U.S. patent application number 10/877035 was filed with the patent office on 2005-03-24 for optimization of immunomodulatory properties of genetic vaccines.
This patent application is currently assigned to Maxygen, Inc., a Delaware corporation. Invention is credited to Howard, Russell, Punnonen, Juha, Stemmer, Willem P.C., Whalen, Robert G..
Application Number | 20050064464 10/877035 |
Document ID | / |
Family ID | 34317325 |
Filed Date | 2005-03-24 |
United States Patent
Application |
20050064464 |
Kind Code |
A1 |
Punnonen, Juha ; et
al. |
March 24, 2005 |
Optimization of immunomodulatory properties of genetic vaccines
Abstract
This invention provides methods for obtaining molecules that can
modulate an immune response, and immunomodulatory molecules
obtained using the methods. The molecules find use, for example, in
the tailoring of an immune response induced by a genetic vaccine
for a desired purpose.
Inventors: |
Punnonen, Juha; (Belmont,
CA) ; Stemmer, Willem P.C.; (Los Gatos, CA) ;
Whalen, Robert G.; (Foster City, CA) ; Howard,
Russell; (Los Altos Hills, CA) |
Correspondence
Address: |
MAXYGEN, INC.
INTELLECTUAL PROPERTY DEPARTMENT
515 GALVESTON DRIVE
RED WOOD CITY
CA
94063
US
|
Assignee: |
Maxygen, Inc., a Delaware
corporation
|
Family ID: |
34317325 |
Appl. No.: |
10/877035 |
Filed: |
June 25, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10877035 |
Jun 25, 2004 |
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09724869 |
Nov 28, 2000 |
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09724869 |
Nov 28, 2000 |
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09248716 |
Feb 10, 1999 |
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60074294 |
Feb 11, 1998 |
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Current U.S.
Class: |
435/6.18 ;
435/6.1; 435/91.2; 514/44R |
Current CPC
Class: |
C07K 2319/74 20130101;
C07K 2319/40 20130101; C07K 14/005 20130101; C07K 14/24 20130101;
C12N 15/1027 20130101; C12N 15/1093 20130101; A61K 39/00 20130101;
C12N 15/1034 20130101; C40B 40/02 20130101; C12N 2730/10122
20130101; C12N 15/1037 20130101; C12N 2740/16222 20130101; A61K
2039/53 20130101; C07K 2319/02 20130101 |
Class at
Publication: |
435/006 ;
435/091.2; 514/044 |
International
Class: |
C12Q 001/68; C12P
019/34; A61K 048/00 |
Goverment Interests
[0002] This invention was made with Government support under Grant
No. N65236-98-1-5401, awarded by the Defense Advanced Projects
Agency. The Government has certain rights in this invention.
Claims
What is claimed is:
1. A method for obtaining a polynucleotide that has a modulatory
effect on an immune response, or encodes a polypeptide that has a
modulatory effect on an immune response, that is induced by a
genetic vaccine vector, the method comprising: creating a library
of recombinant polynucleotides; and screening the library to
identify an optimized recombinant 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
recombinant polynucleotide or the polypeptide encoded by the
recombinant polynucleotide exhibits an enhanced ability to modulate
an immune response compared to a non-recombinant polynucleotide
from which the library was created.
2. The method of claim 1, wherein the optimized recombinant
polynucleotide is incorporated into a genetic vaccine vector.
3. The method of claim 1, wherein the optimized recombinant
polynucleotide, or a polypeptide encoded by the optimized
recombinant polynucleotide, is administered in conjunction with a
genetic vaccine vector.
4. The method of claim 1, wherein the library of recombinant
polynucleotides is created by a process selected from the group
consisting of DNA shuffling, error-prone PCR,
oligonucleotide-directed mutagenesis, uracil-mediated mutagenesis,
and repair-deficient host mutagenesis.
5. The method of claim 1, wherein the polynucleotide that has a
modulatory effect on an immune response is obtained by: (1)
recombining 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 recombinant
polynucleotides; and (2) screening the library to identify at least
one optimized recombinant polynucleotide that exhibits, either by
itself or through the encoded molecule, an enhanced ability to
modulate an immune response than a form of the nucleic acid from
which the library was created.
6. The method of claim 5, wherein the method further comprises the
steps of: (3) recombining at least one optimized recombinant
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 recombinant polynucleotides; (4) screening the
further library to identify at least one further optimized
recombinant 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 recombinant polynucleotide
exhibits an further enhanced ability to modulate an immune response
than a form of the nucleic acid from which the library was
created.
7. The method of claim 1, wherein the optimized recombinant
polynucleotide encodes a peptide or polypeptide that can interact
with a cellular receptor involved in mediating an immune response,
wherein the peptide or polypeptide acts as an agonist or antagonist
of the receptor.
8. The method of claim 7, wherein the cellular receptor is a
macrophage scavenger receptor.
9. The method of claim 7, wherein the cellular receptor is selected
from the group consisting of a cytokine receptor and a chemokine
receptor.
10. The method of claim 9, wherein the chemokine receptor is
CCR6.
11. The method of claim 7, wherein the peptide or polypeptide
mimics the activity of a natural ligand for the receptor but does
not induce immune reactivity to the natural ligand.
12. The method of claim 7, wherein the library is screened by:
expressing the recombinant 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.
13. The method of claim 12, wherein the replicable genetic package
is selected from the group consisting of a bacteriophage, a cell, a
spore, and a virus.
14. The method of claim 13, wherein the replicable genetic package
is an Ml 3 bacteriophage and the protein is encoded by geneIII or
geneVII.
15. The method of claim 7, which method further comprises
introducing the optimized recombinant 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.
16. The method of claim 7, which method further comprises producing
the peptide or polypeptide encoded by the optimized recombinant
polynucleotide and introducing the peptide or polypeptide into a
mammal in conjunction with a genetic vaccine vector.
17. The method of claim 7, wherein the optimized recombinant
polynucleotide is inserted into an antigen-encoding nucleotide
sequence of a genetic vaccine vector.
18. The method of claim 17, wherein the optimized recombinant
polypeptide is introduced into a nucleotide sequence that encodes
an M-loop of an HBsAg polypeptide.
19. The method of claim 1, wherein the optimized recombinant
polynucleotide comprises a nucleotide sequence rich in unmethylated
CpG.
20. The method of claim 1, wherein the optimized recombinant
polynucleotide encodes a polypeptide that inhibits an allergic
reaction.
21. The method of claim 20, 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.
22. The method of 1, wherein the optimized recombinant
polynucleotide encodes an antagonist of IL-10.
23. The method of claim 22, wherein the antagonist of IL-10 is
soluble or defective IL-10 receptor or IL-20/MDA-7.
24. The method of claim 1, wherein the optimized recombinant
polynucleotide encodes a costimulator.
25. The method of claim 24, wherein the costimulator is B7-1 (CD80)
or B7-2 (CD86) and the screening step involves selecting variants
with altered activity through CD28 or CTLA-4.
26. The method of claim 24, wherein the costimulator is CD1, CD40,
CD154 (ligand for CD40) or CD150 (SLAM).
27. The method of claim 24, wherein the costimulator is a
cytokine.
28. The method of claim 27, 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-17,
IL-18, GM-CSF, G-CSF, TNF-.alpha., IFN-.alpha., IFN-.gamma., and
IL-20 (MDA-7).
29. The method of 28, wherein the library of recombinant
polynucleotides is screened by testing the ability of cytokines
encoded by the recombinant polynucleotides to activate cells which
contain a receptor for the cytokine.
30. The method of claim 29, wherein the cells contain a
heterologous nucleic acid that encodes the receptor for the
cytokine.
31. The method of 28, 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.
32. The method of 28, wherein the cytokine is interferon-.alpha.
and the screening is performed by: expressing the recombinant
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 B cells; and identifying phage library
members that are capable of inhibiting proliferation of the B
cells.
33. The method of claim 28, 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..
34. The method of claim 27, wherein the cytokine encoded by the
optimized recombinant 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 recombinant polynucleotide
into a mammal and determining whether an immune response is induced
against the cytokine.
35. The method of claim 24, wherein the costimulator is B7-1 (CD80)
or B7-2 (CD86) and the cell is tested for ability to costimulate an
immune response.
36. The method of claim 1, wherein the optimized recombinant
polynucleotide encodes a cytokine antagonist.
37. The method of claim 36, 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.
38. The method of claim 36, wherein the cytokine antagonist is
selected from the group consisting of .DELTA.IL-10R and
.DELTA.IL-4R.
39. The method of claim 1, wherein the optimized recombinant
polynucleotide encodes a polypeptide capable of inducing a
predominantly T.sub.H1 immune response.
40. The method of claim 1, wherein the optimized recombinant
polynucleotide encodes a polypeptide capable of inducing a
predominantly T.sub.H2 immune response.
41. A method for obtaining a polynucleotide that encodes an
accessory molecule that improves the transport or presentation of
antigens by a cell, the method comprising: creating a library of
recombinant polynucleotides by subjecting to recombination nucleic
acids that encode all or part of the accessory molecule; and
screening the library to identify an optimized recombinant
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.
