U.S. patent application number 12/508859 was filed with the patent office on 2010-02-11 for enrichment method for variant proteins with altered binding properties.
This patent application is currently assigned to Genentech, Inc.. Invention is credited to Steven Bass, Lisa J. GARRARD, Ronald Greene, Dennis J. Henner, Henry B. Lowman, David J. Matthews, James A. Wells.
Application Number | 20100035236 12/508859 |
Document ID | / |
Family ID | 27505166 |
Filed Date | 2010-02-11 |
United States Patent
Application |
20100035236 |
Kind Code |
A1 |
GARRARD; Lisa J. ; et
al. |
February 11, 2010 |
ENRICHMENT METHOD FOR VARIANT PROTEINS WITH ALTERED BINDING
PROPERTIES
Abstract
A method for selecting novel proteins such as growth hormone and
antibody fragment variants having altered binding properties for
their respective receptor molecules is provided. The method
comprises fusing a gene encoding a protein of interest to the
carboxy terminal domain of the gene III coat protein of the
filamentous phage M13. The gene fusion is mutated to form a library
of structurally related fusion proteins that are expressed in low
quantity on the surface of a phagemid particle. Biological
selection and screening are employed to identify novel ligands
useful as drug candidates. Disclosed are preferred phagemid
expression vectors and selected human growth hormone variants.
Inventors: |
GARRARD; Lisa J.;
(Burlingame, CA) ; Henner; Dennis J.; (Pacifica,
CA) ; Bass; Steven; (Hillsborough, CA) ;
Greene; Ronald; (Durham, NC) ; Lowman; Henry B.;
(El Granada, CA) ; Wells; James A.; (Burlingame,
CA) ; Matthews; David J.; (San Francisco,
CA) |
Correspondence
Address: |
GENENTECH, INC.
1 DNA WAY
SOUTH SAN FRANCISCO
CA
94080
US
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
27505166 |
Appl. No.: |
12/508859 |
Filed: |
July 24, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11761180 |
Jun 11, 2007 |
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12508859 |
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11199062 |
Aug 8, 2005 |
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11761180 |
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09717641 |
Nov 21, 2000 |
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11199062 |
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08922345 |
Sep 3, 1997 |
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09717641 |
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08463587 |
Jun 5, 1995 |
5821047 |
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08922345 |
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08050058 |
Apr 30, 1993 |
5750373 |
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PCT/US91/09133 |
Dec 3, 1991 |
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08463587 |
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07743614 |
Aug 9, 1991 |
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08050058 |
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07715300 |
Jun 14, 1991 |
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07743614 |
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07683400 |
Apr 10, 1991 |
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07715300 |
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07621667 |
Dec 3, 1990 |
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07683400 |
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Current U.S.
Class: |
435/5 ;
435/235.1; 530/399 |
Current CPC
Class: |
C07K 14/575 20130101;
G01N 33/74 20130101; Y10S 930/12 20130101; C12N 2750/00011
20130101; C12N 15/62 20130101; G01N 33/6845 20130101; C07K 2319/735
20130101; C07K 14/61 20130101; C12N 15/1037 20130101; Y10S 436/802
20130101; C07K 2319/02 20130101; C12N 2795/14122 20130101; C07K
2319/00 20130101; C07K 2319/75 20130101; G01N 2333/61 20130101;
C12N 2795/14022 20130101; C07K 14/005 20130101; C40B 40/02
20130101 |
Class at
Publication: |
435/5 ; 530/399;
435/235.1 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70; C07K 14/61 20060101 C07K014/61; C12N 7/00 20060101
C12N007/00 |
Claims
1. A method for selecting novel binding polypeptides comprising:
(a) constructing a replicable expression vector comprising a
transcription regulatory element operably linked to a gene fusion
encoding a fusion protein wherein the gene fusion comprises a first
gene encoding a polypeptide, and a second gene encoding at least a
portion of a phage coat protein; (b) mutating the vector at one or
more selected positions within the first gene thereby forming a
family of related plasmids; (c) transforming suitable host cells
with the plasmids; (d) infecting the transformed host cells with a
helper phage having a gene encoding the phage coat protein; (e)
culturing the transformed infected host cells under conditions
suitable for forming recombinant phagemid particles containing at
least a portion of the plasmid and capable of transforming the
host, the conditions adjusted so that no more than a minor amount
of phagemid particles display more than one copy of the fusion
protein on the surface of the particle; (f) contacting the phagemid
particles with a target molecule so that at least a portion of the
phagemid particles bind to the target molecule; and (g) separating
the phagemid particles that bind from those that do not.
2. The method of claim 1 further comprising infecting a suitable
host cells with the phagemid particles that bind and repeating
steps (d) through (g).
3. The method of claim 2 wherein the steps are repeated one or more
times.
4. The method of claim 1 wherein the expression vector further
comprises a secretory signal sequence.
5. The method of claim 1 wherein the transcription regulatory
element is a promoter system selected from the group; lac Z, pho A,
tryptophan, tac, .lamda..sub.PL, bacteriophage T7, and combinations
thereof.
6. The method of claim 1 wherein the first gene encodes a mammalian
protein.
7. The method of claim 6 wherein the protein is selected from the
group; growth hormone, human growth hormone (hGH), des-N methionyl
human growth hormone, bovine growth hormone, parathyroid hormone,
thyroxine, insulin A-chain, insulin B-chain, proinsulin, relaxin
A-chain, relaxin B-chain, prorelaxin, follicle stimulating hormone
(FSH), thyroid stimulating hormone (TSH), leutinizing hormone (LH),
glycoprotein hormone receptors, calcitonin, glucagon, factor VIII,
an antibody, lung surfactant, urokinase, streptokinase, human
tissue-type plasminogen activator (t-PA), bombesin, factor IX,
thrombin, hemopoietic growth factor, tumor necrosis factor-alpha
and -beta, enkephalinase, human serum albumin, mullerian-inhibiting
substance, mouse gonadotropin-associated peptide, .beta.-lactamase,
tissue factor protein, inhibin, activin, vascular endothelial
growth factor, integrin receptors, thrombopoietin, protein A or D,
rheumatoid factors, NGF-.beta., platelet-growth factor,
transforming growth factor; TGF-alpha and TGF-beta, insulin-like
growth factor-I and -II, insulin-like growth factor binding
proteins, CD-4, DNase, latency associated peptide, erythropoietin,
HER2 ligands, osteoinductive factors, interferon-alpha, -beta, and
-gamma, colony stimulating factors (CSFs), M-CSF, GM-CSF, and
G-CSF, interleukins (ILs), IL-1, IL-2, IL-3, IL-4, superoxide
dismutase; decay accelerating factor, viral antigen, HIV envelope
proteins GP120 and GP140, atrial natriuretic peptides A, B, or C,
or immuno globulins, and fragments of the above-listed
proteins.
8. The method of claim 7 wherein the protein is a human
protein.
9. The method of claim 8 wherein the protein comprises more than
about 100 amino acid residues.
10. The method of claim 1 wherein the protein comprises a plurality
of rigid secondary structures displaying amino acids capable of
interacting with the target, and the mutations are primarily
produced at positions corresponding to codons encoding the amino
acids.
11. The method of claim 10 wherein the rigid secondary structures
comprise structures selected from the group; .alpha.-(3.6.sub.13)
helix, 3.sub.10 helix, 4.4.sub.16) helix, parallel and
anti-parallel .beta.-pleated sheets, reverse turns, and non-ordered
structures.
12. The method of claim 10 wherein the mutations are produced at
more than one codon.
13. The method of claim 12 wherein the mutations are produced on
more than one rigid secondary structure.
14. The method of claim 1 wherein the helper phage is selected from
the group M13KO7, M13R408, M13-VCS, and Phi X 174.
15. The method of claim 14 wherein the helper phage is M13KO7 and
the coat protein is the M13 phage gene III coat protein.
16. The method of claim 15 wherein the host is E. coli.
17. The method of claim 16 wherein the plasmid is under tight
control of the transcription regulatory element.
18. The method of claim 17 wherein the amount is less than about
1%.
19. The method of claim 18 wherein the amount is less than 20% the
amount of phagemid particles displaying a single copy of the fusion
protein.
20. The method of claim 19 wherein the amount is less than 10%.
21. The method of claim 1 further comprising in step (a), inserting
a DNA triplet, encoding an mRNA suppressible terminator codon
between said first gene encoding a polypeptide, and said second
gene encoding at least a portion of a phage coat protein.
22. The method of claim 21 wherein said mRNA suppressible
terminator codon is selected from the following: UAG (amber), UAA
(ocher) and UGA (opel).
23. The method of claim 22 wherein said suppressible mutation
results in the detectable production of a fusion polypeptide
containing sadi polypeptide and said coat protein when said
expression vector is grown in a suppressor host cell; and, when
grown in a non-suppressor host cell said polypeptide is synthesized
substantially without fusion to said phage coat protein.
24. A human growth hormone variant wherein hGH amino acids 172,
174, 176 and 178 respectively are as a group sequentially selected
from one of the following: (1) R,S,F,R; (2) R,A,Y,R; (3) K,T,Y,K;
(4) R,S,Y,R; (5) K,A,Y,R; (6) R,F,F,R; (7) K,Q,Y,R; (8) R,T,Y,H;
(9) Q,R,Y,R; (10) K,K,Y,K; (11) R,S,F,S; and (12) K,S,N,R.
25. A phagemid comprising a replicable expression vector comprising
a transcription regulatory element operably linked to a gene fusion
encoding a fusion protein wherein the gene fusion comprises a first
gene encoding a polypeptide, and a second gene encoding at least a
portion of a phage coat protein, wherein a DNA triplet codon
encoding an mRNA suppressible terminator codon selected from UAG,
UAA and UGA is inserted between the fused ends of the first and
second genes, or is substituted for an amino acid encoding triplet
codon adjacent to the gene fusion junction.
26. The phagemid of claim 25 wherein said first gene encodes a
mammalian protein.
27. The phagemid of claim 26 wherein the protein is selected from
the group: growth hormone, human growth hormone (hGH),
des-N-methionyl human growth hormone, bovine growth hormone,
parathyroid hormone, thyroxine, insulin A-chain, insulin B-chain,
proinsulin, relaxin A-chain, relaxin B-chain, prorelaxin, follicle
stimulating hormone (FSH), thyroid stimulating hormone (TSH),
leutinizing hormone (LH), glycoprotein hormone receptors,
calcitonin, glucagon, factor VIII, an antibody, lung surfactant,
urokinase, streptokinase, human tissue-type plasminogen activator
(t-PA), bombesin, factor IX, thrombin, hemopoietic growth factor,
tumor necrosis factor-alpha and -beta, enkephalinase, human serum
albumin, mullerian-inhibiting substance, mouse
gonadotropin-associated peptide, .beta.-lactamase, tissue factor
protein, inhibin, activin, vascular endothelial growth factor,
integrin receptors, thrombopoietin, protein A or D, rheumatoid
factors, NGF-.beta. platelet-growth factor, transforming growth
factor; TGF-alpha and TGF-beta, insulin-like growth-I and -II,
insulin-like growth factor binding proteins, CD-4, DNase, latency
associated peptide, erythropoietin, osteoinductive factors,
interferon-alpha, -beta, and -gamma, colony stimulating factors
(CSFs), M-CSF, GM-CSF, and G-CSF, interleukins (ILs), IL-1, IL-2,
IL-3, IL-4, superoxide dismutase; decay accelerating factor, viral
antigen, HIV envelope proteins GP120 and GP140, atrial natriuretic
peptides A, B or C immuno globulins, and fragments of the
above-listed proteins.
28. The phagemid of claim 27 wherein said protein is a human
protein.
29. The phagemid of claim 28 wherein the protein comprises more
than about 100 amino acid residues.
30. The phagemid of claim 25 wherein said protein comprises a
plurality of rigid secondary structures displaying amino acids
capable of interacting with the target.
31. The phagemid of claim 30 wherein said rigid secondary
structures comprises structures selected from the group;
.alpha.-(3.6.sub.13) helix, 3.sub.10 helix, .pi.-(4.4.sub.16)
helix, parallel and anti-parallel .beta.-pleated sheets, reverse
turns, and non-ordered structures.
32. The phagemid of claim 25 wherein the helper phage is selected
from the group M13KO7, M13R408, M13-VCS, and Phi X 174.
33. The phagemid of claim 32 wherein the helper phage is M13KO7 and
the coat protein is the M13 phage gene III coat protein.
34. The phagemid of claim 33 wherein the host is the E. coli wild
type or suppressor type.
35. The phagemid of claim 34 wherein the plasmid is under tight
control of the transcription regulatory element.
36. The phagemid of claim 35 wherein the number of phagemid
particles displaying more than one copy of the fusion protein on
the surface of the particles is less than 1%.
37. The phagemid of claim 36 wherein said number of phagemid
particles is less than about 10%.
38. The phagemid of claim 37 wherein the number of phagemid
particles is less than about 20%.
39. A human growth variant wherein hGH amino acids 10, 14, 18, and
21 respectively are as a group sequentially selected from one of
the following: (1) H,G,N,N; (2) A,W,D,N; (3) F,S,F,L; (4) Y,T,V,N
and (5) I,N,I,N.
40. A human growth variant wherein hGH amino acids 174 is serine
and 176 is tyrosine and hGH amino acids 167, 171, 175 and 179
respectively are as a group sequentially selected from one of the
following: (1) N,S,T,T; (2) E,S,T,I; (3) K,S,T,L; (4) N,N,T,T; (5)
R,D,I,I; and (6) N,S,T,Q.
41. A method for selecting novel binding polypeptides comprising
(a) constructing a replicable expression vector comprising a
transcription regulatory element operably linked to DNA encoding a
protein of interest containing one or more subunits, wherein the
DNA encoding at least one of the subunits is fused to the DNA
encoding at least a portion of a phage coat protein; (b) mutating
the DNA encoding the protein of interest at one or more selected
positions thereby forming a family of related vectors; (c)
transforming suitable host cells with the vectors; (d) infecting
the transformed host cells with a helper phage having a gene
encoding the phage coat protein; (e) culturing the transformed
infected host cells under conditions suitable for forming
recombinant phagemid particles containing at least a portion of the
plasmid and capable of transforming the host, the conditions
adjusted so that no more than a minor amount of phagemid particles
display more than one copy of the fusion protein on the surface of
the particle; (f) contacting the phagemid particles with a target
molecule so that at least a portion of the phagemid particles bind
to the target molecule; and (g) separating the phagemid particles
that bind from those that do not.
42. The method of claim 41 wherein the expression vector further
comprises a secretory signal sequence operably linked to the DNA
encoding each subunit of the protein of interest.
43. The method of claim 42 wherein the protein of interest is a
mammalian protein.
44. The method of claim 43 wherein the protein of interest is
selected from the group; insulin, relaxin, follicle stimulating
hormone (FSH), thyroid stimulating hormone (TSH), leutinizing
hormone (LH), glycoprotein hormone receptors, monoclonal and
polyclonal antibodies, lung surfactant, integrin receptors,
insulin-like growth factor-I and -II, and fragments of the
above-listed proteins.
45. The method of claim 44 wherein the protein of interest is a
humanized antibody.
46. The method of claim 45 wherein the protein of interest is a
humanized Fab fragment capable of binding to the HER-2 receptor
(human epidermal growth factor receptor-2).
47. A human growth hormone (hGH) variant wherein hGH amino acid
glutamate.sub.174 is replaced by serine.sub.174 and
phenylalanine.sub.176 is replaced by tyrosine.sub.176 and one or
more of the eight naturally occurring hGH amino acids F10, M14,
H18, H21, R167, D171, T175 and I179 are replaced by another natural
amino acid.
48. The hGH variant of claim 47 wherein the eight naturally
occurring hGH amino acids F10, M14, H18, H21, R167, D171, T175 and
I179 respectively are as a group replaced with a corresponding
amino acid sequentially selected from one of the following groups:
TABLE-US-00027 (1) H, G, N, N, N, S, T, T; (2) H, G, N, N, E, S, T,
I; (3) H, G, N, N, N, N, T, T; (4) A, W, D, N, N, S, T, T; (5) A,
W, D, N, E, S, T, I; (6) A, W, D, N, N, N, T, T; (7) F, S, F, L, N,
S, T, T; (8) F, S, F, L, E, S, T, I; (9) F, S, F, L, N, N, T, T.
(10) H, G, N, N, N, S, T, N; (11) A, N, D, A, N, N, T, N; (12) F,
S, F, G, H, S, T, T; (13) H, Q, T, S, A, D, N, S; (14) H, G, N, N,
N, A, T, T; (15) F, S, F, L, S, D, T, T; (16) A, S, T, N, R, D, T,
I; (17) Q, Y, N, N, H, S, T, T; (18) W, G, S, S, R, D, T, I; (19)
F, L, S, S, K, N, T, V; (20) W, N, N, S, H, S, T, T; (21) A, N, A,
S, N, S, T, T; (22) P, S, D, N, R, D, T, I; (23) H, G, N, N, N, N,
T, S; (24) F, S, T, G, R, D, T, I; (25) M, T, S, N, Q, S, T, T;
(26) F, S, F, L, T, S, T, S; (27) A, W, D, N, R, D, T, I; (28) A,
W, D, N, H, S, T, N; (29) M, Q, M, N, N, S, T, T; (30) H, Y, D, H,
R, D, T, T; (31) L, N, S, H, R, D, T, I; (32) L, N, S, H, T, S, T,
T; (33) A, W, D, N, N, A, T, T; (34) F, S, T, G, R, D, T, I; (35)
A, W, D, N, R, D, T, I; (36) I, Q, E, H, N, S, T, T; (37) F, S, L,
A, N, S, T, V; (38) F, S, F, L, K, D, T, T; (39) M, A, D, N, N, S,
T, T; (40) A, W, D, N, S, S, V, T; (41) H, Q, Y, S, R, D, T, I.
49. The method of claim 48 wherein said human growth hormone
variant (11) further contains leucine.sub.15 replaced by
arginine.sub.15 and lysine.sub.168 replaced by
arginine.sub.168.
50. The method of claim 48 wherein said human growth hormone
variant (40) further contains phenylalanine.sub.176
51. A method for selecting novel binding polypeptides comprising:
(a) constructing a replicable expression vector comprising a
transcription regulatory element operably linked to a gene fusion
encoding a fusion protein wherein the gene fusion comprises a first
gene encoding a polypeptide operable connected to a linking amino
acid sequence, and a second gene encoding at least a portion of a
phage coat protein; (b) mutating the vector at one or more selected
positions within the amino acid linking sequence of the first gene
thereby forming a family of related plasmids; (c) transforming
suitable host cells with the plasmids; (d) infecting the
transformed host cells with a helper phage having a gene encoding
the phage coat protein; (e) culturing the transformed infected host
cells under conditions suitable for forming recombinant phagemid
particles containing at least a portion of the plasmid and capable
of transforming the host, the conditions adjusted so that no more
than a minor amount of phagemid particles display more than one
copy of the fusion protein on the surface of the particle; (f)
contacting the phagemid particles with a target molecule so that at
least a portion of the phagemid particles bind to the target
molecule; and (g) contacting the bound phagemid particles with a
protease capable of hydrolysing the linking a amino acid sequence
of at least a portion of the bound phagemid particles, and (h)
isolating the hydrolyzed phagemid particles.
52. The method of claim 51 further comprising infecting suitable
host cells with the hydrolyzed phagemid particles and repeating
steps (d) through (h).
Description
[0001] This application is a continuation application of Ser. No.
11/761,180 filed Jun. 11, 2007, which is a continuation of Ser. No.
11/199,062 filed Aug. 8, 2005, now abandoned; which is a
continuation of application Ser. No. 09/717,641 filed Nov. 21,
2000, now abandoned; which is a continuation of application Ser.
No. 08/922,345 filed Sep. 3, 1997, now abandoned; which is a
continuation of application Ser. No. 08/463,587 filed Jun. 5, 1995,
now issued as U.S. Pat. No. 5,821,047; which is a divisional of
application Ser. No. 08/050,058 filed Apr. 30, 1993, now issued as
U.S. Pat. No. 5,750,373; which is a 371 of International
Application No. PCT/US91/09133 filed Dec. 3, 1991, which is a
continuation-in-part of application Ser. No. 07/743,614 filed Aug.
9, 1991, now abandoned; which is a continuation-in-part of
application Ser. No. 07/715,300 filed Jun. 14, 1991, now abandoned;
which is a continuation-in-part of application Ser. No. 07/683,400
filed Apr. 10, 1991, now abandoned; which is a continuation-in-part
of application Ser. No. 07/621,667 filed Dec. 3, 1990, now
abandoned. The contents of these applications are incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to the preparation and systematic
selection of novel binding proteins having altered binding
properties for a target molecule. Specifically, this invention
relates to methods for producing foreign polypeptides mimicking the
binding activity of naturally occurring binding partners. In
preferred embodiments, the invention is directed to the preparation
of therapeutic or diagnostic compounds that mimic proteins or
nonpeptidyl molecules such a hormones, drugs and other small
molecules, particularly biologically active molecules such as
growth hormone.
BACKGROUND OF THE INVENTION
[0003] Binding partners are substances that specifically bind to
one another, usually through noncovalent interactions. Examples of
binding partners include ligand-receptor, antibody-antigen,
drug-target, and enzyme-substrate interactions. Binding partners
are extremely useful in both therapeutic and diagnostic fields.
[0004] Binding partners have been produced in the past by a variety
of methods including; harvesting them from nature (e.g.,
antibody-antigen, and ligand-receptor pairings) and by adventitious
identification (e.g. traditional drug development employing random
screening of candidate molecules). In some instances these two
approaches have been combined. For example, variants of proteins or
polypeptides, such as polypeptide fragments, have been made that
contain key functional residues that participate in binding. These
polypeptide fragments, in turn, have been derivatized by methods
akin to traditional drug development. An example of such
derivitization would include strategies such as cyclization to
conformationally constrain a polypeptide fragment to produce a
novel candidate binding partner.
[0005] The problem with prior art methods is that naturally
occurring ligands may not have proper characteristics for all
therapeutic applications. Additionally, polypeptide ligands may not
even be available for some target substances. Furthermore, methods
for making non-naturally occurring synthetic binding partners are
often expensive and difficult, usually requiring complex synthetic
methods to produce each candidate. The inability to characterize
the structure of the resulting candidate so that rational drug
design methods can be applied for further optimization of candidate
molecules further hampers these methods.
[0006] In an attempt to overcome these problems, Geysen (Geysen,
Immun. Today, 6:364-369 [1985]); and (Geysen et al., Mol. Immun.,
23:709-715 [1986]) has proposed the use of polypeptide synthesis to
provide a framework for systematic iterative binding partner
identification and preparation. According to Geysen et al., Ibid,
short polypeptides, such as dipeptides, are first screened for the
ability to bind to a target molecule. The most active dipeptides
are then selected for an additional round of testing comprising
linking, to the starting dipeptide, an additional residue (or by
internally modifying the components of the original starting
dipeptide) and then screening this set of candidates for the
desired activity. This process is reiterated until the binding
partner having the desired properties is identified.
[0007] The Geysen et al. method suffers from the disadvantage that
the chemistry upon which it is based, peptide synthesis, produces
molecules with ill-defined or variable secondary and tertiary
structure. As rounds of iterative selection progress, random
interactions accelerate among the various substituent groups of the
polypeptide so that a true random population of interactive
molecules having reproducible higher order structure becomes less
and less attainable. For example, interactions between side chains
of amino acids, which are sequentially widely separated but which
are spatially neighbors, freely occur. Furthermore, sequences that
do not facilitate conformationally stable secondary structures
provide complex peptide-sidechain interactions which may prevent
sidechain interactions of a given amino acid with the target
molecule. Such complex interactions are facilitated by the
flexibility of the polyamide backbone of the polypeptide
candidates. Additionally, candidates may exist in numerous
conformations making it difficult to identify the conformer that
interacts or binds to the target with greatest affinity or
specificity complicating rational drug design.
[0008] A final problem with the iterative polypeptide method of
Geysen is that, at present, there are no practical methods with
which a great diversity of different peptides can be produced,
screened and analyzed. By using the twenty naturally occurring
amino acids, the total number of all combinations of hexapeptides
that must be synthesized is 64,000,000. Even having prepared such a
diversity of peptides, there are no methods available with which
mixtures of such a diversity of peptides can be rapidly screened to
select those peptides having a high affinity for the target
molecule. At present, each "adherent" peptide must be recovered in
amounts large enough to carry out protein sequencing.
[0009] To overcome many of the problems inherent in the Geysen
approach, biological selection and screening was chosen as an
alternative. Biological selections and screens are powerful tools
to probe protein function and to isolate variant proteins with
desirable properties (Shortle, Protein Engineering, Oxender and
Fox, eds., A.R. Liss, Inc., NY, pp. 103-108 [1988]) and Bowie et
al., Science, 247:1306-1310 [1990)]. However, a given selection or
screen is applicable to only one or a small number of related
proteins.
[0010] Recently, Smith and coworkers (Smith, Science, 228:
1315-1317 [1985]) and Parmley and Smith, Gene, 73:305-318 [1985]
have demonstrated that small protein fragments (10-50 amino acids)
can be "displayed" efficiently on the surface of filamentous phage
by inserting short gene fragments into gene III of the fd phage
("fusion phage"). The gene III minor coat protein (present in about
5 copies at one end of the virion) is important for proper phage
assembly and for infection by attachment to the pili of E. coli
(see Rasched et al., Microbiol. Rev., 50: 401-427 [1986]).
Recently, "fusion phage" have been shown to be useful for
displaying short mutated peptide sequences for identifying peptides
that may react with antibodies (Scott et al., Science 249: 386-390,
[1990]) and Cwirla et al., Proc. Natl. Acad. U.S.A 87: 6378-6382,
[1990]). or a foreign protein (Devlin et al., Science, 249: 404-406
[1990]).
[0011] There are, however, several important limitations in using
such "fusion phage" to identify altered peptides or proteins with
new or enhanced binding properties. First, it has been shown
(Parmley et al., Gene, 73: 305-318, [1988]) that fusion phage are
useful only for displaying proteins of less than 100 and preferably
less than 50 amino acid residues, because large inserts presumably
disrupt the function of gene III and therefore phage assembly and
infectivity. Second, prior art methods have been unable to select
peptides from a library having the highest binding affinity for a
target molecule. For example, after exhaustive panning of a random
peptide library with an anti-.beta. endorphin monoclonal antibody,
Cwirla and co-workers could not separate moderate affinity peptides
(K.sub.d.about.10 .mu.M) from higher affinity peptides
(K.sub.d.about.0.4 .mu.M) fused to phage. Moreover, the parent
.beta.-endorphin peptide sequence which has very high affinity
(K.sub.d.about.7 nM), was not panned from the epitope library.
