U.S. patent application number 17/387399 was filed with the patent office on 2022-03-31 for combination of dr5 agonist and anti-pd-1 antagonist and methods of use.
The applicant listed for this patent is BRISTOL-MYERS SQUIBB COMPANY. Invention is credited to Bryan BARNHART, Maria N. JURE-KUNKEL.
Application Number | 20220098302 17/387399 |
Document ID | / |
Family ID | |
Filed Date | 2022-03-31 |
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United States Patent
Application |
20220098302 |
Kind Code |
A1 |
BARNHART; Bryan ; et
al. |
March 31, 2022 |
COMBINATION OF DR5 AGONIST AND ANTI-PD-1 ANTAGONIST AND METHODS OF
USE
Abstract
Provided are methods and compositions for treating cancer using
an effective amount of a PD-1 antagonist (e.g., an antibody) in
combination with a DR4 or DR5 agonist (e.g., an antibody).
Inventors: |
BARNHART; Bryan; (San
Francisco, CA) ; JURE-KUNKEL; Maria N.; (Plainsboro,
NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BRISTOL-MYERS SQUIBB COMPANY |
Princeton |
NJ |
US |
|
|
Appl. No.: |
17/387399 |
Filed: |
July 28, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15993687 |
May 31, 2018 |
11103579 |
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17387399 |
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14774382 |
Sep 10, 2015 |
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PCT/US2014/024208 |
Mar 12, 2014 |
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15993687 |
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61783184 |
Mar 14, 2013 |
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International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 39/395 20060101 A61K039/395; A61P 35/00 20060101
A61P035/00 |
Claims
1-30. (canceled)
31. A method of treating cancer in a subject, the method comprising
administering to the subject an effective amount of a PD-1
antagonist and a DR4 or DR5 agonist.
32. The method of claim 31, wherein the PD-1 antagonist and the DR4
or DR5 agonist are selected from the group consisting of a ligand,
antibody, and multivalent agent.
33. The method of claim 32, wherein the DR5 agonist is an antibody
selected from the group consisting of Lexatumumab, Tigatuzumab,
Conatumumab, Drozitumab, HGSTR2J/KMTRS, and LBY-135.
34. The method of claim 32, wherein the DR5 agonist is a
multivalent agent comprising TAS266.
35. The method of claim 31, wherein the PD-1 antagonist is an
anti-PD-1 antibody comprising the CDR1, CDR2 and CDR3 domains in a
heavy chain variable region having the sequence set forth in SEQ ID
NO: 13, and the CDR1, CDR2 and CDR3 domains in a light chain
variable region having the sequence set forth in SEQ ID NO: 15.
36. The method of claim 31, wherein the PD-1 antagonist is an
anti-PD-1 antibody comprising: (a) a heavy chain variable region
CDR1 having the sequence set forth in SEQ ID NO: 17; (b) a heavy
chain variable region CDR2 having the sequence set forth in SEQ ID
NO: 18; (c) a heavy chain variable region CDR3 having the sequence
set forth in SEQ ID NO: 19; (d) a light chain variable region CDR1
having the sequence set forth in SEQ ID NO: 20; (e) a light chain
variable region CDR2 having the sequence set forth in SEQ ID NO:
21; and (f) a light chain variable region CDR3 having the sequence
set forth in SEQ ID NO: 22.
37. The method of claim 31, wherein the PD-1 antagonist is an
anti-PD-1 antibody comprising heavy and light chain variable
regions having the sequences set forth in SEQ ID NOs: 13 and 15,
respectively.
38. The method of claim 31, wherein the PD-1 antagonist is an
anti-PD-1 antibody comprising heavy and light chains having the
sequences as set forth in SEQ ID NOs: 11 and 12, respectively.
39. The method of claim 31, wherein the PD-1 antagonist is an
anti-PD-L1 antibody comprising the CDR1, CDR2 and CDR3 domains in a
heavy chain variable region having the sequence set forth in SEQ ID
NO: 1, and the CDR1, CDR2 and CDR3 domains in a light chain
variable region having the sequence set forth in SEQ ID NO: 3.
40. The method of claim 31, wherein the PD-1 antagonist is an
anti-PD-L1 antibody comprising: (a) a heavy chain variable region
CDR1 having the sequence set forth in SEQ ID NO: 5 (b) a heavy
chain variable region CDR2 having the sequence set forth in SEQ ID
NO: 6; (c) a heavy chain variable region CDR3 having the sequence
set forth in SEQ ID NO: 7; (d) a light chain variable region CDR1
having the sequence set forth in SEQ ID NO: 8; (e) a light chain
variable region CDR2 having the sequence set forth in SEQ ID NO: 9;
and (f) a light chain variable region CDR3 having the sequence set
forth in SEQ ID NO: 10.
41. The method of claim 31, wherein the PD-1 antagonist is an
anti-PD-L1 antibody comprising heavy and light chain variable
regions having the sequences set forth in SEQ ID NOs: 1 and 3,
respectively.
42. The method of claim 31, wherein administration of the PD-1
antagonist and DR5 agonist reduces tumor size by at least 50%.
43. A method of treating cancer in a subject, the method comprising
administering to the subject an effective amount of a PD-1
antagonist and a DR5 agonist, wherein: (a) the PD-1 antagonist is
an antibody comprising the CDR1, CDR2 and CDR3 domains in a heavy
chain variable region having the sequence set forth in SEQ ID NO:
13, and the CDR1, CDR2 and CDR3 domains in a light chain variable
region having the sequence set forth in SEQ ID NO: 15 or an
antibody comprising the CDR1, CDR2 and CDR3 domains in a heavy
chain variable region having the sequence set forth in SEQ ID NO:
1, and the CDR1, CDR2 and CDR3 domains in a light chain variable
region having the sequence set forth in SEQ ID NO: 3; and (b) the
DR5 agonist is an antibody.
44. The method of claim 31, wherein the PD-1 antagonist and the DR5
agonist are administered separately.
45. The method of claim 31, wherein the DR5 agonist and the PD-1
antagonist are administered simultaneously.
46. The method of claim 31, wherein the cancer is a cancer selected
from the group consisting of leukemia, lymphoma, blastoma,
carcinoma and sarcoma.
47. The method of claim 31, which comprises administration of an
additional therapeutic agent.
48. A composition comprising a PD-1 antagonist and a DR5
agonist.
49. A method of treating cancer in a subject, comprising
administering the subject the composition of claim 48.
50. A kit for treating a cancer in a subject, the kit comprising:
(a) a dose of a PD-1 antagonist; (b) a dose of a DR5 agonist; and
(c) instructions for using the PD-1 antagonist and DR5 agonist in
the method of claim 31.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/993,687, filed on May 31, 2018 (now
allowed), which is a continuation of U.S. patent application Ser.
No. 14/774,382, filed on Sep. 10, 2015, (now abandoned), which is a
U.S. National Stage Entry of International Application No.
PCT/US2014/024208, filed Mar. 12, 2014, and which claims the
benefit of U.S. Provisional Application No. 61/783,184, filed on
Mar. 14, 2013. The entire contents of these applications are
incorporated herein by reference in their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jul. 27, 2021, is named "MXI_529USCN2_Sequence_Listing.txt" and
is 24,915 bytes in size. The Sequence Listing is being submitted by
EFS Web and is hereby incorporated by reference into the
specification.
BACKGROUND
[0003] The National Cancer Institute has estimated that in the
United States alone, 1 in 3 people will be struck with cancer
during their lifetime. Moreover, approximately 50% to 60% of people
contracting cancer will eventually succumb to the disease. The
widespread occurrence of this disease underscores the need for
improved anticancer regimens for the treatment of malignancy.
[0004] Programmed Cell Death 1 (PD-1) is a cell surface signaling
receptor that plays a critical role in the regulation of T cell
activation and tolerance (Keir M E, et al., Annu Rev Immunol 2008;
26:677-704). It is a type I transmembrane protein and together with
BTLA, CTLA-4, ICOS and CD28, comprise the CD28 family of T cell
co-stimulatory receptors. PD-1 is primarily expressed on activated
T cells, B cells, and myeloid cells (Dong H, et al., Nat Med 1999;
5:1365-1369). It is also expressed on natural killer (NK) cells
(Terme M, et al., Cancer Res 2011; 71:5393-5399). PD-1 is highly
expressed on tumor infiltrating lymphocytes, and its ligands are
up-regulated on the cell surface of many different tumors (Dong H,
et al., Nat Med 2002; 8:793-800). Multiple murine cancer models
have demonstrated that binding of ligand to PD-1 results in immune
evasion. In addition, blockade of this interaction results in
anti-tumor activity.
[0005] Two cell surface glycoprotein ligands for PD-1 have been
identified, PD-L1 and PD-L2, and have been shown to downregulate T
cell activation and cytokine secretion upon binding to PD-1
(Freeman et al. (2000) J Exp Med 192:1027-34; Latchman et al.
(2001) Nat Immunol 2:261-8; Carter et al. (2002) Eur J Immunol
32:634-43; Ohigashi et al. (2005) Clin Cancer Res 11:2947-53). Both
PD-L1 (B7-H1) and PD-L2 (B7-DC) are B7 homologs that bind to PD-1,
but do not bind to other CD28 family members (Blank et al. (2004).
Expression of PD-L1 on the cell surface has also been shown to be
upregulated through IFN-gamma stimulation.
[0006] PD-L1 expression has been found in several murine and human
cancers, including human lung, ovarian and colon carcinoma and
various myelomas (Iwai et al. (2002) PNAS 99:12293-7; Ohigashi et
al. (2005) Clin Cancer Res 11:2947-53). PD-L1 has been suggested to
play a role in tumor immunity by increasing apoptosis of
antigen-specific T-cell clones (Dong et al. (2002) Nat Med
8:793-800). It has also been suggested that PD-L1 might be involved
in intestinal mucosal inflammation and inhibition of PD-L1
suppresses wasting disease associated with colitis (Kanai et al.
(2003) J Immunol 171:4156-63).
[0007] TRAIL (tumor necrosis factor (TNF)-related
apoptosis-inducing ligand) is a member of the TNF superfamily with
the ability to induce apoptosis of tumor cells. At least five
receptors for TRAIL have been identified. DR4 (TRAIL-R1) and DR5
(TRAIL-R2) are apoptosis-inducing receptors, which each contain an
intracellular death domain (see e.g., Pan G, et al., Science. 1997;
276:111-113, Pan G, et al., Science. 1997; 277:815-818, Sheridan J
P, et al., Science. 1997; 277:818-821, and Walczak H, et al., EMBO
J. 1997; 16:5386-5397). Upon receptor activation, DR4 and DR5
recruit FAS associated protein with death domain (FADD) and
caspase-8 to form the death-inducing signaling complex (DISC),
which activates caspase-8, subsequently leading to the activation
of executioner caspases such as caspase-3 that induce apoptosis
(see, e.g., Kischkel F C, et al., Immunity. 2000; 12:611-620,
Thomas L R, et al., J Biol Chem. 2004; 279:32780-32785, Thomas L R,
et al., J Biol Chem. 2004; 279:52479-52486, Varfolomeev E, et al.,
J Biol Chem. 2005; 280:40599-40608, Ashkenazi A., Nat Rev Cancer.
2002; 2:420-430, and Thorburn A. Cell Signal. 2004;
16:139-144).
[0008] TRAIL and agonistic antibodies that recognize TRAIL
receptors preferentially kill tumor cells and induce potent
anti-tumor activity in a variety of experimental models (see,
Griffith T S, et al., Curr Opin Immunol. 1998; 10:559-563,
Ashkenazi A, et al., J Clin Invest. 1999; 104:155-162, Walczak H,
et al, Nat Med. 1999; 5:157-163, Chuntharapai A, et al., J Immunol.
2001; 166:4891-4898, and Ichikawa K, et al., Nat Med. 2001;
7:954-960). Administration of TRAIL to mice bearing human tumors
actively suppressed tumor progression and improved survival of the
animal (Walczak H, et al, Nat Med. 1999; 5:157-163). Accordingly,
agonists against DR4 or DR5 by activating apoptosis are becoming
dramatically meaningful as candidates for the treatment of
cancer.
SUMMARY OF THE INVENTION
[0009] The present inventors have discovered for the first time
that co-administration of a DR5 agonist (e.g., an antibody) and an
anti-PD-1 antagonist (e.g., an antibody) effectively inhibits tumor
growth in vivo, even synergistically. Accordingly, it is an object
of the present invention to provide improved methods for treating
subjects with cancer. Specifically, it is an object of the
invention to provide efficacious combination treatment regimens
wherein a DR5 agonist is combined with an anti-PD-1 antagonist for
the treatment of cancer.
[0010] The present invention provides a method for the treatment of
cancer in a subject by co-administering an effective amount of a
PD-1 antagonist and an agent that induces apoptosis in cancer
cells, e.g., an agent that engages the DR4 or DR5 receptor, such as
a DR4 or DR5 agonist.
[0011] Suitable anti-DR5 agonists for use in the methods of the
invention, include, without limitation, ligands, antibodies (e.g.,
monoclonal antibodies and bispecific antibodies) and multivalent
agents. In one embodiment, the DR5 agonist is an antibody selected
from the group consisting of Lexatumumab (also known as ETR2-ST01),
Tigatuzumab (also known as CS-1008), Conatumumab (also known as AMG
655), Drozitumab, HGSTR2J/KMTRS, and LBY-135. In another
embodiment, the DR5 agonist is a multivalent agent (e.g., TAS266).
In a further embodiment, the DR5 agonist is a ligand (e.g., a
TNF-related apoptosis-inducing ligand (TRAIL), such as a
recombinant human TRAIL, e.g., Dulanermin (also known as
AMG951)).
[0012] Suitable PD-1 antagonists for use in the methods of the
invention, include, without limitation, ligands, antibodies (e.g.,
monoclonal antibodies and bispecific antibodies), and multivalent
agents. In one embodiment, the PD-1 antagonist is a fusion protein,
e.g., an Fc fusion protein, such as AMP-244. In one embodiment, the
PD-1 antagonist is an anti-PD-1 or anti-PD-L1 antibody. In another
embodiment, the PD-1 antagonist is an antibody, such as MK-3475 or
CT-011.
[0013] An exemplary anti-PD-1 antibody is 5C4 (referred to as 5C4
in WO 2006/121168; also known as MDX-1106, ONO-4538, and Nivolumab)
comprising heavy and light chains having the sequences shown in SEQ
ID NOs: 11 and 12, respectively, or antigen binding fragments and
variants thereof. In other embodiments, the antibody comprises the
heavy and light chain CDRs or VRs of 5C4. Accordingly, in one
embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains
of the VH region of 5C4 having the sequence shown in SEQ ID NO: 13,
and the CDR1, CDR2 and CDR3 domains of the VL region of 5C4 having
the sequence shown in SEQ ID NO:15. In another embodiment, the
antibody comprises the heavy chain CDR1, CDR2 and CDR3 domains
having the sequences set forth in SEQ ID NOs: 17, 18, and 19,
respectively, and the light chain CDR1, CDR2 and CDR3 domains
having the sequences set forth in SEQ ID NOs: 20, 21, and 22,
respectively. In another embodiment, the antibody comprises VH
and/or VL regions having the amino acid sequences set forth in SEQ
ID NO: 13 and/or SEQ ID NO: 15, respectively. In another
embodiment, the antibody comprises the heavy chain variable (VH)
and/or light chain variable (VL) regions encoded by the nucleic
acid sequences set forth in SEQ ID NO: 14 and/or SEQ ID NO: 16,
respectively.
[0014] In another embodiment, the antibody competes for binding
with, and/or binds to the same epitope on PD-1 as, the
above-mentioned antibodies. In another embodiment, the antibody has
at least about 90% variable region amino acid sequence identity
with the above-mentioned antibodies (e.g., at least about 90%, 95%
or 99% variable region identity with SEQ ID NO: 13 or SEQ ID NO:
15).
[0015] In one embodiment, the PD-1 antagonist is an anti-PD-L1
antibody, such as MEDI4736 (also known as Anti-B7-H1) or MPDL3280A
(also known as RG7446). An exemplary anti-PD-L1 antibody is 12A4
(referred to as 12A4 in WO 2007/005874 and U.S. Pat. No.
