U.S. patent application number 13/312201 was filed with the patent office on 2013-09-05 for compositions of pd-1 antagonists and methods of use.
This patent application is currently assigned to Amplimmune, Inc.. The applicant listed for this patent is Solomon Langermann, Linda Liu. Invention is credited to Solomon Langermann, Linda Liu.
Application Number | 20130230514 13/312201 |
Document ID | / |
Family ID | 41725778 |
Filed Date | 2013-09-05 |
United States Patent
Application |
20130230514 |
Kind Code |
A1 |
Langermann; Solomon ; et
al. |
September 5, 2013 |
COMPOSITIONS OF PD-1 ANTAGONISTS AND METHODS OF USE
Abstract
Methods of treating cancer and infectious diseases utilizing a
treatment regimen comprising administering a compound that reduces
inhibitory signal transduction in T cells, in combination with a
potentiating agent, such as cyclophosphamide, to produce potent T
cell mediated responses, are described. Compositions comprising the
PD-1 antagonists and potentiating agents useful in the methods of
the invention are also disclosed.
Inventors: |
Langermann; Solomon;
(Baltimore, MD) ; Liu; Linda; (Clarksville,
MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Langermann; Solomon
Liu; Linda |
Baltimore
Clarksville |
MD
MD |
US
US |
|
|
Assignee: |
Amplimmune, Inc.
|
Family ID: |
41725778 |
Appl. No.: |
13/312201 |
Filed: |
December 6, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12547129 |
Aug 25, 2009 |
8114845 |
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13312201 |
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61211697 |
Apr 2, 2009 |
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61091694 |
Aug 25, 2008 |
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61091709 |
Aug 25, 2008 |
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61091705 |
Aug 25, 2008 |
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Current U.S.
Class: |
424/134.1 |
Current CPC
Class: |
A61K 45/06 20130101;
A61P 33/06 20180101; A61P 33/02 20180101; A61K 31/675 20130101;
A61P 35/00 20180101; Y02A 50/48 20180101; A61P 31/22 20180101; C07K
2319/33 20130101; Y02A 50/409 20180101; A61P 31/10 20180101; Y02A
50/402 20180101; Y02A 50/30 20180101; Y02A 50/411 20180101; A61K
38/177 20130101; Y02A 50/478 20180101; A61P 31/20 20180101; A61P
35/02 20180101; A61P 37/04 20180101; C07K 14/70532 20130101; Y02A
50/475 20180101; A61P 31/14 20180101; A61P 31/16 20180101; A61P
31/18 20180101; A61P 31/00 20180101; Y02A 50/423 20180101; A61P
43/00 20180101; A61P 31/04 20180101; A61P 31/12 20180101; A61P
33/00 20180101; A61K 31/675 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/134.1 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61K 45/06 20060101 A61K045/06; A61K 31/675 20060101
A61K031/675 |
Claims
1. A therapeutic composition comprising a compound comprising a
fusion protein comprising first and second peptide portions wherein
said first peptide portion consists of an amino acid sequence
selected from: wild type B7-DC, an amino acid sequence having at
least 98% sequence identity to amino acids 20-221 or 20-121 of SEQ
ID NO: 1 and which competes in vitro with wild-type B7-DC for
binding to PD-1, a fragment of B7-DC which competes in vitro with
wild-type B7-DC for binding to PD-1, and an extracellular domain of
B7-DC and said second peptide portion comprises a portion of an
immunoglobulin (Ig) and a potentiating agent selected from the
group of: cyclophosphamide, an analog of cyclophosphamide,
sunitinib, anti-TGNF.beta., imatinib, anthracyclines, oxaliplatin,
and doxorubicin.
2. The composition of claim 1 wherein said first peptide portion
consists of a wild type B7-DC polypeptide.
3. The composition of claim 1, wherein said B7-DC is a human
B7-DC.
4. The composition of claim 1, wherein said first peptide portion
consists of a fragment of B7-DC which does not comprise any portion
of the transmembrane portion of said B7-DC polypeptide.
5. The composition of claim 1, wherein said first peptide portion
consists of the soluble portion of said B7-DC polypeptide and said
second peptide portion comprises the Fc region of an antibody but
does not comprise any of the variable region of said antibody.
6. The composition of claim 1, wherein said first peptide portion
consists of the amino acid sequence of SEQ ID NO: 3 and said second
polypeptide portion comprises the Fc region of an antibody but does
not comprise any of the variable region of said antibody.
7. The composition of claim 1, wherein said first peptide portion
consists of an amino acid sequence having at least 98% sequence
identity to amino acids 20-221 or 20-121 of SEQ ID NO: 1.
8. The composition of claim 1, wherein said first peptide portion
consists of the amino acid sequence of amino acids 20-221 or 20-121
of SEQ ID NO:
9. The composition of claim 1, wherein said fusion protein
comprises an amino acid sequence having at least 95% identity to
the sequence of SEQ ID NO: 9, 10, 12 or 13.
10. The composition of claim 1, wherein said fusion protein
comprises the amino acid sequence of SEQ ID NO: 9, 10, 12, or
13.
11. The composition of claim 1, wherein said fusion protein is a
monomer.
12. The composition of claim 1, wherein said fusion protein is part
of a dimer.
13. The composition of claim 12, wherein said dimer is a
homodimer.
14. The composition of claim 12, wherein said dimer is a
heterodimer.
15. The composition of claim 1, wherein said fusion protein
comprises a first peptide portion which consists of 20-221 of SEQ
ID NO: 1 and a second peptide portion which consists of the hinge,
C.sub.H2 and C.sub.H3 regions of a human immunoglobulin
C.gamma.1.
16. The composition of claim 1, wherein said second peptide portion
comprises amino acids 245-476 of human IgG1.
17. The composition of claim 1, wherein said potentiating agent is
cyclophosphamide or an analog of cyclophosphamide.
18. The composition of claim 1, said compound and said potentiating
agent are provided as separate medicaments.
19. The composition of claim 1, wherein said first peptide portion
consists of the extracellular domain of B7-DC or a polypeptide
differing therefrom by only conservative amino acid
substitutions.
20. The composition of claim 1, further comprising at least one
additional agent selected from the group consisting of an anti-PD-1
antibody, an anti-CTLA4 antibody, a mitosis inhibitor, an aromatase
inhibitor, an A2AR antagonist, and an angiogenesis inhibitor.
21. The therapeutic composition of claim 1 further comprising a
pharmaceutically acceptable carrier.
22. A kit comprising (a) a dosage supply of a compound according to
claim 1, and (b) a supply of a potentiating agent.
23. The kit of claim 22 further comprising (c) a supply of
pharmaceutically acceptable carrier; and (d) printed instructions
for administering said compound and said potentiating agent.
24. The kit of claim 22, wherein said potentiating agent is
cyclophosphamide or an analog of cyclophosphamide.
25. The kit of claim 24 wherein said cyclophosphamide or an analog
of cyclophosphamide is provided in an amount of about 0.45 mg to
about 4.5 mg.
26. A composition comprising: a pharmaceutical composition
comprising a fusion protein comprising a first and second peptide
portion wherein said first peptide portion consists of amino acids
20-221 of SEQ ID NO: 1 and said second peptide portion comprises
amino acids 245-476 of human IgG1 and a pharmaceutical carrier; and
cyclophosphamide.
27. A method of treating cancer in a human comprising administering
a therapeutically effective amount of a composition of claim 26 to
said human.
Description
[0001] This application is a divisional application of U.S. Ser.
No. 12/547,129 filed August Aug. 25, 2009 which claims priority of
U.S. Provisional Application Ser. No. 61/211,697, filed 2 Apr.
2009, 61/091,694, filed 25 Aug. 2008, 61/091,709 filed 25 Aug.
2008, and 61/091,705 filed 25 Aug. 2008, the disclosures of which
are hereby incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to therapeutic compositions
containing a compound that prevents inhibitory signal transduction
on T cells in combination with potentiating agents and the use of
said components together or separately for the induction of T cell
responses valuable in disease therapy.
BACKGROUND OF THE INVENTION
[0003] The response of T lymphocytes to disease states, such as
infection and chronic diseases like cancer, is complicated and
involves intercellular interactions and the production of soluble
mediators (called cytokines or lymphokines). Activation of T cells
normally depends on an antigen-specific signal following contact of
the T cell receptor (TCR) with an antigenic peptide presented via
the major histocompatibility complex (MHC) while the extent of this
reaction is controlled by positive and negative antigen-independent
signals eminating from a variety of co-stimulatory molecules. The
latter are commonly members of the CD28/B7 family. Conversely,
Programmed Death-1 (PD-1) is a member of the CD28 family of
receptors that delivers a negative immune response when induced on
T cells. Contact between PD-1 and one of its ligands (B7-H1 or
B7-DC) induces an inhibitory response that decreases T cell
multiplication and/or the strength and/or duration of a T cell
response.
[0004] Thus, the T lymphocyte response is regulated by various
factors, including cell surface molecules that act as receptors,
where the latter include both the TCR complex as well as other
surface molecules.
[0005] In sum, an antigen specific T cell response is mediated by
two signals: 1) engagement of the TCR with antigenic peptide
presented in the context of HC (signal 1), and 2) a second
antigen-independent signal delivered by contact between different
receptor/ligand pairs (signal 2). This "second signal" is critical
in determining the type of T cell response (activation vs
tolerance) as well as the strength and duration of that response,
and is regulated by both positive and negative signals from
costimulatory molecules, such as the B7 family of proteins.
[0006] The most extensively characterized T cell costimulatory
pathway is B7-CD28, in which B7-1 (CD80) and B7-2 (CD86) each can
engage the stimulatory CD28 receptor and the inhibitory CTLA-4
(CD152) receptor. In conjunction with signaling through the T cell
receptor, CD28 ligation increases antigen-specific proliferation of
T cells, enhances production of cytokines, stimulates
differentiation and effector function, and promotes survival of T
cells (Lenshow, et al., Annu. Rev. Immunol., 14:233-258 (1996);
Chambers and Allison, Curr. Opin. Immunol., 9:396-404 (1997); and
Rathmell and Thompson, Annu. Rev. Immunol., 17:781-828 (1999)). In
contrast, signaling through CTLA-4 is thought to deliver a negative
signal that inhibits T cell proliferation, IL-2 production, and
cell cycle progression (Krummel and Allison, J. Exp. Med.,
183:2533-2540 (1996); and Walunas, et al., J. Exp. Med.,
183:2541-2550 (1996)). Other members of the B7 family of
costimulatory molecules include B7-H1 (Dong, et al., Nature Med.,
5:1365-1369 (1999); and Freeman, et al., J. Exp. Med., 192:1-9
(2000)), B7-DC (Tseng, et al., J. Exp. Med., 193:839-846 (2001);
and Latchman, et al., Nature Immunol., 2:261-268 (2001)), B7-H2
(Wang, et al., Blood, 96:2808-2813 (2000); Swallow, et al.,
Immunity, 11:423-432 (1999); and Yoshinaga, et al., Nature,
402:827-832 (1999)), B7-H3 (Chapoval, et al., Nature Immunol.,
2:269-274 (2001)) and B7-H4 (Choi, et al., J. Immunol.,
171:4650-4654 (2003); Sica, et al., Immunity, 18:849-861 (2003);
Prasad, et al., Immunity, 18:863-873 (2003); and Zang, et al.,
Proc. Natl. Acad. Sci. U.S.A., 100:10388-10392 (2003)). B7-H5
(described in WO 2006/012232) is a newly discovered member of the
B7 family.
[0007] B7 family molecules have a membrane proximal IgC (constant)
domain and a membrane distal IgV (variable) domain. The CD28-like
family of receptors for these ligands share a common extracellular
IgV-like domain. Interactions of receptor-ligand pairs are mediated
predominantly through residues in the IgV domains of the ligands
and receptors (Schwartz, et al., Nature Immunol., 3:427-434
(2002)). In general, IgV domains are described as having two sheets
that each contains a layer of .beta.-strands (Williams and Barclay,
Annu. Rev. Immunol., 6:381-405 (1988)). The front and back sheets
of CTLA-4 contain strands A'GFC'C and ABEDC, respectively (Ostrov,
et al., Science, 290:816-819 (2000)), whereas the front and back
sheets of the B7 IgV domains are composed of strands AGFCC'C'' and
BED, respectively (Schwartz, et al., Nature, 410:604-608 (2001);
Stamper, et al., Nature, 410:608-611 (2001); and Ikemizu, et al.,
Immunity, 12:51-60 (2000)). Crystallographic analysis revealed that
the CTLA-4/B7 binding interface is dominated by the interaction of
the CDR3-analogous loop from CTLA-4, composed of a MYPPPY motif,
with a surface on B7 formed predominately by the G, F, C, C' and
C'' strands (Schwartz, et al., Nature, 410:604-608 (2001); and
Stamper, et al., Nature, 410:608-611 (2001)). Data from amino acid
homologies, mutation, and computer modeling provide support for the
concept that this motif also is a major B7-binding site for CD28
(Bajorath, et al., J. Mol. Graph. Model., 15:135-139 (1997)).
Although the MYPPPY motif is not conserved in ICOS, the receptor
for B7-H2, studies have indicated that a related motif having the
sequence FDPPPF and located at the analogous position is a major
determinant for binding of ICOS to B7-H2 (Wand, et al., J. Exp.
Med., 195:1033-1041 (2002)).
[0008] B7-DC (also called PD-L2 or CD273) is a relatively new
member of the B7 family, and has an amino acid sequence that is
about 34% identical to B7-H1 (also called PD-L1). Human and mouse
B7-DC orthologues share about 70% amino acid identity. While B7-H1
and B7-DC transcripts are found in various tissues (Dong, et al.,
Nature Med., 5:1365-1369 (1999); Latchman, et al., Nature Immunol.,
2:261-268 (2001); and Tamura, Blood, 97:1809-1816 (2001)), the
expression profiles of the proteins are quite distinct. B7-H1 is
broadly expressed on a wide variety of tissue and cell types, while
B7-DC expression is predominantly restricted to activated dendritic
cells (DC) and macrophages.
[0009] It has been shown that both B7-H1 and B7-DC bind to PD-1
(Freeman, et al., J. Exp. Med., 192:1027-1034 (2000)), a distant
member of the CD28 family with an immunoreceptor tyrosine-based
inhibitory motif (ITIM) in its cytoplasmic domain (Ishida, et al.,
EMBO J., 11:3887-3895 (1992)). PD-1, a member of the CD28 family of
receptors, is inducibly expressed on activated T cells, B cells,
natural killer (NK) cells, monocytes, DC, and macrophages (Keir, et
al Curr. Opin. Immunol. 19:309-314 (2007)).
[0010] The primary result of PD-1 ligation by its ligands is to
inhibit signaling downstream of the T cell Receptor (TCR).
Therefore, signal transduction via PD-1 usually provides a
suppressive or inhibitory signal to the T cell that results in
decreased T cell proliferation or other reduction in T cell
activation. B7-H1 is the predominant PD-1 ligand causing inhibitory
signal transduction in T cells. The present invention solves the
problem of undesired T cell inhibition by providing agents that
bind to PD-1 and thus prevent inhibitory signal transduction, or
else bind to ligands of PD-1 such as B7-H1, thereby preventing the
ligand from binding to PD-1 to deliver an inhibitory signal. In
either case, T cell responses, such as T cell proliferation or
activation, are stimulated.
[0011] B7-H1 is the predominant PD-1 ligand, likely due to its
broader distribution and higher expression levels. PD-1 inhibition
occurs only when PD-1 and TCR are ligated in close proximity to
each other, in the context of the immune synapse. PD-1 and its
ligands have been the topic of several review articles.
[0012] B7-H1 is also over expressed in many cancers (including
breast cancer, colon cancer, esophageal cancer, gastric cancer,
glioma, leukemia, lung cancer, melanoma, multiple myeloma, ovarian
cancer, pancreatic cancer, renal cell carcinoma, and urothelial
cancer), and has been linked to poor prognosis. B7-H1 is expressed
by many tumor cell lines, especially following stimulation with
interferon gamma (IFN-.gamma.), and is also upregulated on tumor
infiltrating myeloid derived suppressor cells (MDSC). For example,
PD-1 is up-regulated on tumor specific CD8 T cells and is
associated with functional impairment, anergy, exhaustion, and
apoptosis. PD-1 upregulation has also been associated with
dysfunctional and/or suppressive phenotypes on additional cell
types, such as regulatory T cells (Treg) and natural killer T (NKT)
cells.
[0013] The present invention makes use of such molecular functions
by providing treatment regimens for treating diseases through
increased T cell activity, especially cancer and infectious
diseases.
BRIEF SUMMARY OF THE INVENTION
[0014] In one aspect, the present invention relates to a method of
increasing T cell responses, for example, to an antigen, in a
mammal in need of such increase, comprising administering to said
mammal a compound that reduces inhibitory signal transduction in
immune cells, especially T cells, and a potentiating agent, wherein
said treatment regimen is effective to increase the T cell response
of said mammal.
[0015] Compounds useful in the treatment regimen of the invention
include those that bind to and block PD-1 receptors on T cells
without triggering inhibitory signal transduction, compounds that
bind to PD-1 ligands to prevent their binding to PD-1, compounds
that do both and compounds that prevent expression of genes that
encode either PD-1 or natural ligands of PD-1. Such compounds are
referred to herein as "PD-1 antagonists." Compounds that bind to
natural ligands of PD-1 include PD-1 itself, as well as active
fragments of PD-1, and in the case of the B7-H1 ligand, B7.1
proteins and fragments. Such antagonists include proteins,
antibodies, anti-sense molecules and small organics. In a preferred
embodiment, said T cell response is greater than that produced by
either of said PD-1 antagonist or said potentiating agent when
either is administered without the other.
[0016] In another embodiment, compounds useful in the methods of
the invention are those that bind to T cell surface molecules such
as CTLA4 to prevent the inhibitory signals triggered by binding of
natural ligands thereof or that bind to said natural ligands. Such
antagonists include proteins, antibodies, anti-sense molecules and
small organics.
[0017] In a general embodiment, compounds useful in treatment
regimens and compositions of the present invention include those
that bind to PD-1 without triggering, inducing, increasing,
facilitating and/or permitting co-ligation of PD-1 with TCR.
[0018] Preferred compounds that prevent inhibitory signal
transduction through PD-1 and thus act as PD-1 antagonists include,
but are not limited to, B7-DC polypeptides, especially soluble
portions of these, including active fragments of these, variants
and homologs of these, as well as fusion proteins incorporating any
of the foregoing, that bind to PD-1 without triggering inhibitory
signal transduction. In preferred embodiments, B7-DC comprises the
amino acid sequence of SEQ ID NO: 1, 2, 3 or 4. Preferred such
compounds are those incorporating the soluble domain of B7-DC
(i.e., without transmembrane sequence). Suitable fragments of B7-DC
polypeptides include fragments containing the IgV and/or IgC
domains or fragments containing only the IgV domain, with the
latter being a preferred embodiment, with amino acids 20-121 of SEQ
ID NO: 1 being a preferred example of an IgV domain.
[0019] Preferred PD-1 antagonists also include, but are not limited
to, active fragments of natural ligands of PD-1, such as B7-H1
polypeptides (disclosed in U.S. Pat. No. 6,803,192, incorporated by
reference herein in its entirety), especially soluble portions of
these, including variants and homologs of these, as well as fusion
proteins incorporating any of the foregoing, that bind to PD-1
without triggering inhibitory signal transduction.
[0020] Preferred compounds of the invention also include, but are
not limited to, compounds, including active fragments, variants and
homologs, that bind to natural ligands of PD-1, such as fragments
of B7-1 that bind to B7-H1, as well as fusion proteins
incorporating any of the foregoing, that bind to ligands of PD-1 to
prevent the latter from binding to PD-1 to trigger inhibitory
signal transduction.
[0021] In another embodiment, the compositions and methods of use
thereof, include a combination of a PD-1 receptor antagonist that
binds to and blocks the PD-1 receptor, and a separate PD-1 receptor
antagonist that binds to and blocks PD-1 receptor ligands. Another
embodiment of the present invention provides PD-1 receptor
antagonists that bind to the PD-1 receptor without triggering
inhibitory signal transduction through the PD-1 receptor and also
have the ability to bind and antagonize PD-1 receptor ligands, such
as B7-H1, that would otherwise trigger inhibitory signal
transduction through the PD-1 receptor. Other contemplated PD-1
receptor antagonists include bi-specific antibodies that can bind
both the PD-1 receptor and PD-1 receptor ligands.
[0022] Preferred embodiments of compounds useful in the present
invention also include antibodies that bind to PD-1 or CTLA4,
thereby reducing, or abolishing, inhibitory signal transduction
mediated by these sources.
[0023] Preferred compounds for use in the methods of the invention
also include, but are not limited to, active fragments of ligands
of CTLA4 (such as B7-1 and B7-2) that bind to CTLA4 to reduce
subsequent inhibitory signals yet do not bind to CD28 or otherwise
inhibit positive signal transduction by CD28.
[0024] Preferred compounds that prevent inhibitory signal
transduction through PD-1 and thus act as PD-1 antagonists include,
but are not limited to, B7-DC antagonists, especially soluble
portions of these, including active fragments of these, variants
and homologs of these, as well as fusion proteins incorporating any
of the foregoing, that bind to B7-DC.
[0025] In one embodiment, B7-DC polypeptides, fragments or variants
thereof are coupled to other polypeptides to form fusion proteins
that antagonize the PD-1 receptor by binding to the PD-1 receptor
without causing inhibitory signal transduction through PD-1,
thereby reducing, or interfering with, ligand binding to PD-1,
particularly B7-H1 binding, and thereby interfering with inhibitory
signal transduction through the PD-1 receptor. Examples of such
fusion proteins are polypeptides comprising the amino acid sequence
of SEQ ID NO: 9, 10, 12 or 13, as well as homologs thereof. In one
preferred embodiment, all or a portion of the extracellular domain
(ECD) of B7-DC is part of a fusion protein wherein it is linked to
a second polypeptide containing an Fc portion of an immunoglobulin.
A preferred example of this is B7-DC-Ig, especially where this
structure is part of a homodimer wherein two B7-DC-Ig molecules are
linked to each other, such as by a disulfide linkage.
[0026] In specific embodiments, fragments useful in the compounds
of the invention consist of at least 10, 15, 25, 50, 75, 100, 150,
200 or more contiguous amino acids of a polypeptide having the
desired antagonist activity. Such fragments are also commonly part
of fusion proteins for use in the invention.
[0027] In another aspect, the present invention relates to a method
of increasing T cell responses in a mammal in need thereof,
comprising administering to said mammal an effective treatment
regimen comprising an anti-PD-1 antibody and a potentiating agent,
wherein said treatment regimen is effective to increase the T cell
response of said mammal.
[0028] In another aspect, the present invention relates to a method
of increasing T cell responses in a mammal in need thereof,
comprising administering to said mammal an effective treatment
regimen comprising an immunomodulator, and a potentiating agent,
wherein said treatment regimen is effective to increase the T cell
response of said mammal. Such immunomodulators include molecules
that antagonize other CD28 family receptors (such as CTLA4) that
inhibit T cell responses. A preferred embodiment uses an anti-CTLA4
antibody and a potentiating agent. Additional immunomodulators
include: molecules that agonize CD28 family receptors (such as CD28
and ICOS) that activate T cell responses; molecules that antagonize
B7 family ligands (such as B7-H1, B7-DC, B7-H4) that inhibit T cell
responses; and molecules that agonize B7 family ligands (such as
B7.1 and B7.2) that activate T cell responses.
[0029] In additional embodiments of any of the methods of the
invention, the treatment regimen of a PD-1 antagonist compound and
a potentiating agent further comprises at least one additional
therapeutic agent. Additional therapeutic agents contemplated
include immunomodulatory agents. Exemplary immunomodulating agents
for such methods include anti-PD-1 and anti-CTLA4 antibodies.
[0030] In one embodiment, the potentiating agent is selected from
cyclophosphamide and analogs of cyclophosphamide, Sunitinib
(Sutent), anti-TGF.beta. and Imatinib (Gleevac), a mitosis
inhibitor, such as paclitaxel, an aromatase inhibitor, such as
letrozole, an A2a adenosine receptor (A2AR) antagonist, an
angiogenesis inhibitor, anthracyclines, oxaliplatin, doxorubicin,
TLR4 antagonists, and IL-18 antagonists. Some of these agents
reduce the number of Tregs (i.e., regulatory T lymphocytes or
T-regs) within the tumor microenvironment.
