Bispecific CAR-T cell targeting BCMA and CD19

Abstract

A CAR-T cell that targets BCMA and CD19 is provided. Specifically, the invention provides a structure which simultaneously aims at parallel expression of a B cell maturation antigen (BCMA) and a chimeric antigen receptor (CAR) of a CD19 antigen molecule, and optimizes a motif of a costimulatory domain CD28. The use of the chimeric antigen receptor for adoptive T-cell therapy of B-cell related conditions is also provided.

Description

Bispecific CAR-T cells targeting BCMA and CD19 Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and in particular, to B19CAR-T cells targeting BCMA and CD19. Background Art

[0002] B cell maturation antigen (BCMA), also known as CD269, is a member of the tumor necrosis factor receptor superfamily member 17 (TNFRS17). It consists of 185 amino acid residues and is a type III transmembrane protein with a cysteine-rich extracellular domain that is relatively conserved. As a cell surface protein expressed exclusively on B cells, BCMA, along with two other members of the TNF receptor superfamily: the B-cell activation factor (BAFF) receptor and the transmembrane activator and calcium-modulator and cyclophilin ligand (TACI), play crucial roles in regulating B cell proliferation, survival, maturation, and differentiation into plasma cells. These three receptors support the long-term survival of B cells at various stages of development by binding to BAFF and / or their cognate ligand, APRIL. Studies in BCMA knockout mice have further demonstrated that, while BCMA is not essential for overall B cell homeostasis, it is crucial for the survival of long-lived plasma cells. At the same time, BCMA is highly expressed in malignant MM plasma cells, and BCMA expression increases with disease progression. Gene and protein expression profiling analysis confirmed that BCMA is the most selectively expressed cell surface receptor on MM cell lines, making it a very promising ideal target in CAR-T cell therapy for MM.

[0003] CD19 is a 95 kDa glycoprotein expressed on the surface of B cells, beginning early in B cell development and continuing until differentiation into plasma cells. CD19 is a member of the immunoglobulin (Ig) superfamily and, as a component of the B cell surface signaling complex, participates in regulating B cell receptor signaling. In CD19-deficient mouse models, the number of B cells in peripheral lymphoid tissues is significantly reduced, as are responses to vaccines and mitogens, accompanied by decreased serum Ig levels. It is generally believed that CD19 expression is restricted to the B-cell lineage and not expressed on the surface of pluripotent hematopoietic stem cells. However, CD19 is also expressed on the surface of most B-cell lymphomas, mantle cell lymphomas, ALLs, CLLs, hairy cell leukemias, and some acute myeloid leukemia cells. Therefore, CD19 is a valuable immunotherapy target for the treatment of leukemia / lymphoma. Most normal cells, such as pluripotent hematopoietic stem cells, do not express CD19 on their surface. This characteristic makes CD19 a safe therapeutic target, minimizing the risk of autoimmune disease or irreversible myelotoxicity in patients. Anti-CD19 antibodies or scFv fragments have been developed and have demonstrated promising results in mouse models and humans / primates. Treatment with CD19 CAR-T cells expressing CD28 or 4-1BB has resulted in approximately 90% complete remission rates in pediatric and adult patients with relapsed or refractory acute B-cell lymphoma. Recently, CD19 CAR-T cell therapy has demonstrated overall remission rates of 50%-100% in diffuse large B-cell lymphoma, follicular lymphoma, or chronic lymphocytic lymphoma. CD19 CAR-T cells have shown clinical advantages in the treatment of multiple myeloma, as terminally differentiated plasma cells do not express CD19, allowing malignant plasma cells to continue to be generated from malignant B-cell precursors.

[0004] In addition, for CD19-negative recurrence and B cell / plasma cell tumors with low CD19 expression, the development of CAR-T cells with dual targets of BCMA and CD19 can solve the problems of CD19's easy mutation, low expression and immune escape.

[0005] Therefore, there is a need in the art to develop CAR-T cells that simultaneously target BCMA and CD19.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to provide a CAR-T cell with a parallel structure targeting BCMA and CD19, and to modify the intracellular CD28 costimulatory domain and its application.

[0008] The present invention jointly targets the dual tumor targets of BCMA and CD19, has the characteristics of strong specificity and high targeting, can effectively improve and prolong the therapeutic effect of CAR-T, and has better therapeutic effects on multiple myeloma, leukemia or B-cell lymphoma that are positive for surface antigens BCMA and CD19, effectively avoids off-target escape of single targets, and improves the safety of CAR-T drugs.

[0009] In a first aspect of the present invention, an engineered immune cell is provided, wherein the engineered immune cell expresses BCMA CAR and CD19-mutant CAR in parallel, wherein the amino acid sequence of the BCMA CAR is shown in SEQ ID NO: 15; the amino acid sequence of the CD19-mutant CAR is shown in SEQ ID NO: 16; wherein the intracellular costimulatory domain of the CD19-mutant CAR comprises a CD28 mutant as shown in SEQ ID NO: 14, wherein the wild-type motif YMNM (SEQ ID NO: 18) in the CD28 mutant is mutated to YMFM (SEQ ID NO: 19).

[0010] The intracellular costimulatory domain CD28-YMFM of the CD19-mutant CAR is a mutant of the wild-type CD28-YMNM, and the amino acid sequence is shown in SEQ ID NO: 14.

[0011] In another preferred embodiment, the immune cells are NK cells, macrophages or T cells, preferably T cells.

[0012] In another preferred embodiment, the BCMA CAR and CD19-mutant CAR are located on the cell membrane of the immune cell.

[0013] In a second aspect of the present invention, a method for preparing the engineered immune cells according to the first aspect of the present invention is provided, comprising the following steps:

[0014] (A) providing an immune cell to be modified; and

[0015] (B) Transforming the immune cells so that the immune cells express the BCMA CAR and CD19-mutant CAR, thereby obtaining the engineered immune cells as described in the first aspect of the present invention.

[0016] In another preferred embodiment, step (A) further includes isolating and / or activating the immune cells to be modified.

[0017] In another preferred embodiment, in step (B), it includes (B1) introducing a first expression cassette expressing a first CAR targeting the BCMA into the immune cell; and (B2) introducing a second expression cassette expressing a second CAR targeting CD19 into the immune cell; wherein the step (B1) can be performed before, after, simultaneously with, or alternately with step (B2).

[0018] In another preferred embodiment, in step (B), the first expression cassette and / or the second expression cassette is introduced into the nucleus of the immune cell.

[0019] In another preferred embodiment, the immune cells are NK cells, macrophages or T cells.

[0020] In another preferred embodiment, the first expression cassette contains a nucleic acid sequence encoding the BCMA CAR.

[0021] In another preferred embodiment, the second expression cassette contains a nucleic acid sequence encoding the CD19-mutant CAR.

[0022] In another preferred embodiment, the first expression cassette and the second expression cassette are located on the same or different vectors.

[0023] In another preferred embodiment, the first expression cassette and the second expression cassette are located in the same vector.

[0024] In another preferred embodiment, the first expression cassette and the second expression cassette are located in different vectors.

[0025] In another preferred embodiment, the vector is a viral vector.

[0026] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, other gene transfer systems, or a combination thereof.

[0027] In another preferred embodiment, the vector is a lentiviral vector.

[0028] In another preferred embodiment, the method further comprises the step of performing functional and effectiveness testing on the obtained engineered immune cells.

[0029] In the third aspect of the present invention, a pharmaceutical composition is provided, which contains the engineered immune cells as described in the first aspect of the present invention, and a pharmaceutically acceptable carrier, diluent or excipient.

[0030] In another preferred embodiment, the pharmaceutical composition is a liquid preparation.

[0031] In another preferred embodiment, the dosage form of the pharmaceutical composition includes injection.

[0032] In another preferred embodiment, the concentration of the engineered immune cells in the pharmaceutical composition is 1×10 3 -1×10 8 cells / mL, preferably 1×10 4 -1×10 7 cells / mL.

[0033] In another preferred embodiment, the pharmaceutical composition further contains other drugs for treating cancer or tumors (such as emerging antibody drugs, other CAR-T drugs or chemotherapy drugs).