42. The method of claim 41, wherein the screening involves:
introducing the library of recombinant 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.
43. The method of claim 41, wherein the accessory molecule
comprises a proteasome or a TAP polypeptide.
44. The method of claim 41, wherein the accessory molecule
comprises a cytotoxic T-cell inducing sequence.
45. The method of claim 44, wherein the cytotoxic T-cell inducing
sequence is obtained from a hepatitis B surface antigen.
46. The method of claim 41, wherein the accessory molecule
comprises an immunogenic agonist sequence.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims benefit of U.S. Provisional
Application No. 60/074,294, filed Feb. 11, 1998, which application
is incorporated herein by reference for all purposes.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] This invention pertains to the field of modulation of immune
responses such as those induced by genetic vaccines.
[0005] 2. Background
[0006] 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. 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.
[0007] 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.
[0008] 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-.gamma. 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-.gamma. 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.H2 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).
[0009] 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.H1 phenotype is observed in
tuberculoid (resistant) form of leprosy and T.sub.H2 phenotype in
lepromatous, multibacillary (susceptible) lesions (Yamamura et al.
(1991) 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-.alpha. 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-.alpha. production were more likely to
survive the infection (Westendorp et al. (1997) Lancet 349:
170-173).
[0010] 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-.gamma. 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).
[0011] 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.
SUMMARY OF THE INVENTION
[0012] 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 recombinant polynucleotides; and screening the library
to identify at least one optimized recombinant 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 immmune response,
or a predominantly T.sub.H1 immune response.
[0013] In some embodiments, the polynucleotide that has a
modulatory effect on an immune response is obtained by: (1)
recombining 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 recombinant
polynucleotides; and (2) screening the library to identify at least
one optimized recombinant 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) recombining at least
one optimized recombinant 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 recombinant
polynucleotides; (4) screening the further library to identify at
least one further optimized recombinant 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 recombinant polynucleotide exhibits an further enhanced
ability to modulate an immune response than a form of the nucleic
acid from which the library was created.
[0014] In some embodiments of the invention, the library of
recombinant polynucleotides is screened by: expressing the
recombinant 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.
[0015] 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 recombinant polynucleotides by
subjecting to recombination nucleic acids that encode all or part
of the accessory molecule; and screening the library to identify an
optimized recombinant 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 recombinant
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.
[0016] 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-.alpha. 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.
[0017] The invention provides methods of using DNA shuffling 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.
[0018] 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 .DELTA.IL-10R and .DELTA.IL-4R,
and the like.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 shows an example of a cytotoxic T-cell inducing
sequence (CTIS) obtained from HBsAg polypeptide (PreS2 plus S
regions).
[0020] FIG. 2 shows a CTIS having heterologous epitopes attached to
the cytoplasmic portion.
[0021] FIG. 3 shows the derivation of immunogenic agonistic
sequences (IAS) as described in Example 3. Specific killing
(percent) is shown for an effector: target (E:T) ratio of five.
[0022] FIG. 4 shows 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
DNA shuffling to obtain a nucleic acid encoding a poly-epitope
region that contains potential agonist sequences.
[0023] FIG. 5 shows a scheme for improving immunostimulatory
sequences by DNA shuffling.
[0024] FIG. 6 is a diagram of a procedure by which recombinant
libraries of human IL-12 genes can be screened to identify shuffled
IL-12 genes that encode recombinant IL-12 having increased ability
to induce T cell proliferation.
[0025] FIG. 7 shows the results of a high-throughput functional
assay for vectors that encode variants of IL-12 obtained using the
methods of the invention.
[0026] FIG. 8 shows the induction of T cell proliferation upon
transfection of the T cells by individual vectors that encode IL-12
variants.
[0027] FIG. 9 shows results of an experiment which demonstrates
that a shuffled IL-12 chimera obtained using the methods of the
invention exhibits improved ability to activate human T cells.
[0028] FIG. 10 shows 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.
[0029] FIG. 11 shows a method for using DNA shuffling to obtain
CD80/CD86 variants that have improved capacity to induce T cell
activation or anergy.
[0030] FIG. 12 shows results obtained in a screening assay for
altered function of B7.
[0031] FIG. 13 provides experimental results which demonstrate that
shuffled B7 chimeras provide potent T cell activation.
[0032] FIG. 14 presents an alignment of the nucleotide sequences
for human and mouse IL-10 receptor sequences.
[0033] FIG. 15 shows an alignment of the nucleotide sequences of
B7-1 (CD80) genes from human, rhesus monkey, and rabbit.
DETAILED DESCRIPTION
[0034] Definitions
[0035] 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.
[0036] The term "screening" describes, in general, a process that
identifies optimal immunomodulatory molecules. Several properties
of the respective molecules can be used in selection and screening
including, for example, ability to induce a desired immune response
in a test system. 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 genetic vaccines that
include immunomodulatory molecules 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.
[0037] A "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
heterologous 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 DNA shuffling. 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.
Exogenous DNA segments are expressed to yield exogenous
polypeptides.
[0038] 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.
[0039] 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. 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.
[0040] The term "naturally-occurring" is used to describe an object
that can be found in nature as distinct from being artificially
produced by man. 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.
[0041] 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.
[0042] "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.
[0043] 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.
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.
[0044] 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.6 M.sup.-1
or greater.
[0045] 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.
[0046] 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.
[0047] A "recombinant polynucleotide" or a "recombinant
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.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] 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).
[0052] 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.ncbi.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. 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).
[0053] 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.
[0054] Another indication that two nucleic acid sequences are
substantially identical is that the two molecules hybridize to each
other tinder 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.
[0055] "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. 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, New York. 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.
[0056] 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.sub.m 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 1 mg of heparin at 42.degree. C., with the
hybridization being carried out overnight. An example of highly
stringent wash conditions is 0.15M NaCl at 72.degree. C. for about
15 minutes. An example of stringent wash conditions is a
0.2.times.SSC wash at 65.degree. 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 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.
[0057] 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.
[0058] 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.
[0059] "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.
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. 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.
[0060] 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:
[0061] Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine
(L), Isoleucine (I);
[0062] Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan
(W);
[0063] Sulfur-containing: Methionine (M), Cysteine (C);
[0064] Basic: Arginine (R), Lysine (K), Histidine (H);
[0065] Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine
(N), Glutamine (Q). 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".
[0066] 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.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0067] 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.
[0068] A. Creation of Recombinant Libraries
[0069] 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.
[0070] The substrate nucleic acids used for the recombination can
vary depending upon the particular application. For example, where
a polynucleotide that encodes a cytokine, chemokine, or other
accessory molecule is to be optimized, different forms of nucleic
acids that encode all or part of the cytokine, chemokine, or other
accessory molecule are subjected to recombination. The methods
require at least two variant forms of a starting substrate. 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 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
recombination.
[0071] Often, improvements are achieved after one round of
recombination and selection. However, recursive sequence
recombination can be employed to achieve still further improvements
in a desired property. Sequence recombination can be achieved in
many different formats and permutations of formats, as described in
further detail below. These formats share some common principles.
Recursive sequence recombination entails successive cycles of
recombination to generate molecular diversity. That is, one creates
a family of nucleic acid molecules showing some sequence identity
to each other but differing in the presence of mutations. In any
given cycle, recombination can occur in vivo or in vitro,
intracellular or extracellular. Furthermore, diversity resulting
from recombination 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 for
recombination. In some instances, a new or improved property or
characteristic can be achieved after only a single cycle of in vivo
or in vitro recombination, 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.
[0072] In a presently preferred embodiment, the recombinant
libraries are prepared using DNA shuffling. The shuffling and
screening or selection can be used to "evolve" individual genes,
whole plasmids or viruses, multigene clusters, or even whole
genomes (Stemmer (1995) Bio/Technology 13:549-553). Reiterative
cycles of recombination 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. Shuffling allows
the recombination of large numbers of mutations in a minimum number
of selection cycles, in contrast to traditional, pairwise
recombination events. Thus, the sequence recombination techniques
described herein provide particular advantages in that they provide
recombination between mutations in any or all of these, 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
recombination, provides opportunities for modification of the
technique.
[0073] Exemplary formats and examples for sequence recombination,
sometimes referred to as DNA shuffling, evolution, or molecular
breeding, have been described by the present inventors and
co-workers in co-pending applications U.S. patent application Ser.
No. 08/198,431, filed Feb. 17, 1994, Serial No. PCT/US95/02126,
filed, Feb. 17, 1995, Ser. No. 08/425,684, filed Apr. 18, 1995,
Ser. No. 08/537,874, filed October 30, 1.995, Ser. No. 08/564,955,
filed Nov. 30, 1995, Ser. No. 08/621,859, filed Mar. 25, 1996, Ser.
No. 08/621,430, filed Mar. 25, 1996, Serial No. PCT/US96/05480,
filed Apr. 18, 1996, Ser. No. 08/650,400, filed May 20, 1996, Ser.
No. 08/675,502, filed Jul. 3, 1996, Ser. No. 08/721, 824, filed
Sep. 27, 1996, Serial No. PCT/US97/17300, filed Sep. 26, 1997, and
Serial No. PCT/US97/24239, filed Dec. 17, 1997; Stemmer, Science
270:1510 (1995); Stemmer et al., Gene 164:49-53 (1995); Stemmer,
Bio/Technology 13:549-553 (1995); Stemmer, Proc. Natl. Acad. Sci.
