U.S. patent application number 17/006954 was filed with the patent office on 2020-12-17 for compositions, articles of manufacture and methods for treating cancer.
This patent application is currently assigned to Biokine Therapeutics Ltd.. The applicant listed for this patent is Biokine Therapeutics Ltd., BioLineRx Ltd.. Invention is credited to Amnon PELED, Yaron PEREG.
Application Number | 20200390855 17/006954 |
Document ID | / |
Family ID | 1000005059677 |
Filed Date | 2020-12-17 |
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United States Patent
Application |
20200390855 |
Kind Code |
A1 |
PELED; Amnon ; et
al. |
December 17, 2020 |
COMPOSITIONS, ARTICLES OF MANUFACTURE AND METHODS FOR TREATING
CANCER
Abstract
A method of treating cancer in a subject in need thereof is
provided. The method comprising administering to the subject a
therapeutically effective amount of a peptide having an amino acid
sequence as set forth in SEQ ID NO: 1 or an analog or derivative
thereof; and an anti-cancer agent, thereby treating the cancer in
the subject.
Inventors: |
PELED; Amnon; (Tel-Aviv,
IL) ; PEREG; Yaron; (Shoham, IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Biokine Therapeutics Ltd.
BioLineRx Ltd. |
Nes Ziona
Modiln |
|
IL
IL |
|
|
Assignee: |
Biokine Therapeutics Ltd.
Nes Ziona
IL
BioLineRx Ltd.
ModiIn
IL
|
Family ID: |
1000005059677 |
Appl. No.: |
17/006954 |
Filed: |
August 31, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15570342 |
Oct 29, 2017 |
10786547 |
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PCT/IL2016/050765 |
Jul 14, 2016 |
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17006954 |
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62193201 |
Jul 16, 2015 |
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62259182 |
Nov 24, 2015 |
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62291039 |
Feb 4, 2016 |
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62291006 |
Feb 4, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/395 20130101;
A61K 39/3955 20130101; A61K 47/646 20170801; A61K 2039/55522
20130101; A61K 39/12 20130101; C07K 16/2827 20130101; A61P 35/00
20180101; C07K 16/30 20130101; C07K 2317/76 20130101; A61K 38/1761
20130101; C07K 16/2818 20130101; A61K 38/04 20130101; A61K 38/10
20130101 |
International
Class: |
A61K 38/17 20060101
A61K038/17; A61K 38/04 20060101 A61K038/04; A61K 47/64 20060101
A61K047/64; A61P 35/00 20060101 A61P035/00; A61K 38/10 20060101
A61K038/10; A61K 39/12 20060101 A61K039/12; A61K 39/395 20060101
A61K039/395; C07K 16/28 20060101 C07K016/28; C07K 16/30 20060101
C07K016/30 |
Claims
1. An article of manufacture identified for use in treating cancer,
comprising a packaging material, packaging a peptide having an
amino acid sequence as set forth in SEQ ID NO: 1 and an immune
checkpoint regulator, wherein said immune checkpoint regulator is
selected from the group consisting of B7H2, B7H3, B7H4, BTLA-4,
HVEM, CD80, CD86, CD19, OX40L, 4-1BBL, CD70, CD40L, ICOSL and
VISTA.
2. The article of manufacture of claim 1, wherein said peptide and
said immune checkpoint regulator are in separate containers.
3. The article of manufacture of claim 1, wherein said peptide and
said immune checkpoint regulator are in a co-formulation.
4. The article of manufacture of claim 1, wherein said cancer is a
non-solid tumor.
5. The article of manufacture of claim 1, wherein said cancer is a
solid tumor.
6. A method of treating cancer in a subject in need thereof, the
method comprising administering to the subject a therapeutically
effective amount of a peptide having an amino acid sequence as set
forth in SEQ ID NO: 1 and an immune checkpoint regulator, wherein
said immune checkpoint regulator is selected from the group
consisting of B7H2, B7H3, B7H4, BTLA-4, HVEM, CD80, CD86, CD19,
OX40L, 4-1BBL, CD70, CD40L, ICOSL and VISTA.
7. The method of claim 6, wherein said peptide and said immune
checkpoint regulator are in separate containers.
8. The method of claim 6, wherein said peptide and said immune
checkpoint regulator are in a co-formulation.
9. The method of claim 6, wherein said cancer is a non-solid
tumor.
10. The method of claim 6, wherein said cancer is a solid tumor.
Description
RELATED APPLICATIONS
[0001] This application is a Division of U.S. patent application
Ser. No. 15/570,342 filed on Oct. 29, 2020, which is a National
Phase of PCT Patent Application No. PCT/IL2016/050765 having
International Filing Date of Jul. 14, 2016, which claims the
benefit of priority under 35 USC .sctn. 119(e) of U.S. Provisional
Patent Application Nos. 62/193,201 filed on Jul. 16, 2015;
62/259,182 filed on Nov. 24, 2015; 62/291,039 filed on Feb. 4,
2016; and 62/291,006 filed on Feb. 4, 2016. The contents of the
above applications are all incorporated by reference as if fully
set forth herein in their entirety.
SEQUENCE LISTING STATEMENT
[0002] The ASCII file, entitled 83822SequenceListing.txt, created
on Aug. 27, 2020, comprising 39,930 bytes, submitted concurrently
with the filing of this application is incorporated herein by
reference. The sequence listing submitted herewith is identical to
the sequence listing forming part of the international
application.
FIELD AND BACKGROUND OF THE INVENTION
[0003] The present invention, in some embodiments thereof, relates
to methods of treating cancer and, more particularly, but not
exclusively, to the use of a CXCR4 antagonistic peptide and an
anti-cancer agent in the treatment of cancer.
[0004] Cancer is the second leading cause of death in the US. The
estimates for 2014 are that approximately 585,000 people will die
of cancer and 1.6 million new cases will be diagnosed (American
Cancer Society, Cancer Facts & FIGS. 2014). For early stage
cancers, surgical removal is a very effective treatment. However,
for more advanced cases and non-solid hematological malignancies,
standard, non-specific cancer treatments such as chemotherapy and
radiotherapy are typically used. These treatments affect many
healthy cells and result in elevated toxicity and effective in only
a minor percentage of treated individuals. Moreover, even
individuals that initially respond to therapy are at risk for
relapses, and often develop resistance.
[0005] Significant progress in understanding the underlying
principles of tumor biology as well as the basic mechanisms of the
immune response to cancer have led to the development of new
immunotherapies aimed at employing the adaptive immune system to
eradicate cancer with enhanced efficacy and reduced toxicity.
Current immunotherapy strategies include cytokines, monoclonal
antibodies against tumor cells or immune regulatory molecules,
cancer vaccines and cell-based therapies such as adoptive transfer
of ex-vivo activated T cells and natural killer (NK) cells.
[0006] Thus, for example, monoclonal antibodies have become part of
the therapeutic repertoire for several type of cancers with the
anti-CD20 mAb, Rituximab, and the anti-human HER2, Trastuzumab,
routinely used for the treatment of breast cancer; and the
anti-human EGFR, Cetuximab, routinely used for the treatment of
head and neck cancer and colorectal carcinoma [Kirkwood et al.
(2012) CA Cancer J Clin. 62(5): 309-335]. In addition, several
recombinant IFN.alpha. [IFN-.alpha.2 (Intron-A, Merck); Roferon-A
(Roche)] and IL-2 (aldesleukin, Proleukin; Prometheus Inc.) were
approved by the United States Food and Drug Administration for the
treatment of e.g. melanoma; and dendritic cell vaccines have shown
safety and efficacy in several solid tumors, for example
sipuleucel-T (Provenge; Dendreon Corporation) in prostate cancer
and dendritic cells loaded with four melanoma peptides (gp100,
melan-A/MART-1, tyrosinase and MAGE-3), KLH and flu matrix peptide
in metastatic melanoma [Kirkwood et al. (2012) CA Cancer J Clin.
62(5): 309-335; and Banchereau (2001) Cancer Research 61:
6451-58].
[0007] 4F-benzoyl-TN14003 (also known as BKT140, hereinafter
BL-8040), is a 14-residue bio stable synthetic peptide developed as
a specific CXCR4 antagonist. It has been shown that BL-8040 binds
the CXCR4 receptor with high affinity and long receptor occupancy.
Studies in mice demonstrated that a single BL-8040 injection
mobilized long term repopulating stem cells sufficient for
transplantation. [Abraham M et al., Stem Cells (2007); 25:2158-66]
Results from a study in multiple myeloma patients showed that
combined treatment of BL-8040 and G-CSF enabled the collection of
high number of CD34+ hematopoietic stem/progenitor cells (HSPC) in
a single aphaeresis procedure.[Peled A et al. Clin Cancer Res;
(2013) 20(2); 469-79] In addition, BL-8040 was found to be toxic
against several tumors such as myeloid leukemia, hematopoietic
tumors and non-small cell lung cancer (International Patent
Application No. IL2014/050939 and International Patent Application
Publication Nos. WO2013/160895 and WO2008/075370).
[0008] Additional background art includes:
[0009] International Patent Application Publication No.
WO2014/155376;
[0010] International Patent Application Publication No.
WO2012/095849;
[0011] International Patent Application Publication No.
WO2002/020561;
[0012] International Patent Application Publication No.
WO2004/020462;
[0013] International Patent Application Publication No.
WO2008/075369;
[0014] International Patent Application Publication No.
WO2008/075370;
[0015] International Patent Application Publication No.
WO2008/075371;
[0016] International Patent Application Publication No.
WO2010/146578;
[0017] International Patent Application Publication No.
WO2010/146584;
[0018] International Patent Application Publication No.
WO2003/072599;
[0019] International Patent Application Publication no. WO
2015/019284; and
[0020] U.S. Patent Application Publication No. US 2012/0082687.
SUMMARY OF THE INVENTION
[0021] According to an aspect of some embodiments of the present
invention there is provided a method of treating cancer in a
subject in need thereof, the method comprising administering to the
subject a therapeutically effective amount of a peptide having an
amino acid sequence as set forth in SEQ ID NO: 1 or an analog or
derivative thereof; and an anti-cancer agent selected from the
group consisting of:
[0022] (i) a vaccine selected from the group consisting of
IMCgp100, Prophage G-100 & G-200, GV-1001, IMA-950, CV-9201,
CV-9104, Ad-RTS-hIL-12, ETBX-011, Cavatak, JX-594, ColoAd1,
GL-ONC1, ONCOS-102, CRS-207, ADU-623, Dorgenmeltucel-L, HyperAcute
Prostate, FANG vaccine, MGN-1601, HPV vaccine and Tarmogens such as
GI-4000;
[0023] (ii) anti-cancer reactive mononuclear blood cells
(MNBCs);
[0024] (iii) a cytokine capable of inducing activation and/or
proliferation of a T cell;
[0025] (iv) an immune-check point regulator, wherein the
immune-check point regulator is not a PD1 antagonist, PDL-1
antagonist, CTLA-4 antagonist, LAG-3 antagonist, TIM-3 antagonist,
KIR antagonist, IDO antagonist, OX40 agonist, CD137 agonist, CD27
agonist, CD40 agonist, GITR agonist, CD28 agonist or ICOS
agonist;
[0026] (v) an agent capable of binding an immune-check point
protein expressed on a cancer cell;
[0027] (vi) a colony stimulating factor-1 receptor (CSF1R)
antagonist;
[0028] (vii) a CXCR2 antagonist;
[0029] (viii) a STAT3 antagonist;
[0030] (ix) PV-10; and
[0031] (x) Cotara,
[0032] thereby treating the cancer in the subject.
[0033] According to some embodiments of the invention, the
administering comprises multiple administrations of the
peptide.
[0034] According to some embodiments of the invention, the
administering comprises multiple administrations of the agent.
[0035] According to some embodiments of the invention, the
administering the peptide and the administering the agent are
effected sequentially.
[0036] According to some embodiments of the invention, the
administering the peptide is effected prior to the administering
the agent.
[0037] According to some embodiments of the invention, the
administering the peptide is effected following the administering
the agent.
[0038] According to some embodiments of the invention, the
administering the peptide is effected concomitantly with the
administering the agent.
[0039] According to some embodiments of the invention, the peptide
is administered at a dose of 0.5-1 mg/kg.
[0040] According to some embodiments of the invention, the peptide
is administered subcutaneously.
[0041] According to an aspect of some embodiments of the present
invention there is provided an article of manufacture identified
for use in treating cancer, comprising a packaging material
packaging a peptide having an amino acid sequence as set forth in
SEQ ID NO: 1 or an analog or derivative thereof and an anti-cancer
agent selected from the group consisting of:
[0042] (i) a vaccine selected from the group consisting of
IMCgp100, Prophage G-100 & G-200, GV-1001, IMA-950, CV-9201,
CV-9104, Ad-RTS-hIL-12, ETBX-011, Cavatak, JX-594, ColoAd1,
GL-ONC1, ONCOS-102, CRS-207, ADU-623,
[0043] Dorgenmeltucel-L, HyperAcute Prostate, FANG vaccine,
MGN-1601, HPV vaccine and Tarmogens such as GI-4000;
[0044] (ii) anti-cancer reactive mononuclear blood cells
(MNBCs);
[0045] (iii) a cytokine capable of inducing activation and/or
proliferation of a T cell;
[0046] (iv) an immune-check point regulator, wherein the
immune-check point regulator is not a PD1 antagonist, PDL-1
antagonist, CTLA-4 antagonist, LAG-3 antagonist, TIM-3 antagonist,
KIR antagonist, IDO antagonist, OX40 agonist, CD137 agonist, CD27
agonist, CD40 agonist, GITR agonist, CD28 agonist or ICOS
agonist;
[0047] (v) an agent capable of binding an immune-check point
protein expressed on a cancer cell;
[0048] (vi) a colony stimulating factor-1 receptor (CSF1R)
antagonist;
[0049] (vii) a CXCR2 antagonist;
[0050] (viii) a STAT3 antagonist;
[0051] (ix) PV-10; and
[0052] (x) Cotara.
[0053] According to some embodiments of the invention, the peptide
and the agent are in separate containers.
[0054] According to some embodiments of the invention, the peptide
and the agent are in a co-formulation.
[0055] According to an aspect of some embodiments of the present
invention there is provided a pharmaceutical composition comprising
as active ingredients a peptide having an amino acid sequence as
set forth in SEQ ID NO: 1 or an analog or derivative thereof and an
anti-cancer agent selected from the group consisting of:
[0056] (i) a vaccine selected from the group consisting of
IMCgp100, Prophage G-100 & G-200, GV-1001, IMA-950, CV-9201,
CV-9104, Ad-RTS-hIL-12, ETBX-011, Cavatak, JX-594, ColoAd1,
GL-ONC1, ONCOS-102, CRS-207, ADU-623, Dorgenmeltucel-L, HyperAcute
Prostate, FANG vaccine, MGN-1601, HPV vaccine and Tarmogens such as
GI-4000;
[0057] (ii) anti-cancer reactive mononuclear blood cells
(MNBCs);
[0058] (iii) a cytokine capable of inducing activation and/or
proliferation of a T cell;
[0059] (iv) an immune-check point regulator, wherein the
immune-check point regulator is not a PD1 antagonist, PDL-1
antagonist, CTLA-4 antagonist, LAG-3 antagonist, TIM-3 antagonist,
KIR antagonist, IDO antagonist, OX40 agonist, CD137 agonist, CD27
agonist, CD40 agonist, GITR agonist, CD28 agonist or ICOS
agonist;
[0060] (v) an agent capable of binding an immune-check point
protein expressed on a cancer cell;
[0061] (vi) a colony stimulating factor-1 receptor (CSF1R)
antagonist;
[0062] (vii) a CXCR2 antagonist;
[0063] (viii) a STAT3 antagonist;
[0064] (ix) PV-10; and
[0065] (x) Cotara,
[0066] and a pharmaceutically acceptable carrier or diluent.
[0067] According to some embodiments of the invention, the MNBCs
are derived from a subject not treated with a peptide having an
amino acid sequence as set forth in SEQ ID NO: 1 or an analog or
derivative thereof.
[0068] According to some embodiments of the invention, the MNBCs
are selected from the group consisting of T cells, NK cells and
dendritic cells.
[0069] According to some embodiments of the invention, the MNBCs
comprise T cells.
[0070] According to some embodiments of the invention, the MNBCs
comprise dendritic cells.
[0071] According to some embodiments of the invention, the
dendritic cells comprise a dendritic cells vaccine.
[0072] According to some embodiments of the invention, the cytokine
is selected from the group consisting of IFN.alpha., IFN.gamma.,
IL-1, IL-2, IL-6, IL-12, IL-15, IL-21 and TNF.alpha..
[0073] According to some embodiments of the invention, the
immune-check point regulator targets an immune check-point protein
selected from the group consisting of B7-H3, CD19 and CD70.
[0074] According to some embodiments of the invention, the
immune-check point regulator is selected from the group consisting
of an antibody, a peptide and a small molecule.
[0075] According to some embodiments of the invention, the binding
of the agent to the immune-check point protein expressed on the
cancer cell results in at least one of:
[0076] (i) cell cycle arrest of the cancer cell;
[0077] (ii) apoptosis of the cancer cell;
[0078] (iii) sensitization of the cancer cell to a cytotoxic drug;
and
[0079] (iv) activation of an immune response against the cancer
cell.
[0080] According to some embodiments of the invention, the
immune-check point protein expressed on the cancer cell is selected
from the group consisting of LAG3, CD19 and CD70 and CEACAM1.
[0081] According to some embodiments of the invention, the agent
capable of binding the immune-check point protein is an antibody or
a T cell.
[0082] According to some embodiments of the invention, the T cell
comprises a T cell transduced with a T cell receptor (TCR) or a
chimeric antigen receptor (CAR).
[0083] According to some embodiments of the invention, the analog
or derivative has an amino acid sequence as set forth in formula
(I) or a salt thereof:
##STR00001##
[0084] wherein:
[0085] A.sub.1 is an arginine, lysine, ornithine, citrulline,
alanine or glutamic acid residue or a N-.alpha.-substituted
derivative of these amino acids, or A.sub.1 is absent;
[0086] A.sub.2 represents an arginine or glutamic acid residue if
A.sub.1 is present, or A.sub.2 represents an arginine or glutamic
acid residue or a N-.alpha.-substituted derivative of these amino
acids if A.sub.1 is absent;
[0087] A.sub.3 represents an aromatic amino acid residue;
[0088] A.sub.4, A.sub.5 and A.sub.9 each independently represents
an arginine, lysine, ornithine, citrulline, alanine or glutamic
acid residue;
[0089] A.sub.6 represents a proline, glycine, ornithine, lysine,
alanine, citrulline, arginine or glutamic acid residue;
[0090] A.sub.7 represents a proline, glycine, ornithine, lysine,
alanine, citrulline or arginine residue;
[0091] A.sub.8 represents a tyrosine, phenylalanine, alanine,
naphthylalanine, citrulline or glutamic acid residue;
[0092] A.sub.10 represents a citrulline, glutamic acid, arginine or
lysine residue;
[0093] A.sub.11 represents an arginine, glutamic acid, lysine or
citrulline residue wherein the C-terminal carboxyl may be
derivatized;
[0094] and the cysteine residue of the 4-position or the
13-position can form a disulfide bond, and the amino acids can be
of either L or D form.
[0095] According to some embodiments of the invention, the peptide
is selected from the group consisting of SEQ ID NOs: 1-72.
[0096] According to some embodiments of the invention, the peptide
is as set forth in SEQ ID NO: 1.
[0097] According to some embodiments of the invention, the cancer
is a solid tumor cancer.
[0098] According to some embodiments of the invention, the solid
tumor is selected from the group consisting of lung cancer, glioma,
colon cancer, ovarian cancer, renal cancer, melanoma cancer,
hepatocellular cancer, gastric or stomach cancer, glioblastoma,
cervical cancer, bladder cancer, breast cancer, colorectal cancer,
prostate cancer, thyroid cancer, head and neck and pancreatic
cancer.
[0099] According to some embodiments of the invention, the cancer
is a non-solid tumor cancer.
[0100] According to some embodiments of the invention, the
non-solid tumor is selected from the group consisting of multiple
myeloma and leukemia.
[0101] Unless otherwise defined, all technical and/or scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which the invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of
embodiments of the invention, exemplary methods and/or materials
are described below. In case of conflict, the patent specification,
including definitions, will control. In addition, the materials,
methods, and examples are illustrative only and are not intended to
be necessarily limiting.
DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION
[0102] The present invention, in some embodiments thereof, relates
to methods of treating cancer and, more particularly, but not
exclusively, to the use of a CXCR4 antagonistic peptide and an
anti-cancer agent in the treatment of cancer.
[0103] Before explaining at least one embodiment of the invention
in detail, it is to be understood that the invention is not
necessarily limited in its application to the details set forth in
the following description or exemplified by the Examples. The
invention is capable of other embodiments or of being practiced or
carried out in various ways.
[0104] Treatment of most types of cancer involves cytotoxic
treatments such as chemotherapy and radiotherapy that may at least
in part affect many healthy cells and thus result in elevated
toxicity.
[0105] In addition, these treatments are effective in only a small
percentage of cancer affected patients. Immunotherapy strategies
for cancer therapy, aiming at harnessing the immune system to fight
cancer, include cytokines, monoclonal antibodies against tumor
cells or immune regulatory molecules, cancer vaccines as well as
cell-based therapies such as adoptive transfer of ex-vivo activated
T cells and natural killer (NK) cells.
[0106] 4F-benzoyl-TN14003 (SEQ ID NO: 1, also known as BKT140,
hereinafter BL-8040) is a CXCR4 peptide antagonist. It has been
shown that BL-8040 induces mobilization of CD34+ hematopoietic
stem/progenitor cells (HSPC) that can be further used for
transplantation. In addition, BL-8040 was found to be toxic against
several tumors such as myeloid leukemia, hematopoietic tumors and
non-small cell lung cancer.
