WO2020025039A1 - T cell expressing chimeric antigen receptor, chimeric antigen-related expression vector and use thereof - Google Patents

Abstract

Provided is a T cell expressing a chimeric antigen receptor. The chimeric antigen receptor comprises an extracellular domain, and the extracellular domain recognizes a target antigen on the surface of a target cell, thereby mediating the killing of the target cell by the T cell. The T cell itself also expresses the target antigen, and in order to prevent the T cells from killing each other, the expression of the target antigen in the T cell is down-regulated. An expression vector comprising a sequence coding the chimeric antigen receptor is also provided. The T cell provided can be used to treat diseases caused by abnormal proliferation of CS1 positive cells by targeting CS1 antigen, meanwhile, the T cells are prevented from killing each other due to down-regulation of CS1 expression of the T cell, facilitating expansion and survival of the T cell in vitro and in vivo.

Description

T cell expressing chimeric antigen receptor, the chimeric antigen-related expression vector and application thereof Technical field

The present invention relates to T cells (CAR T cells) expressing a Chimeric Antigen Receptor (CAR), expression vectors for transforming T cells, and their use in the treatment of multiple myeloma.

Background technique

The development of chimeric antigen receptors for targets expressed in multiple myeloma (MM) cells is of great interest, as data suggest that infusion of CD19-CAR in patients with refractory B-cell-associated tumors T cells to induce lasting complete remission ^1,2. There are several candidate antigen targets for CAR T cells for treating MM, including CD19 and BCMA, but CD19 is only expressed in a very small number of malignant plasma cells. Phase I clinical trial data of BCMA-CAR T cells show that some patients produce partial and complete responses after treatment, but there are also some problems, including low or uneven expression of BCMA on myeloma cells in enrolled patients, and post-treatment Outbreaks of BCMA-negative or BCMA-low-expressing myeloma cells ³ . These data highlight that the existing CAR targets still have some deficiencies in the treatment of myeloma, and CAR targets with broader applicability to this disease need to be found and verified.

SLAMF7 (also known as CD319, CRACC, or CS1) is a member of the transmembrane receptor signal transduction lymphocyte activation molecule family, which is expressed at high levels in myeloma cells and is involved in regulating the mutual adhesion of myeloma cells and bone marrow stromal cells effect. Immunohistochemical analysis of a series of lymphomas and leukemias revealed that CS1, although present in all myeloma cases, is not expressed in most acute leukemias, B-cell lymphomas, and classic Hodgkin lymphomas. Currently, researchers have investigated the feasibility of CS1 as a CAR T target. CS1-CAR T cells target CS1-expressing multiple myeloma cell lines (such as NCI-H929, IM9, MM1S) and primary tumor cells isolated from patients with multiple myeloma. Compared with untransduced CAR control T cells, CS1-CAR T cells had significantly enhanced cytokine IFN-γ and IL-2 secretion, and significantly increased the number of killed multiple myeloma cell lines or multiple myeloma patients. Generation of tumor cells ⁴ . These results demonstrate that the effect of CS1-CAR T cells is CS1-dependent. Importantly, CS1-CAR T cells can prolong the survival time of NSG mice transplanted with MM1S cells. In general, CS1 has great potential as a CAR target for the treatment of multiple myeloma, and data on CS1 as a CAR target in vitro and in vivo function data have been reported in the literature, but some researchers have pointed out that CS1 as the subject of technical difficulties CAR target and there is concern aspects ^5.

CS1 is highly expressed on multiple myeloma cells, but on NK cells, T cells, B cells, and mature dendritic cells also expressed ^6,7. T cells are divided into helper T cells (CD4 +) and cytotoxic (CD8 +) T cells. In general, CD4 + T cells can proliferate to activate other types of immune cells that produce a direct immune response. CD8 + T cells can kill target cells that produce antigenic responses. Some researchers have investigated the function of CS1 in CD8 + T cells in patients with systemic lupus erythematosus (SLE). The impaired function of CD8 + T cells in SLE patients leads to a decrease in their ability to resist infection, while the expression of CS1 in CD8 + T cells in SLE patients is down-regulated. The activation of CS1 by specific antibodies can restore CD8 + T cells in defective SLE patients, enabling them to act on viral antigens ⁸ . CS1 in NK cells also play an important role in the interaction of activated NK cell function ⁹ by the same tropism of CS1. NK cells express various activating and inhibiting receptors that recognize ligands on potential target cells. The balance between the signals from these receptors determines whether NK cells will be activated, killing target cells and secreting cytokines. Antibodies and ligand-mediated stimulation experiments have demonstrated that CS1 plays an activating role in NK ^cells9,10,11 . The ability of CS1-deficient NK cells to kill CS1 + target cells is impaired. When encountering target cells, the ability of CS1-deficient NK cells to secrete interferon (IFN) also decreased. At the same time, the cytotoxicity of CS1-deficient NK cells to CS1-target cells also decreased. This study indicates that CS1 is involved in the interaction between NK cells and NK cells, and has the potential to promote NK cell function. Although CS1 may have beneficial functions in T cells and NK cells, the expression of CS1 in T cells may cause difficulties in the preparation and application of CS1-CAR T cells.

Flow cytometry was used to detect the expression of CS1 in the common cell subsets in peripheral blood of patients with multiple myeloma. It was found that CS1 protein was expressed in NK cells, T cells, B cells and monocytes, among which CD4 + T cells CS1 protein expression level is lower than the amount of expression in CD8 + T cells in ^5. Since CS1 is also expressed in some T cells and other immune cells, CS1-CAR T may cause these cells to be selectively killed and eliminated. Some researchers analyzed the ability of CS1-CAR T cells to recognize normal lymphocytes and found that they selectively kill CS1 + / high NK cells, CD4 + and CD8 + T cells and B cells. At the same time, CS1-CAR T cells showed a significant reduction in CS1 protein-positive CD4 + and CD8 + cells compared to control T cells after several days of culture. The study also found that when CS1-CAR T cells were co-cultured with a normal lymphocyte population, compared to CD19-CAR T cells, CD4 + T cells, CD8 + T cells, and NK cells co-cultured with CS1-CAR T cells. The percentage of viable cells decreased, with the most significant decrease in the rate of viable cells of NK cells ⁵ .

The suicide of CS1-CAR T cells caused only CS1-negative or low-expressing cells to survive in CS1-CAR T cells, which made it difficult for CS1-CAR T cells to expand; and because CD8 + T cells had more than CD4 + T cells The expression of CS1 will be more cleared, resulting in an abnormal ratio of CD4: CD8 in CS1-CAR T cells, reducing its cytotoxic activity in vitro and in vivo.

Summary of the invention

The invention provides, in one aspect, a T cell expressing a chimeric antigen receptor, the chimeric antigen receptor comprising an extracellular domain that recognizes a target antigen on the surface of a target cell, thereby mediating Killing of the target cell by the T cell; the T cell itself also expresses the target antigen, and in order to prevent the T cell from killing each other, the expression of the target antigen by the T cell is down-regulated.

In some embodiments, the extracellular domain of the chimeric antigen receptor includes a single chain antibody derived from an antibody against the target antigen.

In some embodiments, the target cells are tumor cells, especially multiple myeloma cells.

In some specific embodiments, the target antigen is CS1.

In some embodiments, the T cell down-regulates expression of the target antigen by expressing siRNA.

In some embodiments, the siRNA is produced from a shRNA expressed by the T cell.

In some embodiments, the target nucleic acid sequence of the shRNA includes a nucleotide sequence as shown in SEQ ID NO: 29.

In some specific embodiments, the coding sequence of the shRNA includes a nucleotide sequence as shown in SEQ ID NO: 28.

In some embodiments, the single chain antibody is derived from an anti-CS1 antibody and has an amino acid sequence as shown in SEQ ID NO: 20.

In some specific embodiments, the amino acid sequence of the chimeric antigen receptor includes the CD8α signal peptide, the single chain antibody, the CD8 hinge region, the CD28 transmembrane region, and the CD28 intracellular co-stimulatory domain in order from the N-terminus to the C-terminus. And 4-1BB intracellular co-stimulatory domain and CD3ζ intracellular signaling domain.

In some embodiments, the T cells are transformed with an expression vector including the coding sequence of the chimeric antigen receptor and an expression vector including the coding sequence of the shRNA, or are encoded by the chimeric antigen receptor. The expression vector of the sequence and the coding sequence of the shRNA is transformed.

