WO2020123806A1 - Modulation de pd-1 - Google Patents

Modulation de pd-1 Download PDF

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WO2020123806A1
WO2020123806A1 PCT/US2019/065983 US2019065983W WO2020123806A1 WO 2020123806 A1 WO2020123806 A1 WO 2020123806A1 US 2019065983 W US2019065983 W US 2019065983W WO 2020123806 A1 WO2020123806 A1 WO 2020123806A1
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antibody
shp
seq
amino acid
polypeptide
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Vassiliki A. Boussiotis
Nikolaos PATSUOKIS
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Beth Israel Deaconess Medical Center Inc
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Beth Israel Deaconess Medical Center Inc
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    • C12Y301/03048—Protein-tyrosine-phosphatase (3.1.3.48)
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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    • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)
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    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

Definitions

  • the disclosure is directed to compositions and methods for modulating immune checkpoint genes, for example, programmed death receptor-1 (PD-1).
  • PD-1 programmed death receptor-1
  • Immune checkpoint therapy which often targets regulatory pathways in T cells to enhance anti-tumor immune responses, has led to important clinical advances and provides a new defense, at least, against cancer.
  • the programmed death receptor (PD-1) has been shown to be involved in regulating the balance between T cell activation and T cell tolerance.
  • PD-1 programmed death receptor
  • No currently-available PD-1-related therapeutic compounds has been tested for its ability to trigger PD-1 dimerization or disrupt PD-1 dimerization. Accordingly, there exists a need for therapeutics that either disrupt PD-1 dimerization for enhancing a T cell response or trigger PD-1 dimerization for suppressing T cell responses.
  • the present disclosure fulfills these needs and further provides other related advantages. I
  • Programmed cell death-1 is an inhibitory checkpoint receptor of the B7-CD28 family, which is upregulated upon T cell activation and plays a key role in peripheral T cell tolerance (/. e., an inhibition of T cell activity). PD-1 also restrains anti-viral and anti-tumor T cell responses and is used by tumors to evade immune attack. PD-1 -mediated inhibition relies on its interaction with SHP-2 by a previously-unknown mechanism.
  • the cytoplasmic tail of PD-1 has one immunoreceptor tyrosine-based inhibitory motif (ITIM) at Y223 and one immunoreceptor tyrosine-based switch motif (ITSM) at Y248, which has an indispensable role in PD-1 -mediated inhibitory function, yet their function in relation to SHP-2 remained unclear.
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • ITMS immunoreceptor tyrosine-based switch motif
  • SHP-2 via its amino terminal (N)-SH2 and carboxy terminal (C)-SH2 domains - bridges a first phosphorylated ITSM-Y248 residue on one PD-1 polypeptide and a second phosphorylated ITSM-Y248 residue on a second PD-1 polypeptide, thereby, forming a PD-1 : PD-1 dimer.
  • SHP-2's interaction with the two phosphorylated ITSM-Y248 residues (but not with two ITIM-Y223 residues or with one ITI M-Y223 residue and one ITSM-Y248 residue) activates SHP-2 and inhibits IL-2 production.
  • the present invention relates, in part, to exploiting the geometry of the PD-1 : SHP-2 interaction in methods, compounds, and compositions, e.g., for treating cancer, which enhance T cell responses by disrupting PD-1 dimerization and in methods, compounds, and compositions, e.g., for treating an autoimmune disease or disorder of for treating inflammation, which suppress T cell responses (/.e., inducing T cell tolerance) by triggering PD-1 dimerization.
  • the present invention provides a method for treating cancer in a patient in need thereof, comprising: (a) selecting an agent which decreases SHP-2-mediated PD-1 dimerization and/or reduces PD-1 dimer activity; and (b) administering the agent to the patient.
  • the agent which decreases SHP-2-mediated PD-1 dimerization and/or reduces PD-1 dimer activity is a small molecule or peptide agent.
  • the small molecule or peptide agent is capable of disrupting an interaction between a PD-1 polypeptide and a SHP-2 polypeptide, e.g., is capable of disrupting an interaction between a PD-1 polypeptide and a SH2 domain of SHP-2.
  • the small molecule or peptide agent is capable of disrupting an interaction between a PD-1 polypeptide and a SH2 domain of SHP-2 by preventing binding of the SH2 domain with a motif of the PD-1 polypeptide comprising a tyrosine 248, e.g., a phosphorylated tyrosine 248, e.g., a motif comprising an immunoreceptor tyrosine-based switch motif (ITSM).
  • ITSM immunoreceptor ty
  • the small molecule or peptide agent is capable of disrupting an interaction between a PD-1 polypeptide and a SH2 domain of SHP-2 by preventing binding of an amino terminal SH2 domain of SHP-2 (N-SH2; SEQ ID NO: 19) with the PD-1 polypeptide and/or by preventing binding of a I
  • the small molecule or peptide agent is capable of disrupting an interaction between a first PD-1 polypeptide and an amino terminal SH2 domain of SHP-2 (N-SH2; SEQ ID NO: 19) and is capable of disrupting an interaction between a second PD-1 polypeptide and an carboxy terminal SH2 domain of SHP-2 (C-SH2; SEQ ID NO: 20).
  • the small molecule or peptide agent is capable of disrupting an interaction between the first PD-1 polypeptide and the N-SH2 domain by preventing binding of the N-SH2 domain with a motif of the first PD-1 polypeptide comprising a tyrosine 248, e.g., a phosphorylated tyrosine 248, and is capable of disrupting an interaction between the second PD-1 polypeptide and the C-SH2 domain by preventing binding of the C-SH2 domain with a motif of the second PD-1 polypeptide comprising a tyrosine 248, e.g., a phosphorylated tyrosine 248.
  • the motif of the first PD-1 polypeptide comprising a tyrosine 248, e.g., a phosphorylated tyrosine 248, and the motif of the second PD-1 polypeptide comprising a tyrosine 248, e.g., a phosphorylated tyrosine 248, each comprises a portion of immunoreceptor tyrosine- based switch motif (ITSM).
  • ITSM immunoreceptor tyrosine- based switch motif
  • portion includes a full-length ITSM or a fraction thereof in which the fraction retains the ability to bind a SH2 domain.
  • the tyrosine 248 position is relative to the wild-type, human PD-1 polypeptide sequence: Accession Number: Q151 16; SEQ ID NO: 21.
  • the small molecule or peptide agent is capable of binding to a SH2 domain of SHP-2.
  • the small molecule or peptide agent is capable of binding to an amino terminal SH2 domain of SHP-2 (N-SH2; SEQ ID NO: 19) or to a carboxy terminal SH2 domain of SHP-2 (C-SH2; SEQ ID NO: 20).
  • the peptide agent is capable of binding to an amino terminal SH2 domain of SHP-2 (N-SH2; SEQ ID NO: 19) and to a carboxy terminal SH2 domain of SHP-2 (C-SH2; SEQ ID NO: 20).
  • the peptide agent comprises at least one motif of a PD-1 polypeptide comprising a tyrosine 248, e.g., a phosphorylated tyrosine 248.
  • the motif comprises a portion of an immunoreceptor tyrosine-based switch motif (ITSM).
  • the peptide agent comprises at least two motifs of a PD-1 polypeptide comprising a tyrosine 248, e.g., a phosphorylated tyrosine 248, wherein the at least two motifs are separated by a linker.
  • each of the at least two motifs of a PD-1 polypeptide comprising a tyrosine 248, e.g., a phosphorylated tyrosine 248, comprises a portion of an immunoreceptor tyrosine- based switch motif (ITSM).
  • ITSM immunoreceptor tyrosine- based switch motif
  • portion of an ITSM includes a full-length ITSM or a fraction thereof in which the fraction retains the ability to bind a SH2 domain.
  • the linker comprises at least 3 amino acids and/or the linker is at least about 35 Angstroms long.
  • the peptide agent e.g., that is capable of binding two SH2 domains, comprises: (a) a first amino acid sequence of SEQ ID NO: 1 , 22, or 23, optionally comprising a mutation of 1 , 2, 3, 4, or 5 amino acids, (b) a second amino acid sequence of SEQ ID NO: 1 , 22, or 23, optionally comprising a mutation of 1 , 2, 3, 4, or 5 amino acids, and (c) a linker comprising at least 3 amino acids between the first amino acid sequence and the second amino acid sequence, optionally, the linker separates the two amino acid sequences by at least about 35 Angstroms.
  • the peptide agent comprises the amino acid sequence of SEQ ID NO: 3, 9, 10, or 1 1 , optionally comprising a mutation of 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.
  • the mutation is not of the tyrosine 248, with reference to SEQ ID NO: 21.
  • the peptide agent comprises or consists of SEQ ID NO: 3, 9, 10, or 1 1.
  • the peptide agent comprises the amino acid sequence of SEQ ID NO: 3, 9, 10, 1 1 , or 24, optionally comprising a mutation of 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.
  • the mutation is not of the tyrosine 248, with reference to SEQ ID NO: 21.
  • the peptide agent comprises or consists of SEQ ID NO: 3, 9, 10, 1 1 , or 24.
  • the peptide agent which reduces PD-1 dimer activity is capable of binding to at least a first PD-1 polypeptide comprising a tyrosine 248, e.g., a phosphorylated tyrosine 248; in this embodiment, the peptide agent lacks a phosphatase domain or comprises a non-functional phosphatase domain.
  • the small molecule or peptide agent is capable of binding to the at least first PD-1 polypeptide at or near its immunoreceptor tyrosine-based switch motif (ITSM).
  • the peptide agent is capable of binding to the first PD-1 polypeptide and to an at least second PD-1 polypeptide.
  • the peptide agent is capable of binding to the first PD-1 polypeptide and to the at least second PD-1 polypeptide at or near each polypeptide's immunoreceptor tyrosine-based switch motif (ITSM).
  • the peptide agent comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 19 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 20.
  • the amino acid sequence that is at least 95% identical to SEQ ID NO: 19 comprises an arginine at its amino acid position 32, relative to SEQ ID NO: 19, or the amino acid sequence that is at least 95% identical to SEQ ID NO: 20 comprises an arginine at its amino acid position 28, relative to SEQ ID NO: 20.
  • the peptide agent comprises a first amino acid sequence that is at least 95% identical to SEQ ID NO: 19 and a second amino acid sequence that is at least 95% identical to SEQ ID NO: 20.
  • the amino acid sequence that is at least 95% identical to SEQ ID NO: 19 comprises an arginine at its amino acid position 32, relative to SEQ ID NO: 19
  • the amino acid sequence that is at least 95% identical to SEQ ID NO: 20 comprises an arginine at its amino acid position 28, relative to SEQ ID NO: 20.
  • the first amino acid sequence and the second amino acid sequence are separated by a linker, e.g., a linker comprising at least 3 amino acids and/or a linker that is at least about 35 Angstroms long.
  • a linker e.g., a linker comprising at least 3 amino acids and/or a linker that is at least about 35 Angstroms long.
  • SEQ ID NO: 18 has the amino acid sequence of:
  • SEQ ID NO: 19 has the amino acid sequence of:
  • SEQ ID NO: 20 has the amino acid sequence of:
  • SEQ ID NO: 21 has the amino acid sequence of:
  • the patient is undergoing treatment with an immune checkpoint immunotherapy selected from an agent that modulates one or more PD-1 , programmed death-ligand 1 (PD-L1 ), or programmed death-ligand 2 (PD-L2).
  • an immune checkpoint immunotherapy selected from an agent that modulates one or more PD-1 , programmed death-ligand 1 (PD-L1 ), or programmed death-ligand 2 (PD-L2).
  • the method further comprises administering an agent that modulates one or more of PD-1 , PD-L1 , or PD-L2.
  • the administering of the agent that modulates one or more of PD-1 , PD-L1 , or PD-L2 and the administering of the agent which decreases SPIP-2-mediated PD-1 dimerization and/or reduces PD-1 dimer activity is sequential or simultaneous.
  • the agent that modulates PD-1 is an antibody or antibody format specific for PD-1.
  • the antibody or antibody format specific for PD-1 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific I
  • the antibody or antibody format specific for PD-1 is selected from nivolumab, pembrolizumab, and pidilizumab.
  • the agent that modulates PD-L1 is an antibody or antibody format specific for PD-L1.
  • the antibody or antibody format specific for PD-L1 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.
  • the antibody or antibody format specific for PD-L1 is selected from atezolizumab, avelumab, durvalumab, and BMS-936559.
  • the agent that modulates PD-L2 is an antibody or antibody format specific for PD-L2.
  • the antibody or antibody format specific for PD-L2 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.
  • the administering of the agent which decreases SFIP-2-mediated PD-1 dimerization and/or reduces PD-1 dimer activity is by intratumoral, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or direct injection into cancer tissue.
  • the administering of the agent that modulates one or more of PD-1 , PD-L1 , or PD-L2 is by intratumoral, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or direct injection into cancer tissue.
  • the patient is predicted to be poorly responsive or non-responsive to the immune checkpoint immunotherapy or has presented as poorly responsive or non-responsive to the immune checkpoint immunotherapy.
  • the method reduces and/or mitigates one or more side effects of the immune checkpoint immunotherapy.
  • the side effect is selected from decreased appetite, rashes, fatigue, pneumonia, pleural effusion, pneumonitis, pyrexia, nausea, dyspnea, cough, constipation, diarrhea, immune-mediated pneumonitis, colitis, hepatitis, endocrinopathies, hypophysitis, iridocyclitis, and nephritis.
  • the method reduces the dose of the immune checkpoint immunotherapy.
  • the method reduces number of administrations of the immune checkpoint immunotherapy.
  • the method increases a therapeutic window of the immune checkpoint immunotherapy.
  • the method elicits a potent immune response in less-immunogenic tumors.
  • the method converts a tumor with reduced inflammation ("cold tumor”) to a responsive, inflamed tumor ("hot tumor”).
  • the method makes the cancer responsive or more responsive to a combination therapy of the immune checkpoint immunotherapy and one or more chemotherapeutic agents and/or radiotherapy.
  • the chemotherapeutic agent is selected from one or more of daunorubicin, doxorubicin, epirubicin, idarubicin, adriamycin, vincristine, carmustine, cisplatin, 5-fluorouracil, tamoxifen, prodasone, sandostatine, mitomycin C, foscarnet, paclitaxel, docetaxel, gemcitabine, fludarabine, carboplatin, leucovorin, tamoxifen, goserelin, ketoconazole, leuprolide flutamide, vinblastine, vindesine, vinorelbine, camptothecin, topotecan, irinotecan hydrochloride, etoposide, mitoxantrone, teniposide, amsacrine, merbarone, piroxantrone hydrochloride, methotrexate, 6-mercaptopurine, 6-thioguanine,
  • the patient is predicted to be poorly responsive or non- responsive to the immune checkpoint immunotherapy based on expression of one or more of PD-1 , PD-L1 , or PD-L2, in the patient's biological specimen.
  • the patient is predicted to be poorly responsive or non-responsive to an agent that modulates one or more of PD-1 , PD-L1 , and PD-L2 based on low on expression of PD-1 , PD-L1 , and PD-L2 in a tumor specimen from the patient.
  • the patient is predicted to be poorly responsive or non- responsive to an agent that modulates one or more of PD-1 , PD-L1 , and PD-L2 based on a tumor proportion score (TPS) of less than about 49% for PD-L1 staining.
  • TPS tumor proportion score
  • the cancer is selected from one or more of basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung); melanoma; myeloma; neuroblastoma; oral cavity cancer (lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer;
  • NHL diffuse NHL
  • high grade immunoblastic NHL high grade lymphoblastic NHL
  • high grade small non-cleaved cell NHL bulky disease NHL
  • mantle cell lymphoma mantle cell lymphoma
  • AIDS-related lymphoma Waldenstrom's Macroglobulinemia
  • chronic lymphocytic leukemia CLL
  • acute lymphoblastic leukemia ALL
  • Hairy cell leukemia chronic myeloblastic leukemia
  • PTLD post-transplant lymphoproliferative disorder
  • abnormal vascular proliferation associated with phakomatoses, edema e.g., that associated with brain tumors
  • Meigs' syndrome abnormal vascular proliferation associated with phakomatoses, edema (e.g., that associated with brain tumors), and Meigs' syndrome.
  • the present invention provides a method for treating an autoimmune disease or disorder or for treating inflammation in a patient in need thereof, comprising: (a) selecting an agent which increases SHP-2-mediated PD-1 dimerization and/or increases PD-1 dimer activity; and (b) administering the agent to the patient.
  • the agent which increases SHP-2-mediated PD-1 dimerization and/or increases PD-1 dimer activity is a small molecule or peptide agent.
  • the small molecule or peptide agent is capable of stimulating an interaction between a PD-1 polypeptide and a SHP-2 polypeptide.
  • the small molecule or peptide agent is capable of stimulating an interaction between a first PD-1 polypeptide and an amino terminal SH2 domain of SHP-2 (N-SH2; SEQ ID NO: 19) and an interaction between a second PD-1 polypeptide and a carboxy terminal SH2 domain of SHP-2 (C-SH2; SEQ ID NO: 20).
  • the peptide agent is capable of binding to a first PD-1 polypeptide comprising a tyrosine 248, e.g., a phosphorylated tyrosine 248, and to an at least second PD-1 polypeptide comprising a tyrosine 248, e.g., a phosphorylated tyrosine 248.
  • the peptide agent is capable of binding to the first PD-1 polypeptide and to the at least second PD-1 polypeptide at or near each polypeptide's immunoreceptor tyrosine-based switch motif (ITSM).
  • ITSM immunoreceptor tyrosine-based switch motif
  • the peptide agent comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 19 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 20.
  • the peptide agent comprises a first amino acid sequence that is at least 95% identical to SEQ ID NO: 19 and a second amino acid sequence that is at least 95% identical to SEQ ID NO: 20.
  • the amino acid sequence that is at least 95% identical to SEQ ID NO: 19 comprises an arginine at its amino acid position 32, relative to SEQ ID NO: 19, and the amino acid sequence that is at least 95% identical to SEQ ID NO: 20 comprises an arginine at its amino acid position 28, relative to SEQ ID NO: 20.