[0012] Ladner WO 90/02802 discloses a method for selecting novel
binding proteins displayed on the outer surface of cells and viral
particles where it is contemplated that the heterologous proteins
may have up to 164 amino acid residues. The method contemplates
isolating and amplifying the displayed proteins to engineer a new
family of binding proteins having desired affinity for a target
molecule. More specifically, Ladner discloses a "fusion phage"
displaying proteins having "initial protein binding domains"
ranging from 46 residues (crambin) to 164 residues (T4 lysozyme)
fused to the M13 gene III coat protein. Ladner teaches the use of
proteins "no larger than necessary" because it is easier to arrange
restriction sites in smaller amino acid sequences and prefers the
58 amino acid residue bovine pancreatic trypsin inhibitor (BPTI).
Small fusion proteins, such as BPTI, are preferred when the target
is a protein or macromolecule, while larger fusion proteins, such
as T4 lysozyme, are preferred for small target molecules such as
steroids because such large proteins have clefts and grooves into
which small molecules can fit. The preferred protein, BPTI, is
proposed to be fused to gene III at the site disclosed by Smith et
al. or de la Cruz et al., J. Biol. Chem., 263: 4318-4322 [1988], or
to one of the terminii, along with a second synthetic copy of gene
III so that "some" unaltered gene III protein will be present.
Ladner does not address the problem of successfully panning high
affinity peptides from the random peptide library which plagues the
biological selection and screening methods of the prior art.
[0013] Human growth hormone (hGH) participates in much of the
regulation of normal human growth and development. This 22,000
dalton pituitary hormone exhibits a multitude of biological effects
including linear growth (somatogenesis), lactation, activation of
macrophages, insulin-like and diabetogenic effects among others
(Chawla, R, K. (1983) Ann. Rev. Med. 34, 519; Edwards, C. K. et al.
(1988) Science 239, 769; Thorner, M. O., et al. (1988) J. Clin.
Invest. 81 745). Growth hormone deficiency in children leads to
dwarfism which has been successfully treated for more than a decade
by exogenous administration of hGH. hGH is a member of a family of
homologous hormones that include placental lactogens, prolactins,
and other genetic and species variants or growth hormone (Nicoll,
C. S., et al., (1986) Endocrine Reviews 7, 169). hGH is unusual
among these in that it exhibits broad species specificity and binds
to either the cloned somatogenic (Leung, D. W., et al., [I987]
Nature 330, 537) or prolactin receptor (Boutin, J. M., et al.,
[I988] Ce; 53, 69). The cloned gene for hGH has been expressed in a
secreted form in Escherichia coli (Chang, C. N., et al., [I987]
Gene 55, I89) and its DNA and amino acid sequence has been reported
(Goeddel, et al., [I979] Nature 281, 544; Gray, et al., [I985] Gene
39, 247). The three-dimensional structure of hGH is not available.
However, the three-dimensional folding pattern for porcine growth
hormone (pGH) has been reported at moderate resolution and
refinement (Abdel-Meguid, S. S., et al., [I987] Proc. Natl. Acad.
Sci. USA 84, 6434). Human growth hormone's receptor and antibody
epitopes have been identified by homolog-scanning mutagenesis
(Cunningham et al., Science 243: 1330, 1989). The structure of
novel amino terminal methionyl bovine growth hormone containing a
spliced-in sequence of human growth hormone including histidine 18
and histidine 21 has been shown (U.S. Pat. No. 4,880,910)
[0014] Human growth hormone (hGH) causes a variety of physiological
and metabolic effects in various animal models including linear
bone growth, lactation, activation of macrophages, insulin-like and
diabetogenic effects and others (R. K. Chawla et al., Annu. Rev.
Med. 34, 519 (1983); O. G. P. Isaksson et al., Annu. Rev. Physiol.
47, 483 (1985); C. K. Edwards et al., Science 239, 769 (1988); M.
O. Thorner and M. L. Vance, J. Clin. Invest 82, 745 (1988); J. P.
Hughes and H. G. Friesen, Ann. Rev. Physiol. 47, 469 (1985)). These
biological effects derive from the interaction between hGH and
specific cellular receptors.
[0015] Accordingly, it is an object of this invention to provide a
rapid and effective method for the systematic preparation of
candidate binding substances.
[0016] It is another object of this invention to prepare candidate
binding substances displayed on surface of a phagemid particle that
are conformationally stable.
[0017] It is another object of this invention to prepare candidate
binding substances comprising fusion proteins of a phage coat
protein and a heterologous polypeptide where the polypeptide is
greater than 100 amino acids in length and may be more than one
subunit and is displayed on a phagemid particle where the
polypeptide is encoded by the phagemid genome.
[0018] It is a further object of this invention to provide a method
for the preparation and selection of binding substances that is
sufficiently versatile to present, or display, all peptidyl
moieties that could potentially participate in a noncovalent
binding interaction, and to present these moieties in a fashion
that is sterically confined.
[0019] Still another object of the invention is the production of
growth hormone variants that exhibit stronger affinity for growth
hormone receptor and binding protein.
[0020] It is yet another object of this invention to produce
expression vector phagemids that contain a suppressible termination
codon functionally located between the heterologous polypeptide and
the phage coat protein such that detectable fusion protein is
produced in a host suppressor cell and only the heterologous
polypeptide is produced in a non-suppressor host cell.
[0021] Finally, it is an object of this invention to produce a
phagemid particle that rarely displays more than one copy of
candidate binding proteins on the outer surface of the phagemid
particle so that efficient selection of high affinity binding
proteins can be achieved.
[0022] These and other objects of this invention will be apparent
from consideration of the invention as a whole.
SUMMARY OF THE INVENTION
[0023] These objectives have been achieved by providing a method
for selecting novel binding polypeptides comprising: (a)
constructing a replicable expression vector comprising a first gene
encoding a polypeptide, a second gene encoding at least a portion
of a natural or wild-type phage coat protein wherein the first and
second genes are heterologous, and a transcription regulatory
element operably linked to the first and second genes, thereby
forming a gene fusion encoding a fusion protein; (b) mutating the
vector at one or more selected positions within the first gene
thereby forming a family of related plasmids; (c) transforming
suitable host cells with the plasmids; (d) infecting the
transformed host cells with a helper phage having a gene encoding
the phage coat protein; (e) culturing the transformed infected host
cells under conditions suitable for forming recombinant phagemid
particles containing at least a portion of the plasmid and capable
of transforming the host, the conditions adjusted so that no more
than a minor amount of phagemid particles display more than one
copy of the fusion protein on the surface of the particle; (f)
contacting the phagemid particles with a target molecule so that at
least a portion of the phagemid particles bind to the target
molecule; and (g) separating the phagemid particles that bind from
those that do not. Preferably, the method further comprises
transforming suitable host cells with recombinant phagemid
particles that bind to the target molecule and repeating steps (d)
through (g) one or more times.
[0024] Additionally, the method for selecting novel binding
proteins where the proteins are composed of more than one subunit
is achieved by selecting novel binding peptides comprising
constructing a replicable expression vector comprising a
transcription regulatory element operably linked to DNA encoding a
protein of interest containing one or more subunits, wherein the
DNA encoding at least one of the subunits is fused to the DNA
encoding at least a portion of a phage coat protein; mutating the
DNA encoding the protein of interest at one or more selected
positions thereby forming a family of related vectors; transforming
suitable host cells with the vectors; infecting the transformed
host cells with a helper phage having a gene encoding the phage
coat protein; culturing the transformed infected host cells under
conditions suitable for forming recombinant phagemid particles
containing at least a portion of the plasmid and capable of
transforming the host, the conditions adjusted so that no more than
a minor amount of phagemid particles display more than one copy of
the fusion protein on the surface of the particle; contacting the
phagemid particles with a target molecule so that at least a
portion of the phagemid particles bind to the target molecule; and
separating the phagemid particles that bind from those that do
not.
[0025] Preferably in the method of this invention the plasmid is
under tight control of the transcription regulatory element, and
the culturing conditions are adjusted so that the amount or number
of phagemid particles displaying more than one copy of the fusion
protein on the surface of the particle is less than about 1%. Also
preferably, amount of phagemid particles displaying more than one
copy of the fusion protein is less than 10% the amount of phagemid
particles displaying a single copy of the fusion protein. Most
preferably the amount is less than 20%.
[0026] Typically, in the method of this invention, the expression
vector will further contain a secretory signal sequences fused to
the DNA encoding each subunit of the polypeptide, and the
transcription regulatory element will be a promoter system.
Preferred promoter systems are selected from; Lac Z,
.lamda..sub.PL, TAC, T 7 polymerase, tryptophan, and alkaline
phosphatase promoters and combinations thereof.
[0027] Also typically, the first gene will encode a mammalian
protein, preferably the protein will be selected from; human growth
hormone (hGH), N-methionyl human growth hormone, bovine growth
hormone, parathyroid hormone, thyroxine, insulin A-chain, insulin
B-chain, proinsulin, relaxin A-chain, relaxin B-chain, prorelaxin,
glycoprotein hormones such as follicle stimulating hormone (FSH),
thyroid stimulating hormone (TSH), and leutinizing hormone (LH),
glycoprotein hormone receptors, calcitonin, glucagon, factor VIII,
an antibody, lung surfactant, urokinase, streptokinase, human
tissue-type plasminogen activator (t-PA), bombesin, factor IX,
thrombin, hemopoietic growth factor, tumor necrosis factor-alpha
and -beta, enkephalinase, human serum albumin, mullerian-inhibiting
substance, mouse gonadotropin-associated peptide, a microbial
protein, such as betalactamase, tissue factor protein, inhibin,
activin, vascular endothelial growth factor, receptors for hormones
or growth factors; integrin, thrombopoietin, protein A or D,
rheumatoid factors, nerve growth factors such as NGF-.beta.
platelet-growth factor, transforming growth factors (TGF) such as
TGF-alpha and TGF-beta, insulin-like growth factor-I and -II,
insulin-like growth factor binding proteins, CD-4, DNase, latency
associated peptide, erythropoietin, osteoinductive factors,
interferons such as interferon-alpha, -beta, and -gamma, colony
stimulating factors (CSFs) such as M-CSF, GM-CSF, and G-CSF,
interleukins (ILs) such as IL-1, IL-2, IL-3, IL-4, superoxide
dismutase; decay accelerating factor, viral antigen, HIV envelope
proteins such as GP120, GP140, atrial natriuretic peptides A, B or
C, immunoglobulins, and fragments of any of the above-listed
proteins.
[0028] Preferably the first gene will encode a polypeptide of one
or more subunits containing more than about 100 amino acid residues
and will be folded to form a plurality of rigid secondary
structures displaying a plurality of amino acids capable of
interacting with the target. Preferably the first gene will be
mutated at codons corresponding to only the amino acids capable of
interacting with the target so that the integrity of the rigid
secondary structures will be preserved.
[0029] Normally, the method of this invention will employ a helper
phage selected from; 13KO7, M13R408, M13-VCS, and Phi X 174. The
preferred helper phage is M13KO7, and the preferred coat protein is
the M13 Phage gene III coat protein. The preferred host is E. coli,
and protease deficient strains of E. coli. Novel hGH variants
selected by the method of the present invention have been detected.
Phagemid expression vectors were constructed that contain a
suppressible termination codon functionally located between the
nucleic acids encoding the polypeptide and the phage coat
protein.
BRIEF DESCRIPTION OF THE FIGURES
[0030] FIG. 1. Strategy for displaying large proteins on the
surface of filamentous phage and enriching for altered receptor
binding properties. A plasmid, phGH-M13gIII was constructed that
fuses the entire coding sequence of hGH to the carboxyl terminal
domain of M13 gene III. Transcription of the fusion protein is
under control of the lac promoter/operator sequence, and secretion
is directed by the stII signal sequence. Phagemid particles are
produced by infection with the "helper" phage, M13KO7, and
particles displaying hGH can be enriched by binding to an affinity
matrix containing the hGH receptor. The wild-type gene III (derived
from the M13KO7 phage) is diagramed by 4-5 copies of the multiple
arrows on the tip of the phage, and the fusion protein (derived
from the phagemid, phGH-M13gIII) is indicated schematically by the
folding diagram of hGH replacing the arrow head.
[0031] FIG. 2. Immunoblot of whole phage particles shows that hGH
comigrates with phage. Phagemid particles purified in a cesium
chloride gradient were loaded into duplicate wells and
electrophoresed through a 1% agarose gel in 375 mM Tris, 40 mM
glycine pH 9.6 buffer. The gel was soaked in transfer buffer (25 mM
Tris, pH 8.3, 200 mM glycine, 20% methanol) containing 2% SDS and
2% .beta.-mercaptoethanol for 2 hours, then rinsed in transfer
buffer for 6 hours. The proteins in the gel were then
electroblotted onto immobilon membranes (Millipore). The membrane
containing one set of samples was stained with Coomassie blue to
show the position of the phage proteins (A). The duplicate membrane
was immuno-stained for hGH by reacting the membrane with polyclonal
rabbit anti-hGH antibodies followed by reaction with horseradish
peroxidase conjugated goat anti-rabbit IgG antibodies (B). Lane 1
contains the M13KO7 parent phage and is visible only in the
Coomassie blue stained membrane, since it lacks hGH. Lanes 2 and 3
contain separate preparations of the hormone phagemid particles
which is visible both by Coomassie and hGH immuno-staining. The
difference in migration distance between the parent M13KO7 phage
and hormone phagemid particles reflects the different size genomes
that are packaged within (8.7 kb vs. 5.1 kb, respectively).
[0032] FIG. 3. Summary diagram of steps in the selection process
for an hGH-phage library randomized at codons 172, 174, 176, and
178. The template molecules, pH0415, containing a unique KpnI
restriction site and the hGH(R178G, I179T) gene was mutagenized as
described in the text and electrotransformed into E. coli strain
WJM101 to obtain the initial phagemid library, Library 1. An
aliquot (approximately 2%) from Library 1 was used directly in an
initial selection round as described in the text to yield Library
1G. Meanwhile, double-stranded DNA (dsDNA) was prepared from
Library I, digested with restriction enzyme KpnI to eliminate
template background, and electrotransformed into WJM101 to yield
Library 2. Subsequent rounds of selection (or KpnI digestion,
shaded boxes) followed by phagemid propagation were carried out as
indicated by the arrows, according to the procedure described in
the text. Four independent clones from Library 4G.sup.4 and four
independent clones from Library 5G.sup.6 were sequenced by dideoxy
sequencing. All of these clones had the identical DNA sequence,
corresponding to the hGH mutant (Glu 174 Ser, Phe 176 Tyr).
[0033] FIG. 4. Structural model of hGH derived from a 2.8 .ANG.
folding diagram of porcine growth hormone determined
crystallographically. Location of residues in hGH that strongly
modulate its binding to the hGH-binding protein are within the
shaded circle. Alanine substitutions that cause a greater than
tenfold reduction ( ), a four- to tenfold reduction ( ), or
increase (.largecircle.), or a two- to fourfold reduction ( ), in
binding affinity are indicated. Helical wheel projections in the
regions of .alpha.-helix reveal their amphipathic quality.
Blackened, shaded, or nonshaded residues are charged, polar, or
nonpolar, respectively. In helix-4 the most important residues for
mutation are on the hydrophilic face.
[0034] FIG. 5. Amino acid substitutions at positions 172, 174, 176
and 178 of hGH (The notation, e.g. KSYR, denotes hGH mutant
172K/174S/176Y/178R) found after sequencing a number of clones from
rounds 1 and 3 of the selection process for the pathways indicated
(hGH elution; Glycine elution; or Glycine elution after
pre-adsorption). Non-functional sequences (i.e. vector background,
or other prematurely terminated and/or frame-shifted mutants) are
shown as "NF". Functional sequences which contained a non-silent,
spurious mutation (i.e. outside the set of target residues) are
marked with a "+". Protein sequences which appeared more than once
among all the sequenced clones, but with different DNA sequences,
are marked with a "#". Protein sequences which appeared more than
once among the sequenced clones and with the same DNA sequence are
marked with a "*". Note that after three rounds of selection, 2
different contaminating sequences were found; these clones did not
correspond to cassette mutants, but to previously constructed
hormone phage. The pS0643 contaminant corresponds to wild-type
hGH-phage (hGH "KEFR" (SEQ ID NO:44)). The pH0457 contaminant,
which dominates the third-round glycine-selected pool of phage,
corresponds to a previously identified mutant of hGH, "KSYR." The
amplification of these contaminants emphasizes the ability of the
hormone-phage selection process to select for rarely occurring
mutants. The convergence of sequences is also striking in all three
pathways: R or K occurs most often at positions 172 and 178; Y or F
occurs most often at position 176; and S, T, A, and other residues
occur at position 174.
[0035] FIG. 6. Sequences from phage selected on hPRLbp-beads in the
presence of zinc. The notation is as described in FIG. 5. Here, the
convergence of sequences is not predictable, but there appears to
be a bias towards hydrophobic sequences under the most stringent
(Glycine) selection conditions; L, W and P residues are frequently
found in this pool.
[0036] FIG. 7. Sequences from phage selected on hPRLbp-beads in the
absence of zinc. The notation is as described in FIG. 5. In
contrast to the sequences of FIG. 6, these sequences appear more
hydrophilic. After 4 rounds of selection using hGH elution, two
clones (ANHQ (SEQ ID NO:45), and TLDT/171V (SEQ ID NO:108))
dominate the pool.
[0037] FIG. 8. Sequences from phage selected on blank beads. The
notation is as described in FIG. 5. After three rounds of selection
with glycine elution, no siblings were observed and a background
level of non-functional sequences remained.
[0038] FIG. 9. Construction of phagemid fl ori from pHO415. This
vector for cassette mutagenesis and expression of the hGH-gene III
fusion protein was constructed as follows. Plasmid pS0643 was
constructed by oligonucleotide-directed mutagenesis of pS0132,
which contains pBR322 and f1 origins of replication and expresses
an hGH-gene III fusion protein (hGH residues 1-191, followed by a
single Gly residue, fused to Pro-198 of gene III) under the control
of the E. coli phoA promoter. Mutagenesis was carried out with the
oligonucleotide
5'-GGC-AGC-TGT-GGC-TTC-TAG-AGT-GGC-GGC-GGC-TCT-GGT-3' (SEQ ID
NO:1), which introduced a XbaI site (underlined) and an amber stop
codon (TAG) following Phe-191 of hGH.
[0039] FIG. 10. A. Diagram of plasmid pDH188 insert containing the
DNA encoding the light chain and heavy chain (variable and constant
domain 1) of the F.sub.ab humanized antibody directed to the HER-2
receptor. V.sub.L and V.sub.H are the variable regions for the
light and heavy chains, respectively. C.sub.k is the constant
region of the human kappa light chain. CH1.sub.G1 is the first
constant region of the human gamma 1 chain. Both coding regions
start with the bacterial st II signal sequence. B. A schematic
diagram of the entire plasma pDH188 containing the insert described
in 5A. After transformation of the plasmid into E. coli SR101 cells
and the addition of helper phage, the plasmid is packaged into
phage particles. Some of these particles display the F.sub.ab-p III
fusion (where p III is the protein encoded by the M13 gene III
DNA). The segments in the plasmid figure correspond to the insert
shown in 5A.
[0040] FIG. 11. A through C are collectively referred to here as
FIG. 11. The nucleotide (Seq. ID No: 24) sequence of the DNA
encoding the 4D5 F.sub.ab molecule expressed on the phagemid
surface. The amino acid sequence of the light chain is also shown
(Seq. ID No: 25), as is the amino acid sequence of the heavy chain
p III fusion (Seq. ID No:26).
[0041] FIG. 12. Enrichment of wild-type 4D5 F.sub.ab phagemid from
variant F.sub.ab phagemid. Mixtures of wild-type phagemid and
variant 4D5 F.sub.ab phagemid in a ratio of 1:1,000 were selected
on plates coated with the extra-cellular domain protein of the
HER-2 receptor. After each round of selection, a portion of the
eluted phagemid were infected into E. coli and plasmid DNA was
prepared. This plasmid DNA was then digested with Eco RV and Pst I,
separated on a 5% polyacrylamide gel, and stained with ethidium
bromide. The bands were visualized under UV light. The bands due to
the wild-type and variant plasmids are marked with arrows. The
first round of selection was eluted only under acid conditions;
subsequent rounds were eluted with either an acid elution (left
side of Figure) or with a humanized 4D5 antibody wash step prior to
acid elution (right side of Figure) using methods described in
Example VIII. Three variant 4D5 F.sub.ab molecules were made: H91A
(amino acid histidine at position 91 on the V.sub.L chain mutated
to alanine; indicated as `A` lanes in Figure), Y49A (amino acid
tyrosine at position 49 on the V.sub.L chain mutated to alanine;
indicated as `B` lanes in the Figure), and Y92A (amino acid
tyrosine at position 92 on the V.sub.L chain mutated to alanine;
indicated as `C` lanes in the Figure). Amino acid position
numbering is according to Kabat et al., (Sequences of proteins of
immunological interest, 4th ed., U.S. Dept of Health and Human
Services, Public Health Service, Nat'l. Institute of Health,
Bethesda, Md. [1987]).
[0042] FIG. 13. The Scatchard analysis of the RIA affinity
determination described in Experimental Protocols is shown here.
The amount of labeled ECD antigen that is bound is shown on the
x-axis while the amount that is bound divided by the amount that is
free is shown on the y-axis. The slope of the line indicates the
K.sub.a; the calculated K.sub.d is 1/K.sub.a.
DETAILED DESCRIPTION OF THE INVENTION
[0043] The following discussion will be best understood by
referring to FIG. 1. In its simplest form, the method of the
instant invention comprises a method for selecting novel binding
polypeptides, such as protein ligands, having a desired, usually
high, affinity for a target molecule from a library of structurally
related binding polypeptides. The library of structurally related
polypeptides, fused to a phage coat protein, is produced by
mutagenesis and, preferably, a single copy of each related
polypeptide is displayed on the surface of a phagemid particle
containing DNA encoding that polypeptide. These phagemid particles
are then contacted with a target molecule and those particles
having the highest affinity for the target are separated from those
of lower affinity. The high affinity binders are then amplified by
infection of a bacterial host and the competitive binding step is
repeated. This process is reiterated until polypeptides of the
desired affinity are obtained.
[0044] The novel binding polypeptides or ligands produced by the
method of this invention are useful per se as diagnostics or
therapeutics (eg. agonists or antagonists) used in treatment of
biological organisms. Structural analysis of the selected
polypeptides may also be used to facilitate rational drug
design.
[0045] By "binding polypeptide" as used herein is meant any
polypeptide that binds with a selectable affinity to a target
molecule. Preferably the polypeptide will be a protein that most
preferably contains more than about 100 amino acid residues.
Typically the polypeptide will be a hormone or an antibody or a
fragment thereof.
[0046] By "high affinity" as used herein is meant an affinity
constant (K.sub.d) of <10.sup.-5 M and preferably <10.sup.-7M
under physiological conditions.
[0047] By "target molecule" as used herein is meant any molecule,
not necessarily a protein, for which it is desirable to produce a
ligand. Preferably, however, the target will be a protein and most
preferably the target will be a receptor, such as a hormone
receptor.
[0048] By "humanized antibody" as used herein is meant an antibody
in which the complementarity-determining regions (CDRs) of a mouse
or other non-human antibody are grafted onto a human antibody
framework. By human antibody framework is meant the entire human
antibody excluding the CDRs.
I. Choice of Polypeptides for Display on the Surface of a Phage
[0049] The first step in the method of this invention is to choose
a polypeptide having rigid secondary structure exposed to the
surface of the polypeptide for display on the surface of a
phage.
[0050] By "polypeptide" as used herein is meant any molecule whose
expression can be directed by a specific DNA sequence. The
polypeptides of this invention may comprise more than one subunit,
where each subunit is encoded by a separate DNA sequence.
[0051] By "rigid secondary structure" as used herein is meant any
polypeptide segment exhibiting a regular repeated structure such as
is found in; .alpha.-helices, 3.sub.10 helices, .pi.-helices,
parallel and antiparallel .beta.-sheets, and reverse turns. Certain
"non-ordered" structures that lack recognizable geometric order are
also included in the definition of rigid secondary structure
provided they form a domain or "patch" of amino acid residues
capable of interaction with a target and that the overall shape of
the structure is not destroyed by replacement of an amino acid
within the structure. It is believed that some non-ordered
structures are combinations of reverse turns. The geometry of these
rigid secondary structures is well defined by .phi. and .psi.
torsional angles about the .alpha.-carbons of the peptide
"backbone".
[0052] The requirement that the secondary structure be exposed to
the surface of the polypeptide is to provide a domain or "patch" of
amino acid residues that can be exposed to and bind with a target
molecule. It is primarily these amino acid residues that are
replaced by mutagenesis that form the "library" of structurally
related (mutant) binding polypeptides that are displayed on the
surface of the phage and from which novel polypeptide ligands are
selected. Mutagenesis or replacement of amino acid residues
directed toward the interior of the polypeptide is generally
avoided so that the overall structure of the rigid secondary
structure is preserved. Some replacement of amino acids on the
interior region of the rigid secondary structures, especially with
hydrophobic amino acid residues, may be tolerated since these
conservative substitutions are unlikely to distort the overall
structure of the polypeptide.
[0053] Repeated cycles of "polypeptide" selection are used to
select for higher and higher affinity binding by the phagemid
selection of multiple amino acid changes which are selected by
multiple selection cycles. Following a first round of phagemid
selection, involving a first region or selection of amino acids in
the ligand polypeptide, additional rounds of phagemid selection in
other regions or amino acids of the ligand polypeptide are
conducted. The cycles of phagemid selection are repeated until the
desired affinity properties of the ligand polypeptide are achieved.
To illustrate this process, Example VIII phagemid selection of hGH
was conducted in cycles. In the first cycle hGH amino acids 172,
174, 176 and 178 were mutated and phagemid selected. In a second
cycle hGH amino acids 167, 171, 175 and 179 were phagemid selected.
In a third cycle hGH amino acids 10, 14, 18 and 21 were phagemid
selected. Optimum amino acid changes from a previous cycle may be
incorporated into the polypeptide before the next cycle of
selection. For example, hGH amino acids substitution 174 (serine)
and 176 (tyrosine) were incorporated into the hGH before the
phagemid selection of hGH amino acids 167, 171, 175 and 179.
[0054] From the forgoing it will be appreciated that the amino acid
residues that form the binding domain of the polypeptide will not
be sequentially linked and may reside on different subunits of the
polypeptide. That is, the binding domain tracks with the particular
secondary structure at the binding site and not the primary
structure. Thus, generally, mutations will be introduced into
codons encoding amino acids within a particular secondary structure
at sites directed away from the interior of the polypeptide so that
they will have the potential to interact with the target. By way of
illustration, FIG. 2 shows the location of residues in hGH that are
known to strongly modulate its binding to the hGH-binding protein
(Cunningham et al., Science 247:1461-1465 [1990]). Thus
representative sites suitable for mutagenesis would include
residues 172, 174, 176, and 178 on helix-4, as well as residue 64
located in a "non-ordered" secondary structure.
