U.S. patent application number 17/695081 was filed with the patent office on 2022-09-22 for psgl-1 antagonists and uses thereof.
This patent application is currently assigned to Bristol-Myers Squibb Company. The applicant listed for this patent is Bristol-Myers Squibb Company, Five Prime Therapeutics, Inc.. Invention is credited to Robert J. Johnston, Arathi Krishnakumar, Arvind Rajpal, Andrew Rankin, Paul O. Sheppard.
Application Number | 20220298258 17/695081 |
Document ID | / |
Family ID | 1000006391452 |
Filed Date | 2022-09-22 |
United States Patent
Application |
20220298258 |
Kind Code |
A1 |
Johnston; Robert J. ; et
al. |
September 22, 2022 |
PSGL-1 Antagonists and Uses Thereof
Abstract
Methods of identifying and using PSGL-1 antagonists are
provided. Such methods include, but are not limited to, methods of
treating cancer. PSGL-1 antagonists include, but are not limited
to, antibodies that bind PSGL-1 and antibodies that bind VISTA,
wherein the antibodies inhibit PSGL-1 binding to VISTA, e.g., at
acidic pH (e.g., pH 6.0), as well as PSGL-1 and VISTA extracellular
domain polypeptides.
Inventors: |
Johnston; Robert J.; (San
Mateo, CA) ; Rankin; Andrew; (Redwood City, CA)
; Krishnakumar; Arathi; (Chesterfield, NJ) ;
Sheppard; Paul O.; (Granite Falls, WA) ; Rajpal;
Arvind; (San Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Bristol-Myers Squibb Company
Five Prime Therapeutics, Inc. |
Princeton
Thousand Oaks |
NJ
CA |
US
US |
|
|
Assignee: |
Bristol-Myers Squibb
Company
Princeton
NJ
Five Prime Therapeutics, Inc.
Thousand Oaks
CA
|
Family ID: |
1000006391452 |
Appl. No.: |
17/695081 |
Filed: |
March 15, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16476814 |
Jul 9, 2019 |
11306150 |
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PCT/US2018/013171 |
Jan 10, 2018 |
|
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17695081 |
|
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62445071 |
Jan 11, 2017 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2319/30 20130101;
C07K 2317/55 20130101; C07K 2317/56 20130101; C07K 2317/54
20130101; C07K 2317/24 20130101; C07K 2319/32 20130101; C07K
16/2896 20130101; A61K 45/06 20130101; C07K 2317/76 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Claims
1. A method of treating cancer and/or of inhibiting binding of
PSGL-1 to VISTA in a subject, comprising administering to the
subject an effective amount of at least one PSGL-1 antagonist.
2. The method of claim 1, wherein the method further comprises
administering to the subject an effective amount of a therapeutic
agent selected from chemotherapeutic agents, anti-angiogenesis
agents, growth inhibitory agents, immune-oncology agents, and
anti-neoplastic compositions.
3. The method of claim 1, wherein the PSGL-1 antagonist is selected
from a PSGL-1 antibody and a VISTA antibody.
4.-5. (canceled)
6. The method of claim 3, wherein the antibody is selected from a
chimeric antibody, a humanized antibody, a human antibody, and an
antibody fragment selected from an Fv, a single-chain Fv (scFv), a
Fab, a Fab', and a (Fab').sub.2.
7.-8. (canceled)
9. The method of claim 3, wherein the PSGL-1 antagonist is an
antibody that blocks binding of PSGL-1 to VISTA, at a pH in the
range of pH 5.5 to pH 6.5.
10. The method of claim 3, wherein the PSGL-1 antagonist is an
antibody that binds to VISTA at a pH in the range of pH 5.5 to pH
6.5 or an antibody that binds to PSGL 1 at a pH in the range of pH
5.5 to pH 6.5.
11. The method of claim 3, wherein the antibody binds to VISTA or
PSGL-1 with a K.sub.D of 10 nM or less.
12.-38. (canceled)
39. A method of inhibiting binding of PSGL-1 to VISTA on a cell
comprising contacting the cell with at least one PSGL-1
antagonist.
40. The method of claim 39, wherein the cell is in vitro.
41. The method of claim 39, wherein the PSGL-1 antagonist is
selected from a PSGL-1 antibody and a VISTA antibody.
42.-43. (canceled)
44. The method of claim 41, wherein the antibody is selected from a
chimeric antibody, a humanized antibody, a human antibody, and an
antibody fragment selected from an Fv, a single-chain Fv (scFv), a
Fab, a Fab', and a (Fab').sub.2.
45.-46. (canceled)
47. The method of claim 41, wherein the PSGL-1 antagonist is an
antibody that blocks binding of PSGL-1 to VISTA at a pH in the
range of pH 5.5 to pH 6.5.
48. The method of claim 41, wherein the PSGL-1 antagonist is an
antibody that binds to VISTA at a pH in the range of pH 5.5 to pH
6.5 or an antibody that binds to PSGL-1 at a pH in the range of pH
5.5 to pH 6.5.
49. The method of claim 41, wherein the antibody binds to VISTA or
PSGL-1 with a K.sub.D of 10 nM or less.
50.-65. (canceled)
66. A PSGL-1 antagonist, which inhibits at least partially the
binding of VISTA to PSGL-1, at a pH in the range of pH 5.5 to pH
6.5.
67. The PSGL-1 antagonist of claim 66, which inhibits by at least
90% the binding of VISTA to PSGL-1, at a pH in the range of pH 5.5
to pH 6.5.
68.-69. (canceled)
70. The PSGL-1 antagonist of claim 66, wherein the antagonist is
selected from a PSGL-1 antibody and a VISTA antibody.
71.-72. (canceled)
73. The PSGL-1 antagonist of claim 70, wherein the antibody is
selected from a chimeric antibody, a humanized antibody, a human
antibody, and an antibody fragment selected from an Fv, a
single-chain Fv (scFv), a Fab, a Fab', and a (Fab').sub.2.
74.-75. (canceled)
76. A composition comprising a PSGL-1 antagonist of claim 66.
77.-78. (canceled)
Description
[0001] This application is a divisional of U.S. patent application
Ser. No. 16/476,814, filed Jul. 9, 2019, which is a national stage
application of International Patent Application No.
PCT/US2018/013171, filed Jan. 10, 2018, which claims priority to
U.S. Provisional Application No. 62/445,071 filed Jan. 11, 2017,
each of which is incorporated in their entirety by reference
herein.
TECHNICAL FIELD
[0002] Methods of identifying and using PSGL-1 antagonists are
provided. Such methods include, but are not limited to, methods of
treating cancer. PSGL-1 antagonists include, but are not limited
to, antibodies that bind PSGL-1 and antibodies that bind VISTA,
wherein the antibodies inhibit PSGL-1 binding to VISTA, e.g., at
acidic pH (e.g., pH 6.0).
SEQUENCE LISTING
[0003] The present application is filed with a Sequence Listing in
electronic format. The Sequence Listing is provided as a file
entitled "2018-01-10_01134-0058-00PCT_Final_Seq_List ST25.txt"
created on Jan. 10, 2018, which is 49,152 bytes in size. The
information in the electronic format of the sequence listing is
incorporated herein by reference in its entirety.
BACKGROUND
[0004] V-region Immunoglobulin-containing Suppressor of T cell
Activation (VISTA) is a cell surface-expressed protein that
negatively regulates the activity of T cells (Wang et al., 2011,
JEM 208(3) 577). VISTA is a single pass type-I transmembrane
protein with a single extracellular IgV domain. Notably, the
extracellular domain of VISTA bears homology to B7 family members
such as PDL1, which also plays a role in modulating immune
responses (Wang et al., 2011, JEM 208(3) 577). VISTA expression is
restricted to hematopoietic cells and is present on monocytes, T
cells and a fraction of dendritic cells (Wang et al., 2011, JEM
208(3) 577 & Flies et al., 2011, JI 187:1537). Treatment with
VISTA:Ig in vitro suppresses proliferation and cytokine production
by CD4+ T cells (Wang et al., 2011, JEM 208(3) 577). A VISTA
specific monoclonal antibody can elicit enhanced T cell division in
response to antigens presented by VISTA-expressing dendritic cells
(Wang et al., 2011, JEM 208(3) 577). In vivo treatment of
tumor-bearing animals with an anti-VISTA monoclonal antibody
elicited an immune mediated anti-tumor response that inhibited
tumor growth (Wang et al., 2011, JEM 208(3) 577). Collectively,
these results highlight the importance of VISTA as a regulator of T
cell-driven immune responses such as those observed during immune
mediated tumor rejection. The cognate binding partner for VISTA is
currently unknown.
[0005] Identification of binding partners for VISTA would assist in
the understanding of VISTA-mediated inhibition of T-cell
activation, and provide many advantages to drug development
including selection of therapeutically effective and safe
therapeutics, biomarkers for patient selection and companion
diagnostics, targets for combination therapy, and new targets for
developing cancer immunotherapeutic agents.
SUMMARY
[0006] In some embodiments, methods of identifying PSGL-1
antagonists are provided. In some embodiments, a method comprises
contacting a candidate molecule with a VISTA molecule (e.g., a
mature VISTA protein or a fragment thereof) and a PSGL-1 molecule
(e.g., a mature PSGL-1 protein or fragment thereof), wherein the
VISTA molecule comprises VISTA, a VISTA extracellular domain
("ECD"), or a VISTA ECD fusion molecule (e.g., excluding a signal
sequence; i.e., a mature VISTA or fragment thereof), and the PSGL-1
molecule comprises PSGL-1, PSGL-1 ECD, or PSGL-1 ECD fusion
molecule (e.g., excluding a signal sequence; i.e., a mature PSGL-1
or fragment thereof), wherein the contacting occurs in acidic pH,
e.g., pH<7.0, .ltoreq.6.8, .ltoreq.6.5 or .ltoreq.6.3 or at pH
5.5 to 6.5, 6.0-6.5, 6.5-7.0 or 6.0-7.0, e.g., pH 6.0. In some
embodiments, a method comprises forming a composition comprising a
candidate molecule, a VISTA molecule, and PSGL-1 molecule, wherein
the VISTA molecule comprises VISTA, a VISTA ECD, or a VISTA ECD
fusion molecule, and the PSGL-1 molecule comprises PSGL-1, PSGL-1
ECD, or PSGL-1 ECD fusion molecule, e.g., at acidic pH, e.g., pH
<7.0, .ltoreq.6.8, .ltoreq.6.5 or .ltoreq.6.3 or at pH 5.5 to
6.5, 6.0-6.5, 6.5-7.0 or 6.0-7.0, e.g., pH 6.0. In some
embodiments, a method further comprises detecting binding of the
VISTA molecule to the PSGL-1 molecule. In some embodiments, a
reduction in the binding of the VISTA molecule to the PSGL-1
molecule in the presence of the candidate molecule as compared to
the binding of the VISTA molecule to the PSGL-1 molecule in the
absence of the candidate molecule indicates that the candidate
molecule is PSGL-1 antagonist. In some embodiments, binding of the
VISTA molecule to the PSGL-1 molecule is reduced by at least 30%,
at least 40%, at least 50%, at least 60%, at least 70%, or at least
80% in the presence of the candidate molecule. In some embodiments,
binding of the VISTA molecule to the PSGL-1 molecule is detected by
a method selected from surface plasmon resonance, ELISA, amplified
luminescent proximity homogeneous assay (ALPHA), and flow
cytometry.
[0007] In any of the methods of identifying PSGL-1 antagonists
described herein, the PSGL-1 antagonist may be an antibody that
binds to VISTA, e.g., at acidic pH, e.g., pH<7.0, .ltoreq.6.8,
.ltoreq.6.5 or .ltoreq.6.3 or at pH 5.5 to 6.5, 6.0-6.5, 6.5-7.0 or
6.0-7.0, e.g., pH 6.0. In any of the methods of identifying PSGL-1
antagonists described herein, the PSGL-1 antagonist may be an
antibody that binds PSGL-1. In any of the methods of identifying
PSGL-1 antagonists described herein, the PSGL-1 antagonist may be a
small molecule. In any of the methods of identifying PSGL-1
antagonists described herein, the PSGL-1 antagonist may be a small
peptide.
[0008] In some embodiments, methods of determining whether a VISTA
antibody is PSGL-1 antagonist are provided. In some embodiments, a
method comprises contacting the VISTA antibody with a VISTA
molecule and PSGL-1 molecule, wherein the VISTA molecule comprises
VISTA, a VISTA ECD, or a VISTA ECD fusion molecule, and the PSGL-1
molecule comprises PSGL-1, PSGL-1 ECD, or PSGL-1 ECD fusion
molecule, e.g., at acidic pH, e.g., pH<7.0, .ltoreq.6.8,
.ltoreq.6.5 or .ltoreq.6.3 or at pH 5.5 to 6.5, 6.0-6.5, 6.5-7.0 or
6.0-7.0, e.g., pH 6.0. In some embodiments, a method comprises
forming a composition comprising the VISTA antibody, a VISTA
molecule, and PSGL-1 molecule, wherein the VISTA molecule comprises
VISTA, a VISTA ECD, or a VISTA ECD fusion molecule, and the PSGL-1
molecule comprises PSGL-1, PSGL-1 ECD, or PSGL-1 ECD fusion
molecule, e.g., acidic pH, e.g., pH<7.0, .ltoreq.6.8,
.ltoreq.6.5 or .ltoreq.6.3 or at pH 5.5 to 6.5, 6.0-6.5, 6.5-7.0 or
6.0-7.0, e.g., pH 6.0. In some embodiments, a method further
comprises detecting the binding of the VISTA molecule to the PSGL-1
molecule. In some embodiments, a reduction in the binding of the
VISTA molecule to the PSGL-1 molecule in the presence of the VISTA
antibody as compared to the binding of the VISTA molecule to the
PSGL-1 molecule in the absence of the VISTA antibody indicates that
the VISTA antibody is PSGL-1 antagonist. In some embodiments,
binding of the VISTA molecule to the PSGL-1 molecule is reduced by
at least 30%, at least 40%, at least 50%, at least 60%, at least
70%, or at least 80% in the presence of the VISTA antibody. In some
embodiments, binding of the VISTA molecule to the PSGL-1 molecule
is detected by a method selected from surface plasmon resonance,
ELISA, amplified luminescent proximity homogeneous assay, and flow
cytometry.
[0009] In any of the methods of identifying PSGL-1 antagonists
described herein, the VISTA molecule may be VISTA expressed on the
surface of a cell and/or the PSGL-1 molecule may be PSGL-1
expressed on the surface of a cell.
[0010] In some embodiments, methods of inhibiting binding of PSGL-1
to VISTA in a subject are provided. In some embodiments, a method
comprise administering to the subject at least one PSGL-1
antagonist. In some embodiments, methods of inhibiting binding of
PSGL-1 to VISTA on a cell are provided. In some embodiments, a
method comprises contacting the cell with at least one PSGL-1
antagonist, e.g., at acidic pH, e.g., pH<7.0, .ltoreq.6.8,
.ltoreq.6.5 or .ltoreq.6.3 or at pH 5.5 to 6.5, 6.0-6.5, 6.5-7.0 or
6.0-7.0, e.g., pH 6.0. In some embodiments, the cell is in
vitro.
[0011] In some embodiments, methods of treating cancer are
provided. In some embodiments, a method comprises administering to
a subject with cancer an effective amount of at least one PSGL-1
antagonist. In some embodiments, the method further comprises
administering to the subject an effective amount of a therapeutic
agent selected from chemotherapeutic agents, anti-angiogenesis
agents, growth inhibitory agents, immune-oncology agents, and
anti-neoplastic compositions. In any of the embodiments described
herein, PSGL-1 antagonist may block binding of PSGL-1 to VISTA.
[0012] In any of the embodiments described herein, a method may
comprise administering a PSGL-1 antagonist selected from PSGL-1
antibody and a VISTA antibody, wherein the antibody inhibits
binding of PSGL-1 to VISTA, e.g., at acidic pH, e.g., pH<7.0,
.ltoreq.6.8, .ltoreq.6.5 or .ltoreq.6.3 or at pH 5.5 to 6.5,
6.0-6.5, 6.5-7.0 or 6.0-7.0, e.g., pH 6.0. In any of the
embodiments described herein, a method may comprise administering a
PSGL-1 antibody that inhibits binding of PSGL-1 to VISTA, e.g., at
acidic pH, e.g., pH<7.0, .ltoreq.6.8, .ltoreq.6.5 or .ltoreq.6.3
or at pH 5.5 to 6.5, 6.0-6.5, 6.5-7.0 or 6.0-7.0, e.g., pH 6.0. In
any of the embodiments described herein, an antibody may be
selected from a chimeric antibody, a humanized antibody, and a
human antibody. In any of the embodiments described herein, an
antibody may be an antibody fragment. In some embodiments, the
antibody fragment is selected from an IgG (e.g., IgG1, IgG2 or
IgG4), Fv, a single-chain Fv (scFv), a Fab, a Fab', and a
(Fab').sub.2.
[0013] In some embodiments, uses of PSGL-1 antagonists for treating
cancer in subjects are provided. In any of the uses described
herein, the PSGL-1 antagonist may be a PSGL-1 antibody or a VISTA
antibody, wherein the antibody inhibits binding of PSGL-1 to VISTA,
e.g., at acidic pH, e.g., pH<7.0, .ltoreq.6.8, .ltoreq.6.5 or
.ltoreq.6.3 or at pH 5.5 to 6.5, 6.0-6.5, 6.5-7.0 or 6.0-7.0, e.g.,
pH 6.0. In some embodiments, the antibody is selected from a
chimeric antibody, a humanized antibody, and a human antibody. In
some embodiments, the antibody is an antibody fragment. In some
embodiments, the antibody fragment is selected from an IgG (e.g.,
IgG1, IgG2 or IgG4), Fv, a single-chain Fv (scFv), a Fab, a Fab',
and a (Fab').sub.2.
[0014] Any embodiment described herein or any combination thereof
applies to any and all methods of the invention described
herein.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1 shows flow cytometry results of VISTA Dextramer
binding to T cells at various pH, showing enhanced binding at lower
pH.
[0016] FIG. 2A shows binding of anti-PSGL-1 to B cells, T cells and
NK cells, as described in Example 2. FIG. 2B shows binding of VISTA
Dextramer to B cells, T cells and NK cells, as also described in
Example 2.
[0017] FIG. 3 shows the relative VISTA Dextramer binding to
activated human CD4+ T cells in the presence of a control antibody
(.circle-solid.), hPSGL-1-Fc () or p-selectin (diamonds).
[0018] FIGS. 4A and 4B show that PSGL-1 binds to 293T-hVISTA cells
at pH 6.0. FIG. 4B shows the binding of PSGL-1-Fc to 293T-hVISTA
cells at pH 6.0 (dots/filled circles) and the lack of significant
binding at pH 7.2 (filled squares), as well as the lack of binding
at pH 6.0 and 7.2 of PSGL-1-Fc to 293T cells that do not express
hVISTA (diamonds and triangles, respectively, partially hidden
behind the squares). FIG. 4A provides the rough data supporting
FIG. 4B, and showing binding of PSGL-1 to 293T-hVISTA cells at pH
6.0.
[0019] FIG. 5 shows the percentage of human VISTA binding to T
cells ("control gRNA") and to PSGL-1 CRISPR knockout T cells
("PSGL-1 gRNA#1" and PSGL-1 gRNA#2'', which are two clones obtained
using two different guide RNAs).
DETAILED DESCRIPTION
[0020] The present inventors have identified PSGL-1 as a binding
partner for VISTA, wherein binding occurs preferentially in acidic
pH. Targeting the interaction between VISTA and PSGL-1 may enhance
the immune response to cancer cells by inhibiting the immune
inhibitory effects of VISTA. Targeting molecules include antibodies
that bind PSGL-1 and antibodies that bind VISTA, wherein the
antibodies block the binding of VISTA to PSGL-1. Exemplary
targeting molecules are molecules that bind to VISTA or PSGL-1 at
acidic pH, e.g., at acidic pH, e.g., pH<7.0, .ltoreq.6.8,
.ltoreq.6.5 or .ltoreq.6.3 or at pH 5.5 to 6.5, 6.0-6.5, 6.5-7.0 or
6.0-7.0, e.g., pH 6.0. Such targeting molecules are provided as
therapeutic agents for treating cancer.
[0021] All references cited herein, including patent applications
and publications, are incorporated by reference herein in their
entirety.
[0022] The section headings used herein are for organizational
purposes only and are not to be construed as limiting the subject
matter described.
Definitions
[0023] Unless otherwise defined, scientific and technical terms
used in connection with the present invention shall have the
meanings that are commonly understood by those of ordinary skill in
the art. Further, unless otherwise required by context, singular
terms shall include pluralities and plural terms shall include the
singular.
[0024] Exemplary techniques used in connection with recombinant
DNA, oligonucleotide synthesis, tissue culture and transformation
(e.g., electroporation, lipofection), enzymatic reactions, and
purification techniques are known in the art. Many such techniques
and procedures are described, e.g., in Sambrook et al. Molecular
Cloning: A Laboratory Manual (3rd ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. (2001)), among other
places. In addition, exemplary techniques for chemical syntheses,
chemical analyses, pharmaceutical preparation, formulation, and
delivery, and treatment of patients are also known in the art.
[0025] In this application, the use of "or" means "and/or" unless
stated otherwise. In the context of a multiple dependent claim, the
use of "or" refers back to more than one preceding independent or
dependent claim in the alternative only. Unless otherwise
indicated, the term "include" has the same meaning as "include, but
are not limited to," the term "includes" has the same meaning as
"includes, but is not limited to," and the term "including" has the
same meaning as "including, but not limited to." Similarly, the
term "such as" has the same meaning as the term "such as, but not
limited to." Also, terms such as "element" or "component" encompass
both elements and components comprising one unit and elements and
components that comprise more than one subunit unless specifically
stated otherwise.
[0026] As utilized in accordance with the present disclosure, the
following terms, unless otherwise indicated, shall be understood to
have the following meanings:
[0027] The terms "nucleic acid molecule" and "polynucleotide" may
be used interchangeably, and refer to a polymer of nucleotides.
Such polymers of nucleotides may contain natural and/or non-natural
nucleotides, and include, but are not limited to, DNA, RNA, and
PNA. "Nucleic acid sequence" refers to the linear sequence of
nucleotides that comprise the nucleic acid molecule or
polynucleotide.
[0028] The terms "polypeptide" refers to a polymer of amino acid
residues, and are not limited to a minimum length. Such polymers of
amino acid residues may contain natural or non-natural amino acid
residues, and include, but are not limited to, peptides,
oligopeptides, dimers, trimers, and multimers of amino acid
residues. Both full-length proteins and fragments thereof are
encompassed by the definition. The terms also include
post-expression modifications of the polypeptide, for example,
glycosylation, sialylation, acetylation, phosphorylation, and the
like. Furthermore, for purposes of the present invention, a
"polypeptide" refers to a protein that includes modifications, such
as deletions, additions, and substitutions (generally conservative
in nature), to the native sequence, as long as the protein
maintains the desired activity. These modifications may be
deliberate, as through site-directed mutagenesis, or may be
accidental, such as through mutations of hosts that produce the
proteins or errors due to PCR amplification. A "protein" includes
polypeptides and complexes of 2 or more polypeptides, e.g., dimers
and polymers. A "small peptide" refers to a peptide having 50 or
fewer amino acids. In some embodiments, a small peptide has 40 or
fewer, or 35 or fewer, or 30 or fewer, or 25 or fewer amino acids.
In some embodiments, a small peptide has 10 to 50 amino acids or 15
to 30 amino acids.
[0029] A "native sequence" polypeptide comprises a polypeptide
having the same amino acid sequence as a polypeptide found in
nature. Thus, a native sequence polypeptide can have the amino acid
sequence of naturally occurring polypeptide from any mammal. Such
native sequence polypeptide can be isolated from nature or can be
produced by recombinant or synthetic means. The term "native
sequence" polypeptide specifically encompasses naturally occurring
truncated or secreted forms of the polypeptide (e.g., an
extracellular domain sequence), naturally occurring variant forms
(e.g., alternatively spliced forms) and naturally occurring allelic
variants of the polypeptide.
[0030] A polypeptide "variant" means a biologically active
polypeptide having at least about 80% amino acid sequence identity
with the native sequence polypeptide after aligning the sequences
and introducing gaps, if necessary, to achieve the maximum percent
sequence identity, and not considering any conservative
substitutions as part of the sequence identity. Such variants
include, for instance, polypeptides wherein one or more amino acid
residues are added, or deleted, at the N- or C-terminus of the
polypeptide. In some embodiments, a variant will have at least
about 80% amino acid sequence identity. In some embodiment, a
variant will have at least about 90% amino acid sequence identity.
In some embodiment, a variant will have at least about 95% amino
acid sequence identity with the native sequence polypeptide. In
some embodiment, a variant will have at least about 97% amino acid
sequence identity with the native sequence polypeptide.
[0031] As used herein, "Percent (%) amino acid sequence identity"
and "homology" with respect to a peptide, polypeptide or antibody
sequence are defined as the percentage of amino acid residues in a
candidate sequence that are identical with the amino acid residues
in the specific peptide or polypeptide sequence, after aligning the
sequences and introducing gaps, if necessary, to achieve the
maximum percent sequence identity, and not considering any
conservative substitutions as part of the sequence identity.
Alignment for purposes of determining percent amino acid sequence
identity can be achieved in various ways that are within the skill
in the art, for instance, using publicly available computer
software such as BLAST, BLAST-2, ALIGN or MEGALIGN.TM. (DNASTAR)
software. Those skilled in the art can determine appropriate
parameters for measuring alignment, including any algorithms needed
to achieve maximal alignment over the full length of the sequences
being compared.
[0032] The terms "P-Selectin Glycoprotein Ligand 1" and "PSGL-1"
are used interchangeably to refer to a native, PSGL-1 unless
otherwise indicated. Thus, reference to PSGL-1 has the same meaning
as reference to human PSGL-1 throughout the text, unless the
context clarifies otherwise (e.g. by discussing non-human, or
murine or cynomolgus PSGL-1). A non-human PSGL-1 may be from any
vertebrate source, including mammals such as primates (e.g.
cynomolgus monkeys) and rodents (e.g., mice and rats).
