U.S. patent application number 14/270678 was filed with the patent office on 2015-01-01 for synthetic polypeptide libraries and methods for generating naturally diversified polypeptide variants.
The applicant listed for this patent is NovImmune S.A.. Invention is credited to Nicolas Fischer, Franck Gueneau, Marie Kosco-Vilbois, Ulla Ravn, Sophie Venet-Bonnot.
Application Number | 20150005201 14/270678 |
Document ID | / |
Family ID | 44011762 |
Filed Date | 2015-01-01 |
United States Patent
Application |
20150005201 |
Kind Code |
A1 |
Fischer; Nicolas ; et
al. |
January 1, 2015 |
SYNTHETIC POLYPEPTIDE LIBRARIES AND METHODS FOR GENERATING
NATURALLY DIVERSIFIED POLYPEPTIDE VARIANTS
Abstract
The invention provides compositions and methods for generating
libraries of DNA sequences encoding homologous polypeptides, and
uses of the libraries to identify naturally diversified polypeptide
variants. The invention also provides compositions and methods for
generating collections of synthetic antibody fragments in which one
or several complementary determining regions (CDR) are replaced by
a collection of the corresponding CDR captured from a natural
source. The invention further provides compositions and methods for
diversifying a portion of a polypeptide by inserting a diversified
sequence of synthetic or natural origin without the need for
modification of the original polypeptide coding sequence.
Inventors: |
Fischer; Nicolas; (Geneva,
CH) ; Kosco-Vilbois; Marie; (Minzier, FR) ;
Ravn; Ulla; (Geneva, CH) ; Gueneau; Franck;
(Saint-Julien-En-Genevois, FR) ; Venet-Bonnot;
Sophie; (Saint-Julien-En-Genevois, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NovImmune S.A. |
Geneva |
|
CH |
|
|
Family ID: |
44011762 |
Appl. No.: |
14/270678 |
Filed: |
May 6, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12952659 |
Nov 23, 2010 |
8716196 |
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14270678 |
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12784190 |
May 20, 2010 |
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12952659 |
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61179850 |
May 20, 2009 |
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61287336 |
Dec 17, 2009 |
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61379571 |
Sep 2, 2010 |
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61314794 |
Mar 17, 2010 |
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Current U.S.
Class: |
506/26 |
Current CPC
Class: |
C07K 16/005 20130101;
C12P 19/34 20130101; C40B 50/06 20130101; C07K 2317/565 20130101;
C07K 2317/622 20130101; C40B 40/08 20130101; C07K 2317/76 20130101;
C07K 2317/21 20130101; C12N 15/66 20130101; C07K 2317/64
20130101 |
Class at
Publication: |
506/26 |
International
Class: |
C12P 19/34 20060101
C12P019/34 |
Claims
1. A method for producing a collection of nucleic acids, wherein
each nucleic acid encodes a human immunoglobulin variable domain
comprising a plurality of complementarity determining region 3
(CDR3) sequences isolated separately from the immunoglobulin
variable domain repertoire from a mammalian species, the method
comprising: (a) providing a plurality of Acceptor Framework nucleic
acid sequences encoding distinct human immunoglobulin variable
domains, each Acceptor Framework nucleic acid sequence comprising a
first framework region (FR1), a second framework region (FR2), a
third framework region (FR3), and a fourth framework region (FR4),
wherein the FR1 and FR2 regions are interspaced by a
complementarity determining region 1 (CDR1), the FR2 and FR3
regions are interspaced by a complementarity determining region 2
(CDR2), and the FR3 and FR4 regions are interspaced by a stuffer
nucleic acid sequence comprising at least two Type IIs restriction
enzyme recognition sites interspaced by a random nucleic acid
sequence encodes a polypeptide that performs the function of a
variable immunoglobulin CDR3 region; (b) providing a plurality of
diversified nucleic acid sequences encoding complementarity
determining region 3 (CDR3) sequences isolated from the mammalian
species immunoglobulin repertoire wherein each of the plurality of
diversified nucleic acid sequences comprises a Type IIs restriction
enzyme recognition site at each extremity; (c) digesting each of
the plurality of nucleic acid sequences encoding the CDR3 regions
using a Type IIs restriction enzyme that binds to the Type IIs
restriction enzyme recognition site of step (b) and digesting the
stuffer nucleic acid sequence of step (a) from the Acceptor
Framework using a Type IIs restriction enzyme that binds to the
Type IIs restriction enzyme recognition site of step (a); and (d)
ligating the digested nucleic acid sequences encoding the CDR3
regions or the amino acid sequences of step (c) into the digested
Acceptor Framework of step (c) such that the FR3 and FR4 regions
are interspaced by the nucleic acid sequences encoding the CDR3
region or the amino acid sequence that can fulfill the role of a
CDR3 region and a complete immunoglobulin variable domain encoding
sequences that do not contain the Type IIs restriction enzyme
recognition sites of steps (a) and (b) are restored.
2. The method of claim 1, wherein step (b) is performed by
amplifying the CDR3 sequence from a mammalian species using
oligonucleotide primers containing a Type IIs restriction site.
3. The method of claim 2, wherein the oligonucleotide primer is
designed to enhance compatibility between the mammalian CDR3
sequence and the Acceptor Framework encoding a human immunoglobulin
variable domain.
4. The method of claim 3, wherein the oligonucleotide primer is
designed to modify a nucleic acid sequence at a boundary of the
mammalian CDR3 sequence to produce a compatible cohesive nucleotide
sequence in the Acceptor Framework encoding a human immunoglobulin
variable domain.
5. The method of claim 1, wherein the mammalian species is human,
non-human primate, rodent, canine, feline, sheep, goat, cattle,
horse, a member of the Camelidae family, llama, camel, dromedary,
or pig.
6. The method of claim 1, wherein the Type IIs restriction enzyme
recognition sites of step (a) and step (b) are recognized by a
different Type IIs restriction enzyme.
7. The method of claim 6, wherein the Type IIs restriction enzyme
recognition sites are BsmBI recognition sites, BsaI recognition
sites, FokI recognition sites or a combination thereof.
8. The method of claim 1, wherein the diversified nucleic acid
sequences encoding CDR3 sequences encode heavy chain CDR3 (CDR H3)
sequences, light chain CDR3 (CDR L3) sequences or a combination
thereof.
9. The method of claim 1, wherein the Acceptor Framework nucleic
acid sequence comprises a human heavy chain variable gene sequence
selected from VH1-2, VH1-69, VH1-18, VH3-30, VH3-48, VH3-23, and
VH5-51.
10. The method of claim 1, wherein the Acceptor Framework nucleic
acid sequence comprises a human kappa light chain variable gene
sequence.
11. The method of claim 10, wherein the human kappa light chain
variable gene sequence is selected from VK1-33, VK1-39, VK3-11,
VK3-15, and VK3-20.
12. The method of claim 1, wherein the Acceptor Framework nucleic
acid sequence comprises a human lambda light chain variable gene
sequence.
13. The method of claim 12, wherein the human lambda light chain
variable gene sequence is selected from VL 1-44 and VL 1-51.
14. The method of claim 1, wherein the plurality of Acceptor
Framework nucleic acid sequences comprises a mixture of at least
one variable heavy chain (VH) Acceptor Framework nucleic acid
sequence and at least one variable light chain Acceptor Framework
nucleic acid sequence.
15. The method of claim 1, further comprising the steps of (e)
cloning the library of nucleic acids encoding immunoglobulin
variable domains of step (d) into an expression vector and (f)
transforming the expression vector of step (e) into a host cell and
culturing the host cell under conditions sufficient to express a
plurality of immunoglobulin variable domain encoded by the
library.
16. The method of claim 15, wherein the host cell is E. coli.
17. The method according to claim 16, wherein the expression vector
is a phagemid or a phage vector.
18. A method for producing a collection of nucleic acids, wherein
each nucleic acid encodes a human immunoglobulin variable domain
comprising a plurality of complementarity determining region 3
(CDR3) sequences isolated separately from immunoglobulin variable
domains from an immunized non-human mammal, the method comprising:
(a) providing a plurality of Acceptor Framework nucleic acid
sequences encoding distinct human immunoglobulin variable domains,
each Acceptor Framework nucleic acid sequence comprising a first
framework region (FR1), a second framework region (FR2), a third
framework region (FR3), and a fourth framework region (FR4),
wherein the FR1 and FR2 regions are interspaced by a
complementarity determining region 1 (CDR1), the FR2 and FR3
regions are interspaced by a complementarity determining region 2
(CDR2), and the FR3 and FR4 regions are interspaced by a stuffer
nucleic acid sequence at least two Type IIs restriction enzyme
recognition sites interspaced by a random nucleic acid sequence
encodes a polypeptide that performs the function of a variable
immunoglobulin CDR3 region; (b) providing a plurality of
diversified nucleic acid sequences encoding complementarity
determining region 3 (CDR3) sequences isolated from the immunized
non-human mammal wherein each of the plurality of diversified
nucleic acid sequences comprises a Type IIs restriction enzyme
recognition site at each extremity; (c) digesting each of the
plurality of nucleic acid sequences encoding the CDR3 regions using
a Type IIs restriction enzyme that binds to the Type IIs
restriction enzyme recognition site of step (b) and digesting the
stuffer nucleic acid sequence of step (a) from the Acceptor
Framework using a Type IIs restriction enzyme that binds to the
Type IIs restriction enzyme recognition site of step (a); and (d)
ligating the digested nucleic acid sequences encoding the CDR3
regions or the amino acid sequences of step (c) into the digested
Acceptor Framework of step (c) such that the FR3 and FR4 regions
are interspaced by the nucleic acid sequences encoding the CDR3
region or the amino acid sequence that can fulfill the role of a
CDR3 region and a complete immunoglobulin variable domain encoding
sequences that do not contain the Type IIs restriction enzyme
recognition sites of steps (a) and (b) are restored.
19. The method of claim 18, wherein step (b) is performed by
amplifying the CDR3 sequence from the immunized non-human mammal
using oligonucleotide primers containing a Type IIs restriction
site.
20. The method of claim 19, wherein the oligonucleotide primer is
designed to enhance compatibility between the mammalian CDR3
sequence and the Acceptor Framework encoding a human immunoglobulin
variable domain.
21. The method of claim 20, wherein the oligonucleotide primer is
designed to modify a nucleic acid sequence at a boundary of the
mammalian CDR3 sequence to produce a compatible cohesive nucleotide
sequence in the Acceptor Framework encoding a human immunoglobulin
variable domain.
22. The method of claim 18, wherein step (b) is performed by
amplifying the CDR H3 sequence from the non-human mammal using
oligonucleotide primers containing a FokI IIs restriction site.
23. The method of claim 18, wherein the non-human mammal is
non-human primate, rodent, canine, feline, sheep, goat, cattle,
horse, llama, camel, dromedary, or pig.
24. The method of claim 18, wherein the Type IIs restriction enzyme
recognition sites of step (a) and step (b) are recognized by a
different Type IIs restriction enzyme.
25. The method of claim 18, wherein the Type IIs restriction enzyme
recognition sites are BsmBI recognition sites, BsaI recognition
sites, FokI recognition sites or a combination thereof.
26. The method of claim 18, wherein the diversified nucleic acid
sequences encoding CDR3 sequences encode heavy chain CDR3 (CDR H3)
sequences, light chain CDR3 (CDR L3) sequences or a combination
thereof.
27. The method of claim 18, wherein the Acceptor Framework nucleic
acid sequence comprises a human heavy chain variable gene sequence
selected from VH1-2, VH1-69, VH1-18, VH3-30, VH3-48, VH3-23, and
VH5-51.
28. The method of claim 18, wherein the Acceptor Framework nucleic
acid sequence comprises a human kappa light chain variable gene
sequence.
29. The method of claim 28, wherein the human kappa light chain
variable gene sequence is selected from VK1-33, VK1-39, VK3-11,
VK3-15, and VK3-20.
30. The method of claim 18, wherein the Acceptor Framework nucleic
acid sequence comprises a human lambda light chain variable gene
sequence.
31. The method of claim 30, wherein the human lambda light chain
variable gene sequence is selected from VL 1-44 and VL 1-51.
32. The method of claim 18, wherein the plurality of Acceptor
Framework nucleic acid sequences comprises a mixture of at least
one variable heavy chain (VH) Acceptor Framework nucleic acid
sequence and at least one variable light chain Acceptor Framework
nucleic acid sequence.
33. The method of claim 18, further comprising the steps of (e)
cloning the library of nucleic acids encoding immunoglobulin
variable domains of step (d) into an expression vector and (f)
transforming the expression vector of step (e) into a host cell and
culturing the host cell under conditions sufficient to express a
plurality of immunoglobulin variable domain encoded by the
library.
34. The method of claim 33, wherein the host cell is E. coli.
35. The method according to claim 33, wherein the expression vector
is a phagemid or a phage vector.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/952,659, filed Nov. 23, 2010, which is a
continuation-in-part of U.S. patent application Ser. No.
12/784,190, filed May 20, 2010, which claims the benefit of U.S.
Provisional Application Nos. 61/179,850, filed May 20, 2009,
61/287,336, filed Dec. 17, 2009 and 61/314,794, filed Mar. 17,
2010, and this application claims the benefit of U.S. Provisional
Application No. 61/379,571, filed Sep. 2, 2010, the contents of
each of which are hereby incorporated by reference in their
entirety.
INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING
[0002] The contents of the text file named
"418CIPCONUSSeqList_ST25.txt," which was created on May 5, 2014 and
is 156 KB in size, are hereby incorporated by reference in their
entirety.
FIELD OF THE INVENTION
[0003] The invention relates to the generation of libraries of DNA
sequences encoding homologous polypeptides and to the use of such
libraries. This invention in particular relates to the generation
of collections of synthetic antibody fragments in which one or
several complementary determining regions (CDR) are replaced by a
collection of the corresponding CDR captured from a natural source.
The invention further relates to the generation of collections of
antibody fragments containing CDR derived from an immunized animal
and their use as a better source to derive high affinity antibody
fragments. The invention further relates to the diversification of
a portion of a polypeptide by inserting a diversified sequence of
synthetic or natural origin without the need for modification of
the original polypeptide coding sequence.
BACKGROUND OF THE INVENTION
[0004] An antibody is composed of four polypeptides: two heavy
chains and two light chains. The antigen binding portion of an
antibody is formed by the light chain variable domain (VL) and the
heavy chain variable domain (VH). At one extremity of these domains
six loops form the antigen binding site and also referred to as the
complementarity determining regions (CDR). Three CDRs are located
on the VH domain (H1, H2 and H3) and the three others are on the VL
domain (L1, L2 and L3). During B cell development a unique
immunoglobulin region is formed by somatic recombination known as
V(D)J recombination. The variable region of the immunoglobulin
heavy or light chain is encoded by different gene segments. The
heavy chain is encoded by three segments called variable (V),
diversity (D) and joining (J) segments whereas the light chain
variable is formed by the recombination of only two segments V and
J. A large number of antibody paratopes can be generated by
recombination between one of the multiple copies of the V, D and J
segments that are present in the genome. The V segment encodes the
CDR1 and CDR2 whereas the CDR3 is generated by the recombination
events. During the course of the immune response further diversity
is introduced into the antigen binding site by a process called
somatic hypermutation (SHM). During this process point mutations
are introduced in the variable genes of the heavy and light chains
and in particular into the regions encoding the CDRs. This
additional variability allows for the selection and expansion of B
cells expressing antibody variants with improved affinity for their
cognate antigen.
[0005] In recent years several display technologies have emerged
and allow for the screening of large collections of proteins or
peptides. These include phage display, bacterial display, yeast
display and ribosome display (Smith G P. Science. 1985 Jun. 14;
228(4705):1315-7; Hanes J and Pluckthun A. Proc Natl Acad Sci USA.
1997 May 13; 94(10):4937-42; Daugherty P S et al., Protein Eng.
1998 September; 11(9):825-32; Boder E T and Wittrup K D. Nat
Biotechnol. 1997 June; 15(6):553-7). In particular these methods
have been applied extensively to antibodies and fragments thereof.
A number of methods have been described to generate libraries of
polypeptides and to screen for members with desired binding
properties.
[0006] A first approach is to capture by gene amplification
rearranged immunoglobulin genes from natural repertoires using
either tissues or cells from humans or other mammals as a source of
genetic diversity. These collections of rearranged heavy and light
chains (VH and VL) are then combined to generate libraries of
binding pairs that can be displayed on bacteriophage or on other
display packages such as bacteria, yeast or mammalian cells. In
this case a large fraction of the immunoglobulin repertoire found
in the donor is captured. Thus all of the frameworks encoded by the
donor germline genes can be found in such repertoires as well as
diversity generated both by V(D)J recombination and by somatic
hypermutation (Marks J D et al., J Mol Biol. 1991 Dec. 5;
222(3):581-97; McCaffety U.S. Pat. No. 5,969,108).
[0007] A limitation of natural repertoires is that naturally
occurring antibodies can be based on frameworks with low intrinsic
stability that limit their expression levels, shelf life and their
usefulness as reagents or therapeutic molecules. In order to
overcome these limitations a number of methods have been developed
to generate synthetic antibody libraries. In these approaches, a
unique or a limited number of selected antibody framework encoded
by their corresponding germline genes are selected. The selection
of these frameworks is commonly based on their biochemical
stability and/or their frequency of expression in natural antibody
repertoires. In order to generate a collection of binding proteins,
synthetic diversity is then introduced in all or a subset of CDRs.
Typically either the whole or part of the CDR is diversified using
different strategies. In some cases diversity was introduced at
selected positions within the CDRs (Knappik A et al., J Mol Biol.
2000 Feb. 11; 296(1):57-86). Targeted residues can be those
frequently involved in antigen contact, those displaying maximal
diversity in natural antibody repertoires or even residues that
would be preferentially targeted by the cellular machinery involved
in generating somatic hypermutations during the natural affinity
maturation process (Balint R F, Larrick J W. Gene. 1993 Dec. 27;
137(1):109-18.).
[0008] Several methods have been used to diversify the antibody
CDRs. Overlapping PCR using degenerate oligonucleotides have been
extensively used to assemble framework and CDR elements to
reconstitute antibody genes. In another approach, unique
restriction enzyme sites have been engineered into the framework
regions at the boundary of each CDR allowing for the introduction
of diversified CDRs by restriction enzyme mediated cloning. In any
case, as all the members of the library are based on frameworks
with selected and preferred characteristics, it is anticipated that
the antibodies derived from these repertoires are more stable and
provide a better source of useful reagents. (Knappik, U.S. Pat. No.
6,696,248; Sidhu S S, et al., Methods Enzymol. 2000; 328:333-63;
Lee C V et al., J Mol Biol. 2004 Jul. 23; 340(5):1073-93).
[0009] However, an important limitation of these synthetic
libraries is that a significant proportion of the library members
are not expressed because the randomly diversified sequences do not
allow for proper expression and/or folding of the protein. This
problem is particularly significant for the CDR3 of the heavy
chain. Indeed, this CDR often contributes to most of the binding
energy to the antigen and is highly diverse in length and sequence.
While the other CDR (H1, H2, L1, L2 and L3) can only adopt a
limited number of three dimensional conformations, known as
canonical folds, the number of conformations that can be adopted by
the heavy chain CDR3 remains too diverse to be predicted
(Al-Lazikani B et al., J Mol Biol. 1997 Nov. 7; 273(4):927-48). In
addition, the use of long degenerate oligonucleotides used to cover
long CDR H3 often introduces single base-pair deletions. These
factors significantly reduce the functional size of synthetic
repertoires.
[0010] Both natural and synthetic repertoires have advantages and
limitations. On one hand, strategies relying on the capture of
naturally rearranged antibody variable genes are not optimal as
they include potentially less favorable frameworks within the
library. A positive aspect is that these rearranged variable genes
include CDRs which are compatible with proper domain folding as
they have been expressed in context of a natural antibody. On the
other hand, strategies based on selecting frameworks and inserting
synthetic diversity benefit from the improved stability of the
frameworks but are limited by the large number of CDR sequences
that are not compatible with folding and/or expression and can
destabilize the overall domain (FIG. 1A). There is therefore a need
for novel approaches that could combine the benefits of using
selected frameworks with desirable characteristics and combine them
with properly folded CDRs for instance derived from natural
repertoires.
[0011] All described approaches to generate antibody libraries
either by capturing naturally rearranged antibody sequences or by
generating diversity by synthetic means are limited by the
occurrence of frame shift mutations leading to non-functional
antibody sequences. These mutations can appear at multiple steps of
the molecular handling of the DNA encoding the antibodies such as
PCR amplification and DNA fragment assembly as well as molecular
cloning. The frequency of non-functional members in antibody
libraries typically ranges from 15% to 45% depending of the
strategies employed to capture or generate the antibody diversity
(Persson M A et al., Proc Natl Acad Sci USA. 1991 Mar. 15;
88(6):2432-6; Schoonbroodt S, et al., Nucleic Acids Res. 2005 May
19; 33(9):e81; Soderling E et al., Nat Biotechnol. 2000 August;
18(8):852-6; Rothe et al., J Mol Biol. 2008 Feb. 29;
376(4):1182-200). The frequency of sequences encoding non
functional antibodies has a major impact on the antibody
identification process. First, the functional size of the library
is reduced and, because non-functional clones often have a growth
advantage during the propagation of the libraries, they expand
faster and can compromise the identification process of antibody
candidates (De Bruin R et al., Nat Biotechnol 1999 Apr. 17:
397-399). These issues are recognized as serious limitations for
fully exploiting the potential of antibody libraries. The
generation of highly functional libraries remains a challenge in
the field and has prompted many efforts to improve the process. For
instance, multiple diversification strategies aiming at mimicking
the amino acids usage found in natural CDR sequences have been used
in order to more effectively sample the huge diversity of possible
sequence combination encoded by synthetic CDRs (de Kruif J et al.,
J Mol Biol. 1995 Apr. 21; 248(1):97-105; Sidhu S S et al., J Mol
Biol. 2004 Apr. 23; 338(2):299-310). Another approach is to clean
up the initial library in order to remove nonfunctional clones at
the potential expense of diversity loss. This has been applied to
the pre-selection of synthetic repertoires by binding the antibody
library to a generic ligand. This step allowed for the enrichment
of library members that are able to express and to fold properly
and can be used to recreate a more functional library (Winter and
Tomlinson, U.S. Pat. No. 6,696,245 B2). Regardless of the approach
the quality of any library is dependent on the efficiency of the
molecular biology methods applied to generate the library and
generally lead to 15% to 45% non-functional members of the library.
There is therefore a need for novel and highly efficient approaches
that minimize the frequency on non-functional genes due to frame
shifts introduced during the molecular cloning steps and that
maximize the functionality of libraries by capturing CDR regions
having a high propensity of being correctly folded into antibody
frameworks with desirable properties. Furthermore, there is a need
for approaches that allow the capture of the CDR sequences from an
animal immune repertoire into a therapeutically useful context such
as human antibody frameworks in order to improve the generation
process of high affinity antibodies.
SUMMARY OF THE INVENTION
[0012] The present invention provides methods of generating
libraries of nucleic acid sequences that combine the benefits of
stable framework selection and the insertion of naturally encoded
complementarity determining regions (CDRs) or amino acid sequences
that can fulfill the role of a CDR that have been selected in a
natural context of a functional polypeptide such as an antibody.
The method allows for the recovery of long CDRs or amino acid
sequences that can fulfill the role of a CDR that are very
difficult to encode using synthetic approaches. This invention, by
combining stable frameworks and properly folded CDRs or amino acid
sequences that can fulfill the role of a CDR, maximizes the
proportion of functional antibodies in the library and therefore
the performance of the selection process and the quality of
selected clones. The invention provides a method to capture
naturally expressed CDRs from different species and to insert them
into a human antibody framework. This allows for the use of CDR H3
repertoires that differ significantly in length and composition
when compared to the human repertoire. The invention enables the
generation of human antibody fragments featuring structural
repertoires derived from other species and thus the capacity to
sample different structural spaces. The present methods are also
used to introduce CDRs of synthetic origin or amino acid sequences
that can fulfill the role of a CDR with a higher success frequency
than alternative methods introducing fewer errors causing frame
shifts in the coding sequence. Libraries generated using the
present methods contain a high frequency of functional variants.
Libraries of variants generated according to this method are used
for selection and screening with any described display, selection
and screening technology.
[0013] The analysis of immune repertoires from different species
or, within a species, at different development stages has revealed
some striking differences in the characteristics of CDR H3
composition and length. For instance the average CDRH3 length in
humans is longer in adult when compared to fetal life or to
newborns (Schroeder Jr, H W et al., 2001 Blood 98; 2745-2751).
Interestingly despite large similarities between human and primate
antibody germline genes, the evolution of the CDRH3 length during
development differs (Link J M et al., Molecular Immunol. 2005 42;
943-955). The comparison of CDR H3 sequences found in mice and
humans clearly shows that the average length is significantly
shorter in mice (Rock E P et al., J Exp Med 1994 179; 323-328).
During early B cell development in the bone marrow, the average CDR
H3 length increases in mice whereas it tends to decrease in humans
and in addition the amino acid composition of the murine and human
CDRH3 repertoires differ (Zemlin M et al., 2003 J Mol Biol 334;
733-749; Ivanov I et al., 2005 J Immunol 174; 7773-7780). These
examples indicate that different species express different ranges
of CDR H3 repertoires despite the fact that they are globally
exposed to similar classes of antigens and the biological
significance of these observations remain to be further studied. It
has been demonstrated that the shape of the combining site of
antibodies directed against small antigens such as haptens or
peptides differ from those directed against large proteins and the
shape of the combining site is dictated by the length and
composition of the CDRs (Collis A et al., J Mol Biol 2003 325;
337-354). From these finding it can be anticipated that the CDR H3
repertoire expressed by different species have varying propensities
to react efficiently against different target classes.
[0014] The methods and antibody libraries provided herein are
designed to exploit the various repertoires expressed by different
species for the generation of therapeutic antibodies. These
repertoires that explore different tridimensional spaces might
allow for the generation of antibodies against a wider variety of
target classes and epitopes. Methods to generate libraries form
naive or immunized animals are well described and these methods
allow for the capturing of the corresponding repertoires and the
generation of antibodies. However, antibodies derived from these
libraries are not of human origin and are therefore not well suited
for human therapy without performing further engineering work such
as humanization. There is therefore a need for novel methods to
harness the diversity expressed in the repertoire from different
species and to exploit this diversity in the therapeutically useful
context of a human antibody.
[0015] The methods and antibody libraries provided herein address
several of the limitations described above and are an improvement
over the current art. First, the methods provided herein combine
the benefits of stable framework selection and the insertion of
naturally encoded CDRs that have been selected in a natural context
of a functional antibody. Second, the methods allow for a highly
efficient insertion of synthetic or natural CDRs sequences into an
antibody framework that significantly minimizes the number of frame
shifts in the library and therefore improves its quality. Finally,
the invention allows for a novel way to use naturally occurring
antibody structural diversity by capturing naturally expressed CDR
H3 repertoires from different species and to insert them into human
antibody frameworks. It is thus possible to exploit these
structurally diverse repertoires in a productive way for the
generation of antibodies for human therapy.
[0016] The methods provided herein generate antibodies that contain
a stable framework and correctly folded CDRs or amino acid
sequences that can fulfill the role of a CDR. The methods capture
the natural diversity of sequences in stable frameworks.
[0017] In the methods provided herein, the germline sequences for
framework regions 1, 2 and 3 (FR1, FR2 and FR3) are selected from
the desired organism, for example, from the human genome (see e.g.,
FIGS. 2 and 6). In one embodiment of this method, selected antibody
variable domains are modified by introducing a stuffer sequence
that will serve as an integration site for diversified sequences.
Diversity is introduced into the sequence outside of the
immunoglobulin coding region by introducing restriction enzyme
recognition sites, for example, Type IIs restriction sites, at a
desired location such as the variable heavy chain complementarity
determining region 3 (CDR H3), the variable light chain
complementarity determining region 3 (CDR L3), the variable heavy
chain complementarity determining region 1 (CDR H1), the variable
light chain complementarity determining region 1 (CDR L1), the
variable heavy chain complementarity determining region 2 (CDR H2)
or the variable light chain complementarity determining region 2
(CDR L2). While the examples provided herein demonstrate diversity
at the CDR3 region (in the variable heavy chain region and/or
variable light chain region), it is understood that diversity can
be achieved at any desired location, such as, but not limited to,
the CDR1 region (in the variable heavy chain region and/or variable
light chain region) or the CDR2 region (in the variable heavy chain
region and/or variable light chain region). Diversified DNA
sequences are generated with flanking sequences that include Type
IIs restriction sites. In the methods provided herein, the cohesive
ends generated by the restriction enzymes are compatible and the
reading frame is maintained, thus allowing the diversified DNA
fragments to be ligated into an acceptor framework.
[0018] The methods provided herein are also useful for generating
amino acid sequences having diversified regions encoded therein.
For example, in the methods provided herein, the sequences for the
non-diversified portions of the encoded amino acid are selected
from the desired organism, for example, from the human sequence. A
portion of the encoded amino acid sequence is modified by
introducing a stuffer sequence that will serve as an integration
site for diversified sequences. Diversity is introduced into the
sequence at the desired location(s) by introducing restriction
enzyme recognition sites, for example, Type IIs restriction sites,
at a desired location within the encoded amino acid sequence.
Diversified DNA sequences are generated with flanking sequences
that include Type IIs recognition sites. In the methods provided
herein, the cohesive ends generated by the restriction enzymes are
compatible and the reading frame is maintained, thus allowing the
diversified DNA fragments to be ligated into an acceptor
framework.
[0019] The methods provided herein are also useful for generating
libraries of diverse nucleic acids that encode a higher percentage
of polypeptides that can fold properly and be expressed as a
functional entity such as, e.g., an immunoglobulin.
[0020] A number of factors can significantly impact the quality of
a polypeptide repertoire--such as an antibody library--and
therefore the likelihood of identifying polypeptides with desired
properties. The size and diversity of the repertoire are obviously
critical, and studies have demonstrated the correlation between the
size of an antibody repertoire and the affinity of the antibodies
isolated from that repertoire (Griffiths et al., EMBO J. 1994 Jul.
15; 13(14):3245-60). The size of a library is typically determined
by the number of transformants obtained during construction and the
diversity is estimated by sequencing a limited number of library
members. This type of analysis only provides a superficial
assessment of the library quality. In particular, the sequence
information cannot reliably indicate whether a diversified
polypeptide can fold properly and be expressed as a functional
entity. Therefore, depending on the source of diversity or on the
strategy that is applied to diversify a polypeptide, the functional
size of repertoire can differ significantly from its theoretical
size (based on size and diversity assessment). Ideally, a
repertoire should only contain functional members that can produce
a polypeptide having potentially the desired characteristics. In
addition, members of the library encoding non-functional
polypeptides represent not only useless diversity but they can also
have a major negative impact during the selection process.
[0021] As described above, the quality of any library is dependent
on the efficiency of the molecular biology methods applied to
generate the library and many methods generally lead to about 15%
to 45% non-functional members of the library. It is therefore
important during the cloning or diversification steps of library
construction to maximize the number of sequences that are in frame
and ideally encode polypeptides that can fold into a functional
polypeptide. Methods based on preselecting library members for
proper folding via binding to proteins such as Protein A or Protein
L, have been described. (See e.g., Winter and Tomlinson, U.S. Pat.
No. 6,696,245 B2). In addition, as errors leading to frame shifts
in the coding sequence can be introduced at each cloning step, it
is important to minimize the number of cloning or DNA assembly
steps and to develop efficient cloning strategies. The Type IIS
restriction cloning approach described in the invention leads to a
high number of in frame inserts (>90%) but does not ensure that
the diversified DNA sequences that are cloned encode a polypeptide
that allow proper folding of an immunoglobulin variable domain and
can fulfill the function of a CDR.
[0022] Thus, the invention provides methods for addressing these
limitations and generating libraries of diversified nucleic acids
that encode a higher percentage of functional members. One
embodiment of the invention provides methods to select functional
diversity introduced into one of the antibody variable domains by
expressing the diversified heavy or light chain variable domains in
the context of a constant heavy or variable domain (dummy chains)
and selecting for library members that can be expressed and
displayed at the surface of a display system such as phage. This
pre-selection step is achieved by expressing the diversified
polypeptide repertoire using a helper phage that does not encode a
wild type pIII protein. In this system, phage assembly relies on
the polypeptide-pIII fusion protein that therefore has to able to
be expressed and sufficiently folded to be integrated into a phage
particle. This pIII deficient helper phage called "Hyperphage" has
been described as a way to select for open reading frames. (See
e.g., Hust M et al., Biotechniques 2006 September; 41(3):335-42). A
limitation of this technique, however, is that, after
pre-selection, the common variable chain that was expressed in
conjunction with the diversified repertoire has to be replaced by
another variable repertoire to obtain a library with diversified
heavy and light chains using standard restriction cloning of the
entire variable domain.
[0023] In order to combine the benefit of the invention for
diversification of the CDR3 region by capture of different sources
of natural or synthetic diversity using a Type IIS restriction
enzyme and the use of a common chain for repertoire pre-selection,
another embodiment of the invention provides methods to identify
common--or dummy--variable domains that contain a stuffer DNA
fragment used for diversity cloning that can also fulfill the
function of a functional CDR3. This allows for the generation of
Acceptor libraries that contain pre-selected and functional
diversified light chain variable domains that can directly be used
for the insertion of captured CDRH3 as shown in FIG. 30. The
Examples provided herein describe methods of identifying such
sequences, as well as several examples of such stuffer DNA
fragments that must accommodate three major constrains: 1) include
two Type IIS restriction sites; 2) maintain the reading frame
between FR3 and FR4 regions and 3) encode a heavy variable domain
CDR3 that allows the folding and expression of an antibody variable
domain.
[0024] Libraries generated using the method provided therein have
an increased frequency of potentially functional members by
reducing or eliminating out of frame sequences. Such preselected
libraries contain at least 90% of sequences that are in frame and
thus have the potential to encode a functional polypeptide.
[0025] In the methods provided herein, an "Acceptor Framework" is
generated using a "stuffer fragment" of DNA that contain and are,
preferably, bordered by two Type IIs restriction enzyme sites. (See
e.g., FIG. 6). Preferably, these two Type IIs restriction enzyme
sites digest sequences at the boundary of the site at which
diversity is desired, such as, for example, the CDR H3 region, the
CDR L3 region, the CDR H1 region, the CDR L1 region, the CDR H2
region or the CDR L2 region. As used herein, the term "Acceptor
Framework" refers to a nucleic acid sequence that include the
nucleic acid sequences encoding the FR1, FR2, FR3 and FR4 regions,
the nucleic acid sequences encoding two CDRs or amino acid
sequences that can fulfill the role of these CDRs, and a "stuffer
fragment" that serves as the site of integration for diversified
nucleic acid sequence. For example, in embodiments where diversity
at the CDR3 region (in the variable heavy chain region and/or the
variable light chain region) is desired, the Acceptor Framework
includes the nucleic acid sequences encoding the FR1, FR2, FR3 and
FR4 regions, the nucleic acid sequences encoding the CDR1 and CDR2
regions, and a "stuffer fragment" that serves as the site of
integration for diversified nucleic acid sequence. For example, in
embodiments where diversity at the CDR2 region (in the variable
heavy chain region and/or the variable light chain region) is
desired, the Acceptor Framework includes the nucleic acid sequences
encoding the FR1, FR2, FR3 and FR4 regions, the nucleic acid
sequences encoding the CDR1 and CDR3 regions, and a "stuffer
fragment" that serves as the site of integration for diversified
nucleic acid sequence. For example, in embodiments where diversity
at the CDR1 region (in the variable heavy chain regions and/or the
variable light chain regions) is desired, the Acceptor Framework
includes the nucleic acid sequences encoding the FR1, FR2, FR3 and
FR4 regions, the nucleic acid sequences encoding the CDR2 and CDR3
regions, and a "stuffer fragment" that serves as the site of
integration for diversified nucleic acid sequence.
[0026] The terms "stuffer fragment", "stuffer DNA fragment" and
"stuffer sequence" or any grammatical variation thereof are used
interchangeably herein to refer to a nucleic acid sequence that
includes at least two Type IIs recognition sites and a diversified
sequence. The Acceptor Framework can be a variable heavy chain (VH)
Acceptor Framework or a variable light chain (VL) Acceptor
Framework. The use of the Acceptor Frameworks and the stuffer
fragments contained therein allow for the integration of a CDR
sequence (natural or synthetic) or an amino acid sequence that can
fulfill the role of the CDR into the acceptor framework with no
donor framework nucleotides or residues contained therein or needed
for integration. For example, the use of the Acceptor Frameworks
and the stuffer fragments contained therein allow for the
integration of a CDR sequence (natural or synthetic) selected from
CDR H3, CDR L3, CDR H2, CDR L2, CDR H1 and CDR L1, or an amino acid
sequence that can fulfill the role of a CDR selected from CDR H3,
CDR L3, CDR H2, CDR L2, CDR H1 and CDR L1 into the acceptor
framework with no donor framework nucleotides or residues contained
therein or needed for integration. Thus, upon integration, the
stuffer fragment is removed in full, and the coding region of the
acceptor protein and the inserted proteins fragments (i.e., the
CDRs) are intact.
[0027] In some embodiments, the stuffer fragment includes two Type
IIS restriction sites, maintains the reading frame between FR3 and
FR4 regions and encodes a heavy variable domain CDR3 that allows
the folding and expression of an antibody variable domain.
[0028] The methods provided herein use primers that are designed to
contain cleavage sites for Type IIs restriction enzymes at the
boundary of the site of at which diversity is desired, for example,
the CDR H3 region, the CDR L3 region, the CDR H2 region, the CDR
L2, the CDR H1 region or the CDR L1 region. Random, naturally
occurring CDR clones (see e.g., FIG. 10) or synthetic CDR sequences
(see e.g., Example 6) or amino acid sequences that can fulfill the
role of the CDR are captured in the Acceptor Frameworks used
herein. For example, in embodiments where diversity at the CDR3
region (in the variable heavy chain region and/or the variable
light chain region) is desired, random, naturally occurring CDR3
clones (see e.g., FIG. 10) or synthetic CDR3 sequences (see e.g.,
Example 6) or amino acid sequences that can fulfill the role of a
CDR3 are captured in the Acceptor Frameworks used herein. For
example, in embodiments where diversity at the CDR2 region (in the
variable heavy chain region and/or the variable light chain region)
is desired, random, naturally occurring CDR2 clones (see e.g.,
methods shown in FIG. 10) or synthetic CDR2 sequences (see e.g.,
methods shown in Example 6) or amino acid sequences that can
fulfill the role of a CDR2 are captured in the Acceptor Frameworks
used herein. For example, in embodiments where diversity at the
CDR1 region (in the variable heavy chain region and/or the variable
light chain region) is desired, random, naturally occurring CDR1
clones (see e.g., methods shown in FIG. 10) or synthetic CDR1
sequences (see e.g., methods shown in Example 6) or amino acid
sequences that can fulfill the role of a CDR1 are captured in the
Acceptor Frameworks used herein. As an example, oligonucleotides
primers specific for flanking regions of the DNA sequence encoding
the CDR H3 of immunoglobulins, i.e., specific for the FR3 and FR4
of the variable region, were designed. Oligonucleotide primers
specific for flanking regions of the DNA sequences encoding other
regions, such as, for example, the CDR L3, CDR H1, CDR L1, CDR H2,
or CDR L2, can also be designed. These oligonucleotides contain at
their 5' end a site for a Type IIs restriction enzyme whereas their
3' portion matches the targeted DNA sequence.
[0029] In some embodiments, the primer is a nucleic acid selected
from the group consisting of SEQ ID NOs: 120-254.
[0030] The methods provided herein use Type IIs restriction
enzymes, such as, for example, FokI, to insert natural CDR
sequences, such as, for example, natural CDR H3, CDR L3, CDR H1,
CDR L1, CDR H2, or CDR L2 sequences into the acceptor frameworks
described herein. The methods provided herein use Type IIs
restriction enzymes, such as, for example, FokI, to insert
synthetic CDR sequences, such as, for example, synthetic CDR H3,
CDR L3, CDR H1, CDR L1, CDR H2, or CDR L2 sequences into the
acceptor frameworks described herein. The methods provided herein
use Type IIs restriction enzymes, such as, for example, FokI, to
insert amino acid sequences that can fulfill the role of a desired
CDR region, such as, for example, an amino acid sequence that can
fulfill the role of a natural or synthetic CDR H3, CDR L3, CDR H1,
CDR L1, CDR H2, or CDR L2 region into the acceptor frameworks
described herein. The Type IIs restriction enzymes are enzymes that
cleave outside of their recognition sequence to one side. These
enzymes are intermediate in size, typically 400-650 amino acids in
length, and they recognize sequences that are continuous and
asymmetric. Suitable Type IIs restriction enzymes, also known as
Type IIs restriction endonucleases, and the sequences they identify
are described, for example, in Szybalski et al., "Class-IIS
Restriction Enzymes--a Review." Gene, vol. 100: 13-26 (1991), the
contents of which are hereby incorporated in their entirety by
reference.
[0031] Primary Libraries include a VH Acceptor Framework and a
fixed VL sequence (also referred to as a "dummy VL" sequence) or a
VL Acceptor Framework and a fixed VH sequence (also referred to as
a "dummy VH" sequence). Thus, Primary Libraries exhibit diversity
in only one of the heavy or light chains. Secondary Libraries are
generated by ligating a VH Acceptor Framework and a VL Acceptor
Framework together (see e.g., Example 7). Secondary Libraries have
diversity in both the heavy and light chains.
[0032] The invention provides methods for producing a collection of
nucleic acids, wherein each nucleic acid encodes a human
immunoglobulin heavy chain variable domain containing a plurality
of heavy chain complementarity determining region 3 (CDR H3)
isolated from the immunoglobulin variable domain repertoire from a
non-human species. In some embodiments, the method includes the
steps of: (a) providing a plurality of Acceptor Framework nucleic
acid sequences encoding distinct human immunoglobulin heavy chain
variable domains, each Acceptor Framework nucleic acid sequence
containing a first framework region (FR1), a second framework
region (FR2), a third framework region (FR3), and a fourth
framework region (FR4), wherein the FR1 and FR2 regions are
interspaced by a complementarity determining region 1 (CDR1), the
FR2 and FR3 regions are interspaced by a complementarity
determining region 2 (CDR2), and the FR3 and FR4 regions are
interspaced by a stuffer nucleic acid sequence including at least
two Type IIs restriction enzyme recognition sites interspaced by a
random nucleic acid sequence; (b) providing a plurality of
diversified nucleic acid sequences encoding heavy chain
complementarity determining region 3 (CDR H3) sequences isolated
from a non-human species immunoglobulin repertoire wherein each of
the plurality of diversified nucleic acid sequences includes a Type
IIs restriction enzyme recognition site at each extremity; (c)
digesting each of the plurality of nucleic acid sequences encoding
the CDR H3 regions using a Type IIs restriction enzyme that binds
to the Type IIs restriction enzyme recognition site of step (b) and
digesting the stuffer nucleic acid sequence of step (a) from the
Acceptor Framework using a Type IIs restriction enzyme that binds
to the Type IIs restriction enzyme recognition site of step (a);
and (d) ligating the digested nucleic acid sequences encoding the
CDR H3 regions or the amino acid sequences of step (c) into the
digested Acceptor Framework of step (c) such that the FR3 and FR4
regions are interspaced by the nucleic acid sequences encoding the
CDR H3 region or the amino acid sequence that can fulfill the role
of a CDR3 region and a complete immunoglobulin variable domain
encoding sequences that do not contain the Type IIs restriction
enzyme recognition sites of steps (a) and (b) are restored.