[0034] In the fourth aspect of the present invention, there is provided a use of the engineered immune cells as described in the first aspect of the present invention or the pharmaceutical composition as described in the third aspect of the present invention for preparing a drug or preparation for preventing and / or treating a disease, wherein the disease is selected from the group consisting of hematological tumors, solid tumors, autoimmune diseases or a combination thereof.

[0035] In another preferred embodiment, the blood tumor is selected from the following group: acute myeloid leukemia, acute lymphocytic leukemia, acute monocytic leukemia, acute granulocytic leukemia, acute myeloid-monocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma (MM), myelodysplastic syndrome, POEMS syndrome, systemic amyloidosis, Waldenstrom's macroglobulinemia, monoclonal gammopathy of unknown origin, or a combination thereof.

[0036] In another preferred embodiment, the autoimmune disease is selected from the following groups: systemic autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, scleroderma, pemphigus, myasthenia gravis, rheumatoid arthritis, Sjögren's syndrome and dermatomyositis; organ-specific autoimmune diseases, including myasthenia gravis, ulcerative colitis, pernicious anemia with chronic atrophic gastritis, Goodpasture's syndrome, pemphigoid, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis, neuromyelitis optica, inflammatory bowel disease, or a combination thereof.

[0037] In another preferred embodiment, the solid tumor is selected from the group consisting of prostate cancer, liver cancer, head and neck cancer, melanoma, non-Hodgkin's lymphoma, bladder cancer, glioblastoma, cervical cancer, lung cancer, chondrosarcoma, thyroid cancer, kidney cancer, mesothelioma, osteosarcoma, bile duct cancer, ovarian cancer, gastric cancer, bladder cancer, meningioma, pancreatic cancer, multiple squamous cell carcinoma, esophageal cancer, small cell lung cancer, colorectal cancer, breast cancer, medulloblastoma, breast cancer, nasopharyngeal cancer, thymic cancer, or a combination thereof.

[0038] In another preferred embodiment, the blood tumor is multiple myeloma, preferably relapsed and / or refractory multiple myeloma.

[0039] In a fifth aspect of the present invention, a method for inhibiting tumor cells in vitro is provided, comprising:

[0040] Tumor cells are contacted with the engineered immune cells according to the first aspect of the present invention, or the pharmaceutical composition according to the third aspect of the present invention, thereby inhibiting the tumor cells.

[0041] In another preferred embodiment, the tumor cells are selected from the group consisting of myeloid leukemia cells, lymphoma cells, and myeloma cells.

[0042] In another preferred embodiment, the tumor cells are selected from: H929 cells, H929-CD19 (OE) cells, and Nalm6 cells.

[0043] In a sixth aspect of the present invention, a method for preventing and / or treating a disease is provided, comprising:

[0044] A safe and effective amount of the engineered immune cells described in the first aspect of the present invention or the pharmaceutical composition described in the third aspect of the present invention is administered to a subject in need of treatment.

[0045] In another preferred embodiment, the subject includes a human or a non-human mammal.

[0046] In another preferred embodiment, the non-human mammals include rodents (such as mice, rats, rabbits) and primates (such as monkeys).

[0047] In another preferred embodiment, the method further comprises administering other drugs for treating cancer or tumors to the subject in need of treatment.

[0048] In another preferred embodiment, the other drugs include CAR-T drugs.

[0049] In another preferred embodiment, the disease is cancer or tumor.

[0050] In another preferred embodiment, the tumor includes a tumor that highly expresses CD19 and / or BCMA.

[0051] In another preferred embodiment, the tumor includes a tumor that expresses both CD19 and BCMA.

[0052] In another preferred embodiment, the tumor that highly expresses CD19 and / or BCMA is multiple myeloma, preferably relapsed and / or refractory multiple myeloma.

[0053] In a seventh aspect of the present invention, a fusion protein is provided, comprising BCMA CAR and CD19-mutant CAR sequences connected by a self-cleavage sequence, wherein the amino acid sequence of the BCMA CAR is shown in SEQ ID NO: 15; the amino acid sequence of the CD19-mutant CAR is shown in SEQ ID NO: 16; wherein the intracellular costimulatory domain of the CD19-mutant CAR comprises a CD28 mutant as shown in SEQ ID NO: 14, wherein the wild-type motif YMNM in the CD28 mutant is mutated to YMFM.

[0054] In another preferred embodiment, the self-cleavage sequence is a T2A sequence.

[0055] In the eighth aspect of the present invention, a polynucleotide is provided, which encodes the fusion protein as described in the seventh aspect of the present invention.

[0056] In another preferred embodiment, the polynucleotide is DNA or RNA.

[0057] In the ninth aspect of the present invention, a vector is provided, wherein the vector comprises the polynucleotide according to the eighth aspect of the present invention.

[0058] In another preferred embodiment, the vector is selected from the group consisting of a plasmid, a viral vector, a transposon, or a combination thereof.

[0059] In another preferred embodiment, the vector is selected from the following group: a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, or a combination thereof.

[0060] In another preferred embodiment, the vector comprises one or more promoters, which are operably connected to the nucleic acid sequence, enhancer, intron, transcription termination signal, polyadenylation sequence, replication origin, selective marker, nucleic acid restriction site, and / or homologous recombination site.

[0061] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 shows a schematic diagram of the structures of B19 CAR, B19-wildtype CAR, BCMA CAR, CD19-mutant CAR, and CD19-wildtype CAR.

[0063] Figure 2 shows the B19 CAR plasmid map.

[0064] Figure 3 shows the B19-wildtype CAR plasmid map.

[0065] Figure 4 shows the CD19-mutant CAR plasmid map.

[0066] Figure 5 shows the CD19-wildtype CAR plasmid map.

[0067] Figure 6 shows the BCMA CAR plasmid map.

[0068] FIG7 shows the detection results of CD19 antigen and BMCA antigen on the cell membrane surface of 7 tumor cells.

[0069] Figure 8 shows the mechanism of B19 CAR-T killing BCMA+ and / or CD19+ tumor cells.

[0070] Figure 9 shows the experimental flow chart.

[0071] Figure 10 shows the expansion curves of the four CAR-T cells before repeated stimulation, where A, B, and C correspond to donors A, E, and C, respectively.

[0072] Figure 11 shows the viability of CAR-T cells during the production phase (donor C).

[0073] Figure 12 shows the test results of CAR-T preparation success rate (A) and CAR-T positive rate (B).

[0074] Figures 13A and 13B show the CAR positivity rates of five CAR-T cell lines during the production phase. Figures 13A and B correspond to donors A and B, respectively.

[0075] FIG14 shows the CAR positivity rate before repeated stimulation (A) and after three rounds of repeated stimulation of H929-luc+Nalm6-luc (B).

[0076] Figure 15 shows the CAR-T cell subtypes before repeated stimulation (A), after 3 rounds of repeated stimulation of H929-luc+Raji-luc (B), and after 3 rounds of repeated stimulation of H929-luc+Nalm6-luc (C).

[0077] FIG16 shows the percentages of CD4+ and CD8+ T cells before (A) and after 3 rounds of H929-luc+Nalm6-luc repeated stimulation (B).

[0078] Figure 17 shows the expression ratios of CAR-T cell inhibitory receptors Tim-3 and LAG-3 after three rounds of repeated stimulation with different tumor cells.

[0079] Figure 18 shows the expansion curves of four types of CAR-T cells after repeated stimulation. (A) H929-luc repeated stimulation, donor A; (B) H929-luc + Nalm6-luc repeated stimulation, donor A; (C) H929-luc + Nalm6-luc repeated stimulation, donor E; (D) H929-CD19(OE)-luc repeated stimulation, donor E.

[0080] Figure 19 shows the killing effects of CAR-T cells at different effector-target ratios after co-incubation with various tumor cell lines before repeated stimulation (A) and after two rounds of repeated stimulation of Nalm6-luc (B).

[0081] Figures 20A and 20B show the amount of CAR-T cell factor release before (A) and after (B) repeated stimulation (donor A).

[0082] Figures 21A and 21B show the amount of CAR-T cell factor release before (A) and after (B) repeated stimulation (donor B).

[0083] Figure 22 shows the changes in fluorescence values ​​of tumor-bearing mice in the efficacy test of CD19-mutant CAR-T and CD19-wildtype CAR-T cells (dose: 1.50E+6 cells / mouse) in the Nalm6-luc tumor-bearing model.

