U.S. patent application number 14/344354 was filed with the patent office on 2014-12-25 for t cell receptors recognizing hla-a1- or hla-cw7-restricted mage.
This patent application is currently assigned to The United States of America, as represented by the Secretary, Dept. of Health and Human Services. The applicant listed for this patent is The United States of America, as represented by the Secretary, Dept. of Health and Human Services, The United States of America, as represented by the Secretary, Dept. of Health and Human Services. Invention is credited to Steven A. Feldman, Richard A. Morgan, Paul F. Robbins, Steven A. Rosenberg, Shiqui Zhu.
Application Number | 20140378389 14/344354 |
Document ID | / |
Family ID | 46881176 |
Filed Date | 2014-12-25 |
United States Patent
Application |
20140378389 |
Kind Code |
A1 |
Robbins; Paul F. ; et
al. |
December 25, 2014 |
T CELL RECEPTORS RECOGNIZING HLA-A1- OR HLA-CW7-RESTRICTED MAGE
Abstract
The invention provides an isolated or purified T cell receptor
(TCR) having antigenic specificity for a) melanoma antigen family A
(MAGE A)-3 in the context of HLA-A1 or b) MAGE-A12 in the context
of HLA-Cw7. The invention further provides related polypeptides and
proteins, as well as related nucleic acids, recombinant expression
vectors, host cells, and populations of cells. Further provided by
the invention are antibodies, or an antigen binding portion
thereof, and pharmaceutical compositions relating to the TCRs of
the invention. Methods of detecting the presence of cancer in a
host and methods of treating or preventing cancer in a host are
further provided by the invention.
Inventors: |
Robbins; Paul F.; (Chevy
Chase, MD) ; Rosenberg; Steven A.; (Potomac, MD)
; Zhu; Shiqui; (Potomac, MD) ; Feldman; Steven
A.; (Washington, DC) ; Morgan; Richard A.;
(Columbia, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Dept. of Health and Human Services |
Bethesda |
MD |
US |
|
|
Assignee: |
The United States of America, as
represented by the Secretary, Dept. of Health and Human
Services
Bethesda
MD
|
Family ID: |
46881176 |
Appl. No.: |
14/344354 |
Filed: |
September 11, 2012 |
PCT Filed: |
September 11, 2012 |
PCT NO: |
PCT/US12/54623 |
371 Date: |
April 22, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61535086 |
Sep 15, 2011 |
|
|
|
Current U.S.
Class: |
514/19.3 ;
435/320.1; 435/325; 435/7.23; 530/350; 530/387.9; 530/389.1;
536/23.5 |
Current CPC
Class: |
A61K 2039/5158 20130101;
C07K 14/7051 20130101; A61P 35/02 20180101; G01N 33/57407 20130101;
G01N 33/574 20130101; C07K 2317/73 20130101; C07K 2318/20 20130101;
C07K 16/2803 20130101; A61P 35/00 20180101; C07K 14/70503
20130101 |
Class at
Publication: |
514/19.3 ;
530/350; 530/389.1; 536/23.5; 530/387.9; 435/320.1; 435/325;
435/7.23 |
International
Class: |
C07K 14/705 20060101
C07K014/705; G01N 33/574 20060101 G01N033/574; C07K 16/28 20060101
C07K016/28 |
Claims
1. An isolated or purified T cell receptor (TCR) having antigenic
specificity for a) melanoma antigen family A (MAGE A)-3 in the
context of HLA-A1 or b) MAGE-A12 in the context of HLA-Cw7.
2. The isolated or purified TCR of claim 1 having antigenic
specificity for a) a MAGE-A3 epitope comprising EVDPIGHLY (SEQ ID
NO: 2) or b) a MAGE-A12 epitope comprising VRIGHLYIL (SEQ ID NO:
4).
3. The isolated or purified TCR of claim 2, comprising the amino
acid sequences of SEQ ID NOs: 5-10, 16-21, 26-31, or 36-41.
4. The isolated or purified TCR of claim 3, comprising the amino
acid sequences of SEQ ID NOs: 11-12, 22-23, 32-33, or 42-43.
5. The isolated or purified TCR of claim 4, comprising the amino
acid sequences of SEQ ID NOs: 13-14, 24-25, 34-35, or 44-45.
6. An isolated or purified polypeptide comprising a functional
portion of the TCR of claim 1, wherein the functional portion
comprises the amino acid sequences of SEQ ID NOs: 5-10, 16-21,
26-31, or 36-41.
7. The isolated or purified polypeptide of claim 6, wherein the
portion comprises the amino acid sequences of SEQ ID NOs: 11-12,
22-23, 32-33, or 42-43.
8. The isolated or purified polypeptide of claim 7, wherein the
portion comprises the amino acid sequences of SEQ ID NOs: 13-14,
24-25, 34-35, or 44-45.
9. An isolated or purified protein comprising at least one of the
polypeptides of claim 6.
10. The isolated or purified protein of claim 9, comprising: a
first polypeptide chain comprising the amino acid sequence of SEQ
ID NOs: 5-7, 16-18, 26-28, or 36-38; and a second polypeptide chain
comprising the amino acid sequence of SEQ ID NOs: 8-10, 19-21,
29-31, or 39-41.
11. The isolated or purified protein of claim 10, comprising: a
first polypeptide chain comprising the amino acid sequence of SEQ
ID NO: 11, 22, 32, or 42; and a second polypeptide chain comprising
the amino acid sequence of SEQ ID NO: 12, 23, 33, or 43.
12. The isolated or purified protein of claim 11, comprising: a
first polypeptide chain comprising the amino acid sequence of SEQ
ID NO: 13, 24, 34, or 44; and a second polypeptide chain comprising
the amino acid sequence of SEQ ID NO: 14, 25, 35, or 45.
13. The protein of claim 9, wherein the protein is a fusion
protein.
14. The protein of claim 9, wherein the protein is a recombinant
antibody.
15. An isolated or purified nucleic acid comprising a nucleotide
sequence encoding the TCR of claim 1.
16. The nucleic acid of claim 15, comprising a nucleotide sequence
comprising any of SEQ ID NOs: 46-49.
17. An isolated or purified nucleic acid comprising a nucleotide
sequence which is complementary to the nucleotide sequence of the
nucleic acid of claim 16.
18. An isolated or purified nucleic acid comprising a nucleotide
sequence which hybridizes under stringent conditions to the
nucleotide sequence of the nucleic acid of claim 15.
19. A recombinant expression vector comprising the nucleic acid of
claim 15.
20. An isolated host cell comprising the recombinant expression
vector of claim 19.
21. The isolated host cell of claim 20, wherein the cell is a
peripheral blood lymphocyte (PBL).
22. The isolated host cell of claim 21, wherein the PBL is a T
cell.
23. The isolated host cell of claim 20, wherein the cell is a tumor
infiltrating lymphocyte (TIL).
24. A population of cells comprising at least one host cell of
claim 20.
25. An antibody, or antigen binding portion thereof, which
specifically binds to a functional portion of the TCR of claim 1,
wherein the functional portion comprises the amino acid sequences
of SEQ ID NOs: 5-10, 16-21, 26-31, or 36-41.
26. A pharmaceutical composition comprising the TCR of claim 1, and
a pharmaceutically acceptable carrier.
27. A method of detecting the presence of cancer in a host,
comprising: (i) contacting a sample comprising cells of the cancer
with the TCR of claim 1, thereby forming a complex, and (ii)
detecting the complex, wherein detection of the complex is
indicative of the presence of cancer in the host.
28. A method of treating or preventing cancer in a host, comprising
administering to the host the TCR of claim 1, in an amount
effective to treat or prevent cancer in the host.
29. The method of claim 27, wherein the cancer is melanoma, breast
cancer, leukemia, thyroid cancer, gastric cancer, pancreatic
cancer, liver cancer, lung cancer, ovarian cancer, multiple
myeloma, esophageal cancer, kidney cancer, head cancers, neck
cancers, prostate cancer, or urothelial cancer.
30. The method of claim 27, wherein the host cell is a cell that is
autologous to the host.
31. The method of claim 27, wherein the cells of the population are
cells that are autologous to the host.
32-35. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims priority to U.S. Application No.
61/535,086, filed on Sep. 15, 2011, which is incorporated by
reference herein in its entirety.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
[0002] Incorporated by reference in its entirety herein is a
computer-readable nucleotide/amino acid sequence listing submitted
concurrently herewith and identified as follows: One 52,162 Byte
ASCII (Text) file named "710922ST25.TXT," dated Aug. 22, 2012.
BACKGROUND OF THE INVENTION
[0003] Adoptive cell therapy (ACT) involves the transfer of
reactive T cells into patients, including the transfer of
tumor-reactive T cells into cancer patients. Adoptive cell therapy
using T-cells that target human leukocyte antigen (HLA)-A2
restricted T-cell epitopes has been successful in causing the
regression of tumors in some patients. However, patients that lack
HLA-A2 expression cannot be treated with T-cells that target HLA-A2
restricted T-cell epitopes. Such a limitation creates an obstacle
to the widespread application of adoptive cell therapy.
Accordingly, there exists a need for improved immunological
compositions and methods for treating cancer.
BRIEF SUMMARY OF THE INVENTION
[0004] The invention provides an isolated or purified T cell
receptor (TCR) having antigenic specificity for a) melanoma antigen
family A (MAGE A)-3 in the context of HLA-A1 or b) MAGE-A12 in the
context of HLA-Cw7. The invention further provides related
polypeptides and proteins, as well as related nucleic acids,
recombinant expression vectors, host cells, and populations of
cells. Further provided by the invention are antibodies, or antigen
binding portions thereof, and pharmaceutical compositions relating
to the TCRs of the invention.
[0005] Methods of detecting the presence of cancer in a host and
methods of treating or preventing cancer in a host are further
provided by the invention. The inventive method of detecting the
presence of cancer in a host comprises (i) contacting a sample
comprising cells of the cancer with any of the inventive TCRs,
polypeptides, proteins, nucleic acids, recombinant expression
vectors, host cells, populations of host cells, or antibodies, or
antigen binding portions thereof, described herein, thereby forming
a complex, and (ii) detecting the complex, wherein detection of the
complex is indicative of the presence of cancer in the host.
[0006] The inventive method of treating or preventing cancer in a
host comprises administering to the host any of the TCRs,
polypeptides, or proteins described herein, any nucleic acid or
recombinant expression vector comprising a nucleotide sequence
encoding any of the TCRs, polypeptides, proteins described herein,
or any host cell or population of host cells comprising a
recombinant vector which encodes any of the TCRs, polypeptides, or
proteins described herein, in an amount effective to treat or
prevent cancer in the host.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
[0007] FIG. 1A is a bar graph showing interferon (IFN)-.gamma.
secretion (pg/ml) of untransduced (UT) cells (black bars) or cells
transduced with anti-MAGE-A3 TCR A10 (SEQ ID NO: 46) (unshaded
bars) or anti-MAGE-A3 TCR 13-18 (SEQ ID NO: 48) (grey bars) in
response to co-culture with various tumor cell lines.
[0008] FIG. 1B is a bar graph showing IFN-.gamma. secretion (pg/ml)
of UT cells or cells transduced with anti-MAGE-A3 TCR A10 (SEQ ID
NO: 46) or anti-MART-1 TCR DMF5 in response to co-culture with
HLA-A1+/MAGE-A3+ fresh tumors FrTu 2767 (black bars), FrTu 3178
(grey bars), FrTu 2823 (unshaded bars) or FrTu 3068 (diagonal lined
bars) or HLA-A*0201+/MART-1+ fresh tumors FrTu 2851 (horizontal
lined bars) or FrTu 3242 (vertical lined bar). Checkered bars
indicate cells co-cultured with no tumor cells.
[0009] FIGS. 2A and 2B are bar graphs showing IFN-.gamma. secretion
(pg/ml) of cells from first (FIG. 2A) and second (FIG. 2B) donors
transduced with a control construct encoding the truncated human
low affinity nerve growth factor receptor (NGFR) (black bars),
anti-MAGE-A12 TCR 502 (SEQ ID NO: 47) (unshaded bars), or
anti-MAGE-A12 TCR FM8 (SEQ ID NO: 49) (grey bars) in response to
co-culture with various tumor cell lines.
[0010] FIG. 3A is a bar graph illustrating the cumulative
percentage of the normal Caucasian population that expresses HLA-A1
(unshaded portion of bar), HLA-A2 (grey portion of bar), and/or
HLA-Cw7 (black portion of bar).
[0011] FIGS. 3B and 3C are bar graphs illustrating the cumulative
percentage of the human melanoma (FIG. 3B) and synovial cell
sarcoma (FIG. 3C) patient populations that would be expected to
express HLA-A2 and NY-ESO-1 (diagonal lined portion of bar); HLA-A1
and MAGE-A3 (unshaded portion of bar); HLA-A2, MAGE-A3, and
MAGE-A12 (grey portion of bar); and/or HLA-Cw7 and MAGE-A12 (black
portion of bar).
[0012] FIG. 4 is a bar graph showing IFN-.gamma. secretion (pg/ml)
of cells transduced with NGFR (black bars), anti-MAGE-A12 TCR 502
(SEQ ID NO: 47) (unshaded bars), or anti-MAGE-A12 TCR FM8 (SEQ ID
NO: 49) (grey bars) in response to co-culture with HLA-Cw*0701 and
HLA-Cw*0702 target cells pulsed with peptides from MAGE-A12
(VRIGHLYIL; SEQ ID NO: 4), MAGE-A2 (VPISHLYIL; SEQ ID NO: 50),
MAGE-A3 (DPIGHLYIF; SEQ ID NO: 51), MAGE-A6 (DPIGHVYIF; SEQ ID NO:
52), or control peptide (EDGCPAAEK; SEQ ID NO: 53).
[0013] FIGS. 5A-5D are line graphs showing percent lysis of 397 mel
(A), 624 mel (B), 2984 mel (C), and 2661 RCC (D) cells by PBMC that
were untransduced (closed circles) or transduced with anti-MAGE-A12
TCR 502 (SEQ ID NO: 47) (), anti-MAGE-A12 TCR FM8 (SEQ ID NO: 49)
(diamonds), anti-MAGE-A3 TCR A10 (SEQ ID NO: 46) (squares),
anti-MAGE-A3 TCR 13-18 (SEQ ID NO: 48) (.tangle-solidup.), or
anti-MAGE-A3 TCR 112-120 (open circles) at the indicated effector
to target (E:T) ratios. Representative results from one of two
independent experiments are presented.
[0014] FIG. 6A is a bar graph showing IFN-.gamma. secretion (pg/ml)
of untransduced (control) cells (striped bars) or cells transduced
with anti-MAGE-A3 TCR A10 (SEQ ID NO: 46) (shaded bars) or
anti-MAGE-A3 TCR 13-18 (SEQ ID NO: 48) (checkered bars) in response
to co-culture with various tumor cell lines. Representative results
from two of three independent experiments assessing responses of T
cells transduced with these TCRs are presented.
[0015] FIG. 6B is a bar graph showing estimated relative copies of
vector DNA measured for cells transduced with anti-MAGE-A3 TCR A10
(SEQ ID NO: 46), anti-MAGE-A3 TCR 13-18 (SEQ ID NO: 48),
anti-MAGE-A12 TCR 502 (SEQ ID NO: 47), or anti-MAGE-A12 TCR FM8
(SEQ ID NO: 49).
[0016] FIG. 6C is a line graph showing the amount of IFN-gamma
secreted by cells transduced with anti-MAGE-A3 TCR A10 (SEQ ID NO:
46) (circles) or anti-MAGE-A3 TCR 13-18 (SEQ ID NO: 48) (squares)
in response to co-culture with target cells incubated with various
concentrations of MAGE-A3 168-176 peptide.
[0017] FIG. 6D is a bar graph showing IFN-.gamma. secretion (pg/ml)
of cells transduced with anti-MAGE-A12 TCR 502 (SEQ ID NO: 47)
(shaded bars) or anti-MAGE-A12 TCR FM8 (SEQ ID NO: 49) (checkered
bars) in response to co-culture with various tumor cell lines.
Representative results from two of three independent experiments
assessing responses of T cells transduced with these TCRs are
presented.
[0018] FIG. 6E is a line graph showing the amount of IFN-gamma
secreted by cells transduced with anti-MAGE-A12 TCR 502 (SEQ ID NO:
47) (circles) or anti-MAGE-A12 TCR FM8 (SEQ ID NO: 49) (squares) in
response to co-culture with target cells incubated with various
concentrations of MAGE-A12:170-178 peptide.
[0019] FIG. 6F is a bar graph showing IFN-.gamma. secretion (pg/ml)
of cells untransduced (control) (striped bars) or transduced with
anti-MAGE-A12 TCR 502 (SEQ ID NO: 47) (shaded bars) or
anti-MAGE-A12 TCR FM8 (SEQ ID NO: 49) (checkered bars) in response
to co-culture with various tumor cell lines. Representative results
from two of three independent experiments assessing responses of T
cells transduced with these TCRs are presented.
[0020] FIG. 7A is a bar graph showing IFN-.gamma. secretion (pg/ml)
of untransduced cells (control) (striped bars) or cells transduced
with anti-MAGE-A3 TCR A10 (SEQ ID NO: 46) (shaded bars) or
anti-MAGE-A3 TCR 13-18 (checkered bars) in response to co-culture
with various fresh uncultured tumors. Representative results from
one of three independent experiments assessing responses of T cells
transduced with these TCRs are presented.
[0021] FIG. 7B is a bar graph showing IFN-.gamma. secretion (pg/ml)
of untransduced cells (control) (striped bars) or cells transduced
with anti-MAGE-A12 TCR 502 (SEQ ID NO: 47) (shaded bars) or
anti-MAGE-A12 TCR FM8 (SEQ ID NO: 49) (checkered bars) in response
to co-culture with various fresh uncultured tumors. Representative
results from one of three independent experiments assessing
responses of T cells transduced with these TCRs are presented.
[0022] FIG. 8A is a bar graph showing IFN-.gamma. secretion (pg/ml)
of untransduced cells (control) (striped bars) or cells transduced
with anti-MAGE-A3 TCR A10 (SEQ ID NO: 46) (shaded bars) or
anti-MAGE-A3 TCR 13-18 (checkered bars) co-cultured with target
cells transfected with HLA-A*01 plus either MAGE-A3, A1, A2, A4,
A6, A9, A10 or A12 overnight.
[0023] FIG. 8B is a bar graph showing IFN-.gamma. secretion (pg/ml)
of untransduced cells (control) (striped bars) or cells transduced
with anti-MAGE-A12 TCR 502 (SEQ ID NO: 47) (shaded bars) or
anti-MAGE-A12 TCR FM8 (SEQ ID NO: 49) (checkered bars) co-cultured
with target cells transfected with HLA-C*07:02 plus either MAGE-A3,
A1, A2, A4, A6, A9, A10 or A12 overnight.
[0024] FIG. 8C is a bar graph showing IFN-.gamma. secretion (pg/ml)
of untransduced cells (control) (striped bars) or cells transduced
with anti-MAGE-A12 TCR 502 (SEQ ID NO: 47) (shaded bars) or
anti-MAGE-A12 TCR FM8 (SEQ ID NO: 49) (checkered bars) co-cultured
with target cells transfected with HLA-C*07:01 plus either MAGE-A3,
A1, A2, A4, A6, A9, A10 or A12 overnight.