[0033] In some embodiments, step (b) as set forth above is
performed by amplifying the CDR H3 sequence from a non human
species using oligonucleotide primers containing a Type IIs
restriction site. In some embodiments, step (b) as set forth above
is performed by amplifying the CDR H3 sequence from a non human
species using oligonucleotide primers containing a FokI IIs
restriction site. In some embodiments, the non-human species is
non-human primate, rodent, canine, feline, sheep, goat, cattle,
horse, or pig.
[0034] The invention provides methods for producing a library of
nucleic acids, wherein each nucleic acid encodes an immunoglobulin
variable domain by (a) providing a plurality of Acceptor Framework
nucleic acid sequences encoding distinct immunoglobulin variable
domains, each Acceptor Framework nucleic acid sequence including a
first framework region (FR1), a second framework region (FR2), a
third framework region (FR3), and a fourth framework region (FR4),
wherein the FR1 and FR2 regions are interspaced by a
complementarity determining region 1 (CDR1), the FR2 and FR3
regions are interspaced by a complementarity determining region 2
(CDR2), and the FR3 and FR4 regions are interspaced by a stuffer
nucleic acid sequence containing at least two Type IIs restriction
enzyme recognition sites interspaced by a random nucleic acid
sequence; (b) providing a plurality of diversified nucleic acid
sequences encoding complementarity determining region 3 (CDR3)
regions or encoding amino acid sequences that can fulfill the role
of a CDR3 region, wherein each of the plurality of diversified
nucleic acid sequences includes a Type IIs restriction enzyme
recognition site at each extremity; (c) digesting each of the
plurality of nucleic acid sequences encoding the CDR3 regions or
amino acid sequences that can fulfill the role of a CDR3 region
using a Type IIs restriction enzyme that binds to the Type IIs
restriction enzyme recognition site of step (b) and digesting the
stuffer nucleic acid sequence of step (a) from the Acceptor
Framework using a Type IIs restriction enzyme that binds to the
Type IIs restriction enzyme recognition site of step (a); and (d)
ligating the digested nucleic acid sequences encoding the CDR3
regions or the amino acid sequences that can fulfill the role of a
CDR3 region of step (c) into the digested Acceptor Framework of
step (c) such that the FR3 and FR4 regions are interspaced by the
nucleic acid sequences encoding the CDR3 region or the amino acid
sequence that can fulfill the role of a CDR3 region and a complete
immunoglobulin variable domain encoding sequences that do not
contain the Type IIs restriction enzyme recognition sites of steps
(a) and (b) are restored.
[0035] In some embodiments, the Type IIs restriction enzyme
recognition sites of step (a) and step (b) are recognized by the
same Type IIs restriction enzyme. In some embodiments, the Type IIs
restriction enzyme recognition sites of step (a) and step (b) are
recognized by different Type IIs restriction enzymes. For example,
the Type IIs restriction enzyme recognition sites are FokI
recognition sites, BsaI recognition sites, and/or BsmBI recognition
sites.
[0036] In some embodiments, the Acceptor Framework nucleic acid
sequence is derived from a human gene sequence. For example, the
human sequence is a human heavy chain variable gene sequence or a
sequence derived from a human heavy chain variable gene sequence.
In some embodiments, the human heavy chain variable gene sequence
is selected from VH1-2, VH1-69, VH1-18, VH3-30, VH3-48, VH3-23, and
VH5-51. In some embodiments, the human sequence is a human kappa
light chain variable gene sequence or a sequence derived from a
human kappa light chain variable gene sequence. For example, the
human kappa light chain variable gene sequence is selected from
VK1-33, VK1-39, VK3-11, VK3-15, and VK3-20. In some embodiments,
the human sequence is a human lambda light chain variable gene
sequence or a sequence derived from a human lambda light chain
variable gene sequence. For example, the human lambda light chain
variable gene sequence is selected from VL1-44 and VL1-51.
[0037] In one embodiment, the plurality of diversified nucleic
acids encodes CDR3 regions, and the plurality of diversified
nucleic acids includes naturally occurring sequences or sequences
derived from immunized animals.
[0038] In one embodiment, the plurality of diversified nucleic
acids includes or is derived from sequences selected from naturally
occurring CDR3 sequences, naturally occurring Ig sequences from
humans, naturally occurring Ig sequences from a mammal, naturally
occurring sequences from a loop region of a T cell receptor in a
mammal, and other naturally diversified polypeptide
collections.
[0039] In one embodiment, the plurality of diversified nucleic
acids encodes CDR3 regions, and the plurality of diversified
nucleic acids includes or is derived from immunoglobulin sequences
that occur naturally in humans that have been exposed to a
particular immunogen or sequences derived from animals that have
been identified as having been exposed to a particular antigen.
[0040] In one embodiment, the plurality of diversified nucleic
acids encodes amino acid sequences that can fulfill the role of a
CDR3 region, and the plurality of diversified nucleic acids
includes synthetic sequences.
[0041] In another embodiment, the plurality of diversified nucleic
acids encodes amino acid sequences that can fulfill the role of a
CDR3 region, and the plurality of diversified nucleic acids
includes synthetic sequences.
[0042] In some embodiments, the plurality of Acceptor Framework
nucleic acid sequences include a mixture of at least one variable
heavy chain (VH) Acceptor Framework nucleic acid sequence and at
least one variable light chain Acceptor Framework nucleic acid
sequence.
[0043] In some embodiments, the methods provided include the
additional step of (e) transforming the expression vector of step
(d) into a host cell and culturing the host cell under conditions
sufficient to express the plurality of Acceptor Framework
sequences. For example, the host cell is E. coli. In some
embodiments, the expression vector is a phagemid vector. For
example, the phagemid vector is pNDS1.
[0044] The invention also provides methods for producing a library
of nucleic acids, wherein each nucleic acid encodes an
immunoglobulin variable domain, by (a) providing a plurality of
Acceptor Framework nucleic acid sequences encoding distinct
immunoglobulin variable domains, each Acceptor Framework nucleic
acid sequence including a first framework region (FR1), a second
framework region (FR2), a third framework region (FR3), and a
fourth framework region (FR4), wherein the FR1 and FR2 regions are
interspaced by a stuffer nucleic acid sequence including at least
two Type IIs restriction enzyme recognition sites interspaced by a
random nucleic acid sequence, the FR2 and FR3 regions are
interspaced by a complementarity determining region 2 (CDR2), and
the FR3 and FR4 regions are interspaced by a complementarity
determining region 3 (CDR3); (b) providing a plurality of
diversified nucleic acid sequences encoding complementarity
determining region 1 (CDR1) regions or encoding amino acid
sequences that can fulfill the role of a CDR1 region, wherein each
of the plurality of diversified nucleic acid sequences includes a
Type IIs restriction enzyme recognition site at each extremity; (c)
digesting each of the plurality of nucleic acid sequences encoding
the CDR1 regions or amino acid sequences that can fulfill the role
of a CDR1 region using a Type IIs restriction enzyme that binds to
the Type IIs restriction enzyme recognition site of step (b) and
digesting the stuffer nucleic acid sequence of step (a) from the
Acceptor Framework using a Type IIs restriction enzyme that binds
to the Type IIs restriction enzyme recognition site of step (a);
and (d) ligating the digested nucleic acid sequences encoding the
CDR1 regions or the amino acid sequences that can fulfill the role
of a CDR1 region of step (c) into the digested Acceptor Framework
of step (c) such that the FR1 and FR2 regions are interspaced by
the nucleic acid sequences encoding the CDR1 region or the amino
acid sequence that can fulfill the role of a CDR1 region and a
complete immunoglobulin variable domain encoding sequences that do
not contain the Type IIs restriction enzyme recognition sites of
steps (a) and (b) are restored.
[0045] In some embodiments, the Type IIs restriction enzyme
recognition sites of step (a) and step (b) are recognized by the
same Type IIs restriction enzyme. In some embodiments, the Type IIs
restriction enzyme recognition sites of step (a) and step (b) are
recognized by different Type IIs restriction enzymes. For example,
the Type IIs restriction enzyme recognition sites are FokI
recognition sites, BsaI recognition sites, and/or BsmBI recognition
sites.
[0046] In some embodiments, the Acceptor Framework nucleic acid
sequence is derived from a human gene sequence. For example, the
human sequence is a human heavy chain variable gene sequence or a
sequence derived from a human heavy chain variable gene sequence.
In some embodiments, the human heavy chain variable gene sequence
is selected from VH1-2, VH1-69, VH1-18, VH3-30, VH3-48, VH3-23, and
VH5-51. In some embodiments, the human sequence is a human kappa
light chain variable gene sequence or a sequence derived from a
human kappa light chain variable gene sequence. For example, the
human kappa light chain variable gene sequence is selected from
VK1-33, VK1-39, VK3-11, VK3-15, and VK3-20. In some embodiments,
the human sequence is a human lambda light chain variable gene
sequence or a sequence derived from a human lambda light chain
variable gene sequence. For example, the human lambda light chain
variable gene sequence is selected from VL1-44 and VL1-51.
[0047] In one embodiment, the plurality of diversified nucleic
acids encodes CDR1 regions, and the plurality of diversified
nucleic acids includes naturally occurring sequences or sequences
derived from immunized animals.
[0048] In one embodiment, the plurality of diversified nucleic
acids includes or is derived from sequences selected from naturally
occurring CDR1 sequences, naturally occurring Ig sequences from
humans, naturally occurring Ig sequences from a mammal, naturally
occurring sequences from a loop region of a T cell receptor in a
mammal, and other naturally diversified polypeptide
collections.
[0049] In one embodiment, the plurality of diversified nucleic
acids encodes CDR1 regions, and the plurality of diversified
nucleic acids includes or is derived from immunoglobulin sequences
that occur naturally in humans that have been exposed to a
particular immunogen or sequences derived from animals that have
been identified as having been exposed to a particular antigen.
[0050] In one embodiment, the plurality of diversified nucleic
acids encodes amino acid sequences that can fulfill the role of a
CDR1 region, and the plurality of diversified nucleic acids
includes synthetic sequences.
[0051] In another embodiment, the plurality of diversified nucleic
acids encodes amino acid sequences that can fulfill the role of a
CDR1 region, and the plurality of diversified nucleic acids
includes synthetic sequences.
[0052] In some embodiments, the plurality of Acceptor Framework
nucleic acid sequences include a mixture of at least one variable
heavy chain (VH) Acceptor Framework nucleic acid sequence and at
least one variable light chain Acceptor Framework nucleic acid
sequence.
[0053] In some embodiments, the methods provided include the
additional steps of (e) cloning the library of nucleic acids
encoding immunoglobulin variable domains of step (d) into an
expression vector and (f) transforming the expression vector of
step (e) into a host cell and culturing the host cell under
conditions sufficient to express a plurality of immunoglobulin
variable domain encoded by the library. For example, the host cell
is E. coli. In some embodiments, the expression vector is a
phagemid vector. For example, the phagemid vector is pNDS1.
[0054] The invention also provides methods for producing a library
of nucleic acids, wherein each nucleic acid encodes an
immunoglobulin variable domain, by (a) providing a plurality of
Acceptor Framework nucleic acid sequences encoding distinct
immunoglobulin variable domains, each Acceptor Framework nucleic
acid sequence including a first framework region (FR1), a second
framework region (FR2), a third framework region (FR3), and a
fourth framework region (FR4), wherein the FR1 and FR2 regions are
interspaced by a complementarity determining region 1 (CDR1), the
FR2 and FR3 regions are interspaced by a stuffer nucleic acid
sequence including at least two Type IIs restriction enzyme
recognition sites interspaced by a random nucleic acid sequence,
and the FR3 and FR4 regions are interspaced by a complementarity
determining region 3 (CDR3); (b) providing a plurality of
diversified nucleic acid sequences encoding complementarity
determining region 2 (CDR2) regions or encoding amino acid
sequences that can fulfill the role of a CDR2 region, wherein each
of the plurality of diversified nucleic acid sequences includes a
Type IIs restriction enzyme recognition site at each extremity; (c)
digesting each of the plurality of nucleic acid sequences encoding
the CDR2 regions or amino acid sequences that can fulfill the role
of a CDR2 region using a Type IIs restriction enzyme that binds to
the Type IIs restriction enzyme recognition site of step (b) and
digesting the stuffer nucleic acid sequence of step (a) from the
Acceptor Framework using a Type IIs restriction enzyme that binds
to the Type IIs restriction enzyme recognition site of step (a);
and (d) ligating the digested nucleic acid sequences encoding the
CDR2 regions or the amino acid sequences that can fulfill the role
of a CDR2 region of step (c) into the digested Acceptor Framework
of step (c) such that the FR2 and FR3 regions are interspaced by
the nucleic acid sequences encoding the CDR2 region or the amino
acid sequence that can fulfill the role of a CDR2 region and a
complete immunoglobulin variable domain encoding sequences that do
not contain the Type IIs restriction enzyme recognition sites of
steps (a) and (b) are restored.
[0055] In some embodiments, the Type IIs restriction enzyme
recognition sites of step (a) and step (b) are recognized by the
same Type IIs restriction enzyme. In some embodiments, the Type IIs
restriction enzyme recognition sites of step (a) and step (b) are
recognized by different Type IIs restriction enzymes. For example,
the Type IIs restriction enzyme recognition sites are FokI
recognition sites, BsaI recognition sites, and/or BsmBI recognition
sites.
[0056] In some embodiments, the Acceptor Framework nucleic acid
sequence is derived from a human gene sequence. For example, the
human sequence is a human heavy chain variable gene sequence or a
sequence derived from a human heavy chain variable gene sequence.
In some embodiments, the human heavy chain variable gene sequence
is selected from VH1-2, VH1-69, VH1-18, VH3-30, VH3-48, VH3-23, and
VH5-51. In some embodiments, the human sequence is a human kappa
light chain variable gene sequence or a sequence derived from a
human kappa light chain variable gene sequence. For example, the
human kappa light chain variable gene sequence is selected from
VK1-33, VK1-39, VK3-11, VK3-15, and VK3-20. In some embodiments,
the human sequence is a human lambda light chain variable gene
sequence or a sequence derived from a human lambda light chain
variable gene sequence. For example, the human lambda light chain
variable gene sequence is selected from VL1-44 and VL1-51.
[0057] In one embodiment, the plurality of diversified nucleic
acids encodes CDR2 regions, and the plurality of diversified
nucleic acids includes naturally occurring sequences or sequences
derived from immunized animals.
[0058] In one embodiment, the plurality of diversified nucleic
acids includes or is derived from sequences selected from naturally
occurring CDR2 sequences, naturally occurring Ig sequences from
humans, naturally occurring Ig sequences from a mammal, naturally
occurring sequences from a loop region of a T cell receptor in a
mammal, and other naturally diversified polypeptide
collections.
[0059] In one embodiment, the plurality of diversified nucleic
acids encode CDR2 regions, and the plurality of diversified nucleic
acids includes or is derived from immunoglobulin sequences that
occur naturally in humans that have been exposed to a particular
immunogen or sequences derived from animals that have been
identified as having been exposed to a particular antigen.
[0060] In another embodiment, the plurality of diversified nucleic
acids encodes amino acid sequences that can fulfill the role of a
CDR2 region, and the plurality of diversified nucleic acids include
synthetic sequences.
[0061] In some embodiments, the plurality of Acceptor Framework
nucleic acid sequences include a mixture of at least one variable
heavy chain (VH) Acceptor Framework nucleic acid sequence and at
least one variable light chain Acceptor Framework nucleic acid
sequence.
[0062] In some embodiments, the methods provided include the
additional steps of (e) cloning the library of nucleic acids
encoding immunoglobulin variable domains of step (d) into an
expression vector and (f) transforming the expression vector of
step (e) into a host cell and culturing the host cell under
conditions sufficient to express a plurality of immunoglobulin
variable domain encoded by the library. For example, the host cell
is E. coli. In some embodiments, the expression vector is a
phagemid vector. For example, the phagemid vector is pNDS1.
[0063] The invention also provides methods for making a
target-specific antibody, antibody variable region or a portion
thereof, by (a) providing a plurality of Acceptor Framework nucleic
acid sequences encoding distinct immunoglobulin variable domains,
each Acceptor Framework nucleic acid sequence including a first
framework region (FR1), a second framework region (FR2), a third
framework region (FR3), and a fourth framework region (FR4),
wherein the FR1 and FR2 regions are interspaced by a
complementarity determining region 1 (CDR1), the FR2 and FR3
regions are interspaced by a complementarity determining region 2
(CDR2), and the FR3 and FR4 regions are interspaced by a stuffer
nucleic acid sequence including at least two Type IIs restriction
enzyme recognition sites interspaced by a random nucleic acid
sequence; (b) providing a plurality of diversified nucleic acid
sequences encoding complementarity determining region 3 (CDR3)
regions or encoding amino acid sequences that can fulfill the role
of a CDR3 region, wherein each of the plurality of diversified
nucleic acid sequences includes a Type IIs restriction enzyme
recognition site at each extremity; (c) digesting each of the
plurality of nucleic acid sequences encoding the CDR3 regions or
amino acid sequences that can fulfill the role of a CDR3 region
using a Type IIs restriction enzyme that binds to the Type IIs
restriction enzyme recognition site of step (b) and digesting the
stuffer nucleic acid sequence of step (a) using a Type IIs
restriction enzyme that binds to the Type IIs restriction enzyme
recognition site of step (a); (d) cloning the digested nucleic acid
sequences encoding the CDR3 regions or the amino acid sequences
that can fulfill the role of a CDR3 region into an expression
vector and ligating the digested nucleic acid sequences encoding
the CDR3 regions or the amino acid sequences that can fulfill the
role of a CDR3 region of step (c) into the Acceptor Framework such
that the FR3 and FR4 regions are interspaced by the nucleic acid
sequences encoding the CDR3 region or the amino acid sequence that
can fulfill the role of a CDR3 region and a complete immunoglobulin
variable gene encoding sequence is restored; (e) transforming the
expression vector of step (e) into a host cell and culturing the
host cell under conditions sufficient to express the plurality of
Acceptor Framework sequences; (f) contacting the host cell with a
target antigen; and (g) determining which expressed Acceptor
Framework sequences bind to the target antigen.
[0064] In some embodiments, the Type IIs restriction enzyme
recognition sites of step (a) and step (b) are recognized by the
same Type IIs restriction enzyme. In some embodiments, the Type IIs
restriction enzyme recognition sites of step (a) and step (b) are
recognized by different Type IIs restriction enzymes. For example,
the Type IIs restriction enzyme recognition sites are FokI
recognition sites, BsaI recognition sites, and/or BsmBI recognition
sites.
[0065] In some embodiments, the Acceptor Framework nucleic acid
sequence is derived from a human gene sequence. For example, the
human sequence is a human heavy chain variable gene sequence or a
sequence derived from a human heavy chain variable gene sequence.
In some embodiments, the human heavy chain variable gene sequence
is selected from VH1-2, VH1-69, VH1-18, VH3-30, VH3-48, VH3-23, and
VH5-51. In some embodiments, the human sequence is a human kappa
light chain variable gene sequence or a sequence derived from a
human kappa light chain variable gene sequence. For example, the
human kappa light chain variable gene sequence is selected from
VK1-33, VK1-39, VK3-11, VK3-15, and VK3-20. In some embodiments,
the human sequence is a human lambda light chain variable gene
sequence or a sequence derived from a human lambda light chain
variable gene sequence. For example, the human lambda light chain
variable gene sequence is selected from VL1-44 and VL1-51.
[0066] In one embodiment, the plurality of diversified nucleic
acids encodes CDR3 regions, and the plurality of diversified
nucleic acids includes naturally occurring sequences or sequences
derived from immunized animals.
[0067] In one embodiment, the plurality of diversified nucleic
acids includes or is derived from sequences selected from naturally
occurring CDR3 sequences, naturally occurring Ig sequences from
humans, naturally occurring Ig sequences from a mammal, naturally
occurring sequences from a loop region of a T cell receptor in a
mammal, and other naturally diversified polypeptide
collections.
[0068] In one embodiment, the plurality of diversified nucleic
acids encodes CDR3 regions, and the plurality of diversified
nucleic acids includes or is derived from immunoglobulin sequences
that occur naturally in humans that have been exposed to a
particular immunogen or sequences derived from animals that have
been identified as having been exposed to a particular antigen.
[0069] In another embodiment, the plurality of diversified nucleic
acids encodes amino acid sequences that can fulfill the role of a
CDR3 region, and the plurality of diversified nucleic acids
includes synthetic sequences.
[0070] In some embodiments, the plurality of Acceptor Framework
nucleic acid sequences include a mixture of at least one variable
heavy chain (VH) Acceptor Framework nucleic acid sequence and at
least one variable light chain Acceptor Framework nucleic acid
sequence.
[0071] In some embodiments, the expression vector is a phagemid
vector. For example, the phagemid vector is pNDS1. In some
embodiments, the host cell is E. coli.
[0072] In some embodiments, the method includes the additional step
of (i) sequencing the immunoglobulin variable domain encoding
sequences that bind the target antigen.
[0073] The invention also provides methods for making a
target-specific antibody, antibody variable region or a portion
thereof, by (a) providing a plurality of Acceptor Framework nucleic
acid sequences encoding distinct immunoglobulin variable domains,
each Acceptor Framework nucleic acid sequence including a first
framework region (FR1), a second framework region (FR2), a third
framework region (FR3), and a fourth framework region (FR4),
wherein the FR1 and FR2 regions are interspaced by a stuffer
nucleic acid sequence including at least two Type IIs restriction
enzyme recognition sites interspaced by a random nucleic acid
sequence, the FR2 and FR3 regions are interspaced by a
complementarity determining region 2 (CDR2), and the FR3 and FR4
regions are interspaced by a complementarity determining region 3
(CDR3); (b) providing a plurality of diversified nucleic acid
sequences encoding complementarity determining region 1 (CDR1)
regions or encoding amino acid sequences that can fulfill the role
of a CDR1 region, wherein each of the plurality of diversified
nucleic acid sequences includes a Type IIs restriction enzyme
recognition site at each extremity; (c) digesting each of the
plurality of nucleic acid sequences encoding the CDR1 regions or
amino acid sequences that can fulfill the role of a CDR1 region
using a Type IIs restriction enzyme that binds to the Type IIs
restriction enzyme recognition site of step (b) and digesting the
stuffer nucleic acid sequence of step (a) using a Type IIs
restriction enzyme that binds to the Type IIs restriction enzyme
recognition site of step (a); (d) cloning the digested nucleic acid
sequences encoding the CDR1 regions or the amino acid sequences
that can fulfill the role of a CDR1 region into an expression
vector and ligating the digested nucleic acid sequences encoding
the CDR1 regions or the amino acid sequences that can fulfill the
role of a CDR1 region of step (c) into the Acceptor Framework such
that the FR1 and FR2 regions are interspaced by the nucleic acid
sequences encoding the CDR1 region or the amino acid sequence that
can fulfill the role of a CDR1 region and a complete immunoglobulin
variable gene encoding sequence is restored; (e) transforming the
expression vector of step (e) into a host cell and culturing the
host cell under conditions sufficient to express the plurality of
Acceptor Framework sequences; (f) contacting the host cell with a
target antigen; and (g) determining which expressed Acceptor
Framework sequences bind to the target antigen.
[0074] In some embodiments, the Type IIs restriction enzyme
recognition sites of step (a) and step (b) are recognized by the
same Type IIs restriction enzyme. In some embodiments, the Type IIs
restriction enzyme recognition sites of step (a) and step (b) are
recognized by different Type IIs restriction enzymes. For example,
the Type IIs restriction enzyme recognition sites are FokI
recognition sites BsaI recognition sites, and/or BsmBI recognition
sites.
[0075] In some embodiments, the Acceptor Framework nucleic acid
sequence is derived from a human gene sequence. For example, the
human sequence is a human heavy chain variable gene sequence or a
sequence derived from a human heavy chain variable gene sequence.
In some embodiments, the human heavy chain variable gene sequence
is selected from VH1-2, VH1-69, VH1-18, VH3-30, VH3-48, VH3-23, and
VH5-51. In some embodiments, the human sequence is a human kappa
light chain variable gene sequence or a sequence derived from a
human kappa light chain variable gene sequence. For example, the
human kappa light chain variable gene sequence is selected from
VK1-33, VK1-39, VK3-11, VK3-15, and VK3-20. In some embodiments,
the human sequence is a human lambda light chain variable gene
sequence or a sequence derived from a human lambda light chain
variable gene sequence. For example, the human lambda light chain
variable gene sequence is selected from VL1-44 and VL1-51.
[0076] In one embodiment, the plurality of diversified nucleic
acids encodes CDR1 regions, and the plurality of diversified
nucleic acids includes naturally occurring sequences or sequences
derived from immunized animals.
[0077] In one embodiment, the plurality of diversified nucleic
acids includes or is derived from sequences selected from naturally
occurring CDR1 sequences, naturally occurring Ig sequences from
humans, naturally occurring Ig sequences from a mammal, naturally
occurring sequences from a loop region of a T cell receptor in a
mammal, and other naturally diversified polypeptide
collections.
[0078] In one embodiment, the plurality of diversified nucleic
acids encodes CDR1 regions, and the plurality of diversified
nucleic acids includes or is derived from immunoglobulin sequences
that occur naturally in humans that have been exposed to a
particular immunogen or sequences derived from animals that have
been identified as having been exposed to a particular antigen.
[0079] In another embodiment, the plurality of diversified nucleic
acids encodes amino acid sequences that can fulfill the role of a
CDR1 region, and the plurality of diversified nucleic acids
includes synthetic sequences.
[0080] In some embodiments, the plurality of Acceptor Framework
nucleic acid sequences include a mixture of at least one variable
heavy chain (VH) Acceptor Framework nucleic acid sequence and at
least one variable light chain Acceptor Framework nucleic acid
sequence.
[0081] In some embodiments, the expression vector is a phagemid
vector. For example, the phagemid vector is pNDS1. In some
embodiments, the host cell is E. coli.
[0082] In some embodiments, the method includes the additional step
of (i) sequencing the immunoglobulin variable domain encoding
sequences that bind the target antigen.
[0083] The invention provides methods for making a target-specific
antibody, antibody variable region or a portion thereof, by (a)
providing a plurality of Acceptor Framework nucleic acid sequences
encoding distinct immunoglobulin variable domains, each Acceptor
Framework nucleic acid sequence including a first framework region
(FR1), a second framework region (FR2), a third framework region
(FR3), and a fourth framework region (FR4), wherein the FR1 and FR2
regions are interspaced by a complementarity determining region 1
(CDR1), the FR2 and FR3 regions are interspaced by a stuffer
nucleic acid sequence including at least two Type IIs restriction
enzyme recognition sites interspaced by a random nucleic acid
sequence, and the FR3 and FR4 regions are interspaced by a
complementarity determining region 3 (CDR3); (b) providing a
plurality of diversified nucleic acid sequences encoding
complementarity determining region 2 (CDR2) regions or encoding
amino acid sequences that can fulfill the role of a CDR2 region,
wherein each of the plurality of diversified nucleic acid sequences
includes a Type IIs restriction enzyme recognition site at each
extremity; (c) digesting each of the plurality of nucleic acid
sequences encoding the CDR2 regions or amino acid sequences that
can fulfill the role of a CDR2 region using a Type IIs restriction
enzyme that binds to the Type IIs restriction enzyme recognition
site of step (b) and digesting the stuffer nucleic acid sequence of
step (a) from the Acceptor Framework using a Type IIs restriction
enzyme that binds to the Type IIs restriction enzyme recognition
site of step (a); (d) ligating the digested nucleic acid sequences
encoding the CDR2 regions or the amino acid sequences that can
fulfill the role of a CDR2 region of step (c) into the digested
Acceptor Framework of step (c) such that the FR2 and FR3 regions
are interspaced by the nucleic acid sequences encoding the CDR2
region or the amino acid sequence that can fulfill the role of a
CDR2 region and complete immunoglobulin variable domain encoding
sequences that do not contain the Type IIs restriction enzyme
recognition sites of steps (a) and (b) are restored; (e) cloning
the library of nucleic acids encoding immunoglobulin variable
domains of step (d) into an expression vector; (f) transforming the
expression vector of step (e) into a host cell and culturing the
host cell under conditions sufficient to express a plurality of
immunoglobulin variable domains encoded by the library; (g)
contacting the plurality of immunoglobulin variable domains of step
(f) with a target antigen; and (h) determining which expressed
immunoglobulin variable domain encoding sequences bind to the
target antigen.
[0084] In some embodiments, the Type IIs restriction enzyme
recognition sites of step (a) and step (b) are recognized by the
same Type IIs restriction enzyme. In some embodiments, the Type IIs
restriction enzyme recognition sites of step (a) and step (b) are
recognized by different Type IIs restriction enzymes. For example,
the Type IIs restriction enzyme recognition sites are FokI
recognition sites, BsaI recognition sites, and/or BsmBI recognition
sites.
[0085] In some embodiments, the Acceptor Framework nucleic acid
sequence is derived from a human gene sequence. For example, the
human sequence is a human heavy chain variable gene sequence or a
sequence derived from a human heavy chain variable gene sequence.
In some embodiments, the human heavy chain variable gene sequence
is selected from VH1-2, VH1-69, VH1-18, VH3-30, VH3-48, VH3-23, and
VH5-51. In some embodiments, the human sequence is a human kappa
light chain variable gene sequence or a sequence derived from a
human kappa light chain variable gene sequence. For example, the
human kappa light chain variable gene sequence is selected from
VK1-33, VK1-39, VK3-11, VK3-15, and VK3-20. In some embodiments,
the human sequence is a human lambda light chain variable gene
sequence or a sequence derived from a human lambda light chain
variable gene sequence. For example, the human lambda light chain
variable gene sequence is selected from VL1-44 and VL1-51.
[0086] In one embodiment, the plurality of diversified nucleic
acids encodes CDR2 regions, and the plurality of diversified
nucleic acids includes naturally occurring sequences or sequences
derived from immunized animals.
[0087] In one embodiment, the plurality of diversified nucleic
acids includes or is derived from sequences selected from naturally
occurring CDR2 sequences, naturally occurring Ig sequences from
humans, naturally occurring Ig sequences from a mammal, naturally
occurring sequences from a loop region of a T cell receptor in a
mammal, and other naturally diversified polypeptide
collections.
[0088] In one embodiment, the plurality of diversified nucleic
acids encodes CDR2 regions, and the plurality of diversified
nucleic acids includes or is derived from immunoglobulin sequences
that occur naturally in humans that have been exposed to a
particular immunogen or sequences derived from animals that have
been identified as having been exposed to a particular antigen.
[0089] In one embodiment, the plurality of diversified nucleic
acids encodes amino acid sequences that can fulfill the role of a
CDR2 region, and the plurality of diversified nucleic acids
includes synthetic sequences.
[0090] In another embodiment, the plurality of diversified nucleic
acids encodes amino acid sequences that can fulfill the role of a
CDR2 region, and the plurality of diversified nucleic acids
includes synthetic sequences.
[0091] In some embodiments, the plurality of Acceptor Framework
nucleic acid sequences include a mixture of at least one variable
heavy chain (VH) Acceptor Framework nucleic acid sequence and at
least one variable light chain Acceptor Framework nucleic acid
sequence.
[0092] In some embodiments, the host cell is E. coli. In some
embodiments, the expression vector is a phagemid vector. For
example, the phagemid vector is pNDS1.
[0093] In some embodiments, the method includes the additional step
of (i) sequencing the immunoglobulin variable domain encoding
sequences that bind the target antigen.
[0094] The invention also provides methods for producing a library
of nucleic acids, wherein each nucleic acid encodes an
immunoglobulin variable domain. These methods include the steps of
(a) providing a plurality of Ig Acceptor Framework nucleic acid
sequences into which a source of diversity is introduced at a
single complementarity determining region (CDR) selected from the
group consisting of complementarity determining region 1 (CDR1),
complementarity determining region 2 (CDR2), and complementarity
determining region 3 (CDR3), wherein the Ig Acceptor Framework
sequence includes a stuffer nucleic acid sequence including at
least two Type IIs restriction enzyme recognition sites, and
wherein the source of diversity is a CDR selected from naturally
occurring CDR sequences that contain Type IIs restriction enzyme
recognition sites outside the CDR region, (b) introducing the
source of diversity within each Ig Acceptor Framework by digesting
both the source of diversity and the Ig Acceptor Frameworks with a
Type IIs restriction enzyme; and (c) ligating the digested source
of diversity into the Ig Acceptor Framework such that a complete
immunoglobulin variable domain encoding sequences that do not
contain the Type IIs restriction enzyme recognition sites of steps
(a) and (b) are restored.
[0095] The naturally occurring CDR region sequences are
substantially unaltered from their wild-type, i.e., natural state.
These naturally occurring CDR region sequences are flanked by amino
acid sequences that have been engineered (or otherwise artificially
manipulated) to contain two Type IIs restriction enzyme recognition
sites, with one Type IIs restriction enzyme recognition site on
each of side of the naturally occurring CDR region sequence. The
Type IIS restriction enzyme recognition sites are outside the CDR
encoding region. The sequence of CDR regions are unaltered at the
boundaries of the CDR encoding region--the restriction enzymes
recognize and splice at a region that is up to the boundary of the
CDR encoding region, but does not splice within the CDR encoding
region.
[0096] In some embodiments, the Type IIs restriction enzyme
recognition sites within the stuffer nucleic acid sequences and
flanking the naturally occurring CDR sequences are recognized by
the same Type IIs restriction enzyme. In some embodiments, the Type
IIs restriction enzyme recognition sites within the stuffer nucleic
acid sequences and flanking the naturally occurring CDR sequences
are recognized by different Type IIs restriction enzymes. For
example, the Type IIs restriction enzyme recognition sites are FokI
recognition sites, BsaI recognition sites, and/or BsmBI recognition
sites.
[0097] In some embodiments, the Ig Acceptor Framework nucleic acid
sequence is derived from a human gene sequence. For example, the
human sequence is a human heavy chain variable gene sequence or a
sequence derived from a human heavy chain variable gene sequence.
In some embodiments, the human heavy chain variable gene sequence
is selected from VH1-2, VH1-69, VH1-18, VH3-30, VH3-48, VH3-23, and
VH5-51. In some embodiments, the human sequence is a human kappa
light chain variable gene sequence or a sequence derived from a
human kappa light chain variable gene sequence. For example, the
human kappa light chain variable gene sequence is selected from
VK1-33, VK1-39, VK3-11, VK3-15, and VK3-20. In some embodiments,
the human sequence is a human lambda light chain variable gene
sequence or a sequence derived from a human lambda light chain
variable gene sequence. For example, the human lambda light chain
variable gene sequence is selected from VL1-44 and VL1-51.
[0098] In some embodiments, the set of naturally occurring nucleic
acids includes or is derived from sequences selected from naturally
occurring CDR3 sequences, naturally occurring Ig sequences from
humans, naturally occurring Ig sequences from a mammal, naturally
occurring sequences from a loop region of a T cell receptor in a
mammal, and other naturally diversified polypeptide
collections.
[0099] In some embodiments, the set of naturally occurring nucleic
acids encode CDR3 regions, and the set of naturally occurring
nucleic acids include immunoglobulin sequences that occur naturally
in humans that have been exposed to a particular immunogen or
sequences derived from animals that have been identified as having
been exposed to a particular antigen.
[0100] In some embodiments, the set of naturally occurring nucleic
acids includes or is derived from sequences selected from naturally
occurring CDR1 sequences, naturally occurring Ig sequences from
humans, naturally occurring Ig sequences from a mammal, naturally
occurring sequences from a loop region of a T cell receptor in a
mammal, and other naturally diversified polypeptide
collections.
[0101] In some embodiments, the set of naturally occurring nucleic
acids encode CDR1 regions, and the set of naturally occurring
nucleic acids includes or is derived from immunoglobulin sequences
that occur naturally in humans that have been exposed to a
particular immunogen or sequences derived from animals that have
been identified as having been exposed to a particular antigen.
[0102] In some embodiments, the set of naturally occurring nucleic
acids includes or is derived from sequences selected from naturally
occurring CDR2 sequences, naturally occurring Ig sequences from
humans, naturally occurring Ig sequences from a mammal, naturally
occurring sequences from a loop region of a T cell receptor in a
mammal, and other naturally diversified polypeptide
collections.
[0103] In some embodiments, the set of naturally occurring nucleic
acids encodes CDR2 regions, and the set of naturally occurring
nucleic acids includes immunoglobulin sequences that occur
naturally in humans that have been exposed to a particular
immunogen or sequences derived from animals that have been
identified as having been exposed to a particular antigen.
[0104] In some embodiments, the plurality of Ig Acceptor Framework
nucleic acid sequences include a mixture of at least one variable
heavy chain (VH) Acceptor Framework nucleic acid sequence and at
least one variable light chain (VL) Acceptor Framework nucleic acid
sequence.
[0105] In some embodiments, the methods provided include the
additional steps of (e) cloning the library of nucleic acids
encoding immunoglobulin variable domains of step (d) into an
expression vector and (f) transforming the expression vector of
step (e) into a host cell and culturing the host cell under
conditions sufficient to express a plurality of immunoglobulin
variable domain encoded by the library. For example, the host cell
is E. coli. In some embodiments, the expression vector is a
phagemid vector. For example, the phagemid vector is ANDS 1.
[0106] The invention also provides methods for producing a library
of nucleic acids, wherein each nucleic acid encodes an
immunoglobulin variable domain. These methods include the steps of
(a) providing a plurality of Ig Acceptor Framework nucleic acid
sequences into which a source of diversity is introduced at a
single complementarity determining region (CDR) selected from the
group consisting of complementarity determining region 1 (CDR1),
complementarity determining region 2 (CDR2), and complementarity
determining region 3 (CDR3), where the Ig Acceptor Framework
sequence includes a stuffer nucleic acid sequence including at
least two Type IIs restriction enzyme recognition sites, and
wherein the source of diversity is a CDR selected from
synthetically produced CDR sequences that contain Type IIs
restriction enzyme recognition sites outside the CDR region, (b)
introducing the source of diversity within each Ig Acceptor
Framework by digesting both the source of diversity and the Ig
Acceptor Framework with a Type IIs restriction enzyme; and (c)
ligating the digested source of diversity into the Ig Acceptor
Framework such that a complete immunoglobulin variable domain
encoding sequences that do not contain the Type IIs restriction
enzyme recognition sites of steps (a) and (b) are restored.
[0107] In some embodiments, the Type IIs restriction enzyme
recognition sites within the stuffer nucleic acid sequences and the
synthetically produced CDR sequences are recognized by the same
Type IIs restriction enzyme. In some embodiments, the Type IIs
restriction enzyme recognition sites within the stuffer nucleic
acid sequences and the synthetically produced CDR sequences are
recognized by different Type IIs restriction enzymes. For example,
the Type IIs restriction enzyme recognition sites are FokI
recognition sites, BsaI recognition sites, and/or BsmBI recognition
sites.
[0108] In some embodiments, the Ig Acceptor Framework nucleic acid
sequence is derived from a human sequence. For example, the human
sequence is a human heavy chain variable gene sequence or a
sequence derived from a human heavy chain variable gene sequence.
In some embodiments, the human heavy chain variable gene sequence
is selected from VH1-2, VH1-69, VH1-18, VH3-30, VH3-48, VH3-23, and
VH5-51. In some embodiments, the human sequence is a human kappa
light chain variable gene sequence or a sequence derived from a
human kappa light chain variable gene sequence. For example, the
human kappa light chain variable gene sequence is selected from
VK1-33, VK1-39, VK3-11, VK3-15, and VK3-20. In some embodiments,
the human sequence is a human lambda light chain variable gene
sequence or a sequence derived from a human lambda light chain
variable gene sequence. For example, the human lambda light chain
variable gene sequence is selected from VL1-44 and VL1-51.
[0109] In some embodiments, the plurality of diversified nucleic
acids encodes amino acid sequences that can fulfill the role of a
CDR3 region, and the plurality of diversified nucleic acids
includes synthetic sequences.
[0110] In some embodiments, the plurality of diversified nucleic
acids encodes amino acid sequences that can fulfill the role of a
CDR1 region, and the plurality of diversified nucleic acids
includes synthetic sequences.
[0111] In some embodiments, the plurality of diversified nucleic
acids encode amino acid sequences that can fulfill the role of a
CDR2 region, and the plurality of diversified nucleic acids
includes synthetic sequences.
[0112] In some embodiments, the plurality of Ig Acceptor Framework
nucleic acid sequences includes a mixture of at least one variable
heavy chain (VH) Acceptor Framework nucleic acid sequence and at
least one variable light chain Acceptor Framework nucleic acid
sequence.
[0113] In some embodiments, the methods provided include the
additional steps of (e) cloning the library of nucleic acids
encoding immunoglobulin variable domains of step (d) into an
expression vector and (f) transforming the expression vector of
step (e) into a host cell and culturing the host cell under
conditions sufficient to express a plurality of immunoglobulin
variable domain encoded by the library. For example, the host cell
is E. coli. In some embodiments, the expression vector is a
phagemid vector. For example, the phagemid vector is pNDS1.
[0114] The invention also provides methods for making an
immunoglobulin polypeptide. These methods include the steps of (a)
providing a plurality of Ig Acceptor Framework nucleic acid
sequences into which a source of diversity is introduced at a
single complementarity determining region (CDR) selected from the
group consisting of complementarity determining region 1 (CDR1),
complementarity determining region 2 (CDR2), and complementarity
determining region 3 (CDR3), wherein the Ig Acceptor Framework
sequence includes a stuffer nucleic acid sequence including at
least two Type IIs restriction enzyme recognition sites, and
wherein the source of diversity is a CDR selected from naturally
occurring CDR sequences that contain Type IIs restriction enzyme
recognition sites outside the CDR region, (b) introducing the
source of diversity within each Ig Acceptor Framework by digesting
both the source of diversity and the Ig Acceptor Frameworks with a
Type IIs restriction enzyme; (c) ligating the digested source of
diversity into the Ig Acceptor Framework such that a complete
immunoglobulin variable gene encoding sequence is restored; and (d)
cloning the complete immunoglobulin variable gene encoding sequence
from step (c) into an expression vector; and (e) transforming the
expression vector of step (d) into a host cell and culturing the
host cell under conditions sufficient to express the complete
immunoglobulin gene encoding sequences that do not contain the Type
IIs restriction enzyme recognition sites are restored.
