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WO2024006889A2 - Modulation de lymphocytes t gamma delta ciblant cd20 - Google Patents

Modulation de lymphocytes t gamma delta ciblant cd20 Download PDF

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Publication number
WO2024006889A2
WO2024006889A2 PCT/US2023/069345 US2023069345W WO2024006889A2 WO 2024006889 A2 WO2024006889 A2 WO 2024006889A2 US 2023069345 W US2023069345 W US 2023069345W WO 2024006889 A2 WO2024006889 A2 WO 2024006889A2
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domain
cells
cell
protein
amino acid
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WO2024006889A3 (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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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Definitions

  • compositions and methods including heterodimeric proteins that find use in the treatment of disease, such as immunotherapies for cancer.
  • the instant application contains a sequence listing, which has been submitted in XML format via EFS- Web.
  • Gamma Delta T cells are capable of directly recognizing and killing transformed cells independently of HLA-antigen presentation.
  • the Vy9V ⁇ 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, Gamma Delta T cells represent highly promising effector cell compartment for immunotherapy. However, these cells occur in exceedingly small amounts.
  • the present disclosure relates to Gamma Delta T cell-based compositions and methods, e.g., immunotherapies for cancer, or autoimmune diseases or disorders.
  • the present disclosure relates to a heterodimeric protein, or a nucleic acid encoding the same, comprising an alpha chain and a beta chain, wherein the alpha chain and the beta chain comprise a general structure: N terminus -(b)-(p/n)-(b)-(n/p)-(t)-C terminus wherein (b) is a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1 , optionally wherein the fragment comprises an extracellular domain (ECD) or a variable domain, (t) is a second domain comprising a targeting domain directed to CD20, optionally wherein the targeting domain comprises a single-chain variable fragment (scFv); and (p/n)-(b)-(n/p) is a linker that adjoins the first and second domains, wherein p is a peptide comprising positively charged amino acids and
  • the present disclosure relates to a method for treating 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) 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
  • the present disclosure relates to method for reducing the number of B cells and/or reducing the activity B cells 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) 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 ;
  • the present disclosure relates to method for inducing killing of B cells and/or B cell lymphoma cells 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) 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
  • 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 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 y ⁇ (gamma delta) T cell.
  • the gamma delta T cell expresses Vy4 or Vy9 ⁇ 2.
  • the first domain comprises BTN2A1 and it modulates a Vy4-expressing T cell.
  • the first domain modulates a Vy9 ⁇ 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 y ⁇ (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 killing of B cells and/or B cell lymphoma comprises a decrease in the amount and/or activity of one or more B cells and/or B cell lymphoma cells.
  • the decrease in the amount and/or activity of B cells comprises a reduction in the numbers and/or activity of one or more B cells.
  • the B cells are hCD45+CD3-CD19+CD20+lgD+.
  • 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., CD45+CD3-CD19-), Vd2+ T cells (e.g., CD45+CD3+Vd2+), and/or total T cells (e.g., CD45+CD3+CD19-). In embodiments, the method does not increase and/or stimulate non-B/T cells (e.g., CD45+CD3-CD19-), Vd2+ T cells (e.g., CD45+CD3+Vd2+), and/or total T cells (e.g., CD45+CD3+CD19-).
  • non-B/T cells e.g., CD45+CD3-CD19-
  • Vd2+ T cells e.g., CD45+CD3+Vd2+
  • total T cells e.g., CD45+CD3+CD19-
  • the decreasing the amount and/or activity of B cell lymphoma cells comprises a reduction in the numbers and/or activity of one or more B cell lymphoma cells.
  • the B-cell lymphoma is selected from diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), Mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, Burkitt-like lymphoma, lymphoplasmacytic lymphoma (Waldenstrom macroglobulinemia), hairy cell leukemia (HCL), primary central nervous system (CNS) lymphoma and primary intraocular lymphoma (lymphoma of the eye).
  • DLBCL diffuse large B-cell lymphoma
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • MCL Mantle cell lymphoma
  • 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, Sjogren'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).
  • 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. 1 B shows, without wishing to be bound by theory, a schematic illustration of the mechanism of killing of target-cell 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.
  • Another gamma delta T cell molecule (without limitation, e.g., NKG2D or CD28), without wishing to be 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 Vy9V ⁇ 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 killing of CD20+ lymphoma cells using human Daudi Burkitt’s lymphoma cells (CD20+ lymphoma cell line).
  • FIG. 2C is a graph showing an in vitro killing of normal B cells purified from peripheral blood mononuclear cells ("PBMCs”) of a healthy donor.
  • PBMCs peripheral blood mononuclear cells
  • FIG. 3A to FIG. 3F show the in vivo killing of human B cell by the BTN2A1/3A1 -Fc-CD20scFv heterodimeric protein in PBMC-humanized mice.
  • FIG. 3A shows a schematic representation of experimental design.
  • FIG. 3B shows the killing of B cells (hCD45+CD3-CD19+CD20+lgD+) at Effector (E, V ⁇ 2+ T cells) to Target (T, B cells) ratios of 1 :2 (top panel) and 1 :42 (bottom panel).
  • E Effector
  • T Target
  • 3C demonstrates that the non-B/T cells (hCD45+CD3-CD19-) are not killed at E:T ratios of ratios of 1 :42 (bottom panel) or 1 :2 (top panel).
