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WO2025069022A1 - Immunomodulating peptides and uses thereof - Google Patents

Immunomodulating peptides and uses thereof Download PDF

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Publication number
WO2025069022A1
WO2025069022A1 PCT/IL2024/050951 IL2024050951W WO2025069022A1 WO 2025069022 A1 WO2025069022 A1 WO 2025069022A1 IL 2024050951 W IL2024050951 W IL 2024050951W WO 2025069022 A1 WO2025069022 A1 WO 2025069022A1
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Prior art keywords
peptide
amino acid
seq
pharmaceutical composition
amino acids
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PCT/IL2024/050951
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French (fr)
Inventor
Jonathan E. COHEN
Assaf Friedler
Dana GRUNHAUS
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Hadasit Medical Research Services and Development Co
Yissum Research Development Co of Hebrew University of Jerusalem
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Hadasit Medical Research Services and Development Co
Yissum Research Development Co of Hebrew University of Jerusalem
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Publication of WO2025069022A1 publication Critical patent/WO2025069022A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to novel peptides having immunomodulatory activity and use thereof.
  • SLAMF6 Signaling lymphocytic activation molecule 6
  • Lyl08 or NTB-A is a homotypic surface receptor expressed on T cells, natural killer (NK) cells, B cells, dendritic cells (DC) and myeloid-derived suppressor cells (MDSC).
  • SLAMF6 is largely considered an inhibitory receptor, although certain cell and context- specific effects were determined for this receptor, leading to its characterization as a dual receptor, capable of exerting either activating or inhibitory effects in the context of immune modulation. Engagement of SLAMF6 on human T cells can substitute the CD28 co-stimulatory pathway and induce polarization toward a Thl phenotype.
  • CD4-positive T cells from Ly-108 knockout mice show impairment in IL-4 production, suggesting a role of SLAMF6 in Th2 polarization. The reason for this discrepancy is not fully elucidated.
  • Activation of SLAMF6 on human NK cells stimulates cytotoxicity and proliferation, as well as IFN-y and TNF-a production.
  • SLAMF6 is a natural ligand of SLAMF6 itself.
  • Valdez et al (J Biol Chem 2004, 279(18), pp. 18662-18669) teach that SLAMF6 activates T cells by homotypic interactions, and specifically enhances Thl properties.
  • US 2009/017014 to Valdez et al is directed to the PR020080 polypeptide (having an amino acid sequence corresponding to that of canonical SLAMF6), the extracellular portion thereof, homologs, agonists and antagonists thereof, which are suggested as putative modulators of immune diseases.
  • Uzana et al. J Immunol 2012, 188, pp. 632-640 disclose that SLAMF6 blockade on antigen presenting cells (APC) by specific antibodies inhibited cytokine secretion from CD8 + lymphocytes.
  • APC antigen presenting cells
  • Immunosuppressants are medicines that inhibit or decrease the intensity of the immune response in the body, often used in the management of graft rejection and certain autoimmune disorders.
  • currently available immunosuppressants generally have a broad spectrum of activity leading to significant undesirable effects.
  • some widely employed agents have very long half-lives and lack antidotes, thus leading to extended immune-suppression and risk of severe infections.
  • the present invention relates to novel peptides having immunomodulatory activity, compositions comprising same and use thereof.
  • synthetic peptides capable of inhibiting or preventing excessive immune activation and pathologies associated therewith.
  • compositions and methods for use in inhibiting or reducing immune-related adverse events and in the treatment of diseases and conditions associated with the inappropriate or excessive immune response including, but not limited to autoimmune diseases, graft versus host disease, and graft rejection.
  • the invention is based, in part, on the development of immunomodulating peptides having exceptionally advantageous therapeutic properties.
  • isolated SLAMF6-derived peptides and modified peptides comprising various deletions, substitutions and derivatization by D-amino acids, were synthesized and characterized.
  • peptides of the invention as detailed below were identified to suppress manifestations of immune cell activation in various models, including activation-induced secretion of cytokines and cytolytic granule components, in a SLAMF6-dependent manner.
  • synthetic peptides characterized by improved pharmacokinetic properties, including enhanced stability and resistance to serum proteases is disclosed. The peptides were demonstrated to exhibit potent anti-inflammatory properties in vivo and significantly reduced the severity of colitis in mice.
  • the invention relates to an isolated peptide comprising the amino acid sequence X0TWX1FNX2X3X4X5A set forth as SEQ ID NO: 1 or a retro-inverso sequence thereof.
  • the isolated peptide comprises the amino acid sequence X0TWX1FNX2X3X4X5A (SEQ ID NO: 1) or a retro-inverso sequence thereof, wherein X o and X 5 are each independently a hydrophobic amino acid, Xi , X 2 , X 3 and X 4 are each independently any amino acid and wherein the peptide consists of from 11 to 50 amino acids.
  • the isolated peptide is characterized in that:
  • X o and X 5 are each independently selected from Leu, Vai and He;
  • Xi is selected from Leu, He, Vai, Met, Ser and Thr;
  • X 2 is selected from Gly, Ala, Asp and Glu;
  • X 3 is selected from Ser, Thr, Lys and Arg;
  • X 4 is selected from Ser, Asn, Gin, and Thr (SEQ ID NO: 17).
  • the isolated peptide is characterized in that:
  • XQ and X 5 are each independently selected from Leu, Vai and He;
  • Xi is selected from Thr, and Leu;
  • X 2 is selected from Gly, Glu and Ala;
  • X 3 is selected from Lys and Thr;
  • X 4 is selected from Ser and Thr, set forth as SEQ ID NO: 18.
  • the isolated peptide comprises the amino acid sequence I TWTFNGKSLA (SEQ ID NO: 2).
  • peptides in accordance of the invention are advantageously characterized by the inclusion of at least one D-amino acid.
  • at least 2 amino acids are D-amino acids.
  • 2, 3, 4 or 5 amino acids are D-amino acids, wherein each possibility represents a separate embodiment of the invention.
  • At least 2 of the amino acids at positions 3, 5, 8 and 11 of the sequence X 0 TWX 4 FNX 2 X 3 X 4 X 5 A are D-amino acids.
  • the amino acids at positions 3, 5, 8 and 11 are D-amino acids and the peptide comprises the amino acid sequence X 0 TwX 4 fNX 2 x 3 X 4 X 5 a (SEQ ID NO: 6, wherein D-amino acids are represented by low-case letters).
  • the peptide comprises the amino acid sequence I TwT fNGkSLa (SEQ ID NO: 7).
  • said peptide consists of the amino acid sequence I TwT fNGkSLa (SEQ ID NO: 7).
  • the invention relates to a conjugate of an isolated peptide as disclosed herein.
  • the conjugate comprises said peptide conjugated with polyethylene glycol (PEG).
  • a pharmaceutical composition comprising at least one peptide or conjugate as disclosed herein, and a pharmaceutically acceptable carrier and/or excipient.
  • the peptide comprises or consists of an amino acid sequence selected from I TWTFNGKSLA (SEQ ID NO: 2) and I TwT fNGkSLa (SEQ ID NO: 7). Each possibility represents a separate embodiment of the invention.
  • the pharmaceutical composition is for use in inhibiting or reducing an inappropriate or excessive immune response in a subject in need thereof.
  • the use comprises inhibiting or reducing immune-related adverse events (irAEs).
  • the irAEs are selected from the group consisting of gastrointestinal, endocrine, and dermatologic toxicities, and combinations thereof.
  • the irAEs may comprise toxicities affecting the pancreas and/or thyroid gland.
  • said irAEs are associated with administration of an immuno stimulatory treatment.
  • said immuno stimulatory treatment is a cancer immunotherapy.
  • said irAEs are associated with administration of a cancer immunotherapy.
  • said cancer immunotherapy comprises administration of an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor targets PD-1, PD-L1, PD-L2, CTLA-4, LAG3, TIM3, and/or IDO.
  • the use comprises treating a disease or a condition associated with the inappropriate or excessive immune response.
  • the condition is a T-cell mediated inflammatory or autoimmune condition.
  • the disease or condition is selected from the group consisting of graft versus host disease, an autoimmune disease, and graft rejection.
  • the autoimmune disease is selected from rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), multiple sclerosis (MS), psoriasis, psoriatic arthritis, autoimmune hepatitis and primary biliary cholangitis (PBC).
  • RA rheumatoid arthritis
  • SLE systemic lupus erythematosus
  • IBD inflammatory bowel disease
  • MS multiple sclerosis
  • psoriasis psoriatic arthritis
  • autoimmune hepatitis and primary biliary cholangitis (PBC).
  • said autoimmune disease
  • the pharmaceutical composition comprises at least one isolated peptide comprising the amino acid sequence X0TWX1FNX2X3X4X5A (SEQ ID NO: 1) or a retro-inverso sequence thereof, wherein Xo and X5 are each independently a hydrophobic amino acid, Xi, X2, X3 and X4 are each independently any amino acid and wherein the peptide consists of from 11 to 50 amino acids.
  • the peptide comprises or consists of an amino acid sequence selected from I TWTFNGKSLA (SEQ ID NO: 2) and I TwT fNGkSLa (SEQ ID NO: 7).
  • the method comprises inhibiting or reducing irAEs (e.g. associated with treatments as disclosed herein), in some embodiments, the irAEs are selected from the group consisting of gastrointestinal, endocrine, and dermatologic toxicities, and combinations thereof. In other embodiments, the irAEs comprise toxicities affecting the pancreas and/or thyroid gland. In another embodiment said irAEs are associated with administration of a cancer immunotherapy. In another embodiment said cancer immunotherapy comprises administration of an immune checkpoint inhibitor.
  • the immune checkpoint inhibitor targets PD-1, PD-L1, PD-L2, CTLA-4, LAG3, TIM3, and/or IDO.
  • the method comprises treating a disease or a condition associated with the inappropriate or excessive immune response (e.g. the conditions as disclosed herein).
  • the condition is a T-cell mediated inflammatory or autoimmune condition.
  • the autoimmune disease is selected from rheumatoid arthritis, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), multiple sclerosis (MS), psoriasis, psoriatic arthritis, autoimmune hepatitis, primary biliary cholangitis (PBC), insulin dependent diabetes mellitus and autoimmune thyroiditis, wherein each possibility represents a separate embodiment of the invention.
  • said autoimmune disease is IBD.
  • said autoimmune disease is insulin dependent diabetes mellitus or autoimmune thyroiditis.
  • Fig. 1A shows the amino acid sequence of the SLAMF6 receptor (SEQ ID NO: 12) with the initial peptide sequence spanning amino acids 49 to 91 (V49) marked in bold.
  • Fig. IB shows a three-dimensional structure of the SLAMF6 receptor- V49 in dark grey.
  • Fig. 1C shows binding of V49 to SLAMF6.
  • Mean fluorescence intensity (MFI) was measured by flow cytometry.
  • Fig. ID shows the inhibitory effect of V49 on peripheral blood mononuclear cells (PBMC) that were activated with staphylococcal enterotoxin B (SEB). Activated cells were incubated with the V49, V49(ala) or V49(scr) peptides at the indicated concentrations, and Interleukin-2 (IL-2) secretion was measured by ELISA.
  • PBMC peripheral blood mononuclear cells
  • SEB staphylococcal enterotoxin B
  • Fig. IE shows the inhibitory effect of V49 on tumor-infiltrating lymphocytes (TILs) that were activated by incubation with cognate melanoma cells. Activated cells were incubated with peptides as describe in Fig. ID at the indicated concentrations, and interferon gamma (IFNy) secretion was measured via ELISA.
  • TILs tumor-infiltrating lymphocytes
  • IFNy interferon gamma
  • Fig. 2A shows the inhibitory effect of the 153 peptide on TIL in the presence of cognate 526 melanoma cells. Activated cells were incubated with the indicated peptides at the indicated concentrations, and IFNy secretion was measured by ELISA.
  • Fig. 2B shows the inhibitory effect of the 153 peptide on TIL in the presence of cognate 624 melanoma cells. Activated cells were incubated with the indicated peptides at the indicated concentrations, and IFNy secretion was measured by ELISA.
  • Fig. 2C shows the inhibitory effect of peptides on activation-induced IL-2 secretion in PBMC.
  • SEB-activated cells were incubated with the indicated peptides at the indicated concentrations, and IL-2 secretion was measured by ELISA.
  • Fig. 2D shows the inhibitory effect of different concentrations 153 peptide on PBMC function (measured as IL-2 secretion by ELISA) following SEB activation.
  • Fig. 2E shows the inhibitory effect of 153 and 3mut on PBMCs activated with SEB and treated at 0.5uM and 2.5uM peptides.
  • 153 (ala) was a negative control.
  • IL2 secretion levels were measured by ELISA.
  • Fig. 2F shows the inhibitory effect of 153, CRla and 3mut on TILs activated with their cognate melanoma cells.
  • 153 (ala) used as negative control.
  • IFNy secretion levels were measured by ELISA.
  • Fig. 2G shows the inhibitory effect of peptides of 3mut on murine pMEL splenocytes co-cultured with cognate melanoma cells as compared with a scrambled sequence of 3mut as control (scr). IFNy secretion was measured by ELISA.
  • Fig. 3A shows the stability of the 3Mut (SEQ ID NO: 2) peptide as tested by trypsin and chymotrypsin proteolysis.
  • Fig. 3B shows the stability of the WFKA peptide SEQ ID NO: 7) as tested by trypsin and chymotrypsin proteolysis.
  • Fig. 3C shows the inhibitory effect of WFKA on PBMC cells activated using SEB. Activated cells were incubated with the indicated peptides at the indicated concentrations and IL-2 secretion was measured by ELISA.
  • Fig. 3D shows the inhibitory effect of WFKA on melanoma-specific human TILs co-cultured with a specific melanoma identified by the TILs. IFNy secretion was measured by ELISA.
  • Fig. 3E shows the inhibitory effect of WFKA on murine splenocytes co-cultured with cognate melanoma cells (B16 GP100). IFNy secretion was measured by ELISA.
  • Fig. 4A shows the conserved pattern of 3Mut and WFKA peptides (in boxes).
  • Fig. 4B shows dose-response of WFKA on activation-induced Granzyme-B (GZM-b) secretion in the human Jurkat cell line.
  • CD3-activated cells were incubated with the indicated concentrations of the peptide, and GZM-b was measured by ELISA.
  • Fig. 4C shows the effect of WFKA, scrl and scr2 peptides on activation-induced GZM-b secretion in wild-type Jurkat cells (WT) as compared to SLAMF6 knockout Jurkat cells (SLAMF6' 7 ')- CD3- activated cells were incubated with the indicated peptides at the indicated concentrations, and GZM-b was measured by ELISA.
  • Fig. 4D shows the effect of WFKA, scrl and scr2 on murine splenocytes (WT) as compared to Lyl06-knockout splenocytes (SLAMF6' /_ ). IFNy secretion was measured by ELISA.
  • Fig. 4E shows Granzyme B (GZM-b) secretion levels in JURKAT wild type (WT) cells in the presence of the MUT3 peptide or the WFKA peptide, as measured by ELISA. Scrl and Scr2 peptides were used as negative controls. CD3-activated cells were incubated with the indicated concentrations of the peptide, and GZM-b was measured by ELISA.
  • GZM-b Granzyme B secretion levels in JURKAT wild type (WT) cells in the presence of the MUT3 peptide or the WFKA peptide, as measured by ELISA.
  • Fig. 4F shows that in JURKAT SLAMF6 7 ' cells, GZM-b secretion was not inhibited by the MUT3 or WFKA peptides, indicating that the inhibitory effect of these peptides is directly dependent on the presence of the SLAMF6 receptor.
  • Treatments and methods are as in Fig. 4E.
  • Figs. 5A-5D show scores for different parameters in a DSS-induced colitis murine model.
  • Fig. 5A shows weight loss score
  • Fig. 5B shows diarrhea incidence
  • Fig. 5C shows bleeding score
  • Fig. 5D shows DAI score.
  • C57BL/6 mice were induced with colitis by 2% DSS treatment.
  • the administration of DSS inducing colitis was ceased.
  • Group C - naive mice no DSS, no peptide treatment).
  • Figs. 6A-6C show colons of mice of from different groups of the DSS-induced colitis murine model.
  • Fig. 6A shows colon length in naive mice (Group C).
  • Fig. 6B shows colon length in mice treated with DSS only (Group A).
  • Fig. 6C shows colon length in mice treated with DSS and 3 pM WFKA peptide (Group B).
  • Fig. 7 shows a comparison of colon length between WFKA peptide-treated (Group B) and untreated (Group A) mice of the DSS-induced colitis mice as compared to naive mice (Group C).
  • the present invention relates to novel peptides having immunomodulatory activity, compositions comprising same and use thereof. More specifically, disclosed herein are synthetic peptides capable of inhibiting or preventing excessive immune activation and pathologies associated therewith.
  • the compositions and methods as disclosed herein may be used for inhibiting or reducing immune-related adverse events and in the treatment of diseases and conditions associated with the inappropriate or excessive immune response, including, but not limited to autoimmune diseases, graft versus host disease (GVHD), and graft rejection.
  • the present invention is based on discovery of novel stable peptides capable of modulating immune responses. Specifically, disclosed herein is the identification and characterization of synthetic, non-naturally occurring peptides, shown to exhibit a considerable and significant ability to control excessive immune activation in various experimental models employing the use of human or murine-derived lymphocytes. In vivo, the peptides significantly delayed the development of colitis in a murine model for inflammatory bowel disease (IBD), as evaluated by various clinical and inflammatory manifestations.
  • IBD inflammatory bowel disease
  • the invention relates to an isolated peptide comprising the amino acid sequence X0TWX1FNX2X3X4X5A (SEQ ID NO: 1) or a retro-inverso sequence thereof, wherein X o and X 5 are each independently a hydrophobic amino acid, X 1 A X 2 , X 3 and X 4 are each independently any amino acid and wherein the peptide consists of from 11 to 50 amino acids.
  • the isolated peptide comprises or consists of the amino acid sequence selected from I TWTFNGKSLA (SEQ ID NO: 2) and I TwT fNGkSLa (SEQ ID NO: 7).
  • the invention in another aspect, relates to a pharmaceutical composition
  • a pharmaceutical composition comprising at least one peptide isolated peptide as defined herein, or a conjugate thereof.
  • the pharmaceutical composition is for use in inhibiting or reducing an inappropriate or excessive immune response in a subject in need thereof.
  • the invention relates to a pharmaceutical composition comprising an isolated peptide of the amino acid sequence X0TWX1FNX2X3X4X5A (SEQ ID NO: 1) as defined herein or a retro-inverso sequence thereof, for use in inhibiting or reducing an inappropriate or excessive immune response in a subject in need thereof.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an isolated peptide that comprises or consists of the amino acid sequence selected from I TWTFNGKSLA (SEQ ID NO: 2) and I TwT fNGkSLa (SEQ ID NO: 7), for use in inhibiting or reducing an inappropriate or excessive immune response in a subject in need thereof.
  • the pharmaceutical composition is for use in inhibiting or reducing immune-related adverse events (irAEs).
  • the pharmaceutical composition is for use in treating a disease or a condition associated with the inappropriate or excessive immune response.
  • the invention provides a method for inhibiting or reducing an inappropriate or excessive immune response in a subject in need thereof, comprising administering to the subject the pharmaceutical composition (e.g. comprising an isolated peptide as set forth in any one of SEQ ID NOs: 1, 2 and 7 as defined herein).
  • the invention provides a method for inhibiting or reducing irAEs in a subject in need thereof, comprising administering to the subject the pharmaceutical composition (e.g. comprising an isolated peptide as set forth in any one of SEQ ID NOs: 1, 2 and 7 as defined herein).
  • the irAEs are associated with administration of an immune checkpoint inhibitor (or other forms of cancer immunotherapy).
  • the invention provides a method for treating a disease or a condition associated with the inappropriate or excessive immune response in a subject in need thereof, comprising administering to the subject the pharmaceutical composition (e.g. comprising an isolated peptide as set forth in any one of SEQ ID NOs: 1, 2 and 7 as defined herein).
  • the disease or condition is selected from the group consisting of graft versus host disease (GVHD), an autoimmune disease, and graft rejection.
  • the invention provides a method for treating an autoimmune disease in a subject in need thereof, comprising administering to the subject the pharmaceutical composition (e.g. comprising an isolated peptide as set forth in any one of SEQ ID NOs: 1, 2 and 7 as defined herein).
  • the autoimmune disease is selected from rheumatoid arthritis, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), multiple sclerosis (MS), psoriasis, psoriatic arthritis, autoimmune hepatitis, primary biliary cholangitis (PBC), insulin dependent diabetes mellitus and autoimmune thyroiditis.
  • the present invention provides an isolated peptide comprising an amino acid sequence X0TWX1FNX2X3X4X5A set forth as SEQ ID NO: 1 or a retro-inverso sequence thereof, wherein Xo and X5 are each independently a hydrophobic amino acid, wherein X , , X 2 , X 3 and X 4 are each independently any amino acid and wherein the peptide consists of from 11 to 50 amino acids.
  • the peptide comprises the amino acid sequence X0TWX1FNX2X3X4X5A, wherein X o and X 5 are the same hydrophobic amino acid and Xi , X 2 , X 3 and X 4 are each independently any amino acid and wherein the peptide consists of from 11 to 50 amino acids.
  • the term "peptide" as used herein refers to a short chain of amino acid monomers linked by peptide bonds, i.e., the covalent bond formed between carboxyl group of one amino acid and an amino group of another amino acid. Peptides usually comprise up to 50 amino acids. According to some embodiments, the peptide consists of 12 to 45 amino acids.
  • the peptide consists of 15 to 40 amino acids. According to some embodiments, the peptide comprises 17 to 35 amino acids. According to other embodiments, the peptide consists of 20 to 30 amino acids. According to another embodiment, the peptide consists of 11 to 30 amino acids. According to some embodiments, the peptide consists of 11-15, 11-20, 12 to 25, 13 to 23, or 14 to 20, amino acids. Each possibility represents a separate embodiment of the invention. According to some embodiments, the peptide consists of 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.
  • isolated peptide is meant to include peptides which are not naturally occurring and that do not naturally exist outside the context of their complete, full-length source proteins (if any).
  • isolated is also used herein to refer to peptides of polypeptides that are isolated from other cellular proteins and constituents and is meant to encompass chemically synthesized, recombinantly produced and purified peptides and polypeptides. The term further encompasses in some embodiments separation from the source that produced the peptide, such as recombinant cells or residual peptide synthesis reactants.
  • the isolated peptide is optionally “purified”, which means at least: 80%, 85%, 90%, 95%, 98% or 99% purity and optionally pharmaceutical grade purity.
  • amino acid includes both “natural” and “unnatural” or “non-natural” amino acids.
  • Xo is a hydrophobic amino acid.
  • hydrophobic amino acids or related terms such as “hydrophobic moieties provided by the residues of amino acids” is referring to neutral amino acids which comprise to a large extent mainly a hydrophobic moiety apart from their amino and carboxy group.
  • hydrophobic amino acid refers to Gly, Ala, Vai, Leu, Aic, He, Pro, Tyr, Phe, Met, Eaa, naphthylalanine and Trp.
  • the hydrophobic amino acid is selected from Gly, Ala, Vai, Leu, He, Pro, Tyr, Phe, Met and Trp.
  • small hydrophobic amino acid refers to hydrophobic amino acid having non-bulky residue such as Gly and Ala.
  • Xo is selected from Leu, He, Vai and Met amino acids.
  • Xo is selected from Leu, Vai and He.
  • Xo is He.
  • X5 is a hydrophobic amino acid selected from Gly, Ala, Vai, Leu, He, Pro, Tyr, Phe, Met and Trp.
  • X5 is selected from Leu, He, Vai and Met amino acids.
  • X5 is selected from Leu, Vai and He.
  • X5 is Leu.
  • Xo is He and X5 is Leu.
  • Xi is selected from a hydrophobic and a polar amino acid.
  • polar amino acid and “polar uncharged amino acid residue” refers to an amino acid residue having a side chain that is uncharged and has a dipole moment.
  • polar amino acid residues include, but are not limited to glycine, sarcosine, L-serine, L-threonine, L-cysteine, L-tyrosine, L-asparagine, L-glutamine, D-serine, D- threonine, D- cysteine, D-tyrosine, D-asparagine and D-glutamine, N-methyl-L- serine, N- methyl-L-threonine, N-methyl-L-cysteine, N-methyl-L-tyrosine, N-methyl-L-asparagine, N-methyl-L-glutamine, N- methyl-D- serine, N-methyl-D-threonine, N-methyl-D-cysteine, N
  • the polar amino acid refers to an amino acid selected from the group consisting of Asn, Gin, Ser, Thr, Cys and Tyr. According to some embodiments, the polar amino acid is selected from Asn, Gin, Ser, and Thr. According to other embodiments, the polar amino acid is selected from Ser, and Thr.
  • Xi is selected from Leu, He, Vai, Met, Ser and Thr amino acid. According to one embodiment, Xi is selected from Leu and Thr. According to one embodiment, Xi is Thr. According to one embodiment, Xi is Leu. According to some embodiments, Xoand X5 are each independently selected from Leu, He, Vai and Met amino acids, and Xi is selected from Leu, He, Vai, Met, Ser and Thr amino acid. According to some embodiments, Xo is He, X5 is Leu, and Xi is Thr.
  • X2 is selected from a small hydrophobic and negatively charged amino acid.
  • the term “negatively charged amino acid” refers to an amino acid residue having a side chain capable of bearing a negative charge. Examples include, but are not limited to L-aspartic acid, L-glutamic acid, D-aspartic acid, D- glutamic acid, N-methyl-L- aspartic acid, N-methyl-L-glutamic acid, N-methyl-D-aspartic acid and N-methyl-D-glutamic acid.
  • negatively charged amino acid is selected from Asp and Glu.
  • X2 is selected from Gly, Ala, Asp and Glu. According to some embodiments, X2 is Gly. According to some embodiments, X2 is Glu. According to some embodiments, Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids, and X2 is selected from Gly, Ala, Asp and Glu. According to some embodiments, Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids, and X2 is selected from Gly and Glu. According to some embodiments, Xo is He, X5 is Leu, and X2 is Gly. According to some embodiments, Xo is He, X5 is Leu, and X2 is Glu.
  • Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids, Xi is selected from Leu, He, Vai, Met, Ser and Thr amino acid and X2 is selected from Gly, Ala, Asp and Glu.
  • Xo is He, X5 is Leu, Xi is Thr and X2 is Gly.
  • Xo is He, X5 is Leu, Xi is Leu and X2 is Gly.
  • Xo is He, X5 is Leu, Xi is Thr and X2 is Glu.
  • Xo is He, X5 is Leu, Xi is Leu and X2 is Glu.
  • X3 is selected from a polar amino acid and a positively charged amino acid.
  • positively charged amino acid residue refers to an amino acid residue having a side chain capable of bearing a positive charge.
  • examples include, but are not limited to L-lysine, L-arginine, L-histidine, L-ornithine, D-lysine, D- arginine, D-histidine, D- omithine, N-methyl-L-lysine, N-methyl-L-arginine, N-methyl-L- histidine, N-methyl-L-ornithine, N-methyl-D-lysine, N-methyl-D-arginine, N-methyl- D-histidine, N-methyl-D-ornithine, L- diaminobutyric acid (DAB), D-di aminobutyric acid, N-methyl-L-diaminobutyric acid, N-methyl- D-diaminobutyric acid, L-citrulline (CIT), D- citrulline, N-methyl
  • X3 is selected from Ser, Thr, Lys and Arg. According to some embodiments, X3 is Lys. According to some embodiments, X3 is Thr. According to some embodiments, Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids, and X3 is selected from Ser, Thr, Lys and Arg. According to some embodiments, Xo is He, X5 is Leu, and X3 is Lys. According to some embodiments, Xo is He, X5 is Leu, and X3 is Thr.
  • Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids, Xi is selected from Leu, He, Vai, Met, Ser and Thr amino acid, and X3 is selected from Ser, Thr, Lys and Arg.
  • Xo is He, X5 is Leu, Xi is Thr and X3 is Lys.
  • Xo is He, X5 is Leu, Xi is Thr and X3 is Thr.
  • Xoand X5 are each independently selected from Leu, He, Vai and Met amino acids, X2 is selected from Gly, Ala, Asp and Glu, and X3 is selected from Ser, Thr, Lys and Arg.
  • Xo is He, X5 is Leu, X2 is Gly and X3 is Lys.
  • Xo is He, X5 is Leu, X2 is Gly and X3 is Thr.
  • Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids, Xi is selected from Leu, He, Vai, Met, Ser and Thr amino acid, X2 is selected from Gly, Ala, Asp and Glu, and X3 is selected from Ser, Thr, Lys and Arg.
  • Xo is He
  • X5 is Leu
  • Xi is Thr
  • Xo is He
  • X5 is Leu, He, Xi is Thr
  • X2 is Gly and X3 is Thr.
  • X4 is a polar amino acid.
  • X4 is selected from Asn, Gin, Ser and Thr.
  • X4 is Ser.
  • Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids, and X4 is selected from Asn, Gin, Ser and Thr.
  • Xo is He
  • X5 is Leu
  • X4 is Ser.
  • Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids
  • Xi is selected from Leu, He, Vai, Met, Ser and Thr amino acid
  • X4 is selected from Asn, Gin, Ser and Thr.
  • Xo is He, X5 is Leu, Xi is Thr and X4 is Ser.
  • Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids, X2 is selected from Gly, Ala, Asp and Glu and X4 is selected from Asn, Gin, Ser and Thr.
  • Xo is He
  • X5 is Leu
  • X2 is Gly
  • X4 is Ser.
  • Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids
  • X3 is selected from Ser, Thr, Lys and Arg
  • X4 is selected from Asn, Gin, Ser and Thr.
  • Xo is He, X5 is Leu, X3 is Lys and X4 is Ser.
  • Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids, Xi is selected from Leu, He, Vai, Met, Ser and Thr amino acid, X2 is selected from Gly, Ala, Asp and Glu, and X4 is selected from Asn, Gin, Ser and Thr.
  • Xo is He
  • X5 is Leu Xi is Thr
  • X2 is Gly
  • X4 is Ser.
  • Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids, Xi is selected from Leu, He, Vai, Met, Ser and Thr amino acid, X3 is selected from Ser, Thr, Lys and Arg and X4 is selected from Asn, Gin, Ser and Thr. According to some embodiments, Xo is He, X5 is Leu Xi is Thr, X3 is Lys and X4 is Ser.
  • Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids, X2 is selected from Gly, Ala, Asp and Glu, X3 is selected from Ser, Thr, Lys and Arg and X4 is selected from Asn, Gin, Ser and Thr.
  • Xo is He
  • X5 is Leu
  • X2 is Gly
  • X3 is Lys
  • X4 is Ser.
  • Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids, Xi is selected from Leu, He, Vai, Met, Ser and Thr, X2 is selected from Gly, Ala, Asp and Glu, X3 is selected from Ser, Thr, Lys and Arg and X4 is selected from Asn, Gin, Ser and Thr.
  • Xo is He
  • X5 is Leu Xi is Thr
  • X2 is Gly
  • X3 is Lys
  • X4 is Ser.
  • the peptide is characterized in that:
  • Xo and X5 are each independently selected from Leu, Vai and He;
  • Xi is selected from Leu, He, Vai, Met, Ser and Thr;
  • X2 is selected from Gly, Ala, Asp and Glu;
  • X3 is selected from Ser, Thr, Lys and Arg;
  • X4 is selected from Ser, Asp, Gin, and Thr.
  • the peptide is characterized in that:
  • Xo and X5 are each independently selected from Leu, Vai and He; Xi is selected from Thr, and Leu; X2 is selected from Gly, Glu and Ala; X3 is selected from Lys and Thr; X4 is selected from Ser and Thr, set forth as SEQ ID NO: 17.
  • the peptide is characterized in that:
  • Xo and X5 are each independently selected from Leu and He; Xi is selected from Thr, and Leu; X2 is selected from Gly and Glu; X3 is selected from Lys and Thr; X4 is selected from Ser and Thr, set forth as SEQ ID NO: 18.
  • the peptide comprises an amino acid sequence selected from SEQ ID NO: 20-163. According to some embodiments, the peptide comprises the amino acid sequence ITWTFNGKSLA (SEQ ID NO: 2). According to some embodiments, the peptide consists of the amino acid sequence ITWTFNGKSLA (SEQ ID NO: 2).
  • the peptide comprises or consists of the amino acid sequence VNFITWLFNETSLAFIVPHETKSPEIHVTNPKQGKRLNFTQS (SEQ ID NO: 3). According to some embodiments, the peptide comprises or consists of the amino acid sequence VNFITWTFNGKSLAFIVPHETKSPEIHVTNPKQGKRLNFTQS (SEQ ID NO: 5).
  • At least 1 amino acid of the peptide of the present invention may be a D-amino acid.
  • at least 2 amino acids of the peptide of the present invention are D-amino acids.
  • at least 3 amino acids of the peptide of the present invention are D-amino acids.
  • at least 2 and up to 4 or 5 amino acids of the peptide of the present invention are D- amino acids.
  • from 2 to 6 amino acids of the peptide of the present invention are D-amino acid.
  • 2, 3, 4, 5, 6, or 7 amino acids of the peptide of the present invention having an amino acid sequence SEQ ID NO:1 are D-amino acids.
  • 3 amino acids of the peptide of the present invention are D- amino acids.
  • 4 amino acids of the peptide of the present invention are D-amino acids.
  • 5 amino acids of the peptide of the present invention are D-amino acids.
  • D-amino acid refers to an amino acid having the D-configuration around the a-carbon as opposite to native L-amino acid having L-conformation. As used herein, the D-amino acid in the sequence is represented by a lower-case letter.
  • the L-amino acid is represented by a capital letter.
  • the sequence of VyL represent a sequence in which Vai and Leu are native L-amino acids and Tyr is a D-amino acid.
  • At least 2 amino acids at positions 3, 5, 8 and 11 of the sequence X0TWX1FNX2X3X4X5A are D-amino acids.
  • at least 3 amino acids at positions 3, 5, 8 and 11 of the sequence X0TWX1FNX2X3X4X5A are D-amino acids.
  • the present invention provides a peptide having the amino acid sequence XoTwXifNX2X3X4Xsa (SEQ ID NO: 6) wherein Xo and X5 are each independently a hydrophobic amino acid, Xi, X2, and X4 are each independently any amino acid, and X3 is any D-amino acid.
  • the present invention provides a peptide having the amino acid sequence SEQ ID NO: 6 in which amino acids at positions 3, 5, 8 and 11 of the sequence are D-amino acids. According to some embodiments, the present invention provides a peptide comprising an amino acid sequence selected from SEQ ID NO: 20-163 in which amino acids at positions 3, 5, 8 and 11 of the sequence are D-amino acids. According to some embodiments, the present invention provides a peptide comprising an amino acid sequence selected from SEQ ID NO: 20-163 in which amino acids at positions 3, 5, 8 and 11 of the sequence are D-amino acids and having at least 1 additional amino acid being D-amino acid.
  • the present invention provides a peptide comprising the amino acid sequence ITwTfNGkSLa (SEQ ID NO: 7). According to another embodiment, the present invention provides a peptide consisting of the amino acid sequence ITwTfNGkSLa.
  • VNFITwTfNGkSLaFIVPHETKSPEIHVTNPKQGKRLNFTQS (SEQ ID NO: 8).
  • the peptide of the present invention comprises the retro- inverso amino acid sequence of the amino acid sequence X0TWX1FNX2X3X4X5A (SEQ ID NO: 1). According to one embodiment, the peptide of the present invention comprises the retro-inverso amino acid sequence of a peptide sequence as disclosed herein. Each possibility represents a separate embodiment of the invention.
  • the peptide of the present invention comprises a retro- inverso amino acid sequence of an amino acid sequence selected from SEQ ID NO: 2, 16, and 20- 163.
  • the peptide comprises the amino acid sequence alskgnftwti (SEQ ID NO: 9).
  • the peptide has at least 80% sequence identity to a peptide as disclosed herein. In another embodiment the peptide has at least 90% sequence identity to a peptide as disclosed herein.
  • a peptide according to the general formula of SEQ ID NO: 1 which further has 80% or 90% sequence identity to the peptide of SEQ ID NO: 7.
  • the peptide has at least 80% or at least 90% sequence identity to the peptide of SEQ ID NO: 2.
  • peptides of the invention exhibit immunomodulatory properties (and are thus capable of modulating an immune response or components thereof).
  • peptides in accordance with the invention are capable of exhibiting immunosuppressive activity.
  • peptides of the invention were demonstrated to inhibit activation-induced cytokine secretion and cytolytic granule components from various immune cells, including human peripheral blood mononuclear cells (PBMC), human tumor-infiltrating lymphocytes (TIL) and tumor- specific murine splenocytes.
  • PBMC peripheral blood mononuclear cells
  • TIL human tumor-infiltrating lymphocytes
  • a peptide of the invention is capable of inhibiting immune effector functions (such as cytokine and/or granzyme secretion) in activated cells without substantially affecting effector functions in resting immune cells.
  • the activity is SLAMF6-mediated.
  • the present invention provides a conjugate of a peptide comprising an amino acid sequence X0TWX1FNX2X3X4X5A (SEQ ID NO: 1) or a retro-inverso sequence thereof, wherein Xo and X5 are each independently a hydrophobic amino acid, Xi, X2, X3 and X4 are each independently any amino acid and wherein the peptide consists of from 11 to 50 amino acids.
  • the conjugate comprises the peptide of the present invention and PEG molecule.
  • the PEGylation is effected through C-terminus of the peptide.
  • the PEGylation is effected through N-terminus of the peptide.
  • the PEGylation is effected through a side chain of an amino acid of a peptide.
  • peptide as used in the present invention encompasses also the term analog of the peptide.
  • analog refers to an amino acid sequence in which at least one amino acid of the parent sequence is modified when retaining the functionality of the parent peptide. Examples of such modifications of amino acid sequence are substitutions, rearrangements, deletions, additions and/or chemical modifications in the amino acid sequence of the parent peptide.
  • the analog comprises at least one modification selected from a substitution, deletion and addition.
  • the modification is a substitution.
  • the substitution is a conservative substitution.
  • the analog comprises from 1 to 10, 2 to 8, or 3 to 6 modifications. According to one embodiment, the analog comprises from 1 to 6 modifications or 1 to 3 modifications. According to another embodiment, the analog comprises 1, 2, 3, 4, 5 or 6 modifications. According to some embodiments, the modification is a substitution, such as a conservative substitution. According to one embodiment, the analog comprises 1 to 6 conservative substitutions. In another embodiment, the modifications comprise a truncation or deletion of 1-3 amino acids (e.g. of a non-conserved amino acid denoted by any one of Xi to X4), wherein each possibility represents a separate embodiment of the invention.
  • the Asn at position 6 of the peptide comprising an amino acid sequence X0TWX1FNX2X3X4X5A may be replaced by any other polar amino acid such as Ser, Thr or Gin amino acid.
  • the Ala at position 11 of the peptide comprising an amino acid sequence X0TWX1FNX2X3X4X5A may be replaced by any other small hydrophobic amino acid such Gly and Vai amino acid.
  • the peptide is a cyclic peptide, i.e. cyclized.
  • cyclic peptide refers to a peptide having an intramolecular bond between two non-adjacent amino acids.
  • the cyclization can be effected through a covalent or non-covalent bond.
  • Intramolecular bonds include, but are not limited to, backbone to backbone, side-chain to backbone and side-chain to side-chain bonds.
  • the cyclization occurs between the side chains of two cysteine residues of the peptide, analogs of fragments, to form a disulfide bridge.
  • the cyclization occurs between the N- terminal and C-terminal amino acids.
  • the cyclization is effected via a spacer. The cyclization may be performed using any technique known in the art.
  • peptides in accordance with the invention offer a means to inhibit activation of immune cells.
  • Current immune-suppressive agents have a broad spectrum of activity leading to significant undesirable effects.
  • some widely employed agents have very long half-lives and lack antidotes, thus leading to extended immune-suppression and risk of severe infections.
  • peptides of the invention may have a relatively short half-life offering a means to titrate inhibition and reverse it as necessary.
  • SLAMF6 has a dynamic activation dependent expression pattern.
  • peptides disclosed herein exert a stronger inhibitory effect on activated populations of cells, relatively sparing less active populations, and providing a more specific inhibition of pathologically-active immune cells.
  • pathologically-active immune cells may possess a greater capacity to bind with and be affected by peptides of the invention, as a result of higher SLAMF6 surface density.
  • a pharmaceutical composition comprising at least one peptide or conjugate according to any one of the above aspects or embodiments, and a pharmaceutically acceptable carrier and/or excipient, is further contemplated. All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an isolated peptide comprising the amino acid sequence X0TWX1FNX2X3X4X5A or a retro-inverso sequence thereof, and a pharmaceutically acceptable carrier and/or excipient, wherein Xo and X5 are each independently a hydrophobic amino acid, Xi, X2, X3 and X4 are each independently any amino acid and wherein the peptide consists of from 11 to 50 amino acids.
  • the pharmaceutical composition comprises a conjugate of the peptide.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an isolated peptide comprising or consisting of an amino acid sequence selected from ITWTFNGKSLA (SEQ ID NO: 2), ITwTfNGkSLa (SEQ ID NO: 7), SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8 alskgnftwti (SEQ ID NO: 9), SEQ ID NO: 20-163, and SEQ ID NO: 20-163 comprising D-amino acids at positions 3, 5, 8 and 11 , and a pharmaceutically acceptable carrier and/or excipient.
  • the peptides as cyclic peptides.
  • the pharmaceutical composition comprises a conjugate of the peptide.
  • pharmaceutically acceptable carrier or “pharmaceutically acceptable excipient” as used herein refer to any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, glidants, pH adjusting agents, buffering agents, enhancers, wetting agents, solubilizing agents, surfactants, antioxidants the like, that are compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art.
  • the compositions may contain other active agents providing supplemental, additional, or enhanced therapeutic functions.
  • Parenteral administration includes administration intravenously, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, intranasally, by inhalation, intraspinally, intracerebrally, and transdermally.
  • a compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent.
  • Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • the pharmaceutical composition comprising the peptide or the conjugate of the present invention is administered via a systemic administration.
  • the pharmaceutical composition comprising the peptide or the conjugate of the present invention is administered orally, intravenously or transdermally.
  • the composition is administered intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, intranasally or by inhalation.
  • the pharmaceutical composition according to the present invention may be prepared in any known method.
  • the pharmaceutical composition may be formulated using a method known in the art so as to provide rapid, continuous or delayed release of the active ingredient after administration.
  • the pharmaceutical composition is formulated as a solid dosage form selected from tablets, capsules, powder or granules.
  • the pharmaceutical composition is formulated as a liquid or semi-liquid dosage form selected from an elixir, tincture, suspension, syrup, emulsion or gel.
  • the half-life of the peptides in the pharmaceutical composition is as described above.
  • compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and may further comprise one or more agents selected from sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations.
  • Tablets contain the active agent in admixture with non-toxic pharmaceutically acceptable excipients, which are suitable for the manufacture of tablets. These excipients may be, e.g., inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrating agents, e.g., corn starch or alginic acid; binders; and lubricating agents.
  • the tablets are optionally coated utilizing known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide an extended release of the drug over a longer period.
  • the peptide of the present invention has an immunomodulatory activity and according to some embodiments may be used as an immunosuppressant. According to some embodiments, the peptide of the present invention or a conjugate thereof are for use as a medicament. According to any one of the above aspects and embodiments, the peptide, the conjugate or the pharmaceutical composition comprising the peptide and/or a conjugate thereof is for use in suppressing an immune system. According to any one of the above aspects and embodiments, the peptide, the conjugate or the pharmaceutical composition comprising the peptide and/or the conjugate is for use in inhibiting a SLAMF6-mediated (or SLAMF6-modulated) immune cell activation, e.g. in T cells, B cells and/or NK cells.
  • a SLAMF6-mediated immune cell activation e.g. in T cells, B cells and/or NK cells.
  • the peptide of the present invention may be used as a short-term immunosuppressant.
  • the peptide of the present invention may be used as a medium-term immunosuppressant.
  • short-term immunosuppressants are provided, having serum half-life of at least about 1 hour and typically several hours, e.g. 1-2, 1-4, 2-5, 3-7, or in other embodiments 8-14 or 10-16 hours.
  • medium-term immunosuppressants are provided, having serum half-life of at least about 24 hours and typically several days, e.g. 1-2, 1-4, 2-5 or 3-6 days.
  • medium-term immunosuppressants are conveniently used in managing the symptoms of autoimmune disorders or other conditions in which a non-infective etiology had been confirmed.
  • long-term immune suppression may be induced (for example by using conjugates with half-life elongating substances such as Fc moieties).
  • short-term immunosuppressants may be used in subjects in which the presence of an infective agent is suspected, or wherein the subject is at risk of developing an infection. In these cases, rapid discontinuation of treatment upon signs of infection is readily facilitated.
  • immunosuppression refers to the suppression of the immune system and its ability to fight infections and other diseases. Immunosuppression may be deliberately induced with drugs (for example prior to transplantation to prevent graft rejection), or it can result from certain diseases, environmental factors, or as a side effect to other drugs such as anti-cancer drugs. Immunosuppression may involve partial or complete depletion of one or more leukocyte populations, and/or reduction in their reactivity, expansion and/or differentiation. Immunosuppressive drugs, also referred to herein as “immunosuppressants”, refers to drugs used to induce immunosuppression in a subject.
  • Non-limiting examples of conditions in which the use of immunosuppressants is indicated include graft rejection, GVHD and autoimmune diseases.
  • the peptide of the present invention induces immunosuppression.
  • the peptide of the present invention, the conjugate, or the pharmaceutical composition comprising the peptide or conjugate is for use in inhibiting immune cells' activation.
  • the peptide of the present invention, the conjugate, or the pharmaceutical composition comprising the peptide or conjugate is for use in treating a disease or a condition involving activated or overactivated immune system.
  • the peptide of the present invention, the conjugate, or the pharmaceutical composition comprising the peptide or conjugate is for use in treating a disease or a condition requiring immunosuppression.
  • the disease or a condition comprises, immune overactivation, graft versus host disease, an autoimmune disease, immune related adverse effects of checkpoint inhibitors, cytokine storm, and rejection of stem cells transplant and/or organ transplant.
  • the use comprises prevention of rejection of transplanted cells, tissues or organs.
  • the use comprises preventing or inhibiting the side effects of checkpoint inhibitors.
  • GVHD Graft Versus Host Disease
  • the term “Graft Versus Host Disease” or “GVHD” refers to the pathological reaction that occurs between grafted immune cells and their host-recipient.
  • GVHD involves donor-derived alloreactive T lymphocytes recognizing the recipient's tissues as foreign, mounting an inflammatory and destructive response against the recipient.
  • GVHD often occurs following hematopoietic cell transplantation, and rarely after solid organ transplantation, and has a predilection for epithelial tissues, especially skin, liver, and mucosa of the gastrointestinal tract.
  • Transplant patients with GVHD are often treated with powerful immunosuppressant agents, thereby making them more susceptible to opportunistic infections.
  • GVHD may be chronic or acute.
  • transplant rejection refers to rejection of tissue transplanted from a donor individual to a recipient individual.
  • transplant graft rejection e.g. allograft rejection
  • allograft rejection occurs when the donor tissue carries an alloantigen recognized as foreign by the recipient's immune system, thereby inducing an inflammatory and destructive immune response directed to the alloantigen.
  • autoimmune disease is defined as a disease or disorder in which the subject mounts a destructive immune response against its own constituents (e.g. cells or tissues). An autoimmune disease is therefore the result of an inappropriate and excessive response to a self-antigen. Autoimmune disorders can affect almost every organ system in the subject (e.g., human), including, but not limited to, diseases of the nervous, gastrointestinal, and endocrine systems, as well as skin and other connective tissues, eyes, blood and blood vessels.
  • autoimmune diseases include, but are not limited to Hashimoto's thyroiditis, Systemic lupus erythematosus, Sjogren's syndrome, Graves' disease, Scleroderma, Rheumatoid arthritis, Multiple sclerosis, Myasthenia gravis, Crohn’s disease, ulcerative colitis, primary biliary cholangitis, primary biliary sclerosis, psoriasis and Diabetes.
  • cytokine storm (or cytokine release syndrome) as used herein is a form of systemic inflammatory response syndrome (SIRS) that can be triggered by a variety of factors such as infections and certain drugs. It occurs when the immune system causes an uncontrolled and excessive release of pro-inflammatory cytokines. This sudden release in such large quantities can cause multisystem organ failure and death.
  • SIRS systemic inflammatory response syndrome
  • the use is in treating an autoimmune disease e.g., selected from rheumatoid arthritis, lupus, inflammatory bowel disease, including Crohn’s disease and ulcerative colitis, multiple sclerosis, psoriasis or psoriatic arthritis.
  • said disease is autoimmune hepatitis or primary biliary cholangitis (PBC).
  • PBC primary biliary cholangitis
  • the inflammatory bowel disease comprises Crohn’s disease and ulcerative colitis.
  • the use comprises treating rheumatoid arthritis.
  • CD Crohn’s disease
  • Ulcerative colitis is a condition related to CD that involves only the colon, and collectively these diseases are frequently referred to as inflammatory bowel disease (IBD).
  • MS Multiple sclerosis
  • CNS central nervous system
  • MS central nervous system
  • Acute lesions are characterized by perivenular cuffing and infiltration of T lymphocytes and macrophages, along with a few B cells and plasma cells.
  • MS is reportedly an autoimmune disorder, likely triggered by environmental exposure in a genetically susceptible host.
  • SLE is a chronic, recurrent, potentially fatal multisystem inflammatory disorder mainly affecting women. SLE is associated with a large spectrum of autoantibodies, and is diagnosed on the basis of eleven criteria defined by the American College of Rheumatology (ACR).
  • ACR American College of Rheumatology
  • RA is a chronic inflammatory autoimmune disease characterized by joint inflammation, joint swelling, joint tenderness, and destruction of synovial joints, leading to severe disability and premature mortality.
  • Type I Diabetes (T1DM, or Insulin Dependent Diabetes Mellitus) is caused by a deficiency of insulin due to an autoimmune response which leads to the destruction of the beta cells (P cells) in the Islets of Langerhans of the pancreas.
  • An initial phase of T1DM includes an inflammation of the pancreatic islets, known as insulitis, characterized by leukocyte and macrophage infiltration into the islets followed by the actual destruction of pancreatic P cells in an autoimmune attack.
  • Autoimmune thyroiditis is a condition in which the thyroid gland diffusely swells and frequently occurs in young to middle-aged women. Although thyroid function is normal in the early stage of autoimmune thyroiditis, progress of thyroid tissue destruction by an autoimmune mechanism leads to hypothyroidism requiring thyroid hormone replacement therapy. Hypothyroidism is a serious disease in which the functions of various organs in the body including the central nervous system are impaired, causing a major obstacle in social life.
  • the use is in treating chronic inflammation or chronic inflammatory disease.
  • treating refers to taking steps to obtain beneficial or desired results, including clinical results.
  • beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms or parameters associated with the disorder, delaying or slowing of that impairment, amelioration, palliation or stabilization of that impairment, and other beneficial results.
  • a chronic inflammatory disease is characterized by a persistent inflammatory response with pathologic sequelae. This state is typically accompanied by infiltration of mononuclear cells, proliferation of fibroblasts and small blood vessels, increased connective tissue, and tissue destruction.
  • Non-limitative examples include idiopathic or non-infective chronic conditions, e.g. pericarditis or periodontitis.
  • conditions to be treated may include chronic inflammatory diseases of the liver, for example non-alcoholic fatty liver disease (NAFLD) and liver cirrhosis resulting from chronic liver disease.
  • NAFLD non-alcoholic fatty liver disease
  • liver cirrhosis resulting from chronic liver disease.
  • the peptide of the present invention, the conjugate, or the pharmaceutical composition comprising the peptide or conjugate is for use in treating or preventing a T cell-mediated immune disorder (in which the activity of T cells is known to be implicated in the etiology and/or pathology).
  • a T cell-mediated immune disorder in which the activity of T cells is known to be implicated in the etiology and/or pathology.
  • the T-cell-mediated immune disorder is an inflammatory disease, or an autoimmune disorder.
  • the T- cell- mediated immune disorder is chosen from infections (viral, bacterial, fungal and parasitic), endotoxic shock associated with infection, sepsis, arthritis, rheumatoid arthritis, asthma, chronic obstructive pulmonary disease (COPD), inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peyronie's disease, coeliac disease, psoriasis, vasculitis, surgical adhesions, stroke, type I diabetes, meningoencephalitis, autoimmune uveitis, immune mediated inflammatory disorders of the central and peripheral nervous system including multiple sclerosis, lupus (systemic lupus erythematosus) and Guillain-Barre syndrome, atopic dermatitis, autoimmune hepatitis, fibrosing alveolitis, Grave's disease, immunoglobulin (Ig)A nephropathy, idiopathic thrombocytopenic purpura, Meniere's disease, pe
  • the peptide of the present invention, the conjugate, or the pharmaceutical composition comprising the peptide or conjugate is for use in treating or preventing a B cell-mediated immune disorder.
  • the peptide of the present invention, the conjugate, or the pharmaceutical composition comprising the peptide or conjugate is for use in treating or preventing an NK cell-mediated immune disorder.
  • Such disorders encompass conditions (e.g. inflammatory or autoimmune) in which the activity of the recited cell type (e.g. B cell or NK cell) is known to be implicated in the etiology and/or pathology.
  • the peptide of the present invention, the conjugate, or the pharmaceutical composition comprising the peptide or conjugate is for use in treating or preventing conditions involving myeloid-derived suppressor cells (MDSC) dysfunction, e.g. contributing to autoimmunity (MDSC-mediated immune disorders).
  • MDSC myeloid-derived suppressor cells
  • MDSC-mediated immune disorders e.g. contributing to autoimmunity
  • MDSC impairment has been implicated in the development of autoimmune diseases including MS, RA, type I diabetes, SLE and autoimmune uveoretinitis.
  • the MDSC are selected from the group consisting of monocytic MDSC (M-MDSC), polymorphonuclear MDSC (PMN-MDSC) and combinations thereof.
  • the peptide, conjugate or the pharmaceutical composition comprising the peptide or conjugate is co-administered in a combination with an additional immunosuppressant.
  • the peptide, conjugate or the pharmaceutical composition comprising the peptide or conjugate is co-administered in a combination with an immuno stimulatory treatment (e.g. a cancer immunotherapy such as an immune checkpoint inhibitor).
  • an immuno stimulatory treatment e.g. a cancer immunotherapy such as an immune checkpoint inhibitor.
  • coadministration encompasses administration of a first and second agent in an essentially simultaneous manner, such as in a single dosage form, e.g., a capsule or tablet having a fixed ratio of first and second amounts, or in multiple dosage forms for each.
  • the agents can be administered sequentially in either order.
  • co-administration involves the separate administration of each agent, the agents are administered sufficiently close in time to have the desired effect (e.g., complex formation).
  • co-administration comprises administration by separate administration routes.
  • the present invention provides a method for suppressing the immune system in a subject in need thereof comprising administering the peptide according to any one of the above embodiments and aspects or the conjugate or the pharmaceutical composition comprising the peptide or the conjugate.
  • the pharmaceutical composition is for use in inhibiting or reducing an inappropriate or excessive immune response in a subject in need thereof.
  • the phrase "inappropriate or excessive immune response” relates to an undesirable response of a subject's immune system to one or more antigens, that results in the development or progression of a pathology in the subject.
