T cells expressing chimeric receptors

Abstract

Provided is a T cell expressing a chimeric receptor, wherein the chimeric receptor specifically recognizes BCMA, and the endogenous TCR molecules are silenced and the endogenous MHC molecules are silenced. Also provided is the use of the T cell in preparing a drug for treating BCMA-positive tumors.

Description

Technical Field

[0001] The present invention belongs to the field of immune cell therapy, and specifically relates to tumor immunotherapy, and in particular to a genetically engineered immune effector cell. Background Art

[0002] In recent years, tumor immunotherapy has received extensive attention. At present, the T cells used in adoptive immunotherapy are mainly derived from the patient's own body, and are infused back into the patient's body after being modified in vitro. Due to the large individual differences among patients, the treatment effect and safety are difficult to control, and the cost is expensive. In addition, since cell preparation requires a certain period of time, autologous cell therapy cannot be applied when the patient's condition is critical or the patient's own conditions are not suitable for extracting peripheral blood from the body for immune cell therapy.

[0003] Allogeneic immune effector cells refer to cells obtained from other individuals of the same species. They can not only overcome the problem of poor quality of T cell sources, so that all patients with advanced tumors can receive immune cell therapy, but also enable cell products to be better applied to automated production, reduce production costs, and ensure product consistency. However, due to the immunogenetic differences between the donor and the recipient, after the donor T lymphocytes enter the recipient patient's body, they will mistake the normal cells or tissues in the patient's body for foreign targets and attack them, thereby generating a graft-versus-host reaction (GVHD); on the other hand, allogeneic cells, as foreign transplants, may also be recognized and attacked by the patient's immune cells, thereby inhibiting or eliminating allogeneic cells, generating a host-versus-graft reaction (HVGR).

[0004] In patent PCT / EP2016 / 055332, Cellectis knocked out TCR receptors and CD52 in allogeneic T cells to obtain universal CAR-T cells. Although this technology reduces the risk of GVHD, it cannot inhibit the production of HVGR and has certain safety risks. In patent CN201710983276.X, Precision Biotech knocked out TRAC, B2M and CIITA genes at the same time to obtain universal CAR-T cells, but there are problems such as low cell preparation efficiency.

[0005] Therefore, it is necessary to develop a chimeric receptor-modified allogeneic T cell that can reduce GVHD and HVGR immune rejection reactions and is not easily attacked by host NK cells. Summary of the invention

[0006] The object of the present invention is to provide a T cell expressing a chimeric receptor, which can not only suppress the risk of GVHD and HVGR, but also is not easily attacked by NK cells.

[0007] In a first aspect of the present invention, a T cell expressing a chimeric receptor is provided, wherein the chimeric receptor specifically recognizes BCMA, and the endogenous TCR molecule and endogenous MHC molecule of the T cell are silenced.

[0008] In a specific embodiment, the TCR molecule silencing refers to the silencing of genes encoding one or both of the α and β chains of the TCR. In a specific embodiment, the TCR molecule silencing refers to the silencing of genes encoding the α chain of the TCR (i.e., the TRAC gene), which is also referred to as the TRAC gene in this article.

[0009] In a specific embodiment, the TCR molecule silencing refers to silencing of the gene encoding the α chain constant region of the TCR.

[0010] In a specific embodiment, the TCR molecule silencing refers to silencing of the first exon of the gene encoding the α chain constant region of the TCR.

[0011] In a specific embodiment, the MHC molecule silencing refers to MHC class I molecule silencing.

[0012] In a specific embodiment, the MHC molecule is an HLA molecule. Preferably, the MHC molecule is a class I HLA molecule.

[0013] Preferably, the HLA molecule is selected from at least one of molecules encoding HLA-A, HLA-B, HLA-C, B2M and CIITA. More preferably, the HLA molecule is a B2M molecule, and the gene encoding B2M is also referred to as B2M gene in this article.

[0014] In a specific embodiment, gene editing technology is used to silence endogenous TCR molecules and endogenous MHC molecules.

[0015] In a specific embodiment, the gene encoding the constant region of the α and / or β chain of the TCR is gene-edited to silence the gene encoding one or both of the α and β chains of the TCR. In a specific embodiment, the gene encoding the α chain of the TCR is gene-edited. Preferably, the gene encoding the constant region of the α chain of the TCR is gene-edited. More preferably, the first exon of the gene encoding the constant region of the α chain of the TCR is gene-edited.

[0016] In a specific embodiment, the MHC molecule is selected from at least one of HLA-A, HLA-B, HLA-C, B2M and CIITA molecules. In a preferred embodiment, the MHC molecule is a B2M molecule.

[0017] In a specific embodiment, the gene encoding the constant region of B2M is gene-edited to silence the B2M molecule; preferably, the first exon of the gene encoding the constant region of B2M is gene-edited to silence the B2M molecule.

[0018] In a specific embodiment, the gene editing technology is selected from CRISPR / Cas9 technology, artificial zinc finger nuclease (Zinc Finger Nucleases, ZFN) technology, transcription activator-like effector (transcription activator-like effector, TALE) technology or TALE-CRISPR / Cas9 technology; preferably, CRISPR / Cas9 technology is used.

[0019] In a specific embodiment, when CRISPR / Cas9 technology is used to silence the B2M molecule, the sequence of the selected gRNA is shown in SEQ ID NO: 1.

[0020] In a specific embodiment, when CRISPR / Cas9 technology is used to silence the α chain of TCR, the sequence of the selected gRNA is as shown in SEQ ID NO: 2, 27, 28, or 29.

[0021] In a specific embodiment, when silencing the B2M molecule, the sequence of the selected gRNA is shown in SEQ ID NO: 1, and when silencing the α chain of TCR, the sequence of the selected gRNA is shown in SEQ ID NO: 2.

[0022] In a specific embodiment, the chimeric receptor is selected from a chimeric antigen receptor (CAR) or a T cell antigen coupler (TAC).

[0023] In a specific embodiment, the chimeric receptor is a chimeric antigen receptor (CAR)

[0024] In a specific embodiment, the chimeric antigen receptor comprises:

[0025] (i) an antibody or fragment thereof that specifically binds to BCMA, CD28 or the transmembrane region of CD8, the co-stimulatory signaling domain of CD28, and CD3δ; or

[0026] (ii) an antibody or fragment thereof that specifically binds to BCMA, CD28 or the transmembrane region of CD8, the co-stimulatory signaling domain of CD137, and CD3δ; or

[0027] (iii) an antibody or a fragment thereof that specifically binds to BCMA, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD28, the costimulatory signaling domain of CD137, and CD3δ.

[0028] In a specific embodiment, the chimeric receptor is a TAC, comprising: (a) an extracellular domain: the extracellular domain comprises an antibody domain having an antigen binding domain, and a single-chain antibody that binds to CD3; (b) a transmembrane region; (c) an intracellular domain, wherein the intracellular domain is connected to the protein kinase LCK.

[0029] In a specific embodiment, the antibody or fragment thereof that specifically binds to BCMA is a scFv or VHH.

[0030] In a specific embodiment, the antibody that specifically binds to BCMA contains HCDR1 shown in SEQ ID NO: 10, HCDR2 shown in SEQ ID NO: 11, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 13, LCDR2 shown in SEQ ID NO: 14, and LCDR3 shown in SEQ ID NO: 15; in one embodiment, the antibody has a heavy chain variable region shown in SEQ ID NO: 17 and a light chain variable region shown in SEQ ID NO: 18; in a specific embodiment, the antibody that specifically binds to BCMA has a sequence shown in SEQ ID NO: 3.

[0031] In a specific embodiment, the sequence of the chimeric receptor comprises the sequence shown in SEQ ID NO: 4, 5, 6, or 7. In a specific embodiment, the T cell has no reduced in vitro tumor cell killing ability compared to a T cell expressing the chimeric receptor with endogenous TCR and endogenous MHC activity.

[0032] In a specific embodiment, the T cells have low alloreactivity when applied to allogeneic tissues and are not easily rejected by NK cells.

[0033] In a specific embodiment, the T cells have reduced graft versus host reaction (GVHD) and reduced host versus graft reaction (HVGR).

[0034] In the second aspect of the present invention, there is provided a use of the T cells described in the first aspect for preparing a drug for treating BCMA-positive tumors; in a specific embodiment, the BCMA-positive tumor is selected from multiple myeloma.

[0035] In the third aspect of the present invention, a pharmaceutical composition is provided, comprising the cells described in the first aspect and a pharmaceutically acceptable carrier or excipient.

[0036] In the fourth aspect of the present invention, a kit is provided, comprising the cell described in the first aspect or the pharmaceutical composition described in the third aspect.

[0037] Beneficial Effects

[0038] The genetically engineered immune effector cells of the present invention target BCMA, can effectively reduce GVHD and HVGR immune rejection reactions caused by allogeneic cell therapy, and are not easily attacked by NK cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 The results showed that TCR / B2M double negative cells were obtained by magnetic bead sorting, and the detection results showed that the CD3 and B2M negative cells after sorting reached more than 99.6%.

[0040] Figure 2 The expression of CAR positivity rate was shown, and the CAR positivity rate in CAR-T cells without double gene knockout was compared with that in CAR-T cells with double gene knockout of TRAC and B2M. The results showed that the CAR positivity rate of both was above 80%, among which UTD refers to blank T cells, that is, uninfected T cells; BCMA CAR-T refers to normal CAR-T cells with BCMA targeting activity, and BCMA UCAR-T refers to double knockout universal CAR-T cells with BCMA targeting activity.

