U.S. patent application number 11/962295 was filed with the patent office on 2008-05-29 for antibodies that specifically bind pms2.
This patent application is currently assigned to Morphotek, Inc.. Invention is credited to Luigi Grasso, Nicholas C. Nicolaides, Eric Routhier, Philip M. Sass.
Application Number | 20080124744 11/962295 |
Document ID | / |
Family ID | 34676833 |
Filed Date | 2008-05-29 |
United States Patent
Application |
20080124744 |
Kind Code |
A1 |
Grasso; Luigi ; et
al. |
May 29, 2008 |
Antibodies That Specifically Bind PMS2
Abstract
Antibodies against PMS2 and PMS2-134 and cells that produce the
anti-PMS2 and anti-PMS2-134 antibodies are provided. The antibodies
of the invention may be used in methods for detecting a PMS2
protein, including a truncated PMS2, and in methods for detecting
an abnormal condition in a patient.
Inventors: |
Grasso; Luigi; (Bala Cynwyd,
PA) ; Nicolaides; Nicholas C.; (Boothwyn, PA)
; Sass; Philip M.; (Audubon, PA) ; Routhier;
Eric; (Glen Mills, PA) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
CIRA CENTRE, 12TH FLOOR, 2929 ARCH STREET
PHILADELPHIA
PA
19104-2891
US
|
Assignee: |
Morphotek, Inc.
Exton
PA
|
Family ID: |
34676833 |
Appl. No.: |
11/962295 |
Filed: |
December 21, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11007428 |
Dec 8, 2004 |
7332584 |
|
|
11962295 |
|
|
|
|
60528269 |
Dec 8, 2003 |
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Current U.S.
Class: |
435/7.23 ;
435/7.21 |
Current CPC
Class: |
C07K 16/30 20130101;
G01N 33/574 20130101; G01N 33/57419 20130101; C07K 16/18 20130101;
G01N 33/567 20130101; G01N 33/57484 20130101 |
Class at
Publication: |
435/7.23 ;
435/7.21 |
International
Class: |
G01N 33/574 20060101
G01N033/574; G01N 33/567 20060101 G01N033/567 |
Claims
1. A method for detecting a truncated form of a PMS2 protein
comprising preparing a cell lysate from a test cell, exposing said
lysate to an antibody that specifically binds an epitope of SEQ ID
NO: 1, and detecting said antibody.
2. The method of claim 1 wherein said epitope is located between
amino acids 55 and 81 of SEQ ID NO: 1.
3. The method of claim 1 wherein said epitope is located between
amino acids 81 and 133 of SEQ ID NO: 1.
4. A method for detecting an abnormal condition in a patient
expressing a truncated PMS2, said method comprising contacting a
test cell lysate from said patient with an antibody that
specifically binds an epitope of SEQ ID NO: 1 and detecting the
presence or absence of a truncated form of PMS2, wherein the
presence of said truncated PMS2 is indicative of an abnormal
condition.
5. The method of claim 4 wherein said epitope is located between
amino acids 55 and 81 of SEQ ID NO: 1.
6. The method of claim 4 wherein said epitope is located between
amino acids 81 and 133 of SEQ ID NO: 1.
7. The method of claim 4 wherein said abnormal condition is cancer.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional application of U.S. application Ser.
No. 11/007,428, filed Dec. 8, 2004, which claims benefit of U.S.
Provisional Application Ser. No. 60/528,269, filed Dec. 8, 2003,
the entire contents of both of which are incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The invention relates to antibodies against PMS2 and cells
that produce the anti-PMS2 antibodies. The invention also relates
to methods for detecting a PMS2 protein and methods for detecting
an abnormal condition in a patient using the antibodies of the
invention.
BACKGROUND OF THE INVENTION
[0003] PMS2 is a protein involved in mismatch repair (MMR). The
process of MMR, also called mismatch proofreading, is carried out
by protein complexes in cells ranging from bacteria to mammalian
cells. A MMR gene is a gene that encodes for one of the proteins of
such a mismatch repair complex. The MMR complex is believed to
detect distortions of the DNA helix resulting from
non-complementary pairing of nucleotide bases. The
non-complementary base on the newer DNA strand is excised, and the
excised base is replaced with the appropriate base, which is
complementary to the older DNA strand. In this way, cells eliminate
many mutations that occur as a result of mistakes in DNA
replication.
[0004] Dominant negative alleles of mismatch repair genes have been
shown to cause a MMR-defective phenotype even in the presence of a
wild-type allele in the same cell. An example of a dominant
negative allele of a MMR gene is the human gene hPMS2-134, which
carries a truncating mutation at codon 134. The mutation causes the
product of this gene to abnormally terminate at the position of the
134th amino acid, resulting in a shortened polypeptide containing
the N-terminal 133 amino acids. Such a mutation causes an increase
in the rate of mutations, which accumulate in cells after DNA
replication. Expression of a dominant negative allele of a mismatch
repair gene results in impairment of mismatch repair activity, even
in the presence of the wild-type allele. Any allele which produces
such effect can be used in this invention. Dominant negative
alleles of a MMR gene can be obtained from the cells of humans,
animals, yeast, bacteria, or other organisms.
[0005] Antibodies to detect PMS2 and truncation mutants thereof
would be useful in biological assays for studying mismatch repair,
and in diagnostic applications for detecting the presence of a form
of PMS2 which may predispose a patient to cancer.
SUMMARY OF THE INVENTION
[0006] The invention relates to novel antibodies that specifically
bind PMS2. The antibodies specifically recognize a portion of the
amino-terminal portion of PMS2, such that truncation mutants of
PMS2 may also be detected.
[0007] The antibodies of the invention may be used in immunological
assays to detect the presence of PMS2 in a sample. The methods may
also be used to detect truncated forms of PMS2. Such assays
include, but are not limited to radioimmunoassay, Western blot,
ELISA, immunoprecipitation, and the like.
[0008] The antibodies of the invention may be used in a method for
detecting a predisposition to cancer in a patient, wherein a
truncated form of PMS2 is detected in a patient sample in a
screening assay and correlated to a risk of cancer in the
patient.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 shows reactivity in a Western blot of antibody from a
349-29.5.2 cell supernate and an HRP-conjugated purified antibody
from 349-29.5.2 against human PMS2-134 expressed in a human cell
line.
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0010] The reference works, patents, patent applications, and
scientific literature, including accession numbers to GenBank
database sequences that are referred to herein establish the
knowledge of those with skill in the art and are hereby
incorporated by reference in their entirety to the same extent as
if each was specifically and individually indicated to be
incorporated by reference. Any conflict between any reference cited
herein and the specific teachings of this specification shall be
resolved in favor of the latter. Likewise, any conflict between an
art-understood definition of a word or phrase and a definition of
the word or phrase as specifically taught in this specification
shall be resolved in favor of the latter.