  • FIG. 3D shows that the V ⁇ 2+ T cells (hCD45+CD3+ V ⁇ 2+) are not killed at E:T ratios of 1 :2 (top panel) and 1 :46 (bottom panel).
  • FIG. 3E shows that the total T cells (hCD45+CD3+CD19-) are not killed at E:T ratios of 1 :2 (top panel) and 1 :46 (bottom panel). Top and bottom panels in FIG. 3B to FIG.
  • FIG. 3E represent animals engrafted with PBMCs derived from two different donors, containing high and low input ratios of V62+ T cells (Effector, E) and B cells (Target, T), respectively.
  • FIG. 3F are box plots showing the changes in B cells (left panel) and V ⁇ 2+ T cells (Right panel) as a function of the dose of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein in PBMC- humanized mice.
  • 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. 40 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. 4D shows the tumor growth in mice treated with a CD3xCD20 engager, which was used as a positive control.
  • the present disclosure is based, in part, on the observation that a BTN2A1/3A1 -Fc-CD20scFv heterodimeric protein induces a gamma delta T cell mediated killing of B cells and B cell lymphoma cells.
  • the present disclosure relates to a method for treating 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) 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
  • the present disclosure relates to method for reducing the number of B cells and/or reducing the activity B cells 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) 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 ;
  • the present disclosure relates to method for inducing killing of B cells and/or B cell lymphoma cells 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) 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
  • the present disclosure relates to a method for decreasing the amount and/or activity of B cells and/or B cell lymphoma cells 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
  • 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 .
  • 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 a variable region present in an amino acid sequence selected from 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 domain.
  • the linker comprises a hinge-CH2-CH3 Fc domain.
  • the hinge- CH2-CH3 Fc domain is derived from lgG1 , optionally from human lgG1 .
  • the hinge-CH2- CH3 Fc domain is derived from lgG4, optionally from human lgG4.
  • 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 5 (gamma delta) T cell.
  • the gamma delta T cell expresses Vy4 or Vy962.
  • the first domain comprises BTN2A1 and it modulates a Vy4-expressing T cell.
  • the first domain modulates a Vy9 ⁇ 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 y ⁇ (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 killing of B cells and/or B cell lymphoma comprises a decrease in the amount and/or activity of one or more B cells and/or B cell lymphoma cells.
  • the decrease in the amount and/or activity of B cells comprises a reduction in the numbers and/or activity of one or more B cells.
  • the B cells are hCD45+CD3-CD19+CD20+lgD+.
  • 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., CD45+CD3-CD19-), Vd2+ T cells (e.g., CD45+CD3+Vd2+), and/or total T cells (e.g., CD45+CD3+CD19-). In embodiments, the method does not increase and/or stimulate non-B/T cells (e.g., CD45+CD3-CD19-), Vd2+ T cells (e.g., CD45+CD3+Vd2+), and/or total T cells (e.g., CD45+CD3+CD19-).
  • non-B/T cells e.g., CD45+CD3-CD19-
  • Vd2+ T cells e.g., CD45+CD3+Vd2+
  • total T cells e.g., CD45+CD3+CD19-
  • the decreasing the amount and/or activity of B cell lymphoma cells comprises a reduction in the numbers and/or activity of one or more B cell lymphoma cells.
  • the B-cell lymphoma is selected from diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), Mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, Burkitt-like lymphoma, lymphoplasmacytic lymphoma (Waldenstrom macroglobulinemia), hairy cell leukemia (HCL), primary central nervous system (CNS) lymphoma and primary intraocular lymphoma (lymphoma of the eye).
  • DLBCL diffuse large B-cell lymphoma
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • MCL Mantle cell lymphoma
  • 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 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).
  • 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, Sjogren'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).
  • 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 DELta T cell ENgager
  • 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, or a nucleic acid encoding the same, comprising an alpha chain and a beta chain, wherein the alpha chain and the beta chain comprise a general structure:
  • (b) is a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophiiin family protein being BTN2A1 and/or BTN3A1 , optionally wherein the fragment comprises an extracellular domain (ECD) or a variable domain
  • (t) is a second domain comprising a targeting domain directed to CD20, optionally wherein the targeting domain comprises a single-chain variable fragment (scFv); and
  • (p/n)-(b)-(n/p) is a linker that adjoins the first and second domains, wherein p is a peptide comprising positively charged amino acids and n is a peptide comprising negatively charged amino acids, and (b) is a CH2-CH3-Fc domain, wherein the first targeting domain and the second targeting domain have different binding specificities and/or sequences.
  • the present disclosure is a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophiiin family
  • the present disclosure relates to a heterodimeric protein, or a nucleic acid encoding the same, comprising an alpha chain and a beta chain, wherein the alpha chain and the beta chain comprise a general structure:
  • the present disclosure relates to a heterodimeric protein, or a nucleic acid encoding the same, comprising an alpha chain and a beta chain, wherein the alpha chain and the beta chain comprise a general structure:
  • (b) is a first domain comprising a butyrophilin family protein, or a fragment thereof, the butyrophilin family protein being BTN2A1 and/or BTN3A1
  • (t) is a second domain comprising a targeting domain directed to CD20: and
  • (p/n)-(b)-(n/p) is a linker that adjoins the first and second domains, wherein p is a peptide comprising positively charged amino acids and n is a peptide comprising negatively charged amino acids, and (b) is a CH2-CH3-Fc domain, wherein the first targeting domain and the second targeting domain have different binding specificities and/or sequences, wherein the alpha and/or beta chains comprise at least one glycosylation selected from N -linked glycosylation and 0 -linked glycosylation.
