JP6938564B2 - VISTA antagonist and method of use - Google Patents

Description

Related application

Cross-reference to related applications [0001] This application is a partial continuation of 13 / 925,094 filed June 24, 2013, which is a US provisional application filed June 25, 2012. US provisional application number 61/927 filed on January 14, 2014, claiming priority over numbers 61 / 663,969 and US provisional application number 61 / 663,431 filed June 22, 2012. , 061's precedence is also claimed and the contents of each (including sequence listings) are incorporated herein by reference in their entirety.

The sequence listing file, having a size of 688 bytes and named "43260o1003" generated on November 6, 2014, is incorporated herein by reference in its entirety.

The immune system is tightly regulated by co-stimulating and co-inhibiting ligands and receptors. These molecules provide not only a second signal of T cell activation, but also a balanced network of positive and negative signals that maximize the immune response to infection while limiting self-immunity.

Induction of an immune response requires T cell proliferation, differentiation, contraction and establishment of T cell memory. T cells encounter antigen-presenting cells (APCs) and exchange information on the APCs through T cell receptor (TCR) / major histocompatibility complex (MHC) interactions. Once the TCR / MHC interaction is established, co-stimulation via another set of receptor-ligand contacts between T cells and APCs, namely CD154 / CD40 and CD28 / B7.1-B7.2. Needed. The synergy between these contacts can lead to a productive immune response that can eliminate pathogens and tumors and induce autoimmunity.

Another level of regulation, regulatory T cells ( _Tregs ), has been identified. This particular subset of T cells is produced in the thymus and delivered into the periphery, allowing for sustained and inducible control of T cell responses. Sakaguchi (2000) Cell 101 (5): 455-8; Shevac (2000) Annu. Rev.
Immunol. 18: 423-49; Bluestone and Abbas
(2003) Nat. Rev. Immunol. 3 (3): 2 53-7. T _reg is represented by CD4 + CD25 + phenotype, a high level of cytotoxic T lymphocyte-associated antigen -4 (CTLA-4), OX -40,4-1BB and glucocorticoid-induced TNF receptor-related protein (GITR) Is also expressed. McHugh, et al. (2002) Immunity 16 (2): 311-23; Shimazu, et al. (2002) Nat. Immun. 3 (2): 135-42.5 Day 25 Thymectomy or _{elimination of Treg} cells by antibody depletion using anti-CD25 results in autoimmune pathology and T cell response to foreign and self-antigens Induces exacerbations (including enhanced anti-tumor response). Sakaguchi, et al. (1985) J.M. Exp. Med. 161 (1): 72-87; Sakaguchi, et al. (1995) J.M. Immunol. 155 (3): 1151-64; Jones, et al. (2002) Cancer Immuno. 2: 1. _{In addition, Tregs} are also involved in the induction and maintenance of _{transplantation tolerance, as depletion of Tregs} by anti-CD25 monoclonal antibodies results in elimination of transplantation tolerance and acute transplant rejection. Jarvinen, et al. (2003) Transpl
application 76: 1375-9. Ligation of GITR on the surface of agonistic monoclonal antibodies and Treg resulted in a rapid termination of T _reg activity, because it provides the autoimmune pathologies and graft tolerance removed, among the receptors expressed by T _reg, GITR Seems to be an important component.

Co-stimulatory and co-inhibiting ligands and receptors are not only the "second signal" of T cell activation, but also positive and negative signals that maximize the immune response to infection while limiting self-immunity. It also provides a well-balanced network. The most well-characterized co-stimulatory ligands are B7.1 and B7.2, which are expressed by professional APCs whose receptors are CD28 and CTLA-4. Greenwald, et al. (2005) Annu Rev Immunol 23, 515-548; Sharpe and Freeman (2002) Nat Rev
Immunol 2, 116-126. CD28 is expressed by naive T cells and activated T cells and is important for optimal T cell activation. In contrast, CTLA-4 is induced during T cell activation and inhibits T cell activation by binding to B7.1 / B7.2, thus impairing CD28-mediated co-stimulation. CTLA-4 also transmits negative signaling through its cytoplasmic ITIM motif. Teft, et al. (2006). Annu Rev Immunol 24, 65-97. B7.1 / B7.2 KO mice have an abnormal adaptive immune response (Borriello, et al. (1997) Immunity 6, 303-313; Freeman,
et al. (1993) Science 262, 907-909), but CTLA-4 KO mice are unable to adequately control inflammation and develop systemic autoimmune disease. Chambers, et al. (1997) Immunity 7, 885-895; Tivol, et al. (1995) Immunity 3,
541-547; Waterhouse, et al. (1995) Science 270, 985-988. The B7 family ligands have expanded to include co-stimulating B7-H2 (ICOS ligand) and B7-H3, as well as co-inhibiting B7-H1 (PD-L1), B7-DC (PD-L2). , B7-H4 (B7S1 or B7x) and B7-H6. Brandt, et al. (2009) J Exp Med 206, 1495-1503; Greenwald, et al. (2005) Annu Rev Immunol 23: 515-548.

Inducible co-stimulation (ICOS) molecules are expressed on activated T cells and bind to B7-H2. Yoshinaga, et al. (1999) See Nature 402, 827-832. ICOS is important for T cell activation, differentiation and function, as well as for T helper cell-induced B cell activation, Ig class switching and germinal center (GC) formation. Dong, et al. (2001) Nature 409, 97-101; Tafuri, et al. (2001) Nature 409, 105-109; Yoshinaga, et al. (1999) Nature 402, 827-832. Programmed death 1 (PD-1), on the other hand, negatively regulates T cell response. PD-1 KO mice develop lupus-like autoimmune disease or autoimmune dilated cardiomyopathy depending on their genetic background. Nishimura, et al. (1999) Immunity 11, 141-151. Nishimura, et al. (2001) Science 291: 319-322. Autoimmunity is most likely resulting from a lack of signaling by both ligands PD-L1 and PD-L2. Recently, CD80 has been identified as a second receptor for PD-L1 that transmits an inhibitory signal into T cells. Butte, et al. (2007) Immunity 27: 111-122. Receptors for B7-H3 and B7-H4 remain unknown.

The most well-characterized co-stimulatory ligands are B7.1 and B7.2, which belong to the Ig superfamily and are expressed on professional APCs whose receptors are CD28 and CTLA-. 4 (Greenwald, et al. (2005) Annu. Rev. Immunol. 23: 515-548). CD28 is expressed by naive T cells and activated T cells and is important for optimal T cell activation. In contrast, CTLA-4 is induced during T cell activation and inhibits T cell activation by binding to B7.1 / B7.2, impairing CD28-mediated co-stimulation. B7.1 and B7.2 KO mice have an abnormal adaptive immune response (Borriello, et al. (1997) Immunity 6: 303-313), whereas CTLA-4 knockout mice adequately control inflammation. Unable to develop systemic autoimmune disease (Tivol, et al. (1995) Immunity 3: 541-547; Waterhouse, et al. (1995) Science 270: 985-988; Chambers, et al. ( 1997) Immunity 7: 885-895).

B7 family ligands have expanded to include co-stimulatory B7-H2 (inducible T cell co-stimulatory factor (ICOS) ligand) and B7-H3, as well as co-inhibitory B7-H1 (PD). -L1), B7-DC (PD-L2), B7-H4 (B7S1 or B7x) and B7-H6 are included (Greenwald, et al. (2005) above; Brandt, et al. (2009) J. Exp. Med. 206: 1495-1503). Therefore, additional CD28 family receptors have been identified. ICOS is expressed on activated T cells and binds to B7-H2 (Yoshinaga, et al. (1999) Nature 402: 827-832). ICOS is a positive co-regulator, which is important for T cell activation, differentiation and function (Yoshinaga, et al. (1999) above; Dong, et al.
(2001) Nature 409: 97-101). In contrast, PD-1 (programmed death 1) negatively regulates T cell response. PD-1 knockout mice develop lupus-like autoimmune disease or autoimmune dilated cardiomyopathy (Nishimura, et al. (1999) Immunity 11: 141-151; Nishimura, et al. (2001) Science 291). : 319-322). This autoimmunity is most likely resulting from a lack of signaling by both ligands PD-L1 and PD-L2. Recently, CD80 has been identified as a second receptor for PD-L1 that transmits an inhibitory signal into T cells (Butte, et al. (2007) Immunoty 27: 111-122).

These two inhibitory B7 family ligands (PD-L1 and PD-L2) have distinct expression patterns. PD-L2 is inducibly expressed on DCs and macrophages, whereas PD-L1 is widely expressed on both hematopoietic and non-hematopoietic cell types (Okazaki & Honjo (2006) Immunology 27: 195-201; Keira, et al. (2008) Annu. Rev. Immunol. 26: 677-704). Consistent with the immunosuppressive role of PD-1 receptors, ^{from studies using PD-L1 − / −} and PD-L2 ^{− / −} mice, both ligands overlap in inhibiting T cell proliferation and cytokine production. (Keira, et al. (2006) J. Exp. Med.
203: 883-895). PD-L1 deficiency promotes disease progression in both non-obese diabetes models of autoimmune diabetes and mouse models of multiple sclerosis (experimental autoimmune encephalomyelitis; Anasari, et al. (2003)). J. Exp. Med. 198: 63-69; Salama, et al. (2003) J. Exp. Med. 198: 71-78; Ratchman, et al. (2004) Proc. Natl. Acad. Sci. USA. 101: 10
691-10696 (EAE)). PD-L1 ^{− / −} T cells increase levels of pro-inflammatory cytokines in both disease models. In addition, bone marrow chimera experiments demonstrated that tissue expression of PD-L1 (ie, in the pancreas) uniquely contributes to the ability to control inflammation locally (Keira, et al. (2006), supra). Keira, et al. (2007) J. Immunol. 179: 5064-5070; Gravie, et al. (2007) Organization. 116: 2062-2071). PD-L1 is also highly expressed on the placental syncytiotrophobic cell layer, which decisively regulates the maternal immune response to allogeneic fetal (Gulleria, et al. (2005) J. Exp. Med. 202: 231-237).

Consistent with its immunosuppressive role, PD-L1 strongly suppresses antitumor immune responses and helps tumors evade immune surveillance. PD-L1 ^{can induce apoptosis of infiltrating cytotoxic CD8 +} T cells, which express high levels of PD-1 (Dong, et al. (2002) Nature 409: 97-101; Dong & Chen (2003) J. Mol. Med. 81: 281-287). Studies have shown that blocking the PD-L1-PD-1 signaling pathway in combination with other immunotherapies prevents tumor progression by promoting antitumor cytotoxic T lymphocyte activity and cytokine production. (Iwai, et al. (2002) Proc. Natl. Acad. Cy. USA 99: 12293-12297;
Blank, et al. (2004) Cancer Res. 64: 1140-1145; Blank, et al. (2005) Cancer Immunol. Immunother. 54: 307-314; Geng, et al. (2006) Int. J. Cancer. 118: 2657-2664). In addition, PD-L1 expression on dendritic cells promotes the induction of adaptive Foxp3 ⁺ CD4 ⁺ regulatory T cells _{(aT reg} cells), PD-L1 is a powerful induction of _{aT reg} cells within the tumor microenvironment It has been shown to be an agent (Wang, et al. (2008) Proc. Natl. Acad. Sci. USA. 105: 9331-9336).

An additional immunomodulatory ligand called V-domain Ig suppressor (VISTA) for T cell activation or PD-L3 has recently been identified as a molecule that is upregulated in the T cell transcription profiling screen ( Wang, et al.
(2011) J.M. Exp. Med. 208: 577; WO 2011/120013). The extracellular Ig domain of VISTA has been shown to share significant sequence homology with the B7 family ligands PD-L1 and PD-L2, but with unique structural features that distinguish it from B7 family members. There is.

VISTA is expressed primarily on hematopoietic cells, and VISTA expression is highly regulated on myeloid antigen presenting cells (APCs) and T cells. Expression of VISTA on antigen-presenting cells (APCs) suppresses T cell responses by engaging its counterreceptors on T cells during homologous interactions between T cells and APCs. VISTA blockade promotes T cell-mediated immunity in autoimmune disease models and is unique and non-redundant in the regulation of autoimmunity when compared to other inhibitory B7 family ligands (such as PD-L1 and PD-L2). Suggest a role. In addition, VISTA blockade promotes anti-tumor immunity and tumor growth suppression in preclinical mouse tumor models (WO2011 / 120013). In this regard, therapeutic interventions in the VISTA inhibitory pathway represent a novel approach to regulate T cell-mediated immunity for the treatment of diseases (such as viral infections and cancer).

According to the present invention, a peptide having the same amino acid sequence as SEQ ID NO: 1 (Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-Hi)
s), or an isolated VISTA antagonist comprising a multimer, conjugate, analog, derivative or imitation thereof.

In one embodiment, the isolated VISTA antagonist is a peptide identical to the amino acid sequence of SEQ ID NO: 1 (Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His). ), Or a peptide having an amino acid sequence different from SEQ ID NO: 1 with at most two amino acid residues, or a multimer, conjugate, analog, derivative or mimic thereof thereof. In another embodiment, the isolated VISTA antagonist comprises a peptide having an amino acid sequence different from SEQ ID NO: 1 at most one amino acid residue, or a multimer, conjugate, analog, derivative or imitation thereof. In yet another embodiment, the isolated VISTA antagonist is a peptide identical to the amino acid sequence of SEQ ID NO: 1 (Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His). , Or its multimer or conjugate. In certain embodiments, the isolated VISTA antagonist consists of the amino acid sequence of SEQ ID NO: 1 (Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His).

[0016] In one embodiment, positions 4 and 11 of SEQ ID NO: 1 (Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His), or those in a variant of the peptide. Cysteine residues at the corresponding positions of form a disulfide bridge.

In another embodiment, the isolated VISTA antagonist is modified to improve binding affinity and / or stability. In certain embodiments, the isolated VISTA antagonist is modified by PEG, acetylation, XTEN, albumin and / or multimerization.

In another embodiment, the isolated VISTA antagonist is added directly or indirectly to the immunoglobulin polypeptide or fragment thereof. Immunoglobulin polypeptides can include human IgG1, IgG2, IgG3 or IgG4 constant regions or fragments thereof. Preferably, the immunoglobulin polypeptide comprises a human IgG1 constant region or a fragment thereof.

In yet another embodiment, the isolated VISTA antagonist comprises a plurality of (ie, 2, 3, 4, 5, 6, 7 or more) copies of the peptide.

In a further embodiment, the isolated VISTA antagonist comprises another moiety that targets the peptide to the target site. The targeting moiety can be selected from an antibody or ligand that binds to the antigen, a receptor expressed by the target cell, or an infectious pathogen.

[0021] In a further embodiment, the isolated VISTA antagonist is added to another moiety or another copy of the antagonist via a linker. The linker can be a peptide that allows the antagonist to interact with VISTA expressed on the surface of the target cell.

