Bispecific cd19 / cd20 targeted chimeric antigen receptors and uses thereof

Abstract

Various embodiments of the present application provide binding molecules for B-lymphocyte antigens (e.g., CD19 and CD20), including antigen recognizing receptors (e.g., CARs) that target B-lymphocyte antigens (e.g., CD19 and CD20). Embodiments of the present application also provide cells expressing such binding molecules, therapeutic compositions comprising such cells, and methods for treating diseases or disorders associated with B-lymphocyte antigens (e.g., CD19 and CD20), including hematological cancers, autoimmune diseases (e.g., multiple sclerosis and systemic lupus erythematosus).

Description

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 587,646, filed October 3, 2023, the disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] sequence list This application includes an electronic sequence list, which has been submitted with this application in XML file format, the entire contents of which are incorporated herein by reference. The sequence list XML file submitted with this application is named "14183-016-228_SEQLISTING.xml", was created on September 30, 2024, and has a size of 71,327 bytes. 1. Technical Field Embodiments of the present invention relate to bispecific chimeric antigen receptors (CARs) targeting CD19 and CD20, cells containing CARs, and methods for treating B-lymphocyte antigen-related diseases (e.g., hematologic malignancies and autoimmune diseases) using such CARs and cells. 2. Background Technology Hematologic malignancies represent some of the most common cancers occurring in both children and adults. For example, approximately 4,000 new cases of aggressive B-cell lineage malignancy, B-cell acute lymphoblastic leukemia (B-ALL), are diagnosed annually in the United States, representing the most common malignancy in children. Gene mutations that induce abnormal arrest of normal lymphocyte maturation, escape from apoptosis, and uncontrolled cell proliferation lead to an overproduction of B-cell lymphoblasts. In adults, more than 6,000 new cases of acute lymphoblastic leukemia (ALL) occur annually. (Hanahan D. et al.) Cell (2000);100:57-70; Teitell M. et al. Annu Rev Pathol (2009);4:175-198). Furthermore, lymphomas (e.g., growths in lymphoid tissue) account for approximately 5% of all cancer cases in the United States. The primary category is malignant tumors of lymphocytes, cell types found in both lymph and blood. In this way, both lymphoma and leukemia are malignant tumors of hematopoietic and lymphoid tissues (e.g., tumors). As lymphoproliferative disorders, lymphomas and lymphoblastic leukemias are so closely related that some of them can be referred to by either name (e.g., adult T-cell leukemia / lymphoma).

[0005] Autoimmune diseases are the third most prevalent disease in the United States, affecting approximately 50 million Americans. Autoimmune diseases occur when the immune system mistakenly attacks the body's own tissues, leading to inflammation and tissue damage. Three of the most well-known autoimmune diseases include multiple sclerosis (MS), systemic lupus erythematosus (SLE), and rheumatoid arthritis (RA). In MS, the immune system targets the central nervous system, particularly the myelin sheath that protects nerve cells, leading to nerve damage. In RA, the immune system primarily attacks the joints, causing chronic inflammation and joint destruction. SLE, on the other hand, is a systemic disease affecting multiple organs, driven by autoantibodies targeting nuclear components within cells.

[0006] B cells, a type of white blood cell, play a central role in the pathogenesis of these and other autoimmune diseases. In RA and SLE, B cells produce autoantibodies that mistakenly target body tissues, exacerbating inflammation and tissue destruction. B cells also contribute to disease progression through antigen presentation and the production of pro-inflammatory cytokines (which amplify the immune response). In MS, B cells are involved in producing antibodies that attack the central nervous system, including the brain, leading to demyelination and neurodegeneration. In other neurodegenerative diseases such as Guillain-Barré syndrome (GBS), chronic inflammatory demyelinating polyneuropathy (CIDP), and multifocal motor neuropathy (MMN), B cells produce autoantibodies against peripheral nerves, which attack the myelin sheath of neurons in peripheral nerves, such as those present in the limbs.

[0007] To address the role of B cells in autoimmune diseases, several therapies have been developed to target and deplete them. One of the most widely used therapies is rituximab, a monoclonal antibody that depletes B cells by targeting the CD20 protein on their surface. This therapy is effective in treating rheumatoid arthritis (RA) and is also used in some cases of MS and SLE. Other therapies include belimumab, which targets B cell activating factor (BAFF), a protein essential for B cell survival, and newer CD20-targeting therapies such as ofamumab and oxotuzumab.

[0008] While these therapies offer significant improvements in disease control, they also have distinct limitations. For example, the extensive depletion of B cells weakens the immune system's ability to fight infection, leading to adverse reactions such as increased susceptibility to infection. Furthermore, lack of efficacy remains a challenge in some patients, with some individuals showing incomplete responses or relapses despite treatment. In addition, long-term use of B-cell therapy may lead to the development of treatment resistance or other immune complications. Therefore, there is a need for improved compositions and therapies for treating the aforementioned and other autoimmune diseases.

[0009] Cell-based immunotherapy is a potentially curative treatment for cancer and autoimmune diseases. T cells and other immune cells can be modified to target tumor antigens by introducing genetic material encoding artificial or synthetic receptors that target antigens (called chimeric antigen receptors (CARs)), which are specific to the selected antigens. CAR-targeted T-cell therapies have recently shown clinical success in treating hematologic malignancies. However, despite positive results from early trials, infusion of CD19-targeted CAR T cells into patients still results in a number of side effects of varying severity. These side effects include tumor lysis syndrome (TLS), cytokine release syndrome (CRS), macrophage activation syndrome, CNS transport, prolonged B-cell hypoplasia, and immune escape. Furthermore, due to significant challenges in accessing the central nervous system, current B-cell-targeting cell-based immunotherapies (e.g., via the CD20 antigen) may not halt the progression of autoimmune diseases in cases of MS and other related conditions. Therefore, given the aforementioned long-standing and unmet needs, there is a need for improved therapies for hematologic malignancies and autoimmune diseases. 3. Summary of the Invention Various embodiments of the present invention provide chimeric antigen receptors (CARs) targeting CD19 and CD20, cells containing CARs, and uses of the cells, such as for treating B-lymphocyte antigen-related diseases or conditions.

[0011] In a first aspect, embodiments of the present invention provide a chimeric antigen receptor (CAR) targeting CD19 and CD20. In some embodiments, the CAR comprises: (a) an extracellular domain comprising (i) an antigen-binding domain that binds to CD19 and (ii) a second antigen-binding domain that binds to CD20; (b) a transmembrane domain comprising a first CD28 polypeptide; and (c) an intracellular domain comprising a modified CD3ζ polypeptide containing natural ITAM1, ITAM2 variants, and ITAM3 variants, wherein each of the ITAM2 and ITAM3 variants comprises two loss-of-function mutations.

[0012] In some embodiments, the first anti-CD19 antigen-binding domain includes: a first heavy chain variable region (V H ) and the first light chain variable region (V L ), where the first V H V containing the amino acid sequence shown in SEQ ID NO: 15 H V H CDR1, V H CDR2 and V H CDR3. In some implementations, the first V LV containing the amino acid sequence shown in SEQ ID NO: 16 L V L CDR1, V L CDR2 and V L CDR3.

[0013] In some implementations, the first V H Contains V containing the amino acid sequence shown in SEQ ID NO: 9 or its conserved modifications. H CDR1, containing the amino acid sequence shown in SEQ ID NO: 10 or its conserved modifications thereof, V H CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 11 or its conserved modifications H CDR3. In some implementations, the first V L Contains V containing the amino acid sequence shown in SEQ ID NO: 12 or its conserved modifications. L CDR1, containing the amino acid sequence shown in SEQ ID NO: 13 or its conserved modifications thereof, V L CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 14 or its conserved modifications L CDR3.

[0014] In some implementations, the first V H V containing the amino acid sequence shown in SEQ ID NO: 9 H CDR1, containing the amino acid sequence shown in SEQ ID NO: 10, V H CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 11 H CDR3. In some implementations, the first V L V containing the amino acid sequence shown in SEQ ID NO: 12 L CDR1, containing the amino acid sequence shown in SEQ ID NO: 13, V L CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 14 L CDR3.

[0015] In some implementations, the first V HContains an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical or homologous to the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the first V L It contains amino acid sequences that are at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical or homologous to the amino acid sequence shown in SEQ ID NO: 16.

[0016] In some implementations, the first V H It contains the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the first V L It contains the amino acid sequence shown in SEQ ID NO: 16.

[0017] In some embodiments, the first anti-CD19 antigen binding domain includes a first V H With the first V L The first linker between them. In some embodiments, the first linker comprises the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the first V H And the first V L Positioning from the N-terminus to the C-terminus: V L -V H .

[0018] In some embodiments, the first anti-CD19 antigen-binding domain includes a first single-stranded variable fragment (scFv) that binds to anti-CD19. In some embodiments, the first anti-CD19 scFv includes the amino acid sequence shown in SEQ ID NO: 17.

[0019] In some embodiments, the second anti-CD20 antigen-binding domain includes: a second V H Second V L The second V H V containing the amino acid sequence shown in SEQ ID NO: 27 H V H CDR1, V H CDR2 and V H CDR3. In some implementations, the second V LV containing the amino acid sequence shown in SEQ ID NO: 28 L V L CDR1, V L CDR2 and V L CDR3.

[0020] In some implementations, the second V H Contains V containing the amino acid sequence shown in SEQ ID NO: 21 or its conserved modifications. H CDR1, containing the amino acid sequence shown in SEQ ID NO: 22 or its conserved modifications thereof, V H CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 23 or its conserved modifications H CDR3. In some implementations, the second V L Contains V containing the amino acid sequence shown in SEQ ID NO: 24 or its conserved modifications. L CDR1, containing the amino acid sequence shown in SEQ ID NO: 25 or its conserved modifications thereof, V L CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 26 or its conserved modifications L CDR3.

[0021] In some implementations, the second V H V containing the amino acid sequence shown in SEQ ID NO: 21 H CDR1, containing the amino acid sequence shown in SEQ ID NO: 22, V H CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 23 H CDR3. In some implementations, the second V L V containing the amino acid sequence shown in SEQ ID NO: 24 L CDR1, containing the amino acid sequence shown in SEQ ID NO: 25, V L CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 26 L CDR3.

[0022] In some implementations, the second V HContains an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical or homologous to the amino acid sequence shown in SEQ ID NO: 27. In some embodiments, the second V L It contains amino acid sequences that are at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical or homologous to the amino acid sequence shown in SEQ ID NO: 28.

[0023] In some implementations, the second V H It contains the amino acid sequence shown in SEQ ID NO: 27. Additionally, in some embodiments, the second V... L It contains the amino acid sequence shown in SEQ ID NO: 28.

[0024] In some embodiments, the second anti-CD20 antigen-binding domain includes a second V H With the second V L A second linker between them. In some embodiments, the second linker comprises the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the second V H Second V L Positioning from the N-terminus to the C-terminus: V H -V L .

[0025] In some embodiments, the second anti-CD20 antigen-binding domain comprises a second anti-CD20 single-chain variable fragment (scFv) that binds to CD20. In some embodiments, the second anti-CD20 scFv comprises the amino acid sequence shown in SEQ ID NO: 29.

[0026] In some implementations, V H CDR1, V H CDR2, V H CDR3, V L CDR1, V L CDR2 and V L CDR3 is based on the Kabat numbering system, Chothia numbering system, AbM numbering system, Contact numbering system, or IMGT information system. ®It is used for identification. In some implementations, V H CDR1, V H CDR2, V H CDR3, V L CDR1, V L CDR2 and V L CDR3 is identified according to the Kabat numbering system.

[0027] In some embodiments, the CD19 / CD20-targeted CAR further includes a third linker between the first anti-CD19 antigen-binding domain and the second anti-CD20 antigen-binding domain. In some embodiments, the third linker includes the amino acid sequence shown in SEQ ID NO:3. In some embodiments, the first anti-CD19 antigen-binding domain is located downstream of the second anti-CD20 antigen-binding domain.

[0028] In some embodiments, the leader sequence is covalently attached to the N-terminus of the second anti-CD20 antigen binding domain. In some embodiments, the leader sequence comprises a CD8 polypeptide. In some embodiments, the CD8 polypeptide comprises the amino acid sequence shown in SEQ ID NO: 33.

[0029] In some embodiments, the first CD28 polypeptide is a transmembrane domain sequence of CD28. In some embodiments, the first CD28 polypeptide comprises amino acids 153 to 179 of SEQ ID NO: 35.

[0030] In some embodiments, the CD19 / CD20-targeted CAR further comprises a hinge domain. In some embodiments, the hinge domain comprises a second CD28 peptide. In some embodiments, the second CD28 peptide comprises amino acids 114 to 152 of SEQ ID NO: 35.

[0031] In some embodiments, each of the two loss-of-function mutations is located at a tyrosine amino acid residue. In some embodiments, the ITAM2 variant comprises the amino acid sequence shown in SEQ ID NO: 45. In some embodiments, the ITAM3 variant comprises the amino acid sequence shown in SEQ ID NO: 49. In some embodiments, the native ITAM1 comprises the amino acid sequence shown in SEQ ID NO: 39. In some embodiments, the modified CD3ζ polypeptide comprises the amino acid sequence shown in SEQ ID NO: 51.

[0032] In some embodiments, the intracellular domain further includes a co-stimulatory signaling region, which may include a third CD28 polypeptide in one or more embodiments. In some embodiments, the third CD28 polypeptide is a CD28 signaling domain sequence. Additionally, in some embodiments, the third CD28 polypeptide includes amino acids 180 to 220 of SEQ ID NO: 35 in one or more embodiments.

[0033] In some embodiments, the CAR contains the amino acid sequence shown in SEQ ID NO: 54.

[0034] In another embodiment, embodiments of the present invention provide a nucleic acid encoding a CD19 / CD20-targeted CAR disclosed herein. In some embodiments, the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO: 55.

[0035] In another aspect, embodiments of the present invention provide vectors comprising the nucleic acids disclosed herein. In some embodiments, the vector is a viral vector, specifically a retroviral vector.

[0036] In another aspect, embodiments of the present invention provide cells comprising the CAR, nucleic acid, or vector disclosed herein. In some embodiments, the cells are transduced using a CAR, nucleic acid, or vector. In some embodiments, the CAR is constitutively expressed on the surface of the cell.

[0037] In some embodiments, the cells are immune-response cells or immune-effect cells. In some embodiments, the cells are lymphoid lineage cells or myeloid lineage cells. In some embodiments, lymphoid lineage cells are selected from the group consisting of: T cells, natural killer (NK) cells, B cells, and stem cells from which they can differentiate into lymphoid cells. In some embodiments, myeloid lineage cells are selected from the group consisting of: monocytes, macrophages, neutrophils, dendritic cells, basophils, neutrophils, eosinophils, megakaryocytes, mast cells, erythrocytes, platelets, and stem cells from which they can differentiate into myeloid cells.

[0038] In some embodiments, the cells are selected from the group consisting of: T cells, B cells, natural killer (NK) cells, macrophages, innate lymphoid cells (ILCs), cytokine-induced killer (CIK) cells, lymphokine-activated killer (LAK) cells, stem cells from which they can differentiate into lymphoid cells, stem cells from which they can differentiate into myeloid cells, and combinations thereof. In some embodiments, the cells are T cells. In some embodiments, the T cells are selected from the group consisting of: helper T cells, cytotoxic T cells (CTLs), memory T cells, regulatory T cells, tumor-infiltrating lymphocytes (TILs), natural killer T cells, mucosa-associated inert T cells, αβ T cells, and γδ T cells. In some embodiments, the T cells are central memory T cells. In some embodiments, the T cells are positive for CD45RO and CD62L.

[0039] In some embodiments, the stem cells are pluripotent stem cells. In some embodiments, the pluripotent stem cells are embryo-like stem cells or induced pluripotent stem cells.

[0040] In some embodiments, the cells are cytotoxic T lymphocytes (CTLs). In some embodiments, the cells are CTLs sensitized with viral antigens. In some embodiments, the viral antigen is selected from the group consisting of Epstein-Barr virus (EBV), cytomegalovirus (CMV), BK virus (BKV), John Cunningham virus (JCV), pituitary RNA virus, hepatotropic DNA virus, hepatitis C virus, delta virus, hepatitis E virus, or any combination thereof. In some embodiments, the cells are EBV-sensitized CTLs.

[0041] In another aspect, embodiments of the present invention provide compositions comprising the cells disclosed herein, said compositions for treating various cancers, autoimmune diseases, and related conditions. In some embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier. In some embodiments, the composition contains about 1 × 10⁻⁶ cells. 6 With approximately 5×10 8 Between cells. In some embodiments, the composition contains about 1 × 10 6 With approximately 1×10 8 Between cells. In some embodiments, the composition contains about 1 × 10 6 With approximately 5×10 7 Between cells. In some embodiments, the composition contains about 1 × 10 6 With approximately 1×10 7 Cells between. In these and related implementations, the dosage of cells can be titrated based on one or more of the patient's weight, age, and condition (e.g., leukemia, lymphoma, MS, SLE, etc.).

[0042] In another aspect, embodiments of the present invention provide a method for inhibiting the growth of target cells expressing at least one B lymphocyte antigen. In some embodiments, the method includes contacting the target cells with cells or compositions disclosed herein.

[0043] In some embodiments, the at least one B lymphocyte antigen is selected from the group consisting of CD19, CD20, and combinations thereof. In some embodiments, the target cells express detectable levels of both CD19 and CD20. In some embodiments, the target cells express detectable levels of CD20 and low or undetectable levels of CD19. In some embodiments, the target cells express detectable levels of CD19 and low or undetectable levels of CD20. In some embodiments, the target cells are autologous to the CAR-expressing cells. In some embodiments, the target cells are allogeneic to the CAR-expressing cells. In some embodiments, the target cells are tumor cells. In some embodiments, the tumor is cancer. In some embodiments, the cancer is a blood cancer. In some implementations, the blood cancers are selected from the group consisting of: acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, mantle cell lymphoma (MCL), and follicular lymphoma (FL). In some implementations, the target cells are B cells. In some implementations, the B cells are CD19 cells. + CD20 + CD19 + CD20 - Or CD19 - CD20 + In some implementations, the target cells are T cells. In some implementations, the T cells are CD19 cells. + CD20 + CD19 + CD20 - Or CD19 - CD20 + In some implementations, T cells are CD20. + .

[0044] In some embodiments, the cells or composition exhibit cytotoxic and / or cytolytic activity against the target cells. In some embodiments, the cytotoxic and / or cytolytic activity is dose-dependent. In some embodiments, the cytotoxic and / or cytolytic activity is long-lasting. In some embodiments, the cytotoxic and / or cytolytic activity persists for at least 24 hours, at least 72 hours, at least one week, at least one month, or at least two months after initial contact of the target cells with the cells or composition. In some embodiments, the cytotoxic and / or cytolytic activity persists for approximately 40 days after initial contact of the target cells with the cells or composition. In some embodiments, the cytotoxic and / or cytolytic activity persists for approximately 60 days after initial contact of the target cells with the cells or composition.

[0045] In another aspect, embodiments of the present invention provide methods for treating B-lymphocyte antigen-related diseases such as various blood cancers and autoimmune diseases in humans or other subjects. In some embodiments, the method includes administering to a subject the cells disclosed herein and / or pharmaceutical compositions comprising the cells disclosed herein.

[0046] In some embodiments, the B lymphocyte antigen is selected from the group consisting of CD19, CD20, and combinations thereof. In some embodiments, the B lymphocyte antigen is either CD19 or CD20. In some embodiments, the B lymphocyte antigen-associated disease is cancer. In some embodiments, the cancer is a blood cancer. In some embodiments, the blood cancer is selected from the group consisting of: acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and follicular lymphoma (FL).

[0047] In some implementations, B-lymphocyte antigen-associated diseases are autoimmune diseases. In some implementations, autoimmune diseases are selected from the group consisting of: multiple sclerosis, systemic lupus erythematosus (SLE), Sjögren's syndrome, AL amyloidosis, IgG4-associated disease (IgG4-RD), inflammatory myopathy (e.g., myositis), scleroderma, pemphigus vulgaris, chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barré syndrome (GBS), stiff-person syndrome, rheumatoid arthritis, neuromyelitis optica spectrum disorder (NMOSD), myasthenia gravis, N-methyl... -D-aspartate receptor (NMDAR) encephalitis, anti-myelin oligodendrocyte glycoprotein (MOG) syndrome, MOGAD (MOGAD) disease, membranous nephropathy (MN), IgA nephropathy (IgAN), type 1 diabetes, anti-neutrophil cytoplasmic antibody (ANCA) vasculitis, thrombocytopenic purpura, hemolytic anemia (AIHA), multifocal motor neuropathy (MMN), inflammatory bowel disease (IBD), celiac disease, Crohn's disease, ulcerative colitis, and ankylosing spondylitis. In some implementations, the autoimmune disease is multiple sclerosis (MS). In some implementations, the autoimmune disease is systemic lupus erythematosus (SLE).

[0048] In some embodiments, the cells or composition exhibit cytotoxic and / or cytolytic activity against target cells in a subject, wherein the target cells are positive for B lymphocyte antigens. In some embodiments, the target cells express: i) detectable levels of CD19 and detectable levels of CD20; ii) detectable levels of CD20 and low or undetectable levels of CD19; or iii) detectable levels of CD19 and low or undetectable levels of CD20.

[0049] In some embodiments, the target cells are tumor cells. In some embodiments, the tumor is cancer. In some embodiments, the cancer is a blood cancer. In some embodiments, the blood cancer is selected from the group consisting of: acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell leukemia, multiple myeloma, Hodgkin's lymphoma, non-Hodgkin's lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma, mantle cell lymphoma (MCL), and follicular lymphoma (FL). In some embodiments, the target cells are B cells. In some embodiments, the B cells are CD19 cells. + CD20 + CD19 + CD20 - Or CD19 - CD20+ In some implementations, the target cells are T cells. In some implementations, the T cells are CD19 cells. + CD20 + CD19 + CD20 - Or CD19 - CD20 + In some implementations, T cells are CD20. + .

[0050] In some embodiments, the cytotoxic and / or cytolytic activity is dose-dependent. In some embodiments, the cytotoxic and / or cytolytic activity is long-lasting. In some embodiments, the cytotoxic and / or cytolytic activity persists for at least 24 hours, at least 72 hours, at least one week, at least one month, or at least two months after initial administration of the cells or composition. In some embodiments, the cytotoxic and / or cytolytic activity persists for about 40 days after initial administration of the cells or composition. In some embodiments, the cytotoxic and / or cytolytic activity persists for about 60 days after initial administration of the cells or composition. In some embodiments, the cells or composition do not induce graft-versus-host disease.

[0051] In some embodiments, the cells or composition do not induce or induce minimal allotropic reactivity against HLA-mismatched cells in the subject that are positive for B lymphocyte antigens. In some embodiments, the cells or composition do not exhibit or exhibit minimal cytotoxic and / or cytolytic activity against off-target cells in the subject, wherein the off-target cells are negative for B lymphocyte antigens. In some embodiments, the off-target cells are selected from the group consisting of CD19. - CD20 - T cells, natural killer T cells (NK T cells), natural killer cells (NK cells), and combinations thereof. In some embodiments, the cells or composition induce cytokine secretion. In some embodiments, the cytokines are selected from the group consisting of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and combinations thereof.

[0052] In some embodiments, the cells are T cells. In some embodiments, the T cells are EBV-sensitized CTLs. In some embodiments, the cells persist in the subject for at least 24 hours, at least 72 hours, at least one week, at least one month, or at least two months after initial administration of the cells or composition. In some embodiments, the cells persist in the subject for approximately 40 days after initial administration of the cells or composition. In some embodiments, the cells persist in the subject for approximately 60 days after initial administration of the cells or composition.

[0053] In another aspect, embodiments of the present invention provide a method for treating a subject with blood cancers. In some embodiments, the method includes administering to the subject the cells or compositions disclosed herein.

[0054] In some implementations, blood cancers are selected from the group consisting of: acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, mantle cell lymphoma (MCL), and follicular lymphoma (FL).

[0055] In some embodiments, the cells or composition exhibit cytotoxic and / or cytolytic activity against target cells in a subject, wherein the target cells express: i) detectable levels of CD19 and detectable levels of CD20; ii) detectable levels of CD20 and low or undetectable levels of CD19; iii) detectable levels of CD19 and low or undetectable levels of CD20. In some embodiments, the target cells are CD19. + CD20 + CD19 + CD20 - Or CD19 - CD20 + .

[0056] In some embodiments, cytotoxicity and / or cytolytic activity is dose-dependent. In some embodiments, cytotoxicity and / or cytolytic activity is long-lasting. In some embodiments, cytotoxicity and / or cytolytic activity persists for at least one month or at least two months after initial administration of the cells or composition. In some embodiments, cytotoxicity and / or cytolytic activity persists for about 40 days after initial administration of the cells or composition. In some embodiments, cytotoxicity and / or cytolytic activity persists for about 60 days after initial administration of the cells or composition. In some embodiments, the cells or composition do not induce graft-versus-host disease.

[0057] In some embodiments, the cells or composition do not induce or induce minimal allogeneic reactivity against HLA-mismatched cells in the subject that are positive for CD19 and / or CD20. In some embodiments, the cells or composition do not exhibit or exhibit minimal cytotoxic and / or cytolytic activity against off-target cells in the subject, wherein the off-target cells are negative for CD19 and CD20.

[0058] In some embodiments, the cells are T cells. In some embodiments, the T cells are EBV-sensitized CTLs. In some embodiments, the cells persist in the subject for at least one month or at least two months after initial administration of the cells or composition. In some embodiments, the cells persist in the subject for approximately 40 days after initial administration of the cells or composition. In some embodiments, the cells persist in the subject for approximately 60 days after initial administration of the cells or composition.

[0059] In another aspect, embodiments of the present invention provide a method for treating an autoimmune disease in a subject. In some embodiments, the method includes administering to the subject the cells disclosed herein and / or a pharmaceutical composition comprising the cells disclosed herein.

[0060] In some implementations, autoimmune diseases are selected from the group consisting of: multiple sclerosis, systemic lupus erythematosus (SLE), Sjögren's syndrome, AL amyloidosis, IgG4-related disease (IgG4-RD), inflammatory myopathy (e.g., myositis), scleroderma, pemphigus vulgaris, chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barré syndrome (GBS), stiff-person syndrome, rheumatoid arthritis, neuromyelitis optica spectrum disorder (NMOSD), and myasthenia gravis. Asthenia, N-methyl-D-aspartate receptor (NMDAR) encephalitis, anti-myelin oligodendrocyte glycoprotein (MOG) syndrome, MOGAD (MOGAD) disease, membranous nephropathy (MN), IgA nephropathy (IgAN), type 1 diabetes, anti-neutrophil cytoplasmic antibody (ANCA) vasculitis, thrombocytopenic purpura, hemolytic anemia (AIHA), multifocal motor neuropathy (MMN), inflammatory bowel disease (IBD), celiac disease, Crohn's disease, ulcerative colitis, and ankylosing spondylitis. In some implementations, the autoimmune disease is multiple sclerosis. In some implementations, the autoimmune disease is systemic lupus erythematosus (SLE).

[0061] In some embodiments, the cells or composition exhibit cytotoxic and / or cytolytic activity against target cells in a subject, wherein the target cells express i) detectable levels of CD19 and detectable levels of CD20; ii) detectable levels of CD20 and low or undetectable levels of CD19; or iii) detectable levels of CD19 and low or undetectable levels of CD20. In some embodiments, the target cells are CD19. + CD20 + CD19 + CD20 - Or CD19 - CD20 +In some embodiments, the target cells are B cells. In some embodiments, the B cells are CD19 cells. + CD20 + CD19 + CD20 - Or CD19 - CD20 + In some implementations, the target cells are T cells. In some implementations, the T cells are CD19 cells. + CD20 + CD19 + CD20 - Or CD19 - CD20 + In some implementations, T cells are CD20. + In some embodiments, the cells or composition exhibit cytotoxic and / or cytolytic activity against a first target cell and a second target cell in a subject, wherein the first and second target cells each express i) a detectable level of CD19 and a detectable level of CD20; ii) a detectable level of CD20 and a low or undetectable level of CD19; or iii) a detectable level of CD19 and a low or undetectable level of CD20. In some embodiments, the first and second target cells are each CD19. + CD20 + CD19 + CD20 - Or CD19 - CD20 + In some embodiments, the first target cell is a B cell. In some embodiments, the second target cell is a T cell. In some embodiments, the T cell is a CD20 cell. + In some embodiments, the cytotoxic and / or cytolytic activity is dose-dependent. In some embodiments, the cytotoxic and / or cytolytic activity is long-lasting. In some embodiments, the cytotoxic and / or cytolytic activity persists for at least 24 hours or at least 72 hours after initial administration of the cells or composition. In some embodiments, the cells or composition do not induce graft-versus-host disease.

[0062] In some embodiments, the cells or composition do not induce or induce minimal allotropic reactivity against HLA-mismatched cells in the subject that are positive for CD19 and / or CD20. In some embodiments, the cells or composition do not exhibit or exhibit minimal cytotoxicity and / or cytolytic activity against off-target cells in the subject, wherein the off-target cells are negative for both CD19 and CD20. In some embodiments, the off-target cells are selected from the group consisting of: CD19 - CD20- T cells, natural killer T cells (NK T cells), natural killer cells (NK cells), and combinations thereof. In some embodiments, the cells or composition induce cytokine secretion. In some embodiments, the cytokines are selected from the group consisting of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and combinations thereof. In some embodiments, the cells are T cells. In some embodiments, the T cells are EBV-sensitized CTLs.

[0063] In some embodiments, the subject is a human subject. In some embodiments, the cells are autologous to the subject. In some embodiments, the cells are allogeneic to the subject.

[0064] In some embodiments, the cells or compositions disclosed herein are used in a therapy. In some embodiments, the cells or compositions disclosed herein are used in a method of inhibiting the growth of target cells expressing at least one B lymphocyte antigen. In some embodiments, the cells or compositions disclosed herein are used in a method of treating a subject with a B lymphocyte antigen-related disease. In some embodiments, the cells or compositions disclosed herein are used in a method of treating a subject with a blood cancer. In some embodiments, the cells or compositions disclosed herein are used in a method of treating a subject with an autoimmune disease.

[0065] In another aspect, embodiments of the present invention provide a method for generating one or more cells comprising the CD19 / CD20-targeting CAR disclosed herein. In some embodiments, the method includes introducing the CD19 / CD20-targeting CAR disclosed herein, a nucleic acid encoding the CD19 / CD20-targeting CAR disclosed herein, or a vector disclosed herein into cells. 4. Description of the attached drawings Figure 1 The structures of various bispecific CD19 / CD20-targeting CARs, including the CARs disclosed in this invention, are described. “TM” indicates “transmembrane domain”.

[0067] Figures 2A-2E The CD19 and CD20 specificity activities of the bispecific CD19 / CD20-targeting CARs were characterized in consecutive challenge assays. The “20-19 1XX” CAR or “20-19 BB06z” CAR was transduced and expressed in allogeneic EBV-sensitized CTLs derived from donor 40 using the proprietary manufacturing process MP1. Figure 2A This demonstrates the effect of CAR-T cells on CD19. + CD20 - The result of antigen-specific cell lysis of K562 cells. Figure 2BThis demonstrates the effect of CAR-T cells on CD19. - CD20 + The result of antigen-specific cell lysis of K562 cells. Figure 2C This demonstrates the effect of CAR-T cells on CD19. + CD20 + The result of antigen-specific cell lysis of K562 cells. Figure 2D This demonstrates the effect of CAR-T cells on CD19. 低 CD20 + The result of antigen-specific cell lysis of Raji cells. Figure 2E This demonstrates the effect of CAR-T cells on wild-type (WT) CD19. - CD20 - The results of antigen-specific cell lysis of K562 cells. "401XX" indicates allogeneic EBV-sensitized CTLs expressing the "20-19 1XX" CAR derived from donor 40. "40 BB06z" indicates allogeneic EBV-sensitized CTLs expressing the "20-19 BB06z" CAR derived from donor 40. "40 NTD" indicates non-transduced allogeneic EBV-sensitized CTLs (not expressing CAR) derived from donor 40. "Target Only" indicates cases where no allogeneic EBV-sensitized CTLs were added to the assay, i.e., only target cells were present as a negative control.

