A chimeric antigen receptor complex, a CAR-T cell comprising the same, a preparation method and use thereof

Abstract

This invention relates to a chimeric antigen receptor complex, CAR-T cells comprising the same, a preparation method therein, and its uses. The chimeric antigen receptor complex comprises a chimeric antigen receptor and a viral protein. The chimeric antigen receptor comprises an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain. The viral protein is selected from at least one of the following proteins: US2 protein, US6 protein, US11 protein, K3 protein, and Nef protein. The chimeric antigen receptor complex of this invention can selectively downregulate HLA-I molecules on the surface of T cells, such as HLA-A, HLA-B, and HLA-C, reducing the host's T cell rejection of allogeneic CAR-T cells, while not affecting HLA-E molecule expression, thus reducing the host's NK cell rejection of allogeneic CAR-T cells and improving the persistence of allogeneic CAR-T cells in the host.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more particularly to a chimeric antigen receptor complex, CAR-T cells containing the complex, preparation methods, and uses. Background Technology

[0002] Since the world's first CAR-T cell therapy (Chimeric Antigen Receptor T Cell Therapy) was approved for marketing in 2017, a new chapter in cell therapy has begun, with marketed CAR-T cell therapies achieving remarkable clinical results in hematologic malignancies. Although several CAR-T therapy products have been approved for marketing, all are autologous CAR-T therapies. Autologous CAR-T therapies involve obtaining T cells from the patient, genetically engineering them in vitro to express chimeric antigen receptors targeting cancer antigens on the T cell surface, and then infusing the proliferated cells back into the patient. Therefore, the widespread application of autologous CAR-T cell drugs still faces many limitations.

[0003] First, autologous CAR-T cell therapy is a personalized product, requiring a single, one-time administration, which hinders large-scale and standardization efforts and results in long waiting times for patients. Second, some patients, due to their health status, prior treatments, and other factors, cannot provide enough T cells, leading to a high failure rate in CAR-T cell preparation and preventing them from benefiting from this groundbreaking technology. Finally, due to the personalized and complex nature of autologous CAR-T cell products, both hardware and software costs are quite high, resulting in expensive pricing and limited patient accessibility. Currently, the international price of CAR-T cell therapy is generally between $300,000 and $500,000, while the prices of several CAR-T products launched in China are around $1 million.

[0004] Given the limitations and challenges of autologous CAR-T cell therapies, universal CAR-T (UCAR-T) therapy from allogeneic donors has demonstrated significant advantages. Universal CAR-T therapy involves collecting T cells from healthy donors, modifying them in vitro using genetic engineering techniques, and then scaling them up to produce readily available cell therapies. Because it originates from healthy donors, it is not affected by the quantity or quality of the patient's T cells, resulting in a high success rate. A single batch can meet the needs of hundreds of patients, offering significant advantages in terms of time and price, enabling more patients to benefit.

[0005] Despite the many advantages of allogeneic CAR-T therapy, two challenges remain to be addressed. Firstly, the donor's T cell receptor (TCR) recognizes human lymphocyte antigen (HLA) on the patient's cell surface, potentially triggering graft-versus-host disease (GvHD) and causing organ damage. Secondly, the HLA expressed on donor T cells is recognized by the host's immune system (T cells and NK cells), leading to host-versus-graft (HvG) reactions. This makes allogeneic CAR-T cells easily eliminated in vivo, affecting their survival and anti-tumor efficacy.

[0006] To address the GvHD risk associated with allogeneic CAR-T cells, the industry currently focuses on gene editing of TCRαβ in allogeneic T cells, or selecting different types of T cells, such as virus-specific T cells (VST), γδ T cells, and invariant NKT cells (iNKT), etc.

[0007] Strategies to improve HvG in allogeneic CAR-T cells primarily involve knocking out MHC class I / II molecules to prevent rejection by host T cells. This can be achieved by gene editing β2M on universal CAR-T cells to block the expression of HLA class I molecules, effectively reducing the immune rejection response of patient T cells. However, HLA molecules are major ligand inhibitors for NK cells. If CAR-T cells completely lack HLA class I molecules, it can lead to the activation of NK cells in the patient, killing allogeneic CAR-T cells. To further prevent NK cell immune rejection of allogeneic CAR-T cells, CAR-T cells can express non-classical HLA molecules (such as HLA-E and HLA-G) or overexpress siglec 7 and siglec 9 ligands to protect universal CAR-T cells from NK cell killing. However, this method requires multi-gene modification of CAR-T cells and overexpression of molecules. Multi-gene editing introduces risks to product safety and significantly increases the difficulty of product development.

[0008] Whether it's knocking out all HLA-I molecules by knocking out B2M, or partially knocking out HLA-A, HLA-B, and other molecules, it involves the knockout of multiple genes. Editing multiple genes on T cells carries the safety risk of chromosomal translocation, which is a huge challenge for the development of universal CAR-T products. Summary of the Invention

[0009] This invention provides a chimeric antigen receptor complex, CAR-T cells containing the complex, a preparation method, and applications. The inventors have discovered that selective expression of virus-derived proteins in CAR-T cells can selectively downregulate HLA molecules on the surface of T cells, reduce rejection by host T cells and NK cells, and improve the persistence of allogeneic CAR-T cells in the host.

[0010] This invention provides a chimeric antigen receptor complex comprising a chimeric antigen receptor and a viral protein; wherein the chimeric antigen receptor comprises an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain.

[0011] The viral protein is selected from at least one of the following proteins:

[0012] US2 protein, US6 protein, US11 protein, K3 protein, Nef protein.

[0013] In some embodiments of the chimeric antigen receptor complex described above, the amino acid sequences of the US2 protein, US6 protein, US11 protein, K3 protein, and Nef protein are shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively.

[0014] In some embodiments of the chimeric antigen receptor complex described above, the chimeric antigen receptor targets one or more antigens selected from the following: CD19, CD20, CD22, BCMA, GPRC5D, CLL1, CD7, CD5, GPC3, DLL3, Trop2, and ROR1.

[0015] Optionally, the chimeric antigen receptor targets CD19 / BCMA.

[0016] In some embodiments of the chimeric antigen receptor complex described above, the extracellular antigen recognition domain includes an anti-BCMA extracellular antigen recognition domain and an anti-CD19 extracellular antigen recognition domain.

[0017] The anti-BCMA extracellular antigen recognition domain includes BCMA VH and BCMA VL, wherein the amino acid sequence of BCMA VH is shown in SEQ ID NO:11 and the amino acid sequence of BCMA VL is shown in SEQ ID NO:12.

[0018] The anti-CD19 extracellular antigen recognition domain includes CD19 VH and CD19 VL, wherein the amino acid sequence of CD19 VH is shown in SEQ ID NO:13 and the amino acid sequence of CD19 VL is shown in SEQ ID NO:14.

[0019] In some embodiments of the chimeric antigen receptor complex described above, the extracellular antigen recognition domain sequentially comprises CD19 VL, a first linker sequence, BCMA VL, a second linker sequence, BCMA VH, a third linker sequence, and CD19 VH.

[0020] Optionally, the first linking sequence, the second linking sequence, and the third linking sequence are independently selected from one or more of the following sequences: SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17;

[0021] Further optionally, the extracellular antigen recognition domain includes an amino acid sequence as shown in SEQ ID NO:18.

[0022] In some embodiments of the chimeric antigen receptor complex described above, the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α; optionally, the hinge region is derived from CD8α; more preferably, the amino acid sequence of the hinge region is as shown in SEQ ID NO:19; and / or

[0023] The transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70; optionally, the transmembrane region is derived from CD8α; more preferably, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO:20.

[0024] In some embodiments of the chimeric antigen receptor complex described above, the intracellular domain includes an intracellular signal transduction region; optionally, it also includes a co-stimulatory signal transduction region.

[0025] Further optionally, the intracellular signal transduction region is derived from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk; optionally, the intracellular signal transduction region is derived from CD3ζ; more preferably, the amino acid sequence of the intracellular signal transduction region is as shown in SEQ ID NO:22; and / or

[0026] Further optionally, the co-stimulatory signal transduction region is derived from one, two, or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, and MyD88; optionally, the co-stimulatory signal transduction region is derived from 4-1BB; more preferably, the amino acid sequence of the co-stimulatory signal transduction region is as shown in SEQ ID NO:23.

[0027] In some embodiments, the chimeric antigen receptor complex further comprises a guide peptide located at the N-terminus of the chimeric antigen receptor amino acid sequence; optionally, the guide peptide is derived from CD8α; more preferably, the amino acid sequence of the guide peptide is as shown in SEQ ID NO:26.

[0028] The present invention also provides a universal CAR-T cell comprising the above-described chimeric antigen receptor complex.

