U.S. patent application number 15/442083 was filed with the patent office on 2017-08-03 for antibodies against t cell immunoglobulin domain and mucin domain 1 (tim-1) antigen and uses thereof.
The applicant listed for this patent is Amgen Fremont Inc., Celldex Therapeutics, Inc.. Invention is credited to Binyam Bezabeh, Francine Chen, Ian Foltz, Michael Jeffers, Nikolia Khramtsov, Gregory M. Landes, Mehdi Mesri, Peter Mezes, Gary Starling, Kam Fai Tse.
Application Number | 20170218067 15/442083 |
Document ID | / |
Family ID | 33098138 |
Filed Date | 2017-08-03 |
United States Patent
Application |
20170218067 |
Kind Code |
A1 |
Landes; Gregory M. ; et
al. |
August 3, 2017 |
Antibodies Against T Cell Immunoglobulin Domain and Mucin Domain 1
(TIM-1) Antigen and Uses Thereof
Abstract
The invention described herein is related to antibodies directed
to the antigen TIM-1 and uses of such antibodies. In particular,
there are provided fully human monoclonal antibodies directed to
the antigen TIM-1. Isolated polynucleotide sequences encoding, and
amino acid sequences comprising, heavy and light chain
immunoglobulin molecules, particularly sequences corresponding to
contiguous heavy and light chain sequences spanning the framework
regions (FR's) and/or complementarity determining regions (CDR's),
specifically from FR1 through FR4 or CDR1 through CDR3, are
provided. Hybridomas or other cell lines expressing such
immunoglobulin molecules and monoclonal antibodies are also
provided.
Inventors: |
Landes; Gregory M.; (The
Woodlands, TX) ; Chen; Francine; (San Francisco,
CA) ; Bezabeh; Binyam; (Oakland, CA) ; Foltz;
Ian; (Burnaby, CA) ; Tse; Kam Fai; (Clinton,
CT) ; Jeffers; Michael; (Branford, CT) ;
Mesri; Mehdi; (Branford, CT) ; Starling; Gary;
(Clinton, CT) ; Mezes; Peter; (Old Lyme, CT)
; Khramtsov; Nikolia; (Branford, CT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Celldex Therapeutics, Inc.
Amgen Fremont Inc. |
Hampton
Fremont |
NJ
CA |
US
US |
|
|
Family ID: |
33098138 |
Appl. No.: |
15/442083 |
Filed: |
February 24, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13305898 |
Nov 29, 2011 |
9580503 |
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15442083 |
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10805177 |
Mar 19, 2004 |
8067544 |
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13305898 |
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60456652 |
Mar 19, 2003 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/50 20130101;
G01N 2333/70503 20130101; C07K 16/2809 20130101; A61P 13/12
20180101; A61P 35/00 20180101; C07K 2317/33 20130101; A61P 13/08
20180101; C07K 2317/34 20130101; C07K 2317/31 20130101; G01N 33/574
20130101; C07K 2317/76 20130101; A61P 13/10 20180101; C07K 2317/622
20130101; C07K 16/2803 20130101; A61K 51/1039 20130101; C07K
2317/73 20130101; A61P 43/00 20180101; A61P 29/00 20180101; C07K
2317/56 20130101; A61K 2039/505 20130101; C07K 2317/92 20130101;
C07K 16/28 20130101; C07K 2317/21 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; G01N 33/574 20060101 G01N033/574; A61K 51/10 20060101
A61K051/10 |
Claims
1. An isolated human antibody, or binding fragment thereof, that
specifically binds to T cell, immunoglobulin domain and mucin
domain 1 (TIM-1).
2. The antibody of claim 1, wherein said TIM-1 comprises the amino
acid sequence shown in SEQ ID NO:54.
3. The antibody of claim 1, wherein said TIM-1 comprises the amino
acid sequence of SEQ ID NO:87.
4. The antibody of claim 1, wherein said antibody is a monoclonal
antibody.
5. The antibody of claim 1, wherein said binding fragment comprises
a Fab, Fab', F(ab').sub.2, or Fv fragment of said antibody.
6. The antibody of claim 1, wherein said antibody is a single chain
antibody.
7. The antibody, or binding fragment, of claim 1, wherein said
antibody or binding fragment is associated with a pharmaceutically
acceptable carrier or diluent.
8. The antibody, or binding fragment of claim 1, wherein the
antibody or binding fragment is conjugated to a therapeutic
agent.
9. The antibody, or binding fragment of claim 8, wherein the
therapeutic agent is a toxin.
10. The antibody or binding fragment of claim 8, wherein the
therapeutic agent is a radioactive isotope.
11. The antibody or binding fragment of claim 8, wherein the
therapeutic agent is a chemotherapeutic agent.
12. A hybridoma cell line producing the antibody, or binding
fragment, of claim 1.
13. A transformed cell comprising a gene encoding the antibody, or
binding fragment, of claim 1.
14. The transformed cell of claim 13, wherein the cell is a Chinese
hamster ovary (CHO) cell.
15. A method of inhibiting cell proliferation associated with the
expression of TIM-1, comprising treating cells expressing TIM-1
with an effective amount of the antibody of claim 1.
16. A method of effectively treating renal cancer comprising:
identifying an animal in need of treatment for renal cancer;
administering to said animal a therapeutically effective dose of
the antibody of claim 1.
17. A method of effectively treating ovarian cancer comprising:
identifying an animal in need of treatment for ovarian cancer;
administering to said animal a therapeutically effective dose of
the antibody of claim 1.
18. An article of manufacture comprising a container, a composition
contained therein, and a package insert or label indicating that
the composition can be used to treat cancer characterized by the
overexpression of TIM-1, wherein the composition comprises the
antibody, or binding fragment, of claim 1.
19. An assay kit for the detection of TIM-1 in mammalian tissues or
cells in order to screen for lung, colon, gastric, kidney, renal,
prostate or ovarian carcinomas, the TIM-1 being an antigen
expressed by lung, colon, gastric, kidney, renal, prostate or
ovarian carcinomas, the kit comprising the antibody of claim 1 and
means for indicating the reaction of the antibody with the antigen,
if present.
20. The assay kit of claim 19, wherein the antibody is labeled.
21. The assay kit of claim 19, wherein the antibody is an unlabeled
first antibody and the means for indicating the reaction comprises
a labeled second antibody that is anti-immunoglobulin.
22. The assay kit of claim 21, wherein the antibody that binds the
antigen is labeled with a marker selected from the group consisting
of a fluorochrome, an enzyme, a radionuclide and a radiopaque
material.
23. The assay kit of claim 22, wherein the second antibody is
labeled with a marker selected from the group consisting of a
fluorochrome, an enzyme, a radionuclide and a radiopaque material.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/305,898, filed Nov. 29, 2011 to issue on
Feb. 28, 2017 as U.S. Pat. No. 9,580,503, which is a division of
U.S. patent application Ser. No. 10/805,177, filed Mar. 19, 2004
and now issued as U.S. Pat. No. 8,067,544, which claims the benefit
of priority under 35 U.S.C. .sctn.119(e) to U.S. Provisional
Application No. 60/456,652, filed Mar. 19, 2003, each of which is
hereby expressly incorporated by reference.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING
[0002] The contents of the text file named "Cura965C4US.txt," which
was created on Jun. 5, 2012 and is 144 KB in size, are hereby
incorporated by reference in their entirety.
BACKGROUND OF THE INVENTION
[0003] Field of the Invention
[0004] The invention disclosed herein is related to antibodies
directed to the antigen T cell, immunoglobulin domain and mucin
domain 1 (TIM-1) proteins and uses of such antibodies. In
particular, there are provided fully human monoclonal antibodies
directed to the antigen TIM-1. Nucleotide sequences encoding, and
amino acid sequences comprising, heavy and light chain
immunoglobulin molecules, particularly sequences corresponding to
contiguous heavy and light chain sequences spanning the framework
regions and/or complementarity determining regions (CDRs),
specifically from FR1 through FR4 or CDR1 through CDR3, are
provided. Hybridomas or other cell lines expressing such
immunoglobulin molecules and monoclonal antibodies are also
provided.
[0005] Description of the Related Art
[0006] A new family of genes encoding T cell, immunoglobulin domain
and mucin domain (TIM) proteins (three in humans and eight in mice)
have been described recently with emerging roles in immunity.
Kuchroo et al., Nat Rev Immunol 3:454-462 (2003); McIntire et al.,
Nat Immunol 2:1109-1116 (2001). The TIM gene family members reside
in chromosomal regions, 5q33.2 in human and 11B1.1 in mouse, and
have been linked to allergy and autoimmune diseases. Shevach, Nat
Rev Immunol 2:389-400 (2002); Wills-Karp et al., Nat Immunol
4:1050-1052 (2003).
[0007] One TIM family member, TIM-1, is also known as Hepatitis A
virus cellular receptor (HAVcr-1) and was originally discovered as
a receptor for Hepatitis A virus (HAV) (Kaplan et al, EMBO J
15(16):4282-96 (1996)). This gene was later cloned as kidney injury
molecule 1 (KIM-1) (Ichimura et al., J Biol Chem 273:4135-4142
(1998); Han et al., Kidney Int 62:237-244 (2002)).
[0008] Kaplan et al. isolated the cellular receptor for hepatitis A
virus from a cDNA library from a primary African Green Monkey
Kidney (AGMK) cell line expressing the receptor. See U.S. Pat. No.
5,622,861. The disclosed utility of the polypeptides and nucleic
acids was to diagnose infection by hepatitis A virus, to separate
hepatitis A virus from impurities in a sample, to treat infection
as well as to prevent infection by hepatitis A virus. Furthermore,
the polypeptides could be expressed in transformed cells and used
to test efficacy of compounds in an anti-hepatitis A virus binding
assay.
[0009] The human homolog, hHAVcr-1 (aka TIM-1), was described by
Feigelstock et al., J Virology 72(8): 6621-6628 (1998). The same
molecules were described in PCT Publication Nos: WO 97/44460 and WO
98/53071 and U.S. Pat. No. 6,664,385 as Kidney Injury-related
Molecules (KIM) that were found to be upregulated in renal tissue
after injury to the kidney. The molecules were described as being
useful in a variety of therapeutic interventions, specifically,
renal disease, disorder or injury. For example, PCT Publication No.
WO 02/098920 describes antibodies to KIM and describes antibodies
that inhibit the shedding of KIM-1 polypeptide from KIM-1
expressing cells e.g., renal cells, or renal cancer cells.
[0010] TIM-1 is a type 1 membrane protein that contains a novel
six-cysteine immunoglobulin-like domain and a mucin
threonine/serine.proline-rich (T/S/P) domain. TIM-1 was originally
identified in rat. TIM-1 has been found in mouse, African green
monkey, and humans (Feigelstock et al., J Virol 72(8):6621-8
(1998). The African green monkey ortholog is most closely related
to human TIM-1 showing 77.6% amino acid identity over 358 aligned
amino acids. Rat and mouse orthologs exhibit 50% (155/310) and
45.6% (126/276) amino acid identity respectively, although over
shorter segments of aligned sequence than for African green monkey.
Monoclonal antibodies to the Ig-like domain of TIM-1 have been
shown to be protective against Hepatitis A Virus infection in
vitro. Silberstein et al., J Virol 75(2):717-25 (2001). In
addition, Kim-1 was shown to be expressed at low levels in normal
kidney but its expression is increased dramatically in postischemic
kidney. Ichimura et al., J Biol Chem 273(7):4135-42 (1998). HAVCR-1
is also expressed at elevated levels in clear cell carcinomas and
cancer cell lines derived from the same.
[0011] TIM-1 shows homology to the P-type "trefoil" domain
suggesting that it may have similar biological activity to other
P-type trefoil family members. Some trefoil domain containing
proteins have been shown to induce cellular scattering and invasion
when used to treat kidney, colon and breast tumor cell lines. Prest
et al., FASEB J 16(6):592-4 (2002). In addition, some trefoil
containing proteins confer cellular resistance to anoikis, an
anchorage-related apoptosis phenomenon in epithelium. Chen et al.,
Biochem Biophys Res Commun 274(3):576-82 (2000).
[0012] TIM-1 maps to a region of human chromosome 5 known as Tapr
in the murine sytenic region that has been implicated in asthma.
Tapr, a major T cell regulatory locus, controls the development of
airway hyperreactivity. Wills-Karp, Nature Immunology 2:1095-1096
(2001); McIntire et al., Nature Immunology 2:1109-1116 (2001).
SUMMARY OF THE INVENTION
[0013] Embodiments of the invention described herein are based upon
the development of human monoclonal antibodies, or binding
fragements thereof, that bind TIM-1 and affect TIM-1 function.
TIM-1 is expressed at elevated levels in pathologies, such as
neoplasms and inflammatory diseases. Inhibition of the biological
activity of TIM-1 can thus prevent inflammation and other desired
effects, including TIM-1 induced cell proliferation. Embodiments of
the invention are based upon the generation and identification of
isolated antibodies, or binding fragments thereof, that bind
specifically to TIM-1.
[0014] Accordingly, one embodiment of the invention includes
isolated antibodies, or fragments of those antibodies, that
specifically bind to TIM-1. As known in the art, the antibodies can
advantageously be, for example, monoclonal, chimeric and/or fully
human antibodies. Embodiments of the invention described herein
also provide cells for producing these antibodies.
[0015] Some embodiments of the invention described herein relate to
monoclonal antibodies that bind TIM-1 and affect TIM-1 function.
Other embodiments relate to fully human anti-TIM-1 antibodies and
anti-TIM-1 antibody preparations with desirable properties from a
therapeutic perspective, including strong binding affinity for
TIM-1, the ability to neutralize TIM-1 in vitro and in vivo, and
the ability to inhibit TIM-1 induced cell proliferation.
[0016] In a preferred embodiment, antibodies described herein bind
to TIM-1 with very high affinities (Kd). For example a human,
rabbit, mouse, chimeric or humanized antibody that is capable of
binding TIM-1 with a Kd less than, but not limited to, 10.sup.-7,
10.sup.-8, 10.sup.-9, 10.sup.-10, 10.sup.-11, 10.sup.-12,
10.sup.-13 or 10.sup.-14 M, or any range or value therein. Affinity
and/or avidity measurements can be measured by KinExA.RTM. and/or
BIACORE.RTM., as described herein.
[0017] In one embodiment, the invention provides an isolated
antibody that specifically binds to T cell, immunoglobulin domain
and mucin domain 1 (TIM-1). In some embodiments, the isolated
antibody has a heavy chain polypeptide comprising an amino acid
sequence selected from the group consisting of SEQ ID NOs: 2, 6,
10, 14, 18, 22, 26, 30, 34, 38, 42, 46, and 50.
[0018] In another embodiment, the invention provides an isolated
antibody that specifically binds to T cell, immunoglobulin domain
and mucin domain 1 (TIM-1) and has a light chain polypeptide
comprising an amino acid sequence selected from the group
consisting of SEQ ID NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44,
48, and 52.
[0019] In yet another embodiment, the invention provides an
isolated antibody that specifically binds to TIM-1 and has a heavy
chain polypeptide comprising an amino acid sequence selected from
the group consisting of SEQ ID NOs: 2, 6, 10, 14, 18, 22, 26, 30,
34, 38, 42, 46, and 50 and has a light chain polypeptide comprising
an amino acid sequence selected from the group consisting of SEQ ID
NOs: 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, and 52.
[0020] Another embodiment of the invention is a fully human
antibody that specifically binds to TIM-1 and has a heavy chain
polypeptide comprising an amino acid sequence comprising the
complementarity determining region (CDR) with one of the sequences
shown in Table 4. It is noted that CDR determinations can be
readily accomplished by those of ordinary skill in the art. See for
example, Kabat et al., Sequences of Proteins of Immunological
Interest, Fifth Edition, NIH Publication 91-3242, Bethesda Md.
[1991], vols. 1-3.
[0021] Yet another embodiment is an antibody that specifically
binds to TIM-1 and has a light chain polypeptide comprising an
amino acid sequence comprising a CDR comprising one of the
sequences shown in Table 5. In certain embodiments the antibody is
a fully human monoclonal antibody.
[0022] A further embodiment is an antibody that binds to TIM-1 and
comprises a heavy chain polypeptide comprising an amino acid
sequence comprising one of the CDR sequences shown in Table 4 and a
light chain polypeptide comprising an amino acid sequence
comprising one of the CDR sequences shown in Table 5. In certain
embodiments the antibody is a fully human monoclonal antibody.
[0023] Another embodiment of the invention is a fully human
antibody that binds to orthologs of TIM-1. A further embodiment
herein is an antibody that cross-competes for binding to TIM-1 with
the fully human antibodies described herein.
[0024] Other embodiments includes methods of producing high
affinity antibodies to TIM-1 by immunizing a mammal with human
TIM-1, or a fragment thereof, and one or more orthologous sequences
or fragments thereof.
[0025] It will be appreciated that embodiments of the invention are
not limited to any particular form of an antibody. For example, the
anti-TIM-1 antibody can be a full length antibody (e.g., having an
intact human Fc region) or an antibody fragment (e.g., a Fab, Fab',
F(ab').sub.2, Fv, or single chain antibodies). In addition, the
antibody can be manufactured from a hybridoma that secretes the
antibody, or from a recombinantly produced cell that has been
transformed or transfected with a gene or genes encoding the
antibody.
[0026] Some embodiments of the invention include isolated nucleic
acid molecules encoding any of the anti-TIM-1 antibodies described
herein, vectors having an isolated nucleic acid molecule encoding
the anti-TIM-1 antibody, and a host cell transformed with such a
nucleic acid molecule. In addition, one embodiment of the invention
is a method of producing an anti-TIM-1 antibody by culturing host
cells under conditions wherein a nucleic acid molecule is expressed
to produce the antibody followed by recovering the antibody from
the host cell.
[0027] In other embodiments the invention provides compositions,
including an antibody, or functional fragment thereof, and a
pharmaceutically acceptable carrier.
[0028] In some embodiments, the invention includes pharmaceutical
compositions having an effective amount of an anti-TIM-1 antibody
in admixture with a pharmaceutically acceptable carrier or diluent.
In yet other embodiments, the anti-TIM-1 antibody, or a fragment
thereof, is conjugated to a therapeutic agent. The therapeutic
agent can be, for example, a toxin, a radioisotope, or a
chemotherapeutic agent. Preferably, such antibodies can be used for
the treatment of pathologies, including for example, tumors and
cancers, such as ovarian, stomach, endometrial, salivary gland,
lung, kidney, colon, colorectal, thyroid, pancreatic, prostate and
bladder cancer, as well as other inflammatory conditions. More
preferably, the antibodies can be used to treat renal and ovarian
carcinomas.
[0029] In still further embodiments, the antibodies described
herein can be used for the preparation of a medicament for the
effective treatment of TIM-1 induced cell proliferation in an
animal, wherein said monoclonal antibody specifically binds to
TIM-1.
[0030] Yet another embodiment is the use of an anti-TIM-1 antibody
in the preparation of a medicament for the treatment of diseases
such as neoplasms and inflammatory conditions. In one embodiment,
the neoplasm includes, without limitation, tumors and cancers, such
as ovarian, stomach, endometrial, salivary gland, lung, kidney,
colon, colorectal, thyroid, pancreatic, prostate and bladder
cancer.
[0031] In yet another aspect, the invention includes a method for
effectively treating pathologies associated with the expression of
TIM-1. These methods include selecting an animal in need of
treatment for a condition associated with the expression of TIM-1,
and administering to said animal a therapeutically effective dose
of a fully human monoclonal antibody, wherein said antibody
specifically binds to TIM-1.
[0032] Preferably a mammal and, more preferably, a human, receives
the anti-TIM-1 antibody. In a preferred embodiment, neoplasms are
treated, including, without limitation, renal and pancreatic
tumors, head and neck cancer, ovarian cancer, gastric (stomach)
cancer, melanoma, lymphoma, prostate cancer, liver cancer, lung
cancer, renal cancer, bladder cancer, colon cancer, esophageal
cancer, and brain cancer.
[0033] Further embodiments of the invention include the use of an
antibody of in the preparation of medicament for the effective
treatment of neoplastic disease in an animal, wherein said
monoclonal antibody specifically binds to TIM-1. Treatable
neoplastic diseases include, for example, ovarian cancer, bladder
cancer, lung cancer, glioblastoma, stomach cancer, endometrial
cancer, kidney cancer, colon cancer, pancreatic cancer, and
prostrate cancer.
[0034] In some embodiments, the invention includes a method for
inhibiting cell proliferation associated with the expression of
TIM-1. These methods include selecting an animal in need of
treatment for TIM-1 induced cell proliferation and administering to
said animal a therapeutically effective dose of a fully human
monoclonal antibody, wherein the antibody specifically binds TIM-1.
In other embodiments, cells expressing TIM-1 are treated with an
effective amount of an anti-TIM-1 antibody or a fragment thereof.
The method can be performed in vivo.
[0035] The methods can be performed in vivo and the patient is
preferably a human patient. In a preferred embodiment, the methods
concern the treatment of neoplastic diseases, for example, tumors
and cancers, such as renal (kidney) cancer, pancreatic cancer, head
and neck cancer, ovarian cancer, gastric (stomach) cancer,
melanoma, lymphoma, prostate cancer, liver cancer, breast cancer,
lung cancer, bladder cancer, colon cancer, esophageal cancer, and
brain cancer.
[0036] In some embodiments, the anti-TIM-1 antibody is administered
to a patient, followed by administration of a clearing agent to
remove excess circulating antibody from the blood.
[0037] In some embodiments, anti-TIM-1 antibodies can be modified
to enhance their capability of fixing complement and participating
in complement-dependent cytotoxicity (CDC). In one embodiment,
anti-TIM-1 antibodies can be modified, such as by an amino acid
substitution, to alter their clearance from the body.
Alternatively, some other amino acid substitutions can slow
clearance of the antibody from the body.
[0038] In another embodiment, the invention provides an article of
manufacture including a container. The container includes a
composition containing an anti-TIM-1 antibody, and a package insert
or label indicating that the composition can be used to treat
neoplastic or inflammatory diseases characterized by the
overexpression of TIM-1.
[0039] Yet another embodiment provides methods for assaying the
level of TIM-1 in a patient sample, comprising contacting an
anti-TIM-1 antibody with a biological sample from a patient, and
detecting the level of binding between said antibody and TIM-1 in
said sample. In more specific embodiments, the biological sample is
blood.
[0040] In one embodiment, the invention includes an assay kit for
detecting TIM-1 and TIM-1 orthologs in mammalian tissues or cells
to screen for neoplastic diseases or inflammatory conditions. The
kit includes an antibody that binds to TIM-1 and a means for
indicating the reaction of the antibody with TIM-1, if present.
Preferably the antibody is a monoclonal antibody. In one
embodiment, the antibody that binds TIM-1 is labeled. In another
embodiment the antibody is an unlabeled first antibody and the kit
further includes a means for detecting the first antibody. In one
embodiment, the means includes a labeled second antibody that is an
anti-immunoglobulin. Preferably the antibody is labeled with a
marker selected from the group consisting of a fluorochrome, an
enzyme, a radionuclide and a radiopaque material.
[0041] Another embodiment of the invention includes a method of
diagnosing diseases or conditions in which an antibody prepared as
described herein is utilized to detect the level of TIM-1 in a
patient sample. In one embodiment, the patient sample is blood or
blood serum. In further embodiments, methods for the identification
of risk factors, diagnosis of disease, and staging of disease is
presented which involves the identification of the overexpression
of TIM-1 using anti-TIM-1 antibodies.
[0042] Embodiments of the invention described herein also pertain
to variants of a TIM-1 protein that function as either TIM-1
agonists (mimetics) or as TIM-1 antagonists.
[0043] Another embodiment of the invention is the use of monoclonal
antibodies directed against the TIM-1 antigen coupled to cytotoxic
chemotherapic agents or radiotherapic agents such as anti-tumor
therapeutics.
[0044] One embodiment provides an isolated antibody that blocks
simultaneous binding to TIM-1 antigen by an antibody having a heavy
chain sequence comprising an the amino acid sequence selected from
the group consisting of SEQ ID NOS: 2, 6, 10, 14, 18, 22, 26, 30,
34, 38, 42, 46, and 50. Another embodiment provides an isolated
antibody that binds to TIM-1 antigen and that cross reacts with an
antibody having a heavy chain sequence comprising the amino acid
sequence from the group consisting of SEQ ID NOS: 2, 6, 10, 14, 18,
22, 26, 30, 34, 38, 42, 46, and 50.
[0045] Another embodiment of the invention provides an isolated
antibody that binds to an epitope of SEQ ID NO: 87 on the TIM-1
antigen of SEQ ID NO. 54, and that cross reacts with an antibody
having a heavy chain sequence comprising the amino acid sequence
selected from the group consisting of SEQ ID NOS: 2, 6, 10, 14, 18,
22, 26, 30, 34, 38, 42, 46, and 50. In still another embodiment,
the invention provides an isolated antibody that binds to an
epitope of SEQ ID NO: 87 on the TIM-1 antigen of SEQ ID NO. 54,
wherein said antibody blocks simultaneous binding to TIM-1 antigen
by an antibody having a heavy chain sequence comprising the amino
acid sequence selected from the group comprising SEQ ID NOS: 2, 6,
10, 14, 18, 22, 26, 30, 34, 38, 42, 46, and 50.
BRIEF DESCRIPTION OF THE DRAWINGS
[0046] FIG. 1 is a bar graph of the results of an ELISA assay of
anti-TIM-1 monoclonal antibodies 1.29, 2.56.2, 2.59.2, and 2.45.1
against the TIM-1 antigen.
[0047] FIG. 2 is a bar graph of the results of an ELISA assay of
anti-TIM-1 monoclonal antibodies 1.29, 2.56.2, 2.59.2, and 2.45.1
against irrelevant protein.
[0048] FIG. 3A shows staining of Renal Cell Cancer, and FIG. 3B
shows Pancreatic Cancer with the anti-TIM-1 mAb 2.59.2.
[0049] FIG. 4 is a bar graph of clonogenic assay results of
anti-TIM-1 monoclonal antibody mediated toxin killing in the ACHN
kidney cancer cell line.
[0050] FIG. 5 is a bar graph of clonogenic assay results of
anti-TIM-1 monoclonal antibody mediated toxin killing in the BT549
breast cancer cell line.
[0051] FIG. 6 is a bar graph of the results of a clonogenic assay
of CAKI-1 cells treated with Auristatin E (AE) conjugated
antibodies.
[0052] FIG. 7 is a bar graph of the results of a clonogenic assay
of BT549 cells treated with Auristatin E (AE) conjugated
antibodies.
[0053] FIG. 8 is a bar graph showing that anti-TIM-1 monoclonal
antibodies 2.59.2, 2.56.2 and 2.45.1 significantly inhibit IL-4
release from Th1 cells compared to the control PK16.3 mAb.
[0054] FIG. 9 is a bar graph showing that anti-TIM-1 monoclonal
antibodies 2.59.2 and 2.45.1 significantly inhibit IL-4 release
from Th2 cells compared to control PK16.3 mAb.
[0055] FIG. 10 is a bar graph showing that anti-TIM-1 monoclonal
antibody 2.59.2 significantly inhibited IL-5 release from Th1 cells
compared to control PK16.3 mAb.
[0056] FIG. 11 is a bar graph showing that anti-TIM-1 monoclonal
antibodies 2.59.2 and 1.29 significantly inhibited IL-5 release
from Th2 cells compared to control PK16.3 mAb.
[0057] FIG. 12 is a bar graph showing that anti-TIM-1 monoclonal
antibodies 2.59.2, 1.29 and 2.56.2 significantly inhibited IL-10
release from Th1 cells compared to control PK16.3 mAb.
[0058] FIG. 13 is a bar graph showing that anti-TIM-1 monoclonal
antibodies 2.59.2, 1.29 and 2.45.1 significantly inhibited IL-10
release from Th2 cells compared to control PK16.3 mAb.
[0059] FIG. 14 is a bar graph showing that anti-TIM-1 monoclonal
antibodies 2.59.2, 1.29 and 2.56.2 significantly inhibited IL-13
release from Th1 cells compared to control PK16.3 mAb.
[0060] FIG. 15 is a bar graph showing that anti-TIM-1 monoclonal
antibodies 2.59.2 and 1.29 significantly inhibited IL-13 release
from Th2 cells compared to control PK16.3 mAb.
[0061] FIG. 16 is a bar graph showing that anti-TIM-1 monoclonal
antibodies did not inhibit IFN.gamma. release from Th1 cells
compared to control PK16.3 mAb.
[0062] FIG. 17 is a bar graph showing that anti-TIM-1 monoclonal
antibodies 2.59.2 and 2.45.1 significantly inhibited IFN.gamma.
release from Th2 cells compared to control PK16.3 mAb.
[0063] FIGS. 18A-18T are bar graphs showing BrdU incorporation
assay results from experiments in which the neutralization of
various human anti-TIM-1 monoclonal antibodies was assessed.
[0064] FIGS. 19A-19D are line graphs showing the results of
antibody conjugate studies performed using the plant toxin Saporin
conjugated to TIM-1-specific antibodies and irrelevant antibodies
(FIG. 19A-19C). Additional negative controls included irrelevant
antibodies alone without toxin (FIG. 19D).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0065] Embodiments of the invention described herein are based upon
the generation and identification of isolated antibodies that bind
specifically to T cell, immunoglobulin domain and mucin domain 1
(TIM-1). As discussed below, TIM-1 is expressed at elevated levels
in clear cell carcinomas and cancer cell lines derived from the
same. Accordingly, antibodies that bind to TIM-1 are useful for the
treatment and inhibition of carcinomas. In addition, antibodies
that bind TIM-1 are also useful for reducing cell migration and
enhancing apoptosis of kidney cancer cells.
[0066] Accordingly, embodiments of the invention described herein
provide isolated antibodies, or fragments of those antibodies, that
bind to TIM-1. As known in the art, the antibodies can
advantageously be, e.g., monoclonal, chimeric and/or human
antibodies. Embodiments of the invention described herein also
provide cells for producing these antibodies.
[0067] Another embodiment of the invention provides for using these
antibodies for diagnostic or therapeutic purposes. For example,
embodiments of the invention provide methods and antibodies for
inhibiting the expression of TIM-1 associated with cell
proliferation. Preferably, the antibodies are used to treat
neoplasms such as renal and pancreatic tumors, head and neck
cancer, ovarian cancer, gastric (stomach) cancer, melanoma,
lymphoma, prostate cancer, liver cancer, breast cancer, lung
cancer, renal cancer, bladder cancer, colon cancer, esophageal
cancer, and brain cancer. In association with such treatment,
articles of manufacture comprising these antibodies are provided.
Additionally, an assay kit comprising these antibodies is provided
to screen for cancers or tumors.
[0068] Additionally, the nucleic acids described herein, and
fragments and variants thereof, may be used, by way of nonlimiting
example, (a) to direct the biosynthesis of the corresponding
encoded proteins, polypeptides, fragments and variants as
recombinant or heterologous gene products, (b) as probes for
detection and quantification of the nucleic acids disclosed herein,
(c) as sequence templates for preparing antisense molecules, and
the like. Such uses are described more fully in the following
disclosure.
[0069] Furthermore, the TIM-1 proteins and polypeptides described
herein, and fragments and variants thereof, may be used, in ways
that include (a) serving as an immunogen to stimulate the
production of an anti-TIM-1 antibody, (b) a capture antigen in an
immunogenic assay for such an antibody, (c) as a target for
screening for substances that bind to a TIM-1 polypeptide described
herein, and (d) a target for a TIM-1 specific antibody such that
treatment with the antibody affects the molecular and/or cellular
function mediated by the target. TIM-1 polypeptide expression or
activity can promote cell survival and/or metastatic potential.
Conversely, a decrease in TIM-1 polypeptide expression or
inhibition of its function reduces tumor cell survival and
invasiveness in a therapeutically beneficial manner.
[0070] Single chain antibodies (scFv's) and bispecific antibodies
specific for TIM-1 are useful particularly because it may more
readily penetrate a tumor mass due to its smaller size relative to
a whole IgG molecule. Studies comparing the tumor penetration
between whole IgG molecules and scFv's have been have been
described in the literature. The scFv can be derivatized with a
toxin or radionuclide in order to destroy tumor cells expressing
the TIM-1 antigen, in a manner similar to the IgG2 or IgG4
anti-TIM-1 toxin labeled or radionuclide derivatized whole
antibodies already discussed, but with the advantage of being able
to penetrate the tumor more fully, which may translate into
increased efficacy in eradicating the tumor. A specific example of
a biologically active anti-TIM-1 scFv is provided herein.
Sequence Listing
[0071] The heavy chain and light chain variable region nucleotide
and amino acid sequences of representative human anti-TIM-1
antibodies are provided in the sequence listing, the contents of
which are summarized in Table 1 below.
TABLE-US-00001 TABLE 1 mAb SEQ ID ID No.: Sequence NO: 1.29
Nucleotide sequence encoding the variable region and a portion of
the 1 constant region of the heavy chain Amino acid sequence of the
variable region of the heavy chain 2 Nucleotide sequence encoding
the variable region and a portion of the 3 constant region of the
light chain Amino acid sequence of the variable region of the light
chain 4 1.37 Nucleotide sequence encoding the variable region and a
portion of the 5 constant region of the heavy chain Amino acid
sequence of the variable region of the heavy chain 6 Nucleotide
sequence encoding the variable region and a portion of the 7
constant region of the light chain Amino acid sequence of the
variable region of the light chain 8 2.16 Nucleotide sequence
encoding the variable region and a portion of the 9 constant region
of the heavy chain Amino acid sequence of the variable region of
the heavy chain 10 Nucleotide sequence encoding the variable region
and a portion of the 11 constant region of the light chain Amino
acid sequence of the variable region of the light chain 12 2.17
Nucleotide sequence encoding the variable region and a portion of
the 13 constant region of the heavy chain Amino acid sequence of
the variable region of the heavy chain 14 Nucleotide sequence
encoding the variable region and a portion of the 15 constant
region of the light chain Amino acid sequence of the variable
region of the light chain 16 2.24 Nucleotide sequence encoding the
variable region and a portion of the 17 constant region of the
heavy chain Amino acid sequence of the variable region of the heavy
chain 18 Nucleotide sequence encoding the variable region and a
portion of the 19 constant region of the light chain Amino acid
sequence of the variable region of the light chain 20 2.45
Nucleotide sequence encoding the variable region and a portion of
the 21 constant region of the heavy chain Amino acid sequence of
the variable region of the heavy chain 22 Nucleotide sequence
encoding the variable region and a portion of the 23 constant
region of the light chain Amino acid sequence of the variable
region of the light chain 24 2.54 Nucleotide sequence encoding the
variable region and a portion of the 25 constant region of the
heavy chain Amino acid sequence of the variable region of the heavy
chain 26 Nucleotide sequence encoding the variable region and a
portion of the 27 constant region of the light chain Amino acid
sequence of the variable region of the light chain 28 2.56
Nucleotide sequence encoding the variable region and a portion of
the 29 constant region of the heavy chain Amino acid sequence of
the variable region of the heavy chain 30 Nucleotide sequence
encoding the variable region and a portion of the 31 constant
region of the light chain Amino acid sequence of the variable
region of the light chain 32 2.59 Nucleotide sequence encoding the
variable region and a portion of the 33 constant region of the
heavy chain Amino acid sequence of the variable region of the heavy
chain 34 Nucleotide sequence encoding the variable region and a
portion of the 35 constant region of the light chain Amino acid
sequence of the variable region of the light chain 36 2.61
Nucleotide sequence encoding the variable region and a portion of
the 37 constant region of the heavy chain Amino acid sequence of
the variable region of the heavy chain 38 Nucleotide sequence
encoding the variable region and a portion of the 39 constant
region of the light chain Amino acid sequence of the variable
region of the light chain 40 2.70 Nucleotide sequence encoding the
variable region and a portion of the 41 constant region of the
heavy chain Amino acid sequence of the variable region of the heavy
chain 42 Nucleotide sequence encoding the variable region and a
portion of the 43 constant region of the light chain Amino acid
sequence of the variable region of the light chain 44 2.76
Nucleotide sequence encoding the variable region and a portion of
the 45 constant region of the heavy chain Amino acid sequence of
the variable region of the heavy chain 46 Nucleotide sequence
encoding the variable region and a portion of the 47 constant
region of the light chain Amino acid sequence of the variable
region of the light chain 48 2.70.2 Nucleotide sequence encoding
the variable region and a portion of the 49 constant region of the
heavy chain Amino acid sequence of the variable region and a
porition of the 50 constant region of the heavy chain Nucleotide
sequence encoding the variable region and a portion of the 51
constant region of the light chain Amino acid sequence of the
variable region and a porition of the 52 constant region of the
light chain
Definitions
[0072] Unless otherwise defined, scientific and technical terms
used in connection with the invention described herein 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)), which is incorporated herein by
reference. 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.
[0073] As utilized in accordance with the present disclosure, the
following terms, unless otherwise indicated, shall be understood to
have the following meanings:
[0074] The term "TIM-1" refers to T cell, immunoglobulin domain and
mucin domain 1. In one embodiment, TIM-1 refers to a polypeptide
comprinsing the amino acid sequence of SEQ ID NO: 54.
[0075] 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. Preferred polypeptides in accordance with
the invention comprise human heavy chain immunoglobulin molecules
and human kappa light chain immunoglobulin molecules, as well as
antibody molecules formed by combinations comprising the heavy
chain immunoglobulin molecules with light chain immunoglobulin
molecules, such as the kappa light chain immunoglobulin molecules,
and vice versa, as well as fragments and analogs thereof.
[0076] The term "polynucleotide" as referred to herein means a
polymeric form 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.
[0077] 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.
[0078] 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 murine proteins, (3) is expressed by a
cell from a different species, or (4) does not occur in nature.
[0079] The term "oligonucleotide" referred to herein includes
naturally occurring, and modified nucleotides linked together by
naturally occurring, and non-naturally occurring oligonucleotide
linkages. Oligonucleotides are a polynucleotide subset generally
comprising a length of 200 bases or fewer. Preferably
oligonucleotides are 10 to 60 bases in length and most preferably
12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length.
