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WO2025029484A1 - Procédés d'expansion et de greffe de cellules in vivo à médiation par des cellules sanguines - Google Patents

Procédés d'expansion et de greffe de cellules in vivo à médiation par des cellules sanguines Download PDF

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Publication number
WO2025029484A1
WO2025029484A1 PCT/US2024/038360 US2024038360W WO2025029484A1 WO 2025029484 A1 WO2025029484 A1 WO 2025029484A1 US 2024038360 W US2024038360 W US 2024038360W WO 2025029484 A1 WO2025029484 A1 WO 2025029484A1
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Prior art keywords
cell
car
administered
subject
immune cell
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Farzad Haerizadeh
Laurence J.N. Cooper
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Bio4t2 LLC
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Bio4t2 LLC
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    • 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
    • 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
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • 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
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4254—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • 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
    • C07K14/7051—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
    • 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
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K40/00
    • A61K2239/38—Indexing codes associated with cellular immunotherapy of group A61K40/00 characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • 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
    • 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
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • aspects of the present disclosure relate generally to a method of administering an immunotherapy.
  • the method comprises administration of a cell or composition containing or coding for the CAR targeting CEACAM6.
  • the CAR comprises at least one binding site for a second target or additional targets.
  • the CAR protein has a single target and is capable of neutrophil and/or granulocyte activation.
  • CAR T cell therapies such as chimeric antigen receptor (CAR) T cell therapies
  • CAR T cell therapies involve the use of genetically engineered T cells expressing receptors targeted to cancer-associated cell surface markers and other antigens, enabling directed killing of cancer cells while minimally affecting normal cells in a patient.
  • CAR T cell therapies for CD 19+ B cell lymphoma cancers.
  • the CAR which is made up of an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain, enables directed killing of cancer cells based on cell surface antigen expression while minimally affecting normal cells that are not expressing the targeted antigen.
  • the extracellular antigen binding domain is often made up of an antibody or a binding fragment or derivative thereof, such as a single chain variable fragment (scFv) or single domain antibody (sdAb).
  • scFv single chain variable fragment
  • sdAb single domain antibody
  • a chimeric antigen receptor (CAR) protein with at least one target.
  • the CAR protein has a single target and is capable of neutrophil and/or granulocyte activation.
  • the target is CEACAM6 (also referred to as “CEA6,” “CD66c,” “CEAL,” “NCA,” “carcinoembryonic antigen related cell adhesion molecule 6,” and “CEA cell adhesion molecule 6”).
  • the CAR protein has at least two targets.
  • the CAR protein comprises a first binding site is capable of binding CEACAM6; and a second binding site is capable of binding an antigen expressed in an abnormal cell or tissue.
  • the abnormal cell and/or abnormal tissue is a cancer cell or tissue. In some embodiments, the abnormal cell and/or abnormal tissue is from a diseased cell or tissue. In some embodiments, the diseased cell or tissue is selected from an autoimmune disease, myocardial disease, viral infection, rheumatoid arthritis, fibrosis, cardiac fibrosis, HIV, COVID-19, Chronic hepatitis C virus (HCV), human cytomegalovirus (HCMV), Influenza, cell senescence targets (selectively clear senescent cells (SC)), liver fibrosis, lung fibrosis, atherosclerosis, diabetes, osteoarthritis, obesity, and/or aging.
  • autoimmune disease myocardial disease
  • viral infection rheumatoid arthritis
  • fibrosis fibrosis
  • cardiac fibrosis HIV
  • COVID-19 Chronic hepatitis C virus
  • HCV chronic hepatitis C virus
  • HCMV human cytomegalovirus
  • the diseased cell or tissue is a viral infection, senescing cell, or fibriosis. In some embodiments, the abnormal cell or tissue is not blood bome. In some embodiments, the abnormal cell or tissue is a solid tumor.
  • Non-limiting example antigens that the second binding site may be capable of binding to in an embodiment include: AFP, ANTXR1, AXL, avP3, avP6, B7-H3, CAIX, CD171, CD20, CD32A, CD46, CD47, CD56, CD80/86, CEA, Claudin 18.2, DLL-3, DR5, EGFR, EGFRIII, EGFR806, EpCAM, EpHA2, FAP, FR-alpha, GD2, Glypican-2, Glypican-3, gplOO, GSPG4, GUCY2C, HBV surface antigen (HBsAg), HER2, IL-13R- alpha2, Ll-CAM, Lewis Y, LMP1, MAGE-A1/3/3/
  • the abnormal cell or tissue is selected from: brain, breast, central nervous system, cervical, colon, colorectal, epidermis, gastric, glioblastoma, glioma, hepatoma, liver, lung, nasopharyngeal, neuroblastoma, ovarian, pancreatic, pediatric glioma, prostate, rectum, renal, and stomach cells and/or tissue.
  • nucleotide sequence encoding the CAR protein of any one of the embodiments of the present disclosure.
  • Also disclosed herein is a vector encoding the CAR protein of any one of the embodiments of the present disclosure, and/ or the nucleotide of any one of the embodiments of the present disclosure.
  • the cell capable of expressing the CAR protein of any one of the embodiments of the present disclosure, and/or the nucleotide of any one of the embodiments of the present disclosure, and/or the vector of any one of the embodiments of the present disclosure.
  • the cell is an immune cell.
  • the immune cell is B4T2-001.
  • the immune cell is a TIL cell.
  • the immune cell is a T cell, NK cell, or macrophage.
  • compositions comprising the CAR protein of any one of the embodiments of the present disclosure, and/or the cell of any one of the embodiments of the present disclosure.
  • the composition is formulated for administering to a subject.
  • the subject is a mammal and/or human.
  • the administration is performed through an infusion.
  • the administration is performed locally, intraperitoneally, intravenously, intratumorally, or intramurally.
  • the administration is performed more than once.
  • the administration is performed without a lymphodepletion step.
  • the use is for stimulating a blood cell in a subject.
  • the subject is a mammal and/or human.
  • the blood cell is a neutrophil and/or a granulocyte.
  • the cell of any one of the embodiments of the present disclosure or a cell expressing the CAR of any one of the embodiments of the present disclosure undergoes a significant population expansion.
  • the cell undergoes a significant activation and/or a potent population expansion.
  • the significant activation and/or potent population expansion is at least a 50-fold increase in cell population.
  • the subject has a disease or disorder, such as cancer which is treated by the CAR.
  • treatment by the CAR does not require a lymphodepletion step prior to administration of the CAR.
  • Also disclosed herein is a method of treating a disease or disorder in a subject in need thereof.
  • the method comprising administering to the subject any one of the CAR proteins of any one of the embodiments of the present disclosure, and/or the cell of any one of the embodiments of the present disclosure, and/or the composition of any one of the embodiments of the present disclosure.
  • the subject is a mammal and/or human.
  • the method further comprises the CAR protein, cell, and/or composition stimulating a blood cell within the subject.
  • the blood cell is a neutrophil and/or a granulocyte.
  • the cell of any one of the embodiments of the present disclosures and/or a cell expressing the CAR of any one of the embodiments of the present disclosures undergoes a significant population expansion.
  • the cell undergoes a significant activation and/or a potent population expansion.
  • the significant activation and/or potent population expansion is at least a 50-fold increase in cell population.
  • the method of treating a disease or disorder in a subject in need thereof comprises administering a CAR-immune cell comprising a first binding site for CEACAM6 and a second binding site for a disease or disorder.
  • the disease or disorder is any one of: an Autoimmune Disease, myocardial disease, viral infection, rheumatoid arthritis, fibrosis, cardiac fibrosis, HIV, COVID- 19, Chronic hepatitis C virus (HCV), human cytomegalovirus (HCMV), Influenza, cell senescence targets (selectively clear senescent cells (SC)), liver fibrosis, lung fibrosis, atherosclerosis, diabetes, osteoarthritis, obesity, and/or aging.
  • HCV chronic hepatitis C virus
  • HCMV human cytomegalovirus
  • Influenza cell senescence targets (selectively clear senescent cells (SC)), liver fibrosis, lung fibrosis, atherosclerosis, diabetes
  • the disease or disorder is a cancer.
  • the cancer is not blood borne.
  • the cancer is a solid tumor.
  • the second binding site is capable of binding to at least any one of: AFP, ANTXR1, AXL, av[33, av 6, B7-H3, CAIX, CD171, CD20, CD32A, CD46, CD47, CD56, CD80/86, CEA, Claudin 18.2, DLL-3, DR5, EGFR, EGFRIII, EGFR8O6, EpCAM, EpHA2, FAP, FR-alpha, GD2, Glypican-2, Glypican-3, gplOO, GSPG4, GUCY2C, HBV surface antigen (HBsAg), HER2, IL-13R-alpha2, Ll-CAM, Lewis Y, LMP1, M2c, MAGE-A1/3/4, mesothelin, c-MET, MUC1, MUC16,
  • the cancer is present in a cell and/or tissue that is selected from: brain, breast, central nervous system, cervical, colon, colorectal, epidermis, gastric, glioblastoma, glioma, hepatoma, liver, lung, nasopharyngeal, neuroblastoma, ovarian, pancreatic, pediatric glioma, prostate, rectum, renal, and stomach cells and/or tissue.
