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WO2025076485A1 - Nouvelles compositions de lymphocytes t tueurs naturels invariants et d'activateurs de lymphocytes t et méthodes d'utilisation associées - Google Patents

Nouvelles compositions de lymphocytes t tueurs naturels invariants et d'activateurs de lymphocytes t et méthodes d'utilisation associées Download PDF

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WO2025076485A1
WO2025076485A1 PCT/US2024/050139 US2024050139W WO2025076485A1 WO 2025076485 A1 WO2025076485 A1 WO 2025076485A1 US 2024050139 W US2024050139 W US 2024050139W WO 2025076485 A1 WO2025076485 A1 WO 2025076485A1
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cancer
seq
pharmaceutical composition
cells
cell
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Marc VAN DIJK
Eleni CHANTZOURA
Martyna POPIS
Efrat ALTMAN-SHARONI
Magdalena Niedzielska
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Mink Therapeutics Inc
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Mink Therapeutics Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/15—Natural-killer [NK] cells; Natural-killer T [NKT] cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
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    • 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
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    • 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
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    • C—CHEMISTRY; METALLURGY
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39541—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/39558—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against tumor tissues, cells, antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/526—CH3 domain
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • T cells including allogeneic invariant natural killer T cells, and T cell engagers and methods of using such cells and T cell engagers for the treatment of cancer.
  • Invariant natural killer T (iNKT) cells also known as type I or classical NKT cells, function during innate and adaptive immunity and are rare in the human blood pool, comprising just 0.01-1% of peripheral blood mononuclear cells (PBMCs).
  • PBMCs peripheral blood mononuclear cells
  • iNKT cells express a unique TCR containing an invariant Va24-Jal8 a-chain and a limited number of P chains.
  • iNKT cells respond and are activated by glycolipid antigens presented by the MHC class lb related molecule, CD Id.
  • the CD3 binding domain comprises a variable light chain or binding fragment thereof with an amino acid sequence at least about 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% or more identical to the amino acid sequences of the variable light chain of one of HuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, L2K, and I2C.
  • the NKT cells are allogeneic cells.
  • NKT cells e.g., iNKT cells
  • iNKT cells are stimulated and expanded by culturing in the presence of (i) IL-2, IL-7, IL- 15, IL- 12, and/or TNE-alpha and (ii) irradiated PBMCs loaded with aGalCer or irradiated artificial antigen-presenting cells (aAPCs) expressing CDl-d loaded with aGalCer.
  • the NKT cells e.g., iNKT cells
  • the NKT cells e.g., iNKT cells
  • the NKT cells are engineered to express another molecule (e.g., a cytokine or ligand) capable of enhancing one or more properties (e.g., survival/persistence, immune interaction with other cells such as macrophages and/or dendritic cells, disrupt immunosuppressive tumor microenvironment) of the cells.
  • a cytokine or ligand e.g., a cytokine or ligand
  • the NKT cells are engineered to express any suitable armoring molecule known in the art.
  • the invention includes NKT cells (e.g., iNKT cells) engineered to express IL-15, IL-2, IL-12, CD40L, 4- 1BBL, IL- 18, IL-7, IL-33, constitutively active Akt (caAkt), hybrid IL-4/IL-7 receptor, checkpoint inhibitors such as anti-PDl antibodies, nanobodies targeting CD47, or bispecific T-cell engagers (BiTEs).
  • a subject in need of treatment is administered a therapeutically effective amount of a pharmaceutical composition comprising NKT cells (e.g., iNKT cells) and a therapeutically effective amount of a pharmaceutical composition comprising a T cell engager.
  • NKT cells e.g., iNKT cells
  • T cell engager a pharmaceutical composition comprising a T cell engager.
  • the NKT cells e.g., iNKT cells
  • T cell engagers are separately formulated (i.e., two separate pharmaceutical compositions) but are administered to the subject at the same time.
  • the NKT cells (e.g., iNKT cells) administered to the subject are allogeneic NKT cells (e.g., allogeneic iNKT cells).
  • the NKT cells are not autologous NKT cells (for instance, not autologous iNKT cells).
  • lymphodepletion agents are not administered to the subject prior to the administration of NKT cells (e.g., iNKT cells) and T cell engager and are not administered concurrently with the NKT cells (e.g., iNKT cells) and T cell engager.
  • the subject does not receive treatment with bendamustine, fludarabine, or cyclophosphamide prior to, or concurrent with, the treatment with allogeneic iNKT cells and T cell engager.
  • the cancer is a blood malignancy. In certain methods of the invention, the cancer is a B cell malignancy. In certain methods of the invention, the cancer is In some embodiments the cancer is diffuse large B-cell lymphoma, follicular lymphoma, marginal zone B-cell lymphoma, mucosa-associated lymphatic tissue lymphoma, small lymphocytic lymphoma (also known as chronic lymphocytic leukemia, CLL), mantle cell lymphoma, primary mediastinal (thymic) large B cell lymphoma, T cell/histiocyte-rich large B-cell lymphoma, primary cutaneous diffuse large B-cell lymphoma, EBV positive diffuse large B-cell lymphoma, Burkitt’s lymphoma, lymphoplasmacytic lymphoma, nodal marginal zone B cell lymphoma, splenic marginal zone lymphoma, intravascular large B-cell lymphoma, primary effusion lymph
  • the cancer comprises a blood cancer.
  • the blood cancer comprises myeloma, leukemia, lymphoma, Non-Hodgkin lymphoma, Hodgkin lymphoma, a myeloid neoplasm, a lymphoid neoplasm, acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), or chronic myelogenous leukemia (CML).
  • ALL acute lymphoblastic leukemia
  • AML acute myelogenous leukemia
  • CLL chronic lymphocytic leukemia
  • CML chronic myelogenous leukemia
  • FIGURE 5 shows the cytolytic activity of a CD19-CD3 bispecific antibody against CD19+ and CD19- cancer cells.
  • CFSE labeled target cells A375 (A), A375-CD19 (B), C1R (C), or Raji (D) were used for the iNKT cytotoxicity assay.
  • expanded 1st or 2nd stim iNKT cells from two donor (effector cells) were incubated with target cells at 4: 1 E:T ratio in presence or absence of CD19-CD3 engager or HIV-CD3 for 48 h in Xuri based medium with 100 lU/ml IL-2.
  • FIGURE 7 shows Mucl6-CD3 mediated iNKT cytotoxicity.
  • the assay was performed in 2D adherent cell cultures using Mucl6-CD3 engager.
  • iNKT cells from six donors were used as effector cells.
  • CFSE labeled OVCAR-3 cell line was used as target cells.
  • Effector and target cells ware co-cultured at 5:1, 1:1 and 1:5 E:T ratio in presence or absence of Mucl6-CD3 engager for 24h, 48h and 72h. Percentage- specific lysis of the target cells was determined using flow cytometry. Cells were acquired by FACS Canto and analyzed in Flow Jo.
  • FIGURES 11A-11D compare engager-mediated killing with iNKT versus T- cells.
  • FIGURES 11A-11D show CD3-engager mediated iNKT and T cell cytotoxicity. Assay was performed in 2D adherent cell cultures using Mucl6-CD3 engager molecule and DLL3-, Her3-, and Cldnl8.2-CD3 engager molecules (all purchased from Proteogenix). Expanded 2nd stim iNKT cells from two donors were used as effector cells. CFSE labeled OVCAR-3, SHP77, SKBR3, and PATU8988S cell lines was used as target cells.
  • Effector and target cells ware co-cultured at 5:1, 1:1 and 1:5 E:T ratio in presence or absence of CD3 engagers in RPMI-1640 based medium with 50 lU/ml IL-2 for 24h, 48h and 72h. Percentage- specific lysis of the target cells was determined using flow cytometry. Cells were harvested following the incubation period, stained with a live dead dye and activation markers. Cells were acquired by FACS Canto and analyzed in Flow Jo.
  • iNKT cells are capable of complexing with T cell engagers and “cross-link” tumor cells.
  • the T cell engager binds CD3 on an iNKT cell (via its CD3 binding domain).
  • the T cell engager binds an overexpressed cell surface tumor antigen on the surface of the tumor cell (via its tumor antigen binding domain), and an iNKT cell binds tumor cells via its invariant T cell receptors.