[0107] While reducing the present invention to practice, the
present inventors have found that in-vivo administration of BL-8040
induces rapid mobilization of a variety of immune cells including
immature stem/progenitor cells as well as fully differentiated T
cells and NK cells. The present findings therefore can be harnessed
to the use of BL-8040 to induce the mobilization and dissemination
of ImDCs and T effector and memory cells into tumor sites and thus
can augment the anti-tumor effect of anti-cancer agents such as
immunotherapeutics.
[0108] Consequently, the present teachings and the protocols
presented in Example 1, suggest the use of a peptide having an
amino acid sequence as set forth in SEQ ID NO: 1 or an analog or
derivative thereof in combination with several anti-cancer agents
for the treatment of cancer.
[0109] Thus, according to a first aspect of the present invention
there is provided a method of treating cancer in a subject in need
thereof, the method comprising administering to the subject a
therapeutically effective amount of a peptide having an amino acid
sequence as set forth in SEQ ID NO: 1 or an analog or derivative
thereof; and an anti-cancer agent selected from the group
consisting of:
[0110] (i) a vaccine selected from the group consisting of
IMCgp100, Prophage G-100 & G-200, GV-1001, IMA-950, CV-9201,
CV-9104, Ad-RTS-hIL-12, ETBX-011, Cavatak, JX-594, ColoAd1,
GL-ONC1, ONCOS-102, CRS-207, ADU-623, Dorgenmeltucel-L, HyperAcute
Prostate, FANG vaccine, MGN-1601, HPV vaccine and Tarmogens such as
GI-4000;
[0111] (ii) anti-cancer reactive mononuclear blood cells
(MNBCs);
[0112] (iii) a cytokine capable of inducing activation and/or
proliferation of a T cell;
[0113] (iv) an immune-check point regulator, wherein said
immune-check point regulator is not a PD1 antagonist, PDL-1
antagonist, CTLA-4 antagonist, LAG-3 antagonist, TIM-3 antagonist,
KIR antagonist, IDO antagonist, OX40 agonist, CD137 agonist, CD27
agonist, CD40 agonist, GITR agonist, CD28 agonist or ICOS
agonist;
[0114] (v) an agent capable of binding an immune-check point
protein expressed on a cancer cell;
[0115] (vi) a colony stimulating factor-1 receptor (CSF1R)
antagonist;
[0116] (vii) a CXCR2 antagonist;
[0117] (viii) a STAT3 antagonist;
[0118] (ix) PV-10; and
[0119] (x) Cotara,
[0120] thereby treating the cancer in the subject.
[0121] As used herein, the terms "treating" or "treatment" refers
to inhibiting, preventing or arresting the development of a
pathology (e.g. cancer) and/or causing the reduction, remission, or
regression of a pathology. Those of skill in the art will
understand that various methodologies and assays can be used to
assess the development of a pathology, and similarly, various
methodologies and assays may be used to assess the reduction,
remission or regression of a pathology.
[0122] As used herein the phrase "subject in need thereof" refers
to a mammalian male or female subject (e.g., human being) who is
diagnosed with cancer. In a specific embodiment, this term
encompasses individuals who are at risk to develop cancer.
Veterinary uses are also contemplated. The subject may be of any
gender or at any age including neonatal, infant, juvenile,
adolescent, adult and elderly adult.
[0123] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth.
[0124] Cancers which can be treated by the method of this aspect of
some embodiments of the invention can be any solid or non-solid
cancer and/or cancer metastasis.
[0125] According to a specific embodiment, the cancer is a solid
tumor.
[0126] According another specific embodiment, the cancer is a
non-solid tumor.
[0127] Examples of cancer include but are not limited to,
carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More
particular examples of such cancers include 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), melanoma cancer, 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
and various types of head and neck cancer, as well as 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; acute myeloblastic
leukemia; Multiple Myeloma; and post-transplant lymphoproliferative
disorder (PTLD), as well as abnormal vascular proliferation
associated with phakomatoses, edema (such as that associated with
brain tumors), and Meigs' syndrome. Preferably, the cancer is
selected from the group consisting of breast cancer, colorectal
cancer, rectal cancer, non-small cell lung cancer, non-Hodgkins
lymphoma (NHL), acute lymphoblastic leukemia (ALL); chronic
myeloblastic leukemia (CML); acute myeloblastic leukemia (AML);
renal cell cancer, prostate cancer, liver cancer, pancreatic
cancer, soft-tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma,
head and neck cancer, melanoma, ovarian cancer, mesothelioma, and
multiple myeloma. The cancerous conditions amenable for treatment
of the invention include metastatic cancers.
[0128] According to specific embodiments the cancer is selected
from the group consisting of lung cancer, glioma, colon cancer,
ovarian cancer, renal cancer, melanoma cancer, hepatocellular
cancer, gastric or stomach cancer, glioblastoma, cervical cancer,
bladder cancer, breast cancer, colorectal cancer, prostate cancer,
thyroid cancer, head and neck and pancreatic cancer.
[0129] According to specific embodiments, the cancer is selected
from the group consisting of lung cancer, glioma, colon cancer and
pancreatic cancer.
[0130] According to specific embodiments, the cancer is selected
from the group consisting of multiple myeloma, Lymphoma and
leukemia.
[0131] According to other specific embodiments, the cancer is
selected from the group consisting of multiple myeloma and
leukemia.
[0132] As used herein, the term "peptide" encompasses native
peptides (either degradation products, synthetically synthesized
peptides or recombinant peptides) and peptidomimetics (typically,
synthetically synthesized peptides), as well as peptoids and
semipeptoids which are peptide analogs, which may have, for
example, modifications rendering the peptides more stable while in
a body or more capable of penetrating into cells.
[0133] According to a specific embodiment, the peptide is no more
than 100 amino acids in length. According to a specific embodiment,
the peptide is 5-100 amino acids in length. According to a specific
embodiment, the peptide is 5-50 amino acids in length. According to
a specific embodiment, the peptide is 5-20 amino acids in length.
According to a specific embodiment, the peptide is 5-15 amino acids
in length. According to a specific embodiment, the peptide is 10-20
amino acids in length. According to a specific embodiment, the
peptide is 10-15 amino acids in length.
[0134] As used herein the term "peptide having an amino acid
sequence as set forth in SEQ ID NO: 1 or an analog or derivative
thereof" refers to 4F-benzoyl-TN14003 (SEQ ID NO: 1, also known as
BKT140, hereinafter BL-8040) peptide and functional analogs or
derivatives thereof. The peptides of the present invention are
structurally and functionally related to the peptides disclosed in
patent applications WO2002/020561 and WO2004/020462, also known as
"T-140 analogs", as detailed hereinbelow. The peptide of the
present invention is a CXCR4-antagonistic peptide i.e. it reduces
CXCR-4 activation by at least 10% as compared to same in the
absence of the peptide antagonist. According to a specific
embodiment the peptide antagonist is a competitive inhibitor.
According to a specific embodiment the peptide antagonist is a
non-competitive inhibitor.
[0135] According to specific embodiments, a functional CXCR4
antagonistic peptide, as used herein, is capable of inducing
mobilization and dissemination of ImDCs, NK cells, B cells,
monocytes/macrophages and T effector and memory cells into a tumor
of a subject upon administration.
[0136] According to other specific embodiments, a functional CXCR4
antagonistic peptide, as used herein, is capable of enhancing an
immune-response to a tumor.
[0137] In various particular embodiments, the peptide analog or
derivative has an amino acid sequence as set forth in the following
formula (I) or a salt thereof:
##STR00002##
[0138] wherein:
[0139] A.sub.1 is an arginine, lysine, ornithine, citrulline,
alanine or glutamic acid residue or a N-.alpha.-substituted
derivative of these amino acids, or A.sub.1 is absent;
[0140] A.sub.2 represents an arginine or glutamic acid residue if
A.sub.1 is present, or A.sub.2 represents an arginine or glutamic
acid residue or a N-.alpha.-substituted derivative of these amino
acids if A.sub.1 is absent;
[0141] A.sub.3 represents an aromatic amino acid residue;
[0142] A.sub.4, A.sub.5 and A.sub.9 each independently represents
an arginine, lysine, ornithine, citrulline, alanine or glutamic
acid residue;
[0143] A.sub.6 represents a proline, glycine, ornithine, lysine,
alanine, citrulline, arginine or glutamic acid residue;
[0144] A.sub.7 represents a proline, glycine, ornithine, lysine,
alanine, citrulline or arginine residue;
[0145] A.sub.8 represents a tyrosine, phenylalanine, alanine,
naphthylalanine, citrulline or glutamic acid residue;
[0146] A.sub.10 represents a citrulline, glutamic acid, arginine or
lysine residue;
[0147] A.sub.11 represents an arginine, glutamic acid, lysine or
citrulline residue wherein the C-terminal carboxyl may be
derivatized;
[0148] and the cysteine residue of the 4-position or the
13-position can form a disulfide bond, and the amino acids can be
of either L or D form.
[0149] Exemplary peptides according to formula (I) are peptides
having an amino acid sequence as set forth in any one of SEQ ID
NOs: 1-72, as presented in Table 1 hereinbelow.
TABLE-US-00001 TABLE 1 T-140 and currently preferred T-140 analogs
SEQ ID Analog NO: Amino acid sequence 4F-benzoyl- 1
4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-
TN14003 NH.sub.2 AcTC14003 2
Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
AcTC14005 3
Ac-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OH
AcTC14011 4
Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OH
AcTC14013 5
Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-OH
AcTC14015 6
Ac-Cit-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
AcTC14017 7
Ac-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OH
AcTC14019 8
Ac-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Cit-Cit-Cys-Arg-OH
AcTC14021 9
Ac-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-OH
AcTC14012 10
Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH.sub.2
AcTC14014 11
Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-NH.sub.2
AcTC14016 12
Ac-Cit-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH.sub.2
AcTC14018 13
Ac-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH.sub.2
AcTC14020 14
Ac-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Cit-Cit-Cys-Arg-NH.sub.2
AcTC14022 15
Ac-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Cit-Cit-Cys-Arg-NH.sub.2
TE14001 16
H-DGlu-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
TE14002 17
H-Arg-Glu-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
TE14003 18
H-Arg-Arg-Nal-Cys-Tyr-Glu-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
TE14004 19
H-Arg-Arg-Nal-Cys-Tyr-Arg-Glu-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
TE14005 20
H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-OH
TE14006 21
H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Glu-Cit-Cys-Arg-OH
TE14007 22
H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Glu-OH
TE14011 23
H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH.s-
ub.2 TE14012 24
H-Arg-Arg-Nal-Cys-Tyr-DGlu-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH.-
sub.2 TE14013 25
H-Arg-Arg-Nal-Cys-Tyr-DGlu-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH.-
sub.2 TE14014 26
H-DGlu-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH.-
sub.2 TE14015 27
H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-DGlu-Arg-Cit-Cys-Arg-NH.-
sub.2 TE14016 28
H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-DGlu-Cys-Arg-NH.-
sub.2 AcTE14014 29
Ac-DGlu-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH.sub.2
AcTE14015 30
Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-DGlu-Arg-Cit-Cys-Arg-NH.sub.2
AcTE14016 31
Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-DGlu-Cys-Arg-NH.sub.2
TF1: 32
Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH.sub-
.2 AcTE14011 TF2: guanyl- 33
guanyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH.sub.2
TE14011 TF3: TMguanyl- 34
TMguanyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH.sub.-
2 TE14011 TF4: TMguanyl- 35
TMguanyl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH.sub.2
TE14011 (2-14) TF5: 4F- 36
4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-
benzoyl- NH.sub.2 TE14011 TF6: 2F- 37
2F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-
benzoyl- NH.sub.2 TE14011 TF7: APA- 38
APA-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH.sub.2
TE14011 (2-14) TF8: desamino- 39
desamino-R-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH.sub.2
R-TE14011 (2-14) TF9: guanyl- 40
Guanyl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH.sub.2
TE14011 (2-14) TF10: succinyl- 41
succinyl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH.sub.2
TE14011 (2-14) TF11: glutaryl- 42
glutaryl-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH.sub.2
TE14011 (2-14) TF12: 43
deaminoTMG-APA-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-A-
rg- deaminoTMG- NH.sub.2 APA-TE14011 (2-14) TF15: H-Arg- 44
R-CH2-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH.sub.2
CH2NH- RTE14011 (2- 14) TF17: TE14011 45
H-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-NH.sub.2
(2-14) TF18: 46
TMguanyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-
-NH.sub.2 TMguanyl- TC14012 TF19: ACA- 47
ACA-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH.sub.2
TC14012 TF20: ACA- 48
ACA-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
T140 TZ14011 49
H-Arg-Arg-Nal-Cys-Tyr-Cit-Arg-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH.s-
ub.2 AcTZ14011 50
Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Arg-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH.sub.2
AcTN14003 51
Ac-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH.sub.2
AcTN14005 52
Ac-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH.sub.2
4F-benzoyl- 53
4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-
TN14011-Me NHMe 4F-benzoyl- 54
4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-
TN14011-Et NHEt 4F-benzoyl- 55
4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-
TN14011-iPr NHiPr 4F-benzoyl- 56
4F-benzoyl-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DGlu-Pro-Tyr-Arg-Cit-Cys-Arg-
TN14011- tyramine tyramine TA14001 57
H-Ala-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
TA14005 58
H-Arg-Arg-Nal-Cys-Tyr-Ala-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
TA14006 59
H-Arg-Arg-Nal-Cys-Tyr-Arg-Ala-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
TA14007 60
H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DAla-Pro-Tyr-Arg-Cit-Cys-Arg-OH
TA14008 61
H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Ala-Tyr-Arg-Cit-Cys-Arg-OH
TA14009 62
H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Ala-Arg-Cit-Cys-Arg-OH
TA14010 63
H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Ala-Cit-Cys-Arg-OH
TC14001 64
H-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
TC14003 65
H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
TN14003 66
H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-NH.s-
ub.2 TC14004 67
H-Arg-Arg-Nal-Cys-Tyr-Arg-Cit-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
TC14012 68
H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH.s-
ub.2 T-140 69
H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH
TC14011 70
H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OH
TC14005 71
H-Arg-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-OH
TC14018 72
H-Cit-Arg-Nal-Cys-Tyr-Arg-Lys-DCit-Pro-Tyr-Arg-Cit-Cys-Arg-NH.s-
ub.2
[0150] According to a specific embodiment, in each one of SEQ ID
NOs: 1-72, two cysteine residues are coupled in a disulfide
bond.
[0151] In another embodiment, the analog or derivative has an amino
acid sequence as set forth in SEQ ID NO: 65
(H-Arg-Arg-Nal-Cys-Tyr-Cit-Lys-DLys-Pro-Tyr-Arg-Cit-Cys-Arg-OH;
TC14003).
[0152] In another embodiment, the peptide used in the compositions
and methods of the invention consists essentially of an amino acid
sequence as set forth in SEQ ID NO: 1. In another embodiment, the
peptide used in the compositions and methods of the invention
comprises an amino acid sequence as set forth in SEQ ID NO: 1. In
another embodiment, the peptide is at least 60%, at least 70% or at
least 80% homologous to SEQ ID NO: 1. In another embodiment, the
peptide is at least 90% homologous to SEQ ID NO: 1. In another
embodiment, the peptide is at least about 95% homologous to SEQ ID
NO: 1. Each possibility represents a separate embodiment of the
present invention.
[0153] In various other embodiments, the peptide is selected from
SEQ ID NOs: 1-72, wherein each possibility represents a separate
embodiment of the present invention.
[0154] In another embodiment, the peptide has an amino acid
sequence as set forth in any one of SEQ ID NOs: 1-4, 10, 46, 47,
51-56, 65, 66, 68, 70 and 71. In another embodiment, the peptide
has an amino acid sequence as set forth in any one of SEQ ID NOs:
4, 10, 46, 47, 68 and 70. In another embodiment, the peptide has an
amino acid sequence as set forth in any one of SEQ ID NOs: 1, 2,
51, 65 and 66. In another embodiment, the peptide has an amino acid
sequence as set forth in any one of SEQ ID NOs: 53-56.
[0155] In an embodiment, the peptide has an amino acid sequence as
set forth in SEQ ID NO: 1. According to a specific embodiment, the
peptide is as set forth in SEQ ID NO: 1. In another embodiment, the
peptide has an amino acid sequence as set forth in SEQ ID NO: 2. In
another embodiment, the peptide has an amino acid sequence as set
forth in SEQ ID NO: 51. In another embodiment, the peptide has an
amino acid sequence as set forth in SEQ ID NO: 66.
[0156] According to a specific embodiment the peptide is as set
forth in SEQ ID NO: 1 and any embodiment described herein should be
read as if specifically reading over this peptide.
[0157] The peptides of some embodiments of the invention may be
synthesized by any techniques that are known to those skilled in
the art of peptide synthesis. For solid phase peptide synthesis, a
summary of the many techniques may be found in J. M. Stewart and J.
D. Young, Solid Phase Peptide Synthesis, W. H. Freeman Co. (San
Francisco), 1963 and J. Meienhofer, Hormonal Proteins and Peptides,
vol. 2, p. 46, Academic Press (New York), 1973. For classical
solution synthesis see G. Schroder and K. Lupke, The Peptides, vol.
1, Academic Press (New York), 1965.
[0158] In general, these methods comprise the sequential addition
of one or more amino acids or suitably protected amino acids to a
growing peptide chain. Normally, either the amino or carboxyl group
of the first amino acid is protected by a suitable protecting
group. The protected or derivatized amino acid can then either be
attached to an inert solid support or utilized in solution by
adding the next amino acid in the sequence having the complimentary
(amino or carboxyl) group suitably protected, under conditions
suitable for forming the amide linkage. The protecting group is
then removed from this newly added amino acid residue and the next
amino acid (suitably protected) is then added, and so forth. After
all the desired amino acids have been linked in the proper
sequence, any remaining protecting groups (and any solid support)
are removed sequentially or concurrently, to afford the final
peptide compound. By simple modification of this general procedure,
it is possible to add more than one amino acid at a time to a
growing chain, for example, by coupling (under conditions which do
not racemize chiral centers) a protected tripeptide with a properly
protected dipeptide to form, after deprotection, a pentapeptide and
so forth. Further description of peptide synthesis is disclosed in
U.S. Pat. No. 6,472,505.
[0159] Large scale peptide synthesis is described by Andersson
Biopolymers 2000; 55(3):227-50.
[0160] According to specific embodiments, the CXCR4 antagonistic
peptide is administered to the subject in combination with one or
more white blood cell mobilizing agents. For example, the peptide
may be administered in sequential or concomitant combination with
one or more other growth factors or cytokines that affect
mobilization such as, but not limited to, G-CSF, GM-CSF and
SCF.
[0161] As mentioned, the peptide of the present invention is
administered in combination with an anti-cancer agent.
[0162] As used herein, the term "anti-cancer agent" refers to an
agent effective in inhibiting, slowing or arresting the growth or
metastasis of a cancerous cell or which exhibits a cytotoxic effect
on a cancerous cell. According to specific embodiments, the
anti-cancer agent is an anti-cancer immune modulator agent which is
capable of eliciting an immune response (e.g. T cell, NK cell, B
cell, complement) against a cancerous cell.
[0163] The anti-cancer agent of the present invention is selected
from the group consisting of:
[0164] (i) a vaccine selected from the group consisting of
IMCgp100, Prophage G-100 & G-200, GV-1001, IMA-950, CV-9201,
CV-9104, Ad-RTS-hIL-12, ETBX-011, Cavatak, JX-594, ColoAd1,
GL-ONC1, ONCOS-102, CRS-207, ADU-623, Dorgenmeltucel-L, HyperAcute
Prostate, FANG vaccine, MGN-1601, HPV vaccine and Tarmogens such as
GI-4000;
[0165] (ii) anti-cancer reactive mononuclear blood cells
(MNBCs);
[0166] (iii) a cytokine capable of inducing activation and/or
proliferation of a T cell;
[0167] (iv) an immune-check point regulator, wherein said
immune-check point regulator is not a PD1 antagonist, PDL-1
antagonist, CTLA-4 antagonist, LAG-3 antagonist, TIM-3 antagonist,
KIR antagonist, IDO antagonist, OX40 agonist, CD137 agonist, CD27
agonist, CD40 agonist, GITR agonist, CD28 agonist or ICOS
agonist;
[0168] (v) an agent capable of binding an immune-check point
protein expressed on a cancer cell;
[0169] (vi) a colony stimulating factor-1 receptor (CSF1R)
antagonist;
[0170] (vii) a CXCR2 antagonist;
[0171] (viii) a STAT3 antagonist;
[0172] (ix) PV-10; and
[0173] (x) Cotara,
[0174] wherein each possibility represents a separate embodiment of
the present invention.
[0175] As used herein, the term "vaccine" refers to a composition
used to provoke a specific immune response against a cancerous cell
(e.g. the production of anti-cancer antibodies, eliciting a cell
mediated immune-response) following administration.