[0011] Standard reference works setting forth the general
principles of recombinant DNA technology known to those of skill in
the art include Ausubel et al. CURRENT PROTOCOLS IN MOLECULAR
BIOLOGY, John Wiley & Sons, New York (1998); Sambrook et al.
MOLECULAR CLONING: A LABORATORY MANUAL, 2D ED., Cold Spring Harbor
Laboratory Press, Plainview, N.Y. (1989); Kaufman et al., Eds.,
HANDBOOK OF MOLECULAR AND CELLULAR METHODS IN BIOLOGY AND MEDICINE,
CRC Press, Boca Raton (1995); McPherson, Ed., DIRECTED MUTAGENESIS:
A PRACTICAL APPROACH, IRL Press, Oxford (1991).
[0012] As used herein, the term "epitope" refers to the portion of
an antigen to which a monoclonal antibody specifically binds.
[0013] As used herein, the term "conformational epitope" refers to
a discontinuous epitope formed by a spatial relationship between
amino acids of an antigen other than an unbroken series of amino
acids.
[0014] As used herein, the term "about" refers to an approximation
of a stated value within an acceptable range. Preferably the range
is +/-5% of the stated value.
[0015] The antibodies of the invention specifically bind to PMS2
and truncated fragments thereof. The antibodies include those in
which the epitope is found within the amino acid sequence of SEQ ID
NO: 1 or SEQ ID NO:2. In other embodiments, the epitope comprises
the sequence of SEQ ID NO: 1 or SEQ ID NO:2. In specific
embodiments, the antibody is 349-22.1.3. In other embodiments, the
antibody is 349-29.5.2.
[0016] In some embodiments the antibody is produced in a host cell
other than a hybridoma cell. In these cases the antibody genes are
cloned out of the hybridomas 349.22.1.3 and/or 349-29.5.2 and
placed in an expression vector, operably linked to expression
control sequences such that a functional antibody is produced.
[0017] Preferred antibodies and antibodies suitable for use in the
methods of the invention include, for example, fully human
antibodies, human antibody homologs, humanized antibody homologs,
chimeric antibody homologs, Fab, Fab', F(ab').sub.2 and F(v)
antibody fragments, single chain antibodies, and monomers or dimers
of antibody heavy or light chains or mixtures thereof.
[0018] The antibodies of the invention may include intact
immunoglobulins of any isotype including types IgA, IgG, IgE, IgD,
IgM (as well as subtypes thereof). The light chains of the
immunoglobulin may be kappa or lambda. Class switching may be
induced or may be engineered through recombinant techniques known
in the art using the antibodies expressed in the hybridoma cells
349-22.1.3 and 349-29.5.2.
[0019] The antibodies of the invention include portions of intact
antibodies that retain antigen-binding specificity, for example,
Fab fragments, Fab' fragments, F(ab').sub.2 fragments, F(v)
fragments, heavy chain monomers or dimers, light chain monomers or
dimers, dimers consisting of one heavy and one light chain, and the
like. Thus, antigen-binding fragments as well as full-length
dimeric or trimeric polypeptides derived from the above-described
antibodies are themselves useful.
[0020] The expression cells of the invention include any insect
expression cell line known, such as, for example, Spodoptera
frugiperda cells. The expression cell lines may also be bacterial
or fungal cell lines. The expression cell lines also may be yeast
cell lines, such as, for example, Saccharomyces cerevisiae and
Schizosaccharomyces pombe cells. The expression cells may also be
mammalian cells such as, for example, Chinese hamster ovary, baby
hamster kidney cells, human embryonic kidney line 293, normal dog
kidney cell lines, normal cat kidney cell lines, monkey kidney
cells, African green monkey kidney cells, COS cells, and
non-tumorigenic mouse myoblast G8 cells, fibroblast cell lines,
myeloma cell lines, mouse NIH/3T3 cells, LMTK31 cells, mouse
sertoli cells, human cervical carcinoma cells, buffalo rat liver
cells, human lung cells, human liver cells, mouse mammary tumor
cells, TRI cells, MRC 5 cells, and FS4 cells.
[0021] A "chimeric antibody" is an antibody produced by recombinant
DNA technology in which all or part of the hinge and constant
regions of an immunoglobulin light chain, heavy chain, or both,
have been substituted for the corresponding regions from another
animal's immunoglobulin light chain or heavy chain. In this way,
the antigen-binding portion of the parent monoclonal antibody is
grafted onto the backbone of another species' antibody. One
approach, described in EP 0239400 to Winter et al. describes the
substitution of one species' complementarity determining regions
(CDRs) for those of another species, such as substituting the CDRs
from human heavy and light chain immunoglobulin variable region
domains with CDRs from mouse variable region domains. These altered
antibodies may subsequently be combined with human immunoglobulin
constant regions to form antibodies that are human except for the
substituted murine CDRs which are specific for the antigen. Methods
for grafting CDR regions of antibodies may be found, for example in
Riechmann et al. (1988) Nature 332:323-327 and Verhoeyen et al.
(1988) Science 239:1534-1536.
[0022] Chimeric antibodies were thought to circumvent the problem
of eliciting an immune response in humans as chimeric antibodies
contain less murine amino acid sequence. It was found that the
direct use of rodent monoclonal antibodies (MAbs) as human
therapeutic agents led to human anti-rodent antibody ("HARA")
responses which occurred in a significant number of patients
treated with the rodent-derived antibody (Khazaeli, et al. (1994)
Immunother. 15:42-52).
[0023] As a non-limiting example, a method of performing CDR
grafting may be performed by sequencing the mouse heavy and light
chains of the antibody of interest that binds to the target antigen
(e.g., PMS2), genetically engineering the CDR DNA sequences, and
imposing these amino acid sequences to corresponding human V
regions by site-directed mutagenesis. Human constant region gene
segments of the desired isotype are added, and the "humanized"
heavy and light chain genes are co-expressed in mammalian cells to
produce soluble humanized antibody. A typical expression cell is a
Chinese Hamster Ovary (CHO) cell. Suitable methods for creating the
chimeric antibodies may be found, for example, in Jones et al.
(1986) Nature 321:522-525; Riechmann (1988) Nature 332:323-327;
Queen et al. (1989) Proc. Nat. Acad. Sci. USA 86:10029; and Orlandi
et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833.
[0024] Further refinement of antibodies to avoid the problem of
HARA responses led to the development of "humanized antibodies."
Humanized antibodies are produced by recombinant DNA technology, in
which at least one of the amino acids of a human immunoglobulin
light or heavy chain that is not required for antigen binding has
been substituted for the corresponding amino acid from a nonhuman
mammalian immunoglobulin light or heavy chain. For example, if the
immunoglobulin is a mouse monoclonal antibody, at least one amino
acid that is not required for antigen binding is substituted using
the amino acid that is present on a corresponding human antibody in
that position. Without wishing to be bound by any particular theory
of operation, it is believed that the "humanization" of the
monoclonal antibody inhibits human immunological reactivity against
the foreign immunoglobulin molecule.