[0055] There is no requirement that the polypeptide chosen as a
ligand to a target normally bind to that target. Thus, for example,
a glycoprotein hormone such as TSH can be chosen as a ligand for
the FSH receptor and a library of mutant TSH molecules are employed
in the method of this invention to produce novel drug
candidates.
[0056] This invention thus contemplates any polypeptide that binds
to a target molecule, and includes antibodies. Preferred
polypeptides are those that have pharmaceutical utility. More
preferred polypeptides include; a growth hormone, including human
growth hormone, des-N-methionyl human growth hormone, and bovine
growth hormone; parathyroid hormone; thyroid stimulating hormone;
thyroxine; insulin A-chain; insulin B-chain; proinsulin; follicle
stimulating hormone; calcitonin; leutinizing hormone; glucagon;
factor VIII; an antibody; lung surfactant; a plasminogen activator,
such as urokinase or human tissue-type plasminogen activator
(t-PA); bombesin; factor IX, thrombin; hemopoietic growth factor;
tumor necrosis factor-alpha and -beta; enkephalinase; a serum
albumin such as human serum albumin; mullerian-inhibiting
substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse
gonadotropin-associated peptide; a microbial protein, such as
betalactamase; tissue factor protein; inhibin; activin; vascular
endothelial growth factor; receptors for hormones or growth
factors; integrin; thrombopoietin; protein A or D; rheumatoid
factors; nerve growth factor such as NGF-.beta.; platelet-derived
growth factor; fibroblast growth factor such as aFGF and bFGF;
epidermal growth factor; transforming growth factor (TGF) such as
TGF-alpha and TGF-beta; insulin-like growth factor-I and -II;
insulin-like growth factor binding proteins; CD-4; DNase; latency
associated peptide; erythropoietin; osteoinductive factors; an
interferon such as interferon-alpha, -beta, and -gamma; colony
stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF;
interleukins (ILs), e.g., IL-1, IL-2, IL-3, IL-4, etc.; superoxide
dismutase; decay accelerating factor; atrial natriuretic peptides
A, B or C; viral antigen such as, for example, a portion of the HIV
envelope; immunoglobulins; and fragments of any of the above-listed
polypeptides. In addition, one or more predetermined amino acid
residues on the polypeptide may be substituted, inserted, or
deleted, for example, to produce products with improved biological
properties. Further, fragments of these polypeptides, especially
biologically active fragments, are included. Yet more preferred
polypeptides of this invention are human growth hormone and atrial
naturetic peptides A, B, and C, endotoxin, subtilisin, trypsin and
other serine proteases.
[0057] Still more preferred are polypeptide hormones that can be
defined as any amino acid sequence produced in a first cell that
binds specifically to a receptor on the same cell type (autocrine
hormones) or a second cell type (non-autocrine) and causes a
physiological response characteristic of the receptor-bearing cell.
Among such polypeptide hormones are cytokines, lymphokines,
neurotrophic hormones and adenohypophyseal polypeptide hormones
such as growth hormone, prolactin, placental lactogen, luteinizing
hormone, follicle-stimulating hormone, thyrotropin, chorionic
gonadotropin, corticotropin, or .beta.-melanocyte-stimulating
hormone, .beta.-lipotropin gamma-lipotropin and the endorphins;
hypothalmic release-inhibiting hormones such as
corticotropin-release factor, growth hormone release-inhibiting
hormone, growth hormone-release factor; and other polypeptide
hormones such as atrial natriuretic peptides A, B or C.
II. Obtaining a First Gene (Gene 1) Encoding the Desired
Polypeptide
[0058] The gene encoding the desired polypeptide (i.e., a
polypeptide with a rigid secondary structure) can be obtained by
methods known in the art (see generally, Sambrook et al., Molecular
Biology: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring
Harbor, N.Y. [1989]). If the sequence of the gene is known, the DNA
encoding the gene may be chemically synthesized (Merrfield, J. Am.
Chem. Soc., 85:2149 [1963]). If the sequence of the gene is not
known, or if the gene has not previously been isolated, it may be
cloned from a cDNA library (made from RNA obtained from a suitable
tissue in which the desired gene is expressed) or from a suitable
genomic DNA library. The gene is then isolated using an appropriate
probe. For cDNA libraries, suitable probes include monoclonal or
polyclonal antibodies (provided that the cDNA library is an
expression library), oligonucleotides, and complementary or
homologous cDNAs or fragments thereof. The probes that may be used
to isolate the gene of interest from genomic DNA libraries include
cDNAs or fragments thereof that encode the same or a similar gene,
homologous genomic DNAs or DNA fragments, and oligonucleotides.
Screening the cDNA or genomic library with the selected probe is
conducted using standard procedures as described in chapters 10-12
of Sambrook et al., supra.
[0059] An alternative means to isolating the gene encoding the
protein of interest is to use polymerase chain reaction methodology
(PCR) as described in section 14 of Sambrook et al., supra. This
method requires the use of oligonucleotides that will hybridize to
the gene of interest; thus, at least some of the DNA sequence for
this gene must be known in order to generate the
oligonucleotides.
[0060] After the gene has been isolated, it may be inserted into a
suitable vector (preferably a plasmid) for amplification, as
described generally in Sambrook et al., supra.
III. Constructing Replicable Expression Vectors
[0061] While several types of vectors are available and may be used
to practice this invention, plasmid vectors are the preferred
vectors for use herein, as they may be constructed with relative
ease, and can be readily amplified. Plasmid vectors generally
contain a variety of components including promoters, signal
sequences, phenotypic selection genes, origin of replication sites,
and other necessary components as are known to those of ordinary
skill in the art.
[0062] Promoters most commonly used in prokaryotic vectors include
the lac Z promoter system, the alkaline phosphatase pho A promoter,
the bacteriophage .lamda.PL promoter (a temperature sensitive
promoter), the tac promoter (a hybrid trp-lac promoter that is
regulated by the lac repressor), the tryptophan promoter, and the
bacteriophage T7 promoter. For general descriptions of promoters,
see section 17 of Sambrook et al. supra. While these are the most
commonly used promoters, other suitable microbial promoters may be
used as well.
[0063] Preferred promoters for practicing this invention are those
that can be tightly regulated such that expression of the fusion
gene can be controlled. It is believed that the problem that went
unrecognized in the prior art was that display of multiple copies
of the fusion protein on the surface of the phagemid particle lead
to multipoint attachment of the phagemid with the target. It is
believed this effect, referred to as the "chelate effect", results
in selection of false "high affinity" polypeptides when multiple
copies of the fusion protein are displayed on the phagemid particle
in close proximity to one another so that the target was
"chelated". When multipoint attachment occurs, the effective or
apparent Kd may be as high as the product of the individual Kds for
each copy of the displayed fusion protein. This effect may be the
reason Cwirla and coworkers supra were unable to separate moderate
affinity peptides from higher affinity peptides.
[0064] It has been discovered that by tightly regulating expression
of the fusion protein so that no more than a minor amount, i.e.
fewer than about 1%, of the phagemid particles contain multiple
copies of the fusion protein the "chelate effect" is overcome
allowing proper selection of high affinity polypeptides. Thus,
depending on the promoter, culturing conditions of the host are
adjusted to maximize the number of phagemid particles containing a
single copy of the fusion protein and minimize the number of
phagemid particles containing multiple copies of the fusion
protein.
[0065] Preferred promoters used to practice this invention are the
lac Z promoter and the pho A promoter. The lac Z promoter is
regulated by the lac repressor protein lac i, and thus
transcription of the fusion gene can be controlled by manipulation
of the level of the lac repressor protein. By way of illustration,
the phagemid containing the lac Z promoter is grown in a cell
strain that contains a copy of the lac i repressor gene, a
repressor for the lac Z promoter. Exemplary cell strains containing
the lac i gene include JM 101 and XL1-blue. In the alternative, the
host cell can be cotransfected with a plasmid containing both the
repressor lac i and the lac Z promoter. Occasionally both of the
above techniques are used simultaneously, that is, phagemid
particles containing the lac Z promoter are grown in cell strains
containing the lac i gene and the cell strains are cotransfected
with a plasmid containing both the lac Z and lac i genes. Normally
when one wishes to express a gene, to the transfected host above
one would add an inducer such as isopropylthiogalactoside (IPTG).
In the present invention however, this step is omitted to (a)
minimize the expression of the gene III fusion protein thereby
minimizing the copy number (i.e. the number of gene III fusions per
phagemid number) and to (b) prevent poor or improper packaging of
the phagemid caused by inducers such as IPTG even at low
concentrations. Typically, when no inducer is added, the number of
fusion proteins per phagemid particle is about 0.1 (number of bulk
fusion proteins/number of phagemid particles). The most preferred
promoter used to practice this invention is pho A. This promoter is
believed to be regulated by the level of inorganic phosphate in the
cell where the phosphate acts to down-regulate the activity of the
promoter. Thus, by depleting cells of phosphate, the activity of
the promoter can be increased. The desired result is achieved by
growing cells in a phosphate enriched medium such as 2YT or LB
thereby controlling the expression of the gene III fusion.
[0066] One other useful component of vectors used to practice this
invention is a signal sequence. This sequence is typically located
immediately 5' to the gene encoding the fusion protein, and will
thus be transcribed at the amino terminus of the fusion protein.
However, in certain cases, the signal sequence has been
demonstrated to be located at positions other 5' to the gene
encoding the protein to be secreted. This sequence targets the
protein to which it is attached across the inner membrane of the
bacterial cell. The DNA encoding the signal sequence may be
obtained as a restriction endonuclease fragment from any gene
encoding a protein that has a signal sequence. Suitable prokaryotic
signal sequences may be obtained from genes encoding, for example,
LamB or OmpF (Wong et al., Gene, 68:193 [1983]), MalE, PhoA and
other genes. A preferred prokaryotic signal sequence for practicing
this invention is the E. coli heat-stable enterotoxin II (STII)
signal sequence as described by Chang et al., Gene, 55: 189
[1987].
[0067] Another useful component of the vectors used to practice
this invention is phenotypic selection genes. Typical phenotypic
selection genes are those encoding proteins that confer antibiotic
resistance upon the host cell. By way of illustration, the
ampicillin resistance gene (amp), and the tetracycline resistance
gene (tet) are readily employed for this purpose.
[0068] Construction of suitable vectors comprising the
aforementioned components as well as the gene encoding the desired
polypeptide (gene 1) are prepared using standard recombinant DNA
procedures as described in Sambrook et al. supra. Isolated DNA
fragments to be combined to form the vector are cleaved, tailored,
and ligated together in a specific order and orientation to
generate the desired vector.
[0069] The DNA is cleaved using the appropriate restriction enzyme
or enzymes in a suitable buffer. In general, about 0.2-1 .mu.g of
plasmid or DNA fragments is used with about 1-2 units of the
appropriate restriction enzyme in about 20 .mu.l of buffer
solution. Appropriate buffers, DNA concentrations, and incubation
times and temperatures are specified by the manufacturers of the
restriction enzymes. Generally, incubation times of about one or
two hours at 37.degree. C. are adequate, although several enzymes
require higher temperatures. After incubation, the enzymes and
other contaminants are removed by extraction of the digestion
solution with a mixture of phenol and chloroform, and the DNA is
recovered from the aqueous fraction by precipitation with
ethanol.
[0070] To ligate the DNA fragments together to form a functional
vector, the ends of the DNA fragments must be compatible with each
other. In some cases, the ends will be directly compatible after
endonuclease digestion. However, it may be necessary to first
convert the sticky ends commonly produced by endonuclease digestion
to blunt ends to make them compatible for ligation. To blunt the
ends, the DNA is treated in a suitable buffer for at least 15
minutes at 15.degree. C. with 10 units of the Klenow fragment of
DNA polymerase I (Klenow) in the presence of the four
deoxynucleotide triphosphates. The DNA is then purified by
phenol-chloroform extraction and ethanol precipitation.
[0071] The cleaved DNA fragments may be size-separated and selected
using DNA gel electrophoresis. The DNA may be electrophoresed
through either an agarose or a polyacrylamide matrix. The selection
of the matrix will depend on the size of the DNA fragments to be
separated. After electrophoresis, the DNA is extracted from the
matrix by electroelution, or, if low-melting agarose has been used
as the matrix, by melting the agarose and extracting the DNA from
it, as described in sections 6.30-6.33 of Sambrook et al.,
supra.
[0072] The DNA fragments that are to be ligated together
(previously digested with the appropriate restriction enzymes such
that the ends of each fragment to be ligated are compatible) are
put in solution in about equimolar amounts. The solution will also
contain ATP, ligase buffer and a ligase such as T4 DNA ligase at
about 10 units per 0.5 .mu.g of DNA. If the DNA fragment is to be
ligated into a vector, the vector is at first linearized by cutting
with the appropriate restriction endonuclease(s). The linearized
vector is then treated with alkaline phosphatase or calf intestinal
phosphatase. The phosphatasing prevents self-ligation of the vector
during the ligation step.
[0073] After ligation, the vector with the foreign gene now
inserted is transformed into a suitable host cell. Prokaryotes are
the preferred host cells for this invention. Suitable prokaryotic
host cells include E. coli strain JM101, E. coli K12 strain 294
(ATCC number 31,446), E. coli strain W3110 (ATCC number 27,325), E.
coli X1776 (ATCC number 31,537), E. coli XL-1 Blue (stratagene),
and E. coli B; however many other strains of E. coli, such as
HB101, NM522, NM538, NM539, and many other species and genera of
prokaryotes may be used as well. In addition to the E. coli strains
listed above, bacilli such as Bacillus subtilis, other
enterobacteriaceae such as Salmonella typhimurium or Serratia
marcesans, and various Pseudomonas species may all be used as
hosts.
[0074] Transformation of prokaryotic cells is readily accomplished
using the calcium chloride method as described in section 1.82 of
Sambrook et al., supra. Alternatively, electroporation (Neumann et
al., EMBO J., 1:841 [1982]) may be used to transform these cells.
The transformed cells are selected by growth on an antibiotic,
commonly tetracycline (tet) or ampicillin (amp), to which they are
rendered resistant due to the presence of tet and/or amp resistance
genes on the vector.
[0075] After selection of the transformed cells, these cells are
grown in culture and the plasmid DNA (or other vector with the
foreign gene inserted) is then isolated. Plasmid DNA can be
isolated using methods known in the art. Two suitable methods are
the small scale preparation of DNA and the large-scale preparation
of DNA as described in sections 1.25-1.33 of Sambrook et al.,
supra. The isolated DNA can be purified by methods known in the art
such as that described in section 1.40 of Sambrook et al., supra.
This purified plasmid DNA is then analyzed by restriction mapping
and/or DNA sequencing. DNA sequencing is generally performed by
either the method of Messing et al. Nucleic Acids Res., 9:309
[1981] or by the method of Maxam et al. Meth. Enzymol., 65: 499
[1980].
IV. Gene Fusion
[0076] This invention contemplates fusing the gene enclosing the
desired polypeptide (gene 1) to a second gene (gene 2) such that a
fusion protein is generated during transcription. Gene 2 is
typically a coat protein gene of a phage, and preferably it is the
phage M13 gene III coat protein, or a fragment thereof. Fusion of
genes 1 and 2 may be accomplished by inserting gene 2 into a
particular site on a plasmid that contains gene 1, or by inserting
gene 1 into a particular site on a plasmid that contains gene
2.
[0077] Insertion of a gene into a plasmid requires that the plasmid
be cut at the precise location that the gene is to be inserted.
Thus, there must be a restriction endonuclease site at this
location (preferably a unique site such that the plasmid will only
be cut at a single location during restriction endonuclease
digestion). The plasmid is digested, phosphatased, and purified as
described above. The gene is then inserted into this linearized
plasmid by ligating the two DNAs together. Ligation can be
accomplished if the ends of the plasmid are compatible with the
ends of the gene to be inserted. If the restriction enzymes are
used to cut the plasmid and isolate the gene to be inserted create
blunt ends or compatible sticky ends, the DNAs can be ligated
together directly using a ligase such as bacteriophage T4 DNA
ligase and incubating the mixture at 16.degree. C. for 1-4 hours in
the presence of ATP and ligase buffer as described in section 1.68
of Sambrook et al., supra. If the ends are not compatible, they
must first be made blunt by using the Klenow fragment of DNA
polymerase I or bacteriophage T4 DNA polymerase, both of which
require the four deoxyribonucleotide triphosphates to fill-in
overhanging single-stranded ends of the digested DNA.
Alternatively, the ends may be blunted using a nuclease such as
nuclease S1 or mung-bean nuclease, both of which function by
cutting back the overhanging single strands of DNA. The DNA is then
religated using a ligase as described above. In some cases, it may
not be possible to blunt the ends of the gene to be inserted, as
the reading frame of the coding region will be altered. To overcome
this problem, oligonucleotide linkers may be used. The linkers
serve as a bridge to connect the plasmid to the gene to be
inserted. These linkers can be made synthetically as double
stranded or single stranded DNA using standard methods. The linkers
have one end that is compatible with the ends of the gene to be
inserted; the linkers are first ligated to this gene using ligation
methods described above. The other end of the linkers is designed
to be compatible with the plasmid for ligation. In designing the
linkers, care must be taken to not destroy the reading frame of the
gene to be inserted or the reading frame of the gene contained on
the plasmid. In some cases, it may be necessary to design the
linkers such that they code for part of an amino acid, or such that
they code for one or more amino acids.
[0078] Between gene 1 and gene 2, DNA encoding a termination codon
may be inserted, such termination codons are UAG (amber), UAA
(ocher) and UGA (opel). (Microbiology, Davis et al. Harper &
Row, New York, 1980, pages 237, 245-47 and 274). The termination
codon expressed in a wild type host cell results in the synthesis
of the gene 1 protein product without the gene 2 protein attached.
However, growth in a suppressor host cell results in the synthesis
of detectable quantities of fused protein. Such suppressor host
cells contain a tRNA modified to insert an amino acid in the
termination codon position of the mRNA thereby resulting in
production of detectable amounts of the fusion protein. Such
suppressor host cells are well known and described, such as E. coli
suppressor strain (Bullock et al., BioTechniques 5, 376-379
[1987]). Any acceptable method may be used to place such a
termination codon into the mRNA encoding the fusion
polypeptide.
[0079] The suppressible codon may be inserted between the first
gene encoding a polypeptide, and a second gene encoding at least a
portion of a phage coat protein. Alternatively, the suppressible
termination codon may be inserted adjacent to the fusion site by
replacing the last amino acid triplet in the polypeptide or the
first amino acid in the phage coat protein. When the phagemid
containing the suppressible codon is grown in a suppressor host
cell, it results in the detectable production of a fusion
polypeptide containing the polypeptide and the coat protein. When
the phagemid is grown in a non-suppressor host cell, the
polypeptide is synthesized substantially without fusion to the
phage coat protein due to termination at the inserted suppressible
triplet encoding UAG, UAA, or UGA. In the non-suppressor cell the
polypeptide is synthesized and secreted from the host cell due to
the absence of the fused phage coat protein which otherwise
anchored it to the host cell.
V. Alteration (Mutation) of Gene 1 at Selected Positions
[0080] Gene 1, encoding the desired polypeptide, may be altered at
one or more selected codons. An alteration is defined as a
substitution, deletion, or insertion of one or more codons in the
gene encoding the polypeptide that results in a change in the amino
acid sequence of the polypeptide as compared with the unaltered or
native sequence of the same polypeptide. Preferably, the
alterations will be by substitution of at least one amino acid with
any other amino acid in one or more regions of the molecule. The
alterations may be produced be a variety of methods known in the
art. These methods include but are not limited to
oligonucleotide-mediated mutagenesis and cassette mutagenesis.
[0081] A. Oligonucleotide-Mediated Mutagenesis
[0082] Oligonucleotide-mediated mutagenesis is preferred method for
preparing substitution, deletion, and insertion variants of gene 1.
This technique is well known in the art as described by Zoller et
al. Nucleic Acids Res. 10: 6487-6504 [1987]. Briefly, gene 1 is
altered by hybridizing an oligonucleotide encoding the desired
mutation to a DNA template, where the template is the
single-stranded form of the plasmid containing the unaltered or
native DNA sequence of gene 1. After hybridization, a DNA
polymerase is used to synthesize an entire second complementary
strand of the template will thus incorporate the oligonucleotide
primer, and will code for the selected alteration in gene 1.
[0083] Generally, oligonucleotides of at least 25 nucleotides in
length are used. An optimal oligonucleotide will have 12 to 15
nucleotides that are completely complementary to the template on
either side of the nucleotide(s) coding for the mutation. This
ensures that the oligonucleotide will hybridize properly to the
single-stranded DNA template molecule. The oligonucleotides are
readily synthesized using techniques known in the art such as that
described by Crea et al. Proc. Nat'l. Acad. Sci. USA, 75: 5765
[1978].
[0084] The DNA template can only be generated by those vectors that
are either derived from bacteriophage M13 vectors (the commercially
available M13mp18 and M13mp19 vectors are suitable), or those
vectors that contain a single-stranded phage origin of replication
as described by Viera et al. Meth. Enzymol., 153: 3 [1987]. Thus,
the DNA that is to be mutated must be inserted into one of these
vectors in order to generate single-stranded template. Production
of the single-stranded template is described in sections 4.21-4.41
of Sambrook et al., supra.
[0085] To alter the native DNA sequence, the oligonucleotide is
hybridized to the single stranded template under suitable
hybridization conditions. A DNA polymerizing enzyme, usually the
Klenow fragment of DNA polymerase I, is then added to synthesize
the complementary strand of the template using the oligonucleotide
as a primer for synthesis. A heteroduplex molecule is thus formed
such that one strand of DNA encodes the mutated form of gene 1, and
the other strand (the original template) encodes the native,
unaltered sequence of gene 1. This heteroduplex molecule is then
transformed into a suitable host cell, usually a prokaryote such as
E. Coli JM101. After growing the cells, they are plated onto
agarose plates and screened using the oligonucleotide primer
radiolabelled with 32-Phosphate to identify the bacterial colonies
that contain the mutated DNA.
[0086] The method described immediately above may be modified such
that a homoduplex molecule is created wherein both strands of the
plasmid contain the mutation(s). The modifications are as follows:
The single-stranded oligonucleotide is annealed to the
single-stranded template as described above. A mixture of three
deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine
(dGTP), and deoxyribothymidine (dTTP), is combined with a modified
thio-deoxyribocytosine called dCTP-(aS) (which can be obtained from
Amersham). This mixture is added to the template-oligonucleotide
complex. Upon addition of DNA polymerase to this mixture, a strand
of DNA identical to the template except for the mutated bases is
generated. In addition, this new strand of DNA will contain
dCTP-(aS) instead of dCTP, which serves to protect it from
restriction endonuclease digestion. After the template strand of
the double-stranded heteroduplex is nicked with an appropriate
restriction enzyme, the template strand can be digested with ExolII
nuclease or another appropriate nuclease past the region that
contains the site(s) to be mutagenized. The reaction is then
stopped to leave a molecule that is only partially single-stranded.
A complete double-stranded DNA homoduplex is then formed using DNA
polymerase in the presence of all four deoxyribonucleotide
triphosphates, ATP, and DNA ligase. This homoduplex molecule can
then be transformed into a suitable host cell such as E. coli
JM101, as described above.
[0087] Mutants with more than one amino acid to be substituted may
be generated in one of several ways. If the amino acids are located
close together in the polypeptide chain, they may be mutated
simultaneously using one oligonucleotide that codes for all of the
desired amino acid substitutions. If, however, the amino acids are
located some distance from each other (separated by more than about
ten amino acids), it is more difficult to generate a single
oligonucleotide that encodes all of the desired changes. Instead,
one of two alternative methods may be employed.
[0088] In the first method, a separate oligonucleotide is generated
for each amino acid to be substituted. The oligonucleotides are
then annealed to the single-stranded template DNA simultaneously,
and the second strand of DNA that is synthesized from the template
will encode all of the desired amino acid substitutions. The
alternative method involves two or more rounds of mutagenesis to
produce the desired mutant. The first round is as described for the
single mutants: wild-type DNA is used for the template, an
oligonucleotide encoding the first desired amino acid
substitution(s) is annealed to this template, and the heteroduplex
DNA molecule is then generated. The second round of mutagenesis
utilizes the mutated DNA produced in the first round of mutagenesis
as the template. Thus, this template already contains one or more
mutations. The oligonucleotide encoding the additional desired
amino acid substitution(s) is then annealed to this template, and
the resulting strand of DNA now encodes mutations from both the
first and second rounds of mutagenesis. This resultant DNA can be
used as a template in a third round of mutagenesis, and so on.
[0089] B. Cassette Mutagenesis
[0090] This method is also a preferred method for preparing
substitution, deletion, and insertion variants of gene 1. The
method is based on that described by Wells et al. Gene, 34:315
[1985]. The starting material is the plasmid (or other vector)
comprising gene 1, the gene to be mutated. The codon(s) in gene 1
to be mutated are identified. There must be a unique restriction
endonuclease site on each side of the identified mutation site(s).
If no such restriction sites exist, they may be generated using the
above-described oligonucleotide-mediated mutagenesis method to
introduce them at appropriate locations in gene 1. After the
restriction sites have been introduced into the plasmid, the
plasmid is cut at these sites to linearize it. A double-stranded
oligonucleotide encoding the sequence of the DNA between the
restriction sites but containing the desired mutation(s) is
synthesized using standard procedures. The two strands are
synthesized separately and then hybridized together using standard
techniques. This double-stranded oligonucleotide is referred to as
the cassette. This cassette is designed to have 3' and 5' ends that
are compatible with the ends of the linearized plasmid, such that
it can be directly ligated to the plasmid. This plasmid now
contains the mutated DNA sequence of gene 1.
VI. Obtaining DNA Encoding the Desired Protein
[0091] In an alternative embodiment, this invention contemplates
production of variants of a desired protein containing one or more
subunits. Each subunit is typically encoded by separate gene. Each
gene encoding each subunit can be obtained by methods known in the
art (see, for example, Section II). In some instances it may be
necessary to obtain the gene encoding the various subunits using
separate techniques selected from any of the methods described in
Section II.
[0092] When constructing a replicable expression vector where the
protein of interest contains more than one subunit, all subunits
can be regulated by the same promoter, typically located 5' to the
DNA encoding the subunits, or each may be regulated by separate
promoter suitably oriented in the vector so that each promoter is
operably linked to the DNA it is intended to regulate. Selection of
promoters is carried out as described in Section III above.
