Artificial immune cells, compositions and methods thereof for treating disease

Abstract

The present invention relates to the treatment or management of autoimmune diseases, organ rejection and cancer utilizing CAR constructs that bind to T cell surface antigens or B cell or plasma surface antigens or both.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This non-provisional application claims the benefit of U.S. Provisional Application No. 63 / 428,259, filed November 28, 2022, which is hereby incorporated by reference in its entirety for all purposes.

[0002] FIELD OF THE INVENTION The present invention relates to the treatment or management of autoimmune diseases, organ rejection and cancer. [Background technology]

[0003] background T cells, a type of lymphocyte, play a central role in cell-mediated immunity. They are distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of T cell receptors (TCRs) on their surface.

[0004] Autoimmune diseases occur when a subject's immune system attacks the subject's own healthy cells, tissues, and organs. In some cases, these disorders result from the aberrant recognition of antigens on the subject's own tissues ("self-antigens") by T lineage cells ("autoreactive T cells") via the T cell receptor (TCR). A similar process occurs in hosts after receiving organs from a donor; this is also known as "organ rejection."

[0005] For example, type 1 diabetes (T1D) is an autoimmune disease in which autoreactive T cells destroy insulin-producing beta cells in the pancreatic islets of Langerhans, resulting in insulin deficiency and hyperglycemia. This is primarily mediated by T cells specific to autoantigens in the islets. There is currently no cure for T1D, and its incidence increases by approximately 2-3% each year. In the United States, the disease affects approximately 22.9 people per 100,000 under the age of 65. T1D is diagnosed when fasting blood glucose levels are 126 mg / dL or higher and random blood glucose levels are 200 mg / dL or higher. Patients with T1D often experience hypoglycemia and ketoacidosis, which can be life-threatening.

[0006] Microvascular complications of T1D manifest as retinopathy, neuropathy, nephropathy, cognitive impairment, and increased risk of atherosclerosis and thrombosis. Standard clinical management aims to lower HbA1C and reduce the risk of hyperglycemia.

[0007] Globally, type 1 disease (T1D) represents an emerging public health burden, with onset most common in those under 15 years of age. Unfortunately, no disease-modifying prevention or intervention strategies to halt T1D progression are routinely used in clinical practice. Patients rely on exogenous insulin administration to maintain glucose homeostasis, a practice that has been in place since insulin was first used in humans approximately 100 years ago. However, despite evidence demonstrating the importance of glycemic control in reducing complications, lifelong adherence to routine management regimens remains challenging for individuals and results are suboptimal. Therefore, developing methods to maintain or restore endogenous insulin secretion is the ultimate goal of T1D research. Given the lack of groundbreaking improvements over the past century, there is a clear and ongoing unmet clinical need in this area. An ideal innovative treatment for diabetes would potentially halt and reverse β-cell mass destruction without significant side effects, resulting in long-term independence from exogenous insulin.

[0008] Similarly, treatment protocols for IBD (inflammatory bowel disease) have remained largely unchanged for decades, focusing on clinical management of the disease. Therefore, there is an ongoing clinical need to provide innovative, more effective, safer, and longer-lasting treatments for IBD patients.

[0009] Many autoimmune diseases, including cancer, plague people, yet there are few treatment options, all of which are associated with serious side effects. There is a great need for safer and more effective therapies for autoimmune diseases and cancer.

[0010] Therefore, existing treatments for autoimmune diseases are not always effective in inducing or maintaining remission and may be associated with undesirable side effects. Thus, a clear and present need for additional therapies for the treatment and management of autoimmune diseases has been reaffirmed. The novel invention herein resets the immune system by depleting disease-causing autologous T cells, B cells, and plasma cells and repopulating lymphocyte populations from bone marrow stem cells. Summary of the Invention [Problem to be solved by the invention]

[0011] Summary of the Invention The present invention relates to methods of treating or managing a subject with an autoimmune disease or organ rejection utilizing a CAR that binds to an antigen present on the surface of a T cell, including but not limited to CD2, CD3, CD4, CD5, CD7, and an antigen present on the surface of a B cell or plasma cell, including but not limited to CD19, CD20, CD22, CD38, CS1 (CD319, SLAMF7), GPRC5D (G protein-coupled receptor, class C, group 5, member D), and BCMA. In a preferred embodiment, the dual CAR has one CAR unit that targets T cells by selecting at least one target antigen from the following group; and the other CAR unit that targets B cells by selecting at least one target antigen from the following group: CD2, CD3, CD4, CD5, and CD7; and the other CAR unit that targets B cells by selecting at least one target antigen from the following group: CD19, CD20, CD22, or for plasma cells, at least one from the following group or target antigens: BCMA, CD38, CS1 (CD319, SLAMF7), GPRC5D, CD138.

[0012] In another embodiment, dual CARs, which may be bispecific tandem CARs, compound CARs, cistronic chimeric antigen receptor CARs, and bispecific CARs, are utilized in the methods. Compound CAR (cCAR) structures and methods for producing cCARs are described in PCT / US2016 / 039306, PCT / US2016 / 068349, and PCT / US2018 / 038529, all of which are incorporated herein by reference in their entireties.

[0013] In one embodiment, the present disclosure provides a method of treating a cell proliferative disorder, the method comprising administering to a patient in need thereof a therapeutically effective amount of engineered cells expressing a CAR polypeptide having a CD2, CD3, CD4, CD5, CD7, CD8, or CD52 antigen recognition domain.

[0014] In one embodiment, the present disclosure provides a method of treating an autoimmune disease, the method comprising administering to a patient in need thereof a therapeutically effective amount of engineered cells expressing a CAR polypeptide having a CD2, CD3, CD4, CD5, CD7, CD8, or CD52 antigen recognition domain.

[0015] In one embodiment, the invention provides a method of treating or managing an autoimmune disease, the method using a CAR configured by selecting one of the target antigens from the following group: CD2, CD3, CD4, CD5, or CD7. In a further embodiment, the CAR T cells deplete autoreactive T cells against host tissue.

[0016] In a preferred embodiment, the autoimmune disease is caused by T lineage cells (e.g., autoreactive T lineage cells). In a preferred embodiment, the T lineage cells, e.g., autoreactive T lineage cells, are T cells having a TCR directed against healthy tissues, including pancreatic β cells. In a further embodiment, the CAR T cells deplete autoreactive T cells against healthy host tissues.

[0017] In another embodiment, the autoimmune disease is caused by T lineage cells (e.g., autoreactive T lineage cells) causing inflammation and damage to healthy tissue, such as infiltration of the lamina propria by inflammatory CD4+ T cell populations in IBD (Crohn's disease, ulcerative colitis). In a further embodiment, the CAR T cells deplete autoreactive T cells from healthy host tissue.

[0018] In another embodiment, the autoimmune disease is caused by T lineage cells (e.g., autoreactive T lineage cells) causing inflammation and damage to healthy tissue, such as infiltration by autoreactive T cells in psoriasis. In a further embodiment, the CAR T cells deplete autoreactive T cells from healthy host tissue.

[0019] In another embodiment, graft-versus-host disease caused by donor T-cell lineage cells leads to inflammation and damage to healthy tissues of the recipient. In a further embodiment, the CAR T cells deplete donor autoreactive T cells against healthy host tissues.

[0020] In a preferred embodiment, the autoimmune disease is caused by both T-lineage cells (e.g., autoreactive T-lineage cells) and autoreactive antibodies produced by B cells or plasma cells. In another embodiment, the autoimmune disease is an organ transplant autorejection caused by autoreactive T cells or autoreactive antibodies produced by B cells or plasma cells.

[0021] In another embodiment, the autorejection of an organ transplant is caused by both autoreactive T cells and autoreactive antibodies produced by B cells or plasma cells.

[0022] In another embodiment, the present disclosure provides an artificial cell having at least one chimeric antigen receptor polypeptide and an enhancer.

[0023] In one embodiment, the present disclosure provides an engineered cell having at least two different chimeric antigen receptor polypeptides and an enhancer.

[0024] As used herein, enhancers include biological molecules that promote or enhance the activity of artificial cells having chimeric antigen receptor polypeptides. Enhancers include cytokines. In another embodiment, enhancers include IL-2, IL-7, IL-12, IL-15, IL-18, IL-10, IL-21, PD-1, PD-L1, CSF1R, CTAL-4, TIM-3, and TGFRβ, their receptors, TNF-α, IL-15 or IL-15 / IL-15sushi or IL-15 / IL-15sushi anchor, and functional fragments thereof.

[0025] In one embodiment, the present disclosure provides an artificial T cell, NK cell, having an enhancer selected from the following: IL-15 or IL-15 / IL-15sushi or IL-15 / IL-15sushi anchor. In this further embodiment, the additional enhancer promotes proliferation and persistence of the CAR T cell.

[0026] In another embodiment, the present disclosure provides a method for reducing the number of autoreactive T-lineage cells or autoreactive B-lineage cells thereof, comprising administering to said host in need thereof a composition comprising (i) engineered or modified immune cells and (ii) IL-7, IL-15, IL- / IL-15 sushi, IL-15 / IL-15 sushi anchor, IL-10, CCL-119, or CCL-21. The most common cells of the immune system include, but are not limited to, lymphocytes (T cells, B cells, NK cells), neutrophils, and monocytes / macrophages.

[0027] In another embodiment, the present disclosure provides a method for reducing the number of autoreactive T lineage cells or autoreactive B lineage cells thereof, comprising administering to said host in need thereof a composition comprising (i) engineered or modified NK cells or T cells, and (ii) IL-7, IL-15, IL- / IL-15sushi, IL-15 / IL-15sushi anchor, CCL-119, or CCL-21.

[0028] In yet another embodiment, a method for ex vivo expansion of NK cells and T cells, comprising: 1) isolation of NK cells or T cells; 2) introduction of at least one CAR; 3) introduction of at least one enhancer selected from the group of IL-7, IL-15, IL-15sushi, IL-15 / IL-15anchor, CCL-19 (CCL19) and CCL-21 (CCL21), and 3) expansion of NK cells or T cells. In some embodiments, the autoimmune disease is selected from T1D, MS, IBD, celiac disease, asthma, systemic lupus erythematosus, IgA nephropathy, IgG4-related disease, membranous nephropathy, myasthenia gravis, neuromyelitis optica, pemphigus vulgaris, anti-PAD4-activated rheumatoid arthritis, sensitized / pre-existing antibodies in solid organ transplantation, Guillain-Barré syndrome (acute inflammatory demyelinating polyneuropathy - AIDP), chronic inflammatory demyelinating polyneuropathy (CIDP), immune thrombocytopenic purpura, rheumatoid arthritis, and ANCA-associated vasculitis (AAV). In preferred embodiments, the autoimmune disease is a T cell-mediated autoimmune disease.

[0029] In some embodiments, the autoimmune disease is newly diagnosed (e.g., newly diagnosed T1D, MS, or IBD). In some embodiments, the autoimmune disease is relapsing or refractory (e.g., relapsing or refractory T1D, MS, IBD).

[0030] In another embodiment, CARs targeting the CD7 surface antigen can deplete autoreactive immune cells expressing the CD7 surface antigen. The unexpected discovery was that the CD7+ population is depleted (to approximately 90 or more T lymphocytes), while the CD7- population of T lymphocytes expands to maintain the total number of T cells and prevent infection. This phenomenon acts as an immune system reset for the T cell immune system, treating T cell-mediated autoreactive diseases.

[0031] In another embodiment, a CD7 monoclonal antibody targeting the CD7 surface antigen can deplete autoreactive immune cells expressing the CD7 surface antigen. The CD7+ population is depleted (approximately 90 or more T lymphocytes), and the CD7- population of T lymphocytes expands to maintain the total T cell population and prevent infection, revealing an unexpected finding. This phenomenon acts as a reset of the T cell immune system and treats T cell-mediated autoimmune diseases.

[0032] In another embodiment, anti-CD7CAR targets T lymphocyte lineage cells, particularly T regulatory cells (Treg cells), and enhances T cell proliferation. In a further embodiment, CD7CAR can be used as a pretreatment for CAR T cell therapy. In such an embodiment, there is an unexpected finding that CD7CAR has low toxicity and does not significantly reduce the number of T cells in the treated subject.

[0033] In another embodiment, anti-CD7CAR targets T lymphocyte lineage cells, particularly T regulatory cells (Treg cells), and enhances T cell expansion. In a further embodiment, CD7CAR can be used as a conditioning treatment for CAR T cell therapy in combination with conditioning medications (cyclophosphamide, fludarabine). In such an embodiment, the unexpected finding is that CD7CAR has low toxicity and does not significantly reduce the number of T cells in treated subjects.

[0034] In another embodiment, a subject may be pre-administered an anti-CD7 CAR to deplete T cells and Tregs that express the CD7 surface antigen, and then administered the targeted CAR. In some embodiments, a subject may be pre-administered an anti-CD7 CAR to improve CAR T or NK kinetics and initial response and reduce rejection.

[0035] In another embodiment, subjects can be pretreated with an anti-CD7 monoclonal antibody in combination with cyclophosphamide and fludarabine to improve CAR T cell and NK cell expansion and reduce rejection.

[0036] In another embodiment, when a subject is administered an anti-CD7 monoclonal antibody, the combination of cyclophosphamide and fludarabine can improve CAR T cell and NK cell proliferation and reduce rejection.

[0037] In a preferred embodiment, the autoimmune disease is caused by T lineage cells (e.g., autoreactive T lineage cells). In a preferred embodiment, the T lineage cells, e.g., autoreactive T lineage cells, are T cells having a TCR directed against healthy tissues, including pancreatic β cells. In a further embodiment, autoreactive T lineage cells against pancreatic β cells can be depleted by anti-CD7 monoclonal antibodies.

[0038] In another embodiment, a dual CAR comprising a CD7CAR and a targeted CAR in the construct can be administered to a subject.

[0039] In further embodiments, anti-CD7 CARs elucidated the unexpected finding that greater than 90% of CD7-positive T-lineage lymphocytes were depleted, while a minority population (2-10%) of CD7-negative T-lineage lymphocytes expanded along with the CAR T-cell population. The unexpected finding of normal T-lineage lymphocyte population numbers following anti-CD7 CAR treatment provided subjects with T-cell immune function and protection against infection.

[0040] In certain embodiments, the CD7CAR can be combined with a CAR requiring CAR T cell expansion. The CD7CAR can be combined with a CAR selected from the following target antigens: GD2, GD3, ROR1, PSMA, PSCA (prostate stem cell antigen), MAGE A3, glycolipids, glypican 3, F77, GD-2, WT1, CEA, HER-2 / neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, MUC1, MUC2, MUC3, MUC4, MUC5, KIF20A, Survivin, AFP-1, gp100, MUC1, PAP-10, PAP-5, TRP2-1, SART-1, VEGFR1, VEGFR2, NEIL3, MPHOSPH1, DEPDC1, FOXM1, CDH3, TTK, TOMM34, URLC10, KOC1, UBE2T, TOPK, ECT2, MESOTHELIN, NKG2D, P1 A, GM2, CD30, MMG49 epitope, EGFRvIII, CD33, CD123, CLL-1, immunoglobulin kappa and lambda, CD38, CD52, CD47, CD200, CD70, CD19, CD20, CD22, CD38, BCMA, CS1, NKG2D receptor, April receptor, BAFF receptor, TACI, CD3, CD4, CD8, CD5, CD2, GPRC5D (G protein-coupled receptor, class C, group 5, member D), and CD138. Target antigens also include viral and fungal antigens such as human papillomavirus (HPV) antigens and EBV (Epstein-Barr virus) antigens E6 and E7.

[0041] In another embodiment, the present disclosure provides an artificial cell having at least one of recombinant IL-15, IL-15RA, IL-15sushi, IL-15 / IL-15RA, IL15-RA / IL-15, IL-15 / IL-15sushi, IL15sushi / IL-15, functional fragments thereof, or combinations thereof; and an antigen recognition domain selected from the group consisting of NY-ESO-1, alpha-fetoprotein (AFP), glypican-3 (GPC3), BCMA, BAFF-R, BCMA, TACI, LeY, CD5, CD7, CD2, CD3, CD4, CD45, CD13, CD14, CD15, CD19, CD20, CD22, CD33, CD41, CD61, CD64, CD68, CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor, ROR1, PSMA, At least one distinct CAR polypeptide, including MAGE A3, glycolipid, F77, GD-2, WT1, CEA, HER-2 / neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, CD30, EGFRvIII, immunoglobulins kappa and lambda, CD38, CS1. Target antigens can also include viral or fungal antigens, such as E6 and E7 from human papillomavirus (HPV) or EBV (Epstein-Barr virus) antigens. In further embodiments, antigen-recognizing polypeptides (scFvs) and corresponding polynucleotides against CD2, CD3, CD5, CD7, and CD52, as well as IL-15 / IL-15sushi and IL-15sushi, are described in more detail in PCT Application Nos. PCT / US2016 / 39306 and PCT / US2016 / 019953, the contents of which are incorporated herein by reference.

[0042] In another embodiment, the present disclosure provides cells engineered to further express other regulators of immune function, such as CCL21, IL-2, IL-4, IL-12, IL-13, IL-17, IL-18, IP-10, CCL4, Flt3L, interferon-γ, MIP-1α, GM-CSF, M-CSF, TGF-β, and TNF-α.

[0043] In another embodiment, the present disclosure provides an artificial cell having at least one of recombinant IL-15, IL-15RA, IL-15sushi, IL-15 / IL-15RA, IL15-RA / IL-15, IL-15 / IL-15sushi, IL15sushi / IL-15, functional fragments thereof, or combinations thereof; and an antigen recognition domain selected from the group consisting of NY-ESO-1, alpha-fetoprotein (AFP), glypican-3 (GPC3), BCMA, BAFF-R, BCMA, TACI, LeY, CD5, CD7, CD2, CD3, CD4, CD45, CD13, CD14, CD15, CD19, CD20, CD22, CD33, CD41, CD61, CD64, CD68, CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor, ROR1, PSMA, At least one distinct CAR polypeptide, including MAGE A3, glycolipid, F77, GD-2, WT1, CEA, HER-2 / neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, CD30, EGFRvIII, immunoglobulins kappa and lambda, CD38, CS1. Target antigens can also include viral or fungal antigens, such as E6 and E7 from human papillomavirus (HPV) or EBV (Epstein-Barr virus) antigens. In further embodiments, antigen-recognizing polypeptides (scFvs) and corresponding polynucleotides against CD2, CD3, CD5, CD7, and CD52, as well as IL-15 / IL-15sushi and IL-15sushi, are described in more detail in PCT Application Nos. PCT / US2016 / 39306 and PCT / US2016 / 019953, the contents of which are incorporated herein by reference.

[0044] In another embodiment, the present disclosure provides cells engineered to further express other regulators of immune function, such as CCL21, IL-2, IL-4, IL-12, IL-13, IL-17, IL-18, IP-10, CCL4, Flt3L, interferon-γ, MIP-1α, GM-CSF, M-CSF, TGF-β, and TNF-α.

[0045] In another embodiment, a subject can be administered an anti-CD7 CAR to deplete T cells and Tregs that express the CD7 surface antigen, followed by administration of a targeted CAR having at least one target antigen selected from, but not limited to, this group: GD2, GD3, ROR1, PSMA, PSCA (prostate stem cell antigen), MAGE A3, glycolipids, glypican 3, F77, GD-2, WT1, CEA, HER-2 / neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, MUC1, MUC2, MUC3, MUC4, MUC5, KIF20A, Survivin, AFP-1, gp100, MUC1, PAP-10, PAP- 5, TRP2-1, SART-1, VEGFR1, VEGFR2, NEIL3, MPHOSPH1, DEPDC1, FOXM1, CDH3, TTK, TOMM34, URLC10, KOC1, UBE2T, TOPK, ECT 2, mesothelin, NKG2D, P1A, GM2, CD30, MMG49 epitope, EGFRvIII, CD33, CD123, CLL-1, immunoglobin kappa and lambda, CD38, CD52, CD47, CD200, CD70, CD19, CD20, CD22, CD38, BCMA, CS1, NKG2D receptor, April receptor, BAFF receptor, TACI, CD3, CD4, CD8, CD5, CD2, and CD 138. Target antigens can also include viral or fungal antigens, such as human papillomavirus (HPV) or EBV (Epstein-Barr virus) antigens E6 and E7.

[0046] One aspect provided is a method for generating or isolating CD7-negative T cells or NK cells (CD7- T or NK cells) for use in CAR T cell or NK cell therapy. The method consists of (1) natural selection of T cells or NK cells transduced with a CD7 CAR construct, (2) isolation of peripheral CD7-negative T cells or NK cells, (3) an anti-CD7 scFv fused to the ER (endoplasmic reticulum) retention sequence KDEL to confine intracellular CD7 protein within the secretory pathway, and (4) CD7 gene knockout in T cells or NK cells.

[0047] In certain embodiments, the CD7CAR can be combined with another CAR in a single construct to enhance the expansion of CAR T cells or NK cells (see Figure 8). The CD7CAR generates a CD7-negative T or NK population, enhancing CAR T or NK cell killing and persistence, while reducing adverse toxicity events associated with CAR T or NK cell expansion. The CD7CAR can be combined with a CAR whose CAR target is selected from the following target antigens: GD2, GD3, ROR1, PSMA, PSCA (prostate stem cell antigen), MAGE A3, glycolipids, glypican 3, F77, GD-2, WT1, CEA, HER-2 / neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, MUC1, MUC2, MUC3, MUC4, MUC5, KIF20A, Survivin, AFP-1, gp100, MUC1, PAP-10, PAP-5, TRP2-1, SART -1, VEGFR1, VEGFR2, NEIL3, MPHOSPH1, DEPDC1, FOXM1, CDH3, TTK, TOMM34, URLC10, KOC1, UBE2T, TOPK, ECT2, MESOTHELIN, NKG2D, P1A, GM2, CD 30, MMG49 epitope, EGFRvIII, CD33, CD123, CLL-1, delta-like protein 3 (DLL3), immunoglobulin kappa and lambda, CD38, CD52, CD47, CD200, CD70, CD19, CD20, CD22, CD38, BCMA, CS1, NKG2D receptor, April receptor, BAFF receptor, TACI, CD3, CD4, CD8, CD5, CD2, GPRC5D (G protein-coupled receptor, class C, group 5, member D), and CD138. Target antigens can also include viral or fungal antigens, such as human papillomavirus (HPV) or EBV (Epstein-Barr virus) antigens E6 and E7.

[0048] In one embodiment, a CD7 negative population of T cells or NK cells is generated by a natural self-selection approach by directly introducing a CD7 CAR, and the CD7 negative population is used to generate a CAR that targets cancer or an autoimmune disease.

[0049] In one embodiment, CD7-negative T cell or NK cell populations are used to generate CARs that target hematopoietic malignancies or solid tumors to reduce CAR T associated cytokine release syndrome and neurotoxicity.

[0050] In one embodiment, a CD7-negative T cell or NK cell population is used to generate a CAR that targets a hematological malignancy or solid tumor, and the CD7-negative T cells or NK cells enhance the killing effect of the CAR.

[0051] In one embodiment, CD7-negative T cell or NK cell populations are used to generate CARs that target hematologic malignancies or solid tumors, and the CD7-negative T cells or NK cells enhance the persistence or penetration of the CAR into the tumor or tissue.

[0052] In certain embodiments, CD7-negative T cells or NK cells are used to generate CARs with target antigens selected from, but not limited to, the following: GD2, GD3, ROR1, PSMA, PSCA (prostate stem cell antigen), MAGE A3, glycolipids, glypican 3, F77, GD-2, WT1, CEA, HER-2 / neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, MUC1, MUC2, MUC3, MUC4, MUC5, KIF20A, Survivin, AFP-1, gp100, MUC1, PAP-10, PAP-5, TRP2-1, SART-1, VEGFR1, VEGFR2, NEIL3, MPHOSPH1, DEPDC1, FOXM1, Delta-like protein 3 (DLL3), CDH3, TTK, TOMM34, URLC10, KOC1, UBE2T, TOPK, ECT2, mesothelin, NKG2D, P1A, GM2, CD30, MMG49 epitope, EGFRvIII, CD33, CD123, CLL-1, immunoglobulin kappa and lambda, CD38, CD52, CD47, CD200, CD70, CD19, claudin 18, 2, CD20, CD22, CD38, BCMA, CS1, NKG2D receptor, April receptor, BAFF receptor, TACI, CD3, CD4, CD8, CD5, CD2, GPRC5D (G protein-coupled receptor, class C, group 5, member D), and CD138. Target antigens can also include viral or fungal antigens, such as human papillomavirus (HPV) or EBV (Epstein-Barr virus) antigens E6 and E7.

[0053] In one embodiment, there is provided a method for treating or preventing a disease or condition in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition, a CAR T cell of the invention.

[0054] In one embodiment, the CD7 negative T cells or NK cells enhance the CAR to target cancer, and the cancer is selected from the group consisting of gastric cancer, colon cancer, lung cancer, hepatocellular carcinoma, melanoma, breast cancer, bladder cancer, ovarian cancer, cervical cancer, renal cell carcinoma, glioma, glioblastoma and other solid cancers, B cell lymphoma and leukemia, Hodgkin's lymphoma, multiple myeloma, T cell lymphoblastic leukemia and lymphoma, T cell lymphoma and leukemia, acute myeloid leukemia, chronic myelogenous leukemia, myelodysplastic syndrome, chronic myeloproliferative neoplasm, NK cell neoplasm and other liquid tumors.

[0055] In another embodiment, a composite chimeric antigen receptor dual CAR is utilized, where one chimeric antigen receptor (CAR) unit targets T cells by selecting one of the target antigens from the following group: CD2, CD3, CD4, CD5, and CD7; and the other CAR unit targets B cells by selecting one of the target antigens CD19, CD20, CD22; or the target antigens BCMA, CD38, CS1, GPRC5D, and CD138. [Brief explanation of the drawings]

