U.S. patent application number 14/930597 was filed with the patent office on 2016-06-09 for methods and biomarkers for predicting efficacy and evaluation of an ox40 agonist treatment.
This patent application is currently assigned to GENENTECH, INC.. The applicant listed for this patent is GENENTECH, INC.. Invention is credited to Mahrukh HUSENI.
Application Number | 20160160290 14/930597 |
Document ID | / |
Family ID | 54479025 |
Filed Date | 2016-06-09 |
United States Patent
Application |
20160160290 |
Kind Code |
A1 |
HUSENI; Mahrukh |
June 9, 2016 |
METHODS AND BIOMARKERS FOR PREDICTING EFFICACY AND EVALUATION OF AN
OX40 AGONIST TREATMENT
Abstract
The present disclosure provides methods for predicting
responsiveness of a subject having cancer to an OX40 agonist
treatment by measuring the expression level of one or more
biomarkers. Also provided are methods for monitoring
pharmacodynamic activity of or responsiveness to an OX40 agonist
treatment by measuring the expression level of one or more
biomarkers. Further provided are methods related thereto for
treating or delaying progression of cancer in a subject by
administering an effective amount of an OX40 agonist to a subject.
Specific biomarkers for all such methods are described herein.
Inventors: |
HUSENI; Mahrukh; (Union
City, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GENENTECH, INC. |
South San Francisco |
CA |
US |
|
|
Assignee: |
GENENTECH, INC.
South San Francisco
CA
|
Family ID: |
54479025 |
Appl. No.: |
14/930597 |
Filed: |
November 2, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62074612 |
Nov 3, 2014 |
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Current U.S.
Class: |
424/133.1 ;
424/143.1; 435/29; 435/6.11; 435/6.12; 435/7.1; 506/9;
514/19.3 |
Current CPC
Class: |
A61P 43/00 20180101;
C07K 2317/24 20130101; G01N 2800/52 20130101; A61P 35/00 20180101;
C12Q 1/6886 20130101; G01N 33/57484 20130101; H04M 3/5238 20130101;
H04M 2203/402 20130101; C12Q 2600/158 20130101; C07K 16/2878
20130101; C12Q 2600/106 20130101; C07K 2317/75 20130101; H04M 3/51
20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07K 16/28 20060101 C07K016/28 |
Claims
1. A method for predicting responsiveness of a subject having
cancer to an OX40 agonist treatment, comprising: (a) measuring the
expression level of one or more marker genes in a sample comprising
leukocytes obtained from the subject, wherein said one or more
marker genes are selected from the group consisting of CD8a, CD8b,
H2-d, CTLA4, CD64, CXCL9, IFNg, IDO1, GZMA, GZMB, PRF1, PDCA1,
KLRK1, PTPRC, CXCL1, ITGAM, and IL7R; and (b) classifying the
subject as a responsive or non-responsive subject based on the
expression level of said one or more marker genes in the sample
obtained from the subject, as compared with a reference, wherein an
increased expression level of the one or more marker genes as
compared with the reference indicates the subject may be responsive
to an OX40 agonist treatment.
2. A method for treating or delaying progression of cancer in a
subject, comprising: (a) measuring the expression level of one or
more marker genes in a sample comprising leukocytes obtained from
the subject, wherein said one or more marker genes are selected
from the group consisting of CD8a, CD8b, H2-d, CTLA4, CD64, CXCL9,
IFNg, IDO1, GZMA, GZMB, PRF1, PDCA1, KLRK1, PTPRC, CXCL1, ITGAM,
and IL7R; and (b) if the expression level of said one or more
marker genes in the sample obtained from the subject is higher than
a reference, administering to the subject an effective amount of an
OX40 agonist.
3. A method for treating or delaying progression of cancer in a
subject, comprising administering to the subject an effective
amount of an OX40 agonist, wherein a sample comprising leukocytes
obtained from the subject has increased expression of one or more
marker genes are selected from the group consisting of CD8a, CD8b,
H2-d, CTLA4, CD64, CXCL9, IFNg, IDO1, GZMA, GZMB, PRF1, PDCA1,
KLRK1, PTPRC, CXCL1, ITGAM, and IL7R, as compared with a
reference.
4-6. (canceled)
7. A method for predicting responsiveness of a subject having
cancer to an OX40 agonist treatment, comprising: (a) measuring the
expression level of one or more marker genes in a sample comprising
leukocytes obtained from the subject, wherein said one or more
marker genes are selected from the group consisting of CSF2, CCL22,
EPCAM, GATA3, IL13, and VTCN1; and (b) classifying the subject as a
responsive or non-responsive subject based on the expression level
of said one or more marker genes in the sample obtained from the
subject, as compared with a reference, wherein a decreased
expression level of the one or more marker genes as compared with
the reference indicates the subject may be responsive to an OX40
agonist treatment.
8. A method for treating or delaying progression of cancer in a
subject, comprising (a) measuring the expression level of one or
more marker genes in a sample comprising leukocytes obtained from
the subject, wherein said one or more marker genes are selected
from the group consisting of CSF2, CCL22, EPCAM, GATA3, IL13, and
VTCN1; and (b) if the expression level of said one or more marker
genes in the sample obtained from the subject is lower than a
reference, administering to the subject an effective amount of an
OX40 agonist.
9. A method for treating or delaying progression of cancer in a
subject, comprising administering to the subject an effective
amount of an OX40 agonist, wherein a sample comprising leukocytes
obtained from the subject has decreased expression of one or more
marker genes are selected from the group consisting of CSF2, CCL22,
EPCAM, GATA3, IL13, and VTCN1, as compared with a reference.
10. A method for monitoring pharmacodynamic activity of an OX40
agonist treatment, comprising: (a) measuring the expression level
of one or more marker genes in a sample comprising leukocytes
obtained from the subject, wherein the subject has been treated
with an OX40 agonist, and wherein said one or more marker genes are
selected from the group consisting of ARG1, CCL2, CCL22, CCL5,
CCR5, CD226, CD27, CD274, CD28, CD3E, CD40, CD8A, CD8b, CXCL10,
CXCL9, EOMES, FasL, Fcgr1/CD64, FOXP3, GZMA, GZMB, HAVCR2, ICAM1,
IDO1, IFNg, IL10, IL12A (TDO2), IL13, IL2, IL7R, ITGAM, KLRK1,
LAG3, MAP4K1, MS4A1, PDCD1, PDCD1LG2, PRF1, PTPRC, TNF, TNFRSF14,
TNFRSF9, and TNFSF4; and (b) determining the treatment as
demonstrating pharmacodynamic activity based on the expression
level of said one or more marker genes in the sample obtained from
the subject, as compared with a reference, wherein an increased
expression level of the one or more marker genes as compared with
the reference indicates pharmacodynamic activity to the OX40
agonist treatment.
11. (canceled)
12. A method for monitoring responsiveness of a subject to an OX40
agonist treatment, comprising: (a) measuring the expression level
of one or more marker genes in a sample comprising leukocytes
obtained from the subject, wherein the subject has been treated
with an OX40 agonist, and wherein said one or more marker genes are
selected from the group consisting of BTLA, CD4, CD69, CD80, CD83,
CD86, CSF2, CTLA4, CXCR3, Fcgr2b/CD32, Fcgr3/CD16, H2-aa, H2-d,
H2-k, ICOS, IL10, PDCA1, and TNFRSF18; and (b) classifying the
subject as responsive or non-responsive to said treatment based on
the expression level of said one or more marker genes in the sample
obtained from the subject, as compared with a reference, wherein an
increased expression level of the one or more marker genes as
compared with the reference indicates a responsive subject.
13. (canceled)
14. The method of claim 12, wherein responsiveness comprises immune
activation and/or treatment efficacy.
15. The method of claim 2, wherein the leukocytes are in a tumor
sample obtained from the subject.
16. The method of claim 2, wherein the leukocytes are in a
peripheral blood sample obtained from the subject.
17. The method of claim 2, wherein the expression level of said one
or more marker genes is normalized to the expression level of a
reference gene in the sample.
18. (canceled)
19. The method of claim 2, wherein the expression level of said one
or more marker genes is mRNA expression level.
20. (canceled)
21. The method of claim 2, wherein the expression level of said one
or more marker genes is protein expression level.
22. (canceled)
23. The method of claim 2, wherein the cancer is selected from the
group consisting of colorectal cancer, non-small cell lung cancer,
renal cell carcinoma, bladder cancer, ovarian cancer, glioblastoma,
neuroblastoma, melanoma, triple-negative breast carcinoma, gastric
cancer, and hepatocellular carcinoma.
24. (canceled)
25. The method of claim 2, wherein the OX40 agonist is an
antibody.
26-30. (canceled)
31. The method of claim 25, wherein the antibody comprises (a)
HVR-H1 comprising the amino acid sequence of SEQ ID NO:2, 8 or 9;
(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3, 10,
11, 12, 13, or 14; (c) HVR-H3 comprising the amino acid sequence of
SEQ ID NO:4, 15 or 19; (d) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6; and (f) HVR-L3 comprising an amino acid
sequence selected from SEQ ID NO:7, 22, 23, 24, 25, 26, 27 or
28.
32-34. (canceled)
35. The method of claim 25, wherein the antibody is MEDI6469 or
MEDI0562.
36. The method of claim 2, wherein the OX40 agonist comprises one
or more extracellular domains of OX40L.
37. The method of claim 2, wherein the OX40 agonist is MEDI6383.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority benefit of U.S.
Provisional Application Ser. No. 62/074,612, filed on Nov. 3, 2014,
which is incorporated herein by reference in its entirety.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002] The content of the following submission on ASCII text file
is incorporated herein by reference in its entirety: a computer
readable form (CRF) of the Sequence Listing (file name:
146392028900SEQLIST.TXT, date recorded: Nov. 2, 2015, size: 185
KB).
FIELD
[0003] The present disclosure relates to methods of predicting or
monitoring responsiveness to treatment with an OX40 agonist, as
well as methods of treating cancer related thereto.
BACKGROUND
[0004] OX40 (also known as CD34, TNFRSF4 and ACT35) is a member of
the tumor necrosis factor receptor superfamily. OX40 is not
constitutively expressed on naive T cells, but is induced after
engagement of the T cell receptor (TCR). The ligand for OX40,
OX40L, is predominantly expressed on antigen presenting cells. OX40
is highly expressed by activated CD4+ T cells, activated CD8+ T
cells, memory T cells, and regulatory T cells. OX40 signaling can
provide costimulatory signals to CD4 and CD8 T cells, leading to
enhanced cell proliferation, survival, effector function and
migration. OX40 signaling also enhances memory T cell development
and function.
[0005] Regulatory T cells (Treg) cells are highly enriched in
tumors and tumor draining lymph nodes derived from multiple cancer
indications, including melanoma, NSCLC, renal, ovarian, colon,
pancreatic, hepatocellular, and breast cancer. In a subset of these
indications, increased intratumoral T reg cell densities are
associated with poor patient prognosis, suggesting that these cells
play an important role in suppressing antitumor immunity. OX40
positive tumor infiltrating lymphocytes have been described.
[0006] It is clear that there continues to be a need for
diagnostic, prognostic, and predictive methods to identify patients
that are more likely to benefit from anti-tumor treatments that
modulate OX40 activity. The invention described herein meets these
needs and provides other benefits.
[0007] All references cited herein, including patent applications,
patent publications, and UniProtKB/Swiss-Prot Accession numbers are
herein incorporated by reference in their entirety, as if each
individual reference were specifically and individually indicated
to be incorporated by reference.
BRIEF SUMMARY
[0008] The present disclosure describes methods and biomarkers for
predicting efficacy and evaluation of an OX40 agonist treatment,
including methods for predicting responsiveness, monitoring
pharmacodynamic activity or responsiveness, and methods of treating
or delaying progression of cancer.
[0009] In certain aspects, the present disclosure provides a method
for predicting responsiveness of a subject having cancer to an OX40
agonist treatment, comprising: (a) measuring the expression level
of one or more marker genes in a sample comprising leukocytes
obtained from the subject, wherein said one or more marker genes
are selected from the group consisting of CD8a, CD8b, H2-d, CTLA4,
CD64, CXCL9, IFNg, IDO1, GZMA, GZMB, PRF1, PDCA1, KLRK1, PTPRC,
CXCL1, ITGAM, and IL7R; and (b) classifying the subject as a
responsive or non-responsive subject based on the expression level
of said one or more marker genes in the sample obtained from the
subject, as compared with a reference, wherein an increased
expression level of the one or more marker genes as compared with
the reference indicates the subject may be responsive to an OX40
agonist treatment.
[0010] In certain aspects, the present disclosure provides a method
for treating or delaying progression of cancer in a subject,
comprising: (a) measuring the expression level of one or more
marker genes in a sample comprising leukocytes obtained from the
subject, wherein said one or more marker genes are selected from
the group consisting of CD8a, CD8b, H2-d, CTLA4, CD64, CXCL9, IFNg,
IDO1, GZMA, GZMB, PRF1, PDCA1, KLRK1, PTPRC, CXCL1, ITGAM, and
IL7R; and (b) if the expression level of said one or more marker
genes in the sample obtained from the subject is higher than a
reference, administering to the subject an effective amount of an
OX40 agonist.
[0011] In certain aspects, the present disclosure provides a method
for treating or delaying progression of cancer in a subject,
comprising administering to the subject an effective amount of an
OX40 agonist, wherein a sample comprising leukocytes obtained from
the subject has increased expression of one or more marker genes
are selected from the group consisting of CD8a, CD8b, H2-d, CTLA4,
CD64, CXCL9, IFNg, IDO1, GZMA, GZMB, PRF1, PDCA1, KLRK1, PTPRC,
CXCL1, ITGAM, and IL7R, as compared with a reference.
[0012] In some embodiments, said one or more marker genes are
selected from the group consisting of CD8a, CD8b, IFNg, GZMA, GZMB,
PRF1, and PDCA1. In some embodiments, said one or more marker genes
are selected from the group consisting of H2-d, CTLA4, CXCL9,
PTPRC, IL7R, KLRK1, and CXCL1. In some embodiments, said one or
more marker genes are selected from the group consisting of CD64,
IDO1, and ITGAM.
[0013] In certain aspects, the present disclosure provides a method
for predicting responsiveness of a subject having cancer to an OX40
agonist treatment, comprising: (a) measuring the expression level
of one or more marker genes in a sample comprising leukocytes
obtained from the subject, wherein said one or more marker genes
are selected from the group consisting of CSF2, CCL22, EPCAM,
GATA3, IL13, and VTCN1; and (b) classifying the subject as a
responsive or non-responsive subject based on the expression level
of said one or more marker genes in the sample obtained from the
subject, as compared with a reference, wherein a decreased
expression level of the one or more marker genes as compared with
the reference indicates the subject may be responsive to an OX40
agonist treatment.
[0014] In certain aspects, the present disclosure provides a method
for treating or delaying progression of cancer in a subject,
comprising: (a) measuring the expression level of one or more
marker genes in a sample comprising leukocytes obtained from the
subject, wherein said one or more marker genes are selected from
the group consisting of CSF2, CCL22, EPCAM, GATA3, IL13, and VTCN1;
and (b) if the expression level of said one or more marker genes in
the sample obtained from the subject is lower than a reference,
administering to the subject an effective amount of an OX40
agonist.
[0015] In certain aspects, the present disclosure provides a method
for treating or delaying progression of cancer in a subject,
comprising administering to the subject an effective amount of an
OX40 agonist, wherein a sample comprising leukocytes obtained from
the subject has decreased expression of one or more marker genes
are selected from the group consisting of CSF2, CCL22, EPCAM,
GATA3, IL13, and VTCN1, as compared with a reference.
[0016] In certain aspects, the present disclosure provides a method
for monitoring pharmacodynamic activity of an OX40 agonist
treatment, comprising: (a) measuring the expression level of one or
more marker genes in a sample comprising leukocytes obtained from
the subject, wherein the subject has been treated with an OX40
agonist, and wherein said one or more marker genes are selected
from the group consisting of ARG1, CCL2, CCL22, CCL5, CCR5, CD226,
CD27, CD274, CD28, CD3E, CD40, CD8A, CD8b, CXCL10, CXCL9, EOMES,
FasL, Fcgr1/CD64, FOXP3, GZMA, GZMB, HAVCR2, ICAM1, IDO1, IFNg,
IL10, IL12A (TDO2), IL13, IL2, IL7R, ITGAM, KLRK1, LAG3, MAP4K1,
MS4A1, PDCD1, PDCD1LG2, PRF1, PTPRC, TNF, TNFRSF14, TNFRSF9, and
TNFSF4; and (b) determining the treatment as demonstrating
pharmacodynamic activity based on the expression level of said one
or more marker genes in the sample obtained from the subject, as
compared with a reference, wherein an increased expression level of
the one or more marker genes as compared with the reference
indicates pharmacodynamic activity to the OX40 agonist treatment.
In some embodiments, said one or more marker genes are selected
from the group consisting of CD3, CD8, IFNg, GZMA, GZMB, PRF1,
TNFa, PDCD1, and CD274.
[0017] In certain aspects, the present disclosure provides a method
for monitoring responsiveness of a subject to an OX40 agonist
treatment, comprising: (a) measuring the expression level of one or
more marker genes in a sample comprising leukocytes obtained from
the subject, wherein the subject has been treated with an OX40
agonist, and wherein said one or more marker genes are selected
from the group consisting of BTLA, CD4, CD69, CD80, CD83, CD86,
CSF2, CTLA4, CXCR3, Fcgr2b/CD32, Fcgr3/CD16, H2-aa, H2-d, H2-k,
ICOS, IL10, PDCA1, and TNFRSF18; and (b) classifying the subject as
responsive or non-responsive to said treatment based on the
expression level of said one or more marker genes in the sample
obtained from the subject, as compared with a reference, wherein an
increased expression level of the one or more marker genes as
compared with the reference indicates a responsive subject. In some
embodiments, said one or more marker genes are selected from the
group consisting of CD80, CD86, ICOS, H2-aa, and CXCR3. In some
embodiments, responsiveness comprises immune activation and/or
treatment efficacy.
[0018] In some embodiments, the sample comprising leukocytes is
from a tumor sample obtained from the subject. In some embodiments,
the sample comprising leukocytes is from a peripheral blood sample
obtained from the subject. In some embodiments, the expression
level of said one or more marker genes is normalized to the
expression level of a reference gene in the sample. In some
embodiments, the reference gene is a housekeeping gene. In some
embodiments, the expression level of said one or more marker genes
is mRNA expression level. In some embodiments, the mRNA expression
level is measured by an assay selected from the group consisting of
quantitative PCR, semi-quantitative PCR, nucleotide microarray,
RNA-seq, in situ hybridization, and Northern blotting. In some
embodiments, the expression level of said one or more marker genes
is protein expression level. In some embodiments, the protein
expression level is measured by Western blotting, peptide
microarray, immunohistochemistry, flow cytometry, or mass
spectrometry. In some embodiments, the cancer is selected from the
group consisting of colorectal cancer, non-small cell lung cancer,
renal cell carcinoma, bladder cancer, ovarian cancer, glioblastoma,
neuroblastoma, melanoma, breast carcinoma, gastric cancer, and
hepatocellular carcinoma. In some embodiments, the breast carcinoma
is triple-negative breast carcinoma. In some embodiments, the OX40
agonist is an agonist anti-human OX40 antibody. In some
embodiments, the antibody is a monoclonal antibody. In some
embodiments, the antibody is a humanized or human antibody. In some
embodiments, the antibody comprises an IgG1 Fc region. In some
embodiments, the antibody comprises an IgG4 Fc region. In some
embodiments, the antibody comprises an Fc region comprising a
mutation that decreases binding to an Fc receptor. In some
embodiments, the antibody comprises (a) HVR-H1 comprising the amino
acid sequence of SEQ ID NO:2, 8 or 9; (b) HVR-H2 comprising the
amino acid sequence of SEQ ID NO:3, 10, 11, 12, 13, or 14; (c)
HVR-H3 comprising the amino acid sequence of SEQ ID NO:4, 15 or 19;
(d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f)
HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:7,
22, 23, 24, 25, 26, 27 or 28. In some embodiments, the antibody
comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3;
(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; (d)
HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f)
HVR-L3 comprising an amino acid sequence selected from SEQ ID NO:7.
In some embodiments, the antibody comprises (a) HVR-H1 comprising
the amino acid sequence of SEQ ID NO:2; (b) HVR-H2 comprising the
amino acid sequence of SEQ ID NO:3; (c) HVR-H3 comprising the amino
acid sequence of SEQ ID NO:4; (d) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6; and (f) HVR-L3 comprising an amino acid
sequence selected from SEQ ID NO:26. In some embodiments, the
antibody comprises (a) HVR-H1 comprising the amino acid sequence of
SEQ ID NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ
ID NO:3; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:4; (d) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5;
(e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and
(f) HVR-L3 comprising an amino acid sequence selected from SEQ ID
NO:27. In some embodiments, the antibody is MEDI6469 or MEDI0562.
In some embodiments, the OX40 agonist comprises one or more
extracellular domains of OX40L. In some embodiments, the OX40
agonist is MEDI6383.
[0019] It is to be understood that one, some, or all of the
properties of the various embodiments described above and herein
may be combined to form other embodiments of the present invention.
These and other aspects of the invention will become apparent to
one of skill in the art. These and other embodiments of the
invention are further described by the detailed description that
follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIGS. 1A & 1B show the effects of anti-OX40 treatment on
T cell function. The graphs depict T cell proliferation (FIG. 1A)
and IFN.gamma. production (FIG. 1B) in response to activation with
anti-CD3 and anti-OX40 treatment.
[0021] FIG. 2 shows the effect of anti-OX40 treatment on Treg
activity in an in vitro Treg suppression assay, as compared to
control treatment.
[0022] FIGS. 3A-3C show the effect of anti-OX40 treatment on tumor
volume in syngeneic mouse tumor models. Results from the EMT6
breast (FIG. 3A), Cloudman melanoma (FIG. 3B), and CT26 CRC (FIG.
3C) tumor models are shown.
[0023] FIGS. 4A-4C show the effect of anti-OX40 treatment on tumor
volume in syngeneic mouse tumor models. Results from the MC38 CRC
(FIG. 4A), 51Blim10 CRC (FIG. 4B), and JC breast (FIG. 4C) tumor
models are shown.
[0024] FIG. 5 provides a heat-map of gene expression in mouse tumor
models (as labeled) prior to anti-OX40 treatment.
[0025] FIGS. 6A & 6B demonstrate a dose-dependent effect of
anti-OX40 treatment on peripheral blood cells in EMT6 tumor-bearing
mice. Shown are the percentages of Tregs (FIG. 6A) and CD8 T cells
(FIG. 6B) in peripheral blood following treatment with the labeled
concentration of anti-OX40 antibody.
[0026] FIGS. 7A & 7B show the effect of anti-OX40 treatment on
numbers of Treg (FIG. 7A) and CD8 (FIG. 7B) cell infiltrates in
EMT6 tumors, compared to control treatment.
[0027] FIGS. 8A & 8B show the effect of anti-OX40 treatment on
numbers of Treg (FIG. 8A) and CD8 (FIG. 8B) cell infiltrates in JC
tumors, compared to control treatment.
[0028] FIGS. 9A-9D show genes that are upregulated following
anti-OX40 treatment, compared to control treatment, in both EMT6
and JC tumor models. Shown are relative expression levels of IFNg
(FIG. 9A), granzyme A (FIG. 9B), perforin (FIG. 9C), and TNFa (FIG.
9D).
[0029] FIGS. 10A-10D show genes that are upregulated following
anti-OX40 treatment, compared to control treatment, in an EMT6
tumor model. Shown are relative expression levels of H2-aa (FIG.
10A), CD86 (FIG. 10B), ICOS (FIG. 10C), and CXCR3 (FIG. 10D).
DETAILED DESCRIPTION
I. General Techniques
[0030] The techniques and procedures described or referenced herein
are generally well understood and commonly employed using
conventional methodology by those skilled in the art, such as, for
example, the widely utilized methodologies described in Sambrook et
al., Molecular Cloning: A Laboratory Manual 3d edition (2001) Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.; Current
Protocols in Molecular Biology (F. M. Ausubel, et al. eds.,
(2003)); the series Methods in Enzymology (Academic Press, Inc.):
PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G.
R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A
Laboratory Manual, and Animal Cell Culture (R. I. Freshney, ed.
(1987)); Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods
in Molecular Biology, Humana Press; Cell Biology: A Laboratory
Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell
Culture (R. I. Freshney), ed., 1987); Introduction to Cell and
Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press;
Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B.
Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons;
Handbook of Experimental Immunology (D. M. Weir and C. C.
Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.
Miller and M. P. Calos, eds., 1987); PCR: The Polymerase Chain
Reaction, (Mullis et al., eds., 1994); Current Protocols in
Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in
Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A.
Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997);
Antibodies: A Practical Approach (D. Catty., ed., IRL Press,
1988-1989); Monoclonal Antibodies: A Practical Approach (P.
Shepherd and C. Dean, eds., Oxford University Press, 2000); Using
Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring
Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.
D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer:
Principles and Practice of Oncology (V. T. DeVita et al., eds.,
J.B. Lippincott Company, 1993).
II. Definitions
[0031] Before describing the invention in detail, it is to be
understood that this invention is not limited to particular
compositions or biological systems, which can, of course, vary. It
is also to be understood that the terminology used herein is for
the purpose of describing particular embodiments only, and is not
intended to be limiting.
[0032] As used herein and in the appended claims, the singular
forms "a," "or," and "the" include plural referents unless the
context clearly dictates otherwise.
[0033] Reference to "about" a value or parameter herein includes
(and describes) variations that are directed to that value or
parameter per se. For example, description referring to "about X"
includes description of "X".
[0034] It is understood that aspects and variations of the
invention described herein include "consisting" and/or "consisting
essentially of" aspects and variations.
[0035] The term "dysfunction" in the context of immune dysfunction,
refers to a state of reduced immune responsiveness to antigenic
stimulation.
[0036] The term "dysfunctional", as used herein, also includes
refractory or unresponsive to antigen recognition, specifically,
impaired capacity to translate antigen recognition into downstream
T-cell effector functions, such as proliferation, cytokine
production (e.g., gamma interferon) and/or target cell killing.
[0037] "Enhancing T cell function" means to induce, cause or
stimulate an effector or memory T cell to have a renewed, sustained
or amplified biological function. Examples of enhancing T-cell
function include: increased secretion of .gamma.-interferon from
CD8.sup.+ effector T cells, increased secretion of
.gamma.-interferon from CD4+ memory and/or effector T-cells,
increased proliferation of CD4+ effector and/or memory T cells,
increased proliferation of CD8+ effector T-cells, increased antigen
responsiveness (e.g., clearance), relative to such levels before
the intervention. In one embodiment, the level of enhancement is at
least 50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%,
200%. The manner of measuring this enhancement is known to one of
ordinary skill in the art.
[0038] "Tumor immunity" refers to the process in which tumors evade
immune recognition and clearance. Thus, as a therapeutic concept,
tumor immunity is "treated" when such evasion is attenuated, and
the tumors are recognized and attacked by the immune system.
Examples of tumor recognition include tumor binding, tumor
shrinkage and tumor clearance.
[0039] "Immunogenicity" refers to the ability of a particular
substance to provoke an immune response. Tumors are immunogenic and
enhancing tumor immunogenicity aids in the clearance of the tumor
cells by the immune response.
[0040] An "acceptor human framework" for the purposes herein is a
framework comprising the amino acid sequence of a light chain
variable domain (VL) framework or a heavy chain variable domain
(VH) framework derived from a human immunoglobulin framework or a
human consensus framework, as defined below. An acceptor human
framework "derived from" a human immunoglobulin framework or a
human consensus framework may comprise the same amino acid sequence
thereof, or it may contain amino acid sequence changes. In some
embodiments, the number of amino acid changes are 10 or less, 9 or
less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or
less, or 2 or less. In some embodiments, the VL acceptor human
framework is identical in sequence to the VL human immunoglobulin
framework sequence or human consensus framework sequence.
[0041] "Affinity" refers to the strength of the sum total of
noncovalent interactions between a single binding site of a
molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (Kd).
Affinity can be measured by common methods known in the art,
including those described herein. Specific illustrative and
exemplary embodiments for measuring binding affinity are described
in the following.
[0042] An "agonist antibody," as used herein, is an antibody which
activates a biological activity of the antigen it binds.
[0043] An "anti-angiogenic agent" refers to a compound which
blocks, or interferes with to some degree, the development of blood
vessels. An anti-angiogenic agent may, for instance, be a small
molecule or antibody that binds to a growth factor or growth factor
receptor involved in promoting angiogenesis. In one embodiment, an
anti-angiogenic agent is an antibody that binds to vascular
endothelial growth factor (VEGF), such as bevacizumab
(AVASTIN).
[0044] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC"
refers to a form of cytotoxicity in which secreted immunoglobulin
bound onto Fc receptors (FcRs) present on certain cytotoxic cells
(e.g. NK cells, neutrophils, and macrophages) enable these
cytotoxic effector cells to bind specifically to an antigen-bearing
target cell and subsequently kill the target cell with cytotoxins.
The primary cells for mediating ADCC, NK cells, express
Fc.gamma.RIII only, whereas monocytes express Fc.gamma.RI,
Fc.gamma.RII, and Fc.gamma.RIII. FcR expression on hematopoietic
cells is summarized in Table 3 on page 464 of Ravetch and Kinet,
Annu. Rev. Immunol 9:457-92 (1991). To assess ADCC activity of a
molecule of interest, an in vitro ADCC assay, such as that
described in U.S. Pat. No. 5,500,362 or 5,821,337 or U.S. Pat. No.
6,737,056 (Presta), may be performed. Useful effector cells for
such assays include PBMC and NK cells. Alternatively, or
additionally, ADCC activity of the molecule of interest may be
assessed in vivo, e.g., in an animal model such as that disclosed
in Clynes et al. PNAS (USA) 95:652-656 (1998). An exemplary assay
for assessing ADCC activity is provided in the examples herein.
[0045] The terms "anti-OX40 antibody" and "an antibody that binds
to OX40" refer to an antibody that is capable of binding OX40 with
sufficient affinity such that the antibody is useful as a
diagnostic and/or therapeutic agent in targeting OX40. In one
embodiment, the extent of binding of an anti-OX40 antibody to an
unrelated, non-OX40 protein is less than about 10% of the binding
of the antibody to OX40 as measured, e.g., by a radioimmunoassay
(RIA). In certain embodiments, an antibody that binds to OX40 has a
dissociation constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM,
.ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM, or
.ltoreq.0.001 nM (e.g. 10.sup.-8M or less, e.g. from 10.sup.-8M to
10.sup.-13M, e.g., from 10.sup.-9M to 10.sup.-13 M). In certain
embodiments, an anti-OX40 antibody binds to an epitope of OX40 that
is conserved among OX40 from different species.
[0046] As use herein, the term "binds", "specifically binds to" or
is "specific for" refers to measurable and reproducible
interactions such as binding between a target and an antibody,
which is determinative of the presence of the target in the
presence of a heterogeneous population of molecules including
biological molecules. For example, an antibody that binds to or
specifically binds to a target (which can be an epitope) is an
antibody that binds this target with greater affinity, avidity,
more readily, and/or with greater duration than it binds to other
targets. In one embodiment, the extent of binding of an antibody to
an unrelated target is less than about 10% of the binding of the
antibody to the target as measured, e.g., by a radioimmunoassay
(RIA). In certain embodiments, an antibody that specifically binds
to a target has a dissociation constant (Kd) of .ltoreq.1 .mu.M,
.ltoreq.100 nM, .ltoreq.10 nM, .ltoreq.1 nM, or .ltoreq.0.1 nM. In
certain embodiments, an antibody specifically binds to an epitope
on a protein that is conserved among the protein from different
species. In another embodiment, specific binding can include, but
does not require exclusive binding.
[0047] The term "antibody" herein is used in the broadest sense and
encompasses various antibody structures, including but not limited
to monoclonal antibodies, polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments so
long as they exhibit the desired antigen-binding activity.
[0048] An "antibody fragment" refers to a molecule other than an
intact antibody that comprises a portion of an intact antibody that
binds the antigen to which the intact antibody binds. Examples of
antibody fragments include but are not limited to Fv, Fab, Fab',
Fab'-SH, F(ab').sub.2; diabodies; linear antibodies; single-chain
antibody molecules (e.g. scFv); and multispecific antibodies formed
from antibody fragments.
[0049] An "antibody that binds to the same epitope" as a reference
antibody refers to an antibody that blocks binding of the reference
antibody to its antigen in a competition assay by 50% or more, and
conversely, the reference antibody blocks binding of the antibody
to its antigen in a competition assay by 50% or more. An exemplary
competition assay is provided herein.
[0050] The term "binding domain" refers to the region of a
polypeptide that binds to another molecule. In the case of an FcR,
the binding domain can comprise a portion of a polypeptide chain
thereof (e.g. the alpha chain thereof) which is responsible for
binding an Fc region. One useful binding domain is the
extracellular domain of an FcR alpha chain.
[0051] A polypeptide with a variant IgG Fc with "altered" FcR, ADCC
or phagocytosis activity is one which has either enhanced or
diminished FcR binding activity (e.g, Fc.gamma.R) and/or ADCC
activity and/or phagocytosis activity compared to a parent
polypeptide or to a polypeptide comprising a native sequence Fc
region.
[0052] The term "OX40," as used herein, refers to any native OX40
from any vertebrate source, including mammals such as primates
(e.g. humans) and rodents (e.g., mice and rats), unless otherwise
indicated. The term encompasses "full-length," unprocessed OX40 as
well as any form of OX40 that results from processing in the cell.
The term also encompasses naturally occurring variants of OX40,
e.g., splice variants or allelic variants. The amino acid sequence
of an exemplary human OX40 is shown in SEQ ID NO:1.
[0053] "OX40 activation" refers to activation, of the OX40
receptor. Generally, OX40 activation results in signal
transduction.
[0054] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. Examples of cancer include but are not
limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or
lymphoid malignancies. More particular examples of such cancers
include, but not limited to, squamous cell cancer (e.g., epithelial
squamous cell cancer), lung cancer including small-cell lung
cancer, non-small cell lung cancer, adenocarcinoma of the lung and
squamous carcinoma of the lung, cancer of the peritoneum,
hepatocellular cancer, gastric or stomach cancer including
gastrointestinal cancer and gastrointestinal stromal cancer,
pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer,
liver cancer, bladder cancer, cancer of the urinary tract,
hepatoma, breast cancer, colon cancer, rectal cancer, colorectal
cancer, endometrial or uterine carcinoma, salivary gland carcinoma,
kidney or renal cancer, prostate cancer, vulval cancer, thyroid
cancer, hepatic carcinoma, anal carcinoma, penile carcinoma,
melanoma, superficial spreading melanoma, lentigo maligna melanoma,
acral lentiginous melanomas, nodular melanomas, multiple myeloma
and B-cell lymphoma; chronic lymphocytic leukemia (CLL); acute
lymphoblastic leukemia (ALL); hairy cell leukemia; chronic
myeloblastic leukemia; and post-transplant lymphoproliferative
disorder (PTLD), as well as abnormal vascular proliferation
associated with phakomatoses, edema (such as that associated with
brain tumors), Meigs' syndrome, brain, as well as head and neck
cancer, and associated metastases. In certain embodiments, cancers
that are amenable to treatment by the antibodies of the invention
include breast cancer, colorectal cancer, rectal cancer, non-small
cell lung cancer, glioblastoma, non-Hodgkins lymphoma (NHL), renal
cell cancer, prostate cancer, liver cancer, pancreatic cancer,
soft-tissue sarcoma, kaposi's sarcoma, carcinoid carcinoma, head
and neck cancer, ovarian cancer, mesothelioma, and multiple
myeloma. In some embodiments, the cancer is selected from:
non-small cell lung cancer, glioblastoma, neuroblastoma, melanoma,
breast carcinoma (e.g. triple-negative breast cancer), gastric
cancer, colorectal cancer (CRC), and hepatocellular carcinoma. Yet,
in some embodiments, the cancer is selected from: non-small cell
lung cancer, colorectal cancer, glioblastoma and breast carcinoma
(e.g. triple-negative breast cancer), including metastatic forms of
those cancers.
[0055] The term "tumor" refers to all neoplastic cell growth and
proliferation, whether malignant or benign, and all pre-cancerous
and cancerous cells and tissues. The terms "cancer," "cancerous,"
"cell proliferative disorder," "proliferative disorder" and "tumor"
are not mutually exclusive as referred to herein.
[0056] The terms "cell proliferative disorder" and "proliferative
disorder" refer to disorders that are associated with some degree
of abnormal cell proliferation. In one embodiment, the cell
proliferative disorder is cancer.
[0057] The term "chimeric" antibody refers to an antibody in which
a portion of the heavy and/or light chain is derived from a
particular source or species, while the remainder of the heavy
and/or light chain is derived from a different source or
species.
[0058] The "class" of an antibody refers to the type of constant
domain or constant region possessed by its heavy chain. There are
five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and
several of these may be further divided into subclasses (isotypes),
e.g., IgG.sub.1, IgG.sub.2, IgG.sub.3, IgG.sub.4, IgA.sub.1, and
IgA.sub.2. The heavy chain constant domains that correspond to the
different classes of immunoglobulins are called .alpha., .delta.,
.epsilon., .gamma., and .mu., respectively.
[0059] "Complement dependent cytotoxicity" or "CDC" refers to the
lysis of a target cell in the presence of complement. Activation of
the classical complement pathway is initiated by the binding of the
first component of the complement system (C1q) to antibodies (of
the appropriate subclass), which are bound to their cognate
antigen. To assess complement activation, a CDC assay, e.g., as
described in Gazzano-Santoro et al., J. Immunol. Methods 202:163
(1996), may be performed. Polypeptide variants with altered Fc
region amino acid sequences (polypeptides with a variant Fc region)
and increased or decreased C1q binding capability are described,
e.g., in U.S. Pat. No. 6,194,551 B1 and WO 1999/51642. See also,
e.g., Idusogie et al. J. Immunol. 164: 4178-4184 (2000).
[0060] The term "cytostatic agent" refers to a compound or
composition which arrests growth of a cell either in vitro or in
vivo. Thus, a cytostatic agent may be one which significantly
reduces the percentage of cells in S phase. Further examples of
cytostatic agents include agents that block cell cycle progression
by inducing G0/G1 arrest or M-phase arrest. The humanized anti-Her2
antibody trastuzumab (HERCEPTIN.RTM.) is an example of a cytostatic
agent that induces G0/G1 arrest. Classical M-phase blockers include
the vincas (vincristine and vinblastine), taxanes, and
topoisomerase II inhibitors such as doxorubicin, epirubicin,
daunorubicin, etoposide, and bleomycin. Certain agents that arrest
G1 also spill over into S-phase arrest, for example, DNA alkylating
agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine,
cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further
information can be found in Mendelsohn and Israel, eds., The
Molecular Basis of Cancer, Chapter 1, entitled "Cell cycle
regulation, oncogenes, and antineoplastic drugs" by Murakami et al.
(W.B. Saunders, Philadelphia, 1995), e.g., p. 13. The taxanes
(paclitaxel and docetaxel) are anticancer drugs both derived from
the yew tree. Docetaxel (TAXOTERE.RTM., Rhone-Poulenc Rorer),
derived from the European yew, is a semisynthetic analogue of
paclitaxel (TAXOL.RTM., Bristol-Myers Squibb). Paclitaxel and
docetaxel promote the assembly of microtubules from tubulin dimers
and stabilize microtubules by preventing depolymerization, which
results in the inhibition of mitosis in cells.
[0061] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents a cellular function and/or
causes cell death or destruction. Cytotoxic agents include, but are
not limited to, radioactive isotopes (e.g., At.sup.211, I.sup.131,
I.sup.125, Y.sup.90, Re.sup.186, Re.sup.188, Sm.sup.153,
Bi.sup.212, P.sup.32, Pb.sup.212 and radioactive isotopes of Lu);
chemotherapeutic agents or drugs (e.g., methotrexate, adriamicin,
vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,
melphalan, mitomycin C, chlorambucil, daunorubicin or other
intercalating agents); growth inhibitory agents; enzymes and
fragments thereof such as nucleolytic enzymes; antibiotics; toxins
such as small molecule toxins or enzymatically active toxins of
bacterial, fungal, plant or animal origin, including fragments
and/or variants thereof; and the various antitumor or anticancer
agents disclosed below.
[0062] A "depleting anti-OX40 antibody," is an anti-OX40 antibody
that kills or depletes OX40-expressing cells. Depletion of OX40
expressing cells can be achieved by various mechanisms, such as
antibody-dependent cell-mediated cytotoxicity and/or phagocytosis.
Depletion of OX40-expressing cells may be assayed in vitro, and
exemplary methods for in vitro ADCC and phagocytosis assays are
provided herein. In some embodiments, the OX40-expressing cell is a
human CD4+ effector T cell. In some embodiments, the
OX40-expressing cell is a transgenic BT474 cell that expresses
human OX40.
[0063] "Effector functions" refer to those biological activities
attributable to the Fc region of an antibody, which vary with the
antibody isotype. Examples of antibody effector functions include:
C1q binding and complement dependent cytotoxicity (CDC); Fc
receptor binding; antibody-dependent cell-mediated cytotoxicity
(ADCC); phagocytosis; down regulation of cell surface receptors
(e.g. B cell receptor); and B cell activation.
[0064] An "effective amount" of an agent, e.g., a pharmaceutical
formulation, refers to an amount effective, at dosages and for
periods of time necessary, to achieve the desired therapeutic or
prophylactic result.
[0065] "Fc receptor" or "FcR" describes a receptor that binds to
the Fc region of an antibody. In some embodiments, an FcR is a
native human FcR. In some embodiments, an FcR is one which binds an
IgG antibody (a gamma receptor) and includes receptors of the
Fc.gamma.RI, Fc.gamma.RII, and Fc.gamma.RIII subclasses, including
allelic variants and alternatively spliced forms of those
receptors. Fc.gamma.RII receptors include Fc.gamma.RIIA (an
"activating receptor") and Fc.gamma.RIIB (an "inhibiting
receptor"), which have similar amino acid sequences that differ
primarily in the cytoplasmic domains thereof. Activating receptor
Fc.gamma.RIIA contains an immunoreceptor tyrosine-based activation
motif (ITAM) in its cytoplasmic domain. Inhibiting receptor
Fc.gamma.RIIB contains an immunoreceptor tyrosine-based inhibition
motif (ITIM) in its cytoplasmic domain. (see, e.g., Daeron, Annu.
Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example,
in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et
al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab.
Clin. Med. 126:330-41 (1995). Other FcRs, including those to be
identified in the future, are encompassed by the term "FcR" herein.
The term "Fc receptor" or "FcR" also includes the neonatal
receptor, FcRn, which is responsible for the transfer of maternal
IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim
et al., J. Immunol. 24:249 (1994)) and regulation of homeostasis of
immunoglobulins. Methods of measuring binding to FcRn are known
(see, e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997);
Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton
et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004/92219
(Hinton et al.). Binding to human FcRn in vivo and serum half life
of human FcRn high affinity binding polypeptides can be assayed,
e.g., in transgenic mice or transfected human cell lines expressing
human FcRn, or in primates to which the polypeptides with a variant
Fc region are administered. WO 2000/42072 (Presta) describes
antibody variants with improved or diminished binding to FcRs. See
also, e.g., Shields et al. J. Biol. Chem. 9(2):6591-6604
(2001).
[0066] The term "Fc region" herein is used to define a C-terminal
region of an immunoglobulin heavy chain that contains at least a
portion of the constant region. The term includes native sequence
Fc regions and variant Fc regions. In one embodiment, a human IgG
heavy chain Fc region extends from Cys226, or from Pro230, to the
carboxyl-terminus of the heavy chain. However, the C-terminal
lysine (Lys447) of the Fc region may or may not be present. Unless
otherwise specified herein, numbering of amino acid residues in the
Fc region or constant region is according to the EU numbering
system, also called the EU index, as described in Kabat et al.,
Sequences of Proteins of Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md.,
1991.
[0067] A "functional Fc region" possesses an "effector function" of
a native sequence Fc region. Exemplary "effector functions" include
C1q binding; CDC; Fc receptor binding; ADCC; phagocytosis; down
regulation of cell surface receptors (e.g. B cell receptor; BCR),
etc. Such effector functions generally require the Fc region to be
combined with a binding domain (e.g., an antibody variable domain)
and can be assessed using various assays as disclosed, for example,
in definitions herein.
[0068] "Human effector cells" refer to leukocytes that express one
or more FcRs and perform effector functions. In certain
embodiments, the cells express at least Fc.gamma.RIII and perform
ADCC effector function(s). Examples of human leukocytes which
mediate ADCC include peripheral blood mononuclear cells (PBMC),
natural killer (NK) cells, monocytes, cytotoxic T cells, and
neutrophils. The effector cells may be isolated from a native
source, e.g., from blood.
[0069] "Framework" or "FR" refers to variable domain residues other
than hypervariable region (HVR) residues. The FR of a variable
domain generally consists of four FR domains: FR1, FR2, FR3, and
FR4. Accordingly, the HVR and FR sequences generally appear in the
following sequence in VH (or VL):
FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.
[0070] The terms "full length antibody," "intact antibody," and
"whole antibody" are used herein interchangeably to refer to an
antibody having a structure substantially similar to a native
antibody structure or having heavy chains that contain an Fc region
as defined herein.
[0071] The terms "host cell," "host cell line," and "host cell
culture" are used interchangeably and refer to cells into which
exogenous nucleic acid has been introduced, including the progeny
of such cells. Host cells include "transformants" and "transformed
cells," which include the primary transformed cell and progeny
derived therefrom without regard to the number of passages. Progeny
may not be completely identical in nucleic acid content to a parent
cell, but may contain mutations. Mutant progeny that have the same
function or biological activity as screened or selected for in the
originally transformed cell are included herein.
[0072] A "human antibody" is one which possesses an amino acid
sequence which corresponds to that of an antibody produced by a
human or a human cell or derived from a non-human source that
utilizes human antibody repertoires or other human
antibody-encoding sequences. This definition of a human antibody
specifically excludes a humanized antibody comprising non-human
antigen-binding residues.
[0073] A "human consensus framework" is a framework which
represents the most commonly occurring amino acid residues in a
selection of human immunoglobulin VL or VH framework sequences.
Generally, the selection of human immunoglobulin VL or VH sequences
is from a subgroup of variable domain sequences. Generally, the
subgroup of sequences is a subgroup as in Kabat et al., Sequences
of Proteins of Immunological Interest, Fifth Edition, NIH
Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In one
embodiment, for the VL, the subgroup is subgroup kappa I as in
Kabat et al., supra. In one embodiment, for the VH, the subgroup is
subgroup III as in Kabat et al., supra.
[0074] A "humanized" antibody refers to a chimeric antibody
comprising amino acid residues from non-human HVRs and amino acid
residues from human FRs. In certain embodiments, a humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the HVRs (e.g., CDRs) correspond to those of a non-human
antibody, and all or substantially all of the FRs correspond to
those of a human antibody. A humanized antibody optionally may
comprise at least a portion of an antibody constant region derived
from a human antibody. A "humanized form" of an antibody, e.g., a
non-human antibody, refers to an antibody that has undergone
humanization.
[0075] The term "hypervariable region" or "HVR" as used herein
refers to each of the regions of an antibody variable domain which
are hypervariable in sequence ("complementarity determining
regions" or "CDRs") and/or form structurally defined loops
("hypervariable loops") and/or contain the antigen-contacting
residues ("antigen contacts"). Generally, antibodies comprise six
HVRs: three in the VH (H1, H2, H3), and three in the VL (L1, L2,
L3). Exemplary HVRs herein include:
[0076] (a) hypervariable loops occurring at amino acid residues
26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and
96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917
(1987));
[0077] (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56
(L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat
et al., Sequences of Proteins of Immunological Interest, 5th Ed.
Public Health Service, National Institutes of Health, Bethesda, Md.
(1991));
[0078] (c) antigen contacts occurring at amino acid residues 27c-36
(L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101
(H3) (MacCallum et al. J. Mol. Biol. 262: 732-745 (1996)); and
[0079] (d) combinations of (a), (b), and/or (c), including HVR
amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2),
26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102
(H3).
[0080] Unless otherwise indicated, HVR residues and other residues
in the variable domain (e.g., FR residues) are numbered herein
according to Kabat et al., supra.
[0081] An "immunoconjugate" is an antibody conjugated to one or
more heterologous molecule(s), including but not limited to a
cytotoxic agent.
[0082] An "individual" or "subject" is a mammal. Mammals include,
but are not limited to, domesticated animals (e.g., cows, sheep,
cats, dogs, and horses), primates (e.g., humans and non-human
primates such as monkeys), rabbits, and rodents (e.g., mice and
rats). In certain embodiments, the individual or subject is a
human.
[0083] "Promoting cell growth or proliferation" means increasing a
cell's growth or proliferation by at least 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, 95%, or 100%.
