Bispecific CAR-T cells targeting BCMA and CD19

Abstract

This invention relates to CAR-T cells targeting BCMA and CD19. Specifically, the invention provides a structure for the parallel expression of a chimeric antigen receptor (CAR) that simultaneously targets B cell maturation antigen (BCMA) and CD19 antigen molecules, and optimizes the motif of the co-stimulatory domain CD28. The invention further provides applications of the chimeric antigen receptor for adoptive T cell therapy for B cell-related conditions.

Description

[Technical Field]

[0001] This invention relates to the field of biopharmaceuticals, and more specifically, to B19 CAR-T cells that target BCMA and CD19. [Background technology]

[0002] CD269, a B cell maturation antigen (BCMA), is a member of the tumor necrosis factor receptor superfamily member 17 (TNFRS17). Composed of 185 amino acid residues, it is a type III transmembrane protein with a cysteine-rich extracellular domain and exhibits certain conservation. As a cell surface protein expressed only in B cell lineages, it plays a crucial regulatory role in B cell proliferation, survival, maturation, and differentiation into plasma cells, along with two other TNF receptor superfamily members: the B-cell activation factor (BAFF) receptor and transmembrane activator, as well as the calcium modulator and cyclophilin ligand (TACI). These three receptors support the long-term survival of B cells at various developmental stages by binding to BAFF and / or its homologous ligand, APRIL. Studies using BCMA knockout mice further demonstrate that BCMA is not essential for overall B cell homeostasis, but is important for the survival of long-lived plasma cells. Simultaneously, BCMA is highly expressed in malignant MM plasma cells, and its expression increases as the disease progresses. Gene and protein expression profiling analyses confirm that BCMA is the most selectively expressed cell surface receptor in MM cell lines, making it a very promising and ideal target for CAR-T cell therapy against MM.

[0003] CD19 is a 95kDa glycoprotein found on the surface of B cells, expressed from the early stages of B cell development 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, is involved in regulating B cell receptor signaling processes. In CD19-deficient mouse models, the number of B cells in peripheral lymphoid tissue is significantly reduced, along with decreased responses to vaccines and mitogens, and simultaneously, decreased serum Ig levels. CD19 expression is generally considered to be limited to B-cell lineages and not expressed on the surface of pluripotent hematopoietic stem cells. 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 highly valuable immunotherapy target in the treatment of leukemia / lymphoma. Most normal cells, such as pluripotent hematopoietic stem cells, do not express CD19 on their surface. This characteristic allows CD19 to function as a safe therapeutic target, minimizing the risk of patients developing autoimmune disease or irreversible myelotoxic injury. Antibodies or scFv fragments of CD19 are currently being developed, and their application has been demonstrated as promising in mouse models and human / primate animals. After treatment with CD19 CAR-T cells expressing CD28 or 4-1BB, pediatric and adult patients with relapsed or refractory acute B-cell lymphoma show a complete remission rate of approximately 90%. Recently, the overall remission rate of CD19 CAR-T cell therapy in diffuse large B-cell lymphoma, follicular lymphoma, or chronic lymphoma has been 50% to 100%. Because terminally differentiated plasma cells do not express CD19, and malignant B cell precursors continue to generate malignant plasma cells, CD19 CAR-T cells have a clinical advantage in the treatment of multiple myeloma.

[0004] Furthermore, for CD19-negative relapsed and low-CD19-expressing B-cell / plasmacytic neoplasms, the development of dual-target BCMA and CD19 CAR-T cells may address the issues of CD19 being prone to mutations, having low expression levels, and being immune to evasion. Therefore, in this field, it is necessary to develop CAR-T cells that simultaneously target BCMA and CD19. [Overview of the project] [Problems that the invention aims to solve]

[0005] The object of the present invention is to provide CAR-T cells that target the parallel structure of BCMA and CD19, as well as modifications to the intracellular CD28 costimulatory domain and their applications.

[0006] The present invention jointly targets the dual tumor targets of BCMA and CD19, possessing strong specificity and high targetability, effectively improving and extending the therapeutic effect of CAR-T therapy, exhibiting excellent therapeutic efficacy against multiple myeloma, leukemia, or B-cell lymphoma that are positive for surface antigens BCMA and CD19, effectively avoiding off-target escape of single targets, and improving the safety of CAR-T drugs. [Means for solving the problem]

[0007] A first aspect of the present invention provides engineered immune cells that express BCMA CAR and CD19-mutant CAR in parallel, wherein the amino acid sequence of the BCMA CAR is as shown in SEQ ID NO:15, and the amino acid sequence of the CD19-mutant CAR is as shown in SEQ ID NO:16, wherein the intracellular costimulatory domain of the CD19-mutant CAR includes a CD28 mutant as shown in SEQ ID NO:14, and the wild-type motif YMNM (SEQ ID NO:18) in the CD28 mutant is mutated to YMFM (SEQ ID NO:19).

[0008] The intracellular costimulatory domain CD28-YMFM of the aforementioned CD19-mutant CAR is a mutant of wild-type CD28-YMNM, and its amino acid sequence is as shown in SEQ ID NO:14.

[0009] In another preferred example, the immune cells are NK cells, macrophages, or T cells, preferably T cells.

[0010] In another preferred example, the BCMA CAR, CD19-mutant CAR is located on the cell membrane of the immune cell.

[0011] A second aspect of the present invention is: (A) The step of providing immune cells to be modified, and (B) A method for preparing engineered immune cells according to a first aspect of the present invention is provided, comprising the step of modifying the immune cells to express the BCMA CAR and the CD19-mutant CAR, thereby obtaining engineered immune cells according to a first aspect of the present invention.

[0012] In another preferred example, step (A) further includes isolating and / or activating the immune cells to be modified.

[0013] In another preferred example, step (B) includes (B1) introducing a first expression cassette expressing a first CAR targeting the BCMA into the immune cells, and (B2) introducing a second expression cassette expressing a second CAR targeting CD19 into the immune cells, wherein step (B1) can be performed before, after, simultaneously with, or in conjunction with step (B2).

[0014] In another preferred example, in step (B), the first expression cassette and / or the second expression cassette are introduced into the cell nucleus of the immune cells.

[0015] In another preferred example, the immune cells are NK cells, macrophages or T cells.

[0016] In another preferred example, the first expression cassette contains a nucleic acid sequence encoding the BCMA CAR.

[0017] In another preferred example, the second expression cassette contains a nucleic acid sequence encoding the CD19-mutant CAR.

[0018] In another preferred example, the first expression cassette and the second expression cassette are located on the same or different vectors.

[0019] In another preferred example, the first expression cassette and the second expression cassette are located on the same vector.

[0020] In another preferred example, the first expression cassette and the second expression cassette are located on different vectors.

[0021] In another preferred example, the vector is a viral vector.

[0022] In another preferred example, the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adenoviral vector, retroviral vector, transposon, other gene delivery systems, or combinations thereof.

[0023] In another preferred example, the vector is a lentiviral vector.

[0024] In another preferred example, the method further includes a step of detecting the function and efficacy of the obtained engineered immune cells.

[0025] A third aspect of the present invention provides a pharmaceutical composition comprising engineered immune cells as described in the first aspect of the present invention, as well as a pharmaceutically acceptable carrier, diluent, or excipient.

[0026] In another preferred example, the pharmaceutical composition is a liquid formulation.

[0027] In another preferred example, the dosage form of the pharmaceutical composition includes an injectable preparation.

[0028] In another preferred example, the concentration of the manipulated immune cells in the pharmaceutical composition is 1 × 10 3 ~1 × 10 8 cells / mL, preferably 1 × 10⁶ 4 ~1 × 10 7 The concentration is cells / mL.

[0029] In another preferred example, the pharmaceutical composition further comprises other drugs for treating cancer or tumors (e.g., emerging antibody drugs, other CAR-T drugs, or chemotherapy drugs).

[0030] A fourth aspect of the present invention provides a use for 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, used in the preparation of a drug or formulation for the prevention and / or treatment of a disease, wherein the disease is selected from the group consisting of hematological malignancies, solid tumors, autoimmune diseases or combinations thereof.

