CN112469734A - T cell expressing chimeric antigen receptor, expression vector related to chimeric antigen and application of T cell and expression vector - Google Patents

Abstract

The present invention provides a T cell expressing a 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 cells themselves also express the target antigen, and to prevent the T cells from killing each other, the expression of the target antigen by the T cells is down-regulated. The invention also provides an expression vector containing the chimeric antigen receptor coding sequence. The T cells provided by the invention can be used for treating diseases caused by abnormal proliferation of CS1 positive cells by targeting CS1 antigen, and prevent the T cells from killing each other due to the fact that the expression of CS1 of the T cells is down-regulated, thereby being beneficial to the expansion and survival of the T cells in vitro and in vivo.

Description

T cell expressing chimeric antigen receptor, expression vector related to chimeric antigen and application of T cell and expression vector 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 multiple myeloma treatment.

Background

The development of chimeric antigen receptors against targets expressed in Multiple Myeloma (MM) cells is of great interest, as there is data that suggests that infusion of CD19-CAR T cells can induce sustained complete remission in patients with refractory relapsed B-cell associated tumors^1,2. There are currently several candidate antigen targets for CAR T cells for MM therapy, including CD19 and BCMA, but CD19 is expressed in only a very few malignant plasma cells. Phase I clinical trial data on BCMA-CAR T cells showed that partial and complete responses occurred after treatment in some patients, but that there was alsoSeveral problems include low or uneven expression of BCMA on myeloma cells of patients in the cohort, and outbreaks of BCMA negative or low expressing myeloma cells after treatment³. These data highlight that existing CAR targets have some drawbacks for the treatment of myeloma, and there is also a need to find and validate CAR targets with broader applicability for this disease.

SLAMF7 (also known as CD319, CRACC, or CS1) is a member of a family of signaling lymphocyte activating molecules that are transmembrane receptors, which are expressed at high levels in myeloma cells and are involved in regulating the adhesion of myeloma cells to bone marrow stromal cells. By immunohistochemical analysis of a series of lymphomas and leukemias, the results indicated that CS1, although present in all myeloma cases, was not expressed in the vast majority of acute leukemias, B-cell lymphomas, and classical hodgkin lymphomas. Currently, researchers have investigated the feasibility of CS1 as a CAR T target. CS1-CAR T cells target CS 1-expressing multiple myeloma cell lines (e.g., NCI-H929, IM9, MM1S) and primary tumor cells isolated from multiple myeloma patients. CS1-CAR T cells have significantly enhanced secretion of cytokines IFN- γ and IL-2 compared to control T cells that have not been transduced with CAR, and significantly increase the proportion of killed multiple myeloma cell lines or primary tumor cells from multiple myeloma patients⁴. These results demonstrate that the effect of CS1-CAR T cells is CS1 dependent. Importantly, CS1-CAR T cells were able to prolong the survival of NSG mice transplanted with MM1S cells. Overall, CS1 has great potential as a CAR target for the treatment of multiple myeloma, and in vitro function of CS1 as a CAR target and in vivo function data of mice have been reported in the literature, but researchers have pointed out the technical difficulties and concerns of CS1 as a CAR target⁵。

CS1 is highly expressed on multiple myeloma cells, but is also expressed on NK cells, T cells, B cells, and mature dendritic cells^6,7. T cells are classified as helper T cells (CD4+) and cytotoxic (CD8+) T cells. In general, CD4+ T cells can proliferate to activate other types of productionImmune cells that are directly immunoreactive. CD8+ T cells can kill target cells that produce an antigenic response. The function of CS1 in CD8+ T cells of patients with Systemic Lupus Erythematosus (SLE) was studied by researchers. Impaired function of CD8+ T cells in SLE patients results in a reduced ability to fight infection, whereas CS1 expression in CD8+ T cells in SLE patients is down-regulated. Activation of CS1 by specific antibodies restores CD8+ T cells in defective SLE patients, enabling the role of viral antigens⁸. CS1 also plays an important role in NK cells, activating NK cell function through homophilic interaction of CS1⁹. NK cells express a variety of activating and inhibitory receptors that recognize ligands on potential target cells. The balance between the signals from these receptors determines whether NK cells will be activated, kill target cells and secrete cytokines. Antibody and ligand mediated stimulation experiments demonstrated that CS1 plays an activating role in NK cells^9,10,11. CS 1-deficient NK cells have an impaired ability to kill CS1+ target cells. Upon encountering target cells, CS 1-deficient NK cells also have a reduced ability to secrete Interferon (IFN). Meanwhile, the cytotoxicity of CS 1-deficient NK cells on CS 1-target cells was also reduced. This study suggests that CS1 is involved in NK cell-NK cell interaction and is likely to promote NK cell function. Although CS1 may have beneficial functions in T cells and NK cells, expression of CS1 in T cells may present difficulties in the preparation and use of CS1-CAR T cells.

The expression of CS1 in a cell subset common in peripheral blood of patients with multiple myeloma is detected by flow cytometry, and the expression of CS1 protein is found in NK cells, T cells, B cells and monocytes, wherein the expression amount of CS1 protein in CD4+ T cells is lower than that in CD8+ T cells⁵. Since CS1 is also expressed in some T cells and other immune cells, CS1-CAR T may lead to selective killing and clearance of these cells. Researchers have analyzed the ability of CS1-CAR T cells to recognize normal lymphocytes and found that they selectively kill T cells and B cells of CS1+/high NK cells, CD4+, and CD8 +. Meanwhile, CS1-CAR T cells were cultured for several days, and then compared with controlCompared with T cells, the CS1 protein positive CD4+ and CD8+ cells are obviously reduced. The study also found that when CS1-CAR T cells were co-cultured with normal lymphocyte populations, the live cell rates of CD4+ T cells, CD8+ T cells, and NK cells co-cultured with CS1-CAR T cells all decreased compared to CD19-CAR T cells, with the most significant decrease in the live cell rate of NK cells⁵。

Suicide of CS1-CAR T cells caused only CS 1-negative or low-expression cells of CS1-CAR T cells to survive, leading to difficulties in expansion of CS1-CAR T cells; and since CD8+ T cells express more CS1 than CD4+ T cells and are therefore more cleared, CD 4: abnormal proportion of CD8 reduces its cytotoxic activity in vitro and in vivo.

Disclosure of Invention

The present invention provides in one aspect a T cell expressing a 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 cells themselves also express the target antigen, and to prevent the T cells from killing each other, the expression of the target antigen by the T cells is down-regulated.

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

In some embodiments, the target cell is a tumor cell, particularly a multiple myeloma cell.

In some embodiments, the target antigen is CS 1.

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

In some embodiments, the siRNA is generated from an shRNA expressed by the T cell.

In some embodiments, the target nucleic acid sequence of the shRNA comprises a sequence as set forth in SEQ ID NO: 29.

In some embodiments, the coding sequence for the shRNA comprises the sequence set forth in SEQ ID NO: 28.

In some embodiments, the single chain antibody is derived from an anti-CS 1 antibody and has the amino acid sequence set forth in SEQ ID NO: 20, or a pharmaceutically acceptable salt thereof.

In some embodiments, the amino acid sequence of the chimeric antigen receptor comprises, in order from N-terminus to C-terminus, a CD8 a signal peptide, the single chain antibody, a CD8 hinge region, a CD28 transmembrane region, a CD28 intracellular co-stimulatory domain and a 4-1BB intracellular co-stimulatory domain, and a CD3 ζ intracellular signaling domain.

In some embodiments, the T cell is transformed with an expression vector comprising the coding sequence for the chimeric antigen receptor and an expression vector comprising the coding sequence for the shRNA, or an expression vector comprising the coding sequence for the chimeric antigen receptor and the coding sequence for the shRNA.

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

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

In some embodiments, the coding sequence for 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 cell is a tumor cell, particularly a multiple myeloma cell.

In a specific embodiment, the expression vector uses pLVX-EF1 alpha-IRES-Puro as a backbone vector.