Oligonucleotides are usually single stranded, e.g. for probes;
although oligonucleotides may be double stranded, e.g. for use in
the construction of a gene mutant. Oligonucleotides described
herein can be either sense or antisense oligonucleotides.
[0080] Similarly, unless specified otherwise, the lefthand end of
single-stranded polynucleotide sequences is the 5' end; the
lefthand direction of double-stranded polynucleotide sequences is
referred to as the 5' direction. The direction of 5' to 3' addition
of nascent RNA transcripts is referred to as the transcription
direction; sequence regions on the DNA strand having the same
sequence as the RNA and which are 5' to the 5' end of the RNA
transcript are referred to as upstream sequences; sequence regions
on the DNA strand having the same sequence as the RNA and which are
3' to the 3' end of the RNA transcript are referred to as
downstream sequences.
[0081] 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.
[0082] The term "naturally occurring nucleotides" referred to
herein includes deoxyribonucleotides and ribonucleotides. The term
"modified nucleotides" referred to herein includes nucleotides with
modified or substituted sugar groups and the like. The term
"oligonucleotide linkages" referred to herein includes
oligonucleotides linkages such as phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phoshoraniladate, phosphoroamidate, and the
like. See, e.g., LaPlanche et al., Nucl. Acids Res. 14:9081 (1986);
Stec et al., J. Am. Chem. Soc. 106:6077 (1984); Stein et al., Nucl.
Acids Res. 16:3209 (1988); Zon et al., Anti-Cancer Drug Design
6:539 (1991); Zon et al., Oligonucleotides and Analogues: A
Practical Approach, pp. 87-108 (F. Eckstein, ed., Oxford University
Press, Oxford England (1991)); Stec et al., U.S. Pat. No.
5,151,510; Uhlmann and Peyman, Chemical Reviews 90:543 (1990), the
disclosures of which are hereby incorporated by reference. An
oligonucleotide can include a label for detection, if desired.
[0083] 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.
[0084] 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.
[0085] The term "selectively hybridize" referred to herein means to
detectably and specifically bind. Polynucleotides, oligonucleotides
and fragments thereof described herein selectively hybridize to
nucleic acid strands under hybridization and wash conditions that
minimize appreciable amounts of detectable binding to nonspecific
nucleic acids. High stringency conditions can be used to achieve
selective hybridization conditions as known in the art and
discussed herein. Generally, the nucleic acid sequence homology
between the polynucleotides, oligonucleotides, and fragments
described herein and a nucleic acid sequence of interest will be at
least 80%, and more typically with preferably increasing homologies
of at least 85%, 90%, 95%, 99%, and 100%.
[0086] Two amino acid sequences are homologous if there is a
partial or complete identity between their sequences. For example,
85% homology means that 85% of the amino acids are identical when
the two sequences are aligned for maximum matching. Gaps (in either
of the two sequences being matched) are allowed in maximizing
matching; gap lengths of 5 or less are preferred with 2 or less
being more preferred. Alternatively and preferably, two protein
sequences (or polypeptide sequences derived from them of at least
30 amino acids in length) are homologous, as this term is used
herein, if they have an alignment score of at more than 5 (in
standard deviation units) using the program ALIGN with the mutation
data matrix and a gap penalty of 6 or greater. See Dayhoff, M. O.,
in Atlas of Protein Sequence and Structure, pp. 101-110 (Volume 5,
National Biomedical Research Foundation (1972)) and Supplement 2 to
this volume, pp. 1-10. The two sequences or parts thereof are more
preferably homologous if their amino acids are greater than or
equal to 50% identical when optimally aligned using the ALIGN
program.
[0087] The term "corresponds to" is used herein to mean that a
polynucleotide sequence is homologous (i.e., is identical, not
strictly evolutionarily related) to all or a portion of a reference
polynucleotide sequence, or that a polypeptide sequence is
identical to a reference polypeptide sequence.
[0088] In contradistinction, the term "complementary to" is used
herein to mean that the complementary sequence is homologous to all
or a portion of a reference polynucleotide sequence. For
illustration, the nucleotide sequence "TATAC" corresponds to a
reference sequence "TATAC" and is complementary to a reference
sequence "GTATA."
[0089] The following terms are used to describe the sequence
relationships between two or more polynucleotide or amino acid
sequences: "reference sequence," "comparison window," "sequence
identity," "percentage of sequence identity," and "substantial
identity." A "reference sequence" is a defined sequence used as a
basis for a sequence comparison; a reference sequence may be a
subset of a larger sequence, for example, as a segment of a
full-length cDNA or gene sequence given in a sequence listing or
may comprise a complete cDNA or gene sequence. Generally, a
reference sequence is at least 18 nucleotides or 6 amino acids in
length, frequently at least 24 nucleotides or 8 amino acids in
length, and often at least 48 nucleotides or 16 amino acids in
length. Since two polynucleotides or amino acid sequences may each
(1) comprise a sequence (i.e., a portion of the complete
polynucleotide or amino acid sequence) that is similar between the
two molecules, and (2) may further comprise a sequence that is
divergent between the two polynucleotides or amino acid sequences,
sequence comparisons between two (or more) molecules are typically
performed by comparing sequences of the two molecules over a
comparison window to identify and compare local regions of sequence
similarity. A "comparison window," as used herein, refers to a
conceptual segment of at least 18 contiguous nucleotide positions
or 6 amino acids wherein a polynucleotide sequence or amino acid
sequence may be compared to a reference sequence of at least 18
contiguous nucleotides or 6 amino acid sequences and wherein the
portion of the polynucleotide sequence in the comparison window may
comprise additions, deletions, substitutions, and the like (i.e.,
gaps) of 20 percent or less as compared to the reference sequence
(which does not comprise additions or deletions) for optimal
alignment of the two sequences. Optimal alignment of sequences for
aligning a comparison window may be conducted by the local homology
algorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981), by
the homology alignment algorithm of Needleman and Wunsch, J. Mol.
Biol., 48:443 (1970), by the search for similarity method of
Pearson and Lipman, Proc. Natl. Acad. Sci. (U.S.A.), 85:2444
(1988), by computerized implementations of these algorithms (GAP,
BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software
Package Release 7.0, (Genetics Computer Group, 575 Science Dr.,
Madison, Wis.), Geneworks, or MacVector software packages), or by
inspection, and the best alignment (i.e., resulting in the highest
percentage of homology over the comparison window) generated by the
various methods is selected.
[0090] The term "sequence identity" means that two polynucleotide
or amino acid sequences are identical (i.e., on a
nucleotide-by-nucleotide or residue-by-residue basis) over the
comparison window. The term percentage of sequence identity is
calculated by comparing two optimally aligned sequences over the
window of comparison, determining the number of positions at which
the identical nucleic acid base (e.g., A, T, C, G, U, or I) or
residue occurs in both sequences to yield the number of matched
positions, dividing the number of matched positions by the total
number of positions in the comparison window (i.e., the window
size), and multiplying the result by 100 to yield the percentage of
sequence identity. The terms "substantial identity" as used herein
denotes a characteristic of a polynucleotide or amino acid
sequence, wherein the polynucleotide or amino acid comprises a
sequence that has at least 85 percent sequence identity, preferably
at least 90 to 95 percent sequence identity, more usually at least
99 percent sequence identity as compared to a reference sequence
over a comparison window of at least 18 nucleotide (6 amino acid)
positions, frequently over a window of at least 24-48 nucleotide
(8-16 amino acid) positions, wherein the percentage of sequence
identity is calculated by comparing the reference sequence to the
sequence which may include deletions or additions which total 20
percent or less of the reference sequence over the comparison
window. The reference sequence may be a subset of a larger
sequence.
[0091] As used herein, the twenty conventional amino acids and
their abbreviations follow conventional usage. See Immunology-A
Synthesis (2.sup.nd Edition, E. S. Golub and D. R. Gren, Eds.,
Sinauer Associates, Sunderland, Mass. (1991)), which is
incorporated herein by reference. 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 described herein.
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 lefthand direction is the amino terminal direction and
the righthand direction is the carboxy-terminal direction, in
accordance with standard usage and convention.
[0092] 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. Preferably, residue positions which are not identical
differ by conservative amino acid substitutions. 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.
[0093] As discussed herein, minor variations in the amino acid
sequences of antibodies or immunoglobulin molecules are
contemplated as being encompassed by the invention described
herein, providing that the variations in the amino acid sequence
maintain at least 75%, more preferably at least 80%, 90%, 95%, and
most preferably 99% sequence identity to the antibodies or
immunoglobin molecules described herein. 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=aspartate, glutamate; (2) basic=lysine, arginine, histidine;
(3) non-polar=alanine, valine, leucine, isoleucine, proline,
phenylalanine, methionine, tryptophan; and (4) uncharged
polar=glycine, asparagine, glutamine, cysteine, serine, threonine,
tyrosine. More preferred families are: serine and threonine are
aliphatic-hydroxy family; asparagine and glutamine are an
amide-containing family; alanine, valine, leucine and isoleucine
are an aliphatic family; and phenylalanine, tryptophan, and
tyrosine are an 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 described herein.
[0094] 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 al., Nature, 354:105 (1991),
which are each incorporated herein by reference.
[0095] The term "polypeptide fragment" as used herein refers to a
polypeptide that has an amino-terminal and/or carboxy-terminal
deletion, but where the remaining amino acid sequence is identical
to the corresponding positions in the naturally-occurring sequence
deduced, for example, from a full-length cDNA sequence. Fragments
typically are at least 5, 6, 8 or 10 amino acids long, preferably
at least 14 amino acids long, more preferably at least 20 amino
acids long, usually at least 50 amino acids long, and even more
preferably at least 70 amino acids long. The term "analog" as used
herein refers to polypeptides which are comprised of a segment of
at least 25 amino acids that has substantial identity to a portion
of a deduced amino acid sequence and which has at least one of the
following properties: (1) specific binding to a TIM-1, under
suitable binding conditions, (2) ability to block appropriate TIM-1
binding, or (3) ability to inhibit the growth and/or survival of
TIM-1 expressing cells in vitro or in vivo. Typically, polypeptide
analogs comprise a conservative amino acid substitution (or
addition or deletion) with respect to the naturally occurring
sequence. Analogs typically are at least 20 amino acids long,
preferably at least 50 amino acids long or longer, and can often be
as long as a full-length naturally-occurring polypeptide.
[0096] Peptide analogs are commonly used in the pharmaceutical
industry as non-peptide drugs with properties analogous to those of
the template peptide. These types of non-peptide compounds are
termed peptide mimetics or peptidomimetics. Fauchere, J. Adv. Drug
Res., 15:29 (1986); Veber and Freidinger, TINS, p.392 (1985); and
Evans et al., J. Med. Chem., 30:1229 (1987), which are incorporated
herein by reference. Such compounds are often developed with the
aid of computerized molecular modeling. Peptide mimetics that are
structurally similar to therapeutically useful peptides may be used
to produce an equivalent therapeutic or prophylactic effect.
Generally, peptidomimetics are structurally similar to a paradigm
polypeptide (i.e., a polypeptide that has a biochemical property or
pharmacological activity), such as human antibody, but have one or
more peptide linkages optionally replaced by a linkage selected
from the group consisting of: --CH.sub.2NH--, --CH.sub.2S--,
--CH.sub.2-CH.sub.2--, --CH.dbd.CH--(cis and trans),
--COCH.sub.2--, --CH(OH)CH.sub.2--, and --CH.sub.2SO--, by methods
well known in the art. Systematic substitution of one or more amino
acids of a consensus sequence with a D-amino acid of the same type
(e.g., D-lysine in place of L-lysine) may be used to generate more
stable peptides. In addition, constrained peptides comprising a
consensus sequence or a substantially identical consensus sequence
variation may be generated by methods known in the art (Rizo and
Gierasch, Ann. Rev. Biochem., 61:387 (1992), incorporated herein by
reference); for example, by adding internal cysteine residues
capable of forming intramolecular disulfide bridges which cyclize
the peptide.
[0097] "Antibody" or "antibody peptide(s)" refer to an intact
antibody, or a binding fragment thereof that competes with the
intact antibody for specific binding. Binding fragments are
produced by recombinant DNA techniques, or by enzymatic or chemical
cleavage of intact antibodies. Binding fragments include Fab, Fab',
F(ab').sub.2, Fv, and single-chain antibodies. An antibody other
than a bispecific or bifunctional antibody is understood to have
each of its binding sites identical. An antibody substantially
inhibits adhesion of a receptor to a counterreceptor when an excess
of antibody reduces the quantity of receptor bound to
counterreceptor by at least about 20%, 40%, 60% or 80%, and more
usually greater than about 85% (as measured in an in vitro
competitive binding assay).
[0098] Digestion of antibodies with the enzyme, papain, results in
two identical antigen-binding fragments, known also as "Fab"
fragments, and a "Fc" fragment, having no antigen-binding activity
but having the ability to crystallize. Digestion of antibodies with
the enzyme, pepsin, results in the a "F(ab').sub.2" fragment in
which the two arms of the antibody molecule remain linked and
comprise two-antigen binding sites. The F(ab').sub.2 fragment has
the ability to cros slink antigen.
[0099] "Fv" when used herein refers to the minimum fragment of an
antibody that retains both antigen-recognition and antigen-binding
sites.
[0100] "Fab" when used herein refers to a fragment of an antibody
which comprises the constant domain of the light chain and the CH1
domain of the heavy chain.
[0101] 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. An antibody is said to
specifically bind an antigen when the dissociation constant is
.ltoreq.1 .mu.M, preferably .ltoreq.100 nM and most preferably
.ltoreq.10 nM.
[0102] The term "agent" is used herein to denote a chemical
compound, a mixture of chemical compounds, a biological
macromolecule, or an extract made from biological materials.
[0103] 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.
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)), incorporated herein by reference).
[0104] The term "antineoplastic agent" is used herein to refer to
agents that have the functional property of inhibiting a
development or progression of a neoplasm in a human, particularly a
malignant (cancerous) lesion, such as a carcinoma, sarcoma,
lymphoma, or leukemia. Inhibition of metastasis is frequently a
property of antineoplastic agents.
[0105] 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.
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.
[0106] "Active" or "activity" in regard to a TIM-1 polypeptide
refers to a portion of a TIM-1 polypeptide which has a biological
or an immunological activity of a native TIM-1 polypeptide.
"Biological" when used herein refers to a biological function that
results from the activity of the native TIM-1 polypeptide. A
preferred biological activity includes, for example, regulation of
cellular growth.
[0107] "Label" or "labeled" as used herein refers to the addition
of a detectable moiety to a polypeptide, for example, a radiolabel,
fluorescent label, enzymatic label chemiluminescent labeled or a
biotinyl group. Radioisotopes or radionuclides may include .sup.3H,
.sup.14C, .sup.15N, .sup.35S, .sup.90Y, .sup.99Tc, .sup.111In,
.sup.125I, .sup.131I, fluorescent labels may include rhodamine,
lanthanide phosphors or FITC and enzymatic labels may include
horseradish peroxidase, .beta.-galactosidase, luciferase, alkaline
phosphatase.
[0108] "Mammal" when used herein refers to any animal that is
considered a mammal. Preferably, the mammal is human.
[0109] "Liposome" when used herein refers to a small vesicle that
may be useful for delivery of drugs that may include the TIM-1
polypeptide described herein or antibodies to such a TIM-1
polypeptide to a mammal.
[0110] The term "patient" includes human and veterinary
subjects.
Antibody Structure
[0111] The basic whole 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 domain 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. Human
light chains are classified as kappa and lambda light chains. Human
heavy chains are classified as mu, delta, gamma, alpha, or epsilon,
and define the antibody's isotype as IgM, IgG, IgA, and IgE,
respectively. Within light and heavy chains, the variable and
constant regions are joined by a "J" region of about 12 or more
amino acids, with the heavy chain also including a "D" region of
about 10 more amino acids. See generally, Fundamental Immunology
Ch. 7 (Paul, W., ed., 2d ed. Raven Press, N.Y. (1989))
(incorporated by reference in its entirety for all purposes). The
variable regions of each light/heavy chain pair form the antibody
binding site.
[0112] The variable domains all exhibit the same general structure
of relatively conserved framework regions (FR) joined by three
hyper variable regions, also called complementarity determining
regions or CDRs. The CDRs from the heavy and light chains of each
pair are aligned by the framework regions, enabling binding to a
specific epitope. From N-terminal to C-terminal, both light and
heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3
and FR4. The assignment of amino acids to each region 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).
[0113] A bispecific or bifunctional antibody is an artificial
hybrid antibody having two different heavy/light chain pairs and
two different binding sites. Bispecific antibodies can be produced
by a variety of methods including fusion of hybridomas or linking
of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin.
Exp. Immunol. 79: 315-321 (1990), Kostelny et al., J. Immunol.
148:1547-1553 (1992). Bispecific antibodies do not exist in the
form of fragments having a single binding site (e.g., Fab, Fab',
and Fv).
[0114] It will be appreciated that such bifunctional or bispecific
antibodies are contemplated and encompassed by the invention. A
bispecific single chain antibody with specificity to TIM-1 and to
the CD3 antigen on cytotoxic T lymphocytes can be used to direct
these T cells to tumor cells expressing TIM-1 and cause apoptosis
and eradication of the tumor. Two bispecific scFv constructs for
this purpose are described herein. The scFv components specific for
TIM-1 can be derived from anti-TIM-1 antibodies described herein.
In some embodiments, the anti-TIM-1 antibody components disclosed
in Tables 4 and 5 can be used to generate a biologically active
scFv directed against TIM-1. In a preferred embodiment, the scFv
components are derived from mAb 2.70. The anti-CD3 scFv component
of the therapeutic bispecific scFv was derived from a sequence
deposited in Genbank (accession number CAE85148). Alternative
antibodies known to target CD3 or other T cell antigens may
similarly be effective in treating malignancies when coupled with
anti-TIM-1, whether on a single-chain backbone or a full IgG.
Human Antibodies and Humanization of Antibodies
[0115] Embodiments of the invention described herein contemplate
and encompass human antibodies. Human antibodies avoid certain of
the problems associated with antibodies that possess murine or rat
variable and/or constant regions. The presence of such murine or
rat derived proteins can lead to the rapid clearance of the
antibodies or can lead to the generation of an immune response
against the antibody by a mammal other than a rodent.
Human Antibodies
[0116] The ability to clone and reconstruct megabase-sized human
loci in YACs and to introduce them into the mouse germline provides
a powerful approach to elucidating the functional components of
very large or crudely mapped loci as well as generating useful
models of human disease. An important practical application of such
a strategy is the "humanization" of the mouse humoral immune
system. Introduction of human immunoglobulin (Ig) loci into mice in
which the endogenous Ig genes have been inactivated offers the
opportunity to develop human antibodies in the mouse. Fully human
antibodies are expected to minimize the immunogenic and allergic
responses intrinsic to mouse or mouse-derivatized Mabs and thus to
increase the efficacy and safety of the antibodies administered to
humans. The use of fully human antibodies can be expected to
provide a substantial advantage in the treatment of chronic and
recurring human diseases, such as inflammation, autoimmunity, and
cancer, which require repeated antibody administrations.
[0117] One approach toward this goal was to engineer mouse strains
deficient in mouse antibody production with large fragments of the
human Ig loci in anticipation that such mice would produce a large
repertoire of human antibodies in the absence of mouse antibodies.
This general strategy was demonstrated in connection with our
generation of the first XenoMouse.RTM. strains as published in
1994. See Green et al., Nature Genetics 7:13-21 (1994). The
XenoMouse.RTM. strains were engineered with yeast artificial
chromosomes (YACs) containing 245 kb and 190 kb-sized germline
configuration fragments of the human heavy chain locus and kappa
light chain locus, respectively, which contained core variable and
constant region sequences. Id. The XENOMOUSE.RTM. strains are
available from Abgenix, Inc. (Fremont, Calif.). Greater than
approximately 80% of the human antibody repertoire has been
introduced through introduction of megabase sized, germline
configuration YAC fragments of the human heavy chain loci and kappa
light chain loci, respectively, to produce XenoMouse.RTM. mice.
[0118] The production of the XENOMOUSE.RTM. is further discussed
and delineated in U.S. patent application Ser. No. 07/466,008,
filed Jan. 12, 1990, Ser. No. 07/610,515, filed Nov. 8, 1990, Ser.
No. 07/919,297, filed Jul. 24, 1992, Ser. No. 07/922,649, filed
Jul. 30, 1992, filed Ser. No. 08/031,801, filed Mar. 15,1993, Ser.
No. 08/112,848, filed Aug. 27, 1993, Ser. No. 08/234,145, filed
Apr. 28, 1994, Ser. No. 08/376,279, filed Jan. 20, 1995, Ser. No.
08/430, 938, Apr. 27, 1995, Ser. No. 08/464,584, filed Jun. 5,
1995, Ser. No. 08/464,582, filed Jun. 5, 1995, Ser. No. 08/463,191,
filed Jun. 5, 1995, Ser. No. 08/462,837, filed Jun. 5, 1995, Ser.
No. 08/486,853, filed Jun. 5, 1995, Ser. No. 08/486,857, filed Jun.
5, 1995, Ser. No. 08/486,859, filed Jun. 5, 1995, Ser. No.
08/462,513, filed Jun. 5, 1995, Ser. No. 08/724,752, filed Oct. 2,
1996, and Ser. No. 08/759,620, filed Dec. 3, 1996 and U.S. Pat.
Nos. 6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598 and
Japanese Patent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2.
See also Mendez et al., Nature Genetics 15:146-156 (1997) and Green
and Jakobovits, J. Exp. Med. 188:483-495 (1998). See also European
Patent No. EP 0 463 151 B1, grant published Jun. 12, 1996,
International Patent Application No., WO 94/02602, published Feb.
3, 1994, International Patent Application No., WO 96/34096,
published Oct. 31, 1996, WO 98/24893, published Jun. 11, 1998, WO
00/76310, published Dec. 21, 2000. The disclosures of each of the
above-cited patents, applications, and references are hereby
incorporated by reference in their entirety. [000117] Alternative
approaches have utilized a "minilocus" approach, in which an
exogenous Ig locus is mimicked through the inclusion of pieces
(individual genes) from the Ig locus. Thus, one or more V.sub.H
genes, one or more D.sub.H genes, one or more J.sub.H genes, a mu
constant region, and a second constant region (preferably a gamma
constant region) are formed into a construct for insertion into an
animal. This approach is described in U.S. Pat. No. 5,545,807 to
Surani et al. and U.S. Pat. Nos. 5,545,806, 5,625,825, 5,625,126,
5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,877,397,
5,874,299, and 6,255,458 each to Lonberg and Kay, U.S. Pat. No.
5,591,669 and 6,023,010 to Krimpenfort and Berns, U.S. Pat. Nos.
5,612,205, 5,721,367, and 5,789,215 to Berns et al., and U.S. Pat.
No. 5,643,763 to Choi and Dunn, and GenPharm International U.S.
patent application Ser. No. 07/574,748, filed Aug. 29, 1990, Ser.
No. 07/575,962, filed Aug. 31, 1990, Ser. No. 07/810,279, filed
Dec. 17, 1991, Ser. No. 07/853,408, filed Mar. 18, 1992, Ser. No.
07/904,068, filed Jun. 23, 1992, Ser. No. 07/990,860, filed Dec.
16, 1992, Ser. No. 08/053,131, filed Apr. 26, 1993, Ser. No.
08/096,762, filed Jul. 22, 1993, Ser. No. 08/155,301, filed Nov.
18, 1993, Ser. No. 08/161,739, filed Dec. 3, 1993, Ser. No.
08/165,699, filed Dec. 10, 1993, Ser. No. 08/209,741, filed Mar. 9,
1994, the disclosures of which are hereby incorporated by
reference. See also European Patent No. 0 546 073 B1, International
Patent Application Nos. WO 92/03918, WO 92/22645, WO 92/22647, WO
92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO
97/13852, and WO 98/24884 and U.S. Pat. No. 5,981,175, the
disclosures of which are hereby incorporated by reference in their
entirety. See further Taylor et al., 1992, Chen et al., 1993,
Tuaillon et al., 1993, Choi et al., 1993, Lonberg et al., (1994),
Taylor et al., (1994), and Tuaillon et al., (1995), Fishwild et
al., (1996), the disclosures of which are hereby incorporated by
reference in their entirety.
[0119] While chimeric antibodies have a human constant region and a
murine variable region, it is expected that certain human
anti-chimeric antibody (HACA) responses will be observed,
particularly in chronic or multi-dose utilizations of the antibody.
Thus, it would be desirable to provide fully human antibodies
against TIM-1 in order to vitiate concerns and/or effects of human
anti-mouse antibody (HAMA) or HACA response.
Humanization and Display Technologies
[0120] Antibodies with reduced immunogenicity can be generated
using humanization and library display techniques. It will be
appreciated that antibodies can be humanized or primatized using
techniques well known in the art. See e.g., Winter and Harris,
Immunol Today 14:43-46 (1993) and Wright et al., Crit, Reviews in
Immunol. 12:125-168 (1992). The antibody of interest can be
engineered by recombinant DNA techniques to substitute the CH1,
CH2, CH3, hinge domains, and/or the framework domain with the
corresponding human sequence (see WO 92/02190 and U.S. Pat. Nos.
5,530,101, 5,585,089, 5,693,761, 5,693,792, 5,714,350, and
5,777,085). Also, the use of Ig cDNA for construction of chimeric
immunoglobulin genes is known in the art (Liu et al., P.N.A.S.
84:3439 (1987) and J. Immunol.139:3521 (1987)). mRNA is isolated
from a hybridoma or other cell producing the antibody and used to
produce cDNA. The cDNA of interest can be amplified by the
polymerase chain reaction using specific primers (U.S. Pat. Nos.
4,683,195 and 4,683,202). Alternatively, an expression library is
made and screened to isolate the sequence of interest encoding the
variable region of the antibody is then fused to human constant
region sequences. The sequences of human constant regions genes can
be found in Kabat et al., "Sequences of Proteins of Immunological
Interest," N.I.H. publication no. 91-3242 (1991). Human C region
genes are readily available from known clones. The choice of
isotype will be guided by the desired effector functions, such as
complement fixation, or activity in antibody-dependent cellular
cytotoxicity. Preferred isotypes are IgG1, IgG2 and IgG4. Either of
the human light chain constant regions, kappa or lambda, can be
used. The chimeric, humanized antibody is then expressed by
conventional methods. Expression vectors include plasmids,
retroviruses, YACs, EBV derived episomes, and the like.
[0121] Antibody fragments, such as Fv, F(ab').sub.2 and Fab can be
prepared by cleavage of the intact protein, e.g., by protease or
chemical cleavage. Alternatively, a truncated gene is designed. For
example, a chimeric gene encoding a portion of the F(ab').sub.2
fragment would include DNA sequences encoding the CH1 domain and
hinge region of the H chain, followed by a translational stop codon
to yield the truncated molecule.
[0122] Consensus sequences of H and L J regions can be used to
design oligonucleotides for use as primers to introduce useful
restriction sites into the J region for subsequent linkage of V
region segments to human C region segments. C region cDNA can be
modified by site directed mutagenesis to place a restriction site
at the analogous position in the human sequence.
[0123] Expression vectors include plasmids, retroviruses, YACs, EBV
derived episomes, and the like. A convenient vector is one that
encodes a functionally complete human CH or CL immunoglobulin
sequence, with appropriate restriction sites engineered so that any
VH or VL sequence can be easily inserted and expressed. In such
vectors, splicing usually occurs between the splice donor site in
the inserted J region and the splice acceptor site preceding the
human C region, and also at the splice regions that occur within
the human CH exons. Polyadenylation and transcription termination
occur at native chromosomal sites downstream of the coding regions.
The resulting chimeric antibody can be joined to any strong
promoter, including retroviral LTRs, e.g., SV-40 early promoter,
(Okayama et al., Mol. Cell. Bio. 3:280 (1983)), Rous sarcoma virus
LTR (Gorman et al., P.N.A.S. 79:6777 (1982)), and moloney murine
leukemia virus LTR (Grosschedl et al., Cell 41:885 (1985)). Also,
as will be appreciated, native Ig promoters and the like can be
used.
[0124] Further, human antibodies or antibodies from other species
can be generated through display-type technologies, including,
without limitation, phage display, retroviral display, ribosomal
display, and other techniques, using techniques well known in the
art and the resulting molecules can be subjected to additional
maturation, such as affinity maturation, as such techniques are
well known in the art. Wright and Harris, supra., Hanes and
Plucthau, PNAS USA 94:4937-4942 (1997) (ribosomal display), Parmley
and Smith, Gene 73:305-318 (1988) (phage display), Scott, TIBS
17:241-245 (1992), Cwirla et al., PNAS USA 87:6378-6382 (1990),
Russel et al., Nucl. Acids Res. 21:1081-1085 (1993), Hoganboom et
al., Immunol. Reviews 130:43-68 (1992), Chiswell and McCafferty,
TIBTECH 10:80-84 (1992), and U.S. Pat. No. 5,733,743. If display
technologies are utilized to produce antibodies that are not human,
such antibodies can be humanized as described above.
[0125] Using these techniques, antibodies can be generated to TIM-1
expressing cells, TIM-1 itself, forms of TIM-1, epitopes or
peptides thereof, and expression libraries thereto (see e.g. U.S.
Pat. No. 5,703,057) which can thereafter be screened as described
above for the activities described above.
Antibody Therapeutics
[0126] In certain respects, it can be desirable in connection with
the generation of antibodies as therapeutic candidates against
TIM-1 that the antibodies be capable of fixing complement and
participating in complement-dependent cytotoxicity (CDC). Such
antibodies include, without limitation, the following: murine IgM,
murine IgG2a, murine IgG2b, murine IgG3, human IgM, human IgG1, and
human IgG3. It will be appreciated that antibodies that are
generated need not initially possess such an isotype but, rather,
the antibody as generated can possess any isotype and the antibody
can be isotype switched thereafter using conventional techniques
that are well known in the art. Such techniques include the use of
direct recombinant techniques (see, e.g., U.S. Patent No.
4,816,397), cell-cell fusion techniques (see, e.g., U.S. Patent
Nos. 5,916,771 and 6,207,418), among others.
[0127] In the cell-cell fusion technique, a myeloma or other cell
line is prepared that possesses a heavy chain with any desired
isotype and another myeloma or other cell line is prepared that
possesses the light chain. Such cells can, thereafter, be fused and
a cell line expressing an intact antibody can be isolated.
[0128] By way of example, the TIM-1 antibody discussed herein is a
human anti-TIM-1 IgG2 antibody. If such antibody possessed desired
binding to the TIM-1 molecule, it could be readily isotype switched
to generate a human IgM, human IgG1, or human IgG3 isotype, while
still possessing the same variable region (which defines the
antibody's specificity and some of its affinity). Such molecule
would then be capable of fixing complement and participating in
CDC.
Design and Generation of Other Therapeutics
[0129] Due to their association with renal and pancreatic tumors,
head and neck cancer, ovarian cancer, gastric (stomach) cancer,
melanoma, lymphoma, prostate cancer, liver cancer, breast cancer,
lung cancer, renal cancer, bladder cancer, colon cancer, esophageal
cancer, and brain cancer, antineoplastic agents comprising
anti-TIM-1 antibodies are contemplated and encompassed by the
invention.
[0130] Moreover, based on the activity of the antibodies that are
produced and characterized herein with respect to TIM-1, the design
of other therapeutic modalities beyond antibody moieties is
facilitated. Such modalities include, without limitation, advanced
antibody therapeutics, such as bispecific antibodies, immunotoxins,
and radiolabeled therapeutics, generation of peptide therapeutics,
gene therapies, particularly intrabodies, antisense therapeutics,
and small molecules.
[0131] In connection with the generation of advanced antibody
therapeutics, where complement fixation is a desirable attribute,
it can be possible to sidestep the dependence on complement for
cell killing through the use of bispecifics, immunotoxins, or
radiolabels, for example.
[0132] For example, in connection with bispecific antibodies,
bispecific antibodies can be generated that comprise (i) two
antibodies one with a specificity to TIM-1 and another to a second
molecule that are conjugated together, (ii) a single antibody that
has one chain specific to TIM-1 and a second chain specific to a
second molecule, or (iii) a single chain antibody that has
specificity to TIM-1 and the other molecule. Such bispecific
antibodies can be generated using techniques that are well known
for example, in connection with (i) and (ii) see, e.g., Fanger et
al., Immunol Methods 4:72-81 (1994) and Wright and Harris, supra
and in connection with (iii) see, e.g., Traunecker et al., Int. J.
Cancer (Suppl.) 7:51-52 (1992). In each case, the second
specificity can be made to the heavy chain activation receptors,
including, without limitation, CD16 or CD64 (see, e.g., Deo et al.,
18:127 (1997)) or CD89 (see, e.g., Valerius et al., Blood
90:4485-4492 (1997)). Bispecific antibodies prepared in accordance
with the foregoing would be likely to kill cells expressing TIM-1,
and particularly those cells in which the TIM-1 antibodies
described herein are effective.
[0133] With respect to immunotoxins, antibodies can be modified to
act as immunotoxins utilizing techniques that are well known in the
art. See, e.g., Vitetta, Immunol Today 14:252 (1993). See also U.S.
Pat. No. 5,194,594. In connection with the preparation of
radiolabeled antibodies, such modified antibodies can also be
readily prepared utilizing techniques that are well known in the
art. See, e.g., Junghans et al., in Cancer Chemotherapy and
Biotherapy 655-686 (2d ed., Chafner and Longo, eds., Lippincott
Raven (1996)). See also U.S. Pat. Nos. 4,681,581, 4,735,210,
5,101,827, 5,102,990 (RE 35,500), U.S. Pat. No. 5,648,471, and
5,697,902. Each of immunotoxins and radiolabeled molecules would be
likely to kill cells expressing TIM-1, and particularly those cells
in which the antibodies described herein are effective.
[0134] In connection with the generation of therapeutic peptides,
through the utilization of structural information related to TIM-1
and antibodies thereto, such as the antibodies described herein (as
discussed below in connection with small molecules) or screening of
peptide libraries, therapeutic peptides can be generated that are
directed against TIM-1. Design and screening of peptide
therapeutics is discussed in connection with Houghten et al.,
Biotechniques 13:412-421 (1992), Houghten, PNAS USA 82:5131-5135
(1985), Pinalla et al., Biotechniques 13:901-905 (1992), Blake and
Litzi-Davis, BioConjugate Chem. 3:510-513 (1992). Immunotoxins and
radiolabeled molecules can also be prepared, and in a similar
manner, in connection with peptidic moieties as discussed above in
connection with antibodies.
[0135] Assuming that the TIM-1 molecule (or a form, such as a
splice variant or alternate form) is functionally active in a
disease process, it will also be possible to design gene and
antisense therapeutics thereto through conventional techniques.
Such modalities can be utilized for modulating the function of
TIM-1. In connection therewith the antibodies, as described herein,
facilitate design and use of functional assays related thereto. A
design and strategy for antisense therapeutics is discussed in
detail in International Patent Application No. WO 94/29444. Design
and strategies for gene therapy are well known. However, in
particular, the use of gene therapeutic techniques involving
intrabodies could prove to be particularly advantageous. See, e.g.,
Chen et al., Human Gene Therapy 5:595-601 (1994) and Marasco, Gene
Therapy 4:11-15 (1997). General design of and considerations
related to gene therapeutics is also discussed in International
Patent Application No. WO 97/38137.
[0136] Small molecule therapeutics can also be envisioned. Drugs
can be designed to modulate the activity of TIM-1, as described
herein. Knowledge gleaned from the structure of the TIM-1 molecule
and its interactions with other molecules, as described herein,
such as the antibodies described herein, and others can be utilized
to rationally design additional therapeutic modalities. In this
regard, rational drug design techniques such as X-ray
crystallography, computer-aided (or assisted) molecular modeling
(CAMM), quantitative or qualitative structure-activity relationship
(QSAR), and similar technologies can be utilized to focus drug
discovery efforts. Rational design allows prediction of protein or
synthetic structures which can interact with the molecule or
specific forms thereof which can be used to modify or modulate the
activity of TIM-1. Such structures can be synthesized chemically or
expressed in biological systems. This approach has been reviewed in
Capsey et al., Genetically Engineered Human Therapeutic Drugs
(Stockton Press, NY (1988)). Further, combinatorial libraries can
be designed and synthesized and used in screening programs, such as
high throughput screening efforts.
TIM-1 Agonists And Antagonists
[0137] Embodiments of the invention described herein also pertain
to variants of a TIM-1 protein that function as either TIM-1
agonists (mimetics) or as TIM-1 antagonists. Variants of a TIM-1
protein can be generated by mutagenesis, e.g., discrete point
mutation or truncation of the TIM-1 protein. An agonist of the
TIM-1 protein can retain substantially the same, or a subset of,
the biological activities of the naturally occurring form of the
TIM-1 protein. An antagonist of the TIM-1 protein can inhibit one
or more of the activities of the naturally occurring form of the
TIM-1 protein by, for example, competitively binding to a
downstream or upstream member of a cellular signaling cascade which
includes the TIM-1 protein. Thus, specific biological effects can
be elicited by treatment with a variant of limited function. In one
embodiment, treatment of a subject with a variant having a subset
of the biological activities of the naturally occurring form of the
protein has fewer side effects in a subject relative to treatment
with the naturally occurring form of the TIM-1 protein.
[0138] Variants of the TIM-1 protein that function as either TIM-1
agonists (mimetics) or as TIM-1 antagonists can be identified by
screening combinatorial libraries of mutants, e.g., truncation
mutants, of the TIM-1 protein for protein agonist or antagonist
activity. In one embodiment, a variegated library of TIM-1 variants
is generated by combinatorial mutagenesis at the nucleic acid level
and is encoded by a variegated gene library. A variegated library
of TIM-1 variants can be produced by, for example, enzymatically
ligating a mixture of synthetic oligonucleotides into gene
sequences such that a degenerate set of potential TIM-1 sequences
is expressible as individual polypeptides, or alternatively, as a
set of larger fusion proteins (e.g., for phage display) containing
the set of TIM-1 sequences therein. There are a variety of methods
which can be used to produce libraries of potential TIM-1 variants
from a degenerate oligonucleotide sequence. Chemical synthesis of a
degenerate gene sequence can be performed in an automatic DNA
synthesizer, and the synthetic gene then ligated into an
appropriate expression vector. Use of a degenerate set of genes
allows for the provision, in one mixture, of all of the sequences
encoding the desired set of potential TIM-1 variant sequences.