[0033] The term PSGL-1 includes full-length, unprocessed PSGL-1 as
well as any form of PSGL-1 that results from processing in the cell
or any fragment thereof that retains the ability to specifically
bind VISTA, e.g., with an affinity (Kd) of less than .ltoreq.1
.mu.M, .ltoreq.100 nM, or .ltoreq.10 nM. The term also encompasses
naturally occurring variants of PSGL-1, e.g., splice variants or
allelic variants. In some embodiments, PSGL-1 comprises the amino
acid sequence of SEQ ID NO: 1 (human isoform 1 precursor, with
signal peptide) or SEQ ID NO: 2 (human isoform 1 mature, without
signal peptide) or the amino acid sequence of SEQ ID NO: 14 (human
isoform 2 precursor, with signal peptide) or SEQ ID NO: 15 (human
isoform 2 mature, without signal peptide).
[0034] The term "PSGL-1" also includes full-length PSGL-1, PSGL-1
fragments, and PSGL-1 variants, with or without a signal peptide.
The term "full-length PSGL-1", as used herein, refers to
full-length, unprocessed PSGL-1 as well as any form of PSGL-1 that
results from processing in the cell or any fragment thereof that
retains the ability to specifically bind VISTA, e.g., with an
affinity (Kd) of less than .ltoreq.1 .mu.M, .ltoreq.100 nM, or
.ltoreq.10 nM. In some embodiments, a full-length PSGL-1 has the
amino acid sequence of SEQ ID NO: 1 (isoform 1 precursor, with
signal peptide) or SEQ ID NO: 2 (isoform 1 mature, without signal
peptide) or SEQ ID NO: 14 (isoform 2 precursor, with signal
peptide) or SEQ ID NO: 15 (isoform 2 mature, without signal
peptide). As used herein, the term "PSGL-1 fragment" refers to
PSGL-1 having one or more residues deleted from the N- and/or
C-terminus of the full-length PSGL-1 and that retains the ability
to bind VISTA. The PSGL-1 fragment may or may not include an
N-terminal signal peptide. As used herein, the term "PSGL-1
variant" refers to PSGL-1 that contains amino acid additions,
deletions, and substitutions and that remain capable of binding to
VISTA. Such variants may be at least 80%, 85%, 90%, 92%, 95%, 97%,
98%, or 99% identical to the parent PSGL-1. The % identity of two
polypeptides can be measured by a similarity score determined by
comparing the amino acid sequences of the two polypeptides using
the Bestfit program with the default settings for determining
similarity. Bestfit uses the local homology algorithm of Smith and
Waterman, Advances in Applied Mathematics 2:482-489 (1981) to find
the best segment of similarity between two sequences.
[0035] The terms "V-domain Ig Suppressor of T cell Activation,"
"platelet receptor Gi24 isoform 1 precursor," "B7115," and "VISTA"
refer herein to a native, human VISTA unless specified otherwise.
Thus, the expressions "VISTA" and "human VISTA" have the same
meaning unless the context clarifies otherwise (e.g. by referring
specifically to a non-human VISTA species). If the VISTA is
non-human, it may be from any vertebrate source, including mammals
such as primates (e.g. cynomolgus monkeys) and rodents (e.g., mice
and rats). The term includes full-length, unprocessed VISTA as well
as any form of VISTA that results from processing in the cell or
any fragment thereof that retains the ability to specifically bind
PSGL-1, e.g., with an affinity (Kd) of less than .ltoreq.10M,
.ltoreq.100 nM, or .ltoreq.10 nM. The term also encompasses
naturally occurring variants of VISTA, e.g., splice variants or
allelic variants. In some embodiments, VISTA comprises the amino
acid sequence of SEQ ID NO: 5 (precursor, with signal peptide) or
SEQ ID NO: 6 (mature, without signal peptide). A nonlimiting
exemplary non-human VISTA is mouse VISTA, which has the amino acid
sequence of SEQ ID NO: 7 (precursor, with signal peptide) or SEQ ID
NO: 8 (mature, without signal peptide).
[0036] The term "antagonist" is used in the broadest sense, and
includes any molecule that partially or fully inhibits or
neutralizes a biological activity of a polypeptide, such as PSGL-1
or VISTA, or that partially or fully inhibits the transcription or
translation of a nucleic acid encoding the polypeptide. Exemplary
antagonist molecules include, but are not limited to, antagonist
antibodies, small peptides, oligopeptides, organic molecules
(including small molecules), aptamers, and antisense nucleic acids.
In some embodiments, an antagonist agent may be referred to as a
blocking agent (such as a blocking antibody).
[0037] The term "PSGL-1 antagonist" refers to a molecule that
interacts with PSGL-1 or VISTA and inhibits PSGL-1 and/or
VISTA-mediated signaling. Exemplary PSGL-1 antagonists include
antibodies that bind PSGL-1 and antibodies that bind VISTA. In some
embodiments, PSGL-1 antagonist is an antibody to PSGL-1. In some
embodiments, PSGL-1 antagonist blocks binding of PSGL-1 to
VISTA.
[0038] A PSGL-1 antagonist is considered to "block binding of
PSGL-1 to VISTA" when it reduces the amount of detectable binding
of PSGL-1 to VISTA by at least 50%. In some embodiments, a PSGL-1
antagonist reduces the amount of detectable binding of PSGL-1 to
VISTA by at least 60%, at least 70%, at least 80%, or at least 90%.
In some such embodiments, the antagonist is said to block ligand
binding by at least 50%, at least 60%, at least 70%, etc.
[0039] The terms "inhibition" or "inhibit" refer to a decrease or
cessation of any phenotypic characteristic or to the decrease or
cessation in the incidence, degree, or likelihood of that
characteristic. In some embodiments, by "reduce" or "inhibit" is
meant the ability to cause a decrease of 20% or greater. In another
embodiment, by "reduce" or "inhibit" is meant the ability to cause
a decrease of 50% or greater. In yet another embodiment, by
"reduce" or "inhibit" is meant the ability to cause an overall
decrease of 75%, 85%, 90%, 95%, or greater.
[0040] The term "PSGL-1 antibody" or "antibody that binds PSGL-1,"
as used herein, refers to an antibody that binds to PSGL-1, e.g. at
acidic pH, e.g., pH<7.0, .ltoreq.6.8, .ltoreq.6.5 or .ltoreq.6.3
or at pH 5.5 to 6.5, 6.0-6.5, 6.5-7.0 or 6.0-7.0, e.g., pH 6.0. In
some embodiments, PSGL-1 antibody inhibits PSGL-1 and/or
VISTA-mediated signaling. In some embodiments, PSGL-1 antibody
blocks binding of PSGL-1 to VISTA, e.g., at acidic pH, e.g.,
pH<7.0, .ltoreq.6.8, .ltoreq.6.5 or .ltoreq.6.3 or at pH 5.5 to
6.5, 6.0-6.5, 6.5-7.0 or 6.0-7.0, e.g., pH 6.0. In some
embodiments, PSGL-1 antibody refers to an antibody that is capable
of binding PSGL-1 with sufficient affinity such that the antibody
is useful as a diagnostic and/or therapeutic agent in targeting
PSGL-1. In some embodiments, the extent of binding of PSGL-1
antibody to an unrelated, non-PSGL-1 protein is less than about 10%
of the binding of the antibody to PSGL-1 as measured, e.g., by a
radioimmunoassay (RIA). In some embodiments, PSGL-1 antibody binds
to an epitope of PSGL-1 that is conserved among PSGL-1 from
different species. In some embodiments, PSGL-1 antibody binds to
the same epitope as a human or humanized PSGL-1 antibody that binds
PSGL-1.
[0041] The term "VISTA antibody" or "antibody that binds VISTA," as
used herein, refers to an antibody that binds to VISTA, e.g., at
acidic pH, e.g., pH<7.0, .ltoreq.6.8, .ltoreq.6.5 or .ltoreq.6.3
or a pH 6.0-6.5, 6.5-7.0 or 6.0-7.0. In some embodiments, a VISTA
antibody inhibits PSGL-1 and/or VISTA-mediated signaling. In some
embodiments, a VISTA antibody blocks binding of PSGL-1 to VISTA, as
defined above, e.g., at acidic pH, e.g., pH<7.0, .ltoreq.6.8,
.ltoreq.6.5 or .ltoreq.6.3 or at pH 5.5 to 6.5, 6.0-6.5, 6.5-7.0 or
6.0-7.0, e.g., pH 6.0. Thus, in some embodiments, a VISTA antibody
is PSGL-1 antagonist. In some embodiments, a VISTA antibody refers
to an antibody that is capable of binding VISTA with sufficient
affinity such that the antibody is useful as a diagnostic and/or
therapeutic agent in targeting VISTA. In some embodiments, the
extent of binding of a VISTA antibody to an unrelated, non-VISTA
protein is less than about 10% of the binding of the antibody to
VISTA as measured, e.g., by a radioimmunoassay (MA). In some
embodiments, a VISTA antibody binds to an epitope of VISTA that is
conserved among VISTA from different species. In some embodiments,
a VISTA antibody binds to the same epitope as a human or humanized
VISTA antibody that binds human VISTA.
[0042] The term "antibody" herein is used in the broadest sense and
encompasses various antibody structures, including but not limited
to monoclonal antibodies, polyclonal antibodies, multispecific
antibodies (e.g., bispecific antibodies), and antibody fragments so
long as they exhibit the desired antigen-binding activity. The term
"antibody" as used herein further refers to a molecule comprising
complementarity-determining region (CDR) 1, CDR2, and CDR3 of a
heavy chain and CDR1, CDR2, and CDR3 of a light chain, wherein the
molecule is capable of binding to antigen. The term antibody
includes, but is not limited to, fragments that are capable of
binding antigen, such as Fv, single-chain Fv (scFv), Fab, Fab', and
(Fab')2. The term antibody also includes, but is not limited to,
chimeric antibodies, humanized antibodies, and antibodies of
various species such as mouse, human, cynomolgus monkey, etc.
[0043] In some embodiments, an antibody comprises a heavy chain
variable region and a light chain variable region. In some
embodiments, an antibody comprises at least one heavy chain
comprising a heavy chain variable region and at least a portion of
a heavy chain constant region, and at least one light chain
comprising a light chain variable region and at least a portion of
a light chain constant region. In some embodiments, an antibody
comprises two heavy chains, wherein each heavy chain comprises a
heavy chain variable region and at least a portion of a heavy chain
constant region, and two light chains, wherein each light chain
comprises a light chain variable region and at least a portion of a
light chain constant region. As used herein, a single-chain Fv
(scFv), or any other antibody that comprises, for example, a single
polypeptide chain comprising all six CDRs (three heavy chain CDRs
and three light chain CDRs) is considered to have a heavy chain and
a light chain. In some such embodiments, the heavy chain is the
region of the antibody that comprises the three heavy chain CDRs
and the light chain in the region of the antibody that comprises
the three light chain CDRs.
[0044] The term "heavy chain variable region" as used herein refers
to a region comprising heavy chain CDR1, framework (FR) 2, CDR2,
FR3, and CDR3. In some embodiments, a heavy chain variable region
also comprises at least a portion of an FR1, which is N-terminal to
CDR1, and/or at least a portion of an FR4, which is C-terminal to
CDR3.
[0045] The term "heavy chain constant region" as used herein refers
to a region comprising at least three heavy chain constant domains,
CH1, CH2, and CH3. Nonlimiting exemplary heavy chain constant
regions include .gamma., .delta., and .alpha.. Nonlimiting
exemplary heavy chain constant regions also include and .mu.. Each
heavy constant region corresponds to an antibody isotype. For
example, an antibody comprising a y constant region is an IgG
antibody, an antibody comprising a .delta. constant region is an
IgD antibody, and an antibody comprising an .alpha. constant region
is an IgA antibody. Further, an antibody comprising a .mu. constant
region is an IgM antibody, and an antibody comprising an constant
region is an IgE antibody. Certain isotypes can be further
subdivided into subclasses. For example, IgG antibodies include,
but are not limited to, IgG1 (comprising a .gamma..sub.1 constant
region), IgG2 (comprising a .gamma..sub.2 constant region), IgG3
(comprising a .gamma..sub.3 constant region), and IgG4 (comprising
a .gamma..sub.4 constant region) antibodies; IgA antibodies
include, but are not limited to, IgA1 (comprising an .alpha..sub.1
constant region) and IgA2 (comprising an .alpha..sub.2 constant
region) antibodies; and IgM antibodies include, but are not limited
to, IgM1 and IgM2.
[0046] The term "heavy chain" as used herein refers to a
polypeptide comprising at least a heavy chain variable region, with
or without a leader sequence. In some embodiments, a heavy chain
comprises at least a portion of a heavy chain constant region. The
term "full-length heavy chain" as used herein refers to a
polypeptide comprising a heavy chain variable region and a heavy
chain constant region, with or without a leader sequence, and with
or without a C-terminal lysine.
[0047] The term "light chain variable region" as used herein refers
to a region comprising light chain CDR1, framework (FR) 2, CDR2,
FR3, and CDR3. In some embodiments, a light chain variable region
also comprises an FR1 and/or an FR4.
[0048] The term "light chain constant region" as used herein refers
to a region comprising a light chain constant domain, CL.
Nonlimiting exemplary light chain constant regions include .lamda.,
and .kappa..
[0049] The term "light chain" as used herein refers to a
polypeptide comprising at least a light chain variable region, with
or without a leader sequence. In some embodiments, a light chain
comprises at least a portion of a light chain constant region. The
term "full-length light chain" as used herein refers to a
polypeptide comprising a light chain variable region and a light
chain constant region, with or without a leader sequence.
[0050] An "antibody that binds to the same epitope" as a reference
antibody as determined by an antibody competition assay, refers to
an antibody that blocks binding of the reference antibody to its
antigen in a competition assay by 50% or more, and conversely, the
reference antibody blocks binding of the antibody to its antigen in
a competition assay by 50% or more. The term "compete" when used in
the context of an antibody that compete for the same epitope means
competition between antibodies is determined by an assay in which
an antibody being tested prevents or inhibits specific binding of a
reference antibody to a common antigen (e.g., PSGL-1 or VISTA).
Numerous types of competitive binding assays can be used, for
example: solid phase direct or indirect radioimmunoassay (RIA),
solid phase direct or indirect enzyme immunoassay (EIA), sandwich
competition assay (see, e.g., Stahli et al., 1983, Methods in
Enzymology 9:242-253); solid phase direct biotin-avidin EIA (see,
e.g., Kirkland et al., 1986, J. Immunol. 137:3614-3619) solid phase
direct labeled assay, solid phase direct labeled sandwich assay
(see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual,
Cold Spring Harbor Press); solid phase direct label RIA using 1-125
label (see, e.g., Morel et al., 1988, Molec. Immunol. 25:7-15);
solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al.,
1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer et
al., 1990, Scand. J. Immunol. 32:77-82). Typically, such an assay
involves the use of purified antigen bound to a solid surface or
cells bearing either of these, an unlabeled test antigen binding
protein and a labeled reference antibody. Competitive inhibition is
measured by determining the amount of label bound to the solid
surface or cells in the presence of the test antibody. Usually the
test antibody is present in excess. Antibodies identified by
competition assay (competing antibodies) include antibodies binding
to the same epitope as the reference antibodies and antibodies
binding to an adjacent epitope sufficiently proximal to the epitope
bound by the reference antibody for steric hindrance to occur. In
some embodiments, when a competing antibody is present in excess,
it will inhibit specific binding of a reference antibody to a
common antigen by at least 40%, 45%, 50%, 55%, 60%, 65%, 70% or
75%. In some instance, binding is inhibited by at least 80%, 85%,
90%, 95%, or 97% or more.
[0051] The term "antigen" refers to a molecule or a portion of a
molecule capable of being bound by a selective binding agent, such
as an antibody or immunologically functional fragment thereof, and
additionally capable of being used in a mammal to produce
antibodies capable of binding to that antigen. An antigen may
possess one or more epitopes that are capable of interacting with
antibodies.
[0052] The term "epitope" is the portion of a molecule that is
bound by a selective binding agent, such as an antibody or a
fragment thereof. The term includes any determinant capable of
specifically binding to an antibody. An epitope can be contiguous
or non-contiguous (e.g., in a polypeptide, amino acid residues that
are not contiguous to one another in the polypeptide sequence but
that within in context of the molecule are bound by the antigen
binding protein). In some embodiments, epitopes may be mimetic in
that they comprise a three dimensional structure that is similar to
an epitope used to generate the antibody, yet comprise none or only
some of the amino acid residues found in that epitope used to
generate the antibody. Epitope determinants may include chemically
active surface groupings of molecules such as amino acids, sugar
side chains, phosphoryl or sulfonyl groups, and may have specific
three dimensional structural characteristics, and/or specific
charge characteristics. In some embodiments, an "epitope" is
defined by the method used to determine it. For example, in some
embodiments, an antibody binds to the same epitope as a reference
antibody, if they bind to the same region of the antigen, as
determined by hydrogen-deuterium exchange (HDX). In certain
embodiments, an antibody binds to the same epitope as a reference
antibody if they bind to the same region of the antigen, as
determined by X-ray crystallography.
[0053] A "chimeric antibody" as used herein refers to an antibody
comprising at least one variable region from a first species (such
as mouse, rat, cynomolgus monkey, etc.) and at least one constant
region from a second species (such as human, cynomolgus monkey,
chicken, etc.). In some embodiments, a chimeric antibody comprises
at least one mouse variable region and at least one human constant
region. In some embodiments, a chimeric antibody comprises at least
one cynomolgus variable region and at least one human constant
region. In some embodiments, all of the variable regions of a
chimeric antibody are from a first species and all of the constant
regions of the chimeric antibody are from a second species.
[0054] A "humanized antibody" as used herein refers to an antibody
in which at least one amino acid in a framework region of a
non-human variable region (such as mouse, rat, cynomolgus monkey,
chicken, etc.) has been replaced with the corresponding amino acid
from a human variable region. In some embodiments, a humanized
antibody comprises at least one human constant region or fragment
thereof. In some embodiments, a humanized antibody is an Fab, an
scFv, a (Fab').sub.2, etc.
[0055] A "CDR-grafted antibody" as used herein refers to a
humanized antibody in which one or more complementarity determining
regions (CDRs) of a first (non-human) species have been grafted
onto the framework regions (FRs) of a second (human) species.
[0056] A "human antibody" as used herein refers to antibodies
produced in humans, antibodies produced in non-human animals that
comprise human immunoglobulin genes, such as XenoMouse.RTM., and
antibodies selected using in vitro methods, such as phage display,
wherein the antibody repertoire is based on a human immunoglobulin
sequences.
[0057] The term "PSGL-1 extracellular domain" ("PSGL-1 ECD")
includes full-length PSGL-1 ECDs, PSGL-1 ECD fragments, and PSGL-1
ECD variants, and refers to PSGL-1 polypeptide that lacks the
intracellular and transmembrane domains, with or without a signal
peptide. The PSGL-1 ECD polypeptide is a native, human ECD unless
specified otherwise. The term "full-length PSGL-1 ECD", as used
herein, refers to PSGL-1 ECD that extends to the last amino acid of
the extracellular domain, and may or may not include an N-terminal
signal peptide, and includes natural splice variants in the
extracellular domain. A non-limiting exemplary PSGL-1 ECD comprises
amino acids 1 to 241 of SEQ ID NO: 1 (with signal sequence), or
amino acids 23 to 241 of SEQ ID NO: 1 (without signal sequence),
amino acids 1 to 219 of SEQ ID NO: 2), SEQ ID NO: 3, or SEQ ID NO:
4. Another exemplary PSGL-1 ECD comprises amino acids 1 to 241 of
SEQ ID NO: 14 (with signal sequence), or amino acids 23 to 241 of
SEQ ID NO: 14 (without signal sequence), or amino acids 1 to 219 of
SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, or SEQ ID NO: 18.
As used herein, the term "PSGL-1 ECD fragment" refers to PSGL-1 ECD
having one or more residues deleted from the N- and/or C-terminus
of the full-length ECD and that retains the ability to bind VISTA.
The PSGL-1 ECD fragment may or may not include an N-terminal signal
peptide. As used herein, the term "PSGL-1 ECD variants" refers to
PSGL-1 ECDs that contain amino acid additions, deletions, and
substitutions and that remain capable of binding to VISTA. Such
variants may be at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99%
identical to the parent PSGL-1 ECD. The % identity of two
polypeptides can be measured by a similarity score determined by
comparing the amino acid sequences of the two polypeptides using
the Bestfit program with the default settings for determining
similarity. Bestfit uses the local homology algorithm of Smith and
Waterman, Advances in Applied Mathematics 2:482-489 (1981) to find
the best segment of similarity between two sequences.
[0058] The term "PSGL-1 ECD fusion molecule" refers to a molecule
comprising PSGL-1 ECD, and one or more "fusion partners." In some
embodiment, the PSGL-1 ECD and the fusion partner are covalently
linked ("fused"). If the fusion partner is also a polypeptide ("the
fusion partner polypeptide"), the PSGL-1 ECD and the fusion partner
polypeptide may be part of a continuous amino acid sequence, and
the fusion partner polypeptide may be linked to either the
N-terminus or the C-terminus of the PSGL-1 ECD. In such cases, the
PSGL-1 ECD and the fusion partner polypeptide may be translated as
a single polypeptide from a coding sequence that encodes both the
PSGL-1 ECD and the fusion partner polypeptide (the "PSGL-1 ECD
fusion protein"). In some embodiments, the PSGL-1 ECD and the
fusion partner are covalently linked through other means, such as,
for example, a chemical linkage other than a peptide bond. Many
known methods of covalently linking polypeptides to other molecules
(for example, fusion partners) may be used. In other embodiments,
the PSGL-1 ECD and the fusion partner may be fused through a
"linker," which is comprised of at least one amino acid or chemical
moiety. SEQ ID NO: 19 provides a linker used in a PSGL-1 Fc fusion
molecule of the examples herein. A nonlimiting exemplary PSGL-1 ECD
fusion molecule comprises a fusion molecule described in T. Pouyani
et al., Cell 83: 333-343 (1995). Nonlimiting exemplary PSGL-1 ECD
fusion molecules may comprise a PSGL-1 ECD in combination with an
Fc such as a combination of (a) amino acids 1 to 241 of SEQ ID NO:
1 (with signal sequence), or amino acids 23 to 241 of SEQ ID NO: 1
(without signal sequence), amino acids 1 to 219 of SEQ ID NO: 2),
SEQ ID NO: 3, or SEQ ID NO: 4, or amino acids 1 to 241 of SEQ ID
NO: 14 (with signal sequence), or amino acids 23 to 241 of SEQ ID
NO: 14 (without signal sequence), or amino acids 1 to 219 of SEQ ID
NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17, or SEQ ID NO: 18; with (b)
an Fc, such as an Fc of SEQ ID NO: 11, 12, or 13, or an Fc domain
from a human IgG1.
[0059] In some embodiments, the PSGL-1 polypeptide and the fusion
partner are noncovalently linked. In some such embodiments, they
may be linked, for example, using binding pairs. Exemplary binding
pairs include, but are not limited to, biotin and avidin or
streptavidin, an antibody and its antigen, etc.
[0060] Exemplary fusion partners include, but are not limited to,
an immunoglobulin Fc domain, albumin, and polyethylene glycol. The
amino acid sequences of nonlimiting exemplary Fc domains are shown
in SEQ ID NOs: 11 to 13.
[0061] As noted above, a PSGL-1 ECD is derived from a native, human
PSGL-1 sequence unless specified otherwise. In some embodiments,
however, PSGL-1 ECD amino acid sequence is derived from that of a
non-human mammal. In such embodiments, the non-human PSGL-1 ECD
amino acid sequence may be derived from mammals including, but not
limited to, rodents (including mice, rats, hamsters), rabbits,
simians, felines, canines, equines, bovines, porcines, ovines,
caprines, mammalian laboratory animals, mammalian farm animals,
mammalian sport animals, and mammalian pets. PSGL-1 ECD fusion
molecules incorporating a non-humPSGL-1 ECD are termed
"non-humPSGL-1 ECD fusion molecules" or "non-human PSGL-1 ECD
fusion molecules." Similar to human PSGL-1 ECD fusion molecules,
non-human fusion molecules may comprise a fusion partner, optional
linker, and a non-human PSGL-1 ECD. Such non-human fusion molecules
may also include a signal peptide.
[0062] A "PSGL-1 ECD fragment" refers to a PSGL-1 ECD having one or
more residues deleted from the N- and/or C-terminus of a
full-length, human ECD and that retains the ability to bind to
human VISTA. A "non-humPSGL-1 ECD fragment" refers to a
non-humPSGL-1 ECD having one or more residues deleted from the N-
and/or C-terminus of the full-length ECD and that retains the
ability to bind to VISTA of the non-human animal from which the
sequence was derived. This is in contrast to a "PSGL-1 ECD
variant," refers to a PSGL-1 ECD that contain amino acid additions,
deletions, and substitutions compared to a native, human PSGL-1 ECD
and that remain capable of binding to human VISTA.
[0063] A "non-humPSGL-1 ECD variant" refers to non-human PSGL-1
ECDs that contain amino acid additions, deletions, and
substitutions and that remain capable of binding to VISTA from the
animal from which the sequence was derived. This is in contrast to
a "PSGL-1 ECD variant," which refers to a human PSGL-1 ECD that
contains amino acid additions, deletions, and substitutions and
that remains capable of binding to human VISTA.
[0064] In any of the embodiments described herein, PSGL-1,
including but not limited to, full-length PSGL-1, PSGL-1 fragments,
PSGL-1 variants, PSGL-1 ECDs, and PSGL-1 ECD fusion proteins, may
further comprise a tag. Nonlimiting exemplary tags include FITC,
His6, biotin, and other labels and tags known in the art.
[0065] The term "VISTA extracellular domain" ("VISTA ECD") includes
full-length VISTA ECDs, VISTA ECD fragments, and VISTA ECD
variants, and refers to a VISTA polypeptide that lacks the
intracellular and transmembrane domains, with or without a signal
peptide. The polypeptide is a native, human ECD unless specified
otherwise. The term "full-length VISTA ECD", as used herein, refers
to a VISTA ECD that extends to the last amino acid of the
extracellular domain, and may or may not include an N-terminal
signal peptide, and includes natural splice variants in the
extracellular domain.
