CN117778328A

CN117778328A - Proliferation method of universal BCMA CAR-T cells

Info

Publication number: CN117778328A
Application number: CN202211199033.4A
Authority: CN
Inventors: 张云龙; 路佳兴; 周立; 吕璐璐
Original assignee: Heyuan Kanghua Pharmaceutical Technology Beijing Co ltd
Current assignee: Heyuan Kanghua Pharmaceutical Technology Beijing Co ltd
Priority date: 2022-09-29
Filing date: 2022-09-29
Publication date: 2024-03-29

Abstract

The invention provides a proliferation method of general BCMA CAR-T cells, which comprises the following steps: preparing universal BCMA CAR-T cells and proliferating the universal BCMA CAR-T cells; wherein: the step of proliferation of the universal BCMA CAR-T cell comprises the steps of: and activating the universal BCMA CAR-T cells by adopting BCMA proteins and CD86 proteins after the first subculture stage, and harvesting the proliferated universal BCMA CAR-T cells after the second subculture stage.

Description

Proliferation method of universal BCMA CAR-T cells

Technical Field

The application relates to the field of biological medicine, in particular to a proliferation method of universal BCMA CAR-T cells.

Background

CAR-T cell therapeutics are methods that utilize genetic engineering to cause T cells to express a CAR protein, which is a core component of a CAR cell therapeutic, which can include a targeting moiety (e.g., a moiety that binds a Tumor-associated antigen (Tumor-Associated Antigen, TAA)), a hinge region, a transmembrane region, and an intracellular domain. Such CAR proteins have the ability to recognize intact proteins on the membrane surface without reliance on antigen presentation, thereby causing activation and functional effects of T cells.

The CAR-T cell therapeutic drugs approved for marketing use are autologous CAR-T cell drugs that are transfected in vitro and post-proliferation for reinfusion into patients, but the autologous CAR-T cell drugs have the following problems: some patients' autologous T cells do not meet the requirements, autologous CAR-T products cannot be prepared, or even if the patient autologous T cells meet the requirements, the individualized CAR-T cells are long in preparation time, complex in preparation process and very high in cost. However, the use of allogeneic CAR-T cells requires immune-typing and involves the risk of immune rejection, GVHD, and the like, and has not yet been used. If the universal CAR-T cell therapeutic drug can be prepared so that the universal CAR-T cell therapeutic drug has patient population universality, the universal CAR-T cell therapeutic drug can be subjected to large-scale industrial production, and a standardized universal product can be obtained. At the same time, standardized universal CAR-T cell therapeutics can be frozen for immediate use anywhere in the world, the so-called "ready-to-use". This concept is expected to greatly shorten the CAR-T preparation process and reduce the production cost, and is expected to solve the difficulties encountered in current autologous CAR-T cell therapies.

Currently, it is possible to prepare Universal CAR-T (UCAR-T), an allogeneic CAR-T cell therapy, by gene editing techniques that do not require the preparation of patient-individualized CAR-T cells, but rather directly genetically modify T cells from young healthy donors. Allogenic CAR-T cell UCART19 developed by Cellectis corporation using TALEN technology has successfully cured one example of recurrent Acute Lymphoblastic Leukemia (ALL) infant, which has targeted knockout of the Trac gene (removal of T cell receptor of the cell itself, prevention of GVHD), CD52 gene (resistance of the cell to alemtuzumab), demonstrating the possibility of combining gene editing technology with CAR-T therapy.

However, as a new therapeutic attempt, there are still some problems in UCAR-T technology that need to be solved, such as: after gene is knocked out by genome editing, the proliferation capacity of T cells is greatly reduced, but the T cells which can be provided by a single donor are limited, and the T cells of different donors cannot be mixed, so that the influence of the gene editing on the proliferation capacity of the T cells restricts the productivity of UCAR-T; on the other hand, the exogenous CAR gene carried by CAR-T cells may itself have an effect on their proliferative capacity. The proliferation capacity of T cells is critical for subsequent treatment, cost of the overall therapy, industrialization of technology. Therefore, it is important to increase the proliferation level of T cells after genome editing operations.

Disclosure of Invention

The application provides a method for proliferation of universal BCMA CAR-T cells, which adopts a mode of activating BCMA protein and CD86 protein and performing subculture twice in the step of proliferation of the universal BCMA CAR-T cells, and can greatly improve the proliferation level of the universal BCMA CAR-T cells on the premise of not affecting the functions (such as CAR expression rate, cytokine release, killing effect, cell depletion level and GVHD reaction).

A method of proliferation of universal BCMA CAR-T cells comprising the steps of: preparing universal BCMA CAR-T cells and proliferating the universal BCMA CAR-T cells; wherein: the step of proliferation of the universal BCMA CAR-T cell comprises the steps of:

and activating the universal BCMA CAR-T cells by adopting BCMA proteins and CD86 proteins after the first subculture stage, and harvesting the proliferated universal BCMA CAR-T cells after the second subculture stage.

In certain embodiments, the time for the first subculture stage is: 12-14 days from the first subculture stage of the universal BCMA CAR-T cells; the time of the second subculture stage is as follows: the activation treatment of the universal BCMA CAR-T cells with BCMA protein and CD86 protein is performed for 9 to 14 days. Optionally, the time of the first subculture stage is: 12 days from the first subculture stage for universal BCMA CAR-T cells; the time of the second subculture stage is as follows: activation of universal BCMA CAR-T cells with BCMA protein and CD86 protein was performed for 9 days.

In certain embodiments, the general-purpose BCMA CAR-T cells are activated using BCMA protein and CD86 protein by: mixing a carrier loaded with BCMA protein and a carrier loaded with CD86 protein with universal BCMA CAR-T cells to perform activation treatment; alternatively, the BCMA protein-loaded cargo and the CD86 protein-loaded cargo are the same cargo; further alternatively, the carrier loaded with BCMA protein and CD86 protein simultaneously is magnetic beads or a polymer material.

In certain embodiments, the method for preparing a carrier loaded with BCMA protein and CD86 protein simultaneously comprises the steps of: every 5×10 ⁷ Adding 20-30ug of BCMA protein capable of being loaded on the magnetic beads and 20-30ug of CD86 protein capable of being loaded on the magnetic beads into each magnetic bead for mixed incubation; alternatively, every 5×10 ⁷ Adding 25ug of BCMA protein capable of being loaded on the magnetic beads and 25ug of CD86 protein capable of being loaded on the magnetic beads into each magnetic bead for mixed incubation; further alternatively, BCMA protein and CD86 protein are biotinylated corresponding proteins, and the magnetic beads are labeled with anti-biotin antibodies. As used herein, anti-Biotin MACSiBead Particles, MACIBead, miltenyi Inc. are all ^TM The average diameter is 3.5 mu m, and the anti-biotin antibody is coupled on the surface, so that the anti-biotin antibody is a magnetic bead which is specially designed for activating, amplifying and differentiating primary cells of different types. MACIBead ^TM Because the cell surface antigen is similar to the cell in size, biotinylated protein can be loaded on the magnetic beads through the anti-biotin antibody, so that the cell surface antigen crosslinking can be efficiently promoted, and the cell surface antigen can be simulatedActivation of the cell-cell interactions within, thereby inducing activation and expansion of the cells.

In certain embodiments, the ratio of the number of magnetic beads to the number of universal BCMA CAR-T cells is 0.4 to 2:1, a step of; optionally 0.5:1.

in certain embodiments, the method of preparing a universal BCMA CAR-T cell is selected from one of the following 2 methods:

the method one comprises the following steps:

a. activating T cells by adopting CD3 protein and CD28 protein;

b. transferring a nucleic acid molecule encoding a chimeric antigen receptor targeting BCMA into T cells to prepare CAR-T cells;

c. introducing a gene editing tool into the CAR-T cell to obtain a universal BCMA CAR-T cell, the gene editing tool being capable of disabling normal expression of the Trac gene on chromosome 14 and/or the CD52 gene on chromosome 1 of the CAR-T cell;

The second method comprises the following steps:

a. introducing a gene editing tool into the T cell to produce a genetically edited T cell, wherein the gene editing tool is capable of disabling normal expression of the Trac gene on chromosome 14 and/or the CD52 gene on chromosome 1 of the T cell;

b. activating the T cells subjected to gene editing by adopting a CD3 protein and a CD28 protein;

c. transferring a nucleic acid molecule encoding a chimeric antigen receptor targeting BCMA into T cells subjected to gene editing and activation treatment to obtain universal BCMA CAR-T cells;

in certain embodiments, the method of preparing a universal BCMA CAR-T cell is selected from method one.

In certain embodiments, the subculture medium for culturing T cells, CAR-T cells and/or universal BCMA CAR-T cells is: adding an OpTmizer culture medium of IL-7 with a final concentration of 8-12ng/ml and IL-15 with a final concentration of 4-6 ng/ml; optionally: add the OpTmizer medium with IL-7 at a final concentration of 10ng/ml and IL-15 at a final concentration of 5 ng/ml.

In certain embodiments, the method for proliferation further comprises the step of removing residual cd3+ cells from the proliferated cells during the step of proliferating the universal BCMA CAR-T cells; alternatively, residual cd3+ cells in the proliferating cells are removed on days 6 to 8 of the first subculture stage; further alternatively, residual cd3+ cells in the proliferating cells are removed on day 8 of the first subculture stage.

In certain embodiments, the method of proliferation described above employs the CD3 protein and CD28 protein to activate T cells in the following manner: activating T cells by adopting a carrier loaded with CD3 protein and a carrier loaded with CD28 protein; alternatively, the CD3 protein-loaded and CD28 protein-loaded loads are the same load; further alternatively, the carrier loaded with both CD3 protein and CD28 protein is a magnetic bead or a polymeric material.

In certain embodiments, the above proliferation methods, the gene editing tool is selected from one of a CRISPR/Cas system, a zinc finger nuclease system, a transcription activator-like effector nuclease system.

In certain embodiments, the proliferation methods described above, the gene editing tool is selected from a CRISPR/Cas system.

In certain embodiments, the gene editing tool prevents normal expression of the Trac gene by gene editing one or more of exon 1, exon 2, exon 3, and exon 4 of the Trac gene on chromosome 14 of the T cell or CAR-T cell.