Methods for synthesizing degenerate oligonucleotides are known in
the art (see, e.g., Narang, Tetrahedron 39:3 (1983); Itakura et
al., Annu. Rev. Biochem. 53:323 (1984); Itakura et al., Science
198:1056 (1984); Ike et al., Nucl. Acid Res. 11:477 (1983).
Radioimmuno & Immunochemotherapeutic Antibodies
[0139] Cytotoxic chemotherapy or radiotherapy of cancer is limited
by serious, sometimes life-threatening, side effects that arise
from toxicities to sensitive normal cells because the therapies are
not selective for malignant cells. Therefore, there is a need to
improve the selectivity. One strategy is to couple therapeutics to
antibodies that recognize tumor-associated antigens. This increases
the exposure of the malignant cells to the ligand-targeted
therapeutics but reduces the exposure of normal cells to the same
agent. See Allen, Nat. Rev. Cancer 2(10):750-63 (2002).
[0140] The TIM-1 antigen is one of these tumor-associated antigens,
as shown by its specific expression on cellular membranes of tumor
cells by FACS and IHC. Therefore one embodiment of the invention is
to use monoclonal antibodies directed against the TIM-1 antigen
coupled to cytotoxic chemotherapic agents or radiotherapic agents
as anti-tumor therapeutics.
[0141] Radiolabels are known in the art and have been used for
diagnostic or therapeutic radioimmuno conjugates. Examples of
radiolabels includes, but are not limited to, the following:
radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc,
111In, 1251, 1311, 177Lu, Rhenium-186, Rhenium-188, Samarium-153,
Copper-64, Scandium-47). For example, radionuclides which have been
used in radioimmunoconjugate guided clinical diagnosis include, but
are not limited to: 131 I, 125 I, 123 I, 99 Tc, 67 Ga, as well as
111 In. Antibodies have also been labeled with a variety of
radionuclides for potential use in targeted immunotherapy (see
Peirersz et al., 1987). Monoclonal antibody conjugates have also
been used for the diagnosis and treatment of cancer (e.g., Immunol.
Cell Biol. 65:111-125). These radionuclides include, for example,
188 Re and 186 Re as well as 90 Y, and to a lesser extent 199 Au
and 67 Cu. I-(131) have also been used for therapeutic purposes.
U.S. Pat. No. 5,460,785 provides a listing of such radioisotopes.
Radiotherapeutic chelators and chelator conjugates are known in the
art. See U.S. Pat. Nos. 4,831,175, 5,099,069, 5,246,692, 5,286,850,
and 5,124,471.
[0142] Immunoradiopharmaceuticals utilizing anti-TIM-1 antibodies
can be prepared utilizing techniques that are well known in the
art. See, e.g., Junghans et al., in Cancer Chemotherapy and
Biotherapy 655-686 (2d ed., Chafner and Longo, eds., Lippincott
Raven (1996)), U.S. Pat. Nos. 4,681,581, 4,735,210, 5,101,827, RE
35,500, U.S. Pat. No. 5,648,471, and 5,697,902.
[0143] Cyotoxic immunoconjugates are known in the art and have been
used as therapeutic agents. Such immunoconjugates may for example,
use maytansinoids (U.S. Pat. No. 6,441,163), tubulin polymerization
inhibitor, auristatin (Mohammad et al., Int. J. Oncol. 15(2):367-72
(1999); Doronina et al., Nature Biotechnology 21(7):778-784
(2003)), dolastatin derivatives (Ogawa et al., Toxicol Lett.
121(2):97-106 (2001); 21(3)778-784), Mylotarg.RTM. (Wyeth
Laboratories, Philidelphia, Pa.); maytansinoids (DM1), taxane or
mertansine (ImmunoGen Inc.). Immunotoxins utilizing anti-TIM-1
antibodies may be prepared by techniques that are well known in the
art. See, e.g., Vitetta, Immunol Today 14:252 (1993); U.S. Pat. No.
5,194,594.
[0144] Bispecific antibodies may be generated using techniques that
are well known in the art for example, see, e.g., Fanger et al.,
Immunol Methods 4:72-81 (1994); Wright and Harris, supra;
Traunecker et al., Int. J. Cancer (Suppl.) 7:51-52 (1992). In each
case, the first specificity is to TIM-1, the second specificity may
be made to the heavy chain activation receptors, including, without
limitation, CD16 or CD64 (see, e.g., Deo et al., 18:127 (1997)) or
CD89 (see, e.g., Valerius et al., Blood 90:4485-4492 (1997)).
Bispecific antibodies prepared in accordance with the foregoing
would kill cells expressing TIM-1.
[0145] Depending on the intended use of the antibody, i.e., as a
diagnostic or therapeutic reagent, radiolabels are known in the art
and have been used for similar purposes. For example, radionuclides
which have been used in clinical diagnosis include, but are not
limited to: .sup.131 I, .sup.125 I, .sup.123 I, .sup.99 Tc, .sup.67
Ga, as well as .sup.111 In. Antibodies have also been labeled with
a variety of radionuclides for potential use in targeted
immunotherapy. See Peirersz et al., (1987). Monoclonal antibody
conjugates have also been used for the diagnosis and treatment of
cancer. See, e.g., Immunol. Cell Biol. 65:111-125. These
radionuclides include, for example, .sup.188 Re and .sup.186 Re as
well as .sup.90 Y, and to a lesser extent .sup.199 Au and .sup.67
Cu. I-(131) have also been used for therapeutic purposes. U.S. Pat.
No. 5,460,785 provides a listing of such radioisotopes.
[0146] Patents relating to radiotherapeutic chelators and chelator
conjugates are known in the art. For example, U.S. Pat. No.
4,831,175 of Gansow is directed to polysubstituted
diethylenetriaminepentaacetic acid chelates and protein conjugates
containing the same, and methods for their preparation. U.S. Pat.
Nos. 5,099,069, 5,246,692, 5,286,850, and 5,124,471 of Gansow also
relate to polysubstituted DTPA chelates.
[0147] Cytotoxic chemotherapies are known in the art and have been
used for similar purposes. For example, U.S. Pat. No. 6,441,163
describes the process for the production of cytotoxic conjugates of
maytansinoids and antibodies. The anti-tumor activity of a tubulin
polymerization inhibitor, auristatin PE, is also known in the art.
Mohammad et al., Int. J. Oncol. 15(2):367-72 (Aug 1999).
Preparation of Antibodies
[0148] Briefly, XenoMouse.RTM. lines of mice were immunized with
TIM-1 protein, lymphatic cells (such as B-cells) were recovered
from the mice that express antibodies and were fused with a
myeloid-type cell line to prepare immortal hybridoma cell lines,
and such hybridoma cell lines were screened and selected to
identify hybridoma cell lines that produce antibodies specific to
TIM-1. Alternatively, instead of being fused to myeloma cells to
generate hybridomas, the recovered B cells, isolated from immunized
XenoMouse.RTM. lines of mice, with reactivity against TIM-1
(determined by e.g. ELISA with TIM-1-His protein), were then
isolated using a TIM-1-specific hemolytic plaque assay. Babcook et
al., Proc. Natl. Acad. Sci. USA, 93:7843-7848 (1996). In this
assay, target cells such as sheep red blood cells (SRBCs) were
coated with the TIM-1 antigen. In the presence of a B cell culture
secreting the anti-TIM-1 antibody and complement, the formation of
a plaque indicates specific TIM-1-mediated lysis of the target
cells. Single antigen-specific plasma cells in the center of the
plaques were isolated and the genetic information that encodes the
specificity of the antibody isolated from single plasma cells.
[0149] Using reverse-transcriptase PCR, the DNA encoding the
variable region of the antibody secreted was cloned and inserted
into a suitable expression vector, preferably a vector cassette
such as a pcDNA, more preferably the pcDNA vector containing the
constant domains of immunglobulin heavy and light chain. The
generated vector was then be transfected into host cells,
preferably CHO cells, and cultured in conventional nutrient media
modified as appropriate for inducing promoters, selecting
transformants, or amplifying the genes encoding the desired
sequences.
[0150] In general, antibodies produced by the above-mentioned cell
lines possessed fully human IgG2 heavy chains with human kappa
light chains. The antibodies possessed high affinities, typically
possessing Kd's of from about 10-6 through about 10-11 M, when
measured by either solid phase and solution phase. These mAbs can
be stratified into groups or "bins" based on antigen binding
competition studies, as discussed below.
[0151] As will be appreciated, antibodies, as described herein, can
be expressed in cell lines other than hybridoma cell lines.
Sequences encoding particular antibodies can be used for
transformation of a suitable mammalian host cell. Transformation
can be by any known method for introducing polynucleotides into a
host cell, including, for example packaging the polynucleotide in a
virus (or into a viral vector) and transducing a host cell with the
virus (or vector) or by transfection procedures known in the art,
as exemplified by U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461,
and 4,959,455 (which patents are hereby incorporated herein by
reference). The transformation procedure used depends upon the host
to be transformed. Methods for introduction of heterologous
polynucleotides into mammalian cells are well known in the art and
include dextran-mediated transfection, calcium phosphate
precipitation, polybrene mediated transfection, protoplast fusion,
electroporation, encapsulation of the polynucleotide(s) in
liposomes, and direct microinjection of the DNA into nuclei.
[0152] Mammalian cell lines available as hosts for expression are
well known in the art and include many immortalized cell lines
available from the American Type Culture Collection (ATCC),
including but not limited to Chinese hamster ovary (CHO) cells,
HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells
(COS), human hepatocellular carcinoma cells (e.g., Hep G2), and a
number of other cell lines. Cell lines of particular preference are
selected through determining which cell lines have high expression
levels and produce antibodies with constitutive TIM-1 binding
properties.
Therapeutic Administration and Formulations
[0153] The compounds of the invention are formulated according to
standard practice, such as prepared in a carrier vehicle. The term
"pharmacologically acceptable carrier" means one or more organic or
inorganic ingredients, natural or synthetic, with which the mutant
proto-oncogene or mutant oncoprotein is combined to facilitate its
application. A suitable carrier includes sterile saline although
other aqueous and non-aqueous isotonic sterile solutions and
sterile suspensions known to be pharmaceutically acceptable are
known to those of ordinary skill in the art. In this regard, the
term "carrier" encompasses liposomes and the antibody (See Chen et
al., Anal. Biochem. 227: 168-175 (1995) as well as any plasmid and
viral expression vectors.
[0154] Any of the novel polypeptides of this invention may be used
in the form of a pharmaceutically acceptable salt. Suitable acids
and bases which are capable of forming salts with the polypeptides
of the present invention are well known to those of skill in the
art, and include inorganic and organic acids and bases.
[0155] A compound of the invention is administered to a subject in
a therapeutically-effective amount, which means an amount of the
compound which produces a medically desirable result or exerts an
influence on the particular condition being treated. An effective
amount of a compound of the invention is capable of ameliorating or
delaying progression of the diseased, degenerative or damaged
condition. The effective amount can be determined on an individual
basis and will be based, in part, on consideration of the physical
attributes of the subject, symptoms to be treated and results
sought. An effective amount can be determined by one of ordinary
skill in the art employing such factors and using no more than
routine experimentation.
[0156] The compounds of the invention may be administered in any
manner which is medically acceptable. This may include injections,
by parenteral routes such as intravenous, intravascular,
intraarterial, subcutaneous, intramuscular, intratumor,
intraperitoneal, intraventricular, intraepidural, or others as well
as oral, nasal, ophthalmic, rectal, or topical. Sustained release
administration is also specifically included in the invention, by
such means as depot injections or erodible implants. Localized
delivery is particularly contemplated, by such means as delivery
via a catheter to one or more arteries, such as the renal artery or
a vessel supplying a localized tumor.
[0157] Biologically active anti-TIM-1 antibodies as described
herein can be used in a sterile pharmaceutical preparation or
formulation to reduce the level of serum TIM-1 thereby effectively
treating pathological conditions where, for example, serum TIM-1 is
abnormally elevated. Anti-TIM-1 antibodies preferably possess
adequate affinity to potently suppress TIM-1 to within the target
therapeutic range, and preferably have an adequate duration of
action to allow for infrequent dosing. A prolonged duration of
action will allow for less frequent and more convenient dosing
schedules by alternate parenteral routes such as subcutaneous or
intramuscular injection.
[0158] When used for in vivo administration, the antibody
formulation must be sterile. This is readily accomplished, for
example, by filtration through sterile filtration membranes, prior
to or following lyophilization and reconstitution. The antibody
ordinarily will be stored in lyophilized form or in solution.
Therapeutic antibody compositions generally are placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having an adapter that allows retrieval of the
formulation, such as a stopper pierceable by a hypodermic injection
needle.
[0159] The route of antibody administration is in accord with known
methods, e.g., injection or infusion by intravenous,
intraperitoneal, intracerebral, intramuscular, intraocular,
intraarterial, intrathecal, inhalation or intralesional routes, or
by sustained release systems as noted below. The antibody is
preferably administered continuously by infusion or by bolus
injection.
[0160] An effective amount of antibody to be employed
therapeutically will depend, for example, upon the therapeutic
objectives, the route of administration, and the condition of the
patient. Accordingly, it is preferred that the therapist titer the
dosage and modify the route of administration as required to obtain
the optimal therapeutic effect. Typically, the clinician will
administer antibody until a dosage is reached that achieves the
desired effect. The progress of this therapy is easily monitored by
conventional assays or by the assays described herein.
[0161] Antibodies, as described herein, can be prepared in a
mixture with a pharmaceutically acceptable carrier. This
therapeutic composition can be administered intravenously or
through the nose or lung, preferably as a liquid or powder aerosol
(lyophilized). The composition can also be administered
parenterally or subcutaneously as desired. When administered
systemically, the therapeutic composition should be sterile,
pyrogen-free and in a parenterally acceptable solution having due
regard for pH, isotonicity, and stability. These conditions are
known to those skilled in the art. Briefly, dosage formulations of
the compounds described herein are prepared for storage or
administration by mixing the compound having the desired degree of
purity with physiologically acceptable carriers, excipients, or
stabilizers. Such materials are non-toxic to the recipients at the
dosages and concentrations employed, and include buffers such as
TRIS HCl, phosphate, citrate, acetate and other organic acid salts;
antioxidants such as ascorbic acid; low molecular weight (less than
about ten residues) peptides such as polyarginine, proteins, such
as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers
such as polyvinylpyrrolidinone; amino acids such as glycine,
glutamic acid, aspartic acid, or arginine; monosaccharides,
disaccharides, and other carbohydrates including cellulose or its
derivatives, glucose, mannose, or dextrins; chelating agents such
as EDTA; sugar alcohols such as mannitol or sorbitol; counterions
such as sodium and/or nonionic surfactants such as TWEEN, PLURONICS
or polyethyleneglycol.
[0162] Sterile compositions for injection can be formulated
according to conventional pharmaceutical practice as described in
Remington: The Science and Practice of Pharmacy (20.sup.th ed,
Lippincott Williams & Wilkens Publishers (2003)). For example,
dissolution or suspension of the active compound in a vehicle such
as water or naturally occurring vegetable oil like sesame, peanut,
or cottonseed oil or a synthetic fatty vehicle like ethyl oleate or
the like can be desired. Buffers, preservatives, antioxidants and
the like can be incorporated according to accepted pharmaceutical
practice.
[0163] Suitable examples of sustained-release preparations include
semipermeable matrices of solid hydrophobic polymers containing the
polypeptide, which matrices are in the form of shaped articles,
films or microcapsules. Examples of sustained-release matrices
include polyesters, hydrogels (e.g.,
poly(2-hydroxyethyl-methacrylate) as described by Langer et al., J.
Biomed Mater. Res., (1981) 15:167-277 and Langer, Chem. Tech.,
(1982) 12:98-105, or poly(vinylalcohol)), polylactides (U.S. Pat.
No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma
ethyl-L-glutamate (Sidman et al., Biopolymers, (1983) 22:547-556),
non-degradable ethylene-vinyl acetate (Langer et al., supra),
degradable lactic acid-glycolic acid copolymers such as the LUPRON
Depot.TM. (injectable microspheres composed of lactic acid-glycolic
acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid (EP 133,988).
[0164] While polymers such as ethylene-vinyl acetate and lactic
acid-glycolic acid enable release of molecules for over 100 days,
certain hydrogels release proteins for shorter time periods. When
encapsulated proteins remain in the body for a long time, they can
denature or aggregate as a result of exposure to moisture at
37.degree. C., resulting in a loss of biological activity and
possible changes in immunogenicity. Rational strategies can be
devised for protein stabilization depending on the mechanism
involved. For example, if the aggregation mechanism is discovered
to be intermolecular S--S bond formation through disulfide
interchange, stabilization can be achieved by modifying sulfhydryl
residues, lyophilizing from acidic solutions, controlling moisture
content, using appropriate additives, and developing specific
polymer matrix compositions.
[0165] Sustained-released compositions also include preparations of
crystals of the antibody suspended in suitable formulations capable
of maintaining crystals in suspension. These preparations when
injected subcutaneously or intraperitonealy can produce a sustained
release effect. Other compositions also include liposomally
entrapped antibodies. Liposomes containing such antibodies are
prepared by methods known per se: U.S. Pat. No. DE 3,218,121;
Epstein et al., Proc. Natl. Acad. Sci. USA, (1985) 82:3688-3692;
Hwang et al., Proc. Natl. Acad. Sci. USA, (1980) 77:4030-4034; EP
52,322; EP 36,676; EP 88,046; EP 143,949; 142,641; Japanese patent
application 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and
EP 102,324.
[0166] The dosage of the antibody formulation for a given patient
will be determined by the attending physician taking into
consideration various factors known to modify the action of drugs
including severity and type of disease, body weight, sex, diet,
time and route of administration, other medications and other
relevant clinical factors. Therapeutically effective dosages can be
determined by either in vitro or in vivo methods.
[0167] An effective amount of the antibodies, described herein, to
be employed therapeutically will depend, for example, upon the
therapeutic objectives, the route of administration, and the
condition of the patient. Accordingly, it is preferred for the
therapist to titer the dosage and modify the route of
administration as required to obtain the optimal therapeutic
effect. A typical daily dosage might range from about 0.001 mg/kg
to up to 100 mg/kg or more, depending on the factors mentioned
above. Typically, the clinician will administer the therapeutic
antibody until a dosage is reached that achieves the desired
effect. The progress of this therapy is easily monitored by
conventional assays or as described herein.
[0168] It will be appreciated that administration of therapeutic
entities in accordance with the compositions and methods herein
will be administered with suitable carriers, excipients, and other
agents that are incorporated into formulations to provide improved
transfer, delivery, tolerance, and the like. These formulations
include, for example, powders, pastes, ointments, jellies, waxes,
oils, lipids, lipid (cationic or anionic) containing vesicles (such
as Lipofectin.TM.) DNA conjugates, anhydrous absorption pastes,
oil-in-water and water-in-oil emulsions, emulsions carbowax
(polyethylene glycols of various molecular weights), semi-solid
gels, and semi-solid mixtures containing carbowax. Any of the
foregoing mixtures can be appropriate in treatments and therapies
in accordance with the present invention, provided that the active
ingredient in the formulation is not inactivated by the formulation
and the formulation is physiologically compatible and tolerable
with the route of administration. See also Baldrick P.
"Pharmaceutical excipient development: the need for preclinical
guidance." Regul. Toxicol. Pharmacol. 32(2):210-8 (2000), Wang W.
"Lyophilization and development of solid protein pharmaceuticals."
Int. J. Pharm. 203(1-2):1-60 (2000), Charman W N "Lipids,
lipophilic drugs, and oral drug delivery-some emerging concepts." J
Pharm Sci .89(8):967-78 (2000), Powell et al. "Compendium of
excipients for parenteral formulations" PDA J Pharm Sci Technol.
52:238-311 (1998) and the citations therein for additional
information related to formulations, excipients and carriers well
known to pharmaceutical chemists.
[0169] It is expected that the antibodies described herein will
have therapeutic effect in treatment of symptoms and conditions
resulting from TIM-1 expression. In specific embodiments, the
antibodies and methods herein relate to the treatment of symptoms
resulting from TIM-1 expression including symptoms of cancer.
Further embodiments, involve using the antibodies and methods
described herein to treat cancers, such as cancer of the lung,
colon, stomach, kidney, prostrate, or ovary.
Diagnostic Use
[0170] TIM-1 has been found to be expressed at low levels in normal
kidney but its expression is increased dramatically in postischemic
kidney. Ichimura et al., J. Biol. Chem. 273(7):4135-42 (1998). As
immunohistochemical staining with anti-TIM-1 antibody shows
positive staining of renal, kidney, prostate and ovarian carcinomas
(see below), TIM-1 overexpression relative to normal tissues can
serve as a diagnostic marker of such diseases.
[0171] Antibodies, including antibody fragments, can be used to
qualitatively or quantitatively detect the expression of TIM-1
proteins. As noted above, the antibody preferably is equipped with
a detectable, e.g., fluorescent label, and binding can be monitored
by light microscopy, flow cytometry, fluorimetry, or other
techniques known in the art. These techniques are particularly
suitable if the amplified gene encodes a cell surface protein,
e.g., a growth factor. Such binding assays are performed as known
in the art.
[0172] In situ detection of antibody binding to the TIM-1 protein
can be performed, for example, by immunofluorescence or
immunoelectron microscopy. For this purpose, a tissue specimen is
removed from the patient, and a labeled antibody is applied to it,
preferably by overlaying the antibody on a biological sample. This
procedure also allows for determining the distribution of the
marker gene product in the tissue examined. It will be apparent for
those skilled in the art that a wide variety of histological
methods are readily available for in situ detection.
Epitope Mapping
[0173] The specific part of the protein immunogen recognized by an
antibody may be determined by assaying the antibody reactivity to
parts of the protein, for example an N terminal and C terminal
half. The resulting reactive fragment can then be further
dissected, assaying consecutively smaller parts of the immunogen
with the antibody until the minimal reactive peptide is defined.
Anti-TIM-1 mAb 2.70.2 was assayed for reactivity against
overlapping peptides designed from the antigen sequence and was
found to specifically recognize the amino acid sequence PLPRQNHE
(SEQ ID NO:96) corresponding to amino acids 189-202 of the TIM-1
immunogen (SEQ ID NO:54). Furthermore using an alanine scanning
technique, it has been determined that the second proline and the
asparagine residues appear to be important for mAb 2.70.2
binding.
[0174] Alternatively, the epitope that is bound by the anti-TIM-1
antibodies of the invention may be determined by subjecting the
TIM-1 immunogen to SDS-PAGE either in the absence or presence of a
reduction agent and analyzed by immunoblotting. Epitope mapping may
also be performed using SELDI. SELDI ProteinChip.RTM. (LumiCyte)
arrays used to define sites of protein-protein interaction. TIM-1
protein antigen or fragments thereof may be specifically captured
by antibodies covalently immobilized onto the PROTEINCHIP array
surface. The bound antigens may be detected by a laser-induced
desorption process and analyzed directly to determine their
mass.
[0175] The epitope recognized by anti-TIM-1 antibodies described
herein may be determined by exposing the PROTEINCHIP Array to a
combinatorial library of random peptide 12-mer displayed on
Filamentous phage (New England Biolabs). Antibody-bound phage are
eluted and then amplified and taken through additional binding and
amplification cycles to enrich the pool in favor of binding
sequences. After three or four rounds, individual binding clones
are further tested for binding by phage ELISA assays performed on
antibody-coated wells and characterized by specific DNA sequencing
of positive clones.
EXAMPLES
[0176] The following examples, including the experiments conducted
and results achieved are provided for illustrative purposes only
and are not to be construed as limiting upon the invention
described herein.
Example 1
Preparation of Monoclonal Antibodies that Bind TIM-1
[0177] The soluble extracellular domain of TIM-1 was used as the
immunogen to stimulate an immune response in XenoMouse.RTM.
animals. A DNA (CG57008-02), which encodes the amino acid sequence
for the TIM-1 extracellular domain (minus the predicted N-terminal
signal peptide) was subcloned to the baculovirus expression vector,
pMelV5His (CuraGen Corp., New Haven, Conn.), expressed using the
pBlueBac baculovirus expression system (Invitrogen Corp., Carlsbad,
Calif.), and confirmed by Western blot analyses. The nucleotide
sequence below encodes the polypeptide used to generate
antibodies.
TABLE-US-00002 (SEQ ID NO: 53)
TCTGTAAAGGTTGGTGGAGAGGCAGGTCCATCTGTCACACTACCCTGCCA
CTACAGTGGAGCTGTCACATCAATGTGCTGGAATAGAGGCTCATGTTCTC
TATTCACATGCCAAAATGGCATTGTCTGGACCAATGGAACCCACGTCACC
TATCGGAAGGACACACGCTATAAGCTATTGGGGGACCTTTCAAGAAGGGA
TGTCTCTTTGACCATAGAAAATACAGCTGTGTCTGACAGTGGCGTATATT
GTTGCCGTGTTGAGCACCGTGGGTGGTTCAATGACATGAAAATCACCGTA
TCATTGGAGATTGTGCCACCCAAGGTCACGACTACTCCAATTGTCACAAC
TGTTCCAACCGTCACGACTGTTCGAACGAGCACCACTGTTCCAACGACAA
CGACTGTTCCAACGACAACTGTTCCAACAACAATGAGCATTCCAACGACA
ACGACTGTTCCGACGACAATGACTGTTTCAACGACAACGAGCGTTCCAAC
GACAACGAGCATTCCAACAACAACAAGTGTTCCAGTGACAACAACGGTCT
CTACCTTTGTTCCTCCAATGCCTTTGCCCAGGCAGAACCATGAACCAGTA
GCCACTTCACCATCTTCACCTCAGCCAGCAGAAACCCACCCTACGACACT
GCAGGGAGCAATAAGGAGAGAACCCACCAGCTCACCATTGTACTCTTACA
CAACAGATGGGAATGACACCGTGACAGAGTCTTCAGATGGCCTTTGGAAT
AACAATCAAACTCAACTGTTCCTAGAACATAGTCTACTG
[0178] The amino acid sequence encoded thereby is as follows:
TABLE-US-00003 (SEQ ID NO: 54)
SVKVGGEAGPSVTLPCHYSGAVTSMCWNRGSCSLFTCQNGIVWTNGTHVT
YRKDTRYKLLGDLSRRDVSLTIENTAVSDSGVYCCRVEHRGWFNDMKITV
SLEIVPPKVTTTPIVTTVPTVTTVRTSTTVPTTTTVPTTTVPTTMSIPTT
TTVPTTMTVSTTTSVPTTTSIPTTTSVPVTTTVSTFVPPMPLPRQNHEPV
ATSPSSPQPAETHPTTLQGAIRREPTSSPLYSYTTDGNDTVTESSDGLWN
NNQTQLFLEHSLL
[0179] To facilitate purification of recombinant TIM-1, the
expression construct can incorporate coding sequences for the V5
binding domain V5 and a HIS tag. Fully human IgG2 and IgG4
monoclonal antibodies (mAb), directed against TIM-1 were generated
from human antibody-producing XenoMouse.RTM. strains engineered to
be deficient in mouse antibody production and to contain the
majority of the human antibody gene repertoire on megabase-sized
fragments from the human heavy and kappa light chain loci as
previously described in Yang et al., Cancer Res. (1999). Two
XenoMouse.RTM. strains, an hIgG2 (xmg-2) strain and an IgG4 (3C-1)
strain, were immunized with the TIM-1 antigen (SEQ ID NO: 54). Both
strains responded well to immunization (Tables 2 and 3).
TABLE-US-00004 TABLE 2 Serum titer of XENOMOUSE .RTM. hIgG.sub.2
strain immunized with TIM-1 antigen. Group 1: 5 mice (hIgG.sub.2
strain); mode of immunization = footpad Reactivity to TIM-1 Titers
via hIgG Mouse ID Bleed After 4 inj. Bleed After 6 inj. M716-1
600,000 600,000 M716-2 600,000 500,000 M716-3 200,000 400,000
M716-4 300,000 200,000 M716-5 400,000 400,000 Negative Control 75
110 Positive Control -- 600,000
TABLE-US-00005 TABLE 3 Serum titer of XENOMOUSE .RTM. IgG.sub.4
strain immunized with TIM-1 antigen Group 2: 5 mice (IgG.sub.4
strain); mode of immunization = footpad Reactivity to TIM-1 Titers
via hIgG Mouse ID Bleed After 4 inj. Bleed After 6 inj. M326-2
15,000 73,000 M326-3 7,500 60,000 M329-1 27,000 30,000 M329-3 6,500
50,000 M337-1 2,500 16,000 Negative Control <100 90 Positive
Control -- 600,000
[0180] Hybridoma cell lines were generated from the immunized mice.
Selected hybridomas designated 1.29, 1.37, 2.16, 2.17, 2.24, 2.45,
2.54 2.56, 2.59, 2.61, 2.70, and 2.76 (and subclones thereof) were
further characterized. The antibodies produced by cell lines 1.29
and 1.37 possess fully human IgG2 heavy chains with human kappa
light chains while those antibodies produced by cell lines 2.16,
2.17, 2.24, 2.45, 2.54 2.56, 2.59, 2.61, 2.70, and 2.76 possess
fully human IgG4 heavy chains with human kappa light chains.
[0181] The amino acid sequences of the heavy chain variable domain
regions of twelve anti-TIM-1 antibodies with their respective
germline sequences are shown in Table 4 below. The corresponding
light chain variable domain regions amino acid sequence is shown in
Table 5 below. "X" indicates any amino acid, preferably the
germline sequence in the corresponding amino acid position. The
CDRs (CDR1, CDR2, and CDR3) and FRs (FR1, FR2, and FR3) in the
immunoglobulins are shown under the respective column headings.
TABLE-US-00006 TABLE 4 Heavy Chain Analysis SEQ ID mAb NO: D FR1
CDR1 FR2 CDR2 55 Germline QVQLVESGGGVVQP GFTFSSYGMH WVRQAPGKG
VIWYDGSNKYYADSVKG GRSLRLSCAAS LEWVA 2.54 26 VH3-33/--/JH4b
QVQLEQSGGGVVQP GFTFTNYGLH WVRQAPGKG VIWYDGSHKFYADSVKG GRSLRLSCAAS
LDWVA 56 Germline QVQLVESGGGVVQP GFTFSSYGMH WVRQAPGKG
VIWYDGSNKYYADSVKG GRSLRLSCAAS LEWVA 2.76 46 VH3-33/D3-22/JH6b
XXXXEQSGGGVVQP GFTFSSYGMY WVRQAPGKG VIWYDGSNKYYADSVKG GRSLRLSCAAS
LEWVA 57 Germline QVQLQESGPGLVKP GGSISSGGYYWS WIRQHPGKG
YIYYSGSTYYNPSLKS SQTLSLTCTVS LEWIG 2.59 34 VH4-31/D6-13/JH4b
XXXXXQSGPRLVKP GGSISSDGYYWS WIRQHPGKG YIYYSGSTFYNPSLKS SQTLSLTCTVS
LEWIG 58 Germline QVQLVESGGGVVQP GFTFSSYGMH WVRQAPGKG
VIWYDGSNKYYADSVKG GRSLRLSCAAS LEWVA 2.70 42 VH3-33/D3-22/JH4b
QVQLVESGGGVVQP GFIFSRYGMH WVRQAPGKG VIWYDGSNKLYADSVKG GRSLRLSCAAS
LKWVA 2.24 18 QVQLEQSGGGVVQP GFTFSRYGMH WVRQAPGKG VIWYDGSNKLYADSVKG
GRSLRLSCAAS LKWVA 2.61 38 QVQLVEAGGGVVQP GFTFRSYGMH WVRQAPGKG
VIWYDGSNKYYTDSVKG GRSLRLSCAAS LKWVA 2.56 30 QVQLVESGGGVVQP
GFTFSSYGMH WVRQAPGKG VIWYDGSHKYYADSVKG GRSLRLSCAAS LEWVA 59
Germline EVQLVESGGGLVKP GFTFSNAWMS WVRQAPGKG RIKSKTDGGTTDYAAPVKG
GGSLRLSCAAS LEWVG 2.16 10 VH3-15/D3-16/JH4b XXXXEQSGGGVVKP
GFTFSNAWMT WVRQAPGKG RIKRRTDGGTTDYAAPVKG GGSLRLSCAAS LEWVG 60
Germline QVQLQESGPGLVKP GGSVSSGGYYWS WIRQPPGKG YIYYSGSTNYNPSLKS
SETLSLTCTVS LEWIG 1.29 2 VH4-61/D1-7/JH4b QVQLQESGPGLVKP
GGSVSSGGYYWS WIRQPPGKG FIYYTGSTNYNPSLKS SETLSLTCTVS LEWIG 61
Germline EVQLVESGGGLVKP GFTFSNAWMS WVRQAPGKG RIKSKTDGGTTDYAAPVKG
GGSLRLSCAAS LEWVG 2.45 22 VH3-15/D6-19/JH4b XXXXXQSGGGLVKP
GFTFSNAWMT WVRQAPGKG RIKRKTDGGTTDYAAPVKG GGSLRLSCAAS LEWVG 62
Germline EVQLVESGGGLVQP GFTFSSYWMS WVRQAPGKG NIKQDGSEKYYVDSVKG
GGSLRLSCAAS LEWVA 1.37 6 VH3-7/--/JH4b EVQLVESGGGLVQP GFTFTNYWMS
WVRQAPGKG NIQQDGSEKYYVDSVRG GGSLRLSCAAS LEWVA 63 Germline
EVQLVESGGGLVQP GFTFSSYSMN WVRQAPGKG YISSSSSTIYYADSVKG GGSLRLSCAAS
LEWVS 2.17 14 VH3-48/--/JH4b QVQLEQSGGGLVQP GFTFSTYSMN WVRQAPGKG
YIRSSTSTIYYAESLKG GGSLRLSCAAS LEWVS SEQ ID mAb NO: FR3 CDR3 J 55
RFTISRDNSKNTLYLQMN XXDY WGQGTLVTVSSA SLRAEDTAVYYCAR 2.54 26
RFTISRDNSKNTLFLQMN DLDY WGQGTLVTVSSA SLRAEDTAVYYCTR 56
RFTISRDNSKNTLYLQMN XXYDSSXXXYGMDV WGQGTTVTVSSA SLRAEDTAVYYCAX 2.76
46 RFTISRDNSKNTLYLQMN DFYDSSRYHYGMDV WGQGTTVTVSSA SLRAEDTAVYYCAR 57
RVTISVDTSKNQFSLKLS XXXXSSSWYXXFDY WGQGTLVTVSSA SVTAADTAVYYCAR 2.59
34 RVAISVDTSKNQFSLKLS ESPHSSNWYSGFDC WGQGTLVTVSSA SVTAADTAVYYCAR 58
RFTISRDNSKNTLYLQMN DYYDSSXXXXXFDY WGQGTLVTVSSA SLRAEDTAVYYCAR 2.70
42 RFTISRDNSKNTLYLQMN DYYDNSRHHWGFDY WGQGTLVTVSSA SLRAEDTAVYYCAR
2.24 18 RFTISRDNSKNTLYLQMN DYYDNSRHHWGFDY WGQGTLVTVSSA
SLRAEDTAVYYCAR 2.61 38 RFTISRDNSKNTLYLQMN DYYDNSRHHWGFDY
WGQGTLVTVSSA SLRAEDTAVYYCVR 2.56 30 RFTISRDNSKNTLYLQMN
DYYDTSRHHWGFDC WGQGTLVTVSSA SLRAEDTAVYYSAR 59 RFTISRDDSKNTLYLQMN
XDXXXDY WGQGTLVTVSSA SLKTEDTAVYYCTX 2.16 10 RFTISRDDSKNTLYLQMN
VDNDVDY WGQGTLVTVSSA NLKNEDTAVYYCTS 60 RVTISVDTSKNQFSLKLS
XXXWXXXFDY WGQGTLVTVSSA SVTAADTAVYYCAR 1.29 2 RVSISVDTSKNQFSLKLS
DYDWSFHFDY WGQGTLVTVSSA SVTAADAAVYYCAR 61 RFTISRDDSKNTLYLQMN
XXXSGDY WGQGTLVTVSSA SLKTEDTAVYYCTT 2.45 22 RFTISRDDSENTLYLQMN
VDNSGDY WGQGTLVTVSSA SLETEDTAVYYCTT 62 RFTISRDNAKNSLYLQMN XDY
WGQGTLVTVSSA SLRAEDTAVYYCAR 1.37 6 RFTISRDNAKNSLYLQMN WDY
WGQGTLVTVSSA SLRAEDSAVYYCAR 63 RFTISRDNAKNSLYLQMN XFDY WGQGTLVTVSSA
SLRDEDTAVYYCAX 2.17 14 RFTISSDNAKNSLYLQMN DFDY WGQGTLVTVSSA
SLRDEDTAVYYCAR
TABLE-US-00007 TABLE 5 Light Chain Analysis SEQ ID mAb NO: J FR1
CDR1 FR2 CDR2 64 Germline EIVLTQSPGTLSLS RASQSVSSSYLA WYQQKPGQAPR
GASSRAT PGERATLSC LLIY 2.54 28 A27/JK4 ETQLTQSPGTLSLS RASQSVSNNYLA
WYQQKPGQAPR GASSRAT PGERVTLSC LLIY 65 Germline DIVMTQSPLSLPVT
RSSQSLLHSNGYN WYLQKPGQSPQ LGSNRAS PGEPASISC YLD LLIY 2.16 12 A3/JK4
XXXLTQSPLSLPVT RSSQSLLHSNGYN WYLQKPGQSPQ LGSNRAS PGEPASISC YLD LLIY
2.45 24 XXXXTQSPLSLPVT RSSQSLLHSNGYN WYLQKPGQSPQ LGSNRAS PGEPASISC
YLD LLIY 66 Germline DIQMTQSPSSLSAS RASQGIRNDLG WYQQKPGKAPK AASSLQS
VGDRVTITC RLIY 1.29 4 A30/JK4 DIQMTQSPSSLSAS RASQGIRNDLG
WYQQKPGKAPK AASSLQS IGDRVTITC RLIY 67 Germline DIVMTQTPLSSPVT
RSSQSLVHSDGNT WLQQRPGQPPR KISNRFS LGQPASISC YLS LLIY 2.17 16
A23/JK5 EIQLTQSPLSSPVT RSSQSLVHSDGDT WLQQRPGQPPR KISTRFS LGQPASISC
YLN LLIY 68 Germline DIQMTQSPSSLSAS RASQSISSYLN WYQQKPGKAPK AASSLQS
VGDRVTITC LLIY 2.24 20 O12/JK1 DIQLTQSPSSLSAS RASQSIYSYLN
WYQQKPGKAPK AASSLQS VGDRVTITC LLIY 69 Germline DIVMTQTPLSSPVT
RSSQSLVHSDGNT WLQQRPGQPPR KISNRFS LGQPASISC YLS LLIY 1.37 8 A23/JK1
DIVMTQTPLSSTVI RSSQSLVHSDGNT WLQQRPGQPPR MISNRFS LGQPASISC YLN LLIY
70 Germline DIVMTQTPLSLPVT RSSQSLLDSDDGN WYLQKPGQSPQ TLSYRAS
PGEPASISC TYLD LLIY 2.70 44 O1/JK5 DIVMTQTPLSLPVT RSSRSLLDSDDGN
WYLQKPGQSPQ TLSYRAS PGEPASISC TYLD LLIY 2.56 32 EIVMTQTPLSLPVT
RSSQSLLDSEDGN WYLQKPGQSPQ TLSHRAS PGEPASISC TYLD LLIY 2.76 48
XXXXTQCPLSLPVT RSSQSLLDSDDGN WYLQKPGQSPQ TVSYRAS PGEPASISC TYLD
LLIY 71 Germline EIVLTQSPDFQSVT RASQSIGSSLH WYQQKPDQSPK YASQSFS
PKEKVTITC LLIK 2.59 36 A26/JK3 XXXXTQSPDFQSVT RASQSIGSRLH
WYQQKPDQSPK YASQSFS PKEKVTITC LLIK 72 Germline DIQMTQSPSSLSAS
RASQGIRNDLG WYQQKPGKAPK AASSLQS VGDRVTITC RLIY 2.61 40 A30/JK2
DIQMTQSPSSRCAS RASQGIRNDLA WYQQKPGKAPK AASSLQS VGDRVTITC RLIY SEQ
ID mAb NO: FR3 CDR3 J 64 GIPDRFSGSGSGTDFTLTISRL QQYGSSXXLT
FGGGTKVEIKR EPEDFAVYYC 2.54 28 GIPDRFSGSGSGTDFTLTISRL QQYGSSLPLT
FGGGTKVEIKR EPEDCAECYC 65 GVPDRFSGSGSGTDFTLKISRV MQALQTXXT
FGGGTKVEIKR EAEDVGVYYC 2.16 12 GVPDRFSGSGSGTDFTLKISRV MQALQTPLT
FGGGTKVDIKR EAEDIGLYYC 2.45 24 GVPDRFSGSGSGTDFTLKISRV MQALQTPLT
FGGGTKVEIKR EAEDVGVYYC 66 GVPSRFSGSGSGTEFTLTISSL LQHNSYPLT
FGGGTKVEIKR QPEDFATYYC 1.29 4 GVPSRFSGSGSGTEFTLTISSL LQHNSYPLT
FGGGTKVEIKR QPEDFATYYC 67 GVPDRFSGSGAGTDFTLKISRV MQATQFPXIT
FGQGTRLEIKR EAEDVGVYYC 2.17 16 GVPDRFSGSGAGTDFTLKISRV MQTTQIPQIT
FGQGTRLEIKR ETDDVGIYYC 68 GVPSRFSGSGSGTDFTLTISSL QQSYSTPPT
FGQGTKVEIKR QPEDFATYYC 2.24 20 GVPSRFSGSGSGTDFTLTISSL QQSYSTPPT
FGQGTKVEIKR QPEDFATYYC 69 GVPDRFSGSGAGTDFTLKISRV MQATQFPQT
FGQGTKVEIKR EAEDVGVYYC 1.37 8 GVPDRFSGSGAGTDFTLKISRV MQATESPQT
FGQGTKVEIKR EAEDVGVYYC 70 GVPDRFSGSGSGTDFTLKISRV MQRIEFPIT
FGQGTRLEIKR EAEDVGVYYC 2.70 44 GVPDRFSGSGSGTDFTLKISRV MQRVEFPIT
FGQGTRLEIKR EAEDVGVYYC 2.56 32 GVPDRFSGSGSGTDFTLKISRV MQRVEFPIT
FGQGTRLEIKR EAEDVGVYCC 2.76 48 GVPDRFSGSGSGTDFTLKISRV MQRIEFPIT
FGQGTRLEIKR EAEDVGVYYC 71 GVPSRFSGSGSGTDFTLTINSL HQSSSLPFT
FGPGTKVDIKR EAEDAATYYC 2.59 36 GVPSRFSGSGSGTDFTLTINSL HQSSNLPFT
FGPGTKVDIKR EAEDAATYYC 72 GVPSRFSGSGSGTEFTLTISSL LQHNSYPXX
FGQGTKLEIKR QPEDFATYYC 2.61 40 GVPSRFSGSRSGTEFTLTISSL LQHNSYPPS
FGQGTKLEIKR QPEDFAAYYC
[0182] Human antibody heavy chain VH3-33 was frequently selected in
productive rearrangement for producing antibody successfully
binding to TIM-1. Any variants of a human antibody VH3-33 germline
in a productive rearrangement making antibody to TIM-1 is within
the scope of the invention. Other heavy chain V regions selected in
TIM-1 binding antibodies included: VH4-31, VH3-15, VH4-61, VH3-7
and VH3-48. The light chain V regions selected included: A27, A3,
A30, A23, O12, O1, and A26. It is understood that the
.lamda..kappa. XenoMouse.RTM. may be used to generate anti-TIM-1
antibodies utilizing lambda V regions.