[0093] In constructing a replicable expression vector containing
DNA encoding the protein of interest having multiple subunits, the
reader is referred to FIG. 10 where, by way of illustration, a
vector is diagrammed showing DNA encoding each subunit of an
antibody fragment. This figure shows that, generally, one of the
subunits of the protein of interest will be fused to a phage coat
protein such as M13 gene III. This gene fusion generally will
contain its own signal sequence. A separate gene encodes the other
subunit or subunits, and it is apparent that each subunit generally
has its own signal sequence. FIG. 10 also shows that a single
promoter can regulate the expression of both subunits.
Alternatively, each subunit may be independently regulated by a
different promoter. The protein of interest subunit-phage coat
protein fusion construct can be made as described in Section IV
above.
[0094] When constructing a family of variants of the desired
multi-subunit protein, DNA encoding each subunit in the vector may
mutated in one or more positions in each subunit. When
multi-subunit antibody variants are constructed, preferred sites of
mutagenesis correspond to codons encoding amino acid residues
located in the complementarity-determining regions (CDR) of either
the light chain, the heavy chain, or both chains. The CDRs are
commonly referred to as the hypervariable regions. Methods for
mutagenizing DNA encoding each subunit of the protein of interest
are conducted essentially as described in Section V above.
VII. Preparing a Target Molecule and Binding with Phagemid
[0095] Target proteins, such as receptors, may be isolated from
natural sources or prepared by recombinant methods by procedures
known in the art. By way of illustration, glycoprotein hormone
receptors may be prepared by the technique described by McFarland
et al., Science 245:494-499 [1989], nonglycosylated forms expressed
in E. coli are described by Fuh et al. J. Biol. Chem 265:3111-3115
[1990] Other receptors can be prepared by standard methods.
[0096] The purified target protein may be attached to a suitable
matrix such as agarose beads, acrylamide beads, glass beads,
cellulose, various acrylic copolymers, hydroxylalkyl methacrylate
gels, polyacrylic and polymethacrylic copolymers, nylon, neutral
and ionic carriers, and the like. Attachment of the target protein
to the matrix may be accomplished by methods described in Methods
in Enzymology 44 [1976], or by other means known in the art.
[0097] After attachment of the target protein to the matrix, the
immobilized target is contacted with the library of phagemid
particles under conditions suitable for binding of at least a
portion of the phagemid particles with the immobilized target.
Normally, the conditions, including pH, ionic strength, temperature
and the like will mimic physiological conditions.
[0098] Bound phagemid particles ("binders") having high affinity
for the immobilized target are separated from those having a low
affinity (and thus do not bind to the target) by washing. Binders
may be dissociated from the immobilized target by a variety of
methods. These methods include competitive dissociation using the
wild-type ligand, altering pH and/or ionic strength, and methods
known in the art.
[0099] Suitable host cells are infected with the binders and helper
phage, and the host cells are cultured under conditions suitable
for amplification of the phagemid particles. The phagemid particles
are then collected and the selection process is repeated one or
more times until binders having the desired affinity for the target
molecule are selected.
[0100] Optionally the library of phagemid particles may be
sequentially contacted with more than one immobilized target to
improve selectivity for a particular target. For example, it is
often the case that a ligand such as hGH has more than one natural
receptor. In the case of hGH, both the growth hormone receptor and
the prolactin receptor bind the hGH ligand. It may be desirable to
improve the selectivity of hGH for the growth hormone receptor over
the prolactin receptor. This can be achieved by first contacting
the library of phagemid particles with immobilized prolactin
receptor, eluting those with a low affinity (i.e. lower than wild
type hGH) for the prolactin receptor and then contacting the low
affinity prolactin "binders" or non-binders with the immobilized
growth hormone receptor, and selecting for high affinity growth
hormone receptor binders. In this case an hGH mutant having a lower
affinity for the prolactin receptor would have therapeutic utility
even if the affinity for the growth hormone receptor were somewhat
lower than that of wild type hGH. This same strategy may be
employed to improve selectivity of a particular hormone or protein
for its primary function receptor over its clearance receptor.
[0101] In another embodiment of this invention, an improved
substrate amino acid sequence can be obtained. These may be useful
for making better "cut sites" for protein linkers, or for better
protease substrates/inhibitors. In this embodiment, an
immobilizable molecule (e.g. hGH-receptor, biotin-avidin, or one
capable of covalent linkage with a matrix) is fused to gene III
through a linker. The linker will preferably be from 3 to 10 amino
acids in length and will act as a substrate for a protease. A
phagemid will be constructed as described above where the DNA
encoding the linker region is randomly mutated to produce a
randomized library of phagemid particles with different amino acid
sequences at the linking site. The library of phagemid particles
are then immobilized on a matrix and exposed to a desired protease.
Phagemid particles having preferred or better substrate amino acid
sequences in the liner region for the desired protease will be
eluted, first producing an enriched pool of phagemid particles
encoding preferred linkers. These phagemid particles are then
cycled several more times to produce an enriched pool of particles
encoding consense sequence(s) (see examples XIII and XIV).
VIII. Growth Hormone Variants and Methods of Use
[0102] The cloned gene for hGH has been expressed in a secreted
form in Eschericha coli (Chang, C. N>, et al., [1987] Gene 55,
189) and its DNA and amino acid sequence has been reported
(Goeddel, et al. [1979] Nature 281, 544; Gray et al., [1985] Gene
39, 247). The present invention describes novel hGH variants
produced using the phagemid selection methods. Human growth hormone
variants containing substitutions at positions 10, 14, 18, 21, 167,
171, 172, 174, 175, 176, 178 and 179 have been described. Those
having higher binding affinities are described in Tables VII, XIII
and XIV. The amino acid nomenclature for describing the variants is
shown below. Growth hormone variants may be administered and
formulated in the same manner as regular growth hormone. The growth
hormone variants of the present invention may be expressed in any
recombinant system which is capable of expressing native or met
hGH.
[0103] Therapeutic formulations of hGH for therapeutic
administration are prepared for storage by mixing hGH having the
desired degree of purity with optional physiologically acceptable
carriers, excipients, or stabilizers (Remington's Pharmaceutical
Sciences, 16th edition, Osol, A., Ed., (1980), in the form of
lyophilized cake or aqueous solutions. Acceptable carriers,
excipients or stabilizers are nontoxic to recipients at the dosages
and concentrations employed, and include buffers such as phosphate,
citrate, and other organic acids; antioxidants including ascorbic
acid; low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine, or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; divalent metal ions such as zinc, cobalt or copper; sugar
alcohols such as mannitol or sorbitol; salt-forming counterions
such as sodium; and/or nonionic surfactants such as Tween,
Pluronics or polyethylene glycol (PEG). Formulations of the present
invention may additionally contain a pharmaceutically acceptable
buffer, amino acid, bulking agent and/or non-ionic surfactant.
These include, for example, buffers, chelating agents,
antioxidants, preservatives, cosolvents, and the like; specific
examples of these could include, trimethylamaine salts ("Tris
buffer"), and disodium edetate. The phagemids of the present
invention may be used to produce quantities of the hGH variants
free of the phage protein. To express hGH variants free of the gene
III portion of the fusion, pS0643 and derivatives can simply be
grown in a non-suppressor strain such as 16C9. In this case, the
amber codon (TAG) leads to termination of translation, which yields
free hormone, without the need for an independent DNA construction.
The hGH variant is secreted from the host and may be isolated from
the culture medium.
[0104] One or more of the eight hGH amino acids F10, M14, H18, H21,
R167, D171, T175 and I179 may be replaced by any amino acid other
than the one found in that position in naturally occurring hGH as
indicated. Therefore, 1, 2, 3, 4, 5, 6, 7, or all 8 of the
indicated amino acids, F10, M14, H18, H21, R167, D171, T175 and
I179, may be replaced by any of the other 19 amino acids out of the
20 amino acids listed below. In a preferred embodiment, all eight
listed amino acids are replaced by another amino acid. The most
preferred eight amino acids to be substituted are indicated in
Table XIV in Example XII.
Amino Acid Nomenclature
[0105] Ala (A) [0106] Arg (R) [0107] Asn (N) [0108] Asp (D) [0109]
Cys (C) [0110] Gln (Q) [0111] Glu (E) [0112] Gly (G) [0113] His (H)
[0114] Ile (I) [0115] Leu (L) [0116] Lys (K) [0117] Met (M) [0118]
Phe (F) [0119] Pro (P) [0120] Ser (S) [0121] Thr (T) [0122] Trp (W)
[0123] Tyr (Y) [0124] Val (V) The one letter hGH variant
nomenclature first gives the hGH amino acid deleted, for example
glutamate 179; then the amino acid inserted; for example, serine;
resulting in (E1795S).
EXAMPLES
[0125] Without further description, it is believed that one of
ordinary skill in the art can, using the preceding description and
illustrative examples, make and utilize the present invention to
the fullest extent. The following working examples therefore
specifically point out preferred embodiments of the present
invention, and are not to be construed as limiting in any way of
the remainder of the disclosure.
Example I
Plasmid Constructions and Preparation of hGH-Phagemid Particles
[0126] The plasmid phGH-M13gIII (FIG. 1), was constructed from
M13KO7.sup.7 and the hGH producing plasmid, pBO473 (Cunningham, B.
C., et al., Science, 243:1330-1336, [1989]). A synthetic
oligonucleotide
5'-AGC-TGT-GGC-TTC-GGG-CCC-TTA-GCA-TTT-AAT-GCG-GTA-3' (SEQ ID NO:2)
was used to introduce a unique ApaI restriction site (underlined)
into pBO473 after the final Phe191 codon of hGH. The
oligonucleotide 5'-TTC-ACA-AAC-GAA-GGG-CCC-CTA-ATT-AAA-GCC-AGA-3'
(SEQ ID NO:3) was used to introduce a unique ApaI restriction site
(underlined), and a Glu197-to-amber stop codon (bold lettering)
into M13KO7 gene III. The oligonucleotide
5'-CAA-TAA-TAA-CGG-GCT-AGC-CAA-AAG-AAC-TGG-3' (SEQ ID NO:4)
introduces a unique NheI site (underlined) after the 3' end of the
gene III coding sequence. The resulting 650 base pair (bp)
ApaI-NheI fragment from the doubly mutated M13KO7 gene III was
cloned into the large ApaI-NheI fragment of pBO473 to create the
plasmid, pSO132. This fuses the carboxyl terminus of hGH (Phe191)
to the Pro198 residue of the gene III protein with the insertion of
a glycine residue encoded from the ApaI site and places the fusion
protein under control of the E. coli alkaline phosphatase (phoA)
promoter and stII secretion signal sequence (Chang, C. N., et al.,
Gene, 55:189-196, [1987]). For inducible expression of the fusion
protein in rich media, we replaced the phoA promoter with the lac
promoter and operator. A 138 bp EcoRI-XbaI fragment containing the
lac promoter, operator, and Cap binding site was produced by PCR of
plasmid pUC119 using the oligonucleotides
5'-CACGACAGAATTCCCGACTGGAAA-3' (SEQ ID NO:5) and 5'-CTGTT
TCTAGAGTGAAATTGTTA-3' (SEQ ID NO:6) that flank the desired lac
sequences and introduce the EcoRI and XbaI restriction sites
(underlined). This lac fragment was gel purified and ligated into
the large EcoRI-XbaI fragment of pSO132 to create the plasmid,
phGH-M13gIII. The sequences of all tailored DNA junctions were
verified by the dideoxy sequence method (Sanger, F., et al. Proc.
Natl. Acad. Sci. U.S.A. 74:5463-5467, [1977]). The R64A variant hGH
phagemid was constructed as follows: the NsiI-BglII mutated
fragment of hGH (Cunningham et al. supra) encoding the Arg64 to Ala
substitution (R64A) (Cunningham, B. C., Wells, J. A., Science,
244:1081-1085, [1989]) was cloned between the corresponding
restriction sites in the phGH-M13gIII plasmid (FIG. 1) to replace
the wild-type hGH sequence. The R64A hGH phagemid particles were
propagated and titered as described below for the wild-type
hGH-phagemid.
[0127] Plasmids were transformed into a male strain of E. coli
(JM101) and selected on carbenicillin plates. A single transformant
was grown in 2 ml 2YT medium for 4 h at 37.degree. C. and infected
with 50 .mu.l of M13KO7 helper phage. The infected culture was
diluted into 30 ml 2YT, grown overnight, and phagemid particles
were harvested by precipitation with polyethylene glycol (Vierra,
J., Messing, J., Methods in Enzymology, 153:3-11, [1987]). Typical
phagemid particle titers ranged from 2 to 5.times.10.sup.11 cfu/ml.
The particles were purified to homogeneity by CsCl density
centrifugation (Day, L. A. J. Mol. Biol., 39:265-277, [1969]) to
remove any fusion protein not attached to virions.
Example II
Immunochemical Analyses of hGH on the Fusion Phage
[0128] Rabbit polyclonal antibodies to hGH were purified with
protein A, and coated onto microtiter plates (Nunc) at a
concentration of 2 .mu.g/ml in 50 mM sodium carbonate buffer (pH
10) at 4.degree. C. for 16-20 hours. After washing in PBS
containing 0.05% Tween 20, hGH or hGH-phagemid particles were
serially diluted from 2.0-0.002 nM in buffer A (50 mM Tris (pH
7.5), 50 mM NaCl, 2 mM EDTA, 5 mg/ml bovine serum albumin, and
0.05% Tween 20). After 2 hours at room temperature (rt), the plates
were washed well and the indicated Mab (Cunningham et al. supra)
was added at 1 .mu.g/ml in buffer A for 2 hours at rt. Following
washing, horseradish peroxidase conjugated goat anti-mouse IgG
antibody was bound at rt for 1 hour. After a final wash, the
peroxidase activity was assayed with the substrate,
o-phenylenediamine.
Example III
Coupling of the hGH Binding Protein to Polyacrylamide Beads and
Binding Enrichments
[0129] Oxirane polyacrylamide beads (Sigma) were conjugated to the
purified extracellular domain of the hGH receptor (hGHbp) (Fuh, G.,
et al., J. Biol. Chem., 265:3111-3115 [1990]) containing an extra
cysteine residue introduced by site-directed mutagenesis at
position 237 that does not affect binding of hGH (J. Wells,
unpublished). The hGHbp was conjugated as recommended by the
supplier to a level of 1.7 pmol hGHbp/mg dry oxirane bead, as
measured by binding of [.sup.125I] hGH to the resin. Subsequently,
any unreacted oxirane groups were blocked with BSA and Tris. As a
control for non-specific binding of phagemid particles, BSA was
similarly coupled to the beads. Buffer for adsorption and washing
contained 10 mM Tris.HCl (pH 7.5), 1 mM EDTA, 50 mM NaCl, 1 mg/ml
BSA, and 0.02% Tween 20. Elution buffers contained wash buffer plus
200 nM hGH or 0.2 M glycine (pH 2.1). Parental phage M13KO7 was
mixed with hGH phagemid particles at a ratio of nearly 3000:1
(original mixture) and tumbled for 8-12 h with a 5 .mu.l aliquot
(0.2 mg of acrylamide beads) of either absorbent in a 50 .mu.l
volume at room temperature. The beads were pelleted by
centrifugation and the supernate carefully removed. The beads were
resuspended in 200 .mu.l wash buffer and tumbled at room
temperature for 4 hours (wash 1). After a second wash (wash 2), the
beads were eluted twice with 200 nM hGH for 6-10 hours each (eluate
1, eluate 2). The final elution was with a glycine buffer (pH 2.1)
for 4 hours to remove remaining hGH phagemid particles (eluate 3).
Each fraction was diluted appropriately in 2YT media, mixed with
fresh JM101, incubated at 37.degree. C. for 5 minutes, and plated
with 3 ml of 2YT soft agar on LB or LB carbenicillin plates.
Example IV
Construction of hGH-Phagemid Particles with a Mixture of Gene III
Products
[0130] The gene III protein is composed of 410 residues divided
into two domains that are separated by a flexible linker sequence
(Armstrong, J., et al., FEBS Lett., 135:167-172, [1981]). The
amino-terminal domain is required for attachment to the pili of E.
coli, while the carboxyl-terminal domain is imbedded in the phage
coat and required for proper phage assembly (Crissman, J. W.,
Smith, G. P., Virology, 132:445-455, [1984]). The signal sequence
and amino-terminal domain of gene III was replaced with the stII
signal and entire hGH gene (Chang et al. supra) by fusion to
residue 198 in the carboxyl-terminal domain of gene III (FIG. 1).
The hGH-gene III fusion was placed under control of the lac
promoter/operator in a plasmid (phGH-M13gIII; FIG. 1) containing
the pBR322 .beta.-lactamase gene and Col E1 replication origin, and
the phage f1 intergenic region. The vector can be easily maintained
as a small plasmid vector by selection on carbenicillin, which
avoids relying on a functional gene III fusion for propagation.
Alternatively, the plasmid can be efficiently packaged into virions
(called phagemid particles) by infection with helper phage such as
M13KO7 (Viera et al. supra) which avoids problems of phage
assembly. Phagemid infectivity titers based upon transduction to
carbenicillin resistance in this system varied from
2-5.times.10.sup.11 colony forming units (cfu)/ml. The titer of the
M13KO7 helper phage in these phagemid stocks is .about.10.sup.10
plaque forming units (pfu)/ml.
[0131] With this system we confirmed previous studies (Parmley,
Smith supra) that homogeneous expression of large proteins fused to
gene III is deleterious to phage production (data not shown). For
example, induction of the lac promoter in phGH-M13gIII by addition
of IPTG produced low phagemid titers. Moreover, phagemid particles
produced by co-infection with M13KO7 containing an amber mutation
in gene III gave very low phagemid titers (<10.sup.10 cfu/ml).
We believed that multiple copies of the gene III fusion attached to
the phagemid surface could lead to multiple point attachment (the
"chelate effect") of the fusion phage to the immobilized target
protein. Therefore to control the fusion protein copy number we
limited transcription of the hGH-gene III fusion by culturing the
plasmid in E. coli JM101 (lacI.sup.Q) which contains a
constitutively high level of the lac repressor protein. The E. coli
JM101 cultures containing phGH-M13gIII were best propagated and
infected with M13KO7 in the absence of the lac operon inducer
(IPTG); however, this system is flexible so that co-expression of
other gene III fusion proteins can be balanced. We estimate that
about 10% of the phagemid particles contain one copy of the hGH
gene III fusion protein from the ratio of the amount of hGH per
virion (based on hGH immuno-reactive material in CsCl gradient
purified phagemid). Therefore, the titer of fusion phage displaying
the hGH gene III fusion is about 2-5.times.10.sup.10/ml. This
number is much greater than the titer of E. coli (.about.10.sup.8
to 10.sup.9/ml) in the culture from which they are derived. Thus,
on average every E. coli cell produces 10-100 copies of phage
decorated with an hGH gene III fusion protein.
Example V
Structural Integrity of the hGH-Gene III Fusion
[0132] Immunoblot analysis (FIG. 2) of the hGH-gene III phagemid
show that hGH cross-reactive material comigrates with phagemid
particles in agarose gels. This indicates that the hGH is tightly
associated with phagemid particles. The hGH-gene III fusion protein
from the phagemid particles runs as a single immuno-stained band
showing that there is little degradation of the hGH when it is
attached to gene III. Wild-type gene III protein is clearly present
because about 25% of the phagemid particles are infectious. This is
comparable to specific infectivity estimates made for wild-type M13
phage that are similarly purified (by CsCl density gradients) and
concentrations estimated by UV absorbance (Smith, G. P. supra and
Parmley, Smith supra) Thus, both wild-type gene III and the
hGH-gene III fusion proteins are displayed in the phage pool.
[0133] It was important to confirm that the tertiary structure of
the displayed hGH was maintained in order to have confidence that
results from binding selections will translate to the native
protein. We used monoclonal antibodies (Mabs) to hGH to evaluate
the structural integrity of the displayed hGH gene III fusion
protein (Table I).
TABLE-US-00001 TABLE I Binding of Eight Different Monoclonal
Antibodies (Mab's) to hGH and hGH Phagemid Particles* IC.sub.50
(nM) Mab hGH hGH-phagemid 1 0.4 0.4 2 0.04 0.04 3 0.2 0.2 4 0.1 0.1
5 0.2 >2.0 6 0.07 0.2 7 0.1 0.1 8 0.1 0.1 *Values given
represent the concentration (nM) of hGH or hGH-phagemid particles
to give half-maximal binding to the particular Mab. Standard errors
in these measurements are typically at or below .+-.30% of the
reported value. See Materials and Methods for further details.
[0134] The epitopes on hGH for these Mabs have been mapped
(Cunningham et al. supra) and binding for 7 of 8 Mabs requires that
hGH be properly folded. The IC.sub.50 values for all Mabs were
equivalent to wild-type hGH except for Mab 5 and 6. Both Mabs 5 and
6 are known to have binding determinants near the carboxyl-terminus
of hGH which is blocked in the gene III fusion protein. The
relative IC.sub.50 value for Mab1 which reacts with both native and
denatured hGH is unchanged compared to the conformationally
sensitive Mabs 2-5, 7 and 8. Thus, Mab1 serves as a good internal
control for any errors in matching the concentration of the hGH
standard to that of the hGH-gene III fusion.
Example VI
Binding Enrichments on Receptor Affinity Beads
[0135] Previous workers (Parmley, Smith supra; Scott, Smith supra;
Cwirla et al. supra; and Devlin et al. supra) have fractionated
phage by panning with streptavidin coated polystyrene petri dishes
or microtiter plates. However, chromatographic systems would allow
more efficient fractionation of phagemid particles displaying
mutant proteins with different binding affinities. We chose
non-porous oxirane beads (Sigma) to avoid trapping of phagemid
particles in the chromatographic resin. Furthermore, these beads
have a small particle size (1 .mu.m) to maximize the surface area
to mass ratio. The extracellular domain of the hGH receptor (hGHbp)
(Fuh et al., supra) containing a free cysteino residue was
efficiently coupled to these beads and phagemid particles showed
very low non-specific binding to beads coupled only to bovine serum
albumin (Table II).
TABLE-US-00002 TABLE II Specific Binding of Hormone Phage to
hGHbp-coated Beads Provides an Enrichment for hGH-phage over M13KO7
Phage* Sample Absorbent.sup..dagger-dbl. Total pfu Total cfu Ratio
(cfu/pfu) Enrichment.sup..sctn. Original mixture.sup..dagger. 8.3
.times. 10.sup.11 2.9 .times. 10.sup.8 3.5 .times. 10.sup.-4 (1)
Supernatant BSA 7.4 .times. 10.sup.11 2.8 .times. 10.sup.8 3.8
.times. 10.sup.-4 1.1 hGHbp 7.6 .times. 10.sup.11 3.3 .times.
10.sup.8 4.3 .times. 10.sup.-4 1.2 Wash 1 BSA 1.1 .times. 10.sup.10
6.0 .times. 10.sup.6 5.5 .times. 10.sup.-4 1.6 hGHbp 1.9 .times.
10.sup.10 1.7 .times. 10.sup.7 8.9 .times. 10.sup.-4 2.5 Wash 2 BSA
5.9 .times. 10.sup.7 2.8 .times. 10.sup.4 4.7 .times. 10.sup.-4 1.3
hGHbp 4.9 .times. 10.sup.7 2.7 .times. 10.sup.6 5.5 .times.
10.sup.-2 1.6 .times. 10.sup.2 Eluate 1 (hGH) BSA 1.1 .times.
10.sup.6 1.9 .times. 10.sup.3 1.7 .times. 10.sup.-3 4.9 hGHbp 1.2
.times. 10.sup.6 2.1 .times. 10.sup.6 1.8 5.1 .times. 10.sup.3
Eluate 2 (hGH) BSA 5.9 .times. 10.sup.5 1.2 .times. 10.sup.3 2.0
.times. 10.sup.-3 5.7 hGHbp 5.5 .times. 10.sup.5 1.3 .times.
10.sup.6 2.4 6.9 .times. 10.sup.3 Eluate 3 (pH 2.1) BSA 4.6 .times.
10.sup.5 2.0 .times. 10.sup.3 4.3 .times. 10.sup.-3 12.3 hGHbp 3.8
.times. 10.sup.5 4.0 .times. 10.sup.6 10.5 3.0 .times. 10.sup.4
*The titers of M13KO7 and hGH-phagemid particles in each fraction
was determined by multiplying the number of plaque forming units
(pfu) or carbenicillin resistant colony forming units (cfu) by the
dilution factor, respectively. See Example IV for details.
.sup..dagger.The ratio of M13KO7 to hGH-phagemid particles was
adjusted to 3000:1 in the original mixture.
.sup..dagger-dbl.Absorbents were conjugated with BSA or hGHbp.
.sup..sctn.Enrichments are calculated by dividing the cfu/pfu ratio
after each step by cfu/pfu ratio in the original mixture.
[0136] In a typical enrichment experiment (Table II), one part of
hGH phagemid was mixed with >3,000 parts M13KO7 phage. After one
cycle of binding and elution, 10.sup.6 phage were recovered and the
ratio of phagemid to M13KO7 phage was 2 to 1. Thus, a single
binding selection step gave >5000-fold enrichment. Additional
elutions with free hGH or acid treatment to remove remaining
phagemids produced even greater enrichments. The enrichments are
comparable to those obtained by Smith and coworkers using batch
elution from coated polystyrene plates (Smith, G. P. supra and
Parmely, Smith supra) however much smaller volumes are used on the
beads (200 .mu.l vs. 6 ml). There was almost no enrichment for the
hGH phagemid over M13KO7 when we used beads linked only to BSA. The
slight enrichment observed for control beads (.about.10-fold for pH
2.1 elution; Table 2) may result from trace contaminants of bovine
growth hormone binding protein present in the BSA linked to the
bead. Nevertheless these data show the enrichments for the hGH
phage depend upon the presence of the hGHbp on the bead suggesting
binding occurs by specific interaction between hGH and the
hGHbp.
[0137] We evaluated the enrichment for wild-type hGH over a weaker
binding variant of the hGH on fusion phagemids to further
demonstrate enrichment specificity, and to link the reduction in
binding affinity for the purified hormones to enrichment factors
after panning fusion phagemids. A fusion phagemid was constructed
with an hGH mutant in which Arg64 was substituted with Ala (R64A).
The R64A variant hormone is about 20-fold reduced in receptor
binding affinity compared to hGH (Kd values of 7.1 nM and 0.34 nM,
respectively [Cunningham, Wells, supra]). The titers of the R64A
hGH-gene III fusion phagemid were comparable to those of wild-type
hGH phagemid. After one round of binding and elution (Table III)
the wild-type hGH phagemid was enriched from a mixture of the two
phagemids plus M13KO7 by 8-fold relative to the phagemid R64A, and
.about.10.sup.4 relative to M13KO7 helper phage.