  • the first amino acid sequence and the second amino acid sequence are separated by a linker, e.g., a linker comprising at least 3 amino acids and/or a linker that is at least about 35 Angstroms long.
  • the peptide agent comprises: (a) a first amino acid sequence of SEQ ID NO: 19 or 20, optionally comprising a mutation of 1 , 2, 3, 4, or 5 amino acids, (b) a second amino acid sequence of SEQ ID NO: 19 or 20, optionally comprising a mutation of 1 , 2, 3, 4, or 5 amino acids, and (c) a linker comprising at least 3 amino acids I
  • the linker separates the two amino acid sequences by at least about 35 Angstroms.
  • the peptide agent further comprises a functional phosphatase domain.
  • the functional phosphatase domain comprises a protein tyrosine phosphatase (PTP) domain.
  • PTP protein tyrosine phosphatase
  • a phosphatase domain is any portion, e.g., a catalytic domain, of a phosphatase enzyme that is capable of removing a phosphate group from a phosphorylated amino acid, e.g., a phosphorylated tyrosine.
  • the peptide agent comprises the amino acid sequence of SEQ ID NO: 18. In embodiments, the peptide agent does not comprise of the amino acid sequence of SEQ ID NO: 18.
  • the autoimmune disease or disorder is selected from multiple sclerosis, diabetes mellitus, lupus, celiac disease, Crohn's disease, ulcerative colitis, Guillain-Barre syndrome, scleroderms, Goodpasture's syndrome, Wegener's granulomatosis, autoimmune epilepsy, Rasmussen's encephalitis, Primary biliary sclerosis, Sclerosing cholangitis, Autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis, Fibromyalgia, Menier's syndrome; transplantation rejection (e.g., prevention of allograft rejection) pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome, and
  • the inflammation is acute inflammation, chronic inflammation, respiratory disease, atherosclerosis, restenosis, asthma, allergic rhinitis, atopic dermatitis, septic shock, rheumatoid arthritis, inflammatory bowel disease, inflammatory pelvic disease, pain, ocular inflammatory disease, celiac disease, Leigh Syndrome, Glycerol Kinase Deficiency, Familial eosinophilia (FE), autosomal recessive spastic ataxia, laryngeal inflammatory disease; Tuberculosis, Chronic cholecystitis, Bronchiectasis, Silicosis and other pneumoconioses.
  • FE Familial eosinophilia
  • the patient is undergoing treatment or has undergone treatment with an immunosuppressive agent.
  • the method further comprises administering an immunosuppressive agent.
  • the administering of the agent which increases SHP-2-mediated PD-1 dimerization and/or increases PD-1 dimer activity and the administering of the immunosuppressive agent is sequential or simultaneous.
  • the immunosuppressive agent is a steroidal anti-inflammatory agent or a non-steroidal anti inflammatory agent (NSAID), selected from salicylic acid, acetyl salicylic acid, methyl salicylate, glycol salicylate, salicylmides, benzyl-2, 5-diacetoxybenzoic acid, ibuprofen, fulindac, naproxen, ketoprofen, etofenamate, phenylbutazone, and indomethacin.
  • NSAID non-steroidal anti inflammatory agent
  • the immunosuppressive agent is a steroid, such as a corticosteroids selected from hydroxyltriamcinolone, alpha-methyl dexamethasone, beta-methyl betamethasone, beclomethasone dipropionate, betamethasone benzoate, betamethasone dipropionate, betamethasone valerate, I
  • a corticosteroids selected from hydroxyltriamcinolone, alpha-methyl dexamethasone, beta-methyl betamethasone, beclomethasone dipropionate, betamethasone benzoate, betamethasone dipropionate, betamethasone valerate, I
  • clobetasol valerate desonide, desoxymethasone, dexamethasone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, fluclorolone acetonide, flumethasone pivalate, fluosinolone acetonide, fluocinonide, flucortine butylester, fluocortolone, fluprednidene (fluprednylidene) acetate, flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, fluradrenolone acetonide, medrysone, amcinafel, amcinafide, betamethasone and the balance of its esters, chloropred
  • the immunosuppressive agent is a cytostatic such as alkylating agents, anti metabolites (e.g., azathioprine, methotrexate), cytotoxic antibiotics, antibodies (e.g., basiliximab, daclizumab, and muromonab), anti-immunophilins (e.g., cyclosporine, tacrolimus, sirolimus), inteferons, opioids, TNF binding proteins, mycophenolates, and small biological agents (e.g., fingolimod, myriocin).
  • cytostatic such as alkylating agents, anti metabolites (e.g., azathioprine, methotrexate), cytotoxic antibiotics, antibodies (e.g., basiliximab, daclizumab, and muromonab), anti-immunophilins (e.g., cyclosporine, tacrolimus, sirolimus), inteferons, opioids,
  • the present invention provides a method of making an agent effective for the treatment of a cancer, comprising: (a) identifying the agent by screening for a disruption or decrease of SHP-2-mediated programmed cell death protein-1 (PD-1 ) dimerization or of PD-1 dimer activity; and (b) formulating the agent for administration to a patient having a cancer.
  • PD-1 programmed cell death protein-1
  • the agent is a small molecule or peptide agent.
  • the small molecule or peptide agent disrupts or decreases an interaction between a PD-1 polypeptide and a SHP-2 polypeptide.
  • the small molecule or peptide agent disrupts or decreases an interaction between a PD-1 polypeptide and a SH2 domain of SHP-2.
  • the small molecule or peptide agent disrupts or decreases an interaction between a PD-1 polypeptide and a SH2 domain of SHP-2 which prevents their interaction at the immunoreceptor tyrosine-based switch motif (ITSM) of the PD-1 polypeptide.
  • ITSM immunoreceptor tyrosine-based switch motif
  • the small molecule or peptide agent disrupts or decreases an interaction between a PD-1 polypeptide and a SH2 domain of SHP-2 which prevents their interaction at tyrosine 248 of PD-1.
  • the small molecule or peptide agent disrupts or decreases an interaction between a PD-1 polypeptide and an amino terminal SH2 domain of SHP-2 (N-SH2; SEQ ID NO: 19), e.g., at the arginine at amino acid position 32 of SEQ ID NO: 19, or disrupts or decreases an interaction between the PD-1 polypeptide and a carboxy terminal SH2 domain of SHP-2 (C-SH2; SEQ ID NO: 20), e.g., at the arginine at amino acid position 28 of SEQ ID NO: 20.
  • the small molecule or peptide agent disrupts or decreases an interaction between a first PD-1 polypeptide and an amino terminal SH2 domain of SHP-2 (N-SH2; SEQ ID NO: 19), e.g., at the arginine at amino acid position 32 of SEQ ID NO: 19, and disrupts or decreases an interaction between a second PD-1 polypeptide and an carboxy terminal SH2 domain of SHP-2 (C-SH2; SEQ ID NO: 20), e.g., at the arginine at amino acid position 28 of SEQ ID NO: 20.
  • N-SH2 amino terminal SH2 domain of SHP-2
  • C-SH2 carboxy terminal SH2 domain of SHP-2
  • the peptide agent that disrupts or decreases PD-1 dimer activity is capable of binding to at least a first PD-1 polypeptide comprising a tyrosine 248, e.g., a phosphorylated tyrosine 248; in this embodiment, I
  • the peptide agent lacks a phosphatase domain or comprises a non-functional phosphatase domain.
  • the peptide agent is capable of binding to the at least first PD-1 polypeptide at or near its immunoreceptor tyrosine- based switch motif (ITSM).
  • the peptide agent is capable of binding to the first PD-1 polypeptide and to an at least second PD-1 polypeptide.
  • the peptide agent is capable of binding to the first PD-1 polypeptide and to the at least second PD-1 polypeptide at or near each polypeptide's immunoreceptor tyrosine-based switch motif (ITSM)
  • the peptide agent comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 19 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 20.
  • the amino acid sequence that is at least 95% identical to SEQ ID NO: 19 comprises an arginine at its amino acid position 32, relative to SEQ ID NO: 19, or the amino acid sequence that is at least 95% identical to SEQ ID NO: 20 comprises an arginine at its amino acid position 28, relative to SEQ ID NO: 20.
  • the peptide agent comprises a first amino acid sequence that is at least 95% identical to SEQ ID NO: 19 and a second amino acid sequence that is at least 95% identical to SEQ ID NO: 20.
  • the amino acid sequence that is at least 95% identical to SEQ ID NO: 19 comprises an arginine at its amino acid position 32, relative to SEQ ID NO: 19, and the amino acid sequence that is at least 95% identical to SEQ ID NO: 20 comprises an arginine at its amino acid position 28, relative to SEQ ID NO: 20.
  • the first amino acid sequence and the second amino acid sequence are separated by a linker, e.g., a linker comprising at least 3 amino acids and/or a linker that is at least about 35 Angstroms long.
  • the present invention provides a method of making an agent effective for the treatment of an autoimmune disease or disorder of for the treatment of inflammation, comprising: (a) identifying the agent by screening for a stimulation or increase of SHP-2-mediated PD-1 dimerization and/or increases PD-1 dimer activity; and (b) formulating the agent for administration to a patient having a autoimmune disease or disorder or inflammation.
  • the agent which stimulates or increases SHP-2-mediated PD-1 dimerization and/or increases PD-1 dimer activity is a small molecule or peptide agent.
  • the small molecule or peptide agent is capable of stimulating an interaction between a PD-1 polypeptide and a SHP-2 polypeptide.
  • the small molecule or peptide agent is capable of stimulating an interaction between a first PD-1 polypeptide and an amino terminal SH2 domain of SHP-2 (N-SH2; SEQ ID NO: 19) and an interaction between a second PD-1 polypeptide and a carboxy terminal SH2 domain of SHP-2 (C-SH2; SEQ ID NO: 20).
  • the peptide agent is capable of binding to a first PD-1 polypeptide comprising a tyrosine 248, e.g., a phosphorylated tyrosine 248, and to an at least second PD- 1 polypeptide comprising a tyrosine 248, e.g., a phosphorylated tyrosine 248.
  • the peptide agent is capable of binding to the first PD-1 polypeptide and to the at least second PD-1 polypeptide at or near each polypeptide's immunoreceptor tyrosine-based switch motif (ITSM).
  • the peptide agent comprises an I
  • the peptide agent comprises a first amino acid sequence that is at least 95% identical to SEQ ID NO: 19 and a second amino acid sequence that is at least 95% identical to SEQ ID NO: 20.
  • the amino acid sequence that is at least 95% identical to SEQ ID NO: 19 comprises an arginine at its amino acid position 32, relative to SEQ ID NO: 19, and the amino acid sequence that is at least 95% identical to SEQ ID NO: 20 comprises an arginine at its amino acid position 28, relative to SEQ ID NO: 20.
  • the first amino acid sequence and the second amino acid sequence are separated by a linker, e.g., a linker comprising at least 3 amino acids and/or a linker that is at least about 35 Angstroms long.
  • the peptide agent comprises: (a) a first amino acid sequence of SEQ ID NO: 19 or 20, optionally comprising a mutation of 1 , 2, 3, 4, or 5 amino acids, (b) a second amino acid sequence of SEQ ID NO: 19 or 20, optionally comprising a mutation of 1 , 2, 3, 4, or 5 amino acids, and (c) a linker comprising at least 3 amino acids between the first amino acid sequence and the second amino acid sequence, optionally, the linker separates the two amino acid sequences by at least about 35 Angstroms.
  • the peptide agent further comprises a functional phosphatase domain.
  • the functional phosphatase domain comprises a protein tyrosine phosphatase (PTP) domain.
  • a phosphatase domain is any portion of a phosphatase enzyme that is capable of removing a phosphate groups from phosphorylated amino acid, e.g., a phosphorylated tyrosine.
  • the peptide agent comprises the amino acid sequence of SEQ ID NO: 18. In embodiments, the peptide agent does not comprise the amino acid sequence of SEQ ID NO: 18.
  • the present invention provides a method for predicting a cancer patient response to an immune checkpoint immunotherapy, comprising determining the presence of SHP-2-mediated PD-1 dimerization in a biological sample from the patient, wherein the presence of SHP-2-mediated PD-1 dimerization is indicative of an inhibitory immune signal and a likelihood of responding to the immune checkpoint immunotherapy.
  • the immune checkpoint immunotherapy is an agent that modulates one or more of PD-1 , PD-L1 , or PD-L2.
  • the agent that modulates PD-1 is an antibody or antibody format specific for PD-1.
  • the antibody or antibody format specific for PD-1 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.
  • the antibody or antibody format specific for PD-1 is selected from nivolumab, pembrolizumab, and pidilizumab.
  • the agent that modulates PD-L1 is an antibody or antibody format specific for PD-L1.
  • I is an antibody or antibody format specific for PD-L1.
  • the antibody or antibody format specific for PD-L1 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.
  • the antibody or antibody format specific for PD-L1 is selected from atezolizumab, avelumab, durvalumab, and B MS-936559.
  • the agent that modulates PD-L2 is an antibody or antibody format specific for PD-L2.
  • the antibody or antibody format specific for PD-L2 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.
  • the present invention provides method for treating cancer, comprising: (a) evaluating a subject's likelihood of response to an immune checkpoint immunotherapy, comprising evaluating a level of SFIP-2-mediated PD- 1 dimerization in a biological sample from the patient, wherein a presence or high level of SFIP-2-mediated PD-1 dimerization is indicative of a cancer that is suitable for immune checkpoint immunotherapy; and (b) administering an immune checkpoint immunotherapy to the patient.
  • the immune checkpoint immunotherapy is an agent that modulates one or more of PD-1 , PD-L1 , or PD-L2.
  • the agent that modulates PD-1 is an antibody or antibody format specific for PD-1.
  • the antibody or antibody format specific for PD-1 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.
  • the antibody or antibody format specific for PD-1 is selected from nivolumab, pembrolizumab, and pidilizumab.
  • the agent that modulates PD-L1 is an antibody or antibody format specific for PD-L1.
  • the antibody or antibody format specific for PD-L1 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.
  • the antibody or antibody format specific for PD-L1 is selected from atezolizumab, avelumab, durvalumab, and BMS-936559.
  • the agent that modulates PD-L2 is an antibody or antibody format specific for PD-L2.
  • the antibody or antibody format specific for PD-L2 is selected from one or more of a monoclonal I
  • antibody polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.
  • FIG. 1A to FIG. 1 D show that phosphorylation of PD-1 immunoreceptor tyrosine-based switch motif (ITSM) Y248 by TCR proximal Src family kinases Fyn and Lck but not ZAP-70 is required for interaction with SHP-2.
  • FIG. 1 A Jurkat- PD1 cells were left unstimulated or stimulated with aCD3/CD28/lgG or aCD3/CD28/PDL1 -lg beads for the indicated times, lysates were prepared followed by immunoprecipitation with anti-PD-1 antibody, SDS PAGE and western blot with antibodies for SHP-2 and PD-1. SHP-2 expression in whole cell lysates was examined (bottom panel).
  • FIG. 1 A shows that phosphorylation of PD-1 immunoreceptor tyrosine-based switch motif (ITSM) Y248 by TCR proximal Src family kinases Fyn and Lck but not ZAP-70 is required for
  • J-PD1 Jurkat-PD-1
  • J-PD1 -Y248 Jurkat-PD-1 -Y248F cells were either left unstimulated or stimulated with aCD3/CD28/lgG or aCD3/CD28/PDL1 -lg beads for 5 minutes, cell lysates were prepared followed by immunoprecipitation with anti-PD-1 antibody, SDS-PAGE and western blot with antibodies specific for pPD-1 -Y248, SHP-2 and PD-1.
  • FIG. 1 D left panel, COS cells were co-transfected with SHP-2, Fyn kinase active and either PD-1 wild type, PD-1 Y223F, PD-1 Y248F or PD-1 Y223F/Y248F.
  • PD-1 immunoprecipitation was performed in cell lysates followed by SDS-PAGE and immunoblot with the indicated antibodies.
  • FIG. 1 D Right panel shows expression of the same proteins in the same whole cell lysates was assessed. Results are representative of five independent experiments.
  • FIG. 2A to FIG. 2H shows that interaction of both SH2 domains of SHP-2 is required for SHP-2 binding to PD-1.
  • FIG. 2A is a schematic representation of the GST-SHP-2 fusion proteins: SHP-2 full-length (GST-SHP-2-FL); GST-SHP-2- PTP, which contains only the protein tyrosine phosphatase (PTP) domain, GST-SHP-2- DNSH2, which lacks the N- terminal SH2 domain; GST-SHP-2-N-SH2 and GST-SHP-2-C-SH2, which contain only the N-SH2 and the C-SH2 domain, respectively.
  • GST-SHP-2-N-SH2 and GST-SHP-2-C-SH2 which contain only the N-SH2 and the C-SH2 domain, respectively.
  • Jurkat-PD-1 cells were either left unstimulated or were stimulated with aCD3/CD28/lgG or aCD3/CD28/PDL1-lg beads for 5 minutes at 37°C followed by lysate preparation. Pull down assays were performed with the indicated GST-SHP-2 fusion proteins followed by SDS-PAGE and PD-1 immunoblot. Pull down with GST alone served as negative control.
  • FIG. 2C COS cells were transfected with kinase active Fyn, PD- 1 WT and either SHP-2-WT, SHP-2-R32A or SHP-2-R138A mutants FLAG-tagged (top two panels); Fyn, PD-1 -Y223F I
  • SHP-2 tandem-SFI2 SHP-2 amino acids 1 -225, which only contains the SHP-2 N-SH2 and C-SH2 domains in their natural tandem sequence (referred to as SHP-2 tandem-SFI2), was used.
  • FIG. 2E is a model of PD-1 : SHP-2 interaction after PD-1 phosphorylation.
  • FIG. 2F is a gel showing binding of ITIM-pY223 or ITSM-pY248 phosphopeptide to 1 SHP-2 SH2 domains by native PAGE binding assay.
  • FIG. 2G is a model showing that PD-1 ITSM-pY248 serves as the high affinity binding site for one of the SHP-2 SH2 domains thereby being a pre-requisite for the binding of the second SH2 domain on PD-1 ITI M Y223 that serves the low affinity interaction site.
