U.S. patent application number 14/534793 was filed with the patent office on 2015-08-20 for vista antagonist and methods of use.
The applicant listed for this patent is Sabrina CEERAZ, Isabelle LeMERCIER, Janet LINES, Randolph J. NOELLE, Elizabeth NOWAK, Mark SPALLER, Li WANG. Invention is credited to Sabrina CEERAZ, Isabelle LeMERCIER, Janet LINES, Randolph J. NOELLE, Elizabeth NOWAK, Mark SPALLER, Li WANG.
Application Number | 20150231215 14/534793 |
Document ID | / |
Family ID | 57276452 |
Filed Date | 2015-08-20 |
United States Patent
Application |
20150231215 |
Kind Code |
A1 |
NOELLE; Randolph J. ; et
al. |
August 20, 2015 |
VISTA Antagonist and Methods of Use
Abstract
The present invention is directed to a peptide, multimer,
conjugate, analog, derivative or mimetic thereof that inhibits the
activity of VISTA. The invention further contemplates therapeutic
use of the VISTA antagonist peptide, multimer, conjugate,
derivative or mimetic thereof, including treating or preventing
cancer, bacterial infections, viral infections, parasitic
infections and fungal infections, as well as research uses of the
antagonist.
Inventors: |
NOELLE; Randolph J.;
(Plainfield, NH) ; CEERAZ; Sabrina; (Lebanon,
NH) ; LeMERCIER; Isabelle; (Enfield, NH) ;
NOWAK; Elizabeth; (West Lebanon, NH) ; LINES;
Janet; (London, GB) ; WANG; Li; (Norwich,
VT) ; SPALLER; Mark; (Lebanon, NH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NOELLE; Randolph J.
CEERAZ; Sabrina
LeMERCIER; Isabelle
NOWAK; Elizabeth
LINES; Janet
WANG; Li
SPALLER; Mark |
Plainfield
Lebanon
Enfield
West Lebanon
London
Norwich
Lebanon |
NH
NH
NH
NH
VT
NH |
US
US
US
US
GB
US
US |
|
|
Family ID: |
57276452 |
Appl. No.: |
14/534793 |
Filed: |
November 6, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13925094 |
Jun 24, 2013 |
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14534793 |
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61663969 |
Jun 25, 2012 |
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61663431 |
Jun 22, 2012 |
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Current U.S.
Class: |
424/178.1 ;
424/185.1; 530/327; 530/362; 530/391.7 |
Current CPC
Class: |
C07K 2317/74 20130101;
Y02A 50/491 20180101; A61K 9/0019 20130101; A61K 38/10 20130101;
Y02A 50/465 20180101; C07K 2317/76 20130101; Y02A 50/393 20180101;
Y02A 50/419 20180101; Y02A 50/402 20180101; A61K 38/00 20130101;
A61K 45/06 20130101; Y02A 50/30 20180101; Y02A 50/464 20180101;
Y02A 50/475 20180101; C07K 7/08 20130101; C07K 16/2827 20130101;
C07K 2319/30 20130101; Y02A 50/409 20180101; Y02A 50/58 20180101;
Y02A 50/423 20180101; Y02A 50/467 20180101; Y02A 50/385 20180101;
Y02A 50/463 20180101; Y02A 50/488 20180101; Y02A 50/411 20180101;
Y02A 50/421 20180101; Y02A 50/476 20180101; Y02A 50/478 20180101;
G01N 33/566 20130101; A61K 39/0005 20130101; Y02A 50/387
20180101 |
International
Class: |
A61K 39/00 20060101
A61K039/00; A61K 45/06 20060101 A61K045/06; C07K 7/08 20060101
C07K007/08 |
Claims
1. An isolated VISTA antagonist comprising a peptide which is
identical to the amino acid sequence of SEQ ID NO:1
(Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His), or a multimer,
conjugate, analog, derivative or mimetic thereof.
2. An isolated VISTA antagonist according to claim 1, comprising a
peptide which is identical to the amino acid sequence of SEQ ID
NO:1 (Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His), or which
comprises a peptide having an amino acid sequence that differs from
SEQ ID NO:1 by at most 2 amino acid residues, or an multimer,
conjugate, analog, derivative or mimetic thereof.
3-5. (canceled)
6. The isolated VISTA antagonist according to claim 1, wherein the
cysteine residues at positions 4 and 11 of SEQ ID NO:1
(Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His), or their
corresponding position in a variant of said peptide, form a
disulfide bridge.
7. An isolated VISTA antagonist according to claim 1, which has
been modified to improve binding affinity and/or stability.
8. The isolated VISTA antagonist of claim 7, wherein the
modification is selected from the group consisting of PEG,
acetylation, XTEN, albumin and multimerization.
9. The isolated VISTA antagonist according to claim 1, which is
directly or indirectly attached to an immunoglobulin polypeptide or
a fragment thereof.
10. The isolated antagonist of claim 9, wherein said immunoglobulin
polypeptide comprises a human IgG1, IgG2, IgG3 or IgG4 constant
region or fragment thereof.
11-12. (canceled)
13. An isolated VISTA antagonist according to claim 1, which
comprises another moiety that targets said peptide to a target
site.
14-20. (canceled)
21. A composition suitable for therapeutic, prophylactic or
diagnostic use comprising a therapeutically, prophylactically or
diagnostically effective amount of the isolated VISTA antagonist of
claim 1.
23. The composition of claim 21, further comprising another
therapeutic agent.
24. The composition of claim 23, wherein the other therapeutic
agent is an anti-cancer agent, an anti-viral agent, a cytokine or
an immune agonist.
25. The composition of claim 21, wherein the other therapeutic
agent is selected from CTLA-4-Ig, anti-PD-1, PD-L1 or PD-L2 fusion
proteins, and EGFR antagonists.
26-32. (canceled)
33. A method for stimulating an immune response in a subject,
comprising administering to the subject in need thereof an
effective amount of an isolated VISTA antagonist according to claim
1 optionally comprising another therapeutic agent selected from an
anti-cancer agent, an anti-viral agent, a cytokine or an immune
agonist.
34. The method of claim 33, wherein the subject has an infection
selected from the group consisting of bacterial, viral, parasitic
and fungal infections.
35-39. (canceled)
40. The method of claim 33, for treating cancer in a subject.
41. The method of claim 33, which is for enhancing anti-cancer or
anti-tumor immunity, comprising administering to a subject in need
thereof an effective amount of an isolated VISTA antagonist
according to claim 1 and optionally comprising another therapeutic
agent selected from an anti-cancer agent, an anti-viral agent, a
cytokine or an immune agonist.
42. The method of claim 33, which is for treating or preventing
cancer, inhibiting tumor invasion and/or cancer metastasis,
comprising administering to a subject in need thereof an effective
amount of said VISTA antagonist and optionally comprising another
therapeutic agent selected from an anti-cancer agent, an anti-viral
agent, a cytokine or an immune agonist.
44. The method of claim 33, which is for treating or preventing a
viral infection, comprising administering to a subject in need
thereof an effective amount of said VISTA antagonist and optionally
comprising another therapeutic agent selected from an anti-cancer
agent, an anti-viral agent, a cytokine or an immune agonist.
45-50. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of Ser. No.
13/925,094, filed Jun. 24, 2013, which claims priority to U.S.
Provisional Ser. No. 61/663,969, filed Jun. 25, 2012, and U.S.
Provisional Ser. No. 61/663,431, filed Jun. 22, 2012, and also
claims priority to U.S. Provisional Ser. No. 61/927,061, filed on
Jan. 14, 2014, the contents of each, including the sequence
listing, are incorporated herein by reference in their
entireties.
[0002] The sequence listing file named "43260o1003", having a size
of 688 bytes and created Nov. 6, 2014, is hereby incorporated by
reference in its entirety.
BACKGROUND
[0003] The immune system is tightly controlled by co-stimulatory
and co-inhibitory ligands and receptors. These molecules provide
not only a second signal for T cell activation but also a balanced
network of positive and negative signals to maximize immune
responses against infection while limiting immunity to self.
[0004] Induction of an immune response requires T cell expansion,
differentiation, contraction and establishment of T cell memory. T
cells must encounter antigen presenting cells (APCs) and
communicate via T cell receptor (TCR)/major histocompatibility
complex (MHC) interactions on APCs. Once the TCR/MHC interaction is
established, other sets of receptor-ligand contacts between the T
cell and the APC are required, i.e. co-stimulation via CD154/CD40
and CD28/B7.1-B7.2. The synergy between these contacts results in a
productive immune response capable of clearing pathogens and
tumors, and may be capable of inducing autoimmunity.
[0005] Another level of control has been identified, namely
regulatory T cells (T.sub.reg). This specific subset of T cells is
generated in the thymus, delivered into the periphery, and is
capable of constant and inducible control of T cells responses.
Sakaguchi (2000) Cell 101(5):455-8; Shevach (2000) Annu. Rev.
Immunol. 18:423-49; Bluestone and Abbas (2003) Nat. Rev. Immunol.
3(3):253-7. T.sub.reg are represented by a CD4+CD25+ phenotype and
also express high levels of cytotoxic T lymphocyte-associated
antigen-4 (CTLA-4), OX-40, 4-1BB and the glucocorticoid inducible
TNF receptor-associated protein (GITR). McHugh, et al. (2002)
Immunity 16(2):311-23; Shimizu, et al. (2002) Nat. Immun.
3(2):135-42. Elimination of T.sub.reg cells by 5 day neonatal
thymectomy or antibody depletion using anti-CD25, results in the
induction of autoimmune pathology and exacerbation of T cells
responses to foreign and self-antigens, including heightened
anti-tumor responses. Sakaguchi, et al. (1985) J. Exp. Med.
161(1):72-87; Sakaguchi, et al. (1995) J. Immunol. 155(3):1151-64;
Jones, et al. (2002) Cancer Immun. 2:1. In addition, T.sub.reg have
also been involved in the induction and maintenance of
transplantation tolerance, since depletion of T.sub.reg with
anti-CD25 monoclonal antibodies results in ablation of
transplantation tolerance and rapid graft rejection. Jarvinen, et
al. (2003) Transplantation 76:1375-9. Among the receptors expressed
by T.sub.reg GITR seems to be an important component since ligation
of GITR on the surface of Treg with an agonistic monoclonal
antibody results in rapid termination of T,.sub.g activity,
resulting in autoimmune pathology and ablation of transplantation
tolerance.
[0006] Costimulatory and co-inhibitory ligands and receptors not
only provide a "second signal" for T cell activation, but also a
balanced network of positive and negative signal to maximize immune
responses against infection while limiting immunity to self. The
best characterized costimulatory ligands are B7.1 and B7.2, which
are expressed by professional APCs, and whose receptors are CD28
and CTLA-4. Greenwald, et al. (2005) Annu Rev Immunol 23, 515-548;
Sharpe and Freeman (2002) Nat Rev Immunol 2, 116-126. CD28 is
expressed by naive and activated T cells and is critical for
optimal T cell activation. In contrast, CTLA-4 is induced upon T
cell activation and inhibits T cell activation by binding to
B7.1/B7.2, thus impairing CD28-mediated costimulation. CTLA-4 also
transduces negative signaling through its cytoplasmic ITIM motif.
Teft, et al. (2006). Annu Rev Immunol 24, 65-97. B7.1/B7.2 KO mice
are impaired in adaptive immune response (Borriello, et al. (1997)
Immunity 6, 303-313; Freeman, et al. (1993) Science 262, 907-909),
whereas CTLA-4 KO mice can not adequately control inflammation and
develop systemic autoimmune diseases. Chambers, et al. (1997)
Immunity 7, 885-895; Tivol, et al. (1995) Immunity 3, 541-547;
Waterhouse, et al. (1995) Science 270, 985-988. The B7 family
ligands have expanded to include costimulatory B7-H2 (ICOS Ligand)
and B7-H3, as well as co-inhibitory B7-H1 (PD-L1), B7-DC (PD-L2),
B7-H4 (B7S1 or B7x), and B7-H6. See Brandt, et al. (2009) J Exp Med
206, 1495-1503; Greenwald, et al. (2005) Annu Rev Immunol 23:
515-548.
[0007] Inducible costimulatory (ICOS) molecule is expressed on
activated T cells and binds to B7-H2. See Yoshinaga, et al. (1999)
Nature 402, 827-832. ICOS is important for T cell activation,
differentiation and function, as well as essential for
T-helper-cell-induced B cell activation, Ig class switching, and
germinal center (GC) formation. Dong, et al. (2001) Nature 409,
97-101; Tafuri, et al. (2001) Nature 409, 105-109; Yoshinaga, et
al. (1999) Nature 402, 827-832. Programmed Death 1 (PD-1) on the
other hand, negatively regulates T cell responses. PD-1 KO mice
develop lupus-like autoimmune disease, or autoimmune dilated
cardiomyopathy depending upon the genetic background. Nishimura, et
al. (1999) Immunity 11, 141-151. Nishimura, et al. (2001) Science
291: 319-322. The autoimmunity most likely results from the loss of
signaling by both ligands PD-L1 and PD-L2. Recently, CD80 was
identified as a second receptor for PD-L1 that transduces
inhibitory signals into T cells. Butte, et al. (2007) Immunity 27:
111-122. The receptor for B7-H3 and B7-H4 still remain unknown.
[0008] The best characterized co-stimulatory ligands are B7.1 and
B7.2, which belong to the Ig superfamily and are expressed on
professional APCs and whose receptors are CD28 and CTLA-4
(Greenwald, et al. (2005) Annu. Rev. Immunol. 23:515-548). CD28 is
expressed by naive and activated T cells and is critical for
optimal T cell activation. In contrast, CTLA-4 is induced upon T
cell activation and inhibits T cell activation by binding to
B7.1/B7.2, impairing CD28-mediated co-stimulation. B7.1 and B7.2 KO
mice are impaired in adaptive immune response (Borriello, et al.
(1997) Immunity 6:303-313), whereas CTLA-4 knockout mice cannot
adequately control inflammation and develop systemic autoimmune
diseases (Tivol, et al. (1995) Immunity 3:541-547; Waterhouse, et
al. (1995) Science 270:985-988; Chambers, et al. (1997) Immunity
7:885-895).
[0009] The B7 family ligands have expanded to include
co-stimulatory B7-H2 (inducible T cell co-stimulator (ICOS) ligand)
and B7-H3, as well as co-inhibitory B7-H1 (PD-L1), B7-DC (PD-L2),
B7-H4 (B7S1 or B7x), and B7-H6 (Greenwald, et al. (2005) supra;
Brandt, et al. (2009) J. Exp. Med. 206:1495-1503). Accordingly,
additional CD28 family receptors have been identified. ICOS is
expressed on activated T cells and binds to B7-H2 (Yoshinaga, et
al. (1999) Nature 402:827-832). ICOS is a positive coregulator,
which is important for T cell activation, differentiation, and
function (Yoshinaga, et al. (1999) supra; Dong, et al. (2001)
Nature 409:97-101). In contrast, PD-1 (programmed death 1)
negatively regulates T cell responses. PD-1 knockout mice develop
lupus-like autoimmune disease or autoimmune dilated cardiomyopathy
(Nishimura, et al. (1999) Immunity 11:141-151; Nishimura, et al.