In a specific embodiment, the target antigen is CS 1.

In a specific embodiment, the target nucleic acid sequence of the shRNA comprises the sequence set forth in SEQ ID NO: 29.

In a specific embodiment, the coding sequence of the shRNA comprises the sequence set forth in SEQ ID NO: 28.

In a specific embodiment, the single chain antibody is derived from an anti-CS 1 antibody and has the amino acid sequence as set forth in SEQ ID NO: 20, or a pharmaceutically acceptable salt thereof.

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

In another aspect, the invention provides a method of preventing T cells expressing a chimeric antigen receptor from killing each other, 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 CS 1.

In some embodiments, the downregulating comprises allowing the T cells to express an shRNA that produces an siRNA that inhibits expression of the target antigen in the T cells.

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

In another aspect, the invention provides a method of treating multiple myeloma in a subject, comprising administering to the subject a T cell that expresses a chimeric antigen receptor that targets CS1 on the surface of the multiple myeloma cell, and that also expresses an shRNA for inhibiting expression of CS1 in the T cell.

In another aspect, the invention provides the use of a T cell or an expression vector as described above in the manufacture of a medicament for the treatment of 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 invention can be used for treating diseases (such as multiple myeloma) caused by abnormal proliferation of CS1 positive cells by targeting CS1 antigen, and simultaneously prevent the T cells from killing each other due to the fact that the expression of CS1 of the T cells is down-regulated, thereby being beneficial to the expansion and survival of the T cells in vitro and in vivo.

Drawings

FIG. 1 is a schematic representation of the composition of the mock-5.3-CAR element. The CAR consists of: a CD8 a signal peptide, a BCMA-specific scfv (BCMA scfv), a CD8 hinge region, a CD28 transmembrane region (TM), a CD28 intracellular co-stimulatory domain, a 4-1BB intracellular co-stimulatory domain, and a CD3 ζ intracellular signaling domain.

FIG. 2 is a schematic representation of the composition of mock-CS1-CAR elements. The CAR consists of: a CD8 a signal peptide, a CS1 specific scFv (CS1scFv), a CD8 hinge region, a CD28 transmembrane domain (TM), a CD28 intracellular co-stimulatory domain, a 4-1BB intracellular co-stimulatory domain, and a CD3 ζ intracellular signaling domain.

FIG. 3 is a schematic representation of the composition of SH3-5.3-CAR elements. It consists of a gene encoding SH3shRNA (SH3) capable of knocking down CS1 expression and mock-5.3-CAR shown in figure 1. Transcription of the shRNA was initiated by the H1 promoter.

FIG. 4 is a schematic representation of the composition of SH3-CS1-CAR elements. It consists of a gene encoding SH3shRNA (SH3) capable of knocking down CS1 expression and mock-CS1-CAR shown in fig. 2. Transcription of the shRNA was initiated by the H1 promoter.

FIG. 5 is a schematic representation of the BCMA-T2A-puro construct overexpressing BCMA, comprising BCMA, T2A, and puromycin resistance gene (puro).

FIG. 6 is a schematic representation of the CS1-IRES-puro construct overexpressing CS1, comprising CS1, IRES and puromycin resistance gene (puro).

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

FIG. 8 shows the expression of CS1 on the cell surface of C-K562 cells as determined by flow cytometry, with the left peak being unstained C-K562 cells and the right peak being C-K562 cells stained with Human CRACC/SLAMF7 APC-conjugated antibodies.

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

Figure 10 shows proliferation of CAR T cells and control T cells. Since the surface of the T cell expresses CS1, suicide can occur during the culture of CS1-CAR T, and the cell is difficult 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 SH 3-5.3). Expansion curves for mock-5.3 and SH3-5.3 cells also showed no significant effect on cell proliferation after knockdown of CS 1.

Figure 11 shows the subpopulation composition of various CAR T cells. Knockdown of expression of CS1 did not affect the proportion of cell subsets in CAR T cells.

CM and EM refer to

T cell, T_CMCells and T_EMA cell;

figure 12 shows the in vitro killing ability of various CAR T cells against different target cells. mock-5.3 cells and SH3-5.3 cells were able to specifically kill BCMA-positive target cells (MM1S and B-K562), but the killing effect of SH3-5.3 cells on MM1S was slightly reduced compared to mock-5.3 cells. It is possible that knocking down CS1 has some effect on the killing function of T cells, but further experiments are still needed to confirm. mock-CS1 cells and SH3-CS1 cells can kill CS1 positive target cells (MM1S and C-K562) specifically, and no phenomenon that the tumor killing effect of SH3-CS1 cells is weakened is observed, and the adverse effect of knocking down CS1 on the killing function of T cells is counteracted by reducing the suicide of CS1-CAR T cells by knocking down CS 1.

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 reduction in degranulation levels of SH3-CS1 cells was observed, it is still possible that knocking down CS1 results in reduced suicide of CS1-CAR T cells, counteracting the adverse effects thereof.

Figure 14 shows the expression of immune checkpoint molecules in various CAR T cells. PD-1 and CTLA-4 (top panel) and TIM-3 and LAG3 (bottom panel) immune checkpoints are markers of T cell depletion, with lower expression of the four above markers for the SH3-CS1 group, suggesting that CAR T cells with CS1 knockdown are less depleted due to reduced suicide, suggesting that CS1-CAR T with CS1 knockdown 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 an immunoglobulin secreted by plasma cells (effector B cells) that is used by the body's immune system to identify and neutralize foreign substances (polypeptides, viruses, bacteria, etc.). The foreign substance is accordingly referred to as an antigen. The basic structure of an antibody molecule is a 4-mer consisting of 2 identical heavy chains and 2 identical light chains. Heavy and light chains are divided into variable regions at the amino terminus and constant regions at the carboxy terminus, based on conservative differences in amino acid sequence. The variable regions of one heavy chain and one light chain interact to form one antigen binding site. Thus, an intact antibody molecule comprises two antigen binding sites.

As used herein, the term "single chain antibody (scFv)" refers to a single peptide chain formed by linking an antibody heavy chain variable region and a light chain variable region via a short peptide. When correctly folded, the heavy chain variable region and the light chain variable region form an antigen binding site through non-covalent interaction, and the affinity activity of the source antibody to the antigen can be well reserved.

As used herein, the term "chimeric antigen receptor" refers to an engineered protein receptor molecule that confers a desired specificity to immune effector cells (e.g., T cells), for example, the ability to bind to a particular tumor antigen. These receptors are called "chimeric" because they are fusion proteins, consisting of components of different origins. Chimeric antigen receptors generally 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 sequence of scFv that is responsible for recognizing and binding a particular antigen on the target cell (the target antigen). The intracellular domain typically includes an Immunoreceptor Tyrosine Activation Motif (ITAM), such as the signaling domain derived from the CD3 ζ molecule, responsible for activating immune effector cells, which may increase cytotoxicity, proliferative capacity, and prolong T cell survival. In addition, the chimeric antigen receptor may also include a signal peptide at the amino terminus responsible for the 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 product of interest (e.g., a protein or RNA) in a cell, as compared to normal levels. This can be achieved in a variety of ways, for example, by inhibiting transcription initiation, interfering with mRNA translation, promoting mRNA degradation, or promoting degradation of expressed proteins, among others.

As used herein, the term "sirna (small interfering RNA)" refers to double stranded RNA molecules of about 21nt in length that can complementarily bind to homologous sequences in the target mRNA, resulting in the mRNA becoming dysfunctional or degraded and thus unable to be translated to produce a protein. One way to introduce siRNA into a cell is to express the corresponding short hairpin RNA (shRNA) molecule in the cell, where the "loop" of the short hairpin RNA molecule is degraded by nucleases (e.g., Dicer enzyme) in the cell to form siRNA, which acts to down-regulate the expression of the target gene.

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

When referring to CAR T cells, the term "suicide" herein refers to the situation where the CAR T cell population is in a suicide state as seen overall, since the CAR T cells themselves also express the target antigen, resulting in killing by mutual recognition between cells within the CAR T cell population.

