Hardy et al, 1992; Bartel et al, 1993; and
Vojtek et al, 1993). However, variations of the two-hybrid method
have been used to identify mutations of a known protein that affect
its binding to a second known protein (Li and Fields, 1993; Lalo et
al, 1993; Jackson et al, 1993; and Madura et al, 1993). Two-hybrid
systems have also been used to identify interacting structural
domains of two known proteins (Bardwell et al, 1993; Chakrabarty et
al, 1992; Staudinger et al, 1993; and Milne and Weaver 1993) or
domains responsible for oligomerization of a single protein
(Iwabuchi et al, 1993; Bogerd et al, 1993). Variations of
two-hybrid systems have been used to study the in vivo activity of
a proteolytic enzyme (Dasmahapatra et al, 1992). Alternatively, an
E. coli/BCCP interactive screening system (Germino et al, 1993;
Guarente, 1993) can be used to identify interacting protein
sequences (i.e., protein sequences which heterodimerize or form
higher order heteromultimers). Sequences selected by a two-hybrid
system can be pooled and shuffled and introduced into a two-hybrid
system for one or more subsequent rounds of screening to identify
polypeptide sequences which bind to the hybrid containing the
predetermined binding sequence. The sequences thus identified can
be compared to identify consensus sequence(s) and consensus
sequence kernals.
[1881] In general, standard techniques of recombination DNA
technology are described in various publications (e.g. Sambrook et
al, 1989; Ausubel et al, 1987; and Berger and Kimmel, 1987; each of
which is incorporated herein in its entirety by reference.
Polynucleotide modifying enzymes were used according to the
manufacturer's recommendations. Oligonucleotides were synthesized
on an Applied Biosystems Inc. Model 394 DNA synthesizer using ABI
chemicals. If desired, PCR amplimers for amplifying a predetermined
DNA sequence may be selected at the discretion of the
practitioner.
[1882] One microgram samples of template DNA are obtained and
treated with U.V. light to cause the formation of dimers, including
TT dimers, particularly purine dimers. U.V. exposure is limited so
that only a few photoproducts are generated per gene on the
template DNA sample. Multiple samples are treated with U.V. light
for varying periods of time to obtain template DNA samples with
varying numbers of dimers from U.V. exposure.
[1883] A random priming kit which utilizes a non-proofreading
polymease (for example, Prime-It II Random Primer Labeling kit by
Stratagene Cloning Systems) is utilized to generate different size
polynucleotides by priming at random sites on templates which are
prepared by U.V. light (as described above) and extending along the
templates. The priming protocols such as described in the Prime-It
II Random Primer Labeling kit may be utilized to extend the
primers. The dimers formed by U.V. exposure serve as a roadblock
for the extension by the non-proofreading polymerase. Thus, a pool
of random size polynucleotides is present after extension with the
random primers is finished.
[1884] The present invention is further directed to a method for
generating a selected mutant polynucleotide sequence (or a
population of selected polynucleotide sequences) typically in the
form of amplified and/or cloned polynucleotides, whereby the
selected polynucleotide sequences(s) possess at least one desired
phenotypic characteristic (e.g., encodes a polypeptide, promotes
transcription of linked polynucleotides, binds a protein, and the
like) which can be selected for. One method for identifying hybrid
polypeptides that possess a desired structure or functional
property, such as binding to a predetermined biological
macromolecule (e.g., a receptor), involves the screening of a large
library of polypeptides for individual library members which
possess the desired structure or functional property conferred by
the amino acid sequence of the polypeptide.