7,943,743). In one embodiment, the antibody comprises the heavy and
light chain CDRs or VRs of 12A4. Accordingly, in one embodiment,
the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH
region of 12A4 having the sequence shown in SEQ ID NO: 1, and the
CDR1, CDR2 and CDR3 domains of the VL region of 5C4 having the
sequence shown in SEQ ID NO: 3. In another embodiment, the antibody
comprises the heavy chain CDR1, CDR2 and CDR3 domains having the
sequences set forth in SEQ ID NOs: 5, 6, and 7, respectively, and
the light chain CDR1, CDR2 and CDR3 domains having the sequences
set forth in SEQ ID NOs: 8, 9, and 10, respectively. In another
embodiment, the antibody comprises VH and/or VL regions having the
amino acid sequences set forth in SEQ ID NO: 1 and/or SEQ ID NO: 3,
respectively. In another embodiment, the antibody comprises the
heavy chain variable (VH) and/or light chain variable (VL) regions
encoded by the nucleic acid sequences set forth in SEQ ID NO: 2
and/or SEQ ID NO: 4, respectively. In another embodiment, the
antibody competes for binding with, and/or binds to the same
epitope on PD-L1 as, the above-mentioned antibodies. In another
embodiment, the antibody has at least about 90% variable region
amino acid sequence identity with the above-mentioned antibodies
(e.g., at least about 90%, 95% or 99% variable region identity with
SEQ ID NO: 1 or SEQ ID NO: 3).
[0016] In one embodiment, the invention provides a method of
treating cancer in a subject, the method comprising administering
to the subject an effective amount of a PD-1 antagonist and a DR5
agonist, wherein
(a) the PD-1 antagonist is an anti-PD-1 antibody comprising the
CDR1, CDR2 and CDR3 domains in a heavy chain variable region having
the sequence set forth in SEQ ID NO: 13, and the CDR1, CDR2 and
CDR3 domains in a light chain variable region having the sequence
set forth in SEQ ID NO: 15; and (b) the DR5 agonist is an
antibody.
[0017] In another embodiment, the invention provides a method
cancer in a subject, the method comprising administering to the
subject an effective amount of a PD-1 antagonist and a DR5 agonist,
wherein
(a) the PD-1 antagonist is an anti-PD-L1 antibody comprising the
CDR1, CDR2 and CDR3 domains in a heavy chain variable region having
the sequence set forth in SEQ ID NO: 1, and the CDR1, CDR2 and CDR3
domains in a light chain variable region having the sequence set
forth in SEQ ID NO: 3; and (b) the DR5 agonist is an antibody.
[0018] The efficacy of the treatment methods provided herein can be
assessed using any suitable means. In one embodiment, the treatment
produces at least one therapeutic effect selected from the group
consisting of reduction in size of a tumor, reduction in number of
metastasic lesions over time, complete response, partial response,
and stable disease. In another embodiment, administration of a PD-1
antagonist and a DR5 agonist results in at least a 1, 1.25, 1.50,
1.75, 2, 2.25, 2.50, 2.75, 3, 3.25, 3.5, 3.75, or 4-fold reduction
in tumor volume, e.g., relative to treatment with the PD-1
antagonist or DR5 agonist alone, or relative to tumor volume before
initiation of treatment. In another embodiment, administration of
the PD-1 antagonist and DR5 agonist results in at least a 1-fold,
2-fold, or more preferably a 3-fold reduction in tumor volume,
e.g., relative to treatment with the PD-1 antagonist or DR5 agonist
alone, or relative to tumor volume before initiation of treatment.
In a further embodiment, administration of a PD-1 antagonist and a
DR5 agonist results in tumor growth inhibition of at least 50%,
60%, 70% or 80%, e.g., relative to treatment with the PD-1
antagonist or DR5 agonist alone, or relative to tumor volume before
initiation of treatment. In certain embodiments, tumor volume is
reduced by 50%, 60%, 70%, 80%, 90% or more, e.g., relative to tumor
size before initiation of the treatment.
[0019] The PD-1 antagonist and DR5 agonist can be administered
accordingly to a suitable dosage, route (e.g., intravenous,
intraperitoneal, intramuscular, intrathecal or subcutaneous). The
antagonist and agonist can also be administered according to any
suitable schedule. For example, the antagonist and agonist can be
simultaneously administered in a single formulation. Alternatively,
the antagonist and agonist can be formulated for separate
administration, wherein they are administered concurrently or
sequentially. In one embodiment, the PD-1 antagonist is
administered prior to administration of the DR5 agonist. In another
embodiment, the DR5 agonist is administered prior to administration
of the PD-1 antagonist. In a further embodiment, the DR5 agonist
and the PD-1 antagonist are administered simultaneously.
[0020] In one embodiment, the cancer is a cancer selected from the
group consisting of leukemia, lymphoma, blastoma, carcinoma and
sarcoma. In another embodiment, the cancer is selected from the
group consisting of chronic myeloid leukemia, acute lymphoblastic
leukemia, Philadelphia chromosome positive acute lymphoblastic
leukemia (Ph+ ALL), squamous cell carcinoma, small-cell lung
cancer, non-small cell lung cancer, glioma, gastrointestinal
cancer, renal cancer, ovarian cancer, liver cancer, colorectal
cancer, endometrial cancer, kidney cancer, prostate cancer, thyroid
cancer, neuroblastoma, pancreatic cancer, glioblastoma multiforme,
cervical cancer, stomach cancer, bladder cancer, hepatoma, breast
cancer, colon carcinoma, and head and neck cancer, gastric cancer,
germ cell tumor, pediatric sarcoma, sinonasal natural killer,
multiple myeloma, acute myelogenous leukemia (AML), and chronic
lymphocytic leukemia (CML).
[0021] Additional agents and therapies can be administered in
combination with the agonists and antagonists described herein. In
one embodiment, the methods comprise administration of an
additional therapeutic agent (e.g., a cyotoxin or chemotherapeutic
agent.
[0022] Also provided herein are compositions comprising a PD-1
antagonist and a DR5 agonist. In one embodiment, the antagonist
and/or agonist is a ligand, antibody (e.g., monoclonal antibody or
bispecific antibody) or multivalent agent. In another embodiment,
the PD-1 antagonist is an anti-PD-1 antibody comprising the heavy
and light chain CDRs or VRs of 5C4. In another embodiment, the PD-1
antagonist is an anti-PD-L1 antibody comprising the heavy and light
chain CDRs or VRs of 12A4.
[0023] Further provided are kits for treating a cancer in a
subject, the kit comprising:
[0024] (a) a dose of a PD-1 antagonist;
[0025] (b) a dose of a DR5 agonist; and
[0026] (c) instructions for using the PD-1 antagonist and DR5
agonist in the methods described herein. In one embodiment, the DR5
agonist is an antibody. In another embodiment, the PD-1 antagonist
is an antibody. In particular embodiment, the PD-1 antagonist is an
anti-PD-1 antibody comprising the CDR1, CDR2 and CDR3 domains in a
heavy chain variable region having the sequence set forth in SEQ ID
NO: 13, and the CDR1, CDR2 and CDR3 domains in a light chain
variable region having the sequence set forth in SEQ ID NO: 15. In
another particular embodiment, the PD-1 antagonist is an anti-PD-L1
antibody comprising antibody comprises the CDR1, CDR2 and CDR3
domains in a heavy chain variable region having the sequence set
forth in SEQ ID NO: 1, and the CDR1, CDR2 and CDR3 domains in a
light chain variable region having the sequence set forth in SEQ ID
NO: 3.
BRIEF DESCRIPTION OF THE DRAWINGS
[0027] FIG. 1 is a graph depicting the median tumor volume in mice
(mm.sup.3) after administration of a control, an anti-DR5 antibody,
an anti-PD-1 antibody, or combination of both an anti-DR5 antibody,
an anti-PD-1 antibody, up to 23 days post implant.
[0028] FIGS. 2A-2H depict the tumor volume in individual mice
administered a control (FIG. 2A), an anti-PD-1 antibody on day 6
post-implant (FIG. 2B), an anti-PD-1 antibody on day 8 post-implant
(FIG. 2C), an anti-PD-1 antibody on day 9 post-implant (FIG. 2D),
an anti-DR5 antibody on day 8 post-implant (FIG. 2E), an anti-DR5
antibody on day 8 post-implant in combination with an anti-PD-1
antibody on day 6 post-implant (FIG. 2F), an anti-DR5 antibody on
day 8 post-implant in combination with an anti-PD-1 antibody on day
8 post-implant (FIG. 2G), and an anti-DR5 antibody on day 8
post-implant in combination with an anti-PD-1 antibody on day 9
post-implant (FIG. 2H).
[0029] FIG. 3 is a graph depicting the percent body weight change
following administration of a control, an anti-DR5 monoclonal
antibody, an anti-PD-1 antibody, or combination of both an anti-DR5
monoclonal antibody and anti-PD-1 antibody, up to 24 days post
implant.
DETAILED DESCRIPTION OF THE INVENTION
[0030] As described herein, the invention is based on the discovery
that co-administration of a DR5 agonist (e.g., an antibody) and a
PD-1 antagonist (e.g., an antibody) effectively inhibits tumor
growth in vivo, even synergistically. Accordingly, the present
invention provides a method for the treatment of cancer in a
subject which comprises administering to a subject (e.g., human) an
effective amount of a PD-1 antagonist and a DR5 agonist.
I. Definitions
[0031] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by the
skilled artisan. Although any methods and compositions similar or
equivalent to those described herein can be used in practice or
testing of the present invention, the preferred methods and
compositions are described herein.
[0032] As used herein, the term "subject" or "patient" is a human
patient (e.g., a patient having cancer).
[0033] A "solid tumor" includes, for example, sarcoma, melanoma,
carcinoma, prostate carcinoma, lung carcinoma, colon carcinoma, or
other solid tumor cancer.
[0034] The terms "cancer", "cancerous", or "malignant" refer to or
describe the physiological condition in mammals that is typically
characterized by unregulated cell growth. Examples of cancer
include, for example, leukemia, lymphoma, blastoma, carcinoma and
sarcoma. More particular examples of such cancers include chronic
myeloid leukemia, acute lymphoblastic leukemia, Philadelphia
chromosome positive acute lymphoblastic leukemia (Ph+ ALL),
squamous cell carcinoma, small-cell lung cancer, non-small cell
lung cancer, glioma, gastrointestinal cancer, renal cancer, ovarian
cancer, liver cancer, colorectal cancer, endometrial cancer, kidney
cancer, prostate cancer, thyroid cancer, neuroblastoma, pancreatic
cancer, glioblastoma multiforme, cervical cancer, stomach cancer,
bladder cancer, hepatoma, breast cancer, colon carcinoma, and head
and neck cancer, gastric cancer, germ cell tumor, pediatric
sarcoma, sinonasal natural killer, multiple myeloma, acute
myelogenous leukemia (AML), and chronic lymphocytic leukemia
(CML).
[0035] As used herein, "effective treatment" refers to treatment
producing a beneficial effect, e.g., amelioration of at least one
symptom of a disease or disorder. A beneficial effect can take the
form of an improvement over baseline, i.e., an improvement over a
measurement or observation made prior to initiation of therapy
according to the method. A beneficial effect can also take the form
of arresting, slowing, retarding, or stabilizing of a deleterious
progression of a marker of cancer. Effective treatment may refer to
alleviation of at least one symptom of cancer. Such effective
treatment may, e.g., reduce patient pain, reduce the size and/or
number of lesions, may reduce or prevent metastasis of a tumor,
and/or may slow tumor growth.
[0036] The term "effective amount" refers to an amount of an agent
that provides the desired biological, therapeutic, and/or
prophylactic result. That result can be reduction, amelioration,
palliation, lessening, delaying, and/or alleviation of one or more
of the signs, symptoms, or causes of a disease, or any other
desired alteration of a biological system. In reference to solid
tumors, an effective amount comprises an amount sufficient to cause
a tumor to shrink and/or to decrease the growth rate of the tumor
(such as to suppress tumor growth) or to prevent or delay other
unwanted cell proliferation. In some embodiments, an effective
amount is an amount sufficient to delay tumor development. In some
embodiments, an effective amount is an amount sufficient to prevent
or delay tumor recurrence. An effective amount can be administered
in one or more administrations. The effective amount of the drug or
composition may: (i) reduce the number of cancer cells; (ii) reduce
tumor size; (iii) inhibit, retard, slow to some extent and may stop
cancer cell infiltration into peripheral organs; (iv) inhibit
(i.e., slow to some extent and may stop tumor metastasis; (v)
inhibit tumor growth; (vi) prevent or delay occurrence and/or
recurrence of tumor; and/or (vii) relieve to some extent one or
more of the symptoms associated with the cancer. In one example, an
"effective amount" is the amount of a PD-1 antagonist (e.g., an
antibody) and DR5 agonist antibody (e.g., an antibody), in
combination, to effect a significant decrease in cancer or slowing
of progression of cancer, such as an advanced solid tumor.
[0037] As used herein, the term "antagonist" refers to a molecule
which blocks (e.g., reduces or prevents) a biological activity.
[0038] As used herein, the term "agonist" refers to a molecule that
triggers (e.g., initiates or promotes), partially or fully
enhances, stimulates or activates one or more biological
activities. Agonists often mimic the action of a naturally
occurring substance. Whereas an agonist causes an action, an
antagonist blocks the action of the agonist.
[0039] As used herein, the term "ligand" refers to a molecule that
forms a complex with a biomolecule (e.g., a receptor) to serve a
biological purpose. In a narrower sense, is a signal triggering
molecule, binding to a site on a target protein. The binding occurs
by intermolecular forces, such as ionic bonds, hydrogen bonds and
van der Waals forces. The docking (association) is usually
reversible (dissociation). Actual irreversible covalent binding
between a ligand and its target molecule is rare in biological
systems. Ligand binding to a receptor (receptor protein) alters its
chemical conformation (three dimensional shape). The conformational
state of a receptor protein determines its functional state.
[0040] As used herein, the terms "synergy", "therapeutic synergy",
and "synergistic effect" refer to a phenomenon where treatment of
patients with a combination of therapeutic agents (e.g., PD-1
antagonist in combination with DR5 agonist) manifests a
therapeutically superior outcome to the outcome achieved by each
individual constituent of the combination used at its optimum dose
(see, e.g., T. H. Corbett et al., 1982, Cancer Treatment Reports,
66, 1187). In this context a therapeutically superior outcome is
one in which the patients either a) exhibit fewer incidences of
adverse events while receiving a therapeutic benefit that is equal
to or greater than that where individual constituents of the
combination are each administered as monotherapy at the same dose
as in the combination, or b) do not exhibit dose-limiting
toxicities while receiving a therapeutic benefit that is greater
than that of treatment with each individual constituent of the
combination when each constituent is administered in at the same
doses in the combination(s) as is administered as individual
components. In xenograft models, a combination, used at its maximum
tolerated dose, in which each of the constituents will be present
at a dose generally not exceeding its individual maximum tolerated
dose, manifests therapeutic synergy when decrease in tumor growth
achieved by administration of the combination is greater than the
value of the decrease in tumor growth of the best constituent when
the constituent is administered alone.
[0041] As used herein, the term "antibody" includes whole
antibodies and any antigen binding fragment (i.e., "antigen-binding
fragments" (also known as "antigen-binding portions")) or single
chains thereof. Whole antibodies are glycoproteins comprising at
least two heavy (H) chains and two light (L) chains inter-connected
by disulfide bonds. Each heavy chain is comprised of a heavy chain
variable region (abbreviated herein as VH) and a heavy chain
constant region. The heavy chain constant region is comprised of
three domains, C.sub.H1, C.sub.H2 and C.sub.H3. Each light chain is
comprised of a light chain variable region (abbreviated herein as
V.sub.L) and a light chain constant region. The light chain
constant region is comprised of one domain, C.sub.L. The V.sub.H
and V.sub.L regions can be further subdivided into regions of
hypervariability, termed complementarity determining regions (CDR),
interspersed with regions that are more conserved, termed framework
regions (FR). Each V.sub.H and V.sub.L is composed of three CDRs
and four FRs, arranged from amino-terminus to carboxy-terminus in
the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The
variable regions of the heavy and light chains contain a binding
domain that interacts with an antigen. The constant regions of the
antibodies can mediate the binding of the immunoglobulin to host
tissues or factors, including various cells of the immune system
(e.g., effector cells) and the first component (C1q) of the
classical complement system. The term "antibody" also encompasses
chimeric antibodies, humanized antibodies, fully human antibodies,
as well as multimeric forms of antibodies, such as minibodies,
bis-scFv, diabodies, triabodies, tetrabodies and chemically
conjugated Fab' multimers.