(2001) Science 291:319-322). The autoimmunity most likely results
from the loss of signaling by both ligands PD-L1 and PD-L2.
Recently, CD80 was identified as a second receptor for PD-L1 that
transduces inhibitory signals into T cells (Butte, et al. (2007)
Immunity 27:111-122).
[0010] The two inhibitory B7 family ligands, PD-L1 and PD-L2, have
distinct expression patterns. PD-L2 is inducibly expressed on DCs
and macrophages, whereas PD-L1 is broadly expressed on both
hematopoietic cells and nonhematopoietic cell types (Okazaki &
Honjo (2006) Immunology 27:195-201; Keir, et al. (2008) Annu. Rev.
Immunol. 26:677-704). Consistent with the immune-suppressive role
of PD-1 receptor, a study using PD-L1.sup.-/- and PD-L2.sup.-/-
mice has shown that both ligands have overlapping roles in
inhibiting T cell proliferation and cytokine production (Keir, et
al. (2006) J. Exp. Med. 203:883-895). PD-L1 deficiency enhances
disease progression in both the non-obese diabetic model of
autoimmune diabetes and the mouse model of multiple sclerosis
(experimental autoimmune encephalomyelitis (EAE); Anasari, et al.
(2003) J. Exp. Med. 198:63-69; Salama, et al. (2003) J. Exp. Med.
198:71-78; Latchman, et al. (2004) Proc. Natl. Acad. Sci. USA.
101:10691-10696). PD-L1.sup.-/- T cells produce elevated levels of
the proinflammatory cytokines in both disease models. In addition,
bone marrow chimera experiments have demonstrated that the tissue
expression of PD-L1 (i.e., within pancreas) uniquely contributes to
its capacity of regionally controlling inflammation (Keir, et al.
(2006) supra; Keir, et al. (2007) J. Immunol. 179:5064-5070;
Grabie, et al. (2007) Circulation. 116:2062-2071). PD-L1 is also
highly expressed on placental syncytiotrophoblasts, which
critically control the maternal immune responses to allogeneic
fetus (Guleria, et al. (2005) J. Exp. Med. 202:231-237).
[0011] Consistent with its immune-suppressive role, PD-L1 potently
suppresses antitumor immune responses and helps tumors evade immune
surveillance. PD-L1 can induce apoptosis of infiltrating cytotoxic
CD8.sup.+ T cells, which express a high level of PD-1 (Dong, et al.
(2002) Nature 409:97-101; Dong & Chen (2003) J. Mol. Med.
81:281-287). Studies have shown that blocking the PD-L1-PD-1
signaling pathway, in conjunction with other immune therapies,
prevents tumor progression by enhancing antitumor cytotoxic T
lymphocyte activity and cytokine production (Iwai, et al. (2002)
Proc. Natl. Acad. Sci. USA 99:12293-12297; Blank, et al. (2004)
Cancer Res. 64:1140-1145; Blank, et al. (2005) Cancer Immunol.
Immunother. 54:307-314; Geng, et al. (2006) Int. J. Cancer.
118:2657-2664). In addition, it has been shown that PD-L1
expression on dendritic cells promotes the induction of adaptive
Foxp3.sup.+CD4.sup.+ regulatory T cells (aT.sub.reg cells), and
PD-L1 is a potent inducer of aT.sub.reg cells within the tumor
microenvironment (Wang, et al. (2008) Proc. Natl. Acad. Sci. USA.
105:9331-9336).
[0012] An additional immune regulatory ligand, referred to as
V-domain Ig suppressor of T cell activation (VISTA) or PD-L3, has
been recently identified as an upregulated molecule in a T cell
transcriptional profiling screen. (Wang, et al. (2011) J. Exp. Med.
208:577; WO 2011/120013). It has been shown that the extracellular
Ig domain of VISTA shares significant sequence homology with the B7
family ligands PD-L1 and PD-L2, albeit with unique structural
features that distinguish it from the B7 family members.
[0013] VISTA is primarily expressed on hematopoietic cells, and
VISTA expression is highly regulated on myeloid antigen-presenting
cells (APCs) and T cells. Expression of VISTA on antigen presenting
cells (APCs) suppresses T cell responses by engaging its
counter-receptor on T cells during cognate interactions between T
cells and APCs. VISTA blockade enhances T cell-mediated immunity in
an autoimmune disease model, suggesting its unique and
non-redundant role in controlling autoimmunity when compared with
other inhibitory B7 family ligands such as PD-L1 and PD-L2. In
addition, VISTA blockade enhances anti-tumor immunity and
suppressed tumor growth in preclinical murine tumor models (WO
2011/120013). In this regard, therapeutic intervention of the VISTA
inhibitory pathway represents a novel approach to modulate T
cell-mediated immunity for treating diseases such as viral
infection and cancer.
SUMMARY OF THE INVENTION
[0014] The present invention provides an isolated VISTA antagonist
that comprises a peptide that is identical to the amino acid
sequence of SEQ ID NO:1
(Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His), or a multimer,
conjugate, analog, derivative or mimetic thereof.
[0015] In one embodiment, the isolated VISTA antagonist comprises a
peptide which is identical to the amino acid sequence of SEQ ID
NO:1 (Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His), or which
comprises a peptide having an amino acid sequence that differs from
SEQ ID NO:1 by at most 2 amino acid residues, or an multimer,
conjugate, analog, derivative or mimetic thereof. In another
embodiment, the isolated VISTA antagonist comprises a peptide
having an amino acid sequence that differs from SEQ ID NO:1 by at
most 1 amino acid residue, or an multimer, conjugate, analog,
derivative or mimetic thereof. In yet another embodiment, the
isolated VISTA antagonist comprises a peptide which is identical to
the amino acid sequence of SEQ ID NO:1
(Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His), or a multimer,
or conjugate thereof. In a specific embodiment, the isolated VISTA
antagonist consists of the amino acid sequence of SEQ ID NO:1
(Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His).
[0016] In one embodiment, the cysteine residues at positions 4 and
11 of SEQ ID NO:1
(Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His), or their
corresponding positions in a variant of said peptide, form a
disulfide bridge.
[0017] In another embodiment, the isolated VISTA antagonist has
been modified to improve binding affinity and/or stability. In a
specific embodiment, the isolated VISTA antagonist has been
modified by PEG, acetylation, XTEN, albumin and/or
multimerization.
[0018] In another embodiment, the isolated VISTA antagonist is
directly or indirectly attached to an immunoglobulin polypeptide or
a fragment thereof. The immunoglobulin polypeptide may comprise a
human IgG1, IgG2, IgG3 or IgG4 constant region or fragment thereof.
Preferably, the immunoglobulin polypeptide comprises a human IgG1
constant region or fragment thereof.
[0019] In yet another embodiment, the isolated VISTA antagonist
comprises multiple, i.e., 2, 3, 4, 5, 6, 7 or more, copies of said
peptide.
[0020] In a further embodiment, the isolated VISTA antagonist
comprises another moiety that targets said peptide to a target
site. The targeting moiety may be selected from an antibody or
ligand that binds to an antigen, a receptor expressed by the target
cell or an infectious agent.
[0021] In yet a further embodiment, the isolated VISTA antagonist
is attached to another moiety or another copy of said antagonist
via a linker. The linker may be a peptide that permits the
antagonist to interact with VISTA expressed on the surface of a
target cell.
[0022] In a further embodiment, the isolated VISTA antagonist is
directly or indirectly attached to a detectable label or
therapeutic agent.
[0023] In several of the embodiments, the isolated VISTA antagonist
binds to the extracellular domain of VISTA and disrupts its
interaction with a VISTA receptor and/or reduces or inhibits
VISTA-mediated T cell suppression.
[0024] In one embodiment, the isolated VISTA antagonist elicits
anti-tumor and/or anti-viral activity.
[0025] Additionally, the invention contemplates a composition
suitable for therapeutic, prophylactic or diagnostic use comprising
a therapeutically, prophylactically or diagnostically effective
amount of the isolated VISTA antagonist.
[0026] In one embodiment, the composition further comprises a
pharmaceutically acceptable carrier, diluent, solubilizer,
preservative or mixture thereof.
[0027] In another embodiment, the composition further comprises
another therapeutic agent, e.g., an anti-cancer agent, an
anti-viral agent, a cytokine or an immune agonist. In a particular
embodiment, the other therapeutic agent is selected from CTLA-4-Ig,
anti-PD-1, PD-L1 or PD-L2 fusion proteins, and EGFR
antagonists.
[0028] In one embodiment, the composition is suitable for
subcutaneous administration or intravenous administration.
[0029] Moreover, the present invention further contemplates an
isolated nucleic acid sequence encoding a VISTA antagonist peptide,
analog, derivative or mimetic thereof disclosed herein, a vector
containing the isolated nucleic acid sequence, and a host cell
comprising the isolated nucleic acid sequence or the vector.
[0030] In one embodiment, the host cell is a mammalian cell, a
bacterial cell, a fungal cell, a yeast cell, an avian cell or an
insect cell.
[0031] The present invention further contemplates a method of
expressing a VISTA antagonist peptide, analog, derivative or
mimetic thereof comprising culturing the host cell under conditions
that provide for expression of said peptide, analog, derivative or
mimetic thereof.
[0032] Furthermore, the present invention contemplates various uses
of the isolated VISTA antagonist.
[0033] In one embodiment, the invention provides a method for
blocking, inhibiting or neutralizing VISTA-mediated T cell
suppression, comprising administering to a subject in need thereof
an effective amount of an isolated VISTA antagonist disclosed
herein or a composition containing said isolated VISTA
antagonist.
[0034] In another embodiment, the invention provides a method for
stimulating an immune response in a subject, comprising
administering to the subject in need thereof an effective amount of
an isolated VISTA antagonist disclosed herein or a composition
containing said isolated VISTA antagonist. Such a method may be
used for treating cancer in a subject.
[0035] The subject may have cancer and/or an infection selected
from the group consisting of bacterial, viral, parasitic and fungal
infections.
[0036] The bacterial infection may be caused by at least one
bacterium selected from the group consisting of Bordetella,
Borrelia, Brucella, Burkholderia, Campylobacter, Chlamydia,
Clostridium, Corynebacterium, Enterobacter, Enterococcus, Erwinia,
Escherichia, Francisella, Haemophilus, Heliobacter, Legionella,
Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria,
Pasteurella, Pelobacter, Pseudomonas, Rickettsia, Salmonella,
Serratia, Shigella, Staphylococcus, Streptococcus, Treponema,
Vibrio, Yersinia and Xanthomonas.
[0037] The viral infection may be caused by at least one virus
selected from the group consisting of Adenoviridae,
Papillomaviridae, Polyomaviridae, Herpesviridae, Poxviridae,
Hepadnaviridae, Parvoviridae, Astroviridae, Caliciviridae,
Picornaviridae, Coronoviridae, Flaviviridae, Retroviridae,
Togaviridae, Arenaviridae, Bunyaviridae, Filoviridae,
Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, and Reoviridae.
More specifically, the virus may be adenovirus, herpes simplex type
I, herpes simplex type 2, Varicella-zoster virus, Epstein-barr
virus, cytomegalovirus, herpesvirus type 8, papillomavirus, BK
virus, JC virus, smallpox, Hepatitis B, bocavirus, parvovirus B19,
astrovirus, Norwalk virus, coxsackievirus, Hepatitis A, poliovirus,
rhinovirus, severe acute respiratory syndrome virus, Hepatitis C,
yellow fever, dengue virus, West Nile virus, rubella, Hepatitis E,
human immunodeficiency virus (HIV), influenza, guanarito virus,
Junin virus, Lassa virus, Machupo virus, Sabia virus, Crimean-Congo
hemorrhagic fever virus, ebola virus, Marburg virus, measles virus,
mumps virus, parainfluenza, respiratory syncytial virus, human
metapneumovirus, Hendra virus, Nipah virus, rabies, Hepatitis D,
rotavirus, orbivirus, coltivirus or Banna virus.
[0038] The fungal infection may be selected from the group
consisting of thrush, candidiasis, cryptococcosis, histoplasmosis,
blastomycosis, aspergillosis, coccidioidomycosis,
paracoccidiomycosis, sporotrichosis, zygomycosis,
chromoblastomycosis, lobomycosis, mycetoma, onychomycosis, piedra
pityriasis versicolor, tinea barbae, tinea capitis, tinea corporis,
tinea cruris, tinea favosa, tinea nigra, tinea pedis, otomycosis,
phaeohyphomycosis, or rhinosporidiosis.
[0039] The parasitic infection may be caused by at least one
parasite selected from the group consisting of Entamoeba
hystolytica, Giardia lamblia, Cryptosporidium muris,
Trypanosomatida gambiense, Trypanosomatida rhodesiense,
Trypanosomatida crusi, Leishmania mexicana, Leishmania
braziliensis, Leishmania tropica, Leishmania donovani, Toxoplasma
gondii, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae,
Plasmodium falciparum, Trichomonas vaginalis, Histomonas
meleagridis; Secementea; Trichuris trichiura, Ascaris lumbricoides,
Enterobius vermicularis, Ancylostoma duodenale, Necator americanus,
Strongyloides stercoralis, Wuchereria bancrofti, Dracunculus
medinensis; blood flukes, liver flukes, intestinal flukes, lung
flukes; Schistosoma mansoni, Schistosoma haematobium, Schistosoma
japonicum, Fasciola hepatica, Fasciola gigantica, Heterophyes
heterophyes, and Paragonimus westermani.
[0040] In another embodiment, the invention provides a method for
enhancing anti-cancer or anti-tumor immunity, comprising
administering to a subject in need thereof an effective amount of
an isolated VISTA antagonist disclosed herein or a composition
containing said isolated VISTA antagonist.
[0041] In another embodiment, the invention provides a method for
treating or preventing cancer, inhibiting tumor invasion and/or
cancer metastasis, comprising administering to a subject in need
thereof an effective amount of an isolated VISTA antagonist
disclosed herein or a composition containing said isolated VISTA
antagonist.
[0042] The cancer may be selected from the group consisting of
carcinoma, lymphoma, blastoma, sarcoma, leukemia, lymphoid
malignancies, melanoma, squamous cell cancer, lung cancer
(including small-cell lung cancer, non-small cell lung cancer,
adenocarcinoma of the lung, and squamous carcinoma of the lung),
cancer of the peritoneum, hepatocellular cancer, gastric or stomach
cancer (including gastrointestinal cancer), 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 or renal cancer, liver cancer, prostate cancer, vulval
cancer, thyroid cancer, hepatic carcinoma, head and neck cancer,
B-cell lymphoma (including low grade/follicular non-Hodgkin's
lymphoma (NHL); small lymphocytic (SL) NHL; intermediate
grade/follicular NHL; intermediate grade diffuse NHL; high grade
immunoblastic NHL; high grade lymphoblastic NHL; high grade small
non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma;
AIDS-related lymphoma; and Waldenstrom's Macroglobulinemia);
chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia
(ALL); Hairy cell leukemia; chronic myeloblastic leukemia;
post-transplant lymphoproliferative disorder (PTLD), abnormal
vascular proliferation associated with phakomatoses, edema (such as
that associated with brain tumors), and Meigs' syndrome.