U.S.A. 91:10747-10751 (1994); Stemmer, Nature 370:389-391 (1994);
Crameri et al., Nature Medicine 2(1):1-3 (1996); Crameri et al.,
Nature Biotechnology 14:315-319 (1996), each of which is
incorporated by reference in its entirety for all purposes.
[0074] Other methods for obtaining recombinant polynucleotides
and/or for obtaining diversity in nucleic acids used as the
substrates for shuffling include, for example, homologous
recombination (PCT/US98/05223; Publ. No. WO98/42727);
oligonucleotide-directed mutagenesis (for review see, 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 Grundstrom et al., Nucl. Acids Res. 13:
3305-3316 (1985). Kits for mutagenesis are commercially available
(e.g., Bio-Rad, Amersham International, Anglian Biotechnology).
[0075] B. Screening Methods
[0076] A recombination cycle is usually followed by at least one
cycle of screening or selection for molecules having a desired
property or characteristic. If a recombination cycle is performed
in vitro, the products of recombination, 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.
Alternatively, products of recombination generated in vitro are
sometimes packaged as viruses before screening. If recombination is
performed in vivo, recombination products can sometimes be screened
in the cells in which recombination occurred. In other
applications, recombinant segments are extracted from the cells,
and optionally packaged as viruses, before screening.
[0077] 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 recombination
(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 recombined 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.
[0078] 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. Later rounds, and other types of screening
which are not amenable to screening in bacterial cells, are
performed 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 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.
[0079] 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 identified as components of cells, components of viruses or in
free form. More than one round of screening or selection can be
performed after each round of recombination.
[0080] 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 recombination. These recombinant segments can be recombined with
each other or with exogenous segments representing the original
substrates or further variants thereof. Again, recombination 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 recombination in vivo, or
can be subjected to further recombination in vitro, or can be
isolated before performing a round of in vitro recombination.
Conversely, if the previous screening step identifies desired
recombinant segments in naked form or as components of viruses,
these segments can be introduced into cells to perform a round of
in vivo recombination. The second round of recombination,
irrespective how performed, generates further recombinant segments
which encompass additional diversity than is present in recombinant
segments resulting from previous rounds.
[0081] The second round of recombination 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. Additional
rounds of recombination and screening can then be performed until
the recombinant segments have sufficiently evolved to acquire the
desired new or improved property or function.
[0082] Various screening methods for particular applications are
described herein. In several instances, screening involves
expressing the recombinant peptides or polypeptides encoded by the
recombinant 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 & Smith, Science 249:
386-388 (1990); Ladner et al., U.S. Pat. No. 5,571,698. Other
replicable genetic packages include, for example, bacteria,
eukaryotic viruses, yeast, and spores.
[0083] 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, WO91/18989; 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.
[0084] 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.
[0085] 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. 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.
[0086] C. Evolution of Improved Immunomodulatory Sequences
[0087] 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.
[0088] Of particular interest are IFN-.alpha. and IL-12, which skew
immune responses towards a T helper 1 (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 techniques of the invention provide much faster solutions
than rational design.
[0089] The methods of the invention typically involve the use of
DNA shuffling or other methods to create a library of recombinant
polynucleotides. The library is then screened to identify
recombinant 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 recombinant polynucleotides into mammalian cells and
determining whether the cells, or culture medium obtained by
growing the cells, is capable of modulating an immune response.
[0090] Optimized recombinant vector modules obtained through
polynucleotide recombination 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 DNA shuffling methods of the invention
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.
[0091] 1. Immunostimulatory DNA Sequences
[0092] 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 (Id.). 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.).
[0093] According to the invention, a library is generated by
subjecting to recombination 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 recombined to produce a
library of recombinant polynucleotides.
[0094] The library is then screened to identify those recombinant
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 in FIG.
5. 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-.gamma.. One can also
test for changes in ratios of IL-4/IFN-.gamma., IL-4/IL-2,
IL-5/IFN-.gamma., IL-5/IL-2, IL-13/IFN-.gamma., 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 CD14.
[0095] Other useful screens include identifying recombinant
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.
[0096] Libraries of recombinant polynucleotides can also be
screened for improved CTL and antibody responses in vivo and for
improved protection from infection, cancer, allergy or
autoimmunity. Recombinant polynucleotides that exhibit the desired
property can be recovered from the cell and, if further improvement
is desired, the shuffling 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.
[0097] 2. Cytokines, Chemokines, and Accessory Molecules
[0098] 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.
[0099] 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. 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-.alpha./.gamma., IL-10, IL-12, and antagonists
of IL-4 and IL-13.
[0100] The optimized immunomodulators, or optimized recombinant
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.
[0101] In the methods of the invention, a library of recombinant
polynucleotides that encode immunomodulators is created by
subjecting substrate nucleic acids to a recombination protocol,
such as DNA shuffling 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.
[0102] Cytokines are among the immunomodulators that can be
improved using the 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-15, IL-16, IL-17, IL-18, G-CSF, GM-CSF,
IFN-.alpha., IFN-.gamma., TGF-.beta., TNF-.alpha., TNF-.beta.,
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.
[0103] In some embodiments, the invention provides methods of
obtaining optimized immunomodulators 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-.alpha. 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-.gamma.
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. For example, where increased
mucosal immunity is desired, including protective immunity,
enhancing the T.sub.H2 response can lead to increased antibody
production, particularly IgA.
[0104] 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-.gamma. 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-.gamma.
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: 241), 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).
[0105] 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 shuffling and selection.
[0106] 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-.alpha. (IFN-.alpha.) and
interleukin-12 (IL-12) are preferred substrates for recombination
and selection in order to obtain maximal specific activity and
capacity to act as adjuvants in genetic vaccinations. IFN-.alpha.
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-.alpha. was the first cytokine to be used in
clinical practice. Today, IFN-.alpha. is used for a wide variety of
applications, including several types of cancers and viral
diseases. IFN-.alpha. 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. The
species difference was recently explained by data indicating that,
like IL-12, IFN-.alpha. 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).
[0107] Family DNA shuffling is a preferred method for optimizing
IFN-.alpha., using as substrates the mammalian IFN-.alpha. genes,
which are 85%-97% homologous. Greater than 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. Recombinants
with improved potency and selectivity profiles are being
selectively bred for improved activity. Variants which demonstrate
improved binding to IFN-.alpha. 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-.alpha. mutants to induce IL-2 and IFN-.gamma.
production in in vitro human T lymphocyte cultures can be studied
by cytokine-specific ELISA and cytoplasmic cytokine staining and
flow cytometry.
[0108] 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. 158: 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; Stern et al. (1990) Proc. Nat'l.
Acad. Sci. USA 87: 6808). Recently Lieschke et al. ((1997) Nature
Biotech. 15: 35) 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 shuffle IL-12 genes as one entity, which is
beneficial in designing the shuffling protocol. 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 shuffled IL-12 molecules.
[0109] Incorporation of evolved IFN-.alpha. and/or IL-12 genes into
genetic vaccine vectors is expected to be safe. The safety of
IFN-.alpha. 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
aims 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). DNA shuffling
may allow selection for a shorter half-life, thereby reducing the
toxicity even after high bolus doses.
[0110] 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.
[0111] 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 shuffling of IL-4 or soluble IL-4
receptor. The IL-4 receptor consists of an IL-4R .alpha.-chain (140
kD high-affinity binding unit) and an IL-2R y-chain (these cytokine
receptors share a common .gamma.-chain). The IL-4R .alpha.-chain is
shared by IL-4 and IL-13 receptor complexes. Both IL-4 and IL-13
induce phosphorylation of the IL-4R .alpha.-chain, but expression
of IL-4R .alpha.-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 DNA
shuffling 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 shuffling 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 shuffled IL-4R .alpha.-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.
[0112] IL-2 and IL-15 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-.alpha. or IFN-.gamma.. 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 DNA shuffling methods of the invention
increases the advantageous effects compared to wild-type IL-2 and
IL-15.
[0113] 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. IL-6 has also been shown to enhance
IL-4-induced IgE 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 DNA shuffling, will have more beneficial effects than
the wild-type IL-6.
[0114] 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
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-.alpha., IFN-.gamma. 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 DNA shuffling methods of the invention
to obtain IL-8 with improved specific activity and/or with improved
expression in target cells.
[0115] 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.
[0116] 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 Ig
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 IgE synthesis, by autocrine mechanisms.
[0117] The invention provides methods of evolving an IL-S
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 shuffled 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-S dependent cells lines cultured in the presence of recombinant
wild-type IL-5. Alternatively, shuffled IL-5R .alpha.-chains are
screened for improved binding to IL-5.