[0029] This invention also provides a method for preparing universal CAR-T cells, comprising:

[0030] 1) Preparation of T cells;

[0031] 2) Gene editing tools and donor templates are introduced into T cells to prepare CAR-T cells;

[0032] The donor template contains a nucleotide sequence encoding a chimeric antigen receptor complex, the nucleotide sequence including a nucleotide sequence encoding an extracellular antigen recognition domain, a nucleotide sequence encoding a hinge region, a nucleotide sequence encoding a transmembrane region, a nucleotide sequence encoding an intracellular domain, and at least one of the following genes: US2 gene, US6 gene, US11 gene, K3 gene, and Nef gene.

[0033] In some embodiments of the above preparation method, the US2 gene, US6 gene, US11 gene, K3 gene, and Nef gene encode amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5, respectively; optionally, the nucleotide sequences of the US2 gene, US6 gene, US11 gene, K3 gene, and Nef gene are shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, respectively.

[0034] In some embodiments of the above preparation method, the gene editing tool is selected from one of the CRISPR / Cas system, zinc finger nuclease system, and transcription activator-like effector nuclease system;

[0035] Optionally, the gene-editing tool is selected from the CRISPR / Cas system;

[0036] Alternatively, the CRISPR / Cas system includes the Cas protein and sgRNA.

[0037] In some embodiments of the above preparation method, the Cas protein includes any one of the following: spCas9, AsCas12a, or LbCas12a;

[0038] The sgRNA targets the TRAC site;

[0039] Optionally, the sgRNA sequence targeting the TRAC site is shown in SEQ ID NO:34.

[0040] In some embodiments of the above preparation method, the donor template is plasmid DNA, dsDNA, or ssDNA;

[0041] Optionally, the donor template includes, in the 5'-3' direction, a right homologous arm sequence, a promoter sequence, a nucleotide sequence encoding an extracellular antigen recognition domain, a nucleotide sequence encoding a hinge region, a nucleotide sequence encoding a transmembrane region, a nucleotide sequence encoding an intracellular domain, a nucleotide sequence encoding T2A, at least one of the following gene sequences: US2, US6, US11, K3, Nef, polyA, and a left homologous arm sequence.

[0042] Further optionally, the donor template further comprises a nucleotide sequence encoding a guide peptide, and the nucleotide sequence encoding the guide peptide is located between the promoter sequence and the nucleotide sequence encoding an extracellular antigen recognition domain.

[0043] In some embodiments of the above preparation method, the promoter is selected from the SFFV promoter, CMV promoter, or EF1α promoter; optionally, the nucleotide sequence of the EF1α promoter is shown in SEQ ID NO:35.

[0044] The polyA is a BGH polyA signal sequence; optionally, the nucleotide sequence of the BGH polyA signal sequence is shown in SEQ ID NO:37.

[0045] In some embodiments of the above preparation method, the nucleotide sequence encoding the extracellular antigen recognition domain includes: a nucleotide sequence encoding CD19 VL, the first linker sequence, BCMA VL, the second linker sequence, BCMA VH, the third linker sequence, and CD19 VH; and / or

[0046] The nucleotide sequence encoding the hinge region is the nucleotide sequence encoding CD8α; and / or

[0047] The nucleotide sequence encoding the transmembrane region is the same as the nucleotide sequence encoding CD8α; and / or

[0048] Nucleotide sequences encoding intracellular domains include those encoding CD3ζ and those encoding 4-1BB.

[0049] In some embodiments of the above preparation method, the left homologous arm sequence and the right homologous arm sequence in the donor template are shown as SEQ ID NO:38 and SEQ ID NO:39, respectively.

[0050] The present invention also provides a pharmaceutical composition comprising the above-described universal CAR-T cells and pharmaceutically acceptable excipients;

[0051] Optionally, pharmaceutically acceptable excipients include protective agents;

[0052] Alternatively, pharmaceutically acceptable excipients include cell cryopreservation solutions;

[0053] Alternatively, the pharmaceutical composition may be an intravenous injection.

[0054] The present invention also provides the use of the above-mentioned chimeric antigen receptor complex, the above-mentioned universal CAR-T cells and the above-mentioned pharmaceutical composition in the preparation of medicaments for treating tumor diseases and autoimmune diseases;

[0055] Optionally, the tumor disease is B-cell acute lymphoblastic leukemia (B-ALL), B-cell non-Hodgkin lymphoma (B-NHL), follicular lymphoma (FL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), or multiple myeloma (MM), and the autoimmune disease is systemic lupus erythematosus (SLE), lupus nephritis (LN), systemic lupus erythematosus-associated immune thrombocytopenic purpura (SLE-ITP), neuromyelitis optica spectrum disorder (NMOSD), myasthenia gravis (MG), myasthenia gravis (MS), or systemic sclerosis (SSc).

[0056] The beneficial effects of this invention are as follows:

[0057] Selectively expressing virus-derived proteins, such as US2, US6, and US11 from human cytomegalovirus (HCMV), K3 from human herpesvirus (HHV), and Nef from human immunodeficiency virus (HIV), in allogeneic CAR-T cells can selectively downregulate HLA-I molecules on the surface of T cells, such as HLA-A, HLA-B, and HLA-C. This reduces the host's T cells' rejection of allogeneic CAR-T cells without affecting HLA-E molecule expression, thus reducing the host's NK cells' rejection of allogeneic CAR-T cells and improving the persistence of allogeneic CAR-T cells in the host. Attached Figure Description

[0058] Figure 1 This graph shows the CD19-CAR positivity rate of cells at different time points. From left to right, they represent 02G-Q33, 02G-Q33-US2, 02G-Q33-US6, 02G-Q33-US11, 02G-Q33-K3, and 02G-Q33-Nef, respectively.

[0059] Figure 2 The graph shows the BCMA-CAR positivity rate of cells at different time points. From left to right, they represent 02G-Q33, 02G-Q33-US2, 02G-Q33-US6, 02G-Q33-US11, 02G-Q33-K3, and 02G-Q33-Nef, respectively.

[0060] Figure 3 The graph shows the expression of HLA-ABC in CAR-positive cells (row 1) and CAR-negative cells (row 2).

[0061] Figure 4 This is a graph showing the expression of B2M in CAR-positive cells (row 1) and CAR-negative cells (row 2).

[0062] Figure 5 The graph shows the expression of HLA-E in CAR-positive cells (row 1) and CAR-negative cells (row 2).

[0063] Figure 6 This is a graph showing the expression of HLA-A in CAR-positive cells (row 1) and CAR-negative cells (row 2).

[0064] Figure 7 This is a graph showing the expression of HLA-B in CAR-positive cells (row 1) and CAR-negative cells (row 2).

[0065] Figure 8The graph shows the expression of HLA-C in CAR-positive cells (row 1) and CAR-negative cells (row 2).

[0066] Figure 9 The expression of HLA-DRDPDQ in CAR-positive cells (row 1) and CAR-negative cells (row 2) is shown.

[0067] Figure 10 The in vitro killing activity against positive target cells Nalm6 is shown. From left to right, they represent UTD (untransduced CAR T cells), 02G-Q33, 02G-Q33-US2, 02G-Q33-US11, and 02G-Q33-Nef.

[0068] Figure 11 The in vitro killing activity against positive target cells MM.1S is shown. From left to right, they represent UTD (untransduced CAR T cells), 02G-Q33, 02G-Q33-US2, 02G-Q33-US11, and 02G-Q33-Nef. Detailed Implementation

[0069] It should be noted that, unless otherwise defined, the technical or scientific terms used in this application shall have the ordinary meaning as understood by one of ordinary skill in the art.

[0070] In this application, the terms "gene editing" and "gene editing technology" have their common meaning in the art, referring to a new technology for site-specific modification of the genome. This technology allows for precise targeting to a specific site on the genome, where target DNA fragments can be cut, or target gene fragments can be knocked in or knocked out. As a molecular biology technique, gene editing technology enables precise modification of chromosomes, thereby altering the existing functions of cells. Compared to cell lines commonly used in basic research, T cells, as primary cells, are not particularly unique except for their inability to proliferate long-term, and can also be edited using gene editing technology. Currently, there are three main gene editing technologies: zinc finger nuclease (ZFN) technology, transcription activator-like effector nuclease (TALEN) technology, and RNA-guided CRISPR / Cas nuclease technology. Compared to traditional gene targeting techniques, this new gene editing technology retains the characteristic of site-specific modification, can be applied to more species and cells, is more efficient, has a shorter construction time, and is less costly.