[0068] Figure 3 The experimental setup for an in vivo xenograft mouse model with Burkitt lymphoma (Raji cells) was described.

[0069] Figures 4A-4C This study depicts the in vivo tumor growth inhibition of 20-19 1XX CAR-T cells and 20-19 BB06z CAR-T cells in a therapeutic model of Burkitt lymphoma (Raji cells). Mice received 3 × 10⁻⁶ CAR-T cells. 6 One CAR-T cell. Figure 4A The tumor burden of mice receiving non-transduced allogeneic EBV-sensitized CTLs (not expressing CAR) derived from donor 40 is shown. Figure 4B It shows that it accepts 3×10 6 Tumor burden in mice sensitized with allogeneic EBV and expressing “20-19 BB06z” CAR derived from donor 40. Figure 4C It shows that it accepts 3×10 6Tumor burden of mice with allogeneic EBV-sensitized CTLs expressing the “20-19 1XX” CAR derived from donor 40. PBS was used as a negative control. “40 1XX” indicates an allogeneic EBV-sensitized CTL expressing the “20-19 1XX” CAR derived from donor 40. “40 BB06z” indicates an allogeneic EBV-sensitized CTL expressing the “20-19 BB06z” CAR derived from donor 40. “40 NTD” indicates a non-transduced allogeneic EBV-sensitized CTL (not expressing CAR) derived from donor 40.

[0070] Figure 5A and 5B The association between CAR-T cell expansion and overall survival in treated mice was depicted. Figure 5A It showed in vivo CAR-T amplification. Figure 5B It shows that it accepts 3×10 6 Overall survival of CAR-T cells in treated mice. "401XX" indicates allogeneic EBV-sensitized CTLs expressing the "20-19 1XX" CAR derived from donor 40. "40 BB06z" indicates allogeneic EBV-sensitized CTLs expressing the "20-19 BB06z" CAR derived from donor 40. "40 NTD" indicates non-transduced allogeneic EBV-sensitized CTLs (not expressing CAR) derived from donor 40. "Tumor only" indicates cases where no allogeneic EBV-sensitized CTLs were added to the assay, i.e., only target cells were present as a negative control.

[0071] Figures 6A-6E The CD19 and CD20 specificity activities of the bispecific CD19 / CD20-targeting CARs were characterized in consecutive challenge assays. The “20-19 1XX” CAR or “20-19 BB06z” CAR was transduced and expressed in allogeneic EBV-sensitized CTLs derived from donor 14 using the proprietary manufacturing process MP2. Figure 6A This demonstrates the effect of CAR-T cells on CD19. + CD20 - The result of antigen-specific cell lysis of K562 cells. Figure 6B This demonstrates the effect of CAR-T cells on CD19. - CD20 + The result of antigen-specific cell lysis of K562 cells. Figure 6C This demonstrates the effect of CAR-T cells on CD19. + CD20 + The result of antigen-specific cell lysis of K562 cells. Figure 6D This demonstrates the effect of CAR-T cells on CD19. 低 CD20+ The result of antigen-specific cell lysis of Raji cells. Figure 6E This demonstrates the effect of CAR-T cells on wild-type (WT) CD19. - CD20 - The results of antigen-specific cell lysis of K562 cells. "141XX" indicates allogeneic EBV-sensitized CTLs expressing the "20-19 1XX" CAR derived from donor 14. "14 BB06z" indicates allogeneic EBV-sensitized CTLs expressing the "20-19 BB06z" CAR derived from donor 14. "14 NTD" indicates non-transduced allogeneic EBV-sensitized CTLs (not expressing CAR) derived from donor 14. "Target Only" indicates cases where no allogeneic EBV-sensitized CTLs were added to the assay, i.e., only target cells were present as a negative control.

[0072] Figures 7A-7E The CD19 and CD20 specific activities of the bispecific CD19 / CD20-targeting CARs were characterized in consecutive challenge assays. The “20-19 1XX” CAR or “20-19 BB06z” CAR was transduced and expressed in allogeneic EBV-sensitized CTLs derived from donor 40 using the proprietary manufacturing process MP2. Figure 7A This demonstrates the effect of CAR-T cells on CD19. + CD20 - The result of antigen-specific cell lysis of K562 cells. Figure 7B This demonstrates the effect of CAR-T cells on CD19. - CD20 + The result of antigen-specific cell lysis of K562 cells. Figure 7C This demonstrates the effect of CAR-T cells on CD19. + CD20 + The result of antigen-specific cell lysis of K562 cells. Figure 7D This demonstrates the effect of CAR-T cells on CD19. 低 CD20 + The result of antigen-specific cell lysis of Raji cells. Figure 7E This demonstrates the effect of CAR-T cells on wild-type (WT) CD19. - CD20 -The results of antigen-specific cell lysis of K562 cells. "401XX" indicates allogeneic EBV-sensitized CTLs expressing the "20-19 1XX" CAR derived from donor 40. "40 BB06z" indicates allogeneic EBV-sensitized CTLs expressing the "20-19 BB06z" CAR derived from donor 40. "40 NTD" indicates non-transduced allogeneic EBV-sensitized CTLs (not expressing CAR) derived from donor 40. "Target Only" indicates cases where no allogeneic EBV-sensitized CTLs were added to the assay, i.e., only target cells were present as a negative control.

[0073] Figure 8 The structure of the baseline CD19 / CD20-targeted CAR tested in Example 4 is depicted.

[0074] Figure 9 The experimental setup for an in vivo xenograft model with Burkitt lymphoma (Raji cells) was described.

[0075] Figures 10A-10C The antitumor efficacy of allogeneic EBV-sensitized CTLs expressing “20-19 1XX” CAR and autologous T cells expressing baseline CD19 / CD20-targeting CARs in attacking an in vivo lymphoma model was described. Figure 10A The tumor burden of mice that received the tested T cells is shown. “CD20 / 19 Autologous Benchmark” indicates autologous T cells expressing the benchmark CD19 / CD20-targeted CAR described in Example 4. Figure 10B The blood cell counts of mice treated with CAR-T cells are shown. Figure 10C The number of vector copies per cell (VCN) for CAR-T cells is shown.

[0076] Figure 11A-11C The CAR expression and T cell phenotype of allogeneic EBV-sensitized T cells expressing the “20-19 1XX” CAR were depicted. The “20-19 1XX” CAR T cells were generated from three different donors. Figure 11A The results show CAR expression and vector copy number (VCN) as analyzed by flow cytometry. Figure 11B The CD4 and CD8 distributions in the product “20-19 1XX” are shown. Figure 11C Flow cytometry analysis of memory markers CD45RO and CD62L is shown. Bars represent the standard error of the mean.

[0077] Figure 12A and 12BThe CD19 and CD20-specific activities of “20-19 1XX” CAR T cells in serial challenge assays were characterized. This was achieved against various non-Hodgkin lymphoma (NHL) cell lines expressing CD19 and CD20 (including Jeko-1). Figure 12A ) and Su-DHL-4 ( Figure 12B Using a 5:1 effector (E) CAR+): The target (T) (E:T) ratio was measured using continuous stimulation. The vertical dashed line represents repeated continuous stimulation.

[0078] Figure 13A and 13B The allogeneic reactivity of "20-19 1XX" CAR T cells was described. Regarding T cells and CD69... + / 4-1 BB + T cells, HLA matching (auto) and non-matching (allo) CD20 + / CD19 + Cell lysis of the target B lymphoblast-like cell line (BLCL) was used to test the allogeneic reaction potential of “20-19 1XX”. Figure 13A The activation of T cells was shown after two days of co-culturing with “20-19 1XX” CAR T cells. Figure 13B BLCL cell lysis is shown after two days of co-culture with “20-19 1XX” CAR T cells. Maintenance of “20-19 1XX” EBV specificity was assessed using flow cytometry. Target cell lysis was measured using xCELLigence RTCA. Error bars represent SD. The symbol “****” indicates successful two-way ANOVA (P < 0.0001).

[0079] Figures 14A-14E The in vivo antitumor activity of “20-19 1XX” CAR T cells was described in a dose study. Figure 14A The injection of 1×10 showed 6 Tumor burden of mice with “20-19 1XX” or NTD EBV T cells. Figure 14B The injection of 3×10 showed 6 Tumor burden of mice with “20-19 1XX” or NTD EBV T cells. Figure 14C The injection of 9×10 showed 6 Tumor burden in mice with “20-191XX” or NTD EBV T cells. Vertical dashed lines indicate injection. Tumor burden was measured by bioluminescence imaging (BLI). Figure 14D The loop of human hCD45 was displayed. + Absolute count of T cells. Figure 14EThe measurement of the carrier copy number is displayed.

[0080] Figures 15A-15H The study depicted "20-19 1XX" CAR T cells at 3 × 10⁶ cells per mouse. 6 The in vivo antitumor activity of CAR T cells at a dose of 1.5 × 10⁵ was measured. 6 Raji CD19 低 Geometric mean of BLI in NSG mice (cells) Figure 15A ), mean absolute count of circulating human T cells ( Figure 15B ), average VCN ( Figure 15C ) and survival rate ( Figure 15D ). Measurement of intravenous implantation of 0.5 × 10 6 Raji CD19 高 Geometric mean of BLI in NSG mice (cells) Figure 15E ), mean absolute count of circulating human T cells ( Figure 15F ), average VCN ( Figure 15G ) and survival rate ( Figure 15H Vertical dashed lines indicate injection. Tumor burden is measured by BLI. Error bars represent SD. * indicates P < 0.1, and ** indicates P < 0.01. One outlier from “20-19 1XX” and NTD is excluded from the geometric mean calculation. Log-rank tests are performed separately for “20-19 1XX”.

[0081] Figure 16 The experimental setup for single-shot kill determination is described.

[0082] Figures 17A-17D The CD19 and CD20 specific activities of the bispecific CD19 / CD20-targeted CAR were depicted in a single kill assay. Figure 17A This demonstrates the effect of CAR-T cells on CD19. + CD20 - The result of antigen-specific cell lysis of K562 cells. Figure 17B This demonstrates the effect of CAR-T cells on CD19. - CD20 + The result of antigen-specific cell lysis of K562 cells. Figure 17C This demonstrates the effect of CAR-T cells on CD19. + CD20 + The result of antigen-specific cell lysis of K562 cells. Figure 17D This demonstrates the effect of CAR-T cells on wild-type (WT) CD19. - CD20 -The result of antigen-specific cell lysis of K562 cells. "20-19 1XX" indicates allogeneic EBV-sensitized T cells from donor 104 expressing "20-19 1XX" CAR. "NTD" indicates non-transduced allogeneic EBV-sensitized T cells (not expressing CAR) from donor 104.

[0083] Figure 18 The experimental setup for single-shot kill determination was described.

[0084] Figures 19A-19C The CD19 and CD20 specific activities of the bispecific CD19 / CD20-targeting CAR were depicted in a series of challenge assays. CAR-T cells and target cells were used at a 5:1 effector ratio (E... CAR+): Target (T) ratio co-culture. The “20-19 1XX” CAR was transduced and expressed in allogeneic EBV-sensitized T cells derived from donor 14 or donor 23. Figure 19A This demonstrates the effect of CAR-T cells on knockout (KO) CD19. - The result of antigen-specific cell lysis of Raji cells. Figure 19B This demonstrates the effect of CAR-T cells on CD19. 低 The result of antigen-specific cell lysis of Raji cells. Figure 19C This demonstrates the effect of CAR-T cells on CD19. 高 The results of antigen-specific cell lysis of Raji cells. "14 20-19 1XX" indicates allogeneic EBV-sensitized T cells expressing the "20-19 1XX" CAR derived from donor 14. "23 20-19 1XX" indicates allogeneic EBV-sensitized T cells expressing the "20-19 1XX" CAR derived from donor 23. "14 NTD" indicates non-transduced allogeneic EBV-sensitized T cells (not expressing CAR) derived from donor 14. "23 NTD" indicates non-transduced allogeneic EBV-sensitized T cells (not expressing CAR) derived from donor 23. "Target Only" indicates cases where no allogeneic EBV-sensitized T cells were added to the assay, i.e., only target cells were present as a negative control.

[0085] Figures 20A-20C The CD19 and CD20 specific activities of the bispecific CD19 / CD20-targeting CAR were depicted in a series of challenge assays. CAR-T cells and target cells were used at a 1:1 effector ratio (E... CAR+): Target (T) ratio co-culture. The “20-19 1XX” CAR was transduced and expressed in allogeneic EBV-sensitized T cells derived from donor 14 or donor 23. Figure 20A This demonstrates the effect of CAR-T cells on knockout (KO) CD19.- The result of antigen-specific cell lysis of Raji cells. Figure 20B This demonstrates the effect of CAR-T cells on CD19. 低 The result of antigen-specific cell lysis of Raji cells. Figure 20C This demonstrates the effect of CAR-T cells on CD19. 高 The results of antigen-specific cell lysis of Raji cells. "14 20-19 1XX" indicates allogeneic EBV-sensitized T cells expressing the "20-19 1XX" CAR derived from donor 14. "23 20-19 1XX" indicates allogeneic EBV-sensitized T cells expressing the "20-19 1XX" CAR derived from donor 23. "14 NTD" indicates non-transduced allogeneic EBV-sensitized T cells (not expressing CAR) derived from donor 14. "23 NTD" indicates non-transduced allogeneic EBV-sensitized T cells (not expressing CAR) derived from donor 23. "Target Only" indicates cases where no allogeneic EBV-sensitized T cells were added to the assay, i.e., only target cells were present as a negative control.

[0086] Figures 21A-21D The CD19 and CD20 specific activities of bispecific CD19 / CD20-targeting CARs were characterized in serial challenge assays against various NHL cell lines. CAR-T cells and target cells were compared at a 1:1 effector ratio (E... CAR+): Target (T) ratio co-culture. The “20-19 1XX” CAR was transduced and expressed in allogeneic EBV-sensitized T cells derived from donor 14 or donor 104. Figure 21A The results show the antigen-specific cell lysis of Raji cells (Burkit lymphoma) by CAR-T cells. Figure 21B The results show the antigen-specific cell lysis of Jeko-1 cells (mantle cell lymphoma (MCL)) by CAR-T cells. Figure 21C The results show the antigen-specific cell lysis of Su-DHL-4 cells (diffuse large B-cell lymphoma (DLBCL)) by CAR-T cells. Figure 21DThis diagram shows the results of antigen-specific cell lysis of MEC-1 cells (chronic lymphocytic leukemia (CLL)) by CAR-T cells. "14 20-19 1XX" indicates allogeneic EBV-sensitized T cells expressing the "20-19 1XX" CAR derived from donor 14. "104 20-19 1XX" indicates allogeneic EBV-sensitized T cells expressing the "20-19 1XX" CAR derived from donor 104. "14 NTD" indicates untransduced allogeneic EBV-sensitized T cells (not expressing CAR) derived from donor 14. "104 NTD" indicates untransduced allogeneic EBV-sensitized T cells (not expressing CAR) derived from donor 104. "Target Only" indicates cases where no allogeneic EBV-sensitized T cells were added to the assay, i.e., only target cells were present as a negative control.

[0087] Figures 22A-22D This study describes the antitumor efficacy of allogeneic EBV-sensitized T cells expressing the “20-19 1XX” CAR in attacking an in vivo Burkitt lymphoma model (Raji cells). “14 20-19 1XX” represents allogeneic EBV-sensitized T cells expressing the “20-19 1XX” CAR derived from donor 14. “14 NTD” represents untransduced allogeneic EBV-sensitized T cells (without CAR expression) derived from donor 14. “23 20-19 1XX” represents allogeneic EBV-sensitized T cells expressing the “20-19 1XX” CAR derived from donor 23. “23 NTD” represents untransduced allogeneic EBV-sensitized T cells (without CAR expression) derived from donor 23. Figure 22A The tumor burden of mice that underwent the test with T cells is shown. Figure 22B The blood cell counts of mice treated with CAR-T cells are shown. Figure 22C The number of vector copies per cell (VCN) for CAR-T cells is shown. Figure 22D No serious toxicity was detected in mice treated with the tested CAR T cells.

[0088] Figure 23 The experimental setup for an in vivo xenograft model with mantle cell lymphoma (MCL) (Jeko-1 cells) was described.

[0089] Figures 24A-24D The dose-dependent antitumor efficacy of allogeneic EBV-sensitized T cells expressing the “20-19 1XX” CAR in attacking an in vivo mantle cell lymphoma (MCL) model (Jeko-1 cells) was described. Figure 24A The tumor burden of mice that received the CAR-T cells for testing is shown. Figure 24BThe blood cell counts of mice treated with the tested CAR-T cells are shown. Figure 24C The number of vector copies per cell (VCN) for the tested CAR-T cells is shown. Figure 24D No serious toxicity was detected in mice treated with the tested CAR T cells.

[0090] Figure 25A and 25B Characterization of peripheral blood mononuclear cells (PBMCs) from multiple sclerosis (MS) donors was described. Figure 25A CD20 from the PBMC of the MS donor is shown. + The percentage of T cells. Figure 25B CD19 from the PBMC of the MS donor is shown. + / CD20 + Percentage of B cells. **<0.005 and *<0.05 were obtained using two-factor ANOVA.

[0091] Figure 26 An experimental setup for an in vitro cytotoxicity assay of multiple sclerosis (MS) B cells was described.

[0092] Figures 27A-27C The dose-dependent cytotoxic activity of T cells containing “20-19 1XX” CAR against MS donor B cells was depicted after 24 hours. Figure 27A The study demonstrated the cytotoxicity of CAR-T cells against B cells derived from MS donor A. Figure 27B The study demonstrated the cytotoxicity of CAR-T cells against B cells derived from MS donor B. Figure 27C The study demonstrated the cytotoxicity of CAR-T cells against B cells derived from MS donor C.

[0093] Figures 28A-28D The cytotoxic activity of T cells containing “20-19 1XX” CAR against MS donor B cells was depicted over 72 hours. Figure 28A The study demonstrated the cytotoxicity of CAR-T cells against B cells derived from MS donor A. Figure 28B The study demonstrated the cytotoxicity of CAR-T cells against B cells derived from MS donor B. Figure 28C The study demonstrated the cytotoxicity of CAR-T cells against B cells derived from MS donor C. Figure 28D The study showed the cytotoxicity of CAR-T cells against B cells from MS donors A, B, and C, as measured at 48 hours.

[0094] Figure 29A-29I The study showed that T cells containing “20-19 1XX” lacked off-target cytotoxic activity against non-B cells derived from MS donor PBMCs. Figure 29A , 29D 29G showed that CAR-T cells lacked off-target cell lysis against non-B cells of donor A. Figure 29B , 29E The results showed that CAR-T cells lacked off-target cell lysis against non-B cells of donor B within 72 hours. Figure 29C , 29F 29I showed that CAR-T cells lacked off-target cell lysis against non-B cells of donor C within 48 hours.

[0095] Figure 30A and 30B The study depicted the response of "20-19 1XX" CAR T cells to CD20 from PBMCs derived from MS donors. + T cell cytolytic activity. Figure 30A The results showed that CAR-T cells targeted CD20 from MS donors A, B, and C at 48 hours. + T-cell cytotoxicity. Figure 30B The results showed that CAR-T cells targeted CD20 in MS donors A and B at 72 hours. + T cell cytotoxicity. Error bars represent standard deviation (SD). Through two-way ANOVA, **** < 0.0001, *** = 0.0001, and ** < 0.005.

[0096] Figure 31A and 31B The secretion of IFN-γ and TNF-α by T cells containing “20-19 1XX” CARs co-cultured with PBMCs from MS donors was described. Figure 31A The secretion of IFN-γ and TNF-α was measured over 24 hours via a flow cytometry bead array. Figure 31B The secretion of IFN-γ and TNF-α, measured by flow cytometry via a microbead array, is shown. Error bars represent SD. By two-way ANOVA, ***=0.0001.

[0097] Figures 32A-32H The proliferation of T cells containing “20-19 1XX” CARs co-cultured with PBMCs from MS donors was depicted. Figure 32A and 32E The proliferation of donor 14 "20-19 1XX" CAR T cells and donor 23 "20-19 1XX" CAR T cells co-cultured with PBMCs from MS donor A for 48 hours is shown. Figure 32B and 32FThe proliferation of donor 14"20-19 1XX" CAR T cells and donor 23"20-19 1XX" CAR T cells co-cultured with PBMCs from MS donor B for 72 hours is shown. Figure 32C and 32G The proliferation of donor 14"20-19 1XX" CAR T cells and donor 23"20-19 1XX" CAR T cells co-cultured with PBMCs from MS donor C for 72 hours is shown. Figure 32D and 32H The proliferation of donor 14"20-19 1XX" CAR T cells and donor 23"20-19 1XX" CAR T cells co-cultured with NTD EBV T cells for 72 hours was shown.

[0098] Figure 33 An experimental setup for an in vitro cytotoxicity assay of systemic lupus erythematosus (SLE) B cells was described.

[0099] Figures 34A-34F The cytotoxic activity of T cells containing “20-19 1XX” CAR against B cells from SLE donors was described. Figure 34A The antigen-specific cell lysis of T cells containing the “20-19 1XX” CAR is shown. Figure 34B and 34C The cell count of CTV+ effector T cells is shown. Figures 34D to 34F The image shows CD56 in PBMCs of SLE donors as detected by flow cytometry. + NK cells ( Figure 34D CD56 + / CD3 + NK T cells ( Figure 34E ) and CD3 + T cells ( Figure 34F Cell count.

[0100] Figure 35 The study describes the allogeneic responsiveness of allogeneic EBV-sensitized T cells expressing the “20-19 1XX” CAR to an HLA-mismatched target. 5. Detailed Implementation Various embodiments of the present invention provide CARs that target both CD19 and CD20 (referred to as "bispecific CD19 / CD20-targeted CARs"). Various embodiments of the present invention also provide cells comprising such bispecific CARs. According to one or more embodiments, the cells may be immune-responding cells and / or immune-effect cells, such as genetically modified immune-responding cells (e.g., T cells or NK cells), or stem cells (e.g., pluripotent stem cells) that can differentiate into immune-responding cells and / or immune-effect cells.

[0102] The subject matter disclosed in this invention is based at least on the following findings: The bispecific CD19 / CD20-targeting CAR disclosed in this invention has a novel and unique structure, including but not limited to a G4S linker connecting the anti-CD19 antigen-binding domain and the anti-CD20 antigen-binding domain, and a linker connecting the anti-CD19 heavy chain variable region (V H ) and anti-CD19 light chain variable region (V L The Whitlow linker, along with the intracellular domain containing the modified CD3ζ signaling domain and the CD28 co-stimulatory signaling region, exhibited superior antitumor efficacy both in vitro and in vivo compared to other bispecific CARs targeting CD19 / 20 with different structures.

[0103] Non-limiting embodiments of this disclosure are described in this specification and examples.

[0104] For the purpose of clarity and transparency, rather than through restriction, the detailed description is divided into the following sections: 5.1. Definition; 5.2. B lymphocyte antigen; 5.3. Chimeric antigen receptor (CAR); 5.4. Cells; 5.5. Nucleic acids and vectors; 5.6. Preparation and application; and 5.7. How to use.

[0105] 5.1. definition Unless otherwise defined, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the subject matter of this invention pertains.

[0106] As used herein, the term "about" or "approximately" refers to an acceptable margin of error for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to practice in the art, "about" may mean within three or more standard deviations. Alternatively, "about" may mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and even more preferably up to 1% of a given value. Alternatively, particularly for biological systems or processes, the term may mean within an order of magnitude of the value, preferably within five times, more preferably within two times.

[0107] As used herein, the term "immune response cell" refers to a cell or its progenitor or progeny that plays a role in an immune response. In some embodiments, the immune response cell is a lymphoid lineage cell. Non-limiting examples of lymphoid lineage cells include T cells, natural killer (NK) cells, B cells, and stem cells from which lymphocytes can differentiate. In some embodiments, the immune response cell is a myeloid lineage cell.

[0108] As used herein, the term "activated immune response cells" refers to the induction of signal transduction or alteration of protein expression in cells, thereby initiating an immune response. For example, a signal transduction cascade is generated when the CD3 chain aggregates in response to ligand binding and immunoreceptor tyrosine-based inhibitory motifs (ITAMs). In some embodiments, when a CAR binds to an antigen, the formation of an immune synapse occurs, which includes binding to receptors (e.g., CD4 or CD8, CD3). Numerous molecules aggregate near the CD3 chain. This aggregation of membrane-bound signaling molecules allows the ITAM motif contained within the CD3 chain to be phosphorylated. This phosphorylation, in turn, initiates T cell activation pathways, ultimately activating transcription factors such as NF-κB and AP-1. These transcription factors induce global gene expression on T cells to increase IL-2 production, thereby proliferating and expressing master-regulating T cell proteins to initiate T cell-mediated immune responses.

[0109] As used herein, the term "stimulated immune response cells" refers to a signal that results in a robust and sustained immune response. In some embodiments, this occurs after activation of immune response cells (e.g., T cells) or is mediated concurrently by receptors, including but not limited to CD28, 4-1BB, OX40, CD40, and ICOS. Receiving multiple stimulating signals can be important for generating robust and long-lasting T cell-mediated immune responses. T cells can rapidly become suppressed and unresponsive to antigens. While the effects of these co-stimulating signals may differ, they generally lead to increased gene expression to produce long-lived, proliferative, and anti-apoptotic T cells that respond strongly to antigens for complete and sustained eradication.

[0110] As used herein, the term "antigen-binding fragment" refers to any derivative of an antibody that is less than full-length. In exemplary embodiments, the antigen-binding fragment retains at least a substantial portion of the specific binding ability of the full-length antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab')2, scFv, Fv, dsFv biantibodies, Fc, and Fd fragments. Antigen-binding fragments can be generated by any means. For example, antigen-binding fragments can be enzymatically or chemically generated by fragmentation of a complete antibody, can be generated by genetic recombination encoding a portion of the antibody sequence, or can be generated entirely or partially synthetically. Antigen-binding fragments may optionally be single-chain antibody fragments. Alternatively, the fragment may comprise, for example, multiple chains linked together by disulfide bonds. The fragment may also optionally be a multi-molecular complex.

[0111] As used herein, the term "Fab fragment" refers to an antibody fragment containing an antigen-binding site produced by cleaving an antibody with papain, which cleaves at the N-terminus of the inter-heavy chain disulfide bond in the hinge region and produces two Fab fragments from one antibody molecule.

[0112] As used herein, the term "F(ab')2 fragment" refers to an antibody fragment containing two antigen-binding sites, which is produced by cleaving an antibody molecule with pepsin at the C-terminus of the inter-heavy chain disulfide bond in the hinge region.

[0113] As used herein, the term "Fc fragment" refers to an antibody fragment containing the heavy chain constant domain of the antibody.

[0114] As used herein, the term "Fv fragment" refers to an antibody fragment containing variable domains of both the heavy and light chains of the antibody.

[0115] The term "single-chain variable fragment" or "scFv" refers to a variable fragment (Fv) in which a heavy chain domain is linked to a light chain domain. The heavy chain and light chain domains are joined directly or via a linker that connects the N-terminus of the heavy chain domain to the C-terminus of the light chain domain, or vice versa. As used herein, the term "linker" is recognized in the art and refers to a molecule or group of molecules that connects two compounds, such as two polypeptides. A linker may consist of a single linker molecule or may contain a linker molecule and a spacer molecule designed to separate the linker molecule and the compound at a specific distance. Linkers are typically enriched with glycine for flexibility and serine or threonine for solubility.

[0116] In some embodiments, the adapter is a Whitlow adapter. In some embodiments, the adapter comprises or consists of the amino acid sequence shown in SEQ ID NO: 1 provided below.

[0117] GSTSGSGKPGSGEGSTKG[SEQ ID NO: 1] In some embodiments, the adapter is a G4S adapter. In some embodiments, the adapter comprises or consists of the amino acid sequence shown in SEQ ID NO: 2 provided below.

[0118] GGGGSGGGGSGGGGS [SEQ ID NO: 2] In some embodiments, the connector comprises or consists of the amino acid sequence shown in SEQ ID NO: 3 provided below.

[0119] GGGGSGGGGSGGGGSGGGGSGGGGS [SEQ ID NO: 3] In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 4 provided below.

[0120] GGCTCCACGAGCGGCAGCGGCAAACCCGGCAGCGGCGAGGGCAGCACCAAGGGC[SEQ ID NO: 4] In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 2 is shown in SEQ ID NO: 5 provided below.

[0121] GGTGGAGGGGGCTCTGGCGGTGGGGGGTCCGGCGGTGGCGGATCT [SEQ ID NO: 5] In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 3 is shown in SEQ ID NO: 6 provided below.

[0122] GGCGGTGGCGGGTCTGGCGGCGGTGGATCCGGTGGTGGTGGATCTGGAGGAGGGGGCTCCGGGGGCGGTGGCAGC [SEQ ID NO: 6] As used herein, the "complementarity-determining region" or "CDR" is defined as the amino acid sequence of the complementarity-determining region of an antibody, which is a hypervariable region of the heavy and light chains of an immunoglobulin. CDRs can be identified according to many known numbering systems. In some implementations, CDRs are identified according to the Kabat numbering system. The Kabat CDR is based on sequence variability and is the most commonly used (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). CDRs can also be identified according to the Chothia numbering system. Chothia refers to the location of a structural loop (see, for example, Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). CDRs can also be identified according to the AbM numbering system. The AbM hypervariable region represents a compromise between the Kabat CDR and the Chothia structural ring and is used by the AbM antibody modeling software from Oxford Molecular (see, for example, Martin, in Antibody Engineering, Vol. 2, Chapter 3, Springer Verlag). CDRs can be identified according to the Contact numbering system (see, for example, MacCallum RM et al., 1996, J MolBiol 5: 732-745). Contact CDRs are based on the analysis of available complex crystal structures. CDRs can be determined according to ImMunoGeneTics (IMGT). ® Information systems are used for identification. A universal numbering system, IMGT, has been developed and widely adopted. ® Information Systems (Lefranc et al., Dev. Comp. Immunol. 27(1):55-77 (2003)). IMGT ® It is an integrated information system specifically designed for the study of immunoglobulins (IG), T cell receptors (TR), and major histocompatibility complexes (MHC) in humans and other vertebrates.

[0123] The term "amino acid sequence" refers to a list of abbreviations, letters, characters, or words representing amino acid residues. The amino acid abbreviations used in this article are the standard single-letter codes for amino acids and are represented as follows: A, alanine; B, asparagine or aspartic acid; C, cysteine; D, aspartic acid; E, glutamate, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; Y, tyrosine; Z, glutamine or glutamic acid.

[0124] The terms “peptide,” “protein,” and “polypeptide” are used interchangeably to refer to a natural or synthetic molecule containing two or more amino acids linked by the carboxyl group of one amino acid to the α-amino group of another amino acid.

[0125] When used to refer to a specific polypeptide, the term "peptide fragment" or "fragment" refers to a polypeptide lacking amino acid residues compared to the reference polypeptide itself, but whose remaining amino acid sequence is generally identical to that of the reference polypeptide. Such deletions can occur at the N-terminus or C-terminus of the reference polypeptide, or both. Fragments are typically at least about 5, 6, 8, or 10 amino acids long, at least about 14 amino acids long, at least about 20, 30, 40, or 50 amino acids long, at least about 75 amino acids long, or at least about 100, 150, 200, 300, 500, or more amino acids long. Fragments may retain one or more biological activities of the reference polypeptide. In various embodiments, the fragment may contain enzymatic activities and / or interaction sites of the reference polypeptide. In some embodiments, the fragment may have immunogenic properties.

[0126] As used herein, the terms "substantially identical" or "substantially homologous" refer to a polypeptide or nucleic acid molecule that exhibits at least about 50% identity or homology with a reference amino acid sequence (e.g., any amino acid sequence described herein) or a reference nucleotide sequence (e.g., any nucleotide sequence described herein). In some embodiments, such a sequence is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or at least about 100% identity or homology with the amino acid or nucleotide sequence used for comparison.