[0066] As used herein, the term "VISTA ECD fragment" refers to a
VISTA ECD having one or more residues deleted from the N- and/or
C-terminus of the full-length ECD and that retains the ability to
bind PSGL-1. The VISTA ECD fragment may or may not include an
N-terminal signal peptide. As used herein, the term "VISTA ECD
variants" refers to VISTA ECDs that contain amino acid additions,
deletions, and substitutions and that remain capable of binding to
PSGL-1. Such variants may be at least 80%, 85%, 90%, 92%, 95%, 97%,
98%, or 99% identical to the parent VISTA ECD.
[0067] The % identity of two polypeptides can be measured by a
similarity score determined by comparing the amino acid sequences
of the two polypeptides using the Bestfit program with the default
settings for determining similarity. Bestfit uses the local
homology algorithm of Smith and Waterman, Advances in Applied
Mathematics 2:482-489 (1981) to find the best segment of similarity
between two sequences. In some embodiments, a VISTA ECD comprising
a His tag has the amino acid sequence of SEQ ID NO: 10. In some
embodiments, a VISTA ECD without a His tag has the amino acid
sequence of amino acids 1-202 of SEQ ID NO: 10 (corresponding to
the full sequence of SEQ ID NO: 10 minus the last six His residues
of that sequence).
[0068] The term "VISTA ECD fusion molecule" refers to a molecule
comprising a VISTA ECD, and one or more "fusion partners." In some
embodiment, the VISTA ECD and the fusion partner are covalently
linked ("fused"). If the fusion partner is also a polypeptide ("the
fusion partner polypeptide"), the VISTA ECD and the fusion partner
polypeptide may be part of a continuous amino acid sequence, and
the fusion partner polypeptide may be linked to either the
N-terminus or the C-terminus of the VISTA ECD. In such cases, the
VISTA ECD and the fusion partner polypeptide may be translated as a
single polypeptide from a coding sequence that encodes both the
VISTA ECD and the fusion partner polypeptide (the "VISTA ECD fusion
protein"). In some embodiments, the VISTA ECD and the fusion
partner are covalently linked through other means, such as, for
example, a chemical linkage other than a peptide bond. Many known
methods of covalently linking polypeptides to other molecules (for
example, fusion partners) may be used. In other embodiments, the
VISTA ECD and the fusion partner may be fused through a "linker,"
which is comprised of at least one amino acid or chemical moiety. A
nonlimiting exemplary VISTA ECD fusion molecule comprises the
sequence of SEQ ID NO: 9. Another exemplary VISTA ECD fusion
molecule comprises the sequence of amino acids 1-202 of SEQ ID NO:
10 plus an Fc sequence of SEQ ID NOs: 11, 12, or 13.
[0069] In some embodiments, the VISTA polypeptide and the fusion
partner are noncovalently linked. In some such embodiments, they
may be linked, for example, using binding pairs. Exemplary binding
pairs include, but are not limited to, biotin and avidin or
streptavidin, an antibody and its antigen, etc.
[0070] Exemplary fusion partners include, but are not limited to,
an immunoglobulin Fc domain, albumin, and polyethylene glycol. The
amino acid sequences of nonlimiting exemplary Fc domains are shown
in SEQ ID NOs: 11 to 13.
[0071] Again, unless specified otherwise, a VISTA ECD amino acid
sequence is derived from that of a human. In some embodiments,
however, a VISTA ECD amino acid sequence is derived from that of a
non-human mammal. In such embodiments, the VISTA ECD amino acid
sequence may be derived from mammals including, but not limited to,
rodents (including mice, rats, hamsters), rabbits, simians,
felines, canines, equines, bovines, porcines, ovines, caprines,
mammalian laboratory animals, mammalian farm animals, mammalian
sport animals, and mammalian pets. VISTA ECD fusion molecules
incorporating a non-human VISTA ECD are termed "non-human VISTA ECD
fusion molecules." Similar to the human VISTA ECD fusion molecules,
non-human fusion molecules may comprise a fusion partner, optional
linker, and a VISTA ECD. Such non-human fusion molecules may also
include a signal peptide. A "VISTA ECD fragment" in contrast,
refers to a native, VISTA ECD having one or more residues deleted
from the N- and/or C-terminus of a full-length human ECD and that
retains the ability to bind to human PSGL-1.
[0072] A "non-human VISTA ECD fragment" refers to a non-human VISTA
ECD having one or more residues deleted from the N- and/or
C-terminus of the full-length ECD and that retains the ability to
bind to PSGL-1 of the non-human animal from which the sequence was
derived. A "VISTA ECD variant" in contrast, refers to VISTA ECDs
that contain amino acid additions, deletions, and substitutions
compared to native, human VISTA ECD and that remain capable of
binding to human PSGL-1. A "non-human VISTA ECD variant" refers to
VISTA ECDs that contain amino acid additions, deletions, and
substitutions compared to their parent VISTA ECD and that remain
capable of binding to PSGL-1 from the animal from which the
sequence was derived.
[0073] In any of the embodiments described herein, VISTA, including
but not limited to, full-length VISTA, VISTA fragments, VISTA
variants, VISTA ECDs, and VISTA ECD fusion proteins, may further
comprise a tag. Nonlimiting exemplary tags include FITC, His.sub.6,
biotin, and other labels and tags known in the art.
[0074] The term "signal peptide" refers to a sequence of amino acid
residues located at the N-terminus of a polypeptide that
facilitates secretion of a polypeptide from a mammalian cell. A
signal peptide may be cleaved upon export of the polypeptide from
the mammalian cell, forming a mature protein. Signal peptides may
be natural or synthetic, and they may be heterologous or homologous
to the protein to which they are attached. Exemplary signal
peptides include, but are not limited to, the signal peptides of
PSGL-1 and VISTA. Exemplary signal peptides also include signal
peptides from heterologous proteins. A "signal sequence" refers to
a polynucleotide sequence that encodes a signal peptide.
[0075] The term "vector" is used to describe a polynucleotide that
may be engineered to contain a cloned polynucleotide or
polynucleotides that may be propagated in a host cell. A vector may
include one or more of the following elements: an origin of
replication, one or more regulatory sequences (such as, for
example, promoters and/or enhancers) that regulate the expression
of the polypeptide of interest, and/or one or more selectable
marker genes (such as, for example, antibiotic resistance genes and
genes that may be used in colorimetric assays, e.g.,
.beta.-galactosidase). The term "expression vector" refers to a
vector that is used to express a polypeptide of interest in a host
cell.
[0076] A "host cell" refers to a cell that may be or has been a
recipient of a vector or isolated polynucleotide. Host cells may be
prokaryotic cells or eukaryotic cells. Exemplary eukaryotic cells
include mammalian cells, such as primate or non-primate animal
cells; fungal cells, such as yeast; plant cells; and insect cells.
Nonlimiting exemplary mammalian cells include, but are not limited
to, NSO cells, PER.C6.RTM. cells (Crucell), and 293 and CHO cells,
and their derivatives, such as 293-6E and DG44 cells,
respectively.
[0077] The term "isolated" as used herein refers to a molecule that
has been separated from at least some of the components with which
it is typically found in nature or has been separated from at least
some of the components with which it is typically produced. For
example, a polypeptide is referred to as "isolated" when it is
separated from at least some of the components of the cell in which
it was produced. Where a polypeptide is secreted by a cell after
expression, physically separating the supernatant containing the
polypeptide from the cell that produced it is considered to be
"isolating" the polypeptide. Similarly, a polynucleotide is
referred to as "isolated" when it is not part of the larger
polynucleotide (such as, for example, genomic DNA or mitochondrial
DNA, in the case of a DNA polynucleotide) in which it is typically
found in nature, or is separated from at least some of the
components of the cell in which it was produced, e.g., in the case
of an RNA polynucleotide. Thus, a DNA polynucleotide that is
contained in a vector inside a host cell may be referred to as
"isolated" so long as that polynucleotide is not found in that
vector in nature.
[0078] The terms "subject" and "patient" are used interchangeably
herein to refer to a human. In some embodiments, methods of
treating other mammals, including, but not limited to, rodents,
simians, felines, canines, equines, bovines, porcines, ovines,
caprines, mammalian laboratory animals, mammalian farm animals,
mammalian sport animals, and mammalian pets, are also provided. In
some instances, a "subject" or "patient" refers to a subject or
patient in need of treatment for a disease or disorder.
[0079] The term "sample" or "patient sample" as used herein, refers
to material that is obtained or derived from a subject of interest
that contains a cellular and/or other molecular entity that is to
be characterized and/or identified, for example based on physical,
biochemical, chemical and/or physiological characteristics. For
example, the phrase "disease sample" and variations thereof refers
to any sample obtained from a subject of interest that would be
expected or is known to contain the cellular and/or molecular
entity that is to be characterized. By "tissue or cell sample" is
meant a collection of similar cells obtained from a tissue of a
subject or patient. The source of the tissue or cell sample may be
solid tissue as from a fresh, frozen and/or preserved organ or
tissue sample or biopsy or aspirate; blood or any blood
constituents; bodily fluids such as sputum, cerebral spinal fluid,
amniotic fluid, peritoneal fluid, or interstitial fluid; cells from
any time in gestation or development of the subject. The tissue
sample may also be primary or cultured cells or cell lines.
Optionally, the tissue or cell sample is obtained from a disease
tissue/organ. The tissue sample may contain compounds which are not
naturally intermixed with the tissue in nature such as
preservatives, anticoagulants, buffers, fixatives, nutrients,
antibiotics, or the like.
[0080] A "reference sample", "reference cell", or "reference
tissue", as used herein, refers to a sample, cell or tissue
obtained from a source known, or believed, not to be afflicted with
the disease or condition for which a method or composition of the
invention is being used to identify. In one embodiment, a reference
sample, reference cell or reference tissue is obtained from a
healthy part of the body of the same subject or patient in whom a
disease or condition is being identified using a composition or
method of the invention. In one embodiment, a reference sample,
reference cell or reference tissue is obtained from a healthy part
of the body of at least one individual who is not the subject or
patient in whom a disease or condition is being identified using a
composition or method of the invention. In some embodiments, a
reference sample, reference cell or reference tissue was previously
obtained from a patient prior to developing a disease or condition
or at an earlier stage of the disease or condition.
[0081] A condition "has previously been characterized as having [a
characteristic]" when such characteristic of the condition has been
shown in at least a subset of patients with the condition, or in
one or more animal models of the condition. In some embodiments,
such characteristic of the condition does not have to be determined
in the patient to be treated one or more PSGL-1 antagonists of the
present invention. The presence of the characteristic in a specific
patient who is to be treated using the present methods and/or
compositions need not have been determined in order for the patient
to be considered as having a condition that has previously been
characterized as having the characteristic.
[0082] A "disorder" or "disease" is any condition that would
benefit from treatment with one or more PSGL-1 antagonists of the
invention. This includes chronic and acute disorders or diseases
including those pathological conditions that predispose the mammal
to the disorder in question. Nonlimiting examples of disorders to
be treated herein include cancers.
[0083] The term "cancer" is used herein to refer to a group of
cells that exhibit abnormally high levels of proliferation and
growth. A cancer may be benign (also referred to as a benign
tumor), pre-malignant, or malignant. Cancer cells may be solid
cancer cells (i.e., forming solid tumors) or leukemic cancer cells.
The term "cancer growth" is used herein to refer to proliferation
or growth by a cell or cells that comprise a cancer that leads to a
corresponding increase in the size or extent of the cancer.
[0084] Examples of cancer include but are not limited to,
carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More
particular nonlimiting examples of such cancers include squamous
cell cancer, small-cell lung cancer, pituitary cancer, esophageal
cancer, astrocytoma, soft tissue sarcoma, non-small cell lung
cancer, adenocarcinoma of the lung, squamous carcinoma of the lung,
cancer of the peritoneum, hepatocellular cancer, gastrointestinal
cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian
cancer, liver cancer, bladder cancer, hepatoma, breast cancer,
colon cancer, colorectal cancer, endometrial or uterine carcinoma,
salivary gland carcinoma, kidney cancer, renal cancer, liver
cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic
carcinoma, brain cancer, endometrial cancer, testis cancer,
cholangiocarcinoma, gallbladder carcinoma, gastric cancer,
melanoma, and various types of head and neck cancer.
[0085] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer. Examples of chemotherapeutic agents
include, but are not limited to, alkylating agents such as thiotepa
and Cytoxan.RTM. cyclosphosphamide; alkyl sulfonates such as
busulfan, improsulfan and piposulfan; aziridines such as benzodopa,
carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
trietylenephosphoramide, triethiylenethiophosphoramide and
trimethylolomelamine; acetogenins (especially bullatacin and
bullatacinone); a camptothecin (including the synthetic analogue
topotecan); bryostatin; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogues);
cryptophycins (particularly cryptophycin 1 and cryptophycin 8);
dolastatin; duocarmycin (including the synthetic analogues, KW-2189
and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;
spongistatin; nitrogen mustards such as chlorambucil,
chlornaphazine, cholophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, and ranimnustine; antibiotics
such as the enediyne antibiotics (e.g., calicheamicin, especially
calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew,
Chem Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including
dynemicin A; bisphosphonates, such as clodronate; an esperamicin;
as well as neocarzinostatin chromophore and related chromoprotein
enediyne antiobiotic chromophores), aclacinomysins, actinomycin,
authramycin, azaserine, bleomycins, cactinomycin, carabicin,
carminomycin, carzinophilin, chromomycinis, dactinomycin,
daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine,
Adriamycin.RTM. doxorubicin (including morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogues such
as denopterin, methotrexate, pteropterin, trimetrexate; purine
analogs such as fludarabine, 6-mercaptopurine, thiamiprine,
thioguanine; pyrimidine analogs such as ancitabine, azacitidine,
6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine,
enocitabine, floxuridine; androgens such as calusterone,
dromostanolone propionate, epitiostanol, mepitiostane,
testolactone; anti-adrenals such as aminoglutethimide, mitotane,
trilostane; folic acid replenisher such as frolinic acid;
aceglatone; aldophosphamide glycoside; aminolevulinic acid;
eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate;
defofamine; demecolcine; diaziquone; elfornithine; elliptinium
acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea;
lentinan; lonidainine; maytansinoids such as maytansine and
ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine;
pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic
acid; 2-ethylhydrazide; procarbazine; PSK.RTM. polysaccharide
complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;
sizofiran; spirogermanium; tenuazonic acid; triaziquone;
2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2
toxin, verracurin A, roridin A and anguidine); urethan; vindesine;
dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman;
gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa;
taxoids, e.g., Taxol.RTM. paclitaxel (Bristol-Myers Squibb
Oncology, Princeton, N.J.), Abraxane.RTM. Cremophor-free,
albumin-engineered nanoparticle formulation of paclitaxel (American
Pharmaceutical Partners, Schaumberg, Ill.), and Taxotere.RTM.
doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;
Gemzar.RTM. gemcitabine; 6-thioguanine; mercaptopurine;
methotrexate; platinum analogs such as cisplatin, oxaliplatin and
carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide;
mitoxantrone; vincristine; Navelbine.RTM. vinorelbine; novantrone;
teniposide; edatrexate; daunomycin; aminopterin; xeloda;
ibandronate; irinotecan (Camptosar, CPT-11) (including the
treatment regimen of irinotecan with 5-FU and leucovorin);
topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO);
retinoids such as retinoic acid; capecitabine; combretastatin;
leucovorin (LV); oxaliplatin, including the oxaliplatin treatment
regimen (FOLFOX); inhibitors of PKC-alpha, Raf, H-Ras, EGFR (e.g.,
erlotinib))(Tarceva.RTM. and VEGF-A that reduce cell proliferation
and pharmaceutically acceptable salts, acids or derivatives of any
of the above.
[0086] Further nonlimiting exemplary chemotherapeutic agents
include anti-hormonal agents that act to regulate or inhibit
hormone action on cancers such as anti-estrogens and selective
estrogen receptor modulators (SERMs), including, for example,
tamoxifen (including Nolvadex.RTM. tamoxifen), raloxifene,
droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,
onapristone, and Fareston.RTM. toremifene; aromatase inhibitors
that inhibit the enzyme aromatase, which regulates estrogen
production in the adrenal glands, such as, for example,
4(5)-imidazoles, aminoglutethimide, Megase.RTM. megestrol acetate,
Aromasin.RTM. exemestane, formestanie, fadrozole, Rivisor.RTM.
vorozole, Femara.RTM. letrozole, and Arimidex.RTM. anastrozole; and
anti-androgens such as flutamide, nilutamide, bicalutamide,
leuprolide, and goserelin; as well as troxacitabine (a
1,3-dioxolane nucleoside cytosine analog); antisense
oligonucleotides, particularly those which inhibit expression of
genes in signaling pathways implicated in abherant cell
proliferation, such as, for example, PKC-alpha, Ralf and H-Ras;
ribozymes such as a VEGF expression inhibitor (e.g., Angiozyme.RTM.
ribozyme) and a HER2 expression inhibitor; vaccines such as gene
therapy vaccines, for example, Allovectin.RTM. vaccine,
Leuvectin.RTM. vaccine, and Vaxid.RTM. vaccine; Proleukin.RTM.
rIL-2; Lurtotecan.RTM. topoisomerase 1 inhibitor; Abarelix.RTM.
rmRH; and pharmaceutically acceptable salts, acids or derivatives
of any of the above.
[0087] An "anti-angiogenesis agent" or "angiogenesis inhibitor"
refers to a small molecular weight substance, a polynucleotide
(including, e.g., an inhibitory RNA (RNAi or siRNA)), a
polypeptide, an isolated protein, a recombinant protein, an
antibody, or conjugates or fusion proteins thereof, that inhibits
angiogenesis, vasculogenesis, or undesirable vascular permeability,
either directly or indirectly. It should be understood that the
anti-angiogenesis agent includes those agents that bind and block
the angiogenic activity of the angiogenic factor or its receptor.
For example, an anti-angiogenesis agent is an antibody or other
antagonist to an angiogenic agent, e.g., antibodies to VEGF-A
(e.g., bevacizumab (Avastin.RTM.)) or to the VEGF-A receptor (e.g.,
KDR receptor or Flt-1 receptor), anti-PDGFR inhibitors such as
Gleevec.RTM. (Imatinib Mesylate), small molecules that block VEGF
receptor signaling (e.g., PTK787/ZK2284, SU6668,
Sutent.RTM./SU11248 (sunitinib malate), AMG706, or those described
in, e.g., international patent application WO 2004/113304).
Anti-angiogensis agents also include native angiogenesis inhibitors
, e.g., angiostatin, endostatin, etc. See, e.g., Klagsbrun and
D'Amore (1991) Annu. Rev. Physiol. 53:217-39; Streit and Detmar
(2003) Oncogene 22:3172-3179 (e.g., Table 3 listing anti-angiogenic
therapy in malignant melanoma); Ferrara & Alitalo (1999) Nature
Medicine 5(12):1359-1364; Tonini et al. (2003) Oncogene
22:6549-6556 (e.g., Table 2 listing known anti-angiogenic factors);
and, Sato (2003) Int. J. Clin. Oncol. 8:200-206 (e.g., Table 1
listing anti-angiogenic agents used in clinical trials).
[0088] A "growth inhibitory agent" as used herein refers to a
compound or composition that inhibits growth of a cell (such as a
cell expressing VEGF) either in vitro or in vivo. Thus, the growth
inhibitory agent may be one that significantly reduces the
percentage of cells (such as a cell expressing VEGF) in S phase.
Examples of growth inhibitory agents include, but are not limited
to, agents that block cell cycle progression (at a place other than
S phase), such as agents that induce G1 arrest and M-phase arrest.
Classical M-phase blockers include the vincas (vincristine and
vinblastine), taxanes, and topoisomerase II inhibitors such as
doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin.
Those agents that arrest G1 also spill over into S-phase arrest,
for example, DNA alkylating agents such as tamoxifen, prednisone,
dacarbazine, mechlorethamine, cisplatin, methotrexate,
5-fluorouracil, and ara-C. Further information can be found in
Mendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter
1, entitled "Cell cycle regulation, oncogenes, and antineoplastic
drugs" by Murakami et al. (W.B. Saunders, Philadelphia, 1995),
e.g., p. 13. The taxanes (paclitaxel and docetaxel) are anticancer
drugs both derived from the yew tree. Docetaxel (Taxotere.RTM.,
Rhone-Poulenc Rorer), derived from the European yew, is a
semisynthetic analogue of paclitaxel (Taxol.RTM., Bristol-Myers
Squibb). Paclitaxel and docetaxel promote the assembly of
microtubules from tubulin dimers and stabilize microtubules by
preventing depolymerization, which results in the inhibition of
mitosis in cells.
[0089] The term "anti-neoplastic composition" refers to a
composition useful in treating cancer comprising at least one
active therapeutic agent. Examples of therapeutic agents include,
but are not limited to, e.g., chemotherapeutic agents, growth
inhibitory agents, cytotoxic agents, agents used in radiation
therapy, anti-angiogenesis agents, cancer immunotherapeutic agents
(also referred to as immuno-oncology agents), apoptotic agents,
anti-tubulin agents, and other-agents to treat cancer, such as
anti-HER-2 antibodies, anti-CD20 antibodies, an epidermal growth
factor receptor (EGFR) antagonist (e.g., a tyrosine kinase
inhibitor), RERVEGFR inhibitor (e.g., erlotinib (Tarceva), platelet
derived growth factor inhibitors (e.g., Gleevec.RTM. (Imatinib
Mesylate)), a COX-2 inhibitor (e.g., celecoxib), interferons, CTLA4
inhibitors (e.g., anti-CTLA antibody ipilimumab (YERVOY.RTM.)),
PD-1 inhibitors (e.g., anti-PD1 antibodies, BMS-936558), PDL1
inhibitors (e.g., anti-PDL1 antibodies, MPDL3280A), PDL2 inhibitors
(e.g., anti-PDL2 antibodies), VISTA inhibitors (e.g., anti-VISTA
antibodies), cytokines, antagonists (e.g., neutralizing antibodies)
that bind to one or more of the following targets ErbB2, ErbB3,
ErbB4, PDGFR-beta, BlyS, APRIL, BCMA, PD-1, PDL1, PDL2, CTLA4,
VISTA, or VEGF receptor(s), TRAIL/Apo2, and other bioactive and
organic chemical agents, etc. Combinations thereof are also
included in the invention.
[0090] "Treatment," as used herein, refers to therapeutic
treatment, for example, wherein the object is to slow down (lessen)
the targeted pathologic condition or disorder as well as, for
example, wherein the object is to inhibit recurrence of the
condition or disorder. "Treatment," as used herein, covers any
administration or application of a therapeutic for a disease (also
referred to herein as a "disorder" or a "condition") in a mammal,
including a human, and includes inhibiting the disease or
progression of the disease, inhibiting or slowing the disease or
its progression, arresting its development, partially or fully
relieving the disease, partially or fully relieving one or more
symptoms of a disease, or restoring or repairing a lost, missing,
or defective function; or stimulating an inefficient process. The
term "treatment" also includes reducing the severity of any
phenotypic characteristic and/or reducing the incidence, degree, or
likelihood of that characteristic. Those in need of treatment
include those already with the disorder as well as those at risk of
recurrence of the disorder or those in whom a recurrence of the
disorder is to be prevented or slowed down.
[0091] The term "effective amount" or "therapeutically effective
amount" refers to an amount of a drug effective to treat a disease
or disorder in a subject. In some embodiments, an effective amount
refers to an amount effective, at dosages and for periods of time
necessary, to achieve the desired therapeutic or prophylactic
result. A therapeutically effective amount of PSGL-1 antagonist of
the invention may vary according to factors such as the disease
state, age, sex, and weight of the individual, and the ability of
the antagonist to elicit a desired response in the individual. A
therapeutically effective amount encompasses an amount in which any
toxic or detrimental effects of PSGL-1 antagonist are outweighed by
the therapeutically beneficial effects.
[0092] A "prophylactically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired prophylactic result. Typically, but not necessarily,
since a prophylactic dose is used in subjects prior to or at an
earlier stage of disease, the prophylactically effective amount
would be less than the therapeutically effective amount.
[0093] A "pharmaceutically acceptable carrier" refers to a
non-toxic solid, semisolid, or liquid filler, diluent,
encapsulating material, formulation auxiliary, or carrier
conventional in the art for use with a therapeutic agent that
together comprise a "pharmaceutical composition" for administration
to a subject. A pharmaceutically acceptable carrier is non-toxic to
recipients at the dosages and concentrations employed and is
compatible with other ingredients of the formulation. The
pharmaceutically acceptable carrier is appropriate for the
formulation employed. For example, if the therapeutic agent is to
be administered orally, the carrier may be a gel capsule. If the
therapeutic agent is to be administered subcutaneously, the carrier
ideally is not irritable to the skin and does not cause injection
site reaction.
[0094] An "article of manufacture" is any manufacture (e.g., a
package or container) or kit comprising at least one reagent, e.g.,
a medicament for treatment of a disease or disorder, or a probe for
specifically detecting a biomarker described herein. In some
embodiments, the manufacture or kit is promoted, distributed, or
sold as a unit for performing the methods described herein.
Therapeutic Compositions and Methods
Methods of Treating Diseases
[0095] PSGL-1 antagonists are provided for use in methods of
treating humans and other mammals. Methods of treating a disease
comprising administering PSGL-1 antagonists to humans and other
mammals are provided.
Methods of Treating Cancer
[0096] In some embodiments, methods for treating or preventing a
cancer are provided, comprising administering an effective amount
of PSGL-1 antagonist to a subject in need of such treatment.
[0097] The present inventors have identified PSGL-1 as a binding
partner for VISTA. VISTA is a receptor on the surface of various
immune cells (such as T cells, dendritic cells, natural killer
cells, monocytes, and macrophages) that serves as an inhibitor of
active immune responses. Expression of PSGL-1 and/or VISTA on the
surface of cancer and/or immune cells (e.g., T cells and NK cells)
cells may inhibit immune responses by engaging PSGL-1 and/or VISTA
on immune cells. Inhibition of the VISTA-PSGL-1 interaction may
enhance immune-mediated killing of cancer cells.