[0031] In another embodiment, the methods and/or compositions of
the invention specifically contemplate use of any suitable adjuvant
as part of said method and/or composition.
[0032] In accordance with the invention, T cells can be contacted
with PD-1 receptor antagonist and/or compositions thereof
containing a potentiating agent in vitro, ex vivo or in vivo.
Contacting T cells using PD-1 receptor antagonists and/or
compositions thereof containing a potentiating agent can occur
before, during or after activation of the T cell.
[0033] In a specific embodiment, a molecule that prevents or
reduces inhibitory signal transduction through PD-1 and the
potentiating agent are administered at different times, such as
where the potentiating agent is administered prior to administering
the PD-1 antagonist. Such administration may be in conjunction with
an additional therapeutic agent.
[0034] In specific embodiments of any of the methods of the
invention, the treatment regimen includes administration of the
potentiating agent at least 1 hour, or at least 2 hours, or at
least 3 hours, or at least 5 hours, or at least 10 hours, or at
least 15 hours, or at least 20 hours, or at least 24 hours, or at
least 30 hours or even longer before administering any or all of
the PD-1 antagonist, the anti-PD-1 antibody, the anti-CTLA4
antibody, and/or additional therapeutic agents. Administration of
the potentiating agent may also occur after administering any or
all of the PD-1 antagonist, the anti-PD-1 antibody, the anti-CTLA4
antibody and/or additional therapeutic agents, such as no more than
1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 15 hours, 20 hours, 24
hours, or even up to 30 hours after administering a PD-1
antagonist, or may occur in conjunction with administering the PD-1
antagonist.
[0035] The increased T cell response achieved as a result of the
methods of the invention is sufficient to treat a disease,
including one or more of cancer, viral infection, bacterial
infection and parasitic infection. Where the disease is cancer,
such cancer is any one or more of bladder, brain, breast, cervical,
colo-rectal, esophageal, kidney, liver, lung, nasopharangeal,
pancreatic, prostate, skin, stomach, uterine, ovarian, testicular,
or hematologic cancer.
[0036] In another aspect, the present invention includes
compositions of the antagonists used in the methods of the
invention, in a pharmaceutically acceptable carrier and wherein
said PD-1 binding molecule and said potentiating agent are each
present in an amount effective to produce increased T cell
stimulation.
[0037] In one preferred embodiment, the invention includes medical
kits comprising containers holding one or more of the agents for
use in the invention together with pharmaceutical carriers for
dilution thereof and instructions for administration. In addition,
both of said PD-1 receptor antagonist and potentiating agent may be
present as components in a single container, in a pharmaceutically
acceptable carrier, when said components are to be administered at
the same time.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1 shows that B7-DC-Ig binds to PD-1. Labeled B7-DC-Ig
was incubated at various concentrations with a CHO cell line
constitutively expressing PD-1 or parent CHO cells that do not
express PD-1. Binding was analyzed by flow cytometry. The median
fluorescence intensity (MFI) of B7-DC-Ig (y-axis) is shown as a
function of the concentration of probe (x-axis). B7-DC-Ig binds to
CHO.PD-1 cells (solid circle) but not untransfected CHO cells (gray
triangle).
[0039] FIG. 2 shows that B7-DC-Ig competes with B7-H1 for binding
to PD-1. Unlabeled B7-DC-Ig at various concentrations was first
incubated with a CHO cell line constitutively expressing PD-1
before adding labeled B7-H1-Ig to the cell mixture. The median
fluorescence intensity (MFI) of B7-H1-Ig (y-axis) is shown as a
function of the concentration of unlabeled B7-DC-Ig competitor
(x-axis) added. As the concentration of unlabeled B7-DC-Ig is
increased the amount of labeled B7-H1-Ig bound to CHO cells
decreases, demonstrating that B7-DC-Ig competes with B7-H1 for
binding to PD-1.
[0040] FIG. 3 shows the results of experiments wherein the
combination of cyclophosphamide (CTX or Cytoxan.RTM.) and dimeric
murine B7-DC-Ig resulted in eradication of established CT26 tumors
(colon carcinoma) in mice. Graph A shows tumor volume (mm.sup.3)
versus days post tumor challenge in mice treated with 100 mg/kg of
CTX on Day 10 while Graph B shows tumor volume (mm.sup.3) versus
days post tumor challenge in mice treated with CTX on Day 10
followed a day later by the first B7-DC-Ig administration. Each
line in each graph represents one mouse. Black arrow stands for
B7-DC-Ig administration. Graph C shows average tumor volume.
[0041] FIG. 4 shows the results of experiments wherein the
combination of CTX and dimeric murine B7-DC-Ig eradicated
established CT26 tumors (colon carcinoma) in mice and protected
against re-challenge with CT26. Mice that were treated with CTX and
B7-DC-Ig and found to be free of tumor growth on day 44 following
tumor inoculation were rechallenged with tumors. The mice were
later rechallenged again on on Day 70. None of the mice displayed
tumor growth by day 100.
[0042] FIG. 5 shows CTX and B7-DC-Ig treatment resulted in
generation of tumor specific memory CTL. Mice eradicated
established CT26 subcutenous tumors post CTX and B7-DC-Ig treatment
were re-challenged with CT26 cells. Seven days later, splenocytes
were isolated and pulsed with either ovalbumin, an irrelevant
peptide, or AH1, a CT26 specific peptide. Cells were stained with
anti-CD8 antibody first followed by intracellular staining with
anti-IFN.gamma. antibody prior to FACS analysis.
[0043] FIG. 6 shows the effects of different doses of B7-DC-Ig in
combination with CTX on the eradication of established CT26 tumors
in mice. Balb/C mice at age of 9 to 11 weeks were implanted
subcutaneously with 1E05 CT26 cells. On Day 9, mice were injected
IP with 100 mg/kg of CTX. Twenty four hours later, on Day 10, mice
were treated with 30, 100, or 300 ug of B7-DC-Ig followed by 2
injections every week up to total 8 treatments. Tumor growth was
measured two times per week.
[0044] FIG. 7 shows the results of experiments wherein the
combination of CTX and anti-PD-1 antibody resulted in eradication
of established CT26 tumors (colon carcinoma) in mice. Graph A shows
tumor volume (mm.sup.3) versus days post tumor challenge in
untreated mice (i.e., mice treated with vehicle alone), Graph B
shows tumor volume (mm.sup.3) versus days post tumor challenge in
mice treated with anti-PD-1 alone starting on Day 11 at 300 .mu.g
per injection, 3 times per week, up to 12 injections and Graph C
shows tumor volume (mm.sup.3) versus days post tumor challenge in
mice treated with CTX on Day 11 and the first anti-PD-1
administration on Day 12 at 300 .mu.g per injection, 3 times per
week, up to 12 injections. Each line in each graph represents one
mouse. Black arrow stands for anti-PD-1 administration.
[0045] FIG. 8 shows the results of experiments wherein the
combination of CTX and anti-CTLA4 antibody resulted in eradication
of established CT26 tumors (colon carcinoma) in mice. Here, Graph A
shows tumor volume (mm.sup.3) versus days post tumor challenge in
mice treated with 100 mg/kg of CTX on Day 11 while Graph B shows
tumor volume (mm.sup.3) versus days post tumor challenge in mice
treated with CTX on Day 11 and anti-CTLA4 on Day 12 at 100 .mu.g
per injection, 2 times per week, up to 8 injections. Each line in
each graph represents one mouse. Black arrow stands for anti-CTLA-4
administration.
[0046] FIG. 9 shows the results of experiments wherein Balb/C mice
at age of 9 to 11 weeks of age were implanted with 1.times.10.sup.5
CT26 cells subcutaneously. On Day 9, mice were injected with 100
mg/kg of CTX, IP. Twenty four hours later, on Day 10, mice were
treated with 100 ug of B7-DC-Ig. There were 5 groups: naive mice
that did not receive any tumor cells, vehicle injected, CTX alone,
CTX+B7-DC-Ig or B7-DC-Ig alone. Two naive mice and 4 mice from
other groups were removed from the study on Day 11 (2 days post
CTX) and Day 16 (7 days post CTX) for T cell analysis. Left panel
shows on Day 11, 2 days post CTX injection, Treg in the spleen of
the mice with CTX treatment was significantly lower than the one in
the mice with tumor implantation and injected with vehicle. Right
panel shows that on Day 16, 7 days post CTX and 6 days post
B7-DC-Ig treatment, B7-DC-Ig significantly lowered the CD4+ T cells
expressing high PD-1. This was observed in both the B7-DC-Ig
treated and CTX+B7-DC-Ig treated mice. Mice implanted with tumor
cells intended to have more PD-1+/CD4+ T cells in the draining LN
compared with naive mice.
[0047] FIG. 10 shows the results of experiments wherein the
combination of CTX and B7-DC-Ig resulted in increased survival in
mice with tail vein injection of a mouse prostate tumor cell line.
SP-1 cells were isolated from mouse lungs that were metastasized
from TRAMP prostate tumor cell injection. B10.D2 mice were first
injected with 3.times.105 SP-1 cells via tail vein injection. On
Day 5, 12 and 19, mice were injected with 50 mg/kg of CTX where was
indicated. On Day 6, 13 and 20, mice were administered with 5 mg/kg
of B7-DC-Ig were it was indicated. Here, "NT" refers to "not
treated".
[0048] FIG. 11. Balb/C mice at age of 11-13 weeks were given
isolated hepatic metastases using a hemispleen injection technique.
The spleens of anesthetized mice were divided into two halves and
the halves were clipped. CT26 cells (1E05) were injected into one
hemispleen, and after 30 seconds, that hemispleen was resected and
the splenic draining vein was clipped. On Day 10, mice received 1
injection of CTX at 50 mg/kg, IP. Twenty four hours later, on Day
11, mice were treated with recombinant Listeria carrying AH1
peptide, an immunodominant epitope of CT26, at 0.1.times.LD.sub.50
(1.times.10.sup.7 CFU), then on Day 14 and 17. Mice were also
treated with B7-DC-Ig on Day 11 and then on Day 18. Mouse overall
survival was monitored.
DEFINITIONS
[0049] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
skill in the art to which the disclosed invention belongs. In
particular, the following terms and phrases have the following
meaning.
[0050] The term "inhibitory signal transduction" is intended to
mean any signal transduction having the effect of abolishing, or
otherwise reducing, T cell responses against an antigen, whether by
reducing T cell proliferation or by any other inhibitory mechanism,
whereby the extent or duration of an immunogenic T cell response is
decreased. Such inhibitory signal transduction may be due to PD-1
binding to a natural ligand, such as binding of PD-1 by B7-H1 or
some other member of this class of ligands, B7-DC, or may be due to
binding of CTLA4 to ligands, such as B7-1 or B7-2. In general,
compounds of the invention reduce such inhibitory signal
transduction and include, but are not limited to, PD-1 antagonists
and CTLA4 antagonists.
[0051] The term "PD-1 antagonist" means any molecule that
attenuates inhibitory signal transduction mediated by PD-1, found
on the surface of T cells, B cells, natural killer (NK) cells,
monocytes, DC, and macrophages. Such an antagonist includes a
molecule that disrupts any inhibitory signal generated by a PD-1
molecule on a T cell. In specific examples of the invention, a PD-1
antagonist is a molecule that inhibits, reduces, abolishes or
otherwise reduces inhibitory signal transduction through the PD-1
receptor signaling pathway. Such decrease may result where: (i) the
PD-1 antagonist of the invention binds to a PD-1 receptor without
triggering signal transduction, to reduce or block inhibitory
signal transduction; (ii) the PD-1 antagonist binds to a ligand
(e.g. an agonist) of the PD-1 receptor, preventing its binding
thereto (for example, where said agonist is B7-H1); (iii) the PD-1
antagonist binds to, or otherwise inhibits the activity of, a
molecule that is part of a regulatory chain that, when not
inhibited, has the result of stimulating or otherwise facilitating
PD-1 inhibitory signal transduction; or (iv) the PD-1 antagonist
inhibits expression of a PD-1 receptor or expression ligand
thereof, especially by reducing or abolishing expression of one or
more genes encoding PD-1 or one or more of its natural ligands.
Thus, a PD-1 antagonist of the invention is a molecule that effects
a decrease in PD-1 inhibitory signal transduction, thereby
increasing T cell response to one or more antigens.
[0052] As used herein, the term "CTLA4 antagonist" means a compound
that reduces CTLA4-mediated inhibition of T cell reactions. For
example, in an T cell, CTLA4 delivers an inhibitory impulse upon
binding of B7 ligands, such B7-1 and B7-2. A CTLA4 antagonist is
one that disrupts binding of said ligands to CTLA4 on activated T
cells. In one embodiment, the antagonist is an anti-CTLA4 antibody
that binds CTLA4 to prevent ligand binding. I
[0053] As used herein, the term "active fragment" refers to a
portion of a natural polypeptide, or a polypeptide with high
sequence homology (for example, at least 80%, 85%, 90%, 95%, 98%,
or 99% amino acid sequence identity) to a natural polypeptide and
that exhibits PD-1 antagonist activity, for example, by binding
PD-1 or by binding to a ligand of PD-1. In preferred embodiments,
such a fragment would consist of the extracellular domain (ECD) of
a B7-DC protein that binds to PD-1, such as SEQ ID NO: 3,
preferably amino acids 20 to 221 thereof. In the case of PD-1
polypeptide, an active fragment would be a portion of said
polypeptide comprising a binding domain that binds to a natural
ligand of PD-1 to prevent stimulation of PD-1 mediated inhibitory
signal transduction by said ligand. Active fragments may be
identified by their ability to compete with the molecule they are
derived from for binding to a natural binding site. For example,
active fragments will compete with wild-type B7-DC for binding to
PD-1.
[0054] With respect to an antibody, the term "active fragment"
means an antigen binding portion of an antibody that is less than
an entire immunoglobulin. Such fragments include Fab and
F(ab.sub.2)' fragments, capable of reacting with and binding to any
of the polypeptides disclosed herein as being receptors or ligands.
These Fab and F(ab').sub.2 fragments lack the Fc portion of an
intact antibody, clear more rapidly from the circulation, and may
have less non-specific tissue binding than an intact antibody (Wahl
et al., J. Nuc. Med. 24:316-325 (1983)). Also included are Fv
fragments (Hochman, J. et al. (1973) Biochemistry 12:1130-1135;
Sharon, J. et al. (1976) Biochemistry 15:1591-1594). These various
fragments are produced using conventional techniques such as
protease cleavage or chemical cleavage (see, e.g., Rousseaux et
al., Meth. Enzymol., 121:663-69 (1986)).
[0055] As used herein, the term "soluble portion" of a PD-1
antagonist means that portion of the full length polypeptide that
does not include any part of the transmembrane portion or segment.
For example, with respect to B7-DC, a soluble portion would include
the extracellular portion (with or without the N-terminal signal
sequence) but would not include any part of the transmembrane
portion (or, at least, not enough to reduce solubility). Thus, the
ECD of human B7-DC is shown as SEQ ID NO: 3 and consists of both
the IgV-like and IgC-like domains of the full length molecule
(i.e., amino acids 20-221 of the full length sequence (SEQ ID NO:
1).
[0056] As used herein, a "co-stimulatory polypeptide" is a
polypeptide that, upon interaction with a cell-surface molecule on
T cells, modulates the activity of the T cell. Thus, the response
of the T cell can be an effector (e.g., CTL or antibody-producing B
cell) response, a helper response providing help for one or more
effector (e.g., CTL or antibody-producing B cell) responses, or a
suppressive response.
[0057] As used herein, the term "treatment regimen" refers to a
treatment of a disease or a method for achieving a desired
physiological change, such as increased or decreased response of
the immune system to an antigen or immunogen, such as an increase
or decrease in the number or activity of one or more cells, or cell
types, that are involved in such response, wherein said treatment
or method comprises administering to an animal, such as a mammal,
especially a human being, a sufficient amount of two or more
chemical agents or components of said regimen to effectively treat
a disease or to produce said physiological change, wherein said
chemical agents or components are administered together, such as
part of the same composition, or administered separately and
independently at the same time or at different times (i.e.,
administration of each agent or component is separated by a finite
period of time from one or more of the agents agents or components)
and where administration of said one or more agents or components
achieves a result greater than that of any of said agents or
components when administered alone or in isolation.
[0058] As used herein the term "isolated" is meant to describe a
compound of interest (e.g., either a polynucleotide or a
polypeptide) that is in an environment different from that in which
the compound naturally occurs e.g. separated from its natural
milieu such as by concentrating a peptide to a concentration at
which it is not found in nature. "Isolated" is meant to include
compounds that are within samples that are substantially enriched
for the compound of interest and/or in which the compound of
interest is partially or substantially purified.
[0059] As used herein, the term "polypeptide" refers to a chain of
amino acids of any length, regardless of modification (e.g.,
phosphorylation or glycosylation). A polypeptide of the present
invention may be a recombinant polypeptide, a natural polypeptide
or a synthetic polypeptide, preferably a recombinant
polypeptide.
[0060] As used herein, a "variant" polypeptide contains at least
one amino acid sequence alteration as compared to the amino acid
sequence of the corresponding wild-type polypeptide.
[0061] As used herein, an "amino acid sequence alteration" can be,
for example, a substitution, a deletion, or an insertion of one or
more amino acids.
[0062] As used herein, the terms "portion," "segment," and
"fragment," when used in relation to polypeptides, refer to a
continuous sequence of residues, such as amino acid residues, which
sequence forms a subset of a larger sequence. For example, if a
polypeptide were subjected to treatment with any of the common
endopeptidases, such as trypsin or chymotrypsin, the oligopeptides
resulting from such treatment would represent portions, segments or
fragments of the starting polypeptide. A "fragment" of a
polypeptide thus refers to any subset of the polypeptide that is a
shorter polypeptide of the full length protein. Generally,
fragments will be five or more amino acids in length.
[0063] A derivative, analog or homolog, of a polypeptide (or
fragment thereof) of the invention may be (i) one in which one or
more of the amino acid residues are substituted with a conserved or
non-conserved amino acid residue (preferably a conserved amino acid
residue) and such substituted amino acid residue may or may not be
one encoded by the genetic code, or (ii) one in which one or more
of the amino acid residues includes a substituent group, or (iii)
one in which the mature polypeptide is fused with another compound,
such as a compound to increase the half-life of the polypeptide
(for example, polyethylene glycol), or (iv) one in which the
additional amino acids are fused to the mature polypeptide, such as
a leader or secretory sequence or a sequence which is employed for
purification of the mature polypeptide or a proprotein sequence.
Such derivatives and analogs are deemed to be within the scope of
those skilled in the art from the teachings herein.
[0064] As used herein, "valency" refers to the number of binding
sites available per molecule.
[0065] In accordance with the present invention, the term "percent
identity" or "percent identical," when referring to a sequence,
means that a sequence is compared to a claimed or described
sequence after alignment of the sequence to be compared (the
"Compared Sequence") with the described or claimed sequence (the
"Reference Sequence"). The Percent Identity is then determined
according to the following formula:
Percent Identity=100[1-(C/R)]
wherein C is the number of differences between the Reference
Sequence and the Compared Sequence over the length of alignment
between the Reference Sequence and the Compared Sequence wherein
(i) each base or amino acid in the Reference Sequence that does not
have a corresponding aligned base or amino acid in the Compared
Sequence and (ii) each gap in the Reference Sequence and (iii) each
aligned base or amino acid in the Reference Sequence that is
different from an aligned base or amino acid in the Compared
Sequence, constitutes a difference; and R is the number of bases or
amino acids in the Reference Sequence over the length of the
alignment with the Compared Sequence with any gap created in the
Reference Sequence also being counted as a base or amino acid. If
an alignment exists between the Compared Sequence and the Reference
Sequence for which the percent identity as calculated above is
about equal to or greater than a specified minimum Percent Identity
then the Compared Sequence has the specified minimum percent
identity to the Reference Sequence even though alignments may exist
in which the hereinabove calculated Percent Identity is less than
the specified Percent Identity.
[0066] As used herein, the term "conservative amino acid
substitution" means a substitution wherein the substituted amino
acid has similar structural or chemical properties, and
"non-conservative" amino acid substitutions are those in which the
charge, hydrophobicity, or bulk of the substituted amino acid is
significantly altered. Non-conservative substitutions will differ
more significantly in their effect on maintaining (a) the structure
of the peptide backbone in the area of the substitution, for
example, as a sheet or helical conformation, (b) the charge or
hydrophobicity of the molecule at the target site, or (c) the bulk
of the side chain. Examples of conservative amino acid
substitutions include those in which the substitution is within one
of the five following groups: 1) small aliphatic, nonpolar or
slightly polar residues (Ala, Ser, Thr, Pro, Gly); 2) polar,
negatively charged residues and their amides (Asp, Asn, Glu, Gln);
polar, positively charged residues (His, Arg, Lys); large
aliphatic, nonpolar residues (Met, Leu, Ile, Val, Cys); and large
aromatic resides (Phe, Tyr, Trp). Examples of non-conservative
amino acid substitutions are those where 1) a hydrophilic residue,
e.g., seryl or threonyl, is substituted for (or by) a hydrophobic
residue, e.g., leucyl, isoleucyl, phenylalanyl, valyl, or alanyl;
2) a cysteine or proline is substituted for (or by) any other
residue; 3) a residue having an electropositive side chain, e.g.,
lysyl, arginyl, or histidyl, is substituted for (or by) an
electronegative residue, e.g., glutamyl or aspartyl; or 4) a
residue having a bulky side chain, e.g., phenylalanine, is
substituted for (or by) a residue that does not have a side chain,
e.g., glycine.
[0067] The terms "individual", "host", "subject", and "patient" are
used interchangeably herein, and refer to a mammal, including, but
not limited to, primates, for example, human beings, as well as
rodents, such as mice and rats, and other laboratory animals.
[0068] As used herein the term "effective amount" or
"therapeutically effective amount" means a dosage sufficient to
treat, inhibit, or alleviate one or more symptoms of a disease
state being treated or to otherwise provide a desired pharmacologic
and/or physiologic effect, especially enhancing T cell response to
a selected antigen. The precise dosage will vary according to a
variety of factors such as subject-dependent variables (e.g., age,
immune system health, etc.), the disease, and the treatment being
administered.
[0069] As used herein "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active compound, use thereof in the therapeutic compositions is
contemplated. Supplementary active compounds can also be
incorporated into the compositions.
[0070] The term "antibody" is meant to include both intact
molecules as well as fragments thereof that include the
antigen-binding site. Whole antibody structure is often given as
H.sub.2L.sub.2 and refers to the fact that antibodies commonly
comprise 2 light (L) amino acid chains and 2 heavy (H) amino acid
chains. Both chains have regions capable of interacting with a
structurally complementary antigenic target. The regions
interacting with the target are referred to as "variable" or "V"
regions and are characterized by differences in amino acid sequence
from antibodies of different antigenic specificity. The variable
regions of either H or L chains contains the amino acid sequences
capable of specifically binding to antigenic targets. Within these
sequences are smaller sequences dubbed "hypervariable" because of
their extreme variability between antibodies of differing
specificity. Such hypervariable regions are also referred to as
"complementarity determining regions" or "CDR" regions. These CDR
regions account for the basic specificity of the antibody for a
particular antigenic determinant structure. The CDRs represent
non-contiguous stretches of amino acids within the variable regions
but, regardless of species, the positional locations of these
critical amino acid sequences within the variable heavy and light
chain regions have been found to have similar locations within the
amino acid sequences of the variable chains. The variable heavy and
light chains of all antibodies each have 3 CDR regions, each
non-contiguous with the others (termed L1, L2, L3, H1, H2, H3) for
the respective light (L) and heavy (H) chains. The accepted CDR
regions have been described by Kabat et al, J. Biol. Chem.