[0127] The term "identity" refers to the sequence identity between two nucleic acid molecules or polypeptides. Identity can be determined by comparing positions in each sequence, which can be aligned for comparative purposes. When positions in the compared sequences are occupied by the same bases, the molecules are identical at that position. The degree of similarity or identity between nucleic acid or amino acid sequences depends on the number of identical or matching nucleotides at shared positions in the nucleotide sequences. Various alignment algorithms and / or procedures can be used to calculate the identity between two sequences, including FASTA or BLAST, which are available as part of the GCG sequence analysis package and can be used, for example, with default settings. When using BLASTP, the similarity percentage is based on the BLASTP positive score, and the sequence identity percentage is based on the BLASTP identity score. BLASTP "identity" shows the number and score of the total number of identical residues in high-scoring sequence pairs; and BLASTP "positive" shows the number and score of residues with positive alignment scores that are similar to each other. This disclosure considers and covers amino acid sequences that have these degrees of identity or similarity or any intermediate degree of identity or similarity with the amino acid sequences disclosed herein. The polynucleotide sequences of similar polypeptides are deduced using the genetic code and can be obtained by conventional methods, particularly by reverse translating their amino acid sequences using the genetic code.

[0128] The term "nucleic acid" refers to a natural or synthetic molecule that contains a single nucleotide or two or more nucleotides linked to the 5' end of another nucleotide by a phosphate group at the 3' position of one nucleotide. Nucleic acids are not limited in length, and therefore can include deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).

[0129] The term "operably linked to" refers to a functional relationship between one nucleic acid and another. Promoters, enhancers, transcription and translation termination sites, and other signaling sequences are examples of nucleotide sequences that are operably linked to other sequences. For example, the operable linking of DNA to a transcriptional control element refers to the physical and functional relationship between DNA and a promoter, such that transcription of this DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to, and transcribes this DNA.

[0130] As used herein, the term “signal sequence” or “leader sequence” refers to a peptide sequence (e.g., 5, 10, 15, 20, 25, or 30 amino acids) present at the N-terminus of a protein that guides the protein into the secretion pathway.

[0131] As used herein, the term "conserved sequence modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of the bispecific CD19 / CD20-targeted CAR disclosed in this invention. Conserved modifications can include amino acid substitutions, additions, and deletions. Modifications can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Amino acids can be classified according to their physicochemical properties, such as charge and polarity. A conserved amino acid substitution is a substitution in which an amino acid residue is replaced by an amino acid within the same group. For example, amino acids can be classified by charge: positively charged amino acids include lysine, arginine, and histidine; negatively charged amino acids include aspartic acid and glutamic acid; and neutrally charged amino acids include alanine, asparagine, cysteine, glutamine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine. Furthermore, amino acids can be classified by polarity: polar amino acids include arginine (basic polar), asparagine, aspartic acid (acidic polar), glutamic acid (acidic polar), glutamine, histidine (basic polar), lysine (basic polar), serine, threonine, and tyrosine; nonpolar amino acids include alanine, cysteine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. Therefore, one or more amino acid residues within the CDR region can be replaced with other amino acid residues from the same group, and the retained function of the altered antibody can be tested using the functional assays described herein. In some embodiments, no more than one, two, three, four, or five residues within the specified sequence or CDR region may be altered.

[0132] The term "vector" refers to a nucleic acid that can transport another nucleic acid linked to a vector sequence into a cell. The term "expression vector" includes any vector (e.g., plasmid, granulosome, or phage chromosome) that contains a gene construct (e.g., linked to a transcriptional control element) in a form suitable for expression by cells.

[0133] The term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that, within reasonable medical judgment, are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit / risk ratio.

[0134] The term "treatment" refers to the medical administration of a subject with the aim of curing, improving, stabilizing, or preventing a disease, pathological condition, or symptom. This term includes active therapy, which is treatment specifically designed to improve a disease, pathological condition, or symptom, and also includes etiological therapy, which is treatment aimed at eliminating the cause of the disease, pathological condition, or symptom. Furthermore, this term includes palliative care, which is treatment designed to relieve symptoms rather than cure a disease, pathological condition, or symptom; preventative care, which is treatment aimed at minimizing or partially or completely inhibiting the development of a disease, pathological condition, or symptom; and supportive care, which is treatment used to complement another specific therapy aimed at improving a disease, pathological condition, or symptom.

[0135] In this document, "individual" or "subject" refers to a vertebrate, such as a human, or a non-human animal, such as a mammal. Mammals include, but are not limited to, humans, primates, farm animals, treadmills, rodents, and pets. Non-limiting examples of non-human animal subjects include rodents such as mice, rats, hamsters and guinea pigs, rabbits, dogs, cats, sheep, pigs, goats, cattle, horses, and non-human primates such as apes and monkeys. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human.

[0136] As used herein, the term "B lymphocyte antigen" refers to one or both of CD-19 and CD20. Furthermore, the term "B lymphocyte antigen," particularly "CD19" or "CD20," is intended to include fragments, variants (e.g., allele variants), and derivatives of antigen molecules (e.g., CD19 or CD20 molecules). For example, but not limited to, in some embodiments, CD19 is wild-type CD19 or mutant CD19. For example, but not limited to, in some embodiments, CD20 is wild-type CD20 or mutant CD20. In some such embodiments, the B lymphocyte antigen is expressed on the cell surface (e.g., on the surface of precancerous or malignant cells). Cells expressing the B lymphocyte antigen may include, but are not limited to, B cells, T cells, and tumor cells (e.g., cancer cells, such as hematologic malignancies). In some embodiments, the cells expressing the B lymphocyte antigen are B cells. In some embodiments, the cells expressing the B lymphocyte antigen are T cells. In some embodiments, the cells expressing the B lymphocyte antigen are tumor cells. In some embodiments, the cells expressing the B lymphocyte antigen are cancer cells. In some implementations, the cells expressing B lymphocyte antigens are blood cancer cells.

[0137] Anti-CD19 antibodies (and their scFv forms) suitable for binding CD19 are well known in the art, and include, for example, but not limited to, antibodies FMC63, SJ25C1 (JCAR015) and HD37 (Bonatumab).

[0138] Anti-CD20 antibodies (and their scFv forms) suitable for binding CD20 are well known in the art and include, for example, but not limited to, antibodies Leu16, rituximab, oxotuzumab, oxofamumab, and TG-1101 (ublituximab).

[0139] The term “comprises / comprising” is intended to have the broad meaning given to them under U.S. patent law and can mean “including”, “containing”, etc.

[0140] 5.1.1. By incorporating through reference.

[0141] Various patents, patent applications, publications, product specifications, schemes and serial numbers are cited throughout this application, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0142] 5.2. B lymphocyte antigen 5.2.1 CD19 In some implementations, the B lymphocyte antigen is CD19. CD19 is also known as, for example, the CD19 molecule (differentiation cluster 19), B lymphocyte surface antigen B4, T cell surface antigen Leu-12, and common variable immunodeficiency 3 antigen (CVID3). CD19 is a 95 kDa type I transmembrane glycoprotein in the immunoglobulin superfamily (IgSF). CD19 is expressed in all B lineage cells during B cell development. Most B-cell malignancies express normal to high levels of CD19, making CD19 a promising tumor marker and a potential molecular target for therapeutic interventions in B-cell cancers, particularly hematologic malignancies and lymphomas.

[0143] The CAR disclosed in this invention binds to CD19. In some embodiments, CD19 is human CD19. In some embodiments, CD19 is wild-type human CD19. In some embodiments, CD19 comprises or consists of the amino acid sequence of Uniprot reference number: P15391-1 (SEQ ID NO: 7) provided below.

[0144] MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIWLFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNR SSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARP VLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKRKRMTDPTRRFFKVTPPPGSGPQNQYGNVLSLPTPTSGLGRAQRWAAGLGGTAPSYGNPSSDVQADGALGSRSPPGVGPEEEEGEGYEEPDSEED SEFYENDSNLGQDQLSQDGSGYENPEDEPLGPEDEDSFSNAESYENEDEELTQPVARTMDFLSPHGSAWDPSREATSLGSQSYEDMRGILYAAPQLRSIRGQPGPNHEEDADSYENMDNPDGPDPAWGGGGRMGTWSTR [SEQ ID NO: 7] In some embodiments, CD19 contains or is composed of at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% of the amino acid sequence or fragment thereof shown in SEQ ID NO: 7.

[0145] In some embodiments, CD19 comprises an extracellular domain, a transmembrane domain, and a cytoplasmic domain. In some embodiments, the extracellular domain of CD19 comprises amino acids 20 to 291 of SEQ ID NO: 7. In some embodiments, the transmembrane domain of CD19 comprises amino acids 292 to 313 of SEQ ID NO: 7. In some embodiments, the cytoplasmic domain of CD19 comprises amino acids 314 to 556 of SEQ ID NO: 7.

[0146] In some embodiments, the CAR disclosed in this invention binds to a fragment of CD19 (e.g., a fragment of human CD19). In some embodiments, the CAR disclosed in this invention binds to a fragment of the amino acid sequence shown in SEQ ID NO: 7. In some embodiments, the CAR disclosed in this invention binds to the extracellular domain of CD19 (e.g., human CD19). In some embodiments, the CAR disclosed in this invention binds to amino acids 20 to 291 of SEQ ID NO: 7. In some embodiments, the CAR disclosed in this invention binds to a fragment of amino acids 20 to 291 of SEQ ID NO: 7.

[0147] 5.2.2 CD20 In some implementations, the B lymphocyte antigen is CD20. CD20 is also known as, for example, the CD20 molecule (differentiation cluster 20), transmembrane 4-domain A1 (MS4A1), B lymphocyte surface antigen B1, Bp35 antigen, S7 antigen, FMC7 antigen, leukocyte surface antigen Leu-16, and common variable immunodeficiency 5 antigen (CVID5). CD20 is a non-glycosylated 33–37 kDa phosphoprotein member of the MS4A family that forms a four-transmembrane binding protein. CD20 is expressed on B cells throughout B cell differentiation, prior to terminal differentiation into plasma cells. In particular, CD20 has been found on B-cell lymphoma, hairy cell leukemia, B-cell chronic lymphocytic leukemia, and melanoma cancer stem cells, making CD20 a promising tumor marker and a potential therapeutic target for B-cell malignancies, particularly hematologic malignancies and lymphomas.

[0148] The CAR disclosed in this invention binds to CD20. In some embodiments, CD20 is human CD20. In some embodiments, CD20 is wild-type human CD20. In some embodiments, CD20 comprises or consists of the amino acid sequence of Uniprot reference number: P11836-1 (SEQ ID NO: 8) provided below.

[0149] MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAVQIMNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSLLAATEKNSRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISHFLKME SLNFIRAHTPYINIYNCEPANPSEKNSPSTQYCYSIQSLFLGILSVMLIFAFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEEVVGLTETTSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSP[SEQ ID NO: 8] In some embodiments, CD20 contains or is composed of at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% of the amino acid sequence or fragment thereof shown in SEQ ID NO: 8.

[0150] In some embodiments, CD20 comprises four transmembrane domains, two extracellular domains, and intracellular N- and C-terminal domains. In some embodiments, the extracellular domains of CD20 comprise amino acids 79 to 84 of SEQ ID NO: 8. In some embodiments, the extracellular domains of CD20 comprise amino acids 142 to 188 of SEQ ID NO: 8. In some embodiments, the transmembrane domains of CD20 comprise amino acids 57 to 78 of SEQ ID NO: 8. In some embodiments, the transmembrane domains of CD20 comprise amino acids 85 to 105 of SEQ ID NO: 8. In some embodiments, the transmembrane domains of CD20 comprise amino acids 121 to 141 of SEQ ID NO: 8. In some embodiments, the transmembrane domains of CD20 comprise amino acids 189 to 209 of SEQ ID NO: 8. In some embodiments, the N-terminal intracellular domain of CD20 comprises amino acids 1 to 45 of SEQ ID NO: 8. In some embodiments, the C-terminal intracellular domain of CD20 contains amino acids 210 to 297 of SEQ ID NO: 8.

[0151] In some embodiments, the CAR disclosed in this invention binds to a fragment of CD20 (e.g., a fragment of human CD20). In some embodiments, the CAR disclosed in this invention binds to a fragment of the amino acid sequence shown in SEQ ID NO: 8. In some embodiments, the CAR disclosed in this invention binds to the extracellular domain of CD20 (e.g., human CD20). In some embodiments, the CAR disclosed in this invention binds to amino acids 79 to 84 of SEQ ID NO: 8. In some embodiments, the CAR disclosed in this invention binds to a fragment of amino acids 79 to 84 of SEQ ID NO: 8. In some embodiments, the CAR disclosed in this invention binds to amino acids 142 to 188 of SEQ ID NO: 8. In some embodiments, the CAR disclosed in this invention binds to a fragment of amino acids 142 to 188 of SEQ ID NO: 8.

[0152] 5.3. Chimeric antigen receptor (CAR) The CAR disclosed in this invention binds to CD19 and CD20. CAR is an engineered receptor that grafts or confers specificity of interest onto or confers specificity on immune effector cells. CAR can be used to specifically graft monoclonal antibodies onto cells, such as immune-responding cells, such as T cells or NK cells; this is done by transferring their coding sequences, which is facilitated, for example, by a retroviral vector.

[0153] In some embodiments, the CAR disclosed in this invention comprises an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a first antigen-binding domain targeting CD19 and a second antigen-binding domain targeting CD20. In some embodiments, the transmembrane domain (TD) links the extracellular domain (ED) to the intracellular domain (ID) and is located within the cell membrane when expressed by a cell (e.g., an immune-responding cell or an immune effector cell). In some embodiments, the transmembrane domain includes a first CD28 polypeptide. In some embodiments, the intracellular domain includes a signal transduction domain (SD) that transmits an activation signal to the cell upon antigen recognition. The “signal transduction domain (SD)” typically includes an immune receptor tyrosine-based activation motif (ITAM) that, when phosphorylated, triggers an activation signal transduction cascade. SD can be a polypeptide selected from the group consisting of: CD8, CD3ζ, CD3δ, CD3γ, CD3ε, FcγRI-γ, FcyRIII-γ, FcεRIβ, FcεRIγ, DAP10, DAP12, CD32, CD79a, CD79b, and mutants thereof. In some embodiments, SD comprises a modified CD3ζ polypeptide. In some embodiments, the modified CD3ζ polypeptide comprises natural ITAM1, ITAM2 variants, and ITAM3 variants. In some embodiments, each of the ITAM2 and ITAM3 variants comprises two loss-of-function mutations. In some embodiments, the intracellular domain further comprises a co-stimulatory signal transduction region (CSR). The term "co-stimulatory signal transduction region (CSR)" refers to an intracellular signal transduction domain derived from co-stimulatory protein receptors such as CD28, 41BB, inducible co-stimulatory factor (ICOS), CD80, CD86, OX40, DAP10, DAP12, MyD88, CD7, CD27, CD4, B2C, BTNL3, NKG2D, NKp30, and their variants, which can enhance the activation of immune response cells or immune effector cells (e.g., T cells).

[0154] In some implementations, the disclosed CAR is defined by the following formula: LP–ED–HD–TM–CSR–ISD, where “LP” represents an optional leader peptide, “ED” represents an extracellular domain containing a first antigen-binding domain targeting CD19 and a second antigen-binding domain targeting CD20, “HD” represents an optional hinge domain, “TM” represents a transmembrane domain, “CSR” represents one or more co-stimulatory signal transduction regions, “SD” represents a signal transduction domain, and “–” represents a peptide bond or linker.

[0155] In some implementations, the first anti-CD19 antigen-binding domain is linked to HG.

[0156] Other CAR constructs are described, for example, by Fresnak et al. Nat. Rev. Cancer 16(9):566-81(2016), which is incorporated into the whole for teaching these CAR models by reference.

[0157] In some implementations, the CAR may be, for example (but not limited to), a TRUCK, a general-purpose CAR, a self-driving CAR, an armored CAR, a self-destructing CAR, a conditional CAR, or a marked CAR.

[0158] TRUCK (redirected T cells for universal cytokine killing) co-expresses chimeric antigen receptor (CAR) and anti-tumor cytokines. Cytokine expression can be constitutive or induced by T cell activation. Through CAR-specific targeting, the local production of pro-inflammatory cytokines recruits endogenous immune cells to the tumor site and can enhance the anti-tumor response.

[0159] Universal allogeneic CAR T cells are engineered to no longer express endogenous T cell receptor (TCR) and / or major histocompatibility complex (MHC) molecules, thereby preventing graft-versus-host disease (GVHD) or rejection, respectively.

[0160] The self-driven CAR co-expresses CAR and a chemokine receptor, which binds to tumor ligands to enhance tumor homing.

[0161] CAR T cells engineered to resist immunosuppression (armored CARs) can be genetically modified to no longer express various immune checkpoint molecules (e.g., cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) or programmed cell death protein 1 (PD-1)). Exemplary “knockdown” and “knockout” techniques include, but are not limited to, RNA interference (RNAi) (e.g., asRNA, miRNA, shRNA, siRNA, etc.) and CRISPR interference (CRISPRi) (e.g., CRISPR-Cas9). In some embodiments, CAR T cells are engineered to express a dominant-negative form of a checkpoint molecule. In some embodiments, the extracellular ligand-binding domain of an immune checkpoint molecule is fused to a transmembrane membrane to compete for ligand binding. For example, the extracellular ligand-binding domain of PD-1 can be fused to the CD8 transmembrane domain to compete for PD-1 ligands from target cells. In some embodiments, CAR T cells are engineered to express an immune checkpoint switching receptor to utilize inhibitory immune checkpoint ligands present on target cells. In some embodiments, the extracellular ligand-binding domain of an immune checkpoint molecule is fused with a signaling, stimulatory, and / or co-stimulatory domain. For example, the extracellular ligand-binding domain of PD-1 may be fused with a CD28 domain to provide CD28 co-stimulation while blocking PD-1 signaling. In some embodiments, CAR T cells may be administered together with an aptamer or monoclonal antibody that blocks immune checkpoint signaling. In some embodiments, CAR T cells (e.g., CAR T cell therapy) are combined with a PD-1 blocking approach, such as administration with a PD-1 / PD-L1 antagonistic aptamer or anti-PD-1 / PD-L1 antibody. In some embodiments, CAR T cells and a PD-1 pathway blocking antibody are administered in combination. In some embodiments, CAR T cells are engineered to express or express and secrete immune checkpoint blocking antibodies, such as anti-PD-1 or anti-PD-L1 or fragments thereof. In some embodiments, CAR T cells are administered together with a vector (e.g., an engineered virus) that expresses the immune checkpoint blocking molecule described herein.

[0162] Self-destructing CARs can be designed using RNA encoded by electroporation. Alternatively, induced apoptosis of T cells can be achieved based on ganciclovir binding to thymidine kinase in genetically modified lymphocytes or, as recently described, by activating human caspase 9 via a small dimer.

[0163] Conditional CAR T cells are unresponsive by default, or "off," until a small molecule is added to complete the circuit, enabling full transduction of both signal 1 and signal 2, thereby activating the CAR T cells. Alternatively, T cells can be engineered to express an adaptor-specific receptor with affinity for a subsequently administered second antibody against the target antigen.

[0164] Labeled CAR T cells express CAR plus tumor epitopes that bind to existing monoclonal antibody agents. In cases of intolerable adverse reactions, administration of monoclonal antibodies clears CAR T cells and alleviates symptoms without additional extratumor effects.

[0165] There are three generations of CARs. "First-generation" CARs typically consist of an extracellular antigen-binding domain (e.g., scFv) fused to a transmembrane domain, which in turn fuses to an intracellular domain. "First-generation" CARs can provide de novo antigen recognition via their CD3ζ chain signaling domain within a single fusion molecule and induce CD4+ α-receptor activation. + and CD8 + Both T cell activation and activation are achieved independently of HLA-mediated antigen presentation. "Second-generation" CARs add intracellular signaling domains from co-stimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40) to the intracellular domains of the CAR to provide additional signaling to the cell. "Second-generation" CARs contain those that provide both co-stimulation (e.g., CD28 or 4-1BB) and activation (CD3ζ). "Third-generation" CARs combine multiple signaling domains to further enhance potency, for example, by combining multiple signaling domains (such as CD3ζ-CD28-OC40 or CD3ζ-CD28-4-1BB) to enhance potency through stronger cytokine production and killing ability. In some embodiments, the CAR disclosed in this invention is a second-generation CAR. In some embodiments, the CAR disclosed in this invention is a third-generation CAR.

[0166] 5.3.1. Extracellular domains of CAR The extracellular domain of the CAR disclosed in this invention comprises a first antigen-binding domain targeting CD19 and a second antigen-binding domain targeting CD20. Such antigen-binding domains are typically derived from antibodies. In some embodiments, the first and / or second antigen-binding domains comprise either a functional antibody fragment or a derivative thereof (e.g., a single-chain variable fragment (scFv) or Fab, or any suitable antigen-binding fragment of an antibody).

[0167] In some embodiments, the primary anti-CD19 antigen-binding domain comprises a primary anti-CD19 scFv. In some embodiments, the primary anti-CD19 scFv is derived from a monoclonal antibody (mAb). The primary anti-CD19 scFv can be a human scFv, a humanized scFv, or a mouse scFv. In some embodiments, the primary anti-CD19 scFv is a mouse scFv. In some embodiments, the primary anti-CD19 scFv is a mouse scFv. The primary anti-CD19 scFv can be derived from the heavy chain variable region (V) of a fusion antibody. H ) and light chain variable region (V L Alternatively or additionally, the primary anti-CD19 scFv may be derived from Fab (rather than from an antibody, for example, from a Fab library).

[0168] In some embodiments, the second anti-CD20 antigen-binding domain comprises a second anti-CD20 scFv. In some embodiments, the second anti-CD20 scFv is derived from a monoclonal antibody (mAb). The second anti-CD20 scFv can be a human scFv, a humanized scFv, or a mouse scFv. In some embodiments, the second anti-CD20 scFv is a mouse scFv. In some embodiments, the second anti-CD20 scFv is a mouse scFv. The second anti-CD20 scFv can be derived from the V of the fusion antibody. H and V L Alternatively or additionally, the secondary anti-CD20 scFv may be derived from Fab (instead of from an antibody, for example, from a Fab library).

[0169] Alternatively, the first antigen-binding domain and / or the second antigen-binding domain may comprise either Fab or F(ab)2.

[0170] In some embodiments, the first anti-CD19 antigen binding domain includes a first V H And the first V L In some implementations, the first V H V containing the amino acid sequence of SEQ ID NO: 15 H V H CDR1, V H CDR2 and V H CDR3. In some implementations, the first V L V containing the amino acid sequence of SEQ ID NO: 16 L V L CDR1, V L CDR2 and V LCDR3. SEQ ID NO: 15 and SEQ ID NO: 16 are disclosed in Table 1.

[0171] In some implementations, the first V H Contains V containing the amino acid sequence shown in SEQ ID NO: 9 or its conserved modifications. H CDR1, containing the amino acid sequence shown in SEQ ID NO: 10 or its conserved modifications thereof, V H CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 11 or its conserved modifications H CDR3. In some implementations, the first V H V containing the amino acid sequence shown in SEQ ID NO: 9 H CDR1, containing the amino acid sequence shown in SEQ ID NO: 10, V H CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 11 H CDR3. SEQ ID NO: 9-11 is provided in Table 1.

[0172] In some implementations, the first V H Contains an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identical or homologous to the amino acid sequence shown in SEQ ID NO: 15. For example, the first V H Contains approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% identical or homologous amino acid sequences to the amino acid sequence shown in SEQ ID NO: 15. In some embodiments, the first V H It contains the amino acid sequence shown in SEQ ID NO: 15.

[0173] In some implementations, the first V L Contains V containing the amino acid sequence shown in SEQ ID NO: 12 or its conserved modifications. L CDR1, containing the amino acid sequence shown in SEQ ID NO: 13 or its conserved modifications thereof, V L CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 14 or its conserved modifications L CDR3. In some implementations, the first V LV containing the amino acid sequence shown in SEQ ID NO: 12 L CDR1, containing the amino acid sequence shown in SEQ ID NO: 13, V L CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 14 L CDR3. SEQ ID NO: 12-14 are provided in Table 1.

[0174] In some implementations, the first V L Contains an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identical or homologous to the amino acid sequence shown in SEQ ID NO: 16. For example, the first V L Contains approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% identical or homologous amino acid sequences to the amino acid sequence shown in SEQ ID NO: 16. In some embodiments, the first V L It contains the amino acid sequence shown in SEQ ID NO: 16.

[0175] In some implementations, the first V H Contains V containing the amino acid sequence shown in SEQ ID NO: 9 or its conserved modifications. H CDR1, containing the amino acid sequence shown in SEQ ID NO: 10 or its conserved modifications thereof, V H CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 11 or its conserved modifications H CDR3; and the first V L Contains V containing the amino acid sequence shown in SEQ ID NO: 12 or its conserved modifications. L CDR1, containing the amino acid sequence shown in SEQ ID NO: 13 or a conserved modified V L CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 14 or its conserved modifications L CDR3. In some implementations, the first V H V containing the amino acid sequence shown in SEQ ID NO: 9 H CDR1, containing the amino acid sequence shown in SEQ ID NO: 10, V HCDR2 and V containing the amino acid sequence shown in SEQ ID NO: 11 H CDR3; and the first V L V containing the amino acid sequence shown in SEQ ID NO: 12 L CDR1, containing the amino acid sequence shown in SEQ ID NO: 13, V L CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 14 L CDR3.

[0176] In some implementations, the first V H Contains the amino acid sequence shown in SEQ ID NO: 15, and the first V L Contains the amino acid sequence shown in SEQ ID NO: 16. First V H And the first V L They can be connected one after another, for example, via connectors. In some implementations, the first V H And the first V L The connection is made via a first linker. In some embodiments, the first linker comprises or consists of the amino acid sequence shown in SEQ ID NO: 1. The variable region from the N-terminus to the C-terminus can be V... H -V L or V L -V H In some implementations, the first V L Located at the N-end of the variable region, i.e., the first V H And the first V L The region from the N-terminus to the C-terminus is defined as V. L -V H .

[0177] In some embodiments, the first anti-CD19 antigen-binding domain is a first anti-CD19 scFv. In some embodiments, the first anti-CD19 scFv comprises the first V disclosed herein. H And the first V published in this article L In some embodiments, the first anti-CD19 scFv comprises or consists of the amino acid sequence shown in SEQ ID NO: 17. SEQ ID NO: 17 is provided in Table 1. In some embodiments, the first anti-CD19 scFv is derived from the mouse anti-CD19 antibody FCM63, which binds to human CD19. FCM63 is disclosed in Zola et al. Immunology and Cell Biology (1991);69:411-422 and Nicholson et al. Molecular Immunology(1997);34(16-17):1157-1165, which is incorporated into this paper in its entirety by reference.

[0178] In some implementations, the CDR is identified according to the Kabat numbering system. In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 15 is shown in SEQ ID NO: 18 provided below.

[0179] GAAGTAAAACTGCAAGAGAGTGGCCCCGGTCTTGTGGCCCCTTCTCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGCGTGAGCTGGATCAGACAGCCCCCTAGAAAGGGCCTGGAGTGGCTCGGTGTGATCTGGGGCAGCGAGACCACCTACTACAAC AGCGCCCTGAAGAGCAGACTGACCATCATCAAGGACAACAGCAAGAGCCAAGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGTGCCAAGCATTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAAGGCACAAGCGTCACCGTCAGCAGC [SEQ ID NO:18] In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 16 is shown in SEQ ID NO: 19 provided below.

[0180] GATATACAGATGACACAGACCACAAGCAGCCTGAGCGCGTCGTTAGGTGACAGAGTGACCATCAGCTGCCGCGCTAGCCAAGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACAAGCAGAC TGCACAGCGGCGTACCATCTCGGTTCTCTGGCAGCGGCTCTGGAACCGACTACTCGCTGACCATCAGTAATCTGGAGCAAGAGGACATCGCCACCTACTTCTGTCAGCAAGGCAACACCCTGCCCTACACATTCGGCGGCGGTACGAAGCTGGAGATAACC [SEQ ID NO: 19] In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 17 is shown in SEQ ID NO: 20 provided below.

[0181] GATATACAGATGACACAGACCACAAGCAGCCTGAGCGCGTCGTTAGGTGACAGAGTGACCATCAGCTGCCGCGCTAGCCAAGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAGCCCGACGGCACCGTGAAGCTGCTGATCTACCACACAAGCAGACTGCACAGCGGCGTACCATCTCGG TTCTCTGGCAGCGGCTCTGGAACCGACTACTCGCTGACCATCAGTAATCTGGAGCAAGAGGACATCGCCACCTACTTCTGTCAGCAAGGCAACACCCTGCCCTACACATTCGGCGGCGGTACGAAGCTGGAGATAACCGGCTCCACGAGCGGCAGCGGCAAACCCGGCAGCGGCGAGGGCAGCA CCAAGGGCGAAGTAAAACTGCAAGAGAGTGGCCCCGGTCTTGTGGCCCCTTCTCAGAGCCTGAGCGTGACCTGCACCGTGAGCGGCGTGAGCCTGCCCGACTACGGGCTGAGCTGGATCAGACAGCCCCCTAGAAAGGGCCTGGAGTGGCTCGGTGTGATCTGGGGCAGCGAGACCACCTACTA CAACAGCGCCCTGAAGAGCAGACTGACCATCATCAAGGACAACAGCAAGAGCCAAGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCGCCATCTACTACTGTGCCAAGCATTACTACTACGGCGGCAGCTACGCCATGGACTACTGGGGCCAAGGCACAAGCGTCACCGTCAGCAGC [SEQ ID NO: 20] In some embodiments, the second anti-CD20 antigen-binding domain includes a second V H Second V L .

[0182] In some implementations, the second V H V containing the amino acid sequence of SEQ ID NO: 27 H V H CDR1, V H CDR2 and V H CDR3. In some implementations, the second V LV containing the amino acid sequence of SEQ ID NO: 28 L V L CDR1, V L CDR2 and V L CDR3. SEQ ID NO: 27 and SEQ ID NO: 28 are disclosed in Table 2.

[0183] In some implementations, the second V H Contains V containing the amino acid sequence shown in SEQ ID NO: 21 or its conserved modifications. H CDR1, containing the amino acid sequence shown in SEQ ID NO: 22 or its conserved modifications thereof, V H CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 23 or its conserved modifications H CDR3. In some implementations, the second V H V containing the amino acid sequence shown in SEQ ID NO: 21 H CDR1, containing the amino acid sequence shown in SEQ ID NO: 22, V H CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 23 H CDR3. SEQ ID NO: 21-23 is provided in Table 2.

[0184] In some implementations, the second V H Contains an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identical or homologous to the amino acid sequence shown in SEQ ID NO: 27. For example, the second V H Contains amino acid sequences that are approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% identical or homologous to the amino acid sequence shown in SEQ ID NO: 27. In some embodiments, the second V H It contains the amino acid sequence shown in SEQ ID NO: 27.

[0185] In some implementations, the second V L Contains V containing the amino acid sequence shown in SEQ ID NO: 24 or its conserved modifications. L CDR1, containing the amino acid sequence shown in SEQ ID NO: 25 or its conserved modifications thereof, V LCDR2 and V containing the amino acid sequence shown in SEQ ID NO: 26 or its conserved modifications L CDR3. In some implementations, the second V L V containing the amino acid sequence shown in SEQ ID NO: 24 L CDR1, containing the amino acid sequence shown in SEQ ID NO: 25, V L CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 26 L CDR3. SEQ ID NO: 24-26 are provided in Table 2.

[0186] In some implementations, the second V L Contains an amino acid sequence that is at least about 80% (e.g., at least about 85%, at least about 90%, or at least about 95%) identical or homologous to the amino acid sequence shown in SEQ ID NO: 28. For example, the second V L Contains amino acid sequences that are approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100% identical or homologous to the amino acid sequence shown in SEQ ID NO: 28. In some embodiments, the second V L It contains the amino acid sequence shown in SEQ ID NO: 28.