[0041] Figure 3 It was shown to kill RPMI-8226 and NCI-H929 tumor cells in vitro. The multiple myeloma cell lines RPMI-8226 and NCI-H929 were cultured in vitro as tumor target cells, and the CAR-T cells before and after double gene knockout targeting BCMA acted on tumor cells. The results showed that the CAR-T cells before and after gene knockout could effectively kill RPMI-8226 and NCI-H929 tumor cells, and the killing ability was comparable.

[0042] Figure 4 The double-knockout CAR-T cells targeting BCMA showed an anti-tumor effect in vivo.

[0043] Figure 5 The in vivo survival effect of double-gene knockout CAR-T cells targeting BCMA was shown.

[0044] Figure 6 The results show the changes in mouse body weight in the GVHD model, indicating that BCMA UCAR-T cells can significantly reduce the side effects caused by GVHD reaction.

[0045] Figure 7 The in vivo survival of BCMA UCAR-T cells in the GVHD model was shown. The results showed that BCMA UCAR-T cells did not cause GVHD reaction in vivo and had good safety.

[0046] Figure 8 The killing ability of NK-92 cells on T cells is demonstrated. The results in the figure show that the T cell lysis efficiency in the CAR-T cells before and after the double gene knockout targeting BCMA is comparable, indicating that the CAR-T targeting BCMA after the double gene knockout will not cause abnormal rejection of NK-92 cells, and the genetically engineered immune effector cells targeting BCMA prepared by the present invention can effectively survive in the patient's body. DETAILED DESCRIPTION

[0047] After extensive and in-depth research, the inventor unexpectedly found that by silencing endogenous TCR molecules and B2M molecules in T lymphocytes, T cells modified with universal chimeric receptors for allogeneic cells can be prepared, thereby enabling application to different individuals. On this basis, the present invention was completed.

[0048] Similar or equivalent methods and materials described herein can be used in the practice or testing of the present invention. Publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict, this specification shall prevail. In addition, unless otherwise specified, materials, methods and embodiments are illustrative only and are not intended to be limiting.

[0049] Unless otherwise indicated, the practice of the present invention will employ conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA and immunology, which are within the skill of the art. These techniques are fully explained in the literature. See, for example, Current Protocols in Molecular Biology (Frederick M. AUSUBEL, 2000, Wiley and Son Inc., Library of Congress, USA); Molecular Cloning: A Laboratory Manual, Third Edition, (Sambrook et al., 2001, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press); Oligonucleotide Synthesis (MJ Gaited., 1984); Mullis et al. US Pat. No. 4,683,195; Nucleic AcidHybridization (BD Harries&S.J.Higginseds.1984); Transcription And Translation (BDHames&S.J.Higginseds.1984); Culture Of Animal Cells (RI Freshney, Alan R. Liss, Inc., 1987);

[0050] Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the series, Methods In ENZYMOLOGY (J. Abelson and M. Simon, eds.-in-chief, Academic Press, Inc., New York), especially Vols. 154 and 155 (Wuetal. eds.) and Vol. 185, "Gene Expression Technology" (D. Goeddel, ed.); GeneTransfer Vectors For Mammalian Cells (JHMiller and MP Caloseds., 1987, Cold Spring Harbor Laboratory); Immunochemicals In Cell And Molecular Method Biology (Mayer and Walker, eds., Academic Press, London, 1987); Hand book OfExperimental Immunology, Volumes I-IV (DM Weir and CC Blackwell, eds., 1986); and Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986).

[0051] the term

[0052] Unless specifically defined herein, all technical and scientific terms used have the same meanings as commonly understood by those skilled in the fields of gene therapy, biochemistry, genetics, and molecular biology.

[0053] T cell receptor (TCR) is a cell surface receptor that participates in T cell activation in response to the presentation of antigens. TCR is usually composed of two chains, α and β, which can be assembled to form a heterodimer and associated with the CD3 transduction subunit to form a T cell receptor complex present on the cell surface. The α chain and β chain of TCR are composed of the following: N-terminal variable region (V) and constant region (C) of immunoglobulin-like, hydrophobic transmembrane domain and short cytoplasmic region. For immunoglobulin molecules, the variable regions of α chain and β chain are produced by V (D) J recombination, so that a large number of diverse antigen specificities are produced in the population of T cells. However, in contrast to immunoglobulins that recognize complete antigens, T cells are activated by processed peptide fragments associated with MHC molecules, and extra dimensions are introduced into antigen recognition by T cells, which is called MHC restriction. The potential development of cell proliferation and GVHD is caused by the recognition of MHC differences between donors and recipients by T cell receptors. It has been shown that normal surface expression of the TCR depends on the coordinated synthesis and assembly of all seven components of the complex (Ashwell and Klusner 1990).

[0054] Inactivation of either the α chain of the TCR or the β chain of the TCR can lead to the elimination of the TCR from the surface of T cells, thereby preventing recognition of alloantigens and the resulting GVHD.

[0055] The term "MHC" is a histocompatibility complex, which is a general term for all gene groups that encode biocompatible complex antigens. MHC antigens are expressed in the tissues of all higher vertebrates and are called HLA antigens in human cells. They play an important role in transplantation reactions, and rejection is mediated by T cells that respond to histocompatibility antigens on the surface of the implanted tissue. MHC proteins play a vital role in T cell stimulation. Antigen presenting cells (usually dendritic cells) display peptides that are degradation products of foreign proteins on the cell surface on MHC. In the presence of co-stimulatory signals, T cells are activated and act on target cells that also display the same peptide / MHC complex.

[0056] For example, stimulated T helper cells will target macrophages that display antigens bound to their MHC, or cytotoxic T cells (CTLs) will act on virus-infected cells that display foreign viral peptides. MHC antigens are divided into MHC class I antigens and MHC class II antigens.

[0057] In humans, the class I HLA gene cluster includes three major loci: HLA-A, HLA-B, and HLA-C, as well as several minor loci. The class II HLA cluster also includes three major loci: HLA-DP, HLA-DQ, and HLA-DR.

[0058] The term "human leukocyte antigen" (HLA) is the gene encoding the human major histocompatibility complex, located on chromosome 6 (6p21.31), including a series of tightly linked loci, which are closely related to the human immune system function. The genes encoding HLA include class I, class II and class III gene parts. The antigens expressed by class I and class II genes of HLA are located on the cell membrane, which are MHC-I (HLA-A, HLA-B, HLA-C site encoding) and MHC-II (HLA-D region encoding). Class I is distributed on the surface of almost all cells in the body. It is a heterodimer composed of heavy chain (α chain) and β2 microglobulin (B2M). Class II is mainly a glycoprotein located on the surface of macrophages and B lymphocytes.

[0059] The term "B2M" is beta-2 microglobulin, also known as B2M, which is the light chain of the MHC class I molecule. In humans, B2M is encoded by the b2m gene located on chromosome 15, opposite the other MHC genes located as a gene cluster on chromosome 6. Mouse models deficient in beta-2 microglobulin have shown that B2M is necessary for cell surface expression of MHC class I and stability of the peptide binding groove. It has been further shown that hematopoietic transplants from mice lacking normal cell surface MHC I expression due to targeted mutations in the beta-2 microglobulin gene were rejected by NK1.1+ cells in normal mice, indicating that defective expression of MHC I molecules renders bone marrow cells susceptible to rejection by the host immune system (Bix et al. 1991).

[0060] Therefore, in order to provide T cells with lower alloreactivity, the T cells provided by the present invention include T cells in which one TCR gene and one HLA gene are inactivated or mutated.

[0061] The term "inactive" means that the target gene (such as TCR gene) is no longer expressed in the form of a functional protein.

[0062] In a specific embodiment, the method of inactivating the target gene may include deleting, frameshifting or mutating the target gene, such as introducing a rare-cutting endonuclease that can break the target gene into the cell. In a specific embodiment, the cell can be transfected with a nucleic acid encoding a rare-cutting endonuclease that can break the target gene, so that the rare-cutting endonuclease is expressed in the cell. The rare-cutting endonuclease can be a giant nuclease, a zinc finger nuclease, a CRISPR / Cas9 nuclease, an MBBBD-nuclease or a TALEN-nuclease. In a preferred embodiment, the rare-cutting endonuclease is a CRISPR / Cas9 nuclease, a TALEN-nuclease.

[0063] The "TCR is inactive" refers to the inactivation of at least one subunit gene of the endogenous TCR, especially the inactivation of the TCRα and / or TCRβ genes, more preferably the TCRα gene (also referred to as TRAC gene in this article).

[0064] The "MHC is inactive" refers to the inactivation of at least one subunit gene of the endogenous MHC, especially the inactivation of the MHC I gene, more preferably, the B2M gene.

[0065] The term "gene editing" refers to a technology that integrates exogenous genes into a certain site on the target cell genome through homologous recombination to achieve the purpose of modifying a gene on the chromosome. It overcomes the blindness and contingency of random integration and is an ideal method for modifying and transforming biological genetic material, but its targeting efficiency is extremely low. In recent years, the technology of genome targeted modification guided by nucleases has developed rapidly. This type of nuclease is usually composed of a DNA recognition domain and a non-specific nuclease domain. The target site of the DNA recognition domain is used to locate the nuclease to the genomic region that needs to be edited, and then the non-specific nuclease cuts the DNA double strand, causing the DNA break self-repair mechanism, thereby inducing mutations in the gene sequence and promoting the occurrence of homologous recombination. The latest development technologies include CRISPR / Cas technology, ZFN technology, TALE technology and TALE-CRISPR / Cas technology.