[0176] According to specific embodiments of the present invention
the vaccine includes: IMCgp100 [a soluble affinity enhanced T cell
receptor (TCR) specific for the melanoma-associated antigen gp100,
fused to an anti-CD3 specific antibody fragment (scFv), e.g.,
produced by Immunocore]; the antigen based vaccines Prophage G-100
& G-200 (HSPPC-96, e.g., produced by Agenus), GV-1001 (e.g.,
produced by KAEL-GemVax), IMA-950 (e.g., produced by Immatics
Biotechnologies), CV-9201 (e.g., produced by CureVac) and CV-9104
(CV-9103, e.g., produced by CureVac); the viral vectors vaccines
Ad-RTS-hIL-12 (INXN-2001, e.g., produced by Ziopharm), ETBX-011
(Ad5 [E1-, E2b-]-CEA(6D), e.g., produced by Etubics), Cavatak
(Coxsackievirus A21, e.g., produced by Viralytics), JX-594 (e.g.,
produced by Jennerex Biopharmaceuticals/Transgene), ColoAd1 (e.g.,
produced by PsiOxus Therapeutics), GL-ONC1 (e.g., produced by
Genelux) and ONCOS-102 (CGTG-102, e.g., produced by Oncos
Therapeutics); the bacterial vectors vaccines CRS-207 (an
attenuated form of Listeria monocytogenes genetically modified to
express the tumor associated antigen mesothelin, e.g., produced by
Aduro BioTech/BioSante Pharmaceuticals) and ADU-623 (a
live-attenuated, double-deleted strain of the Gram-positive
bacterium Listeria monocytogenes encoding a mutant form of the
tumor-associated antigens, epidermal growth factor receptor
(EGFRvIII) and the cancer/testis antigen NY-ESO-1, e.g., produced
by Aduro BioTech); the tumor cell vaccines Dorgenmeltucel-L (e.g.,
produced by NewLink Genetics), HyperAcute Prostate (e.g., produced
by NewLink Genetics), FANG vaccine (e.g., produced by Gradalis),
HPV vaccine and MGN-1601 (e.g., produced by Mologen AG); and
Tarmogens such as GI-4000 (e.g., produced by Globelmmune)
heat-inactivated S. cerevisiae yeast expressing a unique
combination of three Ras mutations, collectively targeting seven of
the most common Ras mutations observed in human cancers.
[0177] According to a specific embodiment, the vaccine is a human
papiloma virus (HPV, e.g., HPV 16 vaccine) typically targeting E6
and/or E7. The vaccine may be a preventive vaccine or a therapeutic
vaccine. Detailed examples of HPV vaccines which can be used along
with the present teachings can be found in Lin et al. J Formos Med
Assoc. 2010 January; 109(1): 4-24; and Rice et al. Cancer Gene
Therapy 22, 454-462.
[0178] According to a specific embodiment, the preventive vaccines
utilize the capsid proteins L1 and L2 as target antigens, inducing
antibodies to neutralize and prevent entry of HPV into cells.
Expression of recombinant L1, the major component of the capsid, in
various cell types results in spontaneous assembly of virus-like
particles (VLPs), which are immunologically and morphologically
similar to HPV virions.
[0179] According to another specific embodiment, the vaccines is
Gardasil.TM. or Cervarix.TM. Gardasil is a quadrivalent vaccine
containing recombinant L1 VLPs for HPV genotypes 6, 11, 16 and 18
whereas the bivalent vaccine Cervarix contains L1 VLPs for HPV-16
and 18.
[0180] According to another specific embodiment, the vaccine is a
monovalent HPV-16 L1 vaccine with an aluminium hydroxyphosphate
sulfate adjuvant.
[0181] Exemplary, non-limiting, therapeutic vaccines comprise HPV
E6 and E7 antigens. These represent ideal targets for therapeutic
vaccines since these are constitutively expressed in HPV-infected
cells and not healthy cells. E6 and E7 are essential to the
induction and maintenance of cellular transformation, and thus are
unlikely to be lost in an attempt to evade the immune system.
[0182] According to a specific embodiment, the therapeutic vaccines
target E6 and/or E7.
[0183] Therapeutic vaccines typically include:
[0184] Live vector vaccines--Vector-based vaccines can deliver the
antigens E6 and E7 to the dendritic cells (DCs), stimulating
antigen expression through MHC class I (to CD8+ cytotoxic T cells)
and MHC class II (to CD4+ helper T cells). Viral vectors used
adenovirus, adeno-associated virus, vaccinia virus and
alphaviruses, such as the Venezuelan equine encephalitis (VEE)
virus;
[0185] Peptide/protein-based vaccines--Administered peptides and
proteins derived from HPV antigens (e.g., E6 and/or E7) are taken
up by DCs, processed and expressed via MHC II and/or I to the
appropriate CD4+/CD8+ T cells;
[0186] Cell-based vaccines--dendritic cell-based or tumor cell
based vaccines; and
[0187] Nucleic acid-based vaccines e.g., naked DNA based vaccines
(e.g., ZYC-101 and ZYC-101a), naked RNA replicon vaccines.
[0188] The vaccine may be administered with an adjuvant, such as,
but not limited to, incomplete Freund's Adjuvant, aluminum salts,
oil-in-water emulsion (MF59), and nontoxic derivatives from
Salmonella (MPL), water-in-oil emulsions (e.g. Montanide ISA 51 and
ISA 720), saponins (e.g. ISCOM, QS-21, ASO1 and AS02), SRL-172,
histamine dihydrochloride, thymocartin, Tio-TEPA,
monophosphoryl-lipid A/micobacteria compositions, alum, Montanide
ISA, Ribi Adjuvant System, TiterMax adjuvant, syntex adjuvant
formulations, immune-stimulating complexes (ISCOMs), Gerbu.sup.R
adjuvant, CpG oligodeoxynucleotides, lipopolysaccharide, and
polyinosinic:polycytidylic acid.
[0189] As used herein, the phrase "anti-cancer reactive mononuclear
blood cells (MNBCs)" refers to blood cells having a single nucleus.
According to a specific embodiment MNBCs include lymphocytes,
monocytes and dendritic cells (DCs) which are capable of eliciting
an immune response (e.g. T cell, NK cell) against a cancerous
cell.
[0190] According to specific embodiments the MNBCs are selected
from the group consisting of dendritic cells (DCs), T cells, B
cells, NK cells and NKT cells.
[0191] As used herein, the term "T cell" refers to a differentiated
lymphocyte with a CD3.sup.+, T cell receptor (TCR).sup.+ having
either CD4.sup.+ or CD8.sup.+ phenotype. The T cell may be either
an effector or a regulatory T cells.
[0192] As used herein, the term "effector T cells" refers to a T
cell that activates or directs other immune cells e.g. by producing
cytokines or has a cytotoxic activity e.g., CD4+, Th1/Th2, CD8+
cytotoxic T lymphocyte.
[0193] As used herein, the term "regulatory T cell" or "Treg"
refers to a T cell that negatively regulates the activation of
other T cells, including effector T cells, as well as innate immune
system cells. Treg cells are characterized by sustained suppression
of effector T cell responses. According to a specific embodiment,
the Treg is a CD4+CD25+Foxp3+ T cell.
[0194] According to specific embodiments, MNBCs comprise T
cells.
[0195] As used herein the term "B cell" refers to a lymphocyte with
a B cell receptor (BCR)+, CD19+ and or B220+ phenotype. B cells are
characterized by their ability to bind a specific antigen and
elicit a humoral response.
[0196] As used herein the term "natural killer cell (NK cell)"
refers to a differentiated lymphocyte with a CD16+ CD56+ and/or
CD57+ TCR- phenotype. NKs are characterized by their ability to
bind to and kill cells that fail to express "self" MHC/HLA antigens
by the activation of specific cytolytic enzymes, the ability to
kill tumor cells or other diseased cells that express a ligand for
NK activating receptors, and the ability to release protein
molecules called cytokines that stimulate or inhibit the immune
response.
[0197] As used herein the term "NKT cells" refers to a specialized
population of T cells that express a semi-invariant .alpha..beta.
T-cell receptor, but also express a variety of molecular markers
that are typically associated with NK cells, such as NK1.1. NKT
cells include NK1.1.sup.+ and NK1.1.sup.-, as well as CD4.sup.+,
CD4.sup.-, CD8.sup.+ and CD8.sup.- cells. The TCR on NKT cells is
unique in that it recognizes glycolipid antigens presented by the
MHC I-like molecule CD1d. NKT cells can have either protective or
deleterious effects due to their abilities to produce cytokines
that promote either inflammation or immune tolerance.
[0198] As used herein the term "dendritic cell (DC)" refers to an
antigen presenting cell capable of sensitizing HLA-restricted T
cells. DCs include DCs derived from bone marrow hematopoietic cells
such as plasmacytoid dendritic cells, myeloid dendritic cells,
Langerhans cells and interdigitating cells; and follicular DCs.
Dendritic cells may be recognized by function, or by phenotype,
particularly by cell surface phenotype. These cells are
characterized by their distinctive morphology having veil-like
projections on the cell surface, intermediate to high levels of
surface HLA-class II expression and ability to present antigen to T
cells, particularly to naive T cells (See Steinman R, et al., Ann.
Rev. Immunol. 1991; 9:271-196.). Typically, cell surface phenotype
of DCs include CD1a+, CD4+, CD86+, or HLA-DR. The term DCs
encompasses both immature and mature DCs.
[0199] According to specific embodiments, the MNBCs comprise
dendritic cells.
[0200] According to specific embodiments the MNBCs do not comprise
CD34+ hematopoietic stem/progenitor cells.
[0201] The MNBCs used according to specific embodiments of the
present invention may be autologous or non-autologous; they can be
syngeneic or non-syngeneic: allogeneic or xenogeneic. Each
possibility represents a separate embodiment of the present
invention.
[0202] As used herein, the term "autologous" means that the donor
subject is the recipient subject. Thus, in autologous
transplantation the cells have been removed and re-introduced e.g.,
re-infused to the subject.
[0203] As used herein, the term "non-autologous" means that the
donor subject is not the recipient subject.
[0204] As used herein, the term "syngeneic" means that the donor
subject is essentially genetically identical with the recipient
subject. Examples of syngeneic transplantation include
transplantation of cells derived from the subject (also referred to
in the art as "autologous"), a clone of the subject, or a
homozygotic twin of the subject.
[0205] As used herein, the term "allogeneic" means that the donor
is of the same species as the recipient, but which is substantially
non-clonal with the recipient. Typically, outbred, non-zygotic twin
mammals of the same species are allogeneic with each other. It will
be appreciated that an allogeneic donor may be HLA identical or HLA
non-identical with respect to the subject.
[0206] As used herein, the term "xenogeneic" means that the donor
subject is from a different species relative to the recipient
subject.
[0207] Methods of obtaining and/or developing anti-cancer reactive
MNBCs that can be used according to specific embodiments of the
present invention are well known in the art (see e.g. Hildebrandt
et al. Cytotherapy. 2014 16(40): S120-S129; Leen et al. Immunol
Rev. 2014; 258(1): 12-29; Qian et al. Journal of Immunology
Research Volume 2014, Article ID 525913, 9 pages; Martelli et al.
Blood. 2014; 123(7):967-973; Ophir and Reisner Front Immunol. 2012
3:93; Lask et al. Blood. 2013; 121(15):3033-3040; Galluzzi et al.
Oncoimmunology. 2012; 1(3):306-315; Itzhaki et al. Immunotherapy.
2013; 5(1):79-90; Rosenberg et al. Clinical cancer research: an
official journal of the American Association for Cancer Research.
2011; 17(13):4550-4557; Bouquie, et al. Cancer Immunol Immunother.
2009; 58:553-66; Lu et al. Journal of immunology. 2013;
190(12):6034-6042; Robbins et al. Journal of clinical oncology:
official journal of the American Society of Clinical Oncology.
2011; 29(7):917-924; June J. Clin Invest. (2007) 117(6): 1466-1476;
Greenberg, R. & Riddell, S. (1999) Science 285: 546-51); and
Shtivelman et al. Oncotarget. 2014 Apr. 15; 5(7):1701-52; the
contents of each of which are fully incorporated herein by
reference).
[0208] Thus, for example, a peripheral blood sample is collected
from a subject by methods well known in the art such as drawing
whole blood from the subject and collection in a container
containing an anti-coagulant (e.g. heparin or citrate); and
apheresis. Following, at least one type of MNBCs is purified from
the peripheral blood and enriched for at least one type of MNBCs
effective for the treatment of cancer. There are several methods
and reagents known to those skilled in the art for purifying MNBCs
from whole blood such as leukapheresis, sedimentation, density
gradient centrifugation (e.g. ficoll), centrifugal elutriation,
fractionation, chemical lysis of e.g. red blood cells (e.g. by
ACK), selection of specific cell types using cell surface markers
(using e.g. FACS sorter or magnetic cell separation techniques),
and depletion of specific cell types by methods such as eradication
(e.g. killing) with specific antibodies or by affinity based
purification based on negative selection (using e.g. magnetic cell
separation techniques, FACS sorter and/or capture ELISA labeling).
Such methods are described for example in THE HANDBOOK OF
EXPERIMENTAL IMMUNOLOGY, Volumes 1 to 4, (D. N. Weir, editor) and
FLOW CYTOMETRY AND CELL SORTING (A. Radbruch, editor, Springer
Verlag, 2000).
[0209] Enriching for an anti-cancer reactive MNBCs can be effected
by any method known in the art (see e.g. Hildebrandt et al.
Cytotherapy. 2014 16(40): S120-S129; Leen et al. Immunol Rev. 2014;
258(1): 12-29; Qian et al. Journal of Immunology Research Volume
2014, Article ID 525913, 9 pages; Palucka and Banchereau Immunity.
2013; 39(1): 38-48; and Pizzurro and Barrio Front Immunol. 2015
Mar. 3; 6:91) and include for example, activating the specifically
desired population of cells; promoting presentation of a specific
antigen leading to enrichment of a population of antigen presenting
cells presenting the specific antigen and/or activation and/or
proliferation of antigen-specific population of cells (e.g. T
cells); promoting presentation of a specific receptor leading to
enrichment of a population of antigen-specific population of cells
(e.g. T cells, B cells).
[0210] As a non-limiting example, for the generation of T cells
that can be used for adoptive T cells transfer for cancer therapy
the peripheral blood or purified population thereof comprising
CD3+, CD4+ or CD8+ T cells or tumor-associated lymphocytes (TALs)
selected for T-cell receptor (TCR) specificity (see e.g. Cancer
Immunol Immunother. 2009; 58: 553-66) is activated and expanded
ex-vivo by incubation with mature DCs preloaded with tumor antigens
or transfected with mRNA coding for cancer antigens. Alternatively,
the peripheral blood or a purified population thereof comprising
both APCs (e.g. DCs) and T cells (e.g. CD3+, CD4+ or CD8+ T cells
or TALs selected for TCR specificity) is contacted with tumor
antigens or transfected with mRNA coding for cancer antigens under
conditions which allow capturing and presentation of the cancer
antigen by the APC leading to activation and expansion of
anti-cancer antigen-specific T cells.
[0211] A specific non-limiting example of an autologous T cell
therapy that can be used according to some embodiments of the
present invention is C ntego tumor-infiltrating lymphocytes (TILs,
produced by Lion Biotechnologies).
[0212] Other methods of expanding and processing T cells for
anti-cancer adoptive cell transfer include redirecting T cell
specificity by promoting presentation of an anti-cancer receptor by
way of transducing with a T cell receptor (TCR) or a chimeric
antigen receptor (CAR).
[0213] According to specific embodiments, the MNBCs comprise T
cells transduced with a T cell receptor (TCR) or a chimeric antigen
receptor (CAR).
[0214] As used herein "transduction with a TCR" refers to cloning
of variable .alpha.- and .beta.-chains are from T cells with
specificity against a cancer antigen presented in the context of
MHC. Method of transducing with a TCR are known in the art and are
disclosed e.g. in Nicholson et al. Adv Hematol. 2012; 2012:404081;
Wang and Riviere Cancer Gene Ther. 2015 March; 22(2):85-94); and
Lamers et al., Cancer Gene Therapy (2002) 9, 613-623.
[0215] As used herein "transducing with a CAR" refers to cloning of
a nucleic acid sequence encoding a chimeric antigen receptor (CAR),
wherein the CAR comprises an antigen recognition moiety and a
T-cell activation moiety. A chimeric antigen receptor (CAR) is an
artificially constructed hybrid protein or polypeptide containing
an antigen binding domain of an antibody (e.g., a single chain
variable fragment (scFv)) linked to T-cell signaling or T-cell
activation domains. Method of transducing with a CAR are known in
the art and are disclosed e.g. in Davila et al. Oncoimmunology.
2012 Dec. 1; 1(9):1577-1583; Wang and Riviere Cancer Gene Ther.
2015 March; 22(2):85-94); and Maus et al. Blood. 2014 Apr. 24;
123(17):2625-35.
[0216] Another non-limiting example is the generation of dendritic
cells vaccine that can be used according to some embodiments of the
present invention.
[0217] Typically, for the generation of an anti-cancer DC-based
vaccine DCs are expanded ex vivo and contacted with a cancer
antigen or a cancer cell lysate to thereby induce presentation of
the cancer antigen (see e.g. Nestle, F. et al. (1998) Nature
Medicine 4: 328-332). Alternatively, promoting presentation of a
cancer antigen by a DC comprises transfecting the DC with a DNA,
cDNA or an mRNA coding for a cancer antigen. Non-limiting examples
of cancer antigens include MAGE-AI, MAGE-A2, MAGE-A3, MAGE-A4,
MAGE-AS, MAGE-A6, MAGE-A7, MAGE-AS, MAGE-A9, MAGE-AIO, MAGE-All,
MAGE-A12, GAGE-I, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7,
GAGE-8, BAGE-1, RAGE-1, LB33/MUM-1, PRAME, NAG, MAGE-Xp2 (MAGE-B2),
MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-Cl/CT7, MAGE-C2,
NY-ESO-1, LAGE-1, SSX-1, SSX-2(HOM-MEL-40), SSX-3, SSX-4, SSX-5,
SCP-1 and XAGE, melanocyte differentiation antigens, p53, ras, CEA,
MUCI, PMSA, PSA, tyrosinase, Melan-A, MART-I, gplOO, gp75,
alphaactinin-4, Bcr-Abl fusion protein, Casp-8, beta-catenin,
cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1
fusion protein, LDLR-fucosyltransferaseAS fusion protein, HLA-A2,
HLA-All, hsp70-2, KIAA0205, Mart2, Mum-2, and 3, neo-PAP, myosin
class I, OS-9, pml-RAR alpha fusion protein, PTPRK, K-ras, N-ras,
Triosephosphate isomerase, GnTV, Herv-K-mel, NA-88, SP17, and
TRP2-Int2, (MART-I), E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein
Barr virus antigens, EBNA, human papillomavirus (HPV) antigens E6
and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p1SOerbB-3,
c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa,
K-ras, alpha.-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA
27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5,
0250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\170K,
NYCO-I, RCASI, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin
C-associated protein), TAAL6, TAG72, TLP, TPS, tyrosinase related
proteins, TRP-1, or TRP-2 and tumor derived heat shock
proteins.
[0218] Other tumor antigens that may be expressed include
out-of-frame peptide-MHC complexes generated by the non-AUG
translation initiation mechanisms employed by "stressed" cancer
cells (Malarkannan et al. Immunity 1999).
[0219] Other tumor antigens that may be expressed are well-known in
the art (see for example WO00/20581; Cancer Vaccines and
Immunotherapy (2000) Eds Stern, Beverley and Carroll, Cambridge
University Press, Cambridge). The sequences of these tumor antigens
are readily available from public databases but are also found in
WO 1992/020356, WO 1994/005304, WO 1994/023031, WO 1995/020974, WO
1995/023874 & WO 1996/026214.
[0220] For EBV-associated lymphoma, EBV-specific antigens can be
used as the cancer antigen. Other cancer antigens may include the
proteins from viruses implicated in human cancers such a Human
Papilloma Viruses (HPV), Hepatitis Viruses (HBV and HCV) and
Kaposi's Herpes Sarcoma Virus (KHSV).
[0221] Alternatively or additionally a tumor antigen may be
identified using cancer cells obtained from the subject by e.g.
biopsy.
[0222] DCs can also be fused directly to tumor cells for the
purposes of immunization (see e.g. Kugler, A. et al. (2000) Nature
Medicine 6:332-336).
[0223] Specific non-limiting examples of DC-based vaccines that can
be used according to specific embodiments of the present invention
include: BPX-201 (produced by Bellicum Pharmaceuticals), AV-0113
(Trivax, produced by Activartis Biotech), ICT-107 (produced by
ImmunoCellular Therapeutics) and Ad.p53-DC vaccine (produced by
Medvax).
[0224] The anti-cancer reactive MNBCs used according to some
embodiments of the present invention can be freshly isolated cells;
stored e.g., cryopreserved (i.e. frozen) at e.g. liquid nitrogen
temperature cells; and cell lines.
[0225] According to specific embodiments the MNBCs are derived from
a subject not treated with a peptide having an amino acid sequence
as set forth in SEQ ID NO: 1 or an analog or derivative
thereof.
[0226] According to specific embodiments, the anti-cancer agent is
a cytokine capable of inducing activation and/or proliferation of a
T cell. Non-limiting examples of cytokines capable of inducing
activation and/or proliferation of a T cell include, but are not
limited to IL-2, IFN.alpha., IL-12, IFN-gamma, TNF-.alpha., IL-15,
IL-6 and IL-1, IL-21.
[0227] According to specific embodiments, the cytokine is selected
from the group consisting of IFN.alpha., IFN.gamma., IL-1, IL-2,
IL-6, IL-12, IL-15, IL-21 and TNF.alpha..
[0228] According to other specific embodiments, the cytokine is
selected from the group consisting of IFN.alpha., IL-2, IL-12,
IL-21 and IL-15.