TABLE-US-00003 TABLE III hGHbp-coated Beads Select for hGH
Phagemids Over a Weaker Binding hGH Variant Phagemid Control beads
hGHbp beads WT phagemid enrichment WT phagemid enrichment Sample
total phagemid for WT/R64A total phagemid for WT/R64A Original
mixture 8/20 (1) 8/20 (1) Supernatant ND -- 4/10 1.0 Elution 1
(hGH) 7/20 0.8 17/20 .sup. 8.5.sup..dagger-dbl. Elution 2 (pH 2.1)
11/20 1.8 21/27 5.2 *The parent M13KO7 phage, wild-type hGH
phagemid and R64A phagemid particles were mixed at a ratio of
10.sup.4:0.4:0.6. Binding selections were carried out using beads
linked with BSA (control beads) or with the hGHbp (hGHbp beads) as
described in Table II and the Materials and Methods After each
step, plasmid DNA was isolated (Birnboim, H. C., Doly, J., Nucleic
Acids Res., 7: 1513-1523, [1979]) from carbenicillin resistant
colonies and analyzed by restriction analysis to determine if it
contained the wild-type hGH or the R64A hGH gene III fusion.
.sup..dagger.The enrichment for wild-type hGH phagemid over R64A
mutant was calculated from the ratio of hGH phagemid present after
each step to that present in the original mixture (8/20), divided
by the corresponding ratio for R64A phagemids. WT = wild-type; ND =
not determined. .sup..dagger-dbl.The enrichment for phagemid over
total M13KO7 parental phage was ~10.sup.4 after this step.
CONCLUSIONS
[0138] By displaying a mixture of wild-type gene III and the gene
III fusion protein on phagemid particles one can assemble and
propagate virions that display a large and proper folded protein as
a fusion to gene III. The copy number of the gene III fusion
protein can be effectively controlled to avoid "chelate effects"
yet maintained at high enough levels in the phagemid pool to permit
panning of large epitope libraries (>10.sup.10). We have shown
that hGH (a 22 kD protein) can be displayed in its native folded
form. Binding selections performed on receptor affinity beads
eluted with free hGH, efficiently enriched for wild-type hGH
phagemids over a mutant hGH phagemid shown to have reduced receptor
binding affinity. Thus, it is possible to sort phagemid particles
whose binding constants are down in the nanomolar range.
[0139] Protein-protein and antibody-antigen interactions are
dominated by discontinuous epitopes (Janin, J., et al., J. Mol.
Biol., 204:155-164, [1988]; Argos, P., Prot. Eng., 2:101-113,
[1988]; Barlow, D. J., et al., Nature, 322:747-748, [1987]; and
Davies, D. R., et al., J. Biol. Chem., 263:10541-10544, [1988]);
that is the residues directly involved in binding are close in
tertiary structure but separated by residues not involved in
binding. The screening system presented here should allow one to
analyze more conveniently protein-receptor interactions and isolate
discontinuous epitopes in proteins with new and high affinity
binding properties.
Example VII
Selection of hGH Mutants from a Library Randomized at hGH Codons
172, 174, 176, 178
Construction of Template
[0140] A mutant of the hGH-gene III fusion protein was constructed
using the method of Kunkel, et al. Meth. Enzymol. 154, 367-382
[1987]. Template DNA was prepared by growing the plasmid pS0132
(containing the natural hGH gene fused to the carboxy-terminal half
of M13 gene III, under control of the alkaline phosphatase
promoter) in CJ236 cells with M13-K07 phage added as helper.
Single-stranded, uracil-containing DNA was prepared for mutagenesis
to introduce (1) a mutation in hGH which would greatly reduce
binding to the hGH binding protein (hGHbp); and (2) a unique
restriction site (KpnI) which could be used for assaying for--and
selecting against--parental background phage.
Oligonucleotide-directed mutagenesis was carried out using T7 DNA
polymerase and the following oligodeoxy-nucleotide (SEQ ID
NO:7):
TABLE-US-00004 hGH codon: Gly Thr 178 179 5'-G ACA TTC CTG GGT ACC
GTG CAG T-3' < KpnI >
[0141] This oligo introduces the KpnI site as shown, along with
mutations (R178G, I179T) in hGH. These mutations are predicted to
reduce binding of hGH to hGHbp by more than 30-fold. Clones from
the mutagenesis were screened by KpnI digestion and confirmed by
dideoxy DNA sequencing. The resulting construct, to be used as a
template for random mutagenesis, was designated pHO415.
Random Mutagenesis within Helix-4 of hGH
[0142] Codons 172, 174, 176, 178 were targeted for random
mutagenesis in hGH, again using the method of Kunkel.
Single-stranded template from pH0415 was prepared as above and
mutagenesis was carried out using the following pool of oligos (SEQ
ID NO:8):
TABLE-US-00005 hGH codon: 172 174 5'-GC TTC AGG AAG GAC ATG GAC NNS
GTC NNS ACA-- Ile 176 178 179 NNS CTG NNS ATC GTG CAG TGC CGC TCT
GTG G-3'
As shown, this oligo pool reverts codon 179 to wild-type (Ile),
destroys the unique KpnI site of pH0415, and introduces random
codons (NNS, where N=A, G, C, or T and S=G or C) at positions 172,
174, 176, and 178. Using this codon selection in the context of the
above sequence, no additional KpnI sites can be created. The choice
of the NNS degenerate sequence yields 32 possible codons (including
one "stop" codon, and at least one codon for each amino acid) at 4
sites, for a total of (32).sup.4=1,048,576 possible nucleotide
sequences (12% of which contain at least one stop codon), or
(20).sup.4=160,000 possible polypeptide sequences plus 34,481
prematurely terminated sequences (i.e. sequences containing at
least one stop codon).
Propagation of the Initial Library
[0143] The mutagenesis products were extracted twice with
phenol:chloroform (50:50) and ethanol precipitated with an excess
of carrier tRNA to avoid adding salt that would confound the
subsequent electroporation step. Approximately 50 ng (15 fmols) of
DNA was electroporated into WJM101 cells (2.8.times.10.sup.10
cells/mL) in 45 .mu.L total volume in a 0.2 cm cuvette at a voltage
setting of 2.49 kV with a single pulse (time constant=4.7
msec.).
[0144] The cells were allowed to recover 1 hour at 37.degree. C.
with shaking, then mixed with 25 mL 2YT medium, 100 .mu.g/mL
carbenicillin, and M13-K07 (multiplicity of infection=1000).
Plating of serial dilutions from this culture onto
carbenicillin-containing media indicated that 8.2.times.10.sup.6
electrotransformants were obtained. After 10' at 23.degree. C., the
culture was incubated overnight (15 hours) at 37.degree. C. with
shaking.
[0145] After overnight incubation, the cells were pelleted, and
double-stranded DNA (dsDNA), designated pLIB1, was prepared by the
alkaline lysis method. The supernatant was spun again to remove any
remaining cells, and the phage, designated phage pool .phi.1, were
PEG-precipitated and resuspended in 1 mL STE buffer (10 mM Tris, pH
7.6, 1 mM EDTA, 50 mM NaCl). Phage titers were measured as
colony-forming units (CFU) for the recombinant phagemid containing
hGH-g3p gene III fusion (hGH-g.sup.3) plasmid, and plaque-forming
units (PFU) for the M13-K07 helper phage.
Binding Selection Using Immobilized hGHbp
[0146] 1. BINDING: An aliquot of phage pool .phi.1
(6.times.10.sup.9 CFU, 6.times.10.sup.7 PFU) was diluted 4.5-fold
in buffer A (Phosphate-buffered saline, 0.5% BSA, 0.05% Tween-20,
0.01% thimerosal) and mixed with a 5 .mu.L suspension of
oxirane-polyacrylamide beads coupled to the hGHbp containing a
Ser237 Cys mutation (350 fmols) in a 1.5 mL silated polypropylene
tube. As a control, an equivalent aliquot of phage were mixed in a
separate tube with beads that had been coated with BSA only. The
phage were allowed to bind to the beads by incubating 3 hours at
room temperature (23.degree. C.) with slow rotation (approximately
7 RPM). Subsequent steps were carried out with a constant volume of
200 .mu.L and at room temperature.
[0147] 2. WASH: The beads were spun 15 sec., and the supernatant
was removed (Sup. 1). To remove phage/phagemid not specifically
bound, the beads were washed twice by resuspending in buffer A,
then pelleting. A final wash consisted of rotating the beads in
buffer A for 2 hours.
[0148] 3. hGH ELUTION: Phage/phagemid binding weakly to the beads
were removed by stepwise elution with hGH. In the first step, the
beads were rotated with buffer A containing 2 nM hGH. After 17
hours, the beads were pelleted and resuspended in buffer A
containing 20 nM hGH and rotated for 3 hours, then pelleted. In the
final hGH wash, the beads were suspended in buffer A containing 200
nM hGH and rotated for 3 hours then pelleted.
[0149] 4. GLYCINE ELUTION: To remove the tightest-binding phagemid
(i.e. those still bound after the hGH washes), beads were suspended
in Glycine buffer (1 M Glycine, pH 2.0 with HCl), rotated 2 hours
and pelleted. The supernatant (fraction "G"; 200 .mu.L) was
neutralized by adding 30 .mu.L of 1 M Tris base.
[0150] Fraction G eluted from the hGHbp-beads (1.times.10.sup.6
CFU, 5.times.10.sup.4 PFU) was not substantially enriched for
phagemid over K07 helper phage. We believe this resulted from the
fact that K07 phage packaged during propagation of the recombinant
phagemid display the hGH-g3p fusion.
[0151] However, when compared with fraction G eluted from the
BSA-coated control beads, the hGHbp-beads yielded 14 times as many
CFU's. This reflects the enrichment of tight-binding hGH-displaying
phagemid over nonspecifically-binding phagemid.
[0152] 5. PROPAGATION: An aliquot (4.3.times.10.sup.5 CFU) of
fraction G eluted from the hGHbp-beads was used to infect log-phase
WJM101 cells. Transductions were carried out by mixing 100 .mu.L
fraction G with 1 mL WJM101 cells, incubating 20 min. at 37.degree.
C., then adding K07 (multiplicity of infection=1000). Cultures (25
mL 2YT plus carbenicillin) were grown as described above and the
second pool of phage (Library 1G, for first glycine elution) were
prepared as described above.
[0153] Phage from library 1G (FIG. 3) were selected for binding to
hGHbp beads as described above. Fraction G eluted from hGHbp beads
contained 30 times as many CFU's as fraction G eluted from
BSA-beads in this selection. Again, an aliquot of fraction G was
propagated in WJM101 cells to yield library 1G.sup.2 (indicating
that this library had been twice selected by glycine elution).
Double-stranded DNA (pLIB 1G.sup.2) was also prepared from this
culture.
KpnI Assay and Restriction-Selection of dsDNA
[0154] To reduce the level of background (KpnI.sup.+) template, an
aliquot (about 0.5 .mu.g) of pLIB 1G.sup.2 was digested with KpnI
and electroporated into WJM101 cells. These cells were grown in the
presence of K07 (multiplicity of infection=100) as described for
the initial library, and a new phage pool, pLIB 3, was prepared
(FIG. 3).
[0155] In addition, an aliquot (about 0.5 .mu.g) of dsDNA from the
initial library (pLIB1) was digested with KpnI and electroporated
directly into WJM101 cells. Transformants were allowed to recover
as above, infected with M13-K07, and grown overnight to obtain a
new library of phage, designated phage Library 2 (FIG. 3).
Successive Rounds of Selection
[0156] Phagemid binding, elution, and propagation were carried out
in successive rounds for phagemid derived from both pLIB 2 and pLIB
3 (FIG. 3) as described above, except that (1) an excess (10-fold
over CFU) of purified K07 phage (not displaying hGH) was added in
the bead-binding cocktail, and (2) the hGH stepwise elutions were
replaced with brief washings of buffer A alone. Also, in some
cases, XL1-Blue cells were used for phagemid propagation.
[0157] An additional digestion of dsDNA with KpnI was carried out
on pLIB 2G.sup.3 and on pLIB 3G.sup.5 before the final round of
bead-binding selection (FIG. 3).
DNA Sequencing of Selected Phagemids
[0158] Four independently isolated clones from LIB 4G.sup.4 and
four independently isolated clones from LIB 5G.sup.6 were sequenced
by dideoxy sequencing. All eight of these clones had identical DNA
sequences (SEQ ID NO:9):
TABLE-US-00006 hGH codon: 172 174 176 178 5'-AAG GTC TCC ACA TAC
CTG AGG ATC-3'
Thus, all these encode the same mutant of hGH: (E174S, F176Y).
Residue 172 in these clones is Lys as in wild-type. The codon
selected for 172 is also identical to wild-type hGH. This is not
surprising since AAG is the only lysine-codon possible from a
degenerate "NNS" codon set. Residue 178-Arg is also the same as
wild-type, but here, the codon selected from the library was AAG
instead of CGC as is found in wild-type hGH, even though the latter
codon is also possible using the "NNS" codon set.
Multiplicity of K07 Infection
[0159] The multiplicity of infection of K07 infection is an
important parameter in the propagation of recombinant phagemids.
The K07 multiplicity of infection must be high enough to insure
that virtually all cells transformed or transfected with phagemid
are able to package new phagemid particles. Furthermore, the
concentration of wild-type gene III in each cell should be kept
high to reduce the possibility of multiple hGH-gene III fusion
molecules being displayed on each phagemid particle, thereby
reducing chelate effects in binding. However, if the K07
multiplicity of infection is too high, the packaging of K07 will
compete with that of recombinant phagemid. We find that acceptable
phagemid yields, with only 1-10% background K07 phage, are obtained
when the K07 multiplicity of infection is 100.
TABLE-US-00007 TABLE IV Enrichment hGHbp/BSA Phage Pool moi (K07)
CFU/PFU beads Fraction Kpnl LIB 1 1000 ND 14 0.44 LIB 1G 1000 ND 30
0.57 LIB 3 100 ND 1.7 0.26 LIB 3G.sup.3 10 ND 8.5 0.18 LIB 3G.sup.4
100 460 220 0.13 LIB 5 100 ND 15 ND LIB 2 100 ND 1.7 <0.05 LIB
2G 10 ND 4.1 <0.10 LIB 2G.sup.2 100 1000 27 0.18 LIB 4 100 170
38 ND
[0160] Phage pools are labeled as shown (FIG. 3). The multiplicity
of infection (moi) refers to the multiplicity of K07 infection
(PFU/cells) in the propagation of phagemid. The enrichment of CFU
over PFU is shown in those cases where purified K07 was added in
the binding step. The ratio of CFU eluting from hGHbp-beads over
CFU eluting from BSA-beads is shown. The fraction of
KpnI-containing template (i.e., pH0415) remaining in the pool was
determined by digesting dsDNA with KpnI plus EcoRI, running the
products on a 1% agarose gel, and laser-scanning a negative of the
ethidium bromide-stained DNA.
Receptor-Binding Affinity of the Hormone hGH(E174S, F176Y)
[0161] The fact that a single clone was isolated from two different
pathways of selection (FIG. 3) suggested that the double mutant
(E174S, F176Y) binds strongly to hGHbp. To determine the affinity
of this mutant of hGH for hGHbp, we constructed this mutant of hGH
by site-directed mutagenesis, using a plasmid (pBO720) which
contains the wild-type hGH gene as template and the following
oligonucleotide which changes codons 174 and 176 (SEQ ID NO:10)
TABLE-US-00008 hGH codon: 172 174 176 178 Lys Ser Tyr Arg 5'-ATG
GAC AAG GTG TCG ACA TAC CTG CGC ATC GTG-3'
The resulting construct, pH0458B, was transformed into E. coli
strain 16C9 for expression of the mutant hormone. Scatchard
analysis of competitive binding of hGH(E174S, F176Y) versus
.sup.125I-hGH to hGHbp indicated that the (E174S, F176Y) mutant has
a binding affinity at least 5.0-fold tighter than that of wild-type
hGH.
Example VIII
Selection of hGH Variants from a Helix-4 Random Cassette Library of
Hormone-Phage
[0162] Human growth hormone variants were produced by the method of
the present invention using the phagemid described in FIG. 9.
Construction of a De-Fusable Hormone-Phage Vector
[0163] We designed a vector for cassette mutagenesis (Wells et al.,
Gene 34, 315-323 [1985]) and expression of the hGH-gene III fusion
protein with the objectives of (1) improving the linkage between
hGH and the gene III moiety to more favorably display the hGH
moiety on the phage (2) limiting expression of the fusion protein
to obtain essentially "monovalent display," (3) allowing for
restriction nuclease selection against the starting vector, (4)
eliminating expression of fusion protein from the starting vector,
and (5) achieving facile expression of the corresponding free
hormone from a given hGH-gene III fusion mutant.
[0164] Plasmid pS0643 was constructed by oligonucleotide-directed
mutagenesis (Kunkel et al., Methods Enzymol. 154, 367-382 [1987])
of pS0132, which contains pBR322 and f1 origins of replication and
expresses an hGH-gene III fusion protein (hGH residues 1-191,
followed by a single Gly residue, fused to Pro-198 of gene III)
under the control of the E. coli phoA promoter (Bass et al.,
Proteins 8, 309-314 [1990])(FIG. 9). Mutagenesis was carried out
with the oligonucleotide
5'-GGC-AGC-TGT-GGC-TTC-TAG-AGT-GGC-GGC-GGC-TCT-GGT-3' (SEQ ID
NO:1), which introduces a XbaI site (underlined) and an amber stop
codon (TAG) following Phe-191 of hGH. In the resulting construct,
pS0643, a portion of gene III was deleted, and two silent mutations
(underlined) occurred, yielding the following junction between hGH
and gene III (SEQ ID NOS: 11 AND 12):
TABLE-US-00009 ---hGH --------------> gene III -------- 187 188
189 190 191 am* 249 250 251 252 253 254 Gly Ser Cys Gly Phe Glu Ser
Gly Gly Gly Ser Gly GGC AGC TGT GGA TTC TAG AGT GGC GGT GGC TCT
GGT
[0165] This shortens the total size of the fusion protein from 401
residues in pS0132 to 350 residues in pS0643. Experiments using
monoclonal antibodies against hGH have demonstrated that the hGH
portion of the new fusion protein, assembled on a phage particle,
is more accessible than was the previous, longer fusion.
[0166] For propagation of hormone-displaying phage, pS0643 and
derivatives can be grown in a amber-suppressor strain of E. coli,
such as JM101 or XL1-Blue (Bullock et al., BioTechniques 5, 376-379
[1987]). Shown above is substitution of Glu at the amber codon
which occurs in supE suppressor strains. Suppression with other
amino acids is also possible in various available strains of E.
coli well known and publically available.
[0167] To express hGH (or mutants) free of the gene III portion of
the fusion, pS0643 and derivatives can simply be grown in a
non-suppressor strain such as 16C9. In this case, the amber codon
(TAG) leads to termination of translation, which yields free
hormone, without the need for an independent DNA construction.
[0168] To create sites for cassette mutagenesis, pS0643 was mutated
with the oligonucleotides (1)
5'-CGG-ACT-GGG-CAG-ATA-TTC-AAG-CAG-ACC-3' (SEQ ID NO:13), which
destroys the unique BglII site of pS0643; (2)
5'-CTC-AAG-AAC-TAC-GGG-TTA-CCC-TGA-CTG-CTT-CAG-GAA-GG-3' (SEQ ID
NO:14), which inserts a unique BstEII site, a single-base
frameshift, and a non-amber stop codon (TGA); and (3)
5'-CGC-ATC-GTG-CAG-TGC-AGA-TCT-GTG-GAG-GGC-3' (SEQ ID NO:15), which
introduces a new BglII site, to yield the starting vector, pH0509.
The addition of a frameshift along with a TGA stop codon insures
that no geneIII-fusion can be produced from the starting vector.
The BstEII-BglII segment is cut out of pH0509 and replaced with a
DNA cassette, mutated at the codons of interest. Other restriction
sites for cassette mutagenesis at other locations in hGH have also
been introduced into the hormone-phage vector.
Cassette Mutagenesis within Helix 4 of hGH
[0169] Codons 172, 174, 176 and 178 of hGH were targeted for random
mutagenesis because they all lie on or near the surface of hGH and
contribute significantly to receptor-binding (Cunningham and Wells,
Science 244, 1081-1085 [1989]); they all lie within a well-defined
structure, occupying 2 "turns" on the same side of helix 4; and
they are each substituted by at least one amino acid among known
evolutionary variants of hGH.
[0170] We chose to substitute NNS(N=A/G/C/T; S=G/C) at each of the
target residues. The choice of the NNS degenerate sequence yields
32 possible codons (including at least one codon for each amino
acid) at 4 sites, for a total of (32).sup.4=1,048,576 possible
nucleotide sequences, or (20).sup.4=160,000 possible polypeptide
sequences. Only one stop codon, amber (TAG), is allowed by this
choice of codons, and this codon is suppressible as Glu in supE
strains of E. coli.
[0171] Two degenerate oligonucleotides, with NNS at codons 172,
174, 176, and 178, were synthesized, phosphorylated, and annealed
to construct the mutagenic cassette:
5'-GT-TAC-TCT-ACT-GCT-TTC-AGG-AAG-GAC-ATG-GAC-NNS-GTC-NNS-ACA-NNS-CTG-NNS-
-ATC-GTG-CAG-TGC-A-3' (SEQ ID NO:16), and
5'-GA-TCT-GCA-CTG-CAC-GAT-SNN-CAG-SNN-TGT-SNN-GAC-SNN-GTC-CAT-GTC-CTT-CCT-
-GAA-GCA-GTA-GA-3' (SEQ ID NO: 17).
[0172] The vector was prepared by digesting pH0509 with BstEII
followed by BglII. The products were run on a 1% agarose gel and
the large fragment excised, phenol-extracted, and ethanol
precipitated. This fragment was treated with calf intestinal
phosphatase (Boehringer), then phenol:chloroform extracted, ethanol
precipitated, and resuspended for ligation with the mutagenic
cassette.
Propagation of the Initial Library in XL1-Blue Cells
[0173] Following ligation, the reaction products were again
digested with BstEII, then phenol:chloroform extracted, ethanol
precipitated and resuspended in water. (A BstEII recognition site
(GGTNACC) is created within cassettes which contain a G at position
3 of codon 172 and an ACC (Thr) codon at 174. However, treatment
with BstEII at this step should not select against any of the
possible mutagenic cassettes, because virtually all cassettes will
be heteroduplexes, which cannot be cleaved by the enzyme).
Approximately 150 ng (45 fmols) of DNA was electroporated into
XL1-Blue cells (1.8.times.10.sup.9 cells in 0.045 mL) in a 0.2 cm
cuvette at a voltage setting of 2.49 kV with a single pulse (time
constant=4.7 msec.).
[0174] The cells were allowed to recover 1 hour at 37.degree. C. in
S.O.C media with shaking, then mixed with 25 mL 2YT medium, 100
mg/mL carbenicillin, and M13-K07 (moi=100). After 10' at 23.degree.
C., the culture was incubated overnight (15 hours) at 37.degree. C.
with shaking. Plating of serial dilutions from this culture onto
carbenicillin-containing media indicated that 3.9.times.10.sup.7
electrotransformants were obtained.
[0175] After overnight incubation, the cells were pelleted, and
double-stranded DNA (dsDNA), designated pH0529E (the initial
library), was prepared by the alkaline lysis method. The
supernatant was spun again to remove any remaining cells, and the
phage, designated phage pool .phi.H0529E (the initial library of
phage), were PEG-precipitated and resuspended in 1 mL STE buffer
(10 mM Tris, pH 7.6, 1 mM EDTA, 50 mM NaCl). Phage titers were
measured as colony-forming units (CFU) for the recombinant phagemid
containing hGH-g3p. Approximately 4.5.times.10.sup.13 CFU were
obtained from the starting library.
Degeneracy of the Starting Library
[0176] From the pool of electrotransformants, 58 clones were
sequenced in the region of the BstEII-BglII cassette. Of these, 17%
corresponded to the starting vector, 17% contained at least one
frame shift, and 7% contained a non-silent (non-terminating)
mutation outside the four target codons. We conclude that 41% of
the clones were defective by one of the above measures, leaving a
total functional pool of 2.0.times.10.sup.7 initial transformants.
This number still exceeds the possible number of DNA sequences by
nearly 20-fold. Therefore, we are confident of having all possible
sequences represented in the starting library.
[0177] We examined the sequences of non-selected phage to evaluate
the degree of codon bias in the mutagenesis (Table V). The results
indicated that, although some codons (and amino acids) are under-
or over-represented relative to the random expectation, the library
is extremely diverse, with no evidence of large-scale "sibling"
degeneracy (Table VI).
TABLE-US-00010 TABLE V Codon distribution (per 188 codons) of
non-selected hormone phage. Residue Number expected Number found
Found/Expected Leu 17.6 18 1.0 Ser 17.6 26 1.5 Arg 17.6 10 0.57 Pro
11.8 16 1.4 Thr 11.8 14 1.2 Ala 11.8 13 1.1 Gly 11.8 16 1.4 Val
11.8 4 0.3 Ile 5.9 2 0.3 Met 5.9 1 0.2 Tyr 5.9 1 0.2 His 5.9 2 0.3
Trp 5.9 2 0.3 Phe 5.9 5 0.9 Cys 5.9 5 0.9 Gln 5.9 7 1.2 Asn 5.9 14
2.4 Lys 5.9 11 1.9 Asp 5.9 9 1.5 Glu 5.9 6 1.0 amber* 5.9 6 1.0
Clones were sequenced from the starting library (pH0529E). All
codons were tabulated, including those from clones which contained
spurious mutations and/or frameshifts. *Note: the amber stop codon
(TAG) is suppressed as Glu in XL1-Blue cells. Highlighted codons
were over/under-represented by 50% or more.