[0056] Brief Description of the Drawings [Figure 1]Figures 1A-1D. Generation of CD4CAR T cells. (1A) Experimental design. (1B) CB buffy coat cells were activated with anti-CD3 antibody and IL-2 for 2 days. After 7 days of incubation, cells were analyzed by flow cytometry using biotin-conjugated goat anti-mouse Fab2 or goat IgG antibodies and streptavidin-PE. Untransduced, labeled CB cells are shown on the left. (1C) CD4CAR T cells deplete the CD4+ population during T cell expansion. CB buffy coat cells were activated with anti-CD3 antibody and IL-2 for 2 days. Two T cell subsets are present in the CB buffy coat: CD8+ cytotoxic T cells and CD4+ helper T cells (left). Cells were transduced with GFP (center) or CD4CAR (right) lentiviral supernatant. After 3 days of culture, cells were analyzed by flow cytometry using mouse anti-human CD4 (FITC) and CD8 (APC) antibodies. Non-transfected PMBCs were also labeled (left). (1D) Most CD4CAR T cells have a central memory-like phenotype. CB buffy coat cells were activated with anti-CD3 antibody for 2 days. Cells were transduced with CD4CAR lentiviral supernatant. After 6 days of expansion, CD8+ cells were analyzed by flow cytometry for CD62L, CD45RO, and CD45RA phenotypes (N=3). [Figure 2]Figures 2A-2D. CD4CAR T cells eliminate T-cell leukemia cells in coculture assays. (2A) CD4CAR T cells eliminate KARPAS 299 T-cell leukemia cells in coculture. Activated human CB buffy coat cells transduced with GFP (center) or CD4CAR (right) lentiviral supernatant were incubated with KARPAS 299 cells at a 2:1 ratio. After 24 hours of coculture, cells were stained with mouse anti-human CD4 (APC) and CD8 (PerCp) antibodies and analyzed by flow cytometry for T-cell subsets (N=3). (2B) and (2C) CD4CAR T cells eliminate primary T-cell leukemia cells in coculture. Activated human CB buffy coat cells transduced with GFP (center) or CD4CAR (right) lentiviral supernatant were incubated with primary T-cell leukemia cells from Sézary syndrome and PTCL (2C) at a 2:1 ratio. After 24 hours of coculture, cells were analyzed by flow cytometry using mouse anti-human CD4 (FITC) and CD8 (APC) antibodies (N=3). Human primary cells alone were also labeled (left). (2D) CD4CAR T cells failed to lyse CD4-negative lymphoma cells (SP53, a B-cell lymphoma cell line). Activated human CB buffy coat cells transduced with GFP (center) or CD4CAR (right) lentiviral supernatant were incubated at a 2:1 ratio with SP53 mantle cell lymphoma cells prestained with the membrane dye CMTMR. After 24 hours of coculture, cells were stained with mouse anti-human CD3 (PerCp) and analyzed by flow cytometry (N=2). SP53 cells alone, prestained with CMTMR, were also labeled (left). [Figure 3]Figures 3A-3B. PBMC-derived CD4CAR T cells are highly enriched for CD8+ T cells and specifically kill CD4-expressing leukemia cell lines. (3A) PBMC-derived CD4CAR T cells are highly enriched for CD8+ T cells. PMBC buffy coat cells, which comprise T cells, CD8+ and CD4+ (left), were activated with anti-CD3 antibody and IL-2 for 2 days and then transduced with either GFP (center) or CD4CAR (right) lentiviral supernatant. After 3 days of culture, cells were labeled and analyzed by flow cytometry for T cell subsets. Non-transfected PMBC were also labeled (left). (3B) CD4CAR T cells specifically kill KARPAS 299 cells. PMBC T cells transfected with GFP control or CD4CAR lentiviral supernatant were incubated with CFSE-stained KARPAS 299 cells at a ratio of 2:1, 5:1, or 10:1, respectively. After overnight incubation at 37°C, the dye 7AAD was added and the cells were analyzed by flow cytometry. The killing rate of target cells was measured by comparing the viability of target cells to that of negative control cells (SP53 cells, a B-cell lymphoma cell line stained with CMTMR). [Figure 4] Figures 4A-4D. CD4CAR T cells efficiently mediate anti-leukemia effects in vivo through different modes. NSG mice received 2.5 Gy of sublethal irradiation. 24 hours after irradiation, the mice were subcutaneously injected with 1 x 106 (4A) or 0.5 x 106 (4B and 4C) KARPAS 299 cells. The injected mice were treated with different courses and schedules of CD4CAR T cells or control T cells. N=5 per group. In (4A), a low dose of 2 x 106 CD4CAR T cells was injected on day 3, and after accelerated tumor growth was observed, a high dose of 8 x 106 CD4CAR T cells was injected on day 22. In (4B), two high doses of CD4CAR T cells, 8 x 106 and 5.5 x 106, were injected on days 3 and 10, respectively. (4C) Repeated low doses (2.5 x 10) of CD4CAR T cells were injected every 5 days for a total of four times. (4D) Overall survival of mice treated with labeled CD4CAR T cells or control GFP T cells. N=10. [Figure 5] Coculture specificity and dose-response killing curves. CD4CAR NK cells specifically lyse CD4-expressing leukemia cell lines in a dose-dependent manner. CD4CAR NK cells and vector control cells were cultured with CFSE-stained "on-target" (Karpas 299 or CCRF-CEM) cells and CMTMR-stained "off-target" MOLT4 cells at effector / target ratios of 1:4, 1:2, or 1:1. After 24 hours, 7-AAD dye was added, and remaining viable cells were analyzed by flow cytometry. Target cell killing was measured by comparing the viability of CD4+ Karpas 299 or CCRF-CEM cells in CD4CAR NK cell cocultures with that in vector control NK cell cocultures. [Figure 6] Figures 6A-6B. CD4CAR NK cells eliminate CD4+ T cells isolated from human umbilical cord blood at a 2:1 effector-to-target ratio without affecting the production of the hematopoietic stem / progenitor compartment. (6A) Co-culture assays were performed at a 2:1 effector-to-target ratio for 24 hours, after which cells were stained with mouse anti-human CD56 and CD4 antibodies. Target cells were cultured alone as a control (left). NK cells were transduced with vector control (center) or CD4CAR (right) lentiviral supernatant and incubated with CD4+ T cells obtained from human umbilical cord blood. (N=2) (6B) CD4CAR NK cells were incubated with 500 CD34+ cord blood cells at co-culture effector:target ratios of 2:1 and 5:1, respectively, in NK cell medium supplemented with IL-2 for 24 hours. Controls included CD34+ cells alone, and nontransformed NK cells were cocultured with CD34+ CB cells at effector:target ratios of 2:1 and 5:1, respectively. The output of the hematopoietic compartment was assessed by the formation of burst-forming units-erythroid (BFU-E) and the number of colony-forming units-granulocyte / monocyte (CFU-GM) on day 16. Statistical analysis of CFUs was performed by two-way ANOVA with an alpha of 0.05. [Figure 7]Figures 7A-7D. CD4CAR NK cells demonstrate anti-leukemia effects in vivo. NSG mice were irradiated with sublethal doses and intradermally injected with luciferase-expressing Karpas 299 cells (day 0) to induce measurable tumor formation. Mice were intravenously injected with 5 x 106 CD4CAR NK cells or vector-control NK control cells on days 1 and every 5 for a total of six courses. (7A) On days 7, 14, and 21, mice were subcutaneously injected with RediJect D-Luciferin and imaged using IVIS. (7B) The mean light intensity of CD4CAR NK-injected mice was compared with that of vector-control NK-injected mice. (7C) Tumor area was measured on day 1 and every other day thereafter, and the mean tumor size between the two groups was compared. (7D) Mouse survival was measured and compared between the two groups. [Figure 8]Figures 8A-8C. Construction of CD5CAR. The DNA gene structure and translated protein structure of CD5CAR, the anchored CD5 scFv antibody, and a diagram showing the construction and function of CD5CAR. The DNA construct of the third-generation CD5CAR construct, read from 5' to 3', includes the leader sequence, the anti-CD5 extracellular single-chain variable fragment (anti-CD5 ScFv), the hinge region, the transmembrane region, and the three intracellular signaling domains (CD28, 4-1BB, and CD3ζ) that define this construct as a third-generation CD5CAR. The DNA construct of the anchored CD5 scFv antibody is identical to the CD5CAR construct, excluding the intracellular signaling domain, and the translated protein product of the anchored CD5 scFv antibody is also identical. The translated protein construct contains the anti-CD5 ScFv that binds to the CD5 target, the hinge region that allows for proper positioning of the anti-CD5 ScFv for optimal binding, and the transmembrane region. The complete CD5CAR protein also contains two costimulatory domains and the intracellular domain of the CD3 zeta chain. This construct is considered a third-generation CAR: CD28, 4-1BB, and CD3ζ (8A). Western blot analysis demonstrated CD5CAR expression in HEK293 cells. HEK293 cells transduced with GFP (as a negative control) or CD5CAR lentivirus for 48 hours were used to measure CD5CAR expression by Western blot analysis using a CD3ζ antibody. The left lane shows HEK293 cells transduced with GFP (as a negative control), and as expected, no band is observed. The right lane shows a band at approximately 50 kDa, the molecular weight expected for the CD5CAR construct (8B). Flow cytometry analysis of CD5CAR expression on the T cell surface of lentivirally transduced CD5CAR T cells. This analysis was performed on doubly transfected CD5CAR T cells 8 days after the second lentiviral transduction. Flow cytometry using goat anti-mouse F(AB')2-PE demonstrated a 20.53% response in the isotype control T cell population (negative control) versus transduced T cells expressing the CD5 CAR (8C). [Figure 9]Comparison of downregulation of T cell surface CD5 expression following single versus double transduction of CD5CAR lentivirus. Downregulation of extracellular CD5 protein over 8 days following lentiviral transduction was analyzed relative to GFP T cell controls. Single-transduced CD5CAR T cells did not show complete downregulation of CD5 from the cell surface by day 8, with the maximal decrease in CD5 protein expression occurring at day 6. In the double-transduced population, the absolute number of CD5+, CD3+ double-positive CD5CAR T cells decreased over time, from 24.44% on day 0 to a near-complete decrease in CD5 expression by day 4. In contrast, the GFP T cell control maintained a CD5+, CD3+ double-positive population of >95% from days 2 through 8. [Figure 10] Figures 10A-10B. CD5CAR cells effectively lyse CD5-expressing T-ALL cell lines but not T-leukemia cell lines that do not express CD5. (10A) Flow cytometry analysis of T-ALL cell lines alone (left column), coculture with GFP vector-transduced T cells (middle column), and coculture with CD5CAR-transduced T cells (right column). The top and middle columns are CD5+ T-ALL cell lines (CCRF-CEM and Molt-4), and the bottom column is a CD5-negative cell line (KARPAS 299). KARPAS 299 is a CD5-negative T-cell lymphoma. All cocultures were cultured for 24 hours at an effector:target cell ratio of 5:1. Cell lysis rates compared to the GFP control were 78% or higher for both CD5 T-ALL leukemia cell lines. (10B) This bar graph shows the T-cell lysis achieved by CD5CAR T cells compared to the GFP T-cell coculture described in Figure 10A. No lysis was observed in CD5 CAR T cells co-cultured with KARPAS 299, which is CD5 negative (n=3 independent experiments performed in duplicate). [Figure 11]Figures 11A-11D. CD5CAR cells effectively lyse T-cell acute lymphoblastic leukemia cells from patient samples expressing CD5. (11A) Flow cytometry analysis of T-ALL cells alone (left column), co-cultured with GFP T cells (middle column), and co-cultured with CD5CAR T cells (right column). Each patient's cells were assigned a row and numbered to protect patient privacy. All co-cultures were cultured for 24 hours, with an effector-to-target cell ratio of 5:1. Cell lysis compared to the GFP control was greater than 71.3% for T-ALL-1. Other cell lines also showed similar cytolysis, but to a lesser extent, ranging from 33-47%. This may be related to the CD5 expression of each leukemia sample, as described below. (11B) This bar graph shows the T-cell lysis achieved by CD5CAR T cells compared to the GFP T-cell co-culture. T-ALL-1 and T-ALL-3 have different CD5 positivity rates. (11C) Differences in mean fluorescence intensity (MFI) were determined by flow cytometry analysis. (11D) Flow cytometry analysis of CD5 expression levels in a panel of four patient sample T-ALL cell populations. [Figure 12] Detailed analysis of the killing ability of CD5CAR T cells against patient T-ALL cells (T-ALL-8). Flow cytometry analysis demonstrating the killing ability of CD5CAR T cells against patient T-ALL cells. Co-cultures of control GFP-T cells and T-ALL-8 cells are shown on the left, and co-cultures of CD5CAR and T-ALL-8 cells are shown on the right. CD5-positive cells were actively lysed, along with CD34-positive cells (circled in the right column) and CD34-negative cells (circled in the left column, T cells), but CD5-negative cells were not lysed. Compared to the GFP control, CD5CAR T cells lyse at least 93.1% of CD5-positive T-ALL-8 cells. Experiments were performed in duplicate. Furthermore, CD5CAR T cells essentially eliminate a T-cell population (CD5+CD34-, circled). [Figure 13]Figures 13A-13B. CD5CAR T cells effectively eliminate normal GFP-labeled T cells. (13A) CD5CAR T cells kill normal T cells in a dose-dependent manner. CD5CAR T cells or CD123CAR T cells (control) were co-cultured with GFP-labeled T cells at effector-to-target ratios of 0.25:1, 0.5:1, or 1:1. After 24 hours, remaining viable GFP T cells were analyzed by flow cytometry. Because T cells do not express CD123, target cell killing was measured by comparing the viability of GFP T cells in CD5 co-cultures with that in control CD123CAR T cells. (13B) Co-culture killing curve based on the data in 13A. [Figure 14] Figures 14A-14C. Coculture assays were performed to determine whether CD5 expression was maintained when normal T cells were cocultured with CD5CAR or anchored CD5 scFv T cells or CD123CAR (control) at a 1:1 ratio for 2 days (Figures 14A and 14B) or 4 days (Figure 14C). CCRF-CEM or Molt-4 T ALL cells were transduced with lentivirus expressing CD5CAR or anchored CD5 scFv. After the 2nd transduction, transduced leukemia cells were analyzed for CD5 expression by flow cytometry. [Figure 15]Figures 15A-15D. CD5CAR T cells demonstrate significant anti-leukemia efficacy in vivo. NSG mice were sublethally irradiated and 24 hours later, 1 x 106 luciferase-expressing CCRF-CEM cells (day 0) were intravenously administered to induce measurable tumor formation. On days 3 and 4, mice were intravenously injected with 5 x 106 CD5CAR T cells or vector control T cells. These injections were repeated on days 6 and 7, for a total of 2.0 x 107 cells per mouse. On days 5, 8, 10, and 13, mice were subcutaneously injected with RediJect D-Luciferin and imaged with IVIS. (15A) On days 5, 8, 10, and 13, mice were subcutaneously injected with RediJect D-Luciferin and imaged with IVIS. (15B) The mean light intensity of CD5CAR T-injected mice was compared to that of vector control T-injected mice. (15C) Tumor cell killing rate in mice treated with CD5CAR T cells versus control. (15D) Peripheral blood was collected from mice on day 15, and the percentage of leukemia cells was measured and compared with that in vector control or normal injected mice. [Figure 16] Figures 16A-16C. CD5CAR NK cells (NK-92) effectively eliminate the CCRF-CEM T-ALL cell line in vitro. (16A) and (16B) The CD5-expressing T lymphoblastoid cell line, CCRF-CEM, was co-cultured with CD5CAR NK cells at the indicated E:T (effector:target) cell ratios for 24 hours. Target populations were quantified by flow cytometry after separating NK-CAR and target cell populations using CD56 and CD5, respectively. Cell viability is expressed relative to transduced vector control NK cells, and each bar represents the mean statistic of N=2 duplicate samples. (16C) CD5CAR NK cells dose-dependently eliminate CCRF-CEM cells. The CD5-expressing T lymphoblastoid cell line, CCRF-CEM, was co-cultured with CD5CAR NK cells at decreasing E:T (effector:target) ratios. Saturation was reached at an E:T ratio of 2:1, and co-culture at lower ratios resulted in dose-dependent elimination of CD5. CCRF-CEM was completely eliminated at a 5:1 ratio. [Figure 17] Figures 17A-17B. CD5CAR NK cells exert potent anti-leukemia effects in vivo. NSG mice were irradiated with sublethal doses and 24 hours later, 1 x 106 luciferase-expressing CCRF-CEM cells were intravenously administered (day 0) to induce measurable tumor formation. On days 3 and 4, mice were intravenously administered 5 x 106 CD5CAR NK cells or vector control NK cells. These administrations were repeated on days 6 and 7, for a total of 2.0 x 107 cells per mouse. (17A) On day 5, mice were subcutaneously injected with RediJect D-Luciferin and subjected to IVIS imaging. (17B) Tumor cell death in mice treated with CD5CAR NK cells relative to controls. [Figure 18] Figures 18A-18B. Construction and expression of CD3CAR. (18A) Schematic diagram of the construction of CD3CAR in a lentiviral vector. CAR expression is driven by the SFFV (spleen focus-forming virus) promoter. As a third-generation construct, it contains a leader sequence, anti-CD3 scFv, hinge domain (H), transmembrane domain (TM), two costimulatory domains (CD28 and 4-BB), and the intracellular signaling domain of CD3 zeta. (18B) HEK-293FT cells were transfected with lentiviral plasmids encoding GFP (lane 1) and CD3CAR (lane 2). Western blot analysis was performed 48 hours post-transfection and probed with a mouse anti-human CD3 zeta antibody (18B). [Figure 19]Figures 19A-19D. CD3CAR NK cells eliminate CD3-expressing T-ALL cell lines in vitro. (19A) Jurkat, a T-lymphoblastoid cell line expressing approximately 80% CD3, was cocultured with CD3CAR NK cells at the indicated E:T (effector:target) cell ratio for 6 hours. (19B) Sorted (CCRF-CD3) or unsorted (CCRF-CEM) cells were cocultured with CD3CAR NK cells for 24 hours. Target cell populations were quantified by flow cytometry using CD56 and CD3 to separate NK-CAR and target cell populations, respectively. Cell viability is expressed relative to transduced vector control NK cells, and each bar represents the mean statistic of duplicate samples from N=2 experiments. (19C) CD3CAR NK cells demonstrate robust killing ability against primary CD3+ leukemia cells from patient samples. SPT-1 (Sézary syndrome) patient cells were CD3 positive and co-cultured with CD3CAR NK cells at the indicated E:T (effector:target) cell ratio for 24 hours. The target cell population was quantified by flow cytometry using CD56 and CD3 to separate the NK-CAR and target cell populations, respectively. While SPT-1 is a heterogeneous cell population, a broad population of cells expressing CD3+ is eliminated by the CD3NK-CAR. (19D) PT4 (unsorted PTCL) patient cells were CD3+CD7- and co-cultured with CD3CAR NK cells at the E:T (effector:target) cell ratio for 24 hours. The target population was gated and quantified as shown. PT4 leukemia cells are CD3+CD7- and are effectively eliminated by CD3CAR NK cells. A broad CD3+ population is also affected by CD3CAR NK cells. [Figure 20]Figures 20A–20C. CD3CAR NK cells demonstrate significant anti-leukemia efficacy in vivo. NSG mice were irradiated with sublethal doses and 24 hours later, 1 x 106 luciferase-expressing Jurkat cells were intravenously injected (day 0) to induce measurable tumor formation. On days 3 and 4, mice received daily intravenous injections of 5 x 106 CD3CAR NK cells or vector control NK cells. These injections were repeated on days 6, 7, and 10, for a total of 2.5 x 107 cells per mouse. On days 4, 7, 9, and 13, mice were subcutaneously injected with RediJect D-Luciferin and subjected to IVIS imaging (20A). (20B) The mean light intensity measured in mice injected with CD3CAR NK cells was compared to that in mice injected with vector control NK cells. (20C) Percentage of tumor cells killed in mice treated with CD3CAR NK cells compared to controls. [Figure 21] Three pairs of sgRNAs were designed for each gene using CHOPCHOP to target CD2, CD3, CD5, and CD7. The gene-specific sgRNAs were then cloned into a lentiviral vector (Lenti U6-sgRNA-SFFV-Cas9-puro-wpre) expressing human Cas9 and a puromycin resistance gene linked by an E2A self-cleaving linker. The U6-sgRNA cassette precedes the Cas9 element. Expression of the sgRNA and Cas9puro is driven by the U6 promoter and SFFV promoter, respectively. [Figure 22]Figures 22A-22D. Generation of stable CD5-deficient CCRF-CEM and MOLT-4 T cells using the CRISPR / Cas9 lentiviral system. (22A) Flow cytometry analysis showing loss of CD5 expression in CCRF-CEM T cells after puromycin selection by CRISPR / Cas9 knockdown using two different sgRNAs, Lenti-U6-sgCD5a-SFFV-Cas9puro (sgCD5A) and Lenti-U6-sgCD5b-SFFV-Cas9puro (sgCD5B). The leftmost scatter plot shows the wild-type control. Because the CRISPR / Cas9 knockdown method using sgRNA CD5A was more successful in downregulating CD5 protein, this population (indicated by the circle and arrow) was chosen for sorting, purification, and analysis in Figure 22B. (22B) Flow cytometry analysis data showing the percentage of purely sorted, stable CD5-negative CCRF-CEM cells transduced using scCD5A CRISPR / Cas9 technology. The purity of CD45-positive, CD5-negative CCRF sgCD5A T cells is >99%. (22C) Flow cytometry analysis showing the loss of CD5 expression after puromycin treatment in MOLT-4 T cells subjected to CRISPR / Cas9 knockdown using two different sgRNA sequences (sequences CD5A and CD5B, middle and right columns). The leftmost scatter plot represents the wild-type control. Because the CRISPR / Cas9 knockdown method using primer CD5A was more successful in downregulating CD5 protein, this population (indicated by the circle and arrow) was selected for sorting, purification, and analysis in Figure 22D. (22D) Flow cytometry analysis data showing the percentage of pure, sorted, stable CD5-negative MOLT-4 cells transduced using scCD5A CRISPR / Cas9 technology. The purity of CD45-positive, CD5-negative MOLT-4 sgCD5A T cells is greater than 99%. [Figure 23]Figures 23A-23D. Creation of stable CD7 deficiency in CCRF-CEM or NK-92 cells using the CRISPR / Cas9 lentiviral system and cell sorting. The percentage of CD7 deficiency in CCRF-CEM (Figures 23A and 23B) or NK-92 (Figures 23C and 23D) cells using sgCD7A (Lenti-U6-sgCD7a-SFFV-Cas9-puro) and sgCD7B (Lenti-U6-sgCD7b-SFFV-Cas9-puro) was determined by flow cytometry analysis using CD45 and CD7 antibodies after puromycin treatment. The values ​​in the figures indicate the percentage of positive and negative cells expressing CD45 or CD7. The right panel shows the percentage purity of sorted stable CD7-negative cells in CCRF-CEM (23C) or NK-92 cells prepared from CD7-negative cells transduced with sgCD7A or sgCD7D CRISPR lentivirus (23D). [Figure 24]Figures 24A-24C. CD2CAR NK cells eliminate T-cell leukemia cells in coculture assays. (24A) CD2CAR NK cells eliminate leukemia cells from T-ALL patient cells in coculture. NK-92 cells transduced with GFP (top) or CD2CAR (bottom) lentiviral supernatant were incubated with primary human T-ALL cells, SAMPL1 (PT1), at a ratio of 5:1 (1:100,000 cells). After 24 hours of coculture, cells were stained with mouse anti-human CD2 (APC) antibody and analyzed by flow cytometry (N=2). (24B) Elimination of leukemia cells from the T-ALL cell line, CCRF, by CD2CAR NK cells in coculture. NK-92 cells transduced with GFP (top) or CD2CAR (bottom) lentiviral supernatant were incubated with CCRF cells at a ratio of 5:1 (1:100,000 cells). CCRF cells were pre-stained with a cell tracker dye (CMTMR). After 24 hours of co-culture, cells were stained with mouse anti-human CD2 (APC) antibody and analyzed by flow cytometry (N=2). (24C) Percentage of lysed target cells (CCRF or PT1) compared to GFP NK experimental controls. When co-cultured at a 5:1 ratio for 24 hours, CD2CAR NK cells were able to eliminate approximately 60% of CD2-positive leukemia cells in the co-culture assay. [Figure 25] Figures 25A-25B. CD7CAR NK 7- -92 cells effectively lyse CD7-expressing T-cell ALL cell line T cells. To avoid self-killing, CD7-deficient NK-92 (NK 7- -92) cells were generated and transduced with CD7CAR. Killing ability was tested using two types of CD7CAR-transduced NK 7- -92 cells (#A and #B). (25A) Flow cytometry analysis of CCRF-CEM cells alone (left column), co-culture with GFP NK 7- -92 cells (middle column), and co-culture with CD7CAR-NK-92 cells #A and #B (right column). (25B) Bar graph based on data obtained in A. [Figure 26]Schematic diagram of a recombinant lentiviral vector encoding CD7CAR (also called CD7RTXCAR). CD7CAR consists of a humanized anti-CD7 scFv, CD8 hinge and transmembrane regions, and a CD28 coactivator fused to the CD3zeta signaling domain. The hinge region of CD7CAR also contains two RTX-binding epitopes. [Figure 27] Figures 27A-27D. (27A) Characterization of CD7CAR. Staining with goat-anti-mouse F(Ab')2-Pe revealed approximately 70% CAR expression 8 days after viral infection. (27B) Staining with anti-human CD34 (used to detect the RTX-binding epitope) also demonstrated approximately 80% transduction efficiency. (27C) Staining with anti-human CD3 and anti-human CD7 shows that CD7CAR T cells retain CD3 expression but lose CD7 expression. (27D) Despite losing CD7 expression, CD7CAR T cells are able to proliferate at a rate similar to control T cells. [Figure 28] CD7CAR exhibits potent cytotoxicity against CD7+ cell lines in vitro. CEM-CCRF cells are approximately 90% CD7+ (lower right panel). Control T cells (left panel) or RTX T cells (right panel) were co-cultured with CEM-CCRF cells at an E:T ratio of 1:1 (first row) or 2:1 (second row) for 18 hours. Target CD7+ cells are circled in each panel. Results from the co-culture experiments showed that CD7CAR T cells lysed 99.88% and 99.82% of CEM-CCRF cells relative to the control at 1:1 and 2:1 ratios, respectively. [Figure 29]Figures 29A-29D. CD7CAR improves prognosis in an in vivo model of T-ALL. (29A) NSG mice were sublethally irradiated and intravenously injected with 1.0 × 10 luciferase-expressing CEM-CCRF cells on day 1. Five days later, mice were injected with 10 × 10 control or CD7CAR T cells. Mice were injected with RediJect D-Luciferin on days 5, 10, 13, 16, and 19 and imaged by IVIS. Dorsal view. (29B) Total flux (photons / second) was measured and demonstrated a statistically significant difference in tumor burden between the two groups as early as day 8. (29C) Flux in CD7CAR-treated mice showed a 41.3% (dorsal) tumor reduction by day 8 and increased to 99.6% (dorsal) by day 19. (29D) Control mice required euthanasia due to hind limb paralysis and hunchback by days 24-26, whereas CD7CAR-treated mice had a significantly longer survival time, surviving until day 43. Kaplan-Meier survival analysis curve (p = 0.0026). [Figure 30] Figure 30 shows the patient's leukemic blast distribution before CAR T-cell therapy. Peripheral blood yield by flow cytometry was 0.39%, bone marrow morphology was 24%, and bone marrow yield by flow cytometry was 6.13%. The patient received standard preconditioning therapy with fludarabine and cytarabine. Four days after preconditioning, the first dose of CD7 CAR T-cell therapy was administered at 1x10^6 / kg, followed by doses of 2.0x10^6 / kg over the following two days. The patient achieved complete remission. Leukemic blasts in peripheral blood and bone marrow were MRD (minimal residual disease) negative. [Figure 31] As shown in Figure 31, 6 days before CAR T cell therapy (left panel), the T cell population was positive for the CD7 surface protein. After CAR T cell therapy, the T cell population consisted of almost entirely CD3+ CD7-negative cells (right three panels). [Figure 32]Summary shows absolute CBC (complete blood count), lymphocyte subtypes, chimerism, and percentage of CAR T expression in BM and PB. See figure / box. Bottom hexagon indicates absence of leukemic cells (right and center panels). Bottom right shows bone marrow flow cytometry analysis showing near-complete absence of CD7 surface protein on all T cells. WBC: white blood cells; NEU: neutrophils; LYM: lymphocytes; HGB: hemoglobin; PLT: platelets; CIK: cytokine-induced killer cells; PB: peripheral blood; BM: bone marrow; GVHD: graft-versus-host disease. [Figure 33] Construction of anti-TAA-anit-CD3-ENG for expression of anti-TAA-anti-CD3 engager. The viral construct contains tandem cDNAs encoding an anti-TAA scFv with a short serine-glycine linker and myc tag, and an immunoglobulin heavy chain leader peptide preceding an anti-CD3 scFv. TAA is called tumor-associated antigen. G4S linker: GGGGS [Figure 34]Construction of CD7 CARs and CARs X required for CAR T expansion and reduced toxicity. CD7 CARs can be combined with CARs X selected from the following target antigens: at least one of this group, but not limited to, GD2, GD3, ROR1, PSMA, PSCA (prostate stem cell antigen), MAGE A3, glycolipids, glypican 3, F77, GD-2, WT1, CEA, HER-2 / neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, MUC1, MUC2, MUC3, MUC4, MUC5, KIF20A, Survivin, AFP-1, gp100, MUC1, PAP-10, PAP-5, TRP2-1, S ART-1, VEGFR1, VEGFR2, NEIL3, MPHOSPH1, DEPDC1, FOXM1, CDH3, TTK, TOMM34, URLC10, KOC1, UBE2T, TOPK, ECT2, MESOTHELIN, NKG2D, P1A, GM2, CD30, MMG49 epitope, EGFRvIII, CD33, CD123, CLL-1 (CD371), immunoglobulin kappa and lambda, CD38, CD52, CD47, CD200, CD70, CD19, CD20, CD22, CD38, BCMA, CS1, NKG2D receptor, April receptor, BAFF receptor, TACI, CD3, CD4, CD8, CD5, CD2, GPRC5D (G protein-coupled receptor, class C, group 5, member D), and CD138. Target antigens also include viral and fungal antigens, such as human papillomavirus (HPV) antigens and Epstein-Barr virus (EBV) antigens E6 and E7. Schematic diagram of CD7-conjugated CAR (cCAR). This construct consists of a promoter driving the expression of two CAR units linked by a cleavage site, such as P1A, E2A, T2A, or F2A. Upon cleavage of the linker peptide, the cCAR splits and binds to targets expressing CD7 and other antigens. For novel CD7 cCAR constructs, the costimulatory domains of the construct include, but are not limited to, 4-1BB and CD28. The costimulatory domains of each CAR can be the same or different.CD7CAR consists of an anti-CD7 scFv, CD8 hinge (H) and transmembrane (TM) regions, and a coactivator fused to the CD3zeta signaling domain. The hinge region of CD7CAR also contains two CD20 RTX-binding epitopes. Expression is driven by a single promoter. [Figure 35] Schematic diagram of the cCAR construct (CD7-CD19 cCAR). This construct consists of a single promoter driving the expression of two modular CARs linked by a P2A peptide. Upon cleavage of the linker, the cCAR splits and binds to targets expressing CD7 and / or CD19. As a novel cCAR construct, the costimulatory domains of the construct include, but are not limited to, the 4-1BB domain on the CD7 CAR segment and the CD28 domain on the CD19 CAR. The costimulatory domains of each CAR can be identical or different. Each CAR consists of a costimulator fused to an scFv, CD8 hinge (H) and transmembrane (TM) regions, and a CD3zeta signaling domain. The hinge region of the CD7 CAR also contains two CD20 RTX-binding epitopes. The CD7-CD19 cCAR is designed to target autoreactive T cells expressing CD7 and autoreactive B cells expressing CD19. [Figure 36]Figures 36A-36D. (36A) Expression of BCMA-CD19-VAC (BVMA-CD19-IL-15 / IL15sushi) cCAR T cells. Flow cytometry analysis showed approximately 28.18% CAR expression after staining with goat anti-mouse F(Ab')2-Pe. Virus was obtained from the BCMA-CD19 CAR stable producing cell line RD114. (36B): Five days after tumor cell injection, three mice per group were intravenously injected with 10x106 BCMA-CD19-IL-15 / IL-15sushi T cells or vector control T cells. On days 5, 8, 11, 14, and 17, mice were subcutaneously injected with RediJect D-Luciferin (Perkin Elmer) and underwent IVIS imaging to measure tumor burden. (36C): Five days after REH tumor cell injection, three mice each were intravenously injected with 10 x 106 BCMA-CD19-IL-15 / IL-15sushi T cells or vector control T cells. On days 5, 11, 14, 17, and 20, mice were subcutaneously injected with RediJect D-Luciferin (Perkin Elmer) and IVIS imaging was performed to measure tumor burden. (36D). Quantification of autoantibody levels by ELISA. Disease-causing autoantibody levels (D-7 to D360) and complement levels (D-7 to D360) were as follows: (A) AHA; (B) ANA; (C) anti-U1-snRNP; (D) anti-nucleosome; (E) anti-dsDNA; (F) anti-ribosome; (G) anti-SSA / Ro52; (H) anti-Sm; (I) anti-SSA / Ro60; (J) complement C3; (K) complement C4. Note: D: Day. [Figure 37]Figures 37A-37D. Expression of CLL1-CD33 VAC (also called CLL1-CD33-IL-15 / IL15sush) cCAR T cells (37A). Expression of BCMA-CD19 VAC (also called BCMA-CD19-IL-15 / IL15sushi) cCAR (37B). Expression of CD7 RTX CAR (37C). Flow cytometry analysis by staining with goat-anti-mouse F(Ab')2-Pe showed that ~31.32% of T cells expressed CLL-1-CD33 VAC cCAR, ~61% of T cells expressed BCMA-CD19 VAC cCAR, and 70% of T cells expressed CD7 RTX CAR. The CD7+ T cell population was completely depleted (37D, lower right panel). Expression was driven by the MMLV promoter. CAR expression in control cells (top left panel) and transduced T cells (circle, right) using F(Ab')2 antibody. [Figure 38] Schematic diagram of the BCMA-CD7-IL-15 / IL-15sushi cCAR construct. Linkage by P2A and T2A is a schematic diagram for generating a cCAR co-expressing IL-15 / IL-15sushi in a single construct. This construct consists of two complete CAR units (cCARs) and a promoter driving the expression of secreted IL-15 / IL-15sushi. Upon cleavage of the linker (P2A and T2A), the CD7 CAR and BCMA CAR split and bind to the target(s). Each CAR contains at least six domains, including a leader sequence (signal domain), scFv, hinge region, transmembrane domain, costimulatory domain (including, but not limited to, CD28 and 4-1BB), and the CD3 zeta chain for intracellular signaling. CD7-negative T cells or NK cells can be generated by natural selection using the CD7 CAR construct. Secreted IL-15 / IL-15sushi enhances CAR T proliferation and persistence. The CD7 CAR has a safety switch (rituximab) built into the hinge region. [Figure 39]Figures 39A-39B. (39A) Example of generation of cCAR-expressing CD7-negative T cells. Flow cytometry results of activated human T cells transduced with the BCMA-CD7Q-IL15 / IL15sushi retroviral vector. Three days after transduction (and five days after transduction), untransduced T cells (left panel) and transduced T cells (right panel) were harvested, washed, and labeled with goat anti-mouse F(Ab')2 antibody, mouse anti-human CD3 antibody, and CD7 antibody. Cells were fixed with 2% formalin and analyzed by flow cytometry (NovoCyte, Agilent). After transduction, almost all positive T cells became CD7-negative T cells with CAR expression. Top panel: Approximately 42% of CAR T cells expressed the F(Ab'2) phenotype. Lower panels: Compared to controls, after CAR transduction, all T cells were CD7 negative (right), and most T cells were CD7 positive (left). (39B) Coculture experiments comparing lysis of BCMA-CD7Q-IL-15 / IL15sushi CAR T cells with U-BCMA (U-BCMA)-expressing cells from a human tumor cell line (U937) that synthetically expresses the CD269 (BCMA) antigen on its cell surface were performed at E:T ratios of 2:1 (upper panel) and 5:1 (lower panel) for 24 hours. CCRF-CEM cells were pre-labeled with the membrane dye CMTMR to phenotypically distinguish them from T cells. Target cells only are shown on the left. Cells were analyzed by flow cytometry using anti-CD7 and anti-CD3 labeling. The circled population highlights target cell lysis. In 24-hour coculture experiments, BCMA-CD7Q-IL-15 / IL15sushi CAR T cells demonstrated profound killing (nearly 90%) of target U-BCMA cells at an effector:target ratio of 2:1. [Figure 40]Schematic diagram of the CD7-CD19-IL-15 / IL-15sushi cCAR construct. Linkage by P2A and T2A is a schematic diagram for generating a cCAR co-expressing IL-15 / IL-15sushi in a single construct. This construct consists of two complete CAR units and a promoter driving the expression of secreted IL-15 / IL-15sushi. Upon cleavage of the linker (P2A and T2A), the CD7 CAR and CD19 CAR separate and bind to their targets. Each CAR contains at least six domains, including a leader sequence (signal domain), scFv, hinge region, transmembrane domain, costimulatory domain (including, but not limited to, CD28 or 4-1BB), and the CD3 zeta chain for intracellular signaling. CD7-negative T cells or NK cells can be generated by natural selection using the CD7 CAR construct. Secreted IL-15 / IL-15sushi enhances the proliferation and persistence of CAR T cells in vivo. The CD7 CAR has a safety switch (rituximab) built into the hinge region. [Figure 41] Another example of the generation of CD7-negative T cells expressing cCAR. Flow cytometry results of activated human T cells transduced with the CD19-CD7-IL15 / IL15sushi (also known as CD7-CD19-Vac) retroviral vector. Three days after transduction (and five days after transduction), untransduced T cells (left panel) and transduced T cells (right panel) were harvested, washed, and labeled with goat anti-mouse F(Ab')2 antibody, mouse anti-human CD3 antibody, and CD7 antibody. Cells were fixed with 2% formalin and analyzed by flow cytometry (NovoCyte, Agilent). After transduction, nearly all T cells became CD7-negative T cells expressing CAR. Top panel: Approximately 71% of CAR T cells express the F(Ab'2) phenotype, bottom panel: shows that after CAR transduction all T cells are CD7 negative (right) and most T cells are CD7 positive (left) compared to the control. [Figure 42]CD7-negative T cells transduced with a CD19 CAR exhibited significant killing activity. Activated CD7-positive T cells were transduced with CD7-CD19-IL-15 / IL-15sushi CAR T cells, resulting in CD7-negative T cells containing both a CD19 CAR and a CD7 CAR. These CD7-negative T cells containing a CD19 CAR demonstrated nearly complete elimination of the CD19 antigen-expressing tumor cell line, REH, even at effector T cell to target tumor cell ratios as low as 1:1. Flow cytometry analysis of control T cells (center panel) and CD7-CD19-IL-15 / IL-15sushi CAR T cells (right panel) against the REH cell line after 18 hours of coculture at effector to target cell ratios of 1:1 (top panel) and 2:1 (bottom panel). T cells are shown in red. Target cells are circled. REH cells alone are on the far left. [Figure 43] CD7-negative T cells transfected with a CD7 CAR exhibited significant killing activity. Activated CD7-positive T cells were transfected with CD7-CD19-IL-15 / IL-15sushi CAR T cells, and the resulting CD7-positive T cells were CD7-negative, carrying the CD7 CAR as well as the CD19 CAR. These CD7-negative T cells carrying the CD7 CAR demonstrate nearly complete elimination of the CD7 antigen-expressing tumor cell line CCRF-CEM, even at effector T:target tumor cell ratios as low as 1:1. Flow cytometry analysis of control T cells (center panel) and CD7-CD19-IL-15 / IL-15sushi CAR T cells (right panel) after 18 hours of coculture with the CCRF-CEM cell line at effector:target ratios of 1:1 (upper panel) and 2:1 (lower panel). T cells are shown in red. Target cells are circled. CCRF-CEM cells alone are on the far left. [Figure 44]CD7 CAR and CAR,X constructions require CAR T expansion and reduced toxicity. CD7CARs can be combined with another CAR,X within a single construct to target antigens selected from the following: at least one of this group (but not limited to), GD2, GD3, ROR1, PSMA, PSCA (prostate stem cell antigen), MAGE A3, glycolipids, glypican 3, F77, GD-2, WT1, CEA, HER-2 / neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, MUC1, MUC2, MUC3, MUC4, MUC5, KIF20A, Survivin, AFP-1, gp100, MUC1, PAP-10, PAP-5, TRP2-1, S ART-1, VEGFR1, VEGFR2, NEIL3, MPHOSPH1, DEPDC1, FOXM1, CDH3, TTK, TOMM34, URLC10, KOC1, UBE2T, TOPK, ECT2, MESOTHELIN, NKG2D, P1A, GM2, CD30, MMG49 epitope, EGFRvIII, CD33, CD123, CLL-1 (CD371), immunoglobulin kappa and lambda, CD38, CD52, CD47, CD200, CD70, CD19, CD20, CD22, CD38, BCMA, CS1, NKG2D receptor, April receptor, BAFF receptor, TACI, CD3, CD4, CD8, CD5, CD2, GPRC5D (G protein-coupled receptor, class C, group 5, member D), and CD138. Target antigens also include viral and fungal antigens, such as human papillomavirus (HPV) antigens and Epstein-Barr virus (EBV) antigens E6 and E7. Schematic diagram of CD7-conjugated CAR (cCAR). This construct consists of a promoter driving the expression of two CAR units linked by a cleavage site, such as P1A, E2A, T2A, or F2A. Upon cleavage of the linker peptide, the cCAR splits and binds to targets expressing CD7 and other antigens. For the novel CD7 cCAR construct, the costimulatory domains of the construct include, but are not limited to, 4-1BB or CD28. The costimulatory domains of each CAR can be the same or different.CD7CAR consists of an anti-CD7 scFv, CD8 hinge (H) and transmembrane (TM) regions, and a coactivator fused to the CD3zeta signaling domain. The hinge region of CD7CAR also contains two CD20 RTX-binding epitopes. Expression is driven by a single promoter. CD7-negative T cells and NK cells can be generated by a natural selection approach by introducing a cCAR containing the CD7 CAR construct. [Figure 45] Anti-BCMA VHH discovery strategy. Overview of the anti-BCMA VHH discovery workflow. [Figure 46] Figures 46A-46C. Protein binding assay using ELISA. Interaction of BCMA VHHs antibodies, BCMA4 (BC4) and BCMA5 (BC5), with NIH-CoVnb-112 and BCMA (human) or Cyno BCMA (platypus) proteins (46A and 46B). TACI and BFFR were used as negative controls (46C). [Figure 47] Cell-based assay of BC4 and BC5 binding to surface BCMA protein using CHO cells. The binding affinity of cell surface BCMA VHHs was analyzed by flow cytometry analysis using BCMA-expressing CHO cells. [Figure 48] Schematic diagram illustrating the construct (BC4-RTX-IL-15 / IL-15sushi). The complete BCMA CAR unit (using only the variable heavy chain domain (VHH) as the antigen recognition sequence), the rituximab-binding domain in the hinge region, and the IL-15 / IL15sushi domain of the IL-15α receptor were assembled on an expression vector, and their expression was driven by a retroviral promoter. The anti-BC4 CAR was linked to the IL-15 / IL15sushi domain via a P2A self-cleaving sequence. The IL-15 / IL15sushi portion consists of an IL-2 signal peptide fused to IL-15, linked to the sushi domain via a 26-amino acid polyproline linker. [Figure 49]Figures 49A-49B. Co-culture experiments comparing lysis of U937-BCMA cells, a human tumor cell line that synthetically expresses BCMA (CD269) on its cell surface, by BC4-RTX-IL-15 / IL-15sushi CAR T cells were performed for 24 hours at E:T ratios of 2:1 (top) and 5:1 (bottom). U937-BCMA cells were pre-labeled with the membrane dye CMTMR to distinguish them from T cells (49A). Cells were analyzed by flow cytometry using anti-CD269 and anti-CD3 labeling. The boxed population highlights target cell lysis. (49B) During the 24-hour co-culture experiment, BC4-RTX-IL-15 / IL-15sushi CAR T cells demonstrated profound killing (84%) of target U937-BCMA cells at an effector:target ratio of 2:1. [Figure 50] Figures 50A-50B. Co-culture experiments comparing lysis of U937-BCMA cells, a human tumor cell line synthetically expressing BCMA (CD269) on the cell surface, by BC5-RTX-IL-15 / IL-15sushi CAR T cells were performed at E:T ratios of 2:1 (top) and 5:1 (bottom) for 24 hours (50A). U937-BCMA cells were pre-labeled with the membrane dye CMTMR to better distinguish them from T cells. Cells were analyzed by flow cytometry using anti-CD269 and anti-CD3 labeling. The circled population highlights target cell lysis. In the 24-hour co-culture experiment, BC5-RTX-IL-15 / IL-15sushi CAR T cells demonstrated profound killing (82%) of target U937-BCMA cells at an effector:target ratio of 5:1. The BC5-RTX-IL-15 / IL-15sushi construct was prepared using a method similar to that used for BC4-RTX-IL-15 / IL-15sushi (50B). [Figure 51]Figures 51A-51C. Co-culture assays of BC4-CD19c-tan-CAR-T cells with BCMA-expressing U937 cells were performed for 22 hours at an effector-to-target ratio of 2:1. (51A) To clearly distinguish between effector and target cells, target cells were pre-stained with a cell tracking dye (CMTMR). Circled dots indicate target cells remaining in the co-culture assay with effector T cells. (51B) The bar graph on the right shows the % target cell lysis of BCMA-expressing U937 (replicating) T cells compared to control T cells in the co-culture assay. (51C). BC4-CD19c-tan-CAR-transduced cells eliminated the endogenous CD19-positive cell population after recovery 2 days after intracellular CAR-virus transduction. Flow cytometry analysis revealed a CD19-positive population in the control cell sample (circled dots on the left panel). In contrast, no CD19-positive population was detected in the CAR virus-transduced sample (right panel). To assess whether BC4-CD19c-tan-CAR-transduced T cells could eliminate endogenous CD19-positive cells, particularly B cells, in donor cells after CAR virus transduction, we performed flow cytometry analysis on control and CAR-transduced T cells, labeled with CD45-PerCP, CD3-PE, and CD19-APC antibodies, two days after recovery (day 6 after T cell activation). After gating on the CD45-positive population and FSC, the data were plotted against CD19 and CD45 to confirm the presence of a CD19-positive cell population. Flow cytometry analysis demonstrated that the control cell sample contained a CD19-positive population (circled dot in the left panel). In contrast, no CD19-positive population was detected in the CAR virus-transduced sample (right panel). [Figure 52]Figures 52A-52C. (52A) CD19-based CARs deplete Reh cells in vivo, and IL-15 / IL-15sushi conjugates enhance antitumor responses. Mice were injected with luciferase-expressing Reh tumor cells (0.5x106 cells / mouse) on day 1. IVIS imaging was performed on day 3 to measure the appearance of circulating Reh cells. On day 4, control T cells, CD19b CAR, and CD19b-IL15 / IL-15sushi CAR T cells were injected (~7.5x106 cells / mouse). IVIS imaging was performed from day 6 to day 22 to measure semiquantitative assessment of tumor burden, subsequent tumor depletion, and T cell-mediated control of cell proliferation. Here, both CAR T treatments demonstrated similar efficacy, with the IL-15-secreting CAR suppressing Reh tumor growth to a similar or greater extent than standard CART19 cells. (52B) Long-term comparison of CD19b-CAR-T and CD19b-IL-15 / IL-15sushi CAR-T on REH cells. (TOP) Similar experimental scheme using the same IVIS method as above, but mice were followed until signs of tumor recurrence were observed. Here, we observed that after day 30, standard CART19-treated mice began to develop aggressive Reh tumor recurrence. Most CART19 mice showed tumor clusters (circled areas on mice imaged by IVIS), and one CD19b-IL-15 / IL-15sushi CART-treated mouse also showed tumor growth by day 22. However, after day 30, all CART19 mice showed signs of severe tumor recurrence, while CD19b-IL-15 / IL-15sushi CART-treated mice showed no signs of tumor growth. Even mice that relapsed on day 22 were tumor-free by day 32, indicating that CD19b-IL-15 / IL-15sushi CART cells were still circulating effectively. (52C). Line graph summarizing IVIS trend values ​​estimating tumor growth over time for each treatment cohort. After day 30, tumor burden in standard CD19b CAR (CART19)-treated mice rose sharply, resulting in a highly significant increase in tumor burden compared to the CD19b-IL-15 / IL-15sushi CART-treated group, which remained largely tumor-free.Values ​​are shown for both views of the mouse (dorsal image acquisition view). [Figure 53] Figures 53A-53B. pX-CD19c-CD7Q tandem CAR (Q, also known as RTX). (53A) Activated human T cells transduced with CD19c-CD7Q retroviral vector, 3 days after transduction. PBMCs from a healthy donor were activated for 48 hours in the presence of mouse anti-human CD3 antibody. Cells (1.0 x 10e6) were transduced 1 mL of control supernatant (left panel) or transfected cell supernatant (right panel) at a 1:1 ratio and incubated for 24 hours. A second identical transfection was performed for another 24 hours. After the second transduction, T cells were washed and suspended in T cell medium supplemented with 300 IU / mL IL-2. After 24 hours of culture after the second transduction, T cells were labeled for flow cytometry analysis, detecting T cell and CD7 phenotypes with mouse anti-human CD3 and CD7 antibodies, respectively. CD7 expression on control T cells (circle, bottom left) and transduced T cells (circle, bottom right). (53B) Co-culture experiment comparing lysis of CD19-expressing REH cells by the same CD19c-CD7Q CAR T cells as in panel A was performed for 16 hours at an effector:target cell ratio of 2:1. Cells were analyzed by flow cytometry using anti-CD19 and anti-CD3 labeling. The boxed population highlights target cell lysis. [Figure 54]Figures 54A-54B. CD19b-CD7Q-IL-15 / IL15sushi tandem CAR. (54A) Activated human T cells transduced with the pX-CD19b-CD7Q-IL-15 / IL15sushi retroviral vector from transfected H29 cells, 3 days after transfection. PBMCs from a healthy donor were activated for 48 hours in the presence of mouse anti-human CD3 antibody. Cells (1.0 x 10e6) were transduced 1:1 with 1 mL of control supernatant (left panel) or supernatant from transfected cells (right panel) and incubated for 24 hours. A second identical transfection was performed for another 24 hours. After the second transfection, T cells were washed and suspended in T cell medium supplemented with 300 IU / mL IL-2. After 24 hours of culture following the second transduction, T cells were labeled for flow cytometry analysis, detecting T cell and CD7 phenotypes with mouse anti-human CD3 and CD7 antibodies, respectively. CD7 expression on control T cells (circled, bottom left) and transduced T cells (circled, bottom right). (54B) A co-culture experiment comparing lysis of CD19-expressing REH cells by the same CD19b-CD7Q-IL-15 / IL15sushi CAR T cells as in panel A was performed for 16 hours at an effector:target cell ratio of 2:1. Cells were analyzed by flow cytometry using anti-CD19 and anti-CD3 labeling. The boxed population highlights target cell lysis. [Figure 55] Figure 55. CD19b-BC4 tandem CAR. Activated human T cells transduced with a CD19c-BC4 CAR retroviral vector, 3 days post-transduction. Co-culture experiments comparing lysis of CD19-expressing REH cells by the same CD19b-BC4 CAR T cells were performed for 16 hours at an effector:target cell ratio of 2:1. Cells were analyzed by flow cytometry using anti-CD19 and anti-CD3 labeling. The target cell population is highlighted and boxed. The CD19b-BC4 tandem CAR depleted CD19-expressing REH cells. DETAILED DESCRIPTION OF THE INVENTION