[0183] The heavy chain variable domain germ line usage of the
twelve anti-TIM-1 antibodies is shown in Table 6. The light chain
variable domain germ line usage is shown in Table 7 (below).
TABLE-US-00008 TABLE 6 Germ Line Usage of the Heavy Chain Variable
Domain Regions V D2 mAb V Heavy Sequence #N's N D1 D1 Sequence #N's
N D2 Sequence 2.16 VH3-15 TGTACC 5 TCA D3-16 CGATAA -N.A- -N.A-
-N.A- -N.A- (1-285) GT (291-296) 2.70 VH3-33 GAGAGA 0 D3-22
TTACTATGAT -N.A- -N.A- -N.A- -N.A- (1-290) (291-306) AATAGT (SEQ ID
NO: 73) 2.59 VH4-31 GAGAGA 8 ATC D6-13 ATAGCAGCAA -N.A- -N.A- -N.A-
-N.A- (2-284) CCC (293-309) CTGGTAC TC (SEQ ID NO: 75) 2.24 VH3-33
GAGAGA 0 D3-22 TTACTATGAT -N.A- -N.A- -N.A- -N.A- (1-296) (297-312)
AATAGT (SEQ ID NO: 76) 1.29 VH4-61 GAGAGA 5 TTA D1-7 ACTGGA -N.A-
-N.A- -N.A- -N.A- (1-293) TG (299-304) 2.61 VH3-33 GAGAGA 0 D3-22
TTACTATGAT -N.A- -N.A- -N.A- -N.A- (1-296) (297-312) AATAGT (SEQ ID
NO: 78) 2.76 VH3-33 TGCGAG 6 GGA D3-22 CTATGATAGT -N.A- -N.A- -N.A-
-N.A- (1-281) TTT (288-300) AGT (SEQ ID NO: 80) 2.54 VH3-33 GCGAGA
-N.A- -N.A- -N.A- -N.A- -N.A- -N.A- -N.A- -N.A- (1-296) 1.37 VH3-7
(7- GCGAGA -N.A- -N.A- -N.A- -N.A- -N.A- -N.A- -N.A- -N.A- 300)
2.17 VH3-48 TGTGCG -N.A- -N.A- -N.A- -N.A- -N.A- -N.A- -N.A- -N.A-
(2-291) 2.45 VH3-15 CCACAG 7 TCG D6-19 CAGTGG -N.A- -N.A- -N.A-
-N.A- (2-286) ATAA (294-299) 2.56 VH3-33 GAGAGA 0 D3-22 TTACTATGAT
-N.A- -N.A- -N.A- -N.A- (1-290) (291-301) A (SEQ ID NO: 81) J
Constant mAb #N's N JH Sequence Region CDR1 CDR2 CDR3 2.16 7
TGACGTG JH4b (304-343) GACTAC G4 (344-529) 64-93 136-192 289-309
2.70 15 AGACATCA JH4b (322-364) TTTGAC G4 (365-502) 70-99 142-192
289-330 CTGGGGG (SEQ ID NO: 74) 2.59 5 TCGGG JH4b (315-358) CTTTGA
G4 (359-545) 61-96 139-186 283-324 2.24 15 AGACATCA JH4b (328-370)
TTTGAC G4 (371-568) 76-105 148-198 295-336 CTGGGGG (SEQ ID NO: 77)
1.29 6 GCTTCC JH4b (311-355) ACTTTG G2 (356-491) 70-105 148-195
292-321 2.61 15 AGACATCA JH4b (328-370) TTTGAC G4 (371-534) 76-105
148-198 295-336 CTGGGGG (SEQ ID NO: 79) 2.76 7 CGTTACC JH6b
(308-358) ACTACG G4 (359-544) 64-93 136-186 283-324 2.54 2 TC JH4b
(299-340) TTGACT G4 (341-537) 76-105 148-198 295-306 1.37 3 TGG
JH4b (304-343) GACTAC G2 (344-469) 82-111 154-204 301-309 2.17 5
CGGGA JH4b (297-340) CTTTGA G4 (341-538) 76-105 148-198 295-306
2.45 0 JH4b (300-340) TGACTA G4 (341-526) 61-90 133-189 286-306
2.56 20 CGAGTCGG JH4b (322-364) TTTGAC G4 (365-527) 70-99 142-192
289-330 CATCACTG GGGG (SEQ ID NO: 82)
TABLE-US-00009 TABLE 7 Germ Line Usage of the Light Chain Variable
Domain Regions Constant mAb VL V Sequence #N's N JL J Sequence
Region CDR1 CDR2 CDR3 2.70 O1 (46-348) TTTCCT 0 JK5 (349-385)
ATCACC IGKC (386-522) 115-165 211-231 328-354 2.59 A26 (1-272)
TTTACC 0 JK3 (273-310) ATTCAC IGKC (311-450) 58-90 136-156 253-279
2.24 O12 (1-287) CCCTCC 0 JK1 (288-322) GACGTT IGKC (323-472)
70-102 148-168 265-291 1.29 A30 (46-331) ACCCTC 0 JK4 (332-367)
TCACTT IGKC (368-504) 115-147 193-213 310-336 2.56 O1 (46-348)
TTTCCT 0 JK5 (349-385) ATCACC IGKC (386-521) 115-165 211-231
328-354 2.61 A30 (1-287) CCCTCC 3 CAG JK2 (291-322) TTTTGG IGKC
(323-470) 70-102 148-168 265-291 2.76 O1 (1-290) GTTTCC 0 JK5
(291-328) GATCAC IGKC (329-419) 58-108 154-174 271-297 1.37 A23
(43-344) TCCTCA 0 JK1 (345-379) GACGTT IGKC (380-454) 112-159
205-225 322-348 2.17 A23 (1-302) TCCTCA 1 A JK5 (304-340) ATCACC
IGKC (341-490) 70-117 163-183 280-309 2.54 A27 (1-286) GCTCAC 4
TCCC JK4 (291-328) GCTCAC IGKC (329-480) 70-105 151-171 268-297
2.16 A3 (2-290) AACTCC 2 GC JK4 (293-328) TCACTT IGKC (329-447)
61-108 154-174 271-297 2.45 A3 (1-287) AACTCC 2 GC JK4 (290-325)
TCACTT IGKC (326-465) 58-105 151-171 268-294
[0184] The sequences encoding monoclonal antibodies 1.29, 1.37,
2.16, 2.17, 2.24, 2.45, 2.54 2.56, 2.59, 2.61, 2.70, and 2.76,
respectively, including the heavy chain nucleotide sequence (A),
heavy chain amino acid sequence (B) and the light chain nucleotide
sequence (C) with the encoded amino acid sequence (D) are provided
in the sequence listing as summarized in Table 1 above. A
particular monoclonal antibody, 2.70, was further subcloned and is
designated 2.70.2, see Table 1.
Example 2
Antibody Reactivity with Membrane Bound TIM-1 Protein by FACS.
[0185] Fluorescent Activated Cell Sorter (FACS) analysis was
performed to demonstrate the specificity of the anti-TIM-1
antibodies for cell membrane-bound TIM-1 antigen and to identify
preferred antibodies for use as a therapeutic or diagnostic agent.
The analysis was performed on two renal cancer cell lines, ACHN
(ATCC#:CRL-1611) and CAKI-2 (ATCC#:HTB-47). A breast cancer cell
line that does not express the TIM-1 antigen, BT549, was used as a
control. Table 8 shows that both antibodies 2.59.2 and 2.70.2
specifically bound to TIM-1 antigen expressed on ACHN and CAKI-2
cells, but not antigen negative BT549 cells. Based on the Geo Mean
Ratios normalized to the irrelevant antibody isotype control
(pK16), ACHN cells had a higher cell surface expression of TIM-1
protein than CAKI-2 cells.
TABLE-US-00010 TABLE 8 Geo Mean Ratio (relative to negative
control) Antibody BIN ACHN CAKI-2 BT549 2.59.2 1 15.2 7.7 1.4
2.70.2 6 19.4 8.8 1.8 1.29 1 17.9 1.2 2.16.1 2 7.9 1.5 2.56.2 5
12.2 1.5 2.45.1 8 4.3 1.1
Example 3
Specificity of the Anti-TIM-1 Monoclonal Antibodies
[0186] The anti-TIM-1 antibodies bound specifically to TIM-1
protein but not an irrelevant protein in an ELISA assay. TIM-1
antigen (with a V5-HIS tag) specific binding results for four of
the anti-TIM-1 monoclonal antibodies (1.29, 2.56.2, 2.59.2, and
2.45.1) as well as an isotype matched control mAb PK16.3 are shown
in FIG. 1. The X axis depicts the antibodies used in the order
listed above and the Y axis is the optical density. The respective
binding of these antibodies to the irrelevant protein (also with a
V5-HIS tag) is shown in FIG. 2.
ELISA Protocol.
[0187] A 96-well high protein binding ELISA plate (Corning Costar
cat. no. 3590) was coated with 50 .mu.L of the TIM-1 antigen at a
concentration of 5 .mu.g/mL diluted in coating buffer (0.1M
Carbonate, pH9.5), and incubated overnight at 4 oC. The wells were
then washed five times with 200-300 .mu.L of 0.5% Tween-20 in PBS.
Next, plates were blocked with 200 .mu.L of assay diluent
(Pharmingen, San Diego, Calif., cat. no. 26411E) for at least 1
hour at room temperature. Anti-TIM-1 monoclonal antibodies were
then diluted in assay diluent with the final concentrations of 7,
15, 31.3, 62.5, 125, 250, 500 and 1000 ng/mL. An anti-V5-HRP
antibody was used at 1:1000 to detect the V5 containing peptide as
the positive control for the ELISA. Plates were then washed again
as described above. Next 50 .mu.L of each antibody dilution was
added to the proper wells, then incubated for at least 2 hours at
room temp. Plates were washed again as described above, then 50
.mu.L of secondary antibody (goat anti-human-HRP) was added at
1:1000 and allowed to incubate for 1 hour at room temp. Plates were
washed again as described above then developed with 100 .mu.L of
TMB substrate solution/well (1:1 ratio of solution A+B)
(Pharmingen, San Diego, Calif., cat. no. 2642KK). Finally, the
reaction was stopped with 50 .mu.L sulfuric acid and the plates
read at 450 nm with a correction of 550 nm.
Example 4
Antibody Sequences
[0188] In order to analyze structures of antibodies, as described
herein, genes encoding the heavy and light chain fragments out of
the particular hybridoma were cloned. Gene cloning and sequencing
was accomplished as follows. Poly(A)+ mRNA was isolated from
approximately 2.times.105 hybridoma cells derived from immunized
XenoMouse.RTM. mice using a Fast-Track kit (Invitrogen). The
generation of random primed cDNA was followed by PCR. Human VH or
human V.kappa. family specific variable domain primers (Marks et.
al., 1991) or a universal human VH primer, MG-30
(CAGGTGCAGCTGGAGCAGTCIGG) (SEQ ID NO:83) were used in conjunction
with primers specific for the human:
[0189] C.gamma.2 constant region (MG-40d; 5'-GCT GAG GGA GTA GAG
TCC TGA GGA-3' (SEQ ID NO:84));
[0190] C.gamma.1 constant region (HG1; 5' CAC ACC GCG GTC ACA TGG C
(SEQ ID NO:85)); or
[0191] C.gamma.3 constant region (HG3; 5' CTA CTC TAG GGC ACC TGT
CC (SEQ ID NO:86)) or the human C.kappa. constant domain
(h.kappa.P2; as previously described in Green et al., 1994).
Sequences of human MAbs-derived heavy and kappa chain transcripts
from hybridomas were obtained by direct sequencing of PCR products
generated from poly(A.sup.+) RNA using the primers described above.
PCR products were also cloned into pCRII using a TA cloning kit
(Invitrogen) and both strands were sequenced using Prism
dye-terminator sequencing kits and an ABI 377 sequencing machine.
All sequences were analyzed by alignments to the "V BASE sequence
directory" (Tomlinson et al., MRC Centre for Protein Engineering,
Cambridge, UK) using MacVector and Geneworks software programs.
[0192] In each of Tables 4-7 above, CDR domains were determined in
accordance with the Kabat numbering system. See Kabat, Sequences of
Proteins of Immunological Interest (National Institutes of Health,
Bethesda, Md. (1987 and 1991)).
Example 5
Epitope Binning and BiaCore.RTM. Affinity Determination
Epitope Binning
[0193] Certain antibodies, described herein were "binned" in
accordance with the protocol described in U.S. Patent Application
Publication No. 20030157730, published on Aug. 21, 2003, entitled
"Antibody Categorization Based on Binding Characteristics."
[0194] MxhIgG conjugated beads were prepared for coupling to
primary antibody. The volume of supernatant needed was calculated
using the following formula: (n+10).times.50 .mu.L (where n=total
number of samples on plate). Where the concentration was known, 0.5
.mu.g/mL was used. Bead stock was gently vortexed, then diluted in
supernatant to a concentration of 2500 of each bead per well or
0.5.times.105 /mL and incubated on a shaker in the dark at room
temperature overnight, or 2 hours if at a known concentration of
0.5 .mu.g/mL. Following aspiration, 50 .mu.L of each bead was added
to each well of a filter plate, then washed once by adding 100
.mu.L/well wash buffer and aspirating. Antigen and controls were
added to the filter plate 50 .mu.L/well then covered and allowed to
incubate in the dark for 1 hour on shaker. Following a wash step, a
secondary unknown antibody was added at 50 .mu.L/well using the
same dilution (or concentration if known) as used for the primary
antibody. The plates were then incubated in the dark for 2 hours at
room temperature on shaker followed by a wash step. Next, 50
.mu.L/well biotinylated mxhIgG diluted 1:500 was added and allowed
to incubate in the dark for 1 hour on shaker at room temperature.
Following a wash step, 50 .mu.L/well Streptavidin-PE was added at
1:1000 and allowed to incubate in the dark for 15 minutes on shaker
at room temperature. Following a wash step, each well was
resuspended in 80 .mu.L blocking buffer and read using a Luminex
system.
[0195] Table 9 shows that the monoclonal antibodies generated
belong to eight distinct bins. Antibodies bound to at least three
distinct epitopes on the TIM-1 antigen.
Determination of Anti-TIM-1 mAb Affinity using BiaCore.RTM.
Analysis
[0196] BiaCore.RTM. analysis was used to determine binding affinity
of anti-TIM-1 antibody to TIM-1 antigen. The analysis was performed
at 25.degree. C. using a BiaCore.RTM. 2000 biosensor equipped with
a research-grade CM5 sensor chip. A high-density goat a human
antibody surface over a CM5 BiaCore.RTM. chip was prepared using
routine amine coupling. Antibody supernatents were diluted to
.about.5 .mu.g/mL in HBS-P running buffer containing 100 .mu.g/mL
BSA and 10 mg/mL carboxymethyldextran. The antibodies were then
captured individually on a separate surface using a 2 minute
contact time, and a 5 minute wash for stabilization of antibody
baseline.
[0197] TIM-1 antigen was injected at 292 nM over each surface for
75 seconds, followed by a 3-minute dissociation. Double-referenced
binding data were obtained by subtracting the signal from a control
flow cell and subtracting the baseline drift of a buffer inject
just prior to the TIM-1 injection. TIM-1 binding data for each mAb
were normalized for the amount of mAb captured on each surface. The
normalized, drift-corrected responses were also measured. The
kinetic analysis results of anti-TIM-1 mAB binding at 25.degree. C.
are listed in Table 9 below.
TABLE-US-00011 TABLE 9 Competition Bins and KDS for TIM-1-specific
mAbs Affinity nM Bin Antibody by BIAcore 1 2.59 0.38 1.29 3.64 2
2.16 0.79 3 2.17 2.42 4 1.37 2.78 2.76 0.57 2.61 1.0 5 2.24 2.42
2.56 1.1 6 2.70 2.71 7 2.54 3.35 8 2.45 1.15
Example 6
Epitope Mapping
[0198] Anti-TIM-1 mAb 2.70.2 was assayed for reactivity against
overlapping peptides designed from the TIM-1 antigen sequence.
Assay plates were coated with the TIM-1 fragment peptides, using
irrelevant peptide or no peptide as controls. Anti-TIM-1 mAb 2.70.2
was added to the plates, incubated, washed and then bound antibody
was detected using anti-human Ig HRP conjugate. Human antibody not
specific to TIM-1, an isotype control antibody or no antibody
served as controls. Results showed that mAb 2.70.2 specifically
reacted with a peptide having the amino acid sequence
PMPLPRQNHEPVAT (SEQ ID NO:87), corresponding to amino acids 189-202
of the TIM-1 immunogen (SEQ ID NO:54).
[0199] Specificity of mAb 2.70.2 was further defined by assaying
against the following peptides:
TABLE-US-00012 A) (SEQ ID NO: 87) PMPLPRQNHEPVAT B) (SEQ ID NO: 88)
PMPLPRQNHEPV C) (SEQ ID NO: 89) PMPLPRQNHE D) (SEQ ID NO: 90)
PMPLPRQN E) (SEQ ID NO: 91) PMPLPR F) (SEQ ID NO: 92) PLPRQNHEPVAT
G) (SEQ ID NO: 93) PRQNHEPVAT H) (SEQ ID NO: 94) QNHEPVAT I) (SEQ
ID NO: 95) HEPVAT
[0200] Results showed mAb 2.70.2 specifically bound to peptides A,
B, C, and F, narrowing the antibody epitope to PLPRNHE (SEQ ID
NO:96)
[0201] As shown in Table 10, synthetic peptides were made in which
each amino acid residue of the epitope was replace with an alanine
and were assayed for reactivity with mAb 2.70.2. In this
experiment, the third proline and the asparagines residues were
determined to be critical for mAb 2.70.2 binding. Furthermore,
assays of peptides with additional N or C terminal residues removed
showed mAb 2.70.2 binding was retained by the minimal epitope
LPRQNH (SEQ ID NO:97)
TABLE-US-00013 TABLE 10 SEQ ID mAb 2.70.2 NO: Reactivity P M P L P
R Q N H E 89 + P M P A P R Q N H E 98 + P M P L A R Q N H E 99 - P
M P L P A Q N H E 100 + P M P L P R A N H E 101 + P M P L P R Q A H
E 102 - P M P L P R Q N A E 103 + P L P R Q N H E 104 + L P R Q N H
E 105 + P L P R Q N H E 106 + L P R Q N H E 107 +
Example 7
Immunohistochemical (MC) Analysis of TIM-1 Expression in Normal and
Tumor Tissues
[0202] Immunohistochemical (IHC) analysis of TIM-1 expression in
normal and tumor tissue specimens was performed with techniques
known in the art. Biotinylated fully human anti-TIM-1 antibodies
2.59.2, 2.16.1 and 2.45.1 were analyzed. Streptavidin-HRP was used
for detection.
[0203] Briefly, tissues were deparaffinized using conventional
techniques, and then processed using a heat-induced epitope
retrieval process to reveal antigenic epitopes within the tissue
sample. Sections were incubated with 10% normal goat serum for 10
minutes. Normal goat serum solution was drained and wiped to remove
excess solution. Sections were incubated with the biotinylated
anti-TIM-1 mAb at 5 .mu.g/mL for 30 minutes at 25.degree. C., and
washed thoroughly with PBS. After incubation with streptavidin-HRP
conjugate for 10 minutes, a solution of diaminobenzidine (DAB) was
applied onto the sections to visualize the immunoreactivity. For
the isotype control, sections were incubated with a biotinylated
isotype matched negative control mAb at 5 .mu.g/mL for 30 minutes
at 25.degree. C. instead of biotinylated anti-TIM-1 mAb. The
results of the IHC studies are summarized in Tables 11 and 12.
[0204] The specimens were graded on a scale of 0-3, with a score of
1+ indicating that the staining is above that observed in control
tissues stained with an isotype control irrelevant antibody. The
corresponding histological specimens from one renal tumor and the
pancreatic tumor are shown in FIGS. 3A and 3B. In addition to these
the renal and pancreatic tumors, specimens from head and neck
cancer, ovarian cancer, gastric cancer, melanoma, lymphoma,
prostate cancer, liver cancer, breast cancer, lung cancer, bladder
cancer, colon cancer, esophageal cancer, and brain cancer, as well
the corresponding normal tissues were stained with anti-TIM-1 mAb
2.59.2. Overall, renal cancer tissue samples and pancreatic cancer
tissue samples highly positive when stained with anti-TIM-1 mAb
2.59.2. No staining in normal tissues was seen. These results
indicate that TIM-1 is a marker of cancer in these tissues and that
anti-TIM-1 mAb can be used to differentiate cancers from normal
tissues and to target TIM-1 expressing cells in vivo.
TABLE-US-00014 TABLE 11 Immunohistology Renal tumors expression of
TIM-1 protein detected by anti-TIM-1 mAb 2.59.2 Specimen Cell Type
Histology Score 1 Malignant cells Not known 0 1 Other Not cell
associated 2 2 Malignant cells Clear Cell 2 3 Malignant cells Clear
Cell 0 4 Malignant cells Clear Cell 3 5 Malignant cells Clear Cell
2 (occasional) 6 Malignant cells Not known 2 7 Malignant cells
Clear Cell 2 8 Malignant cells Clear Cell 0 9 Malignant cells Clear
Cell 2 (occasional) 10 Malignant cells Clear Cell 1-2 11 Malignant
cells Not known 3 (many) 12 Malignant cells Clear Cell 1-2 12 Other
Not cell associated 2 13 Malignant cells Clear Cell 2 (occasional)
14 Malignant cells Clear Cell 1-2 15 Malignant cells Clear Cell 3-4
16 Malignant cells Not known 1-2 17 Malignant cells Not known 4
(occasional) 18 Malignant cells Not known 1-2 19 Malignant cells
Clear Cell 0 20 Malignant cells Clear Cell 3-4 21 Malignant cells
Clear Cell 2 (occasional) 22 Malignant cells Clear Cell 3 23
Malignant cells Clear Cell 2 24 Malignant cells Not known 3-4
occasional 25 Malignant cells Not known 2-3 26 Malignant cells Not
known 3 27 Malignant cells Clear Cell 2 27 Other Not cell
associated 2 28 Malignant cells Not known 2 29 Malignant cells
Clear Cell 2-3 30 Malignant cells Clear Cell 2 31 Malignant cells
Clear Cell 2-3 32 Malignant cells Clear Cell 0 33 Malignant cells
Clear Cell 0 34 Malignant cells Clear Cell 2 34 Other Not cell
associated 2 35 Malignant cells Clear Cell 2-3 36 Malignant cells
Clear Cell 3 37 Malignant cells Not known 3 38 Malignant cells
Clear Cell 3 39 Malignant cells Not known 2 40 Malignant cells
Clear Cell 2-3
TABLE-US-00015 TABLE 12 Normal Human Tissue Immunohistology with
anti-TIM-1 mAb 2.59.2 Score Tissue Specimen 1 Specimen 2 Adrenal
Cortex 0 0 Adrenal Medulla 0 1 Bladder: Smooth muscle 0 0 Bladder:
Transitional Epithelium 3 0 Brain cortex: Blia 0 0 Brain cortex:
Neurons 0 0 Breast: Epithelium 0 0 Breast: Stroma 0 0 Colon:
Epithelium 0 0 Colon: Ganglia 0 NA Colon: Inflammatory compartment
3-4 (occasional) 3 (occasional) Colon: Smooth muscle 1 (occasional)
0 Heart: Cardiac myocytes 0 0 Kidney cortex: Glomeruli 2-3 2 Kidney
cortex: Tubular epithelium 2 2-3 Kidney medulla: Tubular 2 0
epithelium Kidney medulla: other NA 2-3 Liver: Bile duct epithelium
0 0 Liver: Hepatocytes 1-2 1 Liver: Kupffer cells 0 0 Lung: Airway
epithelium 0 0 Lung: Alveolar macrophages 2 (occasional)-3 2-3
(occasional) Lung: other 3 NA Lung: Pneumocytes 2-3 (occasional)
2-3 (occasional) Ovary: Follicle 2 (occasional) 1-2 Ovary: Stroma 1
1 (occasional) Pancreas: Acinar epithelium 0 1 (occasional)
Pancreas: Ductal epithelium 0 0 Pancreas: Islets of Langerhans 0 0
Placenta: Stroma 0 0 Placenta: Trophoblasts 0 0 Prostate:
Fibromuscular stroma 0 0 Prostate: Glandular epithelium 0 0
Skeletal muscle: Myocytes 0 0 Skin: Dermis 0 0 Skin: Epidermis 0 0
Small intestine: Epithelium 0 0 Small intestine: Ganglion 0 0 Small
intestine: Inflammatory 0 0 compartment Small intestine: Smooth
muscle 0 0 cells Spleen: Red pulp 0 2 (rare) Spleen: white pulp 0 0
Stomach: Epithelium 0 0 Stomach: Smooth Muscle Cells 0 0 Tstis:
Leydig cells 2 1-2 Testis: Seminiferous epithelium 1 2 Thymus:
Epithelium 0 0 Thymus: Lymphocytes 2 (rare) 2 (occasional) Thyroid:
Follicular epithelium 0 0 Tonsil: Epithelium 0 0 Tonsil:
Lymphocytes 3 (occasional) 2 (occasional) Uterus: Endometrium 0 0
Uterus: Myometrium 0 0
Example 8
Antibody Mediated Toxin Killing
[0205] A clonogenic assay as described in the art was used to
determine whether primary antibodies can induce cancer cell death
when used in combination with a saporin toxin conjugated secondary
antibody reagent. Kohls and Lappi, Biotechniques, 28(1):162-5
(2000).
Assay Protocol
[0206] ACHN and BT549 cells were plated onto flat bottom tissue
culture plates at a density of 3000 cells per well. On day 2 or
when cells reached .about.25% confluency, 100 ng/well secondary
mAb-toxin (goat anti-human IgG-saporin; Advanced Targeting Systems;
HUM-ZAP; cat. no. IT-22) was added. A positive control anti-EGFR
antibody, mAb 2.7.2, mAb 2.59.2, or an isotype control mAb was then
added to each well at the desired concentration (typically 1 to 500
ng/mL). On day 5, the cells were trypsinized, transferred to a 150
mm tissue culture dish, and incubated at 37.degree. C. Plates were
examined daily. On days 10-12, all plates were Giemsa stained and
colonies on the plates were counted. Plating efficiency was
determined by comparing the number of cells prior to transfer to
150 mm plates to the number of colonies that eventually formed.
[0207] The percent viability in antigen positive ACHN and antigen
negative BT549 cell lines are presented in FIG. 4 and FIG. 5
respectively. In this study, the cytotoxic chemotherapy reagent 5
Flurouracil (5-FU) was used as the positive control and induced
almost complete killing, whereas the saporin conjugated-goat
anti-human secondary antibody alone had no effect. A monoclonal
antibody (NeoMarkers MS-269-PABX) generated against the EGF
receptor expressed by both cell lines was used to demonstrate
primary antibody and secondary antibody-saporin conjugate specific
killing. The results indicate that both cell lines were susceptible
to EGFR mAb mediated toxin killing at 100 ng/mL. At the same dose,
both the anti-TIM-1 mAb 2.59.2 and the anti-TIM-1 mAb 2.70.2
induced over 90% ACHN cell death as compared to 0% BT549 cell
death.
Antibody Toxin Conjugate Mediated Killing: Clonogenic Assay
[0208] CAKI-1 and BT549 cells were plated onto flat bottom tissue
culture plates at a density of 3000 cells per well. On day 2 or
when cells reach .about.25% confluency, various concentrations
(typically 1 to 1000 ng/ml) of unconjugated and Auristatin E
(AE)-conjugated mAb, which included anti-EGFR, anti-TIM-1 mAb
2.7.2, anti-TIM-1 mAb 2.59.2 or isotype control mAb, were added to
cells. Each of these antibodies was conjugated to AE. The
monoclonal antibody (NeoMarkers MS-269-PABX) generated against the
EGF receptor, which is expressed by both cell lines, was used as a
positive control to demonstrate specific killing mediated by
AE-conjugated antibody. On day 5, the cells were trypsinized,
transferred to a 150 mm tissue culture dish, and incubated at
37.degree. C. Plates were examined daily. On days 10-12, all plates
were Giemsa stained and colonies on the plates were counted.
Plating efficiency was determined by counting the cells prior to
transfer to 150 mm plates and compared to the number of colonies
that eventually formed.
[0209] The percent viability in antigen positive CAKI-1 and antigen
negative BT549 cell lines are presented in FIG. 6 and FIG. 7,
respectively.
[0210] The results indicate that unconjugated and AE-conjugated
isotype control mAb had no effect on growth of both CAKI-1 and
BT549 cells. However, both cell lines were susceptible to AE-EGFR
mAb mediated toxin killing in a dose-dependent fashion. At the
maximum dose, both anti-TIM-1 mAbs (2.59.2 and 2.70.2) induced over
90% CAKI-1 cell death when compared to their unconjugated
counterparts. The response was dose dependent. At the same dose
range, both anti-TIM-1 mAbs 2.59.2 and 2.70.2 did not affect the
survival of BT549 cells.
Example 9
Human Tumor Xenograft Growth Delay Assay
[0211] A tumor growth inhibition model is used according to
standard testing methods. Geran et al., Cancer Chemother. Rep.
3:1-104 (1972). Athymic nude mice (nu/nu) are implanted with either
tumor cells or tumor fragments from an existing host, in
particular, renal (CaKi-1) or ovarian (OVCAR) carcinoma tumor
fragments are used. These animals are then treated with an
anti-TIM-1 antibody immunotoxin conjugate, for example, mAb 2.70.2
AE conjugate at doses ranging from 1 to 20 mg/kg body weight, twice
weekly for a period of 2 weeks. Tumor volume for treated animals is
assessed and compared to untreated control tumors, thus determining
the tumor growth delay.
[0212] After reaching a volume of 100 mm3 animals are randomized
and individually identified in groups of 5 individuals per cage.
Protein or antibody of interest is administered via conventional
routes (intraperitoneal, subcutaneous, intravenous, or
intramuscular) for a period of 2 weeks. Twice weekly, the animals
are evaluated for tumor size using calipers. Daily individual
animal weights are recorded throughout the dosing period and twice
weekly thereafter. Tumor volume is determined using the formula:
Tumor volume (in
mm3)=(length.times.width.times.height).times.0.536. The volume
determinations for the treated groups are compared to the untreated
tumor bearing control group. The difference in time for the treated
tumors to reach specific volumes is calculated for 500 1000, 1500
and 2000 mm3. Body weights are evaluated for changes when compared
to untreated tumor bearing control animals. Data are reported as
tumor growth in volume plotted against time. Body weights for each
experimental group are also plotted in graph form.
[0213] Results show that the treatment is well tolerated by the
mice. Treatment with anti-TIM-1 mAb AE conjugate inhibits tumor
growth of established CaKi-1 and OVCAR tumors.
Example 10
Treatment of Renal Carcinoma with anti-TIM-1 Antibodies
[0214] A patient in need of treatment for a renal carcinoma is
given an intravenous injection of anti-TIM-1 antibodies coupled to
a cytotoxic chemotherapic agent or radiotherapic agent. The
progress of the patient is monitored and additional administrations
of anti-TIM-1 antibodies are given as needed to inhibit growth of
the renal carcinoma. Following such treatment, the level of
carcinoma in the patient is decreased.
Example 11
FACS Analysis of Expression of TIM-1 Protein on CD4+ T Cells
[0215] Mononuclear cells were isolated from human blood diluted 1:1
in PBS, by spinning over Ficoll for 20 minutes. The mononuclear
cells were washed twice at 1000 rpm with PBS --Mg and Ca and
re-suspended in Miltenyi buffer (Miltenyi Biotec Inc., Auburn,
Calif.); PBS, 0.5% BSA, 5 mM EDTA at approximately 108 cells/mL. 20
.mu.L of CD4 Miltenyi beads were added per 107 cells and incubated
for 15 minutes on ice. Cells were washed with a 10-fold excess
volume of Miltenyi buffer. A positive selection column (type VS+)
(Miltenyi Biotec Inc., Auburn, Calif.) was washed with 3 mL of
Miltenyi buffer. The pelleted cells were re-suspended at 108 cells
per mL of Miltenyi buffer and applied to the washed VS column. The
column was then washed three times with 3 mL of Miltenyi buffer.