TABLE-US-00011 TABLE VI K.epsilon. NT (SEQ ID NO: 46) TWGS (SEQ ID
NO: 47) P.epsilon. ER (SEQ ID NO: 48) LPPS (SEQ ID NO: 49) SLDP
(SEQ ID NO: 50) QQSN (SEQ ID NO: 51) GSKT (SEQ ID NO: 52) TPVT (SEQ
ID NO: 53) RSRA (SEQ ID NO: 54) LCGL (SEQ ID NO: 55) TGRL (SEQ ID
NO: 56) AKAS (SEQ ID NO: 57) GNDD (SEQ ID NO: 58) KTEQ (SEQ ID NO:
59) NNCR (SEQ ID NO: 60) FPCL (SEQ ID NO: 61) NSDF (SEQ ID NO: 62)
HRPS (SEQ ID NO: 63) LSLE (SEQ ID NO: 64) NGSK (SEQ ID NO: 65) LTTE
(SEQ ID NO: 66) PSGG (SEQ ID NO: 67) LWFP (SEQ ID NO: 68) PAGS (SEQ
ID NO: 69) GRAK (SEQ ID NO: 70) GTNG (SEQ ID NO: 71) CVLQ (SEQ ID
NO: 72) EASL (SEQ ID NO: 73) SSKE (SEQ ID NO: 74) ALLL (SEQ ID NO:
75) PSHP (SEQ ID NO: 76) SYAP (SEQ ID NO: 77) ASNG (SEQ ID NO: 78)
EANN (SEQ ID NO: 79) KNAK (SEQ ID NO: 80) SRGK (SEQ ID NO: 81) GLDG
(SEQ ID NO: 82) NDPI (SEQ ID NO: 83) Non-selected (pHO529E) clones
with an open reading frame. The notation, e.g. TWGS, denotes the
hGH mutant 172T/174W/176G/178S. Amber (TAG) codons, translated as
Glu in XL1 -Blue cells are shown as {tilde over (.quadrature.)}
Preparation of Immobilized hGHbp and hPRLbp
[0178] Immobilized hGHbp ("hGHbp-beads") was prepared as described
(Bass et al., Proteins 8, 309-314 [1990]), except that wild-type
hGHbp (Fuh et al., J. Biol. Chem. 265, 3111-3115 [1990]) was used.
Competitive binding experiments with [.sup.125I] hGH indicated that
58 fmols of functional hGHbp were coupled per .mu.L of bead
suspension.
[0179] Immobilized hPRLbp ("hPRLbp-beads") was prepared as above,
using the 211-residue extracellular domain of the prolactin
receptor (Cunningham et al., Science 250, 1709-1712 [1990]).
Competitive binding experiments with [.sup.125I] hGH in the
presence of 50 .mu.M zinc indicated that 2.1 fmols of functional
hPRLbp were coupled per .mu.L of bead suspension.
[0180] "Blank beads" were prepared by treating the
oxirane-acrylamide beads with 0.6 M ethanolamine (pH 9.2) for 15
hours at 4.degree. C.
Binding Selection Using Immobilized hGHbp and hPRLbp
[0181] Binding of hormone-phage to beads was carried out in one of
the following buffers: Buffer A (PBS, 0.5% BSA, 0.05% Tween 20,
0.01% thimerosal) for selections using hGHbp and blank beads;
Buffer B (50 mM tris pH 7.5, 10 mM MgCl.sub.2, 0.5% BSA, 0.05%
Tween 20, 100 mM ZnCl.sub.2) for selections using hPRLbp in the
presence of zinc (+Zn.sup.2+); or Buffer C (PBS, 0.5% BSA, 0.05%
Tween 20, 0.01% thimerosal, 10 mM EDTA) for selections using hPRLbp
in the absence of zinc (+EDTA). Binding selections were carried out
according to each of the following paths: (1) binding to blank
beads, (2) binding to hGHbp-beads, (3) binding to hPRLbp-beads
(+Zn.sup.2+), (4) binding to hPRLbp-beads (+EDTA), (5)
pre-adsorbing twice with hGHbp beads then binding the non-adsorbed
fraction to hPRLbp-beads ("-hGHbp, +hPRLbp" selection), or (6)
pre-adsorbing twice with hPRLbp-beads then binding the non-adsorbed
fraction to hGHbp-beads ("-hPRLbp, +hGHbp" selection). The latter
two procedures are expected to enrich for mutants binding hPRLbp
but not hGHbp, or for mutants binding hGHbp but not hPRLbp,
respectively. Binding and elution of phage was carried out in each
cycle as follows:
[0182] 1. BINDING: An aliquot of hormone phage (typically
10.sup.9-10.sup.10 CFU) was mixed with an equal amount of
non-hormone phage (pCAT), diluted into the appropriate buffer (A,
B, or C), and mixed with a 10 mL suspension of hGHbp, hPRLbp or
blank beads in a total volume of 200 mL in a 1.5 mL polypropylene
tube. The phage were allowed to bind to the beads by incubating 1
hour at room temperature (23.degree. C.) with slow rotation
(approximately 7 RPM). Subsequent steps were carried out with a
constant volume of 200 .mu.L and at room temperature.
[0183] 2. WASHES: The beads were spun 15 sec., and the supernatant
was removed. To reduce the number of phage not specifically bound,
the beads were washed 5 times by resuspending briefly in the
appropriate buffer, then pelleting.
[0184] 3. hGH ELUTION: Phage binding weakly to the beads were
removed by elution with hGH. The beads were rotated with the
appropriate buffer containing 400 nM hGH for 15-17 hours. The
supernatant was saved as the "hGH elution" and the beads. The beads
were washed by resuspending briefly n buffer and pelleting.
[0185] 4. GLYCINE ELUTION: To remove the tightest-binding phage
(i.e. those still bound after the hGH wash), beads were suspended
in Glycine buffer (Buffer A plus 0.2 M Glycine, pH 2.0 with HCl),
rotated 1 hour and pelleted. The supernatant ("Glycine elution";
200 .mu.L) was neutralized by adding 30 mL of 1 M Tris base and
stored at 4.degree. C.
[0186] 5. PROPAGATION: Aliquots from the hGH elutions and from the
Glycine elutions from each set of beads under each set of
conditions were used to infect separate cultures of log-phase
XL1-Blue cells. Transductions were carried out by mixing phage with
1 mL XL1-Blue cells, incubating 20 min. at 37.degree. C., then
adding K07 (moi=100). Cultures (25 mL 2YT plus carbenicillin) were
grown as described above and the next pool of phage was prepared as
described above.
[0187] Phage binding, elution, and propagation were carried out in
successive rounds, according to the cycle described above. For
example, the phage amplified from the hGH elution from hGHbp-beads
were again selected on hGHbp-beads and eluted with hGH, then used
to infect a new culture of XL1-Blue cells. Three to five rounds of
selection and propagation were carried out for each of the
selection procedures described above.
DNA Sequencing of Selected Phagemids
[0188] From the hGH and Glycine elution steps of each cycle, an
aliquot of phage was used to inoculate XL1-Blue cells, which were
plated on LB media containing carbenicillin and tetracycline to
obtain independent clones from each phage pool. Single-stranded DNA
was prepared from isolated colony and sequenced in the region of
the mutagenic cassette. The results of DNA sequencing are
summarized in terms of the deduced amino acid sequences in FIGS. 5,
6, 7, and 8.
Expression and Assay of hGH Mutants
[0189] To determine the binding affinity of some of the selected
hGH mutants for the hGHbp, we transformed DNA from sequenced clones
into E. coli strain 16C9. As described above, this is a
non-suppressor strain which terminates translation of protein after
the final Phe-191 residue of hGH. Single-stranded DNA was used for
these transformations, but double-stranded DNA or even whole phage
can be easily electroporated into a non-suppressor strain for
expression of free hormone.
[0190] Mutants of hGH were prepared from osmotically shocked cells
by ammonium sulfate precipitation as described for hGH (Olson et
al., Nature 293, 408-411 [1981]), and protein concentrations were
measured by laser densitomoetry of Coomassie-stained
SDS-polyacrylamide gel electrophoresis gels, using hGH as standard
(Cunningham and Wells, Science 244, 1081-1085 [1989]).
[0191] The binding affinity of each mutant was determined by
displacement of .sup.125I hGH as described (Spencer et al., J.
Biol. Chem. 263, 7862-7867 [1988]; Fuh et al., J. Biol. Chem. 265,
3111-3115 [1990]), using an anti-receptor monoclonal antibody
(Mab263).
[0192] The results for a number of hGH mutants, selected by
different pathways (FIG. 6) are shown in Table VII. Many of these
mutants have a tighter binding affinity for hGHbp than wild-type
hGH. The most improved mutant, KSYR (SEQ ID NO:84), has a binding
affinity 5.6 times greater than that of wild-type hGH. The weakest
selected mutant, among those assayed was only about 10-fold lower
in binding affinity than hGH.
[0193] Binding assays may be carried out for mutants selected for
hPRLbp-binding.
TABLE-US-00012 TABLE VII Competitive binding to hGHbp Mutant Kd
(nM) Kd(mut)/Kd(hGH) Pool KSYR (6) (SEQ ID NO: 84) 0.06 + 0.01 0.18
1G, 3G RSFR (SEQ ID NO: 85) 0.10 + 0.05 0.30 3G RAYR (SEQ ID NO:
86) 0.13 + 0.04 0.37 3* KTYK (2) (SEQ ID NO: 87) 0.16 + 0.04 0.47
H, 3G RSYR (3) (SEQ ID NO: 88) 0.20 + 0.07 0.58 1G, 3H, 3G KAYR (3)
(SEQ ID NO: 89) 0.22 + 0.03 0.66 3G RFFR (2) (SEQ ID NO: 90) 0.26 +
0.05 0.76 3H KQYR (SEQ ID NO: 91) 0.33 + 0.03 1.0 3G KEFR = wt (9)
0.34 + 0.05 1.0 3H, 3G, 3* RTYH (SEQ ID NO: 92) 0.68 + 0.17 2.0 3H
QRYR (SEQ ID NO: 93) 0.83 + 0.14 2.5 3* KKYK (SEQ ID NO: 94) 1.1 +
0.4 3.2 3* RSFS (2) (SEQ ID NO: 95) 1.1 + 0.2 3.3 3G, * KSNR (SEQ
ID NO: 96) 3.1 + 0.4 9.2 3* The selected pool in which each mutant
was found is indicated as 1G (first glycine selection), 3G (third
glycine selection), 3H (third hGH selection), 3* (third selection,
not binding to hPRLbp, but binding to hGHbp). The number of times
each mutant occurred among all sequenced clones is shown ( ).
Additive and Non-Additive Effects on Binding
[0194] At some residues, substitution of a particular amino acid
has essentially the same effect independent of surrounding
residues. For example, substitution of F176Y in the background of
172R/174S reduces binding affinity by 2.0-fold (RSFR (SEQ ID NO:85)
vs. RSYR (SEQ ID NO:88)). Similarly, in the background of 172K/174A
the binding affinity of the F176Y mutant (KAYR (SEQ ID NO:89)) is
2.9-fold weaker than the corresponding 176F mutant (KAFR;
Cunningham and Wells, 1989).
[0195] On the other hand, the binding constants determined for
several selected mutants of hGH demonstrate non-additive effects of
some amino acid substitutions at residues 172, 174, 176, and 178.
For example, in the background of 172K/176Y, the substitution E174S
results in a mutant (KSYR (SEQ ID NO:84)) which binds hGHbp
3.7-fold tighter than the corresponding mutant containing E174A
(KAYR (SEQ ID NO:89)). However, in the background of 172R/176Y, the
effects of these E174 substitutions are reversed. Here, the E174A
mutant (RAYR (SEQ ID NO:86)) binds 1.5-fold tighter than the E174S
mutant (RSYR (SEQ ID NO:88)).
[0196] Such non-additive effects on binding for substitutions at
proximal residues illustrate the utility of protein-phage binding
selection as a means of selecting optimized mutants from a library
randomized at several positions. In the absence of detailed
structural information, without such a selection process, many
combinations of substitutions might be tried before finding the
optimum mutant.
Example IX
Selection of hGH Variants from a Helix-1 Random Cassette Library of
Hormone-Phage
[0197] Using the methods described in Example VIII, we targeted
another region of hGH involved in binding to the hGHbp and/or
hPRLbp, helix 1 residues 10, 14, 18, 21, for random mutagenesis in
the phGHam-g3p vector (also known as pS0643; see Example VIII).
[0198] We chose to use the "amber" hGH-g3 construct (called
phGHam-g3p) because it appears to make the target protein, hGH,
more accessible for binding. This is supported by data from
comparative ELISA assays of monoclonal antibody binding. Phage
produced from both pS0132 (S. Bass, R. Greene, J. A. Wells,
Proteins 8, 309 (1990)) and phGHam-g3 were tested with three
antibodies (Medix 2, 1B5.G2, and 5B7.C10) that are known to have
binding determinants near the carboxyl-terminus of hGH [B. C.
Cunningham, P. Jhurani, P. Ng, J. A. Wells, Science 243, 1330
(1989); B. C. Cunningham and J. A. Wells, Science 244, 1081 (1989);
L. Jin and J. Wells, unpublished results], and one antibody (Medix
1) that recognizes determinants in helices 1 and 3 ([B. C.
Cunningham, P. Jhurani, P. Ng, J. A. Wells, Science 243, 1330
(1989); B. C. Cunningham and J. A. Wells, Science 244, 1081
(1989)]). Phagemid particles from phGHam-g3 reacted much more
strongly with antibodies Medix 2, 1B5.G2, and 5B7.C10 than did
phagemid particles from pS0132. In particular, binding of pS0132
particles was reduced by >2000-fold for both Medix 2 and 5B7.C10
and reduced by >25-fold for 1B5.G2 compared to binding to Medix
1. On the other hand, binding of phGHam-g3 phage was weaker by only
about 1.5-fold, 1.2-fold, and 2.3-fold for the Medix 2, 1B5.G2, and
5B7.C10 antibodies, respectively, compared with binding to MEDIX
1.
Construction of the Helix 1 Library by Cassette Mutagenesis
[0199] We mutated residues in helix 1 that were previously
identified by alanine-scanning mutagenesis [B. C. Cunningham, P.
Jhurani, P. Ng, J. A. Wells, Science 243, 1330 (1989); B. C.
Cunningham and J. A. Wells, Science 244, 1081 (1989), 15, 16) to
modulate the binding of the extracellular domains of the hGH and/or
hPRL receptors (called hGHbp and hPRLbp, respectively). Cassette
mutagenesis was carried out essentially as described [J. A. Wells,
M. Vasser, D. B. Powers, Gene 34, 315 (1985)]. This library was
constructed by cassette mutagenesis that fully mutated four
residues at a time (see Example VIII) which utilized a mutated
version of phGHam-g3 into which unique KpnI (at hGH codon 27) and
XhoI (at hGH codon 6) restriction sites (underlined below) had been
inserted by mutagenesis [T. A. Kunkel, J. D. Roberts, R. A. Zakour,
Methods Enzymol. 154, 367-382] with the oligonucleotides 5'-GCC TTT
GAC AGG TAC CAG GAG TTT G-3' (SEQ ID NO:18) and 5'-CCA ACT ATA CCA
CTC TCG AGG TCT ATT CGA TAA C-3' (SEQ ID NO:19), respectively. The
later oligo also introduced a +1 frameshift (italicized) to
terminate translation from the starting vector and minimize
wild-type background in the phagemid library. This strating vector
was designated pH0508B. The helix 1 library, which mutated hGH
residues 10, 14, 18, 21, was constructed by ligating to the large
XhoI-KpnI fragment of pH0508B a cassette made from the
complementary oligonucleotides 5'-pTCG AGG CTC NNS GAC AAC GCG NNS
CTG CGT GCT NNS CGT CTT NNS CAG CTG GCC TTT GAC ACG TAC-3' (SEQ ID
NO:20) and 5'-pGT GTC AAA GGC CAG CTG SNN AAG ACG SNN AGC ACG CAG
SNN CGC GTT GTC SNN GAG CC-3' (SEQ ID NO:21). The KpnI site was
destroyed in the junction of the ligation product so that
restriction enzyme digestion could be used for analysis of
non-mutated background.
[0200] The library contained at least 10.sup.7 independent
transformants so that if the library were absolutely random
(10.sup.6 different combinations of codons) we would have an
average of about 10 copies of each possible mutated hGH gene.
Restriction analysis using KpnI indicated that at least 80% of
helix 1 library constructs contained the inserted cassette.
[0201] Binding enrichments of hGH-phage from the libraries was
carried out using hGHbp immobilized on oxirane-polyacrylamide beads
(Sigma Chemical Co.) as described (Example VIII). Four residues in
helix 1 (F10, M14, H18, and H21) were similarly mutated and after 4
and 6 cycles a non-wild-type consensus developed (Table VIII).
Position 10 on the hydrophobic face of helix 1 tended to be
hydrophobic whereas positions 21 and 18 on the hydrophillic face
tended were dominated by Asn; no obvious consensus was evident for
position 14 (Table IX).
[0202] The binding constants for these mutants of hGH to hGHbp was
determined by expressing the free hormone variants in the
non-suppressor E. coli strain 16C9, purifying the protein, and
assaying by competitive displacement of labelled wt-hGH from hGHbp
(see Example VIII). As indicated, several mutants bind tighter to
hGHbp than does wt-hGH.
TABLE-US-00013 TABLE VIII Selection of hGH helix 1 mutants Gly
elution F10 M14 H18 H21 4 Cycles H G N N A W D N (2) Y T V N I N I
N L N S H F S F G 6 Cycles H G N N (6) F S F L Consensus: H G N N
Variants of hGH (randomly mutated at residues F10, M14, H18, H21)
expressed on phagemid particles were selected by binding to
hGHbp-beads and eluting with hGH (0.4 mM) buffer followed by
glycine (0.2 M, pH 2) buffer(see Example VIII).
TABLE-US-00014 TABLE IX Consensus sequences from the selected helix
1 library Observed frequency is fraction of all clones sequenced
with the indicated amino acid. The nominal frequency is calculated
on the basis of NNS 32 codon degeneracy. The maximal enrichment
factor varies from 11 to 32 depending upon the nominal frequency
value for a given residue. Values of [K.sub.d(Ala mut)/K.sub.d(wt
hGH)] for single alanine mutations were taken from B. C. Cunningham
and J. A. Wells, Science 244, 1081 (1989); B. C. Cunningham, D. J.
Henner, J. A. Wells, Science 247, 1461 (1990); B. C. Cunningham and
J. A. Wells, Proc. Natl. Acad. Sci. USA 88, 3407 (1991). Wild type
residue K d ( Ala mut ) K d ( wt hGH ) ##EQU00001## Selected
residue Frequency observed nominal Enrich- ment F10 5.9 H 0.50
0.031 17 F 0.14 0.031 5 A 0.14 0.062 2 M14 2.2 G 0.50 0.062 8 W
0.14 0.031 5 N 0.14 0.031 5 S 0.14 0.093 2 H18 1.6 N 0.50 0.031 17
D 0.14 0.031 5 F 0.14 0.031 5 H21 0.33 N 0.79 0.031 26 H 0.07 0.031
2
TABLE-US-00015 TABLE X Binding of purified hGH helix 1 mutants to
hGHbp Sequence position 10 14 18 21 P Kd (nM\f(Kd mut) Kd(Wt hGH))
H G N N 0.50 0.14 .+-. 0.04 0.42 A W D N 0.14 0.10 .+-. 0.03 0.30
wt= F M H H 0 0.34 .+-. 0.05 (1) F S F L 0.07 0.68 .+-. 0.19 2.0 Y
T V N 0.07 0.75 .+-. 0.19 2.2 L N S H 0.07 0.82 .+-. 0.20 2.4 I N I
N 0.07 1.2 .+-. 0.31 3.4 Competition binding experiments were
performed using [.sup.125I] hGH (wild-type), hGHbp (containing the
extracellular receptor domain, residues 1-238), and Mab263 [B. C.
Cunningham, P. Jhurani, P. Ng, J. A. Wells, Science 243, 1330
(1989)];. The number P indicates the fractional occurrence of each
mutant among all the clones sequenced after one or more rounds of
selection.
Example X
Selection of hGH Variants from a Helix-4 Random Cassette Library
Containing Previously Found Mutations by Enrichment of
Hormone-Phage
[0203] Design of Mutant Proteins with Improved Binding Properties
by Iterative Selection Using Hormone-Phage
[0204] Our experience with recruiting non-binding homologs of hGH
evolutionary variants suggests that many individual amino acid
substitutions can be combined to yield cumulatively improved
mutants of hGH with respect to binding a particular receptor [B. C.
Cunningham, D. J. Henner, J. A. Wells, Science 247, 1461 (1990); B.
C. Cunningham and J. A. Wells, Proc. Natl. Acad. Sci. USA 88, 3407
(1991); H. B. Lowman, B. C. Cunningham, J. A. Wells, J. Biol. Chem.
266, in press (1991)].
[0205] The helix 4b library was constructed in an attempt to
further improve the helix 4 double mutant (E174S/F176Y) selected
from the helix 4a library that we found bound tighter to the hGH
receptor (see Example VIII). With the E174S/F176Y hGH mutant as the
background starting hormone, residues were mutated that surrounded
positions 174 and 176 on the hydrophilic face of helix 4 (R167,
D171, T175 and I179).
Construction of the Helix 4b Library by Cassette Mutagenesis
[0206] Cassette mutagenesis was carried out essentially as
described [J. A. Wells, M. Vasser, D. B. Powers, Gene 34, 315
(1985)]. The helix 4b library, which mutated residues 167, 171, 175
and 179 within the E174S/F176Y background, was constructed using
cassette mutagenesis that fully mutated four residues at a time
(see Example VIII) and which utilized a mutated version of
phGHam-g3 into which unique BstEII and BglII restriction sites had
been inserted previously (Example VIII). Into the BstEII-BglII
sites of the vector was inserted a cassette made from the
complementary oligonucleotides 5'-pG TTA CTC TAC TGC TTC NNS AAG
GAC ATG NNS AAG GTC AGC NNS TAC CTG CGC NNS GTG CAG TGC A-3' (SEQ
ID NO:22) and 5'-pGA TCT GCA CTG CAC SNN GCG CAG GTA SNN GCT GAC
CTT SNN CAT GTC CTT SNN GAA GCA GTA GA-3' (SEQ ID NO:23). The
BstEII site was eliminated in the ligated cassette. From the helix
4b library, 15 unselected clones were sequenced. Of these, none
lacked a cassette insert, 20% were frame-shifted, and 7% had a
non-silent mutation.
Results of hGHbp Enrichment
[0207] Binding enrichments of hGH-phage from the libraries was
carried out using hGHbp immobilized on oxirane-polyacrylamide beads
(Sigma Chemical Co.) as described (Example VIII). After 6 cycles of
binding a reasonably clear consensus developed (Table XI).
Interestingly, all positions tended to contain polar residues,
notably Ser, Thr and Asn (XII).
Assay of hGH Mutants
[0208] The binding constants for some of these mutants of hGH to
hGHbp was determined by expressing the free hormone variants in the
non-suppressor E. coli strain 16C9, purifying the protein, and
assaying by competitive displacement of labelled wt-hGH from hGHbp
(see Example VIII). As indicated, the binding affinities of several
helix-4b mutants for hGHbp were tighter than that of wt-hGH Table
XIII).
Receptor-Selectivity of hGH Variants
[0209] Finally, we have begun to analyze the binding affinity of
several of the tighter hGHbp binding mutants for their ability to
bind to the hPRLbp. The E174S/F176Y mutant binds 200-fold weaker to
the hPRLbp than hGH. The E174T/F176Y/R178K and
R167N/D171S/E174S/F176Y/I179T mutants each bind >500-fold weaker
to the hPRLbp than hGH. Thus, it is possible to use the produce new
receptor selective mutants of hGH by phage display technology.
Hormone-Phagemid Selection Identifies the Information-Content of
Particular Residues
[0210] Of the 12 residues mutated in three hGH-phagemid libraries
(Examples VIII, IX, X), 4 showed a strong, although not exclusive,
conservation of the wild-type residues (K172, T175, F176, and
R178). Not surprisingly, these were residues that when converted to
Ala caused the largest disruptions (4- to 60-fold) in binding
affinity to the hGHbp. There was a class of 4 other residues (F10,
M14, D171, and I179) where Ala substitutions caused weaker effects
on binding (2- to 7-fold) and these positions exhibited little
wild-type consensus. Finally the other 4 residues (H18, H21, R167,
and E174), that promote binding to the hPRLbp but not the hGHbp,
did not exhibit any consensus for the wild-type hGH sequence by
selection on hGHbp-beads. In fact two residues (E174 and H21),
where Ala substitutions enhance binding affinity to the hGHbp by 2-
to 4-fold [B. C. Cunningham, P. Jhurani, P. Ng, J. A. Wells,
Science 243, 1330 (1989); B. C. Cunningham and J. A. Wells, Science
244, 1081 (1989); B. C. Cunningham, D. J. Henner, J. A. Wells,
Science 247, 1461 (1990); B. C. Cunningham and J. A. Wells, Proc.
Natl. Acad. Sci. USA 88, 3407 (1991)]. Thus, the alanine-scanning
mutagenesis data correlates reasonably well with the flexibility to
substitute each position. In fact, the reduction in binding
affinity caused by alanine substitutions [B. C. Cunningham, P.
Jhurani, P. Ng, J. A. Wells, Science 243, 1330 (1989); B. C.
Cunningham and J. A. Wells, Science 244, 1081 (1989)], B. C.
Cunningham, D. J. Henner, J. A. Wells, Science 247, 1461 (1990); B.
C. Cunningham and J. A. Wells, Proc. Natl. Acad. Sci. USA 88, 3407
(1991)] is a reasonable predictor of the percentage that the
wild-type residue is found in the phagemid pool after 3-6 rounds of
selection. The alanine-scanning information is useful for targeting
side-chains that modulate binding, and the phage selection is
appropriate for optimizing them and defining the flexibility of
each site (and/or combinations of sites) for substitution. The
combination of scanning mutational methods [B. C. Cunningham, P.
Jhurani, P. Ng, J. A. Wells, Science 243, 1330 (1989); B. C.
Cunningham and J. A. Wells, Science 244, 1081 (1989)] and phage
display is a powerful approach to designing receptor-ligand
interfaces and studying molecular evolution in vitro.
Variations on Iterative Enrichment of Hormone-Phagemid
Libraries
[0211] In cases where combined mutations in hGH have additive
effects on binding affinity to receptor, mutations learned through
hormone-phagemid enrichment to improve binding can be combined by
simple cutting and ligation of restriction fragments or mutagenesis
to yield cumulatively optimized mutants of hGH.
[0212] On the other hand, mutations in one region of hGH which
optimize receptor binding may be structurally or functionally
incompatible with mutations in an overlapping or another region of
the molecule. In these cases, hormone phagemid enrichment can be
carried out by one of several variations on the iterative
enrichment approach (1) random DNA libraries can be generated in
each of two (or perhaps more) regions of the molecule by cassette
or another mutagenesis method. Thereafter, a combined library can
be created by ligation of restriction fragments from the two DNA
libraries; (2) an hGH variant, optimized for binding by mutation in
one region of the molecule, can be randomly mutated in a second
region of the molecule as in the helix-4b library example; (3) two
or more random libraries can be partially selected for improved
binding by hormone-phagemid enrichment; after this "roughing-in" of
the optimized binding site, the still-partially-diverse libraries
can be recombined by ligation of restriction fragments to generate
a single library, partially diverse in two or more regions of the
molecules, which in turn can be further selected for optimized
binding using hormone-phagemid enrichment.