  • iNKT cells are capable of binding stress ligands on tumor cells via NK receptors such as NKG2D or DNAM1.
  • NK receptors such as NKG2D or DNAM1.
  • the binding of the iNKT cells to tumor cells activates iNKT cells, resulting in the release of cytokines and tumor cell lysis.
  • the cytokine release profile of activated iNKT cells is different from that of activated T cells.
  • the cytokine release profile of activated iNKT cells shows less TNFa as compared to that of activated T cells.
  • natural killer T or “NKT” cells constitute a small population of non-conventional T lymphocytes defined by the expression of both aP T-cell receptors (TCR) and some lineage markers of NK cells.
  • TCR T cell receptor
  • TCR T cell receptor
  • iNKT cells invariant natural killer T cell
  • iNKT cells encompass both native and engineered invariant natural killer T cells.
  • iNKT cells can be harvested from the body, for instance, isolated from peripheral blood and expanded.
  • iNKT cells are harvested from healthy donors.
  • iNKT cells are isolated from the blood or PBMCs of healthy doors and subsequently activated and expanded by culturing the iNKT cells in the presence of one or more activating agents.
  • the invention includes iNKT cells which are isolated from donor cells and cultured in the presence of aGalCer -pulsed irradiated PBMCs and interleukin-2 (IL-2).
  • IL-2 interleukin-2
  • TAMs CD Id-positive tumor-associated macrophages
  • IL-6 interleukin-6
  • TAMs are known to be a major producer of interleukin-6 (IL-6) that promotes proliferation of many solid tumors, including neuroblastomas and breast and prostate carcinomas (See Song et al., J Clin Invest, 119: 1524-1536, 2009; Hong et al, Cancer, 110: 1911-1928, 2007).
  • IL-6 interleukin-6
  • Direct CD Id-dependent cytotoxic activity of iNKT cells against TAMs suggests that important alternative indirect pathways exist by which iNKT cells can mediate antitumor immunity, especially against solid tumors that do not express CD Id.
  • the recognition unit the CD Id molecule
  • the recognition unit has a structure closely resembling that of the MHC class I molecule, including the presence of beta-2 microglobulin. It is characterized by a deep cleft bordered by two alpha chains and containing highly hydrophobic residues, which accepts lipid chains. The cleft is open at both extremities, allowing it to accommodate longer chains.
  • the canonical ligand for CD Id is the synthetic alpha galactosylceramide (“alpha GalCer”).
  • NKT cells e.g., iNKT cells
  • the cells are isolated from a healthy donor and the recipient is a subject suffering from a cancer.
  • donor cells are activated and expanded prior to administering to an unrelated subject.
  • iNKT cells harvested from the peripheral blood of a single healthy donor can be subsequently expanded, formulated, and optionally cryopreserved, for use for treatment of several hundred subjects.
  • autologous is intended to refer to NKT cells (e.g., iNKT cells) that are isolated from a subject and returned to the same subject.
  • the invention includes isolating iNKT cells from a subject, expanding the cells and using the cells in conjunction with a T cell engager to treat the subject.
  • the iNKT cells of the pharmaceutical compositions and methods are not autologous (i.e., the cells are allogeneic).
  • isolated for example, with respect to cells and/or nucleic acids means altered or removed from the natural state through human intervention.
  • Isolated iNKT cells are iNKT cells that have been removed from surrounding cells, for instance, isolation of iNKT cells from a sample of PBMCs or leukopak.
  • the iNKT activator may be a GalCer derivative in which galactose, a key element of iNKT TCR recognition, is substituted with a nonglycosidic substitutions, for example, as described in Silk et al., Cutting Edge: Nonglycosidic CD Id Lipid Ligands Activate Human and Murine Invariant NKT Cells, J Immunol 2008; 180:6452- 6456.
  • Exemplary GalCer variants include threitolceramide arabinitolceramide, and glycerolceramide.
  • the iNKT activator is threitolceramide modified to have restricted flexibility in the sugar headgroup, for example, by incorporating the threitol unit into a carbocycle such as six- or seven-membered ring, for example, as described in Jukes et al., Non-glycosidic compounds can stimulate both human and mouse iNKT cells, Eur. J. Immunol. 2016. 46: 1224-1234.
  • the iNKT activator is a GalCer variant comprising
  • the iNKT activator is a GalCer variant comprising carbohydrate and sphingoid base modifications in an a-galactosyl ceramide, for instance, as described in Chennamadhavuni et al., Dual Modifications of a-Galactosylceramide Synergize to Promote Activation of Human Invariant Natural Killer T Cells and Stimulate Anti-tumor Immunity, 2018, Cell Chemical Biology 25, 571-584.
  • the iNKT activator is a GalCer variant comprising an
  • E-alkene linker between the sugar and lipid moieties for example, GCK152, which has an aromatic ring in the tail of the acyl chain.
  • the iNKT activator comprises glyco sphingolipid, a- glyco(Gal/Glc)diacylglycerol, P-glucosylsphingosine, phosphatidylinositol, phosphatidylethanolamine, phosphatidylglycerol, diphosphati-dylglycerol, lysophosphatidylcholine, or phenyl 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonate.
  • the iNKT activator is a naturally occurring glycolipid antigen derived from a microbial pathogen that acts as an activator of iNKT cells, for instance, glycosylceramides from Sphingomonadaceae bacterial species, including, for example, GSL-1, GSL-3 and GSL-4; galactosyl diacylglycerols isolated from Borrelia burgdorferi; galactosyl diacylglycerols from Streptococcus pneumoniae; a-Linked glucosyl DAGs and a disaccharide Gal-Glu-DAG of the protozoan Entamoeba histolytica; asperamide B from Aspergillus spp.; a-mannnosyll-3 (6'-O-acyl a-mannosyl)-l-l monoacylglycerol from Saccharopolyspora; cholesteryl 6'-O-acyl a-mannoside,
  • T cell engager refers to an engineered antibody comprising at least one tumor antigen binding domain and at least one CD3 binding domain.
  • the invention includes bispecific and multi- specific T cell engagers.
  • a “tumor antigen domain” or “tumor antigen binding domain” refers to a binding domain that specifically binds a tumor antigen.
  • a “CD3 binding domain” refers to a binding domain that specifically binds CD3.
  • the T cell engagers of the invention may be configured in any bispecific or multi- specific antibody format known in the art.
  • the T cell engagers of the invention may comprise additional domains or regions.
  • the T cell engager is an engineered antibody selected from the group consisting of knobs-into-holes (including knobs- into holes with cognate light chain and knobs-into-holes with common light chain), scFv-Ig, BiTE, BiTE-Fc, DART, DART-Fc, Tetravalent DART, Diabody, BiKE, TriKE, scFv-scFv- scFv, TandAb, TrioMab, CrossMabFab, CrossMab VH-VL, 2:1 CrossMab, 2:2 CrossMab, DVD-Ig, IgG-IgG, and Fab-scFv-Fc.
  • the invention includes T cell engagers in the antibody formats as described in Wang Q, Chen Y, Park J, Liu X, Hu Y, Wang T, McFarland K, Betenbaugh MJ. Design and Production of Bispecific Antibodies. Antibodies (Basel). 2019 Aug 2;8(3):43 and in Ma J, Mo Y, Tang M, Shen J, Qi Y, Zhao W, Huang Y, Xu Y and Qian C (2021) Bispecific Antibodies: From Research to Clinical Application. Front. Immunol. 12:626616, each of which is hereby incorporated by reference in its entirety.
  • a heterodimer T cell engager is produced using strand-exchange engineered domain (“SEED”) heterodimerization.
  • SEED heterodimerization utilizes complementarity of alternating sequences derived from IgG and IgA CH3 domains also known as AG SEED CH3 and GA SEED CH3.
  • the IgG and IgA CH3 derivatives generate complementary sequences so that the two complementary heavy-chain heterodimers are assembled while excluding the assembly of homodimers lacking complementarity.
  • SEEDbodies Fusion proteins based on strand-exchange engineered domain (SEED) CH3 heterodimers in an Fc analogue platform for asymmetric binders or immunofusions and bispecific antibodies. Protein Eng. Des. Sei. PEDS. 2010;23:195-202.)
  • the T cell engager comprises a variable heavy chain that specifically binds to the tumor antigen, a variable heavy chain that specifically binds to CD3, and a common variable light chain.