[0118] Tumor necrosis factors (.alpha. and .beta.) and their
receptors are also suitable targets for modification and use in
genetic vaccines. TNF-.alpha., 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-.alpha. 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-.alpha.
plays a major role in the pathogenesis of endotoxin shock. A
membrane-bound form of TNF-.alpha. (mTNF-.alpha.), which is
involved in interactions between B- and T-cells, is rapidly
upregulated within four hours of T cell activation. mTNF-.alpha.
plays a role in the polyclonal B cell activation observed in
patients infected with HIV. Monoclonal antibodies specific for
mTNF-.alpha. or the p55 TNF-.alpha. receptor strongly inhibit IgE
synthesis induced by activated CD4.sup.+ T cell clones or their
membranes. Mice deficient for p55 TNF-.alpha.R are resistant to
endotoxic shock, and soluble TNF-AR prevents autoimmune diabetes
mellitus in NOD mice. Phase III trials using sTNF-.alpha.R in the
treatment of rheumatoid arthritis are in progress, after promising
results obtained in the phase II trials.
[0119] The methods of the invention can be used to, for example,
evolve a soluble TNF-.alpha.R that has improved affinity, and thus
is capable of acting as an antagonist for TNF activity. Nucleic
acids that encode TNF-.alpha.R and exhibit sequence diversity, such
as the natural diversity observed in cDNA libraries from activated
T cells of human and other primates, are shuffled. The shuffled
nucleic acids are expressed, e.g., on phage, after which mutants
are selected that bind to TNF-.alpha. with improved affinity. If
desired, the improved mutants can be subjected to further assays
using biological activity, and the shuffled genes can be subjected
to one or more rounds of shuffling and screening.
[0120] Another target of interest for application of the methods of
the invention is interferon-.gamma., and the evolution of
antagonists of this cytokine. The receptor for IFN-.gamma. 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. The mouse
IFN-.gamma. receptor is 53% identical to that of mouse at the amino
acid level. The human and mouse receptors only bind human and mouse
IFN-.gamma., respectively. Vaccinia, cowpox and camelpox viruses
have homologues of sIFN-.gamma.R, which have relatively low amino
acid sequence similarity (.about.20%), but are capable of efficient
neutralization of IFN-.gamma. in vitro. These homologues bind
human, bovine, rat (but not mouse) IFN-.gamma., and may have in
vivo activity as IFN-.gamma. antagonists. All eight cysteines are
conserved in human, mouse, myxoma and Shope fibroma virus (6 in
vaccinia virus) IFN-.gamma.R polypeptides, indicating similar 3-D
structures. An extracellular portion of mIFN-.gamma.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-.gamma. receptor (100
mg/three times a week i.p.) inhibits the onset of
glomerulonephritis. All mice treated with sIFN-.gamma. or
anti-IFN-.gamma. mAbs were alive 4 weeks after the treatment was
discontinued, compared with 50% in a placebo group, and 78% of
IFN-.gamma.-treated mice died.
[0121] The methods of the invention can be used to evolve soluble
IFN-.gamma.R receptor polypeptides with improved affinity, and to
evolve IFN-.gamma. 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 recombination and screened to identify those
recombinant nucleic acids that encode a polypeptide having improved
activity. In the case of shuffled IFN-.gamma.R, the library of
shuffled nucleic acids can be expressed on phage, which are
screened to identify mutants that bind to IFN-.gamma. with improved
affinity. In the case of IFN-.gamma., the shuffled 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-.gamma.
molecules can improve the efficacy of vaccinations (e.g. when used
as adjuvants).
[0122] Diseases that can be treated using high-affinity
sIFN-.gamma.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-.gamma. in the brain
of the patients, and increased production of IFN-.gamma. by
patients' T cells in vitro. IFN-.gamma. treatment has been shown to
significantly exacerbate the disease (in contrast to EAE in
mice).
[0123] Transforming growth factor (TGF)-.beta. is another cytokine
that can be optimized for use in genetic vaccines using the methods
of the invention. TGF-.beta. 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-.beta. inhibits proliferation of both B and T cells, and it
also suppresses development of and differentiation of cytotoxic T
cells and NK cells. TGF-.beta. 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-.beta. can specifically induce
IgA switching.
[0124] Due to its capacity to direct IgA switching, TGF-.beta. 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-.beta. is useful as a
component of therapeutical cancer vaccines. TGF-.beta. with
improved specific activity and/or with improved expression
levels/kinetics will have increased beneficial effects compared to
the wild-type TGF-.beta..
[0125] 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 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.
[0126] 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 shuffled to
create a library of shuffled 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 shuffled 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. Shuffled genes from these cells are
isolated; if desired, these genes can be used for additional rounds
of shuffling and selection.
[0127] 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-11/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. 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,
gp130, and a LIF-.beta. receptor. The CNTFR .alpha.-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 gp130. 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 (HSVI), cough, increased oral
secretions). Antibodies against CNTF were detected in almost all
patients, thus illustrating the need for alternative CNTF with
different immunological properties.
[0128] The recombination and screening methods of the invention can
be used to obtain modified CNTF polypeptides that exhibit decreased
immunogenicity in vivo; higher specific activity is also obtainable
using the methods. Shuffling is conducted using nuclei acids
encoding CNTF. In a preferred embodiment, an IL-6/LIF/(CNTF) hybrid
is obtained by shuffling using an excess of oligonucleotides 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.
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 shuffled CNTF polypeptides can be tested
to identify those that exhibit reduced immunogenicity upon
administration to a mammal.
[0129] Another way in which the recombination 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.
[0130] Target diseases for treatment with optimized CNTF, using
either the shuffled 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.
[0131] 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, IP10)
(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.
[0132] Genetic vaccine vectors can also include optimized
recombinant 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 CD150 (SLAM), can be subjected to DNA shuffling
to obtain variants have altered and/or improved activities.
[0133] Optimized recombinant 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 MHC molecules.
Importantly, CD1 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 DNA shuffling methods of the
invention.
[0134] 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.sup.+
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.
DNA shuffling can be used to further improve the properties,
including adjuvant activity, of heat shock proteins, such as
HSP70.
[0135] 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.
[0136] 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 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-.gamma. and IL-12 direct differentiation of T.sub.H1
cells (which produce high levels of IL-2 and IFN-.gamma.), 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.
[0137] Large libraries of vectors, generated by gene shuffling 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).
[0138] 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.
[0139] For a T.sub.H1 response, for example, the library is
screened to identify recombinant polynucleotides that are capable
of inducing T cells to produce IL-2 and IFN-.gamma., while
screening for induction of T cell production of IL-4, IL-5, and
IL-13 is performed to identify recombinant polynucleotides that
favor a T.sub.H2 response.
[0140] 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.
[0141] 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.
[0142] 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).
[0143] 3. Agonists or Antagonists of Cellular Receptors
[0144] The invention also provides methods for obtaining optimized
recombinant polynucleotides that encode a peptide or polypeptide
that can interact with a cellular receptor that is involved in
mediating an immune response. The optimized recombinant
polynucleotides can act as an agonist or an antagonist of the
receptor.
[0145] Cytokine antagonists can be used as components of genetic
vaccine cocktails. 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.
[0146] 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 gene
shuffling to generate a recombinant library of polynucleotides
which are then screened to identify those that encode an improved
antagonist. As substrates for the DNA shuffling, 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 recombination 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
recombinant polynucleotides is then screened to identify those that
encode cytokine antagonists with the desired affinity and
biological activity.
[0147] 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 DNA shuffling
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-.alpha. 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. 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).
[0148] 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 a role for IL-10 in the
pathogenesis of B cell malignancies.
[0149] 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 shuffling 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.
[0150] 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.
[0151] To obtain an IL-10 antagonist that has sufficient affinity
and antagonistic activity to function in vivo, DNA shuffling 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 shuffled using homologous cDNAs
encoding IL-10R derived from human and other mammalian species. An
alignment of human and mouse IL-10 receptor sequences is shown in
FIG. 14 to illustrate the feasibility of family DNA shuffling when
evolving IL-10 receptors with improved affinity. A phage library of
IL-10 receptor recombinants can be screened for improved binding of
shuffled IL-10R to human or viral IL-10. Wild-type IL-10 and/or
viral IL-10 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.
[0152] 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 shuffled 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 shuffling. 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
.alpha.-chain but has minimal IL-4-like agonistic activity.
[0153] Another example of an IL-10 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 13th European Immunology Meeting,
Amsterdam, 22-25 Jun. 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-.alpha. and IL-6.
IL-20/mda-7 also enhances production of IFN-.gamma. 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.
[0154] 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.
[0155] 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.
[0156] The optimized agonist or antagonist peptides or polypeptides
are obtained by generating a library of recombinant 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
DNA shuffling 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.
[0157] The optimized recombinant 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.
[0158] 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.
[0159] 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.alpha., 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.
[0160] 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. 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.
[0161] 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 a-helical coil and a collagen-like triple helix (see, Kodama et
al., Nature 343: 531-535 (1990)). Therefore, screening of the
library of recombinant 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. The optimized recombinant
polynucleotides identified by this method can be incorporated into
antigen-encoding sequences to evaluate their modulatory effect on
the immune response.
[0162] 4. Costimulatory Molecules Capable of Inhibiting or
Enhancing Activation, Differentiation, or Anergy of
Antigen-Specific T Cells
[0163] Also provided are methods of obtaining optimized recombinant
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. 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).
[0164] 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.
[0165] 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.
[0166] 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. DNA
shuffling 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.