In a further embodiment, the isolated VISTA antagonist is added directly or indirectly to a detectable label or therapeutic agent.
In some of the embodiments, the isolated VISTA antagonist binds to the extracellular domain of VISTA, disrupts its interaction with the VISTA receptor, and / or produces VISTA-mediated T cell suppression. Reduce or inhibit.

In one embodiment, the isolated VISTA antagonist induces antitumor and / or antiviral activity.
In addition, the present invention provides compositions suitable for therapeutic, prophylactic or diagnostic use, comprising therapeutically, prophylactically or diagnostically effective amounts of isolated VISTA antagonists. Plan.

In one embodiment, the composition further comprises a pharmaceutically acceptable carrier, diluent, solubilizer, preservative or mixture thereof.
In another embodiment, the composition further comprises other therapeutic agents (eg, anti-cancer agents, antiviral agents, cytokines or immunoagonists). In certain embodiments, the other therapeutic agent is selected from CTLA-4-Ig, anti-PD-1, PD-L1 fusion protein or PD-L2 fusion protein, and EGFR antagonists.

In one embodiment, the composition is suitable for subcutaneous or intravenous administration.
Further, the present invention comprises an isolated nucleic acid sequence encoding a VISTA antagonist peptide, an analog thereof, a derivative or a mimic thereof disclosed herein, a vector containing the isolated nucleic acid sequence. And host cells containing the isolated nucleic acid sequences or vectors thereof are further contemplated.

In one embodiment, the host cell is a mammalian cell, a bacterial cell, a fungal cell, a yeast cell, an avian cell or an insect cell.
The present invention expresses a peptide, analog, derivative or imitation thereof, which comprises culturing a host cell under conditions that provide expression of the VISTA antagonist peptide, analog, derivative or imitation thereof. The method is further planned.

Further, the present invention contemplates various uses of isolated VISTA antagonists.
[0033] In one embodiment, the invention administers an effective amount of an isolated VISTA antagonist disclosed herein or a composition comprising the isolated VISTA antagonist to a subject in need. Provided are methods of blocking, inhibiting or neutralizing VISTA-mediated T cell suppression, including.

In another embodiment, the invention administers an effective amount of an isolated VISTA antagonist disclosed herein or a composition comprising the isolated VISTA antagonist to a subject in need. Provided is a method of stimulating an immune response in a subject, including the above. Such methods can be used for the treatment of cancer in a subject.

Subjects may have cancer and / or infections selected from the group consisting of bacterial, viral, parasitic and fungal infections.
[0036] Bacterial infections, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Chlamydia, Clostridium, Corynebacterium, Enterobacter, Enterococcus, Erwinia, Escherichia, Francisella, Haemophilus, Heliobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pasteurella , Pelobacter, P
At least one bacterium selected from the group consisting of seudomonas, Rickettsia, Salmonella, Serratia, Shigella, Staphylococcus, Streptococcus, Treponema, Vibrio, Yersinia and Xanthomonas, which can be caused by at least one bacterium.

[0037] viral infection, comprising Adenoviridae, Papillomaviridae, Polyomaviridae, Herpesviridae, Poxviridae, Hepadnaviridae, Parvoviridae, Astroviridae, Caliciviridae, Picornaviridae, Coronoviridae, Flaviviridae, Retroviridae, Togaviridae, Arenaviridae, Bunyaviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, from Rhabdoviridae and Reoviridae It can be caused by at least one virus selected from the group. More specifically, the viruses are adenovirus, simple herpes type I, simple herpes type 2, varicella herpes virus, Epstein-bar virus, cytomegalovirus, herpesvirus type 8, papillomavirus, BK virus, JC virus, Natural pox, hepatitis B, bocavirus, parvovirus B19, astrovirus, nowalk virus, coxsackie virus, hepatitis A, poliovirus, rhinovirus, severe acute respiratory syndrome virus, hepatitis C, yellow fever, dengue virus, West Nile virus, wind rash, hepatitis E, human immunodeficiency virus (HIV), influenza, guanaritovirus, funin virus, lassa fever virus, macpovirus, savia virus, Crimea-Congo hemorrhagic fever virus, Ebola virus, Marburg virus , Hashi virus, mumps virus, parainfluenza, respiratory follicles virus, human metapneumonia virus, Hendra virus, nipavirus, mad dog disease, hepatitis D, rotavirus, orbivirus, cortivirus or bannavirus.

Fungal infections include ringworm, candidiasis, cryptococcus, tinea capitis, blastomycetes, aspergillosis, ringworm, paracoccidiosis, sporotricum disease, tinea capitis, tinea capitis, robofungal Diseases, fungal tumors, tinea pedis, tinea pedis, tinea capitis, tinea capitis, tinea capitis, tinea, tinea, tinea, tinea pedis, otofungal disease, pheofihofungal disease or linospoli It can be selected from the group consisting of ringworm.

[0039] parasite infection, Entamoeba hystolytica, Giardia lamblia, Cryptosporidium muris, Trypanosomatida gambiense, Trypanosomatida rhodesiense, Trypanosomatida crusi, Leishmania mexicana, Leishmania braziliensis, Leishmania tropica, Leishmania donovani, Toxoplasma gondii, Plasmodium vivax, Plasmodium
ovale, Plasmodium malariae, Plasmodium falciparum, Trichomonas vaginalis, Histomonas meleagridis; Secementea; Trichuris trichiura, Ascaris lumbricoides, Enterobius vermicularis, Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Wuchereria bancrofti, Dracunculus medinensis; schistosomiasis, liver fluke, intestinal fluke, Pulmonary flukes; Schistosoma mansoni, Schistosoma haematobium, Schistosoma japonicum, Fasciola hepatica, Fasciola gigantic
It can be caused by at least one parasite selected from the group consisting of a, Heterophies heterophyes and Paragonimus westermani.

In another embodiment, the invention administers an effective amount of an isolated VISTA antagonist disclosed herein or a composition comprising the isolated VISTA antagonist to a subject in need. Provided are methods of promoting anti-cancer immunity or anti-tumor immunity, including the above.

In another embodiment, the invention administers an effective amount of an isolated VISTA antagonist disclosed herein or a composition comprising the isolated VISTA antagonist to a subject in need. Provided are methods for treating or preventing cancer, including the inhibition of tumor infiltration and / or cancer metastasis.

Cancers include cancers, lymphomas, blastomas, sarcomas, leukemias, lymphoid malignancies, melanomas, squamous cell carcinomas, lung cancers (small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma). Included), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder Cancer, liver cancer, breast cancer, colon cancer, colonic rectal cancer, endometrial cancer or uterine cancer, salivary adenocarcinoma, kidney cancer (kidney cancer) or kidney cancer (renal cancer), liver cancer, prostate cancer, genital cancer, thyroid Cancer, liver cancer, head and neck cancer, B-cell lymphoma (low grade / follicular non-hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; medium grade / follicular NHL; medium grade diffuse NHL; high Malignant immunoblastic NHL; High-grade lymphoblastic NHL; High-grade small non-cutting nuclear cell NHL; Giant lesion NHL; Mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrem macroglobulinemia Included); Chronic lymphocytic leukemia (CLL); Acute lymphoblastic leukemia (ALL); Hairy cell leukemia; Chronic myeloid leukemia; Post-transplant lymphoproliferative disorder (PTLD), for mother's plaque It can be selected from the group consisting of abnormal angiogenesis, edema (such as those associated with brain tumors), and Maegus syndrome.

[0043] In yet another embodiment, the invention administers an effective amount of an isolated VISTA antagonist disclosed herein or a composition comprising the isolated VISTA antagonist to a subject in need. Provide methods for treating or preventing viral infections, including.

[0044] These methods can further include administration of another therapeutic agent, the peptide and treatment method can be administered simultaneously or at different times, separated or in combination.

In one embodiment, the other therapeutic agent is an anti-cancer agent, antiviral agent or other anti-infective agent, cytokine or immunoagonist. Preferably, the other therapeutic agent is selected from CTLA-4-Ig, anti-PD-1, PD-L1 fusion protein or PD-L2 fusion protein, and EGFR antagonists.

Finally, the present invention is a method of mapping the active site of VISTA, wherein (a) the isolated VISTA fusion protein is the same peptide (Ser-Ser-Ala) as the amino acid sequence of SEQ ID NO: 1. -Peptides containing-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His) or having an amino acid sequence different from SEQ ID NO: 1 at most two amino acid residues or a multimer, conjugate, or similar thereof. The method also comprises incubating with an isolated VISTA antagonist, including a body, derivative or mimic; (b) determining the binding site of the isolated VISTA antagonist.

In one embodiment, the active site of VISTA binds to the VISTA receptor and mediates immunosuppression. In another embodiment, step (b) includes domain deletion, domain swapping, amino acid mutagenesis, foot printing, NMR, X-ray crystallography, or homology modeling.

It is shown that the VISTA antagonist peptide (SEQ ID NO: 1) significantly promotes T cell proliferation as compared to the anti-VISTA antibody (aVISTA) and the anti-PD-L1 antibody (aPDL1). Myelogenous CD11B + APC was incubated with OT2 CD4 + T cells, antigens, and monoclonal antibody (aVISTA or aPDL1) or AP1049. T cell proliferation was measured by tritium uptake at 72 hours. It is shown that the VISTA antagonist peptide (SEQ ID NO: 1) significantly promotes anti-tumor immunity. Meth mouths inoculated with MB49 tumors were treated with either PBS (control) or AP1049. Tumor size was measured by caliper every 2-3 days.

The following detailed description is provided so that the invention described herein can be fully understood. Various embodiments of the invention are described in detail and can be further illustrated by the provided examples.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. In the present invention or tests of the present invention, methods and materials similar or equivalent to those described herein can be used, but suitable methods and materials are described herein. The materials, methods, and examples are merely exemplary and descriptive and are not intended to be limiting.

Definitions [0052] As used herein and throughout the claims, the meanings of "a,""an," and "the" include multiple references unless the context explicitly indicates. included.

[0053] As used herein, "antagonist" refers to a compound (preferably a peptide) that opposes the physiological effects of another compound. For example, at the receptor level, an antagonist is a compound that opposes a receptor-related response normally induced by other agents that bind to the receptor and activate the biological activity of the receptor. Similarly, at the ligand level, an antagonist is a compound that opposes a ligand-related response that is normally induced when the ligand binds to its target receptor and / or accessory factor. In a specific embodiment, the VISTA antagonist binds to VISTA and counters one or more of its biological activities (eg, VISTA-mediated T cell suppression and / or VISTA-mediated anti-tumor immunosuppression). And thereby a compound that promotes T cell-mediated immunity and / or anti-tumor immunity (eg, peptides or analogs, derivatives or mimics thereof).

[0054] As used herein, an "analog" is a compound whose structure is related to that of a given compound (preferably a peptide), but which differs in chemical and biological properties. (Preferably a peptide).

When used herein, an "antigen-presenting cell" is a professional antigen-presenting cell (eg, B lymphocyte, monosphere, dendritic cell, and Langerhans cell) as well as other antigen-presenting cells (eg, Langerhans cells). (Keratinized cells, endothelial cells, stellate cells, fibroblasts and oligodendrocytes) are broadly referred to.

[0056] As used herein, "amino acids" broadly refer to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Point to. Naturally occurring amino acids are those amino acids that are later modified (eg, hydroxyproline, γ-carboxyglutamic acid and O-phosphoserine) in addition to those encoded by the genetic code. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids (ie hydrogen, carboxyl groups, carbons attached to amino groups) and R groups (eg homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium). Point to. Analogs may have a modified R group (eg, norleucine) or a modified peptide backbone, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that differ from the general chemical structure of amino acids but have a structure that functions in a manner similar to naturally occurring amino acids.

[0057] "Allergic disease" as used herein broadly refers to a disease in which an allergic reaction is involved. More specifically, in "allergic disease", an allergen has been identified and there is a strong correlation between exposure to the allergen and the onset of pathological changes, the pathological changes being the immune mechanism. Is defined as a disease that has been proven to have. As used herein, the immune mechanism means that leukocytes exhibit an immune response to allergen stimulation.

[0058] As used herein, "autoimmune disease" broadly refers to a disease or disorder that arises from or co-isolates of an individual's own tissue or co-isolates and is directed to them, or a finding or condition resulting from it. Point to.

[0059] As used herein, "cancer" is any neoplastic disease (invasive or metastatic) characterized by abnormal and uncontrolled cell division (eg, unregulated cell proliferation) that causes malignant growth or tumors. Broadly refers to (regardless of gender).

[0060] A "conservatively modified variant", as used herein, applies to both amino acid and nucleic acid sequences and encodes the same or essentially the same amino acid sequence for a particular nucleic acid sequence. Refers broadly to conservatively modified variants that refer to those nucleic acids, or, if the nucleic acids do not encode an amino acid sequence, to essentially the same sequence. Due to the degeneracy of the genetic code, multiple functionally identical nucleic acids encode any given protein. A "silent variation" is a conservatively modified nucleic acid variant. All nucleic acid sequences encoding a polypeptide herein also describe all silent variations of the nucleic acid. One of ordinary skill in the art will modify each codon in the nucleic acid to result in a functionally identical molecule, except for AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan. You will recognize that you will get.

[0061] "Co-stimulatory receptor" as used herein broadly refers to a receptor that transmits a co-stimulatory signal to immune cells (eg, CD28, ICOS).
[0062] As used herein, "cytoplasmic domain" broadly refers to the portion of a protein that extends into the cytoplasm of a cell.

[0063] A "derivative" or "peptide derivative", as used herein, contains modifications of one or more amino acid residues or linker groups or other covalently linked groups. Non-limiting examples of derivatives are produced by reaction with an N-acyl derivative of an amino-terminated or another free amino group, an ester of a carboxyl-terminated or another free carboxyl group or a hydroxy group, ammonia or a suitable amine. Amido of carboxyl-terminated or another free carboxyl group, sugar chain-added derivative, hydroxylated derivative, nucleotidylated derivative, ADP ribosylated derivative, PEGylated derivative, phosphorylated derivative, Derivatives conjugated to lipophilic moieties and derivatives conjugated to antibodies or other biological ligands are included. Chemical derivatives also include those obtained, for example, by modifying the peptide bond -CO-NH- by reduction to -CH _{2-NH- or alkylation to -CO-N (alkyl)-.} Preferred derivatizations include, but are not limited to, C-terminal amidation and N-terminal acetylation, which either remove the C-terminal negative charge or the N-terminal positive charge, respectively. C-terminal or N-terminal blocking (such as by C-terminal amidation or N-terminal acetylation) can improve stability to proteolysis due to reduced susceptibility to exoproteolysis. Peptide derivatives with a C-terminal amide are represented by _{"NH 2" at the C-terminus.}

[0064] "Diagnosis" as used herein broadly refers to identifying the presence or nature of a pathological condition. Diagnostic methods differ in their sensitivity and specificity. The "susceptibility" of a diagnostic assay is the percentage of diseased individuals who test positive (percentage of "true positives"). Disease individuals not detected by the assay are "false negatives". Subjects who are disease-free and test negative in the assay are referred to as "true negatives." The "specificity" of a diagnostic assay is the 1-false positive rate, and the "false positive" rate is defined as the percentage of those who are disease-free and test positive. Certain diagnostic methods may not provide a definitive diagnosis of the condition, but it is sufficient if this method provides a positive indicator to assist in the diagnosis.

