Method of blood cell mediated in vivo CAR cell expansion and implantation

Abstract

Disclosed herein are methods of administering immunotherapy. In some embodiments, the methods include administering cells to a subject, and subsequent expansion of the cells in peripheral blood. In some embodiments, the methods use blood cells to promote the amplification. The method can be used to treat cancer.

Description

[0001] Related applications and incorporation via citation

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 516773, filed July 31, 2023, which is incorporated herein by reference in its entirety. Background of the Invention

[0003] Invention Field

[0004] This disclosure generally relates to methods of administering immunotherapy. In some embodiments, the method includes administering cells or a composition containing or encoding a CAR targeting CEACAM6. In some embodiments, the CAR includes at least one binding site for a second or additional target. In some embodiments, the CAR protein has a single target and is capable of activating neutrophils and / or granulocytes.

[0005] Related technical descriptions

[0006] Adoptive cell therapies, such as chimeric antigen receptor (CAR) T-cell therapy, have shown great promise in cancer treatment. CAR T-cell therapy involves the use of genetically engineered T cells that express receptors targeting cancer-associated cell surface markers and other antigens, enabling the targeted killing of cancer cells while minimizing impact on the patient's normal cells. For example, Brexucabtagene Autoleucel, Tisagenlecleucel, and Axicabtagene Ciloleucel are FDA-approved CAR T-cell therapies for CD19+ B-cell lymphoma.

[0007] CARs consist of an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain, enabling targeted killing of cancer cells based on cell surface antigen expression while minimally affecting normal cells that do not express the target antigen. The extracellular antigen-binding domain is typically composed of an antibody or its binding fragment or derivative (e.g., single-chain variable fragment (scFv) or single-domain antibody (SDAB)). There is a need to improve CAR administration for the treatment of various cancers and other diseases. Summary of the Invention

[0008] In some embodiments, a chimeric antigen receptor (CAR) protein having at least one target is disclosed. In some embodiments, the CAR protein has a single target and is capable of activating neutrophils and / or granulocytes. In some embodiments, the target is CEACAM6 (also known as “CEA6,” “CD66c,” “CEAL,” “NCA,” “carcinoembryonic antigen-associated cell adhesion molecule 6,” and “CEA cell adhesion molecule 6”). In some embodiments, the CAR protein has at least two targets. In some embodiments, the CAR protein includes a first binding site and a second binding site, the first binding site being capable of binding CEACAM6; the second binding site being capable of binding an antigen expressed in abnormal cells or tissues. In some embodiments, the abnormal cells and / or abnormal tissues are cancer cells or tissues. In some embodiments, the abnormal cells and / or abnormal tissues originate from diseased cells or tissues. In some embodiments, the diseased cells or tissues are selected from autoimmune diseases, cardiomyopathy, viral infections, rheumatoid arthritis, fibrosis, cardiac fibrosis, HIV, COVID-19, chronic hepatitis C virus (HCV), human cytomegalovirus (HCMV), influenza, cellular senescence targets (selective clearance of senescent cells (SC)), liver fibrosis, pulmonary fibrosis, atherosclerosis, diabetes, osteoarthritis, obesity, and / or aging. In some embodiments, the diseased cells or tissues are viral infections, senescent cells, or fibrotic cells. In some embodiments, the abnormal cells or tissues are non-hematogenous. In some embodiments, the abnormal cells or tissues are solid tumors. In the implementation scheme, non-limiting example antigens that the second binding site may be able to bind include: AFP, ANTXR1, AXL, αvβ3, αvβ6, 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-α, GD2, Glypican-2, Glypican-3, gp100, GSPG4, GUCY2C, HBV surface antigen (HBsAg), HER2, IL-13R-α2, L1-CAM, Lewis Y, LMP1, MAGE-A1 / 3 / 4, mesothelin, M2e, c-MET, MUC1, MUC16, MUC3A, Nectin4 / FAP, NKG2D, PAP, PSA, PSCA, PSMA, ROR, TAG-72, Trop2, uPAR and / or VEGFR2.In some embodiments, the abnormal cells or tissues are selected from: brain, breast, central nervous system, cervix, colon, colorectal, epidermis, gastric, glioblastoma, glioma, hepatocellular carcinoma, liver, lung, nasopharynx, neuroblastoma, ovary, pancreas, pediatric glioma, prostate, rectum, kidney, and stomach cells and / or tissues.

[0009] This document also discloses nucleotide sequences that encode the CAR protein of any of the embodiments disclosed herein.

[0010] This document also discloses vectors that encode a CAR protein and / or a nucleotide of any embodiment of this disclosure.

[0011] This document also discloses cells capable of expressing the CAR protein and / or nucleotides and / or vectors of any embodiment of this disclosure. In some embodiments, the cells are immune cells. In some embodiments, the immune cells are B4T2-001. In some embodiments, the immune cells are TIL cells. In some embodiments, the immune cells are T cells, NK cells, or macrophages.

[0012] This document also discloses compositions comprising a CAR protein of any embodiment of this disclosure and / or cells of any embodiment of this disclosure. In some embodiments, the composition is formulated for administration to a subject. In some embodiments, the subject is a mammal and / or a human. In some embodiments, the administration is performed by infusion. In some embodiments, the administration is performed locally, intraperitoneally, intravenously, intratumorally, or intramurally. In some embodiments, the administration is performed more than once. In some embodiments, the administration is performed without a lymphodepletion step.

[0013] This document also discloses the use of the CAR protein of any embodiment of this disclosure and / or the cells of any embodiment of this disclosure and / or the compositions of any embodiment of this disclosure. In some embodiments, the use is for stimulating blood cells of a subject. In some embodiments, the subject is a mammal and / or a human. In some embodiments, the blood cells are neutrophils and / or granulocytes. In some embodiments, after stimulation of the blood cells, the cells of any embodiment of this disclosure or cells expressing the CAR of any embodiment of this disclosure undergo significant population expansion. In some embodiments, the cells undergo significant activation and / or potent population expansion. In some embodiments, the significant activation and / or potent population expansion is at least a 50-fold increase in the cell population. In some embodiments, the subject suffers from a disease or condition, such as cancer, which is treated with the CAR. In some embodiments, the treatment with the CAR does not require a lymphocyte clearance step prior to administration of the CAR.

[0014] This document also discloses methods for treating diseases or conditions in subjects in need. In some embodiments, the method includes applying to the subject any CAR protein of any embodiment of this disclosure, and / or cells of any embodiment of this disclosure, and / or a composition of any embodiment of this disclosure. In some embodiments, the subject is a mammal and / or a human. In some embodiments, the method further includes stimulating the subject's blood cells with the CAR protein, cells, and / or composition. In some embodiments, the blood cells are neutrophils and / or granulocytes. In some embodiments, after stimulation of the blood cells, the cells of any embodiment of this disclosure and / or cells expressing a CAR of any embodiment of this disclosure undergo significant population expansion. In some embodiments, the cells undergo significant activation and / or potent population expansion. In some embodiments, the significant activation and / or potent population expansion is at least a 50-fold increase in the cell population.

[0015] In some embodiments, the method of treating a disease or condition in a subject of need includes administering CAR immune cells containing a first binding site for CEACAM6 and a second binding site for the disease or condition. In some embodiments, the disease or condition is any of the following: autoimmune diseases, cardiomyopathy, viral infections, rheumatoid arthritis, fibrosis, cardiac fibrosis, diseases or conditions caused by HIV, diseases or conditions caused by COVID-19, diseases or conditions caused by chronic hepatitis C virus (HCV), diseases or conditions caused by human cytomegalovirus (HCMV), influenza, targets of cellular senescence (selective clearance of senescent cells (SC)), liver fibrosis, pulmonary fibrosis, atherosclerosis, diabetes, osteoarthritis, obesity, and / or aging. In some embodiments, the disease or condition is cancer. In some embodiments, the cancer is non-hematogenous. In some embodiments, the cancer is a solid tumor. In some embodiments, the second binding site is capable of binding to at least one of the following: AFP, ANTXR1, AXL, αvβ3, αvβ6, B7-H3, CAIX, CD171, CD20, CD32A, CD46, CD47, CD56, CD80 / 86, CEA, sealing protein 18.2, DLL-3, DR5, EGFR, EGFRIII, EGFR806, EpCAM, EpHA2, FAP, FR-α, GD2, phosphatidylinositol proteoglycan-2, phosphatidylinositol proteoglycan-3, gp100, GSPG4, GUCY2C, HBV surface antigen (HBsAg), HER2, IL-13R-α2, L1-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, Trop2, uPAR, and / or VEGFR2. In some embodiments, the cancer is present in cells and / or tissues selected from the following: brain, breast, central nervous system, cervix, colon, colorectal, epidermis, stomach, glioblastoma, glioma, hepatocellular carcinoma, liver, lung, nasopharynx, neuroblastoma, ovary, pancreas, pediatric glioma, prostate, rectum, kidney, and stomach.

[0016] In some embodiments, the method does not require a lymphocyte clearance step. In some embodiments, the subject is a mammal and / or a human. In some embodiments, the method further includes stimulating the subject's blood cells with the CAR immune cells. In some embodiments, the blood cells are neutrophils and / or granulocytes. In some embodiments, after stimulating the blood cells, the CAR immune cells undergo significant population expansion. In some embodiments, the cells undergo significant activation and / or potent population expansion. In some embodiments, the significant activation and / or potent population expansion is at least a 50-fold increase in the cell population. In some embodiments, the CAR immune cells are B4T2-001. In some embodiments, the CAR immune cells are TIL cells. In some embodiments, the CAR immune cells are T cells, NK cells, or macrophages. In some embodiments, the administration is performed by infusion. In some embodiments, the administration is performed locally, intraperitoneally, intravenously, intratumorally, or intramurally. In some embodiments, the method further includes administering the payload multiple times 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 cells is reversible. In some embodiments, the significant activation and / or potent population expansion results in at least 20%, 25%, 30%, 40%, or 50% engraftment of the cells. In some embodiments, the significant population expansion results in at least 20% engraftment of the cells. In some embodiments, the significant activation and / or potent population expansion results in at least 25% engraftment of the cells. In some embodiments, the significant activation and / or potent population expansion results in at least 50% engraftment of the cells. In some embodiments, the cells undergo significant activation and / or potent population expansion in at least one of the subject's peripheral blood, peritoneal fluid, and / or pulmonary fluid. In some embodiments, the method further includes administration of a kill switch activator. In some embodiments, the kill switch activator is an anti-EGFR antibody and / or cetuximab. In some embodiments, the kill switch activator is administered topically, intraperitoneally, intravenously, intratumorally, or intramurally. In some embodiments, at about 0.1 mg / m²... 2 Approximately 1 mg / m 2 Approximately 10 mg / m 2 Approximately 100 mg / m 2 Approximately 250 mg / m 2 Approximately 500 mg / m 2 Approximately 700 mg / m 2 Approximately 750 mg / m 2 The dosage is 0.1 mg / m 2 Up to 750 mg / m2 The kill switch activator is administered in any integer dose. In some embodiments, the kill switch activator is administered more than once. In some embodiments, the kill switch activator is administered more than once locally, intraperitoneally, intravenously, intratumorally, and / or intramurally.