In another aspect, the present invention provides an expression vector for expression in T cells, comprising a coding sequence for a chimeric antigen receptor and a shRNA coding sequence, wherein the chimeric antigen receptor recognizes on the surface of a target cell And the shRNA down-regulates the expression of the target antigen in the T cells through the siRNA produced by the shRNA.

In some embodiments, the chimeric antigen receptor includes an extracellular domain, a transmembrane domain, and an intracellular domain, the extracellular domain comprising a single chain antibody derived from an antibody against the target antigen.

In some embodiments, the coding sequence of the shRNA is under the control of the H1 promoter.

In some embodiments, the expression vector is selected from a lentiviral expression vector, a DNA plasmid expression vector, or a viral expression vector.

In some embodiments, the target cells are tumor cells, especially multiple myeloma cells.

In a specific embodiment, the expression vector uses pLVX-EF1α-IRES-Puro as a backbone vector.

In a specific embodiment, the target antigen is CS1.

In a specific embodiment, the target nucleic acid sequence of the shRNA includes a nucleotide sequence as shown in SEQ ID NO: 29.

In a specific embodiment, the coding sequence of the shRNA includes a nucleotide sequence as shown in SEQ ID NO: 28.

In a specific embodiment, the single chain antibody is derived from an anti-CS1 antibody and has an amino acid sequence as shown in SEQ ID NO: 20.

In another aspect, the present invention provides a method for preparing a T cell expressing a chimeric antigen receptor, which comprises transforming the T cell with the expression vector described above.

In another aspect, the present invention provides a method for preventing mutual killing of T cells expressing a chimeric antigen receptor, comprising down-regulating the expression of a target antigen targeted by the chimeric antigen receptor in the T cells.

In a specific embodiment, the target antigen is CS1.

In some embodiments, the down-regulating comprises allowing the T cells to express shRNA, and the siRNA produced by the shRNA inhibits expression of the target antigen in the T cells.

In some embodiments, the method is achieved by transforming the T cell with an expression vector comprising a coding sequence of the shRNA.

In another aspect, the present invention provides a method for treating multiple myeloma in a subject, comprising administering to the subject T cells expressing a chimeric antigen receptor, the chimeric antigen receptor targets CS1 on the surface of multiple myeloma cells, and the T cells also express shRNAs for inhibiting the expression of CS1 in the T cells.

In another aspect, the present invention provides the use of the above T cell or expression vector in the manufacture of a medicament for treating a disease caused by the proliferation of CS1-positive cells. Preferably, the disease is multiple myeloma or plasma cell leukemia.

The T cells provided by the present invention can be used to treat diseases caused by abnormal proliferation of CS1-positive cells (such as multiple myeloma) by targeting the CS1 antigen, and at the same time prevent the T cells from down-regulating the CS1 expression of the T cells to prevent The T cells kill each other, which is conducive to the expansion and survival of the T cells in vitro and in vivo.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic diagram of the composition of mock-5.3-CAR elements. The CAR consists of the following components: CD8α signal peptide, BCMA-specific scFv (BCMA scFv), CD8 hinge region, CD28 transmembrane region (TM), CD28 intracellular costimulatory domain, 4-1BB intracellular costimulatory domain, and CD3ζ cell Inner signal domain.

Figure 2 is a schematic diagram of the composition of the mock-CS1-CAR element. The CAR consists of the following components: CD8α signal peptide, CS1-specific scFv (CS1scFv), CD8 hinge region, CD28 transmembrane domain (TM), CD28 intracellular costimulatory domain, 4-1BB intracellular costimulatory domain, and CD3ζ intracellular Signal domain.

Figure 3 is a schematic diagram of the SH3-5.3-CAR element structure. It consists of a gene encoding SH3 shRNA (SH3) capable of knocking down CS1 expression and mock-5.3-CAR shown in FIG. 1. Transcription of this shRNA is initiated by the H1 promoter.

Figure 4 is a schematic diagram of the structure of the SH3-CS1-CAR element. It consists of a gene encoding SH3 shRNA (SH3) capable of knocking down CS1 expression and mock-CS1-CAR shown in FIG. 2. Transcription of this shRNA is initiated by the H1 promoter.

FIG. 5 is a schematic diagram of a BCMA-T2A-puro construct overexpressing BCMA, which includes BCMA, T2A, and a puromycin resistance gene (puro).

FIG. 6 is a schematic diagram of a CS1-IRES-puro construct overexpressing CS1, which includes CS1, IRES, and a puromycin resistance gene (puro).

Figure 7 shows the expression of BCMA on the surface of B-K562 cells as determined by flow cytometry. The left peak is un-stained B-K562, and the right peak is B-K562 stained with APC anti-human CD269 (BCMA) Antibody.

Figure 8 shows the expression of CS1 on the surface of C-K562 cells measured by flow cytometry. The left peak is unstained C-K562, and the right peak is C-K562 stained with Human CRACC / SLAMF7 APC-conjugated Antibody.

Figure 9 is a bar graph showing the expression of CS1 protein in CAR T cells and control T cells treated with SH3 knockdown.

Figure 10 shows the proliferation of CAR T cells and control T cells. Because CS1 is expressed on the surface of T cells, suicide will occur during CS1-CAR T culture, making it difficult for cells to expand. After the inventors knocked down the expression of CS1 in CAR T cells, the cells could expand normally as well as control T cells and BCMA-CAR T cells (mock-5.3 and SH3-5.3). The expansion curves of mock-5.3 and SH3-5.3 cells also showed no significant effect on cell proliferation after CS1 knockdown.

CM和EM分别指

T细胞、T _CM细胞和T _EM细胞； Figure 11 shows the composition of various CAR T cell subpopulations. Knockdown of CS1 expression does not affect the proportion of cell subsets in CAR T cells.

CM and EM refer to

T cells, T _CM cells and T _EM cells;

Figure 12 shows the in vitro killing ability of various CAR T cells to different target cells. Mock-5.3 cells and SH3-5.3 cells can specifically kill BCMA-positive target cells (MM1S and B-K562), but compared with mock-5.3 cells, the killing effect of SH3-5.3 cells on MM1S is slightly reduced. It is possible that knocking down CS1 has an effect on the killing function of T cells, but further experiments need to be confirmed. Mock-CS1 cells and SH3-CS1 cells can specifically kill CS1-positive target cells (MM1S and C-K562), and no weakening of the tumor-killing effect of SH3-CS1 cells is observed, which may be through knocking down CS1-CAR of CS1. Reduced T-cell suicides offset the negative effects of knockdown of CS1 on T-cell killing function.

Figure 13 shows the results of CD107a detection on various CAR T cells and control T cells after target cell stimulation. Mock-CS1 cells and SH3-CS1 cells were specifically degranulated when co-cultured with CS1-positive target cells (MM1S and C-K562), and no decrease in degranulation level of SH3-CS1 cells was observed. Low CS1 leads to reduced suicide of CS1-CAR T cells, which offsets its adverse effects.

Figure 14 shows the expression of immune checkpoint molecules in various CAR T cells. PD-1 and CTLA-4 (top) and TIM-3 and LAG3 (bottom) immune checkpoints are signs of T cell depletion. The expression of the above four markers in the SH3-CS1 group is lower, suggesting that knockdown of CS1 CAR T cells are less depleted due to reduced suicide, suggesting that CS1-CAR T that knocks down CS1 may have better therapeutic potential.

detailed description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

As used herein, the term "antibody" refers to immunoglobulins secreted by plasma cells (effector B cells) and used by the body's immune system to identify and neutralize foreign substances (polypeptides, viruses, bacteria, etc.). This foreign substance is accordingly called an antigen. The basic structure of an antibody molecule is a 4-mer consisting of two identical heavy chains and two identical light chains. According to the conservative differences in amino acid sequences, the heavy and light chains are divided into a variable region at the amino terminus and a constant region at the carboxy terminus. The variable regions of a heavy chain and a light chain interact to form an antigen-binding site. Thus, a complete antibody molecule includes two antigen-binding sites.

As used herein, the term "single chain antibody (scFv)" refers to a single peptide chain formed by linking the variable region of an antibody's heavy chain and the variable region of a light chain through a short peptide. When correctly folded, the variable region of the heavy chain and the variable region of the light chain form an antigen-binding site through non-covalent bond interactions, which can better retain the affinity activity of the source antibody for the antigen.