[0025] Queen et al. (1989) Proc. Nat. Acad. Sci. USA 86:10029-10033
and WO 90/07861 describe the preparation of a humanized antibody.
Human and mouse variable framework regions were chosen for optimal
protein sequence homology. The tertiary structure of the murine
variable region was computer-modeled and superimposed on the
homologous human framework to show optimal interaction of amino
acid residues with the mouse CDRs. This led to the development of
antibodies with improved binding affinity for antigen (which is
typically decreased upon making CDR-grafted chimeric antibodies).
Alternative approaches to making humanized antibodies are known in
the art and are described, for example, in Tempest (1991)
Biotechnology 9:266-271.
[0026] "Single chain antibodies" refer to antibodies formed by
recombinant DNA techniques in which immunoglobulin heavy and light
chain fragments are linked to the F(v) region via an engineered
span of amino acids. Various methods of generating single chain
antibodies are known, including those described in U.S. Pat. No.
4,694,778; Bird (1988) Science 242:423-442; Huston et al. (1988)
Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature
334:54454; and Skerra et al. (1988) Science 242:1038-1041.
[0027] The antibodies of the invention may be used alone or as
immunoconjugates with a label. Such labels include enzymes, biotin,
radionuclides, fluorophores, chemiluminescers, paramagnetic
particles, and the like. Suitable labels include, but are not
limited to fluorescein, rhodamine, isothiocyanate, phycoerythrin,
horseradish peroxidase, and colloidal gold.
[0028] The antibodies of the invention include derivatives that are
modified, e.g., by the covalent attachment of any type of molecule
to the antibody such that covalent attachment does not prevent the
antibody from binding to its epitope. Examples of suitable
derivatives include, but are not limited to glycosyled antibodies
and fragments, acetyled antibodies and fragments, pegylated
antibodies and fragments, phosphorylated antibodies and fragments,
and amidated antibodies and fragments. The antibodies and
derivatives thereof of the invention may themselves by derivatized
by known protecting/blocking groups, proteolytic cleavage, linkage
to a cellular ligand or other proteins, and the like. Further, the
antibodies and derivatives thereof of the invention may contain one
or more non-classical amino acids.
[0029] The monoclonal antibodies of the invention may be produced
by immunizing animals with PMS2, truncated fragments thereof, or
peptide fragments thereof. Animals so immunized will produce
antibodies against the protein. Standard methods are known for
creating monoclonal antibodies including, but are not limited to,
the hybridoma technique (see Kohler & Milstein (1975) Nature
256:495-497); the trioma technique; the human B-cell hybridoma
technique (see Kozbor et al. (1983) Immunol Today 4:72) and the EBV
hybridoma technique to produce human monoclonal antibodies (see
Cole, et al. in MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.
Liss, Inc., 1985, pp. 77-96).
[0030] Screening for antibodies that specifically bind to PMS2 or
truncated fragments thereof may be accomplished using an
enzyme-linked immunosorbent assay (ELISA) in which microtiter
plates are coated with the PMS2, for example.
[0031] Confirmation of reactivity of the antibodies to PMS2, or
truncated forms thereof may be accomplished, for example, using a
Western Blot assay in which protein from normal patients or a
patient with Hereditary Non-Polyposis Colon Cancer (HNPCC) are run
on an SDS-PAGE gel under reducing and non-reducing conditions and
subsequently are blotted onto a membrane. The membrane may then be
probed with the putative anti-PMS2 antibodies. Appropriately-sized
bands on Western indicates specificity of the antibodies and the
ability to bind both full-length and truncated forms of PMS2.
[0032] The antibodies and derivatives thereof of the invention have
binding affinities that include a dissociation constant (K.sub.d)
of less than 1.times.10.sup.-2. In some embodiments, the K.sub.d is
less than 1.times.10.sup.-3. In other embodiments, the K.sub.d is
less than 1.times.10.sup.-4. In some embodiments, the K.sub.d is
less than 1.times.10.sup.-5. In still other embodiments, the
K.sub.d is less than 1.times.10.sup.-6. In other embodiments, the
K.sub.d is less than 1.times.10.sup.-7. In other embodiments, the
K.sub.d is less than 1.times.10.sup.-8. In other embodiments, the
K.sub.d is less than 1.times.10.sup.-9. In other embodiments, the
K.sub.d is less than 1.times.10.sup.-10. In still other
embodiments, the K.sub.d is less than 1.times.10.sup.-11. In some
embodiments, the K.sub.d is less than 1.times.10.sup.-12. In other
embodiments, the K.sub.d is less than 1.times.10.sup.-13. In other
embodiments, the K.sub.d is less than 1.times.10.sup.-14. In still
other embodiments, the K.sub.d is less than 1.times.10.sup.-15.
[0033] Antibodies of the invention may be produced in vivo or in
vitro. For in vivo antibody production, animals are generally
immunized with an immunogenic portion of PMS2 (such as an
immunogenic peptide of PMS2). The antigen is generally combined
with an adjuvant to promote immunogenicity. Adjuvants vary
according to the species used for immunization. Examples of
adjuvants include, but are not limited to: Freund's complete
adjuvant ("FCA"), Freund's incomplete adjuvant ("FIA"), mineral
gels (e.g., aluminum hydroxide), surface active substances (e.g.,
lysolecithin, pluronic polyols, polyanions), peptides, oil
emulsions, keyhole limpet hemocyanin ("KLH"), dinitrophenol
("DNP"), and potentially useful human adjuvants such as Bacille
Calmette-Guerin ("BCG") and corynebacterium parvum. Such adjuvants
are also well known in the art.
[0034] Immunization may be accomplished using well-known
procedures. The dose and immunization regimen will depend on the
species of mammal immunized, its immune status, body weight, and/or
calculated surface area, etc. Typically, blood serum is sampled
from the immunized mammals and assayed for anti-PMS2 antibodies
using appropriate screening assays as described below, for
example.
[0035] Splenocytes from immunized animals may be immortalized by
fusing the splenocytes (containing the antibody-producing B cells)
with an immortal cell line such as a myeloma line. Typically,
myeloma cell line is from the same species as the splenocyte donor.
In one embodiment, the immortal cell line is sensitive to culture
medium containing hypoxanthine, aminopterin and thymidine ("HAT
medium"). In some embodiments, the myeloma cells are negative for
Epstein-Barr virus (EBV) infection. In preferred embodiments, the
myeloma cells are HAT-sensitive, EBV negative and Ig expression
negative. Any suitable myeloma may be used. Murine hybridomas may
be generated using mouse myeloma cell lines (e.g., the
P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myeloma lines). These
murine myeloma lines are available from the ATCC. These myeloma
cells are fused to the donor splenocytes polyethylene glycol
("PEG"), preferably 1500 molecular weight polyethylene glycol ("PEG
1500"). Hybridoma cells resulting from the fusion are selected in
HAT medium which kills unfused and unproductively fused myeloma
cells. Unfused splenocytes die over a short period of time in
culture. In some embodiments, the myeloma cells do not express
immunoglobulin genes.