[0031] In another preferred example, the hematological malignancies are selected from the group consisting of acute myeloid leukemia, acute lymphoblastic leukemia, acute monocytic leukemia, acute myeloblastic leukemia, acute myeloid monocytic leukemia, chronic lymphocytic leukemia, chronic myeloblastic leukemia, chronic myeloid leukemia, lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, multiple myeloma (MM), myelodysplastic syndrome, POEMS syndrome, systemic amyloidosis, Waldenstrom macroglobulinemia, monoclonal gammaglobulinemia of unknown cause, or a combination thereof.

[0032] In another preferred example, the solid tumor is selected from the group consisting of prostate cancer, liver cancer, head and neck cancer, melanoma, non-Hodgkin lymphoma, bladder cancer, glioblastoma, cervical cancer, lung cancer, chondrosarcoma, thyroid cancer, kidney cancer, mesothelioma, osteosarcoma, bile duct cancer, ovarian cancer, stomach cancer, bladder cancer, meningioma, pancreatic cancer, multiple squamous cell tumors, esophageal cancer, small cell lung cancer, colorectal cancer, breast cancer, medulloblastoma, breast cancer, nasopharyngeal cancer, thymic cancer, or a combination thereof.

[0033] In another preferred example, the hematological malignancy is multiple myeloma, preferably relapsed and / or refractory multiple myeloma.

[0034] A fifth aspect of the present invention provides a method for inhibiting tumor cells in vitro, comprising contacting tumor cells with manipulated immune cells described in the first aspect of the present invention or a pharmaceutical composition described in the third aspect of the present invention, thereby inhibiting the tumor cells.

[0035] In another preferred example, the tumor cells are selected from myeloid leukemia cells, lymphoma cells, and myeloma cells.

[0036] In another preferred example, the tumor cells are selected from H929 cells, H929-CD19(OE) cells, and Nalm6 cells.

[0037] A sixth aspect of the present invention provides a method for preventing and / or treating a disease, comprising administering to a subject in need of treatment a safe and effective amount of manipulated immune cells according to the first aspect of the present invention, or a pharmaceutical composition according to the third aspect of the present invention.

[0038] In another preferred example, the subject includes humans or non-human mammals.

[0039] In another preferred example, the non-human mammals include rodents (e.g., mice, rats, rabbits) and primates (e.g., monkeys).

[0040] In another preferred example, the method further includes administering to a subject in need of treatment another drug for treating cancer or tumor.

[0041] In another preferred example, the other drugs include CAR-T drugs.

[0042] In another preferred example, the disease is cancer or a tumor.

[0043] In another preferred example, the tumor includes a tumor that highly expresses CD19 and / or BCMA.

[0044] In another preferred example, the tumor includes a tumor that simultaneously expresses CD19 and BCMA.

[0045] In another preferred example, the tumor highly expressing CD19 and / or BCMA is multiple myeloma, preferably relapsed and / or refractory multiple myeloma.

[0046] A seventh aspect of the present invention provides a fusion protein comprising a BCMA CAR and a CD19-mutant CAR sequence linked by a self-cleaving sequence, wherein the amino acid sequence of the BCMA CAR is as shown in SEQ ID NO:15, and the amino acid sequence of the CD19-mutant CAR is as 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, and the wild-type motif YMNM in the CD28 mutant is mutated to YMFM.

[0047] In another preferred example, the self-cleaving sequence is a T2A sequence.

[0048] An eighth aspect of the present invention provides a polynucleotide which encodes a fusion protein as described in the seventh aspect of the present invention.

[0049] In another preferred example, the polynucleotide is DNA or RNA.

[0050] A ninth aspect of the present invention provides a vector comprising a polynucleotide as described in the eighth aspect of the present invention.

[0051] In another preferred example, the vector is selected from the group consisting of plasmids, viral vectors, transposons, or combinations thereof.

[0052] In another preferred example, the vector is selected from the group consisting of lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, or combinations thereof.

[0053] In another preferred example, the vector comprises one or more promoters, the promoters operably linked to the nucleic acid sequence, enhancers, introns, transcription termination signals, polyadenylation sequences, replication origins, selection markers, nucleic acid restriction sites, and / or homologous recombination sites. [Effects of the Invention]

[0054] It should be understood that, within the scope of the present invention, new or preferred technical solutions can be constructed by combining the above-described technical features of the present invention with the technical features specifically described below (e.g., in the examples). Due to space limitations, this will not be repeated here. [Brief explanation of the drawing]

[0055] [Figure 1] Schematic diagrams of the structures of B19 CAR, B19-wildtype CAR, BCMA CAR, CD19-mutant CAR, and CD19-wildtype CAR are shown. [Figure 2] The B19 CAR plasmid map is shown. [Figure 3] This shows a map of the B19-wildtype CAR plasmid. [Figure 4] This shows a CD19-mutant CAR plasmid map. [Figure 5] This shows a map of CD19-wild-type CAR plasmids. [Figure 6] The BCMA CAR plasmid map is shown. [Figure 7] The results of detecting CD19 antigen and BMCA antigen on the surface of seven types of tumor cell membranes are shown. [Figure 8] This diagram illustrates the mechanism by which B19 CAR-T cells kill BCMA+ and / or CD19+ tumor cells. [Figure 9] The flowchart of the experiment is shown below. [Figure 10] The amplification curves of four types of CAR-T cells before repeated stimulation are shown, where A, B, and C correspond to donors A, E, and C, respectively. [Figure 11] This shows the survival rate of CAR-T cells (donor C) during the preparation stage. [Figure 12] The detection results for CAR-T preparation success rate (A) and CAR-T positivity rate (B) are shown. [Figure 13A] The CAR-positive rates of five types of CAR-T cells during the preparation stage are shown. Figure 13A corresponds to donor A. [Figure 13B] The CAR-positive rates of five types of CAR-T cells during the preparation stage are shown. Figure 13B corresponds to donor B. [Figure 14] The CAR positivity rates are shown before repeated stimulation (A) and after 3 rounds of repeated stimulation with H929-luc + Nalm6-luc (B). [Figure 15] The CAR-T cell subtypes are shown before repeated stimulation (A), after 3 rounds of repeated stimulation with H929-luc + Raji-luc (B), and after 3 rounds of repeated stimulation with H929-luc + Nalm6-luc (C). [Figure 16] The percentages of CD4+ and CD8+ T cells before repeated stimulation (A) and after 3 rounds of repeated stimulation with H929-luc + Nalm6-luc (B) are shown. [Figure 17]The expression ratios of CAR-T cell inhibitory receptors Tim-3 and LAG-3 are shown after three rounds of repeated stimulation with different tumor cells. [Figure 18] The amplification curves of four types of CAR-T cells after repeated stimulation are shown. (A) Repeated stimulation with H929-luc, donor A; (B) Repeated stimulation with H929-luc + Nalm6-luc, donor A; (C) Repeated stimulation with H929-luc + Nalm6-luc, donor E; (D) Repeated stimulation with H929-CD19(OE)-luc, donor E. [Figure 19] Co-incubation of CAR-T cells with multiple tumor cell lines before repeated stimulation (A) and after two rounds of repeated stimulation with Nalm6-luc (B) shows killing effects under different effector:target ratios. [Figure 20A] This shows the amount of CAR-T cytokines released before repeated stimulation (A) (Donor A). [Figure 20B] This shows the amount of CAR-T cytokines released after repeated stimulation (B) (donor A). [Figure 21A] This shows the amount of CAR-T cytokines released before repeated stimulation (A) (donor B). [Figure 21B] This shows the amount of CAR-T cytokines released after repeated stimulation (B) (donor B). [Figure 22] This shows the changes in fluorescence values ​​in tumor-bearing mice during efficacy studies of CD19-mutant CAR-T and CD19-wild-type CAR-T cells (dose: 1.50E+6 cells / mice) in a Nalm6-luc tumor model. [Figure 23] This shows the changes in fluorescence values ​​in tumor-bearing mice during a drug efficacy study of Nalm6-luc tumor model using B19 CAR-T and B19-wild-type CAR-T cells (dose: 3.00E+6 cells / mouse). [Figure 24] This shows the changes in fluorescence values ​​in tumor-bearing mice during efficacy studies of Nalm6-luc+H929-luc in a tumor-bearing model using B19 CAR-T and B19-wild-type CAR-T cells (dose: 3.00E+6 cells / mice). [Figure 25]This shows the changes in fluorescence values ​​in tumor-bearing mice during drug efficacy studies of Nalm6-luc+H929-luc in a tumor-bearing model using B19 CAR-T and B19-wild-type CAR-T cells (dose: 1.50E+6 cells / mice). [Modes for carrying out the invention]

[0056] After extensive and thorough research and large-scale screening, the inventors constructed B19 CAR-T cells that simultaneously express BCMA CAR and CD19-mutant CAR. Considering the problems of therapeutic range, immune escape, and safety in existing single-target CAR-T immunotherapy, the present invention constructs T cells expressing dual-target B19 CAR, and the characteristics of these dual-target B19 CAR-T cells are as follows: (1) Activated T cells are transduced using a lentivirus to express BCMA CAR and CD19-mutant CAR in parallel; (2) In the intracellular costimulatory domain of the CD19-mutant CAR, the asparagine residue (N) in the YMNM motif of the intracellular costimulatory domain of normal wild-type CD28 is mutated to a phenylalanine (F) residue. The present invention prepares the CAR-T cells and evaluates CAR-T function in vitro and in vivo in mice. Based on this, the present invention was completed.