  • the present disclosure relates to a heterodimeric protein, or a nucleic acid encoding the same, comprising an alpha chain and a beta chain, wherein the alpha chain and the beta chain comprise a general structure:
  • N terminus -(b)-(p/n)-(b)-(n/p)-(t)-C terminus wherein (b) is a first domain comprising an extracellular domain of BTN2A1 and/or BTN3A1 , (t) is a second domain comprising a targeting domain directed to CD20; and (p/n)-(b)-(n/p) is a linker that adjoins the first and second domains, wherein p is a peptide comprising positively charged amino acids and n is a peptide comprising negatively charged amino acids, and (b) is a CH2-CH3-Fc domain, wherein the first targeting domain and the second targeting domain have different binding specificities and/or sequences, wherein the alpha and/or beta chains comprise at least one glycosylation selected from N -linked glycosylation and 0 -linked glycosylation.
  • the present disclosure relates to a heterodimeric protein, or a nucleic acid encoding the same, comprising an alpha chain and a beta chain, wherein the alpha chain and the beta chain comprise a general structure:
  • N terminus -(b)-(p/n)-(b)-(n/p)-(t)-C terminus wherein (b) is a first domain comprising an extracellular domain of BTN2A1 and/or BTN3A1 , (t) is a second domain comprising anti-CD20 single-chain antibody (scFv); and (p/n)-(b)-(n/p) is a linker that adjoins the first and second domains, wherein p is a peptide comprising positively charged amino acids and n is a peptide comprising negatively charged amino acids, and (b) is a CH2-CH3-Fc domain, wherein the first targeting domain and the second targeting domain have different binding specificities and/or sequences, wherein the alpha and/or beta chains comprise at least one glycosylation selected from N - linked glycosylation and 0 -linked glycosylation.
  • 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 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 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.
  • 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.
  • the heterodimeric protein modulates or is suitable for modulating a y ⁇ (gamma delta) T cell.
  • the heterodimeric protein : (I) modulates or is suitable for modulating a y ⁇ (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 ot a gamma delta T ceil
  • 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 Vy4 or Vy9 ⁇ 2.
  • the first domain comprises BTNL3 and BTNL8 and it modulates a Vy4-expressing T cell.
  • the first domain modulates a Vy9 ⁇ 2-expressing T vii.
  • 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.
  • Any alpha chain may be combined with any beta chain having a different specificity to generate a bispecific heterodimeric fusion protein.
  • BTN2A1-Alpha-CD2O scFv (Leader sequence, which is optionally removed during secretion of the protein, 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):
  • 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:)
  • the BTN2A1 -Alpha-CD20 scFv comprises a polypeptide comprising the complementarity determining regions (CDRs) present in SEQ ID NO: 33.
  • 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: 33 lacking the leader sequence.
  • the BTN2A1 -Aipha-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: 33, wherein the polypeptide comprises the complementarity determining regions (CDRs) present in SEQ ID NO: 33.
  • CDRs complementarity determining regions
  • BTN3A1 -beta-CD20scFv (Leader sequence, which is optionally removed during secretion of the protein, 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):
  • 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.
  • the BTN3A1-beta-CD20scFv comprises a polypeptide comprising the complementarity determining regions (CDRs) present in SEQ ID NO: 34.
  • 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: 34 lacking the leader sequence.
  • 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: 34, wherein the polypeptide comprises the complementarity determining regions (CDRs) present in SEQ ID NO: 34.
  • CDRs complementarity determining regions
  • the alpha chain and the beta chain lack the leader sequence.
  • 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 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. 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.
  • 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.
  • the butyrophilin family protein is butyrophilin subfamily 2 member A1 (BTN2A1 ).
  • the first domain comprises substantially all the extracellular domain of BTN2A1.
  • the first domain is capable of binding a gamma delta T cell receptor (e.g., Vy9 ⁇ 2).
  • 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.
  • 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:
  • the fragment of human BTN2A1 comprises the variable domain of BTN2A1 , which has the following amino acid sequence:
  • 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.
  • 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 about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%
  • 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 Vy9V62 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 y ⁇ T cells. Science 367(6478) :eaay5516 ( 2020).
  • 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., Vy9 ⁇ 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.
  • 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:
  • the fragment of human BTN3A1 comprises the variable domain, which has the following amino acid sequence:
  • 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.
  • 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 about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%
  • 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 y ⁇ T-cell receptor (e.g., y9 ⁇ 2)-bi nding fragment thereof.
  • the first domain comprises a portion of BTN3A1 .
  • the portion of BTN3A1 is an extracellular domain of BTN3A1 , or a y ⁇ T-cell receptor (e.g., y9 ⁇ 2)-bi nding fragment thereof.
  • the first domain comprises a portion of BTN2A1 .
  • the portion of BTN2A1 is an extracellular domain of BTN2A1 , or a y ⁇ T-cell receptor (e.g., y9 ⁇ 2)-binding fragment thereof.
  • the first domain comprises a portion of BTN3A1 .
  • the portion of BTN3A1 is an extracellular domain of BTN3A1 , or a y ⁇ T-cell receptor (e.g., v9 ⁇ 2)-bi nding fragment thereof.