[0066] In this article, "gene editing" is also referred to as "gene knockout."

[0067] The term "molecular silencing" or "gene silencing" refers to the phenomenon that, due to various reasons, genes are not expressed or expressed at a low level without damaging the original DNA. Molecular silencing occurs at two levels. One is molecular silencing at the transcriptional level caused by DNA methylation, heterochromatinization, and position effects. The other is post-transcriptional molecular silencing, that is, gene inactivation at the level of gene transcription by specific inhibition of target RNA, including antisense RNA, co-suppression, gene repression, RNA interference, and microRNA-mediated translation inhibition.

[0068] The term "artificial zinc finger nuclease (Zinc Finger Nuclease, ZFN)" technology is the first generation of nuclease site-directed modification technology, which can specifically recognize the zinc finger motif of triplet DNA fragments instead of bases as the basic unit for recognizing specific DNA sequences. The most classic zinc finger nuclease is a non-specific nuclease FokI fused with a domain containing zinc fingers, and its purpose is naturally to cut a specific sequence. The cut DNA can be repaired by excision to delete the single-stranded part of the cut, and then reconnected together. ZFN technology was once used as the main means to induce genome mutations and improve the efficiency of homologous recombination. However, because ZFN is affected by upstream and downstream dependent effects, the binding between the target DNA sequence is unstable, and all target sequences cannot be located. A lot of screening and identification work is required, and off-target mutations are prone to occur. In addition, a set of commercial ZFNs is expensive, the design is cumbersome, and the effect is unstable.

[0069] The term "transcription activator-like effector (TALE)" has DNA binding specificity, and the modular operation of the recognition code is simple and convenient. The TALE-DNA binding domain is composed of tandem repeat units, most of which contain 3 to 4 amino acids, and the 12th and 13th amino acids of the unit are designed as variable regions (repeat variable residues, RVD). The RVD of TALE recognizes the 4 bases of the DNA sequence with high specificity, and the 13th amino acid directly binds specifically to the base of DNA. It is possible to construct a specific TALEDN recognition and binding domain at any site according to the DNA sequence, which can be widely used in gene sequence mutation modification and gene targeting. Set the DNA target sequence, assemble the TALE-DNA binding domain, and fuse the non-specific DNA cleavage domain of the Fok I endonuclease to assemble into TALE nucleases (transcription activator-like effector nucleases, TALENs). TALENs target and bind to DNA to produce DNA double-strand breaks (DNA double-strand breaks, DSBs). DSBs in eukaryotic cells activate two conservative DNA repair pathways. Non-homologous end joining (NHEJ) can reconnect broken chromosomes. During the connection process, the break site may introduce the loss or insertion of small fragments of bases, thereby affecting gene function or producing gene knockout effects. If a homologous recombination vector is introduced at this time, homology-directed repair (HDR) can use a similar DNA template to guide the repair, replace the DNA sequence around the break point, achieve specific mutations or introduce foreign DNA at a specific site.

[0070] CRISPR / Cas is a third-generation gene editing technology, which has obvious advantages over ZEN and TALEN technologies. It is simple and convenient to construct, has high gene editing efficiency when used, and has low cost. In general, "CRISPR system" refers to transcripts and other elements related to the expression of CRISPR-related ("Cas") genes or guiding their activity, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (such as tracrRNA or active partial tracrRNA), tracr pairing sequences (covering "direct repeats" and partial direct repeats of tracrRNA processing in the context of endogenous CRISPR systems), guide sequences (also called "spacers" in the context of endogenous CRISPR systems), or other sequences and transcripts from CRISPR loci. In general, the CRISPR system is characterized by elements that promote the formation of CRISPR complexes (also called pre-intervals in the context of endogenous CRISPR systems) at the site of the target sequence. In the context of CRISPR complex formation, "target sequence" refers to a sequence to which the guide sequence is designed to be complementary, wherein the hybridization between the target sequence and the guide sequence promotes the formation of CRISPR complexes. Complete complementarity is not required, provided that there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR complex. A target sequence can comprise any polynucleotide, such as a DNA or RNA polynucleotide. In some embodiments, the target sequence is located in the nucleus or cytoplasm of the cell. Currently, there are three main types of CRISPR / Cas systems: Type I, Type II, and Type III. The Type II CRISPR / Cas system is only found in the genome of bacteria, in which Cas9 is a multifunctional protein with a large molecular weight that is involved in the maturation of crRNAs and subsequent interference reactions.

[0071] In general, a guide sequence is any polynucleotide sequence that has sufficient complementarity to a target polynucleotide sequence to hybridize to the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more when optimally aligned using a suitable alignment algorithm. Optimal alignment can be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows WheelerAligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, Inc.), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0072] In general, a tracr mate sequence includes any sequence that has sufficient complementarity to a tracr sequence to promote one or more of the following: (1) excision of a guide sequence flanked by tracr mate sequences in a cell containing the corresponding tracr sequence; and (2) formation of a CRISPR complex at a target sequence, wherein the CRISPR complex comprises the tracr mate sequence hybridized to the tracr sequence. Typically, the degree of complementarity is with respect to optimal alignment of the tracr mate sequence with the tracr sequence along the length of the shorter of the two sequences. The optimal alignment can be determined by any suitable alignment algorithm, and can further account for secondary structure, such as self-complementarity within the tracr sequence or tracr mate sequence. In some embodiments, when optimally aligned, the degree of complementarity between the tracr sequence and the tracr mate sequence along the length of the shorter of the two is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or more.

[0073] In some embodiments, the CRISPR enzyme is part of a fusion protein comprising one or more heterologous protein domains (e.g., about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more domains in addition to the CRISPR enzyme). The CRISPR enzyme fusion protein may comprise any other protein, and optionally a linker sequence between any two domains. Examples of protein domains that may be fused to the CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza virus hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), autofluorescent proteins including blue fluorescent protein (BFP). The CRISPR enzyme can be fused to a gene sequence encoding a protein or protein fragment that binds to a DNA molecule or binds to other cellular molecules, including, but not limited to, maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusion, GAL4 DNA binding domain fusion, and herpes simplex virus (HSV) BP16 protein fusion. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US 20110059502, which is incorporated herein by reference. In one embodiment, the exons of the corresponding coding genes of the constant regions of one or both of the α and β chains of the TCR are knocked out using CRISPER / Cas technology to render the endogenous TCR inactive, preferably the first exon of the constant region of the endogenous TCR α chain is knocked out at a specific point. "Inhibiting" or "suppressing" the expression of B2M or TCR refers to a reduction in the expression of B2M or TCR in a cell by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 1070%, at least 80%, at least 90%, at least 95%, at least 99% or 100%.More specifically, "inhibiting" or "suppressing" the expression of B2M refers to a decrease in the content of B2M in cells by at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% or 100%. The expression or content of the protein in the cell can be determined by any suitable method known in the art, such as ELISA, immunohistochemistry, Western Blotting or flow cytometry using a specific antibody to B2M or TCR.

[0074] The term "modification" used in the present invention refers to a change in the state or structure of the protein or polypeptide of the present invention. The modification can be chemical, structural or functional.

[0075] The term "T CELL ANTIGEN COUPLER (TAC)" includes three functional domains: a tumor targeting domain, including a single-chain antibody, a designed ankyrin repeat protein (DARPin) or other targeting groups2; an extracellular domain, a single-chain antibody that binds to CD3, thereby bringing the TAC receptor into proximity with other TCR receptors; a transmembrane region and an intracellular region of the CD4 co-receptor, wherein the intracellular region is connected to the protein kinase LCK, catalyzing the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR complex as the initial step of T cell activation.

[0076] The term "BCMA" refers to B cell maturation antigen. In a specific embodiment, BCMA refers to human BCMA. It is a type III transmembrane protein composed of 184 amino acid residues (NCBI Reference Sequence: NP_001183.2), and the amino acid sequence is shown in SEQ ID NO: 26.

[0077] The terms "activation" and "activation" used herein are used interchangeably, and they and their grammatical other forms can refer to the process by which a cell changes from a quiescent state to an active state. The process can include a response to an antigen, migration, and / or a phenotypic or genetic change in a functionally active state. For example, the term "activation" can refer to the process by which a T cell is gradually activated.

[0078] For example, T cells may require at least two signals to be fully activated. The first signal may occur after the antigen-MHC complex engages the TCR, and the second signal may occur by the engagement of a co-stimulatory molecule (see the co-stimulatory molecules listed in Table 1). In vitro, anti-CD3 can simulate the first signal, and anti-CD28 can simulate the second signal. For example, engineered T cells can be activated by expressed CAR. T cell activation or T cell triggering used herein may refer to the state of T cells that have been fully stimulated to induce detectable cell proliferation, cytokine production, and / or detectable effector function.