  • An exemplary amino acid sequence of a common variable light chain is set forth below: DIOMTOSPSSLSASVGDRVTITCRASOSISTYLNWYOOKPGKAPKLLIYTASSLQSGV PSRFSGSGTDFTLTISSLQPEDFATYYCQQSYSTPPITFGOGTRLEIK (SEQ ID NO:33; CDRs underlined).
  • the T cell engagers are in the bispecific T-cell engager (BiTE), dual-affinity re-targeting (DARTs) or Tandem diabodies (TandAbs) format.
  • BiTE bispecific T-cell engager
  • DARTs dual-affinity re-targeting
  • TandAbs Tandem diabodies
  • the T cell engager is a modified BiTE antibody.
  • the invention includes BiTE-albumin and BiTE-Fc T cell engagers.
  • BiTE-albumin and BiTE-Fc T cell engagers See Arvedson T.L., Balazs M., Bogner P., Black K., Graham K., Henn A., Friedrich M., Hoffmann P., Kischel R., Kufer P., et al. Abstract 55: Generation of half-life extended anti- CD33 BiTE® antibody constructs compatible with once-weekly dosing. Cancer Res.
  • tumor-associated antigens As used herein, “tumor-associated antigens,” “cell surface tumor antigens,” or
  • the binding domain comprises a variable heavy chain sequence and variable light chain sequence or three light chain complementary determining regions (CDRs) and three heavy chain CDRs from an antibody placed into alternative framework regions (FRs) (e.g., human FRs optionally comprising one or more amino acid substitutions).
  • FRs alternative framework regions
  • the binding domain comprises an scFv.
  • assays are known for identifying binding domains of the present disclosure that specifically 1 bind a particular target, including Western blot, ELISA, phage display library screening, and BIACORE interaction analysis.
  • antibodies or fragments thereof to the aforementioned antigens can be obtained by using methods which are described, e.g., in Harlow and Lane “Antibodies, A Laboratory Manual”, CSH Press, Cold Spring Harbor, 1988. Methods for the production of humanized antibodies are described in, e.g., EP-A1 0 239 400 and WO 90/07861.
  • a further source of antibodies to be utilized in accordance with the present invention are so-called xenogenic antibodies.
  • the general principle for the production of xenogenic antibodies such as human antibodies in mice is described in, e.g., WO 91/10741, WO 94/02602, WO 96/34096 and WO 96/33735.
  • tumor antigen binding domain includes nonimmunoglobulins that are capable of specifically binding a cell surface tumor antigen.
  • the invention includes binding domain polypeptides that are derived from polypeptide ligands such as hormones, cytokines, chemokines, and the like; cell surface or soluble receptors for such polypeptide ligands; lectins; intercellular adhesion receptors such as specific leukocyte integrins, selectins, immunoglobulin gene superfamily members, intercellular adhesion molecules (ICAM-1, -2, -3) and the like; histocompatibility antigens; etc.
  • polypeptide ligands such as hormones, cytokines, chemokines, and the like
  • cell surface or soluble receptors for such polypeptide ligands cell surface or soluble receptors for such polypeptide ligands
  • lectins lectins
  • intercellular adhesion receptors such as specific leukocyte integrins, selectins, immunoglobul
  • Examples of cell surface receptors that may provide a binding domain polypeptide, and that may also be selected as the target molecule or antigen to which a binding domain binds include the following, or the like: HER1 (e.g., GenBank Accession Nos. U48722, SEG_HEGFREXS, KO3193), HER2 (Yoshino et al., 1994 J. Immunol. 152:2393; Disis et al., 1994 Cane. Res. 54:16; see also, e.g., GenBank Acc. Nos. X03363, M17730, SEG_HUMHER20), HER3 (e.g., GenBank Acc. Nos.
  • GenBank Acc. Nos. L07868, T64105 epidermal growth factor receptor
  • EGFR epidermal growth factor receptor
  • vascular endothelial cell growth factor e.g., GenBank No. M32977
  • vascular endothelial cell growth factor receptor e.g., GenBank Acc. Nos. AF022375, 1680143, U48801, X62568, insulin-like growth factor-I (e.g., GenBank Acc. Nos.
  • SEG_MUSMUCIO, M65132, M64928) NY-ESO-1 (e.g., GenBank Acc. Nos. AJ003149, U87459), NA 17-A (e.g., European Patent No. WO 96/40039), Melan- A/MART-1 (Kawakami et al., 1994 Proc. Nat. Acad. Sci. USA 91:3515; see also e.g., GenBank Acc. Nos. U06654, U06452), tyrosinase (Topalian et al., 1994 Proc. Nat. Acad. Sci. USA 91:9461; see also e.g., GenBank Acc. Nos.
  • binding domain of a T cell engager interacts or specifically interacts with a given epitope, target or antigen on the tumor cell or CD3.
  • the interaction between the binding domain of the T cell engager and the epitope or the region comprising the epitope implies that a binding domain exhibits appreciable affinity for the epitope / the region comprising the epitope on a particular protein or antigen and, generally, does not exhibit significant reactivity with proteins or antigens other than the tumor antigen of interest and CD3.
  • a binding domain specifically reacts with or binds to a target can be tested readily by, inter alia, comparing the reaction of said binding domain with a target protein or antigen with the reaction of said binding domain with proteins or antigens other than the tumor antigen of interest or CD3.
  • a binding domain does not essentially or substantially bind to proteins or antigens other than the tumor antigen or CD3 (e.g., the first binding domain is not capable of binding to proteins other than the tumor antigen and the second binding domain is not capable of binding to proteins other than CD3).
  • Antibodies that specifically bind to the antigen or the epitope within the antigen may, however, have cross-reactivity to other related antigens, for example to the same antigen from other species (homologs), such as human or monkey, for example Macaca fascicularis (cynomolgus, cyno) or Pan troglodytes (chimpanzee, chimp). While a monospecific antibody specifically binds one antigen or one epitope, a bispecific antibody specifically binds two distinct antigens or two distinct epitopes.
  • the DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and/or constant domains into a suitable configuration, or to introduce codons, create cysteine residues, modify, add or delete amino acids, etc.
  • Non-limiting examples of antigen-binding fragments include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide), or a constrained FR3- CDR3-FR4 peptide.
  • CDR complementarity determining region
  • epitope is antigenic and thus the term epitope is sometimes also referred to herein as “antigenic structure” or “antigenic determinant.”
  • binding domain is an "antigen interaction site.” Such binding/interaction is also understood to define a "specific recognition.”
  • Epitopes can be formed both by contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of a protein.
  • a “linear epitope” is an epitope where an amino acid primary sequence comprises the recognized epitope.
  • a linear epitope typically includes at least 3 or at least 4, and more usually, at least 5 or at least 6 or at least 7, for example, about 8 to about 10 amino acids in a unique sequence.
  • a “conformational epitope”, in contrast to a linear epitope, is an epitope wherein the primary sequence of the amino acids comprising the epitope is not the sole defining component of the epitope recognized (e.g., an epitope wherein the primary sequence of amino acids is not necessarily recognized by the binding domain).
  • a conformational epitope comprises an increased number of amino acids relative to a linear epitope.
  • the binding domain recognizes a three- dimensional structure of the antigen, preferably a peptide or protein or fragment thereof (in the context of the present invention, the antigenic structure for one of the binding domains is comprised within the target cell surface antigen protein).
  • OKT3 monoclonal antibody See Perez P, Titus JA, Lotze MT, Cuttitta F, Longo DL, Groves ES, et al. Specific lysis of human tumor cells by T cells coated with anti-T3 cross-linked to anti-tumor antibody. J Immunol Oct. (1986) 137:2069-72; Ortho multicenter Transplant Study Group (1985) N. Engl. J. Med.313:337) and derivatives thereof such as OKT3 ala-ala (also referred to as OKT3 AA-FL or OKT3 FL), a humanized, Fc variant with alanine substitutions at positions 234 and 235 (See Herold et al. (2003) J Clin.
  • a IgGl derivative constant region has one or more of the following amino acids mutated: glutamate at position 233(E233), leucine at position 234 (L234), leucine at position 235 (L235), glycine at position 237 (G237), asparagine at position 297 (N297) glutamate at position 318 (E318), lysine at position 320 (K320), lysine at position 322 (K322), or any combination thereof (numbering according to EU).