[0229] Specific non-limiting examples of cytokines and cytokines
agonists that can be used according to some embodiments of the
invention include:
[0230] IL-2 (produced by Roche); IL21 (produced by BMY); ALT-803
(IL15 superagonist combined with a soluble IL-15a receptor,
produced by Altor Bioscience); Darleukin (L19-IL2, human IL-2
conjugated with an antibody (L19) that is specific to the EDB
region of fibronectin, produced by Philogen); Denenicokin
[BMS-982470, a recombinant human peptide homologous to IL-21,
produced by Bristol-Myers Squibb (ZymoGenetics)]; and Immunopulse
(delivery of DNA-based IL-12 leading to localized expression of
IL-12 in the tumor microenvironment, produced by Oncosec
Medical.
[0231] According to specific embodiments the anti-cancer agent is
an immune-check point regulator.
[0232] As used herein the term "immune-check point regulator"
refers to a molecule that modulates the activity of one or more
immune-check point proteins in an agonistic or antagonistic manner
resulting in recruitment of an immune cell to elicit an immune
activity against a cancer cell. The immune-check point regulator
may bind directly the immune-check point or through an intermediary
molecule, it can modulate the activity of a specific immune-check
point protein with no cross reactivity with other immune-check
point proteins or modulate the activity of at least 2, at least 3,
at least 4 immune-check point proteins; each possibility represents
a separate embodiment of the present invention.
[0233] As used herein the term "activation" refers to the process
of stimulating an immune cell (e.g. T cell, NK cell, B cell) that
results in cellular proliferation, maturation, cytokine production
and/or induction of regulatory or effector functions.
[0234] As used herein the term "immune-check point protein" refers
to an antigen independent protein that modulates an immune cell
response (i.e. activation or function). Immune-check point proteins
can be either co-stimulatory proteins [i.e. positively regulating
an immune cell activation or function by transmitting a
co-stimulatory secondary signal resulting in activation of an
immune cell] or inhibitory proteins (i.e. negatively regulating an
immune cell activation or function by transmitting an inhibitory
signal resulting in suppressing activity of an immune cell).
[0235] According to specific embodiments, the immune-check point
protein regulates activation or function of a T cell.
[0236] Numerous check-point proteins are known in the art and
include, but not limited to, PD1, PDL-1, B7H2, B7H3, B7H4, BTLA-4,
HVEM, CTLA-4, CD80, CD86, LAG-3, TIM-3, KIR, IDO, CD19, OX40,
OX40L, 4-1BB (CD137), 4-1BBL, CD27, CD70, CD40, CD40L, GITR, CD28,
ICOS (CD278), ICOSL, VISTA and adenosine A2a receptor.
[0237] Methods of determining signaling of a stimulatory or
inhibitory signal are well known in the art and include, but are
not limited to, binding assay using e.g. BiaCore, HPLC or flow
cytometry, enzymatic activity assays such as kinase activity
assays, and expression of molecules involved in the signaling
cascade using e.g. PCR, Western blot, immunoprecipitation and
immunohistochemistry. Additionally or alternatively, determining
transmission of a signal (co-stimulatory or inhibitory) can be
effected by evaluating immune cell activation or function. Methods
of evaluating immune cell activation or function are well known in
the art and include, but are not limited to, proliferation assays
such as BRDU and thymidine incorporation, cytotoxicity assays such
as chromium release, cytokine secretion assays such as
intracellular cytokine staining ELISPOT and ELISA, expression of
activation markers such as CD25, CD69 and CD69 using flow
cytometry.
[0238] According to specific embodiments, determining the signaling
activity is effected in-vitro or ex-vivo e.g. in a mixed lymphocyte
reaction (MLR).
[0239] For the same culture conditions the signaling activity or
the immune cell activation or function are generally expressed in
comparison to the signaling, activation or function in a cell of
the same species but not contacted with the immune-check point
regulator or contacted with a vehicle control, also referred to as
control.
[0240] According to specific embodiments the immune-check point
regulator is selected from the group consisting of an antibody, a
peptide and a small molecule as further described in details
hereinbelow.
[0241] Depending on the immune-check point protein (i.e.
co-stimulatory or inhibitory) the immune-check point regulator can
be an agonist or antagonist.
[0242] According to specific embodiment the immune-check point
regulator is an antagonist.
[0243] As used herein the term "antagonist" refers to a molecule
that prevents and/or inhibits the biological function and/or
expression of an immune-check point protein.
[0244] According to specific embodiments, the antagonist prevents
and/or inhibits the suppressive effect of an immune-check point
protein on an immune cell (e.g. T cells).
[0245] According to specific embodiments, the antagonist prevents
and/or inhibits signaling to an immune cell (e.g. T cell) by an
immune-check point protein.
[0246] The molecule may be a reversible or an irreversible
antagonist.
[0247] According to specific embodiments, the antagonist completely
prevents the biological function (e.g. signal transduction) of the
immune-check point protein.
[0248] According to other specific embodiments, the antagonist
inhibits the biological function (e.g. signal transduction) of the
immune-check point protein e.g., as detected by e.g. kinase
activity, proliferation assay, cytotoxicity assay or cytokine
secretion assay. The reduction may be by at least a 10%, at least
20%, at least 30%, at least 40%, at least 50%, at least 60%, at
least 70%, at least 80%, at least 90%, at least 95% or at least 99%
as compared to same in the absence of the antagonist.
[0249] Preventing and/or inhibiting the biological function of an
immune-check point protein can be effected at the protein level
(e.g., antibodies, small molecules, inhibitory peptides, enzymes
that cleave the polypeptide, aptamers and the like) but may also be
effected at the genomic (e.g. homologous recombination and site
specific endonucleases) and/or the transcript level using a variety
of molecules which interfere with transcription and/or translation
(e.g., RNA silencing agents) of an inhibitory immune-check point
protein.
[0250] Non limiting examples of agents that can function as
antagonists are described in details hereinbelow.
[0251] Suppressing Biological Function at the Polypeptide Level
[0252] According to specific embodiments, the antagonistic agent is
an antibody.
[0253] According to specific embodiments the antagonistic antibody
is capable of specifically binding an inhibitory immune-check point
protein. According to specific embodiments, the antagonistic
antibody specifically binds at least one epitope of an inhibitory
immune-check point protein.
[0254] As used herein, the term "epitope" refers to any antigenic
determinant on an antigen to which the paratope of an antibody
binds. Epitopic determinants usually consist of chemically active
surface groupings of molecules such as amino acids or carbohydrate
side chains and usually have specific three dimensional structural
characteristics, as well as specific charge characteristics.
[0255] The term "antibody" as used in this invention includes
intact molecules as well as functional fragments thereof, such as
Fab, F(ab')2, Fv, scFv, dsFv, or single domain molecules such as VH
and VL that are capable of binding to an epitope of an antigen.
[0256] The antibody may be mono-specific (capable of recognizing
one epitope or protein), bi-specific (capable of binding two
epitopes or proteins) or multi-specific (capable of recognizing
multiple epitopes or proteins).
[0257] Suitable antibody fragments for practicing some embodiments
of the invention include a complementarity-determining region (CDR)
of an immunoglobulin light chain (referred to herein as "light
chain"), a complementarity-determining region of an immunoglobulin
heavy chain (referred to herein as "heavy chain"), a variable
region of a light chain, a variable region of a heavy chain, a
light chain, a heavy chain, an Fd fragment, and antibody fragments
comprising essentially whole variable regions of both light and
heavy chains such as an Fv, a single chain Fv Fv (scFv), a
disulfide-stabilized Fv (dsFv), an Fab, an Fab', and an
F(ab')2.
[0258] As used herein, the terms "complementarity-determining
region" or "CDR" are used interchangeably to refer to the antigen
binding regions found within the variable region of the heavy and
light chain polypeptides. Generally, antibodies comprise three CDRs
in each of the VH (CDR HI or HI; CDR H2 or H2; and CDR H3 or H3)
and three in each of the VL (CDR LI or LI; CDR L2 or L2; and CDR L3
or L3).
[0259] The identity of the amino acid residues in a particular
antibody that make up a variable region or a CDR can be determined
using methods well known in the art and include methods such as
sequence variability as defined by Kabat et al. (See, e.g., Kabat
et al., 1992, Sequences of Proteins of Immunological Interest, 5th
ed., Public Health Service, NIH, Washington D.C.), location of the
structural loop regions as defined by Chothia et al. (see, e.g.,
Chothia et al., Nature 342:877-883, 1989.), a compromise between
Kabat and Chothia using Oxford Molecular's AbM antibody modeling
software (now Accelrys.RTM., see, Martin et al., 1989, Proc. Natl
Acad Sci USA. 86:9268; and world wide web site
www(dot)bioinf-org(dot)uk/abs), available complex crystal
structures as defined by the contact definition (see MacCallum et
al., J. Mol. Biol. 262:732-745, 1996), the "conformational
definition" (see, e.g., Makabe et al., Journal of Biological
Chemistry, 283:1156-1166, 2008) and IMGT [Lefranc M P, et al.
(2003) IMGT unique numbering for immunoglobulin and T cell receptor
variable domains and Ig superfamily V-like domains. Dev Comp
Immunol 27: 55-77].
[0260] As used herein, the "variable regions" and "CDRs" may refer
to variable regions and CDRs defined by any approach known in the
art, including combinations of approaches.
[0261] Functional antibody fragments comprising whole or
essentially whole variable regions of both light and heavy chains
are defined as follows:
[0262] (i) Fv, defined as a genetically engineered fragment
consisting of the variable region of the light chain (VL) and the
variable region of the heavy chain (VH) expressed as two
chains;
[0263] (ii) single chain Fv ("scFv"), a genetically engineered
single chain molecule including the variable region of the light
chain and the variable region of the heavy chain, linked by a
suitable polypeptide linker as a genetically fused single chain
molecule;
[0264] (iii) disulfide-stabilized Fv ("dsFv"), a genetically
engineered antibody including the variable region of the light
chain and the variable region of the heavy chain, linked by a
genetically engineered disulfide bond;
[0265] (iv) Fab, a fragment of an antibody molecule containing a
monovalent antigen-binding portion of an antibody molecule which
can be obtained by treating whole antibody with the enzyme papain
to yield the intact light chain and the Fd fragment of the heavy
chain which consists of the variable and CH1 domains thereof;
[0266] (v) Fab', a fragment of an antibody molecule containing a
monovalent antigen-binding portion of an antibody molecule which
can be obtained by treating whole antibody with the enzyme pepsin,
followed by reduction (two Fab' fragments are obtained per antibody
molecule);
[0267] (vi) F(ab') 2, a fragment of an antibody molecule containing
a monovalent antigen-binding portion of an antibody molecule which
can be obtained by treating whole antibody with the enzyme pepsin
(i.e., a dimer of Fab' fragments held together by two disulfide
bonds); and
[0268] (vii) Single domain antibodies or nanobodies are composed of
a single VH or VL domains which exhibit sufficient affinity to the
antigen.
[0269] The antibody may be monoclonal or polyclonal.
[0270] Methods of producing polyclonal and monoclonal antibodies as
well as fragments thereof are well known in the art (See for
example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold
Spring Harbor Laboratory, New York, 1988, incorporated herein by
reference).
[0271] Antibody fragments according to some embodiments of the
invention can be prepared by proteolytic hydrolysis of the antibody
or by expression in E. coli or mammalian cells (e.g. Chinese
hamster ovary cell culture or other protein expression systems) of
DNA encoding the fragment. Antibody fragments can be obtained by
pepsin or papain digestion of whole antibodies by conventional
methods. For example, antibody fragments can be produced by
enzymatic cleavage of antibodies with pepsin to provide a 5S
fragment denoted F(ab')2. This fragment can be further cleaved
using a thiol reducing agent, and optionally a blocking group for
the sulfhydryl groups resulting from cleavage of disulfide
linkages, to produce 3.5S Fab' monovalent fragments. Alternatively,
an enzymatic cleavage using pepsin produces two monovalent Fab'
fragments and an Fc fragment directly. These methods are described,
for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647,
and references contained therein, which patents are hereby
incorporated by reference in their entirety. See also Porter, R. R.
[Biochem. J. 73: 119-126 (1959)]. Other methods of cleaving
antibodies, such as separation of heavy chains to form monovalent
light-heavy chain fragments, further cleavage of fragments, or
other enzymatic, chemical, or genetic techniques may also be used,
so long as the fragments bind to the antigen that is recognized by
the intact antibody.
[0272] Fv fragments comprise an association of VH and VL chains.
This association may be noncovalent, as described in Inbar et al.
[Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the
variable chains can be linked by an intermolecular disulfide bond
or cross-linked by chemicals such as glutaraldehyde. Preferably,
the Fv fragments comprise VH and VL chains connected by a peptide
linker. These single-chain antigen binding proteins (sFv) are
prepared by constructing a structural gene comprising DNA sequences
encoding the VH and VL domains connected by an oligonucleotide. The
structural gene is inserted into an expression vector, which is
subsequently introduced into a host cell such as E. coli. The
recombinant host cells synthesize a single polypeptide chain with a
linker peptide bridging the two V domains. Methods for producing
sFvs are described, for example, by [Whitlow and Filpula, Methods
2: 97-105 (1991); Bird et al., Science 242:423-426 (1988); Pack et
al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No. 4,946,778,
which is hereby incorporated by reference in its entirety.
[0273] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing
cells. See, for example, Larrick and Fry [Methods, 2: 106-10
(1991)].
[0274] It will be appreciated that for human therapy or
diagnostics, humanized antibodies are preferably used. Humanized
forms of non-human (e.g., murine) antibodies are chimeric molecules
of immunoglobulins, immunoglobulin chains or fragments thereof
(such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding
subsequences of antibodies) which contain minimal sequence derived
from non-human immunoglobulin. Humanized antibodies include human
immunoglobulins (recipient antibody) in which residues form a
complementary determining region (CDR) of the recipient are
replaced by residues from a CDR of a non-human species (donor
antibody) such as mouse, rat or rabbit having the desired
specificity, affinity and capacity. In some instances, Fv framework
residues of the human immunoglobulin are replaced by corresponding
non-human residues. Humanized antibodies may also comprise residues
which are found neither in the recipient antibody nor in the
imported CDR or framework sequences. In general, the humanized
antibody will comprise substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin consensus sequence. The humanized
antibody optimally also will comprise at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann
et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct.
Biol., 2:593-596 (1992)].
[0275] Methods for humanizing non-human antibodies are well known
in the art.
[0276] Generally, a humanized antibody has one or more amino acid
residues introduced into it from a source which is non-human. These
non-human amino acid residues are often referred to as import
residues, which are typically taken from an import variable domain.
Humanization can be essentially performed following the method of
Winter and co-workers [Jones et al., Nature, 321:522-525 (1986);
Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al.,
Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR
sequences for the corresponding sequences of a human antibody.
Accordingly, such humanized antibodies are chimeric antibodies
(U.S. Pat. No. 4,816,567), wherein substantially less than an
intact human variable domain has been substituted by the
corresponding sequence from a non-human species. In practice,
humanized antibodies are typically human antibodies in which some
CDR residues and possibly some FR residues are substituted by
residues from analogous sites in rodent antibodies.
[0277] Human antibodies can also be produced using various
techniques known in the art, including phage display libraries
[Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et
al., J. Mol. Biol., 222:581 (1991)]. The techniques of Cole et al.
and Boerner et al. are also available for the preparation of human
monoclonal antibodies (Cole et al., Monoclonal Antibodies and
Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J.
Immunol., 147(1):86-95 (1991)]. Similarly, human antibodies can be
made by introduction of human immunoglobulin loci into transgenic
animals, e.g., mice in which the endogenous immunoglobulin genes
have been partially or completely inactivated. Upon challenge,
human antibody production is observed, which closely resembles that
seen in humans in all respects, including gene rearrangement,
assembly, and antibody repertoire. This approach is described, for
example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825;
5,625,126; 5,633,425; 5,661,016, and in the following scientific
publications: Marks et al., Bio/Technology 10: 779-783 (1992);
Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368
812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51
(1996); Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg
and Huszar, Intern. Rev. Immunol. 13, 65-93 (1995).
[0278] Once antibodies are obtained, they may be tested for
activity, for example via ELISA.
[0279] Another agent which can be used as antagonist with some
embodiments of the invention is an aptamer. As used herein, the
term "aptamer" refers to double stranded or single stranded RNA
molecule that binds to specific molecular target, such as a
protein. Various methods are known in the art which can be used to
design protein specific aptamers. The skilled artisan can employ
SELEX (Systematic Evolution of Ligands by Exponential Enrichment)
for efficient selection as described in Stoltenburg R, Reinemann C,
and Strehlitz B (Biomolecular engineering (2007)
24(4):381-403).
[0280] Another agent capable of being an antagonist would be any
molecule which interferes with the immune-check point protein
function (e.g. catalytic or interaction) by binding to and/or
cleaving the immune-check point protein. Such molecules can be, but
are not limited to, small molecules, inhibitory peptides, enzymes
that cleave the immune-check point protein, adnectins, affibodies,
avimers, anticalins, tetranectins, DARPins, and engineered
Kunitz-type inhibitors wherein each possibility is a separate
embodiment of the invention.
[0281] According to a specific embodiment, the antagonist is a
small molecule.
[0282] According to a specific embodiment, the antagonist is a
peptide molecule.
[0283] It will be appreciated that a non-functional analogue of at
least a catalytic or binding portion of an inhibitory peptide can
be also used as an antagonist.
[0284] Suppressing Biological Function at the Nucleic Acid
Level
[0285] Down-regulation at the nucleic acid level is typically
effected using a nucleic acid agent, having a nucleic acid
backbone, DNA, RNA, mimetics thereof or a combination of same. The
nucleic acid agent may be encoded from a DNA molecule or provided
to the cell per se.
[0286] Thus, the antagonist of some embodiments of the invention
can be an RNA silencing agent. As used herein, the phrase "RNA
silencing" refers to a group of regulatory mechanisms [e.g. RNA
interference (RNAi), transcriptional gene silencing (TGS),
post-transcriptional gene silencing (PTGS), quelling,
co-suppression, and translational repression] mediated by RNA
molecules which result in the inhibition or "silencing" of the
expression of a corresponding protein-coding gene. RNA silencing
has been observed in many types of organisms, including plants,
animals, and fungi.
[0287] As used herein, the term "RNA silencing agent" refers to an
RNA which is capable of specifically inhibiting or "silencing" the
expression of a target gene. In certain embodiments, the RNA
silencing agent is capable of preventing complete processing (e.g.,
the full translation and/or expression) of an mRNA molecule through
a post-transcriptional silencing mechanism. RNA silencing agents
include non-coding RNA molecules, for example RNA duplexes
comprising paired strands, as well as precursor RNAs from which
such small non-coding RNAs can be generated. Exemplary RNA
silencing agents include dsRNAs such as siRNAs, miRNAs and
shRNAs.
[0288] In one embodiment, the RNA silencing agent is capable of
inducing RNA interference.
[0289] In another embodiment, the RNA silencing agent is capable of
mediating translational repression.
[0290] According to an embodiment of the invention, the RNA
silencing agent is specific to the target RNA (i.e. an immune-check
point protein) and does not cross inhibit or silence other targets
or a splice variant which exhibits 99% or less global homology to
the target gene, e.g., less than 98%, 97%, 96%, 95%, 94%, 93%, 92%,
91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81% global
homology to the target gene; as determined by PCR, Western blot,
Immunohistochemistry and/or flow cytometry.
[0291] RNA interference refers to the process of sequence-specific
post-transcriptional gene silencing in animals mediated by short
interfering RNAs (siRNAs).
[0292] Following is a detailed description on RNA silencing agents
that can be used according to specific embodiments of the present
invention.
[0293] DsRNA, siRNA and shRNA--
[0294] The presence of long dsRNAs in cells stimulates the activity
of a ribonuclease III enzyme referred to as dicer. Dicer is
involved in the processing of the dsRNA into short pieces of dsRNA
known as short interfering RNAs (siRNAs). Short interfering RNAs
derived from dicer activity are typically about 21 to about 23
nucleotides in length and comprise about 19 base pair duplexes.
[0295] The RNAi response also features an endonuclease complex,
commonly referred to as an RNA-induced silencing complex (RISC),
which mediates cleavage of single-stranded RNA having sequence
complementary to the antisense strand of the siRNA duplex. Cleavage
of the target RNA takes place in the middle of the region
complementary to the antisense strand of the siRNA duplex.
[0296] Accordingly, some embodiments of the invention contemplate
use of dsRNA to downregulate protein expression from mRNA.
[0297] According to one embodiment dsRNA longer than 30 bp are
used. Various studies demonstrate that long dsRNAs can be used to
silence gene expression without inducing the stress response or
causing significant off-target effects--see for example [Strat et
al., Nucleic Acids Research, 2006, Vol. 34, No. 13 3803-3810;
Bhargava A et al. Brain Res. Protoc. 2004; 13:115-125; Diallo M.,
et al., Oligonucleotides. 2003; 13:381-392; Paddison P. J., et al.,
Proc. Natl Acad. Sci. USA. 2002; 99:1443-1448; Tran N., et al.,
FEBS Lett. 2004; 573:127-134].
[0298] According to some embodiments of the invention, dsRNA is
provided in cells where the interferon pathway is not activated,
see for example Billy et al., PNAS 2001, Vol. 98, pages
14428-14433. and Diallo et al., Oligonucleotides, Oct. 1, 2003,
13(5): 381-392. doi:10.1089/154545703322617069.
[0299] According to an embodiment of the invention, the long dsRNA
are specifically designed not to induce the interferon and PKR
pathways for down-regulating gene expression. For example, Shinagwa
and Ishii [Genes & Dev. 17 (11): 1340-1345, 2003] have
developed a vector, named pDECAP, to express long double-strand RNA
from an RNA polymerase II (Pol II) promoter. Because the
transcripts from pDECAP lack both the 5'-cap structure and the
3'-poly(A) tail that facilitate ds-RNA export to the cytoplasm,
long ds-RNA from pDECAP does not induce the interferon
response.