Other terms are described in detail below to describe specific embodiments.

The invention introduces short hairpin RNA against CS1 mRNA in a lentiviral expression vector encoding a chimeric antigen receptor targeting CS1 (CS 1-CAR). After the lentivirus infects T cells and integrates the coding sequences of CS1-CAR and shRNA into the genome of the T cells, the shRNA coding gene is 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 preventing the CS1-CAR T cells from mutual recognition and killing. The inventors also investigated the cell proliferation and subpopulation composition of CAR T cells knockdown (knockdown) CS1 expression, while also comparing their killing function in vitro to CAR T cells not knockdown CS 1.

Example 1 Preparation of chimeric antigen receptor-modified T cells

1. Preparation of chimeric antigen receptor Gene expression vector

1.1 general description

(1) CARs are chimeric proteins that fuse an antigen-binding domain that specifically recognizes a target antigen (e.g., CS1) to an intracellular signaling structure that is capable of activating or stimulating an immune cell. Generally, a CAR includes an extracellular domain, a transmembrane domain, and an intracellular domain. In this example, the extracellular domain includes a single chain antibody (scFv) that specifically binds CS1, a CD8 a signal peptide, and a CD8 hinge region. The hinge region is typically derived from a CD8 or IgG4 molecule, which serves to link intracellular and extracellular proteins. In this example, the inventors used CD8 hinder derived from CD 8. The transmembrane domain is a structure that links the extracellular and intracellular domains of the CAR, and in this example the CD28 transmembrane region is used. The CAR intracellular domain employed in this study was an intracellular signaling domain capable of transducing the information of CS1CAR binding to human CS1 toImmune effector cells to trigger effector cell functions (such as activation, cytokine production, proliferation, and cytotoxic activity). The intracellular signaling domain of the "first generation" CAR comprises only CD3 ζ, and the intracellular signaling domain of the "second generation" CAR comprises one costimulatory molecule (e.g., CD28 or 4-1BB4-1BB) and CD3 ζ. To further improve the design of CARs, many research groups began to look at developing "third generation" CARs. A "third generation" CAR comprises multiple costimulatory molecules (e.g., CD28 and 4-1BB) and CD3 ζ. Different researchers use different targets and co-stimulation signals to carry out research, and the obtained comparison results of the second generation CAR and the third generation CAR have certain difference. Some studies report that recombinant T cells expressing "third generation" CARs all show significant improvements in antitumor activity, survival cycle, and cytokine release^12,13. The results of the Wilkie et al study showed that the MMC 1-targeted second and third generation CAR recombinant T cells did not differ significantly in anti-tumor cytotoxicity, although T cells expressing third generation CAR were able to secrete greater amounts of IFN- γ¹⁴. This study employed a "third generation" CAR whose intracellular signaling domain comprised CD28 and a 4-1BB costimulatory molecule (otherwise known as the intracellular costimulatory domain) and a CD3 zeta stimulatory molecule.

(2) The pLVX-EF1 alpha-IRES-Puro vector (purchased from vast ling plasmid platform) contains relevant elements required for generating lentiviruses, and elements capable of increasing virus titer and increasing transgene expression. For example, the WPRE therein can facilitate RNA processing events and enhance nuclear export of viral RNA, resulting in increased viral titers produced by packaging cells; rev Responsive Element (RRE) enhances transport of unspliced viral RNA from the nucleus, further increasing viral titer; the central polypurine/central termination sequence element (cPPT/CTS) creates a central DNA sheet (flap) that increases nuclear import of the viral genome during infection of target cells, resulting in increased vector integration and more efficient transduction. pLVX-EF1 alpha-IRES-Puro contains restriction enzyme sites and Multiple Cloning Sites (MCS) to facilitate the researcher to subclone the DNA sequence of interest into the vector as desired. MCS is a short DNA sequence containing multiple (up to 20) restriction sites, and is a standard configuration sequence of a vector plasmid commonly used in genetic engineering. In MCS, each restriction enzyme site is usually unique, i.e. they occur only once in a particular vector plasmid, and there may be overlap of the enzyme sites for different enzymes.

1.2 materials and methods

(1) The fusion gene fragment was designed in the following order of the coding genes: CD8 alpha signal peptide (nucleic acid sequence SEQ ID NO: 1; amino acid sequence SEQ ID NO: 2), BCMA scFv (nucleic acid sequence SEQ ID NO: 11; amino acid sequence SEQ ID NO: 12), CD8 Hinger (nucleic acid sequence SEQ ID NO: 3; amino acid sequence SEQ ID NO: 4), CD28 transmembrane region and CD28 intracellular co-stimulatory domain (nucleic acid sequence SEQ ID NO: 5; amino acid sequence SEQ ID NO: 6), 4-1BB intracellular co-stimulatory domain (nucleic acid sequence SEQ ID NO: 7; amino acid sequence SEQ ID NO: 8) and CD3 zeta intracellular signal domain (nucleic acid sequence SEQ ID NO: 9; amino acid sequence SEQ ID NO: 10), and both ends are respectively designed with EcoRI and MluI restriction enzyme digestion sites. The fusion gene fragment was genetically synthesized by Nanjing Kingsrei Biotech. The BCMA scFv is formed by connecting a heavy chain (the nucleic acid sequence is SEQ ID NO: 13, the amino acid sequence is SEQ ID NO: 14) and a light chain (the nucleic acid sequence is SEQ ID NO: 15, the amino acid sequence is SEQ ID NO: 16) through a connecting peptide (the nucleic acid sequence is SEQ ID NO: 17, and the amino acid sequence is SEQ ID NO: 18). The synthesized gene fragment was subcloned into EcoRI and MluI restriction endonuclease cleavage sites of pLVX-EF1 alpha-IRES-Puro vector, and then the product was transformed into E.coli DH5 alpha competent cells (purchased from Takara) and positive colonies were selected by Ampicillin (Ampicillin, Amp). And (3) selecting positive colonies, sending the positive colonies to Wuhan Tianyihuiyuan biotechnology limited for sequencing the complete sequence of the plasmid, inoculating the colonies with the correct sequence into 250mL LB liquid culture medium containing Amp, and shaking the colonies at 37 ℃ and 220rpm for 16-24 h. Plasmids were extracted from the bacterial suspension using the EndoFree Plasmid Giga Kit (purchased from Qiagen) and named "pLVX-mock-5.3-CAR" (FIG. 1).

SEQ ID NO: 2 amino acid sequence of artificially synthesized CD8 alpha signal peptide
MALPVTALLLPLALLLHAARP

SEQ ID NO: 4 amino acid sequence of artificially synthesized CD8 Hinger
TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD

SEQ ID NO: 8 amino acid sequence of artificially synthesized 4-1BB intracellular co-stimulatory domain
KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL

SEQ ID NO: 17 DNA sequence of artificially synthesized connecting peptide for connecting light chain and heavy chain in BCMA scFV
GGCAGCACCAGCGGCTCCGGCAAGCCTGGCTCTGGCGAGGGCAGCACAAAGGGA

SEQ ID NO: 18 amino acid sequence of a synthetic connecting peptide for connecting a light chain and a heavy chain in a BCMA scFV
GSTSGSGKPGSGEGSTKG

(2) The fusion gene fragment was designed in the following order of the coding genes: CD8 alpha signal peptide, CS1scFv (nucleic acid sequence is SEQ ID NO: 19; amino acid sequence is SEQ ID NO: 20), CD8 Hinger, CD28 transmembrane region, CD28, 4-1BB intracellular co-stimulatory domain and CD3 zeta intracellular signal domain, and BamHI and MluI restriction enzyme cutting sites are respectively designed at two ends. The fusion gene fragment was genetically synthesized by Nanjing Kingsrei Biotech. The CS1scFv is formed by connecting a heavy chain (the nucleic acid sequence is SEQ ID NO: 21, the amino acid sequence is SEQ ID NO: 22) and a 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, and the amino acid sequence is SEQ ID NO: 26). The gene fragment synthesized by the above gene was subcloned into BamHI and MluI restriction enzyme cleavage sites of pLVX-EF1 alpha-IRES-Puro vector, and the plasmid pLVX-mock-CS1-CAR was obtained by the method of screening colonies and extracting the plasmid as described above (FIG. 2).

