WO2018218876A1 - 一种基于octs技术的淋系白血病car-t治疗载体及其构建方法和应用 - Google Patents

一种基于octs技术的淋系白血病car-t治疗载体及其构建方法和应用 Download PDF

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WO2018218876A1
WO2018218876A1 PCT/CN2017/110667 CN2017110667W WO2018218876A1 WO 2018218876 A1 WO2018218876 A1 WO 2018218876A1 CN 2017110667 W CN2017110667 W CN 2017110667W WO 2018218876 A1 WO2018218876 A1 WO 2018218876A1
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chain antibody
seq
linker
light chain
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祁伟
俞磊
康立清
林高武
余宙
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上海优卡迪生物医药科技有限公司
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Definitions

  • the invention belongs to the field of medical biology, and particularly relates to a carrier, in particular to a lymphoid leukemia CAR-T therapeutic carrier based on OCTS technology. Furthermore, the invention relates to a method and application of the construction of the vector.
  • tumor immunotherapy The theoretical basis of tumor immunotherapy is that the immune system has the ability to recognize tumor-associated antigens and regulate the body's attack on tumor cells (highly specific cell lysis).
  • Burnet and Thomas proposed the theory of "immuno-monitoring", which believed that the mutant tumor cells that often appear in the body can be cleared by the immune system and lay a theoretical foundation for tumor immunotherapy [Burnet FM.Immunological aspects of malignant disease.Lancet, 1967; 1:171-4].
  • various tumor immunotherapy including cytokine therapy, monoclonal antibody therapy, adoptive immunotherapy, vaccine therapy, and the like are successively applied to the clinic.
  • CAR-T Chimeric Antigen Receptor T-Cell Immunotherapy, chimeric antigen receptor T cell immunotherapy.
  • the therapy is a method of transgenic introduction of a chimeric molecule composed of a promoter, an antigen recognition region, a costimulatory factor, and an effector region into a T cell genome, thereby enabling T cell recognition and signal transduction of target cells.
  • the 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 ].
  • CAR-T therapy is the most clinically advanced Novartis CLT019.
  • CLT019 is used to treat patients with relapsed and refractory acute lymphoblastic leukemia.
  • the progression-free survival rate of tumors is 67% for six months, and the longest response time is more than two years. .
  • Shanghai Youkadi Biomedical Technology Co., Ltd. headquartered in Shanghai, China, cooperated with the hospital.
  • 36 patients with relapsed and refractory acute lymphoblastic leukemia were treated, including 24 patients, with a remission rate of 66.6%. This is a disruptive breakthrough in anti-cancer research.
  • CAR-T cell therapy is probably one of the most likely means of curing cancer, and was named the top of the 2013 Top Ten Technology Breakthrough by Science.
  • CAR-T is currently effective in the treatment of several types of hematological tumors such as B-lymphocytic leukemia, but there are some limitations.
  • a chimeric antigen receptor can only recognize one antigen target, and tumor cells are a complex group. After the tumor cells containing the corresponding antigen are cleared, the tumor cells that do not contain the corresponding antigen rapidly proliferate, and the tumor relapses after a period of time. Therefore, in order for the CAR-T recognition to recognize both antigens simultaneously, there are two options: one is to construct two sets of chimeric antigen receptors into a lentiviral transgenic vector, and the two sets of chimeric antigen receptors are used at one time. Transduction into primary T lymphocytes; second, two lentiviral transgenic vectors were transduced twice, and the two sets of chimeric antigen receptors were transduced into primary T lymphocytes, respectively.
  • protocol 1 takes up the valuable capacity of the lentiviral transgenic vector, which is not conducive to loading other functional elements; the transgenic vector has low packaging efficiency; the gene transduction efficiency is very low, and it is difficult to transduce into the primary T lymphocytes.
  • the disadvantage of the second scheme is that it requires two transductions.
  • the combined efficiency of the two transductions is low, the transduction cycle time is long, and the primary cells are prone to aging, resulting in a decline in proliferative capacity, a decrease in killing function, and an effect on tumor clearance.
  • Human B-lymphocyte antigen CD19 also known as CD19, is a protein encoded by the human CD19 gene.