[0025] FIGS. 9A and 9B are bar graphs showing IFN-gamma secretion
of CD8+ (FIG. 9A) or CD4+ cells (FIG. 9B) that were untransduced
(control) (striped bars) or cells transduced with anti-MAGE-A3 TCR
A10 (SEQ ID NO: 46) (shaded bars) or anti-MAGE-A3 TCR 13-18
(checkered bars) co-cultured with various tumor targets.
Representative results from one of two independent experiments are
presented.
[0026] FIG. 9C is a bar graph showing IFN-gamma secretion of CD8+
cells that were untransduced (control) (striped bars) or cells
transduced with anti-MAGE-A12 TCR 502 (SEQ ID NO: 47) (shaded bars)
or anti-MAGE-A12 TCR FM8 (SEQ ID NO: 49) (checkered bars)
co-cultured with various tumor targets. Representative results from
one of two independent experiments are presented.
DETAILED DESCRIPTION OF THE INVENTION
[0027] An embodiment of the invention provides a T cell receptor
(TCR) having antigenic specificity for a) melanoma antigen family A
(MAGE A)-3 (also known as MAGE-3) in the context of HLA-A1 or b)
MAGE-A12 (also known as MAGE-12) in the context of HLA-Cw7.
[0028] MAGE-A3 and MAGE-A12 are members of the MAGE-A family of
twelve homologous proteins also including MAGE-A1, MAGE-A2,
MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, and
MAGE-A11. The MAGE-A proteins are cancer testis antigens (CTA),
which are expressed only in tumor cells and non-MHC expressing germ
cells of the testis and placenta. MAGE-A proteins are expressed in
a variety of human cancers including, but not limited to, melanoma,
breast cancer, leukemia, thyroid cancer, gastric cancer, pancreatic
cancer, liver cancer (e.g., hepatocellular carcinoma), lung cancer
(e.g., non-small cell lung carcinoma), ovarian cancer, multiple
myeloma, esophageal cancer, kidney cancer, head cancers (e.g.,
squamous cell carcinoma), neck cancers (e.g., squamous cell
carcinoma), prostate cancer, and urothelial cancer.
[0029] The TCRs of the invention provide many advantages, including
when used for adoptive cell transfer. For example, by targeting a)
MAGE-A3 that is presented in the context of HLA-A1 or b) MAGE-A12
that is presented in the context of HLA-Cw7, the inventive TCRs
make it possible to treat patients who are unable to be treated
using TCRs that target MAGE antigens that are presented in the
context of other HLA molecules, e.g., HLA-A2. Because HLA-A1 and
HLA-Cw7 are highly prevalent alleles, the inventive TCRs
advantageously greatly expand the patient population that can be
treated. Additionally, without being bound by a particular theory,
it is believed that because MAGE-A3 and/or MAGE-A12 are expressed
by cells of multiple cancer types, the inventive TCRs
advantageously provide the ability to destroy cells of multiple
types of cancer and, accordingly, treat or prevent multiple types
of cancer. Additionally, without being bound to a particular
theory, it is believed that because the MAGE-A proteins are cancer
testis antigens that are expressed only in tumor cells and non-MHC
expressing germ cells of the testis and placenta, the inventive
TCRs advantageously target the destruction of cancer cells while
minimizing or eliminating the destruction of normal, non-cancerous
cells, thereby reducing, for example, by minimizing or eliminating,
toxicity.
[0030] The phrase "antigenic specificity" as used herein means that
the TCR can specifically bind to and immunologically recognize
MAGE-A3 or MAGE-A12 with high avidity. For example, a TCR may be
considered to have "antigenic specificity" for MAGE-A3 or MAGE-A12
if T cells expressing the TCR secrete at least about 200 pg/ml or
more (e.g., 200 pg/ml or more, 300 pg/ml or more, 400 pg/ml or
more, 500 pg/ml or more, 600 pg/ml or more, 700 pg/ml or more, 1000
pg/ml or more, 5,000 pg/ml or more, 7,000 pg/ml or more, 10,000
pg/ml or more) of IFN-.gamma. upon co-culture with antigen-negative
HLA-A1+ target cells or HLA-Cw7+ target cells, respectively, pulsed
with a low concentration of MAGE-A3 peptide or MAGE-A12 peptide,
respectively (e.g., about 0.05 ng/ml to about 5 ng/ml, 0.05 ng/ml,
0.1 ng/ml, 0.5 ng/ml, 1 ng/ml, or 5 ng/ml). Alternatively or
additionally, a TCR may be considered to have "antigenic
specificity" for MAGE-A3 or MAGE-A12 if T cells expressing the TCR
secrete at least twice as much IFN-.gamma. as the untransduced PBL
background level of IFN-.gamma. upon co-culture with
antigen-negative HLA-A1+ target cells or HLA-Cw7+ target cells,
respectively, pulsed with a low concentration of MAGE-A3 peptide or
MAGE-A12 peptide, respectively. The inventive TCRs may also secrete
IFN-.gamma. upon co-culture with antigen-negative HLA-A1+ target
cells or HLA-Cw7+ target cells pulsed with higher concentrations of
MAGE-A3 peptide or MAGE-A12 peptide, respectively.
[0031] An embodiment of the invention provides a TCR with antigenic
specificity for any MAGE-A3 protein, polypeptide or peptide. The
inventive TCR may have antigenic specificity for a MAGE-A3 protein
comprising, consisting of, or consisting essentially of, SEQ ID NO:
1. In a preferred embodiment of the invention, the TCR has
antigenic specificity for a MAGE-A3 168-176 peptide comprising,
consisting of, or consisting essentially of, EVDPIGHLY (SEQ ID NO:
2).
[0032] The inventive TCRs are able to recognize MAGE-A3 in a human
leukocyte antigen (HLA)-A1-dependent manner. By "HLA-A1-dependent
manner" as used herein means that the TCR elicits an immune
response upon binding to a MAGE-A3 cancer antigen within the
context of an HLA-A1 molecule. The inventive TCRs are able to
recognize MAGE-A3 that is presented by an HLA-A1 molecule and may
bind to the HLA-A1 molecule in addition to MAGE-A3. Exemplary
HLA-A1 molecules, in the context of which the inventive TCRs
recognize MAGE-A3, include those encoded by the HLA-A*0101,
HLA-A*0102, and/or HLA-A*0103 alleles.
[0033] An embodiment of the invention provides a TCR with antigenic
specificity for any MAGE-A12 protein, polypeptide or peptide. The
inventive TCR may have antigenic specificity for a MAGE-A12 protein
comprising, consisting of, or consisting essentially of, SEQ ID NO:
3. In a preferred embodiment of the invention, the TCR has
antigenic specificity for a MAGE-A12 170-178 peptide comprising,
consisting of, or consisting essentially of, VRIGHLYIL (SEQ ID NO:
4).
[0034] The inventive TCRs are able to recognize MAGE-A12 in an
HLA-Cw7-dependent manner. By "HLA-Cw7-dependent manner" as used
herein means that the TCR elicits an immune response upon binding
to a MAGE-A12 cancer antigen within the context of an HLA-Cw7
molecule. The inventive TCRs are able to recognize MAGE-A12 that is
presented by an HLA-Cw7 molecule and may bind to the HLA-Cw7
molecule in addition to MAGE-A12. Exemplary HLA-Cw7 molecules, in
the context of which the inventive TCRs recognize MAGE-A12, include
those encoded by the HLA-Cw*0701 and/or HLA-Cw*0702 alleles.
[0035] The invention provides a TCR comprising two polypeptides
(i.e., polypeptide chains), such as an alpha (.alpha.) chain of a
TCR, a beta (.beta.) chain of a TCR, a gamma (.gamma.) chain of a
TCR, a delta (.delta.) chain of a TCR, or a combination thereof.
Such polypeptide chains of TCRs are known in the art. The
polypeptides of the inventive TCR can comprise any amino acid
sequence, provided that the TCR has antigenic specificity for a)
MAGE-A3 in the context of HLA-A1 or b) MAGE-A12 in the context of
HLA-Cw7.
[0036] In an embodiment of the invention, the TCR comprises two
polypeptide chains, each of which comprises a variable region
comprising a complementarity determining region (CDR) 1, a CDR2,
and a CDR3 of a TCR. In an embodiment of the invention, the TCR has
antigenic specificity for MAGE-A3 168-176 and comprises a first
polypeptide chain comprising a CDR1 comprising the amino acid
sequence of SEQ ID NO: 5 or 16 (CDR1 of a chain), a CDR2 comprising
the amino acid sequence of SEQ ID NO: 6 or 17 (CDR2 of a chain),
and a CDR3 comprising the amino acid sequence of SEQ ID NO: 7 or 18
(CDR3 of a chain), and a second polypeptide chain comprising a CDR1
comprising the amino acid sequence of SEQ ID NO: 8 or 19 (CDR1 of
.beta. chain), a CDR2 comprising the amino acid sequence of SEQ ID
NO: 9 or 20 (CDR2 of .beta. chain), and a CDR3 comprising the amino
acid sequence of SEQ ID NO: 10 or 21 (CDR3 of .beta. chain). In
another embodiment of the invention, the TCR has antigenic
specificity for MAGE-A12 170-178, and comprises a first polypeptide
chain comprising a CDR1 comprising the amino acid sequence of SEQ
ID NO: 26 or 36 (CDR1 of .alpha. chain), a CDR2 comprising the
amino acid sequence of SEQ ID NO: 27 or 37 (CDR2 of .alpha. chain),
and a CDR3 comprising the amino acid sequence of SEQ ID NO: 28 or
38 (CDR3 of .alpha. chain), and a second polypeptide chain
comprising a CDR1 comprising the amino acid sequence of SEQ ID NO:
29 or 39 (CDR1 of .beta. chain), a CDR2 comprising the amino acid
sequence of SEQ ID NO: 30 or 40 (CDR2 of .beta. chain), and a CDR3
comprising the amino acid sequence of SEQ ID NO: 31 or 41 (CDR3 of
.beta. chain). In this regard, the inventive TCR can comprise any
one or more of the amino acid sequences selected from the group
consisting of any one or more of SEQ ID NOs: 5-7, 8-10, 16-18,
19-21, 26-28, 29-31, 36-38, and 39-41. Preferably the TCR comprises
the amino acid sequences of SEQ ID NOs: 5-10, 16-21, 26-31, or
36-41. More preferably the TCR comprises the amino acid sequences
of SEQ ID NOs: 5-10 or 26-31.
[0037] Alternatively or additionally, the TCR can comprise an amino
acid sequence of a variable region of a TCR comprising the CDRs set
forth above. In this regard, the TCR with antigenic specificity for
MAGE-A3 168-176 can comprise the amino acid sequence of SEQ ID NO:
11 or 22 (the variable region of an .alpha. chain) or 12 or 23 (the
variable region of a .beta. chain), both SEQ ID NOs: 11 and 12 or
both SEQ ID NOs: 22 and 23. In another embodiment of the invention,
the TCR has antigenic specificity for MAGE-A12 170-178 and
comprises the amino acid sequence of SEQ ID NO: 32 or 42 (the
variable region of an .alpha. chain) or 33 or 43 (the variable
region of a .beta. chain), both SEQ ID NOs: 32 and 33, or both SEQ
ID NOs: 42 and 43. Preferably, the inventive TCR comprises the
amino acid sequences of both SEQ ID NOs: 11 and 12 or both SEQ ID
NOs: 32 and 33.
[0038] Alternatively or additionally, the TCR can comprise an a
chain of a TCR and a .beta. chain of a TCR. Each of the .alpha.
chain and .beta. chain of the inventive TCR can independently
comprise any amino acid sequence. Preferably, the .alpha. chain
comprises the variable region of an .alpha. chain as set forth
above. In this regard, the inventive TCR with antigenic specificity
for MAGE-A3 168-176 can comprise the amino acid sequence of SEQ ID
NO: 13 or 24 and the inventive TCR with antigenic specificity for
MAGE-A12 170-178 can comprise the amino acid sequence of SEQ ID NO:
34 or 44. An inventive TCR of this type can be paired with any
.beta. chain of a TCR. Preferably, the (3 chain of the inventive
TCR comprises the variable region of a .beta. chain as set forth
above. In this regard, the inventive TCR with antigenic specificity
for MAGE-A3 168-176 can comprise the amino acid sequence of SEQ ID
NO: 14 or 25 and the inventive TCR with antigenic specificity for
MAGE-A12 170-178 can comprise the amino acid sequence of SEQ ID NO:
35 or 45. The inventive TCR, therefore, can comprise the amino acid
sequence of SEQ ID NO: 13, 14, 24, 25, 34, 35, 44, or 45, both SEQ
ID NOs: 13 and 14, both SEQ ID NOs: 24 and 25, both SEQ ID NOs: 34
and 35, or both SEQ ID NOs: 44 and 45. Preferably, the inventive
TCR comprises the amino acid sequences of both SEQ ID NOs: 13 and
14 or both SEQ ID NOs: 34 and 35.
[0039] Also provided by the invention is a polypeptide comprising a
functional portion of any of the TCRs described herein. The term
"polypeptide" as used herein includes oligopeptides and refers to a
single chain of amino acids connected by one or more peptide
bonds.
[0040] With respect to the inventive polypeptides, the functional
portion can be any portion comprising contiguous amino acids of the
TCR of which it is a part, provided that the functional portion
specifically binds to MAGE-A3 or MAGE-A12. The term "functional
portion" when used in reference to a TCR refers to any part or
fragment of the TCR of the invention, which part or fragment
retains the biological activity of the TCR of which it is a part
(the parent TCR). Functional portions encompass, for example, those
parts of a TCR that retain the ability to specifically bind to
MAGE-A3 (e.g., in an HLA-A1-dependent manner) or MAGE-A12 (e.g., in
an HLA-Cw7-dependent manner), or detect, treat, or prevent cancer,
to a similar extent, the same extent, or to a higher extent, as the
parent TCR. In reference to the parent TCR, the functional portion
can comprise, for instance, about 10%, 25%, 30%, 50%, 68%, 80%,
90%, 95%, or more, of the parent TCR.
[0041] The functional portion can comprise additional amino acids
at the amino or carboxy terminus of the portion, or at both
termini, which additional amino acids are not found in the amino
acid sequence of the parent TCR. Desirably, the additional amino
acids do not interfere with the biological function of the
functional portion, e.g., specifically binding to MAGE-A3 or
MAGE-A12; and/or having the ability to detect cancer, treat or
prevent cancer, etc. More desirably, the additional amino acids
enhance the biological activity, as compared to the biological
activity of the parent TCR.
[0042] The polypeptide can comprise a functional portion of either
or both of the .alpha. and .beta. chains of the TCRs of the
invention, such as a functional portion comprising one of more of
CDR1, CDR2, and CDR3 of the variable region(s) of the .alpha. chain
and/or .beta. chain of a TCR of the invention. In this regard, the
polypeptide can comprise a functional portion comprising the amino
acid sequence of SEQ ID NO: 5, 16, 26, or 36 (CDR1 of .alpha.
chain), 6, 17, 27, or 37 (CDR2 of .alpha. chain), 7, 18, 28, or 38
(CDR3 of .alpha. chain), 8, 19, 29, or 39 (CDR1 of .beta. chain),
9, 20, 30, or 40 (CDR2 of .beta. chain), 10, 21, 31, or 41 (CDR3 of
.beta. chain), or a combination thereof. Preferably, the inventive
polypeptide comprises a functional portion comprising SEQ ID NOs:
5-7; 8-10; 16-18; 19-21; 26-28; 29-31; 36-38; 39-41; all of SEQ ID
NOs: 5-10; all of SEQ ID NOs: 16-21; all of SEQ ID NOs: 26-31; or
all of SEQ ID NOs: 36-41. More preferably, the polypeptide
comprises a functional portion comprising the amino acid sequences
of all of SEQ ID NOs: 5-10 or all of SEQ ID NOs: 26-31.
[0043] Alternatively or additionally, the inventive polypeptide can
comprise, for instance, the variable region of the inventive TCR
comprising a combination of the CDR regions set forth above. In
this regard, the polypeptide can comprise the amino acid sequence
of SEQ ID NO: 11, 22, 32, or 42 (the variable region of an .alpha.
chain), SEQ ID NO: 12, 23, 33, or 43 (the variable region of a (3
chain), both SEQ ID NOs: 11 and 12, both SEQ ID NOs: 22 and 23,
both SEQ ID NOs: 32 and 33, or both SEQ ID NOs: 42 and 43.
Preferably, the polypeptide comprises the amino acid sequences of
both SEQ ID NOs: 11 and 12 or both SEQ ID NOs: 32 and 33.
[0044] Alternatively or additionally, the inventive polypeptide can
comprise the entire length of an .alpha. or .beta. chain of one of
the TCRs described herein. In this regard, the inventive
polypeptide can comprise an amino acid sequence of SEQ ID NOs: 13,
14, 24, 25, 34, 35, 44, or 45. Alternatively, the polypeptide of
the invention can comprise .alpha. and .beta. chains of the TCRs
described herein. For example, the inventive polypeptide can
comprise the amino acid sequences of both SEQ ID NOs: 13 and 14;
both SEQ ID NOs: 24 and 25; both SEQ ID NOs: 34 and 35; or both SEQ
ID NOs: 44 and 45. Preferably, the polypeptide comprises the amino
acid sequences of both SEQ ID NOs: 13 and 14 or both SEQ ID NOs: 34
and 35.
[0045] The invention further provides a protein comprising at least
one of the polypeptides described herein. By "protein" is meant a
molecule comprising one or more polypeptide chains.
[0046] In an embodiment, the protein of the invention can comprise
a first polypeptide chain comprising the amino acid sequences of
SEQ ID NOs: 5-7; SEQ ID NOs: 16-18; SEQ ID NOs: 26-28; or SEQ ID
NOs: 36-38 and a second polypeptide chain comprising the amino acid
sequence of SEQ ID NOs: 8-10; SEQ ID NOs: 19-21; SEQ ID NOs: 29-31;
or SEQ ID NOs: 39-41. Alternatively or additionally, the protein of
the invention can comprise a first polypeptide chain comprising the
amino acid sequence of SEQ ID NO: 11, 22, 32, or 42 and a second
polypeptide chain comprising the amino acid sequence of SEQ ID NO:
12, 23, 33, or 43. The protein of the invention can, for example,
comprise a first polypeptide chain comprising the amino acid
sequence of SEQ ID NO: 13, 24, 34, or 44 and a second polypeptide
chain comprising the amino acid sequence of SEQ ID NO: 14, 25, 35,
or 45. In this instance, the protein of the invention can be a TCR.
Alternatively, if, for example, the protein comprises a single
polypeptide chain comprising SEQ ID NO: 13, 24, 34, or 44 and SEQ
ID NO: 14, 25, 35, or 45, or if the first and/or second polypeptide
chain(s) of the protein further comprise(s) other amino acid
sequences, e.g., an amino acid sequence encoding an immunoglobulin
or a portion thereof, then the inventive protein can be a fusion
protein. In this regard, the invention also provides a fusion
protein comprising at least one of the inventive polypeptides
described herein along with at least one other polypeptide. The
other polypeptide can exist as a separate polypeptide of the fusion
protein, or can exist as a polypeptide, which is expressed in frame
(in tandem) with one of the inventive polypeptides described
herein. The other polypeptide can encode any peptidic or
proteinaceous molecule, or a portion thereof, including, but not
limited to an immunoglobulin, CD3, CD4, CD8, an MHC molecule, a CD1
molecule, e.g., CD1a, CD1b, CD1c, CD1d, etc.