[0043] In yet another embodiment, the invention provides a method
for treating or preventing a viral infection, comprising
administering to a subject in need thereof an effective amount of
an isolated VISTA antagonist disclosed herein or a composition
containing said isolated VISTA antagonist.
[0044] These methods may further comprise the administration of
another therapeutic agent, wherein said peptide and therapeutic may
be separately or jointly administered, at the same or different
times.
[0045] In one embodiment, the other therapeutic agent is an
anti-cancer agent, an anti-viral or other anti-infectious agent, a
cytokine or an immune agonist. Preferably, the other therapeutic
agent is selected from CTLA-4-Ig, anti-PD-1, PD-L1 or PD-L2 fusion
proteins, and EGFR antagonists.
[0046] Finally, the present invention also contemplates a method
for mapping the active site of VISTA, comprising: (a) incubating an
isolated VISTA fusion protein with an isolated VISTA antagonist
comprising a peptide that is identical to the amino acid sequence
of SEQ ID NO:1 (Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His),
or which comprises a peptide having an amino acid sequence which
differs from SEQ ID NO:1 by at most 2 amino acid residues or an
multimer, conjugate, analog, derivative or mimetic thereof; and (b)
determining the binding site of the isolated VISTA antagonist.
[0047] In one embodiment, the active site of VISTA binds to a VISTA
receptor and mediates immune suppression. In another embodiment,
step (b) comprises domain deletion, domain swapping, amino acid
mutagenesis, foot printing, NMR, X-ray crystallography or homology
modeling.
BRIEF DESCRIPTION OF THE DRAWINGS
[0048] FIG. 1 shows that a VISTA antagonist peptide (SEQ ID NO:1)
significantly enhances the proliferation of T cells as compared to
an anti-VISTA antibody (aVISTA) and an anti-PD-L1 antibody (aPDL1).
Myeloid CD11B+ APCs were incubated with OT2 CD4+ T cells, antigen,
and a monoclonal antibody (aVISTA or aPDL1) or AP1049.
Proliferation of T cells was measured by tritium incorporation at
72 hours.
[0049] FIG. 2 shows that a VISTA antagonist peptide (SEQ ID NO:1)
significantly enhances anti-tumor immunity. Female mice inoculated
with MB49 tumors were treated with either PBS (control) or AP1049.
Tumor size was measured by caliper every 2-3 days.
DETAILED DESCRIPTION OF THE INVENTION
[0050] In order that the invention herein described may be fully
understood, the following detailed description is set forth.
Various embodiments of the invention are described in detail and
may be further illustrated by the provided examples.
[0051] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as those commonly understood by
one of ordinary skill in the art to which this invention belongs.
Although methods and materials similar or equivalent to those
described herein may be used in the invention or testing of the
present invention, suitable methods and materials are described
herein. The materials, methods and examples are illustrative only,
and are not intended to be limiting.
Definitions
[0052] As used in the description herein and throughout the claims
that follow, the meaning of "a," "an," and "the" includes plural
reference unless the context clearly dictates otherwise.
[0053] "Antagonist," as used herein, refers to a compound
(preferably a peptide) that opposed the physiological effects of
another compound. For example, at the receptor level, an antagonist
is a compound that opposes the receptor-associated response
normally induced by another agent that binds to and activates the
biological activity the receptor. Likewise, at the ligand level, an
antagonist is a compound that opposes the ligand-associated
response normally induced when the ligand binds to its target
receptor and/or accessory factors. In a specific embodiment, a
VISTA antagonist is a compound, e.g., a peptide or analog,
derivative or mimetic thereof, that binds to VISTA and opposes one
or more of its biological activities, e.g., VISTA-mediated T cell
suppression and/or VISTA-mediated suppression of anti-tumor
immunity, thereby enhancing T cell-mediated immunity and/or
anti-tumor immunity.
[0054] "Analog," as used herein, refers to a compound (preferably a
peptide) whose structure is related to that of a given compound
(preferably a peptide) but differs in chemical and biological
properties.
[0055] "Antigen presenting cell," as used herein, refers broadly to
professional antigen presenting cells (e.g., B lymphocytes,
monocytes, dendritic cells, and Langerhans cells) as well as other
antigen presenting cells (e.g., keratinocytes, endothelial cells,
astrocytes, fibroblasts, and oligodendrocytes).
[0056] "Amino acid," as used herein refers broadly to naturally
occurring and synthetic amino acids, as well as amino acid analogs
and amino acid mimetics that function in a manner similar to the
naturally occurring amino acids. Naturally occurring amino acids
are those encoded by the genetic code, as well as those amino acids
that are later modified (e.g., hydroxyproline,
.gamma.-carboxyglutamate, and O-phosphoserine.) Amino acid analogs
refers to compounds that have the same basic chemical structure as
a naturally occurring amino acid (i.e., a carbon that is bound to a
hydrogen, a carboxyl group, an amino group), and an R group (e.g.,
homoserine, norleucine, methionine sulfoxide, methionine methyl
sulfonium.) Analogs may have modified R groups (e.g., norleucine)
or modified peptide backbones, but retain the same basic chemical
structure as a naturally occurring amino acid. Amino acid mimetics
refers to chemical compounds that have a structure that is
different from the general chemical structure of an amino acid, but
that functions in a manner similar to a naturally occurring amino
acid.
[0057] "Allergic disease," as used herein, refers broadly to a
disease involving allergic reactions. More specifically, an
"allergic disease" is defined as a disease for which an allergen is
identified, where there is a strong correlation between exposure to
that allergen and the onset of pathological change, and where that
pathological change has been proven to have an immunological
mechanism. Herein, an immunological mechanism means that leukocytes
show an immune response to allergen stimulation.
[0058] "Autoimmune disease" as used herein, refers broadly to a
disease or disorder arising from and directed against an
individual's own tissues or a co-segregate or manifestation thereof
or resulting condition therefrom.
[0059] "Cancer," as used herein, refers broadly to any neoplastic
disease (whether invasive or metastatic) characterized by abnormal
and uncontrolled cell division causing malignant growth or tumor
(e.g., unregulated cell growth.)
[0060] "Conservatively modified variants," as used herein, applies
to both amino acid and nucleic acid sequences, and with respect to
particular nucleic acid sequences, refers broadly to conservatively
modified variants refers to those nucleic acids which encode
identical or essentially identical amino acid sequences, or where
the nucleic acid does not encode an amino acid sequence, to
essentially identical sequences. Because of the degeneracy of the
genetic code, a large number of functionally identical nucleic
acids encode any given protein. "Silent variations" are one species
of conservatively modified nucleic acid variations. Every nucleic
acid sequence herein which encodes a polypeptide also describes
every possible silent variation of the nucleic acid. One of skill
will recognize that each codon in a nucleic acid (except AUG, which
is ordinarily the only codon for methionine, and TGG, which is
ordinarily the only codon for tryptophan) may be modified to yield
a functionally identical molecule.
[0061] "Costimulatory receptor," as used herein, refers broadly to
receptors which transmit a costimulatory signal to an immune cell,
e.g., CD28 or ICOS.
[0062] "Cytoplasmic domain," as used herein, refers broadly to the
portion of a protein which extends into the cytoplasm of a
cell.
[0063] "Derivative" or "peptide derivative," as used herein,
contain a modification of one or more amino acid residues or a
linker group or other covalently linked group. Non-limiting
examples of derivatives include N-acyl derivatives of the amino
terminal or of another free amino group, esters of the carboxyl
terminal or of another free carboxyl or hydroxy group, amides of
the carboxyl terminal or of another free carboxyl group produced by
reaction with ammonia or with a suitable amine, glycosylated
derivatives, hydroxylated derivatives, nucleotidylated derivatives,
ADP-ribosylated derivatives, pegylated derivatives, phosphorylated
derivatives, derivatives conjugated to lipophilic moieties, and
derivatives conjugated to an antibody or other biological ligand.
Also included among the chemical derivatives are those obtained by
modification of the peptide bond --CO--NH--, for example by
reduction to --CH.sub.2--NH-- or alkylation to --CO--N(alkyl)-.
Preferred derivatisation include, but are not limited tom
C-terminal amidation and N-terminal acetylation, which removes the
negative charge of the C terminus or removes the positive charge at
the N-terminus, respectively. Blocking of the C- or N-terminus,
such as by C-terminal amidation or N-terminal acetylation, may
improve proteolytic stability due to reduced susceptibility to
exoproteolytic digestion. Peptide derivatives having a C-terminal
amide are represented with "NH.sub.2" at the C-terminus.
[0064] "Diagnostic," as used herein, refers broadly to identifying
the presence or nature of a pathologic condition. Diagnostic
methods differ in their sensitivity and specificity. The
"sensitivity" of a diagnostic assay is the percentage of diseased
individuals who test positive (percent of "true positives").
Diseased individuals not detected by the assay are "false
negatives." Subjects who are not diseased and who test negative in
the assay are termed "true negatives." The "specificity" of a
diagnostic assay is 1 minus the false positive rate, where the
"false positive" rate is defined as the proportion of those without
the disease who test positive. While a particular diagnostic method
may not provide a definitive diagnosis of a condition, it suffices
if the method provides a positive indication that aids in
diagnosis.
[0065] "Diagnosing," as used herein refers broadly to classifying a
disease or a symptom, determining a severity of the disease,
monitoring disease progression, forecasting an outcome of a disease
and/or prospects of recovery. The term "detecting" may also
optionally encompass any of the foregoing. Diagnosis of a disease
according to the present invention may, in some embodiments, be
affected by determining a level of a polynucleotide or a
polypeptide of the present invention in a biological sample
obtained from the subject, wherein the level determined can be
correlated with predisposition to, or presence or absence of the
disease. It should be noted that a "biological sample obtained from
the subject" may also optionally comprise a sample that has not
been physically removed from the subject.
[0066] "Effective amount," as used herein, refers broadly to the
amount of a compound, antibody, antigen, or cells that, when
administered to a patient for treating a disease, is sufficient to
effect such treatment for the disease. The effective amount may be
an amount effective for prophylaxis, and/or an amount effective for
prevention. The effective amount may be an amount effective to
reduce, an amount effective to prevent the incidence of
signs/symptoms, to reduce the severity of the incidence of
signs/symptoms, to eliminate the incidence of signs/symptoms, to
slow the development of the incidence of signs/symptoms, to prevent
the development of the incidence of signs/symptoms, and/or effect
prophylaxis of the incidence of signs/symptoms. The "effective
amount" may vary depending on the disease and its severity and the
age, weight, medical history, susceptibility, and pre-existing
conditions, of the patient to be treated. The term "effective
amount" is synonymous with "therapeutically effective amount" for
purposes of this invention.
[0067] "Extracellular domain," as used herein refers broadly to the
portion of a protein that extend from the surface of a cell.
[0068] "Expression vector," as used herein, refers broadly to any
recombinant expression system for the purpose of expressing a
nucleic acid sequence of the invention in vitro or in vivo,
constitutively or inducibly, in any cell, including prokaryotic,
yeast, fungal, plant, insect or mammalian cell. The term includes
linear or circular expression systems. The term includes expression
systems that remain episomal or integrate into the host cell
genome. The expression systems can have the ability to
self-replicate or not, i.e., drive only transient expression in a
cell. The term includes recombinant expression cassettes which
contain only the minimum elements needed for transcription of the
recombinant nucleic acid.
[0069] "Homology," as used herein, refers broadly to a degree of
similarity between a nucleic acid sequence and a reference nucleic
acid sequence or between a polypeptide sequence and a reference
polypeptide sequence. Homology may be partial or complete. Complete
homology indicates that the nucleic acid or amino acid sequences
are identical. A partially homologous nucleic acid or amino acid
sequence is one that is not identical to the reference nucleic acid
or amino acid sequence. The degree of homology can be determined by
sequence comparison. The term "sequence identity" may be used
interchangeably with "homology."
[0070] "Host cell," as used herein, refers broadly to refer to a
cell into which a nucleic acid molecule of the invention, such as a
recombinant expression vector of the invention, has been
introduced. Host cells may be prokaryotic cells (e.g., E. coli), or
eukaryotic cells such as yeast, insect (e.g., SF9), amphibian, or
mammalian cells such as CHO, HeLa, HEK-293, e.g., cultured cells,
explants, and cells in vivo. The terms "host cell" and "recombinant
host cell" are used interchangeably herein. It should be understood
that such terms refer not only to the particular subject cell but
to the progeny or potential progeny of such a cell. Because certain
modifications may occur in succeeding generations due to either
mutation or environmental influences, progeny may not, in fact, be
identical to the parent cell, but are still included within the
scope of the term as used herein.
[0071] "Immune response," as used herein, refers broadly to T
cell-mediated and/or B cell-mediated immune responses that are
influenced by modulation of T cell costimulation. Exemplary immune
responses include B cell responses (e.g., antibody production) T
cell responses (e.g., cytokine production, and cellular
cytotoxicity) and activation of cytokine responsive cells, e.g.,
macrophages. As used herein, the term "down modulation" with
reference to the immune response includes a diminution in any one
or more immune responses, while the term "up modulation" with
reference to the immune response includes an increase in any one or
more immune responses. It will be understood that up modulation of
one type of immune response may lead to a corresponding
downmodulation in another type of immune response. For example, up
modulation of the production of certain cytokines (e.g., IL-10) can
lead to downmodulation of cellular immune responses.
[0072] "Inflammatory disease," as used herein, refers broadly to
chronic or acute inflammatory diseases.
[0073] "Detectable label" as used herein, refers broadly to a
composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, chemical, or other physical means.
[0074] "Mimetic" or "peptidomimetic," as used herein, refers to a
fully or partially synthetic peptide that has the activity of a
given peptide. Such a mimetic or peptidomimetic comprises one or
more amino acid residues that is an artificial chemical mimetic of
a corresponding naturally occurring amino acid, naturally occurring
amino acid polymers and non-naturally occurring amino acid
polymers.
[0075] Modifications of the VISTA and VISTA conjugate polypeptides
described herein include, but are not limited to N-terminus
modification, C-terminus modification, peptide bond modification
(e.g., CH.sub.2--NH, CH.sub.2--S, CH.sub.2--S.dbd.O, O.dbd.C--NH,
CH.sub.2--O, CH.sub.2--CH.sub.2, S.dbd.C--NH, CH.dbd.CH or
CF.dbd.CH), backbone modifications, and residue modification, e.g.,
by the addition of carbohydrate residues to form glycoproteins, by
the addition of chemical residues such as PEG and/or XTEN, etc.