  • CD19 is mainly expressed on the surface of follicular dendritic cells and B cells.
  • CD19 is not expressed on the surface of hematopoietic stem cells. It is present in the developmental stage of Pro-Bcell to Memory B cell and disappears on the surface of plasma cells.
  • CD19 is expressed on the surface of most B cell malignancies, including chronic lymphocytic leukemia (CLL), B acute lymphoblastic leukemia (B-ALL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) Cellular lymphoma (MCL).
  • CLL chronic lymphocytic leukemia
  • B-ALL B acute lymphoblastic leukemia
  • DLBCL diffuse large B-cell lymphoma
  • FL follicular lymphoma
  • MCL Cellular lymphoma
  • CD19 is a good target for tumor cell immunotherapy [Scheuermann RH, Racila E. CD19 antigen in leukemia and lymphoma diagnosis and immunotherapy. Leukemia & Lymphoma. 1995, 18(5-6): 385-97.].
  • CD22 is a type I transmembrane protein expressed mainly on mature B lymphocytes and plays an important role in B cell signaling. As a co-receptor of B cell receptor (BCR), CD22 cross-links CD22 and BCR through antigen, triggering CD22 phosphorylation, which mainly dephosphorylates and inactivates downstream signaling proteins and inhibits BCR signaling. CD22 is ubiquitous in normal B cell and B cell malignancies.
  • BCR B cell receptor
  • CD20 acts as a component of a signal transduction complex that regulates the growth of B lymphocytes, and CD20 is specifically expressed in the differentiation phase of pre B cell to mature B cell, especially on the surface of most lymphoma cells (Mohamed-Rachid) Boulassel and Ahmed Galal. Immunotherapy for B-Cell Neoplasms using T Cells expressing Chimeric Antigen Receptors. Sultan Qaboos University Med J, August 2012, Vol. 12, Iss. 3, pp. 273-285.).
  • CD30 belongs to the tumor necrosis factor receptor family and plays an important role in the apoptosis and proliferation of lymphocytes. It is expressed on almost all Hodgkin's lymphoma and some non-Hodgkin's lymphoma surfaces. It is currently used as a Hodge in the clinic. An important marker for the diagnosis of gold lymphoma and anaplastic large cell lymphoma (Carlos A. Ramos, et al. Chimeric T-Cells for Therapy of CD30+Hodgkin and Non-Hodgkin Lymphomas (HL & NHL). Biology of Blood and MarrowTransplantation, 2016, 22(3): S145-S146.).
  • CD123 is the alpha chain of human interleukin 3 receptor and is expressed on the surface of most acute myeloid leukemia (AML) cells and many hematopoietic cells.
  • AML acute myeloid leukemia
  • CD123 is a very good target for immunotherapy because even in CD123 expression is rare. In the cells, its expression will gradually increase with time, and acute myeloid leukemia is likely due to the clonal evolution of hematopoietic stem cells in the pre-leukemia stage, with CD123 as the target, it can play a role in clearing the marrow ( Saar Gill, Carl H. June et al. Preclinical targeting of human acute myeloid leukemia and myeloablation using chimeric antigen receptor-modified T cells. Blood. 2014; 123(15): 2343-2354.).
  • the IL-6 receptor (IL-6R) consists of an IL6 receptor subunit and two gp130 signal transduction subunits, mainly distributed on hepatocytes, neutrophils, mononuclear cells, macrophages, and lymphocytes; There is also a soluble receptor (sIL-6R), which lacks a transmembrane component and a cytoplasmic component. In signal transduction, activated sIL-6R binds to the membrane-bound gp130 subunit and exerts its effects [RoseJS , Scheller J, Elson G, et al. Interleukin-6biology is coordinated by membrane-bound and soluble receptors: role in in-flammation and cancer.
  • Cytokine storm (CRS) in CAR-T cell therapy is associated with over-activation of the IL-6 signaling pathway, blocking the blocking of IL-6R, which helps block the over-activation of the IL-6 signaling pathway and thereby control the CRS response.
  • One of the technical problems to be solved by the present invention is to provide a lymphoid leukemia CAR-T therapeutic vector based on OCTS technology.