[1885] In one embodiment, the present invention provides a method
for generating libraries of displayed polypeptides or displayed
antibodies suitable for affinity interaction screening or
phenotypic screening. The method comprises (1) obtaining a first
plurality of selected library members comprising a displayed
polypeptide or displayed antibody and an associated polynucleotide
encoding said displayed polypeptide or displayed antibody, and
obtaining said associated polynucleotides or copies thereof wherein
said associated polynucleotides comprise a region of substantially
identical sequences, optimally introducing mutations into said
polynucleotides or copies, (2) pooling the polynucleotides or
copies, (3) producing smaller or shorter polynucleotides by
interrupting a random or particularized priming and synthesis
process or an amplification process, and (4) performing
amplification, preferably PCR amplification, and optionally
mutagenesis to homologously recombine the newly synthesized
polynucleotides.
[1886] It is a particularly preferred object of the invention to
provide a process for producing hybrid polynucleotides which
express a useful hybrid polypeptide by a series of steps
comprising:
[1887] (a) producing polynucleotides by interrupting a
polynucleotide amplification or synthesis process with a means for
blocking or interrupting the amplification or synthesis process and
thus providing a plurality of smaller or shorter polynucleotides
due to the replication of the polynucleotide being in various
stages of completion;
[1888] (b) adding to the resultant population of single- or
double-stranded polynucleotides one or more single- or
double-stranded oligonucleotides, wherein said added
oligonucleotides comprise an area of identity in an area of
heterology to one or more of the single- or double-stranded
polynucleotides of the population;
[1889] (c) denaturing the resulting single- or double-stranded
oligonucleotides to produce a mixture of single-stranded
polynucleotides, optionally separating the shorter or smaller
polynucleotides into pools of polynucleotides having various
lengths and further optionally subjecting said polynucleotides to a
PCR procedure to amplify one or more oligonucleotides comprised by
at least one of said polynucleotide pools;
[1890] (d) incubating a plurality of said polynucleotides or at
least one pool of said polynucleotides with a polymerase under
conditions which result in annealing of said single-stranded
polynucleotides at regions of identity between the single-stranded
polynucleotides and thus forming of a mutagenized double-stranded
polynucleotide chain;
[1891] (e) optionally repeating steps (c) and (d);
[1892] (f) expressing at least one hybrid polypeptide from said
polynucleotide chain, or chains; and
[1893] (g) screening said at least one hybrid polypeptide for a
useful activity.
[1894] In a preferred aspect of the invention, the means for
blocking or interrupting the amplification or synthesis process is
by utilization of uv light, DNA adducts, DNA binding proteins.
[1895] In one embodiment of the invention, the DNA adducts, or
polynucleotides comprising the DNA adducts, are removed from the
polynucleotides or polynucleotide pool, such as by a process
including heating the solution comprising the DNA fragments prior
to further processing.
[1896] Having thus disclosed exemplary embodiments of the present
invention, it should be noted by those skilled in the art that the
disclosures are exemplary only and that various other alternatives,
adaptations and modifications may be made within the scope of the
present invention. Accordingly, the present invention is not
limited to the specific embodiments as illustrated herein.
[1897] Without further elaboration, it is believed that one skilled
in the art can, using the preceding description, utilize the
present invention to its fullest extent. The following examples are
to be considered illustrative and thus are not limiting of the
remainder of the disclosure in any way whatsoever.
EXAMPLE 1
Generation of Random Size Polynucleotides Using U.V. Induced
Photoproducts
[1898] One microgram samples of template DNA are obtained and
treated with U.V. light to cause the formation of dimers, including
TT dimers, particularly purine dimers. U.V. exposure is limited so
that only a few photoproducts are generated per gene on the
template DNA sample. Multiple samples are treated with U.V. light
for varying periods of time to obtain template DNA samples with
varying numbers of dimers from U.V. exposure.
[1899] A random priming kit which utilizes a non-proofreading
polymerase (for example, Prime-It II Random Primer Labeling kit by
Stratagene Cloning Systems) is utilized to generate different size
polynucleotides by priming at random sites on templates which are
prepared by U.V. light (as described above) and extending along the
templates. The priming protocols such as described in the Prime-It
II Random Primer Labeling kit may be utilized to extend the
primers. The dimers formed by U.V. exposure serve as a roadblock
for the extension by the non-proofreading polymerase. Thus, a pool
of random size polynucleotides is present after extension with the
random primers is finished.