Following this, the VS column was removed from the magnetic field
and CD4+ cells were eluted from the column with 5 mL of Miltenyi
buffer. Isolated CD4+ lymphocytes were pelleted and re-suspended in
DMEM 5% FCS plus additives (non-essential amino acids, sodium
pyruvate, mercaptoethanol, glutamine, penicillin, and streptomycin)
at 106 cells/mL. 1.times.106 freshly isolated resting CD4+ T cells
were transferred into flow cytometry tubes and washed with 2
mL/tube FACS staining buffer (FSB) containing PBS, 1% BSA and 0.05%
NaN3. Cells were spun down and supernatant removed. Cells were
blocked with 20% goat serum in FSB for 30 minutes on ice. Cells
were washed as above and incubated with 10 .mu.g/mL of primary
human anti-TIM-1 mAb or control PK16.3 mAb in FSB (200 .mu.L) for
45 minutes on ice followed by washing. Secondary goat anti-human PE
conjugated antibody was added at 1:50 dilution for 45 minutes on
ice in the dark, washed, resuspended in 500 .mu.L of PBS containing
1% formaldehyde and kept at 4.degree. C. until flow cytometry
analysis was performed.
[0216] FACS analysis was performed to determine the expression of
TIM-1 protein as detected with five anti-TIM-1 monoclonal
antibodies (2.59.2, 1.29, 2.70.2, 2.56.2, 2.45.1) on human and
mouse resting CD4+ T cells, as well as human activated and human
polarized CD4+ T cells. These analyses demonstrate that freshly
isolated resting human CD4+ T cells do not express TIM-1, while a
major fraction of polarized human Th2 and Th1 cells do express
TIM-1.
[0217] FACS Analysis of the Expression of the TIM-1 protein on
human CD4+ Th2 cells using five anti-TIM-1 monoclonal antibodies is
shown in Table 13. The experiment is described in the left-hand
column and the labeled antibody is specified along the top row.
Data is reported as the geometric mean of the fluorescence
intensity.
TABLE-US-00016 TABLE 13 FACS Analysis of the Expression of the
TIM-1 protein on human CD4+ Th2 cells Geometric mean of
fluorescence intensity Control Anti-TIM-1 mAb Experiment PK16.3
1.29 2.45.1 2.56.2 2.59.2 2.70.2 Resting Human 4.6 4.7 5.1 6 4.9
N/A CD4+ T cells Polarized Human 8.4 22.3 42.4 564.1 22 27.8 CD4+
Th2 Cells
[0218] Table 14 demonstrates that over the course of 5 days,
continual stimulation of T cells results in an increase in TIM-1
expression, as measured by anti-TIM-1 mAb 2.70.2, as compared to
the control PK16.3 antibody. Furthermore, addition of matrix
metalloproteinase inhibitor (MMPI) did not measurably increase
TIM-1 expression, demonstrating that the receptor is not shed from
T cells under these experimental conditions. Thus, expression of
the TIM-1 protein and specific antibody binding is specific to
activated Th1 and Th2 cells, which in turn, are characteristic of
inflammatory response, specifically asthma.
TABLE-US-00017 TABLE 14 Percent of activated T cells that express
TIM-1 Day 0 Day 1 Day 2 Day 4 Day 5 Control -MMPI 1 3 3 1 1 PK16.3
+MMPI 1 2 6 2 2 TIM-1 -MMPI 1 8 10 5 13 2.70.2 +MMPI 1 10 14 10
19
Example 12
Cytokine Assays
[0219] IL-4, IL-5, IL-10, IL-13, and IFN.gamma. production levels
by activated Th1 and Th2 cell were measured in culture supernatants
treated with anti-TIM-1 antibodies using standard ELISA protocols.
Cytokine production by Th1 or Th2 cells treated with anti-TIM-1
antibodies was compared to Th1 or Th2 cells treated with the
control PK16.3 antibody. In addition, the following samples were
run in parallel as internal controls: i) anti-CD3 treated Th1 or
Th2 cells, where no cytokine production is expected because of the
absence of co-stimulation, ii) anti-CD3/anti-CD28 stimulated Th1 or
Th2 cells, expected to show detectable cytokine production, and
iii) untreated Th1 or Th2 cells. CD4+ T cells were isolated as
described in the Example above. Isolated CD4+ lymphocytes were then
spun down and re-suspended in DMEM 5% FCS plus additives
(non-essential amino acids, sodium pyruvate, mercaptoethanol,
glutamine, penicillin, and streptomycin) at 10.sup.6 cells/mL.
Falcon 6-well non-tissue culture treated plates were pre-coated
overnight with anti-CD3 (2 .mu.g/mL) and anti-CD28 (10 .mu.g/mL)
(600 .mu.L total in Dulbecco's PBS) overnight at 4.degree. C. The
plates were washed with PBS and CD4+ lymphocytes were suspended at
500,000 cells/mL in Th2 medium: DMEM+ 10% FCS plus supplements and
IL-2 5 ng/mL, IL-4 5 ng/mL, anti-IFN gamma 5 .mu.g/mL and cells
were stimulated 4-6 days at 37.degree. C. and 5% CO2 in the
presence of 5 .mu.g/mL of mAb recognizing the TIM-1 protein or
isotype matched negative control mAb PK16.3.
[0220] In another set of experiments, CD4+ lymphocytes were
suspended at 500,000 cells/mL in Th1 medium: DMEM+ 10% FCS plus
supplements and IL-2 5 ng/mL, IL12 5 ng/mL, anti-IL-4 5 .mu.g/mL
and stimulated 4-6 days 37.degree. C. temp and 5% CO2 in the
presence of 5 .mu.g/mL TIM-1 or isotype matched control mAb PK16.3.
Cells were washed two times in DMEM and resuspended in DMEM, 10%
FCS plus supplements and 2 ng/mL IL-2 (500,000 cells/mL) in the
presence of 5 .mu.g/mL TIM-1 mAb or control PK16.3 mAb and cultured
(rested) for 4-6 days at 37.degree. C. and 5% CO2. The process of
activation and resting was repeated at least once more as described
above with the addition of anti-CD95L (anti-FAS ligand) to prevent
FAS-mediated apoptosis of cells. Falcon 96-well non-tissue culture
treated plates pre-coated overnight with anti-CD3 mAb at 500 ng/mL
and costimulatory molecule B7H2 (B7 homolog 2) 5 .mu.g/mL were
washed and 100 .mu.L of TIM-1 mAb treated Th1 or Th2 (200,000
cells) added per well. After 3 days of culture, the supernatants
were removed and IL-4, IL-5, IL-10, IL-13, and IFN.gamma.
.quadrature..quadrature. levels were determined by ELISA
(Pharmingen, San Diego, Calif. or R&D Systems, Minneapolis,
Minn.).
[0221] As demonstrated below, anti-TIM-1 mAb significantly
inhibited release of the tested cytokines by Th1 and Th2 cells (see
FIGS. 8-17). Results where inhibition of cytokine production is
significant (p=0.02-0.008), are marked on the bar graphs with an
asterisk. Tables 15 and 16 summarize the bar graphs in FIGS.
8-17.
TABLE-US-00018 TABLE 15 Cytokine Inhibition in CD4+ Th1 cells using
anti-TIM-1 antibodies in two independent human donors Donor 12 + 17
Cytokines Anti-TIM-1 Percentage of Control Antibody mAbs IL-5 IL-4
IL-10 IL-13 INF .gamma. TH1 2.56.2 100.17 28.49 * 63.76 * 86.45
93.69 2.45.1 90.23 39.78 * 83.98.sup. 96.25 100.6 1.29 94.63
81.05.sup. 60.77 ** .sup. 73.95 *** 93.51 2.59.2 .sup. 66.62 *
31.40 * 68.99 * 54.5 *** 128.12 Experiments that demonstrate
significant inhibition of cytokine production are marked with an
asterisk: P = 0.01 to 0.05 *; P = 0.005 to 0.009 **; P = 0.001 to
0.004 ***
TABLE-US-00019 TABLE 16 Cytokine Inhibition in CD4+ Th2 cells using
anti-TIM-1 antibodies in two independent human donors Donor 12 + 17
Cytokines Anti-TIM-1 Percentage of Control Antibody mAbs IL-5 IL-4
IL-10 IL-13 INF .gamma. TH2 2.56.2 112.07 103.46 93.97.sup. 86.45
88.30.sup. 2.45.1 148.7 25.66 *** 55.97 * 86.81 25.66 * 1.29 80.26
112.54 44.45 * 48.91 ** 112.54 2.59.2 .sup. 23.62 * .sup. 19.17 **
43.86 * .sup. 43.71 *** 19.18 * Experiments that demonstrate
significant inhibition of cytokine production are marked with an
asterisk: P = 0.01 to 0.05 *; P = 0.005 to 0.009 **; P = 0.001 to
0.004 ***
[0222] A summary of Th2 cytokine inhibition data obtained from
multiple experiments with different donors is provided in Table 17.
Each experiment used purified CD4+ cells isolated from whole blood
samples from two independent donors. Cytokine production is
reported as the percent of cytokine production detected using the
control PK16.3 mAb. The anti-TIM-1 mAb used in each experiment is
specified along the bottom row. Results that report significant
cytokine inhibition are underlined in Table 17 below. The use of
"ND" indicates that the experiment was not performed. These results
do reflect donor dependent variability but show that mAbs 2.59.2
and 1.29 reproducibly block one or more of the Th2 cytokines.
TABLE-US-00020 TABLE 17 Summary of Cytokine Inhibition using
anti-TIM-1 mAbs 2.59.2 and 1.29 in 5 independent human donor groups
Results of experiments that report inhibition greater than 50% of
that seen using the control PK16.3 antibody are underlined. Donor
ID Cytokine 12 + 17 12 + 14 13 + 14 14 12 IL-4 19 626 130 ND ND
IL-5 24 5 122 67 2 IL-10 44 83 19 45 109 IL-13 44 ND 17 100 91
Anti-TIM-1 Anti-TIM-1 mAb 1.29 mAb 2.59.2
Example 13
Construction, Expression and Purification of anti-TIM-1 scFv
[0223] The VL and VH domains of mAb 2.70 were used to make a scFv
construct. The sequence of the anti-TIM-1 scFv was synthesized by
methods known in the art.
[0224] The nucleotide sequence of anti-TIM-1 scFv is as
follows:
TABLE-US-00021 (SEQ ID NO: 108)
ATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCGCTGC
CCAGCCGGCCATGGCCGATATTGTGATGACCCAGACTCCACTCTCCCTGC
CCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCGGAGC
CTCTTGGATAGTGATGATGGAAACACCTATTTGGACTGGTACCTGCAGAA
GCCAGGGCAGTCTCCACAGCTCCTGATCTACACGCTTTCCTATCGGGCCT
CTGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACA
CTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGCAT
GCAACGTGTAGAGTTTCCTATCACCTTCGGCCAAGGGACACGACTGGAGA
TTAAACTTTCCGCGGACGATGCGAAAAAGGATGCTGCGAAGAAAGATGAC
GCTAAGAAAGACGATGCTAAAAAGGACCTCCAGGTGCAGCTGGTGGAGTC
TGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAG
CGTCTGGATTCATCTTCAGTCGCTATGGCATGCACTGGGTCCGCCAGGCT
CCAGGCAAGGGGCTGAAATGGGTGGCAGTTATATGGTATGATGGAAGTAA
TAAACTCTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACA
ATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGAC
ACGGCTGTGTATTACTGTGCGAGAGATTACTATGATAATAGTAGACATCA
CTGGGGGTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG
CTAGCGATTATAAGGACGATGATGACAAATAG
[0225] The amino acid sequence of mature anti-TIM-1 scFv is as
follows:
TABLE-US-00022 (SEQ ID NO: 109)
DIVMTQTPLSLPVTPGEPASISCRSSRSLLDSDDGNTYLDWYLQKPGQSP
QLLIYTLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQRVEF
PITFGQGTRLEIKLSADDAKKDAAKKDDAKKDDAKKDLQVQLVESGGGVV
QPGRSLRLSCAASGFIFSRYGMHWVRQAPGKGLKWVAVIWYDGSNKLYAD
SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDYYDNSRHHWGFDY
WGQGTLVTVSSASDYKDDDDK
[0226] The synthesized DNA can be inserted into the pET-20b(+)
expression vector, for periplasmic expression in E. coli. Cells are
grown and the periplasmic proteins prepared using standard
protocols. Purification of the anti-TIM-1 scFv is achieved using an
anti-FLAG M2 affinity column as per the manufacturer's directions.
The predicted molecular weight of the mature protein is 30222.4
daltons. This purified scFv is used in the assays described below
to test for biological activity. The scFv construct is comprised of
a signal peptide (SP), VL (VL1) derived from mAb 2.70, a linker
(L4) based on the 25 amino acid linker 205C, the VH (VH1) derived
from mAb 2.70, and a Tag (in this case the FLAG tag). It will be
obvious to those skilled in the art that other SP, linker and tag
sequences could be utilized to get the same activity as the
anti-TIM-1 scFv antibody described herein.
Example 14
Construction, Expression and Purification of Anti-TIM-1 and
Anti-CD3 Bispecific scFv1
[0227] The basic formula for the construction of this therapeutic
protein is as follows:
SP1-VL1-L1-VH1-L2-VH2-L3-VL2-Tag
[0228] The signal peptide SP1 is the same as IgG kappa signal
peptide VKIII A27 from Medical Research Council (MRC) Centre for
Protein Engineering, University of Cambridge, UK.
[0229] Other signal peptides can also be used and will be obvious
to those skilled in the art. This protein is designed to be
expressed from mammalian cells. The predicted molecular weight of
the mature cleaved protein is 54833.3 dalton. L1 corresponds to the
(Gly4Ser)3 linker, while linker 2 (L2) corresponds to the short
linker sequence: GGGGS. L3 is an 18 amino acid linker. VH2
corresponds to the anti-CD3 variable heavy chain domain from
Genbank (accession number CAE85148) while VL1 corresponds to the
anti-CD3 variable light chain domain from Genbank (accession number
CAE85148). The tag being used for this construct is a His tag to
facilitate purification and detection of this novel protein.
Standard protocols are used to express and purify this His tagged
protein, which is tested for activity and tumor cell killing in the
protocols described below.
[0230] The amino acid and nucleic acid numbering for the components
comprising the anti-TIM-1 and anti-CD3 bispecific scFv1 is as
follows: [0231] SP: -20 to -1 aa; -60 to -1 nt [0232] VL1: 1-113
aa; 1-339nt [0233] L1: 114-128 aa; 340-384nt [0234] VH1: 129-251
aa; 385-753nt [0235] L2: 252-256 aa; 754-768nt [0236] VH2: 257-375
aa; 769-1125nt [0237] L3: 376-393 aa; 1126-1179nt [0238] VL2:
394-499 aa; 1180-1497nt [0239] Tag: 500-505 aa; 1498-1515nt
[0240] The nucleotide sequence of anti-TIM-1 and anti-CD3
bispecific scFv1 is as follows:
TABLE-US-00023 (SEQ ID NO: 110)
ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGA
TACCACCGGAGATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCA
CCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCGGAGCCTCTTG
GATAGTGATGATGGAAACACCTATTTGGACTGGTACCTGCAGAAGCCAGG
GCAGTCTCCACAGCTCCTGATCTACACGCTTTCCTATCGGGCCTCTGGAG
TCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAA
ATCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGCATGCAACG
TGTAGAGTTTCCTATCACCTTCGGCCAAGGGACACGACTGGAGATTAAAG
GTGGTGGTGGTTCTGGCGGCGGCGGCTCCGGTGGTGGTGGTTCCCAGGTG
CAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAG
ACTCTCCTGTGCAGCGTCTGGATTCATCTTCAGTCGCTATGGCATGCACT
GGGTCCGCCAGGCTCCAGGCAAGGGGCTGAAATGGGTGGCAGTTATATGG
TATGATGGAAGTAATAAACTCTATGCAGACTCCGTGAAGGGCCGATTCAC
CATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCC
TGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATTACTATGAT
AATAGTAGACATCACTGGGGGTTTGACTACTGGGGCCAGGGAACCCTGGT
CACCGTCTCCTCAGGAGGTGGTGGATCCGATATCAAACTGCAGCAGTCAG
GGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCAAGACT
TCTGGCTACACCTTTACTAGGTACACGATGCACTGGGTAAAACAGAGGCC
TGGACAGGGTCTGGAATGGATTGGATACATTAATCCTAGCCGTGGTTATA
CTAATTACAATCAGAAGTTCAAGGACAAGGCCACATTGACTACAGACAAA
TCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTC
TGCAGTCTATTACTGTGCAAGATATTATGATGATCATTACTGCCTTGACT
ACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCAGTCGAAGGTGGAAGT
GGAGGTTCTGGTGGAAGTGGAGGTTCAGGTGGAGTCGACGACATTCAGCT
GACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCA
TGACCTGCAGAGCCAGTTCAAGTGTAAGTTACATGAACTGGTACCAGCAG
AAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAAGTGGC
TTCTGGAGTCCCTTATCGCTTCAGTGGCAGTGGGTCTGGGACCTCATACT
CTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGC
CAACAGTGGAGTAGTAACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGA GCTGAAATAG
[0241] The protein sequence of mature anti-TIM-1 and anti-CD3
bispecific scFv1 is as follows:
TABLE-US-00024 (SEQ ID NO: 111)
DIVMTQTPLSLPVTPGEPASISCRSSRSLLDSDDGNTYLDWYLQKPGQSP
QLLIYTLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQRVEF
PITFGQGTRLEIKGGGGSGGGGSGGGGSQVQLVESGGGVVQPGRSLRLSC
AASGFIFSRYGMHWVRQAPGKGLKWVAVIWYDGSNKLYADSVKGRFTISR
DNSKNTLYLQMNSLRAEDTAVYYCARDYYDNSRHHWGFDYWGQGTLVTVS
SGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTMHWVKQRPGQG
LEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSSLTSEDSAVY
YCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQS
PAIMSASPGEKVTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGV
PYRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSNPLTFGAGTKLELK
Example 15
Construction, Expression and Purification of Anti-TIM-1 and
Anti-CD3 Bispecific scFv2
[0242] The basic formula for the construction of this novel
therapeutic protein is as follows:
SP1-VL1-L4-VH1-L2-VH2-L4-VL2-Tag
[0243] The signal peptide SP1 is IgG kappa signal peptide VKIII A27
from Medical Research Council (MRC) Centre for Protein Engineering,
University of Cambridge, UK. For more information see
mrc-cpe.cam.ac.uk/ALIGNMENTS.php?menu=901. Other signal peptides
and linkers could also be used to get additional biologically
active bispecific single chain antibodies. The protein being
described in this example is also designed to be expressed from
mammalian cells and is similar to the anti-TIM-1 and anti-CD3
bispecific scFv1, except that it utilizes a different linker as
indicated in the basic formula above (L4, as described earlier),
and that a Flag tag is used instead of the His tag as in the first
example.
[0244] The predicted molecular weight of the mature cleaved protein
is 58070.0 dalton. The tag being used for this construct is a FLAG
tag to facilitate purification and detection of this novel protein.
Standard protocols are used to express this secreted protein and
purify it, which is tested for activity and tumor cell killing in
the protocols described below.
[0245] The amino acid and nucleic acid numbering for the components
comprising the anti-TIM-1 and anti-CD3 bispecific scFv2 is as
follows: [0246] SP: -20 to -1 aa; -60 to -1int [0247] VL1: 1-113
aa; 1-339nt [0248] L1: 114-138 aa; 340-414nt [0249] VH1: 139-261
aa; 415-783nt [0250] L2: 262-266 aa; 784-798nt [0251] VH2: 267-385
aa; 799-1155nt [0252] L3: 386-410 aa; 1156-1230nt [0253] VL2:
411-516 aa; 1231-1548nt [0254] Tag: 517-524 aa; 1549-1572nt
[0255] The nucleotide sequence of anti-TIM-1 and anti-CD3
bispecific scFv2 is as follows:
TABLE-US-00025 (SEQ ID NO: 112)
ATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTACTCTGGCTCCCAGA
TACCACCGGAGATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCA
CCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCGGAGCCTCTTG
GATAGTGATGATGGAAACACCTATTTGGACTGGTACCTGCAGAAGCCAGG
GCAGTCTCCACAGCTCCTGATCTACACGCTTTCCTATCGGGCCTCTGGAG
TCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAA
ATCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGCATGCAACG
TGTAGAGTTTCCTATCACCTTCGGCCAAGGGACACGACTGGAGATTAAAC
TTTCCGCGGACGATGCGAAAAAGGATGCTGCGAAGAAAGATGACGCTAAG
AAAGACGATGCTAAAAAGGACCTGCAGGTGCAGCTGGTGGAGTCTGGGGG
AGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTG
GATTCATCTTCAGTCGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGC
AAGGGGCTGAAATGGGTGGCAGTTATATGGTATGATGGAAGTAATAAACT
CTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCA
AGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCT
GTGTATTACTGTGCGAGAGATTACTATGATAATAGTAGACATCACTGGGG
GTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGAGGTG
GTGGATCCGATATCAAACTGCAGCAGTCAGGGGCTGAACTGGCAAGACCT
GGGGCCTCAGTGAAGATGTCCTGCAAGACTTCTGGCTACACCTTTACTAG
GTACACGATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGA
TTGGATACATTAATCCTAGCCGTGGTTATACTAATTACAATCAGAAGTTC
AAGGACAAGGCCACATTGACTACAGACAAATCCTCCAGCACAGCCTACAT
GCAACTGAGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAA
GATATTATGATGATCATTACTGCCTTGACTACTGGGGCCAAGGCACCACT
CTCACAGTCTCCTCACTTTCCGCGGACGATGCGAAAAAGGATGCTGCGAA
GAAAGATGACGCTAAGAAAGACGATGCTAAAAAGGACCTGGACATTCAGC
TGACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACC
ATGACCTGCAGAGCCAGTTCAAGTGTAAGTTACATGAACTGGTACCAGCA
GAAGTCAGGCACCTCCCCCAAAAGATGGATTTATGACACATCCAAAGTGG
CTTCTGGAGTCCCTTATCGCTTCAGTGGCAGTGGGTCTGGGACCTCATAC
TCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTG
CCAACAGTGGAGTAGTAACCCGCTCACGTTCGGTGCTGGGACCAAGCTGG
AGCTGAAAGATTATAAGGACGATGATGACAAATAG
[0256] The protein sequence of mature anti-TIM-1 and anti-CD3
bispecific scFv2 is as follows:
TABLE-US-00026 (SEQ ID NO: 113)
DIVMTQTPLSLPVTPGEPASISCRSSRSLLDSDDGNTYLDWYLQKPGQSP
QLLIYTLSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQRVEF
PITFGQGTRLEIKLSADDAKKDAAKKDDAKKDDAKKDLQVQLVESGGGVV
QPGRSLRLSCAASGFIFSRYGMHWVRQAPGKGLKWVAVIWYDGSNKLYAD
SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDYYDNSRHHWGFDY
WGQGTLVTVSSGGGGSDIKLQQSGAELARPGASVKMSCKTSGYTFTRYTM
HWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLS
SLTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSLSADDAKKDAAKKDD
AKKDDAKKDLDIQLTQSPAIMSASPGEKVTMTCRASSSVSYMNWYQQKSG
TSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQQW
SSNPLTFGAGTKLELKDYKDDDDK
Example 16
Anti-TIM-1 scFv Species Biological Activity
ELISA Analysis:
[0257] To determine if the anti-TIM-1 and anti-CD3 bispecific scFv1
and scFv2 antibodies bind to specific antigen, ELISA analysis is
performed. lug/ml of specific antigen (TIM-1 antigen (CG57008-02)
is bound to ELISA plates overnight in carbonate/bicarbonate buffer
(pH approximately 9.2-9.4). Plates are blocked with assay diluent
buffer purchased from Pharmingen San Diego, Calif.), and various
concentrations of the anti-TIM-1 scFv bispecific antibodies are
added for 1 hour at room temp. Plates are washed in 0.01% Tween 20
in PBS, followed by addition of HRP-conjugated mAb to either the
6-His tag (Invitrogen, Carlsbad, Calif.) or the FLAG peptide tag or
(Sigma, St. Louis, Mo.) in assay diluent for 60 minutes at room
temperature. Color is developed with TMB substrate (Pharmingen),
and the reaction stopped with H.sub.250.sub.4. Plates are read at
A450 nm, and the O.D. value taken as a measure of protein
binding.
FACS Analysis
[0258] Binding of the anti-TIM-1 and anti-CD3 bispecific scFv1 and
scFv2 antibodies, as well as the anti-TIM-1 scFv antibody to cells
expressing the antigens recognized by the anti-TIM-1 human mAbs is
examined by FACS analysis. Cells (such as ACHN) are washed in PBS
and resuspended in FACS buffer consisting of ice cold PBS with
addition of 1% BSA or 1% FBS. The resuspended cells are then
incubated on ice with various concentrations of the bispecific
antibody for 30 minutes. Cells are washed to remove non-bound
antibody. Bound antibody is detected by binding of a secondary
labeled mAb (phycoerythrin or FITC labeled) that specifically
recognizes the 6-his tag or the FLAG-tag that is engineered on the
bispecific antibody sequence. Cells are washed and analyzed for
binding of the anti-tag mAb by FACS analysis. Binding of bispecific
mAb plus anti-tag mAb is compared to binding of the anti-tag mAb
alone.
Cytotoxicity Analysis
[0259] To determine if the bispecific antibody has functional
activity as defined by the ability of the bispecific to target T
cells to TIM-1 expressing normal or tumor cells, the bispecific
antibody is tested in a Cytotoxicity assay. T cells are obtained
from the low density cells derived from centrifugation of blood
over density separation medium (specific density 1.077). T cells
can be used in a heterogeneous mix from the peripheral blood
mononuclear cell fraction (which also contains B cells, NK cells
and monocytes) or further purified from the low-density cells using
MACS separation and negative or positive selection. Killing in
assays with T cells derived from the blood directly will have less
cytolytic activity than cells that have been stimulated in vitro
with PHA, cytokines, activating monoclonal antibodies or other
stimulators of polyclonal T cell activation. Therefore, these
activators will be used to further boost the activity of T cells in
the functional assays. Many variations of cytotoxicity assays are
available. Cytotoxicity assays measure the release of natural
products of the cells metabolism upon lysis, such as LDH. Other
assays are based around labeling cells with various agents such as
radioactive chromium (51Cr), DELFIA BATDA, CSFE or similar labeling
agents and detecting release or change in live cells bound by the
agent.
[0260] DELFIA cytotoxicity assays (PerkinElmer Life and Analytical
Sciences, Inc. Boston, Mass.) offer a non-radioactive method to be
used in cell mediated cytotoxicity studies. The method is based on
loading cells with an acetoxymethyl ester of a fluorescence
enhancing ligand. After the ligand has penetrated the cell membrane
the ester bonds are hydrolyzed within the cell to form a
hydrophilic ligand, which no longer passes through the membrane.
After cytolysis the released ligand is introduced to a europium
solution to form a fluorescent chelate. The measured signal
correlates directly with the amount of lysed cells. Target cells
are resuspended to a concentration of 2.times.10.sup.6/ml. 10 .mu.l
of DELFIA BATDA was mixed in a tube with 2 ml of target cells
according to the manufacturers instructions. Various concentrations
of T cells are added to a fixed concentration of labeled target
cells (5000 cells per well) in 96 well U-bottom plates, and
incubated for at least 2 hours at 37.degree. C. The plates are spun
at approximately 200 g, followed by the aspiration of 20 .mu.l of
supernatant, which was then added to a europium solution (200
.mu.l) in a separate plate. The plate is incubated for 15 minutes
at room temperature, followed by analysis on a SAFIRE (Tecan,
Maennedorf, Switzerland) according to the manufacturer's
instructions. Signal in the test wells are compared to signal in
100% lysis well (10% lysis buffer in place of T cells) and cell
with medium alone (spontaneous release), and % specific lysis is
calculated from the formula
%specific lysis=(test-spontaneous release)/100%
lysis.times.100.
BIAcore Kinetic Analysis of scFv Constructs
[0261] Kinetic measurements to determine the affinity for the scFv
constructs (monomer as well as bispecific, containing at least 1
scFv moiety binding to TIM-1) are measured using the methods
described earlier for the whole antibodies of this invention.
scFv-containing antibody protein affinities to TIM-1 are expected
to be within a factor of 10, i.e. between 0.271-27.1 nM, of the
affinity given for mAb 2.70.
Example 17
Ability of Anti-TIM-1 mAb to Inhibit the Proliferation of Human
Ovary Carcinoma Cells
[0262] Several fully human monoclonal antibody clones were isolated
from the immunizations described above and their ability to inhibit
the proliferative potential of OVCAR-5 (human ovary carcinoma)
cells was analyzed using the 5-bromo-2-deoxyuridine (BrdU)
incorporation assay (described in International Patent Application
No. WO 01/25433).
[0263] In the BrdU assay, OVCAR-5 cancer cells (Manassas, Va.) were
cultured in Dulbeccos Modification of Eagles Medium (DMEM)
supplemented with 10% fetal bovine serum or 10% calf serum
respectively. The ovarian cancer cell line was grown to confluence
at 37.degree. C. in 10% CO.sub.2/air. Cells were then starved in
DMEM for 24 hours. Enriched conditioned medium was added (10
.mu.L/100 .mu.L of culture) for 18 hours. BrdU (10 .mu.M) was then
added and incubated with the cells for 5 hours. BrdU incorporation
was assayed by colorimetric immunoassay according to the
manufacturer's specifications (Boehringer Mannheim, Indianapolis,
Ind.).
[0264] The capability of various human anti-TIM-1 monoclonal
antibodies to neutralize was assessed. The results provided in
FIGS. 18A-18T are presented in a bar graph format to assist in
comparing the levels of BrdU incorporation in OVCARS cells upon
exposure to various human anti-TIM-1monoclonal antibodies described
herein. As positive and negative controls, OVCAR5 cells were
cultured in the presence of either complete media (complete) or
restricted serum-containing media (starved). In addition, the
monoclonal antibody PK16.3 was included as a negative treatment
control representing a human IgG antibody of irrelevant
specificity. Human anti-TIM-1 monoclonal antibodies described
herein were used at varying doses (10-1000 ng/mL) as compared to a
control run utilizing varying concentrations.
Example 18
Antibody Conjugate Studies
[0265] Additional antibody conjugate studies were performed using
the plant toxin saporin conjugated to anti-TIM-1-specific mABs
(1.29 and 2.56.2) and various irrelevant antibodies, including,
PK16.3 (FIGS. 19A-19C). Additional negative controls included
anti-TIM-1-specific mAB 2.56.2 and irrelevant antibody PK16.3
without toxin (FIG. 19D). Four cancer cell lines, three kidney
cancer cell lines (ACHN, CAKI, and 786O) and one breast cancer cell
line (BT549), were treated for 72 hours with saporin-antibody
conjugates or antibodies alone, after which time BrdU was added to
monitor proliferation over a 24 hour period. The results are
described in FIGS. 19A-19C for the kidney cancer cell lines and
FIG. 19D for the breast cancer cell line. All three kidney cancer
cell lines were sensitive to treatment with saporin-TIM-1-specific
antibody conjugates as evidenced by a measurable decrease in BrdU
incorporation. Treatment of the same cell lines with conjugated
irrelevant antibodies had little or no effect demonstrating antigen
dependent antiproliferative effects. The same studies performed
with the BT549 cell line showed that the TIM-1-specific antibody
2.56.2 showed no antiproliferative effect either alone or when
conjugated to saporin. The negative controls for these studies
appeared to work well with no cytotoxic effects
Example 19
Sequences
[0266] Below are sequences related to monoclonal antibodies against
TIM-1. With regard to the amino acid sequences, bold indicates
framework regions, underlining indicates CDR regions, and italics
indicates constant regions.