  • it only requires one transduction, and the transduction efficiency is high, which does not affect the therapeutic effect of CAR-T treatment.
  • it does not occupy the valuable capacity of the lentiviral transgenic vector, and is advantageous for loading other functional elements.
  • it can effectively block IL6R, block IL6 signaling pathway, and prevent inflammatory factor storm (CRS) upgrade.
  • CRS inflammatory factor storm
  • the second technical problem to be solved by the present invention is to provide a method for constructing the carrier.
  • the third technical problem to be solved by the present invention is to provide an application of the carrier.
  • a lymphoblastic CAR-T therapeutic vector based on OCTS technology comprising a lentiviral backbone plasmid, a human EF1 ⁇ promoter, an OCTS chimeric receptor domain and an IL6R single chain antibody;
  • the lentiviral backbone plasmid comprises: an ampicillin resistance gene AmpR sequence for large-scale amplification of a strain of interest, as shown in SEQ ID NO. 1; a prokaryotic replicon pUC Ori sequence for plasmid replication, such as SEQ ID NO .2; a viral replicon SV40Ori sequence for enhancing replication in eukaryotic cells, as set forth in SEQ ID NO. 3; a lentiviral packaging cis element for lentiviral packaging; ZsGreen1 green fluorescent protein, such as SEQ ID NO.11; IRES ribosome binding sequence, as shown in SEQ ID NO. 12; eWPRE-enhanced woodchuck hepatitis B virus post-transcriptional regulatory element for enhancing the expression efficiency of the transgene, as set forth in SEQ ID NO. Show
  • the sequence of the human EF1 ⁇ promoter is shown in SEQ ID NO.
  • the OCTS chimeric receptor domain comprises: a CD8 leader chimeric receptor signal peptide as set forth in SEQ ID NO. 15, two sets of single chain antibodies: the first group is selected from any of the following four groups of single chain antibodies; : CD20 single-chain antibody light chain VL as shown in SEQ ID NO. 18, CD20 single-chain antibody heavy chain VH as shown in SEQ ID NO. 19; CD22 single-chain antibody light chain as shown in SEQ ID NO. VL, CD22 single-chain antibody heavy chain VH as shown in SEQ ID NO. 21; CD30 single-chain antibody light chain VL as shown in SEQ ID NO. 22, CD30 single-chain antibody as shown in SEQ ID NO.
  • a chain VH a CD123 single-chain antibody light chain VL as set forth in SEQ ID NO. 24, a CD123 single-chain antibody heavy chain VH as set forth in SEQ ID NO. 25; and a second set as set forth in SEQ ID NO. CD19 single-chain antibody light chain VL and CD19 single-chain antibody heavy chain VH as shown in SEQ ID NO. 17; antibody internal hinge Inner-Linker as shown in SEQ ID NO. 26, as shown in SEQ ID NO. Single-chain antibody inter-linker Inter-Linker, CD8 Hinge chimeric receptor hinge as set forth in SEQ ID NO. 28, CD8 Transmembrane chimeric receptor transmembrane region as set forth in SEQ ID NO. 29, as SEQ ID NO.
  • TCR shown in 32 a receptor T cell activation domain and a chimeric receptor costimulatory factor region;
  • the chimeric receptor costimulatory factor region is selected from the group consisting of 4-1BB, ICOS, CD27, OX40, CD28, MYD88, IL1R1, CD70, TNFRSF19L, TNFRSF27 a combination of any one or more of tumor necrosis factor receptor superfamily (TNFRSF) such as TNFRSF1OD, TNFRSF13B, TNFRSF18, and CD134.
  • TNFRSF tumor necrosis factor receptor superfamily
  • the lentiviral packaging cis element can be a second generation lentiviral vector or a third generation lentiviral vector.
  • the lentiviral packaging cis element comprises a second generation lentiviral vector comprising: a lentiviral 5terminal LTR as set forth in SEQ ID NO. 5, a lentiviral 3terminal Self-Inactivating LTR as set forth in SEQ ID NO. 6, such as SEQ a Gag cis element as shown in ID NO. 7, an RRE cis element as shown in SEQ ID NO. 8, an env cis element as shown in SEQ ID NO. 9, a cPPT as shown in SEQ ID NO. Cis component.