[0079] The term "chimeric receptor" refers to a fusion molecule formed by connecting cDNAs corresponding to DNA fragments or proteins from different sources using genetic recombination technology, including an extracellular domain, a transmembrane domain, and an intracellular domain. Chimeric receptors include, but are not limited to, chimeric antigen receptors (CARs), modified T cell (antigen) receptors (TCRs), T cell fusion proteins (TFPs), and T cell antigen couplers (TACs). The term "co-stimulatory ligand" includes molecules on antigen presenting cells (e.g., aAPCs, dendritic cells, B cells, etc.) that specifically bind to identical co-stimulatory molecules on T cells, thereby providing a signal that mediates T cell responses together with the first signal provided by, for example, the binding of the TCR / CD3 complex to the MHC molecule loaded with a peptide, including but not limited to proliferation, activation, differentiation, etc. Co-stimulatory ligands may include, but are not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L, PD-L2, 4-1BBL, OX40L, inducible co-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3 / TR6, ILT3, ILT4, HVEM, agonists or antibodies that bind to Toll ligand receptors, and ligands that specifically bind to B7-H3. Co-stimulatory ligands also specifically include antibodies that specifically bind to co-stimulatory molecules present on T cells, such as but not limited to CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0080] The term "costimulatory molecule" refers to an identical binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a co-stimulatory response of the T cell, such as but not limited to proliferation. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA, and Toll ligand receptors.

[0081] The term "co-stimulatory signal" refers to a signal that, in combination with a cell-stimulatory signal molecule, such as TCR / CD3, results in T cell proliferation and / or up-regulation or down-regulation of key molecules.

[0082] The term "chimeric antigen receptor" or "CAR" refers to an engineered molecule that can be expressed by immune cells including but not limited to T cells. CAR is expressed in T cells and can redirect T cells to induce killing of target cells with specificity determined by artificial receptors. The extracellular binding domain of CAR can be derived from mouse, humanized or fully human monoclonal antibodies. When it is in immune effector cells, the cells are provided with specificity for target cells (usually cancer cells) and have intracellular signal generation. CAR generally includes at least one extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signaling domain (also referred to herein as "intracellular signaling domain"), which includes functional signaling domains derived from stimulatory molecules and / or co-stimulatory molecules defined as follows. In some aspects, polypeptide groups are adjacent to each other. The polypeptide group includes a dimerization switch that can couple polypeptides to each other in the presence of a dimerization molecule, for example, an antigen binding domain can be coupled to an intracellular signaling domain. In one aspect, the stimulatory molecule is a delta chain bound to a T cell receptor complex. In one aspect, the cytoplasmic signaling domain further includes one or more functional signaling domains derived from at least one costimulatory molecule defined as follows. In one aspect, the costimulatory molecule is selected from the costimulatory molecules described herein, such as 4-1BB (i.e., CD137), CD27, and / or CD28. In one aspect, CAR includes a chimeric fusion protein, which includes an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one aspect, CAR includes a chimeric fusion protein, which includes an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, CAR includes a chimeric fusion protein, which includes an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one aspect, CAR includes a chimeric fusion protein, which includes an extracellular antigen binding domain, a transmembrane domain, and two functional signaling domains derived from one or more costimulatory molecules.

[0083] The term "signaling domain" refers to a functional portion of a protein that acts by transmitting information within a cell to regulate the activity of the cell via a defined signaling pathway by generating second messengers or by acting as an effector in response to such messengers.

[0084] The term "cell" and its grammatical other forms can refer to cells of human or non-human animal origin. Engineered cells can also refer to cells expressing CAR.

[0085] The term "transfection" refers to the introduction of exogenous nucleic acid into eukaryotic cells. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection and biolistics.

[0086] The term "stable transfection" or "stably transfected" refers to the introduction and integration of foreign nucleic acid, DNA or RNA, into the genome of the transfected cell. The term "stable transfectant" refers to a cell that has stably integrated foreign DNA into its genomic DNA.

[0087] The terms "nucleic acid molecule encoding", "coding DNA sequence" and "coding DNA" refer to the order or sequence of deoxyribonucleotides along a deoxyribonucleic acid chain. The order of these deoxyribonucleotides determines the order of amino acids along a polypeptide (protein) chain. Thus, a nucleic acid sequence encodes an amino acid sequence.

[0088] The term "subject" refers to any animal, such as a mammal or marsupial. The subject of the present invention includes, but is not limited to, humans, non-human primates (such as rhesus monkeys or other types of macaques), mice, pigs, horses, donkeys, cattle, sheep, rats, and poultry of any kind.

[0089] The term "peripheral blood mononuclear cell" (PBMC) refers to cells with a single nucleus in peripheral blood, including lymphocytes, monocytes, etc.

[0090] The term "T cell activation" or "T cell activation" and other grammatical forms thereof may refer to the state of T cells that are fully stimulated to induce detectable cell proliferation, cytokine production and / or detectable effector function. In some cases, "complete T cell activation" may be similar to the cytotoxicity of triggering T cells. Various assays known in the art can be used to measure T cell activation. The assay may be an ELISA, ELISPOT, a flow cytometry assay (CD107) for measuring cytokine secretion, a flow cytometry assay for measuring intracellular cytokine expression, a flow cytometry assay for measuring proliferation, and a cytotoxicity assay (51Cr release assay) for determining target cell elimination. The assay is typically compared with a control (non-engineered cell) and an engineered cell (CAR T) to determine the relative activation of the engineered cell compared to the control. In addition, the assay may be compared with engineered cells incubated or contacted with target cells that do not express the target antigen. For example, the comparison may be a comparison of GPC3-CART cells incubated with target cells that do not express GPC3.

[0091] The term "sequence" and its grammatical alternatives as used herein when referring to a nucleotide sequence may include DNA or RNA and may be single-stranded or double-stranded. Nucleic acid sequences may be mutated. Nucleic acid sequences may be of any length.

[0092] As used herein, the term "effective amount" refers to an amount that provides a therapeutic or prophylactic benefit.

[0093] The term "expression vector" as used herein refers to a vector comprising a recombinant polynucleotide, which comprises an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be provided by a host cell or an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0094] The term "slow virus" as used herein refers to the genus of the Retroviridae family. Retroviruses are unique among retroviruses in being able to infect non-dividing cells; they can deliver large amounts of genetic information into the DNA of host cells, so they are one of the most effective methods for gene delivery vectors. HIV, SIV, and FIV are all examples of slow viruses. Vectors derived from slow viruses provide a means of achieving significant levels of gene transfer in vivo.

[0095] The term "vector" as used herein is a composition comprising an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Many vectors are known in the art, including but not limited to linear polynucleotides, polynucleotides associated with ions or amphiphilic compounds, plasmids and viruses. Therefore, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be interpreted as including non-plasmids and non-viral compounds that promote nucleic acid transfer into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include but are not limited to adenoviral vectors, adeno-associated viral vectors, retroviral vectors, etc.

[0096] The term sequence "identity" as used herein is determined by comparing two sequences that are optimally matched over a comparison window (e.g., at least 20 positions) to determine the percentage of identity, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may include additions or deletions (i.e., gaps), such as 20% or less gaps (e.g., 5 to 15%, or 10 to 12%) for the two sequences that are optimally matched compared to a reference sequence (which does not include additions or deletions). Percentages are usually calculated by determining the number of positions at which the same nucleic acid base or amino acid residue occurs in the two sequences to produce the number of correctly matched positions, dividing the number of correctly matched positions by the total number of positions in the reference sequence (i.e., the window size), and multiplying the result by 100 to produce the percentage of sequence identity.

[0097] As used herein, the term "exogenous" refers to a nucleic acid molecule or polypeptide that is not endogenously expressed in a cell, or that is expressed at a level insufficient to achieve the function that it would have if overexpressed. Thus, "exogenous" includes recombinant nucleic acid molecules or polypeptides expressed in a cell, such as exogenous, heterologous, and overexpressed nucleic acid molecules and polypeptides.

[0098] The term "endogenous" refers to a nucleic acid molecule or polypeptide that is derived from a gene within the organism's own genome.

[0099] In some embodiments, the chimeric receptor of the present invention is a chimeric antigen receptor. The term "chimeric antigen receptor (CAR)" used herein refers to a tumor antigen binding domain fused to an intracellular signal transduction domain that can activate T cells. Commonly, the extracellular binding domain of CAR is derived from a mouse or humanized or human monoclonal antibody.

[0100] Chimeric antigen receptors generally include an extracellular antigen binding region. In some embodiments, the extracellular antigen binding region may be completely human. In other cases, the extracellular antigen binding region may be humanized. In other cases, the extracellular antigen binding region may be mouse-derived, or the chimera in the extracellular antigen binding region may be composed of amino acid sequences from at least two different animals. In some embodiments, the extracellular antigen binding region may be non-human.

[0101] A variety of antigen binding regions can be designed. Non-limiting examples include single-chain variable fragments (scFv) derived from antibodies, fragment antigen binding regions (Fab) selected from libraries, single domain fragments, or natural ligands that engage their cognate receptors. In some embodiments, the extracellular antigen binding region may comprise scFv, Fab, or natural ligands, and any derivatives thereof. The extracellular antigen binding region may refer to a molecule other than a complete antibody, which may comprise a portion of a complete antibody and may bind to an antigen bound by the complete antibody. Examples of antibody fragments may include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; bifunctional antibodies, linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed by antibody fragments.

[0102] The extracellular antigen binding region, such as scFv, Fab or natural ligand, can be a part of the CAR that determines the antigen specificity. The extracellular antigen binding region can bind to any complementary target. The extracellular antigen binding region can be derived from an antibody of a known variable region sequence. The extracellular antigen binding region can be obtained from an antibody sequence obtained from an available mouse hybridoma. Alternatively, the extracellular antigen binding region can be obtained from the full external cleavage sequencing of tumor cells or primary cells such as tumor infiltrating lymphocytes (TIL).