In certain embodiments, the gene editing tool prevents normal expression of the CD52 gene by gene editing one or more of exon 1 and exon 2 of the CD52 gene on chromosome 1 of the T cell or CAR-T cell.

In certain embodiments, the gene editing tool prevents normal expression of the Trac and CD52 genes by gene editing the 1 st exon of the Trac gene on chromosome 14 and the 1 st exon of the CD52 gene on chromosome 1 of T cells or CAR-T cells.

In certain embodiments, the targeting sequence of sgRNA or chRDNA in the CRISPR/Cas system for exon 1 of the Trac gene is shown in SEQ ID NO. 17.

In certain embodiments, the sequence of the sgRNA in the CRISPR/Cas system for exon 1 of the Trac gene is shown in SEQ ID NO. 19.

In certain embodiments, the targeting sequence of sgRNA or chRDNA in the CRISPR/Cas system for exon 1 of the CD52 gene is shown in SEQ ID NO. 18.

In certain embodiments, the sequence of the sgRNA in the CRISPR/Cas system for exon 1 of the CD52 gene is set forth in SEQ ID NO. 20.

In certain embodiments, the amount of Cas9 protein in the CRISPR/Cas system is 1 x 10 per Cas9 ⁶ Cells to be gene edited use 1.5ug-5ug Cas9 protein, the molar ratio of sgRNA to Cas9 protein is 2-4:1, a step of; alternatively, the amount of Cas9 protein in the CRISPR/Cas system is per 1 x 10 ⁶ Cells to be gene edited used 3ug Cas9 protein, the molar ratio of sgRNA to Cas9 protein was 2.5:1.

in certain embodiments, the T cells in the T cells or CAR-T cells are derived from allogeneic T lymphocytes.

In certain embodiments, the T cells in the T cells or CAR-T cells are derived from a healthy donor.

In certain embodiments, the BCMA-targeted chimeric antigen receptor comprises an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain.

In certain embodiments of the above proliferation methods, the extracellular antigen-recognition domain comprises BCMA VH and BCMA VL, wherein the amino acid sequences of the CDRs 1, 2, and 3 of the BCMA VH complementarity determining regions comprise the amino acid sequences shown in SEQ ID NO:1, 2, and 3, respectively, and the amino acid sequences of the CDRs 1, 2, and 3 of the BCMA VL complementarity determining regions comprise the amino acid sequences shown in SEQ ID NO:4, 5, and 6, respectively.

In certain embodiments, the BCMA VH sequence comprises the amino acid sequence shown as SEQ ID NO. 7 and the BCMA VL sequence comprises the amino acid sequence shown as SEQ ID NO. 8.

In certain embodiments, the extracellular antigen-recognition domain targeted to BCMA comprises a BCMA scFv antibody as shown in SEQ ID No. 9.

In certain embodiments of the above proliferation methods, the scFv antibody is a humanized antibody.

In certain embodiments, the hinge region is derived from one or more of IgG1, igG4, CD7, CD28, CD84, CD8 a; alternatively, the hinge region is derived from CD8 a; further alternatively, the sequence of the hinge region comprises the amino acid sequence shown as SEQ ID NO. 10.

In certain embodiments, the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8 a, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, fc 70; alternatively, the transmembrane region is derived from CD8 a; further alternatively, the sequence of the transmembrane region comprises the amino acid sequence shown as SEQ ID NO. 11.

In certain embodiments of the above proliferation methods, wherein the intracellular domain comprises an intracellular signaling region; optionally, a costimulatory signaling region is also included.

In certain embodiments of the above proliferation methods, wherein the intracellular signaling region is derived from one or more of CD3 δ, CD3 γ, CD3 δ, CD3 epsilon, CD5, CD22, CD79a, CD79b, fcrγ, fcrβ, CD66d, DAP10, DAP12, syk; alternatively, the intracellular signaling region is derived from cd3δ; further alternatively, the intracellular signaling region comprises the amino acid sequence shown as SEQ ID NO. 12.

In certain embodiments of the above proliferation methods, wherein the costimulatory signaling 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, myD 88; optionally, the costimulatory signaling region is derived from 4-1BB or CD28; further alternatively, the costimulatory signaling region comprises the amino acid sequence depicted as SEQ ID NO. 13.

The above proliferation methods, in certain embodiments, further comprise a leader peptide located N-terminal to the chimeric antigen receptor amino acid sequence; optionally, wherein the guide peptide is derived from CD8 a; further alternatively, the leader peptide comprises the amino acid sequence shown as SEQ ID NO. 14.

In certain embodiments, the amino acid sequence of the BCMA CAR is shown in SEQ ID NO. 15.

In certain embodiments, the nucleotide sequence encoding a BCMA CAR is set forth in SEQ ID NO. 16.

The application also provides a BCMA-targeted universal CAR-T cell obtained by the proliferation method described above.

The application also provides application of the BCMA-targeted general CAR-T cells in preparation of medicines.

In certain embodiments, the medicament is for treating a disease or disorder associated with expression of BCMA.

In certain embodiments, the medicament is for treating Multiple Myeloma (MM).

In certain embodiments of the above use, the medicament is an intravenous injection.

The present application also provides a method of treating a disease or disorder associated with expression of BCMA comprising the steps of: an effective amount of the aforementioned BCMA-targeted universal CAR-T cells is administered to a subject in need of treatment for a disease or disorder associated with expression of BCMA.

In certain embodiments, the above-described methods involve a disease or disorder associated with expression of BCMA comprising: multiple Myeloma (MM).

In certain embodiments, the method is performed by intravenous injection.

In certain embodiments, the method of administering is by administering an effective amount of the universal CAR-T cells to the subject in a single injection or multiple injections.

In certain embodiments, the effective amount of a universal CAR-T cell is 1 x 10 ⁵ Up to 1X 10 ⁷ Metering of individual cells/kg.

The application also provides a BCMA-targeted general CAR-T cell for preparing medicines.

In certain embodiments, the universal CAR-T cells described above are used to treat a disease or disorder associated with expression of BCMA.

In certain embodiments, the universal CAR-T cells described above are used to treat Multiple Myeloma (MM).

In certain embodiments, the universal CAR-T cell described above, the drug is an intravenous injection.

Drawings

Fig. 1 shows a schematic structure of BCMA CAR in example 1 of the present application.

FIG. 2 shows the concentration of cytokine IFN-gamma released after activation of UTD, autologous CAR-T, UCAR-T groups obtained by different activation means by positive target cells in example 3 of the present application.

FIGS. 3A and 3B show the killing effect of UTD, autologous CAR-T, and UCAR-T groups obtained by different activation methods on target cells in example 3 of the present application, wherein in FIG. 3A, the killing effect is shown on MM.1S of positive target cells endogenously expressing BCMA, and in FIG. 3B, the killing effect is shown on K562 of negative control target cells not expressing BCMA.

FIG. 4 shows the expression of UTD, CAR-T, and UCAR-T sets of cell surface immune checkpoint proteins PD1 and Lag3 obtained by different activation methods in example 3 of the present application.

FIG. 5 shows tumor bioluminescence of PBS, UTD, CAR-T, UCAR-T obtained by different activation patterns in mice in example 4 of the present application in each group of mice.

FIGS. 6A and 6B show IFN-. Gamma.and IL2 secretion in CD3+ PBMC and CD3-PBMC in GVHD experiments of example 5 of the present application.

FIG. 7 shows the effect of different T cell culture media and cytokines on UCAR-T cell proliferation in example 6 of the present application.

Detailed Description

Further advantages and effects of the invention of the present application will become apparent to those skilled in the art from the disclosure of the present application, from the following description of specific embodiments.

The present application is further described below: in the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory procedures as used herein are terms and conventional procedures that are widely used in the corresponding arts. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

In the present application, the terms "gene editing" and "gene editing technique" have the ordinary meaning in the art, and refer to a technique for site-directed modification of a genome. By using this technique, it is possible to precisely locate a site in the genome at which a target DNA fragment is sheared, knocked in or knocked out of a target gene fragment. The gene editing technology is used as a molecular biological technology, and can realize accurate modification of chromosomes so as to change the existing functions of cells. Compared with the cell line frequently used in basic research, the T cell is taken as a primary cell, has no specificity except that the T cell cannot proliferate for a long time, and can be edited by using a gene editing technology. Currently there are mainly 3 gene editing technologies, zinc finger nuclease (zinc finger nuclease, ZFN) technology, transcription activator-like effector nuclease (transcription activator-like effector nuclease, TALEN) technology, and RNA-guided CRISPR/Cas nuclease technology, respectively. Compared with the traditional gene targeting technology, the novel gene editing technology retains the characteristic of site-specific modification, can be applied to more species and cells, and has the advantages of higher efficiency, shorter construction time and lower cost.

In this application, the terms "CRISPR/Cas technology", "CRISPR/Cas system" have the meaning common in the art, and are all known as palindromic repeats clusters and their associated proteins (clusterd regularly interspaced short palindromic repeats/CRISPR-associated proteins), a type of acquired immune system found today in most bacteria and all archaea, and can cleave foreign gene fragments in a targeted manner. Different types of CRISPR/Cas systems have been found, wherein the composition of the second type is simpler, with Cas9 protein and guide RNA (gRNA) as core compositions. CRISPR/Cas has the following advantages over earlier ZFN and TALEN technologies: low off-target rate, high efficiency, economy and wide application range. In addition, CRISPR hybrid RNA-DNA (chRDNA) guided technology has also been reported to significantly improve Cas9 protein specificity compared to total RNA guided technology, thereby achieving high levels of desired genome editing in cells, minimizing off-target.

In the present application, the terms "zinc finger nuclease technology", "zinc finger nuclease system" have the meaning common in the art, and the core design concept of the zinc finger nuclease technology is to subtly chimeric two domains with specific functions, namely a specific recognition module and a functional module. The most classical zinc finger nucleases fuse a non-specific endonuclease Fok I with a zinc finger containing domain that recognizes a specific DNA sequence.

In the present application, the terms "transcription activator-like effector nuclease technology", "transcription activator-like effector nuclease system" have the usual meaning in the art, and TALE effectors were originally discovered as an invasive strategy for bacterial infection of plants. Researchers have formed a powerful tool with specific gene editing function, i.e., TALEN proteins, by linking Fok I nuclease to a synthetic TALE with sequence specific binding capacity. A typical TALEN protein consists of an N-terminal domain containing a nuclear localization signal (nuclear localization signal, NLS), a central domain containing a typical tandem TALE repeat that recognizes a specific DNA sequence, and a C-terminal domain with Fok I endonuclease function. The core principle of the TALEN technology is to orderly realize three different functions of guiding into cell nucleus, specifically recognizing target site DNA and cutting target site DNA on the same TALEN protein.