[0167] DNA shuffling or other recombination 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 recombination 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.
[0168] 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.
[0169] 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 or
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 shuffled B7
variant-encoding sequences identified. These selected B7 variants
can then be subjected to new rounds of shuffling and selection,
and/or they can be further analyzed using functional assays as
described below.
[0170] 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.
[0171] 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.
[0172] 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-.gamma. 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.
[0173] 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' T cells (Groux et al. (1997)
Nature 389: 737). DNA shuffling 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.
[0174] D. Optimization of Transport and Presentation of
Antigens
[0175] 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 recombinant
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.
[0176] 1. Proteasomes
[0177] 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.
[0178] The invention provides methods of obtaining proteasomes that
exhibit increased or decreased ability to specifically process MHC
class I epitopes. According to the methods, DNA shuffling 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.
[0179] The methods involve performing DNA shuffling using as
substrates two or more forms of polynucleotides which encode
proteasome components, where the forms of polynucleotides differ in
at least one nucleotide. Shuffling 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., Stohwasser et al.
(1997) Eur. J. Immunol. 27: 1182-1187 and Gaczynska et al: (1996)
J. Biol. Chem. 271: 17275-17280. In a preferred embodiment, family
shuffling is used, in which the different substrates are proteasome
component-encoding polynucleotides from different species.
[0180] After the recombination reaction is completed, the resulting
library of recombinant polynucleotides is screened to identify
those which encode proteasome components having the desired effect
on class I epitope production. For example, the recombinant
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.
[0181] 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 immunosuppressive
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.
[0182] 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 DNA shuffling simultaneously on the two gene families
followed by random combinations of the two in order to discover
appropriate matched proteolytic and transport specificities.
[0183] 2. Antigen Transport
[0184] 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.
[0185] 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, TAP 1 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. TAP1-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.
[0186] TAP genes are a good target for gene shuffling 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 (TAP1, Genbank Accession Nos. AF001154 and AF00157; TAP2,
Genbank Accession Nos. AF001156 and AF00155). 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 rTAP2a
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.
[0187] The methods of the invention involve performing DNA
shuffling 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 shuffling. Natural polymorphism of the genes can
provide additional diversity of substrate. If desired, optimized
TAP genes obtained from one round of shuffling and screening can be
subjected to additional shuffling/screening rounds to obtain
further optimized TAP-encoding polynucleotides.
[0188] 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.
[0189] 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. Shuffled TAP genes can be
transfected into malignant cell lines that express low levels of
MHC class I molecules using retroviral vectors or electroporation.
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. These sequences
can then subjected into new rounds of shuffling, selection and
recovery, if further optimization is desired.
[0190] 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 gene shuffling, 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. Gene shuffling 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.
[0191] 3. Cytotoxic T-Cell Inducing Sequences and Immunogenic
Agonist Sequences
[0192] 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.
[0193] 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 in FIG. 1.
[0194] 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 & Hughes
(1997) Curr. Biol. 7: R552-R555; Craiu et al. (1997) Proc. Nat'l.
Acad. Sci. 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 DNA shuffling
methodology to be applied.
[0195] 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. DNA
shuffling methodology can be used to scan a large range of sequence
possibilities.
[0196] 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 shuffled to create a diverse range of CTL epitope sequences,
some of which should function as IAS. This process is illustrated
in FIG. 4.
[0197] 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 immunization and
CTL induction.
[0198] E. Genetic Vaccine Pharmaceutical Compositions and Methods
of Administration
[0199] 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. 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.
[0200] In presently preferred embodiments, the reagents obtained
using the invention are used in conjunction with a genetic vaccine
vector. The choice of vector and components can also be optimized
for the particular purpose of treating allergy or other conditions.
For example, an antigen associated with treating a particular
condition can be optimized using recombination and selection
methods analogous to those described herein. Such methods, and
antigens appropriate for various conditions, are described in
copending, commonly assigned U.S. patent application Ser. No.
______, entitled "Antigen Library Immunization," which was filed on
Feb. 10, 1999 as TTC Attorney Docket No. 18097-028710US. 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
recombination and selection methods analogous to those described
herein, as described in International Application No.
PCT/US97/17300 (International Publication No. WO 98/13487). 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 (see, e.g., copending, commonly assigned
U.S. patent application Ser. No. ______, entitled "Genetic Vaccine
Vector Engineering," filed on Feb. 10, 1999 as TTC Attorney Docket
No. 18097-030100US). 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 in copending, commonly
assigned U.S. patent application Ser. No. ______, entitled
"Targeting of Genetic Vaccine Vectors," filed on Feb. 10, 1999 as
TTC Attorney Docket No. 18097-030200US.
[0201] Genetic vaccine vectors that include the optimized
recombinant polynucleotides obtained as 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. 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.
[0202] 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: 367-384; 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):1635-1640 (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 160:38-47; Carter et al. (1989) U.S.
Pat. No. 4,797,368; 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., 63:03822-3828), and the like.
[0203] "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.
[0204] 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.
[0205] 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.
[0206] 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 forms can include one or more of
lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn
starch, potato starch, tragacanth, microcrystalline cellulose,
acacia, gelatin, colloidal silicon dioxide, croscarmellose 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. 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.
[0207] 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.
[0208] 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.
[0209] 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. Parenteral administration and
intravenous administration are the preferred methods of
administration. The formulations of packaged nucleic acid can be
presented in unit-dose or multi-dose sealed containers, such as
ampoules and vials.
[0210] 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.
[0211] 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.
[0212] 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.
[0213] 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.
[0214] 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.
[0215] 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.
[0216] 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).
[0217] 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.
[0218] 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.
EXAMPLES
[0219] The following examples are offered to illustrate, but not to
limit the present invention.
Example 1
Altered Ligand Specificity of B7-1 (CD80) and/or B7-2 (CD86) by DNA
Shuffling
[0220] This Example describes the use of the DNA shuffling methods
of the invention to obtain B7-1 and B7-2 polypeptides that have
altered biological activities.
[0221] DNA Shuffling
[0222] DNA shuffling is used to generate a library of B7 (B7-1/CD80
and B7-2/CD86) variants that have altered relative capacity to act
through CD28 and CTLA-4 when compared to wild-type B7 molecules.
Typically, B7 cDNAs from various species are generated by RT-PCR,
and these sequences are shuffled using family DNA shuffling.
Alignments of human, rhesus monkey and rabbit B7-1 nucleotide
sequences are shown in FIG. 15, demonstrating that family DNA
shuffling is a feasible approach when evolving B7 molecules.
[0223] Screening of B7 Variants
[0224] The library is then screened to identify those variants that
are useful in modulating immune responses in autoimmune diseases,
allergy, cancer, infectious disease and vaccination. Any of several
approaches for screening of the variants can be used:
[0225] A. Flow Cytometry-Based Selection System.
[0226] 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 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.
[0227] Soluble CTLA-4 and CD28 molecules are generated to
facilitate the flow cytometry experiments. Typically, these soluble
polypeptides are fused with the Fc portion of IgG molecule to
improve the stability of the molecules and to enable easy staining
by labeled anti-IgG monoclonal antibodies, as described by van der
Merwe et al. ((1997) J. Exp. Med. 185: 393). 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 are sorted. The plasmids are
then recovered and the shuffled sequences identified. These
selected B7 variants can then be subjected to new rounds of
shuffling and selection, and can be further analyzed using
functional assays as described below.
[0228] B. Selection Based on Functional Properties.
[0229] Bacterial colonies that contain plasmids that include 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 are made as to the binding affinities or
specificities to the known ligands, and possibly new activities
through yet to be identified ligands can be found.
[0230] T cell activation can be analyzed by 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, allows analysis of
antigen-specific T cell activation. 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 selected. Results obtained using a
proliferation-based assay is shown are shown in FIG. 13.
[0231] C. Ability to Direct Either T.sub.H1 or T.sub.H2 Cell
Differentiation.
[0232] Because differential roles for B7-1 and B7-2 molecules in
the regulation of T helper cell differentiation have been
identified (Freeman et al. (1995) Immunity 2: 523; Kuchroo et al.
(1995) Cell 80: 707), one can screen for B7 variants that are the
most effective in directing either T.sub.H1 or T.sub.H2 cell
differentiation. 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-.gamma. or IL-2 production can be used as a marker of
T.sub.H1 cell differentiation. B7 variants that altered capacity to
induce T.sub.H1 or T.sub.H2 cell differentiation are likely to be
useful in the treatment of allergic, malignant, autoimmune and
infectious diseases and in vaccination.
[0233] D. Enhanced IL-10 Production.
[0234] 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). Therefore, B7 variants can be screened to identify those that
have enhanced capacity to induce IL-10 production by
antigen-specific T cells. IL-10 production can be measured, for
example, by ELISA or flow cytometry using intracytoplasmic cytokine
stainings. The variants that induce high levels of IL-10 production
are useful in the treatment of allergic and autoimmune
diseases.