[0103] In some cases, the binding specificity of the extracellular antigen binding region can be determined by complementary determining regions or CDRs, such as light chain CDRs or heavy chain CDRs. In many cases, the binding specificity can be determined by light chain CDRs and heavy chain CDRs. Compared with other reference antigens, a given combination of heavy chain CDRs and light chain CDRs can provide a given binding pocket, which can give antigens (eg, GPC3) greater affinity and / or specificity. For example, CDRs specific to glypican-3 can be expressed in the extracellular binding region of CAR, so that CAR targeting GPC3 can target T cells to tumor cells expressing GPC3.

[0104] In some aspects of any embodiment disclosed herein, the extracellular antigen binding region, such as scFv, may include a light chain CDR specific for an antigen. The light chain CDR may be a complementary determining region of an antigen binding unit such as a scFv light chain of a CAR. The light chain CDR may include a continuous sequence of amino acid residues, or two or more continuous sequences of amino acid residues separated by a non-complementary determining region (such as a framework region). In some cases, the light chain CDR may include two or more light chain CDRs, which may be referred to as light chain CDR-1, CDR-2, etc. In some cases, the light chain CDR may include three light chain CDRs, which may be referred to as light chain CDR-1, light chain CDR-2, and light chain CDR-3, respectively. In some instances, a group of CDRs present in a common light chain may be collectively referred to as light chain CDRs.

[0105] In some aspects of any embodiment disclosed herein, the extracellular antigen binding region, such as scFv, may include a heavy chain CDR specific for an antigen. The heavy chain CDR may be a heavy chain complementary determining region of an antigen binding unit such as scFv. The heavy chain CDR may include a continuous sequence of amino acid residues, or a continuous sequence of two or more amino acid residues separated by a non-complementary determining region (such as a framework region). In some cases, the heavy chain CDR may include two or more heavy chain CDRs, which may be referred to as heavy chain CDR-1, CDR-2, etc. In some cases, the heavy chain CDR may include three heavy chain CDRs, which may be referred to as heavy chain CDR-1, heavy chain CDR-2, and heavy chain CDR-3, respectively. In some cases, a group of CDRs present in a common heavy chain may be collectively referred to as heavy chain CDRs.

[0106] By using genetic engineering, the extracellular antigen binding region can be modified in various ways. In some cases, the extracellular antigen binding region can be mutated so that the extracellular antigen binding region can be selected to have a higher affinity to its target. In some cases, the affinity of the extracellular antigen binding region to its target can be optimized for targets that can be expressed at low levels on normal tissues. This optimization can be performed to minimize potential toxicity. In other cases, the clone of the extracellular antigen binding region with a higher affinity for the membrane-bound form of the target can be superior to its soluble counterpart. This modification can be performed because different levels of soluble forms of the target can also be detected, and their targeting can cause undesirable toxicity.

[0107] In some cases, the extracellular antigen binding region includes a hinge or a spacer. The terms hinge and spacer can be used interchangeably. The hinge can be considered as a part of a CAR for providing flexibility to the extracellular antigen binding region. In some cases, the hinge can be used to detect the CAR on the cell surface of the cell, particularly when the antibody detecting the extracellular antigen binding region does not work or is available. For example, the length of the hinge derived from an immunoglobulin may need to be optimized, depending on the location of the epitope on the target of the extracellular antigen binding region targeting target.

[0108] In some cases, the hinge may not belong to an immunoglobulin, but to another molecule, such as the natural hinge of the CD8α molecule. The CD8α hinge may contain cysteine ​​and proline residues known to play a role in the interaction of CD8 co-receptors and MHC molecules. The cysteine ​​and proline residues may affect the performance of the CAR. The CAR hinge may be adjustable in size. This morphology of the immune synapse between T cells and target cells also defines the distance that cannot be functionally bridged by CAR due to membrane-distal epitopes on cell surface target molecules, even if short hinge CARs are used, the synaptic distance cannot reach the approximate value at which the signal can be conducted. Similarly, the membrane proximal CAR target antigen epitope is only observed to output signals in the context of long hinge CARs. The hinge can be adjusted according to the extracellular antigen binding region used. The hinge can be of any length.

[0109] The transmembrane domain can anchor the CAR to the plasma membrane of the cell. The natural transmembrane portion of CD28 can be used for CAR. In other cases, the natural transmembrane portion of CD8α can also be used in CAR. "CD8" can be a protein with at least 85, 90, 95, 96, 97, 98, 99 or 100% identity to NCBI reference number: NP_001759 or a fragment thereof having stimulatory activity. "CD8 nucleic acid molecule" can be a polynucleotide encoding a CD8 polypeptide, in some cases, the transmembrane region can be a natural transmembrane portion of CD28, and "CD28" can refer to a protein with at least 85, 90, 95, 96, 97, 98, 99 or 100% identity to NCBI reference number: NP_006130 or a fragment thereof having stimulatory activity. "CD28 nucleic acid molecule" can be a polynucleotide encoding a CD28 polypeptide. In some cases, the transmembrane portion can include a CD8α region.

[0110] The intracellular signaling domain of CAR can be responsible for activating at least one of the effector functions of the T cell in which CAR has been placed. CAR can induce the effector function of T cells, for example, the effector function is cytolytic activity or auxiliary activity, including the secretion of cytokines. Therefore, the term "intracellular signaling domain" refers to a protein portion that transduces effector function signals and guides cells to perform specific functions. Although the entire intracellular signaling region can usually be used, in many cases, it is not necessary to use the entire chain of the signaling domain. In some cases, a truncated portion of the intracellular signaling region is used. In some cases, the term intracellular signaling domain is therefore intended to include any truncated portion of the intracellular signaling region sufficient to transduce effector function signals.

[0111] Preferred examples of signaling domains used in CARs may include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors that act synergistically to initiate signal transduction following target-receptor binding, as well as any derivative or variant sequences thereof and any synthetic sequences of these sequences having the same functionality.

[0112] In some cases, the intracellular signaling domain may contain a signaling motif of a known immunoreceptor tyrosine-based activation motif (ITAM). Examples of ITAMs containing cytoplasmic signaling sequences include functional signaling domains derived from proteins of TCRδ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, DAP10 of CD66d, or DAP12. However, in a preferred embodiment, the intracellular signaling domain is derived from the CD3δ chain.

[0113] An example of a T cell signaling domain containing one or more ITAM motifs is the CD3 δ domain, also known as the T cell receptor T3 δ chain or CD247. This domain is part of the T cell receptor-CD3 complex and plays an important role in coupling antigen recognition of several intracellular signal transduction pathways with the primary effector activation of T cells. As used herein, CD3 δ refers primarily to human CD3 δ and its isoforms, as known from Swissprot entry P20963, including proteins with substantially the same sequence. As part of a chimeric antigen receptor, it is reiterated that a full T cell receptor T3 δ chain is not required, and any derivative thereof comprising the signaling domain of the T cell receptor T3 δ chain is suitable, including any functional equivalent thereof.

[0114] The intracellular signaling domain can be selected from any one of the domains in Table 1. In some cases, the domain can be modified so that the identity to the reference domain can be from about 50% to about 100%. Any one of the domains in Table 1 can be modified so that the modified form can contain about 50, 60, 70, 80, 90, 95, 96, 97, 98, 99 or up to about 100% identity.

[0115] The intracellular signaling region of CAR may further include one or more costimulatory domains. The intracellular signaling region may include a single costimulatory domain, such as a delta chain (first generation CAR) or it and CD28 or 4-1BB (second generation CAR). In other examples, the intracellular signaling region may include two costimulatory domains, such as CD28 / OX40 or CD28 / 4-1BB (third generation).

[0116] Together with intracellular signaling domains such as CD8, these co-stimulatory domains can produce downstream activation of kinase pathways, thereby supporting gene transcription and functional cellular responses. The co-stimulatory domains of CARs can activate proximal signaling proteins associated with the CD28 (phosphatidylinositol-4,5-bisphosphate 3-kinase) or 4-1BB / OX40 (TNF-receptor-associated factor adaptor protein) pathways as well as MAPK and Akt activation.

[0117] In some cases, the signal generated by CAR may be combined with auxiliary or costimulatory signals.For costimulatory signal domains, chimeric antigen receptor-like complexes can be designed to include several possible costimulatory signal domains.As well known in the art, in naive T cells, the single engagement of T cell receptors is not enough to induce the complete activation of T cells as cytotoxic T cells.Complete productive T cell activation requires a second costimulatory signal.It has been reported that several receptors for costimulation are provided for T cell activation, including but not limited to CD28, OX40, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a / CD18), 4-1BBL, MyD88 and 4-1BB.The signal transduction pathways used by these costimulatory molecules can all synergize with the main T cell receptor activation signal.The signals provided by these costimulatory signal transduction regions can synergize with the main effector activation signals derived from one or more ITAM motifs (such as CD3zeta signal transduction domains), and the requirements for T cell activation can be completed.

[0118] In some cases, adding a co-stimulatory domain to the chimeric antigen receptor-like complex can enhance the efficacy and durability of the engineered cells. In another embodiment, the T cell signaling domain and the co-stimulatory domain are fused to each other to form a signaling region.

[0119] Table 1. Co-stimulatory domains

[0120]

[0121] As used herein, the term "modulate" refers to a positive or negative change. Examples of modulation include 1%, 2%, 10%, 25%, 50%, 75%, or 100% change.