As used herein, the term "chimeric antigen receptor" refers to an engineered protein receptor molecule that can impart a desired specificity to an immune effector cell (such as a T cell), such as the ability to bind to a specific tumor antigen. These receptors are called "chimeric" because they are fusion proteins and are composed of components from different sources. Chimeric antigen receptors typically include an extracellular domain (or extracellular binding domain), a transmembrane domain (or transmembrane region), and an intracellular domain (or intracellular signaling domain). The extracellular domain typically includes a scFv sequence that is responsible for recognizing and binding to specific antigens (target antigens) on target cells. Intracellular domains usually include immunoreceptor tyrosine activation motifs (ITAM), such as signaling domains derived from the CD3ζ molecule, which are responsible for activating immune effector cells, which can increase the cytotoxicity, proliferative capacity, and prolong T cells. Survival time. In addition, the chimeric antigen receptor can also include a signal peptide at the amino terminus responsible for localization of the nascent protein on the cell, and a hinge region between the scFv sequence and the transmembrane domain.

As used herein, the term "downregulating" refers to a reduction in the ability of a gene or coding sequence to express its target product (such as a protein or RNA) in a cell compared to normal levels. This can be achieved in a variety of ways, for example, by inhibiting the initiation of transcription, interfering with mRNA translation, promoting mRNA degradation, or promoting degradation of expressed proteins.

As used herein, the term "siRNA (small interfering RNA)" refers to a double-stranded RNA molecule that is approximately 21 nt in length. They can be complementary to the homologous sequence in the target mRNA, causing the mRNA to lose function or be degraded, so it cannot be translated. protein. One way to introduce siRNA into a cell is to express the corresponding short hairpin RNA (shRNA) molecule in the cell. The "loop" of the short hairpin RNA molecule is replaced by a nuclease (such as Dicer) in the cell. Enzymes) degrade to form siRNA, which plays a role in down-regulating the expression of target genes.

As used herein, the term "transformation" refers to the introduction of an expression vector (e.g., a plasmid expression vector, a viral expression vector) containing a gene of interest into a host cell (e.g., a T cell) and allowing the gene of interest to be expressed in the host cell. Processes include virus-mediated transduction and transfection using liposomes, calcium phosphate, microinjection, electroporation, and the like.

When referring to CAR T cells, the term "suicide" in this context refers to a situation where the CAR T cells themselves also express the target antigen, which results in the mutual recognition and killing of cells within the CAR T cell population, as a whole It appears that this CAR T cell population is in a state of suicide.

Other terms are described in more detail below when describing specific embodiments.

In the present invention, a short hairpin RNA targeting CS1 mRNA is introduced into a lentivirus expression vector encoding a chimeric antigen receptor (CS1-CAR) targeting CS1. After the lentivirus infects T cells and integrates the CS1-CAR and shRNA coding sequences into the T cell genome, the shRNA coding genes are transcribed in the T cells and processed to form siRNA. The siRNA degrades CS1 mRNA through an RNA interference pathway, thereby reducing the expression level of CS1 protein in CS1-CAR T cells, and protecting the CS1-CAR T cells from mutual recognition and killing. The inventors also studied the cell proliferation and cell subpopulation composition of knockdown CS1-expressing CAR T cells, and compared their in vitro killing function with CAR T cells that did not knock-down CS1.

Example 1 Preparation of chimeric antigen receptor-modified T cells

1. Preparation of chimeric antigen receptor gene expression vector

1.1 Overview

(1) CAR is a chimeric protein that fuses an antigen-binding domain that specifically recognizes a target antigen (such as CS1) with an intracellular signal transduction structure capable of activating or stimulating immune cells. In general, CAR includes extracellular domains, transmembrane domains, and intracellular domains. In this example, the extracellular domain includes a single-chain antibody (scFv) capable of specifically binding to CS1, a CD8α signal peptide, and a CD8 hinge region. The hinge region is usually derived from a CD8 or IgG4 molecule, which is used to connect intracellular and extracellular proteins. In this example, the inventors used a CD8 Hinger derived from CD8. The transmembrane domain is a structure that connects the extracellular and intracellular domains of the CAR. In this example, the CD28 transmembrane domain is used. The CAR intracellular domain used in this study is an intracellular signaling domain that is capable of transducing the information of CS1CAR binding to human CS1 into the interior of immune effector cells to trigger effector cell functions such as activation, cytokine production, proliferation And cytotoxic activity). The "first-generation" CAR intracellular signaling domain contains only CD3ζ, and the "second-generation" CAR intracellular signaling domain contains a costimulatory molecule (eg, CD28 or 4-1BB4-1BB) and CD3ζ. In order to further improve the design of CARs, many research groups have begun to focus on the development of "third-generation" CARs. A "third generation" CAR contains multiple costimulatory molecules (eg, CD28 and 4-1BB) and CD3ζ. Different researchers have carried out research with different targets and co-stimulation signals, and the comparison results between the second-generation CAR and the third-generation CAR have some differences. Some studies have reported that recombinant T cells expressing "third-generation" CARs have significantly improved antitumor activity, survival cycle, and cytokine ^release12,13 . The results of Wilkie et al. Showed that there is no significant difference in antitumor cytotoxicity between second-generation CAR and third-generation CAR recombinant T cells targeted to MMC1, although T-cells expressing third-generation CAR can secrete a larger amount of IFN-γ ¹⁴ . In this study, a "third-generation" CAR was used, whose intracellular signal transduction domain contained CD28 and 4-1BB costimulatory molecules (or intracellular costimulatory domains) and CD3ζ costimulatory molecules.

(2) The pLVX-EF1α-IRES-Puro vector (purchased from Miaoling Plasmid Platform) contains relevant elements required for lentivirus production, as well as elements capable of increasing virus titer and increasing transgene expression. For example, WPRE can promote RNA processing events and enhance the nuclear export of viral RNA, leading to increased viral titers produced by packaging cells; Rev response elements (RRE) enhance the transport of unspliced viral RNA from the nucleus, further increasing viral titers; The central polypurine / central termination sequence element (cPPT / CTS) generates a central DNA flap, thereby increasing nuclear import of the viral genome during target cell infection, resulting in improved vector integration and more efficient transduction. pLVX-EF1α-IRES-Puro contains restriction endonuclease sites and multiple cloning sites (MCS), so that researchers can subclon the DNA sequence of interest into this vector as needed. MCS is a short DNA sequence containing multiple (up to 20) restriction sites, and is the standard configuration sequence of vector plasmids commonly used in genetic engineering. In MCS, each restriction site is usually unique, that is, they appear only once in a particular vector plasmid, and the restriction sites of different enzymes may overlap.

1.2 Materials and methods

(1) Design the fusion gene fragments in the sequence of the following coding genes: CD8α signal peptide (nucleic acid sequence is SEQ ID NO: 1; amino acid sequence is SEQ ID NO: 2), BCMA scFv (nucleic acid sequence is SEQ ID NO: 11; amino acid The sequence is SEQ ID NO: 12), CD8 Hinger (nucleic acid sequence is SEQ ID NO: 3; amino acid sequence is SEQ ID NO: 4), CD28 transmembrane region and CD28 intracellular stimulation domain (nucleic acid sequence is SEQ ID NO: 5; amino acid sequence is SEQ ID NO: 6), 4-1BB intracellular co-stimulation domain (nucleic acid sequence is SEQ ID NO: 7; amino acid sequence is SEQ ID NO: 8), and CD3ζ intracellular signal domain (nucleic acid sequence is SEQ ID NO: 9; the amino acid sequence is SEQ ID NO: 10), and EcoRI and MluI restriction enzyme restriction sites are designed at both ends. The fusion gene fragment was gene synthesized by Nanjing Kingsray Biotechnology Company. The above BCMA scFv consists of a heavy chain (the nucleic acid sequence is SEQ ID NO: 13; the amino acid sequence is SEQ ID NO: 14) and the light chain (the nucleic acid sequence is SEQ ID NO: 15; the amino acid sequence is SEQ ID NO: 16) through a linker peptide (The nucleic acid sequence is SEQ ID NO: 17; the amino acid sequence is SEQ ID NO: 18). The synthetic gene fragment was subcloned into the EcoRI and MluI restriction enzyme restriction sites of the pLVX-EF1α-IRES-Puro vector, and the product was transformed into E. coli DH5α competent cells (purchased from Takara). (Ampicillin, Amp) screened for positive colonies. Pick the positive colonies and send them to Wuhan Tianyi Huiyuan Biotechnology Co., Ltd. to sequence the entire sequence of the plasmid. Inoculate the correct colonies into 250 mL of LB liquid medium containing Amp and shake the bacteria for 16-24 h at 37 ° C and 220 rpm. EndoFree Plasmid Giga Kit (purchased from Qiagen) was used to extract the plasmid from the bacterial solution, and the plasmid was named "pLVX-mock-5.3-CAR" (Fig. 1).