Example 2
Evolution Of Cytokines for Improved Specific Activity and/or
Improved Expression Levels
[0235] This example describes a method to evolve a cytokine for
improved specific activity and/or improved expression levels when
the genetic vaccine is transfected into mammalian cells. IL-12 is
the most potent cytokine directing T.sub.H1 responses, and it
improves the efficacy of genetic vaccinations. Evolved IL-12
molecules are useful as components of genetic vaccines. IL-12 is a
heterodimeric cytokine composed of a 35 kD light chain (p35) and a
40 kD heavy chain (p40) (Kobayashi et al. (1989) J. Exp. Med. 170:
827; Stern et al. (1990) Proc. Nat'l. Acad. Sci. USA 87: 6808).
Recently Lieschke et al. (Nature Biotechnol. (1997) 15: 35)
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. Accordingly, an IL-12 gene is shuffled as one
entity that encodes both subunits, which is beneficial in designing
the shuffling protocol. The subunits of IL-12 can also be expressed
separately in the same expression vector, or the subunits can be
expressed separately and screened using cotransfections of the two
vectors, providing additional shuffling strategies.
[0236] IL-12 plays-several roles in the regulation of allergic
responses. For example, IL-12 induces T.sub.H1 cell differentiation
and downregulates the T.sub.H2 response. IL-12 inhibits IgE
synthesis both in vivo and in vitro, and also induces IFN-.gamma.
production. Accordingly, it is desirable to obtain an optimized
IL-12 that better able to carry out these functions upon
administration to a mammal.
[0237] Cytokine genes, including IL-12 genes, from humans and
nonhuman primates are generally 93-99% homologous (Villinger et al.
(1995) J. Immunol. 0.155:3946-3954), providing a good starting
point for family shuffling. A library of shuffled IL-12 genes was
obtained by shuffling p35 and p40 subunits derived from human,
rhesus monkey, cat, dog, cow, pig, and goat, and incorporated into
vectors and the supernatants of these transfectants are analyzed
for biological activity as shown in FIG. 6. Because of its T cell
growth promoting activities, it is possible to use normal human
peripheral blood T cells in the selection of the most active IL-12
genes, enabling directly to select IL-12 mutants with the most
potent activities on human T cells.
[0238] As shown in FIG. 7, a functional screening assay has been
successfully established. In this assay, COS-7 cells were first
transfected with vectors encoding IL-12 subunits. Forty-eight hours
after transfection, the capacity of these culture supernatants to
induce proliferation of activated human peripheral blood T cells
was studied. FIG. 8 indicates the consistency of the level of T
cell proliferation induced in this assay, indicating that the assay
can be used to distinguish the activities between supernatants that
have different capacities to induce T cell activation. In other
words, the assay provides means to screen for improved IL-12-like
activities in culture supernatants of transfected cells. A vector
with an optimized IL-12-encoding polynucleotide was tested for
ability to induce human T cell activation. Results, shown in FIG.
9, show that the shuffled IL-12 has an significantly increased
ability to induce T cell activation compared to wild-type
IL-12.
[0239] FIG. 6 illustrates a general strategy for screening of
evolved cytokine genes. The specific example is given for IL-12 but
similar approach applies to all cytokines when using cell types
sensitive for each cytokine. For example, GM-CSF can be evolved by
the same approach by using the GM-CSF sensitive cell line TF-1 in
the screening. In addition, although in this example the vectors
are transfected into CHO cells, any mammalian cell that can be
transfected in vitro can be used as host cells. In addition to CHO
cell, other good host cells include cell lines WI-26, COS-1, COS-7,
293, U937 and freshly isolated human antigen presenting cells, such
as monocytes, B cells and dendritic cells.
Example 3
Cytotoxic T-Cell Inducing Sequences Derived from Hepatitis B
Surface Antigen and Strongly Immunogenic Agonistic T Cell
Epitopes
[0240] This Example describes the preparation of a polypeptide
sequence capable of efficient presentation of T cell epitopes and a
strategy for the application of DNA shuffling to discover strongly
immunogenic agonistic T cell epitopes.
[0241] The HBsAg polypeptide (PreS2 plus S regions) was truncated
by the introduction of a stop codon at amino acid position 103
(counting from the beginning of the PreS2 initiator methionine),
transforming a cysteine codon TGT into the Stop codon TGA. The
amino acid sequence of the truncated protein was therefore:
MQWNSTTFHQTLQDPRVRGLYFPAGGSSSGTVNPVLTTAS- PLSSIFSRIGDPALNME
NITSGFLGPLLVLQAGFFLLTRILTIPQSLDSWWTSLNFLGGTTV* where the standard
single-letter code for amino acids is used. The methionine residues
at the start of the PreS2 and S regions are underlined, and the
mouse L.sup.d-restricted CTL epitope is double-underlined; the
asterisk (*) represents the artificially introduced Stop codon.
[0242] A likely structure for this truncated polypeptide is shown
in FIG. 1. During protein biosynthesis, the N-terminal region of
the HBsAg polypeptide is transported through the membrane of the
endoplasmic reticulum (ER). The first part of the S region is a
transmembrane structure which locks the polypeptide into the ER.
The remaining C-terminal region of the polypeptide is located in
the cytoplasmic compartment where it can be accessed by the epitope
processing mechanism of the cell. This structure forms what is
referred to as the Cytotoxic T-cell Inducing Sequence (CTIS).
[0243] A CTIS is preferably used in conjunction with an immunogenic
agonistic sequence (IAS). As an example of an IAS, each position of
a 12 amino acid L.sup.d-restricted class I epitope from HBsAg was
replaced with an alanine-encoding codon in the DNA sequence of the
epitope. The results demonstrate that, in some cases, the
reactivity is greater than if the CTL response is induced by the
natural epitope (FIG. 3).
Example 4
Treatment of Obesity, Anorexia, and Cachexia Using Optimized
Immunomodulatory Molecules
[0244] Optimized immunomodulatory molecules that are obtained using
the methods of the invention find use in a wide variety of
applications, in addition to use in vaccination. For example, there
is increasing evidence that certain forms of obesity are associated
with dysfunction of the immune system, and that molecules which
regulate immune responses, e.g. cytokines, can induce or inhibit
obesity. The invention provides methods of optimizing immune
regulatory molecules for the treatment of obesity, anorexia and
cachexia.
[0245] Leptin and ciliary neurotrophic factor (CNTF) are examples
of cytokines that have been shown to play a role in the development
of obesity. Congenital leptin deficiency results in severe
early-onset obesity in human (Montague et al. (1997) Nature 387:
903-908), and CNTF has been shown to correct obesity and diabetes
associated with leptin deficiency and resistance (Gloaguen et al.
(1997) Proc. Nat'l. Acad. Sci. USA 94: 6456-6461). Antagonists of
CNTF and/or leptin may be useful in the treatment of anorexia
and/or cachexia.
[0246] The methods of the invention are used to generate leptin
and/or CNTF molecules that have improved specific activity. The
methods are also useful for obtaining improved cytokines that
exhibit reduced immunogenicity in vivo; immunogenicity is a
particular concern for CNTF, because the wild-type CNTF is highly
antigenic, which results in the production of high levels of
anti-CNTF antibodies when administered to a human. Improved
cytokine molecules prepared using the methods of the invention are
administered as polypeptides, or the shuffled nucleic acids that
encode improved leptin and/or CNTF polypeptides are used in genetic
vaccine vectors. The invention also provides methods of generating
vectors that induce production of increased levels of leptin and/or
CNTF. The methods of the invention can also be used to obtain
reagents that are useful for the treatment of anorexia, cachexia,
and related disorders. In this embodiment, antagonists of leptin
and/or CNTF are evolved using the DNA shuffling methods. For
example, a leptin receptor can be evolved to obtain a soluble form
that has an enhanced affinity for leptin. The receptor for leptin
in mice is found in the hypothalamus (Mercer et al. (1996) FEBS
Lett. 387: 113), a region known to be involved in maintenance of
energy balance, and in the choroid plexus and leptomeninges, which
form part of the blood/brain barrier.
[0247] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference for all purposes.