  • the method does not require a lymphodepletion step.
  • the subject is a mammal and/or human.
  • the method further comprises the CAR-immune cell stimulating a blood cell within the subject.
  • the blood cell is a neutrophil and/or a granulocyte.
  • CAR-immune cell upon stimulating the blood cell, CAR-immune cell undergoes a significant population expansion.
  • the cell undergoes a significant activation and/or a potent population expansion.
  • the significant activation and/or potent population expansion is at least a 50-fold increase in cell population.
  • the CAR-immune cell is B4T2-001.
  • the CAR-immune cell is a TIL cell. In some embodiments, the CAR-immune cell is a T cell, NK cell, or macrophage. In some embodiments, the administration is conducted through an infusion. In some embodiments, the administration is performed locally, intraperitoneally, intravenously, intratumorally, or intramurally. In some embodiments, the method further comprises multiple administrations of the payload to the subject. In some embodiments, the multiple administrations are performed locally, intraperitoneally, intravenously, intratumorally, and/or intramurally. In some embodiments, the stimulation of the blood cell is reversible.
  • the significant activation and/or potent population expansion results in at least 20%, 25%, 30%, 40%, or 50% engraftment of the cell. In some embodiments, the significant population expansion results in at least 20% engraftment of the cell. In some embodiments, the significant activation and/or potent population expansion results in at least 25% engraftment of the cell. In some embodiments, the significant activation and/or potent population expansion results in at least 50% engraftment of the cell. In some embodiments, the cell undergoes significant activation and/or potent population expansion in at least one of the peripheral blood, peritoneal fluid, and/or pulmonary fluid of the subject. In some embodiments, the method further comprises administration of a kill switch activator.
  • the kill switch activator is an anti-EGFR antibody and/or is cetuximab. In some embodiments, the kill switch activator is administered locally, intraperitoneally, intravenously, intratumorally, or intramurally. In some embodiments, the kill switch activator is administered at a dose of about 0.1, 1, 10, 100, 250, 500, 700, 750, or any integer that is between 0.1 and 750, mg/m2. In some embodiments, the kill switch activator is administered more than once. In some embodiments, the more than one administrations of the kill switch activator is performed locally, intraperitoneally, intravenously, intratumorally, and/or intramurally.
  • FIG. 1 depicts a representative graph for qPCR data, screening for the presence of B4t2-001 CAR T cells in both the blood and the peritoneal area of a subject, following intraperitoneal injection.
  • FIG. 2A depicts a representative graph for the percent of CAR T cells found in the blood of a subject over time following intraperitoneal injection, as quantified using fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • FIG. 2B depicts a representative graph for the percent of CAR T cells found in the intraperitoneal fluid sample of a subject over time following intraperitoneal injection, as quantified using fluorescence-activated cell sorting (FACS).
  • FACS fluorescence-activated cell sorting
  • FIG. 3 depicts a representative graph for qPCR data, screening for the presence of B4t2-001 CAR T cells in the blood and the peritoneal area of a subject, following intraperitoneal injection.
  • a “kill switch” is activated to rid the subject of CAR T cells, by administering cetuximab® to the subject.
  • a chimeric antigen receptor (CAR) protein with at least one target.
  • the CAR protein comprises at least two binding sites, at least three, or at least four binding sites.
  • the CAR protein has two binding sites.
  • the CAR construct binds to carcinoembryonic antigen 6 (CEACAM6, or CEA6).
  • CEACAM6, or CEA6 carcinoembryonic antigen 6
  • the CAR protein has a single target and is capable of neutrophil and/or granulocyte activation.
  • the CAR cell is B4T2-001 which is a specific CAR cell configured to bind to CEA6.
  • the CAR cell has multiple targets.
  • the CAR cell has two targets. In some embodiments, the CAR cell is bivalent. In some embodiments, the CAR cell is multi-valent. In some embodiments, the CAR cell comprises a sdAb antibody that binds to CEA6.
  • CAR proteins which target CEA6 were found to stimulate neutrophils which led to rapid expansion and engraftment of CAR-T cells.
  • CAR proteins which target CEA6 plus a second cancer target, even a solid tumor target are capable of rapid expansion and engraftment due to this neutrophil-mediated expansion.
  • lymphodepletion of endogenous white blood cells were not required for expansion and engraftment to take place, and thus CAR cells having a binding site for CEA6 and a second tumor target can be administered to a patient without the need for a lymphodepletion step prior to administration.
  • the CAR protein comprises a first binding site capable of binding CEA6, and a second binding site is capable of binding an antigen expressed in an abnormal cell or tissue.
  • the abnormal cell and/or abnormal tissue is a diseased cell and/or diseased tissue.
  • the diseased cell and/or diseased tissue is any one of: an Autoimmune Disease, myocardial disease, viral infection, rheumatoid arthritis, fibrosis, cardiac fibrosis, HIV, COVID-19, Chronic hepatitis C virus (HCV), human cytomegalovirus (HCMV), Influenza, cell senescence targets (selectively clear senescent cells (SC)), liver fibrosis, lung fibrosis, atherosclerosis, diabetes, osteoarthritis, obesity, and/or aging.
  • the abnormal cell and/or abnormal tissue is a cancer cell or tissue.
  • the abnormal cell or tissue is not blood borne.
  • the abnormal cell or tissue is a solid tumor.
  • the second binding site is capable of binding to any one of: AFP, ANTXR1, AXL, av 3, av06, B7-H3, CAIX, CD171, CD20, CD32A, CD46, CD47, CD56, CD80/86, CEA, Claudin 18.2, DLL-3, DR5, EGFR, EGFRIII, EGFR806, EpCAM, EpHA2, FAP, FR-alpha, GD2, Glypican-2, Glypican-3, gplOO, GSPG4, GUCY2C, HBV surface antigen (HBsAg), HER2, IL-13R-alpha2, Ll-CAM, Lewis Y, LMP1, M2e, MAGE-A1/3/4, mesothelin, c-MET, MUC1, MUC16, MUC3A, Nectin4/FAP, NKG2D, PAP, PSA, PSCA, PSMA, ROR, TAG-72, Tropothelin, c-
  • the abnormal cell or tissue is selected from: brain, breast, central nervous system, cervical, colon, colorectal, epidermis, gastric, glioblastoma, glioma, hepatoma, liver, lung, nasopharyngeal, neuroblastoma, ovarian, pancreatic, pediatric glioma, prostate, rectum, renal, and stomach cells and/or tissue.
  • the second binding site binds to any of the antigens in Tables 1-2.
  • the antigen is expressed in any of the cell and/or tissue types listed in Tables 1-2:
  • Table 1 List of potential antigens expressed on cancer cell/tissues during specific phases of that cancer
  • Table 2 List of cancer cell/tissues, and known antigens on those cell/tissues
  • nucleotide sequence encoding the CAR protein of any one of the embodiments of the present disclosure.
  • Also disclosed herein is a vector encoding the CAR protein of any one of the embodiments of the present disclosure, and/ or the nucleotide of any one of the embodiments of the present disclosure.
  • a cell capable of expressing the CAR protein of any one of the embodiments of the present disclosure, and/or the nucleotide of any one of the embodiments of the present disclosure, and/or the vector of any one of the embodiments of the present disclosure.
  • the cell is an immune cell.
  • the immune cell is B4T2-001.
  • the immune cell is a TIL cell.
  • the immune cell is a T cell, NK cell, or macrophage.
  • a composition comprising the CAR protein of any one of the embodiments of the present disclosure, and/or the cell of any one of the embodiments of the present disclosure.
  • the composition is formulated for administering to a subject.
  • the subject is a mammal and/or human.
  • the administration is performed through an infusion.
  • the administration is performed locally, intraperitoneally, intravenously, intratumorally, or intramurally.
  • the administration is performed more than once.
  • the administration is performed without a lymphodepletion step.
  • the composition is for intraperitoneal administration.
  • the composition is for intravenous administration.
  • the composition is for intratumoral administration.
  • the cell expresses polypeptides that are incorporated into a chimeric antigen receptor cell.
  • the cell is a mononuclear or a polymorphonuclear immune cell.
  • the cell is a lymphocyte and/or a leukocyte.
  • the cell is a tumor-infiltrating lymphocyte (TIL).
  • TIL tumor-infiltrating lymphocyte
  • the cell is a myelocyte.
  • the cell is a B cell, a T cell, or a Natural Killer (NK cell).
  • the cell is an eosinophil, neutrophil, or monocyte.
  • the cell is a macrophage, basophil, or mast cell. In some embodiments, the cell is a memory cell, plasma cell, memory T cell, cytotoxic T cell, or helper T cell. In some embodiments, the chimeric antigen receptor cell is a chimeric antigen receptor T cell (CAR T-cell).
  • CAR T-cell chimeric antigen receptor T cell
  • the cell is combined with an effective amount of at least one checkpoint inhibitor (iCPI).
  • the checkpoint inhibitor is an anti- CEAMCAM6, an anti-CTLA4, an anti-PDl, an anti-PDLl, an anti-PDNR, and/or an anti PD- 1 dominant negative receptor (DNR).