[0187] In some implementations, the second V H Contains V containing the amino acid sequence shown in SEQ ID NO: 21 or its conserved modifications. H CDR1, containing the amino acid sequence shown in SEQ ID NO: 22 or its conserved modifications thereof, V H CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 23 or its conserved modifications H CDR3; and the second V L Contains V containing the amino acid sequence shown in SEQ ID NO: 24 or its conserved modifications. L CDR1, containing the amino acid sequence shown in SEQ ID NO: 25 or a conserved modified V L CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 26 or its conserved modifications L CDR3. In some implementations, the second V H V containing the amino acid sequence shown in SEQ ID NO: 21H CDR1, containing the amino acid sequence shown in SEQ ID NO: 22, V H CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 23 H CDR3; and the second V L V containing the amino acid sequence shown in SEQ ID NO: 24 L CDR1, containing the amino acid sequence shown in SEQ ID NO: 25, V L CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 26 L CDR3.

[0188] In some implementations, the second V H Contains the amino acid sequence shown in SEQ ID NO: 27, and the second V L It contains the amino acid sequence shown in SEQ ID NO: 28.

[0189] Second V H Second V L They can be connected one after another, for example, via connectors. In some implementations, the second V H Second V L The connection is made via a second linker. In some embodiments, the second linker comprises or consists of the amino acid sequence shown in SEQ ID NO: 2. The variable region from the N-terminus to the C-terminus can be V... H -V L or V L -V H In some implementations, the second V H It is located at the N end of the variable region, i.e., the second V. H Second V L The region from the N-terminus to the C-terminus is defined as V. H -V L In some embodiments, the second anti-CD20 antigen-binding domain is a domain containing the second V disclosed herein. H And the second V disclosed in this article L The second anti-CD20 scFv. In some embodiments, the second anti-CD20 scFv comprises or consists of the amino acid sequence shown in SEQ ID NO: 29. SEQ ID NO: 29 is provided in Table 2.

[0190] In some embodiments, the second anti-CD20 scFv is derived from the mouse anti-CD20 antibody Leu16, which binds to human CD20. Leu16 is disclosed in ensen et al. , Biology of Blood Marrow Transplantation(1998);4:75-83, which is incorporated into this paper in its entirety by reference.

[0191] In some implementations, the CDR is identified according to the Kabat numbering system. In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 27 is shown in SEQ ID NO: 30 provided below.

[0192] GAAGTGCAGCTTCAGCAGAGCGGCGCCGAACTGGTGAAGCCCGGCGCTAGCGTGAAGATGAGCTGCAAGGCTAGCGGCTACACTTCACAAGCTATAACATGCACTGGGTGAAGCAGACCCCCGGCCAAGGCCTGGAGTGGATCGGTGCCATCTACCCCGGCAACGGCGACACAAGCTACAAT CAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACAAGCGAGGACAGCGCCGACTACTATTGCGCACGGAGCAACTACTACGGCAGCAGCTACTGGTTCTTCGACGTATGGGGCGCGGGCACCACGGTCACCGTGAGCTCC [SEQ ID NO: 30] In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 28 is shown in SEQ ID NO: 31 provided below.

[0193] GACATCGTGCTGACACAGAGCCCCGCCATCCTAAGCGCCTCCCCCGGTGAAAAAGTGACCATGACCTGCAGAGCTAGCAGCTCTGTGAACTACATGGACTGGTATCAGAAGAAACCTGGCAGCTCTCCAAAGCCGTGGATCTATGCCACAAGCAATCTG GCATCTGGCGTACCCGCTAGATTCAGCGGCAGCGGATCCGGCACATCATACTCCCTGACCATAAGCCGTGTGGAGGCCGAGGACGCCGCCACCTACTACTGTCAGCAGTGGAGCTTCAACCCCCCTACATTCGGAGGGGGCACCAAGCTGGAGATTAAA [SEQ ID NO: 31] In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 29 is shown in SEQ ID NO: 32 provided below.

[0194] GAAGTGCAGCTTCAGCAGAGCGGCGCCGAACTGGTGAAGCCCGGCGCTAGCGTGAAGATGAGCTGCAAGGCTAGCGGCTACACCTTCACAAGCTATAACATGCACTGGGTGAAGCAGACCCCCGGCCAAGGCCTGGAGTGGATCGGTGCCATCTACCCGGCAACGGCGACACAAGCTACAA TCAGAAGTTCAAGGGCAAGGCCACCCTGACCGCCGACAAGAGCAGCAGCACCGCCTACATGCAGCTGAGCAGCCTGACAAGCGAGGACAGCGCCGACTACTATTGCGCACGGAGCAACTACTACGGCAGCAGCTACTGGTTCTTCGACGTATGGGGCGCGGGCACCACGGTCACCGTGAGCT CCGGTGGAGGGGGCTCTGGCGGTGGGGGGTCCGGCGGTGGCGGATCTGACATCGTGCTGACACAGAGCCCCGCCATCCTAAGCGCCTCCCCCGGTGAAAAAGTGACCATGACCTGCAGAGCTAGCAGCTCTGTGAACTACATGGACTGGTATCAGAAGAAACCTGGCAGCTCTCCAAAGCCG TGGATCTATGCCACAAGCAATCTGGCATCTGGCGTACCCGCTAGATTCAGCGGCAGCGGATCCGGCACATCATACTCCCTGACCATAAGCCGTGTGGAGGCCGAGGACGCCGCCACCTACTACTGTCAGCAGTGGAGCTTCAACCCCCCTACATTCGGAGGGGGCACCAAGCTGGAGATTAAA [SEQ ID NO: 32] A first V sequence having at least about 80%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (e.g., about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%) homology or identity with a specific sequence (e.g., SEQ ID NO: 15, SEQ ID NO: 27, SEQ ID NO: 16, or SEQ ID NO: 28). H Second V HFirst V L and / or the second V L The amino acid sequence may contain substitutions (e.g., conserved substitutions), insertions, or deletions relative to a specified sequence, but retains the ability to bind to the target antigen (e.g., CD19 or CD20). In some embodiments, a total of 1 to 10 amino acids are substituted, inserted, and / or deleted in a specific sequence (e.g., SEQ ID NO: 15, SEQ ID NO: 27, SEQ ID NO: 16, or SEQ ID NO: 28). In some embodiments, the substitution, insertion, or deletion occurs in a region outside the CDR of the first antigen-binding domain and / or the second antigen-binding domain (e.g., in the FR).

[0195] In some embodiments, the extracellular domain of the CAR includes a third linker between a first anti-CD19 antigen-binding domain and a second anti-CD20 antigen-binding domain. In some embodiments, the third linker comprises or consists of the amino acid sequence shown in SEQ ID NO: 3.

[0196] Additionally, the CAR may contain a leader peptide or signal peptide that guides the CAR into the endoplasmic reticulum. The leader peptide or signal peptide may allow the CAR to be glycosylated and anchored in the cell membrane. The leader peptide or signal peptide may have a length of about 5, about 10, about 15, about 20, about 25, or about 30 amino acids. In some embodiments, the leader peptide or signal peptide is covalently attached to the N-terminus of an extracellular domain. In some embodiments, the leader peptide or signal peptide is covalently attached to the N-terminus of a second anti-CD20 antigen binding domain. In some embodiments, the leader peptide or signal peptide comprises a CD8 polypeptide. In some embodiments, the leader peptide or signal peptide comprises or consists of the amino acid sequence shown in SEQ ID NO: 33 provided below. In some embodiments, the leader peptide or signal peptide is associated with the V disclosed herein. H The N-terminus is linked. In some embodiments, the leader peptide or signal peptide is linked to the second V disclosed herein. H The N-terminus is linked. In some embodiments, the leader peptide or signal peptide is linked to the V disclosed herein. L The N-terminus is linked. In some embodiments, the leader peptide or signal peptide is linked to the second V disclosed herein. L The N-terminal connection.

[0197] MALPVTALLLPLALLLHA (SEQ ID NO: 33) In some embodiments, an exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 33 is shown in SEQ ID NO: 34 provided below.

[0198] ATGGCCCTGCCCGTCACCGCTCTGCTTCTGCCCCTGGCTCTGCTCTTACATGCC [SEQ ID NO:34] 5.3.2. Transmembrane domains of CAR In some implementations, the transmembrane domain of the CAR contains a hydrophobic α-helix that spans at least a portion of the membrane. Different transmembrane domains result in different receptor stability. After antigen recognition, the receptor cluster and signal are transduced into the cell.

[0199] Transmembrane domains can be derived from natural or synthetic sources. In the case of a natural source, the domain can originate from any membrane-bound or transmembrane protein. For example, transmembrane regions can originate from the α, β, or ζ chains of T cell receptors, including CD28, CD3ε, CD45, CD4, CD5, CD8 (e.g., CD8α, CD8β), CD9, CD16, CD22, CD27, CD33, CD37, CD40, CD64, CD80, CD86, CD134, CD137, CD154, ICAM-1, KIRDS2, OX40, CD2, NKGD2, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD166, CD19, IL2Rβ, IL2Rγ, and IL7R. α, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100(SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG(CD162), LTBR and PAG / Cbp (For example, at least its transmembrane region). In some embodiments, the transmembrane domain of the CAR comprises a natural or modified transmembrane domain of CD8, CD28, CD3ζ, CD4, 4-1BB, OX40, ICOS, CD84, CD166, CD8a, CD8b, ICAM-1, CTLA-4, CD27, CD40, NKGD2, or combinations thereof. Alternatively, the transmembrane domain may be synthetic, in which case it primarily comprises hydrophobic residues such as leucine and valine. In some cases, a triplet of phenylalanine, tryptophan, and valine may be found at each end of the synthetic transmembrane domain. Short oligopeptides or polypeptide linkers, such as those 2 to 10 amino acids in length, may form the link between the transmembrane domain of the CAR and the intracellular domain.

[0200] In some implementations, the CAR includes more than one transmembrane domain, which may be a repeat of the same transmembrane domain or different transmembrane domains.

[0201] In some embodiments, the transmembrane domain of the CAR comprises a CD28 polypeptide (e.g., the transmembrane domain of CD28 or a portion thereof). In some embodiments, the transmembrane domain of the CAR comprises the transmembrane domain of human CD28 or a portion thereof. The CD28 polypeptide may comprise, or consist of, at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100% identical or homologous amino acid sequences to, or composed of, the sequence having NCBI reference number: NP_006130 (SEQ ID NO: 35) or fragments thereof, and / or may optionally comprise at most one, at most two, or at most three conserved amino acid substitutions. In some embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence as a continuous portion of SEQ ID NO: 35, having a length of at least about 20, or at least about 30, or at least about 40, or at least about 50, or at least about 60, and / or at most about 70, at most about 80, at most about 90, at most about 100, at most about 150, at most about 200, or at most about 220 amino acids. In some embodiments, the CD28 polypeptide comprises or consists of an amino acid sequence of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 153 to 179, 150 to 200, 153 to 179, or 200 to 220 of SEQ ID NO: 35. In some embodiments, the transmembrane domain of the CAR comprises or consists of an amino acid sequence of amino acids 153 to 179 of SEQ ID NO: 35. SEQ ID NO: 35 is provided below.

[0202] MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFC KIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS[SEQ ID NO: 35] An exemplary nucleotide sequence encoding amino acids 153 to 179 of SEQ ID NO: 35 is shown in SEQ ID NO: 36 provided below.

[0203] TTCTGGGTTCTGGTCGTGGTGGGCGGCGTCCTGGCCTGCTACAGCCTGCTGGTGACCGTGGCCTTCATCATCTTCTGGGTG [SEQ ID NO: 36] In some implementations, the CAR also includes a spacer / hinge domain connecting the extracellular domain to the transmembrane domain. The hinge domain is a short amino acid sequence that promotes antibody flexibility (see, for example, Woof et al.). Nat. Rev. Immunol. 4(2): 89-99 (2004)). The hinge domain can be a suitable sequence derived from or obtained from any suitable molecule. The hinge domain can be flexible enough to allow the extracellular domain to be oriented in different directions to facilitate antigen recognition while preserving the activation activity of CAR.

[0204] The hinge / spacer domain of a CAR may comprise a natural or modified hinge region of CD8, CD28, CD3ζ, CD40, 4-1BB, OX40, CD84, CD166, CD8a, CD8b, ICOS, ICAM-1, CTLA-4, CD27, CD40, NKGD2, or combinations thereof. The hinge / spacer region may be a hinge region derived from IgG1, or a portion of the CH2CH3 region and CD3 of an immunoglobulin, a portion of a CD28 polypeptide (e.g., a portion of SEQ ID NO: 35), a portion of a CD8 polypeptide, a variant of any of the foregoing that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% identical or homologous to it, or a synthetic spacer sequence.

[0205] In some embodiments, the hinge domain of the CAR comprises a natural or modified hinge region of CD28. In some embodiments, the hinge domain of the CAR comprises a natural hinge region of CD28. In some embodiments, the hinge domain of the CAR comprises amino acids 114 to 152 of SEQ ID NO: 35.

[0206] An exemplary nucleotide sequence encoding amino acids 114 to 152 of SEQ ID NO: 35 is shown in SEQ ID NO: 37 provided below.

[0207] ATCGAGGTGATGTACCCCCCCCCCTACCTGGACAACGAGAAGAGCAACGGCACCATCATCCACGTGAAGGGCAAGCACCTGTGCCCTAGCCCCCTGTTCCCTGGCCCCTCGAAGCCC [SEQ ID NO: 37] 5.3.3. Intracellular domains of CAR In some embodiments, the intracellular domain of the CAR signals the CAR-containing cell (e.g., an immune-responding cell or an immune effector cell) upon antigen recognition, thereby activating at least one normal effector function of the cell. In some embodiments, the effector functions of the T cell are, for example, cytolytic activity or helper activity, including the secretion of cytokines. Therefore, the intracellular domain may comprise an "intracellular signal transduction domain" of the T cell receptor (TCR) and optionally a co-receptor. While the entire intracellular signal transduction domain can generally be used, in many cases it is not necessary to use the entire strand. Regarding the use of a truncated portion of the intracellular signal transduction domain, such a truncated portion can be used instead of the complete strand, as long as it transduces effector function signals.

[0208] Cytoplasmic signaling sequences that regulate primary activation of the TCR complex, which acts in a stimulus-like manner, may contain signaling motifs known as immune receptor tyrosine-based activation motifs (ITAMs). Examples of ITAM-containing cytoplasmic signaling sequences include, but are not limited to, those derived from CD8, CD3ζ, CD3δ, CD3γ, CD3ε, CD32 (FcγRIIa), DAP10, DAP12, CD79a, CD79b, FcγRIγ, FcγRIIIγ, FcεRIβ (FCERIB), and FcεRIγ (FCERIG).

[0209] In some implementations, the intracellular domain is derived from CD3 zeta (CD3ζ) (TCR ζ). The T cell surface glycoprotein CD3 zeta (CD3ζ) chain, also known as the T cell receptor T3ζ chain or CD247 (differentiation cluster 247), is a T cell receptor derived from CD3 zeta (CD3ζ) in humans. CD247 Genetically encoded protein. Wild-type (“natural”) CD3ζ contains three functional ITAMs and three functional basic-rich extension (BRS) regions (BRS1, BRS2, ​​and BRS3). The intracellular tail of the CD3 molecule contains a single ITAM responsible for TCR signal transduction. The intracellular tail of the ζ chain (CD3ζ) contains three (3) ITAMs. The intracellular domains of the CD3ζ chain are the primary signal transducers from the endogenous TCR.

[0210] In some embodiments, the intracellular domain of the CAR comprises a natural CD3ζ polypeptide. In some embodiments, the natural CD3ζ polypeptide comprises, or consists of, an amino acid sequence that is at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% identical or homologous to, the amino acid sequence having NCBI reference number: NP_932170 (SEQ ID NO: 38), and / or may optionally comprise at most one, at most two, or at most three conserved amino acid substitutions. In some embodiments, the natural CD3ζ polypeptide comprises, or consists of, an amino acid sequence as a continuous portion of SEQ ID NO: 38, having a length of at least about 20, or at least about 30, or at least about 40, or at least about 50, and at most about 164 amino acids. In some embodiments, the natural CD3ζ comprises or consists of an amino acid sequence of amino acids 1 to 164, 1 to 50, 50 to 100, 52 to 164, 100 to 150, or 150 to 164 of SEQ ID NO: 38. In some embodiments, the intracellular domain of the CAR comprises a natural CD3ζ polypeptide containing amino acids 52 to 164 of SEQ ID NO: 38 or consisting of them. SEQ ID NO: 38 is provided below.

[0211] MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR [SEQ ID NO: 38] In some embodiments, the intracellular domain of the CAR comprises a modified CD3ζ polypeptide. In some embodiments, the modified CD3ζ polypeptide contains a mutation, such as a point mutation, in at least one ITAM to render that ITAM nonfunctional. In some embodiments, the proximal ITAM (ITAM1), the distal ITAM (C-terminal third ITAM, ITAM3), or both are nonfunctional. In some embodiments, both proximal ITAMs (ITAM1 and ITAM2) or both distal ITAMs (ITAM2 and ITAM3) are nonfunctional. In some embodiments, only ITAM2 is nonfunctional. In some embodiments, the modified CD3ζ polypeptide contains a deletion (e.g., truncation) mutation, resulting in the deletion of at least one ITAM. In some embodiments, the modified CD3ζ polypeptide lacks a proximal ITAM (ITAM1), a distal ITAM (ITAM3), or both. In other embodiments, the modified CD3ζ polypeptide lacks both proximal ITAMs (ITAM1 and ITAM2) or both distal ITAMs (ITAM2 and ITAM3). In some embodiments, the modified CD3ζ peptide lacks ITAM2. Removing at least one ITAM from the introduced CAR can reduce CD3ζ-mediated apoptosis. Alternatively, removing at least one ITAM from the introduced CAR can reduce its size without loss of function.

[0212] In some embodiments, the modified CD3ζ polypeptide comprises one, two, or three ITAMs. In some embodiments, the modified CD3ζ polypeptide comprises natural ITAM1. In some embodiments, natural ITAM1 comprises or consists of the amino acid sequence shown in SEQ ID NO: 39.

[0213] QNQLYNELNLGRREEYDVLDKR [SEQ ID NO: 39] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 39 is shown in SEQ ID NO: 40 provided below.

[0214] CAGAATCAGCTGTACAACGAGCTGAACCTGGGCAGAAGAGAGGAGTACGACGTGCTGGACAAGAGA[SEQ ID NO: 40] In some embodiments, the modified CD3ζ polypeptide comprises an ITAM1 variant containing one or more loss-of-function mutations. In some embodiments, the ITAM1 variant comprises or consists of two loss-of-function mutations. In some embodiments, each of the one or more (e.g., two) loss-of-function mutations comprises a mutation in a tyrosine residue of ITAM1. In some embodiments, the ITAM1 variant consists of two loss-of-function mutations. In some embodiments, the ITAM1 variant comprises or consists of the amino acid sequence shown in SEQ ID NO: 41 provided below.

[0215] QNQLFNELNLGRREEFDVLDKR [SEQ ID NO: 41] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 41 is shown in SEQ ID NO: 42 provided below.

[0216] CAGAACCAGCTCTTTAACGAGCTCAATCTAGGACGAAGAGAGGAGTTCGATGTTTTGGACAAGAGA[SEQ ID NO: 42] In some embodiments, the modified CD3ζ polypeptide comprises natural ITAM2. In some embodiments, natural ITAM2 comprises or consists of the amino acid sequence shown in SEQ ID NO: 43 provided below.

[0217] QEGLYNELQKDKMAEAYSEIGMK [SEQ ID NO: 43] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 43 is shown in SEQ ID NO: 44 provided below.

[0218] CAGGAAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAA[SEQ ID NO: 44] In some embodiments, the modified CD3ζ polypeptide comprises an ITAM2 variant. In some embodiments, the ITAM2 variant comprises or consists of one or more loss-of-function mutations. In some embodiments, the ITAM2 variant comprises or consists of two loss-of-function mutations. In some embodiments, each of the one or more (e.g., two) loss-of-function mutations comprises a mutation of a tyrosine residue in ITAM2. In some embodiments, the ITAM2 variant consists of two loss-of-function mutations. In some embodiments, the ITAM2 variant comprises or consists of the amino acid sequence shown in SEQ ID NO: 45 provided below.

[0219] QEGLFNELQKDKMAEAFSEIGMK [SEQ ID NO: 45] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 45 is shown in SEQ ID NO: 46 provided below.

[0220] CAAGAGGGCCTGTTCAACGAGCTGCAGAAGGACAAGATGGCCGAGGCCTTCAGCGAGATCGGCATGAAG[SEQ ID NO: 46] In some embodiments, the modified CD3ζ polypeptide comprises natural ITAM3. In some embodiments, natural ITAM3 comprises or consists of the amino acid sequence shown in SEQ ID NO: 47 provided below.

[0221] HDGLYQGLSTATKDTYDALHMQ [SEQ ID NO: 47] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 47 is shown in SEQ ID NO: 48 provided below.

[0222] CACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG[SEQ ID NO: 48] In some embodiments, the modified CD3ζ polypeptide comprises an ITAM3 variant. In some embodiments, the ITAM3 variant comprises or consists of two loss-of-function mutations. In some embodiments, each of one or more (e.g., two) loss-of-function mutations comprises a mutation of a tyrosine residue in ITAM3. In some embodiments, the ITAM3 variant comprises or consists of two loss-of-function mutations. In some embodiments, the ITAM3 variant comprises or consists of the amino acid sequence shown in SEQ ID NO: 49 provided below.

[0223] HDGLFQGLSTATKDTFDALHMQ [SEQ ID NO: 49] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 49 is shown in SEQ ID NO: 50 provided below.

[0224] CACGACGCCTGTTCCAAGGCCTGAGCACCGCCACCAAGGACACCTTCGACGCCCTGCACATGCAA[SEQ ID NO: 50] Various modified CD3ζ peptides and CARs containing modified CD3ζ peptides are disclosed in International Patent Application Publication No. WO2019 / 133969, which is incorporated herein by reference in its entirety.

[0225] In some embodiments, the intracellular domain of the CAR comprises a modified CD3ζ polypeptide containing native ITM1, an ITM2 variant containing one or more (e.g., two) loss-of-function mutations or composed thereof, and an ITM3 variant containing one or more (e.g., two) loss-of-function mutations or composed thereof. In some embodiments, the intracellular domain of the CAR comprises a modified CD3ζ polypeptide containing native ITM1, an ITM2 variant composed of two loss-of-function mutations, and an ITM3 variant composed of two loss-of-function mutations. In some embodiments, the intracellular domain of the CAR comprises a modified CD3ζ polypeptide containing native ITM1 composed of the amino acid sequence shown in SEQ ID NO: 39, an ITM2 variant composed of the amino acid sequence shown in SEQ ID NO: 45, and an ITM3 variant composed of the amino acid sequence shown in SEQ ID NO: 49. In some embodiments, the modified CD3ζ polypeptide comprises or is composed of the amino acid sequence shown in SEQ ID NO: 51, which is designated "1XX". SEQ ID NO: 51 is provided below.

[0226] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR [SEQ ID NO: 51] In some embodiments, the intracellular domain of the CAR comprises a modified CD3ζ polypeptide containing at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% of the same amino acid sequence as or composed of a fragment thereof, and / or may optionally contain at most one, at most two, or at most three conserved amino acid substitutions.

[0227] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 51 is shown in SEQ ID NO: 52 provided below.

[0228] AGAGTGAAGTTCAGCAAGCCGACGCCCCCGCCTATCAGCAAGGGCAGAATCAGCTGTACAACGAGCTGAACCTGGGCAGAAGAGAGGAGTACGACGTGCTGGACAAGAGAAGAGGCAGAGACCCCGAGATGGGCGGCAAGCCTAGAAGAAAGAACCCCCAAGAGGG CCTGTTCAACGAGCTGCAGAAGGACAAGATGGCCGAGGCCTTCAGCGAGATCGGCATGAAGGGCGAGAGAAGAAGAGGCAAGGGCCACGACGGCCTGTTCCAAGGCCTGAGCACCGCCACCAAGGACACCTTCGACGCCCTGCACATGCAAGCCCTGCCCCTAGA[SEQ ID NO: 52] In some embodiments, the intracellular domain of the CAR further includes at least one co-stimulatory signal transduction region (CSR). In some embodiments, the at least one CSR includes a co-stimulatory molecule or a portion thereof. In some embodiments, the at least one CSR includes an intracellular domain of at least one co-stimulatory molecule or a portion thereof.

[0229] As used herein, a “co-stimulatory molecule” refers to a cell surface molecule other than an antigen receptor or its ligand that can provide an effective lymphocyte response to an antigen. Co-stimulatory molecules can provide optimal lymphocyte activation. Co-stimulatory molecules can bind to co-stimulatory ligands, which are proteins expressed on the cell surface that, upon binding to their receptors, produce a co-stimulatory response; that is, when a CAR binds to its target antigen (e.g., CD19 and CD20 for the CARs disclosed herein), an intracellular response to the provided stimulus is achieved. As an example, the 4-1BB ligand (i.e., 4-1BBL) can bind to 4-1BB to provide an intracellular signal that, in combination with CAR signaling, induces CAR activation. + T cell effector cell function.

[0230] Non-restricted examples of co-stimulatory molecules include CD28, 4-1BB (CD137), OX40, ICOS, DAP-10, CD27, CD28, CD30, CD40, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligands that specifically bind to CD83, CD8, CD4, B2C, CD80, CD86, DAP12, MyD88, BTNL3, NKG2D, TLR, IL-2Rβ, IL-15Rα, and combinations thereof.

[0231] In some embodiments, the intracellular domain of the CAR includes a co-stimulatory signaling region comprising a CD28 polypeptide, such as the intracellular domain of CD28 or a portion thereof. In some embodiments, the intracellular domain of the CAR includes a co-stimulatory signaling region comprising the intracellular domain of human CD28 or a portion thereof. In some embodiments, the CD28 polypeptide contained in the co-stimulatory signaling region of the CAR comprises or consists of an amino acid sequence that is at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%, or at least about 100% identical or homologous to or composed of the amino acid sequence shown in SEQ ID NO:35 or a fragment thereof, and / or may optionally include at most one, at most two, or at most three conserved amino acid substitutions. In some embodiments, the CD28 polypeptide contained in the co-stimulatory signal transduction region comprises or consists of an amino acid sequence as a continuous portion of SEQ ID NO: 35, having a length of at least about 20, or at least about 30, or at least about 40, or at least about 50, and at most about 220 amino acids. Alternatively or additionally, in some embodiments, the CD28 polypeptide contained in the co-stimulatory signal transduction region comprises or consists of amino acids 1 to 220, 1 to 50, 50 to 100, 100 to 150, 114 to 220, 150 to 200, 180 to 220, or 200 to 220 of SEQ ID NO: 35. In some embodiments, the intracellular domain of the CAR comprises a co-stimulatory signal transduction region containing a CD28 polypeptide comprising amino acids 180 to 220 of SEQ ID NO: 35 or consisting of them.

[0232] An exemplary nucleotide sequence encoding amino acids 180 to 220 of SEQ ID NO: 35 is shown in SEQ ID NO: 53 provided below.

[0233] AGAAGCAAGAGAAGCAGACTGCTGCACAGCGACTACATGAACATGACCCCTAGAAGACCCGGCCCCACAAGAAAGCACTATCAGCCCTACGCCCCCCCTAGAGACTTCGCCGCCTACAGAAGC[SEQ ID NO: 53] 5.3.4. Exemplary CAR "20-19 1XX" CAR like Figure 1 As shown, the “20-19 1XX” CAR contains (a) an extracellular domain, which includes (i) a first anti-CD19 antigen-binding domain, which includes: a first V HIt comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 9, CDR2 containing the amino acid sequence shown in SEQ ID NO: 10, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 11; and a first V L It comprises (ii) a CDR1 containing the amino acid sequence shown in SEQ ID NO: 12, a CDR2 containing the amino acid sequence shown in SEQ ID NO: 13, and a CDR3 containing the amino acid sequence shown in SEQ ID NO: 14, and (ii) a second anti-CD20 antigen binding domain comprising: a second V H It comprises CDR1 containing the amino acid sequence shown in SEQ ID NO: 21, CDR2 containing the amino acid sequence shown in SEQ ID NO: 22, and CDR3 containing the amino acid sequence shown in SEQ ID NO: 23; and the second V L The SEQ ID NO: 24 comprises: (a) a CDR1 containing the amino acid sequence shown in SEQ ID NO: 25, a CDR2 containing the amino acid sequence shown in SEQ ID NO: 25, and a CDR3 containing the amino acid sequence shown in SEQ ID NO: 26; (b) a hinge domain containing a CD28 polypeptide (e.g., a hinge domain of human CD28 or a portion thereof, such as a CD28 polypeptide composed of amino acids 114 to 152 of SEQ ID NO: 35); (c) a transmembrane domain containing a CD28 polypeptide (e.g., a transmembrane domain of human CD28 or a portion thereof, such as a CD28 polypeptide composed of amino acids 153 to 179 of SEQ ID NO: 35); and (d) an intracellular domain containing (i) a CD3ζ polypeptide (e.g., a modified human CD3ζ polypeptide, such as a CD3ζ polypeptide composed of the amino acid sequence shown in SEQ ID NO: 24), and (ii) a CD28 polypeptide (e.g., an intracellular domain of human CD28 or a portion thereof, such as a CD28 polypeptide composed of amino acids 114 to 179 of SEQ ID NO: 25). The co-stimulatory signal transduction region of the CD28 polypeptide (composed of 35 amino acids, ranging from 180 to 220). First V H And the first V L Linked via a first linker consisting of the amino acid sequence shown in SEQ ID NO: 1. In some embodiments, the first V H And the first V L Positioning from the N-terminus to the C-terminus: V L -V H In some implementations, the second V H Second V L Linked via a second linker consisting of the amino acid sequence shown in SEQ ID NO: 2. In some embodiments, the second VH Second V L Positioning from the N-terminus to the C-terminus: V H -V L The CAR also includes a third linker between a first anti-CD19 antigen-binding domain and a second anti-CD20 antigen-binding domain. The third linker consists of the amino acid sequence shown in SEQ ID NO: 3. The first anti-CD19 antigen-binding domain is located downstream of the second anti-CD20 antigen-binding domain. In some embodiments, the CAR also includes a leader peptide comprising a CD8 polypeptide (e.g., a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 33). In some embodiments, the leader peptide is covalently bound to the N-terminus of the second anti-CD20 antigen-binding domain. In some embodiments, the leader peptide is bound to the second V... H The N-terminus is linked. In some embodiments, the “20-19 1XX” CAR contains the amino acid sequence shown in SEQ ID NO: 54 provided below.

[0234] MALPVTALLLPLALLLHAARPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKGGGGSGGGGSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLFNELQKDKMAEAFSEIGMKGERRRGKGHDGLFQGLSTATKDTFDALHMQALPPR[SEQ ID NO: 54] An exemplary nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 54 is shown in SEQ ID NO: 55 provided below.

[0235] 5.4. cell The subject matter disclosed in this invention provides cells comprising a CD19 / CD20-targeting CAR (e.g., the CAR disclosed in Section 5.3). In some embodiments, the cells are selected from the group consisting of lymphoid and myeloid lineage cells. In some embodiments, the cells are immune-responding cells. In some embodiments, the immune-responding cells are lymphoid lineage cells.

[0236] In some embodiments, the cells are lymphoid lineage cells. Lymphoid lineage cells can provide antibody production, regulation of the cellular immune system, detection of foreign substances in the blood, and detection of foreign cells in the host. Non-limiting examples of lymphoid lineage cells include T cells, natural killer (NK) cells, B cells, dendritic cells, and stem cells from which lymphoid cells can differentiate. In some embodiments, the stem cells are pluripotent stem cells (e.g., embryonic stem cells or induced pluripotent stem cells).