  • the lentiviral packaging cis element employs a third generation lentiviral vector comprising: a lentiviral 5terminal LTR as set forth in SEQ ID NO. 5, a lentiviral 3terminal Self-Inactivating LTR as set forth in SEQ ID NO. 6, such as SEQ a Gag cis element as shown in ID NO. 7, an RRE cis element as shown in SEQ ID NO. 8, an env cis element as shown in SEQ ID NO. 9, a cPPT as shown in SEQ ID NO. A cis-element, and an RSV promoter as set forth in SEQ ID NO.
  • the third generation of lentiviral vectors are preferably employed in the present invention.
  • the two sets of single-chain antibodies adopt a series connection method or a corner connection mode; as shown in FIG. 4:
  • the tandem junction is specifically: CD20 single-chain antibody light chain VL and CD19 single-chain antibody light chain VL adopt single-chain antibody inter-linker Inter-Linker Ligation, CD20 single-chain antibody light chain VL and CD20 single-chain antibody heavy chain VH are linked by antibody internal hinge Inner-Linker, CD19 single-chain antibody light chain VL and CD19 single-chain antibody heavy chain VH are linked by antibody internal hinge Inner-Linker, That is, pOCTS2019s (see Fig. 4A, Fig.
  • the corner connection mode is specifically: CD19 single-chain antibody light chain VL and CD19 single-chain antibody heavy chain VH adopt antibody inner hinge Inner-Linker linkage, CD20 single-chain antibody light chain VL
  • the CD19 single-chain antibody heavy chain VH is linked by a single-chain antibody hinge Inter-Linker
  • the CD20 single-chain antibody heavy chain VH and the CD19 single-chain antibody light chain VL are linked by a single-chain antibody inter-linker, ie, pOCTS2019t (see Figure 4B, Figure 4C);
  • the tandem junction is specifically: CD30 single-chain antibody light chain VL and CD19 single-chain antibody light chain VL adopt single-chain antibody inter-linker Inter-Linker Ligation, CD30 single-chain antibody light chain VL and CD30 single-chain antibody heavy chain VH are linked by an antibody internal hinge Inner-Linker, CD19 single-chain antibody light chain VL and CD19 single-chain antibody heavy chain VH are linked by an antibody internal hinge Inner-Linker, That is, pOCTS3019s (see Fig. 4A, Fig.
  • the corner connection mode is specifically: CD19 single-chain antibody light chain VL and CD19 single-chain antibody heavy chain VH adopt antibody inner hinge Inner-Linker linkage, CD30 single-chain antibody light chain VL
  • the CD19 single-chain antibody heavy chain VH is linked by a single-chain antibody hinge Inter-Linker
  • the CD30 single-chain antibody heavy chain VH and the CD19 single-chain antibody light chain VL are linked by a single-chain antibody inter-linker, ie, pOCTS3019t (see Figure 4B, Figure 4C);
  • the tandem junction is specifically: CD22 single-chain antibody light chain VL and CD19 single-chain antibody light chain VL adopt single-chain antibody inter-linker Inter-Linker Ligation, CD22 single-chain antibody light chain VL and CD22 single-chain antibody heavy chain VH are linked by an antibody internal hinge Inner-Linker, CD19 single-chain antibody light chain VL and CD19 single-chain antibody heavy chain VH are linked by an antibody internal hinge Inner-Linker, That is, pOCTS2219s (see Fig. 4A, Fig.
  • CD19 single-chain antibody light chain VL and CD19 single-chain antibody heavy chain VH adopt antibody inner hinge Inner-Linker linkage
  • CD22 single-chain antibody light chain VL The CD19 single-chain antibody heavy chain VH is linked by a single-chain antibody hinge Inter-Linker, and the CD22 single-chain antibody heavy chain VH and the CD19 single-chain antibody light chain VL are linked by a single-chain antibody inter-linker, ie, pOCTS2219t (see Figure 4B, Figure 4C);
  • the tandem junction is specifically: CD123 single-chain antibody light chain VL and CD19 single-chain antibody light chain VL adopt single-chain antibody inter-linker Inter-Linker Ligation, CD123 single-chain antibody light chain VL and CD123 single-chain antibody heavy chain VH are linked by an antibody internal hinge Inner-Linker, CD19 single-chain antibody light chain VL and CD19 single-chain antibody heavy chain VH are linked by an antibody internal hinge Inner-Linker, That is, pOCTS12319s (see Fig. 4A, Fig.