  • the checkpoint inhibitor is pembrolizumab.
  • the at least one iCPI is administered to the subject intraperitoneally.
  • the at least one iCPI is administered to the subject intravenously.
  • the at least one iCPI is administered to the subject intratumorally.
  • the at least one iCPI is administered to the subject intramurally.
  • the cell comprises a kill switch. “Kill switch” is given its standard scientific meaning, and thus refers to a mechanism incorporated into a cell, by which that cell may be targeted for destruction.
  • the immune cell is created to include a target epitope that when bound by an antibody, may kill the immune cell.
  • an immune cell such as a CAR T cell, may express a particular epitope or epitopes of the EGFR protein on its surface.
  • kill switch activator thus refers to any molecule capable of activating a cell’s degradation pathway by interacting with the kill switch of that cell.
  • the kill switch is caspase 9.
  • rimiducid functions as a drug-mediated kill switch within the subject.
  • the kill switch activates protein degradation.
  • lenalidomide functions as a drug- mediated kill switch within the subject.
  • CAR-encoding mRNA functions as a drug-mediated kill switch within the subject.
  • the kill switch is a suicide gene, such as inducible Caspase 9, herpes simplex virus tyrosine kinase, or human thymidylate kinase.
  • haploidentical stem-cell transplants HSCT
  • ganciclovir functions as a drug-mediated kill switch within the subject.
  • rituximab functions as a drug-mediated kill switch within the subject.
  • the kill switch is the multi-epitope RQR8.
  • the kill switch is an EGFR epitope. In some embodiments, the kill switch is a truncated EGFR molecule. In some embodiments, the anti-EGFR molecule functions as a drug-mediated kill switch activator within the subject. In some embodiments, the anti-EGFR molecule is an antibody. In some embodiments, the anti-EGFR molecule is a monoclonal antibody. In some embodiments, the anti-EGFR molecule is cetuximab. In some embodiments, the anti-EGFR molecule is avelumab. In some embodiments, the anti-EGFR molecule is necitumumab. In some embodiments, the anti-EGFR molecule is panitumumab.
  • the kill switch activator is administered to the subject intraperitoneally. In some embodiments, the kill switch activator is administered to the subject intravenously. In some embodiments, the kill switch activator is administered to the subject intratumorally. In some embodiments, the kill switch activator is administered to the subject intramurally.
  • the method comprises administering a cell of any one of the embodiments disclosed herein to the subject.
  • the method of treating a patient with CAR-T comprises controlling CAR-T persistence and anti-tumor effects.
  • CAR-T persistence and/or anti-tumor effects can be increased or decreased (i.e. CAR-T oscillation) by: (1) CAR-T repeat infusion, (2) administration of anti- EGFR antibody drug such as cetuximab or similar to activate the kill switch and control CAR T levels or ablate the CAR T cells in the blood.
  • the concentration and frequency of cetuximab administration can be modulated to control the CAR T concentration.
  • no lymphodepletion chemotherapy may be used with minimal dose level/formulation (as low as 4E4 CD3 positive and CAR T positive T cells/kg up to lE7/kg patient, or a flat dose starting from 5E5 CAR+ T cells/kg).
  • the route of administration such as IP or other routes of administration including IV or intratumoral administration with one or more iCPIs, including anti-PDl and antiPDLl and others to increase the therapeutic efficacy and window.
  • the method may include treatment in malignant cancers including solid tumors such as gastric cancer, colorectal, etc. with or without ascites, and Peritoneal Carcinomatosis.
  • the CAR-T cells may target one or more targets, including T cells, NKs or other immune cell modalities.
  • the method comprises a combined treatment of CAR-T cells with an at least one checkpoint inhibitor. ICIs targeting the PD1/PDL-1 axis can unleash CAR-T cell inhibition. This effect may enhance CAR-T cell cytotoxic activity and consequently promote its antitumor effect.
  • the method comprises administration of CAR-T and/or B4t2-001 with an at least one iCPI, or a genetic construct such as pDNR (dominant negative receptor).
  • the method comprises the combination of one or more iCPIs with CAR-T and/or B4t2-001.
  • the method comprises administration of an anti-PDl, anti-PDLl, and/or anti-CTLA4.
  • the at least one iCPI is administered in at least one dose.
  • the CAR-T is infused at MTD and anti-PDl and/or anti-PDLl is infused as a dose escalation over several doses.
  • iCPI treatment is administered before or after CAR- T dosing.
  • iCPI treatment is administered with CAR-T dosing.
  • the CAR-T cells are administered through IP, IV, or IT routes.
  • the CAR-T is administered with or without lymphodcplction.
  • the CAR-T cells are administered more than once.
  • the disease or disorder is a cancer.
  • the cancer may be any type of cancer.
  • the cancer is any type of malignancy.
  • the subject also has malignant ascites.
  • the cancer may be acute myeloid leukemia (AML), breast cancer, colorectal cancer, kidney cancer, liver cancer, lung cancer, brain cancer, pancreatic cancer, bladder cancer, testicular cancer, prostate cancer, gastric cancer, a hematologic malignancy, or any combination thereof.
  • the hematologic malignancy may comprise leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, hairy cell leukemia, lymphoma, Hodgkin’s disease, Non-Hodgkin lymphoma, or multiple myeloma.
  • the immune cell may be derived from the subject for an autologous treatment. Alternatively, the immune cell may be derived from the same species as the subject for an allogeneic treatment.
  • the subject is a mammal and/or human.
  • the method further comprises the CAR protein, cell, and/or composition stimulating a blood cell within the subject.
  • the blood cell is a neutrophil and/or a granulocyte.
  • the cell of any one of the embodiments of the present disclosures and/or a cell expressing the CAR of any one of the embodiments of the present disclosures undergoes a significant population expansion.
  • the cell undergoes a significant activation and/or a potent population expansion.
  • the significant activation and/or potent population expansion is at least a 50-fold increase in cell population.
  • the disease or disorder is a cancer.
  • the method does not require a lymphodepletion step.
  • the method of treating a disease or disorder in a subject in need thereof comprises administering a CAR-immune cell comprising a first binding site for CEACAM6 and a second binding site for a cancer.
  • the cancer is not blood borne.
  • the cancer is a solid tumor.
  • the second binding site is capable of binding to any one of: AFP, ANTXR 1 , AXL, avP3, avP6, B7-H3, CAIX, CD171, CD20, CD32A, CD46, CD47, CD56, CD80/86, CEA, Claudin 18.2, DLL-3, DR5, EGFR, EGFRIII, EGFR806, EpCAM, EpHA2, FAP, FR-alpha, GD2, Glypican- 2, Glypican-3, gplOO, GSPG4, GUCY2C, HBV surface antigen (HBsAg), HER2, IL-13R- alpha2, LI -CAM, Lewis Y, LMP1, MAGE- A 1/3/4, mesothelin, c-MET, MUC1, MUC16, MUC3A, Nectin4/FAP, NKG2D, PAP, PSA, PSCA, PSMA, ROR, TAG-72, Trop2, and/or V
  • the cancer is present in a cell and/or tissue that is selected from: brain, breast, central nervous system, cervical, colon, colorectal, epidermis, gastric, glioblastoma, glioma, hepatoma, liver, lung, nasopharyngeal, neuroblastoma, ovarian, pancreatic, pediatric glioma, prostate, rectum, renal, and stomach cells and/or tissue.
  • the method does not require a lymphodepletion step.
  • the subject is a mammal and/or human.
  • the method further comprises the CAR-immune cell stimulating a blood cell within the subject.
  • the blood cell is a neutrophil and/or a granulocyte.
  • CAR-immune cell upon stimulating the blood cell, CAR-immune cell undergoes a significant population expansion. In some embodiments, the CAR-immune cell undergoes a significant activation and/or a potent population expansion. In some embodiments, the significant activation and/or potent population expansion is at least a 50-fold increase in cell population. In some embodiments, the CAR- immune cell is B4T2-001. In some embodiments, the CAR-immune cell is a TIL cell. In some embodiments, the CAR-immune cell is a T cell, NK cell, or macrophage. In some embodiments, the administration is conducted through an infusion.
  • the administration is performed locally, intraperitoneally, intravenously, intratumorally, or intramurally.
  • the method further comprises multiple administrations of the payload to the subject.
  • the multiple administrations are performed locally, intraperitoneally, intravenously, intratumorally, and/or intramurally.
  • the stimulation of the blood cell is reversible.
  • the significant activation and/or potent population expansion results in at least 20%, 25%, 30%, 40%, or 50% engraftment of the cell.
  • the significant activation and/or potent population expansion results in at least 20% engraftment of the cell.
  • the significant activation and/or potent population expansion results in at least 25% engraftment of the cell. In some embodiments, the significant activation and/or potent population expansion results in at least 50% cngraftmcnt of the cell. In some embodiments, the cell undergoes significant activation and/or potent population expansion in at least one of the peripheral blood, peritoneal fluid, and/or pulmonary fluid of the subject. In some embodiments, the method further comprises administration of a kill switch activator. In some embodiments, the kill switch activator is an anti-EGFR antibody and/or is cetuximab. In some embodiments, the kill switch activator is administered locally, intraperitoneally, intravenously, intratumorally, or intramurally.