[0017] Brief description of the attached figures

[0018] In addition to the features described above, other features and variations will become apparent from the following accompanying drawings and exemplary embodiments. It should be understood that these drawings depict typical embodiments and are not intended to limit the scope.

[0019] Figure 1 A representative plot of qPCR data was created to screen for the presence of B4t2-001 CAR T cells in the blood and peritoneal region of subjects after intraperitoneal injection.

[0020] Figure 2A A representative plot depicting the percentage of CAR T cells found in the blood of subjects quantified using fluorescence activated cell sorting (FACS) after intraperitoneal injection over time.

[0021] Figure 2B A representative plot depicting the percentage of CAR T cells found in intraperitoneal fluid samples after intraperitoneal injection, quantified using fluorescence activated cell sorting (FACS), over time.

[0022] Figure 3 A representative plot of qPCR data was created to screen for the presence of B4t2-001 CAR T cells in the blood and peritoneal region of the subjects after intraperitoneal injection. On day 15, the “kill switch” was activated to eliminate the subjects’ CAR T cells by administering cetuximab®. Detailed Implementation

[0023] This document discloses chimeric antigen receptor (CAR) proteins having at least one target. In some embodiments, the CAR protein comprises at least two, at least three, or at least four binding sites. In some embodiments, the CAR protein has two binding sites. In some embodiments, the CAR construct binds to carcinoembryonic antigen 6 (CEACAM6 or CEA6). In some embodiments, the CAR protein has a single target and is capable of activating neutrophils and / or granulocytes. In some embodiments, the CAR cell is B4T2-001, which is a specific CAR cell configured to bind to CEA6. In some embodiments, the CAR cell has multiple targets. In some embodiments, the CAR cell has two targets. In some embodiments, the CAR cell is bivalent. In some embodiments, the CAR cell is multivalent. In some embodiments, the CAR cell comprises an sdAb antibody that binds to CEA6.

[0024] CAR proteins targeting CEA6 were found to stimulate neutrophils, leading to rapid expansion and engraftment of CAR-T cells. Therefore, due to this neutrophil-mediated expansion, CAR proteins targeting CEA6 and second cancer targets (even solid tumor targets) can rapidly expand and engraft. Furthermore, it was found that lymphocyte clearance of endogenous leukocytes is not necessary for expansion and engraftment, thus allowing the administration of CAR cells with binding sites targeting CEA6 and second tumor targets to patients without the need for a pre-administration lymphocyte clearance step.

[0025] In some embodiments, the CAR protein includes a first binding site capable of binding to CEA6 and a second binding site capable of binding to antigens expressed in abnormal cells or tissues. In some embodiments, the abnormal cells and / or abnormal tissues are diseased cells and / or diseased tissues. In some embodiments, the diseased cells and / or diseased tissues are any of the following: autoimmune diseases, cardiomyopathy, viral infections, rheumatoid arthritis, fibrosis, cardiac fibrosis, HIV, COVID-19, chronic hepatitis C virus (HCV), human cytomegalovirus (HCMV), influenza, targets of cellular senescence (selective clearance of senescent cells (SC)), liver fibrosis, pulmonary fibrosis, atherosclerosis, diabetes, osteoarthritis, obesity, and / or aging. In some embodiments, the abnormal cells and / or abnormal tissues are cancer cells or tissues. In some embodiments, the abnormal cells or tissues are non-hematogenous. In some embodiments, the abnormal cells or tissues are solid tumors.

[0026] In some implementations, the second binding site can bind to any of the following: AFP, ANTXR1, AXL, αvβ3, αvβ6, B7-H3, CAIX, CD171, CD20, CD32A, CD46, CD47, CD56, CD80 / 86, CEA, sealing protein 18.2, DLL-3, DR5, EGFR, EGFRIII, EGFR806, EpCAM, EpHA2, FAP, FR-α, GD2, phosphatidylinositol proteoglycan-2, phosphatidylinositol proteoglycan-3, gp100, GSPG4, GUCY2C, HBV surface antigen (HBsAg), HER2, IL-13R-α2, L1-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, Trop2, uPAR, and / or VEGFR2. In some embodiments, the abnormal cells or tissues are selected from: brain, breast, central nervous system, cervix, colon, colorectal, epidermis, stomach, glioblastoma, glioma, hepatocellular carcinoma, liver, lung, nasopharynx, neuroblastoma, ovary, pancreas, pediatric glioma, prostate, rectum, kidney, and stomach cells and / or tissues.

[0027] In some embodiments, the second binding site binds to any of the antigens listed in Tables 1 and 2. In some embodiments, the antigen is expressed in any of the cell and / or tissue types listed in Tables 1 and 2. Table 1: List of potential antigens expressed on cancer cells / tissues at specific stages of cancer

[0028] Table 2: List of cancer cells / tissues and known antigens on these cells / tissues

[0029] This document also discloses the nucleotide sequence encoding the CAR protein of any of the embodiments disclosed herein.

[0030] This document also discloses vectors that encode a CAR protein and / or a nucleotide of any embodiment of this disclosure.

[0031] This document also discloses cells capable of expressing the CAR protein of any embodiment of this disclosure, and / or the nucleotides of any embodiment of this disclosure, and / or the vector of any embodiment of this disclosure. In some embodiments, the cells are immune cells. In some embodiments, the immune cells are B4T2-001. In some embodiments, the immune cells are TIL cells. In some embodiments, the immune cells are T cells, NK cells, or macrophages.

[0032] This document also discloses compositions comprising a CAR protein of any embodiment of this disclosure and / or cells of any embodiment of this disclosure. In some embodiments, the composition is formulated for administration to a subject. In some embodiments, the subject is a mammal and / or a human. In some embodiments, administration is by infusion. In some embodiments, administration is performed locally, intraperitoneally, intravenously, intratumorally, or intramurally. In some embodiments, more than one administration is performed. In some embodiments, administration is performed without a lymphocyte clearance step. In some embodiments, the composition is for intraperitoneal administration. In some embodiments, the composition is for intravenous administration. In some embodiments, the composition is for intratumoral administration.

[0033] Some embodiments disclose cells. In some embodiments, the cells express peptides incorporated into the chimeric antigen receptor cells. In some embodiments, the cells are mononuclear or multinucleated immune cells. In some embodiments, the cells are lymphocytes and / or leukocytes. In some embodiments, the cells are tumor-infiltrating lymphocytes (TILs). In some embodiments, the cells are myelocytes. In some embodiments, the cells are B cells, T cells, or natural killer (NK) cells. In some embodiments, the cells are eosinophils, neutrophils, or monocytes. In some embodiments, the cells are macrophages, basophils, or mast cells. In some embodiments, the cells are memory cells, plasma cells, memory T cells, cytotoxic T cells, or helper T cells. In some embodiments, the chimeric antigen receptor cells are chimeric antigen receptor T cells (CAR T cells).

[0034] In some embodiments, the cells are combined with an effective amount of at least one checkpoint inhibitor (iCPI). In some embodiments, the checkpoint inhibitor is anti-CEAMCAM6, anti-CTLA4, anti-PD1, anti-PDL1, anti-PDNR, and / or anti-PD-1 dominant-negative receptor (DNR). In some embodiments, the checkpoint inhibitor is pembrolizumab. In some embodiments, the at least one iCPI is administered intraperitoneally to the subject. In some embodiments, the at least one iCPI is administered intravenously to the subject. In some embodiments, the at least one iCPI is administered intratumorally to the subject. In some embodiments, the at least one iCPI is administered intramurally to the subject. In some embodiments, the at least one iCPI is administered intramurally to the subject.

[0035] In some embodiments, the cell contains a kill switch. The term "kill switch" is given its standard scientific meaning and thus refers to a mechanism incorporated into the cell that allows it to target and destroy the cell. In some embodiments, immune cells containing a target epitope are generated, which can be killed when an antibody binds to that target epitope. For example, immune cells (such as CAR T cells) may express one or more specific epitopes of the EGFR protein on their surface. Therefore, if it is desired to prevent the future proliferation of these CAR T cells in the body, the patient can be treated with an anti-EGFR antibody (which binds to all CAR T cells expressing the EGFR epitope), and these cells can be removed from the patient's body. Therefore, the term "kill switch activator" refers to any molecule capable of activating a cell's degradation pathway by interacting with the cell's kill switch. In some embodiments, the kill switch is caspase 9. In some embodiments, Rimiducid functions as a drug-mediated kill switch within the subject. In some embodiments, the kill switch activates protein degradation. In some embodiments, lenalidomide functions as a drug-mediated kill switch within the subject. In some embodiments, the mRNA encoding the CAR functions as a drug-mediated kill switch within the subject. In some embodiments, the kill switch is a suicide gene, such as inducible caspase 9, herpes simplex virus tyrosine kinase, or human thymidine kinase. In some embodiments, haploidentical stem cell transplantation (HSCT) functions as a drug-mediated kill switch within the subject. In some embodiments, ganciclovir functions as a drug-mediated kill switch within the subject. In some embodiments, rituximab functions as a drug-mediated kill switch within the subject. In some embodiments, the kill switch is the multi-epitope RQR8. In some embodiments, the kill switch is an EGFR epitope. In some embodiments, the kill switch is a truncated EGFR molecule. In some embodiments, an 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.

[0036] In some embodiments, the kill switch activator is administered intraperitoneally to the subject. In some embodiments, the kill switch activator is administered intravenously to the subject. In some embodiments, the kill switch activator is administered intratumorally to the subject. In some embodiments, the kill switch activator is administered intramurally to the subject.