  • the response may proceed to an inappropriate degree (disproportionate response), typically an inappropriately high degree (excessive immune response) and/or may be inappropriate (undesirable) in terms of its type (e.g. Thl vs. Th2), the nature of immune cells involved (e.g. effector vs. regulatory T cells), the timing of the response (e.g.
  • a chronic condition developing following an initial infection and/or the nature of antigens to which the response is directed
  • mitargeted response for example, a response to self-antigens may lead to the development of an autoimmune disease, and type-I hypersensitivity responses to innocuous allergens may lead to the development of allergic conditions such as asthma and atopic dermatitis).
  • inappropriate or excessive immune responses may be involved in the etiology and/or pathology of various autoimmune and inflammatory diseases and conditions as disclosed herein, collectively referred to as "disease or conditions associated with an inappropriate or excessive immune response".
  • a method of inhibiting or reducing an inappropriate or excessive immune response may also be regarded as a method of preventing the development or progression of these autoimmune and inflammatory diseases and conditions, including e.g. irAEs and T-cell mediated conditions as disclosed herein.
  • a method for inhibiting or reducing an inappropriate or excessive immune response in a subject in need thereof comprising administering to the subject the pharmaceutical composition as disclosed herein.
  • the pharmaceutical composition comprises an isolated peptide comprising the amino acid sequence X0TWX1FNX2X3X4X5A (SEQ ID NO: 1) or a retro-inverso sequence thereof, wherein Xo and X5 are each independently a hydrophobic amino acid, Xi, X2, X3 and X4 are each independently any amino acid and wherein the peptide consists of from 11 to 50 amino acids.
  • the peptide comprises or consists of an amino acid sequence selected from ITWTFNGKSLA (SEQ ID NO: 2) and ITwTfNGkSLa (SEQ ID NO: 7).
  • the pharmaceutical composition comprises a conjugate of the peptide.
  • the method is used for inhibiting or reducing immune-related adverse events (irAEs).
  • irAEs are selected from the group consisting of gastrointestinal, endocrine, dermatologic toxicities, and combinations thereof.
  • AE immune-related adverse events
  • the term "adverse event” or "AE” as used herein refers to any noxious, unintended, or untoward medical occurrence that may appear or worsen in a patient following or during administration of a treatment. It may be a new intercurrent illness, a worsening concomitant illness, an injury, or any concomitant impairment of the participant's health, including laboratory test values, regardless of etiology.
  • AE associated with cancer treatment are defined and classified by the Common Terminology Criteria for Adverse Events (CTCAE) published by the NIH as encompassing any abnormal clinical finding temporally associated with the use of a therapy for cancer.
  • CTCAE Common Terminology Criteria for Adverse Events
  • the term “immune-related adverse event” or “irAE” refers to an AE that has a putative immune-related etiology, and include in particular AE associated with immuno stimulatory cancer immunotherapy.
  • the irAE is associated with administration of immune checkpoint inhibitors.
  • irAE typically include toxicities that are autoimmune or autoinflammatory in nature that may affect various organs and systems of the body, and may be of various grades or levels of severity.
  • the irAE affects the endocrine system (“endocrine irAE” or “endocrine toxicities”), the skin (“dermatological irAE” or “dermatologic toxicities”), or the gastrointestinal tract (“GI irAE” or “gastrointestinal toxicities”).
  • Endocrine toxicities include, but are not limited to, immune-related hypothyroidism, immune-related hyperthyroidism, immune- related adrenal insufficiency, immune-related diabetes mellitus, and immune-related hypophysitis.
  • Dermatological toxicities include, but are not limited to, immune-related rash and immune-related severe cutaneous reaction
  • gastrointestinal toxicities include, but are not limited to, immune- related hepatitis, immune-related colitis, and immune-related pancreatitis.
  • toxicities may be cardiac (e.g. myocarditis), pulmonary (pneumonitis), neural (encephalitis, meningitis, myasthenia gravis like, Guillain Barre), or musculoskeletal (arthritis, myositis).
  • the irAE includes iatrogenic multiple autoimmune conditions.
  • the irAE comprises toxicities affecting the pancreas and/or thyroid gland, e.g. conditions involving diabetes or thyroiditis as disclosed above.
  • the irAE is systemic, e.g. may include cytokine release syndrome (CRS) or cytokine storm (CS).
  • irAE involve immune effector cell-associated neurotoxicity syndrome (ICANS), associated with cell therapies CAR-T and TCR-T adoptive cell therapies.
  • the irAE is a low grade irAE, e.g., a Grade 1 AE (Grade 1 irAE, representing a condition that does not require additional therapeutic intervention due to irAE) or Grade 2 AE (Grade 2 irAE, representing a condition that requires drug intervention, etc., due to irAE, but does not require hospitalization treatment or does not require interruption of treatment).
  • the irAE is a high grade irAE, e.g. a Grade 3 AE (Grade 3 irAE, representing a condition that requires drug intervention, etc., accompanied by hospitalization due to irAE and requires interruption of treatment).
  • the irAE is a Grade 4 AE, posing a life-threatening event.
  • irAE are as detailed for each specific AE in the CTCAE v5.0.
  • said irAEs are associated with administration of an immuno stimulatory treatment (that activates or enhances the activity of the immune system or components thereof, in an antigen-dependent and/or independent manner).
  • said immuno stimulatory treatment is a cancer immunotherapy.
  • said cancer immunotherapy comprises administration of an immune checkpoint inhibitor.
  • said immuno stimulatory treatment is a vaccine.
  • said cancer immunotherapy comprises administration of a cell-based therapy either autologous or allogenic, native or engineered (e.g. CAR-T, TCR-T, CAR-NK etc).
  • cancer immunotherapy refers to a cancer treatment that modulates the patient's immune system to induce or enhance an immune response against a tumor (or cancer cells) of the patient.
  • cancer immunotherapy include immune checkpoint modulators (e.g. inhibitors), adoptive cell therapy, cytokines and their recombinant derivatives, adjuvants, and vaccination with small molecules or cells.
  • immune checkpoint modulators e.g. inhibitors
  • adoptive cell therapy e.g. cytokines and their recombinant derivatives, adjuvants, and vaccination with small molecules or cells.
  • the irAE includes iatrogenic multiple autoimmune conditions associated with administration of an immune checkpoint inhibitor.
  • Immune checkpoint inhibitors or ICIs denote a subclass of cancer immunotherapy in which the cellular targets modulated are immune checkpoint molecules.
  • Immune checkpoint molecules are a group of cell-surface molecules expressed on immune cells including in particular T cells (or other immune cells that regulate immune pathways such as NK cells, dendritic cells, macrophages, and MDSC).
  • Immune checkpoint molecules are known to effectively serve as "brakes” to down- modulate or inhibit an anti-tumor immune response, and include, for example, PD-1, PD-L1, PD- L2, CTLA4, LAG3, TIM3, IDO, CD80, CD86, B7-H3, -H4, -H5, BTLA, TIGIT, CD94/NKG2A, and KIR2DL-1, -2, -3 (see, for example, WO 2012/177624).
  • Typical ICIs act by reducing the activity of a target immune checkpoint protein involved in suppressing immune cells to thereby activate immune cells to attack cancer cells.
  • therapeutic agents that can be used as ICIs are antibodies, small molecules, peptides, peptidomimetics, natural ligands, and derivatives of natural ligands, that can either bind and/or inactivate or inhibit immune checkpoint proteins, or fragments thereof; as well as RNA interference, antisense, nucleic acid aptamers, etc. that can downregulate the expression and/or activity of immune checkpoint nucleic acids, or fragments thereof.
  • ICIs include for example anti-CTLA-4 monoclonal antibodies such as ipilimumab and tremelimumab, anti-PD-1 monoclonal antibodies such as nivolumab and pembrolizumab, anti-PD-Ll monoclonal antibodies such as atezolizumab, durvalumab, and avelumab, and anti-LAG3 monoclonal antibodies such as relatlimab.
  • the immune checkpoint inhibitor targets (specifically downregulates the expression and/or activity) PD-1, PD-L1, PD-L2, CTLA-4, LAG3, TIM3, and/or IDO.
  • PD-1 Programmed cell death 1 receptor
  • NK natural killer
  • B lymphocytes macrophages
  • DCs dendritic cells
  • monocytes monocytes. It is categorized as a type I transmembrane protein, structurally belonging to the CD28/CTLA-4 subfamily of the Ig superfamily, and is encoded by the PDCD1 gene.
  • PD-1 and its ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC) belong to the immune checkpoint pathway, which induces immune suppression.
  • the binding of PD-1 to its ligand e.g.
  • PD-L1 downregulates immune reactivity and promotes self-tolerance by suppressing the activity of effector T cells.
  • PD- L1 is also expressed by many tumor cells, thereby facilitating their evasion from immune surveillance and down-regulating anti-tumor immunity.
  • commercially available PD- Ll-targetind ICIs include e.g. atezolizumab, avelumab, durvalumab, dostarlimab and retifanlimab; commercially available PD-l-targetind ICIs include e.g.
  • nivolumab Opdivo
  • pembrolizumab Keytruda
  • cemiplimab Libtayo
  • camrelizumab sintilimab
  • tislelizumab tislelizumab and balstilimab.
  • Cytotoxic T-Lymphocyte Antigen 4 also known as CD152, is constitutively expressed in regulatory T cells but only upregulated in conventional T cells after activation - a phenomenon which is particularly notable in cancers. It induces an inhibitory signal upon binding to CD80 or CD86 on the surface of antigen-presenting cells. It is encoded by the gene CTLA4 in humans.
  • CTLA-4 -targetind ICIs include e.g. ipilimumab, tremelimumab and botensilimab.
  • LAG3 lymphocyte activation gene 3; or CD223 belongs to Ig superfamily and contains four extracellular Ig-like domains, which are followed by a transmembrane helical domain and a cytoplasmic domain.
  • the precursor polypeptide also contains an N' signal peptide.
  • the precursor sequence of human LAG3 may be found in accession no. P18627.
  • Commercially available LAG3 inhibitors include e.g. IMP321, BMS-986016 (relatlimab), LAG525, REGN3767 (finalimab), and TSR-033.
  • TIM-3 T-cell immunoglobulin and mucin domain-containing 3
  • HAVCR2 Hepatitis A virus cellular receptor 2
  • IDO Indoleamine 2,3-dioxygenase
  • Tregs regulatory T cells
  • Various immune cells and stromal cells but also cancer cells express IDO.
  • the method comprises treating a disease or a condition associated with the inappropriate or excessive immune response.
  • the condition is a T-cell mediated inflammatory or autoimmune condition.
  • a T-cell mediated inflammatory or autoimmune condition encompasses inflammatory and autoimmune diseases in which the activity of T cells is known to be implicated in the etiology and/or pathology of the disease.
  • the disease or condition is selected from the group consisting of graft versus host disease, an autoimmune disease, and graft rejection.
  • the pharmaceutical composition is administered systemically.
  • said pharmaceutical composition is administered locally to the site of manifestation of symptoms associated with the condition (e.g. irAEs or T-cell mediated inflammatory or autoimmune condition).
  • the composition may be administered orally in case of gastrointestinal (GI) symptoms, for example in the case of GI inflammatory or autoimmune diseases such as IBD or GI toxicities associated with irAEs.
  • GI gastrointestinal
  • the composition may be administered by inhalation in case of pulmonary symptoms or toxicities.
  • said composition may be administered locally in a prophylactic manner in a subject predisposed to exhibiting symptoms or toxicities in the organ or tissue in question, prior to or concurrently with administration of an immuno stimulatory treatment (e.g. an immune checkpoint inhibitor).
  • an immuno stimulatory treatment e.g. an immune checkpoint inhibitor
  • administration is performed in vivo. In another embodiment administration is performed ex vivo. In another embodiment of the methods of the invention, administration is effected by expressing in cells of the subject at least one isolated peptide of the invention.
  • the methods of the invention may be performed by contacting cells of the subject (e.g. in vivo or ex vivo) with a nucleic acid molecule encoding a peptide of the invention, wherein the nucleic acid molecule is operably linked to a transcription regulating sequence, such that the peptide of the invention is expressed in the cells and administered to said subject.
  • Table 1 Sequences
  • compositions or components may include additional ingredients, but only if the additional ingredients do not materially alter the basic and novel characteristics of the claimed compositions or methods.
  • PBMCs Peripheral derived blood mononuclear cells
  • SEB staphylococcal endotoxin b
  • IL-2 interleukin-2
  • TILs Tumor infiltrating lymphocytes
  • IFN-y interferon gamma
  • Jurkat cells activated with anti-CD3 antibodies, with granzyme B as the readout; SLAMF6-knockout Jurkat cells were used as a control.
  • Murine model Splenocytes were harvested from PMEL mice in which the TCRs recognize the melanoma B16-gp-100 antigen, then co-cultures with B16 melanoma cells, with IFNy as the readout; SLAMF6 knockout PMEL splenocytes were used as a control.
  • VNFITWLFNETSLAFIVPHETKSPEIHVTNPKQGKRLNFTQS SEQ ID NO: 3
  • the location of V49 in the SLAMF6 protein structure is illustrated in Fig. IB in dark grey.
  • SLAMF6 was expressed in M526 melanoma cells that do not natively express this receptor.
  • the V49 peptide was fluorescently labeled by N' conjugation to FITC-Ahx (FITC via an Ahx linker), and incubated with either WT or SLAMF6-expressing M526 cells for two hours. Two additional peptides were tested: V49(ala), having the amino acid sequence
  • VNAITWAFNETSLAFIVPHETKSPEIHVTNPKQGKRLNFTQS SEQ ID NO: 4, a sequence of V49 in which Phe and Leu were replaced by Ala
  • V49(scr) a sequence of V49 in which Phe and Leu were replaced by Ala
  • V49(scr) a sequence of V49 in which Phe and Leu were replaced by Ala
  • V49(scr) SEQ ID NO: 13
  • Mean fluorescence intensity was measured after repeated washing.
  • MFI was significantly higher in SLAMF6-expressing cells incubated with the V49 peptide, compared to in WT cells incubated with this peptide, indicating SLAMF6-mediated binding.
  • substitution of the amino acids at positions 3 and 6 resulted in marked reduction in the binding capacity of the peptide, whereas the scrambled control peptide no longer exhibited any SLAMF6 specificity.
  • PBMC peripheral blood mononuclear cells
  • PBMC peripheral blood mononuclear cells
  • CM complete medium
  • RPMI 1640 supplemented with 10% heat-inactivated human AB serum, 2 mmol/1 L-glutamine, 1 mmol/1 sodium pyruvate, 1% nonessential amino acids, 25 mmol/1 HEPES (pH 7.4), 50 pmol/1 2-ME, and combined antibiotics (all from ThermoFisher)] with recombinant human interleukin-2 (Chiron, CA) at a concentration of 300 IU.
  • PBMCs peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • PBMCs peripheral blood mononuclear cells
  • TILs were activated by incubation with cognate melanoma cells in the presence or absence of the peptides, and IFNy secretion was measured via ELISA, as follows. TILs were released from fresh tumor samples. Melanoma cells used were cell lines 888meZ (HLA- A2-/MART-l + /gpl00 + ) and 624-mel (HLA-A2 + /MART-l + gpl00 + ); human melanoma 526mel is an HLA-A2 + cell line. TILs were thawed and cultured for 3 days in 24 well plates in CM+IL2 (6000IU)/ml.
  • TIL were collected and counted the replated at xlO 5 cells/well (96 well plate-flat), in CM without-IL2 for 3-4 hours. Then peptides were added to the wells and incubated for 4 hours. IxlO 5 melanoma cells/well were then added to yield a 1:1 ratio with TILs and further incubated for 16 hours. Supernatant was then collected for interferon-gamma ELISA (R&D Systems, catalog no. DY285), according to the manufacturer's instructions.
  • CRlalpha CRla, LTWTFNGKNVA, SEQ ID NO: 11
  • IWLFNETSLA adenovirus SLAMF6-binding protein CRlalpha derived from the viral protein
  • 3Mut IWTFNGKSLA, SEQ ID NO: 2 in which the 3 amin acids marked in bold were taken from the viral sequence in to the human one.
  • Fig. 2A 526 cells
  • Fig. 2B 624 cells
  • Fig. 2E The figure shows a significant decrease in IFNy secretion with 153 in two pairs of TIL and cognate melanoma cells.
  • FIG. 2C shows a decrease in IL-2 secretion by PBMC using the test peptides (153, CRla and 3mut) but not the control V49(ala) peptide.
  • Fig. 2C also shows that the effect for the 3Mut peptide was remarkably obtained at a lower concentration as compared with 153 and CRla when PBMC cells are activated using SEB.
  • Fig. 2D shows the inhibitory effect of different concentrations 153 peptide on PBMC function (measured as IL-2 secretion by ELISA) following SEB activation.
  • Splenocytes previously activated for 7 days, were co-cultured (l x 10 5 ) overnight at a 1:1 ratio with the indicated target cells or activated with 1 pg/ml plate-bound anti-CD3 antibody (Biolegend, clone: 145-2 Cl l) as indicated in each experiment.
  • Conditioned medium was collected, and mouse IL-2 and IFN-y secretion were detected by ELISA (Biolegend).
  • Additional experiments were performed in human TILs co-cultured with human melanoma for activation. IFN-y secretion was measured by ELISA after 24 hours. Further experiments were also performed in SEB-activated PBMC, IL2 secretion was measured by ELISA.
  • Fig. 3C-3E WFKA decreases cytokine secretion similarly to 3Mut.
  • Fig. 3C shows a decrease in IL-2 secretion when PBMC cells are activated using SEB in the presence of both peptides.
  • Fig. 3D shows a decrease in IFNy secretion when human melanoma- specific TILs are co-cultured with a specific melanoma identified by the TILs in the presence of the peptides.
  • Fig. 3E shows that the peptides are also active in cells of murine origin expressing the SLAMF6 homologous protein Lyl06, using the murine PMEL model described above.
  • the peptides were added to a co-culture of splenocytes harvested from PMEL mice recognizing the melanoma B16-gp-100 antigen and B16 melanoma cells and IFNy secretion was quantified using ELISA.
  • Fig. 4F shows that in SLAMF6' /_ Jurkat cells, GZM-b secretion was not inhibited by the MUT3 or WFKA peptides, indicating that the inhibitory effect of these peptides is directly dependent on the presence of the SLAMF6 receptor.
  • IFNy secretion (measured essentially as described with respect to Fig. 3E above), decreased when murine splenocytes were activated in the presence of WFKA but not in Lyl06 knockout splenocytes (the murine SLAMF6 homologue), as shown in Fig. 4D.
  • the most prominent effect was seen at much lower concentration, i.e. 500 nM.
  • X0TWX1FNX2X3X4X5A SEQ ID NO: 1
  • Xo and X5 are each independently a hydrophobic amino acid
  • Xi, X2, X3 and X4 are each independently any amino acid.
  • the conservative pattern comprising D amino acids was identified as Xo TwXi fNX2X3X4Xsa (SEQ ID NO: 6), wherein Xo and X5 are each independently a hydrophobic amino acid, Xi, X2, X3 and X4 are each independently any amino acid and low-case letters represent D-amino acids.
  • peptides of the consensus sequence identified herein were shown to exhibit a considerable and significant ability to control excessive immune activation in various experimental models.
  • WFKA peptide led to a reduction of up to about 20% in the secretion of IL-2.
  • WFKA peptide led to a reduction of up to about 20% of granzyme-B secretion.
  • WFKA peptide led to a reduction of up to about 30% of interferon-y secretion. It was further shown that the activity is dependent on SLAMF6 expression, as SLAMF6 knockout led to loss of activity in Jurkat cells and murine splenocytes.
  • the WFKA peptide is disclosed herein as a particularly advantageous peptide characterized by desirable properties. These advantageous characteristics enables its use as a novel means of modulating auto-reactive or otherwise pathologically activated SLAMF6-expressing immune cells, including, but not limited to auto-reactive T-cells in autoimmune diseases and for inhibiting or reducing immune-related adverse events (irAEs).
  • the main aim of this study is to investigate the SLAMF6-derived WFKA peptide described in Example 3 as a therapeutic strategy for modulating immune activation in a DSS colitis model.
  • Sub-aiml Discover the smallest dose required for an effective therapeutic response.
  • Sub-aim 2 Understanding the mechanism through which the inhibition mechanism works.
  • the established model for inflammation-induced colitis works well with C57BL ⁇ 6 strain. The strain is normal and does not suffer from diseases.
  • 192 female mice (160 C57BL ⁇ 6 mice and 32 C57BL ⁇ 6 SLAMF6 7 ' ) 7 weeks old are used according to the following allocation. Methods relevant to all below experiments
  • mice 7-8 weeks age are admitted to the experimental cages.
  • the mice acclimatize for 7 days before the initiation of the experiments.
  • the mice are weighed and marked.
  • the mice receive DSS in the drinking water and at the same time are IP injected with test items.
  • the experimental groups receive the therapeutic substance in the desired dose in a volume of 0.1 ml and the control groups receive injections of saline (25G needle are used).
  • the mice are sacrificed by CO2 inhalation.
  • This study uses a DSS colitis model in mice to induce chronic inflammation and immune activation
  • Mice were divided into two groups: a control group and a group treated with WFKA peptide.
  • the treatment group received intraperitoneal (IP) injections of WFKA peptide for 7 days.
  • IP intraperitoneal
  • the effects of WFKA peptide on immune activation and colitis symptoms is assessed using various methods, including histological analysis of tissue samples and assessment of inflammatory markers in the blood.
  • mice develop acute intestinal inflammation, 10-40% of the mice typically die following the DSS treatment. Mice treated with DSS exhibit weight loss and signs of loose stool, diarrhea, and rectal bleeding.
  • mice's condition is followed daily during the experiment.
  • mice receive drinking water containing 3.5% dextran sodium sulfate (DSS) for 7 days to induce chronic inflammation and immune activation.
  • DSS dextran sodium sulfate
  • mice in the experimental group receive injections of WFKA peptide according to the established protocol.
  • mice are sacrificed, and the rest of the experiment refers to the analysis of the WFKA peptide treatment.
  • tissue samples from the colon are collected from mice at the end of the treatment period, and the length of the intestine are measured. 5. The samples are then fixed in formalin for histological analysis. The tissue samples are embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) for microscopic examination. The severity of inflammation and tissue damage are evaluated using a standardized scoring system.
  • H&E hematoxylin and eosin
  • RNA sequencing RNA sequencing
  • Immune cells are isolated from the blood and spleen of mice at the end of the treatment period. The cells are stained with specific antibodies and analyzed using flow cytometry to evaluate the effects of WFKA peptide treatment on immune cell activation and function.
  • DSS Disease Activity Index
  • DAI score is assessed according to Table 3 and the histochemical score is assessed according to Table 4. Table 3. DAI assessment score
  • Example 5 The experiments were performed essentially as described in Example 5, with changes as indicated below.
  • An acute model for DSS-induced colitis was used, comprising administration of 2% DSS in drinking water for 7 days, followed by a two-days recovery period (in the absence of DSS administration and treatment) prior to sacrifice.
  • the experimental groups and experimental design are presented in Tables 7 and 8, respectively.
  • Figs. 5A-5D present the results of the wight loss (Fig. 5A), diarrhea incidence (Fig. 5B) blood in feces (Fig. 5C) and Disease Activity Index (DAI) score (Fig. 5D) for the three treatment groups.
  • mice treated with the WFKA peptide showed slower weight loss compared to untreated mice (Group A).
  • Colitis development began on day 3 post-DSS induction. From day 5 onward, untreated mice exhibited greater weight loss compared to treated mice, with a 10% difference by day 7.
  • Treated mice (Group B) regained weight faster on days 8 and 9, indicating that WFKA peptide not only inhibited colitis progression but also aided recovery.
  • diarrhea incidence was lower in WFKA peptide-treated mice (Group B) compared to untreated mice (Group A). By days 7 and 8, all untreated mice developed diarrhea, while approximately 50% of the treated group did.
  • FIG. 5C shows that blood in feces was less frequent in WFKA peptide-treated mice (Group B) than in untreated mice (Group A). By days 6 and 7, all untreated mice showed gross bleeding, while treated mice exhibited 25-30% less bleeding on day 6 and 50% less on day 7. Both groups ceased bleeding after returning to regular water, but the treated group showed less severity.
  • Fig. 5D is a composite of data from Figs. 5A-C, showing the overall DAI score.
  • the DAI score remained lower in WFKA peptide-treated mice, with a maximum effect of 30% reduction on day 7 compared to untreated mice.
  • treated mice showed a 23% improvement in DAI score relative to untreated mice, indicating better recovery.
  • FIG. 6A shows colon length in naive mice (Group C); the average measured length approximately was 7-8 cm on day 10.
  • Fig. 6B shows colon length in mice treated with DSS only (Group A). The average measured length was approximately 4-5.8 cm on day 10.
  • Fig. 6C shows colon length in mice treated with DSS and 3 pM WFKA peptide (Group B). The colon lengths were partially preserved having an average measured length of approximately 6-7 cm on day 10.
  • Fig. 7 shows a comparison of colon length between WFKA peptide-treated (Group B) and untreated (Group A) mice. As can be seen, colons from treated mice were approximately 20% longer than those from untreated mice. The difference was statistically significant, with a p-value of 0.0001.
  • the average and SEM of colon length and spleen weight as measured on day 10 are further presented in Table 11 below. Table 11. Colon length and spleen weight
  • mice sera collected in the experiments described in Example 6 were further assayed for the presence of inflammatory cytokines.
  • Serum cytokine levels were analyzed from mice on day 10 using a multiplex assay (MILLIPLEX MAP Mouse High Sensitivity T Cell Panel, Merck) to quantify 15 cytokines: GM-CSF, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 (p70), IL-13, IL-17A, IL-la, IL-ip, TNFa, MCP-1, KC, and IFN-y.
  • MILLIPLEX MAP Mouse High Sensitivity T Cell Panel Merck
  • mice treated with the WFKA peptide showed cytokine profiles more closely resembling those of naive mice, particularly for IL-2, IL-5, IL-12, IL-la, KC, IL-6, and IFN-y. Exemplary results are presented in Table 12 below, in which the experimental groups are as described in Example 6.
  • results and comparisons highlight the therapeutic effect of the WFKA peptide in modulating inflammatory responses and alleviating colitis symptoms in vivo.
  • results suggest that mitigation of colitis by the WFKA peptide involves modulation of key cytokines implicated in inflammatory pathways.
  • mice 111 C57BL ⁇ 6 mice (wild-type) and 23 C57BL ⁇ 6 SLAMF6' /_ mice (in which the expression of murine SLAMF6 is knocked out).
  • the strain is normal and does not suffer from diseases.
  • Mice receive drinking water containing 3 concentrations of DSS for 7 days to induce acute inflammation and immune activation and to determine the best concertation required for later experiments.
  • mice undergo terminal bleeding (using the 'Cardiac Puncture' withdraw following anesthesia with Thiopental). At this point, mice are sacrificed, and the effects of the WFKA peptide treatment are analyzed as follows.
  • Tissue collection the intestines, spleen, liver, heart, kidney, lung and stomach are collected from mice at the end of the treatment period. Spleens are taken out and weighted. Increased weights correlate with the extent of inflammation and anemia.
  • the colon and cecum are isolated by separating them form the small intestine at the ileocecal junction and from the anus at the distal end of rectum, measure length of the straighten colons, and comparing them between the control and test groups. The colons are quickly flushed (5-10 ml syringe with feeding needle (18G-3” Straight 2.25mm ball, Braintree Scientific Inc) using cold PBS to remove feces and blood.
  • the cecum can be discarded as little or no inflammation is induced by DSS in this region. After flushing with PBS, colon weights can be taken. In accordance with observed tissue wasting, severely inflamed colons exhibit reduced weight as both correlate with the severity of acute inflammation.