EXAMPLE 2
Isolation of Random Size Polynucleotides
[1900] Polynucleotides of interest which are generated according to
Example 1 are gel isolated on a 1.5% agarose gel. Polynucleotides
in the 100-300 bp range are cut out of the gel and 3 volumes of 6 M
NaI is added to the gel slice. The mixture is incubated at
50.degree. C. for 10 minutes and 10 .mu.l of glass milk (Bio 101)
is added. The mixture is spun for 1 minute and the supernatant is
decanted. The pellet is washed with 500 .mu.l of Column Wash
(Column Wash is 50% ethanol, 10 mM Tris-HCl pH 7.5, 100 mM NaCl and
2.5 mM EDTA) and spin for 1 minute, after which the supernatant is
decanted. The washing, spinning and decanting steps are then
repeated. The glass milk pellet is resuspended in 20 .mu.l of
H.sub.2O and spun for 1 minute. DNA remains in the aqueous
phase.
EXAMPLE 3
Shuffling of Isolated Random Size 100-300bp Polynucleotides
[1901] The 100-300 bp polynucleotides obtained in Example 2 are
recombined in an annealing mixture (0.2 mM each dNTP, 2.2 MM
MgCl.sub.2, 50 mM KCl, 10 mM Tris-HCl ph 8.8, 0.1% Triton X-100,
0.3.mu.; Taq DNA polymerase, 50 .mu.l total volume) without adding
primers. A Robocycler by Stratagene was used for the annealing step
with the following program: 95.degree. C. for 30 seconds, 25-50
cycles of [95.degree. C. for 30 seconds, 50 -60.degree. C.
(preferably 58.degree. C.) for 30 seconds, and 72.degree. C. for 30
seconds] and 5 minutes at 72.degree. C. Thus, the 100-300 bp
polynucleotides combine to yield double-stranded polynucleotides
having a longer sequence. After separating out the reassembled
double-stranded polynucleotides and denaturing them to form single
stranded polynucleotides, the cycling is optionally again repeated
with some samples utilizing the single strands as template and
primer DNA and other samples utilizing random primers in addition
to the single strands.
EXAMPLE 4
Screening of Polypeptides from Shuffled Polynucleotides
[1902] The polynucleotides of Example 3 are separated and
polypeptides are expressed therefrom. The original template DNA is
utilized as a comparative control by obtaining comparative
polypeptides therefrom. The polypeptides obtained from the shuffled
polynucleotides of Example 3 are screened for the activity of the
polypeptides obtained from the original template and compared with
the activity levels of the control. The shuffled polynucleotides
coding for interesting polypeptides discovered during screening are
compared further for secondary desirable traits. Some shuffled
polynucleotides corresponding to less interesting screened
polypeptides are subjected to reshuffling.
EXAMPLE 5
Directed Evolution an Enzyme by Saturation Mutagenesis
[1903] Site-Saturation Mutagenesis: To accomplish site-saturation
mutagenesis every residue (316) of a dehalogenase enzyme was
converted into all 20 amino acids by site directed mutagenesis
using 32-fold degenerate oligonucleotide primers, as follows:
[1904] 1. A culture of the dehalogenase expression construct was
grown and a preparation of the plasmid was made
[1905] 2. Primers were made to randomize each codon--they have the
common structure X.sub.20NN(G/T)X.sub.20
[1906] 3. A reaction mix of 25 ul was prepared containing .about.50
ng of plasmid template, 125 ng of each primer, 1.times.native Pfu
buffer, 200 uM each dNTP and 2.5 U native Pfu DNA polymerase
[1907] 4. The reaction was cycled in a Robo96 Gradient Cycler as
follows:
[1908] Initial denaturation at 95.degree. C. for 1 min
[1909] 20 cycles of 95.degree. C. for 45 sec, 53.degree. C. for 1
min and 72.degree. C. for 11 min
[1910] Final elongation step of 72.degree. C. for 10 min
[1911] 5. The reaction mix was digested with 10 U of DpnI at
37.degree. C. for 1 hour to digest the methylated template DNA
[1912] 6. Two ul of the reaction mix were used to transform 50 ul
of XL1-Blue MRF' cells and the entire transformation mix was plated
on a large LB-Amp-Met plate yielding 200-1000 colonies
[1913] 7. Individual colonies were toothpicked into the wells of
96-well microtiter plates containing LB-Amp-IPTG and grown
overnight
[1914] 8. The clones on these plates were assayed the following
day
[1915] Screening: Approximately 200 clones of mutants for each
position were grown in liquid media (384 well microtiter plates)
and screened as follows:
[1916] 1. Overnight cultures in 384-well plates were centrifuged
and the media removed. To each well was added 0.06 mL 1 mM
Tris/SO.sub.4.sup.2- pH 7.8.