[0084] An "isolated" antibody is one which has been separated from
a component of its natural environment. In some embodiments, an
antibody is purified to greater than 95% or 99% purity as
determined by, for example, electrophoretic (e.g., SDS-PAGE,
isoelectric focusing (IEF), capillary electrophoresis) or
chromatographic (e.g., ion exchange or reverse phase HPLC). For
review of methods for assessment of antibody purity, see, e.g.,
Flatman et al., J. Chromatogr. B 848:79-87 (2007).
[0085] An "isolated" nucleic acid refers to a nucleic acid molecule
that has been separated from a component of its natural
environment. An isolated nucleic acid includes a nucleic acid
molecule contained in cells that ordinarily contain the nucleic
acid molecule, but the nucleic acid molecule is present
extrachromosomally or at a chromosomal location that is different
from its natural chromosomal location.
[0086] "Isolated nucleic acid encoding an anti-OX40 antibody"
refers to one or more nucleic acid molecules encoding antibody
heavy and light chains (or fragments thereof), including such
nucleic acid molecule(s) in a single vector or separate vectors,
and such nucleic acid molecule(s) present at one or more locations
in a host cell.
[0087] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical and/or bind the same epitope, except for
possible variant antibodies, e.g., containing naturally occurring
mutations or arising during production of a monoclonal antibody
preparation, such variants generally being present in minor
amounts. In contrast to polyclonal antibody preparations, which
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody of a monoclonal
antibody preparation is directed against a single determinant on an
antigen. Thus, the modifier "monoclonal" indicates the character of
the antibody as being obtained from a substantially homogeneous
population of antibodies, and is not to be construed as requiring
production of the antibody by any particular method. For example,
the monoclonal antibodies to be used in accordance with the present
invention may be made by a variety of techniques, including but not
limited to the hybridoma method, recombinant DNA methods,
phage-display methods, and methods utilizing transgenic animals
containing all or part of the human immunoglobulin loci, such
methods and other exemplary methods for making monoclonal
antibodies being described herein.
[0088] A "naked antibody" refers to an antibody that is not
conjugated to a heterologous moiety (e.g., a cytotoxic moiety) or
radiolabel. The naked antibody may be present in a pharmaceutical
formulation.
[0089] "Native antibodies" refer to naturally occurring
immunoglobulin molecules with varying structures. For example,
native IgG antibodies are heterotetrameric glycoproteins of about
150,000 daltons, composed of two identical light chains and two
identical heavy chains that are disulfide-bonded. From N- to
C-terminus, each heavy chain has a variable region (VH), also
called a variable heavy domain or a heavy chain variable domain,
followed by three constant domains (CH1, CH2, and CH3). Similarly,
from N- to C-terminus, each light chain has a variable region (VL),
also called a variable light domain or a light chain variable
domain, followed by a constant light (CL) domain. The light chain
of an antibody may be assigned to one of two types, called kappa
(.kappa.) and lambda (.lamda.), based on the amino acid sequence of
its constant domain. A "native sequence Fc region" comprises an
amino acid sequence identical to the amino acid sequence of an Fc
region found in nature. Native sequence human Fc regions include a
native sequence human IgG1 Fc region (non-A and A allotypes);
native sequence human IgG2 Fc region; native sequence human IgG3 Fc
region; and native sequence human IgG4 Fc region as well as
naturally occurring variants thereof.
[0090] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, combination therapy, contraindications
and/or warnings concerning the use of such therapeutic
products.
[0091] "Percent (%) amino acid sequence identity" with respect to a
reference polypeptide sequence is defined as the percentage of
amino acid residues in a candidate sequence that are identical with
the amino acid residues in the reference polypeptide sequence,
after aligning the sequences and introducing gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering
any conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)
software. Those skilled in the art can determine appropriate
parameters for aligning sequences, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared. For purposes herein, however, % amino acid sequence
identity values are generated using the sequence comparison
computer program ALIGN-2. The ALIGN-2 sequence comparison computer
program was authored by Genentech, Inc., and the source code has
been filed with user documentation in the U.S. Copyright Office,
Washington D.C., 20559, where it is registered under U.S. Copyright
Registration No. TXU510087. The ALIGN-2 program is publicly
available from Genentech, Inc., South San Francisco, Calif., or may
be compiled from the source code. The ALIGN-2 program should be
compiled for use on a UNIX operating system, including digital UNIX
V4.0D. All sequence comparison parameters are set by the ALIGN-2
program and do not vary.
[0092] In situations where ALIGN-2 is employed for amino acid
sequence comparisons, the % amino acid sequence identity of a given
amino acid sequence A to, with, or against a given amino acid
sequence B (which can alternatively be phrased as a given amino
acid sequence A that has or comprises a certain % amino acid
sequence identity to, with, or against a given amino acid sequence
B) is calculated as follows:
100 times the fraction X/Y
where X is the number of amino acid residues scored as identical
matches by the sequence alignment program ALIGN-2 in that program's
alignment of A and B, and where Y is the total number of amino acid
residues in B. It will be appreciated that where the length of
amino acid sequence A is not equal to the length of amino acid
sequence B, the % amino acid sequence identity of A to B will not
equal the % amino acid sequence identity of B to A. Unless
specifically stated otherwise, all % amino acid sequence identity
values used herein are obtained as described in the immediately
preceding paragraph using the ALIGN-2 computer program.
[0093] The term "pharmaceutical formulation" refers to a
preparation which is in such form as to permit the biological
activity of an active ingredient contained therein to be effective,
and which contains no additional components which are unacceptably
toxic to a subject to which the formulation would be
administered.
[0094] A "pharmaceutically acceptable carrier" refers to an
ingredient in a pharmaceutical formulation, other than an active
ingredient, which is nontoxic to a subject. A pharmaceutically
acceptable carrier includes, but is not limited to, a buffer,
excipient, stabilizer, or preservative.
[0095] As used herein, "treatment" (and grammatical variations
thereof such as "treat" or "treating") refers to clinical
intervention in an attempt to alter the natural course of the
individual being treated, and can be performed either for
prophylaxis or during the course of clinical pathology. Desirable
effects of treatment include, but are not limited to, preventing
occurrence or recurrence of disease, alleviation of symptoms,
diminishment of any direct or indirect pathological consequences of
the disease, preventing metastasis, decreasing the rate of disease
progression, amelioration or palliation of the disease state, and
remission or improved prognosis. In some embodiments, antibodies of
the invention are used to delay development of a disease or to slow
the progression of a disease.
[0096] The term "tumor" refers to all neoplastic cell growth and
proliferation, whether malignant or benign, and all pre-cancerous
and cancerous cells and tissues. The terms "cancer," "cancerous,"
"cell proliferative disorder," "proliferative disorder" and "tumor"
are not mutually exclusive as referred to herein.
[0097] The term "variable region" or "variable domain" refers to
the domain of an antibody heavy or light chain that is involved in
binding the antibody to antigen. The variable domains of the heavy
chain and light chain (VH and VL, respectively) of a native
antibody generally have similar structures, with each domain
comprising four conserved framework regions (FRs) and three
hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby
Immunology, 6.sup.th ed., W.H. Freeman and Co., page 91 (2007).) A
single VH or VL domain may be sufficient to confer antigen-binding
specificity. Furthermore, antibodies that bind a particular antigen
may be isolated using a VH or VL domain from an antibody that binds
the antigen to screen a library of complementary VL or VH domains,
respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887
(1993); Clarkson et al., Nature 352:624-628 (1991).
[0098] A "variant Fc region" comprises an amino acid sequence which
differs from that of a native sequence Fc region by virtue of at
least one amino acid modification, preferably one or more amino
acid substitution(s). Preferably, the variant Fc region has at
least one amino acid substitution compared to a native sequence Fc
region or to the Fc region of a parent polypeptide, e.g. from about
one to about ten amino acid substitutions, and preferably from
about one to about five amino acid substitutions in a native
sequence Fc region or in the Fc region of the parent polypeptide.
The variant Fc region herein will preferably possess at least about
80% homology with a native sequence Fc region and/or with an Fc
region of a parent polypeptide, and most preferably at least about
90% homology therewith, more preferably at least about 95% homology
therewith.
[0099] The term "vector," as used herein, refers to a nucleic acid
molecule capable of propagating another nucleic acid to which it is
linked. The term includes the vector as a self-replicating nucleic
acid structure as well as the vector incorporated into the genome
of a host cell into which it has been introduced. Certain vectors
are capable of directing the expression of nucleic acids to which
they are operatively linked. Such vectors are referred to herein as
"expression vectors."
[0100] A "VH subgroup III consensus framework" comprises the
consensus sequence obtained from the amino acid sequences in
variable heavy subgroup III of Kabat et al. In one embodiment, the
VH subgroup III consensus framework amino acid sequence comprises
at least a portion or all of each of the following sequences:
TABLE-US-00001 (SEQ ID NO: 214) EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID
NO: 215) -H1-WVRQAPGKGLEWV (SEQ ID NO: 216)
-H2-RFTISRDNSKNTLYLQMNSLRAEDTAVYYC (SEQ ID NO: 217)
-H3-WGQGTLVTVSS.
[0101] A "VL subgroup I consensus framework" comprises the
consensus sequence obtained from the amino acid sequences in
variable light kappa subgroup I of Kabat et al. In one embodiment,
the VH subgroup I consensus framework amino acid sequence comprises
at least a portion or all of each of the following sequences:
TABLE-US-00002 (SEQ ID NO: 218) DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:
219) -L1-WYQQKPGKAPKLLIY (SEQ ID NO: 220)
-L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 221)
-L3-FGQGTKVEIK.
[0102] The term "cytotoxic agent" as used herein refers to a
substance that inhibits or prevents a cellular function and/or
causes cell death or destruction. Cytotoxic agents include, but are
not limited to, radioactive isotopes (e.g., At211, I131, I125, Y90,
Re186, Re188, Sm153, Bi212, P32, Pb212 and radioactive isotopes of
Lu); chemotherapeutic agents; growth inhibitory agents; enzymes and
fragments thereof such as nucleolytic enzymes; and toxins such as
small molecule toxins or enzymatically active toxins of bacterial,
fungal, plant or animal origin, including fragments and/or variants
thereof. Exemplary cytotoxic agents can be selected from
anti-microtubule agents, platinum coordination complexes,
alkylating agents, antibiotic agents, topoisomerase II inhibitors,
antimetabolites, topoisomerase I inhibitors, hormones and hormonal
analogues, signal transduction pathway inhibitors, non-receptor
tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents,
proapoptotic agents, inhibitors of LDH-A; inhibitors of fatty acid
biosynthesis; cell cycle signalling inhibitors; HDAC inhibitors,
proteasome inhibitors; and inhibitors of cancer metabolism.
[0103] In one embodiment the cytotoxic agent is selected from
anti-microtubule agents, platinum coordination complexes,
alkylating agents, antibiotic agents, topoisomerase II inhibitors,
antimetabolites, topoisomerase I inhibitors, hormones and hormonal
analogues, signal transduction pathway inhibitors, non-receptor
tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents,
proapoptotic agents, inhibitors of LDH-A, inhibitors of fatty acid
biosynthesis, cell cycle signalling inhibitors, HDAC inhibitors,
proteasome inhibitors, and inhibitors of cancer metabolism. In one
embodiment the cytotoxic agent is a taxane. In one embodiment the
taxane is paclitaxel or docetaxel. In one embodiment the cytotoxic
agent is a platinum agent. In one embodiment the cytotoxic agent is
an antagonist of EGFR. In one embodiment the antagonist of EGFR is
N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine
(e.g., erlotinib). In one embodiment the cytotoxic agent is a RAF
inhibitor. In one embodiment, the RAF inhibitor is a BRAF and/or
CRAF inhibitor. In one embodiment the RAF inhibitor is vemurafenib.
In one embodiment the cytotoxic agent is a PI3K inhibitor.
[0104] "Chemotherapeutic agent" includes chemical compounds useful
in the treatment of cancer. Examples of chemotherapeutic agents
include erlotinib (TARCEVA.RTM., Genentech/OSI Pharm.), bortezomib
(VELCADE.RTM., Millennium Pharm.), disulfiram, epigallocatechin
gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin),
radicicol, lactate dehydrogenase A (LDH-A), fulvestrant
(FASLODEX.RTM., AstraZeneca), sunitib (SUTENT.RTM., Pfizer/Sugen),
letrozole (FEMARA.RTM., Novartis), imatinib mesylate (GLEEVEC.RTM.,
Novartis), finasunate (VATALANIB.RTM., Novartis), oxaliplatin
(ELOXATIN.RTM., Sanofi), 5-FU (5-fluorouracil), leucovorin,
Rapamycin (Sirolimus, RAPAMUNE.RTM., Wyeth), Lapatinib
(TYKERB.RTM., GSK572016, Glaxo Smith Kline), Lonafamib (SCH 66336),
sorafenib (NEXAVAR.RTM., Bayer Labs), gefitinib (IRESSA.RTM.,
AstraZeneca), AG1478, alkylating agents such as thiotepa and
CYTOXAN.RTM. cyclosphosphamide; alkyl sulfonates such as busulfan,
improsulfan and piposulfan; aziridines such as benzodopa,
carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
triethylenephosphoramide, triethylenethiophosphoramide and
trimethylomelamine; acetogenins (especially bullatacin and
bullatacinone); a camptothecin (including topotecan and
irinotecan); bryostatin; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogs);
cryptophycins (particularly cryptophycin 1 and cryptophycin 8);
adrenocorticosteroids (including prednisone and prednisolone);
cyproterone acetate; 5.alpha.-reductases including finasteride and
dutasteride); vorinostat, romidepsin, panobinostat, valproic acid,
mocetinostat dolastatin; aldesleukin, talc duocarmycin (including
the synthetic analogs, KW-2189 and CB1-TM1); eleutherobin;
pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards
such as chlorambucil, chlomaphazine, chlorophosphamide,
estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide
hydrochloride, melphalan, novembichin, phenesterine, prednimustine,
trofosfamide, uracil mustard; nitrosoureas such as carmustine,
chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;
antibiotics such as the enediyne antibiotics (e.g., calicheamicin,
especially calicheamicin .gamma.1I and calicheamicin .omega.1I
(Angew Chem. Intl. Ed. Engl. 1994 33:183-186); dynemicin, including
dynemicin A; bisphosphonates, such as clodronate; an esperamicin;
as well as neocarzinostatin chromophore and related chromoprotein
enediyne antibiotic chromophores), aclacinomysins, actinomycin,
authramycin, azaserine, bleomycins, cactinomycin, carabicin,
caminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,
ADRIAMYCIN.RTM. (doxorubicin), morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as
denopterin, methotrexate, pteropterin, trimetrexate; purine analogs
such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine;
pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine,
carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine,
floxuridine; androgens such as calusterone, dromostanolone
propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals
such as aminoglutethimide, mitotane, trilostane; folic acid
replenisher such as frolinic acid; aceglatone; aldophosphamide
glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil;
bisantrene; edatraxate; defofamine; demecolcine; diaziquone;
elfomithine; elliptinium acetate; an epothilone; etoglucid; gallium
nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as
maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamnol;
nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;
podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK.RTM.
polysaccharide complex (JHS Natural Products, Eugene, Oreg.);
razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid;
triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes
(especially T-2 toxin, verracurin A, roridin A and anguidine);
urethan; vindesine; dacarbazine; mannomustine; mitobronitol;
mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C");
cyclophosphamide; thiotepa; taxoids, e.g., TAXOL (paclitaxel;
Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE.RTM.
(Cremophor-free), albumin-engineered nanoparticle formulations of
paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.),
and TAXOTERE.RTM. (docetaxel, doxetaxel; Sanofi-Aventis);
chloranmbucil; GEMZAR.RTM. (gemcitabine); 6-thioguanine;
mercaptopurine; methotrexate; platinum analogs such as cisplatin
and carboplatin; vinblastine; etoposide (VP-16); ifosfamide;
mitoxantrone; vincristine; NAVELBINE.RTM. (vinorelbine);
novantrone; teniposide; edatrexate; daunomycin; aminopterin;
capecitabine (XELODA.RTM.); ibandronate; CPT-11; topoisomerase
inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such
as retinoic acid; and pharmaceutically acceptable salts, acids and
derivatives of any of the above.
[0105] Chemotherapeutic agent also includes (i) anti-hormonal
agents that act to regulate or inhibit hormone action on tumors
such as anti-estrogens and selective estrogen receptor modulators
(SERMs), including, for example, tamoxifen (including
NOLVADEX.RTM.; tamoxifen citrate), raloxifene, droloxifene,
iodoxyfene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,
onapristone, and FARESTON.RTM. (toremifine citrate); (ii) aromatase
inhibitors that inhibit the enzyme aromatase, which regulates
estrogen production in the adrenal glands, such as, for example,
4(5)-imidazoles, aminoglutethimide, MEGASE.RTM. (megestrol
acetate), AROMASIN.RTM. (exemestane; Pfizer), formestanie,
fadrozole, RIVISOR.RTM. (vorozole), FEMARA.RTM. (letrozole;
Novartis), and ARIMIDEX.RTM. (anastrozole; AstraZeneca); (iii)
anti-androgens such as flutamide, nilutamide, bicalutamide,
leuprolide and goserelin; buserelin, tripterelin,
medroxyprogesterone acetate, diethylstilbestrol, premarin,
fluoxymesterone, all transretionic acid, fenretinide, as well as
troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv)
protein kinase inhibitors; (v) lipid kinase inhibitors; (vi)
antisense oligonucleotides, particularly those which inhibit
expression of genes in signaling pathways implicated in aberrant
cell proliferation, such as, for example, PKC-alpha, Ralf and
H-Ras; (vii) ribozymes such as VEGF expression inhibitors (e.g.,
ANGIOZYME.RTM.) and HER2 expression inhibitors; (viii) vaccines
such as gene therapy vaccines, for example, ALLOVECTIN.RTM.,
LEUVECTIN.RTM., and VAXID.RTM.; PROLEUKIN.RTM., rIL-2; a
topoisomerase 1 inhibitor such as LURTOTECAN.RTM.; ABARELIX.RTM.
rmRH; and (ix) pharmaceutically acceptable salts, acids and
derivatives of any of the above.
[0106] Chemotherapeutic agent also includes antibodies such as
alemtuzumab (Campath), bevacizumab (AVASTIN.RTM., Genentech);
cetuximab (ERBITUX.RTM., Imclone); panitumumab (VECTIBIX.RTM.,
Amgen), rituximab (RITUXAN.RTM., Genentech/Biogen Idec), pertuzumab
(OMNITARG.RTM., 2C4, Genentech), trastuzumab (HERCEPTIN.RTM.,
Genentech), tositumomab (Bexxar, Corixia), and the antibody drug
conjugate, gemtuzumab ozogamicin (MYLOTARG.RTM., Wyeth). Additional
humanized monoclonal antibodies with therapeutic potential as
agents in combination with the compounds of the invention include:
apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab
mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol,
cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab,
epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumab
ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab,
lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab,
natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab,
omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab,
pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab,
resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab,
sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab,
tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin,
tucusituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab,
and the anti-interleukin-12 (ABT-874/J695, Wyeth Research and
Abbott Laboratories) which is a recombinant exclusively
human-sequence, full-length IgG1 .lamda. antibody genetically
modified to recognize interleukin-12 p40 protein.
[0107] Chemotherapeutic agent also includes "EGFR inhibitors,"
which refers to compounds that bind to or otherwise interact
directly with EGFR and prevent or reduce its signaling activity,
and is alternatively referred to as an "EGFR antagonist." Examples
of such agents include antibodies and small molecules that bind to
EGFR. Examples of antibodies which bind to EGFR include MAb 579
(ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL
8508), MAb 528 (ATCC CRL 8509) (see, U.S. Pat. No. 4,943,533,
Mendelsohn et al.) and variants thereof, such as chimerized 225
(C225 or Cetuximab; ERBUTIX.RTM.) and reshaped human 225 (H225)
(see, WO 96/40210, Imclone Systems Inc.); IMC-11F8, a fully human,
EGFR-targeted antibody (Imclone); antibodies that bind type II
mutant EGFR (U.S. Pat. No. 5,212,290); humanized and chimeric
antibodies that bind EGFR as described in U.S. Pat. No. 5,891,996;
and human antibodies that bind EGFR, such as ABX-EGF or Panitumumab
(see WO98/50433, Abgenix/Amgen); EMD 55900 (Stragliotto et al. Eur.
J. Cancer 32A:636-640 (1996)); EMD7200 (matuzumab) a humanized EGFR
antibody directed against EGFR that competes with both EGF and
TGF-alpha for EGFR binding (EMD/Merck); human EGFR antibody,
HuMax-EGFR (GenMab); fully human antibodies known as E1.1, E2.4,
E2.5, E6.2, E6.4, E2.11, E6. 3 and E7.6. 3 and described in U.S.
Pat. No. 6,235,883; MDX-447 (Medarex Inc); and mAb 806 or humanized
mAb 806 (Johns et al., J. Biol. Chem. 279(29):30375-30384 (2004)).
The anti-EGFR antibody may be conjugated with a cytotoxic agent,
thus generating an immunoconjugate (see, e.g., EP659,439A2, Merck
Patent GmbH). EGFR antagonists include small molecules such as
compounds described in U.S. Pat. Nos. 5,616,582, 5,457,105,
5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534,
6,521,620, 6,596,726, 6,713,484, 5,770,599, 6,140,332, 5,866,572,
6,399,602, 6,344,459, 6,602,863, 6,391,874, 6,344,455, 5,760,041,
6,002,008, and 5,747,498, as well as the following PCT
publications: WO98/14451, WO98/50038, WO99/09016, and WO99/24037.
Particular small molecule EGFR antagonists include OSI-774
(CP-358774, erlotinib, TARCEVA.RTM. Genentech/OSI Pharmaceuticals);
PD 183805 (CI 1033,2-propenamide,
N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quin-
azolinyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib
(IRESSA.RTM.)
4-(3'-Chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoli-
ne, AstraZeneca); ZM 105180
((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca); BIBX-1382
(N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4--
d]pyrimidine-2,8-diamine, Boehringer Ingelheim); PKI-166
((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol)-
;
(R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimi-
dine); CL-387785
(N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide); EKB-569
(N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(-
dimethylamino)-2-butenamide) (Wyeth); AG1478 (Pfizer); AG1571 (SU
5271; Pfizer); dual EGFR/HER2 tyrosine kinase inhibitors such as
lapatinib (TYKERB.RTM., GSK572016 or N-[3-chloro-4-[(3
fluorophenyl)methoxy]phenyl]-6[5[[[2methylsulfonyl)ethyl]amino]methyl]-2--
furanyl]-4-quinazolinamine).
[0108] Chemotherapeutic agents also include "tyrosine kinase
inhibitors" including the EGFR-targeted drugs noted in the
preceding paragraph; small molecule HER2 tyrosine kinase inhibitor
such as TAK165 available from Takeda; CP-724,714, an oral selective
inhibitor of the ErbB2 receptor tyrosine kinase (Pfizer and OSI);
dual-HER inhibitors such as EKB-569 (available from Wyeth) which
preferentially binds EGFR but inhibits both HER2 and
EGFR-overexpressing cells; lapatinib (GSK572016; available from
Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor;
PKI-166 (available from Novartis); pan-HER inhibitors such as
canertinib (CI-1033; Pharmacia); Raf-1 inhibitors such as antisense
agent ISIS-5132 available from ISIS Pharmaceuticals which inhibit
Raf-1 signaling; non-HER targeted TK inhibitors such as imatinib
mesylate (GLEEVEC.RTM., available from Glaxo SmithKline);
multi-targeted tyrosine kinase inhibitors such as sunitinib
(SUTENT.RTM., available from Pfizer); VEGF receptor tyrosine kinase
inhibitors such as vatalanib (PTK787/ZK222584, available from
Novartis/Schering AG); MAPK extracellular regulated kinase I
inhibitor CI-1040 (available from Pharmacia); quinazolines, such as
PD 153035,4-(3-chloroanilino)quinazoline; pyridopyrimidines;
pyrimidopyrimidines; pyrrolopyrimidines, such as CGP 59326, CGP
60261 and CGP 62706; pyrazolopyrimidines,
4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidines; curcumin (diferuloyl
methane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostines
containing nitrothiophene moieties; PD-0183805 (Warner-Lamber);
antisense molecules (e.g. those that bind to HER-encoding nucleic
acid); quinoxalines (U.S. Pat. No. 5,804,396); tryphostins (U.S.
Pat. No. 5,804,396); ZD6474 (Astra Zeneca); PTK-787
(Novartis/Schering AG); pan-HER inhibitors such as CI-1033
(Pfizer); Affinitac (ISIS 3521; Isis/Lilly); imatinib mesylate
(GLEEVEC.RTM.); PKI 166 (Novartis); GW2016 (Glaxo SmithKline);
CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474
(AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone),
rapamycin (sirolimus, RAPAMUNE.RTM.); or as described in any of the
following patent publications: U.S. Pat. No. 5,804,396; WO
1999/09016 (American Cyanamid); WO 1998/43960 (American Cyanamid);
WO 1997/38983 (Warner Lambert); WO 1999/06378 (Warner Lambert); WO
1999/06396 (Warner Lambert); WO 1996/30347 (Pfizer, Inc); WO
1996/33978 (Zeneca); WO 1996/3397 (Zeneca) and WO 1996/33980
(Zeneca).
[0109] Chemotherapeutic agents also include dexamethasone,
interferons, colchicine, metoprine, cyclosporine, amphotericin,
metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine,
arsenic trioxide, asparaginase, BCG live, bevacuzimab, bexarotene,
cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane,
epoetin alfa, elotinib, filgrastim, histrelin acetate, ibritumomab,
interferon alfa-2a, interferon alfa-2b, lenalidomide, levamisole,
mesna, methoxsalen, nandrolone, nelarabine, nofetumomab,
oprelvekin, palifermin, pamidronate, pegademase, pegaspargase,
pegfilgrastim, pemetrexed disodium, plicamycin, porfimer sodium,
quinacrine, rasburicase, sargramostim, temozolomide, VM-26, 6-TG,
toremifene, tretinoin, ATRA, valrubicin, zoledronate, and
zoledronic acid, and pharmaceutically acceptable salts thereof.
[0110] Chemotherapeutic agents also include hydrocortisone,
hydrocortisone acetate, cortisone acetate, tixocortol pivalate,
triamcinolone acetonide, triamcinolone alcohol, mometasone,
amcinonide, budesonide, desonide, fluocinonide, fluocinolone
acetonide, betamethasone, betamethasone sodium phosphate,
dexamethasone, dexamethasone sodium phosphate, fluocortolone,
hydrocortisone-17-butyrate, hydrocortisone-17-valerate,
aclometasone dipropionate, betamethasone valerate, betamethasone
dipropionate, prednicarbate, clobetasone-17-butyrate,
clobetasol-17-propionate, fluocortolone caproate, fluocortolone
pivalate and fluprednidene acetate; immune selective
anti-inflammatory peptides (ImSAIDs) such as
phenylalanine-glutamine-glycine (FEG) and its D-isomeric form (feG)
(IMULAN BioTherapeutics, LLC); anti-rheumatic drugs such as
azathioprine, ciclosporin (cyclosporine A), D-penicillamine, gold
salts, hydroxychloroquine, leflunomideminocycline, sulfasalazine,
tumor necrosis factor alpha (TNFa) blockers such as etanercept
(Enbrel), infliximab (Remicade), adalimumab (Humira), certolizumab
pegol (Cimzia), golimumab (Simponi), Interleukin 1 (IL-1) blockers
such as anakinra (Kineret), T cell costimulation blockers such as
abatacept (Orencia), Interleukin 6 (IL-6) blockers such as
tocilizumab (ACTEMERA.RTM.); Interleukin 13 (IL-13) blockers such
as lebrikizumab; Interferon alpha (IFN) blockers such as
Rontalizumab; Beta 7 integrin blockers such as rhuMAb Beta7; IgE
pathway blockers such as Anti-M1 prime; Secreted homotrimeric LTa3
and membrane bound heterotrimer LTa1/.beta.2 blockers such as
Anti-lymphotoxin alpha (LTa); radioactive isotopes (e.g., At211,
1131, 1125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and
radioactive isotopes of Lu); miscellaneous investigational agents
such as thioplatin, PS-341, phenylbutyrate, ET-18-OCH3, or farnesyl
transferase inhibitors (L-739749, L-744832); polyphenols such as
quercetin, resveratrol, piceatannol, epigallocatechine gallate,
theaflavins, flavanols, procyanidins, betulinic acid and
derivatives thereof; autophagy inhibitors such as chloroquine;
delta-9-tetrahydrocannabinol (dronabinol, MARINOL.RTM.);
beta-lapachone; lapachol; colchicines; betulinic acid;
acetylcamptothecin, scopolectin, and 9-aminocamptothecin);
podophyllotoxin; tegafur (UFTORAL.RTM.); bexarotene
(TARGRETIN.RTM.); bisphosphonates such as clodronate (for example,
BONEFOS.RTM. or OSTAC.RTM.), etidronate (DIDROCAL.RTM.), NE-58095,
zoledronic acid/zoledronate (ZOMETA.RTM.), alendronate
(FOSAMAX.RTM.), pamidronate (AREDIA.RTM.), tiludronate
(SKELID.RTM.), or risedronate (ACTONEL.RTM.); and epidermal growth
factor receptor (EGF-R); vaccines such as THERATOPE.RTM. vaccine;
perifosine, COX-2 inhibitor (e.g. celecoxib or etoricoxib),
proteosome inhibitor (e.g. PS341); CCI-779; tipifarnib (R11577);
orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium
(GENASENSE.RTM.); pixantrone; farnesyltransferase inhibitors such
as lonafarnib (SCH 6636, SARASAR.TM.); and pharmaceutically
acceptable salts, acids or derivatives of any of the above; as well
as combinations of two or more of the above such as CHOP, an
abbreviation for a combined therapy of cyclophosphamide,
doxorubicin, vincristine, and prednisolone; and FOLFOX, an
abbreviation for a treatment regimen with oxaliplatin
(ELOXATIN.TM.) combined with 5-FU and leucovorin.
[0111] Chemotherapeutic agents also include non-steroidal
anti-inflammatory drugswith analgesic, antipyretic and
anti-inflammatory effects. NSAIDs include non-selective inhibitors
of the enzyme cyclooxygenase. Specific examples of NSAIDs include
aspirin, propionic acid derivatives such as ibuprofen, fenoprofen,
ketoprofen, flurbiprofen, oxaprozin and naproxen, acetic acid
derivatives such as indomethacin, sulindac, etodolac, diclofenac,
enolic acid derivatives such as piroxicam, meloxicam, tenoxicam,
droxicam, lornoxicam and isoxicam, fenamic acid derivatives such as
mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic
acid, and COX-2 inhibitors such as celecoxib, etoricoxib,
lumiracoxib, parecoxib, rofecoxib, rofecoxib, and valdecoxib.
NSAIDs can be indicated for the symptomatic relief of conditions
such as rheumatoid arthritis, osteoarthritis, inflammatory
arthropathies, ankylosing spondylitis, psoriatic arthritis,
Reiter's syndrome, acute gout, dysmenorrhoea, metastatic bone pain,
headache and migraine, postoperative pain, mild-to-moderate pain
due to inflammation and tissue injury, pyrexia, ileus, and renal
colic.
[0112] The term "cytokine" is a generic term for proteins released
by one cell population that act on another cell as intercellular
mediators. Examples of such cytokines are lymphokines, monokines;
interleukins (ILs) such as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5,
IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-15; a tumor necrosis
factor such as TNF-.alpha. or TNF-.beta.; and other polypeptide
factors including LIF and kit ligand (KL) and gamma interferon. As
used herein, the term cytokine includes proteins from natural
sources or from recombinant cell culture and biologically active
equivalents of the native-sequence cytokines, including
synthetically produced small-molecule entities and pharmaceutically
acceptable derivatives and salts thereof.
[0113] The term "PD-1 axis binding antagonist" is a molecule that
inhibits the interaction of a PD-1 axis binding partner with either
one or more of its binding partner, so as to remove T-cell
dysfunction resulting from signaling on the PD-1 signaling
axis--with a result being to restore or enhance T-cell function
(e.g., proliferation, cytokine production, target cell killing). As
used herein, a PD-1 axis binding antagonist includes a PD-1 binding
antagonist, a PD-L1 binding antagonist and a PD-L2 binding
antagonist.
[0114] The term "PD-1 binding antagonists" is a molecule that
decreases, blocks, inhibits, abrogates or interferes with signal
transduction resulting from the interaction of PD-1 with one or
more of its binding partners, such as PD-L1, PD-L2. In some
embodiments, the PD-1 binding antagonist is a molecule that
inhibits the binding of PD-1 to its binding partners. In a specific
aspect, the PD-1 binding antagonist inhibits the binding of PD-1 to
PD-L1 and/or PD-L2. For example, PD-1 binding antagonists include
anti-PD-1 antibodies, antigen binding fragments thereof,
immunoadhesins, fusion proteins, oligopeptides and other molecules
that decrease, block, inhibit, abrogate or interfere with signal
transduction resulting from the interaction of PD-1 with PD-L1
and/or PD-L2. In one embodiment, a PD-1 binding antagonist reduces
the negative co-stimulatory signal mediated by or through cell
surface proteins expressed on T lymphocytes mediated signaling
through PD-1 so as render a dysfunctional T-cell less dysfunctional
(e.g., enhancing effector responses to antigen recognition). In
some embodiments, the PD-1 binding antagonist is an anti-PD-1
antibody. In a specific aspect, a PD-1 binding antagonist is MDX-1
106 described herein. In another specific aspect, a PD-1 binding
antagonist is Merck 3745 described herein. In another specific
aspect, a PD-1 binding antagonist is CT-011 described herein.
[0115] The term "PD-L1 binding antagonists" is a molecule that
decreases, blocks, inhibits, abrogates or interferes with signal
transduction resulting from the interaction of PD-L1 with either
one or more of its binding partners, such as PD-1, B7-1. In some
embodiments, a PD-L1 binding antagonist is a molecule that inhibits
the binding of PD-L1 to its binding partners. In a specific aspect,
the PD-L1 binding antagonist inhibits binding of PD-L1 to PD-1
and/or B7-1. In some embodiments, the PD-L1 binding antagonists
include anti-PD-L1 antibodies, antigen binding fragments thereof,
immunoadhesins, fusion proteins, oligopeptides and other molecules
that decrease, block, inhibit, abrogate or interfere with signal
transduction resulting from the interaction of PD-L1 with one or
more of its binding partners, such as PD-1, B7-1. In one
embodiment, a PD-L1 binding antagonist reduces the negative
co-stimulatory signal mediated by or through cell surface proteins
expressed on T lymphocytes mediated signaling through PD-L1 so as
to render a dysfunctional T-cell less dysfunctional (e.g.,
enhancing effector responses to antigen recognition). In some
embodiments, a PD-L1 binding antagonist is an anti-PD-L1 antibody.
In a specific aspect, an anti-PD-L1 antibody is YW243.55.S70
described herein. In another specific aspect, an anti-PD-L1
antibody is MDX-1 105 described herein. In still another specific
aspect, an anti-PD-L1 antibody is MPDL3280A described herein.
[0116] The term "PD-L2 binding antagonists" is a molecule that
decreases, blocks, inhibits, abrogates or interferes with signal
transduction resulting from the interaction of PD-L2 with either
one or more of its binding partners, such as PD-1. In some
embodiments, a PD-L2 binding antagonist is a molecule that inhibits
the binding of PD-L2 to its binding partners. In a specific aspect,
the PD-L2 binding antagonist inhibits binding of PD-L2 to PD-1. In
some embodiments, the PD-L2 antagonists include anti-PD-L2
antibodies, antigen binding fragments thereof, immunoadhesins,
fusion proteins, oligopeptides and other molecules that decrease,
block, inhibit, abrogate or interfere with signal transduction
resulting from the interaction of PD-L2 with either one or more of
its binding partners, such as PD-1. In one embodiment, a PD-L2
binding antagonist reduces the negative co-stimulatory signal
mediated by or through cell surface proteins expressed on T
lymphocytes mediated signaling through PD-L2 so as render a
dysfunctional T-cell less dysfunctional (e.g., enhancing effector
responses to antigen recognition). In some embodiments, a PD-L2
binding antagonist is an immunoadhesin.
[0117] The term "phagocytosis" means the internalization of cells
or particulate matter by cells. In some embodiments, the phagocytic
cells or phagocytes are macrophages or neutrophils. In some
embodiments, the cells are cells that express human OX40. Methods
for assaying phagocytosis are known in the art and include use of
microscopy to detect the presence of cells internalized within
another cells. In other embodiments, phagocytosis is detected using
FACS, e.g., by detecting presence of a detectably labeled cell
within another cell (which may be detectably labeled, e.g., with a
different label than the first cell).
[0118] The phrase "does not possess substantial activity" or
"substantially no activity" with respect to an antibody, as used
herein, means the antibody does not exhibit an activity that is
above background level (in some embodiments, that is above
background level that is statistically significant). The phrase
"little to no activity" with respect to an antibody, as used
herein, means the antibody does not display a biologically
meaningful amount of a function. The function can be measured or
detected according to any assay or technique known in the art,
including, e.g., those described herein. In some embodiments,
antibody function is stimulation of effector T cell proliferation
and/or cytokine secretion.
[0119] The term "biomarker" or "marker" as used herein refers
generally to a molecule, including a gene, mRNA, protein,
carbohydrate structure, or glycolipid, the expression of which in
or on a tissue or cell or secreted can be detected by known methods
(or methods disclosed herein) and is predictive or can be used to
predict (or aid prediction) for a cell, tissue, or patient's
responsiveness to treatment regimes. In some embodiments, a
biomarker may refer to a gene or protein, e.g., the level of
expression of the gene or protein detected in one or more cells. In
some embodiments, a biomarker may refer to a cell type of interest,
e.g., the number of a cell type of interest detected in one or more
samples.
[0120] By "patient sample" is meant a collection of cells or fluids
obtained from a cancer patient. The source of the tissue or cell
sample may be solid tissue as from a fresh, frozen and/or preserved
organ or tissue sample or biopsy or aspirate; blood or any blood
constituents; bodily fluids such as cerebrospinal fluid, amniotic
fluid, peritoneal fluid, or interstitial fluid; cells from any time
in gestation or development of the subject. The tissue sample may
contain compounds which are not naturally intermixed with the
tissue in nature such as preservatives, anticoagulants, buffers,
fixatives, nutrients, antibiotics, or the like. Examples of tumor
samples herein include, but are not limited to, tumor biopsy, fine
needle aspirate, bronchiolar lavage, pleural fluid, sputum, urine,
a surgical specimen, circulating tumor cells, serum, plasma,
circulating plasma proteins, ascitic fluid, primary cell cultures
or cell lines derived from tumors or exhibiting tumor-like
properties, as well as preserved tumor samples, such as
formalin-fixed, paraffin-embedded tumor samples or frozen tumor
samples.
[0121] The phrase "based on expression of" when used herein means
that information about expression level or presence or absence of
expression (e.g., presence or absence or prevalence of (e.g.,
percentage of cells displaying) of the one or more biomarkers
herein (e.g., presence or absence of or amount or prevalence of
FcR-expressing cells, or e.g., presence or absence or amount or
prevalence of human effector cells) is used to inform a treatment
decision, information provided on a package insert, or
marketing/promotional guidance etc.
[0122] A cancer or biological sample which "has human effector
cells" is one which, in a diagnostic test, has human effector cells
present in the sample (e.g., infiltrating human effector
cells).
[0123] A cancer or biological sample which "has FcR-expressing
cells" is one which, in a diagnostic test, has FcR-expressing
present in the sample (e.g., infiltrating FcR-expressing cells). In
some embodiments, FcR is Fc.gamma.R. In some embodiments, FcR is an
activating Fc.gamma.R.
[0124] The phrase "recommending a treatment" as used herein refers
to using the information or data generated relating to the level or
presence of c-met in a sample of a patient to identify the patient
as suitably treated or not suitably treated with a therapy. In some
embodiments the therapy may comprise c-met antibody (e.g.,
onartuzumab). In some embodiments, the therapy may comprise VEGF
antagonist (e.g., bevacizumab). In some embodiments, the therapy
may comprise anti-human OX40 agonist antibody. The information or
data may be in any form, written, oral or electronic. In some
embodiments, using the information or data generated includes
communicating, presenting, reporting, storing, sending,
transferring, supplying, transmitting, delivering, dispensing, or
combinations thereof. In some embodiments, communicating,
presenting, reporting, storing, sending, transferring, supplying,
transmitting, delivering, dispensing, or combinations thereof are
performed by a computing device, analyzer unit or combination
thereof. In some further embodiments, communicating, presenting,
reporting, storing, sending, transferring, supplying, transmitting,
dispensing, or combinations thereof are performed by an individual
(e.g., a laboratory or medical professional). In some embodiments,
the information or data includes a comparison of the amount or
prevalence of FcR expressing cells to a reference level. In some
embodiments, the information or data includes a comparison of the
amount or prevalence of human effector cells to a reference level.
In some embodiments, the information or data includes an indication
that human effector cells or FcR-expressing cells are present or
absent in the sample. In some embodiments, the information or data
includes an indication that FcR-expressing cells and/or human
effector cells are present in a particular percentage of cells
(e.g., high prevalence). In some embodiments, the information or
data includes an indication that the patient is suitably treated or
not suitably treated with a therapy comprising anti-human OX40
agonist antibody.
[0125] The term "detection" includes any means of detecting,
including direct and indirect detection.
[0126] The "amount" or "level" of a biomarker associated with an
increased clinical benefit to an individual is a detectable level
in a biological sample. These can be measured by methods known to
one skilled in the art and also disclosed herein. The expression
level or amount of biomarker assessed can be used to determine the
response to the treatment.
[0127] "Elevated expression," "elevated expression levels," or
"elevated levels" refers to an increased expression or increased
levels of a biomarker in an individual relative to a control, such
as an individual or individuals who are not suffering from the
disease or disorder (e.g., cancer), a tumor with a known
responsiveness to a treatment (e.g., with an OX40 agonist), an
internal control (e.g., housekeeping biomarker), or a reference
number (e.g., a set threshold amount, such as a threshold based on
clinical outcome data).
[0128] "Reduced expression," "reduced expression levels," or
"reduced levels" refers to a decrease expression or decreased
levels of a biomarker in an individual relative to a control, such
as an individual or individuals who are not suffering from the
disease or disorder (e.g., cancer), a tumor with a known
responsiveness to a treatment (e.g., with an OX40 agonist), an
internal control (e.g., housekeeping biomarker), or a reference
number (e.g., a set threshold amount, such as a threshold based on
clinical outcome data). In some embodiments, reduced expression is
little or no expression.
[0129] The terms "housekeeping gene" or "housekeeping biomarker" as
used herein may refer to any gene or genes thought to be
constitutively expressed in a cell in normal and/or pathological
states. Such a gene may be used, for example, as a reference, since
its expression is detectable at a consistent amount across
different physiological conditions. In some embodiments, a
housekeeping gene may encode a protein required for basic cellular
function and/or maintenance.
[0130] The term "diagnosis" is used herein to refer to the
identification or classification of a molecular or pathological
state, disease or condition (e.g., cancer). For example,
"diagnosis" may refer to identification of a particular type of
cancer. "Diagnosis" may also refer to the classification of a
particular subtype of cancer, e.g., by histopathological criteria,
or by molecular features (e.g., a subtype characterized by
expression of one or a combination of biomarkers (e.g., particular
genes or proteins encoded by said genes)).
[0131] The term "aiding diagnosis" is used herein to refer to
methods that assist in making a clinical determination regarding
the presence, or nature, of a particular type of symptom or
condition of a disease or disorder (e.g., cancer). For example, a
method of aiding diagnosis of a disease or condition (e.g., cancer)
can comprise measuring certain biomarkers in a biological sample
from an individual.
[0132] The term "sample," as used herein, refers to a composition
that is obtained or derived from a subject and/or individual of
interest that contains a cellular and/or other molecular entity
that is to be characterized and/or identified, for example based on
physical, biochemical, chemical and/or physiological
characteristics. For example, the phrase "disease sample" and
variations thereof refers to any sample obtained from a subject of
interest that would be expected or is known to contain the cellular
and/or molecular entity that is to be characterized. Samples
include, but are not limited to, primary or cultured cells or cell
lines, cell supernatants, cell lysates, platelets, serum, plasma,
vitreous fluid, lymph fluid, synovial fluid, follicular fluid,
seminal fluid, amniotic fluid, milk, whole blood, blood-derived
cells, urine, cerebrospinal fluid, saliva, sputum, tears,
perspiration, mucus, tumor lysates, and tissue culture medium,
tissue extracts such as homogenized tissue, tumor tissue, cellular
extracts, and combinations thereof.
[0133] By "tissue sample" or "cell sample" is meant a collection of
similar cells obtained from a tissue of a subject or individual.
The source of the tissue or cell sample may be solid tissue as from
a fresh, frozen and/or preserved organ, tissue sample, biopsy,
and/or aspirate; blood or any blood constituents such as plasma;
bodily fluids such as cerebral spinal fluid, amniotic fluid,
peritoneal fluid, or interstitial fluid; cells from any time in
gestation or development of the subject. The tissue sample may also
be primary or cultured cells or cell lines. Optionally, the tissue
or cell sample is obtained from a disease tissue/organ. The tissue
sample may contain compounds which are not naturally intermixed
with the tissue in nature such as preservatives, anticoagulants,
buffers, fixatives, nutrients, antibiotics, or the like.
[0134] As used herein, a "section" of a tissue sample is meant a
single part or piece of a tissue sample, e.g. a thin slice of
tissue or cells cut from a tissue sample. It is understood that
multiple sections of tissue samples may be taken and subjected to
analysis according to the present invention, provided that it is
understood that the present invention comprises a method whereby
the same section of tissue sample is analyzed at both morphological
and molecular levels, or is analyzed with respect to protein or
nucleic acid.
[0135] By "correlate" or "correlating" is meant comparing, in any
way, the performance and/or results of a first analysis or protocol
with the performance and/or results of a second analysis or
protocol. For example, one may use the results of a first analysis
or protocol in carrying out a second protocols and/or one may use
the results of a first analysis or protocol to determine whether a
second analysis or protocol should be performed. With respect to
the embodiment of polypeptide analysis or protocol, one may use the
results of the polypeptide expression analysis or protocol to
determine whether a specific therapeutic regimen should be
performed. With respect to the embodiment of polynucleotide
analysis or protocol, one may use the results of the polynucleotide
expression analysis or protocol to determine whether a specific
therapeutic regimen should be performed.
[0136] "Individual response" or "response" can be assessed using
any endpoint indicating a benefit to the individual, including,
without limitation, (1) inhibition, to some extent, of disease
progression (e.g., cancer progression), including slowing down and
complete arrest; (2) a reduction in tumor size; (3) inhibition
(i.e., reduction, slowing down or complete stopping) of cancer cell
infiltration into adjacent peripheral organs and/or tissues; (4)
inhibition (i.e. reduction, slowing down or complete stopping) of
metastasis; (5) relief, to some extent, of one or more symptoms
associated with the disease or disorder (e.g., cancer); (6)
increase or extend in the length of survival, including overall
survival and progression free survival; and/or (9) decreased
mortality at a given point of time following treatment.
[0137] An "effective response" of a patient or a patient's
"responsiveness" to treatment with a medicament and similar wording
refers to the clinical or therapeutic benefit imparted to a patient
at risk for, or suffering from, a disease or disorder, such as
cancer. In one embodiment, such benefit includes any one or more
of: extending survival (including overall survival and progression
free survival); resulting in an objective response (including a
complete response or a partial response); or improving signs or
symptoms of cancer.
[0138] By "extending survival" is meant increasing overall or
progression free survival in a treated patient relative to an
untreated patient (i.e. relative to a patient not treated with the
medicament), or relative to a patient who does not express a
biomarker at the designated level, and/or relative to a patient
treated with an approved anti-tumor agent. An objective response
refers to a measurable response, including complete response (CR)
or partial response (PR).
III. OX40 Agonists
[0139] Provided herein are methods for predicting responsiveness of
a subject having cancer to an OX40 agonist treatment. These methods
are based in part on the discovery described herein that the
expression level of specific biomarkers correlates with
responsiveness to OX40 agonist treatment. For example, an increased
expression level of genes such as CD8a, CD8b, H2-d, CTLA4, CD64,
CXCL9, IFNg, IDO1, GZMA, GZMB, PRF1, PDCA1, KLRK1, PTPRC, CXCL1,
ITGAM, and IL7R correlates with responsiveness to OX40 agonist
treatment. Additionally, a decreased expression level of genes such
as CSF2, CCL22, EPCAM, GATA3, IL13, and VTCN1 was also found to
correlate with responsiveness to OX40 agonist treatment.