[0057] Detailed Description of the Invention The term " autoimmune disease " used herein is defined as a disease caused by autoimmune reaction.Autoimmune disease is the result of inappropriate and excessive reaction to autoantigen.Examples of autoimmune disease include but are not limited to achalasia, Addison's disease, acute inflammatory demyelinating polyneuropathy (AIDP), adult Still's disease, agamaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, anti-PAD4 activated rheumatoid arthritis, antiphospholipid syndrome, asthma, atopic dermatitis, autoimmune angioedema, autoimmune dysautonomia, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune labyrinthitis (AIED), autoimmune myocarditis, autoimmune ovarian hyperplasia. inflammation, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenia, autoimmune urticaria, axonal-neuropathic neuropathy (AMAN), Baro's disease, Behçet's disease, benign mucous membrane pemphigoid, bullous pemphigoid, Castleman's disease (CD), celiac disease, Chagas' disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis (EGPA), cicatricial pemphigoid, Cogan's syndrome, cold agglutinin disease, congenital heart disease Block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, dermatitis, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), diabetes, discoid lupus, Dresier's syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, Goodpasture's syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, Hashimoto's thyroiditis, autoimmune Epidemic hemolytic anemia, Henoch-Schonlein purpura (HSP), herpes gestationis or pemphigus gestationis (PG), hidradenitis suppurativa (HS) (acne inversa), hypogammaglobulinemia, idiopathic membranous nephropathy, idiopathic thrombocytopenic purpura, IgA nephropathy, IgG4-related disease, IgG4-related sclerosing disease, IgG neuropathy, IgM polyneuropathy, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), inflammatory bowel disease (IBD), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis (JM), Kawasaki disease,Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen rubra planus, lichen sclerosus, conjunctivitis, linear immunoglobulin A disease (LAD), lupus, chronic Lyme disease, membranous nephropathy, Meniere's disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatoid arthritis (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parry syndrome Romberg's syndrome, parasquamous cellulitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, pemphigus vulgaris, pemphigus foliaceus, peripheral neuropathy, perivascular encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyarthritis types I, II and III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progestational dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's syndrome, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis , rheumatic fever, rheumatoid arthritis, juvenile rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, sensitizing / preformed antibodies in solid organ transplantation, Sjögren's syndrome, sperm-testicular autoimmunity, stiff-person syndrome (SPS), systemic lupus erythematosus (SLE), subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmopathy (SO), Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura, thrombotic thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), rhabdomyositis, type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease; and Wegener's disease. Guillain-Barr syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus, multiple sclerosis, N-methyl-D-aspartate receptor (NMDAR) encephalitis, myelin-oligodendrocyte glycoprotein (MOG) spectrum disorder (MOGSD), neuromyelitis optica spectrum disorder (NMOSD), myasthenia gravis,Pemphigus vulgaris, psoriasis, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Sjögren's syndrome, spondyloarthropathy, thyroiditis, vasculitis, vitiligo, myxedema, pernicious anemia, and ulcerative colitis. In a preferred embodiment, the autoimmune disease is not an IgG4-related disease. In a preferred embodiment, the autoimmune disease is AAV (e.g., relapsed or refractory AAV), SLE (e.g., relapsed or refractory SLE), or rheumatoid arthritis (e.g., relapsed or refractory rheumatoid arthritis). In a particularly preferred embodiment, the autoimmune disease is AAV (e.g., relapsed or refractory AAV) or rheumatoid arthritis (e.g., relapsed or refractory rheumatoid arthritis).

[0058] Immune or immunoregulatory cells, including but not limited to T cells, macrophages, NK cells, and NK T cells, have been used to treat autoimmune diseases, autoreactive cell targeting of cells, tissues, and organs, infectious diseases, and cancer. In a preferred embodiment, T cells engineered to express a disease-targeting CAR unit or units have demonstrated deep specificity and cytotoxicity. In another embodiment, immune or immunoregulatory cells, including but not limited to T cells, macrophages, NK cells, and dendritic cells, engineered to express a disease-targeting CAR unit or units have demonstrated deep specificity and cytotoxicity.

[0059] In certain embodiments, the NK cells are derived from human peripheral blood mononuclear cells (PBMCs), white blood cell products (PBSCs), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, or umbilical cord blood. Potential drawbacks of using NK cells in cell therapy include a lack of persistence, which may reduce long-term efficacy.

[0060] In one embodiment, the present disclosure includes a method of modifying NK cells with long life span or long-term persistence in vivo to treat disease. Surprisingly, it has been found that NK cells co-expressing IL-15 / IL-15sushi or the IL-15 / IL-15sushi anchor can extend long-term survival.

[0061] IL-15 is a pleiotropic cytokine that is involved in a wide range of immunological processes and plays important roles in both adaptive and innate immunity.

[0062] In a preferred embodiment, the target is an autoimmune disease, such as T1D, which targets β cells and causes insulin deficiency and subsequent systemic disease. While anti-CD3 mAb treatments have been developed, there is a clinical need for them: high doses of anti-CD3 mAb cause severe toxicity and AEs, and low doses of anti-CD3 mAb are ineffective. Furthermore, comprehensive killing of the CD3+ surface antigen population depletes the subject's T cells, exposing them to serious infections and making them unsuitable for life support. Furthermore, autoimmune diseases can be caused by autoreactive T cells, B cells, and plasma cells. In view of this unmet clinical need, a preferred embodiment of a dual CAR is one CAR unit, a CD7 CAR, that targets T cells, and the other CAR unit that targets B cells by selecting one of the target antigens from the following group: CD19, CD20, CD22, or plasma cells by selecting one of the target antigens from the following group: BCMA, CD38, CD138, CS1. In a further embodiment, the dual CAR can be a compound (cCAR) described in more detail in PCT Application Nos. PCT / US2016 / 039306, PCT / US2016 / 068349, and PCT / US2018 / 038529, the contents of which are incorporated herein by reference.

[0063] In a further embodiment, the dual CAR may be a bispecific tandem CAR (tandem CAR), as described in more detail in a publication (ONCOLOGY REPORTS 00: OR-227153 Mohanty, 0000), the contents of which are incorporated herein by reference. Dual CARs are considered dual-target chimeric antigen receptors and include composite CARs (cCARs) and bispecific tandem CARs. cCARs are created by cloning two complete CAR units into the same vector, and the two CAR constructs are expressed from the same vector. Each unit of the CAR contains an extracellular ligand-binding domain (e.g., an antigen-binding domain), a hinge region, a transmembrane domain, at least one costimulatory domain, and a CD3 zeta signaling domain.

[0064] Bispecific tandem CARs have dual domains that bind to two different antigens within a single Bi-CAR. The dual domain structure is formed by scFv1-scFv2 or VHH1-VHH2. Bispecific tandem CARs share the costimulatory domain and CD3 zeta chain.

[0065] In some embodiments, in a dual CAR, one CAR unit targets T cells by selecting one target antigen from the group of CD2, CD3, CD4, CD5, and CD7, and the other CAR unit targets B cells by selecting one target antigen from the group of CD19, CD20, CD22, or targets plasma cells by selecting one target antigen from the group of BCMA, CD38, CD138, CS1, GPRC5D.

[0066] In another embodiment, the present invention provides artificial T cells, macrophages, NK cells, and dendritic cells that have one or two CAR units and co-express immune function enhancers, and have high immune-inducing activity against autoimmune diseases, infectious diseases, and cancer. The present disclosure also provides methods for producing artificial T cells, macrophages, NK cells, and dendritic cells that can secrete immune function enhancers that reprogram the immune system to fight infectious diseases and cancer.

[0067] In a preferred embodiment, the construct consists of a promoter driving expression of a cCAR, one CAR unit targeting T cells by selecting one of the target antigens from the group CD2, CD3, CD4, CD5, and CD7, and the other CAR unit targeting B cells by selecting one of the target antigens CD19, CD20, or CD22, or plasma cells by selecting one of the target antigens BCMA, CS1, CD138, GPRC5D, or CD38, linked by a P2A, T2A, T2P, or F2A self-cleaving peptide, which, upon cleavage, separates the CAR unit from the other CAR unit. In another embodiment, the construct comprises a promoter driving the expression of a cCAR, wherein one CAR unit targets T cells by selecting one of the following target antigens from the following group: CD2, CD3, CD4, CD5, and CD7, and the other CAR unit targets B cells by selecting one of the following target antigens from the following group: CD19, CD20, CD22, or plasma cells by selecting one of the following target antigens: BCMA, CD38, CS1, CD138, and GPRC5D, and the units are linked by a self-cleaving peptide. Cleavage of this peptide separates one CAR unit from the other CAR unit. The enhancer, secreted IL-15 / IL-15sushi, is also linked by a self-cleaving peptide to a site flanking either end of the two different CAR units. The self-cleaving peptide can be selected from the group consisting of P2A, T2A, T2P, or F2A. In another embodiment, the construct comprises a promoter driving the expression of a cCAR, with one CAR unit (CD7 CAR) targeting T cells, the other CAR unit targeting B cells with a selected target antigen of CD19, CD20, or CD22, and the other CAR unit targeting plasma cells with a selected target antigen of BCMA, CD38, CD138, CS1, or GPRC5D. These units are linked by a self-cleaving peptide, which, upon cleavage, separates the two CAR units.

[0068] In another embodiment, the construct is composed of a promoter driving the expression of a cCAR in which one CAR unit, CD7 CAR, targets T cells and the other CAR unit, CD19 CAR, targets B cells (CD7-CD19 cCAR), and the units are linked by a P2A self-cleaving peptide. When this P2A peptide is cleaved, one CAR unit and the other CAR unit are separated (see Figure 35). In another embodiment, the construct is composed of a promoter driving the expression of a cCAR in which one CAR unit, CD7 CAR, targets T cells and the other CAR unit, CD20 CAR, targets B cells (CD7-CD20 cCAR), and the units are linked by a P2A self-cleaving peptide. When this P2A peptide is cleaved, one CAR unit and the other CAR unit are separated (see Figure 35).

[0069] In a preferred embodiment, autoimmune diseases are targeted in which autoreactive T cells are targeted by the CD7 CAR.

[0070] In a preferred embodiment, autoimmune diseases are of interest, where autoreactive T cells are targeted by the CD5 CAR.

[0071] In a preferred embodiment, autoimmune diseases are targeted in which autoreactive T cells are targeted by the CD4 CAR.

[0072] In a preferred embodiment, autoimmune diseases are targeted by targeting memory T cells, including central memory T cells (TCM), effector memory T cells (TEM), and tissue-resident memory T cells (TRM), with a CD4 CAR.

[0073] In a preferred embodiment, autoimmune diseases are of interest, in which autoreactive T cells are targeted by the CD3 CAR.

[0074] In preferred embodiments, the promoter driving CAR expression can be SFFV, human elongation factor 11a (EF) promoter, immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter.

[0075] One example of a suitable promoter is the immediate-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked to it. Another example of a suitable promoter is the elongation growth factor-1a (EF-1a) promoter. However, other constitutive promoter sequences can also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, and RSV sarcoma virus promoter, as well as human gene promoters such as the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present disclosure should not be limited to the use of constitutive promoters; inducible promoters are also contemplated as part of this disclosure. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired and turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionine promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter. Expression of a chimeric antigen receptor polynucleotide can be achieved using, for example, an expression vector containing at least one of the following promoters: the SFFV (spleen focus-forming virus) or human elongation factor 11α (EF) promoter, the CAG (chicken β-actin promoter with a CMV enhancer) promoter, and the human elongation factor 1a (EF) promoter.Examples of low-strength / low-expression promoters that may be utilized include, but are not limited to, the simian virus 40 (SV40) early promoter, the cytomegalovirus (CMV) immediate-early promoter, the ubiquitin C (UBC) promoter, and the phosphoglycerate kinase 1 (PGK) promoter, or portions thereof. Inducible expression of the chimeric antigen receptor can be achieved, for example, using a tetracycline-responsive promoter, including, but not limited to, TRE3GV (containing a Tet-response element, all generations, preferably the third generation), an inducible promoter (Clontech Laboratories, Mountain View, CA), or portions or combinations thereof.

[0076] In a preferred embodiment, the CAR unit consists of a leader sequence, a hinge (H) region, and a transmembrane domain (TM). Self-cleaving peptides of the construct include, but are not limited to, P2A, T2A, F2A, and E2A.

[0077] In another embodiment, the secreted proteins of the construct may also include, but are not limited to, IL-15 / IL-15sushi, IL-15, IL-21, IL-18, IL-7, IL-10, and IL-12. Secretion enhancers such as IL-15 / IL-15sushi enhance the expansion and persistence of T cells and NK cells. Soluble IL-15 / IL-15sushi fusions are stable and function as unexpected and potent immunomodulators of T / NK cells and neighboring immune response cells to alleviate disease.

[0078] Soluble IL-15 / IL-15sushi fusions can also enhance T / NK cell persistence and stimulate T / NK cell function for anti-pathogen or anti-tumor activity. Soluble IL-15 / IL-15sushi fusions also provide vaccine-like effects by reprogramming the body's immune system to fight infectious diseases, autoimmune diseases, and cancer.

[0079] Soluble IL-15 / IL-15sushi fusions are stable and function as unexpected potent immunomodulators of T / NK cells and their neighboring tumor immune response cells. Soluble IL-15 / IL-15sushi fusions enhance T / NK cell persistence and stimulate T / NK cell function in anti-pathogen, anti-autoimmune, and anti-tumor activities. By stimulating immune cell expansion and function, soluble IL-15 / IL-15sushi fusions have a vaccine-like effect by reprogramming the body's immune system to fight infections and cancer.

[0080] This construct consists of a promoter driving the expression of the IL-15 / IL-15 sushi anchor (also called anchor), or the IL-15 / IL-15 sushi anchor linked to a CAR via a peptide self-cleavage site. The IL-15 / IL-15 sushi anchor is fused to IL-15 and consists of a signal peptide linked to the sushi domain of the IL-15α receptor via a 26-amino acid polyproline linker, two copies of the rituximab epitope (stop), a hinge (H) region, and a transmembrane (TM) domain. IL-15 / IL-15 sushi is anchored to the surface of T cells and NK cells, resulting in enhanced NK and T cell expansion and persistence.

[0081] IL-15 / IL-15sushi is anchored to the surface of T cells and NK cells, resulting in enhanced expansion and persistence of NK and T cells.

[0082] Schematic diagram showing the P2A and T2A linkages of both cCAR-T and IL-15 / IL-15sushi in a single construct. This construct consists of an SFFV promoter driving the expression of the two modular CAR units and an enhancer for IL-15 / IL-15sushi. Upon cleavage of the linker, the cCAR and IL-15 / IL-15sushi split and bind to targets expressing CD33 and / or CD123. The CD3-zeta signaling domain completes the assembly of this CAR-T. The enhancer includes, but is not limited to, the IL-15 / IL-15sushi on the cCAR.

[0083] In one embodiment, the construct consists of a promoter driving the expression of two modular CAR units, an enhancer via a peptide cleavage site, and IL-15 / IL-15sushi. Cleavage of the linker separates the cCAR and IL-15 / IL-15sushi. Two copies of rituximab are fused to the hinge region. The CD3-zeta signaling domain completes the assembly of this CAR-T. Enhancers include, but are not limited to, IL-15 / IL-15sushi on the cCAR.

[0084] The construct's self-cleaving peptides include, but are not limited to, P2A, T2A, F2A, and E2A. The construct's secreted proteins may also include, but are not limited to, IL-2, IL-15 / IL-15sushi, IL-15, IL-21, IL-18, IL-7, and IL-12. Secretion enhancers such as IL-15 / IL-15sushi enhance the expansion and persistence of T cells or NK cells. Soluble IL-15 / IL-15sushi fusions are stable and function as unexpected potent immunomodulators of T / NK cells and their neighboring tumor immune response cells. Soluble IL-15 / IL-15sushi fusions are stable, enhance the persistence of T / NK cells, and stimulate their function in anti-pathogen or anti-tumor activities. Soluble IL-15 / IL-15sushi fusions provide a vaccine-like effect by reprogramming the body's immune system to fight infectious diseases, autoimmune diseases, and cancer. This construct consists of a promoter that drives the expression of the rituximab safety switch and secretes IL-7 and IL-15 / IL-15 sushi anchor, linked by the self-cleaving peptides P2A and T2A, respectively. Cleavage of the P2A and T2A peptides releases the enhancer, rituximab safety switch protein, IL-7, and IL-15 / IL-15 sushi anchor. The rituximab safety protein consists of a leader sequence, an immunoglobulin FAB light chain tag, two copies of the rituximab epitope, a hinge (H) region, and a transmembrane domain (TM). The secreted IL-7 (enhancer) consists of the leader sequence and IL-7 protein. The IL-15 / IL-15 sushi anchor consists of a signal peptide fused to IL-15, linked to the sushi domain of the IL-15α receptor via a 26-amino acid polyproline linker, a hinge (H) region, and a transmembrane domain (TM). IL-15 / IL-15sushi is anchored to the surface of T cells and NK cells, resulting in enhanced expansion and persistence of NK and T cells.

[0085] Secretion of IL-7 enhances the function of the IL-15 / IL-15sushi anchor in the proliferation and persistence of immune cells.

[0086] Organ rejection, including cell rejection, tissue rejection, and organ rejection, arises from autoreactive immune cells, where one CAR unit, a CD7 CAR, targets T cells, and the other CAR unit targets B cells by selecting one of the target antigens from the following group: CD19, CD20, and CD22; or targets plasma cells by selecting one of the target antigens from the following group or target antigens: BCMA, CD38, CD138, CS1, and GPRC5D.

[0087] Those skilled in the art would expect that anti-CD7 CAR T cell constructs would be incompatible with the subject's lifestyle due to their depletion of the CD7+ T cell population. However, we unexpectedly discovered that while anti-CD7 CAR T cells deplete the CD7+ T cell population by approximately (90% or more), a CD7-negative T cell population (2-10%) still exists and expands to maintain normal T cell numbers at the time of measurement. CD7+ T cells mature to a CD7-negative phenotype, and depletion of CD7+ T cells acts as a reset to alleviate T cell-mediated autoimmune disease, organ rejection, and cell rejection.

[0088] Anti-CD3 monoclonal antibody constructs have limited efficacy, resulting in T cell aplasia, limited systemic distribution, and severe toxicity that prevents life support. Therefore, anti-CD3 monoclonal antibody therapy is inadequate for the treatment of T cell-mediated diseases. Anti-CD7 CAR constructs do not significantly result in T cell aplasia, as administration of CD7 CAR therapy is accompanied by expansion of the CD7-negative T cell population (2-10%), maintaining total T cell numbers sufficient to fight infection and compatible with host life. Anti-CD7 CAR therapy acts as an immune reset, thereby depleting T cells expressing the CD7 surface antigen and providing a treatment for T cell-mediated autoimmune diseases / disorders.

[0089] Allogeneic or autologous NK cells induce rapid immune responses but have a limited lifespan and poor persistence, resulting in their relatively rapid disappearance from the circulation.

[0090] T cells and NK cells are ideal platforms for CAR or dual CAR targeting against autoreactive cells, tumors, and infectious diseases. However, the in vivo lifespan of NK cells is very short, approximately 1–2 weeks. Ideally, NK cell persistence of 1–2 months would be sufficient for therapeutic use. IL-15 functions through the trimeric IL-15R complex, which contains a high-affinity binding α chain (IL-15Rα) and the common IL-2R β and γ chains. IL-15 secreted from cells binds to IL-15Rα bound to the IL-15 receptor β and γ chains on the cell surface.

[0091] Allogeneic or autologous NK cells induce rapid immune responses but have a limited life span and are therefore cleared relatively quickly from the circulation.

[0092] Constitutive expression of high levels of IL-15 in mice can induce leukemia (Fehniger et al., J Exp Med. 2001 Jan 15;193(2):219-31). IL-15Rα (full-length IL-15 receptor α subunit) promotes leukemia in T cells when constitutively coexpressed with IL-15 (Sato et al., Blood. 2011 Apr 14;117(15):4032-4040).

[0093] Because the CD2, CD3, CD5, and CD7 antigens play important roles in T cell- and NK cell-based killing mechanisms, those skilled in the art creating novel CAR cells would not use cells engineered to lack endogenous expression of the CD2, CD3, CD5, and CD7 antigens. Moreover, those skilled in the art would not even be motivated to consider creating CD2-, CD3-, CD5-, or CD7-specific CAR cells that target these antigens involved in immune responses. Thus, it was completely unexpected that T cells lacking expression of the CD2, CD3, CD5, and CD7 antigens would be effective in CAR T cell therapy targeting these antigens.

[0094] The present inventors disclose a method for improving immune cell function while preventing tumor formation.

