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WO2024191989A1 - Treatment of b cell-mediated diseases by activating cd20-targeted gamma delta t cells - Google Patents

Treatment of b cell-mediated diseases by activating cd20-targeted gamma delta t cells Download PDF

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Publication number
WO2024191989A1
WO2024191989A1 PCT/US2024/019515 US2024019515W WO2024191989A1 WO 2024191989 A1 WO2024191989 A1 WO 2024191989A1 US 2024019515 W US2024019515 W US 2024019515W WO 2024191989 A1 WO2024191989 A1 WO 2024191989A1
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domain
cells
cell
protein
amino acid
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French (fr)
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Taylor Schreiber
Suresh DE SILVA
George FROMM
Anne LAI
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Shattuck Labs Inc
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Shattuck Labs Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2887—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD20
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

  • SEQUENCE LISTING The instant application contains a sequence listing, which has been submitted in XML format via EFS- Web. The contents of the XML copy named “SHK-081PC_116981-5081_Sequence_Listing,” which was created on March 5, 2024, and is 41,423 bytes in size, and the contents of which are incorporated herein by reference in their entirety.
  • BACKGROUND Gamma Delta T cells ⁇ T cells or GDT are capable of directly recognizing and killing transformed cells independently of HLA-antigen presentation.
  • the V ⁇ 9V ⁇ 2 T-cell receptor is believed to sense intracellular phosphorylated metabolites, which accumulate in cancer cells as a result of metabolic dysregulation (e.g., dysregulation of the mevalonate pathway) and/or upon pharmaceutical intervention. Therefore, the Gamma Delta T cells represent highly promising effector cell compartment for immunotherapy. However, these cells occur in very small amounts. For example, although these cells can rapidly expand upon stimulation, healthy individuals typically harbor on the order of only 50-500 million V ⁇ 9V ⁇ 2 T-cells in the peripheral blood, which is 1-10% of the total CD3+ T-cell population. No currently available therapeutic agent employs the Gamma Delta T cells for a therapeutic purpose in human.
  • the present disclosure relates to a compositions and methods that harness potential Gamma Delta T cells for immunotherapies for cancer, or autoimmune diseases or disorders.
  • the present disclosure relates to a method for treating a B cell-mediated disease or disorder, comprising administering to the subject about 0.01 mg/kg to about 100 mg/kg of a heterodimeric protein, or a nucleic acid encoding the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; (b) a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof, the target domain being directed to CD20; and (c) a linker that adjoins the first and second domains; and wherein the beta chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof,
  • the present disclosure relates to a method for increasing and/or stimulating B cell depletion in a subject suffering from a B cell-mediated disease or disorder, comprising administering to the subject about 0.01 mg/kg to about 100 mg/kg of a heterodimeric protein, or a nucleic acid encoding the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; (b) a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof, the target domain being directed to CD20; and (c) a linker that adjoins the first and second domains; and wherein the beta chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and
  • the heterodimeric protein is administered at a frequency of about 1 day, or about 2 days, or about 3 days, or about 4 days, or about 5 days, or about 6 days, or about one week, or about 10 days, or about 15 days, or about 21 days, or more. In embodiments, the heterodimeric protein is administered at a frequency of about 3 days. In embodiments, the heterodimeric protein is administered at a frequency of about 7 days. In embodiments, the heterodimeric protein is administered at a frequency of about 1 day to about 10 days, about 1 day to about 5 days, or about 2 days to about 10 days, or about 5 days to about 15 days, or about one week to about 3 weeks.
  • the heterodimeric protein is administered at a frequency of about 1 day to about 10 days. In embodiments, the heterodimeric protein is administered so as to form a ratio of effector cells (heterodimeric protein-bound Gamma-Delta T cells) to target cells (without limitations, e.g., cancer cells) is about 1:1, or about 1:2, or about 1:5, or about 1:7, or about 1:10, or about 1:15, or about 1:20 or less.
  • the heterodimeric protein is administered so as to form a ratio of effector cells (heterodimeric protein-bound Gamma-Delta T cells) to target cells (without limitations, e.g., cancer cells) is about 1:1 to about 1:5, or about 1:2 to about 1:8, or about 1:10 to about 1: 20.
  • the targeting domain is capable of binding CD20 on the surface of a B cell and/or a cancer cell.
  • the targeting domain comprises an Fv domain.
  • the targeting domain is an antibody-like molecule, or antigen binding fragment thereof.
  • the targeting domain comprises a single-chain variable fragment (scFv), or a fragment thereof. In embodiments, the targeting domain comprises different scFvs. In embodiments, the targeting domain specifically binds human CD20.
  • the nucleic acid is or comprises a DNA or In embodiments, the nucleic acid is or comprises an expression vector. In embodiments, the method modulates or is suitable for modulating a ⁇ (gamma delta) T cell. In embodiments, the gamma delta T cell expresses V ⁇ 4 or V ⁇ 9 ⁇ 2.
  • the first domain comprises BTN2A1 and it modulates a V ⁇ 4-expressing T cell. In embodiments, the first domain modulates a V ⁇ 9 ⁇ 2-expressing T cell.
  • the modulation of a gamma delta T cell is activation of a gamma delta T cell.
  • the method induces killing of a target cell.
  • the e target cell is a human cell.
  • the method : (i) modulates or is suitable for modulating a ⁇ (gamma delta) T cell, and/or (ii) stimulates or promotes localizing to a tumor, and/or binding to a tumor cell, and/or engaging a tumor cell, and/or promoting an immune stimulatory signal, and/or inhibiting an immune inhibitory signal, and/or forming an immune synapse
  • the immune synapse is a synapse between a gamma delta T cell and a tumor cell and/or the chimeric protein is capable of contemporaneous activation and targeting of gamma delta T cells to tumor cells.
  • the B cell depletion comprises a reduction in the numbers and/or activity of one or more B cells.
  • the B cells are hCD45+CD3-CD19+CD20+IgD+.
  • the method does not reduce and/or suppress a non-B cell population of cells.
  • the method does not reduce and/or suppress non-B/T cells (e.g., hCD45+CD3-CD19-), V ⁇ 2+ T cells (e.g., hCD45+CD3+V ⁇ 2+), and/or total T cells (e.g., hCD45+CD3+CD19-).
  • the method does not increase and/or stimulate non-B/T cells (e.g., hCD45+CD3-CD19-), V ⁇ 2+ T cells (e.g., hCD45+CD3+V ⁇ 2+), and/or total T cells (e.g., hCD45+CD3+CD19-).
  • the B cell-mediated disease or disorder is a cancer.
  • the cancer is a cancer expressing CD20.
  • the cancer is a B-cell malignancy.
  • the B-cell DB1/ 145191832.2 3 Attorney Docket No.: SHK-081PC/116981-5081 malignancy is a lymphoma or leukemia.
  • the B-cell malignancy is non-Hodgkin’s lymphoma (NHL) or Hodgkin’s lymphoma (HL).
  • the e B-cell malignancy is advanced stage follicular lymphoma, chronic lymphocytic leukemias (CLL), Burkitt’s lymphoma, or diffuse large B- cell lymphoma.
  • the B-cell mediated disease or disorder is an autoimmune disease or disorder.
  • the autoimmune disease or disorder is one or more of rheumatoid arthritis, systemic lupus erythematosus, diabetes mellitus, ankylosing spondylitis, Sjögren's syndrome, inflammatory bowel diseases (e.g., colitis ulcerosa, Crohn's disease), multiple sclerosis, sarcoidosis, psoriasis, Grave's disease, Hashimoto's thyroiditis, psoriasis, hypersensitivity reactions (e.g., allergies, hay fever, asthma, and acute edema cause type I hypersensitivity reactions), pemphigus, and vasculitis (e.g., Granulomatosis with polyangiitis).
  • rheumatoid arthritis systemic lupus erythematosus
  • diabetes mellitus ankylosing spondylitis
  • Sjögren's syndrome inflammatory bowel diseases
  • inflammatory bowel diseases e.g., colitis
  • FIG.1A is non-limiting, schematic illustration of a BTN2A1/3A1-Fc-CD20scFv heterodimeric protein disclosed herein (also referred to herein as a BTN2A1/3A1-Fc-CD20scFv GAmma DELta T cell ENgager (GADLEN)).
  • FIG.1B shows, without wishing to be bound by theory, a schematic illustration of the mechanism of target-cell depletion mediated by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein.
  • the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein engages a target cell via the scFv fragment, and a gamma delta T cell via the butyrophilin heterodimer.
  • FIG.2A to FIG.2C illustrate the an in vitro activity of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein.
  • FIG.2A shows the degranulation of V ⁇ 9V ⁇ 2 + T cells induced by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein in a plate-based assay.
  • FIG.2B is a graph showing an in vitro depletion of CD20+ lymphoma cells using human Daudi Burkitt’s lymphoma cells (CD20+ lymphoma cell line).
  • FIG.2C is a graph showing an in vitro depletion of CD20+ lymphoma cells using CD20+ B cells purified from peripheral blood mononuclear cells (“PBMCs”) of a healthy donor.
  • PBMCs peripheral blood mononuclear cells
  • FIG. 3E show the in vivo human B cell depletion by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein in PBMC-humanized mice.
  • FIG. 3A shows a schematic representation of experimental design.
  • FIG.3B shows B cells (hCD45+CD3-CD19+CD20+IgD+).
  • FIG.3C shows non-B/T cells (hCD45+CD3-CD19-),
  • FIG.3D shows V ⁇ 2+ T cells (hCD45+CD3+V ⁇ 2+), and
  • FIG.3E shows the total T cells (hCD45+CD3+CD19-) by flow cytometry.
  • FIG.3B to FIG.3E represent animals engrafted with PBMCs derived from two different donors, containing high and low input ratios of V ⁇ 2+ T cells (Effector, E) and B cells (Target, T), respectively.
  • FIG.4A to FIG.4E show the in vivo anti-tumor activity of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein in a mouse model.
  • FIG.4A shows a schematic representation of experimental design.
  • FIG.4B shows the tumor growth in vehicle-only treated mice.
  • FIG.4C shows the tumor growth in mice treated with the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein.
  • FIG.4D shows the tumor growth in mice treated with the BTN2A1/3A1-Fc-B7H3scFv heterodimeric protein, which was used as a negative control.
  • FIG.4E shows the tumor growth in mice treated with a CD3 ⁇ CD20 engager, which was used as a positive control.
  • FIG.5A to FIG.5N are graphs showing the effects of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein in cynomolgus macaques.
  • FIG.5A is a line graph showing the level of the B cell occupancy by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein as the function of time in the indicated cynomolgus monkeys.
  • FIG.5B shows the comparison of pre-dose and post dose occupancy of the CD3- CD19 + target cells by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein in monkey 4501 as determined by flow cytometry.
  • FIG.5C is a line graph showing the level of the Gamma Delta T cell occupancy by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein as the function of time in the indicated cynomolgus monkeys.
  • FIG.5D shows the comparison of pre-dose and post dose occupancy of the V ⁇ 9 + target cells by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein in monkey 4501 as determined by flow cytometry.
  • FIG.5E is a line graph showing the kinetics of change in the levels of B cells in total DB1/ 145191832.2 5 Attorney Docket No.: SHK-081PC/116981-5081 lymphocytes in monkeys treated with the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein at the indicated doses.
  • FIG.5F is a line graph showing the kinetics of change in the levels of CD4 + cells in total lymphocytes in monkeys treated with the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein at the indicated doses.
  • FIG.5G is a line graph showing the kinetics of change in the levels of V ⁇ 9 + cells in total lymphocytes in monkeys treated with the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein at the indicated doses.
  • FIG.5H is a line graph showing the kinetics of B cell depletion in monkeys treated with the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein at the indicated doses.
  • FIG.5I are line graphs showing the pharmacokinetic profile of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein from left to right after first dose, second dose and third dose, respectively.
  • FIG.5J is a bar graph showing the distribution of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein in indicated tissues 48 hours after Dose 3.
  • FIG.5K are line graphs showing the kinetics of receptor occupancy (RO) of B cells by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein from left to right after first dose, second dose and third dose, respectively.
  • RO receptor occupancy
  • FIG.5L are line graphs showing the kinetics of receptor occupancy (RO) of gamma delta T cells by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein from left to right after first dose, second dose and third dose, respectively.
  • FIG.5M is box chart showing the changes in the levels of peripheral B cells after Dose 1 and Dose 3, compared to pre-dose levels.
  • FIG.5N is box chart showing the changes in the levels of peripheral gamma delta T cells after Dose 1 and Dose 3, compared to pre- dose levels.
  • the present disclosure relates to a method for increasing and/or stimulating B cell depletion in a subject suffering from a B cell-mediated disease or disorder, comprising administering to the subject a heterodimeric protein, or a nucleic acid encoding the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises: a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof, the target domain being directed to CD20; and a linker that adjoins the first and second domains; and wherein the beta chain comprises: a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; a second domain comprising
  • the fragment comprises an extracellular domain (ECD).or a variable domain.
  • ECD extracellular domain
  • DB1/ 145191832.2 6 Attorney Docket No.: SHK-081PC/116981-5081
  • the first domains of alpha chain and beta chain comprise ECDs, variable domains, or a combination of an ECD and a variable domain of the same butyrophilin family protein.
  • the first domains of alpha chain and beta chain comprise ECDs, variable domains, or a combination of an ECD and a variable domain of different butyrophilin family proteins.
  • the butyrophilin family protein of the alpha chain and beta chain is independently selected from human BTN2A1 and human BTN3A1.
  • the first domains of alpha chain and beta chain comprise extracellular domains and/or variable domains of BTN2A1 and BTN3A1. In embodiments, the first domains of alpha chain and beta chain comprise variable domains of BTN2A1 and BTN3A1.
  • the first domain of alpha chain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence selected from SEQ ID NOs: 13- 16; and the first domain of beta chain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence selected from SEQ ID NOs: 13-16.
  • the first domains of alpha chain and beta chain comprise a polypeptide having an amino acid sequence of: any one of SEQ ID NOs: 13-16; and any one of SEQ ID NOs: 13-16, respectively.
  • the targeting domain is capable of binding CD20 on the surface of a B cell and/or a cancer cell.
  • the targeting domain comprises an Fv domain.
  • the targeting domain is an antibody-like molecule, or antigen binding fragment thereof.
  • the targeting domain comprises a single-chain variable fragment (scFv), or a fragment thereof.
  • the targeting domain comprises different scFvs.
  • the targeting domain specifically binds human CD20.
  • the targeting domain comprises an scFv that specifically binds human CD20.
  • the targeting domain comprises a polypeptide comprising the complementarity determining regions (CDRs) present in SEQ ID NOs: 17-20.
  • the targeting domain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence selected from SEQ ID NOs: 17-20.
  • the linker comprises a polypeptide selected from a flexible amino acid sequence, an IgG hinge region, and an antibody sequence.
  • the antibody sequence comprises an Fc DB1/ 145191832.2 7 Attorney Docket No.: SHK-081PC/116981-5081 domain.
  • the linker comprises a hinge-CH2-CH3 Fc domain.
  • the hinge- CH2-CH3 Fc domain is derived from IgG1, optionally from human IgG1.
  • the hinge-CH2- CH3 Fc domain is derived from IgG4, optionally from human IgG4.
  • the hinge-CH2-CH3 Fc domain comprises a polypeptide having an amino acid sequence with at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide selected from SEQ ID NOs: 1-12.
  • the nucleic acid is or comprises a DNA or In embodiments, the nucleic acid is or comprises an expression vector.
  • the method modulates or is suitable for modulating a ⁇ (gamma delta) T cell.
  • the gamma delta T cell expresses V ⁇ 4 or V ⁇ 9 ⁇ 2.
  • the first domain comprises BTN2A1 and it modulates a V ⁇ 4-expressing T cell.
  • the first domain modulates a V ⁇ 9 ⁇ 2-expressing T cell.
  • the modulation of a gamma delta T cell is activation of a gamma delta T cell.
  • the method induces killing of a target cell.
  • the e target cell is a human cell.
  • the method : (i) modulates or is suitable for modulating a ⁇ (gamma delta) T cell, and/or (ii) stimulates or promotes localizing to a tumor, and/or binding to a tumor cell, and/or engaging a tumor cell, and/or promoting an immune stimulatory signal, and/or inhibiting an immune inhibitory signal, and/or forming an immune synapse.
  • the immune synapse is a synapse between a gamma delta T cell and a tumor cell and/or the chimeric protein is capable of contemporaneous activation and targeting of gamma delta T cells to tumor cells.
  • the B cell depletion comprises a reduction in the numbers and/or activity of one or more B cells.
  • the B cells are hCD45+CD3-CD19+CD20+IgD+.
  • the method does not reduce and/or suppress a non-B cell population of cells.
  • the method does not reduce and/or suppress non-B/T cells (e.g., hCD45+CD3-CD19-), V ⁇ 2+ T cells (e.g., hCD45+CD3+V ⁇ 2+), and/or total T cells (e.g., hCD45+CD3+CD19-). In embodiments, the method does not increase and/or stimulate non-B/T cells (e.g., hCD45+CD3-CD19-), V ⁇ 2+ T cells (e.g., hCD45+CD3+V ⁇ 2+), and/or total T cells (e.g., hCD45+CD3+CD19-). In embodiments, the B cell-mediated disease or disorder is a cancer.
  • the cancer is a cancer expressing CD20.
  • the cancer is a B-cell malignancy.
  • the B-cell malignancy is a lymphoma or leukemia.
  • the B-cell malignancy is non-Hodgkin’s lymphoma (NHL) or Hodgkin’s lymphoma (HL).
  • the e B-cell malignancy is advanced stage follicular lymphoma, chronic lymphocytic leukemias (CLL), Burkitt’s lymphoma, or diffuse large B- DB1/ 145191832.2 8 Attorney Docket No.: SHK-081PC/116981-5081 cell lymphoma.
  • the cancer is a solid cancer selected from prostate cancer, small cell lung cancer, non-small cell lung cancer, neuroendocrine tumor, renal cancer, bladder cancer, colon cancer, and breast cancer.
  • the cancer has mutations which limit recognition by alpha beta T cells, optionally selected from mutations in MHC I, beta 2 microglobulin, and Transporter associated with antigen processing (TAP).
  • TAP Transporter associated with antigen processing
  • the B cell-mediated disease or disorder is an autoimmune disease or disorder.
  • the autoimmune disease or disorder is one or more of rheumatoid arthritis, systemic lupus erythematosus, diabetes mellitus, ankylosing spondylitis, Sjögren's syndrome, inflammatory bowel diseases (e.g., colitis ulcerosa, Crohn's disease), multiple sclerosis, sarcoidosis, psoriasis, Grave's disease, Hashimoto's thyroiditis, psoriasis, hypersensitivity reactions (e.g., allergies, hay fever, asthma, and acute edema cause type I hypersensitivity reactions), pemphigus, and vasculitis (e.g., Granulomatosis with polyangiitis).
  • rheumatoid arthritis systemic lupus erythematosus
  • diabetes mellitus ankylosing spondylitis
  • Sjögren's syndrome inflammatory bowel diseases
  • inflammatory bowel diseases e.g., colitis
  • heterodimeric proteins e.g., a BTN2A1/3A1-Fc-CD20scFv heterodimeric protein
  • BTN2A1/3A1-Fc- CD20scFv GAmma DELta T cell ENgager (GADLEN) proteins used interchangeably with “GADLEN proteins”.
  • GDLEN proteins used interchangeably with “GADLEN proteins”.
  • the heterodimeric proteins disclosed herein direct gamma delta T cells and other specific effectors of the immune system to target tumor cells, enhancing their cytotoxicity.
  • the heterodimeric proteins provide a higher binding specificity to cancer cells. In embodiments, the heterodimeric proteins enable combining multiple anticancer mechanisms on the same cancer cell. In embodiments, the heterodimeric proteins increase cancer cell specificity and apoptosis, improved antiproliferative effect and decreased the development of resistance.
  • the present disclosure relates to a heterodimeric protein, and methods of use of the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof; (b) a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof; and (c) a linker that adjoins the first and second domains; and wherein the beta chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof; (b) a second domain comprising the targeting domain; and (c) a linker that adjoins the first and second domains.
  • the fragment comprises a variable domain.
  • the alpha chain linker and DB1/ 145191832.2 9 Attorney Docket No.: SHK-081PC/116981-5081 the beta chain linker are charged polarized linkers, wherein one of the alpha chain linker and the beta chain linker is positively charged and the other is negatively charged.
  • the present disclosure relates to a heterodimeric protein, and methods of use of the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises: (a) a variable domain of BTN2A1; (b) a second domain comprising a single-chain antibody (scFv); and (c) a linker that adjoins the first and second domains; and wherein the beta chain comprises: (a) a first domain comprising an extracellular domain (ECD) or a variable domain of BTN3A1; (b) a second domain comprising an anti- CD20 single-chain antibody (scFv); and (c) a linker that adjoins the first and second domains.
  • ECD extracellular domain
  • scFv anti- CD20 single-chain antibody
  • the heterodimeric protein (i) modulates or is suitable for modulating a ⁇ (gamma delta) T cell, and (ii) suitable for localizing to a tumor, and/or binding to a tumor cell, and/or engaging a tumor cell, and/or promoting an immune stimulatory signal, and/or inhibiting an immune inhibitory signal, and/or forming an immune synapse.
  • the modulation of a gamma delta T cell is activation of a gamma delta T cell.
  • the immune synapse is a synapse between a gamma delta T cell and a tumor cell and/or the chimeric protein is suitable for contemporaneous activation and targeting of gamma delta T cells to tumor cells.
  • the gamma delta T cell expresses V ⁇ 4 or V ⁇ 9 ⁇ 2.
  • the first domain comprises BTNL3 and BTNL8 and it modulates a V ⁇ 4-expressing T cell.
  • the first domain modulates a V ⁇ 9 ⁇ 2-expressing T cell.
  • the first domain comprises BTN2A1 and BTN3A1.
  • the present disclosure relates to a heterodimeric protein, and methods of use of the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof; (b) a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof; and (c) a linker that adjoins the first and second domains; and wherein the beta chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof; (b) a second domain comprising the targeting domain; and (c) a linker that adjoins the first and second domains.
  • BTN2A1-Alpha-CD20 scFv (Leader sequence is indicated by a double underlined font, extracellular domain of human BTN2A1 is shown in bold-underlined font, a core domain of the linker is shown in a single underlined font, charged peptides are shown in an italic font and anti-CD20 ScFv sequence is shown in a boldface font): MEFGLSWVFLVAIIKGVQCQFIVVGPTDPILATVGENTTLRCHLSPEKNAEDMEVRWFRSQFSPAVFV YKGGRERTEEQMEEYRGRTTFVSKDISRGSVALVIHNITAQENGTYRCYFQEGRSYDEAILHLVVAGL GSKPLISMRGHEDGGIRLECISRGWYPKPLTVWRDPYGGVAPALKEVSMPDADGLFMVTTAVIIRDK SVRNMSCSINNTLLGQKKESVIFIPESFMPSVSPCAGSGSRKGGKRGSKYGPPCPPCPAPEFLGGPS V
  • the BTN2A1-Alpha-CD20 scFv comprises a polypeptide comprising the complementarity determining regions (CDRs) present in SEQ ID NO: 21.
  • the BTN2A1-Alpha-CD20 scFv comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence of SEQ ID NO: 21.
  • the BTN2A1-Alpha-CD20 scFv comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence of SEQ ID NO: 21, wherein the polypeptide comprises the complementarity determining regions (CDRs) present in SEQ ID NO: 21.
  • CDRs complementarity determining regions
  • the BTN3A1-beta-CD20scFv comprises a polypeptide comprising the complementarity determining regions (CDRs) present in SEQ ID NO: 22.
  • the BTN3A1- beta-CD20scFv comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence of SEQ ID NO: 22.
  • the BTN3A1-beta- CD20scFv comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence of SEQ ID NO: 22, wherein the polypeptide comprises the complementarity determining regions (CDRs) present in SEQ ID NO: 22
  • the First Domain relates to a heterodimeric protein, and methods of use of the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises a first domain comprising DB1/ 145191832.2 12 Attorney Docket No.: SHK-081PC/116981-5081 a butyrophilin family protein, or a fragment thereof; and wherein the beta chain comprises a first domain comprising a butyrophilin family protein, or a fragment thereof.
  • the butyrophilin family protein of the alpha chain and beta chain is independently selected from BTN2A1 and BTN3A1. In embodiments, the butyrophilin family protein of the alpha chain and beta chain is independently selected from human BTN2A1, and human BTN3A1.
  • the first domain of alpha chain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence selected from SEQ ID NOs: 13- 16; and the first domain of beta chain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence selected from SEQ ID NOs: 13-16.
  • the first domains of alpha chain and beta chain comprise a polypeptide having an amino acid sequence of: any one of SEQ ID NOs: 13-16; and any one of SEQ ID NOs: 13-16, respectively.
  • the first domains of alpha chain and beta chain comprise extracellular domains and/or variable domains of BTN2A1 and BTN3A1.
  • the first domains of alpha chain and beta chain comprise extracellular domains of BTN2A1 and BTN3A1, respectively.
  • the first domains of alpha chain and beta chain comprise variable domains of BTN2A1 and BTN3A1; respectively.
  • the first domain comprises two of the same butyrophilin family proteins.
  • the first domain comprises two different butyrophilin family proteins.
  • the butyrophilin family proteins comprise a variable domain. Suitable butyrophilin family proteins or fragments thereof are derived from the native butyrophilin family proteins that comprise a B30.2 domain in the cytosolic tail of the full length protein.
  • the butyrophilin family protein is butyrophilin subfamily 2 member A1 (BTN2A1).
  • BTN2A1 gamma delta T cell receptor
  • BTN2A1 is also known as BT2.1, BTF1.
  • the portion of BTN2A1 is a portion of the extracellular domain of BTN2A1.
  • the present chimeric protein further comprises a domain, e.g., the extracellular domain BTN2A1.
  • the amino acid sequence of extracellular domain of human BTN2A1, which is an illustrative amino acid sequence of human BTN2A1 suitable in the current disclosure is the following: DB1/ 145191832.2 13 Attorney Docket No.: SHK-081PC/116981-5081 QFIVVGPTDPILATVGENTTLRCHLSPEKNAEDMEVRWFRSQFSPAVFVYKGGRERTEEQMEEYRGR TTFVSKDISRGSVALVIHNITAQENGTYRCYFQEGRSYDEAILHLVVAGLGSKPLISMRGHEDGGIRLECI SRGWYPKPLTVWRDPYGGVAPALKEVSMPDADGLFMVTTAVIIRDKSVRNMSCSINNTLLGQKKESVI FIPESFMPSVSPCA (SEQ ID NO:
  • the present chimeric proteins may comprise the extracellular domain of BTN2A1 as described herein, or a variant or functional fragment thereof.
  • the chimeric protein may comprise a sequence of the extracellular domain of BTN2A1 as provided above, or a variant or functional fragment thereof having at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least
  • BTN2A1 derivatives can be constructed from available structural data, including a homology model described by Karunakaran et al., Butyrophilin-2A1 Directly Binds Germline-Encoded Regions of the V ⁇ 9V ⁇ 2 TCR and Is Essential for Phosphoantigen Sensing, Immunity.52(3): 487-498 (2020). Moreover, without wishing to be bound by theory, the protein structure homology-model of BTN2A1 is available at SWISS-MODEL repository. Bienert et al., “The SWISS-MODEL Repository – new features and functionality.” Nucleic Acids Research, 45(D1): D313–D319 (2017). Additional structural insight obtained from mutagenesis.
  • butyrophilin 2A1 is essential for phosphoantigen reactivity by ⁇ T cells. Science 367(6478):eaay5516 ( 2020). DB1/ 145191832.2 14 Attorney Docket No.: SHK-081PC/116981-5081
  • the butyrophilin family protein is butyrophilin subfamily 3 member A1 (BTN3A1).
  • the first domain comprises substantially all the extracellular domain of BTN3A1.
  • the first domain is capable of binding a gamma delta T cell receptor (e.g., V ⁇ 9 ⁇ 2).
  • BTN3A1 is also known as BTF5.
  • the portion of BTN3A1 is a portion of the extracellular domain of BTN3A1.
  • the present chimeric protein further comprises a domain, e.g., the extracellular domain BTN3A1.
  • the amino acid sequence of extracellular domain of human BTN3A1, which is an illustrative amino acid sequence of human BTN3A1 suitable in the current disclosure is the following: QFSVLGPSGPILAMVGEDADLPCHLFPTMSAETMELKWVSSSLRQVVNVYADGKEVEDRQSAPYRGR TSILRDGITAGKAALRIHNVTASDSGKYLCYFQDGDFYEKALVELKVAALGSDLHVDVKGYKDGGIHLEC RSTGWYPQPQIQWSNNKGENIPTVEAPVVADGVGLYAVAASVIMRGSSGEGVSCTIRSSLLGLEKTASI SIADPFFRSAQRWIAALAG (SEQ ID NO: 15)
  • the fragment of human BTN3A1 comprises the variable domain
  • the present chimeric proteins may comprise the extracellular domain of BTN3A1 as described herein, or a variant or functional fragment thereof.
  • the chimeric protein may comprise a sequence of the extracellular domain of BTN3A1 as provided above, or a variant or functional fragment thereof having at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least
  • BTN3A1 derivatives can be constructed from available structural data, including the following: Palakodeti et al., The molecular basis for modulation of human V(gamma)9V(delta)2 T cell responses by CD277/Butyrophilin-3 (BTN3A)-specific antibodies, J Biol Chem 287: 32780-32790 (2012); Vavassori et al., Butyrophilin 3A1 binds phosphorylated antigens and stimulates human gamma delta T cells.
  • the first domain comprises a portion of BTN2A1.
  • the portion of BTN2A1 is an extracellular domain of BTN2A1, or a ⁇ T-cell receptor (e.g., ⁇ 9 ⁇ 2)-binding fragment thereof.
  • the first domain comprises a portion of BTN3A1.
  • the portion of BTN3A1 is an extracellular domain of BTN3A1, or a ⁇ T-cell receptor (e.g., ⁇ 9 ⁇ 2)-binding fragment thereof.
  • the first domain comprises a portion of BTN2A1.
  • the portion of BTN2A1 is an extracellular domain of BTN2A1, or a ⁇ T-cell receptor (e.g., ⁇ 9 ⁇ 2)-binding fragment thereof.
  • the first domain comprises a portion of BTN3A1.
  • the portion of BTN3A1 is an extracellular domain of BTN3A1, or a ⁇ T-cell receptor (e.g., ⁇ 9 ⁇ 2)-binding fragment thereof.
  • the heterodimeric proteins of any of the embodiments disclosed herein comprise an alpha chain and a beta chain, each comprising a targeting domain.
  • the present disclosure relates to a heterodimeric protein, and methods of using the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof; and; and wherein the beta chain comprises a second domain comprising the targeting domain.
  • the targeting domain is capable of binding an antigen on the surface of a cancer cell.
  • the targeting domain is capable of binding a tumor antigen.
  • the targeting domain is an antibody, or an antigen binding fragment thereof.
  • the targeting domain comprises an Fv domain.
  • the targeting domain is an antibody-like molecule, or antigen binding fragment thereof.
  • the targeting domain comprises a single-chain variable fragment (scFv).
  • the targeting domain comprises scFvs or fragments thereof (e.g., half scFvs or Fv fragments).
  • the targeting domain specifically binds CD20.
  • the targeting domain comprises an scFv that specifically binds CD20.
  • the targeting domain comprises a polypeptide comprising the complementarity determining regions (CDRs) present in SEQ ID NOs: 17-20.
  • the targeting domain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence selected from SEQ ID NOs: 17-20.
  • the targeting domain is an antibody-like molecule, or antigen binding fragment thereof.
  • the antibody-like molecule is selected from a single-domain antibody, a recombinant heavy-chain-only antibody (VHH), a single-chain antibody (scFv), a shark heavy-chain-only antibody (VNAR), a microprotein (cysteine knot protein, knottin), a DARPin; a Tetranectin; an Affibody; a Transbody; an Anticalin; an AdNectin; an Affilin; an Affimer, a Microbody; an aptamer; an alterase; a plastic antibody; a phylomer; a stradobody; a maxibody; an evibody; a fynomer, an armadillo repeat protein, a Kunitz domain, an avimer, an atrimer, a probody, an immunobody, a triomab, a troybody; a pepbody; a vaccibody, a UniBody; a Du
  • the antibody-like molecule is an scFv.
  • the targeting domain is capable of binding an antigen on the surface of a cancer cell.
  • the targeting domain specifically binds CD20.
  • the targeting domain specifically binds to CD20.
  • An illustrative targeting domain is CD20scFv-1, which an scFV specific to human CD20, and has the following sequence (the variable regions of the heavy chain (VH) is shown in a boldface font, the variable regions of the light chain (V L ) is indicated in an italics font, and the linker joining V H and V L is shown by an underline): EVQLVESGGGLVQPGRSLRLSCVASGFTFNDYAMHWVRQAPGKGLEWVSVISWNSDSIGYADSVK GRFTISRDNAKNSLYLQMHSLRAEDTALYYCAKDNHYGSGSYYYYQYGMDVWGQGTTVTVSSGGG GSGGGGSGGGGSGGGGSAEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLI DB1/ 145191832.2 17
  • the targeting domain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence of SEQ ID NO: 17.
  • the targeting domain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence of SEQ ID NO: 17, wherein the polypeptide comprises the complementarity determining regions (CDRs) present in SEQ ID NO: 17.
  • CDRs complementarity determining regions
  • An illustrative targeting domain is CD20scFv-2, which an scFV specific to human CD20, and has the following sequence (the variable regions of the heavy clan (V H ) is shown in a boldface font, the variable regions of the light chain (VL) is indicated in an italics font, and the linker joining VH and VL is shown by an underline): EVQLVQSGAEVKKPGESLKISCKGSGRTFTSYNMHWVRQMPGKGLEWMGAIYPLTGDTSYNQKSK LQVTISADKSISTAYLQWSSLKASDTAMYYCARSTYVGGDWQFDVWGKGTTVTVSSGGGGSGGGG SGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASSSVPYIHWYQQKPGQAPRLLIYATSALASGIP DRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWLSHPPTFGQGTKLEIK (SEQ ID NO: 18)
  • the targeting domain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence of SEQ ID NO: 18.
  • the targeting domain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence of SEQ ID NO: 18, wherein the polypeptide comprises the complementarity determining regions (CDRs) present in SEQ ID NO: 18.
  • CDRs complementarity determining regions
  • An illustrative targeting domain is CD20scFv-3, which an scFV specific to human CD20, and has the following sequence (the linker joining the variable regions of the heavy chain (VH) and the variable regions of the light chain (V L ) is shown by an underline): DB1/ 145191832.2 18
  • the targeting domain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence of SEQ ID NO: 19.
  • the targeting domain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence of SEQ ID NO: 19, wherein the polypeptide comprises the complementarity determining regions (CDRs) present in SEQ ID NO: 19.
  • CDRs complementarity determining regions
  • an illustrative targeting domain is CD20scFv-4, which an scFV specific to human CD20, and has the following sequence (the linker joining the variable regions of the heavy clan (V H ) and the variable regions of the light chain (VL) is shown by an underline): EVQLVESGGGLVQPDRSLRLSCAASGFTFHDYAMHWVRQAPGKGLEWVSTISWNSGTIGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSGGGGSGGGGS GGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK (SEQ ID NO: 20)
  • the targeting domain comprises a polypeptide comprising the complementarity determining regions (CDRs) present in SDR
  • the targeting domain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence of SEQ ID NO: 20.
  • the targeting domain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence of SEQ ID NO: 20, wherein the polypeptide comprises the complementarity determining regions (CDRs) present in SEQ ID NO: 20.
  • CDRs complementarity determining regions
  • the linker comprises a polypeptide selected from a flexible amino acid sequence, an IgG hinge region, and an antibody sequence.
  • the antibody sequence comprises an Fc DB1/ 145191832.2 19 Attorney Docket No.: SHK-081PC/116981-5081 domain
  • the linker comprises a hinge-CH2-CH3 Fc domain.
  • the hinge- CH2-CH3 Fc domain is derived from IgG1, optionally from human IgG1.
  • the hinge-CH2- CH3 Fc domain is derived from IgG4, optionally from human IgG4.
  • the hinge-CH2-CH3 Fc domain lacks Fc-effector function.
  • the hinge-CH2-CH3 Fc domain that lacks Fc- effector function is a mutant derivative of IgG4, optionally human IgG4.
  • the hinge-CH2- CH3 Fc domain that lacks Fc-effector function is a mutant derivative of IgG1, optionally human IgG1.
  • the hinge-CH2-CH3 Fc domain has reduced binding to an Fc Gamma Receptor (Fc ⁇ R) selected from Fc ⁇ RI, Fc ⁇ RIIA, Fc ⁇ RIIAB, Fc ⁇ RIIIA and Fc ⁇ RIIIB compared to a wild type hinge-CH2-CH3 Fc domain. Mutant Fc domains lacking Fc effector functions are described in US Publication Nos.
  • the hinge-CH2-CH3 Fc domain comprises a polypeptide having an amino acid sequence with at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide selected from SEQ ID NOs: 1-12.
  • the linker that adjoins the first and second domains comprises a charge polarized core domain.
  • each of the first and second charge polarized core domains comprises proteins having positively or negatively charged amino acid residues at the amino and carboxy terminus of the core domain.
  • the first charge polarized core domain may comprise a protein having positively charged amino acids at the amino terminus which are adjoined by a linker (e.g., a stabilizing domain) to a protein having negatively charged amino acid residues at the carboxy terminus.
  • the second charge polarized core domain may comprise a protein having negatively charged amino acids at the amino terminus which are adjoined by a linker (e.g., a stabilizing domain) to a protein having positively charged amino acid residues at the carboxy terminus.
  • the first charge polarized core domain may comprise a protein having negatively charged amino acids at the amino terminus which are adjoined by a linker (e.g., a stabilizing domain) to a protein having positively charged amino acid residues at the carboxy terminus.
  • the second charge polarized core domain may comprise proteins having positively charged amino acids at the amino terminus which are adjoined by a linker (e.g., a stabilizing domain) to a protein having negatively charged amino acid residues at the carboxy terminus.
  • formation of heterodimeric proteins is driven by electrostatic interactions between the positively charged and negatively charged amino acid residues located at the amino and DB1/ 145191832.2 20 Attorney Docket No.: SHK-081PC/116981-5081 carboxy termini of the first and second charge polarized core domains. Further, formation of homodimeric proteins is prevented by the repulsion between the positively charged amino acid residues or negatively charged amino acid residues located at the amino and carboxy termini of the first and second charge polarized core domains.
  • the protein comprising positively and/or negatively charged amino acid residues at the amino or carboxy terminus of the charge polarized core domains is about 2 to about 50 amino acids long.
  • the protein comprising positively and/or negatively charged amino acid residues at either terminus of the charge polarized core domain may be about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids long.
  • the protein comprising positively charged amino acid residues may include one or more of amino acids selected from His, Lys, and Arg.
  • the protein comprising negatively charged amino acid residues may include one or more amino acids selected from Asp and Glu.
  • each of the first and/or second charge polarized core domains may comprise a protein comprising an amino acid sequence as provided in the TABLE 1 or an amino acid sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto.
  • Illustrative sequences of the first and/or second charge polarized core domains may comprise a protein: SEQ ID Sequence NO e n DB1/ 145191832.2 21 Attorney Docket No.: SHK-081PC/116981-5081 Y n X n CY n X n Y n (where X is a positively charged amino acid such as arginine, 25 histidine or lysine and Y is a spacer amino acid such as serine or glycine, and wh r h n i ind nd ntl n int r 0 t 4) r ns may comprise a peptide comprising the sequence YY n XX n YY n XX n YY n (where X is a positively charged amino acid such as arginine, histidine or lysine and Y is a spacer amino acid such as serine or glycine; SEQ ID NO:
  • Illustrative peptide sequences include, but are not limited to, RKGGKR (SEQ ID NO: 31) or GSGSRKGGKRGS (SEQ ID NO: 32).
  • each of the first and second charge polarized core domains may comprise a peptide comprising the sequence YY n ZZ n YY n ZZ n YY n (where Z is a negatively charged amino acid such as aspartic acid or glutamic acid and Y is a spacer amino acid such as serine or glycine).
  • Illustrative peptide sequences include, but are not limited to, DEGGED (SEQ ID NO: 33) or GSGSDEGGEDGS (SEQ ID NO: 34).
  • the linker comprises (a) a first charge polarized core domain adjoined to a butyrophilin family protein, optionally at the carboxy terminus, and (b) a second charge polarized core domain adjoined to a butyrophilin family protein, optionally at the carboxy terminus.
  • the linker forms a heterodimer through electrostatic interactions between positively charged amino acid residues and negatively charged amino acid residues on the first and second charge polarized core domains.
  • the first and/or second charge polarized core domain comprises a polypeptide linker, optionally selected from a flexible amino acid sequence, IgG hinge region, or antibody sequence.
  • the linker is a synthetic linker, optionally PEG.
  • the linker comprises the hinge-CH2-CH3 Fc domain derived from IgG1, optionally human IgG1. In embodiments, the linker comprises the hinge-CH2-CH3 Fc domain derived from IgG4, optionally human IgG4.
  • the first and/or second charge polarized core domain further comprise peptides having positively and/or negatively charged amino acid residues at the amino and/or carboxy terminus of the charge polarized core domain.
  • the positively charged amino acid residues include one or more of amino acids selected from His, Lys, and Arg. In embodiments, the positively charged amino acid residues are present in a peptide comprising positively charged amino acid residues in the first and/or the second charge polarized core domains.
  • the peptide comprising positively charged amino acid DB1/ 145191832.2 22 Attorney Docket No.: SHK-081PC/116981-5081 residues comprises a sequence selected from YnXnYnXnYn (where X is a positively charged amino acid such as arginine, histidine or lysine and Y is a spacer amino acid such as serine or glycine, and where each n is independently an integer 0 to 4) (SEQ ID NO: 21), YYnXXnYYnXXnYYn (where X is a positively charged amino acid such as arginine, histidine or lysine and Y is a spacer amino acid such as serine or glycine, and where each n is independently an integer 0 to 4) (SEQ ID NO: 23), and YnXnCYnXnYn (where X is a positively charged amino acid such as arginine, histidine or lysine and Y is
  • the peptide comprising positively charged amino acid residues comprises the sequence RKGGKR (SEQ ID NO: 31) or GSGSRKGGKRGS (SEQ ID NO: 32).