[0140] Further provided herein are methods for monitoring
pharmacodynamic activity of an OX40 agonist treatment by measuring
the expression level of one or more marker genes in a sample
containing leukocytes obtained from the subject, where the subject
has been treated with an OX40 agonist, and where the one or more
marker genes are selected from ARG1, CCL2, CCL22, CCL5, CCR5,
CD226, CD27, CD274, CD28, CD3E, CD40, CD8A, CD8b, CXCL10, CXCL9,
EOMES, FasL, Fcgr1/CD64, FOXP3, GZMA, GZMB, HAVCR2, ICAM1, IDO1,
IFNg, IL10, IL12A (TDO2), IL13, IL2, IL7R, ITGAM, KLRK1, LAG3,
MAP4K1, MS4A1, PDCD1, PDCD1LG2, PRF1, PTPRC, TNF, TNFRSF14,
TNFRSF9, and TNFSF4; and determining the treatment as demonstrating
pharmacodynamic activity based on the expression level of said one
or more marker genes in the sample obtained from the subject, as
compared with a reference, wherein an increased expression level of
the one or more marker genes as compared with the reference
indicates pharmacodynamic activity to the OX40 agonist treatment.
These methods are based in part on the discovery described herein
that the expression level of specific biomarkers is upregulated
upon OX40 agonist treatment.
[0141] Yet further provided herein are methods monitoring
responsiveness of a subject to an OX40 agonist treatment by
measuring the expression level of one or more marker genes in a
sample containing leukocytes obtained from the subject, where the
subject has been treated with an OX40 agonist, and where the one or
more marker genes are selected from BTLA, CD4, CD69, CD80, CD83,
CD86, CSF2, CTLA4, CXCR3, Fcgr2b/CD32, Fcgr3/CD16, H2-aa, H2-d,
H2-k, ICOS, IL10, PDCA1, and TNFRSF18; and classifying the subject
as responsive or non-responsive to said treatment based on the
expression level of said one or more marker genes in the sample
obtained from the subject, as compared with a reference, wherein an
increased expression level of the one or more marker genes as
compared with the reference indicates a responsive subject. These
methods are based in part on the discovery described herein that
the expression level of specific biomarkers is upregulated upon
OX40 agonist treatment specifically in tumors responsive to such
treatment.
[0142] Any OX40 agonists known in the art may be used in the
methods described herein. In one aspect, an OX40 agonist is an
isolated antibody that binds to human OX40. Antibodies suitable for
use in the methods of the invention include antibodies that bind to
human OX40. Descriptions of anti-OX40 antibodies (e.g., anti-human
OX40 agonist antibodies) may be found in US PG Pub. No.
US2015/0307617 and International Publication No. WO/2015/153513,
which are each incorporated by reference herein in their
entirety.
[0143] In some embodiments, the anti-human OX40 agonist antibody
binds human OX40 with an affinity of less than or equal to about
0.45 nM. In some embodiments, the anti-human OX40 antibody binds
human OX40 with an affinity of less than or equal to about 0.4 nM.
In some embodiments, the anti-human OX40 antibody binds human OX40
with an affinity of less than or equal to about 0.5 nM. In some
embodiments, the binding affinity is determined using
radioimmunoassay.
[0144] In some embodiments, the anti-human OX40 agonist antibody
binds human OX40 and cynomolgus OX40. In some embodiments, binding
is determined using a FACS assay. In some embodiments, binding to
human OX40 has an EC50 of about 0.2 ug/ml. In some embodiments,
binding to human OX40 has an EC50 of about 0.3 ug/ml or lower. In
some embodiments, binding to cynomolgus OX40 has an EC50 of about
1.5 ug/ml. In some embodiments, binding to cynomolgus OX40 has an
EC50 of about 1.4 ug/ml.
[0145] In some embodiments, the anti-human OX40 agonist antibody
does not bind to rat OX40 or mouse OX40.
[0146] In some embodiments, the anti-human OX40 agonist antibody is
a depleting anti-human OX40 antibody (e.g., depletes cells that
express human OX40). In some embodiments, the human OX40 expressing
cells are CD4+ effector T cells. In some embodiments, the human
OX40 expressing cells are Treg cells. In some embodiments,
depleting is by ADCC and/or phagocytosis. In some embodiments, the
antibody mediates ADCC by binding Fc.gamma.R expressed by a human
effector cell and activating the human effector cell function. In
some embodiments, the antibody mediates phagocytosis by binding
Fc.gamma.R expressed by a human effector cell and activating the
human effector cell function. Exemplary human effector cells
include, e.g., macrophage, natural killer (NK) cells, monocytes,
neutrophils. In some embodiments, the human effector cell is
macrophage. In some embodiments, the human effector cell is NK
cells. In some embodiments, depletion is not by apoptosis.
[0147] In some embodiments, the anti-human OX40 agonist antibody
has a functional Fc region. In some embodiments, effector function
of a functional Fc region is ADCC. In some embodiments, effector
function of a functional Fc region is phagocytosis. In some
embodiments, effector function of a functional Fc region is ADCC
and phagocytosis. In some embodiments, the Fc region is human IgG1.
In some embodiments, the Fc region is human IgG4.
[0148] In some embodiments, the anti-human OX40 agonist antibody
does not induce apoptosis in OX40-expressing cells (e.g., Treg). In
some embodiments, apoptosis is assayed using an antibody
concentration of 30 ug/ml, e.g., by determining whether apoptosis
has occurred using annexin V and proprodium iodide stained
Treg.
[0149] In some embodiments, the anti-human OX40 agonist antibody
enhances CD4+ effector T cell function, for example, by increasing
CD4+ effector T cell proliferation and/or increasing gamma
interferon production by the CD4+ effector T cell (for example, as
compared to proliferation and/or cytokine production prior to
treatment with anti-human OX40 agonist antibody). In some
embodiments, the cytokine is gamma interferon. In some embodiments,
the anti-human OX40 agonist antibody increases number of
intratumoral (infiltrating) CD4+ effector T cells (e.g., total
number of CD4+ effector T cells, or e.g., percentage of CD4+ cells
in CD45+ cells), e.g., as compared to number of intratumoral
(infiltrating) CD4+ T cells prior to treatment with anti-human OX40
agonist antibody. In some embodiments, the anti-human OX40 agonist
antibody increases number of intratumoral (infiltrating) CD4+
effector T cells that express gamma interferon (e.g., total gamma
interferon expressing CD4+ cells, or e.g., percentage of gamma
interferon expressing CD4+ cells in total CD4+ cells), e.g., as
compared to number of intratumoral (infiltrating) CD4+ T cells that
express gamma interferon prior to treatment with anti-human OX40
agonist antibody.
[0150] In some embodiments, the anti-human OX40 agonist antibody
increases number of intratumoral (infiltrating) CD8+ effector T
cells (e.g., total number of CD8+ effector T cells, or e.g.,
percentage of CD8+ in CD45+ cells), e.g., as compared to number of
intratumoral (infiltrating) CD8+ T effector cells prior to
treatment with anti-human OX40 agonist antibody. In some
embodiments, the anti-human OX40 agonist antibody increases number
of intratumoral (infiltrating) CD8+ effector T cells that express
gamma interferon (e.g., percentage of CD8+ cells that express gamma
interferon in total CD8+ cells), e.g., compared to number of
intratumoral (infiltrating) CD8+ T cells that express gamma
interferon prior to treatment with anti-human OX40 agonist
antibody.
[0151] In some embodiments, the anti-human OX40 agonist antibody
enhances memory T cell function, for example by increasing memory T
cell proliferation and/or increasing cytokine production by the
memory cell. In some embodiments, the cytokine is gamma
interferon.
[0152] In some embodiments, the anti-human OX40 agonist antibody
inhibits Treg function, for example, by decreasing Treg suppression
of effector T cell function (e.g., effector T cell proliferation
and/or effector T cell cytokine secretion). In some embodiments,
the effector T cell is a CD4+ effector T cell. In some embodiments,
the anti-human OX40 agonist antibody reduces the number of
intratumoral (infiltrating) Treg (e.g., total number of Treg or
e.g., percentage of Fox3p+ cells in CD4+ cells).
[0153] In some embodiments, the anti-human OX40 agonist antibody is
engineered to increase effector function (e.g., compared to
effector function in a wild-type IgG1). In some embodiments, the
antibody has increased binding to a Fc.gamma. receptor. In some
embodiments, the antibody lacks fucose attached (directly or
indirectly) to the Fc region. For example, the amount of fucose in
such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65%
or from 20% to 40%. In some embodiments, the Fc region comprises
bisected oligosaccharides, e.g., in which a biantennary
oligosaccharide attached to the Fc region of the antibody is
bisected by GlcNAc. In some embodiments, the antibody comprises an
Fc region with one or more amino acid substitutions which improve
ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the
Fc region (EU numbering of residues).
[0154] In some embodiments, the anti-human OX40 agonist antibody
increases OX40 signal transduction in a target cell that expresses
OX40. In some embodiments, OX40 signal transduction is detected by
monitoring NFkB downstream signaling.
[0155] In some embodiments, the anti-human OX40 agonist antibody is
stable after treatment at 40 C for two weeks.
[0156] In some embodiments, the anti-human OX40 agonist antibody
binds human effector cells, e.g., binds Fc.gamma.R (e.g., an
activating Fc.gamma.R) expressed by human effector cells. In some
embodiments, the human effector cell performs (is capable of
performing) ADCC effector function. In some embodiments, the human
effector cell performs (is capable of performing) phagocytosis
effector function.
[0157] In some embodiments, the anti-human OX40 agonist antibody
comprising a variant IgG1 Fc polypeptide comprising a mutation that
eliminates binding to human effector cells (e.g., a DANA mutation)
has diminished activity (e.g., CD4+ effector T cell function, e.g.,
proliferation), relative to anti-human OX40 agonist antibody
comprising native sequence IgG1 Fc portion. In some embodiment, the
anti-human OX40 agonist antibody comprising a variant IgG1 Fc
polypeptide comprising a mutation that eliminates binding to human
effector cells (e.g., a DANA mutation) does not possess substantial
activity (e.g., CD4+ effector T cell function, e.g.,
proliferation).
[0158] In some embodiments, antibody cross-linking is required for
anti-human OX40 agonist antibody function. In some embodiments,
function is stimulation of CD4+ effector T cell proliferation. In
some embodiments, antibody cross-linking is determined by providing
anti-human OX40 agonist antibody adhered on a solid surface (e.g.,
a cell culture plate). In some embodiments, antibody cross-linking
is determined by introducing a mutation in the antibody's IgG1 Fc
portion (e.g., a DANA mutation) and testing function of the mutant
antibody.
[0159] In some embodiments, the anti-human OX40 agonist antibody
competes for binding to human OX40 with OX40L. In some embodiments,
addition of OX40L does not enhance anti-human OX40 antibody
function in an in vitro assay.
[0160] According to another embodiment, the anti-human OX40 agonist
antibodies include any one, any combination, or all of the
following properties: (1) binds human OX40 with an affinity of less
than or equal to about 0.45 nM, in some embodiments, binds human
OX40 with an affinity of less than or equal to about 0.4 nM, in
some embodiments, binds human OX40 with an affinity of less than or
equal to about 0.5 nM, in some embodiments, the binding affinity is
determined using radioimmunoassay; (2) binds human OX40 and
cynomolgus OX40, in some embodiments, binding is determined using a
FACS assay, (3) binds human OX40 with an EC50 of about 0.2 ug/ml,
in some embodiments, binds to human OX40 has an EC50 of about 0.3
ug/ml or lower, in some embodiments, binds to cynomolgus OX40 with
an EC50 of about 1.5 ug/ml, in some embodiments, binds to
cynomolgus OX40 has an EC50 of about 1.4 ug/ml, (4) does not
substantially bind to rat OX40 or mouse OX40, (6) is a depleting
anti-human OX40 antibody (e.g., depletes cells that express human
OX40), in some embodiments, the cells are CD4+ effector T cells
and/or Treg cells, (7) enhances CD4+ effector T cell function, for
example, by increasing CD4+ effector T cell proliferation and/or
increasing gamma interferon production by the CD4+ effector T cell
(for example, as compared to proliferation and/or cytokine
production prior to treatment with anti-human OX40 agonist
antibody), (8) enhances memory T cell function, for example by
increasing memory T cell proliferation and/or increasing cytokine
production by the memory cell, (9) inhibits Treg function, for
example, by decreasing Treg suppression of effector T cell function
(e.g., effector T cell proliferation and/or effector T cell
cytokine secretion). In some embodiments, the effector T cell is a
CD4+ effector T cell, (10) increases OX40 signal transduction in a
target cell that expresses OX40 (in some embodiments, OX40 signal
transduction is detected by monitoring NFkB downstream signaling),
(11) is stable after treatment at 40 C for two weeks, (12) binds
human effector cells, e.g., binds Fc.gamma.R expressed by human
effector cells, (13) anti-human OX40 agonist antibody comprising a
variant IgG1 Fc polypeptide comprising a mutation that eliminates
binding to human effector cells (e.g., N297G) has diminished
activity (e.g., CD4+ effector T cell function, e.g.,
proliferation), relative to anti-human OX40 agonist antibody
comprising native sequence IgG1 Fc portion, in some embodiment, the
anti-human OX40 agonist antibody comprising a variant IgG1 Fc
polypeptide comprising a mutation that eliminates binding to human
effector cells (e.g., N297G) does not possess substantial activity
(e.g., CD4+ effector T cell function, e.g., proliferation), (14)
antibody cross-linking (e.g., by Fc receptor binding) is required
for anti-human OX40 agonist antibody function.
[0161] In one aspect, the invention provides an anti-human OX40
agonist antibody comprising at least one, two, three, four, five,
or six HVRs selected from (a) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:2; (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:3; (c) HVR-H3 comprising the amino acid
sequence of SEQ ID NO:4; (d) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6; and (f) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:7.
[0162] In one aspect, the invention provides an anti-human OX40
agonist antibody comprising at least one, at least two, or all
three VH HVR sequences selected from (a) HVR-H1 comprising the
amino acid sequence of SEQ ID NO:2; (b) HVR-H2 comprising the amino
acid sequence of SEQ ID NO:3; and (c) HVR-H3 comprising the amino
acid sequence of SEQ ID NO:4. In one embodiment, the antibody
comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:4.
In another embodiment, the antibody comprises HVR-H3 comprising the
amino acid sequence of SEQ ID NO:4 and HVR-L3 comprising the amino
acid sequence of SEQ ID NO:7. In a further embodiment, the antibody
comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:4,
HVR-L3 comprising the amino acid sequence of SEQ ID NO:7, and
HVR-H2 comprising the amino acid sequence of SEQ ID NO:3. In a
further embodiment, the antibody comprises (a) HVR-H1 comprising
the amino acid sequence of SEQ ID NO:2; (b) HVR-H2 comprising the
amino acid sequence of SEQ ID NO:3; and (c) HVR-H3 comprising the
amino acid sequence of SEQ ID NO:4.
[0163] In another aspect, the invention provides an anti-human OX40
agonist antibody comprising at least one, at least two, or all
three VL HVR sequences selected from (a) HVR-L1 comprising the
amino acid sequence of SEQ ID NO:5; (b) HVR-L2 comprising the amino
acid sequence of SEQ ID NO:6; and (c) HVR-L3 comprising the amino
acid sequence of SEQ ID NO:7. In one embodiment, the antibody
comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:5; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6;
and (c) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:7.
[0164] In another aspect, an anti-human OX40 agonist antibody of
the invention comprises (a) a VH domain comprising at least one, at
least two, or all three VH HVR sequences selected from (i) HVR-H1
comprising the amino acid sequence of SEQ ID NO:2, (ii) HVR-H2
comprising the amino acid sequence of SEQ ID NO:3, and (iii) HVR-H3
comprising an amino acid sequence selected from SEQ ID NO:4; and
(b) a VL domain comprising at least one, at least two, or all three
VL HVR sequences selected from (i) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:5, (ii) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6, and (c) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:7.
[0165] In another aspect, the invention provides an anti-human OX40
agonist antibody comprising (a) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:2; (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:3; (c) HVR-H3 comprising the amino acid
sequence of SEQ ID NO:4; (d) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6; and (f) HVR-L3 comprising an amino acid
sequence selected from SEQ ID NO:7.
[0166] In one aspect, the invention provides an anti-human OX40
agonist antibody comprising at least one, two, three, four, five,
or six HVRs selected from (a) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:2; (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:3; (c) HVR-H3 comprising the amino acid
sequence of SEQ ID NO:4; (d) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6; and (f) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:26.
[0167] In another embodiment, the antibody comprises HVR-H3
comprising the amino acid sequence of SEQ ID NO:4 and HVR-L3
comprising the amino acid sequence of SEQ ID NO:26. In a further
embodiment, the antibody comprises HVR-H3 comprising the amino acid
sequence of SEQ ID NO:4, HVR-L3 comprising the amino acid sequence
of SEQ ID NO:26, and HVR-H2 comprising the amino acid sequence of
SEQ ID NO:3.
[0168] In another aspect, an antibody of the invention comprises
(a) a VH domain comprising at least one, at least two, or all three
VH HVR sequences selected from (i) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:2, (ii) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:3, and (iii) HVR-H3 comprising an amino acid
sequence selected from SEQ ID NO:4; and (b) a VL domain comprising
at least one, at least two, or all three VL HVR sequences selected
from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5,
(ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, and
(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:26.
[0169] In another aspect, the invention provides an antibody
comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3;
(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; (d)
HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f)
HVR-L3 comprising an amino acid sequence selected from SEQ ID
NO:26.
[0170] In one aspect, the invention provides an anti-human OX40
agonist antibody comprising at least one, two, three, four, five,
or six HVRs selected from (a) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:2; (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:3; (c) HVR-H3 comprising the amino acid
sequence of SEQ ID NO:4; (d) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6; and (f) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:27.
[0171] In another embodiment, the antibody comprises HVR-H3
comprising the amino acid sequence of SEQ ID NO:4 and HVR-L3
comprising the amino acid sequence of SEQ ID NO:27. In a further
embodiment, the antibody comprises HVR-H3 comprising the amino acid
sequence of SEQ ID NO:4, HVR-L3 comprising the amino acid sequence
of SEQ ID NO:27, and HVR-H2 comprising the amino acid sequence of
SEQ ID NO:3.
[0172] In another aspect, an antibody of the invention comprises
(a) a VH domain comprising at least one, at least two, or all three
VH HVR sequences selected from (i) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:2, (ii) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:3, and (iii) HVR-H3 comprising an amino acid
sequence selected from SEQ ID NO:4; and (b) a VL domain comprising
at least one, at least two, or all three VL HVR sequences selected
from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5,
(ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6, and
(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:27.
[0173] In another aspect, the invention provides an antibody
comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:2; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:3;
(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:4; (d)
HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f)
HVR-L3 comprising an amino acid sequence selected from SEQ ID
NO:27.
[0174] In one aspect, the invention provides an anti-human OX40
agonist antibody comprising at least one, two, three, four, five,
or six HVRs selected from (a) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:2, 8 or 9; (b) HVR-H2 comprising the amino
acid sequence of SEQ ID NO:3, 10, 11, 12, 13 or 14; (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:4, 15, or 19; (d)
HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (e)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (f)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:7, 22, 23,
24, 25, 26, 27, or 28.
[0175] In one aspect, the invention provides an antibody comprising
at least one, at least two, or all three VH HVR sequences selected
from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO: 2,
8 or 9; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:
3, 10, 11, 12, 13 or 14; and (c) HVR-H3 comprising the amino acid
sequence of SEQ ID NO: 4, 15, or 19. In one embodiment, the
antibody comprises HVR-H3 comprising the amino acid sequence of SEQ
ID NO: 4, 15, or 19. In another embodiment, the antibody comprises
HVR-H3 comprising the amino acid sequence of SEQ ID NO:4, 15, or 19
and HVR-L3 comprising the amino acid sequence of SEQ ID NO: 7, 22,
23, 24, 25, 26, 27, or 28. In a further embodiment, the antibody
comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:
4, 15, or 19, HVR-L3 comprising the amino acid sequence of SEQ ID
NO: 7, 22, 23, 24, 25, 26, 27, or 28, and HVR-H2 comprising the
amino acid sequence of SEQ ID NO: 3, 10, 11, 12, 13 or 14. In a
further embodiment, the antibody comprises (a) HVR-H1 comprising
the amino acid sequence of SEQ ID NO: 2, 8 or 9; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO: 3, 10, 11, 12, 13
or 14; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO: 4, 15, or 19.
[0176] In another aspect, the invention provides an antibody
comprising at least one, at least two, or all three VL HVR
sequences selected from (a) HVR-L1 comprising the amino acid
sequence of SEQ ID NO: 5; (b) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6; and (c) HVR-L3 comprising the amino acid
sequence of SEQ ID NO: 7, 22, 23, 24, 25, 26, 27, or 28. In one
embodiment, the antibody comprises (a) HVR-L1 comprising the amino
acid sequence of SEQ ID NO:5; (b) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6; and (c) HVR-L3 comprising the amino acid
sequence of SEQ ID NO: 7, 22, 23, 24, 25, 26, 27, or 28.
[0177] In another aspect, an antibody of the invention comprises
(a) a VH domain comprising at least one, at least two, or all three
VH HVR sequences selected from (i) HVR-H1 comprising the amino acid
sequence of SEQ ID NO: 2, 8 or 9, (ii) HVR-H2 comprising the amino
acid sequence of SEQ ID NO: 3, 10, 11, 12, 13 or 14, and (iii)
HVR-H3 comprising an amino acid sequence selected from SEQ ID NO:
4, 15, or 19; and (b) a VL domain comprising at least one, at least
two, or all three VL HVR sequences selected from (i) HVR-L1
comprising the amino acid sequence of SEQ ID NO:5, (ii) HVR-L2
comprising the amino acid sequence of SEQ ID NO:6, and (c) HVR-L3
comprising the amino acid sequence of SEQ ID NO: 7, 22, 23, 24, 25,
26, 27, or 28.
[0178] In another aspect, the invention provides an antibody
comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO: 2, 8 or 9; (b) HVR-H2 comprising the amino acid sequence of SEQ
ID NO: 3, 10, 11, 12, 13 or 14; (c) HVR-H3 comprising the amino
acid sequence of SEQ ID NO: 4, 15, or 19; (d) HVR-L1 comprising the
amino acid sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino
acid sequence of SEQ ID NO:6; and (f) HVR-L3 comprising an amino
acid sequence selected from SEQ ID NO: 7, 22, 23, 24, 25, 26, 27,
or 28.
[0179] In one aspect, the invention provides an anti-human OX40
agonist antibody comprising at least one, two, three, four, five,
or six HVRs selected from (a) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:172; (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:173; (c) HVR-H3 comprising the amino acid
sequence of SEQ ID NO:174; (d) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:5; (e) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6; and (f) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:175. In some embodiment, HVR-H2 is not
DMYPDAAAASYNQKFRE (SEQ ID NO:222). In some embodiments, HVR-H3 is
not APRWAAAA (SEQ ID NO:223). In some embodiments, HVR-L3 is not
QAAAAAAAT (SEQ ID NO:224).
[0180] In one aspect, the invention provides an antibody comprising
at least one, at least two, or all three VH HVR sequences selected
from (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:172; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:173; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:174. In one embodiment, the antibody comprises HVR-H3 comprising
the amino acid sequence of SEQ ID NO:174. In another embodiment,
the antibody comprises HVR-H3 comprising the amino acid sequence of
SEQ ID NO:174 and HVR-L3 comprising the amino acid sequence of SEQ
ID NO:175. In a further embodiment, the antibody comprises HVR-H3
comprising the amino acid sequence of SEQ ID NO:174, HVR-L3
comprising the amino acid sequence of SEQ ID NO:175, and HVR-H2
comprising the amino acid sequence of SEQ ID NO:173. In a further
embodiment, the antibody comprises (a) HVR-H1 comprising the amino
acid sequence of SEQ ID NO:172; (b) HVR-H2 comprising the amino
acid sequence of SEQ ID NO:173; and (c) HVR-H3 comprising the amino
acid sequence of SEQ ID NO:174. In some embodiment, HVR-H2 is not
DMYPDAAAASYNQKFRE (SEQ ID NO:222). In some embodiments, HVR-H3 is
not APRWAAAA (SEQ ID NO:223). In some embodiments, HVR-L3 is not
QAAAAAAAT (SEQ ID NO:224).
[0181] In another aspect, the invention provides an antibody
comprising (a) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:5; (b) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6;
and (c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:175.
In some embodiments, HVR-L3 is not QAAAAAAAT (SEQ ID NO:224).
[0182] In another aspect, an antibody of the invention comprises
(a) a VH domain comprising at least one, at least two, or all three
VH HVR sequences selected from (i) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:172, (ii) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:173, and (iii) HVR-H3 comprising an amino
acid sequence selected from SEQ ID NO:174; and (b) a VL domain
comprising at least one, at least two, or all three VL HVR
sequences selected from (i) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:5, (ii) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:6, and (c) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:175.
[0183] In another aspect, the invention provides an antibody
comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:172; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:173; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:174; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:5; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:6;
and (f) HVR-L3 comprising an amino acid sequence selected from SEQ
ID NO:175. In some embodiment, HVR-H2 is not DMYPDAAAASYNQKFRE (SEQ
ID NO:222). In some embodiments, HVR-H3 is not APRWAAAA (SEQ ID
NO:223). In some embodiments, HVR-L3 is not QAAAAAAAT (SEQ ID
NO:224).
[0184] All possible combinations of the above substitutions are
encompassed by the consensus sequences of SEQ ID NO:172, 173, 174
and 175.
[0185] In one aspect, the invention provides an anti-human OX40
agonist antibody comprising at least one, two, three, four, five,
or six HVRs selected from (a) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:30; (c) HVR-H3 comprising the amino acid
sequence of SEQ ID NO:33; (d) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:37; (e) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:39; and (f) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:42.
[0186] In one aspect, the invention provides an antibody comprising
at least one, at least two, or all three VH HVR sequences selected
from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29;
(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:30; and
(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33. In
one embodiment, the antibody comprises HVR-H3 comprising the amino
acid sequence of SEQ ID NO:33. In another embodiment, the antibody
comprises HVR-H3 comprising the amino acid sequence of SEQ ID NO:33
and HVR-L3 comprising the amino acid sequence of SEQ ID NO:42. In a
further embodiment, the antibody comprises HVR-H3 comprising the
amino acid sequence of SEQ ID NO:33, HVR-L3 comprising the amino
acid sequence of SEQ ID NO:42, and HVR-H2 comprising the amino acid
sequence of SEQ ID NO:30. In a further embodiment, the antibody
comprises (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:30; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:33.
[0187] In another aspect, the invention provides an antibody
comprising at least one, at least two, or all three VL HVR
sequences selected from (a) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:37; (b) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:39; and (c) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:42. In one embodiment, the antibody comprises
(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (b)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:39; and (c)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:42.
[0188] In another aspect, an antibody of the invention comprises
(a) a VH domain comprising at least one, at least two, or all three
VH HVR sequences selected from (i) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:29, (ii) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:30, and (iii) HVR-H3 comprising an amino acid
sequence selected from SEQ ID NO:33; and (b) a VL domain comprising
at least one, at least two, or all three VL HVR sequences selected
from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37,
(ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:39, and
(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:42.
[0189] In another aspect, the invention provides an antibody
comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:30; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:33; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:37; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:39; and (f) HVR-L3 comprising an amino acid sequence selected
from SEQ ID NO:42.
[0190] In one aspect, the invention provides an anti-human OX40
agonist antibody comprising at least one, two, three, four, five,
or six HVRs selected from (a) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:30; (c) HVR-H3 comprising the amino acid
sequence of SEQ ID NO:33; (d) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:37; (e) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:40; and (f) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:42.
[0191] In another aspect, the invention provides an antibody
comprising at least one, at least two, or all three VL HVR
sequences selected from (a) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:37; (b) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:40; and (c) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:42. In one embodiment, the antibody comprises
(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (b)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:40; and (c)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:42.
[0192] In another aspect, an antibody of the invention comprises
(a) a VH domain comprising at least one, at least two, or all three
VH HVR sequences selected from (i) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:29, (ii) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:30, and (iii) HVR-H3 comprising an amino acid
sequence selected from SEQ ID NO:33; and (b) a VL domain comprising
at least one, at least two, or all three VL HVR sequences selected
from (i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37,
(ii) HVR-L2 comprising the amino acid sequence of SEQ ID NO:40, and
(c) HVR-L3 comprising the amino acid sequence of SEQ ID NO:42.
[0193] In another aspect, the invention provides an antibody
comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:30; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:33; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:37; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:40; and (f) HVR-L3 comprising an amino acid sequence selected
from SEQ ID NO:42.
[0194] In one aspect, the invention provides an anti-human OX40
agonist antibody comprising at least one, two, three, four, five,
or six HVRs selected from (a) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:30, 31, or 32; (c) HVR-H3 comprising the
amino acid sequence of SEQ ID NO:33; (d) HVR-L1 comprising the
amino acid sequence of SEQ ID NO:37; (e) HVR-L2 comprising the
amino acid sequence of SEQ ID NO:39, 40 or 41; and (f) HVR-L3
comprising the amino acid sequence of SEQ ID NO:42, 43, or 44.
[0195] In one aspect, the invention provides an antibody comprising
at least one, at least two, or all three VH HVR sequences selected
from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29;
(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO: 30, 31,
or 32; and (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:33. In another embodiment, the antibody comprises HVR-H3
comprising the amino acid sequence of SEQ ID NO:33 and HVR-L3
comprising the amino acid sequence of SEQ ID NO: 42, 43, or 44. In
a further embodiment, the antibody comprises HVR-H3 comprising the
amino acid sequence of SEQ ID NO:33, HVR-L3 comprising the amino
acid sequence of SEQ ID NO: 42, 43, or 44, and HVR-H2 comprising
the amino acid sequence of SEQ ID NO: 39, 40 or 41. In a further
embodiment, the antibody comprises (a) HVR-H1 comprising the amino
acid sequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:30, 31, or 32; and (c) HVR-H3 comprising the
amino acid sequence of SEQ ID NO:33.
[0196] In another aspect, the invention provides an antibody
comprising at least one, at least two, or all three VL HVR
sequences selected from (a) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:37; (b) HVR-L2 comprising the amino acid
sequence of SEQ ID NO: 39, 40 or 41; and (c) HVR-L3 comprising the
amino acid sequence of SEQ ID NO: 42, 43, or 44. In one embodiment,
the antibody comprises (a) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:37; (b) HVR-L2 comprising the amino acid
sequence of SEQ ID NO: 39, 40 or 41; and (c) HVR-L3 comprising the
amino acid sequence of SEQ ID NO: 42, 43, or 44.
[0197] In another aspect, an antibody of the invention comprises
(a) a VH domain comprising at least one, at least two, or all three
VH HVR sequences selected from (i) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:29, (ii) HVR-H2 comprising the amino acid
sequence of SEQ ID NO: 30, 31, or 32, and (iii) HVR-H3 comprising
an amino acid sequence selected from SEQ ID NO:33; and (b) a VL
domain comprising at least one, at least two, or all three VL HVR
sequences selected from (i) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:37, (ii) HVR-L2 comprising the amino acid
sequence of SEQ ID NO: 39, 40 or 41, and (c) HVR-L3 comprising the
amino acid sequence of SEQ ID NO: 42, 43, or 44.
[0198] In another aspect, the invention provides an antibody
comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:
30, 31, or 32; (c) HVR-H3 comprising the amino acid sequence of SEQ
ID NO:33; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:37; (e) HVR-L2 comprising the amino acid sequence of SEQ ID NO:
39, 40 or 41; and (f) HVR-L3 comprising an amino acid sequence
selected from SEQ ID NO: 42, 43, or 44.
[0199] In one aspect, the invention provides an anti-human OX40
agonist antibody comprising at least one, two, three, four, five,
or six HVRs selected from (a) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:29; (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:175; (c) HVR-H3 comprising the amino acid
sequence of SEQ ID NO:33; (d) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:37; (e) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:177; and (f) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:178.
[0200] In one aspect, the invention provides an antibody comprising
at least one, at least two, or all three VH HVR sequences selected
from (a) HVR-H1 comprising the amino acid sequence of SEQ ID NO:29;
(b) HVR-H2 comprising the amino acid sequence of SEQ ID NO:175; and
(c) HVR-H3 comprising the amino acid sequence of SEQ ID NO:33. In
another embodiment, the antibody comprises HVR-H3 comprising the
amino acid sequence of SEQ ID NO:33 and HVR-L3 comprising the amino
acid sequence of SEQ ID NO:177. In a further embodiment, the
antibody comprises HVR-H3 comprising the amino acid sequence of SEQ
ID NO:33, HVR-L3 comprising the amino acid sequence of SEQ ID
NO:178, and HVR-H2 comprising the amino acid sequence of SEQ ID
NO:176. In a further embodiment, the antibody comprises (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:29; (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:176; and (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:33.
[0201] In another aspect, the invention provides an antibody
comprising at least one, at least two, or all three VL HVR
sequences selected from (a) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:37; (b) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:177; and (c) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:177. In one embodiment, the antibody
comprises (a) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:37; (b) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:177; and (c) HVR-L3 comprising the amino acid sequence of SEQ ID
NO:178.
[0202] In another aspect, an antibody of the invention comprises
(a) a VH domain comprising at least one, at least two, or all three
VH HVR sequences selected from (i) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:29, (ii) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:176, and (iii) HVR-H3 comprising an amino
acid sequence selected from SEQ ID NO:33; and (b) a VL domain
comprising at least one, at least two, or all three VL HVR
sequences selected from (i) HVR-L1 comprising the amino acid
sequence of SEQ ID NO:37, (ii) HVR-L2 comprising the amino acid
sequence of SEQ ID NO:177, and (c) HVR-L3 comprising the amino acid
sequence of SEQ ID NO:178.
[0203] In another aspect, the invention provides an antibody
comprising (a) HVR-H1 comprising the amino acid sequence of SEQ ID
NO:29; (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:176; (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:33; (d) HVR-L1 comprising the amino acid sequence of SEQ ID
NO:37; (e) HVR-L2 comprising the amino acid sequence of SEQ ID
NO:177; and (f) HVR-L3 comprising an amino acid sequence selected
from SEQ ID NO:178.
[0204] In any of the above embodiments, an anti-OX40 agonist
antibody is humanized. In one embodiment, an anti-OX40 antibody
comprises HVRs as in any of the above embodiments or for any of the
embodiments in FIG. 11, and further comprises an acceptor human
framework, e.g. a human immunoglobulin framework or a human
consensus framework. In another embodiment, an anti-OX40 antibody
comprises HVRs as in any of the above embodiments, and further
comprises a VH and/or VL comprising an FR sequence shown in FIG.
11.
[0205] In another aspect, an anti-human OX40 agonist antibody
comprises a heavy chain variable domain (VH) sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO:56, 58,
60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92,
94, 96, 98, 100, 108, 114 or 116. In certain embodiments, a VH
sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% identity contains substitutions (e.g., conservative
substitutions), insertions, or deletions relative to the reference
sequence, but an anti-human OX40 agonist antibody comprising that
sequence retains the ability to bind to OX40. In certain
embodiments, a total of 1 to 10 amino acids have been substituted,
inserted and/or deleted in SEQ ID NO:56, 58, 60, 62, 64, 66, 68,
70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100,
108, 114 or 116. In certain embodiments, substitutions, insertions,
or deletions occur in regions outside the HVRs (i.e., in the FRs).
Optionally, the anti-human OX40 agonist antibody comprises the VH
sequence in SEQ ID NO: SEQ ID NO:56, 58, 60, 62, 64, 66, 68, 70,
72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 108,
114 or 116, including post-translational modifications of that
sequence. In a particular embodiment, the VH comprises one, two or
three HVRs selected from: (a) HVR-H1 comprising the amino acid
sequence of SEQ ID NO:2, (b) HVR-H2 comprising the amino acid
sequence of SEQ ID NO:3, and (c) HVR-H3 comprising the amino acid
sequence of SEQ ID NO:4.
[0206] In another aspect, an anti-human OX40 agonist antibody is
provided, wherein the antibody comprises a light chain variable
domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to the amino acid sequence of
SEQ ID NO:57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83,
85, 87, 89, 91, 93, 95, 97, 99, 101, 109, 115 or 117. In certain
embodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g.,
conservative substitutions), insertions, or deletions relative to
the reference sequence, but an anti-human OX40 agonist antibody
comprising that sequence retains the ability to bind to OX40. In
certain embodiments, a total of 1 to 10 amino acids have been
substituted, inserted and/or deleted in SEQ ID NO: 57, 59, 61, 63,
65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97,
99, 101, 109, 115 or 117. In certain embodiments, the
substitutions, insertions, or deletions occur in regions outside
the HVRs (i.e., in the FRs). Optionally, the anti-human OX40
agonist antibody comprises the VL sequence in SEQ ID NO: 57, 59,
61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93,
95, 97, 99, 101, 109, 115 or 117, including post-translational
modifications of that sequence. In a particular embodiment, the VL
comprises one, two or three HVRs selected from (a) HVR-L1
comprising the amino acid sequence of SEQ ID NO:5; (b) HVR-L2
comprising the amino acid sequence of SEQ ID NO:6; and (c) HVR-L3
comprising the amino acid sequence of SEQ ID NO:7.
[0207] In another aspect, an anti-human OX40 agonist antibody
comprises a heavy chain variable domain (VH) sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO:56. In
certain embodiments, a VH sequence having at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the reference sequence, but an anti-human
OX40 agonist antibody comprising that sequence retains the ability
to bind to OX40. In certain embodiments, a total of 1 to 10 amino
acids have been substituted, inserted and/or deleted in SEQ ID
NO:56. In certain embodiments, substitutions, insertions, or
deletions occur in regions outside the HVRs (i.e., in the FRs).
Optionally, the anti-human OX40 agonist antibody comprises the VH
sequence in SEQ ID NO:56, including post-translational
modifications of that sequence. In a particular embodiment, the VH
comprises one, two or three HVRs selected from: (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:2, (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:3, and (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:4.
[0208] In another aspect, an anti-human OX40 agonist antibody is
provided, wherein the antibody comprises a light chain variable
domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to the amino acid sequence of
SEQ ID NO:57. In certain embodiments, a VL sequence having at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity
contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative to the reference sequence, but an
anti-human OX40 agonist antibody comprising that sequence retains
the ability to bind to OX40. In certain embodiments, a total of 1
to 10 amino acids have been substituted, inserted and/or deleted in
SEQ ID NO: 57. In certain embodiments, the substitutions,
insertions, or deletions occur in regions outside the HVRs (i.e.,
in the FRs). Optionally, the anti-human OX40 agonist antibody
comprises the VL sequence in SEQ ID NO: 57, including
post-translational modifications of that sequence. In a particular
embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (c)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:7.
[0209] In another aspect, an anti-human OX40 agonist antibody
comprises a heavy chain variable domain (VH) sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO:94. In
certain embodiments, a VH sequence having at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the reference sequence, but an anti-human
OX40 agonist antibody comprising that sequence retains the ability
to bind to OX40. In certain embodiments, a total of 1 to 10 amino
acids have been substituted, inserted and/or deleted in SEQ ID
NO:94. In certain embodiments, substitutions, insertions, or
deletions occur in regions outside the HVRs (i.e., in the FRs).
Optionally, the anti-human OX40 agonist antibody comprises the VH
sequence in SEQ ID NO:94, including post-translational
modifications of that sequence. In a particular embodiment, the VH
comprises one, two or three HVRs selected from: (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:2, (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:3, and (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:4.
[0210] In another aspect, an anti-human OX40 agonist antibody is
provided, wherein the antibody comprises a light chain variable
domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to the amino acid sequence of
SEQ ID NO:95. In certain embodiments, a VL sequence having at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity
contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative to the reference sequence, but an
anti-human OX40 agonist antibody comprising that sequence retains
the ability to bind to OX40. In certain embodiments, a total of 1
to 10 amino acids have been substituted, inserted and/or deleted in
SEQ ID NO:95. In certain embodiments, the substitutions,
insertions, or deletions occur in regions outside the HVRs (i.e.,
in the FRs). Optionally, the anti-human OX40 agonist antibody
comprises the VL sequence in SEQ ID NO:95, including
post-translational modifications of that sequence. In a particular
embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (c)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:26.
[0211] In another aspect, an anti-human OX40 agonist antibody
comprises a heavy chain variable domain (VH) sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO:96. In
certain embodiments, a VH sequence having at least 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains
substitutions (e.g., conservative substitutions), insertions, or
deletions relative to the reference sequence, but an anti-human
OX40 agonist antibody comprising that sequence retains the ability
to bind to OX40. In certain embodiments, a total of 1 to 10 amino
acids have been substituted, inserted and/or deleted in SEQ ID
NO:96. In certain embodiments, substitutions, insertions, or
deletions occur in regions outside the HVRs (i.e., in the FRs).
Optionally, the anti-human OX40 agonist antibody comprises the VH
sequence in SEQ ID NO:96, including post-translational
modifications of that sequence. In a particular embodiment, the VH
comprises one, two or three HVRs selected from: (a) HVR-H1
comprising the amino acid sequence of SEQ ID NO:2, (b) HVR-H2
comprising the amino acid sequence of SEQ ID NO:3, and (c) HVR-H3
comprising the amino acid sequence of SEQ ID NO:4.
[0212] In another aspect, an anti-human OX40 agonist antibody is
provided, wherein the antibody comprises a light chain variable
domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to the amino acid sequence of
SEQ ID NO:97. In certain embodiments, a VL sequence having at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity
contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative to the reference sequence, but an
anti-human OX40 agonist antibody comprising that sequence retains
the ability to bind to OX40. In certain embodiments, a total of 1
to 10 amino acids have been substituted, inserted and/or deleted in
SEQ ID NO:97. In certain embodiments, the substitutions,
insertions, or deletions occur in regions outside the HVRs (i.e.,
in the FRs). Optionally, the anti-human OX40 agonist antibody
comprises the VL sequence in SEQ ID NO:97, including
post-translational modifications of that sequence. In a particular
embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:5; (b)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:6; and (c)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:27.
[0213] In another aspect, an anti-human OX40 agonist antibody
comprises a heavy chain variable domain (VH) sequence having at
least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
sequence identity to the amino acid sequence of SEQ ID NO: 118,
120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144,
146, 148. In certain embodiments, a VH sequence having at least
90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity
contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative to the reference sequence, but an
anti-human OX40 agonist antibody comprising that sequence retains
the ability to bind to OX40. In certain embodiments, a total of 1
to 10 amino acids have been substituted, inserted and/or deleted in
SEQ ID NO: 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138,
140, 142, 144, 146, 148. In certain embodiments, substitutions,
insertions, or deletions occur in regions outside the HVRs (i.e.,
in the FRs). Optionally, the anti-human OX40 agonist antibody
comprises the VH sequence in SEQ ID NO: SEQ ID NO: 118, 120, 122,
124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148,
including post-translational modifications of that sequence. In a
particular embodiment, the VH comprises one, two or three HVRs
selected from: (a) HVR-H1 comprising the amino acid sequence of SEQ
ID NO: 29, (b) HVR-H2 comprising the amino acid sequence of SEQ ID
NO:30, and (c) HVR-H3 comprising the amino acid sequence of SEQ ID
NO:33.
[0214] In another aspect, an anti-human OX40 agonist antibody is
provided, wherein the antibody comprises a light chain variable
domain (VL) having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99%, or 100% sequence identity to the amino acid sequence of
SEQ ID NO: 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139,
141, 143, 145, 147, 149. In certain embodiments, a VL sequence
having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%
identity contains substitutions (e.g., conservative substitutions),
insertions, or deletions relative to the reference sequence, but an
anti-human OX40 agonist antibody comprising that sequence retains
the ability to bind to OX40. In certain embodiments, a total of 1
to 10 amino acids have been substituted, inserted and/or deleted in
SEQ ID NO: 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139,
141, 143, 145, 147, 149. In certain embodiments, the substitutions,
insertions, or deletions occur in regions outside the HVRs (i.e.,
in the FRs). Optionally, the anti-human OX40 agonist antibody
comprises the VL sequence in SEQ ID NO: 119, 121, 123, 125, 127,
129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, including
post-translational modifications of that sequence. In a particular
embodiment, the VL comprises one, two or three HVRs selected from
(a) HVR-L1 comprising the amino acid sequence of SEQ ID NO:37; (b)
HVR-L2 comprising the amino acid sequence of SEQ ID NO:39; and (c)
HVR-L3 comprising the amino acid sequence of SEQ ID NO:42.
[0215] In one embodiment, the antibody comprises the VH and VL
sequences in SEQ ID NO:56 and SEQ ID NO:57, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:58 and SEQ ID NO:59, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:60 and SEQ ID NO:61, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:62 and SEQ ID NO:63, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:64 and SEQ ID NO:65, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:66 and SEQ ID NO:67, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:68 and SEQ ID NO:69, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:70 and SEQ ID NO:71, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:72 and SEQ ID NO:73, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:74 and SEQ ID NO:75, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:76 and SEQ ID NO:77, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:78 and SEQ ID NO:79, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:80 and SEQ ID NO:81, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:82 and SEQ ID NO:83, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:84 and SEQ ID NO:85, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:86 and SEQ ID NO:87, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:88 and SEQ ID NO:89, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:90 and SEQ ID NO:91, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:92 and SEQ ID NO:93, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:94 and SEQ ID NO:95, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:96 and SEQ ID NO:97, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:98 and SEQ ID NO:99, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:100 and SEQ ID NO:101, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:108 and SEQ ID NO:109, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:114 and SEQ ID NO:115, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:116 and SEQ ID NO:117, respectively, including
post-translational modifications of those sequences.
[0216] In one embodiment, the antibody comprises the VH and VL
sequences in SEQ ID NO:118 and SEQ ID NO:119, respectively,
including post-translational modifications of those sequences. In
one embodiment, the antibody comprises the VH and VL sequences in
SEQ ID NO:120 and SEQ ID NO:121, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:122 and SEQ ID NO:123, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:124 and SEQ ID NO:125, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:126 and SEQ ID NO:127, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:128 and SEQ ID NO:129, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:130 and SEQ ID NO:131, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:132 and SEQ ID NO:133, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:134 and SEQ ID NO:135, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:136 and SEQ ID NO:137, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:138 and SEQ ID NO:139, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:140 and SEQ ID NO:141, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:142 and SEQ ID NO:143, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:144 and SEQ ID NO:145, respectively, including
post-translational modifications of those sequences. In one
embodiment, the antibody comprises the VH and VL sequences in SEQ
ID NO:146 and SEQ ID NO:147, respectively, including
post-translational modifications of those sequences.
[0217] In another aspect, an anti-human OX40 agonist antibody is
provided, wherein the antibody comprises a VH as in any of the
embodiments provided above, and a VL as in any of the embodiments
provided above.
[0218] In some embodiments, the OX40 agonist antibody is MEDI6469.
In some embodiments, the OX40 agonist antibody is MEDI0562.
[0219] In a further aspect, the invention provides an antibody that
binds to the same epitope as an anti-human OX40 antibody provided
herein. In some embodiments, the antibody is an anti-human OX40
agonist antibody.
[0220] In a further aspect of the invention, an anti-OX40 antibody
according to any of the above embodiments is a monoclonal antibody,
including a chimeric, humanized or human antibody. In one
embodiment, an anti-OX40 antibody is an antibody fragment, e.g., a
Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In another
embodiment, the antibody is a full length antibody, e.g., an intact
IgG1 antibody or other antibody class or isotype as defined herein.
In some embodiments, the antibody is a full length intact IgG4
antibody.
[0221] Exemplary amino acid sequences corresponding to OX40
polypeptides and OX40 antibodies are provided below.