252:6609-6616 (1977). The antibodies disclosed according to the
invention may also be wholly synthetic, wherein the polypeptide
chains of the antibodies are synthesized and, possibly, optimized
for binding to the polypeptides disclosed herein as being
receptors. Such antibodies may be chimeric or humanized antibodies
and may be fully tetrameric in structure, or may be dimeric and
comprise only a single heavy and a single light chain.
DETAILED DESCRIPTION OF THE INVENTION
[0071] The present invention provides a treatment regimen, or
combination therapy, for treating disease in mammals comprising a
compound that reduces or abolishes inhibitory signal transduction
in T cells, preferably human T cells, administered in conjunction
with a potentiating agent to increase an immune response.
[0072] The methods of the invention also relate to the use of broad
spectrum immunomodulators and compositions of these. In general,
the increased T cell response resulting from these methods is
greater than any increased T cell response resulting from
administering the same dose of either of said PD-1 antagonist or
said potentiating agent alone.
[0073] The disclosed compositions and regimens are useful to
stimulate or enhance immune responses involving T cells. Thus, the
methods of the invention are most useful in treating a disease
condition that would benefit from an increase in T cell activity
and where the increased T cell response is necessary or sufficient
to treat said disease, even though the disease is not specifically
caused or aggravated by a reduced T cell response. In a preferred
embodiment, the type of disease to be treated or prevented is a
malignant tumor or a chronic infectious disease caused by a
bacterium, virus, protozoan, helminth, or other intracellular
microbial pathogen that is attacked, i.e., by cytotoxic T
lymphocytes. Activation of T cells using the disclosed compositions
is also advantageous to treat or prevent conditions characterized
by immunosuppression.
[0074] In accordance with the present invention, the T cell
response can be regulated by molecules that bind to receptors on
the T cell surface and molecules that bind to ligands of such
receptors. In the case of PD-1, molecules that bind PD-1 to reduce
its inhibitory effect and/or molecules that bind one or more PD-1
ligands to reduce their ability to bind PD-1 have the effect of
reducing the ability of PD-1 to inhibit T cell response, thereby
increasing this response and the immunological effects thereof.
A. PD-1 Receptor Antagonists
[0075] Compositions containing antagonists of PD-1 receptors are
provided and include compounds or agents that either bind to and
block a ligand of PD-1 to interfere with or inhibit the binding of
the ligand to the PD-1 receptor, or bind directly to and block the
PD-1 receptor without inducing inhibitory signal transduction
through the PD-1 receptor. In another embodiment, the PD-1 receptor
antagonist binds directly to the PD-1 receptor without triggering
inhibitory signal transduction and also binds to a ligand of the
PD-1 receptor to reduce or inhibit the ligand from triggering
signal transduction through the PD-1 receptor. By reducing the
number and/or amount of ligands that bind to PD-1 receptor and
trigger the transduction of an inhibitory signal, fewer cells are
attenuated by the negative signal delivered by PD-1 signal
transduction and a more robust immune response can be achieved.
[0076] In accordance with the present invention, PD-1 signaling
requires binding to a PD-1 ligand (such as B7-H1 or B7-DC) in close
proximity to a peptide antigen presented by major
histocompatibility complex (MHC) (see, for example, Freeman Proc.
Natl. Acad. Sci. U.S.A 105:10275-10276 (2008)). Therefore,
proteins, antibodies or small molecules that prevent co-ligation of
PD-1 and TCR on the T cell membrane are useful PD-1 antagonists
contemplated by this invention.
[0077] Exemplary PD-1 receptor antagonists include, but are not
limited to B7-DC polypeptides, including homologs and variants of
these, as well as active fragments of any of the foregoing, and
fusion proteins that incorporate any of these. In a preferred
embodiment, the fusion protein comprises the soluble portion of
B7-DC coupled to the Fc portion of an antibody, such as human IgG,
and does not incorporate all or part of the transmembrane portion
of human B7-DC. The PD-1 receptor antagonists can also be small
molecule antagonists or antibodies that reduce or interfere with
PD-1 receptor signal transduction by binding to ligands of PD-1 or
to PD-1 itself, especially where co-ligation of PD-1 with TCR does
not follow such binding, thereby not triggering inhibitory signal
transduction through the PD-1 receptor.
[0078] The PD-1 receptor antagonists provided herein are generally
useful in vivo and ex vivo as immune response-stimulating
therapeutics. In general, the disclosed antagonist compositions are
useful for treating a subject having or being predisposed to any
disease or disorder to which the subject's immune system mounts an
immune response.
1. B7-DC Polypeptides
[0079] In certain embodiments, B7-DC proteins can be used as PD-1
receptor antagonists. B7-DC is a natural ligand of PD-1 and binds
to PD-1 with higher affinity than B7-H1, and can thus inhibit
B7-H1:PD-1 interactions. Suitable B7-DC polypeptides, including
variants, homologs and fragments thereof, can be obtained from the
following full length human B7-DC polypeptides with (SEQ ID NO:1)
or without (SEQ ID NO:2) the endogenous signal peptide.
TABLE-US-00001 (SEQ ID NO: 1) MIFLLLMLSL ELQLHQIAAL FTVTVPKELY
IIEHGSNVTL ECNFDTGSHV NLGAITASLQ 60 KVENDTSPHR ERATLLEEQL
PLGKASFHIP QVQVRDEGQY QCIIIYGVAW DYKYLTLKVK 120 ASYRKINTHI
LKVPETDEVE LTCQATGYPL AEVSWPNVSV PANTSHSRTP EGLYQVTSVL 180
RLKPPPGRNF SCVFWNTHVR ELTLASIDLQ SQMEPRTHPT WLLHIFIPFC IIAFIFIATV
240 IALRKQLCQK LYSSKDTTKR PVTTTKREVN SAI 273 (SEQ ID NO: 2)
LFTVTVPKEL YIIEHGSNVT LECNFDTGSH VNLGAITASL QKVENDTSPH RERATLLEEQ
60 LPLGKASFHI PQVQVRDEGQ YQCIIIYGVA WDYKYLTLKV KASYRKINTH
ILKVPETDEV 120 ELTCQATGYP LAEVSWPNVS VPANTSHSRT PEGLYQVTSV
LRLKPPPGRN FSCVFWNTHV 180 RELTLASIDL QSQMEPRTHP TWLLHIFIPF
CIIAFIFIAT VIALRKQLCQ KLYSSKDTTK 240 RPVTTTKREV NSAI 254
[0080] The B7 family of molecules, including B7-DC, are expressed
at the cell surface with a membrane proximal constant IgC domain
and a membrane distal IgV domain. Receptors for these ligands share
a common extracellular IgV-like domain. Interactions of
receptor-ligand pairs are mediated predominantly through residues
in the IgV domains of the ligands and receptors. In general, IgV
domains are described as having two sheets that each contains a
layer of .beta.-strands. These .beta.-strands are referred to as
A', B, C, C', C'', D, E, F and G. The structure of such
polypeptides has been described in the literature (See Molnar et
al., Crystal structure of the complex between programmed death-1
(PD-1) and its ligand PD-L2, PNAS, Vol. 105, pp. 10483-10488 (29
Jul. 2008)).
[0081] B7-DC, a transmembrane protein, in its monomeric form,
comprises IgV and IgC domains that make up the extracellular
portion of the molecule (the extracellular domain, or ECD), with
the IgV-like domain being responsible, in whole or in part, for
PD-1 binding as well as other functions recited in the methods of
the invention. For the human protein, the IgV domain is
characterized in that it possesses a disulfide bond linking the B
and F strands (referred to above), which appears to be
characteristic of many IgV domains and possesses a similar
three-dimensional structure with the IgV domains of both B7-1 and
B7-2 (see Molnar et al. (2008), supra).
[0082] In one embodiment the B7-DC variant polypeptides contain
amino acid alterations (i.e., substitutions, deletions or
insertions) within one or more of these .beta.-strands in any
possible combination. In another embodiment, B7-DC variants contain
one or more amino acid alterations (i.e., substitutions, deletions
or insertions) within the A', C, C', C'', D, E, F or G
.beta.-strands. In a preferred embodiment B7-DC variants contain
one or more amino acid alterations in the G .beta.-strand. In
another embodiment, variant B7-DC polypeptide fragments include the
IgC and IgV domains of B7-DC. In another embodiment, variant B7-DC
polypeptide fragments include the IgV domain of B7-DC.
[0083] Human and mouse B7-DC proteins contain a short
intracytoplasmic domain, a single transmembrane domain and an
extracellular domain. The extracellular domain contains two Ig
domains; a membrane proximal IgC domain and a membrane distal IgV
domain. Useful fragments of variant B7-DC polypeptides include
soluble fragments. Soluble B7-DC fragments are fragments of B7-DC
that may be shed, secreted or otherwise extracted from the
producing cells. In one embodiment, variant B7-DC polypeptide
fragments include the entire extracellular domain of B7-DC. The
extracellular domain of B7-DC includes amino acids from about 20 to
about amino acid 221 of murine or human B7-DC or active fragments
thereof. In another embodiment, variant B7-DC polypeptide fragments
include the IgC and IgV domains of B7-DC. In another embodiment,
variant B7-DC polypeptide fragments include the IgV domain of
B7-DC.
[0084] PD-1 signaling is thought to require binding to a PD-1
ligand (typically B7-H1) in close proximity to a peptide antigen
presented by major histocompatibility complex (MHC) (Freeman Proc.
Natl. Acad. Sci. U.S.A 105:10275-10276 (2008)). Therefore,
proteins, antibodies or small molecules that prevent co-ligation of
PD-1 and TCR on the T cell membrane are useful PD-1 antagonists
contemplated by this invention.
[0085] The PD-1 antagonist useful in the methods and compositions
of the invention include fragments of the B7-DC protein
incorporating the ECD. Alternatively, the fragments of B7-DC
include part of the extracellular domain that comprise the an IgV
or IgV-like domain, preferably amino acids 20-221, more preferably
20-121, that are sufficient to bind to the PD-1 receptor to
interfere with, or prevent, or otherwise reduce inhibitory signal
transduction through the PD-1 receptor. In a preferred embodiment
the B7-DC fragment competes with B7-H1 for binding to PD-1
receptors.
[0086] In one embodiment, variant B7-DC polypeptide fragments may
contain a region of the polypeptide that is important for binding
to PD-1. These polypeptide fragments may be useful to compete for
binding to PD-1 and to prevent native B7-DC from binding to PD-1.
By competing for binding to PD-1, these fragments may be useful to
enhance an immune response, as inhibiting interactions of B7-H1 and
B7-DC with PD-1 inhibits the suppression of immune responses that
would otherwise occur. A polypeptide fragment of mouse or human
B7-DC that could competitively bind to PD-1 can contain, for
example, amino acids 101-108 or 110-114. The binding of wild-type
B7-DC to PD-1 typically is inhibited by at least 50 percent, 60
percent, 70 percent, 75 percent, 80 percent, 90 percent, 95
percent, or more than 95 percent as compared to the level of
binding of wild-type B7-DC to PD-1 in the absence of a fragment of
said wild-type B7-DC. Exemplary B7-DC fragments useful in the
methods and/or compositions of the invention include, but are in no
way limited to, the following B7-DC extracellular domains:
TABLE-US-00002 Human B7-DC extracellular domain (ECD): (SEQ ID NO:
3) LFTVTVPKEL YIIEHGSNVT LECNFDTGSH VNLGAITASL QKVENDTSPH
RERATLLEEQ 60 LPLGKASFHI PQVQVRDEGQ YQCIIIYGVA WDYKYLTLKV
KASYRKINTH ILKVPETDEV 120 ELTCQATGYP LAEVSWPNVS VPANTSHSRT
PEGLYQVTSV LRLKPPPGRN FSCVFWNTHV 180 RELTLASIDL QSQMEPRTHP TW 202
and murine B7-DC ECD: (SEQ ID NO: 4) LFTVTAPKEV YTVDVGSSVS
LECDFDRREC TELEGIRASL QKVENDTSLQ SERATLLEEQ 60 LPLGKALFHI
PSVQVRDSGQ YRCLVICGAA WDYKYLTVKV KASYMRIDTR ILEVPGTGEV 120
QLTCQARGYP LAEVSWQNVS VPANTSHIRT PEGLYQVTSV LRLKPQPSRN FSCMFWNAHM
180 KELTSAIIDP LSRMEPKVPR TW 202 Cynomolgus monkey B7-DC ECD: (Seq
ID NO: 15) LFTVTVPKEL YIIEHGSNVT LECNFDTGSH VNLGAITASL QKVENDTSPH
RERATLLEEQ 60 LPLGKASFHI PQVQVRDEGQ YQCIIIYGVA WDYKYLTLKV
KASYRKINTH ILKVPETDEV 120 ELTCQATGYP LAEVSWPNVS VPANTSHSRT
PEGLYQVTSV LRLKPPPGRN FSCVFWNTHV 180 RELTLASIDL QSQMEPRTHP TW
202
[0087] Numerous other primate sequences useful in the methods and
compositions of the invention are provided in Onlamoon et al.,
Immunology, Vol. 124, pp. 277-293 (2008).
[0088] A PD-1 antagonist useful in the compositions and methods of
the invention also includes a fusion protein (as described below)
that comprises first and second polypeptide portions, wherein said
fusion protein, or at least the first polypeptide portion thereof,
possesses PD-1 antagonist activity, especially where said fusion
protein binds to and blocks PD-1 or binds to and blocks a ligand of
PD-1. The first polypeptide portion of such fusion protein can
comprise, or consist of, any of the PD-1 antagonistic polypeptides,
or PD-1 binding fragments thereof, otherwise recited herein for use
as PD-1 antagonists in the methods of the invention. In a preferred
embodiment of such a fusion protein, the recited first polypeptide
portion is N-terminal to the recited second polypeptide portion. In
a separate embodiment, the recited first polypeptide portion is
linked to the recited second polypeptide portion by an oligopeptide
in addition to the amino acids composing the recited first and
second polypeptide portions, where said linking amino acids do not
substantially decrease the PD-1 antagonist activity of said fusion
protein.
[0089] In a preferred dimeric fusion protein, the dimer results
from the covalent bonding of Cys residues in the CH regions of two
of the Ig heavy chains that are the same Cys residues that are
disulfide linked in dimerized normal Ig heavy chains.
[0090] A large number of polypeptide sequences that are routinely
used as fusion protein binding partners are well known in the art.
Examples of useful polypeptide binding partners include, but are
not limited to, green fluorescent protein (GFP), glutathione
S-transferase (GST), polyhistidine, myc, hemaglutinin, Flag.TM. tag
(Kodak, New Haven, Conn.), maltose E binding protein and protein
A.
[0091] Still another embodiment provides a tetramer construct
having a BirA substrate fused to the extracellular domain of a
variant B7-DC polypeptide. Methods for making tetramer constructs
are known in the art (see Pertovas, et al., J. Exp. Med., 203:2281
(2006)).
[0092] Exemplary murine B7-DC fusion proteins contain amino acids
20-221 of murine B7-DC fused to amino acids 237-469 of murine IgG2a
(CAA49868). In one non-limiting example, human B7-DC fusion
proteins contain amino acids 20-221 of human B7-DC fused to amino
acids 245-476 of human IgG1 (AAA02914). The signal peptides for
B7-DC fusion proteins include the endogenous signal peptides or any
other signal peptide that facilitates secretion of the fusion
protein from a host. In another embodiment, the first polypeptide
would include only the IgV domain. Other embodiments may comprise
the hinge and Fc domain of an IgG antibody, such IgG1, with none of
the variable region present. Other embodiments include use of the
hinge and Fc region of IgG2 or IgG4, especially having an N297Q or
other mutation that reduces effector function.
[0093] In accordance with the methods and compositions of the
invention, the polypeptide useful as a PD-1 antagonist, or the
first polypeptide portion of a fusion protein useful as a PD-1
antagonist, comprises an amino acid sequence that has at least 60%,
or at least 65%, or at least 70%, or at least 75%, or at least 80%,
or at least 85%, or at least 90%, or at least 95%, or at least 99%,
identity to amino acids 1-221 of SEQ ID NO: 1, preferably amino
acids 20-221 of SEQ ID NO: 1, or amino acids 26-221 of SEQ ID NO:
1, or amino acids 1-202 of SEQ ID NO: 3 or 4, more preferably amino
acids 20-121 of SEQ ID NO: 1 or amino acids 1-102 of SEQ ID NO: 3
or 4.
[0094] In one embodiment, a polypeptide useful as a PD-1
antagonist, or the first polypeptide portion of a fusion protein
useful as a PD-1 antagonist, consists of amino acids 1-221 of SEQ
ID NO: 1, or consists of amino acids 20-221 of SEQ ID NO: 1, or
consists of amino acids 26-221 of SEQ ID NO: 1, or consists of
amino acids 1-202 of SEQ ID NO: 3 or 4. In one embodiment (SEQ ID
NO: 2), it does not comprise amino acids 1-19 of SEQ ID NO: 1.
[0095] In other specific examples, a PD-1 antagonist polypeptide,
or first polypeptide portion of a PD-1 antagonist fusion protein,
comprises the amino acid sequence 20-121 of SEQ ID NO: 1,
preferably where it comprises the amino acid sequence WDYKY at
residues 110-114 thereof, or where it comprises amino acids 1-102
of SEQ ID NO: 3, preferably where it comprises the amino acid
sequence WDYKY at residues 91-95 thereof.
[0096] In a preferred embodiment, such percent identities are
achieved by reliance on conservative amino acid substitutions as
defined elsewhere herein.
[0097] In one such embodiment, the PD-1 antagonist polypeptide, or
first polypeptide portion of a PD-1 antagonist fusion protein, does
not comprise amino acids 1-19 of SEQ ID NO: 1, or does not comprise
any portion of a transmembrane domain, especially not the entire
such domain, or does not comprise any portion of the intracellular
(or soluble) domain, especially not the entire such domain, of a
PD-1 ligand or other PD-1 antagonist protein. In a preferred
embodiment, such antagonist, or first polypeptide portion,
comprises only the extracellular domain (ECD) of SEQ ID NO:1 and is
thus comprised only of a soluble portion of the polypeptide of said
sequence, or a fragment of said soluble portion.
[0098] In other such embodiments, the PD-1 antagonist polypeptide,
or first polypeptide portion of a PD-1 antagonist fusion protein,
comprises the IgV domain, or IgV-like domain, or PD-1 binding
fragment thereof, of a PD-1 ligand, or consists of the IgV domain,
or IgV-like domain, or PD-1 binding fragment thereof, of a PD-1
ligand. In specific examples, such PD-1 ligand is a wild-type B7-DC
or B7-H1 molecule, preferably mouse or primate, preferably human,
wild-type B7-DC or B7-H1 molecule.
[0099] In other such embodiments, the PD-1 antagonist polypeptide,
or first polypeptide portion of a PD-1 antagonist fusion protein, a
PD-1 binding fragment of the IgV domain, or IgV-like domain, of a
PD-1 ligand, especially where IgV domain, or IgV-like domain,
consists of amino acids 20-121 of SEQ ID NO: 1 or amino acids 1-102
of SEQ ID NO: 3.
[0100] A PD-1 antagonist of the invention also includes a PD-1
binding fragment of amino acids 20-121 of SEQ ID NO: 1 (human full
length), or amino acids 1-102 of SEQ ID NO: 3 (extracellular domain
or ECD).
[0101] In specific embodiments thereof, the polypeptide or PD-1
binding fragment also incorporates amino acids WDYKY at residues
110-114 of SEQ ID NO: 1 or WDYKY at residues 91-95 of SEQ ID NO: 3.
By way of non-limiting examples, such a PD-1 binding fragment
comprises at least 10, or at least 20, or at least 30, or at least
40, or at least 50, or at least 60, or at least 70, or at least 75,
or at least 80, or at least 85, or at least 90, or at least 95, or
at least 100 contiguous amino acids of the sequence of amino acids
20-121 of SEQ ID NO: 1, wherein a preferred embodiment of each such
PD-1 binding fragment would comprise as a sub-fragment the amino
acids WDYKY found at residues 110-114 of SEQ ID NO: 1 or WDYKY at
residues 91-95 of SEQ ID NO: 3.
[0102] Other preferred polypeptides and PD-1 binding fragments
specifically contemplated by the invention include the polypeptide
sequence of amino acids 20-121 of SEQ ID NO: 1 (human full length)
and PD-1 binding fragments thereof, wherein, in such polypeptide or
PD-1 binding fragment, a cysteine is present at residues 42 and/or
102, with a cysteine at both positions being preferred, and/or
wherein a phenylalanine is present at residue 21, and/or wherein a
glutamic acid is present at residue 28, and/or wherein a threonine,
and/or wherein a glutamine is present at residue 60, and/or wherein
a glutamic acid is present at residue 101, and/or wherein
isoleucine is present at residue 103, and/or wherein an isoleucine
is present at residue 105, and/or wherein a glycine is present at
residue 107, and/or wherein valine is present at residue 108,
and/or wherein a tryptophan is present at residue 110, and/or
wherein aspartic acid is present at residue 111, and/or wherein a
tyrosine is present at residue 112, and/or wherein a lysine is
present at residue 113, and/or wherein a tyrosine is present at
residue 114, provided that, in the case of PD-1 binding fragments,
said fragment is large enough to include such amino acid
positions.
[0103] Additional preferred polypeptides and PD-1 binding fragments
specifically contemplated by the invention include the polypeptide
sequence of amino acids 1-102 of SEQ ID NO: 3 (human ECD) or SEQ ID
NO: 4 (murine ECD) and PD-1 binding fragments thereof, wherein, in
such polypeptide or PD-1 binding fragment, a cysteine is present at
residues 23 and/or 83, with a cysteine at both positions being
preferred, and/or wherein a phenylalanine is present at residue 2,
and/or wherein a glutamic acid is present at residue 9, and/or
wherein a threonine or arginine is present at residue 37, with
threonine preferred, and/or wherein a glutamine is present at
residue 41, and/or wherein arginine is present at residue 82,
and/or wherein a leucine is present at residue 84, and/or wherein
an isoleucine is present at residue 86, and/or wherein a glycine is
present at residue 88, and/or wherein an alanine is present at
residue 89, and/or wherein a tryptophan is present at residue 91,
and/or wherein a aspartic acid is present at residue 92, and/or
wherein a tyrosine is present at residue 93, and/or wherein a
lysine is present at residue 94, and/or wherein a tyrosine is
present at residue 95, provided that, in the case of PD-1 binding
fragments, said fragment is large enough to include such amino acid
positions.
[0104] In additional embodiments, any of the above polypeptides may
also incorporate portions or fragments, for example, from 1 to 10
contiguous amino acids, drawn from the signal, transmembrane or
C-terminal domains of the B7-DC or B7-H1 polypeptide, such as that
of mouse or primate, preferably human.
[0105] Such polypeptides and/or PD-1 binding fragments can also be
present in any of the fusion proteins of the invention, for
example, where such polypeptide or PD-1 binding fragment represents
the "first polypeptide" of such fusion protein.
[0106] In specific examples, the molecule, combined with a
potentiating agent for use in a treatment regimen of the invention,
comprises a PD-1 binding fragment of amino acids 20-221 of SEQ ID
NO: 1. In one such embodiment, the fragment is from amino acids
20-121 of SEQ ID NO: 1, preferably where the fragment contains
amino acids 110-114 of SEQ ID NO: 1. In some embodiments, more than
one such fragment is present (as described elsewhere herein) and
the molecule comprises at least 2, 3, 4, 5 or more fragments of a
B7-DC protein, especially where the fragment is part of, or
contains part of, amino acids 20-221 of SEQ ID NO: 1. In a
preferred embodiment thereof, at least one said fragment is from
amino acids 20-121 of SEQ ID NO: 1, more preferrably wherein at
least one said fragment includes amino acids 110-114 of SEQ ID NO:
1 (i.e., the sequence WDYKY (SEQ ID NO: 14)). In preferred
embodiments, the PD-1 binding fragment comprises at least 10, or at
least 25, or at least 50, or at least 75, or at least 100
contiguous amino acids in length.