[0071] In this application, the terms "CRISPR / Cas technology" and "CRISPR / Cas system" have their common meaning in the art. CRISPR stands for clustered regularly interspaced shortpalindromic repeats / CRISPR-associated proteins, an acquired immune system found in most bacteria and all archaea that can directionally cleave foreign gene fragments. Different types of CRISPR / Cas systems have been discovered, with the second type being relatively simple, consisting primarily of the Cas9 protein and guide RNA (gRNA). Compared to earlier ZFN and TALEN technologies, CRISPR / Cas offers advantages such as low off-target rates, high efficiency, affordability, and wide applicability. Furthermore, some literature reports that CRISPR hybrid RNA-DNA (chRDNA) guidance technology significantly improves the specificity of the Cas9 protein compared to whole RNA guidance technology, thereby achieving high levels of intended genome editing in cells and minimizing off-target efficiency.

[0072] In this application, the terms "zinc finger nuclease technology" and "zinc finger nuclease system" have their common meaning in the art. The core design concept of zinc finger nuclease technology is the ingenious integration of two functional domains—a specific recognition module and a functional module. The most classic zinc finger nuclease fuses a non-specific endonuclease, Fok I, with a zinc finger-containing domain, which can recognize specific DNA sequences.

[0073] In this application, the terms "transcription activator-like effector nuclease technology" and "transcription activator-like effector nuclease system" have their common meaning in the art. TALE effectors were initially discovered as an invasion strategy for bacterial infection of plants. Researchers have created a powerful tool with specific gene-editing capabilities—TALEN proteins—by linking the Fok I nuclease to an artificial TALE with sequence-specific binding ability. A typical TALEN protein consists of an N-terminal domain containing a nuclear localization signal (NLS), a central domain containing a typical tandem TALE repeat sequence that recognizes a specific DNA sequence, and a C-terminal domain with Fok I endonuclease function. The core principle of TALEN technology is to achieve three distinct functions—guided entry into the cell nucleus, specific recognition of target DNA, and cleavage of target DNA—in an orderly manner on the same TALEN protein.

[0074] In this application, the term "Chimeric Antigen Receptor" (CAR) is a core component of CAR cell therapy drugs, which may include an extracellular antigen recognition domain (e.g., a portion that binds to tumor-associated antigens (TAAs)), a hinge region, a transmembrane region, and an intracellular domain. CAR-T (Chimeric Antigen Receptor T) cell immunotherapy is considered one of the most promising approaches to combating cancer. CAR-T cells utilize genetic modification to enable T cells to express CAR proteins. These CAR proteins are capable of recognizing intact proteins on the cell membrane surface without antigen presentation, thereby activating and functionally affecting T cells.

[0075] In this application, the term "chimeric antigen receptor complex" refers to a CAR structure formed by attaching one or more functional components (such as viral proteins) to a chimeric antigen receptor.

[0076] In this application, the term "extracellular antigen recognition domain" refers to the antigen recognition domain (ARD). CAR cell therapy products (such as CAR-T cells) rely on extracellular antigen recognition domains to specifically recognize and / or bind to target antigens expressed by tumor cells. To date, antigen recognition domains are derived from the single-chain variable fragment (scFv) of antibodies, or from receptor-ligand interactions, TCR mimics, and variable lymphocyte receptors (VLRs). The most common source to date is the antibody scFv segment, which includes the antibody heavy chain variable region (VH region) and light chain variable region (VL region) linked by a peptide chain, such as the 18-amino acid linker sequence GSTGSGSGKPGSGEGSTKG.

[0077] In this application, the term "hinge region" refers to the connecting segment that acts between the extracellular antigen recognition domain and the transmembrane domain. This region allows the CAR to recognize the antigen by providing a certain range of motion to the antigen recognition domain. Currently used hinge regions are mainly derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α. In addition, typical hinge regions also contain residues that participate in CAR dimerization, which helps to enhance antigen sensitivity.

[0078] In this application, "transmembrane region" refers to a transmembrane domain connecting the intracellular and extracellular components of the CAR structure. Different transmembrane domains can affect CAR expression and stability to some extent, but do not directly participate in signal transduction; however, they can enhance downstream signal transduction through interactions. The transmembrane region may be derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70.

[0079] In this application, the term "intracellular domain" includes intracellular signal transduction regions and may also include co-stimulatory signal transduction regions.

[0080] In this application, the term "intracellular signal transduction region" refers to the activation of at least one normal effector function of an immune effector cell responsible for expressing CAR. The intracellular signal transduction region may originate from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk.

[0081] In this application, the term "co-stimulatory signal transduction region" is used because, in addition to antigen-specific signal stimulation, many immune effector cells require co-stimulation to promote cell proliferation, differentiation, and survival, as well as to activate effector functions. In some embodiments, the CAR may further include one or more co-stimulatory signal transduction regions, wherein the co-stimulatory signal transduction regions may be derived from one, two, or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, and MyD88.

[0082] In this application, the term "guide peptide" refers to a short peptide preceding an extracellular antigen recognition domain (such as the scFv sequence), which guides the export of intracellularly synthesized recombinant proteins to the extracellular space. Commonly used guide peptides include the human CD8α signal peptide or the human GM-CSF receptor α signal peptide.

[0083] In this application, the term "immune effector cell" generally refers to a cell that participates in an immune response, such as promoting an immune effector response. Immune effector cells may be selected from one or more of the following groups: T lymphocytes, natural killer cells (NK cells), peripheral blood mononuclear cells (PBMCs), pluripotent stem cells, T lymphocytes differentiated from pluripotent stem cells, NK cells differentiated from pluripotent stem cells, induced pluripotent stem cells (iPSCs), T cells differentiated from induced pluripotent stem cells (iPSC-T), NK cells differentiated from induced pluripotent stem cells (iPSC-NK), and embryonic stem cells.

[0084] In this application, the term "chimeric antigen receptor T cell" generally refers to CAR-T cells formed by transfecting chimeric antigen receptors (CARs) into T cells. These cells activate T cells by binding to specific antigens on the surface of tumor cells using an antigen-antibody binding mechanism, specifically recognizing and killing tumors (Jackson H et al., Nature Reviews Clinical Oncology, 2016, 13(6):370-383). CAR-T cells recognize tumor antigens without being restricted by human leukocyte antigens (HLA), effectively preventing immune escape by tumor cells through downregulation of major histocompatibility complex (MHC) molecule expression (Fesnak AD et al., Nature Reviews Cancer, 2016, 16(9):566-581).

[0085] In this application, the term "pharmaceutical composition" generally refers to a pharmaceutical composition suitable for use in a patient, which may contain the immune effector cells described in this application, and may also contain one or more pharmaceutically acceptable excipients, such as: carriers, protectants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and preservatives. In some embodiments, pharmaceutically acceptable excipients include protectants, such as cell cryopreservation solutions. In some embodiments, the pharmaceutical composition of this application is a cell suspension or its cryopreserved cells.

[0086] In this application, the term "comprising" generally means including the explicitly specified features, but does not exclude other elements.

[0087] This invention utilizes non-viral site-directed integration technology to prepare universal chimeric antigen receptor T cells. Specifically, the CRISPR-Cas9 gene knock-in method is used to knock exogenous groups into T cells at specific sites.

[0088] CRISPR-Cas9 knock-in (KI) refers to the process where the Cas9 restriction enzyme cuts double-stranded DNA, simultaneously providing a DNA repair template highly homologous to the target gene. The organism then initiates the High-Homogeneous Recombination (HDR) repair pathway, inserting a foreign DNA fragment into the gene at a specific site. The foreign gene in KI can be a protein-coding gene, a DNA element involved in gene regulation, or a non-functional DNA sequence. This repair pathway requires the introduction of a DNA repair template highly homologous to the sequences immediately upstream and downstream of the target editing site, a specific gRNA, and the Cas9 nuclease into the cell. In the presence of a highly homologous DNA template, the HDR mechanism can precisely insert a DNA fragment into a specific genomic site through homologous recombination.

[0089] In this invention, Cas9 protein and sgRNA, along with a donor template containing exogenous groups (dsDNA containing the US2 gene, US6 gene, US11 gene, K3 gene, or Nef gene), are co-transformed into T cells. The transcribed sgRNA guides the Cas9 protein to the insertion site on the T cell, cuts the insertion site, and introduces a DNA double-strand break at the insertion site. Then, homologous recombination is used to knock the exogenous gene into the insertion site, thereby achieving site-specific knock-in of the exogenous gene into the T cell gene.

[0090] In this invention, the sgRNA is designed based on the insertion site sequence of T cells. In this invention, the insertion site refers to any nucleotide site in a gene that a person skilled in the art would like to knock in a foreign gene, such as a TRAC site.