[0115] In these embodiments, the naturally occurring CDR region
sequences are substantially unaltered from their wild-type, i.e.,
natural state. These naturally occurring CDR region sequences are
flanked by amino acid sequences that have been engineered (or
otherwise artificially manipulated) to contain two Type IIs
restriction enzyme recognition sites, with one Type IIs restriction
enzyme recognition site on each of side of the naturally occurring
CDR region sequence.
[0116] In some embodiments, the Type IIs restriction enzyme
recognition sites within the stuffer nucleic acid sequences and
flanking the naturally occurring CDR sequences are recognized by
the same Type IIs restriction enzyme. In some embodiments, the Type
IIs restriction enzyme recognition sites within the stuffer nucleic
acid sequences and flanking the naturally occurring CDR sequences
are recognized by different Type IIs restriction enzymes. For
example, the Type IIs restriction enzyme recognition sites are FokI
recognition sites, BsaI recognition sites, and/or BsmBI recognition
sites.
[0117] In some embodiments, the Acceptor Framework nucleic acid
sequence is derived from a human gene sequence. For example, the
human sequence is a human heavy chain variable gene sequence or a
sequence derived from a human heavy chain variable gene sequence.
In some embodiments, the human heavy chain variable gene sequence
is selected from VH1-2, VH1-69, VH1-18, VH3-30, VH3-48, VH3-23, and
VH5-51. In some embodiments, the human sequence is a human kappa
light chain variable gene sequence or a sequence derived from a
human kappa light chain variable gene sequence. For example, the
human kappa light chain variable gene sequence is selected from
VK1-33, VK1-39, VK3-11, VK3-15, and VK3-20. In some embodiments,
the human sequence is a human lambda light chain variable gene
sequence or a sequence derived from a human lambda light chain
variable gene sequence. For example, the human lambda light chain
variable gene sequence is selected from VL1-44 and VL1-51.
[0118] In some embodiments, the set of naturally occurring nucleic
acids includes or is derived from sequences selected from naturally
occurring CDR3 sequences, naturally occurring Ig sequences from
humans, naturally occurring Ig sequences from a mammal, naturally
occurring sequences from a loop region of a T cell receptor in a
mammal, and other naturally diversified polypeptide
collections.
[0119] In some embodiments, the set of naturally occurring nucleic
acids encode CDR3 regions, and the set of naturally occurring
nucleic acids include immunoglobulin sequences that occur naturally
in humans that have been exposed to a particular immunogen or
sequences derived from animals that have been identified as having
been exposed to a particular antigen.
[0120] In some embodiments, the set of naturally occurring nucleic
acids includes or is derived from sequences selected from naturally
occurring CDR1 sequences, naturally occurring Ig sequences from
humans, naturally occurring Ig sequences from a mammal, naturally
occurring sequences from a loop region of a T cell receptor in a
mammal, and other naturally diversified polypeptide
collections.
[0121] In some embodiments, the set of naturally occurring nucleic
acids encode CDR1 regions, and the set of naturally occurring
nucleic acids includes or is derived from immunoglobulin sequences
that occur naturally in humans that have been exposed to a
particular immunogen or sequences derived from animals that have
been identified as having been exposed to a particular antigen.
[0122] In some embodiments, the set of naturally occurring nucleic
acids includes or is derived from sequences selected from naturally
occurring CDR2 sequences, naturally occurring Ig sequences from
humans, naturally occurring Ig sequences from a mammal, naturally
occurring sequences from a loop region of a T cell receptor in a
mammal, and other naturally diversified polypeptide
collections.
[0123] In some embodiments, the set of naturally occurring nucleic
acids encodes CDR2 regions, and the set of naturally occurring
nucleic acids includes immunoglobulin sequences that occur
naturally in humans that have been exposed to a particular
immunogen or sequences derived from animals that have been
identified as having been exposed to a particular antigen.
[0124] In some embodiments, the plurality of Acceptor Framework
nucleic acid sequences include a mixture of at least one variable
heavy chain (VH) Acceptor Framework nucleic acid sequence and at
least one variable light chain Acceptor Framework nucleic acid
sequence.
[0125] In some embodiments, the expression vector is a phagemid
vector. In some embodiments, the host cell is E. coli.
[0126] In some embodiments, the method also includes the steps of
contacting the host cell with a target antigen, and determining
which expressed complete Ig variable gene encoding sequences bind
to the target antigen, thereby identifying target specific
antibodies, antibody variable regions or portions thereof. In some
embodiments, the method includes the additional step of (i)
sequencing the immunoglobulin variable domain encoding sequences
that bind the target antigen.
[0127] The invention also provides methods for making an
immunoglobulin polypeptide. These methods include the steps of (a)
providing a plurality of Ig Acceptor Framework nucleic acid
sequences into which a source of diversity is introduced at a
single complementarity determining region (CDR) selected from the
group consisting of complementarity determining region 1 (CDR1),
complementarity determining region 2 (CDR2), and complementarity
determining region 3 (CDR3), wherein the Ig Acceptor Framework
sequence includes a stuffer nucleic acid sequence including at
least two Type IIs restriction enzyme recognition sites, and
wherein the source of diversity is a CDR selected from
synthetically produced CDR sequences that contain Type IIs
restriction enzyme recognition sites outside the CDR region, (b)
introducing the source of diversity within each Ig Acceptor
Framework by digesting both the source of diversity and the Ig
Acceptor Framework with a Type IIs restriction enzyme; (c) ligating
the digested source of diversity into the Ig Acceptor Framework
such that a complete immunoglobulin variable gene encoding sequence
is restored; (d) cloning the ligated Ig Acceptor Framework from
step (c) into an expression vector; and (e) transforming the
expression vector of step (d) into a host cell and culturing the
host cell under conditions sufficient to express the complete
immunoglobulin gene encoding sequences that do not contain the Type
IIs restriction enzyme recognition sites are restored.
[0128] In some embodiments, the Type IIs restriction enzyme
recognition sites within the stuffer nucleic acid sequences and the
synthetically produced CDR sequences are recognized by the same
Type IIs restriction enzyme. In some embodiments, the Type IIs
restriction enzyme recognition sites within the stuffer nucleic
acid sequences and the synthetically produced CDR sequences are
recognized by different Type IIs restriction enzymes. For example,
the Type IIs restriction enzyme recognition sites are FokI
recognition sites, BsaI recognition sites, and/or BsmBI recognition
sites.
[0129] In some embodiments, the Ig Acceptor Framework nucleic acid
sequence is derived from a human gene sequence. For example, the
human sequence is a human heavy chain variable gene sequence or a
sequence derived from a human heavy chain variable gene sequence.
In some embodiments, the human heavy chain variable gene sequence
is selected from VH1-2, VH1-69, VH1-18, VH3-30, VH3-48, VH3-23, and
VH5-51. In some embodiments, the human sequence is a human kappa
light chain variable gene sequence or a sequence derived from a
human kappa light chain variable gene sequence. For example, the
human kappa light chain variable gene sequence is selected from
VK1-33, VK1-39, VK3-11, VK3-15, and VK3-20. In some embodiments,
the human sequence is a human lambda light chain variable gene
sequence or a sequence derived from a human lambda light chain
variable gene sequence. For example, the human lambda light chain
variable gene sequence is selected from VL1-44 and VL1-51.
[0130] In some embodiments, the plurality of diversified nucleic
acids encodes amino acid sequences that can fulfill the role of a
CDR3 region, and the plurality of diversified nucleic acids
includes synthetic sequences.
[0131] In some embodiments, the plurality of diversified nucleic
acids encodes amino acid sequences that can fulfill the role of a
CDR1 region, and the plurality of diversified nucleic acids
includes synthetic sequences.
[0132] In some embodiments, the plurality of diversified nucleic
acids encode amino acid sequences that can fulfill the role of a
CDR2 region, and the plurality of diversified nucleic acids
includes synthetic sequences.
[0133] In some embodiments, the plurality of Ig Acceptor Framework
nucleic acid sequences includes a mixture of at least one variable
heavy chain (VH) Acceptor Framework nucleic acid sequence and at
least one variable light chain Acceptor Framework nucleic acid
sequence.
[0134] In some embodiments, the expression vector is a phagemid
vector. In some embodiments, the host cell is E. coli.
[0135] In some embodiments, the method also includes the steps of
contacting the host cell with a target antigen, and determining
which expressed complete Ig variable gene encoding sequences bind
to the target antigen, thereby identifying target specific
antibodies, antibody variable regions or portions thereof. In some
embodiments, the method includes the additional step of (i)
sequencing the immunoglobulin variable domain encoding sequences
that bind the target antigen.
[0136] The invention provides methods for producing a collection of
nucleic acids, wherein each nucleic acid encodes a human
immunoglobulin variable domain including a plurality of
complementarity determining region 3 (CDR3) sequences isolated
separately from the immunoglobulin variable domain repertoire from
a mammalian species. The invention also provides methods for
producing a collection of nucleic acids, wherein each nucleic acid
encodes a human immunoglobulin variable domain including a
plurality of complementarity determining region 2 (CDR2) sequences
isolated separately from the immunoglobulin variable domain
repertoire from a mammalian species. The invention also provides
methods for producing a collection of nucleic acids, wherein each
nucleic acid encodes a human immunoglobulin variable domain
including a plurality of complementarity determining region 1
(CDR1) sequences isolated separately from the immunoglobulin
variable domain repertoire from a mammalian species.
[0137] The invention provides methods for producing a collection of
nucleic acids, wherein each nucleic acid encodes a human
immunoglobulin variable domain including a plurality of
complementarity determining region 3 (CDR3) sequences isolated
separately from the immunoglobulin variable domain repertoire from
a non-human mammalian species. The invention also provides methods
for producing a collection of nucleic acids, wherein each nucleic
acid encodes a human immunoglobulin variable domain including a
plurality of complementarity determining region 2 (CDR2) sequences
isolated separately from the immunoglobulin variable domain
repertoire from a non-human mammalian species. The invention also
provides methods for producing a collection of nucleic acids,
wherein each nucleic acid encodes a human immunoglobulin variable
domain including a plurality of complementarity determining region
1 (CDR1) sequences isolated separately from the immunoglobulin
variable domain repertoire from a non-human mammalian species.
[0138] In some embodiments, the non-human species is non-human
primate, rodent, canine, feline, sheep, goat, cattle, horse, a
member of the Camelidae family, llama, camel, dromedary, or
pig.
[0139] The invention provides methods for producing a collection of
nucleic acids, wherein each nucleic acid encodes a human
immunoglobulin variable domain including a plurality of
complementarity determining region 3 (CDR3) sequences isolated
separately from the immunoglobulin variable domain repertoire from
a human. The invention provides methods for producing a collection
of nucleic acids, wherein each nucleic acid encodes a human
immunoglobulin variable domain including a plurality of
complementarity determining region 2 (CDR2) sequences isolated
separately from the immunoglobulin variable domain repertoire from
a human. The invention provides methods for producing a collection
of nucleic acids, wherein each nucleic acid encodes a human
immunoglobulin variable domain including a plurality of
complementarity determining region 1 (CDR1) sequences isolated
separately from the immunoglobulin variable domain repertoire from
a human.
[0140] The invention provides methods for producing a collection of
nucleic acids, wherein each nucleic acid encodes a human
immunoglobulin variable domain including a plurality of
complementarity determining region 3 (CDR3) sequences isolated
separately from the immunoglobulin variable domain repertoire from
a non-human species.
[0141] In some embodiments, these methods includes the steps of (a)
providing a plurality of Acceptor Framework nucleic acid sequences
encoding distinct human immunoglobulin variable domains, each
Acceptor Framework nucleic acid sequence comprising a first
framework region (FR1), a second framework region (FR2), a third
framework region (FR3), and a fourth framework region (FR4),
wherein the FR1 and FR2 regions are interspaced by a
complementarity determining region 1 (CDR1), the FR2 and FR3
regions are interspaced by a complementarity determining region 2
(CDR2), and the FR3 and FR4 regions are interspaced by a stuffer
nucleic acid sequence comprising at least two Type IIs restriction
enzyme recognition sites interspaced by a random nucleic acid
sequence; (b) providing a plurality of diversified nucleic acid
sequences encoding complementarity determining region 3 (CDR3)
sequences isolated from the mammalian species immunoglobulin
repertoire wherein each of the plurality of diversified nucleic
acid sequences comprises a Type IIs restriction enzyme recognition
site at each extremity; (c) digesting each of the plurality of
nucleic acid sequences encoding the CDR3 regions using a Type IIs
restriction enzyme that binds to the Type IIs restriction enzyme
recognition site of step (b) and digesting the stuffer nucleic acid
sequence of step (a) from the Acceptor Framework using a Type IIs
restriction enzyme that binds to the Type IIs restriction enzyme
recognition site of step (a); and (d) ligating the digested nucleic
acid sequences encoding the CDR3 regions or the amino acid
sequences of step (c) into the digested Acceptor Framework of step
(c) such that the FR3 and FR4 regions are interspaced by the
nucleic acid sequences encoding the CDR3 region or the amino acid
sequence that can fulfill the role of a CDR3 region and a complete
immunoglobulin variable domain encoding sequences that do not
contain the Type IIs restriction enzyme recognition sites of steps
(a) and (b) are restored. These steps may also be performed using a
plurality of diversified nucleic acid sequences encoding
complementarity determining region 2 (CDR2) sequences isolated from
the mammalian species immunoglobulin repertoire. These steps may
also be performed using a plurality of diversified nucleic acid
sequences encoding complementarity determining region 1 (CDR1)
sequences isolated from the mammalian species immunoglobulin
repertoire.
[0142] In some embodiments, step (b) is performed by amplifying the
CDR3 sequence from a mammalian species using oligonucleotide
primers containing a Type IIs restriction site. In some
embodiments, the oligonucleotide primer is designed to enhance
compatibility between the mammalian CDR3 sequence and the Acceptor
Framework encoding a human immunoglobulin variable domain. In some
embodiments, the oligonucleotide primer is designed to modify the
sequence at the boundaries of the mammalian CDR3 sequences to allow
efficient ligation via compatible cohesive ends into the Acceptor
Framework encoding a human immunoglobulin variable domain. In some
embodiments the mammalian DNA sequences flanking the CDR3 regions
might not upon cleavage by Type IIS restriction enzymes generate
cohesive ends compatible with the cohesive ends of the digested
Acceptor Frameworks. In such cases the oligonucleotides used for
amplification are designed to modify the target mammalian sequence
so that after cleavage with a Type IIS restriction enzyme, the
cohesive ends are compatible and efficient ligation can occur.
These steps can also be performed by amplifying the CDR2 sequence
from a mammalian species using oligonucleotide primers containing a
Type IIs restriction site. These steps can also be performed by
amplifying the CDR1 sequence from a mammalian species using
oligonucleotide primers containing a Type IIs restriction site.
[0143] In some embodiments, step (b) is performed by amplifying the
CDR3 sequence from a non human species using oligonucleotide
primers containing a FokI IIs restriction site. These steps can
also be performed by amplifying the CDR2 sequence from a mammalian
species using oligonucleotide primers containing a FokI IIs
restriction site. These steps can also be performed by amplifying
the CDR1 sequence from a mammalian species using oligonucleotide
primers containing a FokI IIs restriction site.
[0144] In some embodiments, the Type IIs restriction enzyme
recognition sites of step (a) and step (b) are recognized by a
different Type IIs restriction enzyme. In some embodiments, the
Type IIs restriction enzyme recognition sites are BsmBI recognition
sites, BsaI recognition sites, FokI recognition sites or a
combination thereof.
[0145] In some embodiments, the diversified nucleic acid sequences
encoding CDR3 sequences encode heavy chain CDR3 (CDR H3) sequences.
In some embodiments, the diversified nucleic acid sequences
encoding CDR3 sequences encode light chain CDR3 (CDR L3) sequences.
In some embodiments, the diversified nucleic acid sequences
encoding CDR2 sequences encode heavy chain CDR2 (CDR H2) sequences.
In some embodiments, the diversified nucleic acid sequences
encoding CDR2 sequences encode light chain CDR2 (CDR L2) sequences.
In some embodiments, the diversified nucleic acid sequences
encoding CDR1 sequences encode heavy chain CDR1 (CDR H1) sequences.
In some embodiments, the diversified nucleic acid sequences
encoding CDR1 sequences encode light chain CDR1 (CDR L1)
sequences.
[0146] In some embodiments, the Acceptor Framework nucleic acid
sequence includes or is derived from at least a portion of a human
heavy chain variable gene sequence selected from VH1-2, VH1-69,
VH1-18, VH3-30, VH3-48, VH3-23, and VH5-51. In some embodiments,
the Acceptor Framework nucleic acid sequence includes is derived
from at least a portion of a human kappa light chain variable gene
sequence. For example, the human kappa light chain variable gene
sequence is selected from VK1-33, VK1-39, VK3-11, VK3-15, and
VK3-20. In some embodiments, the Acceptor Framework nucleic acid
sequence includes or is derived from at least a portion of a human
lambda light chain variable gene sequence. For example, the human
lambda light chain variable gene sequence is selected from VL1-44
and VL1-51.
[0147] In some embodiments, the plurality of Acceptor Framework
nucleic acid sequences comprises a mixture of at least one variable
heavy chain (VH) Acceptor Framework nucleic acid sequence and at
least one variable light chain Acceptor Framework nucleic acid
sequence.
[0148] In some embodiments, the methods described herein also
include the steps of (e) cloning the library of nucleic acids
encoding immunoglobulin variable domains of step (d) into an
expression vector and (f) transforming the expression vector of
step (e) into a host cell and culturing the host cell under
conditions sufficient to express a plurality of immunoglobulin
variable domain encoded by the library. In some embodiments, the
expression vector is a phagemid or phage vector. In some
embodiments, the host cell is E. coli.
[0149] The invention provides methods for producing a collection of
nucleic acids, wherein each nucleic acid encodes a human
immunoglobulin variable domain including a plurality of
complementarity determining region 3 (CDR3) sequences isolated
separately from immunoglobulin variable domains from an immunized
non-human mammal or non-human species. The invention also provides
methods for producing a collection of nucleic acids, wherein each
nucleic acid encodes a human immunoglobulin variable domain
including a plurality of complementarity determining region 2
(CDR2) sequences isolated separately from immunoglobulin variable
domains from an immunized non-human mammal. The invention also
provides methods for producing a collection of nucleic acids,
wherein each nucleic acid encodes a human immunoglobulin variable
domain including a plurality of complementarity determining region
1 (CDR1) sequences isolated separately from immunoglobulin variable
domains from an immunized non-human mammal.
[0150] In some embodiments, the non-human species is non-human
primate, rodent, canine, feline, sheep, goat, cattle, horse, a
member of the Camelidae family, llama, camel, dromedary, or
pig.
[0151] In some embodiments, the methods include the steps of (a)
providing a plurality of Acceptor Framework nucleic acid sequences
encoding distinct human immunoglobulin variable domains, each
Acceptor Framework nucleic acid sequence comprising a first
framework region (FR1), a second framework region (FR2), a third
framework region (FR3), and a fourth framework region (FR4),
wherein the FR1 and FR2 regions are interspaced by a
complementarity determining region 1 (CDR1), the FR2 and FR3
regions are interspaced by a complementarity determining region 2
(CDR2), and the FR3 and FR4 regions are interspaced by a stuffer
nucleic acid sequence comprising at least two Type IIs restriction
enzyme recognition sites interspaced by a random nucleic acid
sequence; (b) providing a plurality of diversified nucleic acid
sequences encoding complementarity determining region 3 (CDR3)
sequences isolated from the immunized non-human mammal wherein each
of the plurality of diversified nucleic acid sequences comprises a
Type IIs restriction enzyme recognition site at each extremity; (c)
digesting each of the plurality of nucleic acid sequences encoding
the CDR3 regions using a Type IIs restriction enzyme that binds to
the Type IIs restriction enzyme recognition site of step (b) and
digesting the stuffer nucleic acid sequence of step (a) from the
Acceptor Framework using a Type IIs restriction enzyme that binds
to the Type IIs restriction enzyme recognition site of step (a);
and (d) ligating the digested nucleic acid sequences encoding the
CDR3 regions or the amino acid sequences of step (c) into the
digested Acceptor Framework of step (c) such that the FR3 and FR4
regions are interspaced by the nucleic acid sequences encoding the
CDR3 region or the amino acid sequence that can fulfill the role of
a CDR3 region and a complete immunoglobulin variable domain
encoding sequences that do not contain the Type IIs restriction
enzyme recognition sites of steps (a) and (b) are restored. These
steps may also be performed using a plurality of diversified
nucleic acid sequences encoding complementarity determining region
2 (CDR2) sequences isolated from the immunized non-human mammal.
These steps may also be performed using a plurality of diversified
nucleic acid sequences encoding complementarity determining region
1 (CDR1) sequences isolated from the immunized non-human
mammal.
[0152] In some embodiments, step (b) is performed by amplifying the
CDR3 sequence from the immunized non-human mammal using
oligonucleotide primers containing a Type IIs restriction site. In
some embodiments, the oligonucleotide primer is designed to enhance
compatibility between the mammalian CDR3 sequence and the Acceptor
Framework encoding a human immunoglobulin variable domain. In some
embodiments, the oligonucleotide primer is designed to modify the
sequence at the boundaries of the mammalian CDR3 sequences to allow
efficient ligation via compatible cohesive ends into the Acceptor
Framework encoding a human immunoglobulin variable domain. In some
embodiments the mammalian DNA sequences flanking the CDR3 regions
might not upon cleavage by Type IIS restriction enzymes generate
cohesive ends compatible with the cohesive ends of the digested
Acceptor Frameworks. In such cases the oligonucleotides used for
amplification are designed to modify the target mammalian sequence
so that after cleavage with a Type IIS restriction enzyme, the
cohesive ends are compatible and efficient ligation can occur.
These steps can also be performed by amplifying the CDR2 sequence
from the immunized non-human mammal using oligonucleotide primers
containing a Type IIs restriction site. These steps can also be
performed by amplifying the CDR1 sequence from the immunized
non-human mammal using oligonucleotide primers containing a Type
IIs restriction site.
[0153] In some embodiments, step (b) is performed by amplifying the
CDR H3 sequence from the non-human mammal using oligonucleotide
primers containing a FokI IIs restriction site. These steps can
also be performed by amplifying the CDR2 sequence from the
non-human mammal using oligonucleotide primers containing a FokI
IIs restriction site. These steps can also be performed by
amplifying the CDR1 sequence from the non-human mammal using
oligonucleotide primers containing a FokI IIs restriction site.
[0154] In some embodiments, the Type IIs restriction enzyme
recognition sites of step (a) and step (b) are recognized by a
different Type IIs restriction enzyme. In some embodiments, the
Type IIs restriction enzyme recognition sites are BsmBI recognition
sites, BsaI recognition sites, FokI recognition sites or a
combination thereof.
[0155] In some embodiments, the diversified nucleic acid sequences
encoding CDR3 sequences encode heavy chain CDR3 (CDR H3) sequences.
In some embodiments, the diversified nucleic acid sequences
encoding CDR3 sequences encode light chain CDR3 (CDR L3) sequences.
In some embodiments, the diversified nucleic acid sequences
encoding CDR2 sequences encode heavy chain CDR2 (CDR H2) sequences.
In some embodiments, the diversified nucleic acid sequences
encoding CDR2 sequences encode light chain CDR2 (CDR L2) sequences.
In some embodiments, the diversified nucleic acid sequences
encoding CDR1 sequences encode heavy chain CDR1 (CDR H1) sequences.
In some embodiments, the diversified nucleic acid sequences
encoding CDR1 sequences encode light chain CDR1 (CDR L1)
sequences.
[0156] In some embodiments, the Acceptor Framework nucleic acid
sequence includes or is derived from at least a portion of a human
heavy chain variable gene sequence selected from VH1-2, VH1-69,
VH1-18, VH3-30, VH3-48, VH3-23, and
VH5-51.
[0157] In some embodiments, the Acceptor Framework nucleic acid
sequence includes or is derived from at least a portion of a human
kappa light chain variable gene sequence. For example, the human
kappa light chain variable gene sequence is selected from VK1-33,
VK1-39, VK3-11, VK3-15, and VK3-20. In some embodiments, the
Acceptor Framework nucleic acid sequence includes or is derived
from at least a portion of a human lambda light chain variable gene
sequence. For example, the human lambda light chain variable gene
sequence is selected from VL1-44 and VL1-51.
[0158] In some embodiments, the plurality of Acceptor Framework
nucleic acid sequences comprises a mixture of at least one variable
heavy chain (VH) Acceptor Framework nucleic acid sequence and at
least one variable light chain Acceptor Framework nucleic acid
sequence.
[0159] In some embodiments, the methods also include the steps of
(e) cloning the library of nucleic acids encoding immunoglobulin
variable domains of step (d) into an expression vector and (f)
transforming the expression vector of step (e) into a host cell and
culturing the host cell under conditions sufficient to express a
plurality of immunoglobulin variable domain encoded by the library.
In some embodiments, the host cell is E. coli. In some embodiments,
the expression vector is a phagemid or phage vector.
BRIEF DESCRIPTION OF THE DRAWINGS
[0160] FIG. 1A is a schematic representation of a protein domain
with a framework and loops providing contact residues with another
protein or molecule. Several situations are depicted: A stable
protein domain with properly folded loop regions; properly folded
loops inserted into a domain of limited intrinsic stability; an
intrinsically stable protein domain which stability is affected by
the loop regions.
[0161] FIG. 1B is a schematic representation of different types of
libraries of protein repertoires generated using different
diversification strategies.
[0162] FIG. 2 is a schematic representation of an antibody variable
Acceptor Framework. Framework regions, CDRs and type IIS-RM
restriction site are indicated.
[0163] FIG. 3 is a schematic representation of a strategy used for
capturing CDRH3 sequences from natural repertoires.
[0164] FIG. 4 is a schematic representation of the benefit of using
primers containing Type IIS-RM restriction enzymes for the
amplification and insertion of natural CDR regions into Acceptor
Frameworks.
[0165] FIG. 5 is an illustration depicting the germline gene
sequences of the variable heavy and light chain domain selected for
the generation of Acceptor Frameworks.
[0166] FIG. 6 is a schematic representation of an amplification
strategy used for the generation of Acceptor Frameworks by addition
to the germline sequences of a stuffer fragment and a FR4
region.
[0167] FIG. 7, top panel, is an illustration depicting the sequence
detail of Stuffer fragments of VH acceptor Framework. DNA sequences
recognized and cleaved by the restriction enzyme BsmBI are boxed in
red and black respectively and indicated in the lower panel of the
figure. The reading frame corresponding to the antibody variable
sequence is underlined.
[0168] FIG. 8 is an illustration depicting the sequences of the 20
Acceptor Frameworks.
[0169] FIG. 9 is a schematic representation of the pNDS1 vector
alone or combined with a dummy heavy chain variable region or a
dummy light variable region.
[0170] FIG. 10 is a table depicting the sequences of CDRH3
sequences that were retrieved from a human cDNA source and inserted
into human Acceptor Frameworks.
[0171] FIG. 11 is a table representing the design of synthetic CDR
sequences for VH, VK and V.lamda.. The positions are numbered
according to the Kabat numbering scheme. The theoretical diversity
of the CDR using a defined codon diversification strategy (NNS,
DVK, NVT, DVT) is indicated. The strategies adopted for VH CDR
synthesis are boxed.
[0172] FIG. 12 is a schematic representation and sequence detail of
synthetic CDR insertion into an Acceptor Framework.
[0173] FIG. 13 is a schematic representation of Primary libraries
and the chain recombination performed to generate Secondary
libraries.
[0174] FIG. 14 is a schematic representation of the generation of
Acceptor VH libraries combined with VL synthetic libraries and the
capture of CDRH3 repertoires of human or non-human origin.
[0175] FIG. 15 is a schematic representation of the MnA, MiB and
MiC library generation using the CDRH3 repertoire from naive mice
or mice immunized with hIFN.gamma. or hCCL5/RANTES as a source of
diversity. The size of the libraries is indicated in the top
panels. The bottom panels show the distribution of CDRH3 lengths
found in these libraries.
[0176] FIG. 16 is a series of graphs depicting phage output
titration during selection against hIFN.gamma. with the secondary
libraries AD1 and AE1.
[0177] FIG. 17 is a series of graphs depicting phage output
titration during selection against monoclonal antibody 5E3 with the
secondary libraries AD1 and AE1.
[0178] FIG. 18 is a series of graphs depicting the frequency of CDR
H3 lengths found in the AE1 and AD1 libraries and after three
rounds of selection against the monoclonal antibody 5E3. The
distribution of each CDR H3 length within the different VH families
is indicated. However, when CDR H3 are longer than 16 amino acids,
the 70 bp sequences delivered by the Illumina Sequencing platform
do not cover enough framework sequence to unambiguously identify
the VH1 family and therefore the VH family is indicated as
undetermined.
[0179] FIG. 19 is a series of graphs depicting dose response ELISA
using purified 6 scFv preparations against mouse 5E3 or an
irrelevant mouse antibody 1A6. The seven clones encode different
scFvs. Clone A6 is a scFv specific for hIFN.gamma. and was used as
a negative control.
[0180] FIG. 20 is a graph that depicts dose response ELISA using
purified scFv preparations against hIFN.gamma. and compared to a
positive scFv specific for hIFN.gamma. (A6).
[0181] FIG. 21 is a graph that depicts the inhibitory effect of
purified scFv preparations in a luciferase reporter gene assay
driven by hIFN.gamma.. The neutralizing activity of two scFv
candidates (AD1R4P1A9 and AE14R3P2E4) was compared to the activity
of a positive control scFv (G9) and a negative control scFv
(D11).
[0182] FIG. 22 is a graph that depicts the inhibitory effect of
purified scFv preparations in a MHCII induction assay in response
to hIFN.gamma.. The neutralizing activity of two scFv candidates
(AD1R4P1A9 and AE14R3P2E4) was compared to the activity of a
negative control scFv (D11).
[0183] FIG. 23 is a series of graphs depicting the inhibitory
effect of the two candidates AD1R4P1A9 and AE14R3P2E4 reformatted
into IgG in a luciferase reporter gene assay driven by hIFN.gamma..
The neutralizing activity of two IgGs was compared to the activity
of an irrelevant IgG directed against human RANTES (NI-0701).
[0184] FIG. 24 is a series of graphs depicting a dose response
ELISA using the IgG G11 and DA4 against mouse 5E3, chimeric rat 5E3
and the corresponding mouse and rat isotype antibodies.
[0185] FIG. 25 is a series of graphs depicting an ELISA for the
detection of mouse 5E3 in different dilutions of mouse serum using
the anti-idiotypic IgGs G11 and DA4 as capture antibodies.
[0186] FIG. 26 is a graph that depicts phage output/input ratios
during selection against hIFN.gamma. with the libraries MnA and
MiB.
[0187] FIG. 27 is a graph depicting the hit rates obtained in a
scFv ELISA screening with clones derived from the MnA, MiB and MiC
libraries after each round of selection against hIFN.gamma.. The
threshold was set to half the signal obtained with the A6 control
scFv.
[0188] FIG. 28 is a graph that represents the distribution
frequency of scFv giving different levels of signal in binding
experiments against hIFN.gamma. obtained with clones derived from
the MnA and MiB libraries.
[0189] FIG. 29 is a graph that depicts dose response ELISA using
purified scFv preparations from clones derived from the MnA and MiB
libraries against hIFN.gamma. and compared to a positive scFv
specific for hIFN.gamma. (A6).
[0190] FIG. 30 is a schematic representation of methods of
generating Acceptor libraries that contain pre-selected and
functional diversified light chain variable domains that can
directly be used for the insertion of captured CDRH3 regions.
[0191] FIG. 31 is an illustration depicting oligonucleotides that
were designed to synthesize a collection of stuffer fragments
containing two BsmBI restriction sites and introducing diversity in
one or two codons.
[0192] FIGS. 32 and 33 are illustrations depicting the
oligonucleotide sequences identified in the selected clones.
DETAILED DESCRIPTION OF THE INVENTION
[0193] Synthetic protein libraries and in particular synthetic
antibody libraries are attractive as it is possible during the
library generation process to select the building blocks composing
these synthetic proteins and include desired characteristics. An
important limitation, however, is that the randomization of
portions of these synthetic proteins to generate a collection of
variants often leads to non-functional proteins and thus can
dramatically decrease the functional library size and its
performance. Another limitation of synthetic diversity is that the
library size needed to cover the theoretical diversity of
randomized amino acid stretches cannot be covered because of
practical limitations. Even with display systems such as ribosome
display a diversity of 10.sup.13 to 10.sup.14 can be generated and
sampled which can maximally cover the complete randomization of
stretches of 9 amino acids. As the average size of natural CDR H3
(also referred to herein as the heavy chain CDR3 or VH CDR3) is
above 9 and can be over 20 amino acids in length, synthetic
diversity is not a practicable approach to generate such CDRs.
[0194] The combination of methods generally used for DNA handling
and that are used in the course of the generation of a library of
protein variants introduces errors in the DNA sequences. These
errors can lead to alterations in the reading frame of the DNA that
will no longer encode a functional polypeptide. Typically, antibody
libraries generated using assembly of DNA fragments by PCR and/or
restriction cloning contain between 15% and 45% sequences that are
not in the correct reading frame for protein translation. These
non-functional library members can compromise the efficiency of the
antibody selection and identification process and are thus
recognized as a limitation in the field. The methods described
allow for a more robust introduction of diversity into an antibody
library by using an alternative cloning strategy. Typically the
frequency of in-frame sequences is approximately 90%. Another
advantage of the invention is that it combines selected acceptor
antibody variable frameworks with CDR loops that have a high
probability of correct folding. It allows for the capture of long
CDRs that are difficult to cover with synthetic randomization
approaches. Furthermore the methods described do not employ any
modification within the coding region of acceptor antibody variable
for cloning of the diversified sequences. Another advantage of this
method is that several sources of diversity can be captured into
the same set of acceptor antibody frameworks. These sources include
but are not limited to: natural antibody CDRs of human or other
mammal origin, CDR from chicken antibodies, CDRs of antibody-like
molecules such as VHH from camelids, IgNARs from sharks, variable
loops from T cell receptors. In addition, natural CDRs can be
derived from naive or immunized animals. In the latter case, the
CDRs retrieved are enriched in sequences that were involved in
recognition of the antigen used for immunization.
[0195] A unique feature of the methods described herein is the
efficient capture of heavy chain CDR3 coding sequences from
non-human species and their insertion into human immunoglobulin
frameworks. Using these methods, it is therefore possible to
generate different antibody combining sites that are shaped by the
captured CDRH3 repertoire from another species and allow for the
sampling of a different tri dimensional space. These methods allow
for the generation of human antibodies with novel specificities
targeting a different range of target classes and epitopes than
those accessible to a human CDRH3 repertoire. Furthermore, these
novel antibodies encode human framework as well as CDR1 and CDR2
regions and thus are suitable for human therapy.
[0196] In this method selected protein domains, as exemplified by
antibody variable domains, are modified by introducing a stuffer
sequence that will serve as an integration site for diversified
sequences. Upon integration, the stuffer fragment is removed in
full, thus leaving intact the coding region of the acceptor protein
and the inserted proteins fragments (i.e., the CDRs). This
integration event is mediated by a the use of Type IIs restriction
enzyme that recognizes a defined site in the DNA sequence but
cleave the DNA at a defined distance from this site. This approach
has two major advantages: (1) it allows for the digestion of
acceptors framework without affecting their coding sequences (no
need to engineer silent restrictions sites); and (2) it allows for
the digestion and cloning of naturally diversified sequences that
by definition do not possess compatible restriction sites.
[0197] As described above, prior attempts to generate libraries
and/or displays of antibody sequences differ from the methods
provided herein. For example, some methods require the grafting of
each CDR, as described for example by U.S. Pat. No. 6,300,064, in
which restriction enzyme sites are engineered at the boundary of
each CDR, not just the CDR H3 region. In other methods, CDR
sequences from natural sources are amplified and rearranged, as
described in, e.g., U.S. Pat. No. 6,989,250. In some methods, such
as those described in US Patent Application Publication No.
20060134098, sequences from a mouse (or other mammal) is added to a
human framework, such that the resulting antibody has CDR1 and CDR2
regions of murine origin and a CDR3 region of human origin. Other
methods, such as those described in US Patent Application
Publication No. 20030232333, generate antibodies that have
synthetic CDR1 and/or CDR1/CDR2 regions along with a natural CDR3
region. However, these methods fail to provide libraries that
contain stable framework regions and correctly folded CDRs.
[0198] The methods provided herein design the antibody acceptor
frameworks for diversity cloning. A strategy was designed to
introduce diversity into the CDR3 of selected human antibody
domains that avoids the modification of the sequence of the
original framework. The strategy relies on the introduction outside
of the immunoglobulin coding region of Type IIs restriction sites.
This class of restriction enzymes recognizes asymmetric and
uninterrupted sequence of 4-7 base pairs but cleave DNA at a
defined distance of up to 20 bases independently of the DNA
sequence found at the cleavage site. In order to take advantage of
this system for cloning of diversified sequences into selected
frameworks, acceptor frameworks containing a stuffer DNA fragment,
instead of the CDR3, that includes two Type IIs restriction sites
were designed. Similarly, diversified DNA sequences are generated
with flanking sequences that include Type IIs. Provided that the
cohesive ends generated by the restriction enzymes are compatible
and that reading frame is maintained, the DNA fragments can be
ligated into the acceptor framework and restore the encoded CDR3 in
the new context of the acceptor antibody framework (FIG. 2).
[0199] The methods provided herein capture natural CDR diversity.
The strategy that was developed to capture naturally diversified
protein fragments as a source of diversity also takes advantage of
Type IIs restriction enzymes. As an example, oligonucleotides
primers specific for flanking regions of the DNA sequence encoding
the CDR H3 of immunoglobulins, i.e., specific for the FR3 and FR4
of the variable region, were designed. These oligonucleotides
contain at their 5' end a site for a Type IIs restriction enzyme
whereas their 3' portion matches the targeted DNA sequence. The
restriction enzyme site used is preferably an enzyme that cleaves
DNA far away from the DNA recognition site such as FokI. This is a
key element of the method as it allows for the efficient
amplification of natural DNA sequences as it maintains a good match
between the 3' end of the primer and the DNA flanking the CDR H3
while allowing for excision of the CDRH3 coding sequence by DNA
cleavage at the boundary between the CDR and framework regions
(FIG. 3). This precise excision of the CDR coding sequence is very
difficult using Type II enzymes that cleave DNA at their
recognition site as the corresponding restriction site is not
present in the natural DNA sequences and that introduction of such
sites during amplification would be difficult due poor primer
annealing. Thus this method allows for the amplification of
diversified protein sequences and their insertion into any the
acceptor antibody framework regardless of origin of amplified
diversity (FIG. 4).
[0200] The methods described herein produce a library of nucleic
acids, wherein each nucleic acid encodes an immunoglobulin variable
domain by: (a) providing a plurality of Acceptor Framework nucleic
acid sequences encoding distinct immunoglobulin variable domains,
each Acceptor Framework nucleic acid sequence including a first
framework region (FR1), a second framework region (FR2), a third
framework region (FR3), and a fourth framework region (FR4),
wherein the FR1 and FR2 regions are interspaced by a
complementarity determining region 1 (CDR1), the FR2 and FR3
regions are interspaced by a complementarity determining region 2
(CDR2), and the FR3 and FR4 regions are interspaced by a stuffer
nucleic acid sequence containing at least two Type IIs restriction
enzyme recognition sites interspaced by a random nucleic acid
sequence; (b) providing a plurality of diversified nucleic acid
sequences encoding complementarity determining region 3 (CDR3)
regions or encoding amino acid sequences that can fulfill the role
of a CDR3 region, wherein each of the plurality of diversified
nucleic acid sequences includes a Type IIs restriction enzyme
recognition site at each extremity; (c) digesting each of the
plurality of nucleic acid sequences encoding the CDR3 regions or
amino acid sequences that can fulfill the role of a CDR3 region
using a Type IIs restriction enzyme that binds to the Type IIs
restriction enzyme recognition site of step (b) and digesting the
stuffer nucleic acid sequence of step (a) from the Acceptor
Framework using a Type IIs restriction enzyme that binds to the
Type IIs restriction enzyme recognition site of step (a); and (d)
ligating the digested nucleic acid sequences encoding the CDR3
regions or the amino acid sequences that can fulfill the role of a
CDR3 region of step (c) into the digested Acceptor Framework of
step (c) such that the FR3 and FR4 regions are interspaced by the
nucleic acid sequences encoding the CDR3 region or the amino acid
sequence that can fulfill the role of a CDR3 region and a complete
immunoglobulin variable domain encoding sequences that do not
contain the Type IIs restriction enzyme recognition sites of steps
(a) and (b) are restored.
[0201] The methods provided herein produce a method for producing a
library of nucleic acids, wherein each nucleic acid encodes an
immunoglobulin variable domain by: (a) providing a plurality of
Acceptor Framework nucleic acid sequences encoding distinct
immunoglobulin variable domains, each Acceptor Framework nucleic
acid sequence including a first framework region (FR1), a second
framework region (FR2), a third framework region (FR3), and a
fourth framework region (FR4), wherein the FR1 and FR2 regions are
interspaced by a stuffer nucleic acid sequence containing at least
two Type IIs restriction enzyme recognition sites interspaced by a
random nucleic acid sequence, the FR2 and FR3 regions are
interspaced by a complementarity determining region 2 (CDR2), and
the FR3 and FR4 regions are interspaced by a complementarity
determining region 3 (CDR3); (b) providing a plurality of
diversified nucleic acid sequences encoding complementarity
determining region 1 (CDR1) regions or encoding amino acid
sequences that can fulfill the role of a CDR1 region, wherein each
of the plurality of diversified nucleic acid sequences includes a
Type IIs restriction enzyme recognition site at each extremity; (c)
digesting each of the plurality of nucleic acid sequences encoding
the CDR1 regions or amino acid sequences that can fulfill the role
of a CDR1 region using a Type IIs restriction enzyme that binds to
the Type IIs restriction enzyme recognition site of step (b) and
digesting the stuffer nucleic acid sequence of step (a) from the
Acceptor Framework using a Type IIs restriction enzyme that binds
to the Type IIs restriction enzyme recognition site of step (a);
and (d) ligating the digested nucleic acid sequences encoding the
CDR1 regions or the amino acid sequences that can fulfill the role
of a CDR1 region of step (c) into the digested Acceptor Framework
of step (c) such that the FR1 and FR2 regions are interspaced by
the nucleic acid sequences encoding the CDR1 region or the amino
acid sequence that can fulfill the role of a CDR1 region and a
complete immunoglobulin variable domain encoding sequences that do
not contain the Type IIs restriction enzyme recognition sites of
steps (a) and (b) are restored.