[0095] In a preferred embodiment, CD7CARs deplete autoreactive T cells expressing the CD7 surface antigen, eliminating the source of T cell-mediated disease. The unexpected discovery is that a small population of T cells negative for CD7 surface expression exists, rapidly expanding to replace a subject's T cell population to relatively normal levels and maintain disease-fighting capabilities. This unexpected discovery acts as an immune cell reset, reducing disease-causing T cells and expanding the non-autoreactive lymphocyte population.

[0096] In a preferred embodiment, the CD7CAR achieves depletion of CD7-expressing T-lineage lymphocytes with an excellent safety profile, without inducing major adverse events.

[0097] In a preferred embodiment, a composite CAR (cCAR) has one CAR unit that targets T cells by selecting one target antigen from the following group: CD2, CD3, CD4, CD5, and CD7; and the other CAR unit that targets B cells by selecting one target antigen from the following group: CD19, CD20, CD22; or for plasma cells, one from the following group or target antigens: BCMA, CD38, CD138, CS1, GPRC5D.

[0098] In another embodiment, a single CAR unit targets T cells by selecting one of the target antigens from the following group: CD2, CD3, CD4, CD5, CD7.

[0099] The 65 amino acid sequence in the extracellular portion of IL-15Rα, called the Sushi domain, is involved in IL-15 binding. The cytoplasmic domain of the IL-15 receptor α chain is known to be important for normal IL-15Rα function.

[0100] The present invention discloses a method for fusing IL-15 to the sushi domain instead of the entire IL-15Rα to form an IL-15 / IL-15sushi fusion. In a further disclosure, the signaling cytoplasmic domain of IL-15Rα is not included in the IL-15 / IL-15sushi fusion. In a further disclosure, the IL-15 / IL-15sushi fusion is expressed and anchored to the cell surface, which is referred to as the IL-15 / IL-15sushi anchor.

[0101] According to the present disclosure, human clinical trials have revealed that cells expressing secreted IL-15 / IL-15sushi do not form leukemia even after observation for more than two years.

[0102] In some embodiments, the IL-15 / IL-15sushi fusion is expressed as a precursor protein that is secreted from the cell.

[0103] Protein precursors are inactive proteins that can be converted to active forms by post-translational modifications.

[0104] IL-15 exerts a vaccine-like effect by promoting the proliferation of innate immune cells, including T cells and NK cells. IL-15 has a very short biological half-life of approximately 2 hours. By adding the sushi domain to form the IL-15 / IL-15sushi complex, the half-life of IL-15 was extended by up to 10-fold, resulting in a longer-lasting effect.

[0105] In some embodiments, low levels and long biological half-life of IL-15 are preferred in vivo.

[0106] Surprisingly, immune cells transduced with IL-15 / IL-15sushi were found to produce only picogram amounts of IL-15 / IL-15sushi without demonstrating autonomous proliferation or leukemic transformation in vitro in human clinical trials observed for at least two years.

[0107] In accordance with the present disclosure, the present inventors have also found that immune cells transduced with secreted IL-15 / IL-15sushi have superior durability and immune induction effects in vivo compared to conventional immune cells.

[0108] To enhance this efficiency, a different leader sequence, IL-2, was used instead of the wild-type IL-15 leader sequence, achieving higher levels of secretion. Furthermore, IL-15 is known to have a short biological half-life. The incorporation of the Sushi domain into the IL-15 / IL-15sushi complex increases the half-life of IL-15 by up to 10-fold, resulting in a longer-lasting effect.

[0109] Prior to this, it was found that constitutive co-expression of IL-15Rα (full-length IL-15 receptor α subunit) with IL-15 promotes the development of T-cell leukemia in a transgenic mouse model (Sato et al., Blood. 2011 Apr 14; 117 (15):4032-4040).

[0110] This disclosure describes an IL-15 / IL-15sushi anchor that expresses IL-15 / IL-15sushi on the surface of immunoregulatory cells and enhances their function. This IL-15 / IL-15sushi anchor is composed of a 65-amino acid portion of the extracellular portion of the IL-15sushi domain involved in IL-15 binding. The present invention lacks the cytoplasmic functional domain and most of the extracellular domain of IL-15Rα to avoid leukemia formation.

[0111] However, this deficiency is compensated for by incorporating either secreted IL-7 or IL-15, or IL-15 / IL-15sushi, into the design, whose secretion can be easily controlled using a safety switch, which can turn off expression under unfavorable conditions.

[0112] In some embodiments, the present invention discloses a method for establishing an NK cell platform or a CAR for universal therapy using a secreted IL-15 / IL-15sushi fusion protein to improve the persistence and killing activity of NK cells. NK cells co-expressing secreted IL-15 / IL-15sushi can be used as a universal platform for the treatment of various diseases. In one embodiment, the present disclosure provides artificial cells expressing the IL-15 / IL-15sushi anchor.

[0113] In a further embodiment, extended persistence of NK cells can be achieved by co-expressing an IL-15 / IL-15sushi anchor.

[0114] In one embodiment, the present disclosure provides an IL-15 / IL-15sushi anchor having an IL-15 / IL-15sushi, signal peptide, hinge region and transmembrane domain (see FIG. 2A).

[0115] CAR configuration A "signal peptide" includes a peptide sequence that directs the transport and localization of a peptide and associated polypeptide within a cell, for example, to a particular organelle (such as the endoplasmic reticulum) and / or to the cell surface.

[0116] A signal peptide is a peptide of any secreted or transmembrane protein that directs the transport of a polypeptide of the present disclosure to the cell membrane and cell surface and provides for correct localization of the polypeptide of the present disclosure. In particular, the signal peptide of the present disclosure directs the polypeptide of the present disclosure to the cell membrane, where the extracellular portion of the polypeptide is displayed on the cell surface, the transmembrane portion spans the cell membrane, and the active domain is in the cytoplasmic portion, i.e., inside the cell.

[0117] In one embodiment, the signal peptide is cleaved after passing through the endoplasmic reticulum (ER), i.e., is a cleavable signal peptide. In one embodiment, the signal peptide is a human type I, type II, type III, or type IV protein. In an embodiment, the signal peptide comprises an immunoglobulin heavy chain signal peptide.

[0118] The hinge sequence can be derived from a suitable sequence of any genera, including, for example, humans or portions thereof. Such hinge regions are known in the art. In one embodiment, the hinge region comprises a hinge region of a human protein, including CD-8α, CD28, 4-1BB, OX40, CD3-zeta, T-cell receptor α or β chain, CD3 zeta chain, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, influenza virus hemagglutinin (HA), glycosylphosphatidylinositol (GPI)-anchored proteins, CD154 and functional derivatives thereof, and combinations thereof.

[0119] In one embodiment, the hinge region comprises a CD8a hinge region. In some embodiments, the hinge region comprises an HA hinge region. In some embodiments, the hinge region comprises, but is not limited to, one selected from immunoglobulins (e.g., IgG1, IgG2, IgG3, IgG4, and IgD).

[0120] Transmembrane domains include hydrophobic polypeptides that span a cell membrane. In particular, transmembrane domains span from one side of the cell membrane (extracellular) to the other side of the cell membrane (intracellular or cytoplasmic). Transmembrane domains may be in the form of an alpha helix, a beta barrel, or a combination thereof. Transmembrane domains may comprise polytopic proteins with many transmembrane segments, each an alpha helix, a beta sheet, or a combination thereof. Transmembrane sequences may be derived from any suitable sequence of any genera, including, for example, humans or portions thereof. Such transmembrane regions are known in the art. In one embodiment, the transmembrane region comprises a transmembrane region of a human protein including a T cell receptor alpha or beta chain, CD3 zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, influenza virus hemagglutinin (HA), and functional derivatives thereof, and combinations thereof.

[0121] In one embodiment, the transmembrane region comprises the CD8a transmembrane region.

[0122] In one embodiment, the transmembrane domain comprises an HA transmembrane domain.

[0123] In some embodiments, the CAR polypeptide further comprises one or more costimulatory domains. In some embodiments, the costimulatory domain is a functional domain from a protein comprising CD27, CD2, CD7, CD28, CD30, CD40, PD-1, CD258, OX40, natural killer group 2 member C (NKG2C), natural killer group 2 member D (NKG2D), B7-H3, a ligand that binds to CD27, natural killer group 2 member D (NKG2D), B7-H3, CD83, ICAM-1, LFA-1 (CD11a / CD18), ICOS and a ligand that binds to 4-1BB (CD137), their active fragments, functional derivatives, and combinations thereof. Such costimulatory domains are known in the art.

[0124] In one embodiment, the signaling domain comprises a polypeptide of a functional domain of CD3 zeta, common FcR gamma (FCER1G), Fc gamma RIIIA, FcR beta (Fc epsilon rib), CD3 gamma, CD3 delta, CD3 epsilon, CD79a, CD79b, DNAX activator protein 10 (DAP10), DNAX activator protein 12 (DAP12), an active fragment thereof, a functional derivative thereof, or a combination thereof. Such signaling domains are known in the art.

[0125] In one embodiment, the antigen-recognition domain comprises a fragment antigen-binding fragment (Fab). In another embodiment, the antigen-recognition domain comprises a single-chain variable fragment (scFv). An scFv is a fusion protein in which the variable regions of the heavy chain (VH) and light chain (VL) of an immunoglobulin are linked by a short linker peptide. Antigen-recognition domains comprising such single-chain variable fragments are known in the art.

[0126] In another embodiment, the antigen recognition domain comprises a camelid single domain antibody or a portion thereof. In one embodiment, camelid single domain antibodies include heavy chain antibodies found in camelids, or VHH antibodies. Camelid (e.g., camel, dromedary, llama, alpaca) VHH antibodies refer to variable fragments of camelid single chain antibodies (see Nguyen et al., 2001; Muyldermans, 2001), and also include isolated camelid VHH antibodies, recombinant camelid VHH antibodies, or synthetic camelid VHH antibodies. Such single domain antibodies are known in the art.

[0127] In some embodiments, NK cells co-expressing IL-15 / IL-15sushi or IL-15 / IL-15sushi anchor can be scaled up and used off-the-shelf.

[0128] In some embodiments, NK cells co-expressing both IL-15 / IL-15sushi and IL-15 / IL-15sushi anchor can be scaled up and used off-the-shelf. In such embodiments, the NK cell enhancer is expressed in a single polypeptide molecule with high-efficiency peptide cleavage sites, including, but not limited to, P2A, T2A, F2A, and E2A. In further embodiments, the NK cell enhancer is expressed in a single open reading frame (ORF) under the control of a strong promoter.

[0129] In some embodiments, NK cells co-expressing both IL-7 and IL-15 / IL-15sushi anchor can be scaled up and used off-the-shelf. In such embodiments, the NK cell enhancer is expressed in a single polypeptide molecule with high-efficiency peptide cleavage sites, including, but not limited to, P2A, T2A, F2A, and E2A. In further embodiments, the NK cell enhancer is expressed in a single open reading frame (ORF) under the control of a strong promoter.

[0130] Examples of high-efficiency cleavage sites include porcine teschovirus-1 2A (P2A), FMDV 2A (abbreviated as F2A herein); equine rhinitis A virus (ERAV) 2A (E2A); and sesame signavirus 2A (T2A), cytoplasmic polyhedrosis virus 2A (BmCPV2A) and flagella virus 2A (BmIFV2A), or combinations thereof. In a preferred embodiment, the high-efficiency cleavage site is P2A. The high-efficiency cleavage site is described in Kim JH, Lee SR, Li LH, Park HJ, Park JH, Lee KY, et al. (2011) High Cleavage Efficiency of a 2A Porcine Teschovirus-1 Derived from a 2A Peptide in Human Cell Lines, Zebrafish, and Mice. PLoS ONE 6(4): e18556, the contents of which are incorporated herein by reference.

[0131] The expression vector may be a bicistronic or multicistronic expression vector, which may contain (1) multiple promoters fused to each of the open reading frames; (2) intergenic splicing signals; fusion of genes whose expression is driven by a single promoter; (3) intergenic proteolytic cleavage sites (self-cleaving peptides); and (iv) intergenic internal ribosome entry sites (IRES).

[0132] In one embodiment, NK cells co-expressing IL-15 / IL-15sushi or IL-15 / IL-15sushi anchor are able to continue supportive cytokine signaling, which is important for survival after infusion in patients.

[0133] In one embodiment, NK cells co-expressing IL-7 or IL-15 / IL-15sushi anchors are able to sustain supportive cytokine signaling important for survival after infusion in patients.

[0134] In a further embodiment, prolonged survival of NK cells may be achieved by co-expressing a cytokine selected from the group of IL-7, IL-15, IL-15 / IL-15 anchor, IL-15 / IL-15RA, IL-12, IL-18 and IL-21.

[0135] Surprisingly, in human clinical trials, immune cells co-expressing IL-15 / IL-15sushi showed a significant increase in CD8+ T cells and NK cells, which was associated with increased anti-tumor activity and reduced disease recurrence.

[0136] In some embodiments, IL-15 can be the IL-15N72D mutant, which is fused to the soluble domain (sushi) of IL-15Rα to form a stable complex in solution, which exhibits increased biological activity compared to uncomplexed IL-15. The mutant IL-15N72D can increase the biological activity of IL-15 (US20177595 A1).

[0137] In some embodiments, NK cells provide various immune defense mechanisms, including: 1) altering NK cell responses to infections and tumors by attacking target cells; 2) increasing NK persistence; and 3) reprogramming the body's immune system to fight infections and cancer.

[0138] In some embodiments, the NK cells express cytokines and / or chemokines.

[0139] Co-expressed cytokines in NK cells can be selected from, but are not limited to, the following group of cytokines: IL-15 / IL-15sushi, IL-15 / IL-15sush anchor, IL-2, IL-4, IL-7, IL-10, IL-12, IL-18, IL-21, GM-CSF, and TGF-β. Co-expressed chemokines in NK cells can also be selected from, but are not limited to, the following group of chemokines: CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL19, CXCL1, CXCL2, CXCL9, CXCL10, or CXCL12 or CCL-21.

[0140] In some embodiments, the NK cells co-express the IL-15 / IL-15 anchor with at least one cytokine selected from the group of cytokines including, but not limited to, IL-15, IL-15 / IL-15sushi, IL-2, IL-4, IL-7, IL-10, IL-12, IL-18, IL-21, GM-CSF, and TGF-β.

[0141] In some embodiments, the artificial cells co-express the IL-15 / IL-15 anchor with IL-15sushi. In some embodiments, the artificial cells co-express the IL-15 / IL-15 anchor with IL-15. In some embodiments, the artificial cells co-express the IL-15 / IL-15 anchor with IL-7.

[0142] T antigen-deficient T cells and NK cells T-cell lymphomas and T-cell leukemias express specific antigens, making them useful targets for these diseases. For example, T-cell lymphomas and leukemias express CD7, CD2, CD3, and CD5. However, CD7, CD2, CD3, and CD5 are also expressed on CAR T cells and NK cells (except for CD3 and CD5), negating their ability to target these antigens. Self-killing may occur in T cells or NK cells armed with CARs targeting any of these antigens. This makes it difficult to engineer CARs targeting these antigens. Therefore, if an endogenous antigen of a T cell or NK cell is used as a target for a CAR, it may be necessary to inactivate that antigen. In another embodiment, engineered cells are further modified to inactivate cell surface polypeptides to prevent the engineered cells from interacting with other engineered cells. For example, one or more of the endogenous CD2, CD3, CD4, CD5, and CD7 genes of the engineered cells can be knocked out or inactivated. In a preferred embodiment, the artificial cell is a natural killer cell in which at least one of the endogenous CD2 and CD7 genes has been knocked out or inactivated. In another preferred embodiment, the artificial cell is a T cell in which at least one of the endogenous CD2, CD3, CD4, CD5, CD7, and CD8 genes has been knocked out or inactivated. In another preferred embodiment, the artificial cell is an NK cell in which at least one of the endogenous CD2 and CD7 genes has been knocked out or inactivated.

[0143] In one embodiment, artificial cells expressing a CAR with a specific antigen recognition domain have the gene expressing that antigen inactivated or knocked out. For example, T cells with a CD2 CAR have the CD2 antigen gene inactivated or knocked out. In another embodiment, artificial cells (e.g., NK cells or T cells) with a CAR with a CD4 antigen recognition domain are modified so that the CD4 antigen is not expressed on their cell surface. In another embodiment, artificial cells (e.g., NK cells or T cells) with a CAR with a CD2 antigen recognition domain and a CAR with a CD7 antigen recognition domain may have both the CD2 antigen gene and the CD7 antigen gene knocked out or inactivated.

[0144] [Table 1]

[0145] Methods for knocking out or inactivating genes are commonly known in the art. For example, the CRISPR / Cas9 system, zinc finger nucleases (ZFNs) and TALE nucleases (TALENs), epitope base editing, and meganucleases can be used to knock out or inactivate CD2, CD3, CD4, CD5, CD7, CD8, and CD52 genes in engineered cells.

[0146] CAR delivery can be achieved by expanding engineered T cells in vitro before administration to patients, either before or after inactivation of CD2, CD5, CD3, or CD7.

[0147] In certain embodiments, inactivation of CD2, CD3, CD5, CD7 may be achieved by any of the following means: (1) Expressing an anti-CD5 scFv linked to a transmembrane domain via a hinge region on the T cell surface may convert CD2- or CD3-positive, or CD5- or CD7-negative, T cells. (2) In some embodiments, scFvs (single-chain antibodies) against CD2, CD3, CD5, or CD7 are derived from monoclonal or polyclonal antibodies that bind to the intracellular protein and inhibit transport of the protein to the cell surface. In a preferred embodiment, anti-CD2, CD3, CD5, or CD7 scFvs are linked to the ER (endoplasmic reticulum) retention sequence, KDEL. When expressed intracellularly and retained in the ER or Golgi apparatus, the anti-scFvs confine their bound proteins to the secretory pathway, thereby preventing the proteins from being properly delivered to the cell surface in T cells. (3) a natural self-selection approach by directly introducing CD2, CD3, CD5, or CD7 CAR constructs.

[0148] Cell Source Artificial cells can be obtained from peripheral blood, umbilical cord blood, bone marrow, tumor-infiltrating lymphocytes, lymph node tissue, thymus tissue, etc. Host cells include placental cells, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, etc. Cells may be obtained from humans, monkeys, chimpanzees, dogs, cats, mice, rats, and transgenic species thereof. Cells may be obtained from established cell lines.

[0149] The cells can be obtained by known methods and can be autologous, allogeneic, or xenogeneic to the recipient of the engineered cells.

[0150] The term "autologous" refers to any material obtained from the same individual that is later reintroduced into that individual.

[0151] The term "allogeneic" refers to material derived from a different animal of the same species as the individual into whom the material is being introduced. Two or more individuals are said to be allogeneic to one another if they are not identical at one or more genetic loci. In some aspects, allogeneic material from individuals of the same species may be sufficiently different genetically to interact antigenically. The term "xenogeneic" refers to a graft derived from an animal of a different species.

[0152] The term syngeneic refers to close genetic similarity or identity, particularly with regard to antigens and immunological responses. Syngeneic systems include, for example, models in which organs or cells (such as cancer cells and their non-cancerous counterparts) are derived from the same individual, or models in which organs or cells are derived from different individual animals of the same inbred strain.

[0153] In certain embodiments, the T cells and NK cells are derived from human peripheral blood mononuclear cells (PBMCs), white blood cell products (PBSCs), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, or umbilical cord blood.

[0154] Potential drawbacks of using NK cells as therapeutic agents include a lack of durability that may reduce long-term efficacy.

[0155] In some embodiments, the engineered cells can be immune or non-immune cells, such as red blood cells, which act as carriers of cytokines and chemokines to infected or cancerous tissue.

[0156] In accordance with the present disclosure, red blood cells as carriers provide readily available cells that are engineered to contain at least one cytokine or chemokine selected from the group of cytokines or chemokines including, but not limited to, IL-15, IL-15 / IL-15sush, IL-15 / IL-15RA (full length IL-15 receptor alpha), IL-15 / IL-15 anchor, IL-2, IL-7, IL-12, IL-18, IL-21, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL19, CXCL1, CXCL2, CXCL9, CXCL10, CXCL12, and CCL-21 polypeptides.

[0157] In one embodiment, the artificial cells comprise natural killer cells. Natural killer cells are well known in the art. In one embodiment, the natural killer cells comprise a cell line such as NK-92 cells. Further examples of NK cell lines include NKG, YT, NK-YS, HANK-1, YTS cells, and NKL cells.

[0158] In one embodiment, the engineered cells include macrophages. NK cells mediate anti-tumor effects without the risk of GvHD and are short-lived compared to T cells. Therefore, NK cells become exhausted soon after destroying target cells, reducing the need for inducible suicide genes to eliminate engineered cells.

[0159] In accordance with the present disclosure, it has surprisingly been found that NK cells provide readily available cells that are engineered to contain at least one cytokine selected from the group of cytokines comprising IL-15, IL-15 / IL-15sush, IL-15 / IL-15RA (full length IL-15 receptor alpha), IL-15 / IL-15 anchor, IL-2, IL-7, IL-12, IL-18 and IL-21 polypeptides disclosed herein.

[0160] Although allogeneic or autologous NK cells induce rapid immune responses, they have a limited lifespan and are therefore cleared from the circulation relatively quickly, thus unexpectedly finding that NK cell-based therapies are less likely to have lasting side effects.

[0161] According to one embodiment of the present invention, NK cells can be transfected with cytokine polynucleotides and expanded according to the present invention. NK cells can be derived from umbilical cord blood, peripheral blood, iPS cells, and embryonic stem cells. According to one embodiment of the present invention, NK-92 cells can be expanded and transfected with cytokine polynucleotides. NK-92 is a continuously growing cell line that possesses the characteristics and signatures of natural killer (NK) cells (Arai, Meagher et al. 2008). The NK-92 cell line is IL-2 dependent and has safety features (Arai, Meagher et al. 2008). A pure population of NK-92 cells bearing a cytokine polynucleotide of interest can be obtained by sorting.

[0162] In some embodiments, the engineered cells contain an inducible suicide gene ("safety switch") or a combination of safety switches, which can be assembled on a vector, such as, but not limited to, a retroviral vector, a lentiviral vector, an adenoviral vector, or a plasmid. The introduction of a "safety switch" significantly improves the safety profile. The "safety switch" may be an inducible suicide gene, such as, but not limited to, a caspase 9 gene, thymidine kinase, cytosine deaminase (CD), or cytochrome P450. Other safety switches for eliminating unwanted modified NK cells or T cells include the expression of CD20 or a CD20 epitope, CD52 or CD19, or a truncated epidermal growth factor receptor in T cells. All possible safety switches are contemplated and embodied in the present invention.

[0163] In one embodiment, the engineered cells contain a rituximab safety switch to eliminate unwanted engineered immune cells. In a further embodiment, two rituximab binding sequences are incorporated into the hinge region of the CAR.

[0164] In one embodiment, the artificial cell co-expresses a rituximab epitope expression construct with IL-15 / IL-15sushi via one peptide cleavage sequence selected from the group consisting of P2A, T2A, E2A, and F2A. In a further embodiment, the rituximab epitope expression construct consists of a signal peptide, two epitope domains of rituximab, a CD8α hinge region, and a CD8α transmembrane domain.

[0165] Rituximab is a chimeric antibody targeting CD20, developed by IDEC Pharmaceuticals, and is a cancer treatment drug.

[0166] How to create artificial cells Any of the polynucleotides disclosed herein can be introduced into the engineered cells by any method known in the art.

[0167] In some embodiments of the present invention, any of the artificial cells disclosed herein may be constructed with a transposon system (also known as "Sleeping Beauty") that integrates genes or DNA into the host genome without the use of a viral vector.

[0168] In one embodiment, to achieve an enhanced safety profile or therapeutic index, any of the artificial cells disclosed herein are constructed as transient DNA- or RNA-modified "biodegradable" versions or derivatives, or combinations thereof. The RNA- or DNA-modified versions of the invention can be electroporated into T cells or NK cells.

[0169] vector Many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene transfer systems. A selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to patient cells either in vivo or ex vivo. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Numerous adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used.

[0170] Viral vector technology is well known in the art and is 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 useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, suitable vectors contain a replication origin that functions in at least one organism, a promoter sequence, a convenient and unique restriction endonuclease site, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0171] Lentiviral vectors are known for their high efficiency in transducing genes into human NK cells. However, expression of the vector-encoded gene depends on an internal promoter driving its expression. A wide variety of promoters are available with varying strengths and cell-type specificity. Gene therapy relies on the cells' ability to express appropriate levels of protein and maintain expression over an extended period of time. The EF-1α promoter is commonly chosen for gene expression.

[0172] The present invention provides an expression vector comprising a strong promoter for high-level gene expression in NK cells or T cells. In a further embodiment, the inventors disclose a strong promoter useful for high-level expression of genes in NK cells or T cells. In a specific embodiment, the strong promoter is related to the SFFV or EF-1α promoter, and is selectively introduced into the expression vector to obtain high-level expression and maintain expression over a long period of time in NK cells or T cells. The expressed genes preferably include cytokines and chemokines used in immunotherapy, and costimulatory factors for NK cells and T cells.

[0173] One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence, which is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked to it.

[0174] In a preferred embodiment, the promoter is an SFFV promoter or a derivative thereof. The SFFV promoter has been unexpectedly discovered to provide stronger expression and greater persistence in cells transduced according to the present disclosure. Another example of a suitable promoter is elongation growth factor-1a (EF-1a). However, other constitutive promoter sequences can also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters such as the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present disclosure should not be limited to the use of constitutive promoters; inducible promoters are also contemplated as part of this disclosure. The use of an inducible promoter provides a molecular switch that can turn on expression of an operably linked polynucleotide sequence when such expression is desired and turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, the metallothionine promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter. Expression of a chimeric antigen receptor polynucleotide can be achieved using, for example, an expression vector including at least one of the SFFV (spleen focus-forming virus) promoter, the human elongation factor 11α (EF) promoter, the CAG (chicken β-actin promoter with CMV enhancer) promoter, and the human elongation factor 1α (EF) promoter, but is not limited to these.Examples of low-strength / low-expressing promoters that may be utilized include, but are not limited to, the simian virus 40 (SV40) early promoter, the cytomegalovirus (CMV) immediate-early promoter, the ubiquitin C (UBC) promoter, and the phosphoglycerate kinase 1 (PGK) promoter, or portions thereof. Inducible expression of the chimeric antigen receptor can be achieved, for example, using a tetracycline-responsive promoter, including, but not limited to, TRE3GV (containing a Tet-response element, all generations, preferably the third generation), an inducible promoter (Clontech Laboratories, Mountain View, CA), or portions or combinations thereof.

[0175] An "expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression. Other elements for expression can be supplied by the host cell or an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating the recombinant polynucleotide. Expression vectors can be bicistronic or multicistronic expression vectors. Bicistronic or multicistronic expression vectors can contain (1) multiple promoters fused to each of the open reading frames; (2) insertion of intergenic splicing signals; fusing genes whose expression is driven by a single promoter; (3) insertion of intergenic proteolytic cleavage sites (self-cleaving peptides); and (iv) insertion of intergenic internal ribosome entry sites (IRES).

[0176] In one embodiment, the present disclosure provides an artificial cell comprising at least one chimeric antigen receptor polypeptide or polynucleotide.

[0177] "Engineered cells" refer to cells of an organism that have been modified, transformed, or manipulated by the addition or modification of a gene, DNA or RNA sequence, or protein or polypeptide. The isolated, host, and genetically engineered cells of the present disclosure include isolated immune cells, such as NK cells and T cells, that contain DNA or RNA sequences encoding a cytokine or chimeric antigen receptor or chimeric antigen receptor complex and express the chimeric receptor on their cell surface. Isolated host cells and engineered cells can be used, for example, to enhance NK cell activity or T lymphocyte activity for the treatment of infectious diseases and cancer.

[0178] In one embodiment, the present disclosure provides lipids or lipid materials used to deliver mRNA encoding a CAR or cytokine. Lipids or lipid materials used to deliver mRNA are described herein or known to those of skill in the art (Liu et al., Front Bioeng Biotechnol. 2021 Jul 27;9:718753).

[0179] The present invention provides methods for the management and treatment of autoimmune diseases. In some embodiments, the present disclosure encompasses administering an anti-CD7 CAR construct to individuals with onset or relapsing / refractory T1D, but who do not meet the diagnostic criteria defined by the American Diabetes Association or the Society for Diabetes Immunology, to prevent or delay the onset of T1D and / or to prevent or delay the need for insulin administration in such patients.

[0180] In further embodiments, high risk factors for identifying predisposed patients include having a first or second degree relative diagnosed with T1D, impaired fasting glucose levels (e.g., at least one fasting (8 hour without food) glucose level of 100-125 mg / dl measured), impaired glucose tolerance to a 75 g OGTT (e.g., at least one 2 hour post-75 g OGTT glucose level of 140-199 mg / dl measured), HLA type DR3, DR4 or DR7 in Caucasian patients, HLA type DR3 or DR4 in individuals of African descent, HLA type DR3, DR4 or DR9 in individuals of Japanese descent, viruses (e.g., A positive diagnosis by art-recognized criteria of at least one other autoimmune disease (e.g., thyroid disease, celiac disease), Coxsackie B virus, enterovirus, adenovirus, rubella, cytomegalovirus, Epstein-Barr virus), and / or detection of autoantibodies, particularly ICA and T1D-associated autoreactive antibodies or T cells, in serum or other tissues. In some embodiments, patients identified as having a predisposition to developing T1D have at least one of the risk factors described herein and / or known in the art. The present disclosure also encompasses the identification of a subject predisposed to developing T1D, wherein the subject exhibits a combination of two or more, three or more, four or more, or five or more of the risk factors disclosed herein or known in the art.

[0181] Serum autoantibodies or autoreactive T cells associated with T1D or a predisposition to developing T1D are pancreatic islet cell autoantibodies (e.g., anti-ICA512 autoantibodies), glutamic acid decarbamylase autoantibodies (e.g., anti-GAD65 autoantibodies), IA2 antibodies, ZnT8 antibodies, and / or anti-insulin autoantibodies or autoreactive T cells.

[0182] Insulin-producing β-cell function before, during, and after treatment can be assessed by the methods described herein or any method known to those skilled in the art. For example, the Diabetes Control and Complications Trial (DCCT) study group has established monitoring of glycosylated hemoglobin (HA1 and HA1c) as a criterion for assessing glycemic control (DCCT, 1993, N. Engl. J. Med. 329:977-986). Alternatively, characterization of daily insulin requirements, C-peptide levels / response, hypoglycemic episodes, and / or FPIR may be used as markers of β-cell function or to establish treatment indicators (see Keymeulen et al., 2005, N. Engl. J. Med. 352:2598-2608; Herold et al., 2005, Diabetes 54:1763-1769; U.S. Patent Application Publication No. 2008). Appl. Pub. No. 2004 / 0038867 A1; and Greenbaum et al., 2001, Diabetes 50:470-476, respectively).

[0183] In some embodiments, a patient predisposed to developing T1D may be a non-diabetic subject who is a relative of a T1D patient. In some embodiments, the non-diabetic subject has two or more diabetes-associated autoantibodies or autoreactive T cells selected from islet cell antibodies (ICA), insulin autoantibodies (IAA), and antibodies against glutamic acid decarboxylase (GAD), tyrosine phosphatase (IA-2 / ICA512), or ZnT8.

[0184] In some embodiments, the non-diabetic subject has abnormal glucose tolerance in an oral glucose tolerance test (OGTT), defined as a fasting glucose level of 110-125 mg / dL, or a 2-hour plasma glucose level of 140 to less than 200 mg / dL, or an intervening glucose level of >200 mg / dL at 30, 60, or 90 minutes in an OGTT.

[0185] Combination therapy The compositions and methods of the present disclosure can be used to generate populations of T lymphocytes or NK cells that deliver both primary and costimulatory signals for use in immunotherapy in the treatment of autoimmune diseases. In further embodiments, the clinical aspects of the present invention are combined with other agents effective in treating autoimmune disorders, such as immunosuppressants and steroids.

[0186] The administration of the artificial cells described herein can be supplemented by co-administration of an enhancing agent. Examples of enhancing agents include, but are not limited to, immunomodulators that enhance immune cell activity, such as agents that target immune checkpoint pathways, and inhibitors of colony-stimulating factor-1 receptor (CSF1R) for better therapeutic outcomes. Agents that target immune checkpoint pathways include small molecules, proteins, or antibodies that bind to the inhibitory immune receptors CTLA-4, PD-1, and PD-L1, resulting in blockade of CTLA-4 and PD-1 / PD-L1. As used herein, enhancing agents include enhancers such as those described above.

[0187] The administration of the artificial cells described herein can be supplemented with co-administration of an enhancer. Examples of enhancers for artificial cells can be selected from the group consisting of anti-CD40 antibodies or CD40 ligands, anti-OX40 antibodies, anti-4-1BB antibodies, TNFR2 blocking antibodies, anti-CTLA4 antibodies, PD-L1 inhibitors, and CpG oligonucleotides (CpG ODN, TLR9 agonists).

[0188] In accordance with the present disclosure, engineered cells can be used to express on their surface a CAR (chimeric antigen receptor) involved in the treatment of disease.