Methods for preparing peptidomimetic compounds are well known in
the art. Martin, (2010).
[0076] "Nucleic acid" or "nucleic acid sequence," as used herein,
refers broadly to a deoxy-ribonucleotide or ribonucleotide
oligonucleotide in either single- or double-stranded form. The term
encompasses nucleic acids, i.e., oligonucleotides, containing known
analogs of natural nucleotides. The term also encompasses
nucleic-acid-like structures with synthetic backbones. Unless
otherwise indicated, a particular nucleic acid sequence also
implicitly encompasses conservatively modified variants thereof
(e.g., degenerate codon substitutions) and complementary sequences,
as well as the sequence explicitly indicated. The term nucleic acid
is used interchangeably with gene, cDNA, mRNA, oligonucleotide, and
polynucleotide.
[0077] "Polypeptide," "peptide" and "protein," are used
interchangeably and refer broadly to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an analog or mimetic of a corresponding
naturally occurring amino acid, as well as to naturally occurring
amino acid polymers. The terms apply to amino acid polymers in
which one or more amino acid residue is an artificial chemical
mimetic of a corresponding naturally occurring amino acid, as well
as to naturally occurring amino acid polymers and non-naturally
occurring amino acid polymer. Polypeptides can be modified, e.g.,
by the addition of carbohydrate residues to form glycoproteins. The
terms "polypeptide," "peptide" and "protein" include glycoproteins,
as well as non-glycoproteins.
[0078] "Prophylactically effective amount," as used herein, refers
broadly to the amount of a compound that, when administered to a
patient for prophylaxis of a disease or prevention of the
reoccurrence of a disease, is sufficient to effect such prophylaxis
for the disease or reoccurrence. The prophylactically effective
amount may be an amount effective to prevent the incidence of signs
and/or symptoms. The "prophylactically effective amount" may vary
depending on the disease and its severity and the age, weight,
medical history, predisposition to conditions, preexisting
conditions, of the patient to be treated.
[0079] "Prophylaxis," as used herein, refers broadly to a course of
therapy where signs and/or symptoms are not present in the patient,
are in remission, or were previously present in a patient.
Prophylaxis includes preventing disease occurring subsequent to
treatment of a disease in a patient. Further, prevention includes
treating patients who may potentially develop the disease,
especially patients who are susceptible to the disease (e.g.,
members of a patent population, those with risk factors, or at risk
for developing the disease).
[0080] "Recombinant" as used herein, refers broadly with reference
to a product, e.g., to a cell, or nucleic acid, protein, or vector,
indicates that the cell, nucleic acid, protein or vector, has been
modified by the introduction of a heterologous nucleic acid or
protein or the alteration of a native nucleic acid or protein, or
that the cell is derived from a cell so modified. Thus, for
example, recombinant cells express genes that are not found within
the native (non-recombinant) form of the cell or express native
genes that are otherwise abnormally expressed, under expressed or
not expressed at all.
[0081] "Sequence identity," as used herein, refers broadly to a
degree of similarity between a nucleic acid sequence and a
reference nucleic acid sequence or between a polypeptide sequence
and a reference polypeptide sequence. Sequence identity (also
synonymous with "homology") may be partial or complete. Complete
sequence identity indicates that the nucleic acid or amino acid
sequences are identical, i.e., 100% sequence identity. A partially
homologous nucleic acid or amino acid sequence is one that is not
identical to the reference nucleic acid or amino acid sequence. The
degree of homology can be determined by sequence comparison, e.g.,
60% identity, 70% identity, 80% identity, 90% identity, 95%
identity, 97% identity, 98% identity, or 99% identity.
[0082] "Signs" of disease, as used herein, refers broadly to any
abnormality indicative of disease, discoverable on examination of
the patient; an objective indication of disease, in contrast to a
symptom, which is a subjective indication of disease.
[0083] "Subject," as used herein, refers broadly to any animal that
is in need of treatment either to alleviate a disease state or to
prevent the occurrence or reoccurrence of a disease state. Also,
"subject" as used herein, refers broadly to any animal that has
risk factors, a history of disease, susceptibility, symptoms, and
signs, was previously diagnosed, is at risk for, or is a member of
a patient population for a disease. The subject may be a clinical
patient such as a human or a veterinary patient such as a
companion, domesticated, livestock, exotic, or zoo animal. The term
"subject" may be used interchangeably with the term "patient."
[0084] "Symptoms" of disease as used herein, refers broadly to any
morbid phenomenon or departure from the normal in structure,
function, or sensation, experienced by the patient and indicative
of disease.
[0085] "T cell," as used herein, refers broadly to CD4+ T cells and
CD8+ T cells. The term T cell also includes both T helper 1 type T
cells and T helper 2 type T cells.
[0086] "Therapy," "therapeutic," "treating," or "treatment", as
used herein, refers broadly to treating a disease, arresting, or
reducing the development of the disease or its clinical symptoms,
and/or relieving the disease, causing regression of the disease or
its clinical symptoms. Therapy encompasses prophylaxis, treatment,
remedy, reduction, alleviation, and/or providing relief from a
disease, signs, and/or symptoms of a disease. Therapy encompasses
an alleviation of signs and/or symptoms in patients with ongoing
disease signs and/or symptoms (e.g., inflammation, pain). Therapy
also encompasses "prophylaxis". The term "reduced", for purpose of
therapy, refers broadly to the clinical significant reduction in
signs and/or symptoms. Therapy includes treating relapses or
recurrent signs and/or symptoms (e.g., inflammation, pain). Therapy
encompasses but is not limited to precluding the appearance of
signs and/or symptoms anytime as well as reducing existing signs
and/or symptoms and eliminating existing signs and/or symptoms.
Therapy includes treating chronic disease ("maintenance") and acute
disease. For example, treatment includes treating or preventing
relapses or the recurrence of signs and/or symptoms (e.g.,
inflammation, pain).
[0087] "Transmembrane domain," as used herein, refers broadly to an
amino acid sequence of about 15 amino acid residues in length which
spans the plasma membrane. More preferably, a transmembrane domain
includes about at least 20, 25, 30, 35, 40, or 45 amino acid
residues and spans the plasma membrane. Transmembrane domains are
rich in hydrophobic residues, and typically have an alpha-helical
structure. In an embodiment, at least 50%, 60%, 70%, 80%, 90%, 95%
or more of the amino acids of a transmembrane domain are
hydrophobic, e.g., leucines, isoleucines, tyrosines, or
tryptophans. Transmembrane domains are described in, for example,
Zagotta, et al. (1996) Annu. Rev. Neurosci. 19:235-263.
[0088] "Tumor," as used herein, refers broadly to at least one cell
or cell mass in the form of a tissue neoformation, in particular in
the form of a spontaneous, autonomous and irreversible excess
growth, which is more or less disinhibited, of endogenous tissue,
which growth is as a rule associated with the more or less
pronounced loss of specific cell and tissue functions. This cell or
cell mass is not effectively inhibited, in regard to its growth, by
itself or by the regulatory mechanisms of the host organism, e.g.,
melanoma or carcinoma. Tumor antigens not only include antigens
present in or on the malignant cells themselves, but also include
antigens present on the stromal supporting tissue of tumors
including endothelial cells and other blood vessel components.
[0089] "Vector," as used herein, refers broadly to a nucleic acid
molecule capable of transporting another nucleic acid molecule to
which it has been linked. One type of vector is a "plasmid", which
refers to a circular double stranded DNA loop into which additional
DNA segments may be ligated. Another type of vector is a viral
vector, wherein additional DNA segments may be ligated into the
viral genome. Certain vectors are capable of autonomous replication
in a host cell into which they are introduced (e.g., bacterial
vectors having a bacterial origin of replication and episomal
mammalian vectors). Other vectors (e.g., non-episomal mammalian
vectors) are integrated into the genome of a host cell upon
introduction into the host cell, and thereby are replicated along
with the host genome. Moreover, certain vectors are capable of
directing the expression of genes to which they are operatively
linked. Vectors are referred to herein as "recombinant expression
vectors" or simply "expression vectors". In general, expression
vectors of utility in recombinant DNA techniques are often in the
form of plasmids. In the present specification, "plasmid" and
"vector" may be used interchangeably as the plasmid is the most
commonly used form of vector. However, the invention is intended to
include such other forms of expression vectors, such as viral
vectors (e.g., replication defective retroviruses, adenoviruses and
adeno-associated viruses), which serve equivalent functions. The
techniques and procedures are generally performed according to
conventional methods well known in the art and as described in
various general and more specific references that are cited and
discussed throughout the present specification. See, e.g.,
Sambrook, et al. (2001) Molec. Cloning: Lab. Manual [3rd Ed] Cold
Spring Harbor Laboratory Press. Standard techniques may be used for
recombinant DNA, oligonucleotide synthesis, and tissue culture, and
transformation (e.g., electroporation, lipofection). Enzymatic
reactions and purification techniques may be performed according to
manufacturer's specifications or as commonly accomplished in the
art or as described herein.
[0090] The nomenclatures utilized in connection with, and the
laboratory procedures and techniques of, analytical chemistry,
synthetic organic chemistry, and medicinal and pharmaceutical
chemistry described herein are those well known and commonly used
in the art. Standard techniques may be used for chemical syntheses,
chemical analyses, pharmaceutical preparation, formulation, and
delivery, and treatment of patients.
VISTA and VISTA Antagonists
[0091] This application relates to a peptide antagonist that can
recognize and suppress the inhibitory activity of VISTA. This
peptide, designated herein as AP1049, was discovered through phage
display and shown to exhibit superior bioactivity when compared to
an anti-VISTA monoclonal antibody. Given its neutralizing activity,
AP1049 can be used to, e.g., treat cancer and/or pathogenic, i.e.,
bacterial, fungal, parasite or viral infections and enhance
anti-tumor immune responses and suppress tumor growth.
[0092] Accordingly, the present invention is a VISTA antagonistic
peptide, as well as multimers, conjugates, analogs, derivatives and
mimetics thereof and methods of using this peptide to inhibit or
suppress the activity of VISTA. As used herein, the term "peptide"
denotes an amino acid polymer that is composed of at least two
amino acids covalently linked by an amide bond. Peptides of the
present invention are desirably 10 to 20 residues in length, or
more desirably 12 to residues in length. In certain embodiments, a
VISTA antagonistic peptide is a 12 to 20 residue peptide containing
the amino acid sequence of SEQ ID NO:1. In other embodiments of the
present invention, the isolated VISTA antagonist comprises a
peptide that is identical to the amino acid sequence of SEQ ID NO:1
(Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His), or which
comprises a peptide having an amino acid sequence that differs from
SEQ ID NO:1 by at most 1 amino acid residue or at most 2 amino acid
residues, or an multimer, conjugate, analog, derivative or mimetic
thereof. In yet other embodiments of the invention, the isolated
VISTA antagonist consists of the amino acid sequence of SEQ ID NO:1
(Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His).
[0093] In certain embodiments of the present invention, cysteine
residues at positions 4 and 11 of the VISTA antagonistic peptide
(or their corresponding positions in a variant of the VISTA
antagonist) form a disulfide bridge.
[0094] In accordance with the present invention, multimers,
conjugates, analogs, derivatives and mimetics of the peptide of the
invention are also provided.
[0095] An analog is a molecule that differs in chemical structure
from a parent compound, for example a homolog (differing by an
increment in the chemical structure, such as a difference one amino
acid residue), a structure that differs by one or more functional
groups, or a change in ionization. Structural analogs are often
found using quantitative structure activity relationships (QSAR),
with techniques such as those disclosed in Remington (The Science
and Practice of Pharmacology, 19th Edition (1995), chapter 28).
[0096] Analogs can be prepared by modifying the amino acids
sequence of SEQ ID NO:1. The simplest modifications involve the
substitution of one or more amino acids for amino acids having
similar physiochemical and/or structural properties. These
so-called conservative substitutions are likely to have minimal
impact on the activity and/or structure of the resultant peptide.
Examples of conservative substitutions include substituting a
serine with a threonine, substituting alanine with a serine or
valine, substituting aspartic acid with glutamic acid, substituting
tryptophan with a tyrosine, substituting isoleucine with leucine or
valine, substituting arginine with lysine, and/or substituting
histidine with arginine or lysine. Conservative substitutions
generally maintain (a) the structure of the peptide backbone in the
area of the substitution, for example, as a helical conformation,
(b) the charge or hydrophobicity of the molecule at the target
site, or (c) the bulk of the side chain.
[0097] Amino acid substitutions are typically classified in one or
more categories, including polar, hydrophobic, acidic, basic and
aromatic, according to their side chains. Examples of polar amino
acids include those having side chain functional groups such as
hydroxyl, sulfhydryl, and amide, as well as the acidic and basic
amino acids. Polar amino acids include, without limitation,
asparagine, cysteine, glutamine, histidine, selenocysteine, serine,
threonine, tryptophan and tyrosine. Examples of hydrophobic or
non-polar amino acids include those residues having non-polar
aliphatic side chains, such as, without limitation, leucine,
isoleucine, valine, glycine, alanine, proline, methionine and
phenylalanine. Examples of basic amino acid residues include those
having a basic side chain, such as an amino or guanidino group.
Basic amino acid residues include, without limitation, arginine,
homolysine and lysine. Examples of acidic amino acid residues
include those having an acidic side chain functional group, such as
a carboxy group. Acidic amino acid residues include, without
limitation aspartic acid and glutamic acid. Aromatic amino acids
include those having an aromatic side chain group. Examples of
aromatic amino acids include, without limitation, biphenylalanine,
histidine, 2-napthylalananine, pentafluorophenylalanine,
phenylalanine, tryptophan and tyrosine. It is noted that some amino
acids are classified in more than one group, for example,
histidine, tryptophan and tyrosine are classified as both polar and
aromatic amino acids. Additional amino acids that are classified in
each of the above groups are known to those of ordinary skill in
the art.
[0098] As used herein, a peptide derivative is a molecule which
retains the primary amino acids of the peptide, however, the
N-terminus, C-terminus, and/or one or more of the side chains of
the amino acids therein have been chemically altered or
derivatized. Such derivatized peptides include, for example,
naturally occurring amino acid derivatives, for example,
4-hydroxyproline for proline, 5-hydroxylysine for lysine,
homoserine for serine, ornithine for lysine, and the like. Other
derivatives or modifications include, e.g., a label, such as
fluorescein or tetramethylrhodamine; or one or more
post-translational modifications such as acetylation, amidation,
formylation, hydroxylation, methylation, phosphorylation,
sulfatation, glycosylation, or lipidation. Indeed, certain chemical
modifications, in particular N-terminal glycosylation, have been
shown to increase the stability of peptides in human serum (Powell
et al. (1993) Pharma. Res. 10:1268-1273). Peptide derivatives also
include those with increased membrane permeability obtained by
N-myristoylation (Brand, et al. (1996) Am. J. Physiol. Cell.