In the present application, the term "gene is not normally expressed" has the meaning common in the art, and gene expression refers to the process of synthesizing genetic information from a gene into a functional gene expression product, which generally refers to a protein.

In this application, the term "GVHD (graft-versus-host disease)" has its ordinary meaning in the art and refers generally to graft versus host disease, in that after transplantation, T lymphocytes in allogeneic donor grafts are subjected to a series of "cytokine storm" stimuli that greatly enhance their immune response to recipient antigens and initiate cytotoxic attacks on recipient target cells, with skin, liver and gut being the primary targets.

In this application, the term "Trac gene" has the usual meaning in the art, and Trac refers to the T cell receptor alpha chain constant region. The signal of the T Cell Receptor (TCR) to recognize antigen is mainly through the TCR-CD3 complex (including the α chain, β chain, cd3γ, cd3δ, etc. of the TCR), which consists of α and β chains, which are assembled into heterodimers, which bind to a plurality of CD3 subtype molecules; in cells, if complete complexes with the CD3 molecule are not formed, the excess TCR may be degraded. After the Trac is knocked out, the formation of TCR-CD3 complex is affected, which means that T cell receptors on the surface of T cells are cleared, avoiding graft versus host reaction (GVHD).

In the present application, the term "antibody" has the meaning conventional in the art and generally refers to an immunoglobulin molecule consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains, which are interconnected by disulfide bonds. By analyzing the amino acid sequences of the heavy and light chains of different antibodies, it was found that the amino acid sequences of the heavy and light chains near the N-terminus varied widely, with the other portions of the amino acid sequences being relatively constant. Thus, regions of the antibody light and heavy chains that vary greatly near the N-terminal amino acid sequence are referred to as variable regions (V), regions near the C-terminal amino acid sequence are referred to as constant regions (C), V regions of the heavy and light chains are abbreviated as VH and VL, respectively, and C regions of the heavy and light chains are abbreviated as CH and CL, respectively. The variation of a small number of amino acid residues within the variable region of an antibody is particularly intense, and the regions of variation in the amino acid residue composition and sequence are more prone to occur are called hypervariable regions (hypervariable regions, HVR); three hypervariable regions are present in each of the V regions of the L and H chains, and these regions are also called complementarity determining regions (complementarity determining region, CDRs) because they are spatially complementary to an epitope. In antibodies, there are Kabat, abM, chothia, contact, IMGT rules for CDR partitioning, which are well known to those skilled in the art, and when a website for executing these rules is applied, the VH and VL sequences are simply inputted and the corresponding rules are selected, so that CDR sequences according to the different rules can be obtained. Those skilled in the art will appreciate that the scope of protection of the present application encompasses combinations of CDR sequences obtained by analysis using different rules. The 6 CDR regions of an antibody together determine the ability and specificity of the antibody to recognize the corresponding antigen. It will be appreciated by those skilled in the art that when the present application defines an amino acid sequence of 6 CDR regions, the ability of an antibody to recognize and to specifically identify a corresponding antigen is contemplated.

In the present application, the term "scFv" has the meaning conventional in the art and refers to a single chain variable region (Single Chain Variable Fragment, abbreviated as scFv) which is an antibody formed by connecting a heavy chain variable region and a light chain variable region of an antibody via a short peptide (linker).

In this application, the term "chimeric antigen receptor" (Chimeric Antigen Receptor, CAR) is a core component of CAR cell therapeutics that can include an extracellular antigen recognition domain (e.g., a portion that binds a Tumor-associated antigen (Tumor-Associated Antigen, TAA)), a hinge region, a transmembrane region, and an intracellular domain. CAR-T (Chimeric Antigen Receptor T) cellular immunotherapy is considered one of the most promising means to combat tumors. The CAR-T cells are characterized in that the T cells express CAR proteins by using a genetic modification method, and the CAR proteins have the capability of recognizing complete proteins on the surface of a membrane under the condition of not depending on antigen presentation, thereby causing activation and functional effects of the T cells.

In the present application, the term "extracellular antigen recognition domain" refers to an antigen recognition domain (Antigen Recognition Domain, ARD). CAR cell therapy products (e.g. CAR-T cells) are capable of specifically recognizing and/or binding to target antigens expressed by tumor cells, relying on extracellular antigen recognition domains, which to date have been derived from single chain variable regions of antibodies (Single Chain Variable Fragment, abbreviated scFv), or from receptor ligand interactions, TCR mimics, variable lymphocyte receptors (Variable Lymphocyte Receptors, VLR). By far the most common source is the scFv fragment of an antibody, the scFv comprising an antibody heavy chain variable region and a light chain variable region, linked by a peptide chain, such as: a linker sequence GSTSGSGKPGSGEGSTKG consisting of 18 amino acids.

In this application, the term "hinge region" refers to a junction between an extracellular antigen recognition domain and a transmembrane domain that allows the CAR to recognize an antigen by imparting a range of motion to the antigen recognition domain. The hinge regions currently in use are derived primarily from one or more of IgG1, igG4, CD7, CD28, CD84, CD8 a. In addition, typical hinge regions also contain residues that are involved in CAR dimerization, helping to enhance antigen sensitivity.

In this application, a "transmembrane region" refers to the transmembrane domain that connects the intracellular and extracellular components of the CAR structure. Different transmembrane domains can affect CAR expression and stability to some extent, but are not directly involved in signaling, and downstream signaling can be enhanced by interactions. The transmembrane region may be derived from one or more of CD3, CD4, CD7, CD8 a, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, fc 70.

Within this application, the term "intracellular domain" includes intracellular signaling regions, and may also include costimulatory signaling regions.

In the present application, the term "intracellular signaling region" refers to activation of at least one normal effector function of an immune effector cell responsible for expressing a CAR. The intracellular signaling region may be derived from one or more of CD3 delta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, fcrgamma, fcrbeta, CD66d, DAP10, DAP12, syk.

In this application, the term "costimulatory signaling region" exists because in addition to stimulation of antigen-specific signals, many immune effector cells require costimulation to promote cell proliferation, differentiation and survival, as well as to activate the effector functions of the cell. In some embodiments, the CAR may further comprise one or more costimulatory signaling regions, wherein the costimulatory signaling regions may be derived from one, two, or more than three of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, myD 88.

In the present application, the term "leader peptide" refers to a short peptide preceding an extracellular antigen recognition domain (e.g., scFv sequence) that functions to direct the export of an intracellular synthesized recombinant protein to the outside of the cell. Typical leader peptides are the human CD 8. Alpha. Signal peptide, or the human GM-CSF receptor. Alpha. Signal peptide.

In this application, the term "humanized antibody" is also referred to as an antibody subjected to humanization engineering, which is prepared by grafting Complementarity Determining Regions (CDRs) of a non-human mammalian antibody, e.g., a mouse antibody, a rat antibody, a rabbit antibody, into CDRs of a human antibody, conventional recombinant DNA techniques for preparing humanized antibodies are known. For example, in the case where the CDRs are obtained from a rabbit antibody, primers can be synthesized which are used to link the CDRs of the rabbit antibody to the Framework Regions (FR) of the human antibody. For human antibody FRs to which CDRs are attached, those are selected that allow the CDR regions to form good antigen binding sites.

In this application, the term "specific recognition and/or binding" refers to recognition and/or binding between a CAR and a specific target, which is binding to the target with greater affinity, avidity, ease, and/or with greater duration than the CAR binds to other targets.

In this application, one of the key factors determining the efficacy of CAR-immune cell therapy is the selection of tumor target antigens. In the present application, the term "BCMA" refers to B cell maturation antigen, a member of the tumor necrosis factor receptor superfamily. Human BCMA is almost exclusively expressed in plasma cells and multiple myeloma cells. BCMA may be a suitable tumor antigen target for immunotherapeutic agents against multiple myeloma.

In this application, the term "isolated" generally refers to those obtained from a natural state by artificial means. If a "isolated" substance or component occurs in nature, it may be that the natural environment in which it is located is altered, or that the substance is isolated from the natural environment, or both. For example, a polynucleotide or polypeptide that has not been isolated naturally occurs in a living animal, and the same polynucleotide or polypeptide that has been isolated from the natural state and is of high purity is said to be isolated. The term "isolated" does not exclude substances which have been obtained from natural sources by artificial means, either by man or by synthesis, nor does it exclude the presence of other impure substances which do not affect the activity of the substance.

In this application, the term "isolated nucleic acid molecule" generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides of any length, which may be isolated from the natural environment or an artificially synthesized analog thereof.

In the present application, the term "pharmaceutical composition" generally refers to a pharmaceutical composition suitable for administration to a patient, which may comprise the immune effector cells described herein, and may further comprise one or more pharmaceutically acceptable excipients, such as: one or more of a carrier, a protective agent, a stabilizer, an excipient, a diluent, a solubilizer, a surfactant, an emulsifier, and a preservative. In some embodiments, pharmaceutically acceptable excipients include protective agents such as: cell cryopreservation solution. In some embodiments, the pharmaceutical composition of the present application is a cell suspension or cryopreserved cells thereof.

In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to mice, rats, cats, dogs, rabbits, horses, pigs, cattle, sheep, or monkeys.

In this application, the term "comprising" is generally intended to include the features specifically recited, but does not exclude other elements.

In this application, the term "about" generally refers to a range of fluctuations that is acceptable to those skilled in the art above or below the specified value, such as: varying within a range of + -0.5% -10%, for example within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% above or below the specified value.

Proliferation method of universal BCMA CAR-T cells

In one aspect, the present application provides a method of proliferation of a universal BCMA CAR-T cell comprising the steps of: preparing universal BCMA CAR-T cells and proliferating the universal BCMA CAR-T cells; wherein: the step of proliferation of the universal BCMA CAR-T cell comprises the steps of:

In the application, the inventor adopts a mode of activating BCMA protein and CD86 protein and performing two subculture stages in the step of proliferating the universal BCMA CAR-T cells, so that the proliferation level of the universal BCMA CAR-T cells can be greatly improved on the premise of not affecting the functions (such as CAR expression rate, cytokine release, killing effect, cell exhaustion level and GVHD reaction-free) of the universal BCMA CAR-T cells.