  • H&E hematoxylin and eosin
  • cytokine assessment array using multiplex (MILLIPLEX MAP Mouse High Sensitivity T Cell Panel - Immunology Multiplex Assay kit, Millipore).
  • MILLIPLEX MAP Mouse High Sensitivity T Cell Panel - Immunology Multiplex Assay kit Millipore.
  • Assessment of inflammatory markers in intestinal tissue Intestinal tissue samples are collected from mice at the end of the treatment period, and RNA is extracted for subsequent mRNA quantification. The levels of various inflammatory markers, including TNF-a, IFN-y, IL-10, and IL-17 are determined using RT-qPCR.
  • RNA sequencing RNA sequencing
  • RNA-seq RNA sequencing
  • the results from both RT-qPCR and RNA-seq are compared between the two groups to evaluate the effects of WFKA peptide treatment on immune activation in the intestine.
  • Flow cytometry analysis of fresh lamina propria cells and splenocytes are isolated from the intestines, spleens and blood of mice at the end of the treatment period. The cells are stained with specific antibodies and analyzed using flow cytometry to evaluate the effects of WFKA peptide treatment on immune cell activation and function.
  • Experiments 1-3 are conducted with the selected DSS concentration while keeping the established parameters of the model and study design with the addition of treatment with the WFKA peptide, by injecting it intraperitoneally on a daily basis with changes as described below.
  • Three experiments are designed to examine and validate the inhibitory properties of the peptide and to mark it as a drug candidate.
  • mice are divided into 4 groups as described in Table 13.
  • An initial effective peptide concentration identified for therapeutic purposes is 3p M. This trial aims to address dose-response, determining the lowest effective concentration required for treatment in mice considering the results received in the previous experiment. Doses lower than 3 pM (3pM, 1.2 pM, 0.5 pM) are tested to establish the minimal dose required for effective result.
  • the experimental groups are shown in Table 14. Schedule (twice or X4 daily) is determined based on experiment 2 results.
  • Example 9 Efficacy of the WFKA peptide in the treatment of ICI-induced autoimmune toxicities.
  • Immune checkpoint inhibitors have revolutionized cancer therapy, significantly extending survival and sometimes curing metastatic disease, but they can also trigger severe autoimmune-related adverse effects.
  • Monotherapy with anti-PDl ICIs leads to about 20% highgrade adverse events (AEs), anti-CTLA4 causes around 30%, and their combination results in nearly 60% severe AEs, including common issues like dermatitis, colitis, and thyroiditis.
  • AEs highgrade adverse events
  • CTLA4 anti-CTLA4
  • irAEs non- obese diabetic (NOD) mouse model was used. This model is prone to autoimmune pathologies and is particularly responsive to ICI-induced complications, specifically diabetes and thyroiditis. Thus, it may serve as a model for iatrogenic multiple autoimmune conditions associated with administration of ICIs.
  • the main aim of this study is to investigate the SLAMF6-derived WFKA peptide described in Example 3 as a therapeutic strategy for modulating immune activation in a ICI-induced diabetes and thyroiditis model (NOD mice).
  • NOD mice ICI-induced diabetes and thyroiditis model
  • the established model for ICI-induced diabetes and thyroid dysfunction works well with NOD/ ShiLtJ strain.
  • 95 female mice, 4-6 weeks old are used according to the following allocation.
  • mice 3-5 weeks age are admitted to the experimental cages.
  • the mice are acclimatized for 7 days before the initiation of the experiments. On the first day, the mice are weighed and marked. Every week the mice are treated twice weekly with combination (Dual ICI: anti-mouse-PD-1 plus anti-mouse-CTLA-4) at 10 mg/kg/dose intraperitoneally.
  • the experimental group is additionally injected Intraperitoneally with the test items every day.
  • the experimental groups receive the therapeutic substance in the desired dose in a volume of 0.1 ml and the control groups receive injections of saline (25G needle are used).
  • Mice are monitored daily for activity (including signs of neuropathy) and appearance and twice weekly for signs of autoimmunity: weight loss, decreased activity, and glucosuria. Activity and weight loss are assessed according to Table 16.
  • mice daily assessment Mice developing glucosuria are treated with 10 units of subcutaneous NPH insulin every 12 hours. After 4 or 8 weeks of treatment, mice are sacrificed, and multiple tissues (salivary, lacrimal, pancreas, liver, lung, heart, colon, eye, gonad, and thyroid) are evaluated by histology and flow cytometry for the development of autoimmune infiltrates. Prior to sacrifice, the mice undergo terminal bleeding (using the 'Cardiac Puncture' withdraw following anesthesia with Thiopental, followed by sacrificing via cervical dislocation. The experimental design for both experiments is described in Table 17.
  • the experiment aims to evaluate the peptide's activity and effectiveness in a second autoimmune pathology model and to determine the minimal effective dose required for treatment.
  • three dosages of the peptide based on those observed in dose-response experiments with cells and the DSS model, are administered daily.
  • the starting dosage tested in the DSS model (3pM, "high dose"), along with two additional dosages are used: one being the minimal effective dose from the DSS model ("minimal dose", 1.2 pM or 0.5 pM), and another in between these two dosages ("median dose”).
  • Mice are divided into 5 groups as described in Table 18.
  • mice are followed daily for mice activity and twice a week for weight and glucosuria. At the end of the 4-week period, the mice are euthanized, and tissues are collected for post-mortem analysis as described below.
  • Tissue Excision the tissues to be excised include the salivary glands, lacrimal glands, pancreas, liver, lungs, heart, colon, eyes, gonads, and thyroid. These tissues are utilized for histology, as follows: tissue immune infiltration is assessed by evaluating hematoxylin and eosin (H&E)-stained formalin-fixed paraffin-embedded (FFPE) sections. This analysis, performed on all tissues, evaluates autoimmune pathology and is conducted by an expert pathologist.
  • H&E hematoxylin and eosin
  • FFPE formalin-fixed paraffin-embedded
  • This experiment is performed after experiment 1 is completed and effective minimal dose is determined. It aims to test the inhibitory properties of the test item over diabetes and thyroiditis in ICI-treated NOD mice and study its mechanism of action.
  • the mice are divided into 3 groups as described in Table 19.
  • Tissue Excision The tissues to be excised include the salivary glands, lacrimal glands, pancreas, liver, lungs, heart, colon, eyes, gonads, and thyroid. These tissues are utilized for histology, flow cytometry, and scRNAseq analysis. Due to the small size of some tissues, using them for one analysis may leave insufficient material for the other two.
  • Subject Grouping To address this limitation, samples are collected from all major organs (i.e., heart, lungs, colon, liver, and pancreas) for histological analysis, while the remaining portions are allocated to flow cytometry and scRNA-seq. Glands are also divided among three analyses: histology, flow cytometry, and scRNA-seq.
  • Immune markers include at least the following: CD4, CD8, CD44, CD62L, CD25, FoxP3, CD127, CD33, CDl lb, CCR7, CD45RO, CD45RA, TIM3, PDL-1, LAG3, TCF7, IFNy, and TNFa.
  • scRNAseq is performed on isolated CD45 + infiltrating cells from the thyroids. The single cell suspension is stained and sorted using fluorescence-conjugated antibodies to CD45, CD1 lb, CD3, CD4, CD8, and CD19 and viability dye DAPI. Samples for each condition are then pooled to obtain sufficient cells for sequencing and analysis.
  • Serum Analysis Sera from the mice are tested for free thyroxine (FT4) and anti-TPO antibodies using ELISA.
  • Thyroid Tissue Processing Fresh thyroid tissues from ICLtreated mice are perfused with saline to remove circulating peripheral immune cells. The tissues are then dissociated into single-cell suspensions and stained for immune markers for flow cytometry analysis as described above.
  • a murine model is established with C57BL/6 mice immunized with the live attenuated Bacillus Calmette-Guerin (BCG) vaccine over a 14-day period, to induce a tumor-like microenvironment characterized by elevated MDSC levels.
  • BCG Bacillus Calmette-Guerin
  • This model mimics the chronic inflammation seen in cancer and allows for efficient isolation of MDSCs from the spleen and bone marrow.
  • MDSCs are isolated using positive selection (CDl lb + Grl + ), and their suppressive function are evaluated in vitro. These MDSCs are cultured, and the activity of WFKA peptide over their activity is assessed in two concentrations (3 pM and 10 pM).
  • the ability of the peptide to modulate MDSC-mediated immune suppression is evaluated by measuring the proliferation of T cells using CFSE labelling and flow cytometry over 48-72 hours. Changes in T cell proliferation indicate how effectively WFKA modulates MDSC-T cell interactions.
  • the research compares the ability of the peptide to inhibit SLAMF6-mediated and SLAMF6-independent MDSC-T cell suppression. Analysis includes NO secretion levels, arginase- 1 and proliferation assay (with CSFE staining).

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Abstract

The present invention relates to novel peptides having immunomodulatory activity, compositions comprising same and use thereof. More specifically, disclosed herein are synthetic peptides capable of inhibiting or controlling immune activation, and methods of using same in the treatment of autoimmunity, immune-related adverse events and other conditions associated with the inappropriate or excessive immune responses.

Description

IMMUNOMODULATING PEPTIDES AND USES THEREOF
FIELD OF THE INVENTION
The present invention relates to novel peptides having immunomodulatory activity and use thereof.
BACKGROUND OF THE INVENTION
Signaling lymphocytic activation molecule 6 (SLAMF6), also known as Lyl08 or NTB-A, is a homotypic surface receptor expressed on T cells, natural killer (NK) cells, B cells, dendritic cells (DC) and myeloid-derived suppressor cells (MDSC). SLAMF6 is largely considered an inhibitory receptor, although certain cell and context- specific effects were determined for this receptor, leading to its characterization as a dual receptor, capable of exerting either activating or inhibitory effects in the context of immune modulation. Engagement of SLAMF6 on human T cells can substitute the CD28 co-stimulatory pathway and induce polarization toward a Thl phenotype. However, CD4-positive T cells from Ly-108 knockout mice (the murine SLAMF6 ortholog) show impairment in IL-4 production, suggesting a role of SLAMF6 in Th2 polarization. The reason for this discrepancy is not fully elucidated. Activation of SLAMF6 on human NK cells stimulates cytotoxicity and proliferation, as well as IFN-y and TNF-a production. SLAMF6 is a natural ligand of SLAMF6 itself.
Valdez et al (J Biol Chem 2004, 279(18), pp. 18662-18669) teach that SLAMF6 activates T cells by homotypic interactions, and specifically enhances Thl properties. US 2009/017014 to Valdez et al is directed to the PR020080 polypeptide (having an amino acid sequence corresponding to that of canonical SLAMF6), the extracellular portion thereof, homologs, agonists and antagonists thereof, which are suggested as putative modulators of immune diseases. Uzana et al. (J Immunol 2012, 188, pp. 632-640) disclose that SLAMF6 blockade on antigen presenting cells (APC) by specific antibodies inhibited cytokine secretion from CD8+ lymphocytes.
Yigit et al (Cancer Immunol Res July 17, 2019) suggest a role for SLAMF6 as a regulator of exhausted CD8+ T cells in cancer. In particular, the publication demonstrates that a SLAMF6- binding antibody reduced the number of T cells expressing PD-1 and various other exhaustion markers (PD-1+CD3+CD44+CD8+ cells) in the spleen, in addition to its direct effect on tumor progression; both effects were suggested to depend mainly on antibody-dependent cellular cytotoxicity (ADCC).
Many pathologies leading to disease involve detrimental activation of the immune-system.
Examples include: graft versus host disease, rheumatoid arthritis, immune related adverse effects of checkpoint inhibitors to mention a few. Immunosuppressants are medicines that inhibit or decrease the intensity of the immune response in the body, often used in the management of graft rejection and certain autoimmune disorders. However, currently available immunosuppressants generally have a broad spectrum of activity leading to significant undesirable effects. Furthermore, some widely employed agents have very long half-lives and lack antidotes, thus leading to extended immune-suppression and risk of severe infections.
There exists a long-felt need for more effective means of curing or ameliorating chronic inflammatory or autoimmune diseases and for development of potent and controllable immunosuppressive agents. The development of immunosuppressive agents capable of acting in an activation-dependent manner would be particularly advantageous.
SUMMARY OF THE INVENTION
The present invention relates to novel peptides having immunomodulatory activity, compositions comprising same and use thereof. Disclosed herein are synthetic peptides capable of inhibiting or preventing excessive immune activation and pathologies associated therewith. Also disclosed are compositions and methods for use in inhibiting or reducing immune-related adverse events and in the treatment of diseases and conditions associated with the inappropriate or excessive immune response, including, but not limited to autoimmune diseases, graft versus host disease, and graft rejection.
The invention is based, in part, on the development of immunomodulating peptides having exceptionally advantageous therapeutic properties. In particular, isolated SLAMF6-derived peptides and modified peptides comprising various deletions, substitutions and derivatization by D-amino acids, were synthesized and characterized. Remarkably, peptides of the invention as detailed below were identified to suppress manifestations of immune cell activation in various models, including activation-induced secretion of cytokines and cytolytic granule components, in a SLAMF6-dependent manner. In addition, the development of synthetic peptides characterized by improved pharmacokinetic properties, including enhanced stability and resistance to serum proteases, is disclosed. The peptides were demonstrated to exhibit potent anti-inflammatory properties in vivo and significantly reduced the severity of colitis in mice.
In one aspect, the invention relates to an isolated peptide comprising the amino acid sequence X0TWX1FNX2X3X4X5A set forth as SEQ ID NO: 1 or a retro-inverso sequence thereof. In one embodiment, the isolated peptide comprises the amino acid sequence X0TWX1FNX2X3X4X5A (SEQ ID NO: 1) or a retro-inverso sequence thereof, wherein Xo and X5 are each independently a hydrophobic amino acid, Xi , X2 , X3 and X4 are each independently any amino acid and wherein the peptide consists of from 11 to 50 amino acids.
In another embodiment, the isolated peptide is characterized in that:
Xo and X5 are each independently selected from Leu, Vai and He;
Xi is selected from Leu, He, Vai, Met, Ser and Thr;
X2 is selected from Gly, Ala, Asp and Glu;
X3 is selected from Ser, Thr, Lys and Arg; and
X4 is selected from Ser, Asn, Gin, and Thr (SEQ ID NO: 17).
In another embodiment, the isolated peptide is characterized in that:
XQ and X5 are each independently selected from Leu, Vai and He;
Xi is selected from Thr, and Leu;
X2 is selected from Gly, Glu and Ala;
X3 is selected from Lys and Thr;
X4 is selected from Ser and Thr, set forth as SEQ ID NO: 18.
In another embodiment, the isolated peptide comprises the amino acid sequence I TWTFNGKSLA (SEQ ID NO: 2).
In another embodiment, peptides in accordance of the invention are advantageously characterized by the inclusion of at least one D-amino acid. In another embodiment, at least 2 amino acids are D-amino acids. According to exemplary embodiments, 2, 3, 4 or 5 amino acids are D-amino acids, wherein each possibility represents a separate embodiment of the invention.
In another embodiment at least 2 of the amino acids at positions 3, 5, 8 and 11 of the sequence X0TWX4FNX2X3X4X5A (SEQ ID NO: 1) are D-amino acids. In another embodiment, the amino acids at positions 3, 5, 8 and 11 are D-amino acids and the peptide comprises the amino acid sequence X0TwX4 fNX2x3X4X5a (SEQ ID NO: 6, wherein D-amino acids are represented by low-case letters). In another embodiment, the peptide comprises the amino acid sequence I TwT fNGkSLa (SEQ ID NO: 7). In another embodiment said peptide consists of the amino acid sequence I TwT fNGkSLa (SEQ ID NO: 7).
In another aspect, the invention relates to a conjugate of an isolated peptide as disclosed herein. In a particular embodiment, the conjugate comprises said peptide conjugated with polyethylene glycol (PEG).
In another aspect, there is provided a pharmaceutical composition comprising at least one peptide or conjugate as disclosed herein, and a pharmaceutically acceptable carrier and/or excipient. In some embodiments, the peptide comprises or consists of an amino acid sequence selected from I TWTFNGKSLA (SEQ ID NO: 2) and I TwT fNGkSLa (SEQ ID NO: 7). Each possibility represents a separate embodiment of the invention.
In another aspect, the pharmaceutical composition is for use in inhibiting or reducing an inappropriate or excessive immune response in a subject in need thereof.
In another embodiment, the use comprises inhibiting or reducing immune-related adverse events (irAEs). In another embodiment, the irAEs are selected from the group consisting of gastrointestinal, endocrine, and dermatologic toxicities, and combinations thereof. According to exemplary embodiments, the irAEs may comprise toxicities affecting the pancreas and/or thyroid gland. In another embodiment said irAEs are associated with administration of an immuno stimulatory treatment. In another embodiment said immuno stimulatory treatment is a cancer immunotherapy. In another embodiment said irAEs are associated with administration of a cancer immunotherapy. In another embodiment, said cancer immunotherapy comprises administration of an immune checkpoint inhibitor. In another embodiment the immune checkpoint inhibitor targets PD-1, PD-L1, PD-L2, CTLA-4, LAG3, TIM3, and/or IDO.
In another embodiment, the use comprises treating a disease or a condition associated with the inappropriate or excessive immune response. In another embodiment the condition is a T-cell mediated inflammatory or autoimmune condition. In another embodiment, the disease or condition is selected from the group consisting of graft versus host disease, an autoimmune disease, and graft rejection. In another embodiment, the autoimmune disease is selected from rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), multiple sclerosis (MS), psoriasis, psoriatic arthritis, autoimmune hepatitis and primary biliary cholangitis (PBC). In a particular embodiment, said autoimmune disease is IBD. In other particular embodiments, said autoimmune disease is insulin dependent diabetes mellitus or autoimmune thyroiditis.
In another aspect, there is provided a method for inhibiting or reducing an inappropriate or excessive immune response in a subject in need thereof, comprising administering to the subject the pharmaceutical composition as disclosed herein. In another embodiment, the pharmaceutical composition comprises at least one isolated peptide comprising the amino acid sequence X0TWX1FNX2X3X4X5A (SEQ ID NO: 1) or a retro-inverso sequence thereof, wherein Xo and X5 are each independently a hydrophobic amino acid, Xi, X2, X3 and X4 are each independently any amino acid and wherein the peptide consists of from 11 to 50 amino acids. In another embodiment, the peptide comprises or consists of an amino acid sequence selected from I TWTFNGKSLA (SEQ ID NO: 2) and I TwT fNGkSLa (SEQ ID NO: 7). In various other embodiments, the method comprises inhibiting or reducing irAEs (e.g. associated with treatments as disclosed herein), in some embodiments, the irAEs are selected from the group consisting of gastrointestinal, endocrine, and dermatologic toxicities, and combinations thereof. In other embodiments, the irAEs comprise toxicities affecting the pancreas and/or thyroid gland. In another embodiment said irAEs are associated with administration of a cancer immunotherapy. In another embodiment said cancer immunotherapy comprises administration of an immune checkpoint inhibitor. In another embodiment the immune checkpoint inhibitor targets PD-1, PD-L1, PD-L2, CTLA-4, LAG3, TIM3, and/or IDO. In various other embodiments, the method comprises treating a disease or a condition associated with the inappropriate or excessive immune response (e.g. the conditions as disclosed herein). In another embodiment the condition is a T-cell mediated inflammatory or autoimmune condition. In other embodiments, the autoimmune disease is selected from rheumatoid arthritis, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), multiple sclerosis (MS), psoriasis, psoriatic arthritis, autoimmune hepatitis, primary biliary cholangitis (PBC), insulin dependent diabetes mellitus and autoimmune thyroiditis, wherein each possibility represents a separate embodiment of the invention. In another embodiment said autoimmune disease is IBD. In another embodiment said autoimmune disease is insulin dependent diabetes mellitus or autoimmune thyroiditis.
These and other embodiments will be described in greater detail below.
BRIEF DESCRIPTION OF DRAWINGS
Fig. 1A shows the amino acid sequence of the SLAMF6 receptor (SEQ ID NO: 12) with the initial peptide sequence spanning amino acids 49 to 91 (V49) marked in bold.
Fig. IB shows a three-dimensional structure of the SLAMF6 receptor- V49 in dark grey.
Fig. 1C shows binding of V49 to SLAMF6. Binding of FITC-conjugated peptides (V49, V49(ala) or V49(scr)) to wild-type 526 melanoma cells (WT), not expressing SLAMF6, is shown in black, and binding to 526 cells engineered to express SLAMF6 (SLAMF6 526) is shown in grey. Mean fluorescence intensity (MFI) was measured by flow cytometry.
Fig. ID shows the inhibitory effect of V49 on peripheral blood mononuclear cells (PBMC) that were activated with staphylococcal enterotoxin B (SEB). Activated cells were incubated with the V49, V49(ala) or V49(scr) peptides at the indicated concentrations, and Interleukin-2 (IL-2) secretion was measured by ELISA.
Fig. IE shows the inhibitory effect of V49 on tumor-infiltrating lymphocytes (TILs) that were activated by incubation with cognate melanoma cells. Activated cells were incubated with peptides as describe in Fig. ID at the indicated concentrations, and interferon gamma (IFNy) secretion was measured via ELISA.
Fig. 2A shows the inhibitory effect of the 153 peptide on TIL in the presence of cognate 526 melanoma cells. Activated cells were incubated with the indicated peptides at the indicated concentrations, and IFNy secretion was measured by ELISA.
Fig. 2B shows the inhibitory effect of the 153 peptide on TIL in the presence of cognate 624 melanoma cells. Activated cells were incubated with the indicated peptides at the indicated concentrations, and IFNy secretion was measured by ELISA.
Fig. 2C shows the inhibitory effect of peptides on activation-induced IL-2 secretion in PBMC. SEB-activated cells were incubated with the indicated peptides at the indicated concentrations, and IL-2 secretion was measured by ELISA.
Fig. 2D shows the inhibitory effect of different concentrations 153 peptide on PBMC function (measured as IL-2 secretion by ELISA) following SEB activation.
Fig. 2E shows the inhibitory effect of 153 and 3mut on PBMCs activated with SEB and treated at 0.5uM and 2.5uM peptides. 153 (ala) was a negative control. IL2 secretion levels were measured by ELISA.
Fig. 2F shows the inhibitory effect of 153, CRla and 3mut on TILs activated with their cognate melanoma cells. 153 (ala) used as negative control. IFNy secretion levels were measured by ELISA.
Fig. 2G shows the inhibitory effect of peptides of 3mut on murine pMEL splenocytes co-cultured with cognate melanoma cells as compared with a scrambled sequence of 3mut as control (scr). IFNy secretion was measured by ELISA.
Fig. 3A shows the stability of the 3Mut (SEQ ID NO: 2) peptide as tested by trypsin and chymotrypsin proteolysis. The % of intact peptide (uncleaved) as a function of time, as measured by analytical high pressure liquid chromatography (HPLC). Absorbance was detected at a wavelength of 220 nm to identify the presence of peptide bonds.
Fig. 3B shows the stability of the WFKA peptide SEQ ID NO: 7) as tested by trypsin and chymotrypsin proteolysis.
Fig. 3C shows the inhibitory effect of WFKA on PBMC cells activated using SEB. Activated cells were incubated with the indicated peptides at the indicated concentrations and IL-2 secretion was measured by ELISA.
Fig. 3D shows the inhibitory effect of WFKA on melanoma-specific human TILs co-cultured with a specific melanoma identified by the TILs. IFNy secretion was measured by ELISA.
Fig. 3E shows the inhibitory effect of WFKA on murine splenocytes co-cultured with cognate melanoma cells (B16 GP100). IFNy secretion was measured by ELISA. Fig. 4A shows the conserved pattern of 3Mut and WFKA peptides (in boxes).
Fig. 4B shows dose-response of WFKA on activation-induced Granzyme-B (GZM-b) secretion in the human Jurkat cell line. CD3-activated cells were incubated with the indicated concentrations of the peptide, and GZM-b was measured by ELISA.
Fig. 4C shows the effect of WFKA, scrl and scr2 peptides on activation-induced GZM-b secretion in wild-type Jurkat cells (WT) as compared to SLAMF6 knockout Jurkat cells (SLAMF6'7')- CD3- activated cells were incubated with the indicated peptides at the indicated concentrations, and GZM-b was measured by ELISA.
Fig. 4D shows the effect of WFKA, scrl and scr2 on murine splenocytes (WT) as compared to Lyl06-knockout splenocytes (SLAMF6'/_). IFNy secretion was measured by ELISA.
Fig. 4E shows Granzyme B (GZM-b) secretion levels in JURKAT wild type (WT) cells in the presence of the MUT3 peptide or the WFKA peptide, as measured by ELISA. Scrl and Scr2 peptides were used as negative controls. CD3-activated cells were incubated with the indicated concentrations of the peptide, and GZM-b was measured by ELISA.
Fig. 4F shows that in JURKAT SLAMF67' cells, GZM-b secretion was not inhibited by the MUT3 or WFKA peptides, indicating that the inhibitory effect of these peptides is directly dependent on the presence of the SLAMF6 receptor. Treatments and methods are as in Fig. 4E.
Figs. 5A-5D show scores for different parameters in a DSS-induced colitis murine model. Fig. 5A shows weight loss score, Fig. 5B shows diarrhea incidence, Fig. 5C shows bleeding score, and Fig. 5D shows DAI score. C57BL/6 mice were induced with colitis by 2% DSS treatment. At day 7 (marked by an arrow), the administration of DSS inducing colitis was ceased. Group A - DSS- treated mice with no peptide administration. Group B - DSS -treated mice with WFKA peptide administration. Group C - naive mice (no DSS, no peptide treatment).
Figs. 6A-6C show colons of mice of from different groups of the DSS-induced colitis murine model. Fig. 6A shows colon length in naive mice (Group C). Fig. 6B shows colon length in mice treated with DSS only (Group A). Fig. 6C shows colon length in mice treated with DSS and 3 pM WFKA peptide (Group B).
Fig. 7 shows a comparison of colon length between WFKA peptide-treated (Group B) and untreated (Group A) mice of the DSS-induced colitis mice as compared to naive mice (Group C).
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to novel peptides having immunomodulatory activity, compositions comprising same and use thereof. More specifically, disclosed herein are synthetic peptides capable of inhibiting or preventing excessive immune activation and pathologies associated therewith. The compositions and methods as disclosed herein may be used for inhibiting or reducing immune-related adverse events and in the treatment of diseases and conditions associated with the inappropriate or excessive immune response, including, but not limited to autoimmune diseases, graft versus host disease (GVHD), and graft rejection.
Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. In case of conflict, the patent specification, including definitions, will control.
The present invention is based on discovery of novel stable peptides capable of modulating immune responses. Specifically, disclosed herein is the identification and characterization of synthetic, non-naturally occurring peptides, shown to exhibit a considerable and significant ability to control excessive immune activation in various experimental models employing the use of human or murine-derived lymphocytes. In vivo, the peptides significantly delayed the development of colitis in a murine model for inflammatory bowel disease (IBD), as evaluated by various clinical and inflammatory manifestations.
In one aspect, the invention relates to an isolated peptide comprising the amino acid sequence X0TWX1FNX2X3X4X5A (SEQ ID NO: 1) or a retro-inverso sequence thereof, wherein Xo and X5 are each independently a hydrophobic amino acid, X1 A X2 , X3 and X4 are each independently any amino acid and wherein the peptide consists of from 11 to 50 amino acids. In various embodiments, the isolated peptide comprises or consists of the amino acid sequence selected from I TWTFNGKSLA (SEQ ID NO: 2) and I TwT fNGkSLa (SEQ ID NO: 7).
In another aspect, the invention relates to a pharmaceutical composition comprising at least one peptide isolated peptide as defined herein, or a conjugate thereof.
