CN107337736B - OCTS-CAR dual-targeting chimeric antigen receptor, encoding gene, recombinant expression vector and its construction and application - Google Patents

Abstract

The present invention provides an OCTS-CAR dual-targeting chimeric antigen receptor based on OCTS technology, an encoding gene, an OCTS-CAR-T recombinant expression vector, and a construction method and application thereof. The OCTS-CAR dual-targeting chimeric antigen receptor includes CD8 leader membrane receptor signal peptide, dual antigen binding region, CD8 Hinge chimeric receptor hinge, CD8 Transmembrane chimeric receptor transmembrane region, CD28 chimeric Co-receptor co-stimulator, CD134 chimeric receptor co-stimulator, and TCR chimeric receptor T cell activation domain, wherein the dual antigen binding region comprises the heavy chain VH and light chain of two single chain antibodies linked in a certain way VL, Inner-Linker of intra-antibody hinge and Inter-Linker of single-chain antibody hinge, the two single-chain antibodies refer to BCMA single-chain antibody, CD319 single-chain antibody, CD38 single-chain antibody, PDL1 single-chain antibody, CD123 single-chain antibody Any pairwise combination between antibodies. In addition, a gene encoding the OCTS-CAR dual-targeting chimeric antigen receptor, a recombinant expression vector and a construction method and application thereof are also provided.

Description

OCTS-CAR dual-targeting chimeric antigen receptor, encoding gene, recombinant expression vector and its Build and apply

technical field

The invention belongs to the technical field of tumor immunotherapy, and in particular relates to a CAR dual-targeting chimeric antigen receptor, encoding gene, OCTS-CAR recombinant expression vector based on OCTS technology for malignant tumor immunotherapy, and a construction method and application thereof .

Background technique

The theoretical basis of tumor immunotherapy is the ability of the immune system to recognize tumor-associated antigens and regulate the body's attack on tumor cells (eg, highly specific cytolysis). In the 1950s, Burnet and Thomas put forward the theory of "immune surveillance", which believed that the mutated tumor cells that often appeared in the body could be recognized and eliminated by the immune system, which laid a theoretical foundation for tumor immunotherapy [Burnet FM. Immunological aspects of malignantdisease]. . Lancet, 1967;1:1171-4]. Subsequently, various tumor immunotherapies, including cytokine therapy, monoclonal antibody therapy, adoptive immunotherapy, vaccine therapy, etc., were successively applied in clinical practice.

In 2013, a more advanced tumor immunotherapy, CAR-T therapy, was successfully used in the clinic and showed unprecedented clinical efficacy. CAR-T, the full name is Chimeric Antigen Receptor T-Cell Immunotherapy, chimeric antigen receptor T cell immunotherapy. The therapy is to introduce a chimeric molecule composed of promoter, antigen recognition region, costimulatory factor, effector region, etc. into the genome of T cells by means of transgenic, so that T cells can recognize, signal transduce, and transduce target cells. Killing and other functions are integrated to achieve specific killing of target cells [Eleanor J. Cheadle, et al. CAR T cells: driving the road from the laboratory to the clinic. Immunological Reviews 2014. Vol. 257: 91–106]. The most clinically advanced CAR-T therapy is Novartis' CLT019. Using CLT019 to treat patients with relapsed and refractory acute lymphoblastic leukemia, the six-month tumor progression-free survival rate reached 67%, and the longest response time was more than two years. . Headquartered in Shanghai, China, Shanghai Ucardi Biomedical Technology Co., Ltd. cooperated with the hospital. As of February 2017, a total of 36 patients with relapsed and refractory acute lymphoblastic leukemia were treated, of which 24 were complete, and the remission rate reached 66.6%. Therefore, CAR-T cell therapy is a subversive breakthrough in anti-cancer research, and may be one of the most likely means of curing cancer, and was rated as the top ten scientific and technological breakthroughs in 2013 by "Science" magazine.

CAR-T is currently effective in the treatment of several types of hematological tumors such as B-lymphocytic leukemia, but there are also some limitations. For example, currently a chimeric antigen receptor can only recognize one antigen target, but tumor cells are a In a complex population, after the tumor cells containing the corresponding antigen are eliminated, the tumor cells without the corresponding antigen will rapidly proliferate, leading to tumor recurrence after a period of time.

To enable CAR-T recognition to recognize two antigens at the same time, there are two options: one is to construct two sets of chimeric antigen receptors into a lentiviral transgenic vector, and to transduce the two sets of chimeric antigen receptors at one time into primary T lymphocytes; the second is to use two lentiviral transgenic vectors to transduce twice, and the two groups of chimeric antigen receptors are respectively transduced into primary T lymphocytes.

The disadvantage of scheme 1 is that it occupies the valuable capacity of the lentiviral transgenic vector, which is not conducive to loading other functional elements; the packaging efficiency of the transgenic vector is low; the gene transduction efficiency is very low, and it is difficult to transduce into primary T lymphocytes.

The disadvantage of scheme 2 is that two transductions are required, the overall efficiency of the two transductions is low, the transduction cycle is long, and the primary cells are easily senescent, resulting in a decline in proliferation ability, a decline in killing function, and an impact on the efficacy of tumor removal.

As the key research object of tumor immunotherapy, the tumor cells of multiple myeloma originate from plasma cells in the bone marrow, and plasma cells are cells that develop to the final functional stage of B lymphocytes. Malignant plasma cell tumors that are refractory to treatment. Usually the patient's survival period is 3 years, even as short as 6-12 months.

However, studies have found that certain substances exist stably or are highly expressed on the surface of B cells or malignant tumor cells, and can be used as cell markers or targets. like:

As a marker, BCMA (B-cell maturation antigen) is selectively expressed on the surface of B cells (including multiple myeloma cells), and can be used as a target for CAR-T cells to guide CAR-T cells to kill tumor cells (B-cell Maturation Antigen). is a Promising Target for Adoptive T cell Therapy of Multiple Myeloma. Robert O. Carpenter, et al. Clin Cancer Res. 2013 April 15;19(8):2048–2060.).

CD319, also known as CS1 and SLAMF7, is a stable cell surface marker expressed in both normal and malignant plasma cells. CD319 is an extremely stable antigen. Even in malignant plasma cell lines isolated by rough means, even after cryopreservation, the expression can still be stably detected [Frigyesi I, AdolfssonJ, Ali M, Christophersen MK, Johnsson E, Turesson I, Gullberg U, Hansson M, Nilsson B (Feb 2014). "Robust isolation of malignant plasma cells in multiple myeloma". Blood. 123(9): 1336–40.].

CD38, also known as cyclic ADP ribose hydrolase, is detected on the surface of many immune cells, including B cells and NK cells. CD38 is a marker of cell activation that has been linked to HIV infection, leukemia, myeloma, solid tumors, and more. Antibodies targeting CD38 are currently used clinically for the treatment of multiple myeloma [Lokhorst, Henk M.; Plesner, Torben; Laubach, Jacob P.; Nahi, Hareth; Gimsing, Peter; Hansson, Markus; Minnema, Monique C. Lassen, Ulrik; Krejcik, Jakub (2015-09-24). "Targeting CD38 with Daratumumab Monotherapy in Multiple Myeloma". The New England Journal of Medicine. 373(13): 1207–1219.].

CD123 is the alpha chain of the human interleukin 3 receptor and is expressed on the surface of most acute myeloid leukemia (AML) cells and many hematopoietic cells. Its expression will gradually increase over time, and acute myeloid leukemia is probably due to the cloning and evolution of hematopoietic stem cells in the early stage of leukemia, and targeting CD123 can play a role in myeloablation ( SaarGill, Carl H. June et al. Preclinical targeting of human acute myeloid leukemia and myeloablation using chimeric antigen receptor-modified Tcells. Blood. 2014;123(15):2343-2354.).

PD-L1 is overexpressed in most cancer tissues, including NSCLC, melanoma, breast cancer, glioma, lymphoma, leukemia and various urological tumors, digestive tract tumors, germline tumors, etc. [Intlekofer AM, ThompsonCB. At the bench:preclinical rationale for CTLA-4and PD-1blockade as cancerimmunotherapy[J].J Leukoc Biol,2013,94(1):25-39.]. Parsa found that IFN-γ secreted abnormally by T cells in mouse and human tumor cells, IFN-γ can induce high expression of PD-L1 on tumor cells [Ding H, Wu X, Wu J, et al.Delivering PD- 1 inhibitory signal concomitant with blocking ICO Sco-stimulation suppresses lupus-like syndrome in autoimmune BXSB mice[J]. Clin Immunol, 2006, 118(2/3): 258-267.]. The high expression of PD-L1 can regulate the expression of cell cycle checkpoint proteins and cell proliferation-related proteins by inhibiting the RAS and PI3K/AKT signaling pathways, and ultimately lead to the inhibition of T cell proliferation [11]. Dong et al. also found in vitro experiments and mouse models that activation of the PD-1/PD-L1 signaling pathway can induce apoptosis of specific CTLs, reduce the sensitivity of CTLs to cytotoxic killing effects, and promote immune escape of tumor cells [Dong H , Strome SE, Salomao DR, et al. Tumor-associated B7-H1promotes T-cell apoptosis: apotential mechanism of immune evasion[J]. Nat Med, 2002, 8(8):793-800.].

SUMMARY OF THE INVENTION

The present invention is based on the above findings, and aims to provide a CAR dual-targeting chimeric antigen receptor, encoding gene, and OCTS-CAR recombination based on OCTS technology for the immunotherapy of malignant tumors, especially multiple myeloma. Expression vector and its construction method and application.

The full name of OCTS is One CAR with Two ScFvs. Through the connection of tandem OCTS (Series OCTS) or turn OCTS (Turn OCTS), the two scFvs are integrated into a chimeric molecule (shown in Figure 1), giving T lymphocytes The ability to recognize two tumor antigens in an HLA-independent manner, compared with traditional CAR-T cells, can recognize a wider range of targets, further expanding the scope of tumor cell clearance.

The basic design of OCTS includes two tumor-associated antigen (TAA) binding regions (usually derived from the scFv segment of the antigen-binding region of a monoclonal antibody), an extracellular hinge region, a transmembrane region, and two cellular Internal signal transduction domain and a response element domain. The scFv region is a key determinant for the specificity, efficacy, and safety of the genetically engineered T cells themselves.

The OCTS-CAR-T technology used in the present invention is based on the current CAR-T cell therapy, through the optimization and transformation of the chimeric antigen receptor (CAR) structure, so that the chimeric antigen receptor can recognize two antigens , which greatly expands the recognition range of CAR-T cells, clears the tumor population more thoroughly, and has a longer lasting effect.

The first aspect of the present invention provides a CAR dual-targeting chimeric antigen receptor based on OCTS technology, including CD8leader membrane receptor signal peptide, double antigen binding region, CD8Hinge chimeric receptor hinge, CD8Transmembrane connected in series in sequence Chimeric receptor transmembrane domain, CD28 chimeric receptor costimulator, CD134 chimeric receptor costimulator, and TCR chimeric receptor T cell activation domain. Wherein, the double-antigen-binding region includes the heavy chain VH and light chain VL of two single-chain antibodies connected in a certain way, the inner-antibody hinge Inner-Linker, and the single-chain antibody hinge Inter-Linker; the two single-chain antibodies refer to It is any combination of BCMA single-chain antibody, CD319 single-chain antibody, CD38 single-chain antibody, PDL1 single-chain antibody, and CD123 single-chain antibody.

Further, the OCTS-CAR dual-targeting chimeric antigen receptor of the present invention also has the following technical features: two single-chain antibodies are connected in series or corner connection.

The second aspect of the present invention provides a gene encoding the above-mentioned CAR dual-targeting chimeric antigen receptor, and the sequence numbers of the genes encoding the above-mentioned parts are as follows: CD8leader membrane receptor signal peptide (SEQ ID NO.15), BCMA single chain antibody light chain VL (SEQ ID NO. 16), BCMA single chain antibody heavy chain VH (SEQ ID NO. 17), CD319 single chain antibody light chain VL (SEQ ID NO. 18), CD319 single chain antibody heavy chain Chain VH (SEQ ID NO. 19), CD38 single chain antibody light chain VL (SEQ ID NO. 20), CD38 single chain antibody heavy chain VH (SEQ ID NO. 21), PDL1 single chain antibody light chain VL (SEQ ID NO. .22), PDL1 single-chain antibody heavy chain VH (SEQ ID NO.23), CD123 single-chain antibody light chain VL (SEQ ID NO.24), CD123 single-chain antibody heavy chain VH (SEQ ID NO.25), antibody Inner-Linker (SEQ ID NO.26), single-chain antibody inter-hinge Inter-Linker (SEQ ID NO.27), CD8Hinge chimeric receptor hinge (SEQ ID NO.28), CD8Transmembrane chimeric receptor transmembrane region (SEQ ID NO.29), CD28 chimeric receptor costimulator (SEQ ID NO.30), CD134 chimeric receptor costimulator (SEQ ID NO.31), TCR chimeric receptor T cell activation domain ( SEQ ID NO. 32).