[0189] In accordance with the present disclosure, cells can be used that are engineered to express T cell receptors (TCRs) on their surface that are involved in the treatment of disease. TCR-modified T (TCR-T) cells show promise against tumors and infectious agents.

[0190] Based on this, the present disclosure also provides methods for providing long-lasting remission to patients by administering engineered cells having co-expression of a TCR polypeptide disclosed herein and IL-15 / IL-15sushi or IL-15 / IL-15sushi anchor to enhance the sensitivity of TCR recognition of target cancer cells, recruit innate immune cells to cancer cells, or enhance the persistence of TCR T cells.

[0191] Based on this, the present disclosure also provides methods for providing long-lasting remission to patients by administering engineered cells having a CAR polypeptide disclosed herein and co-expression of IL-15 / IL-15sushi or an IL-15 / IL-15sushi anchor to enhance the sensitivity of CAR recognition in target cancer cells, or recruit innate immune cells to cancer cells, or enhance CAR persistence.

[0192] Antigen-directed CAR immunotherapies include, but are not limited to, CD19, CD20, CD22, CD2, CD3, CD4, CD5, CD7, CD52, CD38, CD33, CD30, CD123, GD2, CD45, CLL-1, BCMA, CS1, BAFF, TACI, APRIL CAR, and the like.

[0193] In one embodiment, the target of the antigen recognition domain of the CAR is GD2, GD3, interleukin 6 receptor, ROR1, PSMA, PSCA (prostate stem cell antigen), MAGE A3, glycolipids, glypican 3, F77, GD-2, WT1, CEA, HER-2 / neu, DLL3, EGFR, folate receptor alpha, EpCAM, CD171, CD117, mesothelin, GM2, DR5, EGFR, EpCAM, EpHA2, ER-alpha, gp100, LMP1, IL-13R, VEGFR-2, PSMA, PSCA, PD-L, MAGE-3, MAGE-4, MAGE-5, MAGE-6, alpha-fetoprotein, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, MUC1, MUC2, MUC3, MUC4, MUC5, MMG49 epitope, CD30, EGFRvIII, CLDN, CLDN18, CLDN18, CLDN18.2, CD33, CD123, CLL-1 (CD371), NKG2D, NKG2D receptor, immunoglobulin kappa and lambda, CD38, CD52, CD47, CD200, CD70, CD19, CD20, CD22, CD38, BCMA (CD269), CS1 (SLAMF7, CD319), GPRC5D, BAFF receptor, TACI, CD3, CD4, CD8, CD5, CD7, CD2, and CD138.

[0194] As used herein, the term "patient" includes mammals. A mammal referred to herein may be any mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, rodent mammals, such as mice and hamsters, and logomorpha mammals, such as rabbits. Mammals may belong to the order Carnivora, which includes felines (cats) and canines (dogs). Mammals may also belong to the order Artiodactyla, which includes bovines (cattle) and porcinidae (pigs), and perissodactyla, which includes equidae (horses). Mammals may also belong to the order Primates, ceboids, simians (monkeys), and anthropoids (humans and apes). Preferably, the mammal is a human. Patients include test subjects. Patients in need thereof include those suffering from or at risk of developing a disease that would benefit from the claimed treatment methods.

[0195] For example, a high risk for developing type 1 diabetes is defined as: (i) the presence of at least one diabetes-associated autoantibody, including anti-GAD65, anti-ICA512, anti-insulin (MIAA), and ICA (islet cell antibody), at least once; (ii) impaired glucose tolerance by oral glucose tolerance test (OGTT); (iii) fasting blood glucose level ≥ 110 mg / dL but < 126 mg / dL; (iv) and / or 2-hour blood glucose level ≥ 140 mg / dL but < 200 mg / dL; and (v) and / or 30-, 60-, and 90-minute OGTT values ​​≥ 200 mg / dL.

[0196] In certain embodiments, the patient is a human 0-6 months old, 6-12 months old, 1-5 years old, 5-10 years old, 5-12 years old, 10-15 years old, 15-20 years old, 13-19 years old, 20-25 years old, 25-30 years old, 20-65 years old, 30-35 years old, 35-40 years old, 40-45 years old, 45-50 years old, 50-55 years old, 55-60 years old, 60-65 years old, 65-70 years old, 70-75 years old, 75-80 years old, 80-85 years old, 85-90 years old, 90-95 years old, 95-100 years old.

[0197] As used herein, "effective amount" and "therapeutically effective amount" of engineered cells refer to an amount of engineered cells sufficient to produce a desired therapeutic or physiological effect or result. Such effects or results include the reduction or amelioration of symptoms of a cell-mediated disorder. Undesirable effects, e.g., side effects, may occur along with the desired therapeutic effect. Therefore, one of skill in the art will balance the potential benefits against the potential risks when determining an appropriate "effective amount." The exact amount required will vary from patient to patient, depending on the patient's type, age, general condition, method of administration, etc. Therefore, it may not be possible to specify an exact "effective amount." However, an appropriate "effective amount" in a particular case can be determined by one of skill in the art using only routine experimentation. Generally, engineered cells or engineered cells are administered in an amount and under conditions sufficient to reduce proliferation of the target cells.

[0198] After administration of the delivery system for the treatment, suppression, or prevention of autoimmune disease, the effectiveness of the therapeutic artificial cells can be assessed by a variety of methods well known to those skilled in the art. For example, one skilled in the art will understand that the therapeutic artificial cells administered with a chemical adjuvant are effective in treating or suppressing autoreactive cells in a patient by observing that the therapeutic artificial cells reduce the autoimmune cell load or prevent a further increase in the autoimmune cell load.

[0199] Throughout this specification, quantities are defined by ranges and by lower and upper limits of the ranges. Each lower boundary can be combined with each upper boundary to define a range. Each lower boundary and upper boundary should be considered as a separate entity. Throughout this specification, reference to "one embodiment," "an embodiment," "one example," or "an example" means that a particular feature, structure, or characteristic described in connection with an embodiment or example is included in at least one embodiment of the present embodiments. Thus, the appearance of the phrases "in one embodiment," "in an embodiment," "one example," or "an example" in various places throughout this specification does not necessarily all refer to the same embodiment or example. Furthermore, particular features, structures, or characteristics can be combined in any suitable combinations and / or subcombinations in one or more embodiments or examples. In addition, it is understood that the figures provided herein are for illustrative purposes for persons skilled in the art, and that the drawings are not necessarily drawn to scale.

[0200] As used herein, the terms "comprises," "comprising," "includes," "including," "has," "having," or other variations thereof, are intended to cover a non-exclusive inclusion. For example, a process, article, or apparatus consisting of a list of elements is not necessarily limited to only those elements and may include other elements not expressly listed or inherent to such process, article, or apparatus.

[0201] Further, unless expressly stated to the contrary, "or" means an inclusive "or" and not an exclusive "or." For example, condition A or B is satisfied by any of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or exists), or both A and B are true (or exist).

[0202] Furthermore, the examples or illustrations provided herein are not to be considered as limitations, restrictions, or express definitions of the terms or terms in which they are used. Instead, these examples or illustrations are described with respect to one particular embodiment and are to be considered as illustrative only. Those skilled in the art will understand that the terms or terms in which these examples or illustrations are used encompass other embodiments, whether or not provided herein together or elsewhere, and that all such embodiments are intended to be included within the scope of the term or term. Language designating such non-limiting examples and illustrations includes, but is not limited to, the following: "for example," "for instance," "e.g.,," and "in one embodiment."

[0203] Various parameter groups are described herein as including multiple members. Within a parameter group, each member can be combined with any one or more of the other members to form further subgroups. For example, if the members of a group are a, b, c, d, and e, additional subgroups specifically contemplated include any one, two, three, or four of the members, such as a and c; a, d, and e; b, c, d, and e; etc.

[0204] As used herein, a XXXX antigen recognition domain is a polypeptide selective for XXXX. "XXXX" refers to the target discussed herein and above. For example, a CD7 antigen recognition domain is a polypeptide specific for CD7.

[0205] As used herein, CDXCAR refers to a chimeric antigen receptor having a CDX antigen recognition domain.

[0206] Allogeneic or autologous NK cells induce rapid immune responses but have a limited lifespan and poor persistence, resulting in their relatively rapid disappearance from the circulation.

[0207] NK cells can be an effective platform against autoreactive cells, tumors, and infectious diseases if these cells can persist for a relatively long period of time. However, the lifespan of NK cells in vivo is very short, at 1–2 weeks. Ideally, NK cell persistence of 1–2 months would be sufficient for treatment.

[0208] In certain embodiments, a "subject" or "patient" is a human. In certain embodiments, a "subject" or "patient" is under 18 years of age. In some embodiments, a "subject" or "patient" is 18 years of age or older. In some embodiments, the subject is in need of remission induction. In some embodiments, the subject is in need of remission maintenance.

[0209] As used herein, terms such as "treat," "treating," or "treatment" refer to obtaining a desired pharmacological and / or physiological effect. Preferably, the effect is therapeutic, i.e., the effect partially or completely cures a disease and / or adverse symptoms resulting from the disease. Alternatively, the pharmacological and / or physiological effect may be prophylactic, i.e., an effect that completely or partially prevents a disease or its symptoms.

[0210] As used herein, terms such as "manage," "managing," or "management" refer to inhibiting and / or slowing the progression and / or worsening of a disease and / or adverse symptoms resulting from a disease.

[0211] The present invention relates to the treatment or management of autoimmune diseases using bispecific antibodies that bind to an antigen that binds to CD7 or an antigen that binds to T cells (CD2, CD3, CD4, CD5) and an antigen present on the surface of B cells or plasma cells (e.g., CD19, CD20, BCMA, CS1 (SLAMF7, CD319), GPRC5D, CD138). In some embodiments, the multispecific (e.g., bispecific) antibodies of the invention can selectively bind to cells that cause an autoimmune disorder, e.g., BCMA-expressing cells, CD19-expressing cells, or CD7-expressing cells that cause an autoimmune disorder.

[0212] As used herein, the term "autoreactive T lineage cells" refers to T lineage cells that can recognize antigens on a subject's own tissues ("autoantigens"). Autoreactive T lineage cells may have a TCR that targets healthy tissue. In some embodiments, autoreactive B lineage cells are plasmablasts, plasma cells, memory B cells, or any combination thereof.

[0213] The term "autoimmune disease" used herein is defined as a disease caused by autoimmune reaction.Autoimmune disease is the result of inappropriate and excessive reaction to autoantigen.Examples of autoimmune disease include achalasia, Addison's disease, acute inflammatory demyelinating polyneuropathy (AIDP), adult Still's disease, agamaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, anti-PAD4 activated rheumatoid arthritis, antiphospholipid syndrome, asthma, atopic dermatitis, autoimmune angioedema, autoimmune dysautonomia, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune labyrinthitis (AIED), autoimmune myocarditis, autoimmune ophthalmitis, autoimmune orchitis, autoimmune pancreatitis. , autoimmune retinopathy, autoimmune thrombocytopenia, autoimmune urticaria, axonal-neuropathic neuropathy (AMAN), Baro's disease, Behçet's disease, benign mucous membrane pemphigoid, bullous pemphigoid, Castleman's disease (CD), celiac disease, Chagas' disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis (EGPA), cicatricial pemphigoid, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, dermatitis, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), diabetes, discoid lupus, Dresier's syndrome, endometriosis, eosinophilic esophagitis (EoE), Eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, Goodpasture syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, Hashimoto's thyroiditis, autoimmune hemolytic anemia, Henoch-Schonlein purpura (HSP), herpes gestationis or pemphigus gestationis (PG), hidradenitis suppurativa (HS) (inverse acne), hypogammaglobulinemia, idiopathic membranous nephropathy, idiopathic thrombocytopenic purpura, IgA nephropathy, IgG4-related disease, IgG4-related sclerosing disease, IgG neuropathy, IgM polyneuropathy, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), inflammatory bowel disease (IBD), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome,Leukocytoclastic vasculitis, lichen rubra planus, lichen sclerosus, lichen conjunctivitis, linear immunoglobulin A disease (LAD), lupus, chronic Lyme disease, membranous nephropathy, Meniere's disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatoid arthritis (PR), PA NDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, parasquamous uveitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, pemphigus vulgaris, pemphigus foliaceus, peripheral neuropathy, borderline encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyarthritis syndrome types I, II and III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosis cholangitis, progestational dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's syndrome, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, juvenile rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, sensitizing / precursor antibodies in solid organ transplantation, Sjogren's syndrome, sperm and testicular autoimmunity, stiff-person syndrome Examples of autoimmune diseases include, but are not limited to, systemic lupus erythematosus (SPS), systemic lupus erythematosus (SLE), subacute bacterial endocarditis (SBE), Susac syndrome, sympathetic ophthalmopathy (SO), Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura, thrombotic thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), rhabdomyositis, type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease, Wegener's disease, and the like. In a preferred embodiment, the autoimmune disease is not an IgG4-related disease. In a preferred embodiment, the autoimmune disease is AAV (e.g., relapsed or refractory AAV), SLE (e.g., relapsed or refractory SLE), or rheumatoid arthritis (e.g.,In a particularly preferred embodiment, the autoimmune disease is AAV (e.g., relapsed or refractory AAV) or rheumatoid arthritis (e.g., relapsed or refractory rheumatoid arthritis).

[0214] Autoimmune diseases treatable with the CD7 CARs of the present invention include achalasia, Addison's disease, acute inflammatory demyelinating polyneuropathy (AIDP), adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, anti-PAD4-activated rheumatoid arthritis, antiphospholipid syndrome, asthma, atopic dermatitis, autoimmune angioedema, autoimmune dysautonomia, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune labyrinthitis (AIED), autoimmune myocarditis, autoimmune ophthalmitis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, and autoimmune vasculitis. Epidemic thrombocytopenia, autoimmune urticaria-axonal-neuropathy (AMAN), Baro's disease, Behçet's disease, benign mucous membrane pemphigoid, bullous pemphigoid, Castleman's disease (CD), celiac disease, Chagas' disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis (EGPA), cicatricial pemphigoid, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, dermatitis, dermatitis herpetiformis, dermatomyositis, Devic's disease Neuromyelitis optica, diabetes, discoid lupus, Dresier's syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, Goodpasture's syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, Hashimoto's thyroiditis, autoimmune hemolytic anemia, Henoch-Schonlein purpura (HSP), herpes gestationis or pemphigus gestationis (PG), hidradenitis suppurativa (HS) (acne inversus), hypogammaglobulinemia Phosphatemia, idiopathic membranous nephropathy, idiopathic thrombocytopenic purpura, IgA nephropathy, IgG4-related disease, IgG4-related sclerosing disease, IgG neuropathy, IgM polyneuropathy, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), inflammatory bowel disease (IBD), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, conjunctivitis, linear immunoglobulin A disease (LAD), lupus, chronic Lyme disease, membranous nephropathy, Meniere's disease, microscopic polyangiitis (MPA),Mixed connective tissue disease (MCTD), Mullen's ulcer, Mucha-Habermann disease, multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatoid arthritis (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, parasquamous cellulitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, pemphigus vulgaris, pemphigus foliaceus, peripheral neuropathy, borderline encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyarticular syndromes type I, II and III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone-induced dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA) ), pyoderma gangrenosum, Raynaud's syndrome, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, juvenile rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, sensitizing / precursor antibodies in solid organ transplantation, Sjögren's syndrome, sperm and testicular autoimmunity, stiff-person syndrome (SPS), systemic lupus erythematosus (SLE) These include, but are not limited to, subacute bacterial endocarditis (SBE), Susac syndrome, sympathetic ophthalmopathy (SO), Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura, thrombotic thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease, Wegener's disease, and the like. In a preferred embodiment, the autoimmune disease is not an IgG4-related disease. In a preferred embodiment, the autoimmune disease is AAV (e.g., relapsed or refractory AAV), SLE (e.g., relapsed or refractory SLE), or rheumatoid arthritis (e.g., relapsed or refractory rheumatoid arthritis). In particularly preferred embodiments, the autoimmune disease is AAV (e.g., relapsed or refractory AAV) or rheumatoid arthritis (e.g., relapsed or refractory rheumatoid arthritis).

[0215] Autoimmune diseases that can be treated with the CD7-CD19 cCAR of the present invention include achalasia, Addison's disease, acute inflammatory demyelinating polyneuropathy (AIDP), adult Still's disease, agamaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, anti-PAD4-activated rheumatoid arthritis, antiphospholipid syndrome, asthma, atopic dermatitis, autoimmune angioedema, autoimmune dysautonomia, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune ophthalmitis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, Autoimmune thrombocytopenia, autoimmune urticaria, axonal and neuropathic neuropathy (AMAN), Baro's disease, Behçet's disease, benign mucous membrane pemphigoid, bullous pemphigoid, Castleman's disease (CD), celiac disease, Chagas' disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss syndrome (CSS) or eosinophilic granulomatosis (EGPA), cicatricial pemphigoid, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST syndrome, Crohn's disease, dermatitis, dermatitis herpetiformis, dermatomyositis, Day syndrome Big's disease (neuromyelitis optica), diabetes, discoid lupus, Dresier's syndrome, endometriosis, eosinophilic esophagitis (EoE), eosinophilic fasciitis, erythema nodosum, essential mixed cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, Goodpasture's syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, Hashimoto's thyroiditis, autoimmune hemolytic anemia, Henoch-Schonlein purpura (HSP), herpes gestationis or pemphigus gestationis (PG), hidradenitis suppurativa (HS) (acne inversus), hypogammaglobulinemia IgA nephropathy, idiopathic membranous nephropathy, idiopathic thrombocytopenic purpura, IgA nephropathy, IgG4-related disease, IgG4-related sclerosing disease, IgG neuropathy, IgM polyneuropathy, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), inflammatory bowel disease (IBD), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, conjunctivitis, linear immunoglobulin A disease (LAD), lupus, chronic Lyme disease, membranous nephropathy, Meniere's disease, microscopic polyangiitis (MPA),Mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, multifocal motor neuropathy (MMN) or MMNCB, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neonatal lupus, neuromyelitis optica, neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatoid arthritis (PR), PANDAS, paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parry syndrome Romberg's syndrome, parasquamous cellulitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, pemphigus vulgaris, pemphigus foliaceus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyarthritis types I, II and III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progestational dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's syndrome, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS), retroperitoneal fibrosis , rheumatic fever, rheumatoid arthritis, juvenile rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, sensitizing / preformed antibodies in solid organ transplantation, Sjogren's syndrome, sperm-testicular autoimmunity, stiff-person syndrome (SPS), systemic lupus erythematosus (SLE), subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmopathy (SO), Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura, thrombotic thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome (THS), rhabdomyositis, type 1 diabetes, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, Vogt-Koyanagi-Harada disease; and Wegener's disease. In a preferred embodiment, the autoimmune disease is not an IgG4-related disease. In a preferred embodiment, the autoimmune disease is AAV (e.g., relapsed or refractory AAV), SLE (e.g., relapsed or refractory SLE), or rheumatoid arthritis (e.g., relapsed or refractory rheumatoid arthritis). In a particularly preferred embodiment, the autoimmune disease is AAV (e.g., relapsed or refractory AAV) or rheumatoid arthritis (e.g.,In a further embodiment, the CD7-CD19 cCAR targets CD7-positive T cells and CD19-positive T cells.

[0216] Bispecific antibody T cell engagers (also known as BiTEs) are fusion proteins consisting of two single-chain variable fragments (scFvs), one of which targets a tumor-associated antigen (TAA) expressed on tumor cells, and the other of which contains dual scFv functional domains that target the CD3 chain of T cells. BiTEs can target T cells to tumor cells expressing a specific TAA and lyse the tumor cells (Figure 33).

[0217] Both bispecific T cell engagers and CAR T cells have been widely used to target tumor cells and are believed to have revolutionized cancer treatment. However, some patients still experience recurrence, which may be due to loss of target antigens on tumor cells (also known as antigen escape) or the short lifespan of bispecific antibodies after infusion. Furthermore, manufacturing bispecific antibody T engagers is costly and time-consuming.

[0218] In one embodiment, the present disclosure provides bispecific T cell engagers for use in targeting tumor cells or autoreactive cells. Methods for bispecific T cell engagers are known to those skilled in the art (Blanco et al., J Immunol 2003; 171:1070-1077; Compte et al., Cancer Gene Therapy (2007) 14, 380-388).

[0219] In one embodiment, an engineered cell comprising a CAR-BiTE construct expressing at least one different chimeric antigen receptor polypeptide (CAR) and a secreted bispecific T cell engager. In one embodiment, an engineered cell comprising at least two different chimeric antigen receptor polypeptides (CARs) and a secreted bispecific T cell engager.

[0220] In a preferred embodiment, CAR T cells or NK cells are engineered to secrete soluble bispecific antibody T cell engagers. CAR T cells can kill target cells and continuously produce bispecific antibody T cell engagers. CAR T cells and bispecific T cells can act independently to mount a strong immune response against target cells. In a further embodiment, the target cells can be autoreactive cells or cancer cells.

[0221] The CAR-BiTE construct is a complete CAR fused to a BiTE by a self-cleaving peptide, allowing for independent expression. Expression of activated T cells transduced with CAR-BiTE lentiviral or retroviral vectors can be assayed by flow cytometry (FACS) to reveal CAR expression, and secreted BiTEs can be detected by ELISA or Western blot assays.

[0222] The CAR-BiTE construct is a two-unit CAR (also called a composite CAR or cCAR) in which one complete CAR is fused to another complete CAR via a self-cleaving peptide, allowing for separate and independent expression of both CAR receptors on the T cell surface. The two-unit CAR is also fused to a secreted BiTE via another self-cleaving peptide.

[0223] Creating composite CARs with different CAR units is extremely challenging. (1) CAR-CAR interactions can have detrimental effects, and proper CAR design is key to offsetting this effect. (2) Incorporating composite CARs into a single construct requires a long expression cassette, which can lead to reduced viral titers and protein expression levels. (3) To select a strategy for expressing multiple CARs in a single vector, appropriate design, particularly including various CAR body elements, is required. (4) The hinge region of the CAR must be designed to preferably avoid hinge region interactions between each CAR unit. (5) Expressing two or more CAR units in a cell requires avoiding CAR-CAR interactions. The applicant provides novel and surprising CAR compositions and methods to overcome these hurdles.

[0224] In some embodiments, the chimeric antigen receptor (CAR) is composed of a signal domain (also called a leader sequence), an antigen recognition domain, a hinge domain, a transmembrane domain, and one or more costimulatory signaling domains and a CD3 zeta signaling domain.

[0225] In one embodiment, CD7-negative T cells or NK cells can constitute CARs that can bind to different antigens present on target cells.

[0226] In one embodiment, the CD7-negative T cells or NK cells can contain multiple CARs that can bind to different antigens present on the target cell.

[0227] In one embodiment, the CD7-negative T cells or NK cells can comprise a CAR and at least one of recombinant IL-15, IL-15RA, IL-15sushi, IL-15 / IL-15RA, IL15-RA / IL-15, IL-15 / IL-15sushi, IL15sushi / IL-15, functional fragments thereof, or combinations thereof.

[0228] In another embodiment, the present disclosure provides a combination of a CD7 CAR unit and NY-ESO-1, alpha-fetoprotein (AFP), glypican-3 (GPC3), BCMA, BAFF-R, BCMA, TACI, LeY, CD5, CD7, CD2, CD3, CD4, CD45, CD13, CD14, CD15, CD19, CLL-1, CD20, CD22, CD33, CD41, CD61, CD64, CD68, CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor, ROR1, PSMA, MAGE A3, glycolipids, and claudin 18. 2, F77, GD-2, MART-1, MUC1, MUC2, MUC3, MUC4, MUC5, MMG49 epitope, WT1, CEA, HER-2 / neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, CD30, EGFRvIII, immunoglobulin kappa and lambda, CD38, and other CAR units having antigen recognition domains selected from the group consisting of CS1 (Figure 8). CAR target antigens can also include viral or fungal antigens such as E6 and E7 from human papillomavirus (HPV) or EBV (Epstein-Barr virus) antigens. In a further embodiment, CD7-negative T cells or NK cells are generated by a natural selection approach using a cCAR containing a CD7 CAR construct.

[0229] In another embodiment, the present invention provides a method for the treatment of IL-15, IL-15RA, IL-15sushi, IL-15 / IL-15RA, IL15-RA / IL-15, IL-15 / IL-15sushi, IL15sushi / IL-15, functional fragments thereof, or combinations thereof, comprising administering to a patient a therapeutically effective amount of at least one of recombinant IL-15, IL-15RA, IL-15sushi, IL-15 / IL-15RA, IL-15-RA / IL-15, IL-15 / IL-15sushi, IL-15sushi / IL-15, functional fragments thereof, or combinations thereof, comprising administering to a patient a therapeutically effective amount of at least one of recombinant IL-15, IL-15RA, IL-15sushi, IL-15sushi / IL-15, functional fragments thereof, or combinations thereof, wherein the antigen recognition domain is selected from the group consisting of NY-ESO-1, alpha-fetoprotein (AFP), glypican-3 (GPC3), BCMA, BAFF-R, BCMA, TACI, LeY, CD5, CD7, CD2, CD3, CD4, CD45, CD13, CD14, CD15, CD19, CD20, CD22, CD33, CD41, CD61, CD64, CD68, CD117, CD123, CD138, CD267, CD269, CD38, Flt3 receptor, ROR1, PSMA, MAGE A3, CD7-negative T cells or NK cells are provided that are harboring at least one different CAR polypeptide, including glycolipid, Claudin 18.2, F77, GD-2, WT1, CEA, HER-2 / neu, MAGE-3, MAGE-4, MAGE-5, MAGE-6, CA 19-9, CA 72-4, NY-ESO, FAP, ErbB, c-Met, MART-1, CD30, CLL-1, MART-1, MUC1, MUC2, MUC3, MUC4, MUC5, MMG49 epitopes, EGFRvIII, immunoglobulin kappa and lambda, CD38, or CS1. Target antigens also include viral and fungal antigens, such as human papillomavirus (HPV) antigens and EBV (Epstein-Barr virus) antigens E6 and E7.

[0230] In further embodiments, antigen-recognizing polypeptides (scFvs) and corresponding polynucleotides against CD2, CD3, CD5, CD7, and CD52, as well as IL-15 / IL-15sushi and IL-15sushi, are described in more detail in PCT Application Nos. PCT / US2016 / 39306 and PCT / US2016 / 019953, the contents of which are incorporated herein by reference. In some embodiments, CD7-negative T cells or NK cells can be obtained from peripheral blood and umbilical cord blood, or from induced pluripotent stem cells (also known as iPS cells or iPSCs) or cells differentiated from stem cells. [Example]

[0231] Usage example Targeting CD4-expressing T cells and T-cell malignancies using CD4-specific chimeric antigen receptor (CAR)-modified T cells Materials and Methods Blood donors, primary tumor cells and cell lines Human lymphoma cells and peripheral blood mononuclear cells were obtained from residual samples. Umbilical cord blood cells were obtained from donors at Stony Brook University Hospital. SP53 and KARPAS 299 lymphoma cell lines were obtained from ATCC (Manassas, VA).

[0232] Lentivirus production and transduction into T cells 293FT cells were co-transduced with pMD2G and pSPAX viral packaging plasmids and pRSC.CD4.3G or GFP lentiviral vectors using Lipofectamine 2000 (Life Technologies, Carlsbad, CA) according to the manufacturer's protocol. Prior to lentiviral transduction, umbilical cord or peripheral blood mononuclear buffy coat cells were activated for 2 days in the presence of 300 IU / mL IL-2 and 1 μg / mL anti-human CD3 (Miltenyi Biotec, Germany).

[0233] T cell expansion CAR-transduced T cells were grown for 7 days in T cell medium (50% AIM-V, 40% RPMI 1640, 10% FBS, 1x penicillin / streptomycin; all Gibco) supplemented with IL-2. Cell numbers were counted daily, and T cell numbers were increased to 2 x 10 6 The medium was replenished every 2-3 days to maintain the cell density below 100 cells / mL.

[0234] CAR immunophenotype To analyze the immunophenotype of CAR cells, after 7 days of culture, CD4CAR T cells and GFP control cells were stained with CD45RO, CD45RA, CD62L, and CD8 (all from BD Biosciences) and subjected to flow cytometry analysis.

[0235] Co-culture target cell ablation assay CD4CAR T cells or GFP T cells (control) were incubated with target cells at ratios of 2:1, 5:1, or 10:1 (200,000, 500,000, and 1,000,000 effector cells to 100,000 target cells, respectively) in 1 mL of T cell culture medium without IL-2 for 24 hours. Target cells were CD4-expressing KARPAS 299 cells (CD4-expressing anaplastic large T-cell lymphoma), leukemia cells from a patient with CD4+ T-cell leukemia (Sezary syndrome), and cells from a patient with CD4+ PTCL lymphoma. As a negative control, CD4CAR T cells and GFP T cells were also incubated separately with non-CD4-expressing SP53 (mantle cell lymphoma) cells at the same ratios (1 mL each). After 24 hours of coculture, cells were stained with mouse anti-human CD8 and CD4 antibodies. In experiments using SP53 cells, SP53 cells were labeled with CMTMR (Life Technologies) before co-culture with T cells, and T cells were labeled with mouse anti-human CD3 (PerCp) after co-culture.

[0236] In vivo mouse xenograft model NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl / SzJ) from the Jackson Laboratory were used under a protocol approved by the Stony Brook University IACUC. All mice were male and aged 8 to 12 weeks. Three sets of in vivo experiments were performed without blinding. For each set, 10 mice were irradiated with a sublethal dose (2.5 Gy) of gamma rays and randomly assigned to treatment and control groups. 24 hours later, the mice were irradiated with 0.5 x 10 6 or 1.0 x10 6 KARPAS 299 cells were injected intradermally and allowed to form measurable subcutaneous tumors within 7 days. Tumor area was measured every other day. In the first set, 3 days after injection of 1 million KARPAS 299 cells, mice were intravenously (tail vein injection) administered 2 million CD4CAR T cells (5 mice) or 2 million GFP T control cells (5 mice). On day 22, 8 million cells were injected intravenously. In the second set, 10 NSG mice were irradiated and 0.5 x 10 6 On day 2, mice received a course of 8 million CD4CAR T cells (5 mice) and 8 million GFP T control cells (5 mice) intravenously. On day 10, a second dose of 5.5 million cells was administered intravenously. In the third set, 10 NSG mice were irradiated and received 0.5 x 10 6 On day 1, mice were injected with 2.5 x 10 KARPAS 299 cells. 6 CD4CAR T cells or GFP control T cells were intravenously infused (5 mice per group). Intravenous infusions were repeated every 5 days for a total of 4 cycles.

[0237] result: CD4CAR characteristics Cord blood-derived CD4CAR T cells are highly enriched for CD8+ T cells, most of which have a central memory T cell-like immunophenotype.

[0238] Human umbilical cord blood (CB) is an alternative source for allogeneic T cell therapy. Human CB buffy coat cells were activated and transduced with CD4CAR or control (GFP) lentivirus. After transduction, CD4CAR and GFP T cells were expanded for 7 days, resulting in a 20-fold increase in the number of both CD4CAR and GFP T cells. On day 7, the cells were analyzed for T cell subsets by flow cytometry (Figure 1A). Flow cytometry analysis revealed that approximately 54% of T cells expressed CD4CAR (Figure 1B). Furthermore, we analyzed CD4 and CD8 subsets during T cell expansion after CD4CAR transduction. Consistent with previous findings, a small subset of CD8 cells was induced to express CD4 during T cell activation with anti-CD3 and costimulatory molecules (Figure 1C). As expected, the CD4+ T subset was almost completely depleted within 3–4 days after CD4CAR transduction compared to GFP controls, where ~33% of cells remained CD4+ ( Figure 1C ). These data indicate that CD4CAR T cells exhibit potent anti-CD4 activity in vitro during T cell expansion.

[0239] The immunophenotype of CD4CAR T cells was also assessed at the end of each culture. After stimulation, naive T cells lose CD45RA and acquire CD45RO, becoming central memory T cells. Flow cytometry analysis from three representative experiments showed that 96% of expanded T cells were CD45RO+, ~83% were CD62L+, and ~80% were CD8+CD45RO+CD62L+, whereas <4% were CD45RA+ (Figure 1D). The CD8+CD45RO+CD62L+ immunophenotype is consistent with the acquisition of a central memory-like phenotype, while the low expression of CD45RA+ confirms the loss of naive T cell status.

[0240] Cord blood-derived CD4CAR T cells specifically kill CD4-expressing leukemia / lymphomas, including anaplastic large cell lymphoma, Sézary syndrome, and unclassified PTCL lymphoma.