[0047] The fusion protein can comprise one or more copies of the
inventive polypeptide and/or one or more copies of the other
polypeptide. For instance, the fusion protein can comprise 1, 2, 3,
4, 5, or more, copies of the inventive polypeptide and/or of the
other polypeptide. Suitable methods of making fusion proteins are
known in the art, and include, for example, recombinant methods.
See, for instance, Choi et al., Mol. Biotechnol. 31: 193-202
(2005).
[0048] In some embodiments of the invention, the TCRs,
polypeptides, and proteins of the invention may be expressed as a
single protein comprising a linker peptide linking the .alpha.
chain and the .beta. chain. In this regard, the TCRs, polypeptides,
and proteins of the invention comprising SEQ ID NO: 13, 24, 34, or
44 and SEQ ID NO: 14, 25, 35, or 45 may further comprise a linker
peptide comprising SEQ ID NO: 15 or 54. The linker peptide may
advantageously facilitate the expression of a recombinant TCR,
polypeptide, and/or protein in a host cell. Upon expression of the
construct including the linker peptide by a host cell, the linker
peptide may be cleaved, resulting in separated .alpha. and .beta.
chains.
[0049] The protein of the invention can be a recombinant antibody
comprising at least one of the inventive polypeptides described
herein. As used herein, "recombinant antibody" refers to a
recombinant (e.g., genetically engineered) protein comprising at
least one of the polypeptides of the invention and a polypeptide
chain of an antibody, or a portion thereof. The polypeptide of an
antibody, or portion thereof, can be a heavy chain, a light chain,
a variable or constant region of a heavy or light chain, a single
chain variable fragment (scFv), or an Fc, Fab, or F(ab).sub.2'
fragment of an antibody, etc. The polypeptide chain of an antibody,
or portion thereof, can exist as a separate polypeptide of the
recombinant antibody. Alternatively, the polypeptide chain of an
antibody, or portion thereof, can exist as a polypeptide, which is
expressed in frame (in tandem) with the polypeptide of the
invention. The polypeptide of an antibody, or portion thereof, can
be a polypeptide of any antibody or any antibody fragment,
including any of the antibodies and antibody fragments described
herein.
[0050] Included in the scope of the invention are functional
variants of the inventive TCRs, polypeptides, and proteins
described herein. The term "functional variant" as used herein
refers to a TCR, polypeptide, or protein having substantial or
significant sequence identity or similarity to a parent TCR,
polypeptide, or protein, which functional variant retains the
biological activity of the TCR, polypeptide, or protein of which it
is a variant. Functional variants encompass, for example, those
variants of the TCR, polypeptide, or protein described herein (the
parent TCR, polypeptide, or protein) that retain the ability to
specifically bind to MAGE-A3 or MAGE-A12 for which the parent TCR
has antigenic specificity or to which the parent polypeptide or
protein specifically binds, to a similar extent, the same extent,
or to a higher extent, as the parent TCR, polypeptide, or protein.
In reference to the parent TCR, polypeptide, or protein, the
functional variant can, for instance, be at least about 30%, 50%,
75%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or more identical in amino
acid sequence to the parent TCR, polypeptide, or protein.
[0051] The functional variant can, for example, comprise the amino
acid sequence of the parent TCR, polypeptide, or protein with at
least one conservative amino acid substitution. Conservative amino
acid substitutions are known in the art, and include amino acid
substitutions in which one amino acid having certain physical
and/or chemical properties is exchanged for another amino acid that
has the same chemical or physical properties. For instance, the
conservative amino acid substitution can be an acidic amino acid
substituted for another acidic amino acid (e.g., Asp or Glu), an
amino acid with a nonpolar side chain substituted for another amino
acid with a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu,
Met, Phe, Pro, Trp, Val, etc.), a basic amino acid substituted for
another basic amino acid (Lys, Arg, etc.), an amino acid with a
polar side chain substituted for another amino acid with a polar
side chain (Asn, Cys, Gln, Ser, Thr, Tyr, etc.), etc.
[0052] Alternatively or additionally, the functional variants can
comprise the amino acid sequence of the parent TCR, polypeptide, or
protein with at least one non-conservative amino acid substitution.
In this case, it is preferable for the non-conservative amino acid
substitution to not interfere with or inhibit the biological
activity of the functional variant. Preferably, the
non-conservative amino acid substitution enhances the biological
activity of the functional variant, such that the biological
activity of the functional variant is increased as compared to the
parent TCR, polypeptide, or protein.
[0053] The TCR, polypeptide, or protein can consist essentially of
the specified amino acid sequence or sequences described herein,
such that other components of the functional variant, e.g., other
amino acids, do not materially change the biological activity of
the functional variant. In this regard, the inventive TCR,
polypeptide, or protein can, for example, consist essentially of
the amino acid sequence of SEQ ID NO: 13, 14, 24, 25, 34, 35, 44,
or 45, both SEQ ID NOs: 13 and 14, both SEQ ID NOs: 24 and 25, both
SEQ ID NOs: 34 and 35, or both SEQ ID NOs: 44 and 45. Also, for
instance, the inventive TCRs, polypeptides, or proteins can consist
essentially of the amino acid sequence(s) of SEQ ID NO: 11, 12, 22,
23, 32, 33, 42, or 43, both SEQ ID NOs: 11 and 12, both SEQ ID NOs:
22 and 23, both SEQ ID NOs: 32 and 33, or both SEQ ID NOs: 42 and
43. Furthermore, the inventive TCRs, polypeptides, or proteins can
consist essentially of the amino acid sequence of SEQ ID NO: 5, 16,
26, or 36 (CDR1 of .alpha. chain), SEQ ID NO: 6, 17, 27, or 37
(CDR2 of .alpha. chain), SEQ ID NO: 7, 18, 28, or 38 (CDR3 of
.alpha. chain), SEQ ID NO: 8, 19, 29, or 39 (CDR1 of .beta. chain),
SEQ ID NO: 9, 20, 30, or 40 (CDR2 of .beta. chain), SEQ ID NO: 10,
21, 31, or 41 (CDR3 of .beta. chain), or any combination thereof,
e.g., SEQ ID NOs: 5-7; 8-10; 5-10; 16-18; 19-21; 16-21; 26-28;
29-31; 26-31; 36-38; 39-41; or 36-41.
[0054] The TCRs, polypeptides, and proteins of the invention
(including functional portions and functional variants) can be of
any length, i.e., can comprise any number of amino acids, provided
that the TCRs, polypeptides, or proteins (or functional portions or
functional variants thereof) retain their biological activity,
e.g., the ability to specifically bind to MAGE-A3 or MAGE-A12;
detect cancer in a host; or treat or prevent cancer in a host, etc.
For example, the polypeptide can be in the range of from about 50
to about 5000 amino acids long, such as 50, 70, 75, 100, 125, 150,
175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino
acids in length. In this regard, the polypeptides of the invention
also include oligopeptides.
[0055] The TCRs, polypeptides, and proteins of the invention
(including functional portions and functional variants) of the
invention can comprise synthetic amino acids in place of one or
more naturally-occurring amino acids. Such synthetic amino acids
are known in the art, and include, for example, aminocyclohexane
carboxylic acid, norleucine, .alpha.-amino n-decanoic acid,
homoserine, S-acetylaminomethyl-cysteine, trans-3- and
trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine,
4-chlorophenylalanine, 4-carboxyphenylalanine,
.beta.-phenylserine-.beta.-hydroxyphenylalanine, phenylglycine,
.alpha.-naphthylalanine, cyclohexylalanine, cyclohexylglycine,
indoline-2-carboxylic acid,
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic
acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine,
N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine,
.alpha.-aminocyclopentane carboxylic acid, .alpha.-aminocyclohexane
carboxylic acid, .alpha.-aminocycloheptane carboxylic acid,
.alpha.-(2-amino-2-norbornane)-carboxylic acid,
.alpha.,.gamma.-diaminobutyric acid,
.alpha.,.beta.-diaminopropionic acid, homophenylalanine, and
.alpha.-tert-butylglycine.
[0056] The TCRs, polypeptides, and proteins of the invention
(including functional portions and functional variants) can be
glycosylated, amidated, carboxylated, phosphorylated, esterified,
N-acylated, cyclized via, e.g., a disulfide bridge, or converted
into an acid addition salt and/or optionally dimerized or
polymerized, or conjugated.
[0057] When the TCRs, polypeptides, and proteins of the invention
(including functional portions and functional variants) are in the
form of a salt, preferably, the polypeptides are in the form of a
pharmaceutically acceptable salt. Suitable pharmaceutically
acceptable acid addition salts include those derived from mineral
acids, such as hydrochloric, hydrobromic, phosphoric,
metaphosphoric, nitric, and sulphuric acids, and organic acids,
such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic,
glycolic, gluconic, succinic, and arylsulphonic acids, for example,
p-toluenesulphonic acid.
[0058] The TCR, polypeptide, and/or protein of the invention
(including functional portions and functional variants thereof) can
be obtained by methods known in the art. Suitable methods of de
novo synthesizing polypeptides and proteins are described in
references, such as Chan et al., Fmoc Solid Phase Peptide
Synthesis, Oxford University Press, Oxford, United Kingdom, 2005;
Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker,
Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University
Press, Oxford, United Kingdom, 2000; and U.S. Pat. No. 5,449,752.
Also, polypeptides and proteins can be recombinantly produced using
the nucleic acids described herein using standard recombinant
methods. See, for instance, Sambrook et al., Molecular Cloning: A
Laboratory Manual, 3.sup.rd ed., Cold Spring Harbor Press, Cold
Spring Harbor, N.Y. 2001; and Ausubel et al., Current Protocols in
Molecular Biology, Greene Publishing Associates and John Wiley
& Sons, NY, 1994. Further, some of the TCRs, polypeptides, and
proteins of the invention (including functional portions and
functional variants thereof) can be isolated and/or purified from a
source, such as a plant, a bacterium, an insect, a mammal, e.g., a
rat, a human, etc. Methods of isolation and purification are
well-known in the art. Alternatively, the TCRs, polypeptides,
and/or proteins described herein (including functional portions and
functional variants thereof) can be commercially synthesized by
companies, such as Synpep (Dublin, Calif.), Peptide Technologies
Corp. (Gaithersburg, Md.), and Multiple Peptide Systems (San Diego,
Calif.). In this respect, the inventive TCRs, polypeptides, and
proteins can be synthetic, recombinant, isolated, and/or
purified.
[0059] Included in the scope of the invention are conjugates, e.g.,
bioconjugates, comprising any of the inventive TCRs, polypeptides,
or proteins (including any of the functional portions or variants
thereof), nucleic acids, recombinant expression vectors, host
cells, populations of host cells, or antibodies, or antigen binding
portions thereof. Conjugates, as well as methods of synthesizing
conjugates in general, are known in the art (See, for instance,
Hudecz, F., Methods Mol. Biol. 298: 209-223 (2005) and Kirin et
al., Inorg Chem. 44(15): 5405-5415 (2005)).
[0060] By "nucleic acid" as used herein includes "polynucleotide,"
"oligonucleotide," and "nucleic acid molecule," and generally means
a polymer of DNA or RNA, which can be single-stranded or
double-stranded, synthesized or obtained (e.g., isolated and/or
purified) from natural sources, which can contain natural,
non-natural or altered nucleotides, and which can contain a
natural, non-natural or altered internucleotide linkage, such as a
phosphoroamidate linkage or a phosphorothioate linkage, instead of
the phosphodiester found between the nucleotides of an unmodified
oligonucleotide. It is generally preferred that the nucleic acid
does not comprise any insertions, deletions, inversions, and/or
substitutions. However, it may be suitable in some instances, as
discussed herein, for the nucleic acid to comprise one or more
insertions, deletions, inversions, and/or substitutions.
[0061] Preferably, the nucleic acids of the invention are
recombinant. As used herein, the term "recombinant" refers to (i)
molecules that are constructed outside living cells by joining
natural or synthetic nucleic acid segments to nucleic acid
molecules that can replicate in a living cell, or (ii) molecules
that result from the replication of those described in (i) above.
For purposes herein, the replication can be in vitro replication or
in vivo replication.
[0062] The nucleic acids can be constructed based on chemical
synthesis and/or enzymatic ligation reactions using procedures
known in the art. See, for example, Sambrook et al., supra, and
Ausubel et al., supra. For example, a nucleic acid can be
chemically synthesized using naturally occurring nucleotides or
variously modified nucleotides designed to increase the biological
stability of the molecules or to increase the physical stability of
the duplex formed upon hybridization (e.g., phosphorothioate
derivatives and acridine substituted nucleotides). Examples of
modified nucleotides that can be used to generate the nucleic acids
include, but are not limited to, 5-fluorouracil, 5-bromouracil,
5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,
4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N.sup.6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methyl guanine, 3-methylcytosine,
5-methylcytosine, N.sup.6-substituted adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N.sup.6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine.
Alternatively, one or more of the nucleic acids of the invention
can be purchased from companies, such as Macromolecular Resources
(Fort Collins, Colo.) and Synthegen (Houston, Tex.).
[0063] The nucleic acid can comprise any nucleotide sequence which
encodes any of the TCRs, polypeptides, or proteins, or functional
portions or functional variants thereof described herein. For
example, the nucleic acid can comprise, consist, or consist
essentially of any one or more of the nucleotide sequence SEQ ID
NOs: 46-49.
[0064] The invention also provides a nucleic acid comprising a
nucleotide sequence which is complementary to the nucleotide
sequence of any of the nucleic acids described herein or a
nucleotide sequence which hybridizes under stringent conditions to
the nucleotide sequence of any of the nucleic acids described
herein.
[0065] The nucleotide sequence which hybridizes under stringent
conditions preferably hybridizes under high stringency conditions.
By "high stringency conditions" is meant that the nucleotide
sequence specifically hybridizes to a target sequence (the
nucleotide sequence of any of the nucleic acids described herein)
in an amount that is detectably stronger than non-specific
hybridization. High stringency conditions include conditions which
would distinguish a polynucleotide with an exact complementary
sequence, or one containing only a few scattered mismatches from a
random sequence that happened to have a few small regions (e.g.,
3-10 bases) that matched the nucleotide sequence. Such small
regions of complementarity are more easily melted than a
full-length complement of 14-17 or more bases, and high stringency
hybridization makes them easily distinguishable. Relatively high
stringency conditions would include, for example, low salt and/or
high temperature conditions, such as provided by about 0.02-0.1 M
NaCl or the equivalent, at temperatures of about 50-70.degree. C.
Such high stringency conditions tolerate little, if any, mismatch
between the nucleotide sequence and the template or target strand,
and are particularly suitable for detecting expression of any of
the inventive TCRs. It is generally appreciated that conditions can
be rendered more stringent by the addition of increasing amounts of
form amide.
[0066] The invention also provides a nucleic acid comprising a
nucleotide sequence that is at least about 70% or more, e.g., about
80%, about 90%, about 91%, about 92%, about 93%, about 94%, about
95%, about 96%, about 97%, about 98%, or about 99% identical to any
of the nucleic acids described herein.
[0067] The nucleic acids of the invention can be incorporated into
a recombinant expression vector. In this regard, the invention
provides recombinant expression vectors comprising any of the
nucleic acids of the invention. For purposes herein, the term
"recombinant expression vector" means a genetically-modified
oligonucleotide or polynucleotide construct that permits the
expression of an mRNA, protein, polypeptide, or peptide by a host
cell, when the construct comprises a nucleotide sequence encoding
the mRNA, protein, polypeptide, or peptide, and the vector is
contacted with the cell under conditions sufficient to have the
mRNA, protein, polypeptide, or peptide expressed within the cell.
The vectors of the invention are not naturally-occurring as a
whole. However, parts of the vectors can be naturally-occurring.
The inventive recombinant expression vectors can comprise any type
of nucleotides, including, but not limited to DNA and RNA, which
can be single-stranded or double-stranded, synthesized or obtained
in part from natural sources, and which can contain natural,
non-natural or altered nucleotides. The recombinant expression
vectors can comprise naturally-occurring, non-naturally-occurring
internucleotide linkages, or both types of linkages. Preferably,
the non-naturally occurring or altered nucleotides or
internucleotide linkages does not hinder the transcription or
replication of the vector.
[0068] The recombinant expression vector of the invention can be
any suitable recombinant expression vector, and can be used to
transform or transfect any suitable host. Suitable vectors include
those designed for propagation and expansion or for expression or
both, such as plasmids and viruses. The vector can be selected from
the group consisting of the pUC series (Fermentas Life Sciences),
the pBluescript series (Stratagene, LaJolla, Calif.), the pET
series (Novagen, Madison, Wis.), the pGEX series (Pharmacia
Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto,
Calif.). Bacteriophage vectors, such as .lamda.GT10, .lamda.GT11,
.lamda.ZapII (Stratagene), .lamda.EMBL4, and .lamda.NM1149, also
can be used. Examples of plant expression vectors include pBI01,
pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of
animal expression vectors include pEUK-Cl, pMAM and pMAMneo
(Clontech). Preferably, the recombinant expression vector is a
viral vector, e.g., a retroviral vector.
[0069] The recombinant expression vectors of the invention can be
prepared using standard recombinant DNA techniques described in,
for example, Sambrook et al., supra, and Ausubel et al., supra.
Constructs of expression vectors, which are circular or linear, can
be prepared to contain a replication system functional in a
prokaryotic or eukaryotic host cell. Replication systems can be
derived, e.g., from ColE1, 2.mu. plasmid, .lamda., SV40, bovine
papilloma virus, and the like.
[0070] Desirably, the recombinant expression vector comprises
regulatory sequences, such as transcription and translation
initiation and termination codons, which are specific to the type
of host (e.g., bacterium, fungus, plant, or animal) into which the
vector is to be introduced, as appropriate and taking into
consideration whether the vector is DNA- or RNA-based.
[0071] The recombinant expression vector can include one or more
marker genes, which allow for selection of transformed or
transfected hosts. Marker genes include biocide resistance, e.g.,
resistance to antibiotics, heavy metals, etc., complementation in
an auxotrophic host to provide prototrophy, and the like. Suitable
marker genes for the inventive expression vectors include, for
instance, neomycin/G418 resistance genes, hygromycin resistance
genes, histidinol resistance genes, tetracycline resistance genes,
and ampicillin resistance genes.
[0072] The recombinant expression vector can comprise a native or
normative promoter operably linked to the nucleotide sequence
encoding the TCR, polypeptide, or protein (including functional
portions and functional variants thereof), or to the nucleotide
sequence which is complementary to or which hybridizes to the
nucleotide sequence encoding the TCR, polypeptide, or protein. The
selection of promoters, e.g., strong, weak, inducible,
tissue-specific and developmental-specific, is within the ordinary
skill of the artisan. Similarly, the combining of a nucleotide
sequence with a promoter is also within the skill of the artisan.
The promoter can be a non-viral promoter or a viral promoter, e.g.,
a cytomegalovirus (CMV) promoter, an SV40 promoter, an RSV
promoter, and a promoter found in the long-terminal repeat of the
murine stem cell virus.