[0202] The methods provided herein produce a library of nucleic
acids, wherein each nucleic acid encodes an immunoglobulin variable
domain, by: (a) providing a plurality of Acceptor Framework nucleic
acid sequences encoding distinct immunoglobulin variable domains,
each Acceptor Framework nucleic acid sequence including a first
framework region (FR1), a second framework region (FR2), a third
framework region (FR3), and a fourth framework region (FR4),
wherein the FR1 and FR2 regions are interspaced by a
complementarity determining region 1 (CDR1), the FR2 and FR3
regions are interspaced by a stuffer nucleic acid sequence
including at least two Type IIs restriction enzyme recognition
sites interspaced by a random nucleic acid sequence, and the FR3
and FR4 regions are interspaced by a complementarity determining
region 3 (CDR3); (b) providing a plurality of diversified nucleic
acid sequences encoding complementarity determining region 2 (CDR2)
regions or encoding amino acid sequences that can fulfill the role
of a CDR2 region, wherein each of the plurality of diversified
nucleic acid sequences includes a Type IIs restriction enzyme
recognition site at each extremity; (c) digesting each of the
plurality of nucleic acid sequences encoding the CDR2 regions or
amino acid sequences that can fulfill the role of a CDR2 region
using a Type IIs restriction enzyme that binds to the Type IIs
restriction enzyme recognition site of step (b) and digesting the
stuffer nucleic acid sequence of step (a) from the Acceptor
Framework using a Type IIs restriction enzyme that binds to the
Type IIs restriction enzyme recognition site of step (a); and (d)
ligating the digested nucleic acid sequences encoding the CDR2
regions or the amino acid sequences that can fulfill the role of a
CDR2 region of step (c) into the digested Acceptor Framework of
step (c) such that the FR2 and FR3 regions are interspaced by the
nucleic acid sequences encoding the CDR2 region or the amino acid
sequence that can fulfill the role of a CDR2 region and a complete
immunoglobulin variable domain encoding sequences that do not
contain the Type IIs restriction enzyme recognition sites of steps
(a) and (b) are restored.
[0203] In some embodiments, the Type IIs restriction enzyme
recognition sites of step (a) and step (b) in the methods set forth
above are recognized by a different Type IIs restriction enzyme.
For example, in some embodiments, the Type IIs restriction enzyme
recognition sites are BsmBI recognition sites, BsaI recognition
sites, FokI recognition sites or a combination thereof.
[0204] In some embodiments, the Acceptor Framework nucleic acid
sequence is derived from a human gene sequence. For example, in
some embodiments, the human sequence is a human heavy chain
variable gene sequence or a sequence derived from a human heavy
chain variable gene sequence. In some embodiments, the human heavy
chain variable gene sequence is selected from VH1-2, VH1-69,
VH1-18, VH3-30, VH3-48, VH3-23, and VH5-51.
[0205] In some embodiments, the human sequence is a human kappa
light chain variable gene sequence or a sequence derived from a
human kappa light chain variable gene sequence. For example, in
some embodiments, the human kappa light chain variable gene
sequence is selected from VK1-33, VK1-39, VK3-11, VK3-15, and
VK3-20.
[0206] In some embodiments, the human sequence is a human lambda
light chain variable gene sequence or a sequence derived from a
human lambda light chain variable gene sequence. For example, in
some embodiments, the human lambda light chain variable gene
sequence is selected from VL1-44 and VL1-51.
[0207] In some embodiments, the plurality of diversified nucleic
acids includes or is derived from sequences selected from naturally
occurring CDR3 sequences, naturally occurring Ig sequences from
humans, naturally occurring Ig sequences from a mammal, naturally
occurring sequences from a loop region of a T cell receptor in a
mammal, and other naturally diversified polypeptide
collections.
[0208] In some embodiments, the plurality of diversified nucleic
acids encodes CDR3 regions, and the plurality of diversified
nucleic acids includes or is derived from immunoglobulin sequences
that occur naturally in humans that have been exposed to a
particular immunogen or sequences derived from animals that have
been identified as having been exposed to a particular antigen.
[0209] In some embodiments, the plurality of diversified nucleic
acids encodes amino acid sequences that can fulfill the role of a
CDR3 region, and the plurality of diversified nucleic acids
includes synthetic sequences.
[0210] In some embodiments, the plurality of diversified nucleic
acids includes or is derived from sequences selected from naturally
occurring CDR1 sequences, naturally occurring Ig sequences from
humans, naturally occurring Ig sequences from a mammal, naturally
occurring sequences from a loop region of a T cell receptor in a
mammal, and other naturally diversified polypeptide
collections.
[0211] In some embodiments, the plurality of diversified nucleic
acids encodes CDR1 regions, and the plurality of diversified
nucleic acids includes or is derived from immunoglobulin sequences
that occur naturally in humans that have been exposed to a
particular immunogen or sequences derived from animals that have
been identified as having been exposed to a particular antigen.
[0212] In some embodiments, the plurality of diversified nucleic
acids encodes amino acid sequences that can fulfill the role of a
CDR1 region, and the plurality of diversified nucleic acids
includes synthetic sequences.
[0213] In some embodiments, the plurality of diversified nucleic
acids includes or is derived from sequences selected from naturally
occurring CDR2 sequences, naturally occurring Ig sequences from
humans, naturally occurring Ig sequences from a mammal, naturally
occurring sequences from a loop region of a T cell receptor in a
mammal, and other naturally diversified polypeptide
collections.
[0214] In some embodiments, the plurality of diversified nucleic
acids encodes CDR2 regions, and the plurality of diversified
nucleic acids includes or is derived from immunoglobulin sequences
that occur naturally in humans that have been exposed to a
particular immunogen or sequences derived from animals that have
been identified as having been exposed to a particular antigen.
[0215] In some embodiments, the plurality of diversified nucleic
acids encodes amino acid sequences that can fulfill the role of a
CDR2 region, and the plurality of diversified nucleic acids
includes synthetic sequences.
[0216] In some embodiments, the plurality of Acceptor Framework
nucleic acid sequences includes a mixture of at least one variable
heavy chain (VH) Acceptor Framework nucleic acid sequence and at
least one variable light chain Acceptor Framework nucleic acid
sequence.
[0217] In some embodiments, the methods provided herein further
include the steps of (e) cloning the library of nucleic acids
encoding immunoglobulin variable domains of step (d) into an
expression vector and (f) transforming the expression vector of
step (e) into a host cell and culturing the host cell under
conditions sufficient to express a plurality of immunoglobulin
variable domain encoded by the library.
[0218] In some embodiments, the host cell is E. coli. In some
embodiments, the expression vector is a phagemid vector.
[0219] The methods provided herein generate or otherwise produce a
target-specific antibody, antibody variable region or a portion
thereof, by: (a) providing a plurality of Acceptor Framework
nucleic acid sequences encoding distinct immunoglobulin variable
domains, each Acceptor Framework nucleic acid sequence including a
first framework region (FR1), a second framework region (FR2), a
third framework region (FR3), and a fourth framework region (FR4),
wherein the FR1 and FR2 regions are interspaced by a
complementarity determining region 1 (CDR1), the FR2 and FR3
regions are interspaced by a complementarity determining region 2
(CDR2), and the FR3 and FR4 regions are interspaced by a stuffer
nucleic acid sequence having at least two Type IIs restriction
enzyme recognition sites interspaced by a random nucleic acid
sequence; (b) providing a plurality of diversified nucleic acid
sequences encoding complementarity determining region 3 (CDR3)
regions or encoding amino acid sequences that can fulfill the role
of a CDR3 region, wherein each of the plurality of diversified
nucleic acid sequences includes a Type IIs restriction enzyme
recognition site at each extremity; (c) digesting each of the
plurality of nucleic acid sequences encoding the CDR3 regions or
amino acid sequences that can fulfill the role of a CDR3 region
using a Type IIs restriction enzyme that binds to the Type IIs
restriction enzyme recognition site of step (b) and digesting the
stuffer nucleic acid sequence of step (a) from the Acceptor
Framework using a Type IIs restriction enzyme that binds to the
Type IIs restriction enzyme recognition site of step (a); (d)
ligating the digested nucleic acid sequences encoding the CDR3
regions or the amino acid sequences that can fulfill the role of a
CDR3 region of step (c) into the digested Acceptor Framework of
step (c) such that the FR3 and FR4 regions are interspaced by the
nucleic acid sequences encoding the CDR3 region or the amino acid
sequence that can fulfill the role of a CDR3 region and complete
immunoglobulin variable domain encoding sequences that do not
contain the Type IIs restriction enzyme recognition sites of steps
(a) and (b) are restored; (e) cloning the library of nucleic acids
encoding immunoglobulin variable domains of step (d) into an
expression vector; (f) transforming the expression vector of step
(e) into a host cell and culturing the host cell under conditions
sufficient to express a plurality of immunoglobulin variable
domains encoded by the library; (g) contacting the plurality of
immunoglobulin domains of step (f) with a target antigen; and (h)
determining which expressed immunoglobulin variable domain encoding
sequences bind to the target antigen.
[0220] The methods provided herein generate or otherwise produce a
target-specific antibody, antibody variable region or a portion
thereof, by: (a) providing a plurality of Acceptor Framework
nucleic acid sequences encoding distinct immunoglobulin variable
domains, each Acceptor Framework nucleic acid sequence including a
first framework region (FR1), a second framework region (FR2), a
third framework region (FR3), and a fourth framework region (FR4),
wherein the FR1 and FR2 regions are interspaced by a stuffer
nucleic acid sequence including at least two Type IIs restriction
enzyme recognition sites interspaced by a random nucleic acid
sequence, the FR2 and FR3 regions are interspaced by a
complementarity determining region 2 (CDR2), and the FR3 and FR4
regions are interspaced by a complementarity determining region 3
(CDR3); (b) providing a plurality of diversified nucleic acid
sequences encoding complementarity determining region 1 (CDR1)
regions or encoding amino acid sequences that can fulfill the role
of a CDR1 region, wherein each of the plurality of diversified
nucleic acid sequences includes a Type IIs restriction enzyme
recognition site at each extremity; (c) digesting each of the
plurality of nucleic acid sequences encoding the CDR1 regions or
amino acid sequences that can fulfill the role of a CDR1 region
using a Type IIs restriction enzyme that binds to the Type IIs
restriction enzyme recognition site of step (b) and digesting the
stuffer nucleic acid sequence of step (a) from the Acceptor
Framework using a Type IIs restriction enzyme that binds to the
Type IIs restriction enzyme recognition site of step (a); (d)
ligating the digested nucleic acid sequences encoding the CDR1
regions or the amino acid sequences that can fulfill the role of a
CDR1 region of step (c) into the digested Acceptor Framework of
step (c) such that the FR1 and FR2 regions are interspaced by the
nucleic acid sequences encoding the CDR1 region or the amino acid
sequence that can fulfill the role of a CDR1 region and complete
immunoglobulin variable domain encoding sequences that do not
contain the Type IIs restriction enzyme recognition sites of steps
(a) and (b) are restored; (e) cloning the library of nucleic acids
encoding immunoglobulin variable domains of step (d) into an
expression vector; (f) transforming the expression vector of step
(e) into a host cell and culturing the host cell under conditions
sufficient to express a plurality of immunoglobulin variable
domains encoded by the library; (g) contacting the plurality of
immunoglobulin domains of step (f) with a target antigen; and (g)
determining which expressed immunoglobulin variable domain encoding
sequences bind to the target antigen.
[0221] The methods provided herein generate or otherwise produce a
target-specific antibody, antibody variable region or a portion
thereof, by: (a) providing a plurality of Acceptor Framework
nucleic acid sequences encoding distinct immunoglobulin variable
domains, each Acceptor Framework nucleic acid sequence including a
first framework region (FR1), a second framework region (FR2), a
third framework region (FR3), and a fourth framework region (FR4),
wherein the FR1 and FR2 regions are interspaced by a
complementarity determining region 1 (CDR1), the FR2 and FR3
regions are interspaced by a stuffer nucleic acid sequence
including at least two Type IIs restriction enzyme recognition
sites interspaced by a random nucleic acid sequence, and the FR3
and FR4 regions are interspaced by a complementarity determining
region 3 (CDR3); (b) providing a plurality of diversified nucleic
acid sequences encoding complementarity determining region 2 (CDR2)
regions or encoding amino acid sequences that can fulfill the role
of a CDR2 region, wherein each of the plurality of diversified
nucleic acid sequences includes a Type IIs restriction enzyme
recognition site at each extremity; (c) digesting each of the
plurality of nucleic acid sequences encoding the CDR2 regions or
amino acid sequences that can fulfill the role of a CDR2 region
using a Type IIs restriction enzyme that binds to the Type IIs
restriction enzyme recognition site of step (b) and digesting the
stuffer nucleic acid sequence of step (a) from the Acceptor
Framework using a Type IIs restriction enzyme that binds to the
Type IIs restriction enzyme recognition site of step (a); (d)
ligating the digested nucleic acid sequences encoding the CDR2
regions or the amino acid sequences that can fulfill the role of a
CDR2 region of step (c) into the digested Acceptor Framework of
step (c) such that the FR2 and FR3 regions are interspaced by the
nucleic acid sequences encoding the CDR2 region or the amino acid
sequence that can fulfill the role of a CDR2 region and complete
immunoglobulin variable domain encoding sequences that do not
contain the Type IIs restriction enzyme recognition sites of steps
(a) and (b) are restored; (e) cloning the library of nucleic acids
encoding immunoglobulin variable domains of step (d) into an
expression vector; (f) transforming the expression vector of step
(e) into a host cell and culturing the host cell under conditions
sufficient to express a plurality of immunoglobulin variable
domains encoded by the library; (g) contacting the plurality of
immunoglobulin variable domains of step (f) with a target antigen;
and (h) determining which expressed immunoglobulin variable domain
encoding sequences bind to the target antigen.
[0222] In some embodiments, the methods provided herein further
include the step of (i) sequencing the immunoglobulin variable
domain encoding sequences that bind the target antigen.
[0223] In some embodiments, the Type IIs restriction enzyme
recognition sites of step (a) and step (b) are recognized by a
different Type IIs restriction enzyme.
[0224] In some embodiments, the Type IIs restriction enzyme
recognition sites are BsmBI recognition sites, BsaI recognition
sites, FokI recognition sites or a combination thereof.
[0225] In some embodiments, the Acceptor Framework nucleic acid
sequence is derived from a human gene sequence. For example, in
some embodiments, the human sequence is a human heavy chain
variable gene sequence or a sequence derived from a human heavy
chain variable gene sequence. For example, in some embodiments, the
human heavy chain variable gene sequence is selected from VH1-2,
VH1-69, VH1-18, VH3-30, VH3-48, VH3-23, and VH5-51.
[0226] In some embodiments, the human sequence is a human kappa
light chain variable gene sequence or a sequence derived from a
human kappa light chain variable gene sequence. For example, in
some embodiments, the human kappa light chain variable gene
sequence is selected from VK1-33, VK1-39, VK3-11, VK3-15, and
VK3-20.
[0227] In some embodiments, the human sequence is a human lambda
light chain variable gene sequence or a sequence derived from a
human lambda light chain variable gene sequence. For example, in
some embodiments, the human lambda light chain variable gene
sequence is selected from VL1-44 and VL1-51.
[0228] In some embodiments, the plurality of diversified nucleic
acids includes or is derived from sequences selected from naturally
occurring CDR3 sequences, naturally occurring Ig sequences from
humans, naturally occurring Ig sequences from a mammal, naturally
occurring sequences from a loop region of a T cell receptor in a
mammal, and other naturally diversified polypeptide
collections.
[0229] In some embodiments, the plurality of diversified nucleic
acids encodes CDR3 regions, and the plurality of diversified
nucleic acids includes or is derived from immunoglobulin sequences
that occur naturally in humans that have been exposed to a
particular immunogen or sequences derived from animals that have
been identified as having been exposed to a particular antigen.
[0230] In some embodiments, the plurality of diversified nucleic
acids encodes amino acid sequences that can fulfill the role of a
CDR3 region, and the plurality of diversified nucleic acids
includes synthetic sequences.
[0231] In some embodiments, the plurality of diversified nucleic
acids includes or is derived from sequences selected from naturally
occurring CDR1 sequences, naturally occurring Ig sequences from
humans, naturally occurring Ig sequences from a mammal, naturally
occurring sequences from a loop region of a T cell receptor in a
mammal, and other naturally diversified polypeptide
collections.
[0232] In some embodiments, the plurality of diversified nucleic
acids encodes CDR1 regions, and the plurality of diversified
nucleic acids includes or is derived from immunoglobulin sequences
that occur naturally in humans that have been exposed to a
particular immunogen or sequences derived from animals that have
been identified as having been exposed to a particular antigen.
[0233] In some embodiments, the plurality of diversified nucleic
acids encodes amino acid sequences that can fulfill the role of a
CDR1 region, and the plurality of diversified nucleic acids
includes synthetic sequences.
[0234] In some embodiments, the plurality of diversified nucleic
acids included or is derived from sequences selected from naturally
occurring CDR2 sequences, naturally occurring Ig sequences from
humans, naturally occurring Ig sequences from a mammal, naturally
occurring sequences from a loop region of a T cell receptor in a
mammal, and other naturally diversified polypeptide
collections.
[0235] In some embodiments, the plurality of diversified nucleic
acids encodes CDR2 regions, and the plurality of diversified
nucleic acids includes or is derived from immunoglobulin sequences
that occur naturally in humans that have been exposed to a
particular immunogen or sequences derived from animals that have
been identified as having been exposed to a particular antigen.
[0236] In some embodiments, the plurality of Acceptor Framework
nucleic acid sequences includes a mixture of at least one variable
heavy chain (VH) Acceptor Framework nucleic acid sequence and at
least one variable light chain Acceptor Framework nucleic acid
sequence.
[0237] In some embodiments, the expression vector is a phagemid
vector. In some embodiments, the host cell is E. coli.
[0238] Unless otherwise defined, scientific and technical terms
used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. Further, unless otherwise required by context, singular
terms shall include pluralities and plural terms shall include the
singular. Generally, nomenclatures utilized in connection with, and
techniques of, cell and tissue culture, molecular biology, and
protein and oligo- or polynucleotide chemistry and hybridization
described herein are those well known and commonly used in the art.
Standard techniques are used for recombinant DNA, oligonucleotide
synthesis, and tissue culture and transformation (e.g.,
electroporation, lipofection). Enzymatic reactions and purification
techniques are performed according to manufacturer's specifications
or as commonly accomplished in the art or as described herein. The
foregoing techniques and procedures are generally performed
according to conventional methods well known in the art and as
described in various general and more specific references that are
cited and discussed throughout the present specification. See e.g.,
Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed.,
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
(1989)). The nomenclatures utilized in connection with, and the
laboratory procedures and techniques of, analytical chemistry,
synthetic organic chemistry, and medicinal and pharmaceutical
chemistry described herein are those well known and commonly used
in the art. Standard techniques are used for chemical syntheses,
chemical analyses, pharmaceutical preparation, formulation, and
delivery, and treatment of patients.
[0239] As utilized in accordance with the present disclosure, the
following terms, unless otherwise indicated, shall be understood to
have the following meanings:
[0240] As used herein, the term "antibody" refers to immunoglobulin
molecules and immunologically active portions of immunoglobulin
(Ig) molecules, i.e., molecules that contain an antigen binding
site that specifically binds (immunoreacts with) an antigen. By
"specifically bind" or "immunoreacts with" or "immunospecifically
bind" is meant that the antibody reacts with one or more antigenic
determinants of the desired antigen and does not react with other
polypeptides or binds at much lower affinity
(K.sub.d>10.sup.-6). Antibodies include, but are not limited to,
polyclonal, monoclonal, chimeric, dAb (domain antibody), single
chain, F.sub.ab, F.sub.ab' and F.sub.(ab')2 fragments, scFvs, and
an F.sub.ab expression library.
[0241] The basic antibody structural unit is known to comprise a
tetramer. Each tetramer is composed of two identical pairs of
polypeptide chains, each pair having one "light" (about 25 kDa) and
one "heavy" chain (about 50-70 kDa). The amino-terminal portion of
each chain includes a variable region of about 100 to 110 or more
amino acids primarily responsible for antigen recognition. The
carboxy-terminal portion of each chain defines a constant region
primarily responsible for effector function. In general, antibody
molecules obtained from humans relate to any of the classes IgG,
IgM, IgA, IgE and IgD, which differ from one another by the nature
of the heavy chain present in the molecule. Certain classes have
subclasses as well, such as IgG.sub.1, IgG.sub.2, and others.
Furthermore, in humans, the light chain may be a kappa chain or a
lambda chain.
[0242] The term "monoclonal antibody" (MAb) or "monoclonal antibody
composition", as used herein, refers to a population of antibody
molecules that contain only one molecular species of antibody
molecule consisting of a unique light chain gene product and a
unique heavy chain gene product. In particular, the complementarity
determining regions (CDRs) of the monoclonal antibody are identical
in all the molecules of the population. MAbs contain an antigen
binding site capable of immunoreacting with a particular epitope of
the antigen characterized by a unique binding affinity for it.
[0243] The term "antigen-binding site," or "binding portion" refers
to the part of the immunoglobulin molecule that participates in
antigen binding. The antigen binding site is formed by amino acid
residues of the N-terminal variable ("V") regions of the heavy
("H") and light ("L") chains. Three highly divergent stretches
within the V regions of the heavy and light chains, referred to as
"hypervariable regions," are interposed between more conserved
flanking stretches known as "framework regions," or "FRs". Thus,
the term "FR" refers to amino acid sequences which are naturally
found between, and adjacent to, hypervariable regions in
immunoglobulins. In an antibody molecule, the three hypervariable
regions of a light chain and the three hypervariable regions of a
heavy chain are disposed relative to each other in three
dimensional space to form an antigen-binding surface. The
antigen-binding surface is complementary to the three-dimensional
surface of a bound antigen, and the three hypervariable regions of
each of the heavy and light chains are referred to as
"complementarity-determining regions," or "CDRs." The assignment of
amino acids to each domain is in accordance with the definitions of
Kabat Sequences of Proteins of Immunological Interest (National
Institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia
& Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al. Nature
342:878-883 (1989).
[0244] As used herein, the term "epitope" includes any protein
determinant capable of specific binding to an immunoglobulin, an
scFv, or a T-cell receptor. The term "epitope" includes any protein
determinant capable of specific binding to an immunoglobulin or
T-cell receptor. Epitopic determinants usually consist of
chemically active surface groupings of molecules such as amino
acids or sugar side chains and usually have specific three
dimensional structural characteristics, as well as specific charge
characteristics. For example, antibodies may be raised against
N-terminal or C-terminal peptides of a polypeptide. An antibody is
said to specifically bind an antigen when the dissociation constant
is .ltoreq.1 .mu.M; e.g., .ltoreq.100 nM, preferably .ltoreq.10 nM
and more preferably .ltoreq.1 nM.
[0245] As used herein, the terms "immunological binding," and
"immunological binding properties" refer to the non-covalent
interactions of the type which occur between an immunoglobulin
molecule and an antigen for which the immunoglobulin is specific.
The strength, or affinity of immunological binding interactions can
be expressed in terms of the dissociation constant (KO of the
interaction, wherein a smaller K.sub.d represents a greater
affinity. Immunological binding properties of selected polypeptides
can be quantified using methods well known in the art. One such
method entails measuring the rates of antigen-binding site/antigen
complex formation and dissociation, wherein those rates depend on
the concentrations of the complex partners, the affinity of the
interaction, and geometric parameters that equally influence the
rate in both directions. Thus, both the "on rate constant"
(K.sub.on) and the "off rate constant" (K.sub.off) can be
determined by calculation of the concentrations and the actual
rates of association and dissociation. (See Nature 361:186-87
(1993)). The ratio of K.sub.off/K.sub.on enables the cancellation
of all parameters not related to affinity, and is equal to the
dissociation constant K.sub.d. (See, generally, Davies et al.
(1990) Annual Rev Biochem 59:439-473). An antibody of the present
invention is said to specifically bind to its target, when the
equilibrium binding constant (K.sub.d) is .ltoreq.1 .mu.M, e.g.,
.ltoreq.100 nM, preferably .ltoreq.10 nM, and more preferably
.ltoreq.1 nM, as measured by assays such as radioligand binding
assays or similar assays known to those skilled in the art.
[0246] The term "isolated polynucleotide" as used herein shall mean
a polynucleotide of genomic, cDNA, or synthetic origin or some
combination thereof, which by virtue of its origin the "isolated
polynucleotide" (1) is not associated with all or a portion of a
polynucleotide in which the "isolated polynucleotide" is found in
nature, (2) is operably linked to a polynucleotide which it is not
linked to in nature, or (3) does not occur in nature as part of a
larger sequence. Polynucleotides in accordance with the invention
include the nucleic acid molecules encoding the heavy chain
immunoglobulin molecules, and nucleic acid molecules encoding the
light chain immunoglobulin molecules described herein.
[0247] The term "isolated protein" referred to herein means a
protein of cDNA, recombinant RNA, or synthetic origin or some
combination thereof, which by virtue of its origin, or source of
derivation, the "isolated protein" (1) is not associated with
proteins found in nature, (2) is free of other proteins from the
same source, e.g., free of marine proteins, (3) is expressed by a
cell from a different species, or (4) does not occur in nature.
[0248] The term "polypeptide" is used herein as a generic term to
refer to native protein, fragments, or analogs of a polypeptide
sequence. Hence, native protein fragments, and analogs are species
of the polypeptide genus. Polypeptides in accordance with the
invention comprise the heavy chain immunoglobulin molecules, and
the light chain immunoglobulin molecules described herein, as well
as antibody molecules formed by combinations comprising the heavy
chain immunoglobulin molecules with light chain immunoglobulin
molecules, such as kappa light chain immunoglobulin molecules, and
vice versa, as well as fragments and analogs thereof.
[0249] The term "naturally-occurring" as used herein as applied to
an object refers to the fact that an object can be found in nature.
For example, a polypeptide or polynucleotide sequence that is
present in an organism (including viruses) that can be isolated
from a source in nature and which has not been intentionally
modified by man in the laboratory or otherwise is
naturally-occurring.
[0250] The term "operably linked" as used herein refers to
positions of components so described are in a relationship
permitting them to function in their intended manner. A control
sequence "operably linked" to a coding sequence is ligated in such
a way that expression of the coding sequence is achieved under
conditions compatible with the control sequences.
[0251] The term "control sequence" as used herein refers to
polynucleotide sequences which are necessary to effect the
expression and processing of coding sequences to which they are
ligated. The nature of such control sequences differs depending
upon the host organism in prokaryotes, such control sequences
generally include promoter, ribosomal binding site, and
transcription termination sequence in eukaryotes, generally, such
control sequences include promoters and transcription termination
sequence. The term "control sequences" is intended to include, at a
minimum, all components whose presence is essential for expression
and processing, and can also include additional components whose
presence is advantageous, for example, leader sequences and fusion
partner sequences. The term "polynucleotide" as referred to herein
means a polymeric boron of nucleotides of at least 10 bases in
length, either ribonucleotides or deoxynucleotides or a modified
form of either type of nucleotide. The term includes single and
double stranded forms of DNA.
[0252] As used herein, the twenty conventional amino acids and
their abbreviations follow conventional usage. See Immunology--A
Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer
Associates, Sunderland Mass. (1991)). Stereoisomers (e.g., D-amino
acids) of the twenty conventional amino acids, unnatural amino
acids such as .alpha.-, .alpha.-disubstituted amino acids, N-alkyl
amino acids, lactic acid, and other unconventional amino acids may
also be suitable components for polypeptides of the present
invention. Examples of unconventional amino acids include: 4
hydroxyproline, .gamma.-carboxyglutamate,
.epsilon.-N,N,N-trimethyllysine, .epsilon.-N-acetyllysine,
O-phosphoserine, N-acetylserine, N-formylmethionine,
3-methylhistidine, 5-hydroxylysine, .sigma.-N-methylarginine, and
other similar amino acids and imino acids (e.g., 4-hydroxyproline).
In the polypeptide notation used herein, the left-hand direction is
the amino terminal direction and the right-hand direction is the
carboxy-terminal direction, in accordance with standard usage and
convention.
[0253] As applied to polypeptides, the term "substantial identity"
means that two peptide sequences, when optimally aligned, such as
by the programs GAP or BESTFIT using default gap weights, share at
least 80 percent sequence identity, preferably at least 90 percent
sequence identity, more preferably at least 95 percent sequence
identity, and most preferably at least 99 percent sequence
identity.
[0254] Preferably, residue positions which are not identical differ
by conservative amino acid substitutions.
[0255] Conservative amino acid substitutions refer to the
interchangeability of residues having similar side chains. For
example, a group of amino acids having aliphatic side chains is
glycine, alanine, valine, leucine, and isoleucine; a group of amino
acids having aliphatic-hydroxyl side chains is serine and
threonine; a group of amino acids having amide-containing side
chains is asparagine and glutamine; a group of amino acids having
aromatic side chains is phenylalanine, tyrosine, and tryptophan; a
group of amino acids having basic side chains is lysine, arginine,
and histidine; and a group of amino acids having sulfur-containing
side chains is cysteine and methionine. Preferred conservative
amino acids substitution groups are: valine-leucine-isoleucine,
phenylalanine-tyrosine, lysine-arginine, alanine valine,
glutamic-aspartic, and asparagine-glutamine.
[0256] As discussed herein, minor variations in the amino acid
sequences of antibodies or immunoglobulin molecules are
contemplated as being encompassed by the present invention,
providing that the variations in the amino acid sequence maintain
at least 75%, more preferably at least 80%, 90%, 95%, and most
preferably 99%. In particular, conservative amino acid replacements
are contemplated. Conservative replacements are those that take
place within a family of amino acids that are related in their side
chains. Genetically encoded amino acids are generally divided into
families: (1) acidic amino acids are aspartate, glutamate; (2)
basic amino acids are lysine, arginine, histidine; (3) non-polar
amino acids are alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan, and (4) uncharged polar
amino acids are glycine, asparagine, glutamine, cysteine, serine,
threonine, tyrosine. The hydrophilic amino acids include arginine,
asparagine, aspartate, glutamine, glutamate, histidine, lysine,
serine, and threonine. The hydrophobic amino acids include alanine,
cysteine, isoleucine, leucine, methionine, phenylalanine, proline,
tryptophan, tyrosine and valine. Other families of amino acids
include (i) serine and threonine, which are the aliphatic-hydroxy
family; (ii) asparagine and glutamine, which are the amide
containing family; (iii) alanine, valine, leucine and isoleucine,
which are the aliphatic family; and (iv) phenylalanine, tryptophan,
and tyrosine, which are the aromatic family. For example, it is
reasonable to expect that an isolated replacement of a leucine with
an isoleucine or valine, an aspartate with a glutamate, a threonine
with a serine, or a similar replacement of an amino acid with a
structurally related amino acid will not have a major effect on the
binding or properties of the resulting molecule, especially if the
replacement does not involve an amino acid within a framework site.
Whether an amino acid change results in a functional peptide can
readily be determined by assaying the specific activity of the
polypeptide derivative. Assays are described in detail herein.
Fragments or analogs of antibodies or immunoglobulin molecules can
be readily prepared by those of ordinary skill in the art.
Preferred amino- and carboxy-termini of fragments or analogs occur
near boundaries of functional domains. Structural and functional
domains can be identified by comparison of the nucleotide and/or
amino acid sequence data to public or proprietary sequence
databases. Preferably, computerized comparison methods are used to
identify sequence motifs or predicted protein conformation domains
that occur in other proteins of known structure and/or function.
Methods to identify protein sequences that fold into a known
three-dimensional structure are known. Bowie et al. Science 253:164
(1991). Thus, the foregoing examples demonstrate that those of
skill in the art can recognize sequence motifs and structural
conformations that may be used to define structural and functional
domains in accordance with the invention.
[0257] Preferred amino acid substitutions are those which: (1)
reduce susceptibility to proteolysis, (2) reduce susceptibility to
oxidation, (3) alter binding affinity for forming protein
complexes, (4) alter binding affinities, and (4) confer or modify
other physicochemical or functional properties of such analogs.
Analogs can include various muteins of a sequence other than the
naturally-occurring peptide sequence. For example, single or
multiple amino acid substitutions (preferably conservative amino
acid substitutions) may be made in the naturally-occurring sequence
(preferably in the portion of the polypeptide outside the domain(s)
forming intermolecular contacts. A conservative amino acid
substitution should not substantially change the structural
characteristics of the parent sequence (e.g., a replacement amino
acid should not tend to break a helix that occurs in the parent
sequence, or disrupt other types of secondary structure that
characterizes the parent sequence). Examples of art-recognized
polypeptide secondary and tertiary structures are described in
Proteins, Structures and Molecular Principles (Creighton, Ed., W.
H. Freeman and Company, New York (1984)); Introduction to Protein
Structure (C. Branden and J. Tooze, eds., Garland Publishing, New
York, N.Y. (1991)); and Thornton et at. Nature 354:105 (1991).
[0258] As used herein, the terms "label" or "labeled" refers to
incorporation of a detectable marker, e.g., by incorporation of a
radiolabeled amino acid or attachment to a polypeptide of biotinyl
moieties that can be detected by marked avidin (e.g., streptavidin
containing a fluorescent marker or enzymatic activity that can be
detected by optical or calorimetric methods). In certain
situations, the label or marker can also be therapeutic. Various
methods of labeling polypeptides and glycoproteins are known in the
art and may be used. Examples of labels for polypeptides include,
but are not limited to, the following: radioisotopes or
radionuclides (e.g., .sup.3H, .sup.14C, .sup.15N, .sup.35S,
.sup.90Y, .sup.99Tc, .sup.111In, .sup.125I, .sup.131I) fluorescent
labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic
labels (e.g., horseradish peroxidase, p-galactosidase, luciferase,
alkaline phosphatase), chemiluminescent, biotinyl groups,
predetermined polypeptide epitopes recognized by a secondary
reporter (e.g., leucine zipper pair sequences, binding sites for
secondary antibodies, metal binding domains, epitope tags). In some
embodiments, labels are attached by spacer arms of various lengths
to reduce potential steric hindrance. The term "pharmaceutical
agent or drug" as used herein refers to a chemical compound or
composition capable of inducing a desired therapeutic effect when
properly administered to a patient.
[0259] Other chemistry terms herein are used according to
conventional usage in the art, as exemplified by The McGraw-Hill
Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San
Francisco (1985)).
[0260] As used herein, "substantially pure" means an object species
is the predominant species present (i.e., on a molar basis it is
more abundant than any other individual species in the
composition), and preferably a substantially purified fraction is a
composition wherein the object species comprises at least about 50
percent (on a molar basis) of all macromolecular species
present.
[0261] Generally, a substantially pure composition will comprise
more than about 80 percent of all macromolecular species present in
the composition, more preferably more than about 85%, 90%, 95%, and
99%. Most preferably, the object species is purified to essential
homogeneity (contaminant species cannot be detected in the
composition by conventional detection methods) wherein the
composition consists essentially of a single macromolecular
species.
[0262] The term patient includes human and veterinary subjects.
[0263] Antibodies are purified by well-known techniques, such as
affinity chromatography using protein A or protein G, which provide
primarily the IgG fraction of immune serum. Subsequently, or
alternatively, the specific antigen which is the target of the
immunoglobulin sought, or an epitope thereof, may be immobilized on
a column to purify the immune specific antibody by immunoaffinity
chromatography. Purification of immunoglobulins is discussed, for
example, by D. Wilkinson (The Scientist, published by The
Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000),
pp. 25-28).
[0264] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLES
Example 1
Cloning of Immunoglobulin Variable Germline Genes
[0265] Seven human heavy chain variable germline genes (VH1-2,
VH1-69, VH1-18, VH3-30, VH3-48, VH3-23, VH5-51), five human kappa
light chain variable germline genes (VK1-33, VK1-39, VK3-11,
VK3-15, VK3-20) and two human lambda light chain variable germline
genes (VL1-44, VL1-51) were selected to construct the libraries
(Lefranc, M.-P. et al., 1999 Nucleic Acids Research, 27, 209-212).
These genes were selected because they are often used in human
expressed antibody repertoires and the frameworks they encode show
favorable stability and expression profiles as individual domains
or in the context of a VH/VL pair (Ewert S et al., J Mol Biol. 2003
Jan. 17; 325(3):531-53). Two sets of specific primers were used to
amplify these genes from human genomic DNA by nested PCR. This
approach was necessary as the 5' sequences of germline genes of the
same family are identical or very similar. For each gene, a first
pair of primers, called genomic locators, was designed to be
specific to the 5' and 3' untranslated regions flanking the
germline gene. The second pair was designed to be specific for the
beginning of the framework 1 region (FR1) and the end of the FR2.
The 14 independent PCR products were cloned into pGEMT-easy
(Promega, Madison Wis.) and their identity and integrity were
verified by sequencing. The amino acid sequence of the selected
germline genes is shown in FIG. 5.
[0266] The primers and primer combination used are indicated
below.
TABLE-US-00001 Genomic locators (SEQ ID NO: 120) 5 K1-33
TGTTTCTAATCGCAGGTGCCAGATG (SEQ ID NO: 121) 3 K1-33
ATTTATGTTATGACTTGTTACACTG (SEQ ID NO: 122) 5 K1-39
TATTTGTTTTTATGTTTCCAATCTC (SEQ ID NO: 123) 3 K1-39
CCTTGGAGGTTTATGTTATGACTTG (SEQ ID NO: 124) 5 K3-11
TTATTTCCAATTTCAGATACCACCG (SEQ ID NO: 125) 3 K3-11
TTGTTGGGGTTTTTGTTTCATGTGG (SEQ ID NO: 126) 5 K3-15
TATTTCCAATTTCAGATACCACTGG (SEQ ID NO: 127) 3 K3-15
ATGTTGAATCACTGTGGGAGGCCAG (SEQ ID NO: 128) 5 K3-20
TTATTTCCAATCTCAGATACCACCG (SEQ ID NO: 129) 3 K3-20
TTTTGTTTCAAGCTGAATCACTGTG (SEQ ID NO: 130) 5 L1-44
ATGTCTGTGTCTCTCTCACTTCCAG (SEQ ID NO: 131) 3 L1-44
TTCCCCATTGGCCTGGAGCACTGTG (SEQ ID NO: 132) 5 L1-51
GTGTCTGTGTCTCTCCTGCTTCCAG (SEQ ID NO: 133) 3 L1-51
CTTGTCTCAGTTCCCCATTGGGCTG (SEQ ID NO: 134) 5 H1-2
ATCTCATCCACTTCTGTGTTCTCTC (SEQ ID NO: 135) 3 H1-2
TTGGGTTTCTGACACCCTCAGGATG (SEQ ID NO: 136) 5 H1-18
CAGGCCAGTCATGTGAGACTTCACC (SEQ ID NO: 137) 3 H1-18
CTGCCTCCTCCCTGGGGTTTCTGAA (SEQ ID NO: 138) 5 H1-69
CCCCTGTGTCCTCTCCACAGGTGTC (SEQ ID NO: 139) 3 H1-69
CCGGCACAGCTGCCTTCTCCCTCAG (SEQ ID NO: 140) 5 DP-47
GAGGTGCAGCTGTTGGAG (SEQ ID NO: 141) 5 H3-23
TCTGACCAGGGTTTCTTTTTGTTTGC (SEQ ID NO: 142) 3 H3-23
TTGTGTCTGGGCTCACAATGACTTC (SEQ ID NO: 143) 5 H3-30
TGGCATTTTCTGATAACGGTGTCC (SEQ ID NO: 144) 3 H3-30
CTGCAGGGAGGTTTGTGTCTGGGCG (SEQ ID NO: 145) 5 H3-48
ATATGTGTGGCAGTTTCTGACCTTG (SEQ ID NO: 146) 3 H3-48
GGTTTGTGTCTGGTGTCACACTGAC (SEQ ID NO: 147) 5 H5-a
GAGTCTGTGCCGGAAGTGCAGCTGG Specific for coding sequence (SEQ ID NO:
148) 5 VH1 TATCAGGTGCAGCTGGTGCAG (SEQ ID NO: 149) 5 VH3
TATCAGGTGCAGCTGGTGGAG (SEQ ID NO: 150) 5 VH5 TATGAGGTGCAGCTGGTGCAG
(SEQ ID NO: 151) 3 VH1/3 ATATCTCTCGCACAGTAATACAC (SEQ ID NO: 152) 3
VH3 ATATCTCTCGCACAGTAATATAC (SEQ ID NO: 153) 3 VH5
ATATGTCTCGCACAGTAATACAT (SEQ ID NO: 154) 5 VK1
TATGACATCCAGATGACCCAGTCTCCATCCTC (SEQ ID NO: 155) 3 DPK9
ATAGGAGGGGTACTGTAACT (SEQ ID NO: 156) 3 DPK1 ATAGGAGGGAGATTATCATA
(SEQ ID NO: 157) 5 DPK22_L6 TATGAAATTGTGTTGACGCAGTCT (SEQ ID NO:
158) 3 DPK22 ATAGGAGGTGAGCTACCATACTG (SEQ ID NO: 159) 5 DPK21
TATGAAATAGTGATGACGCAGTCT (SEQ ID NO: 160) 3 DPK21
ATAGGAGGCCAGTTATTATACTG (SEQ ID NO: 161) 3 L6
CAGCGTAGCAACTGGCCTCCTAT (SEQ ID NO: 162) 5 DPL2
TACAGTCTGTGCTGACTCAG (SEQ ID NO: 163) 3 DPL2
ATAGGACCATTCAGGCTGTCATC (SEQ ID NO: 164) 5 DPL5
TATCAGTCTGTGTTGACGCAG (SEQ ID NO: 165) 3 DPL5
ATAGGAGCACTCAGGCTGCTAT
Primer Combinations Used to Amplify Selected Germline Genes.