  • the negatively charged amino acid residues may include one or more amino acids selected from Asp and Glu.
  • the negatively charged amino acid residues are present in a peptide comprising negatively charged amino acid residues in the first and/or the second charge polarized core domains.
  • the peptide comprising negatively charged amino acid residues comprises a sequence selected from YnZnYnZnYn (where Z is a negatively charged amino acid such as aspartic acid or glutamic acid and Y is a spacer amino acid such as serine or glycine, and where each n is independently an integer 0 to 4) (SEQ ID NO: 22), YY n ZZ n YY n ZZ n YY n (where Z is a negatively charged amino acid such as aspartic acid or glutamic acid and Y is a spacer amino acid such as serine or glycine, and where each n is independently an integer 0 to 4) (SEQ ID NO: 24), and Y n Z n CY n Z n Y n (where Z is a negatively charged amino acid such as aspartic acid or glutamic acid and Y is a spacer amino acid such as serine or glycine, and where each n is independently an integer 0 to 0 to
  • the linker comprises a knob-in-hole (KIH) mutations.
  • the KIH mutations comprise the mutations that replace amino acids with small side chain with ones with larger side chains, thereby creating a knob or protrusion in one chain and vice versa to create a hole or socket in the partner chain.
  • a T366Y mutation in one CH3 domain in alpha chain creates a knob while an Y407T mutation in the CH3 domain in beta chain gives rise to a hole.
  • the KIH mutations establish intermolecular interactions and promote the heterodimer formation due to knob/hole pairing and bring about the association of two different Fc domains to give rise heterodimer formation.
  • the KIH mutations complement existing knob/hole mutations with F405A and T394W on the knob and hole side, respectively, yielding higher level of molecules as heterodimers.
  • Illustrative Fc domains having knob-in-hole mutations are present in SEQ ID NOs: 5-6 and 9-12.
  • the linker comprises a knob-in-hole (KIH) mutations and FcRn mutations SEQ ID NOs: 11-12).
  • the current disclosure provides a heterodimeric protein comprising (a) a first domain comprising one or more butyrophilin family proteins, or a fragment thereof; (b) a second domain DB1/ 145191832.2 23 Attorney Docket No.: SHK-081PC/116981-5081 comprising a targeting domain, the targeting domain being selected from an antibody, antibody-like molecule, and antigen binding fragment thereof; and (c) a linker that adjoins the first and second domains.
  • the heterodimeric protein comprises two individual polypeptide chains which self- associate.
  • the linker facilitates heterodimerization.
  • the heterodimeric protein comprises two of the same butyrophilin family proteins or two different butyrophilin family proteins.
  • the butyrophilin family proteins comprise a V-type domain and/or a B30.2 domain.
  • the first domain is a butyrophilin-like (BTNL) family protein, such as BTN2A1, BTN3A1, and a fragment thereof.
  • BTNL butyrophilin-like family protein
  • the first polypeptide chain and the second polypeptide chain heterodimers through electrostatic interactions between positively charged amino acid residues and negatively charged amino acid residues on the first and second charge polarized core domains.
  • the positively charged amino acid residues may include one or more of amino acids selected from His, Lys, and Arg.
  • the negatively charged amino acid residues may include one or more amino acids selected from Asp and Glu. Accordingly, in embodiments, each of the first and/or second charge polarized core domains comprises proteins having positively or negatively charged amino acid residues at the amino and carboxy terminus of the core domain.
  • the first charge polarized core domain may comprise a protein having positively charged amino acids at the amino terminus which are adjoined by a linker (e.g., a stabilizing domain) to a protein having negatively charged amino acid residues at the carboxy terminus.
  • the second charge polarized core domain may comprise a protein having negatively charged amino acids at the amino terminus which are adjoined by a linker (e.g., a stabilizing domain) to a protein having positively charged amino acid residues at the carboxy terminus.
  • the first charge polarized core domain may comprise a protein having negatively charged amino acids at the amino terminus which are adjoined by a linker (e.g., a stabilizing domain) to a protein having positively charged amino acid residues at the carboxy terminus.
  • the second charge polarized core domain may comprise proteins having positively charged amino acids at the amino terminus which are adjoined by a linker (e.g., a stabilizing domain) to a protein having negatively charged amino acid residues at the carboxy terminus.
  • each of the first and/or second charge polarized core domains further comprise a linker (e.g., a stabilizing domain) which adjoins the proteins having positively or negatively charged amino acids.
  • the linker e.g., a stabilizing domain
  • the linker (e.g., a stabilizing domain) comprises the hinge-CH2-CH3 Fc domain derived from IgG1, optionally human IgG1.
  • DB1/ 145191832.2 24 Attorney Docket No.: SHK-081PC/116981-5081
  • the linker (e.g., a stabilizing domain) comprises the hinge-CH2-CH3 Fc domain derived from IgG4, optionally human IgG4.
  • the core domain comprises the following sequence: SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPP SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVLHEALHNHYTQKSLSLSLGKIEGRMD (SEQ ID NO: 1).
  • sequence of an illustrative charge polarized core domain (positive – negative) is provided below (peptide comprising positively charged amino acids is shown with an underline, peptide comprising negatively charged amino acids is shown with italic font and IgG4 hinge-CH2-CH3 is shown in boldface font.
  • sequence of an illustrative charge polarized core domain (negative - positive) is provided below (peptide comprising positively charged amino acids is shown with an underline, peptide comprising negatively charged amino acids is shown with italic font and IgG4 hinge-CH2-CH3 is shown in boldface font.
  • the core domain comprises the following sequence (IgG4 hinge-CH2-CH3 is shown in boldface font, rest of the sequence is a joining linker disclosed herein): CPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSC SVLHEALHNHYTQKSLSLSLGK (SEQ ID NO: 4).
  • the core domain comprises a KIHT22Y protein comprising the following sequence (the knob in hole motif mutations are indicated by boldface, underlined font): EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5).
  • the core domain is a KIHY86T protein comprising the following sequence (the knob in hole motif mutations are indicated by boldface, underlined font): EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 6).
  • the core domain comprises a IgG1 hinge-CH2-CH3 protein comprising the following sequence: VPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQ PREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMA KDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCS VLHEGLHNHHTEKSLSHSPGI (SEQ ID NO: 7).
  • the core domain comprises the following sequence (IgG4 hinge-CH2-CH3 is shown in boldface font, rest of the sequence is a joining linker disclosed herein): CPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSC SVLHEALHNHYTQKSLSLSLGK (SEQ ID NO: 8).
  • the protein comprising the charged amino acid residues may further comprise one or more cysteine residues to facilitate disulfide bonding between the electrostatically charged core domains as an additional method to stabilize the heterodimer.
  • each of the first and second charge polarized core domains comprises a linker sequence which may optionally function as a stabilizing domain.
  • 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 e.g., a stabilizing domain
  • the linker is a synthetic linker such as PEG.
  • the linker e.g., a stabilizing domain
  • the linker (e.g., a stabilizing domain) is less than about 500 amino acids long, about 450 amino acids long, about 400 amino acids long, about 350 amino acids long, about 300 amino acids long, about 250 amino acids DB1/ 145191832.2 27 Attorney Docket No.: SHK-081PC/116981-5081 long, about 200 amino acids long, about 150 amino acids long, or about 100 amino acids long.
  • the linker (e.g., a stabilizing domain) may be less than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids long.
  • the linker (e.g., a stabilizing domain) is substantially comprised of glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97% glycines and serines).
  • the linker (e.g., a stabilizing domain) is a hinge region of an antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2).
  • the hinge region acts as a flexible spacer, allowing the Fab portion to move freely in space.
  • the hinge domains are structurally diverse, varying in both sequence and length among immunoglobulin classes and subclasses. For example, the length and flexibility of the hinge region varies among the IgG subclasses.
  • the hinge region of IgG1 encompasses amino acids 216-231 and, because it is freely flexible, the Fab fragments can rotate about their axes of symmetry and move within a sphere centered at the first of two inter-heavy chain disulfide bridges.
  • IgG2 has a shorter hinge than IgG1, with 12 amino acid residues and four disulfide bridges.
  • the hinge region of IgG2 lacks a glycine residue, is relatively short, and contains a rigid poly- proline double helix, stabilized by extra inter-heavy chain disulfide bridges. These properties restrict the flexibility of the IgG2 molecule.
  • IgG3 differs from the other subclasses by its unique extended hinge region (about four times as long as the IgG1 hinge), containing 62 amino acids (including 21 prolines and 11 cysteines), forming an inflexible poly-proline double helix. In IgG3, the Fab fragments are relatively far away from the Fc fragment, giving the molecule a greater flexibility.
  • the elongated hinge in IgG3 is also responsible for its higher molecular weight compared to the other subclasses.
  • the hinge region of IgG4 is shorter than that of IgG1 and its flexibility is intermediate between that of IgG1 and IgG2.
  • the flexibility of the hinge regions reportedly decreases in the order IgG3>IgG1>IgG4>IgG2.
  • the linker may be derived from human IgG4 and contain one or more mutations to enhance dimerization (including S228P) or FcRn binding. According to crystallographic studies, the immunoglobulin hinge region can be further subdivided functionally into three regions: the upper hinge region, the core region, and the lower hinge region.
  • the core hinge region of wild-type human IgG1 contains the sequence Cys-Pro-Pro-Cys which, when dimerized by disulfide bond formation, results in a cyclic octapeptide believed to act as a pivot, thus conferring flexibility.
  • the present linker e.g., a stabilizing domain
  • the hinge region may also contain one or more glycosylation sites, which include a number of structurally distinct types of sites for carbohydrate attachment.
  • IgA1 contains five glycosylation sites within a 17-amino-acid segment of the hinge region, conferring resistance of the hinge region polypeptide to intestinal proteases, considered an advantageous property for a secretory immunoglobulin.
  • the linker e.g., a stabilizing domain of the current disclosure comprises one or more glycosylation sites.
  • the linker (e.g., a stabilizing domain) comprises an Fc domain of an antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)).
  • the linker (e.g., a stabilizing domain) comprises a hinge-CH2-CH3 Fc domain derived from a human IgG4 antibody.
  • the linker (e.g., a stabilizing domain) comprises a hinge-CH2-CH3 Fc domain derived from a human IgG1 antibody.
  • the Fc domain exhibits increased affinity for and enhanced binding to the neonatal Fc receptor (FcRn).
  • the Fc domain includes one or more mutations that increases the affinity and enhances binding to FcRn. Without wishing to be bound by theory, it is believed that increased affinity and enhanced binding to FcRn increases the in vivo half-life of the present heterodimeric proteins.
  • the Fc domain contains one or more amino acid substitutions at amino acid residue 250, 252, 254, 256, 308, 309, 311, 428, 433 or 434 (in accordance with Kabat numbering), or equivalents thereof.
  • the amino acid substitution at amino acid residue 250 is a substitution with glutamine.
  • the amino acid substitution at amino acid residue 252 is a substitution with tyrosine, phenylalanine, tryptophan or threonine.
  • the amino acid substitution at amino acid residue 254 is a substitution with threonine.
  • the amino acid substitution at amino acid residue 256 is a substitution with serine, arginine, glutamine, glutamic acid, aspartic acid, or threonine.
  • the amino acid substitution at amino acid residue 308 is a substitution with threonine.
  • the amino acid substitution at amino acid residue 309 is a substitution with proline.
  • the amino acid substitution at amino acid residue 311 is a substitution with DB1/ 145191832.2 29 Attorney Docket No.: SHK-081PC/116981-5081 serine.
  • the amino acid substitution at amino acid residue 385 is a substitution with arginine, aspartic acid, serine, threonine, histidine, lysine, alanine or glycine.
  • the amino acid substitution at amino acid residue 386 is a substitution with threonine, proline, aspartic acid, serine, lysine, arginine, isoleucine, or methionine.
  • the amino acid substitution at amino acid residue 387 is a substitution with arginine, proline, histidine, serine, threonine, or alanine.
  • the amino acid substitution at amino acid residue 389 is a substitution with proline, serine or asparagine.
  • the amino acid substitution at amino acid residue 428 is a substitution with leucine.
  • the amino acid substitution at amino acid residue 433 is a substitution with arginine, serine, isoleucine, proline, or glutamine.
  • the amino acid substitution at amino acid residue 434 is a substitution with histidine, phenylalanine, or tyrosine.
  • the Fc domain (e.g., comprising an IgG constant region) comprises one or more mutations such as substitutions at amino acid residue 252, 254, 256, 433, 434, or 436 (in accordance with Kabat numbering).
  • the IgG constant region includes a triple M252Y/S254T/T256E mutation or YTE mutation.
  • the IgG constant region includes a triple H433K/N434F/Y436H mutation or KFH mutation.
  • the IgG constant region includes an YTE and KFH mutation in combination.
  • the modified humanized antibodies of the invention comprise an IgG constant region that contains one or more mutations at amino acid residues 250, 253, 307, 310, 380, 428, 433, 434, and 435.
  • Illustrative mutations include T250Q, M428L, T307A, E380A, I253A, H310A, M428L, H433K, N434A, N434F, N434S, and H435A.
  • the IgG constant region comprises a M428L/N434S mutation or LS mutation.
  • the IgG constant region comprises a T250Q/M428L mutation or QL mutation.
  • the IgG constant region comprises an N434A mutation. In another embodiment, the IgG constant region comprises a T307A/E380A/N434A mutation or AAA mutation. In another embodiment, the IgG constant region comprises an I253A/H310A/H435A mutation or IHH mutation. In another embodiment, the IgG constant region comprises a H433K/N434F mutation. In another embodiment, the IgG constant region comprises a M252Y/S254T/T256E and a H433K/N434F mutation in combination. In various embodiments, mutations are introduced to increase stability and/or half-life of the Fc domain. An illustrative Fc stabilizing mutant is S228P.
  • Additional illustrative Fc half-life extending mutants are T250Q, M428L, V308T, L309P, and Q311S and the present linkers (e.g., stabilizing domains) may comprise 1, or 2, or 3, or 4, or 5 of these mutants.
  • the linker may be flexible, including without limitation highly flexible.
  • the linker may be rigid, including without limitation a rigid alpha helix.
  • the linker may be functional.
  • the linker may function to improve the folding and/or stability, improve the expression, improve the pharmacokinetics, and/or improve the bioactivity of the present heterodimeric protein.
  • the linker may function to target the heterodimeric protein to a particular cell type or location.
  • the current disclosure provides a heterodimeric protein, and methods of use of the same, comprising: (a) a first domain comprising one or more butyrophilin family proteins, or a fragment thereof; (b) a second domain comprising a targeting domain, the targeting domain being selected from an antibody, antibody-like molecule, and antigen binding fragment thereof; and (c) a linker that adjoins the first and second domains.
  • the heterodimeric protein is a complex of two polypeptide chains.
  • the heterodimeric protein comprises an alpha chain and a beta chain wherein the alpha chain and the beta chain each independently comprise (a) a first domain comprising a butyrophilin family protein, or fragment thereof; (b) a second domain comprising a targeting domain, the targeting domain being selected from an antibody, antibody-like molecule, and antigen binding fragment thereof; and (c) a linker that adjoins the first and second domains.
  • the alpha chain and the beta chain self-associate to form the heterodimer.
  • the first domain comprises two of the same butyrophilin family proteins. In embodiments, wherein the first domain comprises two different butyrophilin family proteins.
  • the butyrophilin family proteins comprise a V-type domain.
  • the butyrophilin family proteins or fragments thereof are derived from the native butyrophilin family proteins that comprise a B30.2 domain in the cytosolic tail. DB1/ 145191832.2 31 Attorney Docket No.: SHK-081PC/116981-5081
  • the butyrophilin family proteins are selected from BTN2A1, BTN3A1, and a fragment thereof.
  • the first domain comprises: (a) BTN2A1, BTN3A1, and a fragment thereof; and (b) BTN2A1, BTN3A1, and a fragment thereof.
  • the first domain comprises a fragment of butyrophilin family proteins, wherein the fragment is capable of binding a gamma delta T cell receptor and is optionally an extracellular domain, optionally comprising one or more of an immunoglobulin V (IgV)- and IgC-like domain.
  • the first domain comprises a fragment of butyrophilin family proteins, wherein the fragment is capable of binding a V ⁇ 9 ⁇ 2 gamma delta T cell receptor.
  • the first domain comprises a polypeptide having an amino acid sequence of: (a) any one of SEQ ID NOs: 13, 15, or a fragment thereof; and (b) any one of SEQ ID NOs: 13, 15, or a fragment thereof.
  • the first domain comprises a polypeptide having (a) an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 15 or SEQ ID NO: 16, and an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 13 or SEQ ID NO: 14.
  • the targeting domain is an antibody, or antigen binding fragment thereof. In embodiments, the targeting domain is an antibody-like molecule, or antigen binding fragment thereof.
  • the antibody-like molecule is selected from a single-domain antibody, a recombinant heavy-chain-only antibody (VHH), a single-chain antibody (scFv), a shark heavy-chain-only antibody (VNAR), a microprotein (cysteine knot protein, knottin), a DARPin; a Tetranectin; an Affibody; a Transbody; an Anticalin; an AdNectin; an Affilin; an Affimer, a Microbody; an aptamer; an alterase; a plastic antibody; a phylomer; a stradobody; a maxibody; an evibody; a fynomer, an armadillo repeat protein, a Kunitz domain, an avimer, an atrimer, a probody, an immunobody, a triomab, a troybody; a pepbody; a vaccibody, a UniBody; a Du
  • the antibody-like molecule is an scFv.
  • the targeting domain is an extracellular domain.
  • the targeting domain is capable of binding an antigen on the surface of a cancer cell.
  • the targeting domain is capable of binding an antigen on the surface of a cancer cell.
  • the targeting domain specifically binds CD20.
  • the targeting domain specifically binds CD20.
  • the targeting domain is a polypeptide having an amino acid sequence with at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence selected from SEQ ID NOs: 62-65.
  • the targeting domain is a polypeptide having an amino acid sequence of selected from SEQ ID NOs: 62- 65.
  • the linker comprises (a) a first charge polarized core domain adjoined to a butyrophilin family protein, optionally at the carboxy terminus, and (b) a second charge polarized core domain adjoined to a butyrophilin family protein, optionally at the carboxy terminus.
  • the linker forms a heterodimer through electrostatic interactions between positively charged amino acid residues and negatively charged amino acid residues on the first and second charge polarized core domains.
  • the first and/or second charge polarized core domain comprises a polypeptide linker, optionally selected from a flexible amino acid sequence, IgG hinge region, or antibody sequence.
  • the linker is a synthetic linker, optionally PEG.
  • the linker comprises the hinge-CH2-CH3 Fc domain derived from IgG1, optionally human IgG1.
  • the linker comprises the hinge-CH2-CH3 Fc domain derived from IgG4, optionally human IgG4.
  • the first and/or second charge polarized core domain further comprise peptides having positively and/or negatively charged amino acid residues at the amino and/or carboxy terminus of the charge polarized core domain.
  • the positively charged amino acid residues include one or more of amino acids selected from His, Lys, and Arg. In embodiments, the positively charged amino acid residues are present in a peptide comprising positively charged amino acid residues in the first and/or the second charge polarized core domains.
  • the peptide comprising positively charged amino acid residues comprises a sequence selected from Y n X n Y n X n Y n (where X is a positively charged amino acid such as arginine, histidine or lysine and Y is a spacer amino acid such as serine or glycine, and where each n is independently an integer 0 to 4) (SEQ ID NO: 21), YY n XX n YY n XX n YY n (where X is a positively charged amino acid such as arginine, histidine or lysine and Y is a spacer amino acid such as serine or glycine, and where each n is independently an integer 0 to 4) (SEQ ID NO: 23), and Y n X n CY n X n Y n (where X is a positively charged amino acid such as arginine, histidine or lysine and Y is
  • the peptide comprising positively charged amino acid residues comprises the sequence RKGGKR (SEQ ID NO: 31) or GSGSRKGGKRGS (SEQ ID NO: 32).
  • the negatively charged amino acid residues may include one or more amino acids selected from Asp and Glu.
  • the negatively charged amino acid residues are present in a peptide comprising negatively charged amino acid residues in the first and/or the second charge polarized core domains.
  • the peptide comprising negatively charged amino acid residues comprises a sequence selected from YnZnYnZnYn (where Z is a negatively charged amino acid such as aspartic acid or glutamic acid and Y is a spacer amino acid such DB1/ 145191832.2 33 Attorney Docket No.: SHK-081PC/116981-5081 as serine or glycine, and where each n is independently an integer 0 to 4) (SEQ ID NO: 22), YY n ZZ n YY n ZZ n YY n (where Z is a negatively charged amino acid such as aspartic acid or glutamic acid and Y is a spacer amino acid such as serine or glycine, and where each n is independently an integer 0 to 4) (SEQ ID NO: 24), and Y n Z n CY n Z n Y n (where Z is a negatively charged amino acid such as aspartic acid or glutamic acid and Y is a
  • the peptide comprising negatively charged amino acid residues comprises the sequence DEGGED (SEQ ID NO: 33) or GSGSDEGGEDGS (SEQ ID NO: 34).
  • the protein comprising the charged amino acid residues may further comprise one or more cysteine residues to facilitate disulfide bonding between the electrostatically charged core domains as an additional method to stabilize the heterodimer.
  • each of the first and second charge polarized core domains comprises a linker sequence which may optionally function as a stabilizing domain.
  • 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 (e.g., a stabilizing domain) is a synthetic linker such as PEG. In other embodiments, the linker (e.g., a stabilizing domain) is a polypeptide. In embodiments, the linker (e.g., a stabilizing domain) is less than about 500 amino acids long, about 450 amino acids long, about 400 amino acids long, about 350 amino acids long, about 300 amino acids long, about 250 amino acids long, about 200 amino acids long, about 150 amino acids long, or about 100 amino acids long.
  • the linker (e.g., a stabilizing domain) may be less than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids long.
  • the linker e.g., a stabilizing domain
  • the linker is substantially comprised of glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97% glycines and serines).
  • the linker e.g., a stabilizing domain
  • the linker is a hinge region of an antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2).
  • the hinge region found in IgG, IgA, IgD, and IgE class antibodies, acts as a flexible spacer, allowing the Fab portion to move freely in space.
  • the hinge domains are structurally diverse, varying in both sequence and length among immunoglobulin classes and subclasses.
  • the length and flexibility of the hinge region varies among the IgG subclasses.
  • the hinge region of IgG1 encompasses amino acids 216-231 and, because it is freely flexible, the Fab fragments can rotate about their axes of symmetry and move within a sphere centered at the first of two inter-heavy chain disulfide bridges.
  • IgG2 has a shorter hinge than IgG1, with 12 amino acid residues and four disulfide bridges.
  • the hinge region of IgG2 lacks a glycine residue, is relatively short, and contains a rigid poly- proline double helix, stabilized by extra inter-heavy chain disulfide bridges. These properties restrict the flexibility of the IgG2 molecule.
  • IgG3 differs from the other subclasses by its unique extended hinge region (about four times as long as the IgG1 hinge), containing 62 amino acids (including 21 prolines and 11 cysteines), forming an inflexible poly-proline double helix. In IgG3, the Fab fragments are relatively far away from the Fc fragment, giving the molecule a greater flexibility.
  • the elongated hinge in IgG3 is also responsible for its higher molecular weight compared to the other subclasses.
  • the hinge region of IgG4 is shorter than that of IgG1 and its flexibility is intermediate between that of IgG1 and IgG2.
  • the flexibility of the hinge regions reportedly decreases in the order IgG3>IgG1>IgG4>IgG2.
  • the linker may be derived from human IgG4 and contain one or more mutations to enhance dimerization (including S228P) or FcRn binding.
  • the immunoglobulin hinge region can be further subdivided functionally into three regions: the upper hinge region, the core region, and the lower hinge region. See Shin et al., 1992 Immunological Reviews 130:87.
  • the upper hinge region includes amino acids from the carboxyl end of CH1 to the first residue in the hinge that restricts motion, generally the first cysteine residue that forms an interchain disulfide bond between the two heavy chains.
  • the length of the upper hinge region correlates with the segmental flexibility of the antibody.
  • the core hinge region contains the inter- heavy chain disulfide bridges, and the lower hinge region joins the amino terminal end of the CH2 domain and includes residues in C H2 . Id.
  • the core hinge region of wild-type human IgG1 contains the sequence Cys-Pro-Pro-Cys which, when dimerized by disulfide bond formation, results in a cyclic octapeptide believed to act as a pivot, thus conferring flexibility.
  • the present linker (e.g., a stabilizing domain) comprises, one, or two, or three of the upper hinge region, the core region, and the lower hinge region of any antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)).
  • the hinge region may also contain one or more glycosylation DB1/ 145191832.2 35 Attorney Docket No.: SHK-081PC/116981-5081 sites, which include a number of structurally distinct types of sites for carbohydrate attachment.
  • IgA1 contains five glycosylation sites within a 17-amino-acid segment of the hinge region, conferring resistance of the hinge region polypeptide to intestinal proteases, considered an advantageous property for a secretory immunoglobulin.
  • the linker (e.g., a stabilizing domain) of the current disclosure comprises one or more glycosylation sites.
  • the linker (e.g., a stabilizing domain) comprises an Fc domain of an antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)).
  • the linker (e.g., a stabilizing domain) comprises a hinge-CH2-CH3 Fc domain derived from a human IgG4 antibody. In various embodiments, the linker (e.g., a stabilizing domain) comprises a hinge-CH2-CH3 Fc domain derived from a human IgG1 antibody. In embodiments, the Fc domain exhibits increased affinity for and enhanced binding to the neonatal Fc receptor (FcRn). In embodiments, the Fc domain includes one or more mutations that increases the affinity and enhances binding to FcRn. Without wishing to be bound by theory, it is believed that increased affinity and enhanced binding to FcRn increases the in vivo half-life of the present heterodimeric proteins.
  • the Fc domain contains one or more amino acid substitutions at amino acid residue 250, 252, 254, 256, 308, 309, 311, 428, 433 or 434 (in accordance with Kabat numbering), or equivalents thereof.
  • the amino acid substitution at amino acid residue 250 is a substitution with glutamine.
  • the amino acid substitution at amino acid residue 252 is a substitution with tyrosine, phenylalanine, tryptophan or threonine.
  • the amino acid substitution at amino acid residue 254 is a substitution with threonine.
  • the amino acid substitution at amino acid residue 256 is a substitution with serine, arginine, glutamine, glutamic acid, aspartic acid, or threonine.
  • the amino acid substitution at amino acid residue 308 is a substitution with threonine.
  • the amino acid substitution at amino acid residue 309 is a substitution with proline.
  • the amino acid substitution at amino acid residue 311 is a substitution with serine.
  • the amino acid substitution at amino acid residue 385 is a substitution with arginine, aspartic acid, serine, threonine, histidine, lysine, alanine or glycine.
  • the amino acid substitution at amino acid residue 386 is a substitution with threonine, proline, aspartic acid, serine, lysine, arginine, isoleucine, or methionine.
  • the amino acid substitution at amino acid residue 387 is a substitution with arginine, proline, histidine, serine, threonine, or alanine.
  • the amino acid substitution at amino acid residue 389 is a substitution with proline, serine or asparagine.
  • the amino acid substitution at amino acid residue 428 is a substitution with leucine.
  • the amino acid substitution at amino acid residue 433 is a substitution DB1/ 145191832.2 36 Attorney Docket No.: SHK-081PC/116981-5081 with arginine, serine, isoleucine, proline, or glutamine.
  • the amino acid substitution at amino acid residue 434 is a substitution with histidine, phenylalanine, or tyrosine.
  • the Fc domain e.g., comprising an IgG constant region
  • the IgG constant region includes a triple M252Y/S254T/T256E mutation or YTE mutation. In another embodiment, the IgG constant region includes a triple H433K/N434F/Y436H mutation or KFH mutation. In a further embodiment, the IgG constant region includes an YTE and KFH mutation in combination. In embodiments, the modified humanized antibodies of the invention comprise an IgG constant region that contains one or more mutations at amino acid residues 250, 253, 307, 310, 380, 428, 433, 434, and 435.
  • Illustrative mutations include T250Q, M428L, T307A, E380A, I253A, H310A, M428L, H433K, N434A, N434F, N434S, and H435A.
  • the IgG constant region comprises a M428L/N434S mutation or LS mutation.
  • the IgG constant region comprises a T250Q/M428L mutation or QL mutation.
  • the IgG constant region comprises an N434A mutation.
  • the IgG constant region comprises a T307A/E380A/N434A mutation or AAA mutation.
  • the IgG constant region comprises an I253A/H310A/H435A mutation or IHH mutation. In another embodiment, the IgG constant region comprises a H433K/N434F mutation. In another embodiment, the IgG constant region comprises a M252Y/S254T/T256E and a H433K/N434F mutation in combination. In various embodiments, mutations are introduced to increase stability and/or half-life of the Fc domain. An illustrative Fc stabilizing mutant is S228P.
  • Additional illustrative Fc half-life extending mutants are T250Q, M428L, V308T, L309P, and Q311S and the present linkers (e.g., stabilizing domains) may comprise 1, or 2, or 3, or 4, or 5 of these mutants.
  • the linker may be flexible, including without limitation highly flexible. In various embodiments, the linker may be rigid, including without limitation a rigid alpha helix. In various embodiments, the linker may be functional. For example, without limitation, the linker may function to improve the folding and/or stability, improve the expression, improve the pharmacokinetics, DB1/ 145191832.2 37 Attorney Docket No.: SHK-081PC/116981-5081 and/or improve the bioactivity of the present heterodimeric protein. In another example, the linker may function to target the heterodimeric protein to a particular cell type or location.
  • the present disclosure relates to a method for treating a B cell-mediated disease or disorder, comprising administering to the subject about 0.01 mg/kg to about 100 mg/kg of a heterodimeric protein, or a nucleic acid encoding the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; (b) a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof, the target domain being directed to CD20; and (c) a linker that adjoins the first and second domains; and wherein the beta chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or B
  • the present disclosure relates to a method for increasing and/or stimulating B cell depletion in a subject suffering from a B cell-mediated disease or disorder, comprising administering to the subject about 0.01 mg/kg to about 100 mg/kg of a heterodimeric protein, or a nucleic acid encoding the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; (b) a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof, the target domain being directed to CD20; and (c) a linker that adjoins the first and second domains; and wherein the beta chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and
  • the present disclosure relates to a method for a method of contemporaneous activation and targeting of gamma delta T cells to cancer cells , comprising administering to the subject about 0.01 mg/kg to about 100 mg/kg of a heterodimeric protein, or a nucleic acid encoding the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; (b) a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof, the target domain being directed to CD20; and (c) a linker DB1/ 145191832.2 38 Attorney Docket No.: SHK-081PC/116981-5081 that adjoins the first and second domains; and wherein the beta chain comprises: (a) a first domain comprising a butyrophil
  • the present disclosure relates to a method for method of modulating a patient’s immune response, comprising administering to the subject about 0.01 mg/kg to about 100 mg/kg of a heterodimeric protein, or a nucleic acid encoding the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; (b) a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof, the target domain being directed to CD20; and (c) a linker that adjoins the first and second domains; and wherein the beta chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; (b)
  • the present disclosure relates to a method for method of treating cancer, comprising administering to a subject in need thereof a pharmaceutical composition , comprising administering to the subject about 0.01 mg/kg to about 100 mg/kg of a heterodimeric protein, or a nucleic acid encoding the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; (b) a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof, the target domain being directed to CD20; and (c) a linker that adjoins the first and second domains; and wherein the beta chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN
  • the B cell-mediated disease or disorder is a cancer.
  • the cancer is a cancer expressing CD20.
  • the cancer is a B-cell malignancy.
  • the B-cell malignancy is a lymphoma or leukemia.
  • the B-cell malignancy is non-Hodgkin’s lymphoma (NHL) or Hodgkin’s lymphoma (HL).
  • the e B-cell malignancy is advanced stage follicular lymphoma, chronic lymphocytic leukemias (CLL), Burkitt’s lymphoma, or diffuse large B- cell lymphoma.
  • the B-cell mediated disease or disorder is an autoimmune disease or disorder.
  • the autoimmune disease or disorder is one or more of rheumatoid arthritis, systemic lupus erythematosus, diabetes mellitus, ankylosing spondylitis, Sjögren's syndrome, inflammatory bowel diseases (e.g., colitis ulcerosa, Crohn's disease), multiple sclerosis, sarcoidosis, psoriasis, Grave's disease, Hashimoto's thyroiditis, psoriasis, hypersensitivity reactions (e.g., allergies, hay fever, asthma, and acute edema cause type I hypersensitivity reactions), pemphigus, and vasculitis (e.g., Granulomatosis with polyangiitis).
  • the current disclosure provides a method of treating cancer, comprising administering to a subject in need thereof a pharmaceutical composition comprising a heterodimeric protein comprising an alpha chain and a beta chain: wherein the alpha chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof; (b) a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof; and (c) a linker that adjoins the first and second domains; and wherein the beta chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof; (b) a second domain comprising the targeting domain; and (c) a linker that adjoins the first and second domains.
  • the butyrophilin family protein of the alpha chain and the beta chain is independently selected from BTN2A1, and BTN3A1. In embodiments, the butyrophilin family protein of the alpha chain and the beta chain is independently selected from human BTN2A1, and human BTN3A1.
  • the antibody-like molecule is an scFv. In embodiments, the antibody-like molecule is an scFv or a fragment thereof, e.g., the light chain and/or heavy chain of the scFv. In embodiments, the antibody-like molecule is an Fv fragment. In embodiments, the targeting domain is capable of binding an antigen on the surface of a cancer cell.
  • the targeting domain is capable of binding an antigen on the surface of a cancer cell. In embodiments, the targeting domain specifically binds CD20. In embodiments, the targeting domain specifically binds to CD20.
  • the cancer is a B cell lymphoma or leukemia. In embodiments, the cancer is an acute myeloid leukemia (AML). In embodiments, the cancer is a solid cancer selected from prostate cancer, small cell lung cancer, non-small cell lung cancer, neuroendocrine tumors, renal cancer, bladder cancer, colon cancer, and breast cancer. In embodiments, the cancer is known to express the antigenic target of the targeting domain disclosed herein. In embodiments, the cancer is suspected to express the antigenic target of the targeting domain disclosed herein.
  • the present disclosure relates to a method of treating a cancer CD20 in a subject in need thereof comprising: administering to the subject a pharmaceutical composition of any of the embodiments DB1/ 145191832.2 40 Attorney Docket No.: SHK-081PC/116981-5081 disclosed herein, thereby causing an in vivo proliferation of gamma delta T cells and/or contacting a cell derived from the subject with a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an ex vivo proliferation of gamma delta T cells.
  • the present disclosure relates to a method of treating a cancer expressing CD20 in a subject in need thereof comprising: administering to the subject a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an in vivo proliferation of gamma delta T cells and/or contacting a cell derived from the subject with a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an ex vivo proliferation of gamma delta T cells.
  • the present disclosure relates to a method of treating a cancer that is CD20 positive in a subject in need thereof comprising: administering to the subject a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an in vivo proliferation of gamma delta T cells and/or contacting a cell derived from the subject with a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an ex vivo proliferation of gamma delta T cells.
  • the present disclosure relates to a method of treating a cancer that is CD20 + , in a subject in need thereof comprising: administering to the subject a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an in vivo proliferation of gamma delta T cells and/or contacting a cell derived from the subject with a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an ex vivo proliferation of gamma delta T cells.
  • the present disclosure relates to a method of treating a CD20 + cancer in a subject in need thereof comprising: administering to the subject a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an in vivo proliferation of gamma delta T cells and/or contacting a cell derived from the subject with a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an ex vivo proliferation of gamma delta T cells.
  • the present disclosure relates to a method of treating a B cell lymphoma/leukemia in a subject in need thereof.
  • the method comprises administering to the subject a pharmaceutical composition comprising a heterodimeric protein comprising an alpha chain and a beta chain, wherein the alpha chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof; (b) a second domain comprising a targeting domain, and (c) a linker that adjoins the first and second domains; and wherein the beta chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof; (b) a second domain comprising the targeting domain; and (c) a linker that adjoins the first and second domains, wherein the targeting domain is capable of specifically binding to CD20.
  • the present disclosure relates to a method of treating a B cell lymphoma/leukemia in a subject in need thereof.
  • the method comprises contacting a cell derived from the subject with a pharmaceutical composition comprising a heterodimeric protein comprising an alpha chain and a beta chain, wherein the alpha chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof; (b) a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof; and (c) a linker that adjoins the first and second domains; and wherein the beta chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof; (b) a second domain comprising the targeting domain; and (c) a linker that adjoins the first and second domains, wherein the targeting domain is capable of
  • the method optionally further comprises proliferating ex vivo the cell derived from the subject that is contacted with the pharmaceutical composition.
  • the method optionally further comprises administering to the subject the cell that is proliferated ex vivo.
  • the present disclosure relates to a method of stimulating proliferation of gamma delta T cells, comprising: administering a pharmaceutical composition of any one of the embodiments disclosed herein, to a subject in need thereof thereby causing an in vivo proliferation of gamma delta T cells and/or contacting a pharmaceutical composition of any one of the embodiments disclosed herein, with a cell derived from a subject in need thereof thereby causing an ex vivo proliferation of gamma delta T cells.
  • the current disclosure provides a method of treating cancer, comprising administering to a subject in need thereof a pharmaceutical composition of any of the embodiments disclosed herein to a subject in need thereof.
  • the subject ’s T cells are activated by the first domain.
  • the subject has a cancer and the gamma delta T cells modulate cells of the tumor.
  • the method further comprises administering to the subject a second pharmaceutical composition that costimulates ⁇ T cells, or contacting a second pharmaceutical composition that costimulates ⁇ T cells, with a cell derived from a subject in need thereof thereby causing an ex vivo proliferation of gamma delta T cells.
  • the present disclosure relates to a method for treating a cancer in a subject in need thereof comprising: (i) administering to the subject a pharmaceutical composition comprising the heterodimeric protein; and (ii) administering to the subject a second pharmaceutical composition that costimulates ⁇ T cells.
  • the first pharmaceutical composition and the second pharmaceutical composition are administered simultaneously or contemporaneously.
  • the first pharmaceutical composition is administered after the second pharmaceutical composition is administered.
  • the first pharmaceutical composition is administered before the second pharmaceutical composition is administered.
  • the second pharmaceutical composition costimulates a receptor selected from CD28, NKG2D, CD27, CD30, 4-1BB (CD137), IL-2R, IL-15R, IL-7R, IL-21R, NKp30, NKp44, DNAM-1 (CD226), IL-2R, IL-7R, IL-15R, dectins, NLRs, killer Ig-like receptors (e.g., KIR2D, KIR3D), C-type lectins (CD94/NKG2A-C, NKG2D), LFA1, CD2, CD46, Junctional Adhesion Molecule-Like (JAML).
  • a receptor selected from CD28, NKG2D, CD27, CD30, 4-1BB (CD137), IL-2R, IL-15R, IL-7R, IL-21R, NKp30, NKp44, DNAM-1 (CD226), IL-2R, IL-7R, IL-15R, dectins,
  • the second pharmaceutical composition comprises a ligand of the receptor, or a receptor-binding portion thereof.
  • the second pharmaceutical composition comprises a fusion protein (without limitation, e.g., an Fc fusion protein or an albumin fusion protein) comprising a co- stimulatory molecule or a binding portion thereof.
  • the second pharmaceutical composition comprises a fusion protein (without limitation, e.g., an Fc fusion protein or an albumin fusion protein) comprising the ligand of the receptor, or receptor-binding portion thereof.
  • the second pharmaceutical composition comprises a fusion protein (without limitation, e.g., an Fc fusion protein or an albumin fusion protein) comprising the receptor, or a ligand-binding portion thereof.
  • the second pharmaceutical composition comprises an antibody, antibody-like molecule or a receptor-binding portion thereof.
  • the second pharmaceutical composition comprises an agonistic antibody.
  • the second pharmaceutical composition costimulates CD28.
  • the second pharmaceutical composition comprises a CD28 ligand, or a CD28-binding portion thereof.
  • the CD28 ligand is selected from CD80 and CD86.
  • the CD28 ligand of is an antibody, an antibody-like molecule, or a binding fragment thereof.
  • the CD28 ligand is a fusion protein (without limitation, e.g., Fc fusion protein) comprising CD80, CD86, or a CD28-binding portion thereof.
  • the CD28 ligand is an Fc fusion protein comprising CD80, CD86, or a CD28-binding portion thereof.
  • the binding fragment is selected from Fab fragment, heavy variable chain, and single chain variable fragments (scFV).
  • the antibody is an agonistic antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is an anti-CD28 monoclonal antibody selected from JJ316, D665, 5.11A1, TGN1412, 37.51, E18, and PV-1.
  • the antibody is an agonistic antibody.
  • the antibody is a monoclonal antibody.
  • the antibody is an anti-NKG2D monoclonal antibody selected from tesnatilimab (also known as JNJ 4500 or JNJ-64304500) 149810, 1D11 and 5C6.
  • the second pharmaceutical composition comprises a heterologous chimeric protein capable of costimulating a receptor selected from CD28, NKG2D, CD27, CD30, 4-1BB (CD137), IL-2R, IL-15R, IL-7R, IL-21R, NKp30, NKp44, DNAM-1 (CD226), IL-2R, IL-7R, IL-15R, dectins, NLRs, killer Ig-like receptors (e.g., KIR2D, KIR3D), C-type lectins (CD94/NKG2A-C, NKG2D), LFA1, CD2, CD46, and Junctional Adhesion Molecule-Like (JAML) and/or inhibiting a receptor selected from a receptor selected from PD-1, PD-L1 and BTLA.