[0036] Hybridomas producing a desired antibody which are detected
by screening assays, such as those described below, may be used to
produce antibodies in culture or in animals. For example, the
hybridoma cells may be cultured in a nutrient medium under
conditions and for a time sufficient to allow the hybridoma cells
to secrete the monoclonal antibodies into the culture medium. These
techniques and culture media are well known by those skilled in the
art. Alternatively, the hybridoma cells may be injected into the
peritoneum of an unimmunized animal. The cells proliferate in the
peritoneal cavity and secrete the antibody, which accumulates as
ascites fluid. The ascites fluid may be withdrawn from the
peritoneal cavity with a syringe as a rich source of the monoclonal
antibody.
[0037] Another non-limiting method for producing human antibodies
is described in U.S. Pat. No. 5,789,650 which describes transgenic
mammals that produce antibodies of another species (e.g., humans)
with their own endogenous immunoglobulin genes being inactivated.
The genes for the heterologous antibodies are encoded by human
immunoglobulin genes. The transgenes containing the unrearranged
immunoglobulin encoding regions are introduced into a non-human
animal. The resulting transgenic animals are capable of
functionally rearranging the transgenic immunoglobulin sequences
and producing a repertoire of antibodies of various isotypes
encoded by human immunoglobulin genes. The B-cells from the
transgenic animals are subsequently immortalized by any of a
variety of methods, including fusion with an immortalizing cell
line (e.g., a myeloma cell).
[0038] Antibodies against PMS2 may also be prepared in vitro using
a variety of techniques known in the art. For example, but not by
way of limitation, fully human monoclonal antibodies against PMS2
may be prepared by using in vitro-primed human splenocytes (Boerner
et al. (1991) J. Immunol. 147:86-95).
[0039] Alternatively, for example, the antibodies of the invention
may be prepared by "repertoire cloning" (Persson et al. (1991)
Proc. Nat. Acad. Sci. USA 88:2432-2436; and Huang and Stollar
(1991) J. Immunol. Methods 141:227-236). Further, U.S. Pat. No.
5,798,230 describes preparation of human monoclonal antibodies from
human B antibody-producing B cells that are immortalized by
infection with an Epstein-Barr virus that expresses Epstein-Barr
virus nuclear antigen 2 (EBNA2). EBNA2, required for
immortalization, is then inactivated resulting in increased
antibody titers.
[0040] In another embodiment, antibodies against PMS2 are formed by
in vitro immunization of peripheral blood mononuclear cells
("PBMCs"). This may be accomplished by any means known in the art,
such as, for example, using methods described in the literature
(Zafiropoulos et al. (1997) J. Immunological Methods
200:181-190).
[0041] In one embodiment of the invention, the procedure for in
vitro immunization is supplemented with directed evolution of the
hybridoma cells in which a dominant negative allele of a mismatch
repair gene such as PMS1, PMS2, PMS2-134, PMSR2, PMSR3, MLH1, MLH2,
MLH3, MLH4, MLH5, MLH6, PMSL9, MSH1, and MSH2 is introduced into
the hybridoma cells after fusion of the splenocytes, or to the
myeloma cells before fusion. Cells containing the dominant negative
mutant will become hypermutable and accumulate mutations at a
higher rate than untransfected control cells. A pool of the
mutating cells may be screened for clones that produce higher
affinity antibodies, or that produce higher titers of antibodies,
or that simply grow faster or better under certain conditions. The
technique for generating hypermutable cells using dominant negative
alleles of mismatch repair genes is described in U.S. Pat. No.
6,146,894, issued Nov. 14, 2000. Alternatively, mismatch repair may
be inhibited using the chemical inhibitors of mismatch repair
described by Nicolaides et al. in WO 02/054856 "Chemical Inhibitors
of Mismatch Repair" published Jul. 18, 2002. The technique for
enhancing antibodies using the dominant negative alleles of
mismatch repair genes or chemical inhibitors of mismatch repair may
be applied to mammalian expression cells expressing cloned
immunoglobulin genes as well. Cells expressing the dominant
negative alleles can be "cured" in that the dominant negative
allele can be turned off if inducible, eliminated from the cell,
and the like, such that the cells become genetically stable once
more and no longer accumulate mutations at the abnormally high
rate.
[0042] The immunogen may be any PMS2, however, mammalian PMS2 is
preferred. Truncated forms of PMS2 may also be used. As the
N-terminus of PMS2 is highly conserved across species, in some
embodiments, antibodies that recognize one species of PMS2 is
expected to also recognize another species. For example, but not by
way of limitation, a monoclonal antibody that binds human PMS2 (SEQ
ID NO:2) in the N-terminal region may also bind the same region in
mouse PMS2 (SEQ ID NO:5) and even Arabidopsis thaliana PMS2 (SEQ ID
NO:6) and in the truncated human PMS2-134 (SEQ ID NO:1). The
immunogen may also be immunogenic peptides of PMS2 or highly
conserved peptides of PMS2. Two such peptides that may be used are:
IQEFADLTQVETFGFR (SEQ ID NO:3) and ELVENSLDAGATNIDLK (SEQ ID
NO:4).
[0043] The invention also provides a method for detecting an
abnormal condition in a patient expressing a truncated PMS2. The
method comprises contacting a test cell lysate from the patient
suspected of having a defect in mismatch repair with a monoclonal
antibody secreted by hybridoma cell 349-29.5.2 or 349-22.1.3 and
detecting the presence or absence of a truncated form of PMS2. The
presence of a truncated form of PMS2 is indicative of an abnormal
condition in mismatch repair which predisposes the patient to
cancer. Such cancers include, but are not limited to hereditary
non-polyposis colon cancer. The presence of the truncated form of
PMS2 may be detected by various means including
immunoprecipitation, western blot, and ELISA.
[0044] The following Examples are provided to illustrate the
present invention, and should not be construed as limiting
thereof.
Example 1
Immunogen Expression
[0045] Five milliliters of IPTG-induced (100 mM) culture of E. coli
BL21(DE3) cells transformed with plasmid p-ET-k-134 (a plasmid that
expresses hPMS2-134 from a T7 promoter, out of frame with His tag,
NB37p46) were obtained. Expression was induced by inoculation of 1
ml (OD.sub.600=0.5) into 45 ml LB-Kan (50 mg/ml). The cells were
lysed by addition of B-PER bacterial protein extraction reagent,
and inclusion bodies were purified from lysates as per
manufacturer's instructions. The inclusion body pellet was
dissolved in 400 .mu.l 2.times. LDS sample buffer, boiled 5 min,
and electrophoresed 125 .mu.l/gel, on 4 gels, of solubilized
inclusion bodies in reducing 12% Bis-Tris 2-D gels in MES buffer.