TABLE-US-00016 TABLE XI Mutant phagemids of hGH selected from helix
4b library after 4 and 6 cycles of enrichment. R167 D171 T175 1179
4 Cycles N S T T K S T T S N T T D S T T D S T T+ D S A T D S A N T
D T T N D T N A N T N A S T T 6 Cycles N S T T (2) N N T T N S T Q
D S S T E S T I K S T L Consensus: N S T T D N Selection of hGH
helix4b mutants (randomly mutated at residues 167, 171, 175, 179),
each containing the E174S/F176Y double mutant, by binding to
hGHbp-beads and eluting with hGH (0.4 mM) buffer followed by
glycine (0.2 M, pH 2) buffer. One mutant (+) contained the spurious
mutation R178H.
TABLE-US-00017 TABLE XII Consensus sequences from the selected
library. Observed frequency is fraction of all clones sequenced
with the indicated amino acid. The nominal frequency is calculated
on the basis of NNS 32 codon degeneracy. The maximal enrichment
factor varies from 11 to 16 to 32 depending upon the nominal
frequency value for a given residue. Values of [K.sub.d(Ala
mut)/K.sub.d(wt hGH)] for single alanine mutations were taken from
refs. below; for position 175 we only have a value for the T175S
mutant [B. C. Cunningham, P. Jhurani, P. Ng, J. A. Wells, Science
243, 1330 (1989); B. C. Cunningham and J. A. Wells, Science 244,
1081 (1989); B. C. Cunningham, D. J. Henner, J. A. Wells, Science
247, 1461 (1990); B. C. Cunningham and J. A. Wells, Proc. Natl.
Acad. Sci. USA 88, 3407 (1991).]. Wild type residue K d ( Ala mut )
K d ( wt hGH ) ##EQU00002## Selected residue Frequency observed
nominal Enrich- ment R167 0.75 N 0.35 0.031 11 D 0.24 0.031 8 K
0.12 0.031 4 A 0.12 0.062 2 D171 7.1 S 0.76 0.093 8 N 0.18 0.031 6
D 0.12 0.031 4 T175 3.5 T 0.88 0.062 14 A 0.12 0.031 4 I179 2.7 T
0.71 0.062 11 N 0.18 0.031 6
TABLE-US-00018 TABLE XIII Binding of purified hGH mutants to hGHbp.
Sequence position * * * * Kd(Ala mut) 167 171 175 179 F Kd (nM)
Kd(wt hGH) N S T T 0.18 0.04 .+-. 0.02 0.12 E S T I 0.06 0.04 .+-.
0.02 0.12 K S T L 0.06 0.05 .+-. 0.03 0.16 N N T T 0.06 0.06 .+-.
0.03 0.17 R D T I 0 0.06 .+-. 0.01 (0.18) N S T Q 0.06 0.26 .+-.
0.11 0.77 Competition binding experiments were performed using
[.sup.125I]hGH (wild-type), hGHbp (containing the extracellular
receptor domain, residues 1-238), and Mab263 (11). The number P
indicates the fractional occurrence of each mutant among all the
clones sequenced after one or more rounds of selection. Note that
the helix 4b mutations (*) are in the background of
hGH(E174S/F176Y). In the list of helix 4b mutants,, the E174S/F176Y
mutant (*), with wt residues at 167, 171, 175, 179, is shown in
bold.
Example XI
Assembly of F.sub.ab Molecule on the Phagemid Surface
Construction of Plasmids
[0213] Plasmid pDH 188 contains the DNA encoding the F.sub.ab
portion of a humanized IgG antibody, called 4D5, that recognizes
the HER-2 receptor. This plasmid is contained in E. coli strain SR
101, and has been deposited with the ATCC in Rockville, Md.
[0214] Briefly, the plasmid was prepared as follows: the starting
plasmid was pS0132, containing the alkaline phosphatase promoter as
described above. The DNA encoding human growth hormone was excised
and, after a series of manipulations to make the ends of the
plasmid compatible for ligation, the DNA encoding 4D5 was inserted.
The 4D5 DNA contains two genes. The first gene encodes the variable
and constant regions of the light chain, and contains at its 5' end
the DNA encoding the st II signal sequence. The second gene
contains four portions: first, at its 5' end is the DNA encoding
the st II signal sequence. This is followed by the DNA encoding the
variable domain of the heavy chain, which is followed by the DNA
encoding the first domain of the heavy chain constant region, which
in turn is followed by the DNA encoding the M13 gene III. The
salient features of this construct are shown in FIG. 10. The
sequence of the DNA encoding 4D5 is shown in FIG. 11.
E. coli Transformation and Phage Production.
[0215] Both polyethylene glycol (PEG) and electroporation were used
to transform plasmids into SR101 cells. (PEG competent cells were
prepared and transformed according to the method of Chung and
Miller (Nucleic Acids Res. 16:3580 [1988]). Cells that were
competent for electroporation were prepared, and subsequently
transformed via electroporation according to the method of
Zabarovsky and Winberg (Nucleic Acids Res. 18:5912 [1990]). After
placing the cells in 1 ml of the SOC media (described in Sambrook
et al., supra), they were grown for 1 hour at 37.degree. C. with
shaking. At this time, the concentration of the cells was
determined using light scattering at OD.sub.600. A titered KO7
phage stock was added to achieve an multiplicity of infection (MOI)
of 100, and the phage were allowed to adhere to the cells for 20
minutes at room temperature. This mixture was then diluted into 25
mls of 2YT broth (described in Sambrook et al., supra) and
incubated with shaking at 37.degree. C. overnight. The next day,
cells were pelleted by centrifugation at 5000.times.g for 10
minutes, the supernatant was collected, and the phage particles
were precipitated with 0.5 M NaCl and 4% PEG (final concentration)
at room temperature for 10 minutes. Phage particles were pelleted
by centrifugation at 10,000.times.g for 10 minutes, resuspended in
1 ml of TEN (10 mM Tris, pH 7.6, 1 mM EDTA, and 150 mM NaCl), and
stored at 4.degree. C.
Production of Antigen Coated Plates.
[0216] Aliquots of 0.5 ml from a solution of 0.1 mg/ml of the
extra-cellular domain of the HER-2 antigen (ECD) or a solution of
0.5 mg/ml of BSA (control antigen) in 0.1 M sodium bicarbonate, pH
8.5 were used to coat one well of a Falcon 12 well tissue culture
plate. Once the solution was applied to the wells, the plates were
incubated at 4.degree. C. on a rocking platform overnight. The
plates were then blocked by removing the initial solution, applying
0.5 ml of blocking buffer (30 mg/ml BSA in 0.1 M sodium
bicarbonate), and incubating at room temperature for one hour.
Finally, the blocking buffer was removed, 1 ml of buffer A (PBS,
0.5% BSA, and 0.05% Tween-20) was added, and the plates were stored
up to 10 days at 4.degree. C. before being used for phage
selection.
Phage Selection Process.
[0217] Approximately 10.sup.9 phage particles were mixed with a
100-fold excess of KO7 helper phage and 1 ml of buffer A. This
mixture was divided into two 0.5 ml aliquots; one of which was
applied to ECD coated wells, and the other was applied to BSA
coated wells. The plates were incubated at room temperature while
shaking for one to three hours, and were then washed three times
over a period of 30 minutes with 1 ml aliquots of buffer A. Elution
of the phage from the plates was done at room temperature by one of
two methods: 1) an initial overnight incubation of 0.025 mg/ml
purified Mu4D5 antibody (murine) followed by a 30 minute incubation
with 0.4 ml of the acid elution buffer (0.2 M glycine, pH 2.1, 0.5%
BSA, and 0.05% Tween-20), or 2) an incubation with the acid elution
buffer alone. Eluates were then neutralized with 1 M Tris base, and
a 0.5 ml aliquot of TEN was added. These samples were then
propagated, titered, and stored at 4.degree. C.
Phage Propagation
[0218] Aliquots of eluted phage were added to 0.4 ml of 2YT broth
and mixed with approximately 10.sup.8 mid-log phase male E. coli
strain SR101. Phage were allowed to adhere to the cells for 20
minutes at room temperature and then added to 5 ml of 2YT broth
that contained 50 .mu.g/ml of carbenicillin and 5 .mu.g/ml of
tetracycline. These cells were grown at 37.degree. C. for 4 to 8
hours until they reached mid-log phase. The OD.sub.600 was
determined, and the cells were superinfected with KO7 helper phage
for phage production. Once phage particles were obtained, they were
titered in order to determine the number of colony forming units
(cfu). This was done by taking aliquots of serial dilutions of a
given phage stock, allowing them to infect mid-log phase SR101, and
plating on LB plates containing 50 .upsilon.g/ml carbenicillin.
RIA Affinity Determination.
[0219] The affinity of h4D5 F.sub.ab fragments and F.sub.ab phage
for the ECD antigen was determined using a competitive receptor
binding RIA (Burt, D. R., Receptor Binding in Drug Research.
O'Brien, R. A. (Ed.). pp. 3-29, Dekker, New York [1986]). The ECD
antigen was labeled with .sup.125-Iodine using the sequential
chloramine-T method (De Larco, J. E. et al., J. Cell. Physiol.
109:143-152 [1981]) which produced a radioactive tracer with a
specific activity of 14 .mu.Ci/.mu.g and incorporation of 0.47
moles of Iodine per mole of receptor. A series of 0.2 ml solutions
containing 0.5 ng (by ELISA) of F.sub.ab or F.sub.ab phage, 50 nCi
of .sup.125I ECD tracer, and a range of unlabeled ECD amounts (6.4
ng to 3277 ng) were prepared and incubated at room temperature
overnight. The labeled ECD-F.sub.ab or ECD-F.sub.ab phage complex
was separated from the unbound labeled antigen by forming an
aggregate complex induced by the addition of an anti-human IgG
(Fitzgerald 40-GH23) and 6% PEG 8000. The complex was pelleted by
centrifugation (15,000.times.g for 20 minutes) and the amount of
labeled ECD (in cpm) was determined by a gamma counter. The
dissociation constant (K.sub.d) was calculated by employing a
modified version of the program LIGAND (Munson, P. and Rothbard,
D., Anal. Biochem. 107:220-239 [1980]) which utilizes Scatchard
analysis (Scatchard, G., Ann. N.Y. Acad. Sci. 51:660-672 [1949]).
The Kd values are shown in FIG. 13.
Competitive Cell Binding Assay
[0220] Murine 4D5 antibody was labeled with 125-I to a specific
activity of 40-50 .mu.Ci/.mu.g using the Iodogen procedure.
Solutions containing a constant amount of labeled antibody and
increasing amounts of unlabeled variant Fab were prepared and added
to near confluent cultures of SK-BR-3 cells grown in 96-well
microtiter dishes (final concentration of labeled antibody was 0.1
nM). After an overnight incubation at 4.degree. C., the supernatant
was removed, the cells were washed and the cell associated
radioactivity was determined in a gamma counter. K.sub.d values
were determined by analyzing the data using a modified version of
the program LIGAND (Munson, P. and Rothbard, D., supra)
[0221] This deposit of plasmid pDH188 ATCC no. 68663 was made under
the provisions of the Budapest Treaty on the International
Recognition of the Deposit of Microorganisms for the Purpose of
Patent Procedure and the Regulations thereunder (Budapest Treaty).
This assures maintenance of a viable culture for 30 years from the
date of deposit. The organisms will be made available by ATCC under
the terms of the Budapest Treaty, and subject to an agreement
between Genentech, Inc. and ATCC, which assures permanent and
unrestricted availability of the progeny of the cultures to the
public upon issuance of the pertinent U.S. patent or upon laying
open to the public of any U.S. or foreign patent application,
whichever comes first, and assures availability of the progeny to
one determined by the U.S. Commissioner of Patents and Trademarks
to be entitled thereto according to 35 USC .sctn.122 and the
Commissioner's rules pursuant thereto (including 37 CFR .sctn.1.14
with particular reference to 886 OG 638).
[0222] The assignee of the present application has agreed that if
the cultures on deposit should die or be lost or destroyed when
cultivated under suitable conditions, they will be promptly
replaced on notification with a viable specimen of the same
culture. Availability of the deposited cultures is not to be
construed as a license to practice the invention in contravention
of the rights granted under the authority of any government in
accordance with its patent laws.
[0223] The foregoing written specification is considered to be
sufficient to enable one skilled in the art to practice the
invention. The present invention is not to be limited in scope by
the cultures deposited, since the deposited embodiments are
intended as separate illustrations of certain aspects of the
invention and any cultures that are functionally equivalent are
within the scope of this invention. The deposit of material herein
does not constitute an admission that the written description
herein contained is inadequate to enable the practice of any aspect
of the invention, including the best mode thereof, nor is it to be
construed as limiting the scope of the claims to the specific
illustrations that it represents. Indeed, various modifications of
the invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description and fall within the scope of the appended claims.
[0224] While the invention has necessarily been described in
conjunction with preferred embodiments, one of ordinary skill,
after reading the foregoing specification, will be able to effect
various changes, substitutions of equivalents, and alterations to
the subject matter set forth herein, without departing from the
spirit and scope thereof. Hence, the invention can be practiced in
ways other than those specifically described herein. It is
therefore intended that the protection granted by Letters Patent
hereon be limited only by the appended claims and equivalents
thereof.
Example XII
Selection of hGH Variants from Combinations of Helix-1 and Helix-4
Hormone-Phage Variants
Construction of Additive Variants of hGH
[0225] According to additivity principles [J. A. Wells,
Biochemistry 29, 8509 (1990)], mutations in different parts of a
protein, if they are not mutually interacting, are expected to
combine to produce additive changes in the free energy of binding
to another molecule (changes are additive in terms of
.DELTA..DELTA.G.sub.binding, or multiplicative in terms of
K.sub.d=exp[-.DELTA.G/RT]). Thus a mutation producing a 2-fold
increase in binding affinity, when combined with a second mutation
causing a 3-fold increase, would be predicted to yield a double
mutant with a 6-fold increased affinity over the starting
variant.
[0226] To test whether multiple mutations obtained from hGH-phage
selections would produce cumulatively favorable effects on hGHbp
(hGH-binding protein; the extracellular domain of the hGH receptor)
binding, we combined mutations found in the three tightest-binding
variants of hGH from the helix-1 library (Example IX:
F10A/M14W/H18D/H21N, F10H/M14G/H18N/H21N, and F10F/M14S/H18F/H21L)
with those found in the three tightest binding variants found in
the helix-4b library (Example X: R167N/D171S/T175/I179T,
R167E/D171S/T175/I179, and R167N/D171N/T175/I179T).
[0227] hGH-phagemid double-stranded DNA (dsDNA) from each of the
one-helix variants was isolated and digested with the restriction
enzymes EcoRI and BstXI. The large fragment from each helix-4b
variant was then isolated and ligated with the small fragment from
each helix-1 variant to yield the new two-helix variants shown in
Table XIII. All of these variants also contained the mutations
E174S/F176Y obtained in earlier hGH-phage binding selections (see
Example X for details).
Construction of Selective Combinatorial Libraries of hGH
[0228] Although additivity principles appear to hold for a number
of combinations of mutations, some combinations (e.g. E174S with
F176Y) are clearly non-additive (see examples VIII and X). In order
to identify with certainty the tightest binding variant with, for
example, 4 mutations in helix-1 and 4 mutations in helix-4, one
would ideally mutate all 8 residues at once and then sort the pool
for the globally tightest binding variant. However, such a pool
would consist of 1.1.times.10.sup.12 DNA sequences (utilizing NNS
codon degeneracy) encoding 2.6.times.10.sup.10 different
polypeptides. Obtaining a random phagemid library large enough to
assure representation of all variants (perhaps 10.sup.13
transformants) is not practical using current transformation
technology.
[0229] We have addressed this difficulty first by utilizing
successive rounds of mutagenesis, taking the tightest binding
variant from one library, then mutating other residues to further
improve binding (Example X). In a second method, we have utilized
the principle of additivity to combine the best mutations from two
independently sorted libraries to create multiple mutants with
improved binding (described above). Here, we further searched
through the possible combinations of mutations at positions 10, 14,
18, 21, 167, 171, 175, and 179 in hGH, by creating combinatorial
libraries of random or partially-random mutants. We constructed
three different combinatorial libraries of hGH-phagemids, using the
pooled phagemids from the helix 1 library (independently sorted for
0, 2, or 4 cycles; Example IX) and the pool from the helix-4b
library (independently sorted for 0, 2, or 4 cycles; Example X) and
sorted the combined variant pool for hGHbp binding. Since some
amount of sequence diversity exists in each of these pools, the
resulting combinatorial library can explore more sequence
combinations than what we might construct manually (e.g. Table
XIII).
[0230] hGH-phagemid double-stranded DNA (dsDNA) from each of the
one-helix library pools (selected for 0, 2, or 4 rounds) was
isolated and digested with the restriction enzymes AccI and BstXI.
The large fragment from each helix-1 variant pool was then isolated
and ligated with the small fragment from each helix-4b variant pool
to yield the three combinatorial libraries pH0707A (unselected
helix 1 and helix 4b pools, as described in examples IX and X),
pH0707B (twice-selected helix-1 pool with twice-selected helix-4b
pool), and pH0707C (4-times selected helix-1 pool with 4-times
selected helix-4b pool). Duplicate ligations were also set up with
less DNA and designated as pH0707D, pH0707E, and pH0707F,
corresponding to the 0-, 2-, and 4-round starting libraries
respectively. All of these variant pools also contained the
mutations E174S/F176Y obtained in earlier hGH-phage binding
selections (see Example X for details).
Sorting Combinatorial Libraries of hGH-Phage Variants
[0231] The ligation products pH0707A-F were processed and
electro-transformed into XL1-Blue cells as described (Example
VIII). Based on colony-forming units (CFU), the number of
transformants obtained from each pool was as follows:
2.4.times.10.sup.6 from pH0707A, 1.8.times.10.sup.6 from pH0707B,
1.6.times.10.sup.6 from pH0707C, 8.times.10.sup.5 from pH0707D,
3.times.10.sup.5 from pH0707E, and 4.times.10.sup.5 from pH0707F.
hGH-phagemid particles were prepared and selected for hGHbp-binding
over 2 to 7 cycles as described in Example VIII.
Rapid Sorting of hGH-Phagemid Libraries
[0232] In addition to sorting phagemid libraries for tight-binding
protein variants, as measured by equilibrium binding affinity, it
is of interest to sort for variants which are altered in either the
on-rate (k.sub.on) or the off-rate (k.sub.off) of binding to a
receptor or other molecule. From thermodynamics, these rates are
related to the equilibrium dissociation constant,
K.sub.d=(k.sub.off/k.sub.on). We envision that certain variants of
a particular protein have similar K.sub.d's for binding while
having very different k.sub.on's and k.sub.off's. Conversely,
changes in K.sub.d from one variant to another may be due to
effects on k.sub.on, effects on k.sub.off, or both. The
pharmacological properties of a protein may be dependent on binding
affinity or on k.sub.on or k.sub.off, depending on the detailed
mechanism of action. Here, we sought to identify hGH variants with
higher on-rates to investigate the effects of changes in k.sub.on.
We envision that the selection could alternatively be weighted
toward k.sub.off by increasing the binding time and increasing the
wash time and/or concentration with cognate ligand (hGH).
[0233] From time-course analysis of wild-type hGH-phagemid binding
to immobilized hGHbp, it appears that, of the total hGH-phagemid
particles that can be eluted in the final pH 2 wash (see Example
VIII for the complete binding and elution protocol), less than 10%
are bound after 1 minute of incubation, while greater than 90% are
bound after 15 minutes of incubation.
[0234] For "rapid-binding selection," phagemid particles from the
pH0707B pool (twice-selected for helices 1 and 4 independently)
were incubated with immobilized hGHbp for only 1 minute, then
washed six times with 1 mL of binding buffer; the hGH-wash step was
omitted; and the remaining hGH-phagemid particles were eluted with
a pH2 (0.2M glycine in binding buffer) wash. Enrichment of
hGH-phagemid particles over non-displaying particles indicated that
even with a short binding period and no cognate-ligand (hGH)
challenge, hGH-phagemid binding selection sorts tight-binding
variants out of a randomized pool.
Assay of hGH Mutants
[0235] The binding constants for some of these mutants of hGH to
hGHbp was determined by expressing the free hormone variants in the
non-suppressor E. coli strain 16C9 or 34B8, purifying the protein,
and assaying by competitive displacement of labelled wt-hGH from
hGHbp (see Example VIII) in a radio-immunoprecipitation assay. In
Table XIII below, all the variants have glutamate.sub.174 replaced
by serine.sub.174 and phenylalanine.sub.176 replaced by
tyrosine.sub.176 (E174S and F1176Y) plus the additional
substitutions as indicated at hGH amino acid positions 10, 14, 18,
21, 167, 171, 175 and 179.
TABLE-US-00019 TABLE XIII-A hGH variants from addition of helix-1
and helix-4b mutations wild-type residue Helix 1 Helix 4 Variant
F10 M14 H18 H21 R167 D171 T175 I179 H0650AD H G N N N S T T H0650AE
H G N N E S T I H0650AF H G N N N N T T H0650BD A W D N N S T T
H0650BE A W D N E S T I H06508F A W D N N N T T H0650CD F S F L N S
T T H0650CD F S F L E S T I H0650CD F S F L N N T T
[0236] In Table XIV below, hGH variants were selected from
combinatorial libraries by the phagemid binding selection process.
All hGH variants in Table XIV contain two background mutations
(E174S/F176Y). hGH-phagemid pools from the libraries pH0707A (Part
A), pH0707B and pH0707E (Part B), or pH0707C (Part C) were sorted
for 2 to 7 cycles for binding to hGHbp. The number P indicates the
fractional occurrence of each variant type among the set of clones
sequenced from each pool.
TABLE-US-00020 TABLE XIV hGH variants from hormone-phagemid binding
selection of combinatorial libraries. wild-type residue: Helix 1
Helix 4 P Variant F10 M14 H18 H21 R167 D171 T175 I179 Part A: 4
cycles: 0.60 H0714A.1 H G N N N S T N 0.40 H0714A.4 A N D A N N T
N* Part B: 2 cycles: 0.13 H0712B.1 F S F G H S T T 0.13 H0712B.2 H
Q T S A D N S 0.13 H0712B.4 H G N N N A T T 0.13 H07128.5 F S F L S
D T T 0.13 H07128.6 A S T N R D T I 0.13 H0712B.7 Q Y N N H S T T
0.13 H0712B.8 W G S S R D T I 0.13 H0712E.1 F L S S K N T V 0.13
H0712E.2 W N N S H S T T 0.13 H0712E.3 A N A S N S T T 0.13
H0712E.4 P S D N R D T I 0.13 H0712E.5 H G N N N N T S 0.13
H0712E.6 F S T G R D T I 0.13 H0712E.7 M T S N Q S T T 0.13
H0712E.8 F S F L T S T S 4 cycles: 0.17 H0714B.1 A W D N R D T I
0.17 H0714B.2 A W D N H S T N 0.17 H0714B.3 M Q M N N S T T 0.17
H0714B.4 H Y D H R D T T 0.17 H0714B.5 L N S H R D T I 0.17
H0714B.6 L N S H T S T T 7 cycles: 0.57 H0717B.1 A W D N N A T T
0.14 H0717B.2 F S T G R D T I 0.14 H0717B.6 A W D N R D T I 0.14
H0717B.7 I Q E H N S T T 0.50 H0717E.1 F S L A N S T V Part C: 4
cycles: 0.67 H0714C.2 F S F L K D T T * = also contained the
mutations L15R, K168R.
[0237] In Table XV below, hGH variants were selected from
combinatorial libraries by the phagemid binding selection process.
All hGH variants in Table XV contain two background mutations
(E174S/F176Y). The number P is the fractional occurrence of a given
variant among all clones sequenced after 4 cycles of rapid-binding
selection.
TABLE-US-00021 TABLE XV hGH variants from RAPID hGHbp binding
selection of an hGH- phagemid combinatorial library wild-type
residue: Helix 1 Helix 4 P Variant F10 M14 H18 H21 R167 D171 T175
I179 0.14 H07BF4.2 W G S S R D T I 0.57 H07BF4.3 M A D N N S T T
0.14 H07BF4.6 A W D N S S V T.dagger-dbl. 0.14 H076F4.7 H D T S R D
T I .dagger-dbl.= also contained the mutation Y176F (wild-type hGH
also contains F176).
[0238] In table XVI below, binding constants were measured by
competitive displacement of .sup.125I-labelled hormone H0650BD or
labelled hGH using hGHbp (1-238) and either Mab5 or Mab263. The
variant H0650BD appears bind more than 30-fold tighter than
wild-type hGH.
TABLE-US-00022 TABLE XVI Equilibrium binding constants of selected
hGH variants. hGH Kd(variant) Kd(variant) Variant Kd(H0650BD)
Kd(hGH) Kd (pM) hGH 32 -1- 340 .+-. 50 H0650BD -1- 0.031 10 .+-. 3
H0650BF 1.5 0.045 15 .+-. 5 H0714B.6 3.4 0.099 34 .+-. 19 H0712B.7
7.4 0.22 74 .+-. 30 H0712E.2 16 0.48 60 .+-. 70
Example XIII
Selective Enrichment of hGH-Phage Containing a Protease Substrate
Sequence Versus Non-Substrate Phage
[0239] As described in Example I, the plasmid pS0132 contains the
gene for hGH fused to the residue Pro198 of the gene III protein
with the insertion of an extra glycine residue. This plasmid may be
used to produce hGH-phage particles in which the hGH-gene III
fusion product is displayed monovalently on the phage surface
(Example IV). The fusion protein comprises the entire hGH protein
fused to the carboxy terminal domain of gene III via a flexible
linker sequence.
[0240] To investigate the feasibility of using phage display
technology to select favourable substrate sequences for a given
proteolytic enzyme, a genetically engineered variant of subtilisin
BPN' was used. (Carter, P. et al., Proteins: Structure, function
and genetics 6:240-248 (1989)). This variant (hereafter referred to
as A64SAL subtilisin) contains the following mutations: Ser24Cys,
His64Ala, Glu156Ser, Gly169Ala and Tyr217Leu. Since this enzyme
lacks the essential catalytic residue His64, its substrate
specificity is greatly restricted so that certain
histidine-containing substrates are preferentially hyrdrolysed
(Carter et al., Science 237:394-399 (1987)).