  • the kill switch activator is administered at a dose of about 0.1, 1, 10, 100, 250, 500, 700, 750, or any integer that is between 0.1 and 750, mg/m2. In some embodiments, the kill switch activator is administered more than once. In some embodiments, the more than one administrations of the kill switch activator is performed locally, intraperitoneally, intravenously, intratumorally, and/or intramurally.
  • the cell is administered at an effective dose to a subject in need thereof.
  • the subject has cancer.
  • the immune cell is administered more than once.
  • the immune cell is administered locally, intraperitoneally, intravenously, intratumorally, or intramurally.
  • the virus particle coding for the CAR, or mRNA LNP coding for the CAR is administered.
  • B4T2-001 is administered more than once.
  • the at least one iCPI is administered more than once.
  • the anti-EGFR molecule is administered more than once.
  • the anti-EGFR, iCPI, and immune cell are administered at different times. In some embodiments, any combination of the anti-EGFR, iCPI, and immune cell are administered together.
  • the immune cell is administered to a subject prior to administration of an anti-EGFR and/or an at least one iCPI.
  • the addition of iCPI treatment is initiated after 1, 2, 3, 4, 5, 10, 15, 20, 24, or any integer between 1 and 24 weeks following immune cell administration.
  • the iCPI treatment is administered at 1 week following immune cell administration.
  • the iCPI treatment is administered at 2 weeks following immune cell administration.
  • the immune treatment is administered at 3 weeks following immune cell administration.
  • the iCPI treatment is administered at 4 weeks following immune cell administration.
  • the iCPI treatment is administered at 5 weeks following immune cell administration.
  • the iCPI treatment is administered at 6 weeks following immune cell administration. In some embodiments, the iCPI treatment is administered at 7 weeks following immune cell administration. In some embodiments, the iCPI treatment is administered at 8 weeks following immune cell administration. In some embodiments, the iCPI treatment is administered at 9 weeks following immune cell administration. In some embodiments, the iCPI treatment is administered at 10 weeks following immune cell administration. In some embodiments, the iCPI treatment is administered at 11 weeks following immune cell administration. In some embodiments, the iCPI treatment is administered at 12 weeks following immune cell administration. In some embodiments, the iCPI treatment is administered at 13 weeks following immune cell administration. In some embodiments, the iCPI treatment is administered at 14 weeks following immune cell administration.
  • the iCPI treatment is administered at 15 weeks following immune cell administration. In some embodiments, the iCPI treatment is administered at 20 weeks following immune cell administration. In some embodiments, the iCPI treatment is administered at 22 weeks following immune cell administration. In some embodiments, the iCPI treatment is administered at 24 weeks following immune cell administration. In some embodiments, the iCPI treatment is administered every other week following immune cell administration. In some embodiments, the iCPI treatment is administered every 2 weeks following immune cell administration. In some embodiments, the iCPI treatment is administered every 3 weeks following immune cell administration. In some embodiments, the iCPI treatment is administered every 4 weeks following immune cell administration. In some embodiments, the iCPI treatment is administered every 5 weeks following immune cell administration.
  • the iCPI treatment is administered every 6 weeks following immune cell administration. In some embodiments, the iCPI treatment is administered every 7 weeks following immune cell administration. In some embodiments, the iCPI treatment is administered every 8 weeks following immune cell administration. In some embodiments, the iCPI treatment is administered every 9 weeks following immune cell administration. In some embodiments, the iCPI treatment is administered every 10 weeks following immune cell administration. In some embodiments, the iCPI treatment is administered every 11 weeks following immune cell administration. In some embodiments, the iCPI treatment is administered every 12 weeks following immune cell administration. In some embodiments, the immune cell is administered at least once. In some embodiments, the immune cell is administered more than once.
  • the immune cell is administered locally, intraperitoneally, intravenously, intratumorally, or intramurally.
  • iCPI treatment is administered at least once. In some embodiments, the iCPI treatment is administered more than once. In some embodiments, the iCPI treatment is administered at a fixed dose. In some embodiments, the iCPI treatment is administered at a dose that is about 0.1, 0.25, 0.5, 0.75, 1, 2.5, 5, 7.5, 10, or any integer that is between 0.1 and 10, mg/kg. In some embodiments, the iCPI treatment is administered at an escalating dose. In some embodiments, the iCPI treatment is administered at a decreasing dose.
  • the addition of kill switch activator treatment is initiated after 1, 2, 3, 4, 5, 10, 15, 20, 24, or any integer between 1 and 24 weeks following immune cell administration.
  • the kill switch activator treatment is administered at 1 week following immune cell administration. In some embodiments, the kill switch activator treatment is administered at 2 weeks following immune cell administration. In some embodiments, the immune treatment is administered at 3 weeks following immune cell administration. In some embodiments, the kill switch activator treatment is administered at 4 weeks following immune cell administration. In some embodiments, the kill switch activator treatment is administered at 5 weeks following immune cell administration. In some embodiments, the kill switch activator treatment is administered at 6 weeks following immune cell administration. In some embodiments, the kill switch activator treatment is administered at 7 weeks following immune cell administration.
  • the kill switch activator treatment is administered at 8 weeks following immune cell administration. In some embodiments, the kill switch activator treatment is administered at 9 weeks following immune cell administration. In some embodiments, the kill switch activator treatment is administered at 10 weeks following immune cell administration. In some embodiments, the kill switch activator treatment is administered at 11 weeks following immune cell administration. In some embodiments, the kill switch activator treatment is administered at 12 weeks following immune cell administration. In some embodiments, the kill switch activator treatment is administered at 13 weeks following immune cell administration. In some embodiments, the kill switch activator treatment is administered at 14 weeks following immune cell administration. In some embodiments, the kill switch activator treatment is administered at 15 weeks following immune cell administration. In some embodiments, the kill switch activator treatment is administered at 20 weeks following immune cell administration.
  • the kill switch activator treatment is administered at 22 weeks following immune cell administration. In some embodiments, the kill switch activator treatment is administered at 24 weeks following immune cell administration. In some embodiments, the kill switch activator treatment is administered every other week following immune cell administration. In some embodiments, the kill switch activator treatment is administered every 2 weeks following immune cell administration. In some embodiments, the kill switch activator treatment is administered every 3 weeks following immune cell administration. In some embodiments, the kill switch activator treatment is administered every 4 weeks following immune cell administration. In some embodiments, the kill switch activator treatment is administered every 5 weeks following immune cell administration. In some embodiments, the kill switch activator treatment is administered every 6 weeks following immune cell administration. In some embodiments, the kill switch activator treatment is administered every 7 weeks following immune cell administration.
  • the kill switch activator treatment is administered every 8 weeks following immune cell administration. In some embodiments, the kill switch activator treatment is administered every 9 weeks following immune cell administration. In some embodiments, the kill switch activator treatment is administered every 10 weeks following immune cell administration. In some embodiments, the kill switch activator treatment is administered every 11 weeks following immune cell administration. In some embodiments, the kill switch activator treatment is administered every 12 weeks following immune cell administration. In some embodiments, the immune cell is administered at least once. In some embodiments, the immune cell is administered more than once. In some embodiments, the immune cell is administered locally, intraperitoneally, intravenously, intratumorally, or intramurally. In some embodiments, kill switch activator treatment is administered at least once.
  • the kill switch activator treatment is administered more than once. In some embodiments, the kill switch activator treatment is administered at a fixed dose. In some embodiments, the kill switch activator treatment is administered at a dose that is about 0.1, 0.25, 0.5, 0.75, 1, 2.5, 5, 7.5, 10, or any integer that is between 0.1 and 10, mg/kg. In some embodiments, the kill switch activator treatment is administered at a dose that is about 0.1, 1, 10, 100, 250, 500, 700, 750, or any integer that is between 0.1 and 750, mg/m2. In some embodiments, the kill switch activator treatment is administered at an escalating dose. In some embodiments, the kill switch activator treatment is administered at a decreasing dose.
  • the method of treatment does not comprise chemotherapy treatment.
  • chemotherapy as used herein is given its standard scientific meaning, and thus refers to a non-cell and non-protein chemical drug that is used to kill cancer cells.
  • the method does not comprise a lymphodepletion step.
  • lymphodepletion as used herein is given its standard scientific meaning, and thus refers to a short course of chemotherapy administered to a subject in order to kill their T cells before, after, or during immunotherapy.
  • the immune cell is administered to the subject by an at least one intraperitoneal infusion.
  • the at least one intraperitoneal infusion is more than one intraperitoneal infusion.
  • the at least one intraperitoneal infusion is administered 2, 3, 4, 5, 10, 20, 25, 30, 40, 50, 100, or any integer that is between 2 and 100, times.
  • ‘about” is meant a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 % to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.
  • “Significant” as referred to herein refers to a reproducible difference in effect. For example, if compound A is said to bind a target significantly more compared to compound B, then there is a reproducibly greater number of compound A bound to the target compared to compound B.
  • Patent is given its typical scientific meaning, and thus refers to a high effectiveness/activity of a molecule per unit of that molecule.