The gels were stained with Gelcode Blue colloidal Coomassie Blue
(Pierce). Fifteen kilodalton bands were excised and sent to St.
Louis University Hybridoma Facility. One gel slice was subjected to
amino acid analysis. Amino acid analysis was consistent with
hPMS2-134 polypeptide. Another gel slice was processed for
MALDI-TOF MS analysis of trpytic peptides (NB37p72). Two peptide
matches to hPMS2-134 were found upon database search
(IQEFADLTQVETFGFR (SEQ ID NO:3) and ELVENSLDAGATNIDLK (SEQ ID
NO:4)). For generation of hybridomas, four mice were immunized. All
four were shown to be reactive to the original immunogen by Western
blotting using mouse sera. Mouse #464 was chosen for lymphocyte
fusion (NB70p3).
Example 2
Cloning of a Second Bacterial Expression Construct and IMAC
Purification of His-hPMS2-134
[0046] A second arabinose-inducible bacterial expression construct
was made in plasmid pBAD-HisA, this time with an N-terminal
6.times. His tag in-frame with hPMS2-134 (NB37 .mu.l). This plasmid
was designated p0126. His-tagged hPMS2-134 was purified from
induced cultures of BL21 carrying p0126 by immobilized metal
affinity chromatography over Talon cobalt affinity resin (Clontech,
NB37p93). A single hybridoma which reacted specifically with
purified hPMS2-134 (clone 349-1) was identified.
Example 3
Screening of Murine Hybridomas and Epitope Mapping
[0047] Clone 349-1 was further subcloned by limiting dilution and
screened again (NB70p8). Twelve subclones from 349-1 were tested
for reactivity by Western blotting. All 12 clones were specifically
reactive to bacterially produced hPMS2-134 (NB70p12). Only clone
349-1.1 was reactive towards hPMS2-134 expressed from CHO-124 or
CHO-125 (CHO transfectants expressing hPMS2-134 or C-terminal
V5-tagged hPMS2-134, respectively, NB70p14). A second set of three
twice-subcloned hybridomas from 349-1 (349-1.2.1 through
349-1.2.3), were obtained, as well as four twice-subcloned
hybridomas from 349-1 (349-1.1.1 through 349-1.1.4) and all were
tested against bacterially expressed hPMS2-134. All retained
reactivity against hPMS2-134. However, only clone 349-1.2.2
displayed specific reactivity towards CHO-expressed hPMS2-134. This
mAb also identified a second band of Mr 120 kD from CHO lysates
(putative hamster PMS2). Two hybridomas were retained from this
screen (349-1.1.3 and 349-1.2.2). IgG was purified from 35 ml of
culture supernate of each by protein G chromatography (NB70p44).
Neither purified mAb specifically reacted with hPMS2-134 expressed
in CHO.
[0048] A second round of fusion, using mouse #480, and screening
was initiated (NB70p48). Seventeen hybridomas were selected (based
on their reactivity towards bacterially expressed hPMS2-134 by the
Yaciuk group) and tested for reactivity towards CHO-expressed
hPMS2-134. None displayed specific reactivity towards hPMS2-134.
Screening against bacterial hPMS2-134 was repeated. Four hybridomas
(349-22, 349-25, 349-29, 349-36) were reactive (NB70p52).
[0049] Deletion studies pointed to the originally isolated mAbs
(349-1.1.3 and 349-1.2.2) sharing an epitope C-terminal to residue
81, while second generation mAbs shared epitopes located between
amino acids 55 and 81. Epitope mapping studies using overlapping
15-mer peptides failed to identify relevant epitopes.
[0050] Second generation hybridomas (from mouse #480) were
subcloned by limiting dilution twice. Culture supernatants were
tested for reactivity towards bacterial hPMS2-134. The majority
displayed reactivity by Western blotting (NB71p7). Of these, clones
349-22.1.3 and 349-29.5.2 were selected for expansion. Further
validation was performed. Horseradish peroxidase (HRP) conjugation
to 349-29.5.2 was conducted, and the results of a Western blot
probed with supernatant fluid from clone 349-29.5.2 and with
HRP-conjugated 349-29.5.2 antibody is shown in FIG. 1. Each well
contained increasing amounts of a human cell line expressing
PMS2-134. The wells shown contained 30,000; 60,000; 90,000; and
120,000 cells/well in lanes 1, 2, 3, and 4, respectively.
Sequence CWU 1
1
61133PRTHomo sapiens 1Met Glu Arg Ala Glu Ser Ser Ser Thr Glu Pro
Ala Lys Ala Ile Lys1 5 10 15Pro Ile Asp Arg Lys Ser Val His Gln Ile
Cys Ser Gly Gln Val Val20 25 30Leu Ser Leu Ser Thr Ala Val Lys Glu
Leu Val Glu Asn Ser Leu Asp35 40 45Ala Gly Ala Thr Asn Ile Asp Leu