[0237] In some embodiments, the cells are immune effector cells. Immune effector cells can be obtained from many sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. Immune effector cells can be obtained from blood collected from a subject using any number of techniques known to those skilled in the art, such as Ficoll™ isolation. For example, cells from an individual's circulating blood can be obtained via apheresis. In some embodiments, immune effector cells are isolated from peripheral blood lymphocytes by lysing red blood cells and consuming mononuclear cells, for example by centrifugation via a PERCOLL™ gradient or by countercurrent centrifugation. Specific subpopulations of immune effector cells can be further isolated using positive or negative selection techniques. For example, immune effector cells can be isolated using a combination of antibodies against surface markers specific to positively selected cells, for example by incubating with antibody-conjugated microbeads for a period sufficient for positive selection of the desired immune effector cells. Alternatively, enrichment of the immune effector cell population can be accomplished by negative selection using a combination of antibodies against surface markers specific to negatively selected cells.

[0238] In some embodiments, immune effector cells comprise any white blood cells involved in protecting the body from infectious diseases and foreign substances. For example, immune effector cells may include lymphocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, or any combination thereof. For example, immune effector cells may include T lymphocytes, preferably cytotoxic T lymphocytes (CTLs).

[0239] In some implementations, the cell is a T cell. T cells can be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells participate in the adaptive immune system. T cells, or T lymphocytes, can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of T cell receptors (TCRs) on their cell surface. Because they mature in the thymus, they are called T cells (although some also mature in the tonsils). Several subgroups of T cells exist, each with a different function.

[0240] T cells can be any type of T cell, including but not limited to helper T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem cell-like memory T cells (or stem cell-like memory T cells)), and two types of effector memory T cells: for example, T cells... EM Cells and T EMRA T cells), regulatory T cells (also known as suppressor T cells or T cells) reg The T cells include tumor-infiltrating lymphocytes (TILs), natural killer T cells (NK-T cells), mucosa-associated inertial T cells, αβ T cells, and γδ T cells. In some embodiments, the T cells are central memory T cells. Additionally, in some embodiments, the T cells are positive for CD45RO and CD62L.

[0241] In some embodiments, the cells comprising the CD19 / CD20-targeted CAR disclosed in this invention are helper T cells or CD4 cells. + T cells. Helper T cells (or T helper cells, or T cells) H These cells (CD4+ cells) assist other white blood cells in the immune process, including the maturation of B cells into plasma cells and memory B cells, and the activation of cytotoxic T cells and macrophages. These cells are also known as CD4+ cells. + T cells are characterized by the expression of the CD4 glycoprotein on their surface. Helper T cells are activated when they present peptide antigens to each other via MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they rapidly divide and secrete small proteins called cytokines, which regulate or assist in active immune responses. These cells can differentiate into one of several subtypes, including T cells. H 1. T H 2. T H 3. T H 17. T H 9 or T FH These subtypes secrete different cytokines to promote different types of immune responses.

[0242] In some embodiments, the cells comprising the CD19 / CD20-targeted CAR disclosed in this invention are cytotoxic T cells or CD8 cells.+ T cells. Cytotoxic T cells (TC cells or CTLs) destroy virus-infected cells and tumor cells, and are also involved in transplant rejection. These cells are also known as CD8+ cells. + T cells are characterized by the expression of the CD8 glycoprotein on their surface. These cells recognize their targets by binding to antigens associated with MHC class I molecules present on the surface of all nucleated cells. CD8 is also recognized by regulatory T cells through the secretion of IL-10, adenosine, and other molecules. + Cells can be inactivated to a non-allergenic state, which can prevent autoimmune diseases.

[0243] In some embodiments, the cells comprising the CD19 / CD20-targeted CAR disclosed in this invention are memory T cells. Memory T cells are a subgroup of antigen-specific T cells that persist long after infection resolution. Upon re-exposure to their homologous antigens, they rapidly proliferate into a large number of effector T cells, thereby providing the immune system with a “memory” against past infections. Memory cells may be CD4+. + or CD8 + Memory T cells typically express the cell surface protein CD45RO.

[0244] In some embodiments, the cells comprising the CD19 / CD20-targeted CAR disclosed in this invention are regulatory T cells. Regulatory T cells (T cells) reg CD4 cells (formerly known as suppressor T cells) are essential for maintaining immune tolerance. Their primary role is to shut down T cell-mediated immunity as the immune response nears its end and to suppress autoreactive T cells that evade negative selection processes in the thymus. Two main classes of CD4 cells have been described. + T reg Cells—naturally occurring T cells reg Cells and adaptive T reg cell.

[0245] In some embodiments, the cells comprising the CD19 / CD20-targeted CAR disclosed in this invention are natural killer T cells. Natural killer T (NKT) cells (not to be confused with natural killer (NK) cells) link the adaptive immune system to the innate immune system. Unlike conventional T cells, which recognize peptide antigens presented by major histocompatibility complex (MHC) molecules, NKT cells recognize glycolipid antigens presented by molecules called CD1d.

[0246] In some embodiments, the cells comprising the CD19 / CD20-targeting CAR disclosed herein are γδ T cells. γδ T cells have a unique T cell receptor (TCR) with one γ chain and one δ chain instead of α and β chains.

[0247] In some embodiments, the cells comprising the CD19 / CD20-targeted CAR disclosed in this invention are innate lymphoid cells (ILCs), cytokine-induced killer (CIK) cells, or lymphokine-activated killer (LAK) cells.

[0248] In some embodiments, the cell is a T cell, and the CD19 / CD20-targeting CAR disclosed herein is integrated at a locus within the T cell genome. Non-limiting examples of loci include the TRAC locus, TRBC locus, TRDC locus, and TRGC locus. In some embodiments, the locus is either the TRAC locus or the TRBC locus. Methods for targeting CARs to sites within the T cell genome are disclosed in WO2017180989 and Eyquem et al. Nature. In (2017 Mar 2);543(7643): 113-117, both are incorporated in their entirety by reference.

[0249] In some implementations, T cells contain CD4 + T cells and CD8 + A mixture of T cells. In some implementations, one or more subgroups of T cells are enriched based on cell surface marker expression profiles.

[0250] In some implementations, the cells are natural killer (NK) cells. NK cells can be lymphocytes, which are part of cell-mediated immunity and play a role during innate immune responses. NK cells do not require prior activation to exert their cytotoxic effect on target cells. (The last sentence appears to be incomplete and possibly refers to a different implementation, possibly related to CD8 cytotoxicity.) + Unlike T cells, NK cells can initiate cytotoxicity against tumor cells without prior sensitization and can also eradicate MHC-I negative cells. In some embodiments, the cells are genetically modified NK cells. In some embodiments, the cells are edited NK cells. In some embodiments, the cells are NK cells derived from stem cells. In some embodiments, the cells are NK cells derived from pluripotent stem cells. In some embodiments, the cells are NK cells derived from induced pluripotent stem cells (iPSCs).

[0251] The cells disclosed in this invention (e.g., T cells or NK cells) can be autologous (e.g., cells derived from / obtained from a subject receiving cells expressing CAR), non-autologous (e.g., allogeneic) (e.g., cells derived from / obtained from a donor who is not receiving cells expressing CAR), or derived in vitro from engineered progenitor cells or stem cells. In some embodiments, cells comprising the CD19 / CD20-targeting CAR disclosed in this invention are obtained from a subject receiving cells expressing CAR (e.g., a subject to be treated) (i.e., autologous). In some embodiments, cells comprising the CD19 / CD20-targeting CAR disclosed in this invention are allogeneic to the subject receiving cells expressing CAR (e.g., a subject to be treated).

[0252] The cells disclosed in this invention can be myeloid lineage cells. Non-limiting examples of myeloid lineage cells include monocytes, macrophages, neutrophils, dendritic cells, basophils, neutrophils, eosinophils, megakaryocytes, mast cells, erythrocytes, platelets, and stem cells from which myeloid cells can differentiate. In some embodiments, the stem cells are pluripotent stem cells (e.g., embryonic stem cells or induced pluripotent stem cells).

[0253] Epstein-Barr virus (EBV)-induced lymphoproliferative disorder (EBV-LPD) and other EBV-related cancers are significant causes of morbidity and mortality in recipients of allogeneic hematopoietic cell transplantation (HCT) or solid organ transplantation (SOT), particularly in those who have already received certain T-cell reactive antibodies for the prevention or treatment of GVHD. Prophylaxis and treatment via adoptive transfer of autologous or allogeneic EBV-specific cytotoxic T cells, followed by long-term restoration of immunity against EBV-related lymphoproliferation, have provided positive outcomes in managing these equally deadly complications of allogeneic tissue transfer. Therefore, in some embodiments, the disclosed immune effector cells comprising one or more of the CAR peptides of this invention are allogeneic or autologous EBV-specific cytotoxic T lymphocytes (CTLs). For example, the generation of EBV-specific cytotoxic T cells may involve isolating PBMCs from an EBV-seropositive autologous or allogeneic donor and enriching the T cells therein by consuming monocytes and NK cells. EBV-specific cytotoxic T cells can also be generated by contacting donor PBMCs or purified donor T cells with “stimulatory” cells that express one or more EBV antigens and present EBV antigens to unstimulated T cells, thereby stimulating and expanding EBV-specific CTLs. EBV antigens include, for example, latent membrane proteins (LMPs) and EBV nuclear antigens (EBNAs), such as LMP-1, LMP-2A, and LMP-2B, and EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, and EBNA-LP. Cytotoxic T cells containing T cell receptors that recognize one or more EBV-specific antigens are considered to have been “sensitized” to those EBV antigens and are therefore referred to herein as “EBV-sensitized cytotoxic T cells.” Known methods for generating allogeneic or autologous EBV-specific cytotoxic T cell populations that may contain one or more of the CAR peptides of this invention are described, for example, by Barker et al. Blood 116(23):5045-49 (2010); Doubrovina et al., Blood 119(11):2644-56 (2012); Koehne et al., Blood 99(5):1730-40(2002); and Smith et al., Cancer Res.72(5):1116-25 (2012), which is incorporated herein by reference for these teachings. Similarly, cytotoxic T cells can be “sensitized” to other viral antigens, including cytomegalovirus (CMV), papillomavirus (e.g., HPV), adenovirus, polyomavirus (e.g., BKV, JCV, and Merkel cell virus), retrovirus (e.g., HTLV-I, also including lentiviruses such as HIV), small RNA viruses (e.g., hepatitis A virus), hepatotropic DNA viruses (e.g., hepatitis B virus), hepatitis C virus (e.g., hepatitis C virus), delta virus (e.g., hepatitis D virus), hepatitis E virus (e.g., hepatitis E virus), etc. In some embodiments, the T cells used to generate the CAR-T cells of the present invention are pluripotent T cells, i.e., those T cells capable of inducing multiple immune effector functions, which provide a more effective immune response to pathogens than cells that generate, for example, only a single immune effector (e.g., a single biomarker such as cytokines or CD107a). Low-functionality, monofunctional, or even "depleted" T cells may dominate the immune response during chronic infection, thus negatively impacting protection against virus-related complications. In some embodiments, the CAR-T cells disclosed in this invention are multifunctional. In some embodiments, at least 50% of the T cells used to generate the CAR-T cells disclosed in this invention are CD4+. + T cells. In some implementations, less than 50% of T cells are CD4+. + T cells. In some implementations, T cells are primarily CD4+ cells. + T cells. In some embodiments, at least 50% of the T cells used to generate the CAR-T cells disclosed in this invention are CD8+. + T cells. In some implementations, less than 50% of T cells are CD8+. + T cells. In some implementations, T cells are primarily CD8+ cells. + T cells. In some implementations, T cells (e.g., the sensitized T cells and / or CAR-T cells described herein) are stored in a cell library or repository before being administered to a subject.

[0254] In some embodiments, cells expressing the CD19 / CD20-targeting CAR disclosed in this invention further express dominant-negative mutations affecting immune checkpoint blockade. Non-limiting examples of immune checkpoint molecules include programmed death 1 (PD-1), cytotoxic T-lymphocyte antigen-4 (CTLA-4), B and T lymphocyte attenuators (BTLA), T-cell immunoglobulin mucin-3 (TIM-3), lymphocyte activating protein 3 (LAG-3), T-cell immune receptor with Ig and ITIM domains (TIGIT), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), natural killer cell receptor 2B4 (2B4), CD160, and transforming growth factor B (TGF-β) receptors. In some embodiments, the immune checkpoint molecule is CTLA-4. In some embodiments, the immune checkpoint molecule is PD-1. A method for engineering dominant-negative forms of CAR-T cells that express inhibitors of cell-mediated immune responses is described in International Patent Publication WO2017 / 040945, which is incorporated herein by reference in its entirety.

[0255] In some embodiments, cells can be transduced with the CD19 / CD20-targeted CAR disclosed in this invention, so that the cells express the CD19 / CD20-targeted CAR.

[0256] 5.5. Nucleic acid and vector The subject matter disclosed in this invention provides nucleic acids encoding the CD19 / CD20-targeting CAR disclosed herein and cells comprising such nucleic acids. In some embodiments, the nucleic acids further comprise a promoter operatively linked to the nucleic acid encoding the CD19 / CD20-targeting CAR disclosed herein.

[0257] In some embodiments, the promoter is endogenous or exogenous. In some embodiments, the exogenous promoter is selected from the group consisting of: elongation factor (EF)-1 promoter, cytomegalovirus immediate early promoter (CMV) promoter, simian virus 40 early promoter (SV40) promoter, phosphoglycerate kinase (PGK) promoter, metallothionein promoter, and ubiquitin C promoter. In some embodiments, the endogenous promoter is selected from the group consisting of: TCR α promoter, TCR β promoter, and β2-microglobulin promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter is selected from the group consisting of: NFAT transcriptional response element (TRE) promoter, CD69 promoter, CD25 promoter, IL-2 promoter, 4-1BB promoter, PD1 promoter, and LAG3 promoter.

[0258] The subject matter of this invention also provides vectors comprising the nucleic acids disclosed herein. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a retroviral vector.

[0259] Nucleic acids can be delivered into cells by methods known in the art or as described herein. Genetic modification of cells can be accomplished by transducing substantially homogeneous cellular compositions with recombinant DNA constructs. In some embodiments, retroviral vectors (e.g., gamma retroviral vectors or lentiviral vectors) are used to introduce the DNA constructs into cells. For example, nucleic acids disclosed in this invention can be cloned into retroviral vectors and expression can be driven from their endogenous promoters, from retroviral long terminal repeat sequences, or from promoters specific to the target cell type of interest.

[0260] Expression of CAR-encoding nucleic acids is typically achieved by operatively linking a nucleic acid encoding a CAR polypeptide to a promoter and incorporating the construct into an expression vector. A typical cloning vector contains transcription and translation terminators, a start sequence, and a promoter that can be used to regulate the expression of the desired nucleic acid sequence.

[0261] The disclosed nucleic acids can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phage particles, phage derivatives, animal viruses, and entrapments. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0262] Furthermore, expression vectors can be provided to cells in the form of viral vectors. Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Typically, a suitable vector contains a replication origin that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers. In some embodiments, the polynucleotide vector is a lentiviral or retroviral vector.

[0263] Numerous virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to the recipient's cells in vivo or in vitro.

[0264] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operatively linked to it. Another example of a suitable promoter is extension growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the simian virus 40 (SV40) early promoter, the MND (myeloproliferative sarcoma virus) promoter, the mouse mammary tumor virus (MMTV), the human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, the MoMuLV promoter, the avian leukosis virus promoter, the Epstein-Barr virus immediate early promoter, the Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Alternatively, the promoter may be an inducible promoter. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0265] Other promoter elements, such as enhancers, regulate the frequency of transcription initiation. These elements are typically located in a region 30–110 bp upstream of the start site, although recent studies have shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is usually flexible, allowing promoter function to be preserved when the elements are inverted or moved relative to each other.

[0266] To assess the expression of CAR peptides or portions thereof, the expression vector to be introduced into cells may also contain a selectable marker gene or a reporter gene, or both, to identify and select expressing cells from a population of cells attempting to be transfected or infected via a viral vector. In other respects, the selectable marker may be carried on a separate DNA fragment and used in co-transfection procedures. Both selectable markers and reporter genes can be side-linked with appropriate regulatory sequences to enable their expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes.

[0267] Reporter genes are used to identify potentially transfected cells and to assess the function of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue; the expression of the polypeptide encoded by this gene is indicated by easily detectable properties, such as enzyme activity. Reporter gene expression is measured at an appropriate time after DNA is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, or green fluorescent protein genes. Suitable expression systems are well-known and can be prepared or commercially available using known techniques. Typically, constructs exhibiting the highest expression level of the reporter gene with a minimum 5' flanking region are identified as promoters. Such promoter regions can be linked to reporter genes and used to assess the ability of these genes to regulate promoter-driven transcription.

[0268] Methods for introducing and expressing genes in cells are known in the art. In the context of expression vectors, vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0269] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York).

[0270] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian cells (e.g., human cells).

[0271] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, microbeads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery medium in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0272] In the case of using non-viral delivery systems, an exemplary delivery medium is liposomes. On the other hand, nucleic acids can associate with lipids. Lipid-associated nucleic acids can be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, attached to liposomes via linker molecules associated with both liposomes and oligonucleotides, embedded in liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained in lipids as a suspension, contained in or complexed with micelles, or otherwise associated with lipids. Compositions of lipids, lipid / DNA, or lipid / expression vector associations are not limited to any particular structure in solution. For example, they can exist in a bilayer structure, as micelles, or have a “collapsed” structure. They can also simply be dispersed in solution, possibly forming aggregates of non-uniform size or shape. Lipids are fatty substances, which can be naturally occurring or synthetic. For example, lipids include lipid droplets naturally occurring in the cytoplasm and classes of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Suitable lipids can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) is available from Sigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) is available from K & K Laboratories (Plainview, NY); cholesterol (“Choi”) is available from Calbiochem-Behring; and dimyristyl phosphatidylglycerol (“DMPG”) and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, Ala.).

[0273] Any targeted genome editing method can also be used to deliver the CD19 / CD20-targeted CAR disclosed in this invention to cells. In some embodiments, a CRISPR system is used to deliver the CD19 / CD20-targeted CAR disclosed in this invention. In some embodiments, zinc finger nucleases are used to deliver the CD19 / CD20-targeted CAR disclosed in this invention. In some embodiments, a TALEN system is used to deliver the CD19 / CD20-targeted CAR disclosed in this invention.

[0274] The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) system is a genome editing tool found in prokaryotic cells. When used for genome editing, this system comprises Cas9 (a protein that uses crRNA as its guide to modify DNA), CRISPR RNA (crRNA, containing RNA that Cas9 uses to guide it to the correct segment of host DNA, and a region that binds to tracrRNA (usually in the form of a hairpin loop), forming an active complex with Cas9), trans-activating crRNA (tracrRNA, which binds to crRNA and forms an active complex with Cas9), and an optional segment of the DNA repair template (DNA that guides the cellular repair process, allowing the insertion of a specific DNA sequence). CRISPR / Cas9 is typically transfected into target cells using plasmids. The crRNA needs to be designed for each application because it is the sequence that Cas9 uses to identify and directly bind to the target DNA in the cell. The repair template carrying the CAR expression cassette also needs to be designed for each application because it must overlap the sequences on both sides of the nick and encode the insert sequence. Multiple crRNAs and tracrRNAs can be packaged together to form a single guide RNA (sgRNA). This sgRNA can be combined with the Cas9 gene and made into a plasmid for transfection into cells.

[0275] Zinc finger nucleases (ZFNs) are artificial restriction endonucleases generated by combining a zinc finger DNA-binding domain with a DNA-cutting domain. The zinc finger domain can be engineered to target specific DNA sequences, allowing the zinc finger nuclease to target desired sequences within the genome. The DNA-binding domain of each ZFN typically contains multiple individual zinc finger repeats, each capable of recognizing multiple base pairs. The most common method for generating novel zinc finger domains is by combining smaller zinc finger “modules” with known specificity. The most common cutting domain in ZFNs is the non-specific cutting domain derived from the type IIs restriction endonuclease FokI. Using endogenous homologous recombination (HR) mechanisms and homologous DNA templates carrying CAR expression cassettes, ZFNs can be used to insert CAR expression cassettes into the genome. When the target sequence is cleaved by a ZFN, the HR mechanism searches for homology between the damaged chromosome and the homologous DNA template, then replicates the template sequence between the two broken ends of the chromosome, thereby integrating the homologous DNA template into the genome.

[0276] Transcription activator-like effector nucleases (TALENs) are restriction endonucleases that can be engineered to cleave specific sequences of DNA. TALEN systems operate on nearly the same principle as ZFNs. They are generated by combining a transcription activator-like effector DNA-binding domain with a DNA-cleaving domain. A transcription activator-like effector (TALE) consists of a 33-34 amino acid repeating motif with two variable positions that strongly recognize specific nucleotides. By assembling arrays of these TALEs, the TALE DNA-binding domain can be engineered to bind to the desired DNA sequence, thereby guiding the nuclease to cleave at a specific location in the genome. cDNA expression in polynucleotide therapy approaches can be directed by any suitable promoter (e.g., human cytomegalovirus (CMV), simian virus 40 (SV40), metallothionein promoter, or ubiquitin C promoter) and regulated by any suitable mammalian regulatory element or intron (e.g., elongation factor 1a enhancer / promoter / intron structure). For example, enhancers known to preferentially direct gene expression in specific cell types can be used to direct nucleic acid expression if desired. The enhancers used may include, but are not limited to, those characterized as tissue- or cell-specific enhancers. Alternatively, if the genomic clone is used as a therapeutic construct, regulation may be mediated by homologous regulatory sequences, or, if desired, by regulatory sequences derived from heterologous sources, including any of the promoters or regulatory elements described above.

[0277] The methods used to deliver genome editing agents / systems can vary as needed. In some embodiments, components of the selected genome editing method are delivered as DNA constructs in one or more plasmids. In some embodiments, components are delivered via viral vectors. Common delivery methods include, but are not limited to, electroporation, microinjection, gene gun, impale infection, hydrostatic pressure, continuous infusion, sonication, magnetic transfection, adeno-associated virus, pseudotyped envelope proteins of viral vectors, cis- and trans-acting elements of vectors with replication capabilities, herpes simplex virus, and chemical vectors (e.g., oligonucleotides, lipid complexes, polymeric vesicles, polymeric complexes, dendritic polymers, inorganic nanoparticles, and cell-penetrating peptides).

[0278] 5.6. Preparation and application The subject matter of this invention further provides compositions comprising the cells disclosed herein, the cells comprising the CD19 / CD20-targeting CARs disclosed herein. In some embodiments, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0279] The compositions disclosed in this invention can be readily provided as sterile liquid preparations, such as isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which can be buffered to a selected pH. Liquid preparations are generally easier to prepare than gels, other viscous compositions, and solid compositions. Additionally, liquid compositions are more convenient to administer, especially by injection. On the other hand, viscous compositions can be formulated within a suitable viscosity range to provide a longer contact time with a specific tissue. Liquid or viscous compositions may contain a carrier, which can be a solvent or dispersion medium containing, for example, water, saline, phosphate-buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof.

[0280] Compositions comprising the cells disclosed herein can be administered systemically or directly to subjects in need. In some embodiments, the compositions disclosed herein are injected directly into the organ of interest. Alternatively, the compositions disclosed herein are administered indirectly to the organ of interest, for example, by administration to the circulatory system (e.g., the tumor vascular system). Amplification and differentiation agents may be provided before, during, or after the administration of the composition to increase the production of cells in vitro or in vivo.

[0281] The amount of CD19 / CD20-targeting CAR cells contained in the compositions disclosed in this invention can vary depending on the treated subject. In some embodiments, the compositions disclosed in this invention contain about 10 4 With about 10 10 Between, in about 10 4 With about 10 7 Between, in about 10 5 With about 10 7 Between, in about 10 5 With about 10 9 Between or around 10 6 With about 10 8 The present invention discloses cells containing CD19 / CD20-targeting CARs. In some embodiments, the compositions disclosed herein contain about 1 × 10⁻⁶ cells. 6 With approximately 5×10 8 The present invention discloses cells containing CD19 / CD20-targeting CARs. In some embodiments, the compositions disclosed herein contain about 1 × 10⁻⁶ cells. 6 With approximately 1×10 8 The present invention discloses cells containing CD19 / CD20-targeting CARs. In some embodiments, the compositions disclosed herein contain about 1 × 10⁻⁶ cells. 6 With approximately 5×10 7The present invention discloses cells containing CD19 / CD20-targeting CARs. In some embodiments, the compositions disclosed herein contain about 1 × 10⁻⁶ cells. 6 With approximately 1×10 7 The present invention discloses cells comprising CD19 / CD20-targeting CARs. More effective cells can be administered in even smaller quantities. Typically, at least about 1 × 10⁻⁶ cells will be administered. 5 10 cells, eventually reaching approximately 1 × 10⁻⁶ cells. 10 Or more. In some embodiments, the compositions disclosed in this invention contain at least about 1 × 10⁻⁶. 5 5×10 5 1×10 6 Approximately 5×10 6 Approximately 1×10 7 Approximately 2.5 × 10 7 Approximately 5×10 7 Approximately 1×10 8 Approximately 1.5 × 10 8 Approximately 2×10 8 Or approximately 5×10 8 This invention discloses a cell containing a CD19 / CD20-targeting CAR. In some embodiments, the composition disclosed in this invention contains about 1 × 10⁻⁶ cells. 6 This invention discloses cells containing CD19 / CD20-targeting CARs. The precise determination of what is considered an effective dose can be based on individual factors for each subject, including their size, age, sex, weight, and the specific condition of the subject. Those skilled in the art can readily determine the dose based on this disclosure and their knowledge in the art. The composition can be administered multiple times at these doses.

[0282] The CD19 / CD20-targeted CAR-modified cells disclosed in this invention can be administered alone or as a pharmaceutical composition in combination with diluents and / or other components such as IL-2, IL-15, or other cytokines or cell populations. In short, the pharmaceutical composition may comprise a target cell population as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may contain buffers such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. In some embodiments, the compositions used in the disclosed methods are formulated for intravenous administration. The amount and frequency of administration will be determined by factors such as the patient's condition and the severity of the patient's disease, although appropriate dosage can be determined through clinical trials.

[0283] The disclosed compositions can be administered in any convenient manner, including by injection, transfusion, or implantation. The compositions disclosed herein can be administered by any method known in the art, including but not limited to intravenous administration, intradermal administration, subcutaneous administration, intranodular administration, intratumoral administration, intramedullary administration, intramuscular administration, intrathecal administration, intrapleural administration, intraosseous administration, intraperitoneal administration, pleural administration, and direct administration to a subject. In some embodiments, the composition is administered intravenously to the subject. The composition can also be injected directly into a tumor, lymph node, or disease site.

[0284] 5.7. How to use The CD19 / CD20-targeted CAR disclosed in this invention can elicit a therapeutically beneficial immune response against B lymphocyte antigens (e.g., CD19 and / or CD20). For example, the anti-tumor immune response induced by cells modified with the disclosed CAR can be an active or passive immune response. Furthermore, the CAR-mediated immune response can be part of an adoptive immunotherapy approach in which CAR-modified cells induce an immune response specific to at least one B lymphocyte antigen (e.g., CD19 and / or CD20).

[0285] Adoptive transfer of CAR-expressing cells is a promising anticancer therapy. After collecting cells from a subject, the cells can be genetically engineered to express the CD19 / CD20-targeting CAR disclosed in this invention, and then infused back into the subject (i.e., autologous to the subject). Alternatively, cells obtained from a donor other than the subject (i.e., allogeneic to the subject) can be genetically engineered to express the CD19 / CD20-targeting CAR disclosed in this invention, and then the CAR-containing cells are infused into the subject. In some embodiments, the cells containing the CD19 / CD20-targeting CAR disclosed in this invention are allogeneic EBV-specific cytotoxic T cells (CTLs).

[0286] Embodiments of the present invention provide methods for using the cells or compositions disclosed herein. According to one or more embodiments, the cells or compositions disclosed herein can be used in therapies or pharmaceuticals. For example, the cells and compositions of the disclosed present invention expressing CD19 / CD20-targeting CARs can be used to induce and / or enhance immune responses in subjects of need. Therefore, the subject matter of this invention provides methods for inducing and / or enhancing immune responses in subjects of need. In some embodiments, the method includes administering to a subject of need cells or compositions comprising the disclosed present invention that contain the CD19 / CD20-targeting CAR.

[0287] Furthermore, in various embodiments, the cells and compositions expressing CD19 / CD20-targeting CARs disclosed in this invention can be used to treat B-lymphocyte antigen-related diseases, conditions, or illnesses, including one or more symptoms of the disease, condition, or illness. Therefore, the subject matter disclosed in this invention provides methods for treating B-lymphocyte antigen-related diseases, conditions, or illnesses. In some embodiments, the method includes administering to a subject in need the cells disclosed in this invention comprising the CD19 / CD20-targeting CAR disclosed in this invention, or a composition comprising the CAR disclosed in this invention.

[0288] Furthermore, in various embodiments, the cells and compositions of the CD19 / CD20-targeting CAR disclosed in this invention can inhibit the growth of target cells expressing at least one B lymphocyte antigen (e.g., CD19, CD20, or a combination thereof). Therefore, the subject matter disclosed in this invention provides a method for inhibiting the growth of target cells expressing at least one B lymphocyte antigen (e.g., CD19, CD20, or a combination thereof). In some embodiments, the method includes contacting the target cells with cells expressing the CD19 / CD20-targeting CAR disclosed in this invention or compositions containing thereof. The target cells may express CD19 and / or CD20 at different levels. In some embodiments, the target cells express detectable levels of CD19 and detectable levels of CD20. In some embodiments, the target cells express detectable levels of CD20 and low or undetectable levels of CD19. In some embodiments, the target cells express detectable levels of CD19 and low or undetectable levels of CD20. In some embodiments, the subject has or has been diagnosed with a CD19 / CD20-related disease, condition, or ailment.

[0289] In some implementations, the target cells are tumor cells, such as cancerous tumor cells. In some implementations, the tumor is cancer. In some implementations, the cancer is a blood cancer. Non-limiting examples of blood cancers include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, mantle cell lymphoma (MCL), and follicular lymphoma (FL).

[0290] In some implementations, the target cells are B cells. In some implementations, the B cells are CD19 cells. + CD20 + CD19 + CD20 - Or CD19 - CD20 + .

[0291] In some implementations, the target cells are T cells. In some implementations, the T cells are CD19 cells. + CD20 + CD19 + CD20 - Or CD19 - CD20 + In some implementations, T cells are CD20. + .

[0292] The cells or compositions disclosed in this invention exhibit cytotoxic and / or cytolytic activity against target cells. In some embodiments, the cytotoxic and / or cytolytic activity is dose-dependent. In some embodiments, the cytotoxic and / or cytolytic activity is long-lasting. For example, the cytotoxic and / or cytolytic activity may persist for at least 24 hours, at least 72 hours, at least one week, at least one month, or at least two months after initial contact of the target cells with the cells or composition. In some embodiments, the cytotoxic and / or cytolytic activity persists for about 40 days (e.g., 35 days, 36 days, 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, or 45 days) after initial contact of the target cells with the cells or composition. In some embodiments, the cytotoxic and / or cytolytic activity persists for about 60 days (e.g., 55 days, 56 days, 57 days, 58 days, 59 days, 60 days, 61 days, 62 days, 63 days, 64 days, or 65 days) after initial contact of the target cells with the cells or composition.