[0084] Figure 23 shows the changes in fluorescence values ​​of tumor-bearing mice in the efficacy test of B19 CAR-T and B19-wildtype CAR-T cells (dose: 3.00E+6 cells / mouse) in the Nalm6-luc tumor-bearing model.

[0085] Figure 24 shows the changes in fluorescence values ​​of tumor-bearing mice in the efficacy test of B19 CAR-T and B19-wildtype CAR-T cells (dose: 3.00E+6 cells / mouse) in the Nalm6-luc+H929-luc tumor-bearing model.

[0086] Figure 25 shows the changes in fluorescence values ​​of tumor-bearing mice in the efficacy test of B19 CAR-T and B19-wildtype CAR-T cells (dose: 1.50E+6 cells / mouse) in the Nalm6-luc+H929-luc tumor-bearing model. DETAILED DESCRIPTION

[0087] After extensive and in-depth research and extensive screening, the inventors have constructed B19 CAR-T cells that simultaneously express BCMA CAR and CD19-mutant CAR. The present invention addresses the problems of therapeutic range, immune escape and safety in existing single-target CAR-T immunotherapy, and constructs T cells expressing dual-target B19 CAR. The characteristics of the dual-target B19 CAR-T cells are as follows: (1) Activated T cells are transduced using lentivirus to express BCMA CAR and CD19-mutant CAR in parallel; (2) In the intracellular costimulatory domain of CD19-mutant CAR, the asparagine residue (N) in the YMNM motif of the intracellular costimulatory domain of the commonly used wild-type CD28 is mutated to a phenylalanine (F) residue. The present invention prepared the CAR-T cells and evaluated the function of CAR-T in vitro and in mice. On this basis, the present invention was completed.

[0088] the term

[0089] In order to more easily understand the present disclosure, some terms are first defined. As used in this application, unless otherwise expressly provided herein, each of the following terms should have the meaning given below. Other definitions are set forth throughout the application.

[0090] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0091] The term "administering" refers to the physical introduction of the product of the invention into a subject using any of a variety of methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, such as by injection or infusion.

[0092] The term "antibody" (Ab) shall include, but is not limited to, immunoglobulins that specifically bind to an antigen and comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding portions thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, CH1, CH2, and CH3.

[0093] It should be understood that the amino acid names herein are identified by internationally accepted single-letter English letters, and the corresponding three-letter abbreviations of the amino acid names are: Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P), Ser (S), Thr (T), Trp (W), Tyr (Y), and Val (V).

[0094] When referring to amino acid or nucleotide sequences, the term "sequence identity" (also referred to as "sequence identity") refers to the amount of identity between two amino acid or nucleotide sequences (e.g., a query sequence and a reference sequence), generally expressed as a percentage. Typically, before calculating the percent identity between two amino acid or nucleotide sequences, the sequences are aligned and gaps (if any) are introduced. For the purposes of the present invention, the publicly available alignment software BLAST (available at the website ncbi.nlm.nih.gov) can be used to obtain optimal sequence alignment and calculate the sequence identity between two amino acid or nucleotide sequences using default settings. In some embodiments, "at least 90% sequence identity" as described herein includes, but is not limited to, at least 95%, at least 98%, at least 99% or even 100% sequence identity.

[0095] BCMA

[0096] BCMA stands for B cell maturation antigen, also known as CD269. It is a member of the TNF receptor superfamily and is only expressed on the surface of mature B cells. It is not expressed in T cells or monocytes. It is an important B cell biomarker. The expression level of BCMA on multiple myeloma cells is significantly higher than that on healthy plasma cells.

[0097] CD19

[0098] CD19 is a 95kDa glycoprotein found on the surface of B cells. It is generally believed that CD19 expression is limited to the B-cell lineage, such as on the surface of most B-cell lymphomas, mantle cell lymphomas, and some acute myeloid leukemia cells. Recent studies have found that plasma cells in multiple myeloma patients also express low levels of CD19, making CD19 a very valuable immunotherapy target.

[0099] Chimeric Antigen Receptor (CAR)

[0100] As used herein, "chimeric antigen receptors (CARs)" are fusion proteins comprising an extracellular domain capable of binding to an antigen, a transmembrane domain derived from a different polypeptide than the extracellular domain, and at least one intracellular domain.

[0101] Specifically, the chimeric antigen receptor (CAR) of the present invention includes the above three structures. The extracellular domain includes a target-specific binding element (also referred to as an antigen binding domain). The intracellular domain includes a costimulatory signaling region and a ζ chain portion. The costimulatory signaling region refers to a portion of the intracellular domain including costimulatory molecules. Costimulatory molecules are cell surface molecules required for the effective response of lymphocytes to antigens, rather than antigen receptors or their ligands.

[0102] A linker can be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR.

[0103] As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that acts to connect a transmembrane domain to an extracellular domain or cytoplasmic domain of a polypeptide chain. The linker may comprise 0-300 amino acids, preferably 2 to 100 amino acids and most preferably 3 to 50 amino acids. Preferably, the linker is a flexible linker, for example, the linker is (G4S)n, where n is 1-4.

[0104] In a preferred embodiment of the present invention, two CARs are included, BCMA CAR and CD19-mutant CAR. When the CAR of the present invention is expressed in T cells, it can recognize antigens based on antigen binding specificity. When it binds to its associated antigen, it affects tumor cells, inhibits tumor cell growth, causes death or is affected in other ways, and causes the patient's tumor burden to be reduced or eliminated. The antigen binding domain is preferably fused with one or more intracellular domains from the costimulatory molecule and the ζ chain. Preferably, the antigen binding domain is fused with the intracellular domain of a combination of 4-1BB and / or CD28 costimulatory domains and CD3ζ signaling domains. As used herein, "antigen binding domain" and "single-chain antibody fragment" all refer to Fab fragments, Fab' fragments, F(ab')2 fragments, or single Fv fragments with antigen binding activity. Fv antibodies contain antibody heavy chain variable regions, light chain variable regions, but no constant region, and are the smallest antibody fragments with all antigen binding sites. Generally, Fv antibodies also include a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding. The antigen-binding domain is typically a single-chain variable fragment (scFv). An scFv is typically one-sixth the size of a complete antibody. A single-chain antibody is preferably a single amino acid sequence encoded by a single nucleotide chain. As a preferred embodiment of the present invention, the scFv comprises an antibody, preferably a single-chain antibody, that specifically recognizes the highly expressed tumor antigens CD19 and / or BCMA.

[0105] With respect to the hinge region and the transmembrane region (transmembrane domain), a CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR.

[0106] As used herein, "B19 CAR-T" refers to the bispecific T cells targeting BCMA and CD19 of the first aspect of the present invention. In the CAR of the present invention, the CD28 costimulatory domain is a mutant, and the asparagine residue (N) in the YMNM motif of the mutant CD28 costimulatory domain is mutated to a phenylalanine (F) residue.

[0107] carrier

[0108] The present invention also provides a DNA construct encoding the CAR sequence of the present invention.

[0109] The nucleic acid sequence encoding the desired molecule can be obtained using recombinant methods known in the art. Alternatively, the gene of interest can be produced synthetically.

[0110] The present invention also provides a vector into which the expression cassette of the present invention is inserted. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer.

[0111] The expression vectors of the present invention can also be constructed using standard gene delivery protocols for nucleic acid immunization and gene therapy. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entireties. In another embodiment, the present invention provides gene therapy vectors.

[0112] The nucleic acid can be cloned into many types of vectors. For example, they include but are not limited to plasmids, phagemids, phage derivatives, animal viruses and cosmids. Specific functional vectors include expression vectors, replication vectors, probe generation vectors and sequencing vectors.

[0113] Further, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. The viruses that can be used as carriers include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and slow viruses. At present, many virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The gene of selection can be inserted into a vector and packaged into retroviral particles using technology known in the art. In some embodiments, adenoviral vectors are used. In some embodiments, slow viral vectors are used.

[0114] Typically, suitable vectors contain at least one origin of replication functional in the organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (eg, WO 01 / 96584; WO 01 / 29058; and US Pat. No. 6,326,193).

[0115] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence that can drive high-level expression of any nucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to 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 (Epstein-Barr) virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, heme promoter, and creatine kinase promoter.