TABLE-US-00002 [0267] 1st PCR 2nd PCR Family germline 5' 3' 5' 3'
VH1 DP-8/75 HV 1-2 5 H1-2 3 H1-2 5 VH1 3 VH1/3 DP-10 HV 1-69 5
H1-69 3 H1-69 5 VH1 3 VH1/3 DP-14 HV 1-18 5 H1-18 3 H1-18 5 VH1 3
VH1/3 VH3 DP-49 HV 3-30 5 H3-30 3 H3-30 5 VH3 3 VH1/3 DP-51 HV 3-48
5 H3-48 3 H3-48 5 VH3 3 VH1/3 DP-47 HV 3-23 5 H3-23 3 H3-23 5 VH3 3
VH3 VH5 HV 5a 5 H5a 3 VH5 5 VH5 3 VH5 VKI DPK-1 KV 1-33 5 K 1-33 3
K 1-33 5 VK1 3 DPK-1 DPK-9 KV 1-39 5 K 1-39 3 K 1-39 5 VK1 3 DPK-9
VKIII L6 KV 3-11 5 K3-11 3 K3-11 5 DPK22_L6 3 L6 DPK-21 KV 3-15 5
K3-15 3 K3-15 5 DPK21 3 DPK21 DPK-22 KV 3-20 5 K3-20 3 K3-20 5
DPK22_L6 3 DPK22 VL1 DPL-2 LV 1-44 5 L1-44 3 L1-44 5 DPL2 3 DPL2
DPL-5 LV 1-51 5 L1-51 3 L1-51 5 DPL5 3 DPL5
Example 2
Generation of Acceptor Frameworks
[0268] The sequences of the selected germline genes were analyzed
for the presence of Type IIs restriction sites. No BsmBI site was
present in the selected antibody variable germline genes. Two BsmBI
sites were found in the backbone of pNDS1, the phagemid vector in
which the Acceptor Framework would be cloned. These two sites were
removed by site-directed mutagenesis so that unique BsmBI sites
could be introduced into the stuffer DNA sequences of the Acceptor
Frameworks. Each germline gene was amplified by multiple nested PCR
in order to add a stuffer DNA sequence at the 3' end of the FR3
sequence followed by a sequence encoding FR4 which is specific for
each corresponding variable segment (VH, Vk, V.lamda.). The amino
acid sequence of VH FR4 corresponds to the FR4 region encoded by
the germline J genes JH1, JH3, JH4 and JH5. The amino acid sequence
of VK FR4 corresponds to the FR4 region encoded by the germline J
genes JK1. The amino acid sequence of V.lamda. FR4 corresponds to
the FR4 region encoded by the germline J genes JL2 and JL3. Two
variants of the Vk FR4 sequence were generated with a single amino
acid substitution at position 106 (Arginine or Glycine). For the
Acceptor Framework based on the germline gene VH3-23, two variants
were also constructed differing by a single amino acid (Lysine to
Arginine) at position 94, the last residue of FR3. During the final
amplification step SfiI/NcoI and XhoI sites were introduced at the
5' and 3' end of the VH, respectively.
[0269] Similarly, SalI and NotI sites were introduced at the 5' and
3' end of the VL, respectively (FIG. 6). The stuffer fragment was
designed so that the translation reading frame was shifted thus
preventing the expression of any functional protein from the
Acceptor Frameworks (FIG. 7). The primers used in this process are
listed below.
TABLE-US-00003 VH 5 VH1 (SEQ ID NO: 166)
CAGCCGGCCATGGCCCAGGTGCAGCTGGTGCAG 5 VH3-30 (SEQ ID NO: 167)
CAGCCGGCCATGGCCCAGGTGCAGCTGGTGGAG 5 VH3-23 (SEQ ID NO: 168)
CAGCCGGCCATGGCCGAGGTGCAGCTGTTGGAG 5 VH3-48 (SEQ ID NO: 169)
CAGCCGGCCATGGCCGAGGTGCAGCTGGTGGAGTCTGGGGGAG 5 VH5-51 (SEQ ID NO:
170) CAGCCGGCCATGGCCGAGGTGCAGCTGGTGCAG 3 VH1/3 (SEQ ID NO: 171)
CTTACCGTTATTCGTCTCATCTCGCACAGTAATACAC 3 VH3-23 (SEQ ID NO: 172)
CTTACCGTTATTCGTCTCATTTCGCACAGTAATATAC 3 VH3-48 (SEQ ID NO: 173)
CTCGCACAGTAATACACAGCCGTGTCCTCGGCTCTCAGGCTG 3 VH5-51 (SEQ ID NO:
174) CTTACCGTTATTCGTCTCATCTCGCACAGTAATACAT 3 VHext1 (SEQ ID NO:
175) CAATACGCGTTTAAACCTGGTAAACCGCCTTACCGTTATTCGTCTCA 3 VHext2 (SEQ
ID NO: 176) GTTCCCTGGCCCCAAGAGACGCGCCTTCCCAATACGCGTTTAAACCTG 3
VHext3 (SEQ ID NO: 177)
CCTCCACCGCTCGAGACTGTGACCAGGGTTCCCTGGCCCCAAGAG VK 5 VK1 (SEQ ID NO:
178) CGGGTCGACGGACATCCAGATGACCCAGTC 5 VK3-11 (SEQ ID NO: 179)
CGGGTCGACGGAAATTGTGTTGACACAGTCTCCAGC 5 VK3-15 (SEQ ID NO: 180)
CGGGTCGACGGAAATAGTGATGACGCAGTCTCCAGC 5 VK3-20 (SEQ ID NO: 181)
CGGGTCGACGGAAATTGTGTTGACGCAGTCTCCAGG 3 VK1-33 (SEQ ID NO: 182)
CCTTACCGTTATTCGTCTCGCTGCTGACAGTAATATGTTGCAATA 3 VK1-39 (SEQ ID NO:
183) CCTTACCGTTATTCGTCTCGCTGCTGACAGTAGTAAGTTGCAAAA 3 VK3 (SEQ ID
NO: 184) CCTTACCGTTATTCGTCTCGCTGCTGACAGTAATAAACTGCAAAATC 3 VKext1
(SEQ ID NO: 185) CCAATACGCGTTTAAACCTGGTAAACCGCCTTACCGTTATTCGTCTC 3
VKext2 (SEQ ID NO: 186)
GGTCCCTTGGCCGAATGAGACGCGCCTTCCCAATACGCGTTTAAAC 3 Vkext3R (SEQ ID
NO: 187) GTGCGGCCGCCCGTTTGATTTCCACCTTGGTCCCTTGGCCGAATG 3 VKext3G
(SEQ ID NO: 188) GTGCGGCCGCCCCTTTGATTTCCACCTTGGTCCCTTGGCCGAATG
V.lamda. 5 VL1-44 (SEQ ID NO: 189) CGGGTCGACGCAGTCTGTGCTGACTCAGCCAC
5 VL1-51 (SEQ ID NO: 190) CGGGTCGACGCAGTCTGTGTTGACGCAGCCGC 3 VL1-44
(SEQ ID NO: 191) CCTTACCGTTATTCGTCTCCTGCTGCACAGTAATAATC 3 VL1-51
(SEQ ID NO: 192) CCTTACCGTTATTCGTCTCCTGTTCCGCAGTAATAATC 3 Vlext2
(SEQ ID NO: 193) CCCTCCGCCGAACACAGAGACGCGCCTTCCCAATACGCGTTTAAAC 3
Vlext3 (SEQ ID NO: 194)
GTGCGGCCGCCCCTAGGACGGTCAGCTTGGTCCCTCCGCCGAACACAGA
[0270] The sequences of the 20 final assembled Acceptor Frameworks
are shown in FIG. 8.
Example 3
Generation of Phagemid Acceptor Vectors Containing an Invariant
Variable Domain
[0271] The phagemid vector pNDS1 used for the expression of scFv
was first modified to remove two BsmBI sites. A VH3-23 domain
containing a defined CDR3 sequence was cloned into the modified
pNDS1 using the SfiI and XhoI restriction sites to obtain the
phagemid vector pNDS_VHdummy. This domain contained a BsmBI site in
the FR4 region, which was corrected by silent site directed
mutagenesis. In parallel, a VK1-39 domain containing a defined CDR3
sequence was then cloned into the modified pNDS1 using the SalI and
NotI restriction sites to obtain the phagemid vector pNDS_VKdummy
(FIG. 9). The 8 VH Acceptor Frameworks were cloned into
pNDS_VKdummy using the SalI and NotI restrictions sites. The 12 VL
Acceptor Frameworks were cloned into pNDS_VHdummy using the SfiI
and XhoI restrictions sites. The resulting 20 pNDS phagemid vectors
that are listed below could at this stage be used for cloning of
diversified CDR3 using the BsmBI sites present in the stuffer DNA
fragments.
[0272] VH Acceptors: pNDS_VH1-2_VKd; pNDS_VH1-18_VKd;
pNDS_VH1-69_VKd; pNDS_VH3-23R_VKd; pNDS_VH3-23K_VKd;
pNDS_VH3-30_VKd; pNDS_VH5-51_VKd; pNDS_VH3-48_VKd.
[0273] VL Acceptors: pNDS_VHd_VK1-33G; pNDS_VHd_VK1-33R;
pNDS_VHd_VK1-39G; pNDS_VHd_VK1-39R; pNDS_VHd_VK3-11G;
pNDS_VHd_VK3-11R; pNDS_VHd_VK3-15G; pNDS_VHd_VK3-15R;
pNDS_VHd_VK3-20G; pNDS_VHd_VK3-20R; pNDS_VHd_VL1-44;
pNDS_VHd_VK1-51.
Example 4
Capturing Natural CDR H3 Diversity from Human Repertoires
[0274] Multiple sources of human cDNA were used as a template for
amplification of CDR H3 sequences. These sources included human
fetal spleen as well as pools of male and female normal adult
peripheral blood purified cells. Several strategies for
amplification have been used in order to recover CDR H3 sequences
originating from rearranged VH cDNA encoded by a specific germline
gene or CDR H3 sequences originating from any VH cDNA.
[0275] First, mixtures of primers matching the 5' coding regions of
the majority of human VH families were used in combination with
primer mixtures matching all the human JH regions. This allowed for
PCR amplification a majority of heavy chain immunoglobulin variable
genes. The expected amplification products of approximately 400
base pairs (bp) were isolated by agarose gel electrophoresis and
purified. This DNA served as template in a second PCR step using
primers with a 13 bp and 14 bp match for the end FR3 region and the
beginning of FR4, respectively. In most cases, the last residue of
the FR3 is either an arginine or a lysine. As the last by matches
are critical for primer extension by the polymerase, two different
5' primers were used: 5 VHR_FOK (SEQ ID NO: 205 shown below) and 5
VHK_FOK (SEQ ID NO: 206 shown below). Importantly, these primers
also contain a FokI restriction site for excision of the CDR H3
sequence (FIG. 4). The primers used in the second PCR step were
biotinylated at their 5' end to facilitate downstream purification
steps (see example 5). This two step approach allows for an
efficient amplification of the CDR H3 sequences despite the limited
number of base pairs matches. Amplifications were performed at
varying annealing temperatures (between 30.degree. C. and
70.degree. C.) and with several thermostable DNA polymerases to
establish optimal conditions. An annealing temperature of
55-58.degree. C. in combination with GoTaq polymerase (Promega) was
found to be optimal for this set of primers. The second
amplification product was separated on a 2% agarose gel and
resulted in a smear in the lower part of the gel corresponding to
CDR H3 of different length. Either the complete DNA smear was
extracted from the gel or a region corresponding to larger DNA
fragments in order to enrich for long CDR H3.
[0276] Alternatively, the first amplification step was performed
using the 5' primer 5 VH3-23H2 (SEQ ID NO: 201 shown below), which
is specific for the sequence encoding the CDR H2 of the germline
VH3-23. As the different germline genes are diverse in this CDR, VH
cDNAs encoded by the selected germline gene can be preferentially
amplified. The subsequent purification and amplification steps were
identical. In this way, it is possible to retrieve CDRs originating
from a specific framework environment and to re-introduce them into
the same, a similar or different framework.
[0277] Below is a list of primers used for the amplification of
natural human CDR H3 repertoires.
TABLE-US-00004 1st PCR step 5 VH1/5 (SEQ ID NO: 195)
CCGCACAGCCGGCCATGGCCCAGGTGCAGCTGGTGCAGTCTGG 5 VH3 (SEQ ID NO: 196)
CCGCACAGCCGGCCATGGCCGAGGTGCAGCTGGTGGAGTCTGG 5 VH2 (SEQ ID NO: 197)
CCGCACAGCCGGCCATGGCCCAGRTCACCTTGCTCGAGTCTGG 5 VH4 (SEQ ID NO: 198)
CCGCACAGCCGGCCATGGCCCAGGTGCAGCTGCAGGAGTCGGG 5 VH4DP64 (SEQ ID NO:
199) CCGCACAGCCGGCCATGGCCCAGCTGCAGCTGCAGGAGTCCGG 5 VH4DP63 (SEQ ID
NO: 200) CCGCACAGCCGGCCATGGCCCAGGTGCAGCTACAGCAGTGGGG 5 VH3-23H2
(SEQ ID NO: 201) TGGAGTGGGTCTCAGCTATTAGTGGTAGTGGT 3 HJ1/2 (SEQ ID
NO: 202) CGATGGGCCCTTGGTGGAGGCTGAGGAGACRGTGACCAGGGTGCC 3 HJ3/6 (SEQ
ID NO: 203) CGATGGGCCCTTGGTGGAGGCTGAAGAGACGGTGACCRTKGTCCC 3 HJ4/5
(SEQ ID NO: 204) CGATGGGCCCTTGGTGGAGGCTGAGGAGACGGTGACCAGGGTTCC 2nd
PCR step 5 VHR_FOK (SEQ ID NO: 205)
GAGCCGAGGACACGGCCGGATGTTACTGTGCGAGA 5 VHK_FOK (SEQ ID NO: 206)
GAGCCGAGGACACGGCCGGATGTTACTGTGCGAAA 3 JH1_FOK (SEQ ID NO: 207)
GAGGAGACGGTGACGGATGTGCCCTGGCCCCA 3 JH2_FOK (SEQ ID NO: 208)
GAGGAGACGGTGACGGATGTGCCACGGCCCCA 3 JH3456_FOK (SEQ ID NO: 209)
GAGGAGACGGTGACGGATGTYCCTTGGCCCCA
Example 5
Generation of Primary Libraries by Cloning Natural Human CDR H3
into Acceptor Frameworks
[0278] The amplified CDR H3 were digested with FokI, and the
cleaved extremities as well as undigested DNA was removed using
streptavidin coated magnetic beads. In parallel, pNDS VH Acceptor
vectors were digested using BsmBI. As the overhangs generated by
these digestions are compatible, the collection of natural CDR H3
was able to be ligated into the VH Acceptor Framework restoring the
appropriate reading frame. The ligated DNA was purified and
concentrated for transformation into competent E. coli XL1 Blue
cells, and random clones analyzed by sequencing in order to check
that CDR H3 sequence had been reconstituted and that junctions
between the CDR and the Framework region are correct (FIG. 10). The
results indicated that all the clones contained CDR H3 sequences
and that the reading frame was restored, thus encoding an
immunoglobulin variable heavy chain. In addition, all the CDRs were
different, indicating that a large diversity of naturally occurring
sequences had been captured by this approach. The length of the CDR
H3 was also variable and relatively long CDRs of 10 to 15 residues
were found, thus underscoring the advantage of this approach for
sampling long CDR sequences that are difficult to cover using
synthetic diversity.
[0279] Using this method, natural CDR H3 sequences, derived either
from pooled human peripheral blood purified cells or human fetal
spleen, were cloned into each of the pNDS VH Acceptor Frameworks
and transformed into electrocompetent E. coli TG1 cells and plated
on 2.times.TYAG Bioassay plates (2.times.TY media containing 100
.mu.g/ml ampicillin and 2% glucose). After overnight incubation at
30.degree. C., 10 ml of 2.times.TYAG liquid medium was added to the
plates and the cells were scraped from the surface and transferred
to a 50 ml polypropylene tube. 2.times.TYAG containing 50% glycerol
was added to the cell suspension to obtain a final concentration of
17% glycerol. Aliquots of the libraries were stored at -80.degree.
C. In this process, 14 primary libraries were generated
representing a total of 8.1.times.10.sup.9 transformants. 180
randomly picked clones were sequenced to determine the quality and
diversity of the libraries. All clones encoded different VH
sequences and >89% were in frame. These primary libraries
contain diversity in the CDR H3 only as they are combined with a
dummy VL domain.
Example 6
Generation of Primary Libraries by Cloning Synthetic CDR3 into
Acceptor Frameworks
[0280] Although the method is of particular interest for retrieving
natural diversity, it can also be applied for the integration of
synthetic diversity into Acceptor Frameworks. Synthetic CDR3
sequences were designed for both the VH and VL. The design took
into account the frequency of CDRs with a given length and the
diversification strategy (NNS, DVK, NVT or DVT codons) that would
allow a complete coverage of the theoretical diversity within a
reasonable number of transformants in a library
(.about.5.times.10.sup.9 transformants) (FIG. 11). Key residues to
maintain the canonical structure of the CDR were kept constant in
the design of CDR3 for VK and V.lamda. chains. For the heavy chain,
only CDR3 with up to 10 diversified positions were generated as the
number of clones required to cover the diversity encoded by longer
CDRs is beyond practical limits of transformation efficiency.
[0281] Degenerate oligonucleotides of different length were
synthesized using NNS, NVT, DVK or DVT randomized codons. For each
CDR H3, two oligonucleotides were synthesized encoding either a
methionine or a phenylalanine at position 100z (FIG. 11). Each
oligonucleotide was extended and amplified with two external
biotinylated primers to generate double stranded DNA fragments
encoding the designed CDRs. These external primers contain BsmBI
restriction sites for subsequent excision of the CDR sequence and
insertion into the Acceptor Frameworks (FIG. 12). The assembled DNA
fragments were processed without gel purification and digested with
BsmBI. The cleaved extremities as well as undigested DNA was
removed using streptavidin coated magnetic beads. The digested DNA
fragments were concentrated by ethanol precipitation and ligated
into the corresponding pNDS VH, VK or V.lamda. Acceptor vectors.
Ligation products were purified and concentrated for transformation
into electrocompetent E. coli TG 1 cells and plated on 2.times.TYAG
Bioassay plates (2.times.TY media containing 100 .mu.g/ml
ampicillin and 2% glucose). After overnight incubation at
30.degree. C., 10 ml of 2.times.TYAG liquid medium was added to the
plates and the cells were scraped from the surface and transferred
to a 50 ml polypropylene tube. 2.times.TYAG containing 50% glycerol
was added to the cell suspension to obtain a final concentration of
17% glycerol Aliquots of the libraries were stored at -80.degree.
C. A total of 24 primary heavy chain libraries were generated
representing a total of 1.6.times.10.sup.10 transformants.
Similarly, 13 primary light chain libraries were generated
representing a total of 6.9.times.10.sup.9 transformants. These
primary libraries contain diversity in the CDR H3 only as they are
combined with a dummy VL domain. A total of 330 randomly picked
clones were sequenced to determine the quality and diversity of the
libraries. All clones encoded different variable domain sequences
and >90% were in frame. This low frequency of sequences
containing shifts in the reading frame is in sharp contrast with
results traditionally obtained during the construction of synthetic
antibody fragment libraries using overlapping PCR approaches which
are more prone to the introduction of insertion, and significant
loss of functional clones (15-45%) has frequently been
reported.
[0282] The diversity in these primary libraries was restricted to
the CDR H3 or CDR L3 as they are combined with a dummy VL or VH
chain, respectively.
[0283] Primers used for synthetic CDR assembly are listed
below.
TABLE-US-00005 5 H3_R_biot (SEQ ID NO: 210)
ATGATGCTGCTGGCACGTCTCCGAGA 3 H3_M_biot (SEQ ID NO: 211)
CCACGTCATCCGATCCGTCTCCCCCAATAATCCAT 3 H3_F_biot (SEQ ID NO: 212)
CCACGTCATCCGATCCGTCTCCCCCAATAATCAAA H3_4nnsF (SEQ ID NO: 213)
GCTGGCACGTCTCCGAGANNSNNSNNSNNSTTTGATTATTGGGGGAGACG H3_4nnsM (SEQ ID
NO: 214) GCTGGCACGTCTCCGAGANNSNNSNNSNNSATGGATTATTGGGGGAGACG
H3_5nnsF (SEQ ID NO: 215)
GCTGGCACGTCTCCGAGANNSNNSNNSNNSNNSTTTGATTATTGGGGGAG ACG H3_5nnsM
(SEQ ID NO: 216) GCTGGCACGTCTCCGAGANNSNNSNNSNNSNNSATGGATTATTGGGGGAG
ACG H3_6nnsF (SEQ ID NO: 217)
GCTGGCACGTCTCCGAGANNSNNSNNSNNSNNSNNSTTTGATTATTGGGG GAGACG H3_6nnsM
(SEQ ID NO: 218) GCTGGCACGTCTCCGAGANNSNNSNNSNNSNNSNNSATGGATTATTGGGG
GAGACG H3_6dvkF (SEQ ID NO: 219)
GCTGGCACGTCTCCGAGADVKDVKDVKDVKDVKDVKTTTGATTATTGGGG GAGACG H3_6dvkM
(SEQ ID NO: 220) GCTGGCACGTCTCCGAGADVKDVKDVKDVKDVKDVKATGGATTATTGGGG
GAGACG H3_7dvkF (SEQ ID NO: 221)
GCTGGCACGTCTCCGAGADVKDVKDVKDVKDVKDVKDVKTTTGATTATTG GGGGAGACG
H3_7dvkM (SEQ ID NO: 222)
GCTGGCACGTCTCCGAGADVKDVKDVKDVKDVKDVKDVKATGGATTATTG GGGGAGACG
H3_7nvtF (SEQ ID NO: 223)
GCTGGCACGTCTCCGAGANVTNVTNVTNVTNVTNVTNVTTTTGATTATTG GGGGAGACG
H3_7nvtM (SEQ ID NO: 224)
GCTGGCACGTCTCCGAGANVTNVTNVTNVTNVTNVTNVTATGGATTATTG GGGGAGACG
H3_8nvtF (SEQ ID NO: 225)
GCTGGCACGTCTCCGAGANVTNVTNVTNVTNVTNVTNVTNVTTTTGATTA TTGGGGGAGACG
H3_8nvtM (SEQ ID NO: 226)
GCTGGCACGTCTCCGAGANVTNVTNVTNVTNVTNVTNVTNVTATGGATTA TTGGGGGAGACG
H3_9nvtF (SEQ ID NO: 227)
GCTGGCACGTCTCCGAGANVTNVTNVTNVTNVTNVTNVTNVTNVTTTTGA TTATTGGGGGAGACG
H3_9nvtM (SEQ ID NO: 228)
GCTGGCACGTCTCCGAGANVTNVTNVTNVTNVTNVTNVTNVTNVTATGGA TTATTGGGGGAGACG
H3_9dvtF (SEQ ID NO: 229)
GCTGGCACGTCTCCGAGADVTDVTDVTDVTDVTDVTDVTDVTDVTTTTGA TTATTGGGGGAGACG
H3_9dvtM (SEQ ID NO: 230)
GCTGGCACGTCTCCGAGADVTDVTDVTDVTDVTDVTDVTDVTDVTATGGA TTATTGGGGGAGACG
H3_10dvtF (SEQ ID NO: 231)
GCTGGCACGTCTCCGAGADVTDVTDVTDVTDVTDVTDVTDVTDVTDVTTT
TGATTATTGGGGGAGACG H3_10dvtM (SEQ ID NO: 232)
GCTGGCACGTCTCCGAGADVTDVTDVTDVTDVTDVTDVTDVTDVTDVTAT
GGATTATTGGGGGAGACG 5 KL3_biot (SEQ ID NO: 233)
CCGGTGTAGCGAAGGCGTCTCAGCAG 3 KL3_ biot (SEQ ID NO: 234)
TAGGGTCGCCTTGATCGTCTCCCGAAGGTCGG K_4nns (SEQ ID NO: 235)
GAAGGCGTCTCAGCAGNNSNNSNNSNNSCCGACCTTCGGGAGACG K_5nns (SEQ ID NO:
236) GAAGGCGTCTCAGCAGNNSNNSNNSNNSCCGNNSACCTTCGGGAGACG K_6nns (SEQ
ID NO: 237) GAAGGCGTCTCAGCAGNNSNNSNNSNNSNNSCCGNNSACCTTCGGGAGAC G 5
L44W_biot (SEQ ID NO: 238) CGGTCAGTCGCAATACGTCTCCAGCATGGGAT 5
L44Y_biot (SEQ ID NO: 239) CGGTCAGTCGCAATACGTCTCCAGCATATGAT 3
L_biot (SEQ ID NO: 240) CAGGACCAGTCTCGTGAGGATCGTCTCAACAC L44W_4nns
(SEQ ID NO: 241) CGTCTCCAGCATGGGATNNSNNSNNSNNSGTGTTGAGACGATCCTC
L44Y_4nns (SEQ ID NO: 242)
CGTCTCCAGCATATGATNNSNNSNNSNNSGTGTTGAGACGATCCTC L44W_5nns (SEQ ID
NO: 243) CGTCTCCAGCATGGGATNNSNNSNNSNNSNNSGTGTTGAGACGATCCTC
L44Y_5nns (SEQ ID NO: 244)
CGTCTCCAGCATATGATNNSNNSNNSNNSNNSGTGTTGAGACGATCCTC L44W_6nns (SEQ ID
NO: 245) CGTCTCCAGCATGGGATNNSNNSNNSNNSNNSNNSGTGTTGAGACGATCC TC
L44Y_6nns (SEQ ID NO: 246)
CGTCTCCAGCATATGATNNSNNSNNSNNSNNSNNSGTGTTGAGACGATCC TC 5 L51W_biot
(SEQ ID NO: 247) CGGTCAGTCGCAATACGTCTCGAACATGGGAT 5 L51Y_biot (SEQ
ID NO: 248) CGGTCAGTCGCAATACGTCTCGAACATATGAT L51W_4nns (SEQ ID NO:
249) CGTCTCGAACATGGGATNNSNNSNNSNNSGTGTTGAGACGATCCTC L51Y_4nns (SEQ
ID NO: 250) CGTCTCGAACATATGATNNSNNSNNSNNSGTGTTGAGACGATCCTC
L51W_5nns (SEQ ID NO: 251)
CGTCTCGAACATGGGATNNSNNSNNSNNSNNSGTGTTGAGACGATCCTC L51Y_5nns (SEQ ID
NO: 252) CGTCTCGAACATATGATNNSNNSNNSNNSNNSGTGTTGAGACGATCCTC
L51W_6nns (SEQ ID NO: 253)
CGTCTCGAACATGGGATNNSNNSNNSNNSNNSNNSGTGTTGAGACGATCC TC L51Y_6nns
(SEQ ID NO: 254) CGTCTCGAACATATGATNNSNNSNNSNNSNNSNNSGTGTTGAGACGATCC
TC
Example 7
Generation of Secondary Libraries
[0284] In order to generate libraries of scFv carrying diversity in
both the heavy and light chains, the Primary synthetic light chain
libraries were combined with either the Primary synthetic heavy
chain libraries or the Primary natural heavy chain libraries (FIG.
13). Phagemid DNA was prepared from each primary library and
digested with XhoI/NotI restriction enzymes. The DNA fragments
corresponding to the linker and light chains from the Primary
synthetic libraries were inserted by ligation into the digested
Primary natural or synthetic heavy chain vectors. Alternatively the
Linker-VL sequence was also amplified with specific primers before
digestion with XhoI/NotI and ligation. The ligation products were
purified by phenol/chloroform extraction and precipitation before
transformation into electrocompetent E. coli TG1 cells and plating
on 2.times.TYAG Bioassay plates (2.times.TY media containing 100
.mu.g/ml ampicillin and 2% glucose). After overnight incubation at
30.degree. C., 10 ml of 2.times.TYAG liquid medium was added to the
plates and the cells were scraped from the surface and transferred
to a 50 ml polypropylene tube. 2.times.TYAG containing 50% glycerol
was added to the cell suspension to obtain a final concentration of
17% glycerol. Aliquots of the libraries were stored at -80.degree.
C. To limit the number of libraries to be recombined, they were
pooled by chain subclasses (i.e., VH1, VH3, VH5, VK1, VK3,
V.lamda.1) and thus 9 library combination were performed for (i.e.,
VH1.times.VK1, VH1.times.VK3, VH1.times.V.lamda.1, VH3.times.VK1,
VH3.times.VK3, VH3.times.V.lamda.1, VH5.times.VK1, VH5.times.VK3,
VH5.times.V.lamda.1). The total size of the Secondary synthetic
libraries (carrying synthetic diversity in both the VH and VL) was
7.3.times.10.sup.9 transformants. The total size of the Secondary
natural libraries (carrying natural diversity in the VH and
synthetic diversity in the VL) was 1.5.times.10.sup.10
transformants.
Example 8
Generation of Human Antibody Libraries Displaying a CDRH3
Repertoire Derived from a Non-Human Species
[0285] In order to utilize alternative sources of diversity that
would allow exploring a different tri-dimensional space within the
antibody combining site, a library was created by capturing the
CDRH3 of mice and introduced them into a collection of human
antibody frameworks. For this approach an acceptor library
containing a collection of VL genes with synthetic CDR L3 diversity
was constructed and combined with a collection of acceptor
sequences containing a stuffer DNA sequence ready suitable for Type
IIS restriction cloning as described in Example 2. This library
represents the starting point for rapid generation of secondary
libraries with multiple sources of natural (human as well as
non-human) or synthetic CDR H3. In this example, natural CDR H3
diversity was captured from naive Balb/c mice and mice that had
been immunized with hIFN.gamma. or hCCL5 (hRANTES).
[0286] The first step was the generation of acceptor libraries by
cloning a collection of VL containing synthetic CDR L3 diversity
into acceptor VH framework vectors (FIG. 14). The VL sequences were
derived from the seven Primary Synthetic Libraries described in
Example 6 by PCR amplification using primers 5'biot-VHdummy and
3'biot-fdtseq. The resulting VL containing fragments of
approximately 400 bp were digested using XhoI/NotI and purified on
spin columns to remove primers and enzymes. Similarly the pNDS VH
acceptor vectors containing a CDRH3 stuffer and a dummy light chain
were digested with XhoI/NotI and SwaI (SwaI cutting inside the VL
dummy) and purified on Chroma Spin TE columns with a cutoff of 1000
bp to get rid of the VL dummy fragment. The digested VL fragments
were then ligated into the VH acceptor vectors (FIG. 14). To limit
the number of libraries to be recombined, VH acceptor vectors and
VL fragments were pooled by chain subclasses (i.e., VH1, VH3, VH5,
VK1, V.kappa.3, V.lamda.1) and thus nine library combinations were
performed (i.e., VH1.times.V.kappa.1, VH1.times.V.kappa.3,
VH1.times.V.lamda.1, VH3.times.V.kappa.1, VH3.times.V.kappa.3,
VH3.times.V.lamda.1, VH5.times.V.kappa.1, VH5.times.V.kappa.3,
VH5.times.V.lamda.1). The ligation products were transformed into
electrocompetent E. coli TG1 cells and plated on 2.times.TYAG
Bioassay plates (2.times.TY medium containing 100 .mu.g/ml
ampicillin and 2% glucose). After overnight incubation at
30.degree. C., 6 ml of 2.times.TYAG liquid medium was added to the
plates and the cells were scraped from the surface and transferred
to a 50 ml polypropylene tube. Glycerol 50% was added to the cell
suspension to obtain a final concentration of 17% glycerol.
Aliquots of the libraries were stored at -80.degree. C. The total
size of this acceptor library, carrying synthetic diversity in the
CDR L3, was 1.9.times.10.sup.9 transformants.
[0287] The next step was to isolate CDRH3 sequences from a
non-human source. Cells were isolated from the spleen of five naive
or immunized Balb/c mice and total RNA was purified. cDNA was
obtained from the extracted RNA by RT-PCR. This cDNA was used as
template to isolate and amplify mouse VH by PCR. A series of PCRs
were performed using 15 different 5' primers (one for each mouse VH
subgroup) specific for the beginning of the FR1 region and a pool
of 3' primers (four primers covering the JH region). These first
PCRs were pooled and purified on a 2% agarose gel. The purified DNA
served as template to perform a second PCR to isolate the mouse CDR
H3 region.
[0288] The 5' and 3' primers for this second PCR target the FR3 and
FR4 regions of mouse VH, respectively. These primers added a FokI
restriction site in order to allow for precise excision of the CDR
H3 and cloning into the human acceptor vectors. However, alignments
of murine VH sequences revealed that sequence at the 5' boundary of
murine CDR-H3 and that are located at the cleavage site of FokI
almost always differ from human sequence by one base, whereas the
3' end matched between these two species. The sequences cleaved by
FokI are boxed in Table 1 below:
TABLE-US-00006 5' sequences 3' sequences (SEQ ID NO: 281) Human:
TTACTGTGC GAGA Human: TGGG GCCAGGGAA (SEQ ID NO: 285) Mouse: Mouse:
(SEQ ID NO: 282) VH1 TTACTGTGC AAGA JH1 TGGG GCGCAGGGA (SEQ ID NO:
286) (SEQ ID NO: 283) TTTCTGTGC AAGA JH2 TGGG GCCAAGGCA (SEQ ID NO:
287) (SEQ ID NO: 284) VH2 CTACTGTGC CAGA JHC TGGG GCCAGGGCA (SEQ ID
NO: 288) (SEQ ID NO: 282) VH3-16 TTACTGTGC AAGA JH4 TGGG GTCAGGGCA
(SEQ ID NO: 289)
[0289] Consequently the base had to be corrected during the second
amplification step in order to generate cohesive ends that are
compatible with the cohesive ends generated upon digestion of the
Acceptor Frameworks. Efficient amplification was observed
suggesting that this conversion occurred readily. At the 3' end,
mouse and human sequences that will be cut by the Type IIS
restriction enzymes are identical thus avoiding any correction
issues.
[0290] Primers for the second amplification were biotinylated at
their 5' ends to facilitate downstream purification steps. The
acceptor vectors were digested with BsmBI and purified on Chroma
Spin TE columns having a cutoff of 1000 bp. After digestion and
purification, the nine different library combinations were pooled
in equimolar ratio for ligation of the captured mouse CDRH3.
[0291] The ligated DNA was purified by phenol/chloroform
extractions and concentrated by precipitation before transformation
into competent E. coli TG1 cells and plated on 2.times.TYAG
Bioassay plates (2.times.TY medium containing 100 .mu.g/ml
ampicillin and 2% glucose). After overnight incubation at
30.degree. C., 6 ml of 2.times.TYAG liquid medium was added to the
plates and the cells were scraped from the surface and transferred
to a 50 ml polypropylene tube. Glycerol 50% was added to the cell
suspension to obtain a final concentration of 17% glycerol.
Aliquots of the libraries were stored at -80.degree. C. Three
libraries of similar size were obtained: MnA, 2.5.times.10.sup.8
transformants (carrying a restricted natural human framework
diversity, naive mouse diversity in the CDR H3 and synthetic
diversity in the CDR L3); MiB, 7.3.times.10.sup.7 transformants
(carrying a restricted natural human framework diversity, immune
mouse diversity against hIFN.gamma. in the CDR H3 and synthetic
diversity in the CDR L3) and MiC, 1.8.times.10.sup.8 transformants
(carrying a restricted natural human framework diversity, immune
mouse diversity against hCCL5 in the CDR H3 and synthetic diversity
in the CDR L3). Random clones were analyzed by sequencing in order
to check that CDR H3 sequence had been reconstituted and that
junctions between the CDR and the Framework regions were correct.
The results indicated that all the clones contained CDR H3
sequences and that the reading frame was restored, thus encoding an
immunoglobulin variable heavy chain. All the CDRs were different
and resembled typical mouse CDR H3 sequences indicating that a
large diversity of naturally occurring mouse CDRH3 sequences had
been captured by this approach. In addition, the analysis of the
CDRH3 length profiles indicated that a Gaussian distribution was
captured in the naive library that corresponds to the expected
distribution of lengths in normal mouse repertoire. In contrast, in
the two immune libraries the profiles were different suggesting
that a different CDRH3 repertoire had been captured (FIG. 15).
Primers Used for CDRH3 Amplification from Mice
TABLE-US-00007 1.sup.st PCR 5' primers: m5 VH1 (SEQ ID NO: 256)
ATGCGGCCCAGCCGGCCATGGCCSAGGTYCAGCTBCAGCAGTC m5 VH2 (SEQ ID NO: 257)
ATGCGGCCCAGCCGGCCATGGCCCAGGTTCACCTGCAGCARTC m5 VH3 (SEQ ID NO: 258)
ATGCGGCCCAGCCGGCCATGGCCCAGGTRCAGCTGAAGGAGTC m5 VH4 (SEQ ID NO: 259)
ATGCGGCCCAGCCGGCCATGGCCCAGGTCCAACTVCAGCARCC m5 VH5 (SEQ ID NO: 260)
ATGCGGCCCAGCCGGCCATGGCCCAGATCCAGTTGGTVCAGTC m5 VH6 (SEQ ID NO: 261)
ATGCGGCCCAGCCGGCCATGGCCCAGGTGCAGCTGAAGSASTC m5 VH7 (SEQ ID NO: 262)
ATGCGGCCCAGCCGGCCATGGCCGAGGTGCAGSKGGTGGAGTC m5 VH8 (SEQ ID NO: 263)
ATGCGGCCCAGCCGGCCATGGCCGAAGTGAARSTTGAGGAGTC m5 VH9 (SEQ ID NO: 264)
ATGCGGCCCAGCCGGCCATGGCCGAKGTSVAGCTTCAGGAGTC m5 VH10 (SEQ ID NO:
265) ATGCGGCCCAGCCGGCCATGGCCGAGGTGAASSTGGTGGAATC m5 VH11 (SEQ ID
NO: 266) ATGCGGCCCAGCCGGCCATGGCCGAGGTGAAGCTGRTGGARTC m5 VH12 (SEQ
ID NO: 267) ATGCGGCCCAGCCGGCCATGGCCGARGTGAAGCTGRTGGAGTC m5 VH13
(SEQ ID NO: 268) ATGCGGCCCAGCCGGCCATGGCCGAAGTGCAGCTGTTGGAGAC m5
VH14 (SEQ ID NO: 269) ATGCGGCCCAGCCGGCCATGGCCGARGTGAAGCTTCTCSAGTC
m5 VH15 (SEQ ID NO: 270) ATGCGGCCCAGCCGGCCATGGCCCARGTTACTCTGAAAGAGT
3' primers: m3 HJ1 (SEQ ID NO: 271)
CCTGAACCGCCGCCTCCGCTCGAGACGGTGACCGTGGTCCC m3 HJ2 (SEQ ID NO: 272)
CCTGAACCGCCGCCTCCGCTCGAGACTGTGAGAGTGGTGCC m3 HJ3 (SEQ ID NO: 273)
CCTGAACCGCCGCCTCCGCTCGAGACAGTGACCAGAGTCCC m3 HJ4 (SEQ ID NO: 274)
CCTGAACCGCCGCCTCCGCTCGAGACGGTGACTGAGGTTCC 2.sup.nd PCR 5' primers:
5 VHR_FOK_biot (SEQ ID NO: 275) GAGCCGAGGACACGGCCGGATGTTACTGTGCGAGA
3' primers: 3'mJH1_Fok_biot (SEQ ID NO: 276)
GGGGCGCAGGGACATCCGTCACCGTCTCCTC 3'mJH2_Fok_biot (SEQ ID NO: 277)
GAGGAGACTGTGAGGGATGTGCCTTGGCCCCA 3'JH1_Fok (SEQ ID NO: 278)
GAGGAGACGGTGACGGATGTGCCCTGGCCCCA 3'mJH4_Fok_biot (SEQ ID NO: 279)
GAGGAGACGGTGACGGATGTTCCTTGACCCCA
Example 9
Phage Rescue of the Libraries
[0292] Each Primary and Secondary library was rescued independently
according to standard phage display procedures briefly summarized
hereafter. A volume of cell from the frozen library aliquots
sufficient to cover at least 10 times the theoretical diversity of
the library was added to 500 ml of 2.times.TYAG and grown at
37.degree. C. with agitation (240 rpm) until an OD600 of 0.3 to 0.5
was reached. The culture was then super-infected with MK13K07
helper phage and incubated for one hour at 37.degree. C. (150 rpm).
The medium was then changed by centrifuging the cells at 2000 rpm
for 10 minutes, removing the medium and resuspending the pellet in
500 ml of 2.times.TY-AK (100 m/ml ampicillin; 50 .mu.g/ml
kanamycin). The culture was then grown overnight at 30.degree. C.
(240 rpm). The culture was centrifuged at 4000 rpm for 20 minutes
to pellet the cells. The supernatant was collected and 30%
(vol/vol) of PEG 8000 (20%)/2.5M NaCl was added to precipitate the
phage particles by incubating the mixture 1 hour on ice. The phage
particles were collected by centrifugation at 10,000 rpm for 30
minutes and resuspended in 10 ml of TE buffer (10 mM tris-HCl pH
8.0; 1 mM EDTA). The resuspended solution was centrifuged at 10,000
rpm to clear the bacterial debris and the precipitation procedure
was repeated. After final resuspension, phage was titrated by
infection of E. coli and absorption at 280 nm. The display level of
scFv at the surface of phage was also evaluated by Western blot
analysis using an anti-c-myc monoclonal antibody. Purified phage
from different libraries was stored frozen at -80.degree. C. after
addition of glycerol to a final concentration of 15% (w/v).
[0293] In order to use a manageable number of libraries during
selection procedures, the purified phage was pooled into 4 working
libraries: AA1--Phage from all Primary synthetic VH libraries;
AB1--Phage from all Primary synthetic VL libraries; AC1--Phage from
all Primary natural VH libraries; AD1--Phage from all Secondary
natural libraries; AE1--Phage from all Secondary synthetic
libraries; MnA--Libraries with diversity captured from naive mice;
MiB--Libraries with diversity captured from mice immunized with
hIFN.gamma.; MiC--Libraries with diversity captured from mice
immunized with hCCL5/RANTES.
Example 10
High Throughput Sequencing of Antibody Libraries
[0294] The quality and diversity of a library can be evaluated by
DNA sequencing of random library members. In most cases a few
hundred clones are sequenced which represent only a very small
fraction of the library (less than 1 in 10,000,000 library
members). In order to analyze the performance of the methods
provide herein, next generation sequencing technology was used to
analyze a more representative number of library members. DNA
isolated from the library AE1 was used as a template for high
throughput sequencing using an illumina Genome Analyzer instrument.