(3) Tal et al designed four siRNAs for knocking down CS1 expression, and published the target sequences of 4 siRNAs and the effect of knocking down CS1 expression¹⁵In the data in the reference literature of the inventors, the 3 rd siRNA target sequence (nucleic acid sequence SEQ ID NO: 29) was selected, SH3shRNA (nucleic acid sequence SEQ ID NO: 28) against CS1 mRNA was designed, and the function of knocking down the expression of CS1 in T cells was verified in example 3 below. H1 promoter (SEQ ID NO: 27) and DNA sequence encoding SH3shRNA were synthesized in order by Nanjing Kingsler Biotech, and ClaI and SphI restriction enzyme sites were designed at both ends. The synthesized DNA fragments were subcloned into ClaI and SphI restriction endonuclease cleavage sites of pLVX-mock-5.3-CAR and pLVX-mock-CS1-CAR, respectively, and plasmids pLVX-SH3-5.3-CAR (FIG. 3) and pLVX-SH were obtained by screening colonies and extracting plasmids as described above3-CS1-CAR (FIG. 4).

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

GGCGGCGGCGGCAGCGGCGGGGGCGGCAGCGGCGGCGGCGGCAGC

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

SEQ ID NO: 29 target DNA sequence of artificially synthesized SH3shRNA
GTCGGGAAACTCCTAACATAT

(4) The pCMV-VSV-G helper plasmid (purchased from vast Ling plasmid platform) and the psPAX2 helper plasmid (purchased from vast Ling plasmid platform) were transformed into E.coli DH 5. alpha. competent cells, and the above plasmids satisfying the lentiviral preparation requirements and having correct sequences were obtained with reference to the above methods for screening colonies and extracting plasmids.

2. Preparation of chimeric antigen receptor lentivirus

2.1 general description of the principles

Lentiviruses are gene therapy vectors developed based on Human Immunodeficiency Virus (HIV), which are infectious to both dividing and non-dividing cells and which are expressed intracellularly for extended periods of time. The lentiviruses used in this study are "suicide" viruses, i.e., viruses that infect the cells of interest do not infect any more cells, nor do they utilize the host cell to produce new viral particles. Part of genes in lentivirus are deleted and replaced by exogenous target genes, belonging to pseudotyped virus. The pCMV-VSV-G vector contains the VSV-G gene and provides an envelope protein required for viral packaging. The psPAX2 vector contains the gag gene of the HIV virus and encodes the major structural proteins of the virus; a pol gene encoding a virus-specific enzyme; rev gene encoding 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 expressing the SV40 large T antigen and are widely used in transient transfection to overexpress various target proteins or for packaging viruses. After the pLVX vector, the pCMV-VSV-G vector and the psPAX2 vector enter 293T cells by a transfection reagent such as Polyethyleneimine (PEI), a lentivirus backbone carried by the pLVX vector is transcribed into viral RNA, and a protein translated from lentivirus-related genes carried by the psPAX2 and the pCMV-VSV-G is assembled into lentivirus.

2.2 materials method

And (3) taking the plasmids extracted in the last step, mixing the vectors (pLVX-mock-5.3-CAR, pLVX-mock-CS1-CAR, pLVX-SH3-5.3-CAR and pLVX-SH3-CS1-CAR) with the pCMV-VSV-G helper plasmid and the psPAX2 helper plasmid respectively according to the ratio of 6:2:3, and co-transfecting 293T cells. After the 293T cells produced virus for 72 hours, the cell culture containing the virus was collected and centrifuged at 3000g for 5min at 4 ℃. The supernatant was filtered through a 0.45 μm filter and the filtrate was centrifuged at 30000g for 2.5h at 4 ℃. Discarding supernatant, resuspending the precipitate with precooled PBS to obtain corresponding LV-mock-5.3, LV-SH3-5.3, LV-mock-CS1 and LV-SH3-CS1 lentivirus concentrated solutions, and storing at-80 ℃ for later use.

3. Lentivirus activity titer detection

3.1 general description

FITC-conjugated affinity needle Anti-Mouse IgG (H + L) (purchased from Jackson ImmunoResearch) is labeled with fluorescein, and it can bind to the single chain antibody in CAR. The fluorescent signal detected by a flow cytometer can indirectly reflect the expression condition of the CAR encoded by the lentivirus vector in 293T cells, positive cells successfully infected by the lentivirus are identified, and the activity titer data of the lentivirus is calculated according to the proportion of the positive cells.

3.2 materials and methods

The 5.0 x10 of the wells are connected into a 6-well plate⁵293T cells are added into each well, 0.1. mu.L, 0.5. mu.L and 1. mu.L of the lentivirus concentrate are added into each well, and 1 negative control without the lentivirus concentrate is arranged. The 6-well plate was placed at 37 ℃ with 5% CO₂Culturing in the incubator. Three days later, 293T cells were harvested from Versene solution (purchased from Gibco), stained with FITC-conjugated affinity paste Goat Anti-Mouse IgG (H + L), detected by flow cytometry (instrument model: Beckman Cytoflex), and the proportion of CAR-positive 293T cells was analyzed by FlowJo (version 7.6.3). According to the formula: (lentivirus activity titer (TU/mL) ═ 293T cell number. times.CAR-positive 293T cell ratio (%)/lentivirus addition amount (uL). times.1000), the activity titers of the above-mentioned LV-mock-5.3, LV-SH3-5.3, LV-mock-CS1, LV-SH3-CS1 lentivirus concentrates were calculated.

Preparation and activation of T lymphocytes

100mL of fresh blood of healthy persons was taken, and peripheral blood mononuclear cells were separated by a lymphocyte separation medium (purchased from the scientific and technical Limited liability company of tertiary ocean biologics, Tianjin) and a density gradient centrifugation method. The cells were labeled with magnetic beads using CD3MicroBeads, human (available from Miltenyi Biotech) and isolated and purified to obtain T lymphocytes. Preparing a T cell culture medium: to 1L CTS^TM OpTmizer ^TMT Cell Expansion basic Medium (available from Gibco) plusInto 26ml CTS^TM OpTmizer ^TMT-cell Expansion supplement (from Gibco), L-glutamine (from Gibco) at a final concentration of 2mM, and IL-2 (from Shuanglu pharmaceutical) at a final concentration of 500 IU/ml. Cells were incubated with magnetic beads Dynabeads^TMHumanT-Activator CD3/CD28 (from Gibco) was programmed with a 1: 1 ratio was added to the T cell culture medium. Magnetic beads Dynabeads^TMThe HumanT-Activator CD3/CD28 is used for stimulating the activation density of about 1 × 10⁶T cells in ml. The T cells were incubated at 37 ℃ with 5% CO₂The culture was carried out overnight in an incubator to obtain activated T cells.

5. Lentivirally transduced T lymphocytes

CAR T cells were obtained by transducing the activated T cells described above with a lentivirus carrying the CAR gene. After T cells are activated for 18h, LV-mock-5.3, LV-SH3-5.3, LV-mock-CS1 and LV-SH3-CS1 lentivirus concentrated solution are added into MOI-5 respectively to infect 5x10⁶T lymphocytes, and the cells were incubated at 37 ℃ with 5% CO₂Incubate overnight in the incubator. After 24h, mock-5.3, SH3-5.3, mock-CS1 and SH3-CS1 cells after lentivirus transduction are replaced by new T cell culture medium for continuous culture, and observation and counting are carried out every day. When the cell concentration reached 2X10⁶At one/mL, fresh medium was added and the scale-up culture continued.