TABLE-US-00003 TABLE 2 Amino acid sequences SEQ Name SEQUENCE ID
NO: Human OX40 LHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQ 1 (lacking
NTVCRPCGPGFYNDVVSSKPCKPCTWCNLRSG the signal
SERKQLCTATQDTVCRCRAGTQPLDSYKPGVD peptide)
CAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQ PASNSSDAICEDRDPPATQPQETQGPPARPIT
VQPTEAWPRTSQGPSTRPVEVPGGRAVAAILG LGLVLGLLGPLAILLALYLLRRDQRLPPDAHK
PPGGGSFRTPIQEEQADAHSTLAKI HVR-H1- DSYMS 2 1A7.gr.1 1A7.gr.2
1A7.gr.3 1A7.gr.4 1A7.gr.5 1A7.gr.6 1A7.gr.7 1A7.gr.NADS
1A7.gr.NADA 1A7.gr.NGDA 1A7.gr.SGDS 1A7.gr.NGSS 1A7.Ala.1 1A7.Ala.2
1A7.Ala.3 1A7.Ala.4 1A7.Ala.5 1A7.Ala.6 1A7.Ala.7 1A7.Ala.8
1A7.Ala.9 1A7.Ala.10 1A7.Ala.11 1A7.Ala.12 1A7.Ala.13 1A7.Ala.14
1A7.Ala.15 1A7.Ala.16 HVR-H2- DMYPDNGDSSYNQKFRE 3 1A7.gr.1 1A7.gr.2
1A7.gr.3 1A7.gr.4 1A7.gr.5 1A7.gr.6 1A7.gr.7 1A7.gr.DA 1A7.gr.ES
1A7.Ala.1 1A7.Ala.2 1A7.Ala.3 1A7.Ala.4 1A7.Ala.5 1A7.Ala.6
1A7.Ala.7 1A7.Ala.8 1A7.Ala.9 1A7.Ala.10 1A7.Ala.11 1A7.Ala.12
1A7.Ala.13 1A7.Ala.14 1A7.Ala.15 1A7.Ala.16 HVR-H3- APRWYFSV 4
1A7.gr.1 1A7.gr.2 1A7.gr.3 1A7.gr.4 1A7.gr.5 1A7.gr.6 1A7.gr.7
1A7.gr.DA 1A7.gr.ES 1A7.gr.NADS 1A7.gr.NADA 1A7.gr.NGDA 1A7.gr.SGDS
1A7.gr.NGSS 1A7.gr.DANA DA 1A7.Ala.1 1A7.Ala.2 1A7.Ala.3 1A7.Ala.4
1A7.Ala.5 1A7.Ala.6 1A7.Ala.7 1A7-Ala.15 1A7.Ala.16 HVR-L1-
RASQDISNYLN 5 1A7.gr.1 1A7.gr.2 1A7.gr.3 1A7.gr.4 1A7.gr.5 1A7.gr.6
1A7.gr.7 1A7.gr.DA 1A7.gr.ES 1A7.gr.NADS 1A7.gr.NADA 1A7.gr.NGDA
1A7.gr.SGDS 1A7.gr.NGSS 1A7.gr.DANA DA 1A7.Ala.1 1A7.Ala.2
1A7.Ala.3 1A7.Ala.4 1A7.Ala.5 1A7.Ala.6 1A7.Ala.7 1A7.Ala.8
1A7.Ala.9 1A7.Ala.10 1A7.Ala.11 1A7.Ala.12 1A7.Ala.13 1A7.Ala.14
1A7.Ala.15 1A7.Ala.16 HVR-L2- YTSRLRS 6 1A7.gr.1 1A7.gr.2 1A7.gr.3
1A7.gr.4 1A7.gr.5 1A7.gr.6 1A7.gr.7 1A7.gr.DA 1A7.gr.ES 1A7.gr.NADS
1A7.gr.NADA 1A7.gr.NGDA 1A7.gr.SGDS 1A7.gr.NGSS 1A7.gr.DANA DA
1A7.Ala.1 1A7.Ala.2 1A7.Ala.3 1A7.Ala.4 1A7.Ala.5 1A7.Ala.6
1A7.Ala.7 1A7.Ala.8 1A7.Ala.9 1A7.Ala.10 1A7.Ala.11 1A7.Ala.12
1A7.Ala.13 1A7.Ala.14 1A7.Ala.15 1A7.Ala.16 HVR-L3- QQGHTLPPT 7
1A7.gr.1 1A7.gr.2 1A7.gr.3 1A7.gr.4 1A7.gr.5 1A7.gr.6 1A7.gr.7
1A7.gr.DA 1A7.gr.ES 1A7.gr.NADS 1A7.gr.NADA 1A7.gr.NGDA 1A7.gr.SGDS
1A7.gr.NGSS 1A7.gr.DANA DA 1A7.Ala.8 1A7.Ala.9 1A7.Ala.10
1A7.Ala.11 1A7.Ala.12 1A7.Ala.13 1A7.Ala.14 1A7.Ala.15 1A7.Ala.16
HVR-H1- DAYMS 8 1A7.gr.DA HVR-H1- ESYMS 9 1A7.gr.ES 1A7.gr.DANA DA
HVR-H2- DMYPDNADSSYNQKFRE 10 1A7.gr.NADS HVR-H2- DMYPDNADASYNQKFRE
11 1A7.gr.NADA 1A7.gr.DANA DA HVR-H2- DMYPDNGDASYNQKFRE 12
1A7.gr.NGDA HVR-H2- DMYPDSGDSSYNQKFRE 13 1A7.gr.SGDS HVR-H2-
DMYPDNGSSSYNQKFRE 14 1A7.gr.NGSS HVR-H3- APRWYFSA 15 1A7.Ala.8
HVR-H3- APRWYASV 16 1A7.Ala.9 HVR-H3- APRWAFSV 17 1A7.Ala.10
HVR-H3- APAWYFSV 18 1A7.Ala.11 HVR-H3- APRWYFAV 19 1A7.Ala.12
HVR-H3- APRAYFSV 20 1A7.Ala.13 HVR-H3- AARWYFSV 21 1A7.Ala.14
HVR-L3- QQGHTLPAT 22 1A7.Ala.1 HVR-L3- QQGHTAPPT 23 1A7.Ala.2
HVR-L3- QQGATLPPT 24 1A7.Ala.3 HVR-L3- QQGHALPPT 25
1A7.Ala.4 HVR-L3- QQAHTLPPT 26 1A7.Ala.5 HVR-L3- QQGHTLAPT 27
1A7.Ala.6 HVR-L3- QAGHTLPPT 28 1A7.Ala.7 HVR-H1- NYLIE 29 3C8.gr.1
3C8.gr.2 3C8.gr.3 3C8.gr.4 3C8.gr.5 3C8.gr.5.SG 3C8.gr.5.EG
3C8.gr.5.QG 3C9.gr.5.DQ 3C8.gr.5.DA 3C8.gr.6 3C8.gr.7 3C8.gr.8
3C8.gr.9 3C8.gr.10 3C8.gr.11 3C8.A.1 3C8.A.2 3C8.A.3 3C8.A.4
3C8.A.5 3C8.A.6 3C8.A.7 3C8.A.8 3C8.A.9 3C8.A.10 HVR-H2-
VINPGSGDTYYSEKFKG 30 3C8.gr.1 3C8.gr.2 3C8.gr.3 3C8.gr.4 3C8.gr.5
3C8.gr.5.SG 3C8.gr.5.EG 3C8.gr.5.QG 3C8.gr.6 3C8.gr.7 3C8.gr.8
3C8.gr.9 3C8.gr.10 3C8.gr.11 3C8.A.1 3C8.A.2 3C8.A.3 3C8.A.4
3C8.A.5 3C8.A.6 3C8.A.7 3C8.A.8 3C8.A.9 3C8.A.10 HVR-H2-
VINPGSGDAYYSEKFKG 31 3C8.gr.5.DA HVR-H2- VINPGSGDQYYSEKFKG 32
3C8.gr.5.DQ HVR-H3- DRLDY 33 3C8.gr.1 3C8.gr.2 3C8.gr.3 3C8.gr.4
3C8.gr.5 3C8.gr.5.SG 3C8.gr.5.EG 3C8.gr.5.QG 3C8.gr.5.DA
3C8.gr.5.DQ 3C8.gr.6 3C8.gr.7 3C8.gr.8 3C8.gr.9 3C8.gr.10 3C8.gr.11
3C8.A.1 3C8.A.2 3C8.A.3 3C8.A.4 3C8.A.5 3C8.A.6 3C8.A.7 HVR-H3-
ARLDY 34 3C8.A.8 HVR-H3- DALDY 35 3C8.A.9 HVR-H3- DRADY 36 3C8.A.10
HVR-L1- HASQDISSYIV 37 3C8.gr.1 3C8.gr.2 3C8.gr.3 3C8.gr.4 3C8.gr.5
3C8.gr.5.SG 3C8.gr.5.EG 3C8.gr.5.QG 3C8.gr.5.DA 3C8.gr.5.DQ
3C8.gr.6 3C8.gr.7 3C8.gr.8 3C8.gr.9 3C8.gr.10 3C8.gr.11 3C8.A.1
3C8.A.2 3C8.A.3 3C8.A.4 3C8.A.5 3C8.A.6 3C8.A.7 3C8.A.8 3C8.A.9
3C8.A.10 HVR-L2- HGTNLED 38 3C8.gr.1 3C8.gr.2 3C8.gr.3 3C8.gr.4
3C8.gr.5 3C8.gr.5.DA 3C8.gr.5.DQ 3C8.gr.6 3C8.gr.7 3C8.gr.8
3C8.gr.9 3C8.gr.10 3C8.gr.11 3C8.A.1 3C8.A.2 3C8.A.3 3C8.A.4
3C8.A.5 3C8.A.6 3C8.A.7 3C8.A.8 3C8.A.9 3C8.A.10 HVR-L2- HGTNLES 39
3C8.gr5.SG HVR-L2- HGTNLEE 40 3C8.gr.5.EG HVR-L2- HGTNLEQ 41
3C8.gr.5.QG HVR-L3 VHYAQFPYT 42 3C8.gr.1 3C8.gr.2 3C8.gr.3 3C8.gr.4
3C8.gr.5 3C8.gr.5.SG 3C8.gr.5.EG 3C8.gr.5.QG 3C8.gr.5.DA
3C8.gr.5.DQ 3C8.gr.6 3C8.gr.7 3C8.gr.8 3C8.gr.9 3C8.gr.10 3C8.gr.11
3C8.A.8 3C8.A.9 3C8.A.10 HVR-L3- AHYAQFPYT 43 3C8.A.1 HVR-L3-
VAYAQFPYT 44 3C8.A.2 HVR-L3- VHAAQFPYT 45 3C8.A.3 HVR-L3- VHYAAFPYT
46 3C8.A.4 HVR-L3- VHYAQAPYT 47 3C8.A.5 HVR-L3- VHYAQFAYT 48
3C8.A.6 HVR-L3- VHYAQFPAT 49 3C8.A.7 HVR-H1- DYGVL 50 1D2.gr.1
1D2.gr.2 1D2.gr.3 HVR-H2- MIWSGGTTDYNAAFIS 51 1D2.gr.1 1D2.gr.2
1D2.gr.3 HVR-H3- EEMDY 52 1D2.gr.1 1D2.gr.2 1D2.gr.3 HVR-L1-
RASQDISNFLN 53 1D2.gr.1 1D2.gr.2 1D2.gr.3 HVR-L2- YTSRLHS 54
1D2.gr.1 1D2.gr.2 1D2.gr.3 HVR-L3- QQGNTLPWT 55 1D2.gr.1 1D2.gr.2
1D2.gr.3 1A7.gr.1 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 56 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKF RERVTITRDTSTSTAYLELSSLRSEDTAVYYC
VLAPRWYFSVWGQGTLVTVSS 1A7.gr.1 DIQMTQSPSSLSASVGDRVTITCRASQDISNY 57
V.sub.L LNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGHTLPP TFGQGTKVEIK 1A7.gr.2
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 58 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKF
RERVTITVDTSTSTAYLELSSLRSEDTAVYYC VLAPRWYFSVWGQGTLVTVSS 1A7.gr.2
DIQMTQSPSSLSASVGDRVTITCRASQDISNY 59 V.sub.L
LNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSG SGSGTDFTLTISSLQPEDFATYYCQQGHTLPP
TFGQGTKVEIK 1A7.gr.3 EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 60 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKF RERVTLTVDTSTSTAYLELSSLRSEDTAVYYC
VLAPRWYFSVWGQGTLVTVSS 1A7.gr.3 DIQMTQSPSSLSASVGDRVTITCRASQDISNY 61
V.sub.L LNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGHTLPP TFGQGTKVEIK 1A7.gr.4
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 62 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKF RERVTITVDTSTSTAYLELSSLRSEDTAVYYC
VLAPRWYFSVWGQGTLVTVSS 1A7.gr.4 DIQMTQSPSSLSASVGDRVTITCRASQDISNY 63
V.sub.L LNWYQQKPGKTVKLLIYYTSRLRSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGHTLPP TFGQGTKVEIK 1A7.gr.5
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 64 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKF RERVTITVDTSTSTAYLELSSLRSEDTAVYYC
VLAPRWYFSVWGQGTLVTVSS 1A7.gr.5 DIQMTQSPSSLSASVGDRVTITCRASQDISNY 65
VL LNWYQQKPGKTVKLLIYYTSRLRSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGHTLPP TFGQGTKVEIK 1A7.gr.6
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 66 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKF RERVTITVDTSTSTAYLELSSLRSEDTAVYYC
VLAPRWYFSVWGQGTLVTVSS 1A7.gr.6 DIQMTQSPSSLSASVGDRVTITCRASQDISNY 67
V.sub.L LNWYQQKPGKTVKLLIYYTSRLRSGVPSRFSG
SGSGKDYTLTISSLQPEDFATYFCQQGHTLPP TFGQGTKVEIK 1A7.gr.7
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 68 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKF RERVTITVDTSTSTAYLELSSLRSEDTAVYYC
VLAPRWYFSVWGQGTLVTVSS 1A7.gr.7 DIQMTQSPSSLSASVGDRVTITCRASQDISNY 69
V.sub.L LNWYQQKPGKTVKLLIYYTSRLRSGVPSRFSG
SGSGKDYTLTISSLQPEDFATYFCQQGHTLPP TFGQGTKVEIK 1A7.gr.DA
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDA 70 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKF RERVTITRDTSTSTAYLELSSLRSEDTAVYYC
VLAPRWYFSVWGQGTLVTVSS 1A7.gr.DA DIQMTQSPSSLSASVGDRVTITCRASQDISNY 71
V.sub.L LNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGHTLPP TFGQGTKVEIK 1A7.gr.ES
EVQLVQSGAEVKKPGASVKVSCKASGYTFTES 72 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKF RERVTITRDTSTSTAYLELSSLRSEDTAVYYC
VLAPRWYFSVWGQGTLVTVSS 1A7.gr.ES DIQMTQSPSSLSASVGDRVTITCRASQDISNY 73
V.sub.L LNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGHTLPP TFGQGTKVEIK 1A7.gr.NADS
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 74 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNADSSYNQKF RERVTITRDTSTSTAYLELSSLRSEDTAVYYC
VLAPRWYFSVWGQGTLVTVSS 1A7.gr.NADS DIQMTQSPSSLSASVGDRVTITCRASQDISNY
75 V.sub.L LNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGHTLPP TFGQGTKVEIK 1A7.gr.NADA
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 76 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNADASYNQKF RERVTITRDTSTSTAYLELSSLRSEDTAVYYC
VLAPRWYFSVWGQGTLVTVSS 1A7.gr.NADA DIQMTQSPSSLSASVGDRVTITCRASQDISNY
77 V.sub.L LNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGHTLPP TFGQGTKVEIK 1A7.gr.NGDA
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 78 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGDASYNQKF RERVTITRDTSTSTAYLELSSLRSEDTAVYYC
VLAPRWYFSVWGQGTLVTVSS 1A7.gr.NGDA DIQMTQSPSSLSASVGDRVTITCRASQDISNY
79 V.sub.L LNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGHTLPP TFGQGTKVEIK 1A7.gr.SGDS
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 80 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDSGDSSYNQKF RERVTITRDTSTSTAYLELSSLRSEDTAVYYC
VLAPRWYFSVWGQGTLVTVSS 1A7.gr.SGDS DIQMTQSPSSLSASVGDRVTITCRASQDISNY
81 V.sub.L LNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGHTLPP TFGQGTKVEIK 1A7.gr.NGSS
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 82 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGSSSYNQKF RERVTITRDTSTSTAYLELSSLRSEDTAVYYC
VLAPRWYFSVWGQGTLVTVSS 1A7.gr.NGSS DIQMTQSPSSLSASVGDRVTITCRASQDISNY
83 V.sub.L LNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGHTLPP TFGQGTKVEIK 1A7.gr.DANA
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDA 84 DA
YMSWVRQAPGQGLEWIGDMYPDNADASYNQKF V.sub.H
RERVTITRDTSTSTAYLELSSLRSEDTAVYYC VLAPRWYFSVWGQGTLVTVSS 1A7.gr.DANA
DIQMTQSPSSLSASVGDRVTITCRASQDISNY 85 DA
LNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSG V.sub.L
SGSGTDFTLTISSLQPEDFATYYCQQGHTLPP TFGQGTKVEIK 1A7.Ala.1
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 86 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKF RERVTITRDTSTSTAYLELSSLRSEDTAVYYC
VLAPRWYFSVWGQGTLVTVSS 1A7.Ala.1 DIQMTQSPSSLSASVGDRVTITCRASQDISNY 87
V.sub.L LNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGHTLPA TFGQGTKVEIK 1A7.Ala.2
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 88 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKF RERVTITRDTSTSTAYLELSSLRSEDTAVYYC
VLAPRWYFSVWGQGTLVTVSS 1A7.Ala.2 DIQMTQSPSSLSASVGDRVTITCRASQDISNY 89
V.sub.L LNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGHTAPP TFGQGTKVEIK 1A7.Ala.3
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 90 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKF RERVTITRDTSTSTAYLELSSLRSEDTAVYYC
VLAPRWYFSVWGQGTLVTVSS 1A7.Ala.3 DIQMTQSPSSLSASVGDRVTITCRASQDISNY 91
V.sub.L LNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGATLPP TFGQGTKVEIK 1A7.Ala.4
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 92 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKF RERVTITRDTSTSTAYLELSSLRSEDTAVYYC
VLAPRWYFSVWGQGTLVTVSS 1A7.Ala.4 DIQMTQSPSSLSASVGDRVTITCRASQDISNY 93
V.sub.L LNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGHALPP TFGQGTKVEIK 1A7.Ala.5
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 94 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKF RERVTITRDTSTSTAYLELSSLRSEDTAVYYC
VLAPRWYFSVWGQGTLVTVSS 1A7.Ala.5 DIQMTQSPSSLSASVGDRVTITCRASQDISNY 95
V.sub.L LNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQAHTLPP TFGQGTKVEIK 1A7.Ala.6
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 96 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKF RERVTITRDTSTSTAYLELSSLRSEDTAVYYC
VLAPRWYFSVWGQGTLVTVSS 1A7.Ala.6 DIQMTQSPSSLSASVGDRVTITCRASQDISNY 97
V.sub.L LNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGHTLAP TFGQGTKVEIK 1A7.Ala.7
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 98 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKF RERVTITRDTSTSTAYLELSSLRSEDTAVYYC
VLAPRWYFSVWGQGTLVTVSS 1A7.Ala.7 DIQMTQSPSSLSASVGDRVTITCRASQDISNY 99
V.sub.L LNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQAGHTLPP TFGQGTKVEIK 1A7.Ala.8
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 100 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKF RERVTITRDTSTSTAYLELSSLRSEDTAVYYC
VLAPRWYFSAWGQGTLVTVSS 1A7.Ala.8 DIQMTQSPSSLSASVGDRVTITCRASQDISNY
101 V.sub.L LNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGHTLPP TFGQGTKVEIK 1A7.Ala.9
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 102 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKF RERVTITRDTSTSTAYLELSSLRSEDTAVYYC
VLAPRWYASVWGQGTLVTVSS 1A7.Ala.9 DIQMTQSPSSLSASVGDRVTITCRASQDISNY
103 V.sub.L LNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGHTLPP TFGQGTKVEIK 1A7.Ala.10
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 104 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKF RERVTITRDTSTSTAYLELSSLRSEDTAVYYC
VLAPRWAFSVWGQGTLVTVSS 1A7.Ala.10 DIQMTQSPSSLSASVGDRVTITCRASQDISNY
105 V.sub.L LNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGHTLPP TFGQGTKVEIK 1A7.Ala.11
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 106 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKF RERVTITRDTSTSTAYLELSSLRSEDTAVYYC
VLAPAWYFSVWGQGTLVTVSS 1A7.Ala.11 DIQMTQSPSSLSASVGDRVTITCRASQDISNY
107 V.sub.L LNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGHTLPP TFGQGTKVEIK 1A7.Ala.12
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 108 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKF
RERVTITRDTSTSTAYLELSSLRSEDTAVYYC
VLAPRWYFAVWGQGTLVTVSS 1A7.Ala.12 DIQMTQSPSSLSASVGDRVTITCRASQDISNY
109 V.sub.L LNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGHTLPP TFGQGTKVEIK 1A7.Ala.13
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 110 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKF RERVTITRDTSTSTAYLELSSLRSEDTAVYYC
VLAPRAYFSVWGQGTLVTVSS 1A7.Ala.13 DIQMTQSPSSLSASVGDRVTITCRASQDISNY
111 V.sub.L LNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGHTLPP TFGQGTKVEIK 1A7.Ala.14
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 112 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKF RERVTITRDTSTSTAYLELSSLRSEDTAVYYC
VLAARWYFSVWGQGTLVTVSS 1A7.Ala.14 DIQMTQSPSSLSASVGDRVTITCRASQDISNY
113 V.sub.L LNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGHTLPP TFGQGTKVEIK 1A7.Ala.15
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 114 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKF RERVTITRDTSTSTAYLELSSLRSEDTAVYYC
ALAPRWYFSVWGQGTLVTVSS 1A7.Ala.15 DIQMTQSPSSLSASVGDRVTITCRASQDISNY
115 V.sub.L LNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGHTLPP TFGQGTKVEIK 1A7.Ala.16
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 116 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKF RERVTITRDTSTSTAYLELSSLRSEDTAVYYC
VAAPRWYFSVWGQGTLVTVSS 1A7.Ala.16 DIQMTQSPSSLSASVGDRVTITCRASQDISNY
117 V.sub.L LNWYQQKPGKAPKLLIYYTSRLRSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGHTLPP TFGQGTKVEIK 3C8.gr.1
EVQLVQSGAEVKKPGASVKVSCKASGYAFTNY 118 V.sub.H
LIEWVRQAPGQGLEWIGVINPGSGDTYYSEKF KGRVTITRDTSTSTAYLELSSLRSEDTAVYYC
ARDRLDYWGQGTLVTVSS 3C8.gr.1 DIQMTQSPSSLSASVGDRVTITCHASQDISSY 119
V.sub.L IVWYQQKPGKAPKLLIYHGTNLEDGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCVHYAQFPY TFGQGTKVEIK 3C8.gr.2
EVQLVQSGAEVKKPGASVKVSCKASGYAFTNY 120 V.sub.H
LIEWVRQAPGQGLEWIGVINPGSGDTYYSEKF KGRVTITADTSTSTAYLELSSLRSEDTAVYYC
ARDRLDYWGQGTLVTVSS 3C8.gr.2 DIQMTQSPSSLSASVGDRVTITCHASQDISSY 121
V.sub.L IVWYQQKPGKAPKLLIYHGTNLEDGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCVHYAQFPY TFGQGTKVEIK 3C8.gr.3
EVQLVQSGAEVKKPGASVKVSCKASGYAFTNY 122 V.sub.H
LIEWVRQAPGQGLEWIGVINPGSGDTYYSEKF KGRVTLTADTSTSTAYLELSSLRSEDTAVYYC
ARDRLDYWGQGTLVTVSS 3C8.gr.3 DIQMTQSPSSLSASVGDRVTITCHASQDISSY 123
V.sub.L IVWYQQKPGKAPKLLIYHGTNLEDGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCVHYAQFPY TFGQGTKVEIK 3C8.gr.4
EVQLVQSGAEVKKPGASVKVSCKASGYAFTNY 124 VH
LIEWVRQAPGQGLEWIGVINPGSGDTYYSEKF KGRVTITADTSTSTAYLELSSLRSEDTAVYYC
ARDRLDYWGQGTLVTVSS 3C8.gr.4 DIQMTQSPSSLSASVGDRVTITCHASQDISSY 125 VL
IVWYQQKPGKSFKGLIYHGTNLEDGVPSRFSG SGSGTDFTLTISSLQPEDFATYYCVHYAQFPY
TFGQGTKVEIK 3C8.gr.5 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNY 126 VH
LIEWVRQAPGQGLEWIGVINPGSGDTYYSEKF KGRVTLTADTSTSTAYLELSSLRSEDTAVYYC
ARDRLDYWGQGTLVTVSS 3C8.gr.5 DIQMTQSPSSLSASVGDRVTITCHASQDISSY 127 VL
IVWYQQKPGKSFKGLIYHGTNLEDGVPSRFSG SGSGTDFTLTISSLQPEDFATYYCVHYAQFPY
TFGQGTKVEIK 3C8.gr.5.SG EVQLVQSGAEVKKPGASVKVSCKASGYAFTNY 128 VH
LIEWVRQAPGQGLEWIGVINPGSGDTYYSEKF KGRVTLTADTSTSTAYLELSSLRSEDTAVYYC
ARDRLDYWGQGTLVTVSS 3C8.gr.5.SG DIQMTQSPSSLSASVGDRVTITCHASQDISSY 129
VL IVWYQQKPGKSFKGLIYHGTNLESGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCVHYAQFPY TFGQGTKVEIK 3C8.gr.5.EG
EVQLVQSGAEVKKPGASVKVSCKASGYAFTNY 130 VH
LIEWVRQAPGQGLEWIGVINPGSGDTYYSEKF KGRVTLTADTSTSTAYLELSSLRSEDTAVYYC
ARDRLDYWGQGTLVTVSS 3C8.gr.5.EG DIQMTQSPSSLSASVGDRVTITCHASQDISSY 131
VL IVWYQQKPGKSFKGLIYHGTNLEEGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCVHYAQFPY TFGQGTKVEIK 3C8.gr.5.QG
EVQLVQSGAEVKKPGASVKVSCKASGYAFTNY 132 VH
LIEWVRQAPGQGLEWIGVINPGSGDTYYSEKF KGRVTLTADTSTSTAYLELSSLRSEDTAVYYC
ARDRLDYWGQGTLVTVSS 3C8.gr.5.QG DIQMTQSPSSLSASVGDRVTITCHASQDISSY 133
V.sub.L IVWYQQKPGKSFKGLIYHGTNLEQGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCVHYAQFPY TFGQGTKVEIK 3C8.gr.6
EVQLVQSGAEVKKPGASVKVSCKASGYAFTNY 134 V.sub.H
LIEWVRQAPGQGLEWIGVINPGSGDTYYSEKF KGRVTITADTSTSTAYLELSSLRSEDTAVYYC
ARDRLDYWGQGTLVTVSS 3C8.gr.6 DIQMTQSPSSLSASVGDRVTITCHASQDISSY 135
V.sub.L IVWYQQKPGKSFKGLIYHGTNLEDGVPSRFSG
SGSGADYTLTISSLQPEDFATYYCVHYAQFPY TFGQGTKVEIK 3C8.gr.7
EVQLVQSGAEVKKPGASVKVSCKASGYAFTNY 136 V.sub.H
LIEWVRQAPGQGLEWIGVINPGSGDTYYSEKF KGRVTLTADTSTSTAYLELSSLRSEDTAVYYC
ARDRLDYWGQGTLVTVSS 3C8.gr.7 DIQMTQSPSSLSASVGDRVTITCHASQDISSY 137
V.sub.L IVWYQQKPGKSFKGLIYHGTNLEDGVPSRFSG
SGSGADYTLTISSLQPEDFATYYCVHYAQFPY TFGQGTKVEIK 3C8.gr.8
EVQLVQSGAEVKKPGASVKVSCKASGYAFTNY 138 V.sub.H
LIEWVRQAPGQGLEWIGVINPGSGDTYYSEKF KGRVTLTRDTSTSTAYLELSSLRSEDTAVYYC
ARDRLDYWGQGTLVTVSS 3C8.gr.8 DIQMTQSPSSLSASVGDRVTITCHASQDISSY 139
V.sub.L IVWYQQKPGKSFKGLIYHGTNLEDGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCVHYAQFPY TFGQGTKVEIK 3C8.gr.9
EVQLVQSGAEVKKPGASVKVSCKASGYAFTNY 140 V.sub.H
LIEWVRQAPGQGLEWIGVINPGSGDTYYSEKF KGRVTLTRDTSTSTAYLELSSLRSEDTAVYYC
ARDRLDYWGQGTLVTVSS 3C8.gr.9 DIQMTQSPSSLSASVGDRVTITCHASQDISSY 141
V.sub.L IVWYQQKPGKSPKLLIYHGTNLEDGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCVHYAQFPY TFGQGTKVEIK 3C8.gr.10
EVQLVQSGAEVKKPGASVKVSCKASGYAFTNY 142 V.sub.H
LIEWVRQAPGQGLEWIGVINPGSGDTYYSEKF KGRVTLTRDTSTSTAYLELSSLRSEDTAVYYC
ARDRLDYWGQGTLVTVSS 3C8.gr.10 DIQMTQSPSSLSASVGDRVTITCHASQDISSY 143
V.sub.L IVWYQQKPGKAFKLLIYHGTNLEDGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCVHYAQFPY TFGQGTKVEIK 3C8.gr.11
EVQLVQSGAEVKKPGASVKVSCKASGYAFTNY 144 V.sub.H
LIEWVRQAPGQGLEWIGVINPGSGDTYYSEKF KGRVTLTRDTSTSTAYLELSSLRSEDTAVYYC
ARDRLDYWGQGTLVTVSS 3C8.gr.11 DIQMTQSPSSLSASVGDRVTITCHASQDISSY 145
V.sub.L IVWYQQKPGKAPKGLIYHGTNLEDGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCVHYAQFPY TFGQGTKVEIK 3C8.A.1
EVQLVQSGAEVKKPGASVKVSCKASGYAFTNY 146 V.sub.H
LIEWVRQAPGQGLEWIGVINPGSGDTYYSEKF KGRVTLTADTSTSTAYLELSSLRSEDTAVYYC
ARDRLDYWGQGTLVTVSS 3C8.A.1 DIQMTQSPSSLSASVGDRVTITCHASQDISSY 147
V.sub.L IVWYQQKPGKSFKGLIYHGTNLEDGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCAHYAQFPY TFGQGTKVEIK 3C8.A.2
EVQLVQSGAEVKKPGASVKVSCKASGYAFTNY 148 V.sub.H
LIEWVRQAPGQGLEWIGVINPGSGDTYYSEKF KGRVTLTADTSTSTAYLELSSLRSEDTAVYYC
ARDRLDYWGQGTLVTVSS 3C8.A.2 DIQMTQSPSSLSASVGDRVTITCHASQDISSY 149
V.sub.L IVWYQQKPGKSFKGLIYHGTNLEDGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCVAYAQFPY TFGQGTKVEIK 3C8.A.3
EVQLVQSGAEVKKPGASVKVSCKASGYAFTNY 150 V.sub.H
LIEWVRQAPGQGLEWIGVINPGSGDTYYSEKF KGRVTLTADTSTSTAYLELSSLRSEDTAVYYC
ARDRLDYWGQGTLVTVSS 3C8.A.3 DIQMTQSPSSLSASVGDRVTITCHASQDISSY 151
V.sub.L IVWYQQKPGKSFKGLIYHGTNLEDGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCVHAAQFPY TFGQGTKVEIK 3C8.A.4
EVQLVQSGAEVKKPGASVKVSCKASGYAFTNY 152 V.sub.H
LIEWVRQAPGQGLEWIGVINPGSGDTYYSEKF KGRVTLTADTSTSTAYLELSSLRSEDTAVYYC
ARDRLDYWGQGTLVTVSS 3C8.A.4 DIQMTQSPSSLSASVGDRVTITCHASQDISSY 153
V.sub.L IVWYQQKPGKSFKGLIYHGTNLEDGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCVHYAAFPY TFGQGTKVEIK 3C8.A.5
EVQLVQSGAEVKKPGASVKVSCKASGYAFTNY 154 V.sub.H
LIEWVRQAPGQGLEWIGVINPGSGDTYYSEKF KGRVTLTADTSTSTAYLELSSLRSEDTAVYYC
ARDRLDYWGQGTLVTVSS 3C8.A.5 DIQMTQSPSSLSASVGDRVTITCHASQDISSY 155
V.sub.L IVWYQQKPGKSFKGLIYHGTNLEDGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCVHYAQAPY TFGQGTKVEIK 3C8.A.6
EVQLVQSGAEVKKPGASVKVSCKASGYAFTNY 156 V.sub.H
LIEWVRQAPGQGLEWIGVINPGSGDTYYSEKF KGRVTLTADTSTSTAYLELSSLRSEDTAVYYC
ARDRLDYWGQGTLVTVSS 3C8.A.6 DIQMTQSPSSLSASVGDRVTITCHASQDISSY 157
V.sub.L IVWYQQKPGKSFKGLIYHGTNLEDGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCVHYAQFAY TFGQGTKVEIK 3C8.A.7
EVQLVQSGAEVKKPGASVKVSCKASGYAFTNY 158 V.sub.H
LIEWVRQAPGQGLEWIGVINPGSGDTYYSEKF KGRVTLTADTSTSTAYLELSSLRSEDTAVYYC
ARDRLDYWGQGTLVTVSS
3C8.A.7 DIQMTQSPSSLSASVGDRVTITCHASQDISSY 159 V.sub.L
IVWYQQKPGKSFKGLIYHGTNLEDGVPSRFSG SGSGTDFTLTISSLQPEDFATYYCVHYAQFPA
TFGQGTKVEIK 3C8.A.8 EVQLVQSGAEVKKPGASVKVSCKASGYAFTNY 160 V.sub.H
LIEWVRQAPGQGLEWIGVINPGSGDTYYSEKF KGRVTLTADTSTSTAYLELSSLRSEDTAVYYC
ARARLDYWGQGTLVTVSS 3C8.A.8 DIQMTQSPSSLSASVGDRVTITCHASQDISSY 161
V.sub.L IVWYQQKPGKSFKGLIYHGTNLEDGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCVHYAQFPY TFGQGTKVEIK 3C8.A.9
EVQLVQSGAEVKKPGASVKVSCKASGYAFTNY 162 V.sub.H
LIEWVRQAPGQGLEWIGVINPGSGDTYYSEKF KGRVTLTADTSTSTAYLELSSLRSEDTAVYYC
ARDALDYWGQGTLVTVSS 3C8.A.9 DIQMTQSPSSLSASVGDRVTITCHASQDISSY 163
V.sub.L IVWYQQKPGKSFKGLIYHGTNLEDGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCVHYAQFPY TFGQGTKVEIK 3C8.A.10
EVQLVQSGAEVKKPGASVKVSCKASGYAFTNY 164 V.sub.H
LIEWVRQAPGQGLEWIGVINPGSGDTYYSEKF KGRVTLTADTSTSTAYLELSSLRSEDTAVYYC
ARDRADYWGQGTLVTVSS 3C8.A.10 DIQMTQSPSSLSASVGDRVTITCHASQDISSY 165
V.sub.L IVWYQQKPGKSFKGLIYHGTNLEDGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCVHYAQFPY TFGQGTKVEIK 1D2.gr.1
EVQLVESGPGLVKPSETLSLTCTVSGFSLTDY 166 V.sub.H
GVLWIRQPPGKGLEWIGMIWSGGTTDYNAAFI SRVTISVDTSKNQFSLKLSSVTAADTAVYYCV
REEMDYWGQGTLVTVSS 1D2.gr.1 DIQMTQSPSSLSASVGDRVTITCRASQDISNF 167
V.sub.L LNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGNTLPW TFGQGTKVEIK 1D2.gr.2
EVQLVESGPGLVKPSETLSLTCTVSGFSLTDY 168 V.sub.H
GVLWIRQPPGKGLEWIGMIWSGGTTDYNAAFI SRVTISKDTSKNQVSLKLSSVTAADTAVYYCV
REEMDYWGQGTLVTVSS 1D2.gr.2 DIQMTQSPSSLSASVGDRVTITCRASQDISNF 169
V.sub.L LNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGNTLPW TFGQGTKVEIK 1D2.gr.3
EVQLVESGPGLVKPSETLSLTCTVSGFSLTDY 170 V.sub.H
GVLWVRQPPGKGLEWLGMIWSGGTTDYNAAFI SRLTISKDTSKNQVSLKLSSVTAADTAVYYCV
REEMDYWGQGTLVTVSS 1D2.gr.3 DIQMTQSPSSLSASVGDRVTITCRASQDISNF 171
V.sub.L LNWYQQKPGKAPKLLIYYTSRLHSGVPSRFSG
SGSGTDFTLTISSLQPEDFATYYCQQGNTLPW TFGQGTKVEIK CON1 (1A7)
X.sub.1X.sub.2YMS, wherein X.sub.1 is D or E, 172 HVR-H1 and
X.sub.2 is S or A CON1 (1A7)
DMYPDX.sub.1X.sub.2X.sub.3X.sub.4SYNQKFRE, wherein X.sub.1 173
HVR-H2 is N or S, X.sub.1 is A or G, X.sub.3 is D or S, and X.sub.4
is A or S CON1 (1A7) APRWX.sub.1X.sub.2X.sub.3X.sub.4, wherein
X.sub.1 is Y or 174 HVR-H3 A, X.sub.2 is A or F, X.sub.3 is S or A,
and X.sub.4 is A or V. CON1 (1A7)
QX.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7T, wherein
X.sub.1 is A 175 HVR-L3 or Q, X.sub.2 is A or G, X.sub.3 is A or H,
X.sub.4 is A or T, X.sub.5 is A or L, X.sub.6 is A or P, and
X.sub.7 is A or P. CON2 (3C8) VINPGSGDX.sub.1YYSEKFKG, wherein
X.sub.1 is 176 HVR-H2 T, A or Q. CON2 (3C8) HGTNLEX.sub.1, wherein
X.sub.1 is S, E, 177 HVR-L2 or Q. CON2 (3C8)
X.sub.1X.sub.2YAQFPYX.sub.3, wherein X.sub.1 is V or 178 HVR-L3 A,
X.sub.2 is H or A, and X.sub.3 is Y or A. 1A7 V.sub.L
DIQMTQTTSSLSASLGDRVTISCRASQDISNY 179
LNWYQQKPDGTVKLLIYYTSRLRSGVPSRFSG SGSGKDYFLTISNLEQEDVAAYFCQQGHTLPP
TFGGGTKLEIK 1A7 V.sub.H EVQLQQSGPELVKPGASVKISCKASGYTFTDS 180
YMSWVKQSHGKTLEWIGDMYPDNGDSSYNQKF REKVTLTVDKSSTTAYMEFRSLTSEDSAVYYC
VLAPRWYFSVWGTGTTVTVSS 3C8 V.sub.L DILMTQSPSSMSVSLGDTVSITCHASQDISSY
181 IVWLQQKPGKSFRGLIYHGTNLEDGIPSRFSG
SGSGADYSLTISSLESEDFADYYCVHYAQFPY TFGGGTKLEIK 3C8 V.sub.H
QVQLQQSGAELVRPGTSVKVSCKASGYAFTNY 182
LIEWVKQRPGQGLEWIGVINPGSGDTYYSEKF KGKVTLTADKSSSTAYMQLSSLTSEDSAVYFC
ARDRLDYWGQGTTLTVSS 1A7.gr.5' EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 225
V.sub.H YMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKF
RERVTLTVDTSTSTAYLELSSLRSEDTAVYYC VLAPRWYFSVWGQGTLVTVSS 1A7.gr.7'
EVQLVQSGAEVKKPGASVKVSCKASGYTFTDS 226 V.sub.H
YMSWVRQAPGQGLEWIGDMYPDNGDSSYNQKF RERVTLTVDTSTSTAYLELSSLRSEDTAVYYC
VLAPRWYFSVWGQGTLVTVSS
[0222] In some embodiments, the agonist anti-human OX40 antibody is
an anti-OX40 antibody described in U.S. Pat. No. 7,550,140. In some
embodiments, the agonist anti-human OX40 antibody comprises a heavy
chain comprising the sequence of
TABLE-US-00004 (SEQ ID NO: 183)
EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYTMNWVRQAPGKGLEW
VSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVY
YCAKDRYSQVHYALDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG
GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS
VVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF
YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPGK
and/or a light chain comprising the sequence of
DIVMTQSPDSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKAGQSPQLLIYLGSNRA
SGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQQYYNHPTTFGQGTKLEIKRTVAAPS
VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY
SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:184). In some
embodiments, the antibody comprises at least one, two, three, four,
five or six hypervariable region (HVR) sequences of antibody 008 as
described in U.S. Pat. No. 7,550,140. In some embodiments, the
antibody comprises a heavy chain variable region sequence and/or a
light chain variable region sequence of antibody 008 as described
in U.S. Pat. No. 7,550,140.
[0223] In some embodiments, the agonist anti-human OX40 antibody is
an anti-OX40 antibody described in U.S. Pat. No. 7,550,140. In some
embodiments, the agonist anti-human OX40 antibody comprises the
sequence of
MAEVQLVESGGGLVQPGGSLRLSCAASGFTFSNYTMNWVRQAPGKGLEWVSAISGSG
GSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRYSQVHYALDYWG
QGTLVTVLEGTGGSGGTGSGTGTSELDIQMTQSPDSLPVTPGEPASISCRSSQSLLHSNGY
NYLDWYLQKAGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCQ
QYYNHPTTFGQGTKLEIKRAA (SEQ ID NO:185). In some embodiments, the
antibody comprises at least one, two, three, four, five or six
hypervariable region (HVR) sequences of antibody SC02008 as
described in U.S. Pat. No. 7,550,140. In some embodiments, the
antibody comprises a heavy chain variable region sequence and/or a
light chain variable region sequence of antibody SC02008 as
described in U.S. Pat. No. 7,550,140.
[0224] In some embodiments, the agonist anti-human OX40 antibody is
an anti-OX40 antibody described in U.S. Pat. No. 7,550,140. In some
embodiments, the agonist anti-human OX40 antibody comprises a heavy
chain comprising the sequence of
EVQLVESGGGLVHPGGSLRLSCAGSGFTFSSYAMHWVRQAPGKGLEWVSAIGTGGGTY
YADSVMGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYDNVMGLYWFDYWGQGT
LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP
AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK
PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:186) and/or a
light chain comprising the sequence of
EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA
RFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPPAFGGGTKVEIKRTVAAPSVFIFPPS
DEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL
TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:187). In some
embodiments, the antibody comprises at least one, two, three, four,
five or six hypervariable region (HVR) sequences of antibody 023 as
described in U.S. Pat. No. 7,550,140. In some embodiments, the
antibody comprises a heavy chain variable region sequence and/or a
light chain variable region sequence of antibody 023 as described
in U.S. Pat. No. 7,550,140.
[0225] In some embodiments, the agonist anti-human OX40 antibody is
an anti-OX40 antibody described in U.S. Pat. No. 7,960,515. In some
embodiments, the agonist anti-human OX40 antibody comprises a heavy
chain variable region comprising the sequence of
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYISSSSSTID
YADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARESGWYLFDYWGQGTLVTV SS (SEQ
ID NO:188) and/or a light chain variable region comprising the
sequence of
DIQMTQSPSSLSASVGDRVTITCRASQGISSWLAWYQQKPEKAPKSLIYAASSLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSYPPTFGGGTKVEIK (SEQ ID NO:189). In
some embodiments, the antibody comprises at least one, two, three,
four, five or six hypervariable region (HVR) sequences of antibody
11D4 as described in U.S. Pat. No. 7,960,515. In some embodiments,
the antibody comprises a heavy chain variable region sequence
and/or a light chain variable region sequence of antibody 11D4 as
described in U.S. Pat. No. 7,960,515.
[0226] In some embodiments, the agonist anti-human OX40 antibody is
an anti-OX40 antibody described in U.S. Pat. No. 7,960,515. In some
embodiments, the agonist anti-human OX40 antibody comprises a heavy
chain comprising the sequence of
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSGISWNSGSI
GYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDQSTADYYFYYGMDVW
GQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPC
PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKT
KPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQV
YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:190) and/or a
light chain comprising the sequence of
EIVVTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPA
RFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTKVEIKRTVAAPSVFIFPPSD
EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO:191). In some
embodiments, the antibody comprises at least one, two, three, four,
five or six hypervariable region (HVR) sequences of antibody 18D8
as described in U.S. Pat. No. 7,960,515. In some embodiments, the
antibody comprises a heavy chain variable region sequence and/or a
light chain variable region sequence of antibody 18D8 as described
in U.S. Pat. No. 7,960,515.
[0227] In some embodiments, the agonist anti-human OX40 antibody is
an anti-OX40 antibody described in WO2012/027328. In some
embodiments, the agonist anti-human OX40 antibody comprises a heavy
chain variable region comprising the sequence of
QVQLVQSGSELKKPGASVKVSCKASGYTFTDYSMHWVRQAPGQGLKWMGWINTETG
EPTYADDFKGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCANPYYDYVSYYAMDYWGQ GTTVTVSS
(SEQ ID NO:192) and/or a light chain variable region comprising the
sequence of
DIQMTQSPSSLSASVGDRVTITCKASQDVSTAVAWYQQKPGKAPKLLIYSASYLYTGVP
SRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYSTPRTFGQGTKLEIK (SEQ ID NO:193).
In some embodiments, the antibody comprises at least one, two,
three, four, five or six hypervariable region (HVR) sequences of
antibody hu106-222 as described in WO2012/027328. In some
embodiments, the antibody comprises a heavy chain variable region
sequence and/or a light chain variable region sequence of antibody
hu106-222 as described in WO2012/027328.
[0228] In some embodiments, the agonist anti-human OX40 antibody is
an anti-OX40 antibody described in WO2012/027328. In some
embodiments, the agonist anti-human OX40 antibody comprises a heavy
chain variable region comprising the sequence of
EVQLVESGGGLVQPGGSLRLSCAASEYEFPSHDMSWVRQAPGKGLELVAAINSDGGST
YYPDTMERRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHYDDYYAWFAYWGQGT MVTVSS
(SEQ ID NO:194) and/or a light chain variable region comprising the
sequence of
EIVLTQSPATLSLSPGERATLSCRASKSVSTSGYSYMHWYQQKPGQAPRLLIYLASNLES
GVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRELPLTFGGGTKVEIK (SEQ ID
NO:195). In some embodiments, the antibody comprises at least one,
two, three, four, five or six hypervariable region (HVR) sequences
of antibody Hu119-122 as described in WO2012/027328. In some
embodiments, the antibody comprises a heavy chain variable region
sequence and/or a light chain variable region sequence of antibody
Hu119-122 as described in WO2012/027328.
[0229] In some embodiments, the agonist anti-human OX40 antibody is
an anti-OX40 antibody described in WO2013/028231. In some
embodiments, the agonist anti-human OX40 antibody comprises a heavy
chain comprising the sequence of
MYLGLNYVFIVFLLNGVQSEVKLEESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVR
QSPEKGLEWVaEIRSKANNHATYYAESVNGRFTISRDDSKSSVYLQMNSLRAEDTGIYY
CTWGEVFYFDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPV
TVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYITCNVNHKPSNTKVD
KKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE
VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL
PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K (SEQ
ID NO:196) and/or a light chain comprising the sequence of
MRPSIQFLGLLLFWLHGAQCDIQMTQSPSSLSASLGGKVTITCKSSQDINKYIAWYQHKP
GKGPRLLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLLTFGAGTK
LELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES
VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID
NO:197). In some embodiments, the anti-human OX40 agonist antibody
comprises a heavy chain variable region comprising the sequence of
MYLGLNYVFIVFLLNGVQSEVKLEESGGGLVQPGGSMKLSCAASGFTFSDAWMDWVRQSPEKG
LEWVAEIRSKANNHATYYAESVNGRFTISRDDSKSSVYLQMNSLRAEDTGIYYCTWGEVFYFDY
WGQGTTLTVSS (SEQ ID NO:198) and/or a light chain variable region
comprising the sequence of
MRPSIQFLGLLLFWLHGAQCDIQMTQSPSSLSASLGGKVTITCKSSQDINKYIAWYQHKPGKGPR
LLIHYTSTLQPGIPSRFSGSGSGRDYSFSISNLEPEDIATYYCLQYDNLLTFGAGTKLELK (SEQ
ID NO:199). In some embodiments, the antibody comprises at least
one, two, three, four, five or six hypervariable region (HVR)
sequences of antibody Mab CH 119-43-1 as described in
WO2013/028231. In some embodiments, the antibody comprises a heavy
chain variable region sequence and/or a light chain variable region
sequence of antibody Mab CH 119-43-1 as described in
WO2013/028231.
[0230] In some embodiments, the agonist anti-human OX40 antibody is
an anti-OX40 antibody described in WO2013038191. In some
embodiments, the agonist anti-human OX40 antibody comprises a heavy
chain variable region comprising the sequence of
EVQLQQSGPELVKPGASVKMSCKASGYTFTSYVMHWVKQKPGQGLEWIGYINPYNDG
TKYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCANYYGSSLSMDYWGQGTSV TVSS
(SEQ ID NO:200) and/or a light chain variable region comprising the
sequence of
DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLHSGVPS
RFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIKR (SEQ ID NO:201).
In some embodiments, the antibody comprises at least one, two,
three, four, five or six hypervariable region (HVR) sequences of
antibody clone 20E5 as described in WO2013038191. In some
embodiments, the antibody comprises a heavy chain variable region
sequence and/or a light chain variable region sequence of antibody
clone 20E5 as described in WO2013038191.