[1917] 2. Made 2 assay plates from each parent growth plate
consisting of 0.02 mL cell suspension.
[1918] 3. One assay plate was placed at room temperature and the
other at elevated temperature (initial screen used 55.degree. C.)
for a period of time (initially 30 minutes).
[1919] 4. After the prescribed time 0.08 mL room temperature
substrate (TCP saturated 1 mM Tris/SO.sub.4.sup.2- pH 7.8 with 1.5
mM NaN.sub.3 and 0.1 mM bromothymol blue) was added to each
well.
[1920] 5. Measurements at 620 nm were taken at various time points
to generate a progress curve for each well.
[1921] 6. Data were analyzed and the kinetics of the cells heated
to those not heated were compared. Each plate contained 1-2 columns
(24 wells) of unmutated 20F12 controls.
[1922] 7. Wells that appeared to have improved stability were
re-grown and tested under the same conditions.
[1923] Following this procedure nine single site mutations appeared
to confer increased thermal stability on the enzyme. Sequence
analysis was performed to determine of the exact amino acid changes
at each position that were specifically responsible for the
improvement. In sum, the improvement was conferred at 7 sites by
one amino acid change alone, at an eighth site by each of two amino
acid changes, and at a ninth site by each of three amino acid
changes. Several mutants were then made each having a plurality of
these nine beneficial site mutations in combination; of these two
mutants proved superior to all the other mutants, including those
with single point mutations.
EXAMPLE 6
Direct Expression Cloning using End-Selection
[1924] An esterase gene was amplified using 5' phosphorylated
primers in a standard PCR reaction (10 ng template; PCR conditions:
3' 94 C.; [1' 94 C.; 1' 50 C.; 1'30" 68 C.].times.30; 10' 68 C.
91 Forward Primer = 9511TopF (CTAGAAGGGAGGAGAATTACATGAAG-
CGGCTTTTAGCCC) Reverse Primer = 9511TopR
(AGCTAAGGGTCAAGGCCGCACCCGAGG)
[1925] The resulting PCR product (ca.1000 bp) was gel purified and
quantified.
[1926] A vector for expression cloning, pASK3 (Institut fuer
Bioanalytik, Goettingen, Germany), was cut with Xba I and Bgl II
and dephosphorylated with CIP.
[1927] 0.5 pmoles Vaccina Topoisomerase I (Invitrogen, Carlsbad,
Calif.) was added to 60 ng (ca. 0.1 pmole) purified PCR product for
5' 37 C. in buffer NEB I (New England Biolabs, Beverly, Mass.) in 5
.mu.l total volume.
[1928] The topogated PCR product was cloned into the vector pASK3
(5 .mu.l, ca. 200 ng in NEB I) for 5' at room temperature.
[1929] This mixture was dialyzed against H.sub.2O for 30'.
[1930] 2 .mu.l were used for electroporation of DH10B cells (Gibco
BRL, Gaithersburg, Md.).