[0241] CD4CAR T cells highly enriched for CD8+ T cells were generated (Figure 1C). These cells were then tested in vitro for their anti-leukemia function using the KARPAS 299 cell line. The KARPAS 299 cell line was originally established from the peripheral blood of a patient with CD4-expressing anaplastic large T-cell lymphoma. Cytogenetic analysis previously showed that KARPAS 299 cells harbor numerous cytogenetic abnormalities. In coculture experiments, CD4CAR cells demonstrated profound leukemia cell killing ability (Figure 2A). First, we tested whether CB-derived CD4CAR T cells could eliminate CD4+ KARPAS 299 cells. Indeed, after 24 hours of culture at a low E:T (effector:target) ratio of 2:1, CD4CAR cells successfully eliminated KARPAS 299 cells. As a control, CD4CAR T cells were also tested for their ability to ablate CD4-negative lymphoma cells. The SP53 mantle cell lymphoma cell line is a human B-cell lymphoma cell line that does not express CD4. Flow cytometry analysis showed that CD4CAR T cells were unable to lyse or eliminate SP53 mantle cell lymphoma (Figure 2D).

[0242] Studies were also performed using patient samples. Patient 1 presented with Sezary syndrome, an aggressive form of CD4+ T-cell leukemia, which failed to respond to standard chemotherapy. Patient 2 presented with CD4+ PTCL lymphoma, not otherwise specified. Flow cytometry analysis of both patient samples revealed strong and uniform CD4 expression, with almost all leukemic cells expressing CD4 (Figure 2B and 2C). As visualized by flow cytometry analysis, coculture of patient samples with CD4CAR T cells for 24 hours resulted in rapid and robust resection of CD4+ malignancies. Approximately 98% resection was observed in both Sezary syndrome and PTCL cocultures, consistent with the previously demonstrated resection with KARPAS (Figure 2B and 2C). Thus, in coculture assays, CD4CAR T cells were shown to efficiently eliminate two different types of aggressive CD4+ lymphoma / leukemia cells directly from patient samples, even at E:T ratios as low as 2:1 (Figure 2B and 2C). These data support CD4 as a promising therapeutic target for CD4-positive T-cell leukemias and lymphomas, similar to the role of CD19 in targeting B-cell malignancies with anti-CD19 CARs. Thus, our patient sample and CD4CAR coculture assays extend the concept of using CARs to target CD4-positive malignancies.

[0243] PBMC-derived CD4CAR T cells specifically kill CD4-expressing tumor cell lines. Because autologous adoptive CAR-T therapy is common in clinical settings, we next tested CD4CAR T cells derived from peripheral blood mononuclear cells (PBMCs). PBMCs were activated and transduced with CD4CAR lentivirus. CD4 and CD8 T cells were monitored by flow cytometry during cell expansion and compared with those of control GFP-transduced cells. As observed with CB-derived CD4CAR T cells, PBMC-derived CD4CAR T cells were highly enriched for CD8+ T cells (Figure 3A), demonstrating the role of CD4CAR in CD4+ depletion. PBMC-derived CD4CAR cells were then tested for their ability to eliminate CD4+ leukemia / lymphoma cells using the KARPAS 299 cell line. For the ablation assay, CD4CAR T cells or GFP T cells were cocultured with KARPAS 299 cells and the negative control SP53 mantle cell lymphoma cell line. The reaction was stopped after 24 hours, dead cells were stained with 7-AAD (7-aminoactinomycin D), and live cells were analyzed by flow cytometry. KARPAS 299 cells incubated overnight with CD4 CAR T cells were eliminated at rates of 38%, 62%, and 85% at E:T ratios of 2:1, 5:1, and 10:1, respectively (Figure 3B). Together, these data demonstrate a strong dose-response relationship. No killing of KARPAS 299 cells was observed when target cells were incubated with GFP control T cells. These results demonstrate that CD4 CAR T cell ablation is specific to CD4+ targeting.

[0244] CD4CAR T cells demonstrated significant antitumor activity in vivo. To evaluate in vivo antitumor activity, we developed a xenogeneic mouse model using the KARPAS 299 cell line. Several different settings were used to validate the efficacy of CD4CAR T cells in vivo. First, we administered a single low dose of CD4CAR T cells to NSG mice and tested their ability to delay the appearance of leukemia. Before injection, the modified T cells contained approximately 40–50% CD4CAR expression, as demonstrated by flow cytometry analysis. Mice received an intradermal injection of KARPAS 299 cells followed by a single systemic injection (intravenous) of a low dose (2 million CD4CAR T cells). Systemic administration of a single low dose of CD4CAR T cells to leukemia-infected mice resulted in transient regression of the leukemia or merely delayed the appearance of leukemic nodules (Figure 4A). Leukemia growth began to accelerate at 8 x 10 6 Additional administration of CD4CAR T cells significantly halted leukemia growth (Figure 4A).

[0245] To further validate the efficacy of CD4CAR anti-leukemia activity, two courses of relatively large amounts of CD4CAR T cells were administered. 6 Two injections of CD4CAR T cells caused a more pronounced leukemia growth arrest compared to a lower dose of CD4CAR, although the leukemia cell population eventually recovered (Figure 4B). Finally, a lower dose of CD4CAR T cells (2.5 × 10 each) 6 We investigated the efficacy of multiple courses of CD4CAR T cell injections. Mice with subcutaneous leukemia were intravenously injected with CD4CAR T cells every 4 or 5 days for a total of four times. After four courses of CD4CAR T cell injection, one of the four mice was tumor-free and no toxicity was observed. Mice receiving multiple CD4CAR T cell injections showed a more pronounced anti-leukemia effect compared with single injections (Figures 4C and 4A). Furthermore, treatment with CD4CAR T cells significantly extended the survival of KARPAS 299 lymphoma-bearing mice compared with treatment with GFP-transduced control T cells (Figure 4D).

[0246] Anti-CD4 chimeric antigen receptor (CD4CAR) NK cells efficiently target T cell malignancies in preclinical models Method Materials Primary tumor cells and cell lines Human leukemia cells were obtained from residual samples under a protocol approved by the Stony Brook University Institutional Review Board. Umbilical cord blood cells were also obtained from donors at Stony Brook University Hospital under a protocol approved by the Institutional Review Board. Written informed consent was obtained from all donors. Karpas 299, HL-60, CCRF-CEM, MOLT4, and NK-92 cell lines were obtained from ATCC (Manassas, VA). NK-92 cells were cultured in ribonucleoside- and deoxyribonucleoside-free α-MEM supplemented with 2 mM L-glutamine, 1.5 g / L sodium bicarbonate, 12.5% ​​heat-inactivated horse serum, 12.5% ​​heat-inactivated FBS, 1X Pen / Strep, 0.2% inositol, 0.02% folic acid, 50 μM β-mercaptoethanol, and IL-2 (300 IU / mL). Karpas 299, CCRF-CEM, and MOLT4 cell lines were cultured in RPMI, 10% FBS, 1x Pen / Strep (Gibco, Waltham, MA, USA). HL-60 cells were cultured in IMDM, 10% FBS, 1x Pen / Strep (Gibco, Waltham, MA, USA).

[0247] Generation of CAR constructs The CD4-specific CAR (pRSC.SFFV.CD4.3G) was engineered to contain an intracellular CD28 domain upstream of the 4-1BB and CD3zeta domains, making it a third-generation CAR.

[0248] Lentivirus production and transduction To produce viral supernatants, 293F T cells were cotransfected with pMD2G and pSPAX viral packaging plasmids containing either pRSC.SFFV.CD4.3G or a GFP lentiviral vector control using Lipofectamine 2000 (Life Technologies, Carlsbad, CA) according to the manufacturer's protocol.

[0249] NK cells were cultured in the presence of 300 IU / mL IL-2 for a minimum of 2 days before transduction with viral supernatant. See Supplemental Data for transfection and transduction procedures.

[0250] Detection of CAR in transduced NK cells To measure CAR expression, NK cells were washed 3 days after transfection and suspended in FACS buffer (0.2% BSA in DPBS). Normal goat IgG (Jackson Immunoresearch, West Grove, PA) was used to block nonspecific binding. Each NK cell sample was probed with biotin-conjugated polyclonal goat anti-mouse F(Ab')2 (1:250, Jackson Immunoresearch, West Grove, PA) for 30 minutes at 4°C. Cells were washed once and resuspended in FACS buffer. Cells were then stained with PE-conjugated streptavidin (1:250, Jackson Immuno Research, West Grove, PA) for 30 minutes at 4°C. Cells were washed with FACS buffer and resuspended in 2% formalin. Flow cytometry was performed using a FACS Calibur instrument (Becton Dickinson, Franklin Lakes, NJ), and results were analyzed using Kaluza software (Beckman Coulter, Brea, CA).

[0251] Co-culture assay CD4CAR or vector control NK cells were incubated at 2:1 and 5:1 ratios with CD4-expressing Karpas 299 cells (anaplastic large T-cell lymphoma), HL-60 cells (acute promyelocytic leukemia), CCRF-CEM cells (T-cell acute lymphoblastic leukemia), CD4+ T cells isolated from human umbilical cord blood, or CD4-expressing primary human leukemia cells (adult Sézary syndrome and pediatric T-ALL) in 1 mL of NK cell culture medium without IL-2 at 2:1 and 5:1 ratios (100,000 target cells to 200,000 and 500,000 effector cells, respectively). After 24 hours of co-culture, the remaining viable cells were harvested and stained with mouse anti-human CD56 and CD4 antibodies and incubated at 4°C for 30 minutes. CD56+ alone indicated NK cells, and CD4+ alone indicated target cells. All cells were washed with FACs buffer, suspended in 2% formalin, and analyzed by flow cytometry.

[0252] Cytotoxicity assay CD4CAR or vector control NK cells were incubated in 1 mL of NK cell culture medium with a 50:50 mixture of on-target cells (CFSE-stained Karpas 299 cells and CMTMR-stained CCRF-CEM cells) and off-target CMTR-labeled MOLT4 cells at effector:target ratios of 1:1, 1:2, or 1:4 without IL-2. After 24 hours, cells were stained with 7-AAD (BioLegend, San Diego, CA), washed with FACS buffer, and viable 7-AAD-negative cells were analyzed by flow cytometry.

[0253] Xenogeneic mouse models Twelve-week-old male NSG mice (NOD.Cg-Prkdcsid Il2rgtm1Wjl / SzJ) were purchased from The Jackson Laboratory (Bar Harbor, ME) and used according to a protocol approved by the Stony Brook University IACUC. NSG mice were irradiated with a sublethal dose (2.5 Gy) of gamma radiation. 24 hours later, 0.5 × 10 stably transduced luciferase-expressing mice were used to develop measurable subcutaneous tumors. 6 Karpas 299 cells were injected intradermally into mice. On day 1, 24 hours after Karpas 299 cell injection, mice were injected with 5 x 10 6 CD4CAR NK cells or vector control NK cells were injected intravenously via the tail vein (N = 4 per group). Intravenous injections were repeated every 5 days for a total of 6 courses. Tumor area was measured every other day. On days 7, 14, and 21 after Karpas 299 cell injection, mice were injected subcutaneously with 100 μL of RediJect D-Luciferin (PerkinElmer, Waltham, MA) and subjected to IVIS imaging (PerkinElmer, Waltham, MA). Images were analyzed using Caliper Life Sciences software (PerkinElmer, Waltham, MA).

[0254] statistics The sample size for the heterogeneous model was estimated using a two-sample, two-sided, equal-power analysis (90% power, significance level less than 5%). The unpaired Student's t-test was used to determine the significance of tumor size area and light intensity. Survival curves were generated using the Kaplan-Meier method, and statistical analysis of survival rates was performed using the log-rank (Mantel-Cox) test, with P < 0.05 considered significant. Statistical analysis was performed using GraphPad Prism 6 software. Variances were considered similar between treatment and control groups before unpaired Student's test.

[0255] result: CD4CAR NK cells specifically lyse CD4-expressing tumor cell lines in a dose-dependent manner. CD4CAR NK cells specifically lysed CD4+ Karpas 299 and CCRF-CEM leukemia cell lines in vitro at effector:target ratios of 1:4, 1:2, and 1:1 in a dose-dependent manner (Figure 5). For each coculture E:T ratio, CD4CAR NK effector cells or vector-control NK effector cells were incubated with equal numbers of on-target CD4+ cells, CFSE-stained Karpas 299 or CFSE-stained CCRF-CEM, and "off-target" CMTMR-stained CD4-, CD5+ MOLT4 acute lymphoblastic leukemia cells. MOLT4 cells were added to account for variability in starting cell numbers and natural target cell death. After 24 hours, viable cells were analyzed by flow cytometry. Target cell lysis rates were measured by comparing CD4+ target cell viability in CD4CAR NK cocultures with that in vector-control NK cocultures. Karpas 299 cells were eliminated at 67%, 95%, and 100% effector-to-target ratios of 1:4, 1:2, and 1:1, respectively (Fig. 5), and CCRF-CEM cells were eliminated at 39%, 58%, and 69%, respectively, at the same E:T ratios (Fig. 5).

[0256] CD4 isolated from umbilical cord blood + Additional co-culture studies were performed with CD4CAR NK cells. In these experiments, CD4CAR NK cells were co-cultured with CD4 T cells at an effector:target ratio of 2:1 after 24 hours of co-culture. + Completely depleted T cells, with 0.0% CD4 + As expected, CD4+ cord blood cells were compared with the corresponding vector control NK cells (CD56 + , CD4-) and + The CD4 population remained largely intact (Figure 6A), whereas the CD4 + Specific and robust CD4CAR NK-mediated depletion of the population was further confirmed.

[0257] CD4CAR NK cells do not affect stem cell production in the hematopoietic compartment.

[0258] Colony-forming unit (CFU) assay analysis revealed that CD4CAR NK cells did not significantly affect CD34+ cord blood stem cell production in the hematopoietic compartment. Hematopoietic compartment output was assessed by the presence of erythroid progenitors and granulocyte / macrophage progenitors on day 0 and determined by the number of erythroid burst-forming units (BFU-E) and granulocyte / monocyte colony-forming units (CFU-GM) on day 16 (Figure 6B). This finding is consistent with the specific targeting of the mature T cell marker CD4, limited effects on hematopoietic stem cells and early progenitor cells, and lack of lineage bias, an indicator of therapeutic safety.

[0259] CD4CAR NK cells exhibit significant antitumor activity in vivo. To evaluate the in vivo antitumor activity of CD4CAR NK cells, we developed a xenogeneic mouse model in which NSG mice were sublethally irradiated and intradermally injected with luciferase-expressing Karpas 299 cells to induce measurable tumor formation. On day 1, 24 hours after Karpas 299 cell injection, mice were injected with 5 x 10 cells per injection, followed every 5 days for a total of 6 courses. 6CD4CAR NK cells or vector-control NK control cells were intravenously injected. On days 7, 14, and 21, mice were subcutaneously injected with RediJect D-Luciferin and underwent IVIS imaging to measure tumor burden (Figure 7A). The mean light intensity of CD4CAR NK-injected mice was compared with that of vector-control NK-injected mice (Figure 7B). By day 21, CD4CAR NK-injected mice had significantly reduced light intensity and tumor burden compared to the vector control group (p < 0.01). Tumor size was measured on day 1 and every other day thereafter, and the mean tumor size between the two groups was compared (Figure 7C). Unpaired student t-test analysis showed that the mean tumor size of CD4CAR NK-injected mice was significantly smaller than that of vector-control NK-injected mice from day 17 (p < 0.05), continuing through days 19–25 (p < 0.01). Next, mouse survival rates were compared between the two groups (Figure 7D). All mice injected with CD4CAR NK survived until day 30. However, survival of mice injected with vector control NK began to decline at day 17 and ceased by day 23. Collectively, these in vivo data demonstrate that CD4CAR NK cells significantly reduce tumor burden and prolong survival in Karpas 299-injected NSG mice.

[0260] Anti-CD5 chimeric antigen receptor (CD5CAR) T cells efficiently target CD5-positive hematologic malignancies Two examples of mechanisms, including natural internalization and knockout, that result in downregulation of antibody-targeted surface proteins in immune cells are elucidated below. CD5 CAR is used as an example. A CD5 anchor was generated to demonstrate internalization by an anti-CD5 antibody on the T cell surface. The DNA construct of the anchored CD5 scFv antibody is identical to the CD5CAR construct, minus the intracellular signaling domain, as is the translated protein product of the CD5 scFv antibody anchored on the T cell surface (Figure 8).

[0261] Generation of third-generation CD5CARs The CD5CAR construct and CD5-anchored scFv antibody were designed to test the function and mechanism of CD5CAR T cells, both in terms of antibody-mediated internalization and lysis of CD5-expressing cells and the ability of CD5CAR T cells to downregulate CD5 expression within their own CD5CAR T cell population (Figure 8A). To validate the CD5CAR construct, we transduced HEK293 cells with the constructed CD5CAR lentivirus. After 48 hours of treatment with CD5CAR or GFP-lentivirus, CD5CAR expression in HEK293 cells was confirmed by Western blot analysis using a CD3zeta antibody that recognizes the C-terminal region of the CD5CAR protein (Figure 8B). The resulting band was of the expected size for CD5CAR protein in CD5CAR-transduced HEK293 cells, whereas GFP-transduced HEK293 cells showed no specific band in Western blot analysis. To assess the function of the CD5CAR protein for future experiments, we transduced the CD5CAR lentivirus into activated human T cells. Expression of CD5CAR on the T cell surface was assessed by flow cytometry analysis using a goat anti-mouse F(ab') antibody that recognizes the scFv region of the CD5CAR protein. Flow cytometry analysis showed that CD5CAR expression was approximately 20% higher in CD5CAR-transduced T cells compared to isotype controls (Figure 8C). These results demonstrated that CD5CAR-expressing T cells were successfully generated for the following experiments. The DNA construct of the anchored CD5 scFv antibody was the same as the CD5CAR construct, excluding the intracellular signaling domain, and the translated protein product of the anchored CD5 scFv antibody was also the same.

[0262] Downregulation of CD5 expression in CAR therapy Prior to co-culture or animal assays with CD5CAR T cells, CD5 expression on the surface of CD5CAR T cells naturally declines to prevent self-killing within the CD5CAR T cell population. CD5 downregulation prevents self-killing within the CAR T cell population, and CD5 downregulation is associated with increased T cell killing capacity. CARs produced in T cells that lack CD5 expression can be hyperfunctional CARs, regardless of the CAR construct itself. The CD5CAR T cell generation procedure and a comparison of CD5 downregulation following single or double transduction of CD5CAR lentiware are shown in Figure 9. CD5CAR T cells single-transduced with unenriched lenti-CD5CAR virus did not show complete downregulation of CD5 protein from the cell surface by day 8, with a maximum CD5-negative population of 46% observed by day 6 (Figure 9). In the double-transfected population, approximately 90% of transfected T cells were CD5-negative by day 4 of culture. In contrast, GFP T cell controls maintained a CD5+, CD3+ double positive population of over 95% from days 2 to 8 (Figure 9).

[0263] CD5CAR T cells efficiently lyse T-cell ALL cell lines.

[0264] As shown in Figures 10A and 10B, the killing ability of CD5CAR T cells was first tested against the established T-cell ALL cell lines CCRF-CEM and MOLT-4, and the anaplastic large cell leukemia cell line KARPAS 299. When compared to the GFP control, phagocytic killing was observed in both CD5+ cell lines, with target cell lysis exceeding 75% in both lines. The CD5-negative anaplastic large cell line KARPAS 299 achieved 0% lysis.

[0265] CD5CAR T cells effectively lyse T-cell ALL cells in human samples. The ability of CD5CAR to lyse T-ALL cells from patient samples was also evaluated using multiple patient samples, and CD5CAR cell cocultures are shown in Figures 11 and 12. Strong cytolysis was observed in T-ALL 1 patient leukemia cells, similar to the CD5 target cytolysis observed when CD5CAR cells targeted T-cell ALL cell lines, whereas target cytolysis was relatively weak in the other three patient leukemia cells (Figures 11A and 11B).

[0266] CD5CAR T cells exhibit specificity and potent target cell killing. As a control, CD5CAR T cells were also tested for their ability to eliminate CD5-negative leukemic T cells (anaplastic large T-cell lymphoma, a cell line that does not express CD5). Flow cytometry analysis showed that CD5CAR T cells were unable to lyse or eliminate KARPAS 299 cells, as shown in the bottom panel of Figure 11A.

[0267] A patient sample (T-ALL-8) with high CD5 expression was obtained from a mildly affected T-ALL patient. Co-culture with CD5CAR T cells was performed and analyzed in detail, as shown in Figure 12. Three cell populations, including CD5+ normal T cells, CD5+CD34+ T-ALL cells, and CD5-CD34+ T-ALL cells, were co-cultured and evaluated by flow cytometry. CD5CAR demonstrated >93% CD5+ cell lysis in all CD5+ cell populations compared with the GFP control, demonstrating specificity and potent target cell lysis. CD5CAR killed leukemia cells as efficiently as CD5 normal T cells. No killing was observed in the CD5-negative population. CD5CAR T cells essentially eliminated the T cell population (CD5+CD34-).

[0268] CD5CAR T cells effectively eliminate normal T cells. In coculture assays, CD5CAR T cells effectively eliminated normal T cells in a dose-dependent manner at effector-to-target ratios as low as 0.25:1, 0.5:1, and 1:1 (Figure 13). CD5CAR T cells or CD123CAR-T (control) effector cells were incubated with GFP-labeled T cells. Target cell killing was measured by comparing the viability of GFP T cells in CD5CAR-T cocultures with that in CD123CAR-T control cocultures. Normal GFP T cells were eliminated in a dose-responsive manner by CD5CAR T cells. CD5CAR T cells effectively eliminated all GFP T cells at an effector-to-target ratio of 1:1 (Figure 13). Because CD5CAR-T cells effectively eliminated all normal T cells, the feasibility of CD5CAR-T therapy must depend on providing a transient, rather than permanent, effect. CD5CAR-T cells have the potential to be used as a novel conditioning regimen or as a "bridge" to hematopoietic cell transplantation.

[0269] T cells maintained CD5 expression when cocultured with CD5CAR or anchored CD5 scFv T cells. Next, transduced CD5CAR or CD5 anchor scFv and CD123CAR T cells were co-cultured with GFP-labeled T cells at a 1:1 (E:T) ratio for 2 or 4 days. As shown in Figures 14A and 14B, CD5CAR T cells effectively eliminated all GFP-T cells. As expected, transduced CD5 anchor scFv or CD123CAR T cells were unable to lyse GFP T cells. Furthermore, GFP T cells expressed CD5 even when co-cultured with transduced CD5 anchor scFv or CD123CAR T cells. These studies demonstrated that CD5 antigen escape is unlikely when CD5CAR is used for immunotherapy.

[0270] Transduction of T-ALL cells with lenti-CD5CAR or CD5-anchored scFv virus reduced CD5 expression. Next, we examined whether transfection of T-ALL cells with CD5CAR or anchored CD5CAR lentivirus downregulated CD5 expression. CCRF-CEM and MOLT-4 T-ALL cells were transfected with CD5CAR or anchored CD5 scFv lentivirus. CD5CAR or anchored CD5 scFv significantly downregulated or reduced surface CD5 expression on these leukemia cells (Figure 14C).

[0271] CD5CAR T cells exhibit potent antitumor activity in vivo. To evaluate the in vivo antitumor activity of CD5CAR T cells as a predictor of therapeutic efficacy in patients, NSG mice were irradiated sublethally (2.0 Gy) and 1.0x10 6 We developed a xenograft mouse model in which firefly luciferase-expressing CCRF-CEM cells (CD5+) were intravenously injected to induce measurable tumor formation. Three days after CCRF-CEM-Luc+ cell injection, mice were treated with 5 x 10 6 CD5CAR T cells or vector control T cells were injected intravenously. These injections were repeated on days 4, 6, and 7, for a total of 20 x 10 T cells per mouse. On days 5, 8, 10, and 13, mice were subcutaneously injected with RediJect D-Luciferin (Perkin-Elmer) and IVIS imaging (Caliper LifeSciences) was performed to measure tumor burden (Figure 15). Paired t-test analysis revealed a highly significant difference between the two groups by day 13. The CD5CAR-T-injected group had lower light intensity and therefore lower tumor burden compared to the control group (p <0.0012).

[0272] Anti-CD5 chimeric antigen receptor (CD5CAR) NK cells efficiently eliminate CD5-positive hematologic malignancies. Generation of CD5NK-CAR This anti-CD5 molecule is a third-generation CAR with a modular design consisting of a single-chain variable fragment (scFv) with CD28 and 4-1BB domains fused to the CD3zeta signaling domain for improved signal transduction. To efficiently express the CD5CAR molecule on the surface of NK cells, a strong spleen focus-forming virus (SFFV) promoter and a CD8 leader sequence were incorporated into the construct. The anti-CD5 scFv is linked to the intracellular signaling domain via the hinge (H) and transmembrane (TM) regions derived from CD8. The CD5CAR construct was cloned into a lentiviral plasmid.

[0273] Generation of CD5CAR NK cells The CD5CAR transduction efficiency was determined by flow cytometry analysis. To enrich for CD5CAR+ NK cells, high-expressing NK cells were collected using flow cytometry. After sorting, CD5CAR-high NK cells were expanded for in vitro and in vivo efficacy studies.

[0274] CD5CAR NK cells effectively eliminate human T-cell acute lymphoblastic leukemia (T-ALL) cell lines. The anti-T-ALL activity of CD5CAR NK cells was tested in vitro using CCRF-CEM, MOLT-4, and Jurkat cell lines, all of which highly express CD5.

[0275] In coculture experiments, CD5CAR NK cells profoundly killed CCRF-CEM cells at effector to target cell (E:T) ratios as low as 2:1 and 5:1. At these ratios, CD5CAR NK cells virtually eliminated CCRF-CEM cells (Figure 16A). In vitro, CD5CAR NK cells lysed CCRF-CEM leukemia cells in a dose-dependent manner at effector-to-target cell ratios of 0.25:1, 0.5:1, 1:1, 2:1, and 5:1 (Figure 16B).

[0276] CD5CAR NK cells exhibit potent anti-leukemic activity in vivo. Using a similar strategy to CD5CAR T cells, animal studies were conducted to investigate the in vivo antitumor activity of CD5CAR NK cells. Sublethally irradiated NSG mice were treated with 1.0 x 10 NK cells to induce measurable tumor formation. 6 Firefly luciferase-expressing CCRF-CEM cells were injected intravenously. Three days after CCRF-CEM-Luc+ cell injection, mice were treated with 5 x 10 6 CD5CAR NK cells or vector control T cells were injected intravenously. These injections were repeated on day 4 for a total of 10 x 10 per mouse. 6 T cells were injected into the treated NSG mice. On day 5, mice were subcutaneously injected with RediJect D-Luciferin and tumor burden was measured by IVIS imaging (Figure 17A). The mean light intensity of CD5CAR NK cell-injected mice was compared to that of vector control NK cell-injected mice (Figure 17B). Five days after tumor injection, tumor burden in the treatment group was reduced by two-thirds. A paired T-test revealed a highly significant difference (P=0.0302) between the two groups. These in vivo data demonstrate that CD5CAR NK cells rapidly and significantly reduce tumor burden compared to vector control NK cells in NSG mice injected with CCRF-CEM.

[0277] Anti-CD3 chimeric antigen receptor (CD3CAR) NK cells efficiently lyse CD3-positive hematologic malignancies Generation of CD3CAR This anti-CD3 molecule is a third-generation CAR with a modular design consisting of a single-chain variable fragment (scFv) with CD28 and 4-1BB domains fused to the CD3zeta signaling domain for improved signal transduction. To efficiently express the CD3CAR molecule on the surface of NK cells (NK-92), we used the strong spleen focus-forming virus (SFFV) promoter and incorporated a CD8 leader sequence into the construct. The anti-CD3 scFv is linked to the intracellular signaling domain via the hinge (H) and transmembrane (TM) regions derived from CD8 (Figure 18A). This CD3CAR construct was cloned into a lentiviral plasmid.

[0278] CD3CAR characteristics Western blot analysis was performed on HEK293-F T cells transfected with the CD3CAR lentiviral plasmid and vector control plasmid. Immunoblots with anti-CD3zeta monoclonal antibody showed a band of the expected size for the CD3CAR-CD3zeta fusion protein, but no band for the vector control protein (Figure 18B).

[0279] Generation of CD3CAR NK cells using NK-92 cells The transduction efficiency of CD3CAR was determined by flow cytometry analysis. To enrich for CD3CAR NK cells, high-expressing NK cells were collected using fluorescence-activated cell sorting (FACS). Sorting yielded NK cells with relatively high CD3CAR expression. After flow cytometry selection, CD3CAR expression remained stable at approximately 30% of CAR expression during subsequent NK cell expansion and cryopreservation.

[0280] CD3CAR NK cells effectively eliminate CD3+ leukemia cells from human samples. The killing ability of CD3CAR NK cells was also tested using patient samples. Flow cytometry analysis of both patient samples confirmed strong and uniform CD3 expression. Co-culture of Sézary syndrome patient samples with CD3CAR T cells effectively lysed approximately 80% of leukemic cells at a low E:T ratio of 2:1, as analyzed by flow cytometry (Figure 19A). Co-culture of unsorted PTCL from a patient sample with CD3CAR NK cells for 24 hours resulted in virtual elimination of CD3+ malignant cells (Figure 19B). CD3CAR NK cells also affected a broad range of CD3+ populations.

[0281] CD3CAR NK cells exhibit potent anti-leukemic activity in vivo To determine the in vivo antitumor efficacy of CD3CAR NK cells, sublethally irradiated NSG mice were treated with 1.0 x 10 CD3-positive (approximately 80%) firefly luciferase-expressing Jurkat cells. 6 were injected intravenously, and measurable tumor formation was detected by day 3 or 4. Three days after Jurkat-Luc+ cell injection, mice were injected with 5 x 10 CD3CAR NK cells or vector control NK cells per mouse. 6 These injections were repeated on days 3, 6, 7, and 10, for a total of 25 x 10 per mouse. 6 T cells were injected. Mice were subjected to IVIS imaging to measure tumor burden on days 4, 7, 9, and 13 (Figure 20A). Two treated mice died from the injection treatment on day 13. A paired T-test analysis revealed a highly significant difference (P=0.0137) between the two groups. We concluded that these in vivo data demonstrate that CD3CAR NK cells significantly reduce tumor burden and prolong survival in Jurkat-injected NSG mice compared to vector control NK cells.

[0282] CRISPR / Cas nucleases target CD2, CD3, CD5, and CD7, which are expressed on T cells and NK cells. T cells and NK cells appear to share surface antigens, such as CD2, CD3, CD5, and CD7, with leukemias and lymphomas. CD2, CD3, CD5, and CD7 are expressed on most T-cell leukemias / lymphomas, making them good targets for T cells and NK cells.

[0283] Therefore, if one of the surface antigens CD2, CD3, CD5, or CD7 is chosen as a target, and the T cells or NK cells used to generate the CAR share this antigen, it will need to be deleted or downregulated to avoid self-killing within the CAR T cell or NK cell population.

[0284] Three pairs of sgRNAs targeting CD2, CD3, CD5, and CD7 were designed using CHOPCHOP. The gene-specific sgRNAs (Figure 21) were then cloned into a lentiviral vector (Lenti U6-sgRNA-SFFV-Cas9-puro-wpre) expressing human Cas9 and a puromycin resistance gene linked by an E2A self-cleaving linker. The U6-sgRNA cassette precedes the Cas9 element. Expression of the sgRNA and Cas9puro is driven by the U6 promoter and the SFFV promoter, respectively.

[0285] CRISPR / Cas nucleases target CD5 on T cell lines. CCRF-CEM and MOLT cells were transduced with lentivirus carrying gene-specific sgRNAs. Initially, loss of CD5 expression was observed in both of these T cell lines using two different CDISPR / Cas9 sgRNA sequences (Figures 22A and 22C). For each cell line, the most successful population in terms of loss of CD5 expression was selected. These cells were then sorted and expanded normally, revealing greater than 99% purity of CD45+ and CD5- (Figures 22B and 22D).

[0286] CRISPR / Cas nucleases target CD7 on T cell lines and NK cells. CCRF-CEM, MOLT, and NK cells were transduced with lentiviruses carrying gene-specific sgRNAs (Figure 23). Flow cytometry analysis demonstrated the loss of CD7 expression in CCRF-CEM and NK-92 cells using the CRISPR / Cas9 approach with two different sgRNAs (Figures 23A and 23B). This population (indicated by the circle and arrow) was selected for sorting, expansion, and analysis in Figure 23B. Flow cytometry analysis also demonstrated the loss of CD5 expression in NK-92 cells using a similar approach described above using CD7-targeting CRISPR / Cas nucleases (Figures 23C and 23D). Sorted CD7-negative NK-92 cells (Figure 23D) were used to generate CD7CAR NK cells to eliminate CD7-positive leukemia cells.