[0065] As used herein, "diagnosis" broadly refers to the classification of a disease or sign, determination of the severity of a disease, monitoring of disease progression, prediction of disease outcome and / or prediction of recovery. The term "detection" can optionally include any of the aforementioned. The diagnosis of the disease described in the present invention can be influenced in some embodiments by determining the level of the polynucleotide or polypeptide of the invention in a biological sample obtained from a subject, and the determined level is , Predisposition to disease or may correlate with the presence or absence of disease. It should be pointed out that the "biological sample obtained from the subject" can optionally include a sample that has not been physically removed from the subject.

An "effective amount", as used herein, is an amount of a compound, antibody, sufficient to achieve such treatment for a disease when administered to a patient for the treatment of the disease. Broadly refers to an antigen or cell. The effective amount can be an effective amount for prophylaxis and / or an effective amount for prevention. Effective doses are effective amounts for reducing or preventing the appearance of signs / symptoms, reducing the severity of the appearance of signs / symptoms, eliminating the appearance of signs / symptoms, slowing the development of the appearance of signs / symptoms, signs / symptoms. It can be an effective amount to prevent the development of the appearance of and / or the prevention of the appearance of signs / symptoms. The "effective amount" may vary depending on the disease and its severity, as well as the age, weight, history, susceptibility and pre-existing condition of the patient being treated. The term "effective amount" is synonymous with "therapeutically effective amount" for the purposes of the present invention.

[0067] As used herein, "extracellular domain" broadly refers to a portion of a protein that extends from the surface of a cell.
[0068] When used herein, an "expression vector" is used in any cell, including prokaryotic cells, yeast cells, fungal cells, plant cells, insect cells or mammalian cells. Broadly refers to any recombinant expression system for the purpose of constitutively or inducibly expressing a nucleic acid sequence in vitro or in vivo. The term includes linear or cyclic expression systems. The term includes expression systems that remain episomal or integrate into the host cell genome. The expression system may or may not have the ability to self-replicate, i.e. it can only drive transient expression in the cell. The term includes a recombinant expression cassette containing only the smallest elements required for transcription of a recombinant nucleic acid.

[0069] As used herein, "homogeneity" broadly refers to the degree of similarity between a nucleic acid sequence and a reference nucleic acid sequence or between a polypeptide sequence and a reference polypeptide sequence. Homology can be partial or complete. Full homology indicates that the nucleic acid or amino acid sequences are identical. A partially homologous nucleic acid or amino acid sequence is not identical to the reference nucleic acid or amino acid sequence. The degree of homology can be determined by sequence comparison. The term "sequence identity" can be used interchangeably with "homology".

[0070] "Host cell" as used herein broadly refers to a cell into which a nucleic acid molecule of the invention (such as a recombinant expression vector of the invention) has been introduced. Host cells are prokaryotic cells (eg E. coli), or eukaryotic cells (yeast cells, insect cells (eg SF9), amphibian cells or mammalian cells (CHO, HeLa, HEK-293, etc.), eg cultured cells, explants. Pieces and cells in vivo, etc.). The terms "host cell" and "recombinant host cell" are used interchangeably herein. It should be understood that such term refers not only to a particular cell of interest but also to progeny or potential progeny of such cell. Descendants are not actually identical to the parent cell, as certain modifications can occur in the next generation due to either mutations or environmental influences, but when used herein, the term is still used. Included within range.

“Immune response” as used herein broadly refers to a T cell-mediated and / or B cell-mediated immune response that is affected by modification of T cell co-stimulation. Exemplary immune responses include activation of B cell responses (eg, antibody production), T cell responses (eg, cytokine production and cell-mediated cell injury), and cytokine-responsive cells (eg, macrophages). As used herein, the term "downmodulation" with respect to an immune response includes a decrease in any one or more immune responses, while the term "upmodulation" with respect to an immune response is optional. Includes an increase in one or more immune responses. It will be appreciated that up-modulation of one type of immune response can result in corresponding down-modulation of another type of immune response. For example, up-modulation of the production of certain cytokines (eg IL-10) can result in down-modulation of the cell-mediated immune response.

[0072] As used herein, "inflammatory disease" broadly refers to chronic or acute inflammatory disease.
[0073] As used herein, "detectable label" is by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means. Broadly refers to detectable compositions.

[0074] "mimetic" or "peptide mimetic" as used herein refers to a fully or partially synthetic peptide having the activity of a given peptide. Such mimetics or peptide mimetics include one or more amino acid residues that are artificial chemical mimetics of the corresponding naturally occurring amino acids, naturally occurring amino acid polymers, and non-naturally occurring amino acid polymers. ..

Modifications of VISTA and VISTA conjugated polypeptides described herein include N-terminal modifications, C-terminal modifications, and peptide bond modifications (eg, CH _2- NH, CH _2- S, CH _2- S =. O, O = C-NH, CH _2- O, CH _2- CH ₂ , S = C-NH, CH = CH or CF = CH), backbone modification, and addition of carbohydrate residues that form eg, glycoproteins. , Residue modification by addition of chemical residues (PEG and / or XTEN, etc.), but is not limited thereto. Methods of preparing peptide mimetic compounds are well known in the art. Martin, (2010).

[0076] "Nucleic acid" or "nucleic acid sequence" as used herein broadly refers to either single-stranded or double-stranded deoxyribonucleotides or oligonucleotides of ribonucleotides. The term includes nucleic acids (ie, oligonucleotides) that contain known analogs of natural nucleotides. The term also includes nucleic acid-like structures with synthetic backbones. Unless otherwise specified, a particular nucleic acid sequence implicitly includes its conservatively modified variants (eg, degenerate codon substitutions) and complementary sequences, in addition to the clearly shown sequences. The term nucleic acid is used interchangeably with genes, cDNAs, mRNAs, oligonucleotides and polynucleotides.

[0077] "Polypeptides", "peptides" and "proteins" are used interchangeably and broadly refer to polymers of amino acid residues. The term applies to amino acid polymers in which one or more amino acid residues are analogs or imitations of the corresponding naturally occurring amino acids, in addition to the naturally occurring amino acid polymers. The term refers to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers, as well as amino acid polymers in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids. Applies. Polypeptides can be modified, for example, by the addition of carbohydrate residues to form glycoproteins. The terms "polypeptide", "peptide" and "protein" include glycoproteins in addition to non-glycoproteins.

[0078] A "prophylactically effective amount", as used herein, to achieve such prevention for a disease or recurrence when administered to a patient to prevent the disease or recurrence of the disease. Broadly refers to the amount of sufficient compound. A prophylactically effective amount can be an amount effective in preventing the appearance of signs and / or symptoms. The "prophylactically effective amount" may vary depending on the disease and its severity, as well as the age, weight, medical history, predisposition to the condition, and pre-existing condition of the patient being treated.

[0079] As used herein, "prevention" broadly refers to the course of therapy if signs and / or symptoms are absent, in remission, or previously present in a patient. .. Prevention includes preventing the disease that follows after treatment of the disease in the patient. In addition, prevention involves treating patients who may develop the disease, especially those who are susceptible to the disease (eg, members of the patient population, who have risk factors or are at risk of developing the disease). Is included.

[0080] When used herein, "recombinant" refers to the introduction or innate of a cell, nucleic acid, protein or vector of a heterologous nucleic acid or protein with respect to a product (eg, cell, or nucleic acid, protein or vector). Broadly refers to indicating that a cell has been modified by a modification of its nucleic acid or protein, or that the cell is derived from such modified cell. Thus, for example, a recombinant cell expresses a gene that is not found within the cell's native (non-recombinant) form, or otherwise overexpresses or underexpresses the native gene. Or it does not appear at all.

[0081] As used herein, "sequence identity" broadly refers to the degree of similarity between a nucleic acid sequence and a reference nucleic acid sequence or between a polypeptide sequence and a reference polypeptide sequence. Sequence identity (also synonymous with "homology") can be partial or complete. Complete sequence identity indicates that the nucleic acid or amino acid sequence is identical (ie, 100% sequence identity). A partially homologous nucleic acid or amino acid sequence is not identical to the reference nucleic acid or amino acid sequence. The degree of homology can be determined by sequence comparison, eg 60% identity, 70% identity, 80% identity, 90% identity, 95% identity, 97% identity, 98% identity or 99% identity.

[0087] A "symptom" of a disease, as used herein, broadly refers to any abnormality exhibited by the disease that is observed upon examination of the patient; as opposed to a symptom (a subjective indicator of the disease). It is an objective index of disease.

[0083] As used herein, "subject" broadly refers to any animal in need of treatment, either alleviating a disease state or preventing the onset or recurrence of a disease state. Also, as used herein, a "subject" has a risk factor, history, susceptibility, symptoms and signs, has been previously diagnosed with the disease, is at risk of the disease, or is a patient with the disease. Broadly refers to any animal that is a member of a population. The subject can be a clinical patient (such as a human) or a veterinary patient (such as a pet animal, a domestic animal, a domestic animal, an exotic animal or a zoo animal). The term "subject" can be used interchangeably with the term "patient".

The “symptom” of a disease, as used herein, broadly refers to any pathological phenomenon, or deviation from the usual in structure, function, or sensation that is experienced by a patient and indicates the disease. ..

[805] As used herein, "T cell" broadly refers to CD4 + T cell and CD8 + T cell. The term T cell includes both T helper type 1 T cells and T helper type 2 T cells.

When used herein, "therapy," "treatment," "treating," or "treatment" treats, stops, or reduces the onset of the disease or its clinical manifestations. And / or alleviating the disease that causes the disease or its clinical manifestations to recede. Therapy includes the provision of prevention, treatment, correction, reduction, alleviation, and / or alleviation of disease, signs and / or symptoms of the disease. Therapy includes relief of signs and / or symptoms in patients with signs and / or symptoms of ongoing disease (eg, inflammation, pain). Therapy also includes "prevention". The term "reduced" for therapeutic purposes broadly refers to a clinically significant reduction in signs and / or symptoms. Therapy includes treating signs and / or symptoms of relapse or relapse (eg, inflammation, pain). Therapy includes reducing existing signs and / or symptoms and eliminating existing signs and / or symptoms, in addition to eliminating the appearance of signs and / or symptoms at any time. Not limited. Therapy includes treating chronic and acute illnesses (“maintenance”). For example, treatment includes treating or preventing relapse or recurrence of signs and / or symptoms (eg, inflammation, pain).

[0087] The "transmembrane domain" as used herein broadly refers to the amino acid sequence of approximately 15 amino acid residues in length across the plasma membrane. More preferably, the transmembrane domain comprises at least about 20, 25, 30, 35, 40 or 45 amino acid residues and spans the plasma membrane. The transmembrane domain is rich in hydrophobic residues and typically has an α-helix structure. In one embodiment, at least 50%, 60%, 70%, 80%, 90%, 95% or more of the amino acids in the transmembrane domain are hydrophobic (eg, leucine, isoleucine, tyrosine or tryptophan). .. Transmembrane domains are described, for example, in Zagotta, et al. (1996) Annu. Rev. Neurosci. 19: 235-263.

[0088] When used herein, a "tumor" is the shape of a tissue neoplasm, particularly the spontaneous, autonomous and irreversible excess, more or less uninhibited growth shape of endogenous tissue (its growth). Broadly refers to at least one cell or cell mass (usually with a more or less significant loss of specific cell and tissue function). This cell or cell mass is not effectively inhibited for its proliferation by itself or by the regulatory mechanisms of the host organism (eg, melanoma or carcinoma). Tumor antigens include not only antigens that are present in or on the malignant cells themselves, but also antigens that are present on the interstitial support tissue of the tumor, which contains endothelial cells and other vascular components. Is done.

[089] As used herein, "vector" broadly refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors with a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episome mammalian vectors) integrate into the host cell's genome upon introduction into the host cell, thereby replicating with the host genome. In addition, certain vectors can direct the expression of genes to which they are operably linked. Vectors are referred to herein as "recombinant expression vectors" or simply "expression vectors". In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. As used herein, a "plasmid" and a "vector" can be used interchangeably because the plasmid is the most commonly used form of the vector. However, the present invention is intended to include other forms of expression vectors (eg, replication-deficient retroviruses, adenoviruses and adeno-associated viruses) that provide equivalent functionality, such as viral vectors. The techniques and procedures are generally described according to conventional methods well known in the art and in various general references and more specific references cited and discussed throughout this specification. Is carried out. For example, Sambook, et al. (2001) Molec. Cloning: Lab. See Manual [3rd Edition] Cold Spring Harbor Laboratory Press. Standard techniques can be used for recombinant DNA, oligonucleotide synthesis and tissue culture and transformation (eg, electroporation, lipofection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's specifications, as performed in the art or as described herein.

[0090] Utilized nomenclature related to analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry, as described herein, and their experimental procedures and techniques are well known and commonly used in the art. It is a thing. Standard techniques can be used for chemical synthesis, chemical analysis, drug preparation, formulation and delivery, and treatment of patients.

VISTA and VISTA Antagonists [0091] The present application relates to peptide antagonists capable of recognizing VISTA and suppressing its inhibitory activity. This peptide (denoted as AP1049 herein) was found via phage display and was shown to exhibit superior biological activity when compared to anti-VISTA monoclonal antibodies. Given its neutralizing activity, AP1049 is used to treat, for example, cancer and / or pathogenic (ie, bacterial, fungal, parasitic or viral) infections, and promote antitumor immune responses. And can suppress tumor growth.

[0092] Accordingly, the present invention is in addition to the VISTA antagonistic peptide, its multimers, conjugates, analogs, derivatives and mimetics, and in methods of inhibiting or suppressing the activity of VISTA using this peptide. be. As used herein, the term "peptide" refers to an amino acid polymer composed of at least two amino acids covalently linked by an amide bond. The peptides of the invention are preferably 10 to 20 residues in length, or more preferably 12 to 15 residues in length. In certain embodiments, the VISTA antagonistic peptide is a 12-20 residue peptide containing the amino acid sequence of SEQ ID NO: 1. In another embodiment of the invention, the isolated VISTA antagonist is a peptide identical to the amino acid sequence of SEQ ID NO: 1 (Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys- Includes a peptide containing (His) or having at most one amino acid residue or at most two amino acid residues having an amino acid sequence different from SEQ ID NO: 1, or a multimer, conjugate, analog, derivative or mimetic thereof. .. In yet another embodiment of the invention, the isolated VISTA antagonist is from the amino acid sequence of SEQ ID NO: 1 (Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His). Become.