[0042] The term "antibody fragment" (also referred to as
"antigen-binding fragment" or "antigen-binding portion"), as used
herein, refers to one or more fragments of an antibody that retain
the ability to specifically bind to an antigen. It has been shown
that the antigen-binding function of an antibody can be performed
by fragments of a full-length antibody. Examples of binding
fragments encompassed within the term "antigen-binding fragment" of
an antibody include (i) a Fab fragment, a monovalent fragment
consisting of the V.sub.L, V.sub.H, C.sub.L and C.sub.H1 domains;
(ii) a F(ab').sub.2 fragment, a bivalent fragment is essentially a
Fab with part of the hinge region (see, FUNDAMENTAL IMMUNOLOGY
(Paul ed., 3.sup.rd ed. 1993); (iv) a Fd fragment consisting of the
V.sub.H and C.sub.H1 domains; (v) a Fv fragment consisting of the
V.sub.L and V.sub.H domains of a single arm of an antibody, (vi) a
dAb fragment (Ward et al., (1989) Nature 341:544-546), which
consists of a V.sub.H domain; (vii) an isolated complementarity
determining region (CDR); and (viii) a nanobody (also known as a
single-domain antibody (sdAb)), which is a heavy chain variable
region containing a single variable domain and two constant
domains.
[0043] Single domain antibodies include V.sub.HH fragments
(single-domain antibodies engineered from heavy-chain antibodies
found in camelids, as well as VNAR fragments (single-domain
antibodies obtained from heavy-chain antibodies (IgNAR,
`immunoglobulin new antigen receptor`) of cartilaginous
fishes).
[0044] "Antigen binding scaffolds" are proteins that bind
specifically to a target (or antigen) or epitope, such as proteins
comprising an Ig fold or an Ig-like fold, e.g., the DR5 binding
proteins described in WO2009/058379 and WO2011/130328, Antibodies
or antigen binding fragments thereof are also antigen binding
scaffolds. Antigen binding scaffolds can be monovalent,
multivalent, e.g., bivalent, trivalent, tetravalent, or bind 5, 6
or more epitopes. Multivalent antigen binding scaffolds can be
monospecific or multispecific, i.e., binding to multiple (at least
2, 3, 4 or 5) epitopes that are different from one another. For
example, a multivalent monospecific antigen binding scaffold is a
protein that binds to at least 2, 3, 4 or 5 identical epitopes, and
may be a protein comprising at least 2, 3, 4 or 5 identical antigen
binding portions. For example, DR5 binding scaffolds may comprise
2-10, e.g., 2-6, 2-5, 2-4 or 2-3 DR5 binding portions, which may be
the same or different from one another.
[0045] A multivalent antibody includes antibodies comprising at
least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antigen binding portions
of antibodies, which antigen binding portions may comprise a
portion of a heavy chain and a portion of a light chain. An antigen
binding portion may be on a single polypeptide or comprise more
than one polypeptide. For example, a multivalent antibody may
comprise from 2-10 antigen binding portions, which may be the same
or different from each other. A multivalent antibody may be
monospecific or multispecific. A multispecific antibody may be
bispecific, trispecific, tetraspecific or bind to 5 or more
different epitopes.
[0046] Furthermore, although the two domains of the Fv fragment,
V.sub.L and V.sub.H, are coded for by separate genes, they can be
joined, using recombinant methods, by a synthetic linker that
enables them to be made as a single protein chain in which the
V.sub.L and V.sub.H regions pair to form monovalent molecules
(known as single chain Fv (scFv); see e.g., Bird et al. (1988)
Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad.
Sci. USA 85:5879-5883). Such single chain antibodies are also
intended to be encompassed within the term "antigen-binding
fragment" of an antibody. These antibody fragments are obtained
using conventional techniques known to those with skill in the art,
and the fragments are screened for utility in the same manner as
are intact antibodies.
[0047] As used herein, an antigen binding scaffold that
"specifically binds" to an antigen or epitope thereof is an antigen
binding scaffold that binds to the antigen or epitope thereof with
a K.sub.D of 10.sup.-7 M, 5.times.10.sup.-8 M, 10.sup.-8 M,
5.times.10.sup.-9 M, 10.sup.-9 M, 5.times.10.sup.-10 M, 10.sup.-10
M, 5.times.10.sup.-11 M, 10.sup.-11 M, 5.times.10.sup.-12 M,
10.sup.-12 M or less. For example, an antigen binding scaffold that
specifically binds to DR5 is an antigen binding scaffold that binds
to DR5 with a K.sub.D of 10.sup.-7 M, 5.times.10.sup.-8 M,
10.sup.-8 M, 5.times.10.sup.-9 M, 10.sup.-9 M, 5.times.10.sup.-10
M, 10.sup.-10 M, 5.times.10.sup.-11 M, 10.sup.-11 M,
5.times.10.sup.-12 M, 10.sup.-12 M or less. For example, an
antibody that "specifically binds to human PD-1" or "specifically
binds to human PD-L1" is intended to refer to an antibody that
binds to human PD-1 or PD-L1, respectively, with a K.sub.D of
10.sup.-7 M, 5.times.10.sup.-8 M, 10.sup.-8 M, 5.times.10.sup.-9 M,
10.sup.-9 M, 5.times.10.sup.-10 M, 10.sup.-10 M, 5.times.10.sup.-11
M, 10.sup.-11 M, 5.times.10.sup.-12 M, 10.sup.-12 M or less. An
antigen binding scaffold that comprises 2 or more regions binding
to an antigen or epitope may bind specifically to the antigen or
epitope even it has a lower affinity of binding to the antigen or
epitope than the ranges provided above, as it will bind to the
antigen or epitope with increased avidity.
[0048] A "bispecific" or "bifunctional antibody" is an artificial
hybrid antibody having two different heavy/light chain pairs and
two different binding sites. Bispecific antibodies can be produced
by a variety of methods including fusion of hybridomas or linking
of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin.
Exp. Immunol. 79:315-321 (1990); Kostelny et al., J. Immunol. 148,
1547-1553 (1992).
[0049] The term "monoclonal antibody" or "monoclonal antibody
composition," as used herein, refers to an antibody or a
composition of antibodies that displays a single binding
specificity and affinity for a particular epitope. Accordingly, the
term "human monoclonal antibody" or "monoclonal antibody
composition" refers to an antibody or a composition of antibodies
which displays a single binding specificity and which has variable
and optional constant regions derived from human germline
immunoglobulin sequences. In one embodiment, human monoclonal
antibodies are produced by a hybridoma which includes a B cell
obtained from a transgenic non-human animal, e.g., a transgenic
mouse, having a genome comprising a human heavy chain transgene and
a light chain transgene fused to an immortalized cell.
[0050] The term "epitope" or "antigenic determinant" refers to a
site on an antigen to which an immunoglobulin or antibody
specifically binds. Epitopes can be formed both from contiguous
amino acids or noncontiguous amino acids juxtaposed by tertiary
folding of a protein. Epitopes formed from contiguous amino acids
are typically retained on exposure to denaturing solvents, whereas
epitopes formed by tertiary folding are typically lost on treatment
with denaturing solvents. An epitope typically includes at least 3,
4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique
spatial conformation. Methods of determining spatial conformation
of epitopes include techniques in the art and those described
herein, for example, x-ray crystallography and 2-dimensional
nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in
Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed.
(1996)).
[0051] The term "epitope mapping" refers to the process of
identification of the molecular determinants for antibody-antigen
recognition.
[0052] The term "binds to the same epitope," with reference to two
or more antibodies, means that the antibodies compete for binding
to an antigen and bind to the same, overlapping, or encompassing
continuous or discontinuous segments of amino acids. Those of skill
in the art understand that the phrase "binds to the same epitope"
does not necessarily mean that the antibodies bind to exactly the
same amino acids. The precise amino acids to which the antibodies
bind can differ. For example, a first antibody can bind to a
segment of amino acids that is completely encompassed by the
segment of amino acids bound by a second antibody. In another
example, a first antibody binds one or more segments of amino acids
that significantly overlap the one or more segments bound by the
second antibody. For the purposes herein, such antibodies are
considered to "bind to the same epitope."
[0053] Accordingly, also, encompassed by the present invention are
antibodies that bind to an epitope which comprises all or a portion
of an epitope recognized by the particular antibodies described
herein (e.g., the same or an overlapping region or a region between
or spanning the region).
[0054] Also encompassed by the present invention are antibodies
that bind the same epitope and/or antibodies that compete for
binding with the antibodies described herein.
[0055] Antibodies that recognize the same epitope or compete for
binding can be identified using routine techniques. Such techniques
include, for example, an immunoassay, which shows the ability of
one antibody to block the binding of another antibody to a target
antigen, i.e., a competitive binding assay. Competitive binding is
determined in an assay in which the immunoglobulin under test
inhibits specific binding of a reference antibody to a common
antigen. Numerous types of competitive binding assays are known,
for example: solid phase direct or indirect radioimmunoassay (RIA),
solid phase direct or indirect enzyme immunoassay (EIA), sandwich
competition assay (see Stahli et al., Methods in Enzymology 9:242
(1983)); solid phase direct biotin-avidin EIA (see Kirkland et al.,
J. Immunol. 137:3614 (1986)); solid phase direct labeled assay,
solid phase direct labeled sandwich assay (see Harlow and Lane,
Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988));
solid phase direct label RIA using I-125 label (see Morel et al.,
Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidin EIA
(Cheung et al., Virology 176:546 (1990)); and direct labeled RIA.
(Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)). Typically,
such an assay involves the use of purified antigen bound to a solid
surface or cells bearing either of these, an unlabeled test
immunoglobulin and a labeled reference immunoglobulin. Competitive
inhibition is measured by determining the amount of label bound to
the solid surface or cells in the presence of the test
immunoglobulin. Usually the test immunoglobulin is present in
excess. Usually, when a competing antibody is present in excess, it
will inhibit specific binding of a reference antibody to a common
antigen by at least 50-55%, 55-60%, 60-65%, 65-70% 70-75% or
more.
[0056] Other techniques include, for example, epitope mapping
methods, such as, x-ray analyses of crystals of antigen:antibody
complexes which provides atomic resolution of the epitope. Other
methods monitor the binding of the antibody to antigen fragments or
mutated variations of the antigen where loss of binding due to a
modification of an amino acid residue within the antigen sequence
is often considered an indication of an epitope component. In
addition, computational combinatorial methods for epitope mapping
can also be used. These methods rely on the ability of the antibody
of interest to affinity isolate specific short peptides from
combinatorial phage display peptide libraries. The peptides are
then regarded as leads for the definition of the epitope
corresponding to the antibody used to screen the peptide library.
For epitope mapping, computational algorithms have also been
developed which have been shown to map conformational discontinuous
epitopes.
[0057] Chimeric molecules (or fusion molecules) comprising an
antigen binding domain, or equivalent, fused to another polypeptide
or molecule are also encompassed by the present invention. For
example, the polypeptides may be fused or conjugated to an antibody
Fc region, or portion thereof (e.g., an Fc fusion protein). The
antibody portion fused to a polypeptide may comprise the constant
region, hinge region, CH1 domain, CH2 domain, and CH3 domain or any
combination of whole domains or portions thereof. The polypeptides
may also be fused or conjugated to the above antibody portions to
form multimers. For example, Fc portions fused to the polypeptides
of the present invention can form dimers through disulfide bonding
between the Fc portions. Higher multimeric forms can be made by
fusing the polypeptides to portions of IgA and IgM. Methods for
fusing or conjugating the polypeptides of the present invention to
antibody portions are known in the art. See, e.g., U.S. Pat. Nos.
5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and
5,112,946; EP 307,434; EP 367,166; PCT Publication Nos. WO 96/04388
and WO 91/06570; Ashkenazi et al., Proc. Natl. Acad. Sci. USA,
88:10535-10539 (1991); Zheng et al., J. Immunol., 154:5590-5600
(1995); and Vil et al., Proc. Natl. Acad. Sci. USA, 89:11337-11341
(1992).
[0058] As used herein, the term "immunoconjugate" refers to an
antibody linked to a therapeutic moiety, such as a cytotoxin, a
drug or a radioisotope. When conjugated to a cytotoxin, these
antibody conjugates are referred to as "immunotoxins." A cytotoxin
or cytotoxic agent includes any agent that is detrimental to (e.g.,
kills) cells. Examples include taxol, cytochalasin B, gramicidin D,
ethidium bromide, emetine, mitomycin, etoposide, tenoposide,
vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,
dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin
D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine,
lidocaine, propranolol, and puromycin and analogs or homologs
thereof. Therapeutic agents include, but are not limited to,
antimetabolites (e.g., methotrexate, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating
agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,
carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,
dibromomannitol, streptozotocin, mitomycin C, and
cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines
(e.g., daunorubicin (formerly daunomycin) and doxorubicin),
antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin,
mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.,
vincristine and vinblastine). Antibodies use in the present
invention can be conjugated to a radioisotope, e.g., radioactive
iodine, to generate cytotoxic radiopharmaceuticals for treating
cancer.
[0059] Immunoconjugates can be used to modify a given biological
response, and the drug moiety is not to be construed as limited to
classical chemical therapeutic agents. For example, the drug moiety
may be a protein or polypeptide possessing a desired biological
activity. Such proteins may include, for example, an enzymatically
active toxin, or active fragment thereof, such as abrin, ricin A,
Pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor
necrosis factor or interferon-.gamma.; or, biological response
modifiers such as, for example, lymphokines, interleukin-1
("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"),
granulocyte macrophage colony stimulating factor ("GM-CSF"),
granulocyte colony stimulating factor ("G-CSF"), or other growth
factors.
[0060] Techniques for conjugating such therapeutic moiety to
antibodies are well known, see, e.g., Arnon et al., "Monoclonal
Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in
Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.),
pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies
For Drug Delivery", in Controlled Drug Delivery (2nd Ed.), Robinson
et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987); Thorpe,
"Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A
Review", in Monoclonal Antibodies '84: Biological And Clinical
Applications, Pinchera et al. (eds.), pp. 475-506 (1985);
"Analysis, Results, And Future Prospective Of The Therapeutic Use
Of Radiolabeled Antibody In Cancer Therapy", in Monoclonal
Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.),
pp. 303-16 (Academic Press 1985), and Thorpe et al., "The
Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates",
Immunol. Rev., 62:119-58 (1982).
[0061] As used herein, the term "multivalent" refers to a
recombinant molecule that incorporates more than two biologically
active segments. The protein fragments forming the multivalent
molecule optionally may be linked through a polypeptide linker
which attaches the constituent parts and permits each to function
independently.
[0062] "About" as used herein when referring to a measurable value
such as an amount, a temporal duration, and the like, is meant to
encompass variations of .+-.20% or .+-.10%, more preferably .+-.5%,
even more preferably .+-.1%, and still more preferably .+-.0.1%
from the specified value, as such variations are appropriate to
perform the disclosed methods.
[0063] "Percent (%) amino acid sequence identity" herein is defined
as the percentage of amino acid residues in a candidate sequence
that are identical with the amino acid residues in a selected
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, ALIGN-2
or Megalign (DNASTAR) software. Those skilled in the art can
determine appropriate parameters for measuring alignment, including
any algorithms needed to achieve maximal alignment over the
full-length of the sequences being compared.
[0064] For purposes herein, 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 a
sequence alignment program, such as BLAST, BLAST-2, ALIGN, ALIGN-2
or Megalign (DNASTAR), 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.
II. PD-1 Antagonists
[0065] As used herein, the terms "Programmed Death 1," "Programmed
Cell Death 1," "Protein PD-1," "PD-1," PD1," "PDCD1," "hPD-1" and
"hPD-I" are used interchangeably, and include variants, isoforms,
species homologs of human PD-1, and analogs having at least one
common epitope with human PD-1. The complete human PD-1 sequence
can be found under GenBank Accession No. U64863 (SEQ ID NO:23).
[0066] As used herein, the terms "Programmed Cell Death 1 Ligand
1", "PD-L1", "PDL1", "PDCD1L1", "PDCD1LG1", "CD274", "B7 homolog
1", "B7-H1", "B7-H", and "B7H1" are used interchangeably, and
include variants, isoforms, species homologs of human PDL-1, and
analogs having at least one common epitope with human PDL-1. The
complete human PD-L1 amino acid sequence--isoform a precursor--can
be found under GenBank Accession No. NP_054862.1 (SEQ ID NO:24).