  • % w/w or “% wt/wt” means a percentage expressed in terms of the weight of the ingredient or agent over the total weight of the composition multiplied by 100.
  • nucleic acid or “nucleic acid molecule” as used herein refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • PCR polymerase chain reaction
  • Nucleic acid molecules can be composed of monomers that are naturally-occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., enantiomeric forms of naturally-occurring nucleotides), or a combination of both. Nucleic acid monomers can be linked by phosphodicstcr bonds or analogs of such linkages. A nucleic acid or nucleic acids can be contained in a nucleic acid vector or nucleic acid construct (e.g.
  • plasmid plasmid, virus, bacteriophage, cosmid, fosmid, phagemid, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), or human artificial chromosome (HAC)) that can be used for amplification and/or expression of the nucleic acid or nucleic acids in various biological systems.
  • BAC bacterial artificial chromosome
  • YAC yeast artificial chromosome
  • HAC human artificial chromosome
  • the vector or construct will also contain elements including but not limited to promoters, enhancers, terminators, inducers, ribosome binding sites, translation initiation sites, start codons, stop codons, polyadenylation signals, origins of replication, cloning sites, multiple cloning sites, restriction enzyme sites, epitopes, reporter genes, selection markers, antibiotic selection markers, targeting sequences, peptide purification tags, or accessory genes, or any combination thereof.
  • elements including but not limited to promoters, enhancers, terminators, inducers, ribosome binding sites, translation initiation sites, start codons, stop codons, polyadenylation signals, origins of replication, cloning sites, multiple cloning sites, restriction enzyme sites, epitopes, reporter genes, selection markers, antibiotic selection markers, targeting sequences, peptide purification tags, or accessory genes, or any combination thereof.
  • a nucleic acid or nucleic acid molecule can comprise one or more sequences encoding different peptides, polypeptides, or proteins. These one or more sequences can be joined in the same nucleic acid or nucleic acid molecule adjacently, or with extra nucleic acids in between, e.g. linkers, repeats or restriction enzyme sites, or any other sequence that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, or any length in a range defined by any two of the aforementioned lengths.
  • downstream on a nucleic acid as used herein refers to a sequence being after the 3 ’-end of a previous sequence, on the strand containing the encoding sequence (sense strand) if the nucleic acid is double stranded.
  • upstream on a nucleic acid as used herein refers to a sequence being before the 5 ’-end of a subsequent sequence, on the strand containing the encoding sequence (sense strand) if the nucleic acid is double stranded.
  • grouped on a nucleic acid as used herein refers to two or more sequences that occur in proximity either directly or with extra nucleic acids in between, e.g.
  • linkers repeats, or restriction enzyme sites, or any other sequence that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, or any length in a range defined by any two of the aforementioned lengths, but generally not with a sequence in between that encodes for a functioning or catalytic polypeptide, protein, or protein domain.
  • peptide polypeptide
  • protein as used herein refers to macromolecules comprised of amino acids linked by peptide bonds.
  • peptides, polypeptides, and proteins are known in the art, and include but are not limited to enzymes, structure, transport, defense, hormones, or signaling.
  • Peptides, polypeptides, and proteins are often, but not always, produced biologically by a ribosomal complex using a nucleic acid template, although chemical syntheses are also available.
  • nucleic acid template By manipulating the nucleic acid template, peptide, polypeptide, and protein mutations such as substitutions, deletions, truncations, additions, duplications, or fusions of more than one peptide, polypeptide, or protein can be performed.
  • fusions of more than one peptide, polypeptide, or protein can be joined in the same molecule adjacently, or with extra amino acids in between, e.g. linkers, repeats, epitopes, or tags, or any other sequence that is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, or 300 bases long, or any length in a range defined by any two of the aforementioned lengths.
  • the term “downstream” on a polypeptide as used herein refers to a sequence being after the C-terminus of a previous sequence.
  • upstream on a polypeptide as used herein refers to a sequence being before the N-terminus of a subsequent sequence.
  • nucleic acid or peptide sequences presented herein and used in the examples are functional in various biological systems including but not limited to humans, mice, rats, monkeys, primates, cats, dogs, rabbits, E. coli, yeast, and mammalian cells.
  • nucleic acid or peptide sequences sharing at least or lower than 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similarity, or any percentage within a range defined by any two of the aforementioned percentages similarity to the nucleic acid or peptide sequences presented herein and used in the examples can also be used with no effect on the function of the sequences in biological systems.
  • similarity refers to a nucleic acid or peptide sequence having the same overall order of nucleotide or amino acids, respectively, as a template nucleic acid or peptide sequence with specific changes such as substitutions, deletions, repetitions, or insertions within the sequence.
  • two nucleic acid sequences sharing as low as 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% similarity can encode for the same polypeptide by comprising different codons that encode for the same amino acid during translation.
  • sequences having a percent homology to any of the sequences disclosed herein are envisioned and may be used.
  • the term “% homology” refers to the degree of conservation between two sequences when considering their three-dimensional structure. For example, homology between two protein sequences may be dependent on structural motifs, such as beta strands, alpha helices, and other folds, as well as their distribution throughout the sequence. Homology may be determined through structural determination, either empirically or in silico.
  • any sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology to any of the sequences disclosed herein may be used.
  • any sequence having at least 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 substitutions, deletions, or additions relative to any of the sequences disclosed herein, which may or may not affect the overall percent homology, may be used.
  • sequences having a certain “percent similarity” or “percent identity” to any of the sequence disclosed herein are envisioned and may be used.
  • these sequences may include peptide sequences, nucleic acid sequences, CDR sequences, variable region sequences, or heavy or light chain sequences.
  • similarity refers to the comparison of amino acids based on their properties, including but not limited to size, polarity, charge, pK, aromaticity, hydrogen bonding properties, or presence of functional groups (e.g. hydroxyl, thiol, amine, carboxyl, and the like).
  • % similarity refers to the percentage of units (i.e.
  • substitution matrices include BLOSUM45, BLOSUM62, BLOSUM80, PAM 100, PAM 120, PAM 160, PAM200, PAM250, but other substitution matrices or approaches may be used as considered appropriate by the skilled person.
  • a certain substitution matrix may be preferential over the others when considering aspects such as stringency, conservation and/or divergence of related sequences (e.g. within the same species or broader), and length of the sequences in question.
  • a peptide sequence having a certain percent similarity to another sequence will have up to that percent of amino acids that are either identical or an acceptable substitution as governed by the method of similarity determination used.
  • a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence similarity to any of the sequences disclosed herein may be used.
  • any sequence having at least 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 similar substitutions relative to any of the sequences disclosed herein may be used.
  • these similar substitutions may apply to antigen-binding regions (i.e. CDRs) or regions that do not bind to antigens or are only secondary to antigen binding (i.e. framework regions).
  • sequences having a certain “percent identity” to any of the sequence disclosed herein are envisioned and may be used.
  • the term to “percent identity” refers to the percent similarity between two or more sequences. In some embodiments, any sequence having at least 60%, 70%, 80%, 85%, 90%, 95%, 99%, 100%, or any integer that is between 60 and 100% identity, to any of the sequences disclosed herein may be used.
  • consensus sequence refers to the generalized sequence representing all of the different combinations of permissible amino acids at each location of a group of sequences.
  • a consensus sequence may provide insight into the conserved regions of related sequences where the unit (e.g. amino acid or nucleotide) is the same in most or all of the sequences, and regions that exhibit divergence between sequences.
  • the consensus sequence of a CDR may indicate amino acids that are important or dispensable for antigen binding. It is envisioned that consensus sequences may be prepared with any of the sequences provided herein, and the resultant various sequences derived from the consensus sequence can be validated to have similar effects as the template sequences.
  • the term "antibody” denotes the meaning ascribed to it by one of skill in the ail, and further it is intended to include any polypeptide chain-containing molecular structure with a specific shape that fits to and recognizes an epitope, where one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope.
  • the term "compete,” as used herein with regard to an antibody or binding polypeptide, means that a first antibody or binding polypeptide, or an antigen-binding portion thereof, binds to an epitope in a manner sufficiently similar to the binding of a second antibody or binding polypeptide, or an antigen-binding portion thereof, such that the result of binding of the first antibody or binding polypeptide with its cognate epitope is detectably decreased in the presence of the second antibody or binding polypeptide compared to the binding of the first antibody or binding polypeptide in the absence of the second antibody or binding polypeptide.
  • An antibody or binding polypeptide that "preferentially binds" or “specifically binds” (used interchangeably herein) to an epitope is a term well understood in the art, and methods to determine such specific or preferential binding are also well known in the art.
  • a molecule is said to exhibit "specific binding” or “preferential binding” if it reacts or associates more frequently, and/or more rapidly, and/or with greater duration and/or with greater affinity with a particular cell or substance than it does with alternative cells or substances.
  • An antibody or binding polypeptide "specifically binds” or “preferentially binds” to a target if it binds with greater affinity, and/or avidity, and/or more readily, and/or with greater duration than it binds to other substances.
  • non-human antibodies are hybrid immunoglobulins, immunoglobulin chains or fragments thereof which contain minimal sequence derived from non-human immunoglobulin.