The third aspect of the present invention provides an OCTS-CAR recombinant expression vector, which has the following technical features: including an expression vector, a human EF1α promoter, and a gene encoding a CAR-T dual-targeting chimeric antigen receptor. Wherein, the gene sequence of the human EF1α promoter is shown in SEQ ID NO.14, and the genes encoding each part of the CAR-T dual-targeting chimeric antigen receptor are as described above. Wherein, the gene sequence of the antigen-binding region includes the gene sequences of the light chain VL and heavy chain VH of any two single-chain antibodies of BCMA, CD319, CD38, PDL1, and CD123.

Further, the present invention also provides another OCTS-CAR recombinant expression vector. Compared with the above-mentioned recombinant expression vector, the recombinant expression vector also includes a gene encoding PDL1 single-chain antibody, and its gene sequence is shown in SEQ ID NO.33 .

The expression vector backbone used in the present invention is the third-generation lentiviral vector (shown in Figure 2A), and the 3'SIN LTR removes the U3 region, eliminating the possibility of self-replication of the lentiviral vector, and greatly improving the safety; The cPPT and WPRE elements improve the transduction efficiency and the expression efficiency of the transgene; the RSV promoter ensures the continuous and efficient transcription of the core RNA during lentiviral vector packaging; the human EF1α promoter is used to enable the CAR gene to grow in the human body Time continues to express.

The lentiviral expression vector includes ampicillin resistance gene AmpR sequence, prokaryotic replicon pUC Ori sequence, viral replicon SV40Ori sequence, RSV promoter, lentivirus 5terminal LTR, lentivirus 3terminal Self-Inactivating LTR, Gag cis element, RRE cis-element, env cis-element, cPPT cis-element, ZsGreen1 green fluorescent protein, IRES ribosomal binding sequence, eWPRE enhanced woodchuck hepatitis B virus post-transcriptional regulatory element.

The present invention combines human EF1α promoter, CD8leader chimeric receptor signal peptide, any two single-chain antibody light chain VL and heavy chain VH of BCMA, CD319, CD38, PDL1 and CD123, the inner hinge of the antibody Inner-Linker, single chain antibody Chain Antibody Inter-Linker, CD8Hinge Chimeric Receptor Hinge, CD8Transmembrane Chimeric Receptor Transmembrane Region, CD28 Chimeric Receptor Costimulator, CD134 Chimeric Receptor Costimulator, TCR Chimeric Receptor T Cell Activation Domain, and the expression gene of PDL1 single-chain antibody were constructed into a recombinant lentiviral vector, and the expression of the entire OCTS structural gene was promoted by the human EF1α promoter.

The CD8leader chimeric receptor signal peptide is located at the N-terminus of the OCTS protein, which is used to guide the localization of the OCTS protein to the cell membrane; the heavy and light chains of any two groups of single-chain antibodies are combined with each other to form a dual-antigen recognition region, which is used to recognize the corresponding target antigen; The CD8Hinge chimeric receptor hinge is used to anchor the scFv to the outside of the cell membrane; the CD8Transmembrane chimeric receptor transmembrane region is used to fix the entire chimeric receptor on the cell membrane; the CD28 chimeric receptor costimulator is used to stimulate T lymphocytes Cell activation in vitro and tumor cell killing in vivo; CD134 chimeric receptor costimulator is used to promote T lymphocyte proliferation and factor secretion, enhance tumor immunity, and facilitate long-term survival of memory T cells; TCR chimeric receptor T cell activation The PDL1 domain is used to activate the expression of downstream signaling pathways; the single-chain antibody of PDL1 can effectively block PDL1 and block the immune negative regulation signaling pathway, which can be clinically used to inhibit the immune escape of tumors and improve the efficacy of CAR-T cell immunotherapy.

When the antigen recognition region binds to the target antigen, the signal is transmitted into the cell through the chimeric receptor, resulting in a series of T cell proliferation, increased cytokine secretion, increased anti-apoptotic protein secretion, delayed cell death, and lysis of target cells. biological effects.

In the present invention, BCMA single-chain antibody light chain VL, BCMA single-chain antibody heavy chain VH, CD319 single-chain antibody light chain VL, CD319 single-chain antibody heavy chain VH, CD38 single-chain antibody light chain VL, CD38 single-chain antibody heavy chain VH, PDL1 scFv light chain VL, PDL1 scFv heavy chain VH, CD123 scFv light chain VL, CD123 scFv heavy chain VH, PDL1 scFv with nucleotide sequence >=80 as indicated Nucleotide sequences of % homology (preferably, >= 90% homology; etc. preferably, >= 95% homology; most preferably, >= 97% homology;).

In the present invention, BCMA single-chain antibody light chain VL, BCMA single-chain antibody heavy chain VH, CD319 single-chain antibody light chain VL, CD319 single-chain antibody heavy chain VH, CD38 single-chain antibody light chain VL, CD38 single-chain antibody heavy chain VH, PDL1 scFv light chain VL, PDL1 scFv heavy chain VH, CD123 scFv light chain VL, CD123 scFv heavy chain VH, PDL1 scFv with nucleotide sequences corresponding to the indicated nucleotide sequences Amino acid sequence >= 80% homology (preferably, >= 90% homology; etc. preferably, >= 95% homology; most preferably, >= 97% homology).

In the present invention, BCMA single-chain antibody light chain VL, BCMA single-chain antibody heavy chain VH, CD319 single-chain antibody light chain VL, CD319 single-chain antibody heavy chain VH, CD38 single-chain antibody light chain VL, CD38 single-chain antibody heavy chain VH, PDL1 single-chain antibody light chain VL, PDL1 single-chain antibody heavy chain VH, CD123 single-chain antibody light chain VL, CD123 single-chain antibody heavy chain VH, and PDL1 single-chain antibody have all undergone humanization transformation, which can effectively reduce the amount of human in vivo. The production of anti-mouse antibodies (HAMA) prolongs the half-life and effect of scFv.

The transgenic vector used in the present invention is a recombined replication-defective lentiviral vector, which can integrate exogenous fragments into the host gene, can be used once, cannot be replicated and propagated, and is safe and reliable.

The costimulatory factor region in the OCTS chimeric receptor of the present invention can be 4-1BB, ICOS, CD27, OX40, CD28, MYD88, IL1R1, CD70, TNFRSF19L, TNFRSF27, TNFRSF1OD, TNFRSF13B, TNFRSF18 and other tumor necrosis factor super A combination of one, two, three, several, and dozens of tumor necrosis factor receptor superfamily (TNFRSF).

The replication-defective lentiviral transgenic vector used in the present invention can be a second- or third-generation lentiviral transgenic vector.

A fourth aspect of the present invention provides a method for constructing an OCTS-CAR recombinant expression vector, comprising the following steps:

A. Ampicillin resistance gene AmpR sequence (SEQ ID NO.1), prokaryotic replicon pUC Ori sequence (SEQ ID NO.2), viral replicon SV40Ori sequence (SEQ ID NO.3), RSV promoter ( SEQ ID NO.4), lentivirus 5terminal LTR (SEQ ID NO.5), lentivirus 3terminal Self-Inactivating LTR (SEQ ID NO.6), Gag cis-element (SEQ ID NO.7), RRE cis-element ( SEQ ID NO.8), env cis-element (SEQ ID NO.9), cPPT cis-element (SEQ ID NO.10), ZsGreen1 green fluorescent protein (SEQ ID NO.11), IRES ribosome binding sequence (SEQ ID NO.11) NO.12), eWPRE-enhanced woodchuck hepatitis B virus post-transcriptional regulatory element (SEQ ID NO.13) is stored on the third-generation lentiviral backbone plasmid pLenti-3G basic, the method has been developed and constructed by our company, and published in " A CAR-T transgenic vector based on replication-defective recombinant lentivirus and its construction method and application "201610008360.5 patent;

B. Will encode CD8leader membrane receptor signal peptide, dual antigen binding region, CD8Hinge chimeric receptor hinge, CD8Transmembrane chimeric receptor transmembrane region, CD28 chimeric receptor costimulator, CD134 chimeric receptor costimulator and The gene of the T cell activation domain of the TCR chimeric receptor was cloned into the lentiviral backbone plasmid pLenti-3G basic through enzyme digestion, ligation and recombination reaction, and the third-generation OCTS-based recombinant lentiviral plasmids were obtained, such as pOCTS123BCMAs, pOCTS319BCMAs, pOCTS38BCMAs , pOCTS-PDL1BCMAs, pOCTS123BCMAt, pOCTS319BCMAt, pOCTS38BCMAt, pOCTS-PDL1BCMAt, etc., wherein the last letter "s" represents the tandem connection of two scFv segments, and the letter "t" represents the corner connection of two segments of scFv.

C. The recombinant lentiviral plasmids obtained in step B were co-transfected into HEK293T/17 cells with lentiviral packaging plasmids pPac-GP, pPac-R and membrane protein pEnv-G, and gene transcription was performed in HEK293T/17 cells After expression, the successfully packaged recombinant lentiviral vector will be released into the cell culture supernatant, and the supernatant of the contained recombinant lentiviral vector will be collected;

D. Purify the obtained recombinant lentivirus supernatant by suction filtration, adsorption, and elution column purification to obtain CAR-T recombinant lentivirus expression vectors, respectively (named lvOCTS123BCMAs, lvOCTS319BCMAs, lvOCTS38BCMAs, lvOCTS-PDL1BCMAs, lvOCTS123BCMAt , lvOCTS319BCMAt, lvOCTS38BCMAt, lvOCTS-PDL1BCMAt).

In addition, in step B, the gene encoding PDL1 single-chain antibody can also be cloned into the lentiviral backbone plasmid pLenti-3G basic through enzyme digestion, ligation, and recombination reactions together with other genes to obtain a third-generation OCTS-based recombination design. Lentiviral plasmids.

The fifth aspect of the present invention provides an OCTS-CAR-T cell, the OCTS-CAR-T cell is an OCTS-CAR recombinant expression vector introduced into the genome or an OCTS-based CAR dual-targeting chimeric antigen receptor Modified T lymphocytes.

The OCTS-CAR-T cells used in the present invention can be used in human clinical experiments after being produced in a GMP-level workshop.

The sixth aspect of the present invention provides the application of OCTS-CAR-T cells in the preparation of medicines for the treatment of malignant tumors.

Further, the malignant tumor therapeutic drug is a drug for treating malignant plasma cell tumor, acute myeloid leukemia, melanoma, breast cancer, glioma, lymphoma, urinary system tumor, digestive tract tumor or germline tumor.

The role and effect of the invention

The present invention adopts OCTS technology on the basis of the current traditional CAR-T cell therapy, through the optimization and transformation of the chimeric antigen receptor (CAR) structure, so that the chimeric antigen receptor can recognize two antigens, on the one hand, greatly expands the CAR - The recognition range of T cells, the elimination of tumor groups is more thorough, and the efficacy is more durable; on the other hand, batch cultivation of CAR-T cells is avoided, which greatly saves costs, thereby avoiding multiple re-infusion of different targeted CAR-T cells to patients. It saves the patient's economic expenditure, reduces the probability of recurrence, and indirectly improves the patient's quality of life.

Through the connection of series OCTS (Series OCTS) or turn OCTS (Turn OCTS), the two segments of scFv are integrated into a chimeric molecule, which not only endows T lymphocytes with the ability to recognize two tumor antigens in an HLA-independent manner, but also has the ability to recognize two tumor antigens in an HLA-independent manner. Traditional CAR-T cells can identify a wider range of targets and further expand the scope of tumor cell clearance. As OCTS-CAR-T is about to enter the clinical research stage, it marks that CAR-T cell therapy is about to enter the 2.0 era.

In addition, the BCMA single-chain antibody light chain VL, BCMA single-chain antibody heavy chain VH, CD319 single-chain antibody light chain VL, CD319 single-chain antibody heavy chain VH, CD38 single-chain antibody light chain VL, CD38 single-chain antibody heavy chain of the present invention are Chain VH, PDL1 single-chain antibody light chain VL, PDL1 single-chain antibody heavy chain VH, CD123 single-chain antibody light chain VL, CD123 single-chain antibody heavy chain VH, PDL1 single-chain antibody have all undergone humanization transformation, which can effectively reduce The production of human anti-mouse antibodies (HAMA) in vivo prolongs the half-life and effect of scFv, and increases the existence time of OCTS-CAR-T cells.

In addition, one or several combinations of co-stimulatory factors used in the present invention can increase the proliferation rate, survival time, killing efficiency, immune memory and other properties of the transduced cells.

Therefore, the OCTS-CAR-T cells of the present invention will provide a reliable guarantee for tumor cell therapy.

Description of drawings

1 is a schematic diagram of the CAR dual-targeting chimeric antigen receptor (OCTS) of the present invention, wherein (A) is a schematic diagram of a series OCTS (Series OCTS), and (B) is a schematic diagram of a turn OCTS (Turn OCTS).

Figure 2 is a schematic structural diagram of the lentiviral vector of the present invention; wherein (A) is a schematic structural diagram of the third-generation lentiviral vector used in the present invention, and (B) is a schematic structural comparison of the second-generation and third-generation lentiviral vectors.

Fig. 3 is the construction flow chart of the recombinant lentiviral vector of the present invention; wherein, Fig. (A) is a schematic structural diagram of the lentiviral backbone plasmid pLenti-3G basic; Fig. (B) is a schematic diagram of 8 OCTS plasmids; (C) Fig. is the structural schematic diagram of the lentiviral packaging plasmid pPac-GP plasmid; (D) is the structural schematic diagram of the lentiviral packaging plasmid pPac-R plasmid; (E) is the structural schematic diagram of the membrane protein plasmid pEnv-G.