  • any one or more of these amino acids can be changed to alanine.
  • the IgGl derivative constant region has a deletion of a glycine at position 236 (G236del).
  • the T cell engager comprises one or more of the following mutations in the constant region, N297A, E233P, F234V, L235A, G236del, and K322A.
  • the derivative constant region comprises an N297A mutation; an N297A and K322A mutations; or E233P, F234V, L235A, and G236del mutations.
  • T cell engagers comprising an Fc domain typically contain one or more mutations in the Fc region to reduce or eliminate FcyR binding.
  • the T cell engager comprises one or more of the following mutations in the Fc region, N297A, E233P, F234V, L235A, G236del, and K322A.
  • the Fc region comprises an N297A mutation; an N297A and K322A mutations; or E233P, F234V, L235A, and G236del mutations.
  • Cytokine release or "cytokine storm” or “infusion reaction” refers to the release of cytokines from T-cells.
  • systemic symptoms such as fever, nausea, chills, hypotension, tachycardia, asthenia, headache, rash, scratchy throat, and dyspnea can result.
  • Some patients may experience severe, lifethreatening reactions that result from massive release of cytokines.
  • the polypeptide or amino acid sequence which is derived from a particular sequence has an amino acid sequence that is essentially identical to the starting sequence or a portion thereof, wherein the portion consists of at least 10-20 amino acids, at least 20-30 amino acids, or at least 30-50 amino acids, or at least 50-150 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the starting sequence.
  • a binding domain can be derived from an antibody, e.g., a Fab, F(ab')2, Fab', scFv, single domain antibody (sdAb), etc.
  • nucleic acid refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the terms encompass nucleic acids containing analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated.
  • expression vector refers to a nucleic acid molecule, linear or circular, comprising one or more expression units.
  • an expression vector can also include additional nucleic acid segments such as, for example, one or more origins of replication or one or more selectable markers.
  • Expression vectors are generally derived from plasmid or viral DNA, or can contain elements of both.
  • the pharmaceutical composition comprises a therapeutically effective amount of engineered iNKT cells capable of expressing an exogenous TCR, a T cell engager, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition comprises a therapeutically effective amount of engineered iNKT cells capable of expressing a CAR (e.g., BCMA CAR or FAP CAR), a T cell engager, and a pharmaceutically acceptable carrier.
  • a CAR e.g., BCMA CAR or FAP CAR
  • T cell receptor and “TCR” are used interchangeably and refer to molecules comprising CDRs or variable regions from a[3 or y5 T cell receptors.
  • TCRs include, but are not limited to, full-length TCRs, antigenbinding fragments of TCRs, soluble TCRs lacking transmembrane and cytoplasmic regions, single-chain TCRs containing variable regions of TCRs attached by a flexible linker, TCR chains linked by an engineered disulfide bond, single TCR variable domains, single peptide- MHC-specific TCRs, multi- specific TCRs (including bispecific TCRs), TCR fusions, TCRs comprising co-stimulatory regions, human TCRs, humanized TCRs, chimeric TCRs, recombinantly produced TCRs, and synthetic TCRs.
  • the exogenous TCRs are able to recognize TAA antigens in a major histocompatibility complex (MHC) class I-dependent manner.
  • MHC class I-dependent manner means that the engineered TCR elicits an immune response upon binding to TAA antigens within the context of an MHC class I molecule.
  • the MHC class I molecule is an HLA- A molecule.
  • the NKT cells e.g., iNKT cells
  • the NKT cells are engineered to express another molecule (e.g., a cytokine or ligand) capable of enhancing one or more properties (e.g., survival/persistence, immune interaction with other cells such as macrophages and/or dendritic cells, disrupt immunosuppressive tumor microenvironment) of the genetically modified cells.
  • a cytokine or ligand capable of enhancing one or more properties (e.g., survival/persistence, immune interaction with other cells such as macrophages and/or dendritic cells, disrupt immunosuppressive tumor microenvironment) of the genetically modified cells.
  • the NKT cells can be engineered to express any suitable armoring molecule known in the art, e.g., armoring molecule described by Yeku et al., Armored CAR T-cells: utilizing cytokines and pro- inflammatory ligands to enhance CAR T-cell anti-tumour efficacy, Biochem Soc Trans. 2016 Apr 15; 44(2): 412-418; Hawkins et al., Armored CAR T-Cells: The Next Chapter in T-Cell Cancer Immunotherapy, Biologies. 2021; 15: 95-105).
  • armoring molecule described by Yeku et al., Armored CAR T-cells: utilizing cytokines and pro- inflammatory ligands to enhance CAR T-cell anti-tumour efficacy, Biochem Soc Trans. 2016 Apr 15; 44(2): 412-418; Hawkins et al., Armored CAR T-Cells: The Next Chapter in T-Cell Cancer Immunotherapy, Biologie
  • the terms “treat,” “treating,” and “treatment” refer to therapeutic or preventative measures described herein.
  • the methods of “treatment” employ administration of iNKT cells and T cell engagers, optionally with alpha a-galactosylceramide (a-GalCer), to a subject having a disease or disorder, or predisposed to having such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment.
  • a-GalCer alpha a-galactosylceramide
  • the term “effective amount” or “therapeutically effective amount” in the context of the administration of a therapy to a subject refers to the amount of a therapy that achieves a desired prophylactic or therapeutic effect.
  • the T cell engager binds human CD3s and is capable of binding CD3 on iNKT cells.
  • the iNKT cells and T cell engagers form a complex in solution such that the CD3 binding domain of the T cell engagers specifically binds CD3 on the iNKT cells.
  • “complexed” means that the iNKT cell is specifically bound or attached to the T cell engager.
  • the CD3 binding domain of the T cell engager may be bound to the CD3 on an iNKT cell.
  • antibodies selective for iNKT cells may be used to isolate them, for example, an anti- Va24Jal8 CDR3 loop antibody, as described in Exley et al, Selective activation, expansion, and monitoring of human iNKT cells with a monoclonal antibody specific for the TCR a- chain CDR3 loop, Eur. J. Immunol. 2008. 38: 1756-1766.
  • such methods further include expanding the iNKT cells prior to the administering step. In some embodiments, such methods further include contacting the donor iNKT cells with RGI-2001 prior to the administering step.
  • the NKT cells are purified cells.
  • the invention includes compositions comprising about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or more iNKT cells as compared to other cell types (e.g., T cells).
  • the pharmaceutical composition comprises about 10 2 iNKT cells, about 10 3 , iNKT cells, about 10 4 iNKT cells, about 10 5 iNKT cells, about 10 6 iNKT cells, about 10 7 iNKT cells, about 10 8 iNKT cells, about 10 9 iNKT cells, about IO 10 iNKT cells, 1O 2 -1O 10 iNKT cells, about 1O 3 -1O 10 iNKT cells, about 10 4 -10 5 iNKT cells, about 10 4 - 10 6 iNKT cells, about 10 4 -10 7 iNKT cells, about 10 4 -10 8 iNKT cells, about 10 5 -10 6 iNKT cells, about 10 5 -10 7 iNKT cells, about 10 5 -10 8 iNKT cells, 1O 5 -1O 10 iNKT cells, or about 10 6 - 1O 10 iNKT cells.
  • the composition comprises about 1.5 x 10 7 to 2.0 x 10 8 allogen
  • the NKT cells are engineered cells.
  • the NKT cells e.g., iNKT cells
  • the NKT cells are engineered to express TCR.
  • suitable antigens for expression of a TCR are PRAME and NY- ESO-1.
  • the antigen to be recognized by the engineered iNKT-CARs of the invention can be the same cell surface antigen recognized by the T cell engager.
  • the NKT cells e.g., iNKT cells
  • the engineered TCR described herein are also engineered to express IL- 15.
  • the genetically modified cells expressing the engineered TCR described herein are also engineered to express soluble IL-15 (sIL-15).
  • the NKT cells e.g., iNKT cells
  • the CAR described herein are also engineered to express IL- 15.
  • the genetically modified cells expressing the CAR described herein are also engineered to express soluble IL-15 (sIL-15).
  • the T cell engager of the pharmaceutical compositions of the invention can be any T cell engager disclosed herein.