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

1. Preparation of antigen overexpression vectors

1.1 general description

(1) puro is puromycin resistance gene. Puromycin is an aminoglycoside antibiotic produced by fermentation and metabolism of Streptomyces albonensis (Streptomyces alboneger), and kills gram-positive bacteria, various animal and insect cells by inhibiting protein synthesis. Puromycin is commonly used for screening and maintaining stably transfected cells in mammals containing puro resistance genes. "puro" in the name of the vector means that the vector contains a puro resistance gene. In this example, K562 stably expressing BCMA or CS1 was selected using puro resistance gene (nucleic acid sequence SEQ ID NO: 37, amino acid sequence SEQ ID NO: 38) and puro characteristics.

(2) T2A: 2A peptide is a kind of amino acid sequence, when ribosome translates its mRNA, it will trigger ribosome jump, resulting in the stop of the peptide segment being translated, and starting the synthesis of a new peptide segment from the next amino acid codon, thus realizing that one mRNA can generate two proteins at the same time when translated, and it is commonly used to construct polycistronic vector. T2A is the 2A element from the Leuconostoc β -tetrad virus (Thosea asigna virus). In this example, the inventors linked BCMA and puromycin resistance genes using T2A, co-expressed BCMA and puromycin resistance genes in K562, and obtained K562, hereinafter referred to as B-K562, stably expressing BCMA protein by puromycin screening.

(3) IRES (internal ribosome entry site) is found to initiate translation independently by recruiting ribosomes to translate mRNA and fusing IRES to exogenous cDNA. Because of its properties, IRES has been widely used in the construction of binary expression vectors. In this example, IRES (nucleic acid sequence SEQ ID NO: 36) was used to co-express CS1 and puromycin resistance gene, and K562, hereinafter referred to as C-K562, stably expressing CS1 protein was obtained by puromycin screening.

1.2 materials method

(1) The fusion gene fragments were designed in the following order: CD8 alpha signal peptide (the nucleic acid sequence is SEQ ID NO: 1, the amino acid sequence is SEQ ID NO: 2), BCMA (the nucleic acid sequence is SEQ ID NO: 30, the amino acid sequence is SEQ ID NO: 31), T2A (the nucleic acid sequence is SEQ ID NO: 34, the amino acid sequence is SEQ ID NO: 35), purOR (the nucleic acid sequence is SEQ ID NO: 37, the amino acid sequence is SEQ ID NO: 38), and XbaI and MluI restriction endonuclease sites are designed at two ends. The fusion gene fragment was synthesized by Nanjing Kingsler Biotech, and subcloned into XbaI and MluI restriction enzyme sites of pLVX-EF1 α -IRES-Puro vector, and plasmid pLVX-BCMA-T2A-Puro was obtained according to the method of screening colonies and extracting plasmid in example 1 (FIG. 5).

(2) EcoRI and BamHI restriction enzyme sites were designed at both ends of the gene fragment CS1 (nucleic acid sequence is SEQ ID NO: 32; amino acid sequence is SEQ ID NO: 33), CS1 gene fragment was synthesized by Nanjing Kinsley Biotech, and subcloned into EcoRI and BamHI restriction enzyme sites of pLVX-EF1 α -IRES-Puro vector, and plasmid pLVX-CS1-IRES-Puro was obtained by screening colonies and extracting the plasmid in example 1 (FIG. 6).

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

SEQ ID NO: 35 synthetic T2A amino acid sequence
EGRGSLLTCGDVEENPGP

2. Preparation of antigen-overexpressing lentiviruses

2.1 materials 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, to co-transfect 293T cells. After 72h of transfection, cell culture supernatants containing the virus were collected. Centrifuge at 3000g for 5min at 4 ℃. After the supernatant was filtered through a 0.45 μm filter, the virus solution was centrifuged at 30000g for 2.5h at 4 ℃. Discarding supernatant, resuspending and dissolving the precipitate with precooled PBS to obtain LV-BCMA or LV-CS1 lentivirus concentrated solution, and storing at-80 deg.C for use.

3. Lentivirus activity titer

3.1 general description

Human CRACC/SLAMF7 APC-conjugated antibodies and APC anti-Human CD269(BCMA) antibodies are labeled with fluorescein, which binds to CS1 protein and BCMA protein, respectively. The fluorescent signal detected by the flow cytometry can indirectly reflect the expression of BCMA or CS1 in 293T cells, positive cells successfully infected by the lentivirus are identified, and the activity titer of the lentivirus is calculated according to the proportion of the positive cells.

3.2 materials method

The 5.0 x10 of the wells are connected into a 6-well plate⁵293T cells and lentivirus concentrate (LV-BCMA or LV-CS1) were added in an amount of 0.1. mu.L, 0.5. mu.L, and 1. mu.L per well, respectively, and 1 negative control without lentivirus was set. The cells were placed at 37 ℃ with 5% CO₂Culturing in an incubator. Three days later, 293T cells were harvested using Versene solution using Human CRACC/SLAMF7 APC-conjugated antibodies (from R)&D Systems) and APC anti-human CD269(BCMA) Antibody (purchased from Biolegend) were used to measure the proportion of CS1 or BCMA positive 293T cells, and the titer of the activity of the LV-BCMA or LV-CS1 lentivirus concentrate was calculated according to the formula in example 1.

4. Transduction and screening of target cells

4.1 materials method

K562 cells (purchased from the China academy of sciences type culture Collection cell Bank) were subjected to 6X10⁴One/well was seeded in 24-well plates and K562 cells were cultured using RPMI 1640 medium (purchased from GIBCO). The cells were placed at 37 ℃ with 5% CO₂After further culturing in the incubator for 24 hours, the above-mentioned concentrated lentivirus solutions were added at an MOI of 1, respectively. After the virus infected cells for 72h, cells were screened in RPMI 1640 medium containing 2. mu.g/mL puromycin (purchased from GIBCO) to obtain stably transfected cells B-K562 and C-K562. Staining B-K562 and C-K562 cells with Human CRACC/SLAMF7 APC-conjugated Antibody and APC anti-Human CD269(BCMA) Antibody, respectively, and flow cytometryAnd (5) analyzing the detection result by using FlowJo software.

4.2 results

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

Example 3 knockdown efficiency analysis of SH3shRNA

1. Overview

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

2. Materials and methods

The lentivirus-transduced T cells obtained in example 1 (mock-CS1, SH3-CS1, mock-5.3 and SH3-5.3) and non-lentivirus-transduced control T cells (Table 1) were stained with FITC-conjugated affinity Goat Anti-Mouse IgG (H + L) and Human CRACCC/SLAMF 7 APC-conjugated Antibody, and the expression of the above cell surface CAR and CS1 was examined by flow cytometry.

TABLE 1 SH3shRNA knockdown efficiency fruit design for experiments

Cell name	Target of CAR	Whether or not it is knocked-down for expression of CS1
T	-	Whether or not
Mock-5.3	BCMA	Whether or not
SH3-5.3	BCMA	Is that
Mock-CS1	CS1	Whether or not
SH3-CS1	CS1	Is that

3. Results

The expression rates of CS1 on the surfaces of the non-lentivirus-transduced control T cells and lentivirus-transduced mock-5.3, SH3-5.3, mock-CS1 and SH3-CS1 cells were 56%, 55.6%, 27.6%, 12.7% and 4.21%, respectively.

4. Discussion of the related Art

The expression rate of SH3-5.3 cell surface CS1 is lower than mock-5.3, which shows that SH3shRNA can effectively knock down the expression of T cell surface CS 1. The expression rate of CS1 on the cell surface of mock-CS1 was lower than that of control T cells not transduced with lentivirus and mock-5.3 and SH3-5.3 transduced with lentivirus, probably because CS1-CAR T cells killed CS1 positive cells in the population, resulting in a reduction in the number of CS1 positive cells. Compared with mock-CS1 cells, the expression rate of SH3-CS1 cell surface CS1 is reduced, and the fact that SH3shRNA can effectively knock down the expression of T cell surface CS1 is also shown (FIG. 9).