Figure 4 is a schematic diagram of the element sequence of a CAR dual-targeting chimeric antigen receptor (OCTS), wherein, (A) is a schematic diagram of the structure of a series OCTS (Series OCTS), and (B) is the structure of a turn OCTS (Turn OCTS) Schematic.

Figure 5 is the recombinant lentiviral plasmids pOCTS123BCMAs (A), pOCTS319BCMAs (B), pOCTS38BCMAs (C), pOCTS-PDL1BCMAs (D), pOCTS123BCMAt (E), pOCTS319BCMAt (F), pOCTS38BCMAt (G), pOCTS-PDL1BCMAt (H) The enzyme digestion prediction map and the enzyme digestion agarose gel electrophoresis map. Among them, in Figures A to H, lane1 is the predicted cleavage map of the 1kb DNA ladder Marker; lane2 is the predicted cleavage map of the above recombinant lentiviral plasmids; lane3 is the 1kb DNA ladder Marker digestion agarose gel electrophoresis map; Lane4 is the agarose gel electrophoresis image of each recombinant lentiviral plasmid above.

Figure 6 is the titer detection result of the recombinant lentiviral vector.

Figure 7 is a schematic diagram of the construction process of the OCTS-CAR-T cell of the present invention, including the stages of isolation and culture, activation, gene transduction, and OCTS-CAR-T cell identification.

Figure 8 is the mycoplasma detection result of OCTS-CAR-T cells, lane1 is the DL2000 marker, and the bands from top to bottom are: 2kb, 1kb, 750bp, 500bp, 250bp, 100bp; lane2 is the positive control; lane3 is negative control; lane4 is PBS; lane5 is lysate; lane6 is OCTS123BCMAs-CAR-T cells; lane7 is OCTS319BCMAs-CAR-T cells; lane8 is OCTS38BCMAs-CAR-T cells; lane9 is OCTS-PDL1BCMAs-CAR-T cells cells; lane10 is OCTS123BCMAt-CAR-T cells; lane11 is OCTS319BCMAt-CAR-T cells; lane12 is OCTS38BCMAt-CAR-T cells; lane13 is OCTS-PDL1BCMAt-CAR-T cells.

Figure 9 shows the transduction efficiency and immunophenotyping results of OCTS-CAR-T cells detected by flow cytometry. Panel A shows the results of transduction efficiency of OCTS123BCMAs-CAR-T cells; Panel B shows the results of immunophenotyping of OCTS123BCMAs-CAR-T cells; Panel C shows the results of transduction efficiency of OCTS319BCMAs-CAR-T cells; Panel D shows the results of transduction efficiency of OCTS319BCMAs-CAR-T cells The results of immunophenotyping of CAR-T cells; Figure E shows the results of the transduction efficiency of OCTS38BCMAs-CAR-T cells; Figure F shows the results of immunophenotyping of OCTS38BCMAs-CAR-T cells; Figure G shows the results of OCTS-PDL1BCMAs-CAR-T Cell transduction efficiency results; Figure H shows the immunophenotyping results of OCTS-PDL1BCMAs-CAR-T cells; Figure I shows the transduction efficiency results of OCTS123BCMAt-CAR-T cells; Figure J shows the immunization results of OCTS123BCMAt-CAR-T cells Typing results; Figure K shows the results of transduction efficiency of OCTS319BCMAt-CAR-T cells; Figure L shows the results of immunophenotyping of OCTS319BCMAt-CAR-T cells; Figure M shows the results of transduction efficiency of OCTS38BCMAt-CAR-T cells; Figure N represents the results of immunophenotyping of OCTS38BCMAt-CAR-T cells; Figure O represents the results of transduction efficiency of OCTS-PDL1BCMAt-CAR-T cells; Figure P represents the results of immunophenotyping of OCTS-PDL1BCMAt-CAR-T cells.

Figure 10 is a bar graph showing the killing efficiency of OCTS-CAR-T cells on target cells under different target conditions: Figure A shows the killing results of OCTS123BCMAs-CAR-T cells and OCTS123BCMAt-CAR-T cells on different target cells; Figure B shows The killing results of OCTS319BCMAs-CAR-T cells and OCTS319BCMAt-CAR-T cells on different target cells; Figure C shows the killing results of OCTS38BCMAs-CAR-T cells and OCTS38BCMAt-CAR-T cells on different target cells; Figure D shows the killing results of OCTS- Killing results of different target cells by PDL1BCMAs-CAR-T cells and OCTS-PDL1BCMAt-CAR-T cells.

Detailed ways

The following examples are only used to illustrate the present invention, but not to limit the scope of the present invention. The experimental methods that do not specify specific conditions in the examples are usually in accordance with conventional conditions, or in accordance with the conditions suggested by the manufacturer.

The example takes the combination of BCMA single-chain antibody and CD319, CD38, PDL1, CD123 single-chain antibody to form dual antigen recognition regions as an example, the construction of CAR dual-targeting chimeric antigen receptor, OCTS-CAR-T cell construction and function The test method is explained. In each combination, the two segments of scFv were connected in tandem and turn connections, respectively, forming a total of eight dual antigen recognition regions.

The light chain and heavy chain of the single-chain antibody of the other four substances can also be combined into a dual antigen recognition region. The construction of the CAR dual-targeting chimeric antigen receptor, the construction of OCTS-CAR-T cells, and the functional testing methods are the same as those in this implementation. The method described in the example is the same.

The experimental materials used in the examples are as follows:

(1) Lentiviral backbone plasmid pLenti-3G basic, lentiviral packaging plasmids pPac-GP, pPac-R and membrane protein pEnv-G, HEK293T/17 cells, homologous recombinase, Oligo Annealing Buffer, mycoplasma detection kit, Endotoxin detection kit, PSCA+K562, PDL1+K562, PDL1+PSCA+K562, K562 cells were purchased from Shiao (Shanghai) Biomedical Technology Co., Ltd.;

(2) Fresh human peripheral blood is provided by healthy donors;

(3) OCTS123BCMAs, OCTS319BCMAs, OCTS38BCMAs, OCTS-PDL1BCMAs, OCTS123BCMAt, OCTS319BCMAt, OCTS38BCMAt, OCTS-PDL1BCMAt DNA sequences were designed by themselves (see Table 1), and handed over to Shanghai Jierui Bioengineering Co., Ltd. Store in dry powder or plasmid form;

Table 1 Lentiviral recombinant plasmid design

(4) Tool enzymes Cla I, Pst I, BsrG I, Nde I, EcoR I, BamH I, ApaL I, Spe I, and T4 DNA ligase were purchased from NEB Company;

(5) 0.22μm-0.8μm PES filters were purchased from Millipore Company; D-PBS(-), 0.4% trypan blue, mesh, various types of cell culture dishes, culture bags, and culture plates were purchased from Corning Company;

(6) Opti-MEM, Pen-Srep, Hepes, FBS, AIM-V, RPMI 1640, DMEM, lipofectamine3000 were purchased from Invitrogen; Biotinylated protein L was purchased from GeneScript; LDH detection kit was purchased from Promega; Ficoll lymphocytes Separation solution was purchased from GE company; 20% human albumin injection was purchased from Jet Behring company; CryoPremium cryopreservation solution and sorting buffer were self-configured; 21 was purchased from peprotech company; CD3 monoclonal antibody, CD28 monoclonal antibody, CD3/CD28 magnetic beads CD4/CD8 magnetic beads were purchased from Germany Miltenyi company;

(7) The refrigerated centrifuge was purchased from Thermo Scientific Company of the United States; the FACS flow cytometer was purchased from Thermo Company; and the fluorescence inverted microscope was purchased from Olympus Company.

(8) CD4-FITC and CD8-APC were purchased from BioLegend Company; 0.9% saline was purchased from Jinmai Company; ProteinL Magnetic Beads were purchased from BioVision Company; PrimeSTAR and RetroNectin were purchased from Takara Company; phycoerythrin (PE)-conjugated streptavidin was purchased from BD Bioscience company; plasmid extraction kit and agarose gel recovery kit were purchased from MN company; competent cell TOP10 was purchased from tiangen company; NaCl, KCl, Na ₂ HPO ₄ .12H ₂ O, KH ₂ PO ₄ , Trypsin, EDTA, CaCl ₂ , NaOH, and PEG6000 were purchased from Shanghai Shenggong.

(9) DNeasy kit was purchased from Shanghai Jierui Company; SA-HRP was purchased from Shanghai Yisheng Company;

(10) Primers: The primers needed to amplify DNA fragments and target sites are designed according to the primer design principle. The primers are synthesized by Shanghai Biotechnology Co., Ltd.

EF1α-F: 5'-attcaaaattttatcgatgctccggtgcccgtcagt-3' (SEQ ID NO. 34)

EF1α-R: 5'-TCACGACACCTGAAATGGAAGA-3' (SEQ ID NO. 35)

OCTS-F: CATTTCAGGTGTCGTGAGGATCCGCCACCATGGCGCTGCCGGTGAC (SEQ ID NO. 36)

OCTS-R: GGGGAGGGAGAGGGGCTTAGCGCGGCGGCAGCG (SEQ ID NO. 37)

IRES-F: GCCCCTCTCCCTCCCCC (SEQ ID NO. 38)

IRES-R: ATTATCATCGTGTTTTTCAAAGGAA (SEQ ID NO. 39)

PDL1scab-F: AAAACACGATGATAATGCCACCATGAACTCCTTCTCCACAAGCG (SEQ ID NO. 40)

PDL1scab-R: AATCCAGAGGTTGATTGTCGACGAATTCTCATTTGCCCGGGCTCAG (SEQ ID NO. 41)

WPRE-QPCR-F: 5'-CCTTTCCGGGACTTTCGCTTT-3' (SEQ ID NO. 42)

WPRE-QPCR-R: 5'-GCAGAATCCAGGTGGCAACA-3' (SEQ ID NO. 43)

Actin-QPCR-F: 5'-CATGTACGTTGCTATCCAGGC-3' (SEQ ID NO. 44)

Actin-QPCR-R: 5'-CTCCTTAATGTCACGCACGAT-3' (SEQ ID NO. 45).

Example 1 Construction of OCTS-CAR-T cells

1. Construction, purification and detection of recombinant lentiviral vectors lvOCTS123BCMAs, lvOCTS319BCMAs, lvOCTS38BCMAs, lvOCTS-PDL1BCMAs, lvOCTS123BCMAt, lvOCTS319BCMAt, lvOCTS38BCMAt, lvOCTS-PDL1BCMAt

As shown in Figure 3, the construction method of the recombinant lentiviral vector of the present invention is as follows:

1. Human EF1α promoter, OCTS-based CAR dual-targeting chimeric antigen receptor (OCTS123BCMAs, OCTS319BCMAs, OCTS38BCMAs, OCTS-PDL1BCMAs, OCTS123BCMAt, OCTS319BCMAt, OCTS38BCMAt, OCTS-PDL1BCMAt), PDL1 single-chain antibody cloned into lentivirus The backbone plasmid pLenti-3G basic was used to obtain recombinant lentiviral plasmids pOCTS123BCMAs, pOCTS319BCMAs, pOCTS38BCMAs, pOCTS-PDL1BCMAs, pOCTS123BCMAt, pOCTS319BCMAt, pOCTS38BCMAt, and pOCTS-PDL1BCMAt, respectively.

(1) The lentiviral backbone plasmid pLenti-3G basic was double digested with Cla I and EcoR I restriction enzymes, and the product was subjected to 1.5% agarose gel electrophoresis to confirm the 5823bp fragment V1, and the gel was recovered and placed in In the Eppendorf tube, the corresponding fragments (see Table 2) were recovered with the agarose gel recovery kit of MN Company, and the purity and concentration of the product were determined;

Table 2 Agarose gel recovery steps

step specific operation Sol Add the sol solution at a ratio of 200 μl NTI/100 mg gel, and place in a water bath at 50°C for 5-10 minutes. bind DNA Centrifuge at 11000g for 30 seconds and discard the filtrate. Wash the membrane 700 μl of NT3 was added, centrifuged at 11,000 g for 30 seconds, and the filtrate was discarded. Wash the membrane Repeat step 3 once to dry Centrifuge at 11,000g for 1 minute, replace with a new collection tube, and place at room temperature for 1 minute. eluted DNA Add 15-30 μl NE, leave at room temperature for 1 minute, centrifuge at 11000g for 1 minute, and collect the filtrate.

(2) Use primers EF1α-F and EF1α-R to synthesize SEQ ID NO. 14 as template, use the system in Table 3, PCR cycle conditions are: 98°C for 3 min, (98°C for 10sec, 55°C for 15sec, 72°C for 15 seconds) 2min)*35cycle, 72℃ for 10min. The product was subjected to 1.5% agarose gel electrophoresis to confirm the fragment a of 1208 bp, and the gel was recovered and placed in an Eppendorf tube. The corresponding fragment was recovered with the agarose recovery kit of MN Company (see Table 2), and the content of the product was determined. Purity and concentration.