[0122] The term "treatment" as used herein refers to clinical intervention in an attempt to change the disease process caused by an individual or a cell, which can be either preventive or intervention in the clinical pathological process. The therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the progression of the disease, improving or alleviating the condition, alleviating or improving the prognosis, etc.

[0123] Genetically engineered T cells

[0124] The genetically engineered T cells described herein refer to T cells that target BCMA and are modified by genetic engineering. The endogenous TCR gene and MHC gene encoding of the genetically engineered T cells of the present invention are silenced.

[0125] According to one aspect of the present invention, the present invention also includes nucleic acids encoding the chimeric receptor. The present invention also relates to variants of the above polynucleotides, which encode polypeptides or polypeptide fragments, analogs and derivatives having the same amino acid sequence as the present invention.

[0126] The present invention also provides a vector comprising the nucleic acid encoding the chimeric receptor protein expressed on the surface of T cells.

[0127] In a specific embodiment, the vector used in the present invention is a lentiviral plasmid vector PRRLSIN-cPPT.EF-1α. It should be understood that other types of viral vectors and non-viral vectors can also be used.

[0128] The present invention also includes viruses comprising the above-mentioned vectors. The viruses of the present invention include viruses that are packaged and have infectious power, and also include viruses to be packaged that contain the necessary components for packaging into infectious viruses. Other viruses known in the art that can be used to transduce foreign genes into T cells and their corresponding plasmid vectors can also be used in the present invention.

[0129] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. The selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of transgenes and their propagation in daughter cells. Lentiviral vectors have additional advantages over vectors derived from retroviruses such as murine leukemia viruses because they can transduce non-proliferating cells. They also have the additional advantage of low immunogenicity. The advantage of adenoviral vectors is that they do not fuse into the genome of the target cell, thereby bypassing negative integration-related events.

[0130] Cells can be transfected with a transgenic encoding the chimeric receptor. Transgenic concentrations can be from about 100 picograms to about 50 micrograms. In some cases, the amount of nucleic acid (e.g., ssDNA, dsDNA, or RNA) introduced into cells can be changed to optimize transfection efficiency and / or cell viability. For example, 1 microgram of dsDNA can be added to each cell sample for electroporation. In some cases, the amount of nucleic acid (e.g., double-stranded DNA) required for optimal transfection efficiency and / or cell viability varies according to cell type. In some cases, the amount of nucleic acid (e.g., dsDNA) used for each sample can directly correspond to transfection efficiency and / or cell viability. For example, a series of transfection concentrations. Transgenics encoded by a vector can be integrated into the cell genome. In some cases, transgenics encoded by a vector are integrated forward. In other cases, reverse integration of transgenics encoded by a vector.

[0131] In some cases, the immunoreactive cell can be a stem memory TSCM cell composed of CD45RO(-), CCR7(+), CD45RA(+), CD62L+ (L-selectin), CD27+, CD28+ and / or IL-7Rα+, which can also express CD95, IL-2Rβ, CXCR3 and / or LFA-1, and show many functional attributes different from the stem memory cells. Alternatively, the immunoreactive cell can also be a central memory TCM cell containing L-selectin and CCR7, wherein the central memory cell can secrete, for example, IL-2, but not IFNγ or IL-4. The immunoreactive cell can also be an effector memory TEM cell containing L-selectin or CCR7, and produces, for example, effector cytokines such as IFNγ and IL-4.

[0132] The vector is delivered in vivo, typically by administration to an individual patient, either systemically (e.g., intravenously, intraperitoneally, intramuscularly, subcutaneously, or intracranial infusion) or topically, as described below. Alternatively, the vector can be delivered ex vivo to cells, such as cells removed from an individual patient (e.g., lymphocytes, T cells, bone marrow aspirates, tissue biopsies), which are then typically reimplanted into the patient after selecting cells that have incorporated the vector. Prior to or after selection, the cells can be expanded.

[0133] The T cells can be obtained from many sources, including PBMC, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue and tissue from infection sites, ascites, pleural effusion, spleen tissue and tumors. In some cases, any number of techniques known to those skilled in the art, such as FicollTM separation, can be used to obtain T cells from blood collected from an individual. In one embodiment, cells from the circulating blood of an individual are obtained by single blood sampling. Single blood sampling products generally contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells and platelets. In one embodiment, the cells collected by single blood sampling can be washed to remove the plasma fraction and the cells are placed in a suitable buffer or culture medium for subsequent processing steps. Alternatively, cells can be derived from healthy donors, from patients diagnosed with cancer.

[0134] In some embodiments, the cells may be part of a mixed population of cells having different phenotypic characteristics. Cell lines may also be obtained from transformed T cells according to the aforementioned methods. Cells may also be obtained from a cell therapy bank.

[0135] In some cases, suitable primary cells include peripheral blood mononuclear cells (PBMC), peripheral blood lymphocytes (PBL) and other blood cell subsets, such as but not limited to T cells, natural killer cells, monocytes, natural killer T cells, monocyte precursor cells, hematopoietic stem cells or non-pluripotent stem cells. In some cases, the cell can be any T cell such as tumor infiltrating cells (TIL), such as CD3+T cells, CD4+T cells, CD8+T cells or any other type of T cells. T cells can also include memory T cells, memory stem T cells or effector T cells. T cells can also be selected from a large number of populations, such as selecting T cells from whole blood. T cells can also be expanded from a large number of populations. T cells may also tend to specific populations and phenotypes. For example, T cells can be inclined to phenotypes including CD45RO (-), CCR7 (+), CD45RA (+), CD62L (+), CD27 (+), CD28 (+) and / or IL-7R α (+). Suitable cells can be selected from one or more markers in the following list: CD45RO(-), CCR7(+), CD45RA(+), CD62L(+), CD27(+), CD28(+) and / or IL-7Rα(+). Suitable cells also include stem cells, such as, for example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neuronal stem cells and mesenchymal stem cells. Suitable cells can include any number of primary cells, such as human cells, non-human cells and / or mouse cells. Suitable cells can be progenitor cells. Suitable cells can be derived from a subject to be treated (e.g., a patient).

[0136] The amount of therapeutically effective cells required in the patient can vary according to the viability of the cell and the efficiency of the genetic modification of the cell (e.g., the efficiency of the transgene being integrated into one or more cells, or the expression level of the protein encoded by the transgene). In some cases, the product of the cell viability after genetic modification (e.g., multiplication) and the efficiency of transgene integration can correspond to the therapeutic amount of cells that can be used to give the subject. In some cases, the increase in cell viability after genetic modification may correspond to the reduction in the amount of necessary cells that are effective for the patient to be treated. In some cases, the increase in the efficiency of transgene integration into one or more cells can correspond to the reduction in the number of necessary cells that are effective for treatment in the patient. In some cases, determining the amount of therapeutically effective cells required can include determining the function associated with the cell changing over time. In some cases, determining the amount of therapeutically effective cells required can include determining the function corresponding to the efficiency change of integrating the transgene into one or more cells according to time-related variables (e.g., cell culture time, electroporation time, cell stimulation time). In some cases, therapeutically effective cells can be cell populations that include expression of chimeric receptors from about 30% to about 100% on the cell surface. In some cases, therapeutically effective cells may express about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more than about 99.9% of the chimeric receptor on the cell surface as measured by flow cytometry.

[0137] Nucleic Acids

[0138] Nucleic acids described herein have the meanings conventionally understood by those skilled in the art. Specifically, nucleic acids of the present invention refer to nucleic acids used to modify T cells by genetic engineering means, such as nucleic acids encoding chimeric antigen receptors, and nucleic acids used to inactivate endogenous T cell receptors (TCR) and B2M of T cells, such as nucleic acids used to knock out endogenous T cell receptors (TCR) and B2M of T cells.

[0139] Pharmaceutical composition

[0140] The T cells of the present invention can be used to prepare pharmaceutical compositions. In addition to an effective amount of T cells, the pharmaceutical compositions may also contain a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means that when the molecular entities and compositions are appropriately administered to animals or humans, they will not produce adverse, allergic or other adverse reactions.

[0141] Specific examples of some substances that can serve as pharmaceutically acceptable carriers or components thereof are antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphate buffered saline, among others.

[0142] The composition of the present invention can be prepared into various dosage forms as required, and the physician can determine the dosage that is beneficial to the patient according to factors such as the patient type, age, weight, general disease condition, and administration method. The administration method can be, for example, parenteral administration (such as injection) or other treatment methods.

[0143] "Parenteral" administration of the composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im) or intrasternal injection or infusion techniques. The preparation containing the T cell population administered to an individual contains a plurality of T cells effective for treating and / or preventing a particular indication or disease. Thus, a therapeutically effective population of immunoreactive cells can be administered to an individual. Typically, administration contains about 1×10 4 About 1×10 10 In most cases, the preparation will contain approximately 1 × 10 5 About 1×10 9 Immunoreactive cells, about 5×10 5 About 5×10 8 immunoreactive cells, or about 1×10 6 About 1×10 7 The number of CAR immunoreactive cells administered to an individual will vary between a wide range, depending on the location, source, identity, degree and severity of the cancer, the age and physical condition of the individual to be treated, etc. The doctor will ultimately determine the appropriate dose to be used. In some embodiments, a chimeric antigen receptor is used to stimulate an immune cell-mediated immune response. For example, a T cell-mediated immune response is an immune response involving T cell activation. Activated antigen-specific cytotoxic T cells are able to induce apoptosis in target cells displaying exogenous antigen epitopes on the surface, such as cancer cells displaying tumor antigens. In another embodiment, a chimeric antigen receptor is used to provide anti-tumor immunity in a mammal. Due to the T cell-mediated immune response, the subject will develop anti-tumor immunity.