  • CD19 single-chain antibody light chain VL and CD19 single-chain antibody heavy chain VH adopts antibody internal hinge Inner-Linker linkage, CD123 single-chain antibody light chain VL
  • the CD19 single-chain antibody heavy chain VH is linked by a single-chain antibody Hinge Inter-Linker, and the CD123 single-chain antibody heavy chain VH and the CD19 single-chain antibody light chain VL are linked by a single-chain antibody inter-linker, ie, pOCTS12319t (see Figure 4B, Figure 4C).
  • sequence of the IL6R single chain antibody is set forth in SEQ ID NO.
  • the eWPRE-enhanced woodchuck hepatitis B virus post-transcriptional regulatory element has a 6-nucleotide enhancing mutation, specifically: g.396G>A, g.397C>T, g.398T>C, g. 399G>A, g.400A>T, g.411A>T.
  • the entire OCTS structural gene expression is initiated by the human EF1 ⁇ promoter, which is located at the N-terminus of the OCTS coding sequence, for directing the OCTS protein localization to the cell membrane;
  • the antibodies are combined into a dual antigen recognition region for recognizing the corresponding target antigen;
  • the CD8 Hinge chimeric receptor hinge is used to anchor the scFv to the outside of the cell membrane;
  • the CD8 Transmembrane chimeric receptor transmembrane region is used to embed the entire target
  • the receptor is immobilized on a cell membrane;
  • the CD28 chimeric receptor costimulatory factor is used to stimulate T lymphocyte activation in vitro and in vivo tumor cell killing;
  • the CD134 chimeric receptor costimulatory factor is used to promote T lymphocyte proliferation And factor secretion, enhance tumor immunity, and facilitate long-term survival of memory T cells;
  • the TCR chimeric receptor T cell activation domain is used to activate expression of a downstream signaling pathway;
  • the chimeric receptor costimulatory factor region employs a CD28 chimeric receptor costimulatory factor as set forth in SEQ ID NO. 30 and a CD134 chimeric receptor costimulatory factor combination as set forth in SEQ ID NO. .
  • the CD19 single-chain antibody light chain VL, CD19 single-chain antibody heavy chain VH, CD20 single-chain antibody light chain VL, CD20 single-chain antibody heavy chain VH, CD30 single-chain antibody light chain VL, CD30 single-chain antibody heavy The chain VH, CD123 single-chain antibody light chain VL, CD123 single-chain antibody heavy chain VH, IL6R single-chain antibody were all humanized.
  • a lymphoblastic CAR-T therapeutic vector based on the above OCTS technology comprising the steps of:
  • a ampicillin-resistant gene AmpR sequence as shown in SEQ ID NO. 1 a prokaryotic replicon pUC Ori sequence as shown in SEQ ID NO. 2, a viral replicon as shown in SEQ ID NO. SV40Ori sequence, lentiviral packaging cis element for lentiviral packaging, ZsGreen1 green fluorescent protein as set forth in SEQ ID NO. 11, an IRES ribosome binding sequence as set forth in SEQ ID NO. 12, as SEQ ID NO.
  • the eWPRE-enhanced woodchuck hepatitis B virus post-transcriptional regulatory element shown in 13 is stored on the lentiviral backbone plasmid;
  • the obtained recombinant lentiviral plasmid was co-transfected into HEK293T/17 cells together with the lentiviral packaging plasmids pPac-GP, pPac-R and membrane protein granule pEnv-G, and then expressed in HEK293T/17 cells.
  • the successfully packaged lentiviral vector is released into the cell culture supernatant, and the supernatant of the recombinant lentiviral vector contained is collected;
  • the obtained recombinant lentiviral supernatant was purified by column filtration using suction filtration, adsorption and elution to obtain recombinant lentiviral vectors, respectively.