  • the Targeting Domain e.g., v9 ⁇ 2
  • 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. In embodiments, 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 a variable region present in an amino acid sequence selected from SEQ ID NOs: 17-20 or a variant thereof having 1 , 2, 3, or more amino acid mutations independently selected from substitutions, insertions, and deletions.
  • the targeting domain comprises a polypeptide comprising the complementarity determining regions (CDRs) present in a variable region present in an amino acid sequence selected from 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 variable domain present in a polypeptide having an amino acid sequence selected from 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 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
  • 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.
  • CD20scFv-1 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):
  • the targeting domain comprises a polypeptide comprising the complementarity determining regions (CDRs) present in SEQ ID NO: 17 or a variant thereof having 1 , 2, 3, or more amino acid mutations independently selected from substitutions, insertions, and deletions.
  • the targeting domain comprises a polypeptide comprising the complementarity determining regions (CDRs) present in 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 variable domain present in 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 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 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 (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):
  • the targeting domain comprises a polypeptide comprising the complementarity determining regions (CDRs) present in SEQ ID NO: 18 or a variant thereof having 1 , 2, 3, or more amino acid mutations independently selected from substitutions, insertions, and deletions.
  • the targeting domain comprises a polypeptide comprising the complementarity determining regions (CDRs) present in 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 variable domain present in 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 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 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 (VL) is shown by an underline):
  • the targeting domain comprises a polypeptide comprising the complementarity determining regions (CDRs) present in SEQ ID NO: 19 or a variant thereof having 1 , 2, 3, or more amino acid mutations independently selected from substitutions, insertions, and deletions.
  • the targeting domain comprises a polypeptide comprising the complementarity determining regions (CDRs) present in 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 variable domain present in 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 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 an amino acid sequence of SEQ ID NO: 19, wherein the polypeptide comprises the complementarity determining regions (CDRs) present in SEQ ID NO: 19
  • 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):
  • the targeting domain comprises a polypeptide comprising the complementarity determining regions (CDRs) present in SEQ ID NO: 20 or a variant thereof having 1 , 2, 3, or more amino acid mutations independently selected from substitutions, insertions, and deletions.
  • the targeting domain comprises a polypeptide comprising the complementarity determining regions (CDRs) present in 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 variable domain present in 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 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 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 comprises a polypeptide selected from a flexible amino acid sequence, an IgG hinge region, and an antibody sequence.
  • the antibody sequence comprises an Fc domain
  • the linker comprises a hinge-CH2-CH3 Fc domain.
  • the hinge- CH2-CH3 Fc domain is derived from lgG1 , optionally from human lgG1 .
  • the hinge-CH2- CH3 Fc domain is derived from lgG4, optionally from human lgG4. in embodiments, 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 lgG4, optionally human igG4.
  • the hinge-CH2- CH3 Fc domain that lacks Fc -effector function is a mutant derivative of IgGI , optionally human lgG1 .
  • the hinge-CH2-CH3 Fc domain has reduced binding to an Fc Gamma Receptor (FcyR) selected from FcyRi , FcyRIIA, FcvRIIAB, FcyRIIIA and FcyRIl IB compared to a wild type hinge-CH2-CH3 Fc domain.
  • FcyR Fc Gamma Receptor
  • 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.
  • a linker e.g., a stabilizing domain
  • 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 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. In various embodiments, 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:
  • each of the first and second charge polarized core domains 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: 23).
  • Illustrative peptide sequences include, but are not limited to, RKGGKR (SEQ ID NO: 27) or GSGSRKGGKRGS (SEQ ID NO: 28).
  • 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: 30) or GSGSDEGGEDGS (SEQ ID NO: 31).
  • 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 lgG1 , optionally human lgG1. In embodiments, the linker comprises the hinge-CH2-CH3 Fc domain derived from lgG4, optionally human lgG4. 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.
  • 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 XnY n XnY 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 XnCY n X
  • the peptide comprising positively charged amino acid residues comprises the sequence RKGGKR (SEQ ID NO: 27) or GSGSRKGGKRGS (SEQ ID NO: 28).
  • 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 Y n Z n Y 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 O 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
  • 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 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 selfassociate.
  • 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.
  • 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 is optionally selected from a flexible amino acid sequence, IgG hinge region, or antibody sequence.
  • the linker e.g., a stabilizing domain
  • the linker comprises the hinge-CH2-CH3 Fc domain derived from lgG4, optionally human lgG4.
  • the core domain comprises the following sequence:
  • 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 lgG4 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 lgG4 hinge-CH2-CH3 is shown in boldface font. Rest of the sequences are joining linkers disclosed herein):
  • the core domain comprises the following sequence (lgG4 hinge-CH2-CH3 is shown in boldface font, rest of the sequence is a joining linker disclosed herein):
  • the core domain comprises a KIHT22Y protein comprising the following sequence (the knob in hole motif mutations are indicated by boldface, underlined font):
  • the core domain is a KIHY86T protein comprising the following sequence (the knob in hole motif mutations are indicated by boldface, underlined font):
  • the core domain comprises a lgG1 hinge-CH2-CH3 protein comprising the following sequence:
  • the core domain comprises the following sequence (lgG4 hinge-CH2-CH3 is shown in boldface font, rest of the sequence is a joining linker disclosed herein):
  • 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 etal., (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) 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., lgG1 , lgG2, lgG3, and lgG4, and lgA1 and lgA2).
  • 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 lgG1 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.
  • lgG2 has a shorter hinge than lgG1 , with 12 amino acid residues and four disulfide bridges.