[0073] The inventive recombinant expression vectors can be designed
for either transient expression, for stable expression, or for
both. Also, the recombinant expression vectors can be made for
constitutive expression or for inducible expression. Further, the
recombinant expression vectors can be made to include a suicide
gene.
[0074] As used herein, the term "suicide gene" refers to a gene
that causes the cell expressing the suicide gene to die. The
suicide gene can be a gene that confers sensitivity to an agent,
e.g., a drug, upon the cell in which the gene is expressed, and
causes the cell to die when the cell is contacted with or exposed
to the agent. Suicide genes are known in the art (see, for example,
Suicide Gene Therapy: Methods and Reviews, Springer, Caroline J.
(Cancer Research UK Centre for Cancer Therapeutics at the Institute
of Cancer Research, Sutton, Surrey, UK), Humana Press, 2004) and
include, for example, the Herpes Simplex Virus (HSV) thymidine
kinase (TK) gene, cytosine daminase, purine nucleoside
phosphorylase, and nitroreductase.
[0075] Another embodiment of the invention further provides a host
cell comprising any of the recombinant expression vectors described
herein. As used herein, the term "host cell" refers to any type of
cell that can contain the inventive recombinant expression vector.
The host cell can be a eukaryotic cell, e.g., plant, animal, fungi,
or algae, or can be a prokaryotic cell, e.g., bacteria or protozoa.
The host cell can be a cultured cell or a primary cell, i.e.,
isolated directly from an organism, e.g., a human. The host cell
can be an adherent cell or a suspended cell, i.e., a cell that
grows in suspension. Suitable host cells are known in the art and
include, for instance, DH5a E. coli cells, Chinese hamster ovarian
cells, monkey VERO cells, COS cells, HEK293 cells, and the like.
For purposes of amplifying or replicating the recombinant
expression vector, the host cell is preferably a prokaryotic cell,
e.g., a DH5.alpha. cell. For purposes of producing a recombinant
TCR, polypeptide, or protein, the host cell is preferably a
mammalian cell. Most preferably, the host cell is a human cell.
While the host cell can be of any cell type, can originate from any
type of tissue, and can be of any developmental stage, the host
cell preferably is a peripheral blood lymphocyte (PBL) or a
peripheral blood mononuclear cell (PBMC). More preferably, the host
cell is a T cell.
[0076] For purposes herein, the T cell can be any T cell, such as a
cultured T cell, e.g., a primary T cell, or a T cell from a
cultured T cell line, e.g., Jurkat, SupTi, etc., or a T cell
obtained from a mammal. If obtained from a mammal, the T cell can
be obtained from numerous sources, including but not limited to
blood, bone marrow, lymph node, the thymus, or other tissues or
fluids. T cells can also be enriched for or purified. Preferably,
the T cell is a human T cell. More preferably, the T cell is a T
cell isolated from a human. The T cell can be any type of T cell
and can be of any developmental stage, including but not limited
to, CD4.sup.+/CD8.sup.+ double positive T cells, CD4.sup.+ helper T
cells, e.g., Th.sub.1 and Th.sub.2 cells, CD8.sup.+ T cells (e.g.,
cytotoxic T cells), tumor infiltrating lymphocytes (TILs), memory T
cells (e.g., central memory T cells and effector memory T cells),
naive T cells, and the like. Preferably, the T cell is a CD8.sup.+
T cell or a CD4.sup.+ T cell.
[0077] Also provided by the invention is a population of cells
comprising at least one host cell described herein. The population
of cells can be a heterogeneous population comprising the host cell
comprising any of the recombinant expression vectors described, in
addition to at least one other cell, e.g., a host cell (e.g., a T
cell), which does not comprise any of the recombinant expression
vectors, or a cell other than a T cell, e.g., a B cell, a
macrophage, a neutrophil, an erythrocyte, a hepatocyte, an
endothelial cell, an epithelial cells, a muscle cell, a brain cell,
etc. Alternatively, the population of cells can be a substantially
homogeneous population, in which the population comprises mainly of
host cells (e.g., consisting essentially of) comprising the
recombinant expression vector. The population also can be a clonal
population of cells, in which all cells of the population are
clones of a single host cell comprising a recombinant expression
vector, such that all cells of the population comprise the
recombinant expression vector. In one embodiment of the invention,
the population of cells is a clonal population comprising host
cells comprising a recombinant expression vector as described
herein.
[0078] The invention further provides an antibody, or antigen
binding portion thereof, which specifically binds to a functional
portion of any of the TCRs described herein. Preferably, the
functional portion specifically binds to the cancer antigen, e.g.,
the functional portion comprising the amino acid sequence SEQ ID
NO: 5, 16, 26, or 36 (CDR1 of .alpha. chain), 6, 17, 27, or 37
(CDR2 of .alpha. chain), 7, 18, 28, or 38 (CDR3 of .alpha. chain),
8, 19, 29, or 39 (CDR1 of .beta. chain), 9, 20, 30, or 40 (CDR2 of
.beta. chain), 10, 21, 31, or 41 (CDR3 of .beta. chain), SEQ ID NO:
11, 22, 32, or 42 (variable region of .alpha. chain), SEQ ID NO:
12, 23, 33, or 43 (variable region of .beta. chain), or a
combination thereof, e.g., 5-7; 8-10; 5-10; 16-18, 19-21; 16-21;
26-28; 29-31; 26-31; 36-38; 39-41; or 36-41. More preferably, the
functional portion comprises the amino acid sequences of SEQ ID
NOs: 5-10 or SEQ ID NOs: 26-31. In a preferred embodiment, the
antibody, or antigen binding portion thereof, binds to an epitope
which is formed by all 6 CDRs (CDR1-3 of the alpha chain and CDR1-3
of the beta chain). The antibody can be any type of immunoglobulin
that is known in the art. For instance, the antibody can be of any
isotype, e.g., IgA, IgD, IgE, IgG, IgM, etc. The antibody can be
monoclonal or polyclonal. The antibody can be a naturally-occurring
antibody, e.g., an antibody isolated and/or purified from a mammal,
e.g., mouse, rabbit, goat, horse, chicken, hamster, human, etc.
Alternatively, the antibody can be a genetically-engineered
antibody, e.g., a humanized antibody or a chimeric antibody. The
antibody can be in monomeric or polymeric form. Also, the antibody
can have any level of affinity or avidity for the functional
portion of the inventive TCR. Desirably, the antibody is specific
for the functional portion of the inventive TCR, such that there is
minimal cross-reaction with other peptides or proteins.
[0079] Methods of testing antibodies for the ability to bind to any
functional portion of the inventive TCR are known in the art and
include any antibody-antigen binding assay, such as, for example,
radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation,
and competitive inhibition assays (see, e.g., Janeway et al.,
infra, and U.S. Patent Application Publication No. 2002/0197266
A1).
[0080] Suitable methods of making antibodies are known in the art.
For instance, standard hybridoma methods are described in, e.g.,
Kohler and Milstein, Eur. J. Immunol., 5, 511-519 (1976), Harlow
and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988),
and C. A. Janeway et al. (eds.), Immunobiology, 5.sup.th Ed.,
Garland Publishing, New York, N.Y. (2001)). Alternatively, other
methods, such as EBV-hybridoma methods (Haskard and Archer, J.
Immunol. Methods, 74(2), 361-67 (1984), and Roder et al., Methods
Enzymol., 121, 140-67 (1986)), and bacteriophage vector expression
systems (see, e.g., Huse et al., Science, 246, 1275-81 (1989)) are
known in the art. Further, methods of producing antibodies in
non-human animals are described in, e.g., U.S. Pat. Nos. 5,545,806,
5,569,825, and 5,714,352, and U.S. Patent Application Publication
No. 2002/0197266 A1.
[0081] Phage display furthermore can be used to generate the
antibody of the invention. In this regard, phage libraries encoding
antigen-binding variable (V) domains of antibodies can be generated
using standard molecular biology and recombinant DNA techniques
(see, e.g., Sambrook et al. (eds.), Molecular Cloning, A Laboratory
Manual, 3.sup.rd Edition, Cold Spring Harbor Laboratory Press, New
York (2001)). Phage encoding a variable region with the desired
specificity are selected for specific binding to the desired
antigen, and a complete or partial antibody is reconstituted
comprising the selected variable domain. Nucleic acid sequences
encoding the reconstituted antibody are introduced into a suitable
cell line, such as a myeloma cell used for hybridoma production,
such that antibodies having the characteristics of monoclonal
antibodies are secreted by the cell (see, e.g., Janeway et al.,
supra, Huse et al., supra, and U.S. Pat. No. 6,265,150).
[0082] Antibodies can be produced by transgenic mice that are
transgenic for specific heavy and light chain immunoglobulin genes.
Such methods are known in the art and described in, for example
U.S. Pat. Nos. 5,545,806 and 5,569,825, and Janeway et al.,
supra.
[0083] Methods for generating humanized antibodies are well known
in the art and are described in detail in, for example, Janeway et
al., supra, U.S. Pat. Nos. 5,225,539, 5,585,089 and 5,693,761,
European Patent No. 0239400 B1, and United Kingdom Patent No.
2188638. Humanized antibodies can also be generated using the
antibody resurfacing technology described in, for example, U.S.
Pat. No. 5,639,641 and Pedersen et al., J. Mol. Biol., 235, 959-973
(1994).
[0084] The invention also provides antigen binding portions of any
of the antibodies described herein. The antigen binding portion can
be any portion that has at least one antigen binding site, such as
Fab, F(ab').sub.2, dsFv, sFv, diabodies, and triabodies.
[0085] A single-chain variable region fragment (sFv) antibody
fragment, which consists of a truncated Fab fragment comprising the
variable (V) domain of an antibody heavy chain linked to a V domain
of a light antibody chain via a synthetic peptide, can be generated
using routine recombinant DNA technology techniques (see, e.g.,
Janeway et al., supra). Similarly, disulfide-stabilized variable
region fragments (dsFv) can be prepared by recombinant DNA
technology (see, e.g., Reiter et al., Protein Engineering, 7,
697-704 (1994)). Antibody fragments of the invention, however, are
not limited to these exemplary types of antibody fragments.
[0086] Also, the antibody, or antigen binding portion thereof, can
be modified to comprise a detectable label, such as, for instance,
a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate
(FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase,
horseradish peroxidase), and element particles (e.g., gold
particles).
[0087] The inventive TCRs, polypeptides, proteins, (including
functional portions and functional variants thereof), nucleic
acids, recombinant expression vectors, host cells (including
populations thereof), and antibodies (including antigen binding
portions thereof), can be isolated and/or purified. The term
"isolated" as used herein means having been removed from its
natural environment. The term "purified" as used herein means
having been increased in purity, wherein "purity" is a relative
term, and not to be necessarily construed as absolute purity. For
example, the purity can be at least about 50%, can be greater than
60%, 70%, 80%, 90%, 95%, or can be 100%.
[0088] The inventive TCRs, polypeptides, proteins (including
functional portions and variants thereof), nucleic acids,
recombinant expression vectors, host cells (including populations
thereof), and antibodies (including antigen binding portions
thereof), all of which are collectively referred to as "inventive
TCR materials" hereinafter, can be formulated into a composition,
such as a pharmaceutical composition. In this regard, the invention
provides a pharmaceutical composition comprising any of the TCRs,
polypeptides, proteins, functional portions, functional variants,
nucleic acids, expression vectors, host cells (including
populations thereof), and antibodies (including antigen binding
portions thereof), and a pharmaceutically acceptable carrier. The
inventive pharmaceutical compositions containing any of the
inventive TCR materials can comprise more than one inventive TCR
material, e.g., a polypeptide and a nucleic acid, or two or more
different TCRs. Alternatively, the pharmaceutical composition can
comprise an inventive TCR material in combination with another
pharmaceutically active agents or drugs, such as a chemotherapeutic
agents, e.g., asparaginase, busulfan, carboplatin, cisplatin,
daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea,
methotrexate, paclitaxel, rituximab, vinblastine, vincristine,
etc.
[0089] Preferably, the carrier is a pharmaceutically acceptable
carrier. With respect to pharmaceutical compositions, the carrier
can be any of those conventionally used and is limited only by
chemico-physical considerations, such as solubility and lack of
reactivity with the active compound(s), and by the route of
administration. The pharmaceutically acceptable carriers described
herein, for example, vehicles, adjuvants, excipients, and diluents,
are well-known to those skilled in the art and are readily
available to the public. It is preferred that the pharmaceutically
acceptable carrier be one which is chemically inert to the active
agent(s) and one which has no detrimental side effects or toxicity
under the conditions of use.
[0090] The choice of carrier will be determined in part by the
particular inventive TCR material, as well as by the particular
method used to administer the inventive TCR material. Accordingly,
there are a variety of suitable formulations of the pharmaceutical
composition of the invention. The following formulations for oral,
aerosol, parenteral, subcutaneous, intravenous, intramuscular,
intraarterial, intrathecal, and interperitoneal administration are
exemplary and are in no way limiting. More than one route can be
used to administer the inventive TCR materials, and in certain
instances, a particular route can provide a more immediate and more
effective response than another route.
[0091] Topical formulations are well-known to those of skill in the
art. Such formulations are particularly suitable in the context of
the invention for application to the skin.
[0092] Formulations suitable for oral administration can consist of
(a) liquid solutions, such as an effective amount of the inventive
TCR material dissolved in diluents, such as water, saline, or
orange juice; (b) capsules, sachets, tablets, lozenges, and
troches, each containing a predetermined amount of the active
ingredient, as solids or granules; (c) powders; (d) suspensions in
an appropriate liquid; and (e) suitable emulsions. Liquid
formulations may include diluents, such as water and alcohols, for
example, ethanol, benzyl alcohol, and the polyethylene alcohols,
either with or without the addition of a pharmaceutically
acceptable surfactant. Capsule forms can be of the ordinary hard-
or soft-shelled gelatin type containing, for example, surfactants,
lubricants, and inert fillers, such as lactose, sucrose, calcium
phosphate, and corn starch. Tablet forms can include one or more of
lactose, sucrose, mannitol, corn starch, potato starch, alginic
acid, microcrystalline cellulose, acacia, gelatin, guar gum,
colloidal silicon dioxide, croscarmellose sodium, talc, magnesium
stearate, calcium stearate, zinc stearate, stearic acid, and other
excipients, colorants, diluents, buffering agents, disintegrating
agents, moistening agents, preservatives, flavoring agents, and
other pharmacologically compatible excipients. Lozenge forms can
comprise the inventive TCR material in a flavor, usually sucrose
and acacia or tragacanth, as well as pastilles comprising the
inventive TCR material in an inert base, such as gelatin and
glycerin, or sucrose and acacia, emulsions, gels, and the like
containing, in addition to, such excipients as are known in the
art.
[0093] The inventive TCR material, alone or in combination with
other suitable components, can be made into aerosol formulations to
be administered via inhalation. These aerosol formulations can be
placed into pressurized acceptable propellants, such as
dichlorodifluoromethane, propane, nitrogen, and the like. They also
may be formulated as pharmaceuticals for non-pressured
preparations, such as in a nebulizer or an atomizer. Such spray
formulations also may be used to spray mucosa.
[0094] Formulations suitable for parenteral administration include
aqueous and non-aqueous, isotonic sterile injection solutions,
which can contain anti-oxidants, buffers, bacteriostats, and
solutes that render the formulation isotonic with the blood of the
intended recipient, and aqueous and non-aqueous sterile suspensions
that can include suspending agents, solubilizers, thickening
agents, stabilizers, and preservatives. The inventive TCR material
can be administered in a physiologically acceptable diluent in a
pharmaceutical carrier, such as a sterile liquid or mixture of
liquids, including water, saline, aqueous dextrose and related
sugar solutions, an alcohol, such as ethanol or hexadecyl alcohol,
a glycol, such as propylene glycol or polyethylene glycol,
dimethylsulfoxide, glycerol, ketals such as
2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, poly(ethyleneglycol)
400, oils, fatty acids, fatty acid esters or glycerides, or
acetylated fatty acid glycerides with or without the addition of a
pharmaceutically acceptable surfactant, such as a soap or a
detergent, suspending agent, such as pectin, carbomers,
methylcellulose, hydroxypropylmethylcellulose, or
carboxymethylcellulose, or emulsifying agents and other
pharmaceutical adjuvants.
[0095] Oils, which can be used in parenteral formulations include
petroleum, animal, vegetable, or synthetic oils. Specific examples
of oils include peanut, soybean, sesame, cottonseed, corn, olive,
petrolatum, and mineral. Suitable fatty acids for use in parenteral
formulations include oleic acid, stearic acid, and isostearic acid.
Ethyl oleate and isopropyl myristate are examples of suitable fatty
acid esters.
[0096] Suitable soaps for use in parenteral formulations include
fatty alkali metal, ammonium, and triethanolamine salts, and
suitable detergents include (a) cationic detergents such as, for
example, dimethyl dialkyl ammonium halides, and alkyl pyridinium
halides, (b) anionic detergents such as, for example, alkyl, aryl,
and olefin sulfonates, alkyl, olefin, ether, and monoglyceride
sulfates, and sulfosuccinates, (c) nonionic detergents such as, for
example, fatty amine oxides, fatty acid alkanolamides, and
polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents
such as, for example, alkyl-.beta.-aminopropionates, and
2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures
thereof.
[0097] The parenteral formulations will typically contain from
about 0.5% to about 25%, or more, by weight of the inventive TCR
material in solution. Preservatives and buffers may be used. In
order to minimize or eliminate irritation at the site of injection,
such compositions may contain one or more nonionic surfactants
having a hydrophile-lipophile balance (HLB) of from about 12 to
about 17. The quantity of surfactant in such formulations will
typically range from about 5% to about 15% by weight. Suitable
surfactants include polyethylene glycol sorbitan fatty acid esters,
such as sorbitan monooleate and the high molecular weight adducts
of ethylene oxide with a hydrophobic base, formed by the
condensation of propylene oxide with propylene glycol. The
parenteral formulations can be presented in unit-dose or multi-dose
sealed containers, such as ampoules and vials, and can be stored in
a freeze-dried (lyophilized) condition requiring only the addition
of the sterile liquid excipient, for example, water, for
injections, immediately prior to use. Extemporaneous injection
solutions and suspensions can be prepared from sterile powders,
granules, and tablets of the kind previously described.
[0098] Injectable formulations are in accordance with the
invention. The requirements for effective pharmaceutical carriers
for injectable compositions are well-known to those of ordinary
skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice,
J. B. Lippincott Company, Philadelphia, Pa., Banker and Chalmers,
eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs,
Toissel, 4th ed., pages 622-630 (1986)). Preferably, when
administering cells, e.g., T cells, the cells are administered via
injection.
[0099] It will be appreciated by one of skill in the art that, in
addition to the above-described pharmaceutical compositions, the
inventive TCR materials of the invention can be formulated as
inclusion complexes, such as cyclodextrin inclusion complexes, or
liposomes.
[0100] For purposes of the invention, the amount or dose of the
inventive TCR material administered should be sufficient to effect,
e.g., a therapeutic or prophylactic response, in the subject or
animal over a reasonable time frame. For example, the dose of the
inventive TCR material should be sufficient to bind to a cancer
antigen, or detect, treat or prevent cancer in a period of from
about 2 hours or longer, e.g., 12 to 24 or more hours, from the
time of administration. In certain embodiments, the time period
could be even longer. The dose will be determined by the efficacy
of the particular inventive TCR material and the condition of the
animal (e.g., human), as well as the body weight of the animal
(e.g., human) to be treated.