[0098] In some embodiments, methods of treating cancer are
provided, wherein the methods comprise administering PSGL-1
antagonist to a subject with cancer. In some embodiments, use of
PSGL-1 antagonist for treating cancer is provided. Nonlimiting
exemplary cancers that may be treated with PSGL-1 antagonists are
provided herein, including carcinoma, lymphoma, blastoma, sarcoma,
and leukemia. More particular non-limiting examples of such cancers
include squamous cell cancer, small-cell lung cancer, pituitary
cancer, esophageal cancer, astrocytoma, soft tissue sarcoma,
non-small cell lung cancer, adenocarcinoma of the lung, squamous
carcinoma of the lung, cancer of the peritoneum, hepatocellular
cancer, gastrointestinal cancer, pancreatic cancer, glioblastoma,
cervical cancer, ovarian cancer, liver cancer, bladder cancer,
hepatoma, breast cancer, colon cancer, colorectal cancer,
endometrial or uterine carcinoma, salivary gland carcinoma, kidney
cancer, renal cancer, liver cancer, prostate cancer, vulval cancer,
thyroid cancer, hepatic carcinoma, brain cancer, endometrial
cancer, testis cancer, cholangiocarcinoma, gallbladder carcinoma,
gastric cancer, melanoma, and various types of head and neck
cancer. In some embodiments, lung cancer is non-small cell lung
cancer or lung squamous cell carcinoma. In some embodiments,
leukemia is acute myeloid leukemia or chronic lymphocytic leukemia.
In some embodiments, breast cancer is breast invasive carcinoma. In
some embodiments, ovarian cancer is ovarian serous
cystadenocarcinoma. In some embodiments, kidney cancer is kidney
renal clear cell carcinoma. In some embodiments, colon cancer is
colon adenocarcinoma. In some embodiments, bladder cancer is
bladder urothelial carcinoma.
[0099] In some embodiments, the PSGL-1 antagonist is selected from
a PSGL-1 antibody and a VISTA antibody. In some embodiments, the
PSGL-1 antagonist is a PSGL-1 antibody. A PSGL-1 antagonist for
treating cancer may also be a non-antibody protein, such as PSGL-1
or VISTA or a portion thereof (e.g., the ECD) that inhibits the
interaction between PSGL-1 and VISTA, optionally further comprising
a fusion partner and in the form of a fusion molecule. Various
exemplary PSGL-1 antagonists are described in more detail in the
sections that follow.
Routes of Administration and Carriers
[0100] In various embodiments, PSGL-1 antagonists may be
administered subcutaneously or intravenously. In some embodiments,
PSGL-1 antagonist may be administered in vivo by various routes,
including, but not limited to, oral, intra-arterial, parenteral,
intranasal, intramuscular, intracardiac, intraventricular,
intratracheal, buccal, rectal, intraperitoneal, by inhalation,
intradermal, topical, transdermal, and intrathecal, or otherwise,
e.g., by implantation. The subject compositions may be formulated
into preparations in solid, semi-solid, liquid, or gaseous forms;
including, but not limited to, tablets, capsules, powders,
granules, ointments, solutions, suppositories, enemas, injections,
inhalants, and aerosols. In some embodiments, PSGL-1 antagonist is
delivered using gene therapy. As a nonlimiting example, a nucleic
acid molecule encoding PSGL-1 antagonist may be coated onto gold
microparticles and delivered intradermally by a particle
bombardment device, or "gene gun," e.g., as described in the
literature (see, e.g., Tang et al., Nature 356:152-154 (1992)).
[0101] In various embodiments, compositions comprising PSGL-1
antagonist are provided in formulations with a wide variety of
pharmaceutically acceptable carriers (see, e.g., Gennaro,
Remington: The Science and Practice of Pharmacy with Facts and
Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al.,
Pharmaceutical Dosage Forms and Drug Delivery Systems, 7.sup.th
ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook
of Pharmaceutical Excipients, 3rd ed., Pharmaceutical Press
(2000)). Various pharmaceutically acceptable carriers, which
include vehicles, adjuvants, and diluents, are available. Moreover,
various pharmaceutically acceptable auxiliary substances, such as
pH adjusting and buffering agents, tonicity adjusting agents,
stabilizers, wetting agents and the like, are also available.
Nonlimiting exemplary carriers include saline, buffered saline,
dextrose, water, glycerol, ethanol, and combinations thereof.
[0102] In various embodiments, compositions comprising PSGL-1
antagonist may be formulated for injection, including subcutaneous
administration, by dissolving, suspending, or emulsifying them in
an aqueous or nonaqueous solvent, such as vegetable or other oils,
synthetic aliphatic acid glycerides, esters of higher aliphatic
acids, or propylene glycol; and if desired, with conventional
additives such as solubilizers, isotonic agents, suspending agents,
emulsifying agents, stabilizers and preservatives. In various
embodiments, the compositions may be formulated for inhalation, for
example, using pressurized acceptable propellants such as
dichlorodifluoromethane, propane, nitrogen, and the like. The
compositions may also be formulated, in various embodiments, into
sustained release microcapsules, such as with biodegradable or
non-biodegradable polymers. A nonlimiting exemplary biodegradable
formulation includes poly lactic acid-glycolic acid polymer. A
nonlimiting exemplary non-biodegradable formulation includes a
polyglycerin fatty acid ester. Certain methods of making such
formulations are described, for example, in EP 1 125 584 A1.
[0103] Pharmaceutical dosage packs comprising one or more
containers, each containing one or more doses of PSGL-1 antagonist,
are also provided. In some embodiments, a unit dosage is provided
wherein the unit dosage contains a predetermined amount of a
composition comprising PSGL-1 antagonist, with or without one or
more additional agents. In some embodiments, such a unit dosage is
supplied in single-use prefilled syringe for injection. In various
embodiments, the composition contained in the unit dosage may
comprise saline, sucrose, or the like; a buffer, such as phosphate,
or the like; and/or be formulated within a stable and effective pH
range. Alternatively, in some embodiments, the composition may be
provided as a lyophilized powder that may be reconstituted upon
addition of an appropriate liquid, for example, sterile water. In
some embodiments, the composition comprises one or more substances
that inhibit protein aggregation, including, but not limited to,
sucrose and arginine. In some embodiments, a composition of the
invention comprises heparin and/or a proteoglycan.
[0104] Pharmaceutical compositions are administered in an amount
effective for treatment or prophylaxis of the specific indication.
The therapeutically effective amount is typically dependent on the
weight of the subject being treated, his or her physical or health
condition, the extensiveness of the condition to be treated, or the
age of the subject being treated. In some embodiments, PSGL-1
antagonist may be administered in an amount in the range of about
50 .mu.g/kg body weight to about 50 mg/kg body weight per dose. In
some embodiments, PSGL-1 antagonist may be administered in an
amount in the range of about 100 .mu.g/kg body weight to about 50
mg/kg body weight per dose. In some embodiments, PSGL-1 antagonist
may be administered in an amount in the range of about 100 .mu.g/kg
body weight to about 20 mg/kg body weight per dose. In some
embodiments, PSGL-1 antagonist may be administered in an amount in
the range of about 0.5 mg/kg body weight to about 20 mg/kg body
weight per dose.
[0105] In some embodiments, PSGL-1 antagonist may be administered
in an amount in the range of about 10 mg to about 1,000 mg per
dose. In some embodiments, PSGL-1 may be administered in an amount
in the range of about 20 mg to about 500 mg per dose. In some
embodiments, PSGL-1 antagonist may be administered in an amount in
the range of about 20 mg to about 300 mg per dose. In some
embodiments, PSGL-1 antagonist may be administered in an amount in
the range of about 20 mg to about 200 mg per dose.
[0106] The PSGL-1 antagonist compositions may be administered as
needed to subjects. In some embodiments, an effective dose of
PSGL-1 antagonist is administered to a subject one or more times.
In various embodiments, an effective dose of PSGL-1 antagonist is
administered to the subject once a month, less than once a month,
such as, for example, every two months, every three months, or
every six months. In other embodiments, an effective dose of PSGL-1
antagonist is administered more than once a month, such as, for
example, every two weeks, every week, twice per week, three times
per week, daily, or multiple times per day. An effective dose of
PSGL-1 antagonist is administered to the subject at least once. In
some embodiments, the effective dose of PSGL-1 antagonist may be
administered multiple times, including for periods of at least a
month, at least six months, or at least a year. In some
embodiments, PSGL-1 antagonist is administered to a subject
as-needed to alleviate one or more symptoms of a condition.
Combination Therapy
[0107] PSGL-1 antagonist according to the invention, including any
functional fragments thereof, may be administered to a subject in
need thereof in combination with other biologically active
substances or other treatment procedures for the treatment of
diseases. For example, PSGL-1 antagonists may be administered alone
or with other modes of treatment. They may be provided before,
substantially contemporaneous with, or after other modes of
treatment, such as radiation therapy.
[0108] For treatment of cancer, the PSGL-1 antagonist may be
administered in conjunction with one or more of anti-cancer agents,
such as the chemotherapeutic agent, growth inhibitory agent,
anti-angiogenesis agent or anti-neoplastic composition. Nonlimiting
examples of chemotherapeutic agent, growth inhibitory agent,
anti-angiogenesis agent and anti-neoplastic composition that can be
used in combination with one or more PSGL-1 antagonists of the
present invention are provided herein under "Definitions."
[0109] In certain embodiments, PSGL-1 antagonist that specifically
binds to PSGL-1 (an "PSGL-1 binding antagonist"), e.g., PSGL-1
antagonist antibody, is administered with an antagonist that
specifically binds to VISTA (a "VISTA binding antagonist"), e.g., a
VISTA antagonist antibody, to a subject having a disease in which
the stimulation of the immune system would be beneficial, e.g.,
cancer or infectious diseases. The two antagonists may be
administered simultaneously or consecutively, e.g., as described
below for the combination of PSGL-1 antagonist with an
immuno-oncology agent. One or more additional therapeutics, e.g.,
checkpoint modulators may be added to a treatment with PSGL-1
binding antagonist and a VISTA binding antagonist, e.g., for cancer
or infectious diseases.
[0110] In certain embodiments, PSGL-1 antagonist is administered
with another treatment, either simultaneously, or consecutively, to
a subject, e.g., a subject having cancer. For example, PSGL-1
antagonist may be administered with one of more of: radiotherapy,
surgery, or chemotherapy, e.g., targeted chemotherapy or
immunotherapy. Immunotherapy, e.g., cancer immunotherapy includes
cancer vaccines and immuno-oncology agents. PSGL-1 antagonist may
be, e.g., a protein, an antibody, antibody fragment or a small
molecule, that binds to PSGL-1. PSGL-1 antagonist may be an
antibody or antigen binding fragment thereof that specifically
binds to PSGL-1. PSGL-1 antagonist may be, e.g., a protein, an
antibody, antibody fragment or a small molecule, that binds to
VISTA. PSGL-1 antagonist may be an antibody or antigen binding
fragment thereof that specifically binds to VISTA.
[0111] In certain embodiments, a method of treatment of a subject
having cancer comprises administering to the subject having the
cancer PSGL-1 antagonist, e.g., PSGL-1 antibody or a VISTA
antibody, and one or more immuno-oncology agents. Immunotherapy,
e.g., therapy with an immuno-oncology agent, is effective to
enhance, stimulate, and/or upregulate immune responses in a
subject. In one aspect, the administration of PSGL-1 antagonist
with an immuno-oncology agent has a synergic effect in the
treatment of cancer, e.g., in inhibiting tumor growth.
[0112] For the description herein of combinations of PSGL-1
antagonist with another agent, e.g., an immuno-oncology agent, if
PSGL-1 antagonist is PSGL-1 binding antagonist, then an
immuno-oncology agent may be a VISTA binding antagonist, and if
PSGL-1 antagonist is a VISTA binding antagonist, then an
immuno-oncology agent may be PSGL-1 binding antagonist.
[0113] In one aspect, PSGL-1 antagonist is sequentially
administered prior to administration of the immuno-oncology agent.
In one aspect, PSGL-1 antagonist is administered concurrently with
the immunology-oncology agent. In yet one aspect, PSGL-1 antagonist
is sequentially administered after administration of the
immuno-oncology agent. The administration of the two agents may
start at times that are, e.g., 30 minutes, 60 minutes, 90 minutes,
120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48
hours, 3 days, 5 days, 7 days, or one or more weeks apart, or
administration of the second agent may start, e.g., 30 minutes, 60
minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24
hours, 36 hours, 48 hours, 3 days, 5 days, 7 days, or one or more
weeks after the first agent has been administered.
[0114] In certain aspects, PSGL-1 antagonist and an immuno-oncology
agent are administered simultaneously, e.g., are infused
simultaneously, e.g., over a period of 30 or 60 minutes, to a
patient. PSGL-1 antagonist may be co-formulated with an
immuno-oncology agent.
[0115] Immuno-oncology agents include, for example, a small
molecule drug, antibody or fragment thereof, or other biologic or
small molecule. Examples of biologic immuno-oncology agents
include, but are not limited to, antibodies, antibody fragments,
vaccines and cytokines. In one aspect, the antibody is a monoclonal
antibody. In certain aspects, the monoclonal antibody is humanized
or human antibody.
[0116] In one aspect, the immuno-oncology agent is (i) an agonist
of a stimulatory (including a co-stimulatory) molecule (e.g.,
receptor or ligand) or (ii) an antagonist of an inhibitory
(including a co-inhibitory) molecule (e.g., receptor or ligand) on
immune cells, e.g., T cells, both of which result in amplifying
antigen-specific T cell responses. In certain aspects, an
immuno-oncology agent is (i) an agonist of a stimulatory (including
a co-stimulatory) molecule (e.g., receptor or ligand) or (ii) an
antagonist of an inhibitory (including a co-inhibitory) molecule
(e.g., receptor or ligand) on cells involved in innate immunity,
e.g., NK cells, and wherein the immuno-oncology agent enhances
innate immunity. Such immuno-oncology agents are often referred to
as immune checkpoint regulators, e.g., immune checkpoint inhibitor
or immune checkpoint stimulator.
[0117] In certain embodiments, an immuno-oncology agent targets a
stimulatory or inhibitory molecule that is a member of the
immunoglobulin super family (IgSF). For example, an immuno-oncology
agent may be an agent that targets (or binds specifically to) a
member of the B7 family of membrane-bound ligands, which includes
B7-1, B7-2, B7-H1 (PD-L1), B7-DC (PD-L2), B7-H2 (ICOS-L), B7-H3,
B7-H4, B7-H5, and B7-H6, or a co-stimulatory or co-inhibitory
receptor binding specifically to a B7 family member. An
immuno-oncology agent may be an agent that targets a member of the
TNF family of membrane bound ligands or a co-stimulatory or
co-inhibitory receptor binding specifically thereto, e.g., a TNF
receptor family member. Exemplary TNF and TNFR family members that
may be targeted by immuno-oncology agents include CD40 and CD40L,
OX-40, OX-40L, GITR, GITRL, CD70, CD27L, CD30, CD3OL, 4-1BBL, CD137
(4-1BB), TRAIL/Apo2-L, TRAILR1/DR4, TRAILR2/DR5, TRAILR3, TRAILR4,
OPG, RANK, RANKL, TWEAKR/Fn14, TWEAK, BAFFR, EDAR, XEDAR, TACI,
APRIL, BCMA, LT.beta.R, LIGHT, DcR3, HVEM, VEGI/TL1A, TRAMP/DR3,
EDAR, EDA1, XEDAR, EDA2, TNFR1, Lymphotoxin .alpha./TNF.beta.,
TNFR2, TNF.alpha., LT.beta.R, Lymphotoxin .alpha. 1.beta.2, FAS,
FASL, RELT, DR6, TROY and NGFR. An immuno-oncology agent that may
be used in combination with PSGL-1 antagonist agent for treating
cancer may be an agent, e.g., an antibody, targeting an IgSF
member, such as a B7 family member, a B7 receptor family member, a
TNF family member or a TNFR family member, such as those described
above.
[0118] In one aspect, PSGL-1 antagonist is administered with one or
more of (i) an antagonist of a protein that inhibits T cell
activation (e.g., immune checkpoint inhibitor) such as CTLA-4,
PD-1, PD-L1, PD-L2, LAG-3, TIM3, Galectin 9, CEACAM-1, BTLA, CD69,
Galectin-1, TIGIT, CD113, GPR56, VISTA, B7-H3, B7-H4, 2B4, CD48,
GARP, PD1H, LAIR1, TIM-1, TIM-4, and PSGL-1 and (ii) an agonist of
a protein that stimulates T cell activation such as B7-1, B7-2,
CD28, 4-1BB (CD137), 4-1BBL, ICOS, ICOS-L, OX40, OX40L, GITR,
GITRL, CD70, CD27, CD40, CD40L, DR3 and CD28H.
[0119] In one aspect, an immuno-oncology agent is an agent that
inhibits (i.e., an antagonist of) a cytokine that inhibits T cell
activation (e.g., IL-6, IL-10, TGF-.beta., VEGF, and other
immunosuppressive cytokines) or is an agonist of a cytokine, such
as IL-2, IL-7, IL-12, IL-15, IL-21 and IFN.alpha. (e.g., the
cytokine itself) that stimulates T cell activation, and stimulates
an immune response.
[0120] Other agents that can be combined with PSGL-1 antagonist for
stimulating the immune system, e.g., for the treatment of cancer
and infectious diseases, include antagonists of inhibitory
receptors on NK cells or agonists of activating receptors on NK
cells. For example, Anti-PSGL-1 antagonist can be combined with an
antagonist of KIR.
[0121] Yet other agents for combination therapies include agents
that inhibit or deplete macrophages or monocytes, including but not
limited to CSF-1R antagonists such as CSF-1R antagonist antibodies
including RG7155 (WO11/70024, WO11/107553, WO11/131407, WO13/87699,
WO13/119716, WO13/132044) or FPA008 (WO11/140249; WO13169264;
WO14/036357).
[0122] Immuno-oncology agents also include agents that inhibit
TGF-.beta. signaling.
[0123] Additional agents that may be combined with PSGL-1
antagonist include agents that enhance tumor antigen presentation,
e.g., dendritic cell vaccines, GM-CSF secreting cellular vaccines,
CpG oligonucleotides, and imiquimod, or therapies that enhance the
immunogenicity of tumor cells (e.g., anthracyclines).
[0124] Yet other therapies that may be combined with PSGL-1
antagonist include therapies that deplete or block Treg cells,
e.g., an agent that specifically binds to CD25.
[0125] Another therapy that may be combined with PSGL-1 antagonist
is a therapy that inhibits a metabolic enzyme such as indoleamine
dioxigenase (IDO), dioxigenase, arginase, or nitric oxide
synthetase.
[0126] Another class of agents that may be used includes agents
that inhibit the formation of adenosine or inhibit the adenosine
A2A receptor.
[0127] Other therapies that may be combined with PSGL-1 antagonist
for treating cancer include therapies that reverse/prevent T cell
anergy or exhaustion and therapies that trigger an innate immune
activation and/or inflammation at a tumor site.
[0128] PSGL-1 antagonist may be combined with more than one
immuno-oncology agent, and may be, e.g., combined with a
combinatorial approach that targets multiple elements of the immune
pathway, such as one or more of the following: a therapy that
enhances tumor antigen presentation (e.g., dendritic cell vaccine,
GM-CSF secreting cellular vaccines, CpG oligonucleotides,
imiquimod); a therapy that inhibits negative immune regulation
e.g., by inhibiting CTLA-4 and/or PD1/PD-L1/PD-L2 pathway and/or
depleting or blocking Treg or other immune suppressing cells; a
therapy that stimulates positive immune regulation, e.g., with
agonists that stimulate the CD-137, OX-40 and/or GITR pathway
and/or stimulate T cell effector function; a therapy that increases
systemically the frequency of anti-tumor T cells; a therapy that
depletes or inhibits Tregs, such as Tregs in the tumor, e.g., using
an antagonist of CD25 (e.g., daclizumab) or by ex vivo anti-CD25
bead depletion; a therapy that impacts the function of suppressor
myeloid cells in the tumor; a therapy that enhances immunogenicity
of tumor cells (e.g., anthracyclines); adoptive T cell or NK cell
transfer including genetically modified cells, e.g., cells modified
by chimeric antigen receptors (CAR-T therapy); a therapy that
inhibits a metabolic enzyme such as indoleamine dioxigenase (IDO),
dioxigenase, arginase or nitric oxide synthetase; a therapy that
reverses/prevents T cell anergy or exhaustion; a therapy that
triggers an innate immune activation and/or inflammation at a tumor
site; administration of immune stimulatory cytokines or blocking of
immuno repressive cytokines.
[0129] For example, PSGL-1 antagonist can be used with one or more
agonistic agents that ligate positive costimulatory receptors; one
or more antagonists (blocking agents) that attenuate signaling
through inhibitory receptors, such as antagonists that overcome
distinct immune suppressive pathways within the tumor
microenvironment (e.g., block PD-L1/PD-1/PD-L2 interactions); one
or more agents that increase systemically the frequency of
anti-tumor immune cells, such as T cells, deplete or inhibit Tregs
(e.g., by inhibiting CD25); one or more agents that inhibit
metabolic enzymes such as IDO; one or more agents that
reverse/prevent T cell anergy or exhaustion; and one or more agents
that trigger innate immune activation and/or inflammation at tumor
sites.
[0130] In one embodiment, a subject having a disease that may
benefit from stimulation of the immune system, e.g., cancer or an
infectious disease, is treated by administration to the subject of
PSGL-1 antagonist and an immuno-oncology agent, wherein the
immuno-oncology agent is a CTLA-4 antagonist, such as an
antagonistic CTLA-4 antibody. Suitable CTLA-4 antibodies include,
for example, YERVOY (ipilimumab) or tremelimumab.
[0131] In one embodiment, a subject having a disease that may
benefit from stimulation of the immune system, e.g., cancer or an
infectious disease, is treated by administration to the subject of
PSGL-1 antagonist and an immuno-oncology agent, wherein the
immuno-oncology agent is a PD-1 antagonist, such as an antagonistic
PD-1 antibody. Suitable PD-1 antibodies include, for example,
OPDIVO (nivolumab), KEYTRUDA (pembrolizumab), or MEDI-0680
(AMP-514; WO2012/145493). The immuno-oncology agent may also
include pidilizumab (CT-011), though its specificity for PD-1
binding has been questioned. Another approach to target the PD-1
receptor is the recombinant protein composed of the extracellular
domain of PD-L2 (B7-DC) fused to the Fc portion of IgG1, called
AMP-224.
[0132] In one embodiment, a subject having a disease that may
benefit from stimulation of the immune system, e.g., cancer or an
infectious disease, is treated by administration to the subject of
PSGL-1 antagonist and an immuno-oncology agent, wherein the
immuno-oncology agent is a PD-L1 antagonist, such as an
antagonistic PD-L1 antibody. Suitable PD-L1 antibodies include, for
example, MPDL3280A (RG7446; WO2010/077634), durvalumab (MEDI4736),
BMS-936559 (WO2007/005874), MSB0010718C (WO2013/79174) or
rHigM12B7.
[0133] In one embodiment, a subject having a disease that may
benefit from stimulation of the immune system, e.g., cancer or an
infectious disease, is treated by administration to the subject of
PSGL-1 antagonist and an immuno-oncology agent, wherein the
immuno-oncology agent is a LAG-3 antagonist, such as an
antagonistic LAG-3 antibody. Suitable LAG3 antibodies include, for
example, BMS-986016 (WO10/19570, WO14/08218), or IMP-731 or IMP-321
(WO08/132601, WO09/44273).
[0134] In one embodiment, a subject having a disease that may
benefit from stimulation of the immune system, e.g., cancer or an
infectious disease, is treated by administration to the subject of
PSGL-1 antagonist and an immuno-oncology agent, wherein the
immuno-oncology agent is a CD137 (4-1BB) agonist, such as an
agonistic CD137 antibody. Suitable CD137 antibodies include, for
example, urelumab or PF-05082566 (WO12/32433).
[0135] In one embodiment, a subject having a disease that may
benefit from stimulation of the immune system, e.g., cancer or an
infectious disease, is treated by administration to the subject of
PSGL-1 antagonist and an immuno-oncology agent, wherein the
immuno-oncology agent is a GITR agonist, such as an agonistic GITR
antibody. Suitable GITR antibodies include, for example, TRX-518
(WO06/105021, WO09/009116), MK-4166 (WO11/028683) or a GITR
antibody disclosed in WO2015/031667.
[0136] In one embodiment, a subject having a disease that may
benefit from stimulation of the immune system, e.g., cancer or an
infectious disease, is treated by administration to the subject of
PSGL-1 antagonist and an immuno-oncology agent, wherein the
immuno-oncology agent is an OX40 agonist, such as an agonistic OX40
antibody. Suitable OX40 antibodies include, for example, MEDI-6383,
MEDI-6469 or MOXR0916 (RG7888; WO06/029879).
[0137] In one embodiment, a subject having a disease that may
benefit from stimulation of the immune system, e.g., cancer or an
infectious disease, is treated by administration to the subject of
PSGL-1 antagonist and an immuno-oncology agent, wherein the
immuno-oncology agent is a CD40 agonist, such as an agonistic CD40
antibody. In certain embodiments, the immuno-oncology agent is a
CD40 antagonist, such as an antagonistic CD40 antibody. Suitable
CD40 antibodies include, for example, lucatumumab (HCD122),
dacetuzumab (SGN-40), CP-870,893 or Chi Lob 7/4.
[0138] In one embodiment, a subject having a disease that may
benefit from stimulation of the immune system, e.g., cancer or an
infectious disease, is treated by administration to the subject of
PSGL-1 antagonist and an immuno-oncology agent, wherein the
immuno-oncology agent is a CD27 agonist, such as an agonistic CD27
antibody. Suitable CD27 antibodies include, for example, varlilumab
(CDX-1127).