SEQ ID NO: 2 Amino acid sequence of synthetic CD8α signal peptide MALPVTALLLPLALLLHAARP

SEQ ID NO: 4 Amino acid sequence of synthetic CD8 Hinger TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

SEQ ID NO: 8 Amino acid sequence of artificially synthesized 4-1BB intracellular costimulatory domain KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

SEQ ID NO: 17 DNA sequence of a synthetic linker peptide linking light and heavy chains in BCMA scFV GGCAGCACCAGCGGCTCCGGCAAGCCTGGCTCTGGCGAGGGCAGCACAAAGGGA

SEQ ID: NO: 18 Amino acid sequence of a synthetic linker peptide linking light and heavy chains in BCMA scFV GSTSGSGKPGSGEGSTKG

(2) Design fusion gene fragments according to the sequence of the following coding genes: CD8α signal peptide, CS1scFv (nucleic acid sequence is SEQ ID NO: 19; amino acid sequence is SEQ ID NO: 20), CD8 Hinger, CD28 transmembrane region and CD28, 4 The -1BB intrastimulatory domain and the CD3ζ intracellular signaling domain are designed with restriction sites for BamHI and MluI restriction enzymes at each end. The fusion gene fragment was gene synthesized by Nanjing Kingsray Biotechnology Company. The above CS1scFv consists of a heavy chain (the nucleic acid sequence is SEQ ID NO: 21; the amino acid sequence is SEQ ID NO: 22) and the light chain (the nucleic acid sequence is SEQ ID NO: 23; the amino acid sequence is SEQ ID NO: 24) through a connecting peptide ( The nucleic acid sequence is SEQ ID NO: 25; the amino acid sequence is SEQ ID NO: 26). The gene fragment synthesized by the above gene was subcloned into the BamHI and MluI restriction enzyme restriction sites of the pLVX-EF1α-IRES-Puro vector, and the plasmid pLVX-mock-CS1 was obtained by referring to the method for screening colonies and extracting plasmids above. -CAR (Figure 2).

(3) designed like a four Tal siRNA knockdown for expression of CS1, published 4 expression results of each target sequence and siRNA knockdown CS1 in his article ^15, the inventors refer to data in the literature, the third selection SiRNA target sequence (nucleic acid sequence is SEQ ID NO: 29), SH3 shRNA (nucleic acid sequence is SEQ ID NO: 28) was designed for CS1 mRNA, and its knockdown in T cells was verified in Example 3 below CS1 Expressed Function. H1 promoter (nucleic acid sequence is SEQ ID NO: 27) and DNA sequence encoding SH3 shRNA were synthesized by Nanjing Kingsray Biotechnology Company in sequence, and ClaI and SphI restriction endonuclease restriction sites were designed at both ends. . The synthetic DNA fragments were subcloned into the ClaI and SphI restriction enzyme restriction sites of pLVX-mock-5.3-CAR and pLVX-mock-CS1-CAR, respectively. Plasmids pLVX-SH3-5.3-CAR (Figure 3) and pLVX-SH3-CS1-CAR (Figure 4).

SEQ ID: NO: 25 DNA sequence of a synthetic linker peptide linking heavy and light chains in CS1scFv

GGCGGCGGCGGCAGCGGCGGGGGCGGCAGCGGCGGCGGCGGCAGC

SEQ ID: NO: 26 Amino acid sequence of a synthetic linker peptide linking heavy and light chains in CS1scFv GGGGSGGGGSGGGGS

SEQ ID NO: 29 Target DNA sequence of synthetic SH3 shRNA GTCGGGAAACTCCTAACATAT

(4) Transform the pCMV-VSV-G helper plasmid (purchased from Miaoling Plasmid Platform) and psPAX2 helper plasmid (purchased from Miaoling Plasmid Platform) into E. coli DH5α competent cells. Refer to the above method for screening colonies and extracting plasmid To obtain the above-mentioned plasmids that meet the requirements for lentivirus preparation and have the correct sequence.

2. Preparation of chimeric antigen receptor lentivirus

2.1 Principle Overview

Lentivirus is a gene therapy vector developed based on human immunodeficiency virus (HIV). It has the ability to infect dividing cells and non-dividing cells and can be expressed in cells for a long period of time. The lentivirus used in this study is a "suicide" virus, that is, the virus will no longer infect other cells after infecting the target cell, nor will it use the host cell to generate new virus particles. Some genes in the lentivirus have been deleted and replaced by genes for exogenous purposes, which are pseudotyped viruses. The pCMV-VSV-G vector contains the VSV-G gene and provides the envelope protein required for viral packaging. The psPAX2 vector contains the gag gene of the HIV virus, which encodes the main structural protein of the virus; the pol gene, which encodes a virus-specific enzyme; the rev gene, which encodes a regulator that regulates the expression of the gag and pol genes.

293T cells are human embryonic kidney epithelial cell lines derived from 293 cells and expressing the SV40 large T antigen. They are widely used for transient transfection to overexpress various target proteins, or for packaging viruses. After the pLVX vector, pCMV-VSV-G vector, and psPAX2 vector were introduced into 293T cells using transfection reagents such as polyethyleneimine (PEI), the lentiviral backbone carried on the pLVX vector was transcribed into viral RNA, and combined with psPAX2 and pCMV- Proteins translated from lentivirus-related genes carried on VSV-G are assembled into lentiviruses.

2.2 Material method

Take the plasmid extracted in the previous step, and separate each vector (pLVX-mock-5.3-CAR, pLVX-mock-CS1-CAR, pLVX-SH3-5.3-CAR, pLVX-SH3-CS1-CAR) with pCMV-VSV- G helper plasmid and psPAX2 helper plasmid were mixed at a ratio of 6: 2: 3 and co-transfected 293T cells. After the 293T cells produced virus for 72 hours, the cell culture medium containing the virus was collected and centrifuged at 4 ° C and 3000 g for 5 min. The supernatant was filtered through a 0.45 μm filter, and the filtrate was centrifuged at 30000 g at 4 ° C. for 2.5 h. Discard the supernatant and resuspend the pellet in pre-chilled PBS to obtain the corresponding LV-mock-5.3, LV-SH3-5.3, LV-mock-CS1, LV-SH3-CS1 lentiviral concentrates, and store at -80 ° C. spare.

3. Lentiviral activity titer detection

3.1 Overview

FITC-conjugated AffiniPure Anti-Mouse IgG (H + L) (purchased from Jackson ImmunoResearch) is labeled with fluorescein, and it can bind to single-chain antibodies in CAR. The fluorescence signal detected by flow cytometry can indirectly reflect the expression of CAR encoded by lentiviral vector in 293T cells, identify positive cells successfully infected by lentivirus, and calculate lentivirus based on the proportion of positive cells Active titer data.

3.2 Materials and methods

Into a 6-well plate, 5.0 × 10 ⁵ cells / well 293T cells were inserted, and 0.1 μL, 0.5 μL, and 1 μL of lentivirus concentrated solution were added to each well, and a negative control was added without lentivirus concentrated solution. The 6-well plate was cultured in an incubator containing 5% CO ₂ at 37 ° C. Three days later, 293T cells were collected with Versene solution (purchased from Gibco), stained with FITC-conjugated AffiniPure Goat Anti-Mouse IgG (H + L), and detected by flow cytometry (instrument model: Beckman Cytoflex). Version 7.6.3) Analyze the proportion of CAR-positive 293T cells. According to the formula: (lentivirus active titer (TU / mL) = 293T cell number × CAR positive 293T cell ratio (%) / lentivirus addition amount (uL) × 1000), calculate the above LV-mock-5.3, LV-SH3 -5.3, active titers of LV-mock-CS1, LV-SH3-CS1 lentivirus concentrate.