The complete human PD-L1 amino acid sequence--isoform b
precursor--can be found under GenBank Accession No. NP_001254635.1
(SEQ ID NO:25).
[0067] The protein Programmed Death 1 (PD-1) is an inhibitory
member of the CD28 family of receptors, that also includes CD28,
CTLA-4, ICOS and BTLA. PD-1 is expressed on activated B cells, T
cells, and myeloid cells (Agata et al., supra; Okazaki et al.
(2002) Curr. Opin. Immunol. 14: 391779-82; Bennett et al. (2003) J
Immunol 170:711-8). The initial members of the family, CD28 and
ICOS, were discovered by functional effects on augmenting T cell
proliferation following the addition of monoclonal antibodies
(Hutloff et al. (1999) Nature 397:263-266; Hansen et al. (1980)
Immunogenics 10:247-260). PD-1 was discovered through screening for
differential expression in apoptotic cells (Ishida et al. (1992)
EMBO J 11:3887-95). The other members of the family, CTLA-4 and
BTLA, were discovered through screening for differential expression
in cytotoxic T lymphocytes and TH1 cells, respectively. CD28, ICOS
and CTLA-4 all have an unpaired cysteine residue allowing for
homodimerization. In contrast, PD-1 is suggested to exist as a
monomer, lacking the unpaired cysteine residue characteristic in
other CD28 family members.
[0068] The PD-1 gene is a 55 kDa type I transmembrane protein that
is part of the Ig gene superfamily (Agata et al. (1996) Int Immunol
8:765-72). PD-1 contains a membrane proximal immunoreceptor
tyrosine inhibitory motif (ITIM) and a membrane distal
tyrosine-based switch motif (ITSM) (Thomas, M. L. (1995) J Exp Med
181:1953-6; Vivier, E and Daeron, M (1997) Immunol Today
18:286-91). Although structurally similar to CTLA-4, PD-1 lacks the
MYPPPY motif (SEQ ID NO: 27) that is critical for B7-1 and B7-2
binding.
[0069] Consistent with PD-1 being an inhibitory member of the CD28
family, PD-1 deficient animals develop various autoimmune
phenotypes, including autoimmune cardiomyopathy and a lupus-like
syndrome with arthritis and nephritis (Nishimura et al. (1999)
Immunity 11:141-51; Nishimura et al. (2001) Science 291:319-22).
Additionally, PD-1 has been found to play a role in autoimmune
encephalomyelitis, systemic lupus erythematosus, graft-versus-host
disease (GVHD), type I diabetes, and rheumatoid arthritis (Salama
et al. (2003) J Exp Med 198:71-78; Prokunina and Alarcon-Riquelme
(2004) Hum Mol Genet 13:R143; Nielsen et al. (2004) Lupus 13:510).
In a murine B cell tumor line, the ITSM of PD-1 was shown to be
essential to block BCR-mediated Ca.sup.2+-flux and tyrosine
phosphorylation of downstream effector molecules (Okazaki et al.
(2001) PNAS 98:13866-71).
[0070] Two ligands for PD-1 have been identified, PD-L1 and PD-L2,
that have been shown to downregulate T cell activation upon binding
to PD-1 (Freeman et al. (2000) J Exp Med 192:1027-34; Latchman et
al. (2001) Nat Immunol 2:261-8; Carter et al. (2002) Eur J Immunol
32:634-43). Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1,
but do not bind to other CD28 family members. PD-L1 is abundant in
a variety of human cancers (Dong et al. (2002) Nat. Med. 8:787-9).
The interaction between PD-1 and PD-L1 results in a decrease in
tumor infiltrating lymphocytes, a decrease in T-cell receptor
mediated proliferation, and immune evasion by the cancerous cells
(Dong et al. (2003) J. Mol. Med. 8:281-7; Blank et al. (2005)
Cancer Immunol. Immunother. 54:307-314; Konishi et al. (2004) Clin.
Cancer Res. 10:5094-100). Immune suppression can be reversed by
inhibiting the local interaction of PD-1 with PD-L1, and the effect
is additive when the interaction of PD-1 with PD-L2 is blocked as
well (Iwai et al. (2002) Proc. Nat'l. Acad. Sci. USA 99:12293-7;
Brown et al. (2003) J. Immunol. 170:1257-66).
[0071] The methods of the present invention involve the use of a
PD-1 antagonist (e.g., an antibody in combination with a DR5
agonist (e.g., an antibody), for treating cancer. Accordingly, PD-1
antagonists of the invention bind to ligands of PD-1 and interfere
with, reduce, or inhibit the binding of one or more ligands to the
PD-1 receptor, or bind directly to the PD-1 receptor, without
engaging in signal transduction through the PD-1 receptor. In one
embodiment, the PD-1 antagonist binds directly to PD-1 and blocks
PD-1 inhibitory signal transduction. In another embodiment the PD-1
antagonist binds to one or more ligands of PD-1 (e.g., PD-L1 and
PD-L2) and reduces or inhibits the ligand(s) from triggering
inhibitory signal transduction through the PD-1. In one embodiment,
the PD-1 antagonist binds directly to PD-L1, inhibiting or
preventing PD-L1 from binding to PD-1, thereby blocking PD-1
inhibitory signal transduction.
[0072] PD-1 antagonists used in the methods and compositions of the
present invention include PD-1 binding scaffold proteins and
include, but are not limited to, PD-1 ligands, antibodies and
multivalent agents. In a particular embodiment, the antagonist is a
fusion protein, such as AMP-224. In another embodiment, the
antagonist is an anti-PD-1 antibody ("PD-1 antibody").
Anti-human-PD-1 antibodies (or VH and/or VL domains derived
therefrom) suitable for use in the invention can be generated using
methods well known in the art. Alternatively, art recognized
anti-PD-1 antibodies can be used. For example, antibodies MK-3475
or CT-011 can be used. Additionally, monoclonal antibodies 5C4,
17D8, 2D3, 4H1, 4A11, 7D3, and 5F4, described in WO 2006/121168,
the teachings of which are hereby incorporated by reference, can be
used. Antibodies that compete with any of these art-recognized
antibodies for binding to PD-1 also can be used.
[0073] An exemplary anti-PD-1 antibody is 5C4 comprising heavy and
light chains having the sequences shown in SEQ ID NOs: 11 and 12,
respectively, or antigen binding fragments and variants thereof. In
other embodiments, the antibody comprises the heavy and light chain
CDRs or variable regions of 5C4. Accordingly, in one embodiment,
the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH
of 5C4 having the sequence set forth in SEQ ID NO: 13, and the
CDR1, CDR2 and CDR3 domains of the VL of 5C4 having the sequences
set forth in SEQ ID NO: 15. In another embodiment, the antibody
comprises CDR1, CDR2 and CDR3 domains having the sequences set
forth in SEQ ID NOs: 17, 18, and 19, respectively, and CDR1, CDR2
and CDR3 domains having the sequences set forth in SEQ ID NOs: 20,
21, and 22, respectively. In another embodiment, the antibody
comprises VH and/or VL regions having the amino acid sequences set
forth in SEQ ID NO: 13 and/or SEQ ID NO: 15, respectively. In
another embodiment, the antibody comprises the heavy chain variable
(VH) and/or light chain variable (VL) regions encoded by the
nucleic acid sequences set forth in SEQ ID NO: 14 and/or SEQ ID NO:
16, respectively. In another embodiment, the antibody competes for
binding with and/or binds to the same epitope on PD-1 as the
above-mentioned antibodies. In another embodiment, the antibody has
at least about 90% variable region amino acid sequence identity
with the above-mentioned antibodies (e.g., at least about 90%, 95%
or 99% variable region identity with SEQ ID NO: 13 or SEQ ID NO:
15).
[0074] In certain embodiments, the PD1 antibodies exhibit one or
more desirable functional properties, such as high affinity binding
to PD-1, e.g., binding to human PD-1 with a K.sub.D of 10.sup.-7 M
or less; lack of significant cross-reactivity to other CD28 family
members, e.g., CD28, CTLA-4 and ICOS; the ability to stimulate T
cell proliferation in a mixed lymphocyte reaction (MLR) assay; the
ability to increase IFN-7 and/or IL-2 secretion in an MLR; the
ability to inhibit binding of one or more PD-1 ligands (e.g., PD-L1
and/or PD-L2) to PD-1; the ability to stimulate antigen-specific
memory responses; the ability to stimulate antibody responses
and/or the ability to inhibit growth of tumor cells in vivo.
[0075] In another embodiment, the PD-1 antagonist is an anti-PD-L1
antibody. Anti-human-PD-L1 antibodies (or VH and/or VL domains
derived therefrom) suitable for use in the invention can be
generated using methods well known in the art. Alternatively, art
recognized anti-PD-L1 antibodies can be used. For example, MEDI4736
(also known as Anti-B7-H1) or MPDL3280A (also known as RG7446) can
be used. Additionally, monoclonal antibodies 12A4, 3G10, 10A5, 5F8,
10H10, 1B12, 7H1, 11E6, 12B7, and 13G4 described in WO 2007/005874
and U.S. Pat. No. 7,943,743, the teachings of which are hereby
incorporated by reference, can be used. Antibodies that compete
with any of these art-recognized antibodies for binding to PD-L1
also can be used.
[0076] An exemplary anti-PD-L1 antibody is 12A4 (WO 2007/005874 and
U.S. Pat. No. 7,943,743). In one embodiment, the antibody comprises
the heavy and light chain CDRs or VRs of 12A4. Accordingly, in one
embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains
of the VH region of 12A4 having the sequence shown in SEQ ID NO: 1
and the CDR1, CDR2 and CDR3 domains of the VL region of 12A4 having
the sequence shown in SEQ ID NO: 3. In another embodiment, the
antibody comprises the heavy chain CDR1, CDR2 and CDR3 domains
having the sequences set forth in SEQ ID NOs: 5, 6, and 7,
respectively, and the light chain CDR1, CDR2 and CDR3 domains
having the sequences set forth in SEQ ID NOs: 8, 9, and 10,
respectively. In another embodiment, the antibody comprises VH
and/or VL regions having the amino acid sequences set forth in SEQ
ID NO: 1 and/or SEQ ID NO: 3, respectively. In another embodiment,
the antibody comprises the heavy chain variable (VH) and/or light
chain variable (VL) regions encoded by the nucleic acid sequences
set forth in SEQ ID NO: 2 and/or SEQ ID NO: 4, respectively. In
another embodiment, the antibody competes for binding with, and/or
binds to the same epitope on PD-L1 as, the above-mentioned
antibodies. In another embodiment, the antibody has at least about
90% variable region amino acid sequence identity with the
above-mentioned antibodies (e.g., at least about 90%, 95% or 99%
variable region identity with SEQ ID NO: 1 or SEQ ID NO: 3).
[0077] Anti-PD-1 or anti-PD-L1 antibodies may bind to PD-1 or
PD-L1, respectively, with a K.sub.D of 10.sup.-7 M,
5.times.10.sup.-8 M, 10.sup.-8 M, 5.times.10.sup.-9 M, 10.sup.-9 M,
5.times.10.sup.-10 M, 10.sup.-10 M or less.
III. DR5 Agonists
[0078] Provided herein are methods for treating cancer comprising
administering to a subject in need thereof (e.g., a subject having
cancer), a therapeutically effective amount of an agent that
induces apoptosis in a cancer cell and a PD-1 antagonist. Exemplary
apoptosis inducing agents include DR proteins, such as DR4 and
DR5.
[0079] As used herein, the terms "DR5" and "death receptor 5", also
known as "tumor necrosis factor receptor superfamily member 10b",
"TNFRSF10B", "CD262", "KILLER", "TRICK2", "TRICKB", "ZTNFR9",
"TRAILR", "TRAILR2", "Apo-2" "TRICK2A", "TRICK2B", "TRAIL-R2",
"KILLER", "KILLER/DR5", "TR6", "Tango-63", "hAPO8", and TRICK2
(see, e.g., Sheridan et al., Science, 277:818-821 (1997); Pan et
al., Science, 277:815-818 (1997), WO98/51793; WO98/41629; Screaton
et al., Curr. Biol., 7:693-696 (1997); Walczak et al., EMBO J.,
16:5386-5387 (1997); Wu et al., Nature Genetics, 17:141-143 (1997);
WO98/35986; EP870,827; WO98/46643; WO99/02653; WO99/09165;
WO99/11791; US 2002/0072091; US 2002/0098550; U.S. Pat. No.
6,313,269; US 2001/0010924; US 2003/01255540; US 2002/0160446, US
2002/0048785; U.S. Pat. Nos. 6,342,369; 6,569,642, 6,072,047,
6,642,358; 6,743,625) are used interchangeably, and include
variants, isoforms, species homologs of human DR5, and analogs
having at least one common epitope with DR5. The complete human DR5
sequence can be found under GenBank Accession No. AAC01565.1 (SEQ
ID NO: 26).
[0080] DR5 is a member of the tumor necrosis factor (TNF) receptor
superfamily. TNF ligands are known to be among the most pleiotropic
cytokines, inducing a large number of cellular responses, including
cytotoxicity, anti-viral activity, immunoregulatory activities, and
the transcriptional regulation of several genes. Cellular responses
to TNF-family ligands include not only normal physiological
responses, but also diseases associated with increased apoptosis or
the inhibition of apoptosis. Apoptosis (i.e., programmed cell
death) is a physiological mechanism involved in the deletion of
peripheral T lymphocytes of the immune system, and its
dysregulation can lead to a number of different pathogenic
processes. Diseases associated with increased cell survival, or the
inhibition of apoptosis, include cancers, autoimmune disorders,
viral infections, inflammation, graft versus host disease, acute
graft rejection, and chronic graft rejection. Diseases associated
with increased apoptosis include AIDS, neurodegenerative disorders,
myelodysplastic syndromes, ischemic injury, toxin-induced liver
disease, septic shock, cachexia and anorexia.
[0081] The death receptors are characterized by their cysteine rich
domains in the extracellular region and death domains (DD) in the
intracellular region. Death domain endows death receptor with
function of inducing cell death by apoptosis, but sometime it also
mediates other signals. Tumor necrosis factor-related
apoptosis-inducing ligand, TRAIL (Wiley S R, Schooley K, Smolak P,
et al., Immunity, 1995, 3:673-682) in combination with its death
domains triggers two cell death signaling pathways, i.e., death
receptor pathway and mitochondrion pathway, to kill various tumor
cells, but is nontoxic to most normal human cells.
[0082] Five TRAIL receptors, i.e., DR4 (death receptor 4 or named
as TRAIL-R1), DR5, DcR1 (decoy receptor 1 or named as
TRID/TRAIL-R3/LIT), DcR2 (TRAIL-R4 or named as TRUNDD), and
osteoprotegerin (OPG), have been identified. Like DR4, DR5 contains
three cysteine-rich domains in its extracellular portion and a
single cytoplasmic death domain and be capable of signaling
apoptosis upon ligand binding (or upon binding a molecule, such as
an agonist (e.g., antibody), which mimics the activity of the
ligand).
[0083] The term "agonist" as used with reference to DR5 refers to
any molecule that partially or fully enhances, stimulates or
activates one or more biological activities of DR5, in vitro, in
situ, or in vivo. Examples of such biological activities binding of
Apo2L/TRAIL to DR5, include apoptosis as well as those further
reported in the literature. DR5 agonists may function in a direct
or indirect manner. For example, the DR5 agonist may function to
partially or fully enhance, stimulate or activate one or more
biological activities of DR5, in vitro, in situ, or in vivo as a
result of its direct binding to DR5, which causes receptor
activation or signal transduction. The DR5 agonist may also
function indirectly to partially or fully enhance, stimulate or
activate one or more biological activities of DR5, in vitro, in
situ, or in vivo as a result of, e.g., stimulating another effector
molecule which then causes DR5 activation or signal transduction.
It is contemplated that an agonist may act as an enhancer molecule
which functions indirectly to enhance or increase DR5 activation or
activity.
[0084] A DR5 agonist may be any molecule that directly or
indirectly enhances the activity of DR5 and reduces tumor growth,
whether on its own or in combination with another treatment, such
as a PD-1 antagonist. Exemplary DR5 agonists include DR5 binding
scaffolds, such as anti-DR5 antibodies ("DR5 antibodies"), e.g.,
chimeric, humanized or fully human antibodies, an antigen binding
portion thereof, or molecules that are based on or derived from any
of these. DR5 agonists may also be non-antibody proteins. DR5
agonist also include DR5 ligands, e.g., TRAIL and molecules that
are derived from or based on TRAIL.