In some embodiments, the time for the first subculture stage is: 12-14 days from the first subculture stage of the universal BCMA CAR-T cells; the time of the second subculture stage is as follows: activating the universal BCMA CAR-T cells by using BCMA protein and CD86 protein for 9-14 days; optionally, the time of the first subculture stage is: 12 days from the first subculture stage for universal BCMA CAR-T cells; the time of the second subculture stage is as follows: activation of universal BCMA CAR-T cells with BCMA protein and CD86 protein was performed for 9 days.

In the present application, "first subculture stage" and "second subculture stage" do not mean that the cells are passaged only twice during proliferation, but means two subculture stages in each of which a round of passaging is performed according to conventional 2-3 days so that the cells can be continuously expanded to a prescribed number of days. Since T cells are primary cells and cannot be proliferated for a long period of time, it is necessary to maintain the functionality of the cells obtained by proliferation in addition to the quantitative requirements at the time of proliferation, and therefore it is important to select appropriate proliferation culture conditions. By selecting proliferation times for the first and second subculture stages, BCMA UCAR-T cells of different killing functionality can be obtained. For example, although UCAR-T two d14/14 group cells and UCAR-T two d12/9 group cells are functional while maintaining proliferation (based on the number of living cells passing through the first subculture stage, UCAR-T two d14/14 group cells can be expanded 101.2 times and UCAR-T two d12/9 group cells can be expanded 69.5 times), the killing ability of UCAR-T two d14/14 group cells relative to UCAR-T two d12/9 group cells is reduced, and the amount of UCAR-T two d14/14 group cells used is 2 times the amount of UCAR-T two d12/9 group cells used under the condition that the same killing rate is achieved. The killing effect of cells of UCAR-T two d12/9 group in the in vitro killing experiment is equivalent to, even slightly higher than that of the CAR-T group, while cells of UCAR-T one group and UCAR-T two d14/14 group in the in vitro killing experiment is 5: the 1-way target ratio was significantly lower than the CAR-T group.

In some embodiments, the general BCMA CAR-T cells are activated using BCMA protein and CD86 protein by: mixing a carrier loaded with BCMA protein and a carrier loaded with CD86 protein with universal BCMA CAR-T cells to perform activation treatment; alternatively, the BCMA protein-loaded cargo and the CD86 protein-loaded cargo are the same cargo; further alternatively, the carrier loaded with BCMA protein and CD86 protein simultaneously is magnetic beads or a polymer material.

The proliferation methods described above are described in someIn an embodiment, the preparation method of the carrier loaded with the BCMA protein and the CD86 protein simultaneously comprises the following steps: every 5×10 ⁷ Adding 20-30ug of BCMA protein capable of being loaded on the magnetic beads and 20-30ug of CD86 protein capable of being loaded on the magnetic beads into each magnetic bead for mixed incubation; alternatively, every 5×10 ⁷ Adding 25ug of BCMA protein capable of being loaded on the magnetic beads and 25ug of CD86 protein capable of being loaded on the magnetic beads into each magnetic bead for mixed incubation; further alternatively, BCMA protein and CD86 protein are biotinylated corresponding proteins, and the magnetic beads are labeled with anti-biotin antibodies.

In some embodiments, the ratio of the number of magnetic beads to the number of universal BCMA CAR-T cells is 0.4-2:1, a step of; optionally 0.5:1.

In some embodiments, the method of preparing a universal BCMA CAR-T cell is selected from one of the following 2 methods:

the method one comprises the following steps:

a. activating T cells by adopting CD3 protein and CD28 protein;

the second method comprises the following steps:

in some embodiments, the method of preparing a universal BCMA CAR-T cell is selected from method one.

In the present application, nucleic acid molecules encoding chimeric antigen receptors targeting BCMA may be transferred into cells by viral or non-viral methods such as: vectors carrying nucleic acid molecules encoding chimeric antigen receptors targeting BCMA can be transferred into T cells. The term "vector" generally refers to a nucleic acid delivery vehicle that is transformed, transduced or transfected into a host cell to allow expression of the genetic material elements carried thereby within the host cell. For example, the carrier comprises: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosome (YAC), bacterial Artificial Chromosome (BAC) or P1-derived artificial chromosome (PAC); phages such as lambda phage or M13 phage, animal viruses, etc. Animal virus species used as vectors are retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex viruses), poxviruses, baculoviruses, papillomaviruses, papilloma-virus-papilloma-vacuolated viruses (e.g., SV 40). A vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain a replication origin. It is also possible for the vector to include components that assist it in entering the cell, such as viral particles, liposomes or protein shells, but not just these. The non-viral method may be: transposon dependent gene transfer, mRNA mediated gene transduction, and the like; the term "transposon" refers to a discrete piece of DNA that has the ability to migrate and carry genetic information between chromosomal sites, such as: sleep beauty SB system and PB system derived from lepidopteran insects; in some embodiments, mRNA can also be transduced into T cells using electrotransformation methods.

In some embodiments, the subculture medium for culturing T cells, CAR-T cells, and/or universal BCMA CAR-T cells is: adding an OpTmizer culture medium of IL-7 with a final concentration of 8-12ng/ml and IL-15 with a final concentration of 4-6 ng/ml; optionally: add the OpTmizer medium with IL-7 at a final concentration of 10ng/ml and IL-15 at a final concentration of 5 ng/ml. In the present application, opTmizer medium means that it is to be commercializedGIBCO OpTmizer ^TM CTS ^TM T cell expansion medium and commercial Gibco CTS immune cell serum replacement (CTS) ^TM Immune Cell SR human serum surrogate), wherein: GIBCO OptmIzer ^TM CTS ^TM T cell expansion Medium was obtained from Optmizer ^TM T cell expansion basal medium and Optmizer ^TM T cell expansion additive. Before use, the three materials are mixed together according to the commodity recommended proportion, and the specific volume proportion is Optmizer ^TM T cell expansion basal medium: optmizer ^TM T cell expansion additive: immune cell serum replacement (CTS) ^TM Immunecell SR human serum surrogate = 1000:26:50. a commercial T cell expansion serum-free culture medium, which is basically free of protein (only contains human serum albumin), is a special formula culture medium with definite chemical components. Other T cell culture media in the market all need to be added with 5-10% human serum to obtain ideal results, while the Optmizer culture media are different, and can also obtain good cell culture results under the conditions of no human serum or low serum concentration. In this application, the inventors have determined that by optimizing the complete lymphocyte culture broth, namely: the BCMA UCAR-T cells obtained proliferate better and younger through the selection of the type and concentration of the added cytokines.

In some embodiments, the method for proliferation further comprises the step of removing residual cd3+ cells from the proliferated cells; alternatively, residual cd3+ cells in the proliferating cells are removed on days 6 to 8 of the first subculture stage; further alternatively, residual cd3+ cells in the proliferating cells are removed on day 8 of the first subculture stage. In the process of gene editing, although the editing efficiency can reach 90% -98%, there are a small number of T cells or Trac genes in CAR-T cells which are not knocked out, so that residual CD3+ cells exist in the universal BCMA CAR-T cells. In order to avoid GVHD and other phenomena to the greatest extent, the residual CD3+ cells in the universal BCMA CAR-T cells need to be removed, and if the removal step is carried out before the first subculture stage, larger losses are easy to generate due to the smaller amount of cells to be treated; if the removal step is performed at an excessively high proliferation factor, the cells to be treated are also excessively increased. According to example 6, UCAR-T cells proliferated in large amounts on days 9-12 of the first subculture stage, so that the removal step was selected on days 6-8 of the first subculture stage, and the amount of cells to be treated was moderate, and both of the above drawbacks could be avoided.

In some embodiments, the T cells are activated by using the CD3 protein and the CD28 protein in the following ways: activating T cells by adopting a carrier loaded with CD3 protein and a carrier loaded with CD28 protein; alternatively, the CD3 protein-loaded and CD28 protein-loaded loads are the same load; further alternatively, the carrier loaded with both CD3 protein and CD28 protein is a magnetic bead or a polymeric material.

In some embodiments, the above proliferation methods, the gene editing tool is selected from one of a CRISPR/Cas system, a zinc finger nuclease system, a transcription activator-like effector nuclease system.

In some embodiments, the proliferation methods described above, the gene editing tool is selected from a CRISPR/Cas system.

In some embodiments, the gene editing tool prevents normal expression of the Trac gene by gene editing one or more of exon 1, exon 2, exon 3, and exon 4 of the Trac gene on chromosome 14 of the T cell or CAR-T cell.

In some embodiments, the gene editing tool prevents normal expression of the CD52 gene by gene editing one or more of exon 1 and exon 2 of the CD52 gene on chromosome 1 of the T cell or CAR-T cell.

In some embodiments, the gene editing tool prevents normal expression of the Trac and CD52 genes by gene editing the 1 st exon of the Trac gene on chromosome 14 and the 1 st exon of the CD52 gene on chromosome 1 of T cells or CAR-T cells.

In some embodiments, the targeting sequence of sgRNA or chRDNA in the CRISPR/Cas system for exon 1 of the Trac gene is shown in SEQ ID NO: 17.

In some embodiments, the sequence of the sgRNA in the CRISPR/Cas system for exon 1 of the Trac gene is shown in SEQ ID NO. 19.

In some embodiments, the targeting sequence of sgRNA or chRDNA in the CRISPR/Cas system for exon 1 of the CD52 gene is shown in SEQ ID NO. 18.

In some embodiments, the sequence of the sgRNA in the CRISPR/Cas system for exon 1 of the CD52 gene is set forth in SEQ ID NO. 20.

In some embodiments, the amount of Cas9 protein in the CRISPR/Cas system is 1 x 10 per Cas9 ⁶ Cells to be gene edited use 1.5ug-5ug Cas9 protein, the molar ratio of sgRNA to Cas9 protein is 2-4:1, a step of; alternatively, the amount of Cas9 protein in the CRISPR/Cas system is per 1 x 10 ⁶ Cells to be gene edited used 3ug Cas9 protein, the molar ratio of sgRNA to Cas9 protein was 2.5:1.

in some embodiments, the T cells in the T cells or CAR-T cells are derived from allogeneic T lymphocytes.