In another aspect, the pharmaceutical composition is for use in inhibiting or reducing an inappropriate or excessive immune response in a subject in need thereof. In another aspect, the invention relates to a pharmaceutical composition comprising an isolated peptide of the amino acid sequence X0TWX1FNX2X3X4X5A (SEQ ID NO: 1) as defined herein or a retro-inverso sequence thereof, for use in inhibiting or reducing an inappropriate or excessive immune response in a subject in need thereof. In another aspect, the invention relates to a pharmaceutical composition comprising an isolated peptide that comprises or consists of the amino acid sequence selected from I TWTFNGKSLA (SEQ ID NO: 2) and I TwT fNGkSLa (SEQ ID NO: 7), for use in inhibiting or reducing an inappropriate or excessive immune response in a subject in need thereof. In another embodiment the pharmaceutical composition is for use in inhibiting or reducing immune-related adverse events (irAEs). In another embodiment the pharmaceutical composition is for use in treating a disease or a condition associated with the inappropriate or excessive immune response. In another aspect the invention provides a method for inhibiting or reducing an inappropriate or excessive immune response in a subject in need thereof, comprising administering to the subject the pharmaceutical composition (e.g. comprising an isolated peptide as set forth in any one of SEQ ID NOs: 1, 2 and 7 as defined herein).
In another aspect the invention provides a method for inhibiting or reducing irAEs in a subject in need thereof, comprising administering to the subject the pharmaceutical composition (e.g. comprising an isolated peptide as set forth in any one of SEQ ID NOs: 1, 2 and 7 as defined herein). In another embodiment the irAEs are associated with administration of an immune checkpoint inhibitor (or other forms of cancer immunotherapy).
In another aspect the invention provides a method for treating a disease or a condition associated with the inappropriate or excessive immune response in a subject in need thereof, comprising administering to the subject the pharmaceutical composition (e.g. comprising an isolated peptide as set forth in any one of SEQ ID NOs: 1, 2 and 7 as defined herein). In some embodiments, the disease or condition is selected from the group consisting of graft versus host disease (GVHD), an autoimmune disease, and graft rejection.
In another aspect the invention provides a method for treating an autoimmune disease in a subject in need thereof, comprising administering to the subject the pharmaceutical composition (e.g. comprising an isolated peptide as set forth in any one of SEQ ID NOs: 1, 2 and 7 as defined herein). In various embodiments, the autoimmune disease is selected from rheumatoid arthritis, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), multiple sclerosis (MS), psoriasis, psoriatic arthritis, autoimmune hepatitis, primary biliary cholangitis (PBC), insulin dependent diabetes mellitus and autoimmune thyroiditis.
These and other aspects and embodiments of the invention are described in further detail hereinbelow.
Peptides
According to one aspect, the present invention provides an isolated peptide comprising an amino acid sequence X0TWX1FNX2X3X4X5A set forth as SEQ ID NO: 1 or a retro-inverso sequence thereof, wherein Xo and X5 are each independently a hydrophobic amino acid, wherein X , , X2 , X3 and X4 are each independently any amino acid and wherein the peptide consists of from 11 to 50 amino acids. According to some embodiments, the peptide comprises the amino acid sequence X0TWX1FNX2X3X4X5A, wherein Xo and X5 are the same hydrophobic amino acid and Xi , X2 , X3 and X4 are each independently any amino acid and wherein the peptide consists of from 11 to 50 amino acids. The term "peptide" as used herein refers to a short chain of amino acid monomers linked by peptide bonds, i.e., the covalent bond formed between carboxyl group of one amino acid and an amino group of another amino acid. Peptides usually comprise up to 50 amino acids. According to some embodiments, the peptide consists of 12 to 45 amino acids. According to another embodiment, the peptide consists of 15 to 40 amino acids. According to some embodiments, the peptide comprises 17 to 35 amino acids. According to other embodiments, the peptide consists of 20 to 30 amino acids. According to another embodiment, the peptide consists of 11 to 30 amino acids. According to some embodiments, the peptide consists of 11-15, 11-20, 12 to 25, 13 to 23, or 14 to 20, amino acids. Each possibility represents a separate embodiment of the invention. According to some embodiments, the peptide consists of 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.
It is a well-known fact that a short peptide when extracted or isolated from the context of the protein typically loses its structure and native activity. In contradistinction, peptides in accordance with the invention unexpectedly exert therapeutic properties as disclosed herein.
The term “isolated peptide” is meant to include peptides which are not naturally occurring and that do not naturally exist outside the context of their complete, full-length source proteins (if any). The term “isolated” is also used herein to refer to peptides of polypeptides that are isolated from other cellular proteins and constituents and is meant to encompass chemically synthesized, recombinantly produced and purified peptides and polypeptides. The term further encompasses in some embodiments separation from the source that produced the peptide, such as recombinant cells or residual peptide synthesis reactants. Thus, the isolated peptide is optionally “purified”, which means at least: 80%, 85%, 90%, 95%, 98% or 99% purity and optionally pharmaceutical grade purity. The term "amino acid" includes both "natural" and "unnatural" or "non-natural" amino acids.
According to some embodiments, Xo is a hydrophobic amino acid. The term “hydrophobic amino acids” or related terms such as “hydrophobic moieties provided by the residues of amino acids” is referring to neutral amino acids which comprise to a large extent mainly a hydrophobic moiety apart from their amino and carboxy group. In some embodiments, the term "hydrophobic amino acid” refers to Gly, Ala, Vai, Leu, Aic, He, Pro, Tyr, Phe, Met, Eaa, naphthylalanine and Trp. In some embodiments, the hydrophobic amino acid is selected from Gly, Ala, Vai, Leu, He, Pro, Tyr, Phe, Met and Trp. The term "small hydrophobic amino acid" refers to hydrophobic amino acid having non-bulky residue such as Gly and Ala. According to some embodiments, Xo is selected from Leu, He, Vai and Met amino acids. According to other embodiments, Xo is selected from Leu, Vai and He. According to yet another embodiment, Xo is He. According to some embodiments, X5 is a hydrophobic amino acid selected from Gly, Ala, Vai, Leu, He, Pro, Tyr, Phe, Met and Trp. According to some embodiments, X5 is selected from Leu, He, Vai and Met amino acids. According to other embodiments, X5 is selected from Leu, Vai and He. According to yet another embodiment, X5 is Leu. According to a further embodiment, Xo is He and X5 is Leu. According to some embodiment, Xi is selected from a hydrophobic and a polar amino acid.
As used herein, the terms "polar amino acid" and “polar uncharged amino acid residue” refers to an amino acid residue having a side chain that is uncharged and has a dipole moment. Examples of polar amino acid residues, include, but are not limited to glycine, sarcosine, L-serine, L-threonine, L-cysteine, L-tyrosine, L-asparagine, L-glutamine, D-serine, D- threonine, D- cysteine, D-tyrosine, D-asparagine and D-glutamine, N-methyl-L- serine, N- methyl-L-threonine, N-methyl-L-cysteine, N-methyl-L-tyrosine, N-methyl-L-asparagine, N-methyl-L-glutamine, N- methyl-D- serine, N-methyl-D-threonine, N-methyl-D-cysteine, N-methyl-D-tyrosine, N-methyl- D-asparagine, N-methyl-D-glutamine, L-homoarginine, D-homoarginine, N-methyl-L- homoarginine, N-methyl-D-homoarginine, L-O- methyltyrosine, D-O-methyltyrosine, N-methyl- L-O-methyltyrosine, N-methyl-D-O- methyltyrosine, L-homotyrosine, D-homotyrosine, N- methyl-L-homotyrosine, N-methyl- D-homotyrosine, L-O-methylhomotyrosine, D-O- methylhomotyrosine, N-methyl-L-O- methylhomo tyro sine and N-methyl-D-O- methylhomo tyro sine. According to some embodiment, the polar amino acid refers to an amino acid selected from the group consisting of Asn, Gin, Ser, Thr, Cys and Tyr. According to some embodiments, the polar amino acid is selected from Asn, Gin, Ser, and Thr. According to other embodiments, the polar amino acid is selected from Ser, and Thr.
According to some embodiments, Xi is selected from Leu, He, Vai, Met, Ser and Thr amino acid. According to one embodiment, Xi is selected from Leu and Thr. According to one embodiment, Xi is Thr. According to one embodiment, Xi is Leu. According to some embodiments, Xoand X5 are each independently selected from Leu, He, Vai and Met amino acids, and Xi is selected from Leu, He, Vai, Met, Ser and Thr amino acid. According to some embodiments, Xo is He, X5 is Leu, and Xi is Thr.
According to further embodiments, X2 is selected from a small hydrophobic and negatively charged amino acid. As used herein, the term “negatively charged amino acid” refers to an amino acid residue having a side chain capable of bearing a negative charge. Examples include, but are not limited to L-aspartic acid, L-glutamic acid, D-aspartic acid, D- glutamic acid, N-methyl-L- aspartic acid, N-methyl-L-glutamic acid, N-methyl-D-aspartic acid and N-methyl-D-glutamic acid. According to some embodiments, negatively charged amino acid is selected from Asp and Glu.
According to some embodiments, X2 is selected from Gly, Ala, Asp and Glu. According to some embodiments, X2 is Gly. According to some embodiments, X2 is Glu. According to some embodiments, Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids, and X2 is selected from Gly, Ala, Asp and Glu. According to some embodiments, Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids, and X2 is selected from Gly and Glu. According to some embodiments, Xo is He, X5 is Leu, and X2 is Gly. According to some embodiments, Xo is He, X5 is Leu, and X2 is Glu.
According to some embodiments, Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids, Xi is selected from Leu, He, Vai, Met, Ser and Thr amino acid and X2 is selected from Gly, Ala, Asp and Glu. According to some embodiments, Xo is He, X5 is Leu, Xi is Thr and X2 is Gly. According to some embodiments, Xo is He, X5 is Leu, Xi is Leu and X2 is Gly. According to some embodiments, Xo is He, X5 is Leu, Xi is Thr and X2 is Glu. According to some embodiments, Xo is He, X5 is Leu, Xi is Leu and X2 is Glu. According to some embodiments, X3 is selected from a polar amino acid and a positively charged amino acid.
As used herein, the term “positively charged amino acid residue” refers to an amino acid residue having a side chain capable of bearing a positive charge. Examples include, but are not limited to L-lysine, L-arginine, L-histidine, L-ornithine, D-lysine, D- arginine, D-histidine, D- omithine, N-methyl-L-lysine, N-methyl-L-arginine, N-methyl-L- histidine, N-methyl-L-ornithine, N-methyl-D-lysine, N-methyl-D-arginine, N-methyl- D-histidine, N-methyl-D-ornithine, L- diaminobutyric acid (DAB), D-di aminobutyric acid, N-methyl-L-diaminobutyric acid, N-methyl- D-diaminobutyric acid, L-citrulline (CIT), D- citrulline, N-methyl-L-citrulline, N-methyl-D- citrulline, L-homoarginine, D- homoarginine, N-methyl-L-homoarginine and N-methyl-D- homoarginine. According to some embodiments, positively charged amino acid is selected from Lys and Arg
According to some embodiments, X3 is selected from Ser, Thr, Lys and Arg. According to some embodiments, X3 is Lys. According to some embodiments, X3 is Thr. According to some embodiments, Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids, and X3 is selected from Ser, Thr, Lys and Arg. According to some embodiments, Xo is He, X5 is Leu, and X3 is Lys. According to some embodiments, Xo is He, X5 is Leu, and X3 is Thr. According to some embodiments, Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids, Xi is selected from Leu, He, Vai, Met, Ser and Thr amino acid, and X3 is selected from Ser, Thr, Lys and Arg. According to some embodiments, Xo is He, X5 is Leu, Xi is Thr and X3 is Lys. According to some embodiments, Xo is He, X5 is Leu, Xi is Thr and X3 is Thr.
According to some embodiments, Xoand X5 are each independently selected from Leu, He, Vai and Met amino acids, X2 is selected from Gly, Ala, Asp and Glu, and X3 is selected from Ser, Thr, Lys and Arg. According to some embodiments, Xo is He, X5 is Leu, X2 is Gly and X3 is Lys. According to some embodiments, Xo is He, X5 is Leu, X2 is Gly and X3 is Thr.
According to some embodiments, Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids, Xi is selected from Leu, He, Vai, Met, Ser and Thr amino acid, X2 is selected from Gly, Ala, Asp and Glu, and X3 is selected from Ser, Thr, Lys and Arg. According to some embodiments, Xo is He, X5 is Leu, He, Xi is Thr, X2 is Gly and X3 is Lys. According to some embodiments, Xo is He, X5 is Leu, He, Xi is Thr, X2 is Gly and X3 is Thr.
According to another embodiment, wherein X4 is a polar amino acid. According to one embodiment, X4 is selected from Asn, Gin, Ser and Thr. According to one embodiment, X4 is Ser. According to some embodiments, Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids, and X4 is selected from Asn, Gin, Ser and Thr. According to some embodiments, Xo is He, X5 is Leu, and X4 is Ser. According to some embodiments, Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids, Xi is selected from Leu, He, Vai, Met, Ser and Thr amino acid and X4 is selected from Asn, Gin, Ser and Thr. According to some embodiments, Xo is He, X5 is Leu, Xi is Thr and X4 is Ser.
According to some embodiments, Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids, X2 is selected from Gly, Ala, Asp and Glu and X4 is selected from Asn, Gin, Ser and Thr. According to some embodiments, Xo is He, X5 is Leu, X2 is Gly and X4 is Ser. According to some embodiments, Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids, X3 is selected from Ser, Thr, Lys and Arg, and X4 is selected from Asn, Gin, Ser and Thr. According to some embodiments, Xo is He, X5 is Leu, X3 is Lys and X4 is Ser. According to some embodiments, Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids, Xi is selected from Leu, He, Vai, Met, Ser and Thr amino acid, X2 is selected from Gly, Ala, Asp and Glu, and X4 is selected from Asn, Gin, Ser and Thr. According to some embodiments, Xo is He, X5 is Leu Xi is Thr, X2 is Gly, and X4 is Ser. According to some embodiments, Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids, Xi is selected from Leu, He, Vai, Met, Ser and Thr amino acid, X3 is selected from Ser, Thr, Lys and Arg and X4 is selected from Asn, Gin, Ser and Thr. According to some embodiments, Xo is He, X5 is Leu Xi is Thr, X3 is Lys and X4 is Ser. According to some embodiments, Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids, X2 is selected from Gly, Ala, Asp and Glu, X3 is selected from Ser, Thr, Lys and Arg and X4 is selected from Asn, Gin, Ser and Thr. According to some embodiments, Xo is He, X5 is Leu, X2 is Gly, X3 is Lys and X4 is Ser. According to some embodiments, Xo and X5 are each independently selected from Leu, He, Vai and Met amino acids, Xi is selected from Leu, He, Vai, Met, Ser and Thr, X2 is selected from Gly, Ala, Asp and Glu, X3 is selected from Ser, Thr, Lys and Arg and X4 is selected from Asn, Gin, Ser and Thr. According to some embodiments, Xo is He, X5 is Leu Xi is Thr, X2 is Gly, X3 is Lys and X4 is Ser.
In another embodiment, the peptide is characterized in that:
Xo and X5 are each independently selected from Leu, Vai and He;
Xi is selected from Leu, He, Vai, Met, Ser and Thr;
X2 is selected from Gly, Ala, Asp and Glu;
X3 is selected from Ser, Thr, Lys and Arg; and
X4 is selected from Ser, Asp, Gin, and Thr.
In another embodiment, the peptide is characterized in that:
Xo and X5 are each independently selected from Leu, Vai and He; Xi is selected from Thr, and Leu; X2 is selected from Gly, Glu and Ala; X3 is selected from Lys and Thr; X4 is selected from Ser and Thr, set forth as SEQ ID NO: 17.
In another embodiment, the peptide is characterized in that:
Xo and X5 are each independently selected from Leu and He; Xi is selected from Thr, and Leu; X2 is selected from Gly and Glu; X3 is selected from Lys and Thr; X4 is selected from Ser and Thr, set forth as SEQ ID NO: 18.
According to some embodiments, the peptide comprises an amino acid sequence selected from SEQ ID NO: 20-163. According to some embodiments, the peptide comprises the amino acid sequence ITWTFNGKSLA (SEQ ID NO: 2). According to some embodiments, the peptide consists of the amino acid sequence ITWTFNGKSLA (SEQ ID NO: 2).
As used herein the terms "comprise(s)," "include(s)," "having," "has," "contain(s)," and variants thereof, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structure. The term “comprising” always includes the term “consisting” and when the term “comprising” appears in an embodiment of the disclosure, this same embodiment wherein the term “consisting” replaces the term “comprising” is always also an embodiment of the disclosure.
According to some embodiments, the peptide comprises or consists of the amino acid sequence VNFITWLFNETSLAFIVPHETKSPEIHVTNPKQGKRLNFTQS (SEQ ID NO: 3). According to some embodiments, the peptide comprises or consists of the amino acid sequence VNFITWTFNGKSLAFIVPHETKSPEIHVTNPKQGKRLNFTQS (SEQ ID NO: 5).
According to any one of the above embodiments, at least 1 amino acid of the peptide of the present invention may be a D-amino acid. According to some embodiments, at least 2 amino acids of the peptide of the present invention are D-amino acids. According to other embodiments, at least 3 amino acids of the peptide of the present invention are D-amino acids. According to further embodiments, at least 2 and up to 4 or 5 amino acids of the peptide of the present invention are D- amino acids. According to some embodiments, from 2 to 6 amino acids of the peptide of the present invention are D-amino acid. According to some embodiments, 2, 3, 4, 5, 6, or 7 amino acids of the peptide of the present invention having an amino acid sequence SEQ ID NO:1 are D-amino acids. According to some embodiments, 3 amino acids of the peptide of the present invention are D- amino acids. According to some embodiments, 4 amino acids of the peptide of the present invention are D-amino acids. According to some embodiments, 5 amino acids of the peptide of the present invention are D-amino acids. The term “D-amino acid” refers to an amino acid having the D-configuration around the a-carbon as opposite to native L-amino acid having L-conformation. As used herein, the D-amino acid in the sequence is represented by a lower-case letter. In addition, when sequences comprising both D-amino acids and L-amino acids are discussed in this specification, the L-amino acid is represented by a capital letter. As such, the sequence of VyL represent a sequence in which Vai and Leu are native L-amino acids and Tyr is a D-amino acid.
According to some embodiments, at least 2 amino acids at positions 3, 5, 8 and 11 of the sequence X0TWX1FNX2X3X4X5A (SEQ ID NO: 1) are D-amino acids. According to some embodiments, at least 3 amino acids at positions 3, 5, 8 and 11 of the sequence X0TWX1FNX2X3X4X5A are D-amino acids.
According to some embodiments, the present invention provides a peptide having the amino acid sequence XoTwXifNX2X3X4Xsa (SEQ ID NO: 6) wherein Xo and X5 are each independently a hydrophobic amino acid, Xi, X2, and X4 are each independently any amino acid, and X3 is any D-amino acid.
According to some embodiments, the present invention provides a peptide having the amino acid sequence SEQ ID NO: 6 in which amino acids at positions 3, 5, 8 and 11 of the sequence are D-amino acids. According to some embodiments, the present invention provides a peptide comprising an amino acid sequence selected from SEQ ID NO: 20-163 in which amino acids at positions 3, 5, 8 and 11 of the sequence are D-amino acids. According to some embodiments, the present invention provides a peptide comprising an amino acid sequence selected from SEQ ID NO: 20-163 in which amino acids at positions 3, 5, 8 and 11 of the sequence are D-amino acids and having at least 1 additional amino acid being D-amino acid.
According to one embodiment, the present invention provides a peptide comprising the amino acid sequence ITwTfNGkSLa (SEQ ID NO: 7). According to another embodiment, the present invention provides a peptide consisting of the amino acid sequence ITwTfNGkSLa.
According to one embodiment, the present invention provides a peptide comprising or consisting of the amino acid sequence
VNFITwTfNGkSLaFIVPHETKSPEIHVTNPKQGKRLNFTQS (SEQ ID NO: 8).
According to one embodiment, the peptide of the present invention comprises the retro- inverso amino acid sequence of the amino acid sequence X0TWX1FNX2X3X4X5A (SEQ ID NO: 1). According to one embodiment, the peptide of the present invention comprises the retro-inverso amino acid sequence of a peptide sequence as disclosed herein. Each possibility represents a separate embodiment of the invention.
The terms “retro-inverso peptide” and “retro-inverse peptide” are used herein interchangeably and refer to a reverse peptide composed of D-amino acids (retro=reversed; as described e.g., in Regenmortel and Muller: Current Opinion in Biotechnology 9, pp 377-382 ,1998). According to one embodiment, the peptide of the present invention comprises a retro- inverso amino acid sequence of an amino acid sequence selected from SEQ ID NO: 2, 16, and 20- 163. According to some embodiments, the peptide comprises the amino acid sequence alskgnftwti (SEQ ID NO: 9).
In another embodiment the peptide has at least 80% sequence identity to a peptide as disclosed herein. In another embodiment the peptide has at least 90% sequence identity to a peptide as disclosed herein. For example, disclosed herein is a peptide according to the general formula of SEQ ID NO: 1 which further has 80% or 90% sequence identity to the peptide of SEQ ID NO: 7. According to another embodiment, the peptide has at least 80% or at least 90% sequence identity to the peptide of SEQ ID NO: 2. Each possibility represents a separate embodiment of the invention.
The peptides of the invention exhibit immunomodulatory properties (and are thus capable of modulating an immune response or components thereof). As demonstrated herein, peptides in accordance with the invention are capable of exhibiting immunosuppressive activity. For example, peptides of the invention were demonstrated to inhibit activation-induced cytokine secretion and cytolytic granule components from various immune cells, including human peripheral blood mononuclear cells (PBMC), human tumor-infiltrating lymphocytes (TIL) and tumor- specific murine splenocytes. In some embodiments, a peptide of the invention is capable of inhibiting immune effector functions (such as cytokine and/or granzyme secretion) in activated cells without substantially affecting effector functions in resting immune cells. In another embodiment, the activity is SLAMF6-mediated.
According to some embodiments, the peptides of the present invention have half-life of at least about 1 hour in serum. According to some embodiments, the peptides of the present invention have a half-life of from 1 to 2 hours, from 1 to 4 hours, from 2 to 5 hours, from 3 to 7 hours, from 8 to 14, or from 10 to 16 hours in serum. According to some embodiments, the peptides of the present invention have a half-life of at least about 24 hours. According to some embodiments, the peptides of the present invention have a half-life of from 1 to 2 days, from 1 to 4 day, from 2 to 5 days or from 3 to 6 days in serum.
According to another aspect, the present invention provides a conjugate of a peptide according to any one of the above aspects and embodiments. All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well.
According to some embodiments, the present invention provides a conjugate of a peptide comprising an amino acid sequence X0TWX1FNX2X3X4X5A (SEQ ID NO: 1) or a retro-inverso sequence thereof, wherein Xo and X5 are each independently a hydrophobic amino acid, Xi, X2, X3 and X4 are each independently any amino acid and wherein the peptide consists of from 11 to 50 amino acids.
According to some embodiment, the present invention provides a conjugate of a peptide comprising or consisting of an amino acid sequence selected from ITWTFNGKSLA (SEQ ID NO: 2), ITwTfNGkSLa (SEQ ID NO: 7), SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8, alskgnftwti (SEQ ID NO: 9), and SEQ ID NO: 20-163.
The term “conjugate” as used herein refers to the association of a peptide with another moiety. According to some embodiments, the moiety is a non-peptidic moiety. According to some embodiment, the peptide is conjugated with polyethylene glycol (PEG), Poly(N-vinylpyrrolidone), polyglycerol, a permeability enhancing moiety and polysaccharides such as glycosyl. In other embodiments, said moiety is an immunoglobulin Fc domain or a portion thereof, an albumin polypeptide (e.g. BSA) or keyhole limpet hemocyanin (KLH). According to another embodiment, the peptide is conjugated with a marker (e.g. a fluorescent or radioactive marker). According to some embodiments, the marker is a fluorescent marker such as fluorescein.
The term “permeability-enhancing moiety” refers to any moiety known in the art to facilitate actively or passively or enhance permeability of the compound through body barriers or into the cells. Non-limitative examples of permeability-enhancing moieties include: hydrophobic moieties such as fatty acids, steroids and bulky aromatic or aliphatic compounds; moieties which may have cell-membrane receptors or carriers, such as steroids, vitamins and sugars, natural and non-natural amino acids and transporter peptides, nanoparticles and liposomes.
In another embodiment the peptide of the invention is conjugated to an exogeneous moiety, e.g. a carrier or a half-life elongating substance. For example, without limitation, peptides of the invention may be fused to or conjugated with immunoglobulin Fc groups or biologically suitable polymers or copolymers (such as PEG or polypropylene glycol) that may prolong the serum halflife of the peptide, or in-frame fusion partners such as epitope tags, structural- stabilizing carriers or targeting moieties. Yet in other embodiments, peptides of the invention may be used in purified peptide form and provide advantageous serum half-life without fusion or conjugation to stabilizing moieties.
According to some embodiments, the conjugate comprises the peptide of the present invention and PEG molecule. According to some embodiments, the PEGylation is effected through C-terminus of the peptide. According to other embodiments, the PEGylation is effected through N-terminus of the peptide. According to further embodiments, the PEGylation is effected through a side chain of an amino acid of a peptide.
According to some embodiments, the PEG molecule has molecular weight (Mw) between about 300 Dalton to about 100,000 Dalton. According to other embodiments, the PEG molecule has a Mw of from about 400 to about 10,000, about 1000 to about 8,000, about 2000 to about 6,000 and about 3,000 to about 5,000 Dalton. According to other embodiments, PEG molecule have molecular weight selected from about 10,000 Da to about 20,000 Da, from about 20,000 Da to about 30,000 Da, from about 30,000 Da to about 40,000 Da, from about 40,000 Da to about 50,000 Da, from about 50,000 Da to about 60,000 Da, from about 60,000 Da to about 70,000 Da, and from about 70,000 Da to about 80,000 Da. Non-limiting examples of average molecular weights of the PEG moieties are about 350, about 400 Da, about 600 Da, about 1,000 Da, about 2,000 Da, about 6,000 Da, about 8,000 Da, about 10,000 Da, about 20,000 Da, about 30,000 Da, about 40,000 Da, about 50,000 Da, about 60,000 Da, about 70,000 Da, and about 80,000 Da. According to some embodiments, the PEG has from 5 to 200 ethylene glycol monomers. According to other embodiments, PEG has from 6 to 150 ethylene glycol monomers. According to other embodiments, PEG has from 8 to 120, from 10 to 100, from 15 to 80 from 20 to 60 or from 30 to 50 ethylene glycol monomers.
The term peptide as used in the present invention encompasses also the term analog of the peptide. The term “analog” refers to an amino acid sequence in which at least one amino acid of the parent sequence is modified when retaining the functionality of the parent peptide. Examples of such modifications of amino acid sequence are substitutions, rearrangements, deletions, additions and/or chemical modifications in the amino acid sequence of the parent peptide. According to one embodiment, the analog comprises at least one modification selected from a substitution, deletion and addition. According to some embodiments, the modification is a substitution. According to one embodiment, the substitution is a conservative substitution. The term “conservative substitution” as used herein denotes the replacement of an amino acid residue by another, without altering the overall conformation and biological activity of the peptide, including, but not limited to, replacement of an amino acid with one having similar properties (such as, for example, polarity, hydrogen bonding potential, acidic, basic, shape, hydrophobic, aromatic, and the like). Amino acids with similar properties are well known in the art. For example, according to one table known in the art, the following six groups each contain amino acids that are conservative substitutions for one another: (1) Alanine (A), Serine (S), Threonine (T); (2) Aspartic acid (D), Glutamic acid (E); (3) Asparagine (N), Glutamine (Q); (4) Arginine (R), Lysine (K); (5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and (6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
According to some embodiments, the substitution is not limited to natural amino acids and may be effected with non-natural amino acids. The term “non-natural amino acids” refers to amino acids having structures different from those of natural amino acid species. In one embodiment, the non-natural amino acid is a D-amino acid. Another examples of non-natural amino acids include ornithine, 3-substituted tyrosine, azidoalanine, azidohomoalanine, norleucine, norvaline, 4- aminotryptophan, 7-azatryptophan, 6-methyltryptophan, acetyllysine, s-Boc-lysine, 8- methyllysine, 1 -naphthylalanine, 2-naphthylalanine, styrylalanine, diphenylalanine, thiazolylalanine, 2-pyridylalanine, 3 -pyridylalanine, 4-pyridylalanine, anthrylalanine, 2-amino-5- hexynoic acid, furylalanine, benzothienylalanine, thienylalanine, allylglycine, propargylglycine, phosphorylserine, phosphorylthreonine, and 2,3-diaminopropionic acid.