[093] In certain embodiments of the invention, the cysteine residues at positions 4 and 11 of the VISTA antagonistic peptide (or their corresponding positions in the variant of the VISTA antagonist) form disulfide bridges.

According to the present invention, multimers, conjugates, analogs, derivatives and mimetics of the peptides of the invention are also provided.
Analogs are molecules that differ in chemical structure from the parent compound, eg, homologs (different in increments in the chemical structure, such as differences in one amino acid residue), and structures that differ in one or more functional groups. Or a change in ionization. Structural analogs are Remington (The)
Quantitative structure-activity relationships (QSAR) are often found by techniques such as those disclosed in Science and Practice of Pharmacology, 19th Edition (1995), Chapter 28).

[0906] The analog can be prepared by modifying the amino acid sequence of SEQ ID NO: 1. The simplest modifications include substitution of one or more amino acids for amino acids with similar physiochemical and / or structural properties. These so-called conservative substitutions are likely to have the least effect on the activity and / or structure of the resulting peptide. Examples of conservative substitutions include replacement of serine with threonine, substitution of alanine with serine or valine, substitution of aspartic acid with glutamate, substitution of tryptophan with tyrosine, substitution of isoleucine with leucine or valine, substitution of arginine with lysine, and / Or the substitution of histidine with arginine or lysine is included. Conservative substitutions generally include (a) the structure of the peptide backbone in the region of substitution (eg, as a helical conformation), (b) the charge or hydrophobicity of the molecule at the target site, or ( c) Maintain the bulk of the side chain.

[097] Amino acid substitutions are typically classified according to their side chains into one or more categories, including polar, hydrophobic, acidic, basic, and aromatic. Examples of polar amino acids include acidic amino acids and basic amino acids, in addition to those having side chain functional groups (hydroxyl, sulfhydryl, amide, etc.). Polar amino acids include, but are not limited to, asparagine, cysteine, glutamine, histidine, selenocysteine, serine, threonine, tryptophan and tyrosine. Examples of hydrophobic or non-polar amino acids include those of residues having a non-polar aliphatic side chain, such as, but not limited to, leucine, isoleucine, valine, glycine, alanine, proline, methionine and phenylalanine. Is done. Examples of basic amino acid residues include those having a basic side chain (amino group, guanidino group, etc.). Basic amino acid residues include, but are not limited to, arginine, homolysine and lysine. Examples of acidic amino acid residues include those having an acidic side chain functional group (carboxy group, etc.). Acidic amino acid residues include, but are not limited to, aspartic acid and glutamic acid. Aromatic amino acids include those having an aromatic side chain group. Examples of aromatic amino acids include, but are not limited to, biphenylalanine, histidine, 2-naphthylalanine, pentafluorophenylalanine, phenylalanine, tryptophan and tyrosine. It has been pointed out that some amino acids fall into two or more groups, for example histidine, tryptophan and tyrosine are classified as both polar and aromatic amino acids. Additional amino acids classified into each of the above groups are known to those of skill in the art in the art.

[0998] As used herein, a peptide derivative is a molecule that retains the primary amino acid of the peptide, but one or more of the N-terminal, C-terminal, and / or side chains of the amino acids therein. Has been chemically modified or derivatized. Such derivatized peptides include, for example, naturally occurring amino acid derivatives (eg, 4-hydroxyproline for proline, 5-hydroxylysine for lysine, homoserine for lysine, ornithine for lysine, and the like). included. Other derivatives or modifications include, for example, labels (such as fluorescein or tetramethylrhodamine); or one or more post-translational modifications (acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation, sulfation, etc.) Sugar chain addition or lipid addition, etc.) is included. In fact, certain chemical modifications (particularly N-terminal glycosylation) have been shown to increase the stability of peptides in human serum (Powell et al. (1993) Pharma. Res. 10: 1268-1273). .. Peptide derivatives also include those with increased membrane permeability obtained by N-myristoylation (Brand, et al. (1996) Am. J. Physiol. Cell. Physiol. 270: C1362-C1369).

[00099] In addition, the peptide derivatives of the invention may comprise a cell permeation sequence that facilitates, promotes, or increases transmembrane transport or intracellular delivery of the peptide into the cell. can. For example, a variety of proteins have been shown to facilitate the transport of proteins into cells, including the HIV-1 Tat transcription factor, the Drosophila antennapedia transcription factor, as well as the herpes simplex virus VP22 protein. (Wadia and Protein (2002) Curr. Opin. Bioprotein. 13: 52-56). In addition, arginine-rich peptide (Futaki (2002) Int. J. Pharma. 245: 1-7), polylysine peptide-containing Tat PTD (Hashida, et al. (2004) Br. J. Cancer 90 (6): 1252-8). ), Pep-1 (Deshayes, et
al. (2004) Biochemistry 43 (6): 1449-57) or the HSP70 protein or fragment thereof (WO00 / 31113) is suitable for facilitating intracellular delivery of the peptides or mimetics of the invention into cells. ..

[00100] The peptides of the invention can be derivatized by one of the modifications shown above, but the peptides of the invention contain two or more of the above modifications within the same peptide. It is understood that it is also good.

A mimetic or peptide mimetic refers to a synthetic chemical compound having substantially the same structural and / or functional characteristics as the peptides of the invention. The mimic can be composed entirely of synthetic unnatural amino acid analogs, or can be a chimeric molecule comprising one or more natural peptide amino acids and one or more unnatural amino acid analogs. The mimic can also incorporate conservative substitutions of any number of natural amino acids, as long as such substitutions do not disrupt the activity of the mimetic. Routine testing can be used to determine if the mimic has essential activity that can inhibit the activity of, for example, VISTA.

[00102] When used with respect to a mimetic or peptide mimetic, the expression "substantially the same" means that the mimic or peptide mimetic is the activity or function of one or more of the referenced molecules (eg, T cells). It means having the ability to promote proliferation).

There are obvious advantages to the use of imitations of a given peptide. For example, peptide mimetics can be administered orally as compared to parenteral administration of peptides, resulting in significant cost savings and improved patient compliance associated with peptide mimetics. Moreover, the production of peptide mimetics is much cheaper than peptides.

Thus, the peptides of the invention have utility in the development of such small chemical compounds with similar biological activity and thus similar therapeutic utility. Techniques for developing peptide mimetics are conventional. For example, peptide bonds can be replaced by non-peptide bonds or non-natural amino acids that allow peptide mimetics to incorporate similar structures, and thus biological activity, into the original peptide. Further modifications can be made by replacing the chemical groups of the amino acids with chemical groups of other similar structures. The development of peptide mimetics can be assisted by determining the tertiary structure of the original peptide by NMR spectroscopy, crystallography and / or computer-assisted molecular modeling. These techniques support the development of new compositions with higher efficacy and / or higher bioavailability and / or higher stability than the original peptide (Dean).
(1994) BioEssays 16: 683-687; Cohen & Schatzmiller (1993) J.Le. Mol. Graph. 11: 166-173; Wiley & Rich (1993) Med. Res. Rev. 13: 327-384; Moore (1994) Trends Pharmacol. Sci. 15: 124-129; Hruby (1993) Biopolymers 33: 107 3-1082; Bug, et al. (1993) Sci. Am. 269: 92-98). Once a potential peptidomimetic compound has been identified, it can be synthesized and assayed using the assays described herein or other suitable assays for monitoring VISTA activity.

The peptide mimicking composition can contain any combination of non-natural structural components, which typically remains other than the three structural groups, i.e. the natural amide bond (“peptide bond”) linkage. Group-linking groups; non-natural residues in place of naturally occurring amino acid residues; inducing secondary structural mimicry, ie secondary structures (eg, β-turns, γ-turns, β-sheets, α-helix conformations, And the like) from residues that induce or stabilize; or other alterations that confer resistance to proteolysis. For example, a peptide can be characterized as a mimic if one or more of the residues are linked by chemical means other than an amide bond. The individual peptide mimic residues are amide bonds, non-natural non-amide chemical bonds, other chemical bonds or coupling means (eg, glutaaldehyde, N-hydroxysuccinimide ester, bifunctional maleimide, N, N'-dicyclohexyl). It can be linked by carbodiimide (DCC) or N, N'-diisopropyl-carbodiimide (DIC)). Alternative linking groups for amide bond, for example, ketomethylene (e.g., -C (= O) -C instead of _{-NH- (= O) -CH 2 -} ), aminomethylene _(CH 2 -NH), ethylene, olefin (CH = CH), ether _(CH 2 -O), thioether _(CH 2 -S), tetrazole _(CN 4 -), thiazole, retro amides, thioamide, or ester (e.g. Chemistry and Biochemistry of Amino Acids, Peptides and
(See Proteins, 7: 267-357, "Peptide and Backbone Modifications", Marcel Dekker, Putty in NY (1983)).

As discussed, peptides can be characterized as mimics by containing one or more unnatural residues in place of naturally occurring amino acid residues. Non-natural residues are known in the art. Certain non-limiting examples of non-natural residues useful as mimics of natural amino acid residues are mimics of aromatic amino acids, such as D-naphylanine or L-nafil. Alanine; D-phenylglycine or L-phenylglycine; D-2 thienailalanine or L-2 thienailalanine; D-1-pyreneylalanine, D-2-pyreneylalanine, D- 3-Pyreneal Alanine, D-4-Pyreneal Alanine, L-1-Pyreneal Alanine, L-2-Pyreneal Alanine, L-3-Pyreneal Alanine, or L-4-Pyreneal Alanine; D-3 Thienail alanine or L-3 thienail alanine; D- (2-pyridinyl) -alanine or L- (2-pyridinyl) -alanine; D- (3-pyridinyl) -alanine or L- (3-pyridinyl) -alanine D- (2-pyrazinyl) -alanine or L- (2-pyrazinyl) -alanine; D- (4-isopropyl) -phenylglycine or L- (4-isopropyl) -phenylglycine; D- (trifluoromethyl) -Phenylglycine; D- (trifluoromethyl) -phenylalanine; Dp-fluoro-phenylalanine; Dp-biphenylphenylalanine or Lp-biphenylphenylalanine; Dp-methoxy-biphenyl-phenylalanine or Lp- Methoxy-biphenyl-phenylalanine; and D-2-indole (alkyl) alanine or L-2-indole (alkyl) alanine (in the formula, alkyl is substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, (Can be isopropyl, iso-butyl or sec-isotyl, iso-pentyl or a non-acidic amino acid). Aromatic rings of unnatural amino acids that can be used in place of natural aromatic rings include, for example, thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl and pyridyl aromatic rings.

Mimics of acidic amino acids can be produced by substitution with non-carboxylic acid amino acids; (phosphono) alanine; and sulfated threonine while maintaining a negative charge. Carboxyl side groups (eg aspartyl or glutamyl) are also carbodiimides (R'-NC-N-R') (eg 1-cyclohexyl-3 (2-morpholinyl- (4-ethyl) carbodiimide or 1-ethyl-3). It can be selectively modified by reaction with (4-azonia-4,4-dimetholpentyl) including carbodiimide). Aspartyl or glutamil groups are also converted to asparaginyl and glutaminyl groups by reaction with ammonium ions. can do.

A lysine mimic can be produced by reacting lysinyl with a succinic anhydride or other carboxylic acid anhydride (and the amino-terminal residue can be modified). Mimics of lysine and other α-amino-containing residues are imide esters (methyl picolinimide acid, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzene sulfonic acid, O-methylisourea, 2,4, pentandione, etc.) It can also be produced by a reaction with and a transamidase-catalyzed reaction with glyoxylate.

One or more residues can also be replaced by amino acids (or peptide mimetic residues) of reverse chirality. Thus, any naturally occurring amino acid in the L-configuration, which can also be referred to as R or S, depending on the structure of the chemical, is the same amino acid or mimic but of reverse chirality. Can be replaced by one (referred to as the D-amino acid, which can additionally be referred to as the R or S form).

As will be appreciated by those skilled in the art, the peptide mimetics of the invention are one or more of the modifications described herein for derivatized peptides, such as detectable labels (effector labels). Or radionuclides, etc.), therapeutic agents (chemotherapeutic agents, etc.), one or more post-translational modifications, or cell permeation sequences.

[00111] For example, the VISTA antagonists described herein are post-translated and modified to produce effector labels (such as chemical linkers), detectable labels (eg, fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive substances and Chemiluminescent labels, etc.) or functional labels (eg, streptavidin, avidin, biotin, cytotoxins, cytotoxic agents, radioactive substances, etc.) can be added. Further exemplary enzymes include, but are not limited to, horseradish peroxidase, acetylcholinesterase, alkaline phosphatase, β-galactosidase and luciferase. Further exemplary fluorophores include, but are not limited to, rhodamine, fluorescein, fluorescein isothiocyanate, umbelliferone, dichlorotriazinylamine, phycoerythrin and dancil chloride. Further exemplary chemiluminescent labels include, but are not limited to, luminol. Further exemplary bioluminescent materials include, but are not limited to, luciferin, luciferase and aequorin. Further exemplary radioactive materials include ytterbium 213 ( ²¹³ Bs), carbon 14 ( ¹⁴ C), carbon 11 ( ¹¹ C), chlorine 18 (Cl ¹⁸ ), strontium 51 ( ⁵¹ Cr), cobalt 57 ( ⁵⁷ Co). ), Cobalt-60 ( ⁶⁰ Co), Copper 64 ( ⁶⁴ Cu), Copper 67 ( ⁶⁷ Cu), Strontium 165 ( ¹⁶⁵ Dy), Yttrium-169 ( ¹⁶⁹ Er), Fluorine 18 ( ¹⁸ F), Gallium 67 ( ⁶⁷ Ga) , Gallium 68 ( ⁶⁸ Ga), Germanium 68 ( ⁶⁸ Ge), Strontium 166 ( ¹⁶⁶ Ho), Indium 111 ( ¹¹¹ In), Iodine 125 ( ¹²⁵ I), Iodine 123 ( ¹²⁴ I), Iodine 124 ( ¹²⁴ I), Iodine 131 ( ¹³¹ I), ytterbium 192 ( ¹⁹² Ir), iron 59 ( ⁵⁹ Fe), krypton 81 ( ⁸¹ Kr), lead 212 ( ²¹² Pb), strontium 177 ( ¹⁷⁷ Lu), molybdenum 99 ( ⁹⁹ Mo), nitrogen 13 ( ¹³ N), oxygen 15 ( ¹⁵ O), palladium 103 ( ¹⁰³ Pd), phosphorus 32 ( ³² P), potassium 42 ( ⁴² K), renium 186 ( ¹⁸⁶ Re), renium 188 ( ¹⁸⁸ Re), rubidium 81 ( ⁸¹ Rb), Ytterbium 82 ( ⁸² Rb), Yttrium-153 ( ¹⁵³ Sm), Selenium 75 ( ⁷⁵ Se), Sodium 24 ( ²⁴ Na), Strontium 82 ( ⁸² Sr), Strontium 89 ( ⁸⁹ Sr), Strontium 35 ( ³⁵ S), technetium 99 m ( ⁹⁹ Tc), strontium 201 ( ²⁰¹ Tl), tritium ( ³ H), xenon 133 ( ¹³³ Xe), ytterbium 169 ( ¹⁶⁹ Yb), ytterbium 177 ( ¹⁷⁷ Yb) and yttrium-90 ( ⁹⁰ Y). ), But not limited to [00112] In addition, the VISTA antagonists provided herein include chemotherapeutic agents (carboplatin, cisplatin, paclitaxel, gemcitabine, calikeamycin, doxorubicin, 5-fluorouracil, mitomycin). C, Actinomycin D, Cyclophosphamide, Vincristine, Breomycin, VEGF antagonist, EGFR antagonist, Platin, Taxol, Yttrium-90, 5-Fluorouracil, Gemcita Bin (gemcytabine), leucovorine, steroids, cyclophosphamide, melphalan, binca alkaloids (eg vinblastine, vincristine, vincristine and vinorelbine), mustin, tyrosine kinase inhibitors, radiotherapy, sex hormone antagonists, selective androgen Receptor modifiers, selective estrogen receptor modifiers, PDGF antagonists, TNF antagonists, IL-1 antagonists, interleukins (eg IL-12 or IL-2), IL-12R antagonists, toxin-conjugated monoclonal antibodies, tumor antigens Includes specific monoclonal antibodies, Erbitux®, Avastin®, partszumab, anti-CD20 antibody, Rituxan®, oclerizumab, ofatumumab, DXL625, Herceptin®, or any combination thereof) However, it can be modified to add therapeutic agents not limited to these. Toxic enzymes from plants and bacteria (such as ricin, diphtheria toxin and Pseudomonas toxin) can be conjugated to VISTA antagonists to produce cell-type-specific killing reagents. Youule, et al. (1980) Proc. Nat'l Acad. Sci. USA 77 5843; Gilliand, et al. (1980) Proc. Nat'l Acad. Sci.
USA 77 4539; Crolic, et al. (1980) Proc. Nat'l Acad. Sci. USA 77 5419.