[0057] term To facilitate understanding of this disclosure, we first define certain terms. Unless otherwise specified herein, each of the following terms should have the meanings set forth below, as used in this invention. Other definitions are explained throughout the invention. The term "approximately" can refer to a specific value or configuration within a set acceptable margin of error determined by those skilled in the art, which depends in part on how the value or configuration is measured or measured. The term "administration" refers to the physical introduction of the product of the present invention into a target area using any of the various methods and delivery systems known to those skilled in the art, including intravenous, intramuscular, subcutaneous, intraperitoneal, bone marrow, or other parenteral routes of administration (e.g., by injection or infusion). The term "antibody" (Ab) includes, but is not limited to, immunoglobulins comprising at least two heavy (H) chains and two light (L) chains, or their antigen-binding moieties, that specifically bind to an antigen and are interconnected by disulfide bonds. 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.

[0058] In this specification, amino acid names are denoted by a single English letter that is commonly used internationally, and the three-letter abbreviations for the corresponding amino acid names are Ala(A), Arg(R), Asn(N), Asp(D), Cys(C), Gln(Q), Glu(E), Gly(G), His(H), I1e(I), Leu(L), Lys(K), Met(M), Phe(F), Pro(P), Ser(S), Thr(T), Trp(W), Tyr(Y), and Val(V). When referring to amino acid or nucleotide sequences, the term “sequence identity” (also called “sequence identity”) refers to the degree of similarity between two amino acid or nucleotide sequences (e.g., a query sequence and a reference sequence), and is generally expressed as a percentage. Typically, before calculating the percentage of similarity between two amino acid or nucleotide sequences, a sequence alignment and any gaps (if any) are first introduced. For the purposes of this invention, the publicly available alignment software BLAST (available on the website ncbi.nlm.nih.gov) is used, with default settings, to obtain the optimal sequence alignment and calculate the sequence identity between the two amino acid or nucleotide sequences. In some embodiments, the “at least 90% sequence identity” as described in this application includes, but is not limited to, situations of at least 95%, at least 98%, at least 99%, or even 100% sequence identity.

[0059] BCMA BCMA, also known as CD269, is a B cell maturation antigen. It belongs to the TNF receptor superfamily and is expressed only on the surface of mature B cells, not on T cells or monocytes. It is an important B cell biomarker, and the expression level of BCMA on multiple myeloma cells is significantly higher than that on healthy plasma cells.

[0060] CD19 CD19 is a 95kDa glycoprotein found on the surface of B cells, and its expression is typically thought to be limited to B-cell lineages, such as the surface of tumor cells in most B-cell lymphomas, mantle cell lymphomas, and some acute myeloid leukemia cells. Several recent studies have also found low CD19 expression in plasma cells of multiple myeloma patients, making CD19 a very valuable immunotherapy target.

[0061] Chimeric antigen receptor (CAR) 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 polypeptide different from the extracellular domain, and at least one intracellular domain.

[0062] Specifically, the chimeric antigen receptor (CAR) of the present invention comprises the three structures described above. The extracellular domain includes a target-specific binding element (also called 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 that contains costimulatory molecules. Costimulatory molecules are cell surface molecules necessary for an effective lymphocyte response to an antigen, and are neither antigen receptors nor their ligands.

[0063] A linker can be incorporated between the extracellular domain and the transmembrane domain of a CAR, or between the cytoplasmic domain and the transmembrane domain of a CAR.

[0064] As used herein, the term “linker” typically refers to any oligopeptide or polypeptide that helps to link a transmembrane domain to the extracellular or cytoplasmic domain of a polypeptide chain. A linker may contain 0 to 300 amino acids, preferably 2 to 100 amino acids, most preferably 3 to 50 amino acids. Preferably, the linker is a flexible linker, for example, said linker is (G4S)n, where n is 1 to 4.

[0065] A preferred embodiment of the present invention includes two types of CARs, a BCMA CAR and a CD19-mutant CAR. When the CAR of the present invention is expressed on T cells, it can identify antigens based on antigen-binding specificity. When it binds to its associated antigen, it affects tumor cells, inhibits tumor cell growth, causes death, or otherwise affects them, resulting in a reduction or elimination of the patient's tumor burden. The antigen-binding domain is preferably fused to a costimulatory molecule and one or more intracellular domains in the ζ chain. Preferably, the antigen-binding domain is fused to an intracellular domain in combination with a 4-1BB and / or CD28 costimulatory domain and a CD3ζ signaling domain. As used herein, “antigen-binding domain” and “single-chain antibody fragment” all refer to a Fab fragment, Fab' fragment, F(ab')2 fragment, or single Fv fragment having antigen-binding activity. An Fv antibody is the smallest antibody fragment that includes an antibody heavy chain variable region and a light chain variable region, but does not include a constant region, and has all antigen-binding sites. Typically, Fv antibodies further contain a polypeptide linker between the VH and VL domains and can form a structure necessary for antigen binding. The antigen-binding domain is usually an scFv (single-chain variable fragment). The size of an scFv is usually 1 / 6 the size of a complete antibody. A single-chain antibody is preferably a single amino acid chain sequence encoded by a single nucleotide chain. In a preferred embodiment of the present invention, the scFv comprises an antibody, preferably a single-chain antibody, that specifically recognizes highly expressed tumor antigens CD19 and / or BCMA.

[0066] Regarding the hinge region and transmembrane region (transmembrane domain), CARs can be designed to include a transmembrane domain fused to the extracellular domain of the CAR.

[0067] As used herein, “B19 CAR-T” refers to a bispecific T cell targeting BCMA and CD19 according to a first aspect of the present invention. In the CAR of the present invention, the CD28 costimulatory domain is mutant, and the asparagine residue (N) in the YMNM motif of the mutant CD28 costimulatory domain is mutated to a phenylalanine (F) residue.

[0068] vector The present invention further provides DNA constructs encoding the CAR sequence of the present invention.

[0069] Nucleic acid sequences encoding desired molecules can be obtained using recombinant methods known in the art. Selectable and interesting genes can be synthesized and produced.

[0070] 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 long-term gene transfer.

[0071] The expression vectors of the present invention can also be constructed using standard gene delivery protocols for nucleic acid immunotherapy and gene therapy. Methods of gene delivery are known in the art, for example, see U.S. Patents 5,399,346, 5,580,859, 5,589,466, etc., which are incorporated herein by reference in their entirety. In another embodiment, the present invention provides gene therapy vectors.

[0072] The nucleic acid can be cloned into various types of vectors. These include, but are not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Specific functional vectors include expression vectors, replication vectors, probe-generating vectors, and sequencing vectors.

[0073] Furthermore, expression vectors can be delivered to cells in the form of viral vectors. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Manual of Experiments, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology handbooks. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Currently, many virus-based systems for gene delivery into mammalian cells are being developed. For example, retroviruses provide a convenient platform for gene delivery systems. Using techniques known in the art, selected genes can be inserted into vectors and packaged into retroviral particles. In some embodiments, adenovirus vectors are used. In some embodiments, lentiviral vectors are used.

[0074] Typically, a suitable vector includes a replication origin that functions in at least one organism, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers (e.g., WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193).

[0075] One example of a suitable promoter is the pre-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a potent constitutive promoter sequence that can drive high-level expression of any nucleotide sequence operably ligated to it. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences can also be used. These include, but are not limited to, the Simian virus 40 (SV40) early promoter, mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus pre-early promoter, Ruth sarcoma virus promoter, and human gene promoters such as, but are not limited to, the actin promoter, myosin promoter, heme promoter, and creatine kinase promoter.