[0293] In some embodiments, cells are administered in an effective amount to treat one or more diseases or conditions, such as cancer and autoimmune diseases as described herein. As used herein, the term "effective amount" refers to an amount sufficient to affect a beneficial or desired clinical outcome at the time of treatment. An effective amount may be administered to a subject in one or more doses, once or in a series of administrations, and / or by bolus or continuous perfusion. In some embodiments, an effective amount may be an amount sufficient to alleviate, improve, stabilize, reverse, or slow disease progression or otherwise reduce the pathological consequences of the disease. An effective amount may be determined by a physician on a case-by-case basis and within the skill of a person skilled in the art. Several factors are typically considered when determining the appropriate dose to achieve an effective amount. An effective amount may be approximately 10 4 With about 10 10 Between, in about 10 4 With about 10 7 Between, in about 10 5 With about 10 7 Between, in about 10 5 With about 10 9 Between or around 10 6With about 10 8 The present invention discloses cells containing CD19 / CD20-targeting CARs. In some embodiments, the effective amount is about 1 × 10⁻⁶. 6 With approximately 5×10 8 The present invention discloses cells containing CD19 / CD20-targeting CARs. In some embodiments, the effective amount is about 1 × 10⁻⁶. 6 With approximately 1×10 8 The present invention discloses cells containing CD19 / CD20-targeting CARs. In some embodiments, the effective amount is about 1 × 10⁻⁶. 6 With approximately 5×10 7 The present invention discloses cells containing CD19 / CD20-targeting CARs. In some embodiments, the effective amount is about 1 × 10⁻⁶. 6 With approximately 1×10 7 The present invention discloses cells comprising CD19 / CD20-targeting CARs. In some embodiments, the effective amount is at least about 1 × 10⁻⁶. 5 5×10 5 1×10 6 Approximately 5×10 6 Approximately 1×10 7 Approximately 2.5 × 10 7 Approximately 5×10 7 Approximately 1×10 8 Approximately 1.5 × 10 8 Approximately 2×10 8 Or approximately 5×10 8 This invention discloses a cell containing a CD19 / CD20-targeting CAR. In some embodiments, the effective amount is about 1 × 10⁻⁶. 6 This invention discloses cells comprising CD19 / CD20-targeted CARs. These factors include the subject's age, sex, and weight, the condition being treated, the severity of the condition, and the form and effective concentration of the cells administered.

[0294] In some embodiments, the B-lymphocyte antigen-associated disease or condition expresses CD19, CD20, or both. In some embodiments, the B-lymphocyte antigen-associated disease or condition is a tumor. In some embodiments, the B-lymphocyte antigen-associated disease or condition is cancer. In some embodiments, the B-lymphocyte antigen-associated disease or condition is a blood cancer. In some embodiments, the blood cancer is selected from the group consisting of: acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, mantle cell lymphoma (MCL), and follicular lymphoma (FL).

[0295] In some implementations, B-lymphocyte antigen-related disease can be any vegetation or tumor of hematopoietic and / or lymphoid tissue. Therefore, B-lymphocyte antigen-related disease can be any malignancy affecting the blood, bone marrow, lymph, and / or lymphoid system; and any such disease that causes unregulated myeloproliferation and / or lymphoid tissue proliferation. Non-limiting examples of B-lymphocyte antigen-related diseases or conditions include leukemias (including, but not limited to, acute leukemia, chronic leukemia, lymphocytic leukemia, and myeloid leukemia), preleukemic conditions, and lymphomas (including, but not limited to, Hodgkin's lymphoma and non-Hodgkin's lymphoma), such as EBV-related lymphoproliferative disorders, mature B-cell tumors, mature T-cell tumors and natural killer (NK) cell tumors, precursor lymphoid tumors, and immunodeficiency-associated lymphoproliferative disorders.

[0296] In some implementations, B-lymphocyte antigen-related diseases are selected from the group consisting of: acute lymphoblastic leukemia (ALL), precursor B acute lymphoblastic leukemia, precursor T acute lymphoblastic leukemia, Burkitt leukemia, acute biphenotypic leukemia, chronic lymphocytic leukemia (CLL), B-cell prolymphoblastic leukemia, acute myeloid leukemia (AML), acute promyelocytic leukemia (PML), acute myeloblastic leukemia, acute megakaryoblastic leukemia (AMKL), chronic myeloid leukemia (CML), chronic myelomonocytic leukemia, hairy cell leukemia (HCL), and T-cell prolymphoblastic leukemia. Leukemia (T-PLL), large granular lymphocytic leukemia, adult T-cell leukemia, clonal eosinophilia, B-cell chronic lymphocytic leukemia / small cell lymphoma, lymphoplasmacytic lymphoma (such as Waldenström macroglobulinemia), splenic marginal zone lymphoma, plasma cell tumors such as plasma cell myeloma (multiple myeloma), plasmacytoma, monoclonal immunoglobulin deposition disease, heavy chain disease, extranodal marginal zone B-cell lymphoma (MALT lymphoma), intranodal marginal zone B-cell lymphoma, follicular lymphoma (FL), primary cutaneous follicular center lymphoma, mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL) (with or without chronic inflammation), Epstein-Barr virus-positive DLBCL in the elderly, lymphomatoid granulomatosis, primary mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, ALK + Large B-cell lymphoma, plasmablastic lymphoma, primary exudative lymphoma, large B-cell lymphoma caused by HHV8-associated multicentric Castleman disease, Burkitt lymphoma, T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, aggressive NK-cell leukemia, adult T-cell leukemia / lymphoma, extranodal NK / T-cell lymphoma, enteropathy-associated T-cell lymphoma, hepatocellular and splenic T-cell lymphoma, blastic NK-cell lymphoma, mycosis fungoides / Cezari syndrome, primary cutaneous CD30-positive T-cell lymphoproliferative disorder, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma, anaplastic large B-cell lymphoma Cytokine lymphoma, nonspecific B-cell lymphoblastic leukemia / lymphoma, B-cell lymphoblastic leukemia / lymphoma with relapsed genetic abnormalities, T-cell lymphoblastic leukemia / lymphoma; classical Hodgkin lymphoma, such as tuberous sclerosis form, mixed cellular Hodgkin lymphoma, lymphocytic Hodgkin lymphoma, lymphocyte-consuming or non-consuming Hodgkin lymphoma, nodular predominantly lymphocyte Hodgkin lymphoma, and those associated with primary immune disorders, human immunodeficiency virus (HIV), methotrexate therapy, post-transplant immunodeficiency-associated lymphoid tissue proliferative disorders, and primary central nervous system lymphoma.

[0297] In some implementations, the subject is a human subject. The subject may have an advanced form of the disease, in which case treatment goals may include alleviating or reversing disease progression and / or improving side effects. The subject may have a history of having received prior treatment for their condition, in which case treatment goals typically include reducing or delaying the risk of relapse.

[0298] As a result of the surface expression of the CD19 / CD20-targeted CAR disclosed in this invention, the adopted cells are endowed with enhanced and selective cytolytic activity at the tumor site. Furthermore, after they localize to the tumor and proliferate, these cells transform the tumor site into a highly conductive environment for a wide range of cells involved in physiological antitumor responses.

[0299] In some embodiments, the cells and compositions disclosed herein are administered to a subject in combination with any number of relevant therapeutic modalities (e.g., before, during, or after them). In some embodiments, the cells and compositions disclosed herein may be used in combination with chemotherapy, radiation, immunosuppressants, immune scavengers, cytotoxins, and cytokines.

[0300] According to one or more embodiments, the cells and compositions disclosed in this invention can be used in combination with any compound, portion, or group having cytotoxic or cell-inhibiting effects. The pharmaceutical portion includes chemotherapeutic agents that can be used as microtubule inhibitors, mitosis inhibitors, topoisomerase inhibitors, or DNA intercalators, particularly those used in cancer therapy.

[0301] The cells and compositions disclosed in this invention can be used in combination with checkpoint inhibitors. Two known inhibitory checkpoint pathways involve signaling via the cytotoxic T-lymphocyte antigen-4 (CTLA-4) and programmed death 1 (PD-1) receptors. These proteins are members of the CD28-B7 family of co-signaling molecules, which play important roles in all stages of T cell function. The PD-1 receptor (also known as CD279) is expressed on the surface of activated T cells. Its ligands PD-L1 (B7-H1; CD274) and PD-L2 (B7-DC; CD273) are expressed on the surface of APCs such as dendritic cells or macrophages. PD-L1 is the major ligand, while PD-L2 has a more restricted expression pattern. When the ligand binds to PD-1, an inhibitory signal is transmitted to the T cell, which reduces cytokine production and inhibits T cell proliferation. Checkpoint inhibitors include, but are not limited to, antibodies that block PD-1 (e.g., nivolumab (BMS-936558 or MDX1106), CT-011, MK-3475), PD-L1 (e.g., MDX-1105 (BMS-936559), MPDL3280A, MSB0010718C), PD-L2 (e.g., rHIgM12B7), CTLA-4 (e.g., ipilimumab (MDX-010), trimemumab (e.g., CP-675,206)), IDO, B7-H3 (e.g., MGA271), B7-H4, TIM3, and LAG-3 (e.g., BMS-986016). Techniques for combining CARs with checkpoint inhibitors in immune effector cells and their use in the treatment of various conditions are described, for example, in WO 2017 / 040945, which is incorporated herein by reference.

[0302] T-cell receptor activation coupled with co-stimulation can help generate optimal “killing” CD8 T-cell responses, which can be provided through linkages with members of the tumor necrosis factor receptor family, including OX40 (CD134) and 4-1BB (CD137). OX40 is of particular interest because treatment with activating (agonist) anti-OX40 mAbs enhances T-cell differentiation and cytolysis, leading to enhanced anti-tumor immunity against a variety of tumors.

[0303] In some embodiments, the method disclosed in this invention includes administering a second therapeutic agent to a subject. In some embodiments, the second therapeutic agent is an antimetabolite, such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, dacarbazine, hydroxyurea, asparaginase, gemcitabine, or cladribine.

[0304] In some implementations, the second therapeutic agent is an alkylating agent, such as nitrogen mustard, thiotepa, chlorambucil, melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin, and other platinum derivatives, such as carboplatin.

[0305] In some implementations, the second therapeutic agent is an antimitotic agent, such as taxanes, like docetaxel and paclitaxel, and vinblastine alkaloids, such as vincristine, vinblastine, vinorelbine, and vinorelbine.

[0306] In some implementations, the second therapeutic agent is a topoisomerase inhibitor, such as topotecan or irinotecan, or a cell-inhibiting agent, such as etoposide and teniposide.

[0307] In some implementations, the second therapeutic agent is a growth factor inhibitor, such as an inhibitor of ErbB1 (EGFR) (such as EGFR antibodies, such as zarumumab, cetuximab, panitumumab, or nimotuzumab, or other EGFR inhibitors, such as gefitinib or erlotinib), another inhibitor of ErbB2 (HER2 / neu) (such as HER2 antibodies, such as trastuzumab, trastuzumab DM1, or pertuzumab), or an inhibitor of both EGFR and HER2, such as lapatinib.

[0308] In some implementations, the second therapeutic agent is a tyrosine kinase inhibitor, such as imatinib (Gleevec STI571) or lapatinib.

[0309] In some embodiments, the second therapeutic agent is a cytokine, growth factor, chemokine, or a combination thereof. Non-limiting examples of cytokines and growth factors include IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFN-α (e.g., INFα2b), IFN-γ, GM-CSF, CD40L, Flt3 ligand, stem cell factor, ansistatin, and TNFα. Non-limiting examples of chemokines include Glu-Leu-Arg (ELR)-negative chemokines, such as IP-10, MCP-3, MIG, and SDF-1a from the human CXC and CC chemokine families. Suitable cytokines include cytokine derivatives, cytokine variants, cytokine fragments, and cytokine fusion proteins.

[0310] In some embodiments, the second therapeutic agent is a cell cycle control / apoptosis regulator (or “regulator”). The cell cycle control / apoptosis regulator may include molecules that target and regulate cell cycle control / apoptosis, such as (i) cdc-25 (e.g., NSC 663284), (ii) cyclin-dependent kinases that overstimulate the cell cycle (e.g., vrapin (L868275, HMR1275), 7-hydroxyastrosporin (UCN-01, KW-2401), and roscovitine (R-roscovitine, CYC202)), and (iii) telomerase regulators (e.g., BIBR1532, SOT-095, GRN163, and compositions described, for example, in US 6,440,735 and US 6,713,055). Non-limiting examples of molecules that interfere with the apoptosis pathway include TNF-associated apoptosis-inducing ligand (TRAIL) / apoptosis-2 ligand (Apo-2L), antibodies that activate the TRAIL receptor, IFN, and antisense Bcl-2.

[0311] In some implementations, the second therapeutic agent is a hormone modulator, such as agents that can be used for both anti-androgen and anti-estrogen therapy. Examples of such hormone modulators include tamoxifen, edoxifene, fulvestrant, droloxifen, toremifene, raloxifene, diethylstilbestrol, ethinyl estradiol, anti-androgens (such as flutaminde / eulexin), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate / provera, megace), corticosteroids (such as hydrocortisone, prednisone), luteinizing hormone-releasing hormone (and its analogues and other LHRH agonists, such as buserreline and gosereline), aromatase inhibitors (such as anastrozole / arimidex, aminoglutethimide / cytraden, exemestane), or hormone inhibitors (such as octreotide / sandostatin).

[0312] In some implementations, the second therapeutic agent is an anticancer nucleic acid or an anticancer repressive RNA molecule.

[0313] As mentioned above, combined administration can be simultaneous, separate, or sequential. For simultaneous administration, the agents can be administered as a single composition or as separate compositions, depending on the circumstances.

[0314] In some embodiments, the cells and compositions disclosed in this invention are administered in combination with radiotherapy. Radiotherapy may include the administration of radiation or the delivery of a radiopharmaceutical to a subject. The radiation source may be external or internal to the patient being treated (radiotherapy may take the form of, for example, external beam radiotherapy (EBRT) or brachytherapy (BT)). Radioactive elements that can be used to practice such methods include, for example, radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodine-123, iodine-131, and indium-111.

[0315] In some embodiments, the cells and compositions disclosed in this invention are administered in combination with surgery.

[0316] According to various embodiments, the CAR-expressing cells (e.g., CAR-T cells) disclosed in this invention can be engineered in several ways to achieve one or more of the following: enhanced tumor cell toxicity and specificity, evasion of tumor immunosuppression, avoidance of host rejection, and prolongation of their therapeutic half-life. For example, TRUCK (T cells redirected for universal cytokine killing) T cells possess CARs but are also engineered to release cytokines that promote tumor killing (such as IL-12). Because these cells are engineered to release molecular payloads upon CAR activation once localized to the tumor environment, these CAR-T cells are sometimes referred to as “armored CARs.” Several cytokines as cancer therapies are being investigated preclinically and clinically and have shown potential when similarly incorporated into CAR-T therapies in the form of TRUCK cells. These include IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, M-CSF, GM-CSF, IFN-α, IFN-γ, TNF-α, TGF-β, TRAIL, FLT3 ligand, and lymphocyte chemokines. "Self-driven" or "homing" CAR-T cells are engineered to express chemokine receptors in addition to their CAR. Since certain chemokines can be upregulated in tumors, the incorporation of chemokine receptors facilitates tumor transport to and infiltration by adoptive T cells, thereby enhancing the specificity and function of CAR-T. Universal CAR-T cells also possess a CAR, but are engineered to not express endogenous TCR (T cell receptor) or MHC (major histocompatibility complex) proteins. Removing these two proteins from the signal transduction repertoire of adoptive T cell therapy prevents graft-versus-host disease and rejection, respectively. Armored CAR-T cells are also named for their ability to evade tumor immunosuppression and tumor-induced CAR-T dysfunction. These specific CAR-Ts possess a CAR and can be engineered to not express checkpoint inhibitors. Alternatively, these CAR-Ts can be co-administered with monoclonal antibodies (mAbs) that block checkpoint signaling. Administration of anti-PD-L1 antibodies significantly restores the killing ability of CAR-T tumor-infiltrating lymphocytes (TILs). While the PD1-PDL1 and CTLA-4-CD80 / CD86 signaling pathways have been investigated, other immune checkpoint signaling molecules, including LAG-3, Tim-3, IDO-1, 2B4, and KIR, can be targeted in the design of armored CAR-Ts. Other intracellular inhibitors of TILs include phosphatase (SHP1), ubiquitin ligase (i.e., cbl-b), and kinase (i.e., diacylglycerol kinase). Armored CAR-Ts can also be engineered to express proteins or receptors that protect them from or resistant to tumor-secreted cytokines.For example, CTLs (cytotoxic T lymphocytes) transduced with a double-negative form of the TGF-β receptor are resistant to the immunosuppression of TGF-β secreted by lymphoma. When compared with their control counterparts, these transduced cells exhibit significantly increased in vivo antitumor activity.

[0317] Additionally, in some implementations, B-lymphocyte antigen-related diseases are autoimmune diseases. Non-limiting examples of autoimmune diseases include multiple sclerosis, systemic lupus erythematosus (SLE), Sjögren's syndrome, AL amyloidosis, IgG4-related disease (IgG4-RD), inflammatory myopathy (e.g., myositis), scleroderma, pemphigus vulgaris, chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barré syndrome (GBS), stiff-person syndrome, rheumatoid arthritis, neuromyelitis optica spectrum disorder (NMOSD), myasthenia gravis, N- Methyl-D-aspartate receptor (NMDAR) encephalitis, anti-myelin oligodendrocyte glycoprotein (MOG) syndrome, MOGAD (MOGAD) disease, membranous nephropathy (MN), IgA nephropathy (IgAN), type 1 diabetes mellitus, anti-neutrophil cytoplasmic antibody (ANCA) vasculitis, thrombocytopenic purpura, hemolytic anemia (AIHA), multifocal motor neuropathy (MMN), inflammatory bowel disease (IBD), celiac disease, Crohn's disease, ulcerative colitis, and ankylosing spondylitis. In some implementations, the autoimmune disease is multiple sclerosis (MS). In some implementations, the autoimmune disease is systemic lupus erythematosus (SLE).

[0318] Cell-based immunotherapies targeting B cells have shown early promise in the treatment of autoimmune diseases. In autoimmune diseases, B cells can act as immunomodulators, for example, in multiple sclerosis (MS). MS is a demyelinating disease characterized by active neurodegeneration associated with lymphocyte infiltration in the brain. While the cause of MS remains unknown, the results of anti-CD20 therapy for B cell depletion in MS highlight the central, antibody-independent role of B cells in MS attack, particularly by modulating immune cell responses in the peripheral and CNS (Li R, Bar-Or A, ColdSpring Harb Perspect Med (2019);9(4)). B cells contribute to the pathogenesis of MS through several mechanisms, including the release of inflammatory cytokines / chemokines, activation of peripheral T cells, and driving chronic infiltration of pro-inflammatory myeloid and T cells into the CNS. As the disease progresses, the proliferation of locally resident CNS immune cells contributes to further neuroinflammation and demyelination (Li R, Bar-Or A, Cold Spring Harb Perspect Med(2019);9(4) Epub Date2018 / 04 / 18).

[0319] Currently, immunomodulatory therapies targeting B cells via CD20 are effective in preventing relapse but cannot halt disease progression, likely due to challenges in adequate access to the CNS. Autologous chimeric antigen receptor (CAR) T-cell therapy targeting CD19 is highly effective in eliminating pathogenic B cells in trans-B-cell driven autoimmune diseases, attenuating disease activity and significantly improving symptoms in patients with systemic lupus erythematosus, systemic sclerosis, myositis, and antisynthetic enzyme syndrome. (Bergmann C, et al.) Ann Rheum (2023);82(8):1117–1120; Müller F, et al. , N Engl J Med (2024); 390(8):687-700; Pecher AC, et al. JAMA (2023);329(24):2154-2162). CD19-targeted CAR T cells have also shown early promise in the treatment of progressive MS; two patients treated with autologous CD19 CAR T cells showed CAR T amplification in cerebrospinal fluid (CSF), and one patient had reduced oligoclonal bands (OCB) and intrathecal antibodies. No CAR T cell-related neurotoxicity was reported in any of the patients (Fischbach F, et al.). Med (2024);5(6):550-558.e552). Compared with other mAbs that target B cells (e.g., anti-CD20 rituximab), CD19-directed CAR T cells can provide more complete B cell depletion in the CNS because they are able to cross the blood-brain barrier and target CD19-expressing plasmablasts that have lost CD20 expression.

[0320] CD20 + T cells (which have recently been shown to acquire CD20 from B cells via cytokinesis) have been shown to exacerbate the course of disease in animal models of MS (experimental autoimmune encephalomyelitis, EAE) (Ochs J, et al.). Sci Transl Med (2022);14(638):eabi4632). Furthermore, recent studies have reported a higher frequency of CD8 in PPMS patients. + CD20 + The association between T cells and demyelination (von Essen MR, et al.) , Neurol Neuroimmunol Neuroinflamm (2023);10(5)). Recent reports indicate that CD20 can be depleted in MS patients through anti-CD20 antibody therapy (e.g., oligrinumab and olfamolumab). + T cells (Gingele S, et al.) Cells (2018);8(1); von Essen MR, et al. , Neurol Neuroimmunol Neuroinflamm (2022);9(4)). CD20 + The potential role of T cells in the pathophysiology of MS supports additional mechanisms through which targeting CD20 is effective for treating autoimmune diseases (e.g., MS).

[0321] This disclosure provides a method for treating autoimmune diseases in subjects using cells and compositions expressing CD19 / CD20-targeting CARs disclosed in this invention. The cells and compositions disclosed in this invention target both CD19 and CD20 to eliminate pathogenic B cells (e.g., by killing or depleting CD19). + B cells, CD20 + B cells) and T cells (e.g., killing or consuming CD19) + T cells, CD20 + T cells can be used to provide therapeutic benefits in patients with autoimmune diseases such as multiple sclerosis and systemic lupus erythematosus.

[0322] The subject matter disclosed in this invention provides various methods of using the cells or compositions disclosed herein. The cells or compositions disclosed herein can be used in therapies or pharmaceuticals. For example, the cells and compositions of the CD19 / CD20-targeting CAR disclosed herein can be used to manufacture a medicament for inhibiting the growth of target cells expressing at least one B lymphocyte antigen. The cells and compositions of the CD19 / CD20-targeting CAR disclosed herein can be used to manufacture a medicament for treating blood cancers in a subject. The cells and compositions of the CD19 / CD20-targeting CAR disclosed herein can be used to manufacture a medicament for treating an autoimmune disease in a subject. Various methods include administering an effective amount of the cells or compositions disclosed herein to a subject in need. The cells and compositions disclosed herein do not induce graft-versus-host disease. The cells and compositions disclosed herein do not induce or induce minimal allogeneic reactivity against HLA-mismatched cells in a subject that are positive for B lymphocyte antigens. Additionally or alternatively, the cells and compositions disclosed in this invention do not exhibit or exhibit minimal cytotoxicity and / or cytolytic activity against off-target cells in a subject, wherein the off-target cells are negative for B lymphocyte antigens. In some embodiments, the off-target cells are negative for CD19 or CD20. In some embodiments, the off-target cells are selected from the group consisting of: CD19 - CD20 -T cells, natural killer T cells (NK T cells), natural killer cells (NK cells), and combinations thereof. In some embodiments, the cells and compositions disclosed herein induce cytokine secretion. In some embodiments, the cytokine is interferon-γ (IFN-γ). In some embodiments, the cytokine is tumor necrosis factor-α (TNF-α).

[0323] According to one or more embodiments, to better control CAR-T therapy and prevent unwanted side effects, various features can be engineered into the CAR-T cell embodiments disclosed herein, including one or more of a shut-off switch, safety mechanisms, and condition control mechanisms. For example, both self-destructing and labeled / tagged CAR-T cells are engineered to have a “shut-off switch” that promotes the clearance of CAR-expressing T cells. Self-destructing CAR-T cells contain CAR but are also engineered to express a pro-apoptotic suicide gene or “elimination gene” that can be induced upon administration of exogenous molecules. Several suicide genes can be used for this purpose, including HSV-TK (herpes simplex virus thymidine kinase), Fas, iCasp9 (inducible cysteine ​​9), MYC tags, and truncated epidermal growth factor receptor (EGFRt) peptides (endothelial growth factor receptor). For example, HSK converts the prodrug ganciclovir (GCV) into GCV-triphosphate, which incorporates itself into replicating DNA, ultimately leading to cell death. iCasp9 is a chimeric protein containing a component of the FK506-binding protein, which binds to the small molecule AP1903, leading to caspase 9 dimerization and apoptosis. In some embodiments, the suicide gene is an EGFRt polypeptide. The EGFRt polypeptide can achieve T cell elimination by administration of an anti-EGFR monoclonal antibody (e.g., cetuximab). EGFRt can be covalently bound upstream to a CD19 / CD20-targeting CAR. The suicide gene can be contained within a vector comprising nucleic acid encoding the CD19 / CD20-targeting CAR disclosed herein. In this way, administration of a prodrug designed to activate the suicide gene (e.g., a prodrug that can activate iCasp-9 (e.g., AP1903)) during malignant T cell transformation (e.g., GVHD) triggers apoptosis in cells expressing the CD19 / CD20-targeting CAR that have the suicide gene activated. Incorporating the suicide gene into the CD19 / CD20-targeting CAR disclosed herein improves the level of safety and enables the elimination of most receptor-expressing cells within a very short time period. The cells disclosed in this invention, which incorporate suicide genes, can be eliminated at a given time point after cell infusion or eradicated when the earliest signs of toxicity appear.

[0324] According to one or more embodiments, the cells disclosed in this invention can be programmed to express the CD19 / CD20-targeting CAR and the selected marker-tagged CAR-T cells disclosed in this invention. Administration of an mAb targeting this selected marker can promote the elimination of CAR-T cells. Truncated EGFR is one such antigen that can be targeted by an anti-EGFR mAb, and the administration of cetuximab is used to promote the elimination of CAR-T cells. The resulting CARs with these characteristics are also referred to as "switchable CARs" (sCARs) and "tunable CARs" (RCARs).

[0325] Furthermore, in various embodiments, the cells disclosed in this invention may further comprise an inhibitory CAR (iCAR), which includes an extracellular antigen-binding domain that binds to normal tissue and one or more intracellular signaling domains of one or more inhibitory molecules (Fedorov, et al.). , Sci Transl Med (2013);5(215): 215). Non-limiting examples of inhibitory molecules include CTLA-3, PD-1, CD45, PD-L1, B7-1, CD160, PIH, 2B4, CEACAM (CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG-3, TIGIT, BTLA, LAIR1, and TGFβ-R. In the presence of normal tissue, iCAR can inhibit the CD19 / CD20-targeting CARs disclosed in this invention. The enhanced safety of CAR-T engineering improves the specificity of CAR-T cells to tumor tissues and is advantageous in cases where very low levels of tumor-associated antigens may be expressed in certain normal tissues (which would lead to off-target effects of standard CARs).

[0326] Conditional CAR-T cells express an extracellular antigen-binding domain linked to both an intracellular stimulatory domain and a separate intracellular costimulatory domain. The costimulatory and stimulatory domain sequences are engineered so that, upon administration of the exogenous molecule, the resulting protein aggregates intracellularly to maintain the integrity of the CAR circuit. In this way, CAR-T activation can be modulated and even “fine-tuned” or personalized for specific patients. When inactive in a conditional CAR, the stimulatory and costimulatory domains are physically separated; for this reason, these are also referred to as “split CARs.”

[0327] 6. Example Unless otherwise stated, the practice of the subject matter disclosed in this invention employs conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the knowledge of those skilled in the art.

[0328] The following embodiments are provided to provide a complete disclosure and description to those skilled in the art regarding how to prepare and use the cells and compositions disclosed in this invention, and are not intended to limit the scope of the invention as the inventors believe.

[0329] Example 1. Generation of bispecific CD19 / CD20-targeting CAR-T cells Two CARs were constructed, named "20-19 1XX" and "20-19 BB06z". The structure and sequence of the "20-19 1XX" CAR are described in Section 5.3.4. Furthermore... Figure 1 As shown in the image. Figure 1 As shown, the “20-19 BB06z” CAR comprises: (i) an extracellular domain including (a) an anti-CD19 antigen-binding domain, which is an scFv derived from the anti-CD19 antibody FMC63, and (b) an anti-CD20 antigen-binding domain, which is an scFv derived from the anti-CD20 antibody Leu16, (ii) a CD8 hinge region, (iii) a CD8 transmembrane domain, and (iv) an intracellular domain including a native CD3ζ polypeptide and a co-stimulatory signaling domain, which includes a mutant CD28 polypeptide named mut06 and a 4-1BB signaling domain. Mut06 is disclosed in Boucher et al. Cancer Immunol Res Boucher et al. (2021);9:62-74, which are incorporated herein by reference in their entirety. To optimize CD28 function in CAR cells, Boucher et al. incorporated null mutations into the CD28 ITAM subdomains (YMNM, PRRP, or PYAP). Combined mutations of YMNM to FMNM and PRRP to ARRA (mut06) improved functional persistence of CAR-T cells (Boucher et al., (2021)). Furthermore, to enhance CAR T cell persistence, Roselli et al. combined the mut06 costimulatory domain with the 4-1BB costimulatory domain (known to increase persistence compared to CD28) and found that the bispecific CD19 / CD20-targeting CAR with both the 4-1BB and mut06 costimulatory domains had improved in vivo antitumor activity, increased persistence, and reduced depletion compared to the bispecific CD19 / CD20-targeting CAR with only the mut06 costimulatory domain (Roselli et al.). J. Immunother. Cancer (2021);9:1-15). The “20-19 BB06z” CAR contains a V-band connected to anti-CD20 scFv. H and V LThe G4S linker (i.e., (G4S)3 consisting of the amino acid sequence shown in SEQ ID NO: 2), and the G4S linker connecting anti-CD20 scFv and anti-CD19 scFv (i.e., (G4S)5 consisting of the amino acid sequence shown in SEQ ID NO: 3), and the V linker connecting anti-CD19 scFv. H and V L The Whitlow linker (i.e., the linker consisting of the amino acid sequence shown in SEQ ID NO: 1).

[0330] The CARs “20-19 1XX” and “20-19 BB06z” were introduced into the backbone of the retroviral vector SFG-1xx-FMC63 for downstream leader construct determination. The CARs “20-19 1XX” and “20-19 BB06z” were successfully transduced into host T cells and expressed by them. The binding specificity of these two CARs was assessed using an in vitro binding assay. T cells containing both CARs were found to specifically bind to both CD19 and CD20 in the assay (data not shown).

[0331] Example 2. In vivo analysis of allogeneic EBV-sensitized T cells expressing CD19 / CD20-targeting CARs generated by MP1. In vitro and in vivo activity The in vitro and in vivo cytotoxic activities of “20-19 1XX” and “20-19 BB06z” CARs were evaluated using continuous stimulation assays.

[0332] Allogeneic cytotoxic T cells (CTLs) from donors (e.g., “Donor 13”, “Donor 14”, “Donor 15”, and “Donor 40”) were transduced to express either the “20-19 1XX” CAR or the “20-19 BB06z” CAR. Donor CTLs were stimulated against EBV antigens using the proprietary manufacturing process MP1 by co-culturing them with EBV-infected B lymphoblast-like cell lines (BLCLs). CAR expression, vector copy number (VCN), and viability are shown in Table 3. CAR expression was measured by flow cytometry using a fluorescently labeled anti-idiotypic antibody against FMC63 scFv. VCN was measured using droplet digital PCR. Viability was measured using a cell counter. In Table 3, “40 1XX” indicates an allogeneic EBV-sensitized CTL from donor 40 expressing the “20-19 1XX” CAR. “40BB06z” indicates an allogeneic EBV-sensitized CTL derived from donor 40 expressing “20-19 BB06z” CAR. “40 NTD” indicates a non-transduced allogeneic EBV-sensitized CTL derived from donor 40 (not expressing CAR). Next, the in vitro cytotoxic activity of CAR-T cells against cells expressing CD19, CD20, or both was evaluated in a serial challenge assay. In short, CAR-T cells and target cells (K562 cells or Raji cells) were matched at a 5:1 effector ratio (E... CAR+): Target (T) ratio co-culture. K562 cells were engineered to express human CD19, human CD20, or both. Raji B-cell lymphoma cells naturally express both CD19 and CD20. To further elucidate the effects of CD19 and CD20, a group of Raji cells expressing different levels of CD19 were used as target cells, including those without CD19 expression (Raji-CD19). KO ), with low levels (Raji-CD19) 低 ) and high level (Raji-CD19) 高 CD19 cells were selected. Cells were transferred to 96-well round-bottom tissue culture plates for fluorescence activated cell sorting (FACS) analysis. Phenotypic analysis was performed using FlowJo, and the number of live target cells was counted. Additionally, target cells were added to the wells for restimulation. Results are shown in Table 4 and Figures 2A-2E In the middle. As shown in Table 4 and Figures 2A-2E As shown, CTLs sensitized by EBV from donor 40 expressing “20-19 1XX” CAR surprisingly exhibited more potent cytotoxic activity than “20-19BB06z” CAR. In addition, the in vivo antitumor activity of CAR-T cells against Burkitt lymphoma was evaluated.