[0116] Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered part of the present invention. The use of inducible promoters provides a molecular switch that turns on expression of a nucleotide sequence operably linked to the inducible promoter when expression is desired, or turns off expression when expression is not desired.

[0117] In order to evaluate the expression of the CAR polypeptide or portion thereof, the expression vector introduced into the cell may also contain either or both a selectable marker gene or a reporter gene to facilitate the identification and selection of expressing cells from the population of cells sought to be transfected or infected by the viral vector.

[0118] Methods for introducing genes into cells and expressing genes in cells are known in the art. In the context of expression vectors, the vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0119] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, microbeads; and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0120] Biological methods for introducing polynucleotides into host cells include the use of viral vectors, particularly retroviral vectors, which have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0121] In a preferred embodiment of the present invention, the vector is a lentiviral vector.

[0122] The DNA construct further comprises a signal peptide coding sequence. Preferably, the signal peptide sequence is connected upstream of the nucleic acid sequence of the antigen binding domain.

[0123] Therapeutic applications

[0124] The present invention provides a method for T cell-mediated immune response, which has the function of immune response to a target cell population or tissue of a mammal, comprising the following steps: administering the CAR-T cells of the present invention to the mammal.

[0125] In one embodiment, the present invention includes a type of cell therapy in which T cells are genetically modified to express the CAR of the present invention, and the CAR-T cells are infused into a recipient in need. The infused cells are able to kill tumor cells in the recipient. Unlike antibody therapies, CAR-T cells are able to replicate in vivo, resulting in long-term persistence that can lead to sustained tumor control.

[0126] In one embodiment, the CAR-T cells of the present invention can undergo robust in vivo T cell expansion and can sustain an extended period of time. Additionally, CAR-mediated immune responses can be part of an adoptive immunotherapy step, where CAR-modified T cells can induce an immune response specific for the antigen binding domain in the CAR.

[0127] Although the data disclosed herein specifically disclose lentiviral vectors comprising an anti-CD19 / BCMA scFv, hinge and transmembrane regions, and a co-stimulatory domain and a CD3ζ signaling domain, the invention should be construed to encompass any number of variations to each of the construct's components.

[0128] Suitable diseases that can be treated include CD19 and / or BCMA-associated cancers or tumors, such as CD19 and / or BCMA-positive tumors or cancers. CD19 and / or BCMA-associated cancers or tumors can include solid tumors, particularly hepatocellular carcinoma, melanoma, ovarian cancer, squamous cell lung carcinoma, gastric cancer, breast cancer, or a combination thereof.

[0129] The types of cancers treated with the CAR of the present invention include, but are not limited to, carcinoma, blastoma, sarcoma, melanoma, and certain benign tumors. According to the age of the patient, they are divided into adult tumors / cancers and pediatric tumors / cancers.

[0130] Solid tumors are abnormal masses of tissue that do not usually contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell types that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcomas, myxosarcoma, liposarcoma, mesothelioma, lymphoid malignancies, pancreatic cancer, and ovarian cancer.

[0131] The CAR-modified T cells of the present invention can also be used as a vaccine type for ex vivo immunization and / or in vivo therapy of mammals. Preferably, the mammal is a human.

[0132] For ex vivo immunization, at least one of the following occurs in vitro prior to administering the cells into a mammal: i) expanding the cells, ii) introducing a nucleic acid encoding a CAR into the cells, and / or iii) cryopreserving the cells.

[0133] In addition to the use of cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against an antigen in a patient.

[0134] Typically, cells activated and expanded as described herein can be used to treat and prevent diseases that occur in immunocompromised individuals. In particular, the CAR-modified T cells of the present invention are used to treat tumors. In certain embodiments, the cells of the present invention are used to treat patients at risk of developing tumors. Therefore, the present invention provides a method for treating or preventing tumors, comprising administering a therapeutically effective amount of a CAR-modified T cell of the present invention to a subject in need thereof.

[0135] The CAR modified T cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or with other components such as IL-2, IL-17 or other cytokines or cell groups. Briefly, the pharmaceutical composition of the present invention may include a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. The composition of the present invention is preferably formulated for intravenous administration.

[0136] The pharmaceutical compositions of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition, and the type and severity of the patient's disease, although appropriate dosages can be determined by clinical trials.

[0137] When an "effective amount," "anti-tumor effective amount," "tumor-inhibitory effective amount," or "therapeutic amount" is indicated, the precise amount of the composition of the present invention to be administered can be determined by a physician, who takes into account individual differences in the patient's (subject's) age, weight, tumor size, degree of infection or metastasis, and condition. It can generally be stated that a pharmaceutical composition comprising the T cells described herein can be administered in an amount of 10 4 to 10 9 The dose of cells / kg body weight is preferably 10 5 to 10 6 The T cell composition can be administered at a dose of 10 cells / kg body weight (including all integer values ​​within those ranges). The T cell composition can also be administered multiple times at these doses. The cells can be administered using infusion techniques well known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676, 1988). The optimal dose and treatment regimen for a specific patient can be easily determined by a person skilled in the art of medicine by monitoring the patient's disease signs and adjusting the treatment accordingly.

[0138] Administration of the subject composition can be carried out in any convenient manner, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to the patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous (iv) injection or intraperitoneally. In one embodiment, the T cell composition of the present invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by iv injection. The composition of the T cell can be injected directly into the tumor, lymph node or infection site.

[0139] In certain embodiments of the invention, cells activated and expanded using the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are administered to a patient in combination (e.g., before, simultaneously, or after) with any number of relevant treatment modalities, including but not limited to treatment with other preventive and / or therapeutic drugs for tumors (particularly multiple myeloma). Other preventive and / or therapeutic compounds can be administered simultaneously with the main active ingredient, or even in the same composition.

[0140] The main advantages of the present invention include:

[0141] a) The preparation success rate of the intracellular costimulatory domain CD28 mutant B19 CAR-T of the present invention is higher than that of B19-wildtype CAR-T, better meeting the acute needs of patients.

[0142] b) The intracellular costimulatory domain CD28 mutant B19 CAR-T of the present invention is superior to B19-wildtype CAR-T, CD19-mutant CAR-T and CD19-wildtype CAR-T in the preparation stage and the expansion after repeated stimulation of tumor cells.

[0143] c) The B19 CAR-T cells of the present invention have a wider range of tumor cell killing capabilities. The B19 CAR-T cells of the present invention can kill BCMA+ single-positive, CD19+ single-positive, and CD19+BCMA+ double-positive tumor cells, expanding the therapeutic range of CAR-T cells and reducing the risk of immune escape caused by downregulation or loss of antigen expression during single-target CAR-T cell therapy, thereby enhancing the therapeutic efficacy of B19 CAR-T cells. In vivo efficacy studies have shown that B19 CAR-T cells can effectively eliminate the aforementioned tumor cells.

[0144] d) The B19 CAR-T of the present invention is better than B19-wildtype CAR-T in killing tumor cells Nalm6-luc, H929-luc, MM1S-luc and Nalm6-luc+H929-luc.

[0145] e) The B19 CAR-T of the present invention is more effective in killing CD19+BCMA- tumor cells Nalm6-luc than CD19-mutant CAR-T and CD19-wildtype CAR-T.

[0146] f) After the B19 CAR-T with a mutated co-stimulatory domain CD28 of the present invention was co-incubated with tumor cells, the release of cytokines IL-2, TNF-α, and IFN-γ was significantly higher than that of B19-wildtype CAR-T, CD19-mutant CAR-T, CD19-wildtype CAR-T, and BCMA CAR-T.

[0147] g) The mutation of the co-stimulatory domain CD28 in the present invention unexpectedly improved the anti-tumor effect of single-target CD19-mutant CAR-T and dual-target B19 CAR-T in mice, and improved the safety of B19 CAR-T

[0148] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0149] Example 1 Plasmid Construction

[0150] The anti-BCMA scFv antibody (clone number C11D5.3, SEQ ID NO: 2) and the anti-CD19 scFv antibody (clone number FMC63, SEQ ID NO: 9) were selected and combined with the corresponding hinge region, transmembrane region, costimulatory domain, and CD3ζ domain to form BCMA CAR, CD19-mutant CAR, and CD19-wildtype CAR. The CD19 and BCMA monospecific CAR coding sequences were connected by T2A sequence to construct B19 CAR and B19-wildtype CAR coding sequences. Suzhou Jinweizhi Biotechnology Co., Ltd. completed the synthesis of B19 CAR, B19-wildtype CAR, CD19-mutant CAR, CD19-wildtype CAR, and BCMA CAR sequences and the preparation of lentiviral vector plasmids.