4. Preparation and activation of T lymphocytes

100 mL of healthy human fresh blood was taken, and peripheral blood mononuclear cells were separated by lymphocyte separation solution (purchased from Tianjin Yeyang Biological Products Technology Co., Ltd.) and density gradient centrifugation. CD3 MicroBeads, human (purchased from Miltenyi Biotech) were used to magnetically label the cells, and T lymphocytes were isolated and purified. Preparation of T cell culture medium: To 1 L of CTS ^TM OpTmizer ^TM T Cell Expansion Basal Medium (purchased from Gibco) was added 26 ml of CTS ^TM OpTmizer ^TM T-cell Expansion supplement (purchased from Gibco), and L-glutamine was added to a final concentration of 2 mM (Purchased from Gibco), and IL-2 (purchased from Shuanglu Pharmaceutical) was added at a final concentration of 500 IU / ml. Cells and magnetic beads Dynabeads ^™ HumanT-Activator CD3 / CD28 (purchased from Gibco) were added to the T cell culture medium at a ratio of 1: 1. Magnetic beads Dynabeads ^™ HumanT-Activator CD3 / CD28 is used to stimulate T cells with an activation density of about 1 × 10 ⁶ / ml. T cells were cultured at 37 ° C in a 5% CO ₂ incubator overnight to obtain activated T cells.

5. Lentivirus transduced T lymphocytes

CAR T cells were obtained by transducing the activated T cells described above using a lentivirus carrying a CAR gene. After 18 hours of T cell activation, LV-mock-5.3, LV-SH3-5.3, LV-mock-CS1, and LV-SH3-CS1 lentiviral concentrates were added at MOI = 5 to infect 5 × ^{10 6} T lymphocytes. The cells were incubated in a 37 ° C, 5% CO ₂ incubator overnight. After 24 hours, the lentiviral-transduced mock-5.3, SH3-5.3, mock-CS1, and SH3-CS1 cells were replaced with new T cell culture medium and cultured, and observed and counted daily. When the cell concentration reached 2 × 10 ⁶ cells / mL, add fresh medium and continue to expand the culture.

Example 2 Preparation of target cells (B-K562 and C-K562)

1. Preparation of an antigen overexpression vector

1.1 Overview

(1) puro is a puromycin resistance gene. Puromycin is an aminoglycoside antibiotic produced by the fermentation and metabolism of Streptomyces alboniger, which kills Gram-positive bacteria, various animal and insect cells by inhibiting protein synthesis. Puromycin is commonly used to screen and maintain stable transfection of mammals containing puro resistance genes. The "puro" in the vector name means that the vector contains a puro resistance gene. In this example, the characteristics of puro resistance gene (nucleic acid sequence is SEQ ID NO: 37 and amino acid sequence is SEQ ID NO: 38) and puro are used to select K562 that stably expresses BCMA or CS1.

(2) T2A: 2A peptide is a type of amino acid sequence. When the ribosome translates its mRNA, it will trigger ribosome jumping, which will cause the suspension of the peptide being translated and start the synthesis of a new peptide from the next amino acid codon. Thus, two proteins can be produced at the same time when one mRNA is translated, which is often used to construct a polycistronic vector. T2A is a 2A element from the Thalassima virus. In this example, the inventors used T2A to link BCMA and puromycin resistance genes, co-express BCMA and puromycin resistance genes in K562, and then screened through puromycin to obtain K562, which stably expresses BCMA protein, below Called B-K562.

(3) IRES (internal ribosome entry site, internal ribosome entry site) can recruit ribosomes to translate mRNA, fuse IRES with foreign cDNA, and find that IRES can initiate translation independently. Because of this characteristic, IRES has been widely used in the construction of binary expression vectors. In this example, IRES (nucleotide sequence: SEQ ID NO: 36) was used to co-express CS1 and puromycin resistance genes, and then screened through puromycin to obtain K562, which is hereinafter referred to as C-K562.

1.2 Material method

(1) Design the fusion gene fragments in the following order: CD8α signal peptide (nucleic acid sequence is SEQ ID NO: 1; amino acid sequence is SEQ ID NO: 2), BCMA (nucleic acid sequence is SEQ ID NO: 30; amino acid sequence is SEQ ID NO: 31), T2A (nucleic acid sequence is SEQ ID NO: 34; amino acid sequence is SEQ ID NO: 35), puroR (nucleic acid sequence is SEQ ID NO: 37; amino acid sequence is SEQ ID NO: 38), and both ends Designed with XbaI and MluI restriction enzyme sites. The fusion gene fragment was synthesized by Nanjing Kingsley Biotechnology Company, and then subcloned into the XbaI and MluI restriction enzyme sites of the pLVX-EF1α-IRES-Puro vector. The colonies were screened and the plasmid was extracted in reference to Example 1. Method to obtain plasmid pLVX-BCMA-T2A-puro (Figure 5).

(2) Gene fragment CS1 (nucleic acid sequence is SEQ ID NO: 32; amino acid sequence is SEQ ID NO: 33) EcoRI and BamHI restriction endonuclease sites are designed at both ends, and synthesized by Nanjing Kingsray Biotechnology Co., Ltd. The CS1 gene fragment was subcloned into the EcoRI and BamHI restriction endonuclease sites of the pLVX-EF1α-IRES-Puro vector. Refer to the method for screening colonies and extracting plasmids in Example 1 to obtain the plasmid pLVX-CS1-IRES -puro (Figure 6).

SEQ ID NO: 34 DNA sequence of artificially synthesized T2A GAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCCGGCCCT

SEQ ID NO: 35 Amino acid sequence of synthetic T2A EGRGSLLTCGDVEENPGP

2. Preparation of antigen overexpression lentivirus

2.1 Material method

Two expression vectors (pLVX-BCMA-T2A-puro and pLVX-CS1-IRES-puro) were mixed with pCMV-VSV-G helper plasmid and psPAX2 helper plasmid at a ratio of 6: 2: 3, respectively, and co-transfected 293T cells. 72 hours after transfection, the cell culture supernatant containing the virus was collected. Centrifuge at 3000 g for 5 min at 4 ° C. After the supernatant was filtered through a 0.45 μm filter, the virus solution was centrifuged at 30,000 g at 4 ° C. for 2.5 h. The supernatant was discarded, and the pellet was resuspended and dissolved in pre-chilled PBS to obtain a LV-BCMA or LV-CS1 lentivirus concentrate, which was stored at -80 ° C until use.

3. Lentiviral active titer

3.1 Overview

Human CRACC / SLAMF7 APC-conjugated Antibody and APC anti-human CD269 (BCMA) Antibody are labeled with fluorescein, which can bind to CS1 protein and BCMA protein, respectively. The fluorescence signal detected by flow cytometry can indirectly reflect the expression of BCMA or CS1 in 293T cells, identify positive cells successfully infected by lentivirus, and calculate the active titer of lentivirus based on the proportion of positive cells .

3.2 Material method

Add 5.0 × 10 ⁵ cells / well 293T cells to a 6-well plate. Add 0.1 μL, 0.5 μL, and 1 μL of lentivirus concentrate (LV-BCMA or LV-CS1) to each well, and set one without lentivirus. Negative control. The cells were cultured in a 37 ° C incubator containing 5% CO ₂ . Three days later, 293T cells were collected with Versene solution, and the proportion of CS1 or BCMA-positive 293T cells was detected using Human CRACC / SLAMF7 APC-conjugated Antibody (purchased from R & D Systems) and APC anti-human CD269 (BCMA) Antibody (purchased from Biolegend). The active titer of the LV-BCMA or LV-CS1 lentivirus concentrated solution was calculated by referring to the formula in Example 1.

4. Transduction and screening of target cells

4.1 Material method

K562 cells (purchased from the Chinese Academy of Sciences Type Culture Collection Committee Cell Bank) were seeded into 24-well plates at ⁶ × 10 ⁴ cells / well, and K562 cells were cultured using RPMI 1640 medium (purchased from GIBCO). After the cells were cultured in a 37 ° C incubator containing 5% CO _{2 for} 24 hours, the above lentivirus concentrates were added at MOI = 1. 72 hours after the virus infected the cells, the cells were screened with RPMI 1640 medium containing 2 μg / mL puromycin (purchased from GIBCO) to obtain stable transfected cells B-K562 and C-K562. B-K562 and C-K562 cells were stained with Human CRACC / SLAMF7 APC-conjugated Antibody and APC anti-human CD269 (BCMA) Antibody, respectively, detected by flow cytometry, and analyzed by FlowJo software.

4.2 Results

The expression rate of BCMA on the surface of B-K562 cells was 97.4% (Figure 7), and the expression rate of CS1 on the surface of C-K562 cells was 97.4% (Figure 8). Thus, it was shown that the antigen-overexpressing cell lines B-K562 and C-K562 have been successfully prepared.