Physiol. 270:C1362-C1369).
[0099] In addition, a peptide derivative of the invention can
include a cell-penetrating sequence which facilitates, enhances, or
increases the transmembrane transport or intracellular delivery of
the peptide into a cell. For example, a variety of proteins,
including the HIV-1 Tat transcription factor, Drosophila
Antennapedia transcription factor, as well as the herpes simplex
virus VP22 protein have been shown to facilitate transport of
proteins into the cell (Wadia and Dowdy (2002) Curr. Opin.
Biotechnol. 13:52-56). Further, an arginine-rich peptide (Futaki
(2002) Int. J. Pharm. 245:1-7), a polylysine peptide containing Tat
PTD (Hashida, et al. (2004) Br. J. Cancer 90(6):1252-8), Pep-1
(Deshayes, et al. (2004) Biochemistry 43(6):1449-57) or an HSP70
protein or fragment thereof (WO 00/31113) is suitable for enhancing
intracellular delivery of a peptide or mimetic of the invention
into the cell.
[0100] While a peptide of the invention can be derivatized with by
one of the above indicated modifications, it is understood that a
peptide of this invention may contain more than one of the above
described modifications within the same peptide.
[0101] A mimetic or peptidomimetic refers to a synthetic chemical
compound which has substantially the same structural and/or
functional characteristics of a peptide of the invention. The
mimetic can be entirely composed of synthetic, non-natural amino
acid analogues, or can be a chimeric molecule including one or more
natural peptide amino acids and one or more non-natural amino acid
analogs. The mimetic can also incorporate any number of natural
amino acid conservative substitutions as long as such substitutions
do not destroy the activity of the mimetic. Routine testing can be
used to determine whether a mimetic has the requisite activity,
e.g., that it can inhibit the activity of VISTA.
[0102] The phrase "substantially the same," when used in reference
to a mimetic or peptidomimetic, means that the mimetic or
peptidomimetic has one or more activities or functions of the
referenced molecule, e.g., the ability to enhance T cell
proliferation.
[0103] There are clear advantages for using a mimetic of a given
peptide. For example, there are considerable cost savings and
improved patient compliance associated with peptidomimetics, since
they can be administered orally compared with parenteral
administration for peptides. Furthermore, peptidomimetics are much
cheaper to produce than peptides.
[0104] Thus, a peptide of this invention has utility in the
development of such small chemical compounds with similar
biological activities and therefore with similar therapeutic
utilities. The techniques of developing peptidomimetics are
conventional. For example, peptide bonds can be replaced by
non-peptide bonds or non-natural amino acids that allow the
peptidomimetic to adopt a similar structure, and therefore
biological activity, to the original peptide. Further modifications
can also be made by replacing chemical groups of the amino acids
with other chemical groups of similar structure. The development of
peptidomimetics can be aided by determining the tertiary structure
of the original peptide by NMR spectroscopy, crystallography and/or
computer-aided molecular modeling. These techniques aid in the
development of novel compositions of higher potency and/or greater
bioavailability and/or greater stability than the original peptide
(Dean (1994) BioEssays 16:683-687; Cohen & Shatzmiller (1993)
J. Mol. Graph. 11:166-173; Wiley & Rich (1993) Med. Res. Rev.
13:327-384; Moore (1994) Trends Pharmacol. Sci. 15:124-129; Hruby
(1993) Biopolymers 33:1073-1082; Bugg, et al. (1993) Sci. Am.
269:92-98). Once a potential peptidomimetic compound is identified,
it may be synthesized and assayed using an assay described herein
or any other appropriate assay for monitoring VISTA activity.
[0105] Peptide mimetic compositions can contain any combination of
non-natural structural components, which are typically from three
structural groups: residue linkage groups other than the natural
amide bond ("peptide bond") linkages; non-natural residues in place
of naturally occurring amino acid residues; residues which induce
secondary structural mimicry, i.e., induce or stabilize a secondary
structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix
conformation, and the like; or other changes which confer
resistance to proteolysis. For example, a peptide can be
characterized as a mimetic when one or more of the residues are
joined by chemical means other than an amide bond. Individual
peptidomimetic residues can be joined by amide bonds, non-natural
and non-amide chemical bonds other chemical bonds or coupling means
including, for example, glutaraldehyde, N-hydroxysuccinimide
esters, bifunctional maleimides, N,N'-dicyclohexylcarbodiimide
(DCC) or N,N'-diisopropyl-carbodiimide (DIC). Linking groups
alternative to the amide bond include, for example, ketomethylene
(e.g., --C(.dbd.O)--CH.sub.2-- for --C(.dbd.O)--NH--),
aminomethylene (CH.sub.2--NH), ethylene, olefin (CH.dbd.CH), ether
(CH.sub.2--O), thioether (CH.sub.2--S), tetrazole (CN.sub.4--),
thiazole, retroamide, thioamide, or ester (see, e.g., Spatola
(1983) in Chemistry and Biochemistry of Amino Acids, Peptides and
Proteins, 7:267-357, "Peptide and Backbone Modifications," Marcel
Decker, NY).
[0106] As discussed, a peptide can be characterized as a mimetic by
containing one or more non-natural residues in place of a naturally
occurring amino acid residue. Non-natural residues are known in the
art. Particular non-limiting examples of non-natural residues
useful as mimetics of natural amino acid residues are mimetics of
aromatic amino acids include, for example, D- or L-naphylalanine;
D- or L-phenylglycine; D- or L-2 thieneylalanine; D- or L-1, -2,
3-, or 4-pyreneylalanine; D- or L-3 thieneylalanine; D- or
L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- or
L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;
D-(trifluoromethyl)-phenylglycine;
D-(trifluoromethyl)-phenylalanine; D-p-fluoro-phenylalanine; D- or
L-p-biphenylphenylalanine; D- or
L-p-methoxy-biphenyl-phenylalanine; and D- or
L-2-indole(alkyl)alanines, where alkyl can be substituted or
unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl,
isopropyl, iso-butyl, sec-isotyl, iso-pentyl, or a non-acidic amino
acid. Aromatic rings of a non-natural amino acid that can be used
in place a natural aromatic ring include, for example, thiazolyl,
thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl,
and pyridyl aromatic rings.
[0107] Mimetics of acidic amino acids can be generated by
substitution with non-carboxylate amino acids while maintaining a
negative charge; (phosphono)alanine; and sulfated threonine.
Carboxyl side groups (e.g., aspartyl or glutamyl) can also be
selectively modified by reaction with carbodiimides
(R'--N--C--N--R') including, for example,
1-cyclohexyl-3(2-morpholinyl-(4-ethyl)carbodiimide or
1-ethyl-3(4-azonia-4,4-dimetholpentyl)carbodiimide. Aspartyl or
glutamyl groups can also be converted to asparaginyl and glutaminyl
groups by reaction with ammonium ions.
[0108] Lysine mimetics can be generated (and amino terminal
residues can be altered) by reacting lysinyl with succinic or other
carboxylic acid anhydrides. Lysine and other alpha-amino-containing
residue mimetics can also be generated by reaction with
imidoesters, such as methyl picolinimidate, pyridoxal phosphate,
pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,
O-methylisourea, 2,4, pentanedione, and transamidase-catalyzed
reactions with glyoxylate.
[0109] One or more residues can also be replaced by an amino acid
(or peptidomimetic residue) of the opposite chirality. Thus, any
amino acid naturally occurring in the L-configuration (which can
also be referred to as R or S, depending upon the structure of the
chemical entity) can be replaced with the same amino acid or a
mimetic, but of the opposite chirality, referred to as the D-amino
acid, but which can additionally be referred to as the R- or
S-form.
[0110] As will be appreciated by one skilled in the art, a
peptidomimetic of the present invention can also include one or
more of the modifications described herein for derivatized
peptides, e.g., a detectable label (such as an effector label or a
radionuclide), a therapeutic agent (such as a chemotherapeutic
agent), one or more post-translational modifications, or
cell-penetrating sequence.
[0111] For example, the VISTA antagonists described herein may be
modified post-translationally to add effector labels such as
chemical linkers, detectable labels such as for example fluorescent
dyes, enzymes, substrates, bioluminescent materials, radioactive
materials, and chemiluminescent labels, or functional labels such
as for example streptavidin, avidin, biotin, a cytotoxin, a
cytotoxic agent, and radioactive materials. Further exemplary
enzymes include, but are not limited to, horseradish peroxidase,
acetylcholinesterase, alkaline phosphatase, .beta.-galactosidase
and luciferase. Further exemplary fluorescent materials include,
but are not limited to, rhodamine, fluorescein, fluorescein
isothiocyanate, umbelliferone, dichlorotriazinylamine,
phycoerythrin and dansyl chloride. Further exemplary
chemiluminescent labels include, but are not limited to, luminol.
Further exemplary bioluminescent materials include, but are not
limited to, luciferin, luciferase, and aequorin. Further exemplary
radioactive materials include, but are not limited to, bismuth-213
(.sup.213Bs), carbon-14 (.sup.14C), carbon-11 (.sup.11C),
chlorine-C18 (Cl.sup.18), chromium-51 (.sup.51Cr), cobalt-57
(.sup.57Co), cobalt-60 (.sup.60Co), copper-64 (.sup.64Cu),
copper-67 (.sup.67Cu), dysprosium-165 (.sup.165Dy), erbium-169
(.sup.169Er), fluorine-18 (.sup.18F), gallium-67 (.sup.67Ga),
gallium-68 (.sup.68Ga), germanium-68 (.sup.68Ge), holmium-166
(.sup.166Ho), indium-111 (.sup.111In), iodine-125 (.sup.125I),
iodine-123 (.sup.124I), iodine-124 (.sup.124I), iodine-131
(.sup.131I), iridium-192 (.sup.192Ir), iron-59 (.sup.59Fe),
krypton-81 (.sup.81Kr), lead-212 (.sup.212Pb), lutetium-177
(.sup.177Lu), molybdenum-99 (.sup.99Mo), nitrogen-13 (.sup.13N),
oxygen-15 (.sup.15O), palladium-103 (.sup.103Pd), phosphorus-32
(.sup.32P), potassium-42 (.sup.42K), rhenium-186 (.sup.186Re),
rhenium-188 (.sup.188Re), rubidium-81 (.sup.81Rb), rubidium-82
(.sup.82Rb), samarium-153 (.sup.153Sm) , selenium-75 (.sup.75Se),
sodium-24 (.sup.24Na), strontium-82 (.sup.82Sr), strontium-89
(.sup.89Sr), sulfur 35 (.sup.35S), technetium-99m (.sup.99 Tc),
thallium-201 (.sup.201Tl), tritium (.sup.3H), xenon-133
(.sup.133Xe), ytterbium-169 (.sup.169Yb), ytterbium-177
(.sup.177Yb), and yttrium-90 (.sup.90Y).
[0112] Additionally, the VISTA antagonists provided herein may be
modified to add a therapeutic agent including, but not limited to,
chemotherapeutic agents such as carboplatin, cisplatin, paclitaxel,
gemcitabine, calicheamicin, doxorubicin, 5-fluorouracil, mitomycin
C, actinomycin D, cyclophosphamide, vincristine, bleomycin, VEGF
antagonists, EGFR antagonists, platins, taxols, irinotecan,
5-fluorouracil, gemcytabine, leucovorine, steroids,
cyclophosphamide, melphalan, vinca alkaloids (e.g., vinblastine,
vincristine, vindesine and vinorelbine), mustines, tyrosine kinase
inhibitors, radiotherapy, sex hormone antagonists, selective
androgen receptor modulators, selective estrogen receptor
modulators, PDGF antagonists, TNF antagonists, IL-1 antagonists,
interleukins (e.g., IL-12 or IL-2), IL-12R antagonists, Toxin
conjugated monoclonal antibodies, tumor antigen specific monoclonal
antibodies, Erbitux.RTM., Avastin.RTM., Pertuzumab, anti-CD20
antibodies, Rituxan.RTM., ocrelizumab, ofatumumab, DXL625,
Herceptin.RTM., or any combination thereof. Toxic enzymes from
plants and bacteria such as ricin, diphtheria toxin and Pseudomonas
toxin may be conjugated to the VISTA antagonists to generate
cell-type-specific-killing reagents. Youle, et al. (1980) Proc.
Nat'l Acad. Sci. USA 77:5483; Gilliland, et al. (1980) Proc. Nat'l
Acad. Sci. USA 77:4539; Krolick, et al. (1980) Proc. Nat'l Acad.
Sci. USA 77:5419.
[0113] Furthermore, the VISTA antagonists described herein may be
conjugated to a radionuclide that emits alpha or beta particles
(e.g., radioimmunoconjuagtes). Such radioactive isotopes include
but are not limited to beta-emitters such as phosphorus-32
(.sup.32P), scandium-47 (.sup.47Sc), copper-67 (.sup.67Cu),
gallium-67 (.sup.67Ga), yttrium-88 (.sup.HY), yttrium-90
(.sup.90Y), iodine-125 (.sup.125I), iodine-131 (.sup.131I),
samarium-153 (.sup.153Sm), lutetium-177 (.sup.177Lu), rhenium-186
(.sup.186Re), rhenium-188 (.sup.188Re), and alpha-emitters such as
astatine-211 (.sup.211At), lead-212 (.sup.212Pb), bismuth-212
(.sup.212Bi), bismuth-213 (.sup.213Bi) or actinium-225
(.sup.225Ac).
[0114] Methods are known in the art for conjugating a VISTA
antagonist described herein to a label, such as those methods
described by Hunter, et al (1962) Nature 144: 945; David, et al.
(1974) Biochemistry 13: 1014; Pain, et al. (1981) J. Immunol. Meth.
40: 219; and Nygren (1982) Histochem and Cytochem, 30: 407.
[0115] Additionally, the VISTA antagonists described herein may
comprise another moiety, i.e., a "targeting moiety," that targets
the antagonist peptide to a target site (such as a cancer cell, a
tumor, a virally-infected cell, etc). The targeting moiety may be
selected from an antibody or ligand that binds to an antigen, a
receptor expressed by the target cell or an infectious agent.
[0116] The VISTA antagonist (as well as multimers, conjugates,
analogs, derivatives and mimetics thereof) may also be directly or
indirectly attached to an immunoglobulin polypeptide or a fragment
thereof, e.g., a antibody constant region.
[0117] A "conjugate," as used herein, refers to a compound having
at least one isolated VISTA antagonist peptide and one
immunoglobulin polypeptide or a fragment thereof, e.g., antibody
constant region, joined at the polypeptide level, with or without
the use of a linker. A conjugate may be a fusion polypeptide
produced as the result of joining at the nucleic acid level of
genes encoding at least one natriuretic peptide and one antibody
constant region, with or without a coding sequence for a peptide
linker.