[0037] This document also discloses methods for treating diseases or conditions in subjects in need. In some embodiments, the methods include administering cells of any of the embodiments disclosed herein to the subject. In some embodiments, methods of treating patients with CAR-T include controlling CAR-T persistence and antitumor effects. In some embodiments, CAR-T persistence and / or antitumor effects can be increased or decreased (i.e., CAR-T oscillation) by: (1) repeated CAR-T infusions, (2) administration of anti-EGFR antibody drugs, such as cetuximab or analogues, to activate the killing switch and control CAR T levels in the blood or ablate CAR T cells in the blood. The concentration and frequency of cetuximab administration can be adjusted to control CAR T concentrations. Furthermore, very low dose levels / formulations (as low as 4E4 CD3-positive and CAR T-positive T cells / kg, up to 1E7 / kg of patients, or a flat dose starting from 5E5 CAR+ T cells / kg) may be used without concurrent lymphocyte-clearing chemotherapy. Administration routes, such as IP or other administration routes (including IV or intratumoral administration), may be combined with one or more iCPIs (including anti-PD1 and anti-PDL1 and others) to improve therapeutic efficacy and window of action. The method may include treatment of malignant cancers, including solid tumors such as gastric cancer with or without ascites, colorectal cancer, and peritoneal cancer. CAR-T cells may target one or more targets, including T cells, NK cells, or other forms of immune cells.

[0038] In some embodiments, the method includes combination therapy of CAR-T cells with at least one checkpoint inhibitor. ICIs targeting the PD1 / PDL-1 axis can unblock CAR-T cell inhibition. This effect can enhance the cytotoxic activity of CAR-T cells and thus promote their anti-tumor activity. In some embodiments, the method includes administering CAR-T and / or B4t2-001 with at least one iCPI or gene construct, such as pDNR (dominant-negative receptor). In some embodiments, the method includes a combination of one or more iCPIs with CAR-T and / or B4t2-001. In some embodiments, the method includes administering anti-PD1, anti-PDL1, and / or anti-CTLA4. In some embodiments, the at least one iCPI is administered in at least one dose. In some embodiments, CAR-T is infused at an MTD and anti-PD1 and / or anti-PDL1 are infused in multiple doses in escalating increments. In some embodiments, iCPI treatment is administered before or after CAR-T administration. In some embodiments, iCPI treatment is administered concurrently with CAR-T administration. In some embodiments, CAR-T cells are administered via IP, IV, or IT routes. In some embodiments, CAR-T cells are administered with or without lymphocyte clearance. In some embodiments, CAR-T cells are administered more than once. In some embodiments, at least one iCPI is administered at any integer dose from 0.1 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 0.75 mg / kg, 1 mg / kg, 2.5 mg / kg, 5 mg / kg, 7 mg / kg, 7.5 mg / kg, 10 mg / kg, or 0.1 mg / kg to 10 mg / kg. In some embodiments, the disease or condition is cancer. In some embodiments, the cancer can be any type of cancer. In some embodiments, the cancer is any type of malignant tumor. In some embodiments, the subject also suffers from malignant ascites. In some implementations, the cancer can 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, stomach cancer, hematologic malignancies, or any combination thereof. In some implementations, hematologic malignancies can include leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, hairy cell leukemia, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, or multiple myeloma. Immune cells can be derived from the object used for autologous treatment. Alternatively, immune cells can be derived from the same species as the object used for allogeneic treatment.

[0039] In some embodiments, the subject is a mammal and / or a human. In some embodiments, the method further includes stimulating blood cells within the subject with CAR proteins, cells, and / or compositions. In some embodiments, the blood cells are neutrophils and / or granulocytes. In some embodiments, after stimulation of the blood cells, the cells of any embodiment of this disclosure and / or cells expressing the CAR of any embodiment of this disclosure undergo significant population expansion. In some embodiments, the cells undergo significant activation and / or potent population expansion. In some embodiments, significant activation and / or potent population expansion is at least a 50-fold increase in the cell population. In some embodiments, the disease or condition is cancer. In some embodiments, the method does not require a lymphocyte clearance step.

[0040] In some embodiments, a method of treating a disease or condition in a subject of need includes administering CAR immune cells containing a first binding site for CEACAM6 and a second binding site for cancer. In some embodiments, the cancer is non-hematogenous. In some embodiments, the cancer is a solid tumor. In some implementations, the second binding site can bind to any of the following: AFP, ANTXR1, AXL, αvβ3, αvβ6, B7-H3, CAIX, CD171, CD20, CD32A, CD46, CD47, CD56, CD80 / 86, CEA, sealing protein 18.2, DLL-3, DR5, EGFR, EGFRIII, EGFR806, EpCAM, EpHA2, FAP, FR-α, GD2, phosphatidylinositol proteoglycan-2, phosphatidylinositol proteoglycan-3, gp100, GSPG4, GUCY2C, HBV surface antigen (HBsAg), HER2, IL-13R-α2, L1-CAM, Lewis Y, LMP1, MAGE-A1 / 3 / 4, mesothelin, c-MET, MUC1, MUC16, MUC3A, Nectin4 / FAP, NKG2D, PAP, PSA, PSCA, PSMA, ROR, TAG-72, Trop2, and / or VEGFR2. In some embodiments, the cancer is present in cells and / or tissues selected from the following: brain, breast, central nervous system, cervix, colon, colorectal, epidermis, stomach, glioblastoma, glioma, hepatocellular carcinoma, liver, lung, nasopharynx, neuroblastoma, ovary, pancreas, pediatric glioma, prostate, rectum, kidney, and stomach. In some embodiments, the method does not require a lymphocyte removal step. In some embodiments, the subject is a mammal and / or human. In some embodiments, the method further includes CAR immune cell stimulation of blood cells within the subject. In some embodiments, the blood cells are neutrophils and / or granulocytes.

[0041] In some embodiments, CAR immune cells undergo significant population expansion after stimulation of hematopoietic cells. In some embodiments, CAR immune cells undergo significant activation and / or potent population expansion. In some embodiments, significant activation and / or potent population expansion is at least a 50-fold increase in cell population. In some embodiments, the CAR immune cells are B4T2-001. In some embodiments, the CAR immune cells are TIL cells. In some embodiments, the CAR immune cells are T cells, NK cells, or macrophages. In some embodiments, administration is performed by infusion. In some embodiments, administration is performed locally, intraperitoneally, intravenously, intratumorally, or intramurally. In some embodiments, the method further includes 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 hematopoietic cells is reversible. In some embodiments, significant activation and / or potent population expansion results in at least 20%, 25%, 30%, 40%, or 50% cell engraftment. In some embodiments, significant activation and / or potent population expansion results in at least 20% cell engraftment. In some embodiments, significant activation and / or potent population expansion results in at least 25% cell engraftment. In some embodiments, significant activation and / or potent population expansion results in at least 50% cell engraftment. In some embodiments, the cells undergo significant activation and / or potent population expansion in at least one of the subject's peripheral blood, peritoneal fluid, and / or pulmonary fluid. In some embodiments, the method further includes administration of a killer switch activator. In some embodiments, the killer switch activator is an anti-EGFR antibody and / or cetuximab. In some embodiments, the killer switch activator is administered topically, intraperitoneally, intravenously, intratumorally, or intramurally. In some embodiments, it is administered at approximately 0.1 mg / m². 2 Approximately 1 mg / m 2 Approximately 10 mg / m 2 Approximately 100 mg / m 2 Approximately 250 mg / m 2 Approximately 500 mg / m 2 Approximately 700 mg / m 2 Approximately 750 mg / m 2 The dosage is 0.1 mg / m 2 Up to 750 mg / m 2 The kill switch activator is administered in any integer dose. In some embodiments, the kill switch activator is administered more than once. In some embodiments, the kill switch activator is administered more than once locally, intraperitoneally, intravenously, intratumorally, and / or intramurally.

[0042] In some embodiments, cells are administered to the recipient at an effective dose. In some embodiments, the recipient has cancer. In some embodiments, immune cells are administered more than once. In some embodiments, immune cells are administered locally, intraperitoneally, intravenously, intratumorally, or intramurally. In some embodiments, viral particles encoding a CAR or mRNA LNPs encoding a CAR are administered. In some embodiments, B4T2-001 is administered more than once. In some embodiments, at least one iCPI is administered more than once. In some embodiments, an anti-EGFR molecule is administered more than once. In some embodiments, anti-EGFR, iCPI, and immune cells are administered at different times. In some embodiments, any combination of anti-EGFR, iCPI, and immune cells is administered together.