[0107] The endogenous human signal peptide has the following
sequence MIFLLLMLSL ELQLHQIAA (SEQ ID NO:5) and represents the
first 19 amino acids of SEQ ID NO: 1. In certain embodiments, the
polypeptide fragments of B7-DC can include 1, 2, 3, 4, 5, 6, 7, 8,
9, or 10 contiguous amino acids of the endogenous or heterologous
signal peptide (which can be used to produce a recombinant B7-DC
polypeptide by expression in and secretion from a transformed
cell). It will also be appreciated that a useful B7-DC polypeptide
can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous amino acids
of the transmembrane domain of B7-DC, and/or 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10 contiguous amino acids of the cytoplasmic domain, or
combinations thereof provided the B7-DC fragment retains the
ability to antagonize the PD-1 receptor.
[0108] The phenotypes of PD-1-/- mice provide direct evidence for
PD-1 being a negative regulator of immune responses in vivo. In the
absence of PD-1, mice on the C57BL/6 background slowly develop a
lupus-like glomerulonephritis and progressive arthritis (Nishimura,
et al., Immunity, 11:141-151 (1999)). PD-1-/- mice on the BALB/c
background rapidly develop a fatal autoimmune dilated
cardiomyopathy (Nishimura, et al., Science. 291:319-322 (2001)).
However, substantial evidence indicates that B7-DC can function to
costimulate activate T cell responses. In the presence of
suboptimal TCR signals, B7-DC causes increased proliferation and
production of cytokines in vitro (Tseng, et al., J. Exp. Med.
193:839-846 (2001)). On the other hand, in vitro studies indicate a
negative regulatory role for B7-DC in T cell responses. These
seemingly contradictory data are best interpreted by expression of
additional receptors for B7-DC on T cells other than PD-1.
[0109] Therefore, B7-DC proteins, variants, fragments and fusions
thereof, may have the advantage of directly enhancing T cell
responses by binding to an unknown receptor that activates the T
cell, in addition to enhancing T cell responses by preventing the
PD-1 mediated inhibitory signal transduction.
2. B7-H1 Polypeptides
[0110] In another embodiment, the compound for use in combination
with a potentiating agent in the treatment regimen of the
invention, is, or comprises, a fragment of a mammalian B7-H1,
preferably from mouse or primate, preferably human, wherein said
fragment binds to and blocks PD-1 but does not result in inhibitory
signal transduction through PD-1 and said fragment is at least 10,
or at least 20, or at least 30, or at least 40, or at least 50, or
at least 60, or at least 70, or at least 80, or at least 90, or at
least 100 contiguous amino acids in length. In other embodiments,
the fragment can be of variable length so long as it has the
function of binding to PD-1 but does not produce inhibitory signal
transduction that results in reduced T cell proliferation. Such
B7-H1 fragments also find use as part of the first polypeptide
portion of fusion proteins of the invention.
[0111] B7-H1 sequences are as follows:
TABLE-US-00003 Human B7-H1 Polypeptide (SEQ ID NO. 16): MRIFAVFIFM
TYWHLLNAFT VTVPKDLYVV EYGSNMTIEC KFPVEKQLDL AALIVYWEME 60
DKNIIQFVHG EEDLKVQHSS YRQRARLLKD QLSLGNAALQ ITDVKLQDAG VYRCMISYGG
120 ADYKRITVKV NAPYNKINQR ILVVDPVTSE HELTCQAEGY PKAEVIWTSS
DHQVLSGKTT 180 TTNSKREEKL FNVTSTLRIN TTTNEIFYCT FRRLDPEENH
TAELVIPELP LAHPPNERTH 240 LVILGAILLC LGVALTFIFR LRKGRMMDVK
KCGIQDTNSK KQSDTHLEET 290 Murine B7-H1 (SEQ ID NO: 17) MRIFAGIIFT
ACCHLLRAFT ITAPKDLYVV EYGSNVTMEC RFPVERELDL LALVVYWEKE 60
DEQVIQFVAG EEDLKPQHSN FRGRASLPKD QLLKGNAALQ ITDVKLQDAG VYCCIISYGG
120 ADYKRITLKV NAPYRKINQR ISVDPATSEH ELICQAEGYP EAEVIWTNSD
HQPVSGKRSV 280 TTSRTEGMLL NVTSSLRVNA TANDVFYCTF WRSQPGQNHT
AELIIPELPA THPPQNRTHW 240 VLLGSILLFL IVVSTVLLFL RKQVRMLDVE
KCGVEDTSSK NRNDTQFEET 290 Macaca mulatta PD-L1 (SEQ ID NO: 18)
MRIFAVFIFT IYWHLLNAFT VTVPKDLYVV EYGSNMTIEC RFPVEKQLGL 60
TSLIVYWEME DKNIIQFVHG EEDLKVQHSN YRQRAQLLKD QLSLGNAALR 120
ITDVKLQDAG VYRCMISYGG ADYKRITVKV NAPYNKINQR ILVVDPVTSE 180
HELTCQAEGY PKAEVIWTSS DHQVLSGKTT TTNSKREEKL LNVTSTLRIN 240
TTANEIFYCI FRRLGPEENH TAELVIPELP LALPPNERTH LVILGAIFLL 300
LGVALTFIFY LRKGRMMDMK KSGIRVTNSK KQRDTQLEET 340
[0112] B7-H1-Ig proteins are described in WO/2001/014557 (pub. 1
Mar. 2001) and in WO/2002/079499 (pub. 10 Oct. 2002).
3. PD-1 and Other Polypeptides
[0113] Other useful polypeptides of the invention include those
that bind to the ligands of the PD-1 receptor. These include the
PD-1 receptor protein, or soluble fragments thereof, which can bind
to the PD-1 ligands, such as B7-H1 or B7-DC, and prevent binding to
the endogenous PD-1 receptor, thereby preventing inhibitory signal
transduction. B7-H1 has also been shown to bind the protein B7.1
(Butte et al., Immunity, Vol. 27, pp. 111-122, (2007)). Such
fragments also include the soluble ECD portion of the PD-1 protein
that includes mutations, such as the A99L mutation, that increases
binding to the natural ligands (Molnar et al., Crystal structure of
the complex between programmed death-1 (PD-1) and its ligand PD-L2,
PNAS, Vol. 105, pp. 10483-10488 (29 Jul. 2008)). B7-1 or soluble
fragments thereof, which can bind to the B7-H1 ligand and prevent
binding to the endogenous PD-1 receptor, thereby preventing
inhibitory signal transduction, are also useful.
[0114] PD-1 polypeptides useful in the methods of the invention are
as follows:
TABLE-US-00004 Human PD-1 (SEQ ID NO: 19) MQIPQAPWPV VWAVLQLGWR
PGWFLDSPDR PWNPPTFFPA LLVVTEGDNA TFTCSFSNTS 60 ESFVLNWYRM
SPSNQTDKLA AFPEDRSQPG QDCRFRVTQL PNGRDFHMSV VRARRNDSGT 120
YLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLV VGVVGGLLGS
180 LVLLVWVLAV ICSRAARGTI GARRTGQPLK EDPSAVPVFS VDYGELDFQW
REKTPEPPVP 240 CVPEQTEYAT IVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL
288 Cynomolgus monkey PD-1 (SEQ ID NO: 20) MQIPQAPWPV VWAVLQLGWR
PGWFLESPDR PWNAPTFSPA LLLVTEGDNA TFTCSFSNAS 60 ESFVLNWYRM
SPSNQTDKLA AFPEDRSQPG QDCRFRVTRL PNGRDFHMSV VRARRNDSGT 120
YLCGAISLAP KAQIKESLRA ELRVTERRAE VPTAHPSPSP RPAGQFQTLV VGVVGGLLGS
180 LVLLVWVLAV ICSRAARGTI GARRTGQPLK EDPSAVPVFS VDYGELDFQW
REKTPEPPVP 240 CVPEQTEYAT IVFPSGMGTS SPARRGSADG PRSAQPLRPE DGHCSWPL
288
[0115] In accordance with the invention, because B7-1 and fragments
thereof can also bind to B7-H1 and send inhibitory transduction to
T cells through B7-H1, blocking of this interaction can also reduce
inhibitory signal transduction that occurs through B7-H1. Compounds
for use in the invention include those molecules that block this
type of interaction. Such molecules have been disclosed in Butte et
al (2007), supra, and include anti-B7-H1 antibodies with
dual-specificity that block either the B7-H1:B7-1 and B7-H1:PD-1
interaction as well as antibodies exhibiting mono-specificity that
block the PD-L1:B7-1 interaction. Compounds that block this
interaction by blocking B7-1 are also useful, and include anti-B7-1
antibodies.
4. Variant Polypeptides
[0116] Polypeptides useful in the invention, as described, include
those that are mutated to contain one or more amino acid
substitutions, deletions, or insertions. Methods for mutagenesis
are known in the art. The mutated or variant polypeptides inhibit
or reduce inhibitory signal transduction through PD-1 receptors by
binding to ligands of PD-1. Alternatively, the variants (e.g. B7-DC
polypeptides) can bind to the PD-1 receptor and inhibit, reduce, or
block inhibitory signal transduction through the PD-1 receptor. The
variant polypeptides may be of any species of origin. In one
embodiment, the variant polypeptide is from a mammalian species. In
a preferred embodiment, the variant polypeptide is of murine or
primate, preferably human, origin.
[0117] In one embodiment the variant polypeptide is a B7-DC
polypeptide that has the same binding affinity to PD-1 as wildtype
or non-variant B7-DC but does not have or has less than 10% ability
to trigger inhibitory signal transduction through the PD-1 receptor
relative to a non-mutated B7-DC polypeptide. In other embodiments,
the variant B7-DC polypeptide has 10%, 20%, 30%, 40%, 50%, or 60%
more binding affinity to PD-1 than wildtype B7-DC without
triggering PD-1 inhibitory signaling transduction.
[0118] A variant polypeptide (e.g. a variant B7-DC polypeptide)
includes those having any combination of amino acid substitutions,
deletions or insertions so long as the PD-1 antagonizing activity
is not substantially reduced versus the wild type. However, where
there is such a reduction, this should be by no more than half that
of the wild type so that said variant has at least 50% of the PD-1
antagonist activity of the wild type protein, preferably at least
60%, more preferably at least 80%, most preferably at least 90% or
95%, with at least 100% being especially preferred. Increases in
such activity resulting from said variant is even more desirable.
In one embodiment, isolated B7-DC variant polypeptides have amino
acid alterations such that their amino acid sequence shares at
least 60, 70, 80, 85, 90, 95, 97, 98, 99, 99.5 or 100% identity
with an amino acid sequence of a wild type B7-DC polypeptide,
especially that from a mammal, preferably wild type murine or wild
type primate, preferably human, B7-DC polypeptide.
[0119] Polypeptide sequence identity can be calculated using the
definition of % identity provided hereinabove.
[0120] Amino acid substitutions in polypeptides may be
"conservative" or "non-conservative".
[0121] B7 family molecules, including B7-DC, are expressed at the
cell surface with a membrane proximal constant IgC domain and a
membrane distal IgV domain. Receptors for these ligands share a
common extracellular IgV-like domain. Interactions of
receptor-ligand pairs are mediated predominantly through residues
in the IgV domains of the ligands and receptors. In general, IgV
domains are described as having two sheets that each contains a
layer of .beta.-strands. These .beta.-strands are referred to as
A', B, C, C', C'', D, E, F and G. In one embodiment the B7-DC
variant polypeptides contain amino acid alterations (i.e.,
substitutions, deletions or insertions) within one or more of these
.beta.-strands in any possible combination. In another embodiment,
B7-DC variants contain one or more amino acid alterations (i.e.,
substitutions, deletions or insertions) within the A', C, C', C'',
D, E, F or G .beta.-strands. In one embodiment, B7-DC variants
contain one or more amino acid alterations in the G
.beta.-strand.
[0122] With respect to murine or primate, preferably human, B7-DC,
a variant B7-DC polypeptide can contain, without limitation,
substitutions, deletions or insertions at positions that do not
substantially reduce binding to PD-1 relative to non-mutated
B7-DC.
[0123] It is understood, however, that substitutions at the recited
amino acid positions can be made using any amino acid or amino acid
analog. For example, the substitutions at the recited positions can
be made with any of the naturally-occurring amino acids (e.g.,
alanine, aspartic acid, asparagine, arginine, cysteine, glycine,
glutamic acid, glutamine, histidine, leucine, valine, isoleucine,
lysine, methionine, proline, threonine, serine, phenylalanine,
tryptophan, or tyrosine).
[0124] While the substitutions described herein are with respect to
mouse and primate, especially human, B7-DC, it is noted that one of
ordinary skill in the art could readily make equivalent alterations
in the corresponding polypeptides from other species (e.g., rat,
hamster, guinea pig, gerbil, rabbit, dog, cat, horse, pig, sheep,
cow or non-human primate).
[0125] Preferred fragments include all or part of the extracellular
domain of B7-DC effective to bind to PD-1.
[0126] In one embodiment, variant B7-DC polypeptide fragments are
those that retain the ability to bind to PD-1 without triggering
PD-1 inhibitory signal transduction. One embodiment provides a
variant B7-DC polypeptide that is a fragment of full-length B7-DC
and typically has at least 20 percent, 30 percent, 40 percent, 50
percent, 60 percent, 70 percent, 80 percent, 90 percent, 95
percent, 98 percent, 99 percent, 100 percent, or even more than 100
percent of the PD-1 antagonist activity of the full-length variant
B7-DC polypeptide.
[0127] Useful fragments of variant B7-DC polypeptides include
soluble fragments. Soluble B7-DC fragments are fragments of B7-DC
that may be shed, secreted or otherwise extracted from the
producing cells. In one embodiment, variant B7-DC polypeptide
fragments include the entire extracellular domain of B7-DC. The
extracellular domain of B7-DC includes amino acids from about 20 to
about amino acid 221 of murine or primate, preferably human, B7-DC.
In another embodiment, variant B7-DC polypeptide fragments include
the IgC and IgV domains of B7-DC. In another embodiment, variant
B7-DC polypeptide fragments include the IgV domain of B7-DC.
[0128] In one embodiment, variant B7-DC polypeptide fragments
contain a region of the polypeptide that is important for binding
affinity for PD-1. These polypeptide fragments are useful to bind
to and block the PD-1 receptor to prevent native ligands from
binding to PD-1 receptor, thereby enhancing an immune response.
Inhibiting interactions of native B7-H1 or B7-DC with PD-1 inhibits
the suppression of immune responses that would otherwise occur. A
polypeptide fragment of mouse or primate, preferably human, B7-DC
that binds to PD-1 contains, by way of non-limiting example, amino
acids 101-105, or 111-113. The binding of B7-H1 to PD-1 receptor
typically is inhibited by at least 50 percent, or by at least 60
percent, or by at least 70 percent, or by at least 75 percent, or
by at least 80 percent, or by at least 90 percent, or by at least
95 percent, or more compared to the level of binding of B7-H1 to
PD-1 in the absence of the fragment.
[0129] Human PD-1 mutant A99L binds B7-DC and B7-H1 with higher
affinity than unmutated human PD-1 (Lazar Molnar et al PNAS 105 p.
10483-10488 (2008)). In one embodiment of the invention, the
compound acting to reduce inhibitory signal transduction is a
soluble protein, such as the ECD of PD-1 incorporating this
mutation.
5. Modified Polypeptides
[0130] Polypeptides useful in the invention, as described,
including variants, homologs and fragments thereof, can be modified
by chemical moieties found associated with polypeptides in the
normal cellular environment, for example, by phosphorylation,
methylation, amidation, sulfation, acylation, glycosylation,
sumoylation and ubiquitylation of the polypeptide.
[0131] Such polypeptides may also be modified by chemical moieties
that are not normally part of polypeptides in a cellular
environment. Such modifications can be introduced into the molecule
by reacting targeted amino acid residues of the polypeptide with an
organic derivatizing agent that is capable of reacting with
selected side chains or terminal residues. Another useful
modification is cyclization of the protein. Such modifications also
include introduction of a label capable of providing a detectable
signal, either directly or indirectly, including, but not limited
to, radioisotopes and fluorescent compounds.
[0132] Examples of chemical derivatives of the polypeptides include
lysinyl and amino terminal residues derivatized with succinic or
other carboxylic acid anhydrides. Derivatization with a cyclic
carboxylic anhydride has the effect of reversing the charge of the
lysinyl residues. Other suitable reagents for derivatizing
amino-containing residues include imidoesters such as methyl
picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride;
trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione;
and transaminase-catalyzed reaction with glyoxylate. Carboxyl side
groups, aspartyl or glutamyl, may be selectively modified by
reaction with carbodiimides (R--N.dbd.C.dbd.N--R') such as
1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide or
1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,
aspartyl and glutamyl residues can be converted to asparaginyl and
glutaminyl residues by reaction with ammonia. Polypeptides of the
invention can also include one or more D-amino acids that are
substituted for one or more L-amino acids.
[0133] In other embodiments, the potentiating agent, such as CTX,
may be itself part of the compound that reduces inhibitory signal
transduction, such as where the potentiating agent is chemically
linked to a PD-1 antagonist of the invention.
6. Fusion Proteins
[0134] Fusion polypeptides having a first fusion partner, or
polypeptide portion, comprising all or a part of a PD-1 antagonist
protein, a B7-DC polypeptide for example, (including variants,
homologs and fragments thereof) fused (i) directly to a second
polypeptide or, (ii) optionally, fused to a linker peptide sequence
that is fused to the second polypeptide are also provided. The
presence of the fusion partner can alter, for example, the
solubility, affinity and/or valency of the PD-1 antagonist
polypeptide. The disclosed fusion proteins include any combination
of amino acid alteration (i.e., substitution, deletion or
insertion), fragment, and/or modification of a PD-1 antagonist
polypeptide as described above. In one embodiment, B7-DC fusion
proteins include the extracellular domain of a B7-DC protein as the
first binding partner. In another embodiment, such B7-DC fusion
proteins include the IgV and IgC domain of a B7-DC protein as the
first binding partner. In another embodiment, variant B7-DC fusion
proteins include the IgV domain of a B7-DC protein as the first
binding partner.
[0135] Representative first fusion partners include primate,
preferably human, or murine B7-DC polypeptide, fragments thereof,
and variants thereof disclosed hereinabove. Preferred fragments
include the extracellular domain of B7-DC. As recited, the
extracellular domain can include 1-10 contiguous amino acids of a
signal peptide, B7-DC transmembrane domain, or both.
[0136] In one embodiment, the compositions and/or products and/or
methods of the invention utilize PD-1 receptor antagonist,
especially polypeptides, including variants, homologs and fragments
thereof, that are coupled to other polypeptides to form fusion
proteins that antagonize the PD-1 receptor by binding a PD-1
ligand, such as B7-H1, thereby inhibiting the ligand from
interacting with PD-1. In another embodiment, PD-1 receptor
antagonist polypeptides, or variants thereof, are coupled to other
polypeptides to form fusion proteins that antagonize the PD-1
receptor by binding to and blocking the PD-1 receptor and inhibit
or reduce inhibitory signal transduction through PD-1.
[0137] The second polypeptide binding partner, or second
polypeptide portion, may be N-terminal or C-terminal relative to
the PD-1 antagonist polypeptide. In a preferred embodiment, the
second polypeptide is C-terminal to the PD-1 antagonist
polypeptide.
[0138] In a preferred embodiment, the fusion protein contemplated
for use in the methods and compositions and/or products of the
invention comprises at least a portion of an antibody. With the
advent of methods of molecular biology and recombinant technology,
it is now possible to produce antibody molecules by recombinant
means and thereby generate gene sequences that code for specific
amino acid sequences found in the polypeptide structure of the
antibodies. Such antibodies can be produced by either cloning the
gene sequences encoding the polypeptide chains of said antibodies
or by direct synthesis of said polypeptide chains, with in vitro
assembly of the synthesized chains to form active tetrameric
(H.sub.2L.sub.2) structures with affinity for specific epitopes and
antigenic determinants. This has permitted the ready production of
antibodies having sequences characteristic of neutralizing
antibodies from different species and sources.
[0139] Regardless of the source of the antibodies, or how they are
recombinantly constructed, or how they are synthesized, in vitro or
in vivo, using transgenic animals, such as cows, goats and sheep,
using large cell cultures of laboratory or commercial size, in
bioreactors or by direct chemical synthesis employing no living
organisms at any stage of the process, all antibodies have a
similar overall 3 dimensional structure. This structure is often
given as H.sub.2L.sub.2 and refers to the fact that antibodies
commonly comprise 2 light (L) amino acid chains and 2 heavy (H)
amino acid chains. Both chains have regions capable of interacting
with a structurally complementary antigenic target. The regions
interacting with the target are referred to as "variable" or "V"
regions and are characterized by differences in amino acid sequence
from antibodies of different antigenic specificity.
[0140] In preferred embodiments, the PD-1 receptor antagonist
polypeptides, including fragments, mutants and other variants, have
a first fusion partner having all or a part of a B7-DC protein or
variant thereof fused (i) directly to a second polypeptide or, (ii)
optionally, fused to a linker peptide sequence that is fused to the
second polypeptide. The presence of the fusion partner can alter
the solubility, affinity and/or valency of the B7-DC polypeptide.
In more preferred embodiments, B7-DC polypeptides are fused to one
or more domains of an Ig heavy chain constant region, more
preferably an amino acid sequence corresponding to the hinge,
C.sub.H2 and C.sub.H3 regions of a human immunoglobulin C.gamma.1
chain or to the hinge, C.sub.H2 and C.sub.H3 regions of a murine
immunoglobulin C.gamma.2a chain. In a preferred embodiment, the
constant region preferably includes a mutation (for example N297Q)
to eliminate or reduce Fc receptor binding.
[0141] The hinge, C.sub.H2 and C.sub.H3 regions of a human
immunoglobulin C.gamma.1 chain has the following amino acid
sequence:
TABLE-US-00005 (SEQ ID NO: 6) EPKSCDKTHT CPPCPAPELL GGPSVFLFPP
KPKDTLMISR TPEVTCVVVD VSHEDPEVKF 60 NWYVDGVEVH NAKTKPREEQ
YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT 120 ISKAKGQPRE
PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP 180
PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK. 232
[0142] The hinge, C.sub.H2 and C.sub.H3 regions of a murine
immunoglobulin C.gamma.2a chain has the following amino acid
sequence:
TABLE-US-00006 (SEQ ID NO: 7) EPRGPTIKPC PPCKCPAPNL LGGPSVFIFP
PKIKDVLMIS LSPIVTCVVV DVSEDDPDVQ 60 ISWFVNNVEV HTAQTQTHRE
DYNSTLRVVS ALPIQHQDWM SGKEFKCKVN NKDLPAPIER 120 TISKPKGSVR
APQVYVLPPP EEEMTKKQVT LTCMVTDFMP EDIYVEWTNN GKTELNYKNT 180
EPVLDSDGSY FMYSKLRVEK KNWVERNSYS CSVVHEGLHN HHTTKSFSRT PGK 233
[0143] Exemplary murine B7-DC fusion proteins contain amino acids
20-221 of murine B7-DC fused to amino acids 237-469 of murine IgG2a
(CAA49868). Human B7-DC fusion proteins can contain amino acids
20-221 of human B7-DC fused to amino acids 245-476 of human IgG1
(AAA02914). The signal peptides for B7-DC fusion proteins can be
the endogenous signal peptides or any other signal peptide that
facilitates secretion of the fusion protein from a host.
[0144] A representative murine B7-DC-Ig fusion protein is encoded
by the nucleic acid sequence of SEQ ID NO:8.
[0145] It will be appreciated that the disclosed nucleic acid
sequences can be codon-optimized to increase levels of expression
for synthesizing the fusion proteins useful in the methods and
compositions of the present invention. Methods for codon
optimization are known in the art.