In addition, the VISTA antagonists described herein can be conjugated to radionuclides that emit alpha or beta particles (eg, radioimmune conjugates). Such radioactive isotopes include beta radiators (phosphorus 32 ( ³² P), samarium 47 (47 Sc), copper 67 ( ⁶⁷ Cu), gallium 67 ( ⁶⁷ Ga), yttrium 88 ( ⁸⁸ Y), yttrium 90 ( ⁹⁰ Y). ), Iodine 125 ( ¹²⁵ I), Iodine 131 ( ¹³¹ I), Samarium 153 ( ¹⁵³ Sm), Yttrium 177 ( ¹⁷⁷ Lu), Renium 186 ( ¹⁸⁶ Re), Renium 188 ( ¹⁸⁸ Re), etc.), and alpha emitters. (Includes, but is not limited to, astatine 211 ( ²¹¹ At), lead 212 ( ²¹² Pb), bismuth 212 ( ²¹² Bi), bismuth 213 ( ²¹³ Bi) or yttrium 225 ( ^{225 Ac), etc.).}

[00114] The method is known in the art for conjugating VISTA antagonists to labels as described herein, and Hunter, et.
al (1962) Nature 144: 945; David, et al. (1974) Biochemistry 13: 1014; Pain, et.
al. (1981) J. Immunol. Meth. 40: 219; and Nygren (1982) Histochem and Cytochem,
30: These methods and the like described by 407.

In addition, the VISTA antagonists described herein are another moiety (ie, a "targeted moiety") that targets an antagonist peptide to a target site (cancer cells, tumors, virus-infected cells, etc.). ) Can be included. The targeting moiety can be selected from an antibody or ligand that binds to the antigen, a receptor expressed by the target cell, or an infectious pathogen.

VISTA antagonists (in addition to their multimers, conjugates, analogs, derivatives and mimetics) can also be added directly or indirectly to immunoglobulin polypeptides or fragments thereof (eg, antibody constant regions).

When used herein, a "conjugate" is at least one isolated VISTA antagonist peptide and one immunoglobulin polypeptide linked by polypeptide level with or without the use of a linker. Refers to a compound having the fragment (for example, an antibody constant region). The conjugate can be a fusion polypeptide produced as a result of linking at least one natriuretic peptide and a gene encoding one antibody constant region at the nucleic acid level, with or without a coding sequence for the peptide linker.

Such VISTA antagonist peptide antibody conjugates have higher serum stability (eg, at least 20%, preferably at least 30%, 50%, 80%, 100%, 200% or more) under the same conditions. Serum half-life may be increased when compared to antagonist peptides without antibody constant regions. Human antibodies (eg, human IgG such as IgG1, IgG2, IgG3 or IgG4) are often used as the origin of constant regions or fragments thereof for the purpose of making the natriuretic peptide conjugates of the present invention.

As used herein, "antibody (or immunoglobulin) constant region" refers to a polypeptide that corresponds to at least a portion of the heavy or light chain constant region of an antibody. is (one of the constant domains of the constant region or C _H, for example CL) at least one constant domain include. For example, the "antibody constant regions" used to make the conjugates of the invention can be derived from antibody heavy chains, which include two of the three (for IgA, IgD and IgG). C _H 2 and _C H 3), or three of the four of (for IgE and IgM contains the constant domain of the _C H 2, CH3 and CH4); first constant domain _(C H I) some It may exist in the case, but may be excluded elsewhere. Such antibody constant regions can be obtained, for example, by recombinant or synthetic methods, or by a variety of means such as enzymatic digestion of intact antibodies or heavy or light chains of antibodies (eg, purification following pepsin digestion or papain digestion). Can be done.

As used in this application, further included by this term is the constant region of the heavy or light chain of an antibody or a portion thereof, which is at least one constant domain closest to the C-terminus of the antibody chain. Substantial sequence identity (eg, at least 80%, 85%, 90%, 95% or more) to the corresponding amino acid sequence of (containing) such "antibodies" in the VISTA antagonist peptide-antibody constant region conjugate. A polypeptide that has as long as the presence of a "constant region" provides higher serum stability to the conjugate.

In addition, peptides, multimers, conjugates, analogs, derivatives or mimetics can be modified to increase certain properties (eg, biological half-life). N-terminal modification / conjugation (eg, lipid addition or acetylation), C-terminal modification / conjugation (eg, lipid addition or acetylation), amino acid substitution (ie, non-naturally occurring amino acids (D-configuration, N-) Substitution of natural amino acids by methylation, tetra-substitution, β-amino acid, etc.), peptide main chain modification (eg, chemical modification of peptide bonds (simple reduction or replacement of carbonyl or amide groups with esters and sulfides and alkyls, etc.) )), Side chain modification and / or cyclization (eg, disulfide bond formation), but various approaches are possible.

[00122] In one embodiment, the peptide can be, for example, PEGylated to increase the biological (eg, serum) half-life of the antibody. To PEGylate a peptide, typically polyethylene glycol (PEG) (such as a reactive PEG ester derivative or aldehyde derivative) under conditions that result in the addition of one or more PEG groups to the peptide. And react it. Preferably, PEGylation is carried out via an acylation or alkylation reaction with a reactive PEG molecule (or a similar reactive water-soluble polymer).

Similarly, in another embodiment, peptides, multimers, conjugates, analogs, derivatives or mimetics can be modified by conjugation of polysialic acid (PSA) to increase half-life.

[00124] In addition, peptides, multimers, conjugates, analogs, derivatives or mimetics are modified (eg, genetically fused or chemically conjugated) and extended through a process called XTENification. The recombinant polypeptide (XTEN) that has been modified can be included to improve its half-life. XTEN is a long hydrophilic unstructured protein-based polymer of 864 amino acids. See, for example, WO 2013/130683, which is expressly incorporated herein by reference in its entirety. When added to the molecule of interest, the effective size of the molecule is significantly increased, slowing renal clearance in a manner similar to that of PEG, resulting in prolonged presence in serum. In addition to slowing renal clearance, addition to XTEN can also inhibit receptor-mediated clearance by reducing the affinity of the ligand for its receptor. XTEN coupling chemistry includes, but is not limited to, thiol-XTEN; maleimide-XTEN; alkyne-XTEN; and iodoacetyl-XTEN.

In addition, peptides, multimers, conjugates, analogs, derivatives or mimetics can be modified with recombinant albumin (eg Novozymes Recombumin®) to improve half-life. Peptides can be genetically fused or chemically conjugated to recombinant albumin using standard protocols.

In addition, peptides, multimers, conjugates, analogs, derivatives or mimetics can be modified by the addition of specific amino acids to the peptide and / or the removal of specific amino acids from the peptide. For example, adding some specific amino acids to a peptide thereby strengthening or tightening the molecular structure to make it less susceptible to biodegradation, thus, for example, Zealand's Structure Induced Probe (SIP®) tail technology. It can be used to provide a longer lifespan in the blood.

Yet another exemplary method of improving the stability and therapeutic potential of peptides, analogs, derivatives or mimetics is multimerization. For example, a multimer can include two or more copies of an isolated VISTA antagonist or variant thereof (eg, 2, 3, 4, 5, 6, 7 or more). Multimers include both homomultimers and heteromultimers. Multimerization can result in increased peptide stability, higher binding strength (due to multiple valences in the molecule), and / or improved pharmacokinetic properties.

Another exemplary approach for improving the stability and thus therapeutic potential of VISTA antagonist peptides, multimers, conjugates, analogs, derivatives or mimetics disclosed herein is. Addition of an acetyl group to the N-terminus and / or C-terminus of the peptide. Acetylation protects the peptide from exopeptidase, which can prolong the half-life of the peptide.

Production of VISTA Antagonists [00129] Peptide multimers, conjugates, analogs, derivatives or mimetics can be produced and isolated using any method known in the art. Peptides can be synthesized in whole or in part using chemical methods known in the art (eg, Carusers (1980) Nucleic Acids Res. Symp. Ser. 215-223; Horn (1980) Nucleic). Acids Res. Symp. Ser. 225-232; and Banga (1995) Therapeutic Peptides and Proteins, Formulation, Processing and Delivery Systems, Technology, Technology, Technology, Technology. Peptide synthesis can be performed using a variety of solid phase methods (see, eg, Roberge (1995) Science 269: 202; Merrifield (1997) Methods Enzymol. 289: 3-13), and automatic synthesis is, for example, the manufacturer. It can be achieved using an ABI 431A peptide synthesizer (PerkinElmer) according to the instructions in.

Peptides incorporating individual synthetic residues and mimetics can be synthesized using a variety of procedures and techniques known in the art (eg, Organic Syntheses Collective Volumes, Gilman, et al.). ) John Wiley & Sons, Inc., NY). Peptides and peptide mimetics can also be synthesized using combinatorial techniques. Techniques for the production of libraries of peptides and peptide mimetics are well known and include, for example, multipin techniques, tea bag techniques and split-couple-mix techniques (eg, al-Obeidi (1998) Mol. Biotechnol. 9: 205-223; Hruby (1997) Curr. Opin. Chem.
Biol. 1: 114-119; Ostergard (1997) Mol.
Divers. 3: 17-27; and Ostresh (1996) Methods Enzymol. 2 67: 220-234). Modified peptides can be further produced by chemical modification methods (eg, Belousov (1997)).
Nucleic Acids Res. 25: 3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19: 373-380; and Bloommers (1994) Biochemistry 33: 7886-7896).

Alternatively, the peptides of the invention can be prepared in a recombinant protein system using the polynucleotide sequence encoding the peptide. As an illustration, the nucleic acid molecule encoding the peptide of the invention is introduced into a host cell (such as a bacterial, yeast or mammalian cell) under suitable conditions for expression of the peptide, and the peptide is known in the art. Purified or isolated using the method. For example, Germany et al. (1990) Guide to Protein Purification: Methods in Enzymology Vol. 182, see Academic Press. In certain embodiments, peptides or analogs, derivatives or mimetics thereof) are uniformly isolated and / or purified (eg, with a purity greater than 90%).

It is contemplated that the peptides disclosed herein can be used as lead compounds for the design and synthesis of compounds with improved efficacy, clearance, half-life and the like.

One approach involves structure-activity relationship (SAR) analysis (eg, NMR analysis) to determine specific binding interactions between peptides and VISTA, to develop more effective agents. make it easier. Drugs identified in such SAR analysis or from drug libraries can then be tested, for example, for their ability to reduce the activity of VISTA and / or promote T cell proliferation.

Pharmaceutical Compositions [00134] The VISTA antagonist peptides described herein, their multimers, conjugates, analogs, derivatives and mimetics can be provided in the pharmaceutical compositions.

“Pharmaceutical composition” refers to a chemical or biological composition suitable for administration to mammals. Such compositions can be used in several pathways: buccal, epidermal, epidural, inhalation, intraarterial, intracardiac, intracerebral, intradermal, intramuscular, intranasal, intraocular, intraperitoneal, intraspinal, spinal cord. One of (including, but not limited to, transrectal, subcutaneous, sublingual, sublingual, transdermal, and transmucosal via intracavitary, intravenous, oral, parenteral, subcutaneous, enema or suppository) Alternatively, it can be specifically formulated for administration via a plurality. In addition, administration may be by injection, powder, liquid, gel, drop, or other means of administration.

A "pharmaceutical excipient" or "pharmaceutically acceptable excipient" is a carrier (usually a liquid) into which an active therapeutic agent is formulated. In one embodiment of the invention, the active therapeutic agent is a humanized antibody or one or more fragments thereof described herein. Excipients generally do not provide pharmacological activity and / or biological stability to the formulation, but may provide chemical and release properties. An exemplary formulation can be found, for example, in Greenaro (2005) [ed.] Remington: The Science and Practice of Pharmacy [21st edition].

The pharmaceutical composition should typically be sterilized and stable under manufacturing and storage conditions. The present invention contemplates that the pharmaceutical composition is in a lyophilized form. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentrations. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (eg, glycerol, propylene glycol and liquid polyethylene glycol), and suitable mixtures thereof. The present invention further contemplates inclusion of stabilizers in pharmaceutical compositions.

The VISTA antagonist peptides described herein, their multimers, conjugates, analogs, derivatives and mimetics can be formulated into pharmaceutical compositions in various dosage forms. To prepare the pharmaceutical composition of the present invention, at least one VISTA antagonist as an active ingredient can be densely mixed with suitable carriers and additives according to techniques well known to those skilled in the art of pharmaceutical formulations. Greennaro (2005) [ed.] Remington: The Science and Practice of Pharmacy [21st edition]. For example, the antagonists described herein can be formulated in phosphate buffered saline (pH 7.2) and supplied as a 5.0 mg / mL clear, colorless liquid solution.