[0139] In one embodiment, a subject having a disease that may
benefit from stimulation of the immune system, e.g., cancer or an
infectious disease, is treated by administration to the subject of
PSGL-1 antagonist and an immuno-oncology agent, wherein the
immuno-oncology agent is MGA271 (to B7H3) (WO11/109400).
[0140] In one embodiment, a subject having a disease that may
benefit from stimulation of the immune system, e.g., cancer or an
infectious disease, is treated by administration to the subject of
PSGL-1 antagonist and an immuno-oncology agent, wherein the
immuno-oncology agent is a KIR antagonist, such as lirilumab.
[0141] In one embodiment, a subject having a disease that may
benefit from stimulation of the immune system, e.g., cancer or an
infectious disease, is treated by administration to the subject of
PSGL-1 antagonist and an immuno-oncology agent, wherein the
immuno-oncology agent is an IDO antagonist. Suitable IDO
antagonists include, for example, INCB-024360 (WO2006/122150,
WO07/75598, WO08/36653, WO08/36642), indoximod, NLG-919
(WO09/73620, WO09/1156652, WO11/56652, WO12/142237) or F001287.
[0142] In one embodiment, a subject having a disease that may
benefit from stimulation of the immune system, e.g., cancer or an
infectious disease, is treated by administration to the subject of
PSGL-1 antagonist and an immuno-oncology agent, wherein the
immuno-oncology agent is a Toll-like receptor agonist, e.g., a
TLR2/4 agonist (e.g., Bacillus Calmette-Guerin); a TLR7 agonist
(e.g., Hiltonol or Imiquimod); a TLR7/8 agonist (e.g., Resiquimod);
or a TLR9 agonist (e.g., CpG7909).
[0143] In one embodiment, a subject having a disease that may
benefit from stimulation of the immune system, e.g., cancer or an
infectious disease, is treated by administration to the subject of
PSGL-1 antagonist and an immuno-oncology agent, wherein, the
immuno-oncology agent is a TGF-.beta. inhibitor, e.g., GC1008,
LY2157299, TEW7197 or IMC-TR1.
Exemplary PSGL-1 Antagonists
[0144] In some embodiments, a PSGL-1 antagonist is selected from a
PSGL-1 antibody and a VISTA antibody. In some embodiments, a PSGL-1
antagonist is a PSGL-1 antibody. In some embodiments, it is a VISTA
antibody. A PSGL-1 antagonist for treating cancer may also be a
non-antibody protein, such as PSGL-1 or VISTA or a portion thereof
(e.g., the ECD) that inhibits the interaction between PSGL-1 and
VISTA, optionally further comprising a fusion partner and in the
form of a fusion molecule. The antagonist, in other embodiments,
may also be a small molecule or small peptide. PSGL-1 Antibodies
and VISTA Antibodies
[0145] In some embodiments, antibodies that block binding of PSGL-1
to VISTA, e.g., at acidic pH, e.g., pH<7.0, .ltoreq.6.8,
.ltoreq.6.5 or .ltoreq.6.3 or at pH 5.5 to 6.5, 6.0-6.5, 6.5-7.0 or
6.0-7.0, e.g., pH 6.0, are provided. In some embodiments,
antibodies that inhibit PSGL-1-mediated signaling are provided. In
some such embodiments, the antibody is PSGL-1 antibody. In some
embodiments, PSGL-1 antibody binds to PSGL-1 extracellular domain
(ECD). In some embodiments, PSGL-1 antibody inhibits binding of
PSGL-1 to VISTA. In some embodiments, PSGL-1 antibody inhibits
VISTA-mediated signaling. In some embodiments, PSGL-1 antibody
inhibits PSGL-1-mediated signaling.
[0146] In some embodiments, PSGL-1 antibody has a dissociation
constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM, .ltoreq.10 nM,
.ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM, or .ltoreq.0.001 nM
(e.g. 10.sup.-8 M or less, e.g. from 10.sup.-8 M to 10.sup.-13 M,
e.g., from 10.sup.-9 M to 10.sup.-13 M) for PSGL-1, e.g., for
humPSGL-1. In certain embodiments, PSGL-1 antibody has a
dissociation constant (Kd) of .ltoreq.1 .mu.M, .ltoreq.100 nM,
.ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM, .ltoreq.0.01 nM, or
.ltoreq.0.001 nM (e.g. 10.sup.-8 M or less, e.g. from 10.sup.-8 M
to 10.sup.-13 M, e.g., from 10.sup.-9 M to 10.sup.-13 M) for
PSGL-1, e.g., for humPSGL-1, at acidic pH, e.g., pH<7.0,
.ltoreq.6.8, .ltoreq.6.5 or .ltoreq.6.3 or at pH 5.5 to 6.5,
6.0-6.5, 6.5-7.0 or 6.0-7.0, e.g., pH 6.0.
[0147] In some embodiments, a PSGL-1 antibody having any the
characteristics provided herein inhibits at least 25%, 50%, 75%,
80%, 90% or 100% of the binding of VISTA to PSGL-1.
[0148] In some embodiments, an antibody binds to PSGL-1 from
multiple species. For example, in some embodiments, an antibody
binds to human PSGL-1, and also binds to PSGL-1 from at least one
mammal selected from mouse, rat, dog, guinea pig, and cynomolgus
monkey.
[0149] In some embodiments, the antibody is a VISTA antibody. In
some embodiments, a VISTA antibody binds to VISTA extracellular
domain (ECD). In some embodiments, a VISTA antibody inhibits
binding of VISTA to PSGL-1. In some embodiments, a VISTA antibody
inhibits VISTA-mediated signaling. In some embodiments, a VISTA
antibody inhibits PSGL-1-mediated signaling. In some embodiments, a
VISTA antibody has a dissociation constant (Kd) of .ltoreq.1 .mu.M,
.ltoreq.100 nM, .ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM,
.ltoreq.0.01 nM, or .ltoreq.0.001 nM (e.g. 10.sup.-8 M or less,
e.g. from 10.sup.-8 M to 10.sup.-13 M, e.g., from 10.sup.-9 M to
10.sup.-13 M) VISTA, e.g., for human VISTA. In some embodiments, a
VISTA antibody has a dissociation constant (Kd) of .ltoreq.1 .mu.M,
<100 nM, .ltoreq.10 nM, .ltoreq.1 nM, .ltoreq.0.1 nM,
.ltoreq.0.01 nM, or .ltoreq.0.001 nM (e.g. 10.sup.-8 M or less,
e.g. from 10.sup.-8 M to 10.sup.-13 M, e.g., from 10.sup.-9 M to
10.sup.-13 M) for VISTA, e.g., for human VISTA, at acidic pH, e.g.,
pH<7.0, .ltoreq.6.8, .ltoreq.6.5 or .ltoreq.6.3 or at pH 5.5 to
6.5, 6.0-6.5, 6.5-7.0 or 6.0-7.0, e.g., pH 6.0.
[0150] In some embodiments, a VISTA antibody having any the
characteristics provided herein inhibits at least 25%, 50%, 75%,
80%, 90% or 100% of the binding of VISTA to PSGL-1.
[0151] In some embodiments, an antibody binds to VISTA from
multiple species. For example, in some embodiments, an antibody
binds to human VISTA, and also binds to VISTA from at least one
mammal selected from mouse, rat, dog, guinea pig, and cynomolgus
monkey.
[0152] In some embodiments, multispecific antibodies are provided.
In some embodiments, bispecific antibodies are provided.
Nonlimiting exemplary bispecific antibodies include antibodies
comprising a first arm comprising a heavy chain/light chain
combination that binds a first antigen and a second arm comprising
a heavy chain/light chain combination that binds a second antigen.
A further nonlimiting exemplary multispecific antibody is a dual
variable domain antibody. In some embodiments, a bispecific
antibody comprises a first arm that inhibits binding of PSGL-1 to
VISTA and a second arm that stimulates T cells, e.g., by binding
CD3. In some embodiments, the first arm binds PSGL-1.
Humanized Antibodies
[0153] In some embodiments, PSGL-1 or a VISTA antibody is a
humanized antibody. Humanized antibodies are useful as therapeutic
molecules because humanized antibodies reduce or eliminate the
human immune response to non-human antibodies (such as the human
anti-mouse antibody (HAMA) response), which can result in an immune
response to an antibody therapeutic, and decreased effectiveness of
the therapeutic.
[0154] An antibody may be humanized by any method. Nonlimiting
exemplary methods of humanization include methods described, e.g.,
in U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762;
6,180,370; Jones et al., Nature 321: 522-525 (1986); Riechmann et
al., Nature 332: 323-27 (1988); Verhoeyen et al., Science 239:
1534-36 (1988); and U.S. Publication No. US 2009/0136500.
[0155] As noted above, a humanized antibody is an antibody in which
at least one amino acid in a framework region of a non-human
variable region has been replaced with the amino acid from the
corresponding location in a human framework region. In some
embodiments, at least two, at least three, at least four, at least
five, at least six, at least seven, at least eight, at least nine,
at least 10, at least 11, at least 12, at least 15, or at least 20
amino acids in the framework regions of a non-human variable region
are replaced with an amino acid from one or more corresponding
locations in one or more human framework regions.
[0156] In some embodiments, some of the corresponding human amino
acids used for substitution are from the framework regions of
different human immunoglobulin genes. That is, in some such
embodiments, one or more of the non-human amino acids may be
replaced with corresponding amino acids from a human framework
region of a first human antibody or encoded by a first human
immunoglobulin gene, one or more of the non-human amino acids may
be replaced with corresponding amino acids from a human framework
region of a second human antibody or encoded by a second human
immunoglobulin gene, one or more of the non-human amino acids may
be replaced with corresponding amino acids from a human framework
region of a third human antibody or encoded by a third human
immunoglobulin gene, etc. Further, in some embodiments, all of the
corresponding human amino acids being used for substitution in a
single framework region, for example, FR2, need not be from the
same human framework. In some embodiments, however, all of the
corresponding human amino acids being used for substitution are
from the same human antibody or encoded by the same human
immunoglobulin gene.
[0157] In some embodiments, an antibody is humanized by replacing
one or more entire framework regions with corresponding human
framework regions. In some embodiments, a human framework region is
selected that has the highest level of homology to the non-human
framework region being replaced. In some embodiments, such a
humanized antibody is a CDR-grafted antibody.
[0158] In some embodiments, following CDR-grafting, one or more
framework amino acids are changed back to the corresponding amino
acid in a mouse framework region. Such "back mutations" are made,
in some embodiments, to retain one or more mouse framework amino
acids that appear to contribute to the structure of one or more of
the CDRs and/or that may be involved in antigen contacts and/or
appear to be involved in the overall structural integrity of the
antibody. In some embodiments, ten or fewer, nine or fewer, eight
or fewer, seven or fewer, six or fewer, five or fewer, four or
fewer, three or fewer, two or fewer, one, or zero back mutations
are made to the framework regions of an antibody following CDR
grafting.
[0159] In some embodiments, a humanized antibody also comprises a
human heavy chain constant region and/or a human light chain
constant region.
Chimeric Antibodies
[0160] In some embodiments, PSGL-1 antibody or a VISTA antibody is
a chimeric antibody. In some embodiments, PSGL-1 antibody or a
VISTA antibody comprises at least one non-human variable region and
at least one human constant region. In some such embodiments, all
of the variable regions of PSGL-1 antibody or a VISTA antibody are
non-human variable regions, and all of the constant regions of the
PSGL-1 antibody or VISTA antibody are human constant regions. In
some embodiments, one or more variable regions of a chimeric
antibody are mouse variable regions. The human constant region of a
chimeric antibody need not be of the same isotype as the non-human
constant region, if any, it replaces. Chimeric antibodies are
discussed, e.g., in U.S. Pat. No. 4,816,567; and Morrison et al.
Proc. Natl. Acad. Sci. USA 81: 6851-55 (1984).
Human Antibodies
[0161] In some embodiments, PSGL-1 antibody or a VISTA antibody is
a human antibody. Human antibodies can be made by any suitable
method. Nonlimiting exemplary methods include making human
antibodies in transgenic mice that comprise human immunoglobulin
loci. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA 90:
2551-55 (1993); Jakobovits et al., Nature 362: 255-8 (1993);
Lonberg et al., Nature 368: 856-9 (1994); and U.S. Pat. Nos.
5,545,807; 6,713,610; 6,673,986; 6,162,963; 5,545,807; 6,300,129;
6,255,458; 5,877,397; 5,874,299; and 5,545,806.
[0162] Nonlimiting exemplary methods also include making human
antibodies using phage display libraries. See, e.g., Hoogenboom et
al., J. Mol. Biol. 227: 381-8 (1992); Marks et al., J. Mol. Biol.
222: 581-97 (1991); and PCT Publication No. WO 99/10494.
Human Antibody Constant Regions
[0163] In some embodiments, a humanized, chimeric, or human
antibody described herein comprises one or more human constant
regions. In some embodiments, the human heavy chain constant region
is of an isotype selected from IgA, IgG, and IgD. In some
embodiments, the human light chain constant region is of an isotype
selected from .kappa. and .lamda.. In some embodiments, an antibody
described herein comprises a human IgG constant region, for
example, human IgG1, IgG2, IgG3, or IgG4. In some embodiments, an
antibody or Fc fusion partner comprises a C237S mutation, for
example, in an IgG1 constant region. See, e.g., SEQ ID NO: 17. In
some embodiments, an antibody described herein comprises a human
IgG2 heavy chain constant region. In some such embodiments, the
IgG2 constant region comprises a P331S mutation, as described in
U.S. Pat. No. 6,900,292. In some embodiments, an antibody described
herein comprises a human IgG4 heavy chain constant region. In some
such embodiments, an antibody described herein comprises an S241P
mutation in the human IgG4 constant region. See, e.g., Angal et al.
Mol. Immunol. 30(1): 105-108 (1993). In some embodiments, an
antibody described herein comprises a human IgG4 constant region
and a human .kappa. light chain.
[0164] The choice of heavy chain constant region can determine
whether or not an antibody will have effector function in vivo.
Such effector function, in some embodiments, includes
antibody-dependent cell-mediated cytotoxicity (ADCC) and/or
complement-dependent cytotoxicity (CDC), and can result in killing
of the cell to which the antibody is bound. Typically, antibodies
comprising human IgG1 or IgG3 heavy chains have effector
function.
[0165] In some embodiments, effector function is not desirable. For
example, in some embodiments, effector function may not be
desirable in treatments of inflammatory conditions and/or
autoimmune disorders. In some such embodiments, a human IgG4 or
IgG2 heavy chain constant region is selected or engineered. In some
embodiments, an IgG4 constant region comprises an S241P
mutation.
Exemplary Properties of Antibodies
Exemplary Properties of PSGL-1 Antibodies
[0166] In some embodiments, PSGL-1 antibody binds to PSGL-1, e.g.,
at acidic pH, e.g., pH<7.0, .ltoreq.6.8, .ltoreq.6.5 or
.ltoreq.6.3 or at pH 5.5 to 6.5, 6.0-6.5, 6.5-7.0 or 6.0-7.0, e.g.,
pH 6.0, and inhibits PSGL-1-mediated signaling. In some
embodiments, PSGL-1 antibody blocks binding of PSGL-1 to VISTA. In
some embodiments, PSGL-1 antibody blocks binding of PSGL-1 to
VISTA, e.g., by at least 25%, 50%, 75%, 80%, 90% or 100%. In some
embodiments, PSGL-1 antibody binds to PSGL-1 with a binding
affinity (KD) of less than 50 nM, less than 20 nM, less than 10 nM,
or less than 1 nM. In some embodiments, the extent of binding of
PSGL-1 antibody to an unrelated, non-PSGL-1 protein is less than
about 10% of the binding of the antibody to PSGL-1 as measured,
e.g., by a radioimmunoassay (MA). In some embodiments, PSGL-1
antibody binds to an epitope of PSGL-1 that is conserved among
PSGL-1 from different species. In some embodiments, PSGL-1 antibody
binds to the same epitope as a human or humanized PSGL-1 antibody
that binds humPSGL-1.
Exemplary Properties of VISTA Antibodies
[0167] In some embodiments, a VISTA antibody binds to VISTA, e.g.,
at acidic pH, e.g., pH<7.0, .ltoreq.6.8, .ltoreq.6.5 or
.ltoreq.6.3 or at pH 5.5 to 6.5, 6.0-6.5, 6.5-7.0 or 6.0-7.0, e.g.,
pH 6.0, and inhibits PSGL-1-mediated signaling. In some
embodiments, a VISTA antibody blocks binding of PSGL-1 to VISTA. In
some embodiments, a VISTA antibody blocks binding of PSGL-1 to
VISTA, e.g., by at least 25%, 50%, 75%, 80%, 90% or 100%. In some
embodiments, a VISTA antibody binds to VISTA with a binding
affinity (KD) of less than 50 nM, less than 20 nM, less than 10 nM,
or less than 1 nM. In some embodiments, the extent of binding of a
VISTA antibody to an unrelated, non-VISTA protein is less than
about 10% of the binding of the antibody to VISTA as measured,
e.g., by a radioimmunoassay (MA). In some embodiments, a VISTA
antibody binds to an epitope of VISTA that is conserved among VISTA
from different species. In some embodiments, a VISTA antibody binds
to the same epitope as a human or humanized VISTA antibody that
binds human VISTA.
Antibody Conjugates
[0168] In some embodiments, PSGL-1 or a VISTA antibody is
conjugated to a label. As used herein, a label is a moiety that
facilitates detection of the antibody and/or facilitates detection
of a molecule to which the antibody binds. Nonlimiting exemplary
labels include, but are not limited to, radioisotopes, fluorescent
groups, enzymatic groups, chemiluminescent groups, biotin, epitope
tags, metal-binding tags, etc. One skilled in the art can select a
suitable label according to the intended application.
[0169] In some embodiments, a label is conjugated to an antibody
using chemical methods in vitro. Nonlimiting exemplary chemical
methods of conjugation are known in the art, and include services,
methods and/or reagents commercially available from, e.g., Thermo
Scientific Life Science Research Produces (formerly Pierce;
Rockford, Ill.), Prozyme (Hayward, Calif.), SACRI Antibody Services
(Calgary, Canada), AbD Serotec (Raleigh, N.C.), etc. In some
embodiments, when a label is a polypeptide, the label can be
expressed from the same expression vector with at least one
antibody chain to produce a polypeptide comprising the label fused
to an antibody chain. PSGL-1 and VISTA ECDs, ECD Fusion Molecules,
and Small Peptides
[0170] In some embodiments, the PSGL-1 antagonist is a PSGL-1
polypeptide, such as a full-length PSGL-1, or a fragment of PSGL-1
that inhibits binding of PSGL-1 to VISTA. In some embodiments, the
PSGL-1 antagonist is a PSGL-1 extracellular domain (ECD). In some
embodiments, the PSGL-1 antagonist is a full-length PSGL-1 ECD. In
some embodiments, the PSGL-1 ECD is a PSGL-1 ECD fragment, for
example, comprising at least 80%, at least 85%, at least 90%, or at
least 95% of the full length PSGL-1 ECD amino acid sequence from
which it is derived. In some embodiments the PSGL-1 ECD is a PSGL-1
ECD variant, for example, comprising at least 80%, at least 85%, at
least 90%, at least 92%, at least 95%, at least 97%, at least 98%,
or at least 99% sequence identity with the full length PSGL-1 ECD
from which it is derived. In other embodiments, the PSGL-1 ECD is
from a non-human PSGL-1 ECD and may be either full length, a
fragment, or a variant.
[0171] In some embodiments, the PSGL-1 or PSGL-1 fragment is
combined with at least one fusion partner. Thus, in some such
embodiments the PSGL-1 antagonist may comprise a full length PSGL-1
ECD and at least one fusion partner to form a PSGL-1 ECD fusion
molecule. In some embodiments, the PSGL-1 ECD portion of the fusion
molecule comprises a PSGL-1 ECD fragment, for example, comprising
at least 80%, at least 85%, at least 90%, or at least 95% of the
full length PSGL-1 ECD amino acid sequence from which it is
derived. In some embodiments, the PSGL-1 ECD portion of the fusion
molecule is a PSGL-1 ECD variant, for example, comprising at least
80%, at least 85%, at least 90%, at least 92%, at least 95%, at
least 97%, at least 98%, or at least 99% sequence identity with the
full length PSGL-1 ECD from which it is derived. In other
embodiments, the PSGL-1 ECD component is from a non-human PSGL-1
ECD and may be full length, a fragment, or a variant. In any of the
fusion molecule embodiments above, the fusion partner may comprise
an immunoglobulin Fc molecule, for example, a human Fc molecule, or
in some embodiments an Fc having a sequence chosen from SEQ ID NOs:
11-13. In other embodiments, the fusion partner may be a different
molecule such as albumin or polyethylene glycol (PEG). In some
embodiments, more than one fusion partner may be attached to the
PSGL-1 ECD. In some embodiments, the fusion partner (or partners)
is attached at the C-terminal of the ECD, while other attachments
are also possible such as on an amino acid side-chain or at the
N-terminus. The attachment of a fusion partner to a PSGL-1 ECD may
be direct (i.e. by a covalent bond) or indirect through a linker. A
linker may comprise, for example, at least one intervening amino
acid or some other chemical moiety serving to link the fusion
partner to the ECD either covalently or noncovalently.
[0172] In any of the above embodiments, the PSGL-1 polypeptide may
either include a signal sequence or be in a mature form, i.e., not
including a signal sequence. The signal sequence may be from a
native PSGL-1 molecule or it may be a signal sequence from a
different protein, for example one chosen to enhance expression of
the PSGL-1 polypeptide in cell culture.
[0173] In some embodiments a PSGL-1 ECD may comprise the sequence
of amino acids 1-241 of SEQ ID NO: 1 or 1-241 of SEQ ID NO: 14
(human isoforms 1 and 2, respectively, including signal sequence).
In other embodiments, a PSGL-1 ECD may comprise the sequence of
amino acids 23-241 of SEQ ID NO: 1 or 23-241 of SEQ ID NO: 14 or
amino acids 1-219 of SEQ ID NO: 2 or amino acids 1-219 of SEQ ID
NO: 15 or the amino acid sequence of SEQ ID NO: 3 or of SEQ ID NO:
4 or of SEQ ID NO: 16 or of SEQ ID NO: 17 (human isoforms 1 and 2,
mature forms without signal sequence), or SEQ ID NO: 18 (another
exemplary ECD sequence). In some embodiments, the PSGL-1 ECD may
consist of one of the above amino acid sequences. In any of the
above cases, a PSGL-1 ECD may be part of a fusion molecule such
that the above amino acid sequence may be joined to a fusion
partner either directly or via a linker, such as an Fc, albumin, or
PEG. For example, in some embodiments in which the antagonist is a
PSGL-1 ECD fusion molecule, the fusion molecule may comprise one of
the above sequences plus at least one of SEQ ID NOs: 11-13
(immunoglobulin Fc sequences) or an Fc from human IgG1. A PSGL-1
ECD Fc fusion molecule may be formed by a direct attachment of the
PSGL-1 ECD amino acid sequence to the Fc amino acid sequence or via
a linker (either an intervening amino acid or amino acid sequence
or another chemical moiety). SEQ ID NO: 19, for example, provides a
linker used in a PSGL-1 Fc molecule used in the examples below.
Additional PSGL-1 ECD Fc fusion molecules are described in T.
Pouyani et al., Cell 83: 333-343 (1995).
[0174] In some embodiments, the PSGL-1 antagonist is a VISTA
polypeptide, such as a full-length VISTA or a fragment of VISTA
that inhibits the interaction between VISTA and PSGL-1. For
example, in some embodiments, the PSGL-1 antagonist is a
full-length VISTA polypeptide. In some embodiments, the PSGL-1
antagonist is a VISTA extracellular domain (ECD). In some
embodiments, the PSGL-1 antagonist is a full-length human VISTA
ECD. In some embodiments, the VISTA ECD may be a VISTA ECD
fragment, for example, comprising at least 80%, at least 85%, at
least 90%, or at least 95% of the full length VISTA ECD amino acid
sequence from which it is derived. In some embodiments the VISTA
ECD is a VISTA ECD variant, for example, comprising at least 80%,
at least 85%, at least 90%, at least 92%, at least 95%, at least
97%, at least 98%, or at least 99% sequence identity with the full
length VISTA ECD from which it is derived. In other embodiments,
the VISTA ECD is from a non-human VISTA ECD and may be either full
length, a fragment, or a variant.
[0175] In some embodiments, the VISTA or VISTA fragment is combined
with at least one fusion partner. Thus, in some such embodiments
the PSGL-1 antagonist may comprise a full length VISTA ECD and at
least one fusion partner to form a VISTA ECD fusion molecule. In
some embodiments, the VISTA ECD portion of the fusion molecule
comprises a VISTA ECD fragment, for example, comprising at least
80%, at least 85%, at least 90%, or at least 95% of the full length
VISTA ECD amino acid sequence from which it is derived. In some
embodiments, the VISTA ECD portion of the fusion molecule is a
VISTA ECD variant, for example, comprising at least 80%, at least
85%, at least 90%, at least 92%, at least 95%, at least 97%, at
least 98%, or at least 99% sequence identity with the full length
VISTA ECD from which it is derived. In other embodiments, the VISTA
ECD component is from a non-human VISTA ECD and may be full length,
a fragment, or a variant. In any of the fusion molecule embodiments
above, the fusion partner may comprise an immunoglobulin Fc
molecule, for example a human Fc molecule, in some embodiments
having a sequence chosen from SEQ ID NOs: 11-13. In other
embodiments, the fusion partner may be a different molecule such as
albumin or polyethylene glycol (PEG). In some embodiments, more
than one fusion partner may be attached to the VISTA ECD. In some
embodiments, the fusion partner (or partners) is attached at the
C-terminal of the ECD, while other attachments are also possible
such as on an amino acid side-chain or at the N-terminus. The
attachment of a fusion partner to a VISTA ECD may be direct (i.e.
by a covalent bond) or indirect through a linker. A linker may
comprise, for example, at least one intervening amino acid or some
other chemical moiety serving to link the fusion partner to the ECD
either covalently or noncovalently.