[0043] In some embodiments, immune cells are administered to the subject prior to administration of anti-EGFR and / or at least one iCPI. In some embodiments, iCPI treatment is initiated after 1, 2, 3, 4, 5, 10, 15, 20, 24 weeks, or any integer number of weeks from 1 to 24 weeks following immune cell administration. In some embodiments, iCPI treatment is administered 1 week after immune cell administration. In some embodiments, iCPI treatment is administered 2 weeks after immune cell administration. In some embodiments, immunotherapy is administered 3 weeks after immune cell administration. In some embodiments, iCPI treatment is administered 4 weeks after immune cell administration. In some embodiments, iCPI treatment is administered 5 weeks after immune cell administration. In some embodiments, iCPI treatment is administered 6 weeks after immune cell administration. In some embodiments, iCPI treatment is administered 7 weeks after immune cell administration. In some embodiments, iCPI treatment is administered 8 weeks after immune cell administration. In some embodiments, iCPI treatment is administered 9 weeks after immune cell administration. In some embodiments, iCPI treatment is administered 10 weeks after immune cell administration. In some embodiments, iCPI treatment is administered 11 weeks after immune cell administration. In some embodiments, iCPI treatment is administered 12 weeks after immune cell administration. In some embodiments, iCPI treatment is administered 13 weeks after immune cell administration. In some embodiments, iCPI treatment is administered 14 weeks after immune cell administration. In some embodiments, iCPI treatment is administered 15 weeks after immune cell administration. In some embodiments, iCPI treatment is administered 20 weeks after immune cell administration. In some embodiments, iCPI treatment is administered 22 weeks after immune cell administration. In some embodiments, iCPI treatment is administered 24 weeks after immune cell administration. In some embodiments, iCPI treatment is administered every week after immune cell administration. In some embodiments, iCPI treatment is administered every 2 weeks after immune cell administration. In some embodiments, iCPI treatment is administered every 3 weeks after immune cell administration. In some embodiments, iCPI treatment is administered every 4 weeks after immune cell administration. In some embodiments, iCPI treatment is administered every 5 weeks following immune cell administration. In some embodiments, iCPI treatment is administered every 6 weeks following immune cell administration. In some embodiments, iCPI treatment is administered every 7 weeks following immune cell administration. In some embodiments, iCPI treatment is administered every 8 weeks following immune cell administration. In some embodiments, iCPI treatment is administered every 9 weeks following immune cell administration. In some embodiments, iCPI treatment is administered every 10 weeks following immune cell administration. In some embodiments, iCPI treatment is administered every 11 weeks following immune cell administration. In some embodiments, iCPI treatment is administered every 12 weeks following immune cell administration. In some embodiments, immune cells are administered at least once.In some embodiments, more than one dose of immune cells is administered. In some embodiments, immune cells are administered locally, intraperitoneally, intravenously, intratumorally, or intramurally. In some embodiments, at least one iCPI treatment is administered. In some embodiments, more than one iCPI treatment is administered. In some embodiments, iCPI treatment is administered at a fixed dose. In some embodiments, iCPI treatment is administered at any integer dose from about 0.1 mg / kg, about 0.25 mg / kg, about 0.5 mg / kg, about 0.75 mg / kg, about 1 mg / kg, about 2.5 mg / kg, about 5 mg / kg, about 7.5 mg / kg, about 10 mg / kg, or from 0.1 mg / kg to 10 mg / kg. In some embodiments, iCPI treatment is administered in escalating doses. In some embodiments, iCPI treatment is administered in decreasing doses. In some embodiments, killer switch activator treatment is initiated 1, 2, 3, 4, 5, 10, 15, 20, 24 weeks, or any integer number of weeks from 1 to 24 weeks after immune cell administration. In some embodiments, killer switch activator treatment is administered 1 week after immune cell administration. In some embodiments, killer switch activator treatment is administered 2 weeks after immune cell administration. In some embodiments, immunotherapy is administered 3 weeks after immune cell administration. In some embodiments, killer switch activator treatment is administered 4 weeks after immune cell administration. In some embodiments, killer switch activator treatment is administered 5 weeks after immune cell administration. In some embodiments, killer switch activator treatment is administered 6 weeks after immune cell administration. In some embodiments, killer switch activator treatment is administered 7 weeks after immune cell administration. In some embodiments, killer switch activator treatment is administered 8 weeks after immune cell administration. In some embodiments, killer switch activator treatment is administered 9 weeks after immune cell administration. In some embodiments, the killer switch activator is administered 10 weeks after immune cell administration. In some embodiments, the killer switch activator is administered 11 weeks after immune cell administration. In some embodiments, the killer switch activator is administered 12 weeks after immune cell administration. In some embodiments, the killer switch activator is administered 13 weeks after immune cell administration. In some embodiments, the killer switch activator is administered 14 weeks after immune cell administration. In some embodiments, the killer switch activator is administered 15 weeks after immune cell administration. In some embodiments, the killer switch activator is administered 20 weeks after immune cell administration. In some embodiments, the killer switch activator is administered 22 weeks after immune cell administration. In some embodiments, the killer switch activator is administered 24 weeks after immune cell administration. In some embodiments, the killer switch activator is administered every other week after immune cell administration.In some embodiments, the killer switch activator is administered every 2 weeks after immune cell administration. In some embodiments, the killer switch activator is administered every 3 weeks after immune cell administration. In some embodiments, the killer switch activator is administered every 4 weeks after immune cell administration. In some embodiments, the killer switch activator is administered every 5 weeks after immune cell administration. In some embodiments, the killer switch activator is administered every 6 weeks after immune cell administration. In some embodiments, the killer switch activator is administered every 7 weeks after immune cell administration. In some embodiments, the killer switch activator is administered every 8 weeks after immune cell administration. In some embodiments, the killer switch activator is administered every 9 weeks after immune cell administration. In some embodiments, the killer switch activator is administered every 10 weeks after immune cell administration. In some embodiments, the killer switch activator is administered every 11 weeks after immune cell administration. In some embodiments, the killer switch activator is administered every 12 weeks after immune cell administration. In some embodiments, immune cells are administered at least once. In some embodiments, immune cells are administered more than once. In some embodiments, immune cells are administered locally, intraperitoneally, intravenously, intratumorally, or intramurally. In some embodiments, at least one dose of killer switch activator is administered. In some embodiments, more than one dose of killer switch activator is administered. In some embodiments, killer switch activator is administered at a fixed dose. In some embodiments, killer switch activator is administered at doses of about 0.1 mg / kg, about 0.25 mg / kg, about 0.5 mg / kg, about 0.75 mg / kg, about 1 mg / kg, about 2.5 mg / kg, about 5 mg / kg, about 7.5 mg / kg, about 10 mg / kg, or any integer dose from 0.1 mg / kg to 10 mg / kg. In some embodiments, at about 0.1 mg / m²... 2 Approximately 1 mg / m 2 Approximately 10 mg / m 2 Approximately 100 mg / m 2 Approximately 250 mg / m 2 Approximately 500 mg / m 2 Approximately 700 mg / m 2 Approximately 750 mg / m 2 The dosage is 0.1 mg / m 2 Up to 750 mg / m 2 Treatment involves administering the kill switch activator at any integer dose. In some embodiments, treatment involves administering the kill switch activator at an increasing dose. In some embodiments, treatment involves administering the kill switch activator at a decreasing dose.

[0044] In some implementations, the treatment does not include chemotherapy. The term "chemotherapy" as used herein is given its standard scientific meaning and therefore refers to non-cellular, non-protein chemical drugs used to kill cancer cells.

[0045] In some implementations, the method does not include a lymphocyte clearance step. The term "lymphocyte clearance" as used herein is given its standard scientific meaning and therefore refers to short-term chemotherapy administered to a subject before, after, or during immunotherapy to kill the subject's T cells.

[0046] In some embodiments, immune cells are administered to the subject via at least one intraperitoneal infusion. In some embodiments, the at least one intraperitoneal infusion is more than one intraperitoneal infusion. In some embodiments, the at least one intraperitoneal infusion is any integer number of administrations, from 2 to 100, including 2, 3, 4, 5, 10, 20, 25, 30, 40, 50, 100, or 2 to 100 times.

[0047] definition

[0048] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only, and do not limit any of the claimed subject matter.

[0049] The chapter titles used in this article are for organizational purposes only and are not to be construed as limiting the subject matter.

[0050] The articles “a” and “an” used in this article refer to one or more (e.g., at least one) grammatical objects of the article. For example, “an element” refers to one or more elements.

[0051] "Approximately" means a change of up to 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of a reference quantity, level, value, number, frequency, percentage, size, size, quantity, weight, or length.

[0052] The term "significant" as used in this article refers to reproducible differences in effect. For example, if compound A is described as binding to the target more significantly than compound B, then a greater number of compounds A bind to the target reproducibly compared to compound B.

[0053] "Potential" is given its typical scientific meaning, and therefore refers to the high potency / activity of a molecule per unit.

[0054] Throughout this specification, unless the context otherwise requires, the word "comprising / including" will be understood to imply inclusion of the stated steps or elements or groups of steps or elements, but does not exclude any other steps or elements or groups of steps or elements. "Constitutes of" means to include and is limited to anything following the phrase "consisting of". Therefore, the phrase "consisting of" indicates that the listed elements are necessary or mandatory, and no other elements may be present. "Substantially constitutes of" means to include any element listed following the phrase, and is limited to other elements that do not interfere with or contribute to the activity or effect specified in this disclosure for the listed elements. Therefore, the phrase "substantially constitutes of" indicates that the listed elements are necessary or mandatory, but other elements are optional and may be present or absent depending on whether they substantially affect the activity or effect of the listed elements.

[0055] The terms “% w / w” or “% wt / wt” refer to a percentage expressed as the weight of an ingredient or reagent relative to the total weight of the composition multiplied by 100.

[0056] sequence

[0057] As used herein, the term "nucleic acid" or "nucleic acid molecule" refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments produced by polymerase chain reaction (PCR), and fragments produced by any of the following: ligation, cleavage, endonuclease action, and exonuclease action. Nucleic acid molecules may consist of naturally occurring nucleotide monomers (e.g., DNA and RNA), monomers of naturally occurring nucleotide analogs (e.g., enantiomers of naturally occurring nucleotides), or a combination of both. Nucleic acid monomers may be linked by phosphodiester bonds or analogs of such bonds. One or more nucleic acids may be contained in a nucleic acid vector or nucleic acid construct (e.g., plasmid, virus, bacteriophage, cosmid, fosmid, phage particle, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC), or human artificial chromosome (HAC)) that can be used to amplify and / or express the one or more nucleic acids in various biological systems. Typically, 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 helper genes or any combination thereof.

[0058] Nucleic acids or nucleic acid molecules may contain one or more sequences encoding different peptides, polypeptides, or proteins. These one or more sequences may be linked adjacently within the same nucleic acid or nucleic acid molecule, or have additional nucleic acids between them, such as linkers, repetitive sequences, or restriction enzyme sites, or any other sequence of 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 in length, or any length within any two of the aforementioned lengths. The term "downstream" in this article refers to the sequence following the 3' end of the preceding sequence, or, if the nucleic acid is double-stranded, on the strand containing the coding sequence (sense strand). The term "upstream" in this article refers to the sequence preceding the 5' end of the subsequent sequence, or, if the nucleic acid is double-stranded, on the strand containing the coding sequence (sense strand). As used herein, the term “grouped” in nucleic acids refers to two or more sequences that are adjacent to each other, or that have an additional nucleic acid, such as a linker, repeat sequence, or restriction enzyme site, between them; or any other sequence of 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 in length or any length within any two of the aforementioned lengths, but generally does not contain a sequence encoding a functional or catalytic polypeptide, protein, or protein domain between them.