  • single domain binding polypeptide or “single domain antibody” (sdAb) as used herein refers to a single peptide strand (e.g. not bound to another peptide strand with disulfide bonds) comprising an intact immunoglobulin domain or other protein fold which can recognize antigens.
  • Single domain binding polypeptides or sdAbs may be derived from typical heavy or light immunoglobulin chains, such as from human, or from alternative sources such as dromedaries (e.g. VHH) and cartilaginous fish (e.g. VNAR).
  • the single domain binding polypeptide or sdAb comprises one, two, or three complementarity determining regions (CDRs).
  • the single domain binding polypeptide or sdAb comprises one, two, or three of a CDR1, CDR2, and CDR3.
  • CDRs complementarity determining regions
  • single-chain variable fragment as used herein is a fusion protein comprising the variable regions of the heavy (VH) and light chains (VL) of an immunoglobulin, in which the VH and VL are covalently linked to form a VH: VL heterodimer.
  • the VH and VL are either joined directly or joined by a peptide-encoding linker, which connects the N-terminus of the VH with the C-terminus of the VL, or the C-terminus of the VH with the N-terminus of the VL.
  • the linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility.
  • Single chain Fv polypeptide antibodies can be expressed from a nucleic acid including VH- and VL-encoding sequences.
  • the VH and VL of the scFv each comprises one, two, or three CDRs.
  • the VH and VL of the scFv each comprises one, two, or three of a CDR1, CDR2, and CDR3.
  • definitive delineation of a CDR and identification of residues comprising the binding site of an antibody or binding polypeptide is accomplished by solving the structure of the antibody or binding polypeptide and/or solving the structure of the antibody-ligand complex. In certain embodiments, that can be accomplished by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In certain embodiments, various methods of analysis can be employed to identify or approximate the CDR regions. In certain embodiments, various methods of analysis can be employed to identify or approximate the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the IM GT approach (Lefranc et al., 2003) Dev Comp Immunol. 27:55-77), computational programs such as Paratome (Kunik et al., 2012, Nucl Acids Res. W521-4), the AbM definition, and the conformational definition.
  • the Kabat definition is a standard for numbering the residues in an antibody and is typically used to identify CDR regions. See, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8.
  • the Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account positions of certain structural loop regions. See, e.g., Chothia et al., 1986, J. Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83.
  • the AbM definition uses an integrated suite of computer programs produced by Oxford Molecular Group that model antibody structure.
  • the AbM definition models the tertiary structure of an antibody from primary sequence using a combination of knowledge databases and ab initio methods, such as those described by Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach,” in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198.
  • the contact definition is based on an analysis of the available complex crystal structures.
  • CDRs In another approach, referred to herein as the "conformational definition" of CDRs, the positions of the CDRs may be identified as the residues that make enthalpic contributions to antigen binding. See, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1156- 1166. Still other CDR boundary definitions may not strictly follow one of the above approaches, but will nonetheless overlap with at least a portion of the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues do not significantly impact antigen binding.
  • a CDR may refer to CDRs defined by any approach known in the art, including combinations of approaches.
  • the methods used herein may utilize CDRs defined according to any of these approaches.
  • the CDRs may be defined in accordance with any of Kabat, Chothia, extended, IMGT, Paratome, AbM, and/or conformational definitions, or a combination of any of the foregoing.
  • immunoglobulin fragments or “binding fragments” comprising the epitope binding site (e.g., Fab', F(ab')2, single-chain variable fragment (scFv), diabody, minibody, nanobody, singledomain antibody (sdAb), VHH fragments, VNAR fragments, or other fragments) are useful as antibody moieties in the present invention.
  • Such antibody fragments may be generated from whole immunoglobulins by ricin, pepsin, papain, or other protease cleavage.
  • Minimal immunoglobulins may be designed utilizing recombinant immunoglobulin techniques.
  • Fv immunoglobulins for use in the present invention may be produced by linking a variable light chain region to a variable heavy chain region via a peptide linker (e.g., polyglycine or another sequence which does not form an alpha helix or beta sheet motif).
  • Nanobodies or single-domain antibodies can also be derived from alternative organisms, such as dromedaries, camels, llamas, alpacas, sharks, or cartilaginous fish.
  • antibodies can be conjugates, e.g. pegylated antibodies, drug, radioisotope, or toxin conjugates.
  • Monoclonal antibodies directed against a specific epitope, or combination of epitopes, will allow for the targeting and/or depletion of cellular populations expressing the marker.
  • single-domain antibody refers to a single peptide strand (e.g. not bound to another peptide strand with disulfide bonds) comprising an intact immunoglobulin domain or other protein fold which can recognize antigens.
  • sdAbs may be derived from typical heavy or light immunoglobulin chains, such as from human, or from alternative sources such as dromedaries (e.g. VHH) and cartilaginous fish (e.g. VNAR).
  • CEA6 carcinoembryonic antigen-related cell adhesion molecule 6
  • CEACAM6 carcinoembryonic antigen-related cell adhesion molecule 6
  • the CEA6 binding polypeptides comprise an immunoglobulin heavy chain variable domain comprising a CDR-H1, CDR-H2, and CDR-H3.
  • the CEA6 binding polypeptide comprise an immunoglobulin heavy chain variable domain comprising a CDR-H1, CDR-H2, and CDR-H3, where one or more of these CDRs are defined by a consensus sequence.
  • alternative alignments may be done (e.g. using global or local alignment, or with different algorithms, such as Hidden Markov Models, seeded guide trees, Needleman- Wunsch algorithm, or Smith-Waterman algorithm, or other known methods) and as such, alternative consensus sequences can be derived (including those done with a subset of the sequences provided herein).
  • the CDR-H1 is defined by the formula X1X2X3X4X5X6X7X8, where XI is G; X2 is F, R, S, or Y; X3 is I or T; X4 is F, G, L, S, or Y; X5 is D, G, N, or S; X6 is D, F, I, L, N, S, T, or Y; X7 is D, N, or Y; X8 is D, F, H, E, P, T, V, or Y.
  • the CDR-H1 comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to this consensus sequence. In some embodiments, the CDR-H1 comprises a sequence having 0, 1, 2, 3, 4, 5, or 6 substitutions from this consensus sequence.
  • the CDR-H2 is defined by the formula X1X2X3X4X5X6X7X8X9X10, where XI is no amino acid, S, or T; X2 is I; X3 is N, S, or T; X4 is R, S, T, or W; X5 is D, F, I, L, S, T, or Y; X6 is A, D, G, or S; X7 is A, D, G, or S; X8 is I or S; X9 is T; X10 is no amino acid or Y.
  • the CDR-H2 comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to this consensus sequence. In some embodiments, the CDR-H2 comprises a sequence having 0, 1, 2, 3, 4, 5, or 6 substitutions from this consensus sequence.
  • the CDR-H3 is defined by the formula X 1X2X3X4X5X6X7X8X9X 10X 1 IX 12X 13X 14X 15X 16X 17X 18X 19X20X21X22X23X24 X25X26X27X28X29X30X31X32X33, where XI is no amino acid or A; X2 is no amino acid, A, or V; X3 is no amino acid, A, G, M, Q, S, T, or V; X4 is no amino acid, A, D, E, G, I, M, N, R, S, V, or Y; X5 is no amino acid, A, E, K, M, R, S, T, V, or W; X6 is no amino acid, A, E, M, P, S, or V; X7 is no amino acid, A, F, I, M, P, W, or Y; X8 is no amino acid, D, I, K,
  • the CDR-H3 comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to this consensus sequence. In some embodiments, the CDR-H3 comprises a sequence having 0, 1, 2, 3, 4, 5, or 6 substitutions from this consensus sequence.
  • the CEA6 binding polypeptide is humanized. In some embodiments, the CEA6 binding polypeptide is a single domain antibody (sdAb).
  • the CEA6 binding polypeptide binds to CEA6 with a dissociation constant (KD) of less than 1 nM, 2 nM, 5 nM, 10 nM, 15 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, or 1000 nM, or any KD within a range defined by any two of the aforementioned KD.
  • KD dissociation constant
  • the binding polypeptides disclosed herein may be obtained from an antibody library.
  • the antibody library is an immune antibody library, a naive antibody library, a synthetic antibody library, or a semi-synthetic antibody library.
  • the antibody library comprises antibodies derived from human, or antibodies that are not immunogenic in humans, or both.
  • the antibody library comprises antibodies that are humanized, e.g. from mouse, rat, guinea pig, rabbit, cat, dog, cow, horse, sheep, goat, horse, donkey.
  • the antibody library comprises single domain antibodies (sdAb), nanobodies, VHH fragments, VNAR fragments, single-chain variable fragments (scFv), camelid antibodies, or cartilaginous fish antibodies, or any combination thereof.
  • sdAb single domain antibodies
  • nanobodies VHH fragments, VNAR fragments, single-chain variable fragments (scFv)
  • scFv single-chain variable fragments
  • camelid antibodies or cartilaginous fish antibodies, or any combination thereof.