[0176] In any of the above embodiments, the VISTA polypeptide may
either include a signal sequence or be in a mature form, i.e., not
including a signal sequence. The signal sequence may be from a
native VISTA molecule or it may be a signal sequence from a
different protein, for example one chosen to enhance expression of
the VISTA polypeptide in cell culture.
[0177] In some embodiments a VISTA ECD may comprise the sequence of
amino acids 1-202 of SEQ ID NO: 10. In some embodiments, the VISTA
ECD may consist of the above amino acid sequence. In either of the
above cases, a VISTA ECD may be part of a fusion molecule such that
the above amino acid sequence may be joined to a fusion partner
either directly or via a linker, such as an Fc, albumin, or PEG.
For example, in some embodiments in which the antagonist is a VISTA
ECD fusion molecule, the fusion molecule may comprise the sequence
of amino acids 1-202 of SEQ ID NO: 10 plus at least one of SEQ ID
NOs: 11-13 (immunoglobulin Fc sequences), or the entire fusion
molecule may comprise or consist of the amino acid sequence of SEQ
ID NO: 9 (an exemplary VISTA ECD Fc fusion protein). A VISTA ECD Fc
fusion molecule may be formed by a direct attachment of the VISTA
ECD amino acid sequence to the Fc amino acid sequence or via a
linker (either an intervening amino acid or amino acid sequence or
another chemical moiety).
[0178] In some embodiments, the PSGL-1 antagonist may be a small
molecule or a peptide, e.g., a small peptide. In some embodiments,
the PSGL-1 antagonist may be a small peptide comprising an amino
acid sequence of a PSGL-1 ECD fragment. In some embodiments, the
PSGL-1 antagonist may be a small peptide comprising an amino acid
sequence of a VISTA ECD fragment. In some embodiments, the PSGL-1
antagonist is a small peptide having, e.g., from 3 to 20, e.g., 3
to 15 or 3 to 10 amino acids, which peptide may be linear or
circular, with a sequence comprising a PSGL-1 fragment, a PSGL-1
ECD fragment, a VISTA fragment, or a VISTA ECD fragment, or a
variant of a PSGL-1 fragment, a PSGL-1 ECD fragment, a VISTA
fragment, or a VISTA ECD fragment. Such a variant of a PSGL-1 or
VISTA fragment may have, for example, at least 95%, at least 97%,
at least 99% sequence identity to the native fragment sequence from
which it is derived
Signal Peptides
[0179] In order for some secreted proteins to express and secrete
in large quantities, a signal peptide from a heterologous protein
may be desirable. Employing heterologous signal peptides may be
advantageous in that a resulting mature polypeptide may remain
unaltered as the signal peptide is removed in the ER during the
secretion process. The addition of a heterologous signal peptide
may be required to express and secrete some proteins.
[0180] Nonlimiting exemplary signal peptide sequences are
described, e.g., in the online Signal Peptide Database maintained
by the Department of Biochemistry, National University of
Singapore. See Choo et al., BMC Bioinformatics, 6: 249 (2005); and
PCT Publication No. WO 2006/081430.
Co-Translational and Post-Translational Modifications
[0181] In some embodiments, a polypeptide such as PSGL-1 or a VISTA
antibody or a PSGL-1 or VISTA ECD is differentially modified during
or after translation, for example by glycosylation, sialylation,
acetylation, phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, or linkage to an
antibody molecule or other cellular ligand. Any of numerous
chemical modifications may be carried out by known techniques,
including, but not limited to, specific chemical cleavage by
cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease;
NABH4, acetylation; formylation; oxidation; reduction; and/or
metabolic synthesis in the presence of tunicamycin.
[0182] Additional post-translational modifications encompassed by
the invention include, for example, N-linked or O-linked
carbohydrate chains; processing of N-terminal or C-terminal ends;
attachment of chemical moieties to the amino acid backbone;
chemical modifications of N-linked or O-linked carbohydrate chains;
and addition or deletion of an N-terminal methionine residue as a
result of prokaryotic host cell expression.
Nucleic Acid Molecules Encoding PSGL-1 Antagonists
[0183] Nucleic acid molecules are provided, wherein the nucleic
acid molecules comprise polynucleotides that encode one or more
chains of an antibody described herein, such as PSGL-1 or a VISTA
antibody. In some embodiments, a nucleic acid molecule comprises a
polynucleotide that encodes a heavy chain or a light chain of an
antibody described herein. In some embodiments, a nucleic acid
molecule comprises both a polynucleotide that encodes a heavy chain
and a polynucleotide that encodes a light chain, of an antibody
described herein. In some embodiments, a first nucleic acid
molecule comprises a first polynucleotide that encodes a heavy
chain and a second nucleic acid molecule comprises a second
polynucleotide that encodes a light chain.
[0184] In some such embodiments, the heavy chain and the light
chain are expressed from one nucleic acid molecule, or from two
separate nucleic acid molecules, as two separate polypeptides. In
some embodiments, such as when an antibody is an scFv, a single
polynucleotide encodes a single polypeptide comprising both a heavy
chain and a light chain linked together.
[0185] In some embodiments, a polynucleotide encoding a heavy chain
or light chain of an antibody described herein comprises a
nucleotide sequence that encodes a leader sequence, which, when
translated, is located at the N-terminus of the heavy chain or
light chain. As discussed above, the leader sequence may be the
native heavy or light chain leader sequence, or may be another
heterologous leader sequence.
[0186] Nucleic acids encoding other PSGL-1 antagonists are also
provided, such as fragments or variants of PSGL-1 including PSGL-1
ECD molecules or PSGL-1 ECD fusion molecules and including
fragments or variants of VISTA including VISTA ECD molecules or
VISTA ECD fusion molecules. Nucleic acid molecules may be
constructed using recombinant DNA techniques conventional in the
art. In some embodiments, a nucleic acid molecule is an expression
vector that is suitable for expression in a selected host cell.
Polypeptide Expression and Production
Vectors
[0187] Vectors comprising polynucleotides that encode heavy chains
and/or light chains of the antibodies described herein are
provided. Such vectors include, but are not limited to, DNA
vectors, phage vectors, viral vectors, retroviral vectors, etc. In
some embodiments, a vector comprises a first polynucleotide
sequence encoding a heavy chain and a second polynucleotide
sequence encoding a light chain. In some embodiments, the heavy
chain and light chain are expressed from the vector as two separate
polypeptides. In some embodiments, the heavy chain and light chain
are expressed as part of a single polypeptide, such as, for
example, when the antibody is an scFv.
[0188] In some embodiments, a first vector comprises a
polynucleotide that encodes a heavy chain and a second vector
comprises a polynucleotide that encodes a light chain. In some
embodiments, the first vector and second vector are transfected
into host cells in similar amounts (such as similar molar amounts
or similar mass amounts). In some embodiments, a mole- or
mass-ratio of between 5:1 and 1:5 of the first vector and the
second vector is transfected into host cells. In some embodiments,
a mass ratio of between 1:1 and 1:5 for the vector encoding the
heavy chain and the vector encoding the light chain is used. In
some embodiments, a mass ratio of 1:2 for the vector encoding the
heavy chain and the vector encoding the light chain is used.
[0189] In some embodiments, a vector is selected that is optimized
for expression of polypeptides in CHO or CHO-derived cells, or in
NSO cells. Exemplary such vectors are described, e.g., in Running
Deer et al., Biotechnol. Prog. 20:880-889 (2004).
[0190] In some embodiments, a vector is chosen for in vivo
expression of PSGL-1 antagonist in animals, including humans. In
some such embodiments, expression of the polypeptide or
polypeptides is under the control of a promoter or promoters that
function in a tissue-specific manner. For example, liver-specific
promoters are described, e.g., in PCT Publication No. WO
2006/076288.
Host Cells
[0191] In various embodiments, heavy chains and/or light chains of
the antibodies described herein may be expressed in prokaryotic
cells, such as bacterial cells; or in eukaryotic cells, such as
fungal cells (such as yeast), plant cells, insect cells, and
mammalian cells. Such expression may be carried out, for example,
according to procedures known in the art. Exemplary eukaryotic
cells that may be used to express polypeptides include, but are not
limited to, COS cells, including COS 7 cells; 293 cells, including
293-6E cells; CHO cells, including CHO-S and DG44 cells;
PER.C6.RTM. cells (Crucell); and NSO cells. In some embodiments,
heavy chains and/or light chains of the antibodies described herein
may be expressed in yeast. See, e.g., U.S. Publication No. US
2006/0270045 A1. In some embodiments, a particular eukaryotic host
cell is selected based on its ability to make desired
post-translational modifications to the heavy chains and/or light
chains of PSGL-1 or a VISTA antibody. For example, in some
embodiments, CHO cells produce polypeptides that have a higher
level of sialylation than the same polypeptide produced in 293
cells.
[0192] Introduction of one or more nucleic acids into a desired
host cell may be accomplished by any method, including but not
limited to, calcium phosphate transfection, DEAE-dextran mediated
transfection, cationic lipid-mediated transfection,
electroporation, transduction, infection, etc. Nonlimiting
exemplary methods are described, e.g., in Sambrook et al.,
Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor
Laboratory Press (2001). Nucleic acids may be transiently or stably
transfected in the desired host cells, according to any suitable
method.
[0193] In some embodiments, one or more polypeptides may be
produced in vivo in an animal that has been engineered or
transfected with one or more nucleic acid molecules encoding the
polypeptides, according to any suitable method.
Purification of Polypeptides
[0194] The antibodies described herein may be purified by any
suitable method. Such methods include, but are not limited to, the
use of affinity matrices or hydrophobic interaction chromatography.
Suitable affinity ligands include the antigen and/or epitope to
which the antibody binds, and ligands that bind antibody constant
regions. For example, a Protein A, Protein G, Protein A/G, or an
antibody affinity column may be used to bind the constant region
and to purify an antibody.
[0195] In some embodiments, hydrophobic interactive chromatography,
for example, a butyl or phenyl column, is also used for purifying
some polypeptides. Many methods of purifying polypeptides are known
in the art.
Cell-free Production of Polypeptides
[0196] In some embodiments, an antibody described herein is
produced in a cell-free system. Nonlimiting exemplary cell-free
systems are described, e.g., in Sitaraman et al., Methods Mol.
Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45
(2004); Endo et al., Biotechnol. Adv. 21: 695-713 (2003). Methods
of Identifying PSGL-1 Antagonists
[0197] In some embodiments, methods of identifying PSGL-1
antagonists are provided. In some embodiments, a method comprises
contacting a candidate molecule (i.e., a molecule being tested for
antagonist activity) with VISTA, a VISTA ECD, or a VISTA ECD fusion
molecule (collectively referred to as a "VISTA molecule"), e.g., at
acidic pH, e.g., pH<7.0, .ltoreq.6.8, .ltoreq.6.5 or .ltoreq.6.3
or at pH 5.5 to 6.5, 6.0-6.5, 6.5-7.0 or 6.0-7.0, e.g., pH 6.0. In
some embodiments, a method further comprises contacting the
candidate molecule/VISTA molecule mixture with PSGL-1, PSGL-1 ECD,
or PSGL-1 ECD fusion molecule (collectively referred to as an
"PSGL-1 molecule"). In some embodiments, a method comprises
contacting the candidate molecule with the PSGL-1 molecule, and
then contacting the candidate molecule/PSGL-1 molecule mixture with
a VISTA molecule, e.g., at acidic pH, e.g., pH<7.0, .ltoreq.6.8,
.ltoreq.6.5 or .ltoreq.6.3 or at pH 5.5 to 6.5, 6.0-6.5, 6.5-7.0 or
6.0-7.0, e.g., pH 6.0. In some embodiments, a method comprises
contacting a candidate molecule with a VISTA molecule and PSGL-1
molecule approximately simultaneously. In some embodiments, a
method comprises forming a first composition comprising a VISTA
molecule and PSGL-1 molecule, e.g., at acidic pH, e.g., pH<7.0,
.ltoreq.6.8, .ltoreq.6.5 or .ltoreq.6.3 or at pH 5.5 to 6.5,
6.0-6.5, 6.5-7.0 or 6.0-7.0, e.g., pH 6.0, and then contacting the
candidate molecule with the first composition. One skilled in the
art will recognize that the order in which the components are
contacted with one another may be varied according to the assay
design. In some embodiments, contacting the VISTA molecule, PSGL-1
molecule, and candidate molecule occurs at an acidic pH, or a pH
that is lower than pH 8.0, lower than pH 7.0, lower than pH 6.5 or
lower than pH 6.0 or at pH 5.5 to 6.5, 6.0-6.5, 6.5-7.0 or 6.0-7.0,
e.g., pH 6.0. The pH of the composition may be pH 5.0 to pH 8.0, pH
5.5 to pH 7.0, pH 6.0 to pH 8, or pH 6.5 to pH 8.
[0198] In some embodiments, the VISTA molecule is a full length
VISTA, for example, VISTA expressed on the surface of a cell. In
some embodiments, the VISTA molecule is a soluble VISTA, such as a
VISTA ECD or VISTA ECD fusion molecule. In some embodiments, the
PSGL-1 molecule is a full length PSGL-1, for example, PSGL-1
expressed on the surface of a cell. In some embodiments, the PSGL-1
molecule is a soluble PSGL-1, such as PSGL-1 ECD or PSGL-1 ECD
fusion molecule.
[0199] In some embodiments, after the candidate molecule has been
contacted with the VISTA molecule and/or the PSGL-1 molecule, an
assay or assays are carried out to detect PSGL-1 molecule binding
to the VISTA molecule. Nonlimiting exemplary assays for detecting
PSGL-1 molecule binding to a VISTA molecule include ELISA assays,
surface plasmon resonance assays (e.g., Biacore.RTM.), flow
cytometry-based assays (for example, when one or more components
are bound to beads, or are expressed on the surface of cells),
amplified luminescent proximity homogeneous assay (ALPHA), etc.
Many methods of detecting protein-protein binding are known in the
art, and one skilled in the art can select a suitable assay method.
Further, various reagents may be used for detection as needed,
including antibodies (with or without labels), secondary antibodies
(with or without labels), labeled assay components (including, but
not limited to, labeled PSGL-1 molecule and/or labeled VISTA
molecule), etc.
[0200] In some embodiments, methods of identifying PSGL-1
antagonists comprise comparing the extent of VISTA molecule/PSGL-1
molecule binding in the presence and absence of the candidate
molecule, e.g., at acidic pH, e.g., pH<7.0, .ltoreq.6.8,
.ltoreq.6.5 or .ltoreq.6.3 or at pH 5.5 to 6.5, 6.0-6.5, 6.5-7.0 or
6.0-7.0, e.g., pH 6.0. In some embodiments, when VISTA
molecule/PSGL-1 molecule binding is reduced in the presence of the
candidate molecule relative to the binding in the absence of the
candidate molecule, the candidate molecule is PSGL-1 antagonist. In
some embodiments, binding between the VISTA molecule and the PSGL-1
molecule is reduced by at least 20%, at least 30%, at least 40%, at
least 50%, at least 60%, at least 70%, at least 80%, or at least
90% in the presence of the candidate molecule. In some such
embodiments, the candidate molecule is PSGL-1 antagonist.
[0201] Exemplary classes of candidate molecules include, but are
not limited to, antibodies, peptides, small molecules, and
aptamers. In some embodiments, a candidate molecule is an antibody
that is known to bind to VISTA (i.e., a VISTA antibody). In some
embodiments, a candidate molecule is an antibody that is known to
bind to PSGL-1 (i.e., PSGL-1 antibody).
[0202] In some embodiments, methods of determining whether a VISTA
antibody is a PSGL-1 antagonist are provided. In such embodiments,
the VISTA antibody is tested in the assays described above as the
candidate molecule. In some embodiments, methods of determining
whether a VISTA antibody blocks binding of PSGL-1 to VISTA, e.g.,
at acidic pH, e.g., pH<7.0, .ltoreq.6.8, .ltoreq.6.5 or
.ltoreq.6.3 or at pH 5.5 to 6.5, 6.0-6.5, 6.5-7.0 or 6.0-7.0, e.g.,
pH 6.0, are provided. Such methods comprise, in some embodiments,
contacting a VISTA antibody with a VISTA molecule and PSGL-1
molecule, and detecting binding of the VISTA molecule to the PSGL-1
molecule in the presence of the antibody, e.g., as described above
and herein.
Articles of Manufacture
[0203] In some embodiments, an article of manufacture or a kit
containing materials useful for the detection of a biomarker (e.g.,
PSGL-1 or VISTA) or for the treatment, prevention and/or diagnosis
of the disorders described above is provided. The article of
manufacture comprises a container and a label or package insert on
or associated with the container. Suitable containers include, for
example, bottles, vials, syringes, etc. The containers may be
formed from a variety of materials such as glass or plastic. In
some embodiments, the container holds a composition that is by
itself or combined with another composition effective for treating,
preventing and/or diagnosing the condition and may have a sterile
access port (for example the container may be an intravenous
solution bag or a vial having a stopper pierceable by a hypodermic
injection needle). The label or package insert indicates that the
composition is used for treating the condition of choice. In some
embodiments, the article of manufacture may comprise (a) a first
container with a composition contained therein, wherein the
composition comprises PSGL-1 antagonist of the invention; and (b) a
second container with a composition contained therein, wherein the
composition comprises an additional therapeutic agent. The article
of manufacture may further comprise a package insert indicating
that the compositions can be used to treat a particular condition.
Alternatively, or additionally, the article of manufacture may
further comprise a second (or third) container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
[0204] In some embodiments, the molecules of the present invention
can be packaged alone or in combination with other therapeutic
compounds as a kit. In one embodiment, the therapeutic compound is
an anti-cancer agent. In another embodiment, the therapeutic
compound is an immunosuppressive agent. The kit can include
optional components that aid in the administration of the unit dose
to patients, such as vials for reconstituting powder forms,
syringes for injection, customized IV delivery systems, inhalers,
etc. Additionally, the unit dose kit can contain instructions for
preparation and administration of the compositions. The kit may be
manufactured as a single use unit dose for one patient, multiple
uses for a particular patient (at a constant dose or in which the
individual compounds may vary in potency as therapy progresses); or
the kit may contain multiple doses suitable for administration to
multiple patients ("bulk packaging"). The kit components may be
assembled in cartons, blister packs, bottles, tubes, and the
like.
EXAMPLES
[0205] The examples discussed below are intended to be purely
exemplary of the invention and should not be considered to limit
the invention in any way. The examples are not intended to
represent that the experiments below are all or the only
experiments performed. Efforts have been made to ensure accuracy
with respect to numbers used (for example, amounts, temperature,
etc.) but some experimental errors and deviations should be
accounted for. Unless indicated otherwise, parts are parts by
weight, molecular weight is average molecular weight, temperature
is in degrees Centigrade, and pressure is at or near
atmospheric.
Example 1: The VISTA ECD is Histidine Rich and VISTA Dextramers
Bind Leukocytes Selectively at Acidic pH
[0206] The frequency of histidine residues within the extracellular
domains of immunoglobulin receptor superfamily (IgSF) members was
analyzed and VISTA was found to be exceptionally histidine-rich
relative to that of other receptors or ligands.
[0207] PBMC CD4+ T cells were isolated from blood by
RosetteSep.RTM. (Stem Cell Technologies) and stimulated for 3-4
days with a 1:1 ratio of anti-CD3/CD28 Dynabeads.RTM.
(ThermoFisher) and recombinant human IL-2. After stimulation, the
CD4+ T cells were washed and then incubated with
fluorescently-conjugated human VISTA dextramers (a streptavidin
dextramer loaded with an optimal molar ratio of monobiotinylated
recombinant human VISTA molecules) at a pH ranging between 6.97 and
5.99. Binding was detected by flow cytometry.
[0208] The results, which are shown in FIG. 1, indicate that VISTA
binds T cells selectively at acidic pH, rather than at neutral
pH.
Example 2: Identification of PSGL-1 as a VISTA Counter-Receptor at
Acidic pH
[0209] The LRC-TriCEPS.TM. technology was used to identify VISTA
receptor on T cells (Frei et al. (2013) Nat. Protoc. 8:1321; Frei
and Jeon (2012) Nat. Biotechnol. 30:997 and Omasits et al. (2014)
Bioinformatics 15:884). Human VISTA-Fc was coupled to TriCEPS and
incubated on primary human T cells at pH6.0. Anti-CD3 was also
coupled to the TriCEPS reagent and served as a positive control.
Several proteins were identified in the screen, some of which
appeared to bind only to the Fc portion of VISTA-Fc. Human PSGL-1
was identified as a protein binding to human VISTA. The screening
experiment was repeated one more time in the same conditions, and
PSGL-1 was identified again.
[0210] P-Selectin Glycoprotein Ligand 1 (PSGL-1, or SELPL) is a
ligand for P-, E-, and L-selectin, is heavily glycosylated and
tyrosine sulfated and expressed by most leukocytes, including T
cells. PSGL-1 engagement of selectins is critical to leukocyte
extravasation and trafficking.
Example 3: PSGL-1 Expression Correlates with VISTA Dextramer
Binding
[0211] PBMC were isolated from blood by ficoll-paque gradient
centrifugation, washed, and then incubated in Hank's Balanced Salt
Solution (HBSS) at pH 7.2 with fluorescently-conjugated anti-PSGL-1
antibodies or at pH 6.0 with fluorescently-conjugated human VISTA
dextramers (a streptavidin dextramer loaded with an optimal molar
ratio of monobiotinylated recombinant human VISTA molecules).
Binding was detected by flow cytometry.
[0212] The results, which are shown in FIG. 2A-B, indicate that
PSGL-1 surface expression on the PBMC lymphocytes correlates with
VISTA dextramer binding at acidic pH.
Example 4: Soluble PSGL-1 and P-Selectin Block VISTA Dextramer
Binding to Activated CD4+ T Cells
[0213] The ability of soluble PSGL-1 to disrupt VISTA dextramer
binding to T cells was assessed by incubating activated T cells
with fluorescently-conjugated VISTA dextramers (a streptavidin
dextramer loaded with a sub-optimal molar ratio of monobiotinylated
recombinant human VISTA molecules) in Hanks's Balanced Salt
Solution at pH 6.0 and in the presence of titrated concentrations
of a control (i.e., non-VISTA or PSGL-1 specific) antibody (dots),
recombinant human PSGL-1-Fc fusion protein (triangles), or
recombinant human P-selectin (R&D Systems, diamonds). The
recombinant human PSGL-1 Fc fusion molecule was obtained from
R&D Systems Catalog No. 3345-PS, and comprises the PSGL-1 ECD
sequence shown in SEQ ID NO: 18, comprising positions 42-295 of a
human PSGL-1 sequence of accession number AA50061 coupled at its
C-terminal to an IEGRMD linker sequence (SEQ ID NO: 19) followed by
amino acids P100 to K330 of human IgG1. After incubation, the
binding of the VISTA dextramer to the T cells was measured by flow
cytometry. IC50 values were calculated via nonlinear regression in
Prism software (GraphPad). The results, which are shown in FIG. 3
and Table 1, indicate that PSGL-1 and P-selectin inhibit binding of
VISTA to the activated CD4+ T cells in a dose dependent manner at
acidic pH.
TABLE-US-00001 TABLE 1 Agent IC50 (nM) Control antibody --
PSGL-1-Fc 20.54 p-selectin 100.8
Example 5: PSGL-1-Fc Binds to Cell Surface-Expressed VISTA at
Acidic pH
[0214] The binding of humPSGL-1-Fc to 293 T cells ectopically
expressing human VISTA was tested. 293T cells expressing human
VISTA and GFP (dots and squares) were cultured as well as 293T
cells expressing neither VISTA nor GFP (diamonds and triangles).
The cells were washed and then incubated with PSGL-1-Fc fusion
protein (R&D Systems; Cat. No. 3345) in Hanks's Balanced Salt
Solution at either pH 7.2 (triangles and squares) or pH 6.0
(diamonds and dots curves). After primary incubation, cells were
washed and incubated with fluorescently-conjugated anti-hIgG
secondary detection antibodies. After secondary incubation, cells
were washed and binding was detected by flow cytometry. The
results, which are shown in FIGS. 4A and B, indicate that
humPSGL-1-Fc binds to 293 T cells expressing human VISTA (hVISTA),
but not to 293 T cells that do not express human VISTA. In
addition, the results indicate that the binding of PSGL-1-Fc is pH
dependent, as it binds at pH 6.0 (circles/dots in FIG. 4B and right
curve in FIG. 4A), but not significantly at pH 7.2 (squares in FIG.
4B and left curves in FIG. 4A).
Example 6: PBMC CD4 T cell PSGL-1 CRISPR Ablates VISTA Dextramer
Binding
[0215] Human CD4 T cells were isolated from whole blood and
activated for 2 days with plated coated OKT3 & CD28.2. The T
cells were then transfected with Cas9 ribonuclear proteins (RNPs)
loaded with guide RNAs targeting either CD4, PSGL1, or a gRNA with
no human sequence homology (non-targeting control). Transfections
were done in triplicate. Following transfection, the cells were
reactivated with CD3/CD28 coated Dynabeads.RTM. for 4 days. The
cells were then stained with dextramer loaded with recombinant
human avi-tagged VISTA to assess binding to VISTA. Percent of
maximum VISTA binding was determined by dividing the average VISTA
dextramer mean fluorescence intensity (MFI) of the knock-out
population by the average VISTA dextramer MFI of the non-targeting
control.
[0216] The results are shown in FIG. 5, and indicate that VISTA
binding to T cells was reduced by about half in T cells in which
PSGL-1 was ablated. These results further suggest that PSGL-1 is a
VISTA counter-receptor on T cells.