Similarly, compositions for liquid preparation include suitable carriers and additives (water, alcohols, oils, glycols, preservatives, flavors, colorants and suspending agents, among others. Includes, but is not limited to, solutions, emulsions, dispersions, suspensions, syrups and elixirs. Typical preparations for parenteral administration include active ingredients with carriers such as sterile water or parenterally acceptable oils including, but not limited to, polyethylene glycol, polyvinylpyrrolidone, lecithin, peanut oil or sesame oil. And other additives for solubility or storage support can also be included. In the case of a solution, it can be lyophilized into a powder and then reconstituted just before use. Suitable carriers and additives for dispersions and suspensions include aqueous rubbers, celluloses, silicates or oils.

For each of the listed embodiments, Vista antagonist peptides, their multimers, conjugates, analogs, derivatives and mimetics can be administered in a variety of dosage forms. Any biologically acceptable dosage form and combinations thereof known to those of skill in the art are contemplated. Examples of such dosage forms include reconstituted powders, elixirs, liquids, solutions, suspensions, emulsions, powders, granules, particles, microparticles, dispersible granules, cashews, inhalants, aerosol inhalers, patches, particles. Inhalants, implants, depot implants, injectables (subcutaneous, intramuscular, intravenous and intradermal), infusions, and combinations thereof are included without limitation.

[00141] In many cases, it will be preferable to include an isotonic agent (eg, sugar, polyhydric alcohol (mannitol, sorbitol, etc.), or sodium chloride) in the composition. Prolonged absorption of the injectable composition can be provided by including agents in the composition that delay absorption (eg, monostearate and gelatin). In addition, the compounds described herein can be formulated in a sustained release formulation (eg, in a composition comprising a slow release polymer). VISTA and VISTA conjugates can be prepared with carriers that protect the compound against rapid release, including implants and microencapsulated delivery systems, such as controlled release formulations. Biodegradable biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polymilk, polyglycol copolymers (PLG), etc. be. Many methods are known to those skilled in the art for the preparation of such formulations.

Those skilled in the art will appreciate, for example, routine experiments guided by the disclosure herein, and Goodman, et al. (2011) Goodman &Gilman's The Pharmaceutical Bases of Therapeutics [12th Edition]; Howland, et al. (2005) Lippincott's Illustrated Reviews:
Pharmacology [2nd Edition]; and Golan, (2008) Principles of Pharmacology: The Pathophysiological Basic Bases of Drug Therapy right. See also Greenaro (2005) [ed.] Remington: The Science and Practice of Pharmacy [21st edition].

The compositions described herein are described in the following pathways: buccal cavity, suppository, epidural, infusion, inhalation, intraarterial, intracardiac, intracerebral, intradermal, intramuscular, intranasal. Any of intrarectal, subcutaneous, sublingual, transdermal, and transmucosa via intraocular, intraperitoneal, spinal cord, intrathecal, intravenous, oral, parenteral, lung, subcutaneous, enema or suppository Can be administered in. The preferred route of administration is intravenous injection or infusion. Administration is topical (the composition is administered directly to the site of the disease (eg, tumor), in close proximity to it, in, near, there, around, or in the vicinity of that point). Or systemic (the composition is given to the patient and widely passes through the body, thereby reaching the site of the disease). Topical administration (eg, injection) involves cells, tissues, organs, and / or organ systems, which include and / or suffer from the disease, and / or have active disease signs and / or symptoms. It can be done by administration to the place where it will occur (eg, the tumor site). Administration can be topical with local effects, and the composition is applied directly where the action is desired (eg, tumor site).

For each of the listed embodiments, the compounds can be administered in a variety of dosage forms, as is known in the art. Any biologically acceptable dosage form and combinations thereof known to those of skill in the art are contemplated. Examples of such dosage forms include chewable tablets, fast-dissolving tablets, effervescent tablets, reconstituted powders, elixirs, liquids, solutions, suspensions, emulsions, tablets, multi-layer tablets, double-layer tablets, capsules, soft gelatin capsules. , Hard gelatin capsules, caplets, lozenges, chewable lozenges, beads, powders, rubbers, granules, particles, microparticles, dispersible granules, cashews, injections, suppositories, creams, topical agents, inhalants, aerosol inhalers, patches, Includes, but is not limited to, particle inhalers, implants, depot implants, ingestibles, injectables (including subcutaneous, intramuscular, intravenous and intradermal), instillations, and combinations thereof.

Other compounds that may be contained by the admixture are, for example, medically inert ingredients (eg, solid and liquid diluents) (for tablets or capsules, for lactose, dextrose, saccharose, cellulose, starch or calcium phosphate, soft capsules). Is olive oil or ethyl oleate, and water or vegetable oil for suspensions or emulsions); lubricants (silica, talc, stearic acid, magnesium or calcium stearate and / or polyethylene glycol, etc.); gelling agents (colloidal clay, etc.) Etc.); Thickener (tragacanto rubber or sodium alginate, etc.), binder (starch, gum arabic, gelatin, methyl cellulose, carboxymethyl cellulose or polyvinylpyrrolidone, etc.); disintegrant (starch, alginic acid, alginate or sodium starch glycolate, etc.) Boiling agents; Dyes; Sweeteners; Wetting agents (such as lecithin, polysorbate or lauryl sulfate); and other therapeutically acceptable sub-ingredients (wetting agents, preservatives, buffers) that are known additives for such formulations. And antioxidants, etc.).

The liquid dispersion for oral administration can be a syrup, emulsion, solution or suspension. The syrup can contain syrup as a carrier with, for example, saccharose, or glycerol and / or mannitol and / or rubitol. Suspensions and emulsions can contain carriers (eg, natural rubber, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose, polyvinyl alcohol).

[00147] In a further embodiment, the invention provides a kit comprising one or more containers comprising a pharmaceutical dosing unit containing an effective amount of one or more VISTA antagonists of the invention. The kit may include instructions, instructions, labels, marketing information, warnings or information brochures.

The amount of VISTA antagonist in the therapeutic composition according to any embodiment of the invention includes disease status, age, gender, weight, patient history, risk factors, predisposition to disease, route of administration. It can vary depending on factors such as the existing treatment regime (eg, the potential for interaction with other medications) and the weight of the individual. The dosing regimen can be adjusted to provide a response to the optimal treatment. For example, a single bolus can be administered, divided doses can be administered over time, or the dose can be reduced or increased proportionally depending on the circumstances of the treatment situation. can.

It is particularly advantageous to formulate the parenteral composition in the form of a dosage unit for ease of administration and uniformity of dosage. Dosing unit shape, as used herein, refers to physically separated units adapted as an integrated dosage for the mammalian subject being treated; each unit is with the required pharmaceutical carrier. It contains a predetermined amount of antibody and fragments thereof that are jointly intended to produce the desired therapeutic effect. The specifications for dosage unit shapes of the present invention formulate such antibodies and fragments thereof for the treatment of susceptibility in an individual, as well as the unique properties of the antibody and fragments thereof, as well as the particular therapeutic effect achieved. It is determined by the limitations inherent in the art and depends directly on them. In therapeutic use for the treatment of pathological conditions in mammals (eg, humans) for which the antibodies of the invention and fragments thereof or suitable pharmaceutical compositions thereof are effective, the antibodies of the invention and fragments thereof are administered in effective amounts. be able to. Suitable dosages for the present invention can be compositions, pharmaceutical compositions or any other composition described herein.

Dosing can be administered as a single dose, a double dose, a triple dose, a quadruple dose and / or a quintuple dose. Dosing can be administered alone, simultaneously and continuously.

The dosing shape can be any release shape known to those of skill in the art. The compositions of the present invention may be formulated to provide immediate release of the active ingredient or sustained or controlled release of the active ingredient. In sustained or controlled release preparations, release of the active ingredient can occur at a rate such that blood levels remain within the therapeutic range but below toxic levels over an extended period of time (eg 4-24 hours). .. Preferred dosage forms include immediate release, extended release, pulse release, variable release, controlled release, sustained release, sustained release, delayed release, long-acting form and combinations thereof, as well as those known in the art. Is done.

As defined herein, therapeutically effective amounts (ie, effective dosages) of VISTA antagonist peptides, analogs, derivatives or mimics thereof are about 0.001 to 30 mg / kg body weight. , Preferably about 0.01-25 mg / kg body weight, more preferably about 0.1-20 mg / kg body weight, and even more preferably about 1-10 mg / kg, 2-9 mg / kg, 3-8 mg / kg, It is in the range of 4-7 mg / kg, or 5-6 mg / kg body weight.

Those skilled in the art will appreciate certain factors including, but not limited to, the severity of the disease or disorder, conventional treatment, overall health and / or age of the subject, and other diseases present. You will recognize that it can affect the dosage required for effective treatment of. In addition, treatment of a subject with a therapeutically effective amount of peptide may include a single treatment or preferably a series of treatments.

In a preferred example, the subject is between about 1-10 weeks, preferably between 2-8 weeks, more preferably between about 3-7 weeks, and even more preferably about 4,5. Or for 6 weeks, once per week, treated with peptides, analogs, derivatives or mimics in the range between body weights of about 0.1-20 mg / kg. It will also be recognized that effective dosages of antibodies, proteins or polypeptides used for treatment can be increased or decreased over the course of a particular treatment. The results of diagnostic assays as described herein can result in and reveal dosage changes.

It will be appreciated that the pharmacological activity of the composition can be monitored using standard pharmacological models known in the art. In addition, compositions containing VISTA antagonists can be incorporated or encapsulated in suitable polymer matrices or membranes for site-specific delivery, or functionalized with specific target agents capable of achieving site-specific delivery. It will be recognized that it can be transformed. These techniques as well as other drug delivery techniques are well known in the art. Determining the optimal dosage for a particular situation is within the ability of one of ordinary skill in the art. Greennaro (2005) [ed.] Remington: The Science and Practice of Pharmacy [21st edition].

The peptides or analogs, derivatives or mimics of the invention are co-formulated and / or co-administered with one or more additional therapeutic agents (eg, anticancer agents, antiviral agents, cytokines and / or immunoagonists). can do. Such combination therapies require lower dosages of peptides or analogs, derivatives or mimetics and / or co-administered agents, thus avoiding the toxicity or complications that may be associated with various monotherapy. can. There are several agents that can be advantageously combined with the peptides or analogs, derivatives or mimetics of the invention, and the choice of such agents will depend on the disease or condition intended for treatment. For example, the present invention comprises a combination of a peptide or multimer, conjugate, analog, derivative or mimic of the invention and at least one anticancer agent capable of inducing or facilitating a response to a cancerous or precancerous condition. Therapy is included. Thus, in certain embodiments, the peptides or analogs, derivatives or mimics are used as adjuvant therapies in the treatment of cancer. As another example, the invention provides a combination therapy comprising a peptide or analog, derivative or mimic of the invention and at least one antiviral agent capable of inducing or facilitating a response to a treatment for viral infection. Include. Exemplary therapeutic agents that may be included in compositions containing VISTA antagonist peptides, multimers, conjugates, analogs, derivatives or mimics include, for example, CTLA-4-Ig, anti-PD-1, PD-L1 fusions. Includes proteins or PD-L2 fusion proteins, and EGFR antagonists.

Anticancer agents include cytotoxic agents (bincaalkaloids, taxans, topoisomerase inhibitors, etc.); antisense nucleic acids (Augmerocene / G3139, LY90003 (ISIS 3521), ISIS 2503, OGX-011 (ISIS 112998), LE-AON. / LEraf-AON (liploid encapsulated c-raf antisense oligonucleotide / ISIS-5132), MG98, and other antisense nucleic acids targeting PKCα, crusterin, IGFBP, protein kinase A, cyclin D1 or Bcl-2, etc. ); Anti-cancer nucleic acid (Ribozyme)
Pharmaceuticals), etc.); Nucleic acid encoding tumor suppressor (p53, BRCA1, RB1, BRCA2, DPC4 (Smad4), MSH2, MLH1, DCC, etc.); Cancer-dissolving virus (cancer-dissolving adenovirus, herpesvirus, etc.); Anticancer Immunogen (cancer antigen / tumor-related antigen, eg, epithelial cell adhesion molecule (Ep-CAM / TACSTD1))
, Mutin 1 (MUC1), cancer fetal antigen (CEA), tumor-related glycoprotein 72 (TAG-72), gp100, Melan-A, MART-1, KDR, RCAS1, MDA7, cancer-related virus vaccine, tumor-inducible Heat shock proteins and the like); anticancer cytokines, anticancer chemokines or combinations thereof; inhibitors of angiogenesis, neoangiogenesis, and / or other vascular invasion; and / or any other conventional anticancer agent (Fluoropyrimider carbamate, non-polyglutamateable thymidylylase-producing enzyme inhibitor, nucleoside analog, antifolic acid agent, topoisomerase inhibitor, polyamine analog, mTOR inhibitor, alkylating agent, Lectin inhibitors, vitamin D analogs, carbohydrate processing inhibitors, antimetabolite antagonists, thumidilate synthase inhibitors, antimetabolites, ribonuclease reductase inhibitors, dioxolate nucleoside analogs, and chemically modified Includes, but is not limited to, tetracyclins).

The antiviral agents used in the present invention include protease inhibitors (eg, acyclovir) or antiviral antibodies (eg, anti-gp41 antibodies in the context of HIV treatment; anti-CD4 in the context of CMV treatment). Antibodies, etc.), but are not limited to these. Many other types of antiviral agents are known in the art.

Toxicity and therapeutic efficacy of peptides or analogs, derivatives or mimetics can be determined by standard pharmaceutical procedures in cell culture or laboratory animals. Data obtained from cell culture assays and animal studies can be used in formulations with a range of dosages for use in humans. Dosages of such agents are preferably within the range of circulating concentrations containing _{ED 50 with little or no toxicity.} Dosages can vary within this range depending on the dosage form used and the route of administration used. For any agent used in the methods of the invention, a therapeutically effective dose can be initially estimated from the cell culture assay. A dose may be formulated in animal models, it is possible to achieve a circulating plasma concentration range that includes the IC 50 as determined _(i.e. the concentration of test compound which achieves a half-maximal inhibition of symptoms) in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.

Use of VISTA antagonists and compositions containing them [00160]
The peptides or analogs, derivatives or mimetics of the invention are found to be used in inhibiting the activity of VISTA (ie PD-L3), thereby upregulating the immune response. Upregulation of the immune response can be in the form of promoting an existing immune response or inducing an initial immune response. For example, promoting an immune response through inhibition of VISTA activity is useful in the prevention and / or treatment of infection by microorganisms (eg, bacteria, viruses, or parasites), or in immunosuppression and cancer cases.

Accordingly, the present invention includes prophylactic and therapeutic methods for the prevention and treatment of cancer and infectious diseases. As used herein, terms such as "treat," "treat," and "treat" are effective amounts for the purpose of alleviating, ameliorating, or eradicating (curing) such symptoms or disease states that have already developed. Refers to the delivery of the peptides or analogs, derivatives or mimics of the invention. The terms "prevent," "prevent," and "prevent" refer to effective amounts of peptides or analogs, derivatives, or mimetics of the invention for the purpose of preventing any symptom or disease condition that is to develop. Refers to the delivery of goods. Therefore, these terms are intended to include prophylactic treatment.