  • a heterologous chimeric protein capable of costimulating a receptor selected from CD28, NKG2D, CD27, CD30, 4-1BB (CD137), IL
  • the heterologous chimeric protein comprises (a) a first domain comprising an extracellular domain of a type I membrane protein; (b) a second domain comprising an extracellular domain of Type II transmembrane protein; and (c) a linker linking the first domain and the second domain.
  • the linker comprises a hinge- CH2-CH3 Fc domain.
  • Suitable heterologous chimeric proteins are disclosed in PCT publications WO 2017/059168, WO 2017/059168, WO 2017/059168, WO 2017/059168, WO 2018/157165, WO 2018/157165, WO 2020/047319, WO 2020/047322, WO 2020/047325, WO 2020/047327, WO 2020/047328, WO 2020/047329, WO 2020/176718, WO 2020/232365, and WO 2021/041958, which are hereby incorporated by reference in their entirety.
  • the method comprises: administering to the subject the pharmaceutical composition comprising the heterodimeric protein of any of the embodiments disclosed herein.
  • the method comprises: administering to the subject the pharmaceutical composition comprising the heterodimeric protein of any of the embodiments disclosed herein, and administering to the subject the second pharmaceutical composition.
  • the pharmaceutical composition and the second pharmaceutical composition are administered simultaneously or contemporaneously.
  • the pharmaceutical composition is administered after the second pharmaceutical composition is administered.
  • the pharmaceutical composition is administered before the second pharmaceutical composition is administered.
  • the method further comprises administering to the subject in need thereof gamma delta T cells that are proliferated ex vivo.
  • the method comprises: contacting a pharmaceutical composition comprising the heterodimeric protein of any one of the embodiments disclosed herein, with a cell derived from a subject in need thereof thereby causing an ex vivo proliferation of gamma delta T cells, and administering to the subject in need thereof gamma delta T cells that are proliferated ex vivo.
  • the method comprises: contacting cells derived from a subject in need thereof with a pharmaceutical composition comprising the heterodimeric protein of any one of the embodiments disclosed herein, and the second pharmaceutical composition that costimulates ⁇ T cells of any of the DB1/ 145191832.2 44 Attorney Docket No.: SHK-081PC/116981-5081 embodiments disclosed herein, thereby causing an ex vivo proliferation of gamma delta T cells, and administering to the subject in need thereof gamma delta T cells that are proliferated ex vivo.
  • the cancer is a B cell lymphoma or leukemia.
  • the cancer is an acute myeloid leukemia (AML).
  • the cancer is a solid cancer selected from prostate cancer, small cell lung cancer, non-small cell lung cancer, neuroendocrine tumors, renal cancer, bladder cancer, colon cancer, and breast cancer.
  • the cancer is known to express the antigenic target of the targeting domain disclosed herein.
  • the cancer is suspected to express the antigenic target of the targeting domain disclosed herein.
  • the present disclosure relates to a method of treating a cancer expressing CD20 in a subject in need thereof comprising: administering to the subject a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an in vivo proliferation of gamma delta T cells and/or contacting a cell derived from the subject with a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an ex vivo proliferation of gamma delta T cells.
  • the present disclosure relates to a method of treating a cancer that is CD20 positive in a subject in need thereof comprising: administering to the subject a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an in vivo proliferation of gamma delta T cells and/or contacting a cell derived from the subject with a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an ex vivo proliferation of gamma delta T cells.
  • the present disclosure relates to a method of treating a cancer that is CD20 + , in a subject in need thereof comprising: administering to the subject a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an in vivo proliferation of gamma delta T cells and/or contacting a cell derived from the subject with a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an ex vivo proliferation of gamma delta T cells.
  • the cancer is an epithelial-derived carcinoma.
  • the cancer is known to express the antigenic target of the targeting domain.
  • the cancer is known to express the antigenic target of the targeting domain.
  • the cancer has mutations which limit recognition by alpha beta T cells, optionally selected from mutations in MHC I, beta 2 microglobulin, and Transporter associated with antigen processing (TAP).
  • TAP Transporter associated with antigen processing
  • the subject is further administered autologous or allogeneic gamma delta T cells that were expanded ex vivo.
  • the autologous or allogeneic gamma delta T cells express a Chimeric Antigen Receptor.
  • the subject is further administered autologous or allogeneic T cells that express a Chimeric Antigen Receptor.
  • the current disclosure provides a method of treating an autoimmune disease or disorder, comprising administering a pharmaceutical composition of any of the embodiments disclosed herein to a subject in need thereof, wherein the autoimmune disease or disorder is optionally selected from rheumatoid arthritis, systemic lupus erythematosus, diabetes mellitus, ankylosing spondylitis, Sjögren's syndrome, inflammatory bowel diseases (e.g., colitis ulcerosa, Crohn's disease), multiple sclerosis, sarcoidosis, psoriasis, Grave's disease, Hashimoto's thyroiditis, psoriasis, hypersensitivity reactions (e.g., allergies, hay fever, asthma, and acute edema cause type I hypersensitivity reactions), and pemphigus, and vasculitis (e.g., Granulomatosis with poly
  • the current disclosure pertains to the use of the heterodimeric proteins for the treatment of one or more autoimmune diseases or disorders.
  • the treatment of an autoimmune disease or disorder may involve modulating the immune system with the present heterodimeric proteins to favor immune inhibition over immune stimulation.
  • Illustrative autoimmune diseases or disorders treatable with the present heterodimeric proteins include those in which the body’s own antigens become targets for an immune response, such as, for example, rheumatoid arthritis, systemic lupus erythematosus, diabetes mellitus, ankylosing spondylitis, Sjögren's syndrome, inflammatory bowel diseases (e.g., colitis ulcerosa, Crohn's disease), multiple sclerosis, sarcoidosis, psoriasis, Grave's disease, Hashimoto's thyroiditis, psoriasis, hypersensitivity reactions (e.g., allergies, hay fever, asthma, and acute edema cause type I hypersensitivity reactions), and pemphigus, and vasculitis (e.g., Granulomatosis with polyangiitis).
  • rheumatoid arthritis systemic lupus erythematosus
  • diabetes mellitus ankylosing
  • Illustrative autoimmune diseases or conditions that may be treated or prevented using the heterodimeric protein of the invention include, but are not limited to, 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
  • the current disclosure pertains to cancers and/or tumors; for example, the treatment or prevention of cancers and/or tumors.
  • the treatment of cancer may involve in various embodiments, modulating the immune system with the present heterodimeric proteins to favor immune stimulation over immune inhibition.
  • DB1/ 145191832.2 46 Attorney Docket No.: SHK-081PC/116981-5081 Cancers or tumors refer to an uncontrolled growth of cells and/or abnormal increased cell survival and/or inhibition of apoptosis which interferes with the normal functioning of the bodily organs and systems. Included are benign and malignant cancers, polyps, hyperplasia, as well as dormant tumors or micrometastases.
  • the cancer may be a primary cancer or a metastatic cancer.
  • the primary cancer may be an area of cancer cells at an originating site that becomes clinically detectable, and may be a primary tumor.
  • the metastatic cancer may be the spread of a disease from one organ or part to another non-adjacent organ or part.
  • the metastatic cancer may be caused by a cancer cell that acquires the ability to penetrate and infiltrate surrounding normal tissues in a local area, forming a new tumor, which may be a local metastasis.
  • the cancer may also be caused by a cancer cell that acquires the ability to penetrate the walls of lymphatic and/or blood vessels, after which the cancer cell is able to circulate through the bloodstream (thereby being a circulating tumor cell) to other sites and tissues in the body.
  • the cancer may be due to a process such as lymphatic or hematogeneous spread.
  • the cancer may also be caused by a tumor cell that comes to rest at another site, re-penetrates through the vessel or walls, continues to multiply, and eventually forms another clinically detectable tumor.
  • the cancer may be this new tumor, which may be a metastatic (or secondary) tumor.
  • the cancer may be caused by tumor cells that have metastasized, which may be a secondary or metastatic tumor.
  • the cells of the tumor may be like those in the original tumor.
  • the secondary tumor while present in the liver, is made up of abnormal breast or colon cells, not of abnormal liver cells.
  • the tumor in the liver may thus be a metastatic breast cancer or a metastatic colon cancer, not liver cancer.
  • the cancer may have an origin from any tissue.
  • the cancer may originate from melanoma, colon, breast, or prostate, and thus may be made up of cells that were originally skin, colon, breast, or prostate, respectively.
  • the cancer may also be a hematological malignancy, which may be leukemia or lymphoma.
  • the cancer may invade a tissue such as liver, lung, bladder, or intestinal.
  • the heterodimeric protein is used to treat a subject that has a treatment-refractory cancer. In embodiments, the heterodimeric protein is used to treat a subject that is refractory to one or more immune-modulating agents. For example, in embodiments, the heterodimeric protein is used to treat a subject that presents no response to treatment, or even progress, after 12 weeks or so of treatment.
  • the subject is refractory to a PD-1 and/or PD-L1 and/or PD-L2 agent, including, for example, nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), pidilizumab (CT-011, CURE TECH), MK- DB1/ 145191832.2 47 Attorney Docket No.: SHK-081PC/116981-5081 3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), Ibrutinib (PHARMACYCLICS/ABBVIE), atezolizumab (TECENTRIQ, GENENTECH), and/or MPDL328OA (ROCHE)-refractory patients.
  • nivolumab ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB
  • the subject is refractory to an anti-CTLA-4 agent, e.g., ipilimumab (YERVOY)- refractory patients (e.g., melanoma patients).
  • an anti-CTLA-4 agent e.g., ipilimumab (YERVOY)- refractory patients (e.g., melanoma patients).
  • the current disclosure provides methods of cancer treatment that rescue patients that are non-responsive to various therapies, including monotherapy of one or more immune-modulating agents.
  • the current disclosure provides heterodimeric proteins, and methods of use of the same, which target a cell or tissue within the tumor microenvironment.
  • the cell or tissue within the tumor microenvironment expresses one or more targets or binding partners of the heterodimeric protein.
  • the tumor microenvironment refers to the cellular milieu, including cells, secreted proteins, physiological small molecules, and blood vessels in which the tumor exists.
  • the cells or tissue within the tumor microenvironment are one or more of: tumor vasculature; tumor- infiltrating lymphocytes; fibroblast reticular cells; endothelial progenitor cells (EPC); cancer-associated fibroblasts; pericytes; other stromal cells; components of the extracellular matrix (ECM); dendritic cells; antigen presenting cells; T-cells; regulatory T cells; macrophages; neutrophils; and other immune cells located proximal to a tumor.
  • the present heterodimeric protein targets a cancer cell.
  • the cancer cell expresses one or more of targets or binding partners of the heterodimeric protein.
  • the heterodimeric protein of the invention may target a cell (e.g., cancer cell or immune cell) that expresses any of the receptors as described herein.
  • the heterodimeric protein of the invention may target a cell that expresses any of the receptors for a cytokine, growth factor, and/or hormone as described herein.
  • the present methods provide treatment with the heterodimeric protein in a patient who is refractory to an additional agent, such “additional agents” being described elsewhere herein, inclusive, without limitation, of the various chemotherapeutic agents described herein.
  • the current disclosure is directed toward methods of treating and preventing T cell-mediated diseases and disorders, such as, but not limited to diseases or disorders described elsewhere herein and inflammatory disease or disorder, graft-versus-host disease (GVHD), transplant rejection, and T cell proliferative disorder.
  • T cell-mediated diseases and disorders such as, but not limited to diseases or disorders described elsewhere herein and inflammatory disease or disorder, graft-versus-host disease (GVHD), transplant rejection, and T cell proliferative disorder.
  • the present chimeric agents are used in methods of activating a T cell, e.g., via the extracellular domain having an immune stimulatory signal or antibody binding domain (e.g., CDR3, Fab, scFv domain, etc.) having an immune stimulatory signal.
  • an immune stimulatory signal or antibody binding domain e.g., CDR3, Fab, scFv domain, etc.
  • the present chimeric agents are used in methods of preventing the cellular transmission of an immunosuppressive signal.
  • the present disclosure relates to a method of contemporaneous activation and targeting of gamma delta T cells to cancer cells comprising administering to a subject in need thereof a pharmaceutical composition of any one of the embodiments disclosed herein.
  • the present disclosure relates to a method of modulating a patient’s immune response, comprising administering a pharmaceutical composition of any one of the embodiments disclosed herein to a subject in need thereof.
  • the present disclosure relates to a method of stimulating proliferation of gamma delta T cells, comprising: administering a pharmaceutical composition of any one of the embodiments disclosed herein, to a subject in need thereof thereby causing an in vivo proliferation of gamma delta T cells and/or contacting a pharmaceutical composition of any one of the embodiments disclosed herein, with a cell derived from a subject in need thereof thereby causing an ex vivo proliferation of gamma delta T cells.
  • the subject ’s T cells are activated by the first domain.
  • the subject has a cancer and the gamma delta T cells modulate cells of the tumor.
  • the method further comprises administering to the subject a second pharmaceutical composition that costimulates ⁇ T cells, or contacting a second pharmaceutical composition that costimulates ⁇ T cells, with a cell derived from a subject in need thereof thereby causing an ex vivo proliferation of gamma delta T cells.
  • the method comprises administering to the subject the pharmaceutical composition.
  • the method comprises: administering to the subject the pharmaceutical composition, and administering to the subject the second pharmaceutical composition.
  • the pharmaceutical composition and the second pharmaceutical composition are administered simultaneously or contemporaneously.
  • the pharmaceutical composition is administered after the second pharmaceutical composition is administered.
  • the pharmaceutical composition is administered before the second pharmaceutical composition is administered.
  • the invention provides for heterodimeric proteins, and methods of use of the same, and methods that further comprise administering an additional agent to a subject.
  • the invention pertains to co-administration and/or co-formulation. Any of the compositions described herein may be co-formulated and/or co-administered. DB1/ 145191832.2 49 Attorney Docket No.: SHK-081PC/116981-5081
  • any heterodimeric protein described herein acts synergistically when co-administered with another agent and is administered at doses that are lower than the doses commonly employed when such agents are used as monotherapy.
  • any agent referenced herein may be used in combination with any of the heterodimeric proteins, and methods of use of the same, described herein.
  • any of the heterodimeric proteins disclosed herein may be co-administered with another heterodimeric protein disclosed herein.
  • a combined regimen involving the administration of one or more heterodimeric proteins which induce an innate immune response and one or more heterodimeric proteins which induce an adaptive immune response may provide synergistic effects (e.g., synergistic anti-tumor effects).
  • any heterodimeric protein which induces an innate immune response may be utilized in the current disclosure.
  • any heterodimeric protein which induces an adaptive immune response may be utilized in the current disclosure.
  • the current disclosure pertains to chemotherapeutic agents as additional agents.
  • chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and CYTOXAN cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (e.g., bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); br
  • dynemicin including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo- 5-oxo-L-norleucine, ADRIAMYCIN doxorubicin (including morpholino- doxorubicin, cyanomorpholino- DB1/ 145191832.2 50 Attorney Docket No.: SHK-081PC/116981-5081 dox
  • vinorelbine novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (TYKERB); inhibitors of PKC- ⁇ , Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above.
  • DMFO difluoromethylorni
  • the methods of treatment can further include the use of radiation.
  • the methods of treatment can further include the use of photodynamic therapy.
  • the present additional agent is one or more immune-modulating agents selected from an agent that blocks, reduces and/or inhibits DB1/ 145191832.2 51 Attorney Docket No.: SHK-081PC/116981-5081 PD-1 and PD-L1 or PD-L2 and/or the binding of PD-1 with PD-L1 or PD-L2 (by way of non-limiting example, one or more of nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, Merck), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), atezolizumab (TECENTRIQ, GENENTECH), MPDL328OA (ROCHE),
  • the current disclosure pertains to anti-infectives as additional agents.
  • the anti-infective is an anti-viral agent including, but not limited to, Abacavir, Acyclovir, Adefovir, Amprenavir, Atazanavir, Cidofovir, Darunavir, Delavirdine, Didanosine, Docosanol, Efavirenz, Elvitegravir, Emtricitabine, Enfuvirtide, Etravirine, Famciclovir, and Foscarnet.
  • the anti-infective is an anti-bacterial agent including, but not limited to, cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, and ceftobiprole); fluoroquinolone antibiotics (cipro, Levaquin, floxin, tequin, avelox, and norflox); tetracycline antibiotics (tetracycline, minocycline, oxytetracycline, and doxycycline); penicillin antibiotics (amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin, and methicillin); monobactam antibiotics (aztreonam); and carbapenem antibiotics (ertapenem, doripenem, imipenem/cilastatin, and meropenem).
  • cephalosporin antibiotics ce
  • the anti-infectives include anti-malarial agents (e.g., chloroquine, quinine, mefloquine, primaquine, doxycycline, artemether/lumefantrine, atovaquone/proguanil and sulfadoxine/pyrimethamine), metronidazole, tinidazole, ivermectin, pyrantel pamoate, and albendazole.
  • the additional agent is an immunosuppressive agent.
  • the immunosuppressive agent is an anti-inflammatory agent such as a steroidal anti-inflammatory agent or a non-steroidal anti-inflammatory agent (NSAID).
  • corticosteroids useful in the current disclosure include, without limitation, 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, hydrocortis
  • NSAIDS that may be used in the current disclosure, include but are not limited to, salicylic acid, acetyl salicylic acid, methyl salicylate, glycol salicylate, salicylmides, benzyl-2,5-diacetoxybenzoic acid, ibuprofen, fulindac, naproxen, ketoprofen, etofenamate, phenylbutazone, and indomethacin.
  • the immunosupressive agent may be cytostatics such as alkylating agents, antimetabolites (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, antimetabolites (e.g., azathioprine, methotrexate), cytotoxic antibiotics, antibodies (e.g., basiliximab, daclizumab, and muromonab), anti-immunophilins (e.g., cyclosporine, tacrolimus, sirolimus), inteferons, opioids, T
  • the heterodimeric proteins (and/or additional agents) described herein include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the composition such that covalent attachment does not prevent the activity of the composition.
  • derivatives include composition that have been modified by, inter alia, glycosylation, lipidation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc.
  • the heterodimeric proteins (and/or additional agents) described herein further comprise a cytotoxic agent, comprising, in illustrative embodiments, a toxin, a chemotherapeutic agent, a radioisotope, and an agent that causes apoptosis or cell death.
  • a cytotoxic agent comprising, in illustrative embodiments, a toxin, a chemotherapeutic agent, a radioisotope, and an agent that causes apoptosis or cell death.
  • agents may be conjugated to a composition described herein.
  • heterodimeric proteins (and/or additional agents) described herein may thus be modified post- translationally to add effector moieties such as chemical linkers, detectable moieties such as for example fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, and chemiluminescent moieties, or functional moieties such as for example streptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, and radioactive materials.
  • effector moieties such as chemical linkers, detectable moieties such as for example fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, and chemiluminescent moieties, or functional moieties such as for example streptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, and radioactive materials.
  • heterodimeric proteins (and/or additional agents) described herein can possess a sufficiently basic functional group, which can react with an inorganic or organic acid, or a carboxyl group, which can react with an inorganic or organic base, to form a pharmaceutically acceptable salt.
  • a pharmaceutically acceptable acid addition salt is formed from a pharmaceutically acceptable acid, as is well known in the art.
  • Such salts include the pharmaceutically acceptable salts listed in, for example, Journal of Pharmaceutical Science, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use. P. H. Stahl and C. G.
  • compositions described herein are in the form of a pharmaceutically acceptable salt.
  • any heterodimeric protein (and/or additional agents) described herein can be administered to a subject as a component of a composition that comprises a pharmaceutically acceptable carrier or vehicle.
  • Such compositions can optionally comprise a suitable amount of a pharmaceutically acceptable excipient so as to provide the form for proper administration.
  • Pharmaceutical excipients can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
  • the pharmaceutical excipients can be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like.
  • auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used.
  • the pharmaceutically acceptable excipients are sterile when administered to a subject.
  • Water is a useful excipient when any agent described herein is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, specifically for injectable solutions.
  • Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
  • Any agent described herein, if desired, can also comprise minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • the compositions described herein are resuspended in a saline buffer (including, without limitation TBS, PBS, and the like).
  • the heterodimeric proteins may by conjugated and/or fused with another agent to extend half-life or otherwise improve pharmacodynamic and pharmacokinetic properties.
  • the heterodimeric proteins may be fused or conjugated with one or more of PEG, XTEN (e.g., as rPEG), polysialic acid (POLYXEN), albumin (e.g., human serum albumin or HAS), elastin-like protein (ELP), PAS, HAP, GLK, CTP, transferrin, and the like.
  • each of the DB1/ 145191832.2 54 Attorney Docket No.: SHK-081PC/116981-5081 individual heterodimeric proteins is fused to one or more of the agents described in BioDrugs (2015) 29:215–239, the entire contents of which are hereby incorporated by reference.
  • Administration, Dosing, and Treatment Regimens The current disclosure includes the described heterodimeric protein (and/or additional agents) in various formulations.
  • any heterodimeric protein (and/or additional agents) described herein can take the form of solutions, suspensions, emulsion, drops, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use.
  • DNA or RNA constructs encoding the protein sequences may also be used.
  • the composition is in the form of a capsule (see, e.g., U.S. Patent No. 5,698,155).
  • suitable pharmaceutical excipients are described in Remington’s Pharmaceutical Sciences 1447-1676 (Alfonso R.
  • the formulations comprising the heterodimeric protein (and/or additional agents) can also include a solubilizing agent.
  • the agents can be delivered with a suitable vehicle or delivery device as known in the art.
  • Combination therapies outlined herein can be co-delivered in a single delivery vehicle or delivery device.
  • Compositions for administration can optionally include a local anesthetic such as, for example, lignocaine to lessen pain at the site of the injection.
  • the formulations comprising the heterodimeric protein (and/or additional agents) of the current disclosure may conveniently be presented in unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy.
  • Such methods generally include the step of bringing the therapeutic agents into association with a carrier, which constitutes one or more accessory ingredients.
  • a carrier which constitutes one or more accessory ingredients.
  • the formulations are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into dosage forms of the desired formulation (e.g., wet or dry granulation, powder blends, etc., followed by tableting using conventional methods known in the art).
  • any heterodimeric protein (and/or additional agents) described herein is formulated in accordance with routine procedures as a composition adapted for a mode of administration described herein.
  • Routes of administration include, for example: intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin.
  • the DB1/ 145191832.2 55 Attorney Docket No.: SHK-081PC/116981-5081 administering is effected orally or by parenteral injection. In most instances, administration results in the release of any agent described herein into the bloodstream. Any heterodimeric protein (and/or additional agents) described herein can be administered orally.
  • heterodimeric proteins can also be administered by any other convenient route, for example, by intravenous infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with another biologically active agent. Administration can be systemic or local.
  • Various delivery systems are known, e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer. In specific embodiments, it may be desirable to administer locally to the area in need of treatment.
  • the heterodimeric protein (and/or additional agents) are administered in the tumor microenvironment (e.g., cells, molecules, extracellular matrix and/or blood vessels that surround and/or feed a tumor cell, inclusive of, for example, tumor vasculature; tumor- infiltrating lymphocytes; fibroblast reticular cells; endothelial progenitor cells (EPC); cancer-associated fibroblasts; pericytes; other stromal cells; components of the extracellular matrix (ECM); dendritic cells; antigen presenting cells; T-cells; regulatory T cells; macrophages; neutrophils; and other immune cells located proximal to a tumor) or lymph node and/or targeted to the tumor microenvironment or lymph node.
  • the tumor microenvironment e.g., cells, molecules, extracellular matrix and/or blood vessels that surround and/or feed a tumor cell, inclusive of, for example, tumor vasculature; tumor- infiltrating lymphocytes; fibroblast reticular cells; end
  • the heterodimeric protein for instance in the treatment of cancer, are administered intratumorally.
  • the present heterodimeric protein allows for a dual effect that provides less side effects than are seen in conventional immunotherapy (e.g., treatments with one or more of OPDIVO, KEYTRUDA, YERVOY, and TECENTRIQ).
  • the present heterodimeric proteins reduce or prevent commonly observed immune-related adverse events that affect various tissues and organs including the skin, the gastrointestinal tract, the kidneys, peripheral and central nervous system, liver, lymph nodes, eyes, pancreas, and the endocrine system; such as hypophysitis, colitis, hepatitis, pneumonitis, rash, and rheumatic disease.
  • the present local administration e.g., intratumorally, obviate adverse event seen with standard systemic administration, e.g., IV infusions, as are used with conventional immunotherapy (e.g., treatments with one or more of OPDIVO, KEYTRUDA, YERVOY, and TECENTRIQ).
  • Dosage forms suitable for parenteral administration include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid DB1/ 145191832.2 56 Attorney Docket No.: SHK-081PC/116981-5081 compositions (e.g., lyophilized composition), which can be dissolved or suspended in sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art.
  • any heterodimeric protein (and/or additional agents) described herein as well as the dosing schedule can depend on various parameters, including, but not limited to, the disease being treated, the subject’s general health, and the administering physician’s discretion.
  • Any heterodimeric protein described herein can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concurrently with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of an additional agent, to a subject in need thereof.
  • any heterodimeric protein and additional agent described herein are administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11 hours to 12 hours apart, 1 day apart, 2 days apart, 3 days apart, 4 days apart, 5 days apart, 6 days apart, 1 week apart, 2 weeks apart, 3 weeks apart, or 4 weeks apart.
  • the heterodimeric protein is administered at a frequency of about 1 day, or about 2 days, or about 3 days, or about 4 days, or about 5 days, or about 6 days, or about one week, or about 10 days, or about 15 days, or about 21 days, or more. In embodiments, the heterodimeric protein is administered at a frequency of about 3 days. In embodiments, the heterodimeric protein is administered at a frequency of about 7 days. In embodiments, the heterodimeric protein is administered at a frequency of about 1 day to about 10 days, about 1 day to about 5 days, or about 2 days to about 10 days, or about 5 days to about 15 days, or about one week to about 3 weeks.
  • the heterodimeric protein is administered at a frequency of about 1 day to about 10 days. In embodiments, the heterodimeric protein is administered so as to form a ratio of effector cells (heterodimeric protein-bound Gamma-Delta T cells) to target cells (without limitations, e.g., B cells or CD20 + cancer cells) is about 1:1, or about 1:2, or about 1:5, or about 1:7, or about 1:10, or about 1:15, or about 1:20 or less.
  • effector cells heterodimeric protein-bound Gamma-Delta T cells
  • target cells without limitations, e.g., B cells or CD20 + cancer cells
  • the heterodimeric protein is administered so as to form a ratio of effector cells (heterodimeric protein-bound Gamma-Delta T cells) to target cells (without limitations, e.g., B cells or CD20 + cancer cells) is about 1:1 to about 1:5, or about 1:2 to about 1:8, or about 1:10 to about 1: 20.
  • DB1/ 145191832.2 57 Attorney Docket No.: SHK-081PC/116981-5081
  • the current disclosure relates to the co-administration of a heterodimeric protein which induces an innate immune response and another heterodimeric protein which induces an adaptive immune response.
  • the heterodimeric protein which induces an innate immune response may be administered before, concurrently with, or subsequent to administration of the heterodimeric protein which induces an adaptive immune response.
  • the heterodimeric proteins may be administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11 hours to 12 hours apart, 1 day apart, 2 days apart, 3 days apart, 4 days apart, 5 days apart, 6 days apart, 1 week apart, 2 weeks apart, 3 weeks apart, or 4 weeks apart.
  • the heterodimeric protein which induces an innate immune response and the heterodimeric protein which induces an adaptive response are administered 1 week apart, or administered on alternate weeks (i.e., administration of the heterodimeric protein inducing an innate immune response is followed 1 week later with administration of the heterodimeric protein which induces an adaptive immune response and so forth).
  • the dosage of any heterodimeric protein (and/or additional agents) described herein can depend on several factors including the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the subject to be treated.
  • pharmacogenomic the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic
  • dosage used may affect dosage used.
  • the exact individual dosages can be adjusted somewhat depending on a variety of factors, including the specific combination of the agents being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the particular disease being treated, the severity of the disorder, and the anatomical location of the disorder. Some variations in the dosage can be expected.
  • the dosage may be about 0.1 mg to about 250 mg per day, about 1 mg to about 20 mg per day, or about 3 mg to about 5 mg per day.
  • the dosage of any agent described herein may be about 0.1 mg to about 1500 mg per day, or about 0.5 mg to about 10 mg per day, or about 0.5 mg to about 5 mg per day, or about 200 to about 1,200 mg per day (e.g., about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1,000 mg, about 1,100 mg, about 1,200 mg per day).
  • administration of the heterodimeric protein (and/or additional agents) described herein is by parenteral injection at a dosage of about 0.1 mg to about 1500 mg per treatment, or about 0.5 mg DB1/ 145191832.2 58 Attorney Docket No.: SHK-081PC/116981-5081 to about 10 mg per treatment, or about 0.5 mg to about 5 mg per treatment, or about 200 to about 1,200 mg per treatment (e.g., about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1,000 mg, about 1,100 mg, about 1,200 mg per treatment).
  • a suitable dosage of the heterodimeric protein (and/or additional agents) is in a range of about 0.01 mg/kg to about 100 mg/kg of body weight ,or about 0.01 mg/kg to about 10 mg/kg of body weight of the subject, for example, about 0.01 mg/kg, about 0.02 mg/kg, about 0.03 mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07 mg/kg, about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg, about 1.1 mg/kg, about 1.2 mg/kg, about 1.3 mg/kg, about 1.4 mg/kg, about 1.5 mg/kg, about 1.6 mg/kg, about 1.7 mg/kg, about 1.8 mg/kg,
  • delivery can be in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez- Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989).
  • Any heterodimeric protein (and/or additional agents) described herein can be administered by controlled- release or sustained-release means or by delivery devices that are well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Patent Nos.
  • Such dosage forms can be useful for providing controlled- or sustained-release of one or more active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions.
  • Controlled- or sustained- release of an active ingredient can be stimulated by various conditions, including but not limited to, changes in pH, changes in temperature, stimulation by an appropriate wavelength of light, concentration or availability of enzymes, concentration or availability of water, or other physiological conditions or compounds.
  • polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and DB1/ 145191832.2 59 Attorney Docket No.: SHK-081PC/116981-5081 Peppas, 1983, J. Macromol. Sci. Rev. Macromol.
  • a controlled-release system can be placed in proximity of the target area to be treated, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol.2, pp.115-138 (1984)).
  • Other controlled-release systems discussed in the review by Langer, 1990, Science 249:1527-1533 may be used.
  • Administration of any heterodimeric protein (and/or additional agents) described herein can, independently, be one to four times daily or one to four times per month or one to six times per year or once every two, three, four or five years. Administration can be for the duration of one day or one month, two months, three months, six months, one year, two years, three years, and may even be for the life of the subject.
  • the dosage regimen utilizing any heterodimeric protein (and/or additional agents) described herein can be selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the subject; the severity of the condition to be treated; the route of administration; the renal or hepatic function of the subject; the pharmacogenomic makeup of the individual; and the specific compound of the invention employed.
  • Any heterodimeric protein (and/or additional agents) described herein can be administered in a single daily dose, or the total daily dosage can be administered in divided doses of two, three or four times daily.
  • any heterodimeric protein (and/or additional agents) described herein can be administered continuously rather than intermittently throughout the dosage regimen.
  • the present disclosure relates to a polynucleotide encoding the alpha chain and/or beta chain of the heterodimeric protein of any one of the embodiments disclosed herein.
  • the nucleic acid is DNA or RNA.
  • the RNA is mRNA, which is optionally modified mRNA (mmRNA).
  • the present disclosure relates to an expression vector, comprising a nucleic acid encoding the alpha chain and/or beta chain of the heterodimeric protein of any one of the embodiments disclosed herein.
  • the expression vector is a mammalian expression vector.
  • the expression vector comprises DNA or RNA.
  • the present disclosure relates to a host cell, comprising the expression vector of any one of the embodiments disclosed herein, or the polynucleotide of the embodiments disclosed herein.
  • the current disclosure provides an expression vector, comprising a nucleic acid encoding the first and/or second polypeptide chains of the heterodimeric protein of any of any of the embodiments disclosed herein.
  • the expression vector is a mammalian expression vector.
  • the expression vector comprises DNA or RNA.
  • the current disclosure provides a host cell comprising the expression vector of any one of the embodiments disclosed herein.
  • the current disclosure provides an expression vector, comprising a nucleic acid encoding the heterodimeric protein (e.g., a heterodimeric protein comprising a first and second polypeptide chains) described herein.
  • the expression vector comprises DNA or RNA.
  • the expression vector is a mammalian expression vector. Both prokaryotic and eukaryotic vectors can be used for expression of the heterodimeric protein. Prokaryotic vectors include constructs based on E. coli sequences (see, e.g., Makrides, Microbiol Rev 1996, 60:512-538). Non-limiting examples of regulatory regions that can be used for expression in E.
  • prokaryotic expression vectors may include the ⁇ gt vector series such as ⁇ gt11 (Huynh et al., in “DNA Cloning Techniques, Vol. I: A Practical Approach,” 1984, (D. Glover, ed.), pp.49-78, IRL Press, Oxford), and the pET vector series (Studier et al., Methods Enzymol 1990, 185:60-89).
  • ⁇ gt vector series such as ⁇ gt11 (Huynh et al., in “DNA Cloning Techniques, Vol. I: A Practical Approach,” 1984, (D. Glover, ed.), pp.49-78, IRL Press, Oxford)
  • pET vector series Studdier et al., Methods Enzymol 1990, 185:60-89.
  • Prokaryotic host-vector systems cannot perform much of the post-translational processing of mammalian cells, however. Thus, eukaryotic host- vector systems may be particularly useful.
  • a variety of regulatory regions can be used for expression of the heterodimeric proteins in mammalian host cells.
  • the SV40 early and late promoters the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter can be used.
  • CMV cytomegalovirus
  • RSV-LTR Rous sarcoma virus long terminal repeat
  • Inducible promoters that may be useful in mammalian cells include, without limitation, promoters associated with the metallothionein II gene, mouse mammary tumor virus glucocorticoid responsive long terminal repeats (MMTV-LTR), the ⁇ -interferon gene, and the hsp70 gene (see, Williams et al., Cancer Res 1989, 49:2735-42; and Taylor et al., Mol Cell Biol 1990, 10:165-75). Heat shock promoters or stress promoters also may be advantageous for driving expression of the fusion proteins in recombinant host cells.
  • promoters associated with the metallothionein II gene mouse mammary tumor virus glucocorticoid responsive long terminal repeats (MMTV-LTR), the ⁇ -interferon gene, and the hsp70 gene (see, Williams et al., Cancer Res 1989, 49:2735-42; and Taylor et al., Mol Cell Biol 1990, 10:165-75).
  • expression vectors of the invention comprise a nucleic acid encoding at least the first and/or second polypeptide chains of the heterodimeric proteins (and/or additional agents), or a complement thereof, operably linked to an expression control region, or complement thereof, that is functional in a mammalian cell.
  • the expression control region is capable of driving expression of the operably linked blocking and/or stimulating agent encoding nucleic acid such that the blocking and/or stimulating agent is produced in a human cell transformed with the expression vector.
  • Expression control regions are regulatory polynucleotides (sometimes referred to herein as elements), such as promoters and enhancers, that influence expression of an operably linked nucleic acid.
  • An expression control region of an expression vector of the invention is capable of expressing operably linked encoding nucleic acid in a human cell.
  • the cell is a tumor cell.
  • the cell is a non-tumor cell.
  • the expression control region confers regulatable expression to an operably linked nucleic acid.
  • a signal (sometimes referred to as a stimulus) can increase or decrease expression of a nucleic acid operably linked to such an expression control region.
  • Such expression control regions that increase expression in response to a signal are often referred to as inducible.
  • Such expression control regions that decrease expression in response to a signal are often referred to as repressible.
  • the amount of increase or decrease conferred by such elements is proportional to the amount of signal present; the greater the amount of signal, the greater the increase or decrease in expression.
  • the current disclosure contemplates the use of inducible promoters capable of effecting high level of expression transiently in response to a cue.
  • a cell transformed with an expression vector for the heterodimeric protein (and/or additional agents) comprising such an expression control sequence is induced to transiently produce a high level of the agent by exposing the transformed cell to an appropriate cue.
  • Illustrative inducible expression control regions include those comprising an inducible promoter that is stimulated with a cue such as a small molecule chemical compound. Particular examples can be found, for example, in U.S. Pat. Nos. 5,989,910, 5,935,934, 6,015,709, and 6,004,941, each of which is incorporated herein by reference in its entirety.
  • Expression control regions and locus control regions include full-length promoter sequences, such as native promoter and enhancer elements, as well as subsequences or polynucleotide variants which retain all or part of full-length or non-variant function.
  • the term "functional" and grammatical variants thereof, when used in reference to a nucleic acid sequence, subsequence or fragment, means that the sequence has one or more functions of native nucleic acid sequence (e.g., non-variant or unmodified sequence).
  • operble linkage refers to a physical juxtaposition of the components so described as to permit them to function in their intended manner.
  • an expression control element in operable linkage with a nucleic acid, the relationship is such that the control element modulates expression of the nucleic acid.
  • an expression control region that modulates transcription is juxtaposed near the 5' end of the transcribed nucleic acid (i.e., “upstream”).
  • Expression control regions can also be located at the 3’ end of the transcribed sequence (i.e., “downstream”) or within the transcript DB1/ 145191832.2 62
  • Attorney Docket No.: SHK-081PC/116981-5081 e.g., in an intron.
  • Expression control elements can be located at a distance away from the transcribed sequence (e.g., 100 to 500, 500 to 1000, 2000 to 5000, or more nucleotides from the nucleic acid).
  • a specific example of an expression control element is a promoter, which is usually located 5' of the transcribed sequence.
  • Another example of an expression control element is an enhancer, which can be located 5' or 3' of the transcribed sequence, or within the transcribed sequence.
  • Expression systems functional in human cells are well known in the art, and include viral systems.
  • a promoter functional in a human cell is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence into mRNA.
  • a promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and typically a TATA box located 25-30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site.
  • a promoter will also typically contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box.
  • An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation.
  • promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range.
  • transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence.
  • the 3’ terminus of the mature mRNA is formed by site-specific post- translational cleavage and polyadenylation.
  • transcription terminator and polyadenylation signals include those derived from SV40. Introns may also be included in expression constructs.
  • Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, polymer-based systems, DEAE-dextran, viral transduction, the calcium phosphate precipitation method, etc.
  • liposomes For in vivo gene transfer, a number of techniques and reagents may also be used, including liposomes; natural polymer-based delivery vehicles, such as chitosan and gelatin; viral vectors are also suitable for in vivo transduction.
  • a targeting agent such as an antibody or ligand specific for a tumor cell surface membrane protein.
  • proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g., capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo DB1/ 145191832.2 63 Attorney Docket No.: SHK-081PC/116981-5081 internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life.
  • the technique of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990).
  • gene delivery agents such as, e.g., integration sequences can also be employed.
  • Numerous integration sequences are known in the art (see, e.g., Nunes-Duby et al., Nucleic Acids Res. 26:391-406, 1998; Sadwoski, J. Bacteriol., 165:341-357, 1986; Bestor, Cell, 122(3):322-325, 2005; Plasterk et al., TIG 15:326-332, 1999; Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). These include recombinases and transposases. Examples include Cre (Sternberg and Hamilton, J. Mol.
  • transposases of the mariner family (Plasterk et al., supra), and components for integrating viruses such as AAV, retroviruses, and antiviruses having components that provide for virus integration such as the LTR sequences of retroviruses or lentivirus and the ITR sequences of AAV (Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003).
  • direct and targeted genetic integration strategies may be used to insert nucleic acid sequences encoding the chimeric fusion proteins including CRISPR/CAS9, zinc finger, TALEN, and meganuclease gene- editing technologies.
  • the invention provides expression vectors for the expression of the heterodimeric proteins (and/or additional agents) that are viral vectors.
  • viral vectors useful for gene therapy are known (see, e.g., Lundstrom, Trends Biotechnol., 21: 117, 122, 2003.
  • Illustrative viral vectors include those selected from Antiviruses (LV), retroviruses (RV), adenoviruses (AV), adeno-associated viruses (AAV), and ⁇ viruses, though other viral vectors may also be used.
  • viral vectors that do not integrate into the host genome are suitable for use, such as ⁇ viruses and adenoviruses.
  • ⁇ viruses include Sindbis virus, Venezuelan equine encephalitis (VEE) virus, and Semliki Forest virus (SFV).
  • viral vectors that integrate into the host genome are suitable, such as retroviruses, AAV, and Antiviruses.
  • the invention provides methods of transducing a human cell in vivo, comprising contacting a solid tumor in vivo with a viral vector of the invention.
  • the current disclosure provides a host cell, comprising the expression vector comprising the heterodimeric protein described herein. Expression vectors can be introduced into host cells for producing the present heterodimeric proteins. Cells may be cultured in vitro or genetically engineered, for example.
  • Useful mammalian host cells include, without limitation, cells derived from humans, monkeys, and rodents (see, for example, Kriegler DB1/ 145191832.2 64 Attorney Docket No.: SHK-081PC/116981-5081 in “Gene Transfer and Expression: A Laboratory Manual,” 1990, New York, Freeman & Co.).
  • monkey kidney cell lines transformed by SV40 e.g., COS-7, ATCC CRL 1651
  • human embryonic kidney lines e.g., 293, 293-EBNA, or 293 cells subcloned for growth in suspension culture, Graham et al., J Gen Virol 1977, 36:59
  • baby hamster kidney cells e.g., BHK, ATCC CCL 10
  • Chinese hamster ovary-cells-DHFR e.g., CHO, Urlaub and Chasin, Proc Natl Acad Sci USA 1980, 77:4216
  • DG44 CHO cells CHO-K1 cells, mouse sertoli cells (Mather, Biol Reprod 1980, 23:243-251)
  • mouse fibroblast cells e.g., NIH-3T3
  • monkey kidney cells e.g., CV1 ATCC CCL 70
  • African green monkey kidney cells e.g., African green monkey kidney cells.