  • FIG. 2G is a model showing that PD-1 ITSM-pY248 serves as the high affinity binding site for one of the SHP-2 SH2 domains thereby being a pre-requisite for the binding of the second SH2 domain on PD-1 ITI M Y223 that serves the low affinity interaction site.
  • 2H is a model showing that both SH2 domains interact with ITSM-pY248 and because the PD-1 molecule has only one ITSM-Y248, SHP-2 binds phosphorylated ITSM-pY248 residues in two PD-1 molecules using its N-SH2 domain for one PD-1 and its C-SH2 domain for a second PD-1 to form a PD-1 dimer.
  • FIG. 3A to FIG. 3F shows a surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) analysis of SHP-2 interaction with PD-1 ITSM-pY248.
  • SPR surface plasmon resonance
  • ITC isothermal titration calorimetry
  • FIG. 3B shows binding of the purified N-SH2 (left panel) and C-SH2 (right Panel), domains of SHP-2 on PD-1 phosphotyrosyl ITSM-Y248 peptide that was assessed in FIG. 3A. Results are representative of five experiments with full length SHP-2 proteins and three experiments with single SH2 domains.
  • FIG. 3C PD-1 ITSM-pY248 and PD-1 ITI M-pY223 phosphopeptides coated surface
  • FIG. 3D PD-1 ITIM-pY223-coated surface
  • FIG. 3E shows ITC of the PD-1 cyto-plTI M-plTSM: t-SHP-2 interaction, which occurred at 1 : 1 stoichiometry.
  • FIG. 3F shows ITC of the PD-1 cyto-ITIM-plTSM: t-SHP-2 interaction, which occurred at 2: 1 stoichiometry.
  • FIG. 4A to FIG. 4G shows that SHP-2 bridges two PD-1 polypeptides via PD-1 ITSM pY248.
  • FIG. 4A shows that I
  • NanoBiT proximity assay allows detection of protein: protein interaction in living cells by employing a split luciferase enzyme.
  • PD-1 -LgBiT+PD-1 -SmBiT would induce luciferase activity only if two PD-1 polypeptides stably interact thereby forming an active luciferase enzyme.
  • FIG. 4B shows HEK-293 cells co-transfected with PD-1-SmBiT and PD-1 -LgBiT together with kinase active (+) or inactive (-) Fyn and either SHP-2 WT, control empty vector or the mutants SHP-2- R32A, SHP-2-R138A or double mutant SHP-2-R32A/R138A (DM).
  • DM mutants
  • 4C shows HEK-293 cells that were co-transfected with PD-1-SmBiT and PD-1-LgBiT carrying either PD-1 WT or PD-1 -Y248F, together with SHP-2 WT and either kinase active (+) of kinase inactive (-) Fyn.
  • Complex formation between PD-1 -SmBiT and PD-1 -LgBiT was assessed by luciferase assay.
  • 4D shows HEK- 293 cells that were co-transfected with PD-1 -SmBiT and PD-1 -LgBiT together with SHP-2 WT and either kinase active (+) of kinase inactive (-) Fyn.
  • hPD-L1 -lg (dimer), hPD-L1 monomer or IgG were added in the culture and complex formation between PD-1-SmBiT and PD-1 -LgBiT was assessed by luciferase assay.
  • FIG. 4E shows that when vehicle control (0) or increasing amounts of the allosteric SHP-2 inhibitor SHP099 were added in the culture and complex formation between PD-1 -SmBiT and PD-1 -LgBiT was assessed by luciferase assay.
  • H baseline luciferase activity obtained when cells are transfected with PD-1-SmBiT and PD-1-LgBiT, vector and kinase (-) Fyn).
  • 4G show, respectively, primary human T cells and Jurkat T cells transiently co-transfected with PD-1 NanoBiT constructs PD1 -LgBiT and PD 1 -SmBiT and either SHP-2-WT, SHP-2-R32A, SHP-2-R138A or the double mutant SHP-
  • FIG. 5A to FIG. 5G show that the interaction of SHP-2 SH2 domains with two PD-1 ITSM pY248 residues is required for activation of SHP-2 phosphatase activity.
  • FIG. 5B shows SHP-2-WT incubated with bpITSM phosphotyrosyl peptides containing a 4-, 2- or 1-Ahx spacer; plRSY727 was used as negative control. Phosphatase activity was assessed as in FIG. 5A and FIG. 5C. SHP-2-WT, SHP-2-R32A or SHP-2-R138A was incubated with the indicated concentrations of bpITSM phosphotyrosyl peptide containing a 4-Ahx spacer and phosphatase activity was assessed.
  • FIG. 5B shows SHP-2-WT incubated with bpITSM phosphotyrosyl peptides containing a 4-, 2- or 1-Ahx spacer; plRSY727 was used as negative control. Phosphatase activity was assessed as in FIG. 5A and FIG. 5C. SHP-2-WT, SHP-2-R32A or SHP-2-R138A was incubated with
  • 5D shows that when SHP-2-WT is incubated with 20 mM DiFMUP (6,8-difluoro-4-methylumbelliferyl phosphate) as substrate in the presence of the indicated phosphotyrosyl bpITSM peptide (0.01 mM) either alone or with increasing amounts of monophosphorylated pITIM or monophosphorylated I
  • FIG. 5E shows Jurkat T cells stably expressing PD-1 -WT (J-PD-1 ), PD-1-Y223F (J-PD-1 -Y223F) or PD-1-Y248F (J- PD-1 -Y248F) were co-cultured with Raji-control or Raji-PD-L1 cells loaded with SEE. Where indicated, anti-PD-1 blocking antibody or isotype control was added in the cultures. Culture supernatants were collected at 24 hours and IL-2 production was measured.
  • FIG. 5F is a model for PD-1 : PD-1 bridging by SHP-2 and activation of SHP-2 phosphatase activity:
  • An "inside-out” regulation of PD-1 PD-1 dimer complex formation is initiated by TCR-mediated activation of TCR proximal Src family kinases, such as Fyn or Lck, and requires binding of both SH2 domains of SHP-2 to PD-1 tyrosine phosphorylated on ITSM-pY248 in the cytoplasmic tail bringing together two PD-1 polypeptides.
  • Binding of SHP-2 brings together a PD-1 dimer and results in SHP-2 phosphatase activation proximal to the TCR and inhibition of activated T cell responses. Phosphatase activity was assessed in FIG. 5G, and bplTSM-Ahx10 (SEQ ID NO: 24) caused a higher increase in SHP-2 activation that was measurable only when using 3x lower enzyme concentrations than in all other phosphatase assays.
  • FIG. 6A to FIG. 6C shows the regulation of SHP-2 activation and unique structural properties of SHP-2 SH2 domains.
  • FIG. 6A shows that SHP-2 contains two tandem SH2 domains, N-terminal (N-SH2) and C-terminal SH2 (C-SH2), followed by a single protein tyrosine phosphatase (PTP) domain, and a C-terminal hydrophobic tail with two tyrosine phosphorylation sites.
  • FIG. 6B shows that exposure of cells to a variety of extracellular stimuli triggers the binding of SHP-2 via its SH2 domains to tyrosine phosphorylated receptors for growth factors as well as to tyrosine- phosphorylated docking proteins such as insulin receptor substrates (IRSs).
  • IFSs insulin receptor substrates
  • the N-SH2 domain of SHP-2 binds the phosphatase domain in an auto-inhibitory closed conformation and directly blocks its active phosphatase site. Interaction of the N-SH2 domain with phosphotyrosine peptide disrupts its interaction of N-SH2 with the phosphatase active site and activates the enzyme.
  • the C-SH2 domain contributes binding energy and specificity but does not have a direct role in enzymatic activation.
  • FIG. 6C shows the two SH2 domains of SHP-2 have a relatively fixed and a roughly antiparallel or roughly perpendicular orientation relative to one another, with the phosphopeptide- binding sites lying on the surface of the polypeptide and widely spaced. This relative fixed orientation of SH2 domains is stabilized by a disulphide bond and a small hydrophobic patch within the interphase that separates the phosphopeptide binding sites.
  • FIG. 7A to FIG. 7C show the generation of PD-1 -expressing J-PD-1 and J-PD-1 Y248F cells.
  • FIG. 7A shows Jurkat T cells transfected with human PD-1 cDNA expressed in pEF6 vector and stable PD-1 + cells were generated by antibiotic selection. Cell lines were subcloned and stable clones were generated. Experiments were performed in polyclonal cell lines and clones. Surface expression of PD-1 in subclone J-PD-1 is shown.
  • FIG. 7B shows Jurkat T cells transfected with human PD-1 cDNA in which tyrosine 248 was mutated to phenylalanine. Generation and selection of stable cell I
  • FIG. 7 A the surface expression of PD-1 in subclone J-PD-1 -Y248F is shown.
  • FIG. 8A shows a densitometric analysis of FIG. 1 A.
  • the abundance of SHP-2 normalized to that of immunoprecipitated PD-1 at each time point and expressed as the fold change over the levels obtained in unstimulated cells (defined as 1 ). Fold changes are compared between unstimulated and stimulated cells at each time point (*P ⁇ 0.05) or between cells stimulated with or without PD-1 ligation at each time point (*P ⁇ 0.05). Data are presented as the means ⁇ SEM, n 5 experiments.
  • FIG. 8B shows a densitometric analysis of FIG. 1B.
  • FIG. 8C shows a densitometric analysis of FIG. 1C.
  • FIG. 8D shows a densitometric analysis of FIG. 1D.
  • FIG. 9 shows Jurkat-PD 1 cells that were left unstimulated (0) or stimulated with beads coated with either IgG or PDL1 - Ig for the indicated times, lysates were prepared followed by immunoprecipitation with anti-PD-1 antibody, SDS-PAGE and western blot with antibodies for SHP-2 and PD-1. SHP-2 expression in whole cell lysates was also examined (bottom panel).
  • FIG. 10A to FIG. 10C show that Fyn is required for PD-1 (Y248) phosphorylation and interaction with SHP-2.
  • FIG. 10A shows T cells that were purified from spleens and lymph nodes of WT and Fyn-KO mice and were cultured for 72h with anti-CD3 and anti-CD28 mAb and expression of PD-1 was examined by flow cytometry.
  • FIG. 10B shows that after resting in RPMI and 2% FBS for 10 hours, the cells were either left unstimulated (0) or stimulated with aCD3/CD28/lgG- or aCD3/CD28/PDL1 -19-coated beads for 3 minutes.
  • FIG. 10C shows a densitometric analysis of the immunoprecipitation data shown in FIG. 10B.
  • the abundance of SHP-2 and pPD 1 (Y248) was normalized to that I
  • FIG. 11 A to FIG. 11C show that Lck is required for PD-1 (Y248) phosphorylation and interaction with SHP-2.
  • FIG. 11 A shows that the lck-deficient Jurkat T cell line was transfected with human PD-1 cDNA expressed in pEF6 vector and stable PD-1 + cells lines and clones were generated by antibiotic selection. Comparable expression levels of PD-1 in J-PD1 and JCaM1.6-PD1 stably transfected with human PD-1 was confirmed by flow cytometry. Comparable expression of CD3 and CD28 was also confirmed by flow cytometry (not shown). FIG.
  • FIG. 11 B shows J-PD1 and J-CaM1.6- PD1 cells that were left unstimulated (0) or stimulated with aCD3/CD28/lgG or aCD3/CD28/PDL1 -lg beads for the indicated times; lysates were prepared followed by immunoprecipitation with anti-PD-1 antibody. SDS-PAGE and western blot with antibodies for SHP-2, pPD-1 (Y248) and PD-1.
  • FIG. 11C shows a densitometric analysis of the data shown in FIG. 11 B. The abundance of SHP-2 and pPD-1 (Y248) was normalized to that of immunoprecipitated PD-1 at each time point and was expressed as fold change over the values obtained in unstimulated cells (defined as 1 ).
  • FIG. 12A shows a densitometric analysis of FIG. 2B.
  • Jurkat-PD-1 cells were either left unstimulated or were stimulated with aCD3/CD28/lgG or aCD3/CD28/PDL1 -lg beads for 5 minutes at 37°C followed by lysate preparation.
  • Pull down assays were performed with the indicated GST-SFIP-2 fusion proteins followed by SDS-PAGE and PD-1 immunoblot.
  • the abundance of PD-1 pulled down by SFIP-2-GST in each sample was normalized to that of total PD-1 and expressed as the fold change over the values obtained by GST-SFIP2-FL pull down using lysates from unstimulated cells (defined as 1 ).
  • FIG. 12B shows a densitometric analysis of FIG. 2C.
  • COS cells were transfected with kinase active Fyn, PD-1 WT and either SHP-2-WT, SHP-2-R32A or SHP-2-R138A mutants FLAG-tagged (top panel); Fyn, PD-1 -Y223F and either SHP-2- WT, SHP-2-R32A or SHP-2-R138A (middle panel); Fyn, PD-1 -Y248F and either SHP-2- WT, SHP- 2-R32A or SHP-2-R138A (bottom panel). Immunoprecipitation of cell lysates was performed with anti-PD-1 antibody followed by SDS-PAGE and immunoblot with FLAG- or PD-1 -specific antibodies.
  • FIG. 12C primary human T cells were activated for 72h with aCD3 (100 ng/ml) and aCD28 (300 ng/ml) mAbs. The cells were then transfected with the indicated FLAG-tagged SHP-2 constructs or empty vector as described, and cell lysates were prepared and PD-1 immunoprecipitation was performed followed by SDS-PAGE and immunoblot with FLAG- or PD-1 -specific antibody. Densitometric analysis of FIG. 12C is shown in FIG. 12D. The abundance of SHP-
  • FIG. 13A shows that PD-1 interacts with both SH2 domains of SHP-2 in primary human T cells.
  • Human PBMC were cultured with PHA for 72 hours and T cells were purified, rested overnight in RPM, with 2.5% FBS and subsequently were either left unstimulated or were stimulated with aCD3/CD28/1 gG or aCD3/CD28/PDL1 -lg beads for 5 minutes at 37°C followed by lysate preparation. Pull down assays were performed with the indicated GST-SHP-2 fusion proteins followed by SDS-PAGE and immunoblot with PD-1 -specific antibody. Pull down with GST alone served as negative control.
  • FIG. 13B shows a densitometric analysis of results shown in FIG. 13A.
  • the abundance of PD-1 pulled down by SHP-2-GST in each sample was normalized to that of total PD-1 and was expressed as fold change over the value obtained after pull down with GST-SHP2-FL using lysates from unstimulated cells (defined as 1 ).
  • Fold changes of aCD3/CD28/PDL1 -lg-stimulated samples in the pull down with each different GST-SHP-2 fusion protein are compared to the values of the corresponding unstimulated (*P ⁇ 0.05) or aCD3/aCD28/lg-stimulated sample (*P ⁇ 0.05).
  • FIG. 14A to FIG. 14D shows Surface plasmon resonance (SPR) Biacore analysis of SHP-2 interaction using phosphotyrosyl PD-1 plTSM-82 pY248 peptide and GST-SHP- 2 fusion proteins.
  • FIG. 14A shows Phosphotyrosyl PD- 1 pITSM peptide (KTPEPPVPCVPEQTE(pY)AYIVFP (SEQ ID NO: 1 ) immobilized on negatively charged CM5 Biacore chip of Biacore 3000 (GE Healthcare). Efficient immobilization of the phosphopeptide was confirmed by binding of phosphotyrosine specific 4G10 monoclonal antibody.
  • SPR Surface plasmon resonance
  • the GST fusion proteins GST-SHP-2-WT, GST- SHP-2-R32A, GST-SHP-2-R138A, GST-SHP-2-N-SH2 and GST-SHP-2-C-SH2 were generated, purified by affinity chromatography using glutathione-Sepharose beads and analyzed by SDS PAGE and Coomassie staining.
  • FIG. 14C and FIG. 14D the GST tag was removed by thrombin digestion. The cleaved untagged proteins were collected, concentrated, subjected to FPLC purification and subsequently applied to anion exchange column. The eluted fractions were subjected to SDS PAGE and Coomassie staining and immunoblotting.
  • FIG. 15A and FIG. 15B shows that SHP-2 mediates bridging of two PD-1 polypeptides in the presence of kinase active I
  • FIG. 15A HEK-293 cells were co transfected with PD-1 -SmBiT and PD-1 -LgBiT together with kinase inactive (-) Fyn and either SHP-2 WT, SHP-2-R32A, SHP-2-R138A or double mutant SHP-2-R32A/R138A (DM), or control empty vector.
  • SHP-2 WT SHP-2-R32A
  • SHP-2-R138A SHP-2-R138A
  • DM double mutant SHP-2-R32A/R138A
  • FIG. 16A shows HEK-293 cells, Jurkat T cells, or primary human T cells that were transfected with PD1 Lg and PD1 sm nanobit constructs and expression of PD-1 was assessed by flow cytometry.
  • FIG. 16B shows expression of PD-1 in primary human T cells before and after stimulation for 48 hours with anti-CD3 and anti-CD28 antibodies which was assessed in parallel. Results are representative of three independent experiments.
  • FIG. 17A and FIG. 17B shows the generation of Raji-PD-L1.
  • Raji cells were transfected with human PD-L1 cDNA and stable PD-L1 + cells were generated by antibiotic selection.
  • Cell lines were subcloned and stable clones were generated, percentage of cells and Raji-PD-L1 (FIG. 17B) and Raji-control (FIG. 17A).
  • FIG. 18 shows that bisphosphorylated PD-1 ITSM pY248 induces higher SHP-2 enzymatic activity compared to bisphosphorylated IRS1.
  • 1.6 pg/ml of purified SFIP-2-WT protein was incubated with 20 mM DiFMUP with or without the indicated phosphotyrosyl peptides at a concentration of 20 nM.
  • Monophosphoryl peptide plRSY727 was used as negative control.
  • SHP-2 phosphatase activity was monitored by a fluorescent assay using 6,8-difluoro-4- methylumbelliferone (DiFMUP) as substrate.