[0085] A DR5 agonist may be monovalent or multivalent. In certain
embodiments, a DR5 agonist is bivalent, trivalent, tetravalent, or
binds to 5, 6, 7, 8, 9, 10 or more DR5 epitopes, which may be the
same or different DR5 epitopes. In certain embodiments, a DR5
agonist is a multivalent monospecific DR5 binding scaffold, e.g., a
protein comprising a DR5 binding scaffold that comprises at least
2, 3, 4, 5, 6, 7, 8, 9, 10 or more regions that specifically bind
to the same DR5 epitope, which binding regions may be composed of
the same or a different amino acid sequence. For example, a DR5
agonist may be a DR5 binding scaffold comprising 2, 3, 4, 5, 6, 7,
8, 9, 10 or more repeats of the same DR5 binding region. Multimeric
DR5 binding scaffolds are described, e.g., in WO2009/058379,
WO2011/130328, WO2010/042890 and WO2011/098520.
[0086] In certain embodiments, a DR5 agonist binds specifically to
DR5, but does not bind significantly or specifically to other
members of the TNF receptor superfamily, such as DR4. In other
embodiments, a DR5 agonist binds specifically to DR5 and DR4.
[0087] For example, in one embodiment, the DR5 agonist is a
recombinant human TRAIL (TNF-related apoptosis-inducing ligand),
e.g., Dulanermin (also known as AMG-951; available from
Amgen/Genentech).
[0088] In another embodiment, the DR5 agonist is an antibody, e.g.,
an antibody that binds to human DR5 with a K.sub.D of 10.sup.-7 M,
5.times.10.sup.-8 M, 10.sup.-8 M, 5.times.10.sup.-9 M, 10.sup.-9 M,
5.times.10.sup.-10 M, 10.sup.-10 M or less, wherein the antibody
inhibits tumor growth and/or induces apoptosis of tumor cells.
Numerous antibodies binding to human DR5 are known in the art and
some of them have been used in clinical trials. Any of these
antibodies may be used in combination with a PD-1 antagonist,
provided that their combination results in inhibition of tumor
growth or reduction in tumor size, e.g., in a subject having
cancer. Exemplary antibodies that bind specifically to human DR5
include Conatumumab (a hTRAILR2-specific antibody also known as
AMG655; available from Amgen), Drozitumab (a hTRAILR2-specific
antibody also known as Apomab, DAB4, and PR095780; available from
Genentech), Lexatumumab (a hTRAILR2-specific antibody also known as
HGS-ETR2; available from HGS/Kirin), Tigatuzumab (a humanized
TRAILR2-specific antibody also known as CS-1008 and TRA-8;
available fromDaiichi Sankyo), HGSTR2J (a hTRAILR2-specific
antibody also known as KMTRS), or LBY-135 (a TRAILR2-specific Ab;
available from Novartis) (see, e.g., Ashkenazi et al., Journal of
Clinical Investigation 2008; 118:1979-90). In one embodiment, the
DR5 agonist is a bispecific death receptor agonist antibody, see,
e.g., WO2011/039126; available from Roche Glycart). In another
embodiment, the DR5 agonist is an antibody conjugated to targeting
peptides or a cytotoxin, Fc-human TRAIL ligand fusion (see, e.g.,
WO2011/039126; available from Roche Glycart). In another
embodiment, the DR5 agonist is a high affinity Fc-polypeptides
(see, e.g., WO2011/143614; available from Amgen).
[0089] In another embodiment, the DR5 agonist is a multivalent
agent, such as TAS266 (a tetrameric nanobody agonist targeting DR5,
see, e.g., WO2011/098520 and Cancer Research 2012; 72:Supplement 1;
Abstract 3852; available from Novartis and Ablynx), multimeric Tn3
protein (see, e.g., WO2009/058379, WO2011/130328, and Cancer
Research 2012; 72:Supplement 1; Abstract 239; available from
Medimmune), a multimer (e.g., a polypeptide construct with
trimerizing domain and a polypeptide that binds DR5; see
WO2010/042890; available from Anaphore).
[0090] Agents, which compete for binding to DR5 with any of the
exemplary agents listed herein, and which inhibit tumor growth or
reduces tumor size may also be used. Antibodies having VH and VL
chains comprising an amino acid sequence that is at least 90%, 95%,
98% or 99% identical to those of any of the anti-DR5 antibodies
listed herein may be used. In certain embodiments of the methods
described herein, a DR5 agonist is replaced with a DR4 agonist.
Thus, in certain embodiments, a subject having cancer is treated
with a combination of a DR4 agonist and a PD-1 antagonist.
Generally, any agent that induces apoptosis in tumor cells can be
combined with a PD-1 antagonist for treating cancer. In certain
embodiments, an apoptosis inducing agent is an agent that binds
specifically to DR5 and DR4, such as TRAIL or an agent that mimics
TRAIL. An exemplary DR4 agonist is Mapatumumab (HGS-ETR1), which
has been used in phase 2 clinical trials.
IV. Compositions
[0091] In one aspect, the present invention provides composition
comprising a PD-1 antagonist and a DR5 agonist (e.g., formulated
together in a single composition or separately formulated). In one
embodiment, the composition comprises a PD-1 antagonist and a DR5
agonist, wherein (a) the PD-1 antagonist is an anti-PD-1 antibody
comprising the CDR1, CDR2 and CDR3 domains in a heavy chain
variable region having the sequence set forth in SEQ ID NO: 13, and
the CDR1, CDR2 and CDR3 domains in a light chain variable region
having the sequence set forth in SEQ ID NO: 15; and (b) the DR5
agonist is an antibody. In another embodiment, the composition
comprises a PD-1 antagonist and a DR5 agonist, wherein (a) the PD-1
antagonist is an anti-PD-L1 antibody comprising the CDR1, CDR2 and
CDR3 domains in a heavy chain variable region having the sequence
set forth in SEQ ID NO: 1, and the CDR1, CDR2 and CDR3 domains in a
light chain variable region having the sequence set forth in SEQ ID
NO: 3 and (b) the DR5 agonist is an antibody.
[0092] Pharmaceutical compositions suitable for administration to
human patients are typically formulated for parenteral
administration, e.g., in a liquid carrier, or suitable for
reconstitution into liquid solution or suspension for intravenous
administration.
[0093] In general, such compositions typically comprise a
pharmaceutically acceptable carrier. As used herein, the term
"pharmaceutically acceptable" means approved by a government
regulatory agency or listed in the U.S. Pharmacopeia or another
generally recognized pharmacopeia for use in animals, particularly
in humans. The term "carrier" refers to a diluent, adjuvant,
excipient, or vehicle with which the compound is administered. Such
pharmaceutical carriers can be sterile liquids, such as water and
oils, including those of petroleum, animal, vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil,
glycerol polyethylene glycol ricinoleate, and the like. Water or
aqueous solution saline and aqueous dextrose and glycerol solutions
may be employed as carriers, particularly for injectable solutions.
Liquid compositions for parenteral administration can be formulated
for administration by injection or continuous infusion. Routes of
administration by injection or infusion include intravenous,
intraperitoneal, intramuscular, intrathecal and subcutaneous.
[0094] For oral use, the pharmaceutical compositions of the present
invention, may be administered, for example, in the form of tablets
or capsules, powders, dispersible granules, or cachets, or as
aqueous solutions or suspensions. In the case of tablets for oral
use, carriers which are commonly used include lactose, corn starch,
magnesium carbonate, talc, and sugar, and lubricating agents such
as magnesium stearate are commonly added. For oral administration
in capsule form, useful carriers include lactose, corn starch,
magnesium carbonate, talc, and sugar. When aqueous suspensions are
used for oral administration, emulsifying and/or suspending agents
are commonly added.
[0095] In addition, sweetening and/or flavoring agents may be added
to the oral compositions. For intramuscular, intraperitoneal,
subcutaneous and intravenous use, sterile solutions of the active
ingredient(s) are usually employed, and the pH of the solutions
should be suitably adjusted and buffered. For intravenous use, the
total concentration of the solute(s) should be controlled in order
to render the preparation isotonic.
[0096] For preparing suppositories according to the invention, a
low melting wax such as a mixture of fatty acid glycerides or cocoa
butter is first melted, and the active ingredient is dispersed
homogeneously in the wax, for example by stirring. The molten
homogeneous mixture is then poured into conveniently sized molds
and allowed to cool and thereby solidify.
[0097] Liquid preparations include solutions, suspensions and
emulsions. Such preparations are exemplified by water or
water/propylene glycol solutions for parenteral injection. Liquid
preparations may also include solutions for intranasal
administration.
[0098] Aerosol preparations suitable for inhalation may include
solutions and solids in powder form, which may be in combination
with a pharmaceutically acceptable carrier, such as an inert
compressed gas.
[0099] Also included are solid preparations which are intended for
conversion, shortly before use, to liquid preparations for either
oral or parenteral administration. Such liquid forms include
solutions, suspensions and emulsions.
V. Patient Populations
[0100] Provided herein are effective methods for treating cancer in
a patient, e.g., using a combination of a DR5 agonist and PD-1
antagonist. In one embodiment, the patient suffers from a cancer
selected from the group consisting of leukemia, lymphoma, blastoma,
carcinoma and sarcoma. In another embodiment, the patient suffers
from a cancer selected from the group consisting of chronic myeloid
leukemia, acute lymphoblastic leukemia, Philadelphia chromosome
positive acute lymphoblastic leukemia (Ph+ ALL), squamous cell
carcinoma, small-cell lung cancer, non-small cell lung cancer,
glioma, gastrointestinal cancer, renal cancer, ovarian cancer,
liver cancer, colorectal cancer, endometrial cancer, kidney cancer,
prostate cancer, thyroid cancer, neuroblastoma, pancreatic cancer,
glioblastoma multiforme, cervical cancer, stomach cancer, bladder
cancer, hepatoma, breast cancer, colon carcinoma, and head and neck
cancer, gastric cancer, germ cell tumor, pediatric sarcoma,
sinonasal natural killer, multiple myeloma, acute myelogenous
leukemia (AML), and chronic lymphocytic leukemia (CML).
VI. Additional Agents/Therapies
[0101] The combinations of the present invention (e.g., PD-1
antagonist in combination with DR5 agonist) may also be used in
conjunction with other well known therapies that are selected for
their particular usefulness against the cancer that is being
treated. Combinations of the instant invention may alternatively be
used sequentially with known pharmaceutically acceptable agent(s)
when inappropriate.
[0102] For example, the PD-1 antagonists and DR5 agonists described
herein can further be used in combination (e.g., simultaneously or
separately) with an additional treatment, such as irradiation,
chemotherapy (e.g., using camptothecin (CPT-11), 5-fluorouracil
(5-FU), cisplatin, doxorubicin, irinotecan, paclitaxel,
gemcitabine, cisplatin, paclitaxel, doxorubicin, 5-fu, or
camptothecin+apo21/TRAIL (a 6.times. combo)), one or more
proteasome inhibitors (e.g., bortezomib or MG132), one or more
Bcl-2 inhibitors (e.g., BH3I-2' (bcl-xl inhibitor), AT-101
(R-(-)-gossypol derivative), ABT-263 (small molecule), GX-15-070
(obatoclax), or MCL-1 (myeloid leukemia cell differentiation
protein-1) antagonists), iAP (inhibitor of apoptosis protein)
antagonists (e.g., smac7, smac4, small molecule smac mimetic,
synthetic smac peptides (see Fulda et al., Nat Med 2002; 8:808-15),
ISIS23722 (LY2181308), or AEG-35156 (GEM-640)), HDAC (histone
deacetylase) inhibitors, anti-CD20 antibodies (e.g., rituximab),
angiogenesis inhibitors (e.g., bevacizumab), anti-angiogenic agents
targeting VEGF and VEGFR, synthetic triterpenoids (see Hyer et al.,
Cancer Research 2005; 65:4799-808), c-FLIP (cellular
FLICE-inhibitory protein) modulators (e.g., natural and synthetic
ligands of PPAR.gamma. (peroxisome proliferator-activated receptor
7), 5809354 or 5569100), kinase inhibitors (e.g., Sorafenib),
and/or genotoxic drugs.
[0103] The PD-1 antagonists and DR5 agonists described herein can
further be used in combination with one or more anti-proliferative
cytotoxic agents. Classes of compounds that may be used as
anti-proliferative cytotoxic agents include, but are not limited
to, the following:
[0104] Alkylating agents (including, without limitation, nitrogen
mustards, ethylenimine derivatives, alkyl sulfonates, nitrosoureas
and triazenes): Uracil mustard, Chlormethine, Cyclophosphamide
(CYTOXAN.TM.) fosfamide, Melphalan, Chlorambucil, Pipobroman,
Triethylenemelamine, Triethylenethiophosphoramine, Busulfan,
Carmustine, Lomustine, Streptozocin, Dacarbazine, and
Temozolomide.
[0105] Antimetabolites (including, without limitation, folic acid
antagonists, pyrimidine analogs, purine analogs and adenosine
deaminase inhibitors): Methotrexate, 5-Fluorouracil, Floxuridine,
Cytarabine, 6-Mercaptopurine, 6-Thioguanine, Fludarabine phosphate,
Pentostatine, and Gemcitabine.
[0106] Suitable anti-proliferative agents for use in the methods of
the invention, include, without limitation, taxanes, paclitaxel
(paclitaxel is commercially available as TAXOL.RTM.), docetaxel,
discodermolide (DDM), dictyostatin (DCT), Peloruside A,
epothilones, epothilone A, epothilone B, epothilone C, epothilone
D, epothilone E, epothilone F, furanoepothilone D, desoxyepothilone
B1, [17]-dehydrodesoxyepothilone B, [18]dehydrodesoxyepothilones B,
C12,13-cyclopropyl-epothilone A, C6-C8 bridged epothilone A,
trans-9,10-dehydroepothilone D, cis-9,10-dehydroepothilone D,
16-desmethylepothilone B, epothilone B10, discoderomolide,
patupilone (EPO-906), KOS-862, KOS-1584, ZK-EPO, ABJ-789, XAA296A
(Discodermolide), TZT-1027 (soblidotin), ILX-651 (tasidotin
hydrochloride), Halichondrin B, Eribulin mesylate (E-7389),
Hemiasterlin (HTI-286), E-7974, Cyrptophycins, LY-355703,
Maytansinoid immunoconjugates (DM-1), MKC-1, ABT-751, T1-38067,
T-900607, SB-715992 (ispinesib), SB-743921, MK-0731, STA-5312,
eleutherobin,
17beta-acetoxy-2-ethoxy-6-oxo-B-homo-estra-1,3,5(10)-trien-3-ol,
cyclostreptin, isolaulimalide, laulimalide,
4-epi-7-dehydroxy-14,16-didemethyl-(+)-discodermolides, and
cryptothilone 1, in addition to other microtubuline stabilizing
agents known in the art.
[0107] In cases where it is desirable to render aberrantly
proliferative cells quiescent in conjunction with or prior to
treatment with the chemotherapeutic methods of the invention,
hormones and steroids (including synthetic analogs), such as
17a-Ethinylestradiol, Diethylstilbestrol, Testosterone, Prednisone,
Fluoxymesterone, Dromostanolone propionate, Testolactone,
Megestrolacetate, Methylprednisolone, Methyl-testosterone,
Prednisolone, Triamcinolone, Chlorotrianisene, Hydroxyprogesterone,
Aminoglutethimide, Estramustine, Medroxyprogesteroneacetate,
Leuprolide, Flutamide, Toremifene, ZOLADEX.TM., can also be
administered to the patient. When employing the methods or
compositions of the present invention, other agents used in the
modulation of tumor growth or metastasis in a clinical setting,
such as antimimetics, can also be administered as desired.
[0108] Methods for the safe and effective administration of
chemotherapeutic agents are known to those skilled in the art. In
addition, their administration is described in the standard
literature. For example, the administration of many of the
chemotherapeutic agents is described in the Physicians' Desk
Reference (PDR), e.g., 1996 edition (Medical Economics Company,
Montvale, N.J. 07645-1742, USA); the disclosure of which is
incorporated herein by reference thereto.