[0287] CD7CAR NK-92 cells have potent anti-leukemia activity CD7 is expressed on both NK and T-ALL leukemia cells. To avoid self-killing within the CD7CAR NK-92 population, CD7 expression must first be inactivated. CD7-deficient NK-92 cells (NK7- -92 cells) were generated and expanded as described in (Figure 23D). The expanded NK-92 cells were transduced with a lentivirus expressing CD7CAR. CD7CAR is a third-generation CAR that contains CD28 and 4-BB domains fused to the CD3zeta signaling domain and an anti-CD7 scFV. We tested the ability of CD7CAR NK-92 cells to lyse CD7-expressing leukemia cells. As shown in Figure 25, CD7CAR NK7- -92 cells exhibited potent anti-leukemia activity against the T-ALL cell line CCRF-CEM. Flow cytometry analysis revealed that coculture with CCRF-CEM cells effectively lysed approximately 50% of leukemia cells at an E:T ratio of 5:1 (Figures 25A and 25B).

[0288] CD3CAR is used for graft-versus-host disease (GvHD). The CD3CAR is administered to patients before or after stem cell transplantation, and patients are tested for elevated white blood cell levels.

[0289] CD3CAR is administered to patients before or after a bone marrow transplant, and patients are tested for elevated white blood cell levels.

[0290] The CD3CAR is administered to patients before or after the tissue transplant, and the patients are tested for elevated white blood cell levels.

[0291] organ transplantation CD3CAR is administered to organ transplant patients before transplant surgery. Patients are screened for organ rejection. The following histologic signs are assessed: (1) infiltrating T cells, possibly with infiltrating eosinophils, plasma cells, and neutrophils, particularly in significant proportions; (2) structural damage to tissue anatomy, depending on the type of transplanted tissue; and (3) vascular damage.

[0292] CD3CAR is administered to organ transplant patients after transplant surgery. Patients are screened for organ rejection. The following histological signs are assessed: (1) infiltrating T cells, possibly with infiltrating eosinophils, plasma cells, and neutrophils, particularly in significant proportions; (2) structural damage to tissue anatomy, depending on the type of transplanted tissue; and (3) vascular damage.

[0293] Treating T-cell malignancies with CD2CAR Generation of CD2CAR constructs We engineered a CD2CAR, which consists of an anti-CD2 single-chain variable fragment (scFv) region, CD8-derived hinge (H) and transmembrane (TM) regions, and tandem CD28 and 4-1BB coactivation domains linked to a CD3ζ signaling domain, using the strong spleen focus-forming virus (SFFV) promoter and CD8 leader sequence.

[0294] CD2CAR NK -92 NK cells Because NK-92 cells express the CD2 antigen in low numbers, we investigated whether the CD2CAR of NK-92 cells reacts with the CD2 antigen on leukemia cells. NK-92 cells were transfected with a lentivirus expressing CD2CAR, and the resulting CD2CAR NK-92 cells were used to test their anti-leukemia activity.

[0295] CD2CAR NK-92 NK cells specifically lyse CD2+ T-ALL (T-acute lymphoblastic leukemia) cells. To evaluate the anti-leukemic activity of CD2CAR NK92, we performed coculture assays using the T-ALL cell line CCRF-CEM and human primary T-ALL patient samples. The results demonstrated that CD2CAR NK-92 cells consistently potently lyse leukemic cells. After 24 hours of culture at a low effective target cell ratio (E:T ratio 5:1), CD2CAR NK-92 cells effectively lysed over 60% of CCRF-CEM and human primary T-ALL samples (PT1) (Figures 24A-24C).

[0296] Generation of CD7 CAR T cells targeting CD7-expressing cells Structure of CD7 CAR (also called CD7-RTX CAR). CD7 CAR contains an anti-CD7 scFv, a CD8 hinge region and transmembrane region, and a CD28 costimulatory domain fused to a CD3zeta signaling domain. The hinge region of CD7CAR also contains two RTX-binding epitopes.

[0297] We first characterized the CD7-RTX CAR. After transduction, flow cytometry analysis confirmed the expression of the CAR product, the availability of rituximab binding sites, and CD7 downregulation in transduced cells (Figure 27). Because loss of CD7 may reduce the proliferation capacity of CD7CAR T cells, we compared the proliferation of these cells with control cells. They proliferated at similar rates (Figure 27), indicating that CD7 is not required for T cell proliferation (Figure 27D). Characterization of the CD7CAR and staining with goat anti-mouse F(Ab')2-Pe revealed approximately 70% CAR expression at day 8 postinfection. Staining with anti-human CD34 (used to detect the RTX-binding epitope) also demonstrated approximately 80% transduction efficiency (Figures 26B-27B). Staining with anti-human CD3 and anti-human CD7 revealed that CD7CAR T cells maintained CD3 expression but lost CD7 expression (Figure 27C). CD7CAR T cells are able to proliferate at a similar rate to control T cells despite losing CD7 expression (Figure 27D).

[0298] Next, human peripheral blood-derived second-generation CD7CAR T cells expressing CD28 and CD3zeta signaling sites were co-cultured in vitro with CCRF-CEM, Jurkat, and MOLT-4 cell lines, which consist of CD7+ T-ALL cells. Significant elimination of leukemia cells was observed within 18 hours of culture (Figure 28). CD7CAR exhibits potent cytotoxicity against CD7+ cell lines in vitro. CEM-CCRF cells are approximately 90% CD7+ (lower right panel). Control T cells (left panel) or CD7-RTX T cells (right panel) were co-cultured with CEM-CCRF cells at an E:T ratio of 1:1 (first row) or 2:1 (second row) for 18 hours. Target CD7+ cells are circled in each panel. Co-culture experiments showed that CD7CAR T cells lysed 99.88% and 99.82% of CEM-CCRF cells at a ratio of 1:1 and 2:1 relative to the control, respectively (Figure 28).

[0299] To evaluate in vivo antitumor activity, CCRF-CEM leukemia cells were introduced into xenogeneic NSG mice. Prior to injection, 12 mice were subjected to sublethal gamma irradiation (2.0 Gy) and randomly assigned to treatment and control groups. 24 hours later, mice were injected with 1.0x10 6 CCRF-CEM cells were injected intravenously.

[0300] Five days after engraftment of CCRF-CEM, mice were treated with 10x10 6 CD7CAR or control T cells were intravenously injected. On days 5, 10, 13, 16, and 19, RediJect D-Luciferin (Perkin-Elmer) was injected intraperitoneally to assess tumor burden, and IVIS imaging was performed to quantify luciferase activity. Control mice showed a continuous increase in flux, and thus tumor burden, whereas CD7CAR mice remained at near background levels (Figure 29). While control mice showed significant tumor mass in the peripheral blood, CD7CAR-treated mice showed a substantial reduction comparable to untreated mice (Figure 29). Furthermore, CD7CAR-treated mice showed significantly improved survival (Figure 29). CD7CAR improved prognosis in an in vivo model of T-ALL. NSG mice were irradiated subcutaneously and 1.0x10 RT-PCR was administered on day 1. 6 10x10 luciferase-expressing CEM-CCRF cells were injected intravenously. Five days later, mice were 6Mice were injected with control or CD7CAR T cells. Mice were injected with RediJect D-Luciferin on days 5, 10, 13, 16, and 19 and imaged via IVIS. Dorsal view (Figures 28A-29A). Total photon flux (photons / second) was measured, revealing a statistically significant difference in tumor burden between the two groups as early as day 8. The photon flux in CD7CAR-treated mice was reduced by 41.3% (dorsal) by day 8, and the reduction increased to 99.6% (dorsal) by day 19 (Figures 28C-29C). All control mice required euthanasia due to hindlimb paralysis and hunchback by days 24-26, whereas CD7CAR-treated mice survived significantly longer, surviving until day 43 (Figures 28D-29D). Kaplan-Meier survival analysis (p = 0.0026).

[0301] T cells generated by this CD7 CAR were used to treat T-ALL patients, with unexpected results.

[0302] background: A 31-year-old man presented with left neck swelling, a white blood cell count of 352.27 × 109 / L, and ETV6 mutations (40.8%), NOTCH1 mutations (41.8%), and NRAS mutations (44.6%), with negative chromosomal fusion gene detection. Diagnosed with high-risk T-ALL, the patient initiated a multiline chemotherapy regimen and subsequently underwent a sibling hematopoietic stem cell transplant (human stem cell transplant). Although disease remission was achieved with multiple chemotherapy regimens, including bone marrow transplantation, the patient relapsed on day 83. The patient was enrolled to receive CD7 CAR T-cell therapy.

[0303] This patient's relapsed T-ALL was CD7 surface antigen-positive, making him a candidate for CD7 CAR T-cell therapy (Figure 30). Donor lymphocytes were collected from the original donor who provided the bone marrow stem cells and were collected 6 days before CAR T therapy. Baseline parameters before CAR-T therapy were as follows: bone marrow blasts 24%, cerebrospinal fluid (CSF) 1.77%, and chimerism 68.36%. Fludarabine and cytarabine chemotherapy was administered 4 days before CD7 CAR T-cell therapy. Anti-CD7 CAR T cells were administered twice: 1 x 106 / kg CAR T cells in the first dose (day 0) and 2 x 106 / kg CAR T cells were administered. By day 7, the patient's peripheral blood was negative for CD7+ T-ALL cells. Furthermore, by day 11, CD7-positive T-ALL cells in the peripheral blood and bone marrow were completely reduced by CAR-T cell therapy, and the patient's peripheral blood and bone marrow chimerism rates reached 100%. As a result of CD7 CAR T-cell therapy, this patient experienced rapid leukemia resolution, MRD negativity, and complete remission. Interestingly, CD7-positive T cells were virtually completely lost by day 11 after CAR (Figure 31). CD7CAR T-cell therapy completely depleted CD7+ T cells, and the remaining small percentage of CD7-negative T cells were able to replenish the lost T-cell population to normal levels.

[0304] One month after CAR treatment (Figure 32): The patient's CBC was 7.51 x 10 9 / L, NEU 4.47×10 9 / L, LYM 2.63 × 10 9 / L, HGB 76g / L, PTL 37×10 9 The patient's WBC and LYM counts were within normal ranges. Lymphocyte subtypes were as follows: total T 98.35%, absolute CD3+CD4+ 156 / μL, CD3+CD8+ 1026 / μL, and total T cells 1182 / μL (total T cells were within normal ranges). Chimerism was as follows: total T 99.79%, T cells 99.94%, B cells 99.4%, and NK cells 95.75%. In summary, at follow-up 1 month after CD7 CAR T-cell therapy, high levels of CD7 CARs (more than 70% detected) and all T cells lacked the surface protein CD7. CD7 CAR T-cell therapy depleted all leukemia cells and all T cells expressing the CD7 surface protein. This treatment was effective and safe, and the patient continued treatment with grade 1 cytokine release syndrome (CRS).

[0305] In one exemplary embodiment of the present invention, we discovered that T cells expressing a CD7CAR are effective in treating human patients suffering from CD7-expressing T-cell acute lymphoblastic leukemia (T-ALL). Approximately 98% of the human T cell population is known to be CD7-positive. Therefore, it was thought that administration of a CD7CAR would result in human T cell depletion and death. We unexpectedly discovered that approximately 2% of T cells negative for the CD7 surface antigen rapidly proliferate within a short period of time, replacing the eliminated CD7-positive T cell population to a relatively normal range. As a result, humans administered a CD7CAR tolerate the treatment. At the time of filing, it was believed that the CD7 antigen plays a critical role in the T cell-based killing mechanism, and that elimination of CD7-positive T cells would be fatal to patients. Therefore, it was counter-intuitive to generate CD7CAR T cells according to the claimed invention. Surprisingly, we discovered that despite the absence of the CD7 surface protein, the CAR T cell surface protein remains potent in its killing effect, elucidating this unexpected finding. In this particular embodiment, CD7CAR T cells successfully treated a patient with a high-burden disease, T-cell acute lymphoblastic leukemia. In summary, prior to the advent of state-of-the-art technology and teachings, those skilled in the art would not have created CD7CARs because targeting the CD7 antigen was taught to result in overall T cell depletion or depletion, which could be associated with target toxicity in severe infections and would be incompatible with the patient's life. Approximately 95% of the human T cell population is known to be CD7-positive. We unexpectedly discovered that approximately 2-5% of T cells negative for the CD7 surface antigen rapidly proliferate within a short period of time, replacing the eliminated CD7-positive T cell population to a relatively normal range. As a result, humans administered CD7CARs tolerate the treatment well.

[0306] The following were surprising observations: 1) CD7CAR can be used to treat patients with relapsed / refractory T-ALL, with remarkable therapeutic outcomes; 2) CD7 CAR T cell therapy completely depletes CD7+ T cells, and a small percentage of remaining CD7-negative T cells replenishes the lost T cell population to normal levels; and 3) CD7 CAR T cells that do not express CD7 on their surface in patients exhibit a remarkable effect of depleting CD7+ leukemia cells, while T cell levels in the peripheral blood remain nearly normal.

[0307] In one embodiment, CD7 CARs targeting the CD7 surface antigen can deplete autoreactive immune cells expressing the CD7 surface antigen. The unexpected finding was revealed that the CD7+ population is depleted (approximately 90 or more T lymphocytes), while the CD7- population of T lymphocytes expands to maintain the total T cell population and prevent infection. This phenomenon acts as an immune system reset for the T cell immune system, treating T cell-mediated autoimmune diseases.

[0308] In one embodiment, an engineered cell bearing a CD7-RTX CAR contains a CD7 chimeric antigen receptor polypeptide and two CD20-binding epitopes (also called RTX-binding epitopes) in the hinge region (SEQ ID NO. 1), as well as the corresponding nucleotides (SEQ ID NO. 2). The CD7-RTX CAR can be used to deplete T cells associated with autoimmune diseases and CD7-expressing leukemia / lymphoma, particularly T-acute lymphoblastic leukemia / lymphoma or T-cell lymphoma or acute myeloid leukemia.

[0309] In one embodiment, engineered cells bearing CD7-RTX VAC CARs (also referred to as CD7-RTX-IL-15 / IL15sushi CARs) contain a CD7 chimeric antigen receptor polypeptide, two CD20-binding epitopes (also referred to as RTX-binding epitopes) in the hinge region, secreting IL-15 / IL15sushi (SEQ ID NO. 3), and the corresponding nucleotide sequence (SEQ ID NO. 4). CD7-RTX VAC CARs can be used to deplete T cells associated with autoimmune diseases and CD7-expressing leukemia / lymphomas, particularly T-acute lymphoblastic leukemia / lymphoma or T-cell lymphoma or acute myeloid leukemia.

[0310] In one embodiment, the engineered cells bearing the CD7-RTX-CD19 cCAR comprise a CD7 chimeric antigen receptor peptide with two CD20 epitopes in the hinge region, a CD19 chimeric antigen receptor polypeptide (SEQ ID NO. 5), and the corresponding nucleotide (SEQ ID NO. 6). The CD7-RTX-CD19 cCAR can be used to deplete T cells or B cells associated with patients with autoimmune diseases and CD7- or CD19-expressing leukemia / lymphoma, particularly acute lymphoblastic leukemia / lymphoma, or T-cell or B-cell lymphoma, or acute myeloid leukemia.

[0311] In one embodiment, artificial cells harboring a CD7-RTX-CD19 VAC cCAR comprise a CD7 chimeric antigen receptor peptide with two CD20 epitopes in the hinge region, a CD19 chimeric antigen receptor polypeptide that secretes IL-15 / IL-15sushi (SEQ ID NO. 7), and corresponding nucleotides (SEQ ID NO. 8). In one embodiment, artificial cells harboring a CD7-RTX-CD19 cCAR comprise a CD7 chimeric antigen receptor peptide with two CD20 epitopes in the hinge region, a CD20 chimeric antigen receptor polypeptide (SEQ ID NO. 9), and corresponding nucleotides (SEQ ID NO. 10).

[0312] In one embodiment, the engineered cells harboring the CD7-RTX-CD20 VAC cCAR contain a CD7 chimeric antigen receptor peptide with two CD20 epitopes in the hinge region, a CD20 chimeric antigen receptor polypeptide secreting IL-15 / IL-15sushi (SEQ ID NO. 11), and the corresponding nucleotide sequence (SEQ ID NO. 12).

[0313] In one embodiment, the engineered cells harboring the CD7-RTX-CD33 VAC cCAR contain a CD7 chimeric antigen receptor peptide with two CD20 epitopes in the hinge region, a CD33 chimeric antigen receptor polypeptide secreting IL-15 / IL-15sushi (SEQ ID NO. 13), and the corresponding nucleotide sequence (SEQ ID NO. 14).

[0314] In one embodiment, the engineered cells harboring the CD7-RTX-CLL1 VAC cCAR comprise a CD7 chimeric antigen receptor peptide with two CD20 epitopes in the hinge region, a CLL1 chimeric antigen receptor polypeptide secreting IL-15 / IL-15sushi (SEQ ID NO. 15), and the corresponding nucleotide sequence (SEQ ID NO. 16).

[0315] In one embodiment, an artificial cell harboring a CD7-RTX-CS1 cCAR comprises a CD7 chimeric antigen receptor peptide with two CD20 epitopes in the hinge region, a CS1 chimeric antigen receptor polypeptide (SEQ ID NO. 17), and corresponding nucleotides (SEQ ID NO. 18).

[0316] In one embodiment, an engineered cell bearing a CD7-RTX-BCMA cCAR comprises a CD7 chimeric antigen receptor peptide with two CD20 epitopes in the hinge region, a BCMA chimeric antigen receptor polypeptide (SEQ ID NO. 19), and corresponding nucleotides (SEQ ID NO. 20).

[0317] In one embodiment, the engineered cells bearing the CD7-RTX-BCMA VAC cCAR contain a CD7 chimeric antigen receptor peptide with two CD20 epitopes in the hinge region, a BCMA chimeric antigen receptor polypeptide that secretes IL-15 / IL-15sushi (SEQ ID NO. 21), and the corresponding nucleotide sequence (SEQ ID NO. 22).

[0318] In one embodiment, an artificial cell harboring a CD7-RTX-CD38cCAR (CD7-RTX-CD38a cCAR) comprises a CD7 chimeric antigen receptor peptide with two CD20 epitopes in the hinge region, a CD38 chimeric antigen receptor polypeptide (SEQ ID NO. 23), and the corresponding nucleotide (SEQ ID NO. 24).

[0319] In one embodiment, engineered cells harboring pX-BCMA-CD19-IL15 / IL15sushi, which comprises a chimeric antigen receptor peptide, a CD19-chimeric antigen receptor polypeptide, a secreted IL-15 / IL15sushi fusion (SEQ ID NOS: 25 and 47), and the corresponding nucleotides (SEQ ID NOS: 26 and 48). pX-BCMA-CD19-IL15 / IL15sushi can be used to reduce or deplete plasma cells and / or B cells in patients with autoimmune diseases or leukemias that express BCMA and / or B cells.

[0320] In one embodiment, the pX-BC4-CD7 RTX -15R cCAR comprises a chimeric antigen receptor peptide, a BCMA chimeric antigen receptor polypeptide, a CD19 chimeric antigen receptor polypeptide, and a secreted IL-15 / IL15sushi fusion (SEQ ID NO. 27) and corresponding nucleotide sequence (SEQ ID NO. 28). It can be used to deplete or reduce plasma cells and / or T cells associated with autoimmune disease.

[0321] In one embodiment, engineered cells harboring pX-BC4-CD19c-Vac (also referred to as pX-BC4-CD19c-IL-15 / IL15sushi) comprise a chimeric antigen receptor peptide, a BCMA-chimeric antigen receptor polypeptide, a CD19-chimeric antigen receptor polypeptide, and a secreted IL-15 / IL15sushi fusion ((SEQ ID NO. 29) and corresponding nucleotide (SEQ ID NO. 30)). The pX-BC4-CD19c-Vac CAR can be used to deplete or reduce plasma cells and / or B cells in patients with autoimmune disease or leukemia that express BCMA and / or B cells.

[0322] In one embodiment, engineered cells harboring the CLL1-CD33b-IL15 / IL15sushi cCAR contain a chimeric antigen receptor peptide, a CLL1 chimeric antigen receptor polypeptide, a CD33 chimeric antigen receptor polypeptide, and a secreted IL-15 / IL15sushi fusion (SEQ ID NO. 31) and corresponding nucleotide sequence (SEQ ID NO. 32). The CLL1-CD33b-IL15 / IL15sushi cCAR can be used to treat patients with CLL1- or CD33-expressing acute myeloid leukemia, chronic leukemia, and myelodysplastic syndromes and to prevent tumor antigen escape.

[0323] In one embodiment, artificial cells bearing pX-CD7RTX-CD19c-15R can be used to eliminate T cells and / or B cells associated with autoimmune disease and reset the immune system to all B and T cells.

[0324] In one embodiment, the artificial cell carrying the pX-BC4-CD7 RTX-tandem CAR comprises a CD7 scFv (BCMA-CD7 tandem construct) linked to a BCMA VVH via a linker, a hinge region, a transmembrane domain, a costimulatory domain, and a CD3 signaling domain. The costimulatory domain includes, but is not limited to, CD28 and 4-1BB. The pX-BC4-CD7 RTX-tandem CAR sequence (SEQ ID NO. 35) and corresponding nucleotide sequence (SEQ ID NO. 36) are shown. The pX-BC4-CD7Q-tandem CAR can be used to delete plasma cells and / or T cells associated with autoimmune diseases or lymphoma / leukemia that express either the BCMA and / or CD7 antigens.

[0325] In one embodiment, the artificial cell containing the pX-BC4-CD19-tandem CAR contains a CD7 scFv linked to a BCMA VVH via a linker (BCMA-CD19 tandem construct), a hinge region, a transmembrane domain, a costimulatory domain, and a CD3 signaling domain. The pX-BC4-CD19-tandem CAR has SEQ ID NOs. 37, 39, and 41 and the corresponding nucleotides (SEQ ID NOs. 38, 40, and 42). The pX-BC4-CD19-tandem CAR can be used to eliminate plasma cells and / or B cells associated with autoimmune diseases or to treat lymphoma / leukemia patients who express either the BCMA and / or CD19 antigens.

[0326] In one embodiment, the artificial cell carrying the pX-CD19-CD7 tandem CAR comprises a CD19 scFv linked to a CD7 scFv via a linker (CD19-CD7 tandem construct), a hinge region, a transmembrane domain, a costimulatory domain, and a CD3 signaling domain. The pX-CD19-CD7 tandem CAR comprises SEQ ID NOs. 43 and 45 and the corresponding nucleotides (SEQ ID NOs. 44 and 46). The pX-CD19-CD7 tandem CAR can be used to eliminate B cells and / or T cells associated with autoimmune diseases or lymphoma / leukemia that express either B or T antigens.

[0327] In one embodiment, cells engineered with pX-BC4-RTX Vac CAR (also referred to as pX-BC4-RTX-IL-15 / IL15sushi CAR) contain a chimeric antigen receptor peptide, a BCMA chimeric antigen receptor polypeptide, and a secreted IL-15 / IL15sushi fusion ((SEQ ID NOs. 49, 51) and corresponding nucleotides (SEQ ID NOs. 50 and 52). The pX-BC4-RTX Vac CAR can be used to deplete or reduce plasma cells associated with BCMA-expressing autoimmune diseases and myeloma.

[0328] In one embodiment, cells engineered with pX-BC5-RTX Vac CAR (also referred to as pX-BC4-RTX-IL-15 / IL15sushi CAR) contain a chimeric antigen receptor peptide, a BCMA chimeric antigen receptor polypeptide, and a secreted IL-15 / IL15sushi fusion (SEQ ID NOs. 53, 55) and corresponding nucleotides (SEQ ID NOs. 54 and 56). The pX-BC5-RTX Vac CAR can be used to deplete plasma cells associated with BCMA-expressing autoimmune diseases and myeloma.

[0329] In one embodiment, the engineered cells bearing the pX-BC4-RTX CAR comprise a chimeric antigen receptor peptide, the BCMA chimeric antigen receptor polypeptide (SEQ ID NOs. 57, 59) and corresponding nucleotides (SEQ ID NOs. 58, 60). The pX-BC4-RTX CAR can be used to deplete or reduce plasma cells associated with BCMA-expressing autoimmune diseases or myeloma.

[0330] In one embodiment, the engineered cells bearing the pX-BC5-RTX CAR contain the chimeric antigen receptor peptide, BCMA chimeric antigen receptor polypeptide (SEQ ID NOs. 61, 63) and corresponding nucleotides (SEQ ID NOs. 62, 64). The pX-BC5-RTX CAR can be used to deplete or reduce plasma cells associated with BCMA-expressing autoimmune diseases and myeloma.

[0331] In one embodiment, the specific chimeric antigen receptor (CAR) comprises a BCMA antigen-binding domain comprising the amino acid sequence of SEQ. ID NO: 77 or SEQ. ID NO: 78, a hinge domain, a transmembrane domain, at least one costimulatory domain, and a CD3 zeta signaling domain. In a further embodiment, in the BCMA-specific chimeric antigen receptor, said antigen-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 51, 53, 55, 57, 59, 61, and 63.

[0332] In one embodiment, the BCMA-specific chimeric antigen receptor consists of an antigen-binding domain amino acid sequence that has at least 75%, preferably 80%, 85%, 90%, 95%, the amino acid sequence of SEQ ID NOs: 77 and 78.

[0333] In one embodiment, a BCMA antigen binding domain comprising the amino acid sequence of SEQ ID NO: 77 or SEQ ID NO: 78 can be used to reduce or eliminate plasma cells or BCMA-expressing myeloma cells associated with autoimmune disease.

[0334] In one embodiment, a method for producing an anti-BCMA antibody comprises culturing Chinese hamster ovary (CHO) cells as host cells comprising the amino acid sequence of SEQ ID NO: 77 or SEQ ID NO: 78, and producing and isolating the antibody from the culture. Similar methods for producing and isolating anti-BCMA antibodies are described in Patent No. US 10,239,947.

[0335] In one embodiment, the engineered cell harboring the pX-BC4-CD7 RTX-VAC-tandem CAR (also the pX-BC4-CD7-RTX-IL-15 / IL15sushi tandem CAR) comprises CD7 linked to a BCMA VHH via a linker and a secreted IL-15 / IL15sushi fusion ((SEQ ID NO. 65) and corresponding nucleotide (SEQ ID NO. 66). The pX-BC4-CD7 RTX-VAC-tandem CAR can be used to treat plasma cells and / or T cells associated with autoimmune diseases and myeloma / lymphoma that express BCMA and / or CD7.

[0336] In one embodiment, the engineered cell harboring the pX-BC4-CD19-VAC-tandem CAR comprises a BCMA VHH linked to CD19 via a linker and a secreted IL-15 / IL15sushi fusion ((SEQ ID NOs. 67 69 and 71) and corresponding nucleotides (SEQ ID NOs. 68, 70 and 72). The pX-BC4-CD19 RTX-VAC-tandem CAR can be used to treat plasma cells and B cells associated with autoimmune diseases, BCMA-expressing myeloma and / or CD19-expressing lymphoma / leukemia.

[0337] In one embodiment, an engineered cell bearing a pX-CD19-BC4-VAC-tandem CAR comprises a CD19 scFv linked via a linker to a VHH BCMA and a secreted IL-15 / IL15sushi fusion (SEQ ID NOs. 73 and 75) and corresponding nucleotides (SEQ ID NOs. 74 and 76). The pX-CD19-BC4-VAC-tandem CAR can be used to treat B cells and plasma cells associated with autoimmune diseases and lymphoma / leukemia that express CD19 or / BCMA.

[0338] Disruption of CAR-CAR interactions and increased insert size by conjugated CAR-BiTEs correlates with reduced viral titers. In one embodiment, two or more units of CAR expressed in a cell are required to avoid CAR-CAR interactions.

[0339] In one embodiment, the hinge region is designed to exclude amino acids that may cause undesired intra- or intermolecular interactions. For example, the hinge region can be designed to exclude or minimize cysteine ​​residues to prevent disulfide bond formation. In another embodiment, the hinge region can be designed to exclude or minimize hydrophobic residues to prevent undesired hydrophobic interactions.

[0340] In another embodiment, the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domain to the transmembrane domain of the same or a different surface membrane protein in order to minimize interactions with other members of the receptor complex.

[0341] The novel compound CAR contains a set of two CAR polypeptides with 12 functional domains, which has a larger self-cleaving sequence than a single CAR with six functional domains. As discussed in the previous office action, creating a compound CAR according to the claimed invention was unreasonable at the time of filing, because it was believed that the longer the polypeptide, the less effective it would be at generating virus for the CAR and the less effective it would be at killing the virus. This was because the expected protein expression level would decrease as the size increased. Research has shown that expression levels decrease as the insert size increases (Int J Biochem Mol Biol:201-208). Therefore, those skilled in the art would not be motivated to use it to create large compound CARs or BiTE-containing CARs.

[0342] In clinical trials using compound CARs, it was surprisingly discovered that CAR T cells with CAR efficiencies below or around 5% were still able to eliminate target leukemia cells and induce complete remission, suggesting that alternative / weaker promoters could be used in generating effective cCAR T cells in accordance with the claimed invention.

[0343] A surprising discovery and inventive step to overcome the low titer of viruses carrying large-sized cCAR or cCAR-BiTE It was surprising to discover that retroviral stable producer cell lines could solve this problem associated with low titers of viruses carrying larger sized CARs.

[0344] In certain embodiments, the generation of high titer cCAR or cCAR-BiTE can be achieved by a combination of at least one or more of the following steps: 1. Transduce lenti- or retroviral cCAR or cCAR-BiTE into a stable production cell line with a replaced non-cytotoxic viral envelope, such as RD114. 2. CAR expression in stable producing cell lines is determined by flow cytometry assay using anti-Fab antibody. 3. Sort stable producing cells that highly express cCAR or cCAR-BitTE using fluorescence-activated cell sorting (FACS) with anti-FAB antibody. 4. Selected cells with high CAR expression are cultured and the secreted retrovirus or lentivirus is examined. 5. Cloning of highly expressing cells by limiting dilution.

[0345] High-titer production of large BCMA-CD19 VAC cCAR retrovirus using a stable production cell line The BCMA-CD19 VAC cCAR (also known as the BCMA-CD19 IL-15 / IL-15sushi cCAR) is a two-unit CAR consisting of a complete BCMA-CAR and a complete CD19 CAR fused together by self-cleavage, allowing for the independent expression of both CAR receptors on the T cell surface. Each CAR contains a CD8 leader, a CD8-derived hinge (H) and transmembrane (TM) region, and a coactivation domain linked to a CD3ζ signaling domain. The enhancer, IL-15 / IL-15sushi, is also separated from the first and second CARs by a second cleavage site at the C-terminus. Expression of the BCMA-CD19 VAC cCAR is controlled by the Moloney murine leukemia virus (MMLV) long terminal repeat promoter.

[0346] BCMA-CD19 VAC cCAR (also known as BCMA-CD19b-IL-15 / IL15sushi) was transfected into the H29 cell line (transient), and the virus from the transfected H29 was transfected into the RD114 cell line (293, envelope protein from RD114 retrovirus). This envelope protein was utilized because it is not toxic to cells. Cell sorting was used to identify a high-CAR expressing population. The selected high-CAR expressing cell population was then expanded. Virus secreted from the expanded cells was verified for CAR expression in T cells. Furthermore, a single clone with high CAR expression was cloned by limiting dilution. Virus from the high-expressing clone was examined for CAR expression by flow cytometry. Flow cytometry results showed that BCMA-CD19-VAC cCAR-T was expressed in approximately 28.18% of T cells (Figure 36).

[0347] To evaluate the in vivo antitumor activity of BCMA-CD19b-IL-15 / IL15sushi CAR T cells against BCMA-expressing cells, we developed a xenogeneic mouse model in which NSG mice were sublethally irradiated and intravenously injected with 1x10e6 luciferase-expressing U937-BCMA cells, a human tumor cell line that synthetically expresses BCMA (CD269) on its surface, to induce measurable tumor formation (Figure 36). Five days after tumor cell injection, three mice per group were injected with 10x10 6Mice were intravenously injected with BCMA-CD19b-IL-15 / IL15sushi T cells or control T cells. On days 5, 8, 11, 14, and 17, mice were subcutaneously injected with RediJect D-Luciferin (PerkinElmer) and underwent IVIS imaging to measure tumor burden (Figure 36B). The mean light intensity of mice injected with BCMA-CD19b-IL-15 / IL15sushiCAR T cells was compared with that of mice injected with control T cells to determine the percentage of tumor cells in treated and control mice. Mice injected with CAR T cells showed an 80% reduction in tumor burden compared to controls by day 8, whereas mice injected with BCMA-CD19b-IL-15 / IL15sushiCAR T cells showed a 99% reduction in tumor burden by day 17, when nearly all tumor cells had lysed in the treated mice. Unpaired t-test analysis revealed a highly significant difference (P=0.0045) between the control and two groups by day 14, with the BCMA-CD19b-IL-15 / IL15sushi CAR T cell-treated group demonstrating reduced light intensity and consequently reduced tumor burden compared to the control. Collectively, these in vivo data demonstrate that BCMA-CD19b-IL-15 / IL15sushiCAR T cells significantly reduced tumor burden and shrank tumors in NSG mice injected with U937-BCMA compared to vector control T cells.