[0144] In some cases, the method of treating a subject with cancer may involve administering one or more T cells of the present invention to a subject in need of treatment. The T cells can bind to tumor target molecules and induce cancer cell death. As described above, the present invention also provides a method for treating a pathogen infection in an individual, comprising administering a therapeutically effective amount of the T cells of the present invention to the individual.

[0145] Combination with anticancer drugs

[0146] In some embodiments, the T cells of the present invention can be administered in combination with another therapeutic agent. In some embodiments, the other therapeutic agent is a chemotherapeutic agent. Chemotherapeutic drugs that can be used in combination with the T cells of the present invention include, but are not limited to, mitotic inhibitors (vinca alkaloids), including vincristine, vinblastine, vindesine, and novibin (TM) (vinorelbine, 5'-dehydrogen sulfide); topoisomerase I inhibitors, such as camptothecin compounds, including CamptosarTM (irinotecan HCL), HycamtinTM (topotecan HCL) and other compounds derived from camptothecin and its analogs; podophyllotoxin derivatives, such as etoposide, teniposide, and midoxazolidinone; alkylating agents cisplatin, succinimidyl-1-oxadiazine ... Platinum, cyclophosphamide, nitrogen mustard, trimethylenethiophosphoramide, carmustine, busulfan, chlorambucil, brequin, uracil mustard, cloprofen and dacarbazine; antimetabolites, including cytarabine, fluorouracil, methotrexate, mercaptopurine, azathioprine and procarbazine; antibiotics, including but not limited to doxorubicin, bleomycin, dactinomycin, daunorubicin, mycobactin, mitomycin, sarcomamycin C and daunomycin; and other chemotherapeutic drugs, including but not limited to anti-tumor antibodies, dacarbazine, azacytidine, amsarcan, melphalan, ifosfamide and mitoxantrone.

[0147] In some embodiments, chemotherapeutic drugs that can be used in combination with the T cells of the present invention include, but are not limited to, anti-angiogenic agents, including anti-VEGF antibodies (including humanized and chimeric antibodies, anti-VEGF aptamers and antisense oligonucleotides) and other angiogenesis inhibitors, such as angiostatin, endostatin, interferon, retinoic acid, and tissue inhibitors of metalloproteinases-1 and -2.

[0148] Reagent test kit

[0149] The present invention also provides a kit comprising the T cells of the present invention. The kit can be used to treat or prevent cancer, pathogen infection, immune disorders or allogeneic transplantation. In one embodiment, the kit can include a therapeutic or preventive composition containing an effective amount of T cells comprising one or more unit dosage forms.

[0150] In some embodiments, the kit comprises a sterile container that may contain the therapeutic or prophylactic composition.

[0151] In some cases, the kit may include about 1×10 4 cells to about 1×10 6 In some cases, the kit may include at least about 1×10 5 cells, at least about 1×10 6 cells, at least about 1×10 7 cells, at least about 4×10 7cells, at least about 5×10 7 cells, at least about 6×10 7 cells, at least about 6×10 7 cells, 8×10 7 cells, at least about 9×10 7 cells, at least about 1×10 8 cells, at least about 2×10 8 cells, at least about 3×10 8 cells, at least about 4×10 8 cells, at least about 5×10 8 cells, at least about 6×10 8 cells, at least about 6×10 8 Cells, at least about 8 × 10 8 cells, at least about 9×10 8 Cells, at least about 1×10 9 cells, at least about 2×10 9 cells, at least about 3×10 9 cells, at least about 4×10 9 cells, at least about 5×10 9 cells, at least about 6×10 9 cells, at least about 8×10 9 cells, at least about 9×10 9 cells, at least about 1×10 10 cells, at least about 2×10 10 cells, at least about 3×10 10 cells, at least about 4×10 10 cells, at least about 5×10 10 cells, at least about 6×10 10 cells, at least about 9×10 10 cells, at least about 9×10 10 cells, at least about 1×10 11 cells, at least about 2×10 11 cells, at least about 3×10 11 cells, at least about 4×10 11 cells, at least about 5×10 11 cells, at least about 8×10 11 cells, at least about 9×10 11 cells, or at least about 1 × 10 12 For example, approximately 5×10 10 cells.

[0152] In some cases, the kit may include allogeneic cells. In some cases, the kit may include cells that may include genomic modifications. In some cases, the kit may include "off-the-shelf" cells. In some cases, the kit may include cells that may be expanded for clinical use. In some cases, the kit may include contents for research purposes.

[0153] The preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. The embodiments are provided to better illustrate the contents of the present invention, but the contents of the present invention are not limited to the embodiments. Those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above-mentioned invention contents, which belong to the protection scope of the present invention.

[0154] Example 1: Preparation of BCMA-targeted CAR-T cells

[0155] 1. Preparation of CAR-T cells.

[0156] a. Construction of BCMA chimeric antigen receptor plasmid

[0157] Using PRRLSIN-cPPT.EF-1α (purchased from Addgene) as a vector, a lentiviral plasmid PRRLSIN-cPPT.EF-1α-BCMA-BBZ expressing the second-generation chimeric antigen receptor of BCMA antibody was constructed.

[0158] The BCMA-BBZ sequence consists of CD8α signal peptide (SEQ ID NO: 19), BCMA scFv (SEQ ID NO: 16), CD8hinge (SEQ ID NO: 20) and transmembrane region (SEQ ID NO: 24), CD137 intracellular signaling domain (SEQ ID NO: 25), and CD3ξ (SEQ ID NO: 23).

[0159] b. Preparation of CAR-T cells

[0160] The plasmid PRRLSIN-cPPT.EF-1α-BCMA-BBZ was transfected into 293T cells (purchased from the cell bank of the Chinese Academy of Sciences) to obtain the lentivirus PRRL-BCMA-BBZ.

[0161] Peripheral blood mononuclear cells (PBMC cells) were isolated from the peripheral blood of healthy people. Magnetic beads coupled to CD3 / CD28 antibodies (purchased from Thermo Fisher) were added to PBMC to activate CD3-positive T cells, and cultured and expanded in vitro. After 48 hours of T cell activation and expansion, the cell density was adjusted, and PRRL-BCMA-BBZ lentivirus was added at a ratio of MOI = 4 to infect T cells, which were then expanded and cultured to obtain chimeric antigen receptor-modified BCMA CAR-T cells.

[0162] 2. Knockout of TRAC and B2M genes in CAR-T cells.

[0163] The sequence of the first exon of TRAC (gene encoding the α chain of TCR) is shown in SEQ ID NO:8, and the sequence of the first exon of B2M is shown in SEQ ID NO:9.

[0164] Obtaining TRAC gRNA: According to the sequence of TCR gRNA shown in SEQ ID NO: 2, the corresponding primers were designed, and the corresponding gRNA sequence was transcribed and amplified using an in vitro gRNA transcription kit (purchased from Thermo Fisher).

[0165] Obtaining B2M gRNA: According to the sequence of TCR gRNA shown in SEQ ID NO: 1, the corresponding primers were designed, and the corresponding gRNA sequence was transcribed and amplified using an in vitro gRNA transcription kit (purchased from Thermo Fisher).

[0166] The Cas 9 enzyme (purchased from NEB) and the gRNA of B2M as shown in SEQ ID NO: 1 and the gRNA of TCR as shown in SEQ ID NO: 2 were incubated at room temperature for 10 minutes in a ratio of 1:2 to obtain complex RNPs, and the CAR-T cells were mixed with the RNPs to obtain RNP complexes. The RNP complexes were introduced into the CAR-T cells using an electroporator. On the 7th day after electroporation, the knockout of TRAC and B2M genes was detected by flow cytometry, and the knockout of TRAC and B2M single genes and double genes reached more than 70%.

[0167] In addition, the sequence of TRAC gRNA can also be shown as SEQ ID NO: 27, 28, or 29, and T cells in which TRAC is silenced can also be obtained.

[0168] In addition, the sequence of B2M gRNA can also be shown as SEQ ID NO: 30, 31, or 32, and T cells in which B2M is silenced can also be obtained.

[0169] 3.TCR / B2M double negative cell screening.

[0170] B2M / TCR knockout CAR-T cells were amplified in vitro, the cell density was adjusted, the cells were labeled with anti-TCR and B2M antibodies, and then labeled with phycoerythrin (PE)-coupled magnetic beads. The labeled cells were sorted by a sorting column (purchased from Miltenyi) and TCR and B2M negative cells were collected to obtain TCR and B2M double negative universal CAR-T cells. Flow cytometry detection, the results are as follows Figure 1 As shown, after sorting, the percentage of B2M and TCR-negative cells reached more than 99.6%.

[0171] 4. Expression of CAR in BCMA-targeted genetically engineered T cells.

[0172] CAR-T cells that were amplified in vitro but not knocked out of the TRAC / B2M gene, and universal CAR-T cells that were double negative for TRAC / B2M, were taken. T cells that were not transfected with CAR were used as controls. The cell density was adjusted and labeled with an anti-BCMA single-chain antibody to detect the expression of CAR in the double-gene knockout CAR-T cells. Flow cytometry detection, the results are as follows Figure 2 As shown, the CAR positivity rate in normal CAR-T cells (also referred to as BCMA CAR-T in this article) and gene-knockout CAR-T cells (also referred to as BCMA UCAR-T in this article) was above 80%.