Anti-TIM-1 mAb 1.29
[0267] Nucleotide sequence of heavy chain variable region and a
portion of constant region:
TABLE-US-00027 (SEQ ID NO: 1)
5'TGGGTCCTGTCCCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTG
AAGCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCGT
CAGCAGTGGTGGTTACTACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGG
GACTGGAGTGGATTGGGTTTATCTATTACACTGGGAGCACCAACTACAAC
CCCTCCCTCAAGAGTCGAGTCTCCATATCAGTAGACACGTCCAAGAACCA
GTTCTCCCTGAAGCTGAGCTCTGTGACCGCTGCGGACGCGGCCGTGTATT
ACTGTGCGAGAGATTATGACTGGAGCTTCCACTTTGACTACTGGGGCCAG
GGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTT
CCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGG
GCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAAC
TCAGGCGCTCT3'
[0268] Amino acid sequence of heavy chain variable region and a
portion of constant region encoded by SEQ ID NO:1:
TABLE-US-00028 (SEQ ID NO: 114)
WVLSQVQLQESGPGLVKPSETLSLTCTVSGGSVSSGGYYWSWIRQPPGKG
LEWIGFIYYTGSTNYNPSLKSRVSISVDTSKNQFSLKLSSVTAADAAVYY
CARDYDWSFHFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALG
CLVKDYFPEPVTVSWNSGA
[0269] Nucleotide sequence of light chain variable region and a
portion of constant region:
TABLE-US-00029 (SEQ ID NO: 3)
5'CAGCTCCTGGGGCTCCTGCTGCTCTGGTTCCCAGGTGCCAGGTGTGAC
ATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTATAGGAGACAG
AGTCACCATCACTTGCCGGGCAAGTCAGGGCATTAGAAATGATTTAGGCT
GGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGATCTATGCTGCA
TCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGG
GACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATTTTGCAA
CTTATTACTGTCTACAGCATAATAGTTACCCTCTCACTTTCGGCGGAGGG
ACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTT
CCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCC
TGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGAT AACGCC3'
[0270] Amino acid sequence of light chain variable region and a
portion of constant region encoded by SEQ ID NO:3:
TABLE-US-00030 (SEQ ID NO: 115)
QLLGLLLLWFPGARCDIQMTQSPSSLSASIGDRVTITCRASQGIRNDLGW
YQQKPGKAPKRLIYAASSLQSGVPSRFSGSGSGTEFTLTISSLQPEDFAT
YYCLQHNSYPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL
LNNFYPREAKVQWKVDNA
Anti-TIM-1 mAb 1.37
[0271] Nucleotide sequence of heavy chain variable region and a
portion of constant region:
TABLE-US-00031 (SEQ ID NO: 5)
5'CAGTGTGAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTCCAGCCT
GGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTACTAA
CTATTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGG
TGGCCAACATACAGCAAGATGGAAGTGAGAAATACTATGTGGACTCTGTG
AGGGGCCGATTCACCATCTCCAGAGACAACGCCAAGAACTCACTGTATCT
GCAAATGAACAGCCTGAGAGCCGAGGACTCGGCTGTGTATTACTGTGCGA
GATGGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCC
ACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTC
CGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAAC
CGGTGAGCGGTGTCGTGGAAC3'
[0272] Amino acid sequence of heavy chain variable region and a
portion of constant region encoded by SEQ ID NO:5:
TABLE-US-00032 (SEQ ID NO: 116)
QCEVQLVESGGGLVQPGGSLRLSCAASGFTFTNYWMSWVRQAPGKGLEWV
ANIQQDGSEKYYVDSVRGRFTISRDNAKNSLYLQMNSLRAEDSAVYYCAR
WDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEP VSGVVE
[0273] Nucleotide sequence of light chain variable region and a
portion of constant region:
TABLE-US-00033 (SEQ ID NO: 7)
5'CTTCTGGGGCTGCTAATGCTCTGGGTCCCTGGATCCAGTGGGGATATT
GTGATGACCCAGACTCCACTCTCCTCAACTGTCATCCTTGGACAGCCGGC
CTCCATCTCCTGCAGGTCTAGTCAAAGCCTCGTACACAGTGATGGAAACA
CCTACTTGAATTGGCTTCAGCAGAGGCCAGGCCAGCCTCCAAGACTCCTA
ATTTATATGATTTCTAACCGGTTCTCTGGGGTCCCAGACAGATTCAGTGG
CAGTGGGGCAGGGACAGATTTCACACTGAAAATCAGCAGGGTGGAAGCTG
AGGATGTCGGGGTTTATTACTGCATGCAAGCTACAGAATCTCCTCAGACG
TTCGGCCAAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATC
TGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAAGGGCCT CTGTTG3'
[0274] Amino acid sequence of light chain variable region and a
portion of constant region encoded by SEQ ID NO:7:
TABLE-US-00034 (SEQ ID NO: 117)
LLGLLMLWVPGSSGDIVMTQTPLSSTVILGQPASISCRSSQSLVHSDGNT
YLNWLQQRPGQPPRLLIYMISNRFSGVPDRFSGSGAGTDFTLKISRVEAE
DVGVYYCMQATESPQTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGRAS V
Anti-TIM-1 mAb 2.16
[0275] Nucleotide sequence of heavy chain variable region and a
portion of constant:
TABLE-US-00035 (SEQ ID NO: 9)
5'GAGCAGTCGGGGGGAGGCGTGGTAAAGCCTGGGGGGTCTCTTAGACTC
TCCTGTGCAGCCTCTGGATTCACTTTCAGTAACGCCTGGATGACCTGGGT
CCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGTATTAAAAGGA
GAACTGATGGTGGGACAACAGACTACGCTGCACCCGTGAAAGGCAGATTC
ACCATCTCAAGAGATGATTCAAAAAACACGCTGTATCTGCAAATGAACAA
CCTGAAAAACGAGGACACAGCCGTGTATTACTGTACCTCAGTCGATAATG
ACGTGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTTCC
ACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTC
CGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAAC
CGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACC
TTCCCGGCTGTCCTACAGTCCTCAGGACTCT3'
[0276] Amino acid sequence of heavy chain variable region and a
portion of constant region encoded by SEQ ID NO:9:
TABLE-US-00036 (SEQ ID NO: 118)
XXXXEQSGGGVVKPGGSLRLSCAASGFTFSNAWMTWVRQAPGKGLEWVGR
IKRRTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNNLKNEDTAVYYCTS
VDNDVDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLQSSGL
[0277] Nucleotide sequence of light chain variable region and a
portion of constant region:
TABLE-US-00037 (SEQ ID NO: 11)
5'CTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCC
TCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAA
CTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGA
TCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGC
AGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGA
GGATATTGGTCTTTATTACTGCATGCAAGCTCTACAAACTCCGCTCACTT
TCGGCGGAGGGACCAAGGTGGACATCAAACGAACTGTGGCTGCACCATCT
GTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTC
TGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAG 3'
[0278] Amino acid sequence of light chain variable region and a
portion of constant region encoded by SEQ ID NO:11:
TABLE-US-00038 (SEQ ID NO: 119)
XXXLTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ
LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDIGLYYCMQALQTP
LTFGGGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQ
Anti-TIM-1 mAb 2.17
[0279] Nucleotide sequence of heavy chain variable region and a
portion of constant region:
TABLE-US-00039 (SEQ ID NO: 13)
5'CAGGTGCAGCTGGAGCAGTCGGGGGGAGGCTTGGTACAGCCTGGGGGG
TCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTACCTATAG
CATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTTCAT
ACATTAGAAGTAGTACTAGTACCATATACTATGCAGAGTCCCTGAAGGGC
CGATTCACCATCTCCAGCGACAATGCCAAGAATTCACTATATCTGCAAAT
GAACAGCCTGAGAGACGAGGACACGGCTGTGTATTACTGTGCGCGGGACT
TTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTTCCACC
AAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGA
GAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG
TGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTC
CCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCA3'
[0280] Amino acid sequence of heavy chain variable region and a
portion of constant region encoded by SEQ ID NO:13:
TABLE-US-00040 (SEQ ID NO: 120)
QVQLEQSGGGLVQPGGSLRLSCAASGFTFSTYSMNWVRQAPGKGLEWVSY
IRSSTSTIYYAESLKGRFTISSDNAKNSLYLQMNSLRDEDTAVYYCARDF
DYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPV
TVSWNSGALTSGVHTFPAVLQSSGLYSLS
[0281] Nucleotide sequence of light chain variable region and a
portion of constant region:
TABLE-US-00041 (SEQ ID NO: 15)
5'GAAATCCAGCTGACTCAGTCTCCACTCTCCTCACCTGTCACCCTTGGA
CAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAAAGCCTCGTACACAGTGA
TGGAGACACCTACTTGAATTGGCTTCAGCAGAGGCCAGGCCAGCCTCCAA
GACTCCTAATTTATAAGATTTCTACCCGGTTCTCTGGGGTCCCTGACAGA
TTCAGTGGCAGTGGGGCAGGGACAGATTTCACACTGAAAATCAGCAGGGT
GGAGACTGACGATGTCGGGATTTATTACTGCATGCAAACTACACAAATTC
CTCAAATCACCTTCGGCCAAGGGACACGACTGGAGATTAAACGAACTGTG
GCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATC
TGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGG
CCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTA3'
[0282] Amino acid sequence of light chain variable region and a
portion of constant region encoded by SEQ ID NO:15:
TABLE-US-00042 (SEQ ID NO: 121)
EIQLTQSPLSSPVTLGQPASISCRSSQSLVHSDGDTYLNWLQQRPGQPPR
LLIYKISTRFSGVPDRFSGSGAGTDFTLKISRVETDDVGIYYCMQTTQIP
QITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREA
KVQWKVDNALQSG
Anti-TIM-1 mAb 2.24
[0283] Nucleotide sequence of heavy chain variable region and a
portion of constant region:
TABLE-US-00043 (SEQ ID NO: 17)
5'CAGGTGCAGCTGGAGCAGTCGGGGGGAGGCGTGGTCCAGCCTGGGAGG
TCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTCGCTATGG
CATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGAAATGGGTGGCAG
TTATATGGTATGATGGAAGTAATAAACTCTATGCAGACTCCGTGAAGGGC
CGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAAT
GAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGCGAGAGATT
ACTATGATAATAGTAGACATCACTGGGGGTTTGACTACTGGGGCCAGGGA
ACCCTGGTCACCGTCTCCTCAGCTTCCACCAAGGGCCCATCCGTCTTCCC
CCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCT
GCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCA
GGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTC
AGGACTCTACTCCCTCAGCA
[0284] Amino acid sequence of heavy chain variable region and a
portion of constant region encoded by SEQ ID NO:17:
TABLE-US-00044 (SEQ ID NO: 122)
QVQLEQSGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLKWVAV
IWYDGSNKLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDY
YDNSRHHWGFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC
LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS
[0285] Nucleotide sequence of light chain variable region and a
portion of constant region:
TABLE-US-00045 (SEQ ID NO: 19)
5'GACATCCAGCTGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGA
GACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGTATTTATAGTTATTT
AAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATG
CTGCATCCAGTTTGCAAAGTGGGGTCCCATCCAGGTTCAGTGGCAGTGGA
TCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTT
TGCAACTTACTACTGTCAACAGAGTTACAGTACCCCTCCGACGTTCGGCC
AAGGGACCAAGGTGGAAATCAAACGAACTGTGGCTGCACCATCTGTCTTC
ATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGT
GTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGG
TGGATAACGCCCTCCAATCGGGTA3'
[0286] Amino acid sequence of light chain variable region and a
portion of constant region encoded by SEQ ID NO:19:
TABLE-US-00046 (SEQ ID NO: 123)
DIQL/MT/LQSPSSLSASVGDRVTITCRASQSIYSYLNWYQQKPGKAPKL
LIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPP
TFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVDNALQSG
Anti-TIM-1 mAb 2.45
[0287] Nucleotide sequence of heavy chain variable region and a
portion of constant region:
TABLE-US-00047 (SEQ ID NO: 21)
5'CAGTCGGGGGGAGGCTTGGTAAAGCCTGGGGGGTCCCTTAGACTCTCC
TGTGCAGCCTCTGGATTCACTTTCAGTAACGCCTGGATGACCTGGGTCCG
CCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGTATTAAAAGGAAAA
CTGATGGTGGGACAACAGACTACGCTGCACCCGTGAAAGGCAGATTCACC
ATCTCAAGAGATGATTCAGAAAACACGCTGTATCTGCAAATGAACAGCCT
GGAAACCGAGGACACAGCCGTGTATTACTGTACCACAGTCGATAACAGTG
GTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTTCCACC
AAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGA
GAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG
TGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTC
CCGGCTGTCCTACAGTCCTCAGGACTCTCT3'
[0288] Amino acid sequence of heavy chain variable region and a
portion of constant region encoded by SEQ ID NO:21:
TABLE-US-00048 (SEQ ID NO: 124)
XXXXXQSGGGLVKPGGSLRLSCAASGFTFSNAWMTWVRQAPGKGLEWVGR
IKRKTDGGTTDYAAPVKGRFTISRDDSENTLYLQMNSLETEDTAVYYCTT
VDNSGDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLS
[0289] Nucleotide sequence of light chain variable region and a
portion of constant region:
TABLE-US-00049 (SEQ ID NO: 23)
5'ACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCC
TCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCATAGTAATGGATACAA
CTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGA
TCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGACAGGTTCAGTGGC
AGTGGATCAGGCACAGATTTTACACTGAAAATCAGCAGAGTGGAGGCTGA
GGATGTTGGGGTTTATTACTGCATGCAAGCTCTACAAACTCCGCTCACTT
TCGGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCATCT
GTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTC
TGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGT
GGAAGGTGGATAACGCCCTCA3'
[0290] Amino acid sequence of light chain variable region and a
portion of constant region encoded by SEQ ID NO:23:
TABLE-US-00050 (SEQ ID NO: 125)
XXXXTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQ
LLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTP
LTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNAL
Anti-TIM-1 mAb 2.54
[0291] Nucleotide sequence of heavy chain variable region and a
portion of constant region:
TABLE-US-00051 (SEQ ID NO: 25)
5'CAGGTGCAGCTGGAGCAGTCGGGGGGAGGCGTGGTCCAGCCTGGG
AGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCACTAACTA
TGGCTTGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGATTGGGTGG
CAGTTATATGGTATGATGGAAGTCATAAATTCTATGCAGACTCCGTGAAG
GGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTCTTTCTGCA
AATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTACGCGAG
ATCTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTTCC
ACCAAGGGCCCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTC
CGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAAC
CGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACC
TTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGC3'
[0292] Amino acid sequence of heavy chain variable region and a
portion of constant region encoded by SEQ ID NO:25:
TABLE-US-00052 (SEQ ID NO: 126)
QVQLEQSGGGVVQPGRSLRLSCAASGFTFTNYGLHWVRQAPGKGLDWVAV
IWYDGSHKFYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCTRDL
DYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPV
TVSWNSGALTSGVHTFPAVLQSSGLYSLS
[0293] Nucleotide sequence of light chain variable region and a
portion of constant region:
TABLE-US-00053 (SEQ ID NO: 27)
5'GAAACGCAGCTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCA
GGGGAAAGAGTCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAACAA
CTACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTCA
TCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGTGGC
AGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAGCCTGA
AGATTGTGCAGAGTGTTACTGTCAGCAATATGGTAGCTCACTCCCGCTCA
CTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCA
TCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGC
CTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTAC
AGTGGGAAGGTGGGATAACGCCCTCCAATCGGGTA3'
[0294] Amino acid sequence of light chain variable region and a
portion of constant region encoded by SEQ ID NO:27:
TABLE-US-00054 (SEQ ID NO: 127)
ETQLTQSPGTLSLSPGERVTLSCRASQSVSNNYLAWYQQKPGQAPRLLIY
GASSRATGIPDRFSGSGSGTDFTLTISRLEPEDCAECYCQQYGSSLPLTF
GGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW EGGITPSNRV
Anti-TIM-1 mAb 2.56
[0295] Nucleotide sequence of heavy chain variable region and a
portion of constant region:
TABLE-US-00055 (SEQ ID NO: 29)
5'GTCCAGTGTCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTC
CAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTT
CAGTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGG
AGTGGGTGGCAGTTATATGGTATGATGGAAGTCATAAATACTATGCAGAC
TCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCT
GTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACT
CTGCGAGAGATTACTATGATACGAGTCGGCATCACTGGGGGTTTGACTGC
TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCTGCTTCCACCAAGGGCCC
ATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAG
CCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTG
TCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGC3'
[0296] Amino acid sequence of heavy chain variable region and a
portion of constant region encoded by SEQ ID NO: 29:
TABLE-US-00056 (SEQ ID NO: 128)
VQCQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEW
VAVIWYDGSHKY/LYA/TDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV
YYSARDYYDTSRHHWGFDCWGQGTLVTVSSASTKGPSVFPLAPCSRSTSE
STAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
[0297] Nucleotide sequence of light chain variable region and a
portion of constant region:
TABLE-US-00057 (SEQ ID NO: 31)
5'CAGCTCCTGGGGCTGCTAATGCTCTGGGTCCCTGGATCCAGTGAG
GAAATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGA
GCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCTTGGATAGTGAAG
ATGGAAACACCTATTTGGACTGGTACCTGCAGAAGCCAGGGCAGTCTCCA
CAGCTCCTGATCTATACGCTTTCCCATCGGGCCTCTGGAGTCCCAGACAG
GTTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGG
TGGAGGCTGAGGATGTTGGAGTTTATTGCTGCATGCAACGTGTAGAGTTT
CCTATCACCTTCGGCCAAGGGACACGACTGGAGATTAAACGAACTGTGGC
TGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTG
GAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCC
AAAGTACAGTGGAAGGTGGATAACGC3'
[0298] Amino acid sequence of light chain variable region and a
portion of constant region encoded by SEQ ID NO:31:
TABLE-US-00058 (SEQ ID NO: 129)
QLLGLLMLWVPGSSEEIVMTQTPLSLPVTPGEPASISCRSSQSLLDSEDG
NTYLDWYLQKPGQSPQLLIYTLSHRASGVPDRFSGSGSGTDFTLKISRVE
AEDVGVYCCMQRVEFPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGT
ASVVCLLNNFYPREAKVQWKVDN
Anti-TIM-1 mAb 2.59
[0299] Nucleotide sequence of heavy chain variable region and a
portion of constant region:
TABLE-US-00059 (SEQ ID NO: 33)
5'CAGTCGGGCCCAAGACTGGTGAAGCCTTCACAGACCCTGTCCCTC
ACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTGATGGTTACTACTGGAG
CTGGATCCGCCAGCACCCAGGGAAGGGCCTGGAGTGGATTGGGTACATCT
ATTACAGTGGGAGCACCTTCTACAACCCGTCCCTCAAGAGTCGAGTTGCC
ATATCAGTGGACACGTCTAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGT
GACTGCCGCGGACACGGCCGTGTATTACTGTGCGAGAGAATCCCCTCATA
GCAGCAACTGGTACTCGGGCTTTGACTGCTGGGGCCAGGGAACCCTGGTC
ACCGTCTCCTCAGCTTCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGCC
CTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCA
AGGACTACTTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCT
GACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCT CT3'
[0300] Amino acid sequence of heavy chain variable region and a
portion of constant region encoded by SEQ ID NO:33:
TABLE-US-00060 (SEQ ID NO: 130)
XXXXXQSGPRLVKPSQTLSLTCTVSGGSISSDGYYWSWIRQHPGKGLEWI
GYIYYSGSTFYNPSLKSRVAISVDTSKNQFSLKLSSVTAADTAVYYCARE
SPHSSNWYSGFDCWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALG
CLVKDYFPRTGDGVVELRRPDQRRAHLPGCPTVLRTL
[0301] Nucleotide sequence of light chain variable region and a
portion of constant region:
TABLE-US-00061 (SEQ ID NO: 35)
5'ACTCAGTCTCCAGACTTTCAGTCTGTGACTCCAAAGGAGAAAGTC
ACCATCACCTGCCGGGCCAGTCAGAGCATTGGTAGTAGGTTACACTGGTA
CCAGCAGAAACCAGATCAGTCTCCAAAGCTCCTCATCAAGTATGCTTCCC
AGTCCTTCTCAGGGGTCCCCTCGAGGTTCAGTGGCAGTGGATCTGGGACA
GATTTCACCCTCACCATCAATAGCCTGGAAGCTGAAGATGCTGCAACGTA
TTACTGTCATCAGAGTAGTAATTTACCATTCACTTTCGGCCCTGGGACCA
AAGTGGATATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCG
CCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCT
GAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACG CCCTC3'
[0302] Amino acid sequence of light chain variable region and a
portion of constant region encoded by SEQ ID NO:35:
TABLE-US-00062 (SEQ ID NO: 131)
XXXXTQSPDFQSVTPKEKVTITCRASQSIGSRLHWYQQKPDQSPKLLIKY
ASQSFSGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCHQSSNLPFTFGP
GTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNAL
Anti-TIM-1 mAb 2.61
[0303] Nucleotide sequence of heavy chain variable region and a
portion of constant region:
TABLE-US-00063 (SEQ ID NO: 37)
5'CAGGTGCAGCTGGTGGAGGCTGGGGGAGGCGTGGTCCAGCCTGGG
AGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGAAGCTA
TGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGAAATGGGTGG
CAGTTATATGGTATGATGGAAGTAATAAATACTATACAGACTCCGTGAAG
GGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCA
AATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACTGTGTGAGAG
ATTACTATGATAATAGTAGACATCACTGGGGGTTTGACTACTGGGGCCAG
GGAACCCTGGTCACCGTCTCCTCAGCTTCCACCAAGGGCCCATCCGTCTT
CCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGG
GCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAAC
TCAGGCGCCCTGACCAGGCGGCGTGCACACCTTCCCGGC3'
[0304] Amino acid sequence of heavy chain variable region and a
portion of constant region encoded by SEQ ID NO:37:
TABLE-US-00064 (SEQ ID NO: 132)
QVQLVE/QAGGGVVQPGRSLRLSCAASGFTFRSYGMHWVRQAPGKGLKWV
AVIWYDGSNKY/LYTDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC
VRDYYDNSRHHWGFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTA
ALGCLVKDYFPEPVTVSWNSGALTRRRAHLPG
[0305] Nucleotide sequence of light chain variable region and a
portion of constant region:
TABLE-US-00065 (SEQ ID NO: 39)
5'GACATCCAGATGACCCAGTCTCCATCCTCCCGGTGTGCATCCGTAGG
AGACAGAGTCACCATCACTTGCCGGGCAAGTCAGGGCATCAGAAATGATT
TAGCTTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCGCCTGATCTAT
GCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTAG
ATCTGGGACAGAATTCACTCTCACAATCAGCAGCCTGCAGCCTGAAGATT
TTGCAGCTTATTACTGTCTCCAGCATAATAGTTACCCTCCCAGTTTTGGC
CAGGGGACCAAGCTGGAGATCAAACGAACTGTGGCTGCACCATCTGTCT
TCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCTAGCGTT
GTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGA
AGGTGGATAACGCCCTCCAATCGGG3'
[0306] Amino acid sequence of light chain variable region and a
portion of constant region encoded by SEQ ID NO:39:
TABLE-US-00066 (SEQ ID NO: 133)
DIQMTQSPSSRCASVGDRVTITCRASQGIRNDLAWYQQKPGKAPKRLIYA
ASSLQSGVPSRFSGSRSGTEFTLTISSLQPEDFAAYYCLQHNSYPPSFGQ
GTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQS
Anti-TIM-1 mAb 2.70
[0307] Nucleotide sequence of heavy chain variable region and a
portion of constant region:
TABLE-US-00067 (SEQ ID NO: 41)
5'CATGTGCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCT
GGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCATCTTCAGT
CGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGAAA
TGGGTGGCAGTTATATGGTATGATGGAAGTAATAAACTCTATGCAGACTC
CGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTG
TATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGTGTATTACT
GTGCGAGAGATTACTATGATAATAGTAGACATCACTGGGGGTTTGACTAC
TGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTTCCACCAAGGGC
CCATCCGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGC
ACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTG
ACGGTGTCGTGGAACTCAGGCGCCCTGA3'
[0308] Amino acid sequence of heavy chain variable region and a
portion of constant region encoded by SEQ ID NO:41:
TABLE-US-00068 (SEQ ID NO: 134)
HVQVQLVESGGGVVQPGRSLRLSCAASGFIFSRYGMHWVRQAPGKGLKWV
AVIWYDGSNKLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR
DYYDNSRHHWGFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAAL
GCLVKDYFPEPVTVSWNSGAL
[0309] Nucleotide sequence of light chain variable region and a
portion of constant region:
TABLE-US-00069 (SEQ ID NO: 43)
5'TCAGCTCCTGGGGCTGCTAATGCTCTGGGTCCCTGGATCAGTGAGG
ATATTGTGATGACCCAGACTCCACTCTCCCTGCCCGTCACCCCTGGAGAG
CCGGCCTCCATCTCCTGCAGGTCTAGTCGGAGCCTCTTGGATAGTGATGA
TGGAAACACCTATTTGGACTGGTACCTGCAGAAGCCAGGGCAGTCTCCAC
AGCTCCTGATCTACACGCTTTCCTATCGGGCCTCTGGAGTCCCAGACAGG
TTCAGTGGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGT
GGAGGCTGAGGATGTTGGAGTTTATTACTGCATGCAACGTGTAGAGTTTC
CTATCACCTTCGGCCAAGGGACACGACTGGAGATTAAACGAACTGTGGC
TGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTG
GAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCC
AAAGTACAGTGGAAGGTGGATAACGCCT3'
[0310] Amino acid sequence of light chain variable region and a
portion of constant region encoded by SEQ ID NO:43:
TABLE-US-00070 (SEQ ID NO: 135)
SAPGAANALGPWISEDIVMTQTPLSLPVTPGEPASISCRSSRSLLDSDDG
NTYLDWYLQKPGQSPQLLIYTLSYRASGVPDRFSGSGSGTDFTLKISRVE
AEDVGVYYCMQRVEFPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGT
ASVVCLLNNFYPREAKVQWKVDNA
Anti-TIM-1 mAb 2.70.2
[0311] Nucleotide sequence of heavy chain variable region and a
portion of constant region:
TABLE-US-00071 (SEQ ID NO: 49)
5'CGGCCGCCTATTTACCCAGAGACAGGGAGAGGCTCTTCTGTGTGTA
GTGGTTGTGCAGAGCCTCATGCATCACGGAGCATGAGAAGACATTCCCC
TCCTGCCACCTGCTCTTGTCCACGGTTAGCCTGCTGTAGAGGAAGAAGG
AGCCGTCGGAGTCCAGCACGGGAGGCGTGGTCTTGTAGTTGTTCTCCGG
CTGCCCATTGCTCTCCCACTCCACGGCGATGTCGCTGGGGTAGAAGCCT
TTGACCAGGCAGGTCAGGCTGACCTGGTTCTTGGTCATCTCCTCCTGGGA
TGGGGGCAGGGTGTACACCTGTGGCTCTCGGGGCTGCCCTTTGGCTTTG
GAGATGGTTTTCTCGATGGAGGACGGGAGGCCTTTGTTGGAGACCTTGCA
CTTGTACTCCTTGCCGTTCAGCCAGTCCTGGTGCAGGACGGTGAGGACG
CTGACCACACGGTACGTGCTGTTGAACTGCTCCTCCCGCGGCTTTGTCTT
GGCATTATGCACCTCCACGCCATCCACGTACCAGTTGAACTGGACCTCGG
GGTCTTCCTGGCTCACGTCCACCACCACGCACGTGACCTCAGGGGTCCG
GGAGATCATGAGAGTGTCCTTGGGTTTTGGGGGGAACAGGAAGACTGAT
GGTCCCCCCAGGAACTCAGGTGCTGGGCATGATGGGCATGGGGGACCAT
ATTTGGACTCAACTCTCTTGTCCACCTTGGTGTTGCTGGGCTTGTGATCT
ACGTTGCAGGTGTAGGTCTTCGTGCCCAAGCTGCTGGAGGGCACGGTC
ACCACGCTGCTGAGGGAGTAGAGTCCTGAGGACTGTAGGACAGCCGGG
AAGGTGTGCACGCCGCTGGTCAGGGCGCCTGAGTTCCACGACACCGTC
ACCGGTTCGGGGAAGTAGTCCTTGACCAGGCAGCCCAGGGCGGCTGTG
CTCTCGGAGGTGCTCCTGGAGCAGGGCGCCAGGGGGAAGACGGATGG
GCCCTTGGTGGAAGCTGAGGAGACGGTGACCAGGGTTCCCTGGCCCCA
GTAGTCAAACCCCCAGTGATGTCTACTATTATCATAGTAATCTCTCGCAC
AGTAATACACAGCCGTGTCCTCGGCTCTCAGGCTGTTCATTTGCAGATAC
AGCGTGTTCTTGGAATTGTCTCTGGAGATGGTGAATCGGCCCTTCACGG
AGTCTGCATAGAGTTTATTACTTCCATCATACCATATAACTGCCACCCAT
TTCAGCCCCTTGCCTGGAGCCTGGCGGACCCAGTGCATGCCATAGCG
ACTGAAGATGAATCCAGACGCTGCACAGGAGAGTCTCAGGGACCTCCC
AGGCTGGACCACGCCTCCCCCAGACTCCACCAGCTGCACCTGACACT
GGACACCTTTTAAAATAGCCACAAGAAAAAGCCAGCTCAGCCCAAACTC
CATGGTGGTCGACT3'
[0312] Amino acid sequence of heavy chain variable region and a
portion of constant region encoded by SEQ ID NO:49:
TABLE-US-00072 (SEQ ID NO: 50)
MEFGLSWLFLVAILKGVQCQVQLVESGGGVVQPGRSLRLSCAASGFIFSR
YGMHWVRQAPGKGLKWVAVIWYDGSNKLYADSVKGRFTISRDNSKNTLYL
QMNSLRAEDTAVYYCARDYYDNSRHHWGFDYWGQGTLVTVSSASTKGPSV
FPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPS
CPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY
VDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGL
PSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVM
HEALHNHYTQKSLSLSLGK
[0313] Nucleotide sequence of light chain variable region and a
portion of constant region:
TABLE-US-00073 (SEQ ID NO: 51)
5'AGTCGACCACCATGGAAACCCCAGCGCAGCTTCTCTTCCTCCTGCTA
CTCTGGCTCCCAGATACCACCGGAGATATTGTGATGACCCAGACTCCACT
CTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCT
AGTCGGAGCCTCTTGGATAGTGATGATGGAAACACCTATTTGGACTGGTA
CCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATCTACACGCTTTCCT
ATCGGGCCTCTGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGCAC
TGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTT
ATTACTGCATGCAACGTGTAGAGTTTCCTATCACCTTCGGCCAAGGGACA
CGACTGGAGATTAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCC
GCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGC
TGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAAC
GCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCA
AGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGA
CTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGA
GCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAGGCGGCCG3'
[0314] Amino acid sequence of light chain variable region and
portion constant region by SEQ ID NO:51:
TABLE-US-00074 (SEQ ID NO: 52)
METPAQLLFLLLLWLPDTTGDIVMTQTPLSLPVTPGEPASISCRSSRSLL
DSDDGNTYLDWYLQKPGQSPQLLIYTLSYRASGVPDRFSGSGSGTDFTLK
ISRVEAEDVGVYYCMQRVEFPITFGQGTRLEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS
LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Anti-TIM-1 mAb 2.76
[0315] Nucleotide sequence of heavy chain variable region and a
portion of constant region:
TABLE-US-00075 (SEQ ID NO: 45)
5'GAGCAGTCGGGGGGCGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTC
TCCTGTGCAGCGTCTGGATTCACCTTCAGTAGCTATGGCATGTACTGGGT
CCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTATATGGTAT
GATGGAAGCAATAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCAT
CTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGA
GAGCCGAGGACACGGCTGTGTATTACTGTGCGAGGGATTTCTATGATAGT
AGTCGTTACCACTACGGTATGGACGTCTGGGGCCAAGGGACCACGGTCA
CCGTCTCCTCAGCTTCCACCAAGGGCCCATCCGTCTTCCCCCTGGCGC
CCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGG
TCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGC
CCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGA CTCTCT3'
[0316] Amino acid sequence of heavy chain variable region and a
portion of constant region encoded by SEQ ID NO:45:
TABLE-US-00076 (SEQ ID NO: 136)
XXXXEQSGGGVVQPGRSLRLSCAASGFTFSSYGMYWVRQAPGKGLEWVAV
IWYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDF
YDSSRYHYGMDVWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGC
LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLS
[0317] Nucleotide sequence of light chain variable region and a
portion of constant region:
TABLE-US-00077 (SEQ ID NO: 47)
5'ACTCAGTGTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCC
ATCTCCTGCAGGTCTAGTCAGAGCCTCTTGGATAGTGATGATGGAAACA
CCTATTTGGACTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCT
GATCTATACGGTTTCCTATCGGGCCTCTGGAGTCCCAGACAGGTTCAGT
GGCAGTGGGTCAGGCACTGATTTCACACTGAAAATCAGCAGGGTGGAG
GCTGAGGATGTTGGAGTTTATTACTGCATGCAACGTATAGAGTTTCCGA
TCACCTTCGGCCAAGGGACCCGACTGGAGATTAAACGAACTGTGGCTG
CACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGG
AACTGCCTCTGTTGTGTGCCTGCTGAATAA3'
[0318] Amino acid sequence of light chain variable region and a
portion of constant region encoded by SEQ ID NO:47:
TABLE-US-00078 (SEQ ID NO: 137)
XXXXTQCPLSLPVTPGEPASISCRSSQSLLDSDDGNTYLDWYLQKPGQSP
QLLIYTVSYRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQRIEF
PITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN
Incorporation by Reference
[0319] All references cited herein, including patents, patent
applications, papers, text books, and the like, and the references
cited therein, to the extent that they are not already, are hereby
incorporated herein by reference in their entirety. In addition,
the following references are also incorporated by reference herein
in their entirety, including the references cited in such
references:
Equivalents
[0320] While the preferred embodiment of the invention has been
illustrated and described, it is to be understood that this
invention is capable of variation and modification by those skilled
in the art to which it pertains, and is therefore not limited to
the precise terms set forth, but also such changes and alterations
which may be made for adapting the invention to various usages and
conditions. Accordingly, such changes and alterations are properly
intended to be within the full range of equivalents, and therefore
within the purview of the following claims.
[0321] The invention and the manner and a process of making and
using it has been described in such full, clear, concise and exact
terms so as to enable any person skilled in the art to which it
pertains, or with which it is most nearly connected, to make and
use the same.