[0333] like Figure 3 As shown, 6-7 week old female NSG mice were fed with 5 × 10⁶ mice per mouse. 5 RajiCD19 cells were intravenously transplanted in large quantities. 低 -GFP / nanoluc tumor cells (Raji CD19) 低 CD20 + (Tumor cells). On day 6 after tumor implantation, mice were intravenously injected with 3 × 10⁻⁶ cells per mouse. 6 CAR-T cells were used. Untransduced T cells, designated “NTD”, were used as a negative control. Tumor growth was monitored for 10 weeks. Specifically, tumor size was measured twice weekly after tumor implantation. Clinical symptoms and body weight were assessed at least twice weekly. Furthermore, tumor burden was measured by bioluminescence imaging (BLI) at specified days after CAR-T cell injection. The survival days of mice after CAR-T cell therapy are displayed using Kaplan-Meyer plots.

[0334] The results are shown in Figures 4A-4C and Figures 5A-5B In the middle. For example Figures 4A-4C and Figure 5B As shown, allogeneic EBV-sensitized CTLs from donor 40 expressing the "20-191XX" CAR surprisingly exhibited greater tumor growth inhibition than T cells containing the "20-19 BB06z" CAR. Furthermore, Figure 5A and 5B The expansion of “20-19 1XX” CAR-T cells was shown to be associated with their anti-tumor activity, and compared with “20-19 BB06z” CAR-T cells, it resulted in a statistically significant prolongation of overall survival in treated mice.

[0335] In summary, the "20-19 1XX" CAR demonstrated greater tumor growth inhibition than the "20-19 BB06z" CAR in consecutive re-challenge assays and tumor-bearing animals. Furthermore, "20-19 1XX" CAR-T cells showed activity against CD19. 低 and CD19 KO The antigen-specific cell lysis of the target suggests that the CD20 CAR-mediated mechanism of action is required for the treatment of B-cell tumors with CD19 antigen deficiency (e.g., relapsed / refractory (R / R) B-cell tumors).

[0336] Example 3. In vivo analysis of allogeneic EBV-sensitized T cells expressing CD19 / CD20-targeting CARs generated by MP2. External activity Allogeneic CTLs from donors 14 and 40 were transduced to express either the “20-19 1XX” CAR or the “20-19BB06z” CAR. In this embodiment, different proprietary manufacturing processes, MP2, were used to stimulate donor CTLs against EBV antigens by co-culturing CTLs with EBV-infected BLCLs. CAR expression, vector copy number (VCN), and viability of CAR-T cells from donor 14 are shown in Table 5. CAR expression, vector copy number (VCN), and viability of CAR-T cells from donor 14 are shown in Table 6. CAR expression was measured by flow cytometry using a fluorescently labeled anti-idiotypic antibody against FMC63 scFv. VCN was measured using droplet digital PCR. Viability was measured using a cell counter. In Table 5, “14 1XX” indicates allogeneic EBV-sensitized CTLs from donor 14 expressing the “20-191XX” CAR. “14 BB06z” indicates an allogeneic EBV-sensitized CTL expressing “20-19 BB06z” CAR derived from donor 14. “14 NTD” indicates a non-transduced allogeneic EBV-sensitized CTL (not expressing CAR) derived from donor 14. In Table 6, “40 1XX” indicates an allogeneic EBV-sensitized CTL expressing “20-19 1XX” CAR derived from donor 40. “40 BB06z” indicates an allogeneic EBV-sensitized CTL expressing “20-19 BB06z” CAR derived from donor 40. “40 NTD” indicates a non-transduced allogeneic EBV-sensitized CTL (not expressing CAR) derived from donor 40. Next, following the same protocol used in Example 2, the in vitro cytotoxic activity of CAR-T cells against cells expressing CD19, CD20, or both was evaluated in a serial challenge assay. The results are shown in Table 7 and... Figures 6A-6E and Figures 7A-7E As shown in Table 7 and Figures 6A-6E and Figures 7A-7E As shown, allogeneic EBV-sensitized CTLs expressing “20-19 1XX” CAR from both donors 14 and 40 surprisingly exhibited more potent cytotoxic activity than T cells tested. In summary, similar to the results seen in Example 2, the allogeneic “20-19 1XX” CAR-T cells demonstrated greater tumor growth inhibition than the “20-19 BB06z” CAR-T cells in the serial re-challenge assay. Furthermore, the “20-19 1XX” CAR-T cells showed resistance to CD19. + or CD20 +Antigen-specific cell lysis of the target confirms that the CD19 and CD20-specific CAR-mediated mechanism of action is essential as an early line therapy for B-cell tumors and for preventing relapse due to CD19 antigen escape.

[0337] Example 4. In vivo antitumor activity of CD19 / CD20-targeted CAR-T cells In this embodiment, the in vivo antitumor efficacy of allogeneic EBV-sensitized T cells expressing the "20-19 1XX" CAR was compared with that of autologous T cells expressing a baseline CD19 / CD20-targeting CAR. The structure of the baseline CD19 / CD20-targeting CAR is as follows. Figure 8 As shown. Figure 8 As shown, the benchmark CD19 / CD20-targeted CAR comprises: (i) an extracellular domain including (a) an anti-CD19 antigen-binding domain, which is a scFv derived from the anti-CD19 antibody FMC63, and (b) an anti-CD20 antigen-binding domain, which is a scFv derived from the anti-CD20 antibody Leu16; (ii) a CD28 hinge region; (iii) a CD28 transmembrane domain; and (iv) an intracellular domain including a native CD3ζ polypeptide and a co-stimulatory signaling region, which includes a 4-1BB signaling domain. The benchmark CD19 / CD20-targeted CAR includes a V-shaped structure linked to the anti-CD20 scFv. H and V L The GS18 linker (i.e., the linker consisting of the amino acid sequence shown in SEQ ID NO: 56), and the rigid EAAAK linker (i.e., (EAAAK)3 consisting of the amino acid sequence shown in SEQ ID NO: 57) connecting anti-CD20 scFv and anti-CD19 scFv, and the V linker connecting anti-CD19 scFv. H and V L The Whitlow linker (i.e., the linker consisting of the amino acid sequence shown in SEQ ID NO: 1). SEQ ID NO: 56 and SEQ ID NO: 57 are provided below. Zah et al. disclosed that CD19 / CD20-targeting CARs with similar structures to the benchmark CD19 / CD20-targeting CARs are effective in treating B-cell malignancies (Zah et al., Cancer Immunol Res. (2016);4(6):498-508).

[0338] GSTSGGGGSGGGSGGGGSS [SEQ ID NO: 56] EAAAKEAAAKEAAAK [SEQ ID NO: 57] Allogeneic EBV-sensitized T cells were generated from donor 14, and donor CTLs were stimulated against EBV antigens using MP2 by co-culturing CTLs with EBV-infected BLCLs.

[0339] like Figure 9 As shown, 6-7 week old female NSG mice were fed with 5 × 10⁶ mice per mouse. 5 RajiCD19 cells were intravenously transplanted in large quantities. 低 CD20 + Tumor cells. On day 6 after tumor implantation, mice were intravenously injected with 3 × 10⁻⁶ cells per mouse. 6 CAR-T cells were used. Untransduced T cells, designated “NTD”, were used as a negative control. Tumor growth was monitored for 10 weeks. Specifically, tumor size was measured twice weekly after tumor implantation. Clinical symptoms and body weight were assessed at least twice weekly. Furthermore, tumor burden was measured by bioluminescence imaging (BLI) at specified days after CAR-T cell injection. The survival days of mice after CAR-T cell therapy are displayed using Kaplan-Meyer plots.

[0340] The results are shown in Figures 10A-10C In the middle. For example Figure 10A As shown, allogeneic EBV-sensitized T cells expressing the "20-191XX" CAR surprisingly exhibited superior antitumor efficacy in an in vivo lymphoma model compared to autologous baseline CAR-T cells. Blood cell counts in treated mice were quantified using BD TruCount absolute counting beads (BD Biosciences) via a flow cell, and the results are shown in... Figure 10B The study also measured the vector copy number (VCN) per CAR-T cell, such as... Figure 10C As shown.

[0341] Example 5. Safety of CAR-T cells disclosed in this invention in vivo therapy The safety of allogeneic EBV-sensitized T cells expressing "20-19 1XX" or "20-19 BB06z" CAR for in vivo therapy was assessed by monitoring the change in relative body weight of treated mice compared to day 0. Raji CD19 was intravenously transplanted into 6-7 week old female NSG mice. 低 CD20 + Tumor cells. On day 6 after tumor implantation, mice were intravenously injected with 1×10⁻⁶ cells per mouse. 6 CAR-T cells, 3×10 6 One CAR-T cell or 9×10 6 CAR-T cells. The body weight of the various mice used in the in vivo experiments remained consistent over time in both the treated and untreated groups, demonstrating that no safety issues were observed in vivo.

[0342] Example 6. Characterization of allogeneic EBV-sensitized T cells expressing CD19 / CD20-targeting CARs According to the MP2 manufacturing process, "20-19 1XX" CAR T cells were produced from three different donors. Flow cytometry analysis of CAR expression on the "20-19 1XX" products from the three different donors demonstrated successful and stable transduction of EBV T cells using the "20-19 1XX" retroviral vector. Figure 11A As shown. Vector copy number (VCN) analysis showed that the number of vector copies per genome was consistently less than 5. Furthermore, the CD4 and CD8 distribution in the "20-19 1XX" product showed that CD8 was predominant. + T cells, such as Figure 11B As shown. Figure 11C As shown, flow cytometry analysis of memory markers CD45RO and CD62L revealed that the “20-19 1XX” product maintained a robust central memory population compared to autologous baseline CART cells.

[0343] Furthermore, in studies evaluating the “20-19 1XX” (n=10) research batches, T cells contained a high frequency of CD3. + Lymphocytes (median, 95.8%) and uneven numbers of CD8 + T cells, ranging from 41.1% to 90.4% (median, 85.1%), and CD4+ + T cells, ranging from 1.6% to 53.3% (median, 7.78%). The production of “20-19 1XX” cells showed a median cumulative expansion of 243.5 compared to 377.1 in the non-transduced (NTD) control, indicating that CAR transduction had minimal effect on cell expansion (data not shown).

[0344] Further evaluation was conducted to further characterize the immunophenotype of the “20-19 1XX” study batches. These studies showed that “20-19 1XX” was enriched with a central memory population, with a high double-positive population of CD45RO and CD62L ranging from 76.7% to 92.6%, indicating that it is a central memory T cell population that has experienced the antigen.

[0345] Surface CAR expression across 10 study batches of “20-19 1XX” resulted in CAR protein expression in cells ranging from 57.6% to 84.6%. CAR integration was measured by droplet digital PCR analysis, revealing vector copy number ranges of 2.48–4.41 copies per cell (data not shown).

[0346] Example 7. In vitro activity of allogeneic EBV-sensitized T cells expressing CD19 / CD20-targeting CARs To test the function of "20-19 1XX" in vitro, serial stimulation assays were performed against various non-Hodgkin lymphoma (NHL) cell lines expressing CD19 and CD20. The CD19 and CD20-specific antitumor activity of "20-19 1XX" was assessed for each cell line. A 5:1 effector ratio (E) was used in the serial stimulation assays. CAR+): Target (T) (E:T) ratio and Jeko-1 ( Figure 12A ) and Su-DHL-4 ( Figure 12B Co-culture was used to measure the specific functional activity of “20-19 1XX” against target cell lines expressing CD20 and / or CD19.

[0347] Furthermore, the functional activity of “20-19 1XX” was measured by evaluating CD19 and / or CD20-directed cell lysis and cytokine secretion by CAR-T cells against antigen-positive target cells K562. The “20-19 1XX” effector and luciferase-expressing target cells were incubated together at a 1:1 E:T ratio. Target cell lysis was measured after 24 hours.

[0348] Target cell counts following repeated stimulation (represented by vertical dashed lines) demonstrated durable tumor killing and CD20 and / or CD19-specific functional activity against various non-Hodgkin lymphoma cell lines, such as Figure 12A and 12B As shown, NTD EBV T cells from each product batch did not exhibit specific activity against the tested CD19 and / or CD20 positive or negative targets.

[0349] Example 8. Allogeneic response of T cells sensitized with allogeneic EBV expressing CD19 / CD20-targeting CARs sex After two days of co-culture, T cells and CD69 were used... + / 4-1 BB + T cell activation and HLA matching (auto) and mismatch (allo) CD20 + / CD19 + The allotropic potential of “20-19 1XX” was measured by cytolysis of the target B lymphoblast-like cell line (BLCL). The maintenance of “20-19 1XX” EBV specificity was assessed using flow cytometry.

[0350] “20-19 1XX” exhibits minimal allogeneic reactivity against HLA-mismatched targets and shows in vitro activity against CD20. + / CD19 + HLA-independent activity of the target. Results showed autologous EBV + / CD20 + / CD19 + BLCL is recognized and lysed by EBV T cells via CD20, CD19, and / or HLA-restricted EBV; but allogeneic HLA-mismatched EBV... + / CD20 + / CD19 + BLCL was cleaved solely by "20-19 1XX" via HLA-independent CD20 / CD19 CAR-directed cleavage. In vitro EBV specificity assays demonstrated that "20-19 1XX" killing is mediated via CAR, and that "20-19 1XX" exhibits minimal allogeneic reactivity against an HLA-mismatched target. Figure 13A In this study, although "20-191XX" showed robust activation against HLA-mismatched BLCL targets, NTD cells were not activated in the absence of HLA matching in the target BLCLs. Similarly, in Figure 13B In this study, it was confirmed that “20-19 1XX” killing functioned through HLA-independent recognition of CAR. In the absence of CAR, NTD cells targeted only HLA-matched autologous BLCL targets.

[0351] Example 9. Dosage study of allogeneic EBV-sensitized T cells expressing CD19 / CD20-targeting CARs. In vivo antitumor activity NOD scid-γ (NSG) mice were intravenously implanted with 0.5 × 10⁻⁶ sucralose on day 0. 6 Raji CD19 低 Cells. Mice were randomly assigned to different groups using bioluminescence imaging (BLI) and administered a single IV injection of 1×10⁻⁶ cells. 6 3×10 6 Or 9×10 6 Freshly thawed “20-19 1XX” or NTD EBV T cells were used for treatment, as shown by the vertical dashed line. BLI luminescence was measured in each mouse (n=6) after tumor implantation. The absolute count of circulating human T cells and vector copy number were measured.

[0352] The “20-19 1XX” dosage study demonstrated potent tumor rejection following a single low-dose administration in the absence of exogenous cytokine support. Figures 14A-14C As shown, "20-19 1XX" even when targeting CD19 低 Raji's tumor model also showed strong tumor rejection even at low doses. Figures 14A-14C The study demonstrated dose-dependent antitumor activity of "20-19 1XX". 1 × 10⁻⁶ mice were used. 6 Even the lowest dose of CAR T cells still has some therapeutic effect. For example... Figure 14D and 14EAs shown, hCD45 in peripheral blood + Cellular and VCN assays showed that CAR T cells persisted until the end of the study on day 62 post-treatment. No treatment-related toxicities were observed after any dose (data not shown).

[0353] Example 10. In vivo antitumor activity of allogeneic EBV-sensitized T cells expressing CD19 / CD20-targeting CARs. active In the previous examples, 3 × 10⁻⁶ mice were identified. 6 A sub-therapeutic dose of "20-19 1XX" CAR T cells. Then use CD19. 低 Raji model and CD19 高 In in vivo studies, the Raji model compared "20-19 1XX" cells with autologous baseline dual-CART cells. NSG mice received intravenous implantation of 0.5 × 10⁻⁶ cells on day 0. 6 Raji CD19 低 Cells or 0.5 × 10 6 RajiCD19 高 Cells. Mice were randomly divided into different groups using BLI and administered a single IV injection of 3 × 10⁻⁶ cells. 6 Freshly thawed “20-19 1XX” or NTD EBV T cells were used for treatment, as shown by the vertical dashed line. BLI luminescence was measured in each mouse (n=5) after tumor implantation. The geometric mean of BLI, mean absolute count of circulating human T cells, mean VCN, and survival were measured for each group.

[0354] Compared with autologous baseline CAR T cells, “20-19 1XX” showed superior in vivo antitumor activity and functional persistence. Figures 15A-15D The results showed that even at low CD19 levels, "20-19 1XX" exhibited superior tumor control compared to autologous baseline CAR T cells in both donors. Figure 15E-15H In the study, “20-19 1XX” showed complete control of tumor growth, while autologous baseline CAR T cells exhibited varying levels of tumor growth control. Both types of CAR T cells persisted until the end of the study. Animals treated with autologous baseline CAR T cells showed allogeneic reactive expansion of non-CAR T cells after approximately 40 days. No treatment-related toxicities were observed after all doses (data not shown).

[0355] Example 11. Allogeneic EBV-sensitized T cells expressing CD19 / CD20-targeting CARs in a single kill assay In vitro activity of the target Using the proprietary manufacturing process MP3, the “20-19 1XX” CAR was transduced and expressed in allogeneic EBV-sensitized CTLs derived from donor 104. The in vitro activity of “20-19 1XX” was evaluated in a single kill assay. CAR expression and vector copy number (VCN) are shown in Table 8. In Table 8, “104 20-19 1XX” represents allogeneic EBV-sensitized T cells derived from donor 104 expressing the “20-19 1XX” CAR. “104 NTD” represents non-transduced allogeneic EBV-sensitized T cells derived from donor 104 (without CAR expression). Next, the in vitro cytotoxic activity of CAR-T cells against cells expressing CD19, CD20, or both was evaluated. In short, CAR-T cells and target cells (K562 cells) were used in ratios of 1:4, 1:2, 1:1, 2:1, and 4:1, with effector (E) ratios... CAR+): Target (T) series ratio co-culture. K562 cells were engineered to express human CD19, human CD20, or both. Figure 16 As shown, on day 0, a predetermined number of tumor cells were co-cultured with a predetermined number of "20-19 1XX" CAR-T cells. On day 1 or day 2, the number of live T cells and tumor cells in each setup were analyzed by flow cytometry. Figures 17A-17D As shown, allogeneic EBV-sensitized T cells from donor 104 expressing “20-19 1XX” CAR exhibited CD19 and CD20 specific activity.

[0356] Example 12. Allogeneic EBV-sensitized T cells expressing CD19 / CD20-targeting CARs were subjected to continuous stimulation assays. In vitro activity of the target Using manufacturing process MP3, the “20-19 1XX” CAR was transduced and expressed in allogeneic EBV-sensitized CTLs derived from donors 14 and 23. The in vitro activity of “20-19 1XX” was evaluated in a serial challenge assay. CAR expression and vector copy number (VCN) are shown in Table 9. In Table 9, “14 20-19 1XX” represents allogeneic EBV-sensitized T cells derived from donor 14 expressing the “20-19 1XX” CAR. “14 NTD” represents untransduced allogeneic EBV-sensitized T cells derived from donor 14 (without CAR expression). “23 20-19 1XX” represents allogeneic EBV-sensitized T cells derived from donor 23 expressing the “20-19 1XX” CAR. “23 NTD” represents untransduced allogeneic EBV-sensitized T cells derived from donor 23 (without CAR expression). Next, the in vitro cytotoxic activity of CAR-T cells against cells expressing CD19, CD20, or both was evaluated in a series of challenge assays. Figure 18 As shown, on day 0, a predetermined number of tumor cells were co-cultured with a predetermined number of "20-19 1XX" CAR-T cells. From day 2 to day 10, every 2-3 days, the number of live T cells and tumor cells for each setting was analyzed, and the predetermined number of tumor cells was added to the culture. CAR-T cells and target cells were used at an effector ratio of 5:1 or 1:1. CAR+): Target (T) ratio co-culture. To further elucidate the effects of CD19 and CD20, a group of Raji cells expressing different levels of CD19 were used as target cells, including those without CD19 expression (Raji-CD19). KO ), with low levels (Raji-CD19) 低 ) and high level (Raji-CD19) 高 CD19 cells. (e.g.) Figures 19A-19C and Figures 20A-20C As shown, allogeneic EBV-sensitized T cells from donor 14 or donor 23 expressing “20-19 1XX” CAR exhibited CD19 and CD20 specific activity against Raji cells with different levels of CD19 and CD20 positive expression.

[0357] Furthermore, the in vitro cytotoxic activity of CAR-T cells against various non-Hodgkin lymphoma (NHL) cells was evaluated in a serial attack assay. CAR-T cells and target cells were used at a 1:1 effector ratio (E... CAR+): Target (T) ratio co-culture. Various NHL cell lines were used as target cells, including Raji cells (Burkit lymphoma), Jeko-1 cells (mantle cell lymphoma (MCL)), Su-DHL-4 cells (diffuse large B-cell lymphoma (DLBCL)), and MEC-1 cells (chronic lymphocytic leukemia (CLL)). Figures 21A-21D As shown, allogeneic EBV-sensitized T cells expressing “20-19 1XX” CAR from donor 14 or donor 104 all exhibited CD19 and CD20 specific activity against these NHL cells.

[0358] Example 13. In vivo antitumor activity of CD19 / CD20-targeted CAR-T cells The in vivo anti-tumor efficacy of these CAR-T cells was evaluated. Tumor growth was monitored using bioluminescent tumor cells detectable via IVIS imaging. Blood samples were collected, and hCD45 was detected in the blood. +Cells are used to indicate the presence of injected human cells. Flow cytometry was used to analyze the growth and persistence of injected cells. Mouse body weight was monitored as an indicator of severe toxicity (lower body weight indicated toxicity). "14 20-19 1XX" represents allogeneic EBV-sensitized T cells expressing the "20-19 1XX" CAR derived from donor 14. "14 NTD" represents non-transduced allogeneic EBV-sensitized T cells (not expressing CAR) derived from donor 14. "23 20-19 1XX" represents allogeneic EBV-sensitized T cells expressing the "20-19 1XX" CAR derived from donor 23. "23 NTD" represents non-transduced allogeneic EBV-sensitized T cells (not expressing CAR) derived from donor 23.

[0359] After injecting Raji CD19 低 The in vivo antitumor efficacy of allogeneic EBV-sensitized T cells expressing “20-191XX” CAR was evaluated in a Burkitt lymphoma model of / CD20 tumor cells. Results are shown in Figures 22A-22C In the middle. For example Figure 22A As shown, allogeneic EBV-sensitized T cells expressing "20-19 1XX" CAR from donor 14 or donor 23 attacked an in vivo Burkitt lymphoma model (Raji CD19). 低 Antitumor efficacy was demonstrated in both CD20 cells and other cells. Blood cells from treated mice were quantified using BD TruCount absolute counting beads (BD Biosciences) via flow cell analysis, and the results were shown in… Figure 22B The study also measured the vector copy number (VCN) per CAR-T cell, such as... Figure 22C As shown. Allogeneic EBV-sensitized T cells expressing the "20-19 1XX" CAR derived from the donor demonstrated potent tumor growth inhibition in a Burkitt lymphoma tumor model (Burkitt lymphoma expressing low CD19) in NSG mice, and this inhibition persisted until approximately day 40 of the study for donor 14. No serious toxicities associated with "20-19 1XX" treatment were observed. Figure 22D ).

[0360] Furthermore, the in vivo antitumor efficacy of allogeneic EBV-sensitized T cells expressing "20-19 1XX" CAR was evaluated in a mantle cell lymphoma (MCL) model injected with Jeko-1 CD19 / CD20 tumor cells. Figures 24A-24C As shown, allogeneic EBV-sensitized T cells expressing the "20-19 1XX" CAR demonstrated potent dose-dependent antitumor efficacy in the Jeko-1 MCL model in vivo, persisting until approximately day 40 at the end of the study. No serious toxicities associated with "20-19 1XX" treatment were observed. Figure 24D ).

[0361] Example 14. Characterization of PBMCs from multiple sclerosis donors To investigate the effectiveness of targeting CD20 in treating multiple sclerosis (MS), CD20 from peripheral blood mononuclear cells (PBMCs) was characterized from 4 untreated MS donors and 10 healthy donors. + T cell population. Characterization of CD20 in PBMCs collected from healthy donors (HD; n=7) and untreated MS donors (n=4). + T cells and CD19 + / CD20 + B cells. Calculate total CD3. + CD20 in T cells + T cell percentage, and CD19 in total viable cells were calculated. + / CD20 + B cells%.

[0362] like Figure 25A and 25B As shown, PBMCs from MS donors have a significantly higher percentage of CD20 compared to PBMCs from healthy donors. + T cells. Therefore, the inventors investigated the therapeutic potential of the CD19 / CD20-targeted CAR T cells disclosed in this invention to treat MS by eliminating pathogenic B cells and T cells.

[0363] Example 15. Allogeneic EBV-sensitized T cells expressing CD19 / CD20-targeting CARs generated by MP3 were used for In vitro activity for treating multiple sclerosis The in vitro cytotoxic activity of "20-19 1XX" CAR was evaluated by co-culturing PBMCs and "20-19 1XX" CAR T cells from MS patients. A schematic diagram of the assay is shown below. Figure 26 In short, this study measures the cytotoxic activity of CAR-T cells against B cells derived from MS donors. CAR T cells are labeled with cell trace violet (CTV), a dye that emits signals on flow cytometry. MS donor PBMCs are mixed with CTV-labeled CAR T cells at a 1:1 or 3:1 effector ratio (E). CAR+): Target (T) ratio (CAR+ T cells: CD19) +Co-culture with B cells. Viable cell counts of CTV-labeled CAR T cells and MS patient PBMCs were monitored for 72 hours. Cell lysis was detected by flow cytometry and measured via the percentage of B cell lysis. IFN-γ and TNF-α secretion from CAR-T cells were measured to indicate T cell activation. CD19 or CD20-targeted cell lysis, “20-19 1XX” CAR T cell proliferation, and cytokine secretion were measured after co-culturing PBMCs from three different MS donors (donor A, donor B, and donor C) at a 1:1 ratio for 24, 48, and 72 hours. For the 3:1 ratio setting, data were collected only at 24 hours.

[0364] “20-19 1XX” CAR T cells exhibited dose-dependent B cell killing after 24 hours. For example... Figures 27A-27C As shown, the number of B cells normalized to an E:T ratio of 1:1 or 3:1, “20-19 1XX” responds to CD19 in PBMCs from MS donors (donor A, donor B, and donor C). + B cells exhibited antigen-specific B cell lysis within 24 hours. Higher levels of B cell cytotoxicity were observed with increasing E:T ratios, indicating a CAR-mediated response to CD19 and CD20 on B cells exposed to MS donors. Furthermore, “20-19 1XX” CAR T cells persistently targeted B cells for 72 hours. Normalized to the number of B cells, at a 1:1 E:T ratio, “20-19 1XX” responded to CD19 in PBMCs from MS donors (n=3). + B cells exhibit antigen-specific B cell lysis within 48 hours, such as Figure 28A and 28D As shown, or exhibiting antigen-specific B cell lysis within 72 hours, such as Figure 28B and 28C As shown. Figure 28D As shown, the calculation of the percentage of target B cell lysis demonstrates CAR-mediated killing of CD19 and CD20 positive B cells, while no consistent lysis was observed against CD19 and CD20 negative T cells, NK T cells, and NK cells. Figure 29A-29I As shown. Normalized to the number of B cells, at a 1:1 E:T ratio, the "20-191XX" response in non-B cells in PBMCs of MS donors (donors A, B, and C) at 48 hours ( Figure 29C , 29F and 29I) or 72 hours ( Figure 29A , 29D and 29G; and Figure 29B , 29E Within 29H, no or minimal off-target non-B cell lysis was observed.

[0365] Next, the effects of "20-19 1XX" CAR T cells on CD20 were investigated. + T cell cytolytic activity. Normalized to B cell number, at a 1:1 E:T ratio, in response to CD20 from PBMCs from MS donors (n=3). + T cells, with CD20 detected by flow cytometry at 48 or 72 hours. + T cell depletion. For example... Figure 30A and 30B As shown, CD20 was observed. + T cell lysis indicates CD20 + The T cell population was targeted by “20-19 1XX” CAR T cells. NTD EBV T cells did not show consistent or dose-dependent specific activity against CD19 and / or CD20 positive or negative targets.

[0366] In addition, the secretion of IFN-γ and TNF-α from “20-19 1XX” CAR T cells was evaluated. Co-culture supernatants were harvested at 24 and 72 hours, and cytokines were measured using a bead-based immunoassay (n=3). Figure 31A and 31B As shown, compared with the NTD EBV-T cell control, MS PBMCs incubated with “20-19 1XX” CAR T cells exhibited elevated levels of pro-inflammatory cytokines IFN-γ and TNF-α, demonstrating a CAR-mediated response following exposure to CD19 and CD20 on MS donor B cells. Specifically, the results showed that “20-19 1XX” released high levels of IFN-γ, confirming CAR-mediated T cell activation following exposure to CD19 and CD20 on MS B cells.

[0367] Next, the proliferation of "20-19 1XX" CAR T cells was assessed. For example... Figures 32A-32H As shown, normalized to the number of B cells, at a 1:1 E:T ratio, CTV+ "20-19 1XX" cells responded to CD19 and CD20 from PBMCs from MS donors (n=3). + B cells, after 48 hours ( Figure 32A and 32E ) or 72 hours ( Figure 32B , 32F CTV+ NTD EBV-T cells showed robust proliferation within 32C and 32G cells. Normalized to the number of B cells, at a 1:1 E:T ratio, CTV+ NTD EBV-T cells were a negative control response to CD19 and CD20 from PBMCs (n=3; representative plot) of their own MS patients. +B cells did not show robust proliferation within 72 hours. Figure 32D and 32H ).

[0368] Example 16. Allogeneic EBV-sensitized T cells expressing CD19 / CD20-targeting CARs generated by MP2 were used for In vitro activity for treating systemic lupus erythematosus The in vitro cytotoxic activity of "20-19 1XX" CAR was evaluated by co-culturing PBMCs and "20-19 1XX" CAR T cells derived from systemic lupus erythematosus (SLE) donors. A schematic diagram of the assay is shown below. Figure 33 In short, this study measured the cytotoxicity of CAR-T cells against B cells in PBMCs of SLE donors. CAR-T cells were labeled with cell tracer violet (CTV) that emits signals on flow cytometry. PBMCs from SLE donors were mixed with CTV-labeled CAR T cells at a 1:1 effector ratio (E). CAR+): Target (T) ratio (CAR+ T cells: CD19) + Co-culture with B cells. Monitor the viable cell counts of CTV-labeled CAR T cells and PBMCs from SLE patients for 48 hours.

[0369] The results showed that the “20-19 1XX” CAR-T cells continuously targeted B cells for 48 hours, and had minimal off-target non-B cell targeting over time. Figure 34A The results showed that, normalized to the number of B cells, at an E:T ratio of 1:1, “20-19 1XX” CAR-T cells responded to non-B cells from third-party SLE patient PBMCs and showed no or minimal off-target non-B cell lysis within 48 hours. Figures 34D to 34F This showed no or minimal off-target killing in the PBMCs of SLE donors, such as no or minimal non-B cell killing, including CD56. + NK cells ( Figure 34D CD56 + / CD3 + NKT cells ( Figure 34E ) and CD3 + T cells ( Figure 34F ).