  • the hinge region of lgG2 lacks a glycine residue, is relatively short, and contains a rigid polyproline double helix, stabilized by extra inter-heavy chain disulfide bridges. These properties restrict the flexibility of the Ig G2 molecule.
  • Ig G3 differs from the other subclasses by its unique extended hinge region (about four times as long as the lgG1 hinge), containing 62 amino acids (including 21 prolines and 11 cysteines), forming an inflexible poly-proline double helix.
  • the Fab fragments are relatively far away from the Fc fragment, giving the molecule a greater flexibility.
  • the elongated hinge in lgG3 is also responsible for its higher molecular weight compared to the other subclasses.
  • the hinge region of lgG4 is shorter than that of lgG1 and its flexibility is intermediate between that of lgG1 and lgG2.
  • the linker may be derived from human lgG4 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.
  • the upper hinge region includes amino acids from the carboxyl end of C H1 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 interheavy 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 lgG1 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 carbohy ⁇ rate attachment.
  • lgA1 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 lgG, IgA, IgD, and IgE, inclusive of subclasses (e.g., lgG1 , lgG2, lgG3, and lgG4, and lgA1 and lgA2)).
  • the linker (e.g., a stabilizing domain) comprises a hinge-CH2-CH3 Fc domain derived from a human lgG4 antibody.
  • the linker (e.g., a stabilizing domain) comprises a hinge-CH2-CH3 Fc domain derived from a human lgG1 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, 31 1 , 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 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.
  • 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 Q31 1 S 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.
  • 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.
  • 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. 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.
  • 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 Vy962 gamma delta T cell receptor.
  • the first domain comprises a polypeptide having an amino acid seguence 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.
  • 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
  • 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: 17-20.
  • the targeting domain is a polypeptide having an amino acid sequence of selected from SEQ ID NOs: 17- 20.
  • 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 lgG1 , optionally human lgG1.
  • the linker comprises the hinge-CH2-CH3 Fc domain derived from lgG4, optionally human lgG4.
  • 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.
  • 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 XnY n XnY 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 XnCY n XnY n (where X is a positively charged amino acid such as arginine, histidine or lysine and Y is a spacer amino acid such as se
  • the peptide comprising positively charged amino acid residues comprises the sequence RKGGKR (SEQ ID NO: 27) or GSGSRKGGKRGS (SEQ ID NO: 28).
  • 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 Y n Z n Y 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 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) (SEQ ID NO:
  • 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 etal., (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) 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., lgG1 , lgG2, lgG3, and lgG4, and lgA1 and lgA2).
  • 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 hinge region of lgG1 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.
  • lgG2 has a shorter hinge than lgG1 , with 12 amino acid residues and four disulfide bridges.
  • the hinge region of lgG2 lacks a glycine residue, is relatively short, and contains a rigid polyproline double helix, stabilized by extra inter-heavy chain disulfide bridges. These properties restrict the flexibility of the lgG2 molecule.
  • lgG3 differs from the other subclasses by its unique extended hinge region (about four times as long as the lgG1 hinge), containing 62 amino acids (including 21 prolines and 11 cysteines), forming an inflexible poly-proline double helix.
  • the Fab fragments are relatively far away from the Fc fragment, giving the molecule a greater flexibility.
  • the elongated hinge in lgG3 is also responsible for its higher molecular weight compared to the other subclasses.
  • the hinge region of lgG4 is shorter than that of lgG1 and its flexibility is intermediate between that of lgG1 and lgG2.
  • the linker may be derived from human lgG4 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.
  • the upper hinge region includes amino acids from the carboxyl end of Cm 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 interheavy 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 lgG1 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 carbohy ⁇ rate attachment.
  • lgA1 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., lgG1 , lgG2, lgG3, and lgG4, and lgA1 and lgA2)).
  • the linker (e.g., a stabilizing domain) comprises a hinge-CH2-CH3 Fc domain derived from a human lgG4 antibody.
  • the linker (e.g., a stabilizing domain) comprises a hinge-CH2-CH3 Fc domain derived from a human lgG1 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, 31 1 , 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 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.
  • 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 Q31 1 S 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.
  • 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 present disclosure relates to a method for treating 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) 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
  • the present disclosure relates to method for reducing the number of B cells and/or reducing the activity B cells 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) 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 ;
  • the present disclosure relates to a method for increasing and/or stimulating killing of B cells 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) 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
  • the present disclosure relates to a method of contemporaneous activation and targeting of gamma delta T cells to cancer cells, 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) 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
  • the present disclosure relates to a method of modulating a patient’s immune response, 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) 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 CD
  • the present disclosure relates to a method of treating cancer, comprising administering to a subject in need thereof a pharmaceutical composition, 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) 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
  • 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), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), Mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, Burkitt-like lymphoma, lymphoplasmacytic lymphoma (Waldenstrom macroglobulinemia), hairy cell leukemia (HCL), primary central nervous system (CNS) lymphoma and primary intraocular lymphoma (lymphoma of the eye).
  • the 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, Sjogren'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.
  • 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.
  • the cancer is an acute myeloid leukemia (AML).
  • AML acute myeloid leukemia
  • the cancer is a solid cancer selected from prostate cancer, small cell lung cancer, non-smaii cell lung cancer, neuroendocrine tumors, renai 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 B-cell lymphoma is selected from diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), Mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, Burkitt-like lymphoma, lymphoplasmacytic lymphoma (Waldenstrom macroglobulinemia), hairy cell leukemia (HCL), primary central nervous system (CNS) lymphoma and primary intraocular lymphoma (lymphoma of the eye).