[0059] As used herein, the terms “peptide,” “polypeptide,” and “protein” refer to macromolecules composed of amino acids linked together by peptide bonds. Many functions of peptides, polypeptides, and proteins are known in the art and include, but are not limited to, enzymes, structures, transport, defense, hormones, or signal transduction. While chemical synthesis is also possible, peptides, polypeptides, and proteins are typically (but not always) biologically produced by ribosome complexes using nucleic acid templates. Mutations (e.g., substitutions, deletions, truncations, additions, duplications, or fusions of more than one peptide, polypeptide, or protein) can be performed by manipulating the nucleic acid template. These fusions of more than one peptide, polypeptide, or protein may be linked adjacently within the same molecule or have additional amino acids therebetween, such as linkers, repeat sequences, epitopes, or tags, or any other sequence of 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 in length, or any length within any two of the aforementioned lengths. The term “downstream” as used herein on a polypeptide refers to the sequence following the C-terminus of the preceding sequence. The term "upstream" in this article refers to the sequence preceding the N-terminus of the subsequent sequence.

[0060] In some embodiments, the nucleic acid or peptide sequences presented herein and used in examples are functional in a variety of biological systems, including but not limited to human, mouse, rat, monkey, primate, cat, dog, rabbit, *E. coli*, yeast, and mammalian cells. In other embodiments, nucleic acid or peptide sequences having at least or less 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 any two of the defined ranges of similarity percentages above, may also be used without affecting the function of the sequence in the biological system. As used herein, the term "similarity" means that the nucleic acid or peptide sequence has the same overall nucleotide or amino acid sequence as the template nucleic acid or peptide sequence, with specific alterations within the sequence, such as substitution, deletion, duplication, or insertion. In some implementations, two nucleic acid sequences having similarity as low as 0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% can encode the same polypeptide by including different codons that encode the same amino acid during translation.

[0061] As disclosed herein, sequences having a percentage of homology with any of the sequences disclosed herein are envisioned and can be used. The term "homology %" refers to the degree of conservation between two sequences when considering their three-dimensional structures. For example, homology between two protein sequences may depend on structural motifs, such as β-chains, α-helices, and other folds, and their distribution throughout the sequence. Homology can be determined by structural determination, either empirically or computationally. In some embodiments, 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% homology with any of the sequences disclosed herein can be used. In some implementations, 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 sequence disclosed herein may or may not affect the overall percentage of homology.

[0062] As applied herein, sequences having a certain "similarity percentage" or "identity percentage" with any of the sequences disclosed herein are envisioned and can be used. In some embodiments, these sequences may include peptide sequences, nucleic acid sequences, CDR sequences, variable region sequences, or heavy or light chain sequences. As understood in the art for peptide sequences, "similarity" refers to a comparison of amino acids based on amino acid properties, including but not limited to size, polarity, charge, pK, aromaticity, hydrogen bonding properties, or the presence of functional groups (e.g., hydroxyl, thiol, amine, carboxyl, etc.). The term "similarity %" refers to the percentage of identical units (i.e., amino acids) between two or more sequences relative to the sequence length. When the two or more sequences being compared are of the same length, the similarity percentage will correspond to that length. When the two or more sequences being compared are of different lengths, deletions and / or insertions may be introduced to achieve optimal alignment. The similarity of two amino acids can determine whether a substitution is conserved or non-conserved. Methods for determining the conservation of amino acid substitutions are generally known in the art and may involve a substitution matrix. Commonly used substitution matrices include BLOSUM 45, BLOSUM 62, BLOSUM 80, PAM 100, PAM 120, PAM 160, PAM 200, and PAM 250; however, other substitution matrices or methods may be used where deemed appropriate by those skilled in the art. A particular substitution matrix may be superior to others when considering aspects of the relevant sequences, such as strictness, conservation, and / or variability (e.g., within the same species or more broadly) and the length of the sequence of interest. As used herein, a peptide sequence having a certain percentage of similarity to another sequence will have up to that percentage of the same or acceptable substitutions (as specified by the similarity determination method used) of amino acids. In some embodiments, sequences 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. In some implementations, 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 sequence disclosed herein may be used.When applied to antibody sequences, these similar substitutions can be applied to antigen-binding regions (i.e., CDRs) or regions that do not bind antigens or are just below the antigen-binding region (i.e., frame regions).

[0063] As applied herein, sequences having a certain “percentage of identity” with any of the sequences disclosed herein are envisioned and can be used. The term “percentage of identity” refers to the percentage of similarity between two or more sequences. In some embodiments, any sequence having an identity of at least 60%, 70%, 80%, 85%, 90%, 95%, 99%, 100%, or any integer from 60% to 100% with any of the sequences disclosed herein can be used.

[0064] The term "shared sequence" as used herein refers to a broad sequence representing all the different combinations of amino acids allowed at each position of a set of sequences. Shared sequences can provide insights into conserved regions of related sequences, where units (e.g., amino acids or nucleotides) are identical in most or all sequences; and insights into regions that exhibit differences between sequences. In the case of antibodies, the shared sequence of a CDR can indicate amino acids that are important or indispensable for antigen binding. It is envisioned that shared sequences can be prepared using any of the sequences provided herein, and that the various sequences derived from shared sequences can be verified to have similar effects to the template sequence.

[0065] antigen-binding molecules and antibodies

[0066] As used herein, the term “antibody” means as it is understood by those skilled in the art, and is also intended to include any polypeptide chain-containing molecular structure having a particular shape suitable for recognizing epitopes, wherein one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope.

[0067] The term "competition" as used herein with respect to antibodies or binding peptides means that a first antibody or binding peptide, or its antigen-binding portion, binds to an epitope in a manner sufficiently similar to that of a second antibody or binding peptide, or its antigen-binding portion, such that the binding of the first antibody or binding peptide to its homologous epitope is detectably reduced in the presence of the second antibody or binding peptide compared to the binding of the first antibody or binding peptide to its homologous epitope in the absence of the second antibody or binding peptide. Alternatively, it may be, but is not necessary, for the binding of the second antibody or binding peptide to its epitope to be detectably reduced in the presence of the first antibody or binding peptide. Regardless of the mechanism by which such competition occurs (e.g., steric hindrance, conformational change, or binding to a common epitope or portion thereof), those skilled in the art will understand, based on the teachings provided herein, that including such competing antibodies or binding peptides is useful for the methods disclosed herein.

[0068] Antibodies or binding peptides that “preferentially bind” or “specifically bind” epitopes (used interchangeably herein) are well-known terms in the art, and methods for determining such specificity or preferential binding are also well-known in the art. A molecule is said to “specifically bind” or “preferentially bind” if it reacts or associates with a particular cell or substance more frequently and / or faster and / or more persistently and / or with a higher affinity than it reacts or associates with alternative cells or substances. An antibody or binding peptide is “specifically bound” or “preferentially bound” to a target if it binds to a target with greater affinity and / or affinity and / or more readily and / or more persistently than it binds to other substances.

[0069] The term "humanization" applied to non-human (e.g., rodent or primate) antibodies refers to hybrid immunoglobulins, immunoglobulin chains, or fragments thereof containing very few sequences derived from non-human immunoglobulins.

[0070] As used herein, the terms "single-domain binding peptide" or "single-domain antibody" (sdAb) refer to a single peptide chain containing an intact immunoglobulin domain or an fold of another protein capable of recognizing an antigen (e.g., not bound to another peptide chain via disulfide bonds). Single-domain binding peptides or sdAbs can be derived from typical immunoglobulin heavy or light chains, such as those from humans, or from alternative sources such as dromedary camels (e.g., VHH) and cartilaginous fish (e.g., VNAR). In some embodiments, a single-domain binding peptide or sdAb contains one, two, or three complementarity-determining regions (CDRs). In some embodiments, a single-domain binding peptide or sdAb contains one, two, or three of CDR1, CDR2, and CDR3.

[0071] Unless otherwise stated, the complementary determination regions (CDRs) disclosed herein follow the IMGT definition. However, CDRs may also be interpreted, either alone or in the context of variable domains, by the definitions of Kabat, Chothia, or others as understood by those skilled in the art.

[0072] As used herein, the term "single-chain variable fragment" (scFv) is a fusion protein comprising the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin, wherein the VH and VL are covalently linked to form a VH:VL heterodimer. The VH and VL are directly linked or linked via a linker encoding a peptide that connects the N-terminus of the VH to the C-terminus of the VL, or vice versa. For flexibility, the linker is typically glycine-rich, and for solubility, it is typically serine or threonine-rich. Despite the removal of the constant region and the introduction of the linker, the scFv protein retains the specificity of the original immunoglobulin. Single-chain Fv polypeptide antibodies can be expressed from nucleic acids containing both VH-coding and VL-coding sequences. In some embodiments, the VH and VL of the scFv each contain one, two, or three CDRs. In some embodiments, the VH and VL of the scFv each contain one, two, or three CDRs, CDR1, CDR2, and CDR3.

[0073] In some embodiments, the final delimitation of the CDR and the identification of residues containing the binding site of the antibody or binding peptide are achieved by resolving the structure of the antibody or binding peptide and / or the antibody-ligand complex. In some embodiments, this can be achieved by any of a variety of techniques known to those skilled in the art, such as X-ray crystallography. In some embodiments, various analytical methods can be used to identify or simulate the CDR region. Examples of such methods include, but are not limited to, Kabat definition, Chothia definition, IMGT method (Lefranc et al., 2003 Dev Comp Immunol. 27:55-77), computational programs such as Paratome (Kunik et al., 2012, Nucl Acids Res. W521-4), AbM definition, and conformational definition.

[0074] The Kabat definition is a standard for numbering residues in antibodies and is commonly used to identify CDR regions. See, for example, 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 the location of certain structural loop regions. See, for example, Chothia et al., 1986, J.Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83. The AbM definition uses an integrated computer program developed by the Oxford Molecular Group to model antibody structures. See, for example, Martin et al., 1989, Proc Natl Acad Sci (USA), 86:9268-9272; "AbM.TM., A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd. The AbM definition uses a combination of knowledge databases and ab initio methods (e.g., those described in Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198) to model the tertiary structure of an antibody from its primary sequence. The contact definition is based on the analysis of available complex crystal structures. See, for example, MacCallum et al., 1996, J. Mol. Biol., 5:732-45. In another approach, referred to in this paper as the “conformational definition” of the CDR, the position of the CDR can be identified as the residue that contributes enthalpy to antigen binding. See, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166. Other CDR boundary definitions may not strictly follow one of the methods described above, but will still overlap with at least a portion of the Kabat CDR, although they may be shortened or lengthened based on predictions or experimental findings (i.e., specific residues or groups of residues do not significantly affect antigen binding).As used herein, a CDR can refer to a CDR defined by any method known in the art, including combinations of various methods. The methods used herein can utilize a CDR defined according to any of these methods. For any given embodiment containing more than one CDR, the CDR can be defined according to any one or a combination of Kabat, Chothia, extended, IMGT, Paratome, AbM, and / or conformational definitions.