  • the compositions (and methods) of the invention include a T cell engager that binds CD 19 and CD3, HER2 and CD3, HER3 and CD3, EGFR/HER1 and CD3, MUC16 and CD3, B7-H3 and CD3, BCMA and CD3, FAP and CD3, DLL and CD3, PSMA and CD3, P-cadherin (CDH3) and CD3, mesothelin (MSLN) and CD3, claudin-6 and CD3, CD 123 and CD3, CD20 and CD3, ENPP3 and CD3, EpCAM and CD3, TROP-2 and CD3, CD33 and CD3, human C-type lectin like molecule- 1 (CLL-1) and CD3, WT1 and CD3, GPRC5D and CD3, CD38 and CD3, FCRL5 and CD3, VEGFR and CD3, CEA and CD3, PSA and CD3, EphA2 and CD3, gplOO and CD
  • the pharmaceutical composition comprises a T cell engager with a CD 19 binding domain or a B7-H3 binding domain with the sequences as set forth in Table 1 below.
  • the T cell engager of the pharmaceutical composition of the invention comprises a VH CDR1, VH CDR2, and VH CDR3 and VL CDR1, VL CDR2, and VL CDR3 which are the complementarity-determining regions of an anti-CD19 antibody or B7-H3 antibody.
  • the CD 19 tumor antigen binding domain comprises a variable light chain or binding fragment thereof with an amino acid sequence at least about 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% or more identical to the amino acid sequences of SEQ ID NOG.
  • the CD19 tumor antigen binding domain comprises a variable heavy chain and a variable light chain with an amino acid sequence at least about 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% or more identical to the amino acid sequences of SEQ ID NO:1 and SEQ ID NOG.
  • the CD19 tumor antigen binding domain comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO:1 and a variable light chain comprising the amino acid sequence of SEQ ID NOG.
  • the VH CDR1, VH CDR2, and VH CDR3 and the VL CDR1, VL CDR2, and VL CDR3 of the B7-H3 tumor antigen binding domain of a T cell engager comprise an amino acid sequence as set forth in SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16, respectively.
  • the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a CD 19 x CD3 targeting T cell engager in a heterodimer antibody format, and a pharmaceutically acceptable carrier or excipient.
  • iNKT cells e.g., allogeneic iNKT cells, native or engineered
  • CD 19 x CD3 targeting T cell engager in a heterodimer antibody format e.g., allogeneic iNKT cells, native or engineered
  • a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a MUC16 x CD3 targeting T cell engager in a heterodimer antibody format, and a pharmaceutically acceptable carrier or excipient.
  • the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a B7-H3 x CD3 targeting T cell engager in a heterodimer antibody format, and a pharmaceutically acceptable carrier or excipient.
  • iNKT cells e.g., allogeneic iNKT cells, native or engineered
  • B7-H3 x CD3 targeting T cell engager in a heterodimer antibody format
  • a pharmaceutically acceptable carrier or excipient e.g., a pharmaceutically acceptable carrier or excipient.
  • the T cell engager is in the knobs- into-holes antibody format.
  • the “knobs” and “holes” can be engineered into the CH3 of the Fc region using methods known in the art.
  • the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a T cell engager in the knobs-into-holes antibody format and a pharmaceutically acceptable carrier or excipient.
  • iNKT cells e.g., allogeneic iNKT cells, native or engineered
  • a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a CD 19 x CD3 targeting T cell engager in the knobs-into-holes antibody format, and a pharmaceutically acceptable carrier or excipient.
  • the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a MUC16 x CD3 targeting T cell engager in the knobs-into-holes antibody format, and a pharmaceutically acceptable carrier or excipient.
  • the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a B7-H3 x CD3 targeting T cell engager in the knobs-into-holes antibody format, and a pharmaceutically acceptable carrier or excipient.
  • the T cell engager is in the BiTE antibody or BiTE-Fc antibody format.
  • the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a T cell engager in the BiTE or BiTE-Fc antibody format and a pharmaceutically acceptable carrier or excipient.
  • iNKT cells e.g., allogeneic iNKT cells, native or engineered
  • CD 19 x CD3 targeting T cell engager in the BiTE or BiTE-Fc antibody format e.g., CD 19 x CD3 targeting T cell engager in the BiTE or BiTE-Fc antibody format
  • a pharmaceutically acceptable carrier or excipient e.g., allogeneic iNKT cells, native or engineered
  • the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a MUC16 x CD3 targeting T cell engager in the BiTE or BiTE-Fc antibody format, and a pharmaceutically acceptable carrier or excipient.
  • the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a B7-H3 x CD3 targeting T cell engager in the BiTE or BiTE-Fc antibody format, and a pharmaceutically acceptable carrier or excipient.
  • the T cell engager is in the scFv-Ig antibody format.
  • the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a T cell engager in the scFv-Ig antibody format and a pharmaceutically acceptable carrier or excipient.
  • the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a CD 19 x CD3 targeting T cell engager in the scFv-Ig antibody format, and a pharmaceutically acceptable carrier or excipient.
  • the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a MUC16 x CD3 targeting T cell engager in the scFv-Ig antibody format, and a pharmaceutically acceptable carrier or excipient.
  • the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a B7-H3 x CD3 targeting T cell engager in the scFv-Ig antibody format, and a pharmaceutically acceptable carrier or excipient.
  • the T cell engager is in the DART, DART-Fc, or Tetravalent DART antibody format.
  • the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a T cell engager in the DART, DART-Fc, or Tetravalent DART antibody format and a pharmaceutically acceptable carrier or excipient.
  • iNKT cells e.g., allogeneic iNKT cells, native or engineered
  • a CD 19 x CD3 targeting T cell engager in the DART, DART-Fc, or Tetravalent DART antibody format e.g., allogeneic iNKT cells, native or engineered
  • a pharmaceutically acceptable carrier or excipient e.g., allogeneic iNKT cells, native or engineered
  • the T cell engager is in the scFv-scFv-scFv antibody format.
  • the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a T cell engager in the scFv-scFv-scFv antibody format and a pharmaceutically acceptable carrier or excipient.
  • iNKT cells e.g., allogeneic iNKT cells, native or engineered
  • a CD19 x CD3 targeting T cell engager in the scFv-scFv-scFv antibody format e.g., allogeneic iNKT cells, native or engineered
  • a pharmaceutically acceptable carrier or excipient e.g., allogeneic iNKT cells, native or engineered
  • the T cell engager is in the TrioMab, CrossMabFab, CrossMab VH-VL, 2:1 CrossMab, 2:2 or CrossMab antibody format.
  • the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a T cell engager in the a TrioMab, CrossMabFab, CrossMab VH-VL, 2: 1 CrossMab, 2:2 or CrossMab antibody format and a pharmaceutically acceptable carrier or excipient.
  • the T cell engager is in the DVD-Ig, IgG-IgG, or Fab- scFv-Fc antibody format.
  • the invention includes a pharmaceutical composition comprising iNKT cells (e.g., allogeneic iNKT cells, native or engineered), a T cell engager in the DVD- Ig, IgG-IgG, or Fab-scFv-Fc antibody format and a pharmaceutically acceptable carrier or excipient.
  • the CD3 binding domain comprises a variable heavy chain with a variable heavy chain CDR1, CDR2 and CDR3. In certain embodiments, the CD3 binding domain comprises a variable light chain with a variable light chain CDR1, CDR2, and CDR3. In certain embodiments, the CD3 binding domain comprises a variable light and a variable heavy chain. In certain embodiments, the CD3 binding domain is an scFv.
  • the CD3 binding domain binds human CD3s. In certain embodiments, the CD3 binding domain binds the epitope FSEXE (SEQ ID NO: 37) and QDGNE (SEQ ID NO: 38) of CD3s. FSEXE (SEQ ID NO: 37) and QDGNE (SEQ ID NO: 38) is an epitope on the N terminus of CD3s. In certain embodiments, the CD3 binding domain binds human CD3 and cynomolgus monkey CD3. In certain embodiments, the CD3 binding domain is derived from a CD3 antibody that is cross-reactive to both human and to human CD3 and cynomolgus monkey CD3. In certain embodiments of the invention, the CD3 binding domain is a humanized binding domain derived from SP34. In certain embodiments of the invention, the CD3 binding domain comprises a variable heavy and light chain corresponding to anti-CD3 antibody I2C.