[0231] In some embodiments, the agonist anti-human OX40 antibody is
an anti-OX40 antibody described in WO2013038191. In some
embodiments, the agonist anti-human OX40 antibody comprises a heavy
chain variable region comprising the sequence of
EVQLQQSGPELVKPGASVKISCKTSGYTFKDYTMHWVKQSHGKSLEWIGGIYPNNGGS
TYNQNFKDKATLTVDKSSSTAYMEFRSLTSEDSAVYYCARMGYHGPHLDFDVWGAGT TVTVSP
(SEQ ID NO:202) and/or a light chain variable region comprising the
sequence of
DIVMTQSHKFMSTSLGDRVSITCKASQDVGAAVaWYQQKPGQSPKLLIYWASTRHTGV
PDRFTGGGSGTDFTLTISNVQSEDLTDYFCQQYINYPLTFGGGTKLEIKR (SEQ ID NO:203).
In some embodiments, the antibody comprises at least one, two,
three, four, five or six hypervariable region (HVR) sequences of
antibody clone 12H3 as described in WO2013038191. In some
embodiments, the antibody comprises a heavy chain variable region
sequence and/or a light chain variable region sequence of antibody
clone 12H3 as described in WO2013038191.
[0232] In some embodiments, the agonist anti-human OX40 antibody is
an anti-OX40 antibody described in WO2014148895A1. In some
embodiments, the agonist anti-human OX40 antibody comprises a heavy
chain variable region comprising the sequence of
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWMGYINPYND
GTKYNEKFKGRVTITSDTSASTAYMELSSLRSEDTAVYYCANYYGSSLSMDYWGQGTL VTVSS
(SEQ ID NO:204) and/or a light chain variable region comprising the
sequence of
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPS
RFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKR (SEQ ID NO:205).
In some embodiments, the antibody comprises at least one, two,
three, four, five or six hypervariable region (HVR) sequences of
antibody clone 20E5 as described in WO2014148895A1. In some
embodiments, the antibody comprises a heavy chain variable region
sequence and/or a light chain variable region sequence of antibody
clone 20E5 as described in WO2014148895A1.
[0233] In some embodiments, the agonist anti-human OX40 antibody is
an anti-OX40 antibody described in WO2014148895A1. In some
embodiments, the agonist anti-human OX40 antibody comprises a heavy
chain variable region comprising the sequence of
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWMGYINPYND
GTKYNEKFKGRVTITSDTSASTAYMELSSLRSEDTAVYYCANYYGSSLSMDYWGQGTL VTVSS
(SEQ ID NO:204) and/or a light chain variable region comprising the
sequence of
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSRLHSGVPS
RFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKVEIKR (SEQ ID NO:206).
In some embodiments, the antibody comprises at least one, two,
three, four, five or six hypervariable region (HVR) sequences of
antibody clone 20E5 as described in WO2014148895A1. In some
embodiments, the antibody comprises a heavy chain variable region
sequence and/or a light chain variable region sequence of antibody
clone 20E5 as described in WO2014148895A1.
[0234] In some embodiments, the agonist anti-human OX40 antibody is
an anti-OX40 antibody described in WO2014148895A1. In some
embodiments, the agonist anti-human OX40 antibody comprises a heavy
chain variable region comprising the sequence of
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYINPYNDG
TKYNEKFKGRATITSDTSASTAYMELSSLRSEDTAVYYCANYYGSSLSMDYWGQGTLV TVSS
(SEQ ID NO:207) and/or a light chain variable region comprising the
sequence of
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPS
RFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKR (SEQ ID NO:205).
In some embodiments, the antibody comprises at least one, two,
three, four, five or six hypervariable region (HVR) sequences of
antibody clone 20E5 as described in WO2014148895A1. In some
embodiments, the antibody comprises a heavy chain variable region
sequence and/or a light chain variable region sequence of antibody
clone 20E5 as described in WO2014148895A1.
[0235] In some embodiments, the agonist anti-human OX40 antibody is
an anti-OX40 antibody described in WO2014148895A1. In some
embodiments, the agonist anti-human OX40 antibody comprises a heavy
chain variable region comprising the sequence of
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYINPYNDG
TKYNEKFKGRATITSDTSASTAYMELSSLRSEDTAVYYCANYYGSSLSMDYWGQGTLV TVSS
(SEQ ID NO:207) and/or a light chain variable region comprising the
sequence of
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSRLHSGVPS
RFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKVEIKR (SEQ ID NO:206).
In some embodiments, the antibody comprises at least one, two,
three, four, five or six hypervariable region (HVR) sequences of
antibody clone 20E5 as described in WO2014148895A1. In some
embodiments, the antibody comprises a heavy chain variable region
sequence and/or a light chain variable region sequence of antibody
clone 20E5 as described in WO2014148895A1.
[0236] In some embodiments, the agonist anti-human OX40 antibody is
an anti-OX40 antibody described in WO2014148895A1. In some
embodiments, the agonist anti-human OX40 antibody comprises a heavy
chain variable region comprising the sequence of
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYINPYNDG
TKYNEKFKGRATLTSDKSASTAYMELSSLRSEDTAVYYCANYYGSSLSMDYWGQGTL VTVSS
(SEQ ID NO:208) and/or a light chain variable region comprising the
sequence of
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLHSGVPS
RFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKR (SEQ ID NO:205).
In some embodiments, the antibody comprises at least one, two,
three, four, five or six hypervariable region (HVR) sequences of
antibody clone 20E5 as described in WO2014148895A1. In some
embodiments, the antibody comprises a heavy chain variable region
sequence and/or a light chain variable region sequence of antibody
clone 20E5 as described in WO2014148895A1.
[0237] In some embodiments, the agonist anti-human OX40 antibody is
an anti-OX40 antibody described in WO2014148895A1. In some
embodiments, the agonist anti-human OX40 antibody comprises a heavy
chain variable region comprising the sequence of
QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYVMHWVRQAPGQRLEWIGYINPYNDG
TKYNEKFKGRATLTSDKSASTAYMELSSLRSEDTAVYYCANYYGSSLSMDYWGQGTL VTVSS
(SEQ ID NO:208) and/or a light chain variable region comprising the
sequence of
DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAVKLLIYYTSRLHSGVPS
RFSGSGSGTDYTLTISSLQPEDFATYFCQQGNTLPWTFGQGTKVEIKR (SEQ ID NO:206).
In some embodiments, the antibody comprises at least one, two,
three, four, five or six hypervariable region (HVR) sequences of
antibody clone 20E5 as described in WO2014148895A1. In some
embodiments, the antibody comprises a heavy chain variable region
sequence and/or a light chain variable region sequence of antibody
clone 20E5 as described in WO2014148895A1.
[0238] In some embodiments, the agonist anti-human OX40 antibody is
an anti-OX40 antibody described in WO2014148895A1. In some
embodiments, the agonist anti-human OX40 antibody comprises a heavy
chain variable region comprising the sequence of
QVQLVQSGAEVKKPGSSVKVSCKASGYTFKDYTMHWVRQAPGQGLEWMGGIYPNNG
GSTYNQNFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARMGYHGPHLDFDVWGQ GTTVTVSS
(SEQ ID NO:209) and/or a light chain variable region comprising the
sequence of
DIQMTQSPSSLSASVGDRVTITCKASQDVGAAVaWYQQKPGKAPKLLIYWASTRHTGVP
SRFSGSGSGTDFTLTISSLQPEDFATYYCQQYINYPLTFGGGTKVEIKR (SEQ ID NO:210).
In some embodiments, the antibody comprises at least one, two,
three, four, five or six hypervariable region (HVR) sequences of
antibody clone clone 12H3 as described in WO2014148895A1. In some
embodiments, the antibody comprises a heavy chain variable region
sequence and/or a light chain variable region sequence of antibody
clone 12H3 as described in WO2014148895A1.
[0239] In some embodiments, the agonist anti-human OX40 antibody is
an anti-OX40 antibody described in WO2014148895A1. In some
embodiments, the agonist anti-human OX40 antibody comprises a heavy
chain variable region comprising the sequence of
QVQLVQSGAEVKKPGSSVKVSCKASGYTFKDYTMHWVRQAPGQGLEWMGGIYPNNG
GSTYNQNFKDRVTITADKSTSTAYMELSSLRSEDTAVYYCARMGYHGPHLDFDVWGQ GTTVTVSS
(SEQ ID NO:209) and/or a light chain variable region comprising the
sequence of
DIQMTQSPSSLSASVGDRVTITCKASQDVGAAVaWYQQKPGKAPKLLIYWASTRHTGVP
DRFSGGGSGTDFTLTISSLQPEDFATYYCQQYINYPLTFGGGTKVEIKR (SEQ ID NO:211).
In some embodiments, the antibody comprises at least one, two,
three, four, five or six hypervariable region (HVR) sequences of
antibody clone 12H3 as described in WO2014148895A1. In some
embodiments, the antibody comprises a heavy chain variable region
sequence and/or a light chain variable region sequence of antibody
clone 12H3 as described in WO2014148895A1.
[0240] In some embodiments, the agonist anti-human OX40 antibody is
an anti-OX40 antibody described in WO2014148895A1. In some
embodiments, the agonist anti-human OX40 antibody comprises a heavy
chain variable region comprising the sequence of
QVQLVQSGAEVKKPGSSVKVSCKASGYTFKDYTMHWVRQAPGQGLEWIGGIYPNNGG
STYNQNFKDRVTLTADKSTSTAYMELSSLRSEDTAVYYCARMGYHGPHLDFDVWGQG TTVTVSS
(SEQ ID NO:212) and/or a light chain variable region comprising the
sequence of
DIQMTQSPSSLSASVGDRVTITCKASQDVGAAVaWYQQKPGKAPKLLIYWASTRHTGVP
SRFSGSGSGTDFTLTISSLQPEDFATYYCQQYINYPLTFGGGTKVEIKR (SEQ ID NO:210).
In some embodiments, the antibody comprises at least one, two,
three, four, five or six hypervariable region (HVR) sequences of
antibody clone 12H3 as described in WO2014148895A1. In some
embodiments, the antibody comprises a heavy chain variable region
sequence and/or a light chain variable region sequence of antibody
clone 12H3 as described in WO2014148895A1.
[0241] In some embodiments, the agonist anti-human OX40 antibody is
an anti-OX40 antibody described in WO2014148895A1. In some
embodiments, the agonist anti-human OX40 antibody comprises a heavy
chain variable region comprising the sequence of
QVQLVQSGAEVKKPGSSVKVSCKASGYTFKDYTMHWVRQAPGQGLEWIGGIYPNNGG
STYNQNFKDRVTLTADKSTSTAYMELSSLRSEDTAVYYCARMGYHGPHLDFDVWGQG TTVTVSS
(SEQ ID NO:212) and/or a light chain variable region comprising the
sequence of
DIQMTQSPSSLSASVGDRVTITCKASQDVGAAVaWYQQKPGKAPKLLIYWASTRHTGVP
DRFSGGGSGTDFTLTISSLQPEDFATYYCQQYINYPLTFGGGTKVEIKR (SEQ ID NO:211).
In some embodiments, the antibody comprises at least one, two,
three, four, five or six hypervariable region (HVR) sequences of
antibody clone 12H3 as described in WO2014148895A1. In some
embodiments, the antibody comprises a heavy chain variable region
sequence and/or a light chain variable region sequence of antibody
clone 12H3 as described in WO2014148895A1.
[0242] In some embodiments, the agonist anti-human OX40 antibody is
an anti-OX40 antibody described in WO2014148895A1. In some
embodiments, the agonist anti-human OX40 antibody comprises a heavy
chain variable region comprising the sequence of
QVQLVQSGAEVKKPGSSVKVSCKASGYTFKDYTMHWVRQAPGQGLEWIGGIYPNNGG
STYNQNFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCARMGYHGPHLDFDVWGQG TTVTVSS
(SEQ ID NO:213) and/or a light chain variable region comprising the
sequence of
DIQMTQSPSSLSASVGDRVTITCKASQDVGAAVaWYQQKPGKAPKLLIYWASTRHTGVP
SRFSGSGSGTDFTLTISSLQPEDFATYYCQQYINYPLTFGGGTKVEIKR (SEQ ID NO:210).
In some embodiments, the antibody comprises at least one, two,
three, four, five or six hypervariable region (HVR) sequences of
antibody clone 12H3 as described in WO2014148895A1. In some
embodiments, the antibody comprises a heavy chain variable region
sequence and/or a light chain variable region sequence of antibody
clone 12H3 as described in WO2014148895A1.
[0243] In some embodiments, the agonist anti-human OX40 antibody is
an anti-OX40 antibody described in WO2014148895A1. In some
embodiments, the agonist anti-human OX40 antibody comprises a heavy
chain variable region comprising the sequence of
QVQLVQSGAEVKKPGSSVKVSCKASGYTFKDYTMHWVRQAPGQGLEWIGGIYPNNGG
STYNQNFKDRATLTVDKSTSTAYMELSSLRSEDTAVYYCARMGYHGPHLDFDVWGQG TTVTVSS
(SEQ ID NO:213) and/or a light chain variable region comprising the
sequence of
DIQMTQSPSSLSASVGDRVTITCKASQDVGAAVaWYQQKPGKAPKLLIYWASTRHTGVP
DRFSGGGSGTDFTLTISSLQPEDFATYYCQQYINYPLTFGGGTKVEIKR (SEQ ID NO:211).
In some embodiments, the antibody comprises at least one, two,
three, four, five or six hypervariable region (HVR) sequences of
antibody clone 12H3 as described in WO2014148895A1. In some
embodiments, the antibody comprises a heavy chain variable region
sequence and/or a light chain variable region sequence of antibody
clone 12H3 as described in WO2014148895A1.
[0244] In some embodiments, the agonist anti-human OX40 antibody is
L106 BD (Pharmingen Product #340420). In some embodiments, the
antibody comprises at least one, two, three, four, five or six
hypervariable region (HVR) sequences of antibody L106 (BD
Pharmingen Product #340420). In some embodiments, the antibody
comprises a heavy chain variable region sequence and/or a light
chain variable region sequence of antibody L106 (BD Pharmingen
Product #340420).
[0245] In some embodiments, the agonist anti-human OX40 antibody is
ACT35 (Santa Cruz Biotechnology, Catalog #20073). In some
embodiments, the antibody comprises at least one, two, three, four,
five or six hypervariable region (HVR) sequences of antibody ACT35
(Santa Cruz Biotechnology, Catalog #20073). In some embodiments,
the antibody comprises a heavy chain variable region sequence
and/or a light chain variable region sequence of antibody ACT35
(Santa Cruz Biotechnology, Catalog #20073).
[0246] In some embodiments, the agonist anti-human OX40 antibody is
MEDI6469. In some embodiments, the antibody comprises at least one,
two, three, four, five or six hypervariable region (HVR) sequences
of antibody MEDI6469. In some embodiments, the antibody comprises a
heavy chain variable region sequence and/or a light chain variable
region sequence of antibody MEDI6469.
[0247] In some embodiments, the agonist anti-human OX40 antibody is
MEDI0562. In some embodiments, the antibody comprises at least one,
two, three, four, five or six hypervariable region (HVR) sequences
of antibody MEDI0562. In some embodiments, the antibody comprises a
heavy chain variable region sequence and/or a light chain variable
region sequence of antibody MEDI0562.
[0248] Other OX40 Agonists
[0249] OX40 agonists useful for the methods described herein are in
no way intended to be limited to antibodies. Non-antibody OX40
agonists are contemplated and well known in the art.
[0250] As described above, OX40L (also known as CD134L) serves as a
ligand for OX40. As such, agonists that present part or all of
OX40L may serve as OX40 agonists. In some embodiments, an OX40
agonist may include one or more extracellular domains of OX40L.
Examples of extracellular domains of OX40L may include OX40-binding
domains. In some embodiments, an OX40 agonist may be a soluble form
of OX40L that includes one or more extracellular domains of OX40L
but lacks other, insoluble domains of the protein, e.g.,
transmembrane domains. In some embodiments, an OX40 agonist is a
soluble protein that includes one or more extracellular domains of
OX40L able to bind OX40L. In some embodiments, an OX40 agonist may
be linked to another protein domain, e.g., to increase its
effectiveness, half-life, or other desired characteristics. In some
embodiments, an OX40 agonist may include one or more extracellular
domains of OX40L linked to an immunoglobulin Fc domain.
[0251] In some embodiments, an OX40 agonist may be any one of the
OX40 agonists described in U.S. Pat. No. 7,696,175.
[0252] In some embodiments, an OX40 agonist may be an oligomeric or
multimeric molecule. For example, an OX40 agonist may contain one
or more domains (e.g., a leucine zipper domain) that allows
proteins to oligomerize. In some embodiments, an OX40 agonist may
include one or more extracellular domains of OX40L linked to one or
more leucine zipper domains.
[0253] In some embodiments, an OX40 agonist may be any one of the
OX40 agonists described in European Patent No. EP0672141 B1.
[0254] In some embodiments, an OX40 agonist may be a trimeric OX40L
fusion protein. For example, an OX40 agonist may include one or
more extracellular domains of OX40L linked to an immunoglobulin Fc
domain and a trimerization domain (including without limitation an
isoleucine zipper domain).
[0255] In some embodiments, an OX40 agonist may be any one of the
OX40 agonists described in International Publication No.
WO2006/121810. In some embodiments, the OX40 agonist is
MEDI6383.
[0256] In a further aspect, an anti-OX40 agonist and/or antibody
according to any of the above embodiments may incorporate any of
the features, singly or in combination, as described below.
[0257] 1. Antibody Affinity
[0258] In certain embodiments, an antibody provided herein has a
dissociation constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM,
.ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM, or
.ltoreq.0.001 nM (e.g. 10-8 M or less, e.g. from 10-8 M to 10-13 M,
e.g., from 10-9 M to 10-13 M).
[0259] In one embodiment, Kd is measured by a radiolabeled antigen
binding assay (RIA). In one embodiment, an RIA is performed with
the Fab version of an antibody of interest and its antigen. For
example, solution binding affinity of Fabs for antigen is measured
by equilibrating Fab with a minimal concentration of (1251)-labeled
antigen in the presence of a titration series of unlabeled antigen,
then capturing bound antigen with an anti-Fab antibody-coated plate
(see, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)). To
establish conditions for the assay, MICROTITER.RTM. multi-well
plates (Thermo Scientific) are coated overnight with 5 .mu.g/ml of
a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium
carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine
serum albumin in PBS for two to five hours at room temperature
(approximately 23.degree. C.). In a non-adsorbent plate (Nunc
#269620), 100 pM or 26 pM [125I]-antigen are mixed with serial
dilutions of a Fab of interest (e.g., consistent with assessment of
the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res.
57:4593-4599 (1997)). The Fab of interest is then incubated
overnight; however, the incubation may continue for a longer period
(e.g., about 65 hours) to ensure that equilibrium is reached.
Thereafter, the mixtures are transferred to the capture plate for
incubation at room temperature (e.g., for one hour). The solution
is then removed and the plate washed eight times with 0.1%
polysorbate 20 (TWEEN-20.RTM.) in PBS. When the plates have dried,
150 .mu.l/well of scintillant (MICROSCINT-20 .TM.; Packard) is
added, and the plates are counted on a TOPCOUNT.TM. gamma counter
(Packard) for ten minutes. Concentrations of each Fab that give
less than or equal to 20% of maximal binding are chosen for use in
competitive binding assays.
[0260] According to another embodiment, Kd is measured using a
BIACORE.RTM. surface plasmon resonance assay. For example, an assay
using a BIACORE.RTM.-2000 or a BIACORE.RTM.-3000 (BIAcore, Inc.,
Piscataway, N.J.) is performed at 25.degree. C. with immobilized
antigen CM5 chips at .about.10 response units (RU). In one
embodiment, carboxymethylated dextran biosensor chips (CM5,
BIACORE, Inc.) are activated with
N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC)
and N-hydroxysuccinimide (NHS) according to the supplier's
instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8,
to 5 .mu.g/ml (.about.0.2 .mu.M) before injection at a flow rate of
5 .mu.l/minute to achieve approximately 10 response units (RU) of
coupled protein. Following the injection of antigen, 1 M
ethanolamine is injected to block unreacted groups. For kinetics
measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM)
are injected in PBS with 0.05% polysorbate 20 (TWEEN-20.TM.)
surfactant (PBST) at 25.degree. C. at a flow rate of approximately
25 .mu.l/min. Association rates (kon) and dissociation rates (koff)
are calculated using a simple one-to-one Langmuir binding model
(BIACORE.RTM. Evaluation Software version 3.2) by simultaneously
fitting the association and dissociation sensorgrams. The
equilibrium dissociation constant (Kd) is calculated as the ratio
koff/kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999).
If the on-rate exceeds 106 M-1 s-1 by the surface plasmon resonance
assay above, then the on-rate can be determined by using a
fluorescent quenching technique that measures the increase or
decrease in fluorescence emission intensity (excitation=295 nm;
emission=340 nm, 16 nm band-pass) at 25.degree. C. of a 20 nM
anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of
increasing concentrations of antigen as measured in a spectrometer,
such as a stop-flow equipped spectrophometer (Aviv Instruments) or
a 8000-series SLM-AMINCO.TM. spectrophotometer (ThermoSpectronic)
with a stirred cuvette.
[0261] 2. Antibody Fragments
[0262] In certain embodiments, an antibody provided herein is an
antibody fragment. Antibody fragments include, but are not limited
to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, and other
fragments described below. For a review of certain antibody
fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a
review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology
of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.,
(Springer-Verlag, New York), pp. 269-315 (1994); see also WO
93/16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For
discussion of Fab and F(ab')2 fragments comprising salvage receptor
binding epitope residues and having increased in vivo half-life,
see U.S. Pat. No. 5,869,046.
[0263] Diabodies are antibody fragments with two antigen-binding
sites that may be bivalent or bispecific. See, for example, EP
404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003);
and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448
(1993). Triabodies and tetrabodies are also described in Hudson et
al., Nat. Med. 9:129-134 (2003).
[0264] Single-domain antibodies are antibody fragments comprising
all or a portion of the heavy chain variable domain or all or a
portion of the light chain variable domain of an antibody. In
certain embodiments, a single-domain antibody is a human
single-domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g.,
U.S. Pat. No. 6,248,516 B1).
[0265] Antibody fragments can be made by various techniques,
including but not limited to proteolytic digestion of an intact
antibody as well as production by recombinant host cells (e.g. E.
coli or phage), as described herein.
[0266] 3. Chimeric and Humanized Antibodies
[0267] In certain embodiments, an antibody provided herein is a
chimeric antibody. Certain chimeric antibodies are described, e.g.,
in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad.
Sci. USA, 81:6851-6855 (1984)). In one example, a chimeric antibody
comprises a non-human variable region (e.g., a variable region
derived from a mouse, rat, hamster, rabbit, or non-human primate,
such as a monkey) and a human constant region. In a further
example, a chimeric antibody is a "class switched" antibody in
which the class or subclass has been changed from that of the
parent antibody. Chimeric antibodies include antigen-binding
fragments thereof.
[0268] In certain embodiments, a chimeric antibody is a humanized
antibody. Typically, a non-human antibody is humanized to reduce
immunogenicity to humans, while retaining the specificity and
affinity of the parental non-human antibody. Generally, a humanized
antibody comprises one or more variable domains in which HVRs,
e.g., CDRs, (or portions thereof) are derived from a non-human
antibody, and FRs (or portions thereof) are derived from human
antibody sequences. A humanized antibody optionally will also
comprise at least a portion of a human constant region. In some
embodiments, some FR residues in a humanized antibody are
substituted with corresponding residues from a non-human antibody
(e.g., the antibody from which the HVR residues are derived), e.g.,
to restore or improve antibody specificity or affinity.
[0269] Humanized antibodies and methods of making them are
reviewed, e.g., in Almagro and Fransson, Front. Biosci.
13:1619-1633 (2008), and are further described, e.g., in Riechmann
et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad.
Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337,
7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods
36:25-34 (2005) (describing specificity determining region (SDR)
grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing
"resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005)
(describing "FR shuffling"); and Osbourn et al., Methods 36:61-68
(2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000)
(describing the "guided selection" approach to FR shuffling).
[0270] Human framework regions that may be used for humanization
include but are not limited to: framework regions selected using
the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296
(1993)); framework regions derived from the consensus sequence of
human antibodies of a particular subgroup of light or heavy chain
variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci.
USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623
(1993)); human mature (somatically mutated) framework regions or
human germline framework regions (see, e.g., Almagro and Fransson,
Front. Biosci. 13:1619-1633 (2008)); and framework regions derived
from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem.
272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.
271:22611-22618 (1996)).
[0271] 4. Human Antibodies
[0272] In certain embodiments, an antibody provided herein is a
human antibody. Human antibodies can be produced using various
techniques known in the art. Human antibodies are described
generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:
368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459
(2008).
[0273] Human antibodies may be prepared by administering an
immunogen to a transgenic animal that has been modified to produce
intact human antibodies or intact antibodies with human variable
regions in response to antigenic challenge. Such animals typically
contain all or a portion of the human immunoglobulin loci, which
replace the endogenous immunoglobulin loci, or which are present
extrachromosomally or integrated randomly into the animal's
chromosomes. In such transgenic mice, the endogenous immunoglobulin
loci have generally been inactivated. For review of methods for
obtaining human antibodies from transgenic animals, see Lonberg,
Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Pat. Nos.
6,075,181 and 6,150,584 describing XENOMOUSE.TM. technology; U.S.
Pat. No. 5,770,429 describing HuMab.RTM. technology; U.S. Pat. No.
7,041,870 describing K-M MOUSE.RTM. technology, and U.S. Patent
Application Publication No. US 2007/0061900, describing
VelociMouse.RTM. technology). Human variable regions from intact
antibodies generated by such animals may be further modified, e.g.,
by combining with a different human constant region.
[0274] Human antibodies can also be made by hybridoma-based
methods. Human myeloma and mouse-human heteromyeloma cell lines for
the production of human monoclonal antibodies have been described.
(See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al.,
Monoclonal Antibody Production Techniques and Applications, pp.
51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J.
Immunol., 147: 86 (1991).) Human antibodies generated via human
B-cell hybridoma technology are also described in Li et al., Proc.
Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods
include those described, for example, in U.S. Pat. No. 7,189,826
(describing production of monoclonal human IgM antibodies from
hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268
(2006) (describing human-human hybridomas). Human hybridoma
technology (Trioma technology) is also described in Vollmers and
Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and
Vollmers and Brandlein, Methods and Findings in Experimental and
Clinical Pharmacology, 27(3):185-91 (2005).
[0275] Human antibodies may also be generated by isolating Fv clone
variable domain sequences selected from human-derived phage display
libraries. Such variable domain sequences may then be combined with
a desired human constant domain. Techniques for selecting human
antibodies from antibody libraries are described below.
[0276] 5. Library-Derived Antibodies
[0277] Antibodies of the invention may be isolated by screening
combinatorial libraries for antibodies with the desired activity or
activities. For example, a variety of methods are known in the art
for generating phage display libraries and screening such libraries
for antibodies possessing the desired binding characteristics. Such
methods are reviewed, e.g., in Hoogenboom et al. in Methods in
Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press,
Totowa, N. J., 2001) and further described, e.g., in the McCafferty
et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628
(1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and
Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed.,
Human Press, Totowa, N. J., 2003); Sidhu et al., J. Mol. Biol.
338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093
(2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472
(2004); and Lee et al., J. Immunol. Methods 284(1-2):
119-132(2004).
[0278] In certain phage display methods, repertoires of VH and VL
genes are separately cloned by polymerase chain reaction (PCR) and
recombined randomly in phage libraries, which can then be screened
for antigen-binding phage as described in Winter et al., Ann. Rev.
Immunol., 12: 433-455 (1994). Phage typically display antibody
fragments, either as single-chain Fv (scFv) fragments or as Fab
fragments. Libraries from immunized sources provide high-affinity
antibodies to the immunogen without the requirement of constructing
hybridomas. Alternatively, the naive repertoire can be cloned
(e.g., from human) to provide a single source of antibodies to a
wide range of non-self and also self antigens without any
immunization as described by Griffiths et al., EMBO J, 12: 725-734
(1993). Finally, naive libraries can also be made synthetically by
cloning unrearranged V-gene segments from stem cells, and using PCR
primers containing random sequence to encode the highly variable
CDR3 regions and to accomplish rearrangement in vitro, as described
by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
Patent publications describing human antibody phage libraries
include, for example: U.S. Pat. No. 5,750,373, and US Patent
Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000,
2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and
2009/0002360.
[0279] Antibodies or antibody fragments isolated from human
antibody libraries are considered human antibodies or human
antibody fragments herein.
[0280] 6. Multispecific Antibodies
[0281] In certain embodiments, an antibody provided herein is a
multispecific antibody, e.g. a bispecific antibody. Multispecific
antibodies are monoclonal antibodies that have binding
specificities for at least two different sites. In certain
embodiments, one of the binding specificities is for OX40 and the
other is for any other antigen. In certain embodiments, bispecific
antibodies may bind to two different epitopes of OX40. Bispecific
antibodies may also be used to localize cytotoxic agents to cells
which express OX40. Bispecific antibodies can be prepared as full
length antibodies or antibody fragments.
[0282] Techniques for making multispecific antibodies include, but
are not limited to, recombinant co-expression of two immunoglobulin
heavy chain-light chain pairs having different specificities (see
Milstein and Cuello, Nature 305: 537 (1983)), WO 93/08829, and
Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole"
engineering (see, e.g., U.S. Pat. No. 5,731,168). Multi-specific
antibodies may also be made by engineering electrostatic steering
effects for making antibody Fc-heterodimeric molecules (WO
2009/089004A1); cross-linking two or more antibodies or fragments
(see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science,
229: 81 (1985)); using leucine zippers to produce bi-specific
antibodies (see, e.g., Kostelny et al., J. Immunol.,
148(5):1547-1553 (1992)); using "diabody" technology for making
bispecific antibody fragments (see, e.g., Hollinger et al., Proc.
Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain
Fv (sFv) dimers (see, e.g. Gruber et al., J. Immunol., 152:5368
(1994)); and preparing trispecific antibodies as described, e.g.,
in Tutt et al. J. Immunol. 147: 60 (1991).
[0283] Engineered antibodies with three or more functional antigen
binding sites, including "Octopus antibodies," are also included
herein (see, e.g. US 2006/0025576A1).
[0284] The antibody or fragment herein also includes a "Dual Acting
FAb" or "DAF" comprising an antigen binding site that binds to OX40
as well as another, different antigen (see, US 2008/0069820, for
example).
[0285] 7. Antibody Variants
[0286] In certain embodiments, amino acid sequence variants of the
antibodies provided herein are contemplated. For example, it may be
desirable to improve the binding affinity and/or other biological
properties of the antibody. Amino acid sequence variants of an
antibody may be prepared by introducing appropriate modifications
into the nucleotide sequence encoding the antibody, or by peptide
synthesis. Such modifications include, for example, deletions from,
and/or insertions into and/or substitutions of residues within the
amino acid sequences of the antibody. Any combination of deletion,
insertion, and substitution can be made to arrive at the final
construct, provided that the final construct possesses the desired
characteristics, e.g., antigen-binding.
[0287] a) Substitution, Insertion, and Deletion Variants
[0288] In certain embodiments, antibody variants having one or more
amino acid substitutions are provided. Sites of interest for
substitutional mutagenesis include the HVRs and FRs. Conservative
substitutions are shown in Table A under the heading of "preferred
substitutions." More substantial changes are provided in Table A
under the heading of "exemplary substitutions," and as further
described below in reference to amino acid side chain classes.
Amino acid substitutions may be introduced into an antibody of
interest and the products screened for a desired activity, e.g.,
retained/improved antigen binding, decreased immunogenicity, or
improved ADCC or CDC.
TABLE-US-00005 TABLE A Original Exemplary Preferred Residue
Substitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys;
Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn
Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp
Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val;
Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met;
Ala; Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu
Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)
Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe;
Thr; Ser Phe Val (V) Ile; Leu; Met; Phe; Ala; Norleucine Leu
[0289] Amino acids may be grouped according to common side-chain
properties:
[0290] (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;
[0291] (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;
[0292] (3) acidic: Asp, Glu;
[0293] (4) basic: His, Lys, Arg;
[0294] (5) residues that influence chain orientation: Gly, Pro;
[0295] (6) aromatic: Trp, Tyr, Phe.
[0296] Non-conservative substitutions will entail exchanging a
member of one of these classes for another class.
[0297] One type of substitutional variant involves substituting one
or more hypervariable region residues of a parent antibody (e.g. a
humanized or human antibody). Generally, the resulting variant(s)
selected for further study will have modifications (e.g.,
improvements) in certain biological properties (e.g., increased
affinity, reduced immunogenicity) relative to the parent antibody
and/or will have substantially retained certain biological
properties of the parent antibody. An exemplary substitutional
variant is an affinity matured antibody, which may be conveniently
generated, e.g., using phage display-based affinity maturation
techniques such as those described herein. Briefly, one or more HVR
residues are mutated and the variant antibodies displayed on phage
and screened for a particular biological activity (e.g. binding
affinity).
[0298] Alterations (e.g., substitutions) may be made in HVRs, e.g.,
to improve antibody affinity. Such alterations may be made in HVR
"hotspots," i.e., residues encoded by codons that undergo mutation
at high frequency during the somatic maturation process (see, e.g.,
Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or residues
that contact antigen, with the resulting variant VH or VL being
tested for binding affinity. Affinity maturation by constructing
and reselecting from secondary libraries has been described, e.g.,
in Hoogenboom et al. in Methods in Molecular Biology 178:1-37
(O'Brien et al., ed., Human Press, Totowa, N.J., (2001).) In some
embodiments of affinity maturation, diversity is introduced into
the variable genes chosen for maturation by any of a variety of
methods (e.g., error-prone PCR, chain shuffling, or
oligonucleotide-directed mutagenesis). A secondary library is then
created. The library is then screened to identify any antibody
variants with the desired affinity. Another method to introduce
diversity involves HVR-directed approaches, in which several HVR
residues (e.g., 4-6 residues at a time) are randomized. HVR
residues involved in antigen binding may be specifically
identified, e.g., using alanine scanning mutagenesis or modeling.
CDR-H3 and CDR-L3 in particular are often targeted.
[0299] In certain embodiments, substitutions, insertions, or
deletions may occur within one or more HVRs so long as such
alterations do not substantially reduce the ability of the antibody
to bind antigen. For example, conservative alterations (e.g.,
conservative substitutions as provided herein) that do not
substantially reduce binding affinity may be made in HVRs. Such
alterations may, for example, be outside of antigen contacting
residues in the HVRs. In certain embodiments of the variant VH and
VL sequences provided above, each HVR either is unaltered, or
contains no more than one, two or three amino acid
substitutions.
[0300] A useful method for identification of residues or regions of
an antibody that may be targeted for mutagenesis is called "alanine
scanning mutagenesis" as described by Cunningham and Wells (1989)
Science, 244:1081-1085. In this method, a residue or group of
target residues (e.g., charged residues such as arg, asp, his, lys,
and glu) are identified and replaced by a neutral or negatively
charged amino acid (e.g., alanine or polyalanine) to determine
whether the interaction of the antibody with antigen is affected.
Further substitutions may be introduced at the amino acid locations
demonstrating functional sensitivity to the initial substitutions.
Alternatively, or additionally, a crystal structure of an
antigen-antibody complex to identify contact points between the
antibody and antigen. Such contact residues and neighboring
residues may be targeted or eliminated as candidates for
substitution. Variants may be screened to determine whether they
contain the desired properties.
[0301] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a hundred or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Examples of terminal insertions include an antibody with an
N-terminal methionyl residue. Other insertional variants of the
antibody molecule include the fusion to the N- or C-terminus of the
antibody to an enzyme (e.g. for ADEPT) or a polypeptide which
increases the serum half-life of the antibody.
[0302] b) Glycosylation Variants
[0303] In certain embodiments, an antibody provided herein is
altered to increase or decrease the extent to which the antibody is
glycosylated. Addition or deletion of glycosylation sites to an
antibody may be conveniently accomplished by altering the amino
acid sequence such that one or more glycosylation sites is created
or removed.
[0304] Where the antibody comprises an Fc region, the carbohydrate
attached thereto may be altered. Native antibodies produced by
mammalian cells typically comprise a branched, biantennary
oligosaccharide that is generally attached by an N-linkage to
Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al.
TIBTECH 15:26-32 (1997). The oligosaccharide may include various
carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc),
galactose, and sialic acid, as well as a fucose attached to a
GlcNAc in the "stem" of the biantennary oligosaccharide structure.
In some embodiments, modifications of the oligosaccharide in an
antibody of the invention may be made in order to create antibody
variants with certain improved properties.
[0305] In one embodiment, antibody variants are provided having a
carbohydrate structure that lacks fucose attached (directly or
indirectly) to an Fc region. For example, the amount of fucose in
such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65%
or from 20% to 40%. The amount of fucose is determined by
calculating the average amount of fucose within the sugar chain at
Asn297, relative to the sum of all glycostructures attached to Asn
297 (e. g. complex, hybrid and high mannose structures) as measured
by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for
example. Asn297 refers to the asparagine residue located at about
position 297 in the Fc region (Eu numbering of Fc region residues);
however, Asn297 may also be located about .+-.3 amino acids
upstream or downstream of position 297, i.e., between positions 294
and 300, due to minor sequence variations in antibodies. Such
fucosylation variants may have improved ADCC function. See, e.g.,
US Patent Publication Nos. US 2003/0157108 (Presta, L.); US
2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications
related to "defucosylated" or "fucose-deficient" antibody variants
include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US
2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US
2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO
2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742;
WO2002/031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004);
Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of
cell lines capable of producing defucosylated antibodies include
Lec13 CHO cells deficient in protein fucosylation (Ripka et al.
Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No US
2003/0157108 A1, Presta, L; and WO 2004/056312 A1, Adams et al.,
especially at Example 11), and knockout cell lines, such as
alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,
e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda,
Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and
WO2003/085107).
[0306] Antibodies variants are further provided with bisected
oligosaccharides, e.g., in which a biantennary oligosaccharide
attached to the Fc region of the antibody is bisected by GlcNAc.
Such antibody variants may have reduced fucosylation and/or
improved ADCC function. Examples of such antibody variants are
described, e.g., in WO 2003/011878 (Jean-Mairet et al.); U.S. Pat.
No. 6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.).
Antibody variants with at least one galactose residue in the
oligosaccharide attached to the Fc region are also provided. Such
antibody variants may have improved CDC function. Such antibody
variants are described, e.g., in WO 1997/30087 (Patel et al.); WO
1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).
[0307] c) Fc Region Variants
[0308] In certain embodiments, one or more amino acid modifications
may be introduced into the Fc region of an antibody provided
herein, thereby generating an Fc region variant. The Fc region
variant may comprise a human Fc region sequence (e.g., a human
IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid
modification (e.g. a substitution) at one or more amino acid
positions.
[0309] In certain embodiments, the invention contemplates an
antibody variant that possesses some but not all effector
functions, which make it a desirable candidate for applications in
which the half life of the antibody in vivo is important yet
certain effector functions (such as complement and ADCC) are
unnecessary or deleterious. In vitro and/or in vivo cytotoxicity
assays can be conducted to confirm the reduction/depletion of CDC
and/or ADCC activities. For example, Fc receptor (FcR) binding
assays can be conducted to ensure that the antibody lacks
Fc.gamma.R binding (hence likely lacking ADCC activity), but
retains FcRn binding ability. The primary cells for mediating ADCC,
NK cells, express Fc(RIII only, whereas monocytes express Fc(RI,
Fc(RII and Fc(RIII. FcR expression on hematopoietic cells is
summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev.
Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays
to assess ADCC activity of a molecule of interest is described in
U.S. Pat. No. 5,500,362 (see, e.g. Hellstrom, I. et al. Proc. Nat'l
Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc.
Nat'l Acad. Sci. USA 82:1499-1502 (1985); U.S. Pat. No. 5,821,337
(see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)).
Alternatively, non-radioactive assays methods may be employed (see,
for example, ACTI.TM. non-radioactive cytotoxicity assay for flow
cytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox
96.RTM. non-radioactive cytotoxicity assay (Promega, Madison,
Wis.). Useful effector cells for such assays include peripheral
blood mononuclear cells (PBMC) and Natural Killer (NK) cells.
Alternatively, or additionally, ADCC activity of the molecule of
interest may be assessed in vivo, e.g., in an animal model such as
that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA
95:652-656 (1998). C1q binding assays may also be carried out to
confirm that the antibody is unable to bind C1q and hence lacks CDC
activity. See, e.g., C1q and C3c binding ELISA in WO 2006/029879
and WO 2005/100402. To assess complement activation, a CDC assay
may be performed (see, for example, Gazzano-Santoro et al., J.
Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood
101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood
103:2738-2743 (2004)). FcRn binding and in vivo clearance/half life
determinations can also be performed using methods known in the art
(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769
(2006)).
[0310] In some embodiments, an antibody includes an Fc region with
a mutation that decreases binding to an Fc receptor. Antibodies
with reduced effector function include without limitation those
with substitution of one or more of Fc region residues 238, 265,
269, 270, 297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc
mutants include Fc mutants with substitutions at two or more of
amino acid positions 265, 269, 270, 297 and 327, including the
so-called "DANA" Fc mutant with substitution of residues 265 and
297 to alanine (U.S. Pat. No. 7,332,581).
[0311] Certain antibody variants with improved or diminished
binding to FcRs are described. (See, e.g., U.S. Pat. No. 6,737,056;
WO 2004/056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604
(2001).)
[0312] In certain embodiments, an antibody variant comprises an Fc
region with one or more amino acid substitutions which improve
ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the
Fc region (EU numbering of residues).
[0313] In some embodiments, alterations are made in the Fc region
that result in altered (i.e., either improved or diminished) C1q
binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as
described in U.S. Pat. No. 6,194,551, WO 99/51642, and Idusogie et
al. J. Immunol. 164: 4178-4184 (2000).
[0314] Antibodies with increased half lives and improved binding to
the neonatal Fc receptor (FcRn), which is responsible for the
transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol.
117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are
described in US2005/0014934A1 (Hinton et al.). Those antibodies
comprise an Fc region with one or more substitutions therein which
improve binding of the Fc region to FcRn. Such Fc variants include
those with substitutions at one or more of Fc region residues: 238,
256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360,
362, 376, 378, 380, 382, 413, 424 or 434, e.g., substitution of Fc
region residue 434 (U.S. Pat. No. 7,371,826).
[0315] See also Duncan & Winter, Nature 322:738-40 (1988); U.S.
Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351
concerning other examples of Fc region variants.
[0316] d) Cysteine Engineered Antibody Variants
[0317] In certain embodiments, it may be desirable to create
cysteine engineered antibodies, e.g., "thioMAbs," in which one or
more residues of an antibody are substituted with cysteine
residues. In particular embodiments, the substituted residues occur
at accessible sites of the antibody. By substituting those residues
with cysteine, reactive thiol groups are thereby positioned at
accessible sites of the antibody and may be used to conjugate the
antibody to other moieties, such as drug moieties or linker-drug
moieties, to create an immunoconjugate, as described further
herein. In certain embodiments, any one or more of the following
residues may be substituted with cysteine: V205 (Kabat numbering)
of the light chain; A118 (EU numbering) of the heavy chain; and
S400 (EU numbering) of the heavy chain Fc region. Cysteine
engineered antibodies may be generated as described, e.g., in U.S.
Pat. No. 7,521,541.
[0318] e) Antibody Derivatives
[0319] In certain embodiments, an antibody provided herein may be
further modified to contain additional nonproteinaceous moieties
that are known in the art and readily available. The moieties
suitable for derivatization of the antibody include but are not
limited to water soluble polymers. Non-limiting examples of water
soluble polymers include, but are not limited to, polyethylene
glycol (PEG), copolymers of ethylene glycol/propylene glycol,
carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl
pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane,
ethylene/maleic anhydride copolymer, polyaminoacids (either
homopolymers or random copolymers), and dextran or poly(n-vinyl
pyrrolidone)polyethylene glycol, propropylene glycol homopolymers,
prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated
polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
Polyethylene glycol propionaldehyde may have advantages in
manufacturing due to its stability in water. The polymer may be of
any molecular weight, and may be branched or unbranched. The number
of polymers attached to the antibody may vary, and if more than one
polymer are attached, they can be the same or different molecules.
In general, the number and/or type of polymers used for
derivatization can be determined based on considerations including,
but not limited to, the particular properties or functions of the
antibody to be improved, whether the antibody derivative will be
used in a therapy under defined conditions, etc.
[0320] In another embodiment, conjugates of an antibody and
nonproteinaceous moiety that may be selectively heated by exposure
to radiation are provided. In one embodiment, the nonproteinaceous
moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA
102: 11600-11605 (2005)). The radiation may be of any wavelength,
and includes, but is not limited to, wavelengths that do not harm
ordinary cells, but which heat the nonproteinaceous moiety to a
temperature at which cells proximal to the
antibody-nonproteinaceous moiety are killed.
Recombinant Methods and Compositions
[0321] Antibodies may be produced using recombinant methods and
compositions, e.g., as described in U.S. Pat. No. 4,816,567. In one
embodiment, isolated nucleic acid encoding an anti-OX40 antibody
described herein is provided. Such nucleic acid may encode an amino
acid sequence comprising the VL and/or an amino acid sequence
comprising the VH of the antibody (e.g., the light and/or heavy
chains of the antibody). In a further embodiment, one or more
vectors (e.g., expression vectors) comprising such nucleic acid are
provided. In a further embodiment, a host cell comprising such
nucleic acid is provided. In one such embodiment, a host cell
comprises (e.g., has been transformed with): (1) a vector
comprising a nucleic acid that encodes an amino acid sequence
comprising the VL of the antibody and an amino acid sequence
comprising the VH of the antibody, or (2) a first vector comprising
a nucleic acid that encodes an amino acid sequence comprising the
VL of the antibody and a second vector comprising a nucleic acid
that encodes an amino acid sequence comprising the VH of the
antibody. In one embodiment, the host cell is eukaryotic, e.g. a
Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., Y0, NS0,
Sp20 cell). In one embodiment, a method of making an anti-OX40
antibody is provided, wherein the method comprises culturing a host
cell comprising a nucleic acid encoding the antibody, as provided
above, under conditions suitable for expression of the antibody,
and optionally recovering the antibody from the host cell (or host
cell culture medium).
[0322] For recombinant production of an anti-OX40 antibody, nucleic
acid encoding an antibody, e.g., as described above, is isolated
and inserted into one or more vectors for further cloning and/or
expression in a host cell. Such nucleic acid may be readily
isolated and sequenced using conventional procedures (e.g., by
using oligonucleotide probes that are capable of binding
specifically to genes encoding the heavy and light chains of the
antibody).
[0323] Suitable host cells for cloning or expression of
antibody-encoding vectors include prokaryotic or eukaryotic cells
described herein. For example, antibodies may be produced in
bacteria, in particular when glycosylation and Fc effector function
are not needed. For expression of antibody fragments and
polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237,
5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular
Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N. J.,
2003), pp. 245-254, describing expression of antibody fragments in
E. coli.) After expression, the antibody may be isolated from the
bacterial cell paste in a soluble fraction and can be further
purified.
[0324] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for antibody-encoding vectors, including fungi and yeast strains
whose glycosylation pathways have been "humanized," resulting in
the production of an antibody with a partially or fully human
glycosylation pattern. See Gerngross, Nat. Biotech. 22:1409-1414
(2004), and Li et al., Nat. Biotech. 24:210-215 (2006).
[0325] Suitable host cells for the expression of glycosylated
antibody are also derived from multicellular organisms
(invertebrates and vertebrates). Examples of invertebrate cells
include plant and insect cells. Numerous baculoviral strains have
been identified which may be used in conjunction with insect cells,
particularly for transfection of Spodoptera frugiperda cells.
[0326] Plant cell cultures can also be utilized as hosts. See,
e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978,
and 6,417,429 (describing PLANTIBODIES.TM. technology for producing
antibodies in transgenic plants).
[0327] Vertebrate cells may also be used as hosts. For example,
mammalian cell lines that are adapted to grow in suspension may be
useful. Other examples of useful mammalian host cell lines are
monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic
kidney line (293 or 293 cells as described, e.g., in Graham et al.,
J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse
sertoli cells (TM4 cells as described, e.g., in Mather, Biol.
Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African
green monkey kidney cells (VERO-76); human cervical carcinoma cells
(HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL
3A); human lung cells (W138); human liver cells (Hep G2); mouse
mammary tumor (MMT 060562); TRI cells, as described, e.g., in
Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5
cells; and FS4 cells. Other useful mammalian host cell lines
include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells
(Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and
myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of
certain mammalian host cell lines suitable for antibody production,
see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248
(B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268
(2003).
Assays
[0328] Anti-OX40 antibodies provided herein may be identified,
screened for, or characterized for their physical/chemical
properties and/or biological activities by various assays known in
the art.