[0146] The murine B7-DC-Ig fusion protein encoded by SEQ ID NO:8
has the following amino acid sequence:
TABLE-US-00007 (SEQ ID NO: 9) MLLLLPILNL SLQLHPVAAL FTVTAPKEVY
TVDVGSSVSL ECDFDRRECT ELEGIRASLQ 60 KVENDTSLQS ERATLLEEQL
PLGKALFHIP SVQVRDSGQY RCLVICGAAW DYKYLTVKVK 120 ASYMRIDTRI
LEVPGTGEVQ LTCQARGYPL AEVSWQNVSV PANTSHIRTP EGLYQVTSVL 180
RLKPQPSRNF SCMFWNAHMK ELTSAIIDPL SRMEPKVPRT WEPRGPTIKP CPPCKCPAPN
240 LLGGPSVFIF PPKIKDVLMI SLSPIVTCVV VDVSEDDPDV QISWFVNNVE
VHTAQTQTHR 300 EDYNSTLRVV SALPIQHQDW MSGKEFKCKV NNKDLPAPIE
RTISKPKGSV RAPQVYVLPP 360 PEEEMTKKQV TLTCMVTDFM PEDIYVEWTN
NGKTELNYKN TEPVLDSDGS YFMYSKLRVE 420 KKNWVERNSY SCSVVHEGLH
NHHTTKSFSR TPGK 454
[0147] SEQ ID NO:10 provides the amino acid sequence for murine
B7-DC-Ig fusion protein without the signal sequence.
TABLE-US-00008 (SEQ ID NO: 10) LFTVTAPKEV YTVDVGSSVS LECDFDRREC
TELEGIRASL QKVENDTSLQ SERATLLEEQ 60 LPLGKALFHI PSVQVRDSGQ
YRCLVICGAA WDYKYLTVKV KASYMRIDTR ILEVPGTGEV 120 QLTCQARGYP
LAEVSWQNVS VPANTSHIRT PEGLYQVTSV LRLKPQPSRN FSCMFWNAHM 180
KELTSAIIDP LSRMEPKVPR TWEPRGPTIK PCPPCKCPAP NLLGGPSVFI FPPKIKDVLM
240 ISLSPIVTCV VVDVSEDDPD VQISWFVNNV EVHTAQTQTH REDYNSTLRV
VSALPIQHQD 300 WMSGKEFKCK VNNKDLPAPI ERTISKPKGS VRAPQVYVLP
PPEEEMTKKQ VTLTCMVTDF 360 MPEDIYVEWT NNGKTELNYK NTEPVLDSDG
SYFMYSKLRV EKKNWVERNS YSCSVVHEGL 420 HNHHTTKSFS RTPGK 435
[0148] In one embodiment human B7-DC-Ig is encoded by the nucleic
acid sequence of SEQ ID NO:11, encoding the amino acid sequence for
human B7-DC-Ig:
TABLE-US-00009 (SEQ ID NO: 12) MIFLLLMLSL ELQLHQIAAL FTVTVPKELY
IIEHGSNVTL ECNFDTGSHV NLGAITASLQ 60 KVENDTSPHR ERATLLEEQL
PLGKASFHIP QVQVRDEGQY QCIIIYGVAW DYKYLTLKVK 120 ASYRKINTHI
LKVPETDEVE LTCQATGYPL AEVSWPNVSV PANTSHSRTP EGLYQVTSVL 180
RLKPPPGRNF SCVFWNTHVR ELTLASIDLQ SQMEPRTHPT WEPKSCDKTH TCPPCPAPEL
240 LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK FNWYVDGVEV
HNAKTKPREE 300 QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS NKALPAPIEK
TISKAKGQPR EPQVYTLPPS 360 RDELTKNQVS LTCLVKGFYP SDIAVEWESN
GQPENNYKTT PPVLDSDGSF FLYSKLTVDK 420 SRWQQGNVFS CSVMHEALHN
HYTQKSLSLS PGK 453
[0149] The present invention specifically contemplates embodiments
where the mature fusion protein useful in the methods and
compositions of the invention have the signal sequence removed. In
a preferred embodiment, the signal sequence is completely
removed.
[0150] SEQ ID NO:13 provides the amino acid sequence for human
B7-DC-Ig without the signal sequence.
TABLE-US-00010 (SEQ ID NO: 13) LFTVTVPKEL YIIEHGSNVT LECNFDTGSH
VNLGAITASL QKVENDTSPH RERATLLEEQ 60 LPLGKASFHI PQVQVRDEGQ
YQCIIIYGVA WDYKYLTLKV KASYRKINTH ILKVPETDEV 120 ELTCQATGYP
LAEVSWPNVS VPANTSHSRT PEGLYQVTSV LRLKPPPGRN FSCVFWNTHV 180
RELTLASIDL QSQMEPRTHP TWEPKSCDKT HTCPPCPAPE LLGGPSVFLF PPKPKDTLMI
240 SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV
SVLTVLHQDW 300 LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP
SRDELTKNQV SLTCLVKGFY 360 PSDIAVEWES NGQPENNYKT TPPVLDSDGS
FFLYSKLTVD KSRWQQGNVF SCSVMHEALH 420 NHYTQKSLSL SPGK. 434
[0151] The present invention specifically contemplates embodiments
where the disclosed B7-DC-Ig fusion proteins used in the methods
and compositions disclosed herein have at least about 80%, 85%,
90%, 99% or 100% sequence identity to SEQ ID NO: 9, 10, 12, or
13.
[0152] In another embodiment of the invention, the fusion
polypeptide may have bi-specific function whereby the first fusion
partner binds to a ligand of PD-1, such as B7-H1, and the second
fusion partner binds to the PD-1 receptor without triggering
inhibitory signal transduction through the PD-1 receptor.
[0153] While a polypeptide useful in the invention may be monomeric
or dimeric, the fusion proteins themselves may be present in a
monomeric or an oligomeric form, preferably as a dimer. In specific
embodiments, the fusion proteins useful as PD-1 antagonists in the
methods and compositions of the invention may assemble
spontaneously into oligomeric, especially dimeric, forms or may be
chemically linked to form such oligomers by means well known in the
art. For example, a fusion protein useful in practicing the
invention may itself comprise a portion of a B7-DC polypeptide
fused to a portion of an antibody and these may be further
assembled into a dimer. In one such example, a polypeptide for use
in the invention is fused as a single amino acid chain to the Fc
region of an antibody (such as where this construct is expressed
from a single recombinant polynucleotide), after which two such
fusion products are linked to each other to form a homodimer, such
as by a disulfide linkage between the respective Fc regions.
[0154] Such dimeric products may be homodimers (where both
monomeric fusion proteins are identical) or may be heterodimers
(where two different fusion proteins are linked to each other). The
individual monomers of such dimers may be linked by any means known
in the art, such as by covalent linkage (e.g., a disulfide bond) or
by non-covalent linkage (such as an ionic interaction). The
B7-DC-Ig used in the examples of the invention were present in the
form of a homodimer having 2 copies of SEQ ID NO: 10 linked
together by a disulfide linkage. In addition, the heterodimers of
the invention include bispecific proteins and fusion proteins
wherein one monomeric portion binds to PD-1 and the other binds to
a natural ligand of PD-1. Such heterodimers are formed by coupling
of polypeptides and fusion proteins fully described elsewhere
herein.
[0155] In another useful embodiment of the invention, the PD-1
antagonist is a heterodimer, such as where two fusion proteins are
linked together but they are not of identical amino acid sequence.
In a specific example, each monomer may comprise an Fc portion of
an antibody linked to an active fragment of a B7-DC polypeptide
where these active fragments are from different portions of the
B7-DC polypeptide or where a fusion protein comprising an Fc
portion of an antibody fused to a full length native B7-DC
polypeptide is linked (for example, cross-linked) to a fusion
protein comprising an Fc portion of an antibody and an active
fragment of a full length native B7-DC polypeptide. In each such
case, the portion of the antibody used in forming each monomeric
fusion protein may be different between the two monomeric units.
Any such dimeric combination is specifically contemplated by the
methods and compositions of the invention.
[0156] In a preferred dimeric fusion protein, the dimer results
from the covalent bonding of Cys residue in the CH regions of two
of the Ig heavy chains that are the same Cys residues that are
disulfide linked in dimerized normal Ig heavy chains.
[0157] Still another embodiment provides a tetramer construct
having a BirA substrate fused to the extracellular domain of a
variant B7-DC polypeptide. Methods for making tetramer constructs
are known in the art (see Pertovas, et al., J. Exp. Med., 203:2281
(2006)).
7. Anti-PD-1 and Other Antibodies
[0158] Other PD-1 antagonists contemplated by the methods of this
invention include antibodies that bind to PD-1 or ligands of PD-1,
and other antibodies.
[0159] In one aspect, the present invention relates to a method of
increasing a T cell response in a mammal in need thereof,
comprising administering to said mammal an effective treatment
regimen comprising an anti-PD-1 antibody and a potentiating agent,
wherein said treatment regimen is effective to increase the T cell
response of said mammal to said antigen.
[0160] Anti-PD-1 antibodies useful in the treatment regimens(s) of
the invention include, but are not limited to, those described in
the following publications: [0161] PCT/IL03/00425 (Hardy et al.,
WO/2003/099196) [0162] PCT/JP2006/309606 (Korman et al.,
WO/2006/121168) [0163] PCT/US2008/008925 (Li et al.,
WO/2009/014708) [0164] PCT/JP03/08420 (Honjo et al.,
WO/2004/004771) [0165] PCT/JP04/00549 (Honjo et al.,
WO/2004/072286) [0166] PCT/IB2003/006304 (Collins et al.,
WO/2004/056875) [0167] PCT/US2007/088851 (Ahmed et al.,
WO/2008/083174) [0168] PCT/US2006/026046 (Korman et al.,
WO/2007/005874) [0169] PCT/US2008/084923 (Terrett et al.,
WO/2009/073533) [0170] Berger et al., Clin. Cancer Res., Vol. 14,
pp. 30443051 (2008).
[0171] A specific example of an anti-PD-1 antibody useful in the
methods of the invention is MDX-1106 (see Kosak, US 20070166281
(pub. 19 Jul. 2007) at par. 42), a human anti-PD-1 antibody,
preferably administered at a dose of 3 mg/kg.
[0172] In another aspect, the present invention relates to a method
of increasing a T cell response in a mammal in need thereof,
comprising administering to said mammal an effective treatment
regimen comprising an anti-PD-1 ligand antibody, an anti-B7-H1
antibody for example, and a potentiating agent, wherein said
treatment regimen is effective to increase the T cell response of
said mammal to said antigen.
[0173] Anti-B7-H1 antibodies useful in the treatment regimens(s) of
the invention include, but are not limited to, those described in
the following publications: [0174] PCT/US06/022423 (WO/2006/133396,
pub. 14 Dec. 2006) [0175] PCT/US07/088,851 (WO/2008/083174, pub. 10
Jul. 2008) [0176] US 2006/0110383 (pub. 25 May 2006)
[0177] A specific example of an anti-B7-H1 antibody useful in the
methods of the invention is MDX-1105 (WO/2007/005874, published 11
Jan. 2007)), a human anti-B7-H1 antibody.
For anti-B7-DC antibodies see U.S. Pat. Nos. 7,411,051, 7,052,694,
7,390,888, 20060099203
[0178] Another embodiment of the invention includes a bi-specific
antibody that comprises an antibody that binds to the PD-1 receptor
bridged to an antibody that binds to a ligand of PD-1, such as
B7-H1. In a preferred embodiment, the PD-1 binding portion reduces
or inhibits signal transduction through the PD-1 receptor.
[0179] The antibody for use in the invention need not be an
anti-PD-1 or anti-PD-1 ligand antibody but may be another antibody
useful in mediating the effects of T cells in an immune response.
In this aspect, the present invention relates to a method of
increasing a T cell response to an antigen in a mammal in need
thereof, comprising administering to said mammal an effective
treatment regimen comprising an anti-CTLA4 antibody and a
potentiating agent, wherein said treatment regimen is effective to
increase the T cell response of said mammal to said antigen. An
example of an anti-CTLA4 antibody contemplated for use in the
methods of the invention includes an antibody as described in
PCT/US2006/043690 (Fischkoff et al., WO/2007/056539).
[0180] Specific examples of an anti-CTLA4 antibody useful in the
methods of the invention are Ipilimumab, also known as MDX-010 or
MDX-101, a human anti-CTLA4 antibody, preferably administered at a
dose of 10 mg/kg, and Tremelimumab a human anti-CTLA4 antibody,
preferably administered at a dose of 15 mg/kg.
8. Small Molecule PD-1 Antagonists
[0181] The PD-1 receptor antagonists can also be small molecule
antagonists. The term "small molecule" refers to small organic
compounds having a molecular weight of more than 100 and less than
about 2,500 daltons, preferably between 100 and 2000, more
preferably between about 100 and about 1250, more preferably
between about 100 and about 1000, more preferably between about 100
and about 750, more preferably between about 200 and about 500
daltons. The small molecules often include cyclical carbon or
heterocyclic structures and/or aromatic or polyaromatic structures
substituted with one or more functional groups. The small molecule
antagonists reduce or interfere with PD-1 receptor signal
transduction by binding to ligands of PD-1 such as B7-H1 and B7-DC
and preventing the ligand from interacting with PD-1 or by binding
directly to and blocking the PD-1 receptor without triggering
signal transduction through the PD-1 receptor.
[0182] In one embodiment, such a small molecule may be administered
in combination with another PD-1 antagonist or CTLA4 antagonist,
such as an antibody specific for PD-1 or one of its ligands or an
antibody specific for CTLA4 or one of its ligands. Thus, such small
molecules may be administered as compounds in one or more of the
methods of the invention or may be administered in combination with
other compounds useful in the methods of the invention. For
example, a series of small organic compounds have been shown to
bind to the B7-1 ligand to prevent binding to CTLA4 (see Erbe et
al., J. Biol. Chem., Vol. 277, pp. 7363-7368 (2002). Such small
organics could be administered alone or together with an anti-CTLA4
antibody, in combination with CTX administration, to reduce
inhibitory signal transduction of T cells.
[0183] In one embodiment, PD-1 antagonists or CTLA4 antagonists
contemplated for use in the methods of the invention include
anti-sense nucleic acids, both DNA and RNA, as well as siRNA
molecules. Such anti-sense molecules prevent expression of PD-1 on
T cells as well as production of T cell ligands, such as B7-H1,
PD-L1 and PD-L2. For example, siRNA (for example, of about 21
nucleotides in length, which is specific for the gene encoding
PD-1, or encoding a PD-1 ligand, and which oligonucleotides can be
readily purchased commercially) complexed with carriers, such as
polyethyleneimine (see Cubillos-Ruiz et al., J. Clin. Invest.
119(8): 2231-2244 (2009), are readily taken up by cells that
express PD-1 as well as ligands of PD-1 and reduce expression of
these receptors and ligands to achieve a decrease in inhibitory
signal transduction in T cells, thereby activating T cells.
B. Potentiating Agents
[0184] In accordance with the invention, the activity of the PD-1
antagonist is increased, preferably synergistically, by the
presence of a potentiating agent. The potentiating agent acts to
increase the efficacy of the PD-1 receptor antagonist, possibly by
more than one mechanism, although the precise mechanism of action
is not essential to the broad practice of the present
invention.
[0185] In the preferred embodiment, the potentiating agent is
cyclophosphamide. Cyclophosphamide (CTX, Cytoxan.RTM., or
Neosar.RTM.) is an oxazahosphorine drug and analogs include
ifosfamide (IFO, Ifex), perfosfamide, trophosphamide (trofosfamide;
Ixoten), and pharmaceutically acceptable salts, solvates, prodrugs
and metabolites thereof (US patent application 20070202077 which is
incorporated in its entirety). Ifosfamide (MITOXANA.RTM.) is a
structural analog of cyclophosphamide and its mechanism of action
is considered to be identical or substantially similar to that of
cyclophosphamide. Perfosfamide (4-hydroperoxycyclophosphamide) and
trophosphamide are also alkylating agents, which are structurally
related to cyclophosphamide. For example, perfosfamide alkylates
DNA, thereby inhibiting DNA replication and RNA and protein
synthesis. New oxazaphosphorines derivatives have been designed and
evaluated with an attempt to improve the selectivity and response
with reduced host toxicity (Liang J, Huang M, Duan W, Yu X Q, Zhou
S. Design of new oxazaphosphorine anticancer drugs. Curr Pharm Des.
2007; 13(9):963-78. Review). These include mafosfamide (NSC
345842), glufosfamide (D19575, beta-D-glucosylisophosphoramide
mustard), S-(-)-bromofosfamide (CBM-11), NSC 612567
(aldophosphamide perhydrothiazine) and NSC 613060 (aldophosphamide
thiazolidine). Mafosfamide is an oxazaphosphorine analog that is a
chemically stable 4-thioethane sulfonic acid salt of 4-hydroxy-CPA.
Glufosfamide is IFO derivative in which the isophosphoramide
mustard, the alkylating metabolite of IFO, is glycosidically linked
to a beta-D-glucose molecule. Additional cyclophosphamide analogs
are described in U.S. Pat. No. 5,190,929 entitled "Cyclophosphamide
analogs useful as anti-tumor agents" which is incorporated herein
by reference in its entirety.
[0186] In other embodiments, the potentiating agent is an agent
that reduces activity and/or number of regulatory T lymphocytes
(T-regs), preferably Sunitinib (SUTENT.RTM.), anti-TGF.beta. or
Imatinib (GLEEVAC.RTM.). The recited treatment regimen may also
include administering an adjuvant.
[0187] Useful potentiating agents also include mitosis inhibitors,
such as paclitaxol, aromatase inhibitors (e.g. Letrozole) and
angiogenesis inhibitors (VEGF inhibitors e.g. Avastin, VEGF-Trap)
(see, for example, Li et al., Vascular endothelial growth factor
blockade reduces intratumoral regulatory T cells and enhances the
efficacy of a GM-CSF-secreting cancer immunotherapy. Clin Cancer
Res. 2006 Nov. 15; 12(22):6808-16), anthracyclines, oxaliplatin,
doxorubicin, TLR4 antagonists, and IL-18 antagonists.
C. Pharmaceutical Compositions
[0188] In one aspect, the invention relates to a therapeutic
composition, comprising a molecule that prevents inhibitory signal
transduction through PD-1, or a CTLA4 antagonist, and a
potentiating agent in a pharmaceutically acceptable carrier. The
components of said composition are present in an amount effective
to increase a T cell response in a mammal. In specific embodiments,
the potentiating agent is cyclophosphamide or an analog of
cyclophosphamide, examples of such analogs having been recited
above.
[0189] In other specific examples, the potentiating agent is an
agent that reduces activity of regulatory T lymphocytes (T-regs),
preferably where the activity is reduced due to a decrease in the
number of said T-regs. In preferred non-limiting embodiments, the
agent is Sunitinib (SUTENT.RTM.), anti-TGF.beta. or Imatinib
(GLEEVAC.RTM.).
[0190] The potentiating agent useful in formulating compositions of
the invention also include mitosis inhibitors, such as paclitaxol,
aromatase inhibitors (e.g. Letrozole), agniogenesis inhibitors
(VEGF inhibitors e.g. Avastin, VEGF-Trap), anthracyclines,
oxaliplatin, doxorubicin, TLR4 antagonists, and IL-18
antagonists.
[0191] A therapeutic composition of the invention also optionally
comprises at least one additional agent that may be one or more of
an anti-PD-1 antibody, an anti-CTLA4 antibody, a mitosis inhibitor,
an aromatase inhibitor, an A2a adenosine receptor (A2AR)
antagonist, or an angiogenesis inhibitor.
[0192] Any of the therapeutic compositions of the invention may
also contain one or more adjuvants as described herein.
[0193] A PD-1 antagonist useful as a component of a therapeutic
composition of the invention includes any of the PD-1 antagonists
recited herein for use in any of the methods of the invention. For
example, such PD-1 antagonist includes any of the fusion proteins
recited herein. Such antagonist can also be any of the polypeptides
or PD-1 binding fragments recited herein for use as the first
polypeptide portion of any of the fusion proteins described for use
in any of the methods of the invention. Such antagonist can further
be an antibody, such as any of the known anti-PD-1, -B7-DC or
-B7-H1 antibodies mentioned herein.
[0194] A therapeutic composition of the invention also includes, in
addition to or in place of the aforementioned PD-1 antagonist, an
anti-CTLA4 antibody. Such a composition would therefore contain
such an anti-CTLA4 antibody and a potentiating agent of the kind
already described herein.
[0195] A therapeutic composition of the invention finds use in any
of the methods of the invention disclosed herein. Such composition,
while intended for use as an active treatment of a disease
condition, may also find use as prophylactic compositions to
prevent any of the diseases recited herein.
[0196] In one aspect, the present invention contemplates a
therapeutic composition comprising a PD-1 antagonist and a
potentiating agent in a pharmaceutically acceptable carrier,
wherein the PD-1 antagonist and the potentiating agent are together
present in an amount effective to increase a T cell response in a
mammal.
[0197] Therapeutic compositions within the scope of the invention
include compositions comprising any and all combinations of the
PD-1 antagonists and/or antibodies disclosed herein with any of the
recited potentiating agents. By way of non-limiting examples, a
therapeutic composition of the invention includes a composition
comprising an effective amount of one or more PD-1 antagonists,
such as a combination of any or all of the full length polypeptides
enumerated herein as specific SEQ ID NOs. or homologs thereof
together with one or more fragments of any of said polypeptides,
including where any or all of these are fused to other proteins,
such as being fused to one or more immunoglobulins recited herein,
or not so fused, and comprising one or more potentiating agents,
such as cyclophosphamide alone, or cyclophosphamide plus one or
more analogs thereof, of just one or more analogs of
cyclophosphamide, or the potentiating agent may consist of
cyclophosphamide and an agent that reduces T reg number in a mammal
receiving the composition, or may consist of a cyclophosphamide
analog plus an agent that reduces T reg number or the potentiating
agent may consist only of one or more agents that reduce T reg
number or other Treg activity. All such combinations are
contemplated by the invention so long as the composition comprises
at least one PD-1 anatgonist and/or antibody mediating T cell
activity and at least one potentiating agent.
[0198] The compositions of the invention may also include
additional active agents. In preferred embodiments of any of the
compositions of the invention, the pharmaceutical or therapeutic
composition further comprises at least one additional agent
selected from the group consisting of an anti-PD-1 antibody, an
anti-CTLA4 antibody, a mitosis inhibitor, such as paclitaxel, an
aromatase inhibitor, such as letrozole, an A2AR antagonist, an
angiogenesis inhibitor, anthracyclines, oxaliplatin, doxorubicin,
TLR4 antagonists, and IL-18 antagonists.
[0199] The PD-1 antagonist and/or potentiating agent may be
administered by any suitable means. In a preferred embodiment, the
PD-1 antagonist and/or potentiating agent is administered in an
aqueous solution, by parenteral injection. The formulation may also
be in the form of a suspension or emulsion. In general,
pharmaceutical compositions are provided including effective
amounts of a peptide or polypeptide, and optionally include
pharmaceutically acceptable diluents, preservatives, solubilizers,
emulsifiers, adjuvants and/or carriers. Such compositions include
diluents sterile water, buffered saline of various buffer content
(e.g., Tris-HCl, acetate, phosphate), pH and ionic strength; and
optionally, additives such as detergents and solubilizing agents
(e.g., TWEEN 20, TWEEN 80, Polysorbate 80), anti-oxidants (e.g.,
ascorbic acid, sodium metabisulfite), and preservatives (e.g.,
Thimersol, benzyl alcohol) and bulking substances (e.g., lactose,
mannitol). Examples of non-aqueous solvents or vehicles are
propylene glycol, polyethylene glycol, vegetable oils, such as
olive oil and corn oil, gelatin, and injectable organic esters such
as ethyl oleate. The formulations may be lyophilized and
redissolved/resuspended immediately before use. The formulation may
be sterilized by, for example, filtration through a bacteria
retaining filter, by incorporating sterilizing agents into the
compositions, by irradiating the compositions, or by heating the
compositions.
[0200] Pharmaceutical compositions of the invention may be
administered by parenteral (intramuscular, intraperitoneal,
intravenous (IV) or subcutaneous injection), transdermal (either
passively or using iontophoresis or electroporation), or
transmucosal (nasal, vaginal, rectal, or sublingual) routes of
administration. The methods of the invention do not preclude
administering the PD-1 antagonist and the potentiating agent by
separate and different routes (e.g. topically).
[0201] The PD-1 antagonist and the potentiating agent may be
administered at the same time, or at different times, with the
potentiating agent being administered before or after the PD-1
antagonist. In one embodiment, a potentiating agent is administered
both before and after the PD-1 antagonist. In one such embodiment,
the same potentiating agent is administered before and after the
PD-1 antagonist. In another embodiment, the potentiating agent
administered before the PD-1 antagonist.