  • DiFMUP 6,8-difluoro-4- methylumbelliferone
  • FIG. 20A to FIG. 20C show dose-response curves for SHP-2 wild-type, N-SH2 domain, and C-SH2 domain binding to ITSM-pY248 determined by SPR. All SHP-2-related analytes were passed over the plTSM-pY248 ligand-immobilized I
  • the dissociation constant (k d ) was first determined for each analyte concentration using the BIAeval package and was then used to discriminate the association constant k a during the association phase. Calculated KD was then determined as k d / k a .
  • the Chi-square value, where smaller values equate to better fits, are listed first for k a , then for k d , and were: (FIG. 20A) for SHP-2-FL on ITSM-pY248, 0.06 and 0.06; (FIG. 20B) for N-SH2 on ITSM-pY248. 0.37 and 0.16; (FIG. 20C) for C-SH2 on ITSM-pY248.
  • the presents disclosure relates, in part, to exploiting the geometry of the PD-1 : SHP-2 interaction, which leads to SHP-
  • SHP-2 bridges a first phosphorylated immunoreceptor tyrosine-based switch motif (ITSM)-Y248 residue one one PD-1 polypeptide and a second phosphorylated ITSM-Y248 residue on a second PD-1 polypeptide via its amino terminal (N-SH2) and carboxy terminal (C-SH2) domains; thereby, forming a dimer of the two PD-1 polypeptides (FIG. 2E).
  • IMS immunoreceptor tyrosine-based switch motif
  • N-SH2 amino terminal
  • C-SH2 carboxy terminal
  • the method provides an agent that inhibits PD-1 : SHP-2 interaction and PD-1-mediated activation of SHP-2.
  • agents compete for PD-1 : SHP-2 interaction.
  • agents include a small molecule, for example, a peptide or peptide mimetic.
  • the peptide is a biphosphorylated ITSM.
  • the ITSM induces robust activation of SHP-2.
  • the ITSM is a cell-permeable peptide format to compete and inhibit PD-1 : SHP-2 interaction, and, thus, block PD-1 -mediated inhibitory effects.
  • the method provides an agent that inhibits PD-1 dimerization, which prevents the formation of PD1 -homodimer that is formed by bridging together two PD-1 polypeptides by SHP-2.
  • disruption of PD-1 homodimerization prevents the interaction with SHP-2 and PD-1 mediated inhibition of T cell responses.
  • such compositions are monomeric recombinant PD-L1 or PD-L2 polypeptide.
  • the monomeric recombinant PD-L1 or PD-L2 polypeptide bind on one but not on two PD-1 polypeptides simultaneously thereby preventing PD-1 dimerization, which is required for activation of SHP-2.
  • the method provides an agent that promotes PD-1 dimerization.
  • the PD-1 dimerization promotes the formation of PD-1 homodimer and the interaction of SHP-2 with the phosphorylated ITSM-Y248 residues on the PD-1 homodimer.
  • Immune checkpoints regulate T cell responses to maintain self-tolerance. They deliver costimulatory and coinhibitory signals to T cells.
  • PD-L1 mainly expressed by antigen presenting cells engages its receptor PD-1 on T cells, to provide a growth inhibitory signal.
  • Different tumors express high PD-L1 to evade immune recognition.
  • inhibition of PD-1/PD-L1 and other IC polypeptides have become important targets in cancer immunotherapy.
  • PD-1 is a cell-surface receptor that is a member of the CD28 family of T-cell regulators, within the immunoglobulin superfamily of receptors.
  • the human PD-1 gene is located at chromosome 2q37, and the full-length PD-1 cDNA encodes a protein with 288 amino acid residues with 60% homology to murine PD-1. It is present on CD4- CD8- (double negative) thymocytes during thymic development and is expressed upon activation in mature hematopoietic cells such as T and B cells, NKT cells and monocytes after prolonged antigen exposure.
  • the cytoplasmic tail of PD-1 has one ITIM (V/L/l/XpYXX/L/V), which contains tyrosine 223 (Y223 ⁇ ) residue, and one ITSM (TXpYXXV/l), which contains I
  • a tyrosine 248 (Y248) residue a tyrosine 248 (Y248) residue.
  • the Y223 and Y248 positions are relative to the wild-type, human PD-1 polypeptide sequence: Accession Number: Q151 16; SEQ ID NO: 21. Mutational studies have shown that PD-1 -mediated inhibition relies on the interaction of the ITSM with SHP-2 but the mechanism by which PD-1 induces SHP-2 activation remained unclear. As disclosed herein, phosphorylation of PD-1 by TCR-proximal Src family kinases is required for interaction with SHP-2, which binds to the PD-1 ITSM-Y248 residue.
  • SHP-2 This interaction is mediated by both the amino terminal SH2 (N-SH2) domain and carboxy terminal SH2 (C-SH2) domain of SHP-2, which together bridge two PD-1 polypeptides by binding on each PD-1 polypeptide's ITSM-Y248 residue.
  • SHP-2 bridges two PD-1 polypeptides by PD-1 dimerization in live cells by NanoBiT proximity assays; these data showed that upon PD-1 phosphorylation, PD-1 : PD1 interaction occurs only in the presence of SHP-2 expressing intact N-SH2 domain (SEQ ID NO: 19) and C- SH2 domain (SEQ ID NO: 20). Binding of the SH2 domains by two PD-1 polypeptides is required for activation of SHP-
  • a monomeric PD-1 ITSM-pY248 phosphopeptide does not induce catalytic activation of SHP-2 whereas a phosphopeptide generated by covalently joining two PD-1 ITSM-pY248 peptides with a linker - that meets the spacing requirements between the binding sites of the two SI-IP-2 SH2 domains - induced robust activation of SHP-2.
  • cultures of T cells with a recombinant dimeric PD-L1 but not a monomeric PD-L1 , enhanced PD-1 : PD-1 bridging and inhibited proliferation and IFN-y production.
  • the evidence disclosed herein reveal the mechanisms by which PD-1 : SHP-2 interaction leads to SHP-2 activation. They also have implications for the design and development of PD-1 -binding compounds to selectively suppress T cell responses by dimerizing PD-1 or to prevent the PD-1 inhibitory signal by disrupting PD-1 homodimerization.
  • PD-L1 has also been shown to bind to B7-1 (CD80), an interaction that also suppresses T-cell proliferation and cytokine production. Cancer cells drive high expression levels of PD-L1 on their surface, allowing activation of the inhibitory PD- 1 receptor on any T cells that infiltrate the tumor microenvironment, effectively switching those cells off. Indeed, upregulation of PD-L1 expression levels has been demonstrated in many different cancer types, and high levels of PD- L1 expression have been linked to poor clinical outcomes.
  • the subject is undergoing treatment with an immune checkpoint immunotherapy selected from an agent that modulates PD-L1. In some embodiments, the subject is undergoing treatment with an immune checkpoint immunotherapy selected from an agent that modulates PD-L2.
  • the immune-modulating agent targets one or more immune checkpoint genes including, for example, PD-1 , PD-L1 , and PD-L2.
  • the immune-modulating agent is a PD-1 inhibitor.
  • the immune-modulating agent is an antibody or antigen binding fragment thereof, specific for one or more of PD-1 , PD-L1 , and PD-L2.
  • the immune-modulating agent is an antibody or antigen binding fragment thereof such as, by way of non-limitation, nivolumab, (ONO-4538/B MS-936558, MDX1 106, OPDIVO, BRISTOL MYERS SQUI BB), pembrolizumab (KEYTRUDA, MERCK), pidilizumab (CT-011 , CURE TECH), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), MPDL3280A (ROCHE).
  • the immune-modulating agent targets one or more of CD 137 or CD137L.
  • the immune-modulating agent is an antibody or antigen binding fragment thereof specific for one or more of CD 137 or CD137L.
  • the immune-modulating agent is an antibody or antigen binding fragment thereof such as, by way of non-limitation, urelumab (also known as BMS-663513 and anti-4-1 BB antibody).
  • urelumab also known as BMS-663513 and anti-4-1 BB antibody.
  • the present chimeric protein is combined with urelumab (optionally with one or more of nivolumab, lirilumab, and urelumab) for the treatment of solid tumors and/or B-cell non-Hodgkin's lymphoma and/or head and neck cancer and/or multiple myeloma.
  • compositions and methods described herein are useful for treating autoimmune diseases, alloimmune responses, or any other disease, disorder or condition that involves a T cell response in a patient in need thereof.
  • these are conditions in which the immune system of an individual (e.g., activated T cells) attacks the individual's own tissues and cells, or implanted tissues, cells, or molecules (as in a graft or transplant).
  • diseases and disorders include, e.g., autoimmune disease or disorder (e.g., IBD and rheumatoid arthritis), transplant rejection, graft-versus-host disease (GVHD), inflammation, asthma, allergies, and chronic infection.
  • the present invention provides a method for treating an autoimmune disease or disorder or for treating inflammation in a patient in need thereof, comprising: (a) selecting an agent which increases SHP-2-mediated PD-1 dimerization and/or increases PD-1 dimer activity; and (b) administering the agent to the patient.
  • the present invention provides a method of making an agent effective for the treatment of an autoimmune disease or disorder of for the treatment of inflammation, comprising: (a) identifying the agent by screening for a stimulation or increase of SHP-2-mediated PD-1 dimerization and/or increases PD-1 dimer activity; and (b) formulating the agent for administration to a patient having a autoimmune disease or disorder or inflammation.
  • compounds that promote PD-1 homodimerization will promote the inhibitory function of PD-1 and will be useful for the treatment of autoimmune diseases, inflammation and for prevention of graft rejection after organ or hematopoietic stem cell transplantation.
  • the agent which increases SHP-2-mediated PD-1 dimerization is a small molecule or peptide agent.
  • the small molecule or peptide agent stimulates an interaction between PD-1 and SHP- I
  • the small molecule or peptide agent stimulates an interaction between PD-1 and one or two of the SH2 domains of SHP-2.
  • the small molecule or peptide agent stimulates an interaction between PD-1 and one or two of the SH2 domains of SHP-2 mediated by interactions at the immunoreceptor tyrosine-based switch motif (ITSM) of PD- 1. In some embodiments, the small molecule or peptide agent stimulates an interaction between PD-1 and one or two of the SH2 domains of SHP-2 mediated by interactions at tyrosine 248 of PD-1.
  • ITSM immunoreceptor tyrosine-based switch motif
  • the small molecule or peptide agent stimulates an interaction between PD-1 and one or two of the SH2 domains of SHP-2 mediated by interactions at arginine 32 (located in the N terminal SH2 domain of SHP-2) and/or arginine 138 (located in the C terminal SH2 domain of SHP-2).
  • the small molecule or peptide agent stimulates an interaction between PD-1 and two SH2 domains of SHP-2 mediated by interactions at (a) arginine 32 of the N terminal SH2 domain of SHP-2 and tyrosine 248 of PD-1 and (b) arginine 138 of the C terminal SH2 domain of SHP-2 and tyrosine 248 of PD-1.
  • the arginine 32 and arginine 138 positions of the wild-type, human SHP-2 polypeptide sequence correspond to the arginine at amino acid position 32 in SEQ ID NO: 19 and the arginine at amino acid position 28 in SEQ ID NO: 20, respectively.
  • the peptide agent has two domains which interact with tyrosine 248 of PD-1 and are separated by at least about 35 Angstroms. In some embodiments, the peptide agent two SH2 domains of SHP-2 which interact with tyrosine 248 of PD-1 and are separated by at least about 35 Angstroms.
  • the peptide agent has two domains which interact with tyrosine 248 of PD-1 and are separated by at least about 35 Angstroms. In some embodiments, the peptide agent two SH2 domains of SHP-2 which interact with tyrosine 248 of PD-1 and are separated by at least about 35 Angstroms.
  • the autoimmune disease or disorder is selected from multiple sclerosis, L r diabetes mellitus, lupus, celiac disease, Crohn's disease, ulcerative colitis, Guillain-Barre syndrome, scleroderms, Goodpasture's syndrome, Wegener's granulomatosis, autoimmune epilepsy, Rasmussen's encephalitis, Primary biliary sclerosis, Sclerosing cholangitis, Autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis, Fibromyalgia, Menier's syndrome; transplantation rejection (e.g., prevention of allograft rejection) pernicious anemia, rheumatoid arthritis, systemic lupus erythematosus, dermatomyositis, Sjogren's syndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis, Reiter's syndrome
  • exemplary autoimmune diseases that can be treated with the methods of the present disclosure include, e.g., type I diabetes, multiple sclerosis, thyroiditis (such as Hashimoto's thyroiditis and Ord's thyroiditis), systemic lupus erythematosus, scleroderma, psoriasis, arthritis, rheumatoid arthritis, alopecia greata, ankylosing spondylitis, I
  • autoimmune hemolytic anemia autoimmune hepatitis, Behcet's disease, Crohn's disease, dermatomyositis, glomerulonephritis, Guillain-Barre syndrome, IBD, lupus nephritis, myasthenia gravis, myocarditis, pemphigus/pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, rheumatic fever, sarcoidosis, Sjogren's syndrome, ulcerative colitis, uveitis, vitiligo, and Wegener's granulomatosis.
  • the inflammation is acute inflammation, chronic inflammation, respiratory disease, atherosclerosis, restenosis, asthma, allergic rhinitis, atopic dermatitis, septic shock, rheumatoid arthritis, inflammatory bowel disease, inflammatory pelvic disease, pain, ocular inflammatory disease, celiac disease, Leigh Syndrome, Glycerol Kinase Deficiency, Familial eosinophilia (FE), autosomal recessive spastic ataxia, laryngeal inflammatory disease; Tuberculosis, Chronic cholecystitis, Bronchiectasis, Silicosis and other pneumoconioses.
  • FE Familial eosinophilia
  • compositions disclosed herein can be administered as an "induction therapy” in preparation for a solid organ or stem cell transplant, or as "maintenance therapy” in solid organ or stem cell transplant recipients, and can also be administered to a solid organ or stem cell transplant recipient in order to facilitate early withdrawal of maintenance immunosuppressive therapy.
  • the patient is undergoing treatment with an immunosuppressive agent.
  • the method further comprises administering an immunosuppressive agent.
  • the administering of the agent which increases SFIP-2-mediated PD-1 dimerization and/or increases PD-1 dimer activity and the administering of the immunosuppressive agent is sequential or simultaneous.
  • the immunosuppressive agent is a steroidal anti-inflammatory agent or a non-steroidal anti inflammatory agent (NSAID), selected from salicylic acid, acetyl salicylic acid, methyl salicylate, glycol salicylate, salicylmides, benzyl-2, 5-diacetoxybenzoic acid, ibuprofen, fulindac, naproxen, ketoprofen, etofenamate, phenylbutazone, and indomethacin.
  • NSAID non-steroidal anti inflammatory agent
  • the immunosuppressive agent is a steroid, such as a corticosteroids selected from hydroxyltriamcinolone, alpha-methyl dexamethasone, beta-methyl betamethasone, beclomethasone dipropionate, betamethasone benzoate, betamethasone dipropionate, betamethasone valerate, clobetasol valerate, desonide, desoxymethasone, dexamethasone, diflorasone diacetate, diflucortolone valerate, fluadrenolone, fluclorolone acetonide, flumethasone pivalate, fluosinolone acetonide, fluocinonide, flucortine butylester, fluocortolone, fluprednidene (fluprednylidene) acetate, flurandrenolone, halcinonide, hydrocortisone acetate, hydrocortisone butyrate, methyl
  • the immunosuppressive agent is a cytostatics such as alkylating agents, anti metabolites (e.g., azathioprine, methotrexate), cytotoxic antibiotics, antibodies (e.g., basiliximab, daclizumab, and muromonab), anti- immunophilins (e.g., cyclosporine, tacrolimus, sirolimus), inteferons, opioids, TNF binding proteins, mycophenolates, and small biological agents (e.g, fingolimod, myriocin).
  • cytostatics such as alkylating agents, anti metabolites (e.g., azathioprine, methotrexate), cytotoxic antibiotics, antibodies (e.g., basiliximab, daclizumab, and muromonab), anti- immunophilins (e.g., cyclosporine, tacrolimus, sirolimus), inteferons, opioids, TNF binding
  • a viral disease occurs when an organism's body is invaded by pathogenic viruses, and infectious virus particles (virions) attach to and enter.
  • viruses infectious virus particles
  • compounds that disrupt PD-1 homodimerization will prevent interaction of SHP-2 with PD-1 and block PD-1 -mediated inhibitory function.
  • Such compounds will be useful for the enhancement of immune responses against chronic viral infections such as HIV, Hepatitis C, Cytomegalovirus (CMV) or Epstein Barr Virus (EBV).
  • the disclosure provides a method for enhancement of immune responses against chronic viral infections.
  • viral infections include, but are not limited to: infections caused by DNA Viruses (e.g., Herpes Viruses such as Herpes Simplex viruses, Epstein-Barr virus, Cytomegalovirus; Pox viruses such as Variola (small pox) virus; Hepadnaviruses (e.g, Hepatitis B virus); Papilloma viruses; Adenoviruses); RNA Viruses (e.g., HIV I, II; HTLV I, II; Poliovirus; Hepatitis A; Orthomyxoviruses (e.g., Influenza viruses); Paramyxoviruses (e.g., Measles virus); Rabies virus; Hepatitis C); Rhinovirus, Respiratory Syncytial Virus, West Nile Virus, Yellow Fever, Rift Valley Virus, Lassa Fever Virus, Ebola Virus, Lymp
  • Cancer is a group of diseases characterized by uncontrolled cell division which can lead to abnormal tissue and, in turn, disruption of normal physiologic processes and, possibly, death. Cancers have various etiologies and may be responsive to agents that affect aspects of these etiologies. For example, a reduction or loss of nucleic acids that are linked to cancer development may prove fruitful in the treatment of various cancers, including blood-based cancers and breast cancers. Such treatments may replace or supplement existing treatments. I
  • the present invention provides a method for treating cancer in a patient in need thereof, comprising: (a) selecting an agent which decreases SHP-2-mediated PD-1 dimerization and/or reduces PD-1 dimer activity; and (b) administering the agent to the patient.