  • human cervical carcinoma cells e.g., HELA, ATCC CCL 2
  • canine kidney cells e.g., MDCK, ATCC CCL 34
  • buffalo rat liver cells e.g., BRL 3A, ATCC CRL 1442
  • human lung cells e.g., W138, ATCC CCL 75
  • human liver cells e.g., Hep G2, HB 8065
  • mouse mammary tumor cells e.g., MMT 060562, ATCC CCL51.
  • Illustrative cancer cell types for expressing the fusion proteins described herein include mouse fibroblast cell line, NIH3T3, mouse Lewis lung carcinoma cell line, LLC, mouse mastocytoma cell line, P815, mouse lymphoma cell line, EL4 and its ovalbumin transfectant, E.G7, mouse melanoma cell line, B16F10, mouse fibrosarcoma cell line, MC57, and human small cell lung carcinoma cell lines, SCLC#2 and SCLC#7.
  • Host cells can be obtained from normal or affected subjects, including healthy humans, cancer patients, and patients with an infectious disease, private laboratory deposits, public culture collections such as the American Type Culture Collection, or from commercial suppliers.
  • Cells that can be used for production of the present heterodimeric proteins in vitro, ex vivo, and/or in vivo include, without limitation, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells (e.g., as obtained from bone marrow), umbilical cord blood, peripheral blood, fetal liver, etc.
  • epithelial cells include, without limitation, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils,
  • Fc-containing macromolecules such as Fc fusion proteins
  • production and purification of Fc-containing macromolecules has become a standardized process, with minor modifications between products.
  • Fc containing macromolecules are produced by human embryonic kidney (HEK) cells (or variants thereof) or Chinese Hamster Ovary (CHO) cells (or variants thereof) or in some cases by bacterial or synthetic methods.
  • HEK human embryonic kidney
  • CHO Chinese Hamster Ovary
  • purified Fc containing macromolecules are stored in liquid form for some period of time, frozen for extended periods of time or in some cases lyophilized.
  • DB1/ 145191832.2 65 Attorney Docket No.: SHK-081PC/116981-5081 production of the heterodimeric proteins contemplated herein may have unique characteristics as compared to traditional Fc containing macromolecules.
  • the heterodimeric proteins may be purified using specific chromatography resins, or using chromatography methods that do not depend upon Protein A capture.
  • the heterodimeric proteins may be purified in an oligomeric state, or in multiple oligomeric states, and enriched for a specific oligomeric state using specific methods.
  • these methods could include treatment with specific buffers including specified salt concentrations, pH and additive compositions. In other examples, such methods could include treatments that favor one oligomeric state over another.
  • the heterodimeric proteins obtained herein may be additionally ‘polished’ using methods that are specified in the art.
  • the heterodimeric proteins are highly stable and able to tolerate a wide range of pH exposure (between pH 3-12), are able to tolerate a large number of freeze/thaw stresses (greater than 3 freeze/thaw cycles) and are able to tolerate extended incubation at high temperatures (longer than 2 weeks at 40 degrees C).
  • the heterodimeric proteins are shown to remain intact, without evidence of degradation, deamidation, etc. under such stress conditions.
  • the subject and/or animal is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate, such as a monkey, chimpanzee, or baboon.
  • the subject and/or animal is a non-mammal, such, for example, a zebrafish.
  • the subject and/or animal may comprise fluorescently-tagged cells (with e.g., GFP).
  • the subject and/or animal is a transgenic animal comprising a fluorescent cell.
  • the subject and/or animal is a human.
  • the human is a pediatric human. In other embodiments, the human is an adult human. In other embodiments, the human is a geriatric human. In other embodiments, the human may be referred to as a patient. In certain embodiments, the human has an age in a range of from about 0 months to about 6 months old, from about 6 to about 12 months old, from about 6 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 0
  • kits that can simplify the administration of any agent described herein.
  • An illustrative 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 also further comprise one or more additional agent described herein.
  • the kit comprises a container containing a composition of the invention and another composition, such those described herein.
  • the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the materials, compositions, devices, and methods of this technology.
  • the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.
  • the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the disclosure, the present technology, or embodiments thereof, may alternatively be described using more limiting terms such as “consisting of” or “consisting essentially of” the recited ingredients. Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.
  • Example 1 Construction and Characterization of an Illustrative BTN2A1/3A1-Fc-CD20 scFv Heterodimeric Protein
  • the heterodimeric protein of embodiments of the present disclosure comprises a dimer of two chimeric proteins, each comprising a butyrophilin family member, a core domain, and an antigen-targeting domain.
  • the “BTN2A1-Fc-CD20scFv/ BTN3A1-Fc-CD20scFv” construct included an alpha chain comprising an extracellular domain (ECD) of human BTN2A1 fused to an anti-CD20 scFv via a hinge-CH2-CH3 Fc domain, and a beta chain comprising an ECD of human BTN3A1 fused to the anti-CD20 scFv via a hinge- CH2-CH3 Fc domain.
  • FIG.1A Constructs encoding BTN2A1-Fc-CD20scFv protein (alpha chain) and BTN3A1-Fc-CD20scFv protein (beta chain) were generated.
  • This heterodimeric protein is the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein of some embodiments, and is also referred to herein as GAmma DELta T cell ENgager construct also is referred to herein as the BTN2A1/3A1-Fc-CD20scFv ‘GADLEN’ protein.
  • the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein was produced via a transient co-transfection in ExpiCHO cells of two plasmids encoding 1) the BTN2A1-Fc-CD20scFv protein (alpha chain construct) and 2) BTN3A1-Fc-CD20scFv protein (beta chain construct).
  • the alpha and beta constructs encoded a BTN2A1-Fc-CD20scFv protein (alpha chain) and 2) BTN3A1-Fc-CD20scFv protein (beta chain).
  • the alpha and beta chains contained charged polarized linker domains which facilitated heterodimerization of the desired BTN2A1/3A1-Fc-CD20scFv heterodimeric protein.
  • ExpiCHO cells were transiently transfected with a dual gene expression vector (pVITRO2-Blasti) encoding the alpha and beta chains. The cell culture supernatant from the transient transfection was harvested 6 days following transfection and purified over an FcXL chromatography resin.
  • BTN2A1-Fc-CD19scFv homodimeric protein or BTN3A1-Fc-CD19scFv homodimeric protein were designed using BTN2A1-Fc-CD20scFv protein (alpha chain) and BTN2A1-Fc- CD20scFv protein (beta chain), or BTN3A1-Fc-CD20scFv protein (alpha chain) and BTN3A1-Fc- CD20scFv protein (beta chain), and purified as discussed above.
  • BTN2A1/3A1-Fc-CD20scFv heterodimeric protein was assessed using a plate-based assay. Briefly, increasing amounts of purified BTN2A1-Fc- CD19scFv homodimeric protein or BTN3A1-Fc-CD19scFv homodimeric protein, or BTN2A1/3A1-Fc- CD20scFv heterodimeric protein were added in high-binding 96-well plates (Corning) and 1 ⁇ g /mL of an anti-NKG2D antibody was added to wells containing the BTN2A1-Fc-CD19scFv homodimeric protein, BTN3A1-Fc-CD19scFv homodimeric protein, or BTN2A1/3A1-Fc-CD20scFv heterodimeric protein.
  • BTN2A1-Fc-CD19scFv homodimeric protein or BTN3A1-Fc-CD19scFv homodimeric protein were used as a negative control.
  • the plates were incubated overnight at 4°C.
  • Wells were washed twice with DPBS, and 0.5 to 1 ⁇ 10 5 V ⁇ 9V ⁇ 2+-T cells in RPMI + 10% FBS, supplemented with 100 U/mL hIL-2.
  • BD FastImmune CD107a APC, GolgiStop, and GolgiPlug reagents (BD Biosciences) were added to cell culture according to the manufacturer’s protocols. Cells were incubated at 37°C for 4 hours and stained for CD107a expression for analysis by flow cytometry.
  • the proportion of CD107a was calculated as a fraction of the CD3+V ⁇ 9+ population. As shown in FIG.2A (right panel), a dose-dependent increase in CD107a-expressing V ⁇ 9V ⁇ 2+-T cells was observed when the V ⁇ 9V ⁇ 2+-T cells were induced by the combination of BTN2A1/3A1-Fc-CD20scFv heterodimeric protein and the anti-NKG2D antibody.
  • V ⁇ 9V ⁇ 2+-T cells induced the anti-NKG2A antibody in combination with BTN2A1-Fc-CD19scFv or BTN3A1-Fc-CD19scFv homodimeric protein expressed only a background levels of CD107a (FIG.2A (left and middle panels)).
  • Example 2 In Vitro Depletion of CD20+ Lymphoma Cells and B Cells by a BTN2A1/3A1-Fc-CD20scFv Heterodimeric Protein
  • CD20+ lymphoma cells and B cells were depleted by a BTN2A1/3A1- Fc-CD20scFv heterodimeric protein in an in vitro depletion assay.
  • human Daudi Burkitt’s lymphoma cells (CD20+ lymphoma cell line) were labeled with a fluogenic granzyme B substrate and co-cultured with V ⁇ 9+V ⁇ 2+ T cells (GDT) at a 1:1 ratio in the presence of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein at various concentrations to evaluate the ability of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein to promote ⁇ T cell cytotoxicity by transferring DB1/ 145191832.2 69 Attorney Docket No.: SHK-081PC/116981-5081 granzyme B into target cells.
  • CD20+ B cells purified from PBMC of healthy donor were co-cultured with V ⁇ 9+V ⁇ 2+ T cells at 2:1 ratio in the presence of various concentration of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein to evaluate the ability of the BTN2A1/3A1-Fc- CD20scFv heterodimeric protein to promote target cell killing.
  • the proportion of apoptotic B cells was analyzed by Apoptracker Green staining, which detects translocated phosphatidylserine residues on the cell surface, by flow cytometry.
  • mice were administered with PBS (vehicle control), a BTN2A1/3A1-Fc-CD20scFv heterodimeric protein (50 ⁇ g or 200 ⁇ g), or a BTN2A1/3A1-Fc-CD33scFv heterodimeric protein (200 ⁇ g, non-B cell targeting construct) intravenously, two doses administered once every three days (q3dx2).
  • Peripheral blood and spleens were harvested from animals 24 hours post last dose for quantification of B cells (FIG. 3A).
  • FIG. 3B non-B/T cells
  • hCD45+CD3-CD19- FIG. 3A
  • Top and bottom panels represent animals engrafted with PBMCs at different donors, containing high and low input ratios of V ⁇ 2+ T cells (Effector, E) and B cells (Target, T), respectively.
  • the input E to T ratio for each donor is indicated in the figures.
  • the experiments show the absolute count of human B cells significantly decreased after treatment with the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein, but not BTN2A1/3A1-Fc-CD33scFv heterodimeric protein, in both cohorts of animals engrafted with PBMCs with high or low E to T ratios (FIG.3B).
  • the absolute count of non-B cell populations remained unchanged after treatment (FIG.3C), demonstrating the specificity of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein in targeting CD20+ B cells.
  • V ⁇ 2+ T cells The lack of increase in V ⁇ 2+ T cells suggests, inter alia, the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein induced rapid V ⁇ 2+T cell-mediated killing of target cells in the absence of proliferation, and highlights the ability of gamma/delta ( ⁇ ) T cells that exist in low frequencies in promoting serial killing of target cells.
  • mice 8-10 weeks old immunocompromised NSG-IL15 mice were engrafted varying levels of V ⁇ 2+T cells and B cells on Day 0 as shown in FIG.3A and the Table below. On Days 1 and 4, the mice were intravenously administered with PBS (vehicle control) or the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein (50 ⁇ g). Peripheral blood and spleens were harvested from animals 24 hours post last dose for quantification of B cells.
  • PBS vehicle control
  • BTN2A1/3A1-Fc-CD20scFv heterodimeric protein 50 ⁇ g
  • B cell depletion was calculated based on the ratios of B cells in PBS-treated mice in comparison with the mice treated with the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein and expressed as percentage depletion.
  • the results are shown in the Table below: PBMC %V ⁇ 2 + T Cells (E) % B Cells (T) E:T Ratio %B Cell Depletion Donor (mean ⁇ SD)
  • mice were engrafted fixed levels of cells human CD45+CD3+V ⁇ 2+ T cells (V ⁇ 2+T cells) and B cells on Day 0 as shown in FIG.3A. On Days 1 and 4, the mice were intravenously administered with PBS (vehicle control) or increasing levels of the BTN2A1/3A1-Fc- CD20scFv heterodimeric protein (0.5 ⁇ g, 2 ⁇ g, 10 ⁇ g or 50 ⁇ g).
  • PBS vehicle control
  • BTN2A1/3A1-Fc- CD20scFv heterodimeric protein 0.5 ⁇ g, 2 ⁇ g, 10 ⁇ g or 50 ⁇ g.
  • Peripheral blood and spleens were harvested from animals 24 hours post last dose for quantification of B cells and V ⁇ 2+T cells.
  • the extent DB1/ 145191832.2 71 Attorney Docket No.: SHK-081PC/116981-5081 of B cell depletion was calculated based on the ratios of B cells in PBS-treated mice in comparison with the mice treated with the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein and expressed as percentage depletion.
  • FIG.3E left panel
  • there was dose-dependent depletion of B cells although the lowest dose used in this experiment provided near complete depletion of B cells.
  • Example 4 In Vivo Anti-Tumor Activity of the BTN2A1/3A1-Fc-CD20scFv Heterodimeric Protein An in vivo animal model of lymphoma was developed and treated with the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein as schematically shown in FIG.4A.
  • CD20 + human lymphoma Raji cells and 4 ⁇ 10 6 human PBMCs from one of two human donors were injected subcutaneously in 8-10 weeks old immunocompromised NSG-MHCI/MHCII double knockout mice (FIG. 4A).
  • the PBMCs served as a source of human V ⁇ 2+ T cells (CD45+CD3+V ⁇ 2+T cells).
  • PBMCs aliquots from the two donors were analyzed by flow cytometry to calculate the numbers of CD3 + T cells and V ⁇ 2 + T cells to determine the Effector to Target ratios.
  • mice were then randomly divided in four treatment groups: 1) vehicle only, 2) the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein (100 ⁇ g; 125 nM), 3) the BTN2A1/3A1-Fc-B7H3scFv heterodimeric protein (100 ⁇ g; 125 nM),and 4) a CD3 ⁇ CD20 engager (100 ⁇ g; 100 nM).
  • the mice were treated as above on days 0, 3, 7 and 10 (FIG.4A). Tumor growth was monitored and plotted in each individual mouse. The data are shown in FIG.4B to FIG. 4D.
  • CD3 ⁇ CD20 engager which targets all T cells to CD20 positive tumors, was used as a positive control.
  • the BTN2A1/3A1-Fc-B7H3scFv heterodimeric protein was used as a non-targeting control. See Liu et al, Effective killing of cells expressing CD276 (B7-H3) by a bispecific T cell engager based on a new fully human antibody, Transl Oncol. 2021; 14(12): 101232. As shown in FIG.
  • mice harboring Raji cells and PBMCs from wither of the donors showed comparable progressive tumor growth, with tumors in all but one mice becoming larger than 1500 mm 3 by day 20, indicating that the PBMCs DB1/ 145191832.2 72
  • Attorney Docket No.: SHK-081PC/116981-5081 from Donor 4 or Donor 6 themselves did not affect tumor growth compared to each other.
  • the mice treated with CD3 ⁇ CD20 engager showed a detectable reduction in tumor growth as illustrated by the fact that only two of six CD3 ⁇ CD20 engager-treated mice had tumors larger than 1000 mm 3 and none had tumors larger than 1500 mm 3 on day 20 (compare FIG.4D with FIG.4B).
  • mice treated with BTN2A1/3A1-Fc-B7H3scFv heterodimeric protein showed very little inhibition of tumor growth, if any (FIG. 4D).
  • FIG. 4C surprisingly, the mice treated with BTN2A1/3A1-Fc-CD20scFv heterodimeric protein showed remarkable inhibition of tumor growth compared to the vehicle-treated mice.
  • the mice treated with the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein exhibited better tumor control compared to the mice treated with the CD3 ⁇ CD20 engager (FIG.4E).
  • mice treated with the CD3 ⁇ CD20 engager had 5/6 tumors larger than about 300 mm 3 on day 20
  • none of the tumors in the mice treated with BTN2A1/3A1-Fc-CD20scFv heterodimeric protein were larger than about 300 mm 3 on day 20.
  • Example 5 In Vivo B cell Depletion by the BTN2A1/3A1-Fc-CD20scFv Heterodimeric Protein in Cynomolgus Macaques. The monkeys were infused with increasing doses of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein in a dose range of 0.1 to 25 mg/kg. The study design is shown in the Table below: Dose Schedule Dose Level No.
  • B cells showed a dose-dependent occupancy by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein.
  • Durable binding of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein to B-cells was observed in a dose dependent manner over at least 72 hours (FIG.5A).
  • FIG.5B compared to pre-dose samples the post dose samples exhibited occupancy of the CD3- CD19 + target cells by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein.
  • FIG.5C Gamma Delta T cells showed a dose-dependent occupancy by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein in 2 hours.
  • Gamma Delta T cell occupancy by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein returned to baseline by 72 hours (FIG.5C).
  • FIG.5D compared to pre-dose samples the post dose samples exhibited occupancy of the V ⁇ 9 + Gamma Delta T cell target cells by the BTN2A1/3A1- Fc-CD20scFv heterodimeric protein.
  • FIG.5E a rapid and dose-dependent B-cell depletion was observed in 2 hours in monkeys treated with the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein.
  • FIG.5F no such depletion was observed in other immune cell types such as CD4 + T cells.
  • FIG.5G the levels of V ⁇ 9 + cells did not rapidly decrease, indicating a lack of margination of those cells. Indeed, the levels of V ⁇ 9 + cells did not change during the 72 hours (FIG.5G).
  • the ratios of effector to target cells were calculated and the kinetics of B cell depletion was calculated for various E:T ratios as a function of time.
  • the receptor occupancy (RO) of B cells by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein was determined. Briefly, following doses, blood samples were recovered at various times post-dose, B cells were enriched.
  • the enriched B cells were analyzed using flow cytometry to determine proportion of B DB1/ 145191832.2 74 Attorney Docket No.: SHK-081PC/116981-5081 cells based on detection of a B cell marker.
  • the enriched B cells were analyzed using flow cytometry to determine the proportion the B cells bound to the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein were assayed based on detection of butyrophilin.
  • the proportions the B cells bound to the BTN2A1/3A1-Fc- CD20scFv heterodimeric protein were plotted as a function of time.
  • the enriched gamma delta T cells were analyzed using flow cytometry to determine the proportion the gamma delta T cells bound to the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein were assayed based on detection of CD20scFv.
  • receptor occupancy (RO) of gamma delta T cells by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein is transient, which without wishing to be bound by theory, is likely to be due to low affinity engagement of BTN2A1/3A1 with V ⁇ 9V ⁇ 2 TCR.
  • the number of B cells was determined at 24 hours post dosing.
  • FIG.5M a dose-dependent decrease in B cells was observed compared to the vehicle-alone treated group.
  • the number of gamma delta T cells was determined at 24 hours post dosing.
  • FIG.5N a lack of dose-dependent changes in the levels of gamma delta T level was observed.
  • Gamma Delta T cells represented approximately 1-3% of total T cells in cynomolgus macaques, and E:T ratios ranged from 1:7 to 1:49. All treatments were well tolerated, and no evidence of cytokine release syndrome or any other toxicities were observed.
  • DB1/ 145191832.2 75 Attorney Docket No.: SHK-081PC/116981-5081 Rapid depletion of B cells in less than 2 hours demonstrate, inter alia, and without wishing to be bound by theory, Gamma Delta T cells were not required to achieve B cell depletion. Indeed, over several weeks, the circulating frequencies of cynomolgus V ⁇ 9V ⁇ 2 T cells remained stable, and no evidence of either proliferation or migration out of the blood was observed.
  • V ⁇ 9V ⁇ 2 T cells eliminated the B cell pool by serial killing following treatment with the BTN2A1/3A1-Fc- CD20scFv heterodimeric protein.
  • V ⁇ 9V ⁇ 2 T cells achieved rapid depletion of target cells following treatment with the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein, without evidence of toxicity, even at high doses.
  • the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein could be detected at all times tested. Based on the data, it was determined that the serum half-life of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein is ⁇ 48-72 hours. After 48 hours after Dose 3, the animals were sacrificed and samples of liver, spleen, axillary lymph node (LN), inguinal LN, and mesenteric LN were recovered. These samples were homogenized and the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein were assayed in the homogenate. As shown in FIG.
  • the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein could be detected in all tissues 48 hours after dosing.
  • the study showed, inter alia, that all doses were well tolerated. No clinically relevant changes observed with any hematological parameters, except those noted herein. Liver and kidney function were normal throughout the study as were coagulation, D-Dimer, serum proteins and electrolytes. Minimal cytokine changes were observed and no IL-6 elevation was observed.

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Abstract

The current disclosure relates to, inter alia, compositions and methods, including methods that find use in the treatment of disease, such as immunotherapies for cancer and autoimmunity.

Description

Attorney Docket No.: SHK-081PC/116981-5081 TREATMENT OF B CELL-MEDIATED DISEASES BY ACTIVATING CD20-TARGETED GAMMA DELTA T CELLS FIELD OF THE DISCLOSURE The present disclosure relates to, inter alia, compositions comprising heterodimeric proteins and methods of treatment of disease, such as immunotherapies for cancer or autoimmune disease. PRIORITY This Application claims the benefit of, and priority to, U.S. Provisional Application Nos.63/490,221, filed March 14, 2023, and 63/496,491, filed April 17, 2023, the contents of each of which are hereby incorporated by reference in their entirety. SEQUENCE LISTING The instant application contains a sequence listing, which has been submitted in XML format via EFS- Web. The contents of the XML copy named “SHK-081PC_116981-5081_Sequence_Listing,” which was created on March 5, 2024, and is 41,423 bytes in size, and the contents of which are incorporated herein by reference in their entirety. BACKGROUND Gamma Delta T cells (γδ T cells or GDT) are capable of directly recognizing and killing transformed cells independently of HLA-antigen presentation. For example, without wishing to be bound by theory, the Vγ9Vδ2 T-cell receptor is believed to sense intracellular phosphorylated metabolites, which accumulate in cancer cells as a result of metabolic dysregulation (e.g., dysregulation of the mevalonate pathway) and/or upon pharmaceutical intervention. Therefore, the Gamma Delta T cells represent highly promising effector cell compartment for immunotherapy. However, these cells occur in very small amounts. For example, although these cells can rapidly expand upon stimulation, healthy individuals typically harbor on the order of only 50-500 million Vγ9Vδ2 T-cells in the peripheral blood, which is 1-10% of the total CD3+ T-cell population. No currently available therapeutic agent employs the Gamma Delta T cells for a therapeutic purpose in human. Accordingly, therapeutic options harness the potential of Gamma Delta T cells to treat human diseases are desired. SUMMARY In various aspects, the present disclosure relates to a compositions and methods that harness potential Gamma Delta T cells for immunotherapies for cancer, or autoimmune diseases or disorders. 1 DB1/ 145191832.2 Attorney Docket No.: SHK-081PC/116981-5081 Accordingly, in some aspects, the present disclosure relates to a method for treating a B cell-mediated disease or disorder, comprising administering to the subject about 0.01 mg/kg to about 100 mg/kg of a heterodimeric protein, or a nucleic acid encoding the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; (b) a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof, the target domain being directed to CD20; and (c) a linker that adjoins the first and second domains; and wherein the beta chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; (b) a second domain comprising the targeting domain, the target domain being directed to CD20; and (c) a linker that adjoins the first and second domains. In some aspects, the present disclosure relates to a method for increasing and/or stimulating B cell depletion in a subject suffering from a B cell-mediated disease or disorder, comprising administering to the subject about 0.01 mg/kg to about 100 mg/kg of a heterodimeric protein, or a nucleic acid encoding the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; (b) a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof, the target domain being directed to CD20; and (c) a linker that adjoins the first and second domains; and wherein the beta chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; (b) a second domain comprising the targeting domain, the target domain being directed to CD20; and (c) a linker that adjoins the first and second domains. In embodiments, the heterodimeric protein is administered at a frequency of about 1 day, or about 2 days, or about 3 days, or about 4 days, or about 5 days, or about 6 days, or about one week, or about 10 days, or about 15 days, or about 21 days, or more. In embodiments, the heterodimeric protein is administered at a frequency of about 3 days. In embodiments, the heterodimeric protein is administered at a frequency of about 7 days. In embodiments, the heterodimeric protein is administered at a frequency of about 1 day to about 10 days, about 1 day to about 5 days, or about 2 days to about 10 days, or about 5 days to about 15 days, or about one week to about 3 weeks. In embodiments, the heterodimeric protein is administered at a frequency of about 1 day to about 10 days. In embodiments, the heterodimeric protein is administered so as to form a ratio of effector cells (heterodimeric protein-bound Gamma-Delta T cells) to target cells (without limitations, e.g., cancer cells) is about 1:1, or about 1:2, or about 1:5, or about 1:7, or about 1:10, or about 1:15, or about 1:20 or less. DB1/ 145191832.2 2 Attorney Docket No.: SHK-081PC/116981-5081 In embodiments, the heterodimeric protein is administered so as to form a ratio of effector cells (heterodimeric protein-bound Gamma-Delta T cells) to target cells (without limitations, e.g., cancer cells) is about 1:1 to about 1:5, or about 1:2 to about 1:8, or about 1:10 to about 1: 20. In embodiments, the targeting domain is capable of binding CD20 on the surface of a B cell and/or a cancer cell. In embodiments, the targeting domain comprises an Fv domain. In embodiments, the targeting domain is an antibody-like molecule, or antigen binding fragment thereof. In embodiments, the targeting domain comprises a single-chain variable fragment (scFv), or a fragment thereof. In embodiments, the targeting domain comprises different scFvs. In embodiments, the targeting domain specifically binds human CD20. In embodiments, the nucleic acid is or comprises a DNA or In embodiments, the nucleic acid is or comprises an expression vector. In embodiments, the method modulates or is suitable for modulating a γδ (gamma delta) T cell. In embodiments, the gamma delta T cell expresses Vγ4 or Vγ9δ2. In embodiments, the first domain comprises BTN2A1 and it modulates a Vγ4-expressing T cell. In embodiments, the first domain modulates a Vγ9δ2-expressing T cell. In embodiments, the modulation of a gamma delta T cell is activation of a gamma delta T cell. In embodiments, the method induces killing of a target cell. In embodiments, the e target cell is a human cell. In embodiments, the method: (i) modulates or is suitable for modulating a γδ (gamma delta) T cell, and/or (ii) stimulates or promotes localizing to a tumor, and/or binding to a tumor cell, and/or engaging a tumor cell, and/or promoting an immune stimulatory signal, and/or inhibiting an immune inhibitory signal, and/or forming an immune synapse The method of any one of claims 1-36, wherein the immune synapse is a synapse between a gamma delta T cell and a tumor cell and/or the chimeric protein is capable of contemporaneous activation and targeting of gamma delta T cells to tumor cells. In embodiments, the B cell depletion comprises a reduction in the numbers and/or activity of one or more B cells. In embodiments, the B cells are hCD45+CD3-CD19+CD20+IgD+. In embodiments, the method does not reduce and/or suppress a non-B cell population of cells. In embodiments, the method does not reduce and/or suppress non-B/T cells (e.g., hCD45+CD3-CD19-), Vδ2+ T cells (e.g., hCD45+CD3+Vδ2+), and/or total T cells (e.g., hCD45+CD3+CD19-). In embodiments, the method does not increase and/or stimulate non-B/T cells (e.g., hCD45+CD3-CD19-), Vδ2+ T cells (e.g., hCD45+CD3+Vδ2+), and/or total T cells (e.g., hCD45+CD3+CD19-). In embodiments, the B cell-mediated disease or disorder is a cancer. In embodiments, the cancer is a cancer expressing CD20. In embodiments, the cancer is a B-cell malignancy. In embodiments, the B-cell DB1/ 145191832.2 3 Attorney Docket No.: SHK-081PC/116981-5081 malignancy is a lymphoma or leukemia. In embodiments, the B-cell malignancy is non-Hodgkin’s lymphoma (NHL) or Hodgkin’s lymphoma (HL). In embodiments, the e B-cell malignancy is advanced stage follicular lymphoma, chronic lymphocytic leukemias (CLL), Burkitt’s lymphoma, or diffuse large B- cell lymphoma. In embodiments, the B-cell mediated disease or disorder is an autoimmune disease or disorder. In embodiments, the autoimmune disease or disorder is one or more of rheumatoid arthritis, systemic lupus erythematosus, diabetes mellitus, ankylosing spondylitis, Sjögren's syndrome, inflammatory bowel diseases (e.g., colitis ulcerosa, Crohn's disease), multiple sclerosis, sarcoidosis, psoriasis, Grave's disease, Hashimoto's thyroiditis, psoriasis, hypersensitivity reactions (e.g., allergies, hay fever, asthma, and acute edema cause type I hypersensitivity reactions), pemphigus, and vasculitis (e.g., Granulomatosis with polyangiitis). The details of one or more examples of the disclosure are set forth in the description below. Other features or advantages of the present disclosure will be apparent from the following drawings, detailed description of several examples, and also from the appended claims. The details of the disclosure are set forth in the accompanying description below. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, illustrative methods and materials are now described. Other features, objects, and advantages of the disclosure will be apparent from the description and from the claims. In the specification and the appended claims, the singular forms also include the plural unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. BRIEF DESCRIPTION OF THE DRAWINGS The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee. FIG.1A is non-limiting, schematic illustration of a BTN2A1/3A1-Fc-CD20scFv heterodimeric protein disclosed herein (also referred to herein as a BTN2A1/3A1-Fc-CD20scFv GAmma DELta T cell ENgager (GADLEN)). FIG.1B shows, without wishing to be bound by theory, a schematic illustration of the mechanism of target-cell depletion mediated by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein. Briefly, without wishing to be bound by theory, the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein engages a target cell via the scFv fragment, and a gamma delta T cell via the butyrophilin heterodimer. Another gamma delta T cell molecule (without limitation, e.g., NKG2D or CD28), without wishing to be DB1/ 145191832.2 4 Attorney Docket No.: SHK-081PC/116981-5081 bound by theory, mediates co-stimulation to potentiate signal strength and/or to prevent activation- induced cell death of the gamma delta T cell. FIG.2A to FIG.2C illustrate the an in vitro activity of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein. FIG.2A shows the degranulation of Vγ9Vδ2+ T cells induced by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein in a plate-based assay. FIG.2B is a graph showing an in vitro depletion of CD20+ lymphoma cells using human Daudi Burkitt’s lymphoma cells (CD20+ lymphoma cell line). FIG.2C is a graph showing an in vitro depletion of CD20+ lymphoma cells using CD20+ B cells purified from peripheral blood mononuclear cells (“PBMCs”) of a healthy donor. FIG. 3A to FIG. 3E show the in vivo human B cell depletion by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein in PBMC-humanized mice. FIG. 3A shows a schematic representation of experimental design. FIG.3B shows B cells (hCD45+CD3-CD19+CD20+IgD+). FIG.3C shows non-B/T cells (hCD45+CD3-CD19-), FIG.3D shows Vδ2+ T cells (hCD45+CD3+Vδ2+), and FIG.3E shows the total T cells (hCD45+CD3+CD19-) by flow cytometry. Top and bottom panels in FIG.3B to FIG.3E represent animals engrafted with PBMCs derived from two different donors, containing high and low input ratios of Vδ2+ T cells (Effector, E) and B cells (Target, T), respectively. FIG.4A to FIG.4E show the in vivo anti-tumor activity of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein in a mouse model. FIG.4A shows a schematic representation of experimental design. FIG.4B shows the tumor growth in vehicle-only treated mice. FIG.4C shows the tumor growth in mice treated with the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein. FIG.4D shows the tumor growth in mice treated with the BTN2A1/3A1-Fc-B7H3scFv heterodimeric protein, which was used as a negative control. FIG.4E shows the tumor growth in mice treated with a CD3×CD20 engager, which was used as a positive control. FIG.5A to FIG.5N are graphs showing the effects of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein in cynomolgus macaques. FIG.5A is a line graph showing the level of the B cell occupancy by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein as the function of time in the indicated cynomolgus monkeys. FIG.5B shows the comparison of pre-dose and post dose occupancy of the CD3- CD19+ target cells by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein in monkey 4501 as determined by flow cytometry. FIG.5C is a line graph showing the level of the Gamma Delta T cell occupancy by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein as the function of time in the indicated cynomolgus monkeys. FIG.5D shows the comparison of pre-dose and post dose occupancy of the Vγ9+ target cells by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein in monkey 4501 as determined by flow cytometry. FIG.5E is a line graph showing the kinetics of change in the levels of B cells in total DB1/ 145191832.2 5 Attorney Docket No.: SHK-081PC/116981-5081 lymphocytes in monkeys treated with the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein at the indicated doses. FIG.5F is a line graph showing the kinetics of change in the levels of CD4+ cells in total lymphocytes in monkeys treated with the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein at the indicated doses. FIG.5G is a line graph showing the kinetics of change in the levels of Vγ9+ cells in total lymphocytes in monkeys treated with the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein at the indicated doses. FIG.5H is a line graph showing the kinetics of B cell depletion in monkeys treated with the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein at the indicated doses. FIG.5I are line graphs showing the pharmacokinetic profile of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein from left to right after first dose, second dose and third dose, respectively. FIG.5J is a bar graph showing the distribution of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein in indicated tissues 48 hours after Dose 3. FIG.5K are line graphs showing the kinetics of receptor occupancy (RO) of B cells by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein from left to right after first dose, second dose and third dose, respectively. FIG.5L are line graphs showing the kinetics of receptor occupancy (RO) of gamma delta T cells by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein from left to right after first dose, second dose and third dose, respectively. FIG.5M is box chart showing the changes in the levels of peripheral B cells after Dose 1 and Dose 3, compared to pre-dose levels. FIG.5N is box chart showing the changes in the levels of peripheral gamma delta T cells after Dose 1 and Dose 3, compared to pre- dose levels. DETAILED DESCRIPTION Accordingly, in one aspect, the present disclosure relates to a method for increasing and/or stimulating B cell depletion in a subject suffering from a B cell-mediated disease or disorder, comprising administering to the subject a heterodimeric protein, or a nucleic acid encoding the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises: a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof, the target domain being directed to CD20; and a linker that adjoins the first and second domains; and wherein the beta chain comprises: a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; a second domain comprising the targeting domain, the target domain being directed to CD20; and a linker that adjoins the first and second domains. In embodiments, the fragment comprises an extracellular domain (ECD).or a variable domain. DB1/ 145191832.2 6 Attorney Docket No.: SHK-081PC/116981-5081 In embodiments, the first domains of alpha chain and beta chain comprise ECDs, variable domains, or a combination of an ECD and a variable domain of the same butyrophilin family protein. In embodiments, the first domains of alpha chain and beta chain comprise ECDs, variable domains, or a combination of an ECD and a variable domain of different butyrophilin family proteins. In embodiments, the butyrophilin family protein of the alpha chain and beta chain is independently selected from human BTN2A1 and human BTN3A1. In embodiments, the first domains of alpha chain and beta chain comprise extracellular domains and/or variable domains of BTN2A1 and BTN3A1. In embodiments, the first domains of alpha chain and beta chain comprise variable domains of BTN2A1 and BTN3A1. In embodiments, the first domain of alpha chain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence selected from SEQ ID NOs: 13- 16; and the first domain of beta chain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence selected from SEQ ID NOs: 13-16. In embodiments, the first domains of alpha chain and beta chain comprise a polypeptide having an amino acid sequence of: any one of SEQ ID NOs: 13-16; and any one of SEQ ID NOs: 13-16, respectively. In embodiments, the targeting domain is capable of binding CD20 on the surface of a B cell and/or a cancer cell. In embodiments, the targeting domain comprises an Fv domain. In embodiments, the targeting domain is an antibody-like molecule, or antigen binding fragment thereof. In embodiments, the targeting domain comprises a single-chain variable fragment (scFv), or a fragment thereof. In embodiments, the targeting domain comprises different scFvs. In embodiments, the targeting domain specifically binds human CD20. In embodiments, the targeting domain comprises an scFv that specifically binds human CD20. In embodiments, the targeting domain comprises a polypeptide comprising the complementarity determining regions (CDRs) present in SEQ ID NOs: 17-20. In embodiments, the targeting domain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence selected from SEQ ID NOs: 17-20. In embodiments, the linker comprises a polypeptide selected from a flexible amino acid sequence, an IgG hinge region, and an antibody sequence. In embodiments, the antibody sequence comprises an Fc DB1/ 145191832.2 7 Attorney Docket No.: SHK-081PC/116981-5081 domain. In embodiments, the linker comprises a hinge-CH2-CH3 Fc domain. In embodiments, the hinge- CH2-CH3 Fc domain is derived from IgG1, optionally from human IgG1. In embodiments, the hinge-CH2- CH3 Fc domain is derived from IgG4, optionally from human IgG4. In embodiments, the hinge-CH2-CH3 Fc domain comprises a polypeptide having an amino acid sequence with at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide selected from SEQ ID NOs: 1-12. In embodiments, the nucleic acid is or comprises a DNA or In embodiments, the nucleic acid is or comprises an expression vector. In embodiments, the method modulates or is suitable for modulating a γδ (gamma delta) T cell. In embodiments, the gamma delta T cell expresses Vγ4 or Vγ9δ2. In embodiments, the first domain comprises BTN2A1 and it modulates a Vγ4-expressing T cell. In embodiments, the first domain modulates a Vγ9δ2-expressing T cell. In embodiments, the modulation of a gamma delta T cell is activation of a gamma delta T cell. In embodiments, the method induces killing of a target cell. In embodiments, the e target cell is a human cell. In embodiments, the method: (i) modulates or is suitable for modulating a γδ (gamma delta) T cell, and/or (ii) stimulates or promotes localizing to a tumor, and/or binding to a tumor cell, and/or engaging a tumor cell, and/or promoting an immune stimulatory signal, and/or inhibiting an immune inhibitory signal, and/or forming an immune synapse. In embodiments, the immune synapse is a synapse between a gamma delta T cell and a tumor cell and/or the chimeric protein is capable of contemporaneous activation and targeting of gamma delta T cells to tumor cells. In embodiments, the B cell depletion comprises a reduction in the numbers and/or activity of one or more B cells. In embodiments, the B cells are hCD45+CD3-CD19+CD20+IgD+. In embodiments, the method does not reduce and/or suppress a non-B cell population of cells. In embodiments, the method does not reduce and/or suppress non-B/T cells (e.g., hCD45+CD3-CD19-), Vδ2+ T cells (e.g., hCD45+CD3+Vδ2+), and/or total T cells (e.g., hCD45+CD3+CD19-). In embodiments, the method does not increase and/or stimulate non-B/T cells (e.g., hCD45+CD3-CD19-), Vδ2+ T cells (e.g., hCD45+CD3+Vδ2+), and/or total T cells (e.g., hCD45+CD3+CD19-). In embodiments, the B cell-mediated disease or disorder is a cancer. In embodiments, the cancer is a cancer expressing CD20. In embodiments, the cancer is a B-cell malignancy. In embodiments, the B-cell malignancy is a lymphoma or leukemia. In embodiments, the B-cell malignancy is non-Hodgkin’s lymphoma (NHL) or Hodgkin’s lymphoma (HL). In embodiments, the e B-cell malignancy is advanced stage follicular lymphoma, chronic lymphocytic leukemias (CLL), Burkitt’s lymphoma, or diffuse large B- DB1/ 145191832.2 8 Attorney Docket No.: SHK-081PC/116981-5081 cell lymphoma. In embodiments, the cancer is a solid cancer selected from prostate cancer, small cell lung cancer, non-small cell lung cancer, neuroendocrine tumor, renal cancer, bladder cancer, colon cancer, and breast cancer. In embodiments, the cancer has mutations which limit recognition by alpha beta T cells, optionally selected from mutations in MHC I, beta 2 microglobulin, and Transporter associated with antigen processing (TAP). In embodiments, the B cell-mediated disease or disorder is an autoimmune disease or disorder. In embodiments, the autoimmune disease or disorder is one or more of rheumatoid arthritis, systemic lupus erythematosus, diabetes mellitus, ankylosing spondylitis, Sjögren's syndrome, inflammatory bowel diseases (e.g., colitis ulcerosa, Crohn's disease), multiple sclerosis, sarcoidosis, psoriasis, Grave's disease, Hashimoto's thyroiditis, psoriasis, hypersensitivity reactions (e.g., allergies, hay fever, asthma, and acute edema cause type I hypersensitivity reactions), pemphigus, and vasculitis (e.g., Granulomatosis with polyangiitis). The present disclosure, in various embodiments, provides heterodimeric proteins, and methods of use of the same, for specific targeting of gamma delta T cells to cancer cells. These heterodimeric proteins (e.g., a BTN2A1/3A1-Fc-CD20scFv heterodimeric protein) are also herein referred to as BTN2A1/3A1-Fc- CD20scFv GAmma DELta T cell ENgager (GADLEN) proteins (used interchangeably with “GADLEN proteins”). In embodiments, the heterodimeric proteins disclosed herein direct gamma delta T cells and other specific effectors of the immune system to target tumor cells, enhancing their cytotoxicity. In embodiments, the heterodimeric proteins provide a higher binding specificity to cancer cells. In embodiments, the heterodimeric proteins enable combining multiple anticancer mechanisms on the same cancer cell. In embodiments, the heterodimeric proteins increase cancer cell specificity and apoptosis, improved antiproliferative effect and decreased the development of resistance. The Heterodimeric Proteins Disclosed Herein In one aspect, the present disclosure relates to a heterodimeric protein, and methods of use of the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof; (b) a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof; and (c) a linker that adjoins the first and second domains; and wherein the beta chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof; (b) a second domain comprising the targeting domain; and (c) a linker that adjoins the first and second domains. In embodiments, the fragment comprises a variable domain. In embodiments, the alpha chain linker and DB1/ 145191832.2 9 Attorney Docket No.: SHK-081PC/116981-5081 the beta chain linker are charged polarized linkers, wherein one of the alpha chain linker and the beta chain linker is positively charged and the other is negatively charged. In one aspect, the present disclosure relates to a heterodimeric protein, and methods of use of the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises: (a) a variable domain of BTN2A1; (b) a second domain comprising a single-chain antibody (scFv); and (c) a linker that adjoins the first and second domains; and wherein the beta chain comprises: (a) a first domain comprising an extracellular domain (ECD) or a variable domain of BTN3A1; (b) a second domain comprising an anti- CD20 single-chain antibody (scFv); and (c) a linker that adjoins the first and second domains. In embodiments, the alpha chain linker and the beta chain linker are charged polarized linkers, wherein one of the alpha chain linker and the beta chain linker is positively charged and the other is negatively charged. In these embodiments, the alpha chain and the beta chain self-associate to form the heterodimer of alpha and beta chains, which comprise a BTN2A1/3A1-Fc-CD20scFv. In embodiments, the heterodimeric protein modulates or is suitable for modulating a γδ (gamma delta) T cell. In embodiments, the heterodimeric protein: (i) modulates or is suitable for modulating a γδ (gamma delta) T cell, and (ii) suitable for localizing to a tumor, and/or