[0329] 1. Binding Assays and Other Assays
[0330] In one aspect, an antibody of the invention is tested for
its antigen binding activity, e.g., by known methods such as ELISA,
Western blot, etc. OX40 binding may be determined using methods
known in the art and exemplary methods are disclosed herein. In one
embodiment, binding is measured using radioimmunoassay. An
exemplary radioimmunassay is exemplified in the Examples. OX40
antibody is iodinated, and competition reaction mixtures are
prepared containing a fixed concentration of iodinated antibody and
decreasing concentrations of serially diluted, unlabeled OZ X40
antibody. Cells expressing OX40 (e.g., BT474 cells stably
transfected with human OX40) are added to the reaction mixture.
Following an incubation, cells are washed to separate the free
iodinated OX40 antibody from the OX40 antibody bound to the cells.
Level of bound iodinated OX40 antibody is determined, e.g., by
counting radioactivity associated with cells, and binding affinity
determined using standard methods. In another embodiment, ability
of OX40 antibody to bind to surface-expressed OX40 (e.g., on T cell
subsets) is assessed using flow cytometry. Peripheral white blood
cells are obtained (e.g., from human, cynomolgus monkey, rat or
mouse) and cells are blocked with serum. Labeled OX40 antibody is
added in serial dilutions, and T cells are also stained to identify
T cell subsets (using methods known in the art). Following
incubation of the samples and washing, the cells are sorted using
flow cytometer, and data analyzed using methods well known in the
art. In another embodiment, OX40 binding may be analyzed using
surface plasmon resonance. An exemplary surface plasmon resonance
method is exemplified in the Examples.
[0331] In another aspect, competition assays may be used to
identify an antibody that competes with any of the anti-OX40
antibodies disclosed herein for binding to OX40. In certain
embodiments, such a competing antibody binds to the same epitope
(e.g., a linear or a conformational epitope) that is bound by any
of the anti-OX40 antibodies disclosed herein. Detailed exemplary
methods for mapping an epitope to which an antibody binds are
provided in Morris (1996) "Epitope Mapping Protocols," in Methods
in Molecular Biology vol. 66 (Humana Press, Totowa, N.J.). A
competition assay is exemplified in the Examples.
[0332] In an exemplary competition assay, immobilized OX40 is
incubated in a solution comprising a first labeled antibody that
binds to OX40 (e.g., mab 1A7.gr.1, mab 3C8.gr5) and a second
unlabeled antibody that is being tested for its ability to compete
with the first antibody for binding to OX40. The second antibody
may be present in a hybridoma supernatant. As a control,
immobilized OX40 is incubated in a solution comprising the first
labeled antibody but not the second unlabeled antibody. After
incubation under conditions permissive for binding of the first
antibody to OX40, excess unbound antibody is removed, and the
amount of label associated with immobilized OX40 is measured. If
the amount of label associated with immobilized OX40 is
substantially reduced in the test sample relative to the control
sample, then that indicates that the second antibody is competing
with the first antibody for binding to OX40. See Harlow and Lane
(1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.).
[0333] 2. Activity Assays
[0334] In one aspect, assays are provided for identifying anti-OX40
antibodies thereof having biological activity. Biological activity
may include, e.g., binding OX40 (e.g., binding human and/or
cynomolgus OX40), increasing OX40-mediated signal transduction
(e.g., increasing NFkB-mediated transcription), depleting cells
that express human OX40 (e.g., T cells), depleting cells that
express human OX40 by ADCC and/or phagocytosis, enhancing T
effector cell function (e.g., CD4+ effector T cell), e.g., by
increasing effector T cell proliferation and/or increasing cytokine
production (e.g., gamma interferon) by effector T cells, enhancing
memory T cell function (e.g., CD4+ memory T cell), e.g., by
increasing memory T cell proliferation and/or increasing cytokine
production by memory T cells (e.g., gamma interferon), inhibiting
regulatory T cell function (e.g., by decreasing Treg suppression of
effector T cell function (e.g., CD4+ effector T cell function),
binding human effector cells. Antibodies having such biological
activity in vivo and/or in vitro are also provided.
[0335] In certain embodiments, an antibody of the invention is
tested for such biological activity.
[0336] T cell costimulation may be assayed using methods known in
the art and exemplary methods are disclosed herein. For example, T
cells (e.g., memory or effector T cells) may be obtained from
peripheral white blood cells (e.g., isolated from human whole blood
using Ficoll gradient centrifugation). Memory T cells (e.g., CD4+
memory T cells) or effector T cells (e.g. CD4+ Teff cells) may be
isolated from PBMC using methods known in the art. For example, the
Miltenyi CD4+ memory T cell isolation kit or Miltenyi naive CD4+ T
cell isolation kit may be used. Isolated T cells are cultured in
the presence of antigen presenting cells (e.g., irradiated L cells
that express CD32 and CD80), and activated by addition of anti-CD3
antibody in the presence or absence of OX40 agonist antibody.
Effect of agonist OX40 antibody of T cell proliferation may be
measured using methods well known in the art. For example, the
CellTiter Glo kit (Promega) may be used, and results read on a
Multilabel Reader (Perkin Elmer). Effect of agonist OX40 antibody
on T cell function may also be determined by analysis of cytokines
produced by the T cell. In one embodiment, production of interferon
gamma by CD4+ T cells is determined, e.g., by measurement of
interferon gamma in cell culture supernatant. Methods for measuring
interferon gamma are well-known in the art.
[0337] Treg cell function may be assayed using methods known in the
art and exemplary methods are disclosed herein. In one example, the
ability of Treg to suppress effector T cell proliferation is
assayed. T cells are isolated from human whole blood using methods
known in the art (e.g., isolating memory T cells or naive T cells).
Purified CD4+naive T cells are labeled (e.g., with CFSE) and
purified Treg cells are labeled with a different reagent.
Irradiated antigen presenting cells (e.g., L cells expressing CD32
and CD80) are co-cultured with the labeled purified naive CD4+ T
cells and purified Tregs. The co-cultures are activated using
anti-CD3 antibody and tested in the presence or absence of agonist
OX40 antibody. Following a suitable time (e.g., 6 days of
coculture), level of CD4+naive T cell proliferation is tracked by
dye dilution in reduced label staining (e.g., reduced CFSE label
staining) using FACS analysis.
[0338] OX40 signaling may be assayed using methods well known in
the art and exemplary methods are disclosed herein. In one
embodiment, transgenic cells are generated that express human OX40
and a reporter gene comprising the NFkB promoter fused to a
reporter gene (e.g., beta luciferase). Addition of OX40 agonist
antibody to the cells results in increased NFkB transcription,
which is detected using an assay for the reporter gene.
[0339] Phagocytosis may be assayed, e.g., by using monocyte-derived
macrophages, or U937 cells (a human histiocytic lymphoma cells line
with the morphology and characteristics of mature macrophages).
OX40 expressing cells are added to the monocyte-derived macrophages
or U937 cells in the presence or absence of anti-OX40 agonist
antibody. Following culturing of the cells for a suitable period of
time, the percentage of phagocytosis is determined by examining
percentage of cells that double stain for markers of 1) the
macrophage or U937 cell and 2) the OX40 expressing cell, and
dividing this by the total number of cells that show markers of the
OX40 expressing cell (e.g., GFP). Analysis may be done by flow
cytometry. In another embodiment, analysis may be done by
fluorescent microscopy analysis.
[0340] ADCC may be assayed, e.g., using methods well known in the
art. Exemplary methods are described in the definition section and
an exemplary assay is disclosed in the Examples. In some
embodiments, level of OX40 is characterized on an OX40 expressing
cell that is used for testing in an ADCC assay. The cell may be
stained with a detectably labeled anti-OX40 antibody (e.g., PE
labeled), then level of fluorescence determined using flow
cytometry, and results presented as median fluorescence intensity
(MFI). In another embodiment, ADCC may be analyzed by CellTiter Glo
assay kit and cell viability/cytotoxicity may be determined by
chemioluminescence.
[0341] The binding affinities of various antibodies to
Fc.gamma.RIA, Fc.gamma.RIIA, Fc.gamma.RIIB, and two allotypes of
Fc.gamma.RIIIA (F158 and V158) may be measured in ELISA-based
ligand-binding assays using the respective recombinant Fc.gamma.
receptors. Purified human Fc.gamma. receptors are expressed as
fusion proteins containing the extracellular domain of the receptor
.gamma. chain linked to a Gly/6.times.His/glutathione S-transferase
(GST) polypeptide tag at the C-terminus. The binding affinities of
antibodies to those human Fc.gamma. receptors are assayed as
follows. For the low-affinity receptors, i.e. Fc.gamma.RIIA
(CD32A), Fc.gamma.RIIB (CD32B), and the two allotypes of
Fc.gamma.RIIIA (CD16), F-158 and V-158, antibodies may be tested as
multimers by cross-linking with a F(ab')2 fragment of goat
anti-human kappa chain (ICN Biomedical; Irvine, Calif.) at an
approximate molar ratio of 1:3 antibody:cross-linking F(ab')2.
Plates are coated with an anti-GST antibody (Genentech) and blocked
with bovine serum albumin (BSA). After washing with
phosphate-buffered saline (PBS) containing 0.05% Tween-20 with an
ELx405.TM. plate washer (Biotek Instruments; Winooski, Vt.),
Fc.gamma. receptors are added to the plate at 25 ng/well and
incubated at room temperature for 1 hour. After the plates are
washed, serial dilutions of test antibodies are added as multimeric
complexes and the plates were incubated at room temperature for 2
hours. Following plate washing to remove unbound antibodies, the
antibodies bound to the Fc.gamma. receptor are detected with
horseradish peroxidase (HRP)-conjugated F(ab')2 fragment of goat
anti-human F(ab')2 (Jackson ImmunoResearch Laboratories; West
Grove, Pa.) followed by the addition of substrate,
tetramethylbenzidine (TMB) (Kirkegaard & Perry Laboratories;
Gaithersburg, Md.). The plates are incubated at room temperature
for 5-20 minutes, depending on the Fc.gamma. receptors tested, to
allow color development. The reaction is terminated with 1 M H3PO4
and absorbance at 450 nm was measured with a microplate reader
(SpectraMax.RTM.190, Molecular Devices; Sunnyvale, Calif.).
Dose-response binding curves are generated by plotting the mean
absorbance values from the duplicates of antibody dilutions against
the concentrations of the antibody. Values for the effective
concentration of the antibody at which 50% of the maximum response
from binding to the Fc.gamma. receptor is detected (EC50) were
determined after fitting the binding curve with a four-parameter
equation using SoftMax Pro (Molecular Devices).
[0342] To select for antibodies which induce cell death, loss of
membrane integrity as indicated by, e.g., propidium iodide (PI),
trypan blue or 7AAD uptake may be assessed relative to control. A
PI uptake assay can be performed in the absence of complement and
immune effector cells. OX40 expressing cells are incubated with
medium alone or medium containing of the appropriate monoclonal
antibody at e.g., about 10 .mu.g/ml. The cells are incubated for a
time period (e.g., 1 or 3 days). Following each treatment, cells
are washed and aliquoted. In some embodiments, cells are aliquoted
into 35 mm strainer-capped 12.times.75 tubes (1 ml per tube, 3
tubes per treatment group) for removal of cell clumps. Tubes then
receive PI (10 .mu.g/ml). Samples may be analyzed using a
FACSCAN.TM. flow cytometer and FACSCONVERT.TM. CellQuest software
(Becton Dickinson).
[0343] Cells for use in any of the above in vitro assays include
cells or cell lines that naturally express OX40 or that have been
engineered to express OX40. Such cells include activated T cells,
Treg cells and activated memory T cells that naturally express
OX40. Such cells also include cell lines that express OX40 and cell
lines that do not normally express OX40 but have been transfected
with nucleic acid encoding OX40. Exemplary cell lines provided
herein for use in any of the above in vitro assays include
transgenic BT474 cells (a human breast cancer cell line) that
express human OX40
[0344] It is understood that any of the above assays may be carried
out using an immunoconjugate of the invention in place of or in
addition to an anti-OX40 antibody.
[0345] It is understood that any of the above assays may be carried
out using anti-OX40 antibody and an additional therapeutic
agent.
Immunoconjugates
[0346] The invention also provides immunoconjugates comprising an
anti-OX40 antibody herein conjugated to one or more cytotoxic
agents, such as chemotherapeutic agents or drugs, growth inhibitory
agents, toxins (e.g., protein toxins, enzymatically active toxins
of bacterial, fungal, plant, or animal origin, or fragments
thereof), or radioactive isotopes.
[0347] In one embodiment, an immunoconjugate is an antibody-drug
conjugate (ADC) in which an antibody is conjugated to one or more
drugs, including but not limited to a maytansinoid (see U.S. Pat.
Nos. 5,208,020, 5,416,064 and European Patent EP 0 425 235 B1); an
auristatin such as monomethylauristatin drug moieties DE and DF
(MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588, and
7,498,298); a dolastatin; a calicheamicin or derivative thereof
(see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285,
5,770,701, 5,770,710, 5,773,001, and 5,877,296; Hinman et al.,
Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res.
58:2925-2928 (1998)); an anthracycline such as daunomycin or
doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523
(2006); Jeffrey et al., Bioorganic & Med. Chem. Letters
16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005);
Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000);
Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532
(2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S.
Pat. No. 6,630,579); methotrexate; vindesine; a taxane such as
docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a
trichothecene; and CC1065.
[0348] In another embodiment, an immunoconjugate comprises an
antibody as described herein conjugated to an enzymatically active
toxin or fragment thereof, including but not limited to diphtheria
A chain, nonbinding active fragments of diphtheria toxin, exotoxin
A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A
chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins,
dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and
PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria
officinalis inhibitor, gelonin, mitogellin, restrictocin,
phenomycin, enomycin, and the tricothecenes.
[0349] In another embodiment, an immunoconjugate comprises an
antibody as described herein conjugated to a radioactive atom to
form a radioconjugate. A variety of radioactive isotopes are
available for the production of radioconjugates. Examples include
At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and
radioactive isotopes of Lu. When the radioconjugate is used for
detection, it may comprise a radioactive atom for scintigraphic
studies, for example tc99m or I123, or a spin label for nuclear
magnetic resonance (NMR) imaging (also known as magnetic resonance
imaging, mri), such as iodine-123 again, iodine-131, indium-111,
fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium,
manganese or iron.
[0350] Conjugates of an antibody and cytotoxic agent may be made
using a variety of bifunctional protein coupling agents such as
N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC),
iminothiolane (IT), bifunctional derivatives of imidoesters (such
as dimethyl adipimidate HCl), active esters (such as disuccinimidyl
suberate), aldehydes (such as glutaraldehyde), bis-azido compounds
(such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium
derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine),
diisocyanates (such as toluene 2,6-diisocyanate), and bis-active
fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For
example, a ricin immunotoxin can be prepared as described in
Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled
1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid
(MX-DTPA) is an exemplary chelating agent for conjugation of
radionucleotide to the antibody. See WO94/11026. The linker may be
a "cleavable linker" facilitating release of a cytotoxic drug in
the cell. For example, an acid-labile linker, peptidase-sensitive
linker, photolabile linker, dimethyl linker or disulfide-containing
linker (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No.
5,208,020) may be used.
[0351] The immunuoconjugates or ADCs herein expressly contemplate,
but are not limited to such conjugates prepared with cross-linker
reagents including, but not limited to, BMPS, EMCS, GMBS, HBVS,
LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS,
sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and
sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which
are commercially available (e.g., from Pierce Biotechnology, Inc.,
Rockford, Ill., U.S.A).
IV. Methods for Predicting Responsiveness
[0352] Provided herein are methods for predicting responsiveness of
a subject having cancer to an OX40 agonist treatment and methods
for identifying or selecting a subject having cancer for treating
with an OX40 agonist. In some embodiments, the methods include
measuring the expression level of one or more marker genes in a
sample containing leukocytes obtained from the subject, where the
one or more marker genes are selected from CD8a, CD8b, H2-d, CTLA4,
CD64, CXCL9, IFNg, IDO1, GZMA, GZMB, PRF1, PDCA1, KLRK1, PTPRC,
CXCL1, ITGAM, and IL7R; and classifying the subject as a responsive
or non-responsive subject based on the expression level of the one
or more marker genes in the sample obtained from the subject, as
compared with a reference, where an increased expression level of
the one or more marker genes as compared with the reference
indicates the subject may be responsive to an OX40 agonist
treatment, or where a decreased expression level of the one or more
marker genes as compared with the reference indicates the subject
may not be responsive to an OX40 agonist treatment. In some
embodiments, the one or more marker genes may be selected from
CD8a, CD8b, IFNg, GZMA, GZMB, PRF1, and PDCA1. In some embodiments,
the one or more marker genes may be selected from H2-d, CTLA4,
CXCL9, PTPRC, IL7R, KLRK1, and CXCL1. In some embodiments, the one
or more marker genes may be selected from CD64, IDO1, and
ITGAM.
[0353] In other embodiments, the methods include measuring the
expression level of one or more marker genes in a sample containing
leukocytes obtained from the subject, where the one or more marker
genes are selected from CSF2, CCL22, EPCAM, GATA3, IL13, and VTCN1;
and classifying the subject as a responsive or non-responsive
subject based on the expression level of the one or more marker
genes in the sample obtained from the subject, as compared with a
reference, wherein a decreased expression level of the one or more
marker genes as compared with the reference indicates the subject
may be responsive to an OX40 agonist treatment, or wherein an
increased expression level of the one or more marker genes as
compared with the reference indicates the subject may not be
responsive to an OX40 agonist treatment.
[0354] Certain aspects of the present disclosure relate to
measuring the expression level of one or more marker genes. Any
suitable method for measuring gene expression known in the art may
be used. In some embodiments, expression level may refer to mRNA
expression level. mRNA expression level may be measured by many
methods. Such methods may quantify the copies of a specific mRNA
present in a sample by measuring the amount of hybridization to an
mRNA-specific probe. Other methods may amplify mRNA, or cDNA
generated from mRNA, and quantify the amount of amplicon generated
to extrapolate how much mRNA was present in a sample. Yet other
methods may involve next-generation sequencing of part or all of
mRNA transcripts, or cDNA generated from mRNA, then quantifying the
number of sequences detected that correspond to particular gene(s).
In some embodiments, mRNA expression level is measured by
quantitative PCR, semi-quantitative PCR, nucleotide microarray,
RNA-seq, in situ hybridization, and/or Northern blotting.
[0355] In some embodiments, expression level may refer to protein
expression level. Protein expression level may be measured by many
methods. Such methods may quantify proteins present in a sample by
using a probe that specifically binds to a particular protein, such
as an antibody, then detecting the amount of specific binding in a
sample. Other methods may fragment proteins into short peptides,
then detect these peptides and quantify how many peptides
correspond to particular protein(s). In some embodiments, protein
expression level is measured by Western blotting, peptide
microarray, immunohistochemistry, flow cytometry, and/or mass
spectrometry.
[0356] As described herein, some marker genes may be listed by the
name of a murine homolog. In some embodiments, the expression level
of a human homolog of one or more marker genes described herein may
be measured in a human sample. Methods for determining a human
homolog of a murine gene are known in the art. In some embodiments,
a homolog may be functionally determined, i.e., by performing a
similar cellular function. In some embodiments, a homolog may be
determined by sequence homology, e.g., by using a program such as
BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or ALIGN-2 software as
described herein.
[0357] In some embodiments, the marker gene may be one or more of
the marker genes provided in Table 3 below. Table 3 provides a list
of genes whose expression level was discovered as described herein
to be useful for predicting responsiveness to an OX40 agonist
treatment. In Table 3, positive predictors are those genes that,
when expressed at an increased level, predict responsiveness to an
OX40 agonist treatment. Negative predictors are those genes that,
when expressed at a decreased level, predict responsiveness to an
OX40 agonist treatment. Exemplary gene/protein names or aliases, as
well as an exemplary accession number corresponding to a human
protein homolog, are also provided.
TABLE-US-00006 TABLE 3 NO. Gene Predictor Alias (non-exhaustive)
Accession No 1 CSF2 Negative Granulocyte-macrophage colony-
NM_000749 stimulating factor; GM-CSF; CSF; 2 CCL22 Negative
Chemokine (C-C motif) ligand 22; CC NM_022990 chemokine STCP-1;
small inducible cytokine A22 3 GATA3 Negative GATA-binding factor
3, trans-acting T- NM_001002295 cell-specific transcription factor
GATA3 4 CD8a Positive T-cell surface glycoprotein CD8 alpha
NM_001145873 chain; OKT8 T-cell antigen; T-cell antigen Leu2; CD8
antigen, alpha polypeptide p32 5 CD8b Positive T-cell surface
glycoprotein CD8 beta NM_172213 chain; CD8 antigen, alpha
polypeptide p37 6 H2-d Positive MHC Class I B NM_005514 7 CTLA4
Positive Cytotoxic T-lymphocyte protein 4; NM_005214 CD152 isoform;
celiac disease 3; insulin- dependent diabetes mellitus 12 8 CD64
Positive Fc-gamma RI; Fc-gamma RIA NM_000566 9 CXCL9 Positive
C--X--C motif chemokine 9; small- NM_002416 inducible cytokine B9;
gamma- interferon-induced monokine 10 IFNg Positive Interferon
gamma NM_000619 11 IDO1 Positive Indoleamine 2,3-dioxygenase 1
NM_002164 12 GZMA Positive Granzyme A; h factor; CTL tryptase;
NM_006144 fragmentin-1; cytotoxic T-lymphocyte proteinase 1;
Hanukah factor serine protease 13 GZMB Positive Granzyme B; C11;
CTLA-1; fragmentin- NM_004131 2; cathepsin G-like 1; T-cell serine
protease 1-3E; cytotoxic T-lymphocyte proteinase 2 14 PRF1 Positive
Perforin-1; cytolysin; lymphocyte pore- NM_001083116 forming
protein 15 PDCA1 Positive Bone marrow stromal cell antigen 2;
NM_004335 NPC-A-7; HM1.24 antigen 16 KLRK1 Positive Killer cell
lectin-like receptor subfamily NM_007360 K, member 1; NKG2-D type
II integral membrane protein; NK cell receptor D 17 PTPRC Positive
Receptor-type tyrosine-protein NM_002838 phosphatase C; CD45
antigen; T200 glycoprotein 18 CXCL1 Positive Growth-regulated alpha
protein; C--X--C NM_001511 motif chemokine 1; MGSA alpha;
fibroblast secretory protein; GRO1 oncogene 19 ITGAM Positive
Integrin alpha-M; CR-3 alpha chain; NM_001145808 antigen CD11b
(p170); leukocyte adhesion receptor MO1; neutrophil adherence
receptor alpha-M subunit; macrophage antigen alpha polypeptide 20
IL7R Positive Interleukin-7 receptor subunit alpha; NM_002185 CD127
antigen 21 IL13 Negative Interleukin-13 NM_002188 22 EPCAM Negative
Epithelial cell adhesion molecule; cell NM_002354 surface
glycoprotein Trop-1; adenocarcinoma-associated antigen; GA733-2;
epithelial glycoprotein 314 23 VTCN1 Negative V-set
domain-containing T-cell NM_024626 activation inhibitor 1;
B7-H4
[0358] In some embodiments, expression level of an mRNA or protein
may be normalized to the expression level of a reference gene.
Normalizing the expression level of a particular gene to a
reference is thought to enhance reproducibility across samples by
factoring differences in sample size and/or mRNA/protein
extraction. In these examples, expression level relative to the
reference is measured. In some embodiments, multiple reference
genes may be used, either singly or in aggregate (e.g., by
averaging). In other embodiments, expression level of an mRNA or
protein may refer to absolute expression level.
[0359] In some embodiments, a reference gene may be a housekeeping
gene. A housekeeping gene is thought to be constitutively expressed
in a cell in normal and/or pathological states, such as a gene
encoding a protein required for basic cellular function and/or
maintenance. Housekeeping genes are typically used as a reference
to ensure they will be expressed at a detectable and/or
reproducible level across multiple samples. Exemplary housekeeping
genes and further description of the use of such genes as a
reference may be found, for example, in de Kok, J. B., et al.
(2005) Lab Invest. 85(1):154-9.
[0360] In some embodiments, the expression level of one or more
marker genes described herein is compared to a reference. In some
embodiments, the reference is the average, mean, or median level of
expression of the corresponding marker gene in a sample comprising
leukocytes from subjects that have cancer. In some embodiments, the
reference is the average, mean, or median level of expression of
the corresponding marker gene in samples comprising leukocytes from
other subjects having cancer who are not responsive to the OX40
agonist treatment after receiving treatment. For example, a set of
samples obtained from cancers having a shared characteristic (e.g.,
the same cancer type and/or stage, or exposure to a common
treatment such as an OX40 agonist) may be studied from a
population, such as with a clinical outcome study. This set may be
used to derive a reference, e.g., a reference number, to which a
subject's sample may be compared.
[0361] In some embodiments, responsiveness to treatment may refer
to any one or more of: extending survival (including overall
survival and progression free survival); resulting in an objective
response (including a complete response or a partial response); or
improving signs or symptoms of cancer. In some embodiments,
responsiveness may refer to improvement of one or more factors
according to the published set of RECIST or Immune-Related Response
Criteria guidelines for determining the status of a tumor in a
cancer patient, i.e., responding, stabilizing, or progressing. For
a more detailed discussion of these guidelines, see Eisenhauer et
al., Eur J Cancer 2009; 45: 228-47; Topalian et al., N Engl J Med
2012; 366:2443-54; Wolchok et al., Clin Can Res 2009; 15:7412-20;
and Therasse, P., et al. J. Natl. Cancer Inst. 92:205-16 (2000). A
responsive subject may refer to a subject whose cancer(s) show
improvement, e.g., according to one or more factors based on RECIST
or Immune-Related Response criteria. A non-responsive subject may
refer to a subject whose cancer(s) do not show improvement, e.g.,
according to one or more factors based on RECIST or Immune-Related
Response criteria. In some embodiments, responsiveness may include
immune activation. In some embodiments, responsiveness may include
treatment efficacy. In some embodiments, responsiveness may include
immune activation and treatment efficacy.
[0362] Conventional response criteria may not be adequate to
characterize the anti-tumor activity of immunotherapeutic agents,
which can produce delayed responses that may be preceded by initial
apparent radiological progression, including the appearance of new
lesions. Therefore, modified response criteria have been developed
that account for the possible appearance of new lesions and allow
radiological progression to be confirmed at a subsequent
assessment. Accordingly, in some embodiments, responsiveness may
refer to improvement of one of more factors according to
immune-related response criteria (irRC). See, e.g., Wolchok et al.,
Clin Can Res 2009; 15:7412-20. In some embodiments, new lesions are
added into the defined tumor burden and followed, e.g., for
radiological progression at a subsequent assessment. In some
embodiments, presence of non-target lesions are included in
assessment of complete response and not included in assessment of
radiological progression. In some embodiments, radiological
progression may be determined only on the basis of measurable
disease and/or may be confirmed by a consecutive assessment
.gtoreq.4 weeks from the date first documented.
[0363] In some embodiment, tumor may refer to a physical mass
containing a plurality of cancer cells, e.g., cells showing the
characteristics of any of the cancers described herein. Examples of
tumors may include primary tumors of any of the above types of
cancer or metastatic tumors at a second site derived from any of
the above types of cancer. In some embodiments, a tumor may contain
cancer cells as well as tumor stroma.
[0364] Certain aspects of the present disclosure relate to
measurement of the expression level of one or more genes in a
sample. In some embodiments, a sample may include leukocytes. In
some embodiments, the sample may be a tumor sample. A tumor sample
may include cancer cells, lymphocytes, leukocytes, stroma, blood
vessels, connective tissue, basal lamina, and any other cell type
in association with the tumor. In some embodiments, the sample is a
tumor tissue sample containing tumor-infiltrating leukocytes. As
used herein, any leukocyte associated with a tumor may be
considered a tumor-infiltrating leukocyte. Examples of
tumor-infiltrating leukocytes include without limitation T
lymphocytes (such as CD8+ T lymphocytes and/or CD4+ T lymphocytes),
B lymphocytes, or other bone marrow-lineage cells including
granulocytes (neutrophils, eosinophils, basophils), monocytes,
macrophages, dendritic cells (i.e., interdigitating dendritic
cells), histiocytes, and natural killer cells. In some embodiments,
a tumor-infiltrating leukocyte may be associated with cancer cells
of a tumor. In some embodiments, a tumor-infiltrating leukocyte may
be associated with tumor stroma. In some embodiments, the tumor
samples are enriched for tumor area by macrodissection.
[0365] In some embodiments, the sample may be processed to separate
or isolate one or more cell types (e.g., leukocytes). In some
embodiments, the sample may be used without separating or isolating
cell types. A tumor sample may be obtained from a subject by any
method known in the art, including without limitation a biopsy,
endoscopy, or surgical procedure. In some embodiments, a tumor
sample may be prepared by methods such as freezing, fixation (e.g.,
by using formalin or a similar fixative), and/or embedding in
paraffin wax. In some embodiments, a tumor sample may be sectioned.
In some embodiments, a fresh tumor sample (i.e., one that has not
been prepared by the methods described above) may be used. In some
embodiments, a sample may be prepared by incubation in a solution
to preserve mRNA and/or protein integrity. A tumor sample
containing leukocytes may be assayed by any technique described
herein for measuring marker gene expression level.
[0366] In some embodiments, the sample may be a peripheral blood
sample. A peripheral blood sample may include white blood cells,
PBMCs, and the like. Any technique known in the art for isolating
leukocytes from a peripheral blood sample may be used. For example,
a blood sample may be drawn, red blood cells may be lysed, and a
white blood cell pellet may be isolated and used for the sample. In
another example, density gradient separation may be used to
separate leukocytes (e.g., PBMCs) from red blood cells. Isolated
leukocytes from a peripheral blood sample may be assayed by any
technique described herein for measuring marker gene expression
level.
V. Methods of Monitoring Pharmacodynamic Activity and
Responsiveness
[0367] Provided herein are methods for monitoring pharmacodynamic
activity of an OX40 agonist treatment by measuring the expression
level of one or more marker genes in a sample comprising leukocytes
obtained from the subject, where the subject has been treated with
an OX40 agonist, and where the one or more marker genes are
selected from ARG1, CCL2, CCL22, CCL5, CCR5, CD226, CD27, CD274,
CD28, CD3E, CD40, CD8A, CD8b, CXCL10, CXCL9, EOMES, FasL,
Fcgr1/CD64, FOXP3, GZMA, GZMB, HAVCR2, ICAM1, IDO1, IFNg, IL10,
IL12A (TDO2), IL13, IL2, IL7R, ITGAM, KLRK1, LAG3, MAP4K1, MS4A1,
PDCD1, PDCD1LG2, PRF1, PTPRC, TNF, TNFRSF14, TNFRSF9, and TNFSF4;
and determining the treatment as demonstrating pharmacodynamic
activity based on the expression level of the one or more marker
genes in the sample obtained from the subject, as compared with a
reference, where an increased expression level of the one or more
marker genes as compared with the reference indicates
pharmacodynamic activity to the OX40 agonist treatment. These
methods are based in part on the discovery described herein that
expression of specific marker genes (e.g., ARG1, CCL2, CCL22, CCL5,
CCR5, CD226, CD27, CD274, CD28, CD3E, CD40, CD8A, CD8b, CXCL10,
CXCL9, EOMES, FasL, Fcgr1/CD64, FOXP3, GZMA, GZMB, HAVCR2, ICAM1,
IDO1, IFNg, IL10, IL12A (TDO2), IL13, IL2, IL7R, ITGAM, KLRK1,
LAG3, MAP4K1, MS4A1, PDCD1, PDCD1LG2, PRF1, PTPRC, TNF, TNFRSF14,
TNFRSF9, and/or TNFSF4) is upregulated following treatment with an
OX40 agonist in tumors that are responsive to such treatment and
tumors that are non-responsive to such treatment. Expression level
of a marker gene may be measured by one or more methods as
described herein.
[0368] As used herein, "pharmacodynamic (PD) activity" may refer to
an effect of a treatment (e.g., an OX40 agonist treatment) to the
subject. An example of a PD activity may include modulation of the
expression level of one or more genes. Without wishing to be bound
to theory, it is thought that monitoring PD activity, such as by
measuring expression of a gene marker, may be advantageous during a
clinical trial examining an OX40 agonist. Monitoring PD activity
may be used, for example, to monitor response to treatment,
toxicity, and the like.
[0369] In some embodiments, the expression level of one or more
marker genes may be compared to a reference which may include a
sample from a subject not receiving a treatment (e.g., an OX40
agonist treatment). In some embodiments, a reference may include a
sample from the same subject before receiving a treatment (e.g., an
OX40 agonist treatment). In some embodiments, a reference may
include a reference value from one or more samples of other
subjects receiving a treatment (e.g., an OX40 agonist treatment).
For example, a population of patients may be treated, and a mean,
average, or median value for expression level of one or more genes
may be generated from the population as a whole. A set of samples
obtained from cancers having a shared characteristic (e.g., the
same cancer type and/or stage, or exposure to a common treatment
such as an OX40 agonist) may be studied from a population, such as
with a clinical outcome study. This set may be used to derive a
reference, e.g., a reference number, to which a subject's sample
may be compared. Any of the references described herein may be used
as a reference for monitoring PD activity.
[0370] In some embodiments, the one or more marker genes are
selected from CD3, CD8, IFNg, GZMA, GZMB, PRF1, TNFa, PDCD1, and
CD274. In some embodiments, the marker gene may be one or more of
the marker genes provided in Table 4 below. Table 4 provides a list
of genes whose expression level was discovered as described herein
to be upregulated in response OX40 agonist treatment (e.g., as a
marker of PD activity). Exemplary gene/protein names or aliases, as
well as an exemplary accession number corresponding to a human
protein homolog, are provided.
TABLE-US-00007 TABLE 4 NO. Gene Alias (non-exhaustive) Accession No
1 ARG1 Arginase-1; liver-type arginase NM_001244438 2 CCL2 C-C
motif cytokine 2; small-inducible cytokine 2; NM_002982 monocyte
chemoattractant protein 1 3 CCL22 Chemokine (C-C motif) ligand 22;
CC chemokine NM_022990 STCP-1; small inducible cytokine A22 4 CCL5
C-C motif cytokine 5; beta chemokine RANTES; T- NM_001278736 cell
specific protein p288; eosinophil chemotactic cytokine 5 CCR5 C-C
chemokine receptor 5; HIV-1 fusion coreceptor NM_001100168 6 CD226
DNAX accessory molecule-1; platelet and T cell NM_006656 activation
antigen 1; T lineage-specific activation antigen 1 7 CD27 CD27L
receptor; T cell activation antigen S152; T- NM_001242 cell
activation antigen CD27; tumor necrosis factor receptor
superfamily, member 7 8 CD274 PD-L1; B7-H1; B7-4; B7-H NM_014143 9
CD28 T-cell-specific surface glycoprotein CD28 NM_006139 10 CD3E
T-cell surface glycoprotein CD3 epsilon chain; CD3- NM_000733
epsilon; T-cell surface antigen T3/Leu-4 epsilon chain; CD3e
antigen, epsilon polypeptide (TiT3 complex); T-cell antigen
receptor complex, epsilon subunit of T3 11 CD40 tumor necrosis
factor receptor superfamily member 5; NM_001250 CD40L receptor; B
cell-associated molecule; nerve growth factor receptor-related
B-lymphocyte activation molecule 12 CD8A T-cell surface
glycoprotein CD8 alpha chain; OKT8 NM_001145873 T-cell antigen;
T-cell antigen Leu2; CD8 antigen, alpha polypeptide p32 13 CD8b
T-cell surface glycoprotein CD8 beta chain; CD8 NM_172213 antigen,
alpha polypeptide p37 14 CXCL10 C--X--C motif chemokine 10; gamma
IP10; small- NM_001565 inducible cytokine B10; interferon-inducible
cytokine IP-10; 10 kDa interferon gamma-induced protein; small
inducible cytokine subfamily B (Cys-X-Cys), member 10 15 CXCL9
C--X--C motif chemokine 9; small-inducible cytokine NM_002416 B9;
gamma-interferon-induced monokine 16 EOMES eomesodermin homolog;
T-box brain protein 2 NM_001278182 17 FasL tumor necrosis factor
ligand superfamily member 6; NM_000639 CD95 ligand; fas antigen
ligand; apoptosis (APO-1) antigen ligand 1 18 Fcgr1/CD64 Fc-gamma
RI; Fc-gamma RIA NM_000566 19 FOXP3 forkhead box protein P3;
scurfin; FOXP3delta7; NM_014009 immunodeficiency,
polyendocrinopathy, enteropathy, X-linked 20 GZMA Granzyme A; h
factor; CTL tryptase; fragmentin-1; NM_006144 cytotoxic
T-lymphocyte proteinase 1; Hanukah factor serine protease 21 GZMB
Granzyme B; C11; CTLA-1; fragmentin-2; cathepsin NM_004131 G-like
1; T-cell serine protease 1-3E; cytotoxic T- lymphocyte proteinase
2 22 HAVCR2 hepatitis A virus cellular receptor 2; kidney injury
NM_032782 molecule-3; T-cell membrane protein 3; T cell
immunoglobulin mucin 3; T-cell immunoglobulin mucin receptor 3 23
ICAM1 intercellular adhesion molecule 1; cell surface NM_000201
glycoprotein P3.58; major group rhinovirus receptor 24 IDOL
Indoleamine 2,3-dioxygenase 1 NM_002164 25 IFNg Interferon gamma
NM_000619 26 IL10 Interleukin-10; T-cell growth inhibitory factor;
NM_000572 cytokine synthesis inhibitory factor 27 IL12A
interleukin-12 subunit alpha; CLMF p35; IL-12, NM_000882 (TDO2)
subunit p35; interleukin-12 alpha chain; NF cell stimulatory factor
chain 1; NK cell stimulatory factor chain 1; cytotoxic lymphocyte
maturation factor 1, p35 28 IL13 Interleukin-13 NM_002188 29 IL2
interleukin-2; aldesleukin; T cell growth factor NM_000586 30 IL7R
Interleukin-7 receptor subunit alpha; CD127 antigen NM_002185 31
ITGAM Integrin alpha-M; CR-3 alpha chain; antigen CD11b
NM_001145808 (p170); leukocyte adhesion receptor MO1; neutrophil
adherence receptor alpha-M subunit; macrophage antigen alpha
polypeptide 32 KLRK1 Killer cell lectin-like receptor subfamily K,
member 1; NM_007360 NKG2-D type II integral membrane protein; NK
cell receptor D 33 LAG3 lymphocyte activation gene 3 protein
NM_002286 34 MAP4K1 mitogen-activated protein kinase kinase kinase
kinase NM_001042600 1; MEKKK 1; MEK kinase kinase 1; MAPK/ERK
kinase kinase kinase 1; hematopoietic progenitor kinase 1 35 MS4A1
B-lymphocyte antigen CD20; leukocyte surface NM_021950 antigen
Leu-16; B-lymphocyte cell-surface antigen B1 36 PDCD1 CD279; SLEB2
NM_005018 37 PDCD1LG2 B7-DC; Btdc; CD273; PD-L2 NM_025239 38 PRF1
Perforin-1; cytolysin; lymphocyte pore-forming NM_001083116 protein
39 PTPRC Receptor-type tyrosine-protein phosphatase C; CD45
NM_002838 antigen; T200 glycoprotein 40 TNF tumor necrosis factor;
TNF-a; cachectin; APC1 NM_000594 protein; TNF, monocyte-
macrophage-derived; tumor necrosis factor ligand superfamily member
2 41 TNFRSF14 tumor necrosis factor receptor superfamily member
NM_003820 14; CD40-like protein; herpes virus entry mediator A;
tumor necrosis factor receptor-like gene2 42 TNFRSF9 tumor necrosis
factor receptor superfamily member 9; NM_001561 CD137 antigen; T
cell antigen ILA; 4-1BB ligand receptor; induced by lymphocyte
activation (ILA); interleukin-activated receptor, homolog of mouse
Ly63 43 TNFSF4 tumor necrosis factor ligand superfamily member 4;
NM_003326 CD134 ligand; glycoprotein Gp34; OX40 antigen ligand;
tax-transcriptionally activated glycoprotein 1 (34 kD)
[0371] Provided herein are methods for monitoring responsiveness of
a subject to an OX40 agonist treatment by measuring the expression
level of one or more marker genes in a sample comprising leukocytes
obtained from the subject, where the subject has been treated with
an OX40 agonist, and where the one or more marker genes are
selected from BTLA, CD4, CD69, CD80, CD83, CD86, CSF2, CTLA4,
CXCR3, Fcgr2b/CD32, Fcgr3/CD16, H2-aa, H2-d, H2-k, ICOS, IL10,
PDCA1, and TNFRSF18; and classifying the subject as responsive or
non-responsive to the treatment based on the expression level of
the one or more marker genes in the sample obtained from the
subject, as compared with a reference, where an increased
expression level of the one or more marker genes as compared with
the reference indicates a responsive subject. These methods are
based in part on the discovery described herein that expression of
specific marker genes (e.g., BTLA, CD4, CD69, CD80, CD83, CD86,
CSF2, CTLA4, CXCR3, Fcgr2b/CD32, Fcgr3/CD16, H2-aa, H2-d, H2-k,
ICOS, IL10, PDCA1, and/or TNFRSF18) is upregulated following
treatment with an OX40 agonist specifically in tumors that are
responsive to such treatment. Expression level of a marker gene may
be measured by one or more methods as described herein.
[0372] In some embodiments, a reference for monitoring
responsiveness may include a sample from a subject not receiving a
treatment (e.g., an OX40 agonist treatment). In some embodiments, a
reference for monitoring responsiveness may include a sample from
the same subject before receiving a treatment (e.g., an OX40
agonist treatment). In some embodiments, a reference for monitoring
responsiveness may include a reference value from one or more
samples of other patients receiving a treatment (e.g., an OX40
agonist treatment). For example, a population of patients may be
treated, and a mean, average, or median value for expression level
of one or more genes may be generated from the population as a
whole. A set of samples obtained from cancers having a shared
characteristic (e.g., the same cancer type and/or stage, or
exposure to a common treatment such as an OX40 agonist) may be
studied from a population, such as with a clinical outcome study.
This set may be used to derive a reference, e.g., a reference
number, to which a subject's sample may be compared. Any of the
references described herein may be used as a reference for
monitoring PD activity.
[0373] In some embodiments, the one or more marker genes are
selected from CD80, CD86, ICOS, H2-aa, and CXCR3. In some
embodiments, the marker gene may be one or more of the marker genes
provided in Table 5 below. Table 5 provides a list of genes whose
expression level was discovered as described herein to be
upregulated in response OX40 agonist treatment in tumors that are
responsive to such treatment (e.g., as a marker of responsiveness).
Exemplary gene/protein names or aliases, as well as an exemplary
accession number corresponding to a human protein homolog, are
provided.
TABLE-US-00008 TABLE 5 NO. Gene Alias (non-exhaustive) Accession No
1 BTLA B- and T-lymphocyte attenuator; B- and T- NM_181780
lymphocyte-associated protein 2 CD4 T-cell surface glycoprotein
CD4; CD4 receptor; CD4 NM_000616 antigen (p55); T-cell surface
antigen T4/Leu-3 3 CD69 early activation antigen CD69; leukocyte
surface NM_001781 antigen Leu-23; early T-cell activation antigen
p60; early lymphocyte activation antigen; activation inducer
molecule (AIM/CD69); C-type lectin domain family 2, member C 4 CD80
T-lymphocyte activation antigen CD80; activation NM_005191 B7-1
antigen; costimulatory factor CD80; CTLA-4 counter-receptor B7.1;
B-lymphocyte activation antigen B7; costimulatory molecule variant
IgV- CD80 5 CD83 CD83 antigen; B-cell activation protein; cell
surface NM_004233 protein HB15; cell-surface glycoprotein 6 CD86
T-lymphocyte activation antigen CD86; BU63; FUN- NM_175862 1;
CTLA-4 counter-receptor B7.2; B-lymphocyte activation antigen B7-2
7 CSF2 Granulocyte-macrophage colony-stimulating factor; NM_000749
GM-CSF; CSF; 8 CTLA4 Cytotoxic T-lymphocyte protein 4; CD152
isoform; NM_005214 celiac disease 3; insulin-dependent diabetes
mellitus 12 9 CXCR3 C--X--C chemokine receptor type 3; Mig
receptor; IP10 NM_001504 receptor; G protein-coupled receptor 9;
interferon- inducible protein 10 receptor 10 Fcgr2b/CD32 low
affinity immunoglobulin gamma Fc region NM_004001 receptor II-b;
CDw32; Fc gamma RIIb 11 Fcgr3/CD16 low affinity immunoglobulin
gamma Fc region NM_001127592 receptor III-A; FcgammaRIIIA; CD16a
antigen; neutrophil-specific antigen NA 12 H2-aa MHC Class IIA
NM_002122 13 H2-d MHC Class I B NM_005514 14 H2-k MHC Class IA
NM_002116 15 ICOS inducible T-cell costimulator;
activation-inducible NM_012092 lymphocyte immunomediatory molecule
16 IL10 Interleukin-10; T-cell growth inhibitory factor; NM_000572
cytokine synthesis inhibitory factor 17 PDCA1 Bone marrow stromal
cell antigen 2; NPC-A-7; NM_004335 HM1.24 antigen 18 TNFRSF18 Tumor
necrosis factor receptor superfamily member NM_004195 18;
activation-inducible TNFR family receptor; glucocorticoid-induced
TNFR-related protein; TNF receptor superfamily activation-inducible
protein
[0374] In some embodiment, tumor may refer to a physical mass
containing a plurality of cancer cells, e.g., cells showing the
characteristics of any of the cancers described herein. Examples of
tumors may include primary tumors of any of the above types of
cancer or metastatic tumors at a second site derived from any of
the above types of cancer. In some embodiments, a tumor may contain
cancer cells as well as tumor stroma.
[0375] Certain aspects of the present disclosure relate to
measurement of the expression level of one or more genes in a
sample. In some embodiments, a sample may include leukocytes. In
some embodiments, the sample may be a tumor sample. A tumor sample
may include cancer cells, lymphocytes, leukocytes, stroma, blood
vessels, connective tissue, basal lamina, and any other cell type
in association with the tumor. In some embodiments, the sample is a
tumor tissue sample containing tumor-infiltrating leukocytes. For
example, leukocyte associated with a tumor may be considered a
tumor-infiltrating leukocyte. Examples of tumor-infiltrating
leukocytes include without limitation T lymphocytes (such as CD8+ T
lymphocytes and/or CD4+ T lymphocytes), B lymphocytes, or other
bone marrow-lineage cells including granulocytes (neutrophils,
eosinophils, basophils), monocytes, macrophages, dendritic cells
(i.e., interdigitating dendritic cells), histiocytes, and natural
killer cells. In some embodiments, a tumor-infiltrating leukocyte
may be associated with cancer cells of a tumor. In some
embodiments, a tumor-infiltrating leukocyte may be associated with
tumor stroma.
[0376] In some embodiments, the sample may be processed to separate
or isolate one or more cell types (e.g., leukocytes). In some
embodiments, the sample may be used without separating or isolating
cell types. A tumor sample may be obtained from a subject by any
method known in the art, including without limitation a biopsy,
endoscopy, or surgical procedure. In some embodiments, a tumor
sample may be prepared by methods such as freezing, fixation (e.g.,
by using formalin or a similar fixative), and/or embedding in
paraffin wax. In some embodiments, a tumor sample may be sectioned.
In some embodiments, a fresh tumor sample (i.e., one that has not
been prepared by the methods described above) may be used. In some
embodiments, a sample may be prepared by incubation in a solution
to preserve mRNA and/or protein integrity. A tumor sample
containing leukocytes may be assayed by any technique described
herein for measuring marker gene expression level. In some
embodiments, the tumor samples are enriched for tumor area by
macrodissection.
[0377] In some embodiments, the sample may be a peripheral blood
sample. A peripheral blood sample may include white blood cells,
PBMCs, and the like. Any technique known in the art for isolating
leukocytes from a peripheral blood sample may be used. For example,
a blood sample may be drawn, red blood cells may be lysed, and a
white blood cell pellet may be isolated and used for the sample. In
another example, density gradient separation may be used to
separate leukocytes (e.g., PBMCs) from red blood cells. In some
embodiments, a fresh peripheral blood sample (i.e., one that has
not been prepared by the methods described above) may be used. In
some embodiments, a peripheral blood sample may be prepared by
incubation in a solution to preserve mRNA and/or protein
integrity.
[0378] In some embodiments, responsiveness to treatment may refer
to any one or more of: extending survival (including overall
survival and progression free survival); resulting in an objective
response (including a complete response or a partial response); or
improving signs or symptoms of cancer. In some embodiments,
responsiveness may refer to improvement of one or more factors
according to the published set of RECIST guidelines for determining
the status of a tumor in a cancer patient, i.e., responding,
stabilizing, or progressing. For a more detailed discussion of
these guidelines, see Eisenhauer et al., Eur J Cancer 2009; 45:
228-47; Topalian et al., N Engl J Med 2012; 366:2443-54; Wolchok et
al., Clin Can Res 2009; 15:7412-20; and Therasse, P., et al. J.