[0076] Furthermore, the present invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered part of the present invention. The use of an inducible promoter provides a molecular switch that turns on the expression of a nucleotide sequence operably linked to the inducible promoter when expression is desired, or turns off expression when expression is undesirable.

[0077] To evaluate the expression of a CAR polypeptide or a portion thereof, the expression vector introduced into cells may also include either or both a selectable marker gene or a reporter gene to facilitate the identification and selection of expressing cells from a cell population intended to be transfected or infected with a viral vector.

[0078] Methods for introducing genes into cells and expressing those genes are known in the art. In the case of expression vectors, the vector can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be introduced into host cells by physical, chemical, or biological means.

[0079] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle impact, microinjection, and electroporation. 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 polymer complexes, nanocapsules, microspheres, and microbeads, as well as lipid-based systems such as oil-in-water emulsions, micelles, mixed micelles, and liposomes.

[0080] Biological methods for introducing polynucleotides into host cells, including the use of viral vectors, particularly retroviral vectors, are the most widely used methods 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, for example. See, for example, U.S. Patents 5,350,674 and 5,585,362.

[0081] In a preferred embodiment of the present invention, the vector is a lentiviral vector.

[0082] The DNA construct further comprises a signal peptide coding sequence. Preferably, the signal peptide sequence is ligated upstream of the nucleic acid sequence of the antigen-binding domain.

[0083] Therapeutic applications The present invention provides a method for a T cell-mediated immune response, the method having the function of an immune response against a target cell population or tissue in a mammal, and comprising the step of administering the CAR-T cells of the present invention to a mammal.

[0084] In one embodiment, the present invention comprises a type of cell therapy in which T cells are genetically modified to express the CAR of the present invention and are injected into receptors that require CAR-T cells. The injected cells can kill tumor cells in the body at the receptor. Unlike antibody therapy, CAR-T cells can replicate in vivo, resulting in long-lasting effects that provide sustained control of the tumor.

[0085] In one embodiment, the CAR-T cells of the present invention can induce robust T cell proliferation in vivo and persist for a long period of time. Furthermore, the CAR-mediated immune response may be part of the adoptive immunotherapy stage, where CAR-modified T cells can induce an immune response specific to the antigen-binding domain within the CAR.

[0086] The data disclosed herein specifically disclose lentiviral vectors comprising anti-CD19 / BCMA scFv, hinges and transmembrane regions, as well as co-stimulatory domains and CD3ζ signaling domains, but the present invention should be interpreted as including any number of variations in each construct.

[0087] The indications for which this treatment can be administered include CD19 and / or BCMA-related cancers or tumors, such as CD19 and / or BCMA-positive tumors or cancers. CD19 and / or BCMA-related cancers or tumors may include solid tumors, particularly hepatocellular carcinoma, melanoma, ovarian cancer, squamous cell carcinoma of the lung, gastric cancer, breast cancer, or combinations thereof.

[0088] The types of cancer treated by the CAR of the present invention include, but are not limited to, carcinoma, blastoma, sarcoma, melanoma, and certain benign tumors. Depending on the patient's age, they are classified into adult tumors / cancers and pediatric tumors / cancers.

[0089] Solid tumors are abnormal masses of tissue that typically do not contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. Different types of solid tumors are named after the type of cells that form them (e.g., sarcomas, carcinomas, and lymphomas). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, mesothelioma, lymphoma, pancreatic cancer, and ovarian cancer.

[0090] The CAR-modified T cells of the present invention can also be used as a type of vaccine for ex vivo immunization and / or in vivo therapy in mammals. Preferably, the mammal is human.

[0091] In ex vivo immunization, at least one of the following is performed in vivo before administering cells to a mammal: i) growing the cells, ii) introducing CAR-encoding nucleic acids into the cells, and / or iii) treating the cells.

[0092] 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 induce an immune response to an antigen in a patient.

[0093] Typically, the activated and proliferated cells described herein can be used to treat and prevent diseases 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. Accordingly, the present invention provides a method for treating or preventing tumors, comprising administering a therapeutically effective amount of the CAR-modified T cells of the present invention to a subject in need.

[0094] The CAR-modified T cells of the present invention can be administered alone or as part of a pharmaceutical composition in combination with diluents and / or other components such as IL-2, IL-17, or other cytokines or cell populations. In short, the pharmaceutical compositions of the present invention may contain the target cell populations described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. The compositions of the present invention are preferably formulated for intravenous administration.

[0095] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The dosage and frequency of administration are determined by factors such as the patient's condition, the type and severity of the patient's disease, etc., but the appropriate dose may be determined by clinical trials.

[0096] Where “effective dose,” “antitumor effective dose,” “tumor-inhibitory effective dose,” or “therapeutic dose” is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician, taking into account the patient's age, weight, tumor size, degree of infection or introduction, and individual differences in the disease. Generally, the pharmaceutical compositions containing T cells described herein are 10 4 ~10 9 A dose of cells / kg body weight, preferably 10 5 ~10 6 It can be said that the cells can be administered in doses of cells / kg body weight (including all integer values ​​within that range). 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 (e.g., Rosenberg et al., NewEng. J. of Med. 319:1676, 1988). The optimal dose and treatment plan for a specific patient can be easily determined by a person skilled in the medical field by monitoring the patient's signs of disease and adjusting the treatment accordingly.

[0097] The composition may be administered by any convenient method, including spraying, injection, swallowing, infusion, implantation, or transplantation. The compositions described herein may be administered to a patient by subcutaneous, intradermal, intratumoral, intranodal, intraspinal, intramuscular, intravenous (iv) injection, or intraperitoneal injection. In one embodiment, the T cell composition of the present invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the present invention is preferably administered by intravenous injection. The T cell composition may be injected directly into a tumor, lymph node, or infected site.

[0098] In certain embodiments of the present invention, cells are activated and proliferated using the method described herein or other methods known in the Art for proliferating T cells to therapeutic levels and administered to a patient in combination with any number of relevant therapeutic forms (e.g., before, simultaneously with, or after), the therapeutic forms including, but not limited to, treatment with the agent in combination with other prophylactic and / or therapeutic agents for tumors (in particular multiple myeloma), and other prophylactic and / or therapeutic compounds, even in the same composition, may be administered simultaneously with the main active ingredient.

[0099] The main advantages of this invention are as follows: a) The success rate of preparing the intracellular costimulatory domain CD28 mutant B19 CAR-T according to the present invention is higher than that of B19-wild-type CAR-T, and can better meet the urgent needs of patients. b) The intracellular costimulatory domain CD28 mutant B19 CAR-T of the present invention exhibits superior proliferation during the preparation stage and after repeated stimulation of tumor cells compared to B19-wild-type CAR-T, CD19-mutant CAR-T, and CD19-wild-type CAR-T. c) The B19 CAR-T of the present invention can kill a wider range of tumor cells. The B19 CAR-T 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, reducing the risk of immune escape caused by downregulation or deletion of antigen expression during single-target CAR-T cell therapy, and improving the therapeutic effect of B19 CAR-T. In vivo efficacy studies have shown that B19 CAR-T can effectively eliminate the above-mentioned tumor cells. d) The B19 CAR-T of the present invention is more effective than B19-wild-type CAR-T in killing tumor cells Nalm6-luc, H929-luc, MM1S-luc, and Nalm6-luc+H929-luc. e) The B19 CAR-T of the present invention is more effective than CD19-mutant CAR-T and CD19-wild-type CAR-T in killing CD19+BCMA-tumor cells Nalm6-luc. f) After co-incubating the B19 CAR-T with the CD28 co-stimulatory domain of the present invention with tumor cells, the release of cytokines IL-2, TNF-α, and IFN-γ was significantly higher than that of B19-wild-type CAR-T, CD19-mutant CAR-T, CD19-wild-type CAR-T, and BCMA CAR-T. g) The mutation of the co-stimulatory domain CD28 in the present invention unexpectedly enhances the antitumor efficacy of single-target CD19 mutant CAR-T and dual-target B19 CAR-T in mice, and also improves the safety of B19 CAR-T.