[0202] As used herein the term "effective amount" or
"therapeutically effective amount" means a dosage sufficient to
treat, inhibit, or alleviate one or more symptoms of the disorder
being treated or to otherwise provide a desired pharmacologic
and/or physiologic effect. The precise dosage will vary according
to a variety of factors such as subject-dependent variables (e.g.,
age, immune system health, etc.), the disease, and the treatment
being effected. Therapeutically effective amounts of PD-1 receptor
antagonists and/or antibodies together with a potentiating agents
cause an immune response to be activated or sustained.
[0203] The selected dosage depends upon the desired therapeutic
effect, on the route of administration, and on the duration of the
treatment desired. Generally dosage levels of 0.001 to 50 mg/kg of
body weight daily are administered to mammals. Preferrably, said
dose is 1 to 50 mg/kg, more preferably 1 to 40 mg/kg, or even 1 to
30 mg/kg, with a dose of 2 to 20 mg/kg being also a preferred dose.
Examples of other dosages include 2 to 15 mg/kg, or 2 to 10 mg/kg
or even 3 to 5 mg/kg, with a dose of about 4 mg/kg being a specific
example.
[0204] For treatment regimens using a potentiating agent and an
antibody, such as an anti-PD-1 antibody or an anti-CTLA4 antibody,
dosages are commonly in the range of 0.1 to 100 mg/kg, with shorter
ranges of 1 to 50 mg/kg preferred and ranges of 10 to 20 mg/kg
being more preferred. An appropriate dose for a human subject is
between 5 and 15 mg/kg, with 10 mg/kg of antibody (for example,
human anti-PD-1 antibody, like MDX-1106) most preferred (plus a
suitable dose of cyclophosphamide or other potentiating agent given
up to about 24 hours before the antibody).
[0205] In general, by way of example only, dosage forms based on
body weight for any of the signal transduction antagonists useful
in the methods of the invention include doses in the range of 5-300
mg/kg, or 5-290 mg/kg, or 5-280 mg/kg, or 5-270 mg/kg, or 5-260
mg/kg, or 5-250 mg/kg, or 5-240 mg/kg, or 5-230 mg/kg, or 5-220
mg/kg, or 5-210 mg/kg, or 20 to 180 mg/kg, or 30 to 170 mg/kg, or
40 to 160 mg/kg, or 50 to 150 mg/kg, or 60 to 140 mg/kg, or 70 to
130 mg/kg, or 80 to 120 mg/kg, or 90 to 110 mg/kg, or 95 to 105
mg/kg, with doses of 3 mg/kg, 5 mg/kg, 7 mg/kg, 10 mg/kg, 15 mg/kg,
20 mg/kg, 25 mg/kg, 30 mg/kg, 50 mg/kg and 100 mg/kg being specific
examples of preferred doses. Such doses may, of course, be
repeated. The dose will, of course, be correlated with the identity
of the mammal receiving said dose. Doses in the above-recited mg/kg
ranges are convenient for mammals, including rodents, such as mice
and rats, and primates, especially humans, with doses of about 5
mg/kg, about 10 mg/kg and about 15 mg/kg being especially preferred
for treating humans.
[0206] In accordance with the treatment regimen of the invention,
the potentiating agent, for example cyclophosphamide, is
administered in non-toxic doses that vary depending on the animal.
In specific embodiments, the potentiating agent is administered by
any suitable means of administration, including parenteral or oral,
the former including system administration, such as intravenous.
For example, a potentiating agent like cyclophosphamide is normally
administered orally. Such administration may be at any convenient
dosage, depending on the potentiating agent. The dosage in each
case may be based on body weight or may be administered as a unit
dosage.
[0207] While CTX itself is nontoxic, some of its metabolites are
cytotoxic alkylating agents that induce DNA crosslinking and, at
higher doses, strand breaks. Many cells are resistant to CTX
because they express high levels of the detoxifying enzyme aldehyde
dehydrogenase (ALDH). CTX targets proliferating lymphocytes, as
lymphocytes (but not hematopoietic stem cells) express only low
levels of ALDH, and cycling cells are most sensitive to DNA
alkylation agents.
[0208] Low doses of CTX (<200 mg/kg) can have immune stimulatory
effects, including stimulation of anti-tumor immune responses in
humans and mouse models of cancer (Brode & Cooke Crit Rev.
Immunol. 28:109-126 (2008)). These low doses are sub-therapeutic
and do not have a direct anti-tumor activity. In contrast, high
doses of CTX inhibit the anti-tumor response. Several mechanisms
may explain the role of CTX in potentiation of anti-tumor immune
response: (a) depletion of CD4+CD25+FoxP3+ Treg (and specifically
proliferating Treg, which may be especially suppressive), (b)
depletion of B lymphocytes; (c) induction of nitric oxide (NO),
resulting in suppression of tumor cell growth; (d) mobilization and
expansion of CD11b+Gr-1+ MDSC. These primary effects have numerous
secondary effects; for example following Treg depletion macrophages
produce more IFN-.gamma. and less IL-10. CTX has also been shown to
induce type I IFN expression and promote homeostatic proliferation
of lymphocytes.
[0209] Treg depletion is most often cited as the mechanism by which
CTX potentiates the anti-tumor immune response. This conclusion is
based in part by the results of adoptive transfer experiments. In
the AB1-HA tumor model, CTX treatment at Day 9 gives a 75% cure
rate. Transfer of purified Treg at Day 12 almost completely
inhibited the CTX response (van der Most et al. Cancer Immunol.
Immunother. 58:1219-1228 (2009). A similar result was observed in
the HHD2 tumor model: adoptive transfer of CD4+CD25+ Treg after CTX
pretreatment eliminated therapeutic response to vaccine (Taieb, J.
J. Immunol. 176:2722-2729 (2006)).
[0210] Numerous human clinical trials have demonstrated that low
dose CTX is a safe, well-tolerated, and effective agent for
promoting anti-tumor immune responses (Bas, & Mastrangelo
Cancer Immunol. Immunother. 47:1-12 (1998)).
[0211] The optimal dose for CTX to potentiate an anti-tumor immune
response, is one that lowers overall T cell counts by lowering Treg
levels below the normal range but is subtherapeutic (see Machiels
et al. Cancer Res. 61:3689-3697 (2001)).
[0212] In human clinical trials where CTX has been used as an
immunopotentiating agent, a dose of 300 mg/m.sup.2 has usually been
used. For an average male (6 ft, 170 pound (78 kg) with a body
surface area of 1.98 m.sup.2), 300 mg/m.sup.2 is 8 mg/kg, or 624 mg
of total protein. In mouse models of cancer, efficacy has been seen
at doses ranging from 15-150 mg/kg, which relates to 0.45-4.5 mg of
total protein in a 30 g mouse (Machiels et al. Cancer Res.
61:3689-3697 (2001), Hengst et al Cancer Res. 41:2163-2167 (1981),
Hengst Cancer Res. 40:2135-2141 (1980)).
[0213] For larger mammals, such as a primate, preferably human,
patient, such mg/m.sup.2 doses may be used but unit doses
administered over a finite time interval may be preferred. Such
unit doses may be administered on a daily basis for a finite time
period, such as up to 3 days, or up to 5 days, or up to 7 days, or
up to 10 days, or up to 15 days or up to 20 days or up to 25 days,
are all specifically contemplated by the invention. The same
regimen may be applied for the other potentiating agents recited
herein.
[0214] All such administrations may occur before or after
administration of a PD-1 binding molecule of the invention.
Alternatively, administration of one or more doses of a PD-1
binding molecule of the invention may be temporally staggered with
the administration of potentiating agent to form a uniform or
non-uniform course of treatment whereby one or more doses of
potentiating agent are administered, followed by one or more doses
of a PD-1 binding compound, followed by one or more doses of
potentiating agent, all according to whatever schedule is selected
or desired by the researcher or clinician administering said
agents.
[0215] In other specific embodiments, the treatment regimen
includes multiple administrations of one or more PD-1 antagonists.
In some embodiments, such multiple administrations of PD-1
antagonists are in conjunction with multiple administrations of the
same or different potentiating agents.
[0216] As in other embodiments of the invention, here the
potentiating agent is administered at least 1, 2, 3, 5, 10, 15, 20,
24 or 30 hours prior to or after administering of the
PD-1-antagonist.
[0217] The pharmaceutical compositions useful herein also contain a
pharmaceutically acceptable carrier, including any suitable diluent
or excipient, which includes any pharmaceutical agent that does not
itself induce the production of antibodies harmful to the
individual receiving the composition, and which may be administered
without undue toxicity. Pharmaceutically acceptable carriers
include, but are not limited to, liquids such as water, saline,
glycerol and ethanol, and the like, including carriers useful in
forming sprays for nasal and other respiratory tract delivery or
for delivery to the ophthalmic system. A thorough discussion of
pharmaceutically acceptable carriers, diluents, and other
excipients is presented in REMINGTON'S PHARMACEUTICAL SCIENCES
(Mack Pub. Co., N.J. current edition).
[0218] Vaccine compositions (as discussed below) may further
incorporate additional substances to stabilize pH, or to function
as adjuvants, wetting agents, or emulsifying agents, which can
serve to improve the effectiveness of the vaccine.
[0219] Vaccines are generally formulated for parenteral
administration and are injected either subcutaneously or
intramuscularly. Such vaccines can also be formulated as
suppositories or for oral administration, using methods known in
the art, or for administration through nasal or respiratory
routes.
D. Methods of Manufacture
[0220] Isolated PD-1 antagonist polypeptides, including variants,
homologs and fragments thereof, either wild-type or mutated, and
fusion proteins comprising any of these, all contemplated for use
in the invention, can be obtained by, for example, chemical
synthesis or by recombinant production in a host cell. To
recombinantly produce a costimulatory polypeptide, a nucleic acid
containing a nucleotide sequence encoding the polypeptide can be
used to transform, transduce, or transfect a bacterial or
eukaryotic host cell (e.g., an insect, yeast, or mammalian cell).
It will be appreciated that the nucleotide sequences can be
codon-optimized to increase levels of protein expression in a
particular kind of host cell. Methods for codon optimization are
well known in the art. In general, nucleic acid constructs include
a regulatory sequence operably linked to a nucleotide sequence
encoding a costimulatory polypeptide. Regulatory sequences (also
referred to herein as expression control sequences) typically do
not encode a gene product, but instead affect the expression of the
nucleic acid sequences to which they are operably linked. The
signal peptides used to secrete proteins from a cell can be the
endogenous signal peptides or any other signal peptide that
facilitates secretion of the fusion protein from a host.
[0221] For general molecular biology procedures useful in
practicing the present invention, a number of standard references
are available that contain procedures well known in the art of
molecular biology and genetic engineering and which procedures need
not be further described herein. Useful references include
Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor, N.Y., (1989), Wu et al, Methods in
Gene Biotechnology (CRC Press, New York, N.Y., 1997), and
Recombinant Gene Expression Protocols, in Methods in Molecular
Biology, Vol. 62, (Tuan, ed., Humana Press, Totowa, N.J., 1997),
the disclosures of which are hereby incorporated by reference.
E. Disease Treatment
[0222] Diseases to be treated or prevented by administering a
therapeutic combination provided by the present invention include a
malignant tumor or a chronic infectious disease caused by a
bacterium, virus, protozoan, helminth, or other microbial pathogen
that enters intracellularly. Such diseases are often combatted
through attack by cytotoxic T lymphocytes. Because the present
invention provides combination therapies useful in enhancing T cell
responses, through increased T cell activity, increased T cell
proliferation and reduced T cell inhibitory signals, the
combination therapies of the invention have unique advantage in
treating (or even preventing) such diseases.
[0223] In one embodiment, because viral infections are cleared
primarily by T-cells, an increase in T-cell activity is
therapeutically useful in enhancing clearance of an infective viral
agent from an animal or primate, preferably human, subject. Thus,
the disclosed compounds of the invention, with PD-1 receptor
antagonist activity, together with a potentiating agent work in
combination for the treatment of local or systemic viral
infections. Infections that are to be treated by the compounds of
the invention include, but are not limited to, immunodeficiency
(e.g., HIV), papilloma (e.g., HPV), herpes (e.g., HSV),
encephalitis, influenza (e.g., human influenza virus A), hepatitis
(e.g. HCV, HBV), and common cold (e.g., human rhinovirus) viral
infections. Pharmaceutical formulations of PD-1 receptor
antagonists compositions can also be administered to treat systemic
viral diseases, including, but not limited to, AIDS, influenza, the
common cold, or encephalitis.
[0224] Non-viral infections treatable by the compounds of the
invention include, but are not limited to, infections cause by
microoganisms including, but not limited to, Actinomyces, Anabaena,
Bacillus, Bacteroides, Bdellovibrio, Bordetella, Borrelia,
Campylobacter, Caulobacter, Chlamydia, Chlorobium, Chromatium,
Clostridium, Corynebacterium, Cytophaga, Deinococcus, Escherichia,
Francisella, Halobacterium, Heliobacter, Haemophilus, Hemophilus
influenza type B (HIB), Hyphomicrobium, Legionella, Leptspirosis,
Listeria, Meningococcus A, B and C, Methanobacterium, Micrococcus,
Myobacterium, Mycoplasma, Myxococcus, Neisseria, Nitrobacter,
Oscillatoria, Prochloron, Proteus, Pseudomonas, Phodospirillum,
Rickettsia, Salmonella, Shigella, Spirillum, Spirochaeta,
Staphylococcus, Streptococcus, Streptomyces, Sulfolobus,
Thermoplasma, Thiobacillus, Treponema, Vibrio, Yersinia,
Cryptococcus neoformans, Histoplasma sp. (such as Histoplasma
capsulatum), Candida albicans, Candida tropicalis, Nocardia
asteroides, Rickettsia ricketsii, Rickettsia typhi, Leishmania,
Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis,
Plasmodium sp. (such as Plasmodium falciparum), Trypanosoma brucei,
Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis and
Schistosoma mansoni.
[0225] In one embodiment, the present invention provides methods
and compositions for inducing or enhancing an immune response in
host for treating cancer. The types of cancer that may be treated
with the provided compositions and methods include, but are not
limited to, the following: bladder, brain, breast, cervical,
colo-rectal, esophageal, kidney, liver, lung, nasopharangeal,
pancreatic, prostate, skin, stomach, uterine, ovarian testicular,
and hematologic cancer.
[0226] Malignant tumors which may be treated are classified herein
according to the embryonic origin of the tissue from which the
tumor is derived. Carcinomas are tumors arising from endodermal or
ectodermal tissues such as skin or the epithelial lining of
internal organs and glands. Sarcomas, which arise less frequently,
are derived from mesodermal connective tissues such as bone, fat,
and cartilage. The leukemias and lymphomas are malignant tumors of
hematopoietic cells of the bone marrow. Leukemias proliferate as
single cells, whereas lymphomas tend to grow as tumor masses.
Malignant tumors may show up at numerous organs or tissues of the
body to establish a cancer.
[0227] As a demonstration of the value of the treatment regimens of
the invention, the murine analog of B7-DC-Ig (in which the mouse
B7-DC ECD, which shares 72% sequence identity with the human
protein, is fused to the Fc domain of mouse IgG.sub.2a) tested in
syngeneic mouse tumor models for colon cancer, mastocytoma, and
other tumor types incorporating a cyclophosphamide (CTX)
pre-treatment as described herein.
[0228] The results showed that treatment with a single
subtherapeutic dose of CTX, which acts as an immunopotentiating
agent, followed by murine B7-DC-IG eradicates established CT26
colon carcinoma tumors in up to 80% of the animals. Further, in
CT26 colon carcinoma tumor re-challenge studies, no tumor re-growth
was detected in mice that had previously eradicated tumor following
CTX+ murine B7-DC-Ig treatment. These mice were also shown to have
an increased tumor-specific CTL population relative to naive
mice.
[0229] In one embodiment, the present invention contemplates use of
a compound that reduces inhibitory signal transduction in a T cell,
as described elsewhere herein, in the manufacture of a medicament
for increasing a T cell response by combination therapy wherein
said compound is administered in conjunction with a potentiating
agent. Further, the compound that reduces inhibitory signal
transduction in a T cell and said potentiating agent are provided
as separate medicaments for administration at different times,
preferably where the potentiating agent is administered prior to
the compound that reduces inhibitory signal transduction, for
example, up to 24 hours prior to the inhibitory compound (or other
time intervals recited herein). Preferably, the compound and
potentiating agent are for use in the treatment of an infectious
disease or cancer, including diseases caused by any of the
infectious agents or cancers recited elsewhere herein.
[0230] In a preferred embodiment, a compound useful in these
methods is a recombinant protein composed of the ECD of human B7-DC
fused to the Fc domain of human IgG.sub.1, referred to herein as
B7-DC-Ig.
[0231] In one embodiment, the present invention relates to a
medical kit for administering a compound that reduces inhibitory
signal transduction in a T cell, as disclosed herein, in
combination with a potentiating agent, said kit comprising:
[0232] (a) a dosage supply of a compound that reduces inhibitory
signal transduction in a T cell,
[0233] (b) a supply of a potentiating agent;
[0234] (c) a supply of pharmaceutically acceptable carrier; and
[0235] (d) printed instructions for administering the compound in a
use as described above.
F. Combination Therapies
[0236] Vaccines require strong T cell response to eliminate cancer
cells and infected cells or infectious agents. PD-1 receptor
antagonists described herein can be administered as a component of
a vaccine, along with a potentiating agent, to provide a
costimulatory signal to T cells. Vaccines disclosed herein include
antigens, a PD-1 receptor antagonist and optionally adjuvants and
targeting molecules.
[0237] The antigens against which the T cell response is enhanced
by the methods and composition of the invention includes peptides,
proteins, polysaccharides, saccharides, lipids, nucleic acids, or
combinations thereof. The antigens, in the case of disease, are
present due to the disease process.
[0238] The disclosed PD-1 receptor antagonists compositions may be
administered in conjunction with prophylactic vaccines, which
confer resistance in a subject to subsequent exposure to infectious
agents, or in conjunction with therapeutic vaccines, which can be
used to initiate or enhance a subject's immune response to a
pre-existing antigen, such as a tumor antigen in a subject with
cancer, or a viral antigen in a subject infected with a virus.
[0239] The desired outcome of a prophylactic, therapeutic or
de-sensitized immune response may vary according to the disease,
based on principles well known in the art. For example, an immune
response against an infectious agent may completely prevent
colonization and replication of an infectious agent, affecting
"sterile immunity" and the absence of any disease symptoms.
However, a vaccine against infectious agents may be considered
effective if it reduces the number, severity or duration of
symptoms; if it reduces the number of individuals in a population
with symptoms; or reduces the transmission of an infectious agent.
Similarly, immune responses against cancer, allergens or infectious
agents may completely treat a disease, may alleviate symptoms, or
may be one facet in an overall therapeutic intervention against a
disease. For example, the stimulation of an immune response against
a cancer may be coupled with surgical, chemotherapeutic,
radiologic, hormonal and other immunologic approaches in order to
affect treatment.
[0240] The methods and products of the invention do not preclude
use of an adjuvant in addition to the potentiating agent. Such
adjuvant may be administered, for example, along with the PD-1
antagonist. The adjuvants useful in the compositions and methods of
the invention include, but are not limited to, one or more of the
following: oil emulsions (e.g., Freund's adjuvant); saponin
formulations; virosomes and viral-like particles; bacterial and
microbial derivatives; immunostimulatory oligonucleotides;
ADP-ribosylating toxins and detoxified derivatives; alum; BCG;
mineral-containing compositions (e.g., mineral salts, such as
aluminium salts and calcium salts, hydroxides, phosphates,
sulfates, etc.); bioadhesives and/or mucoadhesives; microparticles;
liposomes; polyoxyethylene ether and polyoxyethylene ester
formulations; muramyl peptides; polyphosphazene; imidazoquinolone
compounds; and surface active substances (e.g. lysolecithin,
pluronic polyols, polyanions, peptides, oil emulsions, keyhole
limpet hemocyanin, and dinitrophenol). Useful adjuvants also
include immunomodulators such as cytokines, interleukins (e.g.,
IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons
(e.g., interferon-.gamma.), macrophage colony stimulating factor,
and tumor necrosis factor.
[0241] Nothing herein precludes the disclosed PD-1 receptor
antagonist, including any of the polypeptides, fragments, variants,
homologs and fusion proteins disclosed herein, from being
administered to a subject in need thereof in combination with one
or more additional therapeutic agents (in addition to the
potentiating agent). The additional therapeutic agents are selected
based on the condition, disorder or disease to be treated. For
example, PD-1 receptor antagonists can be co-administered with one
or more additional agents that function to enhance or promote an
immune response, and which are considered herein as active
agents.
[0242] Such agents include, but are not limited to, amsacrine,
bleomycin, busulfan, capecitabine, carboplatin, carmustine,
chlorambucil, cisplatin, cladribine, clofarabine, crisantaspase,
cytarabine, dacarbazine, dactinomycin, daunorubicin, docetaxel,
doxorubicin, epirubicin, etoposide, fludarabine, fluorouracil,
gemcitabine, hydroxycarbamide, idarubicin, ifosfamide, irinotecan,
leucovorin, liposomal doxorubicin, liposomal daunorubicin,
lomustine, melphalan, mercaptopurine, mesna, methotrexate,
mitomycin, mitoxantrone, oxaliplatin, paclitaxel, pemetrexed,
pentostatin, procarbazine, raltitrexed, satraplatin, streptozocin,
tegafur-uracil, temozolomide, teniposide, thiotepa, tioguanine,
topotecan, treosulfan, vinblastine, vincristine, vindesine,
vinorelbine, or a combination thereof. Representative pro-apoptotic
agents include, but are not limited to fludarabinetaurosporine,
cycloheximide, actinomycin D, lactosylceramide, 15d-PGJ(2) and
combinations thereof.
[0243] The therapies provided by the methods and compositions of
the present invention may also be used in conjunction with other
types of therapies, such as radiation treatments, surgery, and the
like.
G. Assays for Antagonist Activity
[0244] The present invention recites a number specific structures
useful in practicing the methods of the invention. Other compounds
possessing antagonist activity and being useful in the methods of
the invention may also be identified by reference to well known
assay procedures for identifying chemical structures that bind to
PD-1, CTLA4, and ligands of any of these and that also possess the
ability to reduce inhibitory signal transduction in T cells. Some
such assays are binding assays useful in determining if a selected
chemical structure binds to receptors; these are well known in the
art and need not be discussed in detail herein (see, for example,
U.S. 2008/0274490, (pub. 6 Nov. 2008) and U.S. Pat. No. 7,105,328
(issued 12 Sep. 2006), each showing assays for PD-1 signaling
modulators using T cells) the disclosures of which are hereby
incorporated by reference in its entirety. Other assays are used to
determine the effects of agents of the invention, such as active
fragments, to activate T cells by increasing proliferation and/or
production of cytokines. Such assays are also well known in the
art. For example, increased proliferation of cells can be
demonstrated by increased .sup.3H-thymidine incorporation (due to
increased DNA synthesis needed for cellular multiplication) or
ELISA and/or RIA for detecting increased production of cytokines by
T cells in culture.
[0245] In one such experiment, PD-1 binding activity of human
B7-DC-Ig was assessed by ELISA. 96-well ELISA plates were coated
with 100 uL 0.75 ug/mL recombinant human PD-1/Fc (R&D Systems)
diluted in BupH Carbonate/Bicarbonate pH 9.4 buffer (Pierce) for 2
hours and then blocked with BSA solution (Jackson ImmunoResearch)
for 90-120 minutes. Serially diluted human B7-DC-Ig (wild type, as
well as D111S mutein, and K113S mutants that were selected for
reduced binding to PD-1) as well as human IgG1 isotype control were
allowed to bind for 90 minutes. Bound B7-DC-Ig was detected using
100 uL of 0.5 ug/mL biotin conjugated anti-human B7-DC clone MIH18
(eBioscience) followed by 1:1000 diluted HRP-Streptavidin (BD
Bioscience) and TMB substrate (BioFX). Absorbance at 450 nm was
read using a plate reader (Molecular Devices) and data were
analyzed in SoftMax using a 4-parameter logistic fit. The data
showed that human B7-DC-Ig (wildtype) bound to PD-1 but the K113S
and D111S mutants do not bind to PD-1.