[0109] The chemotherapeutic agent(s) and/or radiation therapy can
be administered according to therapeutic protocols well known in
the art. It will be apparent to those skilled in the art that the
administration of the chemotherapeutic agent(s) and/or radiation
therapy can be varied depending on the disease being treated and
the known effects of the chemotherapeutic agent(s) and/or radiation
therapy on that disease. Also, in accordance with the knowledge of
the skilled clinician, the therapeutic protocols (e.g., dosage
amounts and times of administration) can be varied in view of the
observed effects of the administered therapeutic agents on the
patient, and in view of the observed responses of the disease to
the administered therapeutic agents.
VII. Treatment Protocols
[0110] Suitable treatment protocols for treating cancer in a
patient include, for example, administering to the patient an
effective amount of a PD-1 antagonist (e.g., antibody) and a DR5
agonist (e.g., antibody).
[0111] As used herein, adjunctive or combined administration
(co-administration) includes simultaneous administration of the
antagonist and agonist in the same or different dosage form, or
separate administration of the antagonist and agonist (e.g.,
sequential administration). Thus, the PD-1 antagonist (e.g.,
antibody) and DR5 agonist (e.g., antibody) can be simultaneously
administered in a single formulation. Alternatively, the PD-1
antagonist and DR5 agonist can be formulated for separate
administration and are administered concurrently or
sequentially.
[0112] For example, the PD-1 antagonist can be administered first
followed by (e.g., immediately followed by) the administration of
the DR5 agonist, or vice versa. In one embodiment, the PD-1
antagonist is administered prior to administration of the DR5
agonist. In one embodiment, the DR5 agonist is administered prior
to administration of the PD-1 antagonist. Such concurrent or
sequential administration preferably results in both the agonist
and antagonist being simultaneously present in treated patients. In
another embodiment, the DR5 agonist and the PD-1 antagonist are
administered simultaneously.
[0113] In an exemplary treatment, a subject is dosed with a single
dose of a DR5 agonist and at least 2 doses of a PD-1 antagonist,
e.g., an anti-PD-1 or anti-PD-L1 antibody. In certain embodiment, a
subject receives a single dose of a DR5 agonist and at least 2, 3,
4, 5, or more doses of a PD-1 antagonist. The multiple doses of
PD-1 antagonist may be provided as one dose per day, one dose every
2 days, one dose every 3 days, one dose every 4 days, one dose
every 5 days or less frequently. In certain embodiments, in which a
PD-1 antagonist is provided as 1 dose every 1, 2, 3, 4, 5 or more
days, the single dose of DR5 agonist may be provided on a day on
which the PD-1 antagonist is provided or on a day on which it is
not provided. The total number of doses of PD-1 antagonist may be
2, 3, 4, 5, 6, 7, 8, 9, 10 or more.
[0114] In certain embodiments, multiple (e.g., 2, 3, 4, 5, 6, 7, 8,
9, 10 or more) doses of a DR5 agonist and multiple (e.g., 2, 3, 4,
5, 6, 7, 8, 9, 10 or more) doses of a PD-1 antagonist are
administered to a subject in need of treatment. Administration of
the DR5 agonist and the PD-1 antagonist may be on the same day, or
alternatively, the DR5 antagonist may be administered 1 or more
days before or after the PD-1 antagonist.
[0115] Administrations of a DR5 agonist and a PD-1 antagonist may
also be done weekly or monthly, in which regimen, they may be
administered on the same day (e.g., simultaneously), or one after
the other (e.g., one or more days before or after one another)
[0116] In one embodiment, the dose of the PD-1 antagonist and/or
DR5 agonist is varied over time. For example, the PD-1 antagonist
and/or DR5 agonist may be initially administered at a high dose and
may be lowered over time. In another embodiment, the PD-1
antagonist and/or DR5 agonist is initially administered at a low
dose and increased over time.
[0117] In another embodiment, the amount of the PD-1 antagonist
and/or DR5 agonist administered is constant for each dose. In
another embodiment, the amount of the PD-1 antagonist and/or DR5
agonist varies with each dose. For example, the maintenance (or
follow-on) dose of the antagonist and/or agonist can be higher or
the same as the loading dose which is first administered. In
another embodiment, the maintenance dose of the antagonist and/or
agonist can be lower or the same as the loading dose. A clinician
may utilize preferred dosages as warranted by the condition of the
patient being treated. The dose of may depend upon a number of
factors, including stage of disease, etc. The specific dose that
should be administered based upon the presence of one or more of
such factors is within the skill of the artisan. Generally,
treatment is initiated with smaller dosages which are less than the
optimum dose of the compound. Thereafter, the dosage is increased
by small amounts until the optimum effect under the circumstances
is reached. For convenience, the total daily dosage may be divided
and administered in portions during the day if desired.
Intermittent therapy (e.g., one week out of three weeks or three
out of four weeks) may also be used.
[0118] In one embodiment, the DR5 agonist (e.g., antibody) is
administered at a dose of 0.1, 0.3, 1, 2, 3, 4, 5, 6, 7, 8, 9, or
10 mg/kg body weight. In another embodiment, the PD-1 antagonist
(e.g., antibody) is administered at a dose of 0.5, 1, 2, 3, 4, 5,
6, 7, 8, 9, or 10 mg/kg body weight. Generally, 200 .mu.g/mouse is
approximately 10 mg/kg and 100 .mu.g/mouse is approximately 5
mg/kg. Therefore, based on the experiments described herein, one or
more doses of 1-20 mg/kg body weight, 1-10 mg/kg body weight, 5-20
mg/kg body weight or 5-10 mg/kg body weight of a DR5 agonist and
PD-1 antagonist may be administered to a subject. In certain
embodiments, a dose of 0.3 mg/kg to 10 mg/kg body weight of a DR5
agonist is used and a dose of at least 1 mg/kg, e.g., 1-100 mg/kg
body weight of a PD-1 antagonist is used.
VIII. Outcomes
[0119] Patients, e.g., humans, treated according to the methods
disclosed herein preferably experience improvement in at least one
sign of cancer. In one embodiment, improvement is measured by a
reduction in the quantity and/or size of measurable tumor lesions.
In another embodiment, lesions can be measured on chest x-rays or
CT or MRI films. In another embodiment, cytology or histology can
be used to evaluate responsiveness to a therapy.
[0120] In one embodiment, the patient treated exhibits a reduction
in size of a tumor, reduction in number of metastasic lesions over
time, complete response, partial response, and stable disease. In
another embodiment, the patient treated experiences tumor shrinkage
and/or decrease in growth rate, i.e., suppression of tumor growth.
In another embodiment, unwanted cell proliferation is reduced or
inhibited. In yet another embodiment, one or more of the following
can occur: the number of cancer cells can be reduced; tumor size
can be reduced; cancer cell infiltration into peripheral organs can
be inhibited, retarded, slowed, or stopped; tumor metastasis can be
slowed or inhibited; tumor growth can be inhibited; recurrence of
tumor can be prevented or delayed; one or more of the symptoms
associated with cancer can be relieved to some extent.
[0121] In another embodiment, the methods of treatment produce a
comparable clinical benefit rate (CBR=CR (complete response), PR
(partial response) or SD (stable disease).gtoreq.6 months) better
than that achieved by a PD-1 (e.g., antibody) or DR5 agonist (e.g.,
antibody) alone. In other embodiments, the improvement of clinical
benefit rate is about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or
more, e.g., compared to treatment with a PD-1 antagonist or DR5
agonist alone or relative to tumor growth on the first day of
treatment or immediately before initiation of treatment.
[0122] In another embodiment, administration of a PD-1 antagonist
and a DR5 agonist results in at least a three-fold reduction (e.g.,
a 3.5-fold reduction) in tumor volume, e.g., relative to treatment
with the PD-1 antagonist or the DR5 agonist alone or relative to
tumor growth on the first day of treatment or immediately before
initiation of treatment.
[0123] In a further embodiment, administration of a PD-1 antagonist
and a DR5 agonist results in tumor growth inhibition of at least
80%, e.g., relative to treatment with the PD-1 antagonist or DR5
agonist alone or relative to tumor growth on the first day of
treatment or immediately before initiation of treatment.
[0124] In certain embodiments, administration of a PD-1 antagonist
and a DR5 agonist reduces tumor mass by at least 10%, 20%, 30%,
40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% relative to the tumor mass
prior to initiation of the treatment or on the first day of
treatment. In some embodiment, the tumor mass is no longer
detectable following treatment as described herein. In some
embodiments, a subject is in partial or full remission.
IX. Kits and Unit Dosage Forms
[0125] Also provided herein are kits which include a pharmaceutical
composition containing (a) a PD-1 antagonist and (b) a DR5 agonist
and a pharmaceutically-acceptable carrier, in a therapeutically
effective amount adapted for use in the preceding methods. In one
embodiment, the PD-1 antagonist is an antibody (e.g., 5C4 or 12A4,
respectively). In another embodiment, the DR5 agonist is an
antibody. The kits optionally also can include instructions, e.g.,
comprising administration schedules, to allow a practitioner (e.g.,
a physician, nurse, or patient) to administer the composition
contained therein to a patient having cancer. The kit also can
include a syringe.
[0126] Optionally, the kits include multiple packages of the
single-dose pharmaceutical compositions each containing an
effective amount of the PD-1 antagonist and the DR5 agonist for a
single administration in accordance with the methods provided
above. Instruments or devices necessary for administering the
pharmaceutical composition(s) also may be included in the kits. For
instance, a kit may provide one or more pre-filled syringes
containing an amount of the PD-1 antagonist and the DR5
agonist.
[0127] In one embodiment, the present invention provides a kit for
treating cancer in a patient, the kit comprising:
[0128] (a) a dose of a PD-1 antagonist;
[0129] (b) a dose of a DR5 agonist; and
[0130] (c) instructions for using the PD-1 antagonist and DR5
agonist in the method of any one of claims 1-32. In another
embodiment, the DR5 agonist is an antibody. In another embodiment,
the PD-1 antagonist is an antibody. In particular embodiment, the
PD-1 antagonist is an anti-PD-1 antibody comprising the CDR1, CDR2
and CDR3 domains in a heavy chain variable region having the
sequence set forth in SEQ ID NO: 13, and the CDR1, CDR2 and CDR3
domains in a light chain variable region having the sequence set
forth in SEQ ID NO: 15. In another particular embodiment, the PD-1
antagonist is an anti-PD-L1 antibody comprising antibody comprises
the CDR1, CDR2 and CDR3 domains in a heavy chain variable region
having the sequence set forth in SEQ ID NO: 1, and the CDR1, CDR2
and CDR3 domains in a light chain variable region having the
sequence set forth in SEQ ID NO: 3.
[0131] The following examples are merely illustrative and should
not be construed as limiting the scope of this disclosure in any
way as many variations and equivalents will become apparent to
those skilled in the art upon reading the present disclosure.
[0132] The contents of all references, Genbank entries, patents and
published patent applications cited throughout this application are
expressly incorporated herein by reference.
EXAMPLES
Example 1
1. Materials and Methods
[0133] Animals
[0134] Ten to eleven-week-old female C57/BL6 mice (Harlan) were
used in the studies. Mice received food and water ad libitum and
were maintained in a controlled environment according to
Association for Assessment and Accreditation of Laboratory Animal
Care (AAALAC) International regulations. All animal studies have
been approved by the appropriate ethics committee and have
therefore been performed in accordance with the ethical standards
laid down in the 1964 Declaration of Helsinki and its later
amendments.
[0135] Antibodies
[0136] Anti-mouse PD-1 mAb (anti-mPD-1 mAb) clone 4H2, mouse IgG1
isotype was produced and purified by Bristol-Myers Squibb
(Biologics Discovery, CA). Agonist anti-mouse DR5 mAb, clone MD5-1,
hamster IgG isotype, was purchased from BioxCell (West Lebanon,
N.H.). Both antibodies were certified to have <0.5 EU/mg
endotoxin levels, >95% purity and <5% high molecular weight
species. Stock solutions of anti-mPD-1 mAb and anti-mDR5 antibody
were kept at 4.degree. C. prior to use. Dosing solutions of
anti-mPD-1 mAb and anti-mDR5 mAb were prepared in sterile phosphate
buffered saline (pH 7.0) and maintained at 4.degree. C.
[0137] Anti-mPD-1 mAb was administered intraperitoneally at its
optimal dose of 10 mg/kg; anti-DR5 mAb at 5 mg/kg.
[0138] Tumor Model
[0139] The MC38 colon carcinoma tumor line used in this study was
maintained in vitro. Cell suspensions were implanted in the
subcutaneous space of the flank of mice of female C57/BL6 mice
(2.0.times.10.sup.6 MC-38 cells in 0.2 mL Hanks Balanced Salt
Solution).
[0140] Tumor size and body weights were measured twice weekly.
Tumor size (measured as mm.sup.3) was calculated by multiplying the
tumor length by the square of the tumor width divided by 2.
Treatments were initiated when subcutaneous tumors reached a median
size of 200 mm.sup.3 (established model). Antitumor activity,
defined as percentage tumor growth inhibition, was calculated with
the formula % Tumor Growth Inhibition (%
TGI)=100-[(Tt/To)/(Ct/Co)]/100-(Ct/Co), where Tt=median tumor size
of treated group at the end of treatment, To=median tumor size of
treated group at treatment initiation, Ct=median tumor size of
control group at the end of treatment, and Co=median tumor size at
treatment initiation (see Table 3). Complete regressions were
defined as absence of measurable tumor mass for at least 2 tumor
volume doubling times.
[0141] The tumor response endpoint was expressed as tumor growth
delay (T-C value), calculated as the difference in time (days)
between the treated (T) and control (C) groups for the tumor to
reach a predetermined target size. A delay in reaching target size
by the treated groups of >1 times tumor volume doubling time was
considered an active result. Therapeutic synergy was defined as an
antitumor effect in which the combination of agents demonstrated
significant superiority (p<0.05) relative to the activity shown
by each agent alone.
[0142] The antitumor effect of single dose anti-mouse DR5 mAb in
combination an anti-PD-1 mAb given at various dose schedules was
evaluated in MC-38 (murine colon) tumor bearing mice. Six days post
tumor implant, mice were sorted into eight groups of 7 mice with a
mean tumor volume of 200 mm.sup.3. Antibodies were administered
according to the dosing schedules descried in Table 1.
TABLE-US-00001 TABLE 1 Dosing Schedule Treatment Group # mice 1:
Control 7 2: Control + anti-PD-1 mAb; 200 ug/mouse; 7 Q4D .times. 3
dosing initiated day 6 3: Control + anti-PD-1 mAb; 200 ug/mouse; 7
Q4D .times. 3 dosing initiated day 8 4: Control + anti-PD-1 mAb;
200 ug/mouse; 7 Q4D .times. 3 dosing initiated day 9 5: Anti-DR5
mAb; 100 ug/mouse; 7 QD + Control dosing initiated day 8 6:
Anti-DR5 mAb; 100 ug/mouse dosed 7 day 8; QD + anti-PD-1 mAb; 200
ug/mouse; Q4D .times. 3 dosed day 6 7: Anti-DR5 mAb; 100 ug/mouse
dosed 7 day 8; QD + anti-PD-1 mAb; 200 ug/mouse; Q4D .times. 3
dosed day 8 8: Anti-DR5 mAb; 100 ug/mouse dosed 7 day 8; QD +
anti-PD-1 mAb; 200 ug/mouse; Q4D .times. 3 dosed day 9 QD: One dose
administered on only one day. Q4D .times. 3: One dose administered
every four days for a total of 3 doses.
[0143] The combination of DR5 mAb and PD-1 mAb were tested
according to the three schedules set forth in Table 2.
TABLE-US-00002 TABLE 2 Administration Schedules Group No.:
Administration Schedule Group 6 DR5 mAb was administered 2 days
after PD-1 mAb therapy. Group 7 DR5 mAb and PD-1 mAb were
administered on the same day. Group 8 DR5 mAb was administered 1
day before PD-1 mAb therapy.
[0144] It was hypothesized that since PD-1 mab induces IFN-gamma,
which in tumors upregulates DR5 expression on tumor cells, it might
be advantageous to administer the DR5 mAb after administration of
PD-1 mAb therapy (Group 6). Alternatively, it was hypothesized that
DR5 mAb induces tumor cell death, which in turn will prime
antitumor immune responses, and PD-1 mAb will subsequently expand
the induced antitumor immunity. To test this hypothesis, DR5 mAb
was administered before PD-1 mAb therapy (Group 8).