[0100] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are used solely for the purpose of illustrating the present invention and do not limit its scope. In the following examples, experimental methods that do not specify conditions typically follow conventional conditions, such as those described in Sambrook et al., Molecular Cloning: An Experimental Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or conditions proposed by the manufacturer. Unless otherwise specified, percentages and parts are calculated as weight percentages and weight parts.

[0101] Example 1. Plasmid construction Anti-BCMA scFv antibody (clone number C11D5.3, SEQ ID NO:2) and anti-CD19 scFv antibody (clone number FMC63, SEQ ID NO:9) were selected, and their corresponding hinge regions, transmembrane regions, costimulatory domains, and CD3ζ domains were combined with BCMA CAR, CD19-mutant CAR, and CD19-wildtype CAR, respectively. The CD19 and BCMA monospecific CAR coding sequences were ligated via the T2A sequence to construct the B19 CAR and B19-wildtype CAR coding sequences. Suzhou GENEWIZ 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.

[0102] The schematic diagrams of the structures expressing the five CARs mentioned above are shown in Figure 1. Plasmid maps expressing the five CARs mentioned above are shown in Figures 2-6.

[0103] Example 2. Tumor cell culture and detection of cell surface antigens Luciferase-expressing tumor cells Nalm6-luc, Raji-luc, MM1S-luc, H929-luc, H929-CD19(OE)-luc, K562-luc, and H929-BCMA(KO)-luc were resuscitated, and the resuscitated tumor cells were transferred to a culture flask, and an appropriate amount of RPMI 1640 + 10% FBS + 1% P / S was added. Then the culture flask was transferred to a cell incubator, and the culture conditions were 5% CO2, 37°C, and the cell density was maintained at 0.5~2 M / mL.

[0104] Flow cytometry detection: Transfer 0.5-1M tumor cells to a 1.5 mL EP tube, resuspend the cells with 1 mL of rising buffer, centrifuge at 500 g for 5 minutes, discard the supernatant, resuspend the cells with 100 μL of 1× rising buffer, add antibody to each tube, incubate at 4°C in the dark for 30 minutes, wash the cells with 1 mL of rising buffer, centrifuge at 500 g for 5 minutes, discard the supernatant, resuspend the cells with 200 μL of rising buffer, and run the detection test on a machine.

[0105] The results are shown in Figure 7 and Table 1, and the expression status 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+, and K562-luc and H929-BCMA(KO)-luc are CD19-BCMA-. All seven types of tumor cells express the luciferase gene, H929-CD19(OE)-luc is a stably transduced cell line that overexpresses the CD19 antigen, and H929-BCMA(KO)-luc is a stably transduced cell line that knocks out the BCMA antigen. [Table 1]

[0106] Example 3. Preparation of lentivirus PEI transfection reagent is used for lentivirus packaging. To culture 293TS cells under optimal conditions, use CD03 medium containing 6 mM L-glutamine. A fourth-generation lentivirus packaging system is used. Prepare two cell culture shaking flasks and add cell suspension medium in an amount equal to 5% of the total volume of suspended cells. Add the plasmid expressing CAR and the packaging plasmid in a ratio of 4:2:2:1 to cell culture shaking flask No. 1, and add twice the total volume of plasmid transfection reagent PEI to cell culture shaking flask No. 2. Shake to mix uniformly and allow to stand at room temperature for 5 minutes. After standing, use a pipette to add all of the PEI solution from cell culture shaking flask No. 2 to cell culture shaking flask No. 1, gently shaking cell culture shaking flask No. 1 while adding, and once all additions are complete, lid and allow to permeate to mix uniformly, then allow to stand at room temperature for 15 minutes. After the PEI solution has settled, transfer it to a cell culture shaking flask containing 293TS. Then, place the cell culture shaking flask in a carbon dioxide constant temperature shaking incubator and continue culturing for 48 hours under conditions of 37°C, 5% CO2, and 140 rpm with a shaker.

[0107] The lentiviral supernatant is collected from the cell culture flask, small molecular impurities such as HCP and nucleases are removed from the lentiviral supernatant using a hollow fiber column, protein particles of various desired sizes are separated using a Capto Core 700, microbial impurities such as bacteria are removed from the lentiviral supernatant using a 0.22 μm capsule filter, and finally the lentiviral supernatant is concentrated to obtain high-purity, high-titer lentivirus.

[0108] Example 4. Selection and activation of T cells After PBMC resuscitation, the cells are resuspended in 9 times the volume of rinsing buffer, counted, centrifuged at 300g for 10 minutes, the supernatant is discarded, the cells are resuspended according to a specific ratio, and the corresponding volume of CD3 magnetic beads is added simultaneously. After homogeneous mixing, the cells are allowed to stand at 4°C for 30 minutes, and then homogeneously mixed once at 10-minute intervals. After washing and resuspending the cells, they are separated using a MACS column to obtain high-purity CD3+ T cells.

[0109] Using a medium containing 10% FBS (X-VIVO-15 + 10% FBS + 1% P / S + 100 IU / mL IL-2), CD3+ T cells were diluted to 1.00 E+6 / mL, and 2 mL of the cell suspension was placed in a 24-well plate. 20 μL of TransACT activator was added to each well. This date is day 0. The 24-well plate was placed in a 37°C carbon dioxide incubator and incubated for 48 hours, after which the activator was removed by centrifugation.

[0110] Example 5. Transduction of T cells and proliferation of CAR-T cells After collecting T cells activated for 48 hours by centrifugation, they are resuspended to 1 M / mL with X-VIVO 15+IL-2, a 24-well plate is prepared, 1 mL of T cells are added to each well, and B19 CAR, B19-wild-type CAR, CD19-mutant CAR, CD19-wild-type CAR, and BCMA CAR lentivirus are added according to MOI=3 to set up virus-free mock T (MockT) control wells. Add 10 μL of DMSO to each well and culture for 24 hours (day 3). Then, replenish with 0.5 mL of fresh medium X-VIVO 15 + IL-2, and on day 4, replace and subculture the medium with complete medium (medium composition: X-VIVO-15 + 10% FBS + 1% P / S + 100 U / mL IL-2). Maintain the subculture density of CAR-T cells at 5.00E+5 cells / mL to 1.00E+6 cells / mL. Collect CAR-T cells every 2-3 days, count them using a cell counter, and detect cell viability.

[0111] A schematic diagram of B19 CAR-T cells that kill 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. An experimental flowchart for CAR-T cell preparation is shown in Figure 9. Unexpectedly, the amplification factors of cells containing the CD28-YMFM mutant were significantly higher than those of CD28 wild-type (CD28-YMNM) CAR-T cells. As shown in Figure 10(A), the amplification factors for B19 CAR-T, B19 wild-type CAR-T, CD19 mutant CAR-T, and CD19 wild-type CAR-T were 13.35, 10.12, 6.31, and 6.49, respectively. As shown in Figure 10(B), the amplification factors for B19 CAR-T, B19 wild-type CAR-T, CD19 mutant CAR-T, and CD19 wild-type CAR-T were 147.38, 85.08, 47.66, 52.16, and 35.05, respectively. As shown in Figure 10(C), the amplification factors for B19 CAR-T and B19-wildtype CAR-T were 40.19 and 13.02, respectively. This suggests that in vitro proliferation of B19 CAR-T is superior to that of B19-wildtype CAR-T. At the same time, unexpectedly, we found that the CD28-YMFM mutation also has a clear advantage in the viability of CAR-T cells during the preparation phase. As shown in Figure 11, during the CAR-T preparation phase, particularly in the early stages of CAR-T preparation, the viability of B19 CAR-T was 99.01%, which is much higher than the 78.5% of B19-wildtype CAR-T. In summary, mutations in the CD28 intracellular costimulatory domain enhance the amplification factor and viability of B19 CAR-T cells, demonstrating significant advantages.

[0112] Example 6. CAR-T positive rate, preparation success rate, phenotype, and typing Count CAR-T cells before (preparation) or after repeated stimulation, and place 0.5–1.00E+6 cells in 1.5 mL EP tubes. Add 1 mL of 1× Rising Buffer to each tube and mix. Centrifuge at 500 g for 5 minutes, discard the supernatant, and resuspend in 100 μL of 1× Rising Buffer. Add the antibody to each tube according to the recommended dose of the commercial detection antibody, and incubate in the dark at 4°C for 30–40 minutes. Wash the cells in each tube with 1 mL of 1× Rising Buffer, centrifuge at 500 g for 5 minutes, discard the supernatant, resuspend in 300 μL of 1× Rising Buffer, and detect using a flow cytometer.