Example 4 in vitro function of CS1 knockdown CAR T cells

CAR T cell expansion profile

1.1 general description

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

1.2 materials method

SH3-CS1, SH3-5.3, mock-CS1, mock-5.3 and non-lentivirus transduced control T cells (T) were cultured according to the conditions in example 1, and the cell numbers were recorded every 2 to 3 days. The time point for lentiviral transduction was scored as 0 day, and cell numbers from 0 to 10 days were recorded.

1.3 results

The number of initial cells in mock-CS1 group was 7X 10⁶The other groups are 5 × 10⁶In vitro culture for 10 days according to the culture conditions in example 1, except that mock-CS1 group was less than 10-fold amplified, each group was normally amplified 25-to 35-fold.

1.4 discussion

As can be seen from the expansion curve of mock-CS1 group (FIG. 10), suicide occurs during the culture of CS1-CAR T due to the expression of CS1 on the surface of T cells, resulting in difficulty in cell expansion. However, after knockdown of the expression of CAR T cell CS1, the cells could be expanded as normal as control T cells (T) and anti-BCMA-CAR T (mock-5.3 and SH3-5.3) that were not transduced by lentiviruses. Furthermore, the amplification curves for mock-5.3 and SH3-5.3 showed no significant effect on cell proliferation after knockdown of CS 1.

2. Subset composition of CAR T cells after knockdown of CS1

2.1 overview

T cells are a heterogeneous population of cells and there are a variety of methods of classification. According to the difference of cell surface differentiation antigen (CD), the cell surface differentiation antigen can be divided into two subgroups of CD4+ and CD8 +; (ii) differentiation into naive T cells based on response to antigen: (

T cell), activated T cell (activated T cell), and memory T cellCell (memory T cell). CD4+ T cells promote proliferation and differentiation of B cells, T cells and other immune cells, and coordinate interactions between immune cells. CD8+ T cells are primarily responsible for the clearance of target cells by direct killing. Memory T cells are derived from effector T cells and may also be transformed directly from naive T cells. Memory T cells can be further classified as T_CM(Central memory T cell) and T_EM(Effective memory T cells). After being stimulated again by antigen, the central memory T cell can quickly generate effect and up-regulate CD40L expression, can secrete IL-2 in a large amount and multiply, is further differentiated into effector T cells, and can maintain immunological memory for a long time. The effector memory T cells produce an immune effect immediately after being stimulated by an antigen, exert a cytotoxic effect and secrete effector molecules, but have low IL-2 secretion ability and proliferation ability. Thus T_EMThe maintenance of immunological memory is short, and mainly plays a role in the first line of immune defense.

Different types of T-cells, which can be distinguished by differences in surface antigens, are, for example, characterized by CD45RA + CCR7+ CD62L^high，T _CMIs characterized by CD45RA-CCR7+ CD62L^highAnd T is_EMIs characterized by CD45RA-CCR7+ CD62L^low. Based on these characteristics of T cell subsets, researchers can use corresponding flow cytometry antibodies to analyze the proportion of each subset in T cells.

2.2 materials and methods

T cells can be divided into the following four subpopulations based on CD45RA and CCR7 expression: naive T cells, T_CMCells, T_EMCells, T_EMRAA cell. The primary T cells are CD45RA + CCR7+, T_CMThe cells are CD45RA-CCR7+ and T_EMThe cells are CD45RA-CCR 7-T_EMRAThe cells are CD45RA + CCR 7-. The lentivirus-transduced T cells (mock-CS1, SH3-CS1, mock-5.3 and SH3-5.3) and the non-lentivirus-transduced control T cells (T) of example 1 were treated with FITC-conjugated affinity Goat Anti-Mouse IgG (H + L), Pacific Blue^TManti-human CD8 Antibody (available from Biolegend),The cell surface was stained with FITC Mouse Anti-Human CD45RA (from BD Biosciences), PE Anti-Human CD197(CCR7) Antibody (from RND), and the results were analyzed by FlowJo software after detection by flow cytometry.

2.3 results

Analysis of the proportion of CD8+ in each group of T cells, and the initial T cells, T_CMCell and T_EMThe proportion of cells.

The proportion of CD8+ cells of control T cells not transduced with lentivirus was 59.3%, with an initial T cell proportion of 20.4%, T cells_CMCell proportion 24.0%, T_EMThe proportion of cells was 55.6%. The proportion of Mock-5.3 group CD8+ cells was 65.2%, where the initial T cell proportion was 10.7%, T_CMCell proportion 17.9%, T_EMThe proportion of cells was 71.4%. The proportion of SH3-5.3 group CD8+ cells was 66.9%, with an initial T cell proportion of 18.2%, T_CMCell proportion 18.1%, T_EMThe proportion of cells was 63.6%. The proportion of Mock-CS1 group CD8+ cells was 64.7%, with an initial T cell proportion of 2.7%, T_CMCell proportion 15.0%, T_EMThe proportion of cells was 82.3%. The proportion of SH3-CS1 group CD8+ cells was 65.5%, wherein the initial T cell proportion was 8.7%, T_CMCell proportion 14.5%, T_EMThe cell proportion was 81.5% (Table 2).

TABLE 2 composition of cell subsets of each experimental group

2.4 discussion

T in mock-5.3, SH3-5.3, mock-CS1 and SH3-CS1 cells_EMCell proportion was higher than that of control T cells not transduced with lentivirusThe rise, and the levels of increase in mock-CS1 and SH3-CS1 were more pronounced, presumably due to CS1-CAR T-cell suicide leading to such results (FIG. 11).

CAR T cell in vitro killing experiment

3.1 general description

(1) In this example, the tumoricidal effect of CAR T cells was determined by the calcein AM ester (calcein-AM) release method. Calcein-AM is a cell staining reagent that can fluorescently label cells, and its methyl acetate is highly lipophilic and can pass through cell membranes. In living cells, Calcein-AM is cleaved by intracellular esterases to form Calcein, which is then retained in the cells. Calcein can emit green fluorescence. When the target cells are lysed, calcein is released into the supernatant. Response to the tumoricidal Effect of CAR T cells by measuring the fluorescence intensity of calcein in the supernatant¹⁶。

(2) The MM1S cell line expresses CS1 and BCMA, which are positive targets for CS1-CAR T cells and BCMA-CAR T cells. B-K562 is a K562 cell line overexpressing BCMA. C-K562 is a K562 cell line overexpressing CS 1. Neither CS1 nor BCMA was expressed in the K562 cell line.

3.2 materials method

The target cells MM1S (purchased from the cell bank of the Committee for type culture Collection of Chinese academy of sciences) and the target cells prepared in example 2 (B-K56, C-K562 and K562) were taken. After counting, the cells were washed twice with PBS (purchased from GIBCO) containing 5% FBS (purchased from GIBCO). Adjusting the cell density to 1X 10⁶And/ml. Add 10. mu.l of Calcein-AM (from Alatin) to 1ml of cell suspension, mix well, incubate at 37 ℃ for 30min in the dark. Effector cells prepared in example 1 (mock-5.3, SH3-5.3, mock-CS1, and SH3-CS1) and control T cells were counted, after which an appropriate amount of cells were washed twice with PBS containing 5% FBS. CAR + T cell density was adjusted to 2.5X 10 based on measured transfection efficiency⁶And/ml. The effector cells and the target cells are respectively expressed by the following effector cell numbers: the number of target cells was 50: 1. 10: 1. 2:1, co-cultured in 96-well plates. The target cells were co-cultured with PBS as a spontaneous release group, and the target cells were mixed with a lysis solution (20mM sodium borate, 0.1% Tr)iton X-100, pH 9.0) co-cultured group as maximum release group. After 3 hours of incubation, the supernatant was transferred to another 96-well plate by centrifugation. The microplate reader (model: Perkin Elmer VictorX3) parameters, excitation light wavelength 485/20, and emission light wavelength 530/25 were set. Reading the fluorescence value F of each hole by a microplate reader, and calculating the tumor killing efficiency according to the following formula: lysis ═ 100% (F experimental wells-F spontaneous release)/(F maximal release-F spontaneous release).