[1931] Efficiency: Based on the actual clone numbers this method
can produce 2.times.10.sup.6 clones per .mu.g vector. All tested
recombinants showed esterase activity after induction with
anhydrotetracycline.
EXAMPLE 7
Dehalogenase Thermal Stability
[1932] This invention provides that a desirable property to be
generated by directed evolution is exemplified in a limiting
fashion by an improved residual activity (e.g. an enzymatic
activity, an immunoreactivity, an antibiotic acivity, etc.) of a
molecule upon subjection to altered environment, including what may
be considered a harsh enviroment, for a specified time. Such a
harsh environment may comprise any combination of the following
(iteratively or not, and in any order or permutation): an elevated
temperature (including a temperature that may cause denaturation of
a working enzyme), a decreased temperature, an elevated salinity, a
decreased salinity, an elevated pH, a decreased pH, an elevated
pressure, a decreassed pressure, and an change in exposure to a
radiation source (including uv radiation, visible light, as well as
the entire electromagnetic spectrum).
[1933] The following example shows an application of directed
evolution to evolve the ability of an enzyme to regain &/or
retain activity upon exposure to an elevated temperature.
[1934] Every residue (316) of a dehalogenase enzyme was converted
into all 20 amino acids by site directed mutagenesis using 32-fold
degenerate oligonucleotide primers. These mutations were introduced
into the already rate-improved variant Dhla 20F12. Approximately
200 clones of each position were grown in liquid media (384 well
microtiter plates) to be screened. The screening procedure was as
follows:
[1935] 1. Overnight cultures in 384-well plates were centrifuged
and the media removed. To each well was added 0.06 mL 1 mM
Tris/SO.sub.4.sup.2- pH 7.8.
[1936] 2. The robot made 2 assay plates from each parent growth
plate consisting of 0.02 mL cell suspension.
[1937] 3. One assay plate was placed at room temperature and the
other at elevated temperature (initial screen used 55.degree. C.)
for a period of time (initially 30 minutes).
[1938] 4. After the prescribed time 0.08 mL room temperature
substrate (TCP saturated 1 mM Tris/SO.sub.4.sup.2- pH 7.8 with 1.5
mM NaN.sub.3 and 0.1 mM bromothymol blue) was added to each well.
TCP=trichloropropane.
[1939] 5. Measurements at 620 nm were taken at various time points
to generate a progress curve for each well.
[1940] 6. Data were analyzed and the kinetics of the cells heated
to those not heated were compared. Each plate contained 1-2 columns
(24 wells) of un-mutated 20F12 controls.
[1941] 7. Wells that appeared to have improved stability were
regrown and tested under the same conditions.
[1942] Following this procedure nine single site mutations appeared
to confer increased thermal stability on Dhla-20F12. Sequence
analysis showed that the following changes were beneficial:
[1943] D89G
[1944] F91S
[1945] T159L
[1946] G189Q, G189V
[1947] I220L
[1948] N238T
[1949] W251Y
[1950] P302A, P302L, P302S, P302K
[1951] P302R/S306R
[1952] Only two sites (189 and 302) had more than one substitution.
The first 5 on the list were combined (using G189Q) into a single
gene (this mutant is referred to as "Dhla5"). All changes but S306R
were incorporated into another variant referred to as Dhla8.
[1953] Thermal stability was assessed by incubating the enzyme at
the elevated temperature (55.degree. C. and 80.degree. C.) for some
period of time and activity assay at 30.degree. C. Initial rates
were plotted vs. time at the higher temperature. The enzyme was in
50 mM Tris/SO.sub.4 pH 7.8 for both the incubation and the assay.
Product (C1.sup.-) was detected by a standard method using
Fe(NO.sub.3).sub.3 and HgSCN. Dhla 20F12 was used as the de facto
wild type. The apparent half-life (T.sub.1/2) was calculated by
fitting the data to an exponential decay function.
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