[0075] Antigen-binding peptides

[0076] In addition to whole immunoglobulins (or their recombinant counterparts), immunoglobulin fragments or “binding fragments” containing epitope binding sites (e.g., Fab’, F(ab’)2, single-chain variable fragments (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), VHH fragment, VNAR fragment, or other fragments) are also used as antibody portions in this invention. Such antibody fragments can be generated from whole immunoglobulins by cleavage with ricin, pepsin, papain, or other proteases. Minimal immunoglobulins can be designed using recombinant immunoglobulin technology. For example, the “Fv” immunoglobulin used in this invention can be generated by linking a variable light chain region to a variable heavy chain region via a peptide linker (e.g., polyglycine or other sequences that do not form α-helical or β-sheet motifs). Nanobodies or single-domain antibodies can also be derived from alternative organisms such as dromedary camels, camels, llamas, alpacas, sharks, or cartilaginous fish. In some implementations, antibodies can be conjugates, such as pegylated antibodies, drugs, radioisotopes, or toxin conjugates. Monoclonal antibodies targeting specific epitopes or combinations of epitopes will allow for the targeting and / or depletion of cell populations expressing markers.

[0077] As used herein, the term "single-domain antibody" (sdAb) refers to a single peptide chain containing an intact immunoglobulin domain or an fold of another protein capable of recognizing an antigen (e.g., not bound to another peptide chain by disulfide bonds). sdAbs can be derived from typical immunoglobulin heavy or light chains, such as those from humans, or from alternative sources such as dromedary camels (e.g., VHH) and cartilaginous fish (e.g., VNAR).

[0078] This document discloses a carcinoembryonic antigen 6 (carcinoembryonic antigen-associated cell adhesion molecule 6; CEA6; CEACAM6) binding peptide. In some embodiments, the CEA6 binding peptide includes an immunoglobulin heavy chain variable domain comprising CDR-H1, CDR-H2, and CDR-H3.

[0079] In some embodiments, the CEA6-binding polypeptide comprises an immunoglobulin heavy chain variable domain comprising CDR-H1, CDR-H2, and CDR-H3, wherein one or more of these CDRs are defined by a common sequence. However, it is envisioned that alternative alignments can be performed (e.g., using global or local alignments, or with different algorithms, such as hidden Markov models, seeded guide trees, Needleman-Wunsch algorithms, or Smith-Watman algorithms, or other known methods), thus obtaining alternative common sequences (including those common sequences obtained using a subset of the sequences provided herein).

[0080] In some embodiments, CDR-H1 is defined by the formula X1X2X3X4X5X6X7X8, where X1 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; and X8 is D, F, H, L, P, T, V, or Y. In some embodiments, CDR-H1 comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the common sequence. In some embodiments, CDR-H1 comprises a sequence having 0, 1, 2, 3, 4, 5, or 6 substitutions from the common sequence.

[0081] In some embodiments, CDR-H2 is defined by the formula X1X2X3X4X5X6X7X8X9X10, wherein X1 is free of amino acids and is 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; and X10 is free of amino acids or is Y. In some embodiments, CDR-H2 comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the common sequence. In some embodiments, CDR-H2 comprises a sequence having 0, 1, 2, 3, 4, 5, or 6 substitutions from the common sequence.

[0082] In some implementations, CDR-H3 is defined by the formula X1X2X3X4X5X6X7X8X9X10X11X12X13X14X15X16X17X18X19X20X21X22X23X24X25X26X27X28X29X30X31X32X33, where X1 has no amino acid or is A; X2 has no amino acid, is A or V; X3 has no amino acid, is A, G, M, Q, S, T or V; X4 has no amino acid, is A, D, E, G, I, M, N, R, S, V or Y; X5 has no amino acid. X6 has no amino acid, and its amino acid is A, E, K, M, R, S, T, V, or W; X7 has no amino acid, and its amino acid is A, F, I, M, P, W, or Y; X8 has no amino acid, and its amino acid is D, I, K, L, S, T, or V; X9 has no amino acid, and its amino acid is A, K, Q, T, or V; X10 has no amino acid, and its amino acid is A, D, E, or S; X11 has no amino acid, and its amino acid is A, I, L, R, V, or Y; X12 has no amino acid, and its amino acid is A, E, G, L, P, S, or T; X13 has no amino acid, and its amino acid is D, G, I, L, N, P. X14 has no amino acid, and is A, E, F, H, K, L, M, P, Q, R, T, V, or Y; X15 has no amino acid, and is A, D, H, I, L, M, P, Q, R, S, T, or V; X16 has no amino acid, and is A, D, E, H, L, S, T, V, W, or Y; X17 has no amino acid, and is E, F, G, H, L, M, N, Q, S, T, or Y; X18 has no amino acid, and is A, D, G, H, K, M, N, Q, R, S, V, or Y; X19 has no amino acid, and is F, H, Q, or Y; X X20 has no amino acid, and is D, N, Q, S, or Y; X21 has no amino acid, and is A, G, or Y; X22 has no amino acid, and is W or Y; X23 has no amino acid, and is A or R; X24 has no amino acid, and is H or S; X25 has no amino acid, and is D or G; X26 has no amino acid, and is E or K; X27 has no amino acid, and is I or T; X28 has no amino acid, and is F or R; X29 has no amino acid or is Y; X30 has no amino acid or is Y; X31 has no amino acid or is Y; X32 has no amino acid, and is N or S; X33 has no amino acid or is Y.

[0083] In some embodiments, CDR-H3 comprises a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the common sequence. In some embodiments, CDR-H3 comprises sequences with 0, 1, 2, 3, 4, 5, or 6 substitutions from the common sequence.

[0084] In some implementations, the CEA6-binding peptide is humanized. In some implementations, the CEA6-binding peptide is a single-domain antibody (sdAb).

[0085] In some embodiments, the CEA6-binding peptide 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 any two of the above-defined ranges.

[0086] The binding peptides disclosed herein can be obtained from antibody libraries. In some embodiments, the antibody library is an immunoantibody library, a natural antibody library, a synthetic antibody library, or a semi-synthetic antibody library. In some embodiments, the antibody library contains antibodies derived from humans or antibodies that are not immunogenic in humans, or both. In some embodiments, the antibody library contains humanized antibodies, such as those derived from mice, rats, guinea pigs, rabbits, cats, dogs, cattle, horses, sheep, goats, horses, or donkeys. In some embodiments, the antibody library contains single-domain antibodies (sdAbs), nanobodies, VHH fragments, VNAR fragments, single-chain variable fragments (scFvs), camel antibodies, or cartilaginous fish antibodies, or any combination thereof. An exemplary library that can be used is a fully humanized synthetic sdAb library, but any other antibody library that can be prepared or is available can be used for the methods disclosed herein. In some embodiments, the antibody library contains sdAbs. In some implementations, the antibody library contains 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 1,00000 unique antibodies, or any number of antibodies within any two of the aforementioned ranges.

[0087] Antibody libraries can be generated on a computer or using machine learning methods. Exemplary methods for generating antibody libraries on a computer include modifying a generic VHH framework with synthetic diversity in one or more complementarity-determining regions (CDRs) (e.g., CDR1, CDR2, or CDR3, or any combination thereof). CDR diversity is introduced by randomizing the sequence library encoding the antibody with degenerate codons. For example, an NNK codon library can be used, where the NNK codons include N (a 25% mixture of A / T / C / G) and K (a 50% mixture of T / G). In some embodiments, the NNK codon library is constructed with all possible amino acids, or by excluding certain amino acid combinations (e.g., cysteine) and stop codons. Other degenerate codon mixtures can replace the NNK codon library for very small-scale experiments. In other embodiments, antibody libraries can be generated using a trimeric codon mixture, which improves the balanced representation of sense codons while reducing the probability of stop codons, thus increasing the efficiency of antibody generation and testing. In some implementations, artificial intelligence-based predictions can be used to randomize specific binding pockets of antibodies using available binding models or structural data.

[0088] In some embodiments, panning an antibody library includes screening for candidate binding peptides via phage display, yeast display, bacterial display, ribosome display, or mRNA display, or any combination thereof. In some embodiments, panning an antibody library includes one or more rounds of selection where candidate binding peptides are selected for specificity against a cancer-associated antigen (e.g., CEA6) or cells or tissues displaying a cancer-associated antigen. In some embodiments, candidate binding peptides are selected under conditions including, but not limited to, tumor microenvironment-like conditions, immunosuppressive conditions, low or high pH, ​​low or high oxygen concentration, low or high temperature, low or high viscosity, or any combination thereof, or for specificity against modified or derived forms of one or more cancer-associated antigens. In some embodiments, immunosuppressive conditions may include the presence of tumor-associated macrophages (TAMs), myeloid-derived suppressor cells (MDSCs), tumor-associated neutrophils (TANs), cancer-associated fibroblasts (CAFs), or other immunosuppressive cells, or the presence of adenosine, or both.

[0089] In some embodiments, the chimeric antigen receptor cells are derived from a cell line (e.g., Jurkat). In some embodiments, the chimeric antigen receptor cells are derived from the subject. In some embodiments, the subject has cancer. In some embodiments, the subject has cancer, and the cancer expresses any one or more of the cancer-associated antigens disclosed herein (e.g., CEA6). In some embodiments, 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, hematologic malignancy, or any combination thereof. In some embodiments, hematologic malignancy may include leukemia, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, hairy cell leukemia, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma, or multiple myeloma. In some implementations, 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 implementations, the subject is a human.

[0090] Chimeric antigen receptor (CAR)

[0091] This document also discloses chimeric antigen receptors (CARs) comprising any one or more of the CEA6-binding peptides disclosed herein. In some embodiments, the CAR is referred to as a "bi-CAR," meaning that it binds to two different target binding sites.