[0113] The results of this example are shown in Figures 12-13. As shown in Figure 12, donor B was transduced a total of 6 times using B19 CAR lentivirus, and CAR-T was successfully prepared 3 times. Donor B was transduced a total of 6 times using B19-wild-type CAR lentivirus, and CAR-T was successfully prepared only once. Donor D was transduced a total of 2 times using B19 CAR lentivirus, and CAR-T was successfully prepared once. Donor D was transduced a total of 2 times using B19-wild-type CAR lentivirus, and CAR-T was not successfully prepared.

[0114] The results of CAR-T preparation in the four groups showed that the success rate of B19 CAR-T preparation was higher than that of B19-wildtype CAR-T preparation, both with multiple batches of cells from the same donor and with cells from multiple different donors, better meeting the urgent drug needs of patients and providing benefits to patients. The difference between B19 CAR and B19-wildtype CAR is a single amino acid residue mutation in the CD28 costimulatory domain, and therefore, the improvement in the success rate of preparation of this CAR-T is likely due to this single amino acid residue mutation in CD28. Figures 13A and 13B show that the positive rate of the prepared CAR-T was >20% and is used for subsequent experiments.

[0115] Figure 14 shows that the CAR+ ratios for B19 CAR-T, B19-wild-type CAR-T, CD19-mutant CAR-T, CD19-wild-type CAR-T, and BCMA CAR-T all clearly increased after repeated stimulation.

[0116] Figure 15 shows that, before repeated stimulation, i.e., during the preparation stage, there was no significant difference in the percentage of Tn+Tscm+Tcm between B19 CAR-T and the other four types of CAR-T in T cells. However, after repeated stimulation of tumor cells, the percentage of Tn+Tscm+Tcm for B19 CAR-T in T cells was higher than that for the other four types of CAR-T in T cells, suggesting that B19 CAR-T may have a superior antitumor effect in vivo.

[0117] Figure 16 shows that after repeated stimulation, the proportion of CD8+ T cells increased among B19 CAR-T, B19-wild-type CAR-T, CD19-mutant CAR-T, CD19-wild-type CAR-T, and BCMA CAR-T cells.

[0118] Figure 17 shows that there was no significant difference in the expression ratio of Tim-3 to LAG-3 in B19 CAR-T and B19-wildtype CAR-T cells after repeated stimulation of different tumor cells for three rounds. In summary, mutations in the CD28 intracellular co-stimulatory domain improve the success rate of B19 CAR-T preparation and the Tn+Tscm+Tcm ratio after repeated stimulation.

[0119] Example 7. Targeted proliferation of CAR-T cells 1. Preparation of target cells Collect well-developed target cells in a 15 mL centrifuge tube, centrifuge at 300 g for 5 minutes, and discard the supernatant. Add culture medium to resuspend the cells to a density of 1 M / ml, add mitomycin C at a final concentration of 1 ug / mL, and treat overnight. The next day, wash the cells twice with 1x Rising Buffer, centrifuge at 300 g for 5 minutes, and discard the supernatant. Resuspend the cells in X-VIVO-15 medium containing 10% FBS, count them using a CountStar cell counter, and detect cell viability. Dilute the cell density to 1.00 E+06 / mL and store.

[0120] 2. Preparation of effector cells Take B19 CAR-T, B19-wild-type CAR-T, CD19-mutant CAR-T, CD19-wild-type CAR-T, and mock T cells and centrifuge at 200 g for 10 minutes. Resuspend the cells in X-VIVO-15 medium, count them with a counter, adjust the cell density to 1.00E+06 / mL (according to the number of CAR-positive cells, where the number of mock T cells is based on the number of T cells), and store them.

[0121] 3. Co-incubation of effector cells and target cells The treated effector and target cells are added to a 24-well plate in a 2:1 effector:target ratio. 200 μL of target cells (0.20 E+06 / mL) and 400 μL of CAR-T cells (0.40 E+06 / mL) are added to each well. 400 μL of X-VIVO-15 + 10% FBS + 1% P / S medium is added to each well. After adding the samples, the 24-well plate is gently shaken to homogeneously mix the cells, and the cells are cultured at 37°C in a 5% CO2 incubator.

[0122] 4. Multiple stimulation and proliferation of tumor cells Effector cells (CAR-T cells) and target cells (tumor cells) are co-incubated for 48 hours, and the cells in the 24-well plate are uniformly mixed using a pipette. The CAR-T cells are counted, and 300 μL of cells from the previous round's well plate are added to the next round's well plate, with 200 μL of target cells (0.20E+06 / mL) added to each well. Then, 500 μL of X-VIVO-15 + 10% FBS + 1% P / S medium is added to each well to complete the culture system to 1 mL. Simultaneously, the above steps are repeated for 3, 4, and 5 rounds of repeated stimulation experiments, and finally, the remaining tumor cells are observed and the final amplification factor of the CAR-T cells is calculated.

[0123] The results are shown in Figure 18. Following repeated stimulation of tumor cells with H929-luc, H929-luc+Nalm6-luc, and H929-CD19(OE)-luc, the proliferation of B19 CAR-T cells was significantly superior to the amplification of B19-wild-type CAR-T, CD19-mutant CAR-T, and CD19-wild-type CAR-T, indicating that B19 CAR-T has a long-lasting ability to kill tumor cells.

[0124] Example 8. Detection of CAR-T cell killing ability before repeated stimulation 1. Preparation of effector cells Collect B19 CAR-T, B19-wild-type CAR-T, CD19-mutant CAR-T, CD19-wild-type CAR-T, and mock T cells, centrifuge at 200g for 10 minutes, and discard the supernatant. Resuspend the cells using X-VIVO 15 pure medium, count them with a counting star, and adjust the CAR-T cell density 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 according to CAR-positive cells). Set the phenotype:target ratio to 1:1, 1:2, 1:4, 1:8, and 1:16.

[0125] 2. Preparation of target cells Good-growing target cells are collected in a centrifuge tube, centrifuged at 400g for 3 minutes, the supernatant is discarded, the cells are resuspended in X-VIVO 15 pure medium, counted using a CountStar cell counter and cell viability is detected, and finally the cell density is diluted to 2.00E+5 cells / mL and stored.

[0126] 3. Co-incubation of effector cells and target cells Mix 100 μL of target cells and 100 μL of CAR-T cells in a 1:1 ratio (three repeats per sample), add to a 96-well microplate, and co-incubate at 37°C and 5% CO2 for 72 hours. After incubation, centrifuge at 500 g for 5 minutes, aspirate 100 μL of supernatant from each well, and store in a refrigerator at -80°C for subsequent cytokine detection. Then, add 50 μL of ONE-Glo™ luciferase assay system detection substrate to each well. Mix thoroughly and let stand for 5 minutes, then place on a TECAN microplate reader and detect fluorescence (wavelength 560 nm).

[0127] 4. Calculation of CAR-T cell killing rate Killing rate (%) = (control group - experimental group) / control group × 100% The results are shown in Figure 19(A). For CD19+BCMA- cells (Nalm6-luc), under low effector:target ratios, for example, when the E:T ratio is 1:8, the tumor cell killing rates for B19 CAR-T, B19-wild-type CAR-T, CD19-mutant CAR-T, and CD19-wild-type CAR-T were 71.80%, 54.99%, 38.38%, and 38.38%, respectively. Similarly, under conditions where the E:T ratio is 1:16, the tumor cell killing rates for B19 CAR-T, B19-wild-type CAR-T, CD19-mutant CAR-T, and CD19-wild-type CAR-T were 49.04%, 30.61%, 24.60%, and 19.67%, respectively. These results suggest that B19 dual-target CAR-T cells not only expand the range of target cells, but that the killing power of target cells expressing only CD19 is significantly higher than that of CD19 single-target CAR-T cells, and this effect is particularly pronounced at low target-to-target ratios.

[0128] CD28 mutations also have a certain effect on the killing power of CAR-T receptors. Under conditions where the E:T ratio is 1:4 and 1:8, the killing effect of B19 CAR-T on tumor cells H929-luc is significantly superior to that of B19-wildtype CAR-T. Under conditions where the E:T ratio is 1:4, 1:8, and 1:16, the killing effect of B19 CAR-T on tumor cells MM1S-luc is significantly superior to that of B19-wildtype CAR-T. In summary, mutations in the CD28 intracellular costimulatory domain enhance the tumor-killing effect of B19 CAR-T.