Table 3 50μL PCR reaction system

reagent Volume (μL) H₂O 32.5 5×Buffer(with Mg²⁺) 10 dNTPs (2.5mM each) 4 Primer1(+)(10μM) 1 Primer2(-)(10μM) 1 Template 1 PrimeSTAR 0.5

(3) Use primers OCTS-F and OCTS-R to synthesize OCTS123BCMAs as templates, and use the system in Table 3. The PCR cycle conditions are: 98°C for 3 min, (98°C for 10sec, 55°C for 15sec, 72°C for 30sec)*35cycle , 72 ℃ 5min. The product was subjected to 1.5% agarose gel electrophoresis to confirm the fragment b of 2357bp, and the gel was recovered and placed in an Eppendorf tube. The corresponding fragment was recovered with the agarose gel recovery kit of MN Company (see Table 2), and the product was determined. Purity and concentration;

(4) Use primers OCTS-F and OCTS-R to synthesize OCTS319BCMAs as templates, and use the system in Table 3. PCR cycle conditions are: 98°C for 3 min, (98°C for 10sec, 55°C for 15sec, 72°C for 30sec)*35cycle , 72 ℃ 5min. The product was subjected to 1.5% agarose gel electrophoresis to confirm the fragment c of 2375bp, and the gel was recovered and placed in an Eppendorf tube. The corresponding fragment was recovered with the agarose gel recovery kit of MN Company (see Table 2), and the product was determined. Purity and concentration;

(5) Use primers OCTS-F and OCTS-R to synthesize OCTS38BCMAs as templates, and use the system in Table 3. PCR cycle conditions are: 98°C for 3 min, (98°C for 10sec, 55°C for 15sec, 72°C for 30sec)*35cycle , 72 ℃ 5min. The product was subjected to 1.5% agarose gel electrophoresis to confirm the fragment d of 2378 bp, and the gel was recovered and placed in an Eppendorf tube. The corresponding fragment was recovered with the agarose recovery kit of MN Company (see Table 2), and the product was determined. Purity and concentration;

(6) Using the primers OCTS-F and OCTS-R, the synthetic OCTS-PDL1BCMAs were used as templates, and the system in Table 3 was used. *35cycle, 72℃ for 5min. The product was subjected to 1.5% agarose gel electrophoresis to confirm the fragment e of 2372bp, and the gel was recovered and placed in an Eppendorf tube, and the corresponding fragment was recovered with the agarose gel recovery kit of MN Company (see Table 2), and the product was determined. Purity and concentration;

(7) Use primers OCTS-F and OCTS-R to synthesize OCTS123BCMAt as template, and use the system in Table 3. PCR cycle conditions are: 98°C for 3 min, (98°C for 10sec, 55°C for 15sec, 72°C for 30sec)*35cycle , 72 ℃ 5min. The product was subjected to 1.5% agarose gel electrophoresis to confirm the fragment f of 2387bp, and the gel was recovered and placed in an Eppendorf tube, and the corresponding fragment was recovered with the agarose gel recovery kit of MN Company (see Table 2), and the product was determined. Purity and concentration;

(8) Use primers OCTS-F and OCTS-R to synthesize OCTS319BCMAt as template, and use the system in Table 3. PCR cycle conditions are: 98°C for 3min, (98°C for 10sec, 55°C for 15sec, 72°C for 30sec)*35cycle , 72 ℃ 5min. The product was subjected to 1.5% agarose gel electrophoresis to confirm the fragment g of 2403bp, and the gel was recovered and placed in an Eppendorf tube. The corresponding fragment was recovered with the agarose gel recovery kit of MN Company (see Table 2), and the product was determined. Purity and concentration;

(9) Use primers OCTS-F and OCTS-R to synthesize OCTS38BCMAt as template, and use the system in Table 3. PCR cycle conditions are: 98°C for 3 min, (98°C for 10sec, 55°C for 15sec, 72°C for 30sec)*35cycle , 72 ℃ 5min. The product was subjected to 1.5% agarose gel electrophoresis to confirm the fragment h of 2406 bp, and the gel was recovered and placed in an Eppendorf tube. The corresponding fragment was recovered with the agarose gel recovery kit of MN Company (see Table 2), and the product was determined. Purity and concentration;

(10) Use primers OCTS-F and OCTS-R to synthesize OCTS-PDL1BCMAt as a template, and use the system in Table 3. The PCR cycle conditions are: 98°C for 3 min, (98°C for 10sec, 55°C for 15sec, 72°C for 30sec) *35cycle, 72℃ for 5min. The product was subjected to 1.5% agarose gel electrophoresis to confirm the fragment i of 2433bp, and the gel was recovered and placed in an Eppendorf tube. The corresponding fragment was recovered with the agarose gel recovery kit of MN Company (see Table 2), and the product was determined. Purity and concentration;

(11) Use primers IRES-F and IRES-R to synthesize SEQ ID NO. 12 as a template, and use the system in Table 3. The PCR cycle conditions are: 98°C for 3 min, (98°C for 10sec, 55°C for 15sec, 72°C for 15 seconds) 30sec)*35cycle,72℃5min. The product was subjected to 1.5% agarose gel electrophoresis to confirm the fragment j of 575 bp, and the gel was recovered and placed in an Eppendorf tube. The corresponding fragment was recovered with the agarose recovery kit of MN Company (see Table 2), and the product was determined. Purity and concentration;

(12) The primers PDL1scab-F and PDL1scab-R were used to synthesize SEQ ID NO. 33 as the template, and the system in Table 3 was used. 30sec)*35cycle, 72℃ for 5min. The product was subjected to 1.5% agarose gel electrophoresis to confirm the fragment k of 1557bp, and the gel was recovered and placed in an Eppendorf tube. The corresponding fragment was recovered with the agarose gel recovery kit of MN Company (see Table 2), and the product was determined. Purity and concentration;

(16) Add the combination of recombinant lentiviral plasmid DNA fragments (see Table 4) into an Eppendorf tube with a total volume of 5 μl and a molar ratio of 1:1:1:1, add 15 μl of homologous recombinase reaction solution, and mix well at 42 Incubate at ℃ for 30 minutes, transfer to ice for 2-3 minutes, add the reaction solution to 50 μl of TOP10, gently swirl to mix the contents, place on ice for 30 minutes, and place the tube in a pre-warmed 42 ℃ Heat shock in a constant temperature water bath for 90 seconds, quickly transfer the tubes to an ice bath to cool the cells for 2-3 minutes, add 900 μl of LB medium to each tube, then transfer the tubes to a 37°C shaker and incubate for 1 hour to allow the bacteria After recovery, 100 μl of the transformed bacterial solution was spread on an Amp LB agar plate, the plate was inverted, and cultured in a constant temperature incubator at 37° C. for 16 hours.

Table 4 Combination of recombinant lentiviral plasmid DNA fragments

Recombinant lentiviral plasmid Fragment combination pOCTS123BCMAs a, b, j, k pOCTS319BCMAs a, c, j, k pOCTS38BCMAs a, d, j, k pOCTS-PDL1BCMAs a, e, j, k pOCTS123BCMAt a, f, j, k pOCTS319BCMAt a, g, j, k pOCTS38BCMAt a, h, j, k pOCTS-PDL1BCMAt a, i, j, k

Pick clones for colony PCR identification, and the correct clones are the recombinant lentiviral plasmids pOCTS123BCMAs, pOCTS319BCMAs, pOCTS38BCMAs, pOCTS-PDL1BCMAs, pOCTS123BCMAt, pOCTS319BCMAt, pOCTS38BCMAt, pOCTS-PDL1BCMAt, and the correct clones are identified by enzyme digestion (see Figure 5). ), and send the sequencing review results.

As shown in Fig. 5, Panel A represents the predicted digestion map and the digestion agarose gel electrophoresis image of pOCTS123BCMAs. lane1 is the digestion prediction map of 1kb DNA ladder Marker, the bands from top to bottom are: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp; lane2 is the predicted BamH I digestion of pOCTS123BCMAs, the bands from top to bottom are: 9712bp, 1277bp, 1023bp, 511bp; lane3 is the electrophoresis result of 1kb DNA ladder Marker; lane4 is the BamH I digestion electrophoresis result of pOCTS123BCMAs.

Panel B represents the predicted cleavage and agarose gel electrophoresis of pOCTS319BCMAs. lane1 is the digestion prediction map of 1kb DNA ladder Marker, the bands from top to bottom are: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp ; lane2 is the predicted ApaL I digestion of pOCTS319BCMAs: the bands from top to bottom are: 6543bp, 1961bp, 1723bp, 1234bp, 488bp; lane3 is the electrophoresis result of 1kb DNA ladderMarker; lane4 is the ApaL I digestion electrophoresis result of pOCTS319BCMAs.

Panel C represents the predicted restriction map and restriction agarose gel electrophoresis image of pOCTS38BCMAs. lane1 is the digestion prediction map of 1kb DNA ladder Marker, the bands from top to bottom are: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp ; lane2 is the predicted BsrG I digestion of pOCTS38BCMAs, the bands from top to bottom are: 10928bp, 1087bp; lane3 is the electrophoresis result of 1kb DNA ladder Marker; lane4 is the BsrG I digestion electrophoresis result of pOCTS38BCMAs.

Panel D represents the predicted digestion map and digestion agarose gel electrophoresis map of pOCTS-PDL1BCMAs. lane1 is the enzyme digestion prediction map of 1kb DNA ladder Marker, the bands from top to bottom are: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp ; lane2 is the predicted Cla I digestion of pOCTS-PDL1BCMAs, the bands from top to bottom are: 8876bp, 3139bp; lane3 is the electrophoresis result of 1kb DNA ladder Marker; lane4 is the Cla I digestion electrophoresis result of pOCTS-PDL1BCMAs.

Panel E represents the predicted cleavage and agarose gel electrophoresis of pOCTS123BCMAt. lane1 is the digestion prediction map of 1kb DNA ladder Marker, the bands from top to bottom are: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp ; lane2 is the predicted BamH I digestion of pOCTS123BCMAt: the bands from top to bottom are: 9545bp, 1467bp, 977bp; lane3 is the electrophoresis result of 1kb DNA ladder Marker; lane4 is the BamH I digestion electrophoresis result of pOCTS123BCMAt.

Panel F represents the predicted cleavage and agarose gel electrophoresis of pOCTS319BCMAt. lane1 is the digestion prediction map of 1kb DNA ladder Marker, the bands from top to bottom are: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp ; lane2 is the predicted Nde I digestion of pOCTS319BCMAt, the bands from top to bottom are: 9686bp, 2342bp; lane3 is the electrophoresis result of 1kb DNA ladder Marker; lane4 is the Nde I digestion electrophoresis result of pOCTS319BCMAt.

Panel G represents the predicted cleavage of pOCTS38BCMAt and the cleavage agarose gel electrophoresis graph. lane1 is the digestion prediction map of 1kb DNA ladder Marker, the bands from top to bottom are: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp ; lane2 is the Spe I digestion prediction of pOCTS38BCMAt: the bands from top to bottom are: 9976bp, 1926bp; lane3 is the electrophoresis result of the 1kb DNA ladder Marker; lane4 is the Spe I digestion electrophoresis result of pOCTS38BCMAt.

Panel H represents the predicted cleavage map and the cleavage agarose gel electrophoresis image of pOCTS-PDL1BCMAt. lane1 is the prediction map of 1kb DNA ladder Marker, the bands from top to bottom are: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp ; lane2 is the predicted Pst I digestion of pOCTS-PDL1BCMAt, the bands from top to bottom are: 10821bp, 1340bp, 411bp; lane3 is the electrophoresis result of 1kb DNA ladder Marker; lane4 is the Pst I digestion electrophoresis result of pOCTS-PDL1BCMAt .

From the electrophoresis results shown in Figure 5, it can be seen that the electrophoresis results of each recombinant lentiviral plasmid to be tested are basically the same as the predicted results of enzyme digestion, and the construction method of the above-mentioned recombinant lentiviral plasmid is effective.

2. Packaging of recombinant lentiviral vectors lvOCTS123BCMAs, lvOCTS319BCMAs, lvOCTS38BCMAs, lvOCTS-PDL1BCMAs, lvOCTS123BCMAt, lvOCTS319BCMAt, lvOCTS38BCMAt, lvOCTS-PDL1BCMAt

2.1 Solution configuration

(1) Complete medium: take out the preheated fresh medium, add 10% FBS+5ml Pen-Srep, invert up and down and mix;

(2) 1XPBS solution: weigh NaCl 8g, KCl 0.2, Na ₂ HPO ₄ .12H ₂ O 3.58g, KH ₂ PO4 0.24g, put it in a 1000ml beaker, add 900ml Milli-Q grade ultrapure water to dissolve, after the dissolution is complete , use a 1000ml graduated cylinder to dilute to 1000ml, and sterilize at 121°C for 20min by high temperature and humidity;

(3) 0.25% Trypsin solution: Weigh Trypsin 2.5g, EDTA 0.19729g into a 1000ml beaker, add 900ml 1XPBS to dissolve, after the dissolution is completed, use a 1000ml graduated cylinder to dilute to 1000ml, 0.22μM filter sterilization, long-term use can be stored to -20℃ refrigerator;

(4) 0.5M CaCl2 solution: Weigh 36.75g _CaCl2 and dissolve it in 400ml Milli-Q grade ultrapure water; dilute the total volume to 500ml with Milli-Q grade ultrapure water, mix well; sterilize by 0.22μm filtration, Dispense and store in 50ml centrifuge tubes, about 45ml per tube, and store at 4°C.