3.3 results

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

The tumor killing efficiency of SH3-5.3 cells to MM1S cells is 79.89%, 60.30% and 40.15% respectively, the tumor killing efficiency to K562 cells is 4.55%, 7.14% and 3.67% respectively, the tumor killing efficiency to B-K562 cells is 92.52%, 68.68% and 24.64% respectively, and the tumor killing efficiency to C-K562 cells is 1.18%, 4.74% and 0.62% respectively.

The tumor killing efficiency of mock-CS1 on MM1S cells is 85.42%, 66.84% and 52.77% respectively, the tumor killing efficiency on K562 cells is 28.84%, 30.23% and 15.74% respectively, the tumor killing efficiency on B-K562 cells is 7.31%, 2.70% and 1.78% respectively, and the tumor killing efficiency on C-K562 cells is 99.07%, 79.47% and 36.38% respectively according to the three proportions.

The tumor killing efficiency of SH3-CS1 on MM1S cells is 90.58%, 75.65% and 42.52% respectively, the tumor killing efficiency on K562 cells is 21.39%, 17.35% and 5.84% respectively, the tumor killing efficiency on B-K562 cells is 7.36%, 8.11% and 0.71% respectively, and the tumor killing efficiency on C-K562 cells is 100.00%, 76.00% and 24.69% respectively according to the three proportions.

Control T cells that were not transduced with lentivirus had no significant killing effect on all four cell lines. The tumor killing efficiency of the control T cells which are not transduced with lentivirus at three ratios to MM1S cells is 29.98%, 13.90% and 2.87%, the tumor killing efficiency to K562 cells is 12.94%, 8.21% and 4.49%, the tumor killing efficiency to B-K562 cells is 10.20%, 7.37% and 4.99%, and the tumor killing efficiency to C-K562 cells is 9.65%, 5.07% and 1.23%.

TABLE 3 CAR T cell in vitro killing test results

3.4 discussion

mock-5.3 cells and SH3-5.3 cells were able to specifically kill BCMA-positive target cells (MM1S and B-K562), but the killing effect of SH3-5.3 cells on MM1S was slightly reduced compared to mock-5.3 cells. It is likely that knockdown of CS1 has some effect on the killing function of T cells, but further experiments are still needed to confirm. mock-CS1 cells and SH3-CS1 cells can kill CS1 positive target cells (MM1S and C-K562) specifically, and no phenomenon that SH3-CS1 tumoricidal effect is weakened is observed, and the adverse effect () of knocking down CS1 on the killing function of T cells is counteracted by reducing the suicide of CS1-CAR T cells by knocking down CS 1.

Expression of CD107a

4.1 overview

CD8+ T cells contain high concentrations of cytotoxic particles in the form of vesicles containing perforin and granzyme, among others. 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, toxic particles will reach the cytoplasmic surface of the cell membrane and fuse with the cell membrane, causing the release of the particle contents, ultimately leading to the death of the target cells^17,18. As degranulation occurs, CD107a is transported to the cell membrane surface, so that expression of T cell membrane surface CD107a can reflect T cell degranulation level, and detection of T cell surface CD107a can reflect T cell degranulation levelResponding to the effect of T cell lysis target cells.

4.2 materials and methods

CAR T cells prepared in example 1 (mock-5.3, SH3-5.3, mock-CS1 and SH3-CS1), control T cells not transduced with lentivirus, and various target cells were taken. The cells were counted and all cell densities were adjusted to 2X10⁶And/ml. Mu.l of each effector cell was co-cultured with 250. mu.l of a different target cell in a 24-well plate (5X 10 numbers of effector and target cells per well)⁵). Effector cell wells without target cells were set. Use of IL-2 free CTS in Co-culture^TM OpTmizer ^TMAnd (4) a culture medium. anti-CD107a flow antibody (from BD Biosciences) was added at 20. mu.l/ml and incubated in an incubator at 37 ℃. 1h later monesin (from BD Biosciences), 3. mu.l/well, and after further incubation for 3h additional PE/Cy7anti-human CD3 Antibody (from Biolegend), Pacific Blue^TMAnti-Human CD8 Antibody (from Biolegend), PE Mouse Anti-Human CD107a (from BD Biosciences) stained the cells and analyzed for the levels of CD3+ CD8+ cell membrane surface CD107 a.

4.3 results

After cocultivation 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-culturing SH3-5.3 cells with K562, MM1S, B-K562 and C-K562 cells, CD107a expression was 10.7%, 24.1%, 32.4% and 10.5%, respectively. After cocultivation of mock-CS1 cells with K562, MM1S, B-K562 and C-K562 cells, CD107a expression was 12.1%, 32.0%, 11.7% and 34.2%, respectively. CD107a expression after co-culture of SH3-CS1 cells with K562, MM1S, B-K562 and C-K562 cells was 13.2%, 35.8%, 11.3% and 46.4%, respectively

CD107a expression was 12.3%, 49.6%, 57.8% and 15.4% after co-culture of control T cells with K562, MM1S, B-K562 and C-K562, 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 the level of degranulation of SH3-5.3 cells was reduced compared to mock-5.3 cells. mock-CS1 and SH3-CS1 specifically degranulated when co-cultured with CS1 positive target cells (MM1S and C-K562), and no reduction in SH3-CS1 degranulation levels was observed, it is still possible that CS1-CAR T cell suicide, which knockdown CS1, was reduced, offsetting its adverse effects (fig. 13).

5. Expression of immune checkpoint molecules

5.1 overview

Immune checkpoints, which can be simply defined as signaling molecules on the surface of T cells that inhibit T cell activation and prevent T cells from participating in an immune response. T cells, when activated, upregulate the expression of inhibitory receptors such as PD-1 and CTLA-4 or ligands on APC that are upregulated by pro-inflammatory cytokines (e.g., PD-1 against PDL1 and PD-L2) are involved in inhibiting TCR signaling. These negative feedback mechanisms prevent excessive T cell activation and minimize pro-inflammatory damage to the host.

5.2 materials method

CAR T cells prepared in example 1 (mock-5.3, SH3-5.3, mock-CS1 and SH3-CS1) and control T cells not transduced with viruses were treated with PE anti-human CD279(PD-1) Antibody (from Bioleged), PE anti-human CD152(CTLA-4) Antibody (from Bioleged), APC anti-human CD366(Tim-3) Antibody (from Bioleged), APC anti-human CD223(LAG-3) Antibody (from Bioleged), Pacific Blue^TMAnti-human CD8 Antibody and FITC-conjugated affinity Goat Anti-Mouse IgG (H + L) staining, flow cytometry determination, and analysis of data.

5.3 results

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

The PD-1 and CTLA-4 expression of the control T cells not transduced with lentivirus were 25.8% and 50.4%, respectively, and the Mean Fluorescence Intensity (MFI) of TIM-3 of CD8+ cells was 53383 and 17100, respectively.

5.3 discussion

As can be seen from the above results, the expressions of SH3-CS1 in both PD-1 and CTLA-4 groups were lower than those in mock-CS1 group. Since there was no significant difference in the positive rates of CAR + CD8+ T cell surface TIM-3 and LAG3 in mock-CS1 and SH3-CS1 groups, the inventors further compared the MFI of the two groups and found that the MFI of TIM3 and LAG3 in SH3-CS1 groups were lower than that in mock-CS1 group. Immune checkpoints such as PD-1, CTLA-4, TIM-3 and LAG3 are markers of T cell depletion, with lower expression of the four above markers for the SH3-CS1 group, suggesting that CAR T cells with CS1 knockdown are less depleted due to reduced suicide, indicating that CS1-CAR T cells with CS1 knockdown may have better therapeutic potential (figure 14).

While the invention has been described in detail and with reference to specific examples thereof, it will be apparent to one skilled in the art that various changes in the examples or embodiments may be made therein without departing from the spirit and scope of the invention and are therefore considered to be within the scope of the invention.