[0300] Another method of evading the interferon and PKR pathways in
mammalian systems is by introduction of small inhibitory RNAs
(siRNAs) either via transfection or endogenous expression.
[0301] The term "siRNA" refers to small inhibitory RNA duplexes
(generally between 18-30 base pairs) that induce the RNA
interference (RNAi) pathway.
[0302] Typically, siRNAs are chemically synthesized as 21mers with
a central 19 bp duplex region and symmetric 2-base 3'-overhangs on
the termini, although it has been recently described that
chemically synthesized RNA duplexes of 25-30 base length can have
as much as a 100-fold increase in potency compared with 21mers at
the same location. The observed increased potency obtained using
longer RNAs in triggering RNAi is suggested to result from
providing Dicer with a substrate (27mer) instead of a product
(21mer) and that this improves the rate or efficiency of entry of
the siRNA duplex into RISC.
[0303] It has been found that position of the 3'-overhang
influences potency of a siRNA and asymmetric duplexes having a
3'-overhang on the antisense strand are generally more potent than
those with the 3'-overhang on the sense strand (Rose et al., 2005).
This can be attributed to asymmetrical strand loading into RISC, as
the opposite efficacy patterns are observed when targeting the
antisense transcript.
[0304] The strands of a double-stranded interfering RNA (e.g., a
siRNA) may be connected to form a hairpin or stem-loop structure
(e.g., a shRNA). Thus, as mentioned, the RNA silencing agent of
some embodiments of the invention may also be a short hairpin RNA
(shRNA).
[0305] The term "shRNA", as used herein, refers to an RNA agent
having a stem-loop structure, comprising a first and second region
of complementary sequence, the degree of complementarity and
orientation of the regions being sufficient such that base pairing
occurs between the regions, the first and second regions being
joined by a loop region, the loop resulting from a lack of base
pairing between nucleotides (or nucleotide analogs) within the loop
region. The number of nucleotides in the loop is a number between
and including 3 to 23, or 5 to 15, or 7 to 13, or 4 to 9, or 9 to
11.
[0306] Some of the nucleotides in the loop can be involved in
base-pair interactions with other nucleotides in the loop. Examples
of oligonucleotide sequences that can be used to form the loop
include 5'-CAAGAGA-3' and 5'-UUACAA-3' (International Patent
Application Nos. WO2013126963 and WO2014107763). It will be
recognized by one of skill in the art that the resulting single
chain oligonucleotide forms a stem-loop or hairpin structure
comprising a double-stranded region capable of interacting with the
RNAi machinery.
[0307] Synthesis of RNA silencing agents suitable for use with some
embodiments of the invention can be effected as follows. First, the
inhibitory-check point mRNA sequence is scanned downstream of the
AUG start codon for AA dinucleotide sequences. Occurrence of each
AA and the 3' adjacent 19 nucleotides is recorded as potential
siRNA target sites. Preferably, siRNA target sites are selected
from the open reading frame, as untranslated regions (UTRs) are
richer in regulatory protein binding sites. UTR-binding proteins
and/or translation initiation complexes may interfere with binding
of the siRNA endonuclease complex [Tuschl Chem Bio chem.
2:239-245]. It will be appreciated though, that siRNAs directed at
untranslated regions may also be effective, as demonstrated for
GAPDH wherein siRNA directed at the 5' UTR mediated about 90%
decrease in cellular GAPDH mRNA and completely abolished protein
level (www(dot)ambion(dot)com/techlib/tn/91/912(dot)html).
[0308] Second, potential target sites are compared to an
appropriate genomic database (e.g., human, mouse, rat etc.) using
any sequence alignment software, such as the BLAST software
available from the NCBI server
(www(dot)ncbi(dot)nlm(dot)nih(dot)gov/BLAST/). Putative target
sites which exhibit significant homology to other coding sequences
are filtered out.
[0309] Qualifying target sequences are selected as template for
siRNA synthesis.
[0310] Preferred sequences are those including low G/C content as
these have proven to be more effective in mediating gene silencing
as compared to those with G/C content higher than 55%. Several
target sites are preferably selected along the length of the target
gene for evaluation. For better evaluation of the selected siRNAs,
a negative control is preferably used in conjunction. Negative
control siRNA preferably include the same nucleotide composition as
the siRNAs but lack significant homology to the genome. Thus, a
scrambled nucleotide sequence of the siRNA is preferably used,
provided it does not display any significant homology to any other
gene.
[0311] It will be appreciated that, and as mentioned hereinabove,
the RNA silencing agent of some embodiments of the invention need
not be limited to those molecules containing only RNA, but further
encompasses chemically-modified nucleotides and
non-nucleotides.
[0312] According to another embodiment the RNA silencing agent may
be a miRNA.
[0313] The term "microRNA", "miRNA", and "miR" are synonymous and
refer to a collection of non-coding single-stranded RNA molecules
of about 19-28 nucleotides in length, which regulate gene
expression. miRNAs are found in a wide range of organisms
(viruses.fwdarw.humans) and have been shown to play a role in
development, homeostasis, and disease etiology.
[0314] Below is a brief description of the mechanism of miRNA
activity.
[0315] Genes coding for miRNAs are transcribed leading to
production of a miRNA precursor known as the pri-miRNA. The
pri-miRNA may form a hairpin with a stem and loop.
[0316] The hairpin structure of the pri-miRNA is recognized by
Drosha, which is a RNase III endonuclease. Drosha typically
recognizes terminal loops in the pri-miRNA and cleaves the
pri-miRNA with a staggered cut typical of RNase III endonucleases
yielding a pre-miRNA stem loop with a 5' phosphate and .about.2
nucleotide 3' overhang. The pre-miRNA is then actively transported
from the nucleus to the cytoplasm by Ran-GTP and the export
receptor Ex-portin-5.
[0317] The double-stranded stem or the 5' phosphate and 3' overhang
at the base of the stem loop of the pre-miRNA is then recognized by
Dicer, which is also an RNase III endonuclease. Dicer then cleaves
off the terminal loop two helical turns away from the base of the
stem loop leaving an additional 5' phosphate and .about.2
nucleotide 3' overhang. The resulting siRNA-like duplex, which may
comprise mismatches, comprises the mature miRNA and a similar-sized
fragment known as the miRNA*. miRNA* sequences may be found in
libraries of cloned miRNAs but typically at lower frequency than
the miRNAs.
[0318] Although initially present as a double-stranded species with
miRNA*, the miRNA eventually becomes incorporated as a
single-stranded RNA into a ribonucleoprotein complex known as the
RNA-induced silencing complex (RISC) while the miRNA* is removed
and degraded.
[0319] The RISC identifies target nucleic acids based on high
levels of complementarity between the miRNA and the mRNA,
especially by nucleotides 2-7 of the miRNA.
[0320] A number of studies have looked at the base-pairing
requirement between miRNA and its mRNA target for achieving
efficient inhibition of translation (reviewed by Bartel 2004, Cell
116-281). In mammalian cells, the first 8 nucleotides of the miRNA
may be important (Doench & Sharp 2004 Genes Dev 2004-504).
However, other parts of the microRNA may also participate in mRNA
binding. Moreover, sufficient base pairing at the 3' can compensate
for insufficient pairing at the 5' (Brennecke et al., 2005 PLoS
3-e85). Computation studies, analyzing miRNA binding on whole
genomes have suggested a specific role for bases 2-7 at the 5' of
the miRNA in target binding but the role of the first nucleotide,
found usually to be "A" was also recognized (Lewis et at 2005 Cell
120-15). Similarly, nucleotides 1-7 or 2-8 were used to identify
and validate targets by Krek et al. (2005, Nat Genet 37-495).
[0321] The target sites in the mRNA may be in the 5' UTR, the 3'
UTR or in the coding region.
[0322] miRNAs may direct the RISC to downregulate gene expression
by either of two mechanisms: mRNA cleavage or translational
repression. The miRNA may specify cleavage of the mRNA if the mRNA
has a certain degree of complementarity to the miRNA. When a miRNA
guides cleavage, the cut is typically between the nucleotides
pairing to residues 10 and 11 of the miRNA. Alternatively, the
miRNA may repress translation if the miRNA does not have the
requisite degree of complementarity to the miRNA.
[0323] It will be appreciated from the description provided herein
above that contacting cells with a miRNA may be effected by
transfecting/loading the cells with e.g. the mature double stranded
miRNA, the pre-miRNA or the pri-miRNA.
[0324] The pre-miRNA sequence may comprise from 45-90, 60-80 or
60-70 nucleotides.
[0325] The pri-miRNA sequence may comprise from 45-30,000,
50-25,000, 100-20,000, 1,000-1,500 or 80-100 nucleotides.
[0326] Antisense--
[0327] Antisense is a single stranded RNA designed to prevent or
inhibit expression of a gene by specifically hybridizing to its
mRNA. Downregulation of an immune-check point can be effected using
an antisense polynucleotide capable of specifically hybridizing
with an mRNA transcript encoding the immune-check point
protein.
[0328] Design of antisense molecules which can be used to
efficiently downregulate an immune-check point must be effected
while considering two aspects important to the antisense approach.
The first aspect is delivery of the oligonucleotide into the
cytoplasm of the appropriate cells, while the second aspect is
design of an oligonucleotide which specifically binds the
designated mRNA within cells in a way which inhibits translation
thereof.
[0329] The prior art teaches of a number of delivery strategies
which can be used to efficiently deliver oligonucleotides into a
wide variety of cell types [see, for example, Jaaskelainen et al.
Cell Mol Biol Lett. (2002) 7(2):236-7; Gait, Cell Mol Life Sci.
(2003) 60(5):844-53; Martino et al. J Biomed Biotechnol. (2009)
2009:410260; Grijalvo et al. Expert Opin Ther Pat. (2014)
24(7):801-19; Falzarano et al., Nucleic Acid Ther. (2014)
24(1):87-100; Shilakari et al. Biomed Res Int. (2014) 2014: 526391;
Prakash et al. Nucleic Acids Res. (2014) 42(13):8796-807 and
Asseline et al. J Gene Med. (2014) 16(7-8):157-65].
[0330] In addition, algorithms for identifying those sequences with
the highest predicted binding affinity for their target mRNA based
on a thermodynamic cycle that accounts for the energetics of
structural alterations in both the target mRNA and the
oligonucleotide are also available [see, for example, Walton et al.
Biotechnol Bioeng 65: 1-9 (1999)]. Such algorithms have been
successfully used to implement an antisense approach in cells.
[0331] In addition, several approaches for designing and predicting
efficiency of specific oligonucleotides using an in vitro system
were also published [(Matveeva et al., Nature Biotechnology 16:
1374-1375 (1998)].
[0332] Thus, the generation of highly accurate antisense design
algorithms and a wide variety of oligonucleotide delivery systems,
enable an ordinarily skilled artisan to design and implement
antisense approaches suitable for downregulating expression of
known sequences without having to resort to undue trial and error
experimentation.
[0333] Nucleic acid agents can also operate at the DNA level as
summarized infra.
[0334] Suppressing the biological function of an immune-check point
can also be achieved by inactivating the gene via introducing
targeted mutations involving loss-of function alterations (e.g.
point mutations, deletions and insertions) in the gene
structure.
[0335] As used herein, the phrase "loss-of-function alterations"
refers to any mutation in the DNA sequence of a gene which results
in downregulation of the expression level and/or activity of the
expressed product, i.e., the mRNA transcript and/or the translated
protein. Non-limiting examples of such loss-of-function alterations
include a missense mutation, i.e., a mutation which changes an
amino acid residue in the protein with another amino acid residue
and thereby abolishes the enzymatic activity of the protein; a
nonsense mutation, i.e., a mutation which introduces a stop codon
in a protein, e.g., an early stop codon which results in a shorter
protein devoid of the enzymatic activity; a frame-shift mutation,
i.e., a mutation, usually, deletion or insertion of nucleic acid(s)
which changes the reading frame of the protein, and may result in
an early termination by introducing a stop codon into a reading
frame (e.g., a truncated protein, devoid of the enzymatic
activity), or in a longer amino acid sequence (e.g., a readthrough
protein) which affects the secondary or tertiary structure of the
protein and results in a non-functional protein, devoid of the
enzymatic activity of the non-mutated polypeptide; a readthrough
mutation due to a frame-shift mutation or a modified stop codon
mutation (i.e., when the stop codon is mutated into an amino acid
codon), with an abolished enzymatic activity; a promoter mutation,
i.e., a mutation in a promoter sequence, usually 5' to the
transcription start site of a gene, which results in
down-regulation of a specific gene product; a regulatory mutation,
i.e., a mutation in a region upstream or downstream, or within a
gene, which affects the expression of the gene product; a deletion
mutation, i.e., a mutation which deletes coding nucleic acids in a
gene sequence and which may result in a frame-shift mutation or an
in-frame mutation (within the coding sequence, deletion of one or
more amino acid codons); an insertion mutation, i.e., a mutation
which inserts coding or non-coding nucleic acids into a gene
sequence, and which may result in a frame-shift mutation or an
in-frame insertion of one or more amino acid codons; an inversion,
i.e., a mutation which results in an inverted coding or non-coding
sequence; a splice mutation i.e., a mutation which results in
abnormal splicing or poor splicing; and a duplication mutation,
i.e., a mutation which results in a duplicated coding or non-coding
sequence, which can be in-frame or can cause a frame-shift.
[0336] According to specific embodiments loss-of-function
alteration of a gene may comprise at least one allele of the
gene.
[0337] The term "allele" as used herein, refers to any of one or
more alternative forms of a gene locus, all of which alleles relate
to a trait or characteristic. In a diploid cell or organism, the
two alleles of a given gene occupy corresponding loci on a pair of
homologous chromosomes.
[0338] According to other specific embodiments loss-of-function
alteration of a gene comprises both alleles of the gene.
[0339] Methods of introducing nucleic acid alterations to a gene of
interest are well known in the art [see for example Menke D.
Genesis (2013) 51: -618; Capecchi, Science (1989) 244:1288-1292;
Santiago et al. Proc Natl Acad Sci USA (2008) 105:5809-5814;
International Patent Application Nos. WO2014085593, WO2009071334
and WO2011146121; U.S. Pat. Nos. 8,771,945, 8,586,526, 6,774,279
and U.S. Patent Application Publication Nos. 20030232410,
20050026157, US20060014264; the contents of which are incorporated
by reference in their entireties] and include targeted homologous
recombination (e.g. "Hit and run", "double-replacement"), site
specific recombinases (e.g. the Cre recombinase and the Flp
recombinase), PB transposases (e.g. Sleeping Beauty, piggyBac, To12
or Frog Prince), genome editing by engineered nucleases (e.g.
meganucleases, Zinc finger nucleases (ZFNs),
transcription-activator like effector nucleases (TALENs) and
CRISPR/Cas system) and genome editing using recombinant
adeno-associated virus (rAAV) platform. Agents for introducing
nucleic acid alterations to a gene of interest can be designed
publically available sources or obtained commercially from
Transposagen, Addgene and Sangamo Biosciences.
[0340] Methods for qualifying efficacy and detecting sequence
alteration are well known in the art and include, but not limited
to, DNA sequencing, electrophoresis, an enzyme-based mismatch
detection assay and a hybridization assay such as PCR, RT-PCR,
RNase protection, in-situ hybridization, primer extension, Southern
blot, Northern Blot and dot blot analysis.
[0341] Sequence alterations in a specific gene can also be
determined at the protein level using e.g. chromatography,
electrophoretic methods, immunodetection assays such as ELISA and
western blot analysis and immunohistochemistry.
[0342] As mentioned, depending on the immune-check point protein
(i.e. co-stimulatory or inhibitory) the immune-check point
regulator can be an agonist or antagonist. Thus, according to
specific embodiments, the immune-check point regulator is an
agonist.
[0343] As used herein the term "agonist" refers to a molecule that
induces and/or increases the biological function and/or expression
of an immune-check point protein.
[0344] According to specific embodiments, the agonist induces
and/or increases the co-stimulatory effect of an immune-check point
protein on an immune cell (e.g. T cells).
[0345] According to specific embodiments, the agonist induces
and/or increases signaling to an immune cell (e.g. T cell) by an
immune-check point protein. The agonist can be a naturally
occurring activator or a functional derivative thereof; or
non-naturally occurring activator.
[0346] According to specific embodiments, the agonist is a full
agonist, that is, the effect of the agonist is equivalent to the
effect of the naturally occurring activator (i.e. ligand).
[0347] According to other specific embodiments, the agonist is a
partial agonist, that is, the effect of the agonist is lower than
the maximal effect of the naturally occurring activator (i.e.
ligand). The effect of the agonist may be lower by at least 5%, at
least 10%, at least 20%, at least 30%, at least 40% at least 50%,
at least 60%, at least 70%, at least 80% or at least 90% as
compared to the maximal effect of the naturally occurring
activator.
[0348] According to yet other specific embodiments, the agonist is
a super agonist, that is, the effect of the agonist is higher than
the maximal effect of the naturally occurring activator (i.e.
ligand). The effect of the agonist may be higher by at least 5%, at
least 10%, at least 20%, at least 30%, at least 40% at least 50%,
at least 60%, at least 70%, at least 80%, at least 90% or at least
2 fold, at least 4 fold, at least 5 fold or at least 10 fold as
compared to the maximal effect of the naturally occurring
activator.
[0349] According to specific embodiments, the agonist induces
complete activation the biological function (e.g. signal
transduction) of the immune-check point protein.
[0350] According to other specific embodiments, the agonist
increases the biological function (e.g. signal transduction) of the
immune-check point protein e.g., as detected by e.g. kinase
activity, proliferation assay, cytotoxicity assay or cytokine
secretion assay. The increase may be by at least a 10%, at least
20%, at least 30%, at least 40%, at least 50%, at least 60%, at
least 70%, at least 80%, at least 90%, at least 95% or at least 99%
as compared to same in the absence of the agonist.
[0351] According to specific embodiments, the agonist binds
directly the immune-check point protein.
[0352] According to other specific embodiments, the agonist
indirectly binds the immune-check point protein by acting through
an intermediary molecule, for example the agonist binds to or
modulates a molecule that in turn binds to or modulates the
immune-check point protein.
[0353] Activating and/or increasing the biological function of an
immune-check point protein can be effected at the protein level
(e.g., antibodies, small molecules, peptides and the like) but may
also be effected at the genomic level (e.g., activation of
transcription via promoters, enhancers, regulatory elements) and/or
the transcript level using a variety of molecules which promote
transcription and/or translation (e.g., correct splicing,
polyadenylation, activation of translation) of a co-stimulatory
immune-check point protein.
[0354] Non limiting examples of agents that can function as
agonists are described in details hereinbelow.
[0355] Activating and/or Increasing Biological Function at the
Polypeptide Level
[0356] According to specific embodiments, the agonist is the
naturally occurring activator or a functional derivative or variant
thereof which retain the ability to specifically bind to the
immune-check point protein.
[0357] It will be appreciated that a functional analogue of at
least a catalytic or binding portion of a co-stimulatory peptide
can be also used as an agonist. Thus, according to specific
embodiments, the agonist is an exogenous polypeptide including at
least a functional portion (e.g. catalytic or interaction) of the
co-stimulatory immune-check point protein. Thus, for example, the
polypeptide can be a ligand capable of binding and activating the
co-stimulatory immune-check point protein receptor.
[0358] According to specific embodiments, the agonist is an
antibody.
[0359] According to specific embodiments the agonistic antibody is
capable of specifically binding a co-stimulatory immune-check point
protein. According to specific embodiments, the agonistic antibody
specifically binds at least one epitope of a co-stimulatory
immune-check point protein. A detailed description on antibodies
that can be used according to specific embodiments of the present
invention is provided hereinabove.
[0360] Another agent capable of being an agonist would be a
molecule which promotes and/or increases the co-stimulatory
immune-check point protein function (e.g. catalytic or interaction)
by binding to the immune-check point protein or an intermediate
thereof. Such molecules can be, but are not limited to, small
molecules, peptides, aptamers, adnectins, affibodies, avimers,
anticalins, tetranectins and DARPins, wherein each possibility is a
separate embodiment of the invention.
[0361] According to specific embodiments, the agonist is a small
molecule.
[0362] According to specific embodiments, the agonist is a
peptide.
[0363] Activating and/or Increasing Biological Function at the
Nucleic Acid Level
[0364] An agonist can also be a molecule which is capable of
increasing the transcription and/or translation of an endogenous
DNA or mRNA encoding the co-stimulatory immune-check point protein
and thus increasing endogenous co-stimulatory immune-check point
protein activity.
[0365] Another agonistic agent may be an exogenous polynucleotide
(DNA or RNA) sequence designed and constructed to express at least
a functional portion of the co-stimulatory immune-check point
protein.
[0366] Several co-stimulatory immune-check points have been cloned
from human, rat and mouse sources. Thus, coding sequences
information is available from several databases including the
GenBank database available through
www(dot)ncbi(dot)nlm(dot)nih(dot)gov/.
[0367] To express an exogenous co-stimulatory immune-check point
protein in mammalian cells, a polynucleotide sequence encoding a
specific co-stimulatory immune-check point protein or a homologue
thereof which exhibit the desired activity is preferably ligated
into a nucleic acid construct suitable for mammalian cell
expression. Such a nucleic acid construct includes a promoter
sequence for directing transcription of the polynucleotide sequence
in the cell in a constitutive [e.g. cytomegalovirus (CMV) and Rous
sarcoma virus (RSV)] or inducible (e.g. the tetracycline-inducible
promoter) manner.
[0368] According to specific embodiments, the promoter utilized by
the nucleic acid construct of some embodiments of the invention is
active in a specific cell population. Examples of cell
type-specific and/or tissue-specific promoters include promoters
such as, but not limited to lymphoid specific promoters [Calame et
al., (1988) Adv. Immunol. 43:235-275]; in particular promoters of
T-cell receptors [Winoto et al., (1989) EMBO J. 8:729-733] and
immunoglobulins [Banerji et al. (1983) Cell 33729-740].