binding to a tumor cell, and/or engaging a tumor cell, and/or promoting an immune stimulatory signal, and/or inhibiting an immune inhibitory signal, and/or forming an immune synapse. In embodiments, the modulation of a gamma delta T cell is activation of a gamma delta T cell. In embodiments, the immune synapse is a synapse between a gamma delta T cell and a tumor cell and/or the chimeric protein is suitable for contemporaneous activation and targeting of gamma delta T cells to tumor cells. In embodiments, the gamma delta T cell expresses Vγ4 or Vγ9δ2. In embodiments, the first domain comprises BTNL3 and BTNL8 and it modulates a Vγ4-expressing T cell. In embodiments, the first domain modulates a Vγ9δ2-expressing T cell. In embodiments, the first domain comprises BTN2A1 and BTN3A1. In one aspect, the present disclosure relates to a heterodimeric protein, and methods of use of the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof; (b) a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof; and (c) a linker that adjoins the first and second domains; and wherein the beta chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof; (b) a second domain comprising the targeting domain; and (c) a linker that adjoins the first and second domains. DB1/ 145191832.2 10 Attorney Docket No.: SHK-081PC/116981-5081 The sequences of exemplary embodiments of heterodimeric protein fusion proteins are provided below. Any alpha chain may be combined with any beta chain having a different specificity to generate a bispecific heterodimeric protein fusion protein. BTN2A1-Alpha-CD20 scFv (Leader sequence is indicated by a double underlined font, extracellular domain of human BTN2A1 is shown in bold-underlined font, a core domain of the linker is shown in a single underlined font, charged peptides are shown in an italic font and anti-CD20 ScFv sequence is shown in a boldface font): MEFGLSWVFLVAIIKGVQCQFIVVGPTDPILATVGENTTLRCHLSPEKNAEDMEVRWFRSQFSPAVFV YKGGRERTEEQMEEYRGRTTFVSKDISRGSVALVIHNITAQENGTYRCYFQEGRSYDEAILHLVVAGL GSKPLISMRGHEDGGIRLECISRGWYPKPLTVWRDPYGGVAPALKEVSMPDADGLFMVTTAVIIRDK SVRNMSCSINNTLLGQKKESVIFIPESFMPSVSPCAGSGSRKGGKRGSKYGPPCPPCPAPEFLGGPS VFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLT VLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSL SLGKDEGGEDGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGAIY PGNGDTSYNQKFQGRVTITADKSISTAYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGAGTLVTV SSGGGGSGGGGSGGGGSGGGGSQIVLTQSPSSLSASVGDRVTITCRASSSVSYIHWFQQKPGKSPK PLIYATSNLASGVPVRFSGSGSGTDYTLTISSLQPEDFATYYCQQWTSNPPTFGGGTKVEIK (SEQ ID NO: 21) An exemplary the alpha chain, referred to herein as “BTN2A1-Alpha- CD20 scFv” comprises: (a) a first domain comprising an extracellular domain of BTN2A1; (b) a second domain comprising a targeting domain comprising an scFv specific to CD20 (CD20 scFv;) and (c) a linker that adjoins the first and second domains. In embodiments, the BTN2A1-Alpha-CD20 scFv comprises a polypeptide comprising the complementarity determining regions (CDRs) present in SEQ ID NO: 21. In embodiments, the BTN2A1-Alpha-CD20 scFv comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence of SEQ ID NO: 21. In embodiments, the BTN2A1-Alpha-CD20 scFv comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence of SEQ ID NO: 21, wherein the polypeptide comprises the complementarity determining regions (CDRs) present in SEQ ID NO: 21. DB1/ 145191832.2 11 Attorney Docket No.: SHK-081PC/116981-5081 BTN3A1-beta-CD20scFv (Leader sequence is indicated by a double underlined font, extracellular domain of human BTN3A1 is shown in bold-underlined font, a core domain of the linker is shown in a single underlined font, charged peptides are shown in an italic font and anti-CD20 scFv sequence is shown in a boldface font): MEFGLSWVFLVAIIKGVQCQFSVLGPSGPILAMVGEDADLPCHLFPTMSAETMELKWVSSSLRQVVN VYADGKEVEDRQSAPYRGRTSILRDGITAGKAALRIHNVTASDSGKYLCYFQDGDFYEKALVELKVA ALGSDLHVDVKGYKDGGIHLECRSTGWYPQPQIQWSNNKGENIPTVEAPVVADGVGLYAVAASVIM RGSSGEGVSCTIRSSLLGLEKTASISIADPFFRSAQRWIAALAGGSGSDEGGEDGSKYGPPCPPCPA PEFLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS TYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVLHEALH NHYTQKSLSLSLGKRKGGKRGSGSQVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAP GQGLEWMGAIYPGNGDTSYNQKFQGRVTITADKSISTAYMELSSLRSEDTAVYYCARSTYYGGDWY FNVWGAGTLVTVSSGGGGSGGGGSGGGGSGGGGSQIVLTQSPSSLSASVGDRVTITCRASSSVSYI HWFQQKPGKSPKPLIYATSNLASGVPVRFSGSGSGTDYTLTISSLQPEDFATYYCQQWTSNPPTFGG GTKVEIK (SEQ ID NO: 22 An exemplary the beta chain, referred to herein as “BTN3A1-beta-CD20scFv” comprises: (a) a first domain comprising an extracellular domain of BTN3A1; (b) a second domain comprising a targeting domain comprising an scFv specific to CD20 (CD20scFv;) and (c) a linker that adjoins the first and second domains. In embodiments, the BTN3A1-beta-CD20scFv comprises a polypeptide comprising the complementarity determining regions (CDRs) present in SEQ ID NO: 22. In embodiments, the BTN3A1- beta-CD20scFv comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence of SEQ ID NO: 22. In embodiments, the BTN3A1-beta- CD20scFv comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence of SEQ ID NO: 22, wherein the polypeptide comprises the complementarity determining regions (CDRs) present in SEQ ID NO: 22 The First Domain In one aspect, the present disclosure relates to a heterodimeric protein, and methods of use of the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises a first domain comprising DB1/ 145191832.2 12 Attorney Docket No.: SHK-081PC/116981-5081 a butyrophilin family protein, or a fragment thereof; and wherein the beta chain comprises a first domain comprising a butyrophilin family protein, or a fragment thereof. In embodiments, the butyrophilin family protein of the alpha chain and beta chain is independently selected from BTN2A1 and BTN3A1. In embodiments, the butyrophilin family protein of the alpha chain and beta chain is independently selected from human BTN2A1, and human BTN3A1. In embodiments, the first domain of alpha chain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence selected from SEQ ID NOs: 13- 16; and the first domain of beta chain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence selected from SEQ ID NOs: 13-16. In embodiments, the first domains of alpha chain and beta chain comprise a polypeptide having an amino acid sequence of: any one of SEQ ID NOs: 13-16; and any one of SEQ ID NOs: 13-16, respectively. In embodiments, the first domains of alpha chain and beta chain comprise extracellular domains and/or variable domains of BTN2A1 and BTN3A1. In embodiments, the first domains of alpha chain and beta chain comprise extracellular domains of BTN2A1 and BTN3A1, respectively. In embodiments, the first domains of alpha chain and beta chain comprise variable domains of BTN2A1 and BTN3A1; respectively. In embodiments, the first domain comprises two of the same butyrophilin family proteins. In embodiments, wherein the first domain comprises two different butyrophilin family proteins. In embodiments, the butyrophilin family proteins comprise a variable domain. Suitable butyrophilin family proteins or fragments thereof are derived from the native butyrophilin family proteins that comprise a B30.2 domain in the cytosolic tail of the full length protein. In embodiments, the butyrophilin family protein is butyrophilin subfamily 2 member A1 (BTN2A1). In embodiments, the first domain comprises substantially all the extracellular domain of BTN2A1. In embodiments, the first domain is capable of binding a gamma delta T cell receptor (e.g., Vγ9δ2). BTN2A1 is also known as BT2.1, BTF1. In embodiments, the portion of BTN2A1 is a portion of the extracellular domain of BTN2A1. In embodiments, the present chimeric protein further comprises a domain, e.g., the extracellular domain BTN2A1. The amino acid sequence of extracellular domain of human BTN2A1, which is an illustrative amino acid sequence of human BTN2A1 suitable in the current disclosure is the following: DB1/ 145191832.2 13 Attorney Docket No.: SHK-081PC/116981-5081 QFIVVGPTDPILATVGENTTLRCHLSPEKNAEDMEVRWFRSQFSPAVFVYKGGRERTEEQMEEYRGR TTFVSKDISRGSVALVIHNITAQENGTYRCYFQEGRSYDEAILHLVVAGLGSKPLISMRGHEDGGIRLECI SRGWYPKPLTVWRDPYGGVAPALKEVSMPDADGLFMVTTAVIIRDKSVRNMSCSINNTLLGQKKESVI FIPESFMPSVSPCA (SEQ ID NO: 13 In embodiments, the fragment of human BTN2A1 comprises the variable domain of BTN2A1, which has the following amino acid sequence: QFIVVGPTDPILATVGENTTLRCHLSPEKNAEDMEVRWFRSQFSPAVFVYKGGRERTEEQMEEYRGR TTFVSKDISRGSVALVIHNITAQENGTYRCYFQEGRSYDEAILHLV (SEQ ID NO: 14) In embodiments, the present heterodimeric protein comprises the extracellular domain of human BTN2A1 which has the amino acid sequence of SEQ ID NO: 13 or SEQ ID NO: 14. In embodiments, the present chimeric proteins may comprise the extracellular domain of BTN2A1 as described herein, or a variant or functional fragment thereof. For instance, the chimeric protein may comprise a sequence of the extracellular domain of BTN2A1 as provided above, or a variant or functional fragment thereof having at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) sequence identity with the amino acid sequence of the extracellular domain of BTN2A1 as described herein. BTN2A1 derivatives can be constructed from available structural data, including a homology model described by Karunakaran et al., Butyrophilin-2A1 Directly Binds Germline-Encoded Regions of the Vγ9Vδ2 TCR and Is Essential for Phosphoantigen Sensing, Immunity.52(3): 487-498 (2020). Moreover, without wishing to be bound by theory, the protein structure homology-model of BTN2A1 is available at SWISS-MODEL repository. Bienert et al., “The SWISS-MODEL Repository – new features and functionality.” Nucleic Acids Research, 45(D1): D313–D319 (2017). Additional structural insight obtained from mutagenesis. Rigau et al., Butyrophilin 2A1 is essential for phosphoantigen reactivity by γδ T cells. Science 367(6478):eaay5516 ( 2020). DB1/ 145191832.2 14 Attorney Docket No.: SHK-081PC/116981-5081 In embodiments, the butyrophilin family protein is butyrophilin subfamily 3 member A1 (BTN3A1). In embodiments, the first domain comprises substantially all the extracellular domain of BTN3A1. In embodiments, the first domain is capable of binding a gamma delta T cell receptor (e.g., Vγ9δ2). BTN3A1 is also known as BTF5. In embodiments, the portion of BTN3A1 is a portion of the extracellular domain of BTN3A1. In embodiments, the present chimeric protein further comprises a domain, e.g., the extracellular domain BTN3A1. The amino acid sequence of extracellular domain of human BTN3A1, which is an illustrative amino acid sequence of human BTN3A1 suitable in the current disclosure is the following: QFSVLGPSGPILAMVGEDADLPCHLFPTMSAETMELKWVSSSLRQVVNVYADGKEVEDRQSAPYRGR TSILRDGITAGKAALRIHNVTASDSGKYLCYFQDGDFYEKALVELKVAALGSDLHVDVKGYKDGGIHLEC RSTGWYPQPQIQWSNNKGENIPTVEAPVVADGVGLYAVAASVIMRGSSGEGVSCTIRSSLLGLEKTASI SIADPFFRSAQRWIAALAG (SEQ ID NO: 15) In embodiments, the fragment of human BTN3A1 comprises the variable domain, which has the following amino acid sequence: AQFSVLGPSGPILAMVGEDADLPCHLFPTMSAETMELKWVSSSLRQVVNVYADGKEVEDRQSAPYRG RTSILRDGITAGKAALRIHNVTASDSGKYLCYFQDGDFYEKALVELKVA (SEQ ID NO: 16) In embodiments, the present heterodimeric protein comprises the extracellular domain of human BTN3A1 which has the amino acid sequence of SEQ ID NO: 15 or SEQ ID NO: 16. In embodiments, the present chimeric proteins may comprise the extracellular domain of BTN3A1 as described herein, or a variant or functional fragment thereof. For instance, the chimeric protein may comprise a sequence of the extracellular domain of BTN3A1 as provided above, or a variant or functional fragment thereof having at least about 60%, or at least about 61%, or at least about 62%, or at least about 63%, or at least about 64%, or at least about 65%, or at least about 66%, or at least about 67%, or at least about 68%, or at least about 69%, or at least about 70%, or at least about 71%, or at least about 72%, or at least about 73%, or at least about 74%, or at least about 75%, or at least about 76%, or at least about 77%, or at least about 78%, or at least about 79%, or at least about 80%, or at least about 81%, or at least about 82%, or at least about 83%, or at least about 84%, or at least about 85%, or at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99%) sequence identity with the amino acid sequence of the extracellular domain of BTN3A1 as described herein. DB1/ 145191832.2 15 Attorney Docket No.: SHK-081PC/116981-5081 BTN3A1 derivatives can be constructed from available structural data, including the following: Palakodeti et al., The molecular basis for modulation of human V(gamma)9V(delta)2 T cell responses by CD277/Butyrophilin-3 (BTN3A)-specific antibodies, J Biol Chem 287: 32780-32790 (2012); Vavassori et al., Butyrophilin 3A1 binds phosphorylated antigens and stimulates human gamma delta T cells. Nat Immunol 14: 908-916 (2013); Sandstrom et al., The Intracellular B30.2 Domain of Butyrophilin 3A1 Binds Phosphoantigens to Mediate Activation of Human V gamma 9V delta 2 T Cells. Immunity 40: 490-500 (2014); Rhodes et al., Activation of Human Gammadelta T Cells by Cytosolic Interactions of Btn3A1 with Soluble Phosphoantigens and the Cytoskeletal Adaptor Periplakin. J Immunol 194: 2390 (2015); Gu et al., Phosphoantigen-induced conformational change of butyrophilin 3A1 (BTN3A1) and its implication on V gamma 9V delta 2 T cell activation. Proc Natl Acad Sci U S A 114: E7311-E7320 (2017); Salim et al., BTN3A1 Discriminates gamma delta T Cell Phosphoantigens from Nonantigenic Small Molecules via a Conformational Sensor in Its B30.2 Domain. ACS Chem Biol 12: 2631-2643 (2017); Yang et al., A Structural Change in Butyrophilin upon Phosphoantigen Binding Underlies Phosphoantigen-Mediated V gamma 9V delta 2 T Cell Activation. Immunity 50: 1043 (2019). In embodiments, the first domain comprises a portion of BTN2A1. In embodiments, the portion of BTN2A1 is an extracellular domain of BTN2A1, or a γδ T-cell receptor (e.g., γ9δ2)-binding fragment thereof. In embodiments, the first domain comprises a portion of BTN3A1. In embodiments, the portion of BTN3A1 is an extracellular domain of BTN3A1, or a γδ T-cell receptor (e.g., γ9δ2)-binding fragment thereof. In embodiments, the first domain comprises a portion of BTN2A1. In embodiments, the portion of BTN2A1 is an extracellular domain of BTN2A1, or a γδ T-cell receptor (e.g., γ9δ2)-binding fragment thereof. In embodiments, the first domain comprises a portion of BTN3A1. In embodiments, the portion of BTN3A1 is an extracellular domain of BTN3A1, or a γδ T-cell receptor (e.g., γ9δ2)-binding fragment thereof. The Targeting Domain The heterodimeric proteins of any of the embodiments disclosed herein comprise an alpha chain and a beta chain, each comprising a targeting domain. In one aspect, the present disclosure relates to a heterodimeric protein, and methods of using the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof; and; and wherein the beta chain comprises a second domain comprising the targeting domain. In embodiments, the targeting domain is capable of binding an antigen on the surface of a cancer cell. In embodiments, the targeting domain is capable of binding a tumor antigen. DB1/ 145191832.2 16 Attorney Docket No.: SHK-081PC/116981-5081 Additionally or alternatively, in embodiments, the targeting domain is an antibody, or an antigen binding fragment thereof. In embodiments, the targeting domain comprises an Fv domain. In embodiments, the targeting domain is an antibody-like molecule, or antigen binding fragment thereof. In embodiments, the targeting domain comprises a single-chain variable fragment (scFv). In embodiments, the targeting domain comprises scFvs or fragments thereof (e.g., half scFvs or Fv fragments). Additionally or alternatively, in embodiments, the targeting domain specifically binds CD20. In embodiments, the targeting domain comprises an scFv that specifically binds CD20. In embodiments, the targeting domain comprises a polypeptide comprising the complementarity determining regions (CDRs) present in SEQ ID NOs: 17-20. In embodiments, the targeting domain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence selected from SEQ ID NOs: 17-20. In embodiments, the targeting domain is an antibody-like molecule, or antigen binding fragment thereof. In embodiments, the antibody-like molecule is selected from a single-domain antibody, a recombinant heavy-chain-only antibody (VHH), a single-chain antibody (scFv), a shark heavy-chain-only antibody (VNAR), a microprotein (cysteine knot protein, knottin), a DARPin; a Tetranectin; an Affibody; a Transbody; an Anticalin; an AdNectin; an Affilin; an Affimer, a Microbody; an aptamer; an alterase; a plastic antibody; a phylomer; a stradobody; a maxibody; an evibody; a fynomer, an armadillo repeat protein, a Kunitz domain, an avimer, an atrimer, a probody, an immunobody, a triomab, a troybody; a pepbody; a vaccibody, a UniBody; a DuoBody, a Fv, a Fab, a Fab′, and a F(ab′)2. In embodiments, the antibody-like molecule is an scFv. In embodiments, the targeting domain is capable of binding an antigen on the surface of a cancer cell. In embodiments, the targeting domain specifically binds CD20. In embodiments, the targeting domain specifically binds to CD20. An illustrative targeting domain is CD20scFv-1, which an scFV specific to human CD20, and has the following sequence (the variable regions of the heavy chain (VH) is shown in a boldface font, the variable regions of the light chain (VL) is indicated in an italics font, and the linker joining VH and VL is shown by an underline): EVQLVESGGGLVQPGRSLRLSCVASGFTFNDYAMHWVRQAPGKGLEWVSVISWNSDSIGYADSVK GRFTISRDNAKNSLYLQMHSLRAEDTALYYCAKDNHYGSGSYYYYQYGMDVWGQGTTVTVSSGGG GSGGGGSGGGGSGGGGSAEIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQKPGQAPRLLI DB1/ 145191832.2 17 Attorney Docket No.: SHK-081PC/116981-5081 YGASTRATGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQHYINWPLTFGGGTKVEIK (SEQ ID NO: 17) In embodiments, the targeting domain comprises a polypeptide comprising the complementarity determining regions (CDRs) present in SEQ ID NO: 17. In embodiments, the targeting domain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence of SEQ ID NO: 17. In embodiments, the targeting domain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence of SEQ ID NO: 17, wherein the polypeptide comprises the complementarity determining regions (CDRs) present in SEQ ID NO: 17. An illustrative targeting domain is CD20scFv-2, which an scFV specific to human CD20, and has the following sequence (the variable regions of the heavy clan (VH) is shown in a boldface font, the variable regions of the light chain (VL) is indicated in an italics font, and the linker joining VH and VL is shown by an underline): EVQLVQSGAEVKKPGESLKISCKGSGRTFTSYNMHWVRQMPGKGLEWMGAIYPLTGDTSYNQKSK LQVTISADKSISTAYLQWSSLKASDTAMYYCARSTYVGGDWQFDVWGKGTTVTVSSGGGGSGGGG SGGGGSGGGGSEIVLTQSPGTLSLSPGERATLSCRASSSVPYIHWYQQKPGQAPRLLIYATSALASGIP DRFSGSGSGTDFTLTISRLEPEDFAVYYCQQWLSHPPTFGQGTKLEIK (SEQ ID NO: 18) In embodiments, the targeting domain comprises a polypeptide comprising the complementarity determining regions (CDRs) present in SEQ ID NO: 18. In embodiments, the targeting domain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence of SEQ ID NO: 18. In embodiments, the targeting domain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence of SEQ ID NO: 18, wherein the polypeptide comprises the complementarity determining regions (CDRs) present in SEQ ID NO: 18. An illustrative targeting domain is CD20scFv-3, which an scFV specific to human CD20, and has the following sequence (the linker joining the variable regions of the heavy chain (VH) and the variable regions of the light chain (VL) is shown by an underline): DB1/ 145191832.2 18 Attorney Docket No.: SHK-081PC/116981-5081 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGAIYPGNGDTSYNQKFQ GRVTITADKSISTAYMELSSLRSEDTAVYYCARSTYYGGDWYFNVWGAGTLVTVSSGGGGSGGGGSG GGGSGGGGSQIVLTQSPSSLSASVGDRVTITCRASSSVSYIHWFQQKPGKSPKPLIYATSNLASGVPV RFSGSGSGTDYTLTISSLQPEDFATYYCQQWTSNPPTFGGGTKVEIK (SEQ ID NO: 19) In embodiments, the targeting domain comprises a polypeptide comprising the complementarity determining regions (CDRs) present in SEQ ID NO: 19. In embodiments, the targeting domain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence of SEQ ID NO: 19. In embodiments, the targeting domain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence of SEQ ID NO: 19, wherein the polypeptide comprises the complementarity determining regions (CDRs) present in SEQ ID NO: 19. An illustrative targeting domain is CD20scFv-4, which an scFV specific to human CD20, and has the following sequence (the linker joining the variable regions of the heavy clan (VH) and the variable regions of the light chain (VL) is shown by an underline): EVQLVESGGGLVQPDRSLRLSCAASGFTFHDYAMHWVRQAPGKGLEWVSTISWNSGTIGYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTALYYCAKDIQYGNYYYGMDVWGQGTTVTVSSGGGGSGGGGS GGGGSGGGGSEIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGI PARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPITFGQGTRLEIK (SEQ ID NO: 20) In embodiments, the targeting domain comprises a polypeptide comprising the complementarity determining regions (CDRs) present in SEQ ID NO: 20. In embodiments, the targeting domain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence of SEQ ID NO: 20. In embodiments, the targeting domain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence of SEQ ID NO: 20, wherein the polypeptide comprises the complementarity determining regions (CDRs) present in SEQ ID NO: 20. The Linker Domain that Adjoins the First and the Second Domain In embodiments, the linker comprises a polypeptide selected from a flexible amino acid sequence, an IgG hinge region, and an antibody sequence. In embodiments, the antibody sequence comprises an Fc DB1/ 145191832.2 19 Attorney Docket No.: SHK-081PC/116981-5081 domain In embodiments, the linker comprises a hinge-CH2-CH3 Fc domain. In embodiments, the hinge- CH2-CH3 Fc domain is derived from IgG1, optionally from human IgG1. In embodiments, the hinge-CH2- CH3 Fc domain is derived from IgG4, optionally from human IgG4. In embodiments, the hinge-CH2-CH3 Fc domain lacks Fc-effector function. In embodiments, the hinge-CH2-CH3 Fc domain that lacks Fc- effector function is a mutant derivative of IgG4, optionally human IgG4. In embodiments, the hinge-CH2- CH3 Fc domain that lacks Fc-effector function is a mutant derivative of IgG1, optionally human IgG1. In embodiments, the hinge-CH2-CH3 Fc domain has reduced binding to an Fc Gamma Receptor (FcγR) selected from FcγRI, FcγRIIA, FcγRIIAB, FcγRIIIA and FcγRIIIB compared to a wild type hinge-CH2-CH3 Fc domain. Mutant Fc domains lacking Fc effector functions are described in US Publication Nos. 20140051834, 20120251531, and Saunders, Conceptual Approaches to Modulating Antibody Effector Functions and Circulation Half-Life, Front. Immunol., 10:1296 (2019), the entire contents of which are hereby incorporated by reference. In embodiments, the hinge-CH2-CH3 Fc domain comprises a polypeptide having an amino acid sequence with at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide selected from SEQ ID NOs: 1-12. In embodiments, the linker that adjoins the first and second domains comprises a charge polarized core domain. In various embodiments, each of the first and second charge polarized core domains comprises proteins having positively or negatively charged amino acid residues at the amino and carboxy terminus of the core domain. In an illustrative embodiment, the first charge polarized core domain may comprise a protein having positively charged amino acids at the amino terminus which are adjoined by a linker (e.g., a stabilizing domain) to a protein having negatively charged amino acid residues at the carboxy terminus. The second charge polarized core domain may comprise a protein having negatively charged amino acids at the amino terminus which are adjoined by a linker (e.g., a stabilizing domain) to a protein having positively charged amino acid residues at the carboxy terminus. In another illustrative embodiment, the first charge polarized core domain may comprise a protein having negatively charged amino acids at the amino terminus which are adjoined by a linker (e.g., a stabilizing domain) to a protein having positively charged amino acid residues at the carboxy terminus. The second charge polarized core domain may comprise proteins having positively charged amino acids at the amino terminus which are adjoined by a linker (e.g., a stabilizing domain) to a protein having negatively charged amino acid residues at the carboxy terminus. In various embodiments, formation of heterodimeric proteins is driven by electrostatic interactions between the positively charged and negatively charged amino acid residues located at the amino and DB1/ 145191832.2 20 Attorney Docket No.: SHK-081PC/116981-5081 carboxy termini of the first and second charge polarized core domains. Further, formation of homodimeric proteins is prevented by the repulsion between the positively charged amino acid residues or negatively charged amino acid residues located at the amino and carboxy termini of the first and second charge polarized core domains. In various embodiments, the protein comprising positively and/or negatively charged amino acid residues at the amino or carboxy terminus of the charge polarized core domains is about 2 to about 50 amino acids long. For example, the protein comprising positively and/or negatively charged amino acid residues at either terminus of the charge polarized core domain may be about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids long. In various embodiments, the protein comprising positively charged amino acid residues may include one or more of amino acids selected from His, Lys, and Arg. In various embodiments, the protein comprising negatively charged amino acid residues may include one or more amino acids selected from Asp and Glu. In various embodiments, each of the first and/or second charge polarized core domains may comprise a protein comprising an amino acid sequence as provided in the TABLE 1 or an amino acid sequence having at least about 90%, or at least about 93%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity thereto. TABLE 1 Illustrative sequences of the first and/or second charge polarized core domains may comprise a protein: SEQ ID Sequence NO e n
Figure imgf000023_0001
DB1/ 145191832.2 21 Attorney Docket No.: SHK-081PC/116981-5081 YnXnCYnXnYn (where X is a positively charged amino acid such as arginine, 25 histidine or lysine and Y is a spacer amino acid such as serine or glycine, and wh r h n i ind nd ntl n int r 0 t 4) r ns may
Figure imgf000024_0001
comprise a peptide comprising the sequence YYnXXnYYnXXnYYn (where X is a positively charged amino acid such as arginine, histidine or lysine and Y is a spacer amino acid such as serine or glycine; SEQ ID NO: 23). Illustrative peptide sequences include, but are not limited to, RKGGKR (SEQ ID NO: 31) or GSGSRKGGKRGS (SEQ ID NO: 32). In another illustrative embodiment, each of the first and second charge polarized core domains may comprise a peptide comprising the sequence YYnZZnYYnZZnYYn (where Z is a negatively charged amino acid such as aspartic acid or glutamic acid and Y is a spacer amino acid such as serine or glycine). Illustrative peptide sequences include, but are not limited to, DEGGED (SEQ ID NO: 33) or GSGSDEGGEDGS (SEQ ID NO: 34). In embodiments, the linker comprises (a) a first charge polarized core domain adjoined to a butyrophilin family protein, optionally at the carboxy terminus, and (b) a second charge polarized core domain adjoined to a butyrophilin family protein, optionally at the carboxy terminus. In embodiments, the linker forms a heterodimer through electrostatic interactions between positively charged amino acid residues and negatively charged amino acid residues on the first and second charge polarized core domains. In embodiments, the first and/or second charge polarized core domain comprises a polypeptide linker, optionally selected from a flexible amino acid sequence, IgG hinge region, or antibody sequence. In embodiments, the linker is a synthetic linker, optionally PEG. In embodiments, the linker comprises the hinge-CH2-CH3 Fc domain derived from IgG1, optionally human IgG1. In embodiments, the linker comprises the hinge-CH2-CH3 Fc domain derived from IgG4, optionally human IgG4. In embodiments, the first and/or second charge polarized core domain further comprise peptides having positively and/or negatively charged amino acid residues at the amino and/or carboxy terminus of the charge polarized core domain. In embodiments, the positively charged amino acid residues include one or more of amino acids selected from His, Lys, and Arg. In embodiments, the positively charged amino acid residues are present in a peptide comprising positively charged amino acid residues in the first and/or the second charge polarized core domains. In embodiments, the peptide comprising positively charged amino acid DB1/ 145191832.2 22 Attorney Docket No.: SHK-081PC/116981-5081 residues comprises a sequence selected from YnXnYnXnYn (where X is a positively charged amino acid such as arginine, histidine or lysine and Y is a spacer amino acid such as serine or glycine, and where each n is independently an integer 0 to 4) (SEQ ID NO: 21), YYnXXnYYnXXnYYn (where X is a positively charged amino acid such as arginine, histidine or lysine and Y is a spacer amino acid such as serine or glycine, and where each n is independently an integer 0 to 4) (SEQ ID NO: 23), and YnXnCYnXnYn (where X is a positively charged amino acid such as arginine, histidine or lysine and Y is a spacer amino acid such as serine or glycine, and where each n is independently an integer 0 to 4) (SEQ ID NO: 25). In embodiments, the peptide comprising positively charged amino acid residues comprises the sequence RKGGKR (SEQ ID NO: 31) or GSGSRKGGKRGS (SEQ ID NO: 32). In embodiments, the negatively charged amino acid residues may include one or more amino acids selected from Asp and Glu. In embodiments, the negatively charged amino acid residues are present in a peptide comprising negatively charged amino acid residues in the first and/or the second charge polarized core domains. In embodiments, the peptide comprising negatively charged amino acid residues comprises a sequence selected from YnZnYnZnYn (where Z is a negatively charged amino acid such as aspartic acid or glutamic acid and Y is a spacer amino acid such as serine or glycine, and where each n is independently an integer 0 to 4) (SEQ ID NO: 22), YYnZZnYYnZZnYYn (where Z is a negatively charged amino acid such as aspartic acid or glutamic acid and Y is a spacer amino acid such as serine or glycine, and where each n is independently an integer 0 to 4) (SEQ ID NO: 24), and YnZnCYnZnYn (where Z is a negatively charged amino acid such as aspartic acid or glutamic acid and Y is a spacer amino acid such as serine or glycine, and where each n is independently an integer 0 to 4) (SEQ ID NO: 26). In embodiments, the linker comprises a knob-in-hole (KIH) mutations. In embodiments, the KIH mutations comprise the mutations that replace amino acids with small side chain with ones with larger side chains, thereby creating a knob or protrusion in one chain and vice versa to create a hole or socket in the partner chain. In embodiments, a T366Y mutation in one CH3 domain in alpha chain creates a knob while an Y407T mutation in the CH3 domain in beta chain gives rise to a hole. In embodiments, the KIH mutations establish intermolecular interactions and promote the heterodimer formation due to knob/hole pairing and bring about the association of two different Fc domains to give rise heterodimer formation. In embodiments, the KIH mutations complement existing knob/hole mutations with F405A and T394W on the knob and hole side, respectively, yielding higher level of molecules as heterodimers. Illustrative Fc domains having knob-in-hole mutations are present in SEQ ID NOs: 5-6 and 9-12. In embodiments, the linker comprises a knob-in-hole (KIH) mutations and FcRn mutations SEQ ID NOs: 11-12). In one aspect, the current disclosure provides a heterodimeric protein comprising (a) a first domain comprising one or more butyrophilin family proteins, or a fragment thereof; (b) a second domain DB1/ 145191832.2 23 Attorney Docket No.: SHK-081PC/116981-5081 comprising a targeting domain, the targeting domain being selected from an antibody, antibody-like molecule, and antigen binding fragment thereof; and (c) a linker that adjoins the first and second domains. In embodiments, the heterodimeric protein comprises two individual polypeptide chains which self- associate. In embodiments, the linker facilitates heterodimerization. In embodiments, the heterodimeric protein comprises two of the same butyrophilin family proteins or two different butyrophilin family proteins. In embodiments, the butyrophilin family proteins comprise a V-type domain and/or a B30.2 domain. In embodiments, the first domain is a butyrophilin-like (BTNL) family protein, such as BTN2A1, BTN3A1, and a fragment thereof. In embodiments, the first polypeptide chain and the second polypeptide chain heterodimers through electrostatic interactions between positively charged amino acid residues and negatively charged amino acid residues on the first and second charge polarized core domains. In embodiments, the positively charged amino acid residues may include one or more of amino acids selected from His, Lys, and Arg. In embodiments, the negatively charged amino acid residues may include one or more amino acids selected from Asp and Glu. Accordingly, In embodiments, each of the first and/or second charge polarized core domains comprises proteins having positively or negatively charged amino acid residues at the amino and carboxy terminus of the core domain. In an illustrative embodiment, the first charge polarized core domain may comprise a protein having positively charged amino acids at the amino terminus which are adjoined by a linker (e.g., a stabilizing domain) to a protein having negatively charged amino acid residues at the carboxy terminus. In such an embodiment, the second charge polarized core domain may comprise a protein having negatively charged amino acids at the amino terminus which are adjoined by a linker (e.g., a stabilizing domain) to a protein having positively charged amino acid residues at the carboxy terminus. In another illustrative embodiment, the first charge polarized core domain may comprise a protein having negatively charged amino acids at the amino terminus which are adjoined by a linker (e.g., a stabilizing domain) to a protein having positively charged amino acid residues at the carboxy terminus. In such an embodiment, the second charge polarized core domain may comprise proteins having positively charged amino acids at the amino terminus which are adjoined by a linker (e.g., a stabilizing domain) to a protein having negatively charged amino acid residues at the carboxy terminus. In various embodiments, each of the first and/or second charge polarized core domains further comprise a linker (e.g., a stabilizing domain) which adjoins the proteins having positively or negatively charged amino acids. In embodiments, the linker (e.g., a stabilizing domain) is optionally selected from a flexible amino acid sequence, IgG hinge region, or antibody sequence. In an embodiment, the linker (e.g., a stabilizing domain) comprises the hinge-CH2-CH3 Fc domain derived from IgG1, optionally human IgG1. DB1/ 145191832.2 24 Attorney Docket No.: SHK-081PC/116981-5081 In another embodiment, the linker (e.g., a stabilizing domain) comprises the hinge-CH2-CH3 Fc domain derived from IgG4, optionally human IgG4. Illustrative sequences of linkers that adjoins the first and second domains, also referred to herein as a core domain are provided below: In embodiments, the core domain comprises the following sequence: SKYGPPCPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHN AKTKPREEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPP SQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQE GNVFSCSVLHEALHNHYTQKSLSLSLGKIEGRMD (SEQ ID NO: 1). The sequence of an illustrative charge polarized core domain (positive – negative) is provided below (peptide comprising positively charged amino acids is shown with an underline, peptide comprising negatively charged amino acids is shown with italic font and IgG4 hinge-CH2-CH3 is shown in boldface font. Rest of the sequences are joining linkers disclosed herein): GSGSRKGGKRGSKYGPPCPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQ PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR LTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGKDEGGEDGSGS (SEQ ID NO: 2). The sequence of an illustrative charge polarized core domain (negative - positive) is provided below (peptide comprising positively charged amino acids is shown with an underline, peptide comprising negatively charged amino acids is shown with italic font and IgG4 hinge-CH2-CH3 is shown in boldface font. Rest of the sequences are joining linkers disclosed herein): GSGSDEGGEDGSKYGPPCPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQF NWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQ PREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSR LTVDKSRWQEGNVFSCSVLHEALHNHYTQKSLSLSLGKRKGGKRGSGS (SEQ ID NO: 3). In embodiments, the core domain comprises the following sequence (IgG4 hinge-CH2-CH3 is shown in boldface font, rest of the sequence is a joining linker disclosed herein): CPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSC SVLHEALHNHYTQKSLSLSLGK (SEQ ID NO: 4). DB1/ 145191832.2 25 Attorney Docket No.: SHK-081PC/116981-5081 In embodiments, the core domain comprises a KIHT22Y protein comprising the following sequence (the knob in hole motif mutations are indicated by boldface, underlined font): EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLYCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 5). In embodiments, the core domain is a KIHY86T protein comprising the following sequence (the knob in hole motif mutations are indicated by boldface, underlined font): EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 6). In embodiments, the core domain comprises a IgG1 hinge-CH2-CH3 protein comprising the following sequence: VPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQ PREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMA KDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCS VLHEGLHNHHTEKSLSHSPGI (SEQ ID NO: 7). In embodiments, the core domain comprises the following sequence (IgG4 hinge-CH2-CH3 is shown in boldface font, rest of the sequence is a joining linker disclosed herein): CPPCPAPEFLGGPSVFLFPPKPKDQLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPR EEQFNSTYRVVSVLTVLHQDWLSGKEYKCKVSSKGLPSSIEKTISNATGQPREPQVYTLPPSQEEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSC SVLHEALHNHYTQKSLSLSLGK (SEQ ID NO: 8). The sequence of an illustrative Fc domains containing knob-in-hole (KIH) mutations are provided below: EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKIEGRMD (SEQ ID NO: 9). The sequence of an illustrative Fc domains containing knob-in-hole (KIH) mutations are provided below: DB1/ 145191832.2 26 Attorney Docket No.: SHK-081PC/116981-5081 EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTL PPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQ QGNVFSCSVMHEALHNHYTQKSLSLSPGKIEGRMD (SEQ ID NO: 10). The sequence of an illustrative Fc domains containing knob-in-hole (KIH) mutations and FcRn mutations are provided below: EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ QGNVFSCSVLHEALHSHYTQKSLSLSPGKIEGRMD (SEQ ID NO: 11). The sequence of an illustrative Fc domains containing knob-in-hole (KIH) mutations and FcRn mutations are provided below: EPKSCDKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTL PPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQ QGNVFSCSVLHEALHSHYTQKSLSLSPGKIEGRMD (SEQ ID NO: 12). In various embodiments, the protein comprising the charged amino acid residues may further comprise one or more cysteine residues to facilitate disulfide bonding between the electrostatically charged core domains as an additional method to stabilize the heterodimer. In various embodiments, each of the first and second charge polarized core domains comprises a linker sequence which may optionally function as a stabilizing domain. In various embodiments, 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. In embodiments, 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. In embodiments, the linker (e.g., a stabilizing domain) is a synthetic linker such as PEG. In other embodiments, the linker (e.g., a stabilizing domain) is a polypeptide. In embodiments, the linker (e.g., a stabilizing domain) is less than about 500 amino acids long, about 450 amino acids long, about 400 amino acids long, about 350 amino acids long, about 300 amino acids long, about 250 amino acids DB1/ 145191832.2 27 Attorney Docket No.: SHK-081PC/116981-5081 long, about 200 amino acids long, about 150 amino acids long, or about 100 amino acids long. For example, the linker (e.g., a stabilizing domain) may be less than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids long. In various embodiments, the linker (e.g., a stabilizing domain) is substantially comprised of glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97% glycines and serines). In various embodiments, the linker (e.g., a stabilizing domain) is a hinge region of an antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2). The hinge region, found in IgG, IgA, IgD, and IgE class antibodies, acts as a flexible spacer, allowing the Fab portion to move freely in space. In contrast to the constant regions, the hinge domains are structurally diverse, varying in both sequence and length among immunoglobulin classes and subclasses. For example, the length and flexibility of the hinge region varies among the IgG subclasses. The hinge region of IgG1 encompasses amino acids 216-231 and, because it is freely flexible, the Fab fragments can rotate about their axes of symmetry and move within a sphere centered at the first of two inter-heavy chain disulfide bridges. IgG2 has a shorter hinge than IgG1, with 12 amino acid residues and four disulfide bridges. The hinge region of IgG2 lacks a glycine residue, is relatively short, and contains a rigid poly- proline double helix, stabilized by extra inter-heavy chain disulfide bridges. These properties restrict the flexibility of the IgG2 molecule. IgG3 differs from the other subclasses by its unique extended hinge region (about four times as long as the IgG1 hinge), containing 62 amino acids (including 21 prolines and 11 cysteines), forming an inflexible poly-proline double helix. In IgG3, the Fab fragments are relatively far away from the Fc fragment, giving the molecule a greater flexibility. The elongated hinge in IgG3 is also responsible for its higher molecular weight compared to the other subclasses. The hinge region of IgG4 is shorter than that of IgG1 and its flexibility is intermediate between that of IgG1 and IgG2. The flexibility of the hinge regions reportedly decreases in the order IgG3>IgG1>IgG4>IgG2. In other embodiments, the linker may be derived from human IgG4 and contain one or more mutations to enhance dimerization (including S228P) or FcRn binding. According to crystallographic studies, the immunoglobulin hinge region can be further subdivided functionally into three regions: the upper hinge region, the core region, and the lower hinge region. See Shin et al., 1992 Immunological Reviews 130:87. The upper hinge region includes amino acids from the carboxyl end of CH1 to the first residue in the hinge that restricts motion, generally the first cysteine residue DB1/ 145191832.2 28 Attorney Docket No.: SHK-081PC/116981-5081 that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region correlates with the segmental flexibility of the antibody. The core hinge region contains the inter- heavy chain disulfide bridges, and the lower hinge region joins the amino terminal end of the CH2 domain and includes residues in CH2. Id. The core hinge region of wild-type human IgG1 contains the sequence Cys-Pro-Pro-Cys which, when dimerized by disulfide bond formation, results in a cyclic octapeptide believed to act as a pivot, thus conferring flexibility. In various embodiments, the present linker (e.g., a stabilizing domain) comprises, one, or two, or three of the upper hinge region, the core region, and the lower hinge region of any antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). The hinge region may also contain one or more glycosylation sites, which include a number of structurally distinct types of sites for carbohydrate attachment. For example, IgA1 contains five glycosylation sites within a 17-amino-acid segment of the hinge region, conferring resistance of the hinge region polypeptide to intestinal proteases, considered an advantageous property for a secretory immunoglobulin. In various embodiments, the linker (e.g., a stabilizing domain) of the current disclosure comprises one or more glycosylation sites. In various embodiments, the linker (e.g., a stabilizing domain) comprises an Fc domain of an antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). In various embodiments, the linker (e.g., a stabilizing domain) comprises a hinge-CH2-CH3 Fc domain derived from a human IgG4 antibody. In various embodiments, the linker (e.g., a stabilizing domain) comprises a hinge-CH2-CH3 Fc domain derived from a human IgG1 antibody. In embodiments, the Fc domain exhibits increased affinity for and enhanced binding to the neonatal Fc receptor (FcRn). In embodiments, the Fc domain includes one or more mutations that increases the affinity and enhances binding to FcRn. Without wishing to be bound by theory, it is believed that increased affinity and enhanced binding to FcRn increases the in vivo half-life of the present heterodimeric proteins. In embodiments, the Fc domain contains one or more amino acid substitutions at amino acid residue 250, 252, 254, 256, 308, 309, 311, 428, 433 or 434 (in accordance with Kabat numbering), or equivalents thereof. In an embodiment, the amino acid substitution at amino acid residue 250 is a substitution with glutamine. In an embodiment, the amino acid substitution at amino acid residue 252 is a substitution with tyrosine, phenylalanine, tryptophan or threonine. In an embodiment, the amino acid substitution at amino acid residue 254 is a substitution with threonine. In an embodiment, the amino acid substitution at amino acid residue 256 is a substitution with serine, arginine, glutamine, glutamic acid, aspartic acid, or threonine. In an embodiment, the amino acid substitution at amino acid residue 308 is a substitution with threonine. In an embodiment, the amino acid substitution at amino acid residue 309 is a substitution with proline. In an embodiment, the amino acid substitution at amino acid residue 311 is a substitution with DB1/ 145191832.2 29 Attorney Docket No.: SHK-081PC/116981-5081 serine. In an embodiment, the amino acid substitution at amino acid residue 385 is a substitution with arginine, aspartic acid, serine, threonine, histidine, lysine, alanine or glycine. In an embodiment, the amino acid substitution at amino acid residue 386 is a substitution with threonine, proline, aspartic acid, serine, lysine, arginine, isoleucine, or methionine. In an embodiment, the amino acid substitution at amino acid residue 