Example 3 Analysis of Sh3 shRNA knockdown effect

1 Overview

shRNAs (short hairpin RNAs), short hairpin RNAs, are non-coding small RNA molecules designed to form hairpin structures. The inventors introduced a short hairpin RNA targeting CS1 mRNA into a lentiviral expression vector encoding CS1CAR. After the lentivirus infects T cells and integrates the CS1-CAR and shRNA coding genes into the T cell genome, the shRNA coding genes are transcribed and processed to form siRNA. It degrades CS1 mRNA through the RNA interference pathway, thereby inhibiting or down-regulating the expression of CS1 gene in T cells.

2. Materials and methods

Take the lentiviral transduced T cells (mock-CS1, SH3-CS1, mock-5.3, and SH3-5.3) obtained in Example 1 and the control T cells (Table 1) that were not transduced by lentivirus, using FITC- conjugated AffiniPure Goat Anti-Mouse IgG (H + L) and Human CRACC / SLAMF7 APC-conjugated Antibody staining, and the expression of CAR and CS1 on the cell surface were detected by flow cytometry.

Table 1 SH3 knockdown effect experimental design

Cell name Target of CAR Whether CS1 expression is knocked down T - no Mock-5.3 BCMA no SH3-5.3 BCMA Yes Mock-CS1 CS1 no SH3-CS1 CS1 Yes

3. Results

The expression rates of CS1 on the surface of control T cells not transduced with lentivirus and mock-5.3, SH3-5.3, mock-CS1, and SH3-CS1 transduced by lentivirus were 56%, 55.6%, 27.6%, 12.7, respectively. % And 4.21%.

4. Discussion

The expression rate of CS1 on SH3-5.3 cell surface was lower than mock-5.3, indicating that SH3 shRNA can effectively knock down the expression of CS1 on T cell surface. The expression rate of CS1 on the surface of mock-CS1 cells was lower than that of control T cells not transduced by lentivirus and mock-5.3 and SH3-5.3 transduced by lentivirus, probably because CS1-CAR T cells killed CS1 positive in the population The number of CS1 positive cells is reduced. Compared with mock-CS1 cells, the expression rate of CS1 on the surface of SH3-CS1 cells decreased, which also showed that SH3 shRNA can effectively knock down the expression of CS1 on the surface of T cells (Figure 9).

Example 4 In vitro function of CS1 knockdown CAR T cells

1.CAR T cell expansion

1.1 Overview

In a suitable environment, T cells can be expanded in vitro by stimulation with CD3 / CD28 magnetic beads. Through continuous cell counting, the rate of T cell expansion in vitro can be observed, reflecting the T cell expansion in vitro.

1.2 Material method

SH3-CS1, SH3-5.3, mock-CS1, mock-5.3, and control T cells (T) without lentiviral transduction were cultured according to the conditions in Example 1, and the number of cells was recorded every 2 to 3 days. The time point of lentiviral transduction is recorded as 0 days, and the number of cells from 0 to 10 days is recorded.

1.3 Results

The initial number of cells in the mock-CS1 group was 7 × 10 ⁶ , and the remaining groups were 5 × 10 ^6. According to the culture conditions in Example 1, the cells were cultured for 10 days in vitro, except for the mock-CS1 group, which was expanded less than 10 times. The group normally expanded 25 to 35 times.

1.4 Discussion

It can be seen from the amplification curve of the mock-CS1 group (Figure 10) that due to the expression of CS1 on the surface of T cells, suicide will occur during the CS1-CAR T culture, which makes it difficult for the cells to expand. However, after knocking down CS1 expression in CAR T cells, the cells can expand normally as well as control T cells (T) and anti-BCMA-CAR T (mock-5.3 and SH3-5.3) without lentiviral transduction. Moreover, the amplification curves of mock-5.3 and SH3-5.3 showed that knocking down CS1 had no significant effect on cell proliferation.

2. Composition of CAR T cell subsets after CS1 knockdown

2.1 Overview

T cell)、活化的T细胞(activated T cell)和记忆性T细胞(memory T cell)。CD4+T细胞能促进B细胞、T细胞和其它免疫细胞的增殖与分化，协调免疫细胞间的相互作用。CD8+T细胞通过直接杀伤作用，主要负责对靶细胞的清除。记忆性T细胞来源于效应T细胞，还可由初始T细胞直接转化而来。记忆性T细胞又可分为T _CM(中央记忆T细 胞)和T _EM(效应记忆T细胞)。中央记忆T细胞在接受抗原再次刺激后能快速产生效应并上调CD40L表达，并且能大量分泌IL-2和多次增殖，进一步分化为效应T细胞，可长时间维持免疫记忆。效应记忆T细胞受抗原刺激后立即产生免疫效应，发挥细胞毒作用并分泌效应分子，但分泌IL-2的能力和增殖能力低下。因此T _EM维持免疫记忆时间较短，主要在免疫防御的第一线起作用。 T cells are an inhomogeneous cell population and can be classified in many ways. According to the cell surface differentiation antigen (CD), it can be divided into two major subgroups of CD4 + and CD8 +; according to the different response to the antigen, it is divided into initial T cells (

T cells), activated T cells, and memory T cells. CD4 + T cells can promote the proliferation and differentiation of B cells, T cells and other immune cells, and coordinate the interaction between immune cells. CD8 + T cells are mainly responsible for the elimination of target cells through direct killing. Memory T cells are derived from effector T cells and can also be directly transformed from the original T cells. Memory T cells can be divided into T _CM (central memory T cells) and T _EM (effect memory T cells). After being stimulated by the antigen again, the central memory T cells can quickly produce effects and up-regulate the expression of CD40L, and can secrete a large amount of IL-2 and proliferate multiple times, further differentiate into effector T cells, and maintain immune memory for a long time. Effector memory T cells have an immune effect immediately after being stimulated by the antigen, exert cytotoxic effects and secrete effector molecules, but the ability to secrete IL-2 and proliferate is low. Therefore, T _EM maintains immune memory for a short time, and mainly plays a role in the front line of immune defense.

Different types of T cells can be distinguished according to different surface antigens. For example, the initial T cells are characterized by CD45RA + CCR7 + CD62L ^high , T _CM is characterized by CD45RA-CCR7 + CD62L ^high , and T _EM is characterized by CD45RA- CCR7 + CD62L ^low . Based on these characteristics of T cell subgroups, researchers can use the corresponding flow cytometry antibodies to analyze the proportion of each subgroup in T cells.

2.2 Materials and methods

T cells can be divided into the following four subgroups based on the expression of CD45RA and CCR7: initial T cells, T _CM cells, T _EM cells, and T _EMRA cells. The initial T cells were CD45RA + CCR7 +, T _CM cells were CD45RA-CCR7 +, T _EM cells were CD45RA-CCR7-, and T _EMRA cells were CD45RA + CCR7-. Take lentivirus-transduced T cells (mock-CS1, SH3-CS1, mock-5.3, and SH3-5.3) and control T cells (T) that were not transduced by lentivirus in Example 1, and use FITC-conjugated AffiniPure Goat Anti-Mouse IgG (H + L), Pacific Blue ^TM anti-human CD8 Antibody (purchased from Biolegend), FITC Mouse Anti-Human CD45RA (purchased from BD Biosciences), PE anti-human CD197 (CCR7) Antibody (purchased from RND) ) Stain the cell surface and analyze the results with FlowJo software after detection by flow cytometry.

2.3 Results

Multicolor flow cytometry was used to analyze the CD8 + ratio of T cells in each group, as well as the ratio of initial T cells, T _CM cells, and T _EM cells.

The proportion of CD8 + cells without control lentivirus transduction was 59.3%, among which the initial T cell proportion was 20.4%, the T _CM cell proportion was 24.0%, and the T _EM cell proportion was 55.6%. The proportion of CD8 + cells in the Mock-5.3 group was 65.2%, of which the initial T cell ratio was 10.7%, the T _CM cell ratio was 17.9%, and the T _EM cell ratio was 71.4%. The proportion of CD8 + cells in the SH3-5.3 group was 66.9%, of which the initial T cell ratio was 18.2%, the T _CM cell ratio was 18.1%, and the T _EM cell ratio was 63.6%. The proportion of CD8 + cells in the Mock-CS1 group was 64.7%, of which the initial T cell ratio was 2.7%, the T _CM cell ratio was 15.0%, and the T _EM cell ratio was 82.3%. The proportion of CD8 + cells in the SH3-CS1 group was 65.5%, of which the initial T cell ratio was 8.7%, the T _CM cell ratio was 14.5%, and the T _EM cell ratio was 81.5% (Table 2).