This next-generation DNA sequencing system allows for billions of
bases to be read in a few days. The sequencing reads are relatively
short (about 70 bases) but perfectly compatible with our library
design. As the diversity is confined to the CDR3 regions a 70 base
read is sufficient to cover the CDRH3 and part of the framework 3
region for VH family identification. This technology has been
applied to sequence several millions of CDRH3 regions from the AE1
library. 5,078,705 sequences were obtained for a total of
365,666,760 bases. Analysis of the data indicated that 5,007,022
sequences (98.6% of the total) were unique. A total of 4,680,882
sequences could be unambiguously ascribed to a VH family (VH1, VH3
and VH5) and the representation of the VH families in the AE1
library determined (41% VH1; 30% VH3; 29% VH5). An important
finding was that the proportion of in frame inserts ranged between
88 and 91%. This data confirmed in a far more statistical manner
the sequencing results of the 24 primary VH synthetic libraries
described in Example 6. This combined set of sequencing data
demonstrates that the type IIs restriction cloning process used in
this method is very robust, leading to an efficient and productive
insertion in the 24 independent library constructions performed to
generate the VH diversity of the AE1 library.
[0295] The sequencing of millions of library members represents an
unprecedented quality control step for an antibody library. The
results demonstrate that the method allows for the generation of
high quality and high diversity libraries in a reproducible and
robust manner.
Example 11
Phage Display Selections Using Secondary Libraries
[0296] Liquid Phase Selections Against Human Interferon Gamma
(hIFN.gamma.):
[0297] Aliquots of AD1 and AE1 phage libraries (10.sup.11-10.sup.12
Pfu) were blocked with PBS containing 3% (w/v) skimmed milk for one
hour at room temperature on a rotary mixer. Blocked phage was then
deselected on streptavidin magnetic beads (Dynal M-280) for one
hour at room temperature on a rotary mixer. Deselected phage was
then incubated with in vivo biotinylated hIFN.gamma. (100 nM) for
two hours at room temperature on a rotary mixer. Beads were
captured using a magnetic stand followed by four washes with
PBS/0.1% Tween 20 and 3 washes with PBS. Beads were then directly
added to 10 ml of exponentially growing TG1 cells and incubated for
one hour at 37.degree. C. with slow shaking (100 rpm). An aliquot
of the infected TG1 was serial diluted to titer the selection
output. The remaining infected TG1 were spun at 3000 rpm for 15
minutes and re-suspended in 0.5 ml 2.times.TYAG (2.times.TY media
containing 100 .mu.g/ml ampicillin and 2% glucose) and spread on
2.times.TYAG agar Bioassay plates. After overnight incubation at
30.degree. C., 10 ml of 2.times.TYAG was added to the plates and
the cells were scraped from the surface and transferred to a 50 ml
polypropylene tube. 2.times.TYAG containing 50% glycerol was added
to the cell suspension to obtain a final concentration of 17%
glycerol. Aliquots of the selection round were kept at -80.degree.
C. Phage outputs were titrated after each round and the progressive
increase in outputs indicated that the enrichment of clones
specific for the target was occurring (FIG. 16).
[0298] Selections by Panning Against the Rat Monoclonal Antibody
5E3:
[0299] Immunotubes were coated with 5E3 at 10 .mu.g/ml in PBS over
night at 4.degree. C. and immunotubes for phage deselection were
coated with an irrelevant rat antibody under the same conditions.
After washing immunotubes were blocked with PBS containing 3% (w/v)
skimmed milk for one hour at room temperature. Aliquots of AD1 and
AE1 phage libraries (10.sup.11-10.sup.12 Pfu) were blocked with PBS
containing 3% (w/v) skimmed milk for one hour at room temperature
on a rotary mixer. Blocked phage was then deselected in the
immunotubes coated with an irrelevant rat antibody for one hour at
room temperature on a rotary mixer. Deselected phage was then
transferred to the immunotubes coated with 5E3 and incubated for
two hours at room temperature on a rotary mixer. Tubes were washed
five times with PBS/0.1% Tween 20 and 3 times with PBS. Phage was
eluted with TEA 100 mM for 10 minutes and neutralized with 1M Tris
HCl pH 7.5. Phage was added to 10 ml of exponentially growing TG1
cells and incubated for one hour at 37.degree. C. with slow shaking
(100 rpm). An aliquot of the infected TG1 was serial diluted to
titer the selection output. The remaining infected TG1 were spun at
3000 rpm for 15 minutes and re-suspended in 0.5 ml 2.times.TYAG
(2.times.TY media containing 100 .mu.g/ml ampicillin and 2%
glucose) and spread on 2.times.TYAG agar Bioassay plates. After
overnight incubation at 30.degree. C., 10 ml of 2.times.TYAG was
added to the plates and the cells were scraped from the surface and
transferred to a 50 ml polypropylene tube. 2.times.TYAG containing
50% glycerol was added to the cell suspension to obtain a final
concentration of 17% glycerol. Aliquots of the selection round were
kept at -80.degree. C. Rounds of selection were performed by
alternating between rat 5E3 and a chimeric version of 5E3 in which
the variable region were fused to mouse constant domains. These
alternating rounds were performed in order to enrich for clones
specific for the variable region of 5E3 and generate anti-idiotypic
antibodies. Phage outputs were titrated after each round and the
progressive increase in outputs indicated that the enrichment of
clones specific for the target was occurring (FIG. 17).
[0300] Phage Rescue:
[0301] 100 .mu.l of cell suspension obtained from previous
selection rounds were added to 20 ml of 2.times.TYAG and grown at
37.degree. C. with agitation (240 rpm) until an OD600 of 0.3 to 0.5
was reached. The culture was then super-infected with
3.3.times.10.sup.10 MK13K07 helper phage and incubated for one hour
at 37.degree. C. (150 rpm). The medium was then changed by
centrifuging the cells at 2000 rpm for 10 minutes, removing the
medium and resuspending the pellet in 20 ml of 2.times.TY-AK (100
.mu.g/ml ampicillin; 50 .mu.g/ml kanamycin). The culture was then
grown overnight at 30.degree. C. (240 rpm).
[0302] Monoclonal Phage Rescue for ELISA:
[0303] Single clones were picked into a microtiter plate containing
150 .mu.l of 2.times.TYAG media (2% glucose) per well and grown at
37.degree. C. (100-120 rpm) for 5-6 h. M13KO7 helper phage was
added to each well to obtain a multiplicity of infection (MOI) of
10 (i.e., 10 phage for each cell in the culture) and incubated at
37.degree. C. (100 rpm) for 1 h. Following growth, plates were
centrifuged at 3,200 rpm for 10 min. Supernatant was carefully
removed, cells resuspended in 150 .mu.l 2.times.TYAK medium and
grown overnight at 30.degree. C. (120 rpm). For the ELISA, the
phage are blocked by adding 150 .mu.l of 2.times. concentration PBS
containing 5% skimmed milk powder followed by one hour incubation
at room temperature. The plates were then centrifuged 10 minutes at
3000 rpm and the phage containing supernatant used for the
ELISA.
[0304] Phage ELISA:
[0305] ELISA plates (Maxisorb, NUNC) were coated overnight with 2
.mu.g/ml hIFN.gamma. in PBS or 2 .mu.g/ml rat 5E3 in PBs. Control
plates were coated with 2 .mu.g/ml BSA or an irrelevant rat
monoclonal antibody. Plates were then blocked with 3% skimmed
milk/PBS at room temperature for 1 h. Plates were washed 3 times
with PBS 0.05% Tween 20 before transferring the pre-blocked phage
supernatants and incubation for one hour at room temperature.
Plates were then washed 3 times with PBS 0.05% Tween 20. 50 .mu.l
of 3% skimmed milk/PBS containing (HRP)-conjugated anti-M13
antibody (Amersham, diluted 1:10,000) to each well. Following
incubation at room temperature for 1 hr, the plates were washed 5
times with PBS 0.05% Tween 20. The ELISA was then revealed by
adding 50 .mu.l of TMB (Sigma) and 50 .mu.l of 2N H.sub.2SO.sub.4
to stop the reaction. Absorption intensity was read at 450 nm.
Clones specific for hIFN.gamma. could be identified and the hit
rates ranged between 10% and 30% after the third round of
selection. Clones specific for the variable region of 5E3 could
also be identified and the hit rates ranged between 7 and 48% after
the third round of selection.
[0306] Phage Clone Sequencing:
[0307] Single clones were grown in 5 ml of 2.times.TYAG media (2%
glucose) per well and grown at 37.degree. C. (120 rpm) overnight.
The next day phagemid DNA was purified and used for DNA sequencing
using a primer specific for pNDS1: mycseq,
5'-CTCTTCTGAGATGAGTTTTTG. (SEQ ID NO: 255).
[0308] Large Scale scFv Purification:
[0309] A starter culture of 1 ml of 2.times.TYAG was inoculated
with a single colony from a freshly streaked 2.times.TYAG agar
plate and incubated with shaking (240 rpm) at 37.degree. C. for 5
hours. 0.9 ml of this culture was used to inoculate a 400 ml
culture of the same media and was grown overnight at 30.degree. C.
with vigorous shaking (300 rpm).
[0310] The next day the culture was induced by adding 400 .mu.l of
1M IPTG and incubation was continued for an additional 3 hours. The
cells were collected by centrifugation at 5,000 rpm for 10 minutes
at 4.degree. C. Pelleted cells were resuspended in 10 ml of
ice-cold TES buffer complemented with protease inhibitors as
described above. Osmotic shock was achieved by adding 15 ml of 1:5
diluted TES buffer and incubation for 1 hour on ice. Cells were
centrifuged at 10,000 rpm for 20 minutes at 4.degree. C. to pellet
cell debris. The supernatant was carefully transferred to a fresh
tube. Imidazole was added to the supernatant to a final
concentration of 10 mM. 1 ml of Ni-NTA resin (Qiagen), equilibrated
in PBS was added to each tube and incubated on a rotary mixer at
4.degree. C. (20 rpm) for 1 hour. The tubes were centrifuged at
2,000 rpm for 5 minutes and the supernatant carefully removed. The
pelleted resin was resuspended in 10 ml of cold (4.degree. C.) Wash
buffer 1 (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 10 mM imidazole, pH
to 8.0). The suspension was added to a polyprep column (Biorad). 8
ml of cold Wash Buffer 2 (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 20
mM imidazole, pH to 8.0) were used to wash the column by gravity
flow. The scFv were eluted from the column with 2 ml of Elution
buffer (50 mM NaH.sub.2PO.sub.4, 300 mM NaCl, 250 mM imidazole, pH
to 8.0). Fractions were analyzed by absorption at 280 nm and
protein containing fractions were pooled before buffer exchange on
a PD10 desalting column (Amersham) equilibrated with PBS. The scFv
in PBS were analyzed by SDS-PAGE and quantified by absorption at
280 nm. The purified scFv were aliquoted and stored at -20.degree.
C. and at 4.degree. C.
Example 12
Analysis of CDR3 Profiles Obtained after Selection Using High
Throughput Sequencing
[0311] Using next generation sequencing technology as described in
Example 10, the distribution of CDR H3 lengths within each VH
family in the AE1 and AD1 libraries as well as in the output
obtained after the third round of selection was analyzed. The
profiles of the AE 1 and AD1 libraries are clearly different (FIG.
18). The CDR H3 length distribution in the AE1 library corresponds
to the intended library design, with lengths ranging between 9-15
amino acids. In contrast, much longer CDR H3 of up to 22 amino
acids are found in the AD1 library, and the profile corresponds to
the length distribution observed in human natural repertoires.
These results confirm that a human natural CDR H3 repertoire has
been captured during the construction of the AD1 library. A similar
analysis performed after three rounds of selection against 5E3
revealed that completely different CDR H3 length profiles were
selected. In particular, a dramatic enrichment of CDR H3 of 8 and
21 amino acids in length could be observed in the selection
performed with the AD1 library. This set of data demonstrated that
different CDR H3 profiles were enriched from the two libraries
after selection against the same target. Furthermore, this analysis
demonstrates that, using the present invention, long CDR H3 that
are very difficult to cover using synthetic diversity could be
captured into selected human frameworks and selected.
Example 13
Evaluating Identified scFvs in Binding Assays
[0312] Purified scFvs preparations of clones having different
sequences and that were identified positive against the variable
region of 5E3 were tested for binding against chimeric 5E3 in a
dose response ELISA. These preparations were also tested against an
irrelevant mouse antibody (1A6). ELISA plates (Maxisorb, NUNC) were
coated overnight with 2 .mu.g/ml mouse 5E3 in PBS. Control plates
were coated with 2 .mu.g/ml 1A6 monoclonal antibody. Plates were
then blocked with 3% skimmed milk/PBS at room temperature for 1 h.
Plates were washed 3 times with PBS 0.05% Tween 20 before adding
different concentrations of purified scFv and incubation for one
hour at room temperature. Plates were then washed 3 times with PBS
0.05% Tween 20. 50 .mu.l of 3% skimmed milk/PBS containing
(HRP)-conjugated anti-myc antibody to each well. Following
incubation at room temperature for 1 hr, the plates were washed 5
times with PBS 0.05% Tween 20. The ELISA was then revealed by
adding 50 .mu.l of Amplex Red fluorescent substrate and the signal
was read on fluorescence spectrophotometer. The data shows that
most of the clones are highly specific for 5E3 as they do not
recognize 1A6 and that they are directed against the variable
regions of 5E3 (FIG. 19).
[0313] Similarly, purified scFvs preparations of clones having
different sequences and that were identified in phage ELISA as
binders against hIFN.gamma. were tested for binding against
hIFN.gamma. in a dose response experiment. ELISA plates (Maxisorb,
NUNC) were coated overnight with 2 .mu.g/ml hIFN.gamma. in PBS and
control plates were coated with 2 .mu.g/ml BSA in PBS. Plates were
then blocked with 3% skimmed milk/PBS at room temperature for 1 h.
Plates were washed 3 times with PBS 0.05% Tween 20 before adding
different concentration of purified scFv and incubation for one
hour at room temperature. Plates were then washed 3 times with PBS
0.05% Tween 20. 50 .mu.l of 3% skimmed milk/PBS containing
(HRP)-conjugated anti-myc antibody to each well. Following
incubation at room temperature for 1 hr, the plates were washed 5
times with PBS 0.05% Tween 20. The ELISA was then revealed by
adding 50 .mu.l TMB substrate and 50 .mu.l of 2N H.sub.2SO.sub.4 to
stop the reaction. The signal was read on an absorbance
spectrophotometer at 450 nm. The data shows that the selected
clones are binding to hIFN.gamma. in a dose dependent manner and
gave a very good signal when compared to a positive control scFv A6
that has a high affinity for hIFN.gamma. (FIG. 20).
Example 14
ScFv Inhibition of Interferon Gamma-Induced Reporter Gene
Expression
[0314] A panel of selected scFv specific for hIFN.gamma. was
produced and purified as described above and tested for the
capacity to block the biological activity of hIFN.gamma.. A
reporter gene (firefly luciferase), driven by the
IFN.gamma.-inducible GBP1 promoter, was transfected into the human
melanoma cell line, Me67.8. Various concentrations of scFv were
incubated with 2 ng/ml of hIFN.gamma. and then added to the cell
culture. Following a 6 hour incubation time, the luciferase
reporter assay was performed and the intensity of the luminescence
measured. The activity was compared to a scFv isolated from another
human scFv antibody library constructed by traditional capturing of
the VH/VL repertoires form human donors (clone G9). The data shows
that scFv isolated either from synthetic or natural human diversity
libraries (AE1 and AD1) were capable of neutralizing the biological
activity of hIFN.gamma. in a dose dependent manner (FIG. 21). The
neutralization potential of these scFv was superior to the
benchmark scFv clone G9.
Example 15
scFv Inhibition of Interferon Gamma-Induced MHC Class II
Expression
[0315] A flow cytometric assay was implemented to identify fully
human IgG antibodies, or fragments thereof, capable of blocking the
expression of IFN.gamma.-induced MHC class II molecules. Following
the plating of Me67.8 cells, 5 ng/ml recombinant human IFN.gamma.
was added to cultures in the presence of various concentrations of
candidate fully human anti-IFN.gamma. monoclonal antibodies.
Following 48 h in culture, cells were stained with fluorescently
labeled anti-human MHC class II antibody (HLA-DR) and analyzed
using a FACSCalibur.RTM.. Thus, the IC.sub.50 (where 50% of the
IFN.gamma.-induced MHC class II expression is inhibited, i.e., 50%
inhibitory concentration), for each candidate antibody is
measured.
[0316] Purified fully human scFv were produced as described above.
The effect of selected scFv on IFN.gamma.-induced MHC class II
expression on melanoma cells was evaluated using the flow
cytometric cell-based assay described above. These scFv inhibited
IFN.gamma.-induced MHC II expression on melanoma cells (FIG.
22).
Example 16
Reformatting scFv into IgG Format
[0317] The V.sub.H and V.sub.L sequence of selected scFv were
amplified with specific oligonucleotides introducing a leader
sequence and a HindIII restriction site at the 5' end. An ApaI site
was introduced at the 3' end of the heavy whereas an AvrII and a
BsiWI site were introduced at the 3' end of the lambda or kappa
light chain sequences, respectively. The amplified V.sub.H
sequences were digested HindIII/ApaI and cloned into the
pCon_gamma1 expression vector (LONZA, Basel, Switzerland). The
amplified V.sub.L lambda sequences were digested HindIII/AvrII and
cloned into the pCon_lambda2 expression vector and the amplified
V.sub.L kappa sequences were digested HindIII/BsiWI and cloned into
the pCon_kappa expression vector (LONZA, Basel, Switzerland). The
constructions were verified by sequencing before transfection into
mammalian cells.
[0318] The V.sub.H and V.sub.L cDNA sequences in their appropriate
expression vectors were transfected into mammalian cells using the
Fugene 6 Transfection Reagent (Roche, Basel, Switzerland). Briefly,
Peak cells were cultured in 6-well plates at a concentration of
6.times.10.sup.5 cells per well in 2 ml culture media containing
fetal bovine serum. The expression vectors, encoding the candidate
V.sub.H and V.sub.L sequences, were co-transfected into the cells
using the Fugene 6 Transfection Reagent according to manufacturer's
instructions. One day following transfection, the culture media was
aspirated, and 3 ml of fresh serum-free media was added to cells
and cultured for three days at 37.degree. C. Following three days
culture period, the supernatant was harvested for IgG purified on
protein G-Sepharose 4B fast flow columns (Sigma, St. Louis, Mo.)
according to manufacturer's instructions. Briefly, supernatants
from transfected cells were incubated overnight at 4.degree. C.
with ImmunoPure (G) IgG binding buffer (Pierce, Rockford Ill.).
Samples were then passed over Protein G-Sepharose 4B fast flow
columns and the IgG consequently purified using elution buffer. The
eluted IgG fraction was then dialyzed against PBS and the IgG
content quantified by absorption at 280 nm. Purity and IgG
integrity were verified by SDS-PAGE.
Example 17
IgG Inhibition of Interferon Gamma Biological Activity
[0319] Two scFv, AE1-4-R3-P2E4 (2E4) and A2-AD1-R4P1A9 (1A9), that
had confirmed inhibitory activity against hIFN.gamma. in functional
assays were reformatted into IgG as described in Example 16 and
tested in the interferon gamma-induced reporter gene assay
described in Example 14. The results shown in FIG. 23 indicate that
in a IgG format both 1A9 and 2E4 could neutralize the activity of
hIFN.gamma. with IC.sub.50 of 42 nM and 10 nM, respectively whereas
a negative control IgG (NI-0701) had no effect in this assay. Thus
these two candidates isolated from both synthetic and natural
diversity libraries could be reformatted into full IgG and feature
neutralizing activity against the selected target.
Example 18
Development of a Pharmacokinetic Assay for the Detection of 5E3 in
Mouse Serum
[0320] Two scFv candidates AD15E3R3P1_A4 and AD25E3R3P1_G11 that
bind specifically to mouse monoclonal antibody 5E3 (FIG. 19) were
reformatted into full human IgG as described in Example 16. The
specificity of the corresponding IgGs DA4 and G11 was confirmed in
ELISA against mouse 5E3 and a chimeric version of this monoclonal
antibody in which the mouse variable regions have been fused to rat
constant IgG regions. The results shown in FIG. 24 demonstrate that
the IgG DA4 and G11 are specific for the variable region of 5E3 as
they bind to both mouse and chimeric rat 5E3 and not to mouse and
rat isotype controls. These two monoclonals antibodies were used to
develop an assay for the quantification of 5E3 in mouse serum for
pharmacokinetic studies. Several dilutions of mouse serum were
spiked with 5 .mu.g/ml of mouse 5E3 antibody and serially diluted
in such a way that serum concentration was maintained constant
throughout the dilution series. Maxisorb plates (Nunc, Denmark)
were coated overnight with 1 .mu.g/ml of IgG DA4 or IgG G11. After
blocking with PBS; 1% BSA dilution series of the spiked serum
preparations were added to the wells. After incubation and washing,
the signal was revealed using an anti-mouse Kappa light chain
monoclonal antibody coupled to horse radish peroxydase (HRP) and a
fluorescent substrate (Amplex red; Invitrogen). The results show
that both antibodies can be used to specifically detect the mouse
monoclonal 5E3 antibody in mouse serum (FIG. 25). The detection
limit of mouse 5E3 in serum was about 200 ng/ml and the assay was
not significantly affected by the serum concentration indicating
that IgG DA4 and IgG G11 are highly specific for mouse 5E3 and do
not bind to other mouse immunoglobulin. These experiments
demonstrate that highly specific anti-idiotypic antibodies could be
isolated from the natural or synthetic libraries AE 1 and AD1.
Example 19
Phage Selection Using Libraries Containing CDRH3 Diversity Captured
from NaiVe and Immunized Mice
[0321] The MnA, MiB and MiC libraries described in Examples 8 and 9
were used in parallel for phage selections against hIFN.gamma.
following the procedure described in Example 11. During the
selection process a similar enrichment of phage was observed (FIG.
26).
[0322] scFv Expression in Microtiter Plate Format:
[0323] Single clones were picked into a microtiter plate containing
150 .mu.l of 2.times.TYAG media (2% glucose) per well and grown at
37.degree. C. (100-120 rpm) for 5-6 h. Plates were centrifuged at
280 rpm, the medium discarded and the cell pellets resuspended in
100 .mu.l of 2.times.TYA medium containing 1 mM IPTG. The plates
were incubated overnight at 30.degree. C. with shaking (100 rpm).
Following growth, plates were centrifuged at 3,200 rpm for 10 min
and the supernatant carefully transferred to a plate containing
2.times. concentrated PBS containing 5% skimmed milk powder for
blocking.
[0324] scFv ELISA:
[0325] ELISA plates (Maxisorb, NUNC) were coated overnight with 2
.mu.g/ml hIFN.gamma. in PBS. Control plates were coated with 2
.mu.g/ml recombinant BSA (Sigma). Plates were then blocked with 3%
skimmed milk/PBS at room temperature for 1 h. Plates were washed 3
times with PBS 0.05% Tween 20 before transferring the pre-blocked
scFv supernatants and incubation for one hour at room temperature.
Plates were then washed 3 times with PBS 0.05% Tween 20. 50 .mu.l
of 3% skimmed milk/PBS containing (HRP)-conjugated anti-cMyc
antibody (diluted 1:5,000) to each well. Following incubation at
room temperature for 1 hr, the plates were washed 5 times with PBS
0.05% Tween 20. The ELISA was then revealed by adding 50 .mu.l of
Amplex Red (Invitrogen). Fluorescence intensity was measured at 590
nm upon excitation at 530 nm. The frequency of hits giving a signal
of half the intensity of the control A6 clone was evaluated after
each round of selection for the three libraries (FIG. 27). The hit
rate obtained with the MiB library was dramatically higher compared
to the two other libraries and the average level of signal was
superior for the clones derived from the MiB library, indicating
that higher affinity scFv were enriched (FIG. 28). In order to
confirm this observation, positive clones were sequenced, expressed
in larger scale and purified to be tested in dose response binding
experiments according to Example 13. The scFv derived from the MiB
library all had a higher apparent affinity for hIFN.gamma. than
those isolated from the naive MnA library (FIG. 29). The results
indicate that the CDRH3 repertoire from mice immunized with a
protein could be captured into a human antibody framework context
in a productive way to generate at higher frequency high affinity
human antibody fragments. Libraries generated using the present
invention thus represent a powerful mean of generating antibodies
with therapeutic potential.
Example 20
Identification of Stuffer DNA Fragments that can Encode Functional
CDRH3
[0326] A combinatorial approach was used to identify stuffer DNA
fragments that fulfill the following criteria: 1) include two Type
IIS restriction sites; 2) maintain the reading frame between FR3
and FR4 regions and 3) encode a heavy variable domain CDR3 that
allows the folding and expression of an antibody variable domain.
The presence of the two restriction enzyme sites partially defines
the sequence of the CDRH3 at the protein level. To maximize the
chances of finding sequences that could accommodate this
constraint, oligonucleotides were designed to synthesize a
collection of stuffer fragments containing two BsmBI restriction
sites and introducing diversity in one or two codons in order to
explore multiple solutions for two defined CDRH3 lengths (FIG. 31).
These two collections of stuffer in frame (SIF) fragments were
generated by assembly PCR using the following primers:
TABLE-US-00008 5 VHstufIF1 (SEQ ID NO: 290)
ATTACTGTGCGAGAGGAGACGNSNNCGTCTCTTGGGGCCAGGGAAC 5 VHstufIF2 (SEQ ID
NO: 291) ATTACTGTGCGAGAGGAGACGNCGTCTCTTGGGGCCAGGGAACCCT 3 VHIF (SEQ
ID NO: 292) ttatgtgtataggGTTCCCTGGCCCCAAGAGACG 5 VHIF (SEQ ID NO:
293) gtgatctgtacctATTACTGTGCGAGAGGAGACG
[0327] The amplified SIF1 and SIF2 were digested with BsmBI and
cloned into the phagemid vector pNDS_VH3-23-VK dummy acceptor
framework previously digested with BsmBI. The ligation products
were transformed into electrocompetent E. coli TG1 cells and plated
on 2.times.TYAG Bioassay plates (2.times.TY medium containing 100
.mu.g/ml ampicillin and 2% glucose). After overnight incubation at
30.degree. C., 6 ml of 2.times.TYAG liquid medium was added to the
plates and the cells were scraped from the surface and transferred
to a 50 ml polypropylene tube. These small diversity libraries
named IF1 and IF2 were rescued using Hyperphage (Hust M et al.,
Biotechniques 2006 September; 41(3):335-42) so that only library
members encoding scFv compatible with expression as a pIII fusion
protein and assembly into a phage particle can lead to phage
production. The rescued phage was directly used to infect TG1 cells
that were then plated on 2.times.TYAG Bioassay plates. After
scraping of the cells, a second round of rescue and infection was
performed and individual colonies were sequenced to identify
sequences that were enriched in this selection process. A total of
8 SIF2 and 15 SIF1 independent sequences were identified in the
selected clones (FIGS. 32 and 33). Each clone was expressed and
purified independently as a scFv using large scale scFv
purification as described in Example 11 to confirm that the SIF
sequence was compatible with the production of a scFv. The scFv
production yield was determined and the integrity of the protein
assessed by SDS-PAGE. Using these parameters 6 clones containing
different SIF sequences were selected and the corresponding vector
DNA was prepared to test Type IIS cloning efficiency and the
capacity of BsmBI to digest both sites in the context of these SIF
sequences. The sequence of the clone SIF.sub.--2b8 was selected and
integrated in all the VH framework Acceptor sequences.
Example 21
Generation and Clean-Up of Acceptor Libraries Containing a SIF
[0328] The SIF VH acceptors were then combined with VL synthetic
primary libraries as described in Example 8 to generate Acceptor
libraries in which CDRH3 diversity can be introduced by digestion
of the SIF. The VL sequences were derived from the seven Primary
Synthetic Libraries described in Example 6 by PCR amplification
using primers 5'biot-VHdummy and 3'biot-fdtseq. The resulting VL
containing fragments of approximately 400 bp were digested using
XhoI/NotI and purified on spin columns to remove primers and
enzymes. Similarly, the pNDS VH acceptor vectors containing a SIF
stuffer and a dummy light chain were digested with XhoI/NotI and
SwaI (SwaI cutting inside the VL dummy) and purified on Chroma Spin
TE columns with a cutoff of 1000 bp to get rid of the VL dummy
fragment. The digested VL fragments were then ligated into the SIF
VH acceptor vectors. The ligation products were transformed into
electrocompetent E. coli TG1 cells and plated on 2.times.TYAG
Bioassay plates (2.times.TY medium containing 100 .mu.g/ml
ampicillin and 2% glucose). After overnight incubation at
30.degree. C., 6 ml of 2.times.TYAG liquid medium was added to the
plates and the cells were scraped from the surface and transferred
to a 50 ml polypropylene tube. Glycerol 50% was added to the cell
suspension to obtain a final concentration of 17% glycerol.
Aliquots of the Acceptor libraries were stored at -80.degree. C.
The total size of this acceptor library, carrying synthetic
diversity in the CDR L3, was 4.3.times.10.sup.9.
[0329] The libraries were rescued using Hyperphage and used for TG1
infection as described above in order to remove out of frame
sequences and therefore enrich the Acceptor libraries for in frame
inserts. To assess the efficiency of the process, 30 individual
clones from three libraries were picked and sequenced before and
after the clean-up procedure and the frequency of in frame
sequences determined. The results shown below in Table 2 indicate
that the frequency of in frame sequences was significantly
increased by this process and in two libraries all of the 30
sequences were in frame. This process and the use of SIF in the
Acceptor libraries increased the functionality of the Acceptor
library making it a better receptacle for CDRH3 diversity.
TABLE-US-00009 TABLE 2 Frequency of in frame sequences in the SIF
Acceptor libraries before and after clean-up process Before
clean-up After clean-up Libraries in frame sequence (%) VH1-2-VK1
76 100 VH1-2-VK3 77 100 VH1-2-V.lamda. 87 94
Other Embodiments
[0330] While the invention has been described in conjunction with
the detailed description thereof, the foregoing description is
intended to illustrate and not limit the scope of the invention,
which is defined by the scope of the appended claims. Other
aspects, advantages, and modifications are within the scope of the
following claims.