[0370] Example 17. Allogeneic EBV-sensitized T cells expressing CD19 / CD20-targeting CARs targeting HLA mismatch Allogeneic reactivity of the target In addition to the CD19 / CD20 bispecific CAR-mediated function, in vitro cytotoxicity of chromium-labeled HLA-matched and HLA-mismatched targets was measured after co-culturing with “20-19 1XX” CAR T cells. Potential allogeneic reactivity of “20-19 1XX” research batches from four donors was assessed via chromium release from labeled targets.

[0371] like Figure 35 As shown, “20-19 1XX” from four study batches exhibited specific lysis of 30% or more against autologous CD19 / CD20-positive BLCL target cells. None of the study batches showed greater than 30% cytolytic activity against autologous phytohemagglutinin blast cells (PHAb). Co-incubation with completely HLA-mismatched PHA blast cells from eight different donors also failed to produce cytolytic activity higher than 30%.

[0372] in conclusion While various embodiments of this disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, alterations, and substitutions will be apparent to those skilled in the art without departing from this disclosure. For example, various embodiments of CAR and CAR T-cell compositions and associated methods of use (e.g., for the treatment of cancer or autoimmune diseases) can be adapted for a variety of pediatric and veterinary applications, the former including various pediatric leukemias and fibromas (e.g., AML, MDS, CML, and JMML as known in the field of oncology), and the latter including one or more of felines, canines, and equines. Specific modifications to the foregoing may include one or more of a particular CAR, CAR-T cell, and its intended target cells (e.g., B cells or T cells) in the therapeutic composition administered to a patient, the cell dose in the composition, and the carrier solution and various excipients used in the carrier solution. They can also be modified to treat any number of B-cell-mediated cancers, including one or more of leukemia and lymphoma, as well as many B-cell and / or T-cell-mediated and other autoimmune diseases, including one or more of multiple sclerosis, Guillain-Barré syndrome, CIDP, MFMN, systemic lupus erythematosus (SLE), rheumatoid arthritis, IBD, celiac disease, Crohn's disease, ulcerative colitis, and ankylosing spondylitis.

[0373] Elements, features, or actions from one embodiment can be readily recombined with or replaced by one or more elements, features, or actions from other embodiments to form a multitude of other embodiments within the scope of this disclosure. Furthermore, in various embodiments, elements shown or described as combining with other elements, features, steps, or actions may exist as independent elements, features, steps, or actions. Moreover, various embodiments expressly contemplate negative representations of any elements, features, steps, or actions, etc., shown or described in one or more embodiments. Therefore, the scope of this disclosure is not limited to the details of the described embodiments, but is limited only by the appended claims.

Claims

1. A bispecific chimeric antigen receptor (CAR) that binds to CD19 and CD20, said CAR comprising: a) Extracellular domain comprising (i) a first antigen-binding domain that binds to CD19 and (ii) a second antigen-binding domain that binds to CD20; b) Contains a transmembrane domain of the first CD28 polypeptide; and c) An intracellular domain comprising a modified CD3ζ polypeptide containing natural ITAM1, ITAM2 variants and ITAM3 variants, each of the ITAM2 variant and the ITAM3 variant containing two loss-of-function mutations.

2. The CAR of claim 1, wherein the first anti-CD19 antigen-binding domain comprises: a first heavy chain variable region (V H ) and the first light chain variable region (V L ), wherein the first V H V containing the amino acid sequence shown in SEQ ID NO: 15 H V H CDR1, V H CDR2 and V H CDR3, and / or the first V L V containing the amino acid sequence shown in SEQ ID NO: 16 L V L CDR1, V L CDR2 and V L CDR3.

3. The CAR of claim 2, wherein the first V H Contains V containing the amino acid sequence shown in SEQ ID NO: 9 or its conserved modifications. H CDR1, containing the amino acid sequence shown in SEQ ID NO: 10 or its conserved modifications thereof, V H CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 11 or its conserved modifications H CDR3; and / or the first V L Contains V containing the amino acid sequence shown in SEQ ID NO: 12 or its conserved modifications. L CDR1, containing the amino acid sequence shown in SEQ ID NO: 13 or its conserved modifications thereof, V L CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 14 or its conserved modifications L CDR3.

4. The CAR as claimed in claim 2 or 3, wherein the first V H V containing the amino acid sequence shown in SEQ ID NO: 9 H CDR1, containing the amino acid sequence shown in SEQ ID NO: 10, V H CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 11 H CDR3; and / or the first V L V containing the amino acid sequence shown in SEQ ID NO: 12 L CDR1, containing the amino acid sequence shown in SEQ ID NO: 13, V L CDR2 and V containing the amino acid sequence shown in SEQ ID NO:14 L CDR3.

5. The CAR as claimed in any one of claims 2 to 4, wherein the first V H Contains an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical or homologous to the amino acid sequence shown in SEQ ID NO: 15; and / or the first V L It contains amino acid sequences that are at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical or homologous to the amino acid sequence shown in SEQ ID NO:

16.

6. The CAR as claimed in any one of claims 2 to 5, wherein the first V H Contains the amino acid sequence shown in SEQ ID NO: 15; and / or the first V L It contains the amino acid sequence shown in SEQ ID NO:

16.

7. The CAR of any one of claims 1 to 6, wherein the first anti-CD19 antigen-binding domain comprises the first V H With the first V L The first joint between them.

8. The CAR of claim 7, wherein the first connector comprises the amino acid sequence shown in SEQ ID NO:

1.

9. The CAR as claimed in any one of claims 2 to 8, wherein the first V H and the first V L Positioning from the N-terminus to the C-terminus: V L -V H .

10. The CAR of any one of claims 1 to 9, wherein the first anti-CD19 antigen-binding domain comprises a first single-stranded variable fragment (scFv) that binds to anti-CD19.

11. The CAR of claim 10, wherein the first anti-CD19 scFv comprises the amino acid sequence shown in SEQ ID NO:

17.

12. The CAR of claim 1, wherein the second anti-CD20 antigen-binding domain comprises: a second V H Second V L The second V H V containing the amino acid sequence shown in SEQ ID NO: 27 H V H CDR1, V H CDR2 and V H CDR3, and / or the second V L V containing the amino acid sequence shown in SEQ ID NO: 28 L V L CDR1, V L CDR2 and V L CDR3.

13. The CAR of claim 12, wherein the second V H Contains V containing the amino acid sequence shown in SEQ ID NO: 21 or its conserved modifications. H CDR1, containing the amino acid sequence shown in SEQ ID NO: 22 or its conserved modifications thereof, V H CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 23 or its conserved modifications H CDR3; and / or the second V L Contains V containing the amino acid sequence shown in SEQ ID NO: 24 or its conserved modifications. L CDR1, containing the amino acid sequence shown in SEQ ID NO: 25 or its conserved modifications thereof, V L CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 26 or its conserved modifications L CDR3.

14. The CAR of claim 12 or 13, wherein the second V H V containing the amino acid sequence shown in SEQ ID NO: 21 H CDR1, containing the amino acid sequence shown in SEQ ID NO: 22, V H CDR2 and V containing the amino acid sequence shown in SEQ ID NO:23 H CDR3; and / or the second V L V containing the amino acid sequence shown in SEQ ID NO: 24 L CDR1, containing the amino acid sequence shown in SEQ ID NO: 25, V L CDR2 and V containing the amino acid sequence shown in SEQ ID NO: 26 L CDR3.

15. The CAR of any one of claims 12 to 14, wherein the second V H Contains an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical or homologous to the amino acid sequence shown in SEQ ID NO: 27; and / or the second V L It contains amino acid sequences that are at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical or homologous to the amino acid sequence shown in SEQ ID NO:

28.

16. The CAR of any one of claims 12 to 15, wherein the second V H Contains the amino acid sequence shown in SEQ ID NO: 27; and / or the second V L It contains the amino acid sequence shown in SEQ ID NO:

28.

17. The CAR of any one of claims 12 to 16, wherein the second anti-CD20 antigen-binding domain comprises the second V H With the second V L The second joint between.

18. The CAR of claim 17, wherein the second connector comprises the amino acid sequence shown in SEQ ID NO:

2.

19. The CAR of any one of claims 12 to 18, wherein the second V H and the second V L Positioning from the N-terminus to the C-terminus: V H -V L .

20. The CAR of any one of claims 1 to 19, wherein the second anti-CD20 antigen-binding domain comprises a second anti-CD20 single-stranded variable fragment (scFv) that binds to CD20.

21. The CAR of claim 20, wherein the second anti-CD20 scFv comprises the amino acid sequence shown in SEQ ID NO:

29.

22. The CAR of claim 2 or 12, wherein the V H CDR1, V H CDR2, V H CDR3, V L CDR1, V L CDR2 and V L CDR3 is based on the Kabat numbering system, Chothia numbering system, AbM numbering system, Contact numbering system, or IMGT information system. ® It is used for identification.

23. The CAR of claim 22, wherein the V H CDR1, V H CDR2, V H CDR3, V L CDR1, V L CDR2 and V L CDR3 is identified according to the Kabat numbering system.

24. The CAR of any one of claims 1 to 23, further comprising: a third linker between the first anti-CD19 antigen-binding domain and the second anti-CD20 antigen-binding domain.

25. The CAR of claim 24, wherein the third connector comprises the amino acid sequence shown in SEQ ID NO:

3.

26. The CAR of any one of claims 1 to 25, wherein the first anti-CD19 antigen-binding domain is located downstream of the second anti-CD20 antigen-binding domain.

27. The CAR of any one of claims 1 to 26, wherein the leader sequence is covalently bound to the N-terminus of the second anti-CD20 antigen binding domain.

28. The CAR of claim 27, wherein the leader sequence comprises a CD8 polypeptide.

29. The CAR of claim 28, wherein the CD8 polypeptide comprises the amino acid sequence shown in SEQ ID NO:

33.

30. The CAR of any one of claims 1 to 29, wherein the first CD28 polypeptide is a transmembrane domain sequence of CD28.

31. The CAR of any one of claims 1 to 30, wherein the first CD28 polypeptide comprises amino acids 153 to 179 of SEQ ID NO:

35.

32. The CAR as claimed in any one of claims 1 to 31, further comprising: a hinge region.

33. The CAR of claim 32, wherein the hinge region comprises a second CD28 polypeptide.

34. The CAR of claim 33, wherein the second CD28 polypeptide comprises amino acids 114 to 152 of SEQ ID NO:

35.

35. The CAR of any one of claims 1 to 34, wherein each of the two loss-of-function mutations is located at a tyrosine amino acid residue.

36. The CAR of any one of claims 1 to 35, wherein the ITAM2 variant comprises the amino acid sequence shown in SEQ ID NO:

45.

37. The CAR of any one of claims 1 to 36, wherein the ITAM3 variant comprises the amino acid sequence shown in SEQ ID NO:

49.

38. The CAR of any one of claims 1 to 37, wherein the natural ITAM1 comprises the amino acid sequence shown in SEQ ID NO:

39.

39. The CAR of any one of claims 1 to 38, wherein the modified CD3ζ polypeptide comprises the amino acid sequence shown in SEQ ID NO:

51.

40. The CAR of any one of claims 1 to 39, wherein the intracellular signal transduction domain further comprises a co-stimulatory signal transduction region.

41. The CAR of claim 40, wherein the co-stimulatory signal transduction region comprises a third CD28 polypeptide.

42. The CAR of claim 41, wherein the third CD28 polypeptide is a CD28 signal transduction domain sequence.

43. The CAR of claim 42, wherein the third CD28 polypeptide comprises amino acids 180 to 220 of SEQ ID NO:

35.

44. The CAR according to any one of claims 1 to 43, comprising the amino acid sequence shown in SEQ ID NO:

54.

45. A nucleic acid encoding a CAR as described in any one of claims 1 to 44.

46. ​​The nucleic acid of claim 45, comprising the nucleotide sequence shown in SEQ ID NO:

55.

47. A vector comprising the nucleic acid as described in claim 45 or 46.

48. The vector of claim 47, wherein the vector is a viral vector.

49. The vector of claim 48, wherein the viral vector is a retroviral vector.

50. A cell comprising a CAR as claimed in any one of claims 1 to 44, a nucleic acid as claimed in claim 44 or 45, or a vector as claimed in any one of claims 47 to 49.

51. The cell of claim 50, wherein the cell is transduced using the CAR, the nucleic acid, or the vector.

52. The cell of claim 50 or claim 51, wherein the CAR is constitutively expressed on the surface of the cell.

53. The cell according to any one of claims 50 to 52, wherein the cell is an immune response cell or an immune effector cell.

54. The cell of any one of claims 50 to 53, wherein the cell is a lymphoid lineage cell or a myeloid lineage cell.

55. The cell of claim 54, wherein the lymphoid lineage cells are selected from the group consisting of: T cells, natural killer (NK) cells, B cells, and stem cells from which lymphoid cells can differentiate.

56. The cell of claim 54, wherein the myeloid lineage cell is selected from the group consisting of: monocytes, macrophages, neutrophils, dendritic cells, basophils, neutrophils, eosinophils, megakaryocytes, mast cells, erythrocytes, platelets, and stem cells from which myeloid cells can differentiate.

57. The cell of any one of claims 50 to 56, wherein the cell is selected from the group consisting of: T cells, B cells, natural killer (NK) cells, macrophages, innate lymphoid cells (ILCs), cytokine-induced killer (CIK) cells, lymphokine-activated killer (LAK) cells, stem cells from which lymphoid cells can differentiate, stem cells from which myeloid cells can differentiate, and combinations thereof.

58. The cell of claim 57, wherein the cell is a T cell.

59. The cell of any one of claims 55, 57 and 58, wherein the T cell is selected from the group consisting of: helper T cells, cytotoxic T cells (CTLs), memory T cells, regulatory T cells, tumor-infiltrating lymphocytes (TILs), natural killer T cells, mucosa-associated inert T cells, αβ T cells and γδ T cells.

60. The cell of claim 59, wherein the memory T cell is a central memory T cell.

61. The cell of any one of claims 58 to 60, wherein the T cell is positive for CD45RO and CD62L.

62. The cell of any one of claims 55 to 57, wherein the stem cell is a pluripotent stem cell.

63. The cell of claim 62, wherein the pluripotent stem cell is an embryo-like stem cell or an induced pluripotent stem cell.

64. The cell of any one of claims 50 to 61, wherein the cell is a cytotoxic T lymphocyte (CTL).

65. The cell according to any one of claims 50 to 61 and 64, wherein the cell is a CTL sensitized with a viral antigen.

66. The cell of any one of claims 50 to 61, 64 and 65, wherein the viral antigen is selected from the group consisting of Epstein-Barr virus (EBV), cytomegalovirus (CMV), BK virus (BKV), John Cunningham virus (JCV), small RNA virus, hepatotropic DNA virus, hepatitis C virus, delta virus, hepatitis E virus or any combination thereof.

67. The cell of claim 66, wherein the cell is an EBV-sensitized CTL.

68. A composition comprising cells as described in any one of claims 50 to 67.

69. The composition of claim 68, wherein the composition is a pharmaceutical composition and further comprises a pharmaceutically acceptable carrier.

70. The composition of claim 68 or 69, comprising about 1 × 10⁻⁶ 6 With approximately 5×10 8 The cells between.

71. The composition according to any one of claims 68 to 70, comprising about 1 × 10 6 With approximately 1×10 8 The cells between.

72. The composition according to any one of claims 68 to 71, comprising about 1 × 10 6 With approximately 5×10 7 The cells between.

73. The composition according to any one of claims 68 to 72, comprising about 1 × 10 6 With approximately 1×10 7 The cells between.

74. A method for inhibiting the growth of target cells expressing at least one B lymphocyte antigen, the method comprising: The target cells are brought into contact with the cells as described in any one of claims 50 to 67 or the composition as described in any one of claims 68 to 73.

75. The method of claim 74, wherein the at least one B lymphocyte antigen is selected from the group consisting of CD19, CD20, and combinations thereof.

76. The method of claim 74 or 75, wherein the target cells express detectable levels of CD19 and detectable levels of CD20.

77. The method of claim 74 or 75, wherein the target cells express detectable levels of CD20 and low or undetectable levels of CD19.

78. The method of claim 74 or 75, wherein the target cells express detectable levels of CD19 and low or undetectable levels of CD20.

79. The method of any one of claims 74 to 78, wherein the target cell is autologous to the cell.

80. The method of any one of claims 74 to 78, wherein the target cell is an allogeneic cell.

81. The method of any one of claims 74 to 80, wherein the target cell is a tumor cell.

82. The method of claim 81, wherein the tumor is cancer.

83. The method of claim 82, wherein the cancer is a blood cancer.

84. The method of claim 83, wherein the blood cancer is selected from the group consisting of: acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, mantle cell lymphoma (MCL), and follicular lymphoma (FL).

85. The method of any one of claims 74 to 80, wherein the target cell is a B cell.

86. The method of claim 85, wherein the B cell is CD19. + CD20 + CD19 + CD20 - Or CD19 - CD20 + .

87. The method of any one of claims 74 to 80, wherein the target cell is a T cell.

88. The method of claim 87, wherein the T cell is CD19. + CD20 + CD19 + CD20 - Or CD19 - CD20 + .

89. The method of claim 87 or 88, wherein the T cell is CD20. + .

90. The method of any one of claims 74 to 89, wherein the cells or composition exhibit cytotoxic and / or cytolytic activity against the target cells.

91. The method of claim 90, wherein the cytotoxicity and / or cytolytic activity is dose-dependent.

92. The method of claim 90 or 91, wherein the cytotoxicity and / or cytolytic activity is long-lasting.

93. The method of any one of claims 90 to 92, wherein the cytotoxic and / or cytolytic activity persists for at least 24 hours, at least 72 hours, at least one week, at least one month, or at least two months after the target cells have been initially contacted with the cells or composition.

94. The method of any one of claims 90 to 92, wherein the cytotoxic and / or cytolytic activity persists for about 40 days after the target cells have been initially contacted with the cells or composition.

95. The method of any one of claims 90 to 92, wherein the cytotoxic and / or cytolytic activity persists for about 60 days after the target cells have been initially contacted with the cells or composition.

96. A method for treating a subject with a B-lymphocyte antigen-related disease, the method comprising: The subject is given the cells as described in any one of claims 50 to 67 or the composition as described in any one of claims 68 to 73.

97. The method of claim 96, wherein the B lymphocyte antigen is selected from the group consisting of CD19, CD20, and combinations thereof.

98. The method of claim 96 or 97, wherein the B-lymphocyte antigen-associated disease is cancer.

99. The method of claim 98, wherein the cancer is a blood cancer.

100. The method of claim 99, wherein the blood cancer is selected from the group consisting of: acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, mantle cell lymphoma (MCL), and follicular lymphoma (FL).

101. The method of claim 96 or 97, wherein the B lymphocyte antigen-associated disease is an autoimmune disease.

102. The method of claim 101, wherein the autoimmune disease is selected from the group consisting of: multiple sclerosis, systemic lupus erythematosus (SLE), Sjögren's syndrome, AL amyloidosis, IgG4-related disease (IgG4-RD), inflammatory myopathy (e.g., myositis), scleroderma, pemphigus vulgaris, chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barré syndrome (GBS), stiff-person syndrome, rheumatoid arthritis, neuromyelitis optica spectrum disorders (NMO). SD), myasthenia gravis, N-methyl-D-aspartate receptor (NMDAR) encephalitis, anti-myelin oligodendrocyte glycoprotein (MOG) syndrome, myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD), membranous nephropathy (MN), IgA nephropathy (IgAN), type 1 diabetes mellitus, anti-neutrophil cytoplasmic antibody (ANCA) vasculitis, thrombocytopenic purpura, hemolytic anemia (AIHA), multifocal motor neuropathy (MMN), inflammatory bowel disease (IBD), celiac disease, Crohn's disease, ulcerative colitis, and ankylosing spondylitis.

103. The method of claim 102, wherein the autoimmune disease is multiple sclerosis.

104. The method of claim 102, wherein the autoimmune disease is systemic lupus erythematosus (SLE).

105. The method of any one of claims 96 to 104, wherein the cells or composition exhibit cytotoxic and / or cytolytic activity against target cells in the subject, wherein the target cells are positive for the B lymphocyte antigen.

106. The method of any one of claims 96 to 105, wherein the target cells express i) detectable levels of CD19 and detectable levels of CD20; ii) detectable levels of CD20 and low or undetectable levels of CD19; or iii) detectable levels of CD19 and low or undetectable levels of CD20.

107. The method of claim 105 or 106, wherein the target cell is a tumor cell.

108. The method of any one of claims 105 to 107, wherein the target cell is a cancer cell.

109. The method of claim 108, wherein the cancer is a blood cancer.

110. The method of claim 109, wherein the blood cancer is selected from the group consisting of: acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, mantle cell lymphoma (MCL), and follicular lymphoma (FL).

111. The method of claim 105 or 106, wherein the target cell is a B cell.

112. The method of claim 111, wherein the B cell is CD19. + CD20 + CD19 + CD20 - Or CD19 - CD20 + .

113. The method of claim 105 or 106, wherein the target cell is a T cell.

114. The method of claim 113, wherein the T cell is CD19. + CD20 + CD19 + CD20 - Or CD19 - CD20 + .

115. The method of claim 113 or 114, wherein the T cell is CD20. + .

116. The method of any one of claims 105 to 115, wherein the cytotoxicity and / or cell lysis activity is dose-dependent.

117. The method of any one of claims 105 to 116, wherein the cytotoxicity and / or cytolytic activity is long-lasting.

118. The method of any one of claims 105 to 117, wherein the cytotoxic and / or cytolytic activity persists for at least 24 hours, at least 72 hours, at least one week, at least one month, or at least two months after initial application of the cells or composition.

119. The method of any one of claims 105 to 118, wherein the cytotoxic and / or cytolytic activity persists for about 40 days after initial application of the cells or composition.

120. The method of any one of claims 105 to 119, wherein the cytotoxic and / or cytolytic activity persists for about 60 days after initial administration of the cells or composition.

121. The method of any one of claims 96 to 120, wherein the cells or composition do not induce graft-versus-host disease.

122. The method of any one of claims 96 to 121, wherein the cells or composition do not induce or induce minimal allogeneic reactivity in HLA-mismatched cells in the subject that are positive for the B lymphocyte antigen.

123. The method of any one of claims 96 to 122, wherein the cells or composition do not exhibit or exhibit minimal cytotoxic and / or cytolytic activity against off-target cells in the subject, wherein the off-target cells are negative for the B lymphocyte antigen.

124. The method of claim 123, wherein the off-target cells are selected from the group consisting of: CD19 - CD20 - T cells, natural killer T cells (NK T cells), natural killer cells (NK cells), and combinations thereof.

125. The method of any one of claims 96 to 124, wherein the cell or composition induces cytokine secretion.

126. The method of claim 125, wherein the cytokine is selected from the group consisting of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and combinations thereof.

127. The method of any one of claims 96 to 126, wherein the cell is a T cell.

128. The method of claim 127, wherein the T cells are EBV-sensitized CTLs.

129. The method of any one of claims 96 to 128, wherein after initial administration of the cells or composition, the cells persist in the subject for at least 24 hours, at least 72 hours, at least one week, at least one month, or at least two months.

130. The method of any one of claims 96 to 129, wherein the cells persist in the subject for about 40 days after initial administration of the cells or composition.

131. The method of any one of claims 96 to 130, wherein the cells persist in the subject for about 60 days after initial administration of the cells or composition.

132. A method for treating a subject with blood cancer, the method comprising: The subject is given the cells as described in any one of claims 50 to 67 or the composition as described in any one of claims 68 to 73.

133. The method of claim 132, wherein the blood cancer is selected from the group consisting of: acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell leukemia, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), diffuse large B-cell lymphoma (DLBCL), Burkitt lymphoma, mantle cell lymphoma (MCL), and follicular lymphoma (FL).

134. The method of claim 132 or 133, wherein the cells or composition exhibit cytotoxic and / or cytolytic activity against target cells in the subject, wherein the target cells express i) detectable levels of CD19 and detectable levels of CD20; ii) detectable levels of CD20 and low or undetectable levels of CD19; iii) detectable levels of CD19 and low or undetectable levels of CD20.

135. The method of claim 134, wherein the target cell is CD19. + CD20 + CD19 + CD20 - Or CD19 - CD20 + .

136. The method of claim 134 or 135, wherein the cytotoxicity and / or cell lysis activity is dose-dependent.

137. The method of any one of claims 134 to 136, wherein the cytotoxicity and / or cytolytic activity is long-lasting.

138. The method of any one of claims 134 to 137, wherein the cytotoxic and / or cytolytic activity persists for at least one month or at least two months after the initial application of the cells or composition.

139. The method of any one of claims 134 to 138, wherein the cytotoxic and / or cytolytic activity persists for about 40 days after initial administration of the cells or composition.

140. The method of any one of claims 134 to 139, wherein the cytotoxic and / or cytolytic activity persists for about 60 days after initial administration of the cells or composition.

141. The method of any one of claims 132 to 140, wherein the cells or composition do not induce graft-versus-host disease.

142. The method of any one of claims 132 to 141, wherein the cells or composition do not induce or induce minimal allogeneic reactivity in HLA-mismatched cells that are positive for CD19 and / or CD20 in the subject.

143. The method of any one of claims 132 to 142, wherein the cells or composition do not exhibit or exhibit minimal cytotoxic and / or cytolytic activity against off-target cells in the subject, wherein the off-target cells are negative for CD19 and CD20.

144. The method of any one of claims 132 to 143, wherein the cell is a T cell.

145. The method of claim 144, wherein the T cells are EBV-sensitized CTLs.

146. The method of any one of claims 132 to 145, wherein the cells persist in the subject for at least one month or at least two months after initial administration of the cells or composition.

147. The method of any one of claims 132 to 146, wherein the cells persist in the subject for about 40 days after initial administration of the cells or composition.

148. The method of any one of claims 132 to 147, wherein the cells persist in the subject for about 60 days after initial administration of the cells or composition.

149. A method for treating an autoimmune disease in a subject, the method comprising: The subject is given the cells as described in any one of claims 50 to 67 or the composition as described in any one of claims 68 to 73.

150. The method of claim 149, wherein the autoimmune disease is selected from the group consisting of: multiple sclerosis, systemic lupus erythematosus (SLE), Sjögren's syndrome, AL amyloidosis, IgG4-related disease (IgG4-RD), inflammatory myopathy (e.g., myositis), scleroderma, pemphigus vulgaris, chronic inflammatory demyelinating polyneuropathy (CIDP), Guillain-Barré syndrome (GBS), stiff-person syndrome, rheumatoid arthritis, neuromyelitis optica spectrum disorders (NMO). SD), myasthenia gravis, N-methyl-D-aspartate receptor (NMDAR) encephalitis, anti-myelin oligodendrocyte glycoprotein (MOG) syndrome, myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD), membranous nephropathy (MN), IgA nephropathy (IgAN), type 1 diabetes mellitus, anti-neutrophil cytoplasmic antibody (ANCA) vasculitis, thrombocytopenic purpura, hemolytic anemia (AIHA), multifocal motor neuropathy (MMN), inflammatory bowel disease (IBD), celiac disease, Crohn's disease, ulcerative colitis, and ankylosing spondylitis.

151. The method of claim 150, wherein the autoimmune disease is multiple sclerosis.

152. The method of claim 150, wherein the autoimmune disease is systemic lupus erythematosus (SLE).

153. The method of any one of claims 149 to 152, wherein the cells or composition exhibit cytotoxic and / or cytolytic activity against target cells in the subject, wherein the target cells express i) detectable levels of CD19 and detectable levels of CD20; ii) detectable levels of CD20 and low or undetectable levels of CD19; or iii) detectable levels of CD19 and low or undetectable levels of CD20.

154. The method of claim 153, wherein the target cell is CD19. + CD20 + CD19 + CD20 - Or CD19 - CD20 + .

155. The method of claim 153 or 154, wherein the target cell is a B cell.

156. The method of claim 155, wherein the B cell is CD19. + CD20 + CD19 + CD20 - Or CD19 - CD20 + .

157. The method of claim 153 or 154, wherein the target cell is a T cell.

158. The method of claim 157, wherein the T cell is CD19. + CD20 + CD19 + CD20 - Or CD19 - CD20 + .

159. The method of claim 157 or 158, wherein the T cell is CD20. + .

160. The method of any one of claims 149 to 159, wherein the cells or composition exhibit cytotoxic and / or cytolytic activity against a first target cell and a second target cell in the subject, wherein the first cell and the second target cell each express i) a detectable level of CD19 and a detectable level of CD20; ii) a detectable level of CD20 and a low or undetectable level of CD19; or iii) a detectable level of CD19 and a low or undetectable level of CD20.

161. The method of claim 160, wherein the first target cell and the second target cell are each CD19. + CD20 + CD19 + CD20 - Or CD19 - CD20 + .

162. The method of claim 160 or 161, wherein the first target cell is a B cell.

163. The method of any one of claims 160 to 162, wherein the second target cell is a T cell.

164. The method of claim 163, wherein the T cell is CD20. + .

165. The method of any one of claims 153 to 164, wherein the cytotoxicity and / or cytolytic activity is dose-dependent.

166. The method of any one of claims 153 to 165, wherein the cytotoxicity and / or cytolytic activity is long-lasting.

167. The method of any one of claims 153 to 166, wherein the cytotoxic and / or cytolytic activity persists for at least 24 hours or at least 72 hours after initial application of the cells or composition.

168. The method of any one of claims 153 to 17, wherein the cells or composition do not induce graft-versus-host disease.

169. The method of any one of claims 153 to 168, wherein the cells or composition do not induce or induce minimal allogeneic reactivity in HLA-mismatched cells in the subject that are positive for CD19 and / or CD20.

170. The method of any one of claims 153 to 169, wherein the cells or composition do not exhibit or exhibit minimal cytotoxic and / or cytolytic activity against off-target cells in the subject, wherein the off-target cells are negative for CD19 and CD20.

171. The method of claim 170, wherein the off-target cells are selected from the group consisting of: CD19 - CD20 - T cells, natural killer T cells (NK T cells), natural killer cells (NK cells), and combinations thereof.

172. The method of any one of claims 149 to 171, wherein the cell or composition induces cytokine secretion.

173. The method of claim 172, wherein the cytokine is selected from the group consisting of interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and combinations thereof.

174. The method of any one of claims 149 to 173, wherein the cell is a T cell.

175. The method of claim 174, wherein the T cells are EBV-sensitized CTLs.

176. The method of any one of claims 96 to 175, wherein the subject is a human subject.

177. The method of claim 176, wherein the cells are autologous to the subject.

178. The method of claim 176, wherein the cells are allogeneic to the subject.

179. The cells of any one of claims 50 to 67 or the composition of any one of claims 68 to 73, used in a therapeutic manner.

180. The cell of any one of claims 50 to 67 or the composition of any one of claims 68 to 73, in a method for inhibiting the growth of target cells expressing at least one B lymphocyte antigen.

181. The cell of any one of claims 50 to 67 or the composition of any one of claims 68 to 73, in a method of treating a subject with a B-lymphocyte antigen-related disease.

182. The cell of any one of claims 50 to 67 or the composition of any one of claims 68 to 73, in a method of treating a subject with a blood cancer.

183. The cell of any one of claims 50 to 67 or the composition of any one of claims 68 to 73, in a method of treating an autoimmune disease in a subject.

184. Use of the cells of any one of claims 50 to 67 or the compositions of any one of claims 68 to 73 for the manufacture of a medicament for inhibiting the growth of target cells expressing at least one B lymphocyte antigen.

185. Use of the cells of any one of claims 50 to 67 or the compositions of any one of claims 68 to 73 for the manufacture of a medicament for treating a subject with a B-lymphocyte antigen-related disease.

186. Use of the cell or composition as described in any one of claims 50 to 67 for the manufacture of a medicament for treating a subject with blood cancer.

187. Use of the cells of any one of claims 50 to 67 or the compositions of any one of claims 68 to 73 for the manufacture of a medicament for treating an autoimmune disease in a subject.

188. A method for producing cells as described in any one of claims 50 to 67, the method comprising: The CAR as described in any one of claims 1 to 44, the nucleic acid as described in claim 45 or 46, or the vector as described in any one of claims 47 to 49 is introduced into the cell.

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