2. Results
[0145] As shown in Table 3, at least 80% tumor growth inhibition
was achieved in mice treated with a combination of both the DR5 mAb
and PD1 mAb.
TABLE-US-00003 TABLE 3 Tumor Responses Schedule % Complete Dose
(days post Regressions Treatment (mg/kg) implant) % TGI (#/total
mice) Anti-PD-1 mAb 10 Day 6, 10, 14 31 0 (0/7) Anti-PD-1 mAb 10
Day 8, 12, 16 14 0 (0/7) Anti-PD-1 mAb 10 Day 9, 13, 17 -8 0 (0/7)
Anti-DR5 mAb 5 Day 8 -4 0 (0/7) Anti-PD-1 mAb + 10 Day 6, 10, 14 96
43 Anti-DR5 mAb 5 Day 8 (3/7) Anti-PD-1 mAb + 10 Day 8, 12, 16 82
14 Anti-DR5 mAb 5 Day 8 (1/7) Anti-PD-1 mAb + 10 Day 9, 13, 17 87
43 Anti-DR5 mAb 5 Day 8 (3/7)
Moreover, as shown in FIG. 1, the medium tumor volume (measured in
mm.sup.3) in mice treated with a combination of the DR5 mAb and the
PD-1 mAb was significantly reduced, compared to mice treated with a
control or either agent alone. Specifically, there was about a 3.5
fold reduction (e.g., at least a 3 fold reduction) in tumor volume
in mice treated with both the DR5 mAb and the PD-1 mAb, compared to
mice treated with a control or either agent alone. The tumor volume
in individual mice is shown in FIGS. 2A-2H.
[0146] In sum, the combination of the DR5 mAb and the PD-1 mAb
resulted in enhanced activity compared to the activity elicited by
single agents alone, independent of the schedule utilized. This
synergy was statistically significant (p<0.05, Wilcoxon). Out of
the 3 administration schedules tested, a trend for better activity
was observed in the groups which were treated with anti-mDR5 first
or with anti-mPD-1 first. As shown in FIG. 3, the combination
therapy was well-tolerated (no significant body weight loss). In
previous studies, significant body weight loss (>20%) was
observed with multiple doses of the DR5 mAb alone or in
combination.
[0147] Therefore, results from this study demonstrate that a
combination regimen that includes a single dose of anti-mDR5 mAb
and multiple doses of PD-1 mAb is well-tolerated and result in
marked antitumor activity.
TABLE-US-00004 SEQ ID NO: SEQUENCE 1 Heavy Chain Variable Region
(VH) Amino Acid Sequence Anti-PD-L1 mAb (12A4; 12A4 in WO
2007/005874 and U.S. Pat. No. 7,943,743) QVQLVQSGAEVKKPGSSVKVSCKT
SGDTFSTYAISWVRQAPGQGLEWM GGIIPIFGKAHYAQKFQGRVTITA
DESTSTAYMELSSLRSEDTAVYFC ARKFHFVSGSPFGMDVWGQGTTVT VSS 2 Heavy Chain
Variable Region (VH) Nucleotide Sequence Anti-PD-L1 mAb (12A4; 12A4
in WO 2007/005874 and U.S. Pat. No. 7,943,743) cag gtc cag ctg gtg
cag tct ggg gct gag gtg aag aag cct ggg tcc tcg gtg aag gtc tcc tgc
aag act tct gga gac acc ttc agc acc tat gct atc agc tgg gtg cga cag
gcc cct gga caa ggg ctt gag tgg atg gga ggg atc atc cct ata ttt ggt
aaa gca cac tac gca cag aag ttc cag ggc aga gtc acg att acc gcg gac
gaa tcc acg agc aca gcc tac atg gag ctg agc agc ctg aga tct gag gac
acg gcc gtg tat ttt tgt gcg aga aag ttt cac ttt gtt tcg ggg agc ccc
ttc ggt atg gac gtc tgg ggc caa ggg acc acg gtc acc gtc tcc 3 Light
Chain Variable Region (VL) Amino Acid Sequence Anti-PD-Ll mAb
(12A4; 12A4 in WO 2007/005874 and U.S. Pat. No. 7,943,743)
EIVITQSPATLSLSPGERATL SCRASQSVSSYLAWYQQKPCQ APRLLIYDASNRATGIPARFS
GSGSGTDFTLTISSLEPEDFA VYYCQQRSNWPTFGQGTKVEIK 4 Light Chain Variable
Region (VL) Nucleotide Sequence Anti-PD-L1 mAb (12A4; 12A4 in WO
2007/005874 and U.S. Pat. No. 7,943,743) gaa att gtg ttg aca cag
tct cca gcc acc ctg tct ttg tct cca ggg gaa aga gcc acc ctc tcc tgc
agg gcc agt cag agt gtt agc agc tac tta gcc tgg tac caa cag aaa cct
ggc cag gct ccc agg ctc ctc atc tat gat gca tcc aac agg gcc act ggc
atc cca gcc agg ttc agt ggc agt ggg tct ggg aca gac ttc act ctc acc
atc agc agc cta gag cct gaa gat ttt gca gtt tat tac tgt cag cag cgt
agc aac tgg ccg acg ttc ggc caa ggg acc aag gtg gaa atc aaa 5 Heavy
Chain CDR1 Amino Acid Sequence Anti-PD-L1 mAb (12A4; 12A4 in WO
2007/005874 and U.S. Pat. No. 7,943,743) TYAIS 6 Heavy Chain CDR2
Amino Acid Sequence Anti-PD-L1 mAb (12A4; 12A4 in WO 2007/005874
and U.S. Pat. No. 7,943,743) GIIPIFGKAHYAQKFQ 7 Heavy Chain CDR3
Amino Acid Sequence Anti-PD-L1 mAb (12A4; 12A4 in WO 2007/005874
and U.S. Pat. No. 7,943,743) KFHFVSGSPFGMDV 8 Light Chain CDR1
Amino Acid Sequence Anti-PD-L1 mAb (12A4; 12A4 in WO 2007/005874
and U.S. Pat. No. 7,943,743) RASQSVSSYLA 9 Light Chain CDR2 Amino
Acid Sequence Anti-PD-L1 mAb (12A4; 12A4 in WO 2007/005874 and U.S.
Pat. No. 7,943,743) DASNRAT 10 Light Chain CDR3 Amino Acid Sequence
Anti-PD-L1 mAb (12A4; 12A4 in WO 2007/005874 and U.S. Pat. No.
7,943,743) QQRSNWPT 11 Heavy Chain Amino Acid Sequence Anti-PD-1
mAb (5C4 in WO 2006/121168) (variable region underlined; constant
region bold) QVQLVESGGGVVQPGRSLRLDCKA SGITFSNSGMHWVRQAPGKGLEWV
AVIWYDGSKRYYADSVKGRFTISR DNSKNTLFLQMNSLRAEDTAVYYC
ATNDDYWGQGTLVTVSSASTKGPS VFPLAPCSRSTSESTAALGCLVKD
YFPEPVTVSWNSGALTSGVHTFPA VLQSSGLYSLSSVVTVPSSSLGTK
TYTCNVDHKPSNTKVDKRVESKYG PPCPPCPAPEFLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVDVSQEDP EVQFNWYVDGVEVHNAKTKPREEQ
FNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKGLPSSIEKTISKAKGQP
REPQVYTLPPSQEEMTKNQVSLTC LVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSRLTVDKS RWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK 12 Light
Chain Amino Acid Sequence Anti-PD-1 mAb (5C4 in WO 2006/121168)
(variable region underlined; constant region bold)
EIVLTQSPATLSLSPGERATLSCR ASQSVSSYLAWYQQKPGQAPRLII
YDASNRATGIPARFSGSGSGTDFT LTISSLEPEDFAVYYCQQSSNWPR
TFGQGTKVEIKRTVAAPSVFIFPP SDEQLKSGTASVVCLINNFYPREA
KVQWKVDNALQSGNSQESVTEQDS DKSTYSLSSTLTLSKADYEKHKVY
ACEVTHQGLSSPVTKSFNRGEC 13 Heavy Chain Variable Region (VH) Amino
Acid Sequence Anti-PD-1 mAb (5C4 in WO 2006/121168) (SEQ ID NO: 4
from WO 2006/121168) QVQLVESGGGWQPGRSLRLDCK ASGITFSNSGMHWVRQAPGKGL
EWVAVIWYDGSKRYYADSVKGR FTISRDNSKNTLFLQMNSLRAE
DTAVYYCATNDDYWGQGTLVTV SS 14 Heavy Chain Variable Region (VH)
Nucleotide Sequence Anti-PD-1 mAb (5C4 in WO 2006/121168) (SEQ ID
NO: 60 from WO 2006/121168) cag gtg cag ctg gtg gag tct ggg gga ggc
gtg gtc cag cct ggg agg tcc ctg aga ctc gac tgt aaa gcg tct gga atc
acc ttc agt aac tct ggc atg cac tgg gtc cgc cag gct cca ggc aag ggg
ctg gag tgg gtg gca gtt att tgg tat gat gga agt aaa aga tac tat gca
gac tcc gtg aag ggc cga ttc acc atc tcc aga gac aat tcc aag aac acg
ctg ttt ctg caa atg aac agc ctg aga gcc gag gac acg gct gtg tat tac
tgt gcg aca aac gac gac tac tgg ggc cag gga acc ctg gtc acc gtc tcc
tca 15 Light Chain Variable Region (VL) Amino Acid Sequence
Anti-PD-1 mAb (5C4 in WO 2006/121168) (SEQ ID NO: 11 from WO
2006/121168) EIVLTQSPATLSLSPGERATLSCR ASQSVSSYLAWYQQKPGQAPRLII
YDASNRATGIPARFSGSGSGTDFT LTISSLEPEDFAVYYCQQSSNWPR TFGQGTKVEIK 16
Light Chain Variable Region (VL) Nucleotide Sequence Anti-PD-1 mAb
(5C4 in WO 2006/121168) (SEQ ID NO: 67 from WO 2006/121168) gaa att
gtg ttg aca cag tct cca gcc acc ctg tct ttg tct cca ggg gaa aga gcc
acc ctc tcc tgc agg gcc agt cag agt gtt agt agt tac tta gcc tgg tac
caa cag aaa cct ggc cag gct ccc agg ctc ctc atc tat gat gca tcc aac
agg gcc act ggc atc cca gcc agg ttc agt ggc agt ggg tct ggg aca gac
ttc act ctc acc atc agc agc cta gag cct gaa gat ttt gca gtt tat tac
tgt cag cag agt agc aac tgg cct cgg acg ttc ggc caa ggg acc aag gtg
gaa atc aaa 17 Heavy Chain CDR1 Amino Acid Sequence Anti-PD-1 mAb
(5C4 in WO 2006/121168) (SEQ ID NO: 18 from WO 2006/121168) NSGMH
18 Heavy Chain CDR2 Amino
Acid Sequence Anti-PD-1 mAb (5C4 in WO 2006/121168) (SEQ ID NO: 25
from WO 2006/121168) VIWYDGSKRYYADSVKG 19 Heavy Chain CDR3 Amino
Acid Sequence Anti-PD-1 mAb (5C4 in WO 2006/121168) (SEQ ID NO: 32
from WO 2006/121168) NDDY 20 Light Chain CDR1 Amino Acid Sequence
Anti-PD-1 mAb (5C4 in WO 2006/121168) (SEQ ID NO: 39 from WO
2006/121168) RASQSVSSYLA 21 Light Chain CDR2 Amino Acid Sequence
Anti-PD-1 mAb (5C4 in WO 2006/121168) (SEQ ID NO: 46 from WO
2006/121168) DASNRAT 22 Light Chain CDR3 Amino Acid Sequence
Anti-PD-1 mAb (5C4 in WO 2006/121168) (SEQ ID NO: 53 from WO
2006/121168) QQSSNWPRT 23 Complete PD-1 sequence (GenBank Accession
No.: U64863) agtttccctt ccgctcacct ccgcctgagc agtggagaag gcggcactct
ggtggggctg ctccaggcat gcagatccca caggcgccct ggccagtcgt ctgggcggtg
ctacaactgg gctggcggcc aggatggttc ttagactccc cagacaggcc ctggaacccc
cccaccttct tcccagccct gctcgtggtg accgaagggg acaacgccac cttcacctgc
agcttctcca acacatcgga gagcttcgtg ctaaactggt accgcatgag ccccagcaac
cagacggaca agctggccgc cttccccgag gaccgcagcc agcccggcca ggactgccgc
ttccgtgtca cacaactgcc caacgggcgt gacttccaca tgagcgtggt cagggcccgg
cgcaatgaca gcggcaccta cctctgtggg gccatctccc tggcccccaa ggcgcagatc
aaagagagcc tgcgggcaga gctcagggtg acagagagaa gggcagaagt gcccacagcc
caccccagcc cctcacccag gccagccggc cagttccaaa ccctggtggt tggtgtcgtg
ggcggcctgc tgggcagcct ggtgctgcta gtctgggtcc tggccgtcat ctgctcccgg
gccgcacgag ggacaatagg agccaggcgc accggccagc ccctgaagga ggacccctca
gccgtgcctg tgttctctgt ggactatggg gagctggatt tccagtggcg agagaagacc
ccggagcccc ccgtgccctg tgtccctgag cagacggagt atgccaccat tgtctttcct
agcggaatgg gcacctcatc ccccgcccgc aggggctcag ccgacggccc tcggagtgcc
cagccactga ggcctgagga tggacactgc tcttggcccc tctgaccggc ttccttggcc
accagtgttc tgcagaccct ccaccatgag cccgggtcag cgcatttcct caggagaagc
aggcagggtg caggccattg caggccgtcc aggggctgag ctgcctgggg gcgaccgggg
ctccagcctg cacctgcacc aggcacagcc ccaccacagg actcatgtct caatgcccac
agtgagccca ggcagcaggt gtcaccgtcc cctacaggga gggccagatg cagtcactgc
ttcaggtcct gccagcacag agctgcctgc gtccagctcc ctgaatctct gctgctgctg
ctgctgctgc tgctgctgcc tgcggcccgg ggctgaaggc gccgtggccc tgcctgacgc
cccggagcct cctgcctgaa cttgggggct ggttggagat ggccttggag cagccaaggt
gcccctggca gtggcatccc gaaacgccct ggacgcaggg cccaagactg ggcacaggag
tgggaggtac atggggctgg ggactcccca ggagttatct gctccctgca ggcctagaga
agtttcaggg aaggtcagaa gagctcctgg ctgtggtggg cagggcagga aacccctccc
acctttacac atgcccaggc agcacctcag gccctttgtg gggcagggaa gctgaggcag
taagcgggca ggcagagctg gaggcctttc aggccagcca gcactctggc ctcctgccgc
cgcattccac cccagcccct cacaccactc gggagaggga catcctacgg tcccaaggtc
aggagggcag ggctggggtt gactcaggcc cctcccagct gtggccacct gggtgttggg
agggcagaag tgcaggcacc tagggccccc catgtgccca ccctgggagc tctccttgga
acccattcct gaaattattt aaaggggttg gccgggctcc caccagggcc tgggtgggaa
ggtacaggcg ttcccccggg gcctagtacc cccgcgtggc ctatccactc ctcacatcca
cacactgcac ccccactcct ggggcagggc caccagcatc caggcggcca gcaggcacct
gagtggctgg gacaagggat cccccttccc tgtggttcta ttatattata attataatta
aatatgagag catgct 24 Human PD-L1 amino acid sequence-isoform a
precursor (GenBank Accession No. NP_054862.1) MRIFAVFIFM TYWHLLNAFT
VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME DKNIIQFVHG EEDLKVQHSS
YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR
ILVVDPVTSE HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL FNVTSTLRIN
TTTNEIFYCT FRRLDPEENH TAELVIPELP LAHPPNERTH LVILGAILLC LGVALTFIFR
LRKGRMMDVK KCGIQDTNSK KQSDTHLEET 25 Human PD-L1 amino acid
sequence-isoform b precursor (GenBank Accession No. NP_001254635.1)
MRIFAVFIFM TYWHLLNAPY NKINQRILVV DPVTSEHELT CQAEGYPKAE VIWTSSDHQV
LSGKTTTTNS KREEKLFNVT STLRINTTTN EIFYCTFRRL DPEENHTAEL VIPELPLAHP
PNERTHLVIL GAILLCLGVA LTFIFRLRKG RMMDVKKCGI QDTNSKKQSD THLEET 26
Human DR5 amino acid sequence (GenBank Accession No. AAC01565.1)
MEQRGQNAPA ASGARKRHGP GPREARGARP GLRVPKTLVL VVAAVLLLVS AESALITQQD
LAPQQRVAPQ QKRSSPSEGL CPPGHHISED GRDCISCKYG QDYSTHWNDL LFCLRCTRCD
SGEVELSPCT TTRNTVCQCE EGTFREEDSP EMCRKCRTGC PRGMVKVGDC TPWSDIECVH
KESGIIIGVT VAAVVLIVAV FVCKSLLWKK VLPYLKGICS GGGGDPERVD RSSQRPGAED
NVLNEIVSIL QPTQVPEQEM EVQEPAEPTG VNMLSPGESE HLLEPAEAER SQRRRLLVPA
NEGDPTETLR QCFDDFADLV PFDSWEPLMR KLGLMDNEIK VAKAEAAGHR DTLYTMLIKW
VNKTGRDASV HTLLDALETL GERLAKQKIE DHLLSSGKFM YLEGNADSAM S
Sequence CWU 1
1
271123PRTHomo sapiens 1Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser1 5 10 15Ser Val Lys Val Ser Cys Lys Thr Ser Gly
Asp Thr Phe Ser Thr Tyr 20 25 30Ala Ile Ser Trp Val Arg Gln Ala Pro
Gly Gln Gly Leu Glu Trp Met 35 40 45Gly Gly Ile Ile Pro Ile Phe Gly
Lys Ala His Tyr Ala Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr
Ala Asp Glu Ser Thr Ser Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser
Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95Ala Arg Lys Phe
His Phe Val Ser Gly Ser Pro Phe Gly Met Asp Val 100 105 110Trp Gly
Gln Gly Thr Thr Val Thr Val Ser Ser 115 1202366DNAHomo sapiens
2caggtccagc tggtgcagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc
60tcctgcaaga cttctggaga caccttcagc acctatgcta tcagctgggt gcgacaggcc
120cctggacaag ggcttgagtg gatgggaggg atcatcccta tatttggtaa
agcacactac 180gcacagaagt tccagggcag agtcacgatt accgcggacg
aatccacgag cacagcctac 240atggagctga gcagcctgag atctgaggac
acggccgtgt atttttgtgc gagaaagttt 300cactttgttt cggggagccc
cttcggtatg gacgtctggg gccaagggac cacggtcacc 360gtctcc