Table 2 Composition of cell subpopulations in each experimental group

2.4 Discussion

The proportion of T _EM cells in mock-5.3, SH3-5.3, mock-CS1, and SH3-CS1 cells was higher than that of control T cells that were not transduced by lentivirus, and the levels were increased in mock-CS1 and SH3-CS1 More obviously, the inventors speculate that such a result was caused by CS1-CAR T cell suicide (Figure 11).

3. CAR T cells in vitro killing experiment

3.1 Overview

(1) In this example, a calcein-AM ester (calcein-AM) release method was used to measure the tumor killing effect of CAR T cells. Calcein-AM is a cell staining reagent that can fluorescently label cells. Methyl acetate is very lipophilic and can penetrate cell membranes. In living cells, Calcein-AM is cleaved by intracellular esterases to form calcein, which remains in the cells. Calcein can emit green fluorescence. When target cells are lysed, calcein is released into the supernatant. The tumor killing effect of CAR T cells was measured by detecting the fluorescence intensity of calcein in the supernatant ¹⁶ .

(2) The MM1S cell line expresses CS1 and BCMA, and is a positive target cell of CS1-CAR T cells and BCMA-CAR T cells. B-K562 is a K562 cell line that overexpresses BCMA. C-K562 is a K562 cell line that overexpresses CS1. K562 cell line expresses neither CS1 nor BCMA.

3.2 Material method

The target cells MM1S (purchased from the Chinese Academy of Sciences Type Culture Collection Committee Cell Bank) and the target cells (B-K56, C-K562, and K562) prepared in Example 2 were taken. After counting, it was washed twice with 5% FBS (available from GIBCO) in PBS (available from GIBCO). The cell density was adjusted to 1 × 10 ⁶ / ml. Add 10 μl Calcein-AM (purchased from Aladdin) solution to each 1ml of cell suspension, mix well, and incubate at 37 ° C in the dark for 30min. The effector cells (mock-5.3, SH3-5.3, mock-CS1, and SH3-CS1) and control T cells prepared in Example 1 were counted, and then an appropriate amount of cells were taken and washed twice with 5% FBS-containing PBS. Adjust CAR + T cell density to 2.5 × 10 ⁶ / ml according to the measured transfection efficiency. The effector cells and the target cells were co-cultured in a 96-well plate at a ratio of the number of effector cells to the number of target cells of 50: 1, 10: 1, and 2: 1, respectively. The target cell and PBS co-culture group was used as the spontaneous release group, and the target cell and lysate (20 mM sodium borate, 0.1% Triton X-100, pH 9.0) was used as the maximum release group. After 3 hours of incubation, the supernatant was transferred to another 96-well plate by centrifugation. Set the parameters of the microplate reader (type: Perkin Elmer VictorX3), the excitation light wavelength is 485/20, and the emission light wavelength is 530/25. The microplate reader reads the fluorescence value F of each well, and calculates the tumor killing efficiency according to the following formula:% lysis = (F experimental well-F spontaneous release) / (F maximum release-F spontaneous release) × 100%.

3.3 Results

At three ratios (50: 1, 10: 1, 2: 1), the killing efficiency of mock-5.3 cells on MM1S cells was 97.17%, 92.43%, and 66.89%, and the killing efficiency of K562 cells was 21.16%. , 16.50%, 7.57%, the tumor-killing efficiency of B-K562 cells was 100.00%, 87.40%, 36.16%, and the tumor-killing efficiency of C-K562 cells were 12.26%, 7.80%, and 4.57% (Table 3). .

The tumor killing efficiency of SH3-5.3 cells on MM1S cells was 79.89%, 60.30%, and 40.5%, and the killing efficiency of K562 cells was 4.55%, 7.14%, and 3.67%, respectively. The tumor-killing efficiency was 92.52%, 68.68%, and 24.64%, and the tumor-killing efficiency on C-K562 cells was 1.18%, 4.74%, and 0.62%, respectively.

The killing efficiency of mock-CS1 on MM1S cells was 85.42%, 66.84%, and 52.77%, and the killing efficiency of K562 cells was 28.84%, 30.23%, and 15.74%, respectively. The tumor efficiencies were 7.31%, 2.70%, 1.78%, and the tumor-killing efficiencies of C-K562 cells were 99.07%, 79.47%, and 36.38%, respectively.

The killing efficiency of SH3-CS1 on MM1S cells was 90.58%, 75.65%, and 42.52%, and the killing efficiency of K562 cells was 21.39%, 17.35%, and 5.84%, respectively. The tumor efficiencies were 7.36%, 8.11%, and 0.71%, respectively. The tumor-killing efficiencies of C-K562 cells were 100.00%, 76.00%, and 24.69%, respectively.

Untransduced lentivirus control T cells had no significant killing effect on all four cell lines. The tumor killing efficiency of untransduced lentiviral control T cells against MM1S cells was 29.98%, 13.90%, and 2.87%, and the killing efficiency of K562 cells was 12.94%, 8.21%, and 4.49%. The tumor-killing efficiency of B-K562 cells was 10.20%, 7.37%, and 4.99%, and the tumor-killing efficiency of C-K562 cells was 9.65%, 5.07%, and 1.23%, respectively.

Table 3 In vitro killing results of CAR T cells

3.4 Discussion

Mock-5.3 cells and SH3-5.3 cells can specifically kill BCMA-positive target cells (MM1S and B-K562), but compared with mock-5.3 cells, the killing effect of SH3-5.3 cells on MM1S is slightly reduced. It may be that knocking down CS1 has a certain effect on the killing function of T cells, but further experiments need to be confirmed. Mock-CS1 cells and SH3-CS1 cells can specifically kill CS1-positive target cells (MM1S and C-K562), and no weakening of the tumor-killing effect of SH3-CS1 is observed, which may be through knocking down CS1-CAR of T1 Reduced cell suicide, offsetting the negative effect of knockdown of CS1 on T cell killing function ().

4. Expression of CD107a

4.1 Overview

CD8 + T cell cytoplasm contains a high concentration of cytotoxic particles in the form of vesicles, which contain perforin and granzymes. Lysosomal-associated membrane protein-1 (LAMP-1 or CD107a) is a highly glycosylated protein that is distributed on the surface of these vesicles. When CD8 + T kills target cells, the toxic particles will reach the cytoplasmic surface of the cell membrane and fuse with the cell membrane, causing the content of the particles to be released, eventually leading to the death of the target ^cells17,18 . With the occurrence of degranulation, CD107a is transported to the surface of cell membranes, so the expression of CD107a on the surface of T cell membranes can reflect the level of degranulation of T cells, and the detection of CD107a on the surface of T cells can reflect the effect of T cell lysis target cells.

4.2 Materials and methods

CAR T cells (mock-5.3, SH3-5.3, mock-CS1, and SH3-CS1) prepared in Example 1, control T cells not transduced with lentivirus, and various target cells were taken. The cells were counted and all cell densities were adjusted to 2 × 10 ⁶ / ml. 250 μl of each effector cell and 250 μl of different target cells were co-cultured in a 24-well plate (the number of effector cells and target cells in each well was 5 × 10 ⁵ ). Set up effector cells without target cells. For co-cultivation, CTS ^™ OpTmizer ^™ medium without IL-2 was used. Anti-CD107a flow antibody (purchased from BD Biosciences) was added at 20 μl / ml and incubated in a 37 ° C incubator. After 1 h, add monesin (purchased from BD Biosciences), 3 μl / well, and continue incubation for 3 h with PE / Cy7anti-human CD3 Antibody (purchased from Biolegend), Pacific Blue ^TM anti-human CD8 Antibody (purchased from Biolegend), PE Mouse Anti -Human CD107a (purchased from BD Biosciences) stained the cells and analyzed the level of CD107a on the surface of CD3 + CD8 + cell membranes.

4.3 Results

After co-culture of Mock-5.3 cells with K562, MM1S, B-K562 and C-K562 cells, CD107a expression was 12.3%, 49.6%, 57.8% and 15.4%, respectively. After co-culture of SH3-5.3 cells with K562, MM1S, B-K562 and C-K562 cells, the expression of CD107a was 10.7%, 24.1%, 32.4% and 10.5%, respectively. After mock-CS1 cells were co-cultured with K562, MM1S, B-K562, and C-K562 cells, CD107a expression was 12.1%, 32.0%, 11.7%, and 34.2%, respectively. After co-culture of SH3-CS1 cells with K562, MM1S, B-K562, and C-K562 cells, CD107a expression was 13.2%, 35.8%, 11.3%, and 46.4%, respectively.