[0091] In this invention, the 5' and 3' ends of the donor template both contain homologous arm sequences, which are homologous to the T cell target gene. These homologous arm sequences mediate homologous recombination between the exogenous gene and the T cell target gene, thereby achieving the knock-in of the exogenous gene. In this invention, the exogenous gene can be any gene fragment that can be knocked into T cell genes by those skilled in the art. It can be a single-gene fragment, or a multi-gene fragment such as a double-gene fragment, a triple-gene fragment, or a quadruple-gene fragment, for example, the US2 gene, the US6 gene, the US11 gene, the K3 gene, or the Nef gene.

[0092] In this invention, the donor template is selected from plasmid templates, double-stranded DNA templates, linear single-stranded DNA templates, and circular single-stranded DNA templates. In other words, this invention does not have special requirements for the form of the donor template; any form of template can be selected according to specific experimental needs to achieve efficient gene knock-in.

[0093] In this invention, the donor template consists of the target gene to be introduced and homologous sequences (homologous arms) upstream and downstream of the target sequence. The length and position of the homologous arms are determined by the size of the edited sequence. The donor template can insert the target gene into the target site through homologous recombination, thereby precisely inserting a DNA sequence into a specific site.

[0094] Sequence information:

[0095] The amino acid sequence of the US2 protein:

[0096] MNNLWKAWVGLWTSMGPLIRLPDGITKAGEDALRPWKSTAKHPWFEIEDNRCYIDNGKLFARGSIVGNMSRFVFDPKADYGGVGENLYVHADDVEFVPGESLKWNVRNLDVMPIFETLALRLVLQGDVIWLRCVPELRVDYTSSAYMWNMQYGMVRKSYTHVAWTIVFYSINITLLVLFIVYVTVDCNLSMMWMRFFVC(SEQ ID NO:1)

[0097] The nucleotide sequence of the US2 gene:

[0098] ATGAACAATCTCTGGAAAGCCTGGGTGGGTCTTTGGACCTCCATGGGTCCCTTGATCCGCCTGCCCGATGGCATCACTAAAGCCGGGGAAGACGCGCTCCGGCCCTGGAAGTCCACGGCCAAGCACCCCTGGTTTGAGATCGAGGACAACCGGTGCTACATTGACAACGGCAAGTTGTTTGCTCGGGGGAGCATCGTGGGCAACATGAGTCGGTTCGTCTTCGATCCGAAGGCCGATTATGGCGGCGTGGGAGAGAACCTGTACGTACACGCCGACGACGTGGAGTTCGTTCCCGGGGAGTCGTTAAAGTGGAACGTGCGGAACTTAGATGTGATGCCGATCTTCGAGACGCTAGCCCTGCGTCTGGTACTGCAAGGGGATGTGATCTGGCTGCGTTGCGTCCCCGAACTGCGAGTGGATTACACGTCTAGCGCGTACATGTGGAACATGCAGTACGGGATGGTGCGGAAGTCATACACGCATGTGGCCTGGACAATAGTGTTTTACTCCATAAACATTACCCTGTTGGTATTGTTTATCGTGTATGTGACTGTGGACTGTAACTTGTCTATGATGTGGATGCGGTTTTTCGTGTGCTAA(SEQ ID NO:6)

[0099] Amino acid sequence of US6 protein:

[0100] MDLLIRLGFLLMCALPTPGERSSRDPKTLLSLSPRQACVPRTKSHRPVCYNDTGDCTDADDSWKQLGEDFAHQCLQAAKKRPKTHKSRPNDRNLEGRLTCQRVRRLLPCDLDIHPSHRLLTLMNNCVCDGAVWNAFRLIERHGFFAVTLYLCCGITLLVVILALLCSITYESTGRGIRRCGS(SEQ ID NO:2)

[0101] Nucleotide sequence of US6 gene:

[0102] ATGGACCTCTTGATCCGTCTCGGTTTTCTGTTGATGTGTGCGTTGCCGACCCCCGGTGAGCGGTCTTCGCGTGACCCGAAAACCCTTCTCTCTCTGTCTCCGCGACAAGCTTGTGTTCCGAGAACAAAGTCGCACAGACCCGTTTGTTACAACGATACAGGGGACTGCACAGATGCAGATGATAGCTGGAAACAGCTGGGTGAGGACTTTGCGCACCAATGCTTGCAGGCGGCGAAAAAGAGGCCTAAAACGCACAAATCCCGTCCGAACGATAGGAACCTTGAGGGTAGGCTGACCTGTCAACGAGTCCGTCGGCTACTGCCCTGTGATTTGGATATTCATCCTAGCCACCGGTTGTTAACGCTTATGAATAACTGCGTCTGTGACGGGGCCGTTTGGAACGCGTTTCGCTTGATAGAACGACACGGATTCTTCGCTGTGACTTTGTATTTATGTTGCGGGATTACCCTGCTGGTTGTTATTCTAGCATTGCTGTGCAGCATAACATACGAATCGACTGGACGTGGGATTCGACGTTGTGGCTCCTAA(SEQ ID NO:7)

[0103] Amino acid sequence of US11 protein:<000022s>

[0104] MNLVMLILALWAPVAGSMPELSLTLFDEPPPLVETEPLPPLPDVSEYRVEYSEARCVLRSGGRLEALWTLRGNLSVPTPTPRVYYQTLEGYADRVPTPVEDISESLVAKRYWLRDYRVPQRTKLVLFYFSPCHQCQTYYVECEPRCLVPWVPLWSSLEDIERLLFEDRRLMAYYALTIKSAQYTLMMVAVIQVFWGLYVKGWLHRHFPWMFSDQW(SEQID NO:3)

[0105] Nucleotide sequence of US11 gene:

[0106] It should be noted that there seems to be a small error in the original text where "<000022s>" was likely a typo and should be " ". This has been corrected in the translation for consistency.ATGAATCTTGTAATGCTTATTCTAGCCCTCTGGGCCCCGGTCGCGGGTAGTATGCCTGAATTATCCTTGACTCTTTTCGATGAACCTCCGCCCTTGGTGGAGACGGAGCCGTTACCGCCTCTGCCCGATGTTTCGGAGTACCGAGTAGAGTATTCCGAGGCGCGCTGCGTGCTCCGATCGGGCGGTCGACTGGAGGCTCTGTGGACCCTGCGCGGGAACCTGTCCGTGCCCACGCCGACACCCCGGGTGTACTACCAGACGCTGGAGGGCTACGCGGATCGAGTGCCGACGCCGGTGGAGGACATCTCCGAAAGCCTCGTCGCAAAACGCTACTGGCTCCGGGACTATCGTGTTCCCCAACGCACAAAACTCGTGTTGTTCTACTTTTCCCCCTGTCATCAATGCCAAACTTATTATGTAGAGTGCGAACCCCGGTGCCTCGTGCCTTGGGTTCCCCTGTGGAGCTCGTTAGAGGACATCGAACGACTATTGTTCGAAGATCGCCGTCTAATGGCGTACTACGCGCTCACGATTAAGTCGGCGCAGTATACGCTGATGATGGTGGCAGTGATTCAAGTGTTTTGGGGGCTGTATGTGAAAGGTTGGCTGCACCGACATTTTCCCTGGATGTTTTCGGACCAGTGGTAA(SEQ ID NO:8)

[0107] Amino acid sequence of K3 protein:

[0108] MEDEDVPVCWICNEELGNERFRACGCTGELENVHRSCLSTWLTISRNTACQICGVVYNTRVVWRPLREMTLLPRLTYQEGLELIVFIFIMTLGAAGLAAATWVWLYIVGGHDPEIDHVAAAAYYVFFVFYQLFVVFGLGAFFHMMRHVGRAYAAVNTRVEVFPYRPRPTSPECAVEEIELQEILPRGDNQDEEGPAGAAPGDQDGPADGAPVHRDSEESVDEAAGYKEAGEPTHNDGRDDNVEPTAVGCDCNNLGAERYRATYCGGYVGAQSGDGAYSVSCHNKAGPSSLVDILPQGLLGGGYGSMGVIRKRSAVSSALMFH(SEQ ID NO:4)

[0109] Nucleotide sequence of the K3 gene:

[0110] ATGGAAGATGAGGATGTTCCTGTCTGCTGGATTTGCAACGAGGAGCTCGGAAATGAGAGATTTAGAGCCTGTGGATGCACAGGAGAGCTCGAGAACGTCCATAGAAGTTGTTTAAGCACCTGGCTCACTATCTCTAGAAACACGGCCTGTCAGATATGTGGCGTCGTATACAACACGCGCGTGGTCTGGCGACCCTTGCGCGAAATGACGCTATTGCCTCGGCTGACTTACCAGGAGGGTCTGGAACTGATTGTTTTTATTTTCATCATGACATTGGGGGCCGCTGGCCTTGCCGCTGCGACGTGGGTTTGGCTATATATAGTGGGCGGTCATGACCCAGAGATAGATCACGTCGCTGCAGCCGCGTACTACGTTTTTTTCGTGTTTTACCAATTGTTTGTCGTTTTTGGGTTGGGTGCGTTTTTCCACATGATGCGCCACGTGGGGCGGGCATACGCTGCTGTAAACACCCGGGTTGAAGTGTTTCCATATAGACCTCGGCCGACATCACCAGAGTGTGCGGTAGAGGAGATCGAGCTTCAGGAAATTCTTCCCCGTGGGGATAACCAGGACGAGGAGGGGCCCGCGGGGGCAGCTCCAGGCGACCAAGATGGCCCCGCGGATGGCGCTCCTGTGCATCGCGACTCAGAAGAATCCGTGGATGAAGCTGCAGGGTACAAGGAAGCGGGAGAACCAACACATAATGATGGACGTGATGACAATGTAGAGCCAACCGCGGTTGGGTGTGACTGTAACAACTTGGGCGCTGAGCGGTATAGGGCCACTTACTGTGGCGGTTATGTTGGTGCCCAGTCGGGCGATGGGGCTTATAGTGTCTCCTGCCATAATAAGGCTGGACCCTCCTCTCTAGTTGATATCCTTCCACAGGGTTTGCTTGGGGGTGGCTATGGTTCCATGGGCGTGATTAGGAAACGTTCGGCTGTTTCGTCTGCCCTTATGTTTCATTAA(SEQ ID NO:9)

[0111] Amino acid sequence of Nef protein:

[0112] MGGKWSKSSVIGWPTVRERMRRAEPAADRVGAASRDLEKHGAITSSNTAATNAACAWLEAQEEEEVGFPVTPQVPLRPMTYKAAVDLSHFLKEKGGLEGLIHSQRRQDILDLWIYHTQGYFPDWQNYTPGPGVRYPLTFGWCYKLVPVEPDKIEEANKGENTSLLHPVSLHGMDDPEREVLEWRFDSRLAFHHVARELHPEYFKNC(SEQ ID NO:5)

[0113] Nucleotide sequence of Nef gene:

[0114] ATGGGTGGCAAGTGGTCAAAAAGTAGTGTGATTGGATGGCCTACTGTAAGGGAAAGAATGAGACGAGCTGAGCCAGCAGCAGATAGGGTGGGAGCAGCATCTCGAGACCTGGAAAAACATGGAGCAATCACAAGTAGCAATACAGCAGCTACCAATGCTGCTTGTGCCTGGCTAGAAGCACAAGAGGAGGAGGAGGTGGGTTTTCCAGTCACACCTCAGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAAAGAAGACAAGATATCCTTGATCTGTGGATCTACCACACACAAGGCTACTTCCCTGATTGGCAGAACTACACACCAGGGCCAGGGGTCAGATATCCACTGACCTTTGGATGGTGCTACAAGCTAGTACCAGTTGAGCCAGATAAGATAGAAGAGGCCAATAAAGGAGAGAACACCAGCTTGTTACACCCTGTGAGCCTGCATGGGATGGATGACCCGGAGAGAGAAGTGTTAGAGTGGAGGTTTGACAGCCGCCTAGCATTTCATCACGTGGCCCGAGAGCTGCATCCGGAGTACTTCAAGAACTGCTAA(SEQ ID NO:10)

[0115] The amino acid sequence of BCMA VH:

[0116] EVQLVESGGGLVQPGGSLRLSCTASGFSLSTYHMTWVRQAPGKGLEWIG VISSSGSTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDYV IDLWGPGTLVTVSS(SEQ ID NO:11)

[0117] Nucleotide sequence encoding BCMA VH:

[0118] GAAGTGCAGCTGGTGGAGTCCGGCGGTGGACTGGTGCAACCGGGAGGCTCACTCAGATTGTCATGCACCGCCTCTGGCTTTAGTCTCTCCACCTATCATATGACTTGGGTGAGGCAGGCACCCGGCAAGGGCCTGGAATGGATCGGCGTGATCTCTTCCAGCGGTAGCACCTATTA CGCCTCTTGGGCGAAGGGCAGGTTTACCATCAGCCGCGACAACAGCAAGAATACCGTTTACCTGCGATGAATAGCCTGAGGGCCGAAGACACGGCGGTCTATTTCTGTGCACGGGACCTTGACTACGTTATTGACCTGTGGGGCCCTGGGACCCTCGTAACTGTGAGCAGC(SEQ ID NO:21)

[0119] The amino acid sequence of BCMA VL:

[0120] EIVMTQSPSTLSSASVGDRVIINCQSSPSVYNNYLSWYQQKPGKAPKLLIY ETSTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCAGTYVSGDRRAFGQ GTKLTVL(SEQ ID NO:12)

[0121] Nucleotide sequence encoding BCMA VL:

[0122] GAGATCGTGATGACCCAGTCCCCAAGTACACTGAGCGCCTCCGTGGGCGACCGCGTGATCATAAACTGTCAAAGCTCACCCTCTGTTTACAACAATTACCTGTCTTGGTATCAACAGAAGCCCGGTAAGGCCCCCAAACTGCTCATTTACGAGACATCCACCCTGGCATCCGGGGTGCCAAGCCGCTTCTCCGGGAGTGGGTCTGGCGCCGAGTTCACCCTGACCATATCTTCCCTGCAGCCCGACGACTTCGCAACGTACTATTGCGCCGGAACCTATGTAAGTGGGGATAGACGCGCCTTCGGGCAGGGCACGAAGTTGACCGTGCTG(SEQ ID NO:24)

[0123] Amino acid sequence of CD19 VH:

[0124] EVQLVQSGAEVKKPGESLKISCKASGYAFSSYWMNWVRQMPGKGLE WMGQIYPGDGDTNYNGKFKGQVTLSADKSISTAYLQWSSLKASDTAMYFC ARKTISSVVDFYFDYWGQGTTVTVSS(SEQ ID NO:13)

[0125] Nucleotide sequence encoding CD19 VH:

[0126] GAGGTGCAGCTGGTGCAGAGCGGCGCCGAGGTGAAGAAGCCCGGCGAGAGCCTGAAGATCAGCTGCAAAGCTTCCGGCTACGCCTTCAGCAGCTACTGGATGAACTGGGTGAGACAGATGCCCGGCAAGGGCCTGGAGTGGATGGGGCAGATCTACCCCGGCGACGGCGACACCAACTACAACGGCAAGTTCAAGGGCCAAGTGACCCTGAGCGCCGACAAGAGCATCAGCACCGCCTACCTGCAGTGGAGCAGCCTGAAGGCCTCCGACACCGCCATGTACTTCTGCGCTAGAAAGACCATCAGCAGCGTGGTGGACTTCTACTTCGACTACTGGGGCCAAGGCACCACCGTGACCGTGAGCAGC(SEQ IDNO:25)

[0127] The amino acid sequence of CD19 VL:

[0128] EIVMTQSPATLSVSPGERATLSCKASQNVGTNVAWYQQKPGQAPRPLI YSATYRNSGIPARFSGSGSGTEFTLTISSLQSEDFAVYFCQQYNRYPYTFGGG TKLEIK(SEQ ID NO:14)

[0129] Nucleotide sequence encoding CD19 VL:

[0130] GAAATCGTGATGACCCAGTCCCCTGCTACACTGAGCGTGTCCCCAGGCGAGCGGGCCACACTGTCTTGCAAGCCTCCCAAAACGTGGGCACCAACGTGGCCTGGTATCAGCAGAAGCCCGGCCAGGCCCCCAGGCCCCTGATCTACAGCGCCACCTACAGA AACAGCGGCATCCCTGCCAGATTCAGCGGCAGCGGCAGCGGCACCGAGTTCACCCTGACCATCAGCAGCCTGCAGTCCGAGGACTTCGCCGTCTACTTCTGTCAGCAGTACAACAGATACCCCTACACATTCGGCGGGGGGACCAAGCTGGAGATCAAA(SEQ ID NO:27)

[0131] The amino acid sequence of the first linker sequence:

[0132] GGGGS (SEQ ID NO:15)

[0133] Nucleotide sequence encoding the first linker sequence:

[0134] GGCGGAGGGGGCTCA(SEQ ID NO:28)

[0135] The amino acid sequence of the second linker sequence:

[0136] GGGGSGGGGSGGGGS(SEQ ID NO:16)

[0137] Nucleotide sequence encoding the second linker sequence:

[0138] GGCGGAGGGGGCTCAGGAGGTGGCGGTAGCGGAGGAGGCGGCTCA (SEQ ID NO: 29)

[0139] The amino acid sequence of the third linker sequence:

[0140] GGGGS (SEQ ID NO:17)

[0141] Nucleotide sequence encoding the third linker sequence:

[0142] GGTGGCGGTGGCTCG(SEQ ID NO:30)

[0143] Amino acid sequence of the extracellular antigen recognition domain:

[0144] EIVMTQSPATLSVSPGERATLSCKASQNVGTNVAWYQQKPGQAPRPLIYSATYRNSGIPARFSGSGSGTEFTLTISSLQSEDFAVYFCQQYNRYPYTFGGGTKLEIKGGGGSEIVMTQSPS TLSASVGDRVIINCQSSPSVYNNYLSWYQQKPGKAPKLLIYETSTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCAGTYVSGDRRAFGQGTKLTVLGGGGSGGGGSGGGGSEVQLV ESGGGLVQPGGSLRLSCTASGFSLSTYHMTWVRQAPGKGLEWIGVISSSGSTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDYVIDLWGPGTLVTVSSGGGGSEVQLV QSGAEVKKPGESLKISCKASGYAFSSYWMNWVRQMPGKGLEWMGQIYPGDGDTNYNGKFKGQVTLSADKSISTAYLQWSSLKASDTAMYFCARKTISSVVDFYFDYWGQGTTVTVSS(SEQ ID NO:18)

[0145] Nucleotide sequence encoding extracellular antigen recognition domain:

[0146]

[0147] The amino acid sequence of CD8α in the hinge region:

[0148] TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD(SEQ ID NO:19)

[0149] Nucleotide sequence encoding CD8α of the hinge region:

[0150] ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCG CGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGG GGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 32)

[0151] The amino acid sequence of the transmembrane region CD8α:

[0152] IYIWAPLAGTCGVLLLSLVITLYC(SEQ ID NO:20)

[0153] Nucleotide sequence encoding the transmembrane region CD8α:

[0154] ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC(SEQ ID NO:33)

[0155] The amino acid sequence of the intracellular signal transduction region CD3ζ:

[0156] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT KDTYDALHMQALPPR(SEQ ID NO:22)

[0157] Nucleotide sequence encoding the intracellular signal transduction region CD3ζ:

[0158] AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGC CTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA(SEQ ID NO:36)

[0159] The amino acid sequence of the co-stimulatory signal transduction region 4-1BB:

[0160] KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL(SEQ ID NO:23)

[0161] The nucleotide sequence encoding the co-stimulatory signal transduction region 4-1BB is as follows:

[0162] AAACGGGGCAGAAAGAAACTCCTGTATATTCAAACAACCATTTA TGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATT TCCAGAAGAAGAAGAAGGAGGATGTGAACTG(SEQ ID NO:41)

[0163] The amino acid sequence of the guide peptide CD8α:

[0164] MALPVTALLLPLALLLHAARP(SEQ ID NO:26)

[0165] Nucleotide sequence encoding the guide peptide CD8α:

[0166] ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCC ACGCCGCCAGGCCG(SEQID NO:42)

[0167] sgRNA sequence targeting the TRAC site:

[0168] AdGdAdGdTdCUdCdTdCdAGCUGGUdAdCA(SEQ ID NO:34)

[0169] The nucleotide sequence of the EF1α promoter:

[0170] GGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTCCTAGAGAAGGTGGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAG (SEQ ID NO: 35)

[0171] The nucleotide sequence of the BGH polyA signal sequence:

[0172] CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG (SEQ ID NO: 37)

[0173] Nucleotide sequence of the left homologous arm:

[0174] CACGGCAGGGTCAGGGTTCTGGATATCTGTGGGACAAGAGGATCAGGGTTAGGACATGATCTCATTTCCCTCTTTGCCCCAACCCAGGCTGGAGTCCAGATGCCAGTGATGGACAAGGGCGGGGCTCTGTGGGGCTGGCAAGTCACGGTCTCATGCTTTATACGGGAAATAGCATCTTAGAAACCAGCTGCTCGTGATGGACTGGGACTCAGGGACAGGCACAAGCTATCAATCTTGGCCAAGAGGCCATGATTTCAGTGAACGTTCACGGCCAGGCCTGGCCTGCCACTCAAGGAAACCTGAAATGCAGGGCTACTTAATAATACTGCTTATTCTTTTATTTAATAGGATCTTCTTCAAAACCCCAGCAATATAACTCTGGCAGAGTAAAGGCAGGCATGGGAAAAAGGCCCAGCAAAGCAAACTGTACATCTTGGAATCTGGAGTGGTCTCCCCAACTTAGGCTGGGCATTAGCAGAATGGGAGGTTTATGGTATGTTGGCATTAAGTTGGGAAATCTATCACATTACCAGGAGATTGCTCTCTCATTGATAGAGGTTTTGAACTATAAATCAGAACACCTGCGTCTAAGCCCCAGCACTACCGTTTACTCGATATAAGGCCTTGAGCAAGTCACAGCAGCTCCTTACATCTCAGGAATTTCACCTGCAAAATGAATATGGTGCCTCATCCACCTTCCTAGCCAGGCTCTTCTGAGAAAGAAATGAGAGCTTCTCCATATAAACATCTATTTAATAAACTGTAAAGTACCAAACAAATGTTAGTTGGAGCCACTGACCCTG(SEQ ID NO:38)Nucleotide sequence of the right homologous arm:

[0175] TTGGACTTTTCCCAGCTGACAGATGGGCTCCCCCAACTAGAATGGTGCTTCCTCTGGGCACACCCCTCATCTGACTTTTTAATTCCTCCACTTCAACACCTGGTGCATTCATGTGCCGGCACAATCAGTGATTGGTGGGTTAATGAGTGACTGCGTGAGACTGACTTAGTGAGCTGGGAAAGATTTTTTGGCAGACAGGGAGAAATAAGGAGAGGCAACTTGGAGAAGGGGCTTAGAATGAGGCCTAGAAGAGCAGTAAGGGGCAAACAGTCTGAGCAAAGGCAGGCAGGCAGGAACTCAGTTGGAGAGACTGAGGCTGGGCCACGTGCCCTCTCCTGCCACCTTCTCTTCATCTGCTTTTTTCCCGTGTCATTCTCTGGACTGCCAGAACAAGGCTCACTGTTTCTTAGTAAAAAGAGGGTTTTGGTGGCAATGGATAAGGCCGAGACCACCAATCAGAGGAGTTTTAGACATCATTGACCAGAGCTCTGGGCAGAACCTGGCCATTCCTGAAGCAAGGAAACAGCCTGCGAAGGCACCAAAGCTGCCCTTACCTGGGCTGGGGAAGAAGGTGTCTTCTGGAATAATGCTGTTGTTGAAGGCGTTTGCACATGCAAAGTCAGATTTGTTGCTCCAGGCCACAGCACTGTTGCTCTTGAAGTCCATAGACCTCATGTCTAGCACAGTTTTGTCTGTGATATACACATCAGAATCCTTACTTTGTGACACATTTGTTTGAGAATCAAAATCGGTGAATAGGCAGACAGACTTGTCACTGGATTTAGAGTCTCTCAGCTGGT(SEQ ID NO:39)

[0176] Amino acid sequence of T2A:

[0177] EGRGSLLTCGDVEENPGP(SEQ ID NO:43)

[0178] Nucleotide sequence encoding T2A:

[0179] GAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCC GGCCCT(SEQ ID NO:40)

[0180] The nucleotide sequence of 02G-Q33:

[0181]

[0182] The nucleotide sequence of 02G-Q33-US2:

[0183]

[0184] The nucleotide sequence of 02G-Q33-US6:

[0185]

[0186] The nucleotide sequence of 02G-Q33-US11:

[0187]

[0188] The nucleotide sequence of 02G-Q33-K3:

[0189]

[0190] The nucleotide sequence of 02G-Q33-Nef:

[0191]

[0192] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the medicinal materials and reagents used in the following examples are commercially available products.

[0193] Example

[0194] 1. Construction and preparation of dsDNA co-expressing proteins from different viruses

[0195] Following the dual-target CD19 / BCMACAR gene sequence, proteins from different viral sources were expressed via T2A ligation. CAR structures 02G-Q33-US2 (SEQ ID NO:45), 02G-Q33-US6 (SEQ ID NO:46), 02G-Q33-US11 (SEQ ID NO:47), 02G-Q33-K3 (SEQ ID NO:48), and 02G-Q33-Nef (SEQ ID NO:49) were constructed, with 02G-Q33 (SEQ ID NO:44) used as a control.

[0196] 2. Preparation of CD19 / BCMACAR-T using non-viral site-specific integration technology

[0197] CAR-T cells were prepared using non-viral site-directed integration technology, and CAR sequences carrying viral protein genes were site-directedly integrated into the TRAC site. Homologous arm sequences for inserting the TRAC site were added to both sides of the different CAR structures to form dsDNA sequences with different structures.