According to some embodiments, the analog comprises from 1 to 10, 2 to 8, or 3 to 6 modifications. According to one embodiment, the analog comprises from 1 to 6 modifications or 1 to 3 modifications. According to another embodiment, the analog comprises 1, 2, 3, 4, 5 or 6 modifications. According to some embodiments, the modification is a substitution, such as a conservative substitution. According to one embodiment, the analog comprises 1 to 6 conservative substitutions. In another embodiment, the modifications comprise a truncation or deletion of 1-3 amino acids (e.g. of a non-conserved amino acid denoted by any one of Xi to X4), wherein each possibility represents a separate embodiment of the invention.
According to some embodiments, the Thr at position 2 of the peptide comprising an amino acid sequence X0TWX1FNX2X3X4X5A may be replaced by any other polar amino acid such as Ser, Asn or Gin amino acid. According to some embodiments, the Trp at position 3 of the peptide comprising an amino acid sequence X0TWX1FNX2X3X4X5A may be replaced by any bulky hydrophobic or aromatic amino acid such as Phe, He, or Tyr. According to some embodiments, the Phe at position 5 of the peptide comprising an amino acid sequence X0TWX1FNX2X3X4X5A may be replaced by any bulky hydrophobic or aromatic amino acid such as Trp, He, or Tyr amino acid. According to some embodiments, the Asn at position 6 of the peptide comprising an amino acid sequence X0TWX1FNX2X3X4X5A may be replaced by any other polar amino acid such as Ser, Thr or Gin amino acid. According to some embodiments, the Ala at position 11 of the peptide comprising an amino acid sequence X0TWX1FNX2X3X4X5A may be replaced by any other small hydrophobic amino acid such Gly and Vai amino acid.
According to some embodiments of the invention, the peptide is a cyclic peptide, i.e. cyclized. The term “cyclic peptide” refers to a peptide having an intramolecular bond between two non-adjacent amino acids. The cyclization can be effected through a covalent or non-covalent bond. Intramolecular bonds include, but are not limited to, backbone to backbone, side-chain to backbone and side-chain to side-chain bonds. According to some embodiments, the cyclization occurs between the side chains of two cysteine residues of the peptide, analogs of fragments, to form a disulfide bridge. According to other embodiments, the cyclization occurs between the N- terminal and C-terminal amino acids. According to some embodiments, the cyclization is effected via a spacer. The cyclization may be performed using any technique known in the art.
As disclosed herein, peptides in accordance with the invention offer a means to inhibit activation of immune cells. Current immune-suppressive agents have a broad spectrum of activity leading to significant undesirable effects. Furthermore, some widely employed agents have very long half-lives and lack antidotes, thus leading to extended immune-suppression and risk of severe infections. Without wishing to be bound by a specific theory or mechanism of action, peptides of the invention may have a relatively short half-life offering a means to titrate inhibition and reverse it as necessary. Importantly, SLAMF6 has a dynamic activation dependent expression pattern. Thus, without being bound by a theory, peptides disclosed herein exert a stronger inhibitory effect on activated populations of cells, relatively sparing less active populations, and providing a more specific inhibition of pathologically-active immune cells.
For example, while many drugs currently used in immunosuppressive therapy (such as immunosuppressive antibodies) have a long serum half-life of up to several weeks that limits the ability to adjust the level of immunosuppression as needed, the present invention provides in some embodiments immunosuppressive peptides having relatively short serum half-life of hours or days, facilitating real time dosage adaptation to the status of the patient. In comparison, conventional peptides may have a very short serum half-life of several minutes, that may make them less attractive or even impractical to be considered for immunosuppressive treatment.
Since SLAMF6 is upregulated on the surface of activated T cells as compared to resting T cells (activation-dependent expression), pathologically-active immune cells (such as those implicated in the progression of autoimmune diseases, chronic inflammatory diseases and irAE) may possess a greater capacity to bind with and be affected by peptides of the invention, as a result of higher SLAMF6 surface density.
Pharmaceutical compositions
A pharmaceutical composition comprising at least one peptide or conjugate according to any one of the above aspects or embodiments, and a pharmaceutically acceptable carrier and/or excipient, is further contemplated. All terms, embodiments and definitions disclosed in any one of the above aspects apply and are encompassed herein as well.
The terms “pharmaceutical composition” and “pharmaceutically acceptable composition” are used herein interchangeably and refer to a composition comprising the peptide of the present invention or a conjugate thereof, formulated together with one or more pharmaceutically acceptable carriers.
According to another aspect, the present invention provides a pharmaceutical composition comprising an isolated peptide comprising the amino acid sequence X0TWX1FNX2X3X4X5A or a retro-inverso sequence thereof, and a pharmaceutically acceptable carrier and/or excipient, wherein Xo and X5 are each independently a hydrophobic amino acid, Xi, X2, X3 and X4 are each independently any amino acid and wherein the peptide consists of from 11 to 50 amino acids. According to some embodiments, the pharmaceutical composition comprises a conjugate of the peptide.
According to some embodiments, the present invention provides a pharmaceutical composition comprising an isolated peptide comprising or consisting of an amino acid sequence selected from ITWTFNGKSLA (SEQ ID NO: 2), ITwTfNGkSLa (SEQ ID NO: 7), SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 8 alskgnftwti (SEQ ID NO: 9), SEQ ID NO: 20-163, and SEQ ID NO: 20-163 comprising D-amino acids at positions 3, 5, 8 and 11 , and a pharmaceutically acceptable carrier and/or excipient. Each possibility represents a separate embodiment of the invention. According to some embodiments, the peptides as cyclic peptides. According to some embodiments, the pharmaceutical composition comprises a conjugate of the peptide.
The terms “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” as used herein refer to any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, glidants, pH adjusting agents, buffering agents, enhancers, wetting agents, solubilizing agents, surfactants, antioxidants the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may contain other active agents providing supplemental, additional, or enhanced therapeutic functions.
According to any one of the above embodiments, the pharmaceutical composition of the present invention may be administered by any known route of administration. The term “administering” or “administration of’ a substance, a compound (e.g. peptide), an agent (e.g. conjugate) or a pharmaceutical composition to a subject can be carried out using one of a variety of methods known to those skilled in the art. For example, a compound, an agent or a composition can be administered enterally or parenterally. Enterally refers to administration via the gastrointestinal tract including per os, sublingually or rectally. Parenteral administration includes administration intravenously, intradermally, intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, intranasally, by inhalation, intraspinally, intracerebrally, and transdermally. A compound or agent can also appropriately be introduced by rechargeable or biodegradable polymeric devices or other devices, e.g., patches and pumps, or formulations, which provide for the extended, slow or controlled release of the compound or agent. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
In one embodiment, the pharmaceutical composition comprising the peptide or the conjugate of the present invention is administered via a systemic administration. For example, the pharmaceutical composition comprising the peptide or the conjugate of the present invention is administered orally, intravenously or transdermally. Alternatively, the composition is administered intramuscularly, intraperitoneally, subcutaneously, ocularly, sublingually, intranasally or by inhalation. The pharmaceutical composition according to the present invention may be prepared in any known method. In particular, the pharmaceutical composition may be formulated using a method known in the art so as to provide rapid, continuous or delayed release of the active ingredient after administration. In one particular embodiment, the pharmaceutical composition is formulated as a solid dosage form selected from tablets, capsules, powder or granules. In another embodiment, the pharmaceutical composition is formulated as a liquid or semi-liquid dosage form selected from an elixir, tincture, suspension, syrup, emulsion or gel. According to some embodiments, the half-life of the peptides in the pharmaceutical composition is as described above.
Pharmaceutical compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and may further comprise one or more agents selected from sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Tablets contain the active agent in admixture with non-toxic pharmaceutically acceptable excipients, which are suitable for the manufacture of tablets. These excipients may be, e.g., inert diluents such as calcium carbonate, sodium carbonate, lactose, calcium phosphate, or sodium phosphate; granulating and disintegrating agents, e.g., corn starch or alginic acid; binders; and lubricating agents. The tablets are optionally coated utilizing known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide an extended release of the drug over a longer period.
Therapeutic use
The peptide of the present invention has an immunomodulatory activity and according to some embodiments may be used as an immunosuppressant. According to some embodiments, the peptide of the present invention or a conjugate thereof are for use as a medicament. According to any one of the above aspects and embodiments, the peptide, the conjugate or the pharmaceutical composition comprising the peptide and/or a conjugate thereof is for use in suppressing an immune system. According to any one of the above aspects and embodiments, the peptide, the conjugate or the pharmaceutical composition comprising the peptide and/or the conjugate is for use in inhibiting a SLAMF6-mediated (or SLAMF6-modulated) immune cell activation, e.g. in T cells, B cells and/or NK cells.
According to some embodiments, the peptide of the present invention may be used as a short-term immunosuppressant. According to other embodiments, the peptide of the present invention may be used as a medium-term immunosuppressant. In some embodiments, short-term immunosuppressants are provided, having serum half-life of at least about 1 hour and typically several hours, e.g. 1-2, 1-4, 2-5, 3-7, or in other embodiments 8-14 or 10-16 hours. In other embodiments, medium-term immunosuppressants are provided, having serum half-life of at least about 24 hours and typically several days, e.g. 1-2, 1-4, 2-5 or 3-6 days. For example, without limitation, medium-term immunosuppressants are conveniently used in managing the symptoms of autoimmune disorders or other conditions in which a non-infective etiology had been confirmed. In some cases (for example for treating chronic conditions such as IBD), long-term immune suppression may be induced (for example by using conjugates with half-life elongating substances such as Fc moieties). In other embodiments, short-term immunosuppressants may be used in subjects in which the presence of an infective agent is suspected, or wherein the subject is at risk of developing an infection. In these cases, rapid discontinuation of treatment upon signs of infection is readily facilitated.
As used herein, the term “immunosuppression” refers to the suppression of the immune system and its ability to fight infections and other diseases. Immunosuppression may be deliberately induced with drugs (for example prior to transplantation to prevent graft rejection), or it can result from certain diseases, environmental factors, or as a side effect to other drugs such as anti-cancer drugs. Immunosuppression may involve partial or complete depletion of one or more leukocyte populations, and/or reduction in their reactivity, expansion and/or differentiation. Immunosuppressive drugs, also referred to herein as "immunosuppressants", refers to drugs used to induce immunosuppression in a subject. Non-limiting examples of conditions in which the use of immunosuppressants is indicated (referred to herein as conditions requiring immunosuppression) include graft rejection, GVHD and autoimmune diseases. According to some embodiments, the peptide of the present invention induces immunosuppression. According to some embodiments, the peptide of the present invention, the conjugate, or the pharmaceutical composition comprising the peptide or conjugate is for use in inhibiting immune cells' activation.
According to some embodiments, the peptide of the present invention, the conjugate, or the pharmaceutical composition comprising the peptide or conjugate is for use in treating a disease or a condition involving activated or overactivated immune system. According to some embodiments, the peptide of the present invention, the conjugate, or the pharmaceutical composition comprising the peptide or conjugate is for use in treating a disease or a condition requiring immunosuppression. According to some embodiments, the disease or a condition comprises, immune overactivation, graft versus host disease, an autoimmune disease, immune related adverse effects of checkpoint inhibitors, cytokine storm, and rejection of stem cells transplant and/or organ transplant. According to some embodiments, the use comprises prevention of rejection of transplanted cells, tissues or organs. According to some embodiments, the use comprises preventing or inhibiting the side effects of checkpoint inhibitors. As used herein, the term “Graft Versus Host Disease” or “GVHD” refers to the pathological reaction that occurs between grafted immune cells and their host-recipient. In particular, GVHD involves donor-derived alloreactive T lymphocytes recognizing the recipient's tissues as foreign, mounting an inflammatory and destructive response against the recipient. GVHD often occurs following hematopoietic cell transplantation, and rarely after solid organ transplantation, and has a predilection for epithelial tissues, especially skin, liver, and mucosa of the gastrointestinal tract. Transplant patients with GVHD are often treated with powerful immunosuppressant agents, thereby making them more susceptible to opportunistic infections. In various embodiments, GVHD may be chronic or acute.
The term “graft rejection” as used herein, refers to rejection of tissue transplanted from a donor individual to a recipient individual. In transplant graft rejection (e.g. allograft rejection), the transplanted tissue is rejected and destroyed by the recipient's immune system. Typically, allograft rejection occurs when the donor tissue carries an alloantigen recognized as foreign by the recipient's immune system, thereby inducing an inflammatory and destructive immune response directed to the alloantigen.
As used herein, the term “autoimmune disease” is defined as a disease or disorder in which the subject mounts a destructive immune response against its own constituents (e.g. cells or tissues). An autoimmune disease is therefore the result of an inappropriate and excessive response to a self-antigen. Autoimmune disorders can affect almost every organ system in the subject (e.g., human), including, but not limited to, diseases of the nervous, gastrointestinal, and endocrine systems, as well as skin and other connective tissues, eyes, blood and blood vessels. Examples of autoimmune diseases include, but are not limited to Hashimoto's thyroiditis, Systemic lupus erythematosus, Sjogren's syndrome, Graves' disease, Scleroderma, Rheumatoid arthritis, Multiple sclerosis, Myasthenia gravis, Crohn’s disease, ulcerative colitis, primary biliary cholangitis, primary biliary sclerosis, psoriasis and Diabetes.
The term “cytokine storm” (or cytokine release syndrome) as used herein is a form of systemic inflammatory response syndrome (SIRS) that can be triggered by a variety of factors such as infections and certain drugs. It occurs when the immune system causes an uncontrolled and excessive release of pro-inflammatory cytokines. This sudden release in such large quantities can cause multisystem organ failure and death.
According to some embodiments, the use is in treating an autoimmune disease e.g., selected from rheumatoid arthritis, lupus, inflammatory bowel disease, including Crohn’s disease and ulcerative colitis, multiple sclerosis, psoriasis or psoriatic arthritis. In another embodiment said disease is autoimmune hepatitis or primary biliary cholangitis (PBC). According to some embodiments, the inflammatory bowel disease comprises Crohn’s disease and ulcerative colitis. According to some embodiments, the use comprises treating rheumatoid arthritis. In yet other embodiments, the autoimmune disease is selected from RA, SLE, IBD, MS, psoriasis, psoriatic arthritis, autoimmune hepatitis, PBC, type I diabetes (T1DM) and autoimmune thyroiditis. In a particular embodiment the autoimmune disease is IBD (e.g. ulcerative colitis or Crohn’s disease). In another particular embodiment autoimmune disease is T1DM. In another particular embodiment autoimmune disease is autoimmune thyroiditis.
Crohn’s disease (CD) is characterized by ulcerations of the small and/or large intestines, but can affect the digestive system anywhere from the mouth to the anus. Various terms are used to describe CD, and tend to reflect the portion of the gastrointestinal tract affected. For example, involvement of the large intestine (colon) only has been termed Crohn’s colitis or granulomatous colitis, while involvement of the small intestine only has been termed Crohn’s enteritis. Disease in the terminal portion of the small intestine i.e. the ileum, has been termed Crohn’s ileitis. When both the small intestine and the large intestine are involved, the condition has been termed Crohn’ s enterocolitis or ileocolitis. Ulcerative colitis (UC) is a condition related to CD that involves only the colon, and collectively these diseases are frequently referred to as inflammatory bowel disease (IBD).
Multiple sclerosis (MS) is a neuroinflammatory disease of the central nervous system (CNS) characterized by chronic inflammation, demyelination and gliosis. Pathologically, MS is characterized by well-demarcated, macroscopic lesions, called plaques, in the brain white matter and, less frequently, gray matter. Acute lesions are characterized by perivenular cuffing and infiltration of T lymphocytes and macrophages, along with a few B cells and plasma cells. MS is reportedly an autoimmune disorder, likely triggered by environmental exposure in a genetically susceptible host.
SLE is a chronic, recurrent, potentially fatal multisystem inflammatory disorder mainly affecting women. SLE is associated with a large spectrum of autoantibodies, and is diagnosed on the basis of eleven criteria defined by the American College of Rheumatology (ACR).
RA is a chronic inflammatory autoimmune disease characterized by joint inflammation, joint swelling, joint tenderness, and destruction of synovial joints, leading to severe disability and premature mortality.
Diabetes mellitus is a common metabolic disorder associated with abnormally high levels of glucose in the blood. There are two major types of diabetes mellitus, termed type I and type II. Type I Diabetes (T1DM, or Insulin Dependent Diabetes Mellitus) is caused by a deficiency of insulin due to an autoimmune response which leads to the destruction of the beta cells (P cells) in the Islets of Langerhans of the pancreas. An initial phase of T1DM includes an inflammation of the pancreatic islets, known as insulitis, characterized by leukocyte and macrophage infiltration into the islets followed by the actual destruction of pancreatic P cells in an autoimmune attack.
Autoimmune thyroiditis (Hashimoto's disease) is a condition in which the thyroid gland diffusely swells and frequently occurs in young to middle-aged women. Although thyroid function is normal in the early stage of autoimmune thyroiditis, progress of thyroid tissue destruction by an autoimmune mechanism leads to hypothyroidism requiring thyroid hormone replacement therapy. Hypothyroidism is a serious disease in which the functions of various organs in the body including the central nervous system are impaired, causing a major obstacle in social life.
According to some embodiments, the use is in treating chronic inflammation or chronic inflammatory disease. The term "treating” as used herein refers to taking steps to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation or amelioration of one or more symptoms or parameters associated with the disorder, delaying or slowing of that impairment, amelioration, palliation or stabilization of that impairment, and other beneficial results.
A chronic inflammatory disease is characterized by a persistent inflammatory response with pathologic sequelae. This state is typically accompanied by infiltration of mononuclear cells, proliferation of fibroblasts and small blood vessels, increased connective tissue, and tissue destruction. Non-limitative examples include idiopathic or non-infective chronic conditions, e.g. pericarditis or periodontitis. In another example, conditions to be treated may include chronic inflammatory diseases of the liver, for example non-alcoholic fatty liver disease (NAFLD) and liver cirrhosis resulting from chronic liver disease.
According to some embodiments, the peptide of the present invention, the conjugate, or the pharmaceutical composition comprising the peptide or conjugate is for use in treating or preventing a T cell-mediated immune disorder (in which the activity of T cells is known to be implicated in the etiology and/or pathology). In another embodiment the T-cell-mediated immune disorder is an inflammatory disease, or an autoimmune disorder. In another embodiment the T- cell- mediated immune disorder is chosen from infections (viral, bacterial, fungal and parasitic), endotoxic shock associated with infection, sepsis, arthritis, rheumatoid arthritis, asthma, chronic obstructive pulmonary disease (COPD), inflammatory bowel disease, Crohn's disease, ulcerative colitis, Peyronie's disease, coeliac disease, psoriasis, vasculitis, surgical adhesions, stroke, type I diabetes, meningoencephalitis, autoimmune uveitis, immune mediated inflammatory disorders of the central and peripheral nervous system including multiple sclerosis, lupus (systemic lupus erythematosus) and Guillain-Barre syndrome, atopic dermatitis, autoimmune hepatitis, fibrosing alveolitis, Grave's disease, immunoglobulin (Ig)A nephropathy, idiopathic thrombocytopenic purpura, Meniere's disease, pemphigus, primary biliary cirrhosis, sarcoidosis, scleroderma, Wegener's granulomatosis, other autoimmune disorders, pancreatitis, graft-versus-host disease, transplant rejection, heart disease including ischemic diseases e.g. myocardial infarction as well as atherosclerosis, intravascular coagulation, bone resorption, osteoporosis, osteoarthritis, periodontitis and hypochlorhydria.
According to some embodiments, the peptide of the present invention, the conjugate, or the pharmaceutical composition comprising the peptide or conjugate is for use in treating or preventing a B cell-mediated immune disorder. According to some embodiments, the peptide of the present invention, the conjugate, or the pharmaceutical composition comprising the peptide or conjugate is for use in treating or preventing an NK cell-mediated immune disorder. Such disorders encompass conditions (e.g. inflammatory or autoimmune) in which the activity of the recited cell type (e.g. B cell or NK cell) is known to be implicated in the etiology and/or pathology. In yet other embodiments, the peptide of the present invention, the conjugate, or the pharmaceutical composition comprising the peptide or conjugate is for use in treating or preventing conditions involving myeloid-derived suppressor cells (MDSC) dysfunction, e.g. contributing to autoimmunity (MDSC-mediated immune disorders). For example, MDSC impairment has been implicated in the development of autoimmune diseases including MS, RA, type I diabetes, SLE and autoimmune uveoretinitis. In various embodiments, the MDSC are selected from the group consisting of monocytic MDSC (M-MDSC), polymorphonuclear MDSC (PMN-MDSC) and combinations thereof.
According to another embodiment, the peptide, conjugate or the pharmaceutical composition comprising the peptide or conjugate is co-administered in a combination with an additional immunosuppressant. According to another embodiment, the peptide, conjugate or the pharmaceutical composition comprising the peptide or conjugate is co-administered in a combination with an immuno stimulatory treatment (e.g. a cancer immunotherapy such as an immune checkpoint inhibitor). The term “coadministration” encompasses administration of a first and second agent in an essentially simultaneous manner, such as in a single dosage form, e.g., a capsule or tablet having a fixed ratio of first and second amounts, or in multiple dosage forms for each. The agents can be administered sequentially in either order. When coadministration involves the separate administration of each agent, the agents are administered sufficiently close in time to have the desired effect (e.g., complex formation). In other embodiments, co-administration comprises administration by separate administration routes.
According to another aspect, the present invention provides a method for suppressing the immune system in a subject in need thereof comprising administering the peptide according to any one of the above embodiments and aspects or the conjugate or the pharmaceutical composition comprising the peptide or the conjugate. In another aspect, the pharmaceutical composition is for use in inhibiting or reducing an inappropriate or excessive immune response in a subject in need thereof.
As used herein, the phrase "inappropriate or excessive immune response" relates to an undesirable response of a subject's immune system to one or more antigens, that results in the development or progression of a pathology in the subject. The response may proceed to an inappropriate degree (disproportionate response), typically an inappropriately high degree (excessive immune response) and/or may be inappropriate (undesirable) in terms of its type (e.g. Thl vs. Th2), the nature of immune cells involved (e.g. effector vs. regulatory T cells), the timing of the response (e.g. a chronic condition developing following an initial infection) and/or the nature of antigens to which the response is directed (mistargeted response, for example, a response to self-antigens may lead to the development of an autoimmune disease, and type-I hypersensitivity responses to innocuous allergens may lead to the development of allergic conditions such as asthma and atopic dermatitis).
As disclosed herein, inappropriate or excessive immune responses may be involved in the etiology and/or pathology of various autoimmune and inflammatory diseases and conditions as disclosed herein, collectively referred to as "disease or conditions associated with an inappropriate or excessive immune response". Accordingly, a method of inhibiting or reducing an inappropriate or excessive immune response may also be regarded as a method of preventing the development or progression of these autoimmune and inflammatory diseases and conditions, including e.g. irAEs and T-cell mediated conditions as disclosed herein.
In another aspect, there is provided a method for inhibiting or reducing an inappropriate or excessive immune response in a subject in need thereof, comprising administering to the subject the pharmaceutical composition as disclosed herein.
In another embodiment the pharmaceutical composition comprises an isolated peptide comprising the amino acid sequence X0TWX1FNX2X3X4X5A (SEQ ID NO: 1) or a retro-inverso sequence thereof, wherein Xo and X5 are each independently a hydrophobic amino acid, Xi, X2, X3 and X4 are each independently any amino acid and wherein the peptide consists of from 11 to 50 amino acids. In some embodiments, the peptide comprises or consists of an amino acid sequence selected from ITWTFNGKSLA (SEQ ID NO: 2) and ITwTfNGkSLa (SEQ ID NO: 7). In another embodiment the pharmaceutical composition comprises a conjugate of the peptide.
In another embodiment, the method is used for inhibiting or reducing immune-related adverse events (irAEs). In another embodiment, the irAEs are selected from the group consisting of gastrointestinal, endocrine, dermatologic toxicities, and combinations thereof. The term "adverse event" or "AE" as used herein refers to any noxious, unintended, or untoward medical occurrence that may appear or worsen in a patient following or during administration of a treatment. It may be a new intercurrent illness, a worsening concomitant illness, an injury, or any concomitant impairment of the participant's health, including laboratory test values, regardless of etiology. In particular, AE associated with cancer treatment are defined and classified by the Common Terminology Criteria for Adverse Events (CTCAE) published by the NIH as encompassing any abnormal clinical finding temporally associated with the use of a therapy for cancer. As used herein, the term “immune-related adverse event” or “irAE” refers to an AE that has a putative immune-related etiology, and include in particular AE associated with immuno stimulatory cancer immunotherapy. In some embodiments, the irAE is associated with administration of immune checkpoint inhibitors. irAE typically include toxicities that are autoimmune or autoinflammatory in nature that may affect various organs and systems of the body, and may be of various grades or levels of severity. In some embodiments, the irAE affects the endocrine system (“endocrine irAE” or "endocrine toxicities"), the skin (“dermatological irAE” or “dermatologic toxicities”), or the gastrointestinal tract (“GI irAE” or "gastrointestinal toxicities"). Endocrine toxicities include, but are not limited to, immune-related hypothyroidism, immune-related hyperthyroidism, immune- related adrenal insufficiency, immune-related diabetes mellitus, and immune-related hypophysitis. Dermatological toxicities include, but are not limited to, immune-related rash and immune-related severe cutaneous reaction, gastrointestinal toxicities include, but are not limited to, immune- related hepatitis, immune-related colitis, and immune-related pancreatitis. In other embodiments, toxicities may be cardiac (e.g. myocarditis), pulmonary (pneumonitis), neural (encephalitis, meningitis, myasthenia gravis like, Guillain Barre), or musculoskeletal (arthritis, myositis).
In another embodiment the irAE includes iatrogenic multiple autoimmune conditions. In a particular embodiment, the irAE comprises toxicities affecting the pancreas and/or thyroid gland, e.g. conditions involving diabetes or thyroiditis as disclosed above. In yet other embodiments, the irAE is systemic, e.g. may include cytokine release syndrome (CRS) or cytokine storm (CS). In yet other embodiments, irAE involve immune effector cell-associated neurotoxicity syndrome (ICANS), associated with cell therapies CAR-T and TCR-T adoptive cell therapies.
In some embodiments, the irAE is a low grade irAE, e.g., a Grade 1 AE (Grade 1 irAE, representing a condition that does not require additional therapeutic intervention due to irAE) or Grade 2 AE (Grade 2 irAE, representing a condition that requires drug intervention, etc., due to irAE, but does not require hospitalization treatment or does not require interruption of treatment). In another embodiment, the irAE is a high grade irAE, e.g. a Grade 3 AE (Grade 3 irAE, representing a condition that requires drug intervention, etc., accompanied by hospitalization due to irAE and requires interruption of treatment). In another embodiment, the irAE is a Grade 4 AE, posing a life-threatening event. In other embodiments, irAE are as detailed for each specific AE in the CTCAE v5.0.