[0348] To evaluate the in vivo antitumor activity of BCMA-CD19b-IL-15 / IL15sushi CAR T cells against CD19-expressing cells, we developed a xenogeneic mouse model using sublethally irradiated NSG mice intravenously injected with 1 x 10e6 luciferase-expressing REH cells, a B-cell-derived human tumor cell line expressing the CD19 antigen (Figure 36C). Five days after tumor cell injection, three mice per group were injected with 10 x 10 6Mice were intravenously injected with BCMA-CD19b-IL-15 / IL15sushi T cells (the same cells as in the previous experiment) or control T cells. On days 5, 8, 11, 14, 17, and 20, mice were subcutaneously injected with RediJect D-Luciferin (Perkin Elmer) and underwent IVIS imaging to measure tumor burden (Figure 36C). The mean light intensity of mice injected with BCMA-CD19b-IL-15 / IL15sushi CAR T cells was compared with that of mice injected with control T cells to determine the percentage of REH tumor cells in treated and control mice. Mice administered with CAR T cells showed a 72% reduction in tumor burden compared to controls by day 8, whereas mice administered with BCMA-CD19b-IL-15 / IL15sushi CAR T cells showed a 99% reduction in tumor burden by day 17, when nearly all tumor cells had lysed. Unpaired t-test analysis revealed a highly significant difference (P=0.0045) between the control and two groups by day 14, with the BCMA-CD19b-IL-15 / IL15sushi CAR T cell-treated group demonstrating lower light intensity and consequently reduced tumor burden compared to the control. Collectively, these in vivo data demonstrate that BCMA-CD19b-IL-15 / IL15sushi CAR T cells significantly reduced tumor burden and shrank tumors in NSG mice injected with REH compared to vector control T cells.

[0349] Lupus nephritis (LN) is a common and serious complication of systemic lupus erythematosus (SLE). In this phase 1, open-label clinical trial, sequentially randomized patients with biopsy-confirmed (class III-V) LN received 1.5-3x10 6BCMA-CD19b-IL-15 / IL15sushi cCAR cells / kg were administered. A total of 13 SLE patients underwent the intervention (1.5-3 x 106 cCAR cells / kg). All patients were negative for all autoantibodies, including those derived from long-lived plasma cells, 3 months after cCAR. During follow-up for up to 44 months after cCAR, all patients achieved symptom-free, medication-free complete remission (CR). The mean Systemic Lupus Erythematosus Disease Activity Index 2000 (SLEDAI-2K) score decreased from 9.9 (baseline) to 2.4 (3 months) (or perhaps 1.0 at 6 months), and renal function significantly improved in 10 LN patients within 90 days after cCAR. Complete B cell recovery was observed 2-6 months after cCAR. Complete immune reset was confirmed by deep sequencing and flow cytometry analysis of the B cell receptor (BCR). In this phase 1 study, SLE patients receiving cCAR achieved a medication-free complete response (CR) with remarkable safety and toxicity profiles. The depletion of disease-causing autoantibodies derived from B cells and long-lived plasma cells (Figure 36D) indicated a complete immune reset. At 2–4 months posttreatment, all 13 patients were negative for the following autoantibodies: antihistone (AHA), antinuclear (ANA), anti-U1-snRNP, antinucleosome, anti-double-stranded DNA (dsDNA), antiribosome, anti-SSA / Ro52, anti-Sm, and anti-SSA / Ro60. Anticentromere B, anti-exoribonuclease, and anti-SSB / La autoantibody levels were below the pathological threshold in all patients. The reduction of anti-SSA / Ro52 and anti-SSA / Ro60 indicated the absence of disease-causing long-lived plasma cells and plasmablasts. All SLE patients showed recovery of CH50, especially C3 and C4, after cCAR (Figure 36D). This treatment may also be applicable to the treatment of other autoimmune diseases.

[0350] We demonstrate that the large insert size of the CLL1-CD33-VAC cCAR-T construct does not interfere with expression, and single clones obtained by clonal limiting dilution express high levels of the CAR on T cells. The CLL1-CD33 VAC cCAR (also known as the CLL1-CD33 IL-15 / IL-15sushi cCAR or CLL1-CD33b VAC CAR) is a two-unit CAR consisting of a complete CLL1-CAR and a complete CD33 CAR fused together by self-cleavage, allowing for the separate expression of both CAR receptors on the T cell surface. Each CAR contains a CD8 leader, a CD8-derived hinge (H) and transmembrane (TM) region, and a coactivation domain linked to a CD3ζ signaling domain. The enhancer, IL-15 / IL-15sushi, is also separated from the first and second CARs by a second cleavage site at the C-terminus.

[0351] The CLL1-CD33 VAC cCAR construct, driven by the Moloney murine leukemia virus (MMLV) long terminal repeat (LTR) promoter, was transduced into the H29 cell line, and then the H29-produced virus was used to transduce RD114 cells. High-expressing clones of CLL1-CD33 VAC cCAR were then screened by limiting dilution. High-expressing clones were identified by flow cytometry analysis. Unexpectedly, CLL1-CD33 VAC cCAR was expressed in approximately 31.32% of T cells (Figure 37A). Unexpected results were also observed when expression of CLL1-CD33-IL-15 / IL-15sushi was driven by the MMLV promoter (Figure 36A). Compared to the strong SFFV (spleen focus-forming virus) promoter, which drives the expression of a single polypeptide, resulting in the generation of both CARs (Table I), most individuals showed very low levels of cCAR T expression, below 10%. Therefore, the Moloney murine leukemia virus (MMLV) long terminal repeat (LTR) promoter is a better choice for generating cCARs. Surprisingly, administration of cCAR-generated cells carrying a first chimeric antigen receptor polypeptide selective for CLL-1 and a second chimeric antigen receptor polypeptide selective for CD33 resulted in complete remission in 9 of 11 AML patients. Furthermore, IL-15 / IL15sushi levels did not significantly increase after infusion and remained within the pg / mL range. No lymphomas or leukemias were observed as a result of CAR treatment.

[0352] In one embodiment, the engineered cell harboring the CLL1-CD33-IL-15 / IL15sushi cCAR comprises a chimeric antigen receptor peptide, a CLL1 chimeric antigen receptor polypeptide, a CD33 chimeric antigen receptor polypeptide, and a secreted IL-15 / IL15sushi fusion ((SEQ ID NO. 31) and corresponding nucleotide (SEQ ID NO. 32). The CLL1-CD33-IL-15 / IL15sushi cCAR can be used to treat acute myeloid leukemia or myeloid leukemia that expresses CLL1 and / or CD33.

[0353] [Table 2]

[0354] Example: MMLV promoter-driven generation of BCMA-CD19-IL-15sushi (also called BCMA-CD19 VAC cCAR) and CD7 CAR BCMA-CD19 VAC cCAR and CD7 CAR constructs were generated and expressed using the Moloney murine leukemia virus (MMLV) long terminal repeat (LTR) promoter, using a strategy similar to that described above. PBMCs from healthy human donors were activated for 48 hours in the presence of mouse anti-human CD3 antibody. Cells (1.0 x 10e6) were transduced with 1 mL of control supernatant (left panel) or CAR supernatant retrovirus (right panel). Cells were labeled for flow cytometry analysis with goat anti-mouse F(Ab')2 antibody to detect the CAR phenotype, and mouse anti-human CD3 and CD7 to detect a reduction in the T cell phenotype population in transduced cells. Unexpectedly, 2 days after viral infection, BCMA-CD19-IL-15 / IL15sush and CD7 RTX CARs were expressed at approximately 61% (Figure 37B) and 83% (Figure 37C), respectively. The CD7+ T cell population was completely depleted (FIG. 37C, lower right panel).

[0355] Example: Generation of CD7-negative T cells containing BCMA-CD7 cCAR. A compound CAR (cCAR) combination is constructed on an expression vector and its expression is driven by a promoter. The first BCMA CAR and the second CD7 CAR are linked by a P2A self-cleaving sequence. A safety switch (rituximab) is incorporated into the hinge region. The cCAR and the IL-15 / IL15 sushi domain of the IL-15α receptor (BCMA-CD7-IL-15 / IL-15sushi) can be optionally assembled on an expression vector and its expression is driven by a promoter (Figure 38). The first BCMA CAR and the second CD7 CAR are linked by a P2A self-cleaving sequence, and the CD7 CAR is linked to the IL-15 / IL-15sushi domain by a T2A self-cleaving sequence. The IL-15 / IL-15sushi portion consists of an IL-2 signal peptide fused to IL-15, linked to the IL-15α sushi domain via a 26-amino acid polyproline linker. A safety switch (rituximab) is incorporated into the hinge region.

[0356] BCMA-CD7-IL-15 / IL-15sushi was used to generate CD7-negative T cells due to the presence of a CD7 CAR.

[0357] Retrovirus production and generation of stably transduced retrovirus-expressing cells The H29 cell line was first transfected with a plasmid containing the pX-BCMA-CD7-15 / IL15sushi gene using Lipofectamine 2000 (Life Technologies, Carlsbad, CA) according to the manufacturer's protocol. After 3 days of culture, the supernatant was removed and replaced with culture medium containing DMEM and 10% FBS. This supernatant was used to transduce RD114 cells (HEK293 cells stably expressing the gag / pol and vsv genes required for viral replication) with the addition of polybrene. After 24 hours of culture, the supernatant was removed and replaced with a second viral supernatant harvested from the H29 cells. This process was repeated three times. After three transductions, the cells were expanded into larger flasks and sorted by FACS based on goat anti-mouse F(Ab')2 expression. After further expansion, the sorted cells were cloned by limiting serial dilution. The resulting single-cell colonies were analyzed by goat anti-mouse F(Ab')2 flow cytometry (see Figure 39), and the highest-expressing clones were expanded. Viral supernatants were collected from expanded cells, filtered through a 0.45 μM syringe filter, and stored at 4°C until transduction or frozen at -80°C for long-term storage. Prior to retroviral transduction, umbilical cord or peripheral blood mononuclear buffy coat cells were activated for 3 days in the presence of 300 IU / mL IL-2 and 1 μg / mL anti-human CD3 (Miltenyi Biotec, Germany). The cells were washed and suspended in T cell medium containing IL-2 at a concentration of 1 x 10e6 / mL. For transduction, 750 μL of this cell mix was combined with an equal volume of control (untransduced) medium or the BCMA-CD7-15 / IL15sushi CAR retroviral vector at a final 1:1 ratio. CAR transduction and validation of CAR expression in T cells are described in detail in the figures.

[0358] Activated human T cells were transduced with control (left) or BCMA-CD7-15 / IL15sushi CAR (right) viral supernatant from RD114 cells. Forty-eight hours after transduction, cells were harvested, washed, and transferred to tissue culture plates containing fresh medium and IL-2. After an additional 3 days of culture, cells were harvested and stained with a 1:250 goat anti-mouse F(Ab')2 antibody for 45 minutes. Cells were washed and stained with a 1:500 streptavidin-PE conjugate, goat anti-mouse CD3 and CD7 antibodies, washed, suspended in 2% formalin, and analyzed by flow cytometry. Nearly all T cells were CD7-negative. Approximately 42% of CAR T cells expressed the F(Ab'2) phenotype (upper panel) (Figure 39A).

[0359] We performed a 24-hour coculture experiment comparing the lysis of BCMA-CD7Q-IL-15 / IL15sushi CAR T cells with U-BCMA (U-BCMA)-expressing U937 cells, a human tumor cell line (U937) that synthetically expresses the CD269 (BCMA) antigen on its cell surface, at an effector:target ratio of 2:1 (top panel) and 5:1 (bottom panel). CCRF-CEM cells were pre-labeled with the membrane dye CMTMR to phenotypically distinguish them from T cells. Target cells alone are shown on the left. Cells were analyzed by flow cytometry using anti-CD7 and anti-CD3 labeling. The boxed population highlights target cell lysis. During the 24-hour coculture experiment, BCMA-CD7Q-IL-15 / IL15sushi CAR T cells demonstrated profound killing (nearly 90%) of target U-BCMA cells at an effector:target ratio of 2:1 (Figure 39B).

[0360] Example: Generation of CD7-negative T cells with CD7-CD19 cCAR. Expression of the CD7-CD19-15 / IL15 sushiCAR is driven by the Moloney murine leukemia virus (MMLV) long terminal repeat (LTR) promoter. The compound CAR (cCAR) contains two complete CARs, each consisting of a signal domain (leader sequence), scFv domain, hinge domain (H), cross-membrane domain (TM), coactivators CD28 (CD7 CAR only), 4-1BB (CD19 CAR only), and the intracellular signaling domain of CD3zeta. The CD7 CAR has a safety switch just before the hinge domain (Figure 40).

[0361] Retrovirus production and generation of stably transduced retrovirus-expressing cells The H29 cell line was first transfected with a plasmid containing the pX-CD7-CD19-15 / IL15sushi gene using Lipofectamine 2000 (Life Technologies, Carlsbad, CA). After 3 days of culture, the supernatant was removed and replaced with culture medium containing DMEM and 10% FBS. This supernatant was used to transduce RD114 cells (HEK293 cells stably expressing the gag / pol and vsv genes required for viral replication) using polybrene. After 24 hours of culture, the supernatant was removed and replaced with a second viral supernatant harvested from H29 cells. This process was repeated three times. After three transductions, the cells were expanded into larger flasks and sorted by FACS based on goat anti-mouse F(Ab')2 expression. After further expansion, the sorted cells were cloned by limiting serial dilution. The resulting single-cell colonies were analyzed by goat anti-mouse F(Ab')2 flow cytometry, and the highest-expressing clones were expanded to produce virus, which was recovered from the supernatant stored in liquid nitrogen, filtered through a 0.45 μM syringe filter, and stored at 4°C or frozen at -80°C until use in transfections.

[0362] Transduction of human T cells with retroviral vectors from stably transduced retrovirus-expressing cells Prior to retroviral transduction, umbilical cord or peripheral blood mononuclear buffy coat cells were activated for 3 days in the presence of 300 IU / mL IL-2 and 1 μg / mL anti-human CD3 (Miltenyi Biotec, Germany). The cells were washed and suspended in T cell medium containing IL-2 at a concentration of 1 x 10e6 / mL. For transduction, 750 μL of this cell mix was combined with an equal volume of control (untransduced) medium or the CD7-CD19-IL-15 / IL-15sushi retroviral vector at a final 1:1 ratio. CAR transduction and verification of CAR expression in T cells are detailed in the following figure.

[0363] Activated human T cells were transduced with control (left) or CD7-CD19-15 / IL15sushi CAR (right) viral supernatant from RD114 cells. Forty-eight hours after transduction, cells were harvested, washed, and transferred to tissue culture plates containing fresh medium and IL-2. After an additional 3 days of culture, cells were harvested and stained with 1:250 goat anti-mouse F(Ab')2 antibody for 45 minutes. Cells were washed and stained with 1:500 streptavidin-PE conjugate, goat anti-mouse CD3 and CD7 antibodies, washed, suspended in 2% formalin, and analyzed by flow cytometry. Nearly all T cells were CD7-negative, and approximately 72% of CAR T cells expressed the F(Ab'2) phenotype (upper panel) (Figure 41).

[0364] Generally, the range of effective lentiviral vector constructs has been limited by the insert size. Long compound CARs (inserts of approximately 3–4 kb) resulted in much lower lentiviral titers and lower CAR T transduction efficiencies than our single CARs. Surprisingly, this problem can be overcome by using stable viral cell lines. Using longer retroviral vector constructs, we were able to obtain higher viral titers after selecting high-retroviral-producing RD114 cells and isolating single clones, resulting in higher CAR T transduction efficiencies for compound CARs. This method can also be used to generate CARs even longer than cCARs, with three or more complete CAR units. For example, a cCAR consisting of complete anti-BCMA and anti-CD19 CAR units and the IL-15 / IL-15sushi described above. While constructs longer than 5 kb may result in lower initial retroviral vector titers, high CAR T transduction efficiencies can be achieved by sorting RD114 cells using the same method to isolate highly expressing RD114 cells.

[0365] CD7-negative T cells induced by CD19CAR exhibit significant killing activity. We tested the functional activity of each cCAR unit (CD7 or CD19) on CD7-negative T cells using tumor cells expressing CD7 or CD19. Because these cells proliferate extensively, we could more easily measure the effect of cCAR on their proliferation and confirm their ability to lyse cells bearing the target antigen.

[0366] A CD19 antigen-positive cell line (REH) was co-cultured with control T cells or CD7-CD19-IL-15 / IL-15sushi CAR T cells at T cell:target cell ratios of 1:1 and 2:1 (Figure 42). Unexpectedly, after 18 hours of co-culture, nearly all target cells were eliminated at the 2:1 and even 1:1 ratios.

[0367] Using a CD7 marker-positive cell line (CCRF-CEM), these cells were co-cultured with control T cells or CD7-CD19-IL-15 / IL-15sushi CAR T cells at T cell:target cell ratios of 1:1 and 3:1 (Figure 43). Unexpectedly, after 18 hours of co-culture, almost all target cells were eliminated, even at the 3:1 and even 1:1 ratios.

[0368] These results demonstrate the robust lytic ability of each component CAR unit in the CD7-CD19-IL-15 / IL-15sushi cCAR against CD7-negative T cells.

[0369] Anti-BCMA VHH discovery strategy VHH antibodies (or nanobodies) are antigen-binding fragments of heavy chain-only antibodies. Alpacas were immunized for 8 weeks, after which PBMC mRNA was isolated and processed into a VHH gene library. This library was transformed into phage-competent bacteria to generate a bacteriophage library. This library was panned against BCMA to enrich for binding clones. High-quality hits were identified using high-throughput enzyme-linked immunosorbent assays (ELISAs) with individual VHH clones. VHH candidates with binding greater than or equal to two-fold above average background were selected and subjected to Sanger sequencing to identify unique sequences (Figure 45A). Protein binding was measured using an ELISA assay (Figure 46). Interactions of BCMA VHH antibodies with NIH-CoVnb-112 BCMA (human) or Cyno BCMA (platypus) proteins were examined (A and B). TACI and BFFR were used as negative controls (C). The binding affinity and half-maximal binding (EC50) of two BCMA VHHs, BC4 and BC5, were 1.578 and 1.099, respectively. ELISAs were developed using standard streptavidin-HRP and tetramethylbenzidine reactions (Figure 46). To further determine the binding of the newly identified VHHs, BC4, and BC5, to the BCMA protein, cell-based assays were performed using CHO cells engineered to express BCMA. The cell surface binding affinities of the BCMA VHHs, BC4, and BC5, were analyzed by flow cytometry analysis using BCMA-expressing CHO cells. Various concentrations of each antibody were used, and each antibody was detected using a secondary anti-human IgG-FITC antibody. The BC4 and BC5 VHHs particularly strongly bound to the surface BCMA protein (Figure 47). The BC4 and BC5 VHHs were used to generate VHH CAR-T cells or NK cells.

[0370] Characterization of BC4-RTX-IL-15 / IL15sushi CAR targeting plasma cells or BCMA-expressing cells. CAR T cells were generated by transfecting primary peripheral blood T cells with the retroviral construct shown in Figure 48. The translated CAR protein is expressed on the T cell surface and can recognize and bind to target proteins on the surface of tumor cells. The pharmacological action and mechanism of CAR is that antigen recognition by BC4-RTX-IL-15 / IL15sushi CAR T cells induces cytotoxic T cell activity, which is further enhanced by incorporating a CD28 coactivation domain into the construct.

[0371] To assay the ability of BC4-RTX-IL-15 / IL15sushi CAR T cells to target CD269 (BCMA)+ cells, control or BC4-RTX-IL-15 / IL15sushi CAR T cells were co-cultured for 18 hours with the synthetic antigen-expressing tumor cell line U937-BCMA at an E:T ratio of 2:1. The U-BCMA target line, which synthetically expresses the BCMA surface antigen, was robustly eliminated by BC4-RTX-IL-15 / IL15sushi CAR T cells compared to control T cells (84%) over the 18-hour co-culture (Figure 49).

[0372] In a similar manner, we tested BC5-RTX-IL-15 / IL15sushi CAR T cells targeting plasma cells or BCMA+ cells. During 24-hour coculture experiments, BC4-RTX-IL-15 / IL-15sushi CAR T cells significantly killed target U937-BCMA cells (82%) at an effector:target ratio of 5:1.

[0373] Making a tandem car. BCMA-CD19 (also known as BC4-CD19c-Tan or BC5-CD19c-Tan) was generated as leader + VHH + linker + scFv + h + TM + Co + CD3 zeta or leader + scFv1 + linker + scFv2 + h + TM + Co + CD3 zeta. Note: Leader, signal peptide; linker, (GGGS)3 or (GGGS)4; H, hinge region; TM, transmembrane domain; Co, costimulatory domain. Tandem targeting constructs of CAR were also generated in a similar manner. CAR constructs were cloned into a retroviral plasmid backbone under the long terminal repeat (LTR) promoter of the MMLV (Membrane-Like Leukemia) vector. Retroviruses expressing CAR were generated as described above using standard methods.

[0374] The BC4-CD19c-Tan CAR virus was then administered to T cells, and CAR expression was tested. Flow cytometry confirmed the expression of the BC4-CD19c-Tan CAR. BCMA-expressing U937 cells were used to evaluate the functional activity of the BC4-CD19c-Tan CAR. BC4-CD19c-TanCAR consistently demonstrated potent lysis of cells expressing BCMA at an E:T ratio of 2:1 (Figure 51A).

[0375] We evaluated whether BC4-CD19c-tan-CAR-transduced T cells could eliminate endogenous CD19-positive cells, particularly B cells, within the donor cells after CAR transduction (Figure 5B). To address this question, we labeled control T cells on day 2 after recovery and CAR-transduced T cells (day 6 after T cell activation) with CD45-PerCP, CD3-PE, and CD19-APC antibodies and performed flow cytometry analysis. Flow cytometry analysis showed that the control cell sample contained a CD19-positive population (circled dots in the left panel), whereas no CD19-positive population was detected in the CAR-transduced sample group (right panel).

[0376] The following multiple tandem targeting constructs for CAR were also generated in a similar manner as above. CD19c-CD7 Q CAR T cells (also called CD19c-CD7 Q-Tan CAR T cells) were generated to target B cells and B cell tumors, as well as T cells and T cell tumors. Q, also known as RTX, was incorporated into the hinge region of the CAR, containing two RTX-binding epitopes. The CD19c-CD7 Q-Tan CAR virus was then administered to T cells, and CAR expression was tested. Flow cytometry confirmed CAR expression. CD7+ cells were completely eliminated from the transduced T cell population (Figure 53A). In a 16-hour coculture experiment, CD19c-CD7Q CAR T cells completely lysed CD19-expressing REH cells at a 2:1 ratio (Figure 53B), demonstrating the superior efficacy of the CD19 CAR. These results indicate that both CAR units successfully lyse the target populations (B cells and T cells).

[0377] A similar method was also applied to the CD19b-CD7Q VAC CAR (also known as CD19b-CD7Q-IL-15 / IL-15sushiCAR-Tan) construct, which targets B cells or B-cell tumors and T cells or T-cell tumors. This tandem CAR is linked to an IL-15 / IL-15sushi fusion protein. The IL-15 / IL-15sushi fusion enhances immune cell function. PBMCs from healthy donors were activated for 48 hours in the presence of a mouse anti-human CD3 antibody. Cells (1.0 x 10e6) were transduced with a retrovirus expressing the construct. CD7+ cells were completely depleted from the transduced T cell population. In a 16-hour coculture experiment, CD19b-CD7Q-IL-15 / IL15sushi CAR T cells completely lysed CD19-expressing REH cells at a 2:1 ratio, demonstrating the superior efficacy of the CD19 CAR. These results demonstrate that both CAR units successfully lyse the target population (Figure 54).

[0378] Similar findings were observed with the CAR construct CD19b-BC4 tandem CAR. In a 16-hour coculture experiment, CD19b-BC4 CAR T cells completely lysed CD19-expressing REH cells at a 2:1 ratio, demonstrating the superior efficacy of the CD19 CAR unit (Figure 55). In addition, CD19b-BC4 CAR T also robustly lysed BCMA-expressing U937 cells.

[0379] IL-15 / IL-15sushi enhances CAR: As an example, the CD19b-IL-15 / IL-15sushi CART construct has improved durability and biological activity compared to standard CD19 CARs. The CD19-IL-15 / IL-15sushi construct contains a CD19 CAR linked via a self-cleaving peptide to an IL-15 fusion protein. The IL-15 fusion protein combines the soluble domains of IL-15 and IL-15Rα (sushi), resulting in secretion of the soluble IL-15 / IL-15 sushi complex.

[0380] To characterize IL-15 / IL-15sushi-secreting CARs as a potential alternative to the current CAR T / NK cell paradigm, we analyzed three broad factors: 1) the ability to kill target cells (efficacy), 2) improved persistence to enhance bioavailability and surveillance, and 3) expansion of a more potent CAR T phenotype.

[0381] Sequence List A sequence listing conforming to the rules of WIPO Standard ST.26 is incorporated herein by reference. The sequence listing has been submitted to the Patent Center as an XML-encoded electronic document in UTF-8 text. The electronic document was created on November 27, 2023, is named "2541-15_PCT_ST26.xml", and is 244,897 bytes in size.

Claims

1. 1. A method of treating an autoimmune disease in a patient in need thereof, comprising administering to the patient a dual CAR, wherein the dual CAR binds to a first antigen and a second antigen, wherein the first antigen is expressed on a T cell or an NK cell and is selected from the group consisting of CD2, CD3, CD4, CD5, and CD7, and the second antigen is expressed on a B cell or a plasma cell and is selected from the group consisting of CD19, CD20, CD22, BCMA, CD38, CD138, CS1, and GPRC5D, and wherein the dual CAR is either a tandem CAR or a combined CAR (cCAR).

2. A method of treating an autoimmune disease in a patient, comprising administering to the patient a CAR that binds to an antigen expressed on a T cell or NK cell, wherein the antigen is selected from the group consisting of CD2, CD3, CD4, CD5, and CD7.

3. 10. The method of claim 1, wherein the autoimmune disease is T cell mediated and / or is caused by autoreactive T cells.

4. 10. The method of claim 1, wherein the autoimmune disease is B cell mediated and / or is caused by autoreactive B cells.

5. 10. The method of claim 1, wherein the autoimmune disease is T cell and B cell mediated and / or is caused by autoreactive immune cells including T cells, B cells, plasma cells, or a combination thereof.

6. 2. The method of claim 1, wherein the dual CAR is composed of a CD7 CAR and another CAR unit that targets an antigen selected from the group consisting of CD19, CD20, CD22, BCMA, CD38, GPRC5D, and CS1, and administration reduces T cells and B cells or plasma cells, or a combination thereof, that express the CD7 surface antigen in the patient.

7. 2. The method of claim 1, wherein the dual CAR binds to a cell expressing CD7 and / or CD19.

8. 10. The method of claim 1, wherein the dual CAR binds to cells expressing CD7 and / or CD20.

9. 10. The method of claim 1, wherein the dual CAR binds to cells expressing CD7 and / or BCMA.

10. 2. The method of claim 1, wherein the dual CAR binds to cells expressing CD19 and / or CD20.

11. 10. The method of claim 1, wherein the dual CAR binds to cells expressing CD19 and / or BCMA.

12. 1. A method of treating an autoimmune disease in a patient in need thereof, comprising administering to the patient a CD7CAR, wherein the CD7CAR binds to a CD7 antigen expressed on T cells, and wherein the administration results in depletion of T cells in the patient that express the CD7 surface antigen.

13. 10. The method of claim 1, wherein the method further comprises administering to the patient in need thereof a steroid and / or an immune cell inhibitor selected from a B cell inhibitory therapy, a plasma cell inhibitory therapy, or an immunosuppressive therapy.

14. 2. The method of claim 1, wherein the autoimmune disease is selected from type 1 diabetes, multiple sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, celiac disease, myasthenia gravis, pemphigus vulgaris, bullous pemphigoid, Graves' disease, asthma, systemic lupus erythematosus, IgA nephropathy, IgG4-related disease, membranous nephropathy, myasthenia gravis, neuromyelitis optica, pemphigus vulgaris, anti-PAD4-activated rheumatoid arthritis, sensitizing / preformed antibodies in solid organ transplantation, psoriasis, Guillain-Barré syndrome (acute inflammatory demyelinating polyneuropathy - AIDP), chronic inflammatory demyelinating polyneuropathy (CIDP), Evans syndrome, immune thrombocytopenic purpura, rheumatoid arthritis, Sjogren's syndrome, and ANCA-associated vasculitis (AAV).

15. 10. The method of claim 1, wherein the autoimmune disease is selected from type 1 diabetes, multiple sclerosis, inflammatory bowel disease, psoriasis, ulcerative colitis, or Crohn's disease.

16. 2. The method of claim 1, wherein the autoimmune disease is type 1 diabetes.

17. 10. The method of claim 1, wherein the autoimmune disease is multiple sclerosis.

18. 10. The method of claim 1, wherein the autoimmune disease is inflammatory bowel disease, ulcerative colitis, or Crohn's disease.

19. 10. The method of claim 1, wherein the patient is in need of prophylactic treatment of a T cell-mediated autoimmune disease or a combination of a T cell-mediated disease and a B cell-mediated disease.

20. 10. The method of claim 1, wherein the patient is in need of treatment for a relapsed or refractory T cell-mediated autoimmune disease, or a combination of a T cell-mediated disease and a B cell-mediated disease.

21. A chimeric antigen receptor (CAR) comprising a BCMA antigen-binding domain comprising the amino acid sequence of SEQ. ID NO: 77 or SEQ. ID NO: 78, a hinge domain, a transmembrane domain, at least one costimulatory domain, and a CD3 zeta signaling domain.

22. 22. The chimeric antigen receptor of claim 21 , wherein the antigen-binding domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 49, 51, 53, 55, 57, 59, 61, and 63.

23. 1. An ex vivo engineered T cell or NK cell that co-expresses two different chimeric antigen receptor (CAR) units on its cell surface, the engineered T cell or NK cell comprising, from 5′ to 3′, a nucleotide sequence consisting of a first polynucleotide encoding a first chimeric antigen receptor polypeptide (first CAR), a second polynucleotide encoding a second chimeric antigen receptor polypeptide (second CAR), and nucleotides encoding Porcine Teschovirus-1 2A (P2A), Thosea asigna virus 2A (T2A), FMDV 2A (F2A), or Equine Rhinitis A virus (ERAV) 2A (E2A), located between the first and second CAR under the transcriptional control of a single promoter: (i.) the first CAR polypeptide comprises a first antigen recognition domain; a first signal peptide; a first hinge region; a first transmembrane domain; a first costimulatory domain; and a first signaling domain; and (ii.) the second CAR comprises a second antigen recognition domain; a second signal peptide; a second hinge region; a second transmembrane domain; a second costimulatory domain; and a second signaling domain; wherein the first antigen recognition domain and the second antigen recognition domain are different, and the modified T cell or NK cell comprises SEQ ID NO: 5, SEQ ID NO: 9, SEQ ID NO: 17, or SEQ ID NO:

19.

24. An in vitro engineered T cell or NK cell co-expresses two different chimeric antigen receptor (CAR) units on its cell surface, wherein the engineered T cell or NK cell comprises, from 5' to 3', a first polynucleotide encoding a first chimeric antigen receptor polypeptide (first CAR), a second polynucleotide encoding a second chimeric antigen receptor polypeptide (second CAR), and nucleotides encoding porcine Teschovirus type 1 2A (P2A), Thosea asigna virus 2A (T2A), foot-and-mouth disease virus 2A (F2A), or equine rhinitis A virus (ERAV) 2A (E2A), arranged between the first CAR and the second CAR under the transcriptional control of a single promoter, wherein: (i.) the first CAR polypeptide comprises a first antigen recognition domain; a first signal peptide; a first hinge region; a first transmembrane domain; a first costimulatory domain; and a first signaling domain; and (ii.) the second CAR comprises a second antigen recognition domain; a second signal peptide; a second hinge region; a second transmembrane domain; a second costimulatory domain; and a second signaling domain; wherein the first antigen-recognition domain and the second antigen-recognition domain are different; wherein the engineered T cells or NK cells comprise an enhancer selected from the group consisting of IL-15 / IL-15sushi, and wherein the engineered T cells or NK cells comprise SEQ ID NO: 7, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 33, or SEQ ID NO:

47.

25. A tandem CAR-T cell or NK cell comprising a single chimeric antigen polypeptide comprising two different antibody binding domains, wherein the antibody binding domains are connected by a linker and share a hinge region, a transmembrane domain, a signaling domain and at least one costimulatory domain, and wherein the tandem CAR consists of the following: SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, or SEQ ID NO:

75.

26. An ex vivo artificial T cell or NK cell that co-expresses a chimeric antigen receptor (CAR) unit or two different chimeric antigen receptor (CAR) units on its cell surface, wherein the artificial T cell or NK cell comprises a nucleotide sequence comprising a single promoter extending from 5' to 3' of an MMLV promoter, and the artificial T cell or NK cell comprises SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 25, SEQ ID NO: 47, or SEQ ID NO: 31.

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