Sequence CWU 1
1
366198PRTHomo sapiens 1Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Gly Tyr 20 25 30 Tyr Met His Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro
Asn Ser Gly Gly Thr Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg
Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg 298PRTHomo sapiens 2Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Gly Ile Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Trp Ile
Ser Ala Tyr Asn Gly Asn Thr Asn Tyr Ala Gln Lys Leu 50 55 60 Gln
Gly Arg Val Thr Met Thr Thr Asp Thr Ser Thr Ser Thr Ala Tyr 65 70
75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg 398PRTHomo sapiens 3Gln Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val
Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile
Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45
Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50
55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala
Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg 498PRTHomo sapiens 4Gln Val Gln Leu
Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30
Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg 598PRTHomo sapiens 5Glu Val
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20
25 30 Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45 Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Tyr Tyr Ala
Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg 698PRTHomo sapiens
6Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys 798PRTHomo
sapiens 7Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser
Phe Thr Ser Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly
Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser
Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile
Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser
Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg
898PRTHomo sapiens 8Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser
Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu
Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly
Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp
Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Asn Leu Pro Pro 85 90 95
Thr Val 998PRTHomo sapiens 9Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg
Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser
Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Pro 85
90 95 Thr Val 1098PRTHomo sapiens 10Glu Ile Val Leu Thr Gln Ser Pro
Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser
Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp
Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65
70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp
Pro Pro 85 90 95 Thr Val 1198PRTHomo sapiens 11Glu Ile Val Met Thr
Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45 Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu
Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn
Asn Trp Pro Pro 85 90 95 Thr Val 1299PRTHomo sapiens 12Glu Ile Val
Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu
Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25
30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg
Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln
Gln Tyr Gly Ser Ser Pro 85 90 95 Pro Thr Val 1398PRTHomo sapiens
13Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1
5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser
Asn 20 25 30 Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro
Lys Leu Leu 35 40 45 Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val
Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser
Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr
Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Asn Gly 1498PRTHomo
sapiens 14Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser Ala Ala Pro
Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn
Ile Gly Asn Asn 20 25 30 Tyr Val Ser Trp Tyr Gln Gln Leu Pro Gly
Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Glu Asn Asn Lys Arg Pro
Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr
Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65 70 75 80 Thr Gly Asp Glu
Ala Asp Tyr Tyr Cys Gly Thr Trp Asp Ser Ser Leu 85 90 95 Ser Ala
15107DNAHomo sapiens 15tattactgtg cgagatgaga cgaataacgg taaggcggtt
taccaggttt aaacgcgtat 60tgggaaggcg cgtctcttgg ggccagggaa ccctggtcac
agtctcg 1071636PRTHomo sapiens 16Val Leu Leu Cys Glu Met Arg Arg
Ile Thr Val Arg Arg Phe Thr Arg 1 5 10 15 Phe Lys Arg Val Leu Gly
Arg Arg Val Ser Trp Gly Gln Gly Thr Leu 20 25 30 Val Thr Val Ser 35
1733PRTHomo sapiens 17Tyr Tyr Cys Ala Arg Asp Glu Arg Gly Gly Leu
Pro Gly Leu Asn Ala 1 5 10 15 Tyr Trp Glu Gly Ala Ser Leu Gly Ala
Arg Glu Pro Trp Ser Gln Ser 20 25 30 Arg 1835PRTHomo sapiens 18Ile
Thr Val Arg Asp Glu Thr Asn Asn Gly Lys Ala Val Tyr Gln Val 1 5 10
15 Thr Arg Ile Gly Thr Lys Ala Arg Leu Leu Gly Pro Gly Asn Pro Gly
20 25 30 His Ser Leu 35 1914DNAArtificial Sequencechemically
synthesized 19cgtctcnnnn nnnn 142014DNAArtificial
Sequencechemically synthesized 20gcagagnnnn nnnn 1421386DNAHomo
sapiens 21caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc
agtgaaggtc 60tcctgcaagg cttctggata caccttcacc ggctactata tgcactgggt
gcgacaggcc 120cctggacaag ggcttgagtg gatgggatgg atcaacccta
acagtggtgg cacaaactat 180gcacagaagt ttcagggcag ggtcaccatg
accagggaca cgtccatcag cacagcctac 240atggagctga gcaggctgag
atctgacgac acggccgtgt attactgtgc gagatgagac 300gaataacggt
aaggcggttt accaggttta aacgcgtatt gggaaggcgc gtctcttggg
360gccagggaac cctggtcaca gtctcg 38622125PRTHomo sapiens 22Pro Gly
Ala Ala Gly Ala Val Trp Gly Gly Glu Glu Ala Trp Gly Leu 1 5 10 15
Ser Glu Gly Leu Leu Gln Gly Phe Trp Ile His Leu His Arg Leu Leu 20
25 30 Tyr Ala Leu Gly Ala Thr Gly Pro Trp Thr Arg Ala Val Asp Gly
Met 35 40 45 Asp Gln Pro Gln Trp Trp His Lys Leu Cys Thr Glu Val
Ser Gly Gln 50 55 60 Gly His His Asp Gln Gly His Val His Gln His
Ser Leu His Gly Ala 65 70 75 80 Glu Gln Ala Glu Ile Arg His Gly Arg
Val Leu Leu Cys Glu Met Arg 85 90 95 Arg Ile Thr Val Arg Arg Phe
Thr Arg Phe Lys Arg Val Leu Gly Arg 100 105 110 Arg Val Ser Trp Gly
Gln Gly Thr Leu Val Thr Val Ser 115 120 125 23125PRTHomo sapiens
23Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly
Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Trp Ile Asn Pro Asn Ser Gly Gly Thr Asn
Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp
Thr Ser Ile Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Arg Leu Arg
Ser Asp Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Glu Arg
Gly Gly Leu Pro Gly Leu Asn Ala Tyr Trp Glu 100 105 110 Gly Ala Ser
Leu Gly Ala Arg Glu Pro Trp Ser Gln Ser 115 120 125 24123PRTHomo
sapiens 24Pro Arg Cys Ser Trp Cys Ser Leu Gly Leu Arg Arg Ser Leu
Gly Pro 1 5 10 15 Gln Arg Ser Pro Ala Arg Leu Leu Asp Thr Pro Ser
Pro Ala Thr Ile 20 25 30 Cys Thr Gly Cys Asp Arg Pro Leu Asp Lys
Gly Leu Ser Gly Trp Asp 35 40 45 Gly Ser Thr Leu Thr Val Val Ala
Gln Thr Met His Arg Ser Phe Arg 50 55 60 Ala Gly Ser Pro Pro Gly
Thr Arg Pro Ser Ala Gln Pro Thr Trp Ser 65 70 75 80 Ala Gly Asp Leu
Thr Thr Arg Pro Cys Ile Thr Val Arg Asp Glu Thr 85 90 95 Asn Asn
Gly Lys Ala Val Tyr Gln Val Thr Arg Ile Gly Lys Ala Arg 100 105 110
Leu Leu Gly Pro Gly Asn Pro Gly His Ser Leu 115 120 25386DNAHomo
sapiens 25caggtgcagc tggtgcagtc tggagctgag gtgaagaagc ctggggcctc
agtgaaggtc 60tcctgcaagg cttctggtta cacctttacc agctatggta tcagctgggt
gcgacaggcc 120cctggacaag ggcttgagtg gatgggatgg atcagcgctt
acaatggtaa cacaaactat 180gcacagaagc tccagggcag agtcaccatg
accacagaca catccacgag cacagcctac 240atggagctga ggagcctgag
atctgacgac acggccgtgt attactgtgc gagatgagac 300gaataacggt
aaggcggttt accaggttta aacgcgtatt gggaaggcgc gtctcttggg
360gccagggaac cctggtcaca gtctcg 38626125PRTHomo sapiens 26Pro Gly
Ala Ala Gly Ala Val Trp Ser Gly Glu Glu Ala Trp Gly Leu 1 5 10 15
Ser Glu Gly Leu Leu Gln Gly Phe Trp Leu His Leu Tyr Gln Leu Trp 20
25 30 Tyr Gln Leu Gly Ala Thr Gly Pro Trp Thr Arg Ala Val Asp Gly
Met 35 40 45 Asp Gln Arg Leu Gln Trp His Lys Leu Cys Thr Glu Ala
Pro Gly Gln 50 55 60 Ser His His Asp His Arg His Ile His Glu His
Ser Leu His Gly Ala 65 70 75 80 Glu Glu Pro Glu Ile Arg His Gly Arg
Val Leu Leu Cys Glu Met Arg 85 90 95 Arg Ile Thr Val Arg Arg Phe
Thr Arg Phe Lys Val Arg Leu Gly Arg 100 105 110 Arg Val Ser Trp Gly
Gln Gly Thr Leu Val Thr Val Ser 115 120 125 27125PRTHomo sapiens
27Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30 Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Trp Ile Ser Ala Tyr Asn Gly Asn Thr Asn
Tyr Ala Gln Lys Leu 50 55 60 Gln Gly Arg Val Thr Met Thr Thr Asp
Thr Ser Thr Ser Thr Ala Tyr 65 70
75 80 Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr
Cys 85 90 95 Ala Arg Asp Glu Arg Gly Gly Leu Pro Gly Leu Asn Ala
Tyr Trp Glu 100 105 110 Gly Ala Ser Leu Gly Ala Arg Glu Pro Trp Ser
Gln Ser 115 120 125 28122PRTHomo sapiens 28Pro Arg Cys Ser Trp Cys
Ser Leu Glu Leu Arg Arg Ser Leu Gly Pro 1 5 10 15 Gln Arg Ser Pro
Ala Arg Leu Leu Val Thr Pro Leu Pro Ala Met Val 20 25 30 Ser Ala
Gly Cys Asp Arg Pro Leu Asp Lys Gly Leu Ser Gly Trp Asp 35 40 45
Gly Ser Ala Leu Thr Met Val Thr Gln Thr Met His Arg Ser Ser Arg 50
55 60 Ala Glu Ser Pro Pro Gln Thr His Pro Arg Ala Gln Pro Thr Trp
Ser 65 70 75 80 Gly Ala Asp Leu Thr Thr Arg Pro Cys Ile Thr Val Arg
Asp Glu Thr 85 90 95 Asn Asn Gly Lys Ala Val Tyr Gln Val Thr Arg
Ile Gly Lys Ala Arg 100 105 110 Leu Leu Gly Pro Gly Asn Pro His Ser
Leu 115 120 29386DNAHomo sapiens 29caggtgcagc tggtgcagtc tggggctgag
gtgaagaagc ctgggtcctc ggtgaaggtc 60tcctgcaagg cttctggagg caccttcagc
agctatgcta tcagctgggt gcgacaggcc 120cctggacaag ggcttgagtg
gatgggaggg atcatcccta tctttggtac agcaaactac 180gcacagaagt
tccagggcag agtcacgatt accgcggacg aatccacgag cacagcctac
240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc
gagatgagac 300gaataacggt aaggcggttt accaggttta aacgcgtatt
gggaaggcgc gtctcttggg 360gccagggaac cctggtcaca gtctcg
38630126PRTHomo sapiens 30Pro Gly Ala Ala Gly Ala Val Trp Gly Gly
Glu Glu Ala Trp Val Leu 1 5 10 15 Gly Glu Gly Leu Leu Gln Gly Phe
Trp Arg His Leu Gln Gln Leu Cys 20 25 30 Tyr Gln Leu Gly Ala Thr
Gly Pro Trp Thr Arg Ala Val Asp Gly Arg 35 40 45 Asp His Pro Tyr
Leu Trp Tyr Ser Lys Leu Arg Thr Glu Val Pro Gly 50 55 60 Gln Ser
His Asp Tyr Arg Gly Arg Ile His Glu His Ser Leu His Gly 65 70 75 80
Ala Glu Gln Pro Glu Ile Gly His Gly Arg Val Leu Leu Cys Glu Met 85
90 95 Arg Arg Ile Thr Val Arg Arg Phe Thr Arg Phe Lys Arg Val Leu
Gly 100 105 110 Arg Arg Val Ser Trp Gly Gln Gly Thr Leu Val Thr Val
Ser 115 120 125 31125PRTHomo sapiens 31Gln Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser
Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser
Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly
Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55
60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Asp Glu Arg Gly Gly Leu Pro Gly Leu Asn
Ala Tyr Trp Glu 100 105 110 Gly Ala Ser Leu Gly Ala Arg Glu Pro Trp
Ser Gln Ser 115 120 125 32123PRTHomo sapiens 32Pro Arg Cys Ser Trp
Cys Ser Leu Gly Leu Arg Arg Ser Leu Gly Pro 1 5 10 15 Arg Arg Ser
Pro Ala Arg Leu Leu Glu Ala Pro Ser Ala Ala Met Leu 20 25 30 Ser
Ala Gly Cys Asp Arg Pro Leu Asp Lys Gly Leu Ser Gly Trp Glu 35 40
45 Gly Ser Ser Leu Ser Val Gln Gln Thr Thr His Arg Ser Ser Arg Ala
50 55 60 Glu Ser Arg Leu Pro Arg Thr Asn Pro Arg Ala Gln Pro Thr
Trp Ser 65 70 75 80 Ala Ala Asp Leu Arg Thr Arg Pro Cys Ile Thr Val
Arg Asp Glu Thr 85 90 95 Asn Asn Gly Lys Ala Val Tyr Gln Val Thr
Arg Ile Gly Lys Ala Arg 100 105 110 Leu Leu Gly Pro Gly Asn Pro Gly
His Ser Leu 115 120 33386DNAHomo sapiens 33gaggtgcagc tgttggagtc
tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
cacctttagc agctatgcca tgagctgggt ccgccaggct 120ccagggaagg
ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac
180gcagactccg tgaagggccg gttcaccatc tccagagaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggccgtat
attactgtgc gagatgagac 300gaataacggt aaggcggttt accaggttta
aacgcgtatt gggaaggcgc gtctcttggg 360gccagggaac cctggtcaca gtctcg
38634126PRTHomo sapiens 34Arg Gly Ala Ala Val Gly Val Trp Gly Arg
Leu Gly Thr Ala Trp Gly 1 5 10 15 Val Pro Glu Thr Leu Leu Cys Ser
Leu Trp Ile His Leu Gln Leu Cys 20 25 30 His Glu Leu Gly Pro Pro
Gly Ser Arg Glu Gly Ala Gly Val Gly Leu 35 40 45 Ser Tyr Trp Trp
Trp His Ile Leu Arg Arg Leu Arg Glu Gly Pro Val 50 55 60 His His
Leu Gln Arg Gln Phe Gln Glu His Ala Val Ser Ser Ala Asn 65 70 75 80
Glu Gln Pro Glu Ser Arg Gly His Gly Arg Ile Leu Leu Cys Glu Met 85
90 95 Arg Arg Ile Thr Val Arg Arg Phe Thr Arg Phe Lys Arg Val Leu
Gly 100 105 110 Arg Arg Val Ser Trp Gly Gln Gly Thr Leu Val Thr Val
Ser 115 120 125 35125PRTHomo sapiens 35Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser
Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55
60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Asp Glu Arg Gly Gly Leu Pro Gly Leu Asn
Ala Tyr Trp Glu 100 105 110 Gly Ala Ser Leu Gly Ala Arg Glu Pro Trp
Ser Gln Ser 115 120 125 36123PRTHomo sapiens 36Pro Arg Cys Ser Cys
Trp Ser Leu Gly Glu Ala Trp Tyr Ser Leu Gly 1 5 10 15 Gly Pro Asp
Ser Pro Val Gln Pro Leu Asp Ser Pro Leu Ala Ala Met 20 25 30 Pro
Ala Gly Ser Ala Arg Leu Gln Gly Arg Gly Trp Ser Gly Ser Gln 35 40
45 Leu Leu Val Val Val Val Val Ala His Thr Thr Gln Thr Pro Arg Ala
50 55 60 Gly Ser Pro Ser Pro Glu Thr Ile Pro Arg Thr Arg Cys Ile
Cys Lys 65 70 75 80 Thr Ala Glu Pro Arg Thr Arg Pro Tyr Ile Thr Val
Arg Asp Glu Thr 85 90 95 Asn Asn Gly Lys Ala Val Tyr Gln Val Thr
Arg Ile Gly Lys Ala Arg 100 105 110 Leu Leu Gly Pro Gly Asn Pro Gly
His Ser Leu 115 120 37385DNAHomo sapiens 37gaggtgcagc tgttggagtc
tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
cacctttagc agctatgcca tgagctgggt ccgccaggct 120ccagggaagg
ggctggagtg ggtctcagct attagtggta gtggtggtag cacatactac
180gcagactccg tgaagggccg gttcacatct ccagagacaa ttccaagaac
acgctgtatc 240tgcaaatgaa cagcctgaga gccgaggaca cggccgtata
ttactgtgcg aaatgagacg 300aataacggta aggcggttta ccaggtttaa
acgcgtattg ggaaggcgcg tctcttgggg 360ccagggaacc ctggtcacag tctcg
38538125PRTHomo sapiens 38Arg Gly Ala Ala Val Gly Val Trp Gly Arg
Leu Gly Thr Ala Trp Gly 1 5 10 15 Val Pro Glu Thr Leu Leu Cys Ser
Leu Trp Ile His Leu Gln Leu Cys 20 25 30 His Glu Leu Gly Pro Pro
Gly Ser Arg Glu Gly Ala Gly Val Gly Leu 35 40 45 Ser Tyr Trp Trp
Trp His Ile Leu Arg Arg Leu Arg Glu Gly Pro Val 50 55 60 His His
Leu Gln Arg Gln Phe Gln Glu His Ala Val Ser Ala Asn Glu 65 70 75 80
Gln Pro Glu Ser Arg Gly His Gly Arg Ile Leu Leu Cys Glu Met Arg 85
90 95 Arg Ile Thr Val Arg Arg Phe Thr Arg Phe Lys Arg Val Leu Gly
Arg 100 105 110 Arg Val Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125 39125PRTHomo sapiens 39Glu Val Gln Leu Leu Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala
Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Lys Asp Glu Arg Gly Gly Leu Pro Gly Leu Asn
Ala Tyr Trp Glu 100 105 110 Gly Ala Ser Leu Gly Ala Arg Glu Pro Trp
Ser Gln Ser 115 120 125 40123PRTHomo sapiens 40Pro Arg Cys Ser Cys
Trp Ser Leu Gly Glu Ala Trp Tyr Ser Leu Gly 1 5 10 15 Gly Pro Asp
Ser Pro Val Gln Pro Leu Asp Ser Pro Leu Ala Ala Met 20 25 30 Pro
Ala Gly Ser Ala Arg Leu Gln Gly Arg Gly Trp Ser Gly Ser Gln 35 40
45 Leu Leu Val Val Val Val Val Ala His Thr Thr Gln Thr Pro Arg Ala
50 55 60 Gly Ser Pro Ser Pro Glu Thr Ile Pro Arg Thr Arg Cys Ile
Cys Lys 65 70 75 80 Thr Ala Glu Pro Arg Thr Arg Pro Tyr Ile Thr Val
Arg Asn Glu Thr 85 90 95 Asn Asn Gly Lys Ala Val Tyr Gln Val Thr
Arg Ile Gly Lys Ala Arg 100 105 110 Leu Leu Gly Pro Gly Asn Pro Gly
His Ser Leu 115 120 41386DNAHomo sapiens 41caggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cctctggatt
caccttcagt agctatgcta tgcactgggt ccgccaggct 120ccaggcaagg
ggctggagtg ggtggcagtt atatcatatg atggaagtaa taaatactac
180gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgag agctgaggac acggctgtgt
attactgtgc gagatgagac 300gaataacggt aaggcggttt accaggttta
aacgcgtatt gggaaggcgc gtctcttggg 360gccagggaac cctggtcaca gtctcg
38642124PRTHomo sapiens 42Pro Gly Ala Ala Gly Gly Val Trp Gly Arg
Arg Gly Pro Ala Trp Glu 1 5 10 15 Val Pro Glu Thr Leu Leu Cys Ser
Leu Trp Ile His Leu Gln Leu Cys 20 25 30 Tyr Ala Leu Gly Pro Pro
Gly Ser Arg Gln Gly Ala Gly Val Gly Gly 35 40 45 Ser Tyr Ile Ile
Trp Lys Ile Leu Arg Arg Leu Arg Glu Gly Pro Ile 50 55 60 His His
Leu Gln Arg Gln Phe Gln Glu His Ala Val Ser Ala Asn Glu 65 70 75 80
Gln Pro Glu Ser Gly His Gly Cys Val Leu Leu Cys Glu Met Arg Arg 85
90 95 Ile Thr Val Arg Arg Phe Thr Arg Phe Lys Arg Val Leu Gly Arg
Arg 100 105 110 Val Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115
120 43125PRTHomo sapiens 43Gln Val Gln Leu Val Glu Ser Gly Gly Gly
Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Asp Glu Arg Gly Gly Leu Pro Gly Leu Asn Ala Tyr Trp
Glu 100 105 110 Gly Ala Ser Leu Gly Ala Arg Glu Pro Trp Ser Gln Ser
115 120 125 44124PRTHomo sapiens 44Pro Arg Cys Ser Trp Trp Ser Leu
Gly Glu Ala Trp Ser Ser Leu Gly 1 5 10 15 Gly Pro Asp Ser Pro Val
Gln Pro Leu Asp Ser Pro Ser Val Ala Met 20 25 30 Leu Cys Thr Gly
Ser Ala Arg Leu Gln Ala Arg Gly Trp Ser Gly Trp 35 40 45 Gln Leu
Tyr His Met Met Glu Val Ile Asn Thr Thr Gln Thr Pro Arg 50 55 60
Ala Asp Ser Pro Ser Pro Glu Thr Ile Pro Arg Thr Arg Cys Ile Cys 65
70 75 80 Lys Thr Ala Glu Leu Arg Thr Arg Leu Cys Ile Thr Val Arg
Asp Glu 85 90 95 Thr Asn Asn Gly Lys Ala Val Tyr Gln Val Thr Arg
Ile Gly Lys Ala 100 105 110 Arg Leu Leu Gly Pro Gly Asn Pro Gly His
Ser Leu 115 120 45386DNAHomo sapiens 45gaggtgcagc tggtggagtc
tgggggaggc ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt
caccttcagt agctatagca tgaactgggt ccgccaggct 120ccagggaagg
ggctggagtg ggtttcatac attagtagta gtagtagtac catatactac
180gcagactctg tgaagggccg attcaccatc tccagagaca atgccaagaa
ctcactgtat 240ctgcaaatga acagcctgag agccgaggac acggctgtgt
attactgtgc gagatgagac 300gaataacggt aaggcggttt accaggttta
aacgcgtatt gggaaggcgc gtctcttggg 360gccagggaac cctggtcaca gtctcg
38646122PRTHomo sapiens 46Arg Gly Ala Ala Gly Gly Val Trp Gly Arg
Leu Gly Thr Ala Trp Gly 1 5 10 15 Val Pro Glu Thr Leu Leu Cys Ser
Leu Trp Ile His Leu Gln Leu His 20 25 30 Glu Leu Gly Pro Pro Gly
Ser Arg Glu Gly Ala Gly Val Gly Phe Ile 35 40 45 His Tyr His Ile
Leu Arg Arg Leu Cys Glu Gly Pro Ile His His Leu 50 55 60 Gln Arg
Gln Cys Gln Glu Leu Thr Val Ser Ala Asn Glu Gln Pro Glu 65 70 75 80
Ser Arg Gly His Gly Cys Val Leu Leu Cys Glu Met Arg Arg Ile Thr 85
90 95 Val Arg Arg Phe Thr Arg Phe Lys Arg Val Leu Gly Arg Arg Val
Ser 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120
47125PRTHomo sapiens 47Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ser Met Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser
Ser Ser Ser Thr Ile Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Asp Glu Arg Gly Gly Leu Pro Gly Leu Asn Ala Tyr Trp Glu
100 105 110 Gly Ala Ser Leu Gly Ala Arg Glu Pro Trp Ser Gln Ser 115
120 125 48123PRTHomo sapiens 48Pro Arg Cys Ser Trp Trp Ser Leu Gly
Glu Ala Trp
Tyr Ser Leu Gly 1 5 10 15 Gly Pro Asp Ser Pro Val Gln Pro Leu Asp
Ser Pro Ser Val Ala Ile 20 25 30 Ala Thr Gly Ser Ala Arg Leu Gln
Gly Arg Gly Trp Ser Gly Phe His 35 40 45 Thr Leu Val Val Val Val
Val Pro Tyr Thr Thr Gln Thr Leu Arg Ala 50 55 60 Asp Ser Pro Ser
Pro Glu Thr Met Pro Arg Thr His Cys Ile Cys Lys 65 70 75 80 Thr Ala
Glu Pro Arg Thr Arg Leu Cys Ile Thr Val Arg Asp Glu Thr 85 90 95
Asn Asn Gly Lys Ala Val Tyr Gln Val Thr Arg Ile Gly Lys Ala Arg 100
105 110 Leu Leu Gly Pro Gly Asn Pro Gly His Ser Leu 115 120
49290DNAHomo sapiens 49gaggtgcagc tggtgcagtc tggagcagag gtgaaaaagc
ccggggagtc tctgaagatc 60tcctgtaagg gttctggata cagctttacc agctactgga
tcggctgggt gcgccagatg 120cccgggaaag gcctggagtg gatggggatc
atctatcctg gtgactctga taccagatac 180agcccgtcct tccaaggcca
ggtcaccatc tcagccgaca agtccatcag caccgcctac 240ctgcagtgga
gcagcctgaa ggcctcggac accgccatgt attactgtgc 29050126PRTHomo sapiens
50Arg Gly Ala Ala Gly Ala Val Trp Ser Arg Gly Glu Lys Ala Arg Gly 1
5 10 15 Val Ser Glu Asp Leu Leu Gly Phe Trp Ile Gln Leu Tyr Gln Leu
Leu 20 25 30 Asp Arg Leu Gly Ala Pro Asp Ala Arg Glu Arg Pro Gly
Val Asp Gly 35 40 45 Asp His Leu Ser Trp Leu Tyr Gln Ile Gln Pro
Val Leu Pro Arg Pro 50 55 60 Gly His His Leu Ser Arg Gln Val His
Gln His Arg Leu Pro Ala Val 65 70 75 80 Glu Gln Pro Glu Gly Leu Gly
His Arg His Val Leu Leu Cys Glu Met 85 90 95 Arg Arg Ile Thr Val
Arg Arg Phe Thr Arg Phe Lys Arg Val Leu Gly 100 105 110 Arg Arg Val
Ser Trp Gly Gln Gly Thr Leu Val Thr Val Ser 115 120 125
51125PRTHomo sapiens 51Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser
Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln
Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro
Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln
Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu
Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90
95 Ala Arg Asp Glu Arg Gly Gly Leu Pro Gly Leu Asn Ala Tyr Trp Glu
100 105 110 Gly Ala Ser Leu Gly Ala Arg Glu Pro Trp Ser Gln Ser 115
120 125 52125PRTHomo sapiens 52Pro Arg Cys Ser Trp Cys Ser Leu Glu
Gln Arg Lys Ser Pro Gly Ser 1 5 10 15 Leu Arg Ser Pro Val Arg Val
Leu Asp Thr Ala Leu Pro Ala Thr Gly 20 25 30 Ser Ala Gly Cys Ala
Arg Cys Pro Gly Lys Ala Trp Ser Gly Trp Gly 35 40 45 Ser Ser Ile
Leu Val Thr Leu Ile Pro Asp Thr Ala Arg Pro Ser Lys 50 55 60 Ala
Arg Ser Pro Ser Gln Pro Thr Ser Pro Ser Ala Pro Pro Thr Cys 65 70
75 80 Ser Gly Ala Ala Arg Pro Arg Thr Pro Pro Cys Ile Thr Val Arg
Asp 85 90 95 Glu Thr Asn Asn Gly Lys Ala Val Tyr Gln Val Thr Arg
Ile Gly Lys 100 105 110 Ala Arg Leu Leu Gly Pro Gly Asn Pro Gly His
Ser Leu 115 120 125 53374DNAHomo sapiens 53gacatccaga tgacccagtc
tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc aggcgagtca
ggacattagc aactatttaa attggtatca gcagaaacca 120gggaaagccc
ctaagctcct gatctacgat gcatccaatt tggaaacagg ggtcccatca
180aggttcagtg gaagtggatc tgggacagat tttactttca ccatcagcag
cctgcagcct 240gaagatattg caacatatta ctgtcagcag cgagacgaat
aacggtaagg cggtttacca 300ggtttaaacg cgtattggga aggcgcgtct
cattcggcca agggaccaag gtggaaatca 360aaggggcggc cgca 37454122PRTHomo
sapiens 54Gly His Pro Asp Asp Pro Val Ser Ile Leu Pro Val Cys Ile
Cys Arg 1 5 10 15 Arg Gln Ser His His His Leu Pro Gly Glu Ser Gly
His Gln Leu Phe 20 25 30 Lys Leu Val Ser Ala Glu Thr Arg Glu Ser
Pro Ala Pro Asp Leu Arg 35 40 45 Cys Ile Gln Phe Gly Asn Arg Gly
Pro Ile Lys Val Gln Trp Lys Trp 50 55 60 Ile Trp Asp Arg Phe Tyr
Phe His His Gln Gln Pro Ala Ala Arg Tyr 65 70 75 80 Cys Asn Ile Leu
Leu Ser Ala Ala Arg Arg Ile Thr Val Arg Arg Phe 85 90 95 Thr Arg
Phe Lys Arg Val Leu Gly Arg Arg Val Ser Phe Gly Gln Gly 100 105 110
Thr Lys Val Glu Ile Lys Gly Ala Ala Ala 115 120 55122PRTHomo
sapiens 55Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp
Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala
Thr Tyr Tyr Cys Gln Gln Arg Asp Glu Arg Gly Gly 85 90 95 Leu Pro
Gly Leu Asn Ala Tyr Trp Glu Gly Ala Ser His Ser Ala Lys 100 105 110
Gly Pro Arg Trp Lys Ser Lys Gly Arg Pro 115 120 56120PRTHomo
sapiens 56Arg Thr Ser Arg Pro Ser Leu His Pro Pro Cys Leu His Leu
Glu Thr 1 5 10 15 Glu Ser Pro Ser Leu Ala Arg Arg Val Arg Thr Leu
Ala Thr Ile Ile 20 25 30 Gly Ile Ser Arg Asn Gln Gly Lys Pro Leu
Ser Ser Ser Thr Met His 35 40 45 Pro Ile Trp Lys Gln Gly Ser His
Gln Gly Ser Val Glu Val Asp Leu 50 55 60 Gly Gln Ile Leu Leu Ser
Pro Ser Ala Ala Cys Ser Leu Lys Ile Leu 65 70 75 80 Gln His Ile Thr
Val Ser Ser Glu Thr Asn Asn Gly Lys Ala Val Tyr 85 90 95 Gln Val
Thr Arg Ile Gly Lys Ala Arg Leu Ile Arg Pro Arg Asp Gln 100 105 110
Gly Gly Asn Gln Arg Gly Gly Arg 115 120 57374DNAHomo sapiens
57gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc
60atcacttgcc aggcgagtca ggacattagc aactatttaa attggtatca gcagaaacca
120gggaaagccc ctaagctcct gatctacgat gcatccaatt tggaaacagg
ggtcccatca 180aggttcagtg gaagtggatc tgggacagat tttactttca
ccatcagcag cctgcagcct 240gaagatattg caacatatta ctgtcagcag
cgagacgaat aacggtaagg cggtttacca 300ggtttaaacg cgtattggga
aggcgcgtct cattcggcca agggaccaag gtggaaatca 360aacgggcggc cgca
37458122PRTHomo sapiens 58Gly His Pro Asp Asp Pro Val Ser Ile Leu
Pro Val Cys Ile Cys Arg 1 5 10 15 Arg Gln Ser His His His Leu Pro
Gly Glu Ser Gly His Gln Leu Phe 20 25 30 Lys Leu Val Ser Ala Glu
Thr Arg Glu Ser Pro Ala Pro Asp Leu Arg 35 40 45 Cys Ile Gln Phe
Gly Asn Arg Gly Pro Ile Lys Val Gln Trp Lys Trp 50 55 60 Ile Trp
Asp Arg Phe Tyr Phe His His Gln Gln Pro Ala Ala Arg Tyr 65 70 75 80
Cys Asn Ile Leu Leu Ser Ala Ala Arg Arg Ile Thr Val Arg Arg Phe 85
90 95 Thr Arg Phe Lys Arg Val Leu Gly Arg Arg Val Ser Phe Gly Gln
Gly 100 105 110 Thr Lys Val Glu Ile Lys Arg Ala Ala Ala 115 120
59122PRTHomo sapiens 59Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala
Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn
Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Arg Asp Glu Arg Gly Gly 85 90
95 Leu Pro Gly Leu Asn Ala Tyr Trp Glu Gly Ala Ser His Ser Ala Lys
100 105 110 Gly Pro Arg Trp Lys Ser Asn Gly Arg Pro 115 120
60120PRTHomo sapiens 60Arg Thr Ser Arg Pro Ser Leu His Pro Pro Cys
Leu His Leu Glu Thr 1 5 10 15 Glu Ser Pro Ser Leu Ala Arg Arg Val
Arg Thr Leu Ala Thr Ile Ile 20 25 30 Gly Ile Ser Arg Asn Gln Gly
Lys Pro Leu Ser Ser Ser Thr Met His 35 40 45 Pro Ile Trp Lys Gln
Gly Ser His Gln Gly Ser Val Glu Val Asp Leu 50 55 60 Gly Gln Ile
Leu Leu Ser Pro Ser Ala Ala Cys Ser Leu Lys Ile Leu 65 70 75 80 Gln
His Ile Thr Val Ser Ser Glu Thr Asn Asn Gly Lys Ala Val Tyr 85 90
95 Gln Val Thr Arg Ile Gly Lys Ala Arg Leu Ile Arg Pro Arg Asp Gln
100 105 110 Gly Gly Asn Gln Thr Gly Gly Arg 115 120 61374DNAHomo
sapiens 61gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga
cagagtcacc 60atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca
gcagaaacca 120gggaaagccc ctaagctcct gatctatgct gcatccagtt
tgcaaagtgg ggtcccatca 180aggttcagtg gcagtggatc tgggacagat
ttcactctca ccatcagcag tctgcaacct 240gaagattttg caacttacta
ctgtcagcag cgagacgaat aacggtaagg cggtttacca 300ggtttaaacg
cgtattggga aggcgcgtct cattcggcca agggaccaag gtggaaatca
360aaggggcggc cgca 37462122PRTHomo sapiens 62Gly His Pro Asp Asp
Pro Val Ser Ile Leu Pro Val Cys Ile Cys Arg 1 5 10 15 Arg Gln Ser
His His His Leu Pro Gly Lys Ser Glu His Gln Leu Phe 20 25 30 Lys
Leu Val Ser Ala Glu Thr Arg Glu Ser Pro Ala Pro Asp Leu Cys 35 40
45 Cys Ile Gln Phe Ala Lys Trp Gly Pro Ile Lys Val Gln Trp Gln Trp
50 55 60 Ile Trp Asp Arg Phe His Ser His His Gln Gln Ser Ala Thr
Arg Phe 65 70 75 80 Cys Asn Leu Leu Leu Ser Ala Ala Arg Arg Ile Thr
Val Arg Arg Phe 85 90 95 Thr Arg Phe Lys Arg Val Leu Gly Arg Arg
Val Ser Phe Gly Gln Gly 100 105 110 Thr Lys Val Glu Ile Lys Gly Ala
Ala Ala 115 120 63123PRTHomo sapiens 63Asp Ile Gln Met Thr Gln Ser
Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile
Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Ser 20 25 30 Tyr Leu Asn
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55
60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Asp Glu
Arg Gly 85 90 95 Gly Leu Pro Gly Leu Asn Ala Tyr Trp Glu Gly Ala
Ser His Ser Ala 100 105 110 Lys Gly Pro Arg Trp Lys Ser Lys Gly Arg
Pro 115 120 64120PRTHomo sapiens 64Arg Thr Ser Arg Pro Ser Leu His
Pro Pro Cys Leu His Leu Glu Thr 1 5 10 15 Glu Ser Pro Ser Leu Ala
Gly Gln Val Arg Ala Leu Ala Ala Ile Ile 20 25 30 Gly Ile Ser Arg
Asn Gln Gly Lys Pro Leu Ser Ser Ser Met Leu His 35 40 45 Pro Val
Cys Lys Val Gly Ser His Gln Gly Ser Val Ala Val Asp Leu 50 55 60
Gly Gln Ile Ser Leu Ser Pro Ser Ala Val Cys Asn Leu Lys Ile Leu 65
70 75 80 Gln Leu Thr Thr Val Ser Ser Glu Thr Asn Asn Gly Lys Ala
Val Tyr 85 90 95 Gln Val Thr Arg Ile Gly Lys Ala Arg Leu Ile Arg
Pro Arg Asp Gln 100 105 110 Gly Gly Asn Gln Arg Gly Gly Arg 115 120
65374DNAHomo sapiens 65gacatccaga tgacccagtc tccatcctcc ctgtctgcat
ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gagcattagc agctatttaa
attggtatca gcagaaacca 120gggaaagccc ctaagctcct gatctatgct
gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagtg gcagtggatc
tgggacagat ttcactctca ccatcagcag tctgcaacct 240gaagattttg
caacttacta ctgtcagcag cgagacgaat aacggtaagg cggtttacca
300ggtttaaacg cgtattggga aggcgcgtct cattcggcca agggaccaag
gtggaaatca 360aacgggcggc cgca 37466122PRTHomo sapiens 66Gly His Pro
Asp Asp Pro Val Ser Ile Leu Pro Val Cys Ile Cys Arg 1 5 10 15 Arg
Gln Ser His His His Leu Pro Gly Lys Ser Glu His Gln Leu Phe 20 25
30 Lys Leu Val Ser Ala Glu Thr Arg Glu Ser Pro Ala Pro Asp Leu Cys
35 40 45 Cys Ile Gln Phe Ala Lys Trp Gly Pro Ile Lys Val Gln Trp
Gln Trp 50 55 60 Ile Trp Asp Arg Phe His Ser His His Gln Gln Ser
Ala Thr Arg Phe 65 70 75 80 Cys Asn Leu Leu Leu Ser Ala Ala Arg Arg
Ile Thr Val Arg Arg Phe 85 90 95 Thr Arg Phe Lys Arg Val Leu Gly
Arg Arg Val Ser Phe Gly Gln Gly 100 105 110 Thr Lys Val Glu Ile Lys
Arg Ala Ala Ala 115 120 67122PRTHomo sapiens 67Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu
Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Asp
Glu Arg Gly Gly 85 90 95 Leu Pro Gly Leu Asn Ala Tyr Trp Glu Gly
Ala Ser His Ser Ala Lys 100 105 110 Gly Pro Arg Trp Lys Ser Asn Gly
Arg Pro 115 120 68120PRTHomo sapiens 68Arg Thr Ser Arg Pro Ser Leu
His Pro Pro Cys Leu His Leu Glu Thr 1 5 10 15 Glu Ser Pro Ser Leu
Ala Gly Gln Val Arg Ala Leu Ala Ala Ile Ile 20 25 30 Gly Ile Ser
Arg Asn Gln Gly Lys Pro Leu Ser Ser Ser Met Leu His 35 40 45 Pro
Val Cys Lys Val Gly Ser His Gln Gly Ser Val Ala Val Asp Leu 50 55
60 Gly Gln Ile Ser Leu Ser Pro Ser Ala Val Cys Asn Leu Lys Ile Leu
65 70 75 80 Gln Leu Thr Thr Val Ser Ser Glu Thr Asn Asn Gly Lys Ala
Val Tyr 85 90 95 Gln Val Thr Arg Ile Gly Lys Ala Arg Leu Ile Arg
Pro Arg Asp Gln 100 105
110 Gly Gly Asn Gln Thr Gly Gly Arg 115 120 69374DNAHomo sapiens
69gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc
60ctctcctgca gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct
120ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg
catcccagcc 180aggttcagtg gcagtgggtc tgggacagac ttcactctca
ccatcagcag cctagagcct 240gaagattttg cagtttatta ctgtcagcag
cgagacgaat aacggtaagg cggtttacca 300ggtttaaacg cgtattggga
aggcgcgtct cattcggcca agggaccaag gtggaaatca 360aaggggcggc cgca
37470122PRTHomo sapiens 70Gly Asn Cys Val Asp Thr Val Ser Ser His
Pro Val Phe Val Ser Arg 1 5 10 15 Gly Lys Ser His Pro Leu Leu Gln
Gly Gln Ser Glu Cys Gln Leu Leu 20 25 30 Ser Leu Val Pro Thr Glu
Thr Trp Pro Gly Ser Gln Ala Pro His Leu 35 40 45 Cys Ile Gln Gln
Gly His Trp His Pro Ser Gln Val Gln Trp Gln Trp 50 55 60 Val Trp
Asp Arg Leu His Ser His His Gln Gln Pro Arg Ala Arg Phe 65 70 75 80
Cys Ser Leu Leu Leu Ser Ala Ala Arg Arg Ile Thr Val Arg Arg Phe 85
90 95 Thr Arg Phe Lys Arg Val Leu Gly Arg Arg Val Ser Phe Gly Gln
Gly 100 105 110 Thr Lys Val Glu Ile Lys Gly Ala Ala Ala 115 120
71122PRTHomo sapiens 71Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu
Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn
Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu
Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Asp Glu Arg Gly Gly 85 90
95 Leu Pro Gly Leu Asn Ala Tyr Trp Glu Gly Ala Ser His Ser Ala Lys
100 105 110 Gly Pro Arg Trp Lys Ser Lys Gly Arg Pro 115 120
72121PRTHomo sapiens 72Arg Lys Leu Cys His Ser Leu Gln Pro Pro Cys
Leu Cys Leu Gln Gly 1 5 10 15 Lys Glu Pro Pro Ser Pro Ala Gly Pro
Val Arg Val Leu Ala Ala Thr 20 25 30 Pro Gly Thr Asn Arg Asn Leu
Ala Arg Leu Pro Gly Ser Ser Ser Met 35 40 45 Met His Pro Thr Gly
Pro Leu Ala Ser Gln Pro Gly Ser Val Ala Val 50 55 60 Gly Leu Gly
Gln Thr Ser Leu Ser Pro Ser Ala Ala Ser Leu Lys Ile 65 70 75 80 Leu
Gln Phe Ile Thr Val Ser Ser Glu Thr Asn Asn Gly Lys Ala Val 85 90
95 Tyr Gln Val Thr Arg Ile Gly Lys Ala Arg Leu Ile Arg Pro Arg Asp
100 105 110 Gln Gly Gly Asn Gln Arg Gly Gly Arg 115 120
73374DNAHomo sapiens 73gaaattgtgt tgacacagtc tccagccacc ctgtctttgt
ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc agctacttag
cctggtacca acagaaacct 120ggccaggctc ccaggctcct catctatgat
gcatccaaca gggccactgg catcccagcc 180aggttcagtg gcagtgggtc
tgggacagac ttcactctca ccatcagcag cctagagcct 240gaagattttg
cagtttatta ctgtcagcag cgagacgaat aacggtaagg cggtttacca
300ggtttaaacg cgtattggga aggcgcgtct cattcggcca agggaccaag
gtggaaatca 360aacgggcggc cgca 37474122PRTHomo sapiens 74Gly Asn Cys
Val Asp Thr Val Ser Ser His Pro Val Phe Val Ser Arg 1 5 10 15 Gly
Lys Ser His Pro Leu Leu Gln Gly Gln Ser Glu Cys Gln Leu Leu 20 25
30 Ser Leu Val Pro Thr Glu Thr Trp Pro Gly Ser Gln Ala Pro His Leu
35 40 45 Cys Ile Gln Gln Gly His Trp His Pro Ser Gln Val Gln Trp
Gln Trp 50 55 60 Val Trp Asp Arg Leu His Ser His His Gln Gln Pro
Arg Ala Arg Phe 65 70 75 80 Cys Ser Leu Leu Leu Ser Ala Ala Arg Arg
Ile Thr Val Arg Arg Phe 85 90 95 Thr Arg Phe Lys Arg Val Leu Gly
Arg Arg Val Ser Phe Gly Gln Gly 100 105 110 Thr Lys Val Glu Ile Lys
Arg Ala Ala Ala 115 120 75122PRTHomo sapiens 75Glu Ile Val Leu Thr
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala
Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40
45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Asp
Glu Arg Gly Gly 85 90 95 Leu Pro Gly Leu Asn Ala Tyr Trp Glu Gly
Ala Ser His Ser Ala Lys 100 105 110 Gly Pro Arg Trp Lys Ser Asn Gly
Arg Pro 115 120 76121PRTHomo sapiens 76Arg Lys Leu Cys His Ser Leu
Gln Pro Pro Cys Leu Cys Leu Gln Gly 1 5 10 15 Lys Glu Pro Pro Ser
Pro Ala Gly Pro Val Arg Val Leu Ala Ala Thr 20 25 30 Pro Gly Thr
Asn Arg Asn Leu Ala Arg Leu Pro Gly Ser Ser Ser Met 35 40 45 Met
His Pro Thr Gly Pro Leu Ala Ser Gln Pro Gly Ser Val Ala Val 50 55
60 Gly Leu Gly Gln Thr Ser Leu Ser Pro Ser Ala Ala Ser Leu Lys Ile
65 70 75 80 Leu Gln Phe Ile Thr Val Ser Ser Glu Thr Asn Asn Gly Lys
Ala Val 85 90 95 Tyr Gln Val Thr Arg Ile Gly Lys Ala Arg Leu Ile
Arg Pro Arg Asp 100 105 110 Gln Gly Gly Asn Gln Thr Gly Gly Arg 115
120 77374DNAHomo sapiens 77gaaatagtga tgacgcagtc tccagccacc
ctgtctgtgt ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc
agcaacttag cctggtacca gcagaaacct 120ggccaggctc ccaggctcct
catctatggt gcatccacca gggccactgg tatcccagcc 180aggttcagtg
gcagtgggtc tgggacagag ttcactctca ccatcagcag cctgcagtct
240gaagattttg cagtttatta ctgtcagcag cgagacgaat aacggtaagg
cggtttacca 300ggtttaaacg cgtattggga aggcgcgtct cattcggcca
agggaccaag gtggaaatca 360aaggggcggc cgca 37478123PRTHomo sapiens
78Gly Asn Ser Asp Asp Ala Val Ser Ser His Pro Val Cys Val Ser Arg 1
5 10 15 Gly Lys Ser His Pro Leu Leu Gln Gly Gln Ser Glu Cys Gln Gln
Leu 20 25 30 Ser Leu Val Pro Ala Glu Thr Trp Pro Gly Ser Gln Ala
Pro His Leu 35 40 45 Trp Cys Ile His Gln Gly His Trp Tyr Pro Ser
Gln Val Gln Trp Gln 50 55 60 Trp Val Trp Asp Arg Val His Ser His
His Gln Gln Pro Ala Val Arg 65 70 75 80 Phe Cys Ser Leu Leu Leu Ser
Ala Ala Arg Arg Ile Thr Val Arg Arg 85 90 95 Phe Thr Arg Phe Lys
Arg Val Leu Gly Arg Arg Val Ser Phe Gly Gln 100 105 110 Gly Thr Lys
Val Glu Ile Lys Gly Ala Ala Ala 115 120 79122PRTHomo sapiens 79Glu
Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Asn
20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr
Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Arg Asp Glu Arg Gly Gly 85 90 95 Leu Pro Gly Leu Asn Ala
Tyr Trp Glu Gly Ala Ser His Ser Ala Lys 100 105 110 Gly Pro Arg Trp
Lys Ser Lys Gly Arg Pro 115 120 80120PRTHomo sapiens 80Arg Lys Arg
Ser Leu Gln Pro Pro Cys Leu Cys Leu Gln Gly Lys Glu 1 5 10 15 Pro
Pro Ser Pro Ala Gly Pro Val Arg Val Leu Ala Ala Thr Pro Gly 20 25
30 Thr Ser Arg Asn Leu Ala Arg Leu Pro Gly Ser Ser Ser Met Val His
35 40 45 Pro Pro Gly Pro Leu Val Ser Gln Pro Gly Ser Val Ala Val
Gly Leu 50 55 60 Gly Gln Ser Ser Leu Ser Pro Ser Ala Ala Cys Ser
Leu Lys Ile Leu 65 70 75 80 Gln Phe Ile Thr Val Ser Ser Glu Thr Asn
Asn Gly Lys Ala Val Tyr 85 90 95 Gln Val Thr Arg Ile Gly Lys Ala
Arg Leu Ile Arg Pro Arg Asp Gln 100 105 110 Gly Gly Asn Gln Arg Gly
Gly Arg 115 120 81374DNAHomo sapiens 81gaaatagtga tgacgcagtc
tccagccacc ctgtctgtgt ctccagggga aagagccacc 60ctctcctgca gggccagtca
gagtgttagc agcaacttag cctggtacca gcagaaacct 120ggccaggctc