[0369] The nucleic acid construct (also referred to herein as an
"expression vector") of some embodiments of the invention includes
additional sequences which render this vector suitable for
replication and integration in prokaryotes, eukaryotes, or
preferably both (e.g., shuttle vectors). In addition, a typical
cloning vectors may also contain a transcription and translation
initiation sequence, transcription and translation terminator and a
polyadenylation signal. By way of example, such constructs will
typically include a 5' LTR, a tRNA binding site, a packaging
signal, an origin of second-strand DNA synthesis, and a 3' LTR or a
portion thereof. The construct may also include an enhancer element
which can stimulate transcription up to 1,000 fold from linked
homologous or heterologous promoters. The vector may or may not
include a eukaryotic replicon.
[0370] The nucleic acid construct of some embodiments of the
invention can also include a signal sequence for secretion of the
peptide from a host cell in which it is placed. Preferably the
signal sequence for this purpose is a mammalian signal sequence or
the signal sequence of the polypeptide variants of some embodiments
of the invention.
[0371] Polyadenylation sequences can also be added to the
expression vector in order to increase the efficiency of a
co-stimulatory immune-check point mRNA translation. Two distinct
sequence elements are required for accurate and efficient
polyadenylation: GU or U rich sequences located downstream from the
polyadenylation site and a highly conserved sequence of six
nucleotides, AAUAAA, located 11-30 nucleotides upstream.
Termination and polyadenylation signals that are suitable for some
embodiments of the invention include those derived from SV40.
[0372] The expression vector of some embodiments of the invention
can further include additional polynucleotide sequences that allow,
for example, the translation of several proteins from a single mRNA
such as an internal ribosome entry site (IRES) and sequences for
genomic integration of the promoter-chimeric polypeptide.
[0373] Other than containing the necessary elements for the
transcription and translation of the inserted coding sequence, the
expression construct of some embodiments of the invention can also
include sequences engineered to enhance stability, production, or
yield of the expressed peptide.
[0374] It will be appreciated that the individual elements
comprised in the expression vector can be arranged in a variety of
configurations.
[0375] The type of vector used by some embodiments of the invention
will depend on the cell type transformed. The ability to select
suitable vectors according to the cell type transformed is well
within the capabilities of the ordinary skilled artisan and as such
no general description of selection consideration is provided
herein.
[0376] Recombinant viral vectors are useful for in vivo expression
of an immune-check point protein since they offer advantages such
as lateral infection and targeting specificity. Viral vectors can
also be produced that are unable to spread laterally.
[0377] Various methods can be used to introduce the expression
vector of some embodiments of the invention into cells. Such
methods are generally described in Sambrook et al., Molecular
Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New
York (1989, 1992), in Ausubel et al., Current Protocols in
Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989),
Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich.
(1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich.
(1995), Vectors: A Survey of Molecular Cloning Vectors and Their
Uses, Butterworths, Boston Mass. (1988) and Gilboa et at.
[Biotechniques 4 (6): 504-512, 1986].
[0378] Currently preferred in vivo nucleic acid transfer techniques
include transfection with viral or non-viral constructs, such as
adenovirus, lentivirus, Herpes simplex I virus, or adeno-associated
virus (AAV) and lipid-based systems. Useful lipids for
lipid-mediated transfer of the gene are, for example, DOTMA, DOPE,
and DC-Chol [Tonkinson et al., Cancer Investigation, 14(1): 54-65
(1996)]. The most preferred constructs for use in gene therapy are
viruses, most preferably adenoviruses, AAV, lentiviruses, or
retroviruses. Other vectors can be used that are non-viral, such as
cationic lipids, polylysine, and dendrimers.
[0379] According to specific embodiments, the immune-check point
regulator targets an immune check-point protein selected from the
group consisting of B7-H3, CD19 and CD70.
[0380] B7-H3 (gene symbol CD276, also known as CD276), is a B7
family protein. The gene is over-expressed in a variety of solid
tumors, including prostate, pancreatic, melanoma, renal cell,
ovarian, colorectal, gastric, bladder and non-small cell lung
cancers has been reported (e.g. Immune Netw. 2014 December; 14(6):
265-276). According to a specific embodiment, the B7-H3 protein
refers to the human protein, such as provided in the following
GenBank Number NP_001019907.
[0381] CD19 (gene symbol CD19, also known as B-lymphocyte antigen
CD19), is a cell surface molecule that assembles with the antigen
receptor of B lymphocytes in order to decrease the threshold for
antigen receptor-dependent stimulation. CD19 is expressed on
follicular dendritic cells and B cells. Typically, it is present on
B cells from earliest recognizable B-lineage cells during
development to B-cell blasts but is lost on maturation to plasma
cells. CD19 is also expressed on several hematologic e.g. B cell
tumors. According to a specific embodiment, the CD19 protein refers
to the human protein, such as provided in the following GenBank
Number NP_001171569.
[0382] CD70 is the ligand for CD27, which is expressed on activated
lymphocytes. CD70 expression is found in different types of cancers
including renal cell carcinomas, metastatic breast cancers, brain
tumours, leukemias, lymphomas and nasopharyngeal carcinomas.
According to a specific embodiment, the CD70 protein refers to the
human protein, such as provided in the following GenBank Number
NP_001243.
[0383] It should be acknowledged that the present invention does
not contemplate the use of an immune-check point regulator selected
from the group consisting of PD1 antagonist, PDL-1 antagonist,
CTLA-4 antagonist, LAG-3 antagonist, TIM-3 antagonist, KIR
antagonist, IDO antagonist, OX40 agonist, CD137 agonist, CD27
agonist, CD40 agonist, GITR agonist, CD28 agonist and ICOS
agonist.
[0384] PD1 (Programmed Death 1), gene symbol PDCD1, is also known
as CD279. According to a specific embodiment, the PD1 protein
refers to the human protein, such as provided in the following
GenBank Number NP_005009.
[0385] PDL-1, gene symbol CD274, is also known as CD274 and B7-H1.
According to a specific embodiment the PDL-1 protein refers to the
human protein, such as provided in the following GenBank accession
Numbers NP_054862 and NP_054862.
[0386] CTLA4, gene symbol Ctla4 is also known as CD152. According
to a specific embodiment the CTLA-4 protein refers to the human
protein, such as provided in the following GenBank Number
NP_001032720.
[0387] LAG-3 (Lymphocyte-activation gene 3), gene symbol LAG3, is
also known as CD223. According to a specific embodiment the LAG-3
protein refers to the human protein, such as provided in the
following GenBank Number NP_002277.
[0388] TIM-3, gene symbol HAVCR2, is also known as Hepatitis A
Virus Cellular Receptor 2, T Cell Immunoglobulin Mucin 3, T-Cell
Immunoglobulin And Mucin Domain-Containing Protein, T-Cell Membrane
Protein 3 and KIM-3. According to a specific embodiment the TIM-3
protein refers to the human protein, such as provided in the
following GenBank Number NP_116171.
[0389] KIRs (killer cell Ig-like receptors) are cell surface
glycoproteins, comprising one to three extracellular
immunoglobulin-like domains, which are expressed by some T cells as
well as most human NK cells. As used herein, the term "KIR" refers
to a KIR that delivers an inhibitory signal to the cell. A number
of KIRs are well characterized (see, e.g., Carrington and Norman,
The KIR Gene Cluster, May 28, 2003, available through the National
Center for Biotechnology Information (NCBI) web site at
www(dot)ncbi(dot)nlm(dot)nih(dot)gov/books/bookres(dot)fcgi/mono_003/ch1d-
1(dot)pdf). The sequences of human KIR genes and cDNAs, as well as
their protein products, are available in public databases,
including GenBank. Non-limiting exemplary GenBank entries of human
KIRs have the following accession numbers: KIR2DL1: Genbank
accession number U24076, NM_014218, AAR16197, L41267 or NP_055033;
KIR2DL2: Genbank accession number U24075, L76669 or NP_055034;
KIR2DL3: Genbank accession number U24074, L41268 or NP_056952;
KIR2DL4: Genbank accession number X97229; and KIR3DL1: Genbank
accession number L41269.
[0390] IDO (indoleamine 2,3-dioxygenase), EC 1.13.11.52, is a
heme-containing intracellular enzyme that catalyzes the first and
rate-determining step in the degradation of the essential amino
acid L-tryptophan to N-formyl-kynurenine. According to a specific
embodiment, the IDO protein refers to the human protein, such as
provided in the following GenBank Number NP_002155.
[0391] OX40, gene symbol TNFRSF4, is also known as CD134, Tumor
necrosis factor receptor superfamily, member 4, TNFRSF4, TXGP1L,
ACT35 and IMD16. According to a specific embodiment, the OX40
protein refers to the human protein, such as provided in the
following GenBank Number NP_003318.
[0392] CD137, gene symbol TNFRSF9, is also known as 4-1BB and Tumor
Necrosis Factor Receptor Superfamily, Member 9 and TNFRSF9.
According to a specific embodiment the CD137 protein refers to the
human protein, such as provided in the following GenBank Number
NP_001552.
[0393] CD27, gene symbol CD27, is also known as Tumor Necrosis
Factor Receptor Superfamily, Member 7, TNFRSF7 and S152. According
to a specific embodiment, the CD27 protein refers to the human
protein, such as provided in the following GenBank Number
NP_001233.
[0394] CD40, gene symbol CD40, is also known as Tumor Necrosis
Factor Receptor Superfamily, Member 5 and TNFRSF5. According to a
specific embodiment the CD40 protein refers to the human protein,
such as provided in the following GenBank Number NP_001241.
[0395] GITR (glucocorticoid-induced tumor necrosis factor
receptor), gene symbol TNFRSF18 is also known as TNF receptor
superfamily 18, TNFRSF18, AITR and CD357. According to a specific
embodiment the GITR protein refers to the human protein, such as
provided in the following GenBank Numbers NP_004186, NP_683699,
NP_683700.
[0396] CD28, gene symbol CD28, is also known as Tp44. According to
a specific embodiment the CD28 protein refers to the human protein,
such as provided in the following GenBank Numbers NP_001230006,
NP_001230007 and NP_006130.
[0397] ICOS (Inducible T-cell co-stimulator), gene symbol ICOS, is
also known as CD278, AILIM and CVID1. According to a specific
embodiment the ICOS protein refers to the human protein, such as
provided in the following GenBank Number NP_036224.
[0398] It has been demonstrated that multiple cancer cells
overexpress certain types of immune-regulatory proteins. Hence,
according to specific embodiments, the anti-cancer agent may be an
agent capable of binding an immune-check point protein expressed on
a cancer cell.
[0399] According to specific embodiments, the immune-check point
protein expressed on said cancer cell is selected from the group
consisting of PDL-1, CD27, LAG3, CD19, CD70 and CEACAM1.
[0400] According to specific embodiments, binding of the
anti-cancer agent to the immune-check point protein expressed on
the cancer cell results in at least one of:
[0401] (i) cell cycle arrest of said cancer cell;
[0402] (ii) apoptosis of said cancer cell;
[0403] (iii) sensitization of said cancer cell to a cytotoxic drug;
and
[0404] (iv) activation of an immune response against said cancer
cell.
[0405] Examples of agents capable of binding a cancer cell
expressing a specific antigen that can be used according to
specific embodiments of the present invention are described in
details hereinabove.
[0406] According to specific embodiments, the agent capable of
binding said immune-check point protein is an antibody or a T
cell.
[0407] According to specific embodiments, the T cell comprises a T
cell transduced with a T cell receptor (TCR) or a chimeric antigen
receptor (CAR).
[0408] Specific non-limiting examples of immune-check point
regulators and/or agents capable of binding an immune-check point
protein expressed on a cancer cell that can be used according to
some embodiments of the invention include:
[0409] IMP321, a Ig fusion protein comprising a soluble dimeric
recombinant form of LAG-3) designed to activate antigen presenting
cells, induce maturation and migration of DCs to the lymph nodes
and enhanced cross-presentation of antigens to CD8+ T cells [see
e.g. Brignone et al., (2007) J. Immunol. 179:4202-4211].
[0410] An agent targeting B7-H3, such as, MGA271 (produced by
MacroGenics), a humanized IgG1.kappa. monoclonal antibody that
recognizes human B7-H3, B7-H3 antagonist such as disclosed e.g. in
US Application Publication No. US 20130122021 and International
Application Publication Nos. WO 2011109400 and WO 2012004410, or a
B7-H3 agonist such as disclosed e.g. in International Application
Publication No. WO 2004093894, the contents of each of which are
fully incorporated herein by reference.
[0411] An agent capable of binding CD19, such as, MEDI-551
(produced by MedImmune) and SAR3419 (produced by Sanofi),
monoclonal antibodies that target CD19 and CART cells directed at
CD19 such as CTL-019 (produced by Novartis) or such as disclosed
e.g. in US Application Publication No: US 20140271635.
[0412] An agent capable of binding CD70, such as ARGX-110 (produced
by arGEN-X BVBA) a monoclonal antibody that targets CD70 and
induces cytotoxic T cell response and ADCC to cells expressing
CD70.
[0413] As disclosed above the anti-cancer agent can also be
selected from the group consisting of a colony stimulating factor-1
receptor (CSF1R) antagonist, a CXCR2 antagonist, a STAT3
antagonist, PV-10 and Cotara.
[0414] CSF1R (also known as macrophage colony-stimulating factor
receptor, M-CSFR and CD115) is a cell single pass type I membrane
protein acting as the receptor for colony stimulating factor 1, a
cytokine which controls the production, differentiation, and
function of macrophages. According to a specific embodiment, the
CSF1R protein refers to the human protein, such as provided in the
following GenBank Number NP_001275634.
[0415] Specific non-limiting examples of CSF1R antagonists that can
be used according to some embodiments of the invention include the
antibodies FPA008 (produced by Five Prime Therapeutics) and RG7155
(produced by Roche). Tumor associated macrophages (TAM), are
dependent on CSF1R signaling. TAMs are myeloid lineage-derived
cells that are part of the tumor microenvironmental support system.
TAMs are potently angiogenic, remodel the stroma (extracellular
matrix and related components) and are immunosuppressive. The
plethora of critical factors produced by TAM include hypoxia
response proteins and growth factors that drive angiogenesis,
tissue remodeling and immunosuppression, i.e. HIF2a, MMP-9, EGF,
VEGF and TGF-beta, cytokines that can maintain this response in a
chronic state (IL-10, IL-4) and chemokines that attract myeloid
cells and regulatory T cells (CCL22, CCXL8). The TAM population can
be directly regulated by tumor cell secretion of CSF-1, thus the
importance of the CSF1R target.
[0416] CXCR2 (also known as IL-8 receptor, beta) is a
G-protein-coupled chemokine receptor which functions as a receptor
for e.g. IL-8, CXCL1, CXCL2, CXCL3 and CXCL5. According to a
specific embodiment, the CXCR2 protein refers to the human protein,
such as provided in the following GenBank Number NP_001161770.
[0417] A specific non-limiting example of a CXCR2 antagonist that
can be used according to some embodiments of the invention include
AZD5069 (produced by AstraZeneca). Cancer initiation and
progression also depends on escape from host immunosurveillance. In
several solid tumors, immune evasion involves a shift of immune
responses, including imbalance in Th1/Th2 responses and enhancement
of immunosuppressive cells such as myeloid-derived suppressor cells
(MDSCs) and regulatory T cells. The number of MDSCs in the blood
correlates well with clinical cancer stage and metastatic tumor
burden in patients (Diaz-Montero et al., 2009; Cancer Immunology,
Immunotherapy, January 2009, Volume 58, Issue 1, pp 49-59). It is
widely accepted that MDSCs contribute to cancer immune evasion via
suppressing functions of T and natural killer (NK) cells
(Gabrilovich and Nagaraj, 2009 Nature Reviews Immunology 9,
162-174). Accumulation of Myeloid Derived Suppressor Cells (MDSCs)
in the tumor bed limits the efficacy of checkpoint blockade in
cancer. Expansion of MDSCs is a major mechanism of tumor immune
escape. CXCR2 is essential for MDSC infiltration into tumors, which
promotes tumor growth and progression via inhibition of CD8+ T cell
cytotoxic activity.
[0418] Signal transducer and activator of transcription 3 (STAT3)
is a transcription factor encoded by the STAT3 gene. STAT3, is a
member of the STAT protein family of signal transducers and
activators of transcription, which represent a family of proteins
that, when activated by protein tyrosine kinases in the cytoplasm
of the cell, migrate to the nucleus and activate gene
transcription.
[0419] According to a specific embodiment, the STAT3 protein refers
to the human protein, such as provided in the following GenBank
Number NP_003141.
[0420] Specific non-limiting examples of STAT3 antagonists that can
be used according to some embodiments of the invention include
AZD9150 (produced by AstraZeneca), a STAT3 antisense; and STAT3
siRNA such as disclosed e.g. in Herrmann A, et al. J Clin Invest.
(2014) 124(7): 2977-87.
[0421] PV-10 (produced by Provectus Biopharmaceuticals Inc.) is a
composition containing Rose Bengal shown to eradicate tumors and
activate anti-tumor T cell response.
[0422] Cotara (131I-chTNT-1/B Mab, produced by Peregrine
Pharmaceuticals Inc.), is an antibody which is combined with
radioactive iodine. Typically, Cotara binds to proteins within the
nucleus of necrotic (i.e. dead and dying) cells which are present
in most malignant tumors and are primarily found at the center of
the tumor. When the Ab is delivered directly to the tumor, it
remains within the tumor and the attached radioactive iodine
bombards the neighboring living tumor cells with radiation.
[0423] According to specific embodiments, the CXCR4 antagonistic
peptide can be administered to a subject in combination with
several of the anti-cancer agents described hereinabove.
[0424] The order in which the CXCR4 antagonistic peptide and the
anti-cancer agent are administered to the subject can vary
according to the method of treating.
[0425] Thus, according to a specific embodiment, administering the
peptide is effected prior to administering the agent.
[0426] According to another specific embodiment, administering the
peptide is effected following administering the agent.
[0427] According to yet another specific embodiment, administering
the peptide is effected concomitantly with administering the
agent.
[0428] Multiple rounds of administration according to the methods
of the present invention and multiple doses of the CXCR4
antagonistic peptide and the anti-cancer agent can be administered.
According to specific embodiments, administering comprises multiple
administrations of the peptide. Thus, according to specific
embodiments, administration of the anti-cancer agent is effected
following at least one administration of the CXCR4 antagonistic
peptide. According to specific embodiments, administering comprises
multiple administrations of the anti-cancer agent. Thus, according
to specific embodiments, administering the CXCR4 antagonistic
peptide of the present invention is effected following at least one
administration of the agent. According to specific embodiments,
administering the peptide and administering the agent are effected
sequentially.
[0429] According to specific embodiments, the CXCR4 antagonistic
peptide and the anti-cancer agent of the invention can be
administered to a subject in combination with other established or
experimental therapeutic regimen to treat cancer including
analgetics, chemotherapeutic agents, radiotherapeutic agents,
hormonal therapy, immune modulators and other treatment regimens
(e.g., surgery, cell transplantation e.g. hematopoietic stem cell
transplantation) which are well known in the art.
[0430] The CXCR4 antagonistic peptides and/or the anti-cancer agent
described hereinabove can be administered to the subject per se, or
in a pharmaceutical composition where it is mixed with suitable
carriers or excipients.
[0431] Thus, according to an aspect of the present invention there
is provided a pharmaceutical composition comprising as active
ingredients a peptide having an amino acid sequence as set forth in
SEQ ID NO: 1 or an analog or derivative thereof and an anti-cancer
agent selected from the group consisting of:
[0432] (i) a vaccine selected from the group consisting of
IMCgp100, Prophage G-100 & G-200, GV-1001, IMA-950, CV-9201,
CV-9104, Ad-RTS-hIL-12, ETBX-011, Cavatak, JX-594, ColoAd1,
GL-ONC1, ONCOS-102, CRS-207, ADU-623, Dorgenmeltucel-L, HyperAcute
Prostate, FANG vaccine, MGN-1601, HPV vaccine and Tarmogens such as
GI-4000;
[0433] (ii) anti-cancer reactive mononuclear blood cells
(MNBCs);
[0434] (iii) a cytokine capable of inducing activation and/or
proliferation of a T cell;
[0435] (iv) an immune-check point regulator, wherein said
immune-check point regulator is not a PD1 antagonist, PDL-1
antagonist, CTLA-4 antagonist, LAG-3 antagonist, TIM-3 antagonist,
KIR antagonist, IDO antagonist, OX40 agonist, CD137 agonist, CD27
agonist, CD40 agonist, GITR agonist, CD28 agonist or ICOS
agonist;
[0436] (v) an agent capable of binding an immune-check point
protein expressed on a cancer cell;
[0437] (vi) a colony stimulating factor-1 receptor (CSF1R)
antagonist;
[0438] (vii) a CXCR2 antagonist;
[0439] (viii) a STAT3 antagonist;
[0440] (ix) PV-10; and
[0441] (x) Cotara,
and a pharmaceutically acceptable carrier or diluent.
[0442] As used herein a "pharmaceutical composition" refers to a
preparation of one or more of the active ingredients described
herein with other chemical components such as physiologically
suitable carriers and excipients. The purpose of a pharmaceutical
composition is to facilitate administration of a compound to an
organism.
[0443] Herein the term "active ingredient" refers to the CXCR4
antagonistic peptides and/or the anti-cancer agent accountable for
the biological effect.