[0246] In carrying out the procedures of the present invention it
is of course to be understood that reference to particular buffers,
media, reagents, cells, culture conditions and the like are not
intended to be limiting, but are to be read so as to include all
related materials that one of ordinary skill in the art would
recognize as being of interest or value in the particular context
in which that discussion is presented. For example, it is often
possible to substitute one buffer system or culture medium for
another and still achieve similar, if not identical, results. Those
of skill in the art will have sufficient knowledge of such systems
and methodologies so as to be able, without undue experimentation,
to make such substitutions as will optimally serve their purposes
in using the methods and procedures disclosed herein.
[0247] The invention is described in more detail in the following
non-limiting examples. It is to be understood that these methods
and examples in no way limit the invention to the embodiments
described herein and that other embodiments and uses will no doubt
suggest themselves to those skilled in the art.
EXAMPLES
Example 1
[0248] B7-DC-Ig Binds to PD01 Expressing CHO Cells
[0249] B7-DC-Ig was first conjugated with allophycocyanin (APC) and
then incubated at various concentrations with a CHO cell line
constitutively expressing PD-1 or parent CHO cells that do not
express PD-1. Binding was analyzed by flow cytometry. FIG. 1 shows
the median fluorescence intensity (MFI) of B7-DC-Ig-APC as a
function of the concentration of probe .alpha.-axis). B7-DC-Ig-APC
binds to CHO.PD-1 cells (solid circle) but not untransfected CHO
cells (gray triangle).
Example 2
B7-DC-Ig Competes with B7-H1 for Binding to PD-1
[0250] B7-H1-Ig was first conjugated with allophycocyanin (APC).
Unlabeled B7-DC-Ig at various concentrations was first incubated
with a CHO cell line constitutively expressing PD-1 before adding
B7-H1-Ig-APC to the probe and cell mixture. FIG. 2 shows the median
fluorescence intensity (MFI) of B7-H1-Fc-APC is shown as a function
of the concentration of unlabeled B7-DC-Ig competitor (x-axis)
added. As the concentration of unlabeled B7-DC-Ig is increased the
amount of B7-H1-Ig-APC bound to CHO cells decreases, demonstrating
that B7-DC-Ig competes with B7-H1 for binding to PD-1.
Example 3
CT26 Tumor Model
[0251] Mouse colorectal tumor cell line, CT26, was obtained from
ATCC. A master cell bank at Passage 4 was generated following ATCC
guidelines. Cells were tested and confirmed no mycoplasma and other
pathogen contamination. One vial of tumor cells was thawed from the
cryopreserved stocks and grown for two passages prior to
inoculation.
[0252] CT26 cells were split at 1:5 dilution with 30 mL complete
medium (RPMI+10% FBS, 2 mM L-Glu, and 1.times.P/S) for two days
culture or at 1:10 dilution with 30 ml complete medium for 3 days
culture.
[0253] CT26 cells were harvested by aspirating medium, rinsing the
flask with 5 mL PBS, adding 5 mL trypsin, incubating at 37.degree.
C. for 2 min, and then neutralizing with 10 mL complete medium.
After centrifuge at 600.times.g (.about.1000 rpm) for 5 min, media
was sspirateed and the cell pellet was resuspended by pipetting
with 10 ml plain RPMI. This wash step was repeated for three
times.
[0254] Cell number and viability of the inoculated cells were
analyzed by trypan blue dye staining with proper dilution (e.g. 1:5
dilution, 10 .mu.L cells+40 .mu.L trypan blue) and confirmed by
NOVA cell count during the last wash step. Cell viability generally
was greater than 95% for inoculation.
[0255] CT26 cells were diluted to 6.7.times.10.sup.5 cells/mL for
initial inoculation with plain RPMI and stored on ice. Typically
each mouse was inoculated with 150 .mu.L (1.times.10.sup.5
cells).
[0256] On Day 9, all the tumor-bearing mice were first grouped into
a rat cage and randomly divided the mice to experimental groups.
CTX solution was reconstituted by 1.times.PBS to 4 mg/mL. Mice were
intraperitoneally (IP) injected with 0.5 mL of CTX solution
resulting in 2 mg for a 20 gram mouse, i.e. 100 mg/kg.
[0257] On Day 10, mice were IP injected with 0.5 mL of B7-DC-Ig
(0.2 mg/mL) resulting in 0.1 mg for a 20 gram mouse, i.e. 5 mg/kg.
The same dose was given 2 time a week for 4 weeks, total 8 doses.
Tumor growth were monitored by measuring the tumor twice weekly,
starting on the day when giving B7-DC-Ig via a digital caliper.
Tumor volume was calculated as following:
Tumor
volume=.pi.(D.sub.short).sup.2.times.(D.sub.long)/6=.about.0.52.ti-
mes.(D.sub.short).sup.2.times.(D.sub.long)
[0258] Mice were euthanized and taken off the study if the tumor
volume reached 2000 mm.sup.3 or if there were skin ulcers and
infections at the tumor inoculation site.
Example 4
Combination of Cyclophosphamide and B7-DC-Ig can Eradicate
Established Tumors
[0259] Balb/C mice at age of 9 to 11 weeks were implanted
subcutaneously with 1.0.times.10.sup.5 CT26 colorectal tumor cells
as described above. On day 10 post tumor implantation, mice
received 100 mg/kg of cyclophosphamide. B7-DC-Ig treatment started
1 day later, on day 11. Mice were treated with 100 ug of B7-DC-Ig,
2 doses per week, for 4 weeks and total 8 doses. 75% of the mice
that received the CTX+B7-DC-Ig treatment regimen eradicated the
established tumors by Day 44, whereas all mice in the control CTX
alone group died as a result of tumor growth or were euthanized
because tumors exceeded the sizes approved by IACUC (results shown
in FIG. 3). These results demonstrate the effectiveness of the
treatment regimen on established tumors and not mere
prophylaxis.
Example 5
Combination of Cyclophosphamide and B7-DC-Ig can Eradicate
Established Tumors and Protect Against Tumor Re-Challenge
[0260] Mice eradicated established CT26 colorectal tumors from the
above described experiment were rechallenged with 1.times.10.sup.5
CT26 cells on Day 44 and Day 70. No tumors grew out from the
rechallenge suggesting they had developed long term anti-tumor
immunity from the cyclophosphamide and B7-DC-Ig combination
treatment. All mice in the vehicle control group developed tumors
(results shown in FIG. 4). These results show the effectiveness of
the treatment regimen on established tumors and that the
cyclophosphamide and B7-DCIg combination treatment resulted in
memory responses to tumor antigens.
Example 6
Combination of Cyclophosphamide and B7-DC-Ig can Generate Tumor
Specific, Memory Cytotoxic T Lymphocytes
[0261] Mice eradiated established CT26 colorectal tumors from the
above described experiment were rechallenged with
2.5.times.10.sup.5 CT26 cells on Day 44. Seven days later, mouse
spleens were isolated. Mouse splenocytes were pulsed with 5 or 50
ug/mL of ovalbumin (OVA) or AH1 peptides for 6 hours in the
presence of a Golgi blocker (BD BioScience). Memory T effector
cells were analyzed by assessing CD8.sup.+/IFN.gamma..sup.+ T
cells. Results in FIG. 5 show that there were significant amount of
CT26 specific T effector cells in the CT26 tumor-eradicated
mice.
Example 7
Effect of B7-DC-Ig Dose Dependent on Tumor Eradication
[0262] Balb/C mice at age of 9 to 11 weeks were implanted
subcutaneously with 1.0.times.10.sup.5 CT26 colorectal tumor cells.
On day 9 post tumor implantation, mice received a single dose of
cyclophosphamide (100 mg/kg) and started treatment on Day 10 with
30, 100 or 300 .mu.g of B7-DC-Ig, 2 doses per week for 4 weeks,
total 8 doses. FIG. 6 shows there were 70% of the mice eradicated
the tumors at 300 .mu.g, 40% tumor eradication with 100 .mu.g, and
30 .mu.g dose gave rise to 10% tumor eradication.
Example 8
Combination of Cyclophosphamide and Anti-PD-1 can Eradicate
Established Tumors
[0263] Balb/C mice at age of 9 to 11 weeks were challenged
subcutaneously with 1.0.times.10.sup.5 CT26 colorectal tumor cells.
On day 11 post tumor challenge, mice received a single dose of
cyclophosphamide (100 mg/kg) and started treatment with anti-PD-1
antibody (250 ug, Clone G4, Hirano F. et al., 2005 Cancer Research)
which was administered 3 times per week for four weeks. 70% of the
mice that received the CTX+anti-PD-1 regimen eradicated established
CT26 tumors at day 50 after tumor challenge, whereas all mice in
the control and anti-PD-1 alone groups died as a result of tumor
growth or were euthanized because tumors exceeded the sizes
approved by IACUC. These results show the effectiveness of the
treatment regimen on established tumors and not mere prophylaxis.
Results are shown in FIG. 7.
Example 9
Combination of Cyclophosphamide and Anti-CTLA4 can Eradicate
Established Tumors
[0264] Balb/C mice at age of 9 to 11 weeks were challenged
subcutaneously with 1.0.times.10.sup.5 CT26 colorectal tumor cells.
On day 11 post tumor challenge, mice received 100 mg/kg of
cyclophosphamide. Anti-CTLA4 (an anti-mouse CTLA4 from hamster
hybridoma--ATCC deposit UC10-4F10-11) treatment was started 1 day
later, on day 12. Mice were treated with 100 ug of anti-CTLA4, 2
doses per week, for 4 weeks. 56% of the mice that received the
CTX+anti-CTLA4 regimen were tumor free at day 50 after tumor
challenge, whereas all mice in the control group died as a result
of tumor growth or were euthanized because tumors exceeded the
sizes approved by IACUC. Results are shown in FIG. 8. These results
show the effectiveness of the treatment regimen on established
tumors and not mere prophylaxis.
Example 10
Combination of Cyclophosphamide and B7-DC-Ig Regimen Leads to
Reduction of Tregs in the Tumor Microenvironment
[0265] FIG. 9 shows the results of experiments wherein Balb/C mice
at age of 9 to 11 weeks of age were implanted with 1.times.10.sup.5
CT26 cells subcutaneously. On Day 9, mice were injected with 100
mg/kg of CTX, IP. Twenty four hours later, on Day 10, mice were
treated with 100 ug of B7-DC-Ig. There were 5 groups: naive mice
that did not receive any tumor cells, vehicle injected, CTX alone,
CTX+B7-DC-Ig or B7-DC-Ig alone. Two naive mice and 4 mice from
other groups were removed from the study on Day 11 (2 days post
CTX) and Day 16 (7 days post CTX) for T cell analysis. Left panel
shows on Day 11, 2 days post CTX injection, Treg in the spleen of
the mice with CTX treatment was significantly lower than the one in
the mice with tumor implantation and injected with vehicle. Right
panel shows that on Day 16, 7 days post CTX and 6 days post
B7-DC-Ig treatment, B7-DC-Ig significantly lowered the CD4+ T cells
expressing high PD-1. This was observed in both the B7-DC-Ig
treated and CTX+B7-DC-Ig treated mice. Mice implanted with tumor
cells intended to have more PD-1+/CD4+ T cells in the draining LN
compared with naive mice.
Example 11
Combination of Cyclophosphamide and B7-DC-Ig can Promote Mouse
Survival in a Metastatic Prostate Tumor Model
[0266] B10.D2 mice at age of 9 to 11 weeks were injected
intravenously with 3.0.times.10.sup.5 SP-1 cells, which were
isolated from lung metastasis post parent TRAMP cell injection. The
CTX mice received 3 doses of CTX, 50 mg/kg, on Day 5, 12 and 19.
The B7-DC-Ig treated mice received 3 doses of B7-DC-Ig, 5 mg/kg, on
Day 6, 13 and 20. On Day 100, 17% of mice in the control groups,
no-treated, CTX alone, B7-DC-Ig alone survived while 43% of the
mice received combination of CTX and B7-DC-Ig survived. Results are
shown in FIG. 10.
Example 12
Combination of Listeria Cancer Vaccine and B7-DC-Ig can Enhance
Mouse Survival Post CT26 Liver Implantation
[0267] Balb/C mice at age of 11-13 weeks were implanted with CT26
cells using a hemispleen injection technique (Yoshimura K et al.,
2007, Cancer Research). On Day 10, mice received 1 injection of CTX
at 50 mg/kg, IP. Twenty four hours later, on Day 11, mice were
treated with recombinant Listeria carrying AH1 peptide, an
immunodominant epitope of CT26, at 0.1 LD.sub.50 (1.times.10.sup.7
CFU), then on Day 14 and 17. Mice were also treated with B7-DC-Ig
on Day 11 and then on Day 18. FIG. 11 shows mice without any
treatment or treated with CTX and Listeria cancer vaccine all died
before Dady 45. There were 60% of the mice received triple
combination, CTX+ Listeria cancer vaccine and B7-DC-Ig
survived.
1. REFERENCE LIST
[0268] 1. Brode S, Cooke A. Immune-potentiating effects of the
chemotherapeutic drug cyclophosphamide. Crit Rev. Immunol. 2008;
28(2):109-26 [0269] 2. van der Most R G, Currie A J, Mahendran S,
Prosser A, Darabi A, Robinson B W, Nowak A K, Lake R A. Tumor
eradication after cyclophosphamide depends on concurrent depletion
of regulatory T cells: a role for cycling TNFR2-expressing
effector-suppressor T cells in limiting effective chemotherapy.
Cancer Immunol. Immunother. 2009 August; 58(8):1219-28 [0270] 3.
Taieb J, Chaput N, Schartz N, Roux S, Novault S, Menard C,
Ghiringhelli F, Terme M, Carpentier A F, Darrasse-Jeze G, et al.
Chemoimmunotherapy of tumors: cyclophosphamide synergizes with
exosome based vaccines. J. Immunol. 2006 Mar. 1; 176(5):2722-9
[0271] 4. Machiels J P, Reilly R T, Emens L A, Ercolini A M, Lei R
Y, Weintraub D, Okoye F I, Jaffee E M. Cyclophosphamide,
doxorubicin, and paclitaxel enhance the antitumor immune response
of granulocyte/macrophage-colony stimulating factor-secreting
whole-cell vaccines in HER-2/neu tolerized mice. Cancer Res. 2001
May 1; 61(9):3689-97 [0272] 5. Bass K K, Mastrangelo M J.
Immunopotentiation with low-dose cyclophosphamide in the active
specific immunotherapy of cancer. Cancer Immunol. Immunother. 1998
September; 47(1):1-12 [0273] 6. Hengst J C, Mokyr M B, Dray S.
Cooperation between cyclophosphamide tumoricidal activity and host
antitumor immunity in the cure of mice bearing large MOPC-315
tumors. Cancer Res. 1981 June; 41(6):2163-7 [0274] 7. Hengst J C,
Mokyr M B, Dray S. Importance of timing in cyclophosphamide therapy
of MOPC-315 tumor-bearing mice. Cancer Res. 1980 July;
40(7):2135-41 [0275] 8. Tsung K, Meko J B, Tsung Y L, Peplinski G
R, Norton J A. Immune response against large tumors eradicated by
treatment with cyclophosphamide and IL-12. J. Immunol. 1998 Feb. 1;
160(3):1369-77 [0276] 9. Honeychurch J, Glennie M J, Illidge T M.
Cyclophosphamide inhibition of anti-CD40 monoclonal antibody-based
therapy of B cell lymphoma is dependent on CD11b+ cells. Cancer
Res. 2005 Aug. 15; 65(16):7493-501 [0277] 10. Wada S, Yoshimura K,
Hipkiss E L, Harris T J, Yen H R, Goldberg M V, Grosso J F, Getnet
D, Demarzo A M, Netto G J, Anders R, Pardoll D M, Drake C G.
Cyclophosphamide augments antitumor immunity: studies in an
autochthonous prostate cancer model. Cancer Res. 2009 May 15;
69(10):4309-18. [0278] 11. Freeman, G. J. Structures of PD-1 with
its ligands: sideways and dancing cheek to cheek. Proc. Natl. Acad.
Sci. U.S.A 105, 10275-10276 (2008). [0279] 12. Brode, S. &
Cooke, A. Immune-potentiating effects of the chemotherapeutic drug
cyclophosphamide. Crit Rev. Immunol. 28, 109-126 (2008). [0280] 13.
van der Most, R. G. et al. Tumor eradication after cyclophosphamide
depends on concurrent depletion of regulatory T cells: a role for
cycling TNFR2-expressing effector-suppressor T cells in limiting
effective chemotherapy. Cancer Immunol. Immunother. 58, 1219-1228
(2009). [0281] 14. Taieb, J. et al. Chemoimmunotherapy of tumors:
cyclophosphamide synergizes with exosome based vaccines. J.
Immunol. 176, 2722-2729 (2006). [0282] 15. Bass, K. K. &
Mastrangelo, M. J. Immunopotentiation with low-dose
cyclophosphamide in the active specific immunotherapy of cancer.
Cancer Immunol. Immunother. 47, 1-12 (1998). [0283] 16. Machiels,
J. P. et al. Cyclophosphamide, doxorubicin, and paclitaxel enhance
the antitumor immune response of granulocyte/macrophage-colony
stimulating factor-secreting whole-cell vaccines in HER-2/neu
tolerized mice. Cancer Res. 61, 3689-3697 (2001). [0284] 17.
Hengst, J. C., Mokyr, M. B., & Dray, S. Cooperation between
cyclophosphamide tumoricidal activity and host antitumor immunity
in the cure of mice bearing large MOPC-315 tumors. Cancer Res. 41,
2163-2167 (1981). [0285] 18. Hengst, J. C., Mokyr, M. B., &
Dray, S. Importance of timing in cyclophosphamide therapy of
MOPC-315 tumor-bearing mice. Cancer Res. 40, 2135-2141 (1980).
[0286] 19. Tsung, K., Meko, J. B., Tsung, Y. L., Peplinski, G. R.,
& Norton, J. A. Immune response against large tumors eradicated
by treatment with cyclophosphamide and IL-12. J. Immunol. 160,
1369-1377 (1998). [0287] 20. Honeychurch, J., Glennie, M. J., &
Illidge, T. M. Cyclophosphamide inhibition of anti-CD40 monoclonal
antibody-based therapy of B cell lymphoma is dependent on CD11b+
cells. Cancer Res. 65, 7493-7501 (2005). [0288] 21. Wada S,
Yoshimura K, Hipkiss E L, Harris T J, Yen H R, Goldberg M V, Grosso
J F, Getnet D, Demarzo A M, Netto G J, Anders R, Pardoll D M, Drake
C G. Cyclophosphamide augments antitumor immunity: studies in an
autochthonous prostate cancer model. Cancer Res. 2009 May 15;
69(10):4309-18.