  • sdAb single domain antibodies
  • sdAb single domain antibodies
  • scFv single-chain variable fragments
  • the antibody library comprises at least 100, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 500000, or 1000000 unique antibodies, or any number of antibodies within a range defined by any two of the aforementioned number of antibodies.
  • Antibody libraries may be generated computationally or using machine learning processes.
  • An exemplary method of generating an antibody library computationally includes modifying a universal VHH framework with synthetic diversity in one or more complementary determining regions (CDRs), such as CDR1, CDR2, or CDR3, or any combination thereof.
  • CDRs complementary determining regions
  • the diversity of the CDRs are introduced by randomizing the library of sequences encoding for the antibodies with degenerate codons.
  • an NNK codon library can be employed, where the NNK codon comprises N (25% mix of A/T/C/G) and K (50% mix of T/G).
  • the NNK codon library is constructed with all possible amino acids, or with some amino acids (e.g. cysteine) and stop codon combinations excluded.
  • the antibody library can be generated using a trimer codon mix, which improves balanced representation of sense codons while reducing the chance of stop codons, improving efficiency of antibody generation and testing.
  • artificial intelligence-based prediction can be used to randomize specific binding pockets of the antibodies using available binding models or structure data.
  • panning the antibody library comprises screening for the candidate binding polypeptides by phage display, yeast display, bacterial display, ribosome display, or mRNA display, or any combination thereof.
  • panning the antibody library comprises one or more rounds of selection, wherein the candidate binding polypeptides are selected for specificity towards a cancer-associated antigen (e.g. CEA6) or cells or tissues displaying the cancer-associated antigen.
  • a cancer-associated antigen e.g. CEA6
  • the candidate binding polypeptides are selected under conditions including but not limited to tumor microenvironment-like conditions, immunosuppressive conditions, low or high pH, low or high oxygen concentrations, low or high temperatures, low or high viscosity, or any combination thereof, or for specificity towards modified or derivative forms of the one or more cancer-associated antigens.
  • the immunosuppressive conditions may comprise the presence of tumor- associated macrophages (TAMs), mycloid-dcrivcd suppressor cells (MDSCs), tumor-associated neutrophils (TANs), cancer-associated fibroblasts (CAFs), or other immunosuppressive cells, or the presence of adenosine, or both.
  • the chimeric antigen receptor cells are from a cell line (e.g. Jurkat). In some embodiments, the chimeric antigen receptor cells are derived from a subject. In some embodiments, the subject has a cancer. In some embodiments, the subject has a cancer, and that cancer expresses any one or more of the cancer-associated antigens disclosed herein (e.g., CEA6).
  • a cell line e.g. Jurkat
  • the chimeric antigen receptor cells are derived from a subject.
  • the subject has a cancer. In some embodiments, the subject has a cancer, and that cancer expresses any one or more of the cancer-associated antigens disclosed herein (e.g., CEA6).
  • the cancer is acute myeloid leukemia (AML), breast cancer, colorectal cancer, kidney cancer, liver cancer, lung cancer, brain cancer, pancreatic cancer, bladder cancer, testicular cancer, prostate cancer, gastric cancer, ovarian cancer, head and neck cancer, gallbladder cancer, a hematologic malignancy, or any combination thereof.
  • the hematologic malignancy may comprise leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, hairy cell leukemia, lymphoma, Hodgkin’s disease, Non-Hodgkin lymphoma, or multiple myeloma.
  • the subject is a mammal, such as a human, cat, dog, mouse, rat, hamster, rodent, cow, pig, horse, goat, sheep, donkey, or monkey. In some embodiments, the subject is a human.
  • CARs chimeric antigen receptors
  • the CAR is a “bi-CAR” meaning that it binds to two different target binding sites.
  • CAR chimeric antigen receptor
  • An exemplary immune cell in which CARs can be used are T cells, but it is envisioned that CARs can be engineered into any amenable cytotoxic immune cell, including but not limited to T cells, Natural Killer (NK) cells, Natural Killer T (NKT) cells, dendritic cells, or macrophages.
  • NK Natural Killer
  • NKT Natural Killer T
  • dendritic cells dendritic cells
  • macrophages any disclosure pertaining to CAR T cells can also be applied to other immune cells comprising CARs.
  • CARs comprise an extracellular antigen-recognizing domain (e.g. tumor receptor ligand, or antibody), hinge, transmembrane, and intracellular signaling domain (endodomain). Different combinations of these CAR components may result in different specificities and efficacy against certain cancer antigens.
  • the CAR comprises at least two single domain binding polypeptides and the CAR is a multivalent CAR. In some embodiments, the CAR comprises two single domain binding polypeptides and the CAR is a bivalent CAR. In some embodiments, the CAR comprises three single domain binding polypeptides and the CAR is a tri valent CAR.
  • the CAR further comprises one or more signal peptides, linkers with various lengths and composition, hinges, transmembrane domains, costimulatory domains, signaling domains, cytoplasmic domains, functionality signals, proliferation signals, anti-exhaustion signals, anti-inhibitory receptors, tumor/cancer homing proteins, or regulatory molecules, or any combination thereof.
  • the hinges comprise CD3( ⁇ , CD4, CD8 or CD28 hinges, or computationally designed synthetic hinges with various lengths.
  • the transmembrane domains comprise CD3( ⁇ , CD4, CD8 or CD28 transmembrane domains, or computationally designed synthetic transmembrane domains.
  • the costimulatory domains comprise CD8, CD28, ICOS, 4-1BB, 0X40 (CD134), CD27, CD40, CD40L, TLR or other TNFR superfamily member or Ig superfamily member costimulatory domains, or other signaling via cytoplasmic domains of IL-2Rp, IL-15R-a, MyD88, or CD40 or any other Toll-like receptor or IL-1 receptor signaling pathway members.
  • the CARs disclosed herein are constructed by assembling CAR expression constructs from nucleic acids encoding for any one of the single domain binding polypeptides disclosed herein and a mixture of compatible nucleic acids encoding for different CAR modules.
  • different combinations of CARs are produced for use in a CAR library for screening for CAR efficacy (in vitro or in vivo).
  • unique CARs are produced separately.
  • the CARs are specific for one target.
  • the CARs are specific for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 targets.
  • the CARs are bi-specific or tri-specific.
  • the nucleic acids encoding for the single domain binding polypeptides identified by panning of the antibody library are assembled into CAR expression constructs with other CAR modules.
  • the CAR expression constructs arc assembled using multi-fragment assembly reactions known in the art.
  • One exemplary method of assembling CAR expression constructs involves using Type IIS restriction enzymes to generate nucleic acid fragments with compatible overhang sequences and ligating the nucleic acid fragments with a ligase. As Type IIS restriction enzymes cleave outside of their recognition sites, multiple compatible nucleic acid fragments may be prepared simultaneously.
  • the CAR expression constructs can be assembled by overlap extension PCR.
  • the different CAR modules comprise signal peptides, linkers, hinges, transmembrane domains, costimulatory domains, activation domains, signaling domains, cytoplasmic domains, functionality signals, proliferation signals, anti-exhaustion signals, antiinhibitor receptors, cancer homing proteins, or regulatory molecules, or any combination thereof.
  • Some exemplary hinges comprise CD8 hinge, CD28 hinge, IgGl hinge, or IgG4 hinge.
  • Some exemplary transmembrane domains comprise CD3 ⁇ transmembrane domain, CD8a transmembrane domain, CD4 transmembrane domain, CD28 transmembrane domain, or ICOS transmembrane domain.
  • Some exemplary costimulatory domains comprise CD8 costimulatory domain, CD28 costimulatory domain, 4-1BB costimulatory domain, 0X40 (CD134) costimulatory domain, ICOS costimulatory domain, CD27 costimulatory domain, CD40 costimulatory domain, CD40L costimulatory domain, TLR costimulatory domains, MYD88- CD40 costimulatory domain, or KIR2DS2 costimulatory domain.
  • the different CAR modules are derived from CD8, CD28, 4-1BB, CD3 ⁇ , or any combination thereof.
  • the CAR may also be modified with various additions, including but not limited to cytokines, chemokines, cytokine receptors, chemokine receptors, antigen receptors or ligands, antibodies, or enzymes.
  • the terms “individual(s)”, “subject(s)” and “patient(s)” are used interchangeably and mean any animal and/or mammal.
  • the mammal is a human.
  • the mammal is a non-human. None of the terms require or are limited to situations characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly or a hospice worker).
  • a health care worker e.g. a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly or a hospice worker.
  • treating means an approach for obtaining beneficial or desired results in a subject's condition, including clinical results.
  • beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of the extent of a disease, stabilizing (i.e., not worsening) the state of disease, prevention of a disease's transmission or spread, delaying or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission, whether partial or total and whether detectable or undetectable.
  • Treatment as used herein also include prophylactic treatment.
  • Treatment methods comprise administering to a subject a therapeutically effective amount of an active agent.
  • the administering step may consist of a single administration or may comprise a series of administrations.
  • the compositions are administered to the subject in an amount and for a duration sufficient to treat the subject.
  • the length of the treatment period depends on a variety of factors, such as the severity of the condition, the age and genetic profile of the subject, the concentration of active agent, the activity of the compositions used in the treatment, or a combination thereof.