[0151] The schematic diagram of the structure expressing the above five CARs is shown in Figure 1. The plasmid maps expressing the above five CARs are shown in Figures 2-6.

[0152] Example 2 Tumor cell culture and cell surface antigen detection

[0153] Resuscitated luciferase-expressing tumor cells Nalm6-luc, Raji-luc, MM1S-luc, H929-luc, H929-CD19(OE)-luc, K562-luc, and H929-BCMA(KO)-luc were transferred to culture flasks and supplemented with an appropriate amount of RPMI 1640 + 10% FBS + 1% P / S. The culture flasks were then transferred to a cell culture incubator under 5% CO2, 37°C, and the cell density was maintained at 0.5-2M / mL.

[0154] Flow cytometry: Transfer 0.5-1M tumor cells to a 1.5mL EP tube, add 1mL Rising buffer to resuspend the cells, and centrifuge at 500g for 5 minutes; discard the supernatant, add 100μL 1x Rising buffer to resuspend the cells, add antibodies to each tube, incubate at 4°C in the dark for 30 minutes; add 1mL Rising buffer to wash the cells, centrifuge at 500g for 5 minutes, discard the supernatant, add 200μL Rising buffer to resuspend, and analyze on the flow cytometer.

[0155] The results are shown in Figure 7 and Table 1. The expression of the tumor cell surface antigens BCMA and CD19 is as follows: Raji-luc and Nalm6-luc are CD19+BCMA-; MM1S-luc and H929-luc are CD19-BCMA+; H929-CD19(OE)-luc is CD19+BCMA+; K562-luc and H929-BCMA(KO)-luc are CD19-BCMA-. All seven tumor cell lines above express the luciferase gene. H929-CD19(OE)-luc is a stably transfected cell line overexpressing the CD19 antigen, and H929-BCMA(KO)-luc is a stably transfected cell line with BCMA antigen knockout.

[0156] Table 1 Expression of tumor cell markers

[0157] Example 3 Lentivirus Preparation

[0158] Lentivirus was packaged using PEI transfection reagent. 293TS cells were cultured to optimal levels using CD03 medium containing 6 mM L-glutamine. A fourth-generation lentiviral packaging system was used. Two cell culture shake flasks were prepared and the suspension cell culture medium was added at 5% of the total volume of the suspended cells. To flask #1, the CAR-expressing plasmid and packaging plasmid were added in a 4:2:2:1 ratio. To flask #2, PEI transfection reagent was added at a volume twice the total amount of plasmid, vortexed to mix, and allowed to stand at room temperature for 5 minutes. After the vortexing period, the PEI solution in flask #2 was pipetted into flask #1, gently shaking flask #1. Once the solution was completely added, the lid was closed, vortexed to mix, and the incubation was allowed to stand at room temperature for 15 minutes. After the PEI solution was allowed to stand for a while, the PEI solution was transferred to a cell culture shake flask containing 293TS, and then the cell culture shake flask was placed in a carbon dioxide constant temperature shaking incubator at 37° C., 5% CO 2 , and a shaking table at 140 rpm for 48 h.

[0159] The lentiviral supernatant was collected from the cell culture flask, and small molecule impurities such as HCP and nuclease were removed from the lentiviral supernatant using a hollow fiber column. Protein particles of different target sizes were separated using a Capto Core 700. Microbial impurities such as bacteria were removed from the lentiviral supernatant using a 0.22 μm Bursa filter. Finally, the lentiviral supernatant was concentrated to obtain high-purity, high-titer lentivirus.

[0160] Example 4 T cell sorting and activation

[0161] After PBMC recovery, resuspend and count in 9 volumes of Rinsing buffer. Centrifuge at 300g for 10 minutes, discard the supernatant, resuspend the cells according to a specific ratio, and add the corresponding volume of CD3 magnetic beads. Mix thoroughly, and incubate at 4°C for 30 minutes, mixing every 10 minutes. After washing and resuspending the cells, separate them using a MACS column to obtain highly pure CD3+ T cells.

[0162] Dilute CD3+ T cells to 1.00E+6 / mL using culture medium containing 10% FBS (X-VIVO-15 + 10% FBS + 1% P / S + 100 IU / mL IL-2). Transfer 2 mL of the cell suspension to a 24-well plate and add 20 μL of TransACT activator to each well. This date is designated as Day 0. Place the 24-well plate in a 37°C CO2 incubator for 48 hours, then centrifuge to remove the activator.

[0163] Example 5 T cell transduction and CAR-T cell expansion

[0164] After 48 hours of activation, T cells were collected by centrifugation and resuspended to 1M / mL in X-VIVO 15 + IL-2. 1 mL of T cells was added to each well of a 24-well plate. B19 CAR, B19-wildtype CAR, CD19-mutant CAR, CD19-wildtype CAR, and BCMA CAR lentivirus were added at an MOI of 3. A mock T control well without virus was set up. After adding 10 μL of DMSO to each well and culturing for 24 h (Day 3), 0.5 mL of fresh culture medium X-VIVO 15 + IL-2 was added, and on Day 4, the medium was replaced with complete culture medium (culture medium composition: X-VIVO-15 + 10% FBS + 1% P / S + 100 U / mL IL-2) for passage. The CAR-T cell culture density was maintained at 5.00E+5 cells / mL to 1.00E+6 cells / mL; CAR-T cells were collected every 2-3 days, and cell viability was detected using a cell counter.

[0165] A schematic diagram of B19 CAR-T cell killing of tumor cells is shown in Figure 8. B19 CAR-T cells can kill CD19+ single-positive, BCMA+ single-positive, and CD19+BCMA+ double-positive tumor cells. The experimental flow chart for CAR-T preparation is shown in Figure 9.

[0166] Unexpectedly, the expansion fold of cells containing the CD28-YMFM mutant was significantly increased compared to CD28 wild-type (CD28-YMNM) CAR-T cells. As shown in Figure 10(A), the expansion folds of B19 CAR-T, B19-wildtype CAR-T, CD19-mutant CAR-T, and CD19-wildtype CAR-T were 13.35, 10.12, 6.31, and 6.49, respectively. As shown in Figure 10(B), the expansion folds of B19 CAR-T, B19-wildtype CAR-T, CD19-mutant CAR-T, and CD19-wildtype CAR-T were 147.38, 85.08, 47.66, 52.16, and 35.05, respectively. As shown in Figure 10(C), the expansion folds of B19 CAR-T and B19-wildtype CAR-T were 40.19 and 13.02, respectively. This indicates that B19 CAR-T cells exhibit superior in vitro expansion compared to B19-wildtype CAR-T cells. Furthermore, the CD28-YMFM mutation was unexpectedly found to significantly improve the viability of CAR-T cells during the production phase. As shown in Figure 11, during the CAR-T production phase, particularly in the early stages of CAR-T production, the viability of B19 CAR-T cells was 99.01%, significantly higher than the 78.5% observed for B19-wildtype CAR-T cells.

[0167] In summary, mutations in the intracellular co-stimulatory domain of CD28 increased the expansion multiple and activity rate of B19 CAR-T, showing significant advantages.

[0168] Example 6 CAR-T Positive Rate, Preparation Success Rate, Phenotype and Typing

[0169] Count CAR-T cells before or after restimulation (preparation phase) and transfer 0.5-1.00E+6 cells to each 1.5mL EP tube. Add 1mL of 1× Rising buffer to each tube, centrifuge at 500g for 5 minutes, discard the supernatant, and resuspend in 100μL of 1× Rising buffer. Add the recommended dosage of commercially available detection antibody to each tube and incubate at 4°C in the dark for 30-40 minutes. Wash the cells with 1mL of 1× Rising buffer per tube, centrifuge at 500g for 5 minutes, discard the supernatant, and resuspend in 300μL of 1× Rising buffer. Analyze on a flow cytometer.