[0118] Such VISTA antagonist peptide-antibody conjugates may have a
higher serum stability, e.g., at least 20%, preferably at least
30%, 50%, 80%, 100%, 200% or more, increase in the serum half-life
when compared with the antagonist peptide without the antibody
constant region under the same conditions. A human antibody, e.g.,
a human IgG, such as IgG1, IgG2, IgG3 or IgG4, is frequently used
to derive a constant region or a fragment thereof for the purpose
of making a natriuretic peptide conjugate of this invention.
[0119] As used herein, an "antibody (or immunoglobulin) constant
region" refers to a polypeptide that corresponds to at least a
portion of the constant region of an antibody heavy chain or light
chain, such portion including at least one constant domain (e.g.,
the constant domain of CL or one of the constant domains of
C.sub.H). For example, an "antibody constant region" used for
making the conjugates of this invention may be derived from an
antibody heavy chain and include two out of three (C.sub.H2 and
C.sub.H3 for IgA, IgD, and IgG) or three out of four (C.sub.H2,
CH3, and CH4, for IgE and IgM) constant domains; the first constant
domain (C.sub.HI) may be present in some cases but may be excluded
in others. Such an antibody constant region can be obtained by a
variety of means, e.g., by a recombinant method or synthetic
method, or by purification subsequent to enzymatic digestion, for
instance, pepsin or papain digestion of an intact antibody or an
antibody heavy or light chain.
[0120] Further encompassed by this term as used in this application
are polypeptides having a substantial sequence identity (for
instance, at least 80%, 85%, 90%, 95% or more) to the corresponding
amino acid sequence of an antibody heavy or light chain constant
region or a portion thereof that contains at least one constant
domain nearest to the C-terminus of the antibody chain, so long as
the presence of such an "antibody constant region" in a VISTA
antagonist peptide-antibody constant region conjugate renders the
conjugate a higher serum stability.
[0121] Additionally, the peptide, multimer, conjugate, analog,
derivative or mimetic may be modified to increase certain
properties, e.g., biological half life. Various approaches are
possible including, but not limited to, N-terminal
modification/conjugation (e.g., lipidation or acetylation),
C-terminal modification/conjugation (e.g., lipidation or
acetylation), amino acid substitutions (i.e., substitution of
natural amino acid with unnatural amino acids, such as
D-conformation, N-methylation, tetra-substitution, beta-amino
acids, etc.), peptide backbone modifications (e.g., chemical
modification of peptide bonds, such as simple reductions or
replacement of carbonyl or amide groups with esters, sulfides and
alkyls), side chain modifications and/or cyclization (e.g.,
disulfide bond formation).
[0122] In one embodiment, the peptide may be pegylated to, e.g.,
increase the biological (e.g., serum) half life of the antibody. To
pegylate a peptide, typically it is reacted with polyethylene
glycol (PEG), such as a reactive ester or aldehyde derivative of
PEG, under conditions in which one or more PEG groups become
attached to the peptide. Preferably, the pegylation is carried out
via an acylation reaction or an alkylation reaction with a reactive
PEG molecule (or an analogous reactive water-soluble polymer.
[0123] Similarly, in another embodiment, a peptide, multimer,
conjugate, analog, derivative or mimetic may be modified by
conjugation of polysialic acid (PSA) to increase half-life.
[0124] Additionally, the peptide, multimer, conjugate, analog,
derivative or mimetic may be modified, e.g., genetically fused or
chemically conjugated, to comprise extended recombinant polypeptide
(XTEN), through a process called XTENylation, to improve its half
life. XTEN is a long, hydrophilic, and unstructured protein-based
polymer of 864 amino acids. See, e.g., WO 2013/130683 which is
herein expressly incorporated by reference in its entirety. When
attached to a molecule of interest, greatly increases the effective
size of the molecule, thereby prolonging its presence in serum by
slowing kidney clearance in a manner analogous to that of PEG. In
addition to slowing kidney clearance, attachment to XTEN can also
inhibit receptor-mediated clearance by reducing the ligand's
affinity for its receptor. XTEN coupling chemistries include, but
are not limited to, Thiol-XTEN; Maleimide-XTEN; Alkyne-XTEN; and
Iodoacetyl-XTEN.
[0125] Moreover, the peptide, multimer, conjugate, analog,
derivative or mimetic may be modified with recombinant albumin,
e.g., Novozymes Recombumin.RTM., to improve half life. The peptide
can be genetically fused or chemically conjugated to a recombinant
albumin using standard protocols.
[0126] Furthermore, the peptide, multimer, conjugate, analog,
derivative or mimetic may be modified by the addition and/or
removal of specific amino acids to and/or from the peptide. For
example, a number of specific amino acids may be added to the
peptide, thereby strengthening or tightening its molecular
structure to make it less susceptible to biological degradation
and, thus, providing a longer life-span in the blood, using, e.g.,
Zealand Structure Induced Probe (SIP.RTM.) tail technology.
[0127] Yet another exemplary method for improving the stability and
therapeutic potential of peptides, analogs, derivatives or mimetics
is multimerization. For example, a multimer may comprise two or
more copies, e.g., 2, 3, 4, 5, 6, or more, of the isolated VISTA
antagonist or variant thereof. Multimers include both homomultimers
and heteromultimers. Multimerization can result in increased
peptide stability, higher binding strength (due to multiple
valencies in the molecule), and/or improved pharmacokinetic
properties.
[0128] Another exemplary approach for improving the stability and,
thus, therapeutic potential of the VISTA antagonist peptides,
multimers, conjugates, analogs, derivatives or mimetics disclosed
herein is the addition of acetyl groups to the N and/or C terminus
of the peptide. Acetylation may protect the peptide from
exopeptidases, thereby extending the half-life of the peptide.
Production of VISTA Antagonists
[0129] The peptide multimer, conjugate, analog, derivative or
mimetic can be produced and isolated using any method known in the
art. Peptides can be synthesized, whole or in part, using chemical
methods known in the art (see, e.g., Caruthers (1980) Nucleic Acids
Res. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser.
225-232; and Banga (1995) Therapeutic Peptides and Proteins,
Formulation, Processing and Delivery Systems, Technomic Publishing
Co., Lancaster, Pa.). Peptide synthesis can be performed using
various solid-phase techniques (see, e.g., Roberge (1995) Science
269:202; Merrifield (1997) Methods Enzymol. 289:3-13) and automated
synthesis may be achieved, e.g., using the ABI 431A Peptide
Synthesizer (Perkin Elmer) in accordance with the manufacturer's
instructions.
[0130] Individual synthetic residues and peptides incorporating
mimetics can be synthesized using a variety of procedures and
methodologies known in the art (see, e.g., Organic Syntheses
Collective Volumes, Gilman, et al. (Eds) John Wiley & Sons,
Inc., NY). Peptides and peptide mimetics can also be synthesized
using combinatorial methodologies. Techniques for generating
peptide and peptidomimetic libraries are well-known, and include,
for example, multipin, tea bag, and split-couple-mix techniques
(see, for example, al-Obeidi (1998) Mol. Biotechnol. 9:205-223;
Hruby (1997) Curr. Opin. Chem. Biol. 1:114-119; Ostergaard (1997)
Mol. Divers. 3:17-27; and Ostresh (1996) Methods Enzymol.
267:220-234). Modified peptides can be further produced by chemical
modification methods (see, for example, Belousov (1997) Nucleic
Acids Res. 25:3440-3444; Frenkel (1995) Free Radic. Biol. Med.
19:373-380; and Blommers (1994) Biochemistry 33:7886-7896).
[0131] Alternatively, a peptide of this invention can be prepared
in recombinant protein systems using polynucleotide sequences
encoding the peptides. By way of illustration, a nucleic acid
molecule encoding a peptide of the invention is introduced into a
host cell, such as bacteria, yeast or mammalian cell, under
conditions suitable for expression of the peptide, and the peptide
is purified or isolated using methods known in the art. See, e.g.,
Deutscher et al. (1990) Guide to Protein Purification: Methods in
Enzymology Vol. 182, Academic Press. In particular embodiments, the
peptide, or analog, derivative or mimetic thereof is isolated
and/or purified to homogeneity (e.g. greater than 90% purity).
[0132] It is contemplated that the peptide disclosed herein can be
used as a lead compound for the design and synthesis of compounds
with improved efficacy, clearance, half-lives, and the like.
[0133] One approach includes structure-activity relationship (SAR)
analysis (e.g., NMR analysis) to determine specific binding
interactions between the peptide and VISTA to facilitate the
development of more efficacious agents. Agents identified in such
SAR analysis or from agent libraries can then be screened for their
ability to, e.g., decrease the activity of VISTA and/or enhance T
cell proliferation.
Pharmaceutical Compositions
[0134] The VISTA antagonist peptide, multimer, conjugate, analog,
derivative and mimetic thereof described herein can be provided in
a pharmaceutical composition.
[0135] A "pharmaceutical composition" refers to a chemical or
biological composition suitable for administration to a mammal.
Such compositions may be specifically formulated for administration
via one or more of a number of routes, including but not limited to
buccal, epicutaneous, epidural, inhalation, intraarterial,
intracardial, intracerebroventricular, intradermal, intramuscular,
intranasal, intraocular, intraperitoneal, intraspinal, intrathecal,
intravenous, oral, parenteral, rectally via an enema or
suppository, subcutaneous, subdermal, sublingual, transdermal, and
transmucosal. In addition, administration may occur by means of
injection, powder, liquid, gel, drops, or other means of
administration.
[0136] A "pharmaceutical excipient" or a "pharmaceutically
acceptable excipient" is a carrier, usually a liquid, in which an
active therapeutic agent is formulated. In one embodiment of the
invention, the active therapeutic agent is a humanized antibody
described herein, or one or more fragments thereof. The excipient
generally does not provide any pharmacological activity to the
formulation, though it may provide chemical and/or biological
stability, and release characteristics. Exemplary formulations may
be found, for example, in Grennaro (2005) [Ed.] Remington: The
Science and Practice of Pharmacy [21.sup.st Ed.]
[0137] Pharmaceutical compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
invention contemplates that the pharmaceutical composition is
present in lyophilized form. The composition may be formulated as a
solution, microemulsion, liposome, or other ordered structure
suitable to high drug concentration. The carrier may be a solvent
or dispersion medium containing, for example, water, ethanol,
polyol (for example, glycerol, propylene glycol, and liquid
polyethylene glycol), and suitable mixtures thereof. The invention
further contemplates the inclusion of a stabilizer in the
pharmaceutical composition.
[0138] The VISTA antagonist peptide, multimer, conjugate, analog,
derivative and mimetic thereof described herein may be formulated
into pharmaceutical compositions of various dosage forms. To
prepare the pharmaceutical compositions of the invention, at least
one VISTA antagonist as the active ingredient may be intimately
mixed with appropriate carriers and additives according to
techniques well known to those skilled in the art of pharmaceutical
formulations. See Grennaro (2005) [Ed.] Remington: The Science and
Practice of Pharmacy [21.sup.st Ed.] For example, the antagonists
described herein may be formulated in phosphate buffered saline pH
7.2 and supplied as a 5.0 mg/mL clear colorless liquid
solution.
[0139] Similarly, compositions for liquid preparations include
solutions, emulsions, dispersions, suspensions, syrups, and
elixirs, with suitable carriers and additives including but not
limited to water, alcohols, oils, glycols, preservatives, flavoring
agents, coloring agents, and suspending agents. Typical
preparations for parenteral administration comprise the active
ingredient with a carrier such as sterile water or parenterally
acceptable oil including but not limited to polyethylene glycol,
polyvinyl pyrrolidone, lecithin, arachis oil or sesame oil, with
other additives for aiding solubility or preservation may also be
included. In the case of a solution, it may be lyophilized to a
powder and then reconstituted immediately prior to use. For
dispersions and suspensions, appropriate carriers and additives
include aqueous gums, celluloses, silicates, or oils.
[0140] For each of the recited embodiments, the VISTA antagonist
peptides, multimers, conjugates, analogs, derivatives and mimetics
thereof may be administered by a variety of dosage forms. Any
biologically-acceptable dosage form known to persons of ordinary
skill in the art, and combinations thereof, are contemplated.
Examples of such dosage forms include, without limitation,
reconstitutable powders, elixirs, liquids, solutions, suspensions,
emulsions, powders, granules, particles, microparticles,
dispersible granules, cachets, inhalants, aerosol inhalants,
patches, particle inhalants, implants, depot implants, injectables
(including subcutaneous, intramuscular, intravenous, and
intradermal), infusions, and combinations thereof.
[0141] In many cases, it will be preferable to include isotonic
agents, e.g., sugars, polyalcohols such as mannitol, sorbitol, or
sodium chloride in the composition. Prolonged absorption of the
injectable compositions may be brought about by including in the
composition an agent which delays absorption, e.g., monostearate
salts and gelatin. Moreover, the compounds described herein may be
formulated in a time release formulation, e.g. in a composition
that includes a slow release polymer. The VISTA and VISTA conjugate
may be prepared with carriers that will protect the compound
against rapid release, such as a controlled release formulation,
including implants and microencapsulated delivery systems.
Biodegradable, biocompatible polymers may be used, such as ethylene
vinyl acetate, polyanhydrides, polyglycolic acid, collagen,
polyorthoesters, polylactic acid and polylactic, polyglycolic
copolymers (PLG). Many methods for the preparation of such
formulations are known to those skilled in the art.
[0142] A person of skill in the art would be able to determine an
effective dosage and frequency of administration through routine
experimentation, for example guided by the disclosure herein and
the teachings in Goodman, et al. (2011) Goodman & Gilman's The
Pharmacological Basis of Therapeutics [12.sup.th Ed.]; Howland, et
al. (2005) Lippincott's Illustrated Reviews: Pharmacology [2.sup.nd
Ed.]; and Golan, (2008) Principles of Pharmacology: The
Pathophysiologic Basis of Drug Therapy [2.sup.nd Ed.] See, also,
Grennaro (2005) [Ed.] Remington: The Science and Practice of
Pharmacy [21.sup.st Ed.]
[0143] The compositions described herein may be administered in any
of the following routes: buccal, epicutaneous, epidural, infusion,
inhalation, intraarterial, intracardial, intracerebroventricular,
intradermal, intramuscular, intranasal, intraocular,
intraperitoneal, intraspinal, intrathecal, intravenous, oral,
parenteral, pulmonary, rectally via an enema or suppository,
subcutaneous, subdermal, sublingual, transdermal, and transmucosal.
The preferred routes of administration are intravenous injection or
infusion. The administration can be local, where the composition is
administered directly, close to, in the locality, near, at, about,
or in the vicinity of, the site(s) of disease, e.g., tumor, or
systemic, wherein the composition is given to the patient and
passes through the body widely, thereby reaching the site(s) of
disease. Local administration (e.g., injection) may be accomplished
by administration to the cell, tissue, organ, and/or organ system,
which encompasses and/or is affected by the disease, and/or where
the disease signs and/or symptoms are active or are likely to occur
(e.g., tumor site). Administration can be topical with a local
effect, composition is applied directly where its action is desired
(e.g., tumor site).