In some embodiments, the T cells in the T cells or CAR-T cells are derived from a healthy donor.

In some embodiments, the BCMA-targeted chimeric antigen receptor comprises an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain.

In some embodiments, the extracellular antigen-recognition domain comprises BCMA VH and BCMA VL, wherein the amino acid sequences of the CDRs 1, 2 and 3 of the BCMA VH complementarity determining regions comprise the amino acid sequences shown in SEQ ID NO:1, 2 and 3, respectively, and the amino acid sequences of the CDRs 1, 2 and 3 of the BCMA VL complementarity determining regions comprise the amino acid sequences shown in SEQ ID NO:4, 5 and 6, respectively.

In some embodiments, the BCMA VH sequence comprises the amino acid sequence shown as SEQ ID NO. 7 and the BCMA VL sequence comprises the amino acid sequence shown as SEQ ID NO. 8.

In some embodiments, the extracellular antigen recognition domain targeted to BCMA comprises a BCMA scFv antibody as shown in SEQ ID NO. 9.

In some embodiments, the scFv antibody is a humanized antibody.

In some embodiments, the hinge region is derived from one or more of IgG1, igG4, CD7, CD28, CD84, CD8 a; alternatively, the hinge region is derived from CD8 a; further alternatively, the sequence of the hinge region comprises the amino acid sequence shown as SEQ ID NO. 10.

In some embodiments, the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8 a, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, fc 70; alternatively, the transmembrane region is derived from CD8 a; further alternatively, the sequence of the transmembrane region comprises the amino acid sequence shown as SEQ ID NO. 11.

In some embodiments, the proliferation methods described above, wherein the intracellular domain comprises an intracellular signaling region; optionally, a costimulatory signaling region is also included.

In some embodiments, the proliferation methods described above, wherein the intracellular signaling region is derived from one or more of CD3 δ, CD3 γ, CD3 δ, CD3 epsilon, CD5, CD22, CD79a, CD79b, fcrγ, fcrβ, CD66d, DAP10, DAP12, syk; alternatively, the intracellular signaling region is derived from cd3δ; further alternatively, the intracellular signaling region comprises the amino acid sequence shown as SEQ ID NO. 12.

In some embodiments, the proliferation methods described above, wherein the costimulatory signaling region is derived from one, two, or more than three of CD2, CD3, CD7, CD27, CD28, CD30, CD40, CD83, CD244, 4-1BB, OX40, LFA-1, ICOS, LIGHT, NKG2C, NKG2D, DAP10, B7-H3, myD 88; optionally, the costimulatory signaling region is derived from 4-1BB or CD28; further alternatively, the costimulatory signaling region comprises the amino acid sequence depicted as SEQ ID NO. 13.

The above proliferation methods, in some embodiments, further comprise a leader peptide located N-terminal to the chimeric antigen receptor amino acid sequence; optionally, wherein the guide peptide is derived from CD8 a; further alternatively, the leader peptide comprises the amino acid sequence shown as SEQ ID NO. 14.

In some embodiments, the amino acid sequence of the BCMA CAR is shown in SEQ ID NO. 15.

In some embodiments, the nucleotide sequence encoding a BCMA CAR is set forth in SEQ ID NO. 16.

Universal CAR-T cells obtained by the proliferation method described above and uses thereof

In another aspect, the present application also provides a universal CAR-T cell targeted to BCMA, said universal CAR-T cell obtained by the proliferation method described above.

In yet another aspect, the present application also provides the use of the above-described BCMA-targeted universal chimeric antigen receptor-T cell in the preparation of a medicament.

In some embodiments, the medicament is for treating a disease or disorder associated with expression of BCMA.

In some embodiments, the medicament is for treating Multiple Myeloma (MM).

In some embodiments, the medicament is an intravenous injection.

In yet another aspect, the present application also provides a method of treating a disease or disorder associated with expression of BCMA, comprising the steps of: an effective amount of the aforementioned BCMA-targeted universal CAR-T cells is administered to a subject in need of treatment for a disease or disorder associated with expression of BCMA.

In some embodiments, the disease or condition associated with expression of BCMA comprises: multiple Myeloma (MM).

In some embodiments, the administration may be performed by different means, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical, or intradermal administration. For example, the mode of administration may be administered to the subject by intravenous injection.

In some embodiments, the method is a method of administering an effective amount of the universal chimeric antigen receptor-T cells to a subject in a single injection or multiple injections. Such as: once a week, once two weeks, once three weeks, once four weeks, once a month, once 3 months, or once 3-6 months.

In some embodiments, the dosage administered may be different for different indications; the dosage may also vary for patients with varying severity of the condition. The dosage range of administration can be 1×10 ⁵ Individual CAR positive T cells/kg to 1×10 ⁷ Individual CAR positive T cells/kg, e.g., 1×10 ⁵ Individual CAR positive T cells/kg to 1×10 ⁶ CAR positive T cells/kg, 1X 10 ⁶ Individual CAR positive T cells/kg to 1×10 ⁷ Each CAR positive T cell/kg.

In some embodiments, the subject may include humans and non-human animals. For example, the subject may include, but is not limited to, mice, rats, cats, dogs, horses, pigs, cattle, sheep, rabbits, or monkeys.

In yet another aspect, the present application also provides a universal CAR-T cell targeting BCMA for use in the preparation of a medicament.

In some embodiments, the universal CAR-T cells described above are used to treat a disease or disorder associated with expression of BCMA.

In some embodiments, the universal CAR-T cells described above are used to treat Multiple Myeloma (MM).

In some embodiments, the universal CAR-T cell described above is an intravenous injection.

Without intending to be limited by any theory, the following examples are presented merely to illustrate the chimeric antigen receptor, immune effector cells, methods of preparation, uses, and the like of the present application and are not intended to limit the scope of the invention of the present application. Examples do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells. Such methods are well known to those having ordinary skill in the art and are described in numerous publications, including Sambrook, j., fritsch, e.f. and maniis, t. (1989) Molecular Cloning: A Laboratory Manual,2nd edition,Cold Spring Harbor Laboratory Press.

Example 1 preparation of BCMA CAR-T cells

We have 1 BCMA-specific scFv sequence (the VH CDR1, CDR2 and CDR3 are shown as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively, the VL CDR1, CDR2 and CDR3 are shown as SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6 respectively, the VH amino acid sequence is shown as SEQ ID NO: 7, and the VL amino acid sequence is shown as SEQ ID NO: 8). In the construction of CAR structure, we used CD8 alpha guide chain (its amino acid sequence shown as SEQ ID NO: 14) as signal peptide, BCMA scFv (its amino acid sequence shown as SEQ ID NO: 9) as extracellular tumor antigen recognition region, hinge region (its amino acid sequence shown as SEQ ID NO: 10) and transmembrane region (its amino acid sequence shown as SEQ ID NO: 11) as CD8 alpha structure, 4-1BB as intracellular co-stimulatory signal (its amino acid sequence shown as SEQ ID NO: 13), CD3 delta as T cell activating signal (its amino acid sequence shown as SEQ ID NO: 12), CAR structure schematic diagram shown as FIG. 1, CAR-T cell preparation method as follows:

1. Construction of lentiviral vectors

The nucleotide sequence encoding the CAR structure with the above structure and the amino acid sequence shown as SEQ ID NO. 15 (shown as SEQ ID NO. 16) is synthesized artificially, and constructed into an empty modified lentiviral vector (manufacturer: SBI company, cat# CD500-CD800, resistance modification is performed as described in example 1 of WO 2021/121227) to obtain a CAR expression vector, and then the CAR expression vector and three packaging plasmids are transfected together into 293T cells, and the functional lentiviral vector is obtained after collection and purification. The three packaging plasmids were pMD2.0G (from Biovector, product number Biovector 012259), pMDLg-pRRE (from Biovector, product number Biovector 012251), pRSV-Rev (from Biovector, product number Biovector 012253), respectively.

2. Preparation of BCMA CAR-T cells by means of lentiviral transduction

Transduction experiments were performed according to conventional methods known to those skilled in the art, and the brief transduction procedure is as follows:

1) Sorting T cells

Peripheral Blood Mononuclear Cells (PBMCs) were isolated from human apheresis cells and T cells were then sorted from PBMC cells using EasySep Human T cell Isolation Kit kit (Stem cell).

2) Activation of T cells

The isolated T cells were resuspended in complete lymphocyte culture medium (Optmizer medium+final concentration 5ng/ml IL-15+final concentration 10ng/ml IL-7) to a final concentration of (1-2). Times.10 ⁶ The individual cells/ml are activated by adding 1 to 2 mu l of CD3/CD28 magnetic beads (cytocare), and the mixture is placed in an incubator for culture under the culture condition of 37 ℃ plus 5 percent CO ₂ The incubation time is at least 24 hours.

3) Lentivirus transduced T cells

Slowly adding slow virus vector into T cells in activated culture according to MOI=1, mixing, adding carbon dioxide, and continuously culturing at 37deg.C+5% CO ₂ After 2 days of culture, the cells were used for the preparation of BCMA Universal CAR-T cells (abbreviated as UCAR-T in this application).

EXAMPLE 2 preparation of BCMA UCAR-T cells knocked out Trac Gene and CD52 Gene

1. Gene editing within CAR-T cells was achieved using the CRISPR-Cas system 2 days after lentivirus transduction of T cells.

Editing a target gene by using a CRISPR-Cas system, and designing sgRNA reversely complementary with a target sequence shown as SEQ ID NO. 17 on a 1 st exon of a Trac gene in the test; the sgRNA which is reversely complementary to the target sequence shown in SEQ ID NO. 18 on the 1 st exon of the CD52 gene is designed; gene editing was performed to obtain BCMA UCAR-T cells.

The method comprises the following specific steps: taking the CAR-T cells obtained in example 1, placing example 1 in a culture systemAfter removal of the CD3/CD28 beads, the supernatant was removed completely and cells were suspended by adding a mixture of 81.8. 81.8ul Nucleofector solution and 18.2ul of support (available from Lonza under the designation PBP 3-00675). Every 1×10 ⁶ 3ug of Cas9 protein, 1.5ug of sgRNA of targeted Trac gene and 1.5ug of sgRNA of targeted CD52 gene (the mol ratio of the sgRNA to the Cas9 protein is 2.5:1) are added into each cell, and are evenly mixed and subjected to electric shock, so as to obtain BCMA UCAR-T cells.