  • the present invention provides a method of making an agent effective for the treatment of a cancer, comprising: (a) identifying the agent by screening for a disruption or decrease of SHP-2-mediated programmed cell death protein-1 (PD-1 ) dimerization or of PD-1 dimer activity; and (b) formulating the agent for administration to a patient having a cancer.
  • PD-1 programmed cell death protein-1
  • the present invention provides a method for predicting a cancer patient response to an immune checkpoint immunotherapy, comprising determining the presence of SHP-2-mediated PD-1 dimerization in a biological sample from the patient, wherein the presence of SHP-2-mediated PD-1 dimerization is indicative of an inhibitory immune signal and a likelihood of responding to the immune checkpoint immunotherapy.
  • the present invention provides method for treating cancer, comprising: (a) evaluating a subject's likelihood of response to an immune checkpoint immunotherapy, comprising evaluating a level of SHP-2-mediated PD-1 dimerization in a biological sample from the patient, wherein a presence or high level of SHP-2-mediated PD-1 dimerization is indicative of a cancer that is suitable for immune checkpoint immunotherapy; and (b) administering an immune checkpoint immunotherapy to the patient.
  • the present disclosure encompasses methods of treating or preventing cancer and/or a metastasis in a subject in need thereof.
  • representative cancers and/or tumors and/or metastases of the present invention include a basal cell carcinoma, biliary tract cancer; bladder cancer; bone cancer; brain and central nervous system cancer; breast cancer; cancer of the peritoneum; cervical cancer; choriocarcinoma; colon and rectum cancer; connective tissue cancer; cancer of the digestive system; endometrial cancer; esophageal cancer; eye cancer; cancer of the head and neck; gastric cancer (including gastrointestinal cancer); glioblastoma; hepatic carcinoma; hepatoma; intra-epithelial neoplasm; kidney or renal cancer; larynx cancer; leukemia; liver cancer; lung cancer (e.g., small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung);
  • lung cancer e.g
  • CLL chronic lymphocytic leukemia
  • ALL acute lymphoblastic leukemia
  • PTLD post-transplant lymphoproliferative disorder
  • the cancer to be treated or prevented is a blood-based cancer or related disease including, for example, a lymphoma, leukemia, myeloma or myelodysplastic/myeloproliferative neoplasm (MDS/MPN).
  • a lymphoma including, for example, a lymphoma, leukemia, myeloma or myelodysplastic/myeloproliferative neoplasm (MDS/MPN).
  • the term subject or patient refers to any vertebrate including, without limitation, humans and other primates (e.g., chimpanzees and other apes and monkey species), farm animals (e.g., cattle, sheep, pigs, goats, and horses), domestic mammals (e.g., dogs and cats), laboratory animals (e.g., rodents such as mice, rats, and guinea pigs), and birds (e.g., domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, and the like).
  • the subject is a mammal.
  • the subject is a human.
  • the present invention provides a method for treating cancer in a patient in need thereof, comprising: (a) selecting an agent which decreases SHP-2-mediated PD-1 dimerization and/or reduces PD-1 dimer activity; and (b) administering the agent to the patient.
  • the agent which decreases SHP-2-mediated PD-1 dimerization and/or reduces PD-1 dimer activity is a small molecule or peptide agent.
  • the small molecule or peptide agent is capable of disrupting an interaction between a PD-1 polypeptide and a SHP-2 polypeptide, e.g., is capable of disrupting an interaction between a PD-1 polypeptide and a SH2 domain of SHP-2.
  • the small molecule or peptide agent is capable of disrupting an interaction between a PD-1 polypeptide and a SH2 domain of SHP-2 by preventing binding of the SH2 domain with a motif of the PD-1 polypeptide comprising a tyrosine 248, e.g., a phosphorylated tyrosine 248, e.g., a motif comprising an immunoreceptor tyrosine-based switch motif (ITSM).
  • ITSM immunoreceptor ty
  • the small molecule or peptide agent is capable of disrupting an interaction between a PD-1 polypeptide and a SH2 domain of SHP-2 by preventing binding of an amino terminal SH2 domain of SHP-2 (N-SH2; SEQ ID NO: 19) with the PD-1 polypeptide and/or by preventing binding of a carboxy terminal SH2 domain of SHP-2 (C-SH2; SEQ ID NO: 20) with the PD-1 polypeptide.
  • the small molecule or peptide agent is capable of disrupting an interaction between a first PD-1 polypeptide and an amino terminal SH2 domain of SHP-2 (N-SH2; SEQ ID NO: 19) and is capable of disrupting an interaction between a second I
  • the small molecule or peptide agent is capable of disrupting an interaction between the first PD-1 polypeptide and the N-SH2 domain by preventing binding of the N-SH2 domain with a motif of the first PD-1 polypeptide comprising a tyrosine 248, e.g., a phosphorylated tyrosine 248, and is capable of disrupting an interaction between the second PD-1 polypeptide and the C-SH2 domain by preventing binding of the C-SH2 domain with a motif of the second PD-1 polypeptide comprising a tyrosine 248, e.g., a phosphorylated tyrosine 248.
  • the motif of the first PD-1 polypeptide comprising a tyrosine 248, e.g., a phosphorylated tyrosine 248, and the motif of the second PD-1 polypeptide comprising a tyrosine 248, e.g., a phosphorylated tyrosine 248, each comprises a portion of immunoreceptor tyrosine- based switch motif (ITSM).
  • ITSM immunoreceptor tyrosine- based switch motif
  • portion includes a full-length ITSM or a fraction thereof in which the fraction retains the ability to bind a SH2 domain.
  • the tyrosine 248 position is relative to the wild-type, human PD-1 polypeptide sequence: Accession Number: Q151 16; SEQ ID NO: 21.
  • the small molecule or peptide agent is capable of binding to a SH2 domain of SHP-2.
  • the small molecule or peptide agent is capable of binding to an amino terminal SH2 domain of SHP-2 (N-SH2; SEQ ID NO: 19) or to a carboxy terminal SH2 domain of SHP-2 (C-SH2; SEQ ID NO: 20).
  • the peptide agent is capable of binding to an amino terminal SH2 domain of SHP-2 (N-SH2; SEQ ID NO: 19) and to a carboxy terminal SH2 domain of SHP-2 (C-SH2; SEQ ID NO: 20).
  • the peptide agent comprises at least one motif of a PD-1 polypeptide comprising a tyrosine 248, e.g., a phosphorylated tyrosine 248.
  • the motif comprises a portion of an immunoreceptor tyrosine-based switch motif (ITSM).
  • the peptide agent comprises at least two motifs of a PD-1 polypeptide comprising a tyrosine 248, e.g., a phosphorylated tyrosine 248, wherein the at least two motifs are separated by a linker.
  • each of the at least two motifs of a PD-1 polypeptide comprising a tyrosine 248, e.g., a phosphorylated tyrosine 248, comprises a portion of an immunoreceptor tyrosine- based switch motif (ITSM).
  • ITSM immunoreceptor tyrosine- based switch motif
  • portion of an ITSM includes a full-length ITSM or a fraction thereof in which the fraction retains the ability to bind a SH2 domain.
  • the linker comprises at least 3 amino acids and/or the linker is at least about 35 Angstroms long.
  • the peptide agent e.g., that is capable of binding two SH2 domains, comprises: (a) a first amino acid sequence of SEQ ID NO: 1 , 22, or 23, optionally comprising a mutation of 1 , 2, 3, 4, or 5 amino acids, (b) a second amino acid sequence of SEQ ID NO: 1 , 22, or 23, optionally comprising a mutation of 1 , 2, 3, 4, or 5 amino acids, and (c) a linker comprising at least 3 amino acids between the first amino acid sequence and the second amino acid sequence, optionally, the linker separates the two amino acid sequences by at least about 35 Angstroms. In embodiments, the linker separates the two amino acid sequences by at least about 50 Angstroms.
  • the peptide agent comprises the amino acid sequence of SEQ ID NO: 3, 9, 10, or 1 1 , optionally comprising a mutation of 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.
  • the mutation is not of the tyrosine 248, with reference to I
  • the peptide agent comprises or consists of SEQ ID NO: 3, 9, 10, or 1 1.
  • the peptide agent comprises the amino acid sequence of SEQ ID NO: 3, 9, 10, 1 1 , or 24, optionally comprising a mutation of 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids.
  • the mutation is not of the tyrosine 248, with reference to SEQ ID NO: 21.
  • the peptide agent comprises or consists of SEQ ID NO: 3, 9, 10, 1 1 , or 24.
  • the peptide agent which reduces PD-1 dimer activity is capable of binding to at least a first PD-1 polypeptide comprising a tyrosine 248, e.g., a phosphorylated tyrosine 248; in this embodiment, the peptide agent lacks a phosphatase domain or comprises a non-functional phosphatase domain.
  • the small molecule or peptide agent is capable of binding to the at least first PD-1 polypeptide at or near its immunoreceptor tyrosine-based switch motif (ITSM).
  • the peptide agent is capable of binding to the first PD-1 polypeptide and to an at least second PD-1 polypeptide.
  • the peptide agent is capable of binding to the first PD-1 polypeptide and to the at least second PD-1 polypeptide at or near each polypeptide's immunoreceptor tyrosine-based switch motif (ITSM).
  • the peptide agent comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 19 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 20.
  • the amino acid sequence that is at least 95% identical to SEQ ID NO: 19 comprises an arginine at its amino acid position 32, relative to SEQ ID NO: 19, or the amino acid sequence that is at least 95% identical to SEQ ID NO: 20 comprises an arginine at its amino acid position 28, relative to SEQ ID NO: 20.
  • the peptide agent comprises a first amino acid sequence that is at least 95% identical to SEQ ID NO: 19 and a second amino acid sequence that is at least 95% identical to SEQ ID NO: 20.
  • the amino acid sequence that is at least 95% identical to SEQ ID NO: 19 comprises an arginine at its amino acid position 32, relative to SEQ ID NO: 19
  • the amino acid sequence that is at least 95% identical to SEQ ID NO: 20 comprises an arginine at its amino acid position 28, relative to SEQ ID NO: 20.
  • the first amino acid sequence and the second amino acid sequence are separated by a linker, e.g., a linker comprising at least 3 amino acids and/or a linker that is at least about 35 Angstroms long.
  • a linker e.g., a linker comprising at least 3 amino acids and/or a linker that is at least about 35 Angstroms long.
  • the present invention provides a method for treating an autoimmune disease or disorder or for treating inflammation in a patient in need thereof, comprising: (a) selecting an agent which increases SHP-2-mediated PD-1 dimerization and/or increases PD-1 dimer activity; and (b) administering the agent to the patient.
  • the agent which increases SHP-2-mediated PD-1 dimerization and/or increases PD-1 dimer activity is a small molecule or peptide agent.
  • the small molecule or peptide agent is capable of stimulating an I
  • the small molecule or peptide agent is capable of stimulating an interaction between a first PD-1 polypeptide and an amino terminal SH2 domain of SHP-2 (N-SH2; SEQ ID NO: 19) and an interaction between a second PD-1 polypeptide and a carboxy terminal SH2 domain of SHP-2 (C-SH2; SEQ ID NO: 20).
  • the peptide agent is capable of binding to a first PD-1 polypeptide comprising a tyrosine 248, e.g., a phosphorylated tyrosine 248, and to an at least second PD-1 polypeptide comprising a tyrosine 248, e.g., a phosphorylated tyrosine 248.
  • the peptide agent is capable of binding to the first PD-1 polypeptide and to the at least second PD-1 polypeptide at or near each polypeptide's immunoreceptor tyrosine-based switch motif (ITSM).
  • ITSM immunoreceptor tyrosine-based switch motif
  • the peptide agent comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 19 or an amino acid sequence that is at least 95% identical to SEQ ID NO: 20.
  • the peptide agent comprises a first amino acid sequence that is at least 95% identical to SEQ ID NO: 19 and a second amino acid sequence that is at least 95% identical to SEQ ID NO: 20.
  • the amino acid sequence that is at least 95% identical to SEQ ID NO: 19 comprises an arginine at its amino acid position 32, relative to SEQ ID NO: 19, and the amino acid sequence that is at least 95% identical to SEQ ID NO: 20 comprises an arginine at its amino acid position 28, relative to SEQ ID NO: 20.
  • the first amino acid sequence and the second amino acid sequence are separated by a linker, e.g., a linker comprising at least 3 amino acids and/or a linker that is at least about 35 Angstroms long.
  • the peptide agent comprises: (a) a first amino acid sequence of SEQ ID NO: 19 or 20, optionally comprising a mutation of 1 , 2, 3, 4, or 5 amino acids, (b) a second amino acid sequence of SEQ ID NO: 19 or 20, optionally comprising a mutation of 1 , 2, 3, 4, or 5 amino acids, and (c) a linker comprising at least 3 amino acids between the first amino acid sequence and the second amino acid sequence, optionally, the linker separates the two amino acid sequences by at least about 35 Angstroms.
  • the peptide agent further comprises a functional phosphatase domain.
  • the functional phosphatase domain comprises a protein tyrosine phosphatase (PTP) domain.
  • PTP protein tyrosine phosphatase
  • a phosphatase domain is any portion, e.g., a catalytic domain, of a phosphatase enzyme that is capable of removing a phosphate group from a phosphorylated amino acid, e.g., a phosphorylated tyrosine.
  • the peptide agent comprises the amino acid sequence of SEQ ID NO: 18. In embodiments, the peptide agent does not comprise of the amino acid sequence of SEQ ID NO: 18.
  • nucleic acid and polynucleotide are used interchangeably herein to refer to single- or double-stranded RNA, DNA, or mixed polymers.
  • Polynucleotides may include genomic sequences, extra-genomic and plasmid sequences, and smaller engineered gene segments that express, or may be adapted to express polypeptides.
  • An isolated nucleic acid is a nucleic acid that is substantially separated from other genome DNA sequences as well as proteins or complexes such as ribosomes and polymerases, which naturally accompany a native sequence.
  • the term embraces a nucleic acid sequence that has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.
  • a substantially pure nucleic acid includes isolated forms of the nucleic acid. Of course, this refers to the nucleic acid as originally isolated and does not exclude genes or sequences later added to the isolated nucleic acid by the hand of man.
  • polypeptide is used in its conventional meaning, i.e., as a sequence of amino acids.
  • the polypeptides are not limited to a specific length of the product.
  • Peptides, oligopeptides, and proteins are included within the definition of polypeptide, and such terms may be used interchangeably herein unless specifically indicated otherwise.
  • This term also does not refer to or exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • a polypeptide may be an entire protein, or a subsequence thereof.
  • an isolated polypeptide is one that has been identified and separated and/or recovered from a component of its natural environment.
  • the isolated polypeptide will be purified (1 ) to greater than 95% by weight of polypeptide as determined by the Lowry method, and more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, silver stain.
  • Isolated polypeptide includes the polypeptide in situ within recombinant cells since at least one component of the polypeptide's natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.
  • two sequences are said to be “identical” if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence, as described below. Comparisons between two sequences are typically performed by comparing the sequences over a comparison window to identify and compare local regions of sequence similarity.
  • a “comparison window” as used herein, refers to a segment of at least about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are aligned.
  • the term substantial identity means that two peptide sequences, when aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 80 percent sequence identity. In some embodiments, the two peptide sequences share at least 90 percent sequence identity. In some embodiments, the two I
  • peptide sequences share at least 95 percent sequence identity. In some embodiments, the two peptide sequences share at least 99 percent sequence identity.
  • residue positions that are not identical differ by conservative amino acid substitutions.
  • Minor variations in the amino acid sequences of polypeptides are contemplated as being encompassed by the present disclosure, providing that the variations in the amino acid sequence maintain at least 60% amino acid sequence identity to a reference sequence (e.g., the wild-type sequence).
  • the variations in the amino acid sequence maintain at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% amino acid identity to the reference sequence.
  • conservative amino acid replacements are contemplated.
  • Linkers may be of any desired length, one end of which can be covalently attached to specific sites of the small molecule or peptide agent.
  • the other end of the linker or spacer element may be attached to an amino acid or peptide linker.
  • the linker is at least about 1 angstrom, 5 angstroms, 10 angstroms, 15 angstroms, 20 angstroms, 25 angstroms, 30 angstroms, 35 angstroms, 40 angstroms, 45 angstroms, 50 angstroms, 55 angstroms, 60 angstroms, 65 angstroms, 70 angstroms, 75 angstroms, 80 angstroms, 85 angstroms, 90 angstroms, 95 angstroms, or 100 angstroms long, inclusive of all endpoints.
  • the linker may be derived from naturally-occurring multi-domain proteins or are empirical linkers as described, for example, in Chichili et al., (2013), Protein Sci. 22(2): 153-167, Chen et al., (2013), Adv Drug Deliv Rev. 65(10): 1357-1369, the entire contents of which are hereby incorporated by reference.
  • the linker may be designed using linker designing databases and computer programs such as those described in Chen et al., (2013), Adv Drug Deliv Rev. 65(10): 1357-1369 and Crasto et. al., (2000), Protein Eng. 13(5):309-312, the entire contents of which are hereby incorporated by reference.
  • the linker or spacer is not peptide-based.
  • the linker is a synthetic linker such as PEG.
  • the linker is 4 amino hexanoic acid (Ahx). In embodiments, the linker is 10 amino hexanoic acid (Ahx).
  • Mutations contemplated include substitutions, additions, and deletions, or any combination thereof.
  • the mutation converts the mutated amino acid to alanine.
  • the mutation converts the mutated amino acid to another amino acid (e.g., glycine, serine, threonine, cysteine, valine, leucine, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, asparagines, glutamine, histidine, lysine, or arginine).
  • the mutation converts the mutated amino acid to a non-natural amino acid (e.g., selenomethionine). In some embodiments, the mutation converts the mutated amino acid to amino acid mimics (e.g., phosphomimics). In some embodiments, the mutation is a conservative mutation. For example, the mutation I
  • the mutation converts the mutated amino acid to amino acids that resemble the size, shape, charge, polarity, conformation, and/or rotamers of the mutated amino acids (e.g., cysteine/serine mutation, lysine/asparagine mutation, histidine/phenylalanine mutation).
  • the mutation causes a shift in reading frame and/or the creation of a premature stop codon.