In another embodiment said irAEs are associated with administration of an immuno stimulatory treatment (that activates or enhances the activity of the immune system or components thereof, in an antigen-dependent and/or independent manner). In another embodiment said immuno stimulatory treatment is a cancer immunotherapy. In another embodiment said cancer immunotherapy comprises administration of an immune checkpoint inhibitor. In another embodiment said immuno stimulatory treatment is a vaccine. In another embodiment, said cancer immunotherapy comprises administration of a cell-based therapy either autologous or allogenic, native or engineered (e.g. CAR-T, TCR-T, CAR-NK etc).
As used herein, the term "cancer immunotherapy" refers to a cancer treatment that modulates the patient's immune system to induce or enhance an immune response against a tumor (or cancer cells) of the patient. Non-limiting examples of cancer immunotherapy include immune checkpoint modulators (e.g. inhibitors), adoptive cell therapy, cytokines and their recombinant derivatives, adjuvants, and vaccination with small molecules or cells. In another embodiment, the irAE includes iatrogenic multiple autoimmune conditions associated with administration of an immune checkpoint inhibitor.
Immune checkpoint inhibitors or ICIs denote a subclass of cancer immunotherapy in which the cellular targets modulated are immune checkpoint molecules. Immune checkpoint molecules are a group of cell-surface molecules expressed on immune cells including in particular T cells (or other immune cells that regulate immune pathways such as NK cells, dendritic cells, macrophages, and MDSC). Immune checkpoint molecules are known to effectively serve as "brakes" to down- modulate or inhibit an anti-tumor immune response, and include, for example, PD-1, PD-L1, PD- L2, CTLA4, LAG3, TIM3, IDO, CD80, CD86, B7-H3, -H4, -H5, BTLA, TIGIT, CD94/NKG2A, and KIR2DL-1, -2, -3 (see, for example, WO 2012/177624).
Typical ICIs act by reducing the activity of a target immune checkpoint protein involved in suppressing immune cells to thereby activate immune cells to attack cancer cells. Examples of therapeutic agents that can be used as ICIs are antibodies, small molecules, peptides, peptidomimetics, natural ligands, and derivatives of natural ligands, that can either bind and/or inactivate or inhibit immune checkpoint proteins, or fragments thereof; as well as RNA interference, antisense, nucleic acid aptamers, etc. that can downregulate the expression and/or activity of immune checkpoint nucleic acids, or fragments thereof. Commercially available ICIs include for example anti-CTLA-4 monoclonal antibodies such as ipilimumab and tremelimumab, anti-PD-1 monoclonal antibodies such as nivolumab and pembrolizumab, anti-PD-Ll monoclonal antibodies such as atezolizumab, durvalumab, and avelumab, and anti-LAG3 monoclonal antibodies such as relatlimab. In another embodiment the immune checkpoint inhibitor targets (specifically downregulates the expression and/or activity) PD-1, PD-L1, PD-L2, CTLA-4, LAG3, TIM3, and/or IDO.
Programmed cell death 1 receptor (PD-1) is an immune checkpoint receptor expressed on the surface of activated T, natural killer (NK) and B lymphocytes, macrophages, dendritic cells (DCs) and monocytes. It is categorized as a type I transmembrane protein, structurally belonging to the CD28/CTLA-4 subfamily of the Ig superfamily, and is encoded by the PDCD1 gene. PD-1 and its ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC) belong to the immune checkpoint pathway, which induces immune suppression. The binding of PD-1 to its ligand (e.g. PD-L1) downregulates immune reactivity and promotes self-tolerance by suppressing the activity of effector T cells. PD- L1 is also expressed by many tumor cells, thereby facilitating their evasion from immune surveillance and down-regulating anti-tumor immunity. For example, commercially available PD- Ll-targetind ICIs include e.g. atezolizumab, avelumab, durvalumab, dostarlimab and retifanlimab; commercially available PD-l-targetind ICIs include e.g. nivolumab (Opdivo), pembrolizumab (Keytruda), cemiplimab (Libtayo), camrelizumab, sintilimab, tislelizumab and balstilimab.
Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4), also known as CD152, is constitutively expressed in regulatory T cells but only upregulated in conventional T cells after activation - a phenomenon which is particularly notable in cancers. It induces an inhibitory signal upon binding to CD80 or CD86 on the surface of antigen-presenting cells. It is encoded by the gene CTLA4 in humans. For example, commercially available CTLA-4 -targetind ICIs include e.g. ipilimumab, tremelimumab and botensilimab. LAG3 (lymphocyte activation gene 3; or CD223) belongs to Ig superfamily and contains four extracellular Ig-like domains, which are followed by a transmembrane helical domain and a cytoplasmic domain. The precursor polypeptide also contains an N' signal peptide. For example, the precursor sequence of human LAG3 may be found in accession no. P18627. Commercially available LAG3 inhibitors include e.g. IMP321, BMS-986016 (relatlimab), LAG525, REGN3767 (finalimab), and TSR-033.
TIM-3 (T-cell immunoglobulin and mucin domain-containing 3), also known as Hepatitis A virus cellular receptor 2 (HAVCR2), s a protein that in humans is encoded by the HAVCR2 (TIM-3) gene. IDO (Indoleamine 2,3-dioxygenase), is an intracellular enzyme that produces T- cell-inhibiting metabolites. It catalyzes the rate-limiting step in the catabolism of local tryptophan and therefore contributes to anergy of effector T cells and promotion of regulatory T cells (Tregs). Various immune cells and stromal cells but also cancer cells express IDO.
In another embodiment, the method comprises treating a disease or a condition associated with the inappropriate or excessive immune response. In another embodiment the condition is a T-cell mediated inflammatory or autoimmune condition. As referred to herein, a T-cell mediated inflammatory or autoimmune condition encompasses inflammatory and autoimmune diseases in which the activity of T cells is known to be implicated in the etiology and/or pathology of the disease. In another embodiment, the disease or condition is selected from the group consisting of graft versus host disease, an autoimmune disease, and graft rejection. In another embodiment, the autoimmune disease is selected from rheumatoid arthritis, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), multiple sclerosis (MS), psoriasis, psoriatic arthritis, autoimmune hepatitis and primary biliary cholangitis (PBC). In a particular embodiment, said autoimmune disease is IBD.
In another embodiment, the pharmaceutical composition is administered systemically. In another embodiment said pharmaceutical composition is administered locally to the site of manifestation of symptoms associated with the condition (e.g. irAEs or T-cell mediated inflammatory or autoimmune condition). For example, without limitation, the composition may be administered orally in case of gastrointestinal (GI) symptoms, for example in the case of GI inflammatory or autoimmune diseases such as IBD or GI toxicities associated with irAEs. In another example, the composition may be administered by inhalation in case of pulmonary symptoms or toxicities. In other embodiments, said composition may be administered locally in a prophylactic manner in a subject predisposed to exhibiting symptoms or toxicities in the organ or tissue in question, prior to or concurrently with administration of an immuno stimulatory treatment (e.g. an immune checkpoint inhibitor). Each possibility represents a separate embodiment of the invention.
In another embodiment, administration is performed in vivo. In another embodiment administration is performed ex vivo. In another embodiment of the methods of the invention, administration is effected by expressing in cells of the subject at least one isolated peptide of the invention. For example, the methods of the invention may be performed by contacting cells of the subject (e.g. in vivo or ex vivo) with a nucleic acid molecule encoding a peptide of the invention, wherein the nucleic acid molecule is operably linked to a transcription regulating sequence, such that the peptide of the invention is expressed in the cells and administered to said subject. Table 1. Sequences
Figure imgf000035_0001
The terms “a,” “an,” and “the” are used herein interchangeably and mean one or more.
The term “and/or” is used to indicate one or both stated cases may occur, for example A and/or B includes, (A and B) and (A or B).
The term “or,” as used herein, denotes alternatives that may, where appropriate, be combined; that is, the term “or” includes each listed alternative separately as well as their combination if the combination is not mutually exclusive. The terms “comprising”, "comprise(s)", "include(s)", "having", "has" and "contain(s)," are used herein interchangeably and have the meaning of “consisting at least in part of’. When interpreting each statement in this specification that includes the term “comprising”, features other than that or those prefaced by the term may also be present. Related terms such as “comprise” and “comprises” are to be interpreted in the same manner. The terms “have”, “has”, having” and “comprising” may also encompass the meaning of “consisting of’ and “consisting essentially of’, and may be substituted by these terms. The term “consisting of’ excludes any component, step or procedure not specifically delineated or listed. The term “consisting essentially of’ means that the composition or component may include additional ingredients, but only if the additional ingredients do not materially alter the basic and novel characteristics of the claimed compositions or methods.
As used herein, the term “about”, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of +/-10%, or +/-5%, +/-1%, or even +/-0.1% from the specified value.
Having now generally described the invention, the same will be more readily understood through reference to the following examples, which are provided by way of illustration and are not intended to be limiting of the present invention.
EXAMPLES
In the experiments described herein, an array of partly overlapping SLAMF6-derived peptides were identified and evaluated using in-silico modeling of the SLAMF6 binding domain, and tested for binding to the recombinant extracellular SLAMF6 domain. Based on the initial screening results, additional candidate peptides were constructed, and functionally evaluated using several experimental models including: (1) Peripheral derived blood mononuclear cells (PBMCs) activated with the staphylococcal endotoxin b (SEB), with interleukin-2 (IL-2) as the readout. (2) Tumor infiltrating lymphocytes (TILs) co-cultured with cognate melanoma cells, with interferon gamma (IFN-y) as the readout. (3) Jurkat cells activated with anti-CD3 antibodies, with granzyme B as the readout; SLAMF6-knockout Jurkat cells were used as a control.
Murine model: Splenocytes were harvested from PMEL mice in which the TCRs recognize the melanoma B16-gp-100 antigen, then co-cultures with B16 melanoma cells, with IFNy as the readout; SLAMF6 knockout PMEL splenocytes were used as a control.
These extensive in silico and experimental evaluations resulted in the construction of unique synthetic peptides having exceptionally advantageous therapeutic properties, as detailed in the Examples below. Example 1. Identification of a SLAMF6-derived fragment having immunomodulatory properties
Initially a 42-amino acid peptide fragment was identified at positions 49-91 of human SLAMF6, having the amino acid sequence
VNFITWLFNETSLAFIVPHETKSPEIHVTNPKQGKRLNFTQS (SEQ ID NO: 3) and denoted as V49. The sequence of the SLAMF6 receptor presented in Fig. 1A, in which the location of the V49 peptide is in bold. The location of V49 in the SLAMF6 protein structure is illustrated in Fig. IB in dark grey. To test the binding properties of this fragment, SLAMF6 was expressed in M526 melanoma cells that do not natively express this receptor. The V49 peptide was fluorescently labeled by N' conjugation to FITC-Ahx (FITC via an Ahx linker), and incubated with either WT or SLAMF6-expressing M526 cells for two hours. Two additional peptides were tested: V49(ala), having the amino acid sequence
VNAITWAFNETSLAFIVPHETKSPEIHVTNPKQGKRLNFTQS (SEQ ID NO: 4, a sequence of V49 in which Phe and Leu were replaced by Ala), and a scrambled peptide based on the V49 sequence (V49(scr), SEQ ID NO: 13). These peptides were similarly N' conjugated with FITC via an Ahx linker, and used as controls in the binding assay. Mean fluorescence intensity (MFI) was measured after repeated washing. As can be seen in Fig. 1C, MFI was significantly higher in SLAMF6-expressing cells incubated with the V49 peptide, compared to in WT cells incubated with this peptide, indicating SLAMF6-mediated binding. As can be further seen in Fig. 1C, substitution of the amino acids at positions 3 and 6 resulted in marked reduction in the binding capacity of the peptide, whereas the scrambled control peptide no longer exhibited any SLAMF6 specificity.
Further, the effect of the various peptides on PBMC activation was evaluated. To this end, PBMC were activated with SEB in the presence or absence of the peptides (at the concentrations indicated in Fig. ID), and IL-2 secretion was measured by ELISA, as follows.
PBMC were purified from healthy donors' buffy coats and stored at -80°. Cells were thawed and incubated for 3-4 hours at a concentration of 2X106 cells/ml, in 6 well plates in 4ml of complete medium [CM, consisting of RPMI 1640 supplemented with 10% heat-inactivated human AB serum, 2 mmol/1 L-glutamine, 1 mmol/1 sodium pyruvate, 1% nonessential amino acids, 25 mmol/1 HEPES (pH 7.4), 50 pmol/1 2-ME, and combined antibiotics (all from ThermoFisher)] with recombinant human interleukin-2 (Chiron, CA) at a concentration of 300 IU. PBMCs were then collected and centrifuged at 1200 rpm for 5 minutes. The cells were resuspended in 5 ml of CM. Cells were counted and seeded in flat 96 well plates, 1.5xl05 cells/well and incubated for 3 hours before peptides addition. Peptides (as indicated) were added to the medium and cells were further incubated for 2 hours at 37° before stimulation with staphylococcal enterotoxin B (SEB) 200ng/ml and further incubation for 3 days. On day 4, supernatant was collected and levels of IL2 determined by ELISA.
The results are presented in Fig. ID. As can be seen, V49 significantly reduced IL-2 secretion form activated PBMC to a greater extent than the V49(ala) peptide, whereas V49(scr) did not affect SEB-induced PBMC activation.
In a further experiment, TILs were activated by incubation with cognate melanoma cells in the presence or absence of the peptides, and IFNy secretion was measured via ELISA, as follows. TILs were released from fresh tumor samples. Melanoma cells used were cell lines 888meZ (HLA- A2-/MART-l+/gpl00+) and 624-mel (HLA-A2+/MART-l+gpl00+); human melanoma 526mel is an HLA-A2+ cell line. TILs were thawed and cultured for 3 days in 24 well plates in CM+IL2 (6000IU)/ml. TIL were collected and counted the replated at xlO5 cells/well (96 well plate-flat), in CM without-IL2 for 3-4 hours. Then peptides were added to the wells and incubated for 4 hours. IxlO5 melanoma cells/well were then added to yield a 1:1 ratio with TILs and further incubated for 16 hours. Supernatant was then collected for interferon-gamma ELISA (R&D Systems, catalog no. DY285), according to the manufacturer's instructions.
The results are presented in Fig IE for 624 cells; similar results were obtained for 526 cells. In both cases, V49 peptide led to significantly decreased cytokine secretion.
Example 2. Identification and characterization of short synthetic peptides
Using additional in-silico analyses, a homologous region between human SLAMF6 and the adenovirus SLAMF6-binding protein CRlalpha was identified as a putative active region within the V49 fragment. Three 11 amino-acid peptides based on this region were synthesized and characterized: CRlalpha (CRla, LTWTFNGKNVA, SEQ ID NO: 11), derived from the viral protein, 153 (ITWLFNETSLA, SEQ ID NO: 10), derived from the human protein, and 3Mut (ITWTFNGKSLA, SEQ ID NO: 2) in which the 3 amin acids marked in bold were taken from the viral sequence in to the human one.
The ability of the peptides to modulate activation-induced cytokine secretion were first evaluated on TIL in the presence of cognate melanoma cells. To this end, M526 and M624 SLAMF6-expressing melanoma cells were incubated with TIL in the presence or absence of the 153 peptide or V49(ala) control, and the results are presented in Fig. 2A (526 cells) and Fig. 2B (624 cells). The results are further summarized in Fig. 2E. The figure shows a significant decrease in IFNy secretion with 153 in two pairs of TIL and cognate melanoma cells. Next, the effects of the various peptides were tested using the SEB-induced PBMC activation model. Fig. 2C shows a decrease in IL-2 secretion by PBMC using the test peptides (153, CRla and 3mut) but not the control V49(ala) peptide. Fig. 2C also shows that the effect for the 3Mut peptide was remarkably obtained at a lower concentration as compared with 153 and CRla when PBMC cells are activated using SEB. Fig. 2D shows the inhibitory effect of different concentrations 153 peptide on PBMC function (measured as IL-2 secretion by ELISA) following SEB activation.
Summarizing all said above (see also Figs. 2F and 2G), the 3Mut peptide having the amino acid sequence ITWTFNGKSLA (SEQ ID NO: 2) was selected for further development.
Example 3. Design and characterization of D-enantiomer-modified peptide
To increase the stability of the 3Mut peptide (SEQ ID NO: 2), four- amino acids were replaced by corresponding D-amino acids to obtain a peptide having the amino acid sequence ITwTfNGkSLa (SEQ ID NO: 7) and entitled WFKA; low-case letters represent D-amino acids. The stability of 3Mut and WFKA was tested. The results are presented in Fig. 3A and 3B, respectively. As can be seen, the 3Mut peptide was found to be unstable under the test conditions and rapidly degraded in the presence of trypsin and chymotrypsin, while WFKA was remarkably stable and remained at a high level even after 24 hours, showing only modest degradation of about 20%.
Further, the effect of the WFKA peptide on activation-induced cytokine secretion was evaluated. To this end, Pmel-1 mice were asphyxiated using CO2, spleens were harvested and splenocytes isolated. Splenocytes (2 x 106/ml) were activated with 1 pg/ml of mouse gpl0025- 33 peptide for 6 days with IL-2 30 lU/ml. Fresh medium containing IL-2 was added every other day. Splenocytes, previously activated for 7 days, were co-cultured (l x 105) overnight at a 1:1 ratio with the indicated target cells or activated with 1 pg/ml plate-bound anti-CD3 antibody (Biolegend, clone: 145-2 Cl l) as indicated in each experiment. Conditioned medium was collected, and mouse IL-2 and IFN-y secretion were detected by ELISA (Biolegend). Additional experiments were performed in human TILs co-cultured with human melanoma for activation. IFN-y secretion was measured by ELISA after 24 hours. Further experiments were also performed in SEB-activated PBMC, IL2 secretion was measured by ELISA.
As follows from Fig. 3C-3E, WFKA decreases cytokine secretion similarly to 3Mut. Fig. 3C shows a decrease in IL-2 secretion when PBMC cells are activated using SEB in the presence of both peptides. Fig. 3D shows a decrease in IFNy secretion when human melanoma- specific TILs are co-cultured with a specific melanoma identified by the TILs in the presence of the peptides. Fig. 3E shows that the peptides are also active in cells of murine origin expressing the SLAMF6 homologous protein Lyl06, using the murine PMEL model described above. The peptides were added to a co-culture of splenocytes harvested from PMEL mice recognizing the melanoma B16-gp-100 antigen and B16 melanoma cells and IFNy secretion was quantified using ELISA.
Example 4. Structure-function analysis
Several further experiments were performed. The dose dependency was tested for WFKA peptide using two models: Granzyme-B (GZM-b) secretion in Jurkat cells activated using anti- CD3, and IFNy secretion in murine Pmel cells. As controls, two peptides denoted as scrl and scr2 (short for scramble) were used, in which various amino acids were substituted with alanine. In scrl (amino acid sequence IAALAAETSLW, SEQ ID NO: 14), the conserved amino acids between the viral peptides and SLAMF6 (see Fig. 4A) were replaced with alanine. In scr2, (amino acid sequence AAALAAETAAW, SEQ ID NO: 15), the semi-conserved amino acids were additionally replaced by alanine.
As can be seen from Fig. 4B, 3pM was the most active concentration of the WFKA peptide in the GZM-b Jurkat model. The control peptide (scrl) was not effective at this concentration. As can be seen in Figs. 4C, 4E and 4F, the effect was SLAMF6-dependent, as in SLAMF6 knockout cells no decrease in GZM-b secretion was observed in the presence of WFKA peptides. This indicated that the inhibitory effect of these peptides is directly dependent on the presence of the SLAMF6 receptor. As can be seen in Fig. 4E, GZM-b secretion levels in WT Jurkat cells were reduced with the MUT3 peptide and with the WFKA peptide, as measured by ELISA (Human Granzyme B DuoSet ELISA, #DY2906, R&D Systems). Scrl and Scr2 peptides, used as negative controls, showed no significant effect, highlighting the specific inhibitory activity of the MUT3 and WFKA peptides. In comparison, Fig. 4F shows that in SLAMF6'/_ Jurkat cells, GZM-b secretion was not inhibited by the MUT3 or WFKA peptides, indicating that the inhibitory effect of these peptides is directly dependent on the presence of the SLAMF6 receptor.
Similarly, IFNy secretion (measured essentially as described with respect to Fig. 3E above), decreased when murine splenocytes were activated in the presence of WFKA but not in Lyl06 knockout splenocytes (the murine SLAMF6 homologue), as shown in Fig. 4D. Interestingly, the most prominent effect was seen at much lower concentration, i.e. 500 nM.
Based on the above experiments, a consensus sequence determined to be significant for the identified immunosuppressive activity was defined as X0TWX1FNX2X3X4X5A (SEQ ID NO: 1), wherein Xo and X5 are each independently a hydrophobic amino acid and Xi, X2, X3 and X4 are each independently any amino acid. Accordingly, the conservative pattern comprising D amino acids was identified as Xo TwXi fNX2X3X4Xsa (SEQ ID NO: 6), wherein Xo and X5 are each independently a hydrophobic amino acid, Xi, X2, X3 and X4 are each independently any amino acid and low-case letters represent D-amino acids.
To summarize, demonstrated herein is SLAMF6 binding of peptides derived from SLAMF6, and inhibitory activity in-vitro on human derived lymphocytes. In addition to the full- length peptides, shorter 11 residues peptides were designed and determined as functionally active. This was achieved via identification of a homologous sequence within the initial peptide and a viral protein known to bind SEAMF6. Substituting 3 amino acids from the human peptide with the viral homologues led to superior function. The new peptide was found to be stabilized by further replacing 4 amino acids with their D enantiomer. This yielded a stable 11 residue peptide with significant inhibition of lymphocyte function in three models of human derived lymphocytes (PBMCS, TIEs and Jurkat cells) as well as in murine PMEL splenocytes.
Accordingly, peptides of the consensus sequence identified herein were shown to exhibit a considerable and significant ability to control excessive immune activation in various experimental models. In a PBMC model, WFKA peptide led to a reduction of up to about 20% in the secretion of IL-2. Similarly, in Jurkat cells, WFKA peptide led to a reduction of up to about 20% of granzyme-B secretion. In Peml splenocytes, WFKA peptide led to a reduction of up to about 30% of interferon-y secretion. It was further shown that the activity is dependent on SLAMF6 expression, as SLAMF6 knockout led to loss of activity in Jurkat cells and murine splenocytes. Thus, the WFKA peptide is disclosed herein as a particularly advantageous peptide characterized by desirable properties. These advantageous characteristics enables its use as a novel means of modulating auto-reactive or otherwise pathologically activated SLAMF6-expressing immune cells, including, but not limited to auto-reactive T-cells in autoimmune diseases and for inhibiting or reducing immune-related adverse events (irAEs).
Example 5. Efficacy of WFKA peptide in the treatment of colitis
The main aim of this study is to investigate the SLAMF6-derived WFKA peptide described in Example 3 as a therapeutic strategy for modulating immune activation in a DSS colitis model. Sub-aiml: Discover the smallest dose required for an effective therapeutic response. Sub-aim 2: Understanding the mechanism through which the inhibition mechanism works. The established model for inflammation-induced colitis works well with C57BL\6 strain. The strain is normal and does not suffer from diseases. Overall, 192 female mice (160 C57BL\6 mice and 32 C57BL\6 SLAMF67' ) 7 weeks old are used according to the following allocation. Methods relevant to all below experiments
Female mice 7-8 weeks age are admitted to the experimental cages. The mice acclimatize for 7 days before the initiation of the experiments. On the eighth day, the mice are weighed and marked. On day 1 the mice receive DSS in the drinking water and at the same time are IP injected with test items. The experimental groups receive the therapeutic substance in the desired dose in a volume of 0.1 ml and the control groups receive injections of saline (25G needle are used). On day 8 the mice are sacrificed by CO2 inhalation.
Research Methods
This study uses a DSS colitis model in mice to induce chronic inflammation and immune activation Mice were divided into two groups: a control group and a group treated with WFKA peptide. The treatment group received intraperitoneal (IP) injections of WFKA peptide for 7 days. The effects of WFKA peptide on immune activation and colitis symptoms is assessed using various methods, including histological analysis of tissue samples and assessment of inflammatory markers in the blood.
In all the below experiments, the mice develop acute intestinal inflammation, 10-40% of the mice typically die following the DSS treatment. Mice treated with DSS exhibit weight loss and signs of loose stool, diarrhea, and rectal bleeding.
The mice's condition is followed daily during the experiment.
The research methodology for the study involves the following steps:
1. Establishing the DSS colitis model in mice: Mice receive drinking water containing 3.5% dextran sodium sulfate (DSS) for 7 days to induce chronic inflammation and immune activation.
2. Treating mice with WFKA peptide during the formation of inflammation: further to the establishment of the DSS colitis model, mice in the experimental group receive injections of WFKA peptide according to the established protocol.
3. Assessing the effects of WFKA peptide on immune activation and colitis symptoms: The effects of WFKA peptide treatment on immune system activation and colitis symptoms are evaluated using various methods. While the mice are alive use the DAI index to rate the intensity of the inflammation in addition to the weighing that is performed daily.
At this point, the mice are sacrificed, and the rest of the experiment refers to the analysis of the WFKA peptide treatment.
4. Histological analysis of tissue samples: Tissue samples from the colon are collected from mice at the end of the treatment period, and the length of the intestine are measured. 5. The samples are then fixed in formalin for histological analysis. The tissue samples are embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) for microscopic examination. The severity of inflammation and tissue damage are evaluated using a standardized scoring system.
6. Assessment of inflammatory markers in intestinal tissue: Intestinal tissue samples are collected from mice at the end of the treatment period, and RNA is extracted for subsequent mRNA quantification. The levels of various inflammatory markers, including TNF-a, IFN- y, IL-10, and IL-17 are determined using RT-qPCR. Additionally, the extracted RNA is subjected to RNA sequencing (RNA-seq) to perform a comprehensive analysis of gene expression changes in the intestinal tissue. The results from both RT-qPCR and RNA-seq are compared between the two groups to evaluate the effects of WFKA peptide treatment on immune activation in the intestine.
Flow cytometry analysis of immune cells: Immune cells are isolated from the blood and spleen of mice at the end of the treatment period. The cells are stained with specific antibodies and analyzed using flow cytometry to evaluate the effects of WFKA peptide treatment on immune cell activation and function.
Experiment 1. Preliminary examination of the in vivo response to the peptide.
In this experiment, the inhibitory activity of injected WFKA peptide on the immune system is tested in an in-vivo model. The experimental setup is shown in Table 2.
Table 2. Experimental setup
Figure imgf000043_0001
DSS is administered daily in drinking water and the test articles are injected daily for 7 days. The mice are sacrificed on day 8. The colon length and histochemistry measurements are performed. In this experiment, measured are Disease Activity Index (DAI), the length of the intestine after sacrifice, and a general look at histochemical results.
DAI score is assessed according to Table 3 and the histochemical score is assessed according to Table 4. Table 3. DAI assessment score
Figure imgf000044_0001
Table 4. Histological score
Figure imgf000044_0002
In case the observed therapeutic activity is not significant, a higher dose of WFKA peptide is tested.
Experiment 2. Determination of WFKA peptide concentration for colitis treatment
To find the suitable concentration for immune inhibition and amelioration of colitis symptoms by
WFKA peptide the following experiment is performed. The Experimental setup is presented in Table 5.
Table 5. Experimental setup - experiment 2
Figure imgf000044_0003
Experiment 3. Elucidation of the mode of action of WFKA peptide in a colitis model
To characterize the cytokines and components of the immune system and elucidate the peptide's activity blood, internal organs, and parts of the intestine of sacrificed mice from the abovedescribed Experiments 1 and 2 are extracted. Cytokine quantification is performed using qPCR from the intestinal tissue. In addition, crypts from the colon of these mice are isolated and RNA for analysis is purified (RNA seq). The mechanism of the peptide's activity is examined. To this end, a group of transgenic mice that do not express the receptor through which it is hypothesized that the inhibition activity occurs is used. Part of the intestinal tissue is crushed, and the T cells are separated from it to characterize the release of the peptide on them. The Experimental setup is presented in Table 6.