TABLE 1 target genes, target sequences and corresponding sgRNAs when preparing BCMA UCAR-T cells

2. The BCMA UCAR-T cells were subjected to a first subculture stage after electric shock, 1 passage every 2-3 days, with the subculture medium using the complete lymphocyte culture broth given in example 1 at 37 ℃ +5% CO ₂ The method comprises the steps of carrying out a first treatment on the surface of the Removing residual cd3+ cells from the proliferating cells on day 8 after the electric shock; BCMA UCAR-T cells after the first subculture stage were harvested on day 12 post-shock.

3. Preparation of BCMA protein and CD86 protein-loaded magnetic bead particles

The anti-biotin antibody-labeled magnetic beads (available from miltenyi, cat. No. 130-092-357) were taken every 5×10 ⁷ The individual beads were incubated with 25. Mu.g of biotin-labeled BCMA protein and 25. Mu.g of biotin-labeled CD86 protein, mixed and placed in a 4℃refrigerator and rotated slowly upside down on a rotator for 3 hours.

4. Activation proliferation of BCMA UCAR-T cells

Mixing the BCMA UCAR-T cells obtained after the first subculture stage with the obtained BCMA protein-and CD86 protein-loaded magnetic bead particles for activation treatment, wherein the ratio of the number of the magnetic bead particles to the number of the BCMA UCAR-T cells is 0.5:1, mixing BCMA UCAR-T cells with magnetic bead particles, and performing a second subculture stage for 1 generation every 2-3 days, wherein the culture medium adopts the complete lymphocyte culture solution shown in the example 1 under the culture condition of 37 ℃ plus 5 percent CO ₂ Post-activation magnetic bead particleBCMA UCAR-T cells after the second subculture stage were harvested 9 days.

Example 3 comparison of different proliferation modes of BCMA UCAR-T cells

In this application, to obtain more UCAR-T cells, 3 proliferation patterns for UCAR-T cells were compared, while UTD cells and CAR-T cells obtained in example 1 were also used as controls, grouped as follows:

TABLE 2 grouping conditions and corresponding proliferation modes thereof

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T cells as a primary cell cannot proliferate for a long period of time, and the UCAR-T group employing BCMA protein and CD86 protein activation and the second subculture stage needs to maintain its functionality in addition to satisfying the proliferation in number, so that the following experiments of cytokine release, cell killing, and immune checkpoint protein expression were performed, and in vitro function verification was performed on the UCAR-T group employing BCMA protein and CD86 protein activation and the second subculture stage.

1. Multiplication factor, CD3+ cell proportion, CD52+ cell proportion and CAR expression rate of BCMA UCAR-T cells obtained by different multiplication modes

After harvesting each group of cells, the cells were counted, stained with fluorescent-labeled CD3 protein (from Biolegend, cat. No. 300308), fluorescent-labeled CD52 protein (from Biolegend, cat. No. 316008) and fluorescent-labeled BCMA protein (from ACRO, cat. No. BCA-HF 254), and the CD3 in each group of cells was detected by flow cytometry ⁺ Cell proportion (CD 3 for T cells without Trac Gene knockout) ⁺ T cells knocked out of Trac Gene are CD3 ^- )、CD52 ⁺ Cell ratio and CAR expression rate.

The results are shown in Table 3: UTD group, CAR-T group, UCAR-T onThe proliferation condition of the group e is normal; for UCAR-T two groups d12/9 and UCAR-T two groups d14/14 activated by BCMA protein and CD86 protein, the proliferation condition is better, and the BCMA UCAR-T cells in the second subculture stage proliferate 69.5 times and 101.2 times respectively based on the number of living cells of the two groups before the BCMA protein and CD86 protein are activated (namely based on the number of living cells in the first subculture stage); UTD group and CAR-T group CD3 due to untransduced sgRNA and Cas-9 protein ⁺ Cell ratios were 97.9% and 96.7%, respectively, CD52 ⁺ The cell proportion respectively accounts for 99.0 percent and 90.6 percent, which accords with the expectation; CD3 of the remaining 3 UCAR-T groups ⁺ Cell ratios were all below 1%, CD52 ⁺ The cell proportion is also only around 10%; except for the non-transduced CARs of UTD group, the CAR expression rates of each group were higher, especially the CAR expression rates of UCAR-T two d12/9 group and UCAR-T two d14/14 group reached 97.7% and 96.6%, respectively. The above results indicate that: compared with cells which only undergo the first subculture stage, UCAR-T groups which undergo activation of BCMA protein and CD86 protein and the second subculture stage can still proliferate remarkably, and the expression rate of CAR is also improved.

TABLE 3 proliferation fold, CD3+ cell fraction, CD52+ cell fraction, CAR expression rate of UCAR-T cells obtained by different proliferation modes

2. Cytokine release assay with BCMA UCAR-T cells obtained by different proliferation modes

Each group of cells was co-cultured with target cells (K562-BCMA cell line (exogenous BCMA-expressing cells, K562 itself does not express BCMA), MM.1S cell line (multiple myeloma cell line, endogenous BCMA-expressing) in 1:1 total lymphocyte culture (Optmizer medium+5 ng/ml IL-15+10 ng/ml IL-7) for 4 hours, and the concentration of cytokine IFN-gamma in cell supernatant was measured by ELISA method with K562 cell line and UTD system without any target cells as negative control, and experimental results are shown in FIG. 2: when the target cells are K562-BCMA or MM.1S, the concentration of IFN-gamma in the UCAR-T group (i.e., UCAR-T two d12/9 group and UCAR-T two d14/14 group) which underwent activation of BCMA protein and CD86 protein and the second subculture stage was comparable to and higher than that of the UCAR-T group (i.e., UCAR-T one group) which underwent only the first subculture stage. In the negative control UTD group, there was no significant IFN-gamma secretion.

3. Cell killing experiments on BCMA UCAR-T cells obtained by different proliferation modes

Each group of cells was co-cultured with target cells MM.1S-GFP-Luc cells (MM.1S-GFP-Luc cell preparation method: constructing a lentiviral expression vector comprising green fluorescent protein GFP and luciferase Luc coding region, then packaging lentivirus, transducing MM.1S cells with lentivirus, sorting positive monoclonal cells by flow cytometry using GFP signals, identifying by culture expansion, GFP expression, confirming that cell preparation was completed) in complete lymphocyte culture broth (OpTmizer medium+final concentration 5ng/ml IL-15+final concentration 10ng/ml IL-7) for 4-6 hours according to different target ratios (1:1, 5:1, 10:1), respectively, K562 cell line was a negative control. Cell survival ratios were measured by detecting the luciferase activity stably expressed in the target cells and the negative control cells, and the results of the cell killing experiments are shown in fig. 3A and 3B. In FIG. 3A, with the exception of UTD groups, both the CAR-T cells and the UCAR-T cells of each group recognize killer tumor cells under antigen-stimulated (i.e., co-cultured with tumor cells having BCMA expression); effective target ratio 5: in the 1, the killing efficiency of the CAR-T group and the UCAR-T two d12/d9 group is highest and can reach nearly 90%; the effective target ratio is 10: at 1, the killing of BCMA positive target cells mm.1s was nearly 100% for each of the remaining groups except UTD group. Also as expected, in fig. 3B, none of the groups of cells had a significant killing effect on negative target cell K562.

4) Immune checkpoint protein expression on BCMA UCAR-T cell surface obtained by different proliferation modes

The proteins PD1 and Lag3 can be used as markers for cell depletion, and the expression level of the immune checkpoint genes in the depleted T cells can be up-regulated. We stained each group of cells with fluorescently labeled antibodies to PD1 (BioLegend Cat. No. 329914), lag3 (BioLegend Cat. No. 369306), and examined by flow cytometry, and the expression rates of PD1 and Lag3 for each group of cells are shown in FIG. 4. Compared with UTD group, CAR-T group and UCAR-T one group, UCAR-T two d12/d9 group and UCAR-T two d14/d14 group have the lowest Lag3 expression rate and lower PD1 expression rate, which indicates lower cell depletion level.

In summary, UCAR-T groups activated with BCMA protein and CD86 protein and in the second subculture stage can maintain their functionality in addition to satisfying the quantitative proliferation requirements. In terms of proliferation: after the BCMA protein and the CD86 protein are adopted for activation, BCMA UCAR-T cells can still proliferate remarkably in the second subculture stage, the number of living cells which only pass through the first subculture stage is taken as a base number, the amplification factor of the UCAR-T two d12/d9 group is 69.5 times, and the amplification factor of the UCAR-T two d14/d14 group is 101.2 times; the expression rate of CAR is also improved. In vitro cell killing: the killing efficiency of UCAR-T two d12/d9 group is the same as and slightly higher than that of CAR-T group; the killing efficiency of UCAR-T two d14/d14 group is equivalent to that of UCAR-T one group, and the effective target ratio is 5: the killing efficiency of UCAR-T two d14/d14 group and UCAR-T one group is lower than that of CAR-T group at 1, but the effective target ratio is 10: in the 1 st, the killing efficiency of UCAR-T two d14/d14 group and UCAR-T one group can reach the level of CAR-T group. In terms of cytokine secretion: IFN-gamma concentrations in UCAR-T two d12/9 and UCAR-T two d14/14 were comparable to and higher than those in UCAR-T one. In terms of immune checkpoint protein expression: compared with UTD group, CAR-T group and UCAR-T one group, UCAR-T two d12/d9 group and UCAR-T two d14/d14 group have the lowest Lag3 expression rate and lower PD1 expression rate, which indicates lower cell depletion level.