  • the filtration step is to control the supernatant volume to be 200 ml to 2000 ml, and the control vacuum degree is -0.5 MPA to -0.9 MPA to prevent carrier loss due to plugging;
  • the adsorption step To control the pH of the solution at 6-8, prevent the pH change from causing the carrier to be inactivated;
  • the elution step should control the ionic strength of the eluent from 0.5M to 1.0M to prevent the ionic strength change from causing incomplete elution. Or the carrier is inactivated.
  • the use of the vector for the preparation of a medicament for the treatment of lymphoid leukemia is provided.
  • the present invention has the following beneficial effects:
  • the OCTS-CAR-T technology used in the present invention is based on the current conventional CAR-T cell therapy, and the chimeric antigen receptor can recognize two kinds of structures by optimizing the chimeric antigen receptor (CAR) structure.
  • the antigen greatly expands the recognition range of CAR-T cells, and the removal of the tumor population is more thorough and the effect is more durable; avoiding the batch culture of CAR-T cells, greatly saving costs; avoiding multiple return of different targeted CAR-T cells by patients It saves patients' economic expenditure, reduces the chance of recurrence, and indirectly improves the quality of life of patients.
  • the transduction efficiency is high, and the therapeutic effect of CAR-T treatment is not affected; the valuable capacity of the lentiviral transgenic vector is not occupied, which is advantageous for loading other functional elements, and the transgenic vector has high packaging efficiency and high gene transduction efficiency.
  • OCTS The full name of OCTS is One CAR with Two ScFvs, which combines two scFvs into one chimeric molecule (as shown in Figure 1) by tandem OCTS (Series OCTS) or OCTS (Turn OCTS) connection, giving T lymph
  • tandem OCTS Series OCTS
  • OCTS Teurn OCTS
  • TAA tumor-associated antigen
  • the basic design of OCTS includes two tumor-associated antigen (TAA) binding regions (usually derived from the scFv segment of the monoclonal antibody antigen binding region), an extracellular hinge region, a transmembrane region, and two cells. Internal signal transduction zone and an effect element zone.
  • TAA tumor-associated antigen
  • the scFv region is a key determinant of the specificity, effectiveness, and safety of genomic T cells themselves. With the upcoming entry into the clinical research phase of OCTS-CAR-T, CAR-T cell therapy is about to enter the 2.0 era.
  • the vector backbone used in the present invention can be applied to the structure of the third generation lentiviral vector, and can also be applied to the structure of the second generation lentiviral vector.
  • the structural differences between the second and third generation lentiviral vectors are shown in Figure 2B.
  • the third generation lentiviral vector (shown in Figure 2A) is preferred in the present invention.
  • the 3'SIN LTR removes the U3 region, eliminating the possibility of lentiviral vector self-replication, greatly improving safety; adding cPPT and WPRE elements, The transduction efficiency and the expression efficiency of the transgene are improved; the RSV promoter ensures the sustained and efficient transcription of the core RNA when the lentiviral vector is packaged; and the human EF1 ⁇ promoter is used to enable the CAR gene to be continuously expressed in the human body for a long time.
  • the CD19 single-chain antibody light chain VL, CD19 single-chain antibody heavy chain VH, CD20 single-chain antibody light chain VL, CD20 single-chain antibody heavy chain VH, CD22 single-chain antibody light chain VL, CD22 single-chain antibody heavy according to the present invention
  • Chain VH, CD30 single-chain antibody light chain VL, CD30 single-chain antibody heavy chain VH, CD123 single-chain antibody light chain VL, CD123 single-chain antibody heavy chain VH, IL6R single-chain antibody have been humanized and can effectively reduce
  • the production of human anti-mouse antibodies (HAMA) in vivo prolongs the half-life and effect of scFv and increases the survival time of OCTS-CAR-T cells.
  • 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 the like of the cells after transduction.
  • the OCTS-CAR-T cells used in the present invention can be used in human clinical experiments after being produced by a GMP-level workshop.
  • the recombinant lentiviral vector of the invention can realize the dual-targeting chimeric antigen receptor expressing CD19, CD20, CD22, CD30, CD123 and the like on human T lymphocytes, and guide and activate T lymphocytes to CD19, CD20, CD22,
  • the killing effect of CD30, CD123 and other positive cells can be used clinically to treat B lymphocytic leukemia and B lymphoma.