(5) 2XHBS solution: Weigh 4.09g NaCl, 0.269g Na ₂ HPO 4 , 5.96 g Hepes, dissolve with 400 ml Milli-Q grade ultrapure water; after calibrating the pH meter, adjust the pH of the HBS solution to 7.05 with 2M NaOH solution . To adjust the pH of each bottle of HBS, the consumption of 2M NaOH is about 3ml.

2.2 HEK293T/17 cell culture

(1) Take out the cryopreserved HEK293T/17 cells from the liquid nitrogen tank, quickly transfer them to a 37°C water bath, and transfer them to an ultra-clean bench after ^1-2 minutes. In the culture dish, supplement DMEM containing 10% FBS to 8mL/10cm ² dish, observe the cells under microscope after 24h, and the cells are passaged if the degree of cell confluence is greater than 80%;

(2) Select HEK293T/17 cells with good cell condition and no pollution, each 2-6 culture dishes as a group, after the cells are trypsinized, use an electric pipette to suck 4-12ml of complete medium, and add 4-12ml of complete medium to each Add 2ml to the digested petri dish to prevent the petri dish from drying out; use a 1ml pipette to pipet all cells into a single-cell suspension and transfer to a medium bottle;

(3) the remaining cells in the above-mentioned 2-6 culture dishes are transferred to the culture medium bottle, and the culture dish is rinsed again with the culture medium;

(4) Tighten the cap of the medium bottle, invert up and down about 10 times to mix the cell suspension thoroughly, and transfer the cells to 8-24 10cm ² dishes, the cell density of each dish should be about 4×10 ⁶ cells/10ml Complete medium or so. If the cell density differs greatly from the expected one, the cells need to be counted, and then seeded at 4×10 ⁶ cells/dish;

(5) Arrange every 6 petri dishes into a stack, pay attention to maintaining the cooperation between the upper and lower dishes. Shake the dish from side to side and back and forth several times to spread the cells well, and then put it into a 5% CO ₂ incubator. Do the same for the remaining cells;

(6) Check the passaged cells, the cell confluency should be 70-80%, the contour is full, the adherence is good, and the cells are evenly distributed in the cell culture dish;

(7) Change the medium for the cells, replace the medium with fresh complete medium, 9ml per dish, and increase the _CO concentration setting value of the incubator to 8%;

2.3 Cell transfection

(1) Prepare DNA/CaCl ₂ solution according to N+0.5. The amount of transfected plasmids per dish of HEK293T/17 cells was used according to the following ratios: recombinant lentiviral plasmid (20μg), pPac-GP (15μg), pPac-R (10μg), pEnv-G (7.5μg). Take a new 5ml centrifuge tube, add 0.5M CaCl2: 0.25ml, recombinant lentiviral plasmid 20μg: pPac-GP 15μg: pPac-R 10μg: pEnv-G 7.5μg, add ultrapure water to 0.5ml, close the lid and fully mix;

(2) Take another 5ml centrifuge tube and add 0.5ml DNA/CaCl2 solution. Turn on the vortex shaker, hold the upper end of the 5ml centrifuge tube with one hand, make the bottom of the tube touch the vibrating head, and make the liquid flow on the tube wall. The ×HBS solution is slowly added dropwise into the centrifuge tube, and the flow rate is controlled. After adding 2×HBS, continue to shake for 5 seconds, stop shaking, and add it directly to the cells to be transfected;

(3) get a dish of cells, drop the 1mL calcium transfer in the centrifuge tube into it, and distribute the calcium transfer reagent to the entire culture dish as much as possible;

(4) After the calcium transfer solution is added, mark the lid of the dish and return the dish to another 5% CO ₂ incubator. Make sure the petri dishes are placed horizontally, no more than 6 petri dishes per stack. Place (6–8h) in a 5% _CO2 incubator;

(5) Adjust the _CO concentration setting value of the first incubator back to 5%;

(6) After 24 hours, check the cell status. Cells should be around 80–85% confluent and in good condition. Aspirate the medium and replace with 10ml fresh DMEM complete medium;

(7) After 48 hours, the transfection efficiency was observed. The vast majority of cells remain adherent. It can be seen that more than 95% of the cells will have green fluorescence. Collect the same virus packaging supernatant together, and continue to add 10 mL of fresh medium to the petri dish;

(8) After 72 hours, the same virus supernatant was collected again, the viruses collected twice can be put together, and the culture dish was discarded; the supernatant collected at this time contained the recombinant lentiviral vectors lvOCTS123BCMAs, lvOCTS319BCMAs, lvOCTS38BCMAs , lvOCTS-PDL1BCMAs, lvOCTS123BCMAt, lvOCTS319BCMAt, lvOCTS38BCMAt, lvOCTS-PDL1BCMAt.

3. Purification of recombinant lentiviral vector by ion exchange chromatography

(1) Using a Thermo vacuum pump, the collected supernatant is filtered through a PES filter of 0.22 μm-0.8 μm to remove impurities;

(2) Add 1.5M NaCl 250mM Tris-HCl (pH 6-8) to the supernatant at a ratio of 1:1 to 1:10;

(3) 2 ion exchange columns are placed in series, and pass the column successively with 4ml 1M NaOH, 4ml 1M NaCl, 5ml 0.15M NaCl25mM Tris-HCl (pH 6-8) solution;

(4) the solution obtained in step 2 is sampled to the ion exchange column at the speed of 1-10ml/min by peristaltic pump;

(5) After all the supernatant is passed through the column, use 10ml of 0.15M NaCl 25mM Tris-HCl (pH 6-8) solution to wash it once;

(6) Use 1-5ml of 1.5M NaCl 25mM Tris-HCl (pH 6-8) to elute according to the loading amount, and collect the eluate;

(7) Divide the eluate into 25 to 50 μL tubes, and store them in a -80°C refrigerator for long-term storage.

4. Determination of recombinant lentiviral vector titer

(1) Take a 24-well plate and inoculate 293T cells. There are 5×10 ⁴ cells in each well, and the volume of the medium added is 500ul. The growth rate of different types of cells is different, and the cell fusion rate during virus infection is 40%-60%;

(2) Prepare 3 sterile EP tubes, add 90ul of fresh complete medium (high glucose DMEM+10% FBS) to each tube, inoculate cells 24 hours later, take the cells in two wells and count them with a hemocytometer, Determine the actual number of cells at the time of infection, denoted as N;

(3) Take 10ul of the virus stock solution to be determined and add it to the first tube, after gently mixing, add 10ul to the second tube, and then operate in sequence until the last tube; add 410ul to each tube to complete the culture base (high glucose DMEM+10% FBS), the final volume is 500ul;

(4) 20 hours after the infection started, the culture supernatant was removed, replaced with 500 μl of complete medium (high glucose DMEM+10% FBS), and the culture was continued in 5% _CO for 48 hours;

(5) After 72 hours, observe the fluorescent expression situation, under normal circumstances, the number of fluorescent cells decreases correspondingly with the increase of the dilution ratio, and takes pictures;

(6) Digest the cells with 0.2 ml of 0.25% trypsin-EDTA solution, and place at 37°C for 1 minute. The entire cell surface was rinsed with medium and the cells were collected by centrifugation. Genomic DNA was extracted following the instructions of the DNeasy kit. Add 200μl of eluate to each sample tube to wash the DNA and quantify;

(7) Prepare target DNA detection qPCRmix manifold I (QPCR primer sequence is SEQ ID NO.42---SEQ ID NO.43):

Table 5 Components in the qPCRmix main pipe I

2 x TaqMan Master Mix 25μl×n Forward primer (100pmol ml-1) 0.1μl×n Reverse primer (100pmol ml-1) 0.1μl×n Probe(100pmol ml-1) 0.1μl×n H₂O 19.7μl×n

n=number of reactions. For example, if the total number of reactions is 40, mix 1ml 2×TaqMan UniversalPCR Master Mix, 4μl forward primer, 4μl reverse primer, 4μl probe and 788μl H2O, and place on ice after shaking.

(8) Prepare internal reference DNA detection qPCR mix tube II (QPCR primer sequence is SEQ ID NO.44---SEQ ID NO.45):

Table 6 Components in qPCRmix manifold II

2 x TaqMan Master Mix 25μl×n 10×RNaseP primer/probe mix 2.5μl×n H₂O 17.5μl×n

n=number of reactions. For example, if the total number of reactions is 40, mix 1 ml of 2×TaqMan Universal PCR Master Mix, 100 μl of 10× RNaseP primer/probe mix and 700 μl H ₂ O, shake and place on ice.

(9) Complete the establishment of the PCR system on a pre-cooled 96-well PCR plate. 45 µl of each from manifold I was added to the wells of rows A-D, and 45 µl of each of manifold II was added to the wells of rows E-G.

(10) Add 5 μl of plasmid standard and genomic DNA of the sample to be tested to rows A-D, and repeat once for each sample. Another well was added with 5 μl of water as a no-template control.

(11) Add 5 μl of the genomic standard and the genomic DNA of the sample to be tested to rows E-G, and repeat once for each sample. Another well was added with 5 μl of water as a no-template control.

(12) The quantitative PCR instrument used was ABI PRISM 7500 quantitative system. Cycling conditions were set as: 50°C for 2 minutes, 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute.

(13) Data analysis: The number of integrated lentiviral vector copies in the measured DNA samples is demarcated by the number of genomes to obtain the number of integrated virus copies per genome.

The calculation formula of titer (integration units per ml, IU ml ^-1 ) is as follows:

IU ml ^-1 = (C×N×D×1000)/V

where: C = average number of viral copies per genome integration

N = number of cells at infection (approximately 1 x 10 ⁵ )

D = dilution factor of viral vector

V = volume of diluted virus added

Figure 6 shows the titer results of the recombinant lentiviral vectors lvOCTS123BCMAs, lvOCTS319BCMAs, lvOCTS38BCMAs, lvOCTS-PDL1BCMAs, lvOCTS123BCMAt, lvOCTS319BCMAt, lvOCTS38BCMAt, and lvOCTS-PDL1BCMAt.

2. Construction of OCTS-CAR-T cells

Figure 7 shows the OCTS-CAR-T cell construction process, and the construction method of the OCTS-CAR-T cell in this example is as follows:

1. Isolation of PBMCs.

(1) Extract 50ml of fresh peripheral blood from healthy donors;

(2) Spray the blood collection bag with alcohol twice and wipe it dry.

(3) Aspirate the blood cells in the bag with a 50ml syringe and transfer to a new 50ml tube.

(4) 400g, centrifuged at 20°C for 10min.

(5) Transfer the upper plasma to a new 50ml centrifuge tube, inactivate the plasma at 56°C for 30min, return to room temperature, centrifuge at 2000g for 30min, and take the supernatant into a 50ml centrifuge tube for use.

(6) Make up to 50ml with D-PBS(-), tighten the lid, and mix by inversion.

(7) Take two new 50ml centrifuge tubes and add 15ml Ficoll lymphocyte separation solution to each tube.

(8) Carefully add 25ml of blood cell dilution to each tube of Ficoll. Centrifuge at 800g, 20°C for 20min.

(9) The liquid in the centrifuge tube is divided into four layers, from top to bottom: the yellow plasma layer (recovered for use), the buffy coat layer, the colorless and transparent Ficoll layer, and the red and black mixed cell layer.

(10) Carefully pipette the buffy coat layer into a new 50ml centrifuge tube, add D-PBS(-) to 50ml, invert and mix, and centrifuge at 500g at 20°C for 10min.

(11) Add 25ml of 5% human albumin and resuspend the cells, centrifuge at 400g for 10min at 20°C.

(12) Discard the supernatant, add 25ml of 5% human serum albumin to resuspend the cell pellet, pass through a 70um sieve, and count.

(13) Take 1 portion containing 1.25× ^{10 8} cells for activation; the remaining cell suspension is 400 g, centrifuged at 20°C for 10 min, added with CryoPremium and frozen.

2. CD4/CD8 positive T cell sorting.

(1) Count the obtained PBMC, add the sorting buffer at a ratio of 80ul/10 ⁷ cells, and resuspend the cell pellet.

(2) Add CD4/CD8 magnetic beads at a ratio of 20ul/10 ⁷ cells, mix by pipetting, and then incubate at 4°C for 15 minutes.

(3) Take out the magnetic bead-cell mixture, add the sorting buffer at a ratio of 2ml/10 ⁷ cells, invert and mix, centrifuge at 250g for 10min at 4°C.

(4) Add sorting buffer at a ratio of 500ul/10 ⁸ cells, and resuspend the cell pellet.

(5) Use tweezers to pick up the LS separation column on the magnetic stand.

(6) At the same time, two 15ml centrifuge tubes were prepared and labeled: CD4-/CD8- cell fluid (tube A) and CD4+/CD8+ cell fluid (tube B).

(7) Rinse LS with 3ml of separation buffer, and connect the buffer with A tube.

(8) Add the cell-magnetic bead mixture, add 3ml of buffer to rinse the column after dropping (add new liquid every time when no liquid remains), a total of three times, and collect CD4/CD8- cells.

(9) Separate the LS separation column from the magnetic stand, connect the cell suspension with the B tube, add 5 ml of buffer, wash it with a little force in the column plug, collect it as CD4+/CD8+ cells, and sample and count.