[0170] The results of this example are shown in Figures 12-13. As shown in Figure 12, donor B was transduced with the B19 CAR lentivirus for a total of six times, with CAR-T cells successfully generated three times. Donor B was transduced with the B19-wildtype CAR lentivirus for a total of six times, with only one successful CAR-T cell generation. Donor D was transduced with the B19 CAR lentivirus for a total of two times, with one successful CAR-T cell generation. Donor D was transduced with the B19-wildtype CAR lentivirus for a total of two times, with no successful CAR-T cell generation.

[0171] The results of the four groups of CAR-T preparations showed that the success rate of B19 CAR-T preparation was higher than that of B19-wildtype CAR-T preparation, whether for multiple batches of cells from the same donor or for cells from multiple different donors, which can better meet the acute medication needs of patients and benefit them. Because the difference between B19 CAR and B19-wildtype CAR is a mutation of a single amino acid residue in the CD28 costimulatory domain, it is believed that the improved success rate of CAR-T preparation is likely due to the mutation of a single amino acid residue in CD28. Figures 13A and 13B show that the positive rate of successfully prepared CAR-T is greater than 20%, and it is used for subsequent experiments.

[0172] Figure 14 shows that the CAR+ ratios of B19 CAR-T, B19-wildtype CAR-T, CD19-mutant CAR-T, CD19-wildtype CAR-T, and BCMA CAR-T were significantly increased after repeated stimulation.

[0173] Figure 15 shows that before repeated stimulation, i.e., in the preparation stage, there was no significant difference in the percentage (%) of Tn+Tscm+Tcm in T cells between B19 CAR-T and the other four CAR-T types. However, after repeated stimulation with tumor cells, the percentage (%) of Tn+Tscm+Tcm in T cells of B19 CAR-T was higher than that of the other four CAR-T types, indicating that B19 CAR-T may have a better anti-tumor effect in vivo.

[0174] Figure 16 shows that after repeated stimulation, the proportion of CD8+ T cells in B19 CAR-T, B19-wildtype CAR-T, CD19-mutant CAR-T, CD19-wildtype CAR-T, and BCMA CAR-T cells increased.

[0175] Figure 17 shows that there was no significant difference in the expression ratios of Tim-3 and LAG-3 between B19 CAR-T and B19-wildtype CAR-T after three rounds of repeated stimulation with different tumor cells.

[0176] In summary, mutations in the intracellular co-stimulatory domain of CD28 increased the success rate of B19 CAR-T preparation and the ratio of Tn+Tscm+Tcm after repeated stimulation.

[0177] Example 7 Targeted Proliferation of CAR-T Cells

[0178] 1. Target Cell Preparation

[0179] Collect well-growing target cells in a 15ml centrifuge tube and centrifuge at 300g for 5 minutes. Discard the supernatant. Resuspend the cells in culture medium to a density of 1M / ml and treat with Mitomycin C at a final concentration of 1ug / mL overnight. The next day, wash the cells twice with 1× Rising Buffer, centrifuge at 300g for 5 minutes, and discard the supernatant. Resuspend the cells in X-VIVO-15 medium supplemented with 10% FBS, count the cells using a Count Star cell counter, and determine cell viability. Dilute the cells to a density of 1.00E+06 / mL for later use.

[0180] 2. Effector Cell Preparation

[0181] B19 CAR-T, B19-wildtype CAR-T, CD19-mutant CAR-T, CD19-wildtype CAR-T, and Mock T cells were centrifuged at 200g for 10 minutes. The cells were resuspended in X-VIVO-15 medium, counted using a Count Star, and adjusted to a cell density of 1.00E+06 / mL (based on the number of CAR-positive cells; the number of Mock T cells was based on the number of T cells) for later use.

[0182] 3. Co-incubation of effector cells and target cells

[0183] Treated effector and target cells were added to a 24-well plate at an effector:target ratio of 2:1. 200 μL of target cells (0.20E+06 / mL) and 400 μL of CAR-T cells (0.40E+06 / mL) were added to each well. 400 μL of X-VIVO-15 medium supplemented with 10% FBS and 1% P / S was added to each well. After addition, the 24-well plate was gently shaken to mix the cells evenly. The cells were then cultured in a 37°C, 5% CO2 incubator.

[0184] 4. Multiple rounds of tumor cell proliferation stimulation

[0185] Effector cells (CAR-T cells) and target cells (tumor cells) were co-incubated for 48 hours, and the cells in the 24-well plate were mixed with a pipette. The CAR-T cells were counted, and 300 μL of cells were taken from the previous round of wells and added to the next round of wells. 200 μL of target cells (0.20E+06 / mL) were added to each well. 500 μL of X-VIVO-15 + 10% FBS + 1% P / S medium was then added to each well to make up the culture system to 1 mL. At the same time, the above steps were repeated for the third, fourth, and fifth rounds of repeated stimulation experiments. Finally, the remaining tumor cells were observed and the final proliferation times of the CAR-T cells were counted.

[0186] The results are shown in Figure 18. After repeated stimulation with tumor cells H929-luc, H929-luc+Nalm6-luc, and H929-CD19(OE)-luc, the expansion of B19 CAR-T was significantly better than that of B19-wildtype CAR-T, CD19-mutant CAR-T, and CD19-wildtype CAR-T, suggesting that B19 CAR-T has a long-lasting ability to kill tumor cells.

[0187] Example 8 Detection of CAR-T cell killing ability before repeated stimulation

[0188] 1. Effector Cell Preparation

[0189] B19 CAR-T, B19-wildtype CAR-T, CD19-mutant CAR-T, CD19-wildtype CAR-T, and mock T cells were collected and centrifuged at 200g for 10 minutes. The supernatant was discarded. The cells were resuspended in X-VIVO 15 pure culture medium and counted using a Count Star. The CAR-T cell density was adjusted to 2.00E+5 cells / mL, 1.00E+5 cells / mL, 5.00E+4 cells / mL, 2.50E+4 cells / mL, and 1.25E+4 cells / mL (based on the number of CAR-positive cells). The effector-target ratio was set at 1:1, 1:2, 1:4, 1:8, and 1:16.

[0190] 2. Target Cell Preparation

[0191] Collect target cells in good growth condition in a centrifuge tube, centrifuge at 400g for 3 minutes, and discard the supernatant; resuspend the cells in X-VIVO 15 pure culture medium, count and detect cell viability using a Count star cell counter, and finally dilute the cell density to 2.00E+5 cells / mL for use.

[0192] 3. Co-incubation of effector cells and target cells

[0193] 100 μL of target cells and 100 μL of CAR-T cells were mixed at a 1:1 ratio (three replicates per sample) and added to a 96-well microplate. The plates were incubated at 37°C, 5% CO₂ for 72 hours. After incubation, the plates were centrifuged at 500 g for 5 minutes. 100 μL of supernatant was aspirated from each well and stored at -80°C for subsequent cytokine detection. 50 μL of ONE-Glo™ luciferase Assay System substrate was then added to each well. The plates were mixed thoroughly and allowed to stand for 5 minutes before fluorescence was measured on a TECAN microplate reader (wavelength 560 nm).

[0194] 4. Calculation of CAR-T cell killing rate

[0195] Killing rate (%) = (control group - experimental group) / control group x 100%

[0196] The results are shown in Figure 19(A). For CD19+BCMA- cells (Nalm6-luc), at low efficiency to target ratios, such as an E:T ratio of 1:8, the tumor cell killing rates of B19 CAR-T, B19-wildtype CAR-T, CD19-mutant CAR-T, and CD19-wildtype CAR-T were 71.80%, 54.99%, 38.38%, and 38.38%, respectively. Similarly, at an E:T ratio of 1:16, the tumor cell killing rates of B19 CAR-T, B19-wildtype CAR-T, CD19-mutant CAR-T, and CD19-wildtype CAR-T were 49.04%, 30.61%, 24.60%, and 19.67%, respectively. The results suggest that B19 dual-target CAR-T cells not only expand the range of target cells, but also have a significantly higher killing ability against target cells that only express CD19 than CD19 single-target CAR-T cells. This effect is particularly evident at low efficiency target ratios.

[0197] CD28 mutations also have a certain impact on the lethality of CAR-T cells. At E:T ratios of 1:4 and 1:8, B19 CAR-T cells were significantly more effective against H929-luc tumor cells than B19-wildtype CAR-T cells. At E:T ratios of 1:4, 1:8, and 1:16, B19 CAR-T cells were significantly more effective against MM1S-luc tumor cells than B19-wildtype CAR-T cells.