Sequence CWU 1
1
201273PRTHomo sapiens 1Met Ile Phe Leu Leu Leu Met Leu Ser Leu Glu
Leu Gln Leu His Gln 1 5 10 15 Ile Ala Ala Leu Phe Thr Val Thr Val
Pro Lys Glu Leu Tyr Ile Ile 20 25 30 Glu His Gly Ser Asn Val Thr
Leu Glu Cys Asn Phe Asp Thr Gly Ser 35 40 45 His Val Asn Leu Gly
Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn 50 55 60 Asp Thr Ser
Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu 65 70 75 80 Pro
Leu Gly Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp 85 90
95 Glu Gly Gln Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr
100 105 110 Lys Tyr Leu Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile
Asn Thr 115 120 125 His Ile Leu Lys Val Pro Glu Thr Asp Glu Val Glu
Leu Thr Cys Gln 130 135 140 Ala Thr Gly Tyr Pro Leu Ala Glu Val Ser
Trp Pro Asn Val Ser Val 145 150 155 160 Pro Ala Asn Thr Ser His Ser
Arg Thr Pro Glu Gly Leu Tyr Gln Val 165 170 175 Thr Ser Val Leu Arg
Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys 180 185 190 Val Phe Trp
Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp 195 200 205 Leu
Gln Ser Gln Met Glu Pro Arg Thr His Pro Thr Trp Leu Leu His 210 215
220 Ile Phe Ile Pro Phe Cys Ile Ile Ala Phe Ile Phe Ile Ala Thr Val
225 230 235 240 Ile Ala Leu Arg Lys Gln Leu Cys Gln Lys Leu Tyr Ser
Ser Lys Asp 245 250 255 Thr Thr Lys Arg Pro Val Thr Thr Thr Lys Arg
Glu Val Asn Ser Ala 260 265 270 Ile 2254PRTHomo sapiens 2Leu Phe
Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly 1 5 10 15
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val Asn 20
25 30 Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn Asp Thr
Ser 35 40 45 Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu
Pro Leu Gly 50 55 60 Lys Ala Ser Phe His Ile Pro Gln Val Gln Val
Arg Asp Glu Gly Gln 65 70 75 80 Tyr Gln Cys Ile Ile Ile Tyr Gly Val
Ala Trp Asp Tyr Lys Tyr Leu 85 90 95 Thr Leu Lys Val Lys Ala Ser
Tyr Arg Lys Ile Asn Thr His Ile Leu 100 105 110 Lys Val Pro Glu Thr
Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly 115 120 125 Tyr Pro Leu
Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn 130 135 140 Thr
Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val 145 150
155 160 Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys Val Phe
Trp 165 170 175 Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp
Leu Gln Ser 180 185 190 Gln Met Glu Pro Arg Thr His Pro Thr Trp Leu
Leu His Ile Phe Ile 195 200 205 Pro Phe Cys Ile Ile Ala Phe Ile Phe
Ile Ala Thr Val Ile Ala Leu 210 215 220 Arg Lys Gln Leu Cys Gln Lys
Leu Tyr Ser Ser Lys Asp Thr Thr Lys 225 230 235 240 Arg Pro Val Thr
Thr Thr Lys Arg Glu Val Asn Ser Ala Ile 245 250 3202PRTHomo sapiens
3Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile Glu His Gly 1
5 10 15 Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser His Val
Asn 20 25 30 Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn
Asp Thr Ser 35 40 45 Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu
Gln Leu Pro Leu Gly 50 55 60 Lys Ala Ser Phe His Ile Pro Gln Val
Gln Val Arg Asp Glu Gly Gln 65 70 75 80 Tyr Gln Cys Ile Ile Ile Tyr
Gly Val Ala Trp Asp Tyr Lys Tyr Leu 85 90 95 Thr Leu Lys Val Lys
Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu 100 105 110 Lys Val Pro
Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly 115 120 125 Tyr
Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro Ala Asn 130 135
140 Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val
145 150 155 160 Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys
Val Phe Trp 165 170 175 Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser
Ile Asp Leu Gln Ser 180 185 190 Gln Met Glu Pro Arg Thr His Pro Thr
Trp 195 200 4202PRTmurine 4Leu Phe Thr Val Thr Ala Pro Lys Glu Val
Tyr Thr Val Asp Val Gly 1 5 10 15 Ser Ser Val Ser Leu Glu Cys Asp
Phe Asp Arg Arg Glu Cys Thr Glu 20 25 30 Leu Glu Gly Ile Arg Ala
Ser Leu Gln Lys Val Glu Asn Asp Thr Ser 35 40 45 Leu Gln Ser Glu
Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly 50 55 60 Lys Ala
Leu Phe His Ile Pro Ser Val Gln Val Arg Asp Ser Gly Gln 65 70 75 80
Tyr Arg Cys Leu Val Ile Cys Gly Ala Ala Trp Asp Tyr Lys Tyr Leu 85
90 95 Thr Val Lys Val Lys Ala Ser Tyr Met Arg Ile Asp Thr Arg Ile
Leu 100 105 110 Glu Val Pro Gly Thr Gly Glu Val Gln Leu Thr Cys Gln
Ala Arg Gly 115 120 125 Tyr Pro Leu Ala Glu Val Ser Trp Gln Asn Val
Ser Val Pro Ala Asn 130 135 140 Thr Ser His Ile Arg Thr Pro Glu Gly
Leu Tyr Gln Val Thr Ser Val 145 150 155 160 Leu Arg Leu Lys Pro Gln
Pro Ser Arg Asn Phe Ser Cys Met Phe Trp 165 170 175 Asn Ala His Met
Lys Glu Leu Thr Ser Ala Ile Ile Asp Pro Leu Ser 180 185 190 Arg Met
Glu Pro Lys Val Pro Arg Thr Trp 195 200 519PRTHomo sapiens 5Met Ile
Phe Leu Leu Leu Met Leu Ser Leu Glu Leu Gln Leu His Gln 1 5 10 15
Ile Ala Ala 6232PRTHomo sapiens 6Glu Pro Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala 1 5 10 15 Pro Glu Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30 Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45 Val Asp
Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 65
70 75 80 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln 85 90 95 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala 100 105 110 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro 115 120 125 Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser Arg Asp Glu Leu Thr 130 135 140 Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 145 150 155 160 Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175 Lys
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185
190 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys 210 215 220 Ser Leu Ser Leu Ser Pro Gly Lys 225 230
7233PRTmurine 7Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys
Lys Cys Pro 1 5 10 15 Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe
Ile Phe Pro Pro Lys 20 25 30 Ile Lys Asp Val Leu Met Ile Ser Leu
Ser Pro Ile Val Thr Cys Val 35 40 45 Val Val Asp Val Ser Glu Asp
Asp Pro Asp Val Gln Ile Ser Trp Phe 50 55 60 Val Asn Asn Val Glu
Val His Thr Ala Gln Thr Gln Thr His Arg Glu 65 70 75 80 Asp Tyr Asn
Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His 85 90 95 Gln
Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys 100 105
110 Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser
115 120 125 Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu
Glu Met 130 135 140 Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr
Asp Phe Met Pro 145 150 155 160 Glu Asp Ile Tyr Val Glu Trp Thr Asn
Asn Gly Lys Thr Glu Leu Asn 165 170 175 Tyr Lys Asn Thr Glu Pro Val
Leu Asp Ser Asp Gly Ser Tyr Phe Met 180 185 190 Tyr Ser Lys Leu Arg
Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser 195 200 205 Tyr Ser Cys
Ser Val Val His Glu Gly Leu His Asn His His Thr Thr 210 215 220 Lys
Ser Phe Ser Arg Thr Pro Gly Lys 225 230 81365DNAartificialMurine
B7-DC-Ig 8atgctgctcc tgctgccgat actgaacctg agcttacaac ttcatcctgt
agcagcttta 60ttcaccgtga cagcccctaa agaagtgtac accgtagacg tcggcagcag
tgtgagcctg 120gagtgcgatt ttgaccgcag agaatgcact gaactggaag
ggataagagc cagtttgcag 180aaggtagaaa atgatacgtc tctgcaaagt
gaaagagcca ccctgctgga ggagcagctg 240cccctgggaa aggctttgtt
ccacatccct agtgtccaag tgagagattc cgggcagtac 300cgttgcctgg
tcatctgcgg ggccgcctgg gactacaagt acctgacggt gaaagtcaaa
360gcttcttaca tgaggataga cactaggatc ctggaggttc caggtacagg
ggaggtgcag 420cttacctgcc aggctagagg ttatccccta gcagaagtgt
cctggcaaaa tgtcagtgtt 480cctgccaaca ccagccacat caggaccccc
gaaggcctct accaggtcac cagtgttctg 540cgcctcaagc ctcagcctag
cagaaacttc agctgcatgt tctggaatgc tcacatgaag 600gagctgactt
cagccatcat tgaccctctg agtcggatgg aacccaaagt ccccagaacg
660tgggagccaa gaggtcctac gatcaagccc tgcccgcctt gtaaatgccc
agctccaaat 720ttgctgggtg gaccgtcagt ctttatcttc ccgccaaaga
taaaggacgt cttgatgatt 780agtctgagcc ccatcgtgac atgcgttgtg
gtggatgttt cagaggatga ccccgacgtg 840caaatcagtt ggttcgttaa
caacgtggag gtgcataccg ctcaaaccca gacccacaga 900gaggattata
acagcaccct gcgggtagtg tccgccctgc cgatccagca tcaggattgg
960atgagcggga aagagttcaa gtgtaaggta aacaacaaag atctgccagc
gccgattgaa 1020cgaaccatta gcaagccgaa agggagcgtg cgcgcacctc
aggtttacgt ccttcctcca 1080ccagaagagg agatgacgaa aaagcaggtg
accctgacat gcatggtaac tgactttatg 1140ccagaagata tttacgtgga
atggactaat aacggaaaga cagagctcaa ttacaagaac 1200actgagcctg
ttctggattc tgatggcagc tactttatgt actccaaatt gagggtcgag
1260aagaagaatt gggtcgagag aaacagttat agttgctcag tggtgcatga
gggcctccat 1320aatcatcaca ccacaaagtc cttcagccga acgcccggga aatga
13659454PRTartificialMurine B7-DC-Ig 9Met Leu Leu Leu Leu Pro Ile
Leu Asn Leu Ser Leu Gln Leu His Pro 1 5 10 15 Val Ala Ala Leu Phe
Thr Val Thr Ala Pro Lys Glu Val Tyr Thr Val 20 25 30 Asp Val Gly
Ser Ser Val Ser Leu Glu Cys Asp Phe Asp Arg Arg Glu 35 40 45 Cys
Thr Glu Leu Glu Gly Ile Arg Ala Ser Leu Gln Lys Val Glu Asn 50 55
60 Asp Thr Ser Leu Gln Ser Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu
65 70 75 80 Pro Leu Gly Lys Ala Leu Phe His Ile Pro Ser Val Gln Val
Arg Asp 85 90 95 Ser Gly Gln Tyr Arg Cys Leu Val Ile Cys Gly Ala
Ala Trp Asp Tyr 100 105 110 Lys Tyr Leu Thr Val Lys Val Lys Ala Ser
Tyr Met Arg Ile Asp Thr 115 120 125 Arg Ile Leu Glu Val Pro Gly Thr
Gly Glu Val Gln Leu Thr Cys Gln 130 135 140 Ala Arg Gly Tyr Pro Leu
Ala Glu Val Ser Trp Gln Asn Val Ser Val 145 150 155 160 Pro Ala Asn
Thr Ser His Ile Arg Thr Pro Glu Gly Leu Tyr Gln Val 165 170 175 Thr
Ser Val Leu Arg Leu Lys Pro Gln Pro Ser Arg Asn Phe Ser Cys 180 185
190 Met Phe Trp Asn Ala His Met Lys Glu Leu Thr Ser Ala Ile Ile Asp
195 200 205 Pro Leu Ser Arg Met Glu Pro Lys Val Pro Arg Thr Trp Glu
Pro Arg 210 215 220 Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys
Pro Ala Pro Asn 225 230 235 240 Leu Leu Gly Gly Pro Ser Val Phe Ile
Phe Pro Pro Lys Ile Lys Asp 245 250 255 Val Leu Met Ile Ser Leu Ser
Pro Ile Val Thr Cys Val Val Val Asp 260 265 270 Val Ser Glu Asp Asp
Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn 275 280 285 Val Glu Val
His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn 290 295 300 Ser
Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp 305 310
315 320 Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu
Pro 325 330 335 Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser
Val Arg Ala 340 345 350 Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu
Glu Met Thr Lys Lys 355 360 365 Gln Val Thr Leu Thr Cys Met Val Thr
Asp Phe Met Pro Glu Asp Ile 370 375 380 Tyr Val Glu Trp Thr Asn Asn
Gly Lys Thr Glu Leu Asn Tyr Lys Asn 385 390 395 400 Thr Glu Pro Val
Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys 405 410 415 Leu Arg
Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys 420 425 430
Ser Val Val His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe 435
440 445 Ser Arg Thr Pro Gly Lys 450 10435PRTartificialMurine
B7-DC-Ig fusion protein without signal sequence 10Leu Phe Thr Val
Thr Ala Pro Lys Glu Val Tyr Thr Val Asp Val Gly 1 5 10 15 Ser Ser
Val Ser Leu Glu Cys Asp Phe Asp Arg Arg Glu Cys Thr Glu 20 25 30
Leu Glu Gly Ile Arg Ala Ser Leu Gln Lys Val Glu Asn Asp Thr Ser 35
40 45 Leu Gln Ser Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu
Gly 50 55 60 Lys Ala Leu Phe His Ile Pro Ser Val Gln Val Arg Asp
Ser Gly Gln 65 70 75 80 Tyr Arg Cys Leu Val Ile Cys Gly Ala Ala Trp
Asp Tyr Lys Tyr Leu 85 90 95 Thr Val Lys Val Lys Ala Ser Tyr Met
Arg Ile Asp Thr Arg Ile Leu 100 105 110 Glu Val Pro Gly Thr Gly Glu
Val Gln Leu Thr Cys Gln Ala Arg Gly 115 120 125 Tyr Pro Leu Ala Glu
Val Ser Trp Gln Asn Val Ser Val Pro Ala Asn 130 135 140 Thr Ser His
Ile Arg Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val 145 150 155 160
Leu Arg Leu Lys Pro Gln Pro Ser Arg Asn Phe Ser Cys Met Phe Trp 165
170 175 Asn Ala His Met Lys Glu Leu Thr Ser Ala Ile Ile Asp Pro Leu
Ser 180 185
190 Arg Met Glu Pro Lys Val Pro Arg Thr Trp Glu Pro Arg Gly Pro Thr
195 200 205 Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu
Leu Gly 210 215 220 Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys
Asp Val Leu Met 225 230 235 240 Ile Ser Leu Ser Pro Ile Val Thr Cys
Val Val Val Asp Val Ser Glu 245 250 255 Asp Asp Pro Asp Val Gln Ile
Ser Trp Phe Val Asn Asn Val Glu Val 260 265 270 His Thr Ala Gln Thr
Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu 275 280 285 Arg Val Val
Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly 290 295 300 Lys
Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile 305 310
315 320 Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln
Val 325 330 335 Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys
Gln Val Thr 340 345 350 Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu
Asp Ile Tyr Val Glu 355 360 365 Trp Thr Asn Asn Gly Lys Thr Glu Leu
Asn Tyr Lys Asn Thr Glu Pro 370 375 380 Val Leu Asp Ser Asp Gly Ser
Tyr Phe Met Tyr Ser Lys Leu Arg Val 385 390 395 400 Glu Lys Lys Asn
Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val 405 410 415 His Glu
Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr 420 425 430
Pro Gly Lys 435 111362DNAartificialHuman B7-DC-Ig 11atgatctttc
ttctcttgat gctgtctttg gaattgcaac ttcaccaaat cgcggccctc 60tttactgtga
ccgtgccaaa agaactgtat atcattgagc acgggtccaa tgtgaccctc
120gaatgtaact ttgacaccgg cagccacgtt aacctggggg ccatcactgc
cagcttgcaa 180aaagttgaaa acgacacttc acctcaccgg gagagggcaa
ccctcttgga ggagcaactg 240ccattgggga aggcctcctt tcatatccct
caggtgcagg ttcgggatga gggacagtac 300cagtgcatta ttatctacgg
cgtggcttgg gattacaagt atctgaccct gaaggtgaaa 360gcgtcctatc
ggaaaattaa cactcacatt cttaaggtgc cagagacgga cgaggtggaa
420ctgacatgcc aagccaccgg ctacccgttg gcagaggtca gctggcccaa
cgtgagcgta 480cctgctaaca cttctcattc taggacaccc gagggcctct
accaggttac atccgtgctc 540cgcctcaaac cgcccccagg ccggaatttt
agttgcgtgt tttggaatac ccacgtgcga 600gagctgactc ttgcatctat
tgatctgcag tcccagatgg agccacggac tcatccaact 660tgggaaccta
aatcttgcga taaaactcat acctgtcccc cttgcccagc ccccgagctt
720ctgggaggtc ccagtgtgtt tctgtttccc ccaaaaccta aggacacact
tatgatatcc 780cgaacgccgg aagtgacatg cgtggttgtg gacgtctcac
acgaagaccc ggaggtgaaa 840ttcaactggt acgttgacgg agttgaggtt
cataacgcta agaccaagcc cagagaggag 900caatacaatt ccacctatcg
agtggttagt gtactgaccg ttttgcacca agactggctg 960aatggaaaag
aatacaagtg caaagtatca aacaaggctt tgcctgcacc catcgagaag
1020acaatttcta aagccaaagg gcagcccagg gaaccgcagg tgtacacact
cccaccatcc 1080cgcgacgagc tgacaaagaa tcaagtatcc ctgacctgcc
tggtgaaagg cttttaccca 1140tctgacattg ccgtggaatg ggaatcaaat
ggacaacctg agaacaacta caaaaccact 1200ccacctgtgc ttgacagcga
cgggtccttt ttcctgtaca gtaagctcac tgtcgataag 1260tctcgctggc
agcagggcaa cgtcttttca tgtagtgtga tgcacgaagc tctgcacaac
1320cattacaccc agaagtctct gtcactgagc ccaggtaaat ga
136212453PRTArtificialHuman B7-DC-Ig 12Met Ile Phe Leu Leu Leu Met
Leu Ser Leu Glu Leu Gln Leu His Gln 1 5 10 15 Ile Ala Ala Leu Phe
Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile 20 25 30 Glu His Gly
Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser 35 40 45 His
Val Asn Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn 50 55
60 Asp Thr Ser Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu
65 70 75 80 Pro Leu Gly Lys Ala Ser Phe His Ile Pro Gln Val Gln Val
Arg Asp 85 90 95 Glu Gly Gln Tyr Gln Cys Ile Ile Ile Tyr Gly Val
Ala Trp Asp Tyr 100 105 110 Lys Tyr Leu Thr Leu Lys Val Lys Ala Ser
Tyr Arg Lys Ile Asn Thr 115 120 125 His Ile Leu Lys Val Pro Glu Thr
Asp Glu Val Glu Leu Thr Cys Gln 130 135 140 Ala Thr Gly Tyr Pro Leu
Ala Glu Val Ser Trp Pro Asn Val Ser Val 145 150 155 160 Pro Ala Asn
Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val 165 170 175 Thr
Ser Val Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys 180 185
190 Val Phe Trp Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp
195 200 205 Leu Gln Ser Gln Met Glu Pro Arg Thr His Pro Thr Trp Glu
Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu 225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310
315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435
440 445 Leu Ser Pro Gly Lys 450 13434PRTArtificialHuman B7-DC-Ig
without signal sequence 13Leu Phe Thr Val Thr Val Pro Lys Glu Leu
Tyr Ile Ile Glu His Gly 1 5 10 15 Ser Asn Val Thr Leu Glu Cys Asn
Phe Asp Thr Gly Ser His Val Asn 20 25 30 Leu Gly Ala Ile Thr Ala
Ser Leu Gln Lys Val Glu Asn Asp Thr Ser 35 40 45 Pro His Arg Glu
Arg Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly 50 55 60 Lys Ala
Ser Phe His Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln 65 70 75 80
Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu 85
90 95 Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile
Leu 100 105 110 Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln
Ala Thr Gly 115 120 125 Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val
Ser Val Pro Ala Asn 130 135 140 Thr Ser His Ser Arg Thr Pro Glu Gly
Leu Tyr Gln Val Thr Ser Val 145 150 155 160 Leu Arg Leu Lys Pro Pro
Pro Gly Arg Asn Phe Ser Cys Val Phe Trp 165 170 175 Asn Thr His Val
Arg Glu Leu Thr Leu Ala Ser Ile Asp Leu Gln Ser 180 185 190 Gln Met
Glu Pro Arg Thr His Pro Thr Trp Glu Pro Lys Ser Cys Asp 195 200 205
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 210
215 220 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
Ile 225 230 235 240 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu 245 250 255 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His 260 265 270 Asn Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg 275 280 285 Val Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys 290 295 300 Glu Tyr Lys Cys
Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 305 310 315 320 Lys
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 325 330
335 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
340 345 350 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp 355 360 365 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val 370 375 380 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
Ser Lys Leu Thr Val Asp 385 390 395 400 Lys Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His 405 410 415 Glu Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 420 425 430 Gly Lys
145PRTHomo sapiens 14Trp Asp Tyr Lys Tyr 1 5 15202PRTMacaca
fascicularis 15Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile
Glu His Gly 1 5 10 15 Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr
Gly Ser His Val Asn 20 25 30 Leu Gly Ala Ile Thr Ala Ser Leu Gln
Lys Val Glu Asn Asp Thr Ser 35 40 45 Pro His Arg Glu Arg Ala Thr
Leu Leu Glu Glu Gln Leu Pro Leu Gly 50 55 60 Lys Ala Ser Phe His
Ile Pro Gln Val Gln Val Arg Asp Glu Gly Gln 65 70 75 80 Tyr Gln Cys
Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr Lys Tyr Leu 85 90 95 Thr
Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr His Ile Leu 100 105
110 Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln Ala Thr Gly
115 120 125 Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val Pro
Ala Asn 130 135 140 Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln
Val Thr Ser Val 145 150 155 160 Leu Arg Leu Lys Pro Pro Pro Gly Arg
Asn Phe Ser Cys Val Phe Trp 165 170 175 Asn Thr His Val Arg Glu Leu
Thr Leu Ala Ser Ile Asp Leu Gln Ser 180 185 190 Gln Met Glu Pro Arg
Thr His Pro Thr Trp 195 200 16290PRTHomo sapiens 16Met Arg Ile Phe
Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu 1 5 10 15 Asn Ala
Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr 20 25 30
Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu 35
40 45 Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn
Ile 50 55 60 Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln
His Ser Ser 65 70 75 80 Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln
Leu Ser Leu Gly Asn 85 90 95 Ala Ala Leu Gln Ile Thr Asp Val Lys
Leu Gln Asp Ala Gly Val Tyr 100 105 110 Arg Cys Met Ile Ser Tyr Gly
Gly Ala Asp Tyr Lys Arg Ile Thr Val 115 120 125 Lys Val Asn Ala Pro
Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val 130 135 140 Asp Pro Val
Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr 145 150 155 160
Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser 165
170 175 Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe
Asn 180 185 190 Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu
Ile Phe Tyr 195 200 205 Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn
His Thr Ala Glu Leu 210 215 220 Val Ile Pro Glu Leu Pro Leu Ala His
Pro Pro Asn Glu Arg Thr His 225 230 235 240 Leu Val Ile Leu Gly Ala
Ile Leu Leu Cys Leu Gly Val Ala Leu Thr 245 250 255 Phe Ile Phe Arg
Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys 260 265 270 Gly Ile
Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu 275 280 285
Glu Thr 290 17290PRTMus musculus 17Met Arg Ile Phe Ala Gly Ile Ile
Phe Thr Ala Cys Cys His Leu Leu 1 5 10 15 Arg Ala Phe Thr Ile Thr
Ala Pro Lys Asp Leu Tyr Val Val Glu Tyr 20 25 30 Gly Ser Asn Val
Thr Met Glu Cys Arg Phe Pro Val Glu Arg Glu Leu 35 40 45 Asp Leu
Leu Ala Leu Val Val Tyr Trp Glu Lys Glu Asp Glu Gln Val 50 55 60
Ile Gln Phe Val Ala Gly Glu Glu Asp Leu Lys Pro Gln His Ser Asn 65
70 75 80 Phe Arg Gly Arg Ala Ser Leu Pro Lys Asp Gln Leu Leu Lys
Gly Asn 85 90 95 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp
Ala Gly Val Tyr 100 105 110 Cys Cys Ile Ile Ser Tyr Gly Gly Ala Asp
Tyr Lys Arg Ile Thr Leu 115 120 125 Lys Val Asn Ala Pro Tyr Arg Lys
Ile Asn Gln Arg Ile Ser Val Asp 130 135 140 Pro Ala Thr Ser Glu His
Glu Leu Ile Cys Gln Ala Glu Gly Tyr Pro 145 150 155 160 Glu Ala Glu
Val Ile Trp Thr Asn Ser Asp His Gln Pro Val Ser Gly 165 170 175 Lys
Arg Ser Val Thr Thr Ser Arg Thr Glu Gly Met Leu Leu Asn Val 180 185
190 Thr Ser Ser Leu Arg Val Asn Ala Thr Ala Asn Asp Val Phe Tyr Cys
195 200 205 Thr Phe Trp Arg Ser Gln Pro Gly Gln Asn His Thr Ala Glu
Leu Ile 210 215 220 Ile Pro Glu Leu Pro Ala Thr His Pro Pro Gln Asn
Arg Thr His Trp 225 230 235 240 Val Leu Leu Gly Ser Ile Leu Leu Phe
Leu Ile Val Val Ser Thr Val 245 250 255 Leu Leu Phe Leu Arg Lys Gln
Val Arg Met Leu Asp Val Glu Lys Cys 260 265 270 Gly Val Glu Asp Thr
Ser Ser Lys Asn Arg Asn Asp Thr Gln Phe Glu 275 280 285 Glu Thr 290
18290PRTMacaca mulatta 18Met Arg Ile Phe Ala Val Phe Ile Phe Thr
Ile Tyr Trp His Leu Leu 1 5 10 15 Asn Ala Phe Thr Val Thr Val Pro
Lys Asp Leu Tyr Val Val Glu Tyr 20 25 30 Gly Ser Asn Met Thr Ile
Glu Cys Arg Phe Pro Val Glu Lys Gln Leu 35 40 45 Gly Leu Thr Ser
Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile 50 55 60 Ile Gln
Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Asn 65 70 75 80
Tyr Arg Gln Arg Ala Gln Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn 85
90 95 Ala Ala Leu Arg Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val
Tyr 100 105 110 Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg
Ile Thr Val 115 120 125 Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln
Arg Ile Leu Val Val 130 135
140 Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr
145 150 155 160 Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln
Val Leu Ser 165 170 175 Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu
Glu Lys Leu Leu Asn 180 185 190 Val Thr Ser Thr Leu Arg Ile Asn Thr
Thr Ala Asn Glu Ile Phe Tyr 195 200 205 Cys Ile Phe Arg Arg Leu Gly
Pro Glu Glu Asn His Thr Ala Glu Leu 210 215 220 Val Ile Pro Glu Leu
Pro Leu Ala Leu Pro Pro Asn Glu Arg Thr His 225 230 235 240 Leu Val
Ile Leu Gly Ala Ile Phe Leu Leu Leu Gly Val Ala Leu Thr 245 250 255
Phe Ile Phe Tyr Leu Arg Lys Gly Arg Met Met Asp Met Lys Lys Ser 260
265 270 Gly Ile Arg Val Thr Asn Ser Lys Lys Gln Arg Asp Thr Gln Leu
Glu 275 280 285 Glu Thr 290 19288PRTHomo sapiens 19Met Gln Ile Pro
Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln 1 5 10 15 Leu Gly
Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30
Asn Pro Pro Thr Phe Phe Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35
40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe
Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp
Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln
Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp
Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly
Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln
Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg
Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160
Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165
170 175 Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile
Cys 180 185 190 Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr
Gly Gln Pro 195 200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe
Ser Val Asp Tyr Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys
Thr Pro Glu Pro Pro Val Pro 225 230 235 240 Cys Val Pro Glu Gln Thr
Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser
Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270 Ser Ala
Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285
20288PRTMacaca fascicularis 20Met Gln Ile Pro Gln Ala Pro Trp Pro
Val Val Trp Ala Val Leu Gln 1 5 10 15 Leu Gly Trp Arg Pro Gly Trp
Phe Leu Glu Ser Pro Asp Arg Pro Trp 20 25 30 Asn Ala Pro Thr Phe
Ser Pro Ala Leu Leu Leu Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr
Phe Thr Cys Ser Phe Ser Asn Ala Ser Glu Ser Phe Val 50 55 60 Leu
Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70
75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe
Arg 85 90 95 Val Thr Arg Leu Pro Asn Gly Arg Asp Phe His Met Ser
Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys
Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser
Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val
Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly
Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly 165 170 175 Leu Leu
Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys 180 185 190
Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro 195
200 205 Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr
Gly 210 215 220 Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro
Pro Val Pro 225 230 235 240 Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr
Ile Val Phe Pro Ser Gly 245 250 255 Met Gly Thr Ser Ser Pro Ala Arg
Arg Gly Ser Ala Asp Gly Pro Arg 260 265 270 Ser Ala Gln Pro Leu Arg
Pro Glu Asp Gly His Cys Ser Trp Pro Leu 275 280 285
* * * * *