Lys Leu Lys Asp Tyr Gly Val Asp50 55 60Leu Ile Glu Val Ser Asp Asn
Gly Cys Gly Val Glu Glu Glu Asn Phe65 70 75 80Glu Gly Leu Thr Leu
Lys His His Thr Ser Lys Ile Gln Glu Phe Ala85 90 95Asp Leu Thr Gln
Val Glu Thr Phe Gly Phe Arg Gly Glu Ala Leu Ser100 105 110Ser Leu
Cys Ala Leu Ser Asp Val Thr Ile Ser Thr Cys His Ala Ser115 120
125Ala Lys Val Gly Thr1302862PRTHomo sapiens 2Met Glu Arg Ala Glu
Ser Ser Ser Thr Glu Pro Ala Lys Ala Ile Lys1 5 10 15Pro Ile Asp Arg
Lys Ser Val His Gln Ile Cys Ser Gly Gln Val Val20 25 30Leu Ser Leu
Ser Thr Ala Val Lys Glu Leu Val Glu Asn Ser Leu Asp35 40 45Ala Gly
Ala Thr Asn Ile Asp Leu Lys Leu Lys Asp Tyr Gly Val Asp50 55 60Leu
Ile Glu Val Ser Asp Asn Gly Cys Gly Val Glu Glu Glu Asn Phe65 70 75
80Glu Gly Leu Thr Leu Lys His His Thr Ser Lys Ile Gln Glu Phe Ala85
90 95Asp Leu Thr Gln Val Glu Thr Phe Gly Phe Arg Gly Glu Ala Leu
Ser100 105 110Ser Leu Cys Ala Leu Ser Asp Val Thr Ile Ser Thr Cys
His Ala Ser115 120 125Ala Lys Val Gly Thr Arg Leu Met Phe Asp His
Asn Gly Lys Ile Ile130 135 140Gln Lys Thr Pro Tyr Pro Arg Pro Arg
Gly Thr Thr Val Ser Val Gln145 150 155 160Gln Leu Phe Ser Thr Leu
Pro Val Arg His Lys Glu Phe Gln Arg Asn165 170 175Ile Lys Lys Glu
Tyr Ala Lys Met Val Gln Val Leu His Ala Tyr Cys180 185 190Ile Ile
Ser Ala Gly Ile Arg Val Ser Cys Thr Asn Gln Leu Gly Gln195 200
205Gly Lys Arg Gln Pro Val Val Cys Thr Gly Gly Ser Pro Ser Ile
Lys210 215 220Glu Asn Ile Gly Ser Val Phe Gly Gln Lys Gln Leu Gln
Ser Leu Ile225 230 235 240Pro Phe Val Gln Leu Pro Pro Ser Asp Ser
Val Cys Glu Glu Tyr Gly245 250 255Leu Ser Cys Ser Asp Ala Leu His
Asn Leu Phe Tyr Ile Ser Gly Phe260 265 270Ile Ser Gln Cys Thr His
Gly Val Gly Arg Ser Ser Thr Asp Arg Gln275 280 285Phe Phe Phe Ile
Asn Arg Arg Pro Cys Asp Pro Ala Lys Val Cys Arg290 295 300Leu Val
Asn Glu Val Tyr His Met Tyr Asn Arg His Gln Tyr Pro Phe305 310 315
320Val Val Leu Asn Ile Ser Val Asp Ser Glu Cys Val Asp Ile Asn
Val325 330 335Thr Pro Asp Lys Arg Gln Ile Leu Leu Gln Glu Glu Lys
Leu Leu Leu340 345 350Ala Val Leu Lys Thr Ser Leu Ile Gly Met Phe
Asp Ser Asp Val Asn355 360 365Lys Leu Asn Val Ser Gln Gln Pro Leu
Leu Asp Val Glu Gly Asn Leu370 375 380Ile Lys Met His Ala Ala Asp
Leu Glu Lys Pro Met Val Glu Lys Gln385 390 395 400Asp Gln Ser Pro
Ser Leu Arg Thr Gly Glu Glu Lys Lys Asp Val Ser405 410 415Ile Ser
Arg Leu Arg Glu Ala Phe Ser Leu Arg His Thr Thr Glu Asn420 425
430Lys Pro His Ser Pro Lys Thr Pro Glu Pro Arg Arg Ser Pro Leu
Gly435 440 445Gln Lys Arg Gly Met Leu Ser Ser Ser Thr Ser Gly Ala
Ile Ser Asp450 455 460Lys Gly Val Leu Arg Pro Gln Lys Glu Ala Val
Ser Ser Ser His Gly465 470 475 480Pro Ser Asp Pro Thr Asp Arg Ala
Glu Val Glu Lys Asp Ser Gly His485 490 495Gly Ser Thr Ser Val Asp
Ser Glu Gly Phe Ser Ile Pro Asp Thr Gly500 505 510Ser His Cys Ser
Ser Glu Tyr Ala Ala Ser Ser Pro Gly Asp Arg Gly515 520 525Ser Gln
Glu His Val Asp Ser Gln Glu Lys Ala Pro Glu Thr Asp Asp530 535
540Ser Phe Ser Asp Val Asp Cys His Ser Asn Gln Glu Asp Thr Gly
Cys545 550 555 560Lys Phe Arg Val Leu Pro Gln Pro Thr Asn Leu Ala
Thr Pro Asn Thr565 570 575Lys Arg Phe Lys Lys Glu Glu Ile Leu Ser
Ser Ser Asp Ile Cys Gln580 585 590Lys Leu Val Asn Thr Gln Asp Met
Ser Ala Ser Gln Val Asp Val Ala595 600 605Val Lys Ile Asn Lys Lys
Val Val Pro Leu Asp Phe Ser Met Ser Ser610 615 620Leu Ala Lys Arg
Ile Lys Gln Leu His His Glu Ala Gln Gln Ser Glu625 630 635 640Gly
Glu Gln Asn Tyr Arg Lys Phe Arg Ala Lys Ile Cys Pro Gly Glu645 650
655Asn Gln Ala Ala Glu Asp Glu Leu Arg Lys Glu Ile Ser Lys Thr
Met660 665 670Phe Ala Glu Met Glu Ile Ile Gly Gln Phe Asn Leu Gly
Phe Ile Ile675 680 685Thr Lys Leu Asn Glu Asp Ile Phe Ile Val Asp
Gln His Ala Thr Asp690 695 700Glu Lys Tyr Asn Phe Glu Met Leu Gln
Gln His Thr Val Leu Gln Gly705 710 715 720Gln Arg Leu Ile Ala Pro
Gln Thr Leu Asn Leu Thr Ala Val Asn Glu725 730 735Ala Val Leu Ile
Glu Asn Leu Glu Ile Phe Arg Lys Asn Gly Phe Asp740 745 750Phe Val
Ile Asp Glu Asn Ala Pro Val Thr Glu Arg Ala Lys Leu Ile755 760
765Ser Leu Pro Thr Ser Lys Asn Trp Thr Phe Gly Pro Gln Asp Val
Asp770 775 780Glu Leu Ile Phe Met Leu Ser Asp Ser Pro Gly Val Met
Cys Arg Pro785 790 795 800Ser Arg Val Lys Gln Met Phe Ala Ser Arg
Ala Cys Arg Lys Ser Val805 810 815Met Ile Gly Thr Ala Leu Asn Thr
Ser Glu Met Lys Lys Leu Ile Thr820 825 830His Met Gly Glu Met Asp