387 is a substitution with arginine, proline, histidine, serine, threonine, or alanine. In an embodiment, the amino acid substitution at amino acid residue 389 is a substitution with proline, serine or asparagine. In an embodiment, the amino acid substitution at amino acid residue 428 is a substitution with leucine. In an embodiment, the amino acid substitution at amino acid residue 433 is a substitution with arginine, serine, isoleucine, proline, or glutamine. In an embodiment, the amino acid substitution at amino acid residue 434 is a substitution with histidine, phenylalanine, or tyrosine. In embodiments, the Fc domain (e.g., comprising an IgG constant region) comprises one or more mutations such as substitutions at amino acid residue 252, 254, 256, 433, 434, or 436 (in accordance with Kabat numbering). In an embodiment, the IgG constant region includes a triple M252Y/S254T/T256E mutation or YTE mutation. In another embodiment, the IgG constant region includes a triple H433K/N434F/Y436H mutation or KFH mutation. In a further embodiment, the IgG constant region includes an YTE and KFH mutation in combination. In embodiments, the modified humanized antibodies of the invention comprise an IgG constant region that contains one or more mutations at amino acid residues 250, 253, 307, 310, 380, 428, 433, 434, and 435. Illustrative mutations include T250Q, M428L, T307A, E380A, I253A, H310A, M428L, H433K, N434A, N434F, N434S, and H435A. In an embodiment, the IgG constant region comprises a M428L/N434S mutation or LS mutation. In another embodiment, the IgG constant region comprises a T250Q/M428L mutation or QL mutation. In another embodiment, the IgG constant region comprises an N434A mutation. In another embodiment, the IgG constant region comprises a T307A/E380A/N434A mutation or AAA mutation. In another embodiment, the IgG constant region comprises an I253A/H310A/H435A mutation or IHH mutation. In another embodiment, the IgG constant region comprises a H433K/N434F mutation. In another embodiment, the IgG constant region comprises a M252Y/S254T/T256E and a H433K/N434F mutation in combination. In various embodiments, mutations are introduced to increase stability and/or half-life of the Fc domain. An illustrative Fc stabilizing mutant is S228P. Additional illustrative Fc half-life extending mutants are T250Q, M428L, V308T, L309P, and Q311S and the present linkers (e.g., stabilizing domains) may comprise 1, or 2, or 3, or 4, or 5 of these mutants. DB1/ 145191832.2 30 Attorney Docket No.: SHK-081PC/116981-5081 Additional illustrative mutations in the IgG constant region are described, for example, in Robbie, et al., Antimicrobial Agents and Chemotherapy (2013), 57(12):6147-6153, Dall’Acqua et al., JBC (2006), 281(33):23514-24, Dall’Acqua et al., Journal of Immunology (2002), 169:5171-80, Ko et al., Nature (2014) 514:642-645, Grevys et al., Journal of Immunology. (2015), 194(11):5497-508, and U.S. Patent No. 7,083,784, the entire contents of which are hereby incorporated by reference. In various embodiments, the linker may be flexible, including without limitation highly flexible. In various embodiments, the linker may be rigid, including without limitation a rigid alpha helix. In various embodiments, the linker may be functional. For example, without limitation, the linker may function to improve the folding and/or stability, improve the expression, improve the pharmacokinetics, and/or improve the bioactivity of the present heterodimeric protein. In another example, the linker may function to target the heterodimeric protein to a particular cell type or location. The Heterodimeric Proteins In one aspect, the current disclosure provides a heterodimeric protein, and methods of use of the same, comprising: (a) a first domain comprising one or more butyrophilin family proteins, or a fragment thereof; (b) a second domain comprising a targeting domain, the targeting domain being selected from an antibody, antibody-like molecule, and antigen binding fragment thereof; and (c) a linker that adjoins the first and second domains. In embodiments the heterodimeric protein is a complex of two polypeptide chains. In embodiments the heterodimeric protein comprises an alpha chain and a beta chain wherein the alpha chain and the beta chain each independently comprise (a) a first domain comprising a butyrophilin family protein, or fragment thereof; (b) a second domain comprising a targeting domain, the targeting domain being selected from an antibody, antibody-like molecule, and antigen binding fragment thereof; and (c) a linker that adjoins the first and second domains. In embodiments the alpha chain and the beta chain self-associate to form the heterodimer. In embodiments, the first domain comprises two of the same butyrophilin family proteins. In embodiments, wherein the first domain comprises two different butyrophilin family proteins. In embodiments, the butyrophilin family proteins comprise a V-type domain. In embodiments, the butyrophilin family proteins or fragments thereof are derived from the native butyrophilin family proteins that comprise a B30.2 domain in the cytosolic tail. DB1/ 145191832.2 31 Attorney Docket No.: SHK-081PC/116981-5081 In embodiments, the butyrophilin family proteins are selected from BTN2A1, BTN3A1, and a fragment thereof. In embodiments, the first domain comprises: (a) BTN2A1, BTN3A1, and a fragment thereof; and (b) BTN2A1, BTN3A1, and a fragment thereof. In embodiments, the first domain comprises a fragment of butyrophilin family proteins, wherein the fragment is capable of binding a gamma delta T cell receptor and is optionally an extracellular domain, optionally comprising one or more of an immunoglobulin V (IgV)- and IgC-like domain. In embodiments, the first domain comprises a fragment of butyrophilin family proteins, wherein the fragment is capable of binding a Vγ9δ2 gamma delta T cell receptor. In embodiments, the first domain comprises a polypeptide having an amino acid sequence of: (a) any one of SEQ ID NOs: 13, 15, or a fragment thereof; and (b) any one of SEQ ID NOs: 13, 15, or a fragment thereof. In embodiments, the first domain comprises a polypeptide having (a) an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 15 or SEQ ID NO: 16, and an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with SEQ ID NO: 13 or SEQ ID NO: 14. Additionally, or alternatively, in any of the embodiments disclosed herein, the targeting domain is an antibody, or antigen binding fragment thereof. In embodiments, the targeting domain is an antibody-like molecule, or antigen binding fragment thereof. In embodiments, the antibody-like molecule is selected from a single-domain antibody, a recombinant heavy-chain-only antibody (VHH), a single-chain antibody (scFv), a shark heavy-chain-only antibody (VNAR), a microprotein (cysteine knot protein, knottin), a DARPin; a Tetranectin; an Affibody; a Transbody; an Anticalin; an AdNectin; an Affilin; an Affimer, a Microbody; an aptamer; an alterase; a plastic antibody; a phylomer; a stradobody; a maxibody; an evibody; a fynomer, an armadillo repeat protein, a Kunitz domain, an avimer, an atrimer, a probody, an immunobody, a triomab, a troybody; a pepbody; a vaccibody, a UniBody; a DuoBody, a Fv, a Fab, a Fab′, and a F(ab′)2. In embodiments, the antibody-like molecule is an scFv. In embodiments, the targeting domain is an extracellular domain. In embodiments, the targeting domain is capable of binding an antigen on the surface of a cancer cell. In embodiments, the targeting domain is capable of binding an antigen on the surface of a cancer cell. In embodiments, the targeting domain specifically binds CD20. In embodiments, the targeting domain specifically binds CD20. Additionally or alternatively, in embodiments, the targeting domain is a polypeptide having an amino acid sequence with at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence selected from SEQ ID NOs: 62-65. In embodiments, DB1/ 145191832.2 32 Attorney Docket No.: SHK-081PC/116981-5081 the targeting domain is a polypeptide having an amino acid sequence of selected from SEQ ID NOs: 62- 65. Additionally or alternatively, in embodiments, the linker comprises (a) a first charge polarized core domain adjoined to a butyrophilin family protein, optionally at the carboxy terminus, and (b) a second charge polarized core domain adjoined to a butyrophilin family protein, optionally at the carboxy terminus. In embodiments, the linker forms a heterodimer through electrostatic interactions between positively charged amino acid residues and negatively charged amino acid residues on the first and second charge polarized core domains. In embodiments, the first and/or second charge polarized core domain comprises a polypeptide linker, optionally selected from a flexible amino acid sequence, IgG hinge region, or antibody sequence. In embodiments, the linker is a synthetic linker, optionally PEG. In embodiments, the linker comprises the hinge-CH2-CH3 Fc domain derived from IgG1, optionally human IgG1. In embodiments, the linker comprises the hinge-CH2-CH3 Fc domain derived from IgG4, optionally human IgG4. In embodiments, the first and/or second charge polarized core domain further comprise peptides having positively and/or negatively charged amino acid residues at the amino and/or carboxy terminus of the charge polarized core domain. In embodiments, the positively charged amino acid residues include one or more of amino acids selected from His, Lys, and Arg. In embodiments, the positively charged amino acid residues are present in a peptide comprising positively charged amino acid residues in the first and/or the second charge polarized core domains. In embodiments, the peptide comprising positively charged amino acid residues comprises a sequence selected from YnXnYnXnYn (where X is a positively charged amino acid such as arginine, histidine or lysine and Y is a spacer amino acid such as serine or glycine, and where each n is independently an integer 0 to 4) (SEQ ID NO: 21), YYnXXnYYnXXnYYn (where X is a positively charged amino acid such as arginine, histidine or lysine and Y is a spacer amino acid such as serine or glycine, and where each n is independently an integer 0 to 4) (SEQ ID NO: 23), and YnXnCYnXnYn (where X is a positively charged amino acid such as arginine, histidine or lysine and Y is a spacer amino acid such as serine or glycine, and where each n is independently an integer 0 to 4) (SEQ ID NO: 25). In embodiments, the peptide comprising positively charged amino acid residues comprises the sequence RKGGKR (SEQ ID NO: 31) or GSGSRKGGKRGS (SEQ ID NO: 32). In embodiments, the negatively charged amino acid residues may include one or more amino acids selected from Asp and Glu. In embodiments, the negatively charged amino acid residues are present in a peptide comprising negatively charged amino acid residues in the first and/or the second charge polarized core domains. In embodiments, the peptide comprising negatively charged amino acid residues comprises a sequence selected from YnZnYnZnYn (where Z is a negatively charged amino acid such as aspartic acid or glutamic acid and Y is a spacer amino acid such DB1/ 145191832.2 33 Attorney Docket No.: SHK-081PC/116981-5081 as serine or glycine, and where each n is independently an integer 0 to 4) (SEQ ID NO: 22), YYnZZnYYnZZnYYn (where Z is a negatively charged amino acid such as aspartic acid or glutamic acid and Y is a spacer amino acid such as serine or glycine, and where each n is independently an integer 0 to 4) (SEQ ID NO: 24), and YnZnCYnZnYn (where Z is a negatively charged amino acid such as aspartic acid or glutamic acid and Y is a spacer amino acid such as serine or glycine) (SEQ ID NO: 26, and where each n is independently an integer 0 to 4). In embodiments, the peptide comprising negatively charged amino acid residues comprises the sequence DEGGED (SEQ ID NO: 33) or GSGSDEGGEDGS (SEQ ID NO: 34). In various embodiments, the protein comprising the charged amino acid residues may further comprise one or more cysteine residues to facilitate disulfide bonding between the electrostatically charged core domains as an additional method to stabilize the heterodimer. In various embodiments, each of the first and second charge polarized core domains comprises a linker sequence which may optionally function as a stabilizing domain. In various embodiments, 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. In embodiments, 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. In embodiments, the linker (e.g., a stabilizing domain) is a synthetic linker such as PEG. In other embodiments, the linker (e.g., a stabilizing domain) is a polypeptide. In embodiments, the linker (e.g., a stabilizing domain) is less than about 500 amino acids long, about 450 amino acids long, about 400 amino acids long, about 350 amino acids long, about 300 amino acids long, about 250 amino acids long, about 200 amino acids long, about 150 amino acids long, or about 100 amino acids long. For example, the linker (e.g., a stabilizing domain) may be less than about 100, about 95, about 90, about 85, about 80, about 75, about 70, about 65, about 60, about 55, about 50, about 45, about 40, about 35, about 30, about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, or about 2 amino acids long. In various embodiments, the linker (e.g., a stabilizing domain) is substantially comprised of glycine and serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97% glycines and serines). DB1/ 145191832.2 34 Attorney Docket No.: SHK-081PC/116981-5081 In various embodiments, the linker (e.g., a stabilizing domain) is a hinge region of an antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2). The hinge region, found in IgG, IgA, IgD, and IgE class antibodies, acts as a flexible spacer, allowing the Fab portion to move freely in space. In contrast to the constant regions, the hinge domains are structurally diverse, varying in both sequence and length among immunoglobulin classes and subclasses. For example, the length and flexibility of the hinge region varies among the IgG subclasses. The hinge region of IgG1 encompasses amino acids 216-231 and, because it is freely flexible, the Fab fragments can rotate about their axes of symmetry and move within a sphere centered at the first of two inter-heavy chain disulfide bridges. IgG2 has a shorter hinge than IgG1, with 12 amino acid residues and four disulfide bridges. The hinge region of IgG2 lacks a glycine residue, is relatively short, and contains a rigid poly- proline double helix, stabilized by extra inter-heavy chain disulfide bridges. These properties restrict the flexibility of the IgG2 molecule. IgG3 differs from the other subclasses by its unique extended hinge region (about four times as long as the IgG1 hinge), containing 62 amino acids (including 21 prolines and 11 cysteines), forming an inflexible poly-proline double helix. In IgG3, the Fab fragments are relatively far away from the Fc fragment, giving the molecule a greater flexibility. The elongated hinge in IgG3 is also responsible for its higher molecular weight compared to the other subclasses. The hinge region of IgG4 is shorter than that of IgG1 and its flexibility is intermediate between that of IgG1 and IgG2. The flexibility of the hinge regions reportedly decreases in the order IgG3>IgG1>IgG4>IgG2. In other embodiments, the linker may be derived from human IgG4 and contain one or more mutations to enhance dimerization (including S228P) or FcRn binding. According to crystallographic studies, the immunoglobulin hinge region can be further subdivided functionally into three regions: the upper hinge region, the core region, and the lower hinge region. See Shin et al., 1992 Immunological Reviews 130:87. The upper hinge region includes amino acids from the carboxyl end of CH1 to the first residue in the hinge that restricts motion, generally the first cysteine residue that forms an interchain disulfide bond between the two heavy chains. The length of the upper hinge region correlates with the segmental flexibility of the antibody. The core hinge region contains the inter- heavy chain disulfide bridges, and the lower hinge region joins the amino terminal end of the CH2 domain and includes residues in CH2. Id. The core hinge region of wild-type human IgG1 contains the sequence Cys-Pro-Pro-Cys which, when dimerized by disulfide bond formation, results in a cyclic octapeptide believed to act as a pivot, thus conferring flexibility. In various embodiments, the present linker (e.g., a stabilizing domain) comprises, one, or two, or three of the upper hinge region, the core region, and the lower hinge region of any antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). The hinge region may also contain one or more glycosylation DB1/ 145191832.2 35 Attorney Docket No.: SHK-081PC/116981-5081 sites, which include a number of structurally distinct types of sites for carbohydrate attachment. For example, IgA1 contains five glycosylation sites within a 17-amino-acid segment of the hinge region, conferring resistance of the hinge region polypeptide to intestinal proteases, considered an advantageous property for a secretory immunoglobulin. In various embodiments, the linker (e.g., a stabilizing domain) of the current disclosure comprises one or more glycosylation sites. In various embodiments, the linker (e.g., a stabilizing domain) comprises an Fc domain of an antibody (e.g., of IgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., IgG1, IgG2, IgG3, and IgG4, and IgA1 and IgA2)). In various embodiments, the linker (e.g., a stabilizing domain) comprises a hinge-CH2-CH3 Fc domain derived from a human IgG4 antibody. In various embodiments, the linker (e.g., a stabilizing domain) comprises a hinge-CH2-CH3 Fc domain derived from a human IgG1 antibody. In embodiments, the Fc domain exhibits increased affinity for and enhanced binding to the neonatal Fc receptor (FcRn). In embodiments, the Fc domain includes one or more mutations that increases the affinity and enhances binding to FcRn. Without wishing to be bound by theory, it is believed that increased affinity and enhanced binding to FcRn increases the in vivo half-life of the present heterodimeric proteins. In embodiments, the Fc domain contains one or more amino acid substitutions at amino acid residue 250, 252, 254, 256, 308, 309, 311, 428, 433 or 434 (in accordance with Kabat numbering), or equivalents thereof. In an embodiment, the amino acid substitution at amino acid residue 250 is a substitution with glutamine. In an embodiment, the amino acid substitution at amino acid residue 252 is a substitution with tyrosine, phenylalanine, tryptophan or threonine. In an embodiment, the amino acid substitution at amino acid residue 254 is a substitution with threonine. In an embodiment, the amino acid substitution at amino acid residue 256 is a substitution with serine, arginine, glutamine, glutamic acid, aspartic acid, or threonine. In an embodiment, the amino acid substitution at amino acid residue 308 is a substitution with threonine. In an embodiment, the amino acid substitution at amino acid residue 309 is a substitution with proline. In an embodiment, the amino acid substitution at amino acid residue 311 is a substitution with serine. In an embodiment, the amino acid substitution at amino acid residue 385 is a substitution with arginine, aspartic acid, serine, threonine, histidine, lysine, alanine or glycine. In an embodiment, the amino acid substitution at amino acid residue 386 is a substitution with threonine, proline, aspartic acid, serine, lysine, arginine, isoleucine, or methionine. In an embodiment, the amino acid substitution at amino acid residue 387 is a substitution with arginine, proline, histidine, serine, threonine, or alanine. In an embodiment, the amino acid substitution at amino acid residue 389 is a substitution with proline, serine or asparagine. In an embodiment, the amino acid substitution at amino acid residue 428 is a substitution with leucine. In an embodiment, the amino acid substitution at amino acid residue 433 is a substitution DB1/ 145191832.2 36 Attorney Docket No.: SHK-081PC/116981-5081 with arginine, serine, isoleucine, proline, or glutamine. In an embodiment, the amino acid substitution at amino acid residue 434 is a substitution with histidine, phenylalanine, or tyrosine. In embodiments, the Fc domain (e.g., comprising an IgG constant region) comprises one or more mutations such as substitutions at amino acid residue 252, 254, 256, 433, 434, or 436 (in accordance with Kabat numbering). In an embodiment, the IgG constant region includes a triple M252Y/S254T/T256E mutation or YTE mutation. In another embodiment, the IgG constant region includes a triple H433K/N434F/Y436H mutation or KFH mutation. In a further embodiment, the IgG constant region includes an YTE and KFH mutation in combination. In embodiments, the modified humanized antibodies of the invention comprise an IgG constant region that contains one or more mutations at amino acid residues 250, 253, 307, 310, 380, 428, 433, 434, and 435. Illustrative mutations include T250Q, M428L, T307A, E380A, I253A, H310A, M428L, H433K, N434A, N434F, N434S, and H435A. In an embodiment, the IgG constant region comprises a M428L/N434S mutation or LS mutation. In another embodiment, the IgG constant region comprises a T250Q/M428L mutation or QL mutation. In another embodiment, the IgG constant region comprises an N434A mutation. In another embodiment, the IgG constant region comprises a T307A/E380A/N434A mutation or AAA mutation. In another embodiment, the IgG constant region comprises an I253A/H310A/H435A mutation or IHH mutation. In another embodiment, the IgG constant region comprises a H433K/N434F mutation. In another embodiment, the IgG constant region comprises a M252Y/S254T/T256E and a H433K/N434F mutation in combination. In various embodiments, mutations are introduced to increase stability and/or half-life of the Fc domain. An illustrative Fc stabilizing mutant is S228P. Additional illustrative Fc half-life extending mutants are T250Q, M428L, V308T, L309P, and Q311S and the present linkers (e.g., stabilizing domains) may comprise 1, or 2, or 3, or 4, or 5 of these mutants. Additional illustrative mutations in the IgG constant region are described, for example, in Robbie, et al., Antimicrobial Agents and Chemotherapy (2013), 57(12):6147-6153, Dall’Acqua et al., JBC (2006), 281(33):23514-24, Dall’Acqua et al., Journal of Immunology (2002), 169:5171-80, Ko et al., Nature (2014) 514:642-645, Grevys et al., Journal of Immunology. (2015), 194(11):5497-508, and U.S. Patent No. 7,083,784, the entire contents of which are hereby incorporated by reference. In various embodiments, the linker may be flexible, including without limitation highly flexible. In various embodiments, the linker may be rigid, including without limitation a rigid alpha helix. In various embodiments, the linker may be functional. For example, without limitation, the linker may function to improve the folding and/or stability, improve the expression, improve the pharmacokinetics, DB1/ 145191832.2 37 Attorney Docket No.: SHK-081PC/116981-5081 and/or improve the bioactivity of the present heterodimeric protein. In another example, the linker may function to target the heterodimeric protein to a particular cell type or location. Diseases; Methods of Treatment, and Patient Selections In some aspects, the present disclosure relates to a method for treating a B cell-mediated disease or disorder, comprising administering to the subject about 0.01 mg/kg to about 100 mg/kg of a heterodimeric protein, or a nucleic acid encoding the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; (b) a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof, the target domain being directed to CD20; and (c) a linker that adjoins the first and second domains; and wherein the beta chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; (b) a second domain comprising the targeting domain, the target domain being directed to CD20; and (c) a linker that adjoins the first and second domains. In some aspects, the present disclosure relates to a method for increasing and/or stimulating B cell depletion in a subject suffering from a B cell-mediated disease or disorder, comprising administering to the subject about 0.01 mg/kg to about 100 mg/kg of a heterodimeric protein, or a nucleic acid encoding the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; (b) a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof, the target domain being directed to CD20; and (c) a linker that adjoins the first and second domains; and wherein the beta chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; (b) a second domain comprising the targeting domain, the target domain being directed to CD20; and (c) a linker that adjoins the first and second domains. In some aspects, the present disclosure relates to a method for a method of contemporaneous activation and targeting of gamma delta T cells to cancer cells , comprising administering to the subject about 0.01 mg/kg to about 100 mg/kg of a heterodimeric protein, or a nucleic acid encoding the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; (b) a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof, the target domain being directed to CD20; and (c) a linker DB1/ 145191832.2 38 Attorney Docket No.: SHK-081PC/116981-5081 that adjoins the first and second domains; and wherein the beta chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; (b) a second domain comprising the targeting domain, the target domain being directed to CD20; and (c) a linker that adjoins the first and second domains.. In some aspects, the present disclosure relates to a method for method of modulating a patient’s immune response, , comprising administering to the subject about 0.01 mg/kg to about 100 mg/kg of a heterodimeric protein, or a nucleic acid encoding the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; (b) a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof, the target domain being directed to CD20; and (c) a linker that adjoins the first and second domains; and wherein the beta chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; (b) a second domain comprising the targeting domain, the target domain being directed to CD20; and (c) a linker that adjoins the first and second domains.. In some aspects, the present disclosure relates to a method for method of treating cancer, comprising administering to a subject in need thereof a pharmaceutical composition , comprising administering to the subject about 0.01 mg/kg to about 100 mg/kg of a heterodimeric protein, or a nucleic acid encoding the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; (b) a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof, the target domain being directed to CD20; and (c) a linker that adjoins the first and second domains; and wherein the beta chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; (b) a second domain comprising the targeting domain, the target domain being directed to CD20; and (c) a linker that adjoins the first and second domains.. In embodiments, the B cell-mediated disease or disorder is a cancer. In embodiments, the cancer is a cancer expressing CD20. In embodiments, the cancer is a B-cell malignancy. In embodiments, the B-cell malignancy is a lymphoma or leukemia. In embodiments, the B-cell malignancy is non-Hodgkin’s lymphoma (NHL) or Hodgkin’s lymphoma (HL). In embodiments, the e B-cell malignancy is advanced stage follicular lymphoma, chronic lymphocytic leukemias (CLL), Burkitt’s lymphoma, or diffuse large B- cell lymphoma. DB1/ 145191832.2 39 Attorney Docket No.: SHK-081PC/116981-5081 In embodiments, the B-cell mediated disease or disorder is an autoimmune disease or disorder. In embodiments, the autoimmune disease or disorder is one or more of rheumatoid arthritis, systemic lupus erythematosus, diabetes mellitus, ankylosing spondylitis, Sjögren's syndrome, inflammatory bowel diseases (e.g., colitis ulcerosa, Crohn's disease), multiple sclerosis, sarcoidosis, psoriasis, Grave's disease, Hashimoto's thyroiditis, psoriasis, hypersensitivity reactions (e.g., allergies, hay fever, asthma, and acute edema cause type I hypersensitivity reactions), pemphigus, and vasculitis (e.g., Granulomatosis with polyangiitis). In one aspect, the current disclosure provides a method of treating cancer, comprising administering to a subject in need thereof a pharmaceutical composition comprising a heterodimeric protein comprising an alpha chain and a beta chain: wherein the alpha chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof; (b) a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof; and (c) a linker that adjoins the first and second domains; and wherein the beta chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof; (b) a second domain comprising the targeting domain; and (c) a linker that adjoins the first and second domains. In embodiments, the butyrophilin family protein of the alpha chain and the beta chain is independently selected from BTN2A1, and BTN3A1. In embodiments, the butyrophilin family protein of the alpha chain and the beta chain is independently selected from human BTN2A1, and human BTN3A1. In embodiments, the antibody-like molecule is an scFv. In embodiments, the antibody-like molecule is an scFv or a fragment thereof, e.g., the light chain and/or heavy chain of the scFv. In embodiments, the antibody-like molecule is an Fv fragment. In embodiments, the targeting domain is capable of binding an antigen on the surface of a cancer cell. In embodiments, the targeting domain is capable of binding an antigen on the surface of a cancer cell. In embodiments, the targeting domain specifically binds CD20. In embodiments, the targeting domain specifically binds to CD20. In embodiments, the cancer is a B cell lymphoma or leukemia. In embodiments, the cancer is an acute myeloid leukemia (AML). In embodiments, the cancer is a solid cancer selected from prostate cancer, small cell lung cancer, non-small cell lung cancer, neuroendocrine tumors, renal cancer, bladder cancer, colon cancer, and breast cancer. In embodiments, the cancer is known to express the antigenic target of the targeting domain disclosed herein. In embodiments, the cancer is suspected to express the antigenic target of the targeting domain disclosed herein. In one aspect, the present disclosure relates to a method of treating a cancer CD20 in a subject in need thereof comprising: administering to the subject a pharmaceutical composition of any of the embodiments DB1/ 145191832.2 40 Attorney Docket No.: SHK-081PC/116981-5081 disclosed herein, thereby causing an in vivo proliferation of gamma delta T cells and/or contacting a cell derived from the subject with a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an ex vivo proliferation of gamma delta T cells. In one aspect, the present disclosure relates to a method of treating a cancer expressing CD20 in a subject in need thereof comprising: administering to the subject a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an in vivo proliferation of gamma delta T cells and/or contacting a cell derived from the subject with a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an ex vivo proliferation of gamma delta T cells. In one aspect, the present disclosure relates to a method of treating a cancer that is CD20 positive in a subject in need thereof comprising: administering to the subject a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an in vivo proliferation of gamma delta T cells and/or contacting a cell derived from the subject with a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an ex vivo proliferation of gamma delta T cells. In one aspect, the present disclosure relates to a method of treating a cancer that is CD20+, in a subject in need thereof comprising: administering to the subject a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an in vivo proliferation of gamma delta T cells and/or contacting a cell derived from the subject with a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an ex vivo proliferation of gamma delta T cells. In one aspect, the present disclosure relates to a method of treating a CD20+ cancer in a subject in need thereof comprising: administering to the subject a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an in vivo proliferation of gamma delta T cells and/or contacting a cell derived from the subject with a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an ex vivo proliferation of gamma delta T cells. In one aspect, the present disclosure relates to a method of treating a B cell lymphoma/leukemia in a subject in need thereof. The method comprises administering to the subject a pharmaceutical composition comprising a heterodimeric protein comprising an alpha chain and a beta chain, wherein the alpha chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof; (b) a second domain comprising a targeting domain, and (c) a linker that adjoins the first and second domains; and wherein the beta chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof; (b) a second domain comprising the targeting domain; and (c) a linker that adjoins the first and second domains, wherein the targeting domain is capable of specifically binding to CD20. DB1/ 145191832.2 41 Attorney Docket No.: SHK-081PC/116981-5081 In one aspect, the present disclosure relates to a method of treating a B cell lymphoma/leukemia in a subject in need thereof. The method comprises contacting a cell derived from the subject with a pharmaceutical composition comprising a heterodimeric protein comprising an alpha chain and a beta chain, wherein the alpha chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof; (b) a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof; and (c) a linker that adjoins the first and second domains; and wherein the beta chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof; (b) a second domain comprising the targeting domain; and (c) a linker that adjoins the first and second domains, wherein the targeting domain is capable of specifically binding to CD20. The method optionally further comprises proliferating ex vivo the cell derived from the subject that is contacted with the pharmaceutical composition. The method optionally further comprises administering to the subject the cell that is proliferated ex vivo. In one aspect, the present disclosure relates to a method of stimulating proliferation of gamma delta T cells, comprising: administering a pharmaceutical composition of any one of the embodiments disclosed herein, to a subject in need thereof thereby causing an in vivo proliferation of gamma delta T cells and/or contacting a pharmaceutical composition of any one of the embodiments disclosed herein, with a cell derived from a subject in need thereof thereby causing an ex vivo proliferation of gamma delta T cells. In one aspect, the current disclosure provides a method of treating cancer, comprising administering to a subject in need thereof a pharmaceutical composition of any of the embodiments disclosed herein to a subject in need thereof. In embodiments, the subject’s T cells are activated by the first domain. In embodiments, the subject has a cancer and the gamma delta T cells modulate cells of the tumor. In embodiments, the method further comprises administering to the subject a second pharmaceutical composition that costimulates γδ T cells, or contacting a second pharmaceutical composition that costimulates γδ T cells, with a cell derived from a subject in need thereof thereby causing an ex vivo proliferation of gamma delta T cells. In one aspect, the present disclosure relates to a method for treating a cancer in a subject in need thereof comprising: (i) administering to the subject a pharmaceutical composition comprising the heterodimeric protein; and (ii) administering to the subject a second pharmaceutical composition that costimulates γδ T cells. In embodiments, the first pharmaceutical composition and the second pharmaceutical composition are administered simultaneously or contemporaneously. In embodiments, the first pharmaceutical composition is administered after the second pharmaceutical composition is administered. In DB1/ 145191832.2 42 Attorney Docket No.: SHK-081PC/116981-5081 embodiments, the first pharmaceutical composition is administered before the second pharmaceutical composition is administered. In embodiments, the second pharmaceutical composition costimulates a receptor selected from CD28, NKG2D, CD27, CD30, 4-1BB (CD137), IL-2R, IL-15R, IL-7R, IL-21R, NKp30, NKp44, DNAM-1 (CD226), IL-2R, IL-7R, IL-15R, dectins, NLRs, killer Ig-like receptors (e.g., KIR2D, KIR3D), C-type lectins (CD94/NKG2A-C, NKG2D), LFA1, CD2, CD46, Junctional Adhesion Molecule-Like (JAML). In embodiments, the second pharmaceutical composition comprises a ligand of the receptor, or a receptor-binding portion thereof. In embodiments, the second pharmaceutical composition comprises a fusion protein (without limitation, e.g., an Fc fusion protein or an albumin fusion protein) comprising a co- stimulatory molecule or a binding portion thereof. In embodiments, the second pharmaceutical composition comprises a fusion protein (without limitation, e.g., an Fc fusion protein or an albumin fusion protein) comprising the ligand of the receptor, or receptor-binding portion thereof. In embodiments, the second pharmaceutical composition comprises a fusion protein (without limitation, e.g., an Fc fusion protein or an albumin fusion protein) comprising the receptor, or a ligand-binding portion thereof. In embodiments, the second pharmaceutical composition comprises an antibody, antibody-like molecule or a receptor-binding portion thereof. In embodiments, the second pharmaceutical composition comprises an agonistic antibody. In embodiments, the second pharmaceutical composition costimulates CD28. In embodiments, the second pharmaceutical composition comprises a CD28 ligand, or a CD28-binding portion thereof. In embodiments, the CD28 ligand is selected from CD80 and CD86. In embodiments, the CD28 ligand of is an antibody, an antibody-like molecule, or a binding fragment thereof. In embodiments, the CD28 ligand is a fusion protein (without limitation, e.g., Fc fusion protein) comprising CD80, CD86, or a CD28-binding portion thereof. In embodiments, the CD28 ligand is an Fc fusion protein comprising CD80, CD86, or a CD28-binding portion thereof. In embodiments, the binding fragment is selected from Fab fragment, heavy variable chain, and single chain variable fragments (scFV). In embodiments, the antibody is an agonistic antibody. In embodiments, the antibody is a monoclonal antibody. In embodiments, the antibody is an anti-CD28 monoclonal antibody selected from JJ316, D665, 5.11A1, TGN1412, 37.51, E18, and PV-1. Poirier et al., CD28-Specific Immunomodulating Antibodies: What Can Be Learned From Experimental Models?, Am J Transplant 12(7):1682-90 (2012), which are hereby incorporated by reference in their entirety. In embodiments, the antibody is an agonistic antibody. In embodiments, the antibody is a monoclonal antibody. In embodiments, the antibody is an anti-NKG2D monoclonal antibody selected from tesnatilimab (also known as JNJ 4500 or JNJ-64304500) 149810, 1D11 and 5C6. DB1/ 145191832.2 43 Attorney Docket No.: SHK-081PC/116981-5081 In any of the embodiments disclosed herein, the second pharmaceutical composition comprises a heterologous chimeric protein capable of costimulating a receptor selected from CD28, NKG2D, CD27, CD30, 4-1BB (CD137), IL-2R, IL-15R, IL-7R, IL-21R, NKp30, NKp44, DNAM-1 (CD226), IL-2R, IL-7R, IL-15R, dectins, NLRs, killer Ig-like receptors (e.g., KIR2D, KIR3D), C-type lectins (CD94/NKG2A-C, NKG2D), LFA1, CD2, CD46, and Junctional Adhesion Molecule-Like (JAML) and/or inhibiting a receptor selected from a receptor selected from PD-1, PD-L1 and BTLA. In embodiments, the heterologous chimeric protein comprises (a) a first domain comprising an extracellular domain of a type I membrane protein; (b) a second domain comprising an extracellular domain of Type II transmembrane protein; and (c) a linker linking the first domain and the second domain. In embodiments, the linker comprises a hinge- CH2-CH3 Fc domain. Suitable heterologous chimeric proteins are disclosed in PCT publications WO 2017/059168, WO 2017/059168, WO 2017/059168, WO 2017/059168, WO 2018/157165, WO 2018/157165, WO 2020/047319, WO 2020/047322, WO 2020/047325, WO 2020/047327, WO 2020/047328, WO 2020/047329, WO 2020/176718, WO 2020/232365, and WO 2021/041958, which are hereby incorporated by reference in their entirety. In embodiments, the method comprises: administering to the subject the pharmaceutical composition comprising the heterodimeric protein of any of the embodiments disclosed herein. In embodiments, the method comprises: administering to the subject the pharmaceutical composition comprising the heterodimeric protein of any of the embodiments disclosed herein, and administering to the subject the second pharmaceutical composition. In embodiments, the pharmaceutical composition and the second pharmaceutical composition are administered simultaneously or contemporaneously. In embodiments, the pharmaceutical composition is administered after the second pharmaceutical composition is administered. In embodiments, the pharmaceutical composition is administered before the second pharmaceutical composition is administered. In embodiments, the method further comprises administering to the subject in need thereof gamma delta T cells that are proliferated ex vivo. In embodiments, the method comprises: contacting a pharmaceutical composition comprising the heterodimeric protein of any one of the embodiments disclosed herein, with a cell derived from a subject in need thereof thereby causing an ex vivo proliferation of gamma delta T cells, and administering to the subject in need thereof gamma delta T cells that are proliferated ex vivo. In embodiments, the method comprises: contacting cells derived from a subject in need thereof with a pharmaceutical composition comprising the heterodimeric protein of any one of the embodiments disclosed herein, and the second pharmaceutical composition that costimulates γδ T cells of any of the DB1/ 145191832.2 44 Attorney Docket No.: SHK-081PC/116981-5081 embodiments disclosed herein, thereby causing an ex vivo proliferation of gamma delta T cells, and administering to the subject in need thereof gamma delta T cells that are proliferated ex vivo. In embodiments, the cancer is a B cell lymphoma or leukemia. In embodiments, the cancer is an acute myeloid leukemia (AML). In embodiments, the cancer is a solid cancer selected from prostate cancer, small cell lung cancer, non-small cell lung cancer, neuroendocrine tumors, renal cancer, bladder cancer, colon cancer, and breast cancer. In embodiments, the cancer is known to express the antigenic target of the targeting domain disclosed herein. In embodiments, the cancer is suspected to express the antigenic target of the targeting domain disclosed herein. In one aspect, the present disclosure relates to a method of treating a cancer expressing CD20 in a subject in need thereof comprising: administering to the subject a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an in vivo proliferation of gamma delta T cells and/or contacting a cell derived from the subject with a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an ex vivo proliferation of gamma delta T cells. In one aspect, the present disclosure relates to a method of treating a cancer that is CD20 positive in a subject in need thereof comprising: administering to the subject a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an in vivo proliferation of gamma delta T cells and/or contacting a cell derived from the subject with a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an ex vivo proliferation of gamma delta T cells. In one aspect, the present disclosure relates to a method of treating a cancer that is CD20+, in a subject in need thereof comprising: administering to the subject a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an in vivo proliferation of gamma delta T cells and/or contacting a cell derived from the subject with a pharmaceutical composition of any of the embodiments disclosed herein, thereby causing an ex vivo proliferation of gamma delta T cells. In embodiments, the cancer is an epithelial-derived carcinoma. In embodiments, the cancer is known to express the antigenic target of the targeting domain. In embodiments, the cancer is known to express the antigenic target of the targeting domain. In embodiments, the cancer has mutations which limit recognition by alpha beta T cells, optionally selected from mutations in MHC I, beta 2 microglobulin, and Transporter associated with antigen processing (TAP). In embodiments, the subject is further administered autologous or allogeneic gamma delta T cells that were expanded ex vivo. In embodiments, the autologous or allogeneic gamma delta T cells express a Chimeric Antigen Receptor. In embodiments, the subject is further administered autologous or allogeneic T cells that express a Chimeric Antigen Receptor. DB1/ 145191832.2 45 Attorney Docket No.: SHK-081PC/116981-5081 In one aspect, the current disclosure provides a method of treating an autoimmune disease or disorder, comprising administering a pharmaceutical composition of any of the embodiments disclosed herein to a subject in need thereof, wherein the autoimmune disease or disorder is optionally selected from rheumatoid arthritis, systemic lupus erythematosus, diabetes mellitus, ankylosing spondylitis, Sjögren's syndrome, inflammatory bowel diseases (e.g., colitis ulcerosa, Crohn's disease), multiple sclerosis, sarcoidosis, psoriasis, Grave's disease, Hashimoto's thyroiditis, psoriasis, hypersensitivity reactions (e.g., allergies, hay fever, asthma, and acute edema cause type I hypersensitivity reactions), and pemphigus, and vasculitis (e.g., Granulomatosis with polyangiitis). In various embodiments, the current disclosure pertains to the use of the heterodimeric proteins for the treatment of one or more autoimmune diseases or disorders. In various embodiments, the treatment of an autoimmune disease or disorder may involve modulating the immune system with the present heterodimeric proteins to favor immune inhibition over immune stimulation. Illustrative autoimmune diseases or disorders treatable with the present heterodimeric proteins include those in which the body’s own antigens become targets for an immune response, such as, for example, rheumatoid arthritis, systemic lupus erythematosus, diabetes mellitus, ankylosing spondylitis, Sjögren's syndrome, inflammatory bowel diseases (e.g., colitis ulcerosa, Crohn's disease), multiple sclerosis, sarcoidosis, psoriasis, Grave's disease, Hashimoto's thyroiditis, psoriasis, hypersensitivity reactions (e.g., allergies, hay fever, asthma, and acute edema cause type I hypersensitivity reactions), and pemphigus, and vasculitis (e.g., Granulomatosis with polyangiitis). Illustrative autoimmune diseases or conditions that may be treated or prevented using the heterodimeric protein of the invention include, but are not limited to, 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, Grave's disease, and other autoimmune diseases. In various embodiments, the current disclosure pertains to cancers and/or tumors; for example, the treatment or prevention of cancers and/or tumors. As described elsewhere herein, the treatment of cancer may involve in various embodiments, modulating the immune system with the present heterodimeric proteins to favor