TABLE-US-00002 TABLE OF SEQUENCES SEQ ID NO Description Sequence 1
HumPSGL-1 isoform 1 MAVGASGLEG DKMAGAMPLQ LLLLLILLGP GNSLQLWDTW
precursor, with signal ADEAEKALGP LLARDRRQAT EYEYLDYDFL PETEPPEMLR
peptide NSTDTTPLTG PGTPESTTVE PAARRSTGLD AGGAVTELTT NP_001193538
ELANMGNLST DSAAMEIQTT QPAATEAQTT QPVPTEAQTT PLAATEAQTT RLTATEAQTT
PLAATEAQTT PPAATEAQTT QPTGLEAQTT APAAMEAQTT APAAMEAQTT PPAAMEAQTT
QTTAMEAQTT APEATEAQTT QPTATEAQTT PLAAMEALST EPSATEALSM EPTTKRGLFI
PFSVSSVTHK GIPMAASNLS VNYPVGAPDH ISVKQCLLAI LILALVATIF FVCTVVLAVR
LSRKGHMYPV RNYSPTEMVC ISSLLPDGGE GPSATANGGL SKAKSPGLTP EPREDREGDD
LTLHSFLP 2 Human PSGL-1, without LQLWDTW ADEAEKALGP LLARDRRQAT
EYEYLDYDFL signal peptide PETEPPEMLR NSTDTTPLTG PGTPESTTVE
PAARRSTGLD AGGAVTELTT ELANMGNLST DSAAMEIQTT QPAATEAQTT QPVPTEAQTT
PLAATEAQTT RLTATEAQTT PLAATEAQTT PPAATEAQTT QPTGLEAQTT APAAMEAQTT
APAAMEAQTT PPAAMEAQTT QTTAMEAQTT APEATEAQTT QPTATEAQTT PLAAMEALST
EPSATEALSM EPTTKRGLFI PFSVSSVTHK GIPMAASNLS VNYPVGAPDH ISVKQCLLAI
LILALVATIF FVCTVVLAVR LSRKGHMYPV RNYSPTEMVC ISSLLPDGGE GPSATANGGL
SKAKSPGLTP EPREDREGDD LTLHSFLP 3 Human PSGL-1 ECD, MAVGASGLEG
DKMAGAMPLQ LLLLLILLGP GNSLQLWDTW with signal peptide ADEAEKALGP
LLARDRRQAT EYEYLDYDFL PETEPPEMLR NSTDTTPLTG PGTPESTTVE PAARRSTGLD
AGGAVTELTT ELANMGNLST DSAAMEIQTT QPAATEAQTT QPVPTEAQTT PLAATEAQTT
RLTATEAQTT PLAATEAQTT PPAATEAQTT QPTGLEAQTT APAAMEAQTT APAAMEAQTT
PPAAMEAQTT QT 4 Human PSGL-1 ECD LQLWDTW ADEAEKALGP LLARDRRQAT
EYEYLDYDFL without signal peptide PETEPPEMLR NSTDTTPLTG PGTPESTTVE
PAARRSTGLD AGGAVTELTT ELANMGNLST DSAAMEIQTT QPAATEAQTT QPVPTEAQTT
PLAATEAQTT RLTATEAQTT PLAATEAQTT PPAATEAQTT QPTGLEAQTT APAAMEAQTT
APAAMEAQTT PPAAMEAQTT QT 5 Human VISTA precursor, MGVPTALEAG
SWRWGSLLFA LFLAASLGPV AAFKVATPYS with signal peptide LYVCPEGQNV
TLTCRLLGPV DKGHDVTFYK TWYRSSRGEV (UniProtKB Ref. QTCSERRPIR
NLTFQDLHLH HGGHQAANTS HDLAQRHGLE Q9H7M9, 19 Feb. 2014) SASDHHGNFS
ITMRNLTLLD SGLYCCLVVE IRHHHSEHRV HGAMELQVQT GKDAPSNCVV YPSSSQDSEN
ITAAALATGA CIVGILCLPL ILLLVYKQRQ AASNRRAQEL VRMDSNIQGI ENPGFEASPP
AQGIPEAKVR HPLSYVAQRQ PSESGRHLLS EPSTPLSPPG PGDVFFPSLD PVPDSPNFEV I
6 Human mature VISTA, FKVATPYS LYVCPEGQNV TLTCRLLGPV DKGHDVTFYK
without signal peptide TWYRSSRGEV QTCSERRPIR NLTFQDLHLH HGGHQAANTS
HDLAQRHGLE SASDHHGNFS ITMRNLTLLD SGLYCCLVVE IRHHHSEHRV HGAMELQVQT
GKDAPSNCVV YPSSSQDSEN ITAAALATGA CIVGILCLPL ILLLVYKQRQ AASNRRAQEL
VRMDSNIQGI ENPGFEASPP AQGIPEAKVR HPLSYVAQRQ PSESGRHLLS EPSTPLSPPG
PGDVFFPSLD PVPDSPNFEV I 7 Mouse VISTA precursor, MGVPAVPEAS
SPRWGTLLLA IFLAASRGLV AAFKVTTPYS with signal peptide LYVCPEGQNA
TLTCRILGPV SKGHDVTIYK TWYLSSRGEV (NCBI Ref QMCKEHRPIR NFTLQHLQHH
GSHLKANASH DQPQKHGLEL NP_083008.1, 26 Feb. ASDHHGNFSI TLRNVTPRDS
GLYCCLVIEL KNHHPEQRFY 2014) GSMELQVQAG KGSGSTCMAS NEQDSDSITA
AALATGACIV GILCLPLILL LVYKQRQVAS HRRAQELVRM DSSNTQGIEN PGFETTPPFQ
GMPEAKTRPP LSYVAQRQPS ESGRYLLSDP STPLSPPGPG DVFFPSLDPV PDSPNSEAI 8
Mouse mature VISTA, FKVTTPYS LYVCPEGQNA TLTCRILGPV SKGHDVTIYK
without signal peptide TWYLSSRGEV QMCKEHRPIR NFTLQHLQHH GSHLKANASH
DQPQKHGLEL ASDHHGNFSI TLRNVTPRDS GLYCCLVIEL KNHHPEQRFY GSMELQVQAG
KGSGSTCMAS NEQDSDSITA AALATGACIV GILCLPLILL LVYKQRQVAS HRRAQELVRM
DSSNTQGIEN PGFETTPPFQ GMPEAKTRPP LSYVAQRQPS ESGRYLLSDP STPLSPPGPG
DVFFPSLDPV PDSPNSEAI 9 Human VISTA ECD-Fc, FKVATPYSLY VCPEGQNVTL
TCRLLGPVDK GHDVTFYKTW without signal peptide YRSSRGEVQT CSERRPIRNL
TFQDLHLHHG GHQAANTSHD LAQRHGLESA SDHHGNFSIT MRNLTLLDSG LYCCLVVEIR
HHHSEHRVHG AMELQVQTGK DAPSNCVVYP SSSQDSENIT AAAGTSGSSG SGSGGSGSGG
GGRSVPRDSG CKPCICTVPE VSSVFIFPPK PKDVLTITLT PKVTCVVVDI SKDDPEVQFS
WFVDDVEVHT AQTKPREEQI NSTFRSVSEL PIMHQDWLNG KEFKCRVNSA AFPAPIEKTI
SKTKGRPKAP QVYTIPPPKE QMAKDKVSLT CMITNFFPED ITVEWQWNGQ PAENYKNTQP
IMDTDGSYFV YSKLNVQKSN WEAGNTFTCS VLHEGLHNHH TEKSLSHSPG K 10
His-tagged human FKVATPYS LYVCPEGQNV TLTCRLLGPV DKGHDVTFYK VISTA
extracellular TWYRSSRGEV QTCSERRPIR NLTFQDLHLH HGGHQAANTS domain
(ECD) HDLAQRHGLE SASDHHGNFS ITMRNLTLLD SGLYCCLVVE IRHHHSEHRV
HGAMELQVQT GKDAPSNCVV YPSSSQDSEN ITAAALATGA CIVGILCLPL ILLLVYKQRQ
AASNRRAQEL VRMDSNIQGI ENPGFEASPP AQGIPEAKVR HPLSYVAQRQ PSESGRHLLS
EPSTPLSPPG PGDVFFPSLD PVPDSPNFEV IGHHHHHH 11 Fc C237S EPKSSDKTHT
CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF NWYVDGVEVH
NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN KALPAPIEKT ISKAKGQPRE
PQVYTLPPSR DELTKNQVSL TCLVKGFYPS DIAVEWESNG QPENNYKTTP PVLDSDGSFF
LYSKLTVDKS RWQQGNVFSC SVMHEALHNH YTQKSLSLSP GK 12 Exemplary Fc #1
ERKCCVECPP CPAPPVAGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE DPEVQFNWYV
DGVEVHNAKT KPREEQFNST FRVVSVLTVV HQDWLNGKEY KCKVSNKGLP APIEKTISKT
KGQPREPQVY TLPPSREEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPMLD
SDGSFFLYSK LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGK 13 Exemplary
Fc #2 ESKYGPPCPS CPAPEFLGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSQ
EDPEVQFNWY VDGVEVHNAK TKPREEQFNS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL
PSSIEKTISK AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE
NNYKTTPPVL DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 14
Human PSGL-1 isoform MPLQLLLLLI LLGPGNSLQL WDTWADEAEK ALGPLLARDR 2
precursor, with signal RQATEYEYLD YDFLPETEPP EMLRNSTDTT PLTGPGTPES
peptide TTVEPAARRS TGLDAGGAVT ELTTELANMG NLSTDSAAME IQTTQPAATE
AQTTQPVPTE AQTTPLAATE AQTTRLTATE AQTTPLAATE AQTTPPAATE AQTTQPTGLE
AQTTAPAAME AQTTAPAAME AQTTPPAAME AQTTQTTAME AQTTAPEATE AQTTQPTATE
AQTTPLAAME ALSTEPSATE ALSMEPTTKR GLFIPFSVSS VTHKGIPMAA SNLSVNYPVG
APDHISVKQC LLAILILALV ATIFFVCTVV LAVRLSRKGH MYPVRNYSPT EMVCISSLLP
DGGEGPSATA NGGLSKAKSP GLTPEPREDR EGDDLTLHSF LP 15 Human PSGL-1
isoform LQL WDTWADEAEK ALGPLLARDR 2, without signal peptide
RQATEYEYLD YDFLPETEPP EMLRNSTDTT PLTGPGTPES TTVEPAARRS TGLDAGGAVT
ELTTELANMG NLSTDSAAME IQTTQPAATE AQTTQPVPTE AQTTPLAATE AQTTRLTATE
AQTTPLAATE AQTTPPAATE AQTTQPTGLE AQTTAPAAME AQTTAPAAME AQTTPPAAME
AQTTQTTAME AQTTAPEATE AQTTQPTATE AQTTPLAAME ALSTEPSATE ALSMEPTTKR
GLFIPFSVSS VTHKGIPMAA SNLSVNYPVG APDHISVKQC LLAILILALV ATIFFVCTVV
LAVRLSRKGH MYPVRNYSPT EMVCISSLLP DGGEGPSATA NGGLSKAKSP GLTPEPREDR
EGDDLTLHSF LP 16 Human PSGL-1 isoform MPLQLLLLLI LLGPGNSLQL
WDTWADEAEK ALGPLLARDR 2 ECD with signal RQATEYEYLD YDFLPETEPP
EMLRNSTDTT PLTGPGTPES peptide TTVEPAARRS TGLDAGGAVT ELTTELANMG
NLSTDSAAME IQTTQPAATE AQTTQPVPTE AQTTPLAATE AQTTRLTATE AQTTPLAATE
AQTTPPAATE AQTTQPTGLE AQTTAPAAME AQTTAPAAME AQTTPPAAME AQTTQT 17
Human PSGL-1 isoform LQL WDTWADEAEK ALGPLLARDR RQATEYEYLD 2 ECD,
without signal YDFLPETEPP EMLRNSTDTT PLTGPGTPES TTVEPAARRS peptide
TGLDAGGAVT ELTTELANMG NLSTDSAAME IQTTQPAATE AQTTQPVPTE AQTTPLAATE
AQTTRLTATE AQTTPLAATE AQTTPPAATE AQTTQPTGLE AQTTAPAAME AQTTAPAAME
AQTTPPAAME AQTTQT 18 Human PSGL-1 ECD(N- QATEYEYLD YDFLPETEPP
EMLRNSTDTT PLTGPGTPES terminal positions 42 to TTVEPAARRS
TGLDAGGAVT ELTTELANMG NLSTDSAAME 295 of a full length IQTTQPAATE
AQTTPLAATE AQTTRLTATE AQTTPLAATE Human PSGL-1 AQTTPPAATE AQTTQPTGLE
AQTTAPAAME AQTTAPAAME Accession No. AQTTPPAAME AQTTQTTAME
AQTTAPEATE AQTTQPTATE AAC50061) AQTTPLAAME ALSTEPSATE ALSMEPTTKR
GLFIPFSVSS VTHKGIPMAA SNLSV 19 Exemplary fusion protein IEGRMD
linker sequence
Sequence CWU 1
1
191428PRTHomo sapiens 1Met Ala Val Gly Ala Ser Gly Leu Glu Gly Asp
Lys Met Ala Gly Ala1 5 10 15Met Pro Leu Gln Leu Leu Leu Leu Leu Ile
Leu Leu Gly Pro Gly Asn 20 25 30Ser Leu Gln Leu Trp Asp Thr Trp Ala
Asp Glu Ala Glu Lys Ala Leu 35 40 45Gly Pro Leu Leu Ala Arg Asp Arg
Arg Gln Ala Thr Glu Tyr Glu Tyr 50 55 60Leu Asp Tyr Asp Phe Leu Pro
Glu Thr Glu Pro Pro Glu Met Leu Arg65 70 75 80Asn Ser Thr Asp Thr
Thr Pro Leu Thr Gly Pro Gly Thr Pro Glu Ser 85 90 95Thr Thr Val Glu
Pro Ala Ala Arg Arg Ser Thr Gly Leu Asp Ala Gly 100 105 110Gly Ala
Val Thr Glu Leu Thr Thr Glu Leu Ala Asn Met Gly Asn Leu 115 120
125Ser Thr Asp Ser Ala Ala Met Glu Ile Gln Thr Thr Gln Pro Ala Ala
130 135 140Thr Glu Ala Gln Thr Thr Gln Pro Val Pro Thr Glu Ala Gln
Thr Thr145 150 155 160Pro Leu Ala Ala Thr Glu Ala Gln Thr Thr Arg
Leu Thr Ala Thr Glu 165 170 175Ala Gln Thr Thr Pro Leu Ala Ala Thr
Glu Ala Gln Thr Thr Pro Pro 180 185 190Ala Ala Thr Glu Ala Gln Thr
Thr Gln Pro Thr Gly Leu Glu Ala Gln 195 200 205Thr Thr Ala Pro Ala
Ala Met Glu Ala Gln Thr Thr Ala Pro Ala Ala 210 215 220Met Glu Ala
Gln Thr Thr Pro Pro Ala Ala Met Glu Ala Gln Thr Thr225 230 235
240Gln Thr Thr Ala Met Glu Ala Gln Thr Thr Ala Pro Glu Ala Thr Glu
245 250 255Ala Gln Thr Thr Gln Pro Thr Ala Thr Glu Ala Gln Thr Thr
Pro Leu 260 265 270Ala Ala Met Glu Ala Leu Ser Thr Glu Pro Ser Ala
Thr Glu Ala Leu 275 280 285Ser Met Glu Pro Thr Thr Lys Arg Gly Leu
Phe Ile Pro Phe Ser Val 290 295 300Ser Ser Val Thr His Lys Gly Ile
Pro Met Ala Ala Ser Asn Leu Ser305 310 315 320Val Asn Tyr Pro Val
Gly Ala Pro Asp His Ile Ser Val Lys Gln Cys 325 330 335Leu Leu Ala
Ile Leu Ile Leu Ala Leu Val Ala Thr Ile Phe Phe Val 340 345 350Cys
Thr Val Val Leu Ala Val Arg Leu Ser Arg Lys Gly His Met Tyr 355 360
365Pro Val Arg Asn Tyr Ser Pro Thr Glu Met Val Cys Ile Ser Ser Leu
370 375 380Leu Pro Asp Gly Gly Glu Gly Pro Ser Ala Thr Ala Asn Gly
Gly Leu385 390 395 400Ser Lys Ala Lys Ser Pro Gly Leu Thr Pro Glu
Pro Arg Glu Asp Arg 405 410 415Glu Gly Asp Asp Leu Thr Leu His Ser
Phe Leu Pro 420 4252395PRTHomo sapiens 2Leu Gln Leu Trp Asp Thr Trp
Ala Asp Glu Ala Glu Lys Ala Leu Gly1 5 10 15Pro Leu Leu Ala Arg Asp
Arg Arg Gln Ala Thr Glu Tyr Glu Tyr Leu 20 25 30Asp Tyr Asp Phe Leu
Pro Glu Thr Glu Pro Pro Glu Met Leu Arg Asn 35 40 45Ser Thr Asp Thr
Thr Pro Leu Thr Gly Pro Gly Thr Pro Glu Ser Thr 50 55 60Thr Val Glu
Pro Ala Ala Arg Arg Ser Thr Gly Leu Asp Ala Gly Gly65 70 75 80Ala
Val Thr Glu Leu Thr Thr Glu Leu Ala Asn Met Gly Asn Leu Ser 85 90
95Thr Asp Ser Ala Ala Met Glu Ile Gln Thr Thr Gln Pro Ala Ala Thr
100 105 110Glu Ala Gln Thr Thr Gln Pro Val Pro Thr Glu Ala Gln Thr
Thr Pro 115 120 125Leu Ala Ala Thr Glu Ala Gln Thr Thr Arg Leu Thr
Ala Thr Glu Ala 130 135 140Gln Thr Thr Pro Leu Ala Ala Thr Glu Ala
Gln Thr Thr Pro Pro Ala145 150 155 160Ala Thr Glu Ala Gln Thr Thr
Gln Pro Thr Gly Leu Glu Ala Gln Thr 165 170 175Thr Ala Pro Ala Ala
Met Glu Ala Gln Thr Thr Ala Pro Ala Ala Met 180 185 190Glu Ala Gln
Thr Thr Pro Pro Ala Ala Met Glu Ala Gln Thr Thr Gln 195 200 205Thr
Thr Ala Met Glu Ala Gln Thr Thr Ala Pro Glu Ala Thr Glu Ala 210 215
220Gln Thr Thr Gln Pro Thr Ala Thr Glu Ala Gln Thr Thr Pro Leu
Ala225 230 235 240Ala Met Glu Ala Leu Ser Thr Glu Pro Ser Ala Thr
Glu Ala Leu Ser 245 250 255Met Glu Pro Thr Thr Lys Arg Gly Leu Phe
Ile Pro Phe Ser Val Ser 260 265 270Ser Val Thr His Lys Gly Ile Pro
Met Ala Ala Ser Asn Leu Ser Val 275 280 285Asn Tyr Pro Val Gly Ala
Pro Asp His Ile Ser Val Lys Gln Cys Leu 290 295 300Leu Ala Ile Leu
Ile Leu Ala Leu Val Ala Thr Ile Phe Phe Val Cys305 310 315 320Thr
Val Val Leu Ala Val Arg Leu Ser Arg Lys Gly His Met Tyr Pro 325 330
335Val Arg Asn Tyr Ser Pro Thr Glu Met Val Cys Ile Ser Ser Leu Leu
340 345 350Pro Asp Gly Gly Glu Gly Pro Ser Ala Thr Ala Asn Gly Gly
Leu Ser 355 360 365Lys Ala Lys Ser Pro Gly Leu Thr Pro Glu Pro Arg
Glu Asp Arg Glu 370 375 380Gly Asp Asp Leu Thr Leu His Ser Phe Leu
Pro385 390 3953242PRTArtificial sequenceHumPSGL-1 ECD, with signal
peptide 3Met Ala Val Gly Ala Ser Gly Leu Glu Gly Asp Lys Met Ala
Gly Ala1 5 10 15Met Pro Leu Gln Leu Leu Leu Leu Leu Ile Leu Leu Gly
Pro Gly Asn 20 25 30Ser Leu Gln Leu Trp Asp Thr Trp Ala Asp Glu Ala
Glu Lys Ala Leu 35 40 45Gly Pro Leu Leu Ala Arg Asp Arg Arg Gln Ala
Thr Glu Tyr Glu Tyr 50 55 60Leu Asp Tyr Asp Phe Leu Pro Glu Thr Glu
Pro Pro Glu Met Leu Arg65 70 75 80Asn Ser Thr Asp Thr Thr Pro Leu
Thr Gly Pro Gly Thr Pro Glu Ser 85 90 95Thr Thr Val Glu Pro Ala Ala
Arg Arg Ser Thr Gly Leu Asp Ala Gly 100 105 110Gly Ala Val Thr Glu
Leu Thr Thr Glu Leu Ala Asn Met Gly Asn Leu 115 120 125Ser Thr Asp
Ser Ala Ala Met Glu Ile Gln Thr Thr Gln Pro Ala Ala 130 135 140Thr
Glu Ala Gln Thr Thr Gln Pro Val Pro Thr Glu Ala Gln Thr Thr145 150
155 160Pro Leu Ala Ala Thr Glu Ala Gln Thr Thr Arg Leu Thr Ala Thr
Glu 165 170 175Ala Gln Thr Thr Pro Leu Ala Ala Thr Glu Ala Gln Thr
Thr Pro Pro 180 185 190Ala Ala Thr Glu Ala Gln Thr Thr Gln Pro Thr
Gly Leu Glu Ala Gln 195 200 205Thr Thr Ala Pro Ala Ala Met Glu Ala
Gln Thr Thr Ala Pro Ala Ala 210 215 220Met Glu Ala Gln Thr Thr Pro
Pro Ala Ala Met Glu Ala Gln Thr Thr225 230 235 240Gln
Thr4209PRTArtificial sequenceHumPSGL-1 ECD without signal peptide
4Leu Gln Leu Trp Asp Thr Trp Ala Asp Glu Ala Glu Lys Ala Leu Gly1 5
10 15Pro Leu Leu Ala Arg Asp Arg Arg Gln Ala Thr Glu Tyr Glu Tyr
Leu 20 25 30Asp Tyr Asp Phe Leu Pro Glu Thr Glu Pro Pro Glu Met Leu
Arg Asn 35 40 45Ser Thr Asp Thr Thr Pro Leu Thr Gly Pro Gly Thr Pro
Glu Ser Thr 50 55 60Thr Val Glu Pro Ala Ala Arg Arg Ser Thr Gly Leu
Asp Ala Gly Gly65 70 75 80Ala Val Thr Glu Leu Thr Thr Glu Leu Ala
Asn Met Gly Asn Leu Ser 85 90 95Thr Asp Ser Ala Ala Met Glu Ile Gln
Thr Thr Gln Pro Ala Ala Thr 100 105 110Glu Ala Gln Thr Thr Gln Pro
Val Pro Thr Glu Ala Gln Thr Thr Pro 115 120 125Leu Ala Ala Thr Glu
Ala Gln Thr Thr Arg Leu Thr Ala Thr Glu Ala 130 135 140Gln Thr Thr
Pro Leu Ala Ala Thr Glu Ala Gln Thr Thr Pro Pro Ala145 150 155
160Ala Thr Glu Ala Gln Thr Thr Gln Pro Thr Gly Leu Glu Ala Gln Thr
165 170 175Thr Ala Pro Ala Ala Met Glu Ala Gln Thr Thr Ala Pro Ala
Ala Met 180 185 190Glu Ala Gln Thr Thr Pro Pro Ala Ala Met Glu Ala
Gln Thr Thr Gln 195 200 205Thr5311PRTHomo sapiens 5Met Gly Val Pro
Thr Ala Leu Glu Ala Gly Ser Trp Arg Trp Gly Ser1 5 10 15Leu Leu Phe
Ala Leu Phe Leu Ala Ala Ser Leu Gly Pro Val Ala Ala 20 25 30Phe Lys
Val Ala Thr Pro Tyr Ser Leu Tyr Val Cys Pro Glu Gly Gln 35 40 45Asn
Val Thr Leu Thr Cys Arg Leu Leu Gly Pro Val Asp Lys Gly His 50 55
60Asp Val Thr Phe Tyr Lys Thr Trp Tyr Arg Ser Ser Arg Gly Glu Val65
70 75 80Gln Thr Cys Ser Glu Arg Arg Pro Ile Arg Asn Leu Thr Phe Gln
Asp 85 90 95Leu His Leu His His Gly Gly His Gln Ala Ala Asn Thr Ser
His Asp 100 105 110Leu Ala Gln Arg His Gly Leu Glu Ser Ala Ser Asp
His His Gly Asn 115 120 125Phe Ser Ile Thr Met Arg Asn Leu Thr Leu
Leu Asp Ser Gly Leu Tyr 130 135 140Cys Cys Leu Val Val Glu Ile Arg
His His His Ser Glu His Arg Val145 150 155 160His Gly Ala Met Glu
Leu Gln Val Gln Thr Gly Lys Asp Ala Pro Ser 165 170 175Asn Cys Val
Val Tyr Pro Ser Ser Ser Gln Asp Ser Glu Asn Ile Thr 180 185 190Ala
Ala Ala Leu Ala Thr Gly Ala Cys Ile Val Gly Ile Leu Cys Leu 195 200
205Pro Leu Ile Leu Leu Leu Val Tyr Lys Gln Arg Gln Ala Ala Ser Asn
210 215 220Arg Arg Ala Gln Glu Leu Val Arg Met Asp Ser Asn Ile Gln
Gly Ile225 230 235 240Glu Asn Pro Gly Phe Glu Ala Ser Pro Pro Ala
Gln Gly Ile Pro Glu 245 250 255Ala Lys Val Arg His Pro Leu Ser Tyr
Val Ala Gln Arg Gln Pro Ser 260 265 270Glu Ser Gly Arg His Leu Leu
Ser Glu Pro Ser Thr Pro Leu Ser Pro 275 280 285Pro Gly Pro Gly Asp
Val Phe Phe Pro Ser Leu Asp Pro Val Pro Asp 290 295 300Ser Pro Asn
Phe Glu Val Ile305 3106279PRTHomo sapiens 6Phe Lys Val Ala Thr Pro
Tyr Ser Leu Tyr Val Cys Pro Glu Gly Gln1 5 10 15Asn Val Thr Leu Thr
Cys Arg Leu Leu Gly Pro Val Asp Lys Gly His 20 25 30Asp Val Thr Phe
Tyr Lys Thr Trp Tyr Arg Ser Ser Arg Gly Glu Val 35 40 45Gln Thr Cys
Ser Glu Arg Arg Pro Ile Arg Asn Leu Thr Phe Gln Asp 50 55 60Leu His
Leu His His Gly Gly His Gln Ala Ala Asn Thr Ser His Asp65 70 75
80Leu Ala Gln Arg His Gly Leu Glu Ser Ala Ser Asp His His Gly Asn
85 90 95Phe Ser Ile Thr Met Arg Asn Leu Thr Leu Leu Asp Ser Gly Leu
Tyr 100 105 110Cys Cys Leu Val Val Glu Ile Arg His His His Ser Glu
His Arg Val 115 120 125His Gly Ala Met Glu Leu Gln Val Gln Thr Gly
Lys Asp Ala Pro Ser 130 135 140Asn Cys Val Val Tyr Pro Ser Ser Ser
Gln Asp Ser Glu Asn Ile Thr145 150 155 160Ala Ala Ala Leu Ala Thr
Gly Ala Cys Ile Val Gly Ile Leu Cys Leu 165 170 175Pro Leu Ile Leu
Leu Leu Val Tyr Lys Gln Arg Gln Ala Ala Ser Asn 180 185 190Arg Arg
Ala Gln Glu Leu Val Arg Met Asp Ser Asn Ile Gln Gly Ile 195 200
205Glu Asn Pro Gly Phe Glu Ala Ser Pro Pro Ala Gln Gly Ile Pro Glu
210 215 220Ala Lys Val Arg His Pro Leu Ser Tyr Val Ala Gln Arg Gln
Pro Ser225 230 235 240Glu Ser Gly Arg His Leu Leu Ser Glu Pro Ser
Thr Pro Leu Ser Pro 245 250 255Pro Gly Pro Gly Asp Val Phe Phe Pro
Ser Leu Asp Pro Val Pro Asp 260 265 270Ser Pro Asn Phe Glu Val Ile
2757309PRTMus musculus 7Met Gly Val Pro Ala Val Pro Glu Ala Ser Ser
Pro Arg Trp Gly Thr1 5 10 15Leu Leu Leu Ala Ile Phe Leu Ala Ala Ser
Arg Gly Leu Val Ala Ala 20 25 30Phe Lys Val Thr Thr Pro Tyr Ser Leu
Tyr Val Cys Pro Glu Gly Gln 35 40 45Asn Ala Thr Leu Thr Cys Arg Ile
Leu Gly Pro Val Ser Lys Gly His 50 55 60Asp Val Thr Ile Tyr Lys Thr
Trp Tyr Leu Ser Ser Arg Gly Glu Val65 70 75 80Gln Met Cys Lys Glu