His Pro Trp Asn Cys Pro His Gly Arg Pro835 840 845Thr Met Arg His
Ile Ala Asn Leu Gly Val Ile Ser Gln Asn850 855 860316PRTHomo
sapiens 3Ile Gln Glu Phe Ala Asp Leu Thr Gln Val Glu Thr Phe Gly
Phe Arg1 5 10 15417PRTHomo sapiens 4Glu Leu Val Glu Asn Ser Leu Asp
Ala Gly Ala Thr Asn Ile Asp Leu1 5 10 15Lys5859PRTMus musculus 5Met
Glu Gln Thr Glu Gly Val Ser Thr Glu Cys Ala Lys Ala Ile Lys1 5 10
15Pro Ile Asp Gly Lys Ser Val His Gln Ile Cys Ser Gly Gln Val Ile20
25 30Leu Ser Leu Ser Thr Ala Val Lys Glu Leu Ile Glu Asn Ser Val
Asp35 40 45Ala Gly Ala Thr Thr Ile Asp Leu Arg Leu Lys Asp Tyr Gly
Val Asp50 55 60Leu Ile Glu Val Ser Asp Asn Gly Cys Gly Val Glu Glu
Glu Asn Phe65 70 75 80Glu Gly Leu Ala Leu Lys His His Thr Ser Lys
Ile Gln Glu Phe Ala85 90 95Asp Leu Thr Gln Val Glu Thr Phe Gly Phe
Arg Gly Glu Ala Leu Ser100 105 110Ser Leu Cys Ala Leu Ser Asp Val
Thr Ile Ser Thr Cys His Gly Ser115 120 125Ala Ser Val Gly Thr Arg
Leu Val Phe Asp His Asn Gly Lys Ile Thr130 135 140Gln Lys Thr Pro
Tyr Pro Arg Pro Lys Gly Thr Thr Val Ser Val Gln145 150 155 160His
Leu Phe Tyr Thr Leu Pro Val Arg Tyr Lys Glu Phe Gln Arg Asn165 170
175Ile Lys Lys Glu Tyr Ser Lys Met Val Gln Val Leu Gln Ala Tyr
Cys180 185 190Ile Ile Ser Ala Gly Val Arg Val Ser Cys Thr Asn Gln
Leu Gly Gln195 200 205Gly Lys Arg His Ala Val Val Cys Thr Ser Gly
Thr Ser Gly Met Lys210 215 220Glu Asn Ile Gly Ser Val Phe Gly Gln
Lys Gln Leu Gln Ser Leu Ile225 230 235 240Pro Phe Val Gln Leu Pro
Pro Ser Asp Ala Val Cys Glu Glu Tyr Gly245 250 255Leu Ser Thr Ser
Gly Arg His Lys Thr Phe Ser Thr Phe Arg Ala Ser260 265 270Phe His
Ser Ala Arg Thr Ala Pro Gly Gly Val Gln Gln Thr Gly Ser275 280
285Phe Ser Ser Ser Ile Arg Gly Pro Val Thr Gln Gln Arg Ser Leu
Ser290 295 300Leu Ser Met Arg Phe Tyr His Met Tyr Asn Arg His Gln
Tyr Pro Phe305 310 315 320Val Val Leu Asn Val Ser Val Asp Ser Glu
Cys Val Asp Ile Asn Val325 330 335Thr Pro Asp Lys Arg Gln Ile Leu
Leu Gln Glu Glu Lys Leu Leu Leu340 345 350Ala Val Leu Lys Thr Ser
Leu Ile Gly Met Phe Asp Ser Asp Ala Asn355 360 365Lys Leu Asn Val
Asn Gln Gln Pro Leu Leu Asp Val Glu Gly Asn Leu370 375 380Val Lys
Leu His Thr Ala Glu Leu Glu Lys Pro Val Pro Gly Lys Gln385 390 395
400Asp Asn Ser Pro Ser Leu Lys Ser Thr Ala Asp Glu Lys Arg Val
Ala405 410 415Ser Ile Ser Arg Leu Arg Glu Ala Phe Ser Leu His Pro
Thr Lys Glu420 425 430Ile Lys Ser Arg Gly Pro Glu Thr Ala Glu Leu
Thr Arg Ser Phe Pro435 440 445Ser Glu Lys Arg Gly Val Leu Ser Ser
Tyr Pro Ser Asp Val Ile Ser450 455 460Tyr Arg Gly Leu Arg Gly Ser
Gln Asp Lys Leu Val Ser Pro Thr Asp465 470 475 480Ser Pro Gly Asp
Cys Met Asp Arg Glu Lys Ile Glu Lys Asp Ser Gly485 490 495Leu Ser
Ser Thr Ser Ala Gly Ser Glu Glu Glu Phe Ser Thr Pro Glu500 505
510Val Ala Ser Ser Phe Ser Ser Asp Tyr Asn Val Ser Ser Leu Glu
Asp515 520 525Arg Pro Ser Gln Glu Thr Ile Asn Cys Gly Asp Leu Asp
Cys Arg Pro530 535 540Pro Gly Thr Gly Gln Ser Leu Lys Pro Glu Asp
His Gly Tyr Gln Cys545 550 555 560Lys Ala Leu Pro Leu Ala Arg Leu
Ser Pro Thr Asn Ala Lys Arg Phe565 570 575Lys Thr Glu Glu Arg Pro
Ser Asn Val Asn Ile Ser Gln Arg Leu Pro580 585 590Gly Pro Gln Ser
Thr Ser Ala Ala Glu Val Asp Val Ala Ile Lys Met595 600 605Asn Lys
Arg Ile Val Leu Leu Glu Phe Ser Leu Ser Ser Leu Ala Lys610 615
620Arg Met Lys Gln Leu Gln His Leu Lys Ala Gln Asn Lys His Glu
Leu625 630 635 640Ser Tyr Arg Lys Phe Arg Ala Lys Ile Cys Pro Gly
Glu Asn Gln Ala645 650 655Ala Glu Asp Glu Leu Arg Lys Glu Ile Ser
Lys Ser Met Phe Ala Glu660 665 670Met Glu Ile Leu Gly Gln Phe Asn
Leu Gly Phe Ile Val Thr Lys Leu675 680 685Lys Glu Asp Leu Phe Leu
Val Asp Gln His Ala Ala Asp Glu Lys Tyr690 695 700Asn Phe Glu Met
Leu Gln Gln His Thr Val Leu Gln Ala Gln Arg Leu705 710 715 720Ile
Thr Pro Gln Thr Leu Asn Leu Thr Ala Val Asn Glu Ala Val Leu725 730
735Ile Glu Asn Leu Glu Ile Phe Arg Lys Asn Gly Phe Asp Phe Val
Ile740 745 750Asp Glu Asp Ala Pro Val Thr Glu Arg Ala Lys Leu Ile
Ser Leu Pro755 760 765Thr Ser Lys Asn Trp Thr Phe Gly Pro Gln Asp
Ile Asp Glu Leu Ile770 775 780Phe Met Leu Ser Asp Ser Pro Gly Val
Met Cys Arg Pro Ser Arg Val785 790 795 800Arg Gln Met Phe Ala Ser
Arg Ala Cys Arg Lys Ser Val Met Ile Gly805 810 815Thr Ala Leu Asn
Ala Ser Glu Met Lys Lys Leu Ile Thr His Met Gly820 825 830Glu Met
Asp His Pro Trp Asn Cys Pro His Gly Arg Pro Thr Met Arg835 840
845His Val Ala Asn Leu Asp Val Ile Ser Gln Asn850