Construction of a hGH-Substrate-Phage Vector
[0241] The sequence of the linker region in pS0132 was mutated to
create a substrate sequence for A64SAL subtilisin, using the
oligonucleotide
5'-TTC-GGG-CCC-TTC-GCT-GCT-CAC-TAT-ACG-CGT-CAG-TCG-ACT-GAC-CTG-CCT-3'
(SEQ ID NO:27). This resulted in the introduction of the protein
sequence Phe-Gly-Pro-Phe-Ala-Ala-His-Tyr-Thr-Arg-Gln-Ser-Thr-Asp
(SEQ ID NO:107) in the linker region between hGH and the carboxy
terminal domain of gene III, where the first Phe residue in the
above sequence is Phe191 of hGH. The sequence
Ala-Ala-His-Tyr-Thr-Agr-Gln (SEQ ID NO:97) is known to be a good
substrate for A64SAL subtilisin (Carter et al (1989), supra). The
resulting plasmid was designated pS0640.
Selective Enrichment of hGH-Substrate-Phage
[0242] Phagemid particles derived from pS0132 and pS0640 were
constructed as described in Example I. In initial experiments, a 10
.mu.l aliquot of each phage pool was separately mixed with 30 .mu.l
of oxirane beads (prepared as described in Example II) in 100 .mu.l
of buffer comprising 20 mM Tris-HCl pH 8.6 and 2.5M NaCl. The
binding and washing steps were performed as described in example
VII. The beads were then resuspended in 400 .mu.l of the same
buffer, with or without 50 nM of A64SAL subtilisin. Following
incubation for 10 minutes, the supernatants were collected and the
phage titres (cfu) measured. Table XVII shows that approximately 10
times more substrate-containing phagemid particles (pS0640) were
eluted in the presence of enzyme than in the absence of enzyme, or
than in the case of the non-substrate phagemids (pS0132) in the
presence or absence of enzyme. Increasing the enzyme, phagemid or
bead concentrations did not improve this ratio.
Improvement of the Selective Enrichment Procedure
[0243] In an attempt to decrease the non-specific elution of
immobilised phagemids, a tight-binding variant of hGH was
introduced in place of the wild-type hGH gene in pS0132 and pS0640.
The hGH variant used was as described in example XI (pH0650bd) and
contains the mutations Phe10Ala, Met14Trp, His18Asp, His21Asn,
Arg167Asn, Asp171Ser, Glu174Ser, Phe176Tyr and Ile179Thr. This
resulted in the construction of two new phagemids: pDM0390
(containing tight-binding hGH and no substrate sequence) and
pDM0411 (containing tight-binding hGH and the substrate sequence
Ala-Ala-His-Tyr-Thr-Agr-Gln). The binding washing and elution
protocol was also changed as follows:
[0244] (i) Binding: COSTAR 12-well tissue culture plates were
coated for 16 hours with 0.5 ml/well 2 ug/ml hGHbp in sodium
carbonate buffer pH 10.0. The plates were then incubated with 1
ml/well of blocking buffer (phosphate buffered saline (PBS)
containing 0.1% w/v bovine serum albumen) for 2 hours and washed in
an assay buffer containing 10 mM Tris-HCl pH 7.5, 1 mM EDTA and 100
mM NaCl. Phagemids were again prepared as described in Example I:
the phage pool was diluted 1:4 in the above assay buffer and 0.5 ml
of phage incubated per well for 2 hours.
[0245] (ii) Washing: The plates were washed thoroughly with
PBS+0.05% Tween 20 and incubated for 30 minuted with 1 ml of this
wash buffer. This washing step was repeated three times.
[0246] (iii) Elution: The plates were incubated for 10 minutes in
an elution buffer consisting of 20 mM Tris-HCl pH 8.6+100 mM NaCl,
then the phage were eluted with 0.5 ml of the above buffer with or
without 500 nM of A64SAL subtilisin.
[0247] Table XVII shows that there was a dramatic increase in the
ratio of specifically eluted substrate-phagemid particles compared
to the method previously described for pS0640 and pS0132. It is
likely that this is due to the fact that the tight-binding hGH
mutant has a significantly slower off-rate for binding to hGH
binding protein compared to wild-type hGH.
TABLE-US-00023 TABLE XVII Specific elution of substrate-phagemids
by A64SAL subtilisin phagemid +50 nM A64SAL no enzyme (i) Wild-type
hGH gene: binding to hGHbp-oxirane beads pS0640 (substrate) 9
.times. 10.sup.6 cfu/10 .mu.l 1.5 .times. 10.sup.6 cfu/10 .mu.l
pS0132 (non-sub- 6 .times. 10.sup.5 cfu/10 .mu.l 3 .times. 10.sup.5
cfu/10 .mu.l strate) (ii) pH0650bd mutant hGH gene: binding to
hGHbp-coated plates pDM0411 (substrate) 1.7 .times. 10.sup.5 cfu/10
.mu.l 2 .times. 10.sup.3 cfu/10 .mu.l pDM0390 (non-sub- 2 .times.
10.sup.3 cfu/10 .mu.l 1 .times. 10.sup.3 cfu/10 .mu.l strate)
Colony forming units (cfu) were estimated by plating out 10 .mu.l
of 10-fold dilutions of phage on 10 .mu.l spots of XL-1 blue cells,
on LB agar plates containing 50 .mu.g/ml carbenicillinl
Example XIV
Identification of Preferred Substrates for A64SAL Subtilisin Using
Selective Enrichment of a Library of Substrate Sequences
[0248] We sought to employ the selective enrichment procedure
described in Example XIII to identify good substrate sequences from
a library of random substrate sequences.
Construction of a Vector for Insertion of Randomised Substrate
Cassettes
[0249] We designed a vector suitable for introduction of randomised
substrate cassettes. and subsequent expression of a library of
substrate sequences. The starting point was the vector pS0643,
described in Example VIII. Site-directed mutagenesis was carried
out using the oligonucleotide
5'-AGC-TGT-GGC-TTC-GGG-CCC-GCC-GCC-GCG-TCG-ACT-GGC-GGT-GGC-TCT-3'
(SEQ ID NO:28), which introduces ApaI (GGGCCC) and SalI (GTCGAC)
restriction sites between hGH and Gene III. This new construct was
designated pDM0253 (The actual sequence of pDM0253 is
5'-AGC-TGT-GGC-TTC-GGG-CCC-GCC-CCC-GCG-TCG-ACT-GGC-GGT-GGC-TCT-3'
(SEQ ID NO:29), where the underlined base substitution is due to a
spurious error in the mutagenic oligonucleotide). In addition, the
tight-binding hGH variant described in example was introduced by
exchanging a fragment from pDM0411 (example XIII). The resulting
library vector was designated pDM0454.
Preparation of the Library Cassette Vector and Insertion of the
Mutagenic Cassette
[0250] To introduce a library cassette, pDM0454 was digested with
ApaI followed by SalI, then precipitated with 13% PEG 8000+10 mM
MgCl.sub.2, washed twice in 70% ethanol and resuspended This
efficiently precipitates the vector but leaves the small Apa-Sal
fragment in solution (Paithankar, K. R. and Prasad, K. S. N.,
Nucleic Acids Research 19:1346). The product was run on a 1%
agarose gel and the ApaI-SalI digested vector excised, purified
using a Bandprep kit (Pharmacia) and resuspended for ligation with
the mutagenic cassette.
[0251] The cassette to be inserted contained a DNA sequence similar
to that in the linker region of pS0640 and pDM0411, but with the
codons for the histidine and tyrosine residues in the substrate
sequence replaced by randomised codons. We chose to substitute
NNS(N=G/A/T/C; S=G/C) at each of the randomised positions as
described in example VIII. The oligonucleotides used in the
mutagenic cassettes were: 5'-C-TTC-GCT-GCT-NNS-NNS-ACC-CGG-CAA-3'
(coding strand) (SEQ ID NO:30) and
5'-T-CGA-TTG-CCG-GGT-SNN-SNN-AGC-AGC-GAA-GGG-CC-3' (non-coding
strand) (SEQ ID NO:31). This cassette also destroys the SalI site,
so that digestion with SalI may be used to reduce the vector
background. The oligonucleotides were not phosphorylated before
insertion into the Apa-Sal cassette site, as it was feared that
subsequent oligomerisation of a small population of the cassettes
may lead to spurious results with multiple cassette inserts.
Following annealing and ligation, the reaction products were
phenol:chloroform extracted, ethanol precipitated and resuspended
in water. Initially, no digestion with SalI to reduce the
background vector was performed. Approximately 200 ng was
electroporated into XL-1 blue cells and a phagemid library was
prepared as described in example VIII.
Selection of Highly Cleavable Substrates from the Substrate
Library
[0252] The selection procedure used was identical to that described
for pDM0411 and pDM0390 in example XIII. After each round of
selection, the eluted phage were propagated by transducing a fresh
culture of XL-1 blue cells and propagating a new phagemid library
as described for hGH-phage in example VIII. The progress of the
selection procedure was monitored by measuring eluted phage titres
and by sequencing individual clones after each round of
selection.
[0253] Table A shows the successive phage titres for elution in the
presence and absence of enzyme after 1, 2 and 3 rounds of
selection.
[0254] Clearly, the ratio of specifically eluted
phage:non-specifically eluted phage (ie phage eluted with
enzyme:phage eluted without enzyme) increases dramatically from
round 1 to round 3, suggesting that the population of good
substrates is increasing with each round of selection.
[0255] Sequencing of 10 isolates from the starting library showed
them all to consist of the wild-type pDM0464 sequence. This is
attributed to the fact that after digestion with ApaI, the SalI
site is very close to the end of the DNA fragment, thus leading to
low efficiency of digestion. Nevertheless, there are only 400
possible sequences in the library, so this population should still
be well represented.
[0256] Tables B1 and B2 shows the sequences of isolates obtained
after round 2 and round 3 of selection. After 2 rounds of
selection, there is clearly a high incidence of histidine residues.
This is exactly what is expected: as described in example XIII,
A64SAL subtilisin requires a histidine residue in the substrate as
it employs a substrate-assisted catalytic mechanism. After 3 rounds
of selection, each of the 10 clones sequenced has a histidine in
the randomised cassette. Note, however, that 2 of the sequences are
of pDM0411, which was not present in the starting library and is
therefore a contaminant.
TABLE-US-00024 TABLE A Titration of initial phage pools and eluted
phage from 3 rounds of selective enrichment ROUND 1 Starting
library: 3 .times. 10.sup.12 cfu/ml LIBRARY: +500 nM A64SAL 4
.times. 10.sup.3 cfu/10 .mu.l no enzyme 3 .times. 10.sup.3 cfu/10
.mu.l pDM0411: +500 nM A64SAL 2 .times. 10.sup.6 cfu/10 .mu.l
(control) no enzyme 8 .times. 10.sup.3 cfu/10 .mu.l ROUND 2 Round 1
library: 7 .times. 10.sup.12 cfu/ml LIBRARY: +500 nM A64SAL 3
.times. 10.sup.4 cfu/10 .mu.l no enzyme 6 .times. 10.sup.3 cfu/10
.mu.l pDM0411: +500 nM A64SAL 3 .times. 10.sup.6 cfu/10 .mu.l
(control) no enzyme 1.6 .times. 10.sup.4 cfu/10 .mu.l ROUND 3 Round
2 library: 7 .times. 10.sup.11 cfu/ml LIBRARY: +500 nM A64SAL 1
.times. 10.sup.5 cfu/10 .mu.l no enzyme <10.sup.3 cfu/10 .mu.l
pDM0411: +500 nM A64SAL 5 .times. 10.sup.6 cfu/10 .mu.l (control)
no enzyme 3 .times. 10.sup.4 cfu/10 .mu.l Colony forming units
(cfu) were estimated by plating out 10 .mu.l of 10-fold dilutions
of phage on 10 .mu.l spots of XL-1 blue cells, on LB agar plates
containing 50 .mu.g/ml carbenicillin
TABLE-US-00025 TABLE B1 Sequences of eluted phage after 2 rounds of
selective enrichment. No. of Sequence occurrences After round 2: *
* A A H Y T R Q (SEQ ID NO: 97) 2 . . . GCT GCT CAC TAC ACC CGG CAA
. . . (SEQ ID NO: 32) A A H M T R Q (SEQ ID NO: 98) 1 . . . GCT GCT
CAC ATG ACC CGG CAA . . . (SEQ ID NO: 33) A A L H T R Q (SEQ ID NO:
99) 1 . . . GCT GCT CTC CAC ACC CGG CAA . . . (SEQ ID NO: 34) A A L
H T R Q (SEQ ID NO: 99) 1 . . . GCT GCT CTG CAC ACC CGG CAA . . .
(SEQ ID NO: 35) A A H T R Q (SEQ ID NO: 100) 1 # . . . GCT GCT CAC
ACC CGG CAA . . . (SEQ ID NO: 36) A A ? H T R Q (SEQ ID NO: 101) 1
## . . . GCT GCT ??? CAC ACC CGG CAA (SEQ ID NO: 37) . . .
wild-type pDM0454 3 #- spurious deletion of 1 codon within the
cassette ##- ambiguous sequence All protein sequences should be of
the form AA**TRQ, where * represents a randomised codon. In the
table below, the randomised codons and amino acids are underlined
and in bold.
TABLE-US-00026 TABLE B2 Sequences of eluted Dhaqe after 3 rounds of
selective enrichment. No. of Sequence occurrences After round 3: *
* A A H Y T R Q (SEQ ID NO: 97) 2# . . . GCT GCT CAC TAT ACG CGT
CAG . . . (SEQ ID NO: 38) A A L H T R Q (SEQ ID NO: 99) 2 . . . GCT
GCT CTC CAC ACC CGG CAA . . . (SEQ ID NO: 34) A A Q H T R Q (SED ID
NO: 102) 1 . . . GCT GCT CAG CAC ACC CGG CAA . . . (SEQ ID NO: 39)
A A T H T R Q (SEQ ID NO: 103) 1 . . . GCT GCT ACG CAC ACC CGG CAA
. . . (SEQ ID NO: 40) A A H S R Q (SEQ ID NO: 104) 1 . . . GCT GCT
CAC TCC CGG CAA . . . (SEQ ID NO: 41) A A H H T R Q (SEQ ID NO:
105) 1## . . . GCT GCT CAT CAT ACC CGG CAA . . . (SEQ ID NO: 42) A
A H F R Q (SEQ ID NO: 106) 1 . . . GCT GCT CAC TTC CGG CAA . . .
(SEQ ID NO: 43) A A H T R Q (SEQ ID NO: 100) 1 . . . GCT GCT CAC
ACC CGG CAA . . . (SEQ ID NO: 36) #- contaminating sequence from
pDM0411 ##- contains the "illegal" codon CAT - T should not appear
in the 3rd position of a codon. All protein sequences should be of
the form AA**TRQ, where * represents a randomised codon. In the
table below, the randomised codons and amino acids are underlined
and in bold.
Sequence CWU 1
1
43136DNAArtificial sequencesequence is synthesized 1ggcagctgtg
gcttctagag tggcggcggc tctggt 36236DNAArtificial sequencesequence is
synthesized 2agctgtggct tcgggccctt agcatttaat gcggta
36333DNAArtificial sequencesequence is synthesized 3ttcacaaacg
aagggcccct aattaaagcc aga 33430DNAArtificial sequencesequence is
synthesized 4caataataac gggctagcca aaagaactgg 30524DNAArtificial
sequencesequence is synthesized 5cacgacagaa ttcccgactg gaaa
24623DNAArtificial sequencesequence is synthesized 6ctgtttctag
agtgaaattg tta 23721DNAArtificial sequencesequence is synthesized
7acattcctgg gtaccgtgca g 21863DNAArtificial sequencesequence is
synthesized 8gcttcaggaa ggacatggac nnsgtcnnsa cannsctgnn satcgtgcag
50tgccgctctg tgg 63924DNAArtificial sequencesequence is synthesized
9aaggtctcca catacctgag gatc 241033DNAArtificial sequencesequence is
synthesized 10atggacaagg tgtcgacata cctgcgcatc gtg
331136DNAArtificial sequencesequence is synthesized 11ggcagctgtg
gattctagag tggcggtggc tctggt 361212PRTArtificial sequencesequence
is synthesized 12Gly Ser Cys Gly Phe Glu Ser Gly Gly Gly Ser Gly1 5
101327DNAArtificial sequencesequence is synthesized 13cggactgggc
agatattcaa gcagacc 271438DNAArtificial sequencesequence is
synthesized 14ctcaagaact acgggttacc ctgactgctt caggaagg
381530DNAArtificial sequencesequence is synthesized 15cgcatcgtgc
agtgcagatc tgtggagggc 301666DNAArtificial sequencesequence is
synthesized 16gttactctac tgctttcagg aaggacatgg acnnsgtcnn
sacannsctg 50nnsatcgtgc agtgca 661764DNAArtificial sequencesequence
is synthesized 17gatctgcact gcacgatsnn cagsnntgts nngacsnngt
ccatgtcctt 50cctgaagcag taga 641825DNAArtificial sequencesequence
is synthesized 18gcctttgaca ggtaccagga gtttg 251933DNAArtificial
sequencesequence is synthesized 19ccaactatac cactctcgag gtctattcga
taa 332066DNAArtificial sequencesequence is synthesized
20tcgaggctcn nsgacaacgc gnnsctgcgt gctnnscgtc ttnnscagct
50ggcctttgac acgtac 662158DNAArtificial sequencesequence is
synthesized 21gtgtcaaagg ccagctgsnn aagacgsnna gcacgcagsn
ncgcgttgtc 50snngagcc 582265DNAArtificial sequencesequence is
synthesized 22gttactctac tgcttcnnsa aggacatgnn saaggtcagc
nnstacctgc 50gcnnsgtgca gtgca 652364DNAArtificial sequencesequence
is synthesized 23gatctgcact gcacsnngcg caggtasnng ctgaccttsn
ncatgtcctt 50snngaagcag taga 64242178DNAArtificial sequencesequence
is synthesized 24atgaaaaaga atatcgcatt tcttcttgca tctatgttcg
ttttttctat 50tgctacaaac gcgtacgctg atatccagat gacccagtcc ccgagctccc
100tgtccgcctc tgtgggcgat agggtcacca tcacctgccg tgccagtcag
150gatgtgaata ctgctgtagc ctggtatcaa cagaaaccag gaaaagctcc
200gaaactactg atttactcgg catccttcct ctactctgga gtcccttctc
250gcttctctgg atccagatct gggacggatt tcactctgac catcagcagt
300ctgcagccgg aagacttcgc aacttattac tgtcagcaac attatactac
350tcctcccacg ttcggacagg gtaccaaggt ggagatcaaa cgaactgtgg
400ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca gttgaaatct
450ggaactgcct ctgttgtgtg cctgctgaat aacttctatc ccagagaggc
500caaagtacag tggaaggtgg ataacgccct ccaatcgggt aactcccagg
550agagtgtcac agagcaggac agcaaggaca gcacctacag cctcagcagc
600accctgacgc tgagcaaagc agactacgag aaacacaaag tctacgcctg
650cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag agcttcaaca
700ggggagagtg ttaagctgat cctctacgcc ggacgcatcg tggccctagt
750acgcaagttc acgtaaaaag ggtatctaga ggttgaggtg attttatgaa
800aaagaatatc gcatttcttc ttgcatctat gttcgttttt tctattgcta
850caaacgcgta cgctgaggtt cagctggtgg agtctggcgg tggcctggtg
900cagccagggg gctcactccg tttgtcctgt gcagcttctg gcttcaacat
950taaagacacc tatatacact gggtgcgtca ggccccgggt aagggcctgg
1000aatgggttgc aaggatttat cctacgaatg gttatactag atatgccgat
1050agcgtcaagg gccgtttcac tataagcgca gacacatcca aaaacacagc
1100ctacctgcag atgaacagcc tgcgtgctga ggacactgcc gtctattatt
1150gttctagatg gggaggggac ggcttctatg ctatggacta ctggggtcaa
1200ggaaccctgg tcaccgtctc ctcggcctcc accaagggcc catcggtctt
1250ccccctggca ccctcctcca agagcacctc tgggggcaca gcggccctgg
1300gctgcctggt caaggactac ttccccgaac cggtgacggt gtcgtggaac
1350tcaggcgccc tgaccagcgg cgtgcacacc ttcccggctg tcctacagtc
1400ctcaggactc tactccctca gcagcgtggt gactgtgccc tctagcagct
1450tgggcaccca gacctacatc tgcaacgtga atcacaagcc cagcaacacc
1500aaggtggaca agaaagttga gcccaaatct tgtgacaaaa ctcacacagg
1550gcccttcgtt tgtgaatatc aaggccaatc gtctgacctg cctcaacctc
1600ctgtcaatgc tggcggcggc tctggtggtg gttctggtgg cggctctgag
1650ggtggtggct ctgagggtgg cggttctgag ggtggcggct ctgagggagg
1700cggttccggt ggtggctctg gttccggtga ttttgattat gaaaagatgg
1750caaacgctaa taagggggct atgaccgaaa atgccgatga aaacgcgcta
1800cagtctgacg ctaaaggcaa acttgattct gtcgctactg attacggtgc
1850tgctatcgat ggtttcattg gtgacgtttc cggccttgct aatggtaatg
1900gtgctactgg tgattttgct ggctctaatt cccaaatggc tcaagtcggt
1950gacggtgata attcaccttt aatgaataat ttccgtcaat atttaccttc
2000cctccctcaa tcggttgaat gtcgcccttt tgtctttagc gctggtaaac
2050catatgaatt ttctattgat tgtgacaaaa taaacttatt ccgtggtgtc
2100tttgcgtttc ttttatatgt tgccaccttt atgtatgtat tttctacgtt
2150tgctaacata ctgcgtaata aggagtct 217825237PRTArtificial
sequencesequence is synthesized 25Met Lys Lys Asn Ile Ala Phe Leu
Leu Ala Ser Met Phe Val Phe1 5 10 15Ser Ile Ala Thr Asn Ala Tyr Ala
Asp Ile Gln Met Thr Gln Ser 20 25 30Pro Ser Ser Leu Ser Ala Ser Val
Gly Asp Arg Val Thr Ile Thr 35 40 45Cys Arg Ala Ser Gln Asp Val Asn
Thr Ala Val Ala Trp Tyr Gln 50 55 60Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile Tyr Ser Ala Ser 65 70 75Phe Leu Tyr Ser Gly Val Pro Ser
Arg Phe Ser Gly Ser Arg Ser 80 85 90Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro Glu Asp 95 100 105Phe Ala Thr Tyr Tyr Cys Gln
Gln His Tyr Thr Thr Pro Pro Thr 110 115 120Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys Arg Thr Val Ala Ala 125 130 135Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 140 145 150 Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg 155 160 165Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 170 175 180Asn Ser
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 185 190 195Tyr
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 200 205
210Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 215
220 225Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 230 235
26461PRTArtificial sequencesequence is synthesized 26Met Lys Lys
Asn Ile Ala Phe Leu Leu Ala Ser Met Phe Val Phe1 5 10 15Ser Ile Ala
Thr Asn Ala Tyr Ala Glu Val Gln Leu Val Glu Ser 20 25 30Gly Gly Gly
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys 35 40 45Ala Ala Ser
Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val 50 55 60Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr 65 70 75Pro Thr Asn
Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg 80 85 90Phe Thr Ile
Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln 95 100 105Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser 110 115 120Arg
Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 125 130
135Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 140
145 150Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr
155 160 165Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val 170 175 180Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr 185 190 195Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser 200 205 210Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile 215 220 225Cys Asn Val Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys 230 235 240Val Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Gly Pro Phe Val 245 250 255Cys Glu Tyr Gln Gly Gln Ser Ser
Asp Leu Pro Gln Pro Pro Val 260 265 270Asn Ala Gly Gly Gly Ser Gly
Gly Gly Ser Gly Gly Gly Ser Glu 275 280 285Gly Gly Gly Ser Glu Gly
Gly Gly Ser Glu Gly Gly Gly Ser Glu 290 295 300Gly Gly Gly Ser Gly
Gly Gly Ser Gly Ser Gly Asp Phe Asp Tyr 305 310 315Glu Lys Met Ala
Asn Ala Asn Lys Gly Ala Met Thr Glu Asn Ala 320 325 330Asp Glu Asn
Ala Leu Gln Ser Asp Ala Lys Gly Lys Leu Asp Ser 335 340 345Val Ala
Thr Asp Tyr Gly Ala Ala Ile Asp Gly Phe Ile Gly Asp 350 355 360Val
Ser Gly Leu Ala Asn Gly Asn Gly Ala Thr Gly Asp Phe Ala 365 370
375Gly Ser Asn Ser Gln Met Ala Gln Val Gly Asp Gly Asp Asn Ser 380
385 390Pro Leu Met Asn Asn Phe Arg Gln Tyr Leu Pro Ser Leu Pro Gln
395 400 405Ser Val Glu Cys Arg Pro Phe Val Phe Ser Ala Gly Lys Pro
Tyr 410 415 420Glu Phe Ser Ile Asp Cys Asp Lys Ile Asn Leu Phe Arg
Gly Val 425 430 435Phe Ala Phe Leu Leu Tyr Val Ala Thr Phe Met Tyr
Val Phe Ser 440 445 450Thr Phe Ala Asn Ile Leu Arg Asn Lys Glu Ser
455 4602748DNAArtificial sequencesequence is synthesized
27ttcgggccct tcgctgctca ctatacgcgt cagtcgactg acctgcct
482845DNAArtificial sequencesequence is synthesized 28agctgtggct
tcgggcccgc cgccgcgtcg actggcggtg gctct 452945DNAArtificial
sequencesequence is synthesized 29agctgtggct tcgggcccgc ccccgcgtcg
actggcggtg gctct 453025DNAArtificial sequencesequence is
synthesized 30cttcgctgct nnsnnsaccc ggcaa 253133DNAArtificial
sequencesequence is synthesized 31tcgattgccg ggtsnnsnna gcagcgaagg
gcc 333221DNAArtificial sequencesequence is synthesized
32gctgctcact acacccggca a 213321DNAArtificial sequencesequence is
synthesized 33gctgctcaca tgacccggca a 213421DNAArtificial
sequencesequence is synthesized 34gctgctctcc acacccggca a
213521DNAArtificial sequencesequence is synthesized 35gctgctctgc
acacccggca a 213618DNAArtificial sequencesequence is synthesized
36gctgctcaca cccggcaa 183721DNAArtificial sequencesequence is
synthesized 37gctgctnnnc acacccggca a 213821DNAArtificial
sequencesequence is synthesized 38gctgctcact atacgcgtca g
213921DNAArtificial sequencesequence is synthesized 39gctgctcagc
acacccggca a 214021DNAArtificial sequencesequence is synthesized
40gctgctacgc acacccggca a 214118DNAArtificial sequencesequence is
synthesized 41gctgctcact cccggcaa 184221DNAArtificial
sequencesequence is synthesized 42gctgctcatc atacccggca a
214318DNAArtificial sequencesequence is synthesized 43gctgctcact
tccggcaa 18
* * * * *