Sequence CWU 1
1
1991509DNAHomo sapiens 1tgggtcctgt cccaggtgca gctgcaggag tcgggcccag
gactggtgaa gccttcggag 60accctgtccc tcacctgcac tgtctctggt ggctccgtca
gcagtggtgg ttactactgg 120agctggatcc ggcagccccc agggaaggga
ctggagtgga ttgggtttat ctattacact 180gggagcacca actacaaccc
ctccctcaag agtcgagtct ccatatcagt agacacgtcc 240aagaaccagt
tctccctgaa gctgagctct gtgaccgctg cggacgcggc cgtgtattac
300tgtgcgagag attatgactg gagcttccac tttgactact ggggccaggg
aaccctggtc 360accgtctcct cagcctccac caagggccca tcggtcttcc
ccctggcgcc ctgctccagg 420agcacctccg agagcacagc ggccctgggc
tgcctggtca aggactactt ccccgaaccg 480gtgacggtgt cgtggaactc aggcgctct
5092121PRTHomo sapiens 2Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser
Gly Gly Ser Val Ser Ser Gly 20 25 30 Gly Tyr Tyr Trp Ser Trp Ile
Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Phe Ile
Tyr Tyr Thr Gly Ser Thr Asn Tyr Asn Pro Ser 50 55 60 Leu Lys Ser
Arg Val Ser Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser
Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Ala Ala Val Tyr Tyr 85 90
95 Cys Ala Arg Asp Tyr Asp Trp Ser Phe His Phe Asp Tyr Trp Gly Gln
100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala 115 120 3504DNAHomo
sapiens 3cagctcctgg ggctcctgct gctctggttc ccaggtgcca ggtgtgacat
ccagatgacc 60cagtctccat cctccctgtc tgcatctata ggagacagag tcaccatcac
ttgccgggca 120agtcagggca ttagaaatga tttaggctgg tatcagcaga
aaccagggaa agcccctaag 180cgcctgatct atgctgcatc cagtttgcaa
agtggggtcc catcaaggtt cagcggcagt 240ggatctggga cagaattcac
tctcacaatc agcagcctgc agcctgaaga ttttgcaact 300tattactgtc
tacagcataa tagttaccct ctcactttcg gcggagggac caaggtggag
360atcaaacgaa ctgtggctgc accatctgtc ttcatcttcc cgccatctga
tgagcagttg 420aaatctggaa ctgcctctgt tgtgtgcctg ctgaataact
tctatcccag agaggccaaa 480gtacagtgga aggtggataa cgcc 5044108PRTHomo
sapiens 4Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Ile Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly
Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Arg 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 Glu
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala
Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Leu 85 90 95 Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 5 469DNAHomo
sapiens 5cagtgtgagg tgcagctggt ggagtctggg ggaggcttgg tccagcctgg
ggggtccctg 60agactctcct gtgcagcctc tggattcacc tttactaact attggatgag
ctgggtccgc 120caggctccag ggaaggggct ggagtgggtg gccaacatac
agcaagatgg aagtgagaaa 180tactatgtgg actctgtgag gggccgattc
accatctcca gagacaacgc caagaactca 240ctgtatctgc aaatgaacag
cctgagagcc gaggactcgg ctgtgtatta ctgtgcgaga 300tgggactact
ggggccaggg aaccctggtc accgtctcct cagcctccac caagggccca
360tcggtcttcc ccctggcgcc ctgctccagg agcacctccg agagcacagc
ggccctgggc 420tgcctggtca aggactactt ccccgaaccg gtgagcggtg tcgtggaac
4696113PRTHomo sapiens 6Glu 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 Thr Asn Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn Ile Gln Gln
Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60 Arg 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 Ser Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Trp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
100 105 110 Ala 7454DNAHomo sapiens 7cttctggggc tgctaatgct
ctgggtccct ggatccagtg gggatattgt gatgacccag 60actccactct cctcaactgt
catccttgga cagccggcct ccatctcctg caggtctagt 120caaagcctcg
tacacagtga tggaaacacc tacttgaatt ggcttcagca gaggccaggc
180cagcctccaa gactcctaat ttatatgatt tctaaccggt tctctggggt
cccagacaga 240ttcagtggca gtggggcagg gacagatttc acactgaaaa
tcagcagggt ggaagctgag 300gatgtcgggg tttattactg catgcaagct
acagaatctc ctcagacgtt cggccaaggg 360accaaggtgg aaatcaaacg
aactgtggct gcaccatctg tcttcatctt cccgccatct 420gatgagcagt
tgaaatctgg aagggcctct gttg 4548113PRTHomo sapiens 8Asp Ile Val Met
Thr Gln Thr Pro Leu Ser Ser Thr Val Ile Leu Gly 1 5 10 15 Gln Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30
Asp Gly Asn Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Pro 35
40 45 Pro Arg Leu Leu Ile Tyr Met Ile Ser Asn Arg Phe Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Met Gln Ala 85 90 95 Thr Glu Ser Pro Gln Thr Phe Gly Gln
Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg 9529DNAHomo sapiens
9gagcagtcgg ggggaggcgt ggtaaagcct ggggggtctc ttagactctc ctgtgcagcc
60tctggattca ctttcagtaa cgcctggatg acctgggtcc gccaggctcc agggaagggg
120ctggagtggg ttggccgtat taaaaggaga actgatggtg ggacaacaga
ctacgctgca 180cccgtgaaag gcagattcac catctcaaga gatgattcaa
aaaacacgct gtatctgcaa 240atgaacaacc tgaaaaacga ggacacagcc
gtgtattact gtacctcagt cgataatgac 300gtggactact ggggccaggg
aaccctggtc accgtctcct cagcttccac caagggccca 360tccgtcttcc
ccctggcgcc ctgctccagg agcacctccg agagcacagc cgccctgggc
420tgcctggtca aggactactt ccccgaaccg gtgacggtgt cgtggaactc
aggcgccctg 480accagcggcg tgcacacctt cccggctgtc ctacagtcct caggactct
52910119PRTHomo sapiensMISC_FEATURE(1)..(4)Xaa is any amino acid
10Xaa Xaa Xaa Xaa Glu Gln Ser Gly Gly Gly Val Val Lys Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn
Ala 20 25 30 Trp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Gly Arg Ile Lys Arg Arg Thr Asp Gly Gly Thr
Thr Asp Tyr Ala Ala 50 55 60 Pro Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Asn
Leu Lys Asn Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Ser Val
Asp Asn Asp Val Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr
Val Ser Ser Ala 115 11447DNAHomo sapiens 11ctgactcagt ctccactctc
cctgcccgtc acccctggag agccggcctc catctcctgc 60aggtctagtc agagcctcct
gcatagtaat ggatacaact atttggattg gtacctgcag 120aagccagggc
agtctccaca gctcctgatc tatttgggtt ctaatcgggc ctccggggtc
180cctgacaggt tcagtggcag tggatcaggc acagatttta cactgaaaat
cagcagagtg 240gaggctgagg atattggtct ttattactgc atgcaagctc
tacaaactcc gctcactttc 300ggcggaggga ccaaggtgga catcaaacga
actgtggctg caccatctgt cttcatcttc 360ccgccatctg atgagcagtt
gaaatctgga actgcctctg ttgtgtgcct gctgaataac 420ttctatccca
gagaggccaa agtacag 44712113PRTHomo sapiensMISC_FEATURE(1)..(3)Xaa
is any amino acid 12Xaa Xaa Xaa Leu Thr Gln Ser Pro Leu Ser Leu Pro
Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser
Gln Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp
Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr
Leu Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg
Val Glu Ala Glu Asp Ile Gly Leu Tyr Tyr Cys Met Gln Ala 85 90 95
Leu Gln Thr Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys 100
105 110 Arg 13538DNAHomo sapiens 13caggtgcagc tggagcagtc ggggggaggc
ttggtacagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt caccttcagt
acctatagca tgaactgggt ccgccaggct 120ccagggaagg ggctggagtg
ggtttcatac attagaagta gtactagtac catatactat 180gcagagtccc
tgaagggccg attcaccatc tccagcgaca atgccaagaa ttcactatat
240ctgcaaatga acagcctgag agacgaggac acggctgtgt attactgtgc
gcgggacttt 300gactactggg gccagggaac cctggtcacc gtctcctcag
cttccaccaa gggcccatcc 360gtcttccccc tggcgccctg ctccaggagc
acctccgaga gcacagccgc cctgggctgc 420ctggtcaagg actacttccc
cgaaccggtg acggtgtcgt ggaactcagg cgccctgacc 480agcggcgtgc
acaccttccc ggctgtccta cagtcctcag gactctactc cctcagca
53814114PRTHomo sapiens 14Gln Val Gln Leu Glu Gln 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 Thr 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 Arg
Ser Ser Thr Ser Thr Ile Tyr Tyr Ala Glu Ser Leu 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Ser Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Ala Arg Asp Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Ser 100 105 110 Ser Ala 15490DNAHomo sapiens 15gaaatccagc
tgactcagtc tccactctcc tcacctgtca cccttggaca gccggcctcc 60atctcctgca
ggtctagtca aagcctcgta cacagtgatg gagacaccta cttgaattgg
120cttcagcaga ggccaggcca gcctccaaga ctcctaattt ataagatttc
tacccggttc 180tctggggtcc ctgacagatt cagtggcagt ggggcaggga
cagatttcac actgaaaatc 240agcagggtgg agactgacga tgtcgggatt
tattactgca tgcaaactac acaaattcct 300caaatcacct tcggccaagg
gacacgactg gagattaaac gaactgtggc tgcaccatct 360gtcttcatct
tcccgccatc tgatgagcag ttgaaatctg gaactgcctc tgttgtgtgc
420ctgctgaata acttctatcc cagagaggcc aaagtacagt ggaaggtgga
taacgccctc 480caatcgggta 49016114PRTHomo sapiens 16Glu Ile Gln Leu
Thr Gln Ser Pro Leu Ser Ser Pro Val Thr Leu Gly 1 5 10 15 Gln Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30
Asp Gly Asp Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Pro 35
40 45 Pro Arg Leu Leu Ile Tyr Lys Ile Ser Thr Arg Phe Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Thr Asp Asp Val Gly Ile Tyr
Tyr Cys Met Gln Thr 85 90 95 Thr Gln Ile Pro Gln Ile Thr Phe Gly
Gln Gly Thr Arg Leu Glu Ile 100 105 110 Lys Arg 17568DNAHomo
sapiens 17caggtgcagc tggagcagtc ggggggaggc gtggtccagc ctgggaggtc
cctgagactc 60tcctgtgcag cgtctggatt caccttcagt cgctatggca tgcactgggt
ccgccaggct 120ccaggcaagg ggctgaaatg ggtggcagtt atatggtatg
atggaagtaa taaactctat 180gcagactccg tgaagggccg attcaccatc
tccagagaca attccaagaa cacgctgtat 240ctgcaaatga acagcctgag
agccgaggac acggctgtgt attactgtgc gagagattac 300tatgataata
gtagacatca ctgggggttt gactactggg gccagggaac cctggtcacc
360gtctcctcag cttccaccaa gggcccatcc gtcttccccc tggcgccctg
ctccaggagc 420acctccgaga gcacagccgc cctgggctgc ctggtcaagg
actacttccc cgaaccggtg 480acggtgtcgt ggaactcagg cgccctgacc
agcggcgtgc acaccttccc ggctgtccta 540cagtcctcag gactctactc cctcagca
56818124PRTHomo sapiens 18Gln Val Gln Leu Glu Gln 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 Arg Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Lys Trp Val 35 40 45 Ala Val Ile Trp
Tyr Asp Gly Ser Asn Lys Leu 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 Tyr Tyr Asp Asn Ser Arg His His Trp Gly Phe Asp
Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 115
120 19472DNAHomo sapiens 19gacatccagc tgacccagtc tccatcctcc
ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gagtatttat
agttatttaa attggtatca gcagaaacca 120gggaaagccc ctaagctcct
gatctatgct gcatccagtt tgcaaagtgg ggtcccatcc 180aggttcagtg
gcagtggatc tgggacagat ttcactctca ccatcagcag tctgcaacct
240gaagattttg caacttacta ctgtcaacag agttacagta cccctccgac
gttcggccaa 300gggaccaagg tggaaatcaa acgaactgtg gctgcaccat
ctgtcttcat cttcccgcca 360tctgatgagc agttgaaatc tggaactgcc
tctgttgtgt gcctgctgaa taacttctat 420cccagagagg ccaaagtaca
gtggaaggtg gataacgccc tccaatcggg ta 47220108PRTHomo sapiens 20Asp
Ile Gln Leu 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 Tyr 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 Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg 100 105 21528DNAHomo sapiens 21cagtcggggg
gaggcttggt aaagcctggg gggtccctta gactctcctg tgcagcctct 60ggattcactt
tcagtaacgc ctggatgacc tgggtccgcc aggctccagg gaaggggctg
120gagtgggttg gccgtattaa aaggaaaact gatggtggga caacagacta
cgctgcaccc 180gtgaaaggca gattcaccat ctcaagagat gattcagaaa
acacgctgta tctgcaaatg 240aacagcctgg aaaccgagga cacagccgtg
tattactgta ccacagtcga taacagtggt 300gactactggg gccagggaac
cctggtcacc gtctcctcag cttccaccaa gggcccatcc 360gtcttccccc
tggcgccctg ctccaggagc acctccgaga gcacagccgc cctgggctgc
420ctggtcaagg actacttccc cgaaccggtg acggtgtcgt ggaactcagg
cgccctgacc 480agcggcgtgc acaccttccc ggctgtccta cagtcctcag gactctct
52822119PRTHomo sapiensMISC_FEATURE(1)..(5)Xaa is any amino acid
22Xaa Xaa Xaa Xaa Xaa Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn
Ala 20 25 30 Trp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Gly Arg Ile Lys Arg Lys Thr Asp Gly Gly Thr
Thr Asp Tyr Ala Ala 50 55 60 Pro Val Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asp Ser Glu Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser
Leu Glu Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Thr Val
Asp Asn Ser Gly Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr
Val Ser Ser Ala 115 23466DNAHomo sapiens 23actcagtctc cactctccct
gcccgtcacc cctggagagc cggcctccat ctcctgcagg 60tctagtcaga gcctcctgca
tagtaatgga tacaactatt tggattggta cctgcagaag 120ccagggcagt
ctccacagct cctgatctat ttgggttcta atcgggcctc cggggtccct
180gacaggttca gtggcagtgg atcaggcaca gattttacac tgaaaatcag
cagagtggag 240gctgaggatg ttggggttta ttactgcatg caagctctac
aaactccgct cactttcggc 300ggagggacca aggtggagat caaacgaact
gtggctgcac catctgtctt catcttcccg 360ccatctgatg agcagttgaa
atctggaact gcctctgttg tgtgcctgct gaataacttc 420tatcccagag
aggccaaagt
acagtggaag gtggataacg ccctca 46624113PRTHomo
sapiensMISC_FEATURE(1)..(4)Xaa is any amino acid 24Xaa Xaa Xaa Xaa
Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30
Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro Leu Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg 25537DNAHomo sapiens
25caggtgcagc tggagcagtc ggggggaggc gtggtccagc ctgggaggtc cctgagactc
60tcctgtgcag cgtctggatt caccttcact aactatggct tgcactgggt ccgccaggct
120ccaggcaagg ggctggattg ggtggcagtt atatggtatg atggaagtca
taaattctat 180gcagactccg tgaagggccg attcaccatc tccagagaca
attccaagaa cacgctcttt 240ctgcaaatga acagcctgag agccgaggac
acggctgtgt attactgtac gcgagatctt 300gactactggg gccagggaac
cctggtcacc gtctcctcag cttccaccaa gggcccatcc 360gtcttccccc
tggcgccctg ctccaggagc acctccgaga gcacagccgc cctgggctgc
420ctggtcaagg actacttccc cgaaccggtg acggtgtcgt ggaactcagg
cgccctgacc 480agcggcgtgc acaccttccc ggctgtccta cagtcctcag
gactctactc cctcagc 53726114PRTHomo sapiens 26Gln Val Gln Leu Glu
Gln 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 Thr Asn Tyr 20 25 30 Gly
Leu His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val 35 40
45 Ala Val Ile Trp Tyr Asp Gly Ser His Lys Phe Tyr Ala Asp Ser Val
50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Thr Arg Asp Leu Asp Tyr Trp Gly Gln Gly
Thr Leu Val Thr Val Ser 100 105 110 Ser Ala 27480DNAHomo sapiens
27gaaacgcagc tgacgcagtc tccaggcacc ctgtctttgt ctccagggga aagagtcacc
60ctctcctgca gggccagtca gagtgttagc aacaactact tagcctggta ccagcagaaa
120cctggccagg ctcccaggct cctcatctat ggtgcatcca gcagggccac
tggcatccca 180gacaggttca gtggcagtgg gtctgggaca gacttcactc
tcaccatcag cagactggag 240cctgaagatt gtgcagagtg ttactgtcag
caatatggta gctcactccc gctcactttc 300ggcggaggga ccaaggtgga
gatcaaacga actgtggctg caccatctgt cttcatcttc 360ccgccatctg
atgagcagtt gaaatctgga actgcctctg ttgtgtgcct gctgaataac
420ttctatccca gagaggccaa agtacagtgg gaaggtggga taacgccctc
caatcgggta 48028110PRTHomo sapiens 28Glu Thr Gln Leu Thr Gln Ser
Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Val Thr Leu
Ser Cys Arg Ala Ser Gln Ser Val Ser Asn Asn 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 Cys Ala Glu Cys Tyr Cys Gln Gln Tyr Gly Ser
Ser Leu 85 90 95 Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile
Lys Arg 100 105 110 29542DNAHomo sapiens 29gtccagtgtc aggtgcagct
ggtggagtct gggggaggcg tggtccagcc tgggaggtcc 60ctgagactct cctgtgcagc
gtctggattc accttcagta gctatggcat gcactgggtc 120cgccaggctc
caggcaaggg gctggagtgg gtggcagtta tatggtatga tggaagtcat
180aaatactatg cagactccgt gaagggccga ttcaccatct ccagagacaa
ttccaagaac 240acgctgtatc tgcaaatgaa cagcctgaga gccgaggaca
cggctgtgta ttactctgcg 300agagattact atgatacgag tcggcatcac
tgggggtttg actgctgggg ccagggaacc 360ctggtcaccg tctcctctgc
ttccaccaag ggcccatccg tcttccccct ggcgccctgc 420tccaggagca
cctccgagag cacagccgcc ctgggctgcc tggtcaagga ctacttcccc
480gaaccggtga cggtgtcgtg gaactcaggc gccctgacca gcggcgtgca
caccttcccg 540gc 54230124PRTHomo sapiens 30Gln 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 Gly Met
His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser His 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 Ser 85 90 95 Ala Arg Asp Tyr Tyr Asp Thr Ser Arg His His
Trp Gly Phe Asp Cys 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val
Ser Ser Ala 115 120 31521DNAHomo sapiens 31cagctcctgg ggctgctaat
gctctgggtc cctggatcca gtgaggaaat tgtgatgacc 60cagactccac tctccctgcc
cgtcacccct ggagagccgg cctccatctc ctgcaggtct 120agtcagagcc
tcttggatag tgaagatgga aacacctatt tggactggta cctgcagaag
180ccagggcagt ctccacagct cctgatctat acgctttccc atcgggcctc
tggagtccca 240gacaggttca gtggcagtgg gtcaggcact gatttcacac
tgaaaatcag cagggtggag 300gctgaggatg ttggagttta ttgctgcatg
caacgtgtag agtttcctat caccttcggc 360caagggacac gactggagat
taaacgaact gtggctgcac catctgtctt catcttcccg 420ccatctgatg
agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc
480tatcccagag aggccaaagt acagtggaag gtggataacg c 52132114PRTHomo
sapiens 32Glu Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr
Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser
Leu Leu Asp Ser 20 25 30 Glu Asp Gly Asn Thr Tyr Leu Asp Trp Tyr
Leu Gln Lys Pro Gly Gln 35 40 45 Ser Pro Gln Leu Leu Ile Tyr Thr
Leu Ser His Arg Ala Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys 65 70 75 80 Ile Ser Arg Val
Glu Ala Glu Asp Val Gly Val Tyr Cys Cys Met Gln 85 90 95 Arg Val
Glu Phe Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile 100 105 110
Lys Arg 33547DNAHomo sapiens 33cagtcgggcc caagactggt gaagccttca
cagaccctgt ccctcacctg cactgtctct 60ggtggctcca tcagtagtga tggttactac
tggagctgga tccgccagca cccagggaag 120ggcctggagt ggattgggta
catctattac agtgggagca ccttctacaa cccgtccctc 180aagagtcgag
ttgccatatc agtggacacg tctaagaacc agttctccct gaagctgagc
240tctgtgactg ccgcggacac ggccgtgtat tactgtgcga gagaatcccc
tcatagcagc 300aactggtact cgggctttga ctgctggggc cagggaaccc
tggtcaccgt ctcctcagct 360tccaccaagg gcccatccgt cttccccctg
gcgccctgct ccaggagcac ctccgagagc 420acagccgccc tgggctgcct
ggtcaaggac tactttcccc gaaccggtga cggtgtcgtg 480gaactcaggc
gccctgacca gcggcgtgca caccttcccg gctgtcctac agtcctcagg 540actctct
54734125PRTHomo sapiensMISC_FEATURE(1)..(5)Xaa is any amino acid
34Xaa Xaa Xaa Xaa Xaa Gln Ser Gly Pro Arg Leu Val Lys Pro Ser Gln 1
5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser
Asp 20 25 30 Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys
Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr
Phe Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Ala Ile Ser Val
Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val
Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Glu Ser
Pro His Ser Ser Asn Trp Tyr Ser Gly Phe Asp 100 105 110 Cys Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser Ala 115 120 125 35450DNAHomo
sapiens 35actcagtctc cagactttca gtctgtgact ccaaaggaga aagtcaccat
cacctgccgg 60gccagtcaga gcattggtag taggttacac tggtaccagc agaaaccaga
tcagtctcca 120aagctcctca tcaagtatgc ttcccagtcc ttctcagggg
tcccctcgag gttcagtggc 180agtggatctg ggacagattt caccctcacc
atcaatagcc tggaagctga agatgctgca 240acgtattact gtcatcagag
tagtaattta ccattcactt tcggccctgg gaccaaagtg 300gatatcaaac
gaactgtggc tgcaccatct gtcttcatct tcccgccatc tgatgagcag
360ttgaaatctg gaactgcctc tgttgtgtgc ctgctgaata acttctatcc
cagagaggcc 420aaagtacagt ggaaggtgga taacgccctc 45036108PRTHomo
sapiensMISC_FEATURE(1)..(4)Xaa is any amino acid 36Xaa Xaa Xaa Xaa
Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys
Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Arg 20 25 30
Leu His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile 35
40 45 Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser
Leu Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Ser
Ser Asn Leu Pro Phe 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp
Ile Lys Arg 100 105 37534DNAHomo sapiens 37caggtgcagc tggtggaggc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60tcctgtgcag cgtctggatt
caccttcaga agctatggca tgcactgggt ccgccaggct 120ccaggcaagg
ggctgaaatg ggtggcagtt atatggtatg atggaagtaa taaatactat
180acagactccg tgaagggccg attcaccatc tccagagaca attccaagaa
cacgctgtat 240ctgcaaatga acagcctgag agccgaggac acggctgtgt
attactgtgt gagagattac 300tatgataata gtagacatca ctgggggttt
gactactggg gccagggaac cctggtcacc 360gtctcctcag cttccaccaa
gggcccatcc gtcttccccc tggcgccctg ctccaggagc 420acctccgaga
gcacagccgc cctgggctgc ctggtcaagg actacttccc cgaaccggtg
480acggtgtcgt ggaactcagg cgccctgacc aggcggcgtg cacaccttcc cggc
53438124PRTHomo sapiens 38Gln Val Gln Leu Val Glu Ala 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 Arg Ser Tyr 20 25 30 Gly Met His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Lys Trp Val 35 40 45 Ala Val Ile Trp
Tyr Asp Gly Ser Asn Lys Tyr Tyr Thr 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 Val Arg Asp Tyr Tyr Asp Asn Ser Arg His His Trp Gly Phe Asp
Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 115
120 39470DNAHomo sapiens 39gacatccaga tgacccagtc tccatcctcc
cggtgtgcat ccgtaggaga cagagtcacc 60atcacttgcc gggcaagtca gggcatcaga
aatgatttag cttggtatca gcagaaacca 120gggaaagccc ctaagcgcct
gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180aggttcagcg
gcagtagatc tgggacagaa ttcactctca caatcagcag cctgcagcct
240gaagattttg cagcttatta ctgtctccag cataatagtt accctcccag
ttttggccag 300gggaccaagc tggagatcaa acgaactgtg gctgcaccat
ctgtcttcat cttcccgcca 360tctgatgagc agttgaaatc tggaactgct
agcgttgtgt gcctgctgaa taacttctat 420cccagagagg ccaaagtaca
gtggaaggtg gataacgccc tccaatcggg 47040108PRTHomo sapiens 40Asp Ile
Gln Met Thr Gln Ser Pro Ser Ser Arg Cys Ala Ser Val Gly 1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20
25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg 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 Arg Ser Gly Thr Glu Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Ala Tyr Tyr Cys Leu
Gln His Asn Ser Tyr Pro Pro 85 90 95 Ser Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys Arg 100 105 41514DNAHomo sapiens 41catgtgcagg
tgcagctggt ggagtctggg ggaggcgtgg tccagcctgg gaggtccctg 60agactctcct
gtgcagcgtc tggattcatc ttcagtcgct atggcatgca ctgggtccgc
120caggctccag gcaaggggct gaaatgggtg gcagttatat ggtatgatgg
aagtaataaa 180ctctatgcag actccgtgaa gggccgattc accatctcca
gagacaattc caagaacacg 240ctgtatctgc aaatgaacag cctgagagcc
gaggacacgg ctgtgtatta ctgtgcgaga 300gattactatg ataatagtag
acatcactgg gggtttgact actggggcca gggaaccctg 360gtcaccgtct
cctcagcttc caccaagggc ccatccgtct tccccctggc gccctgctcc
420aggagcacct ccgagagcac agccgccctg ggctgcctgg tcaaggacta
cttccccgaa 480ccggtgacgg tgtcgtggaa ctcaggcgcc ctga 51442124PRTHomo
sapiens 42Gln 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 Ile
Phe Ser Arg Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Lys Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser
Asn Lys Leu 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 Tyr Tyr Asp Asn Ser Arg His His Trp Gly Phe Asp Tyr 100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala 115 120
43523DNAHomo sapiens 43tcagctcctg gggctgctaa tgctctgggt ccctggatca
gtgaggatat tgtgatgacc 60cagactccac tctccctgcc cgtcacccct ggagagccgg
cctccatctc ctgcaggtct 120agtcggagcc tcttggatag tgatgatgga
aacacctatt tggactggta cctgcagaag 180ccagggcagt ctccacagct
cctgatctac acgctttcct atcgggcctc tggagtccca 240gacaggttca
gtggcagtgg gtcaggcact gatttcacac tgaaaatcag cagggtggag
300gctgaggatg ttggagttta ttactgcatg caacgtgtag agtttcctat
caccttcggc 360caagggacac gactggagat taaacgaact gtggctgcac
catctgtctt catcttcccg 420ccatctgatg agcagttgaa atctggaact
gcctctgttg tgtgcctgct gaataacttc 480tatcccagag aggccaaagt
acagtggaag gtggataacg cct 52344114PRTHomo sapiens 44Asp Ile Val Met
Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro
Ala Ser Ile Ser Cys Arg Ser Ser Arg Ser Leu Leu Asp Ser 20 25 30
Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln 35
40 45 Ser Pro Gln Leu Leu Ile Tyr Thr Leu Ser Tyr Arg Ala Ser Gly
Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys 65 70 75 80 Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Met Gln 85 90 95 Arg Val Glu Phe Pro Ile Thr Phe Gly
Gln Gly Thr Arg Leu Glu Ile 100 105 110 Lys Arg 45546DNAHomo
sapiens 45gagcagtcgg ggggcggcgt ggtccagcct gggaggtccc tgagactctc
ctgtgcagcg 60tctggattca ccttcagtag ctatggcatg tactgggtcc gccaggctcc
aggcaagggg 120ctggagtggg tggcagttat atggtatgat ggaagcaata
aatactatgc agactccgtg 180aagggccgat tcaccatctc cagagacaat
tccaagaaca cgctgtatct gcaaatgaac 240agcctgagag ccgaggacac
ggctgtgtat tactgtgcga gggatttcta tgatagtagt 300cgttaccact
acggtatgga cgtctggggc caagggacca cggtcaccgt ctcctcagct
360tccaccaagg gcccatccgt cttccccctg gcgccctgct ccaggagcac
ctccgagagc 420acagccgccc tgggctgcct ggtcaaggac tacttccccg
aaccggtgac ggtgtcgtgg 480aactcaggcg ccctgaccag cggcgtgcac
accttcccgg ctgtcctaca gtcctcagga 540ctctct 54646124PRTHomo
sapiensMISC_FEATURE(1)..(4)Xaa is any amino acid 46Xaa Xaa Xaa Xaa
Glu Gln 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 Gly Met Tyr Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val
Ile Trp 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 Phe Tyr Asp Ser Ser Arg Tyr His Tyr
Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val Ser
Ser Ala 115 120 47419DNAHomo sapiens 47actcagtgtc cactctccct
gcccgtcacc cctggagagc cggcctccat ctcctgcagg 60tctagtcaga gcctcttgga
tagtgatgat ggaaacacct atttggactg gtacctgcag 120aagccagggc
agtctccaca gctcctgatc tatacggttt cctatcgggc ctctggagtc
180ccagacaggt tcagtggcag tgggtcaggc actgatttca cactgaaaat
cagcagggtg 240gaggctgagg atgttggagt ttattactgc atgcaacgta
tagagtttcc gatcaccttc 300ggccaaggga cccgactgga gattaaacga
actgtggctg caccatctgt cttcatcttc 360ccgccatctg atgagcagtt
gaaatctgga actgcctctg ttgtgtgcct gctgaataa 41948114PRTHomo
sapiensMISC_FEATURE(1)..(4)Xaa is any amino acid 48Xaa Xaa Xaa Xaa
Thr Gln Cys Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30
Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln 35
40 45 Ser Pro Gln Leu Leu Ile Tyr Thr Val Ser Tyr Arg Ala Ser Gly
Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys 65 70 75 80 Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Met Gln 85 90 95 Arg Ile Glu Phe Pro Ile Thr Phe Gly
Gln Gly Thr Arg Leu Glu Ile 100 105 110 Lys Arg 491428DNAHomo
sapiens 49cggccgccta tttacccaga gacagggaga ggctcttctg tgtgtagtgg
ttgtgcagag 60cctcatgcat cacggagcat gagaagacat tcccctcctg ccacctgctc
ttgtccacgg 120ttagcctgct gtagaggaag aaggagccgt cggagtccag
cacgggaggc gtggtcttgt 180agttgttctc cggctgccca ttgctctccc
actccacggc gatgtcgctg gggtagaagc 240ctttgaccag gcaggtcagg
ctgacctggt tcttggtcat ctcctcctgg gatgggggca 300gggtgtacac
ctgtggctct cggggctgcc ctttggcttt ggagatggtt ttctcgatgg
360aggacgggag gcctttgttg gagaccttgc acttgtactc cttgccgttc
agccagtcct 420ggtgcaggac ggtgaggacg ctgaccacac ggtacgtgct
gttgaactgc tcctcccgcg 480gctttgtctt ggcattatgc acctccacgc
catccacgta ccagttgaac tggacctcgg 540ggtcttcctg gctcacgtcc
accaccacgc acgtgacctc aggggtccgg gagatcatga 600gagtgtcctt
gggttttggg gggaacagga agactgatgg tccccccagg aactcaggtg
660ctgggcatga tgggcatggg ggaccatatt tggactcaac tctcttgtcc
accttggtgt 720tgctgggctt gtgatctacg ttgcaggtgt aggtcttcgt
gcccaagctg ctggagggca 780cggtcaccac gctgctgagg gagtagagtc
ctgaggactg taggacagcc gggaaggtgt 840gcacgccgct ggtcagggcg
cctgagttcc acgacaccgt caccggttcg gggaagtagt 900ccttgaccag
gcagcccagg gcggctgtgc tctcggaggt gctcctggag cagggcgcca
960gggggaagac ggatgggccc ttggtggaag ctgaggagac ggtgaccagg
gttccctggc 1020cccagtagtc aaacccccag tgatgtctac tattatcata
gtaatctctc gcacagtaat 1080acacagccgt gtcctcggct ctcaggctgt
tcatttgcag atacagcgtg ttcttggaat 1140tgtctctgga gatggtgaat
cggcccttca cggagtctgc atagagttta ttacttccat 1200cataccatat
aactgccacc catttcagcc ccttgcctgg agcctggcgg acccagtgca
1260tgccatagcg actgaagatg aatccagacg ctgcacagga gagtctcagg
gacctcccag 1320gctggaccac gcctccccca gactccacca gctgcacctg
acactggaca ccttttaaaa 1380tagccacaag aaaaagccag ctcagcccaa
actccatggt ggtcgact 142850469PRTHomo sapiens 50Met Glu Phe Gly Leu
Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 20 25 30 Pro
Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe 35 40
45 Ser Arg Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60 Lys Trp Val Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys Leu
Tyr Ala 65 70 75 80 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ser Lys Asn 85 90 95 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val 100 105 110 Tyr Tyr Cys Ala Arg Asp Tyr Tyr
Asp Asn Ser Arg His His Trp Gly 115 120 125 Phe Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser Ala Ser 130 135 140 Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr 145 150 155 160 Ser
Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 165 170
175 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
180 185 190 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser 195 200 205 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr
Lys Thr Tyr Thr 210 215 220 Cys Asn Val Asp His Lys Pro Ser Asn Thr
Lys Val Asp Lys Arg Val 225 230 235 240 Glu Ser Lys Tyr Gly Pro Pro
Cys Pro Ser Cys Pro Ala Pro Glu Phe 245 250 255 Leu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 260 265 270 Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 275 280 285 Ser
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 290 295
300 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
305 310 315 320 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
Asp Trp Leu 325 330 335 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu Pro Ser 340 345 350 Ser Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro 355 360 365 Gln Val Tyr Thr Leu Pro Pro
Ser Gln Glu Glu Met Thr Lys Asn Gln 370 375 380 Val Ser Leu Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 385 390 395 400 Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 420
425 430 Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
Ser 435 440 445 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
Ser Leu Ser 450 455 460 Leu Ser Leu Gly Lys 465 51741DNAHomo
sapiens 51agtcgaccac catggaaacc ccagcgcagc ttctcttcct cctgctactc
tggctcccag 60ataccaccgg agatattgtg atgacccaga ctccactctc cctgcccgtc
acccctggag 120agccggcctc catctcctgc aggtctagtc ggagcctctt
ggatagtgat gatggaaaca 180cctatttgga ctggtacctg cagaagccag
ggcagtctcc acagctcctg atctacacgc 240tttcctatcg ggcctctgga
gtcccagaca ggttcagtgg cagtgggtca ggcactgatt 300tcacactgaa
aatcagcagg gtggaggctg aggatgttgg agtttattac tgcatgcaac
360gtgtagagtt tcctatcacc ttcggccaag ggacacgact ggagattaaa
cgaactgtgg 420ctgcaccatc tgtcttcatc ttcccgccat ctgatgagca
gttgaaatct ggaactgcct 480ctgttgtgtg cctgctgaat aacttctatc
ccagagaggc caaagtacag tggaaggtgg 540ataacgccct ccaatcgggt
aactcccagg agagtgtcac agagcaggac agcaaggaca 600gcacctacag
cctcagcagc accctgacgc tgagcaaagc agactacgag aaacacaaag
660tctacgcctg cgaagtcacc catcagggcc tgagctcgcc cgtcacaaag
agcttcaaca 720ggggagagtg ttaggcggcc g 74152240PRTHomo sapiens 52Met
Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro 1 5 10
15 Asp Thr Thr Gly Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro
20 25 30 Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser
Arg Ser 35 40 45 Leu Leu Asp Ser Asp Asp Gly Asn Thr Tyr Leu Asp
Trp Tyr Leu Gln 50 55 60 Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile
Tyr Thr Leu Ser Tyr Arg 65 70 75 80 Ala Ser Gly Val Pro Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp 85 90 95 Phe Thr Leu Lys Ile Ser
Arg Val Glu Ala Glu Asp Val Gly Val Tyr 100 105 110 Tyr Cys Met Gln
Arg Val Glu Phe Pro Ile Thr Phe Gly Gln Gly Thr 115 120 125 Arg Leu
Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe 130 135 140
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys 145
150 155 160 Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp
Lys Val 165 170 175 Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln 180 185 190 Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser 195 200 205 Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr Ala Cys Glu Val Thr His 210 215 220 Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 240
53789DNAHomo sapiens 53tctgtaaagg ttggtggaga ggcaggtcca tctgtcacac
taccctgcca ctacagtgga 60gctgtcacat caatgtgctg gaatagaggc tcatgttctc
tattcacatg ccaaaatggc 120attgtctgga ccaatggaac ccacgtcacc
tatcggaagg acacacgcta taagctattg 180ggggaccttt caagaaggga
tgtctctttg accatagaaa atacagctgt gtctgacagt 240ggcgtatatt
gttgccgtgt tgagcaccgt gggtggttca atgacatgaa aatcaccgta
300tcattggaga ttgtgccacc caaggtcacg actactccaa ttgtcacaac
tgttccaacc 360gtcacgactg ttcgaacgag caccactgtt ccaacgacaa
cgactgttcc aacgacaact 420gttccaacaa caatgagcat tccaacgaca
acgactgttc cgacgacaat gactgtttca 480acgacaacga gcgttccaac
gacaacgagc attccaacaa caacaagtgt tccagtgaca 540acaacggtct
ctacctttgt tcctccaatg cctttgccca ggcagaacca tgaaccagta
600gccacttcac catcttcacc tcagccagca gaaacccacc ctacgacact
gcagggagca 660ataaggagag aacccaccag ctcaccattg tactcttaca
caacagatgg gaatgacacc 720gtgacagagt cttcagatgg cctttggaat
aacaatcaaa ctcaactgtt cctagaacat 780agtctactg 78954263PRTHomo
sapiens 54Ser Val Lys Val Gly Gly Glu Ala Gly Pro Ser Val Thr Leu
Pro Cys 1 5 10 15 His Tyr Ser Gly Ala Val Thr Ser Met Cys Trp Asn
Arg Gly Ser Cys 20 25 30 Ser Leu Phe Thr Cys Gln Asn Gly Ile Val
Trp Thr Asn Gly Thr His 35 40 45 Val Thr Tyr Arg Lys Asp Thr Arg
Tyr Lys Leu Leu Gly Asp Leu Ser 50 55 60 Arg Arg Asp Val Ser Leu
Thr Ile Glu Asn Thr Ala Val Ser Asp Ser 65 70 75 80 Gly Val Tyr Cys
Cys Arg Val Glu His Arg Gly Trp Phe Asn Asp Met 85 90 95 Lys Ile
Thr Val Ser Leu Glu Ile Val Pro Pro Lys Val Thr Thr Thr 100 105 110
Pro Ile Val Thr Thr Val Pro Thr Val Thr Thr Val Arg Thr Ser Thr 115
120 125 Thr Val Pro Thr Thr Thr Thr Val Pro Thr Thr Thr Val Pro Thr
Thr 130 135 140 Met Ser Ile Pro Thr Thr Thr Thr Val Pro Thr Thr Met
Thr Val Ser 145 150 155 160 Thr Thr Thr Ser Val Pro Thr Thr Thr Ser
Ile Pro Thr Thr Thr Ser 165 170 175 Val Pro Val Thr Thr Thr Val Ser
Thr Phe Val Pro Pro Met Pro Leu 180 185 190 Pro Arg Gln Asn His Glu
Pro Val Ala Thr Ser Pro Ser Ser Pro Gln 195 200 205 Pro Ala Glu Thr
His Pro Thr Thr Leu Gln Gly Ala Ile Arg Arg Glu 210 215 220 Pro Thr
Ser Ser Pro Leu Tyr Ser Tyr Thr Thr Asp Gly Asn Asp Thr 225 230 235
240 Val Thr Glu Ser Ser Asp Gly Leu Trp Asn Asn Asn Gln Thr Gln Leu
245 250 255 Phe Leu Glu His Ser Leu Leu 260 55114PRTHomo
sapiensMISC_FEATURE(99)..(100)Xaa is any amino acid 55Gln 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 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ala Val Ile Trp 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 Xaa Xaa Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Ala 56124PRTHomo
sapiensMISC_FEATURE(98)..(100)Xaa is any amino acid 56Gln 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 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ala Val Ile Trp 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 Xaa Xaa Xaa Tyr Asp Ser Ser
Xaa Xaa Xaa Tyr Gly Met Asp Val 100 105 110 Trp Gly Gln Gly Thr Thr
Val Thr Val Ser Ser Ala 115 120 57125PRTHomo
sapiensMISC_FEATURE(100)..(103)Xaa is any amino acid 57Gln Val Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr
Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25
30 Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu
35 40 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn
Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser
Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala
Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Xaa Xaa Xaa Xaa Ser
Ser Ser Trp Tyr Xaa Xaa Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser Ala 115 120 125 58124PRTHomo
sapiensMISC_FEATURE(105)..(109)Xaa is any amino acid 58Gln 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 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ala Val Ile Trp 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 Tyr Tyr Asp Ser Ser
Xaa Xaa Xaa Xaa Xaa Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Ala 115 120 59119PRTHomo
sapiensMISC_FEATURE(100)..(101)Xaa is any amino acid 59Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25
30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Val 35 40 45 Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr
Asp Tyr Ala Ala 50 55 60 Pro Val Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu
Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Xaa Xaa Asp
Xaa Xaa Xaa Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val
Ser Ser Ala 115 60121PRTHomo sapiensMISC_FEATURE(100)..(102)Xaa is
any amino acid 60Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val
Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly
Gly Ser Val Ser Ser Gly 20 25 30 Gly Tyr Tyr Trp Ser Trp Ile Arg
Gln Pro Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile Tyr
Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg
Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95
Cys Ala Arg Xaa Xaa Xaa Trp Xaa Xaa Xaa Phe Asp Tyr Trp Gly Gln 100
105 110 Gly Thr Leu Val Thr Val Ser Ser Ala 115 120 61119PRTHomo
sapiensMISC_FEATURE(101)..(103)Xaa is any amino acid 61Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25
30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr
Ala Ala 50 55 60 Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp
Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr
Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Thr Xaa Xaa Xaa Ser
Gly Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser
Ala 115 62113PRTHomo sapiensMISC_FEATURE(099)..(099)Xaa is any
amino acid 62Glu 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 Trp Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn Ile Lys Gln Asp Gly
Ser Glu Lys Tyr Tyr Val 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 Xaa Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 100 105
110 Ala 63114PRTHomo sapiensMISC_FEATURE(98)..(99)Xaa is any amino
acid 63Glu 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 Asp Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Xaa Xaa
Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser
Ala 64110PRTHomo sapiensMISC_FEATURE(96)..(97)Xaa is any amino acid
64Glu 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 Xaa 85 90 95 Xaa Leu Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 65113PRTHomo
sapiensMISC_FEATURE(100)..(101)Xaa is any amino acid 65Asp Ile Val
Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu
Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25
30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly
Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe
Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Xaa Xaa Thr Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg 66108PRTHomo