According to one embodiment, the invention requires an effective amount of an isolated VISTA antagonist disclosed herein or a composition comprising the isolated VISTA antagonist (mammalian). Provided is a method for treating or preventing a cancer that inhibits tumor infiltration and / or cancer metastasis by administration to a subject, preferably a human subject). Optionally, the subject has one or more precancerous lesions or is predisposed to cancer as a result of, for example, genetic variation, family history or exposure to carcinogens. In another embodiment, the invention provides a method of treating cancer in a subject (such as a mammalian subject, preferably a human subject (such as a human subject optionally having a detectable level of cancer cells)). According to these embodiments, the subject is administered in an amount sufficient to detectively reduce the onset or progression of cancer in the subject with the peptides or analogs, derivatives or mimetics of the invention.

Cancer is generally composed of a single clone or several clones of cells capable of partially independent growth in the host (eg, benign tumor) or completely independent growth in the host (malignant cancer). NS. Cancer cells are cells that divide and replicate abnormally with uncontrolled growth.

Cancer cells arise from host cells via malignant transformation (ie, carcinogenesis). In the present specification, terms relating to the description of cells and / or tissues such as "preneoplastic", "premalignant" and "precancerous" are genetic and / or expressions indicating a significant ability to develop cancer. Refers to a cell or tissue that has a phenotypic profile. Usually such cells can be characterized by one or more differences from their closest counterparts that signal a significant risk of the onset of cancer progression or the onset of cancer progression. Such precancerous changes can usually be treated with excellent results if they are detectable.

In general, the precancerous condition will be accompanied by the appearance of neoplasms (s) or preneoplastic lesions (s). Examples of known and likely preneoplastic tissues include ductal carcinoma in situ (DCIS) growth in breast cancer, cervical intraepithelial neoplasm (CIN) in cervical cancer, and colon in colonic rectal cancer. Includes adenomatous polyps in situ, atypical adenomatous hyperplasia in lung cancer, and ductal carcinoma in situ (AK) in skin cancer. Preneoplastic phenotypes and genotypes for various cancers and methods for assessing the presence of preneoplastic states in cells have been characterized. For example, Medina (2000) J. et al. Mammaly Grand Biol. Neoplasmia 5 (4): 393-407; Krishnamurthy, et al. (2002) Adv. Anat. Pathol. 9 (3): 185-97; Ponten (2001) Euro. J. Cancer Super 8: S97-113; Niklinski, et al. (2001) Euro. J. Cancer Prev. 10 (3): 213-26; Walk, et al. Pathobiology (2000) 68 (1): 9-17; Busch (1998) Cancer Surv. See 32: 149-79.

Gene expression profiles can be increasingly used to distinguish between normal cells, precancerous cells and cancer cells. For example, the familial adenoma-like polyp gene is closely promoted in surveillance for colon cancer; the mutated p53 tumor suppressor gene flags cells that develop into invasive cancer; osteopontin expression levels are elevated in premalignant cells. However, increased telomerase activity can also be a marker of precancerous symptoms (eg, in bladder and lung cancers). In one aspect, the invention relates to the treatment of precancerous cells. In another aspect, the invention relates to the preparation of pharmaceuticals for the treatment of precancerous cells.

Generally, the peptides or analogs, derivatives or mimetics of the invention are used to treat subjects with any stage of cancer (and to prepare pharmaceuticals for reducing, delaying or other treatment of cancer). can do. Effective treatment of cancer (and thus its reduction) can be detected by any variety of suitable methods. Methods for cancer detection and effective cancer treatment include laboratory tests (symptoms may include swelling, palpable lumps, lymphadenopathy, bleeding, visible skin lesions and weight loss); Imaging (X-ray technique, mammography, colonoscopy, computed tomography (CT and / or CAT) scanning, magnetic resonance imaging (MRI), etc.); Immunodiagnostic assays (eg, detection of CEA, AFP, CA125, etc.) ); Antibody-mediated radioimaging; and cell / tissue immunohistochemical analysis are included. Other examples of techniques appropriate for assessing cancerous status and effective cancer treatment include PCR and RT-PCR (eg, cancer cell-related genes or "markers"), biopsy, electron microscopy, positrons. Radiation tomography (PET), computer tomography, magnetic resonance imaging (MRI), nuclear type analysis and other chromosomal analysis, immunoassay / immunocytochemical detection techniques (eg differential antibody recognition), histological and / Or histological pathological assays (eg for cell membrane changes), cell dynamics studies and cell cycle analysis, ultrasonography or other ultrasonography detection techniques, radiation detection techniques, flow cytometry, endoscopy visualization techniques, Also includes physical examination techniques.

In general, delivery of peptides or analogs, derivatives or mimetics of the invention to a subject (either by direct administration or expression from nucleic acids) is used to reduce any aspect of cancer in the subject. Can be treated, prevented, or otherwise ameliorated. In this regard, cancer treatment includes, for example, the rate of normal cells transforming into neoplastic cells (or any embodiment thereof), the rate of proliferation of preneoplastic or neoplastic cells, preneoplasm. Number of cells exhibiting sexual and / or malignant phenotype, physical regions of cell media (eg, cell cultures, tissues or organs) containing preneoplastic cells and / or neoplastic cells, normal cells and / Or the probability that preneoplastic cells will transform into neoplastic cells, the probability that cancer cells will progress to the next aspect of cancer progression (eg, reduced metastatic capacity), or any detectable reduction in any combination thereof. Can be included. Such changes are typical of any of the techniques described above or any of the appropriate counterparts known in the art, which are appropriate times prior to administration of the therapeutic regimen to assess its effectiveness. Can be detected using). Whether or not cancer reduction has occurred, the time and conditions for the assay include multiple factors (type of cancer, peptide being delivered to the host, related composition, or combination composition type and amount. Will depend on.

A variety of cancers can be treated using the methods of the invention. The forms of cancer that can be treated by delivery or administration of the peptides or analogs, derivatives or mimics of the invention and combination therapy containing them include rhabdomyosarcoma, leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia. , B-cell lymphoma, T-cell lymphoma, hodgkin lymphoma, non-hodgkin lymphoma, hairy cell lymphoma, barkit lymphoma, acute or chronic myelogenous leukemia, premyelogenous leukemia, fibrosarcoma, rhabdomyosarcoma, melanoma, sperm epithelium Tumors, teratomas, neuroblastomas, gliomas, stellate cell tumors, neuroblastomas, gliomas, neuromas; fibrosarcoma, rhabdomyosarcoma, osteosarcoma, melanoma, pigmented psoriasis Includes, keratinized spinous cell tumor, spermatic epithelioma, follicular thyroid cancer and teratoma. The compositions of the present invention may also be useful in the treatment of other carcinomas of the bladder, breast, colon, kidney, liver, lung, ovary, prostate, pancreas, stomach, cervix, thyroid or skin. The compositions of the present invention include other hematopoietic tumors of the lymphoid lineage, other hematopoietic tumors of the myeloid lineage, other tumors of interstitial origin, other tumors of the central or peripheral nervous system, and /. Or it may be useful in the treatment of other tumors of inter-hematopoietic origin. Advantageously, the methods of the invention are used in prostate cancer cells, melanoma cells (eg, cutaneous melanoma cells, ocular melanoma cells, and / or lymph node-related melanoma cells), breast cancer cells, colon cancer cells, and lung cancer cells. It can also be useful in reducing cancer progression. The method of the present invention can be used for the treatment of tumorigenic and nontumorogenic cancers (eg, nontumorogenic hematopoietic cancers). The methods of the invention are particularly useful in the treatment of epithelial cancers (eg carcinomas) and / or colonic rectal cancers, breast cancers, lung cancers, vaginal cancers, cervical cancers, and / or squamous epithelial cell carcinomas (eg head and neck). .. Additional potential targets include sarcomas and lymphomas. Additional advantageous targets include solid tumors and / or disseminated tumors (eg, myelogenous and lymphoid tumors, which can be acute or chronic).

The present invention promotes anti-cancer or anti-tumor immunity, comprising administering to a subject in need an effective amount of an isolated VISTA antagonist or a composition comprising the isolated VISTA antagonist. It also provides a way to do it.

In addition to treating cancer, the invention also features methods of treating pathogen infections (ie, bacterial, viral, parasitic or fungal infections) in a subject or host. The method comprises administering or otherwise delivering an effective amount of a peptide or analog, derivative or imitation of the invention to reduce the severity, transmission, symptom or duration of such infection. Such pathogen infections include, but are not limited to, diseases caused by bacteria, protozoa, fungi, parasites or viruses.

[00172] In certain embodiments, viral infections are treated. Any virus normally associated with the activity of effector lymphocytes can be treated by this method. For example, using such a method, hepatitis A virus, hepatitis B virus, hepatitis C virus, influenza virus, varicella virus, adenovirus, simple herpes type I (HSV-1) virus, simple herpes type 2 (HSV). -2) Virus, bovine epidemic virus, rhinovirus, echovirus, rotavirus, respiratory vesicle virus, papillomavirus, papillomavirus, cytomegalovirus (CMV, for example, HCMV)
, Echinovirus, Arbovirus, Huntavirus, Coxsackie virus, Otafukukaze virus, Hashi virus, Eczema virus, Poliovirus, and / or human immunodeficiency virus type I or type 2 (HIV-1, HIV-) Infection with one or more viruses selected from 2) can be treated. Practice of such a method can result in a reduction in virus titer (viral load), a reduction in the number of virus-infected cells, and the like.

In addition to pathogen infections, the peptides or analogs, derivatives or mimetics of the invention are in collaboration with the treatment of immunoproliferative disorders, immunodeficiencies, autoimmune disorders, inflammatory reactions, and / or allergic reactions. It can be administered to a subject or otherwise delivered. In addition, the present invention blocks VISTA-mediated T cell suppression in a subject, comprising administering to a subject in need an effective amount of an isolated VISTA antagonist or a composition comprising the isolated VISTA antagonist. , Inhibiting or neutralizing, and / or stimulating an immune response. Such methods may be useful for treating subjects affected with one or more of bacterial, viral, parasitic and fungal infections and / or cancer.

Although the present invention has been generally described herein, it will be more easily understood by reference to the following examples. These examples are included solely for the purpose of exemplifying certain aspects and embodiments of the invention and are not intended to limit the invention.

Example 1: Materials and Methods [00175] Peptide synthesis. AP1049 (SSACDIKRSCH-amide, in the formula Cys4-Cys11 forms a disulfide bridge; SEQ ID NO: 1 (Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His)) and scramble. The negative control sequence (SSACKSWRDICH-amide, in the formula Cys4-Cys11 forms a disulfide bridge; SEQ ID NO: 2) was prepared using standard Fmoc-based solid phase peptide synthesis (SPPS). Peptides were purified via HPLC and analyzed by mass spectrometry using liquid chromatography-mass spectrometry (LC-MS) and matrix-assisted laser desorption / ionization (MALDI).

[00176] Peptide search using a phage display. The M13 phage peptide library was provided by Dr. Brian Kay (U. Illinois-Chicago). The VISTA proteins required for both phage display biopanning experiments and confirmed Elisa binding experiments were prepared by conventional recombinant protein techniques.

T cell proliferation analysis. The VISTA-Ig fusion protein or control Ig fusion protein was co-absorbed into cell culture plates with polyclonal T cell receptor (TCR) stimulation (ie, anti-CD3 antibody). To assess the activity of VISTA-specific peptides, peptides (VISTA-specific and scrambled controls) were added to the culture on day 0 as soluble reagents and T cell proliferation and cytokine production were analyzed after 3-4 days. ..

T cell priming assay. VISTA is known to suppress T cell priming when expressed on antigen presenting cells (APCs). VISTA-expressing myelogenous APC (Cd11b ^hi MHCII ⁺ bone marrow cells) was purified from mouse spleen, sorted by FACS and irradiated (2500 rad). To test the activity of VISTA-specific peptides, transgenic T cells (such as OT-II) were stimulated with Exvivo with VISTA-expressing APC and the homologous antigen triovoalbumin (15 ng / mL). VISTA-specific peptides or control scrambled peptides were added to the cell culture. T cell proliferation and cytokine production were evaluated 3-5 days after culture. As an additional specificity control, APC purified from VISTA-negative parental cell lineage A20 or VISTA knockout mice was used.

Foxp3 + CD4 + Regulatory T Cell (Treg) Suppression Assay. VISTA plays a role in the inhibitory function of Foxp3 + CD4 + regulatory T cells (Tregs), as VISTA blocking monoclonal antibodies partially restore Treg inhibitory activity in in vitro Treg suppression assays. The assay includes antigen presenting cells, purified Foxp3 + CD4 + Treg, and Foxp3-CD4 + naive T cells, which are stimulated by polyclonal TCR stimulation. To test the activity of VISTA-specific peptides, peptides (VISTA-specific and scrambled controls) were added to the Treg suppression assay on day 0. T cell proliferation and cytokine production were measured on day +3.

A model of experimental autoimmune encephalomyelitis (EAE) (mouse autoimmune inflammatory disease model for human multiple sclerosis). VISTA blocking monoclonal antibodies have been shown to exacerbate the severity of the disease in addition to significantly accelerating the onset of disease in the passively transmitted EAE model. In this model, MOG-specific encephalitis-inducing CD4 + T cells were first primed and then purified in donor mice upon immunization with MOG peptides and then purified in the presence of MOG peptides and cytokines (IL23, TGFβ, IL6 and IL1b). Amplify with Exvivo. Amplified encephalitis-induced CD4 + T cells are transferred into naive recipients to induce disease. To assess the activity of VISTA-specific peptides, peptides (VISTA-specific and scrambled controls) are administered prophylactically (starting on day 2) or therapeutically (+7 days during onset of disease). Administer to mice via intraperitoneal administration either (starting with) and every two days continuously for the entire duration of the experiment. Assess the severity of the disease according to established protocols.

Mouse tumor model. It has been demonstrated that VISTA suppresses tumor-specific T cell responses. VISTA blockade via a VISTA-specific monoclonal antibody significantly promotes an antitumor immune response and inhibits tumor progression in mouse tumor models (such as the B16 melanoma model). The activity of VISTA-specific peptides can be assessed in vivo using this tumor model. On day 0, B16 tumor cells (15,000 cells) are inoculated on the flank of the mouse. Peptides (VISTA-specific and scrambled controls), either prophylactically (starting on day 2) or therapeutically (ie, if the tumor is palpable), and continuously every 2 days. Is administered to mice via intraperitoneal administration for the entire duration of the experiment. Tumor growth is measured with a caliper every 2-3 days.