[0144] For each of the recited embodiments, the compounds can be
administered by a variety of dosage forms as known in the art. Any
biologically-acceptable dosage form known to persons of ordinary
skill in the art, and combinations thereof, are contemplated.
Examples of such dosage forms include, without limitation, chewable
tablets, quick dissolve tablets, effervescent tablets,
reconstitutable powders, elixirs, liquids, solutions, suspensions,
emulsions, tablets, multi-layer tablets, bi-layer tablets,
capsules, soft gelatin capsules, hard gelatin capsules, caplets,
lozenges, chewable lozenges, beads, powders, gum, granules,
particles, microparticles, dispersible granules, cachets, douches,
suppositories, creams, topicals, inhalants, aerosol inhalants,
patches, particle inhalants, implants, depot implants, ingestibles,
injectables (including subcutaneous, intramuscular, intravenous,
and intradermal), infusions, and combinations thereof.
[0145] Other compounds which can be included by admixture are, for
example, medically inert ingredients (e.g., solid and liquid
diluent), such as lactose, dextrosesaccharose, cellulose, starch or
calcium phosphate for tablets or capsules, olive oil or ethyl
oleate for soft capsules and water or vegetable oil for suspensions
or emulsions; lubricating agents such as silica, talc, stearic
acid, magnesium or calcium stearate and/or polyethylene glycols;
gelling agents such as colloidal clays; thickening agents such as
gum tragacanth or sodium alginate, binding agents such as starches,
arabic gums, gelatin, methylcellulose, carboxymethylcellulose or
polyvinylpyrrolidone; disintegrating agents such as starch, alginic
acid, alginates or sodium starch glycolate; effervescing mixtures;
dyestuff; sweeteners; wetting agents such as lecithin, polysorbates
or laurylsulphates; and other therapeutically acceptable accessory
ingredients, such as humectants, preservatives, buffers and
antioxidants, which are known additives for such formulations.
[0146] Liquid dispersions for oral administration can be syrups,
emulsions, solutions, or suspensions. The syrups can contain as a
carrier, for example, saccharose or saccharose with glycerol and/or
mannitol and/or sorbitol. The suspensions and the emulsions can
contain a carrier, for example a natural gum, agar, sodium
alginate, pectin, methylcellulose, carboxymethylcellulose, or
polyvinyl alcohol.
[0147] In further embodiments, the present invention provides kits
including one or more containers comprising pharmaceutical dosage
units comprising an effective amount of one or more VISTA
antagonists of the present invention. Kits may include
instructions, directions, labels, marketing information, warnings,
or information pamphlets.
[0148] The amount of VISTA antagonist in a therapeutic composition
according to any embodiments of this invention may vary according
to factors such as the disease state, age, gender, weight, patient
history, risk factors, predisposition to disease, administration
route, pre-existing treatment regime (e.g., possible interactions
with other medications), and weight of the individual. Dosage
regimens may be adjusted to provide the optimum therapeutic
response. For example, a single bolus may be administered, several
divided doses may be administered over time, or the dose may be
proportionally reduced or increased as indicated by the exigencies
of therapeutic situation.
[0149] It is especially advantageous to formulate parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the
mammalian subjects to be treated; each unit containing a
predetermined quantity of antibodies, and fragments thereof,
calculated to produce the desired therapeutic effect in association
with the required pharmaceutical carrier. The specification for the
dosage unit forms of the invention are dictated by and directly
dependent on the unique characteristics of the antibodies, and
fragments thereof, and the particular therapeutic effect to be
achieved, and the limitations inherent in the art of compounding
such an antibodies, and fragments thereof, for the treatment of
sensitivity in individuals. In therapeutic use for treatment of
conditions in mammals (e.g., humans) for which the antibodies and
fragments thereof of the present invention or an appropriate
pharmaceutical composition thereof are effective, the antibodies
and fragments thereof of the present invention may be administered
in an effective amount. The dosages as suitable for this invention
may be a composition, a pharmaceutical composition or any other
compositions described herein.
[0150] The dosage may be administered as a single dose, a double
dose, a triple dose, a quadruple dose, and/or a quintuple dose. The
dosages may be administered singularly, simultaneously, and
sequentially.
[0151] The dosage form may be any form of release known to persons
of ordinary skill in the art. The compositions of the present
invention may be formulated to provide immediate release of the
active ingredient or sustained or controlled release of the active
ingredient. In a sustained release or controlled release
preparation, release of the active ingredient may occur at a rate
such that blood levels are maintained within a therapeutic range
but below toxic levels over an extended period of time (e.g., 4 to
24 hours). The preferred dosage forms include immediate release,
extended release, pulse release, variable release, controlled
release, timed release, sustained release, delayed release, long
acting, and combinations thereof, and are known in the art.
[0152] As defined herein, a therapeutically effective amount of
VISTA antagonist peptide, analog, derivative or mimetic thereof
(i.e., an effective dosage) ranges from about 0.001 to 30 mg/kg
body weight, preferably about 0.01 to 25 mg/kg body weight, more
preferably about 0.1 to 20 mg/kg body weight, and even more
preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7
mg/kg, or 5 to 6 mg/kg body weight.
[0153] The skilled artisan will appreciate that certain factors may
influence the dosage required to effectively treat a subject,
including but not limited to the severity of the disease or
disorder, previous treatments, the general health and/or age of the
subject, and other diseases present. Moreover, treatment of a
subject with a therapeutically effective amount of a peptide can
include a single treatment or, preferably, can include a series of
treatments.
[0154] In a preferred example, a subject is treated with peptide,
analog, derivative or mimetic thereof in the range of between about
0.1 to 20 mg/kg body weight, one time per week for between about 1
to 10 weeks, preferably between 2 to 8 weeks, more preferably
between about 3 to 7 weeks, and even more preferably for about 4,
5, or 6 weeks. It will also be appreciated that the effective
dosage of antibody, protein, or polypeptide used for treatment may
increase or decrease over the course of a particular treatment.
Changes in dosage may result and become apparent from the results
of diagnostic assays as described herein.
[0155] It will be appreciated that the pharmacological activity of
the compositions may be monitored using standard pharmacological
models that are known in the art. Furthermore, it will be
appreciated that the compositions comprising a VISTA antagonist may
be incorporated or encapsulated in a suitable polymer matrix or
membrane for site-specific delivery, or may be functionalized with
specific targeting agents capable of effecting site specific
delivery. These techniques, as well as other drug delivery
techniques are well known in the art. Determination of optimal
dosages for a particular situation is within the capabilities of
those skilled in the art. See, e.g., Grennaro (2005) [Ed.]
Remington: The Science and Practice of Pharmacy [21.sup.st Ed.]
[0156] A peptide or analog, derivative or mimetic of this invention
can be co-formulated with and/or coadministered with one or more
additional therapeutic agents (e.g., an anti-cancer agent, an
anti-viral agent, a cytokine and/or an immune agonist). Such
combination therapies may require lower dosages of the peptide or
analog, derivative or mimetic and/or the co-administered agents,
thus avoiding possible toxicities or complications associated with
the various monotherapies. There are a number of agents that may be
advantageously combined with peptide or analog, derivative or
mimetic of the invention and the selection of such agents will
depend on the intended disease or condition to be treated. For
example, the present invention includes combination therapies
composed of a peptide or multimer, conjugate, analog, derivative or
mimetic of the invention that is capable of inducing or promoting a
response against a cancerous or pre-cancerous condition and at
least one anti-cancer agent. Accordingly, in particular
embodiments, the instant peptide or analog, derivative or mimetic
is used as an adjuvant therapy in the treatment of cancer. As
another example, the invention embraces combination therapies that
include a peptide or analog, derivative or mimetic of the invention
that is capable of inducing or promoting a therapeutic response
against a viral infection and at least one anti-viral agent.
Exemplary therapeutic agents that may be contained in the
compositions comprising the VISTA antagonist peptide, multimer,
conjugate, analog, derivative or mimetic include, e.g., CTLA-4-Ig,
anti-PD-1, PD-L1 or PD-L2 fusion proteins and EGFR antagonists.
[0157] Anti-cancer agents include, but are not limited to,
cytotoxic agents such as Vinca alkaloid, taxanes, and topoisomerase
inhibitors; antisense nucleic acids such as augmerosen/G3139,
LY900003 (ISIS 3521), ISIS 2503, OGX-011 (ISIS 112989),
LE-AON/LEraf-AON (liposome encapsulated c-raf antisense
oligonucleotide/ISIS-5132), MG98, and other antisense nucleic acids
that target PKC.alpha., clusterin, IGFBPs, protein kinase A, cyclin
D1, or Bcl-2; anticancer nucleozymes such as angiozyme (Ribozyme
Pharmaceuticals); tumor suppressor-encoding nucleic acids such as a
p53, BRCA1, RB1, BRCA2, DPC4 (Smad4), MSH2, MLH1, and DCC;
oncolytic viruses such as oncolytic adenoviruses and herpes
viruses; anti-cancer immunogens such as a cancer
antigen/tumor-associated antigen, e.g., an epithelial cell adhesion
molecule (Ep-CAM/TACSTD1), mucin 1 (MUC1), carcinoembryonic antigen
(CEA), tumor-associated glycoprotein 72 (TAG-72), gp100, Melan-A,
MART-1, KDR, RCAS1, MDA7, cancer-associated viral vaccines,
tumor-derived heat shock proteins, and the like; anti-cancer
cytokines, chemokines, or combination thereof; inhibitors of
angiogenesis, neovascularization, and/or other vascularization;
and/or any other conventional anticancer agent including
fluoropyrimidiner carbamates, non-polyglutamatable thymidylate
synthase inhibitors, nucleoside analogs, antifolates, topoisomerase
inhibitors, polyamine analogs, mTOR inhibitors, alkylating agents,
lectin inhibitors, vitamin D analogs, carbohydrate processing
inhibitors, anti-metabolism folate antagonists, thumidylate
synthase inhibitors, antimetabolites, ribonuclease reductase
inhibitors, dioxolate nucleoside analogs, and chemically modified
tetracyclines.
[0158] Anti-viral agents of use in the invention include, but are
not limited to, protease inhibitors (e.g., acyclovir) in the
context of HIV treatment or an anti-viral antibody (e.g., an
anti-gp41 antibody in the context of HIV treatment; an anti-CD4
antibody in the context of the treatment of CMV, etc.). Numerous
other types of anti-viral agents are known in the art.
[0159] Toxicity and therapeutic efficacy of the peptide or analog,
derivative or mimetic can be determined by standard pharmaceutical
procedures in cell cultures or experimental animals. The data
obtained from the cell culture assays and animal studies can be
used in formulating a range of dosage for use in humans. The dosage
of such agents lies preferably within a range of circulating
concentrations that include the ED.sub.50 with little or no
toxicity. The dosage may vary within this range depending upon the
dosage form employed and the route of administration utilized. For
any agent used in the methods of the invention, the therapeutically
effective dose can be estimated initially from cell culture assays.
A dose may be formulated in animal models to achieve a circulating
plasma concentration range that includes the IC.sub.50 (i.e., the
concentration of the test compound which achieves a half-maximal
inhibition of symptoms) as determined in cell culture. Such
information can be used to more accurately determine useful doses
in humans. Levels in plasma may be measured, for example, by high
performance liquid chromatography.
Use of VISTA Antagonists and Compositions Comprising the Same
[0160] The peptide or analog, derivative or mimetic of this
invention finds use in inhibiting the activity of VISTA (i.e.,
PD-L3) thereby upregulating immune responses. Upregulation of
immune responses can be in the form of enhancing an existing immune
response or eliciting an initial immune response. For example,
enhancing an immune response through inhibition of VISTA activity
is useful in the prevention and/or treatment of infections with
microbes, e.g., bacteria, viruses, or parasites, or in cases of
immunosuppression and cancer.
[0161] Accordingly, the present invention includes prophylactic and
therapeutic methods for the prevention and treatment of cancer and
infectious disease. Terms such as "treat," "treating" and
"treatment" herein refer to the delivery of an effective amount of
a peptide or analog, derivative or mimetic of this invention with
the purpose of easing, ameliorating, or eradicating (curing) such
symptoms or disease states already developed. The terms "prevent,"
"preventing" and "prevention" refer to the delivery of an effective
amount of a peptide or analog, derivative or mimetic of this
invention with the purpose of preventing any symptoms or disease
state to develop. Thus, these terms are meant to include
prophylactic treatment.
[0162] Accordingly to one embodiment, the invention provides a
method of treating or preventing cancer, inhibiting tumor invasion
and/or cancer metastasis by administering to a subject in need
thereof, such as a mammalian subject, preferably a human subject,
an effective amount of an isolated VISTA antagonist disclosed
herein or a composition containing said isolated VISTA antagonist.
Optionally the subject has one or more precancerous lesions or is
predisposed to cancer, e.g., as a result of genetic mutation,
family history or exposure to a carcinogenic agent. In another
embodiment the invention provides a method of treating cancer in
subject, such as a mammalian subject, preferably a human subject,
such as a human subject, who optionally has a detectable level of
cancer cells. In accordance with these embodiments, the subject is
administered a peptide or analog, derivative or mimetic of this
invention in an amount sufficient to detectably reduce the
development or progression of the cancer in the subject.
[0163] Cancers are generally composed of single or several clones
of cells that are capable of partially independent growth in a host
(e.g., a benign tumor) or fully independent growth in a host
(malignant cancer). Cancer cells are cells that divide and
reproduce abnormally with uncontrolled growth.
[0164] Cancer cells arise from host cells via neoplastic
transformation (i.e., carcinogenesis). Terms such as
"preneoplastic," "premalignant" and "precancerous" with respect to
the description of cells and/or tissues herein refer to cells or
tissues having a genetic and/or phenotypic profile that signifies a
significant potential of becoming cancerous. Usually such cells can
be characterized by one or more differences from their nearest
counterparts that signal the onset of cancer progression or
significant risk for the start of cancer progression. Such
precancerous changes, if detectable, can usually be treated with
excellent results.
[0165] In general, a precancerous state will be associated with the
incidence of neoplasm(s) or preneoplastic lesion(s). Examples of
known and likely preneoplastic tissues include ductal carcinoma in
situ (DCIS) growths in breast cancer, cervical intra-epithelial
neoplasia (CIN) in cervical cancer, adenomatous polyps of colon in
colorectal cancers, atypical adenomatous hyperplasia in lung
cancers, and actinic keratosis (AK) in skin cancers. Pre-neoplastic
phenotypes and genotypes for various cancers, and methods for
assessing the existence of a preneoplastic state in cells, have
been characterized. See, e.g., Medina (2000) J. Mammary Gland Biol.
Neoplasia 5(4):393-407; Krishnamurthy, et al. (2002) Adv. Anat.