Example 4 in vivo test

Based on the in vitro detection result, we also detected in mice, in vivo experiments we selected an animal model of tumor-bearing tail vein in immunodeficient mice with mm.1s cells, and the specific method is as follows: according to 5X 10 ⁶ The MM.1S-GFP-Luc cells/0.2 ml/tail vein were inoculated into female B-NDG mice (body weight: 20.+ -.2 g). Mean fluorescence intensity at day 5 after cell inoculationUp to 1X 10 ⁷ Left and right, randomly grouped according to fluorescence signal intensity and weight, the grouping conditions are as shown in table 4:

TABLE 4 grouping conditions for in vivo experiments

Single tail vein injection back infuses cells, groupings and doses are as indicated above. Thereafter, the change in fluorescence signal intensity was measured twice a week, and the change in fluorescence signal intensity from day 12 was shown in FIG. 5 (D0 in FIG. 5 was calculated as the number of days of administration of a single injection of CAR-T, and administration was started 7 days after inoculation, denoted as D0). The results show that: compared with UTD group and PBS group, each experimental group has good tumor inhibiting effect; compared with the CAR-T group cells, the UCAR-T two d12/d9 group and the UCAR-T one group have no obvious difference in tumor inhibition effect. The in vivo test results further demonstrate: UCAR-T groups activated by BCMA protein and CD86 protein and used in the second subculture stage can also maintain the functionality of the UCAR-T groups except for meeting the requirement of proliferation in quantity.

Example 5 GVHD reaction experiment

Equal amounts of PBMCs from three different healthy donors were mixed and the resulting mixed PBMC cells were irradiated with gamma rays of 100Gray to destroy the ability of the PBMC cells to self-secrete cytokines, after which the PBMC cells that had been disrupted in their ability to self-secrete cytokines described above were mixed with UCAR-T two d12/d9 cells from different donors prepared as in example 2 according to 1:1 (i.e. CD 3) ^- PBMC group), or PBMC cells that have been disrupted by their ability to secrete cytokines per se, described above, were combined with CAR-T cells from different donors, prepared as in example 1, according to a procedure of 1:1 (i.e. CD 3) ⁺ PBMC group), and the concentration of cytokines IFN- γ and IL2 in the cell supernatant was detected by ELISA after 24 hours of mixed incubation, and the experimental results are shown in fig. 6A and 6B: CD3 ⁺ IFN-gamma and IL2 secretion was detected in the PBMC group, whereas IFN-gamma and IL2 secretion was not detected in the CD3-PBMC group.

Due to PBMC finenessThe cells and CAR-T cells were from different donors, the PBMC cells and UCAR-T cells were also from different donors, and the ability of the PBMC cells themselves to secrete cytokines had been destroyed, indicating that if cytokine production was to be that of CAR-T cells or UCAR-T cells. CD3 ⁺ IFN-gamma and IL2 secretion was detected in the PBMC group, while CD3 ^- No secretion of IFN- γ and IL2 was detected in the PBMC group, indicating that PBMC cells of healthy donors did not produce GVHD response after mixing with UCAR-T cells, whereas PBMC cells of healthy donors produced a certain GVHD response after mixing with allogeneic CAR-T cells, without any antigen stimulation or co-incubation of positive target cells.

Example 6 optimization of BCMA UCAR-T cell proliferation method

1. Optimization of T cell culture media

BCMA UCAR-T was obtained by the method described in example 1, example 2, step 1, and the BCMA UCAR-T cells were subjected to a first subculture stage after electric shock, 1 passage was carried out every 2-3 days, and the subculture medium used was a whole lymphocyte culture broth under culture conditions of 37℃ +5% CO ₂ The method comprises the steps of carrying out a first treatment on the surface of the Removing residual cd3+ cells from the proliferating cells on day 8 after the electric shock; BCMA UCAR-T cells after the first subculture stage were harvested on day 12 post-shock.

The effect of the basal medium of the complete lymphocyte culture broth used during the proliferation and passage of BCMA UCAR-T cells and added cytokines on BCMA UCAR-T cells was examined, and different complete lymphocyte culture broths were used for subculturing, and the experiments were divided into 4 groups: X-VIVO medium+IL2 group (final concentration 100 IU/ml); X-VIVO medium+IL 7+IL15 group (IL 7 final concentration 10ng/ml, IL15 final concentration 5 ng/ml); optmizer medium+IL2 group (final concentration 100 IU/ml); optmizer medium+IL 7+IL15 group (IL 7 final concentration 10ng/ml, IL15 final concentration 5 ng/ml).

Cells were harvested and counted after the first subculture stage and proliferation of each group was as shown in fig. 7: the number of cell proliferation in the Optmizer medium+IL7+IL15 group was far superior to that of the other groups, and in particular, BCMA UCAR-T cells were proliferated in a large amount on days 9-12 after gene knockout by electric shock.

Each group of cells was stained with PerCP fluorescent-labeled CD45RA antibody (available from Biolegend), PE-labeled CCR7 antibody (available from Biolegend), and the expression of each group of cell surface CD45RA, CCR7 was detected by flow cytometry, and CD4+ T cells and CD8+ T cells were separated into naive T cells (T _N ) (CD45RA+CCR7+), effector T cells (T) _E ) (CD45RA+CCR7-), central memory T cells (T) _CM ) (CD 45 RA-CCR7+) and effector memory T cells (T) _EM ) The (CD 45RA-CCR 7-) subpopulation, results are shown in Table 5: the cells in the Optmizer medium+IL7+IL15 group had the highest CD45RA+CCR7+ ratio, indicating that under this medium condition, BCMA UCAR-T cells were younger after the first subculture stage.

TABLE 5 BCMA UCAR-T cell subsets under different T cell Medium conditions

In summary, the complete lymphocyte broth was optimized by selecting the Optmizer medium as the basal medium and adding IL7 at a final concentration of 10ng/ml and IL15 at a final concentration of 5ng/ml to the basal medium.

2. Selection of BCMA protein and CD86 protein loaded magnetic bead particles and ratio of BCMA UCAR-T cells

Preparation of BCMA UCAR-T was performed according to the methods described in example 1 and example 2, and the ratio of the numbers of bead particles loaded with BCMA protein and CD86 protein to UCAR-T cells was changed to 0.5: 1. 1:1 and 2:1. cells were harvested and counted after the second subculture stage, and each group of cells was stained with fluorescently labeled CD3 protein, CD52 protein and BCMA protein, and the cd3+ cell fraction, cd52+ cell fraction and CAR expression rate in each group of cells were detected by flow cytometry, the differences between each group were insignificant, indicating that at 0.5:1-2:1, the magnetic bead particles loaded with BCMA protein and CD86 protein can obtain better effect, and the selection of 0.5:1 is lower in scale cost.

Sequence description

SEQ ID NO:1：BCMA VH CDR1；

GFSLSTYH

SEQ ID NO:2：BCMA VH CDR2；

ISSSGST

SEQ ID NO:3：BCMA VH CDR3；

ARDLDYVIDL

SEQ ID NO:4：BCMA VL CDR1；

PSVYNNY

SEQ ID NO:5：BCMA VL CDR2；

ETS

SEQ ID NO:6：BCMA VL CDR3；

AGTYVSGDRRA

SEQ ID NO. 7: BCMA VH amino acid sequence;

EVQLVESGGGLVQPGGSLRLSCTASGFSLSTYHMTWVRQAPGKGLEWIGVISSSGSTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDYVIDLWGPGTLVTVSS

SEQ ID NO. 8: BCMA VL amino acid sequence;

EIVMTQSPSTLSASVGDRVIINCQSSPSVYNNYLSWYQQKPGKAPKLLIYETSTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCAGTYVSGDRRAFGQGTKLTVL

SEQ ID NO. 9: BCMA scFv amino acid sequence;

EIVMTQSPSTLSASVGDRVIINCQSSPSVYNNYLSWYQQKPGKAPKLLIYETSTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCAGTYVSGDRRAFGQGTKLTVLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSTYHMTWVRQAPGKGLEWIGVISSSGSTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDYVIDLWGPGTLVTVSS

SEQ ID NO. 10: a hinge region sequence;

TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

SEQ ID NO. 11: a transmembrane region sequence;

IYIWAPLAGTCGVLLLSLVITLYC

SEQ ID NO. 12: an intracellular signaling region sequence;

RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO. 13: a costimulatory signaling region sequence;

KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

SEQ ID NO. 14: a leader peptide sequence;

MALPVTALLLPLALLLHAARP

SEQ ID NO. 15: amino acid sequence of BCMA CAR;

MALPVTALLLPLALLLHAARPEIVMTQSPSTLSASVGDRVIINCQSSPSVYNNYLSWYQQKPGKAPKLLIYETSTLASGVPSRFSGSGSGAEFTLTISSLQPDDFATYYCAGTYVSGDRRAFGQGTKLTVLGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCTASGFSLSTYHMTWVRQAPGKGLEWIGVISSSGSTYYASWAKGRFTISRDNSKNTVYLQMNSLRAEDTAVYFCARDLDYVIDLWGPGTLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

SEQ ID NO. 16: a nucleotide sequence encoding a BCMA CAR amino acid sequence;

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGGAGATCGTGATGACCCAGTCCCCAAGTACACTGAGCGCCTCCGTGGGCGACCGCGTGATCATAAACTGTCAAAGCTCACCCTCTGTTTACAACAATTACCTGTCTTGGTATCAACAGAAGCCCGGTAAGGCCCCCAAACTGCTCATTTACGAGACATCCACCCTGGCATCCGGGGTGCCAAGCCGCTTCTCCGGGAGTGGGTCTGGCGCCGAGTTCACCCTGACCATATCTTCCCTGCAGCCCGACGACTTCGCAACGTACTATTGCGCCGGAACCTATGTAAGTGGGGATAGACGCGCCTTCGGGCAGGGCACGAAGTTGACCGTGCTGGGCGGAGGGGGCTCAGGAGGTGGCGGTAGCGGAGGAGGCGGCTCAGAAGTGCAGCTGGTGGAGTCCGGCGGTGGACTGGTGCAACCGGGAGGCTCACTCAGATTGTCATGCACCGCCTCTGGCTTTAGTCTCTCCACCTATCATATGACTTGGGTGAGGCAGGCACCCGGCAAGGGCCTGGAATGGATCGGCGTGATCTCTTCCAGCGGTAGCACCTATTACGCCTCTTGGGCGAAGGGCAGGTTTACCATCAGCCGCGACAACAGCAAGAATACCGTTTACCTGCAGATGAATAGCCTGAGGGCCGAAGACACGGCGGTCTATTTCTGTGCACGGGACCTTGACTACGTTATTGACCTGTGGGGCCCTGGGACCCTCGTAACTGTGAGCAGCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGATATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGCAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCTAA

SEQ ID NO. 17: an sgRNA or chRDNA targeting sequence in the CRISPR/Cas system for exon 1 of the Trac gene;

TGTACCAGCTGAGAGACTCT

SEQ ID NO. 18: an sgRNA or chRDNA targeting sequence in the CRISPR/Cas system for exon 1 of the CD52 gene;

CCTGGTTATGGTACAGGTAA

SEQ ID NO. 19: an sgRNA sequence for exon 1 of the Trac gene;

AGAGUCUCUCAGCUGGUACA

SEQ ID NO. 20: sgRNA sequence for exon 1 of CD52 gene.