[0101] Many assays for determining an administered dose are known
in the art. For purposes of the invention, an assay, which
comprises comparing the extent to which target cells are lysed or
IFN-.gamma. is secreted by T cells expressing the inventive TCR,
polypeptide, or protein upon administration of a given dose of such
T cells to a mammal among a set of mammals of which is each given a
different dose of the T cells, could be used to determine a
starting dose to be administered to a mammal. The extent to which
target cells are lysed or IFN-.gamma. is secreted upon
administration of a certain dose can be assayed by methods known in
the art.
[0102] The dose of the inventive TCR material also will be
determined by the existence, nature and extent of any adverse side
effects that might accompany the administration of a particular
inventive TCR material. Typically, the attending physician will
decide the dosage of the inventive TCR material with which to treat
each individual patient, taking into consideration a variety of
factors, such as age, body weight, general health, diet, sex,
inventive TCR material to be administered, route of administration,
and the severity of the condition being treated. By way of example
and not intending to limit the invention, the dose of the inventive
TCR material can be about 0.001 to about 1000 mg/kg body weight of
the subject being treated/day or more, from about 0.01 to about 10
mg/kg body weight/day or more, or about 0.01 mg to about 1 mg/kg
body weight/day or more. In an embodiment in which the inventive
TCR material is a population of cells, the number of cells
administered may vary, e.g., from about 1.times.10.sup.6 to about
1.times.10.sup.11 cells or more.
[0103] One of ordinary skill in the art will readily appreciate
that the inventive TCR materials of the invention can be modified
in any number of ways, such that the therapeutic or prophylactic
efficacy of the inventive TCR materials is increased through the
modification. For instance, the inventive TCR materials can be
conjugated either directly or indirectly through a bridge to a
targeting moiety. The practice of conjugating compounds, e.g.,
inventive TCR materials, to targeting moieties is known in the art.
See, for instance, Wadwa et al., J. Drug Targeting 3: 111 (1995)
and U.S. Pat. No. 5,087,616. The term "targeting moiety" as used
herein, refers to any molecule or agent that specifically
recognizes and binds to a cell-surface receptor, such that the
targeting moiety directs the delivery of the inventive TCR
materials to a population of cells on which surface the receptor is
expressed. Targeting moieties include, but are not limited to,
antibodies, or fragments thereof, peptides, hormones, growth
factors, cytokines, and any other natural or non-natural ligands,
which bind to cell surface receptors (e.g., Epithelial Growth
Factor Receptor (EGFR), T-cell receptor (TCR), B-cell receptor
(BCR), CD28, Platelet-derived Growth Factor Receptor (PDGF),
nicotinic acetylcholine receptor (nAChR), etc.). The term "bridge"
as used herein, refers to any agent or molecule that links the
inventive TCR materials to the targeting moiety. One of ordinary
skill in the art recognizes that sites on the inventive TCR
materials, which are not necessary for the function of the
inventive TCR materials, are ideal sites for attaching a bridge
and/or a targeting moiety, provided that the bridge and/or
targeting moiety, once attached to the inventive TCR materials,
do(es) not interfere with the function of the inventive TCR
materials, i.e., the ability to bind to MAGE-A3 or MAGE-A12; or to
detect, treat, or prevent cancer.
[0104] Alternatively, the inventive TCR materials can be modified
into a depot form, such that the manner in which the inventive TCR
materials is released into the body to which it is administered is
controlled with respect to time and location within the body (see,
for example, U.S. Pat. No. 4,450,150). Depot forms of inventive TCR
materials can be, for example, an implantable composition
comprising the inventive TCR materials and a porous or non-porous
material, such as a polymer, wherein the inventive TCR materials is
encapsulated by or diffused throughout the material and/or
degradation of the non-porous material. The depot is then implanted
into the desired location within the body and the inventive TCR
materials are released from the implant at a predetermined
rate.
[0105] It is contemplated that the inventive pharmaceutical
compositions, TCRs, polypeptides, proteins, nucleic acids,
recombinant expression vectors, host cells, or populations of cells
can be used in methods of treating or preventing cancer. Without
being bound to a particular theory, the inventive TCRs are believed
to bind specifically to MAGE-A3 MAGE-A12, such that the TCR (or
related inventive polypeptide or protein) when expressed by a cell
is able to mediate an immune response against a target cell
expressing MAGE-A3 or MAGE-A12. In this regard, the invention
provides a method of treating or preventing cancer in a host,
comprising administering to the host any of the pharmaceutical
compostions, TCRs, polypeptides, or proteins described herein, any
nucleic acid or recombinant expression vector comprising a
nucleotide sequence encoding any of the TCRs, polypeptides,
proteins described herein, or any host cell or population of cells
comprising a recombinant vector which encodes any of the TCRs,
polypeptides, or proteins described herein, in an amount effective
to treat or prevent cancer in the host.
[0106] The terms "treat," and "prevent" as well as words stemming
therefrom, as used herein, do not necessarily imply 100% or
complete treatment or prevention. Rather, there are varying degrees
of treatment or prevention of which one of ordinary skill in the
art recognizes as having a potential benefit or therapeutic effect.
In this respect, the inventive methods can provide any amount of
any level of treatment or prevention of cancer in a host.
Furthermore, the treatment or prevention provided by the inventive
method can include treatment or prevention of one or more
conditions or symptoms of the disease, e.g., cancer, being treated
or prevented. Also, for purposes herein, "prevention" can encompass
delaying the onset of the disease, or a symptom or condition
thereof.
[0107] Also provided is a method of detecting the presence of
cancer in a host. The method comprises (i) contacting a sample
comprising cells of the cancer with any of the inventive TCRs,
polypeptides, proteins, nucleic acids, recombinant expression
vectors, host cells, populations of cells, or antibodies, or
antigen binding portions thereof, described herein, thereby forming
a complex, and detecting the complex, wherein detection of the
complex is indicative of the presence of cancer in the host.
[0108] With respect to the inventive method of detecting cancer in
a host, the sample of cells of the cancer can be a sample
comprising whole cells, lysates thereof, or a fraction of the whole
cell lysates, e.g., a nuclear or cytoplasmic fraction, a whole
protein fraction, or a nucleic acid fraction.
[0109] For purposes of the inventive detecting method, the
contacting can take place in vitro or in vivo with respect to the
host. Preferably, the contacting is in vitro.
[0110] Also, detection of the complex can occur through any number
of ways known in the art. For instance, the inventive TCRs,
polypeptides, proteins, nucleic acids, recombinant expression
vectors, host cells, populations of cells, or antibodies, or
antigen binding portions thereof, described herein, can be labeled
with a detectable label such as, for instance, a radioisotope, a
fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin
(PE)), an enzyme (e.g., alkaline phosphatase, horseradish
peroxidase), and element particles (e.g., gold particles).
[0111] For purposes of the inventive methods, wherein host cells or
populations of cells are administered, the cells can be cells that
are allogeneic or autologous to the host. Preferably, the cells are
autologous to the host.
[0112] With respect to the inventive methods, the cancer can be any
cancer, including any of sarcomas (e.g., synovial sarcoma,
osteogenic sarcoma, leiomyosarcoma uteri, and alveolar
rhabdomyosarcoma), lymphomas (e.g., Hodgkin lymphoma and
non-Hodgkin lymphoma), hepatocellular carcinoma, glioma, head
cancers (e.g., squamous cell carcinoma), neck cancers (e.g.,
squamous cell carcinoma), acute lymphocytic cancer, leukemias
(e.g., acute myeloid leukemia and chronic lymphocytic leukemia),
bone cancer, brain cancer, breast cancer, cancer of the anus, anal
canal, or anorectum, cancer of the eye, cancer of the intrahepatic
bile duct, cancer of the joints, cancer of the neck, gallbladder,
or pleura, cancer of the nose, nasal cavity, or middle ear, cancer
of the oral cavity, cancer of the vulva, chronic myeloid cancer,
colon cancers (e.g., colon carcinoma), esophageal cancer, cervical
cancer, gastric cancer, gastrointestinal carcinoid tumor,
hypopharynx cancer, larynx cancer, liver cancers (e.g.,
hepatocellular carcinoma), lung cancers (e.g., non-small cell lung
carcinoma), malignant mesothelioma, melanoma, multiple myeloma,
nasopharynx cancer, ovarian cancer, pancreatic cancer, peritoneum,
omentum, and mesentery cancer, pharynx cancer, prostate cancer,
rectal cancer, kidney cancers (e.g., renal cell carcinoma), small
intestine cancer, soft tissue cancer, stomach cancer, testicular
cancer, thyroid cancer, and urothelial cancers (e.g., ureter cancer
and urinary bladder cancer).
[0113] The host referred to in the inventive methods can be any
host. Preferably, the host is a mammal. As used herein, the term
"mammal" refers to any mammal, including, but not limited to,
mammals of the order Rodentia, such as mice and hamsters, and
mammals of the order Logomorpha, such as rabbits. It is preferred
that the mammals are from the order Carnivora, including Felines
(cats) and Canines (dogs). It is more preferred that the mammals
are from the order Artiodactyla, including Bovines (cows) and
Swines (pigs) or of the order Perssodactyla, including Equines
(horses). It is most preferred that the mammals are of the order
Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids
(humans and apes). An especially preferred mammal is the human.
[0114] The following examples further illustrate the invention but,
of course, should not be construed as in any way limiting its
scope.
Example 1
[0115] This example demonstrates the cloning of TCR genes from T
cell clones and the generation of TCR constructs.
[0116] Four T cell clones were initially identified that recognized
epitopes of the MAGE-A gene family in the context of the dominant
class I alleles HLA-A*01 and C*07. Approximately 30% of the
melanoma patient population expresses HLA-A*01, and more than 95%
of HLA-A*01.sup.+ individuals express the HLA-A*0101 sub-type,
while more than 50% of melanoma patients express one of the two
dominant HLA-C*07 sub-types, C*07:01 and C* 07:02.
[0117] The expressed TCR .alpha. and .beta. chains were isolated
from two clones, A10 and 13-18, that recognized residues 168-176 of
protein MAGE-A3 (MAGE-A3:168-176) in the context of HLA-A*01. In
addition, HLA-C*07 restricted TCRs recognizing a peptide
corresponding to residues 170-178 of the MAGE-A12 protein
(MAGE-A12:170-178) were isolated from clones 502 and FM8.
[0118] The .alpha. and .beta. chains encoding functional TCRs were
isolated from two MAGE-A12 reactive, HLA-C*07 reactive T cell
clones, PHIN LB831-501D/19, referred to "502" (Heidecker et al., J.
Immunol., 164: 6041-6045 (2000)) and "FM8" (Panelli et al., J.
Immunol., 164: 4382-4392 (2000)), as well as two MAGE-A3 reactive,
HLA-A*01 restricted T cell clones, LAU147 CTL1 or 810/A10, referred
to "A10" (Pannentier et al., Nat. Immunol., 11: 449-454 (2010)) and
NW1000 AVP-1 13-18, referred to "13-18." Briefly, oligo-dT was used
to reverse transcribe total RNA isolated from the T cell clones
into cDNA using the SMART RACE cDNA amplification kit (Clontech,
Mountain View, Calif.). The TCR .alpha. and .beta. chains expressed
by the T cell clones were identified by carrying out 5'-RACE
reactions using a primer 5'-CACTGTTGCTCTTGAA GTCC-3' (SEQ ID NO:
55) that is complementary to the TCR .alpha. chain constant region
and 5'-CAGGCAGTAT CTGGAGTCATTGAG-3' (SEQ ID NO: 56) that is
complementary to the TCR .beta. chain constant region in
combination with adaptor primers from the SMART RNA synthesis kit.
After sequencing of the 5'-RACE products, full length gene products
were amplified using specific primers designed to amplify the
appropriate full length TCR .alpha. and .beta. chains. The A10 TCR
expresses AV12-1/BV24-1, 13-18 expresses AV12-3/BV15, 502 TCR
expresses AV13-1/BV25-1, and FM8 expresses AV38-2/BV4-3.
[0119] Transcripts encoding the paired .alpha. and .beta. chains
for each of the four T cell clones were inserted into the MSGV1
retroviral expression vectors.
Example 2
[0120] This example demonstrates the reactivity of cells expressing
anti-MAGE-A3 TCR-A10 (SEQ ID NOs: 13 and 14) and anti-MAGE-A3 TCR
13-18 (SEQ ID NOs: 24 and 25) in response to HLA-A1+/MAGE-A3+
cells.
[0121] Anti-CD3 stimulated T cells transduced with TCR-A10 (SEQ ID
NO: 46) and TCR 13-18 (SEQ ID NO: 48) were evaluated for their
ability to recognize a panel of HLA-A*01+melanoma cell lines that
express MAGE-A3. Untransduced (UT) and transduced cells were
co-cultured overnight with various tumor cell lines (Tables 1A, 1B
and FIG. 6A), and interferon-gamma (IFN-.gamma.) (pg/ml) was
measured.
TABLE-US-00001 TABLE 1A Tumor HLA-A*01 Copies MAGE-A3 1860 mel +
12,100 397 mel + 32,700 SK23 mel + 18,400 2984 mel + 14,900 2951
mel + 12,300 A375 mel + 3,670 537 mel + 4,270 1300-A1 mel + 7,280
1300 mel -- 13,600 2661 RCC + <1,000
TABLE-US-00002 TABLE 1B Tumor HLA-A*01 MAGE-A3 2984 mel + + 397 mel
+ + 2630 mel + + 2556 mel + + 526 mel -- + 624 mel -- + 2359 mel --
+ 2661 RCC + --
[0122] The results indicate that six of the eight
HLA-A*01+/MAGE-A3+ melanoma cell lines that were evaluated
stimulated higher levels of IFN-.gamma. release from TCR A10 than
from TCR 13-18-transduced T cells (FIG. 1A). Lower levels of
IFN-.gamma. were released following the co-culture of
TCR-transduced T cells with two HLA-A1+ melanoma cell lines that
expressed relatively low levels of MAGE-A3, A375 mel and 537 mel,
but TCR A10-transduced T cells released higher levels of
IFN-.gamma. than TCR 13-18 transduced T cells in response to these
target cells. These responses were restricted by HLA-A1 because
1300 mel, which lacked expression of HLA-A1, failed to stimulate
IFN-.gamma. release from TCR A10 and TCR 13-18-transduced cells,
whereas a cell line generated by transfection of the parental 1300
mel cell line with HLA-A*01, designated 1300-A1, stimulated
IFN-.gamma. release from TCR A10 and TCR 13-18 transduced T cells.
An HLA-A*01+ renal cancer cell line that lacked expression of
MAGE-A3, 2661 RCC, failed to stimulate significant IFN-.gamma.
release from TCR A10 and TCR 13-18 transduced T cells. These
results demonstrate that cells expressing TCR A10 release higher
levels of IFN-.gamma., than cells expressing TCR-13-18 when
co-cultured with MAGE-A3.sup.+/HLA-A1.sup.+ target cells. These
results also demonstrate that TCR A10 and TCR-13-18 are stimulated
in the presence of MAGE-A3+/HLA-A1+ target cells.
[0123] The results of co-culture assays carried out with transduced
PBMC demonstrated that TCR A10-transduced T cells generated high
levels of IFN-gamma in response to the HLA-A*01+/MAGE-A3+ tumor
cell lines 397 mel, 2984 mel, and 2556 mel. The cytokine levels
were between five and ten times those generated from TCR 13-18
transduced T cells (FIG. 6A). The MAGE-A3+ but HLA-A*01 negative
cell lines 562, 624 and 2359 mel, as well as the MAGE-A3 negative
but HLA-A*01+ renal cancer cell line 2661 RCC failed to stimulate
significant levels of cytokine from either TCR A10 or 13-18
transduced T cells (FIG. 6A).
[0124] The levels of transduction of the TCRs were evaluated using
a quantitative PCR assay carried out using genomic DNA with forward
(SEQ ID NO: 58) and reverse (SEQ ID NO: 59) primers and a probe
(SEQ ID NO: 60) designed to specifically detect the MSGV1
retroviral LTR but not human endogenous retroviral sequences.
Levels of the amplified products were normalized to a positive
control sample of PBMC that had been transduced with a TCR directed
against the NY-ESO-1:157-165 epitope that was estimated to contain
approximately 80% transduced T cells by staining with an NY-ESO-1
tetramer.
[0125] The differences in activity of T cells transduced with the
A10 or 13-18 TCR did not appear to be due to differences in the
frequency of transduction with the two TCRs, as they appeared to be
equivalent (FIG. 6B). In addition, T cells transduced with the A10
TCR recognized target cells incubated with a minimum concentration
of 0.5 nM MAGE-A3 168-176 peptide, which was a 10-fold lower
concentration than required for recognition by cells transduced
with the 13-18 TCR (FIG. 6C), indicating that the A10 TCR possessed
a higher functional avidity than the 13-18 TCR.
[0126] The ability of fresh, un-cultured tumor cells to stimulate T
cells transduced with either TCR A10 (SEQ ID NO: 46) or DMF5 (a TCR
directed against the HLA-A*0201/MART-1:27-35 T cell epitope), was
evaluated. Untransduced (UT) and transduced cells were co-cultured
with various fresh tumors, and IFN-.gamma. (pg/ml) was
measured.
[0127] The results indicated that TCR A10 transduced T cells
recognized four of the four HLA-A*01+/MAGE-A3+ fresh tumors that
were tested (FrTu 2767, FrTu 3178, FrTu 2823 and FrTu 3068), and
DMF5-transduced T cells recognized both of the HLA-A*0201+/MART-1+
fresh tumor cells that were tested (FrTu 2851 and FrTu 3242) (FIG.
1B). The TCR A10 transduced T cells failed to recognize HLA-A*0201+
fresh tumors, while DMF5 transduced T cells failed to recognize
HLA-A*01+ fresh tumors, indicating that the IFN-.gamma. secretion
by TCR A10 was a HLA-A1+/MAGE-A3+-specific response.
[0128] T cells that were transduced with TCR A10 and 13-18
recognized five of six MAGE-A3+ and HLA-A*01.sup.+ fresh tumors
(FrTu), FrTu 3178, 2767, 2823, 2830 and 3068, but did not recognize
either FrTu 2685, an HLA-A*01.sup.+ fresh tumor that lacked
expression of MAGE-A3 or the three MAGE-A3.sup.+ fresh tumors, FrTu
2181, 3242 and 2803, that lacked expression of HLA-A*01 (FIG. 7A;
Table 1C).
TABLE-US-00003 TABLE 1C Fresh Tumor HLA-A*01 MAGE-A3 3178 + + 2767
+ + 2823 + + 2830 + + 3068 + + 2268 + + 2685 + -- 2181 -- + 3242 --
+ 2803 -- +
Example 3
[0129] This example demonstrates the reactivity of cells expressing
anti-MAGE-A12 TCR 502 (SEQ ID NOs: 34 and 35) or anti-MAGE-A12 TCR
FM8 (SEQ ID NOs: 44 and 45) in response to co-culture with
HLA-Cw*07+/MAGE-A12+ cells.