(10) Resuspend the cell pellet in AIM-V medium at a cell density of 1×10 ⁶ /ml-4×10 ⁶ /ml, and add 2×10 ⁵ to 1×10 ⁶ U/L IFN-γ factor.

3. T cell activation.

(1) 1×10 ³ ug/L～1×10 ⁴ ug/L CD3 monoclonal antibody and 1×10 ³ ug/L～1×10 ⁴ ug/L CD28 monoclonal antibody were added to the 24-well plate one day in advance, Seal with parafilm and coat overnight at 4°C;

(2) Take out the coated T75 flask, pour out the coating solution, wash once with D-PBS(-), inoculate the sorted cell suspension into the T75 flask, shake well, put it in 37℃, 5 Culture in a %CO ₂ incubator.

4. CAR gene transduction and OCTS-CAR-T cell induction and culture.

(1) Coat 1×10 ³ ug/L～1×10 ⁴ ug/L RetroNectin in a 24-well plate one day in advance, seal with parafilm, and coat overnight at 4°C.

(2) Add lvOCTS123BCMAs, lvOCTS319BCMAs, lvOCTS38BCMAs, lvOCTS-PDL1BCMAs, lvOCTS123BCMAt, lvOCTS319BCMAt, lvOCTS38BCMAt, and lvOCTS-PDL1BCMAt to the 24-well plate according to the amount of cells in each well of 5×10 ⁵ cells at an MOI of 5 to 20. Virus transgenic vector, at the same time adding 2×10 ⁵ ～5×10 ⁵ U/L rIL-2, 5×10 ³ ng/L～1×10 ⁴ ng/L rIL-7, 5×10 ³ ng/L～ 1×10 ⁴ ng/L rIL-15, 5×10 ³ ng/L～1×10 ⁴ ng/L rIL-21 and AIM-V medium containing 10% autologous serum were cultured at 37℃, 5% CO ₂ .

5. OCTS-CAR-T cells were expanded in vitro.

(1) Supplement with equal amount of 2×10 ⁵ ～5×10 ⁵ U/L rIL-2, 5×10 ³ ng/L～1×10 ⁴ ng/L rIL-7, 5×10 ³ every 2 days ng/L～1×10 ⁴ ng/L rIL-15, 5×10 ³ ng/L～1×10 ⁴ ng/L rIL-21 and AIM-V medium containing 10% autologous serum to maintain pH value Between 6.5 and 7.5, the cell density was maintained between 5×10 ⁵ and 2×10 ⁶ /ml, and the culture was continued for 10-14 days at 37°C and 5% CO ₂ .

(2) Around day 7, the cultured OCTS-CAR-T cells were cryopreserved for subsequent detection.

Example 2 OCTS-CAR-T cell pathogen detection and expression detection

1. Endotoxin detection;

(1) Endotoxin working standard is 15EU/piece;

(2) Limulus reagent sensitivity λ=0.25EU/ml, 0.5ml/tube

(3) Dilution of endotoxin standard: Take one endotoxin standard, dilute it with BET water in proportion to dissolve at 4λ and 2λ, seal with parafilm, and shake to dissolve for 15 minutes; each dilution step should be placed in a vortex mixer during dilution. Mix for 30s;

(4) Sample loading: Take several Limulus reagents, add 0.5ml of BET water to each to dissolve, and dispense into several endotoxin-free test tubes, each tube is 0.1ml. Two of them are negative control tubes, add 0.1ml of BET water; two are positive control tubes, add 0.1ml of endotoxin working standard solution of 2λ concentration; two are sample positive control tubes, add 0.1ml of endotoxin standard containing 2λ (1ml of 20-fold diluted sample to be tested + 1 ml of 4λ endotoxin standard solution = 2ml of 40-fold diluted sample containing 2λ endotoxin standard).

Add 0.1mL of sample to the sample tube, the dilution ratio is shown in Table 7, and keep it in a water bath (or incubator) at 37±1℃ for 60±1min;

Table 7 Endotoxin dilution ratio and endotoxin detection results of OCTS-CAR-T cells

As shown in Table 7, the endotoxin content of all cells is less than 2.5EU/ml, which meets the standard of less than 10EU/ml in "Pharmacopoeia of the People's Republic of China".

2. Mycoplasma detection

(1) Three days before the experiment, the cell samples were cultured with antibiotic-free medium;

(2) Collect 1 mL of cell suspension (the number of cells is greater than 1×10 ⁵ ) and place it in a 1.5 mL centrifuge tube;

(3) Centrifuge at 13000g for 1min, collect the precipitate, and discard the culture medium;

(4) Add 500ul of PBS, pipette or vortex, and resuspend the pellet. Centrifuge at 13000g for 5min;

(5) Step 4 is repeated once;

(6) Add 50μl of Cell Lysis Buffer, blow with a pipette tip, mix well, and incubate in a 55°C water bath for 20min;

(7) The sample is placed at 95°C and heated for 5min;

(8) After centrifugation at 13000g for 5min, take 5μl supernatant as template, 25μl PCR reaction system is: ddH20 6.5μl, Myco Mix 1μl, 2x Taq Plus Mix Master (Dye Plus) 12.5μl, template 5μl; PCR cycle conditions: 95 ℃ 30sec, (95℃ 30sec, 56℃ 30sec, 72℃ 30sec)*30cycle, 72℃ 5min.

The mycoplasma detection results are shown in Figure 8, and the positive control, negative control and sample judgment descriptions in Figure 8 are shown in Table 8. As shown in the results in Figure 8, none of the OCTS-CAR-T cells contained mycoplasma.

Table 8 Determination description of positive control, negative control and sample

3. Detection of OCTS gene transduction efficiency and immune typing detection;

(1) Collect virus-transduced T cells, resuspend cells in D-PBS(-) solution containing 1-4% human albumin and adjust to 1×10 ⁶ /ml.

(2) 1 ml of D-PBS(-) solution containing 1-4% human serum albumin was added to the centrifuge tube, mixed well, centrifuged at 350 g for 5 min, and the supernatant was discarded.

(3) Repeat step 2 once.

(4) Resuspend the cells with 0.2ml of D-PBS(-) solution containing 1-4% human serum albumin, and add 1ul of 1mg/ul protein L, 5ul CD4-FITC, 5ul CD8- to the centrifuge tube APC, mixed and incubated at 4°C for 45min.

(5) 1 ml of D-PBS(-) solution containing 1-4% human serum albumin was added to the centrifuge tube, mixed well, centrifuged at 350 g for 5 min, and the supernatant was discarded.

(6) Repeat step 5 twice.

(7) Resuspend the cells with 0.2 ml of D-PBS(-) solution containing 1-4% human albumin, add 0.2 ul PE-SA to the centrifuge tube, mix well, and incubate at 37°C for 15 minutes in the dark.

(8) 1 ml of D-PBS(-) solution containing 1-4% human serum albumin was added to the centrifuge tube, mixed, centrifuged at 350 g for 5 min, and the supernatant was discarded.

(9) Resuspend the cell pellet with 1ml D-PBS(-) solution, centrifuge at 350g for 5min, and discard the supernatant.

(10) Repeat step 9 twice.

(11) Resuspend the cell pellet with 0.4 ml D-PBS(-) solution, and detect by flow cytometer.

The results of the OCTS gene transduction efficiency and immunophenotyping test are shown in Figure 9. The infection efficiency of the prepared OCTS-CAR-T cells is mostly between 37% and 50%, and the proportion of CD4-positive cells and CD8-positive cells is between 37% and 50%. Between 1:3 and 3:1, subsequent functional testing can be performed.

Example 3 Functional detection of OCTS-CAR-T cells

1. Evaluation of target cell killing effect.

(1) Culture target cells [BCMA ⁺ K562, CD319 ⁺ K562, CD38 ⁺ K562, PDL1 ⁺ K562, CD123 ⁺ K562, BCMA ⁺ CD123 ⁺ K562, BCMA ⁺ CD319 ⁺ K562, BCMA ⁺ CD38 ⁺ K562, BCMA ⁺ PDL1 ⁺ K562, K562 cells] and effector cells [OCTS-CAR-T cells], the groupings of effector cells co-incubated with single-target cells and double-target cells, respectively, are shown in Table 9.

Table 9. Grouped list of effector cells co-incubated with single-target cells and dual-target cells, respectively

effector cells target cell 1 target cell 2 target cell 3 OCTS123BCMAs-CAR-T CD123⁺K562 BCMA⁺K562 BCMA⁺CD123⁺K562 OCTS319BCMAs-CAR-T CD319⁺K562 BCMA⁺K562 BMCA⁺CD319⁺K562 OCTS38BCMAs-CAR-T CD38⁺K562 BCMA⁺K562 BCMA⁺CD38⁺K562 OCTS-PDLlBCMAs-CAR-T PDL1⁺K562 BCMA⁺K562 BCMA⁺PDL1⁺K562 OCTS123BCMAt-CAR-T CD123⁺K562 BCMA⁺K562 BCMA⁺CD123⁺K562 OCTS319BCMAt-CAR-T CD319⁺K562 BCMA⁺K562 BMCA⁺CD319⁺K562 OCTS38BCMAt-CAR-T CD38⁺K562 BCMA⁺K562 BCMA⁺CD38⁺K562 OCTS-PDL1BCMAt-CAR-T PDL1⁺K562 BCMA⁺K562 BCMA⁺PDL1⁺K562

(2) Collect 4× ^{10 5} cells of target cells and 2.8×10 ⁶ cells of OCTS-CAR-T cells, centrifuge at 800 g for 6 min, and discard the supernatant;

(3) Resuspend the target cells and effector cells with 1ml D-PBS(-) solution respectively, centrifuge at 800g for 6min, and discard the supernatant;

(4) Repeat step 3 once;

(5) Resuspend effector cells with 700ul medium (AIM-V medium + 1-10% FBS), and resuspend target cells with 2 ml medium (AIM-V medium + 1-10% FBS);

(6) Set the experimental wells with effect-target ratios of 1:1, 5:1, and 10:1, and the grouping situation is shown in Table 9, and set up a control group (K562 cells), with 3 duplicate wells in each group;

(7) 250g, 5min plate centrifugation;

(8) Incubate for 4 hours in a 5% CO2 incubator at 37°C;

(9) 250g, 5min plate centrifugation;

(10) Take 50ul of supernatant from each well into a new 96-well plate, and add 50ul of substrate solution to each well (operation in the dark);

(11) Incubate in the dark for 25min;

(12) Add 50ul stop solution to each well;

(13) Microplate reader detects absorbance at 490nm;

(14) Take the average value of 3 duplicate wells; subtract the mean value of the background absorbance value of the medium from the absorbance values of all experimental wells, target cell wells and effector cell wells; subtract the absorbance value of the maximum target cell value from the volume correction control The mean value of absorbance.

(15) The corrected value obtained in step 14 is brought into the following formula to calculate the percentage of cytotoxicity produced by each effector-target ratio.

Killing efficiency=(experimental well-effector cell well-target cell well)/(target cell largest well-target cell well)×100%

The results are shown in Figure 10. OCTS-CAR-T has a good killing effect on their respective single-target cells and dual-target cells. The killing efficiency of CAR-T cells with Turn OCTS structure on target cells is slightly higher than that of Series OCTS structure. CAR-T cells.

The above experimental results show that the OCTS structure formed by the transformation of the antigen recognition region in the traditional CAR structure can significantly improve the range of OCTS-CAR-T cells to recognize and kill target cells, so OCTS-CAR-T cells will be used in the future BCMA. Positive/CD319 positive/CD38 positive/CD123 positive/PDL1 positive/BCMA, CD319 double positive/BCMA, CD38 double positive/BCMA, CD123 double positive/BCMA, PDL1 double positive multiple myeloma cell therapy huge effect.

The foregoing has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above-mentioned embodiments. The above-mentioned embodiments and descriptions only illustrate the principle of the present invention. Such changes and improvements fall within the scope of the claimed invention. The claimed scope of the present invention is defined by the appended claims and their equivalents.

SEQUENCE LISTING

<110> Shanghai Ucardi Biomedical Technology Co., Ltd.