Example 2: Promotion of T cell proliferation [00182] VISTA ⁺ CD11b ⁺ monocytes were concentrated from naive splenocytes using CD11b magnetic beads (Miltenyi). VISTA ⁺ CD11b ^hi MHCII ⁺ myeloid APC was sorted by FACS, irradiated (2500 rad) and used as antigen presenting cells to stimulate ^{OT-II transgenic CD4 + T cells in the presence of OVA peptides.} Monoclonal antibodies specific for control Ig, VISTA and PD-L1 (30 μg / mL), or VISTA-specific peptides (100 μg / mL) were added as shown. Cell proliferation was measured by tritium uptake during the last 8 hours of the 72 hour assay. This analysis showed that T cell proliferation was enhanced in the presence of VISTA or PD-L1 neutralized monoclonal antibody or AP1049 peptide (Fig. 1). In fact, the AP1049 peptide stimulates cell proliferation much better than any of the monoclonal antibodies, pointing out that this peptide retains strong antagonist activity against VISTA.

Example 3: Promotion of anti-tumor immunity [00183] Female mice were inoculated with an immunogenic bladder cancer tumor (MB49). AP1049 was tested for its ability to slow tumor growth and / or facilitate tumor regression. The measurements in this assay were tumor growth.

MB49 tumors were inoculated into female mice (300k) via intradermal (id) inoculation, which facilitates measurement of tumor size. Mice were treated with either PBS (control) or VISTA antagonist peptide (AP1049) via daily injections around the tumor mass, starting on day + 1 and continuing for 2 weeks. Tumor size was measured by caliper every 2-3 days.

These methods were used to obtain delayed tumor growth and / or tumor regression in mice treated with AP1049 as compared to mice treated with controls.

As shown in FIG. 2, AP1049 treatment reduces tumor growth in the MB49 tumor model, the peptide binds to important / active sites of VISTA and blocks the immunosuppressive function of VISTA. It was pointed out that it could be done.
The scope of claims at the time of international filing of the original application is as follows.
[Item 1] A peptide identical to the amino acid sequence of SEQ ID NO: 1 (Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His), or a multimer, conjugate, analog thereof, An isolated VISTA analog, including a derivative or imitation.
[Item 2] The amino acid sequence of SEQ ID NO: 1 (Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His) contains the same peptide, or at most two amino acid residues remain. An isolated VISTA antagonist comprising a peptide having an amino acid sequence different from SEQ ID NO: 1 in the group, or a multimer, conjugate, analog, derivative or imitation thereof.
[Item 3] Any one of claims 1 or 2, which comprises a peptide having an amino acid sequence different from that of SEQ ID NO: 1 at most one amino acid residue, or a multimer, conjugate, analog, derivative or imitation thereof. The isolated VISTA antagonist according to.
[Item 4] The amino acid sequence of SEQ ID NO: 1 (Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His) contains the same peptide, or a multimer or conjugate thereof. Released VISTA antagonist.
[Item 5] The isolated VISTA antagonist according to claim 1, which comprises the amino acid sequence of SEQ ID NO: 1 (Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His). ..
[Item 6] Positions 4 and 11 of SEQ ID NO: 1 (Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys-Arg-Ser-Cys-His), or their corresponding positions in the variant of the peptide. The isolated VISTA antagonist according to any one of claims 1 to 5, wherein the cysteine residue in the above forms a disulfide bridge.
[Item 7] The isolated VISTA antagonist according to any one of claims 1 to 6, which is modified to improve binding affinity and / or stability.
8. The isolated VISTA antagonist of claim 7, wherein the modification is selected from the group consisting of PEG, acetylation, XTEN, albumin and multimerization.
9. The isolated VISTA antagonist according to any one of claims 1 to 8, which is added directly or indirectly to an immunoglobulin polypeptide or a fragment thereof.
Item 10. The isolated antagonist according to claim 9, wherein the immunoglobulin polypeptide comprises a constant region of human IgG1, IgG2, IgG3 or IgG4 or a fragment thereof.
11. The isolated VISTA antagonist of claim 10, wherein the immunoglobulin polypeptide comprises a human IgG1 constant region or a fragment thereof.
12. The isolated VISTA antagonist of any one of claims 1-11, comprising a copy of 2, 3, 4, 5, 6, 7 or more of the peptide.
[Item 13] The isolated VISTA antagonist according to any one of claims 1 to 11, which comprises another portion that targets the peptide at a target site.
14. The isolated VISTA antagonist of claim 13, wherein the targeting moiety is selected from an antibody or ligand that binds to an antigen, a receptor expressed by a target cell, or an infectious pathogen.
15. The isolated VISTA antagonist of any one of claims 1-14, wherein the antagonist is added to another moiety or another copy of the antagonist via a linker.
16. The isolated VISTA antagonist of claim 15, wherein the linker is a peptide that allows the antagonist to interact with VISTA expressed on the surface of a target cell.
17. The isolated VISTA antagonist of any one of claims 1-16, which is added directly or indirectly to a detectable label or therapeutic agent.
[Item 18] The isolated VISTA antagonist according to any one of claims 1 to 17, which binds to the extracellular domain of VISTA and interrupts its interaction with the VISTA receptor.
19. The isolated VISTA antagonist according to any one of claims 1-18, which reduces or inhibits VISTA-mediated T cell suppression.
Item 20. The isolated VISTA antagonist according to any one of claims 1 to 19, which induces antitumor activity and / or antiviral activity.
21. Therapeutic, prophylactic, or diagnostic use comprising the isolated VISTA antagonist according to any one of claims 1-20, which is a therapeutically, prophylactically or diagnostically effective amount. Suitable composition for.
22. The composition of claim 21, further comprising a pharmaceutically acceptable carrier, diluent, solubilizer, preservative or mixture thereof.
23. The composition of any one of claims 21 or 22, further comprising another therapeutic agent.
24. The composition of claim 23, wherein the other therapeutic agent is an anticancer agent, an antiviral agent, a cytokine or an immunoagonist.
25. The composition of claim 21, wherein the other therapeutic agent is selected from CTLA-4-Ig, anti-PD-1, PD-L1 fusion protein or PD-L2 fusion protein, and an EGFR antagonist. thing.
26. The composition of any one of claims 21-25, which is suitable for subcutaneous or intravenous administration.
[Item 27] An isolated nucleic acid sequence encoding the VISTA antagonist peptide according to any one of claims 1 to 20, an analog thereof, a derivative or a mimic.
28. A vector containing the isolated nucleic acid sequence of claim 27.
29. A host cell comprising the isolated nucleic acid sequence of claim 27 or the vector of claim 28.
Item 30. The host cell according to claim 29, which is a mammalian cell, a bacterial cell, a fungal cell, a yeast cell, an avian cell or an insect cell.
31. A VISTA antagonist comprising culturing the host cell according to any one of claims 29 or 30 under conditions that provide expression of the VISTA antagonist peptide, analogs, derivatives or mimetics thereof. A method for expressing a peptide, an analog thereof, a derivative or an imitation.
32. A method of blocking, inhibiting or neutralizing VISTA-mediated T cell suppression, wherein an effective amount of the isolated VISTA antagonist or claim 21 to 20 of any one of claims 1-20. A method comprising administering to a subject in need thereof a composition comprising the isolated VISTA antagonist according to any one of 26.
33. A method of stimulating an immune response in a subject, wherein an effective amount of the isolated VISTA antagonist according to any one of claims 1 to 20 or any one of claims 21 to 26. A method comprising administering a composition comprising the isolated VISTA antagonist of the description to said subject in need thereof.
34. The method of claim 33, wherein the subject has an infection selected from the group consisting of bacterial, viral, parasitic and fungal infections.
[Claim 35], wherein the subject, Bordetella, Borrelia, Brucella, Burkholderia, Campylobacter, Chlamydia, Clostridium, Corynebacterium, Enterobacter, Enterococcus, Erwinia, Escherichia, Francisella, Haemophilus, Heliobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pasteurella, Pelobacter, Pseudomonas, Rickettsia, Salmonella, Serratia, Shigella, Staphylococcus, Streptococcus, Treptonema, Treponema, Vibrio, Yersina Method.
[Claim 36], wherein the subject, Adenoviridae, Papillomaviridae, Polyomaviridae, Herpesviridae, Poxviridae, Hepadnaviridae, Parvoviridae, Astroviridae, Caliciviridae, Picornaviridae, Coronoviridae, Flaviviridae, Retroviridae, Togaviridae, Arenaviridae, Bunyaviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, from Rhabdoviridae and Reoviridae 35. The method of claim 35, which has a viral infection caused by at least one virus selected from the group.
[Item 37] The virus is adenovirus, simple herpes type I, simple herpes type 2, varicella herpes virus, Epstein-bar virus, cytomegalovirus, herpesvirus type 8, papillomavirus, BK virus, JC virus, natural. Hepatitis B, hepatitis B, bocavirus, parvovirus B19, astrovirus, nowalk virus, coxsackie virus, hepatitis A, poliovirus, rhinovirus, severe acute respiratory syndrome virus, hepatitis C, yellow fever, dengue virus, west Nile virus, wind rash, hepatitis E, human immunodeficiency virus (HIV), influenza, guanaritovirus, funin virus, lassa fever virus, macpovirus, savia virus, Crimea-Congo hemorrhagic fever virus, Ebola virus, Marburg virus, 35. The method described.
[Item 38] The subjects are ringworm, candidiasis, cryptococcus disease, tinea capitis, blastomycetes, aspergillosis, tinea capitis, paracoccidiasis, sporotricum disease, tinea capitis, tinea capitis, and robo. Fungal disease, fungal tumor, tinea pedis, tinea pedis, tinea capitis, tinea capitis, tinea capitis, tinea capitis, tinea capitis, tinea pedis, ear fungal disease, phophihofungal disease or linos 34. The method of claim 34, which has a fungal infection selected from the group consisting of tinea capitis.
[Claim 39], wherein the subject, Entamoeba hystolytica, Giardia lamblia, Cryptosporidium muris, Trypanosomatida gambiense, Trypanosomatida rhodesiense, Trypanosomatida crusi, Leishmania mexicana, Leishmania braziliensis, Leishmania tropica, Leishmania donovani, Toxoplasma gondii, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodium falciparum, Trichomonas vaginalis, Histomonas meleagridis; Secementea; Trichuris trichiura, Ascaris lumbricoides, Enterobius vermicularis, Ancylostoma duodenale, Necator americanus, Strongyloides stercoralis, Wuchereria bancrofti, Dracunculus medinensis; schistosomiasis, liver fluke, intestinal flukes, Paragonimus; Schistosoma mansoni , Schistosoma flukes, Schistosoma japonicum, Fasciolas hepatica, Fasciolas gigantica, Heterophyes flukes and Paragonimus flukes 34.
40. The method of claim 33, for the treatment of cancer in a subject.
41. A method of promoting anti-cancer immunity or anti-tumor immunity, wherein an effective amount of the isolated VISTA antagonist according to any one of claims 1 to 20 or any of claims 21 to 26. A method comprising administering a composition comprising the isolated VISTA antagonist according to paragraph 1 to a subject in need thereof.
42. The isolated VISTA antagonist or claim of any one of claims 1-20, which is a method of treating or preventing cancer, inhibiting tumor infiltration and / or cancer metastasis, in an effective amount. A method comprising administering to a subject in need thereof a composition comprising the isolated VISTA antagonist according to any one of Items 21-26.
[Item 43] The cancers are cancer, lymphoma, blastoma, sarcoma, leukemia, lymphocytic malignant disease, melanoma, squamous epithelial cancer, lung cancer (small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous epithelium). Cancer), peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach cancer (including gastrointestinal cancer), pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer , Bladder cancer, liver cancer, breast cancer, colon cancer, colonic rectal cancer, endometrial cancer or uterine cancer, salivary adenocarcinoma, kidney cancer (kidney cancer) or kidney cancer (renal cancer), liver cancer, prostate cancer, genital cancer , Thyroid cancer, liver cancer, head and neck cancer, B-cell lymphoma (low grade / follicular non-hodgkin lymphoma (NHL); small lymphocytic (SL) NHL; moderate grade / follicular NHL; moderate grade diffuse NHL High-grade immunoblastic NHL; High-grade lymphoblastic NHL; High-grade small non-cutting nuclear cell NHL; Giant lesion NHL; Mantle cell lymphoma; AIDS-related lymphoma; Includes); Chronic lymphocytic leukemia (CLL); Acute lymphoblastic leukemia (ALL); Hairy cell leukemia; Chronic myeloid leukemia; Post-transplant lymphoproliferative disorder (PTLD), Mother spot The method of any one of claims 40 or 42, selected from the group consisting of abnormal vascular growth associated with the disease, edema (such as those associated with brain tumors), and Maegus syndrome.
44. A method of treating or preventing a viral infection, wherein an effective amount of the isolated VISTA antagonist according to any one of claims 1 to 20 or any one of claims 21 to 26. A method comprising administering a composition comprising the isolated VISTA antagonist of the description to a subject in need thereof.
[45] The invention of any one of claims 32 to 44, further comprising administration of another therapeutic agent, wherein the peptide and treatment method can be administered simultaneously or separately or in combination at different times. The method described.
46. The method of claim 45, wherein the other therapeutic agent is an anticancer agent, an antiviral agent or another anti-infective agent, a cytokine or an immunoagonist.
47. The method of claim 45, wherein the other therapeutic agent is selected from CTLA-4-Ig, anti-PD-1, PD-L1 fusion protein or PD-L2 fusion protein, and an EGFR antagonist. ..
[Item 48] A method for mapping the active site of VISTA, wherein (a) the isolated VISTA fusion protein is subjected to the amino acid sequence of SEQ ID NO: 1 (Ser-Ser-Ala-Cys-Asp-Trp-Ile-Lys). -Arg-Ser-Cys-His) contains the same peptide, or at most two amino acid residues have an amino acid sequence different from SEQ ID NO: 1. Peptides or their multimers, conjugates, analogs, derivatives or mimetics thereof. The method comprising incubating with an isolated VISTA antagonist comprising; (b) determining the binding site of the isolated VISTA antagonist.
49. The method of claim 48, wherein the active site of VISTA binds to the VISTA receptor and mediates immunosuppression.
50. The method of claim 48, wherein step (b) comprises domain deletion, domain swapping, amino acid mutagenesis, foot printing, NMR, X-ray crystallography, or homology modeling.

Claims (3)

A cell expressing V ISTA antagonist peptides, the peptide the amino acid sequence of SEQ ID NO: 1 that antagonize VISTA activity bound to VISTA (Ser-Ser-Ala- Cys-Asp-Trp-Ile-Lys-Arg- The cell, which is identical to Ser-Cys-His). A composition comprising an isolated VISTA antagonist peptide and a pharmaceutically acceptable carrier , wherein the peptide binds to VISTA and antagonizes VISTA activity with the amino acid sequence of SEQ ID NO: 1 (Ser-Ser-Ala-Cys). -Asp-Trp-Ile-Lys-Arg-Ser-Cys-His) . The composition according to claim 2 , further comprising a diluent, a solubilizer, a preservative or a mixture thereof.

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