[0173] Example 2 In vitro tumor killing experiment of BCMA-targeted double-gene knockout CAR-T cells

[0174] Multiple myeloma cell lines RPMI-8226 and NCI-H929 (purchased from ATCC) were cultured in vitro as tumor target cells. The cell density was adjusted, and a certain amount of cells were inoculated into a 96-well plate. The corresponding CAR-T cells were inoculated at three ratios of effector T cells: target cells of 1:3, 1:1 and 3:1. RPMI-1640 + 10% FBS was used as the culture medium and incubated in a 37°C, 5% CO2 incubator for 18 hours. Using the cytotox-96 non-radioactive cytotoxicity assay kit (purchased from Promega), the supernatant was taken for the determination of lactate dehydrogenase (LDH) content, and the lysis efficiency of RPMI-8226 and NCI-H929 cells in the three experimental groups of UTD, BCMA CAR-T and BCMA UCAR-T was calculated. The test results are as follows Figure 3As shown in the figure, both BCMA UCAR-T and BCMA CAR-T can effectively kill RPMI-8226 and NCI-H929 cells, and the killing ability is comparable, indicating that BCMA UCAR-T can effectively kill myeloma cells.

[0175] Example 3 In vivo tumor killing effect of CAR-T cells after double gene knockout targeting BCMA

[0176] Multiple myeloma cell line RPMI-8226 (purchased from ATCC) was cultured in vitro and 5×10 6 The cells were inoculated into 25 NPG-deficient mice (purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd.). The tumor volume was measured 12 to 14 days after inoculation and the screening grouping was carried out. The average tumor volume was 150-200mm 3 1×10 6 UTD, BCMA CAR-T and BCMA UCAR-T cells, after injection, body weight was measured 2-3 times a week (including grouping and euthanasia day), and the long and short diameters of the tumor were measured and recorded with a vernier caliper, and the tumor volume was calculated. The tumor growth curve was drawn according to the tumor volume, and the differences in tumor growth curves among the groups were compared (tumor volume: V = 1 / 2 × long diameter × short diameter 2 ). At the end of the experiment, the surviving animals were euthanized and the tumor tissues were removed. The tumor weight was weighed and the difference in tumor weight between the groups was calculated. Figure 4 As shown, the volume and weight of tumors in mice in the BCMA UCAR-T cell and BCMA CAR-T cell groups were significantly inhibited, indicating that BCMA UCAR-T cells have good anti-tumor effects in vivo.

[0177] On the 11th day after the injection of CAR-T cells, peripheral blood was collected from mice for CD4 and CD8 flow cytometry staining and quantification using an absolute counting tube (purchased from BD bioscience). Figure 5 As shown, there were a large number of T cells in the peripheral blood in both the BCMA CAR-T cell group and the BCMA UCAR-T cell group, indicating that BCMA UCAR-T cells were effectively expanded in mice.

[0178] Example 4 In vivo GVHD experiment mediated by double gene knockout CAR-T cells targeting BCMA

[0179] The in vivo GVHD model was established using NPG immunodeficient mice as follows.

[0180] UTD, BCMA CAR-T and BCMA UCAR-T cells were expanded and cultured in vitro, and 1×10 7 UTD, BCMA CAR T and BCMA UCAR-T cells, and the negative control group was injected with normal saline (PBS). After injection, the general clinical symptoms of the mice (physical condition, behavioral condition, death, etc.) were observed every day, and the body weight was measured 2-3 times a week. The weight growth curve was drawn according to the weight of the mice, and the weight differences between the groups were compared. Figure 6 As shown, starting from the 30th day, the body weight of mice in the UTD and BCMA CAR-T injection groups decreased significantly, while the body weight of mice in the BCMA UCAR-T group and PBS group remained at a normal level, indicating that BCMA UCAR-T cells can significantly reduce the adverse reactions caused by GVHD reaction.

[0181] At the same time, on the 7th, 21st, and 42nd day after T cell injection, peripheral blood of mice was collected for flow cytometry to compare the survival of T cells in peripheral blood among the groups. Figure 7 As shown in the figure, as time progressed, the number of T cells in the peripheral blood of mice in the UTD and BCMA CAR-T groups continued to increase, while the number of T cells in the BCMA UCAR-T group gradually decreased, indicating that BCMA UCAR-T cells can significantly reduce the abnormal proliferation of T cells caused by GVHD reaction.

[0182] The above experimental results show that BCMA UCAR-T cells do not cause GVHD reaction in vivo and have good safety.

[0183] Example 5 Rejection of allogeneic NK-92 cells to double-gene knockout CAR-T cells

[0184] Studies have shown that knocking out MHC molecules on normal cells (such as the B2M gene in the present invention) may enhance the killing effect of NK cells on B2M-deficient cells. Therefore, we used the NK-92 cell line to detect its rejection of BCMA UCAR-T cells. Adjust the T cell concentration and inoculate it into a 96-well plate. Inoculate the same volume and number of NK-92 cells at a ratio of 1:1 between NK-92 cells and T cells, and incubate them in an incubator for 24 hours. Take the supernatant for lactate dehydrogenase (LDH) content determination and calculate the lysis efficiency of T cells. The test results are as follows: Figure 8 As shown, the lysis levels of BCMA UCAR-T and BCMA CAR-T were comparable, indicating that BCMA-targeting CAR-T with double gene knockout did not cause abnormal rejection of NK-92 cells.

[0185] The sequence used in the present invention is shown in the following table:

[0186]

[0187]

[0188] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. A T cell expressing a chimeric receptor that specifically recognizes BCMA, characterized in that: The endogenous TCR molecules and endogenous MHC molecules of the T cells are silenced; The TCR molecule silencing refers to the silencing of the gene encoding the α chain of TCR; The MHC molecule is an HLA-I class molecule; Wherein, the TCR molecule silencing refers to the silencing of the first exon of the gene encoding the α chain constant region of the TCR; The HLA-I class molecule is a B2M molecule; The chimeric receptor is selected from a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an antibody that specifically binds to BCMA; The antibody that specifically binds to BCMA has HCDR1 shown in SEQ ID NO: 10, HCDR2 shown in SEQ ID NO: 11, HCDR3 shown in SEQ ID NO: 12, LCDR1 shown in SEQ ID NO: 13, LCDR2 shown in SEQ ID NO: 14, and LCDR3 shown in SEQ ID NO:

15.

2. The T cell according to claim 1, wherein Gene editing technology is used to silence endogenous TCR molecules and endogenous HLA-I class molecules.

3. The T cell according to claim 2, wherein The gene editing technology is selected from CRISPR / Cas technology, artificial zinc finger nuclease (Zinc Finger Nucleases, ZFN) technology, transcription activator-like effector (transcription activator-like effector, TALE) technology or TALE-CRISPR / Cas technology.

4. The T cell according to claim 3, characterized in that The gene editing technology uses CRISPR / Cas technology.

5. The T cell according to claim 3, wherein The gene editing technology uses CRISPR / Cas9 technology.

6. The T cell according to claim 3, wherein When CRISPR / Cas9 technology is used to silence the B2M molecule, the sequence of the selected gRNA is shown in SEQ ID NO:

1.

7. The T cell according to claim 3, characterized in that When CRISPR / Cas9 technology is used to silence the α chain molecule of TCR, the sequence of the selected gRNA is shown in SEQ ID NO: 2, 27, 28, or 29.

8. The T cell according to claim 6 or 7, wherein When silencing the B2M molecule, the sequence of the selected gRNA is shown in SEQ ID NO: 1, and when silencing the α chain molecule of TCR, the sequence of the selected gRNA is shown in SEQ ID NO:

2.

9. The T cell according to claim 1, wherein The chimeric antigen receptor comprises: (i) an antibody or fragment thereof that specifically binds to BCMA, CD28 or the transmembrane region of CD8, the co-stimulatory signaling domain of CD28, and CD3ζ; or (ii) an antibody or fragment thereof that specifically binds to BCMA, CD28 or the transmembrane region of CD8, the co-stimulatory signaling domain of CD137, and CD3ζ; or (iii) an antibody or a fragment thereof that specifically binds to BCMA, CD28 or the transmembrane region of CD8, the costimulatory signaling domain of CD28, the costimulatory signaling domain of CD137, and CD3ζ.

10. The T cell according to claim 9, wherein The antibody or fragment thereof that specifically binds to BCMA includes Fv, Fab, Fab', Fab'-SH, F(ab')2; a bifunctional antibody, a linear antibody; a single-chain antibody molecule; or a multispecific antibody formed by antibody fragments.

11. The T cell according to claim 10, wherein The antibody that specifically binds to BCMA is the heavy chain variable region shown in SEQ ID NO: 17 and the light chain variable region shown in SEQ ID NO:

18.

12. The T cell according to claim 10, wherein The antibody that specifically binds to BCMA has a sequence shown in SEQ ID NO:

3.

13. The T cell according to claim 10, wherein The sequence of the chimeric antigen receptor is the sequence shown in SEQ ID NO: 4, 5, 6 or 7.

14. The T cell according to claim 1, wherein The T cells are obtained from the following sources: PBMC, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue or tumor tissue.

15. Use of the T cell according to any one of claims 1 to 14 for preparing a drug for treating multiple myeloma.

16. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the cell according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier or excipient.

17. A kit comprising the cell according to any one of claims 1 to 14 or the pharmaceutical composition according to claim 16.

Images