[0129] Example 9. Detection of CAR-T cell killing ability after repeated stimulation 1. Preparation of effector cells After repeated stimulation, B19 CAR-T, B19-wild-type CAR-T, CD19-mutant CAR-T, CD19-wild-type CAR-T, and mock T cells were collected, centrifuged at 200g for 10 minutes, and the supernatant was discarded. The cells were resuspended using X-VIVO 15 pure medium and counted with a counting star to adjust the CAR-T cell density 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 CAR-positive cells). Effector:target ratios were set to 1:1, 1:2, 1:4, 1:8, and 1:16, and a control group without effector cells was established.

[0130] 2. Preparation of target cells Good-growing target cells are collected in a centrifuge tube, centrifuged at 500g for 5 minutes, the supernatant is discarded, the cells are resuspended in X-VIVO 15 pure medium, counted using a CountStar cell counter and cell viability is detected, and finally the cell density is diluted to 2.00E+5 cells / mL and stored.

[0131] 3. Co-incubation of effector cells and target cells Mix 100 μL of target cells and 100 μL of CAR-T cells in a 1:1 ratio (three repeats per sample), add to a 96-well microplate, repeat a total of three times, and co-incubate at 37°C and 5% CO2 for 72 hours. After incubation is complete, centrifuge at 500 g for 5 minutes, aspirate 100 μL of supernatant from each well, and store in a refrigerator at -80°C for use in subsequent cytokine detection. Add 50 μL of ONE-Glo™ luciferase assay system detection substrate to each well. Mix uniformly and let stand for 5 minutes, then place on a TECAN microplate reader and detect fluorescence (wavelength 560 nm).

[0132] 5. Calculation of CAR-T cell killing rate Killing rate (%) = (control group - experimental group) / control group × 100% The results are shown in Figure 19(B), and after two rounds of repeated stimulation of tumor cells Nalm6-luc, the killing effect of B19 CAR-T on Nalm6-luc and Nalm6-luc+H929-luc cells is clearly superior to that of B19-wildtype CAR-T.

[0133] The results above indicate that B19 CAR-T has a significantly superior tumor cell killing rate compared to B19-wild-type CAR-T and monospecific CD19 CAR-T, regardless of whether target cells are repeatedly stimulated, demonstrating a superior antitumor effect.

[0134] As described in Examples 8 and 9, the tumor-killing effect of B19 CAR-T cells, including mutant structures of the CD28 intracellular costimulatory domain, is significantly superior to that of B19 wild-type CAR-T cells and other single-target CAR-T cells, regardless of whether the target cells are repeatedly stimulated.

[0135] Example 10. Detection of CAR-T cytokine release ability The supernatants from the -80°C refrigerator before and after repeated stimulation are taken and used for cytokine detection. Take an appropriate amount of 1.5 mL EP tube, add 50 μL of the corresponding cell co-incubation supernatant for each group to each tube, centrifuge at 500 g for 5 minutes, and collect 50 μL of the supernatant to detect cytokines.

[0136] Determine the number of cytokines and samples to detect and prepare the capture beads according to the requirements of the CBA detection kit instructions. Remove the bottles of cytokine-capturing magnetic beads to be detected, mix vigorously and uniformly, take out (number of samples to be detected + number of negative controls) × 15 μL / 6 of capturing magnetic beads from each type of magnetic bead, and mix each type of magnetic bead uniformly by turbo shaking, take out n 1.5 mL EP tubes (n = number of samples + number of controls), add 15 μL of mixed magnetic beads to each tube (sample, negative control), add 15 μL of the corresponding reagent to be tested (sample, control) to each tube, add 15 μL of PE detection reagent to each tube, mix thoroughly and uniformly, incubate in the dark at room temperature for 3 hours, after incubation is complete, add 500 μL of wash buffer to each sample, mix thoroughly and uniformly, centrifuge at 500 g for 5 minutes, discard the supernatant, resuspend in 100 μL of wash buffer, centrifuge at 500 g for 5 minutes, discard the supernatant, and add 100 μL of wash buffer (wash The sample is resuspended in buffer and detected using a flow cytometer.

[0137] The results are shown in Figures 20A, 20B, 21A, and 21B. Cytokine release after CAR-T and tumor cell co-incubation without repeated stimulation by tumor cells is shown in Figure 20A (see Tables 2-4 for specific values). After co-incubation of CAR-T and tumor cells Raji-luc or H929-CD19(OE)-luc, the amount of IL-2 released by B19 CAR-T was significantly greater than that released by B19-wild-type CAR-T, CD19-mutant CAR-T, CD19-wild-type CAR-T, and BCMA CAR-T. Following co-incubation of CAR-T cells and tumor cells H929-CD19(OE)-luc, TNF-α release from B19 CAR-T cells was significantly superior to that from B19-wild-type CAR-T, CD19-mutant CAR-T, CD19-wild-type CAR-T, and BCMA CAR-T cells. Similarly, IFN-γ release from B19 CAR-T cells was significantly superior to that from B19-wild-type CAR-T and CD19-mutant CAR-T cells. This suggests that cytokine release from B19 CAR-T cells can be improved by single amino acid residue mutations in the CD28 co-stimulatory domain of the CAR structure and by designing parallel CAR structures, indicating that B19 CAR-T cells with parallel structures may exhibit superior therapeutic effects in the treatment of diseases.

[0138] The release of cytokines after repeated stimulation of tumor cells with Nalm6-luc cells by CAR-T cells and after co-incubation of tumor cells is shown in Figure 20B (see Tables 5-7 for specific values). After repeated stimulation of tumor cells by CAR-T cells and after co-incubation of tumor cells Raji-luc or H929-CD19(OE)-luc, the amount of IL-2 released by B19 CAR-T cells was significantly greater than that released by B19-wild-type CAR-T cells and other single-target CAR-T cells, and the amount of IL-2 released by CD19-mutant CAR-T cells was significantly greater than that released by CD19-wild-type CAR-T cells. After repeated stimulation of tumor cells by CAR-T cells and after co-incubation of tumor cells H929-CD19(OE)-luc, the release of IFN-γ and TNF-α by B19 CAR-T cells was significantly greater than that released by B19-wild-type CAR-T cells. Figures 21A-21B show similar results (see Tables 8-10 and 11-13 for specific values), and the release of IL-2, TNF-α, and IFN-γ before and after repeated stimulation and after co-incubation of B19 CAR-T and target cells is superior to that of B19-wild-type CAR-T and other single-target CAR-T cells. Therefore, B19 CAR-T has superior proliferative and tumor-killing effects in vitro. In summary, regardless of whether target cells are repeatedly stimulated, mutations in the CD28 intracellular costimulatory domain significantly increase the release of cytokines IL-2, TNF-α, and IFN-γ from B19 CAR-T cells.

[0139] [Table 2]

[0140] [Table 3]

[0141] [Table 4] TIFF2026513241000005.tif12170

[0142] Table 5

[0143] Table 6

[0144] Table 7 TIFF2026513241000009.tif17170

[0145] Table 8 TIFF2026513241000011.tif100170

[0146] Table 9 TIFF2026513241000013.tif101170

[0147] Table 10 TIFF2026513241000015.tif103170

[0148] Table 11 TIFF2026513241000017.tif68170

[0149] Table 12 TIFF2026513241000019.tif88170

[0150]

Table 13

[0151] Example 11. In Vivo Test Select 6 - 8 week - old B - NDG mice and inject tumor cells intravenously (-7 days). One day later (-6 days), measure the tumor graft load. On day 0, divide the mice into groups, and after grouping, inject CAR - T cells intravenously. After CAR - T treatment, evaluate the tumor load of the mice on the set days respectively (Figure 22(A), Figure 23(A), Figure 24(A), Figure 25(A)). Inject 0.3 mg of D - luciferin (YEASEN) intraperitoneally into each mouse, and 4 minutes after injection, take pictures with a Xenogen IVIS imaging system (Perkin Elmer Life Sciences) under isoflurane anesthesia and expose for 30 seconds. Calculate the bioluminescence signal based on the amount of photons emitted, normalize the photon amount by exposure time and surface area, and finally obtain the photon amount / s / cm 2 / steradian (p / s / cm 2 / sr).