Pathol. 9(3):185-97; Ponten (2001) Eur. J. Cancer Suppl 8:S97-113;
Niklinski, et al. (2001) Eur. J. Cancer Prev. 10(3):213-26; Walch,
et al. Pathobiology (2000) 68(1):9-17; Busch (1998) Cancer Surv.
32:149-79.
[0166] Gene expression profiles can increasingly be used to
differentiate between normal, precancerous, and cancer cells. For
example, familial adenomatous polyposis genes prompt close
surveillance for colon cancer; mutated p53 tumor-suppressor gene
flags cells that are likely to develop into aggressive cancers;
osteopontin expression levels are elevated in premalignant cells,
and increased telomerase activity also can be a marker of a
precancerous condition (e.g., in cancers of the bladder and lung).
In one aspect, the invention relates to the treatment of
precancerous cells. In another aspect, the invention relates to the
preparation of medicaments for treatment of precancerous cells.
[0167] In general, a peptide or analog, derivative or mimetic of
this invention can be used to treat subjects suffering from any
stage of cancer (and to prepare medicaments for reduction, delay,
or other treatment of cancer). Effective treatment of cancer (and
thus the reduction thereof) can be detected by any variety of
suitable methods. Methods for detecting cancers and effective
cancer treatment include clinical examination (symptoms can include
swelling, palpable lumps, enlarged lymph nodes, bleeding, visible
skin lesions, and weight loss); imaging (X-ray techniques,
mammography, colonoscopy, computed tomography (CT and/or CAT)
scanning, magnetic resonance imaging (MRI), etc.); immunodiagnostic
assays (e.g., detection of CEA, AFP, CA125, etc.);
antibody-mediated radioimaging; and analyzing cellular/tissue
immunohistochemistry. Other examples of suitable techniques for
assessing a cancerous state and effective cancer treatment include
PCR and RT-PCR (e.g., of cancer cell associated genes or
"markers"), biopsy, electron microscopy, positron emission
tomography (PET), computed tomography, magnetic resonance imaging
(MRI), karyotyping and other chromosomal analysis,
immunoassay/immunocytochemical detection techniques (e.g.,
differential antibody recognition), histological and/or
histopathologic assays (e.g., of cell membrane changes), cell
kinetic studies and cell cycle analysis, ultrasound or other
sonographic detection techniques, radiological detection
techniques, flow cytometry, endoscopic visualization techniques,
and physical examination techniques.
[0168] In general, delivering a peptide or analog, derivative or
mimetic of this invention to a subject (either by direct
administration or expression from a nucleic acid) can be used to
reduce, treat, prevent, or otherwise ameliorate any aspect of
cancer in a subject. In this respect, treatment of cancer can
include, e.g., any detectable decrease in the rate of normal cells
transforming to neoplastic cells (or any aspect thereof), the rate
of proliferation of pre-neoplastic or neoplastic cells, the number
of cells exhibiting a pre-neoplastic and/or neoplastic phenotype,
the physical area of a cell media (e.g., a cell culture, tissue, or
organ) containing pre-neoplastic and/or neoplastic cells, the
probability that normal cells and/or preneoplastic cells will
transform to neoplastic cells, the probability that cancer cells
will progress to the next aspect of cancer progression (e.g., a
reduction in metastatic potential), or any combination thereof.
Such changes can be detected using any of the above-described
techniques or suitable counterparts thereof known in the art, which
typically are applied at a suitable time prior to the
administration of a therapeutic regimen so as to assess its
effectiveness. Times and conditions for assaying whether a
reduction in cancer has occurred will depend on several factors
including the type of cancer, type and amount of peptide, related
composition, or combination composition being delivered to the
host.
[0169] The methods of the invention can be used to treat a variety
of cancers. Forms of cancer that may be treated by the delivery or
administration of a peptide or analog, derivative or mimetic of
this invention and combination therapies containing the same
include squamous cell carcinoma, leukemia, acute lymphocytic
leukemia, acute lymphoblastic leukemia, B-cell lymphoma, T-cell
lymphoma, Hodgkins lymphoma, non-Hodgkins lymphoma, hairy cell
lymphoma, Burketts lymphoma, acute or chronic myelogenous
leukemias, promyelocytic leukemia, fibrosarcoma, rhabdomyoscarcoma,
melanoma, seminoma, teratocarcinoma, neuroblastoma, glioma,
astrocytoma, neuroblastoma, glioma, schwannomas; fibrosarcoma,
rhabdomyoscaroma, osteosarcoma, melanoma, xeroderma pigmentosum,
keratoacanthoma, seminoma, thyroid follicular cancer, and
teratocarcinoma. The compositions of this invention also can be
useful in the treatment of other carcinomas of the bladder, breast,
colon, kidney, liver, lung, ovary, prostate, pancreas, stomach,
cervix, thyroid or skin. Compositions of this invention also may be
useful in treatment of other hematopoietic tumors of lymphoid
lineage, other hematopoietic tumors of myeloid lineage, other
tumors of mesenchymal origin, other tumors of the central or
peripheral nervous system, and/or other tumors of mesenchymal
origin. Advantageously, the methods of the invention also may be
useful in reducing cancer progression in prostate cancer cells,
melanoma cells (e.g., cutaneous melanoma cells, ocular melanoma
cells, and/or lymph node-associated melanoma cells), breast cancer
cells, colon cancer cells, and lung cancer cells. The methods of
the invention can be used to treat both tumorigenic and
non-tumorigenic cancers (e.g., non-tumor-forming hematopoietic
cancers). The methods of the invention are particularly useful in
the treatment of epithelial cancers (e.g., carcinomas) and/or
colorectal cancers, breast cancers, lung cancers, vaginal cancers,
cervical cancers, and/or squamous cell carcinomas (e.g., of the
head and neck). Additional potential targets include sarcomas and
lymphomas. Additional advantageous targets include solid tumors
and/or disseminated tumors (e.g., myeloid and lymphoid tumors,
which can be acute or chronic).
[0170] The present invention also provides methods for enhancing
anti-cancer or anti-tumor immunity, comprising administering to a
subject in need thereof an effective amount of an isolated VISTA
antagonist or a composition containing said isolated VISTA
antagonist.
[0171] In addition to cancer treatment, the present invention also
features a method of treating a pathogen infection, i.e., a
bacterial, viral, parasitic or fungal infection, in a subject or
host. This method involves administering or otherwise delivering an
effective amount of a peptide or analog, derivative or mimetic of
this invention so as to reduce the severity, spread, symptoms, or
duration of such infection. Such pathogen infections include, but
are not limited to diseases caused by bacteria, protozoa, fungi,
parasites, or viruses.
[0172] In particular embodiments, a viral infection is treated. Any
virus normally associated with the activity of effector lymphocytes
can be treated by the method. For example, such a method can be
used to treat infection by one or more viruses selected from
hepatitis type A, hepatitis type B, hepatitis type C, influenza,
varicella, adenovirus, herpes simplex type I (HSV-1), herpes
simplex type 2 (HSV-2), rinderpest, rhinovirus, echovirus,
rotavirus, respiratory syncytial virus, papilloma virus, papilloma
virus, cytomegalovirus (CMV, e.g., HCMV), echinovirus, arbovirus,
huntavirus, coxsackie virus, mumps virus, measles virus, rubella
virus, polio virus, and/or human immunodeficiency virus type I or
type 2 (HIV-1, HIV-2). The practice of such methods may result in a
reduction in the titer of virus (viral load), reduction of the
number of virally infected cells, etc.
[0173] In addition to pathogen infections, a peptide or analog,
derivative or mimetic of this invention can be administered or
otherwise delivered to a subject in association with the treatment
of immunoproliferative diseases, immunodeficiency diseases,
autoimmune diseases, inflammatory responses, and/or allergic
responses. Moreover, the invention also provides methods for
blocking, inhibiting or neutralizing VISTA-mediated T cell
suppression and/or stimulating an immune response in a subject,
comprising administering to the subject in need thereof an
effective amount of an isolated VISTA antagonist or a composition
containing said isolated VISTA antagonist. Such methods may be
useful for treating a subject with a one or more of a bacterial,
viral, parasitic and fungal infections and/or cancer.
EXAMPLES
[0174] The invention now being generally described, it will be more
readily understood by reference to the following examples, which
are included merely for purposes of illustration of certain aspects
and embodiments of the present invention, and are not intended to
limit the invention.
Example 1
Materials and Methods
[0175] Peptide Synthesis. AP1049 (SSACDWIKRSCH-amide, wherein
Cys4-Cysll form a disulfide bridge; SEQ ID NO:1
(Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His)) and scrambled
negative control sequence (SSACKSWRDICH-amide, wherein Cys4-Cysll
form a disulfide bridge; SEQ ID NO:2) were prepared using standard
Fmoc-based solid-phase peptide synthesis (SPPS). The peptides were
purified via HPLC, and analyzed by mass spectrometric using the
liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted
laser desorption/ionization (MALDI)
[0176] Peptide Discovery Using Phage Display. An M13 phage peptide
library was provided by Dr. Brian Kay (U. Illinois-Chicago). The
VISTA protein required for both the phage display biopanning
experiments and the confirmatory ELISA binding experiments was
prepared by conventional recombinant protein techniques.
[0177] T Cell Proliferation Assay. An VISTA-Ig fusion protein or
control Ig fusion protein was co-absorbed to a cell-culture plate
together with the polyclonal T cell receptor (TCR) stimuli (i.e.,
anti-CD3 antibody). To evaluate the activity of VISTA-specific
peptides, peptides (VISTA specific and scrambled control) were
added as a soluble reagent to the culture on day 0, and T cell
proliferation and cytokine production were analyzed after 3-4
days.
[0178] T Cell Priming Assay. VISTA is known to suppress T cell
priming when expressed on antigen-presenting cells (APCs).
VISTA-expressing myeloid APCs (Cd11b.sup.hi MHCII.sup.+ myeloid
cells) were purified from mice spleen, FACS sorted, and irradiated
(2500 rads). To test the activity of VISTA-specific peptides,
transgenic T cells such as OT-II were stimulated ex vivo with
VISTA-expressing APCs and cognate antigen chicken ovalbumin (15
ng/mL). VISTA-specific peptide or control scramble peptide was
added to the cell culture. T cell proliferation and cytokine
production was evaluated after 3-5 days of culture. As an
additional specificity control, VISTA-negative parent cell line A20
or APCs purified from VISTA knockout mice were used.
[0179] Foxp3+CD4+ Regulatory T Cell (Treg) Suppression Assay. VISTA
plays a role in the suppressive function of Foxp3+CD4+ regulatory T
cells (Tregs), as VISTA-blocking monoclonal antibody partially
reverses Treg suppressive activity in the in vitro Treg suppression
assay. This assay includes antigen presenting cells, purified
Foxp3+ CD4+ Tregs, and Foxp3- CD4+ naive T cells, which are
stimulated by the polyclonal TCR stimuli. To examine the activity
of VISTA-specific peptides, peptides (VISTA specific and scrambled
control) were added to the Treg suppression assay on day 0. T cell
proliferation and cytokine production were measured on day +3.
[0180] Model of Experimental Autoimmune Encephalomyelitis (EAE), a
Murine Autoimmune Inflammatory Disease Model for Human Multiple
Sclerosis. It has been shown that VISTA-blocking monoclonal
antibody significantly accelerates disease onset, as well as
exacerbates disease severity in a passive transfer EAE model. In
this model, MOG-specific encephalitogenic CD4+T cells are first
primed in donor mice upon immunization with MOG peptide, and then
purified and ex vivo expanded in the presence of MOG peptide and
cytokines (IL23, TGF.beta., IL6 and IL1b). Expanded encephalogenic
CD4+ T cells are transferred into naive recipients to induce
disease. To evaluate the activity of VISTA-specific peptides,
peptides (VISTA specific and scrambled control) are administered
via intraperitoneal injections to mice either prophylactically
(starting from day 2) or therapeutically (starting from day +7
during disease onset), and continuously every 2 days for the entire
duration of the experiment. Disease severity is evaluated according
to the established protocol.
[0181] Murine Tumor Models. It has been demonstrated that VISTA
suppresses tumor-specific T cell responses. VISTA blockade via
VISTA-specific monoclonal antibody significantly enhances
anti-tumor immune responses and inhibits tumor progression in
murine tumor models such as the B16 melanoma model. The activity of
VISTA-specific peptides can be evaluated in vivo using this tumor
model. Mice are inoculated on the flank with B16 tumor cells
(15,000 cells) on day 0. Peptides (VISTA specific and scrambled
control) are administered via intraperitoneal injections to mice
either prophylactically (starting from day 2) or therapeutically
(i.e., when tumors are palpable), and continuously every 2 days for
the entire duration of the experiment. Tumor growth is measured
every 2-3 days with a caliper.
Example 2
Enhancement of T Cell Proliferation
[0182] VISTA.sup.+CD11b.sup.+ monocytes were enriched from naive
splenocytes using CD11b magnetic beads (Miltenyi).
VISTA.sup.+CD11b.sup.hi MHCII.sup.+ myeloid APCs were FACS sorted,
irradiated (2500 rads), and used as antigen-presenting cells to
stimulate OT-II transgenic CD4.sup.+ T cells in the presence of OVA
peptide. Control-Ig, monoclonal antibody specific for VISTA and
PD-L1 (30 .mu.g/mL), or VISTA-specific peptide (100 .mu.g/mL) were
added as indicated. Cell proliferation was measured by tritium
incorporation during the last 8 hours of a 72-hour assay. This
analysis indicated that T cell proliferation was enhanced in the
presence of VISTA or PD-L1 neutralizing monoclonal antibodies, or
the AP1049 peptide (FIG. 1). In fact, the AP1049 peptide stimulated
T cell proliferation much better than either of the monoclonal
antibodies, indicating that the peptide possesses strong
antagonistic activity against VISTA.
Example 3
Enhancement of Anti-Tumor Immunity
[0183] Immunogenic bladder carcinoma tumors (MB49) were inoculated
in female mice. AP1049 was tested for its ability to slow tumor
growth and/or facilitate tumor regression. The readout for this
assay was tumor growth.
[0184] MB49 tumors were inoculated in female mice (300k) via
intradermal (i.d.) inoculation, which facilitates measurement of
tumor size. Mice were treated with either PBS (control) or VISTA
antagonist peptide (AP1049), via daily injections around tumor mass
starting on day+1 and continuing for 2 weeks. Tumor size was
measured by caliper every 2-3 days.
[0185] Using these methods slowed tumor growth and/or tumor
regression in mice treated with AP1049 was obtained as compared
with mice treated with control.
[0186] As shown in FIG. 2, AP1049 treatment reduced tumor growth in
the MB49 tumor model, indicating that the peptide may bind to the
critical/active site of VISTA and block the immune-suppressive
function of VISTA.
Sequence CWU 1
1
1112PRTArtificial SequenceAP1049 1Ser Ser Ala Cys Asp Trp Ile Lys
Arg Ser Cys His 1 5 10
* * * * *