  • the CD3 binding domain of the T cell engager comprises a variable heavy chain and/or a variable light chain derived from HuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, L2K, or I2C.
  • the T cell engager of the pharmaceutical composition of the invention comprises a VH CDR1, VH CDR2, VH CDR3 and VL CDR1, VL CDR2, and VL CDR3 which are the complementaritydetermining regions of an anti-CD3 antibody selected from the group consisting of HuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, L2K, and I2C.
  • the CD3 binding domain comprises a variable heavy chain and a variable light chain with an amino acid sequence at least about 80% identical, 85% identical, 90% identical, 95% identical, 96% identical, 97% identical, 98% identical, or 99% or more identical to the amino acid sequences of the variable heavy chain and variable light chain of one of HuM291, OKT3, SP34, TR66, UCHT1, G19-4, CRIS7, L2K, and I2C.
  • compositions can be supplied as a kit comprising a container that comprises the pharmaceutical composition as described herein.
  • a pharmaceutical composition can be provided, for example, in the form of an injectable solution for single or multiple doses, or as a sterile powder that will be reconstituted before injection.
  • a kit can include a dry-powder disperser, liquid aerosol generator, or nebulizer for administration of a pharmaceutical composition.
  • Such a kit can further comprise written information on indications and usage of the pharmaceutical composition.
  • a pharmaceutical composition comprising allogeneic iNKT cells as described herein can be administered to a subject in need thereof prior to or after administration of a pharmaceutical composition comprising a T cell engager to the subject.
  • the BiTE or BiTE-Fc T cell engager administered to the subject by continuous infusion which specifically binds human HER2, HER3, EGFR/HER1, CD19, MUC16, B7-H3, BCMA, FAP, DLL3, PSMA, P-cadherin (CDH3), mesothelin (MSLN), claudin 6, CD 123, CD20, ENPP3, EpCAM, TROP-2, CD33, human C-type lectin like molecule- 1 (CLL-1), WT1, GPRC5D, CD38, FCRL5, VEGFR, CEA, PSA, EphA2, gplOO, GD2, MUC1, CD22, FLT3, MUC17, or CEACAM5, Claudin 18.2, NKG2DL or binds CD3s through a CD3 binding domain derived from OKT3, SP34, or TR66.
  • a subject with cancer e.g., acute lymphoblastic leukemia
  • a condition or disease associated with expression of CD19 is administered allogeneic iNKT cells and a T cell engager that specifically binds CD 19 and CD3.
  • a subject with cancer e.g., acute lymphoblastic leukemia
  • a condition or disease associated with expression of CD19 is administered allogeneic iNKT cells (e.g., a pharmaceutical composition comprising allogeneic iNKT cells) on day 1, and on day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14 the subject commences treatment with a CD19 and CD3 T cell engager (e.g., blinatumomab) by continuous infusion.
  • allogeneic iNKT cells e.g., a pharmaceutical composition comprising allogeneic iNKT cells
  • the subject may receive 15 or 28 mcg/day of the T cell engager (e.g., blinatumomab) via continuous infusion for 28 days followed by 14 days of no treatment before the next round of treatment by continuous infusion.
  • the subject receives four rounds of treatment of blinatumomab by continuous infusion.
  • HER2 Human epidermal growth factor receptor 2
  • bladder carcinomas gallbladder
  • extrahepatic cholangiocarcinomas cervical, uterine, testicular, breast, ovarian, lung, gastric, and colon cancers.
  • HER2 expression status in diverse cancers review of results from 37,992 patients. Cancer Metastasis Rev. 2015 Mar;34(l): 157-64.
  • HER3 expression has also been reported in pilocytic astrocytoma, a childhood glioma, and in rhabdomyosarcoma, a pediatric sarcoma. (See, for instance, Gandullo-Sanchez, L., Ocana, A. and Pandeilla, A. HER3 in cancer: from bench to the bedside. J Exp Clin Cancer Res 41,310 (2022).)
  • a subject with cancer e.g., EGFR+ lung or brain cancer
  • a condition or disease associated with expression of EGFR is administered allogeneic iNKT cells (e.g., a pharmaceutical composition comprising allogeneic iNKT cells) on day 1, and on day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14
  • the subject is administered a first dose of an EGFR and CD3 T cell engager.
  • the T cell engager may be administered once a week, for instance, once a week for 3 weeks, once a week for 4 weeks, once a week for 5 weeks, or once a week for 6 weeks.
  • a subject with cancer e.g., a hematologic malignancy such as multiple myeloma (MM), chronic lymphocytic leukemia, acute B-lymphoblastic leukemia, non-Hodgkin lymphoma (NHL), or Hodgkin lymphoma
  • a condition or disease associated with aberrant expression of BCMA is concurrently administered allogeneic iNKT cells and a T cell engager that specifically binds BCMA and CD3.
  • the allogeneic iNKT cells and T cell engager may be administered as a single pharmaceutical composition as described herein.
  • a subject with cancer e.g., BCMA+ blood malignancy
  • a condition or disease associated with expression of BCMA is administered allogeneic iNKT cells (e.g., a pharmaceutical composition comprising allogeneic iNKT cells) on day 1, and on day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14 the subject is administered a first dose of a BCMA targeting T cell engager.
  • the methods of the invention include administering a pharmaceutical composition comprising allogeneic iNKT cells (e.g., a pharmaceutical composition comprising IO 10 cells/70 kg patient and a pharmaceutically acceptable carrier) and a T cell engager such as HPN217 (Harpoon), TNB-383 (TeneoBio), AMG701 (Amgen), AMG420 (Amgen), REGN5458 (Regeneron), Elranatamab, or CC-93269.
  • a pharmaceutical composition comprising allogeneic iNKT cells (e.g., a pharmaceutical composition comprising IO 10 cells/70 kg patient and a pharmaceutically acceptable carrier) and a T cell engager such as HPN217 (Harpoon), TNB-383 (TeneoBio), AMG701 (Amgen), AMG420 (Amgen), REGN5458 (Regeneron), Elranatamab, or CC-93269.
  • a subject with cancer e.g., prostate cancer, hepatocellular carcinoma, renal cell carcinoma, breast cancer, lung cancer, sarcoma, soft tissue tumor
  • a condition or disease associated with aberrant expression of PSMA is concurrently administered allogeneic iNKT cells and a T cell engager that specifically binds PSMA and CD3.
  • the allogeneic iNKT cells and T cell engager may be administered as a single pharmaceutical composition as described herein.
  • Systemic therapy targeting FAP can lead to severe cachexia, including muscle damage, osteo toxicity, and even death.
  • Roberts EW Deonarine A
  • Jones JO Denton AE
  • Feig C Lyons SK
  • therapeutics targeting FAP should be designed to selectively kill FAP+ fibroblasts.
  • Delta-like Canonical Notch Ligand 3 is a protein frequently overexpressed in small-cell lung cancer, the most aggressive phenotype within the spectrum of all lung neuroendocrine tumors.
  • a subject with cancer e.g., a neuroendocrine tumor, including small-cell lung cancer
  • a condition or disease associated with aberrant expression of DLL3 expression is administered allogeneic iNKT cells and a T cell engager that specifically binds DLL3 and CD3.
  • the allogenic iNKT cells and DLL3 targeting T cell engager are administered concurrently (e.g., as a single pharmaceutical composition as described herein).
  • a subject with cancer e.g., small-cell lung cancer or neuroendocrine carcinoma
  • a condition or disease associated with overexpression of DLL3 is administered allogeneic iNKT cells (e.g., a pharmaceutical composition comprising allogeneic iNKT cells) on day 1, and on day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14
  • the subject is administered a first dose of a DLL3 targeting T cell engager.
  • the T cell engager may be administered once a week, for instance, once a week for 3 weeks, once a week for 4 weeks, once a week for 5 weeks, or once a week for 6 weeks.
  • the first dose of the T cell engager may be lower than the maximum dose.
  • Tarlatamab (AMG 757) and BI 764532 are T cell engagers that can be administered with allogeneic iNKT cells to a subject pursuant to the methods of the invention.