<120> OCTS-CAR dual targeting chimeric antigen receptor, encoding gene, recombinant expression vector and its construction and application

<130> Claims Specification

<160> 45

<170> PatentIn version 3.5

<210> 1

<211> 837

<212> DNA

<213> Artificial sequences

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atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct 60

gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca 120

cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc 180

gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc 240

cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg 300

gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta 360

tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc 420

ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt 480

gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg 540

cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctatcc 600

cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg 660

gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc 720

ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg 780

acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcc 837

<210> 2

<211> 674

<212> DNA

<213> Artificial sequences

<400> 2

cccgtagaaa agatcaaagg atcttcttga gatcctttttt ttctgcgcgt aatctgctgc 60

ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca 120

actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta 180

gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct 240

ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg 300

gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc 360

acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta 420

tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg 480

gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt 540

cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg 600

cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg 660

ccttttgctc acat 674

<210> 3

<211> 147

<212> DNA

<213> Artificial sequences

<400> 3

atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt 60

tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga 120

ggcttttttg gaggcctaga cttttgc 147

<210> 4

<211> 228

<212> DNA

<213> Artificial sequences

<400> 4

gtagtcttat gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc 60

cttacaagga gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg 120

tgccttatta ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgcc 180

gcattgcaga gatattgtat ttaagtgcct agctcgatac aataaacg 228

<210> 5

<211> 180

<212> DNA

<213> Artificial sequences

<400> 5

ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac 60

tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt 120

gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca 180

<210> 6

<211> 234

<212> DNA

<213> Artificial sequences

<400> 6

tgctagagat tttccacact gactaaaagg gtctgaggga tctctagtta ccagagtcac 60

acaacagacg ggcacacact acttgaagca ctcaaggcaa gctttattga ggcttaagca 120

gtgggttccc tagttagcca gagagctccc aggctcagat ctggtctaac cagagagacc 180

cagtacaagc aaaaagcaga tcttattttc gttgggagtg aattagccct tcca 234

<210> 7

<211> 353

<212> DNA

<213> Artificial sequences

<400> 7

atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc 60

ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg 120

agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa 180

tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata 240

atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag 300

ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caa 353

<210> 8

<211> 233

<212> DNA

<213> Artificial sequences

<400> 8

aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg cagcctcaat 60

gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc agaacaattt 120

gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg gcatcaagca 180

gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc tcc 233

<210> 9

<211> 489

<212> DNA

<213> Artificial sequences

<400> 9

tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct tggaatgcta 60

gttggagtaa taaatctctg gaacagattg gaatcacacg acctggatgg agtgggacag 120

agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc aaaaccagca 180

agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt ggaattggtt 240

taacataaca aattggctgt ggtatataaa attattcata atgatagtag gaggcttggt 300

aggtttaaga atagttttttg ctgtactttc tatagtgaat agagttaggc agggatattc 360

accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc ccgaaggaat 420

agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga acggatctcg 480

acggttaac 489

<210> 10

<211> 119

<212> DNA

<213> Artificial sequences

<400> 10

ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat 60

agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaatttta 119

<210> 11

<211> 696

<212> DNA

<213> Artificial sequences

<400> 11

atggcccagt ccaagcacgg cctgaccaag gagatgacca tgaagtaccg catggagggc 60

tgcgtggacg gccacaagtt cgtgatcacc ggcgagggca tcggctaccc cttcaagggc 120

aagcaggcca tcaacctgtg cgtggtggag ggcggcccct tgcccttcgc cgaggacatc 180

ttgtccgccg ccttcatgta cggcaaccgc gtgttcaccg agtaccccca ggacatcgtc 240

gactacttca agaactcctg ccccgccggc tacacctggg accgctcctt cctgttcgag 300

gacggcgccg tgtgcatctg caacgccgac atcaccgtga gcgtggagga gaactgcatg 360

taccacgagt ccaagttcta cggcgtgaac ttccccgccg acggccccgt gatgaagaag 420

atgaccgaca actgggagcc ctcctgcgag aagatcatcc ccgtgcccaa gcagggcatc 480

ttgaagggcg acgtgagcat gtacctgctg ctgaaggacg gtggccgctt gcgctgccag 540

ttcgacaccg tgtacaaggc caagtccgtg ccccgcaaga tgcccgactg gcacttcatc 600

cagcacaagc tgacccgcga ggaccgcagc gacgccaaga accagaagtg gcacctgacc 660

gagcacgcca tcgcctccgg ctccgccttg ccctga 696

<210> 12

<211> 575

<212> DNA

<213> Artificial sequences

<400> 12

gcccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt 60

gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc 120

ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag 180

gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac 240

aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc 300

tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc 360

acgttgtgag ttggatagtt gtggaaagag tcaaatggct cacctcaagc gtattcaaca 420

aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt 480

gcacatgctt tacatgtgtt tagtcgaggt taaaaaacgt ctaggccccc cgaaccacgg 540

ggacgtggtt ttcctttgaa aaacacgatg ataat 575

<210> 13

<211> 592

<212> DNA

<213> Artificial sequences

<400> 13

aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct 60

ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt 120

atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta tgaggagttg 180

tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc aacccccact 240

ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt ccccctccct 300

attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg ggctcggctg 360

ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc 420

gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc ttcggccctc 480

aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt 540

cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgcc tg 592

<210> 14

<211> 1178

<212> DNA

<213> Artificial sequences

<400> 14

gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg 60

gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg 120

atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag 180

tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg 240

tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta 300

cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg 360

agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc 420

ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt 480

tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc 540

aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg 600

cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag 660

cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg 720

gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag 780

ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga 840

cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt 900

cctcagccgt cgcttcatgt gactccactg agtaccgggc gccgtccagg cacctcgatt 960

agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg 1020

agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat 1080

tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag 1140

tggttcaaag ttttttttctt ccatttcagg tgtcgtga 1178

<210> 15

<211> 63

<212> DNA

<213> Artificial sequences

<400> 15

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccg 63

<210> 16

<211> 333

<212> DNA

<213> Artificial sequences

<400> 16

gatattgtgc tgacccagag cccgccgagc ctggcgatga gcctgggcaa acgcgcgacc 60

attagctgcc gcgcgagcga aagcgtgacc attctgggca gccatctgat tcattggtat 120

cagcagaaac cgggccagcc gccgaccctg ctgattcagc tggcgagcaa cgtgcagacc 180

ggcgtgccgg cgcgctttag cggcagcggc agccgcaccg attttaccct gaccattgat 240

ccggtggaag aagatgatgt ggcggtgtat tattgcctgc agagccgcac cattccgcgc 300

acctttggcg gcggcaccaa actggaaatt aaa 333

<210> 17

<211> 351

<212> DNA

<213> Artificial sequences

<400> 17

cagattcagc tggtgcagag cggcccggaa ctgaaaaaac cgggcgaaac cgtgaaaatt 60

agctgcaaag cgagcggcta tacctttacc gattatagca ttaactgggt gaaacgcgcg 120

ccgggcaaag gcctgaaatg gatgggctgg attaacaccg aaacccgcga accggcgtat 180

gcgtatgatt ttcgcggccg ctttgcgttt agcctggaaa ccagcgcgag caccgcgtat 240

ctgcagatta acaacctgaa atatgaagat accgcgacct atttttgcgc gctggattat 300

agctatgcga tggattattg gggccagggc accagcgtga ccgtgagcag c 351

<210> 18

<211> 324

<212> DNA

<213> Artificial sequences

<400> 18

gatattcaga tgacccagag cccgagcagc ctgagcgcga gcgtgggcga tcgcgtgacc 60

attacctgca aagcgagcca ggatgtgggc attgcggtgg cgtggtatca gcagaaaccg 120

ggcaaagtgc cgaaactgct gatttattgg gcgagcaccc gccataccgg cgtgccggat 180

cgctttagcg gcagcggcag cggcaccgat tttaccctga ccattagcag cctgcagccg 240

gaagatgtgg cgacctatta ttgccagcag tatagcagct atccgtatac ctttggccag 300

ggcaccaaag tggaaattaa acgc 324

<210> 19

<211> 357

<212> DNA

<213> Artificial sequences

<400> 19

gaagtgcagc tggtggaaag cggcggcggc ctggtgcagc cgggcggcag cctgcgcctg 60

agctgcgcgg cgagcggctt tgattttagc cgctattgga tgagctgggt gcgccaggcg 120

ccgggcaaag gcctggaatg gattggcgaa attaacccgg atagcagcac cattaactat 180

gcgccgagcc tgaaagataa atttattatt agccgcgata acgcgaaaaa cagcctgtat 240

ctgcagatga acagcctgcg cgcggaagat accgcggtgt attattgcgc gcgcccggat 300

ggcaactatt ggtattttga tgtgtggggc cagggcaccc tggtgaccgt gagcagc 357

<210> 20

<211> 321

<212> DNA

<213> Artificial sequences

<400> 20

gaaattgtgt tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60

ctctcctgca gggccagtca gagtgttagc agctacttag cctggtacca acagaaacct 120

ggccaggctc ccaggctcct catctatgat gcatccaaca gggccactgg catcccagcc 180

aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctagagcct 240

gaagattttg cagtttatta ctgtcagcag cgtagcaact ggcctccgac gttcggccaa 300

gggaccaagg tggaaatcaa a 321

<210> 21

<211> 366

<212> DNA

<213> Artificial sequences

<400> 21

gaagtgcagc tgctggaaag cggcggcggc ctggtgcagc cgggcggcag cctgcgcctg 60

agctgcgcgg tgagcggctt tacctttaac agctttgcga tgagctgggt gcgccaggcg 120

ccgggcaaag gcctggaatg ggtgagcgcg attagcggca gcggcggcgg cacctattat 180

gcggatagcg tgaaaggccg ctttaccatt agccgcgata acagcaaaaa caccctgtat 240

ctgcagatga acagcctgcg cgcggaagat accgcggtgt atttttgcgc gaaagataaa 300

attctgtggt ttggcgaacc ggtgtttgat tattggggcc agggcaccct ggtgaccgtg 360

agcagc 366

<210> 22

<211> 333

<212> DNA

<213> Artificial sequences

<400> 22

gatattgtgc tgacccagag cccggcgagc ctggcggtga gcccgggcca gcgcgcgacc 60

attacctgcc gcgcgagcca gagcgtgagc accagcagca gcagctttat gcattggtat 120

cagcagaaac cgggccagcc gccgaaactg ctgattaaat atgcgagcaa cctggaaagc 180

ggcgtgccgg cgcgctttag cggcagcggc agcggcaccg attttaccct gaccattaac 240

ccggtggaag cgaacgatac cgcgaactat tattgccagc atagctggga aattccgtat 300

acctttggcc agggcaccaa actggaaatt aaa 333

<210> 23

<211> 348

<212> DNA

<213> Artificial sequences

<400> 23

gaagtgcagc tggtggaaag cggcggcggc ctggtgaaac cgggcggcag cctgcgcctg 60

agctgcgcgg cgagcggctt tatttttcgc agctatggca tgagctgggt gcgccaggcg 120

ccgggcaaag gcctggaatg ggtggcgagc attagcagcg gcggcagcac ctattatccg 180

gatagcgtga aaggccgctt taccattagc cgcgataacg cgaaaaacag cctgtatctg 240

cagatgaaca gcctgcgcgc ggaagatacc gcggtgtatg attgcgcgcg cggctatgat 300

agcggctttg cgtattgggg ccagggcacc ctggtgaccg tgagcagc 348

<210> 24

<211> 324

<212> DNA

<213> Artificial sequences

<400> 24

gatattgtga tgacccagag cccgagcagc gtgagcgcga gcgtgggcga tcgcgtgacc 60

attacctgcc gcgcgagcca gaacgtggat agcgcggtgg cgtggtatca gcagaaaccg 120

ggcaaagcgc cgaaagcgct gatttatagc gcgagctatc gctatagcgg cgtgccgagc 180

cgctttagcg gccgcggcag cggcaccgat tttaccctga ccattagcag cctgcagccg 240

gaagattttg cgacctatta ttgccagcag tattatagca ccccgtggac ctttggccag 300

ggcaccaaag tggaaattaa acgc 324

<210> 25

<211> 381

<212> DNA

<213> Artificial sequences

<400> 25

gaagtgaaac tggtggaaag cggcggcggc ctggtgcagc cgggccgcag cctgcgcctg 60

agctgcaccg cgagcggctt tacctttacc gattattata tgagctgggt gcgccaggcg 120

ccgggcaaag gcctggaatg ggtgggcctg attcgcagca aagcggatgg ctataccacc 180

gaatatagcg cgagcgtgaa aggccgcttt accattagcc gcgatgatag caaaagcatt 240

ctgtatctgc agatgaacag cctgaaaacc gaagataccg cggtgtatta ttgcgcgcgc 300

gatgcggcgt attatagcta ttatagcccg gaaggcgcga tggattattg gggccagggc 360

accctggtga ccgtgagcag c 381