[0130] Anti-CD3 stimulated CD8+ T cells isolated from two patient
PBMC samples were transduced with a control construct encoding the
truncated human low affinity nerve growth factor receptor (NGFR),
TCR 502 (SEQ ID NO: 47), or TCR FM8 (SEQ ID NO: 49) were evaluated
for their ability to recognize a panel of Cw*07+ melanoma cell
lines that express MAGE-A 12.
[0131] Expression of the MAGE-A12 gene product was evaluated by
Q-PCR using two primers (SEQ ID NOs: 61 and 62) designed to
specifically amplify the MAGE-A12 gene product but not other
members of the MAGE-A gene family as well as a MAGE-A12 specific
probe (SEQ ID NO: 63). Antigen expression was determined using
plasmid controls as standards for estimating copy numbers and using
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for normalization.
Tumor cell lines and fresh tumors expressing greater than 1,000
copies of MAGE-A12 per 106 copies of GAPDH were denoted as positive
for MAGE-A12 expression.
[0132] Transduced cells were co-cultured overnight with various
tumor cell lines (Table 2A; FIGS. 6D and 6F), and IFN-.gamma.
(pg/ml) was measured.
TABLE-US-00004 TABLE 2A Stimulator HLA-C allele MAGE-A12 1910 mel
0701, 0303 + 586 mel 0701 + 2359 mel 0701, 16 + F002 mel 0701, 1203
+ 1300 mel 0702 + 624 mel 0702, 0802 + SK23 mel 0701, 0702 + 1909
mel 0701, 0702 + 1011 mel 0702 -- 397 mel 0701 + 526 mel -- + 2556
mel -- + 2984 mel -- + 2630 mel 0701 --
[0133] The results demonstrated that T-cells transduced with TCR
502 recognized eight of the eight MAGE-A12+ melanoma cell lines
tested that express either HLA-Cw*0701 or 0702, whereas T cells
transduced with TCR FM8 only recognized melanoma cell lines that
express HLA-Cw*0702 (FIGS. 2A and 2B; see also FIG. 6F). In
addition, TCR 502 transduced T cells released higher levels of
IFN-.gamma. in response to the HLA-Cw*0702+ targets SK23 mel, 1300
mel and 624 mel as compared to TCR FM8 transduced T cells (FIGS. 6D
and 6F). The 1011 mel cells, which expressed HLA-Cw*0702 but lacked
expression of MAGE-A12, did not stimulate significant cytokine
release from cells transduced with TCR 502 or TCR FM8. T cells
transduced with a control construct encoding NGFR failed to respond
significantly to any of the targets tested. These results
demonstrate that cells expressing TCR 502 release higher levels of
IFN-.gamma. than cells expressing TCR FM8 when co-cultured with
MAGE-A12+/HLA-Cw7 target cells and that TCR 502 recognizes MAGE-A12
in the context of either HLA-Cw0701 or HLA-Cw0702. These results
also demonstrate that TCR A502 and TCR FM8 are stimulated in the
presence of MAGE-A12+ cells.
[0134] The TCR 502 transduced T cells recognized the HLA-C*0702+,
MAGE-A12+ tumor 624 mel as well as two HLA-C*07:01+, MAGE-A12+
tumors, 397 and 2359 mel, whereas FM8 transduced T cells recognized
the HLA-C*07:02+ tumor cell line 624 mel but failed to recognize
397 and 2359 mel (FIG. 6D). Neither population of transduced T
cells recognized 526, 2556, or 2984 mel, MAGE-A3+ melanoma cell
lines that lacked expression of HLA-C*07, or 2630 mel, an
HLA-C*07:01+ tumor cell line that lacked expression of MAGE-A12
(FIG. 6D). These differences did not appear to be due to
differences in transduction frequencies of the two TCRs (measured
as described in Example 2), which appeared to be similar in cells
transduced with either TCR (FIG. 6B). In addition, cells transduced
with the 502 TCR recognized target cells incubated with a minimum
concentration of 2.5 nM MAGE-A12:170-178, a 100-fold lower
concentration than that required for recognition by cells
transduced with the FM8 TCR (FIG. 6E), indicating that the 502 TCR
possessed a higher functional avidity than the FM8 TCR.
[0135] The T cells that were transduced with MAGE-A12 reactive TCRs
were then evaluated for their responses to enzymatic digests of
fresh, un-cultured tumor cells. The T cells transduced with TCR 502
recognized one of the four MAGE-A12.sup.+ fresh tumors that
expressed HLA-C*0701, FrTu 3068, and TCR 502 as well as FM8
transduced T cells recognized one of the two MAGE-A12.sup.+ tumors
that expressed HLA-C*07:02, FrTu 2181 (FIG. 7B; Table 2B). Neither
population of TCR transduced T cells recognized the
HLA-C*07:01.sup.- and 07:02.sup.- fresh tumors 2767 or 2823, or the
MAGE-A12.sup.- tumors 2685, 3242 and 2803 that lacked expression of
MAGE-A12.
TABLE-US-00005 TABLE 2B Fresh Tumor HLA-C*07 MAGE-A12 3068 01 +
2181 02 + 3178 01 + 2830 01 + 2268 01, 02 + 2767 -- + 2823 -- +
2685 01 -- 3242 01 -- 2803 02 --
Example 4
[0136] This example demonstrates the population that may be treated
using the inventive TCRs.
[0137] Approximately 28% of the patient population expresses
HLA-A*01, and approximately 54% of the patient population expresses
HLA-Cw*07. Two dominant subtypes of HLA-Cw*07, Cw*0701 and Cw*0702,
are expressed by approximately 27% and approximately 31% of the
patient population, respectively. FIG. 3A illustrates the
cumulative percentage of the population that would be expected to
be treatable by the use of TCRs restricted by HLA-A1, HLA-A2,
and/or HLA-Cw7 (based upon the percentages of these alleles in the
normal Caucasian population).
[0138] Because approximately 30% of patients express high levels of
MAGE-A3 and MAGE-A12, the use of the inventive TCRs will allow a
significantly higher percentage of patients to be eligible for
TCR-based adoptive immunotherapies. FIGS. 3B and 3C illustrate the
cumulative percentage of the human melanoma (FIG. 3B) and synovial
cell sarcoma (FIG. 3C) patient populations that would be expected
to be treatable using TCRs that recognize NY-ESO-1 in the context
of HLA-A2; MAGE-A3 in the context of HLA-A1; MAGE-A3 and MAGE-A12
in the context of HLA-A2; and/or MAGE-A12 in the context of
HLA-Cw7.
Example 5
[0139] This example demonstrates the reactivity of cells expressing
TCR 502 or TCR FM8 in response to co-culture with HLA-Cw*0701 or
HLA-Cw*0702-expressing target cells pulsed with peptides of various
proteins from the MAGE family.
[0140] Cells transduced with NGFR, TCR 502 (SEQ ID NO: 47), or TCR
FM8 (SEQ ID NO: 49) were co-cultured with cells expressing
HLA-Cw*0701 or HLA-Cw*0702. IFN-.gamma. (pg/ml) secretion was
measured.
[0141] The results demonstrated that cells transduced with TCR 502
recognize cells expressing HLA-Cw*0701 or HLA-Cw*0702 when pulsed
with MAGE-A12 (VRIGHLYIL; SEQ ID NO: 4), and that cells transduced
with TCR FM8 recognized cells expressing HLA-Cw*0702 when pulsed
with MAGE-A12 (VRIGHLYIL; SEQ ID NO: 4) (FIG. 4). Cells transduced
with NGFR showed no significant reactivity.
Example 6
[0142] This example demonstrates the specificity of the
anti-MAGE-A3 and anti-MAGE-A12 TCRs.
[0143] Following stimulation with anti-CD3 antibody, PMBC from a
single donor were transduced with PBMC that were untransduced or
transduced with anti-MAGE-A12 TCR 502 (SEQ ID NO: 47),
anti-MAGE-A12 TCR FM8 (SEQ ID NO: 49), anti-MAGE-A3 TCR A10 (SEQ ID
NO: 46), anti-MAGE-A3 TCR 13-18 (SEQ ID NO: 48), or anti-MAGE-A3
TCR 112-120. Thirteen days after stimulation, transduced T cells
were incubated with the tumor targets set forth in Table 3 in a
standard 4 hour .sup.51Cr release assay.
TABLE-US-00006 TABLE 3 MAGE-A3 MAGE-A12 HLA-A HLA-C 397 mel + +
01/02 0401/0701 624 mel + + 02/03 0702/0802 2984 mel + + 01/02 06
2661 RCC -- -- 01/02 07
[0144] As shown in FIGS. 5(A)-5(D), anti-MAGE-A12 TCR 502
specifically lysed tumor cells that expressed MAGE-A12 and HLA-Cw7
and did not lyse tumor cells that lacked expression of MAGE-A12 or
HLA-Cw7. Anti-MAGE-A3 TCR A10 specifically lysed tumor cells that
expressed MAGE-A3 and HLA-A1 and did not lyse tumor cells that
lacked expression of MAGE-A3 or HLA-A1.
Example 7
[0145] This example demonstrates the specificity of the
anti-MAGE-A3 and anti-MAGE-A12 TCRs.
[0146] The monkey kidney cell line COS-7 was transiently
transfected with either HLA-A*01, C*07:01 or C*07:02 plus either
MAGE-A3, A1, A2, A4, A6, A9, A10 or A12 overnight. The following
day T cells transduced with either TCR A10, 13-18 or un-transduced
control cells or TCR 502, FM8 or un-transduced control cells were
added and the release of soluble IFN-gamma was evaluated following
an overnight co-culture by ELISA.
[0147] T cells transduced with the MAGE-A3-reactive TCR A10
recognize HLA-A1' target cells transfected with MAGE-A3 but failed
to recognize targets transfected with MAGE-A1, A2, A4, A6, A9, A10
or A12 constructs (FIG. 8A) that encoded peptides that differed at
between one and three positions from the MAGE-A3:170-178 epitope.
The T cells that were transduced with either TCR 502 or FM8
recognized HLA-C*07:02.sup.+ targets transfected with MAGE-A12 but
not MAGE-A3, A1, A2, A4,A6, A9, A10 (FIG. 8B), while T cells
transduced with TCR 502 but not FM8 recognized HLA-C*07:01.sup.+
targets transfected with MAGE-A12 but not the additional MAGE-A
family members tested (FIG. 8C).
Example 8
[0148] This example demonstrates the reactivity of transduced T
cells.
[0149] Purified CD8+ and CD4+ T cells were isolated by negative
selection using CD8 and CD4 T lymphocyte enrichment kits
(Becton/Dickinson, Franklin Lakes, N.J.), followed by positive
selection using CD8 and CD4 magnetic beads (Becton/Dickinson). The
isolated CD8+ and CD4+ cells were estimated by fluorescence
activated cell sorting (FACS) analysis to contain less than 1%
contaminating CD4+ and CD8+ T cells, respectively.
[0150] The responses of separated populations of CD8+ and CD4+ T
cells transduced with TCRs to tumor cell targets was then
evaluated. Highly purified CD4+ T cells transduced with TCR A10
containing fewer than 1% contaminating CD8+ T cells released low
but significant levels of IFN-gamma in response to MAGE-A3+ tumor
cell line 397 mel as well as the MAGE-A3+ tumor cell line 1300A1
mel that was stably transfected with HLA-A*01 (Table 4A; FIG. 9B).
CD8+ T cells transduced with TCR A10 released interferon-gamma in
response to 397 mel or 1300-A1 mel (Table 4A; FIG. 9A). CD8+ T
cells transduced with TCR 502 or TCR FM8 released interferon-gamma
in response to 624 mel, and TCR 502 released interferon-gamma in
response to 397 mel (FIG. 9C and Table 4B).
TABLE-US-00007 TABLE 4A Cell Line HLA-A*01 MAGE-A3 397 mel + + 1300
mel + + 1300-A1 mel + + 624 mel -- + 2359 mel -- + 2661 RCC +
--
TABLE-US-00008 TABLE 4B Cell Line HLA-C*07 MAGE-A12 397 mel 01 +
624 mel 02 + 2359 mel 01 + 526 mel -- + 2661 RCC -- --
[0151] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0152] The use of the terms "a" and "an" and "the" and "at least
one" and similar referents in the context of describing the
invention (especially in the context of the following claims) are
to be construed to cover both the singular and the plural, unless
otherwise indicated herein or clearly contradicted by context. The
use of the term "at least one" followed by a list of one or more
items (for example, "at least one of A and B") is to be construed
to mean one item selected from the listed items (A or B) or any
combination of two or more of the listed items (A and B), unless
otherwise indicated herein or clearly contradicted by context. The
terms "comprising," "having," "including," and "containing" are to
be construed as open-ended terms (i.e., meaning "including, but not
limited to,") unless otherwise noted. Recitation of ranges of
values herein are merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range, unless otherwise indicated herein, and each separate value
is incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0153] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
Sequence CWU 1
1
631314PRTHomo sapiens 1Met Pro Leu Glu Gln Arg Ser Gln His Cys Lys
Pro Glu Glu Gly Leu 1 5 10 15 Glu Ala Arg Gly Glu Ala Leu Gly Leu
Val Gly Ala Gln Ala Pro Ala 20 25 30 Thr Glu Glu Gln Glu Ala Ala
Ser Ser Ser Ser Thr Leu Val Glu Val 35 40 45 Thr Leu Gly Glu Val
Pro Ala Ala Glu Ser Pro Asp Pro Pro Gln Ser 50 55 60 Pro Gln Gly
Ala Ser Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp 65 70 75 80 Ser
Gln Ser Tyr Glu Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser 85 90
95 Thr Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys
100 105 110 Val Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala
Arg Glu 115 120 125 Pro Val Thr Lys Ala Glu Met Leu Gly Ser Val Val
Gly Asn Trp Gln 130 135 140 Tyr Phe Phe Pro Val Ile Phe Ser Lys Ala
Ser Ser Ser Leu Gln Leu 145 150 155 160 Val Phe Gly Ile Glu Leu Met
Glu Val Asp Pro Ile Gly His Leu Tyr 165 170 175 Ile Phe Ala Thr Cys
Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp 180 185 190 Asn Gln Ile
Met Pro Lys Ala Gly Leu Leu Ile Ile Val Leu Ala Ile 195 200 205 Ile
Ala Arg Glu Gly Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu 210 215
220 Leu Ser Val Leu Glu Val Phe Glu Gly Arg Glu Asp Ser Ile Leu Gly
225 230 235 240 Asp Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu
Asn Tyr Leu 245 250 255 Glu Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala
Cys Tyr Glu Phe Leu 260 265 270 Trp Gly Pro Arg Ala Leu Val Glu Thr
Ser Tyr Val Lys Val Leu His 275 280 285 His Met Val Lys Ile Ser Gly
Gly Pro His Ile Ser Tyr Pro Pro Leu 290 295 300 His Glu Trp Val Leu
Arg Glu Gly Glu Glu 305 310 29PRTHomo sapiens 2Glu Val Asp Pro Ile
Gly His Leu Tyr 1 5 3314PRTHomo sapiens 3Met Pro Leu Glu Gln Arg
Ser Gln His Cys Lys Pro Glu Glu Gly Leu 1 5 10 15 Glu Ala Gln Gly
Glu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Ala 20 25 30 Thr Glu
Glu Gln Glu Thr Ala Ser Ser Ser Ser Thr Leu Val Glu Val 35 40 45
Thr Leu Arg Glu Val Pro Ala Ala Glu Ser Pro Ser Pro Pro His Ser 50
55 60 Pro Gln Gly Ala Ser Thr Leu Pro Thr Thr Ile Asn Tyr Thr Leu
Trp 65 70 75 80 Ser Gln Ser Asp Glu Gly Ser Ser Asn Glu Glu Gln Glu
Gly Pro Ser 85 90 95 Thr Phe Pro Asp Leu Glu Thr Ser Phe Gln Val
Ala Leu Ser Arg Lys 100 105 110 Met Ala Glu Leu Val His Phe Leu Leu
Leu Lys Tyr Arg Ala Arg Glu 115 120 125 Pro Phe Thr Lys Ala Glu Met
Leu Gly Ser Val Ile Arg Asn Phe Gln 130 135 140 Asp Phe Phe Pro Val
Ile Phe Ser Lys Ala Ser Glu Tyr Leu Gln Leu 145 150 155 160 Val Phe
Gly Ile Glu Val Val Glu Val Val Arg Ile Gly His Leu Tyr 165 170 175
Ile Leu Val Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp 180