  • a subject with cancer e.g., prostate cancer, medulloblastoma, non-small cell lung cancer, pancreatic cancer, melanoma, glioma, breast, renal cell carcinoma, ovarian carcinoma, endometrial cancer, hepatocellular carcinoma, and colorectal cancer
  • allogeneic iNKT cells e.g., a pharmaceutical composition comprising allogeneic iNKT cells
  • day 1, and on day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14 the subject is administered a first dose of a B7-H3 and CD3 T cell engager.
  • MUC16 also known as CA125
  • cancers including ovarian cancer, breast cancer, pancreatic cancer, non-small-cell lung cancer, intrahepatic cholangiocarcinoma- mass forming type, adenocarcinoma of the uterine cervix, and adenocarcinoma of the gastric tract, and in diseases and conditions including inflammatory bowel disease, liver cirrhosis, cardiac failure, peritoneal infection, and abdominal surgery.
  • Haridas, D. et al., 2014, FASEB J., 28:4183-4199 Expression on cancer cells is shown to protect tumor cells from the immune system.
  • combination therapy of NKT cells e.g., iNKT cells
  • a T cell engager that specifically targets MUC16 and CD3 can be used to treat a subject with MUC16+ cancer such as ovarian cancer, breast cancer, pancreatic cancer, non- small-cell lung cancer, intrahepatic cholangiocarcinoma- mass forming type, adenocarcinoma of the uterine cervix, and adenocarcinoma of the gastric tract.
  • MUC16+ cancer such as ovarian cancer, breast cancer, pancreatic cancer, non- small-cell lung cancer, intrahepatic cholangiocarcinoma- mass forming type, adenocarcinoma of the uterine cervix, and adenocarcinoma of the gastric tract.
  • combination therapy of NKT cells e.g., iNKT cells
  • a T cell engager that specifically binds MUC6 and CD3 can be used to treat a subject with a disease or condition associated with MUC16 overexpression such as inflammatory bowel disease, liver cirrhosis, cardiac failure, peritoneal infection, and abdominal surgery.
  • a subject with cancer e.g., ovarian cancer
  • a condition or disease associated with expression of MUC16 is administered allogeneic iNKT cells and a T cell engager that specifically binds MUC16 and CD3.
  • the first dose of the MUC16 and CD3 T cell engager may be 1 mg, followed a week later with a 20 mg dose (i.e., week 2 of T cell engager dosing), and followed a week later with a 250 mg dose.
  • NKT cells e.g., allogeneic iNKT cells
  • ubamatamab is administered in weekly doses ranging from 1 mg to 800 mg.
  • NKG2D ligands (NKG2DL) on many different tumor types, with limited expression on normal tissues, makes them attractive targets for immunotherapy. It has been estimated that more than 90% of human tumors may express at least one NKG2D ligand (See Spear P, Wu MR, Sentman ML, Sentman CL. NKG2D ligands as therapeutic targets. Cancer Immun. 2013 May 1 ; 13:8). There are two families of NKG2D ligands in humans, MHC class I chain-related proteins A (MICA) and B (MICB) as well as ULI 6 binding proteins (ULBP1 - 6). In certain embodiments of the invention, a NKG2DL is the tumor antigen target for the T cell engager.
  • MICA MHC class I chain-related proteins A
  • MICB ULI 6 binding proteins
  • a subject with a condition or disease associated with the expression of a NKG2DL is administered a therapeutically effective amount of NKT cells (e.g., iNKT cells) and a T cell engager that specifically binds the NKG2DL and a CD3.
  • NKG2DL are commonly expressed in soft tissue sarcoma (STS), but generally absent in healthy tissues.
  • a subject with a cancer e.g., a soft tissue sarcoma
  • a cancer e.g., a soft tissue sarcoma
  • NKT cells e.g., iNKT cells
  • T cell engager comprising a tumor antigen binding domain that specifically binds the overexpressed NKG2DL.
  • the invention includes administration for iNKT cells and a T cell engager that specifically binds MICA to a subject with a disease or condition associated with the overexpression of MICA (e.g., cervical, colon, breast, lung, ovarian, prostate, renal cell, and pancreatic carcinomas and cutaneous and malignant melanomas).
  • a disease or condition associated with the overexpression of MICA e.g., cervical, colon, breast, lung, ovarian, prostate, renal cell, and pancreatic carcinomas and cutaneous and malignant melanomas.
  • a subject with cancer or a condition or disease associated with expression of a NKG2DL is administered allogeneic iNKT cells (e.g., a pharmaceutical composition comprising allogeneic iNKT cells) on day 1, and on day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14 the subject is administered a first dose of a NKG2DL and CD3 T cell engager.
  • a NKG2DL e.g., MICA, MICB, ULBPI - 6
  • allogeneic iNKT cells e.g., a pharmaceutical composition comprising allogeneic iNKT cells
  • a subject with cancer e.g., gastrointestinal, breast or lung cancer
  • a condition or disease associated with expression of CEACAM5 is administered allogeneic iNKT cells (e.g., a pharmaceutical composition comprising allogeneic iNKT cells) on day 1, and on day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14
  • the subject is administered a first dose of a CEACAM5 and CD3 T cell engager.
  • the T cell engager may be administered once a week, for instance, once a week for 3 weeks, once a week for 4 weeks, once a week for 5 weeks, or once a week for 6 weeks.
  • a subject with cancer e.g., Claudin 18.2+ gastrointestinal, prostate or lung cancer
  • a condition or disease associated with expression of Claudin 18.2 is administered allogeneic iNKT cells and a T cell engager that specifically binds Claudin 18.2 and CD3.
  • a subject with cancer e.g., gastrointestinal, prostate or lung cancer
  • a condition or disease associated with expression of Claudin 18.2 is administered allogeneic iNKT cells (e.g., a pharmaceutical composition comprising allogeneic iNKT cells) on day 1, and on day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, or day 14
  • the subject is administered a first dose of a Claudin 18.2 and CD3 T cell engager.
  • the T cell engager may be administered once a week, for instance, once a week for 3 weeks, once a week for 4 weeks, once a week for 5 weeks, or once a week for 6 weeks.
  • a subject in need thereof is administered allogeneic iNKT cells and a T cell engager such as blinatumomab, catumaxomab, mosunetuzumab, tebentafusp, teclistamab, ubamatamab, enoblituzumab, linvoseltamab, odronextamab, tebentafusp, forimtamig, or runimotamab.
  • a T cell engager such as blinatumomab, catumaxomab, mosunetuzumab, tebentafusp, teclistamab, ubamatamab, enoblituzumab, linvoseltamab, odronextamab, tebentafusp, forimtamig, or runimotamab.
  • methods also are provided for increasing the frequency of endogenous iNKT cells in vivo or increasing or sustaining the frequency of infused or endogenous iNKT cells in vivo.
  • Such methods can utilize compounds such as, without limitation, cytokines (e.g., IL-2, IL-17, IL-15) or alpha-galactosyl ceramide.
  • cytokines e.g., IL-2, IL-17, IL-15
  • alpha-galactosyl ceramide e.g., IL-15
  • agonists of iNKT cells can be administered to a subject.
  • Agonists of iNKT cells are known in the art and include, without limitation, synthetic agonists (see, e.g., Cerundolo et al., 2010, Curr. Opin.
  • Certain aspects of the disclosure relate to the treatment of cancer and/or use of the NKT cells (e.g., iNKT cells) in combination with T cell engagers, and compositions of the disclosure, to treat cancer.
  • the cancer to be treated or antigen may be an antigen associated with any cancer known in the art or, for example, epithelial cancer, (e.g., breast, gastrointestinal, lung), prostate cancer, bladder cancer, lung (e.g., small cell lung) cancer, colon cancer, ovarian cancer, brain cancer, gastric cancer, renal cell carcinoma, pancreatic cancer, liver cancer, liver metastasis, esophageal cancer, head and neck cancer, or a colorectal cancer.
  • the cancer to be treated or antigen is from one of the following cancers: adenocortical carcinoma, agnogenic myeloid metaplasia, AIDS-related cancers (e.g., AIDS-related lymphoma), anal cancer, appendix cancer, astrocytoma (e.g., cerebellar and cerebral), basal cell carcinoma, bile duct cancer (e.g., extrahepatic), bladder cancer, bone cancer, (osteosarcoma and malignant fibrous histiocytoma), brain tumor (e.g., glioma, brain stem glioma, cerebellar or cerebral astrocytoma (e.g., pilocytic astrocytoma, diffuse astrocytoma, anaplastic (malignant) astrocytoma), malignant glioma, ependymoma, oligodenglioma, meningioma, meningiosarcoma,
  • the subject is administered antitumor therapy in addition to iNKT cells and a T cell engager.