immune stimulation over immune inhibition. DB1/ 145191832.2 46 Attorney Docket No.: SHK-081PC/116981-5081 Cancers or tumors refer to an uncontrolled growth of cells and/or abnormal increased cell survival and/or inhibition of apoptosis which interferes with the normal functioning of the bodily organs and systems. Included are benign and malignant cancers, polyps, hyperplasia, as well as dormant tumors or micrometastases. Also, included are cells having abnormal proliferation that is not impeded by the immune system (e.g., virus infected cells). The cancer may be a primary cancer or a metastatic cancer. The primary cancer may be an area of cancer cells at an originating site that becomes clinically detectable, and may be a primary tumor. In contrast, the metastatic cancer may be the spread of a disease from one organ or part to another non-adjacent organ or part. The metastatic cancer may be caused by a cancer cell that acquires the ability to penetrate and infiltrate surrounding normal tissues in a local area, forming a new tumor, which may be a local metastasis. The cancer may also be caused by a cancer cell that acquires the ability to penetrate the walls of lymphatic and/or blood vessels, after which the cancer cell is able to circulate through the bloodstream (thereby being a circulating tumor cell) to other sites and tissues in the body. The cancer may be due to a process such as lymphatic or hematogeneous spread. The cancer may also be caused by a tumor cell that comes to rest at another site, re-penetrates through the vessel or walls, continues to multiply, and eventually forms another clinically detectable tumor. The cancer may be this new tumor, which may be a metastatic (or secondary) tumor. The cancer may be caused by tumor cells that have metastasized, which may be a secondary or metastatic tumor. The cells of the tumor may be like those in the original tumor. As an example, if a breast cancer or colon cancer metastasizes to the liver, the secondary tumor, while present in the liver, is made up of abnormal breast or colon cells, not of abnormal liver cells. The tumor in the liver may thus be a metastatic breast cancer or a metastatic colon cancer, not liver cancer. The cancer may have an origin from any tissue. The cancer may originate from melanoma, colon, breast, or prostate, and thus may be made up of cells that were originally skin, colon, breast, or prostate, respectively. The cancer may also be a hematological malignancy, which may be leukemia or lymphoma. The cancer may invade a tissue such as liver, lung, bladder, or intestinal. In embodiments, the heterodimeric protein is used to treat a subject that has a treatment-refractory cancer. In embodiments, the heterodimeric protein is used to treat a subject that is refractory to one or more immune-modulating agents. For example, in embodiments, the heterodimeric protein is used to treat a subject that presents no response to treatment, or even progress, after 12 weeks or so of treatment. For instance, In embodiments, the subject is refractory to a PD-1 and/or PD-L1 and/or PD-L2 agent, including, for example, nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), pidilizumab (CT-011, CURE TECH), MK- DB1/ 145191832.2 47 Attorney Docket No.: SHK-081PC/116981-5081 3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), Ibrutinib (PHARMACYCLICS/ABBVIE), atezolizumab (TECENTRIQ, GENENTECH), and/or MPDL328OA (ROCHE)-refractory patients. For instance, In embodiments, the subject is refractory to an anti-CTLA-4 agent, e.g., ipilimumab (YERVOY)- refractory patients (e.g., melanoma patients). Accordingly, in various embodiments the current disclosure provides methods of cancer treatment that rescue patients that are non-responsive to various therapies, including monotherapy of one or more immune-modulating agents. In various embodiments, the current disclosure provides heterodimeric proteins, and methods of use of the same, which target a cell or tissue within the tumor microenvironment. In embodiments, the cell or tissue within the tumor microenvironment expresses one or more targets or binding partners of the heterodimeric protein. The tumor microenvironment refers to the cellular milieu, including cells, secreted proteins, physiological small molecules, and blood vessels in which the tumor exists. In embodiments, the cells or tissue within the tumor microenvironment are one or more of: tumor vasculature; tumor- infiltrating lymphocytes; fibroblast reticular cells; endothelial progenitor cells (EPC); cancer-associated fibroblasts; pericytes; other stromal cells; components of the extracellular matrix (ECM); dendritic cells; antigen presenting cells; T-cells; regulatory T cells; macrophages; neutrophils; and other immune cells located proximal to a tumor. In various embodiments, the present heterodimeric protein targets a cancer cell. In embodiments, the cancer cell expresses one or more of targets or binding partners of the heterodimeric protein. In various embodiments, the heterodimeric protein of the invention may target a cell (e.g., cancer cell or immune cell) that expresses any of the receptors as described herein. For example, the heterodimeric protein of the invention may target a cell that expresses any of the receptors for a cytokine, growth factor, and/or hormone as described herein. In embodiments, the present methods provide treatment with the heterodimeric protein in a patient who is refractory to an additional agent, such “additional agents” being described elsewhere herein, inclusive, without limitation, of the various chemotherapeutic agents described herein. In still another other aspect, the current disclosure is directed toward methods of treating and preventing T cell-mediated diseases and disorders, such as, but not limited to diseases or disorders described elsewhere herein and inflammatory disease or disorder, graft-versus-host disease (GVHD), transplant rejection, and T cell proliferative disorder. In some aspects, the present chimeric agents are used in methods of activating a T cell, e.g., via the extracellular domain having an immune stimulatory signal or antibody binding domain (e.g., CDR3, Fab, scFv domain, etc.) having an immune stimulatory signal. DB1/ 145191832.2 48 Attorney Docket No.: SHK-081PC/116981-5081 In some aspects, the present chimeric agents are used in methods of preventing the cellular transmission of an immunosuppressive signal. In one aspect, the present disclosure relates to a method of contemporaneous activation and targeting of gamma delta T cells to cancer cells comprising administering to a subject in need thereof a pharmaceutical composition of any one of the embodiments disclosed herein. In one aspect, the present disclosure relates to a method of modulating a patient’s immune response, comprising administering a pharmaceutical composition of any one of the embodiments disclosed herein to a subject in need thereof. In one aspect, the present disclosure relates to a method of stimulating proliferation of gamma delta T cells, comprising: administering a pharmaceutical composition of any one of the embodiments disclosed herein, to a subject in need thereof thereby causing an in vivo proliferation of gamma delta T cells and/or contacting a pharmaceutical composition of any one of the embodiments disclosed herein, with a cell derived from a subject in need thereof thereby causing an ex vivo proliferation of gamma delta T cells. In embodiments, the subject’s T cells are activated by the first domain. In embodiments, the subject has a cancer and the gamma delta T cells modulate cells of the tumor. In embodiments, the method further comprises administering to the subject a second pharmaceutical composition that costimulates γδ T cells, or contacting a second pharmaceutical composition that costimulates γδ T cells, with a cell derived from a subject in need thereof thereby causing an ex vivo proliferation of gamma delta T cells. In embodiments, the method comprises administering to the subject the pharmaceutical composition. In embodiments, the method comprises: administering to the subject the pharmaceutical composition, and administering to the subject the second pharmaceutical composition. In embodiments, the pharmaceutical composition and the second pharmaceutical composition are administered simultaneously or contemporaneously. In embodiments, the pharmaceutical composition is administered after the second pharmaceutical composition is administered. In embodiments, the pharmaceutical composition is administered before the second pharmaceutical composition is administered. Combination Therapies and Conjugation In embodiments, the invention provides for heterodimeric proteins, and methods of use of the same, and methods that further comprise administering an additional agent to a subject. In embodiments, the invention pertains to co-administration and/or co-formulation. Any of the compositions described herein may be co-formulated and/or co-administered. DB1/ 145191832.2 49 Attorney Docket No.: SHK-081PC/116981-5081 In embodiments, any heterodimeric protein described herein acts synergistically when co-administered with another agent and is administered at doses that are lower than the doses commonly employed when such agents are used as monotherapy. In various embodiments, any agent referenced herein may be used in combination with any of the heterodimeric proteins, and methods of use of the same, described herein. In various embodiments, any of the heterodimeric proteins disclosed herein may be co-administered with another heterodimeric protein disclosed herein. Without wishing to be bound by theory, it is believed that a combined regimen involving the administration of one or more heterodimeric proteins which induce an innate immune response and one or more heterodimeric proteins which induce an adaptive immune response may provide synergistic effects (e.g., synergistic anti-tumor effects). In various embodiments, any heterodimeric protein which induces an innate immune response may be utilized in the current disclosure. In various embodiments, any heterodimeric protein which induces an adaptive immune response may be utilized in the current disclosure. In embodiments, inclusive of, without limitation, cancer applications, the current disclosure pertains to chemotherapeutic agents as additional agents. Examples of chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and CYTOXAN cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogenins (e.g., bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (e.g., cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB 1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo- 5-oxo-L-norleucine, ADRIAMYCIN doxorubicin (including morpholino- doxorubicin, cyanomorpholino- DB1/ 145191832.2 50 Attorney Docket No.: SHK-081PC/116981-5081 doxorubicin, 2-pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as minoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (e.g., T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL paclitaxel (Bristol- Myers Squibb Oncology, Princeton, N.J.), ABRAXANE Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, 111.), and TAXOTERE doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE. vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including the treatment regimen of irinotecan with 5-FU and leucovorin); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including the oxaliplatin treatment regimen (FOLFOX); lapatinib (TYKERB); inhibitors of PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib (Tarceva)) and VEGF-A that reduce cell proliferation and pharmaceutically acceptable salts, acids or derivatives of any of the above. In addition, the methods of treatment can further include the use of radiation. In addition, the methods of treatment can further include the use of photodynamic therapy. In various embodiments, inclusive of, without limitation, cancer applications, the present additional agent is one or more immune-modulating agents selected from an agent that blocks, reduces and/or inhibits DB1/ 145191832.2 51 Attorney Docket No.: SHK-081PC/116981-5081 PD-1 and PD-L1 or PD-L2 and/or the binding of PD-1 with PD-L1 or PD-L2 (by way of non-limiting example, one or more of nivolumab (ONO-4538/BMS-936558, MDX1106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, Merck), MK-3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), atezolizumab (TECENTRIQ, GENENTECH), MPDL328OA (ROCHE), an agent that increases and/or stimulates CD137 (4-1BB) and/or the binding of CD137 (4-1BB) with one or more of 4-1BB ligand (by way of non-limiting example, urelumab (BMS-663513 and anti-4-1BB antibody), and an agent that blocks, reduces and/or inhibits the activity of CTLA-4 and/or the binding of CTLA-4 with one or more of AP2M1, CD80, CD86, SHP-2, and PPP2R5A and/or the binding of OX40 with OX40L (by way of non- limiting example GBR 830 (GLENMARK), MEDI6469 (MEDIMMUNE). In embodiments, inclusive of, without limitation, infectious disease applications, the current disclosure pertains to anti-infectives as additional agents. In embodiments, the anti-infective is an anti-viral agent including, but not limited to, Abacavir, Acyclovir, Adefovir, Amprenavir, Atazanavir, Cidofovir, Darunavir, Delavirdine, Didanosine, Docosanol, Efavirenz, Elvitegravir, Emtricitabine, Enfuvirtide, Etravirine, Famciclovir, and Foscarnet. In embodiments, the anti-infective is an anti-bacterial agent including, but not limited to, cephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, and ceftobiprole); fluoroquinolone antibiotics (cipro, Levaquin, floxin, tequin, avelox, and norflox); tetracycline antibiotics (tetracycline, minocycline, oxytetracycline, and doxycycline); penicillin antibiotics (amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin, and methicillin); monobactam antibiotics (aztreonam); and carbapenem antibiotics (ertapenem, doripenem, imipenem/cilastatin, and meropenem). In embodiments, the anti-infectives include anti-malarial agents (e.g., chloroquine, quinine, mefloquine, primaquine, doxycycline, artemether/lumefantrine, atovaquone/proguanil and sulfadoxine/pyrimethamine), metronidazole, tinidazole, ivermectin, pyrantel pamoate, and albendazole. In embodiments, inclusive, without limitation, of autoimmune applications, the additional agent is an immunosuppressive agent. In embodiments, the immunosuppressive agent is an anti-inflammatory agent such as a steroidal anti-inflammatory agent or a non-steroidal anti-inflammatory agent (NSAID). Steroids, particularly the adrenal corticosteroids and their synthetic analogues, are well known in the art. Examples of corticosteroids useful in the current disclosure include, without limitation, 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 DB1/ 145191832.2 52 Attorney Docket No.: SHK-081PC/116981-5081 acetate, hydrocortisone butyrate, methylprednisolone, triamcinolone acetonide, cortisone, cortodoxone, flucetonide, fludrocortisone, difluorosone diacetate, fluradrenolone acetonide, medrysone, amcinafel, amcinafide, betamethasone and the balance of its esters, chloroprednisone, clocortelone, clescinolone, dichlorisone, difluprednate, flucloronide, flunisolide, fluoromethalone, fluperolone, fluprednisolone, hydrocortisone, meprednisone, paramethasone, prednisolone, prednisone, beclomethasone dipropionate. (NSAIDS) that may be used in the current disclosure, include but are not limited to, salicylic acid, acetyl salicylic acid, methyl salicylate, glycol salicylate, salicylmides, benzyl-2,5-diacetoxybenzoic acid, ibuprofen, fulindac, naproxen, ketoprofen, etofenamate, phenylbutazone, and indomethacin. In embodiments, the immunosupressive agent may be cytostatics such as alkylating agents, antimetabolites (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). In embodiments, the heterodimeric proteins (and/or additional agents) described herein, include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the composition such that covalent attachment does not prevent the activity of the composition. For example, but not by way of limitation, derivatives include composition that have been modified by, inter alia, glycosylation, lipidation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications can be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of turicamycin, etc. Additionally, the derivative can contain one or more non-classical amino acids. In still other embodiments, the heterodimeric proteins (and/or additional agents) described herein further comprise a cytotoxic agent, comprising, in illustrative embodiments, a toxin, a chemotherapeutic agent, a radioisotope, and an agent that causes apoptosis or cell death. Such agents may be conjugated to a composition described herein. The heterodimeric proteins (and/or additional agents) described herein may thus be modified post- translationally to add effector moieties such as chemical linkers, detectable moieties such as for example fluorescent dyes, enzymes, substrates, bioluminescent materials, radioactive materials, and chemiluminescent moieties, or functional moieties such as for example streptavidin, avidin, biotin, a cytotoxin, a cytotoxic agent, and radioactive materials. Formulations In one aspect, the present disclosure relates to a pharmaceutical composition, comprising the heterodimeric protein of any one of the embodiments disclosed herein, and a carrier. DB1/ 145191832.2 53 Attorney Docket No.: SHK-081PC/116981-5081 The heterodimeric proteins (and/or additional agents) described herein can possess a sufficiently basic functional group, which can react with an inorganic or organic acid, or a carboxyl group, which can react with an inorganic or organic base, to form a pharmaceutically acceptable salt. A pharmaceutically acceptable acid addition salt is formed from a pharmaceutically acceptable acid, as is well known in the art. Such salts include the pharmaceutically acceptable salts listed in, for example, Journal of Pharmaceutical Science, 66, 2-19 (1977) and The Handbook of Pharmaceutical Salts; Properties, Selection, and Use. P. H. Stahl and C. G. Wermuth (eds.), Verlag, Zurich (Switzerland) 2002, which are hereby incorporated by reference in their entirety. In embodiments, the compositions described herein are in the form of a pharmaceutically acceptable salt. Further, any heterodimeric protein (and/or additional agents) described herein can be administered to a subject as a component of a composition that comprises a pharmaceutically acceptable carrier or vehicle. Such compositions can optionally comprise a suitable amount of a pharmaceutically acceptable excipient so as to provide the form for proper administration. Pharmaceutical excipients can be liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The pharmaceutical excipients can be, for example, saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea and the like. In addition, auxiliary, stabilizing, thickening, lubricating, and coloring agents can be used. In one embodiment, the pharmaceutically acceptable excipients are sterile when administered to a subject. Water is a useful excipient when any agent described herein is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid excipients, specifically for injectable solutions. Suitable pharmaceutical excipients also include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. Any agent described herein, if desired, can also comprise minor amounts of wetting or emulsifying agents, or pH buffering agents. In embodiments, the compositions described herein are resuspended in a saline buffer (including, without limitation TBS, PBS, and the like). In various embodiments, the heterodimeric proteins may by conjugated and/or fused with another agent to extend half-life or otherwise improve pharmacodynamic and pharmacokinetic properties. In embodiments, the heterodimeric proteins may be fused or conjugated with one or more of PEG, XTEN (e.g., as rPEG), polysialic acid (POLYXEN), albumin (e.g., human serum albumin or HAS), elastin-like protein (ELP), PAS, HAP, GLK, CTP, transferrin, and the like. In various embodiments, each of the DB1/ 145191832.2 54 Attorney Docket No.: SHK-081PC/116981-5081 individual heterodimeric proteins is fused to one or more of the agents described in BioDrugs (2015) 29:215–239, the entire contents of which are hereby incorporated by reference. Administration, Dosing, and Treatment Regimens The current disclosure includes the described heterodimeric protein (and/or additional agents) in various formulations. Any heterodimeric protein (and/or additional agents) described herein can take the form of solutions, suspensions, emulsion, drops, tablets, pills, pellets, capsules, capsules containing liquids, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, or any other form suitable for use. DNA or RNA constructs encoding the protein sequences may also be used. In one embodiment, the composition is in the form of a capsule (see, e.g., U.S. Patent No. 5,698,155). Other examples of suitable pharmaceutical excipients are described in Remington’s Pharmaceutical Sciences 1447-1676 (Alfonso R. Gennaro eds., 19th ed.1995), incorporated herein by reference. Where necessary, the formulations comprising the heterodimeric protein (and/or additional agents) can also include a solubilizing agent. Also, the agents can be delivered with a suitable vehicle or delivery device as known in the art. Combination therapies outlined herein can be co-delivered in a single delivery vehicle or delivery device. Compositions for administration can optionally include a local anesthetic such as, for example, lignocaine to lessen pain at the site of the injection. The formulations comprising the heterodimeric protein (and/or additional agents) of the current disclosure may conveniently be presented in unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods generally include the step of bringing the therapeutic agents into association with a carrier, which constitutes one or more accessory ingredients. Typically, the formulations are prepared by uniformly and intimately bringing the therapeutic agent into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into dosage forms of the desired formulation (e.g., wet or dry granulation, powder blends, etc., followed by tableting using conventional methods known in the art). In one embodiment, any heterodimeric protein (and/or additional agents) described herein is formulated in accordance with routine procedures as a composition adapted for a mode of administration described herein. Routes of administration include, for example: intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectally, by inhalation, or topically, particularly to the ears, nose, eyes, or skin. In embodiments, the DB1/ 145191832.2 55 Attorney Docket No.: SHK-081PC/116981-5081 administering is effected orally or by parenteral injection. In most instances, administration results in the release of any agent described herein into the bloodstream. Any heterodimeric protein (and/or additional agents) described herein can be administered orally. Such heterodimeric proteins (and/or additional agents) can also be administered by any other convenient route, for example, by intravenous infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and can be administered together with another biologically active agent. Administration can be systemic or local. Various delivery systems are known, e.g., encapsulation in liposomes, microparticles, microcapsules, capsules, etc., and can be used to administer. In specific embodiments, it may be desirable to administer locally to the area in need of treatment. In one embodiment, for instance in the treatment of cancer, the heterodimeric protein (and/or additional agents) are administered in the tumor microenvironment (e.g., cells, molecules, extracellular matrix and/or blood vessels that surround and/or feed a tumor cell, inclusive of, for example, tumor vasculature; tumor- infiltrating lymphocytes; fibroblast reticular cells; endothelial progenitor cells (EPC); cancer-associated fibroblasts; pericytes; other stromal cells; components of the extracellular matrix (ECM); dendritic cells; antigen presenting cells; T-cells; regulatory T cells; macrophages; neutrophils; and other immune cells located proximal to a tumor) or lymph node and/or targeted to the tumor microenvironment or lymph node. In various embodiments, for instance in the treatment of cancer, the heterodimeric protein (and/or additional agents) are administered intratumorally. In the various embodiments, the present heterodimeric protein allows for a dual effect that provides less side effects than are seen in conventional immunotherapy (e.g., treatments with one or more of OPDIVO, KEYTRUDA, YERVOY, and TECENTRIQ). For example, the present heterodimeric proteins reduce or prevent commonly observed immune-related adverse events that affect various tissues and organs including the skin, the gastrointestinal tract, the kidneys, peripheral and central nervous system, liver, lymph nodes, eyes, pancreas, and the endocrine system; such as hypophysitis, colitis, hepatitis, pneumonitis, rash, and rheumatic disease. Further, the present local administration, e.g., intratumorally, obviate adverse event seen with standard systemic administration, e.g., IV infusions, as are used with conventional immunotherapy (e.g., treatments with one or more of OPDIVO, KEYTRUDA, YERVOY, and TECENTRIQ). Dosage forms suitable for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal, subcutaneous and intra-articular injection and infusion) include, for example, solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid DB1/ 145191832.2 56 Attorney Docket No.: SHK-081PC/116981-5081 compositions (e.g., lyophilized composition), which can be dissolved or suspended in sterile injectable medium immediately before use. They may contain, for example, suspending or dispersing agents known in the art. The dosage of any heterodimeric protein (and/or additional agents) described herein as well as the dosing schedule can depend on various parameters, including, but not limited to, the disease being treated, the subject’s general health, and the administering physician’s discretion. Any heterodimeric protein described herein, can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concurrently with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of an additional agent, to a subject in need thereof. In various embodiments any heterodimeric protein and additional agent described herein are administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11 hours to 12 hours apart, 1 day apart, 2 days apart, 3 days apart, 4 days apart, 5 days apart, 6 days apart, 1 week apart, 2 weeks apart, 3 weeks apart, or 4 weeks apart. In embodiments, the heterodimeric protein is administered at a frequency of about 1 day, or about 2 days, or about 3 days, or about 4 days, or about 5 days, or about 6 days, or about one week, or about 10 days, or about 15 days, or about 21 days, or more. In embodiments, the heterodimeric protein is administered at a frequency of about 3 days. In embodiments, the heterodimeric protein is administered at a frequency of about 7 days. In embodiments, the heterodimeric protein is administered at a frequency of about 1 day to about 10 days, about 1 day to about 5 days, or about 2 days to about 10 days, or about 5 days to about 15 days, or about one week to about 3 weeks. In embodiments, the heterodimeric protein is administered at a frequency of about 1 day to about 10 days. In embodiments, the heterodimeric protein is administered so as to form a ratio of effector cells (heterodimeric protein-bound Gamma-Delta T cells) to target cells (without limitations, e.g., B cells or CD20+ cancer cells) is about 1:1, or about 1:2, or about 1:5, or about 1:7, or about 1:10, or about 1:15, or about 1:20 or less. In embodiments, the heterodimeric protein is administered so as to form a ratio of effector cells (heterodimeric protein-bound Gamma-Delta T cells) to target cells (without limitations, e.g., B cells or CD20+ cancer cells) is about 1:1 to about 1:5, or about 1:2 to about 1:8, or about 1:10 to about 1: 20. DB1/ 145191832.2 57 Attorney Docket No.: SHK-081PC/116981-5081 In various embodiments, the current disclosure relates to the co-administration of a heterodimeric protein which induces an innate immune response and another heterodimeric protein which induces an adaptive immune response. In such embodiments, the heterodimeric protein which induces an innate immune response may be administered before, concurrently with, or subsequent to administration of the heterodimeric protein which induces an adaptive immune response. For example, the heterodimeric proteins may be administered 1 minute apart, 10 minutes apart, 30 minutes apart, less than 1 hour apart, 1 hour apart, 1 hour to 2 hours apart, 2 hours to 3 hours apart, 3 hours to 4 hours apart, 4 hours to 5 hours apart, 5 hours to 6 hours apart, 6 hours to 7 hours apart, 7 hours to 8 hours apart, 8 hours to 9 hours apart, 9 hours to 10 hours apart, 10 hours to 11 hours apart, 11 hours to 12 hours apart, 1 day apart, 2 days apart, 3 days apart, 4 days apart, 5 days apart, 6 days apart, 1 week apart, 2 weeks apart, 3 weeks apart, or 4 weeks apart. In an illustrative embodiment, the heterodimeric protein which induces an innate immune response and the heterodimeric protein which induces an adaptive response are administered 1 week apart, or administered on alternate weeks (i.e., administration of the heterodimeric protein inducing an innate immune response is followed 1 week later with administration of the heterodimeric protein which induces an adaptive immune response and so forth). The dosage of any heterodimeric protein (and/or additional agents) described herein can depend on several factors including the severity of the condition, whether the condition is to be treated or prevented, and the age, weight, and health of the subject to be treated. Additionally, pharmacogenomic (the effect of genotype on the pharmacokinetic, pharmacodynamic or efficacy profile of a therapeutic) information about a particular subject may affect dosage used. Furthermore, the exact individual dosages can be adjusted somewhat depending on a variety of factors, including the specific combination of the agents being administered, the time of administration, the route of administration, the nature of the formulation, the rate of excretion, the particular disease being treated, the severity of the disorder, and the anatomical location of the disorder. Some variations in the dosage can be expected. For administration of any heterodimeric protein (and/or additional agents) described herein by parenteral injection, the dosage may be about 0.1 mg to about 250 mg per day, about 1 mg to about 20 mg per day, or about 3 mg to about 5 mg per day. Generally, when orally or parenterally administered, the dosage of any agent described herein may be about 0.1 mg to about 1500 mg per day, or about 0.5 mg to about 10 mg per day, or about 0.5 mg to about 5 mg per day, or about 200 to about 1,200 mg per day (e.g., about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1,000 mg, about 1,100 mg, about 1,200 mg per day). In embodiments, administration of the heterodimeric protein (and/or additional agents) described herein is by parenteral injection at a dosage of about 0.1 mg to about 1500 mg per treatment, or about 0.5 mg DB1/ 145191832.2 58 Attorney Docket No.: SHK-081PC/116981-5081 to about 10 mg per treatment, or about 0.5 mg to about 5 mg per treatment, or about 200 to about 1,200 mg per treatment (e.g., about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1,000 mg, about 1,100 mg, about 1,200 mg per treatment). In embodiments, a suitable dosage of the heterodimeric protein (and/or additional agents) is in a range of about 0.01 mg/kg to about 100 mg/kg of body weight ,or about 0.01 mg/kg to about 10 mg/kg of body weight of the subject, for example, about 0.01 mg/kg, about 0.02 mg/kg, about 0.03 mg/kg, about 0.04 mg/kg, about 0.05 mg/kg, about 0.06 mg/kg, about 0.07 mg/kg, about 0.08 mg/kg, about 0.09 mg/kg, about 0.1 mg/kg, about 0.2 mg/kg, about 0.3 mg/kg, about 0.4 mg/kg, about 0.5 mg/kg, about 0.6 mg/kg, about 0.7 mg/kg, about 0.8 mg/kg, about 0.9 mg/kg, about 1 mg/kg, about 1.1 mg/kg, about 1.2 mg/kg, about 1.3 mg/kg, about 1.4 mg/kg, about 1.5 mg/kg, about 1.6 mg/kg, about 1.7 mg/kg, about 1.8 mg/kg, 1.9 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg body weight, inclusive of all values and ranges therebetween. In another embodiment, delivery can be in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez- Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989). Any heterodimeric protein (and/or additional agents) described herein can be administered by controlled- release or sustained-release means or by delivery devices that are well known to those of ordinary skill in the art. Examples include, but are not limited to, those described in U.S. Patent Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5,674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; and 5,733,556, each of which is incorporated herein by reference in its entirety. Such dosage forms can be useful for providing controlled- or sustained-release of one or more active ingredients using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or a combination thereof to provide the desired release profile in varying proportions. Controlled- or sustained- release of an active ingredient can be stimulated by various conditions, including but not limited to, changes in pH, changes in temperature, stimulation by an appropriate wavelength of light, concentration or availability of enzymes, concentration or availability of water, or other physiological conditions or compounds. In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and DB1/ 145191832.2 59 Attorney Docket No.: SHK-081PC/116981-5081 Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem.23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol.25:351; Howard et al., 1989, J. Neurosurg.71:105). In another embodiment, a controlled-release system can be placed in proximity of the target area to be treated, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol.2, pp.115-138 (1984)). Other controlled-release systems discussed in the review by Langer, 1990, Science 249:1527-1533) may be used. Administration of any heterodimeric protein (and/or additional agents) described herein can, independently, be one to four times daily or one to four times per month or one to six times per year or once every two, three, four or five years. Administration can be for the duration of one day or one month, two months, three months, six months, one year, two years, three years, and may even be for the life of the subject. The dosage regimen utilizing any heterodimeric protein (and/or additional agents) described herein can be selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the subject; the severity of the condition to be treated; the route of administration; the renal or hepatic function of the subject; the pharmacogenomic makeup of the individual; and the specific compound of the invention employed. Any heterodimeric protein (and/or additional agents) described herein can be administered in a single daily dose, or the total daily dosage can be administered in divided doses of two, three or four times daily. Furthermore, any heterodimeric protein (and/or additional agents) described herein can be administered continuously rather than intermittently throughout the dosage regimen. Cells and Nucleic Acids In one aspect, the present disclosure relates to a polynucleotide encoding the alpha chain and/or beta chain of the heterodimeric protein of any one of the embodiments disclosed herein. In embodiments, the nucleic acid is DNA or RNA. In embodiments, the RNA is mRNA, which is optionally modified mRNA (mmRNA). In one aspect, the present disclosure relates to an expression vector, comprising a nucleic acid encoding the alpha chain and/or beta chain of the heterodimeric protein of any one of the embodiments disclosed herein. In embodiments, the expression vector is a mammalian expression vector. In embodiments, the expression vector comprises DNA or RNA. In one aspect, the present disclosure relates to a host cell, comprising the expression vector of any one of the embodiments disclosed herein, or the polynucleotide of the embodiments disclosed herein. DB1/ 145191832.2 60 Attorney Docket No.: SHK-081PC/116981-5081 In one aspect, the current disclosure provides an expression vector, comprising a nucleic acid encoding the first and/or second polypeptide chains of the heterodimeric protein of any of any of the embodiments disclosed herein. In embodiments, the expression vector is a mammalian expression vector. In embodiments, the expression vector comprises DNA or RNA. In one aspect, the current disclosure provides a host cell comprising the expression vector of any one of the embodiments disclosed herein. In various embodiments, the current disclosure provides an expression vector, comprising a nucleic acid encoding the heterodimeric protein (e.g., a heterodimeric protein comprising a first and second polypeptide chains) described herein. In various embodiments, the expression vector comprises DNA or RNA. In various embodiments, the expression vector is a mammalian expression vector. Both prokaryotic and eukaryotic vectors can be used for expression of the heterodimeric protein. Prokaryotic vectors include constructs based on E. coli sequences (see, e.g., Makrides, Microbiol Rev 1996, 60:512-538). Non-limiting examples of regulatory regions that can be used for expression in E. coli include lac, trp, lpp, phoA, recA, tac, T3, T7 and λPL. Non-limiting examples of prokaryotic expression vectors may include the λgt vector series such as λgt11 (Huynh et al., in “DNA Cloning Techniques, Vol. I: A Practical Approach,” 1984, (D. Glover, ed.), pp.49-78, IRL Press, Oxford), and the pET vector series (Studier et al., Methods Enzymol 1990, 185:60-89). Prokaryotic host-vector systems cannot perform much of the post-translational processing of mammalian cells, however. Thus, eukaryotic host- vector systems may be particularly useful. A variety of regulatory regions can be used for expression of the heterodimeric proteins in mammalian host cells. For example, the SV40 early and late promoters, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat (RSV-LTR) promoter can be used. Inducible promoters that may be useful in mammalian cells include, without limitation, promoters associated with the metallothionein II gene, mouse mammary tumor virus glucocorticoid responsive long terminal repeats (MMTV-LTR), the β-interferon gene, and the hsp70 gene (see, Williams et al., Cancer Res 1989, 49:2735-42; and Taylor et al., Mol Cell Biol 1990, 10:165-75). Heat shock promoters or stress promoters also may be advantageous for driving expression of the fusion proteins in recombinant host cells. In embodiments, expression vectors of the invention comprise a nucleic acid encoding at least the first and/or second polypeptide chains of the heterodimeric proteins (and/or additional agents), or a complement thereof, operably linked to an expression control region, or complement thereof, that is functional in a mammalian cell. The expression control region is capable of driving expression of the operably linked blocking and/or stimulating agent encoding nucleic acid such that the blocking and/or stimulating agent is produced in a human cell transformed with the expression vector. DB1/ 145191832.2 61 Attorney Docket No.: SHK-081PC/116981-5081 Expression control regions are regulatory polynucleotides (sometimes referred to herein as elements), such as promoters and enhancers, that influence expression of an operably linked nucleic acid. An expression control region of an expression vector of the invention is capable of expressing operably linked encoding nucleic acid in a human cell. In an embodiment, the cell is a tumor cell. In another embodiment, the cell is a non-tumor cell. In an embodiment, the expression control region confers regulatable expression to an operably linked nucleic acid. A signal (sometimes referred to as a stimulus) can increase or decrease expression of a nucleic acid operably linked to such an expression control region. Such expression control regions that increase expression in response to a signal are often referred to as inducible. Such expression control regions that decrease expression in response to a signal are often referred to as repressible. Typically, the amount of increase or decrease conferred by such elements is proportional to the amount of signal present; the greater the amount of signal, the greater the increase or decrease in expression. In an embodiment, the current disclosure contemplates the use of inducible promoters capable of effecting high level of expression transiently in response to a cue. For example, when in the proximity of a tumor cell, a cell transformed with an expression vector for the heterodimeric protein (and/or additional agents) comprising such an expression control sequence is induced to transiently produce a high level of the agent by exposing the transformed cell to an appropriate cue. Illustrative inducible expression control regions include those comprising an inducible promoter that is stimulated with a cue such as a small molecule chemical compound. Particular examples can be found, for example, in U.S. Pat. Nos. 5,989,910, 5,935,934, 6,015,709, and 6,004,941, each of which is incorporated herein by reference in its entirety. Expression control regions and locus control regions include full-length promoter sequences, such as native promoter and enhancer elements, as well as subsequences or polynucleotide variants which retain all or part of full-length or non-variant function. As used herein, the term "functional" and grammatical variants thereof, when used in reference to a nucleic acid sequence, subsequence or fragment, means that the sequence has one or more functions of native nucleic acid sequence (e.g., non-variant or unmodified sequence). As used herein, “operable linkage” refers to a physical juxtaposition of the components so described as to permit them to function in their intended manner. In the example of an expression control element in operable linkage with a nucleic acid, the relationship is such that the control element modulates expression of the nucleic acid. Typically, an expression control region that modulates transcription is juxtaposed near the 5' end of the transcribed nucleic acid (i.e., “upstream”). Expression control regions can also be located at the 3’ end of the transcribed sequence (i.e., “downstream”) or within the transcript DB1/ 145191832.2 62 Attorney Docket No.: SHK-081PC/116981-5081 (e.g., in an intron). Expression control elements can be located at a distance away from the transcribed sequence (e.g., 100 to 500, 500 to 1000, 2000 to 5000, or more nucleotides from the nucleic acid). A specific example of an expression control element is a promoter, which is usually located 5' of the transcribed sequence. Another example of an expression control element is an enhancer, which can be located 5' or 3' of the transcribed sequence, or within the transcribed sequence. Expression systems functional in human cells are well known in the art, and include viral systems. Generally, a promoter functional in a human cell is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequence into mRNA. A promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and typically a TATA box located 25-30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct RNA polymerase II to begin RNA synthesis at the correct site. A promoter will also typically contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation. Of particular use as promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter. Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3’ terminus of the mature mRNA is formed by site-specific post- translational cleavage and polyadenylation. Examples of transcription terminator and polyadenylation signals include those derived from SV40. Introns may also be included in expression constructs. There are a variety of techniques available for introducing nucleic acids into viable cells. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, polymer-based systems, DEAE-dextran, viral transduction, the calcium phosphate precipitation method, etc. For in vivo gene transfer, a number of techniques and reagents may also be used, including liposomes; natural polymer-based delivery vehicles, such as chitosan and gelatin; viral vectors are also suitable for in vivo transduction. In some situations, it is desirable to provide a targeting agent, such as an antibody or ligand specific for a tumor cell surface membrane protein. Where liposomes are employed, proteins which bind to a cell surface membrane protein associated with endocytosis may be used for targeting and/or to facilitate uptake, e.g., capsid proteins or fragments thereof tropic for a particular cell type, antibodies for proteins which undergo DB1/ 145191832.2 63 Attorney Docket No.: SHK-081PC/116981-5081 internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis is described, for example, by Wu et al., J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990). Where appropriate, gene delivery agents such as, e.g., integration sequences can also be employed. Numerous integration sequences are known in the art (see, e.g., Nunes-Duby et al., Nucleic Acids Res. 26:391-406, 1998; Sadwoski, J. Bacteriol., 165:341-357, 1986; Bestor, Cell, 122(3):322-325, 2005; Plasterk et al., TIG 15:326-332, 1999; Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). These include recombinases and transposases. Examples include Cre (Sternberg and Hamilton, J. Mol. Biol., 150:467-486, 1981), lambda (Nash, Nature, 247, 543-545, 1974), FIp (Broach, et al., Cell, 29:227- 234, 1982), R (Matsuzaki, et al., J. Bacteriology, 172:610-618, 1990), cpC31 (see, e.g., Groth et al., J. Mol. Biol. 335:667-678, 2004), sleeping beauty, transposases of the mariner family (Plasterk et al., supra), and components for integrating viruses such as AAV, retroviruses, and antiviruses having components that provide for virus integration such as the LTR sequences of retroviruses or lentivirus and the ITR sequences of AAV (Kootstra et al., Ann. Rev. Pharm. Toxicol., 43:413-439, 2003). In addition, direct and targeted genetic integration strategies may be used to insert nucleic acid sequences encoding the chimeric fusion proteins including CRISPR/CAS9, zinc finger, TALEN, and meganuclease gene- editing technologies. In one aspect, the invention provides expression vectors for the expression of the heterodimeric proteins (and/or additional agents) that are viral vectors. Many viral vectors useful for gene therapy are known (see, e.g., Lundstrom, Trends Biotechnol., 21: 117, 122, 2003. Illustrative viral vectors include those selected from Antiviruses (LV), retroviruses (RV), adenoviruses (AV), adeno-associated viruses (AAV), and α viruses, though other viral vectors may also be used. For in vivo uses, viral vectors that do not integrate into the host genome are suitable for use, such as α viruses and adenoviruses. Illustrative types of α viruses include Sindbis virus, Venezuelan equine encephalitis (VEE) virus, and Semliki Forest virus (SFV). For in vitro uses, viral vectors that integrate into the host genome are suitable, such as retroviruses, AAV, and Antiviruses. In one embodiment, the invention provides methods of transducing a human cell in vivo, comprising contacting a solid tumor in vivo with a viral vector of the invention. In various embodiments, the current disclosure provides a host cell, comprising the expression vector comprising the heterodimeric protein described herein. Expression vectors can be introduced into host cells for producing the present heterodimeric proteins. Cells may be cultured in vitro or genetically engineered, for example. Useful mammalian host cells include, without limitation, cells derived from humans, monkeys, and rodents (see, for example, Kriegler DB1/ 145191832.2 64 Attorney Docket No.: SHK-081PC/116981-5081 in “Gene Transfer and Expression: A Laboratory Manual,” 1990, New York, Freeman & Co.). These include monkey kidney cell lines transformed by SV40 (e.g., COS-7, ATCC CRL 1651); human embryonic kidney lines (e.g., 293, 293-EBNA, or 293 cells subcloned for growth in suspension culture, Graham et al., J Gen Virol 1977, 36:59); baby hamster kidney cells (e.g., BHK, ATCC CCL 10); Chinese hamster ovary-cells-DHFR (e.g., CHO, Urlaub and Chasin, Proc Natl Acad Sci USA 1980, 77:4216); DG44 CHO cells, CHO-K1 cells, mouse sertoli cells (Mather, Biol Reprod 1980, 23:243-251); mouse fibroblast cells (e.g., NIH-3T3), monkey kidney cells (e.g., CV1 ATCC CCL 70); African green monkey kidney cells. (e.g., VERO-76, ATCC CRL-1587); human cervical carcinoma cells (e.g., HELA, ATCC CCL 2); canine kidney cells (e.g., MDCK, ATCC CCL 34); buffalo rat liver cells (e.g., BRL 3A, ATCC CRL 1442); human lung cells (e.g., W138, ATCC CCL 75); human liver cells (e.g., Hep G2, HB 8065); and mouse mammary tumor cells (e.g., MMT 060562, ATCC CCL51). Illustrative cancer cell types for expressing the fusion proteins described herein include mouse fibroblast cell line, NIH3T3, mouse Lewis lung carcinoma cell line, LLC, mouse mastocytoma cell line, P815, mouse lymphoma cell line, EL4 and its ovalbumin transfectant, E.G7, mouse melanoma cell line, B16F10, mouse fibrosarcoma cell line, MC57, and human small cell lung carcinoma cell lines, SCLC#2 and SCLC#7. Host cells can be obtained from normal or affected subjects, including healthy humans, cancer patients, and patients with an infectious disease, private laboratory deposits, public culture collections such as the American Type Culture Collection, or from commercial suppliers. Cells that can be used for production of the present heterodimeric proteins in vitro, ex vivo, and/or in vivo include, without limitation, epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T lymphocytes, B lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells (e.g., as obtained from bone marrow), umbilical cord blood, peripheral blood, fetal liver, etc. The choice of cell type depends on the type of tumor or infectious disease being treated or prevented, and can be determined by one of skill in the art. Production and purification of Fc-containing macromolecules (such as Fc fusion proteins) has become a standardized process, with minor modifications between products. For example, many Fc containing macromolecules are produced by human embryonic kidney (HEK) cells (or variants thereof) or Chinese Hamster Ovary (CHO) cells (or variants thereof) or in some cases by bacterial or synthetic methods. Following production, the Fc containing macromolecules that are secreted by HEK or CHO cells are purified through binding to Protein A columns and subsequently ‘polished’ using various methods. Generally speaking, purified Fc containing macromolecules are stored in liquid form for some period of time, frozen for extended periods of time or in some cases lyophilized. In various embodiments, DB1/ 145191832.2 65 Attorney Docket No.: SHK-081PC/116981-5081 production of the heterodimeric proteins contemplated herein may have unique characteristics as compared to traditional Fc containing macromolecules. In certain examples, the heterodimeric proteins may be purified using specific chromatography resins, or using chromatography methods that do not depend upon Protein A capture. In other embodiments, the heterodimeric proteins may be purified in an oligomeric state, or in multiple oligomeric states, and enriched for a specific oligomeric state using specific methods. Without being bound by theory, these methods could include treatment with specific buffers including specified salt concentrations, pH and additive compositions. In other examples, such methods could include treatments that favor one oligomeric state over another. The heterodimeric proteins obtained herein may be additionally ‘polished’ using methods that are specified in the art. In embodiments, the heterodimeric proteins are highly stable and able to tolerate a wide range of pH exposure (between pH 3-12), are able to tolerate a large number of freeze/thaw stresses (greater than 3 freeze/thaw cycles) and are able to tolerate extended incubation at high temperatures (longer than 2 weeks at 40 degrees C). In other embodiments, the heterodimeric proteins are shown to remain intact, without evidence of degradation, deamidation, etc. under such stress conditions. Subjects and/or Animals In embodiments, the subject and/or animal is a mammal, e.g., a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate, such as a monkey, chimpanzee, or baboon. In other embodiments, the subject and/or animal is a non-mammal, such, for example, a zebrafish. In embodiments, the subject and/or animal may comprise fluorescently-tagged cells (with e.g., GFP). In embodiments, the subject and/or animal is a transgenic animal comprising a fluorescent cell. In embodiments, the subject and/or animal is a human. In embodiments, the human is a pediatric human. In other embodiments, the human is an adult human. In other embodiments, the human is a geriatric human. In other embodiments, the human may be referred to as a patient. In certain embodiments, the human has an age in a range of from about 0 months to about 6 months old, from about 6 to about 12 months old, from about 6 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 DB1/ 145191832.2 66 Attorney Docket No.: SHK-081PC/116981-5081 years old, from about 85 to about 90 years old, from about 90 to about 95 years old or from about 95 to about 100 years old. In other embodiments, the subject is a non-human animal, and therefore the invention pertains to veterinary use. In a specific embodiment, the non-human animal is a household pet. In another specific embodiment, the non-human animal is a livestock animal. Kits The invention provides kits that can simplify the administration of any agent described herein. An illustrative kit of the invention comprises any composition described herein in unit dosage form. In one embodiment, 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 also further comprise one or more additional agent described herein. In one embodiment, the kit comprises a container containing a composition of the invention and another composition, such those described herein. As used herein, the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the materials, compositions, devices, and methods of this technology. Similarly, the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features. Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the disclosure, the present technology, or embodiments thereof, may alternatively be described using more limiting terms such as “consisting of” or “consisting essentially of” the recited ingredients. Unless defined otherwise, all technical and scientific terms herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials, similar or equivalent to those described herein, can be used in the practice or testing of the present disclosure, the preferred methods and materials are described herein. All publications, patents, and patent publications cited are incorporated by reference herein in their entirety for all purposes. This disclosure is further illustrated by the following non-limiting examples. DB1/ 145191832.2 67 Attorney Docket No.: SHK-081PC/116981-5081 EXAMPLES The examples herein are provided to illustrate advantages and benefits of the present disclosure and to further assist a person of ordinary skill in the art with preparing or using the chimeric proteins of the present disclosure. The examples herein are also presented in order to more fully illustrate the preferred aspects of the present disclosure. The examples should in no way be construed as limiting the scope of the present disclosure, as defined by the appended claims. The examples can include or incorporate any of the variations, aspects or embodiments of the present disclosure described above. The variations, aspects or embodiments described above may also further each include or incorporate the variations of any or all other variations, aspects or embodiments of the present disclosure. Example 1: Construction and Characterization of an Illustrative BTN2A1/3A1-Fc-CD20 scFv Heterodimeric Protein The heterodimeric protein of embodiments of the present disclosure comprises a dimer of two chimeric proteins, each comprising a butyrophilin family member, a core domain, and an antigen-targeting domain. The “BTN2A1-Fc-CD20scFv/ BTN3A1-Fc-CD20scFv” construct included an alpha chain comprising an extracellular domain (ECD) of human BTN2A1 fused to an anti-CD20 scFv via a hinge-CH2-CH3 Fc domain, and a beta chain comprising an ECD of human BTN3A1 fused to the anti-CD20 scFv via a hinge- CH2-CH3 Fc domain. See, FIG.1A. Constructs encoding BTN2A1-Fc-CD20scFv protein (alpha chain) and BTN3A1-Fc-CD20scFv protein (beta chain) were generated. This heterodimeric protein is the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein of some embodiments, and is also referred to herein as GAmma DELta T cell ENgager construct also is referred to herein as the BTN2A1/3A1-Fc-CD20scFv ‘GADLEN’ protein. The BTN2A1/3A1-Fc-CD20scFv heterodimeric protein was produced via a transient co-transfection in ExpiCHO cells of two plasmids encoding 1) the BTN2A1-Fc-CD20scFv protein (alpha chain construct) and 2) BTN3A1-Fc-CD20scFv protein (beta chain construct). The alpha and beta constructs encoded a BTN2A1-Fc-CD20scFv protein (alpha chain) and 2) BTN3A1-Fc-CD20scFv protein (beta chain). The alpha and beta chains contained charged polarized linker domains which facilitated heterodimerization of the desired BTN2A1/3A1-Fc-CD20scFv heterodimeric protein. ExpiCHO cells were transiently transfected with a dual gene expression vector (pVITRO2-Blasti) encoding the alpha and beta chains. The cell culture supernatant from the transient transfection was harvested 6 days following transfection and purified over an FcXL chromatography resin. DB1/ 145191832.2 68 Attorney Docket No.: SHK-081PC/116981-5081 Constructs encoding BTN2A1-Fc-CD19scFv homodimeric protein or BTN3A1-Fc-CD19scFv homodimeric protein were designed using BTN2A1-Fc-CD20scFv protein (alpha chain) and BTN2A1-Fc- CD20scFv protein (beta chain), or BTN3A1-Fc-CD20scFv protein (alpha chain) and BTN3A1-Fc- CD20scFv protein (beta chain), and purified as discussed above. The ability of BTN2A1/3A1-Fc-CD20scFv heterodimeric protein to promote degranulation by Vγ9Vδ2+ T cells was assessed using a plate-based assay. Briefly, increasing amounts of purified BTN2A1-Fc- CD19scFv homodimeric protein or BTN3A1-Fc-CD19scFv homodimeric protein, or BTN2A1/3A1-Fc- CD20scFv heterodimeric protein were added in high-binding 96-well plates (Corning) and 1 μg /mL of an anti-NKG2D antibody was added to wells containing the BTN2A1-Fc-CD19scFv homodimeric protein, BTN3A1-Fc-CD19scFv homodimeric protein, or BTN2A1/3A1-Fc-CD20scFv heterodimeric protein. BTN2A1-Fc-CD19scFv homodimeric protein or BTN3A1-Fc-CD19scFv homodimeric protein were used as a negative control. The plates were incubated overnight at 4°C. Wells were washed twice with DPBS, and 0.5 to 1×105 Vγ9Vδ2+-T cells in RPMI + 10% FBS, supplemented with 100 U/mL hIL-2. BD FastImmune CD107a APC, GolgiStop, and GolgiPlug reagents (BD Biosciences) were added to cell culture according to the manufacturer’s protocols. Cells were incubated at 37°C for 4 hours and stained for CD107a expression for analysis by flow cytometry. The proportion of CD107a was calculated as a fraction of the CD3+Vγ9+ population. As shown in FIG.2A (right panel), a dose-dependent increase in CD107a-expressing Vγ9Vδ2+-T cells was observed when the Vγ9Vδ2+-T cells were induced by the combination of BTN2A1/3A1-Fc-CD20scFv heterodimeric protein and the anti-NKG2D antibody. By contrast, Vγ9Vδ2+-T cells induced the anti-NKG2A antibody in combination with BTN2A1-Fc-CD19scFv or BTN3A1-Fc-CD19scFv homodimeric protein expressed only a background levels of CD107a (FIG.2A (left and middle panels)). These results demonstrate, inter alia, that the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein, in combination with an anti-NKG2D antibody, can activate Vγ9Vδ2+ T cells. Example 2: In Vitro Depletion of CD20+ Lymphoma Cells and B Cells by a BTN2A1/3A1-Fc-CD20scFv Heterodimeric Protein In the experiments of this example, CD20+ lymphoma cells and B cells were depleted by a BTN2A1/3A1- Fc-CD20scFv heterodimeric protein in an in vitro depletion assay. In the experiments shown in FIG.2B, human Daudi Burkitt’s lymphoma cells (CD20+ lymphoma cell line) were labeled with a fluogenic granzyme B substrate and co-cultured with Vγ9+Vδ2+ T cells (GDT) at a 1:1 ratio in the presence of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein at various concentrations to evaluate the ability of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein to promote γδ T cell cytotoxicity by transferring DB1/ 145191832.2 69 Attorney Docket No.: SHK-081PC/116981-5081 granzyme B into target cells. After 1 hour of culture, the proportion of granzyme B+ lymphoma cells was analyzed by flow cytometry. The BTN2A1/3A1-Fc-CD20scFv heterodimeric protein mediated granzyme B transfer into target cells in a dose-dependent manner. *p<0.05, ***p<0.001, ****p<0.0001 by one-way ANOVA (FIG.2B). In the experiments shown in FIG.2C, CD20+ B cells purified from PBMC of healthy donor were co-cultured with Vγ9+Vδ2+ T cells at 2:1 ratio in the presence of various concentration of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein to evaluate the ability of the BTN2A1/3A1-Fc- CD20scFv heterodimeric protein to promote target cell killing. After 4 hours of culture, the proportion of apoptotic B cells was analyzed by Apoptracker Green staining, which detects translocated phosphatidylserine residues on the cell surface, by flow cytometry. The experiments of this example surprisingly demonstrate how the addition of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein increases apoptosis in cancer cells (i.e., CD20+ lymphoma B cells). **p<0.01, ***p<0.001 by one-way ANOVA (FIG.2C). Example 3: In Vivo Human B Cell Depletion by the BTN2A1/3A1-Fc-CD20scFv Heterodimeric Protein in PBMC-Humanized Mice In the experiments of this example, 8-10 weeks old immunocompromised NSG-IL15 mice were engrafted with 1×107 human PBMCs (FIG.3A). One day after PBMC transfer, mice were administered with PBS (vehicle control), a BTN2A1/3A1-Fc-CD20scFv heterodimeric protein (50µg or 200µg), or a BTN2A1/3A1-Fc-CD33scFv heterodimeric protein (200µg, non-B cell targeting construct) intravenously, two doses administered once every three days (q3dx2). Peripheral blood and spleens were harvested from animals 24 hours post last dose for quantification of B cells (FIG. 3A). (hCD45+CD3- CD19+CD20+IgD+) (FIG. 3B), non-B/T cells (hCD45+CD3-CD19-) (FIG. 3C), Vδ2+ T cells (hCD45+CD3+Vδ2+) (FIG.3D), and total T cells (hCD45+CD3+CD19-) (FIG.3E) by flow cytometry. Top and bottom panels represent animals engrafted with PBMCs at different donors, containing high and low input ratios of Vδ2+ T cells (Effector, E) and B cells (Target, T), respectively. The input E to T ratio for each donor is indicated in the figures. The experiments show the absolute count of human B cells significantly decreased after treatment with the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein, but not BTN2A1/3A1-Fc-CD33scFv heterodimeric protein, in both cohorts of animals engrafted with PBMCs with high or low E to T ratios (FIG.3B). The absolute count of non-B cell populations remained unchanged after treatment (FIG.3C), demonstrating the specificity of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein in targeting CD20+ B cells. *p<0.05 and **p<0.01 by one-way ANOVA. The lack of increase in Vδ2+ T cells suggests, inter alia, the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein induced rapid Vδ2+T cell-mediated killing of target cells in the absence of proliferation, and highlights the ability of gamma/delta (γδ) T cells that exist in low frequencies in promoting serial killing of target cells. DB1/ 145191832.2 70 Attorney Docket No.: SHK-081PC/116981-5081 These results, indicated, inter alia, that the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein, but not the BTN2A1/3A1-Fc-CD33scFv heterodimeric protein (non-targeting control), mediated B cell depletion at both high and low E:T ratios in spleen and peripheral blood (data not shown). Non-B cell compartments remained unchanged after treatment. *p<0.05, **p<0.01 The extent of B cell depletion was evaluated at various ratios of Vδ2+ T cells (hCD45+CD3+Vδ2+; Effector, E) to B cells (Target, T). Briefly, 8-10 weeks old immunocompromised NSG-IL15 mice were engrafted varying levels of Vδ2+T cells and B cells on Day 0 as shown in FIG.3A and the Table below. On Days 1 and 4, the mice were intravenously administered with PBS (vehicle control) or the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein (50µg). Peripheral blood and spleens were harvested from animals 24 hours post last dose for quantification of B cells. The extent of B cell depletion was calculated based on the ratios of B cells in PBS-treated mice in comparison with the mice treated with the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein and expressed as percentage depletion. The results are shown in the Table below: PBMC %Vδ2 + T Cells (E) % B Cells (T) E:T Ratio %B Cell Depletion Donor (mean ± SD)
Figure imgf000073_0001
These results demonstrate, inter alia, a near complete B cell depletion upon the BTN2A1/3A1-Fc- CD20scFv heterodimeric protein treatment at all donor E:T ratios. The extent of B cell depletion and changes in the levels of gamma delta T cells was evaluated at increasing doses of BTN2A1/3A1-Fc-CD20scFv heterodimeric protein. Briefly, 8-10 weeks old immunocompromised NSG-IL15 mice were engrafted fixed levels of cells human CD45+CD3+Vδ2+ T cells (Vδ2+T cells) and B cells on Day 0 as shown in FIG.3A. On Days 1 and 4, the mice were intravenously administered with PBS (vehicle control) or increasing levels of the BTN2A1/3A1-Fc- CD20scFv heterodimeric protein (0.5 µg, 2 µg, 10 µg or 50 µg). Peripheral blood and spleens were harvested from animals 24 hours post last dose for quantification of B cells and Vδ2+T cells. The extent DB1/ 145191832.2 71 Attorney Docket No.: SHK-081PC/116981-5081 of B cell depletion was calculated based on the ratios of B cells in PBS-treated mice in comparison with the mice treated with the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein and expressed as percentage depletion. As shown in FIG.3E (left panel), there was dose-dependent depletion of B cells, although the lowest dose used in this experiment provided near complete depletion of B cells. As shown in FIG.3E (right panel), the numbers of Vδ2+T cells remained essentially unchanged. These results indicate, inter alia, the gamma delta T cells have the ability to promote serial killing of target cells in the absence of proliferation or expansion. Example 4: In Vivo Anti-Tumor Activity of the BTN2A1/3A1-Fc-CD20scFv Heterodimeric Protein An in vivo animal model of lymphoma was developed and treated with the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein as schematically shown in FIG.4A. Briefly, 5×106 CD20+ human lymphoma Raji cells and 4×106 human PBMCs from one of two human donors (Donor 4 and Donor 6) were injected subcutaneously in 8-10 weeks old immunocompromised NSG-MHCI/MHCII double knockout mice (FIG. 4A). The PBMCs served as a source of human Vδ2+ T cells (CD45+CD3+Vδ2+T cells). PBMCs aliquots from the two donors were analyzed by flow cytometry to calculate the numbers of CD3+ T cells and Vδ2+ T cells to determine the Effector to Target ratios. These analyzes showed that the ratio of total CD3+T to Raji cells was 1:1.7, and the ratio of Vδ2+T cells to Raji cells was 1:347 for the mice injected with PBMCs from Donor 4. In case of the mice injected with Raji cells and PBMCs from Donor 6, the ratio of total CD3+T to Raji cells was 1:2, and the ratio of Vδ2+T cells to Raji cells was 1:368. The mice were then randomly divided in four treatment groups: 1) vehicle only, 2) the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein (100 µg; 125 nM), 3) the BTN2A1/3A1-Fc-B7H3scFv heterodimeric protein (100 µg; 125 nM),and 4) a CD3×CD20 engager (100 µg; 100 nM). The mice were treated as above on days 0, 3, 7 and 10 (FIG.4A). Tumor growth was monitored and plotted in each individual mouse. The data are shown in FIG.4B to FIG. 4D. In this experiment, CD3×CD20 engager, which targets all T cells to CD20 positive tumors, was used as a positive control. The BTN2A1/3A1-Fc-B7H3scFv heterodimeric protein was used as a non-targeting control. See Liu et al, Effective killing of cells expressing CD276 (B7-H3) by a bispecific T cell engager based on a new fully human antibody, Transl Oncol. 2021; 14(12): 101232. As shown in FIG. 4B, vehicle treated mice harboring Raji cells and PBMCs from wither of the donors showed comparable progressive tumor growth, with tumors in all but one mice becoming larger than 1500 mm3 by day 20, indicating that the PBMCs DB1/ 145191832.2 72 Attorney Docket No.: SHK-081PC/116981-5081 from Donor 4 or Donor 6 themselves did not affect tumor growth compared to each other. The mice treated with CD3×CD20 engager showed a detectable reduction in tumor growth as illustrated by the fact that only two of six CD3×CD20 engager-treated mice had tumors larger than 1000 mm3 and none had tumors larger than 1500 mm3 on day 20 (compare FIG.4D with FIG.4B). These data indicated, inter alia, that targeting all T cells to tumor cells leads to a detectable tumor control. The mice treated with BTN2A1/3A1-Fc-B7H3scFv heterodimeric protein showed very little inhibition of tumor growth, if any (FIG. 4D). As shown in FIG. 4C, surprisingly, the mice treated with BTN2A1/3A1-Fc-CD20scFv heterodimeric protein showed remarkable inhibition of tumor growth compared to the vehicle-treated mice. Surprisingly, the mice treated with the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein exhibited better tumor control compared to the mice treated with the CD3×CD20 engager (FIG.4E). For example, whereas the mice treated with the CD3×CD20 engager had 5/6 tumors larger than about 300 mm3 on day 20, none of the tumors in the mice treated with BTN2A1/3A1-Fc-CD20scFv heterodimeric protein were larger than about 300 mm3 on day 20. These results are remarkable considering that the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein exhibited such control at effector to target ratios that were over 100 fold lower compared to that in case of the CD3×CD20 engager. These results indicated, inter alia, that the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein achieves remarkable tumor control. These results also indicated, inter alia, that low frequencies of gamma delta T cells can be harnessed to achieve similar target-cell killing potential as the broader CD3+ T cell pool, but with potentially less toxicity. Moreover, these results indicated, inter alia, that the BTN2A1/3A1-Fc- CD20scFv heterodimeric protein safely directed low frequencies of gamma delta T cells to eliminate CD20 expressing cells in vivo, providing a differentiated T-cell engager for depletion of malignant or auto- antibody producing B cells. Example 5: In Vivo B cell Depletion by the BTN2A1/3A1-Fc-CD20scFv Heterodimeric Protein in Cynomolgus Macaques A toxicology study was conducted in cynomolgus macaques. The monkeys were infused with increasing doses of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein in a dose range of 0.1 to 25 mg/kg. The study design is shown in the Table below: Dose Schedule Dose Level No. of Animals
Figure imgf000075_0001
DB1/ 145191832.2 73 Attorney Docket No.: SHK-081PC/116981-5081 2.5 mg/kg 2M/2F
Figure imgf000076_0001
Monkey 1001 and 1501 were control monkeys that did not receive the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein. Monkeys 2001 and 2501 received 0.1 mg/kg of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein; monkeys 3001, 3002, 3501, 3502 received 2.5 mg/kg of the BTN2A1/3A1-Fc- CD20scFv heterodimeric protein; and monkeys 4001 and 4501 received 25 mg/kg of the BTN2A1/3A1- Fc-CD20scFv heterodimeric protein. Peripheral blood was extracted from the animals at various times and the receptor occupancy of CD20 in B cells and of Vγ9 in Gamma Delta cells was measured. The levels of B cells, CD4+ cells, Vγ9+ cells were measured by flow cytometry. As shown in FIG.5A, B cells showed a dose-dependent occupancy by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein. Durable binding of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein to B-cells was observed in a dose dependent manner over at least 72 hours (FIG.5A). As shown in FIG.5B, compared to pre-dose samples the post dose samples exhibited occupancy of the CD3- CD19+ target cells by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein. As shown in FIG.5C, Gamma Delta T cells showed a dose-dependent occupancy by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein in 2 hours. Gamma Delta T cell occupancy by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein returned to baseline by 72 hours (FIG.5C). As shown in FIG.5D, compared to pre-dose samples the post dose samples exhibited occupancy of the Vγ9+ Gamma Delta T cell target cells by the BTN2A1/3A1- Fc-CD20scFv heterodimeric protein. As shown in FIG.5E, a rapid and dose-dependent B-cell depletion was observed in 2 hours in monkeys treated with the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein. On the other hand, as shown in FIG.5F, no such depletion was observed in other immune cell types such as CD4+ T cells. As shown in FIG.5G, the levels of Vγ9+ cells did not rapidly decrease, indicating a lack of margination of those cells. Indeed, the levels of Vγ9+ cells did not change during the 72 hours (FIG.5G). Based on the Vγ9+ cell numbers and B cell numbers, the ratios of effector to target cells (Vγ9+ cells to B cells or E:T ratio) were calculated and the kinetics of B cell depletion was calculated for various E:T ratios as a function of time. As shown in FIG.5H, the highest B-cell depletion observed at 1:7 E:T ratio at the 2.5 mg/kg dose level of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein. The receptor occupancy (RO) of B cells by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein was determined. Briefly, following doses, blood samples were recovered at various times post-dose, B cells were enriched. The enriched B cells were analyzed using flow cytometry to determine proportion of B DB1/ 145191832.2 74 Attorney Docket No.: SHK-081PC/116981-5081 cells based on detection of a B cell marker. The enriched B cells were analyzed using flow cytometry to determine the proportion the B cells bound to the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein were assayed based on detection of butyrophilin. The proportions the B cells bound to the BTN2A1/3A1-Fc- CD20scFv heterodimeric protein were plotted as a function of time. As shown in FIG.5K, near complete receptor occupancy (RO) of B cells was observed at 2.5 mg/kg, and the RO of B cells persisted for >72hrs. The receptor occupancy (RO) of gamma delta T cells by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein was determined. Briefly, following doses, blood samples were recovered at various times post- dose, Gamma delta T cells were enriched. The enriched Gamma delta T cells were analyzed using flow cytometry to determine proportion of gamma delta T cells based on detection of a gamma delta T cell marker. The enriched gamma delta T cells were analyzed using flow cytometry to determine the proportion the gamma delta T cells bound to the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein were assayed based on detection of CD20scFv. The extent of binding of the gamma delta T cells to the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein, expressed as percentages, was plotted as a function of time. As shown in FIG.5L, the percentage of GADLEN-bound cells increased after each dose. These data showed that receptor occupancy (RO) of gamma delta T cells by the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein is transient, which without wishing to be bound by theory, is likely to be due to low affinity engagement of BTN2A1/3A1 with Vγ9Vδ2 TCR. The number of B cells was determined at 24 hours post dosing. As shown in FIG.5M, a dose-dependent decrease in B cells was observed compared to the vehicle-alone treated group. The number of gamma delta T cells was determined at 24 hours post dosing. As shown in FIG.5N, a lack of dose-dependent changes in the levels of gamma delta T level was observed. These results suggest, inter alia, the ability of gamma delta T cells to mediate serial killing of target cells in the presence of the BTN2A1/3A1-Fc- CD20scFv heterodimeric protein. Therefore, this study demonstrated, inter alia, a dose-dependent B cell depletion within 2 hours of infusion. Durable receptor occupancy was observed on CD20+ cells for ≥7 days, whereas transient occupancy was observed on circulating cynomolgus Vγ9Vδ2 T cells. This study also demonstrated dose- dependent B cell depletion within 2 hours of infusion. Durable receptor occupancy was observed on CD20+ cells for ≥7 days, whereas transient occupancy was observed on circulating cynomolgus Vγ9Vδ2 T cells. Gamma Delta T cells represented approximately 1-3% of total T cells in cynomolgus macaques, and E:T ratios ranged from 1:7 to 1:49. All treatments were well tolerated, and no evidence of cytokine release syndrome or any other toxicities were observed. DB1/ 145191832.2 75 Attorney Docket No.: SHK-081PC/116981-5081 Rapid depletion of B cells in less than 2 hours demonstrate, inter alia, and without wishing to be bound by theory, Gamma Delta T cells were not required to achieve B cell depletion. Indeed, over several weeks, the circulating frequencies of cynomolgus Vγ9Vδ2 T cells remained stable, and no evidence of either proliferation or migration out of the blood was observed. These observations, together with the speed at which B cell depletion occurred, suggest, inter alia, and without wishing to be bound by theory, that the Vγ9Vδ2 T cells eliminated the B cell pool by serial killing following treatment with the BTN2A1/3A1-Fc- CD20scFv heterodimeric protein. Despite a Gamma Delta T cells frequency of <5% of total T cells in all these studies, Vγ9Vδ2 T cells achieved rapid depletion of target cells following treatment with the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein, without evidence of toxicity, even at high doses. Collectively, these results indicate, without wishing to be bound by theory, that low frequencies of Gamma Delta T cells can be harnessed to achieve similar target-cell killing potential as the broader CD3+ T cell pool but with potentially less toxicity. The pharmacokinetic profile of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein was determined. Briefly, following doses, blood samples were recovered at various times post-dose, sera were prepared and the amounts of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein were assayed in the sera samples. The serum concentrations were plotted as a function of time. As shown in FIG. 5I, the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein could be detected at all times tested. Based on the data, it was determined that the serum half-life of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein is ~48-72 hours. After 48 hours after Dose 3, the animals were sacrificed and samples of liver, spleen, axillary lymph node (LN), inguinal LN, and mesenteric LN were recovered. These samples were homogenized and the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein were assayed in the homogenate. As shown in FIG. 5J, the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein could be detected in all tissues 48 hours after dosing. The study showed, inter alia, that all doses were well tolerated. No clinically relevant changes observed with any hematological parameters, except those noted herein. Liver and kidney function were normal throughout the study as were coagulation, D-Dimer, serum proteins and electrolytes. Minimal cytokine changes were observed and no IL-6 elevation was observed. These results demonstrate, inter alia, that the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein may be used for reducing the number of B cells to treat a B cell-mediated disease or disorder. These results also demonstrate, inter alia, that the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein may be used for killing DB1/ 145191832.2 76 Attorney Docket No.: SHK-081PC/116981-5081 CD20+ cancer cells, and thereby treating a CD20-positive cancer. These results further demonstrate, inter alia, that less than about 0.1 to more than about 25 mg/kg dose of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein may be effective in reducing the number of B cells to treat a B cell-mediated disease or disorder and/or treating a CD20-positive cancer. INCORPORATION BY REFERENCE All patents and publications referenced herein are hereby incorporated by reference in their entireties. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections. EQUIVALENTS While the disclosure has been disclosed in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure as come within known or customary practice within the art to which the disclosure pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments disclosed specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims. DB1/ 145191832.2 77

Claims

Attorney Docket No.: SHK-081PC/116981-5081 CLAIMS What is claimed is: 1. A method for treating a B cell-mediated disease or disorder, comprising administering to the subject about 0.01 mg/kg to about 100 mg/kg of a heterodimeric protein, or a nucleic acid encoding the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; (b) a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof, the target domain being directed to CD20; and (c) a linker that adjoins the first and second domains; and wherein the beta chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; (b) a second domain comprising the targeting domain, the target domain being directed to CD20; and (c) a linker that adjoins the first and second domains. 2. A method for increasing and/or stimulating B cell depletion in a subject suffering from a B cell- mediated disease or disorder, comprising administering to the subject about 0.01 mg/kg to about 100 mg/kg of a heterodimeric protein, or a nucleic acid encoding the same, comprising an alpha chain and a beta chain: wherein the alpha chain comprises: (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; (b) a second domain comprising a targeting domain selected from an antibody, antibody-like molecule, or antigen binding fragment thereof, the target domain being directed to CD20; and (c) a linker that adjoins the first and second domains; and wherein the beta chain comprises: DB1/ 145191832.2 Attorney Docket No.: SHK-081PC/116981-5081 (a) a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1; (b) a second domain comprising the targeting domain, the target domain being directed to CD20; and (c) a linker that adjoins the first and second domains. 3. The method of claim 1 or claim 2, wherein the fragment comprises an extracellular domain (ECD) and/or a variable domain. 4. The method of any one of claims 1-3, wherein the first domains of the alpha chain and beta chain comprise ECDs, variable domains, or a combination of an ECD and a variable domain of the same butyrophilin family protein. 5. The method of any one of claims 1-4, wherein the first domains of the alpha chain and beta chain comprise ECDs, variable domains, or a combination of an ECD and a variable domain of different butyrophilin family proteins. 6. The method of any one of claims 1-5, wherein the butyrophilin family protein of the alpha chain and beta chain is independently selected from human BTN2A1 and human BTN3A1. 7. The method of any one of claims 1-6, wherein the first domains of the alpha chain and beta chain comprise extracellular domains and/or variable domains of BTN2A1 and BTN3A1. 8. The method of any one of claims 1-7, wherein the first domains of the alpha chain and beta chain comprise variable domains of BTN2A1 and BTN3A1. 9. The method of any one of claims 1-8, wherein the first domain of the alpha chain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence selected from SEQ ID NOs: 13-16; and the first domain of the beta chain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence selected from SEQ ID NOs: 13-16. DB1/
Figure imgf000081_0001
Attorney Docket No.: SHK-081PC/116981-5081 10. The method of claim 9, wherein the first domains of the alpha chain and beta chain comprise a polypeptide having an amino acid sequence of: any one of SEQ ID NOs: 13-14; and any one of SEQ ID NOs: 15-16, respectively. 11. The method of any one of claims 1-10, wherein the targeting domain is capable of binding CD20 on the surface of a B cell and/or a cancer cell. 12. The method of any one of claims 1-11, wherein the targeting domain comprises an Fv domain. 13. The method of any one of claims 1-12, wherein the targeting domain is an antibody-like molecule, or antigen binding fragment thereof. 14. The method of claim 13, wherein the targeting domain comprises a single-chain variable fragment (scFv), or a fragment thereof. 15. The method of claim 14, wherein the targeting domain comprises different scFvs. 16. The method of any one of claims 1-15, wherein the targeting domain specifically binds human CD20. 17. The method of claim 16, wherein the targeting domain comprises an scFv that specifically binds human CD20. 18. The method of any one of claims 1-17, wherein the targeting domain comprises a polypeptide comprising the complementarity determining regions (CDRs) present in SEQ ID NOs: 17-20. 19. The method of claim 18, wherein the targeting domain comprises a polypeptide having an amino acid sequence having at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide having an amino acid sequence selected from SEQ ID NOs: 17-20. 20. The method of any one of claims 1-19, wherein the linker comprises a polypeptide selected from a flexible amino acid sequence, an IgG hinge region, and an antibody sequence. 21. The method of claim 20, wherein the antibody sequence comprises an Fc domain. 22. The method of any one of claims 1-21, wherein the linker comprises a hinge-CH2-CH3 Fc domain. DB1/ 145191832.2 80 Attorney Docket No.: SHK-081PC/116981-5081 23. The method of claim 22, wherein the hinge-CH2-CH3 Fc domain is derived from IgG1, optionally from human IgG1. 24. The method of claim 23, wherein the hinge-CH2-CH3 Fc domain is derived from IgG4, optionally from human IgG4. 25. The method of any one of claims 1-24, wherein the hinge-CH2-CH3 Fc domain comprises a polypeptide having an amino acid sequence with at least about 90%, or at least about 95%, or at least about 97%, or at least about 98%, or at least about 99% identity with a polypeptide selected from SEQ ID NOs: 1- 12. 26. The method of any one of claims 1-25, wherein the nucleic acid is or comprises a DNA or RNA. 27. The method of claim 26, wherein the RNA is mRNA, which is an optionally modified mRNA (mmRNA). 28. The method of any one of claims 1-25, wherein the nucleic acid is or comprises an expression vector. 29. The method of any one of claims 1-28, wherein the method modulates or is suitable for modulating a γδ (gamma delta) T cell. 30. The method of claim 29, wherein the gamma delta T cell expresses Vγ4 or Vγ9δ2. 31. The method of claim 30, wherein the first domain comprises BTN2A1 and it modulates a Vγ4- expressing T cell. 32. The method of claim 31, wherein the first domain modulates a Vγ9δ2-expressing T cell. 33. The method of any one of claims 1-32, wherein the modulation of a gamma delta T cell is activation of a gamma delta T cell. 34. The method of any one of claims 1-33, wherein the method induces killing of a target cell. 35. The method of any one of claims 1-34, wherein the target cell is a human cell. 36. The method of any one of claims 1-35, wherein the method: (i) modulates or is suitable for modulating a γδ (gamma delta) T cell, and/or (ii) stimulates or promotes localizing to a tumor, and/or binding to a tumor cell, and/or engaging a tumor cell, and/or promoting an immune stimulatory signal, and/or inhibiting an immune inhibitory signal, and/or forming an immune synapse. DB1/ 145191832.2 81 Attorney Docket No.: SHK-081PC/116981-5081 37. The method of any one of claims 1-36, wherein the immune synapse is a synapse between a gamma delta T cell and a tumor cell and/or the chimeric protein is capable of contemporaneous activation and targeting of gamma delta T cells to tumor cells. 38. The method of any one of claims 1-37, wherein the B cell depletion comprises a reduction in the numbers and/or activity of one or more B cells. 39. The method of any one of claims 1-37, wherein the B cells are hCD45+CD3-CD19+CD20+IgD+. 40. The method of any one of claims 1-39, wherein the method does not reduce and/or suppress a non- B cell population of cells. 41. The method of any one of claims 1-40, wherein the method does not reduce and/or suppress non- B/T cells (e.g., hCD45+CD3-CD19-), Vδ2+ T cells (e.g., hCD45+CD3+Vδ2+), and/or total T cells (e.g., hCD45+CD3+CD19-). 42. The method of any one of claims 1-41, wherein the method does not increase and/or stimulate non- B/T cells (e.g., hCD45+CD3-CD19-), Vδ2+ T cells (e.g., hCD45+CD3+Vδ2+), and/or total T cells (e.g., hCD45+CD3+CD19-). 43. The method of any one of claims 1-42, wherein the heterodimeric protein is administered at a frequency of about 1 day, or about 2 days, or about 3 days, or about 4 days, or about 5 days, or about 6 days, or about one week, or about 10 days, or about 15 days, or about 21 days, or more. 44. The method of claim 43, wherein the heterodimeric protein is administered at a frequency of about 3 days. 45. The method of claim 43, wherein the heterodimeric protein is administered at a frequency of about 7 days. 46. The method of any one of claims 1-45, wherein the heterodimeric protein is administered at a frequency of about 1 day to about 10 days, about 1 day to about 5 days, or about 2 days to about 10 days, or about 5 days to about 15 days, or about one week to about 3 weeks. 47. The method of claim 46, wherein the heterodimeric protein is administered at a frequency of about 1 day to about 10 days. 48. The method of claim 46, wherein the heterodimeric protein is administered at a frequency of about 5 days to about 15 days. DB1/ 145191832.2 82 Attorney Docket No.: SHK-081PC/116981-5081 49. The method of any one of claims 1-48, wherein the heterodimeric protein is administered at a dose that provides a ratio of heterodimeric protein-bound Gamma-Delta T cells (effector cells) to target cells of about 1:1, or about 1:2, or about 1:5, or about 1:7, or about 1:10, or about 1:15, or about 1:20 or less. 50. The method of any one of claims 1-49, wherein the heterodimeric protein is administered at a dose that provides a ratio of heterodimeric protein-bound Gamma-Delta T cells (effector cells) to target cells of about 1:1 to about 1:5, or about 1:2 to about 1:8, or about 1:10 to about 1: 20. 51. The method of claim 49 or claim 50, wherein the targets cells are B cells. 52. The method of claim 49 or claim 50, wherein the targets cells are CD20+ cancer cells. 53. The method of any one of claims 1-52, wherein the B cell-mediated disease or disorder is a cancer. 54. The method of claim 53, wherein the cancer is a cancer expressing CD20. 55. The method of claim 53, wherein the cancer is a B-cell malignancy. 56. The method of claim 55, wherein the B-cell malignancy is a lymphoma or leukemia. 57. The method of claim 55, wherein the B-cell malignancy is non-Hodgkin’s lymphoma (NHL) or Hodgkin’s lymphoma (HL). 58. The method of claim 55, wherein the B-cell malignancy is advanced stage follicular lymphoma, chronic lymphocytic leukemias (CLL), Burkitt’s lymphoma, or diffuse large B-cell lymphoma. 59. The method any one of claims 1-58, wherein the cancer is a solid cancer selected from prostate cancer, small cell lung cancer, non-small cell lung cancer, neuroendocrine tumor, renal cancer, bladder cancer, colon cancer, and breast cancer. 60. The method of any one of claims 1-59, wherein the cancer has mutations which limit recognition by alpha beta T cells, optionally selected from mutations in MHC I, beta 2 microglobulin, and Transporter associated with antigen processing (TAP). 61. The method of any one of claims 1-60, wherein the B cell-mediated disease or disorder is an autoimmune disease or disorder. 62. The method of claim 61, wherein the autoimmune disease or disorder is one or more of rheumatoid arthritis, systemic lupus erythematosus, diabetes mellitus, ankylosing spondylitis, Sjögren's syndrome, inflammatory bowel diseases (e.g., colitis ulcerosa, Crohn's disease), multiple sclerosis, sarcoidosis, DB1/ 145191832.2 83 Attorney Docket No.: SHK-081PC/116981-5081 psoriasis, Grave's disease, Hashimoto's thyroiditis, psoriasis, hypersensitivity reactions (e.g., allergies, hay fever, asthma, and acute edema cause type I hypersensitivity reactions), pemphigus, and vasculitis (e.g., Granulomatosis with polyangiitis). 63. The method of any one of claims 1-62, wherein the method further comprises administering to the subject a second pharmaceutical composition that costimulates γδ T cells. 64. The method of claim 63, wherein the heterodimeric protein and the second pharmaceutical composition are administered simultaneously or contemporaneously. 65. The method of claim 63, wherein the heterodimeric protein is administered after the second pharmaceutical composition is administered. 66. The method of claim 63, wherein the heterodimeric protein is administered before the second pharmaceutical composition is administered. 67. The method of any one of claims 63-66, wherein the second pharmaceutical composition costimulates a receptor selected from CD28, NKG2D, CD27, CD30, 4-1BB (CD137), IL-2R, IL-15R, IL-7R, IL-21R, NKp30, NKp44, DNAM-1 (CD226), IL-2R, IL-7R, IL-15R, dectins, NLRs, killer Ig-like receptors (e.g., KIR2D, KIR3D), C-type lectins (CD94/NKG2A-C, NKG2D), LFA1, CD2, CD46, Junctional Adhesion Molecule-Like (JAML). 68. The method of any one of claims 63-66, wherein the second pharmaceutical composition comprises an antibody, antibody-like molecule or a receptor-binding portion thereof. 69. The method of claim 68, wherein the second pharmaceutical composition comprises an agonistic antibody. 70. The method of claim 68 or claim 69, wherein the antibody is a monoclonal antibody. 71. The method of any one of claims 68-70, wherein the antibody is selected from: (i) an anti-NKG2D monoclonal antibody selected from tesnatilimab, 149810, 1D11 and 5C6, or (ii) an anti-CD28 monoclonal antibody selected from JJ316, D665, 5.11A1, TGN1412, 37.51, E18, and PV-1. DB1/ 145191832.2 84
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