  • the present invention constructs a recombinant lentiviral vector by recombinant lentiviral vector backbone, OCTS domain, interleukin-6 receptor (IL6R) single chain antibody (single-chain antibody), and the recombinant lentiviral vector obtained in this manner can be realized in human T Single-chain antibody expressing IL-6R in lymphocytes can effectively block IL6R, block IL-6 signaling pathway, and can be used clinically to relieve Cytokine Release Syndrome (CRS) to ensure the patient's treatment during cell therapy. life safety.
  • IL6R interleukin-6 receptor
  • the OCTS-CAR-T cells of the present invention will provide a reliable guarantee for the treatment of tumor cells.
  • FIG. 1 is a schematic diagram of an OCTS chimeric receptor according to the present invention, including a schematic diagram of a series OCTS (Series OCTS) and a corner OCTS (Turn OCTS);
  • Series OCTS series OCTS
  • Turn OCTS corner OCTS
  • FIG. 2 is a schematic diagram showing the structure of a lentiviral vector according to the present invention
  • FIG. 2A is a schematic diagram showing the structure of a third generation lentiviral vector used in the present invention
  • FIG. 2B is a schematic diagram showing a comparison of the structures of the second generation and third generation lentiviral vectors
  • FIG. 3 is a flow chart showing the construction of the recombinant lentiviral vector of the present invention in Example 1 of the present invention.
  • A is a schematic diagram of the structure of the lentiviral backbone plasmid pLenti-3G basic;
  • B is a schematic diagram of 8 OCTS plasmids;
  • C is a schematic diagram of the structure of the pPac-GP plasmid;
  • D) is a pPac Schematic diagram of the structure of the -R plasmid;
  • E is a schematic diagram of the structure of the pEnv-G packaging plasmid;
  • FIG. 4 is a schematic diagram showing the sequence of components of an OCTS structure in Embodiment 1 of the present invention, wherein A is a schematic diagram of a series OCTS (Series OCTS), Figure B is a schematic diagram of a corner OCTS (Turn OCTS), and C is a plasmid of an OCTS structure. a list of numbers (OCTS Symbol);
  • Figure 5 is a diagram showing the restriction enzyme digestion and enzymatic cleavage agarose gel electrophoresis of recombinant lentiviral plasmids pOCTS12319s, pOCTS2219s, pOCTS2019s, pOCTS3019s, pOCTS12319t, pOCTS2219t, pOCTS2019t, pOCTS3019t in Example 1 of the present invention; wherein Figure 5A is the digestion of pOCTS12319s.
  • Schematic diagram of prediction, Fig. 5B is an enzymatic cleavage agarose gel electrophoresis map of pOCTS12319s; Fig.
  • FIG. 5C is a schematic diagram of enzymatic cleavage prediction of pOCTS2219s
  • Fig. 5D is an enzymatic cleavage agarose gel electrophoresis pattern of pOCTS2219s
  • Fig. 5E is a schematic diagram of enzymatic cleavage prediction of pOCTS2019s
  • Fig. 5F is a digestion diagram of pOCTS2019s enzymatically cut agarose gel
  • Fig. 5G is a schematic diagram of enzymatic cleavage prediction of pOCTS3019s
  • Fig. 5H is an enzymatic cleavage agarose gel electrophoresis map of pOCTS3019s
  • 5I is a schematic diagram of enzymatic cleavage prediction of pOCTS12319t
  • Fig. 5I 5J is an enzyme-cut agarose gel electrophoresis map of pOCTS12319t
  • FIG. 5K is a schematic diagram of enzymatic cleavage prediction of pOCTS2219t
  • FIG. 5L is an enzyme-cut agarose gel electrophoresis pattern of pOCTS2219t
  • FIG. 5M is a schematic diagram of enzymatic cleavage prediction of pOCTS2019t
  • FIG. 5O is a schematic diagram of enzymatic cleavage prediction of pOCTS3019t
  • Figure 5P is enzymatic cleavage agarose gel electrophoresis of pOCTS3019t Figure 1
  • lane1 in Figure 5A is a 1 kb DNA ladder Marker: the bands are from top to bottom: 10 kb, 8 kb, 6 kb, 5 kb, 4 kb, 3.5 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb, 750 bp, 500 bp. 250bp
  • 5A is the BamH I restriction of pOCTS12319s: the band is 11384 bp and 818 bp from top to bottom; the lane1 in Fig. 5B is the electrophoresis result of the 1 kb DNA ladder Marker; the lane2 in Fig.