  • mutations cause changes to regulatory regions of genes or loci that affect expression of one or more genes.
  • the mutation can be a change in 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18,
  • compositions comprising an agent which decreases SHP-2-mediated PD-1 dimerization.
  • pharmaceutical compositions may be prepared in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
  • a pharmaceutical composition comprises, an agent which decreases SHP-2-mediated PD-1 dimerization and a pharmaceutically acceptable carrier.
  • An effective dose is an amount sufficient to affect a beneficial or desired clinical result.
  • the disclosure provides a method of making an agent effective for the treatment of a cancer, comprising: (a) identifying the agent by screening for a disruption or decrease of SHP-2-mediated PD-1 dimerization and (b) formulating the agent for administration to a cancer patient.
  • the disclosure provides a method of making an agent effective for the treatment of an autoimmune disease or disorder, comprising: (a) identifying the agent by screening for a stimulation or increase of SHP-2-mediated PD-1 dimerization and (b) formulating the agent for administration to an autoimmune disease or disorder patient.
  • a beneficial or desired clinical result may include, inter alia, a reduction in tumor size and/or tumor growth and/or a reduction of a cancer marker that is associated with the presence of cancer as compared to what is observed without administration of the small molecule or peptide agent.
  • a beneficial or desired clinical result may also include, inter alia, an increased presence of a marker that is associated with a reduction of cancer as compared to what is observed without administration of the small molecule or peptide agent. Also included in a beneficial or desired clinical result is, I
  • the gene comprising a marker linked to cancer etiology may include, for example, an immune checkpoint gene, such as PD-1 , PD-L1 , or PD-L2.
  • the present invention relates to, in various embodiments, agents that may be used in the context of various combination therapies encompassed by the present invention.
  • Additional therapeutic agents may be administered simultaneously or sequentially with the disclosed agents, compounds, or compositions. Sequential administration includes administration before or after the disclosed agent, compound, or composition. In some embodiments, the additional therapeutic agent or agents may be administered in the same composition as the disclosed agent, compound, or composition. In other embodiments, there may be an interval of time between administration of the additional therapeutic agent and the disclosed agent, compound, or composition. In some embodiments, administration of an additional therapeutic agent with a disclosed agent, compound, or composition may allow lower doses of the other therapeutic agents and/or administration at less frequent intervals. When used in combination with one or more other active ingredients, the agents, compounds, or compositions of the present invention and the other active ingredients may be used in lower doses than when each is used singly.
  • compositions of the present invention include those that contain one or more other active ingredients, in addition to an agent, compound, or composition of the present disclosure.
  • the above combinations include combinations of an agent, compound, or composition of the present disclosure not only with one other active compound, but also with two or more other active compounds.
  • the agent, compound, or composition of the disclosure can be combined with a variety of anti-cancer drugs and chemotherapeutics.
  • the method makes the cancer responsive or more responsive to a combination therapy of the immune checkpoint immunotherapy and one or more chemotherapeutic agents and/or radiotherapy.
  • the present disclosure includes various cancer biologies, therapeutics, chemotherapeutics, or drugs known in the art.
  • the following drugs may be used in the present invention: daunorubicin, doxorubicin, epirubicin, idarubicin, adriamycin, vincristine, carmustine, cisplatin, 5-fluorouracil, tamoxifen, prodasone, sandostatine, mitomycin C, foscarnet, paclitaxel, docetaxel, gemcitabine, fludarabine, carboplatin, leucovorin, tamoxifen, goserelin, ketoconazole, leuprolide flutamide, vinblastine, vindesine, vinorelbine, camptothecin, topotecan, irinotecan hydrochloride, etoposide, mitoxantrone, teniposide,
  • adenosine arabinoside (Ara-A), cladribine, ftorafur, UFT (combination of uracil and ftorafur), 5-fluoro-2'-deoxyuridine, 5-fluorouridine, 5'-deoxy-5-fluorouridine, hydroxyurea, dihydrolenchiorambucil, tiazofurin, oxaliplatin, melphalan, thiotepa, busulfan, chlorambucil, plicamycin, dacarbazine, ifosfamide phosphate, cyclophosphamide, pipobroman, 4- ipomeanol, dihydrolenperone, spiromustine, geldenamycin, cytochalasins, depsipeptide, 4'-cyano-3-(4-(e.g., ZOLADEX) and 4'-cyano-3-(4-fluorophenylsul
  • the present compositions and methods find use in combination with checkpoint inhibitors - e.g., in the treatment of various cancers.
  • the present compositions and methods may supplement checkpoint inhibitor-based cancer therapies, e.g., by improving patient response to the same (e.g., by converting non-responders to responders, and/or increasing the magnitude of therapeutic response, and/or reducing the does or regimen needed for therapeutic response, and/or reducing one or more side effects of the checkpoint inhibitor-based cancer therapies).
  • the immune checkpoint immunotherapy agent modulates PD-1.
  • the agent that modulates PD-1 is an antibody or antibody format specific for PD-1.
  • the antibody or antibody format specific for PD-1 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.
  • the antibody or antibody format specific for PD-1 is selected from Nivolumab, Pembrolizumab, and Pidilizumab.
  • an antibody or antibody format specific for PD-1 is Nivolumab and can be administered at 240 mg every 2 weeks.
  • an antibody or antibody format specific for PD-1 is Pembrolizumab and can be administered at 200 mg every 3 weeks.
  • an antibody or antibody format specific for PD-1 is Pidilizumab and can be administered at 200 mg every 3 weeks.
  • the immune checkpoint immunotherapy agent modulates PD-L1.
  • the agent that modulates PD-L1 is an antibody or antibody format specific for PD-L1.
  • the antibody or antibody format specific for PD-L1 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.
  • the antibody or antibody format specific for PD-L1 is selected from Atezolizumab, Avelumab Durvalumab and B MS-936559. In some embodiments, the antibody or antibody format specific for PD-L1 is BMS-936559 and can be administered at 0.1 mg/kg every 2 weeks. In some embodiments, the antibody or antibody format specific for PD-L1 is Atezolizumab and can be administered at 1200 mg every 3 weeks. In some embodiments, the antibody or antibody format specific for PD-L1 is Avelumab and can be administered at 10 I
  • the antibody or antibody format specific for PD-L1 is Durvalumab and can be administered at 10 mg/kg every 2 weeks.
  • the agent that modulates PD-L2 is an antibody or antibody format specific for PD-L2.
  • the antibody or antibody format specific for PD-L2 is selected from one or more of a monoclonal antibody, polyclonal antibody, antibody fragment, Fab, Fab', Fab'-SH, F(ab')2, Fv, single chain Fv, diabody, linear antibody, bispecific antibody, multispecific antibody, chimeric antibody, humanized antibody, human antibody, and fusion protein comprising the antigen-binding portion of an antibody.
  • the present invention provides a method for predicting a cancer patient response to an immune checkpoint immunotherapy, comprising determining the presence of SFIP-2-mediated PD-1 dimerization in a biological sample from the patient, wherein the presence of SFIP-2-mediated PD-1 dimerization is indicative of an inhibitory immune signal and a likelihood of responding to the immune checkpoint immunotherapy.
  • the present invention provides method for treating cancer, comprising: (a) evaluating a subject's likelihood of response to an immune checkpoint immunotherapy, comprising evaluating a level of SFIP-2-mediated PD- 1 dimerization in a biological sample from the patient, wherein a presence or high level of SFIP-2-mediated PD-1 dimerization is indicative of a cancer that is suitable for immune checkpoint immunotherapy; and (b) administering an immune checkpoint immunotherapy to the patient.
  • the administration is by intratumoral, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or direct injection into a cancer tissue.
  • the patient is predicted to be poorly responsive or non-responsive to the immune checkpoint immunotherapy or has presented as poorly responsive or non-responsive to the immune checkpoint immunotherapy.
  • the method reduces and/or mitigates one or more side effects of the immune checkpoint immunotherapy.
  • the side effect is selected from decreased appetite, rashes, fatigue, pneumonia, pleural effusion, pneumonitis, pyrexia, nausea, dyspnea, cough, constipation, diarrhea, immune-mediated pneumonitis, colitis, hepatitis, endocrinopathies, hypophysitis, iridocyclitis, and nephritis.
  • the method reduces the dose of the immune checkpoint immunotherapy. In some embodiments, the method reduces number of administrations of the immune checkpoint immunotherapy. In some embodiments, the method increases a therapeutic window of the immune checkpoint immunotherapy.
  • the method elicits a potent immune response in less-immunogenic tumors.
  • the method converts a tumor with reduced inflammation ("cold tumor”) to a responsive, inflamed tumor ("hot tumor”).
  • the method makes the cancer responsive or more responsive to a combination therapy of the immune checkpoint immunotherapy and one or more chemotherapeutic agents and/or radiotherapy.
  • Aqueous compositions of the present invention comprise an effective amount of the delivery vehicle comprising an agent or compound of the present disclosure, (e.g., liposomes or other complexes or expression vectors) dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
  • an agent or compound of the present disclosure e.g., liposomes or other complexes or expression vectors
  • pharmaceutically acceptable or pharmacologically acceptable refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
  • pharmaceutically acceptable carrier includes solvents, buffers, solutions, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like acceptable for use in formulating pharmaceuticals, such as pharmaceuticals suitable for administration to humans.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions, provided they do not inactivate the vectors or polynucleotides of the compositions.
  • compositions of the present disclosure may include classic pharmaceutical preparations. Administration of these compositions according to the present invention may be via any common route so long as the target tissue is available via that route. This includes oral, nasal, or buccal. Alternatively, administration may be by intratumoral, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection, or by direct injection into cancer tissue. The agents disclosed herein may also be administered by catheter systems. Such compositions would normally be administered as pharmaceutically acceptable compositions as described herein.
  • solutions may be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations may easily be administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
  • parenteral administration in an aqueous solution for example, the solution generally is suitably buffered and the liquid diluent first rendered isotonic with, for example, sufficient saline or glucose.
  • aqueous solutions may be used, for example, for intratumoral, intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media are employed as is I
  • a single dose may be dissolved in 1 ml of isotonic NaCI solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion (see, e.g., Remington's Pharmaceutical Sciences, 15th Edition, pages 1035-1038 and 1570- 1580, the contents of which are hereby incorporated by reference).
  • Some variation in dosage will necessarily occur depending on the condition of the subject being treated.
  • the person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biologies standards.
  • the present disclosure includes an agent of the present disclosure and a second agent that is or comprises at least one other cancer biologic, therapeutic, chemotherapeutic or drug or an anti-inflammatory drug.
  • the first and second agents may be administered in either order (e.g., first then second or second then first) or concurrently.
  • the agent of the present invention can be administered over any suitable period of time, such as a period from about 1 day to about 12 months.
  • the period of administration can be from about 1 day to 90 days; from about 1 day to 60 days; from about 1 day to 30 days; from about 1 day to 20 days; from about 1 day to 10 days; from about 1 day to 7 days.
  • the period of administration can be from about 1 week to 50 weeks; from about 1 week to 50 weeks; from about 1 week to 40 weeks; from about 1 week to 30 weeks; from about 1 week to 24 weeks; from about 1 week to 20 weeks; from about 1 week to 16 weeks; from about 1 week to 12 weeks; from about 1 week to 8 weeks; from about 1 week to 4 weeks; from about 1 week to 3 weeks; from about 1 week to 2 weeks; from about 2 weeks to 3 weeks; from about 2 weeks to 4 weeks; from about
  • the agent which decreases SHP-2-mediated PD-1 dimerization can be administered every day, every other day, every week, every 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 1 1 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, or every 20 weeks, or every month.
  • a therapeutically effective amount of a composition or agent of the present disclosure may be about 0.01 mg/kg per day to about 10 mg/kg per day.
  • dosages can range from about 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 3 mg/kg, 5 mg/kg, 6 mg/kg, 7.5 mg/kg, or about 10 mg/kg.
  • the dose will be in the range of about 0.1 mg/day to about 5 mg/kg; about 0.1 mg/day to about 10 mg/kg; about 0.1 mg/day to about 20 mg/kg; about 0.1 mg to about 30 mg/kg; or about 0.1 mg to about 40 mg/kg.
  • a therapeutically effective amount of a composition or agent of the present disclosure may be about 0.1 mg to about 50 mg/kg or in single, divided, or continuous doses (which dose may be adjusted for the patient's weight in kg, body surface area in m 2 , and age in years).
  • a therapeutically effective amount of the composition or agent of the present disclosure may be about 1 mg/kg to about 1000 mg/kg, about 5 mg/kg to about 950 mg/kg, about 10 mg/kg to about 900 mg/kg, about 15 mg/kg to about 850 mg/kg, about 20 mg/kg to about 800 mg/kg, about 25 mg/kg to about 750 mg/kg, about 30 mg/kg to about 700 mg/kg, about 35 mg/kg to about 650 mg/kg, about 40 mg/kg to about 600 mg/kg, about 45 mg/kg to about 550 mg/kg, about 50 mg/kg to about 500 mg/kg, about 55 mg/kg to about 450 mg/kg, about 60 mg/kg to about 400 mg/kg, about 65 mg/kg to about 350 mg/kg, about 70 mg/kg to about 300 mg/kg, about 75 mg/kg to about 250 mg/kg, about 80 mg/kg to about 200 mg/kg, about 85 mg/kg to about 150 mg/kg, and about 90 mg/kg to about 100
  • a therapeutically effective amount of a composition or agent of the present disclosure 0.01 mg/kg to about 500 mg/kg, for example, about 0.1 mg/kg to about 200 mg/kg (such as about 100 mg/kg), or about 0.1 mg/kg to about 10 mg/kg (such as about 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg, 3 mg/kg, 5 mg/kg, 6 mg/kg, 7.5 mg/kg, or about 10 mg/kg).
  • kits that can simplify the administration of any agent disclosed herein.
  • An exemplary kit of the invention comprises any composition described herein in unit dosage form.
  • the unit dosage form is a container, such as a pre-filled syringe, which can be sterile, containing any agent described herein and a pharmaceutically acceptable carrier, diluent, excipient, or vehicle.
  • the kit can further comprise a label or printed instructions instructing the use of any agent described herein.
  • the kit may also include a lid speculum, topical anesthetic, and a cleaning agent for the administration location.
  • the kit can further comprise one or more additional agent, such as a biologic, therapeutic, chemotherapeutic or drug described herein.
  • the kit comprises a container containing an effective amount of a composition of the invention and an effective amount of another composition, such those described herein.
  • FIG. 2D binds phosphorylated ITSM-Y248 residues in two PD-1 polypeptides using its N-SH2 domain for one PD-1 and its C-SH2 domain for the second PD-1 to form a PD-1 dimer (FIG. 2D).
  • PD-1 dimer binds phosphorylated ITSM-Y248 residues in two PD-1 polypeptides using its N-SH2 domain for one PD-1 and its C-SH2 domain for the second PD-1 to form a PD-1 dimer (FIG. 2D).
  • the below-disclosed data demonstrates that, in live cells, after PD-1 phosphorylation by the TCR-proximal Src family tyrosine kinase Fyn, PD-1 dimers are formed. Such dimers are disrupted by the loss of the active site of either SHP-2 SH2 domain or the absence of Fyn kinase activity.
  • SHP-2 activation by PD-1 may be due to the interaction of the tandem SH2 domains with the phosphorylatedtyrosines in PD-1 ITIM-pY223 and PD-1 ITSM-pY248, similarly to that proposed for IRS, the below-disclosed data shows that the binding preference of SHP-2 tandem SH2 domains is incompatible with such model of SHP-2 activation.
  • the two SH2 domains of SHP-2 have a roughly antiparallel or roughly perpendicular orientation relative to one another with the phosphopeptide binding sites widely separated (FIG. 6C).
  • the distance between the phosphopeptide binding sites of the SH2 domains is 41 A in the crystal structure of the tandem SH2 domains and appears to be critical for phosphoprotein recognition and for enzymatic activation of SHP-2.
  • the below-disclosed data shows that a bisphosphorylated peptide (which meets the spacing requirements for dual SHP-2 SH2 domain binding by covalent joining of one plTIM-Y223 and one plTSM-Y248 phosphopeptide with a 4 amino hexanoic acid (Ahx) spacer) did not induce SHP-2 activity, whereas a similarly-designed bisphosphorylated peptide generated by covalently joining two PD-1 plTSM-Y248 peptides induced robust SHP-2 phosphatase activity.
  • CTLA-4 is a covalent dimer. Its higher avidity for the B7 ligands than for CD28 results from the binding of each CTLA-4 dimer to two divalent B7 polypeptides.
  • the crystal structure of CTLA-4: B7 complexes suggests that CTLA-4 covalent dimer can bind to noncovalent dimers of B7-1 to form a lattice of CTLA-4-B7 interactions. Such a lattice can function to form a stable signaling complex at the T cell: APC interface.
  • PD-1 does not have the conserved cysteine located proximal to the transmembrane domain; thus, it does not appear to be structurally-equipped to form a covalent dimer. Indeed, crystallography studies of the PD-1 : PD-L1 binding regions have shown that PD-1 stays as a I
  • PD-1 can form a PD-1: PD-1 noncovalent dimeric complex via a previously-unidentified mechanism. Formation of this complex is guided by an "inside-out” signaling sequence induced by TCR-mediated phosphorylation of PD-1 by Src family kinases. Without wising to be bound by theory, the below-disclosed data strongly suggests that formation of this PD-1: PD-1 dimeric complex is essential for inhibitory function in live cells because only mutagenesis of the PD-1 phosphotyrosine, which is required both for PD-1 dimer formation and SHP-2 enzymatic activation, abrogated PD-1 -mediated inhibition during antigen-specific stimulation.
  • the below-disclosed data unexpectedly shows that phosphorylation of PD-1 cytoplasmic tail also has an active role in PD-L1 -mediated PD-1 oligomerization.
  • the data shows that PD-1: PD-1 dimer formation induced by dimeric PD-L1 was impaired when phosphorylation of PD-1 cytoplasmic tail was compromised by the expression of a kinase dominant negative Fyn.