185 190 Asn Gln Ile Val Pro Lys Thr Gly Leu Leu Ile Ile Val Leu Ala
Ile 195 200 205 Ile Ala Lys Glu Gly Asp Cys Ala Pro Glu Glu Lys Ile
Trp Glu Glu 210 215 220 Leu Ser Val Leu Glu Ala Ser Asp Gly Arg Glu
Asp Ser Val Phe Ala 225 230 235 240 His Pro Arg Lys Leu Leu Thr Gln
Asp Leu Val Gln Glu Asn Tyr Leu 245 250 255 Glu Tyr Arg Gln Val Pro
Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu 260 265 270 Trp Gly Pro Arg
Ala Leu Val Glu Thr Ser Tyr Val Lys Val Leu His 275 280 285 His Leu
Leu Lys Ile Ser Gly Gly Pro His Ile Ser Tyr Pro Pro Leu 290 295 300
His Glu Trp Ala Phe Arg Glu Gly Glu Glu 305 310 49PRTHomo sapiens
4Val Arg Ile Gly His Leu Tyr Ile Leu 1 5 56PRTHomo sapiens 5Asn Ser
Ala Ser Gln Ser 1 5 66PRTHomo sapiens 6Val Tyr Ser Ser Gly Asn 1 5
79PRTHomo sapiens 7Val Val Asn Arg Asp Asn Asp Met Arg 1 5
85PRTHomo sapiens 8Lys Gly His Asp Arg 1 5 96PRTHomo sapiens 9Ser
Phe Asp Val Lys Asp 1 5 1012PRTHomo sapiens 10Ala Thr Ser Glu Gly
Gly Pro Pro Tyr Glu Gln Tyr 1 5 10 11129PRTHomo sapiens 11Met Ile
Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser 1 5 10 15
Trp Val Trp Ser Gln Arg Lys Glu Val Glu Gln Asp Pro Gly Pro Phe 20
25 30 Asn Val Pro Glu Gly Ala Thr Val Ala Phe Asn Cys Thr Tyr Ser
Asn 35 40 45 Ser Ala Ser Gln Ser Phe Phe Trp Tyr Arg Gln Asp Cys
Arg Lys Glu 50 55 60 Pro Lys Leu Leu Met Ser Val Tyr Ser Ser Gly
Asn Glu Asp Gly Arg 65 70 75 80 Phe Thr Ala Gln Leu Asn Arg Ala Ser
Gln Tyr Ile Ser Leu Leu Ile 85 90 95 Arg Asp Ser Lys Leu Ser Asp
Ser Ala Thr Tyr Leu Cys Val Val Asn 100 105 110 Arg Asp Asn Asp Met
Arg Phe Gly Ala Gly Thr Arg Leu Thr Val Lys 115 120 125 Pro
12132PRTHomo sapiens 12Met Ala Ser Leu Leu Phe Phe Cys Gly Ala Phe
Tyr Leu Leu Gly Thr 1 5 10 15 Gly Ser Met Asp Ala Asp Val Thr Gln
Thr Pro Arg Asn Arg Ile Thr 20 25 30 Lys Thr Gly Lys Arg Ile Met
Leu Glu Cys Ser Gln Thr Lys Gly His 35 40 45 Asp Arg Met Tyr Trp
Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu 50 55 60 Ile Tyr Tyr
Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser 65 70 75 80 Asp
Gly Tyr Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser 85 90
95 Leu Glu Ser Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr
100 105 110 Ser Glu Gly Gly Pro Pro Tyr Glu Gln Tyr Phe Gly Pro Gly
Thr Arg 115 120 125 Leu Thr Val Thr 130 13270PRTHomo sapiens 13Met
Ile Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu Ser 1 5 10
15 Trp Val Trp Ser Gln Arg Lys Glu Val Glu Gln Asp Pro Gly Pro Phe
20 25 30 Asn Val Pro Glu Gly Ala Thr Val Ala Phe Asn Cys Thr Tyr
Ser Asn 35 40 45 Ser Ala Ser Gln Ser Phe Phe Trp Tyr Arg Gln Asp
Cys Arg Lys Glu 50 55 60 Pro Lys Leu Leu Met Ser Val Tyr Ser Ser
Gly Asn Glu Asp Gly Arg 65 70 75 80 Phe Thr Ala Gln Leu Asn Arg Ala
Ser Gln Tyr Ile Ser Leu Leu Ile 85 90 95 Arg Asp Ser Lys Leu Ser
Asp Ser Ala Thr Tyr Leu Cys Val Val Asn 100 105 110 Arg Asp Asn Asp
Met Arg Phe Gly Ala Gly Thr Arg Leu Thr Val Lys 115 120 125 Pro Asn
Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser 130 135 140
Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Ile Asp Ser Gln 145
150 155 160 Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr
Asp Lys 165 170 175 Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser
Asn Ser Ala Val 180 185 190 Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys
Ala Asn Ala Phe Asn Asn 195 200 205 Ser Ile Ile Pro Glu Asp Thr Phe
Phe Pro Ser Pro Glu Ser Ser Cys 210 215 220 Asp Val Lys Leu Val Glu
Lys Ser Phe Glu Thr Asp Thr Asn Leu Asn 225 230 235 240 Phe Gln Asn
Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val 245 250 255 Ala
Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 270
14311PRTHomo sapiens 14Met Ala Ser Leu Leu Phe Phe Cys Gly Ala Phe
Tyr Leu Leu Gly Thr 1 5 10 15 Gly Ser Met Asp Ala Asp Val Thr Gln
Thr Pro Arg Asn Arg Ile Thr 20 25 30 Lys Thr Gly Lys Arg Ile Met
Leu Glu Cys Ser Gln Thr Lys Gly His 35 40 45 Asp Arg Met Tyr Trp
Tyr Arg Gln Asp Pro Gly Leu Gly Leu Arg Leu 50 55 60 Ile Tyr Tyr
Ser Phe Asp Val Lys Asp Ile Asn Lys Gly Glu Ile Ser 65 70 75 80 Asp
Gly Tyr Ser Val Ser Arg Gln Ala Gln Ala Lys Phe Ser Leu Ser 85 90
95 Leu Glu Ser Ala Ile Pro Asn Gln Thr Ala Leu Tyr Phe Cys Ala Thr
100 105 110 Ser Glu Gly Gly Pro Pro Tyr Glu Gln Tyr Phe Gly Pro Gly
Thr Arg 115 120 125 Leu Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro
Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser
His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly
Phe Tyr Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly
Lys Glu Val His Ser Gly Val Ser Thr Asp Pro 180 185 190 Gln Pro Leu
Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser
Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215
220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu
225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val
Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser
Glu Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Ala Thr Ile Leu Tyr
Glu Ile Leu Leu Gly Lys Ala 275 280 285 Thr Leu Tyr Ala Val Leu Val
Ser Ala Leu Val Leu Met Ala Met Val 290 295 300 Lys Arg Lys Asp Ser
Arg Gly 305 310 1527PRTHomo sapiens 15Arg Ala Lys Arg Ser Gly Ser
Gly Ala Thr Asn Phe Ser Leu Leu Lys 1 5 10 15 Gln Ala Gly Asp Val
Glu Glu Asn Pro Gly Pro 20 25 166PRTHomo sapiens 16Asn Ser Ala Phe
Gln Tyr 1 5 176PRTHomo sapiens 17Thr Tyr Ser Ser Gly Asn 1 5
189PRTHomo sapiens 18Ala Met Arg Gly Ala Gln Lys Leu Val 1 5
195PRTHomo sapiens 19Leu Asn His Asn Val 1 5 206PRTHomo sapiens
20Tyr Tyr Asp Lys Asp Phe 1 5 2111PRTHomo sapiens 21Ala Thr Ser Trp
Asp Arg Gly Tyr Glu Gln Tyr 1 5 10 22131PRTHomo sapiens 22Met Met
Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu 1 5 10 15
Ser Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asp Pro Gly Pro 20
25 30 Leu Ser Val Pro Glu Gly Ala Ile Val Ser Leu Asn Cys Thr Tyr
Ser 35 40 45 Asn Ser Ala Phe Gln Tyr Phe Met Trp Tyr Arg Gln Tyr
Ser Arg Lys 50 55 60 Gly Pro Glu Leu Leu Met Tyr Thr Tyr Ser Ser
Gly Asn Lys Glu Asp 65 70 75 80 Gly Arg Phe Thr Ala Gln Val Asp Lys
Ser Ser Lys Tyr Ile Ser Leu 85 90 95 Phe Ile Arg Asp Ser Gln Pro
Ser Asp Ser Ala Thr Tyr Leu Cys Ala 100 105 110 Met Arg Gly Ala Gln
Lys Leu Val Phe Gly Gln Gly Thr Arg Leu Thr 115 120 125 Ile Asn Pro
130 23131PRTHomo sapiens 23Met Gly Pro Gly Leu Leu His Trp Met Ala
Leu Cys Leu Leu Gly Thr 1 5 10 15 Gly His Gly Asp Ala Met Val Ile
Gln Asn Pro Arg Tyr Gln Val Thr 20 25 30 Gln Phe Gly Lys Pro Val
Thr Leu Ser Cys Ser Gln Thr Leu Asn His 35 40 45 Asn Val Met Tyr
Trp Tyr Gln Gln Lys Ser Ser Gln Ala Pro Lys Leu 50 55 60 Leu Phe
His Tyr Tyr Asp Lys Asp Phe Asn Asn Glu Ala Asp Thr Pro 65 70 75 80
Asp Asn Phe Gln Ser Arg Arg Pro Asn Thr Ser Phe Cys Phe Leu Asp 85
90 95 Ile Arg Ser Pro Gly Leu Gly Asp Ala Ala Met Tyr Leu Cys Ala
Thr 100 105 110 Ser Trp Asp Arg Gly Tyr Glu Gln Tyr Phe Gly Pro Gly
Thr Arg Leu 115 120 125 Thr Val Thr 130 24272PRTHomo sapiens 24Met
Met Lys Ser Leu Arg Val Leu Leu Val Ile Leu Trp Leu Gln Leu 1 5 10
15 Ser Trp Val Trp Ser Gln Gln Lys Glu Val Glu Gln Asp Pro Gly Pro
20 25 30 Leu Ser Val Pro Glu Gly Ala Ile Val Ser Leu Asn Cys Thr
Tyr Ser 35 40 45 Asn Ser Ala Phe Gln Tyr Phe Met Trp Tyr Arg Gln
Tyr Ser Arg Lys 50 55 60 Gly Pro Glu Leu Leu Met Tyr Thr Tyr Ser
Ser Gly Asn Lys Glu Asp 65 70 75 80 Gly Arg Phe Thr Ala Gln Val Asp
Lys Ser Ser Lys Tyr Ile Ser Leu 85 90 95 Phe Ile Arg Asp Ser Gln
Pro Ser Asp Ser Ala Thr Tyr Leu Cys Ala 100 105 110 Met Arg Gly Ala
Gln Lys Leu Val Phe Gly Gln Gly Thr Arg Leu Thr 115 120 125 Ile Asn
Pro Asn Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg 130 135 140
Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp 145
150 155 160 Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr
Ile Thr 165 170 175 Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe
Lys Ser Asn Ser 180 185 190 Ala Val Ala Trp Ser Asn Lys Ser Asp Phe
Ala Cys Ala Asn Ala Phe 195 200 205 Asn Asn Ser Ile Ile Pro Glu Asp
Thr Phe Phe Pro Ser Pro Glu Ser 210 215 220 Ser Cys Asp Val Lys Leu
Val Glu Lys Ser Phe Glu Thr Asp Thr Asn 225 230 235 240 Leu Asn Phe
Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu 245 250 255 Lys
Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Arg 260 265
270 25310PRTHomo sapiens 25Met Gly Pro Gly Leu Leu His Trp Met Ala
Leu Cys Leu Leu Gly Thr 1 5 10 15 Gly His Gly Asp Ala Met Val Ile
Gln Asn Pro Arg Tyr Gln Val Thr 20 25 30 Gln Phe Gly Lys Pro Val
Thr Leu Ser Cys Ser Gln Thr Leu Asn His 35 40 45 Asn Val Met Tyr
Trp Tyr Gln Gln Lys Ser Ser Gln Ala Pro Lys Leu 50 55 60 Leu Phe
His Tyr Tyr Asp Lys Asp Phe Asn Asn Glu Ala Asp Thr Pro 65 70 75 80
Asp Asn Phe Gln Ser Arg Arg Pro Asn Thr Ser Phe Cys Phe Leu Asp 85
90 95 Ile Arg Ser Pro Gly Leu Gly Asp Ala Ala Met
Tyr Leu Cys Ala Thr 100 105 110 Ser Trp Asp Arg Gly Tyr Glu Gln Tyr
Phe Gly Pro Gly Thr Arg Leu 115 120 125 Thr Val Thr Glu Asp Leu Lys
Asn Val Phe Pro Pro Glu Val Ala Val 130 135 140 Phe Glu Pro Ser Glu
Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu 145 150 155 160 Val Cys
Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp 165 170 175
Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln 180
185 190 Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu
Ser 195 200 205 Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro
Arg Asn His 210 215 220 Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser
Glu Asn Asp Glu Trp 225 230 235 240 Thr Gln Asp Arg Ala Lys Pro Val
Thr Gln Ile Val Ser Ala Glu Ala 245 250 255 Trp Gly Arg Ala Asp Cys
Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly 260 265 270 Val Leu Ser Ala
Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr 275 280 285 Leu Tyr
Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys 290 295 300
Arg Lys Asp Ser Arg Gly 305 310 266PRTHomo sapiens 26Asp Ser Ala
Ser Asn Tyr 1 5 277PRTHomo sapiens 27Ile Arg Ser Asn Val Gly Glu 1
5 2810PRTHomo sapiens 28Ala Ala Ser Gly Ala Thr Asp Lys Leu Ile 1 5
10 295PRTHomo sapiens 29Met Gly His Asp Lys 1 5 306PRTHomo sapiens
30Ser Tyr Gly Val Asn Ser 1 5 319PRTHomo sapiens 31Ala Ser Ser Gly
Gly His Glu Gln Tyr 1 5 32130PRTHomo sapiens 32Met Thr Ser Ile Arg
Ala Val Phe Ile Phe Leu Trp Leu Gln Leu Asp 1 5 10 15 Leu Val Asn
Gly Glu Asn Val Glu Gln His Pro Ser Thr Leu Ser Val 20 25 30 Gln
Glu Gly Asp Ser Ala Val Ile Lys Cys Thr Tyr Ser Asp Ser Ala 35 40
45 Ser Asn Tyr Phe Pro Trp Tyr Lys Gln Glu Leu Gly Lys Arg Pro Gln
50 55 60 Leu Ile Ile Asp Ile Arg Ser Asn Val Gly Glu Lys Lys Asp
Gln Arg 65 70 75 80 Ile Ala Val Thr Leu Asn Lys Thr Ala Lys His Phe
Ser Leu His Ile 85 90 95 Thr Glu Thr Gln Pro Glu Asp Ser Ala Val
Tyr Phe Cys Ala Ala Ser 100 105 110 Gly Ala Thr Asp Lys Leu Ile Phe
Gly Thr Gly Thr Arg Leu Gln Val 115 120 125 Phe Pro 130
33129PRTHomo sapiens 33Met Thr Ile Arg Leu Leu Cys Tyr Met Gly Phe
Tyr Phe Leu Gly Ala 1 5 10 15 Gly Leu Met Glu Ala Asp Ile Tyr Gln
Thr Pro Arg Tyr Leu Val Ile 20 25 30 Gly Thr Gly Lys Lys Ile Thr
Leu Glu Cys Ser Gln Thr Met Gly His 35 40 45 Asp Lys Met Tyr Trp
Tyr Gln Gln Asp Pro Gly Met Glu Leu His Leu 50 55 60 Ile His Tyr
Ser Tyr Gly Val Asn Ser Thr Glu Lys Gly Asp Leu Ser 65 70 75 80 Ser
Glu Ser Thr Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr 85 90
95 Leu Glu Ser Ala Arg Pro Ser His Thr Ser Gln Tyr Leu Cys Ala Ser
100 105 110 Ser Gly Gly His Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu
Thr Val 115 120 125 Thr 34271PRTHomo sapiens 34Met Thr Ser Ile Arg
Ala Val Phe Ile Phe Leu Trp Leu Gln Leu Asp 1 5 10 15 Leu Val Asn
Gly Glu Asn Val Glu Gln His Pro Ser Thr Leu Ser Val 20 25 30 Gln
Glu Gly Asp Ser Ala Val Ile Lys Cys Thr Tyr Ser Asp Ser Ala 35 40
45 Ser Asn Tyr Phe Pro Trp Tyr Lys Gln Glu Leu Gly Lys Arg Pro Gln
50 55 60 Leu Ile Ile Asp Ile Arg Ser Asn Val Gly Glu Lys Lys Asp
Gln Arg 65 70 75 80 Ile Ala Val Thr Leu Asn Lys Thr Ala Lys His Phe
Ser Leu His Ile 85 90 95 Thr Glu Thr Gln Pro Glu Asp Ser Ala Val
Tyr Phe Cys Ala Ala Ser 100 105 110 Gly Ala Thr Asp Lys Leu Ile Phe
Gly Thr Gly Thr Arg Leu Gln Val 115 120 125 Phe Pro Asn Ile Gln Asn
Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp 130 135 140 Ser Lys Ser Ser
Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser 145 150 155 160 Gln
Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp 165 170
175 Lys Thr Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala
180 185 190 Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala
Phe Asn 195 200 205 Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser
Pro Glu Ser Ser 210 215 220 Cys Asp Val Lys Leu Val Glu Lys Ser Phe
Glu Thr Asp Thr Asn Leu 225 230 235 240 Asn Phe Gln Asn Leu Ser Val
Ile Gly Phe Arg Ile Leu Leu Leu Lys 245 250 255 Val Ala Gly Phe Asn
Leu Leu Met Thr Leu Arg Leu Trp Ser Ser 260 265 270 35308PRTHomo
sapiens 35Met Thr Ile Arg Leu Leu Cys Tyr Met Gly Phe Tyr Phe Leu
Gly Ala 1 5 10 15 Gly Leu Met Glu Ala Asp Ile Tyr Gln Thr Pro Arg
Tyr Leu Val Ile 20 25 30 Gly Thr Gly Lys Lys Ile Thr Leu Glu Cys
Ser Gln Thr Met Gly His 35 40 45 Asp Lys Met Tyr Trp Tyr Gln Gln
Asp Pro Gly Met Glu Leu His Leu 50 55 60 Ile His Tyr Ser Tyr Gly
Val Asn Ser Thr Glu Lys Gly Asp Leu Ser 65 70 75 80 Ser Glu Ser Thr
Val Ser Arg Ile Arg Thr Glu His Phe Pro Leu Thr 85 90 95 Leu Glu
Ser Ala Arg Pro Ser His Thr Ser Gln Tyr Leu Cys Ala Ser 100 105 110
Ser Gly Gly His Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val 115
120 125 Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe
Glu 130 135 140 Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr
Leu Val Cys 145 150 155 160 Leu Ala Thr Gly Phe Phe Pro Asp His Val
Glu Leu Ser Trp Trp Val 165 170 175 Asn Gly Lys Glu Val His Ser Gly
Val Ser Thr Asp Pro Gln Pro Leu 180 185 190 Lys Glu Gln Pro Ala Leu
Asn Asp Ser Arg Tyr Cys Leu Ser Ser Arg 195 200 205 Leu Arg Val Ser
Ala Thr Phe Trp Gln Asn Pro Arg Asn His Phe Arg 210 215 220 Cys Gln
Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr Gln 225 230 235
240 Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp Gly
245 250 255 Arg Ala Asp Cys Gly Phe Thr Ser Val Ser Tyr Gln Gln Gly
Val Leu 260 265 270 Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys
Ala Thr Leu Tyr 275 280 285 Ala Val Leu Val Ser Ala Leu Val Leu Met
Ala Met Val Lys Arg Lys 290 295 300 Asp Ser Arg Gly 305 367PRTHomo