[0152] The in vivo efficacy data in Figure 22 shows that in a Nalm6-luc tumor model efficacy study (tumor cell inoculation: 0.4E+6 Nalm6-luc cells / animal), CD19-mutant CAR-T and CD19-wildtype CAR-T cells administered at 1.50E+6 cells / animal all exhibited tumor-killing effects at different detection time points. On days 5 and 9, CD19-mutant CAR-T cells demonstrated superior tumor-clearing efficacy compared to 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 there was no recurrence on day 14. On day 14, CD19-wildtype CAR-T cells completely cleared the tumor. Fourteen days after administration, one animal died in the solvent group, three animals died in the mock T group (not transfected with CAR), and no animals died in the remaining groups. This indicates that mutations in the co-stimulatory domain CD28 can enhance the antitumor effect of CD19 CARs. During the experiment, the body weight of mice in the solvent and mock T groups was clearly reduced, but the CD19-mutant CAR-T and CD19-wildtype CAR-T groups did not show any toxic side effects that did not affect body weight (Table 14). Therefore, we will prepare BCMA and CD19 dual-target CAR-T cells by combining CD19-mutant CARs and CD19-wildtype CARs with BCMA CARs, respectively, to further confirm whether mutations in the co-stimulatory domain CD28 enhance the antitumor effect of dual CAR-T cells.

[0153] The in vivo efficacy data in Figure 23 shows that in a Nalm6-luc tumor model efficacy study (tumor cell inoculation: 0.4E+6 Nalm6-luc cells / animal), B19 CAR-T cells and B19-wildtype CAR-T cells administered at a dose of 3.00E+6 cells / animal all exhibit tumor-killing effects at different detection time points. As shown in Figures 23(B) and 23(C), on day 7, the tumor-removing effect of B19 CAR-T cells was superior to that of B19-wildtype CAR-T cells, with B19 CAR-T cells almost completely removing the tumor, while B19-wildtype CAR-T cells could not completely remove it. On day 14, both B19 CAR-T and B19-wildtype CAR-T cells completely removed the tumor. Fourteen days after administration, five animals died in the solvent group, four animals died in the mock T group (not transfected with CAR), and no animals died in the remaining groups. During the experiment, the body weight of mice in the solvent group, mock T group, and B19-wildtype CAR-T group clearly decreased, but B19 CAR-T did not exhibit any toxic side effects that did not affect body weight (Table 15). This indicates that mutations in the costimulatory domain CD28 can enhance the killing and antitumor effects of B19 CAR-T against CD19+ tumor cells and reduce the toxic side effects of CAR-T cells.

[0154] The in vivo efficacy data in Figure 24 shows that in a Nalm6-luc+H929-luc tumor model efficacy study (tumor cell inoculation: 0.4E+6 Nalm6-luc cells and 5E+6 H929-luc cells / animal), B19 CAR-T cells and B19-wildtype CAR-T cells administered at a dose of 3.00E+6 cells / animal all exhibit tumor-killing effects at different detection time points. As shown in Figures 24(B) and 24(C), on day 7, the tumor-removing effect of B19 CAR-T cells is superior to that of B19-wildtype CAR-T cells, with B19 CAR-T cells almost completely removing the tumor, while B19-wildtype CAR-T cells are unable to completely remove the tumor. On day 14, both B19 CAR-T and B19-wildtype CAR-T cells completely remove the tumor. Fourteen days after administration, four mice died in the solvent group, four mice died in the mock T group (not transfected with CAR), and no animals died in the remaining groups. During the experiment, the body weight of mice in the solvent group, mock T group, and B19-wild-type CAR-T group decreased significantly, but B19 CAR-T did not show any toxic side effects that did not affect body weight (Table 16). This indicates that mutations in the co-stimulatory domain CD28 can enhance the killing and antitumor effects of B19 CAR-T against CD19+BCMA+ tumor cells and reduce the toxic side effects of CAR-T cells.

[0155] The in vivo efficacy data in Figure 25 shows that in a Nalm6-luc+H929-luc tumor model efficacy study (tumor cell inoculation: 0.4E+6 Nalm6-luc cells and 5E+6 H929-luc cells / animal), B19 CAR-T cells and B19-wildtype CAR-T cells administered at 1.50E+6 cells / animal all exhibit tumor-killing effects at different detection time points. As shown in Figures 25(B) and 25(C), on day 7, the tumor-removing effect of B19 CAR-T cells is superior to that of B19-wildtype CAR-T cells, with B19 CAR-T cells almost completely removing the tumor, while B19-wildtype CAR-T cells are unable to completely remove the tumor. On day 14, both B19 CAR-T and B19-wildtype CAR-T cells completely remove the tumor. Fourteen days after administration, five mice died in the mock T group (not transfected with CAR), while no animals died in the remaining groups. During the experiment, body weight decreased significantly in the solvent and mock T groups, but the B19 CAR-T and B19-wild-type CAR-T groups showed no toxic side effects that did not affect body weight (Table 17). This indicates that mutations in the costimulatory domain CD28 enhance the killing and antitumor effects of B19 CAR-T against CD19+BCMA+ tumor cells.

[0156] The in vivo efficacy studies in Figures 22-25 show that mutations in the costimulatory domain CD28 enhance the killing effect of B19 CAR-T against tumor cells Nalm6-luc and Nalm6-luc+H929-luc. Under conditions of a dose of 3.00E+6 cells / mice, the body weight of mice in the B19-wild-type CAR-T group was significantly reduced, while the body weight of mice in the B19 CAR-T group remained unchanged. This indicates that mutations in the costimulatory domain CD28 can improve the safety of B19 CAR-T. In summary, unexpectedly, mutations in the co-stimulatory domain CD28 can enhance the antitumor effects of single-target CD19-mutant CAR-T and dual-target B19 CAR-T in mice, and also improve the safety of B19 CAR-T.

[0157] [Table 14]

[0158] [Table 15] TIFF2026513241000024.tif25170

[0159] [Table 16] TIFF2026513241000026.tif47170

[0160] [Table 17]

[0161] [Table 18] TIFF2026513241000029.tif173170

[0162] In SEQ ID NO:2 and SEQ ID NO:9, a single underline indicates the light chain variable region of scFv, and a double underline indicates the heavy chain variable region of scFv.

[0163] All documents referenced in this invention are cited as references in this application, as if each document were cited individually. Furthermore, after reading the above teachings of this invention, persons skilled in the art can make various changes or modifications to the invention, and these equivalent forms are also included within the scope defined by the claims appended to this application.

Claims

1. Manipulated immune cells, The manipulated immune cells express BCMA CAR and CD19-mutant CAR in parallel, the amino acid sequence of BCMA CAR is as shown in SEQ ID NO: 15, and the amino acid sequence of CD19-mutant CAR is as shown in SEQ ID NO: 16, wherein the intracellular costimulatory domain of CD19-mutant CAR includes 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 manipulated immune cells according to claim 1, (A) The step of providing immune cells to be modified, and (B) A method for preparing engineered immune cells according to claim 1, comprising the step of modifying the immune cells so that they express the BCMA CAR and the CD19-mutant CAR, thereby obtaining the engineered immune cells according to claim 1.

3. It is a fusion protein, The fusion protein comprises a BCMA CAR and a CD19-mutant CAR sequence linked by a self-cleaving sequence, wherein the amino acid sequence of the BCMA CAR is as shown in SEQ ID NO: 15, and the amino acid sequence of the CD19-mutant CAR is as shown in SEQ ID NO: 16, wherein the intracellular costimulatory domain of the CD19-mutant CAR includes 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. It is a polynucleotide, The polynucleotide is characterized in that it encodes the fusion protein described in claim 3.

5. It is a vector, The vector is characterized by comprising the polynucleotide described in claim 4.

6. A pharmaceutical composition, The pharmaceutical composition is characterized by comprising the manipulated immune cells described in claim 1, and a pharmaceutically acceptable carrier, diluent, or excipient.

7. Uses of the manipulated immune cells described in claim 1 or the pharmaceutical composition described in claim 6, Uses of the manipulated immune cells according to claim 1 or the pharmaceutical composition according to claim 6, characterized in that they are used in the preparation of a drug or formulation for the prevention and / or treatment of a disease, wherein the disease is selected from the group consisting of hematological malignancies, solid tumors, autoimmune diseases or combinations thereof.

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