Natl. Cancer Inst. 92:205-16 (2000). A responsive subject may refer
to a subject whose cancer(s) show improvement, e.g., according to
one or more factors based on RECIST criteria. A non-responsive
subject may refer to a subject whose cancer(s) do not show
improvement, e.g., according to one or more factors based on RECIST
criteria.
[0379] Conventional response criteria may not be adequate to
characterize the anti-tumor activity of immunotherapeutic agents,
which can produce delayed responses that may be preceded by initial
apparent radiological progression, including the appearance of new
lesions. Therefore, modified response criteria have been developed
that account for the possible appearance of new lesions and allow
radiological progression to be confirmed at a subsequent
assessment. Accordingly, in some embodiments, responsiveness may
refer to improvement of one of more factors according to
immune-related response criteria2(irRC). See, e.g., Wolchok et al.,
Clin Can Res 2009; 15:7412-20. In some embodiments, new lesions are
added into the defined tumor burden and followed, e.g., for
radiological progression at a subsequent assessment. In some
embodiments, presence of non-target lesions are included in
assessment of complete response and not included in assessment of
radiological progression. In some embodiments, radiological
progression may be determined only on the basis of measurable
disease and/or may be confirmed by a consecutive assessment
.gtoreq.4 weeks from the date first documented.
[0380] In some embodiments, responsiveness may include immune
activation. In some embodiments, responsiveness may include
treatment efficacy. In some embodiments, responsiveness may include
immune activation and treatment efficacy.
VI. Methods of Treatment
[0381] In one aspect, provided herein are methods for treating or
delaying progression of cancer in an individual comprising
administering to the individual an effective amount of an OX40
agonist. The methods of this disclosure may find use, inter alia,
in treating conditions where enhanced immunogenicity is desired
such as increasing tumor immunogenicity for the treatment of cancer
or T cell dysfunctional disorders. A variety of cancers may be
treated, or their progression may be delayed, by these methods.
[0382] Provided herein are methods for treating or delaying
progression of cancer in a subject by measuring the expression
level of one or more marker genes in a sample containing leukocytes
obtained from the subject, where the one or more marker genes are
selected from CD8a, CD8b, H2-d, CTLA4, CD64, CXCL9, IFNg, IDO1,
GZMA, GZMB, PRF1, PDCA1, KLRK1, PTPRC, CXCL1, ITGAM, and IL7R; and
if the expression level of said one or more marker genes in the
sample obtained from the subject is higher than a reference,
administering to the subject an effective amount of an OX40
agonist. Further provided herein are methods for treating or
delaying progression of cancer in a subject including administering
to the subject an effective amount of an OX40 agonist, where a
sample containing leukocytes obtained from the subject has
increased expression of one or more marker genes are selected from
CD8a, CD8b, H2-d, CTLA4, CD64, CXCL9, IFNg, IDO1, GZMA, GZMB, PRF1,
PDCA1, KLRK1, PTPRC, CXCL1, ITGAM, and IL7R, as compared with a
reference. These methods are based in part on the discovery
described herein that higher expression of certain immune
activation and Th1 markers (e.g., CD8a, CD8b, H2-d, CTLA4, CD64,
CXCL9, IFNg, IDO1, GZMA, GZMB, PRF1, PDCA1, KLRK1, PTPRC, CXCL1,
ITGAM, and/or IL7R) is associated with better responsiveness to
OX40 agonist treatment.
[0383] Yet further provided herein are methods for treating or
delaying progression of cancer in a subject by measuring the
expression level of one or more marker genes in a sample containing
leukocytes obtained from the subject, where the one or more marker
genes are selected from CSF2, CCL22, EPCAM, GATA3, IL13, and VTCN1;
and if the expression level of said one or more marker genes in the
sample obtained from the subject is lower than a reference,
administering to the subject an effective amount of an OX40
agonist. Further provided herein are methods for treating or
delaying progression of cancer in a subject including administering
to the subject an effective amount of an OX40 agonist, where a
sample containing leukocytes obtained from the subject has
decreased expression of one or more marker genes are selected from
CSF2, CCL22, EPCAM, GATA3, IL13, and VTCN1, as compared with a
reference. These methods are based in part on the discovery
described herein that lower expression of certain immune activation
and Th1 markers (e.g., CSF2, CCL22, EPCAM, GATA3, IL13, and/or
VTCN1) is associated with better responsiveness to OX40 agonist
treatment.
[0384] In some embodiments, the OX40 agonist is administered to a
subject wherein a sample containing leukocytes from the subject
have been detected for increased expression of one or more marker
genes selected from the group consisting of CD8a, CD8b, H2-d,
CTLA4, CD64, CXCL9, IFNg, IDO1, GZMA, GZMB, PRF1, PDCA1, KLRK1,
PTPRC, CXCL1, ITGAM, and IL7R, and/or decreased expression of one
or more marker selected from the group consisting of CSF2, CCL22,
EPCAM, GATA3, IL13, and VTCN1.
[0385] A sample or cell that expresses a protein of interest (such
as CD8a, CD8b, H2-d, CTLA4, CD64, CXCL9, IFNg, IDO1, GZMA, GZMB,
PRF1, PDCA1, KLRK1, PTPRC, CXCL1, ITGAM, IL7R, CSF2, CCL22, EPCAM,
GATA3, IL13, and VTCN1) may be one in which mRNA encoding the
protein, or the protein, including fragments thereof, is determined
to be present in the sample or cell.
[0386] A sample, cell, tumor, or cancer which has increased
expression of one or more markers (such as CD8a, CD8b, H2-d, CTLA4,
CD64, CXCL9, IFNg, IDO1, GZMA, GZMB, PRF1, PDCA1, KLRK1, PTPRC,
CXCL1, ITGAM, IL7R) in a type of cancer may be one in which the
level of one or more marker expression may be considered increased
to a skilled person for that type of cancer. For example, such
increase may be at least about 0.5 fold, at least about 1 fold, at
least about 2 fold, or at least about 5 fold relative to the levels
in a population of samples, cells, tumors, or cancers of the same
cancer type.
[0387] A sample, cell, tumor, or cancer which has decreased
expression of one or more markers (such as CSF2, CCL22, EPCAM,
GATA3, IL13, and/or VTCN1) in a type of cancer may be one in which
the level of one or more marker expression may be considered
decreased to a skilled person for that type of cancer. For example,
such decrease may be at least about 20%, at least about 30%, at
least about 40%, at least about 50%, at least about 60%, at least
about 70% decrease relative to the levels in a population of
samples, cells, tumors, or cancers of the same cancer type.
[0388] In some embodiments, a cancer to be treated by the methods
of the present disclosure includes, but is not limited to, squamous
cell cancer (e.g., epithelial squamous cell cancer), lung cancer
including small-cell lung cancer, non-small cell lung cancer,
adenocarcinoma of the lung and squamous carcinoma of the lung,
cancer of the peritoneum, hepatocellular cancer, gastric or stomach
cancer including gastrointestinal cancer and gastrointestinal
stromal cancer, pancreatic cancer, glioblastoma, cervical cancer,
ovarian cancer, liver cancer, bladder cancer, cancer of the urinary
tract, hepatoma, breast cancer, colon cancer, rectal cancer,
colorectal cancer, endometrial or uterine carcinoma, salivary gland
carcinoma, kidney or renal cancer, prostate cancer, vulval cancer,
thyroid cancer, hepatic carcinoma, anal carcinoma, penile
carcinoma, melanoma, superficial spreading melanoma, lentigo
maligna melanoma, acral lentiginous melanomas, nodular melanomas,
multiple myeloma and B-cell lymphoma; chronic lymphocytic leukemia
(CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia;
chronic myeloblastic leukemia; and post-transplant
lymphoproliferative disorder (PTLD), as well as abnormal vascular
proliferation associated with phakomatoses, edema (such as that
associated with brain tumors), Meigs' syndrome, brain, as well as
head and neck cancer, and associated metastases. In some
embodiments, the cancer is colorectal cancer. In some embodiments,
the cancer is selected from non-small cell lung cancer, renal cell
carcinoma, ovarian cancer, bladder cancer, glioblastoma,
neuroblastoma, melanoma, breast carcinoma, gastric cancer, and
hepatocellular carcinoma. In some embodiments, the cancer is
triple-negative breast carcinoma. In some embodiments, the cancer
may be an early stage cancer or a late stage cancer. In some
embodiments, the cancer may be a primary tumor. In some
embodiments, the cancer may be a metastatic tumor at a second site
derived from any of the above types of cancer.
[0389] In some embodiments, examples of cancer further include, but
are not limited to, B-cell lymphoma (including low grade/follicular
non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL;
intermediate grade/follicular NHL; intermediate grade diffuse NHL;
high grade immunoblastic NHL; high grade lymphoblastic NHL; high
grade small non-cleaved cell NHL; bulky disease NHL; mantle cell
lymphoma; AIDS-related lymphoma; and Waldenstrom's
Macroglobulinemia); chronic lymphocytic leukemia (CLL); acute
lymphoblastic leukemia (ALL); Hairy cell leukemia; chronic
myeloblastic leukemia; and post-transplant lymphoproliferative
disorder (PTLD), as well as abnormal vascular proliferation
associated with phakomatoses, edema (such as that associated with
brain tumors), B-cell proliferative disorders, and Meigs' syndrome.
More specific examples include, but are not limited to, relapsed or
refractory NHL, front line low grade NHL, Stage III/IV NHL,
chemotherapy resistant NHL, precursor B lymphoblastic leukemia
and/or lymphoma, small lymphocytic lymphoma, B-cell chronic
lymphocytic leukemia and/or prolymphocytic leukemia and/or small
lymphocytic lymphoma, B-cell prolymphocytic lymphoma, immunocytoma
and/or lymphoplasmacytic lymphoma, lymphoplasmacytic lymphoma,
marginal zone B-cell lymphoma, splenic marginal zone lymphoma,
extranodal marginal zone--MALT lymphoma, nodal marginal zone
lymphoma, hairy cell leukemia, plasmacytoma and/or plasma cell
myeloma, low grade/follicular lymphoma, intermediate
grade/follicular NHL, mantle cell lymphoma, follicle center
lymphoma (follicular), intermediate grade diffuse NHL, diffuse
large B-cell lymphoma, aggressive NHL (including aggressive
front-line NHL and aggressive relapsed NHL), NHL relapsing after or
refractory to autologous stem cell transplantation, primary
mediastinal large B-cell lymphoma, primary effusion lymphoma, high
grade immunoblastic NHL, high grade lymphoblastic NHL, high grade
small non-cleaved cell NHL, bulky disease NHL, Burkitt's lymphoma,
precursor (peripheral) large granular lymphocytic leukemia, mycosis
fungoides and/or Sezary syndrome, skin (cutaneous) lymphomas,
anaplastic large cell lymphoma, angiocentric lymphoma.
[0390] In some embodiments, examples of cancer further include, but
are not limited to, B-cell proliferative disorders, which further
include, but are not limited to, lymphomas (e.g., B-Cell
Non-Hodgkin's lymphomas (NHL)) and lymphocytic leukemias. Such
lymphomas and lymphocytic leukemias include e.g. a) follicular
lymphomas, b) Small Non-Cleaved Cell Lymphomas/Burkitt's lymphoma
(including endemic Burkitt's lymphoma, sporadic Burkitt's lymphoma
and Non-Burkitt's lymphoma), c) marginal zone lymphomas (including
extranodal marginal zone B-cell lymphoma (Mucosa-associated
lymphatic tissue lymphomas, MALT), nodal marginal zone B-cell
lymphoma and splenic marginal zone lymphoma), d) Mantle cell
lymphoma (MCL), e) Large Cell Lymphoma (including B-cell diffuse
large cell lymphoma (DLCL), Diffuse Mixed Cell Lymphoma,
Immunoblastic Lymphoma, Primary Mediastinal B-Cell Lymphoma,
Angiocentric Lymphoma-Pulmonary B-Cell Lymphoma), f) hairy cell
leukemia, g) lymphocytic lymphoma, Waldenstrom's macroglobulinemia,
h) acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia
(CLL)/small lymphocytic lymphoma (SLL), B cell prolymphocytic
leukemia, i) plasma cell neoplasms, plasma cell myeloma, multiple
myeloma, plasmacytoma, and/or j) Hodgkin's disease.
[0391] In some embodiments of any of the methods, the cancer is a
B-cell proliferative disorder. In some embodiments, the B-cell
proliferative disorder is lymphoma, non-Hodgkins lymphoma (NHL),
aggressive NHL, relapsed aggressive NHL, relapsed indolent NHL,
refractory NHL, refractory indolent NHL, chronic lymphocytic
leukemia (CLL), small lymphocytic lymphoma, leukemia, hairy cell
leukemia (HCL), acute lymphocytic leukemia (ALL), or mantle cell
lymphoma. In some embodiments, the B-cell proliferative disorder is
NHL, such as indolent NHL and/or aggressive NHL. In some
embodiments, the B-cell proliferative disorder is indolent
follicular lymphoma or diffuse large B-cell lymphoma.
[0392] In some embodiments, the subject has cancer or is at risk of
developing cancer. In some embodiments, the treatment results in a
sustained response in the subject after cessation of the treatment.
In some embodiments, the subject has cancer that may be at early
stage or late stage. In some embodiments, the subject is a human.
In some embodiments, the subject is a mammal, such as domesticated
animals (e.g., cows, sheep, cats, dogs, and horses), primates
(e.g., humans and non-human primates such as monkeys), rabbits, and
rodents (e.g., mice and rats).
[0393] In some embodiments, provided is a method for treating or
delaying progression of cancer in a subject comprising
administering to the subject an effective amount of an OX40
agonist, and further comprising administering an additional
therapy. The additional therapy may be radiation therapy, surgery
(e.g., lumpectomy and a mastectomy), chemotherapy, gene therapy,
DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow
transplantation, nanotherapy, monoclonal antibody therapy, or a
combination of the foregoing. The additional therapy may be in the
form of adjuvant or neoadjuvant therapy. In some embodiments, the
additional therapy is the administration of small molecule
enzymatic inhibitor or anti-metastatic agent. In some embodiments,
the additional therapy is the administration of side-effect
limiting agents (e.g., agents intended to lessen the occurrence
and/or severity of side effects of treatment, such as anti-nausea
agents, etc.). In some embodiments, the additional therapy is
radiation therapy. In some embodiments, the additional therapy is
surgery. In some embodiments, the additional therapy is a
combination of radiation therapy and surgery. In some embodiments,
the additional therapy is gamma irradiation. In some embodiments,
the additional therapy is therapy targeting PI3K/AKT/mTOR pathway,
HSP90 inhibitor, tubulin inhibitor, apoptosis inhibitor, and/or
chemopreventative agent. The additional therapy may be one or more
of the chemotherapeutic agents described hereabove.
[0394] Such combination therapies noted above encompass combined
administration (where two or more therapeutic agents are included
in the same or separate formulations), and separate administration,
in which case, administration of the OX40 agonist of the invention
can occur prior to, simultaneously, and/or following,
administration of the additional therapeutic agent or agents. In
one embodiment, administration of the OX40 agonist and
administration of an additional therapeutic agent occur within
about one month, or within about one, two or three weeks, or within
about one, two, three, four, five, or six days, of each other. OX40
agonists of the invention can also be used in combination with
radiation therapy.
[0395] In some embodiments, an anti-human OX40 agonist may be
administered in conjunction with a chemotherapy or chemotherapeutic
agent. In some embodiments, an anti-human OX40 agonist may be
administered in conjunction with a radiation therapy or
radiotherapeutic agent. In some embodiments, an anti-human OX40
agonist may be administered in conjunction with a targeted therapy
or targeted therapeutic agent. In some embodiments, an anti-human
OX40 agonist may be administered in conjunction with an
immunotherapy or immunotherapeutic agent, for example a monoclonal
antibody.
[0396] In some embodiments, an OX40 agonist (e.g., an anti-human
OX40 agonist antibody) may be administered in conjunction with in
combination with a PD-1 axis binding antagonist. A PD-1 axis
binding antagonist includes but is not limited to a PD-1 binding
antagonist, a PD-L1 binding antagonist and a PD-L2 binding
antagonist. Alternative names for "PD-1" include CD279 and SLEB2.
Alternative names for "PD-L1" include B7-H 1, B7-4, CD274, and
B7-H. Alternative names for "PD-L2" include B7-DC, Btdc, and CD273.
In some embodiments, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1
and PD-L2. In some embodiments, the PD-1 binding antagonist is a
molecule that inhibits the binding of PD-1 to its ligand binding
partners. In a specific aspect the PD-1 ligand binding partners are
PD-L1 and/or PD-L2. In another embodiment, a PD-L1 binding
antagonist is a molecule that inhibits the binding of PD-L1 to its
binding partners. In a specific aspect, PD-L1 binding partners are
PD-1 and/or B7-1. In another embodiment, the PD-L2 binding
antagonist is a molecule that inhibits the binding of PD-L2 to its
binding partners. In a specific aspect, a PD-L2 binding partner is
PD-1. The antagonist may be an antibody, an antigen binding
fragment thereof, an immunoadhesin, a fusion protein, or
oligopeptide. In some embodiment, the PD-1 binding antagonist is an
anti-PD-1 antibody (e.g., a human antibody, a humanized antibody,
or a chimeric antibody). In some embodiments, the anti-PD-1
antibody is selected from the group consisting of MDX-1 106, Merck
3475 and CT-011. In some embodiments, the PD-1 binding antagonist
is an immunoadhesin (e.g., an immunoadhesin comprising an
extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a
constant region (e.g., an Fc region of an immunoglobulin sequence).
In some embodiments, the PD-1 binding antagonist is AMP-224. In
some embodiments, the PD-L1 binding antagonist is an anti-PD-L1
antibody. In some embodiments, the anti-PD-L1 binding antagonist is
selected from the group consisting of YW243.55.S70, MPDL3280A
(atezolizumab), MEDI4736 (durvalumab), MDX-1105, and MSB0010718C
(avelumab). MDX-1 105, also known as BMS-936559, is an anti-PD-L1
antibody described in WO2007/005874. Antibody YW243.55.S70 (heavy
and light chain variable region sequences shown in SEQ ID Nos. 20
and 21, respectively) is an anti-PD-L1 described in WO 2010/077634
A1. MDX-1 106, also known as MDX-1 106-04, ONO-4538 or BMS-936558,
is an anti-PD-1 antibody described in WO2006/121168. Merck 3745,
also known as MK-3475, SCH-900475, pembrolizumab, and
KEYTRUDA.RTM., is an anti-PD-1 antibody described in WO2009/114335.
CT-011, also known as hBAT, hBAT-1, and pidilizumab, is an
anti-PD-1 antibody described in WO2009/101611. AMP-224, also known
as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in
WO2010/027827 and WO2011/066342. In some embodiments, the anti-PD-1
antibody is MDX-1 106. Alternative names for "MDX-1106" include
MDX-1 106-04, ONO-4538, BMS-936558, Nivolumab, or OPDIVO.RTM.. In
some embodiments, the anti-PD-1 antibody is Nivolumab (CAS Registry
Number: 946414-94-4). In some embodiments, the anti-PD-1 antibody
is selected from the group consisting of MDX-1106 (nivolumab,
OPDIVO.RTM.), Merck 3475 (MK-3475, pembrolizumab, KEYTRUDA.RTM.),
CT-011 (Pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810,
BGB-108, and BGB-A317.
[0397] In some embodiments, an OX40 agonist (e.g., an anti-human
OX40 agonist antibody) may be administered in conjunction with an
antagonist directed against CTLA-4 (also known as CD152), e.g., a
blocking antibody. In some embodiments, an OX40 agonist (e.g., an
anti-human OX40 agonist antibody) may be administered in
conjunction with ipilimumab (also known as MDX-010, MDX-101, or
Yervoy.RTM.). In some embodiments, an OX40 agonist (e.g., an
anti-human OX40 agonist antibody) may be administered in
conjunction with tremelimumab (also known as ticilimumab or
CP-675,206). In some embodiments, an OX40 agonist (e.g., an
anti-human OX40 agonist antibody) may be administered in
conjunction with an antagonist directed against B7-H3 (also known
as CD276), e.g., a blocking antibody. In some embodiments, an OX40
agonist (e.g., an anti-human OX40 agonist antibody) may be
administered in conjunction with MGA271. In some embodiments, an
OX40 agonist (e.g., an anti-human OX40 agonist antibody) may be
administered in conjunction with an antagonist directed against a
TGF beta, e.g., metelimumab (also known as CAT-192), fresolimumab
(also known as GC1008), or LY2157299.
[0398] In some embodiments, an OX40 agonist (e.g., an anti-human
OX40 agonist antibody) may be administered in conjunction with a
treatment comprising adoptive transfer of a T cell (e.g., a
cytotoxic T cell or CTL) expressing a chimeric antigen receptor
(CAR). In some embodiments, an OX40 agonist (e.g., an anti-human
OX40 agonist antibody) may be administered in conjunction with a
treatment comprising adoptive transfer of a T cell comprising a
dominant-negative TGF beta receptor, e.g, a dominant-negative TGF
beta type II receptor. In some embodiments, an OX40 agonist (e.g.,
an anti-human OX40 agonist antibody) may be administered in
conjunction with a treatment comprising a HERCREEM protocol (see,
e.g., ClinicalTrials.gov Identifier NCT00889954).
[0399] In some embodiments, an OX40 agonist (e.g., an anti-human
OX40 agonist antibody) may be administered in conjunction with an
agonist directed against CD137 (also known as TNFRSF9, 4-1BB, or
ILA), e.g., an activating antibody. In some embodiments, an OX40
agonist (e.g., an anti-human OX40 agonist antibody) may be
administered in conjunction with urelumab (also known as
BMS-663513). In some embodiments, an OX40 agonist (e.g., an
anti-human OX40 agonist antibody) may be administered in
conjunction with an agonist directed against CD40, e.g., an
activating antibody. In some embodiments, an OX40 agonist (e.g., an
anti-human OX40 agonist antibody) may be administered in
conjunction with CP-870893. In some embodiments, an OX40 agonist
(e.g., an anti-human OX40 agonist antibody) may be administered in
conjunction with an agonist directed against OX40 (also known as
CD134), e.g., an activating antibody. In some embodiments, an OX40
agonist (e.g., an anti-human OX40 agonist antibody) may be
administered in conjunction with a different anti-OX40 antibody
(e.g., AgonOX). In some embodiments, an OX40 agonist (e.g., an
anti-human OX40 agonist antibody) may be administered in
conjunction with an agonist directed against CD27, e.g., an
activating antibody. In some embodiments, an OX40 agonist (e.g., an
anti-human OX40 agonist antibody) may be administered in
conjunction with CDX-1127. In some embodiments, an OX40 agonist
(e.g., an anti-human OX40 agonist antibody) may be administered in
conjunction with an antagonist directed against
indoleamine-2,3-dioxygenase (IDO). In some embodiments, with the
IDO antagonist is 1-methyl-D-tryptophan (also known as 1-D-MT).
[0400] In some embodiments, an OX40 agonist (e.g., an anti-human
OX40 agonist antibody) may be administered in conjunction with an
antibody-drug conjugate. In some embodiments, the antibody-drug
conjugate comprises mertansine or monomethyl auristatin E (MMAE).
In some embodiments, an OX40 agonist (e.g., an anti-human OX40
agonist antibody) may be administered in conjunction with and
anti-NaPi2b antibody-MMAE conjugate (also known as DNIB0600A or
RG7599). In some embodiments, an OX40 agonist (e.g., an anti-human
OX40 agonist antibody) may be administered in conjunction with
trastuzumab emtansine (also known as T-DM1, ado-trastuzumab
emtansine, or KADCYLA.RTM., Genentech). In some embodiments, an
OX40 agonist (e.g., an anti-human OX40 agonist antibody) may be
administered in conjunction with DMUC5754A. In some embodiments, an
OX40 agonist (e.g., an anti-human OX40 agonist antibody) may be
administered in conjunction with an antibody-drug conjugate
targeting the endothelin B receptor (EDNBR), e.g., an antibody
directed against EDNBR conjugated with MMAE.
[0401] In some embodiments, an OX40 agonist (e.g., an anti-human
OX40 agonist antibody) may be administered in conjunction with an
angiogenesis inhibitor. In some embodiments, an OX40 agonist (e.g.,
an anti-human OX40 agonist antibody) may be administered in
conjunction with an antibody directed against a VEGF, e.g., VEGF-A.
In some embodiments, an OX40 agonist (e.g., an anti-human OX40
agonist antibody) may be administered in conjunction with
bevacizumab (also known as AVASTIN.RTM., Genentech). In some
embodiments, an OX40 agonist (e.g., an anti-human OX40 agonist
antibody) may be administered in conjunction with an antibody
directed against angiopoietin 2 (also known as Ang2). In some
embodiments, an OX40 agonist (e.g., an anti-human OX40 agonist
antibody) may be administered in conjunction with MEDI3617.
[0402] In some embodiments, an OX40 agonist (e.g., an anti-human
OX40 agonist antibody) may be administered in conjunction with an
antineoplastic agent. In some embodiments, an OX40 agonist (e.g.,
an anti-human OX40 agonist antibody) may be administered in
conjunction with an agent targeting CSF-1R (also known as M-CSFR or
CD115). In some embodiments, an OX40 agonist (e.g., an anti-human
OX40 agonist antibody) may be administered in conjunction with
anti-CSF-1R (also known as IMC-CS4). In some embodiments, an OX40
agonist (e.g., an anti-human OX40 agonist antibody) may be
administered in conjunction with an interferon, for example
interferon alpha or interferon gamma. In some embodiments, an OX40
agonist (e.g., an anti-human OX40 agonist antibody) may be
administered in conjunction with Roferon-A (also known as
recombinant Interferon alpha-2a). In some embodiments, an OX40
agonist (e.g., an anti-human OX40 agonist antibody) may be
administered in conjunction with GM-CSF (also known as recombinant
human granulocyte macrophage colony stimulating factor, rhu GM-CSF,
sargramostim, or Leukine.RTM.). In some embodiments, an OX40
agonist (e.g., an anti-human OX40 agonist antibody) may be
administered in conjunction with IL-2 (also known as aldesleukin or
Proleukin.RTM.). In some embodiments, an OX40 agonist (e.g., an
anti-human OX40 agonist antibody) may be administered in
conjunction with IL-12. In some embodiments, an OX40 agonist (e.g.,
an anti-human OX40 agonist antibody) may be administered in
conjunction with an antibody targeting CD20. In some embodiments,
the antibody targeting CD20 is obinutuzumab (also known as GA101 or
Gazyva.RTM.) or rituximab. In some embodiments, an OX40 agonist
(e.g., an anti-human OX40 agonist antibody) may be administered in
conjunction with an antibody targeting GITR. In some embodiments,
the antibody targeting GITR is TRX518.
[0403] In some embodiments, an OX40 agonist (e.g., an anti-human
OX40 agonist antibody) may be administered in conjunction with a
cancer vaccine. In some embodiments, the cancer vaccine is a
peptide cancer vaccine, which in some embodiments is a personalized
peptide vaccine. In some embodiments the peptide cancer vaccine is
a multivalent long peptide, a multi-peptide, a peptide cocktail, a
hybrid peptide, or a peptide-pulsed dendritic cell vaccine (see,
e.g., Yamada et al., Cancer Sci, 104:14-21, 2013). In some
embodiments, an OX40 agonist (e.g., an anti-human OX40 agonist
antibody) may be administered in conjunction with an adjuvant. In
some embodiments, an OX40 agonist (e.g., an anti-human OX40 agonist
antibody) may be administered in conjunction with a treatment
comprising a TLR agonist, e.g., Poly-ICLC (also known as
Hiltonol.RTM.), LPS, MPL, or CpG ODN. In some embodiments, an OX40
agonist (e.g., an anti-human OX40 agonist antibody) may be
administered in conjunction with tumor necrosis factor (TNF) alpha.
In some embodiments, an OX40 agonist (e.g., an anti-human OX40
agonist antibody) may be administered in conjunction with IL-1. In
some embodiments, an OX40 agonist (e.g., an anti-human OX40 agonist
antibody) may be administered in conjunction with HMGB1. In some
embodiments, an OX40 agonist (e.g., an anti-human OX40 agonist
antibody) may be administered in conjunction with an IL-10
antagonist. In some embodiments, an OX40 agonist (e.g., an
anti-human OX40 agonist antibody) may be administered in
conjunction with an IL-4 antagonist. In some embodiments, an OX40
agonist (e.g., an anti-human OX40 agonist antibody) may be
administered in conjunction with an IL-13 antagonist. In some
embodiments, an OX40 agonist (e.g., an anti-human OX40 agonist
antibody) may be administered in conjunction with an HVEM
antagonist. In some embodiments, an OX40 agonist (e.g., an
anti-human OX40 agonist antibody) may be administered in
conjunction with an ICOS agonist, e.g., by administration of
ICOS-L, or an agonistic antibody directed against ICOS. In some
embodiments, an OX40 agonist (e.g., an anti-human OX40 agonist
antibody) may be administered in conjunction with a treatment
targeting CX3CL1. In some embodiments, an OX40 agonist (e.g., an
anti-human OX40 agonist antibody) may be administered in
conjunction with a treatment targeting CXCL9. In some embodiments,
an OX40 agonist (e.g., an anti-human OX40 agonist antibody) may be
administered in conjunction with a treatment targeting CXCL10. In
some embodiments, an OX40 agonist (e.g., an anti-human OX40 agonist
antibody) may be administered in conjunction with a treatment
targeting CCL5. In some embodiments, an OX40 agonist (e.g., an
anti-human OX40 agonist antibody) may be administered in
conjunction with an LFA-1 or ICAM1 agonist. In some embodiments, an
OX40 agonist (e.g., an anti-human OX40 agonist antibody) may be
administered in conjunction with a Selectin agonist.
[0404] In some embodiments, an OX40 agonist (e.g., an anti-human
OX40 agonist antibody) may be administered in conjunction with a
targeted therapy. In some embodiments, an OX40 agonist (e.g., an
anti-human OX40 agonist antibody) may be administered in
conjunction with an inhibitor of B-Raf. In some embodiments, an
OX40 agonist (e.g., an anti-human OX40 agonist antibody) may be
administered in conjunction with vemurafenib (also known as
Zelboraf.RTM.). In some embodiments, an OX40 agonist (e.g., an
anti-human OX40 agonist antibody) may be administered in
conjunction with dabrafenib (also known as Tafinlar.RTM.). In some
embodiments, an OX40 agonist (e.g., an anti-human OX40 agonist
antibody) may be administered in conjunction with erlotinib (also
known as Tarceva.RTM.). In some embodiments, an OX40 agonist (e.g.,
an anti-human OX40 agonist antibody) may be administered in
conjunction with an inhibitor of a MEK, such as MEK1 (also known as
MAP2K1) or MEK2 (also known as MAP2K2). In some embodiments, an
OX40 agonist (e.g., an anti-human OX40 agonist antibody) may be
administered in conjunction with cobimetinib (also known as
GDC-0973 or XL-518). In some embodiments, an OX40 agonist (e.g., an
anti-human OX40 agonist antibody) may be administered in
conjunction with trametinib (also known as Mekinist.RTM.). In some
embodiments, an OX40 agonist (e.g., an anti-human OX40 agonist
antibody) may be administered in conjunction with an inhibitor of
K-Ras. In some embodiments, an OX40 agonist (e.g., an anti-human
OX40 agonist antibody) may be administered in conjunction with an
inhibitor of c-Met. In some embodiments, an OX40 agonist (e.g., an
anti-human OX40 agonist antibody) may be administered in
conjunction with onartuzumab (also known as MetMAb). In some
embodiments, an OX40 agonist (e.g., an anti-human OX40 agonist
antibody) may be administered in conjunction with an inhibitor of
Alk. In some embodiments, an OX40 agonist (e.g., an anti-human OX40
agonist antibody) may be administered in conjunction with AF802
(also known as CH5424802 or alectinib). In some embodiments, an
OX40 agonist (e.g., an anti-human OX40 agonist antibody) may be
administered in conjunction with an inhibitor of a
phosphatidylinositol 3-kinase (PI3K). In some embodiments, an OX40
agonist (e.g., an anti-human OX40 agonist antibody) may be
administered in conjunction with BKM120. In some embodiments, an
OX40 agonist (e.g., an anti-human OX40 agonist antibody) may be
administered in conjunction with idelalisib (also known as GS-1101
or CAL-101). In some embodiments, an OX40 agonist (e.g., an
anti-human OX40 agonist antibody) may be administered in
conjunction with perifosine (also known as KRX-0401). In some
embodiments, an OX40 agonist (e.g., an anti-human OX40 agonist
antibody) may be administered in conjunction with an inhibitor of
an Akt. In some embodiments, an OX40 agonist (e.g., an anti-human
OX40 agonist antibody) may be administered in conjunction with
MK2206. In some embodiments, an OX40 agonist (e.g., an anti-human
OX40 agonist antibody) may be administered in conjunction with
GSK690693. In some embodiments, an OX40 agonist (e.g., an
anti-human OX40 agonist antibody) may be administered in
conjunction with GDC-0941. In some embodiments, an OX40 agonist
(e.g., an anti-human OX40 agonist antibody) may be administered in
conjunction with an inhibitor of mTOR. In some embodiments, an OX40
agonist (e.g., an anti-human OX40 agonist antibody) may be
administered in conjunction with sirolimus (also known as
rapamycin). In some embodiments, an OX40 agonist (e.g., an
anti-human OX40 agonist antibody) may be administered in
conjunction with temsirolimus (also known as CCI-779 or
Torisel.RTM.). In some embodiments, an OX40 agonist (e.g., an
anti-human OX40 agonist antibody) may be administered in
conjunction with everolimus (also known as RAD001). In some
embodiments, an OX40 agonist (e.g., an anti-human OX40 agonist
antibody) may be administered in conjunction with ridaforolimus
(also known as AP-23573, MK-8669, or deforolimus). In some
embodiments, an OX40 agonist (e.g., an anti-human OX40 agonist
antibody) may be administered in conjunction with OSI-027. In some
embodiments, an OX40 agonist (e.g., an anti-human OX40 agonist
antibody) may be administered in conjunction with AZD8055. In some
embodiments, an OX40 agonist (e.g., an anti-human OX40 agonist
antibody) may be administered in conjunction with INK128. In some
embodiments, an OX40 agonist (e.g., an anti-human OX40 agonist
antibody) may be administered in conjunction with a dual PI3K/mTOR
inhibitor. In some embodiments, an OX40 agonist (e.g., an
anti-human OX40 agonist antibody) may be administered in
conjunction with XL765. In some embodiments, an OX40 agonist (e.g.,
an anti-human OX40 agonist antibody) may be administered in
conjunction with GDC-0980. In some embodiments, an OX40 agonist
(e.g., an anti-human OX40 agonist antibody) may be administered in
conjunction with BEZ235 (also known as NVP-BEZ235). In some
embodiments, an OX40 agonist (e.g., an anti-human OX40 agonist
antibody) may be administered in conjunction with BGT226. In some
embodiments, an OX40 agonist (e.g., an anti-human OX40 agonist
antibody) may be administered in conjunction with GSK2126458. In
some embodiments, an OX40 agonist (e.g., an anti-human OX40 agonist
antibody) may be administered in conjunction with PF-04691502. In
some embodiments, an OX40 agonist (e.g., an anti-human OX40 agonist
antibody) may be administered in conjunction with PF-05212384 (also
known as PKI-587).
[0405] An OX40 agonist of the invention (and any additional
therapeutic agent) can be administered by any suitable means,
including parenteral, intrapulmonary, and intranasal, and, if
desired for local treatment, intralesional administration.
Parenteral infusions include intramuscular, intravenous,
intraarterial, intraperitoneal, or subcutaneous administration.
Dosing can be by any suitable route, e.g. by injections, such as
intravenous or subcutaneous injections, depending in part on
whether the administration is brief or chronic. Various dosing
schedules including but not limited to single or multiple
administrations over various time-points, bolus administration, and
pulse infusion are contemplated herein.
[0406] OX40 agonists of the invention would be formulated, dosed,
and administered in a fashion consistent with good medical
practice. Factors for consideration in this context include the
particular disorder being treated, the particular mammal being
treated, the clinical condition of the individual patient, the
cause of the disorder, the site of delivery of the agent, the
method of administration, the scheduling of administration, and
other factors known to medical practitioners. The antibody need not
be, but is optionally formulated with one or more agents currently
used to prevent or treat the disorder in question. The effective
amount of such other agents depends on the amount of antibody
present in the formulation, the type of disorder or treatment, and
other factors discussed above. These are generally used in the same
dosages and with administration routes as described herein, or
about from 1 to 99% of the dosages described herein, or in any
dosage and by any route that is empirically/clinically determined
to be appropriate.
[0407] For the prevention or treatment of disease, the appropriate
dosage of an OX40 agonist of the invention (when used alone or in
combination with one or more other additional therapeutic agents)
will depend on the type of disease to be treated, the type of
antibody, the severity and course of the disease, whether the
antibody is administered for preventive or therapeutic purposes,
previous therapy, the patient's clinical history and response to
the antibody, and the discretion of the attending physician. The
antibody is suitably administered to the patient at one time or
over a series of treatments. Depending on the type and severity of
the disease, about 1 .mu.g/kg to 40 mg/kg of antibody can be an
initial candidate dosage for administration to the patient,
whether, for example, by one or more separate administrations, or
by continuous infusion. One typical daily dosage might range from
about 1 .mu.g/kg to 100 mg/kg or more, depending on the factors
mentioned above. For repeated administrations over several days or
longer, depending on the condition, the treatment would generally
be sustained until a desired suppression of disease symptoms
occurs. Such doses may be administered intermittently, e.g. every
week or every three weeks (e.g. such that the patient receives from
about two to about twenty, or e.g. about six doses of the
antibody). An initial higher loading dose, followed by one or more
lower doses may be administered. However, other dosage regimens may
be useful. The progress of this therapy is easily monitored by
conventional techniques and assays.
[0408] In some embodiments of the methods of the present
disclosure, the cancer has elevated levels of T cell infiltration.
As used herein, T cell infiltration of a cancer may refer to the
presence of T cells, such as tumor-infiltrating lymphocytes (TILs),
within or otherwise associated with the cancer tissue. It is known
in the art that T cell infiltration may be associated with improved
clinical outcome in certain cancers (see, e.g., Zhang et al., N.
Engl. J. Med. 348(3):203-213 (2003)). In some embodiments, the TILs
may be OX40+. In some embodiments, the TILs may be CD4+OX40+Foxp3+
Treg or CD4+OX40+Foxp3-Teff cells.
VII. Kits and Articles of Manufacture
[0409] For use in the applications described or suggested above,
kits or articles of manufacture are also provided by the invention.
Such kits may comprise at least one reagent specific for detecting
expression level of a marker gene described herein (e.g., genes
described in Tables 3-5), and may further include instructions for
carrying out a method described herein.
[0410] In some embodiments, the invention provides compositions and
kits comprising primers and primer pairs, which allow the specific
amplification of the polynucleotides of the marker genes or of any
specific parts thereof, and probes that selectively or specifically
hybridize to nucleic acid molecules described herein or to any part
thereof. Probes may be labeled with a detectable marker, such as,
for example, a radioisotope, fluorescent compound, bioluminescent
compound, a chemiluminescent compound, metal chelator or enzyme.
Such probes and primers can be used to detect the presence of
polynucleotides, such as the polynucleotides corresponding to genes
listed in Tables 3-5, in a sample and as a means for detecting cell
expressing proteins encoded by the polynucleotides. As will be
understood by the skilled artisan, a great many different primers
and probes may be prepared based on the sequences provided herein
and used effectively to amplify, clone and/or determine the
presence and/or levels of mRNAs. In some embodiments, the kits may
further comprise a surface or substrate (such as a microarray) for
capture probes for detecting of amplified nucleic acids. In some
embodiments, the kits comprise at least one pair of primers and a
probe specific for detecting one marker gene expression level using
qRT-PCR.
[0411] The reagents for detecting the protein expression level of a
marker gene may comprise an antibody that specifically binds to the
protein encoded by the marker gene.
[0412] In some embodiments, the kits further comprise an OX40
agonist (e.g., an anti-OX40 agonist antibody). The kits may further
comprise instructions to administering an OX40 agonist if the
patient is identified as responsive to the OX40 agonist
treatment.
[0413] The kits may further comprise a carrier means being
compartmentalized to receive in close confinement one or more
container means such as vials, tubes, and the like, each of the
container means comprising one of the separate elements to be used
in the method. For example, one of the container means may comprise
a probe that is or can be detectably labeled. Such probe may be an
antibody or polynucleotide specific for a marker gene. Where the
kit utilizes nucleic acid hybridization to detect the target
nucleic acid, the kit may also have containers containing
nucleotide(s) for amplification of the target nucleic acid sequence
and/or a container comprising a reporter-means, such as a
biotin-binding protein, such as avidin or streptavidin, bound to a
reporter molecule, such as an enzymatic, florescent, or
radioisotope label.
[0414] The kit of the invention will typically comprise the
container described above and one or more other containers
comprising materials desirable from a commercial and user
standpoint, including buffers, diluents, filters, needles,
syringes, and package inserts with instructions for use. A label
may be present on the container to indicate that the composition is
used for a specific therapy or non-therapeutic application, and may
also indicate directions for either in vivo or in vitro use, such
as those described above.
[0415] The kit can further comprise a set of instructions and
materials for preparing a tissue or cell sample and preparing
nucleic acids (such as mRNA) from the sample.
[0416] The specification is considered to be sufficient to enable
one skilled in the art to practice the invention. Various
modifications of the invention in addition to those shown and
described herein will become apparent to those skilled in the art
from the foregoing description and fall within the scope of the
appended claims.
[0417] All publications, patents, and patent applications cited
herein are hereby incorporated by reference in their entirety for
all purposes.
EXAMPLES
[0418] The invention can be further understood by reference to the
following examples, which are provided by way of illustration and
are not meant to be limiting.
Example 1
OX40 Agonist Treatment Modulates T Cell Activities and Anti-Tumor
Activity
[0419] OX40 is known to be a co-stimulatory molecule expressed on
activated CD4 T cells (Teff) and T regulatory (Treg) cells.
Ligation of OX40 in the presence of TCR stimulation is known to
enhance T effector cell function via dual mechanism of potentiating
activation of Teff cells and inhibiting Treg cells.
[0420] Enhancement of anti-tumor immunity by OX40 agonist treatment
is a promising therapeutic approach to treat cancer. Therefore, in
vitro and in vivo preclinical studies were conducted to
characterize co-stimulatory activity, evaluate anti-tumor efficacy,
and identify predictive and pharmacodynamic (PD) biomarkers of OX40
agonists.
[0421] Materials and Methods
In Vitro T Cell Co-Stimulation Assay
[0422] CD4.sup.+ T cells were isolated from PBMCs from healthy
donors via magnetic enrichment (Miltenyi). Sorted CD4.sup.+ were
stimulated with PHA to induce OX40 expression, followed by resting
in the presence of IL-2, and subsequently restimulated with a fixed
concentration of anti-CD3 and variable anti-OX40 (anti-human OX40
mAb) concentrations in platebound form. T cell proliferation was
measured by quantitation of ATP levels (Promega), and supernatant
IFN-gamma levels were assessed by ELISA.
In Vitro Treg Suppression Assay
[0423] CD4.sup.+CD25.sup.+CD127.sup.- Tregs and naive T cells were
isolated from healthy donors via FACS and magnetic purification,
respectively. Suppression assay cultures were comprised of Tregs
and CFSE labeled naive CD4.sup.+ cells (2 Treg:1 naive), irradiated
CD80.sup.+ CD32.sup.+ L cells as APCs, anti-CD3, and anti-human
OX40 mAb. Suppression of naive T cell proliferation was quantified
via FACS.
In Vivo Tumor Studies
[0424] Syngeneic tumor cells were inoculated either subcutaneously
(CT26, 51Blim10) or orthotopically (EMT6, JC) in C57/B16 or BalbC
mice. For predictive biomarker analysis, tumors were harvested when
they reached a volume of 150-250 mm.sup.3. For efficacy and
pharmacodynamic biomarker analysis, when the tumors reached a
volume of 150-250 mm.sup.3, mice were treated with 10 mg/kg of
either control Ab or murine anti-mouse OX40 mAb 2.times./wk for 3
weeks. Tumors and peripheral blood were harvested on day 2, day 9,
and day 16 post first dose.
Flow Cytometry
[0425] Tumor tissues were cut into small pieces, incubated with
collagenase and DNAse (Roche), and single cell suspensions were
prepared using a gentle MACS Dissociator (Miltenyi) per
manufacturer's protocol. Phenotyping of tumor infiltrating
lymphocytes (TILs) was performed with commercial antibodies against
CD45, CD3, CD4, CD8, CD25, (all BD Biosciences) and Foxp3
(eBiosciences) per manufacturers' instructions. The Live/Dead
Fixable Near-IR Dead Cell Stain Kit (Life Technologies) protocol
was used to exclude dead cells from analysis. Stained cells were
analyzed on a BD FACSCanto flow cytometer.
Gene Expression Analysis
[0426] RNA was extracted from tumor lysates using the mirvana miRNA
Isolation kit (Life Technologies) according to the total RNA
isolation protocol supplied by the manufacturer. RNA quality was
assessed using an Agilent 2100 Bioanalyzer. RNA (200 ng) was
subjected to a one-step cDNA synthesis/preamplification reaction
using the Invitrogen Platinum Taq/Reverse Transcriptase enzyme mix
as per the manufacturer's protocol with the exception that PCR
cycling conditions were changed from a 14 cycle preamplification to
16 cycles (Life Technologies). Following amplification, samples
were diluted one to four with TE and qPCR was conducted on Fluidigm
96.96 Dynamic Arrays using the BioMark.TM. HD system according to
the manufacturer's protocol and as previously described (Shames, D.
S., et al. (2013) PLoS ONE 8:e56765). Samples were run in duplicate
and cycle threshold (Ct) values were converted to relative
expression values (negative delta Ct) by subtracting the mean of
the three reference genes from the mean of each target gene. A list
of all genes evaluated in these studies is provided in Table 6
below.
TABLE-US-00009 TABLE 6 Assay Gene 1 CD3E 2 CD4 3 CD8A 4 CD8b 5
GATA3 6 TBX21 7 RORC 8 FOXP3 9 BTLA 10 CTLA4 11 PDCD1 12 CD28 13
CD27 14 CD69 15 ICOS 16 EOMES 17 CD226 18 LAG3 19 TNFRSF4 20 GZMA
21 GZMB 22 PRF1 23 CD80 24 CD86 25 CD274 26 CD276 27 PDCD1LG2 28
VTCN1 29 IL1B 30 IL2 31 IL4 32 IL6 33 IL7 34 IL7R 35 IL10 36 IL12A
37 IL13 38 IL17A 39 IL33 40 CSF2 41 IFNg 42 TGFB1 43 TNF 44 IL2RA
45 CXCL1 46 CXCL9 47 CXCL10 48 CCL2 49 CCL5 50 CCL22 51 PVR 52 CCR5
53 CCR7 54 CX3CL1 55 CXCR3 56 VEGFA 57 CD40 58 MS4A1 59 Fcgr3/CD16
60 KLRK1 61 TIGIT 62 IFNa 63 NCAM1 64 CD83 65 CD209a 66 PDCA1 67
Zbtb46 68 PTGS2 69 ARG1 70 ITGAM 71 H2-d 72 H2-aa 73 H2-k 74
Fcgr2b/CD32 75 ICAM1 76 VCAM1 77 EPCAM 78 TNFSF4 (OX40L) 79 TNFSF9
(4-1BBL) 80 TNFRSF9 (4-1BB) 81 HAVCR2 82 TNFRSF14 83 FasL 84 CD48
85 TNFRSF18 (GITR) 86 PTPRC 87 PGE2/PGES2 88 PTGER4 89 IDO1 90
LGALS9 91 MAP4K1 92 IL9 93 SP2 94 YWHAZ 95 GUSb 96 Fcgr1/CD64
Data Analysis
[0427] For predictive biomarker evaluation, expression of each gene
was normalized to median expression. Genes that showed at least an
average of 2 fold statistically significant (Student's t-test,
p<0.05) differential expression between more responsive (EMT6
and CT26) and less responsive (JC and 51Blim10) models were
selected for downstream analysis. Hierarchical clustering was
carried out on median-centered data with the complete linkage
method using Cluster v3.0 and visualized using Treeview (Eisen, M.
B., et al. (1998) Proc. Natl. Acad. Sci. 95:14863-8).
[0428] Results
[0429] OX40 is known to be highly expressed on Teff and Treg cells
in mouse and human tumors. As such, modulating OX40 activity may
provide a means to modulate T cell function in cancer, i.e., for
cancer immunotherapy. Therefore, the effect of OX40 agonist
treatment on T cell function in vitro was examined.