[0092] As used herein, the term "chimeric antigen receptor (CAR)" refers to an engineered biological receptor that confers artificial specificity to a particular antigen (e.g., a tumor-associated antigen) on an immune cell. An exemplary immune cell that can use a CAR is the T cell; however, it is envisioned that a CAR can be engineered to any readily treatable cytotoxic immune cell, including but not limited to T cells, natural killer (NK) cells, natural killer T (NKT) cells, dendritic cells, or macrophages. In this regard, any disclosure relating to CAR T cells can also be applied to other immune cells containing CARs. At its core, a CAR contains an extracellular antigen recognition domain (e.g., a tumor receptor ligand or antibody), a hinge, a transmembrane domain, and an intracellular signaling domain (intramidal domain). Different combinations of these CAR components can result in varying specificity and efficacy against certain cancer antigens.

[0093] In some embodiments, the CAR comprises at least two single-domain binding peptides, and the CAR is a multivalent CAR. In some embodiments, the CAR comprises two single-domain binding peptides, and the CAR is a bivalent CAR. In some embodiments, the CAR comprises three single-domain binding peptides, and the CAR is a trivalent CAR.

[0094] In some embodiments, the CAR further comprises one or more signal peptides, linkers of varying lengths and compositions, hinges, transmembrane domains, co-stimulatory domains, signal transduction domains, cytoplasmic domains, functional signaling molecules, proliferative signaling molecules, anti-exhaustion signaling molecules, anti-inhibitory receptors, tumor / cancer homing proteins, or regulatory molecules, or any combination thereof. In some embodiments, the hinge comprises a CD3ζ, CD4, CD8, or CD28 hinge, or a computer-designed synthetic hinge of various lengths. In some embodiments, the transmembrane domain comprises a CD3ζ, CD4, CD8, or CD28 transmembrane domain, or a computer-designed synthetic transmembrane domain. In some implementations, the co-stimulatory domains include co-stimulatory domains of CD8, CD28, ICOS, 4-1BB, OX40 (CD134), CD27, CD40, CD40L, TLR or other TNFR superfamily members or Ig superfamily members, or other signal transduction via cytoplasmic domains of IL-2Rβ, IL-15R-α, MyD88 or CD40 or any other Toll-like receptor or IL-1 receptor signaling pathway member.

[0095] In some embodiments, the CARs disclosed herein are constructed by assembling CAR expression constructs derived from a mixture of nucleic acids encoding any of the single-domain binding peptides disclosed herein and compatible nucleic acids encoding different CAR modules. In some embodiments, different CAR combinations are generated for use in a CAR library to screen CAR efficacy (in vitro or in vivo). In some embodiments, unique CARs are generated individually. In some embodiments, the CAR is specific for one target. In some embodiments, the CAR is specific for at least one, two, three, four, five, six, seven, eight, nine, or ten targets. In some embodiments, the CAR is bispecific or trispecific.

[0096] To construct any of the CARs disclosed herein, a nucleic acid encoding a single-domain binding polypeptide identified by antibody library panning is assembled into a CAR expression construct along with other CAR modules. In some embodiments, the CAR expression construct is assembled using multi-fragment assembly reactions known in the art. An exemplary method for assembling a CAR expression construct involves using an IIS-type restriction enzyme to generate a nucleic acid fragment having a compatible protrusion sequence, and ligating said nucleic acid fragment with a ligase. Because the IIS-type restriction enzyme cleaves outside its recognition site, multiple compatible nucleic acid fragments can be prepared simultaneously. In other embodiments, the CAR expression construct can be assembled by overlap extension PCR. It is contemplated that any other method for assembling a nucleic acid construct from more than one nucleic acid fragment can be employed. In some embodiments, the different CAR modules include signal peptides, linkers, hinges, transmembrane domains, co-stimulatory domains, activation domains, signal transduction domains, cytoplasmic domains, functional signals, proliferation signals, anti-exhaustion signals, anti-inhibitor receptors, cancer homing proteins, or regulatory molecules, or any combination thereof. Some exemplary hinges include CD8 hinges, CD28 hinges, IgG1 hinges, or IgG4 hinges. Some exemplary transmembrane domains include the CD3ζ transmembrane domain, CD8α transmembrane domain, CD4 transmembrane domain, CD28 transmembrane domain, or ICOS transmembrane domain. Some exemplary co-stimulatory domains include the CD8 co-stimulatory domain, CD28 co-stimulatory domain, 4-1BB co-stimulatory domain, OX40 (CD 134) co-stimulatory domain, ICOS co-stimulatory domain, CD27 co-stimulatory domain, CD40 co-stimulatory domain, CD40L co-stimulatory domain, TLR co-stimulatory domain, MYD88-CD40 co-stimulatory domain, or KIR2DS2 co-stimulatory domain. In some embodiments, different CAR modules are derived from any combination of CD8, CD28, 4-1BB, CD3ζ, or more. CARs can also be modified with various additives, including but not limited to cytokines, chemokines, cytokine receptors, chemokine receptors, antigen receptors or ligands, antibodies, or enzymes.

[0097] Use or treatment methods

[0098] As used herein, the terms “individual,” “object,” and “patient” are used interchangeably and refer to any animal and / or mammal. In some embodiments, the mammal is a human being. In some embodiments, the mammal is a non-human being. None of these terms require or are limited to situations characterized by supervision (e.g., continuous or intermittent) by a healthcare worker (e.g., a physician, registered nurse, nursing practitioner, physician assistant, caregiver, or hospice worker).

[0099] As used herein, the terms “treating” or “treatment” (and as is known in the art) mean a method of obtaining a beneficial or desired outcome (including clinical outcomes) in a subject’s condition. Beneficial or desired clinical outcomes may include, but are not limited to, relief or improvement of one or more symptoms or conditions, reduction of disease severity, stabilization (i.e., non-exacerbation) of the disease state, prevention of disease spread or diffusion, delay or slowing of disease progression, improvement or mitigation of the disease state, reduction of disease recurrence, and remission, whether partial or complete, and whether detectable or undetectable. As used herein, “treating” and “treatment” also include preventative treatment. Treatment methods include administering a therapeutically effective amount of an active agent to the subject. Administration steps may consist of a single administration or may include a series of administrations. The composition is 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 subject’s age and genetic characteristics, the concentration of the active agent, the activity of the composition used for treatment, or a combination thereof. It will also be understood that the effective dose of the agent used for treatment or prevention may be increased or decreased during a particular treatment or prevention regimen. Dosage changes can be produced and become apparent using standard diagnostic assays known in the art. In some cases, prolonged administration may be required.

[0100] As used herein, the term "effective amount" or "effective dose" refers to the amount of a composition or compound that results in an observable, specified effect. The actual dose level of the active ingredient in the active compositions of the currently disclosed subject matter can be varied to administer an amount of the active composition or compound that effectively achieves a specified response for a particular subject and / or application. The selected dose level can vary depending on a variety of factors, including but not limited to the activity of the composition, its formulation, route of administration, combination with other drugs or treatments, the severity of the condition being treated, and the physical condition and medical history of the subject being treated. In some embodiments, a minimum effective dose is administered, and the dose is escalated to the minimum effective amount in the absence of dose-limiting toxicity. This document considers the determination and adjustment of the effective dose, and assesses when and how such adjustments should be made.

[0101] As used herein, the term "therapeutic target" refers to a gene or gene product whose activity, once modulated (e.g., by regulating expression, biological activity, etc.), can provide regulation of a disease phenotype. As used throughout the text, "regulation" means an increase or decrease in the indicated phenomenon (e.g., regulation of biological activity means an increase or decrease in biological activity).

[0102] As used herein, the terms “standard care,” “best practice,” and “standard therapy” refer to treatments that are accepted by medical practitioners as appropriate, suitable, effective, and / or widely used for a particular disease. Standard care for a disease depends on many different factors, including the biological effects of the treatment, its area or location in the body, patient status (e.g., age, weight, sex, genetic risk, other disabilities, secondary conditions), toxicity, metabolism, bioaccumulation, therapeutic index, dosage, and other factors known in the art. Determining standard care for a disease also depends on the safety and efficacy established in clinical trials, as standardized by regulatory bodies such as the U.S. Food and Drug Administration, the International Coordinating Council, Health Canada, the European Medicines Agency, the Therapeutic Goods Authority, the Central Drug Standards Control Organization, national drug administrations, drug and medical device agencies, the Ministry of Food and Drug Safety, and the World Health Organization. Standard care for a disease may include, but is not limited to, surgery, radiation therapy, chemotherapy, targeted therapy, or immunotherapy.

[0103] This document also discloses methods for treating cancer in subjects of need. In some embodiments, the method includes administering chimeric antigen receptor cells to the subject. In some embodiments, the method includes administering any of the chimeric antigen receptor cells disclosed herein. In some embodiments, the chimeric antigen receptor cells express and / or contain any of the CEA6 single-domain binding peptides disclosed herein. In some embodiments, the chimeric antigen receptor cells are CAR T cells. In some embodiments, the chimeric antigen receptor cells are CAR NK cells. In some embodiments, the chimeric antigen receptor cells are CAR tumor-infiltrating lymphocytes (TILs). In some embodiments, the chimeric antigen receptor cells are macrophages. In some embodiments, the chimeric antigen receptor cells are derived from the subject and are autologous to the subject. In some embodiments, the chimeric antigen receptor cells are allogeneic to the subject. In some embodiments, the chimeric antigen receptor cells are derived 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, cattle, pig, horse, goat, sheep, donkey, or monkey. In some embodiments, the subject is a human. In some implementations, 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, hematologic malignancy, or any combination thereof. In some implementations, chimeric antigen receptor cells are administered parenterally.

[0104] In some implementations, chimeric antigen receptor cells are administered once daily, twice daily, three times daily, or more frequently. In some implementations, chimeric antigen receptor cells are administered daily, every other day, five days a week, once a week, every other week, two weeks a month, three weeks a month, once a month, twice a month, three times a month, or more frequently. In some implementations, immune cells are 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 longer.

[0105] In some embodiments, the amount of a given agent corresponding to such a quantity varies depending on factors such as the specific compound, the severity of the disease, and the characteristics (e.g., weight) of the person or host requiring treatment, but is still routinely determined in a manner known in the art based on the specific circumstances surrounding the case, including, for example, the specific agent to be administered, the route of administration, and the person or host being treated. In some cases, the desired dose is conveniently presented as a single dose, or as fractional doses administered simultaneously (or over a short period of time) or at appropriate intervals, such as sub-dose twice, three, four, or more times daily.