3663106PRTHomo sapiens 3Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu
Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser
Gln Ser Val Ser Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly
Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp Ala Ser Asn Arg Ala Thr
Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe
Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala Val
Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Thr 85 90 95Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 1054318DNAHomo sapiens 4gaaattgtgt
tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60ctctcctgca
gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct
120ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg
catcccagcc 180aggttcagtg gcagtgggtc tgggacagac ttcactctca
ccatcagcag cctagagcct 240gaagattttg cagtttatta ctgtcagcag
cgtagcaact ggccgacgtt cggccaaggg 300accaaggtgg aaatcaaa
31855PRTHomo sapiens 5Thr Tyr Ala Ile Ser1 5616PRTHomo sapiens 6Gly
Ile Ile Pro Ile Phe Gly Lys Ala His Tyr Ala Gln Lys Phe Gln1 5 10
15714PRTHomo sapiens 7Lys Phe His Phe Val Ser Gly Ser Pro Phe Gly
Met Asp Val1 5 10811PRTHomo sapiens 8Arg Ala Ser Gln Ser Val Ser
Ser Tyr Leu Ala1 5 1097PRTHomo sapiens 9Asp Ala Ser Asn Arg Ala
Thr1 5108PRTHomo sapiens 10Gln Gln Arg Ser Asn Trp Pro Thr1
511440PRTHomo sapiens 11Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val
Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly
Ile Thr Phe Ser Asn Ser 20 25 30Gly Met His Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp Tyr Asp Gly Ser
Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ser Lys Asn Thr Leu Phe65 70 75 80Leu Gln Met Asn Ser
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Thr Asn Asp
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser 100 105 110Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser 115 120
125Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
130 135 140Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr145 150 155 160Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr 165 170 175Ser Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu Gly Thr Lys 180 185 190Thr Tyr Thr Cys Asn Val Asp
His Lys Pro Ser Asn Thr Lys Val Asp 195 200 205Lys Arg Val Glu Ser
Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala 210 215 220Pro Glu Phe
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro225 230 235
240Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
245 250 255Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp
Tyr Val 260 265 270Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln 275 280 285Phe Asn Ser Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln 290 295 300Asp Trp Leu Asn Gly Lys Glu Tyr
Lys Cys Lys Val Ser Asn Lys Gly305 310 315 320Leu Pro Ser Ser Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 325 330 335Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr 340 345 350Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 355 360
365Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
370 375 380Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
Leu Tyr385 390 395 400Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val Phe 405 410 415Ser Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys 420 425 430Ser Leu Ser Leu Ser Leu Gly
Lys 435 44012214PRTHomo sapiens 12Glu Ile Val Leu Thr Gln Ser Pro
Ala Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys
Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln
Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp Ala Ser Asn
Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp
Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg 85 90 95Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105
110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg
Gly Glu Cys 21013113PRTHomo sapiens 13Gln Val Gln Leu Val Glu Ser
Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Asp Cys
Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser 20 25 30Gly Met His Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Val Ile Trp
Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe65 70 75 80Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110Ser14339DNAHomo sapiens 14caggtgcagc tggtggagtc
tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60gactgtaaag cgtctggaat
caccttcagt aactctggca tgcactgggt ccgccaggct 120ccaggcaagg
ggctggagtg ggtggcagtt atttggtatg atggaagtaa aagatactat
180gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa
cacgctgttt 240ctgcaaatga acagcctgag agccgaggac acggctgtgt
attactgtgc gacaaacgac 300gactactggg gccagggaac cctggtcacc gtctcctca
33915107PRTHomo sapiens 15Glu Ile Val Leu Thr Gln Ser Pro Ala Thr
Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala
Ser Gln Ser Val Ser Ser Tyr 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45Tyr Asp Ala Ser Asn Arg Ala
Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp
Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro65 70 75 80Glu Asp Phe Ala
Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg 85 90 95Thr Phe Gly
Gln Gly Thr Lys Val Glu Ile Lys 100 10516321DNAHomo sapiens
16gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc
60ctctcctgca gggccagtca gagtgttagt agttacttag cctggtacca acagaaacct
120ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg
catcccagcc 180aggttcagtg gcagtgggtc tgggacagac ttcactctca
ccatcagcag cctagagcct 240gaagattttg cagtttatta ctgtcagcag
agtagcaact ggcctcggac gttcggccaa 300gggaccaagg tggaaatcaa a
321175PRTHomo sapiens 17Asn Ser Gly Met His1 51817PRTHomo sapiens
18Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val Lys1
5 10 15Gly194PRTHomo sapiens 19Asn Asp Asp Tyr12011PRTHomo sapiens
20Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala1 5 10217PRTHomo
sapiens 21Asp Ala Ser Asn Arg Ala Thr1 5229PRTHomo sapiens 22Gln
Gln Ser Ser Asn Trp Pro Arg Thr1 5232106DNAHomo sapiens
23agtttccctt ccgctcacct ccgcctgagc agtggagaag gcggcactct ggtggggctg
60ctccaggcat gcagatccca caggcgccct ggccagtcgt ctgggcggtg ctacaactgg
120gctggcggcc aggatggttc ttagactccc cagacaggcc ctggaacccc
cccaccttct 180tcccagccct gctcgtggtg accgaagggg acaacgccac
cttcacctgc agcttctcca 240acacatcgga gagcttcgtg ctaaactggt
accgcatgag ccccagcaac cagacggaca 300agctggccgc cttccccgag
gaccgcagcc agcccggcca ggactgccgc ttccgtgtca 360cacaactgcc
caacgggcgt gacttccaca tgagcgtggt cagggcccgg cgcaatgaca
420gcggcaccta cctctgtggg gccatctccc tggcccccaa ggcgcagatc
aaagagagcc 480tgcgggcaga gctcagggtg acagagagaa gggcagaagt
gcccacagcc caccccagcc 540cctcacccag gccagccggc cagttccaaa
ccctggtggt tggtgtcgtg ggcggcctgc 600tgggcagcct ggtgctgcta
gtctgggtcc tggccgtcat ctgctcccgg gccgcacgag 660ggacaatagg
agccaggcgc accggccagc ccctgaagga ggacccctca gccgtgcctg
720tgttctctgt ggactatggg gagctggatt tccagtggcg agagaagacc
ccggagcccc 780ccgtgccctg tgtccctgag cagacggagt atgccaccat
tgtctttcct agcggaatgg 840gcacctcatc ccccgcccgc aggggctcag
ccgacggccc tcggagtgcc cagccactga 900ggcctgagga tggacactgc
tcttggcccc tctgaccggc ttccttggcc accagtgttc 960tgcagaccct
ccaccatgag cccgggtcag cgcatttcct caggagaagc aggcagggtg
1020caggccattg caggccgtcc aggggctgag ctgcctgggg gcgaccgggg
ctccagcctg 1080cacctgcacc aggcacagcc ccaccacagg actcatgtct
caatgcccac agtgagccca 1140ggcagcaggt gtcaccgtcc cctacaggga
gggccagatg cagtcactgc ttcaggtcct 1200gccagcacag agctgcctgc
gtccagctcc ctgaatctct gctgctgctg ctgctgctgc 1260tgctgctgcc
tgcggcccgg ggctgaaggc gccgtggccc tgcctgacgc cccggagcct
1320cctgcctgaa cttgggggct ggttggagat ggccttggag cagccaaggt
gcccctggca 1380gtggcatccc gaaacgccct ggacgcaggg cccaagactg
ggcacaggag tgggaggtac 1440atggggctgg ggactcccca ggagttatct
gctccctgca ggcctagaga agtttcaggg 1500aaggtcagaa gagctcctgg
ctgtggtggg cagggcagga aacccctccc acctttacac 1560atgcccaggc
agcacctcag gccctttgtg gggcagggaa gctgaggcag taagcgggca
1620ggcagagctg gaggcctttc aggccagcca gcactctggc ctcctgccgc
cgcattccac 1680cccagcccct cacaccactc gggagaggga catcctacgg
tcccaaggtc aggagggcag 1740ggctggggtt gactcaggcc cctcccagct
gtggccacct gggtgttggg agggcagaag 1800tgcaggcacc tagggccccc
catgtgccca ccctgggagc tctccttgga acccattcct 1860gaaattattt
aaaggggttg gccgggctcc caccagggcc tgggtgggaa ggtacaggcg
1920ttcccccggg gcctagtacc cccgcgtggc ctatccactc ctcacatcca
cacactgcac 1980ccccactcct ggggcagggc caccagcatc caggcggcca
gcaggcacct gagtggctgg 2040gacaagggat cccccttccc tgtggttcta
ttatattata attataatta aatatgagag 2100catgct 210624290PRTHomo
sapiens 24Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His
Leu Leu1 5 10 15Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val
Val Glu Tyr 20 25 30Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val
Glu Lys Gln Leu 35 40 45Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met
Glu Asp Lys Asn Ile 50 55 60Ile Gln Phe Val His Gly Glu Glu Asp Leu
Lys Val Gln His Ser Ser65 70 75 80Tyr Arg Gln Arg Ala Arg Leu Leu
Lys Asp Gln Leu Ser Leu Gly Asn 85 90 95Ala Ala Leu Gln Ile Thr Asp
Val Lys Leu Gln Asp Ala Gly Val Tyr 100 105 110Arg Cys Met Ile Ser
Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val 115 120 125Lys Val Asn
Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val 130 135 140Asp
Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr145 150
155 160Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu
Ser 165 170 175Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys
Leu Phe Asn 180 185 190Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr
Asn Glu Ile Phe Tyr 195 200 205Cys Thr Phe Arg Arg Leu Asp Pro Glu
Glu Asn His Thr Ala Glu Leu 210 215 220Val Ile Pro Glu Leu Pro Leu
Ala His Pro Pro Asn Glu Arg Thr His225 230 235 240Leu Val Ile Leu
Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr 245 250 255Phe Ile
Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys 260 265
270Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu
275 280 285Glu Thr 29025176PRTHomo sapiens 25Met Arg Ile Phe Ala
Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu1 5 10 15Asn Ala Pro Tyr
Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro 20 25 30Val Thr Ser
Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys 35 40 45Ala Glu
Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys 50 55 60Thr
Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr65 70 75
80Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr
85 90 95Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val
Ile 100 105 110Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr
His Leu Val 115 120 125Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val
Ala Leu Thr Phe Ile 130 135 140Phe Arg Leu Arg Lys Gly Arg Met Met
Asp Val Lys Lys Cys Gly Ile145 150 155 160Gln Asp Thr Asn Ser Lys
Lys Gln Ser Asp Thr His Leu Glu Glu Thr 165 170 17526411PRTHomo
sapiens 26Met Glu Gln Arg Gly Gln Asn Ala Pro Ala Ala Ser Gly Ala
Arg Lys1 5 10 15Arg His Gly Pro Gly Pro Arg Glu Ala Arg Gly Ala Arg
Pro Gly Leu 20 25 30Arg Val Pro Lys Thr Leu Val Leu Val Val Ala Ala
Val Leu Leu Leu 35 40 45Val Ser Ala Glu Ser Ala Leu Ile Thr Gln Gln
Asp Leu Ala Pro Gln 50 55 60Gln Arg Val Ala Pro Gln Gln Lys Arg Ser
Ser Pro Ser Glu Gly Leu65 70 75 80Cys Pro Pro Gly His His Ile Ser
Glu Asp Gly Arg Asp Cys Ile Ser 85 90 95Cys Lys Tyr Gly Gln Asp Tyr
Ser Thr His Trp Asn Asp Leu Leu Phe 100 105 110Cys Leu Arg Cys Thr
Arg Cys Asp Ser Gly Glu Val Glu Leu Ser Pro 115
120 125Cys Thr Thr Thr Arg Asn Thr Val Cys Gln Cys Glu Glu Gly Thr
Phe 130 135 140Arg Glu Glu Asp Ser Pro Glu Met Cys Arg Lys Cys Arg
Thr Gly Cys145 150 155 160Pro Arg Gly Met Val Lys Val Gly Asp Cys
Thr Pro Trp Ser Asp Ile 165 170 175Glu Cys Val His Lys Glu Ser Gly
Ile Ile Ile Gly Val Thr Val Ala 180 185 190Ala Val Val Leu Ile Val
Ala Val Phe Val Cys Lys Ser Leu Leu Trp 195 200 205Lys Lys Val Leu
Pro Tyr Leu Lys Gly Ile Cys Ser Gly Gly Gly Gly 210 215 220Asp Pro
Glu Arg Val Asp Arg Ser Ser Gln Arg Pro Gly Ala Glu Asp225 230 235
240Asn Val Leu Asn Glu Ile Val Ser Ile Leu Gln Pro Thr Gln Val Pro
245 250 255Glu Gln Glu Met Glu Val Gln Glu Pro Ala Glu Pro Thr Gly
Val Asn 260 265 270Met Leu Ser Pro Gly Glu Ser Glu His Leu Leu Glu
Pro Ala Glu Ala 275 280 285Glu Arg Ser Gln Arg Arg Arg Leu Leu Val
Pro Ala Asn Glu Gly Asp 290 295 300Pro Thr Glu Thr Leu Arg Gln Cys
Phe Asp Asp Phe Ala Asp Leu Val305 310 315 320Pro Phe Asp Ser Trp
Glu Pro Leu Met Arg Lys Leu Gly Leu Met Asp 325 330 335Asn Glu Ile
Lys Val Ala Lys Ala Glu Ala Ala Gly His Arg Asp Thr 340 345 350Leu
Tyr Thr Met Leu Ile Lys Trp Val Asn Lys Thr Gly Arg Asp Ala 355 360
365Ser Val His Thr Leu Leu Asp Ala Leu Glu Thr Leu Gly Glu Arg Leu
370 375 380Ala Lys Gln Lys Ile Glu Asp His Leu Leu Ser Ser Gly Lys
Phe Met385 390 395 400Tyr Leu Glu Gly Asn Ala Asp Ser Ala Met Ser
405 410276PRTUnknownsource/note="Description of Unknown CTLA-4
motif peptide" 27Met Tyr Pro Pro Pro Tyr1 5
* * * * *