ccaggctcct catctatggt gcatccacca gggccactgg tatcccagcc
180aggttcagtg gcagtgggtc tgggacagag ttcactctca ccatcagcag
cctgcagtct 240gaagattttg cagtttatta ctgtcagcag cgagacgaat
aacggtaagg cggtttacca 300ggtttaaacg cgtattggga aggcgcgtct
cattcggcca agggaccaag gtggaaatca 360aacgggcggc cgca 37482123PRTHomo
sapiens 82Gly Asn Ser Asp Asp Ala Val Ser Ser His Pro Val Cys Val
Ser Arg 1 5 10 15 Gly Lys Ser His Pro Leu Leu Gln Gly Gln Ser Glu
Cys Gln Gln Leu 20 25 30 Ser Leu Val Pro Ala Glu Thr Trp Pro Gly
Ser Gln Ala Pro His Leu 35 40 45 Trp Cys Ile His Gln Gly His Trp
Tyr Pro Ser Gln Val Gln Trp Gln 50 55 60 Trp Val Trp Asp Arg Val
His Ser His His Gln Gln Pro Ala Val Arg 65 70 75 80 Phe Cys Ser Leu
Leu Leu Ser Ala Ala Arg Arg Ile Thr Val Arg Arg 85 90 95 Phe Thr
Arg Phe Lys Arg Val Leu Gly Arg Arg Val Ser Phe Gly Gln 100 105 110
Gly Thr Lys Val Glu Ile Lys Arg Ala Ala Ala 115 120 83122PRTHomo
sapiens 83Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser
Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser
Val Ser Ser Asn 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Gly Ala Ser Thr Arg Ala Thr
Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu
Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser 65 70 75 80 Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Arg Asp Glu Arg Gly Gly 85 90 95 Leu Pro
Gly Leu Asn Ala Tyr Trp Glu Gly Ala Ser His Ser Ala Lys 100 105 110
Gly Pro Arg Trp Lys Ser Asn Gly Arg Pro 115 120 84119PRTHomo
sapiens 84Arg Lys Arg Ser Leu Gln Pro Pro Cys Leu Cys Leu Gln Gly
Lys Glu 1 5 10 15 Pro Pro Ser Pro Ala Gly Pro Val Arg Val Leu Ala
Ala Thr Pro Gly 20 25 30 Thr Ser Arg Asn Leu Ala Arg Leu Pro Gly
Ser Ser Ser Met Val His 35 40 45 Pro Pro Gly Pro Leu Val Ser Gln
Pro Gly Ser Val Ala Val Gly Leu 50 55 60 Gly Gln Ser Ser Leu Ser
Pro Ser Ala Ala Cys Ser Leu Lys Ile Leu 65 70 75 80 Gln Phe Ile Thr
Val Ser Ser Glu Thr Asn Asn Gly Lys Ala Val Tyr 85 90 95 Gln Val
Thr Arg Ile Gly Lys Ala Arg Leu Ile Arg Pro Asp Gln Gly 100 105 110
Gly Asn Gln Thr Gly Gly Arg 115 85377DNAHomo sapiens 85gaaattgtgt
tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca
gggccagtca gagtgttagc agcagctact tagcctggta ccagcagaaa
120cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac
tggcatccca 180gacaggttca gtggcagtgg gtctgggaca gacttcactc
tcaccatcag cagactggag 240cctgaagatt ttgcagttta ttactgtcag
cagcgagacg aataacggta aggcggttta 300ccaggtttaa acgcgtattg
ggaaggcgcg tctcattcgg ccaagggacc aaggtggaaa 360tcaaaggggc ggccgca
37786124PRTHomo sapiens 86Gly Asn Cys Val Asp Ala Val Ser Arg His
Pro Val Phe Val Ser Arg 1 5 10 15 Gly Lys Ser His Pro Leu Ile Gln
Gly Gln Ser Glu Cys Gln Gln Leu 20 25 30 Leu Ser Leu Val Pro Ala
Glu Thr Trp Pro Gly Ser Gln Ala Pro His 35 40 45 Leu Trp Cys Ile
Gln Gln Gly His Trp His Pro Arg Gln Val Gln Trp 50 55 60 Gln Trp
Val Trp Asp Arg Leu His Ser His His Gln Gln Thr Gly Ala 65 70 75 80
Arg Phe Cys Ser Leu Leu Leu Ser Ala Ala Arg Arg Ile Thr Val Arg 85
90 95 Arg Phe Thr Arg Phe Lys Arg Val Leu Gly Arg Arg Val Ser Phe
Gly 100 105 110 Gln Gly Thr Lys Val Glu Ile Lys Gly Ala Ala Ala 115
120 87123PRTHomo sapiens 87Glu Ile Val Leu Thr Gln Ser Pro Gly Thr
Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg
Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala
Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Asp Glu Arg Gly 85
90 95 Gly Leu Pro Gly Leu Asn Ala Tyr Trp Glu Gly Ala Ser His Ser
Ala 100 105 110 Lys Gly Pro Arg Trp Lys Ser Lys Gly Arg Pro 115 120
88123PRTHomo sapiens 88Arg Lys Leu Cys Arg Ser Leu Gln Ala Pro Cys
Leu Cys Leu Gln Gly 1 5 10 15 Lys Glu Pro Pro Ser Pro Ala Gly Pro
Val Arg Val Leu Ala Ala Ala 20 25 30 Thr Pro Gly Thr Ser Arg Asn
Leu Ala Arg Leu Pro Gly Ser Ser Ser 35 40 45 Met Val His Pro Ala
Gly Pro Leu Ala Ser Gln Thr Gly Ser Val Ala 50 55 60 Val Gly Leu
Gly Gln Thr Ser Leu Ser Pro Ser Ala Asp Trp Ser Leu 65 70 75 80 Lys
Ile Leu Gln Phe Ile Thr Val Ser Ser Glu Thr Asn Asn Gly Lys 85 90
95 Ala Val Tyr Gln Val Thr Arg Ile Gly Lys Ala Arg Leu Ile Arg Pro
100 105 110 Arg Asp Gln Gly Gly Asn Gln Arg Gly Gly Arg 115 120
89377DNAHomo sapiens 89gaaattgtgt tgacgcagtc tccaggcacc ctgtctttgt
ctccagggga aagagccacc 60ctctcctgca gggccagtca gagtgttagc agcagctact
tagcctggta ccagcagaaa 120cctggccagg ctcccaggct cctcatctat
ggtgcatcca gcagggccac tggcatccca 180gacaggttca gtggcagtgg
gtctgggaca gacttcactc tcaccatcag cagactggag 240cctgaagatt
ttgcagttta ttactgtcag cagcgagacg aataacggta aggcggttta
300ccaggtttaa acgcgtattg ggaaggcgcg tctcattcgg ccaagggacc
aaggtggaaa 360tcaaacgggc ggccgca 37790124PRTHomo sapiens 90Gly Asn
Cys Val Asp Ala Val Ser Arg His Pro Val Phe Val Ser Arg 1 5
10 15 Gly Lys Ser His Pro Leu Leu Gln Gly Gln Ser Glu Cys Gln Gln
Leu 20 25 30 Leu Ser Leu Val Pro Ala Glu Thr Trp Pro Gly Ser Gln
Ala Pro His 35 40 45 Leu Trp Cys Ile Gln Gln Gly His Trp His Pro
Arg Gln Val Gln Trp 50 55 60 Gln Trp Val Trp Asp Arg Leu His Ser
His His Gln Gln Thr Gly Ala 65 70 75 80 Arg Phe Cys Ser Leu Leu Leu
Ser Ala Ala Arg Arg Ile Thr Val Arg 85 90 95 Arg Phe Thr Arg Phe
Lys Arg Val Leu Gly Arg Arg Val Ser Phe Gly 100 105 110 Gln Gly Thr
Lys Val Glu Ile Lys Arg Ala Ala Ala 115 120 91123PRTHomo sapiens
91Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1
5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser
Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile
Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr
Tyr Cys Gln Gln Arg Asp Glu Arg Gly 85 90 95 Gly Leu Pro Asp Leu
Asn Ala Tyr Trp Glu Gly Ala Ser His Ser Ala 100 105 110 Lys Gly Pro
Arg Trp Lys Ser Asn Gly Arg Pro 115 120 92123PRTHomo sapiens 92Arg
Lys Leu Cys Arg Ser Leu Gln Ala Pro Cys Leu Cys Leu Gln Gly 1 5 10
15 Lys Glu Pro Pro Ser Pro Ala Gly Pro Val Arg Val Leu Ala Ala Ala
20 25 30 Thr Pro Gly Thr Ser Arg Asn Leu Ala Arg Leu Pro Gly Ser
Ser Ser 35 40 45 Met Val His Pro Ala Gly Pro Leu Ala Ser Gln Thr
Gly Ser Val Ala 50 55 60 Val Gly Leu Gly Gln Thr Ser Leu Ser Pro
Ser Ala Asp Trp Ser Leu 65 70 75 80 Lys Ile Leu Gln Phe Ile Thr Val
Ser Ser Glu Thr Asn Asn Gly Lys 85 90 95 Ala Val Tyr Gln Val Thr
Arg Ile Gly Lys Ala Arg Leu Ile Arg Pro 100 105 110 Arg Asp Gln Gly
Gly Asn Gln Thr Gly Gly Arg 115 120 93380DNAHomo sapiens
93cagtctgtgt tgacgcagcc gccctcagtg tctgcggccc caggacagaa ggtcaccatc
60tcctgctctg gaagcagctc caacattggg aataattatg tatcctggta ccagcagctc
120ccaggaacag cccccaaact cctcatttat gacaataata agcgaccctc
agggattcct 180gaccgattct ctggctccaa gtctggcacg tcagccaccc
tgggcatcac cggactccag 240actggggacg aggccgatta ttactgcgga
acaggagacg aataacggta aggcggttta 300ccaggtttaa acgcgtattg
ggaaggcgcg tctctgtgtt cggcggaggg accaagctga 360ccgtcctagg
ggcggccgca 38094122PRTHomo sapiens 94Ala Val Cys Val Asp Ala Ala
Ala Leu Ser Val Cys Gly Pro Arg Thr 1 5 10 15 Glu Gly His His Leu
Leu Leu Trp Lys Gln Leu Gln His Trp Glu Leu 20 25 30 Cys Ile Leu
Val Pro Ala Ala Pro Arg Asn Ser Pro Gln Thr Pro His 35 40 45 Leu
Gln Ala Thr Leu Arg Asp Ser Pro Ile Leu Trp Leu Gln Val Trp 50 55
60 His Val Ser His Pro Gly His His Arg Thr Pro Asp Trp Gly Arg Gly
65 70 75 80 Arg Leu Leu Leu Arg Asn Arg Arg Arg Ile Thr Val Arg Arg
Phe Thr 85 90 95 Arg Phe Lys Arg Val Leu Gly Arg Arg Val Ser Val
Phe Gly Gly Gly 100 105 110 Thr Lys Leu Thr Val Leu Gly Ala Ala Ala
115 120 95122PRTHomo sapiens 95Gln Ser Val Leu Thr Gln Pro Pro Ser
Val Ser Ala Ala Pro Gly Gln 1 5 10 15 Lys Val Thr Ile Ser Cys Ser
Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Tyr Val Ser Trp Tyr
Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Asp
Asn Asn Lys Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly
Ser Lys Ser Gly Thr Ser Ala Thr Leu Gly Ile Thr Gly Leu Gln 65 70
75 80 Thr Gly Asp Glu Ala Asp Tyr Tyr Cys Gly Thr Gly Asp Glu Arg
Gly 85 90 95 Gly Leu Pro Gly Leu Asn Ala Tyr Trp Glu Gly Ala Ser
Leu Cys Ser 100 105 110 Ala Glu Gly Pro Ser Pro Ser Gly Arg Pro 115
120 96125PRTHomo sapiens 96Arg Ser Leu Cys Arg Ser Arg Pro Gln Cys
Leu Arg Pro Gln Asp Arg 1 5 10 15 Arg Ser Pro Ser Pro Ala Leu Glu
Ala Ala Pro Thr Leu Gly Ile Ile 20 25 30 Met Tyr Pro Gly Thr Ser
Ser Ser Gln Glu Gln Pro Pro Asn Ser Ser 35 40 45 Phe Met Thr Ile
Ile Ser Asp Pro Gln Gly Phe Leu Thr Asp Ser Leu 50 55 60 Ala Pro
Ser Leu Ala Arg Gln Pro Pro Trp Ala Ser Pro Asp Ser Arg 65 70 75 80
Leu Gly Thr Arg Pro Ile Ile Thr Ala Glu Gln Glu Thr Asn Asn Gly 85
90 95 Lys Ala Val Tyr Gln Val Thr Arg Ile Gly Lys Ala Arg Leu Cys
Val 100 105 110 Arg Arg Arg Asp Gln Ala Asp Arg Pro Arg Gly Gly Arg
115 120 125 97380DNAHomo sapiens 97cagtctgtgc tgactcagcc accctcagcg
tctgggaccc ccgggcagag ggtcaccatc 60tcttgttctg gaagcagctc caacatcgga
agtaatactg taaactggta ccagcagctc 120ccaggaacgg cccccaaact
cctcatctat agtaataatc agcggccctc aggggtccct 180gaccgattct
ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag
240tctgaggatg aggctgatta ttactgtgca gcaggagacg aataacggta
aggcggttta 300ccaggtttaa acgcgtattg ggaaggcgcg tctctgtgtt
cggcggaggg accaagctga 360ccgtcctagg ggcggccgca 38098119PRTHomo
sapiens 98Ala Val Cys Ala Asp Ser Ala Thr Leu Ser Val Trp Asp Pro
Arg Ala 1 5 10 15 Glu Gly His His Leu Leu Phe Trp Lys Gln Leu Gln
His Arg Lys Tyr 20 25 30 Cys Lys Leu Val Pro Ala Ala Pro Arg Asn
Gly Pro Gln Thr Pro His 35 40 45 Leu Ser Ala Ala Leu Arg Gly Pro
Pro Ile Leu Trp Leu Gln Val Trp 50 55 60 His Leu Ser Leu Pro Gly
His Gln Trp Ala Pro Val Gly Gly Leu Leu 65 70 75 80 Leu Cys Ser Arg
Arg Arg Ile Thr Val Arg Arg Phe Thr Arg Phe Lys 85 90 95 Arg Val
Leu Gly Arg Arg Val Ser Val Phe Gly Gly Gly Thr Lys Leu 100 105 110
Thr Val Leu Gly Ala Ala Ala 115 99122PRTHomo sapiens 99Gln Ser Val
Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg
Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25
30 Thr Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu
35 40 45 Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg
Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile
Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala
Ala Gly Asp Glu Arg Gly 85 90 95 Gly Leu Pro Gly Leu Asn Ala Tyr
Trp Glu Gly Ala Ser Leu Cys Ser 100 105 110 Ala Glu Gly Pro Ser Pro
Ser Gly Arg Pro 115 120 100124PRTHomo sapiens 100Arg Ser Leu Cys
Leu Ser His Pro Gln Arg Leu Gly Pro Pro Gly Arg 1 5 10 15 Gly Ser
Pro Ser Leu Val Leu Glu Ala Ala Pro Thr Ser Glu Val Ile 20 25 30
Leu Thr Gly Thr Ser Ser Ser Gln Glu Arg Pro Pro Asn Ser Ser Ser 35
40 45 Ile Val Ile Ile Ser Gly Pro Gln Gly Ser Leu Thr Asp Ser Leu
Ala 50 55 60 Pro Ser Leu Ala Pro Gln Pro Pro Trp Pro Ser Val Gly
Ser Ser Leu 65 70 75 80 Arg Met Arg Leu Ile Ile Thr Val Gln Gln Glu
Thr Asn Asn Gly Lys 85 90 95 Ala Val Tyr Gln Val Thr Arg Ile Gly
Lys Ala Arg Leu Cys Val Arg 100 105 110 Arg Arg Asp Gln Ala Asp Arg
Pro Arg Gly Gly Arg 115 120 10119PRTHomo sapiens 101Cys Ala Lys Ala
Phe Arg Pro Asn Trp Gly Ser Arg Val Leu Tyr Phe 1 5 10 15 Asp Tyr
Trp 10212PRTHomo sapiens 102Cys Ala Lys Leu Asn Trp Gly Trp Phe Asp
Pro Trp 1 5 10 10312PRTHomo sapiens 103Cys Ala Lys Gly Trp Glu Gly
Glu Tyr Asp Tyr Trp 1 5 10 10417PRTHomo sapiens 104Cys Ala Lys Asp
Leu Trp Leu Asp Ser Ser Ser Asn Trp Phe Asp Pro 1 5 10 15 Trp
10511PRTHomo sapiens 105Cys Ala Lys Asp Leu Pro Gly Asp Pro His Trp
1 5 10 10613PRTHomo sapiens 106Cys Ala Lys Ala Pro Pro Thr Gly Tyr
Phe Asp Tyr Trp 1 5 10 10714PRTHomo sapiens 107Cys Ala Lys Val Gly
Leu Thr Gly Val His Phe Asp Tyr Trp 1 5 10 10816PRTHomo sapiens
108Cys Ala Lys Asp Ser Tyr Gly Ser Gly Ser Tyr Tyr Asn Asp Tyr Trp
1 5 10 15 10918PRTHomo sapiens 109Cys Ala Lys Val Ala Gly Thr Trp
Gly Arg Val Ala Tyr Tyr Phe Asp 1 5 10 15 Tyr Trp 11015PRTHomo
sapiens 110Cys Ala Lys Val Thr Gly Val Phe Val Gly Asn Phe Asp Tyr
Trp 1 5 10 15 11115PRTHomo sapiens 111Cys Ala Lys Asp Ala Arg Asn
Ser Gly Ser Tyr Phe Asp Tyr Trp 1 5 10 15 11214PRTHomo sapiens
112Cys Ala Lys Gly Val Arg Thr Gly Val Val Phe Asp Tyr Trp 1 5 10
11319PRTHomo sapiens 113Cys Ala Lys Gly Val Val Asn Trp Gly Thr Arg
Arg Lys Gly Trp Phe 1 5 10 15 Asp Pro Trp 11411PRTHomo sapiens
114Cys Ala Lys Trp Gly Gly Trp Phe Asp Pro Trp 1 5 10 11513PRTHomo
sapiens 115Cys Ala Lys Arg Arg Asp Asn Trp Gly Ser Val Asp Trp 1 5
10 11615PRTHomo sapiens 116Cys Ala Lys Gly Ser Gly Phe Ser Ser Gly
Trp Phe Asp Ser Trp 1 5 10 15 11744DNAHomo
sapiensmisc_feature(13)..(14)n is a, t, c or g 117acgtctccga
gannsnnsnn snnsatggat tattggggga gacg 4411814PRTHomo
sapiensMISC_FEATURE(5)..(8)Xaa is Cys, Trp, Arg, Ser or Gly 118Thr
Ser Pro Arg Xaa Xaa Xaa Xaa Met Asp Tyr Trp Gly Arg 1 5 10
11914PRTHomo sapiensMISC_FEATURE(4)..(4)Xaa is Asp or Glu 119Arg
Leu Arg Xaa Xaa Xaa Xaa Xaa Trp Ile Ile Gly Gly Asp 1 5 10
12025DNAArtificial Sequencechemically synthesized 120tgtttctaat
cgcaggtgcc agatg 2512125DNAArtificial Sequencechemically
synthesized 121atttatgtta tgacttgtta cactg 2512225DNAArtificial
Sequencechemically synthesized 122tatttgtttt tatgtttcca atctc
2512325DNAArtificial Sequencechemically synthesized 123ccttggaggt
ttatgttatg acttg 2512425DNAArtificial Sequencechemically
synthesized 124ttatttccaa tttcagatac caccg 2512525DNAArtificial
Sequencechemically synthesized 125ttgttggggt ttttgtttca tgtgg
2512625DNAArtificial Sequencechemically synthesized 126tatttccaat
ttcagatacc actgg 2512725DNAArtificial Sequencechemically
synthesized 127atgttgaatc actgtgggag gccag 2512825DNAArtificial
Sequencechemically synthesized 128ttatttccaa tctcagatac caccg
2512925DNAArtificial Sequencechemically synthesized 129ttttgtttca
agctgaatca ctgtg 2513025DNAArtificial Sequencechemically
synthesized 130atgtctgtgt ctctctcact tccag 2513125DNAArtificial
Sequencechemically synthesized 131ttccccattg gcctggagca ctgtg
2513225DNAArtificial Sequencechemically synthesized 132gtgtctgtgt
ctctcctgct tccag 2513325DNAArtificial Sequencechemically
synthesized 133cttgtctcag ttccccattg ggctg 2513425DNAArtificial
Sequencechemically synthesized 134atctcatcca cttctgtgtt ctctc
2513525DNAArtificial Sequencechemically synthesized 135ttgggtttct
gacaccctca ggatg 2513625DNAArtificial Sequencechemically
synthesized 136caggccagtc atgtgagact tcacc 2513725DNAArtificial
Sequencechemically synthesized 137ctgcctcctc cctggggttt ctgaa
2513825DNAArtificial Sequencechemically synthesized 138cccctgtgtc
ctctccacag gtgtc 2513925DNAArtificial Sequencechemically
synthesized 139ccggcacagc tgccttctcc ctcag 2514018DNAArtificial
Sequencechemically synthesized 140gaggtgcagc tgttggag
1814126DNAArtificial Sequencechemically synthesized 141tctgaccagg
gtttcttttt gtttgc 2614225DNAArtificial Sequencechemically
synthesized 142ttgtgtctgg gctcacaatg acttc 2514324DNAArtificial
Sequencechemically synthesized 143tggcattttc tgataacggt gtcc
2414425DNAArtificial Sequencechemically synthesized 144ctgcagggag
gtttgtgtct gggcg 2514525DNAArtificial Sequencechemically
synthesized 145atatgtgtgg cagtttctga ccttg 2514625DNAArtificial
Sequencechemically synthesized 146ggtttgtgtc tggtgtcaca ctgac
2514725DNAArtificial Sequencechemically synthesized 147gagtctgtgc
cggaagtgca gctgg 2514821DNAArtificial Sequencechemically
synthesized 148tatcaggtgc agctggtgca g 2114921DNAArtificial
Sequencechemically synthesized 149tatcaggtgc agctggtgga g
2115021DNAArtificial Sequencechemically synthesized 150tatgaggtgc
agctggtgca g 2115123DNAArtificial Sequencechemically synthesized
151atatctctcg cacagtaata cac 2315223DNAArtificial
Sequencechemically synthesized 152atatctctcg cacagtaata tac
2315323DNAArtificial Sequencechemically synthesized 153atatgtctcg
cacagtaata cat 2315432DNAArtificial Sequencechemically synthesized
154tatgacatcc agatgaccca gtctccatcc tc 3215520DNAArtificial
Sequencechemically synthesized 155ataggagggg tactgtaact
2015620DNAArtificial Sequencechemically synthesized 156ataggaggga
gattatcata 2015724DNAArtificial Sequencechemically synthesized
157tatgaaattg tgttgacgca gtct 2415823DNAArtificial
Sequencechemically synthesized 158ataggaggtg
agctaccata ctg 2315924DNAArtificial Sequencechemically synthesized
159tatgaaatag tgatgacgca gtct 2416023DNAArtificial
Sequencechemically synthesized 160ataggaggcc agttattata ctg
2316123DNAArtificial Sequencechemically synthesized 161cagcgtagca
actggcctcc tat 2316220DNAArtificial Sequencechemically synthesized
162tacagtctgt gctgactcag 2016323DNAArtificial Sequencechemically
synthesized 163ataggaccat tcaggctgtc atc 2316421DNAArtificial
Sequencechemically synthesized 164tatcagtctg tgttgacgca g
2116522DNAArtificial Sequencechemically synthesized 165ataggagcac
tcaggctgct at 2216633DNAArtificial Sequencechemically synthesized
166cagccggcca tggcccaggt gcagctggtg cag 3316733DNAArtificial
Sequencechemically synthesized 167cagccggcca tggcccaggt gcagctggtg
gag 3316833DNAArtificial Sequencechemically synthesized
168cagccggcca tggccgaggt gcagctgttg gag 3316943DNAArtificial
Sequencechemically synthesized 169cagccggcca tggccgaggt gcagctggtg
gagtctgggg gag 4317033DNAArtificial Sequencechemically synthesized
170cagccggcca tggccgaggt gcagctggtg cag 3317137DNAArtificial
Sequencechemically synthesized 171cttaccgtta ttcgtctcat ctcgcacagt
aatacac 3717237DNAArtificial Sequencechemically synthesized
172cttaccgtta ttcgtctcat ttcgcacagt aatatac 3717342DNAArtificial
Sequencechemically synthesized 173ctcgcacagt aatacacagc cgtgtcctcg
gctctcaggc tg 4217437DNAArtificial Sequencechemically synthesized
174cttaccgtta ttcgtctcat ctcgcacagt aatacat 3717547DNAArtificial
Sequencechemically synthesized 175caatacgcgt ttaaacctgg taaaccgcct
taccgttatt cgtctca 4717648DNAArtificial Sequencechemically
synthesized 176gttccctggc cccaagagac gcgccttccc aatacgcgtt taaacctg
4817745DNAArtificial Sequencechemically synthesized 177cctccaccgc
tcgagactgt gaccagggtt ccctggcccc aagag 4517830DNAArtificial
Sequencechemically synthesized 178cgggtcgacg gacatccaga tgacccagtc
3017936DNAArtificial Sequencechemically synthesized 179cgggtcgacg
gaaattgtgt tgacacagtc tccagc 3618036DNAArtificial
Sequencechemically synthesized 180cgggtcgacg gaaatagtga tgacgcagtc
tccagc 3618136DNAArtificial Sequencechemically synthesized
181cgggtcgacg gaaattgtgt tgacgcagtc tccagg 3618245DNAArtificial
Sequencechemically synthesized 182ccttaccgtt attcgtctcg ctgctgacag
taatatgttg caata 4518345DNAArtificial Sequencechemically
synthesized 183ccttaccgtt attcgtctcg ctgctgacag tagtaagttg caaaa
4518447DNAArtificial Sequencechemically synthesized 184ccttaccgtt
attcgtctcg ctgctgacag taataaactg caaaatc 4718547DNAArtificial
Sequencechemically synthesized 185ccaatacgcg tttaaacctg gtaaaccgcc
ttaccgttat tcgtctc 4718646DNAArtificial Sequencechemically
synthesized 186ggtcccttgg ccgaatgaga cgcgccttcc caatacgcgt ttaaac
4618745DNAArtificial Sequencechemically synthesized 187gtgcggccgc
ccgtttgatt tccaccttgg tcccttggcc gaatg 4518845DNAArtificial
Sequencechemically synthesized 188gtgcggccgc ccctttgatt tccaccttgg
tcccttggcc gaatg 4518932DNAArtificial Sequencechemically
synthesized 189cgggtcgacg cagtctgtgc tgactcagcc ac
3219032DNAArtificial Sequencechemically synthesized 190cgggtcgacg
cagtctgtgt tgacgcagcc gc 3219138DNAArtificial Sequencechemically
synthesized 191ccttaccgtt attcgtctcc tgctgcacag taataatc
3819238DNAArtificial Sequencechemically synthesized 192ccttaccgtt
attcgtctcc tgttccgcag taataatc 3819346DNAArtificial
Sequencechemically synthesized 193ccctccgccg aacacagaga cgcgccttcc
caatacgcgt ttaaac 4619449DNAArtificial Sequencechemically
synthesized 194gtgcggccgc ccctaggacg gtcagcttgg tccctccgcc
gaacacaga 4919543DNAArtificial Sequencechemically synthesized
195ccgcacagcc ggccatggcc caggtgcagc tggtgcagtc tgg
4319643DNAArtificial Sequencechemically synthesized 196ccgcacagcc
ggccatggcc gaggtgcagc tggtggagtc tgg 4319743DNAArtificial
Sequencechemically synthesized 197ccgcacagcc ggccatggcc cagrtcacct
tgctcgagtc tgg 4319843DNAArtificial Sequencechemically synthesized
198ccgcacagcc ggccatggcc caggtgcagc tgcaggagtc ggg
4319943DNAArtificial Sequencechemically synthesized 199ccgcacagcc
ggccatggcc cagctgcagc tgcaggagtc cgg 4320043DNAArtificial
Sequencechemically synthesized 200ccgcacagcc ggccatggcc caggtgcagc
tacagcagtg ggg 4320132DNAArtificial Sequencechemically synthesized
201tggagtgggt ctcagctatt agtggtagtg gt 3220245DNAArtificial
Sequencechemically synthesized 202cgatgggccc ttggtggagg ctgaggagac
rgtgaccagg gtgcc 4520345DNAArtificial Sequencechemically
synthesized 203cgatgggccc ttggtggagg ctgaagagac ggtgaccrtk gtccc
4520445DNAArtificial Sequencechemically synthesized 204cgatgggccc
ttggtggagg ctgaggagac ggtgaccagg gttcc 4520535DNAArtificial
Sequencechemically synthesized 205gagccgagga cacggccgga tgttactgtg
cgaga 3520635DNAArtificial Sequencechemically synthesized
206gagccgagga cacggccgga tgttactgtg cgaaa 3520732DNAArtificial
Sequencechemically synthesized 207gaggagacgg tgacggatgt gccctggccc
ca 3220832DNAArtificial Sequencechemically synthesized
208gaggagacgg tgacggatgt gccacggccc ca 3220932DNAArtificial
Sequencechemically synthesized 209gaggagacgg tgacggatgt yccttggccc
ca 3221026DNAArtificial Sequencechemically synthesized
210atgatgctgc tggcacgtct ccgaga 2621135DNAArtificial
Sequencechemically synthesized 211ccacgtcatc cgatccgtct cccccaataa
tccat 3521235DNAArtificial Sequencechemically synthesized
212ccacgtcatc cgatccgtct cccccaataa tcaaa 3521350DNAArtificial
Sequencechemically synthesized 213gctggcacgt ctccgagann snnsnnsnns
tttgattatt gggggagacg 5021450DNAArtificial Sequencechemically
synthesized 214gctggcacgt ctccgagann snnsnnsnns atggattatt
gggggagacg 5021553DNAArtificial Sequencechemically synthesized
215gctggcacgt ctccgagann snnsnnsnns nnstttgatt attgggggag acg
5321653DNAArtificial Sequencechemically synthesized 216gctggcacgt
ctccgagann snnsnnsnns nnsatggatt attgggggag acg
5321756DNAArtificial Sequencechemically synthesized 217gctggcacgt
ctccgagann snnsnnsnns nnsnnstttg attattgggg gagacg
5621856DNAArtificial Sequencechemically synthesized 218gctggcacgt
ctccgagann snnsnnsnns nnsnnsatgg attattgggg gagacg
5621956DNAArtificial Sequencechemically synthesized 219gctggcacgt
ctccgagadv kdvkdvkdvk dvkdvktttg attattgggg gagacg
5622056DNAArtificial Sequencechemically synthesized 220gctggcacgt
ctccgagadv kdvkdvkdvk dvkdvkatgg attattgggg gagacg
5622159DNAArtificial Sequencechemically synthesized 221gctggcacgt
ctccgagadv kdvkdvkdvk dvkdvkdvkt ttgattattg ggggagacg
5922259DNAArtificial Sequencechemically synthesized 222gctggcacgt
ctccgagadv kdvkdvkdvk dvkdvkdvka tggattattg ggggagacg
5922359DNAArtificial Sequencechemically synthesized 223gctggcacgt
ctccgaganv tnvtnvtnvt nvtnvtnvtt ttgattattg ggggagacg
5922459DNAArtificial Sequencechemically synthesized 224gctggcacgt
ctccgaganv tnvtnvtnvt nvtnvtnvta tggattattg ggggagacg
5922562DNAArtificial Sequencechemically synthesized 225gctggcacgt
ctccgaganv tnvtnvtnvt nvtnvtnvtn vttttgatta ttgggggaga 60cg
6222662DNAArtificial Sequencechemically synthesized 226gctggcacgt
ctccgaganv tnvtnvtnvt nvtnvtnvtn vtatggatta ttgggggaga 60cg
6222765DNAArtificial Sequencechemically synthesized 227gctggcacgt
ctccgaganv tnvtnvtnvt nvtnvtnvtn vtnvttttga ttattggggg 60agacg
6522865DNAArtificial Sequencechemically synthesized 228gctggcacgt
ctccgaganv tnvtnvtnvt nvtnvtnvtn vtnvtatgga ttattggggg 60agacg
6522965DNAArtificial Sequencechemically synthesized 229gctggcacgt
ctccgagadv tdvtdvtdvt dvtdvtdvtd vtdvttttga ttattggggg 60agacg
6523065DNAArtificial Sequencechemically synthesized 230gctggcacgt
ctccgagadv tdvtdvtdvt dvtdvtdvtd vtdvtatgga ttattggggg 60agacg
6523168DNAArtificial Sequencechemically synthesized 231gctggcacgt
ctccgagadv tdvtdvtdvt dvtdvtdvtd vtdvtdvttt tgattattgg 60gggagacg
6823268DNAArtificial Sequencechemically synthesized 232gctggcacgt
ctccgagadv tdvtdvtdvt dvtdvtdvtd vtdvtdvtat ggattattgg 60gggagacg
6823326DNAArtificial Sequencechemically synthesized 233ccggtgtagc
gaaggcgtct cagcag 2623432DNAArtificial Sequencechemically
synthesized 234tagggtcgcc ttgatcgtct cccgaaggtc gg
3223545DNAArtificial Sequencechemically synthesized 235gaaggcgtct
cagcagnnsn nsnnsnnscc gaccttcggg agacg 4523648DNAArtificial
Sequencechemically synthesized 236gaaggcgtct cagcagnnsn nsnnsnnscc
gnnsaccttc gggagacg 4823751DNAArtificial Sequencechemically
synthesized 237gaaggcgtct cagcagnnsn nsnnsnnsnn sccgnnsacc
ttcgggagac g 5123832DNAArtificial Sequencechemically synthesized
238cggtcagtcg caatacgtct ccagcatggg at 3223932DNAArtificial
Sequencechemically synthesized 239cggtcagtcg caatacgtct ccagcatatg
at 3224032DNAArtificial Sequencechemically synthesized
240caggaccagt ctcgtgagga tcgtctcaac ac 3224146DNAArtificial
Sequencechemically synthesized 241cgtctccagc atgggatnns nnsnnsnnsg
tgttgagacg atcctc 4624246DNAArtificial Sequencechemically
synthesized 242cgtctccagc atatgatnns nnsnnsnnsg tgttgagacg atcctc
4624349DNAArtificial Sequencechemically synthesized 243cgtctccagc
atgggatnns nnsnnsnnsn nsgtgttgag acgatcctc 4924449DNAArtificial
Sequencechemically synthesized 244cgtctccagc atatgatnns nnsnnsnnsn
nsgtgttgag acgatcctc 4924552DNAArtificial Sequencechemically
synthesized 245cgtctccagc atgggatnns nnsnnsnnsn nsnnsgtgtt
gagacgatcc tc 5224652DNAArtificial Sequencechemically synthesized
246cgtctccagc atatgatnns nnsnnsnnsn nsnnsgtgtt gagacgatcc tc
5224732DNAArtificial Sequencechemically synthesized 247cggtcagtcg
caatacgtct cgaacatggg at 3224832DNAArtificial Sequencechemically
synthesized 248cggtcagtcg caatacgtct cgaacatggg at
3224946DNAArtificial Sequencechemically synthesized 249cgtctcgaac
atgggatnns nnsnnsnnsg tgttgagacg atcctc 4625046DNAArtificial
Sequencechemically synthesized 250cgtctcgaac atatgatnns nnsnnsnnsg
tgttgagacg atcctc 4625149DNAArtificial Sequencechemically
synthesized 251cgtctcgaac atgggatnns nnsnnsnnsn nsgtgttgag
acgatcctc 4925249DNAArtificial Sequencechemically synthesized
252cgtctcgaac atatgatnns nnsnnsnnsn nsgtgttgag acgatcctc
4925352DNAArtificial Sequencechemically synthesized 253cgtctcgaac
atgggatnns nnsnnsnnsn nsnnsgtgtt gagacgatcc tc 5225452DNAArtificial
Sequencechemically synthesized 254cgtctcgaac atgggatnns nnsnnsnnsn
nsnnsgtgtt gagacgatcc tc 5225521DNAArtificial Sequencechemically
synthesized 255ctcttctgag atgagttttt g 2125643DNAArtificial
Sequencechemically synthesized 256atgcggccca gccggccatg gccsaggtyc
agctbcagca gtc 4325743DNAArtificial Sequencechemically synthesized
257atgcggccca gccggccatg gcccaggttc acctgcagca rtc
4325843DNAArtificial Sequencechemically synthesized 258atgcggccca
gccggccatg gcccaggtrc agctgaagga gtc 4325943DNAArtificial
Sequencechemically synthesized 259atgcggccca gccggccatg gcccaggtcc
aactvcagca rcc 4326043DNAArtificial Sequencechemically synthesized
260atgcggccca gccggccatg gcccagatcc agttggtvca gtc
4326143DNAArtificial Sequencechemically synthesized 261atgcggccca
gccggccatg gcccaggtgc agctgaagsa stc 4326243DNAArtificial
Sequencechemically synthesized 262atgcggccca gccggccatg gccgaggtgc
agskggtgga gtc 4326343DNAArtificial Sequencechemically synthesized
263atgcggccca gccggccatg gccgaagtga arsttgagga gtc
4326443DNAArtificial Sequencechemically synthesized 264atgcggccca
gccggccatg gccgakgtsv agcttcagga gtc 4326543DNAArtificial
Sequencechemically synthesized 265atgcggccca gccggccatg gccgaggtga
asstggtgga atc 4326643DNAArtificial Sequencechemically synthesized
266atgcggccca gccggccatg gccgaggtga agctgrtgga rtc
4326743DNAArtificial Sequencechemically synthesized 267atgcggccca
gccggccatg gccgargtga agctgrtgga gtc 4326843DNAArtificial
Sequencechemically synthesized 268atgcggccca gccggccatg gccgaagtgc
agctgttgga gac 4326943DNAArtificial Sequencechemically synthesized
269atgcggccca gccggccatg gccgargtga agcttctcsa gtc
4327042DNAArtificial Sequencechemically synthesized 270atgcggccca
gccggccatg gcccargtta ctctgaaaga gt 4227141DNAArtificial
Sequencechemically synthesized 271cctgaaccgc cgcctccgct cgagacggtg
accgtggtcc c 4127241DNAArtificial Sequencechemically synthesized
272cctgaaccgc cgcctccgct cgagactgtg agagtggtgc c
4127341DNAArtificial Sequencechemically synthesized 273cctgaaccgc
cgcctccgct cgagacagtg accagagtcc c 4127441DNAArtificial
Sequencechemically synthesized 274cctgaaccgc cgcctccgct cgagacggtg
actgaggttc c 4127535DNAArtificial Sequencechemically synthesized
275gagccgagga cacggccgga tgttactgtg cgaga 3527631DNAArtificial
Sequencechemically synthesized 276ggggcgcagg gacatccgtc accgtctcct
c 3127732DNAArtificial Sequencechemically synthesized 277gaggagactg
tgagggatgt gccttggccc ca 3227832DNAArtificial Sequencechemically
synthesized 278gaggagacgg tgacggatgt gccctggccc ca
3227932DNAArtificial Sequencechemically synthesized 279gaggagacgg
tgacggatgt tccttgaccc ca 3228014PRTHomo
sapiensMISC_FEATURE(4)..(4)Xaa is Ile or Met 280Val Ser Glu Xaa Xaa
Xaa Xaa Xaa Gly Leu Leu Gly Glu Thr 1 5 10 28113DNAArtificial
Sequencechemically synthesized 281ttactgtgcg aga
1328213DNAArtificial Sequencechemically synthesized 282ttactgtgca
aga 1328313DNAArtificial Sequencechemically synthesized
283tttctgtgca aga 1328413DNAArtificial Sequencechemically
synthesized 284ctactgtgcc aga 1328513DNAArtificial
Sequencechemically synthesized 285tggggccagg gaa
1328613DNAArtificial Sequencechemically synthesized 286tggggcgcag
gga 1328713DNAArtificial Sequencechemically synthesized
287tggggccaag gca 1328813DNAArtificial Sequencechemically
synthesized 288tggggccagg gca 1328913DNAArtificial
Sequencechemically synthesized 289tggggtcagg gca
1329046DNAArtificial Sequencechemically synthesized 290attactgtgc
gagaggagac gnsnncgtct cttggggcca gggaac 4629146DNAArtificial
Sequencechemically synthesized 291attactgtgc gagaggagac gncgtctctt
ggggccaggg aaccct 4629234DNAArtificial Sequencechemically
synthesized 292ttatgtgtat agggttccct ggccccaaga gacg
3429334DNAArtificial Sequencechemically synthesized 293gtgatctgta
cctattactg tgcgagagga gacg 3429446DNAArtificial Sequencechemically
synthesized 294taatgacacg ctctcctctg cnsnngcaga gaaccccggt cccttg
4629514PRTArtificial Sequencechemically synthesized 295Tyr Cys Ala
Arg Gly Asp Xaa Xaa Val Ser Trp Gly Gln Gly 1 5 10
29646DNAArtificial Sequencechemically synthesized 296taatgacacg
ctctcctctg cngcagagaa ccccggtccc ttggga 4629714PRTArtificial
Sequencechemically synthesized 297Tyr Cys Ala Arg Gly Asp Xaa Val
Ser Trp Gly Gln Gly Thr 1 5 10 29824DNAArtificial
Sequencechemically synthesized 298gcgaaaggag acgcccccgt ctct
2429924DNAArtificial Sequencechemically synthesized 299cgctttcctc
tgcgggggca gaga 243008PRTArtificial Sequencechemically synthesized
300Ala Lys Gly Asp Ala Pro Val Ser 1 5 30124DNAArtificial
Sequencechemically synthesized 301gcgagaggag acgccttcgt ctct
2430224DNAArtificial Sequencechemically synthesized 302cgctctcctc
tgcggaagca gaga 243038PRTArtificial Sequencechemically synthesized
303Ala Arg Gly Asp Ala Phe Val Ser 1 5 30424DNAArtificial
Sequencechemically synthesized 304gcgagaggag acgagtacgt ctct
2430524DNAArtificial Sequencechemically synthesized 305cgctctcctc
tgcctatgca gaga 243068PRTArtificial Sequencechemically synthesized
306Ala Arg Gly Asp Glu Tyr Val Ser 1 5 30724DNAArtificial
Sequencechemically synthesized 307gcgagaggag acgagctcgt ctct
2430824DNAArtificial Sequencechemically synthesized 308cgctctcctc
tgctcgagca gaga 243098PRTArtificial Sequencechemically synthesized
309Ala Arg Gly Asp Glu Leu Val Ser 1 5 31024DNAArtificial
Sequencechemically synthesized 310gcgagaggag acggctgcgt ctct
2431124DNAArtificial Sequencechemically synthesized 311cgctctcctc
tgccgacgca gaga 243128PRTArtificial Sequencechemically synthesized
312Ala Arg Gly Asp Gly Cys Val Ser 1 5 31324DNAArtificial
Sequencechemically synthesized 313gcgagaggag acgagcccgt ctct
2431424DNAArtificial Sequencechemically synthesized 314cgctctcctc
tgctcgggca gaga 243158PRTArtificial Sequencechemically synthesized
315Ala Arg Gly Asp Glu Pro Val Ser 1 5 31624DNAArtificial
Sequencechemically synthesized 316gcgagaggag acgggatcgt ctct
2431724DNAArtificial Sequencechemically synthesized 317cgctctcctc
tgccctagca gaga 243188PRTArtificial Sequencechemically synthesized
318Ala Arg Gly Asp Gly Ile Val Ser 1 5 31924DNAArtificial
Sequencechemically synthesized 319gcgaaaggag acgggcgcgt ctct
2432024DNAArtificial Sequencechemically synthesized 320cgctttcctc
tgcccgcgca gaga 243218PRTArtificial Sequencechemically synthesized
321Ala Lys Gly Asp Gly Arg Val Ser 1 5 32224DNAArtificial
Sequencechemically synthesized 322gcgagaggag acgacgccgt ctct
2432324DNAArtificial Sequencechemically synthesized 323cgctctcctc
tgctgcggca gaga 243248PRTArtificial Sequencechemically synthesized
324Ala Arg Gly Asp Asp Ala Val Ser 1 5 32524DNAArtificial
Sequencechemically synthesized 325gcgagaggag acgggtccgt ctct
2432624DNAArtificial Sequencechemically synthesized 326cgctctcctc
tgcccaggca gaga 243278PRTArtificial Sequencechemically synthesized
327Ala Arg Gly Asp Gly Ser Val Ser 1 5 32824DNAArtificial
Sequencechemically synthesized 328gcgagaggag acgccgtcgt ctct
2432924DNAArtificial Sequencechemically synthesized 329cgctctcctc
tgcggcagca gaga 243308PRTArtificial Sequencechemically synthesized
330Ala Arg Gly Asp Ala Val Val Ser 1 5 33124DNAArtificial
Sequencechemically synthesized 331gcgaaaggag acgtgtccgt ctct
2433224DNAArtificial Sequencechemically synthesized 332cgctttcctc
tgcacaggca gaga 243338PRTArtificial Sequencechemically synthesized
333Ala Lys Gly Asp Val Ser Val Ser 1 5 33424DNAArtificial
Sequencechemically synthesized 334gcgagaggag acgggcacgt ctct
2433524DNAArtificial Sequencechemically synthesized 335cgctctcctc
tgcccgtgca gaga 243368PRTArtificial Sequencechemically synthesized
336Ala Arg Gly Asp Gly His Val Ser 1 5 33724DNAArtificial
Sequencechemically synthesized 337gcgagaggag acgagaccgt ctct
2433824PRTArtificial Sequencechemically synthesized 338Cys Gly Cys
Thr Cys Thr Cys Cys Thr Cys Thr Gly Cys Thr Cys Thr 1 5 10 15 Gly
Gly Cys Ala Gly Ala Gly Ala 20 3398PRTArtificial Sequencechemically
synthesized 339Ala Arg Gly Asp Glu Thr Val Ser 1 5
34024DNAArtificial Sequencechemically synthesized 340gcgagaggag
acgccatcgt ctct 2434124DNAArtificial Sequencechemically synthesized
341cgctctcctc tgcggtagca gaga 243428PRTArtificial
Sequencechemically synthesized 342Ala Arg Gly Asp Ala Ile Val Ser 1
5 34321DNAArtificial Sequencechemically synthesized 343gcgagaggag
acggcgtctc t 2134421DNAArtificial Sequencechemically synthesized
344cgctctcctc tgccgcagag a 213457PRTArtificial Sequencechemically
synthesized 345Ala Arg Gly Asp Gly Val Ser 1 5 34621DNAArtificial
Sequencechemically synthesized 346gcgagaggag acgacgtctc t
2134721DNAArtificial Sequencechemically synthesized 347cgctctcctc
tgctgcagag a 213487PRTArtificial Sequencechemically synthesized
348Ala Arg Gly Asp Asp Val Ser 1 5 34921DNAArtificial
Sequencechemically synthesized 349gcgagaggag acgccgtctc t
2135021DNAArtificial Sequencechemically synthesized 350cgctctcctc
tgcggcagag a 213517PRTArtificial Sequencechemically synthesized
351Ala Arg Gly Asp Ala Val Ser 1 5 35221DNAArtificial
Sequencechemically synthesized 352gcgaaaggag acgacgtctc t
2135321DNAArtificial Sequencechemically synthesized 353cgctttcctc
tgctgcagag a 213547PRTArtificial Sequencechemically synthesized
354Ala Lys Gly Asp Asp Val Ser 1 5 35521DNAArtificial
Sequencechemically synthesized 355gcgagaggag acgtcgtctc t
2135621DNAArtificial Sequencechemically synthesized 356cgctctcctc
tgcagcagag a 213577PRTArtificial Sequencechemically synthesized
357Ala Arg Gly Asp Val Val Ser 1 5 35821DNAArtificial
Sequencechemically synthesized 358gcgaaaggag acggcgtctc t
2135921DNAArtificial Sequencechemically synthesized 359cgctttcctc
tgccgcagag a 213607PRTArtificial Sequencechemically synthesized
360Ala Lys Gly Asp Gly Val Ser 1 5 36121DNAArtificial
Sequencechemically synthesized 361gcgaaaggag acgccgtctc t
2136221DNAArtificial Sequencechemically synthesized 362cgctttcctc
tgcggcagag a 213637PRTArtificial Sequencechemically synthesized
363Ala Lys Gly Asp Ala Val Ser 1 5 36421DNAArtificial
Sequencechemically synthesized 364gcgaaaggag acgtcgtctc t
2136521DNAArtificial Sequencechemically synthesized 365cgctttcctc
tgcagcagag a 213667PRTArtificial Sequencechemically synthesized
366Ala Lys Gly Asp Val Val Ser 1 5
* * * * *