  • the additional anti-tumor therapy may be administered at the same time as the iNKT cells and T cell engager or sequentially (e.g., on weeks when subject does not receive iNKT and T cell engager therapy).
  • CTLA-4 inhibitor e.g., ipilimumab
  • a TIM3 inhibitor e.g., a BTLA inhibitor, a TIGIT inhibitor, a CD47 inhibitor, a GITR inhibitor, an antagonist of another T cell co-inhibitor or ligand (e.g., an antibody to LAG3, CD-28, 2B4, LY108, LAIR1 , ICOS, CD160 or VISTA)
  • IDO indoleamine-2, 3-dioxygenase
  • VEGF vascular endothelial growth factor
  • VEGF vascular endothelial growth factor
  • VEGF vascular endothelial growth factor
  • the methods of treating a subject do not include lymphodepletion of the subject as a pre-treatment. Lymphodepletion is frequently performed prior to immunotherapies to debulk a tumor, alter tumor phenotype, modify the tumor microenvironment, remove cytokine sinks (e.g., make IL-2, IL-7, and IL- 15 more available) and suppress the host immune system.
  • lymphodepletion has multiple negative effects including, neutropenia, anemia, thrombocytopenia, and immunosuppression, and toxicities associated with lymphodepletion agents such as fludarabine and cyclophosphamide.
  • the subject administered iNKT cells and a T cell engager is not administered a lymphodepletion agent prior to or during treatment.
  • bendamustine, fludarabine and cyclophosphamide are not administered to the subject prior to or during treatment with iNKT cells and a T cell engager.
  • CD3xCD19 and CD3xB7-H3 T cell engager antibodies were constructed in the Knobs-into-Holes (KiH) antibody format.
  • the anti-B7-H3 variable heavy and light chain sequences from enoblituzumab were used to construct the anti-B7-H3 domain (SEQ ID NO:9; SEQ ID NO: 10), and the anti-CD19 variable heavy and light chain sequences from blinatumomab were used to construct the anti-CD19 domain (SEQ ID NO: 1; SEQ ID NO:2).
  • the anti-CD3 variable domain of the OKT3 derivative used in blinatumomab (SEQ ID NO: 17; SEQ ID NO: 18) was used for both T cell engagers.
  • Light chains were expressed under mouse IgK leader peptide and heavy chains were expressed under CD33 leader peptide.
  • ‘Knob” polypeptides and “hole” polypeptides were separately expressed in ExpiCHO cells and purified using a protein A column. Purified polypeptides were reduced in a IM 2-MAE, lOmM Tris pH8, lOOmM NaCl buffer for 2 hours at room temperature. The “knob” and “hole” polypeptides were then mixed in a one-to-one molar ratio and re-oxidated in a 10 mM Sodium Citrate pH 6.0, 115 mM NaCl by buffer exchange for 3 hours at room temperature. Heterodimerization was confirmed by SDS-PAGE gel and analytical SEC.
  • iNKT cells were separated out from the peripheral mononuclear blood cell (PBMC) fraction and then expanded with irradiated aGalCer loaded autologous PBMCs in the presence of human IL-2 for 10-14 days. Cells from the flow-through fraction were cryopreserved for further expansion rounds as well as isolation of conventional T lymphocytes.
  • PBMC peripheral mononuclear blood cell
  • T lymphocytes were isolated from PBMCs by negative selection using EasySepTM (STEMCELL Technologies). For activation, isolated T cells were stimulated in vitro for 72 hours with ImmunoCultTM Human CD3/CD28 T Cell Activator (STEMCELL Technologies) in presence of human IL-2 and IL-7. Afterwards, T cells were expanded for 10-14 days in media containing human IL-2 and IL-7. Medium was refreshed every third or fourth day.
  • Expanded iNKT cells freshly isolated T cells and expanded T cells were stained with anti-CD3s antibody. Briefly, 100,000 cells were plated per well of a 96-well V bottom plate. In order to be able to quantify number of molecules per cell, one drop of Quantibrite beads was plated on one of the wells on the same plate (Quantibrite Beads PE Fluorescence Quantitation Kit, BD, Catalog number PE 340495). Beads were stained and washed in the same way as the cells. Plate was spun down for 300g for 5 minutes at room temperature. Supernatant was removed and cells were washed with 2% FBS in PBS. Cells were spun down at 300g for 5 minutes at room temperature.
  • Cells were gated based on forward and side scatter (height), single cells were gated based on forward scatter (width and height), and life cells were gated based on life/dead dye exclusion.
  • Mean geometric fluorescence for PE signal was calculated for the gated cells and the beads. Specific signal was calculated by subtracting MFI of the isotype stained cells from cells stained with CD3s - PE signal.
  • iNKT cells were incubated with various concentrations of the engager followed by fluorescently tagged anti-human IgG antibody. Binding was determined by flow cytometry analysis. Secondary antibody alone was set as the negative control. (FIGURES 2A and 2B)
  • FIGURE 11B Similar studies were done using iNKT cells or T cells isolated from two donors with DLL3-CD3 engagers for killing SHP333 lung cancer cell (FIGURE 11B), Her2- CD3 engagers for killing SKBR3 breast cancer cells (FIGURE 11C), or Cldnl8.2-CD3 engager for killing PATU89885 liver metastatic cancer cells (FIGURE 11D). Similar results were observed that the various engager mediated iNKT cell killing of the target cells faster than that of the T cells.

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Abstract

La présente invention concerne de nouvelles compositions comprenant des lymphocytes T tueurs naturels ("cellules NKT") et des activateurs de lymphocytes T. Selon certains modes de réalisation, les cellules NKT sont des lymphocytes T tueurs naturels invariants allogéniques ("cellules iNKT") et l'activateur de lymphocytes T est un anticorps bispécifique ou multispécifique comprenant un domaine de liaison à CD3 et un domaine de liaison à l'antigène tumoral. L'invention comprend des méthodes de traitement du cancer qui consistent à administrer à un sujet en ayant besoin des cellules NKT (par exemple, des cellules iNKT allogéniques) et un activateur de lymphocytes T.
PCT/US2024/050139 2023-10-05 2024-10-04 Nouvelles compositions de lymphocytes t tueurs naturels invariants et d'activateurs de lymphocytes t et méthodes d'utilisation associées Pending WO2025076485A1 (fr)

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Citations (3)

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US20220184142A1 (en) * 2019-04-11 2022-06-16 Fate Therapeutics, Inc. CD3 RECONSTITUTION IN ENGINEERED iPSC AND IMMUNE EFFECTOR CELLS
US20220267420A1 (en) * 2018-02-15 2022-08-25 Memorial Sloan Kettering Cancer Center Foxp3 targeting agent compositions and methods of use for adoptive cell therapy
WO2024077104A2 (fr) * 2022-10-04 2024-04-11 Mink Therapeutics, Inc. Cellules car-t tueuse naturelle invariante (inkt) anti-protéine d'activation des fibroblastes (fap) et leurs utilisations

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US20220267420A1 (en) * 2018-02-15 2022-08-25 Memorial Sloan Kettering Cancer Center Foxp3 targeting agent compositions and methods of use for adoptive cell therapy
US20220184142A1 (en) * 2019-04-11 2022-06-16 Fate Therapeutics, Inc. CD3 RECONSTITUTION IN ENGINEERED iPSC AND IMMUNE EFFECTOR CELLS
WO2024077104A2 (fr) * 2022-10-04 2024-04-11 Mink Therapeutics, Inc. Cellules car-t tueuse naturelle invariante (inkt) anti-protéine d'activation des fibroblastes (fap) et leurs utilisations

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MINK: "Mink Therapeutics R&D Day", MINK THERAPEUTICS VOL. 1, 10 November 2022 (2022-11-10), pages 1 - 92, XP093302237, Retrieved from the Internet <URL:https://investor.minktherapeutics.com/static-files/e44e0c29-7510-4e50-844d-a21a70c11fb6> *

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