sapiens 36Thr Ser Glu Ser Asp Tyr Tyr 1 5 378PRTHomo sapiens 37Gln
Glu Ala Tyr Lys Gln Gln Asn 1 5 3813PRTHomo sapiens 38Ala Tyr Arg
Ser Ala Gln Gly Gly Ser Glu Lys Leu Val 1 5 10 395PRTHomo sapiens
39Leu Gly His Asn Ala 1 5 406PRTHomo sapiens 40Tyr Ser Leu Glu Glu
Arg 1 5 4112PRTHomo sapiens 41Ala Ser Ser Gln Thr Thr Tyr Tyr Asn
Glu Gln Phe 1 5 10 42137PRTHomo sapiens 42Met Ala Cys Pro Gly Phe
Leu Trp Ala Leu Val Ile Ser Thr Cys Leu 1 5 10 15 Glu Phe Ser Met
Ala Gln Thr Val Thr Gln Ser Gln Pro Glu Met Ser 20 25 30 Val Gln
Glu Ala Glu Thr Val Thr Leu Ser Cys Thr Tyr Asp Thr Ser 35 40 45
Glu Ser Asp Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln 50
55 60 Met Ile Leu Val Ile Arg Gln Glu Ala Tyr Lys Gln Gln Asn Ala
Thr 65 70 75 80 Glu Asn Arg Phe Ser Val Asn Phe Gln Lys Ala Ala Lys
Ser Phe Ser 85 90 95 Leu Lys Ile Ser Asp Ser Gln Leu Gly Asp Ala
Ala Met Tyr Phe Cys 100 105 110 Ala Tyr Arg Ser Ala Gln Gly Gly Ser
Glu Lys Leu Val Phe Gly Lys 115 120 125 Gly Thr Lys Leu Thr Val Asn
Pro Tyr 130 135 43132PRTHomo sapiens 43Met Gly Cys Arg Leu Leu Cys
Cys Ala Val Leu Cys Leu Leu Gly Ala 1 5 10 15 Val Pro Met Glu Thr
Gly Val Thr Gln Thr Pro Arg His Leu Val Met 20 25 30 Gly Met Thr
Asn Lys Lys Ser Leu Lys Cys Glu Gln His Leu Gly His 35 40 45 Asn
Ala Met Tyr Trp Tyr Lys Gln Ser Ala Lys Lys Pro Leu Glu Leu 50 55
60 Met Phe Val Tyr Ser Leu Glu Glu Arg Val Glu Asn Asn Ser Val Pro
65 70 75 80 Ser Arg Phe Ser Pro Glu Cys Pro Asn Ser Ser His Leu Phe
Leu His 85 90 95 Leu His Thr Leu Gln Pro Glu Asp Ser Ala Leu Tyr
Leu Cys Ala Ser 100 105 110 Ser Gln Thr Thr Tyr Tyr Asn Glu Gln Phe
Phe Gly Pro Gly Thr Arg 115 120 125 Leu Thr Val Leu 130
44277PRTHomo sapiens 44Met Ala Cys Pro Gly Phe Leu Trp Ala Leu Val
Ile Ser Thr Cys Leu 1 5 10 15 Glu Phe Ser Met Ala Gln Thr Val Thr
Gln Ser Gln Pro Glu Met Ser 20 25 30 Val Gln Glu Ala Glu Thr Val
Thr Leu Ser Cys Thr Tyr Asp Thr Ser 35 40 45 Glu Ser Asp Tyr Tyr
Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln 50 55 60 Met Ile Leu
Val Ile Arg Gln Glu Ala Tyr Lys Gln Gln Asn Ala Thr 65 70 75 80 Glu
Asn Arg Phe Ser Val Asn Phe Gln Lys Ala Ala Lys Ser Phe Ser 85 90
95 Leu Lys Ile Ser Asp Ser Gln Leu Gly Asp Ala Ala Met Tyr Phe Cys
100 105 110 Ala Tyr Arg Ser Ala Gln Gly Gly Ser Glu Lys Leu Val Phe
Gly Lys 115 120 125 Gly Thr Lys Leu Thr Val Asn Pro Tyr Ile Gln Asn
Pro Asp Pro Ala 130 135 140 Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser
Asp Lys Ser Val Cys Leu 145 150 155 160 Phe Thr Asp Phe Asp Ser Gln
Thr Asn Val Ser Gln Ser Lys Asp Ser 165 170 175 Asp Val Tyr Ile Thr
Asp Lys Thr Val Leu Asp Met Arg Ser Met Asp 180 185 190 Phe Lys Ser
Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala 195 200 205 Cys
Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe 210 215
220 Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe
225 230 235 240 Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val
Ile Gly Phe 245 250 255 Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn
Leu Leu Met Thr Leu 260 265 270 Arg Leu Trp Ser Ser 275
45311PRTHomo sapiens 45Met Gly Cys Arg Leu Leu Cys Cys Ala Val Leu
Cys Leu Leu Gly Ala 1 5 10 15 Val Pro Met Glu Thr Gly Val Thr Gln
Thr Pro Arg His Leu Val Met 20 25 30 Gly Met Thr Asn Lys Lys Ser
Leu Lys Cys Glu Gln His Leu Gly His 35 40 45 Asn Ala Met Tyr Trp
Tyr Lys Gln Ser Ala Lys Lys Pro Leu Glu Leu 50 55 60 Met Phe Val
Tyr Ser Leu Glu Glu Arg Val Glu Asn Asn Ser Val Pro 65 70 75 80 Ser
Arg Phe Ser Pro Glu Cys Pro Asn Ser Ser His Leu Phe Leu His 85 90
95 Leu His Thr Leu Gln Pro Glu Asp Ser Ala Leu Tyr Leu Cys Ala Ser
100 105 110 Ser Gln Thr Thr Tyr Tyr Asn Glu Gln Phe Phe Gly Pro Gly
Thr Arg 115 120 125 Leu Thr Val Leu Glu Asp Leu Lys Asn Val Phe Pro
Pro Glu Val Ala 130 135 140 Val Phe Glu Pro Ser Glu Ala Glu Ile Ser
His Thr Gln Lys Ala Thr 145 150 155 160 Leu Val Cys Leu Ala Thr Gly
Phe Tyr Pro Asp His Val Glu Leu Ser 165 170 175 Trp Trp Val Asn Gly
Lys Glu Val His Ser Gly Val Ser Thr Asp Pro 180 185 190 Gln Pro Leu
Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu 195 200 205 Ser
Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn 210 215
220 His Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu
225 230 235 240 Trp Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val
Ser Ala Glu 245 250 255 Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser
Glu Ser Tyr Gln Gln 260 265 270 Gly Val Leu Ser Ala Thr Ile Leu Tyr
Glu Ile Leu Leu Gly Lys Ala 275 280 285 Thr Leu Tyr Ala Val Leu Val
Ser Ala Leu Val Leu Met Ala Met Val 290 295 300 Lys Arg Lys Asp Ser
Arg Gly 305 310 461827DNAHomo sapiens 46atgatatcct tgagagtttt
actggtgatc ctgtggcttc agttaagctg ggtttggagc 60caacggaagg aggtggagca
ggatcctgga cccttcaatg ttccagaggg agccactgtc 120gctttcaact
gtacttacag caacagtgct tctcagtctt tcttctggta cagacaggat
180tgcaggaaag aacctaagtt gctgatgtcc gtatactcca gtggtaatga
agatggaagg 240tttacagcac agctcaatag agccagccag tatatttccc
tgctcatcag agactccaag 300ctcagtgatt cagccaccta cctctgtgtg
gtgaaccgcg acaatgacat gcgctttgga 360gcagggacca gactgacagt
aaaaccaaat atccagaacc ctgaccctgc cgtgtaccag 420ctgagagact
ctaaatccag tgacaagtct gtctgcctat tcaccgatat tgattctcaa
480acaaatgtgt cacaaagtaa ggattctgat gtgtatatca cagacaaaac
tgtgctagac 540atgaggtcta tggacttcaa gagcaacagt gctgtggcct
ggagcaacaa atctgacttt 600gcatgtgcaa acgccttcaa caacagcatt
attccagaag acaccttctt ccccagccca 660gaaagttcct gtgatgtcaa
gctggtcgag aaaagctttg aaacagatac gaacctaaac 720tttcaaaacc
tgtcagtgat tgggttccga atcctcctcc tgaaagtggc cgggtttaat
780ctgctcatga cgctgcggct gtggtccagc cgggccaagc ggtccggatc
cggagccacc 840aacttcagcc tgctgaagca ggccggcgac gtggaggaga
accccggccc catggcctcc 900ctgctcttct tctgtggggc cttttatctc
ctgggaacag ggtccatgga tgctgatgtt 960acccagaccc caaggaatag
gatcacaaag acaggaaaga ggattatgct ggaatgttct 1020cagactaagg
gtcatgatag aatgtactgg tatcgacaag acccaggact gggcctacgg
1080ttgatctatt actcctttga tgtcaaagat ataaacaaag gagagatctc
tgatggatac 1140agtgtctctc gacaggcaca ggctaaattc tccctgtccc
tagagtctgc catccccaac 1200cagacagctc tttacttctg tgccaccagt
gagggagggc cgccctacga gcagtacttc 1260gggccgggca ccaggctcac
ggtcacagag gacctgaaaa acgtgttccc acccgaggtc 1320gctgtgtttg
agccatcaga agcagagatc tcccacaccc aaaaggccac actggtgtgc
1380ctggccacag gcttctaccc cgaccacgtg gagctgagct ggtgggtgaa
tgggaaggag 1440gtgcacagtg gggtcagcac agacccgcag cccctcaagg
agcagcccgc cctcaatgac 1500tccagatact gcctgagcag ccgcctgagg
gtctcggcca ccttctggca gaacccccgc 1560aaccacttcc gctgtcaagt
ccagttctac gggctctcgg
agaatgacga gtggacccag 1620gatagggcca aacctgtcac ccagatcgtc
agcgccgagg cctggggtag agcagactgt 1680ggcttcacct ccgagtctta
ccagcaaggg gtcctgtctg ccaccatcct ctatgagatc 1740ttgctaggga
aggccacctt gtatgccgtg ctggtcagtg ccctcgtgct gatggcyatg
1800gtcaagagaa aggattccag aggctag 1827471821DNAHomo sapiens
47atgacatcca ttcgagctgt atttatattc ctgtggctgc agctggactt ggtgaatgga
60gagaatgtgg agcagcatcc ttcaaccctg agtgtccagg agggagacag cgctgttatc
120aagtgtactt attcagacag tgcctcaaac tacttccctt ggtataagca
agaacttgga 180aaaagacctc agcttattat agacattcgt tcaaatgtgg
gcgaaaagaa agaccaacga 240attgctgtta cattgaacaa gacagccaaa
catttctccc tgcacatcac agagacccaa 300cctgaagact cggctgtcta
cttctgtgca gcaagtgggg ccaccgacaa gctcatcttt 360gggactggga
ccagattaca agtctttcca aatatccaga accctgaccc tgccgtgtac
420cagctgagag actctaaatc cagtgacaag tctgtctgcc tattcaccga
ttttgattct 480caaacaaatg tgtcacaaag taaggattct gatgtgtata
tcacagacaa aactgtgcta 540gacatgaggt ctatggactt caagagcaac
agtgctgtgg cctggagcaa caaatctgac 600tttgcatgtg caaacgcctt
caacaacagc attattccag aagacacctt cttccccagc 660ccagaaagtt
cctgtgatgt caagctggtc gagaaaagct ttgaaacaga tacgaaccta
720aactttcaaa acctgtcagt gattgggttc cgaatcctcc tcctgaaagt
ggccgggttt 780aatctgctca tgacgctgcg gctgtggtcc agccgggcca
agcggtccgg atccggagcc 840accaacttca gcctgctgaa gcaggccggc
gacgtggagg agaaccccgg ccccatgact 900atcaggctcc tctgctacat
gggcttttat tttctggggg caggcctcat ggaagctgac 960atctaccaga
ccccaagata ccttgttata gggacaggaa agaagatcac tctggaatgt
1020tctcaaacca tgggccatga caaaatgtac tggtatcaac aagatccagg
aatggaacta 1080cacctcatcc actattccta tggagttaat tccacagaga
agggagatct ttcctccgag 1140tcaacagtct ccagaataag gacggagcat
tttcccctga ccctggagtc tgccaggccc 1200tcacatacct ctcagtacct
ctgtgccagc agtggcgggc acgagcagta cttcgggccg 1260ggcaccaggc
tcacggtcac agaggacctg aaaaacgtgt tcccacccga ggtcgctgtg
1320tttgagccat cagaagcaga gatctcccac acccaaaagg ccacactggt
gtgcctggcc 1380acaggcttct tccctgacca cgtggagctg agctggtggg
tgaatgggaa ggaggtgcac 1440agtggggtca gcacggaccc gcagcccctc
aaggagcagc ccgccctcaa tgactccaga 1500tactgcctga gcagccgcct
gagggtctcg gccaccttct ggcagaaccc ccgcaaccac 1560ttccgctgtc
aagtccagtt ctacgggctc tcggagaatg acgagtggac ccaggatagg
1620gccaaacccg tcacccagat cgtcagcgcc gaggcctggg gtagagcaga
ctgtggcttt 1680acctcggtgt cctaccagca aggggtcctg tctgccacca
tcctctatga gatcctgcta 1740gggaaggcca ccctgtatgc tgtgctggtc
agcgcccttg tgttgatggc tatggtcaag 1800agaaaggatt ccagaggcta g
1821481830DNAHomo sapiens 48atgatgaaat ccttgagagt tttactggtg
atcctgtggc ttcagttaag ctgggtttgg 60agccaacaga aggaggtgga gcaggatcct
ggaccactca gtgttccaga gggagccatt 120gtttctctca actgcactta
cagcaacagt gcttttcaat acttcatgtg gtacagacag 180tattccagaa
aaggccctga gttgctgatg tacacatact ccagtggtaa caaagaagat
240ggaaggttta cagcacaggt cgataaatcc agcaagtata tctccttgtt
catcagagac 300tcacagccca gtgattcagc cacctacctc tgtgcaatgc
ggggagccca gaagctggta 360tttggccaag gaaccaggct gactatcaac
ccaaatatcc agaaccctga ccctgccgtg 420taccagctga gagactctaa
atccagtgac aagtctgtct gcctattcac cgattttgat 480tctcaaacaa
atgtgtcaca aagtaaggat tctgatgtgt atatcacaga caaaactgtg
540ctagacatga ggtctatgga cttcaagagc aacagtgctg tggcctggag
caacaaatct 600gactttgcat gtgcaaacgc cttcaacaac agcattattc
cagaagacac cttcttcccc 660agcccagaaa gttcctgtga tgtcaagctg
gtcgagaaaa gctttgaaac agatacgaac 720ctaaactttc aaaacctgtc
agtgattggg ttccgaatcc tcctcctgaa agtggccggg 780tttaatctgc
tcatgacgct gcggctgtgg tccagacggg ccaagcggtc cggatccgga
840gccaccaact tcagcctgct gaagcaggcc tgcgacgtgg aggagaaccc
cggccccatg 900ggtcctgggc ttctccactg gatggccctt tgtctccttg
gaacaggtca tggggatgcc 960atggtcatcc agaacccaag ataccaggtt
acccagtttg gaaagccagt gaccctgagt 1020tgttctcaga ctttgaacca
taacgtcatg tactggtacc agcagaagtc aagtcaggcc 1080ccaaagctgc
tgttccacta ctatgacaaa gattttaaca atgaagcaga cacccctgat
1140aacttccaat ccaggaggcc gaacacttct ttctgctttc ttgacatccg
ctcaccaggc 1200ctgggggacg cagccatgta cctgtgtgcc acctcctggg
accgagggta cgagcagtac 1260ttcgggccgg gcaccaggct cacggtcaca
gaggacctga aaaacgtgtt cccacccgag 1320gtcgctgtgt ttgagccatc
agaagcagag atctcccaca cccaaaaggc cacactggtg 1380tgcctggcca
caggcttcta ccccgaccac gtggagctga gctggtgggt gaatgggaag
1440gaggtgcaca gtggggtcag cacagacccg cagcccctca aggagcagcc
cgccctcaat 1500gactccagat actgcctgag cagccgcctg agggtctcgg
ccaccttctg gcagaacccc 1560cgcaaccact tccgctgtca agtccagttc
tacgggctct cggagaatga cgagtggacc 1620caggataggg ccaaacctgt
cacccagatc gtcagcgccg aggcctgggg tagagcagac 1680tgtggcttca
cctccgagtc ttaccagcaa ggggtcctgt ctgccaccat cctctatgag
1740atcttgctag ggaaggccac cttgtatgcc gtgctggtca gtgccctcgt
gctgatggct 1800atggtcaaga gaaaggattc cagaggctag 1830491848DNAHomo
sapiens 49atggcatgcc ctggcttcct gtgggcactt gtgatctcca cctgtcttga
atttagcatg 60gctcagacag tcactcagtc tcaaccagag atgtctgtgc aggaggcaga
gaccgtgacc 120ctgagctgca catatgacac cagtgagagt gattattatt
tattctggta caagcagcct 180cccagcaggc agatgattct cgttattcgc
caagaagctt ataagcaaca gaatgcaaca 240gagaatcgtt tctctgtgaa
cttccagaaa gcagccaaat ccttcagtct caagatctca 300gactcacagc
tgggggatgc cgcgatgtat ttctgtgctt ataggagcgc tcagggcgga
360tctgaaaagc tggtctttgg aaagggaacg aaactgacag taaacccata
tatccagaac 420cctgaccctg ccgtgtacca gctgagagac tctaaatcca
gtgacaagtc tgtctgccta 480ttcaccgatt ttgattctca aacaaatgtg
tcacaaagta aggattctga tgtgtatatc 540acagacaaaa ctgtgctaga
catgaggtct atggacttca agagcaacag tgctgtggcc 600tggagcaaca
aatctgactt tgcatgtgca aacgccttca acaacagcat tattccagaa
660gacaccttct tccccagccc agaaagttcc tgtgatgtca agctggtcga
gaaaagcttt 720gaaacagata cgaacctaaa ctttcaaaac ctgtcagtga
ttgggttccg aatcctcctc 780ctgaaagtgg ccgggtttaa tctgctcatg
acgctgcggc tgtggtccag ccgggccaag 840cggtccggat ccggagccac
caacttcagc ctgctgaagc aggccggcga cgtggaggag 900aaccccggcc
ccatgggctg caggctgctc tgctgtgcgg ttctctgtct cctgggagcg
960gtccccatgg aaacgggagt tacgcagaca ccaagacacc tggtcatggg
aatgacaaat 1020aagaagtctt tgaaatgtga acaacatctg ggtcataacg
ctatgtattg gtacaagcaa 1080agtgctaaga agccactgga gctcatgttt
gtctacagtc ttgaagaacg ggttgaaaac 1140aacagtgtgc caagtcgctt
ctcaccggaa tgccccaaca gctctcactt attccttcac 1200ctacacaccc
tgcagccaga agactcggcc ctgtatctct gcgccagcag ccaaactact
1260tactacaatg agcagttctt cgggccaggg acacggctca ccgtgctaga
ggacctgaaa 1320aacgtgttcc cacccgaggt cgctgtgttt gagccatcag
aagcagagat ctcccacacc 1380caaaaggcca cactggtgtg cctggccaca
ggcttctacc ccgaccacgt ggagctgagc 1440tggtgggtga atgggaagga
ggtgcacagt ggggtcagca cagacccgca gcccctcaag 1500gagcagcccg
ccctcaatga ctccagatac tgcctgagca gccgcctgag ggtctcggcc
1560accttctggc agaacccccg caaccacttc cgctgtcaag tccagttcta
cgggctctcg 1620gagaatgacg agtggaccca ggatagggcc aaacccgtca
cccagatcgt cagcgccgag 1680gcctggggta gagcagactg tggcttcacc
tccgagtctt accagcaagg ggtcctgtct 1740gccaccatcc tctatgagat
cttgctaggg aaggccacct tgtatgccgt gctggtcagt 1800gccctcgtgc
tgatggccat ggtcaagaga aaggattcca gaggctag 1848509PRTHomo sapiens
50Val Pro Ile Ser His Leu Tyr Ile Leu 1 5 519PRTHomo sapiens 51Asp
Pro Ile Gly His Leu Tyr Ile Phe 1 5 529PRTHomo sapiens 52Asp Pro
Ile Gly His Val Tyr Ile Phe 1 5 539PRTHomo sapiens 53Glu Asp Gly
Cys Pro Ala Ala Glu Lys 1 5 5427PRTArtificial SequenceSynthetic
54Arg Ala Lys Arg Ser Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys 1
5 10 15 Gln Ala Cys Asp Val Glu Glu Asn Pro Gly Pro 20 25
5520DNAArtificial SequenceSynthetic 55cactgttgct cttgaagtcc
205624DNAArtificial SequenceSynthetic 56caggcagtat ctggagtcat tgag
24574PRTArtificial SequenceSynthetic 57Ser Gly Ser Gly 1
5827DNAArtificial SequenceSynthetic 58tgcaaggcat ggaaaataca taactga
275925DNAArtificial SequenceSynthetic 59cacagatatc ctgtttggcc catat
256020DNAArtificial SequenceSynthetic 60tctctctgtt cctaaccttg
206120DNAArtificial SequenceSynthetic 61tccgtgagga ggcaaggttc
206219DNAArtificial SequenceSynthetic 62gagcctgcgc acccaccaa
196328DNAArtificial SequenceSynthetic 63agtgtgggca ggagctagtg
ctgctccg 28
* * * * *