sapiens 66Asp 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 Gly
Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Arg 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 Glu
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala
Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Leu 85 90 95 Thr Phe
Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 67114PRTHomo
sapiensMISC_FEATURE(101)..(101)Xaa is any amino acid 67Asp Ile Val
Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr Leu Gly 1 5 10 15 Gln
Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25
30 Asp Gly Asn Thr Tyr Leu Ser Trp Leu Gln Gln Arg Pro Gly Gln Pro
35 40 45 Pro Arg Leu Leu Ile Tyr Lys Ile Ser Asn Arg Phe Ser Gly
Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe
Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val
Tyr Tyr Cys Met Gln Ala 85 90 95 Thr Gln Phe Pro Xaa Ile Thr Phe
Gly Gln Gly Thr Arg Leu Glu Ile 100 105 110 Lys Arg 68108PRTHomo
sapiens 68Asp 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 Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 69113PRTHomo
sapiens 69Asp Ile Val Met Thr Gln Thr Pro Leu Ser Ser Pro Val Thr
Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser
Leu Val His Ser 20 25 30 Asp Gly Asn Thr Tyr Leu Ser Trp Leu Gln
Gln Arg Pro Gly Gln Pro 35 40 45 Pro Arg Leu Leu Ile Tyr Lys Ile
Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser
Gly Ala Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu
Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ala 85 90 95 Thr Gln
Phe Pro Gln Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110
Arg 70114PRTHomo sapiens 70Asp Ile Val Met Thr Gln Thr Pro Leu Ser
Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg
Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Asp Gly Asn Thr Tyr
Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln 35 40 45 Ser Pro Gln Leu
Leu Ile Tyr Thr Leu Ser Tyr Arg Ala Ser Gly Val 50 55 60 Pro Asp
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys 65 70 75 80
Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln 85
90 95 Arg Ile Glu Phe Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu
Ile 100 105 110 Lys Arg 71108PRTHomo sapiens 71Glu Ile Val Leu Thr
Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys 1 5 10 15 Glu Lys Val
Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ser Ser 20 25 30 Leu
His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Leu Leu Ile 35 40
45 Lys Tyr Ala Ser Gln Ser Phe Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu
Glu Ala 65 70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys His Gln Ser Ser
Ser Leu Pro Phe 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile
Lys Arg 100 105 72108PRTHomo sapiensMISC_FEATURE(96)..(97)Xaa is
any amino acid 72Asp 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 Gly Ile Arg Asn Asp 20 25 30 Leu Gly Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Arg 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 Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp
Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Xaa 85 90 95
Xaa Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg 100 105 7316DNAHomo
sapiens 73ttactatgat aatagt 167415DNAHomo sapiens 74agacatcact
ggggg 157517DNAHomo sapiens 75atagcagcaa ctggtac 177616DNAHomo
sapiens 76ttactatgat aatagt 167715DNAHomo sapiens 77agacatcact
ggggg 157816DNAHomo sapiens 78ttactatgat aatagt 167915DNAHomo
sapiens 79agacatcact ggggg 158013DNAHomo sapiens 80ctatgatagt agt
138111DNAHomo sapiens 81ttactatgat a 118220DNAHomo sapiens
82cgagtcggca tcactggggg 208323DNAHomo
sapiensmisc_feature(21)..(21)n is inosine 83caggtgcagc tggagcagtc
ngg 238424DNAHomo sapiens 84gctgagggag tagagtcctg agga
248519DNAHomo sapiens 85cacaccgcgg tcacatggc 198620DNAHomo sapiens
86ctactctagg gcacctgtcc 208714PRTHomo sapiens 87Pro Met Pro Leu Pro
Arg Gln Asn His Glu Pro Val Ala Thr 1 5 10 8812PRTHomo sapiens
88Pro Met Pro Leu Pro Arg Gln Asn His Glu Pro Val 1 5 10
8910PRTHomo sapiens 89Pro Met Pro Leu Pro Arg Gln Asn His Glu 1 5
10 908PRTHomo sapiens 90Pro Met Pro Leu Pro Arg Gln Asn 1 5
916PRTHomo sapiens 91Pro Met Pro Leu Pro Arg 1 5 9212PRTHomo
sapiens 92Pro Leu Pro Arg Gln Asn His Glu Pro Val Ala Thr 1 5 10
9310PRTHomo sapiens 93Pro Arg Gln Asn His Glu Pro Val Ala Thr 1 5
10 948PRTHomo sapiens 94Gln Asn His Glu Pro Val Ala Thr 1 5
956PRTHomo sapiens 95His Glu Pro Val Ala Thr 1 5 967PRTHomo sapiens
96Pro Leu Pro Arg Asn His Glu 1 5 976PRTHomo sapiens 97Leu Pro Arg
Gln Asn His 1 5 9810PRTHomo sapiens 98Pro Met Pro Ala Pro Arg Gln
Asn His Glu 1 5 10 9910PRTHomo sapiens 99Pro Met Pro Leu Ala Arg
Gln Asn His Glu 1 5 10 10010PRTHomo sapiens 100Pro Met Pro Leu Pro
Ala Gln Asn His Glu 1 5 10 10110PRTHomo sapiens 101Pro Met Pro Leu
Pro Arg Ala Asn His Glu 1 5 10 10210PRTHomo sapiens 102Pro Met Pro
Leu Pro Arg Gln Ala His Glu 1 5 10 10310PRTHomo sapiens 103Pro Met
Pro Leu Pro Arg Gln Asn Ala Glu 1 5 10 1048PRTHomo sapiens 104Pro
Leu Pro Arg Gln Asn His Glu 1 5 1057PRTHomo sapiens 105Leu Pro Arg
Gln Asn His Glu 1 5 1068PRTHomo sapiens 106Pro Leu Pro Arg Gln Asn
His Glu 1 5 1077PRTHomo sapiens 107Leu Pro Arg Gln Asn His Glu 1 5
108882DNAHomo sapiens 108atgaaatacc tgctgccgac cgctgctgct
ggtctgctgc tcctcgctgc ccagccggcc 60atggccgata ttgtgatgac ccagactcca
ctctccctgc ccgtcacccc tggagagccg 120gcctccatct cctgcaggtc
tagtcggagc ctcttggata gtgatgatgg aaacacctat 180ttggactggt
acctgcagaa gccagggcag tctccacagc tcctgatcta cacgctttcc
240tatcgggcct ctggagtccc agacaggttc agtggcagtg ggtcaggcac
tgatttcaca 300ctgaaaatca gcagggtgga ggctgaggat gttggagttt
attactgcat gcaacgtgta 360gagtttccta tcaccttcgg ccaagggaca
cgactggaga ttaaactttc cgcggacgat 420gcgaaaaagg atgctgcgaa
gaaagatgac gctaagaaag acgatgctaa aaaggacctc 480caggtgcagc
tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc
540tcctgtgcag cgtctggatt catcttcagt cgctatggca tgcactgggt
ccgccaggct 600ccaggcaagg ggctgaaatg ggtggcagtt atatggtatg
atggaagtaa taaactctat 660gcagactccg tgaagggccg attcaccatc
tccagagaca attccaagaa cacgctgtat 720ctgcaaatga acagcctgag
agccgaggac acggctgtgt attactgtgc gagagattac 780tatgataata
gtagacatca ctgggggttt gactactggg gccagggaac cctggtcacc
840gtctcctcag ctagcgatta taaggacgat gatgacaaat ag 882109271PRTHomo
sapiens 109Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr
Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Arg Ser
Leu Leu Asp Ser 20 25 30 Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr
Leu Gln Lys Pro Gly Gln 35 40 45 Ser Pro Gln Leu Leu Ile Tyr Thr
Leu Ser Tyr Arg Ala Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys 65 70 75 80 Ile Ser Arg Val
Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln 85 90 95 Arg Val
Glu Phe Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile
100 105 110 Lys Leu Ser Ala Asp Asp Ala Lys Lys Asp Ala Ala Lys Lys
Asp Asp 115 120 125 Ala Lys Lys Asp Asp Ala Lys Lys Asp Leu Gln Val
Gln Leu Val Glu 130 135 140 Ser Gly Gly Gly Val Val Gln Pro Gly Arg
Ser Leu Arg Leu Ser Cys 145 150 155 160 Ala Ala Ser Gly Phe Ile Phe
Ser Arg Tyr Gly Met His Trp Val Arg 165 170 175 Gln Ala Pro Gly Lys
Gly Leu Lys Trp Val Ala Val Ile Trp Tyr Asp 180 185 190 Gly Ser Asn
Lys Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 195 200 205 Ser
Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 210 215
220 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp Tyr Tyr Asp
225 230 235 240 Asn Ser Arg His His Trp Gly Phe Asp Tyr Trp Gly Gln
Gly Thr Leu 245 250 255 Val Thr Val Ser Ser Ala Ser Asp Tyr Lys Asp
Asp Asp Asp Lys 260 265 270 1101560DNAHomo sapiens 110atggaaaccc
cagcgcagct tctcttcctc ctgctactct ggctcccaga taccaccgga 60gatattgtga
tgacccagac tccactctcc ctgcccgtca cccctggaga gccggcctcc
120atctcctgca ggtctagtcg gagcctcttg gatagtgatg atggaaacac
ctatttggac 180tggtacctgc agaagccagg gcagtctcca cagctcctga
tctacacgct ttcctatcgg 240gcctctggag tcccagacag gttcagtggc
agtgggtcag gcactgattt cacactgaaa 300atcagcaggg tggaggctga
ggatgttgga gtttattact gcatgcaacg tgtagagttt 360cctatcacct
tcggccaagg gacacgactg gagattaaag gtggtggtgg ttctggcggc
420ggcggctccg gtggtggtgg ttcccaggtg cagctggtgg agtctggggg
aggcgtggtc 480cagcctggga ggtccctgag actctcctgt gcagcgtctg
gattcatctt cagtcgctat 540ggcatgcact gggtccgcca ggctccaggc
aaggggctga aatgggtggc agttatatgg 600tatgatggaa gtaataaact
ctatgcagac tccgtgaagg gccgattcac catctccaga 660gacaattcca
agaacacgct gtatctgcaa atgaacagcc tgagagccga ggacacggct
720gtgtattact gtgcgagaga ttactatgat aatagtagac atcactgggg
gtttgactac 780tggggccagg gaaccctggt caccgtctcc tcaggaggtg
gtggatccga tatcaaactg 840cagcagtcag gggctgaact ggcaagacct
ggggcctcag tgaagatgtc ctgcaagact 900tctggctaca cctttactag
gtacacgatg cactgggtaa aacagaggcc tggacagggt 960ctggaatgga
ttggatacat taatcctagc cgtggttata ctaattacaa tcagaagttc
1020aaggacaagg ccacattgac tacagacaaa tcctccagca cagcctacat
gcaactgagc 1080agcctgacat ctgaggactc tgcagtctat tactgtgcaa
gatattatga tgatcattac 1140tgccttgact actggggcca aggcaccact
ctcacagtct cctcagtcga aggtggaagt 1200ggaggttctg gtggaagtgg
aggttcaggt ggagtcgacg acattcagct gacccagtct 1260ccagcaatca
tgtctgcatc tccaggggag aaggtcacca tgacctgcag agccagttca
1320agtgtaagtt acatgaactg gtaccagcag aagtcaggca cctcccccaa
aagatggatt 1380tatgacacat ccaaagtggc ttctggagtc ccttatcgct
tcagtggcag tgggtctggg 1440acctcatact ctctcacaat cagcagcatg
gaggctgaag atgctgccac ttattactgc 1500caacagtgga gtagtaaccc
gctcacgttc ggtgctggga ccaagctgga gctgaaatag 1560111499PRTHomo
sapiens 111Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr
Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Arg Ser
Leu Leu Asp Ser 20 25 30 Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr
Leu Gln Lys Pro Gly Gln 35 40 45 Ser Pro Gln Leu Leu Ile Tyr Thr
Leu Ser Tyr Arg Ala Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys 65 70 75 80 Ile Ser Arg Val
Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln 85 90 95 Arg Val
Glu Phe Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile 100 105 110
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115
120 125 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly
Arg 130 135 140 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe
Ser Arg Tyr 145 150 155 160 Gly Met His Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Lys Trp Val 165 170 175 Ala Val Ile Trp Tyr Asp Gly Ser
Asn Lys Leu Tyr Ala Asp Ser Val 180 185 190 Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 195 200 205 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 210 215 220 Ala Arg
Asp Tyr Tyr Asp Asn Ser Arg His His Trp Gly Phe Asp Tyr 225 230 235
240 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser
245 250 255 Asp Ile Lys Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro
Gly Ala 260 265 270 Ser Val Lys Met Ser Cys Lys Thr Ser Gly Tyr Thr
Phe Thr Arg Tyr 275 280 285 Thr Met His Trp Val Lys Gln Arg Pro Gly
Gln Gly Leu Glu Trp Ile 290 295 300 Gly Tyr Ile Asn Pro Ser Arg Gly
Tyr Thr Asn Tyr Asn Gln Lys Phe 305 310 315 320 Lys Asp Lys Ala Thr
Leu Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr 325 330 335 Met Gln Leu
Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 340 345 350 Ala
Arg Tyr Tyr Asp Asp His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly 355 360
365 Thr Thr Leu Thr Val Ser Ser Val Glu Gly Gly Ser Gly Gly Ser Gly
370 375 380 Gly Ser Gly Gly Ser Gly Gly Val Asp Asp Ile Gln Leu Thr
Gln Ser 385 390 395 400 Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys
Val Thr Met Thr Cys 405 410 415 Arg Ala Ser Ser Ser Val Ser Tyr Met
Asn Trp Tyr Gln Gln Lys Ser 420 425 430 Gly Thr Ser Pro Lys Arg Trp
Ile Tyr Asp Thr Ser Lys Val Ala Ser 435 440 445 Gly Val Pro Tyr Arg
Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser 450 455 460 Leu Thr Ile
Ser Ser Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys 465 470 475 480
Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu 485
490 495 Glu Leu Lys 1121635DNAHomo sapiens 112atggaaaccc cagcgcagct
tctcttcctc ctgctactct ggctcccaga taccaccgga 60gatattgtga tgacccagac
tccactctcc ctgcccgtca cccctggaga gccggcctcc 120atctcctgca
ggtctagtcg gagcctcttg gatagtgatg atggaaacac ctatttggac
180tggtacctgc agaagccagg gcagtctcca cagctcctga tctacacgct
ttcctatcgg 240gcctctggag tcccagacag gttcagtggc agtgggtcag
gcactgattt cacactgaaa 300atcagcaggg tggaggctga ggatgttgga
gtttattact gcatgcaacg tgtagagttt 360cctatcacct tcggccaagg
gacacgactg gagattaaac tttccgcgga cgatgcgaaa 420aaggatgctg
cgaagaaaga tgacgctaag aaagacgatg ctaaaaagga cctgcaggtg
480cagctggtgg agtctggggg aggcgtggtc cagcctggga ggtccctgag
actctcctgt 540gcagcgtctg gattcatctt cagtcgctat ggcatgcact
gggtccgcca ggctccaggc 600aaggggctga aatgggtggc agttatatgg
tatgatggaa gtaataaact ctatgcagac 660tccgtgaagg gccgattcac
catctccaga gacaattcca agaacacgct gtatctgcaa 720atgaacagcc
tgagagccga ggacacggct gtgtattact gtgcgagaga ttactatgat
780aatagtagac atcactgggg gtttgactac tggggccagg gaaccctggt
caccgtctcc 840tcaggaggtg gtggatccga tatcaaactg cagcagtcag
gggctgaact ggcaagacct 900ggggcctcag tgaagatgtc ctgcaagact
tctggctaca cctttactag gtacacgatg 960cactgggtaa aacagaggcc
tggacagggt ctggaatgga ttggatacat taatcctagc 1020cgtggttata
ctaattacaa tcagaagttc aaggacaagg ccacattgac tacagacaaa
1080tcctccagca cagcctacat gcaactgagc agcctgacat ctgaggactc
tgcagtctat 1140tactgtgcaa gatattatga tgatcattac tgccttgact
actggggcca aggcaccact 1200ctcacagtct cctcactttc cgcggacgat
gcgaaaaagg atgctgcgaa gaaagatgac 1260gctaagaaag acgatgctaa
aaaggacctg gacattcagc tgacccagtc tccagcaatc 1320atgtctgcat
ctccagggga gaaggtcacc atgacctgca gagccagttc aagtgtaagt
1380tacatgaact ggtaccagca gaagtcaggc acctccccca aaagatggat
ttatgacaca 1440tccaaagtgg cttctggagt cccttatcgc ttcagtggca
gtgggtctgg gacctcatac 1500tctctcacaa tcagcagcat ggaggctgaa
gatgctgcca cttattactg ccaacagtgg 1560agtagtaacc cgctcacgtt
cggtgctggg accaagctgg agctgaaaga ttataaggac 1620gatgatgaca aatag
1635113524PRTHomo sapiens 113Asp Ile Val Met Thr Gln Thr Pro Leu
Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys
Arg Ser Ser Arg Ser Leu Leu Asp Ser 20 25 30 Asp Asp Gly Asn Thr
Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln 35 40 45 Ser Pro Gln
Leu Leu Ile Tyr Thr Leu Ser Tyr Arg Ala Ser Gly Val 50 55 60 Pro
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys 65 70
75 80 Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met
Gln 85 90 95 Arg Val Glu Phe Pro Ile Thr Phe Gly Gln Gly Thr Arg
Leu Glu Ile 100 105 110 Lys Leu Ser Ala Asp Asp Ala Lys Lys Asp Ala
Ala Lys Lys Asp Asp 115 120 125 Ala Lys Lys Asp Asp Ala Lys Lys Asp
Leu Gln Val Gln Leu Val Glu 130 135 140 Ser Gly Gly Gly Val Val Gln
Pro Gly Arg Ser Leu Arg Leu Ser Cys 145 150 155 160 Ala Ala Ser Gly
Phe Ile Phe Ser Arg Tyr Gly Met His Trp Val Arg 165 170 175 Gln Ala
Pro Gly Lys Gly Leu Lys Trp Val Ala Val Ile Trp Tyr Asp 180 185 190
Gly Ser Asn Lys Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile 195
200 205 Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
Leu 210 215 220 Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Asp
Tyr Tyr Asp 225 230 235 240 Asn Ser Arg His His Trp Gly Phe Asp Tyr
Trp Gly Gln Gly Thr Leu 245 250 255 Val Thr Val Ser Ser Gly Gly Gly
Gly Ser Asp Ile Lys Leu Gln Gln 260 265 270 Ser Gly Ala Glu Leu Ala
Arg Pro Gly Ala Ser Val Lys Met Ser Cys 275 280 285 Lys Thr Ser Gly
Tyr Thr Phe Thr Arg Tyr Thr Met His Trp Val Lys 290 295 300 Gln Arg
Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Ser 305 310 315
320 Arg Gly Tyr Thr Asn Tyr Asn Gln Lys Phe Lys Asp Lys Ala Thr Leu
325 330 335 Thr Thr Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser
Ser Leu 340 345 350 Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg
Tyr Tyr Asp Asp 355 360 365 His Tyr Cys Leu Asp Tyr Trp Gly Gln Gly
Thr Thr Leu Thr Val Ser 370 375 380 Ser Leu Ser Ala Asp Asp Ala Lys
Lys Asp Ala Ala Lys Lys Asp Asp 385 390 395 400 Ala Lys Lys Asp Asp
Ala Lys Lys Asp Leu Asp Ile Gln Leu Thr Gln 405 410 415 Ser Pro Ala
Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr 420 425 430 Cys
Arg Ala Ser Ser Ser Val Ser Tyr Met Asn Trp Tyr Gln Gln Lys 435 440
445 Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Val Ala
450 455 460 Ser Gly Val Pro Tyr Arg Phe Ser Gly Ser Gly Ser Gly Thr
Ser Tyr 465 470 475 480 Ser Leu Thr Ile Ser Ser Met Glu Ala Glu Asp
Ala Ala Thr Tyr Tyr 485 490 495 Cys Gln Gln Trp Ser Ser Asn Pro Leu
Thr Phe Gly Ala Gly Thr Lys 500 505 510 Leu Glu Leu Lys Asp Tyr Lys
Asp Asp Asp Asp Lys 515 520 114169PRTHomo sapiens 114Trp Val Leu
Ser Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val 1 5 10 15 Lys
Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser 20 25
30 Val Ser Ser Gly Gly Tyr Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly
35 40 45 Lys Gly Leu Glu Trp Ile Gly Phe Ile Tyr Tyr Thr Gly Ser
Thr Asn 50 55 60 Tyr Asn Pro Ser Leu Lys Ser Arg Val Ser Ile Ser
Val Asp Thr Ser 65 70 75 80 Lys Asn Gln Phe Ser Leu Lys Leu Ser Ser
Val Thr Ala Ala Asp Ala 85 90 95 Ala Val Tyr Tyr Cys Ala Arg Asp
Tyr Asp Trp Ser Phe His Phe Asp 100 105 110 Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val
Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu 130 135 140 Ser Thr
Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155
160 Val Thr Val Ser Trp Asn Ser Gly Ala 165 115168PRTHomo sapiens
115Gln Leu Leu Gly Leu Leu Leu Leu Trp Phe Pro Gly Ala Arg Cys Asp
1 5 10 15 Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Ile
Gly Asp 20 25 30 Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile
Arg Asn Asp Leu 35 40 45 Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala
Pro Lys Arg Leu Ile Tyr 50 55 60 Ala Ala Ser Ser Leu Gln Ser Gly
Val Pro Ser Arg Phe Ser Gly Ser 65 70 75 80 Gly Ser Gly Thr Glu Phe
Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu 85 90 95 Asp Phe Ala Thr
Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro Leu Thr 100 105 110 Phe Gly
Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro 115 120 125
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 130
135 140 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
Lys 145 150 155 160 Val Gln Trp Lys Val Asp Asn Ala 165
116156PRTHomo sapiens 116Gln Cys Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro 1 5 10 15 Gly Gly Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Thr Phe Thr 20 25 30 Asn Tyr Trp Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 35 40 45 Trp Val Ala Asn
Ile Gln Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp 50 55 60 Ser Val
Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser 65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Ser Ala Val Tyr 85
90 95 Tyr Cys Ala Arg Trp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
Val 100 105 110 Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Cys 115 120 125 Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu
Gly Cys Leu Val Lys 130 135 140 Asp Tyr Phe Pro Glu Pro Val Ser Gly
Val Val Glu 145 150 155 117151PRTHomo sapiens 117Leu Leu Gly Leu
Leu Met Leu Trp Val Pro Gly Ser Ser Gly Asp Ile 1 5 10 15 Val Met
Thr Gln Thr Pro Leu Ser Ser Thr Val Ile Leu Gly Gln Pro 20 25 30
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser Asp Gly 35
40 45 Asn Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Pro Pro
Arg 50 55 60 Leu Leu Ile Tyr Met Ile Ser Asn Arg Phe Ser Gly Val
Pro Asp Arg 65 70 75 80 Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr
Leu Lys Ile Ser Arg 85 90 95 Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Met Gln Ala Thr Glu
100 105 110 Ser Pro Gln Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg Thr 115 120 125 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu 130 135 140 Lys Ser Gly Arg Ala Ser Val 145 150
118180PRTHomo sapiensMISC_FEATURE(1)..(4)Xaa is any amino acid
118Xaa Xaa Xaa Xaa Glu Gln Ser Gly Gly Gly Val Val Lys Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Asn Ala 20 25 30 Trp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Gly Arg Ile Lys Arg Arg Thr Asp Gly Gly
Thr Thr Asp Tyr Ala Ala 50 55 60 Pro Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn
Asn Leu Lys Asn Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Ser
Val Asp Asn Asp Val Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130
135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu 180 119152PRTHomo
sapiensMISC_FEATURE(1)..(3)Xaa is any amino acid 119Xaa Xaa Xaa Leu
Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30
Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Ile Gly Leu Tyr
Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro Leu Thr Phe Gly Gly
Gly Thr Lys Val Asp Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg
Glu Ala Lys Val Gln 145 150 120179PRTHomo sapiens 120Gln Val Gln
Leu Glu Gln 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 Thr 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 Arg Ser Ser Thr Ser Thr Ile Tyr Tyr Ala Glu
Ser Leu 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ser Asp Asn Ala Lys
Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Asp Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Phe Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val Ser 100 105 110 Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Cys Ser 115 120 125 Arg Ser Thr Ser
Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 130 135 140 Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 145 150 155
160 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
165 170 175 Ser Leu Ser 121163PRTHomo sapiens 121Glu Ile Gln Leu
Thr Gln Ser Pro Leu Ser Ser Pro Val Thr Leu Gly 1 5 10 15 Gln Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30
Asp Gly Asp Thr Tyr Leu Asn Trp Leu Gln Gln Arg Pro Gly Gln Pro 35
40 45 Pro Arg Leu Leu Ile Tyr Lys Ile Ser Thr Arg Phe Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ala Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Thr Asp Asp Val Gly Ile Tyr
Tyr Cys Met Gln Thr 85 90 95 Thr Gln Ile Pro Gln Ile Thr Phe Gly
Gln Gly Thr Arg Leu Glu Ile 100 105 110 Lys Arg Thr Val Ala Ala Pro
Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120 125 Glu Gln Leu Lys Ser
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 130 135 140 Phe Tyr Pro
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155 160
Gln Ser Gly 122189PRTHomo sapiens 122Gln Val Gln Leu Glu Gln 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 Arg Tyr 20 25 30 Gly Met His
Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Lys Trp Val 35 40 45 Ala
Val Ile Trp Tyr Asp Gly Ser Asn Lys Leu 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 Tyr Tyr Asp Asn Ser Arg His His Trp
Gly Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro
Cys Ser Arg Ser Thr Ser Glu Ser 130 135 140 Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 180 185
123157PRTHomo sapiensMISC_FEATURE(4)..(4)Xaa is Leu or Met 123Asp
Ile Gln Xaa Xaa 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 Tyr 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 Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155
124181PRTHomo sapiensMISC_FEATURE(1)..(5)Xaa is any amino acid
124Xaa Xaa Xaa Xaa Xaa Gln Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
Asn Ala 20 25 30 Trp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Gly Arg Ile Lys Arg Lys Thr Asp Gly Gly
Thr Thr Asp Tyr Ala Ala 50 55 60 Pro Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asp Ser Glu Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn
Ser Leu Glu Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Thr
Val Asp Asn Ser Gly Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 130
135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Ser 180 125159PRTHomo
sapiensMISC_FEATURE(1)..(4)Xaa is any amino acid 125Xaa Xaa Xaa Xaa
Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro
Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser 20 25 30
Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35
40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val
Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr
Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr
Tyr Cys Met Gln Ala 85 90 95 Leu Gln Thr Pro Leu Thr Phe Gly Gly
Gly Thr Lys Val Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser
Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145 150 155
126179PRTHomo sapiens 126Gln Val Gln Leu Glu Gln 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 Thr Asn Tyr 20 25 30 Gly Leu His Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Asp Trp Val 35 40 45 Ala Val Ile Trp
Tyr Asp Gly Ser His Lys Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Thr Arg Asp Leu Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
Ser 100 105 110 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Cys Ser 115 120 125 Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp 130 135 140 Tyr Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr 145 150 155 160 Ser Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr 165 170 175 Ser Leu Ser
127160PRTHomo sapiens 127Glu Thr Gln Leu Thr Gln Ser Pro Gly Thr
Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Val Thr Leu Ser Cys Arg
Ala Ser Gln Ser Val Ser Asn Asn 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 Cys Ala Glu Cys Tyr Cys Gln Gln Tyr Gly Ser Ser Leu 85
90 95 Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr
Val 100 105 110 Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
Gln Leu Lys 115 120 125 Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe Tyr Pro Arg 130 135 140 Glu Ala Lys Val Gln Trp Glu Gly Gly
Ile Thr Pro Ser Asn Arg Val 145 150 155 160 128180PRTHomo
sapiensMISC_FEATURE(62)..(62)Xaa is Tyr or Leu 128Val Gln Cys Gln
Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln 1 5 10 15 Pro Gly
Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 20 25 30
Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 35
40 45 Glu Trp Val Ala Val Ile Trp Tyr Asp Gly Ser His Lys Xaa Tyr
Xaa 50 55 60 Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn 65 70 75 80 Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala
Glu Asp Thr Ala Val 85 90 95 Tyr Tyr Ser Ala Arg Asp Tyr Tyr Asp
Thr Ser Arg His His Trp Gly 100 105 110 Phe Asp Cys Trp Gly Gln Gly
Thr Leu Val Thr Val Ser Ser Ala Ser 115 120 125 Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr 130 135 140 Ser Glu Ser
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro 145 150 155 160
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 165
170 175 His Thr Phe Pro 180 129173PRTHomo sapiens 129Gln Leu Leu
Gly Leu Leu Met Leu Trp Val Pro Gly Ser Ser Glu Glu 1 5 10 15 Ile
Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly Glu 20 25
30 Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser Glu
35 40 45 Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 50 55 60 Pro Gln Leu Leu Ile Tyr Thr Leu Ser His Arg Ala
Ser Gly Val Pro 65 70 75 80 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Lys Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Val
Gly Val Tyr Cys Cys Met Gln Arg 100 105 110 Val Glu Phe Pro Ile Thr
Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 115 120 125 Arg Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 130 135 140 Gln Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 145 150 155
160 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn 165 170
130187PRTHomo sapiensMISC_FEATURE(1)..(5)Xaa is any amino acid
130Xaa Xaa Xaa Xaa Xaa Gln Ser Gly Pro Arg Leu Val Lys Pro Ser Gln
1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser
Ser Asp 20 25 30 Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly
Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser
Thr Phe Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Ala Ile Ser
Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser
Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg Glu
Ser Pro His Ser Ser Asn Trp Tyr Ser Gly Phe Asp 100 105 110 Cys Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 115
120 125 Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
Glu 130 135 140 Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Arg Thr 145 150 155 160 Gly Asp Gly Val Val Glu Leu Arg Arg Pro
Asp Gln Arg Arg Ala His 165 170 175 Leu Pro Gly Cys Pro Thr Val Leu
Arg Thr Leu 180 185 131154PRTHomo sapiensMISC_FEATURE(1)..(4)Xaa is
any amino acid 131Xaa Xaa Xaa Xaa Thr Gln Ser Pro Asp Phe Gln Ser
Val Thr Pro Lys 1 5 10 15 Glu Lys Val Thr Ile Thr Cys Arg Ala Ser
Gln Ser Ile Gly Ser Arg 20 25 30 Leu His Trp Tyr Gln Gln Lys Pro
Asp Gln Ser Pro Lys Leu Leu Ile 35 40 45 Lys Tyr Ala Ser Gln Ser
Phe Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Asn Ser Leu Glu Ala 65 70 75 80 Glu Asp
Ala Ala Thr Tyr Tyr Cys His Gln Ser Ser Asn Leu Pro Phe 85 90 95
Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys Arg Thr Val Ala Ala 100
105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu 145
150 132178PRTHomo sapiensMISC_FEATURE(6)..(6)Xaa is Glu or Gln
132Gln Val Gln Leu Val Xaa Ala 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 Arg
Ser Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Lys Trp Val 35 40 45 Ala Val Ile Trp Tyr Asp Gly Ser Asn Lys
Xaa Tyr Thr 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 Val Arg Asp Tyr
Tyr Asp Asn Ser Arg His His Trp Gly Phe Asp Tyr 100 105 110 Trp Gly
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125
Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 130
135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Arg Arg
Arg Ala His Leu 165 170 175 Pro Gly 133156PRTHomo sapiens 133Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Arg Cys Ala Ser Val Gly 1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp
20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg
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 Arg Ser Gly Thr Glu Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Ala Tyr Tyr Cys
Leu Gln His Asn Ser Tyr Pro Pro 85 90 95 Ser Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155
134171PRTHomo sapiens 134His Val Gln Val Gln Leu Val Glu Ser Gly
Gly Gly Val Val Gln Pro 1 5 10 15 Gly Arg Ser Leu Arg Leu Ser Cys
Ala Ala Ser Gly Phe Ile Phe Ser 20 25 30 Arg Tyr Gly Met His Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Lys 35 40 45 Trp Val Ala Val
Ile Trp Tyr Asp Gly Ser Asn Lys Leu Tyr Ala Asp 50 55 60 Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr 65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr 85
90 95 Tyr Cys Ala Arg Asp Tyr Tyr Asp Asn Ser Arg His His Trp Gly
Phe 100 105 110 Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr 115 120 125 Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys
Ser Arg Ser Thr Ser 130 135 140 Glu Ser Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu 145 150 155 160 Pro Val Thr Val Ser Trp
Asn Ser Gly Ala Leu 165 170 135174PRTHomo sapiens 135Ser Ala Pro
Gly Ala Ala Asn Ala Leu Gly Pro Trp Ile Ser Glu Asp 1 5 10 15 Ile
Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly Glu 20 25
30 Pro Ala Ser Ile Ser Cys Arg Ser Ser Arg Ser Leu Leu Asp Ser Asp
35 40 45 Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly
Gln Ser 50 55 60 Pro Gln Leu Leu Ile Tyr Thr Leu Ser Tyr Arg Ala
Ser Gly Val Pro 65 70 75 80 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
Asp Phe Thr Leu Lys Ile 85 90 95 Ser Arg Val Glu Ala Glu Asp Val
Gly Val Tyr Tyr Cys Met Gln Arg 100 105 110 Val Glu Phe Pro Ile Thr
Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 115 120 125 Arg Thr Val Ala
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 130 135 140 Gln Leu
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 145 150 155
160 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala 165 170
136186PRTHomo sapiensMISC_FEATURE(1)..(4)Xaa is any amino acid
136Xaa Xaa Xaa Xaa Glu Gln 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 Gly Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val 35 40 45 Ala Val Ile Trp 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 Phe
Tyr Asp Ser Ser Arg Tyr His Tyr Gly Met Asp Val 100 105 110 Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125
Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 130
135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Ser
180 185 137143PRTHomo sapiensMISC_FEATURE(1)..(4)Xaa is any amino
acid 137Xaa Xaa Xaa Xaa Thr Gln Cys Pro Leu Ser Leu Pro Val Thr Pro
Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu
Leu Asp Ser 20 25 30 Asp Asp Gly Asn Thr Tyr Leu Asp Trp Tyr Leu
Gln Lys Pro Gly Gln 35 40 45 Ser Pro Gln Leu Leu Ile Tyr Thr Val
Ser Tyr Arg Ala Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Phe Thr Leu Lys 65 70 75 80 Ile Ser Arg Val Glu
Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln 85 90 95 Arg Ile Glu
Phe Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile 100 105 110 Lys
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 115 120
125 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn 130
135 140 13810PRTHomo sapiens 138Gly Phe Thr Phe Thr Asn Tyr Gly Leu
His 1 5 10 13917PRTHomo sapiens 139Val Ile Trp Tyr Asp Gly Ser His
Lys Phe Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 1404PRTHomo sapiens
140Asp Leu Asp Tyr 1 14112PRTHomo sapiens 141Arg Ala Ser Gln Ser
Val Ser Asn Asn Tyr Leu Ala 1 5 10 1427PRTHomo sapiens 142Gly Ala
Ser Ser Arg Ala Thr 1 5 14310PRTHomo sapiens 143Gln Gln Tyr Gly Ser
Ser Leu Pro Leu Thr 1 5 10 14410PRTHomo sapiens 144Gly Phe Thr Phe
Ser Ser Tyr Gly Met Tyr 1 5 10 14517PRTHomo sapiens 145Val Ile Trp
Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly
14614PRTHomo sapiens 146Asp Phe Tyr Asp Ser Ser Arg Tyr His Tyr Gly
Met Asp Val 1 5 10 14717PRTHomo sapiens 147Arg Ser Ser Gln Ser Leu
Leu Asp Ser Asp Asp Gly Asn Thr Tyr Leu 1 5 10 15 Asp 1487PRTHomo
sapiens 148Thr Val Ser Tyr Arg Ala Ser 1 5 1499PRTHomo sapiens
149Met Gln Arg Ile Glu Phe Pro Ile Thr 1 5 15012PRTHomo sapiens
150Gly Gly Ser Ile Ser Ser Asp Gly Tyr Tyr Trp Ser 1 5 10
15116PRTHomo sapiens 151Tyr Ile Tyr Tyr Ser Gly Ser Thr Phe Tyr Asn
Pro Ser Leu Lys Ser 1 5 10 15 15214PRTHomo sapiens 152Glu Ser Pro
His Ser Ser Asn Trp Tyr Ser Gly Phe Asp Cys 1 5 10 15311PRTHomo
sapiens 153Arg Ala Ser Gln Ser Ile Gly Ser Arg Leu His 1 5 10
1547PRTHomo sapiens 154Tyr Ala Ser Gln Ser Phe Ser 1 5 1559PRTHomo
sapiens 155His Gln Ser Ser Asn Leu Pro Phe Thr 1 5 15610PRTHomo
sapiens 156Gly Phe Ile Phe Ser Arg Tyr Gly Met His 1 5 10
15717PRTHomo sapiens 157Val Ile Trp Tyr Asp Gly Ser Asn Lys Leu Tyr
Ala Asp Ser Val Lys 1 5 10 15 Gly 15814PRTHomo sapiens 158Asp Tyr
Tyr Asp Asn Ser Arg His His Trp Gly Phe Asp Tyr 1 5 10 15917PRTHomo
sapiens 159Arg Ser Ser Arg Ser Leu Leu Asp Ser Asp Asp Gly Asn Thr
Tyr Leu 1 5 10 15 Asp 1607PRTHomo sapiens 160Thr Leu Ser Tyr Arg
Ala Ser 1 5 1619PRTHomo sapiens 161Met Gln Arg Val Glu Phe Pro Ile
Thr 1 5 16210PRTHomo sapiens 162Gly Phe Thr Phe Ser Arg Tyr Gly Met
His 1 5 10 16311PRTHomo sapiens 163Arg Ala Ser Gln Ser Ile Tyr Ser
Tyr Leu Asn 1 5 10 1647PRTHomo sapiens 164Ala Ala Ser Ser Leu Gln
Ser 1 5 1659PRTHomo sapiens 165Gln Gln Ser Tyr Ser Thr Pro Pro Thr
1 5 16610PRTHomo sapiens 166Gly Phe Thr Phe Arg Ser Tyr Gly Met His
1 5 10 16717PRTHomo sapiens 167Val Ile Trp Tyr Asp Gly Ser Asn Lys
Tyr Tyr Thr Asp Ser Val Lys 1 5 10 15 Gly 16811PRTHomo sapiens
168Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Ala 1 5 10 1699PRTHomo
sapiens 169Leu Gln His Asn Ser Tyr Pro Pro Ser 1 5 17010PRTHomo
sapiens 170Gly Phe Thr Phe Ser Ser Tyr Gly Met His 1 5 10
17117PRTHomo sapiens 171Val Ile Trp Tyr Asp Gly Ser His Lys Tyr Tyr
Ala Asp Ser Val Lys 1 5 10 15 Gly 17214PRTHomo sapiens 172Asp Tyr
Tyr Asp Thr Ser Arg His His Trp Gly Phe Asp Cys 1 5 10 17317PRTHomo
sapiens 173Arg Ser Ser Gln Ser Leu Leu Asp Ser Glu Asp Gly Asn Thr
Tyr Leu 1 5 10 15 Asp 1747PRTHomo sapiens 174Thr Leu Ser His Arg
Ala Ser 1 5 17510PRTHomo sapiens 175Gly Phe Thr Phe Ser Asn Ala Trp
Met Thr 1 5 10 17619PRTHomo sapiens 176Arg Ile Lys Arg Arg Thr Asp
Gly Gly Thr Thr Asp Tyr Ala Ala Pro 1 5 10 15 Val Lys Gly
1777PRTHomo sapiens 177Val Asp Asn Asp Val Asp Tyr 1 5 17816PRTHomo
sapiens 178Arg Ser Ser Gln Ser Leu Leu His Ser Asn Gly Tyr Asn Tyr
Leu Asp 1 5 10 15 1797PRTHomo sapiens 179Leu Gly Ser Asn Arg Ala
Ser 1 5 1809PRTHomo sapiens 180Met Gln Ala Leu Gln Thr Pro Leu Thr
1 5 18112PRTHomo sapiens 181Gly Gly Ser Val Ser Ser Gly Gly Tyr Tyr
Trp Ser 1 5 10 18216PRTHomo sapiens 182Phe Ile Tyr Tyr Thr Gly Ser
Thr Asn Tyr Asn Pro Ser Leu Lys Ser 1 5 10 15 18310PRTHomo sapiens
183Asp Tyr Asp Trp Ser Phe His Phe Asp Tyr 1 5 10 18411PRTHomo
sapiens 184Arg Ala Ser Gln Gly Ile Arg Asn Asp Leu Gly 1 5 10
1859PRTHomo sapiens 185Leu Gln His Asn Ser Tyr Pro Leu Thr 1 5
18619PRTHomo sapiens 186Arg Ile Lys Arg Lys Thr Asp Gly Gly Thr Thr
Asp Tyr Ala Ala Pro 1 5 10 15 Val Lys Gly 1877PRTHomo sapiens
187Val Asp Asn Ser Gly Asp Tyr 1 5 18810PRTHomo sapiens 188Gly Phe
Thr Phe Thr Asn Tyr Trp Met Ser 1 5 10 18917PRTHomo sapiens 189Asn
Ile Gln Gln Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val Arg 1 5 10
15 Gly 1903PRTHomo sapiens 190Trp Asp Tyr 1 19116PRTHomo sapiens
191Arg Ser Ser Gln Ser Leu Val His Ser Asp Gly Asn Thr Tyr Leu Asn
1 5 10 15 1927PRTHomo sapiens 192Met Ile Ser Asn Arg Phe Ser 1 5
1939PRTHomo sapiens 193Met Gln Ala Thr Glu Ser Pro Gln Thr 1 5
19410PRTHomo sapiens 194Gly Phe Thr Phe Ser Thr Tyr Ser Met Asn 1 5
10 19517PRTHomo sapiens 195Tyr Ile Arg Ser Ser Thr Ser Thr Ile Tyr
Tyr Ala Glu Ser Leu Lys 1 5 10 15 Gly 1964PRTHomo sapiens 196Asp
Phe Asp Tyr 1 19716PRTHomo sapiens 197Arg Ser Ser Gln Ser Leu Val
His Ser Asp Gly Asp Thr Tyr Leu Asn 1 5 10 15 1987PRTHomo sapiens
198Lys Ile Ser Thr Arg Phe Ser 1 5 19910PRTHomo sapiens 199Met Gln
Thr Thr Gln Ile Pro Gln Ile Thr 1 5 10
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