Reference documents:

1.Kochenderfer,J.N.et al.Chemotherapy-Refractory Diffuse Large B-Cell Lymphoma and Indolent B-Cell Malignancies Can Be Effectively Treated With Autologous T Cells Expressing an Anti-CD19Chimeric Antigen Receptor.Journal of Clinical Oncology 33,540–549(2015).

2.Maude,S.L.et al.Chimeric Antigen Receptor T Cells for Sustained Remissions in Leukemia.New England Journal of Medicine 371,1507–1517(2014).

3.Ali,S.A.et al.T cells expressing an anti-B-cell maturation antigen chimeric antigen receptor cause remissions of multiple myeloma.Blood 128,1688–1700(2016).

4.Chu,J.et al.Genetic Modification of T Cells Redirected toward CS1Enhances Eradication of Myeloma Cells.Clinical Cancer Research 20,3989–4000(2014).

5.Gogishvili,T.et al.SLAMF7-CAR T cells eliminate myeloma and confer selective fratricide of SLAMF7+normal lymphocytes.Blood 130,2838–2847(2017).

6.Boles K.S.et al.Molecular cloning of CS1,a novel human natural killer cell receptor belonging to the CD2 subset of the immunoglobulin superfamily.Immunogenetics 52,302-307(2001).

7.Bouchon,A.et al.Activation of NK cell-mediated cytotoxicity by a SAP-independent receptor of the CD2 family.Journal of Immunology 167,5517-5521(2001).

8.Comte,D.et al.SLAMF7 engagement restores defective effector CD8+T cells activity in response to foreign antigens in systemic lupus erythematosus.Arthritis&Rheumatology 69,1035–1044(2017).

9.Kumaresan,P.R.et al.CS1,a novel member of the CD2 family,is homophilic and regulates NK cell function.Molecular Immunology 39,1-8(2002).

10.Cruzmunoz,M.E.et al.Influence of CRACC,a SLAM family receptor coupled to the adaptor EAT-2,on natural killer cell function.Nature Immunology 10,297-305(2009).

11.Stark,S.,et al.B4(CD244)NTB-A and CRACC(CS1)stimulate cytotoxicity but no proliferation in human NK cells.international immunology 18,241-7(2006).

12.Kiesgen,S.,Chicaybam,L.,Chintala,N.K.&Adusumilli,P.S.Chimeric antigen receptor(CAR)T-cell therapy for thoracic malignancies.Journal of Thoracic Oncology Official Publication of the International Association for the Study of Lung Cancer 13,(2017).

13.Ninomiya,S.et al.Tumor indoleamine 2,3-dioxygenase(IDO)inhibits CD19-CAR T cells and is downregulated by lymphodepleting drugs.Blood 125,3905–3916(2015).

14.Wilkie,S.et al.Retargeting of Human T Cells to Tumor-Associated MUC1:The Evolution of a Chimeric Antigen Receptor.The Journal of Immunology 180,4901–4909 (2008).

15.Tai,Y.-T.et al.Anti-CS1 humanized monoclonal antibody HuLuc63 inhibits myeloma cell adhesion and induces antibody-dependent cellular cytotoxicity in the bone marrow milieu.Blood 112,1329–1337(2008).

16.Biddison,W.E.,Lichtenfels,R.,Adibzadeh,M.&Martin,R.Measurement of Polyclonal and Antigen-Specific Cytotoxic T Cell Function.Current Protocols in Immunology 17,7.17.1-7.17.14(1996).

17.Aktas,E.,Kucuksezer,U.,S.,Erten,G.&Deniz,G.Relationship between CD107a expression and cytotoxic activity.Cellular Immunology 254,149–154(2009).

18.Betts,M.R.et al.Sensitive and viable identification of antigen-specific CD8+T cells by a flow cytometric assay for degranulation.Journal of Immunological Methods 281,65–78(2003).

Claims (31)

1. a T cell expressing a 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 cells themselves also express the target antigen, and to prevent the T cells from killing each other, the expression of the target antigen by the T cells is down-regulated.
2. 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.
3. The T cell of claim 1, wherein the target cell is a tumor cell.
4. The T cell of claim 3, wherein the tumor cell is a multiple myeloma cell.
5. The T cell of claim 1, wherein the target antigen is CS 1.
6. The T cell of any one of claims 1 to 5, wherein the T cell down-regulates expression of the target antigen by expressing siRNA.
7. The T cell of claim 6, wherein the siRNA is generated from an shRNA expressed by the T cell.
8. The T cell of claim 7, wherein the target nucleic acid sequence of the shRNA comprises a sequence as set forth in SEQ ID NO: 29.
9. The T cell of claim 7, wherein the coding sequence of the shRNA comprises the sequence set forth in SEQ ID NO: 28.
10. The T cell of claim 5, wherein the single chain antibody has the amino acid sequence as set forth in SEQ ID NO: 20, or a pharmaceutically acceptable salt thereof.
11. The T cell of claim 2, wherein the amino acid sequence of the chimeric antigen receptor comprises, in order from N-terminus to C-terminus, a CD8 a signal peptide, the single chain antibody, a CD8 hinge region, a CD28 transmembrane region, CD28 and 4-1BB intracellular co-stimulatory domains, and a CD3 ζ intracellular signaling domain.
12. The T cell of claim 7, transformed with an expression vector comprising the coding sequence for the chimeric antigen receptor and an expression vector comprising the coding sequence for the shRNA, or transformed with an expression vector comprising the coding sequence for the chimeric antigen receptor and the coding sequence for the shRNA.
13. An expression vector for expression in a T cell comprising a coding sequence for a chimeric antigen receptor and a coding sequence for an shRNA, wherein the chimeric antigen receptor recognizes a target antigen on the surface of a target cell and the shRNA downregulates expression of the target antigen in the T cell by siRNA produced thereby.
14. The expression vector of claim 13, wherein the chimeric antigen receptor comprises an extracellular domain comprising a single chain antibody derived from an antibody against the target antigen, a transmembrane domain, and an intracellular domain.
15. The expression vector of claim 13, wherein the coding sequence for the shRNA is under the control of an H1 promoter.
16. The expression vector of claim 13, which is a lentiviral expression vector.
17. The expression vector of claim 16, which has pLVX-EF1 a-IRES-Puro as a backbone vector.
18. The expression vector of claim 13, wherein the target cell is a tumor cell.
19. The expression vector of claim 18, wherein the tumor cell is a multiple myeloma cell.
20. The expression vector of claim 13, wherein the target antigen is CS 1.
21. The expression vector of claim 20, wherein the target nucleic acid sequence of the shRNA comprises the sequence set forth in SEQ ID NO: 29.
22. The expression vector of claim 20, wherein the coding sequence of the shRNA comprises the sequence set forth in SEQ ID NO: 28.
23. The expression vector of claim 14, wherein said single chain antibody has the amino acid sequence as set forth in SEQ ID NO: 20, or a pharmaceutically acceptable salt thereof.
24. A method of making a T cell expressing a chimeric antigen receptor comprising transforming a T cell with the expression vector of any one of claims 13 to 23.
25. A method of preventing T cells expressing a chimeric antigen receptor from killing each other, comprising downregulating expression in the T cells of a target antigen targeted by the chimeric antigen receptor.
26. The method of claim 25, wherein the target antigen is CS 1.
27. The method of claim 25 or 26, wherein the downregulating comprises allowing the T cell to express an shRNA that generates an siRNA that inhibits expression of the target antigen in the T cell.
28. The method of claim 27, by transforming the T cell with an expression vector comprising a coding sequence for the shRNA.
29. A method of treating multiple myeloma in a subject, comprising administering to the subject a T cell that expresses a chimeric antigen receptor that targets CS1 on the surface of the multiple myeloma cell, and that further expresses an shRNA for inhibiting expression of CS1 in the T cell.
30. Use of a T cell according to any one of claims 1 to 12 or an expression vector according to any one of claims 13 to 23 in the manufacture of a medicament for the treatment of a disease caused by proliferation of CS1 positive cells.
31. The use of claim 30, wherein the disease is multiple myeloma or plasma cell leukemia.

Images