[0198] In summary, mutations in the intracellular co-stimulatory domain of CD28 improve the tumor-killing effect of B19 CAR-T.

[0199] Example 9 Detection of CAR-T cell killing ability after repeated stimulation

[0200] 1. Effector Cell Preparation

[0201] After repeated stimulation, B19 CAR-T cells, B19-wildtype CAR-T cells, CD19-mutant CAR-T cells, CD19-wildtype CAR-T cells, and mock T cells were collected and centrifuged at 200g for 10 minutes. The supernatant was discarded. The cells were resuspended in X-VIVO 15 pure medium and counted using a Count Star. The CAR-T cell density was adjusted to 2.00E+5 cells / mL, 1.00E+5 cells / mL, 5.00E+4 cells / mL, 2.50E+4 cells / mL, and 1.25E+4 cells / mL (calculated based on the number of CAR-positive cells). The effector-target ratio was 1:1, 1:2, 1:4, 1:8, and 1:16. A control group without effector cells was also established.

[0202] 2. Target Cell Preparation

[0203] Collect target cells in good growth condition in a centrifuge tube, centrifuge at 500g for 5 minutes, and discard the supernatant; resuspend the cells in X-VIVO 15 pure culture medium, count and detect cell viability using a Count star cell counter, and finally dilute the cell density to 2.00E+5 cells / mL for use.

[0204] 3. Co-incubation of effector cells and target cells

[0205] 100 μL of target cells and 100 μL of CAR-T cells were mixed at a 1:1 ratio (three replicates per sample) and added to a 96-well microplate. Three replicates were added and incubated at 37°C, 5% CO2 for 72 hours. After incubation, the plates were centrifuged at 500 g for 5 minutes. 100 μL of supernatant was aspirated from each well and stored in a -80°C refrigerator for subsequent cytokine detection. 50 μL of ONE-Glo™ luciferase Assay System substrate was added to each well. Mix well, let stand for 5 minutes, and then measure fluorescence (wavelength 560 nm) on a TECAN microplate reader.

[0206] 5. Calculation of CAR-T cell killing rate

[0207] Killing rate (%) = (control group - experimental group) / control group x 100%

[0208] The results are shown in Figure 19(B). After two rounds of repeated stimulation of tumor cells Nalm6-luc, B19 CAR-T has a significant advantage in killing Nalm6-luc and Nalm6-luc+H929-luc cells compared with B19-wildtype CAR-T.

[0209] The above results show that regardless of whether the target cells are repeatedly stimulated, the tumor cell killing rate of B19 CAR-T is significantly better than that of B19-wildtype CAR-T and monospecific CD19 CAR-T, and has better anti-tumor effect.

[0210] Based on Examples 8 and 9, regardless of whether target cells were repeatedly stimulated or not, the B19 CAR-T cells containing the mutant structure of the CD28 intracellular co-stimulatory domain were significantly better at killing tumors than the B19 wildtype CAR-T cells and other single-target CAR-T cells.

[0211] Example 10 Detection of CAR-T cell factor release ability

[0212] Remove the supernatant from the -80°C freezer before and after repeated stimulation for cytokine analysis. Add 50 μL of the corresponding cell incubation supernatant from each group to an appropriate number of 1.5 mL EP tubes. Centrifuge at 500 g for 5 minutes and collect 50 μL of the supernatant for cytokine analysis.

[0213] According to the instructions of the CBA detection kit, determine the cytokines and number of samples to be tested, and prepare capture beads: remove the bottle of cytokine capture beads to be tested, mix vigorously, and remove (number of samples to be tested + number of negative controls) × 15 μL / 6 of capture beads for each magnetic bead, and mix each magnetic bead by turbine shaking; take n 1.5 mL EP tubes (n = number of samples + number of controls) and add 15 μL of mixed magnetic beads to each tube (sample, negative control); add 15 μL of the corresponding test agent (sample, control) to each tube; add 15 μL of PE Detection Reagent to each tube, mix thoroughly, and incubate at room temperature for 3 hours in the dark; after incubation, add 500 μL of wash buffer to each sample, mix thoroughly, centrifuge at 500g for 5 minutes, and discard the supernatant; resuspend with 100 μL of wash buffer, centrifuge at 500g for 5 minutes, discard the supernatant, and resuspend the sample with 100 μL of wash buffer; analyze on the flow cytometer.

[0214] The results are shown in Figures 20A, 20B, 21A, and 21B. Cytokine release after co-incubation of CAR-T cells without repeated tumor cell stimulation with tumor cells is shown in Figure 20A (see Tables 2-4 for specific values). After co-incubation of CAR-T cells with Raji-luc or H929-CD19(OE)-luc tumor cells, the amount of IL-2 released by B19 CAR-T cells was significantly greater than that released by B19-wildtype CAR-T, CD19-mutant CAR-T, CD19-wildtype CAR-T, and BCMA CAR-T cells. After co-incubation of CAR-T cells with H929-CD19(OE)-luc tumor cells, the amount of TNF-α released by B19 CAR-T cells was significantly greater than that released by B19-wildtype CAR-T, CD19-mutant CAR-T, CD19-wildtype CAR-T, and BCMA CAR-T cells. After coincubation of CAR-T cells with H929-CD19(OE)-luc tumor cells, the IFN-γ release of B19 CAR-T cells was significantly superior to that of B19-wildtype CAR-T and CD19-mutant CAR-T cells. This indicates that single amino acid residue mutations in the CD28 co-stimulatory domain of the CAR structure and the design of the parallel CAR structure can enhance cytokine release by B19 CAR-T, suggesting that the parallel structure of B19 CAR-T may have better therapeutic efficacy in treating diseases.

[0215] Figure 20B shows the cytokine release of CAR-T cells after repeated stimulation with Nalm6-luc tumor cells and co-incubation with tumor cells (see Tables 5-7 for specific values). After co-incubation of CAR-T cells after repeated stimulation with tumor cells and either Raji-luc or H929-CD19(OE)-luc tumor cells, B19 CAR-T cells released significantly more IL-2 than B19-wildtype CAR-T cells and other single-target CAR-T cells. Furthermore, CD19-mutant CAR-T cells released significantly more IL-2 than CD19-wildtype CAR-T cells. After co-incubation of CAR-T cells after repeated stimulation with H929-CD19(OE)-luc tumor cells, B19 CAR-T cells released significantly more IFN-γ and TNF-α than B19-wildtype CAR-T cells.

[0216] Figures 21A-21B show similar results (see Tables 8-10 and 11-13 for specific values, respectively). Before and after repeated stimulation, the release of IL-2, TNF-α, and IFN-γ by B19 CAR-T cells co-incubated with target cells was superior to that of B19-wildtype CAR-T cells and other single-target CAR-T cells. Therefore, B19 CAR-T cells have better expansion and tumor killing effects in vitro.

[0217] In summary, regardless of repeated stimulation of target cells, mutations in the intracellular co-stimulatory domain of CD28 significantly increased the release of cytokines IL-2, TNF-α, and IFN-γ by B19 CAR-T.

[0218] Table 2

[0219] Table 3

[0220] Table 4

[0221] Table 5

[0222] Table 6

[0223] Table 7

[0224] Table 8

[0225] Table 9

[0226] Table 10

[0227] Table 11

[0228] Table 12

[0229] Table 13

[0230] Example 11 In vivo test

[0231] 6-8 week old B-NDG mice were selected and injected with tumor cells intravenously (Day-7). One day later (Day-6), the tumor graft burden was measured. On Day 0, the mice were divided into groups and then injected with CAR-T cells intravenously. After CAR-T treatment, the tumor burden of the mice was evaluated on the set dates (Figure 22 (A), Figure 23 (A), Figure 24 (A), Figure 25 (A)). Each mouse was injected intraperitoneally with 0.3 mg of D-luciferin (YEASEN), and 4 minutes after the injection, the mouse was photographed under isoflurane anesthesia using a Xenogen IVIS imaging system (Perkin Elmer Life Sciences) for 30 seconds. The bioluminescent signal was calculated based on the amount of photons emitted, and the photon amount was normalized by exposure time and surface area to obtain the photon amount / s / cm. 2 / Solid surface (p / s / cm 2 / sr).