UUACCUGUACCAUAACCAGG

Claims

1. A method of proliferation of universal BCMA CAR-T cells comprising the steps of: preparing universal BCMA CAR-T cells and proliferating the universal BCMA CAR-T cells;

wherein: the step of proliferation of the universal BCMA CAR-T cell comprises the steps of:

2. The proliferation method according to claim 1, wherein the time of the first subculture stage is: 12-14 days from the first subculture stage of the universal BCMA CAR-T cells; the time of the second subculture stage is as follows: activating the universal BCMA CAR-T cells by using BCMA protein and CD86 protein for 9-14 days;

Optionally, the time of the first subculture stage is: 12 days from the first subculture stage for universal BCMA CAR-T cells; the time of the second subculture stage is as follows: activation of universal BCMA CAR-T cells with BCMA protein and CD86 protein was performed for 9 days.

3. The proliferation method of claim 1, wherein the universal BCMA CAR-T cells are activated with BCMA protein and CD86 protein by: mixing a carrier loaded with BCMA protein and a carrier loaded with CD86 protein with universal BCMA CAR-T cells to perform activation treatment;

alternatively, the BCMA protein-loaded cargo and the CD86 protein-loaded cargo are the same cargo;

further alternatively, the carrier loaded with BCMA protein and CD86 protein simultaneously is magnetic beads or a polymer material.

4. The proliferation method according to claim 3, wherein the preparation method of the carrier loaded with both BCMA protein and CD86 protein comprises the steps of: every 5×10 ⁷ Adding 20-30ug of BCMA protein capable of being loaded on the magnetic beads and 20-30ug of CD86 protein capable of being loaded on the magnetic beads into each magnetic bead for mixed incubation;

alternatively, every 5×10 ⁷ Adding 25ug of BCMA protein capable of being loaded on the magnetic beads and 25ug of CD86 protein capable of being loaded on the magnetic beads into each magnetic bead for mixed incubation;

further alternatively, BCMA protein and CD86 protein are biotinylated corresponding proteins, and the magnetic beads are labeled with anti-biotin antibodies.

5. The proliferation method according to claim 3 or 4, wherein the ratio of the number of magnetic beads to the number of universal BCMA CAR-T cells is 0.4-2:1, a step of;

alternatively, the ratio of the number of magnetic beads to the number of universal BCMA CAR-T cells is 0.5:1.

6. the proliferation method of any one of claims 1-5, wherein the method of preparing a universal BCMA CAR-T cell is selected from one of the following 2 methods:

the method one comprises the following steps:

a. activating T cells by adopting CD3 protein and CD28 protein;

the second method comprises the following steps:

alternatively, the method of making a universal BCMA CAR-T cell is selected from method one.

7. The proliferation method of claim 6, wherein the subculture medium for culturing T cells, CAR-T cells and/or universal BCMA CAR-T cells is: adding an OpTmizer culture medium of IL-7 with a final concentration of 8-12ng/ml and IL-15 with a final concentration of 4-6 ng/ml;

alternatively, the subculture medium for culturing T cells, CAR-T cells and/or universal BCMA CAR-T cells is: add the OpTmizer medium with IL-7 at a final concentration of 10ng/ml and IL-15 at a final concentration of 5 ng/ml.

8. The proliferation method according to claim 6, wherein the cd3+ cells remaining in the proliferating cells are removed 6 to 8 days after the introduction of the gene editing tool into the CAR-T cells;

alternatively, residual cd3+ cells in proliferating cells are removed 8 days after introduction of the gene editing tool into CAR-T cells.

9. The proliferation method according to claim 6, wherein the T cells are activated by using CD3 protein and CD28 protein in the following manner: activating T cells by adopting a carrier loaded with CD3 protein and a carrier loaded with CD28 protein;

Alternatively, the CD3 protein-loaded and CD28 protein-loaded loads are the same load;

further alternatively, the carrier loaded with both CD3 protein and CD28 protein is a magnetic bead or a polymeric material.

10. The proliferation method according to claim 6, wherein the gene editing tool prevents the normal expression of the Trac gene by gene editing one or more of exon 1, exon 2, exon 3, and exon 4 of the Trac gene on chromosome 14 of T cells or CAR-T cells;

alternatively, the gene editing tool disables the normal expression of the CD52 gene by gene editing one or more of exon 1 and exon 2 of the CD52 gene on chromosome 1 of the T cell or CAR-T cell;

further alternatively, the gene editing tool can prevent the normal expression of the Trac gene and the CD52 gene by gene editing the 1 st exon of the Trac gene on chromosome 14 and the 1 st exon of the CD52 gene on chromosome 1 of T cells or CAR-T cells.

11. The proliferation method of claim 6, wherein the gene editing tool is selected from one of a CRISPR/Cas system, a zinc finger nuclease system, a transcription activator-like effector nuclease system;

Optionally, the gene editing tool is selected from a CRISPR/Cas system.

12. The proliferation method according to claim 10 or 11, wherein the sgRNA or chRDNA targeting sequence in the CRISPR/Cas system for exon 1 of the Trac gene is shown in SEQ ID No. 17; and/or

The targeting sequence of sgRNA or chRDNA in CRISPR/Cas system aiming at the 1 st exon of CD52 gene is shown as SEQ ID NO. 18;

alternatively, the sequence of the sgRNA in the CRISPR/Cas system for exon 1 of the Trac gene is shown in SEQ ID NO. 19; and/or

Alternatively, the sequence of the sgRNA in the CRISPR/Cas system for exon 1 of the CD52 gene is shown in SEQ ID NO. 20.

13. The proliferation method of claim 12, wherein the amount of Cas9 protein in the CRISPR/Cas system is per 1 x 10 ⁶ Cells to be gene edited use 1.5ug-5ug Cas9 protein, the molar ratio of sgRNA to Cas9 protein is 2-4:1, a step of;

alternatively, the amount of Cas9 protein in the CRISPR/Cas system is per 1 x 10 ⁶ Cells to be gene edited used 3ug Cas9 protein, the molar ratio of sgRNA to Cas9 protein was 2.5:1.

14. the proliferation method of claim 6, wherein the T cells in the T cells or CAR-T cells are derived from allogeneic T lymphocytes;

Alternatively, the T cells in the T cells or CAR-T cells are derived from a healthy donor.

15. The proliferation method of claim 6, wherein the BCMA-targeted chimeric antigen receptor comprises an extracellular antigen recognition domain, a hinge region, a transmembrane region, and an intracellular domain;

alternatively, the extracellular antigen recognition domain comprises BCMA VH and BCMA VL, wherein the amino acid sequences of the CDRs 1, 2 and 3 of the BCMA VH complementarity determining regions comprise the amino acid sequences shown in SEQ ID NO. 1, 2 and 3, respectively, and the amino acid sequences of the CDRs 1, 2 and 3 of the BCMA VL complementarity determining regions comprise the amino acid sequences shown in SEQ ID NO. 4, 5 and 6, respectively;

further alternatively, the BCMA VH sequence comprises an amino acid sequence shown as SEQ ID NO. 7, and the BCMA VL sequence comprises an amino acid sequence shown as SEQ ID NO. 8;

still further alternatively, the extracellular antigen-recognition domain targeted to BCMA comprises a BCMA scFv antibody as shown in SEQ ID No. 9.

Still further alternatively, the scFv antibody is a humanized antibody.

16. The proliferation method of claim 15, wherein the hinge region is derived from one or more of IgG1, igG4, CD7, CD28, CD84, CD8 a;

Alternatively, the hinge region is derived from CD8 a;

further alternatively, the sequence of the hinge region comprises the amino acid sequence shown as SEQ ID NO. 10.

17. The proliferation method of claim 15, wherein the transmembrane region is derived from one or more of CD3, CD4, CD7, CD8 a, CD28, CD80, CD86, CD88, 4-1BB, CD152, OX40, fc 70;

alternatively, the transmembrane region is derived from CD8 a;

further alternatively, the sequence of the transmembrane region comprises the amino acid sequence shown as SEQ ID NO. 11.

18. The proliferation method of claim 15, wherein the intracellular domain comprises an intracellular signaling region;

optionally, a costimulatory signaling region;

further alternatively, the intracellular signaling region is derived from one or more of CD3 δ, CD3 γ, CD3 δ, CD3 epsilon, CD5, CD22, CD79a, CD79b, fcrγ, fcrβ, CD66d, DAP10, DAP12, syk; alternatively, the intracellular signaling region is derived from cd3δ;

still further alternatively, the intracellular signaling region comprises the amino acid sequence shown as SEQ ID NO. 12.

19. The proliferation method of claim 18, wherein the costimulatory signaling 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, myD 88;

Optionally, the costimulatory signaling region is derived from 4-1BB or CD28;

further alternatively, the costimulatory signaling region comprises the amino acid sequence depicted as SEQ ID NO. 13.

20. The proliferation method according to any one of claims 15-19, further comprising a leader peptide at the N-terminus of said chimeric antigen receptor amino acid sequence;

optionally, wherein the guide peptide is derived from CD8 a;

further alternatively, the leader peptide comprises the amino acid sequence shown as SEQ ID NO. 14.

21. The proliferation method according to claim 15, wherein the amino acid sequence of BCMA CAR is shown in SEQ ID No. 15.

22. The proliferation method according to claim 21, wherein the nucleotide sequence encoding BCMA CAR is shown in SEQ ID No. 16.

23. A BCMA-targeted universal CAR-T cell, wherein the universal CAR-T cell is obtained by the proliferation method of any one of claims 1-22.

24. Use of a BCMA-targeted universal CAR-T cell according to claim 23 in the manufacture of a medicament;

optionally, the medicament is for treating a disease or disorder associated with expression of BCMA;

further optionally, the medicament is for treating Multiple Myeloma (MM);

Still further alternatively, the medicament is an intravenous injection.