[0198] The T cell culture medium consisted of Opti-mizer basal medium + 2.6% Supplement + 5% ISR + 1% GlutaMax + IL-15 5 ng / mL + IL-7 10 ng / mL.

[0199] PBMC cell resuscitation was performed on day -1.

[0200] On day 0, T cells in PBMC cells were sorted using magnetic beads, and T cells were activated using Thermo CD3 / CD28 activation magnetic beads.

[0201] On day 2, the activation beads were removed, and Cas9 protein and sgRNA (SEQ ID NO:34) were resuspended in electroporation buffer to form an RNP, which was then incubated for 15 minutes. Donor template dsDNA was added to the RNP. Activated T cells were resuspended in electroporation buffer and mixed with the RNP. The mixture was transferred to an electroporation cuvette, which was then transferred to a Lonza electroporator for electroporation under EH100 conditions. After electroporation, the electroporation mixture was transferred to T cell culture medium, and the cells were cultured in a CO2 incubator at 37°C and 5% CO2.

[0202] Add T cell culture medium every 2-3 days according to cell growth, and harvest CAR-T cells after 13-15 days of culture.

[0203] 3. Flow cytometry detection

[0204] 3.1 CAR Positive Rate Detection

[0205] 3.1.1 During cell culture after electroporation, samples were taken every 2-3 days for flow cytometry analysis. Antibodies included CD19-PE and BCMA-FITC.

[0206] 3.1.2 After incubating the cells with the flow cytometry antibody for 15-20 minutes, wash them with FACS buffer (FBS + 0.25% BSA).

[0207] 3.1.3 The expression of CD19-CAR and BCMA-CAR on the cell surface was detected by flow cytometry.

[0208] 3.2 Detection of HLA molecule expression on the surface of T cells

[0209] 3.2.1 After culturing for 13-15 days, CAR-T cells were harvested and samples were taken for flow cytometry analysis. Antibodies included anti-HLA-ABC, anti-B2M, anti-HLA-E, anti-HLA-A, anti-HLA-B, anti-HLA-C, and anti-HLA-DRDPDQ.

[0210] 3.2.2 After incubating the cells with the flow cytometry antibody for 15-20 minutes, wash them with FACS buffer.

[0211] 3.2.3 The expression of HLA-ABC, B2M, HLA-E, HLA-A, HLA-B, HLA-C, and HLA-DRDPDQ on the cell surface was detected by flow cytometry.

[0212] 4. Validation of CD19 / BCMACAR-T's cytotoxic activity against target cells

[0213] 4.1 Target cell placement

[0214] 4.1.1 Target cell preparation:

[0215] 4.1.1.1 The target cells are in good logarithmic growth phase with a cell viability of over 85%.

[0216] 4.1.1.2 Count the target cells according to standard cell counting procedures, and take 2 × 10⁻⁶ cells. 6 Transfer the target cells to a 15ml centrifuge tube, centrifuge at 300g for 8 minutes, remove the supernatant, and resuspend the target cells in T0 medium (X-Vivo medium + 5% inactivated FBS + 1% GlutaMax) until the cell density is 2×10⁻⁶ cells / mL. 5 per ml.

[0217] 4.1.1.3 Carefully transfer the diluted target cells into a sterile sample loading tray, and use a multi-channel pipette to add the target cells into a black, transparent, flat-bottomed 96-well plate at a rate of 50 μL / well.

[0218] 4.1.2 Effector cell plate

[0219] 4.1.2.1 Effector cell preparation: Based on effector-to-target ratios of 9:1, 3:1, and 1:1, the cell dosage was calculated to be 9 × 10⁻⁶ cells / cells. 4 1, 3×10 4 1×10 4 Count the effector cells, take the required number of CAR-T cell samples into a 15ml centrifuge tube, centrifuge at 300g for 8min, and discard the supernatant.

[0220] 4.1.2.2 Resuspend CAR-T cells in T0 medium (X-Vivo medium + 5% inactivated FBS + 1% GlutaMax) and adjust the effector cell density according to the effector-target ratio.

[0221] 4.1.2.3 Add effector cells to the well plates at ratios of 9:1, 3:1, and 1:1, 50 μL / well.

[0222] 4.1.3 Setting up a target cell control group

[0223] Add 50 μL of target cells and 50 μL of T0 medium (X-Vivo medium + 5% inactivated FBS + 1% GlutaMax) to the wells of the target cell control group to make the total culture volume of the target cell control group consistent with the total culture volume of the co-culture experimental group.

[0224] 4.2 Detecting luc value

[0225] 4.2.1 Cell co-culture: Place the 96-well plate with the added samples in a carbon dioxide incubator and co-culture for 4 ± 0.5 hours at 37°C and 5% CO2.

[0226] 4.2.2 Adding the detection reagent: Melt steadyglo luciferase (commercially available) at 4°C or room temperature in the dark. Dilute the steadyglo required for the experiment with PBS 3 times and mix well. Add 50 μL to each well and place on a 96-well plate shaker. Shake at 100 rpm for 15 min.

[0227] 4.2.3 Instrumental Detection: Turn on the multi-mode microplate reader and computer software. Place the shaken 96-well plate into the multi-mode microplate reader and detect the luc fluorescence intensity. Remove the 96-well plate and turn off the instrument and computer.

[0228] 4.3 Calculation of Cell Killing Activity

[0229] After obtaining the luc values ​​of all wells, calculate the cytotoxic activity using the following formula.

[0230] Cell-killing activity = (average luc value of target cells alone - luc value of co-culture wells) / average luc value of target cells alone × 100%

[0231] 5. Experimental Results

[0232] 5.1 Level of Expression

[0233] like Figure 1 As shown, except for O2G-Q33-K3, all T cells expressed CD19-CAR.

[0234] like Figure 2 As shown, all T cells except O2G-Q33-K3 expressed BCMA-CAR.

[0235] like Figure 3 As shown, CAR-positive T cells with 02G-Q33-US2, 02G-Q33-US6, and 02G-Q33-US11 showed downregulation of HLA-ABC molecules, while CAR-negative T cells with 02G-Q33-US2, 02G-Q33-US6, and 02G-Q33-US11 did not show downregulation of HLA-ABC molecules. This indicates that expressing US2, US6, and US11 in CAR-T cells can downregulate the expression of HLA-ABC molecules.

[0236] like Figure 4 As shown, CAR-positive T cells with 02G-Q33-US2, 02G-Q33-US6, and 02G-Q33-US11 showed downregulation of B2M molecules, while CAR-negative T cells with 02G-Q33-US2, 02G-Q33-US6, and 02G-Q33-US11 did not show downregulation of B2M molecules. This indicates that expressing US2, US6, and US11 in CAR-T cells can downregulate the expression of B2M molecules.

[0237] like Figure 5 As shown, HLA-E molecule expression was unaffected in both CAR-positive and CAR-negative cells across all groups.

[0238] like Figure 6 As shown, CAR-positive T cells with 02G-Q33-US2, 02G-Q33-US6, and 02G-Q33-US11 showed downregulation of HLA-A molecules, while CAR-negative T cells with 02G-Q33-US2, 02G-Q33-US6, and 02G-Q33-US11 did not show downregulation of HLA-A molecules. This indicates that expressing US2, US6, and US11 in CAR-T cells can downregulate the expression of HLA-A molecules.

[0239] like Figure 7 As shown, CAR-positive T cells with 02G-Q33-US2, 02G-Q33-US6, and 02G-Q33-US11 showed downregulation of HLA-B molecules, while CAR-negative T cells with 02G-Q33-US2, 02G-Q33-US6, and 02G-Q33-US11 did not show downregulation of HLA-B molecules. This indicates that expressing US2, US6, and US11 in CAR-T cells can downregulate the expression of HLA-B molecules.

[0240] like Figure 8 As shown, CAR-positive T cells with 02G-Q33-US2, 02G-Q33-US6, and 02G-Q33-US11 showed downregulation of HLA-C molecules, while CAR-negative T cells with 02G-Q33-US2, 02G-Q33-US6, and 02G-Q33-US11 did not show downregulation of HLA-C molecules. This indicates that expressing US2, US6, and US11 in CAR-T cells can downregulate the expression of HLA-C molecules.

[0241] like Figure 9 As shown, the expression of HLA-II molecules (HLA-DRDPDQ) was unaffected in both CAR-positive and CAR-negative cells across all groups.

[0242] 5.2 External Killing

[0243] like Figure 10 As shown, expression of US2, US11, and Nef in CAR-T cells does not affect the in vitro killing activity of CAR-T cells against CD19+ target cells Nalm6.

[0244] like Figure 11 As shown, expression of US2, US11, and Nef in CAR-T cells can enhance the in vitro killing activity of CAR-T cells against BCMA+ target cells MM.1S.