<210> 26

<211> 45

<212> DNA

<213> Artificial sequences

<400> 26

ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatct 45

<210> 27

<211> 57

<212> DNA

<213> Artificial sequences

<400> 27

gcctccacca agggcccatc tgtcttcccc ctggccccca gctcctctgg ctccgga 57

<210> 28

<211> 141

<212> DNA

<213> Artificial sequences

<400> 28

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgatatcta c 141

<210> 29

<211> 66

<212> DNA

<213> Artificial sequences

<400> 29

atctgggcgc ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt 60

tactgc 66

<210> 30

<211> 123

<212> DNA

<213> Artificial sequences

<400> 30

aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60

gggcccaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120

tcc 123

<210> 31

<211> 111

<212> DNA

<213> Artificial sequences

<400> 31

cgccgcgatc agcgcctgcc gccggatgcg cataaaccgc cgggcggcgg cagctttcgc 60

accccgattc aggaagaaca ggcggatgcg catagcaccc tggcgaaaat t 111

<210> 32

<211> 336

<212> DNA

<213> Artificial sequences

<400> 32

cgcgtgaaat ttagccgcag cgcggatgcg ccggcgtatc agcagggcca gaaccagctg 60

tataacgaac tgaacctggg ccgccgcgaa gaatatgatg tgctggataa acgccgcggc 120

cgcgatccgg aaatgggcgg caaaccgcgc cgcaaaaacc cgcaggaagg cctgtataac 180

gaactgcaga aagataaaat ggcggaagcg tatagcgaaa ttggcatgaa aggcgaacgc 240

cgccgcggca aaggccatga tggcctgtat cagggcctga gcaccgcgac caaagatacc 300

tatgatgcgc tgcatatgca ggcgctgccg ccgcgc 336

<210> 33

<211> 1557

<212> DNA

<213> Artificial sequences

<400> 33

atgaactcct tctccacaag cgccttcggt ccagttgcct tctccctggg gctgctcctg 60

gtgttgcctg ctgccttccc tgccccagat attgtgctga cccagagccc ggcgagcctg 120

gcggtgagcc cgggccagcg cgcgaccatt acctgccgcg cgagccagag cgtgagcacc 180

agcagcagca gctttatgca ttggtatcag cagaaaccgg gccagccgcc gaaactgctg 240

attaaatatg cgagcaacct ggaaagcggc gtgccggcgc gctttagcgg cagcggcagc 300

ggcaccgatt ttaccctgac cattaacccg gtggaagcga acgataccgc gaactattat 360

tgccagcata gctgggaaat tccgtatacc tttggccagg gcaccaaact ggaaattaaa 420

ggtggcggtg gctcgggcgg tggtgggtcg ggtggcggcg gatctgaagt gcagctggtg 480

gaaagcggcg gcggcctggt gaaaccgggc ggcagcctgc gcctgagctg cgcggcgagc 540

ggctttattt ttcgcagcta tggcatgagc tgggtgcgcc aggcgccggg caaaggcctg 600

gaatgggtgg cgagcattag cagcggcggc agcacctatt atccggatag cgtgaaaggc 660

cgctttacca ttagccgcga taacgcgaaa aacagcctgt atctgcagat gaacagcctg 720

cgcgcggaag ataccgcggt gtatgattgc gcgcgcggct atgatagcgg ctttgcgtat 780

tggggccagg gcaccctggt gaccgtgagc agcggtggcg gtggctcggg cggtggtggg 840

tcgggtggcg gcggatctga accgaaaagc tgcgacaaaa ctcacacatg cccaccgtgc 900

ccagcacctg aactcctggg gggaccgtca gtcttcctct tccccccaaa acccaaggac 960

accctcatga tctcccggac ccctgaggtc acatgcgtgg tggtggacgt gagccacgaa 1020

gaccctgagg tcaagttcaa ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca 1080

aagccgcggg aggagcagta caacagcacg taccgtgtgg tcagcgtcct caccgtcctg 1140

caccaggact ggctgaatgg caaggagtac aagtgcaagg tctccaacaa agccctccca 1200

gcccccatcg agaaaaccat ctccaaagcc aaagggcagc cccgagaacc acaggtgtac 1260

accctgcccc catcccggga ggagatgacc aagaaccagg tcagcctgac ctgcctggtc 1320

aaaggcttct atcccagcga catcgccgtg gagtgggaga gcaatgggca gccggagaac 1380

aactacaaga ccacgcctcc cgtgctggac tccgacggct ccttcttcct ctacagcaag 1440

ctcaccgtgg acaagagcag gtggcagcag gggaacgtct tctcatgctc cgtgatgcac 1500

gaggctctgc acaaccacta cacgcagaag agcctctccc tgtctccggg taaatga 1557

<210> 34

<211> 36

<212> DNA

<213> Artificial sequences

<400> 34

attcaaaatt ttatcgatgc tccggtgccc gtcagt 36

<210> 35

<211> 22

<212> DNA

<213> Artificial sequences

<400> 35

tcacgacacc tgaaatggaa ga 22

<210> 36

<211> 46

<212> DNA

<213> Artificial sequences

<400> 36

catttcaggt gtcgtgagga tccgccacca tggcgctgcc ggtgac 46

<210> 37

<211> 33

<212> DNA

<213> Artificial sequences

<400> 37

ggggagggag aggggcttag cgcggcggca gcg 33

<210> 38

<211> 17

<212> DNA

<213> Artificial sequences

<400> 38

gcccctctcc ctccccc 17

<210> 39

<211> 25

<212> DNA

<213> Artificial sequences

<400> 39

attatcatcg tgtttttcaa aggaa 25

<210> 40

<211> 44

<212> DNA

<213> Artificial sequences

<400> 40

aaaacacgat gataatgcca ccatgaactc cttctccaca agcg 44

<210> 41

<211> 46

<212> DNA

<213> Artificial sequences

<400> 41

aatccagagg ttgattgtcg acgaattctc atttgcccgg gctcag 46

<210> 42

<211> 21

<212> DNA

<213> Artificial sequences

<400> 42

cctttccggg actttcgctt t 21

<210> 43

<211> 20

<212> DNA

<213> Artificial sequences

<400> 43

gcagaatcca ggtggcaaca 20

<210> 44

<211> 21

<212> DNA

<213> Artificial sequences

<400> 44

catgtacgtt gctatccagg c 21

<210> 45

<211> 21

<212> DNA

<213> Artificial sequences

<400> 45

ctccttaatg tcacgcacga t 21

Claims (9)

1. An OCTS-CAR dual-targeting chimeric antigen receptor, characterized in that it comprises CD8 leader membrane receptor signal peptide, dual antigen binding region, CD8 Hinge chimeric receptor hinge, CD8 Transmembrane chimera connected in series successively Receptor transmembrane domain, CD28 chimeric receptor costimulator, CD134 chimeric receptor costimulator and TCR chimeric receptor T cell activation domain, Wherein, the double-antigen-binding region includes the heavy chain VH and light chain VL of two single-chain antibodies connected in series or at a turn, the intra-antibody hinge Inner-Linker and the single-chain antibody inter-hinge Inter-Linker, The two single-chain antibodies refer to any combination of BCMA single-chain antibody, CD319 single-chain antibody, CD38 single-chain antibody, PDL1 single-chain antibody, and CD123 single-chain antibody, The gene sequence encoding the CD8 leader membrane receptor signal peptide is shown in SEQ ID NO.15; the gene sequence encoding the BCMA single-chain antibody light chain VL is shown in SEQ ID NO.16, encoding the BCMA single chain The gene sequence of the antibody heavy chain VH is shown in SEQ ID NO.17; the gene sequence encoding the CD319 single-chain antibody light chain VL is shown in SEQ ID NO.18, and the gene encoding the CD319 single-chain antibody heavy chain VH is shown in SEQ ID NO.18. The sequence is shown in SEQ ID NO.19; the gene sequence encoding the CD38 single-chain antibody light chain VL is shown in SEQ ID NO.20, and the gene sequence encoding the CD38 chain antibody heavy chain VH is shown in SEQ ID NO.21. The gene sequence encoding the light chain VL of the PDL1 single-chain antibody is shown in SEQ ID NO.22, and the gene sequence encoding the heavy chain VH of the PDL1 single-chain antibody is shown in SEQ ID NO.23; the CD123 encoding the CD123 The gene sequence of the single-chain antibody light chain VL is shown in SEQ ID NO.24, the gene sequence encoding the CD123 single-chain antibody heavy chain VH is shown in SEQ ID NO.25; the gene sequence encoding the inner hinge Inner-Linker of the antibody As shown in SEQ ID NO.26; the gene sequence encoding the single-chain antibody hinge Inter-Linker is shown in SEQ ID NO.27; the gene sequence encoding the CD8 Hinge chimeric receptor hinge is shown in SEQ ID NO.28 The gene sequence encoding the CD8 Transmembrane chimeric receptor transmembrane region is shown in SEQ ID NO.29; the gene sequence encoding the CD28 chimeric receptor costimulator is shown in SEQ ID NO.30; The gene sequence of the CD134 chimeric receptor costimulatory factor is shown in SEQ ID NO.31; the gene sequence encoding the T-cell activation domain of the TCR chimeric receptor is shown in SEQ ID NO.32. 2. an OCTS-CAR recombinant expression vector, is characterized in that, comprises: Expression vector, human EF1α promoter, and gene encoding OCTS-CAR dual-targeting chimeric antigen receptor, Wherein, the gene sequence of the human EF1α promoter is shown in SEQ ID NO. 14, and the gene encoding the OCTS-CAR dual-targeting chimeric antigen receptor is described in claim 1. 3. an OCTS-CAR recombinant expression vector, is characterized in that, comprises: Expression vector, human EF1α promoter, gene encoding OCTS-CAR dual targeting chimeric antigen receptor and gene encoding PDL1 single chain antibody, Wherein, the gene sequence of human EF1α promoter is shown in SEQ ID NO. 14, the gene encoding OCTS-CAR dual-targeting chimeric antigen receptor is described in claim 1, and the gene sequence encoding PDL1 single-chain antibody is shown in SEQ ID NO.33 shows. 4. OCTS-CAR recombinant expression vector according to claim 2 or 3, is characterized in that: Wherein, the expression vector is a lentivirus expression vector, a retrovirus expression vector, an adenovirus expression vector, an adenovirus-related virus expression vector or a plasmid. 5. OCTS-CAR recombinant expression vector according to claim 4, is characterized in that: Wherein, the expression vector is the third-generation lentiviral expression vector pLenti-3G basic, and the lentiviral expression vector includes the ampicillin resistance gene AmpR sequence, the prokaryotic replicon pUC Ori sequence, the viral replicon SV40 Ori sequence, and the RSV promoter. sub, lentiviral 5terminal LTR, lentiviral 3terminal Self-Inactivating LTR, Gag cis-element, RRE cis-element, env cis-element, cPPT cis-element, ZsGreen1 green fluorescent protein, IRES ribosomal binding sequence, eWPRE enhanced soil Post-transcriptional regulatory elements of rat hepatitis B virus. 6. a construction method of OCTS-CAR recombinant expression vector as claimed in claim 3, is characterized in that, comprises the following steps: A. Ampicillin resistance gene AmpR sequence, prokaryotic replicon pUC Ori sequence, viral replicon SV40 Ori sequence, RSV promoter, lentivirus 5terminal LTR, lentivirus 3terminal Self-Inactivating LTR, Gag cis element, RRE cis Form element, env cis element, cPPT cis element, ZsGreen1 green fluorescent protein, IRES ribosome binding sequence, eWPRE enhanced woodchuck hepatitis B virus post-transcriptional regulatory element stored on the third-generation lentiviral backbone plasmid pLenti-3G basic ; B. Will encode CD8 leader membrane receptor signal peptide, double antigen binding region, CD8Hinge chimeric receptor hinge, CD8 Transmembrane chimeric receptor transmembrane region, CD28 chimeric receptor costimulator, CD134 chimeric receptor costimulator And the gene of TCR chimeric receptor T cell activation domain was cloned into the lentiviral backbone plasmid pLenti-3Gbasic through enzyme digestion, ligation and recombination reaction, and the third-generation recombinant lentiviral plasmid based on OCTS design was obtained; C. The recombinant lentiviral plasmids obtained in step B were co-transfected into HEK293T/17 cells with lentiviral packaging plasmids pPac-GP, pPac-R and membrane protein pEnv-G, and gene transcription was performed in HEK293T/17 cells After expression, the successfully packaged recombinant lentiviral vector will be released into the cell culture supernatant, and the supernatant of the contained recombinant lentiviral vector will be collected; D. Purify the obtained recombinant lentiviral supernatant by suction filtration, adsorption, and elution column purification to obtain CAR-T recombinant lentiviral expression vectors respectively. 7. a construction method of OCTS-CAR recombinant expression vector as claimed in claim 4, is characterized in that, comprises the following steps: A. Ampicillin resistance gene AmpR sequence, prokaryotic replicon pUC Ori sequence, viral replicon SV40 Ori sequence, RSV promoter, lentivirus 5terminal LTR, lentivirus 3terminal Self-Inactivating LTR, Gag cis element, RRE cis Form element, env cis element, cPPT cis element, ZsGreen1 green fluorescent protein, IRES ribosome binding sequence, eWPRE enhanced woodchuck hepatitis B virus post-transcriptional regulatory element stored on the third-generation lentiviral backbone plasmid pLenti-3G basic ; B. Will encode CD8 leader membrane receptor signal peptide, double antigen binding region, CD8Hinge chimeric receptor hinge, CD8 Transmembrane chimeric receptor transmembrane region, CD28 chimeric receptor costimulator, CD134 chimeric receptor costimulator , TCR chimeric receptor T cell activation domain and the gene encoding PDL1 single-chain antibody were cloned into the lentiviral backbone plasmid pLenti-3G basic through enzyme digestion, ligation and recombination reaction, and the third-generation recombinant lentiviral plasmid based on OCTS design was obtained. ; C. The recombinant lentiviral plasmids obtained in step B were co-transfected into HEK293T/17 cells with lentiviral packaging plasmids pPac-GP, pPac-R and membrane protein pEnv-G, and gene transcription was performed in HEK293T/17 cells After expression, the successfully packaged recombinant lentiviral vector will be released into the cell culture supernatant, and the supernatant of the contained recombinant lentiviral vector will be collected; D. Purify the obtained recombinant lentiviral supernatant by suction filtration, adsorption, and elution column purification to obtain CAR-T recombinant lentiviral expression vectors respectively. 8. An OCTS-CAR-T cell, which is the OCTS-CAR-T recombinant expression vector of claim 3 or 4 introduced into the genome or modified by the CAR dual-targeting chimeric antigen receptor of claim 1 of T lymphocytes. 9. The application of the OCTS-CAR-T cell of claim 8 in the preparation of a therapeutic drug for malignant plasma cell tumor or acute myeloid leukemia.