Table 6. Experimental setup - experiment 3
Figure imgf000045_0001
Example 6. In vivo WFKA administration slows colitis onset and reduces clinical severity in mice
The experiments were performed essentially as described in Example 5, with changes as indicated below. An acute model for DSS-induced colitis was used, comprising administration of 2% DSS in drinking water for 7 days, followed by a two-days recovery period (in the absence of DSS administration and treatment) prior to sacrifice. The experimental groups and experimental design are presented in Tables 7 and 8, respectively.
Table 7. Experimental groups - DSS model
Figure imgf000045_0002
Table 8. Experiment design - DSS model
Figure imgf000046_0001
Figs. 5A-5D present the results of the wight loss (Fig. 5A), diarrhea incidence (Fig. 5B) blood in feces (Fig. 5C) and Disease Activity Index (DAI) score (Fig. 5D) for the three treatment groups.
As can be seen in Fig. 5A, mice treated with the WFKA peptide (Group B) showed slower weight loss compared to untreated mice (Group A). Colitis development began on day 3 post-DSS induction. From day 5 onward, untreated mice exhibited greater weight loss compared to treated mice, with a 10% difference by day 7. Treated mice (Group B) regained weight faster on days 8 and 9, indicating that WFKA peptide not only inhibited colitis progression but also aided recovery. As can be seen in Fig. 5B, diarrhea incidence was lower in WFKA peptide-treated mice (Group B) compared to untreated mice (Group A). By days 7 and 8, all untreated mice developed diarrhea, while approximately 50% of the treated group did. After switching to regular water, both groups improved, but treated mice maintained a better overall health status, as reflected by their lower diarrhea scores. Fig. 5C shows that blood in feces was less frequent in WFKA peptide-treated mice (Group B) than in untreated mice (Group A). By days 6 and 7, all untreated mice showed gross bleeding, while treated mice exhibited 25-30% less bleeding on day 6 and 50% less on day 7. Both groups ceased bleeding after returning to regular water, but the treated group showed less severity.
Fig. 5D is a composite of data from Figs. 5A-C, showing the overall DAI score. As can be seen, the DAI score remained lower in WFKA peptide-treated mice, with a maximum effect of 30% reduction on day 7 compared to untreated mice. On days 8 and 9, treated mice showed a 23% improvement in DAI score relative to untreated mice, indicating better recovery. The average and SEM of the DAI scores as measured on days 5-10 are summarized in Table 9 below. As can be seen, the differences on days 6-8 were statistically significant, with a p-value =< 0.01.
Table 9. DAI scores
Figure imgf000046_0002
Figure imgf000047_0001
Animal body weight and weight loss are further summarized in Table 10 below.
Table 10. Weight loss
Figure imgf000047_0002
Colons harvested on day 10 were straightened against a ruler for measurement. Evaluation of the length of excised colons further demonstrated marked efficacy of the peptide with respect to DSS-induced intestinal shortening, as can be seen in Figs. 6A-6C and 7.
In particular, Fig. 6A shows colon length in naive mice (Group C); the average measured length approximately was 7-8 cm on day 10. Fig. 6B shows colon length in mice treated with DSS only (Group A). The average measured length was approximately 4-5.8 cm on day 10. Fig. 6C shows colon length in mice treated with DSS and 3 pM WFKA peptide (Group B). The colon lengths were partially preserved having an average measured length of approximately 6-7 cm on day 10.
Fig. 7 shows a comparison of colon length between WFKA peptide-treated (Group B) and untreated (Group A) mice. As can be seen, colons from treated mice were approximately 20% longer than those from untreated mice. The difference was statistically significant, with a p-value of 0.0001. The average and SEM of colon length and spleen weight as measured on day 10 are further presented in Table 11 below. Table 11. Colon length and spleen weight
Figure imgf000048_0001
Thus, the results demonstrate WFKA treatment significantly decelerates DSS-induced intestinal shortening in mice compared to untreated controls.
In summary, WFKA treatment was found to delay the development of colitis in treated mice, affecting both clinical inflammatory expression (DAI score, weight loss) and physical phenotype (colon length). The reduction of the inflammatory outcome was highly pronounced, achieving a reduction of 30% on day 7. Accordingly, the results support a role for the WFKA peptide as a promising drug candidate for autoimmune diseases, and for inflammatory and autoimmune conditions of the gastrointestinal tract in particular. Example 7. In vivo cytokine secretion
Mice sera collected in the experiments described in Example 6 were further assayed for the presence of inflammatory cytokines. Serum cytokine levels were analyzed from mice on day 10 using a multiplex assay (MILLIPLEX MAP Mouse High Sensitivity T Cell Panel, Merck) to quantify 15 cytokines: GM-CSF, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 (p70), IL-13, IL-17A, IL-la, IL-ip, TNFa, MCP-1, KC, and IFN-y. Mice treated with the WFKA peptide showed cytokine profiles more closely resembling those of naive mice, particularly for IL-2, IL-5, IL-12, IL-la, KC, IL-6, and IFN-y. Exemplary results are presented in Table 12 below, in which the experimental groups are as described in Example 6.
Table 12. Serum cytokine levels
Figure imgf000048_0002
Figure imgf000049_0001
As can be seen from Table 12, in vivo treatment with the WFKA peptide resulted in inhibition of about 50% in DSS-induced enhancement in serum cytokine levels. In addition, comparison of cytokine levels relative to Disease Activity Index (DAI) scores and colon length ratios between WFKA peptide-treated and untreated mice were performed. It was found that treated mice exhibited cytokine profiles more closely aligned with naive mice, correlating with improved DAI scores and longer colon lengths compared to untreated mice.
The results and comparisons highlight the therapeutic effect of the WFKA peptide in modulating inflammatory responses and alleviating colitis symptoms in vivo. In addition, without wishing to be bound by a specific theory or mechanism of action, the results suggest that mitigation of colitis by the WFKA peptide involves modulation of key cytokines implicated in inflammatory pathways.
Example 8. DSS-induced colitis in wild-type and SLAMF6-deficient mice
Additional experiments in DSS models were performed essentially as described in Example 5, with the following changes. Overall, 134 female 7 weeks old mice are used: 111 C57BL\6 mice (wild-type) and 23 C57BL\6 SLAMF6'/_ mice (in which the expression of murine SLAMF6 is knocked out). The strain is normal and does not suffer from diseases. Mice receive drinking water containing 3 concentrations of DSS for 7 days to induce acute inflammation and immune activation and to determine the best concertation required for later experiments. On day 8 mice undergo terminal bleeding (using the 'Cardiac Puncture' withdraw following anesthesia with Thiopental). At this point, mice are sacrificed, and the effects of the WFKA peptide treatment are analyzed as follows. Tissue collection: the intestines, spleen, liver, heart, kidney, lung and stomach are collected from mice at the end of the treatment period. Spleens are taken out and weighted. Increased weights correlate with the extent of inflammation and anemia. The colon and cecum are isolated by separating them form the small intestine at the ileocecal junction and from the anus at the distal end of rectum, measure length of the straighten colons, and comparing them between the control and test groups. The colons are quickly flushed (5-10 ml syringe with feeding needle (18G-3” Straight 2.25mm ball, Braintree Scientific Inc) using cold PBS to remove feces and blood. The cecum can be discarded as little or no inflammation is induced by DSS in this region. After flushing with PBS, colon weights can be taken. In accordance with observed tissue wasting, severely inflamed colons exhibit reduced weight as both correlate with the severity of acute inflammation.
For RNAseq or qRT-PCR analysis, the middle portion (50mg) of the intestine and half of the spleens is stored in RNAlater (Sigma, Ref = R0901) until RNA extraction. For longer storage, tissue in RNAlater should be frozen at -20°C. Distal portion (~1 cm) of the colons is then fixed in formalin for histological analysis. The tissue samples are embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) for microscopic examination. The severity of inflammation and tissue damage are evaluated using a standardized scoring system.
All organs, apart from the intestines and spleens, are homogenized and immediately frozen at -80C for further analysis of peptide biodistribution as part of safety testing. A quantitative measurement of cytokine level from the serum is performed by cytokine assessment array using multiplex (MILLIPLEX MAP Mouse High Sensitivity T Cell Panel - Immunology Multiplex Assay kit, Millipore). Assessment of inflammatory markers in intestinal tissue: Intestinal tissue samples are collected from mice at the end of the treatment period, and RNA is extracted for subsequent mRNA quantification. The levels of various inflammatory markers, including TNF-a, IFN-y, IL-10, and IL-17 are determined using RT-qPCR. Additionally, the extracted RNA is subjected to RNA sequencing (RNA-seq) to perform a comprehensive analysis of gene expression changes in the intestinal tissue. The results from both RT-qPCR and RNA-seq are compared between the two groups to evaluate the effects of WFKA peptide treatment on immune activation in the intestine. Flow cytometry analysis of fresh lamina propria cells: and splenocytes are isolated from the intestines, spleens and blood of mice at the end of the treatment period. The cells are stained with specific antibodies and analyzed using flow cytometry to evaluate the effects of WFKA peptide treatment on immune cell activation and function.
Experiments 1-3 are conducted with the selected DSS concentration while keeping the established parameters of the model and study design with the addition of treatment with the WFKA peptide, by injecting it intraperitoneally on a daily basis with changes as described below. Three experiments are designed to examine and validate the inhibitory properties of the peptide and to mark it as a drug candidate.
Experiment 1: Determining administration schedule for peptide injections
Previous results showed that intraperitoneal injections of the peptide at 3 pM, four times daily, achieved a 30% reduction in colitis development by day 7. In this experiment, the goal is to assess whether reducing the number of injections can simplify the treatment and lower the peptide dosage. To this end, mice are divided into 4 groups as described in Table 13.
Table 13. Experimental groups - Experiment 1
Figure imgf000051_0001
Experiment 2: Discovering the minimal effective dose required for inhibition
An initial effective peptide concentration identified for therapeutic purposes is 3p M. This trial aims to address dose-response, determining the lowest effective concentration required for treatment in mice considering the results received in the previous experiment. Doses lower than 3 pM (3pM, 1.2 pM, 0.5 pM) are tested to establish the minimal dose required for effective result. The experimental groups are shown in Table 14. Schedule (twice or X4 daily) is determined based on experiment 2 results.
Table 14. Experimental groups - Experiment 2
Figure imgf000051_0002
Experiment 3: Elucidating the role of the peptide as a direct inhibitor of the SLAMF6 receptor
In this experiment the aim is to compare the activity of SLAMF6-derived peptide between C57BL/6 mice and a knockout strain of C57BL\6 SLAMF6-/\ both after induction of colitis with DSS in autoclaved drinking water. The lowest effective dose as determined in Experiment 2 is used in this experiment and injected to mice of both strains under the same conditions for comparison of its ability to exert anti-inflammatory effects. The experimental groups are shown in
Table 15.
Table 15. experimental groups - Experiment 3
Figure imgf000052_0001
Example 9. Efficacy of the WFKA peptide in the treatment of ICI-induced autoimmune toxicities.
Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy, significantly extending survival and sometimes curing metastatic disease, but they can also trigger severe autoimmune-related adverse effects. Monotherapy with anti-PDl ICIs leads to about 20% highgrade adverse events (AEs), anti-CTLA4 causes around 30%, and their combination results in nearly 60% severe AEs, including common issues like dermatitis, colitis, and thyroiditis. To test the ability of the WFKA peptide to reduce these immune-related adverse effects (irAEs), the non- obese diabetic (NOD) mouse model was used. This model is prone to autoimmune pathologies and is particularly responsive to ICI-induced complications, specifically diabetes and thyroiditis. Thus, it may serve as a model for iatrogenic multiple autoimmune conditions associated with administration of ICIs.
The main aim of this study is to investigate the SLAMF6-derived WFKA peptide described in Example 3 as a therapeutic strategy for modulating immune activation in a ICI-induced diabetes and thyroiditis model (NOD mice). The established model for ICI-induced diabetes and thyroid dysfunction works well with NOD/ ShiLtJ strain. Overall, 95 female mice, 4-6 weeks old are used according to the following allocation.
In the experiments described below, female mice 3-5 weeks age are admitted to the experimental cages. The mice are acclimatized for 7 days before the initiation of the experiments. On the first day, the mice are weighed and marked. Every week the mice are treated twice weekly with combination (Dual ICI: anti-mouse-PD-1 plus anti-mouse-CTLA-4) at 10 mg/kg/dose intraperitoneally. The experimental group is additionally injected Intraperitoneally with the test items every day. The experimental groups receive the therapeutic substance in the desired dose in a volume of 0.1 ml and the control groups receive injections of saline (25G needle are used). Mice are monitored daily for activity (including signs of neuropathy) and appearance and twice weekly for signs of autoimmunity: weight loss, decreased activity, and glucosuria. Activity and weight loss are assessed according to Table 16.
Table 16. Mice daily assessment
Figure imgf000053_0001
Mice developing glucosuria are treated with 10 units of subcutaneous NPH insulin every 12 hours. After 4 or 8 weeks of treatment, mice are sacrificed, and multiple tissues (salivary, lacrimal, pancreas, liver, lung, heart, colon, eye, gonad, and thyroid) are evaluated by histology and flow cytometry for the development of autoimmune infiltrates. Prior to sacrifice, the mice undergo terminal bleeding (using the 'Cardiac Puncture' withdraw following anesthesia with Thiopental, followed by sacrificing via cervical dislocation. The experimental design for both experiments is described in Table 17.
Table 17. Experiment design
Figure imgf000054_0001
Experiment 1: Dose-response of the WFKA peptide in NOD model. (Length 4 weeks)
The experiment aims to evaluate the peptide's activity and effectiveness in a second autoimmune pathology model and to determine the minimal effective dose required for treatment. In this experiment, three dosages of the peptide, based on those observed in dose-response experiments with cells and the DSS model, are administered daily. The starting dosage tested in the DSS model (3pM, "high dose"), along with two additional dosages are used: one being the minimal effective dose from the DSS model ("minimal dose", 1.2 pM or 0.5 pM), and another in between these two dosages ("median dose"). Mice are divided into 5 groups as described in Table 18.
Table 18. Experimental groups - experiment 1
Figure imgf000054_0002
Mice are followed daily for mice activity and twice a week for weight and glucosuria. At the end of the 4-week period, the mice are euthanized, and tissues are collected for post-mortem analysis as described below. Tissue Excision: the tissues to be excised include the salivary glands, lacrimal glands, pancreas, liver, lungs, heart, colon, eyes, gonads, and thyroid. These tissues are utilized for histology, as follows: tissue immune infiltration is assessed by evaluating hematoxylin and eosin (H&E)-stained formalin-fixed paraffin-embedded (FFPE) sections. This analysis, performed on all tissues, evaluates autoimmune pathology and is conducted by an expert pathologist.
Experiment 2: Examination of the WFKA peptide over diabetes and thyroiditis development. (Eength 8 weeks)
This experiment is performed after experiment 1 is completed and effective minimal dose is determined. It aims to test the inhibitory properties of the test item over diabetes and thyroiditis in ICI-treated NOD mice and study its mechanism of action. The mice are divided into 3 groups as described in Table 19.
Table 19. Experimental groups - experiment 2
Figure imgf000055_0001
This experiment replicates Experiment 1 but is extended to 8 weeks to ensure the development of thyroiditis and to allow for significant statistical analysis. Mice are monitored daily and treated as described above. In the end of 8 weeks, mice are sacrificed and post-mortem evaluation is performed as described below.
Tissue Excision: The tissues to be excised include the salivary glands, lacrimal glands, pancreas, liver, lungs, heart, colon, eyes, gonads, and thyroid. These tissues are utilized for histology, flow cytometry, and scRNAseq analysis. Due to the small size of some tissues, using them for one analysis may leave insufficient material for the other two. Subject Grouping: To address this limitation, samples are collected from all major organs (i.e., heart, lungs, colon, liver, and pancreas) for histological analysis, while the remaining portions are allocated to flow cytometry and scRNA-seq. Glands are also divided among three analyses: histology, flow cytometry, and scRNA-seq. Thyroids are exclusively analyzed by flow cytometry to ensure a detailed assessment of tissue inflammation. Naive Mice Grouping: Similarly, a cohort of 15 naive mice is included in the study and divided as described above, to ensure significant statistical analysis. Histology: Tissue immune infiltration is assessed by evaluating hematoxylin and eosin (H&E)-stained formalin-fixed paraffin-embedded (FFPE) sections. This analysis, performed on all tissues, evaluates autoimmune pathology and is conducted by an expert pathologist. Flow Cytometry: Fresh tissues are lysed and live cells are monitored for immune markers using flow cytometry. Immune markers include at least the following: CD4, CD8, CD44, CD62L, CD25, FoxP3, CD127, CD33, CDl lb, CCR7, CD45RO, CD45RA, TIM3, PDL-1, LAG3, TCF7, IFNy, and TNFa. scRNAseq is performed on isolated CD45+ infiltrating cells from the thyroids. The single cell suspension is stained and sorted using fluorescence-conjugated antibodies to CD45, CD1 lb, CD3, CD4, CD8, and CD19 and viability dye DAPI. Samples for each condition are then pooled to obtain sufficient cells for sequencing and analysis. Serum Analysis: Sera from the mice are tested for free thyroxine (FT4) and anti-TPO antibodies using ELISA. Thyroid Tissue Processing: Fresh thyroid tissues from ICLtreated mice are perfused with saline to remove circulating peripheral immune cells. The tissues are then dissociated into single-cell suspensions and stained for immune markers for flow cytometry analysis as described above.
Example 10. Effect on myeloid-derived suppressor cell (MDSC)-mediated immune suppression.
The ability of the WFKA peptide to affect the activity of MDSC and their interaction with T cells is assessed ex vivo on human blood-derived cells and in vivo using animal models.
For the in vivo experiments, a murine model is established with C57BL/6 mice immunized with the live attenuated Bacillus Calmette-Guerin (BCG) vaccine over a 14-day period, to induce a tumor-like microenvironment characterized by elevated MDSC levels. This model mimics the chronic inflammation seen in cancer and allows for efficient isolation of MDSCs from the spleen and bone marrow. After the immunization phase, MDSCs are isolated using positive selection (CDl lb+Grl+), and their suppressive function are evaluated in vitro. These MDSCs are cultured, and the activity of WFKA peptide over their activity is assessed in two concentrations (3 pM and 10 pM). The ability of the peptide to modulate MDSC-mediated immune suppression is evaluated by measuring the proliferation of T cells using CFSE labelling and flow cytometry over 48-72 hours. Changes in T cell proliferation indicate how effectively WFKA modulates MDSC-T cell interactions.
In addition to measuring proliferation, the experiment evaluates the functional consequences of MDSC inhibition by analyzing nitric oxide (NO) secretion (using Griess reagent) and arginase- 1 activity (through the conversion of L- arginine to urea), according to accepted protocols. These assays provide insights into how WFKA affects the metabolic pathways used by MDSCs to suppress T cell function, wherein reduced NO levels and arginase- 1 activity indicate a decrease in MDSC suppressive function in the presence of the peptide and vice versa. The model also includes co-culture experiments with SLAMF6-deficient T cells and MDSCs to explore how SLAMF6 influences MDSC suppression and whether WFKA peptide can modulate these effects. By studying the interactions between MDSCs and SLAMF67' T cells, the research compares the ability of the peptide to inhibit SLAMF6-mediated and SLAMF6-independent MDSC-T cell suppression. Analysis includes NO secretion levels, arginase- 1 and proliferation assay (with CSFE staining).
For the experiments in human cells, an ex vivo model is established to explores the inhibitory activity of the WFKA peptide on MDSC subpopulations and their interactions with cytotoxic T cells (CD8+) in the context of ICI-induced irAEs. Using PBMC samples from patients pre- and post-ICI treatment, with or without irAEs, the study determines how the WFKA peptide impacts MDSC-mediated suppression of T cells. Monocytic MDSCs (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs) are characterized using flow cytometry, with markers for MDSCs (CDl lb+, CD33+, CXCRU) and T cells (CD4+, CD8+) and the abundance and activation status of these subpopulations is evaluated.
Next, co-culture experiments with isolated MDSCs and activated CD8+ T cells from PBMC samples are performed, in which the cells are incubated with the WFKA peptide to assess its impact on T cell suppression. The peptide is tested in varying concentrations as described above to determine its ability to mitigate or otherwise modulate the suppressive activity of MDSCs, which is often linked to immune suppression in cancer and autoimmunity. Readouts include T cell proliferation (measured by CFSE staining and flow cytometry), cytokine secretion (e.g., IFNy, TNFa), and cytotoxicity, essentially as described above. Flow cytometry is employed to monitor changes in T cell activation and the suppression mediated by MDSCs in response to WFKA peptide.
Although the present invention has been described herein above by way of preferred embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims.

Claims

1. An isolated peptide comprising the amino acid sequence X0TWX1FNX2X3X4X5A (SEQ ID NO: 1) or a retro-inverso sequence thereof, wherein Xo and X5 are each independently a hydrophobic amino acid, X4 , X2 , X3 and X4 are each independently any amino acid and wherein the peptide consists of from 11 to 50 amino acids.
2. The isolated peptide of claim 1, characterized in that:
XQ and X5 are each independently selected from Leu, Vai and He;
Xi is selected from Thr, Leu, He, Vai, Met, and Ser;
X2 is selected from Gly, Ala, Asp and Glu;
X3 is selected from Ser, Thr, Lys and Arg; and
X4 is selected from Ser, Asn, Gin, and Thr, set forth as SEQ ID NO: 17.
3. The isolated peptide of claim 1, characterized in that:
XQ and X5 are each independently selected from Leu, Vai and He;
Xi is selected from Thr, and Leu;
X2 is selected from Gly, Glu and Ala;
X3 is selected from Lys and Thr;
X4 is selected from Ser and Thr, set forth as SEQ ID NO: 18.
4. The isolated peptide of claim 1, comprising the amino acid sequence I TWTFNGKSLA (SEQ ID NO: 2).
5. The isolated peptide of any one of claims 1 to 4, wherein at least 2 amino acids are D-amino acids.
6. The isolated peptide of claim 5, wherein 2, 3, 4 or 5 amino acids are D-amino acids.
7. The isolated peptide of claim 5 or 6, wherein at least 2 of the amino acids at positions 3, 5, 8 and 11 of the sequence X0TWX4FNX2X3X4X5A (SEQ ID NO: 1) are D-amino acids.
8. The isolated peptide of claim 7, wherein the amino acids at positions 3, 5, 8 and 11 are D- amino acids and the peptide comprises an amino acid sequence X0TwX4fNX2x3X4X5a set forth as SEQ ID NO: 6.
9. The isolated peptide of claim 8 comprising the amino acid sequence I TwT fNGkSLa (SEQ ID NO: 7).
10. The isolated peptide of claim 9, consisting of the amino acid sequence I TwT fNGkSLa (SEQ ID NO: 7).
11. A conjugate of the isolated peptide of any one of claims 1 to 10.
12. The conjugate of claim 11, wherein the peptide is conjugated with polyethylene glycol (PEG).
13. A pharmaceutical composition comprising at least one isolated peptide as defined in any one of claims 1 to 10, or the conjugate of claims 11 or 12, and a pharmaceutically acceptable carrier and/or excipient.
14. The pharmaceutical composition of claim 13, wherein the isolated peptide comprises or consists of the amino acid sequence selected from I TWTFNGKSLA (SEQ ID NO: 2) and I TwT fNGkSLa (SEQ ID NO: 7).
15. The pharmaceutical composition of claim 13 or 14, for use in inhibiting or reducing an inappropriate or excessive immune response in a subject in need thereof.
16. The pharmaceutical composition for use of claim 15, wherein the use comprises inhibiting or reducing immune-related adverse events (irAEs).
17. The pharmaceutical composition for use of claim 16, wherein the irAEs are selected from the group consisting of gastrointestinal, endocrine, and dermatologic toxicities, and combinations thereof.
18. The pharmaceutical composition for use of claim 16, wherein the irAEs comprise toxicities affecting the pancreas and/or thyroid gland.
19. The pharmaceutical composition for use of claim 14 or 15, wherein said irAEs are associated with administration of a cancer immunotherapy.
20. The pharmaceutical composition for use of claim 19, wherein said cancer immunotherapy comprises administration of an immune checkpoint inhibitor.
21. The pharmaceutical composition for use of claim 20, wherein the immune checkpoint inhibitor targets PD-1, PD-L1, PD-L2, CTLA-4, LAG3, TIM3, and/or IDO.
22. The pharmaceutical composition for use of claim 15, wherein the use comprises treating a disease or a condition associated with the inappropriate or excessive immune response.
23. The pharmaceutical composition for use of claim 22, wherein the condition is a T-cell mediated inflammatory or autoimmune condition.
24. The pharmaceutical composition for use of claim 22, wherein the disease or condition is selected from the group consisting of graft versus host disease, an autoimmune disease, and graft rejection.
25. The pharmaceutical composition for use of claim 24, wherein the autoimmune disease is selected from rheumatoid arthritis, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), multiple sclerosis (MS), psoriasis, psoriatic arthritis, autoimmune hepatitis and primary biliary cholangitis (PBC).
26. The pharmaceutical composition for use of claim 25, wherein said autoimmune disease is IBD.
27. The pharmaceutical composition for use of claim 24, wherein said autoimmune disease is insulin dependent diabetes mellitus or autoimmune thyroiditis.
28. A method for inhibiting or reducing an inappropriate or excessive immune response in a subject in need thereof, comprising administering to the subject a pharmaceutical composition comprising at least one isolated peptide comprising the amino acid sequence X0TWX1FNX2X3X4X5A (SEQ ID NO: 1) or a retro-inverso sequence thereof, wherein Xo and X5 are each independently a hydrophobic amino acid, Xi, X2, X3 and X4 are each independently any amino acid and wherein the peptide consists of from 11 to 50 amino acids.
29. The method of claim 28, wherein the peptide comprises or consists of an amino acid sequence selected from ITWTFNGKSLA (SEQ ID NO: 2) and ITwTfNGkSLa (SEQ ID NO: 7).
30. The method of claim 28, wherein the method comprises inhibiting or reducing immune- related adverse events (irAEs).
31. The method of claim 30, wherein the irAEs are selected from the group consisting of gastrointestinal, endocrine, and dermatologic toxicities, and combinations thereof.
32. The method of claim 31, wherein the irAEs comprise toxicities affecting the pancreas and/or thyroid gland.
33. The method of claim 30, wherein said irAEs are associated with administration of a cancer immunotherapy .
34. The method of claim 33, wherein said cancer immunotherapy comprises administration of an immune checkpoint inhibitor.
35. The method of claim 34, wherein the immune checkpoint inhibitor targets PD-1, PD-L1, PD-L2, CTLA-4, LAG3, TIM3, and/or IDO.
36. The method of claim 28, comprising treating a disease or a condition associated with the inappropriate or excessive immune response.
37. The method of claim 36, wherein the condition is a T-cell mediated inflammatory or autoimmune condition.
38. The method of claim 37, wherein the disease or condition is selected from the group consisting of graft versus host disease, an autoimmune disease, and graft rejection.
39. The method of claim 38, wherein the autoimmune disease is selected from rheumatoid arthritis, systemic lupus erythematosus (SLE), inflammatory bowel disease (IBD), multiple sclerosis (MS), psoriasis, psoriatic arthritis, autoimmune hepatitis, primary biliary cholangitis (PBC), insulin dependent diabetes mellitus and autoimmune thyroiditis.
40. The method of claim 39, wherein said autoimmune disease is IBD.
41. The method of claim 39, wherein said autoimmune disease is insulin dependent diabetes mellitus or autoimmune thyroiditis.
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Citations (1)

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Publication number Priority date Publication date Assignee Title
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Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006037421A2 (en) * 2004-10-02 2006-04-13 Immatics Biotechnologies Gmbh Immunogenic t-helper epitopes from human tumour antigens and immunotherapeutic methods using said epitopes

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