[0232] The in vivo efficacy data in Figure 22 show that in the Nalm6-luc tumor-bearing model (tumor cell inoculation: 0.4E+6 Nalm6-luc cells / mouse), both CD19-mutant CAR-T and CD19-wildtype CAR-T cells had tumor-killing effects at different test time points at a dose of 1.50E+6 cells / mouse. On Day 5 and Day 9, CD19-mutant CAR-T cells were more effective in clearing tumors than CD19-wildtype CAR-T cells. As shown in Figures 22(B) and 22(C), on Day 9, CD19-mutant CAR-T cells completely cleared the tumor, and no recurrence occurred on Day 14. On Day 14, CD19-wildtype CAR-T cells completely cleared the tumor. Fourteen days after administration, one animal died in the vehicle group; three animals died in the non-CAR-transfected mock T group; and no animals died in the other groups. This indicates that mutations in the costimulatory domain CD28 can enhance the anti-tumor effect of CD19 CAR. During the experiment, the body weight of mice in the vehicle group and the Mock T group decreased significantly, while no toxic side effects affecting body weight were observed in the CD19-mutant CAR-T and CD19-wildtype CAR-T groups (Table 14). Therefore, CD19-mutant CAR and CD19-wildtype CAR were combined with BCMA CAR to prepare BCMA and CD19 dual-target CAR-T cells to further confirm whether mutations in the costimulatory domain CD28 enhance the anti-tumor effect of dual CAR-T.

[0233] The in vivo efficacy data in Figure 23 show that in a Nalm6-luc tumor-bearing model (tumor cell inoculation: 0.4E+6 Nalm6-luc cells / animal), both B19 CAR-T cells and B19-wildtype CAR-T cells, administered at a dose of 3.00E+6 cells / animal, demonstrated tumor-killing activity at various time points. As shown in Figures 23(B) and 23(C), on Day 7, B19 CAR-T cells were more effective at clearing tumors than B19-wildtype CAR-T cells. B19 CAR-T cells essentially completely eliminated tumors, while B19-wildtype CAR-T cells failed to do so. On Day 14, both B19 CAR-T and B19-wildtype CAR-T cells completely eliminated tumors. Fourteen days after administration, five animals in the vehicle group died; four animals in the non-CAR-transfected mock T group died; and no animals in the remaining groups died. During the experiment, mice in the vehicle, Mock T, and B19-wildtype CAR-T groups experienced significant weight loss, while B19 CAR-T showed no toxic side effects that affected body weight (Table 15). This suggests that mutations in the costimulatory domain CD28 can enhance B19 CAR-T's ability to kill CD19+ tumor cells and its anti-tumor effects, while mitigating the toxic side effects of CAR-T cells.

[0234] The in vivo efficacy data in Figure 24 show that in the Nalm6-luc + H929-luc tumor-bearing model (tumor cell inoculation: 0.4E+6 Nalm6-luc cells and 5E+6 H929-luc cells / cell), at different test time points, both B19 CAR-T cells and B19-wildtype CAR-T cells, administered at a dose of 3.00E+6 cells / cell, had tumor-killing effects. As shown in Figures 24(B) and 24(C), on Day 7, B19 CAR-T cells were more effective in clearing tumors than B19-wildtype CAR-T cells. B19 CAR-T cells essentially completely cleared the tumor, while B19-wildtype CAR-T cells failed to completely clear the tumor. On Day 14, both B19 CAR-T and B19-wildtype CAR-T cells completely cleared the tumor. Fourteen days after administration, four mice died in the vehicle group; four died in the non-CAR-transfected Mock T group; and no animals died in the other groups. During the experiment, mice in the vehicle, Mock T, and B19-wildtype CAR-T groups experienced significant weight loss, while the B19 CAR-T group showed no toxic side effects that affected body weight (Table 16). This suggests that mutations in the costimulatory domain CD28 can enhance the B19 CAR-T's ability to kill CD19+BCMA+ tumor cells and its anti-tumor effects, while mitigating the toxic side effects of CAR-T cells.

[0235] The in vivo efficacy data in Figure 25 show that in the Nalm6-luc + H929-luc tumor-bearing model (tumor cell inoculation: 0.4E+6 Nalm6-luc cells and 5E+6 H929-luc cells / cell), at different test time points, both B19 CAR-T cells and B19-wildtype CAR-T cells, administered at a dose of 1.50E+6 cells / cell, demonstrated tumor killing. As shown in Figures 25(B) and 25(C), on Day 7, B19 CAR-T cells were more effective at clearing tumors than B19-wildtype CAR-T cells. B19 CAR-T cells essentially completely eliminated the tumor, while B19-wildtype CAR-T cells failed to completely eliminate the tumor. On Day 14, both B19 CAR-T and B19-wildtype CAR-T cells completely eliminated the tumor. Fourteen days after administration, five mice in the non-CAR-transfected Mock T group died, while no animals in the other groups died. During the experiment, mice in the vehicle and Mock T groups experienced significant weight loss, while the B19 CAR-T and B19-wildtype CAR-T groups showed no toxic side effects affecting body weight (Table 17). This suggests that mutations in the costimulatory domain CD28 can enhance the B19 CAR-T's ability to kill CD19+BCMA+ tumor cells and enhance its anti-tumor efficacy.

[0236] The in vivo efficacy tests in Figures 22-25 show that the mutation in the costimulatory domain CD28 improves the killing effect of B19 CAR-T on tumor cells Nalm6-luc and Nalm6-luc+H929-luc; at a dose of 3.00E+6 cells / mouse, the body weight of mice in the B19 CAR-T group decreased significantly compared with that in the B19-wildtype CAR-T group, while there was no significant change in the body weight of mice in the B19 CAR-T group, indicating that the mutation in the costimulatory domain CD28 can improve the safety of B19 CAR-T.

[0237] In summary, the mutation of the costimulatory domain CD28 unexpectedly enhanced the anti-tumor effect of single-target CD19-mutant CAR-T and dual-target B19 CAR-T in mice, and improved the safety of B19 CAR-T.

[0238] Table 14

[0239] Table 15

[0240] Table 16

[0241] Table 17

[0242] Table 18 Sequence Listing

[0243] In SEQ ID NO: 2 and SEQ ID NO: 9, a single underline indicates the light chain variable region of the scFv, and a double underline indicates the heavy chain variable region of the scFv.

[0244] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. An engineered immune cell, characterized in that: The engineered immune cells express BCMA CAR and CD19-mutant CAR in parallel, the amino acid sequence of the BCMA CAR is shown in SEQ ID NO: 15; the amino acid sequence of the CD19-mutant CAR is shown in SEQ ID NO: 16; wherein the intracellular co-stimulatory domain of the CD19-mutant CAR comprises a CD28 mutant as shown in SEQ ID NO: 14, and the wild-type motif YMNM in the CD28 mutant is mutated to YMFM.

2. A method for preparing engineered immune cells according to claim 1, characterized in that: The following steps are involved: (A) providing an immune cell to be modified; and (B) transforming the immune cells so that the immune cells express the BCMA CAR and CD19-mutant CAR, thereby obtaining the engineered immune cells as described in claim 1.

3. A fusion protein, characterized in that The fusion protein comprises BCMA CAR and CD19-mutant CAR sequences connected by a self-cleavage sequence, the amino acid sequence of the BCMA CAR is shown in SEQ ID NO: 15; the amino acid sequence of the CD19-mutant CAR is shown in SEQ ID NO: 16; wherein the intracellular co-stimulatory domain of the CD19-mutant CAR comprises a CD28 mutant as shown in SEQ ID NO: 14, and the wild-type motif YMNM in the CD28 mutant is mutated to YMFM.

4. A polynucleotide, characterized in that The polynucleotide encodes the fusion protein according to claim 3.

5. A carrier, characterized in that The vector comprises the polynucleotide according to claim 4.

6. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the engineered immune cell as claimed in claim 1, and a pharmaceutically acceptable carrier, diluent or excipient.

7. A use of the engineered immune cell according to claim 1 or the pharmaceutical composition according to claim 6, characterized in that: Used for preparing a medicine or preparation for preventing and / or treating a disease, wherein the disease is selected from the following group: a blood tumor, a solid tumor, an autoimmune disease or a combination thereof.

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