  • DLBCL diffuse large B-cell lymphoma
  • CLL chronic lymphocytic leukemia
  • SLL small lymphocytic lymphoma
  • MCL Mantle cell lymphoma
  • marginal zone lymphoma Burkitt lymphoma
  • Burkitt-like lymphoma lymphoplasmacytic lymphoma
  • HCL hairy cell le
  • 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 CD20+ cancer expresses CD20 at a level that is higher than non-cancerous tissue.
  • the CD20+ cancer expresses CD20 at a level that is higher than surrounding non-cancerous tissue.
  • the CD20+ cancer expresses CD20 at a level that is higher than non-cancerous tissue of the same tissue origin as the cancer, in embodiments, the CD20+ cancer expresses CD20 at a level that is higher than non-cancerous B cells. In embodiments, the CD20+ cancer expresses CD20 at a level that is higher than non-cancerous tissue. In embodiments, the CD20+ cancer comprises higher amount of CD20 on blood cells, B cells and/or cancer cells compared to the surrounding non-cancerous tissue. In embodiments, the CD20--- cancer comprises higher amount of CD20 on blood cells. B cells and/or cancer cells compared to the same tissue origin as the cancer. In embodiments, the CD20+ cancer comprises higher amount of CD20 on blood cells, B cells and/or cancer cells compared to the blood cells and/or B cells in an individual not suffering from cancer.
  • 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 ceil 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 expresses CD20 at a level that is higher than non-cancerous tissue.
  • the cancer expresses CD20 at a level that is higher than surrounding non-cancerous tissue.
  • the cancer expresses CD20 at a level that is higher than non-cancerous tissue of the same tissue origin as the cancer. In embodiments, the cancer expresses CD20 at a level that is higher than non-cancerous B cells, in embodiments, the cancer expresses CD20 at a level that is higher than non-cancerous tissue. In embodiments, the cancer comprises higher amount of CD20 on blood cells, B cells and/or cancer cells compared to the surrounding non-cancerous tissue, to embodiments, the cancer comprises higher amount of CD20 on blood cells, B cells and/or cancer ceils compared to the same tissue origin as the cancer. In embodiments, the cancer comprises higher amount of CD20 on biood cells, B cells and/or cancer cells compared to the blood cells and/or B cells in an individual not suffering from cancer.
  • 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 ceils 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 CD20 positive cancer expresses CD20 at a level that is higher than non-cancerous tissue. In embodiments, the CD20 positive cancer expresses CD20 at a level that is higher than surrounding non-cancerous tissue. In embodiments, the CD20 positive cancer expresses CD20 at a level that is higher than non-cancerous tissue of the same tissue origin as the cancer, in embodiments, the CD20 positive cancer expresses CD20 at a level that is higher than non-cancerous B cells.
  • the CD20 positive cancer expresses CD20 at a level that is higher than non-cancerous tissue, in embodiments, the CD20 positive cancer comprises higher amount of CD20 on blood cells, B cells and/or cancer cells compared to the surrounding non-cancerous tissue. In embodiments, the CD20 positive cancer comprises higher amount of CD20 on blood cells, B ceils and/or cancer cells compared to the same tissue origin as the cancer. In embodiments, the CD20 positive cancer comprises higher amount of CD20 on blood cells, B cells and/or cancer cells compared to the blood cells and/or B cells in an individual not suffering from cancer.
  • 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 CD2O 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 ceil 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 CD20+ cancer expresses CD20 at a level that is higher than non-cancerous tissue. In embodiments, the CD20+ cancer expresses CD20 at a level that is higher than surrounding non- cancerous tissue. In embodiments, the CD20+ cancer expresses CD20 at a level that is higher than non- cancerous tissue of the same tissue origin as the cancer. In embodiments, the CD20+ cancer expresses CD20 at a level that is higher than non-cancerous B cells, In embodiments, the CD20+ cancer expresses CD20 at a level that is higher than non-cancerous tissue. In embodiments, the CD20+ cancer comprises higher amount of CD20 on blood cells, B cells and/or cancer ceils compared to the surrounding non- cancerous tissue.
  • the CD20+ cancer comprises higher amount of CD20 on blood cells, B cells and/or cancer cells compared to the same tissue origin as the cancer. In embodiments, the CD20+ cancer comprises higher amount of CD20 on blood cells, B cells and/or cancer cells compared to the blood cells and/or B cells in an individual not suffering from cancer.
  • 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 specifically binding to CD20.
  • the method optionally further comprises proliferating ex vivo the cell derived from the
  • 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 y ⁇ T cells, or contacting a second pharmaceutical composition that costimulates y ⁇ T cells, with a cell derived from a subject in need thereof thereby causing an ex vivo proliferation of gamma delta T cells.
  • 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.
  • 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 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 y ⁇ 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, 4-1 BB (CD137), IL-2R, IL-15R, IL-7R, IL-21 R, 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, 4-1 BB (CD137), IL-2R, IL-15R, IL-7R, IL-21 R, NKp30, NKp44, DNAM-1 (CD226), IL-2R, IL-7R, IL-15R, de
  • 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 costimulatory 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. 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.
  • 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, 1 D1 1 and 5C6.