  • the effective dosage of an agent used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required.
  • an effective amount refers to that amount of a recited composition or compound that results in an observable designated effect.
  • Actual dosage levels of active ingredients in an active composition of the presently disclosed subject matter can be varied so as to administer an amount of the active composition or compound that is effective to achieve the designated response for a particular subject and/or application.
  • the selected dosage level can vary based upon a variety of factors including, but not limited to, the activity of the composition, formulation, route of administration, combination with other drugs or treatments, severity of the condition being treated, and the physical condition and prior medical history of the subject being treated.
  • a minimal dose is administered, and dose is escalated in the absence of dose- limiting toxicity to a minimally effective amount. Determination and adjustment of an effective dose, as well as evaluation of when and how to make such adjustments, arc contemplated herein.
  • the term "therapeutic target” refers to a gene or gene product that, upon modulation of its activity (e.g., by modulation of expression, biological activity, and the like), can provide for modulation of the disease phenotype.
  • modulation is meant to refer to an increase or a decrease in the indicated phenomenon (e.g., modulation of a biological activity refers to an increase in a biological activity or a decrease in a biological activity).
  • standard of care refers to the treatment that is accepted by medical practitioners to be an appropriate, proper, effective, and/or widely used treatment for a certain disease.
  • the standard of care of a certain disease depends on many different factors, including the biological effect of treatment, region or location within the body, patient status (e.g. age, weight, gender, hereditary risks, other disabilities, secondary conditions), toxicity, metabolism, bioaccumulation, therapeutic index, dosage, and other factors known in the art.
  • Determining a standard of care for a disease is also dependent on establishing safety and efficacy in clinical trials as standardized by regulatory bodies such as the US Food and Drug Administration, International Council for Harmonisation, Health Canada, European Medicines Agency, Therapeutics Goods Administration, Central Drugs Standard Control Organization, National Medical Products Administration, Pharmaceuticals and Medical Devices Agency, Ministry of Food and Drug Safety, and the World Health Organization.
  • the standard of care for a disease may include but is not limited to surgery, radiation, chemotherapy, targeted therapy, or immunotherapy.
  • the methods comprise administering a chimeric antigen receptor cell to the subject. In some embodiments, the methods comprise administering any one of the chimeric antigen receptor cells disclosed herein. In some embodiments, the chimeric antigen receptor cell expresses and/or comprises any one of the CEA6 single domain binding polypeptides disclosed herein. In some embodiments, the chimeric antigen receptor cell is a CAR T-cell. In some embodiments, the chimeric antigen receptor cell is a CAR NK cell. In some embodiments, the chimeric antigen receptor cell is a CAR Tumor Infiltrating Lymphocte (TIL) cell.
  • TIL Tumor Infiltrating Lymphocte
  • the chimeric antigen receptor cell is a macrophage. In some embodiments, the chimeric antigen receptor cell is derived from the subject and is autologous to the subject. In some embodiments, the chimeric antigen receptor cell is allogeneic to the subject. In some embodiments, the chimeric antigen receptor cell is from a cell line (e.g. Jurkat). In some embodiments, the subject is a mammal, such as a human, cat, dog, mouse, rat, hamster, rodent, cow, pig, horse, goat, sheep, donkey, or monkey. In some embodiments, the subject is a human.
  • a mammal such as a human, cat, dog, mouse, rat, hamster, rodent, cow, pig, horse, goat, sheep, donkey, or monkey. In some embodiments, the subject is a human.
  • the cancer is acute myeloid leukemia (AML), breast cancer, colorectal cancer, kidney cancer, liver cancer, lung cancer, brain cancer, pancreatic cancer, bladder cancer, testicular cancer, prostate cancer, gastric cancer, a hematologic malignancy, or any combination thereof.
  • AML acute myeloid leukemia
  • breast cancer colorectal cancer
  • liver cancer liver cancer
  • lung cancer brain cancer
  • pancreatic cancer bladder cancer
  • testicular cancer prostate cancer
  • gastric cancer gastric cancer
  • a hematologic malignancy a hematologic malignancy
  • the chimeric antigen receptor cell is administered once per day, twice per day, three times per day or more. In some embodiments, the chimeric antigen receptor cell is administered daily, every day, every alternate day, five days a week, once a week, every other week, two weeks per month, three weeks per month, once a month, twice a month, three times per month, or more. In some embodiments, the immune cell is administered for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, or more.
  • the amount of a given agent that correspond to such an amount varies depending upon factors such as the particular compound, the severity of the disease, the identity (e.g., weight) of the subject or host in need of treatment, but nevertheless is routinely determined in a manner known in the art according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the route of administration, and the subject or host being treated.
  • the desired dose is conveniently presented in a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example as two, three, four or more sub-doses per day.
  • toxicity and therapeutic efficacy of such therapeutic regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between the toxic and therapeutic effects is the therapeutic index and it is expressed as the ratio between LD50 and ED50.
  • Compounds exhibiting high therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies are used in formulating a range of dosage for use in human.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage varies within this range depending upon the dosage form employed and the route of administration utilized.
  • administering includes intraperitoneal administration.
  • Intraperitoneal is given its standard scientific meaning, and thus refers to administration of a substance into a subject’s peritoneal cavity. Intraperitoneal administration thus includes any effective means of delivering a substance into the peritoneal cavity, including injection and infusion into the cavity.
  • Some embodiments of the present disclosure relate to a method of treatment for cancer, comprising administering at least one checkpoint inhibitor.
  • at least two checkpoint inhibitors are administered.
  • the at least two checkpoint inhibitors are administered at the same time.
  • the at least two checkpoint inhibitors are administered at different times.
  • a “checkpoint inhibitor” is a molecule, drug, and/or composition that is functional in inhibiting at least one immune checkpoint.
  • An “immune checkpoint” is a regulator of the immune system in a subject.
  • Non-limiting examples of stimulatory checkpoint molecules include CD27, CD28, CD40, CD122, CD137, 0X40, GITR, and TCOS.
  • Nonlimiting examples of inhibitory checkpoint molecules include A2AR, A2BR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, N0X2, PD-1, PD-L1, PD-L2, TIM-3, VISTA, SIGLEC7, and the PD-1 dominant negative receptor (DNR).
  • the cancer in a subject may avoid targeting by the immune system by altering the function of immune checkpoint targets.
  • Checkpoint inhibitors function to block this altered activity, thus restoring normal immune function. Consequently, cancer cells are predicted to be more susceptible to the immune system in patients that are under checkpoint inhibitor treatment.
  • Non-limiting examples of checkpoint inhibitors include iplimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, cemiplimab, tremelimumab, relatlimab, opdualag, and spartalizumab.
  • Example 1 Treatment of cancer with immune cell through IP injection
  • a human subject with cancer will be administered an effective dose of the CAR T cell B4T2-001 through intraperitoneal injection.
  • the human subject will not undergo any lymphodepletion/chemotherapy treatment prior to or during the CAR T cell administration.
  • Another dose of CAR T cells will be administered through intraperitoneal injection.
  • the subject will be administered the anti-EGFR antibody cetuximab to activate the kill switch and prevent any further effects by the CAR T cells.
  • Cetuximab will be administered at an initial dose of 400mg/m2 body surface area through IV over 120 min, followed by up to four weekly dose of 250 mg/m2 or until clearance of CAR T such as evaluated by qPCR) leads to complete elimination of the genetically modified T cells by antibody-dependent cellular cytotoxicity (ADCC).
  • ADCC antibody-dependent cellular cytotoxicity
  • B4t2-001 CAR T cells were infused into a subject at lE6/kg dose with no lymphodepletion chemotherapy through IP injection. Engraftment of the CAR T cells was monitored over time using qPCR (FIG. 1). The B4t2-001 CAR-T efficiently expanded in the peritoneal area, reached the peak on the second day post infusion, and engrafted efficiently in circulating Blood with a Cmax of 52,546 copies per microgram of genomic DNA.

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Abstract

Sont divulguées des méthodes d'administration d'une immunothérapie. Dans certains modes de réalisation, le procédé comprend l'administration d'une cellule à un patient et l'expansion ultérieure de cette cellule dans le sang périphérique. Dans certains modes de réalisation, le procédé utilise des cellules sanguines pour alimenter l'expansion. Cette méthode peut être utilisée dans le traitement du cancer.
PCT/US2024/038360 2023-07-31 2024-07-17 Procédés d'expansion et de greffe de cellules in vivo à médiation par des cellules sanguines Pending WO2025029484A1 (fr)

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LECLERCQ GABRIELLE, SERVERA LLUCIA ALBERTI, DANILIN SABRINA, CHALLIER JOHN, STEINHOFF NATHALIE, BOSSEN CLAUDIA, ODERMATT ALEX, NIC: "Dissecting the mechanism of cytokine release induced by T-cell engagers highlights the contribution of neutrophils", ONCOIMMUNOLOGY, TAYLOR & FRANCIS, vol. 11, no. 1, 31 December 2022 (2022-12-31), XP093277941, ISSN: 2162-402X, DOI: 10.1080/2162402X.2022.2039432 *

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