Sequence CWU 1
1
6 1 3632 DNA Homo sapiens human interleukin 10 receptor (IL-10R) 1
aaagagctgg aggcgcgcag gccggctccg ctccggcccc ggacgatgcg gcgcgcccag
60 gatgctgccg tgcctcgtag tgctgctggc ggcgctcctc agcctccgtc
ttggctcaga 120 cgctcatggg acagagctgc ccagccctcc gtctgtgtgg
tttgaagcag aatttttcca 180 ccacatcctc cactggacac ccatcccaaa
tcagtctgaa agtacctgct atgaagtggc 240 gctcctgagg tatggaatag
agtcctggaa ctccatctcc aactgtagcc agaccctgtc 300 ctatgacctt
accgcagtga ccttggacct gtaccacagc aatggctacc gggccagagt 360
gcgggctgtg gacggcagcc ggcactccaa ctggaccgtc accaacaccc gcttctctgt
420 ggatgaagtg actctgacag ttggcagtgt gaacctagag atccacaatg
gcttcatcct 480 cgggaagatt cagctaccca ggcccaagat ggcccccgcg
aatgacacat atgaaagcat 540 cttcagtcac ttccgagagt atgagattgc
cattcgcaag gtgccgggaa acttcacgtt 600 cacacacaag aaagtaaaac
atgaaaactt cagcctccta acctctggag aagtgggaga 660 gttctgtgtc
caggtgaaac catctgtcgc ttcccgaagt aacaagggga tgtggtctaa 720
agaggagtgc atctccctca ccaggcagta tttcaccgtg accaacgtca tcatcttctt
780 tgcctttgtc ctgctgctct ccggagccct cgcctactgc ctggccctcc
agctgtatgt 840 gcggcgccga aagaagctac ccagtgtcct gctcttcaag
aagcccagcc ccttcatctt 900 catcagccag cgtccctccc cagagaccca
agacaccatc cacccgcttg atgaggaggc 960 ctttttgaag gtgtccccag
agctgaagaa cttggacctg cacggcagca cagacagtgg 1020 ctttggcagc
accaagccat ccctgcagac tgaagagccc cagttcctcc tccctgaccc 1080
tcacccccag gctgacagaa cgctgggaaa cggggagccc cctgtgctgg gggacagctg
1140 cagtagtggc agcagcaata gcacagacag cgggatctgc ctgcaggagc
ccagcctgag 1200 ccccagcaca gggcccacct gggagcaaca ggtggggagc
aacagcaggg gccaggatga 1260 cagtggcatt gacttagttc aaaactctga
gggccgggct ggggacacac agggtggctc 1320 ggccttgggc caccacagtc
ccccggagcc tgaggtgcct ggggaagaag acccagctgc 1380 tgtggcattc
cagggttacc tgaggcagac cagatgtgct gaagagaagg caaccaagac 1440
aggctgcctg gaggaagaat cgcccttgac agatggcctt ggccccaaat tcgggagatg
1500 cctggttgat gaggcaggct tgcatccacc agccctggcc aagggctatt
tgaaacagga 1560 tcctctagaa atgactctgg cttcctcagg ggccccaacg
ggacagtgga accagcccac 1620 tgaggaatgg tcactcctgg ccttgagcag
ctgcagtgac ctgggaatat ctgactggag 1680 ctttgcccat gaccttgccc
ctctaggctg tgtggcagcc ccaggtggtc tcctgggcag 1740 ctttaactca
gacctggtca ccctgcccct catctctagc ctgcagtcaa gtgagtgact 1800
cgggctgaga ggctgctttt gattttagcc atgcctgctc ctctgcctgg accaggagga
1860 gggccctggg gcagaagtta ggcacgaggc agtctgggca cttttctgca
agtccactgg 1920 ggctggccca gccaggctgc agggctggtc agggtgtctg
gggcaggagg aggccaactc 1980 actgaactag tgcagggtat gtgggtggca
ctgacctgtt ctgttgactg gggccctgca 2040 gactctggca gagctgagaa
gggcagggac cttctccctc ctaggaactc tttcctgtat 2100 cataaaggat
tatttgctca ggggaaccat ggggctttct ggagttgtgg tgaggccacc 2160
aggctgaagt cagctcagac ccagacctcc ctgcttaggc cactcgagca tcagagcttc
2220 cagcaggagg aagggctgta ggaatggaag cttcagggcc ttgctgctgg
ggtcattttt 2280 aggggaaaaa ggaggatatg atggtcacat ggggaacctc
ccctcatcgg gcctctgggg 2340 caggaagctt gtcactggaa gatcttaagg
tatatatttt ctggacactc aaacacatca 2400 taatggattc actgagggga
gacaaaggga gccgagaccc tggatggggc ttccagctca 2460 gaacccatcc
ctctggtggg tacctctggc acccatctgc aaatatctcc ctctctccaa 2520
caaatggagt agcatccccc tggggcactt gctgaggcca agccactcac atcctcactt
2580 tgctgcccca ccatcttgct gacaacttcc agagaagcca tggttttttg
tattggtcat 2640 aactcagccc tttgggcggc ctctgggctt gggcaccagc
tcatgccagc cccagagggt 2700 cagggttgga ggcctgtgct tgtgtttgct
gctaatgtcc agctacagac ccagaggata 2760 agccactggg cactgggctg
gggtccctgc cttgttggtg ttcagctgtg tgattttgga 2820 ctagccactt
gtcagagggc ctcaatctcc catctgtgaa ataaggactc cacctttagg 2880
ggaccctcca tgtttgctgg gtattagcca agctggtcct gggagaatgc agatactgtc
2940 cgtggactac caagctggct tgtttcttat gccagaggct aacagatcca
atgggagtcc 3000 atggtgtcat gccaagacag tatcagacac agccccagaa
gggggcatta tgggccctgc 3060 ctccccatag gccatttgga ctctgccttc
aaacaaaggc agttcagtcc acaggcatgg 3120 aagctgtgag gggacaggcc
tgtgcgtgcc atccagagtc atctcagccc tgcctttctc 3180 tggagcattc
tgaaaacaga tattctggcc cagggaatcc agccatgacc cccacccctc 3240
tgccaaagta ctcttaggtg ccagtctggt aactgaactc cctctggagg caggcttgag
3300 ggaggattcc tcagggttcc cttgaaagct ttatttattt attttgttca
tttatttatt 3360 ggagaggcag cattgcacag tgaaagaatt ctggatatct
caggagcccc gaaattctag 3420 ctctgacttt gctgtttcca gtggtatgac
cttggagaag tcacttatcc tcttggagcc 3480 tcagtttcct catctgcaga
ataatgactg acttgtctaa ttcataggga tgtgaggttc 3540 tgctgaggaa
atgggtatga atgtgccttg aacacaaagc tctgtcaata agtgatacat 3600
gttttttatt ccaataaatt gtcaagacca ca 3632 2 3497 DNA Mus musculus
mouse interleukin 10 receptor (IL-10R) 2 ccattgtgct ggaaagcagg
acgcgccggc cggaggcgta aaggccggct ccagtggacg 60 atgccgctgt
gcgcccagga tgttgtcgcg tttgctccca ttcctcgtca cgatctccag 120
cctgagccta gaattcattg catacgggac agaactgcca agcccttcct atgtgtggtt
180 tgaagccaga tttttccagc acatcctcca ctggaaacct atcccaaacc
agtctgagag 240 cacctactat gaagtggccc tcaaacagta cggaaactca
acctggaatg acatccatat 300 ctgtagaaag gctcaggcat tgtcctgtga
tctcacaacg ttcaccctgg atctgtatca 360 ccgaagctat ggctaccggg
ccagagtccg ggcagtggac aacagtcagt actccaactg 420 gaccaccact
gagactcgct tcacagtgga tgaagtgatt ctgacagtgg atagcgtgac 480
tctgaaagca atggacggca tcatctatgg gacaatccat ccccccaggc ccacgataac
540 ccctgcaggg gatgagtacg aacaagtctt caaggatctc cgagtttaca
agatttccat 600 ccggaagttc tcagaactaa agaatgcaac caagagagtg
aaacaggaaa ccttcaccct 660 cacggtcccc ataggggtga gaaagttttg
tgtcaaggtg ctgccccgct tggaatcccg 720 aattaacaag gcagagtggt
cggaggagca gtgtttactt atcacgacgg agcagtattt 780 cactgtgacc
aacctgagca tcttagtcat atctatgctg ctattctgtg gaatcctggt 840
ctgtctggtt ctccagtggt acatccggca cccggggaag ttgcctacag tcctggtctt
900 caagaagcct cacgacttct tcccagccaa ccctctctgc ccagaaactc
ccgatgccat 960 tcacatcgtg gacctggagg ttttcccaaa ggtgtcacta
gagctgagag actcagtcct 1020 gcatggcagc accgacagtg gctttggcag
tggtaaacca tcacttcaga ctgaagagtc 1080 ccaattcctc ctccctggct
cccaccccca gatacagggg actctgggaa aagaagagtc 1140 tccagggcta
caggccacct gtggggacaa cacggacagt gggatctgcc tgcaggagcc 1200
cggcttacac tccagcatgg ggcccgcctg gaagcagcag cttggatata cccatcagga
1260 ccaggatgac agtgacgtta acctagtcca gaactctcca gggcagccta
agtacacaca 1320 ggatgcatct gccttgggcc atgtctgtct cctagaacct
aaagcccctg aggagaaaga 1380 ccaagtcatg gtgacattcc agggctacca
gaaacagacc agatggaagg cagaggcagc 1440 aggcccagca gaatgcttgg
acgaagagat tcccttgaca gatgcctttg atcctgaact 1500 tggggtacac