  • the second pharmaceutical composition comprises a heterologous chimeric protein capable of costimulating a receptor selected from CD28, NKG2D, CD27, CD30, 4-1 BB (CD137), IL-2R, IL-15R, IL-7R, IL-21 R, 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, 4-1 BB
  • 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. 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.
  • 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 y ⁇ T cells of any of the 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), 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.
  • AML acute myeloid leukemia
  • the cancer is known to express the antige ic 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 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 ceils and/or contacting a ceil 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 ceils.
  • 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 ceil 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 CD2CH, 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 ceil 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 ceils.
  • 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).
  • 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, Sjogren'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
  • 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, Sjogren'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.
  • 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.
  • 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, MDX1 106, OPDIVO, BRISTOL MYERS SQUIBB), pembrolizumab (KEYTRUDA, MERCK), pidilizumab (CT-011 , CURE TECH), MK- 3475 (MERCK), BMS 936559 (BRISTOL MYERS SQUIBB), Ibrutinib (PHARMACYCLICS/ABBVIE), atezolizumab (TECENTRIQ, GENENTECH), and/or MPDL328OA (ROCHE)-refractory patients.
  • nivolumab ONO-4538/BMS-936558, MDX1 106, OPDIVO, BRISTOL MYERS SQUIBB
  • pembrolizumab KEYTRUDA, MERCK
  • 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; tumorinfiltrating 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.
  • a cell e.g., cancer cell or immune cell
  • 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.
  • diseases or disorders described elsewhere herein e.g., GVHD
  • transplant rejection e.g., transplant rejection, 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.
  • GVHD graft-versus-host disease
  • 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 y ⁇ T cells, or contacting a second pharmaceutical composition that costimulates y ⁇ 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.
  • 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
  • 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, car
  • 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, cyanomorpholinodoxorubicin, 2-pyrrolino-doxorubicin and deoxy doxorubicin), epirubicin, 6-diazo- 5-oxo-L-norleucine,
  • the present additional agent is one or more immune-modulating agents selected from an agent that blocks, reduces and/or inhibits 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 (4-1 BB) and/or the binding of CD137 (4-1 BB) with one or more of 4-1 BB ligand (by way of non-limiting example, urelumab (BMS-
  • 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.
  • anti-malarial agents e.g., chloroquine, quinine, mefloquine, primaquine, doxycycline, artemether/lumefantrine, atovaquone/proguanil and sulfadoxine/pyrimethamine
  • metronidazole e.g., chloroquine, quinine, mefloquine, primaquine, doxycycline, artemether/lumefantrine, atovaquone/proguanil and sulfa
  • 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).
  • NSAID non-steroidal anti-inflammatory agent
  • Steroids, particularly the adrenal corticosteroids and their synthetic analogues, are well known in the art.
  • corticosteroids useful in the current disclosure include, without limitation, hy ⁇ roxyltriamcinolone, alphamethyl 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, hy ⁇ rocortisone acetate, hy ⁇ rocortisone butyrate, methylpre
  • 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- transl ationally 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.
  • the present disclosure relates to a pharmaceutical composition, comprising the heterodimeric protein of any one of the embodiments disclosed herein, and a carrier.
  • 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.
  • 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.
  • 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.
  • compositions described herein are resuspended in a saline buffer (including, without limitation TBS, PBS, and the like).
  • 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 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.
  • 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. Gennaro eds., 19th ed. 1995), incorporated herein by reference.
  • 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. 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).
  • 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
  • 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 administering is effected orally or by parenteral injection. In most instances, administration results in the release of any agent described herein into the bloodstream.
  • 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.
  • 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; tumorinfiltrating 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 heterodimeric protein (and/or additional agents) 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 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 e.
  • 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,
  • 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 (/.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).
  • 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
  • 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 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
  • 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).
  • 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, hy ⁇ ropropylmethyl 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 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)
  • 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.
  • 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, Ipp, phoA, recA, tac, T3, T7 and PL.
  • Non-limiting examples of prokaryotic expression vectors may include the Agt vector series such as Agt11 (Huynh et al., in "DNA Cloning Techniques, Vol. I: A Practical Approach,” 1984, (D Glover, ed.), pp.
  • 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.
  • 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 p-interferon gene, and the hspZO gene (see, Williams ef 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.
  • 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.
  • 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.
  • 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.
  • promoter sequences such as native promoter and enhancer elements
  • 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).
  • operable linkage refers to a physical juxtaposition of the components so described as to permit them to function in their intended manner.
  • 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 (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.
  • 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. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter.
  • 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
  • nucleic acids 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.
  • 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 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 geneediting 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 : 1 17, 122, 2003.
  • Illustrative viral vectors include those selected from Antiviruses (LV), retroviruses (RV), adenoviruses (AV), adeno-associated viruses (AAV), and a 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 a viruses and adenoviruses.
  • viruses include Sindbis virus, Venezuelan equine encephalitis (VEE) virus, and Semliki Forest virus (SFV).
  • VEE Venezuelan equine encephalitis
  • SFV Semliki Forest virus
  • 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 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., VERO-76, ATCC CRL-1587
  • human cervical carcinoma cells e.g.
  • 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.
  • Fc-containing macromolecules such as Fc fusion proteins
  • 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
  • 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.
  • 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.
  • 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. 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.
  • 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.
  • 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.
  • the human is an adult human.
  • the human is a geriatric human.
  • the human may be referred to as a patient.
  • 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 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.
  • the subject is a non-human animal, and therefore the invention pertains to veterinary use.