After co-culture of control T cells with K562, MM1S, B-K562, and C-K562, CD107a expression was 12.3%, 49.6%, 57.8%, and 15.4%, respectively.

4.4 Discussion

Mock-5.3 cells and SH3-5.3 cells were specifically degranulated when co-cultured with BCMA-positive target cells (MM1S and B-K562), but compared with mock-5.3 cells, degranulation levels of SH3-5.3 cells were weakened. Mock-CS1 and SH3-CS1 were specifically degranulated when co-cultured with CS1-positive target cells (MM1S and C-K562), and no decrease in SH3-CS1 degranulation was observed. It is still possible to knock down CS1 CS1-CAR T cells reduced suicide, offsetting their adverse effects (Figure 13).

5. Expression of immune checkpoint molecules

5.1 Overview

Immune checkpoints can be simply defined as signal molecules that inhibit the activation of T cells and prevent T cells from participating in immune responses. T cells up-regulate the expression of inhibitory receptors such as PD-1 and CTLA-4 when activated or up-regulate ligands on APC through pro-inflammatory cytokines (such as PD-1 for PDL1 and PD-L2) to participate in the inhibition of TCR signals Conduction. These negative feedback mechanisms prevent excessive T cell activation and minimize pro-inflammatory damage to the host.

5.2 Material method

Take CAR T cells (mock-5.3, SH3-5.3, mock-CS1, and SH3-CS1) prepared in Example 1 and control T cells that have not been transduced with virus, and use PE anti-human CD279 (PD-1) Antibody (Purchased from Biolegend), PE anti-human CD152 (CTLA-4) Antibody (purchased from Biolegend), APC anti-human CD366 (Tim-3) Antibody (purchased from Biolegend), APC anti-human CD223 (LAG-3) Antibody (purchased from Biolegend), Pacific Blue ^™ anti-human CD8 Antibody, and FITC-conjugated AffiniPure Goat Anti-Mouse IgG (H + L) staining, measurement by flow cytometry, and data analysis.

5.3 Results

The PD-1 and CTLA-4 expression of CAR + cells in Mock-5.3 cells were 51.3% and 64.5%, respectively, and the average fluorescence intensity (MFI) of CAR + CD8 + cells was 80429 and 25508, respectively. The expression of PD-1 and CTLA-4 of CAR + cells in SH3-5.3 cells was 58.0% and 55.9%, and the mean fluorescence intensity (MFI) of CAR + CD8 + cells was 87907 and 26866, respectively. In Mock-CS1 cells, the expression of PD-1 and CTLA-4 of CAR + cells were 42.3% and 66.4%, and the average fluorescence intensity (MFI) of CAR + CD8 + cells' TIM-3 was 111750 and 59086, respectively. The expression of PD-1 and CTLA-4 of CAR + cells in SH3-CS1 cells was 31.8% and 39.2%, and the mean fluorescence intensity (MFI) of CAR + CD8 + cells was 99556 and 37902, respectively.

PD-1 expression and CTLA-4 expression of untransduced lentiviral control T cells were 25.8% and 50.4%, respectively, and the average fluorescence intensity (MFI) of TIM-3 of CD8 + cells was 53383 and 17100, respectively.

5.3 Discussion

From the above results, it can be seen that the expression of PD-1 or CTLA-4 in SH3-CS1 group was lower than that in mock-CS1 group. Since there was no significant difference in the positive rates of TIM-3 and LAG3 on the surface of CAR + CD8 + T cells in mock-CS1 and SH3-CS1 groups, the inventors further compared the MFI of the two and found that the MFI of SH3-CS1 and TIM3 were low In mock-CS1 group. Immunity checkpoints such as PD-1, CTLA-4, TIM-3, and LAG3 are signs of T cell depletion, and the expression of the above four markers in the SH3-CS1 group is lower, suggesting that knockdown of the CAR of CS1 due to reduced suicide in T cells Resulting in a lower degree of depletion, showing that CS1-CAR T cells that knock down CS1 may have better therapeutic potential (Figure 14).

The present invention has been described in detail through specific embodiments. It should be noted that, for those skilled in the art, without departing from the spirit and scope of the present invention, several changes can be made to these embodiments or implementations. They should also It is considered to be within the protection scope of the present invention.

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Claims (31)

A T cell expressing a chimeric antigen receptor, the chimeric antigen receptor includes an extracellular domain, the extracellular domain recognizes a target antigen on a target cell surface, thereby mediating the T cell against the Killing of target cells; the T cells themselves also express the target antigen, and in order to prevent the T cells from killing each other, the expression of the target antigen by the T cells is down-regulated. The T cell of claim 1, wherein the extracellular domain of the chimeric antigen receptor comprises a single chain antibody derived from an antibody against the target antigen. The T cell of claim 1, wherein the target cell is a tumor cell. The T cell according to claim 3, wherein the tumor cell is a multiple myeloma cell. The T cell of claim 1, wherein the target antigen is CS1. The T cell according to any one of claims 1 to 5, wherein the T cell down-regulates expression of the target antigen by expressing siRNA. The T cell of claim 6, wherein the siRNA is produced from a shRNA expressed by the T cell. The T cell according to claim 7, wherein the target nucleic acid sequence of the shRNA comprises a nucleotide sequence as shown in SEQ ID NO: 29. The T cell according to claim 7, wherein the coding sequence of the shRNA comprises a nucleotide sequence as shown in SEQ ID NO: 28. The T cell according to claim 5, wherein the single-chain antibody has an amino acid sequence as shown in SEQ ID NO: 20. The T cell according to claim 2, wherein the amino acid sequence of the chimeric antigen receptor comprises a CD8α signal peptide, the single chain antibody, a CD8 hinge region, a CD28 transmembrane region, CD28 and 4-1BB intracellular co-stimulatory domain and CD3ζ intracellular signaling domain. The T cell according to claim 7, transformed with an expression vector including the coding sequence of the chimeric antigen receptor and an expression vector including the coding sequence of the shRNA, or by a The coding sequence and the expression vector of the shRNA coding sequence are transformed. An expression vector for expression in T cells includes a coding sequence of a chimeric antigen receptor and a shRNA coding sequence, wherein the chimeric antigen receptor recognizes a target antigen on a target cell surface, and the shRNA passes The siRNA produced by it down-regulates the expression of the target antigen in the T cells. The expression vector of claim 13, wherein the chimeric antigen receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain, and the extracellular domain comprises an antibody derived from the target antigen Single chain antibody. The expression vector of claim 13, wherein the coding sequence of the shRNA is under the control of the H1 promoter. The expression vector according to claim 13, which is a lentiviral expression vector. The expression vector according to claim 16, wherein pLVX-EF1α-IRES-Puro is used as a backbone vector. The expression vector according to claim 13, wherein the target cell is a tumor cell. The expression vector according to claim 18, wherein the tumor cell is a multiple myeloma cell. The expression vector according to claim 13, wherein the target antigen is CS1. The expression vector according to claim 20, wherein the target nucleic acid sequence of the shRNA comprises a nucleotide sequence as shown in SEQ ID NO: 29. The expression vector according to claim 20, wherein the coding sequence of the shRNA comprises a nucleotide sequence as shown in SEQ ID NO: 28. The expression vector according to claim 14, wherein the single-chain antibody has an amino acid sequence as shown in SEQ ID NO: 20. A method for preparing a T cell expressing a chimeric antigen receptor, comprising transforming the T cell with the expression vector according to any one of claims 13 to 23. A method for preventing mutual killing of T cells expressing a chimeric antigen receptor, comprising down-regulating the expression of a target antigen targeted by the chimeric antigen receptor in the T cells. The method of claim 25, wherein the target antigen is CS1. The method of claim 25 or 26, wherein said down-regulating comprises allowing said T cells to express shRNA, and said siRNA produced by said shRNA inhibits expression of said target antigen in said T cells. The method according to claim 27, which is achieved by transforming the T cells with an expression vector including a coding sequence of the shRNA. A method for treating multiple myeloma in a subject, comprising administering to the subject T cells expressing a chimeric antigen receptor, the chimeric antigen receptor targets CS1 on the surface of the multiple myeloma cell And the T cells also express shRNAs for inhibiting the expression of CS1 in the T cells. Use of the T cell according to any one of claims 1 to 12 or the expression vector according to any one of claims 13 to 23 for the manufacture of a medicament for treating a disease caused by CS1-positive cell proliferation. The use according to claim 30, wherein the disease is multiple myeloma or plasma cell leukemia.

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