[0106] The scope of application is merely suggestive, as there are a great many variables regarding individual treatment regimens, and considerable deviations from these recommended values ​​are not uncommon. Such dosages vary based on multiple variables, including but not limited to the activity of the compound used, the disease or condition to be treated, the method of administration, the individual's requirements, the severity of the disease or condition to be treated, and the practitioner's judgment.

[0107] In some embodiments, the toxicity and therapeutic efficacy of such treatment regimens are determined using standard pharmaceutical procedures in cell culture or laboratory animals, including but not limited to the determination of LD50 (the dose that is lethal to 50% of the population) and ED50 (the dose that is effective to 50% of the population). The dose ratio between toxicity and therapeutic effect is the therapeutic index and is expressed as the ratio of LD50 to ED50. Compounds exhibiting a high therapeutic index are preferred. Data obtained from cell culture assays and animal studies are used to formulate a series of doses for human use. The doses of such compounds are preferably within a range that includes the ED50 and the cyclic concentration with minimal toxicity. The dose varies within this range depending on the dosage form used and the route of administration employed.

[0108] Methods applied to an object

[0109] The term "application" includes intraperitoneal application. "Intraperitoneal" is given its standard scientific meaning and therefore refers to the application of a substance to the peritoneal cavity of a subject. Thus, intraperitoneal application includes any effective method of delivering a substance into the peritoneal cavity, including injection and infusion.

[0110] Checkpoint inhibitors (iCPI)

[0111] Some embodiments of this disclosure relate to cancer treatment methods, including the administration of at least one checkpoint inhibitor. In some embodiments, at least two checkpoint inhibitors are administered. In some embodiments, the at least two checkpoint inhibitors are administered simultaneously. In some embodiments, the at least two checkpoint inhibitors are administered at different times.

[0112] "Checkpoint inhibitors" are molecules, drugs, and / or compositions that function in inhibiting at least one immune checkpoint. An "immune checkpoint" is a regulator of the target immune system. Non-limiting examples of stimulating checkpoint molecules include CD27, CD28, CD40, CD122, CD137, OX40, GITR, and ICOS. Non-limiting examples of inhibitory checkpoint molecules include A2AR, A2BR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, NOX2, PD-1, PD-L1, PD-L2, TIM-3, VISTA, SIGLEC7, and the dominant-negative PD-1 receptor (DNR).

[0113] In some implementations, the cancer in question may evade the immune system's targeting by altering the function of immune checkpoint targets. Checkpoint inhibitors function by blocking this altered activity, thereby restoring normal immune function. Therefore, cancer cells are predicted to be more susceptible to the immune system of patients treated with checkpoint inhibitors.

[0114] Non-limiting examples of checkpoint inhibitors (iCPIs) include iplimumab, pembrolizumab, nivolumab, atezolizumab, avelumab, durvalumab, cemiplimab, tremelimumab, relatlimab, opdualag, and spartalizumab.

[0115] Example

[0116] Some aspects of the embodiments discussed above are further disclosed in detail in the following examples, which are not intended to limit the scope of this disclosure in any way. Those skilled in the art will understand that many other technical solutions also fall within the scope of the invention as described above and in the claims.

[0117] Example 1: Cancer Treatment via IP Injection with Immunocells

[0118] An effective dose of CAR T cells B4T2-001 will be administered to a human subject with cancer via intraperitoneal injection. The human subject will not undergo any lymphocyte clearance / chemotherapy before or during CAR T cell administration. Two weeks later (day 14 of the experiment), another dose of CAR T cells will be administered via intraperitoneal injection. One week later (day 21 of the experiment), the subject will be administered the anti-EGFR antibody cetuximab to activate the killing switch and prevent any further effects of the CAR T cells. Cetuximab will be administered intravenously over 120 minutes at an initial dose of 400 mg / m² body surface area, followed by up to four weekly doses of 250 mg / m², or until CAR T clearance, as evaluated by qPCR, results in the complete elimination of the genetically modified T cells via antibody-dependent cytotoxicity (ADCC).

[0119] One week after initial cetuximab exposure (day 28 of the experiment), participants will be administered an anti-PD1 immune checkpoint inhibitor. One week later (day 35 of the experiment), participants will be administered an anti-PDL1 immune checkpoint inhibitor.

[0120] Example 2: CAR T cell uptake and expression after IP injection

[0121] B4t2-001 CAR T cells were infused into subjects via intraperitoneal injection at a dose of 1E6 / kg without prior lymphocyte ablation chemotherapy. CAR T cell engraftment was monitored over time using qPCR. Figure 1 B4t2-001 CAR-T cells effectively expanded in the peritoneal region, reaching peak levels on the second day after infusion, and were effectively engrafted into the circulating blood. max Each microgram contains 52,546 copies of genomic DNA.

[0122] CAR T cell levels in blood and peritoneal fluid were also measured by FACS ( Figures 2A to 2B FACS data showed that B4t2-001 CAR-T cells effectively expanded in the peritoneal space, reaching 15% of total lymphocytes on day 2 after CAR-T infusion and 45% on day 8. Furthermore, on day 15 after CAR-T infusion, B4t2-001 CAR-T cells effectively engrafted into more than 35% of circulating blood lymphocytes.

[0123] On day 15, cetuximab was administered to subjects at a dose of 400 mg / m², leading to targeted degradation of CAR-T cells. This administration of the killer switch activator resulted in a rapid decrease in CAR-T cells on day 17. Figure 3 ).

[0124] While various aspects and embodiments have been disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for illustrative purposes and are not intended to be limiting; the true scope and spirit are indicated by the appended claims.

[0125] All references cited herein (including, but not limited to, published and unpublished applications, patents, and literature references) are incorporated herein by reference in their entirety and form part of this specification. Where a publication or patent or patent application incorporated by reference contradicts the disclosure contained in this specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

Claims

1. A method for treating a disease or ailment in a person in need, said method comprising: CAR immune cells are administered to the subject, wherein the CAR immune cells stimulate the subject's blood cells. Wherein, after stimulating the blood cells, the CAR immune cells undergo activation and / or population expansion, resulting in at least 20% of the CAR immune cells being implanted into the subject.

2. The method of claim 1, wherein the CAR immune cell contains a first binding site for CEACAM6.

3. The method of claim 2, wherein the CAR immune cell contains a second binding site for an antigen associated with the disease or condition.

4. The method according to any one of claims 1 to 3, wherein the disease or symptom is a tumor, cancer, solid tumor, or any combination thereof.

5. The method according to any one of claims 1 to 4, wherein the disease or condition is an autoimmune disease, cardiomyopathy, viral infection, rheumatoid arthritis, fibrosis, cardiac fibrosis, disease or condition caused by HIV, disease or condition caused by COVID-19, disease or condition caused by chronic hepatitis C virus (HCV), disease or condition caused by human cytomegalovirus (HCMV), influenza, cellular senescence targets (selective clearance of senescent cells (SC)), liver fibrosis, pulmonary fibrosis, atherosclerosis, diabetes, osteoarthritis, obesity, and / or aging.

6. The method according to any one of claims 3 to 5, wherein the second binding site is capable of binding to any one of the following: AFP, ANTXR1, AXL, αvβ3, αvβ6, B7-H3, CAIX, CD171, CD20, CD32A, CD46, CD47, CD56, CD80 / 86, CEA, sealing protein 18.2, DLL-3, DR5, EGFR, EGFRIII, EGFR806, EpCAM, EpHA2, FAP, FR-α, GD2, phosphatidylinositol proteoglycan-2, phosphatidylinositol proteoglycan-3, gp100, GSPG4, GUCY2C, HBV surface antigen (HBsAg), HER2, IL-13R-α2, L1-CAM, Lewis Y, LMP1, MAGE-A1 / 3 / 4, mesothelin, c-MET, M2e, MUC1, MUC16, MUC3A, Nectin4 / FAP, NKG2D, PAP, PSA, PSCA, PSMA, ROR, TAG-72, Trop2, uPAR and / or VEGFR2.

7. The method according to any one of claims 1 to 6, wherein the CAR immune cell is B4T2-001.

8. The method according to any one of claims 1 to 7, wherein the CAR immune cells are TIL cells.

9. The method according to any one of claims 1 to 8, wherein the CAR immune cell is a T cell, NK cell, or macrophage.

10. The method according to any one of claims 1 to 9, wherein the object is a mammal and / or a human.

11. The method according to any one of claims 1 to 10, wherein the blood cells are neutrophils and / or granulocytes.

12. The method according to any one of claims 1 to 11, wherein the activation and / or population expansion is an increase of at least 50-fold in the cell population.

13. The method according to any one of claims 1 to 12, wherein the method does not require a lymphocyte removal step.

14. The method according to any one of claims 1 to 13, wherein the disease or condition is cancer and / or tumor, and wherein the cancer and / or tumor is present in cells and / or tissues selected from: brain, breast, central nervous system, cervix, colon, colorectal, epidermis, stomach, glioblastoma, glioma, hepatocellular carcinoma, liver, lung, nasopharynx, neuroblastoma, ovary, pancreas, pediatric glioma, prostate, rectum, kidney and stomach.

15. The method according to any one of claims 1 to 14, wherein the application is performed by infusion.

16. The method according to any one of claims 1 to 15, wherein the application is performed locally, intraperitoneally, intravenously, intratumorally, or intramurally.

17. The method according to any one of claims 1 to 16, wherein the method further comprises administering the CAR immune cells to the subject multiple times.

18. The method according to any one of claims 1 to 17, wherein the stimulation of the blood cells is reversible.

19. The method according to any one of claims 1 to 18, wherein the CAR immune cells undergo activation and / or population expansion in at least one of the peripheral blood, peritoneal fluid, and / or pulmonary fluid of the subject.

20. The method according to any one of claims 1 to 19, wherein the method further comprises applying a kill switch activator.

21. The method of claim 20, wherein the kill switch activator is an anti-EGFR antibody.

22. The method according to any one of claims 20 or 21, wherein the kill switch activator is applied locally, intraperitoneally, intravenously, intratumorally, or intramurally.

23. The method of any one of claims 20-22, wherein the killer switch activator is administered at a dose of about 0.1 mg / m 2 , about 1 mg / m 2 , about 10 mg / m 2 , about 100 mg / m 2 , about 250 mg / m 2 , about 500 mg / m 2 , about 700 mg / m 2 , about 750 mg / m 2 , or any integer dose from 0.1 mg / m 2 to 750 mg / m 2 .

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