His Arg Pro Ile Arg Asn Phe Thr Leu Gln His 85 90 95Leu Gln His His
Gly Ser His Leu Lys Ala Asn Ala Ser His Asp Gln 100 105 110Pro Gln
Lys His Gly Leu Glu Leu Ala Ser Asp His His Gly Asn Phe 115 120
125Ser Ile Thr Leu Arg Asn Val Thr Pro Arg Asp Ser Gly Leu Tyr Cys
130 135 140Cys Leu Val Ile Glu Leu Lys Asn His His Pro Glu Gln Arg
Phe Tyr145 150 155 160Gly Ser Met Glu Leu Gln Val Gln Ala Gly Lys
Gly Ser Gly Ser Thr 165 170 175Cys Met Ala Ser Asn Glu Gln Asp Ser
Asp Ser Ile Thr Ala Ala Ala 180 185 190Leu Ala Thr Gly Ala Cys Ile
Val Gly Ile Leu Cys Leu Pro Leu Ile 195 200 205Leu Leu Leu Val Tyr
Lys Gln Arg Gln Val Ala Ser His Arg Arg Ala 210 215 220Gln Glu Leu
Val Arg Met Asp Ser Ser Asn Thr Gln Gly Ile Glu Asn225 230 235
240Pro Gly Phe Glu Thr Thr Pro Pro Phe Gln Gly Met Pro Glu Ala Lys
245 250 255Thr Arg Pro Pro Leu Ser Tyr Val Ala Gln Arg Gln Pro Ser
Glu Ser 260 265 270Gly Arg Tyr Leu Leu Ser Asp Pro Ser Thr Pro Leu
Ser Pro Pro Gly 275 280 285Pro Gly Asp Val Phe Phe Pro Ser Leu Asp
Pro Val Pro Asp Ser Pro 290 295 300Asn Ser Glu Ala Ile3058277PRTMus
musculus 8Phe Lys Val Thr Thr Pro Tyr Ser Leu Tyr Val Cys Pro Glu
Gly Gln1 5 10 15Asn Ala Thr Leu Thr Cys Arg Ile Leu Gly Pro Val Ser
Lys Gly His 20 25 30Asp Val Thr Ile Tyr Lys Thr Trp Tyr Leu Ser Ser
Arg Gly Glu Val 35 40 45Gln Met Cys Lys Glu His Arg Pro Ile Arg Asn
Phe Thr Leu Gln His 50 55 60Leu Gln His His Gly Ser His Leu Lys Ala
Asn Ala Ser His Asp Gln65 70 75 80Pro Gln Lys His Gly Leu Glu Leu
Ala Ser Asp His His Gly Asn Phe 85 90 95Ser Ile Thr Leu Arg Asn Val
Thr Pro Arg Asp Ser Gly Leu Tyr Cys 100 105 110Cys Leu Val Ile Glu
Leu Lys Asn His His Pro Glu Gln Arg Phe Tyr 115 120 125Gly Ser Met
Glu Leu Gln Val Gln Ala Gly Lys Gly Ser Gly Ser Thr 130 135 140Cys
Met Ala Ser Asn Glu Gln Asp Ser Asp Ser Ile Thr Ala Ala Ala145 150
155 160Leu Ala Thr Gly Ala Cys Ile Val Gly Ile Leu Cys Leu Pro Leu
Ile 165 170 175Leu Leu Leu Val Tyr Lys Gln Arg Gln Val Ala Ser His
Arg Arg Ala 180 185 190Gln Glu Leu Val Arg Met Asp Ser Ser Asn Thr
Gln Gly Ile Glu Asn 195 200 205Pro Gly Phe Glu Thr Thr Pro Pro Phe
Gln Gly Met Pro Glu Ala Lys 210 215 220Thr Arg Pro Pro Leu Ser Tyr
Val Ala Gln Arg Gln Pro Ser Glu Ser225 230 235 240Gly Arg Tyr Leu
Leu Ser Asp Pro Ser Thr Pro Leu Ser Pro Pro Gly 245 250 255Pro Gly
Asp Val Phe Phe Pro Ser Leu Asp Pro Val Pro Asp Ser Pro 260 265
270Asn Ser Glu Ala Ile 2759411PRTArtificial
sequenceHuman VISTA ECD-Fc, without signal peptide 9Phe Lys Val Ala
Thr Pro Tyr Ser Leu Tyr Val Cys Pro Glu Gly Gln1 5 10 15Asn Val Thr
Leu Thr Cys Arg Leu Leu Gly Pro Val Asp Lys Gly His 20 25 30Asp Val
Thr Phe Tyr Lys Thr Trp Tyr Arg Ser Ser Arg Gly Glu Val 35 40 45Gln
Thr Cys Ser Glu Arg Arg Pro Ile Arg Asn Leu Thr Phe Gln Asp 50 55
60Leu His Leu His His Gly Gly His Gln Ala Ala Asn Thr Ser His Asp65
70 75 80Leu Ala Gln Arg His Gly Leu Glu Ser Ala Ser Asp His His Gly
Asn 85 90 95Phe Ser Ile Thr Met Arg Asn Leu Thr Leu Leu Asp Ser Gly
Leu Tyr 100 105 110Cys Cys Leu Val Val Glu Ile Arg His His His Ser
Glu His Arg Val 115 120 125His Gly Ala Met Glu Leu Gln Val Gln Thr
Gly Lys Asp Ala Pro Ser 130 135 140Asn Cys Val Val Tyr Pro Ser Ser
Ser Gln Asp Ser Glu Asn Ile Thr145 150 155 160Ala Ala Ala Gly Thr
Ser Gly Ser Ser Gly Ser Gly Ser Gly Gly Ser 165 170 175Gly Ser Gly
Gly Gly Gly Arg Ser Val Pro Arg Asp Ser Gly Cys Lys 180 185 190Pro
Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro 195 200
205Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr
210 215 220Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln
Phe Ser225 230 235 240Trp Phe Val Asp Asp Val Glu Val His Thr Ala
Gln Thr Lys Pro Arg 245 250 255Glu Glu Gln Ile Asn Ser Thr Phe Arg
Ser Val Ser Glu Leu Pro Ile 260 265 270Met His Gln Asp Trp Leu Asn
Gly Lys Glu Phe Lys Cys Arg Val Asn 275 280 285Ser Ala Ala Phe Pro
Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys 290 295 300Gly Arg Pro
Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu305 310 315
320Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asn Phe
325 330 335Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln
Pro Ala 340 345 350Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr
Asp Gly Ser Tyr 355 360 365Phe Val Tyr Ser Lys Leu Asn Val Gln Lys
Ser Asn Trp Glu Ala Gly 370 375 380Asn Thr Phe Thr Cys Ser Val Leu
His Glu Gly Leu His Asn His His385 390 395 400Thr Glu Lys Ser Leu
Ser His Ser Pro Gly Lys 405 41010286PRTArtificial
sequenceHis-tagged human VISTA extracellular domain (ECD) 10Phe Lys
Val Ala Thr Pro Tyr Ser Leu Tyr Val Cys Pro Glu Gly Gln1 5 10 15Asn
Val Thr Leu Thr Cys Arg Leu Leu Gly Pro Val Asp Lys Gly His 20 25
30Asp Val Thr Phe Tyr Lys Thr Trp Tyr Arg Ser Ser Arg Gly Glu Val
35 40 45Gln Thr Cys Ser Glu Arg Arg Pro Ile Arg Asn Leu Thr Phe Gln
Asp 50 55 60Leu His Leu His His Gly Gly His Gln Ala Ala Asn Thr Ser
His Asp65 70 75 80Leu Ala Gln Arg His Gly Leu Glu Ser Ala Ser Asp
His His Gly Asn 85 90 95Phe Ser Ile Thr Met Arg Asn Leu Thr Leu Leu
Asp Ser Gly Leu Tyr 100 105 110Cys Cys Leu Val Val Glu Ile Arg His
His His Ser Glu His Arg Val 115 120 125His Gly Ala Met Glu Leu Gln
Val Gln Thr Gly Lys Asp Ala Pro Ser 130 135 140Asn Cys Val Val Tyr
Pro Ser Ser Ser Gln Asp Ser Glu Asn Ile Thr145 150 155 160Ala Ala
Ala Leu Ala Thr Gly Ala Cys Ile Val Gly Ile Leu Cys Leu 165 170
175Pro Leu Ile Leu Leu Leu Val Tyr Lys Gln Arg Gln Ala Ala Ser Asn
180 185 190Arg Arg Ala Gln Glu Leu Val Arg Met Asp Ser Asn Ile Gln
Gly Ile 195 200 205Glu Asn Pro Gly Phe Glu Ala Ser Pro Pro Ala Gln
Gly Ile Pro Glu 210 215 220Ala Lys Val Arg His Pro Leu Ser Tyr Val
Ala Gln Arg Gln Pro Ser225 230 235 240Glu Ser Gly Arg His Leu Leu
Ser Glu Pro Ser Thr Pro Leu Ser Pro 245 250 255Pro Gly Pro Gly Asp
Val Phe Phe Pro Ser Leu Asp Pro Val Pro Asp 260 265 270Ser Pro Asn
Phe Glu Val Ile Gly His His His His His His 275 280
28511232PRTArtificial sequenceFc C237S 11Glu Pro Lys Ser Ser Asp
Lys Thr His Thr Cys Pro Pro Cys Pro Ala1 5 10 15Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 20 25 30Lys Asp Thr Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 35 40 45Val Asp Val
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 50 55 60Asp Gly
Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln65 70 75
80Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala 100 105 110Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro 115 120 125Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr 130 135 140Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser145 150 155 160Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 165 170 175Lys Thr Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 180 185 190Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 195 200
205Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220Ser Leu Ser Leu Ser Pro Gly Lys225 23012228PRTArtificial
sequenceExemplary Fc #1 12Glu Arg Lys Cys Cys Val Glu Cys Pro Pro
Cys Pro Ala Pro Pro Val1 5 10 15Ala Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu 20 25 30Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser 35 40 45His Glu Asp Pro Glu Val Gln
Phe Asn Trp Tyr Val Asp Gly Val Glu 50 55 60Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr65 70 75 80Phe Arg Val Val
Ser Val Leu Thr Val Val His Gln Asp Trp Leu Asn 85 90 95Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ala Pro 100 105 110Ile
Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln 115 120
125Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
130 135 140Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val145 150 155 160Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro 165 170 175Pro Met Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr 180 185 190Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val 195 200 205Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 210 215 220Ser Pro Gly
Lys22513229PRTArtificial sequenceExemplary Fc #2 13Glu Ser Lys Tyr
Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe1 5 10 15Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 20 25 30Leu Met
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 35 40 45Ser
Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val 50 55
60Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser65
70 75 80Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu 85 90 95Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro Ser 100 105 110Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro 115 120 125Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu
Glu Met Thr Lys Asn Gln 130 135 140Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala145 150 155 160Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 165 170 175Pro Pro Val
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu 180 185 190Thr
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser 195 200
205Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
210 215 220Leu Ser Leu Gly Lys22514412PRTHomo sapiens 14Met Pro Leu
Gln Leu Leu Leu Leu Leu Ile Leu Leu Gly Pro Gly Asn1 5 10 15Ser Leu
Gln Leu Trp Asp Thr Trp Ala Asp Glu Ala Glu Lys Ala Leu 20 25 30Gly
Pro Leu Leu Ala Arg Asp Arg Arg Gln Ala Thr Glu Tyr Glu Tyr 35 40
45Leu Asp Tyr Asp Phe Leu Pro Glu Thr Glu Pro Pro Glu Met Leu Arg
50 55 60Asn Ser Thr Asp Thr Thr Pro Leu Thr Gly Pro Gly Thr Pro Glu
Ser65 70 75 80Thr Thr Val Glu Pro Ala Ala Arg Arg Ser Thr Gly Leu
Asp Ala Gly 85 90 95Gly Ala Val Thr Glu Leu Thr Thr Glu Leu Ala Asn
Met Gly Asn Leu 100 105 110Ser Thr Asp Ser Ala Ala Met Glu Ile Gln
Thr Thr Gln Pro Ala Ala 115 120 125Thr Glu Ala Gln Thr Thr Gln Pro
Val Pro Thr Glu Ala Gln Thr Thr 130 135 140Pro Leu Ala Ala Thr Glu
Ala Gln Thr Thr Arg Leu Thr Ala Thr Glu145 150 155 160Ala Gln Thr
Thr Pro Leu Ala Ala Thr Glu Ala Gln Thr Thr Pro Pro 165 170 175Ala
Ala Thr Glu Ala Gln Thr Thr Gln Pro Thr Gly Leu Glu Ala Gln 180 185
190Thr Thr Ala Pro Ala Ala Met Glu Ala Gln Thr Thr Ala Pro Ala Ala
195 200 205Met Glu Ala Gln Thr Thr Pro Pro Ala Ala Met Glu Ala Gln
Thr Thr 210 215 220Gln Thr Thr Ala Met Glu Ala Gln Thr Thr Ala Pro
Glu Ala Thr Glu225 230 235 240Ala Gln Thr Thr Gln Pro Thr Ala Thr
Glu Ala Gln Thr Thr Pro Leu 245 250 255Ala Ala Met Glu Ala Leu Ser
Thr Glu Pro Ser Ala Thr Glu Ala Leu 260 265 270Ser Met Glu Pro Thr
Thr Lys Arg Gly Leu Phe Ile Pro Phe Ser Val 275 280 285Ser Ser Val
Thr His Lys Gly Ile Pro Met Ala Ala Ser Asn Leu Ser 290 295 300Val
Asn Tyr Pro Val Gly Ala Pro Asp His Ile Ser Val Lys Gln Cys305 310
315 320Leu Leu Ala Ile Leu Ile Leu Ala Leu Val Ala Thr Ile Phe Phe
Val 325 330 335Cys Thr Val Val Leu Ala Val Arg Leu Ser Arg Lys Gly
His Met Tyr 340 345 350Pro Val Arg Asn Tyr Ser Pro Thr Glu Met Val
Cys Ile Ser Ser Leu 355 360 365Leu Pro Asp Gly Gly Glu Gly Pro Ser
Ala Thr Ala Asn Gly Gly Leu 370 375 380Ser Lys Ala Lys Ser Pro Gly
Leu Thr Pro Glu Pro Arg Glu Asp Arg385 390 395 400Glu Gly Asp Asp
Leu Thr Leu His Ser Phe Leu Pro 405 41015395PRTHomo sapiens 15Leu
Gln Leu Trp Asp Thr Trp Ala Asp Glu Ala Glu Lys Ala Leu Gly1 5 10
15Pro Leu Leu Ala Arg Asp Arg Arg Gln Ala Thr Glu Tyr Glu Tyr Leu
20 25 30Asp Tyr Asp Phe Leu Pro Glu Thr Glu Pro Pro Glu Met Leu Arg
Asn 35 40 45Ser Thr Asp Thr Thr Pro Leu Thr Gly Pro Gly Thr Pro Glu
Ser Thr 50 55 60Thr Val Glu Pro Ala Ala Arg Arg Ser Thr Gly Leu Asp
Ala Gly Gly65 70 75 80Ala Val Thr Glu Leu Thr Thr Glu Leu Ala Asn
Met Gly Asn Leu Ser 85 90 95Thr Asp Ser Ala Ala Met Glu Ile Gln Thr
Thr Gln Pro Ala Ala Thr 100 105 110Glu Ala Gln Thr Thr Gln Pro Val
Pro Thr Glu Ala Gln Thr Thr Pro 115 120 125Leu Ala Ala Thr Glu Ala
Gln Thr Thr Arg Leu Thr Ala Thr Glu Ala 130 135 140Gln Thr Thr Pro
Leu Ala Ala Thr Glu Ala Gln Thr Thr Pro Pro Ala145 150 155 160Ala
Thr Glu Ala Gln Thr Thr Gln Pro Thr Gly Leu Glu Ala Gln Thr 165 170
175Thr Ala Pro Ala Ala Met Glu Ala Gln Thr Thr Ala Pro Ala Ala Met
180 185 190Glu Ala Gln Thr Thr Pro Pro Ala Ala Met Glu Ala Gln Thr
Thr Gln 195 200 205Thr Thr Ala Met Glu Ala Gln Thr Thr Ala Pro Glu
Ala Thr Glu Ala 210 215 220Gln Thr Thr Gln Pro Thr Ala Thr Glu Ala
Gln Thr Thr Pro Leu Ala225 230 235 240Ala Met Glu Ala Leu Ser Thr
Glu Pro Ser Ala Thr Glu Ala Leu Ser 245 250 255Met Glu Pro Thr Thr
Lys Arg Gly Leu Phe Ile Pro Phe Ser Val Ser 260 265 270Ser Val Thr
His Lys Gly Ile Pro Met Ala Ala Ser Asn Leu Ser Val 275 280 285Asn
Tyr Pro Val Gly Ala Pro Asp His Ile Ser Val Lys Gln Cys Leu 290 295
300Leu Ala Ile Leu Ile Leu Ala Leu Val Ala Thr Ile Phe Phe Val
Cys305 310 315 320Thr Val Val Leu Ala Val Arg Leu Ser Arg Lys Gly
His Met Tyr Pro 325 330 335Val Arg Asn Tyr Ser Pro Thr Glu Met Val
Cys Ile Ser Ser Leu Leu 340 345 350Pro Asp Gly Gly Glu Gly Pro Ser
Ala Thr Ala Asn Gly Gly Leu Ser 355 360 365Lys Ala Lys Ser Pro Gly
Leu Thr Pro Glu Pro Arg Glu Asp Arg Glu 370 375 380Gly Asp Asp Leu
Thr Leu His Ser Phe Leu Pro385 390 39516226PRTArtificial
sequenceHumPSGL-1 isoform 2 ECD, with signal peptide 16Met Pro Leu
Gln Leu Leu Leu Leu Leu Ile Leu Leu Gly Pro Gly Asn1 5 10 15Ser Leu
Gln Leu Trp Asp Thr Trp Ala Asp Glu Ala Glu Lys Ala Leu 20 25 30Gly
Pro Leu Leu Ala Arg Asp Arg Arg Gln Ala Thr Glu Tyr Glu Tyr 35 40
45Leu Asp Tyr Asp Phe Leu Pro Glu Thr Glu Pro Pro Glu Met Leu Arg
50 55 60Asn Ser Thr Asp Thr Thr Pro Leu Thr Gly Pro Gly Thr Pro Glu
Ser65 70 75 80Thr Thr Val Glu Pro Ala Ala Arg Arg Ser Thr Gly Leu
Asp Ala Gly 85 90 95Gly Ala Val Thr Glu Leu Thr Thr Glu Leu Ala Asn
Met Gly Asn Leu 100 105 110Ser Thr Asp Ser Ala Ala Met Glu Ile Gln
Thr Thr Gln Pro Ala Ala 115 120 125Thr Glu Ala Gln Thr Thr Gln Pro
Val Pro Thr Glu Ala Gln Thr Thr 130 135 140Pro Leu Ala Ala Thr Glu
Ala Gln Thr Thr Arg Leu Thr Ala Thr Glu145 150 155 160Ala Gln Thr
Thr Pro Leu Ala Ala Thr Glu Ala Gln Thr Thr Pro Pro 165 170 175Ala
Ala Thr Glu Ala Gln Thr Thr Gln Pro Thr Gly Leu Glu Ala Gln 180 185
190Thr Thr Ala Pro Ala Ala Met Glu Ala Gln Thr Thr Ala Pro Ala Ala
195 200 205Met Glu Ala Gln Thr Thr Pro Pro Ala Ala Met Glu Ala Gln
Thr Thr 210 215 220Gln Thr22517209PRTArtificial sequenceHuman
PSGL-1 isoform 2 ECD, without signal peptide 17Leu Gln Leu Trp Asp
Thr Trp Ala Asp Glu Ala
Glu Lys Ala Leu Gly1 5 10 15Pro Leu Leu Ala Arg Asp Arg Arg Gln Ala
Thr Glu Tyr Glu Tyr Leu 20 25 30Asp Tyr Asp Phe Leu Pro Glu Thr Glu
Pro Pro Glu Met Leu Arg Asn 35 40 45Ser Thr Asp Thr Thr Pro Leu Thr
Gly Pro Gly Thr Pro Glu Ser Thr 50 55 60Thr Val Glu Pro Ala Ala Arg
Arg Ser Thr Gly Leu Asp Ala Gly Gly65 70 75 80Ala Val Thr Glu Leu
Thr Thr Glu Leu Ala Asn Met Gly Asn Leu Ser 85 90 95Thr Asp Ser Ala
Ala Met Glu Ile Gln Thr Thr Gln Pro Ala Ala Thr 100 105 110Glu Ala
Gln Thr Thr Gln Pro Val Pro Thr Glu Ala Gln Thr Thr Pro 115 120
125Leu Ala Ala Thr Glu Ala Gln Thr Thr Arg Leu Thr Ala Thr Glu Ala
130 135 140Gln Thr Thr Pro Leu Ala Ala Thr Glu Ala Gln Thr Thr Pro
Pro Ala145 150 155 160Ala Thr Glu Ala Gln Thr Thr Gln Pro Thr Gly
Leu Glu Ala Gln Thr 165 170 175Thr Ala Pro Ala Ala Met Glu Ala Gln
Thr Thr Ala Pro Ala Ala Met 180 185 190Glu Ala Gln Thr Thr Pro Pro
Ala Ala Met Glu Ala Gln Thr Thr Gln 195 200
205Thr18254PRTArtificial sequenceHuman PSGL-1 ECD (N-terminal
positions 42 to 295 of a full length Human PSGL-1 Accession No.
AAC50061) 18Gln Ala Thr Glu Tyr Glu Tyr Leu Asp Tyr Asp Phe Leu Pro
Glu Thr1 5 10 15Glu Pro Pro Glu Met Leu Arg Asn Ser Thr Asp Thr Thr
Pro Leu Thr 20 25 30Gly Pro Gly Thr Pro Glu Ser Thr Thr Val Glu Pro
Ala Ala Arg Arg 35 40 45Ser Thr Gly Leu Asp Ala Gly Gly Ala Val Thr
Glu Leu Thr Thr Glu 50 55 60Leu Ala Asn Met Gly Asn Leu Ser Thr Asp
Ser Ala Ala Met Glu Ile65 70 75 80Gln Thr Thr Gln Pro Ala Ala Thr
Glu Ala Gln Thr Thr Pro Leu Ala 85 90 95Ala Thr Glu Ala Gln Thr Thr
Arg Leu Thr Ala Thr Glu Ala Gln Thr 100 105 110Thr Pro Leu Ala Ala
Thr Glu Ala Gln Thr Thr Pro Pro Ala Ala Thr 115 120 125Glu Ala Gln
Thr Thr Gln Pro Thr Gly Leu Glu Ala Gln Thr Thr Ala 130 135 140Pro
Ala Ala Met Glu Ala Gln Thr Thr Ala Pro Ala Ala Met Glu Ala145 150
155 160Gln Thr Thr Pro Pro Ala Ala Met Glu Ala Gln Thr Thr Gln Thr
Thr 165 170 175Ala Met Glu Ala Gln Thr Thr Ala Pro Glu Ala Thr Glu
Ala Gln Thr 180 185 190Thr Gln Pro Thr Ala Thr Glu Ala Gln Thr Thr
Pro Leu Ala Ala Met 195 200 205Glu Ala Leu Ser Thr Glu Pro Ser Ala
Thr Glu Ala Leu Ser Met Glu 210 215 220Pro Thr Thr Lys Arg Gly Leu
Phe Ile Pro Phe Ser Val Ser Ser Val225 230 235 240Thr His Lys Gly
Ile Pro Met Ala Ala Ser Asn Leu Ser Val 245 250196PRTArtificial
sequenceExemplary fusion protein linker sequence 19Ile Glu Gly Arg
Met Asp1 5
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