[0245] While the above descriptions are merely examples of specific embodiments of the present invention, those skilled in the art should understand that these are only illustrative, and the scope of protection of the present invention is defined by the appended claims. Those skilled in the art can make various changes or modifications to these embodiments without departing from the principles and essence of the present invention, and all such changes or modifications shall fall within the scope of protection of the present invention.

Claims

1. A chimeric antigen receptor complex comprising a chimeric antigen receptor and a viral protein; The chimeric antigen receptor comprises an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain. The viral protein is selected from at least one of the following proteins: US2 protein, US6 protein, US11 protein, K3 protein, Nef protein.

2. The chimeric antigen receptor complex according to claim 1, wherein the amino acid sequences of the US2 protein, US6 protein, US11 protein, K3 protein and Nef protein are as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively.

3. The chimeric antigen receptor complex according to claim 1 or 2, wherein the chimeric antigen receptor targets one or more antigens selected from the following: CD19, CD20, CD22, BCMA, GPRC5D, CLL1, CD7, CD5, GPC3, DLL3, Trop2, ROR1; Optionally, the chimeric antigen receptor targets CD19 / BCMA.

4. The chimeric antigen receptor complex according to any one of claims 1-3, wherein the extracellular antigen recognition domain comprises an anti-BCMA extracellular antigen recognition domain and an anti-CD19 extracellular antigen recognition domain; The anti-BCMA extracellular antigen recognition domain includes BCMA VH and BCMA VL, wherein the amino acid sequence of BCMA VH is shown in SEQ ID NO:11 and the amino acid sequence of BCMA VL is shown in SEQ ID NO:

12. The anti-CD19 extracellular antigen recognition domain includes CD19 VH and CD19 VL, wherein the amino acid sequence of CD19 VH is shown in SEQ ID NO:13 and the amino acid sequence of CD19 VL is shown in SEQ ID NO:

14.

5. The chimeric antigen receptor complex according to claim 4, wherein the extracellular antigen recognition domain comprises, in sequence, CD19 VL, a first linker sequence, BCMA VL, a second linker sequence, BCMA VH, a third linker sequence, and CD19 VH; Optionally, the first linking sequence, the second linking sequence, and the third linking sequence are independently selected from one or more of the following sequences: SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; Further optionally, the extracellular antigen recognition domain includes an amino acid sequence as shown in SEQ ID NO:

18.

6. The chimeric antigen receptor complex according to any one of claims 1-5, wherein the hinge region is derived from one or more of IgG1, IgG4, CD4, CD7, CD28, CD84, and CD8α; optionally, the hinge region is derived from CD8α; more preferably, the amino acid sequence of the hinge region is as shown in SEQ ID NO:19; and / or The transmembrane region is derived from one or more of CD3, CD4, CD7, CD8α, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, and Fc70; optionally, the transmembrane region is derived from CD8α; more preferably, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO:

20.

7. The chimeric antigen receptor complex according to any one of claims 1-6, wherein the intracellular domain comprises an intracellular signal transduction region; optionally, it further comprises a co-stimulatory signal transduction region; Further optionally, the intracellular signal transduction region is derived from one or more of CD3ζ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, FcRγ, FcRβ, CD66d, DAP10, DAP12, and Syk; optionally, the intracellular signal transduction region is derived from CD3ζ; more preferably, the amino acid sequence of the intracellular signal transduction region is as shown in SEQ ID NO:22; and / or Further optionally, the co-stimulatory signal transduction region is derived from one, two, or more of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, and MyD88; optionally, the co-stimulatory signal transduction region is derived from 4-1BB; more preferably, the amino acid sequence of the co-stimulatory signal transduction region is as shown in SEQ ID NO:

23.

8. The chimeric antigen receptor complex according to any one of claims 1-7, further comprising a guide peptide located at the N-terminus of the chimeric antigen receptor amino acid sequence; optionally, wherein the guide peptide is derived from CD8α; more preferably, the amino acid sequence of the guide peptide is as shown in SEQ ID NO:

26.

9. A universal CAR-T cell comprising a chimeric antigen receptor complex according to any one of claims 1-8.

10. A method for preparing universal CAR-T cells, comprising: 1) Preparation of T cells; 2) Gene editing tools and donor templates are introduced into T cells to prepare CAR-T cells; The donor template contains a nucleotide sequence encoding a chimeric antigen receptor complex, the nucleotide sequence including a nucleotide sequence encoding an extracellular antigen recognition domain, a nucleotide sequence encoding a hinge region, a nucleotide sequence encoding a transmembrane region, a nucleotide sequence encoding an intracellular domain, and at least one of the following genes: US2 gene, US6 gene, US11 gene, K3 gene, and Nef gene.

11. The preparation method according to claim 10, wherein the US2 gene, US6 gene, US11 gene, K3 gene and Nef gene encode amino acid sequences as shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively; optionally, the nucleotide sequences of the US2 gene, US6 gene, US11 gene, K3 gene and Nef gene are shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, respectively.

12. The preparation method according to claim 10 or 11, wherein the gene editing tool is selected from one of the CRISPR / Cas system, zinc finger nuclease system, and transcription activator-like effector nuclease system; Optionally, the gene-editing tool is selected from the CRISPR / Cas system; Alternatively, the CRISPR / Cas system includes the Cas protein and sgRNA.

13. The preparation method according to claim 12, wherein the Cas protein comprises any one of the following: spCas9, AsCas12a, or LbCas12a; The sgRNA targets the TRAC site; Optionally, the sgRNA sequence targeting the TRAC site is shown in SEQ ID NO:

34.

14. The preparation method according to any one of claims 10-13, wherein the donor template is plasmid DNA, dsDNA or ssDNA; Optionally, the donor template includes, in the 5'-3' direction, a right homologous arm sequence, a promoter sequence, a nucleotide sequence encoding an extracellular antigen recognition domain, a nucleotide sequence encoding a hinge region, a nucleotide sequence encoding a transmembrane region, a nucleotide sequence encoding an intracellular domain, a nucleotide sequence encoding T2A, at least one of the following gene sequences: US2, US6, US11, K3, Nef, polyA, and a left homologous arm sequence. Further optionally, the donor template further comprises a nucleotide sequence encoding a guide peptide, and the nucleotide sequence encoding the guide peptide is located between the promoter sequence and the nucleotide sequence encoding an extracellular antigen recognition domain.

15. The preparation method according to claim 14, wherein the promoter is selected from the SFFV promoter, the CMV promoter, or the EF1α promoter; optionally, the nucleotide sequence of the EF1α promoter is shown in SEQ ID NO:35; The polyA is a BGH polyA signal sequence; optionally, the nucleotide sequence of the BGH polyA signal sequence is shown in SEQ ID NO:

37.

16. The preparation method according to any one of claims 10-15, wherein the nucleotide sequence encoding the extracellular antigen recognition domain comprises: Nucleotide sequences encoding CD19 VL, first linker sequence, BCMAVL, second linker sequence, BCMA VH, third linker sequence, and CD19 VH; and / or The nucleotide sequence encoding the hinge region is the nucleotide sequence encoding CD8α; and / or The nucleotide sequence encoding the transmembrane region is the same as the nucleotide sequence encoding CD8α; and / or Nucleotide sequences encoding intracellular domains include those encoding CD3ζ and those encoding 4-1BB.

17. The preparation method according to claim 14, wherein the left homologous arm sequence and the right homologous arm sequence in the donor template are shown as SEQ ID NO:38 and SEQ ID NO:39, respectively.

18. A pharmaceutical composition comprising the universal CAR-T cells of claim 9 and pharmaceutically acceptable excipients; Optionally, pharmaceutically acceptable excipients include protective agents; Alternatively, pharmaceutically acceptable excipients include cell cryopreservation solutions; Alternatively, the pharmaceutical composition may be an intravenous injection.

19. Use of the chimeric antigen receptor complex of any one of claims 1-8, the universal CAR-T cell of claim 9, and the pharmaceutical composition of claim 18 in the preparation of a medicament for treating tumor diseases and autoimmune diseases; Optionally, the tumor disease is B-cell acute lymphoblastic leukemia (B-ALL), B-cell non-Hodgkin lymphoma (B-NHL), follicular lymphoma (FL), mantle cell lymphoma (MCL), diffuse large B-cell lymphoma (DLBCL), or multiple myeloma (MM), and the autoimmune disease is systemic lupus erythematosus (SLE), lupus nephritis (LN), systemic lupus erythematosus-associated immune thrombocytopenic purpura (SLE-ITP), neuromyelitis optica spectrum disorder (NMOSD), myasthenia gravis (MG), myasthenia gravis (MS), or systemic sclerosis (SSc).

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