  • the non-human animal is a household pet.
  • the non-human animal is a livestock animal.
  • 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 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.
  • BTN2A1 -Fc-CD20scFv protein alpha chain
  • BTN3A1-Fc-CD20scFv protein beta chain
  • 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-CD20scFv homodimeric protein or BTN3A1 -Fc-CD20scFv 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- CD20scFv homodimeric protein or BTN3A1-Fc-CD20scFv homodimeric protein, or BTN2A1/3A1-Fc- CD20scFv heterodimeric protein were added in high-binding 96-well plates (CORNING) and 1 pg /mL of an anti-NKG2D antibody was added to wells containing the BTN2A1 -Fc-CD20scFv homodimeric protein, BTN3A1-Fc-CD20scFv homodimeric protein, or BTN2A1/3A1 -Fc-CD20scFv heterodimeric protein.
  • BTN2A1-Fc-CD20scFv homodimeric protein or BTN3A1-Fc-CD20scFv 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 Vy9V ⁇ 2+-T cells in RPMI + 10% PBS, supplemented with 100 U/mL hlL-2.
  • BD Fastlmmune 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+Vy9+ population.
  • a dose-dependent increase in CD107a- expressing Vy9V ⁇ 2+-T cells was observed when the Vy9V ⁇ 2+-T cells were induced by the combination of BTN2A1/3A1-Fc-CD20scFv heterodimeric protein and the anti-NKG2D antibody.
  • Vy9V ⁇ 2+-T cells induced the CD107a in combination with BTN2A1-Fc-CD20scFv or BTN3A1 -Fc- CD20scFv homodimeric protein expressed only a background levels of CD107a (FIG. 2A).
  • CD20+ lymphoma cells and B cells were killed by a BTN2A1/3A1-Fc- CD20scFv heterodimeric protein in an in vitro assay.
  • human Daudi Burkitt's lymphoma cells (CD20+ lymphoma cell line) were labeled with a fluogenic granzyme B substrate and co-cultured with Vy9+V62+ 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 y ⁇ T cell cytotoxicity by transferring granzyme B into target cells.
  • CD20+ B cells purified from PBMC of healthy donor were cocultured with Vy9+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 8-10 weeks old immunocompromised NSG-IL15 mice were engrafted with 1 * 10 7 human PBMCs (FIG. 3A).
  • PBS vehicle control
  • BTN2A1/3A1-Fc-CD20scFv heterodimeric protein 50 g or 200pig
  • BTN2A1/3A1-Fc-CD33scFv heterodimeric protein 200 ig, non-B cell targeting construct
  • Peripheral blood and spleens were harvested from animals 24 hours post last dose for quantification of B cells (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.
  • FIG. 3F are box plots showing the changes in B cells (left panel) and V62+ T cells (Right panel) as a function of the dose of the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein in PBMC-humanized mice.
  • 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 (50pg). 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 50pg
  • gamma delta T cell mediated killing of B cells 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 killing of B cells.
  • the results are shown in the Table below: These results demonstrate, inter alia, gamma delta T cell mediated killing of B cells upon the BTN2A1/3A1-Fc-CD20scFv heterodimeric protein treatment at all donor E:T ratios.
  • mice 8- 10 weeks old immunocompromised NSG-IL15 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 pig, 2 pig, 10 pig or 50 pig).
  • PBS vehicle control
  • BTN2A1/3A1- Fc-CD20scFv heterodimeric protein 0.5 pig, 2 pig, 10 pig or 50 pig.
  • 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 of gamma delta T cell mediated killing of B cells 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 killing of B cells.
  • FIG. 3E left panel
  • there was dosedependent killing of B cells although the lowest dose used in this experiment provided a significant gamma delta T cell mediated killing of B cells.
  • FIG. 3E (right panel), the numbers of V ⁇ 2+T cells remained essentially unchanged.
  • gamma delta T cells have the ability to promote serial killing of target cells in the absence of proliferation or expansion.
  • FIG. 4A 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*10 6 CD20 + human lymphoma Raji cells and 4x10 6 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 V ⁇ 2+ T cells (CD45+CD3+V ⁇ 2+T cells).
  • mice 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. These analyzes showed that the ratio of total CD3+T to Raji cells was 1 :1 .7, and the ratio of V62+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 V ⁇ 2+T cells to Raji cells was 1 :368. The mice were then randomly divided in four treatment groups:
  • a CD3xCD20 engager (100 pg; 100 nM).
  • 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 either 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 from Donor 4 or Donor 6 themselves did not affect tumor growth compared to each other.
  • the mice treated with CD3xCD20 engager showed a detectable reduction in tumor growth as illustrated by the fact that only two of six CD3xCD20 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 CD3xCD20 engager.
  • mice treated with the CD3xCD20 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.

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Abstract

La présente invention concerne, entre autres, des compositions et des méthodes, par exemple, des immunothérapies du cancer et de l'auto-immunité. Les présentes compositions comprennent des protéines hétérodimères comprenant une chaîne alpha et une chaîne bêta, la chaîne alpha et la chaîne bêta comprenant toutes deux une partie de butyrophiline BTN2A1 et/ou BTN3A1 adjointe par l'intermédiaire d'un lieur à un domaine de ciblage dirigé vers CD20.
PCT/US2023/069345 2022-06-29 2023-06-29 Modulation de lymphocytes t gamma delta ciblant cd20 Ceased WO2024006889A2 (fr)

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