8556923PRTArabidopsis thaliana 6Met Gln Gly Asp Ser Ser Pro Ser Pro
Thr Thr Thr Ser Ser Pro Leu1 5 10 15Ile Arg Pro Ile Asn Arg Asn Val
Ile His Arg Ile Cys Ser Gly Gln20 25 30Val Ile Leu Asp Leu Ser Ser
Ala Val Lys Glu Leu Val Glu Asn Ser35 40 45Leu Asp Ala Gly Ala Thr
Ser Ile Glu Ile Asn Leu Arg Asp Tyr Gly50 55 60Glu Asp Tyr Phe Gln
Val Ile Asp Asn Gly Cys Gly Ile Ser Pro Thr65 70 75 80Asn Phe Lys
Val Leu Ala Leu Lys His His Thr Ser Lys Leu Glu Asp85 90 95Phe Thr
Asp Leu Leu Asn Leu Thr Thr Tyr Gly Phe Arg Gly Glu Ala100 105
110Leu Ser Ser Leu Cys Ala Leu Gly Asn Leu Thr Val Glu Thr Arg
Thr115 120 125Lys Asn Glu Pro Val Ala Thr Leu Leu Thr Phe Asp His
Ser Gly Leu130 135 140Leu Thr Ala Glu Lys Lys Thr Ala Arg Gln Ile
Gly Thr Thr Val Thr145 150 155 160Val Arg Lys Leu Phe Ser Asn Leu
Pro Val Arg Ser Lys Glu Phe Lys165 170 175Arg Asn Ile Arg Lys Glu
Tyr Gly Lys Leu Val Ser Leu Leu Asn Ala180 185 190Tyr Ala Leu Ile
Ala Lys Gly Val Arg Phe Val Cys Ser Asn Thr Thr195 200 205Gly Lys
Asn Pro Lys Ser Val Val Leu Asn Thr Gln Gly Arg Gly Ser210 215
220Leu Lys Asp Asn Ile Ile Thr Val Phe Gly Ile Ser Thr Phe Thr
Ser225 230 235 240Leu Gln Pro Val Ser Ile Cys Val Ser Glu Asp Cys
Arg Val Glu Gly245 250 255Phe Leu Ser Lys Pro Gly Gln Gly Thr Gly
Arg Asn Leu Ala Asp Arg260 265 270Gln Tyr Phe Phe Ile Asn Gly Arg
Pro Val Asp Met Pro Lys Val Ser275 280 285Lys Leu Val Asn Glu Leu
Tyr Lys Asp Thr Ser Ser Arg Lys Tyr Pro290 295 300Val Thr Ile Leu
Asp Phe Ile Val Pro Gly Gly Ala Cys Asp Leu Asn305 310 315 320Val
Thr Pro Asp Lys Arg Lys Val Phe Phe Ser Asp Glu Thr Ser Val325 330
335Ile Gly Ser Leu Arg Glu Gly Leu Asn Glu Ile Tyr Ser Ser Ser
Asn340 345 350Ala Ser Tyr Ile Val Asn Arg Phe Glu Glu Asn Ser Glu
Gln Pro Asp355 360 365Lys Ala Gly Val Ser Ser Phe Gln Lys Lys Ser
Asn Leu Leu Ser Glu370 375 380Gly Ile Val Leu Asp Val Ser Ser Lys
Thr Arg Leu Gly Glu Ala Ile385 390 395 400Glu Lys Glu Asn Pro Ser
Leu Arg Glu Val Glu Ile Asp Asn Ser Ser405 410 415Pro Met Glu Lys
Phe Lys Phe Glu Ile Lys Ala Cys Gly Thr Lys Lys420 425 430Gly Glu
Gly Ser Leu Ser Val His Asp Val Thr His Leu Asp Lys Thr435 440
445Pro Ser Lys Gly Leu Pro Gln Leu Asn Val Thr Glu Lys Val Thr
Asp450 455 460Ala Ser Lys Asp Leu Ser Ser Arg Ser Ser Phe Ala Gln
Ser Thr Leu465 470 475 480Asn Thr Phe Val Thr Met Gly Lys Arg Lys
His Glu Asn Ile Ser Thr485 490 495Ile Leu Ser Glu Thr Pro Val Leu
Arg Asn Gln Thr Ser Ser Tyr Arg500 505 510Val Glu Lys Ser Lys Phe
Glu Val Arg Ala Leu Ala Ser Arg Cys Leu515 520 525Val Glu Gly Asp
Gln Leu Asp Asp Met Val Ile Ser Lys Glu Asp Met530 535 540Thr Pro
Ser Glu Arg Asp Ser Glu Leu Gly Asn Arg Ile Ser Pro Gly545 550 555
560Thr Gln Ala Asp Asn Val Glu Arg His Glu Arg Glu His Glu Lys
Pro565 570 575Ile Arg Phe Glu Glu Pro Thr Ser Asp Asn Thr Leu Thr
Lys Gly Asp580 585 590Val Glu Arg Val Ser Glu Asp Asn Pro Arg Cys
Ser Gln Pro Leu Arg595 600 605Ser Val Ala Thr Val Leu Asp Ser Pro
Ala Gln Ser Thr Gly Pro Lys610 615 620Met Phe Ser Thr Leu Glu Phe
Ser Phe Gln Asn Leu Arg Thr Arg Arg625 630 635 640Leu Glu Arg Leu
Ser Arg Leu Gln Ser Thr Gly Tyr Val Ser Lys Cys645 650 655Met Asn
Thr Pro Gln Pro Lys Lys Cys Phe Ala Ala Ala Thr Leu Glu660 665
670Leu Ser Gln Pro Asp Asp Glu Glu Arg Lys Ala Arg Ala Leu Ala
Ala675 680 685Ala Thr Ser Glu Leu Glu Arg Leu Phe Arg Lys Glu Asp
Phe Arg Arg690 695 700Met Gln Val Leu Gly Gln Phe Asn Leu Gly Phe
Ile Ile Ala Lys Leu705 710 715 720Glu Arg Asp Leu Phe Ile Val Asp
Gln His Ala Ala Asp Glu Lys Phe725 730 735Asn Phe Glu His Leu Ala
Arg Ser Thr Val Leu Asn Gln Gln Pro Leu740 745 750Leu Gln Pro Leu
Asn Leu Glu Leu Ser Pro Glu
Glu Glu Val Thr Val755 760 765Leu Met His Met Asp Ile Ile Arg Glu
Asn Gly Phe Leu Leu Glu Glu770 775 780Asn Pro Ser Ala Pro Pro Gly
Lys His Phe Arg Leu Arg Ala Ile Pro785 790 795 800Tyr Ser Lys Asn
Ile Thr Phe Gly Val Glu Asp Leu Lys Asp Leu Ile805 810 815Ser Thr
Leu Gly Asp Asn His Gly Glu Cys Ser Val Ala Ser Ser Tyr820 825
830Lys Thr Ser Lys Thr Asp Ser Ile Cys Pro Ser Arg Val Arg Ala
Met835 840 845Leu Ala Ser Arg Ala Cys Arg Ser Ser Val Met Ile Gly
Asp Pro Leu850 855 860Arg Lys Asn Glu Met Gln Lys Ile Val Glu His
Leu Ala Asp Leu Glu865 870 875 880Ser Pro Trp Asn Cys Pro His Gly
Arg Pro Thr Met Arg His Leu Val885 890 895Asp Leu Thr Thr Leu Leu
Thr Leu Pro Asp Asp Asp Asn Val Asn Asp900 905 910Asp Asp Asp Asp
Asp Ala Thr Ile Ser Leu Ala915 920
* * * * *