  • plTSM-Y248 appears to be required for SHP-2 enzymatic activation; this data explains why TCR- mediated signaling is required concomitantly with PD-1 ligation by its natural ligands to induce PD-1 inhibitory function.
  • PD-1 partitions in the TCR microclusters which consist of TCR and proximal signaling molecules.
  • TCR microclusters consist of TCR and proximal signaling molecules.
  • SHP-2 SHP-2 to the TCR proximal signaling molecules.
  • Recruitment of PD-1 in the TCR microclusters can occur in the absence of ITSM-Y248 phosphorylation but, under these conditions, fails to inhibit T cell activation.
  • the below-disclosed data reveals why TCR signaling is required for the activation of the inhibitory effect of PD-1 and the mechanistic role of PD-1 plTSM-Y248 in PD-1-mediated inhibition. It is further expected that PD-1 dimers can bind to covalent or noncovalent dimers of PD-L1 and PD-L2 to form a lattice of PD-1 : PD-L1 interactions; thereby forming a stable signaling complex at the T cell: APC interface.
  • the below- disclosed data reveals the geometry of PD-1 : SHP-2 interaction that leads to SHP-2 activation; exploiting this interaction will reveal avenues for the development of PD-1 -binding compounds which selectively suppress T cell I
  • Gibco 293H cells (Fisher Scientific) were cultured in DMEM supplemented with 10% heat-inactivated FBS, 10 mM HEPES, 1 % glutamax, 1 % Pen/Strep, 15 pg/ml gentamycin.
  • Jurkat cells were stably transfected with PD-1 and cultured in presence of 5 pg/ml selection antibiotic blasticidin. Before stimulation experiments, Jurkat cells were rested overnight at 37 °C in RPMI- 1640 containing 2% FBS. Raji cells were stably transfected with PD-L1 and cultured in presence of 10 pg/ml blasticidin).
  • Jurkat cells were re-suspended at 100 x 10 6 cells/ml in pre-warmed RPMI 1640 containing 10 mM HEPES and mixed with an equal volume of RPMI/HEPES containing equal numbers of tosyl activated magnetic beads (conjugated either with anti-CD3, anti-CD28 mAbs and IgG or with anti-CD3, anti-CD28 and PD-L1 -lg).
  • the cells-beads mixture was then centrifuged for 1 minute at 300 g at room temperature and placed immediately at 37 °C. At the indicated time points, the reaction was stopped by adding cold PBS, cell-bead pellet disruption by pipetting and placement on ice.
  • Raji cells were resuspended at 1 x 10 6 cells/ml in RPMI complete medium and loaded with 0.5 ng/ml SEE (Toxin Technologies) by 30 min rotation at 37 °C followed by three washes to remove excess SEE.
  • Jurkat cells were cultured in 96-well tissue culture plates, at 10 5 cells/well with equal numbers of Raji cells (with or without SEE loading) in a final volume of 100 pi. When indicated, a PD-1 blocking antibody (clone EH12) or an isotype control IgG was added in the cultures.
  • COS cells were plated 24 hours before transfection on 10 cm dishes at a density of 0.8 x 10 6 cells/10 cm/dish. Next day the cells were transfected by GeneJuice transfection reagent (EMD Millipore Corp., Billerica, MA) according to the manufacturer's instructions. Briefly, for each plate, 0.6 ml plain RPMI was mixed with 18 pi GeneJuice reagent and incubated 10 min at room temperature (RT). 12 pg total plasmid DNA was added in the mixture followed by 30 min incubation at RT. The mixture was added dropwise to the plate and cells were incubated 24 hours at 37 °C, medium was replaced and cells were harvested at 48 hr post-transfection, trypsinized and lysed for subsequent western blot analysis.
  • GeneJuice transfection reagent EMD Millipore Corp., Billerica, MA
  • lysates were washed in PBS and lysed in lysis buffer containing 50 mM Tris-HCI, pH 7.4, 150 mM NaCI, 2 mM MgCI2, 10% glycerol and 1 % NP-40 supplemented with 2 mM sodium orthovanadate, 1 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride (PMSF), and protease Inhibitor Cocktail (Thermo Scientific). Cell lysates were resolved by SDS-PAGE and then analyzed by Western blotting with the indicated antibodies.
  • the mAb against FLAG (clone M2) was from Sigma, St. Louis, MO.
  • the mouse monoclonal anti-PD-1 antibodies clones EH12 and EH33 have been previously described.
  • the rabbit polyclonal anti-363 phospho-Y248 (ITSM) PD-1 antibody was developed. Immunoprecipitations were performed with PD-1 mAb clone EH12 covalently conjugated to Dynabeads protein G (Thermo Scientific). Briefly, 40 pi of beads/sample were incubated for 15 min at RT with gentle rotation with 10 pg PD- 1 antibody in PBS containing 0.02% Tween-20 in a final volume of 200 pi.
  • Beads were then washed in 200 pi conjugation buffer (20 mM NaHP04, 150 mM NaCI, pH 7.5) followed by 30 min gentle rotation at RT with 250 pi 5 mM Bis(Sulfosuccinimidyl) substrate (BS3) (Thermo Scientific) in conjugation buffer. The reaction was stopped by adding 12.5 pi quenching buffer (1 M Tris, pH 7.5) followed by 15 min gentle rotation at RT. Beads were washed in IP buffer (lysis buffer without NP-40) and subsequently incubated with 500 pg of cell lysates overnight at 4 °C with gentle rotation. One sample of antibody conjugated beads without lysate was used as a negative control.
  • Beads were washed and boiled 5 min in western blot denaturing sample buffer followed by quick spin, magnetic bead removal. The supernatant was then analyzed by SDS-PAGE, transferred to a nitrocellulose membrane, western blotted with the indicated antibodies and exposed to digital imager FluorChem E (Proteinsimple, San Jose, CA).
  • SHP-2 cDNA (Addgene, Cambridge, MA) was used to generate Glutathione-S-transferase (GST) fusions to SHP-2 wild type full length (SHP-2-WT-FL) and deletion mutants (FIG. 1A) using the pGEX 4T-3 vector (GE Healthcare Life Sciences, Marlborough, MA) and Flag-tagged mutants using the p3xFLAG-CMV10 vector (Addgene, Cambridge, MA).
  • Human PD-1 and PD-L1 cDNAs were expressed in pEF6 vector.
  • the QuickChange Lightning Site-Directed Mutagenesis kit from Agilent Technologies was used. All mutations were HD cloning kit from Clontech Laboratories, Inc., Mountain View, CA, following the manufacturer's instructions45.
  • Proteins were extracted by B- PER bacterial protein extraction kit (Thermo Scientific, Rockford, IL) and purified on glutathione-sepharose (GE Healthcare Life Sciences, Marlborough, MA). GST and GST fusion proteins (10 pg each) were incubated for 1 hour at 4°C with 50 pi of glutathione-Sepharose in GST-buffer and then incubated with cell extracts (500 pg per sample) overnight followed by SDS-PAGE and Western blotting analysis with the indicated antibodies.
  • the GST tag was removed by incubating 10-15 ml of bacterial lysate (containing the GST-SHP-2 proteins) with 2 ml glutathione-sepharose (1 ml bed volume) for 2 hours and gentle rotation at 4 °C.
  • the approximately 1 ml bed volume of glutathione-sepharose was washed 3 x 10 ml PBS and was mixed with 80 units of thrombin (GE Healthcare Life Sciences, Marlborough, MA) and PBS up to
  • 1 mg protein was diluted in a buffer with final concentration 50 mM NaCI, 20 mM Tris-HCI (pH 8.5), 1 mM dithiothreitol (DTT) and applied to a Bio-Rad Q1 Anion exchange column UNO Q1 equilibrated with 20 mM Tris (pH 8.5), 50 mM NaCI, 1 mM DTT.
  • the protein was eluted with 80 ml gradient from 50-250 mM NaCI (in 20 mM Tris pH 8.5). 2-ml fractions were collected at a flow rate of 2 ml/min and analyzed for SHP-2 enrichment and purity by SDS-PAGE, Coomassie staining and immunoblotting.
  • Enriched fractions were pooled and buffer was exchanged to PBS by PD-10 desalting columns and concentrated by Amicon Ultra-4, 10K centrifugal filters. Protein concentration was measured by a standard Bradford assay (Bio-Rad Laboratories).
  • SHP-2 tandem-SH2 For assessment of immunoreceptor tyrosine-based inhibitory motif (ITIM)-pY223 and the immunoreceptor tyrosine- based switch motif (ITSM)-pY248 binding on SHP-2 SH2 domains, a SHP-2 peptide comprising amino acids 1 -225, which only contains the SHP-2 N-SH2 and C-SH2 domains in their natural tandem sequence (referred to as SHP-2 tandem-SH2), was produced and purified.
  • ITIM immunoreceptor tyrosine-based inhibitory motif
  • ITSM immunoreceptor tyrosine-based switch motif
  • Binding with monophosphotyrosyl ITIM-pY223 (pITIM, KEDPSAVPVFSVD(pY)GELDFQWRE; SEQ ID NO: 2) or monophosphotyrosyl ITSM-pY248 (pITSM, KTPEPPVPCVPEQTE(pY)ATIVFPS; SEQ ID NO: 1 ) peptide was assessed. 20 mM of SHP-2 tandem-SH2 were mixed with either pITI M or pITSM peptide at various molar ratios in 50 mM HEPES, 100 mM NaCI, pH 7.4, in a final volume of 20 mI and incubated for one hour at room temperature. 5 mI 5X Native Sample Buffer were added and Native PAGE was performed at 4oC with 8-16% gradient Tris-Glycine gel (Bio-Rad) followed by Coomassie stain.
  • Phosphotyrosyl peptide pITSM (KTPEPPVPCVPEQTE(pY)ATIVFPS) (SEQ ID NO: 1 ) was synthesized at the Tufts University Protein Synthesis Core Facility. For immobilization, presentation of the peptide at high concentration was used to overcome the difficulties in immobilizing the negatively charged phosphopeptides (pl£3) to the negatively charged sensor chip.
  • HEK-293 For experiments with HEK-293 cells, one day before transfection, HEK-293 was plated as 20,000 cells in 0.1 ml DMEM complete medium per well, in 6 replicates per condition, on a white 96-well tissue culture plate. The following day cells were transfected by Fugene HD (Promega Corporation, Madison, Wl) according to the manufacturer's instructions. Briefly, plasmid DNA was diluted to 12.5 pg/ml in 0.1 ml Opti-MEM I (Fisher Scientific). For 4 plasmids, 3.125 pg/ml of each one in the above mixture was used.
  • the remaining plasmid DNA was supplemented with appropriate amount of empty vector (up to 12.5 pg/ml). 3.75 pi of Fugene were added to each DNA-Opti-MEM I mix and after 10 min incubation at room temperature 8 pi of each mixture were added per well and cells were placed back in the incubator. 30 hours after transfection, 25 pi of 1X Nano-Glo Live Cell Assay Substrate (Promega) was added per well and luminescence was read on a SpectraMax M3 plate reader (Molecular Devices). For experiments in Jurkat cells, transfection was performed by electroporation.
  • SHP-2 Catalytic activity of SHP-2 was monitored by a fluorescent assay using the substrate 6,8- difluoro-4- methylumbelliferone (DiFMUP) as previously with slight modifications. Specifically, the phosphatase reactions were performed in 50 mM HEPES, pH 7.4, 100 mM NaCI and 10 mM DTT in 96-well black polystyrene plate, flat bottom, non-binding surface, using a final reaction volume of 100 pi. 1.6 pg/ml of SHP-2-WT, SHP-2-R32A or SHP-2-R138A I
  • mutant proteins were incubated with 20 mM DiFMUP with the indicated concentrations of phosphotyrosyl peptides or without peptide at 37 °C. Fluorescence signal was monitored every 1 min for 3 min by a SpectraMax5 microplate reader and reaction rate was calculated by the change of fluorescence signal with time using excitation and emission wavelengths of 340 nm and 450 nm, respectively. Under these conditions product formation was linear with respect to the time of incubation. Results were normalized against the basal activity determined for each condition in the absence of peptide and activity was calculated. All peptides were synthesized at the Tufts University Core Facility. The sequence of the phosphopeptides used were as follows, with pY indicating the phosphorylated tyrosine.
  • MMonophosphoryl ITSM peptide KTPEPPVPCVPEQTE(pY)ATIVFPS (SEQ ID NO: 1 ).
  • MMonophosphoryl ITIM peptide KEDPSAVPVFSVD(pY)GELDFQWRE (SEQ ID NO: 2).
  • DBisphosphoryl ITSM peptide ITSM-Ahx4-ITSM: TE(pY)ATIVFP-Ahx4-QTE(pY)ATIVFPS (SEQ ID NO: 3; comprising SEQ ID NO: 22-Ahx4-SEQ ID NO: 23).
  • ITI M-Ahx4-ITIM VD(pY)GELDFQ-Ahx4-SVD(pY)GELDFQW (SEQ ID NO: 4).
  • ITI M-ITSM peptide pITI M-pITSM peptide
  • ITI M-Ahx4-ITSM VD(pY)GELDFQ-Ahx4- QTE(pY)ATIVFPS (SEQ ID NO: 5).
  • Monophosphoryl IRS-Y727 peptide TGD(pY)MNMSPVG (SEQ ID NO: 6).
  • Monophosphoryl IRS-Y1 172 SLN(pY)IDLDLVK (SEQ ID NO: 7).
  • Bisphosphoryl IRS peptide (bpIRS), 1 172-Ahx4-1222: LN(pY)IDLDLV-Ahx4-LST(pY)ASINFQK (SEQ ID NO: 8).
  • KTPEPPVPCVPEQTE(pY)ATIVFPS-Ahx4-KTPEPPVPCVPEQTE(pY)ATIVFPS (SEQ ID NO: 9; comprising SEQ ID NO: 1 -Ahx4-SEQ ID NO: 1 ).
  • KTPEPPVPCVPEQTE(pY)ATIVFPS-Ahx4-TE(pY)ATIVFP (SEQ ID NO: 10; comprising SEQ ID NO: 1 -Ahx4-SEQ ID NO: 22).
  • KTPEPPVPCVPEQTE(pY)ATIVFPS-Ahx4-QTE(pY)ATIVFPS (SEQ ID NO: 1 1 ; comprising SEQ ID NO: 1 -Ahx4-SEQ ID NO: 23).
  • KEDPSAVPVFSVD(pY)GELDFQWRE-Ahx4-SVD(pY)GELDFQW (SEQ ID NO: 14).
  • KTPEPPVPCVPEQTE(pY)ATIVFPS-Ahx4-KEDPSAVPVFSVD(pY)GELDFQWRE (SEQ ID NO: 15).
  • KTPEPPVPCVPEQTE(pY)ATIVFPS-Ahx4-VD(pY)GELDFQ SEQ ID NO: 16
  • KTPEPPVPCVPEQTE(pY)ATIVFPS-Ahx4-SVD(pY)GELDFQW (SEQ ID NO: 17).
  • Bisphosphoryl ITSM peptide plTSM-Ahx10-plTSM (bplTSM-Ahx10): TE(pY)ATIVFP-Ahx10-QTE(pY)ATIVFPS (SEQ ID NO: 24).
  • the cytoplasmic tail of PD-1 contains two tyrosine-based structural motifs, an immunoreceptor tyrosine-based inhibitory motif (ITI M) (V/L/l/XpYXX/LA/) and an immunoreceptor tyrosine-based switch motif (ITSM) (TXpYXXV/l). Mutational studies have shown that PD-1 inhibitory function is mainly dependent on the ITSM phosphotyrosine, which preferentially recruits SHP-2 phosphatase, resulting in dephosphorylation and downregulation of downstream effector molecules.
  • ITMI M immunoreceptor tyrosine-based inhibitory motif
  • ITSM immunoreceptor tyrosine-based switch motif
  • SHP-2 has two tandem SH2 domains, N-terminal (N-SH2) and C-terminal SH2 (C-SH2), followed by a single protein tyrosine phosphatase (PTP) domain, and a C-terminal hydrophobic tail with two tyrosine phosphorylation sites (FIG. 6A).
  • tyrosine phosphorylated receptors for growth factors such as platelet-derived growth factor (PDGF) as well as to tyrosine- phosphorylated docking proteins including insulin receptor substrates (IRSs), signal regulatory protein a (SIRPa; also known as SHP substrate-1 (SHPS-1 ), Grb2- associated binder proteins (Gabs), and fibroblast growth factor receptor substrate (FRS).
  • IRSs insulin receptor substrates
  • SIRPa signal regulatory protein a
  • Gabs Grb2- associated binder proteins
  • FES fibroblast growth factor receptor substrate
  • the N-SH2 domain of SHP-2 binds the phosphatase domain in an auto-inhibitory closed conformation and directly blocks the active phosphatase site. Interaction of the N-SH2 domain with phosphotyrosine peptide disrupts the interaction of N-SH2 with the phosphatase active site and activates the enzyme (FIG. 6B).
  • the C-SH2 domain contributes binding energy and specificity but does not have a direct role in enzymatic activation.
  • the crystal structure of SHP-2 has shown that the two SH2 domains of SHP-2 have a roughly antiparallel or roughly perpendicular orientation relative to one another, with the phosphopeptide binding sites lying fully exposed on the surface of the polypeptide and widely spaced (FIG. 6C).
  • the mechanisms by which PD-1 : SHP-2 interaction results in activation of SHP-2 phosphatase activity was examined.
  • SHP-2 interaction activates SHP-2 phosphatase activity. Since PD-1 is expressed after activation of human or mouse T cells either pre-activation of T cells or lentiviral transduction of PD-1 have been used to induce PD- 1 expression in primary T cells. To avoid changes that can be induced by such approaches in the tyrosine phosphorylation state of key signaling components of the TCR pathway, Jurkat T cells stably expressing human PD-1 (J-PD1 cells) were generated (FIG. 7A).
  • FIG. 7B PD- 1 immunoprecipitation showed that PD-1 ITSM-Y248 phosphorylation and SHP-2 co-precipitation were detected after aCD3/aCD28/PDL1 -lg incubation in J-PD-1 cells but was abrogated in J-PD-1 -Y248F cells (FIG. 1 B and FIG. 8B), indicating that Y248 in PD-1 ITSM has an essential role in PD-1 : SHP-2 interaction.