[0444] Hereinafter, the phrases "physiologically acceptable
carrier" and "pharmaceutically acceptable carrier" which may be
interchangeably used refer to a carrier or a diluent that does not
cause significant irritation to an organism and does not abrogate
the biological activity and properties of the administered
compound. An adjuvant is included under these phrases.
[0445] Herein the term "excipient" refers to an inert substance
added to a pharmaceutical composition to further facilitate
administration of an active ingredient. Examples, without
limitation, of excipients include calcium carbonate, calcium
phosphate, various sugars and types of starch, cellulose
derivatives, gelatin, vegetable oils and polyethylene glycols.
[0446] Techniques for formulation and administration of drugs may
be found in "Remington's Pharmaceutical Sciences," Mack Publishing
Co., Easton, Pa., latest edition, which is incorporated herein by
reference.
[0447] Suitable routes of administration may, for example, include
oral, rectal, transmucosal, especially transnasal, intestinal or
parenteral delivery, including intramuscular, intradermal,
subcutaneous and intramedullary injections as well as intrathecal,
direct intraventricular, intracardiac, e.g., into the right or left
ventricular cavity, into the common coronary artery, intravenous,
intraperitoneal, intranasal, or intraocular injections.
[0448] Conventional approaches for drug delivery to the central
nervous system (CNS) include: neurosurgical strategies (e.g.,
intracerebral injection or intracerebroventricular infusion);
molecular manipulation of the agent (e.g., production of a chimeric
fusion protein that comprises a transport peptide that has an
affinity for an endothelial cell surface molecule in combination
with an agent that is itself incapable of crossing the BBB) in an
attempt to exploit one of the endogenous transport pathways of the
BBB; pharmacological strategies designed to increase the lipid
solubility of an agent (e.g., conjugation of water-soluble agents
to lipid or cholesterol carriers); and the transitory disruption of
the integrity of the BBB by hyperosmotic disruption (resulting from
the infusion of a mannitol solution into the carotid artery or the
use of a biologically active agent such as an angiotensin peptide).
However, each of these strategies has limitations, such as the
inherent risks associated with an invasive surgical procedure, a
size limitation imposed by a limitation inherent in the endogenous
transport systems, potentially undesirable biological side effects
associated with the systemic administration of a chimeric molecule
comprised of a carrier motif that could be active outside of the
CNS, and the possible risk of brain damage within regions of the
brain where the BBB is disrupted, which renders it a suboptimal
delivery method.
[0449] Alternately, one may administer the pharmaceutical
composition in a local rather than systemic manner, for example,
via injection of the pharmaceutical composition directly into a
tissue region of a patient.
[0450] The CXCR4 antagonistic peptide of the invention, the
anti-cancer agent or the pharmaceutical composition comprising same
can be administered in the same route or in separate routes.
[0451] According to a specific embodiment, the CXCR4 antagonistic
peptide of the invention or the pharmaceutical composition
comprising same is administered subcutaneously.
[0452] According to another specific embodiment, the CXCR4
antagonistic peptide of the invention or the pharmaceutical
composition comprising same is administered intravenously.
[0453] According to a specific embodiment, the anti-cancer agent or
the pharmaceutical composition comprising same is administered
intravenously.
[0454] According to a specific embodiment, the anti-cancer agent or
the pharmaceutical composition comprising same is administered via
a subcutaneous route.
[0455] Pharmaceutical compositions of some embodiments of the
invention may be manufactured by processes well known in the art,
e.g., by means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping
or lyophilizing processes.
[0456] Pharmaceutical compositions for use in accordance with some
embodiments of the invention thus may be formulated in conventional
manner using one or more physiologically acceptable carriers
comprising excipients and auxiliaries, which facilitate processing
of the active ingredients into preparations which, can be used
pharmaceutically. Proper formulation is dependent upon the route of
administration chosen.
[0457] For injection, the active ingredients of the pharmaceutical
composition may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hank's solution,
Ringer's solution, or physiological salt buffer. For transmucosal
administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are
generally known in the art.
[0458] For oral administration, the pharmaceutical composition can
be formulated readily by combining the active compounds with
pharmaceutically acceptable carriers well known in the art. Such
carriers enable the pharmaceutical composition to be formulated as
tablets, pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for oral ingestion by a patient.
Pharmacological preparations for oral use can be made using a solid
excipient, optionally grinding the resulting mixture, and
processing the mixture of granules, after adding suitable
auxiliaries if desired, to obtain tablets or dragee cores. Suitable
excipients are, in particular, fillers such as sugars, including
lactose, sucrose, mannitol, or sorbitol; cellulose preparations
such as, for example, maize starch, wheat starch, rice starch,
potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or
physiologically acceptable polymers such as polyvinylpyrrolidone
(PVP). If desired, disintegrating agents may be added, such as
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate.
[0459] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, titanium dioxide, lacquer
solutions and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0460] Pharmaceutical compositions which can be used orally include
push-fit capsules made of gelatin as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules may contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
lubricants such as talc or magnesium stearate and, optionally,
stabilizers. In soft capsules, the active ingredients may be
dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added. All formulations for oral administration
should be in dosages suitable for the chosen route of
administration.
[0461] For buccal administration, the compositions may take the
form of tablets or lozenges formulated in conventional manner.
[0462] For administration by nasal inhalation, the active
ingredients for use according to some embodiments of the invention
are conveniently delivered in the form of an aerosol spray
presentation from a pressurized pack or a nebulizer with the use of
a suitable propellant, e.g., dichlorodifluoromethane,
trichlorofluoromethane, dichloro-tetrafluoroethane or carbon
dioxide. In the case of a pressurized aerosol, the dosage unit may
be determined by providing a valve to deliver a metered amount.
Capsules and cartridges of, e.g., gelatin for use in a dispenser
may be formulated containing a powder mix of the compound and a
suitable powder base such as lactose or starch.
[0463] The pharmaceutical composition described herein may be
formulated for parenteral administration, e.g., by bolus injection
or continuous infusion. Formulations for injection may be presented
in unit dosage form, e.g., in ampoules or in multidose containers
with optionally, an added preservative. The compositions may be
suspensions, solutions or emulsions in oily or aqueous vehicles,
and may contain formulatory agents such as suspending, stabilizing
and/or dispersing agents.
[0464] Pharmaceutical compositions for parenteral administration
include aqueous solutions of the active preparation in
water-soluble form. Additionally, suspensions of the active
ingredients may be prepared as appropriate oily or water based
injection suspensions. Suitable lipophilic solvents or vehicles
include fatty oils such as sesame oil, or synthetic fatty acids
esters such as ethyl oleate, triglycerides or liposomes.
[0465] Aqueous injection suspensions may contain substances, which
increase the viscosity of the suspension, such as sodium
carboxymethyl cellulose, sorbitol or dextran. Optionally, the
suspension may also contain suitable stabilizers or agents which
increase the solubility of the active ingredients to allow for the
preparation of highly concentrated solutions.
[0466] Alternatively, the active ingredient may be in powder form
for constitution with a suitable vehicle, e.g., sterile,
pyrogen-free water based solution, before use.
[0467] The pharmaceutical composition of some embodiments of the
invention may also be formulated in rectal compositions such as
suppositories or retention enemas, using, e.g., conventional
suppository bases such as cocoa butter or other glycerides.
[0468] Alternative embodiments include depots providing sustained
release or prolonged duration of activity of the active ingredient
in the subject, as are well known in the art.
[0469] Pharmaceutical compositions suitable for use in context of
some embodiments of the invention include compositions wherein the
active ingredients are contained in an amount effective to achieve
the intended purpose. More specifically, according to specific
embodiments, a therapeutically effective amount means an amount of
active ingredients effective to prevent, alleviate or ameliorate
symptoms of a disorder (e.g., cancer) or prolong the survival of
the subject being treated.
[0470] Determination of a therapeutically effective amount is well
within the capability of those skilled in the art, especially in
light of the detailed disclosure provided herein.
[0471] For any preparation used in the methods of the invention,
the therapeutically effective amount or dose can be estimated
initially from in vitro and cell culture assays. For example, a
dose can be formulated in animal models to achieve a desired
concentration or titer. Such information can be used to more
accurately determine useful doses in humans.
[0472] Toxicity and therapeutic efficacy of the active ingredients
described herein can be determined by standard pharmaceutical
procedures in vitro, in cell cultures or experimental animals. The
data obtained from these in vitro and cell culture assays and
animal studies can be used in formulating a range of dosage for use
in human.
[0473] The dosage may vary depending upon the dosage form employed
and the route of administration utilized.
[0474] The exact formulation, route of administration and dosage
can be chosen by the individual physician in view of the patient's
condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological
Basis of Therapeutics", Ch. 1 p. 1).
[0475] Dosage amount and interval may be adjusted individually to
provide levels of the active ingredient are sufficient to induce or
suppress the biological effect (minimal effective concentration,
MEC). The MEC will vary for each preparation, but can be estimated
from in vitro data. Dosages necessary to achieve the MEC will
depend on individual characteristics and route of administration.
Detection assays can be used to determine plasma
concentrations.
[0476] Depending on the severity and responsiveness of the
condition to be treated, dosing can be of a single or a plurality
of administrations, with course of treatment lasting from several
days to several weeks or until cure is effected or diminution of
the disease state is achieved.
[0477] The amount of a composition to be administered will, of
course, be dependent on the subject being treated, the severity of
the affliction, the manner of administration, the judgment of the
prescribing physician, etc.
[0478] According to specific embodiments the CXCR4 antagonistic
peptide of the invention or the pharmaceutical composition
comprising same is administered in a dose ranging between 0.1 to 10
mg/kg of body weight, between 0.1 to 2 mg/kg of body weight,
between 0.1 to 1 mg/kg of body weight, between 0.3 to 10 mg/kg of
body weight, between 0.3 to 2 mg/kg of body weight, between 0.3 to
1 mg/kg of body weight or between 0.3 to 0.9 mg/kg of body
weight.
[0479] According to a specific embodiment, the CXCR4 antagonistic
peptide of the invention or the pharmaceutical composition
comprising same is administered in a dose ranging between 0.5-2
mg/kg.
[0480] According to another specific embodiment the CXCR4
antagonistic peptide of the invention or the pharmaceutical
composition comprising same is administered at a dose of 0.5-1
mg/kg.
[0481] According to specific embodiments, the anti-cancer antibody
is administered in a dose ranging between 0.001 to 30 mg/kg body
weight, between 0.001 to 20 mg/kg body weight, between 0.001 to 10
mg/kg body weight, between 0.001 to 1 mg/kg body weight, between
0.01 to 30 mg/kg body weight, between 0.01 to 20 mg/kg body weight,
between 0.01 to 10 mg/kg body weight, between 0.01 to 1 mg/kg body
weight, between 0.1 to 30 mg/kg body weight, between 0.1 to 20
mg/kg body weight, between 0.1 to 10 mg/kg body weight, between 0.1
to 1 mg/kg body weight, between 1 to about 30 mg/kg, between 1 to
about 20 mg/kg or between 1 to about 10 mg/kg.
[0482] The desired dose can be administered at one time or divided
into sub-doses, e.g., 2-4 sub-doses and administered over a period
of time, e.g., at appropriate intervals through the day or other
appropriate schedule.
[0483] According to specific embodiments, the CXCR4 antagonistic
peptide of the invention, the anti-cancer agent or the
pharmaceutical composition comprising same is administered multiple
times e.g. 2-10, over a period of time e.g. for several days to
several weeks at appropriate intervals e.g. once a day, twice a
week, once a week, once every two weeks, once a month, once every 3
to 6 months.
[0484] Compositions of some embodiments of the invention may, if
desired, be presented in a pack or dispenser device, such as an FDA
approved kit, which may contain one or more unit dosage forms
containing the active ingredient. The pack may, for example,
comprise metal or plastic foil, such as a blister pack. The pack or
dispenser device may be accompanied by instructions for
administration. The pack or dispenser may also be accommodated by a
notice associated with the container in a form prescribed by a
governmental agency regulating the manufacture, use or sale of
pharmaceuticals, which notice is reflective of approval by the
agency of the form of the compositions or human or veterinary
administration. Such notice, for example, may be of labeling
approved by the U.S. Food and Drug Administration for prescription
drugs or of an approved product insert. Compositions comprising a
preparation of the invention formulated in a compatible
pharmaceutical carrier may also be prepared, placed in an
appropriate container, and labeled for treatment of an indicated
condition, as is further detailed above.
[0485] According an aspect of the present invention there is
provided an article of manufacture identified for use in treating
cancer, comprising a packaging material packaging a peptide having
an amino acid sequence as set forth in SEQ ID NO: 1 or an analog or
derivative thereof and an anti-cancer agent selected from the group
consisting of:
[0486] (i) a vaccine selected from the group consisting of
IMCgp100, Prophage G-100 & G-200, GV-1001, IMA-950, CV-9201,
CV-9104, Ad-RTS-hIL-12, ETBX-011, Cavatak, JX-594, ColoAd1,
GL-ONC1, ONCOS-102, CRS-207, ADU-623, Dorgenmeltucel-L, HyperAcute
Prostate, FANG vaccine, MGN-1601, HPV vaccine and Tarmogens such as
GI-4000;
[0487] (ii) anti-cancer reactive mononuclear blood cells
(MNBCs);
[0488] (iii) a cytokine capable of inducing activation and/or
proliferation of a T cell;
[0489] (iv) an immune-check point regulator, wherein said
immune-check point regulator is not a PD1 antagonist, PDL-1
antagonist, CTLA-4 antagonist, LAG-3 antagonist, TIM-3 antagonist,
KIR antagonist, IDO antagonist, OX40 agonist, CD137 agonist, CD27
agonist, CD40 agonist, GITR agonist, CD28 agonist or ICOS
agonist;
[0490] (v) an agent capable of binding an immune-check point
protein expressed on a cancer cell;
[0491] (vi) a colony stimulating factor-1 receptor (CSF1R)
antagonist;
[0492] (vii) a CXCR2 antagonist;
[0493] (viii) a STAT3 antagonist;
[0494] (ix) PV-10; and
[0495] (x) Cotara.
[0496] The peptide and the agent may be packaged in the same
container or in separate containers; each possibility represents a
separate embodiment of the present invention.
[0497] According to specific embodiments, the peptide and the agent
are in separate containers.
[0498] According to specific embodiments, the peptide and the agent
are in separate formulations.
[0499] According to other specific embodiments, the peptide and the
agent are in a co-formulation.
[0500] It is expected that during the life of a patent maturing
from this application many relevant anti-cancer agents will be
developed and the scope of the term "anti-cancer agent" is intended
to include all such new technologies a priori.
[0501] As used herein the term "about" refers to .+-.10%.
[0502] The terms "comprises", "comprising", "includes",
"including", "having" and their conjugates mean "including but not
limited to".
[0503] The term "consisting of" means "including and limited
to".
[0504] The term "consisting essentially of" means that the
composition, method or structure may include additional
ingredients, steps and/or parts, but only if the additional
ingredients, steps and/or parts do not materially alter the basic
and novel characteristics of the claimed composition, method or
structure.
[0505] As used herein, the singular form "a", "an" and "the"
include plural references unless the context clearly dictates
otherwise. For example, the term "a compound" or "at least one
compound" may include a plurality of compounds, including mixtures
thereof.
[0506] Throughout this application, various embodiments of this
invention may be presented in a range format. It should be
understood that the description in range format is merely for
convenience and brevity and should not be construed as an
inflexible limitation on the scope of the invention. Accordingly,
the description of a range should be considered to have
specifically disclosed all the possible subranges as well as
individual numerical values within that range. For example,
description of a range such as from 1 to 6 should be considered to
have specifically disclosed subranges such as from 1 to 3, from 1
to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as
well as individual numbers within that range, for example, 1, 2, 3,
4, 5, and 6. This applies regardless of the breadth of the
range.
[0507] Whenever a numerical range is indicated herein, it is meant
to include any cited numeral (fractional or integral) within the
indicated range. The phrases "ranging/ranges between" a first
indicate number and a second indicate number and "ranging/ranges
from" a first indicate number "to" a second indicate number are
used herein interchangeably and are meant to include the first and
second indicated numbers and all the fractional and integral
numerals therebetween.
[0508] As used herein the term "method" refers to manners, means,
techniques and procedures for accomplishing a given task including,
but not limited to, those manners, means, techniques and procedures
either known to, or readily developed from known manners, means,
techniques and procedures by practitioners of the chemical,
pharmacological, biological, biochemical and medical arts.
[0509] When reference is made to particular sequence listings, such
reference is to be understood to also encompass sequences that
substantially correspond to its complementary sequence as including
minor sequence variations, resulting from, e.g., sequencing errors,
cloning errors, or other alterations resulting in base
substitution, base deletion or base addition, provided that the
frequency of such variations is less than 1 in 50 nucleotides,
alternatively, less than 1 in 100 nucleotides, alternatively, less
than 1 in 200 nucleotides, alternatively, less than 1 in 500
nucleotides, alternatively, less than 1 in 1000 nucleotides,
alternatively, less than 1 in 5,000 nucleotides, alternatively,
less than 1 in 10,000 nucleotides.
[0510] It is appreciated that certain features of the invention,
which are, for clarity, described in the context of separate
embodiments, may also be provided in combination in a single
embodiment. Conversely, various features of the invention, which
are, for brevity, described in the context of a single embodiment,
may also be provided separately or in any suitable subcombination
or as suitable in any other described embodiment of the invention.
Certain features described in the context of various embodiments
are not to be considered essential features of those embodiments,
unless the embodiment is inoperative without those elements.
[0511] Various embodiments and aspects of the present invention as
delineated hereinabove and as claimed in the claims section below
find experimental support in the following examples.
EXAMPLES
[0512] Reference is now made to the following examples, which
together with the above descriptions illustrate some embodiments of
the invention in a non limiting fashion.
[0513] Generally, the nomenclature used herein and the laboratory
procedures utilized in the present invention include molecular,
biochemical, microbiological and recombinant DNA techniques. Such
techniques are thoroughly explained in the literature. See, for
example, "Molecular Cloning: A laboratory Manual" Sambrook et al.,
(1989); "Current Protocols in Molecular Biology" Volumes I-III
Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in
Molecular Biology", John Wiley and Sons, Baltimore, Md. (1989);
Perbal, "A Practical Guide to Molecular Cloning", John Wiley &
Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific
American Books, New York; Birren et al. (eds) "Genome Analysis: A
Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory
Press, New York (1998); methodologies as set forth in U.S. Pat.
Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057;
"Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E.,
ed. (1994); "Culture of Animal Cells--A Manual of Basic Technique"
by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current
Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994);
Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition),
Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi
(eds), "Selected Methods in Cellular Immunology", W. H. Freeman and
Co., New York (1980); available immunoassays are extensively
described in the patent and scientific literature, see, for
example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;
3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;
3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and
5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984);
"Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds.
(1985); "Transcription and Translation" Hames, B. D., and Higgins
S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed.
(1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A
Practical Guide to Molecular Cloning" Perbal, B., (1984) and
"Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols:
A Guide To Methods And Applications", Academic Press, San Diego,
Calif. (1990); Marshak et al., "Strategies for Protein Purification
and Characterization--A Laboratory Course Manual" CSHL Press
(1996); all of which are incorporated by reference as if fully set
forth herein. Other general references are provided throughout this
document. The procedures therein are believed to be well known in
the art and are provided for the convenience of the reader. All the
information contained therein is incorporated herein by
reference.
Example 1
Use of BL-8040 for Treating Cancer
[0514] BL-8040 is safe and well tolerated drug that was shown to
induce rapid mobilization of hematopoietic stem/progenitor cells
and mesenchymal stem cells as well as T cells, B cells, NK cells,
NKT cells and ImDC to the peripheral blood. Therefore, BL-8040 can
be used to induce the mobilization and dissemination of immature DC
and T effector and memory cells into tumors.
[0515] Experimental Procedures
[0516] According to one protocol, BL-8040 is injected into a cancer
patient at a dose of 0.5-1 mg/kg for 3-10 days and then 1-3 times a
week in combination with a tumor vaccine such as a dendritic cells
vaccine, IMCgp100, Prophage G-100 & G-200, GV-1001, IMA-950,
CV-9201, CV-9104, Ad-RTS-hIL-12, ETBX-011, Cavatak, JX-594,
ColoAd1, GL-ONC1, ONCOS-102, CRS-207, ADU-623, Dorgenmeltucel-L,
HyperAcute Prostate, FANG vaccine, MGN-1601, HPV vaccine and
Tarmogens such as GI-4000.
[0517] According to another protocol, BL-8040 is injected into a
cancer patient at a dose of 0.5-1 mg/kg for 3-10 days and then 1-3
times a week in combination with immunomodulatory cytokines such as
IL-2, IFN-.alpha., IL-12.
[0518] According to another protocol, BL-8040 is injected into a
cancer patient at a dose of 0.5-1 mg/kg for 3-10 days and then 1-3
times a week in combination with a tumor vaccine such as a
dendritic cells vaccine, IMCgp100, Prophage G-100 & G-200,
GV-1001, IMA-950, CV-9201, CV-9104, Ad-RTS-hIL-12, ETBX-011,
Cavatak, JX-594, ColoAd1, GL-ONC1, ONCOS-102, CRS-207, ADU-623,
Dorgenmeltucel-L, HyperAcute Prostate, FANG vaccine, MGN-1601, HPV
vaccine and Tarmogens such as GI-4000 and immunomodulatory
cytokines such as IL-2, IFN-.alpha. and IL-12.
[0519] According to another protocol, BL-8040 is injected into a
cancer patient at a dose of 0.5-1 mg/kg for 3-10 days and then 1-3
times a week in combination with anti-cancer adoptive T cell
transfer such as T cells transduced with an anti-cancer T cell
receptor (TCR) or a chimeric antigen receptor (CAR).