[0430] Isolated CD4+ T cells were isolated from PBMCs, stimulated
to induce OX40 expression, and then re-stimulated with anti-CD3 in
the presence of the agonist anti-human OX40 mAb, as described
above. FIGS. 1A & 1B demonstrate that T cell proliferation
(FIG. 1A) and IFN.gamma. production (FIG. 1B) were both enhanced by
treatment with anti-OX40, as compared to a control treatment.
[0431] In addition, anti-OX40 treatment was found to reduce Treg
activity in the in vitro Treg suppression assay described above
(FIG. 2). These results demonstrate that OX40 agonist treatment is
able to modulate several critical T cell functions.
[0432] OX40 agonist treatment was next analyzed in several
syngeneic mouse tumor models to examine whether OX40 agonism has an
effect on tumor growth in vivo. Mice were inoculated with various
syngeneic tumor cell types to develop tumors, and once these tumors
reached a threshold size, mice were treated with murine anti-mouse
OX40 mAb or control antibody, as described above.
[0433] As shown in FIGS. 3A-3C, anti-OX40 agonist treatment caused
a dramatic reduction and durable regression in several tumor
models, including EMT6 breast (FIG. 3A), Cloudman melanoma (FIG.
3B), and CT26 colorectal cancer (CRC) (FIG. 3C) tumor models. In
contrast, other mouse tumor models showed less responsiveness to
treatment, such as the MC38 CRC (FIG. 4A), 51Blim10 CRC (FIG. 4B),
and JC breast (FIG. 4C) tumor models. These results demonstrate
differential anti-tumor activity of OX40 agonist treatment in mouse
tumor models. However, OX40 agonist treatment led to anti-tumor
activity, including durable tumor regression, in several tumor
models.
Example 2
Baseline Immune Function Associated with Gene Expression in Tumors
May be Predictive of Differential Anti-Tumor Activity of OX40
Agonist Treatment
[0434] Because of the observation that OX40 agonist treatment leads
to differential responsiveness in various tumor models, the
expression of more than 90 immune-related genes was next examined,
comparing gene expression in tumor models that were found to be
more responsive to OX40 agonist treatment with gene expression in
tumor models that were found to be less responsive. As described
above, these samples were isolated prior to any treatment with OX40
agonist, when tumors reached a threshold size.
[0435] As shown in FIG. 5, higher expression of certain immune
activation and Th1 markers may be associated with better
responsiveness in EMT6 and CT26 tumors. For example, higher
expression of CD8, IFNg, GZMB, PRF1, and PDCA1 may be associated
with better responsiveness to anti-OX40 treatment. In addition,
higher expression of CXCL9, PTPRC, IL7R, KLRK1, and CXCL1 may also
be associated with better responsiveness to anti-OX40
treatment.
[0436] In contrast, higher expression of CCL2, GATA3, IL13, VTCN1,
and CSF2 may be associated with poor responsiveness to an anti-OX40
treatment, but lower expression of these genes may be associated
with better responsiveness to an anti-OX40 treatment. Genes that
are differentially expressed between responsive and non-responsive
tumor types are listed below in Table 7. As shown in Table 7, genes
that were found to correlate with better responsiveness when
expressed at a higher level are classified as positive predictors,
whereas genes that were found to correlate with poor responsiveness
when expressed at a higher level are classified as negative
predictors. These negative predictors may correlate with better
responsiveness when expressed at a lower level in a tumor.
TABLE-US-00010 TABLE 7 Differentially expressed genes for
predictive biomarker evaluation. Gene Predictor CSF2 Negative CCL22
Negative GATA3 Negative CD8a Positive CD8b Positive H2-d Positive
CTLA4 Positive CD64 Positive CXCL9 Positive IFNg Positive IDO1
Positive GZMA Positive GZMB Positive PRF1 Positive PDCA1 Positive
KLRK1 Positive PTPRC Positive CXCL1 Positive ITGAM Positive IL7R
Positive IL13 Negative EPCAM Negative VTCN1 Negative
[0437] In summary, these results indicate that the expression level
of specific genes may be associated with responsiveness to OX40
agonist treatment.
Example 3
Ox40 Agonist Treatment Induces Immune Modulation in Different Tumor
Models
[0438] In addition to gene expression, other parameters of immune
modulation were next examined in various tumor models in response
to the OX40 agonist treatment.
[0439] FIGS. 6A & 6B show the dose-dependent effects of
anti-OX40 treatment on peripheral blood cells in the EMT6 tumor
model. Anti-OX40 treatment using murine anti-mouse OX40 mAb led to
a dose-dependent reduction in peripheral Treg cells (FIG. 6A) and a
dose-dependent increase in peripheral CD8 T cell proliferation
(FIG. 6B).
[0440] T cell sub-populations in tumors were also analyzed.
Anti-OX40 treatment caused a reduction in Treg cells in the EMT6
breast tumor model, which showed better responsiveness to the
anti-OX40 treatment (FIG. 7A). Importantly, the anti-OX40 treatment
induced a sustained increase in CD8 tumor infiltrate in the EMT6
model (FIG. 7B). In the JC breast tumor model, which was less
responsive to the anti-OX40 treatment, the anti-OX40 antibody also
led to a reduction in Treg cells in JC tumors (FIG. 8A). However,
CD8 T cells in JC tumors showed a more slight increase than in EMT6
cells (FIG. 8B).
[0441] Gene expression of specific markers of immune activation was
also examined in these tumor models. As shown in FIGS. 9A-9D,
expression of several gene markers was associated with
pharmacodynamic (PD) activity in both tumor models, with slightly
increased activity in the EMT6 model compared to the JC model.
These gene markers included IFNg (FIG. 9A), granzyme A (FIG. 9B),
perforin (FIG. 9C), and TNFa (FIG. 9D). Table 8 lists the markers
for PD activity identified in these experiments.
TABLE-US-00011 TABLE 8 Markers of PD activity ARG1 CCL2 CCL22 CCL5
CCR5 CD226 CD27 CD274 CD28 CD3E CD40 CD8A CD8b CXCL10 CXCL9 EOMES
FasL Fcgr1/CD64 FOXP3 GZMA GZMB HAVCR2 ICAM1 IDO1 IFNg IL10 IL12A
(TDO2) IL13 IL2 IL7R ITGAM KLRK1 LAG3 MAP4K1 MS4A1 PDCD1 PDCD1LG2
PRF1 PTPRC TNF TNFRSF14 TNFRSF9 TNFSF4
[0442] These results suggest that genes associated with immune
activation are induced by anti-OX40 in both EMT6 and JC tumors, but
this increase in expression may not reach a threshold required for
anti-tumor activity in JC tumors. Importantly, these results
identify genes that could be used as markers for PD activity in
both responsive and non-responsive tumors.
[0443] Other genes associated with antigen presentation,
co-stimulation, and IFNg response were differentially upregulated
in EMT6 tumors (FIGS. 10A-10D). These gene markers included H2-aa
(FIG. 10A), CD86 (FIG. 10B), ICOS (FIG. 10C), and CXCR3 (FIG. 10D).
Table 9 lists the markers for responsiveness to OX40 agonist
treatment identified in these experiments.
TABLE-US-00012 TABLE 9 Markers for responsiveness BTLA CD4 CD69
CD80 CD83 CD86 CSF2 CTLA4 CXCR3 Fcgr2b/CD32 Fcgr3/CD16 H2-aa H2-d
H2-k ICOS IL10 PDCA1 TNFRSF18
[0444] These results indicate that enhanced PD modulation by
anti-OX40 (murine anti-mouse OX40 mAb) in EMT6 tumors compared to
JC tumors may be associated with a better anti-tumor response.
Further, these results identify genes that could be used as markers
for responsiveness to OX40 agonist treatment.
[0445] In summary, these results demonstrate that OX40 agonists
induce potent T cell activation and promote anti-tumor immunity and
efficacy in preclinical systems. In addition, the biomarkers
identified in these studies may be utilized to confirm mechanism of
action, inform dose finding, and guide patient and indication
selection in clinical trials.
[0446] All patents, patent applications, documents, and articles
cited herein are herein incorporated by reference in their
entireties.
Sequence CWU 1
1
2261249PRTHomo sapiens 1Leu His Cys Val Gly Asp Thr Tyr Pro Ser Asn
Asp Arg Cys Cys His1 5 10 15 Glu Cys Arg Pro Gly Asn Gly Met Val
Ser Arg Cys Ser Arg Ser Gln 20 25 30 Asn Thr Val Cys Arg Pro Cys
Gly Pro Gly Phe Tyr Asn Asp Val Val 35 40 45 Ser Ser Lys Pro Cys
Lys Pro Cys Thr Trp Cys Asn Leu Arg Ser Gly 50 55 60 Ser Glu Arg
Lys Gln Leu Cys Thr Ala Thr Gln Asp Thr Val Cys Arg65 70 75 80 Cys
Arg Ala Gly Thr Gln Pro Leu Asp Ser Tyr Lys Pro Gly Val Asp 85 90
95 Cys Ala Pro Cys Pro Pro Gly His Phe Ser Pro Gly Asp Asn Gln Ala
100 105 110 Cys Lys Pro Trp Thr Asn Cys Thr Leu Ala Gly Lys His Thr
Leu Gln 115 120 125 Pro Ala Ser Asn Ser Ser Asp Ala Ile Cys Glu Asp
Arg Asp Pro Pro 130 135 140 Ala Thr Gln Pro Gln Glu Thr Gln Gly Pro
Pro Ala Arg Pro Ile Thr145 150 155 160 Val Gln Pro Thr Glu Ala Trp
Pro Arg Thr Ser Gln Gly Pro Ser Thr 165 170 175 Arg Pro Val Glu Val
Pro Gly Gly Arg Ala Val Ala Ala Ile Leu Gly 180 185 190 Leu Gly Leu
Val Leu Gly Leu Leu Gly Pro Leu Ala Ile Leu Leu Ala 195 200 205 Leu
Tyr Leu Leu Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His Lys 210 215
220 Pro Pro Gly Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu Gln
Ala225 230 235 240 Asp Ala His Ser Thr Leu Ala Lys Ile 245
25PRTArtificial SequenceSynthetic Construct 2Asp Ser Tyr Met Ser1 5
317PRTArtificial SequenceSynthetic Construct 3Asp Met Tyr Pro Asp
Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe Arg1 5 10 15
Glu48PRTArtificial SequenceSynthetic Construct 4Ala Pro Arg Trp Tyr
Phe Ser Val1 5 511PRTArtificial SequenceSynthetic Construct 5Arg
Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn1 5 10 67PRTArtificial
SequenceSynthetic Construct 6Tyr Thr Ser Arg Leu Arg Ser1 5
79PRTArtificial SequenceSynthetic Construct 7Gln Gln Gly His Thr
Leu Pro Pro Thr1 5 85PRTArtificial SequenceSynthetic Construct 8Asp
Ala Tyr Met Ser1 5 95PRTArtificial SequenceSynthetic Construct 9Glu
Ser Tyr Met Ser1 5 1017PRTArtificial SequenceSynthetic Construct
10Asp Met Tyr Pro Asp Asn Ala Asp Ser Ser Tyr Asn Gln Lys Phe Arg1
5 10 15 Glu1117PRTArtificial SequenceSynthetic Construct 11Asp Met
Tyr Pro Asp Asn Ala Asp Ala Ser Tyr Asn Gln Lys Phe Arg1 5 10 15
Glu1217PRTArtificial SequenceSynthetic Construct 12Asp Met Tyr Pro
Asp Asn Gly Asp Ala Ser Tyr Asn Gln Lys Phe Arg1 5 10 15
Glu1317PRTArtificial SequenceSynthetic Construct 13Asp Met Tyr Pro
Asp Ser Gly Asp Ser Ser Tyr Asn Gln Lys Phe Arg1 5 10 15
Glu1417PRTArtificial SequenceSynthetic Construct 14Asp Met Tyr Pro
Asp Asn Gly Ser Ser Ser Tyr Asn Gln Lys Phe Arg1 5 10 15
Glu158PRTArtificial SequenceSynthetic Construct 15Ala Pro Arg Trp
Tyr Phe Ser Ala1 5 168PRTArtificial SequenceSynthetic Construct
16Ala Pro Arg Trp Tyr Ala Ser Val1 5 178PRTArtificial
SequenceSynthetic Construct 17Ala Pro Arg Trp Ala Phe Ser Val1 5
188PRTArtificial SequenceSynthetic Construct 18Ala Pro Ala Trp Tyr
Phe Ser Val1 5 198PRTArtificial SequenceSynthetic Construct 19Ala
Pro Arg Trp Tyr Phe Ala Val1 5 208PRTArtificial SequenceSynthetic
Construct 20Ala Pro Arg Ala Tyr Phe Ser Val1 5 218PRTArtificial
SequenceSynthetic Construct 21Ala Ala Arg Trp Tyr Phe Ser Val1 5
229PRTArtificial SequenceSynthetic Construct 22Gln Gln Gly His Thr
Leu Pro Ala Thr1 5 239PRTArtificial SequenceSynthetic Construct
23Gln Gln Gly His Thr Ala Pro Pro Thr1 5 249PRTArtificial
SequenceSynthetic Construct 24Gln Gln Gly Ala Thr Leu Pro Pro Thr1
5 259PRTArtificial SequenceSynthetic Construct 25Gln Gln Gly His
Ala Leu Pro Pro Thr1 5 269PRTArtificial SequenceSynthetic Construct
26Gln Gln Ala His Thr Leu Pro Pro Thr1 5 279PRTArtificial
SequenceSynthetic Construct 27Gln Gln Gly His Thr Leu Ala Pro Thr1
5 289PRTArtificial SequenceSynthetic Construct 28Gln Ala Gly His
Thr Leu Pro Pro Thr1 5 295PRTArtificial SequenceSynthetic Construct
29Asn Tyr Leu Ile Glu1 5 3017PRTArtificial SequenceSynthetic
Construct 30Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe Lys1 5 10 15 Gly3117PRTArtificial SequenceSynthetic Construct
31Val Ile Asn Pro Gly Ser Gly Asp Ala Tyr Tyr Ser Glu Lys Phe Lys1
5 10 15 Gly3217PRTArtificial SequenceSynthetic Construct 32Val Ile
Asn Pro Gly Ser Gly Asp Gln Tyr Tyr Ser Glu Lys Phe Lys1 5 10 15
Gly335PRTArtificial SequenceSynthetic Construct 33Asp Arg Leu Asp
Tyr1 5 345PRTArtificial SequenceSynthetic Construct 34Ala Arg Leu
Asp Tyr1 5 355PRTArtificial SequenceSynthetic Construct 35Asp Ala
Leu Asp Tyr1 5 365PRTArtificial SequenceSynthetic Construct 36Asp
Arg Ala Asp Tyr1 5 3711PRTArtificial SequenceSynthetic Construct
37His Ala Ser Gln Asp Ile Ser Ser Tyr Ile Val1 5 10
387PRTArtificial SequenceSynthetic Construct 38His Gly Thr Asn Leu
Glu Asp1 5 397PRTArtificial SequenceSynthetic Construct 39His Gly
Thr Asn Leu Glu Ser1 5 407PRTArtificial SequenceSynthetic Construct
40His Gly Thr Asn Leu Glu Glu1 5 417PRTArtificial SequenceSynthetic
Construct 41His Gly Thr Asn Leu Glu Gln1 5 429PRTArtificial
SequenceSynthetic Construct 42Val His Tyr Ala Gln Phe Pro Tyr Thr1
5 439PRTArtificial SequenceSynthetic Construct 43Ala His Tyr Ala
Gln Phe Pro Tyr Thr1 5 449PRTArtificial SequenceSynthetic Construct
44Val Ala Tyr Ala Gln Phe Pro Tyr Thr1 5 459PRTArtificial
SequenceSynthetic Construct 45Val His Ala Ala Gln Phe Pro Tyr Thr1
5 469PRTArtificial SequenceSynthetic Construct 46Val His Tyr Ala
Ala Phe Pro Tyr Thr1 5 479PRTArtificial SequenceSynthetic Construct
47Val His Tyr Ala Gln Ala Pro Tyr Thr1 5 489PRTArtificial
SequenceSynthetic Construct 48Val His Tyr Ala Gln Phe Ala Tyr Thr1
5 499PRTArtificial SequenceSynthetic Construct 49Val His Tyr Ala
Gln Phe Pro Ala Thr1 5 505PRTArtificial SequenceSynthetic Construct
50Asp Tyr Gly Val Leu1 5 5116PRTArtificial SequenceSynthetic
Construct 51Met Ile Trp Ser Gly Gly Thr Thr Asp Tyr Asn Ala Ala Phe
Ile Ser1 5 10 15 525PRTArtificial SequenceSynthetic Construct 52Glu
Glu Met Asp Tyr1 5 5311PRTArtificial SequenceSynthetic Construct
53Arg Ala Ser Gln Asp Ile Ser Asn Phe Leu Asn1 5 10
547PRTArtificial SequenceSynthetic Construct 54Tyr Thr Ser Arg Leu
His Ser1 5 559PRTArtificial SequenceSynthetic Construct 55Gln Gln
Gly Asn Thr Leu Pro Trp Thr1 5 56117PRTArtificial SequenceSynthetic
Construct 56Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly
Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile
Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu
Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 57107PRTArtificial SequenceSynthetic
Construct 57Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp
Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro 85 90 95 Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 58117PRTArtificial
SequenceSynthetic Construct 58Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met
Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg
Glu Arg Val Thr Ile Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Val Leu Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly
Thr Leu 100 105 110 Val Thr Val Ser Ser 115 59107PRTArtificial
SequenceSynthetic Construct 59Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr
Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
60117PRTArtificial SequenceSynthetic Construct 60Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys
Phe 50 55 60 Arg Glu Arg Val Thr Leu Thr Val Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg Trp Tyr Phe Ser
Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
61107PRTArtificial SequenceSynthetic Construct 61Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly
His Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105 62117PRTArtificial SequenceSynthetic Construct
62Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser
Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Val Asp
Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg
Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val
Ser Ser 115 63107PRTArtificial SequenceSynthetic Construct 63Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Thr Val Lys Leu
Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Gly His Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 105 64117PRTArtificial SequenceSynthetic
Construct 64Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly
Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile
Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu
Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 65107PRTArtificial SequenceSynthetic
Construct 65Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp
Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro 85 90 95 Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 66117PRTArtificial
SequenceSynthetic Construct 66Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20
25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn
Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Val Asp Thr Ser
Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg Trp Tyr
Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser
115 67107PRTArtificial SequenceSynthetic Construct 67Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25
30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Thr Val Lys Leu Leu Ile
35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln
Gly His Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys 100 105 68117PRTArtificial SequenceSynthetic Construct
68Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser
Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Val Asp
Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg
Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val
Ser Ser 115 69107PRTArtificial SequenceSynthetic Construct 69Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Thr Val Lys Leu
Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Lys Asp Tyr Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Phe Cys
Gln Gln Gly His Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 105 70117PRTArtificial SequenceSynthetic
Construct 70Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Asp Ala 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly
Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile
Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu
Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 71107PRTArtificial SequenceSynthetic
Construct 71Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp
Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro 85 90 95 Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 72117PRTArtificial
SequenceSynthetic Construct 72Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Glu Ser 20 25 30 Tyr Met Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met
Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg
Glu Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Val Leu Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly
Thr Leu 100 105 110 Val Thr Val Ser Ser 115 73107PRTArtificial
SequenceSynthetic Construct 73Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr
Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
74117PRTArtificial SequenceSynthetic Construct 74Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Asp Met Tyr Pro Asp Asn Ala Asp Ser Ser Tyr Asn Gln Lys
Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg Trp Tyr Phe Ser
Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
75107PRTArtificial SequenceSynthetic Construct 75Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly
His Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105 76117PRTArtificial SequenceSynthetic Construct
76Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Ala Asp Ala Ser
Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Arg Asp
Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg
Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val
Ser Ser 115 77107PRTArtificial SequenceSynthetic Construct 77Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Gly His Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 105 78117PRTArtificial SequenceSynthetic
Construct 78Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly
Asp Ala Ser Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile
Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu
Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 79107PRTArtificial SequenceSynthetic
Construct 79Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp
Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro 85 90 95 Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 80117PRTArtificial
SequenceSynthetic Construct 80Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met
Tyr Pro Asp Ser Gly Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg
Glu Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Val Leu Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly
Thr Leu 100 105 110 Val Thr Val Ser Ser 115 81107PRTArtificial
SequenceSynthetic Construct 81Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr
Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
82117PRTArtificial SequenceSynthetic Construct 82Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Asp Met Tyr Pro Asp Asn Gly Ser Ser Ser Tyr Asn Gln Lys
Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg Trp Tyr Phe Ser
Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
83107PRTArtificial SequenceSynthetic Construct 83Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly
His Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105 84117PRTArtificial SequenceSynthetic Construct
84Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Ala 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Ala Asp Ala Ser
Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Arg Asp
Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg
Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val
Ser Ser 115 85107PRTArtificial SequenceSynthetic Construct 85Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Gly His Thr
Leu Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 86117PRTArtificial SequenceSynthetic Construct 86Glu Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20
25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn
Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Arg Asp Thr Ser
Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg Trp Tyr
Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser
115 87107PRTArtificial SequenceSynthetic Construct 87Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25
30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Gly His Thr Leu Pro Ala 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys 100 105 88117PRTArtificial SequenceSynthetic Construct
88Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser
Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Arg Asp
Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg
Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val
Ser Ser 115 89107PRTArtificial SequenceSynthetic Construct 89Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Gly His Thr Ala Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 105 90117PRTArtificial SequenceSynthetic
Construct 90Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly
Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile
Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu
Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 91107PRTArtificial SequenceSynthetic
Construct 91Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp
Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Gln Gly Ala Thr Leu Pro Pro 85 90 95 Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 92117PRTArtificial
SequenceSynthetic Construct 92Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met
Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg
Glu Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Val Leu Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly
Thr Leu 100 105 110 Val Thr Val Ser Ser 115 93107PRTArtificial
SequenceSynthetic Construct 93Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr
Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Ala Leu Pro Pro
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
94117PRTArtificial SequenceSynthetic Construct 94Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys
Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg Trp Tyr Phe Ser
Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
95107PRTArtificial SequenceSynthetic Construct 95Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala
His Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105 96117PRTArtificial SequenceSynthetic Construct
96Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser
Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Arg Asp
Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg
Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val
Ser Ser 115 97107PRTArtificial SequenceSynthetic Construct 97Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Gly His Thr Leu Ala Pro 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 105 98117PRTArtificial SequenceSynthetic
Construct 98Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly
Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile
Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu
Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110
Val Thr Val Ser Ser 115 99107PRTArtificial SequenceSynthetic
Construct 99Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp
Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys
Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser
Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala
Thr Tyr Tyr Cys Gln Ala Gly His Thr Leu Pro Pro 85 90 95 Thr Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 100117PRTArtificial
SequenceSynthetic Construct 100Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met
Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg
Glu Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Val Leu Ala Pro Arg Trp Tyr Phe Ser Ala Trp Gly Gln Gly
Thr Leu 100 105 110 Val Thr Val Ser Ser 115 101107PRTArtificial
SequenceSynthetic Construct 101Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr
Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
102117PRTArtificial SequenceSynthetic Construct 102Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys
Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg Trp Tyr Ala Ser
Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
103107PRTArtificial SequenceSynthetic Construct 103Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly
His Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105 104117PRTArtificial SequenceSynthetic Construct
104Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser
Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Arg Asp
Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg
Trp Ala Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val
Ser Ser 115 105107PRTArtificial SequenceSynthetic Construct 105Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20
25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Gly His Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys 100 105 106117PRTArtificial SequenceSynthetic
Construct 106Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn
Gly Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr
Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val
Leu Ala Pro Ala Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105
110 Val Thr Val Ser Ser 115 107107PRTArtificial SequenceSynthetic
Construct 107Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 108117PRTArtificial
SequenceSynthetic Construct 108Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met
Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg
Glu Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Val Leu Ala Pro Arg Trp Tyr Phe Ala Val Trp Gly Gln Gly
Thr Leu 100 105 110 Val Thr Val Ser Ser 115 109107PRTArtificial
SequenceSynthetic Construct 109Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr
Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
110117PRTArtificial SequenceSynthetic Construct 110Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys
Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Pro Arg Ala Tyr Phe Ser
Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115
111107PRTArtificial SequenceSynthetic Construct 111Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly
His Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu
Ile Lys 100 105 112117PRTArtificial SequenceSynthetic Construct
112Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn Gly Asp Ser Ser
Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr Ile Thr Arg Asp
Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Leu Ala Ala Arg
Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val
Ser Ser 115 113107PRTArtificial SequenceSynthetic Construct 113Asp
Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10
15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr
20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg Ser Gly Val Pro Ser
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys
Gln Gln Gly His Thr Leu Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 105 114117PRTArtificial SequenceSynthetic
Construct 114Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr
Thr Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met Tyr Pro Asp Asn
Gly Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg Glu Arg Val Thr
Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80 Leu Glu Leu
Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala
Leu Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly Thr Leu 100 105
110 Val Thr Val Ser Ser 115 115107PRTArtificial SequenceSynthetic
Construct 115Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro 85 90 95 Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 116117PRTArtificial
SequenceSynthetic Construct 116Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met
Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg
Glu Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Val Ala Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly
Thr Leu 100 105 110 Val Thr Val Ser Ser 115 117107PRTArtificial
SequenceSynthetic Construct 117Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr
Ser Arg Leu Arg Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly His Thr Leu Pro Pro
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
118114PRTArtificial SequenceSynthetic Construct 118Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser119107PRTArtificial
SequenceSynthetic Construct 119Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
120114PRTArtificial SequenceSynthetic Construct 120Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser121107PRTArtificial
SequenceSynthetic Construct 121Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
122114PRTArtificial SequenceSynthetic Construct 122Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser123107PRTArtificial
SequenceSynthetic Construct 123Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
124114PRTArtificial SequenceSynthetic Construct 124Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser125107PRTArtificial
SequenceSynthetic Construct 125Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
126114PRTArtificial SequenceSynthetic Construct 126Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser127107PRTArtificial
SequenceSynthetic Construct 127Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
128114PRTArtificial SequenceSynthetic Construct 128Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser129107PRTArtificial
SequenceSynthetic Construct 129Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
130114PRTArtificial SequenceSynthetic Construct 130Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser131107PRTArtificial
SequenceSynthetic Construct 131Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Glu Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
132114PRTArtificial SequenceSynthetic Construct 132Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser133107PRTArtificial
SequenceSynthetic Construct 133Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Gln Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
134114PRTArtificial SequenceSynthetic Construct 134Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser135107PRTArtificial
SequenceSynthetic Construct 135Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Ala Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
136114PRTArtificial SequenceSynthetic Construct 136Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser137107PRTArtificial
SequenceSynthetic Construct 137Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Ala Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
138114PRTArtificial SequenceSynthetic Construct 138Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser139107PRTArtificial
SequenceSynthetic Construct 139Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
140114PRTArtificial SequenceSynthetic Construct 140Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser141107PRTArtificial
SequenceSynthetic Construct 141Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Pro Lys Leu Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
142114PRTArtificial SequenceSynthetic Construct 142Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser143107PRTArtificial
SequenceSynthetic Construct 143Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Phe Lys Leu Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
144114PRTArtificial SequenceSynthetic Construct 144Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60
Lys Gly Arg Val Thr Leu Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65
70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly Gln Gly Thr
Leu Val Thr Val 100 105 110 Ser Ser145107PRTArtificial
SequenceSynthetic Construct 145Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
146114PRTArtificial SequenceSynthetic Construct 146Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser147107PRTArtificial
SequenceSynthetic Construct 147Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Ala His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
148114PRTArtificial SequenceSynthetic Construct 148Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser149107PRTArtificial
SequenceSynthetic Construct 149Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val Ala Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
150114PRTArtificial SequenceSynthetic Construct 150Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser151107PRTArtificial
SequenceSynthetic Construct 151Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Ala Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
152114PRTArtificial SequenceSynthetic Construct 152Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser153107PRTArtificial
SequenceSynthetic Construct 153Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Ala Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
154114PRTArtificial SequenceSynthetic Construct 154Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser155107PRTArtificial
SequenceSynthetic Construct 155Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Ala Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
156114PRTArtificial SequenceSynthetic Construct 156Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser157107PRTArtificial
SequenceSynthetic Construct 157Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Ala Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
158114PRTArtificial SequenceSynthetic Construct 158Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser159107PRTArtificial
SequenceSynthetic Construct 159Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Ala
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
160114PRTArtificial SequenceSynthetic Construct 160Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ala Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser161107PRTArtificial
SequenceSynthetic Construct 161Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
162114PRTArtificial SequenceSynthetic Construct 162Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ala Leu Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser163107PRTArtificial
SequenceSynthetic Construct 163Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
164114PRTArtificial SequenceSynthetic Construct 164Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp Thr Ser Thr Ser
Thr Ala Tyr65 70 75 80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Ala Asp Tyr Trp Gly
Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser165107PRTArtificial
SequenceSynthetic Construct 165Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Tyr Gln
Gln Lys Pro Gly Lys Ser Phe Lys Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
166113PRTArtificial SequenceSynthetic Construct 166Glu Val Gln Leu
Val Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15 Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Tyr 20 25 30
Gly Val Leu Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35
40 45 Gly Met Ile Trp Ser Gly Gly Thr Thr Asp Tyr Asn Ala Ala Phe
Ile 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln
Phe Ser Leu65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys Val 85 90 95 Arg Glu Glu Met Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser 100 105 110 Ser167107PRTArtificial
SequenceSynthetic Construct 167Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Ile Ser Asn Phe 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr
Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
168113PRTArtificial SequenceSynthetic Construct 168Glu Val Gln Leu
Val Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15 Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Tyr 20 25 30
Gly Val Leu Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile 35
40 45 Gly Met Ile Trp Ser Gly Gly Thr Thr Asp Tyr Asn Ala Ala Phe
Ile 50 55 60 Ser Arg Val Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln
Val Ser Leu65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys Val 85 90 95 Arg Glu Glu Met Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser 100 105 110 Ser169107PRTArtificial
SequenceSynthetic Construct 169Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Ile Ser Asn Phe 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr
Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
170113PRTArtificial SequenceSynthetic Construct 170Glu Val Gln Leu
Val Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15 Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Asp Tyr 20 25 30
Gly Val Leu Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35
40 45 Gly Met Ile Trp Ser Gly Gly Thr Thr Asp Tyr Asn Ala Ala Phe
Ile 50 55 60 Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Asn Gln
Val Ser Leu65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr Cys Val 85 90 95 Arg Glu Glu Met Asp Tyr Trp Gly Gln
Gly Thr Leu Val Thr Val Ser 100 105 110 Ser171107PRTArtificial
SequenceSynthetic Construct 171Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Ile Ser Asn Phe 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr
Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Trp
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
1725PRTArtificial SequenceSynthetic Construct 172Xaa Xaa Tyr Met
Ser1 5 17317PRTArtificial SequenceSynthetic Construct 173Asp Met
Tyr Pro Asp Xaa Xaa Xaa Xaa Ser Tyr Asn Gln Lys Phe Arg1 5 10 15
Glu1748PRTArtificial SequenceSynthetic Construct 174Ala Pro Arg Trp
Xaa Xaa Xaa Xaa1 5 1759PRTArtificial SequenceSynthetic Construct
175Gln Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr1 5 17617PRTArtificial
SequenceSynthetic Construct 176Val Ile Asn Pro Gly Ser Gly Asp Xaa
Tyr Tyr Ser Glu Lys Phe Lys1 5 10 15 Gly1777PRTArtificial
SequenceSynthetic Construct 177His Gly Thr Asn Leu Glu Xaa1 5
1789PRTArtificial SequenceSynthetic Construct 178Xaa Xaa Tyr Ala
Gln Phe Pro Tyr Xaa1 5 179107PRTArtificial SequenceSynthetic
Construct 179Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala
Ser Leu Gly1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp
Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu Arg
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Lys
Asp Tyr Phe Leu Thr Ile Ser Asn Leu Glu Gln65 70 75 80 Glu Asp Val
Ala Ala Tyr Phe Cys Gln Gln Gly His Thr Leu Pro Pro 85 90 95 Thr
Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 180117PRTArtificial
SequenceSynthetic Construct 180Glu Val Gln Leu Gln Gln Ser Gly Pro
Glu Leu Val Lys Pro Gly Ala1 5 10 15 Ser Val Lys Ile Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val
Lys Gln Ser His Gly Lys Thr Leu Glu Trp Ile 35 40 45 Gly Asp Met
Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg
Glu Lys Val Thr Leu Thr Val Asp Lys Ser Ser Thr Thr Ala Tyr65 70 75
80 Met Glu Phe Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95 Val Leu Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Thr Gly
Thr Thr 100 105 110 Val Thr Val Ser Ser 115 181107PRTArtificial
SequenceSynthetic Construct 181Asp Ile Leu Met Thr Gln Ser Pro Ser
Ser Met Ser Val Ser Leu Gly1 5 10 15 Asp Thr Val Ser Ile Thr Cys
His Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Ile Val Trp Leu Gln
Gln Lys Pro Gly Lys Ser Phe Arg Gly Leu Ile 35 40 45 Tyr His Gly
Thr Asn Leu Glu Asp Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Ala Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Ser65 70 75
80 Glu Asp Phe Ala Asp Tyr Tyr Cys Val His Tyr Ala Gln Phe Pro Tyr
85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105
182114PRTArtificial SequenceSynthetic Construct 182Gln Val Gln Leu
Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Thr Asn Tyr 20 25 30
Leu Ile Glu Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35
40 45 Gly Val Ile Asn Pro Gly Ser Gly Asp Thr Tyr Tyr Ser Glu Lys
Phe 50 55 60 Lys Gly Lys Val Thr Leu Thr Ala Asp Lys Ser Ser Ser
Thr Ala Tyr65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser
Ala Val Tyr Phe Cys 85 90 95 Ala Arg Asp Arg Leu Asp Tyr Trp Gly
Gln Gly Thr Thr Leu Thr Val 100 105 110 Ser Ser183451PRTArtificial
SequenceSynthetic Construct 183Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Thr Met Asn Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile
Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr65 70 75
80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Lys Asp Arg Tyr Ser Gln Val His Tyr Ala Leu Asp Tyr
Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160 Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200
205 Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys
210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
Leu Gly225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325
330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro385 390 395 400 Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445
Pro Gly Lys 450 184219PRTArtificial SequenceSynthetic Construct
184Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Pro Val Thr Pro Gly1
5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His
Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Ala
Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg
Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Lys Ile65 70 75 80 Ser Arg Val Glu Ala Glu Asp
Val Gly Val Tyr Tyr Cys Gln Gln Tyr 85 90 95 Tyr Asn His Pro Thr
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135
140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe
Asn Arg Gly Glu Cys 210 215 185219PRTArtificial SequenceSynthetic
Construct 185Asp Ile Gln Met Thr Gln Ser Pro Asp Ser Leu Pro Val
Thr Pro Gly1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln
Ser Leu Leu His Ser 20 25 30 Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr
Leu Gln Lys Ala Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu
Gly Ser Asn Arg Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile65 70 75 80 Ser Arg Val
Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Tyr 85 90 95 Tyr
Asn His Pro Thr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105
110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val
Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 186450PRTArtificial
SequenceSynthetic Construct 186Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val His Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Gly Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile
Gly Thr Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val Met 50 55 60 Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu65 70 75
80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95 Arg Tyr Asp Asn Val Met Gly Leu Tyr Trp Phe Asp Tyr Trp
Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser145 150 155 160 Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200
205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp
210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
Gly Gly225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325
330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val385 390 395 400 Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445
Gly Lys 450 187214PRTArtificial SequenceSynthetic Construct 187Glu
Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Glu Pro65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Ala Phe Gly Gly Gly Thr
Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe
Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145
150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser
Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
188118PRTArtificial SequenceSynthetic Construct 188Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30
Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35
40 45 Ser Tyr Ile Ser Ser Ser Ser Ser Thr Ile Asp Tyr Ala Asp Ser
Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn
Ser Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Ser Gly Trp Tyr Leu Phe
Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115
189107PRTArtificial SequenceSynthetic Construct 189Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35
40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser
Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Asn Ser Tyr Pro Pro 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys 100 105 190124PRTArtificial SequenceSynthetic Construct
190Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp
Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn Ser Gly Ser Ile Gly
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Lys Asp Gln Ser
Thr Ala Asp Tyr Tyr Phe Tyr Tyr Gly Met Asp 100 105 110 Val Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 191106PRTArtificial
SequenceSynthetic Construct 191Glu Ile Val Val Thr Gln Ser Pro Ala
Thr Leu Ser Leu Ser Pro Gly1 5 10 15 Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala
Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75
80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Thr
85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
192122PRTArtificial SequenceSynthetic Construct 192Gln Val Gln Leu
Val Gln Ser Gly Ser Glu Leu Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30
Ser Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Lys Trp Met 35
40 45 Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asp Asp
Phe 50 55 60 Lys Gly Arg Phe Val Phe Ser Leu Asp Thr Ser Val Ser
Thr Ala Tyr65 70 75 80 Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Pro Tyr Tyr Asp Tyr Val Ser
Tyr Tyr Ala Met Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Thr Val Thr
Val Ser Ser 115 120 193107PRTArtificial SequenceSynthetic Construct
193Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Thr
Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Tyr Leu Tyr Thr Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe
Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr
Cys Gln Gln His Tyr Ser Thr Pro Arg 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105 194120PRTArtificial
SequenceSynthetic Construct 194Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu Arg Leu Ser Cys Ala
Ala Ser Glu Tyr Glu Phe Pro Ser His 20 25 30 Asp Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Leu Val 35 40 45 Ala Ala Ile
Asn Ser Asp Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Met 50 55 60 Glu
Arg Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr65 70 75
80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg His Tyr Asp Asp Tyr Tyr Ala Trp Phe Ala Tyr Trp
Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser 115 120
195111PRTArtificial SequenceSynthetic Construct 195Glu Ile Val Leu
Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15 Glu Arg
Ala Thr Leu Ser Cys Arg Ala Ser Lys Ser Val Ser Thr Ser 20 25 30
Gly Tyr Ser Tyr Met His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 35
40 45 Arg Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro
Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser65 70 75 80 Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr
Cys Gln His Ser Arg 85 90 95 Glu Leu Pro Leu Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys 100 105 110 196469PRTArtificial
SequenceSynthetic Construct 196Met Tyr Leu Gly Leu Asn Tyr Val Phe
Ile Val Phe Leu Leu Asn Gly1 5 10 15 Val Gln Ser Glu Val Lys Leu
Glu Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Gly Gly Ser Met
Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe 35 40 45 Ser Asp Ala
Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu 50 55 60 Glu
Trp Val Ala Glu Ile Arg Ser Lys Ala Asn Asn His Ala Thr Tyr65 70 75
80 Tyr Ala Glu Ser Val Asn Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser
85 90 95 Lys Ser Ser Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
Asp Thr 100 105 110 Gly Ile Tyr Tyr Cys Thr Trp Gly Glu Val Phe Tyr
Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro 130 135 140 Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr145 150 155 160 Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 165 170 175 Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 180 185 190 Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 195 200
205 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Thr Cys Asn Val
210 215 220 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys225 230 235 240 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu 245 250 255 Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr 260 265 270 Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val 275 280 285 Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 290 295 300 Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser305 310 315 320
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 325
330 335 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala 340 345 350 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro 355 360 365 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln 370 375 380 Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala385 390 395 400 Val Glu Trp Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 405 410 415 Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 420 425 430 Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 435 440 445
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 450
455 460 Leu Ser Pro Gly Lys465 197233PRTArtificial
SequenceSynthetic Construct 197Met Arg Pro Ser Ile Gln Phe Leu Gly
Leu Leu Leu Phe Trp Leu His1 5 10 15 Gly Ala Gln Cys Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Leu Gly Gly
Lys Val Thr Ile Thr Cys Lys Ser Ser Gln Asp 35 40 45 Ile Asn Lys
Tyr Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro 50 55 60 Arg
Leu Leu Ile His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser65 70 75
80 Arg Phe Ser Gly Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser
85 90 95 Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln
Tyr Asp 100 105 110 Asn Leu Leu Thr Phe Gly Ala Gly
Thr Lys Leu Glu Leu Lys Arg Thr 115 120 125 Val Ala Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 130 135 140 Lys Ser Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro145 150 155 160 Arg
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 165 170
175 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
180 185 190 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His 195 200 205 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro Val 210 215 220 Thr Lys Ser Phe Asn Arg Gly Glu Cys225
230 198138PRTArtificial SequenceSynthetic Construct 198Met Tyr Leu
Gly Leu Asn Tyr Val Phe Ile Val Phe Leu Leu Asn Gly1 5 10 15 Val
Gln Ser Glu Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln 20 25
30 Pro Gly Gly Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
35 40 45 Ser Asp Ala Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys
Gly Leu 50 55 60 Glu Trp Val Ala Glu Ile Arg Ser Lys Ala Asn Asn
His Ala Thr Tyr65 70 75 80 Tyr Ala Glu Ser Val Asn Gly Arg Phe Thr
Ile Ser Arg Asp Asp Ser 85 90 95 Lys Ser Ser Val Tyr Leu Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr 100 105 110 Gly Ile Tyr Tyr Cys Thr
Trp Gly Glu Val Phe Tyr Phe Asp Tyr Trp 115 120 125 Gly Gln Gly Thr
Thr Leu Thr Val Ser Ser 130 135 199126PRTArtificial
SequenceSynthetic Construct 199Met Arg Pro Ser Ile Gln Phe Leu Gly
Leu Leu Leu Phe Trp Leu His1 5 10 15 Gly Ala Gln Cys Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser 20 25 30 Ala Ser Leu Gly Gly
Lys Val Thr Ile Thr Cys Lys Ser Ser Gln Asp 35 40 45 Ile Asn Lys
Tyr Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro 50 55 60 Arg
Leu Leu Ile His Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser65 70 75
80 Arg Phe Ser Gly Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser
85 90 95 Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Leu Gln
Tyr Asp 100 105 110 Asn Leu Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu
Leu Lys 115 120 125 200119PRTArtificial SequenceSynthetic Construct
200Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala1
5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30 Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys
Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ser Asp
Lys Ser Ser Ser Thr Ala Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Thr
Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Tyr Tyr Gly
Ser Ser Leu Ser Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Ser Val
Thr Val Ser Ser 115 201108PRTArtificial SequenceSynthetic Construct
201Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly1
5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn
Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys
Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu
Thr Ile Ser Asn Leu Glu Gln65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe
Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly
Thr Lys Leu Glu Ile Lys Arg 100 105 202121PRTArtificial
SequenceSynthetic Construct 202Glu Val Gln Leu Gln Gln Ser Gly Pro
Glu Leu Val Lys Pro Gly Ala1 5 10 15 Ser Val Lys Ile Ser Cys Lys
Thr Ser Gly Tyr Thr Phe Lys Asp Tyr 20 25 30 Thr Met His Trp Val
Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile 35 40 45 Gly Gly Ile
Tyr Pro Asn Asn Gly Gly Ser Thr Tyr Asn Gln Asn Phe 50 55 60 Lys
Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr65 70 75
80 Met Glu Phe Arg Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Met Gly Tyr His Gly Pro His Leu Asp Phe Asp Val
Trp Gly 100 105 110 Ala Gly Thr Thr Val Thr Val Ser Pro 115 120
203108PRTArtificial SequenceSynthetic Construct 203Asp Ile Val Met
Thr Gln Ser His Lys Phe Met Ser Thr Ser Leu Gly1 5 10 15 Asp Arg
Val Ser Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Ala Ala 20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35
40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr
Gly 50 55 60 Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn
Val Gln Ser65 70 75 80 Glu Asp Leu Thr Asp Tyr Phe Cys Gln Gln Tyr
Ile Asn Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu
Ile Lys Arg 100 105 204119PRTArtificial SequenceSynthetic Construct
204Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu
Glu Trp Met 35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys
Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Arg Val Thr Ile Thr Ser Asp
Thr Ser Ala Ser Thr Ala Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Tyr Tyr Gly
Ser Ser Leu Ser Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser 115 205108PRTArtificial SequenceSynthetic Construct
205Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Asn
Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu
Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Gly Asn Thr Leu Pro Trp 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 105 206108PRTArtificial
SequenceSynthetic Construct 206Asp Ile Gln Met Thr Gln Ser Pro Ser
Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys
Arg Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr
Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75
80 Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Trp
85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105
207119PRTArtificial SequenceSynthetic Construct 207Gln Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val
Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30
Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35
40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys
Phe 50 55 60 Lys Gly Arg Ala Thr Ile Thr Ser Asp Thr Ser Ala Ser
Thr Ala Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Tyr Tyr Gly Ser Ser Leu Ser
Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser
115 208119PRTArtificial SequenceSynthetic Construct 208Gln Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25
30 Val Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile
35 40 45 Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu
Lys Phe 50 55 60 Lys Gly Arg Ala Thr Leu Thr Ser Asp Lys Ser Ala
Ser Thr Ala Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Tyr Tyr Gly Ser Ser Leu
Ser Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser
Ser 115 209121PRTArtificial SequenceSynthetic Construct 209Gln Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Lys Asp Tyr 20
25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
Met 35 40 45 Gly Gly Ile Tyr Pro Asn Asn Gly Gly Ser Thr Tyr Asn
Gln Asn Phe 50 55 60 Lys Asp Arg Val Thr Ile Thr Ala Asp Lys Ser
Thr Ser Thr Ala Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Met Gly Tyr His Gly
Pro His Leu Asp Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr Thr Val
Thr Val Ser Ser 115 120 210108PRTArtificial SequenceSynthetic
Construct 210Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
Ser Val Gly1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln
Asp Val Gly Ala Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly
Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Ser Thr Arg His
Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro65 70 75 80 Glu Asp Phe
Ala Thr Tyr Tyr Cys Gln Gln Tyr Ile Asn Tyr Pro Leu 85 90 95 Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105
211108PRTArtificial SequenceSynthetic Construct 211Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Ala Ala 20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35
40 45 Tyr Trp Ala Ser Thr Arg His Thr Gly Val Pro Asp Arg Phe Ser
Gly 50 55 60 Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
Leu Gln Pro65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr
Ile Asn Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys Arg 100 105 212121PRTArtificial SequenceSynthetic Construct
212Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Lys Asp
Tyr 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Ile 35 40 45 Gly Gly Ile Tyr Pro Asn Asn Gly Gly Ser Thr
Tyr Asn Gln Asn Phe 50 55 60 Lys Asp Arg Val Thr Leu Thr Ala Asp
Lys Ser Thr Ser Thr Ala Tyr65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Met Gly Tyr
His Gly Pro His Leu Asp Phe Asp Val Trp Gly 100 105 110 Gln Gly Thr
Thr Val Thr Val Ser Ser 115 120 213121PRTArtificial
SequenceSynthetic Construct 213Gln Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ser1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Lys Asp Tyr 20 25 30 Thr Met His Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Gly Ile
Tyr Pro Asn Asn Gly Gly Ser Thr Tyr Asn Gln Asn Phe 50 55 60 Lys
Asp Arg Ala Thr Leu Thr Val Asp Lys Ser Thr Ser Thr Ala Tyr65 70 75
80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Ala Arg Met Gly Tyr His Gly Pro His Leu Asp Phe Asp Val
Trp Gly 100 105 110 Gln Gly Thr Thr Val Thr Val Ser Ser 115 120
21425PRTArtificial SequenceSynthetic Construct 214Glu Val Gln Leu
Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15 Ser Leu
Arg Leu Ser Cys Ala Ala Ser 20 25 21513PRTArtificial
SequenceSynthetic Construct 215Trp Val Arg Gln Ala Pro Gly Lys Gly
Leu Glu Trp Val1 5 10 21630PRTArtificial SequenceSynthetic
Construct 216Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu
Tyr Leu Gln1 5 10 15 Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 20 25 30 21711PRTArtificial SequenceSynthetic Construct
217Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser1 5 10
21823PRTArtificial SequenceSynthetic Construct 218Asp Ile Gln Met
Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15 Asp Arg
Val Thr Ile Thr Cys 20 21915PRTArtificial SequenceSynthetic
Construct 219Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile Tyr1 5 10 15 22032PRTArtificial SequenceSynthetic Construct
220Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr1
5 10 15 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr
Cys 20 25 30 22110PRTArtificial SequenceSynthetic Construct 221Phe
Gly Gln Gly Thr Lys Val Glu Ile Lys1 5 10
22217PRTArtificial SequenceSynthetic Construct 222Asp Met Tyr Pro
Asp Ala Ala Ala Ala Ser Tyr Asn Gln Lys Phe Arg1 5 10 15
Glu2238PRTArtificial SequenceSynthetic Construct 223Ala Pro Arg Trp
Ala Ala Ala Ala1 5 2249PRTArtificial SequenceSynthetic Construct
224Gln Ala Ala Ala Ala Ala Ala Ala Thr1 5 225117PRTArtificial
SequenceSynthetic Construct 225Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met
Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg
Glu Arg Val Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Val Leu Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly
Thr Leu 100 105 110 Val Thr Val Ser Ser 115 226117PRTArtificial
SequenceSynthetic Construct 226Glu Val Gln Leu Val Gln Ser Gly Ala
Glu Val Lys Lys Pro Gly Ala1 5 10 15 Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30 Tyr Met Ser Trp Val
Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Met
Tyr Pro Asp Asn Gly Asp Ser Ser Tyr Asn Gln Lys Phe 50 55 60 Arg
Glu Arg Val Thr Leu Thr Val Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75
80 Leu Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95 Val Leu Ala Pro Arg Trp Tyr Phe Ser Val Trp Gly Gln Gly
Thr Leu 100 105 110 Val Thr Val Ser Ser 115
* * * * *