  • Y248 phosphorylation requires TCR signaling simultaneously with PD-1 ligation (FIG. 1A) and PD-1 lacks intrinsic kinase activity, which TCR-proximal kinases might be able to mediate PD-1 Y248 phosphorylation was examined.
  • COS cells were co-transfected with PD-1 cells, SHP-2, and either kinase-active or dominant negative forms of Fyn, Lck or ZAP-70 cDNAs.
  • constructs having single mutations of PD-1 ITIM (Y223F), ITSM (Y248F) or double mutations of ITI M and ITSM (Y223F/Y248F) were generated and co-transfected into COS cells with each PD-1 mutant or PD-1 wild type and cDNAs of Fyn and SHP-2.
  • PD-1 immunoprecipitation showed that SHP-2 interacted with PD-1-WT, and that mutation of ITI M (Y223F) did not affect this interaction (FIG. 1 D and FIG. 8D).
  • GST-fusion proteins (FIG. 2A) of SHP-2 full-length (GST-SHP-2-FL) and four SHP-2 deletion mutants were generated, GST-SHP-2-PTP, which contains only the protein tyrosine phosphatase (PTP) domain, GST-SHP-2-DNSH2, lacking the N-terminal SH2 domain, and GST-SHP-2-N-SH2 and GST-SHP-2-C- SH2, which contain only SHP-2 N134 SH2 or C-SH2 single domains.
  • GST-fusion proteins FIG. 2A
  • GST-SHP-2-PTP which contains only the protein tyrosine phosphatase (PTP) domain
  • GST-SHP-2-DNSH2 lacking the N-terminal SH2 domain
  • GST-SHP-2-N-SH2 and GST-SHP-2-C- SH2 which contain only SHP-2 N134 SH2 or C-SH2 single domains.
  • COS cells were transfected with SHP-2 constructs in which contained inactivating mutations in the active site of each SH2 domain: specifically SHP-2's arginine 32 residue (which is in the N-SH2 domain and which corresponds to the amino acid at position 32 of SEQ ID NO: 19) and SHP-2's arginine 138 (which is in the C-SH2 domain and which corresponds to the amino acid at position 28 of SEQ ID NO: 20); these residues are critical for mediating binding of each SH2 domain of SHP-2 with phosphotyrosine).
  • PD-1 ITSM-Y248F mutant abrogated interaction of PD-1 with wild type SHP-2 (FIG. 2C and FIG. 12B, bottom panels), consistent with the findings that PD-1 ITSM- Y248 is essential for interaction with SHP-2 (FIG. 1B and FIG. 1D).
  • PD-1 ITSM154 Y248F mutant abrogated interaction of PD-1 with either SHP-2 mutant (FIG. 2C and FIG. 12C, bottom panel), indicating that each SH2 domain of SHP-2 interacts with PD-1 ITSM-Y248 when co-expressed in live cells.
  • FIG. 2E binds phosphorylated ITSM-Y248 residues in two PD-1 polypeptides using its N-SH2 domain for one PD-1 and its C159 SH2 domain for a second PD-1 to form a PD-1 dimeric complex (FIG. 2E). Consistent with this model, the above- described binding studies (FIG.2D) showed that complete shift of SHP-2 from the unbound to the SHP-2: ITSM-pY248 bound form occurs at an ITSM-pY248 to SHP-2 ratio of greater than two.
  • FIG. 2D also indicated that when ITIM-pY223 and ITSM- pY248 phosphopeptides were incubated with SHP-2 SH2 domains at the same molar ratio, ITSM- pY248 is the preferred binding partner of SHP-2. Consistently, compared with a peptide containing both phosphorylated tyrosines of the native PD-1 cytoplasmic tail (FIG.
  • electrophoretic mobility shift of t-SHP-2 was preserved by a peptide containing phosphorylation of Y248 (PD- 1 cyto-ITI M-plTSM), but not by a peptide containing phosphorylation of Y223 (PD-1 cyto-plTIM-ITSM) (FIG. 2F middle and bottom panel).
  • PD-1 ITSM-pY248 serves as the high affinity binding site for one of the SHP-2 SH2 domains thereby being a pre-requisite for the binding of the second SH2 domain on PD- 1 ITIM Y223 that serves the low affinity interaction site (FIG. 2G).
  • This model can provide an explanation as to why PD-1 ITSM-Y248F disrupted the interaction of PD-1 with either SHP-2 WT or each SHP-2 SH2 active site mutant (FIG. 2C, bottom panel) and why ITI M-Y223F did not affect interaction with SHP-2 WT.
  • a second model is that both SH2 domains interact with ITSM- pY248 and because the PD-1 molecule has only one ITSM-Y248, SHP-2 binds phosphorylated ITSM-pY248 residues in two PD-1 molecules using its N-SH2 domain for one PD-1 and its C-SH2 domain for a second PD-1 to form a PD-1 dimer (FIG. 2H).
  • the latter can explain why PD-1 ITSM-Y248F disrupted the interaction of PD-1 with either SHP-2 WT or each SHP-2 SH2 active site mutant (FIG. 2C, bottom panel).
  • This model can also explain why in cells expressing PD-1 ITIM-Y223F the interaction of PD-1 with SHP-2 WT was not affected but the interaction of PD-1 with each SH2 active site mutant was disrupted (FIG. 2C, middle panel).
  • SPR Surface plasmon resonance
  • SHP-2 interaction was assessed by isothermal titration calorimetry (ITC).
  • ITC isothermal titration calorimetry
  • t-SHP-2 The SHP-2 protein construct that contains only the two tandem SH2 domains (t-SHP-2) and a phosphopeptide corresponding to the native PD-1 cytoplasmic tail in which both ITIM-Y223 and ITSM-Y248 tyrosines were phosphorylated (PD-201 1 cyto-plTIM-plTSM) were used to assess how PD-1 : SHP-2 interaction might occur in the presence of both phosphotyrosines.
  • PD-1 cyto-plTIM-plTSM t-SHP-2 interaction occurred at 1 : 1 stoichiometry (FIG. 3E).
  • a phosphopeptide was used corresponding to the native PD-1 cytoplasmic tail in which ITSM- Y248 but not ITI M-Y223, which was phosphorylated (PD-1 cyto-ITI M-pITSM).
  • PD-1 cyto-ITIM-plTSM t-SHP-2 interaction occurred at 2: 1 stoichiometry (FIG. 3F) providing biophysical evidence that interaction of SHP-2 SH2 domains with ITSM-pY248 from two PD-1 molecules to form a PD-1 dimer is feasible.
  • PD-1 SHP-2 interaction can occur in two different ways: 1 ) One PD-1 molecule can bind with SHP-2 tandem SH2 domains likely using both phosphotyrosines, each of which interacts with one SH2 domain; 2) PD-1 : SHP-2 interaction can also occur by binding of SHP-2 SH2 domains solely with ITSM- pY248 phosphotyrosine. In the latter binding mode, ITSM- pY248 on two PD-1 molecules interact with each of the tandem SH2 domains of SHP-2 indicating that SHP-2 can bridge two PD-1 molecules via its N-SH2 and C-SH2 domains, to form a PD-1 dimer.
  • SHP-2 bridges two PD-1 polypeptides via PD-1 ITSM pY248
  • NanoBiT proximity assay (Promega) was used, which permits detection of protein: protein interaction in living cells by employing a split luciferase enzyme.
  • Large BiT LiBiT; 17.6 kDa
  • Small BiT (SmBiT;
  • PD-1-LgBiT in which the PD-1 cytoplasmic domain was linked to the LgBiT peptide sequence
  • PD-1 -SmBiT in which the PD-1 cytoplasmic domain was linked to the SmBiT peptide sequence
  • HEK-293 cells were transfected with PD-1-LgBiT and PD-1 -SmBiT together with SHP-2 and Fyn kinase, to recapitulate the effect of TCR-mediated signaling and interaction with SHP-2 required for PD-1 phosphorylation.
  • Assessment of complex formation between PD-1-SmBiT and PD-1 -LgBiT by measuring luminescent signal showed that in the presence of kinase-active Fyn but not kinase inactive Fyn, SHP-2 induced PD-1 : PD-1 interaction (FIG. 4A, FIG. 15A, and FIG. 15B).
  • the allosteric SHP-2 inhibitor SHP099 (which prevents the conformation change of SHP-2 and the release of the N-SH2 domain from the PTP site that is induced upon interaction of N-SH2 with phosphorylated substrates) was used.
  • PD-1 dimers bridged by SHP-2 are formed in T cells during antigen encounter and PD-1 ligation
  • primary human T cells or Jurkat T cells were transfected with PD-1-LgBiT and PD-1 -SmBiT.
  • the expression level of PD-1 induced by PD-1 -LgBiT and PD-1 -SmBiT transfection was comparable to that induced in activated primary human T cells (FIG. 16A and FIG. 16B), confirming that PD-1 -LgBiT and PD-1 -SmBiT transfection did not induce artificial overexpression of PD-1.
  • the transfected primary human T cells or Jurkat T cells were stimulated with Raji cells which stably expressed human PD-L1 with or without prior loading with SEE (FIG. 17A and FIG. 17B).
  • Co-culture with Raji- PD-L1 loaded with SEE but not without SEE loading resulted in an elevated luminescent signal. This was significantly diminished by expression of SHP-2-R32A or SHP-2- R138A that were mutagenized in the active site of N-SH2 or C- SH2 domain, respectively, or the SHP-2-DM that was mutagenized in the active sites of both N-SH2 and C-SH2 domains (FIG. 4F and FIG. 4G).
  • Tyrosine phosphorylated motifs that interact with SHP-2 SH2 domains have a decisive role in the activation of SHP-2 PTP enzymatic activity.
  • SHP-2 activity measurements were performed using DiFMUP (6,8-difluoro-4-methylumbelliferyl phosphate) substrate.
  • PD-1 ITSM224 pY248 pITSM
  • PD-1 ITIM-pY223 pITI M
  • monophosphorylated peptide IRS-1 -pY1 172 plRSY1 172
  • IRS-1 -pY727 plRSY727
  • SHP-2 phosphatase enzymatic activity showed that the effect of PD-1 pITIM was comparable to the established negative control plRSY727 (FIG. 5A).
  • PD-1 pITSM induced approximately 5-fold increase of SHP-2 activity, which was higher than the activity induced by plRSY1 172 (FIG. 5A), previously established as a monophosphorylated peptide that can induce SHP-2 phosphatase activation.
  • PD-1 ITSM-Y248 would affect SHP-2 phosphatase activity was examined.
  • the two SH2 domains of SHP-2 have a roughly antiparallel or roughly perpendicular orientation relative to one another with the phosphopeptide binding sites widely separated (FIG. 6C).
  • the distance between the phosphopeptide binding sites of the SH2 domains is 41 A in the crystal structure of the tandem SH2 domains and is critical for phosphoprotein recognition and for enzymatic activation of SHP-2.
  • a bisphosphoryl plTSM-Y248 peptide (bpITSM) that matches the spacing between the phosphopeptide binding sites of the SHP-2 SH2 domains was generated, by covalent joining of two plTSM242 Y248 phosphopeptides with a 4 amino hexanoic acid (Ahx) spacer (SEQ ID NO: 3).
  • bpITSM bisphosphoryl plTSM-Y248 peptide
  • Ahx hexanoic acid spacer
  • a similarly designed peptide was generated with two plTI M-Y223 phosphopeptide sequences (bisphosphoryl ITIM:“bpITIM”) by covalent attachment of two plTIM-Y223 with a 4-Ahx spacer to determine whether SHP-2 would interact with two phosphorylated PD-1 ITI M- Y223 (SEQ ID NO: 4).
  • a similarly designed peptide was generated with one pITI M and one plTSM-Y248 covalently attached with a 4-Ahx spacer (bisphosphorylated ITI M-ITSM: "pITI M-pITSM”; SEQ ID NO: 5) to examine whether interaction of SHP-2 with one plTIM-Y223 and one plTSM-Y248 might activate SHP-2 phosphatase activity.
  • Measurements of SHP-2 catalytic activity showed that bpITI M (SEQ ID NO: 4) or pITIM-pITSM (SEQ ID NO: 5) did not induce an increase in the catalytic activity of SHP-2 compared to monophosphoryl pITIM or monophosphoryl pITSM, respectively (FIG.
  • PD-1 bpITSM Compared with bpIRS, an established bisphosphoryl peptide activator of SHP-2 catalytic activity, PD-1 bpITSM was approximately three times more potent in inducing SHP-2 activation (FIG. 18). Loss of the active site of either N-SH2 or C-SH2 domain in SHP-2-R32A and SHP-2-R138A, respectively, abrogated SHP-2 activity caused by bpITSM at peptide concentrations below 1 mM (FIG. 5C). A small induction of SHP-2 activity in the SHP-2- R138A was observed at peptide concentrations above 1 pM.
  • bpITSM the phosphopeptide containing two pITSM regions that could induce SHP-2 activation
  • the reaction supplemented with increasing amounts of monophosphorylated pITIM or monophosphorylated pITSM peptide to assess if they could compete with bpITSM phophopeptide for SHP-2 binding and phosphatase activation.
  • pITI M did not disrupt SHP-2 activation induced by the bpITSM phosphopeptide
  • addition of pITSM decreased SHP-2 phosphatase activity (FIG. 5D).
  • a bisphosphorylated peptide was generated by covalently joining two plTSM-Y248 phosphopeptides with a 10-Ahx spacer (bplTSM-Ahx10; SEQ ID NO: 24), which corresponds to the distance of PD-1 phosphotyrosines.
  • bplTSM-Ahx10 SEQ ID NO: 24
  • experiments disclosed herein demonstrate ITSM-Y248 phosphorylation and also how spatial distribution of phosphorylated PD-1 molecules in proximity to TOR signaling substrates in the TOR microclusters will affect the inhibitory function of PD-1 , because only PD-1 molecules bridged through plTSM-Y248 by the two SH2 domains of SHP-2 at such a distance that can induce SHP-2 conformational change allowing activation of SHP-2 phosphatase will be responsible for inhibition of T cell responses. Moreover, in contrast to the recruitment of PD-1 to the TOR I
  • the 64-kDa protein that associates with the platelet- derived growth factor receptor beta subunit via Tyr-1009 is the SH2-containing phosphotyrosine phosphatase Syp. Proc Natl Acad Sci USA 90, 6939-6943 (1993).
  • the insulin receptor substrate 1 associates with the SH2-containing phosphotyrosine phosphatase Syp. J. Biol. Chem. 268, 1 14791 1481 (1993).
  • Patsoukis, N. ef a/. PD-1 alters T-cell metabolic reprogramming by inhibiting glycolysis and promoting lipolysis and fatty acid oxidation. Nat. Commun. 6, 6692, doi: 10.1038/ncomms7692 (2015).
  • Patsoukis, N. ef a/. RIAM Regulates the Cytoskeletal Distribution and Activation of PLC- ⁇ gamma ⁇ 1 in T Cells. Science signaling 2, ra79 (2009).
  • Boussiotis V. A. ef al. Differential association of protein tyrosine kinases with the T cell receptor is linked to the induction of anergy and its prevention by B7 family-mediated costimulation. J. Exp. Med. 184, 365-376 (1996).
  • PD-1 increases PTEN phosphatase activity while decreasing PTEN protein stability by inhibiting casein kinase 2. Mol. Cell. Biol. 33, 3091 -3098 (2013).

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Abstract

La présente invention concerne des compositions et des procédés de modulation de gènes de points de contrôle immunitaires, par exemple, un récepteur de mort programmée (PD-1).
PCT/US2019/065983 2018-12-14 2019-12-12 Modulation de pd-1 Ceased WO2020123806A1 (fr)

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CN112694535A (zh) * 2021-01-05 2021-04-23 重庆医科大学 用于抗体检测的多功能蛋白分子开关
US11434291B2 (en) 2019-05-14 2022-09-06 Provention Bio, Inc. Methods and compositions for preventing type 1 diabetes
CN115197314A (zh) * 2021-04-11 2022-10-18 中国科学院分子细胞科学卓越创新中心 一种pd1变体及其用途
US12006366B2 (en) 2020-06-11 2024-06-11 Provention Bio, Inc. Methods and compositions for preventing type 1 diabetes
US12565529B2 (en) 2021-05-24 2026-03-03 Provention Bio, Inc. Methods for treating type 1 diabetes

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WO2018084204A1 (fr) * 2016-11-02 2018-05-11 国立大学法人京都大学 Marqueur de détermination d'efficacité dans le traitement de maladie par un inhibiteur de signal pd-1
WO2018160731A1 (fr) * 2017-02-28 2018-09-07 Novartis Ag Compositions d'inhibiteur shp et utilisations pour une thérapie de récepteur d'antigène chimère
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WO2018084204A1 (fr) * 2016-11-02 2018-05-11 国立大学法人京都大学 Marqueur de détermination d'efficacité dans le traitement de maladie par un inhibiteur de signal pd-1
WO2018160731A1 (fr) * 2017-02-28 2018-09-07 Novartis Ag Compositions d'inhibiteur shp et utilisations pour une thérapie de récepteur d'antigène chimère
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11434291B2 (en) 2019-05-14 2022-09-06 Provention Bio, Inc. Methods and compositions for preventing type 1 diabetes
US12006366B2 (en) 2020-06-11 2024-06-11 Provention Bio, Inc. Methods and compositions for preventing type 1 diabetes
CN112694535A (zh) * 2021-01-05 2021-04-23 重庆医科大学 用于抗体检测的多功能蛋白分子开关
CN112694535B (zh) * 2021-01-05 2023-03-31 重庆医科大学 用于抗体检测的多功能蛋白分子开关
CN115197314A (zh) * 2021-04-11 2022-10-18 中国科学院分子细胞科学卓越创新中心 一种pd1变体及其用途
CN115197314B (zh) * 2021-04-11 2025-08-19 中国科学院分子细胞科学卓越创新中心 一种pd1变体及其用途
US12565529B2 (en) 2021-05-24 2026-03-03 Provention Bio, Inc. Methods for treating type 1 diabetes

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