[0520] According to another protocol, BL-8040 is injected into a
cancer patient at a dose of 0.5-1 mg/kg for 3-10 days and then 1-3
times a week in combination with an immune-check point regulator
targeting e.g. B7-H3, CD19 and CD70.
[0521] According to another protocol, BL-8040 is injected into a
cancer patient at a dose of 0.5-1 mg/kg for 3-10 days and then 1-3
times a week in combination with an anti-cancer agent (e.g.
antibody or T cell) capable of binding a cancer cell expressing an
immune-check point protein.
[0522] Although the invention has been described in conjunction
with specific embodiments thereof, it is evident that many
alternatives, modifications and variations will be apparent to
those skilled in the art. Accordingly, it is intended to embrace
all such alternatives, modifications and variations that fall
within the spirit and broad scope of the appended claims.
[0523] All publications, patents and patent applications mentioned
in this specification are herein incorporated in their entirety by
reference into the specification, to the same extent as if each
individual publication, patent or patent application was
specifically and individually indicated to be incorporated herein
by reference. In addition, citation or identification of any
reference in this application shall not be construed as an
admission that such reference is available as prior art to the
present invention. To the extent that section headings are used,
they should not be construed as necessarily limiting.
Sequence CWU 1
1
72114PRTArtificialsynthetic
peptideMOD_RES(1)..(1)4-fluorobenzoyl-arginineMOD_RES(3)..(3)3-((2-naphth-
yl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-lysineMOD_RES(12)..(1-
2)citrullineMOD_RES(14)..(14)C' AMIDATED 1Xaa Arg Xaa Cys Tyr Xaa
Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5 10214PRTArtificialSYNTHETIC
PEPTIDEMOD_RES(1)..(1)N' ACETYLATIONMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-lysineMOD_RES(12)..(12)c-
itrulline 2Arg Arg Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys Arg1
5 10314PRTArtificialSYNTHETIC PEPTIDEMOD_RES(1)..(1)N'
ACETYLATIONMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(8)..(8)D-citrullineMOD_RES(12)..(12)citrulline 3Arg
Arg Xaa Cys Tyr Arg Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
10414PRTArtificialsynthetic peptideMOD_RES(1)..(1)N'
ACETYLATEDMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-citrullineMOD_RES(12)..(-
12)citrulline 4Arg Arg Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys
Arg1 5 10514PRTArtificialsynthetic peptideMOD_RES(1)..(1)N'
ACETYLATEDMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-lysineMOD_RES(11)..(12)c-
itrulline 5Arg Arg Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Xaa Xaa Cys Arg1
5 10614PRTArtificialsynthetic peptideMOD_RES(1)..(1)N' acetylated
citrullineMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-lysineMOD_RES(12)..(12)c-
itrulline 6Xaa Arg Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys Arg1
5 10714PRTArtificialsynthetic peptideMOD_RES(1)..(1)N' acetylated
citrullineMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(8)..(8)D-citrullineMOD_RES(12)..(12)citrulline 7Xaa
Arg Xaa Cys Tyr Arg Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
10814PRTArtificialsynthetic peptideMOD_RES(1)..(1)N'
ACETYLATEDMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(8)..(8)D-citrullineMOD_RES(11)..(12)citrulline 8Arg
Arg Xaa Cys Tyr Arg Lys Xaa Pro Tyr Xaa Xaa Cys Arg1 5
10914PRTArtificialsynthetic peptideMOD_RES(1)..(1)N' ACETYLATED
citrullineMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(8)..(8)D-lysineMOD_RES(11)..(12)citrulline 9Xaa Arg
Xaa Cys Tyr Arg Lys Xaa Pro Tyr Xaa Xaa Cys Arg1 5
101014PRTArtificialsynthetic peptideMOD_RES(1)..(1)N'
ACETYLATEDMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-citrullineMOD_RES(12)..(-
12)citrullineMOD_RES(14)..(14)C' AMIDATED 10Arg Arg Xaa Cys Tyr Xaa
Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5 101114PRTArtificialsynthetic
peptideMOD_RES(1)..(1)N' ACETYLATEDMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-lysineMOD_RES(11)..(12)c-
itrullineMOD_RES(14)..(14)C' AMIDATED 11Arg Arg Xaa Cys Tyr Xaa Lys
Xaa Pro Tyr Xaa Xaa Cys Arg1 5 101214PRTArtificialsynthetic
peptideMOD_RES(1)..(1)N' ACETYLATED
citrullineMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-lysineMOD_RES(12)..(12)c-
itrullineMOD_RES(14)..(14)AMIDATED 12Xaa Arg Xaa Cys Tyr Xaa Lys
Xaa Pro Tyr Arg Xaa Cys Arg1 5 101314PRTArtificialsynthetic
peptideMOD_RES(1)..(1)N' ACETYLATED
citrullineMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(8)..(8)D-citrullineMOD_RES(12)..(12)citrullineMOD_RES(14).-
.(14)C' AMIDATED 13Xaa Arg Xaa Cys Tyr Arg Lys Xaa Pro Tyr Arg Xaa
Cys Arg1 5 101414PRTArtificialsynthetic peptideMOD_RES(1)..(1)N'
ACETYLATEDMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(8)..(8)D-citrullineMOD_RES(11)..(12)citrullineMOD_RES(14).-
.(14)C' AMIDATED 14Arg Arg Xaa Cys Tyr Arg Lys Xaa Pro Tyr Xaa Xaa
Cys Arg1 5 101514PRTArtificialsynthetic peptideMOD_RES(1)..(1)N'
ACETYLATED citrullineMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(8)..(8)D-lysineMOD_RES(11)..(12)citrullineMOD_RES(14)..(14-
)C' AMIDATED 15Xaa Arg Xaa Cys Tyr Arg Lys Xaa Pro Tyr Xaa Xaa Cys
Arg1 5 101614PRTArtificialsynthetic
peptideMOD_RES(1)..(1)D-glutamic acidMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(8)..(8)D-lysineMOD_RES(12)..(12)citrulline 16Xaa Arg
Xaa Cys Tyr Arg Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
101714PRTArtificialsynthetic peptideMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(8)..(8)D-lysineMOD_RES(12)..(12)citrulline 17Arg Glu
Xaa Cys Tyr Arg Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
101814PRTArtificialsynthetic peptideMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(8)..(8)D-lysineMOD_RES(12)..(12)citrulline 18Arg Arg
Xaa Cys Tyr Glu Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
101914PRTArtificialsynthetic peptideMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(8)..(8)D-lysineMOD_RES(12)..(12)citrulline 19Arg Arg
Xaa Cys Tyr Arg Glu Xaa Pro Tyr Arg Xaa Cys Arg1 5
102014PRTArtificialsynthetic peptideMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(8)..(8)D-glutamic acidMOD_RES(12)..(12)citrulline
20Arg Arg Xaa Cys Tyr Arg Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
102114PRTArtificialsynthetic peptideMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(8)..(8)D-lysineMOD_RES(12)..(12)citrulline 21Arg Arg
Xaa Cys Tyr Arg Lys Xaa Pro Tyr Glu Xaa Cys Arg1 5
102214PRTArtificialsynthetic peptideMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(8)..(8)D-lysineMOD_RES(12)..(12)citrulline 22Arg Arg
Xaa Cys Tyr Arg Lys Xaa Pro Tyr Arg Xaa Cys Glu1 5
102314PRTArtificialsynthetic peptideMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-glutamic
acidMOD_RES(12)..(12)citrullineMOD_RES(14)..(14)C' amidated 23Arg
Arg Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
102414PRTArtificialsynthetic peptideMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)D-glutamic
acidMOD_RES(8)..(8)D-citrullineMOD_RES(12)..(12)citrullineMOD_RES(14)..(1-
4)c' amidated 24Arg Arg Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys
Arg1 5 102514PRTArtificialsynthetic
peptideMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)D-glutamic acidMOD_RES(8)..(8)D-glutamic
acidMOD_RES(12)..(12)citrullineMOD_RES(14)..(14)c' amidated 25Arg
Arg Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
102614PRTArtificialsynthetic peptideMOD_RES(1)..(1)D-glutamic
acidMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-glutamic
acidMOD_RES(12)..(12)citrullineMOD_RES(14)..(14)c' amidated 26Xaa
Arg Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
102714PRTArtificialsynthetic peptideMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-glutamic
acidMOD_RES(10)..(10)D-glutamic
acidMOD_RES(12)..(12)citrullineMOD_RES(14)..(14)c' amidated 27Arg
Arg Xaa Cys Tyr Xaa Lys Xaa Pro Xaa Arg Xaa Cys Arg1 5
102814PRTArtificialsynthetic peptideMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-glutamic
acidMOD_RES(12)..(12)D-glutamic acidMOD_RES(14)..(14)c' amidated
28Arg Arg Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
102914PRTArtificialsynthetic peptideMOD_RES(1)..(1)N' ACETYLATED
D-glutamic acidMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-glutamic
acidMOD_RES(12)..(12)citrullineMOD_RES(14)..(14)c' amidated 29Xaa
Arg Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
103014PRTArtificialsynthetic peptideMOD_RES(1)..(1)N'
ACETYLATEDMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-glutamic
acidMOD_RES(10)..(10)D-glutamic
acidMOD_RES(12)..(12)citrullineMOD_RES(14)..(14)c' amidated 30Arg
Arg Xaa Cys Tyr Xaa Lys Xaa Pro Xaa Arg Xaa Cys Arg1 5
103114PRTArtificialsynthetic peptideMOD_RES(1)..(1)N'
ACETYLATEDMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-glutamic
acidMOD_RES(12)..(12)D-glutamic acidMOD_RES(14)..(14)c' amidated
31Arg Arg Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
103214PRTArtificialsynthetic peptideMOD_RES(1)..(1)N'
ACETYLATEDMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-glutamic
acidMOD_RES(12)..(12)citrullineMOD_RES(14)..(14)c' amidated 32Arg
Arg Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
103314PRTArtificialsynthetic
peptideMOD_RES(1)..(1)Guanyl-arginineMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-glutamic
acidMOD_RES(12)..(12)citrullineMOD_RES(14)..(14)c' amidated 33Xaa
Arg Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
103414PRTArtificialsynthetic
peptideMOD_RES(1)..(1)Tetramethylguanyl-arginineMOD_RES(3)..(3)3-((2-naph-
thyl) alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-glutamic
acidMOD_RES(12)..(12)citrullineMOD_RES(14)..(14)c' amidated 34Xaa
Arg Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
103513PRTArtificialsynthetic
peptideMOD_RES(1)..(1)Tetramethylguanyl-arginineMOD_RES(2)..(2)3-((2-naph-
thyl) alanineMOD_RES(5)..(5)citrullineMOD_RES(7)..(7)D-glutamic
acidMOD_RES(11)..(11)citrullineMOD_RES(13)..(13)c' amidated 35Xaa
Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
103614PRTArtificialsynthetic
peptideMOD_RES(1)..(1)4-fluorobenzoyl-arginineMOD_RES(3)..(3)3-((2-naphth-
yl) alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-glutamic
acidMOD_RES(12)..(12)citrullineMOD_RES(14)..(14)amidated 36Xaa Arg
Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
103714PRTArtificialsynthetic
peptideMOD_RES(1)..(1)2-fluorobenzoyl-arginineMOD_RES(3)..(3)3-((2-naphth-
yl) alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-glutamic
acidMOD_RES(12)..(12)citrullineMOD_RES(14)..(14)c' amidated 37Xaa
Arg Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
103813PRTArtificialsynthetic
peptideMOD_RES(1)..(1)5-aminopentanoyl-arginineMOD_RES(2)..(2)3-((2-napht-
hyl) alanineMOD_RES(5)..(5)citrullineMOD_RES(7)..(7)D-glutamic
acidMOD_RES(11)..(11)citrullineMOD_RES(13)..(13)c' amidated 38Xaa
Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
103914PRTArtificialsynthetic
peptideMOD_RES(1)..(1)2-desamino-arginylMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-glutamic
acidMOD_RES(12)..(12)citrullineMOD_RES(14)..(14)c' amidated 39Xaa
Arg Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
104013PRTArtificialsynthetic
peptideMOD_RES(1)..(1)Guanyl-arginineMOD_RES(2)..(2)3-((2-naphthyl)
alanineMOD_RES(5)..(5)citrullineMOD_RES(7)..(7)D-glutamic
acidMOD_RES(11)..(11)citrullineMOD_RES(13)..(13)c' amidated 40Xaa
Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
104113PRTArtificialsynthetic
peptideMOD_RES(1)..(1)Succinyl-arginineMOD_RES(2)..(2)3-((2-naphthyl)
alanineMOD_RES(5)..(5)citrullineMOD_RES(7)..(7)D-glutamic
acidMOD_RES(11)..(11)citrullineMOD_RES(13)..(13)c' amidated 41Xaa
Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
104213PRTArtificialsynthetic
peptideMOD_RES(1)..(1)Glutaryl-arginineMOD_RES(2)..(2)3-((2-naphthyl)
alanineMOD_RES(5)..(5)citrullineMOD_RES(7)..(7)D-glutamic
acidMOD_RES(11)..(11)citrullineMOD_RES(13)..(13)c' amidated 42Xaa
Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
104314PRTArtificialsynthetic
peptideMISC_FEATURE(1)..(1)desaminoTMG-APA (formula IV in the
specification)MOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-glutamic
acidMOD_RES(12)..(12)citrullineMOD_RES(14)..(14)c' amidated 43Xaa
Arg Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
104414PRTArtificialsynthetic peptideMISC_FEATURE(1)..(1)R-CH2 -
formula (V) in the specificationMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-glutamic
acidMOD_RES(12)..(12)citrullineMOD_RES(14)..(14)c' amidated 44Xaa
Arg Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
104513PRTArtificialsynthetic peptideMOD_RES(2)..(2)3-((2-naphthyl)
alanineMOD_RES(5)..(5)citrullineMOD_RES(7)..(7)D-glutamic
acidMOD_RES(11)..(11)citrullineMOD_RES(13)..(13)c' amidated 45Arg
Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
104614PRTArtificialsynthetic
peptideMOD_RES(1)..(1)tetramethylguanyl-arginineMOD_RES(3)..(3)3-((2-naph-
thyl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-citrullineMOD_RES(1-
2)..(12)citrullineMOD_RES(14)..(14)C' AMIDATED 46Xaa Arg Xaa Cys
Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
104714PRTArtificialsynthetic
peptideMOD_RES(1)..(1)6-aminohexanoyl-arginineMOD_RES(3)..(3)3-((2-naphth-
yl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-citrullineMOD_RES(12)-
..(12)citrullineMOD_RES(14)..(14)C' AMIDATED 47Xaa Arg Xaa Cys Tyr
Xaa Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5 104814PRTArtificialsynthetic
peptideMOD_RES(1)..(1)6-aminohexanoyl-arginineMOD_RES(3)..(3)3-((2-naphth-
yl) alanineMOD_RES(8)..(8)D-lysineMOD_RES(12)..(12)citrulline 48Xaa
Arg Xaa Cys Tyr Arg Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
104914PRTArtificialsynthetic peptideMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-lysineMOD_RES(12)..(12)c-
itrullineMOD_RES(14)..(14)C' amidated 49Arg Arg Xaa Cys Tyr Xaa Arg
Xaa Pro Tyr Arg Xaa Cys Arg1 5 105014PRTArtificialsynthetic
peptideMOD_RES(1)..(1)N' ACETYLATEDMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-lysineMOD_RES(12)..(12)c-
itrullineMOD_RES(14)..(14)C' amidated 50Arg Arg Xaa Cys Tyr Xaa Arg
Xaa Pro Tyr Arg Xaa Cys Arg1 5 105114PRTArtificialSYNTHETIC
PEPTIDEMOD_RES(1)..(1)N' ACETYLATEDMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-lysineMOD_RES(12)..(12)c-
itrullineMOD_RES(14)..(14)C' AMIDATED 51Arg Arg Xaa Cys Tyr Xaa Lys
Xaa Pro Tyr Arg Xaa Cys Arg1 5 105214PRTArtificialsynthetic
peptideMOD_RES(1)..(1)N' ACETYLATEDMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(8)..(8)D-citrullineMOD_RES(12)..(12)citrullineMOD_RES(14).-
.(14)C' AMIDATED 52Arg Arg Xaa Cys Tyr Arg Lys Xaa Pro Tyr Arg Xaa
Cys Arg1 5 105314PRTArtificialsynthetic
peptideMOD_RES(1)..(1)4-fluorobenzoyl-arginineMOD_RES(3)..(3)3-((2-naphth-
yl) alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-glutamic
acidMOD_RES(12)..(12)citrullineMISC_FEATURE(14)..(14)derivatization
by a NH-methyl group 53Xaa Arg Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg
Xaa Cys Arg1 5 105414PRTArtificialsynthetic
peptideMOD_RES(1)..(1)4-fluorobenzoyl-arginineMOD_RES(3)..(3)3-((2-naphth-
yl) alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-glutamic
acidMOD_RES(12)..(12)citrullineMISC_FEATURE(14)..(14)derivatization
by a NH-ethyl group 54Xaa Arg Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg
Xaa Cys Arg1 5 105514PRTArtificialsynthetic
peptideMOD_RES(1)..(1)4-fluorobenzoyl-arginineMOD_RES(3)..(3)3-((2-naphth-
yl) alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-glutamic
acidMOD_RES(12)..(12)citrullineMISC_FEATURE(14)..(14)derivatization
by NH-isopropyl 55Xaa Arg Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa
Cys Arg1 5 105614PRTArtificialsynthetic
peptideMOD_RES(1)..(1)4-fluorobenzoyl-arginineMOD_RES(3)..(3)3-((2-naphth-
yl) alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-glutamic
acidMOD_RES(12)..(12)citrullineMISC_FEATURE(14)..(14)derivatization
with a tyramine residue 56Xaa Arg Xaa Cys Tyr Xaa Lys Xaa Pro Tyr
Arg Xaa Cys Arg1 5 105714PRTArtificialsynthetic
peptideMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(8)..(8)D-lysineMOD_RES(12)..(12)citrulline 57Ala Arg
Xaa Cys Tyr Arg Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
105814PRTArtificialsynthetic peptideMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(8)..(8)D-lysineMOD_RES(12)..(12)citrulline 58Arg Arg
Xaa Cys Tyr Ala Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
105914PRTArtificialsynthetic peptideMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(8)..(8)D-lysineMOD_RES(12)..(12)citrulline 59Arg Arg
Xaa Cys Tyr Arg Ala Xaa Pro Tyr Arg Xaa Cys Arg1 5
106014PRTArtificialsynthetic peptideMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(8)..(8)D-alanineMOD_RES(12)..(12)citrulline 60Arg
Arg Xaa Cys Tyr Arg Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
106114PRTArtificialsynthetic peptideMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(8)..(8)D-lysineMOD_RES(12)..(12)citrulline 61Arg Arg
Xaa Cys Tyr Arg Lys Xaa Ala Tyr Arg Xaa Cys Arg1 5
106214PRTArtificialsynthetic peptideMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(8)..(8)D-lysineMOD_RES(12)..(12)citrulline 62Arg Arg
Xaa Cys Tyr Arg Lys Xaa Pro Ala Arg Xaa Cys Arg1 5
106314PRTArtificialsynthetic peptideMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(8)..(8)D-lysineMOD_RES(12)..(12)citrulline 63Arg Arg
Xaa Cys Tyr Arg Lys Xaa Pro Tyr Ala Xaa Cys Arg1 5
106414PRTArtificialsynthetic
peptideMOD_RES(1)..(1)citrullineMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(8)..(8)D-lysineMOD_RES(12)..(12)citrulline 64Xaa Arg
Xaa Cys Tyr Arg Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
106514PRTArtificialsynthetic peptideMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-lysineMOD_RES(12)..(12)c-
itrulline 65Arg Arg Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys
Arg1 5 106614PRTArtificialsynthetic
peptideMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-lysineMOD_RES(12)..(12)c-
itrullineMOD_RES(14)..(14)C' AMIDATED 66Arg Arg Xaa Cys Tyr Xaa Lys
Xaa Pro Tyr Arg Xaa Cys Arg1 5 106714PRTArtificialsynthetic
peptideMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(7)..(7)citrullineMOD_RES(8)..(8)D-lysineMOD_RES(12)..(12)c-
itrulline 67Arg Arg Xaa Cys Tyr Arg Xaa Xaa Pro Tyr Arg Xaa Cys
Arg1 5 106814PRTArtificialsynthetic
peptideMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-citrullineMOD_RES(12)..(-
12)citrullineMOD_RES(14)..(14)C' AMIDATED 68Arg Arg Xaa Cys Tyr Xaa
Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5 106914PRTArtificialsynthetic
peptideMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(8)..(8)D-lysineMOD_RES(12)..(12)citrulline 69Arg Arg
Xaa Cys Tyr Arg Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
107014PRTArtificialsynthetic peptideMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(6)..(6)citrullineMOD_RES(8)..(8)D-citrullineMOD_RES(12)..(-
12)citrulline 70Arg Arg Xaa Cys Tyr Xaa Lys Xaa Pro Tyr Arg Xaa Cys
Arg1 5 107114PRTArtificialsynthetic
peptideMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(8)..(8)D-citrullineMOD_RES(12)..(12)citrulline 71Arg
Arg Xaa Cys Tyr Arg Lys Xaa Pro Tyr Arg Xaa Cys Arg1 5
107214PRTArtificialsynthetic
peptideMOD_RES(1)..(1)citrullineMOD_RES(3)..(3)3-((2-naphthyl)
alanineMOD_RES(8)..(8)D-citrullineMOD_RES(12)..(12)citrullineMOD_RES(14).-
.(14)C' Amidated 72Xaa Arg Xaa Cys Tyr Arg Lys Xaa Pro Tyr Arg Xaa
Cys Arg1 5 10
* * * * *