ctgcaggatg atttggcttg gcctccacca gctctggccg caggttattt 1560
gaaacaggag tctcaaggga tggcttctgc tccaccaggg acaccaagta gacagtggaa
1620 tcaactgacc gaagagtggt cactcctggg tgtggttagc tgtgaagatc
taagcataga 1680 aagttggagg tttgcccata aacttgaccc tctggactgt
ggggcagccc ctggtggcct 1740 cctggatagc cttggctcta acctggtcac
cctgccgttg atctccagcc tgcaggtaga 1800 agaatgacag cggctaagag
ttatttgtat tccagccatg cctgctcccc tccctgtacc 1860 tgggaggctc
aggagtcaaa gaaatatgtg ggtccttttc tgcagaccta ctgtgaccag 1920
ctagccaggc tccacggggc aaggaaaggc catcttgata cacgagtgtc aggtacatga
1980 gaggttgtgg ctagtctgct gagtgagggt ctgtagatac cagcagagct
gagcaggatt 2040 gacagagacc tcctcatgcc tcagggctgg ctcctacact
ggaaggacct gtgtttgggt 2100 gtaacctcag ggctttctgg atgtggtaag
actgtaggtc tgaagtcagc tgagcctgga 2160 tgtctgcgga ggtgttggag
tggctagcct gctacaggat aaagggaagg ctcaagagat 2220 agaagggcag
agcatgagcc aggtttaatt ttgtcctgta gagatggtcc ccagccagga 2280
tgggttactt gtggctggga gatcttgggg tatacaccac cctgaatgat cagccagtca
2340 attcagagct gtgtggcaaa agggactgag acccagaatt tctgttcctc
ttgtgaggtg 2400 tctctgctac ccatctgcag acagacatct tcatcttttt
actatggctg tgtcccctga 2460 attaccagca gtggccaagc cattactccc
tgctgctcac tgttgtgacg tcagaccaga 2520 ccagacgctg tctgtctgtg
ttagtacact accctttagg tggcctttgg gcttgagcac 2580 tggcccaggc
ttaggactta tgtctgcttt tgctgctaat ctctaactgc agacccagag 2640
aacagggtgc tgggctgaca cctccgtgtt cagctgtgtg acctccgacc agcagcttcc
2700 tcaggggact aaaataatga ctaggtcatt cagaagtccc tcatgctgaa
tgttaaccaa 2760 ggtgcccctg gggtgatagt ttaggtcctg caacctctgg
gttggaagga agtggactac 2820 ggaagccatc tgtccccctg gggagcttcc
acctcatgcc agtgtttcag agatcttgtg 2880 ggagcctagg gccttgtgcc
aagggagctg ctagtccctg gggtctaggg ctggtccctg 2940 cctccctata
ctgcgtttga gacctgtctt caaatggagg cagtttgcag cccctaagca 3000
aggatgctga gagaagcagc aaggctgctg atccctgagc ccagagtttc tctgaagctt
3060 tccaaataca gactgtgtga cggggtgagg ccagccatga actttggcat
cctgccgaga 3120 aggtcatgac cctaatctgg tacgagagct ccttctggaa
ctgggcaagc tctttgagac 3180 ccccctggaa cctttattta tttatttgct
cacttattta ttgaggaagc agcgtggcac 3240 aggcgcaagg ctctgggtct
ctcaggaggt ctagatttgc ctgccctgtt tctagctgtg 3300 tgaccttggg
caagtcacgt ttcctcgtgg agcctcagtt ttcctgtctg tatgcaaagc 3360
ttggaaattg aaatgtacct gacgtgctcc atccctagga gtgctgagtc ccactgagaa
3420 agcgggcaca gacgcctcaa atggaaccac aagtggtgtg tgttttcatc
ctaataaaaa 3480 gtcaggtgtt ttgtgga 3497 3 867 DNA Homo sapiens
human B7-1 (CD80) 3 atgggccaca cacggaggca gggaacatca ccatccaagt
gtccatacct caatttcttt 60 cagctcttgg tgctggctgg tctttctcac
ttctgttcag gtgttatcca cgtgaccaag 120 gaagtgaaag aagtggcaac
gctgtcctgt ggtcacaatg tttctgttga agagctggca 180 caaactcgca
tctactggca aaaggagaag aaaatggtgc tgactatgat gtctggggac 240
atgaatatat ggcccgagta caagaaccgg accatctttg atatcactaa taacctctcc
300 attgtgatcc tggctctgcg cccatctgac gagggcacat acgagtgtgt
tgttctgaag 360 tatgaaaaag acgctttcaa gcgggaacac ctggctgaag
tgacgttatc agtcaaagct 420 gacttcccta cacctagtat atctgacttt
gaaattccaa cttctaatat tagaaggata 480 atttgctcaa cctctggagg
ttttccagag cctcacctct cctggttgga aaatggagaa 540 gaattaaatg
ccatcaacac aacagtttcc caagatcctg aaactgagct ctatgctgtt 600
agcagcaaac tggatttcaa tatgacaacc aaccacagct tcatgtgtct catcaagtat
660 ggacatttaa gagtgaatca gaccttcaac tggaatacaa ccaagcaaga
gcattttcct 720 gataacctgc tcccatcctg ggccattacc ttaatctcag
taaatggaat ttttgtgata 780 tgctgcctga cctactgctt tgccccaaga
tgcagagaga gaaggaggaa tgagagattg 840 agaagggaaa gtgtacgccc tgtataa
867 4 867 DNA Macaca mulatta rhesus monkey B7-1 (CD80) 4 atgggccaca
cacggaggca ggaaatatca ccatccaagt gtccatacct caagttcttt 60
cagctcttgg tgctggcttg tctttctcat ttctgttcag gtgttatcca cgtgaccaag
120 gaagtgaaag aagtggcaac gctgtcctgt ggtcacaatg tttctgttga
agagctggca 180 caaactcgca tctactggca aaaggagaag aaaatggtgc
tgactatgat gtctggggac 240 atgaatatat ggcccgagta caagaaccgg
accatctttg atatcacaaa taacctctcc 300 attgtgattc tggctctgcg
cccatctgac gagggcacat acgagtgtgt tgttctgaag 360 tatgaaaaag
atgctttcaa gcgggaacac ctggctgaag tgatgttatc cgtcaaagct 420
gacttcccta cacctagtat aactgactct gaaattccac cttctaacat tagaaggata
480 atttgctcaa actctggagg ttttccagag cctcacctct cctggttgga
aaatggagaa 540 gaattaaatg ccatcagcac aacagtttcc caagatcctg
aaactgagct ctatactgtt 600 agcagcaaac tggatttcaa tatgacaacc
aatcacagtt tcatgtgtct catcaagtat 660 ggacatttaa gagtgaatca
gaccttcaac tggaacacac ccaagcaaga gcattttcct 720 gataacctgc
tcccatcctg ggccattatc ctaatctcag taaatggaat ttttgtgata 780
tgctgcctga cctactgttt tgccccaagg tgcagagaga gaagaaggaa tgagacattg
840 agaagggaaa gtgtacgccc tgtatga 867 5 900 DNA Rabbit (genus and
species unknown) rabbit B7-1 (CD80) 5 atgggccaca cgctgaggcc
gggaactcca ctgcccaggt gtctacacct caagctctgc 60 ctgctcttgg
cgctggcggg tctccacttc tcttcaggta tcagccaggt caccaagtcg 120
gtgaaagaaa tggcagcact gtcctgtgat tacaacattt ctatcgatga actggcgaga
180 atgcgcatat actggcagaa ggaccaacag atggtgctga gcatcatctc
tgggcaagtg 240 gaagtgtggc ctgagtacaa gaaccgcacc ttccccgaca
tcattaacaa cctctccctt 300 atgatcctgg cactgcgcct gtcggacaag
ggcacctaca cctgcgtggt tcagaagaat 360 gagaacgggt ctttcagacg
ggagcacctg acctccgtga cactgtccat cagagctgac 420 ttccctgtcc
ctagcataac tgacattgga catcccgacc ctaatgtgaa aaggataaga 480
tgctccgcct ctggaggttt tccagagcct cgcctcgcct ggatggaaga tggagaagaa
540 ctaaacgccg tcaacacgac ggttgaccag gatttggaca cggagctcta
cagcgtcagc 600 agtgaactgg atttcaatgt gacaaataac cacagcatcg
tgtgtctcat caaatacggg 660 gagctgtcgg tgtcacagat cttcccttgg
agcaaaccca agcaggagcc tcccattgat 720 cagcttccat tctgggtcat
tatcccagta agtggtgctt tggtgctcac tgcggtagtt 780 ctctactgcc
tggcctgcag acatgttgcg aggtggaaaa gaacaagaag gaatgaagag 840
acagtgggaa ctgaaaggct gtcccctatc tacttaggct ctgcgcaatc ctcgggctga
900 6 102 PRT Artificial Sequence Description of Artificial
Sequencetruncated hepatitis B surface antigen (HBsAg) (PreS2 plus S
regions) 6 Met Gln Trp Asn Ser Thr Thr Phe His Gln Thr Leu Gln Asp
Pro Arg 1 5 10 15 Val Arg Gly Leu Tyr Phe Pro Ala Gly Gly Ser Ser
Ser Gly Thr Val 20 25 30 Asn Pro Val Leu Thr Thr Ala Ser Pro Leu
Ser Ser Ile Phe Ser Arg 35 40 45 Ile Gly Asp Pro Ala Leu Asn Met
Glu Asn Ile Thr Ser Gly Phe Leu 50 55 60 Gly Pro Leu Leu Val Leu
Gln Ala Gly Phe Phe Leu Leu Thr Arg Ile 65 70 75 80 Leu Thr Ile Pro
Gln Ser Leu Asp Ser Trp Trp Thr Ser Leu Asn Phe 85 90 95 Leu Gly
Gly Thr Thr Val 100
* * * * *
References