  • 5B is The results of BamH I digestion of pOCTS12319s;
  • the lane1 in Figure 5C is a 1 kb DNA ladder Marker: the bands are from top to bottom: 10 kb, 8 kb, 6 kb, 5 kb, 4 kb, 3.5 kb, 3 kb, 2.5 kb, 2 kb, 1.5 Kb, 1 kb, 750 bp, 500 bp, 250 bp;
  • lane2 in Figure 5C is the Kpn I restriction of pOCTS2219s: the bands are: 7824 bp, 4322 bp from top to bottom;
  • the lane1 in Figure 5D is the electrophoresis result of 1 kb DNA ladder Marker Lane2 in Fig.
  • 5D is the result of Kpn I digestion of pOCTS2219s; lane1 in Fig. 5E is 1 kb DNA ladder Marker: the bands are from top to bottom: 10 kb, 8 kb, 6 kb, 5 kb, 4 kb, 3.5 kb, 3 kb 2.5 kb, 2 kb, 1.5 kb, 1 kb, 750 bp, 500 bp, 250 bp; lane2 in Fig.
  • 5E is pOCTS2019s ApaL I digestion prediction: the bands from top to bottom are: 4883 bp, 3931 bp, 1726 bp, 1246 bp, 497 bp;
  • Figure 5F lane1 is the electrophoresis result of 1 kb DNA ladder Marker;
  • Figure 5F lane2 is the result of ApaL I digestion of pOCTS2019s ;
  • lane1 in Figure 5G is 1kb DNA Ladder Marker: The bands are: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp;
  • the lane2 in Figure 5G is pOCTS3019s BamH I digestion prediction: the bands from top to bottom are: 10268 bp, 1197 bp, 818 bp;
  • the lane1 in Figure 5H is the electrophore
  • 5I is a 1 kb DNA ladder Marker: the bands are from top to bottom: 10 kb, 8 kb, 6 kb, 5 kb, 4 kb, 3.5 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb, 750 bp, 500 bp, 250 bp; lane2 in Fig. 5I is the Sal I restriction of pOCTS12319t: the bands are: 6934 bp and 5389 bp from top to bottom; the lane1 in Fig. 5J is the electrophoresis result of the 1 kb DNA ladder Marker; the lane2 in Fig.
  • 5J is pOCTS12319t The result of Sal I digestion;
  • the lane1 in Figure 5K is a 1 kb DNA ladder Marker: the bands are from top to bottom: 10 kb, 8 kb, 6 kb, 5 kb, 4 kb, 3.5 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb. , 1 kb, 750 bp, 500 bp, 250 bp;
  • lane2 in Figure 5K is the EcoR I restriction of pOCTS2219t: From top to bottom, the bands are: 10626 bp, 1399 bp, 311 bp; the lane1 in Fig.
  • 5L is the electrophoresis result of the 1 kb DNA ladder Marker; the lane2 in Fig. 5L is the result of EcoR I digestion of pOCTS2219t; the lane1 in Fig. 5M is 1 kb.
  • DNA ladder Marker from top to bottom: 10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp;
  • lane2 in Figure 5M is The Pst I restriction of pOCTS2019t was predicted: the bands were: 9231 bp, 2554 bp, and 617 bp from top to bottom; the lane1 in Figure 5N was the electrophoresis result of the 1 kb DNA ladder Marker; the lane2 in Figure 5N was the Pst I digestion of pOCTS2019t. As a result, lane1 in Fig.
  • 5O is a 1 kb DNA ladder Marker: the bands are from top to bottom: 10 kb, 8 kb, 6 kb, 5 kb, 4 kb, 3.5 kb, 3 kb, 2.5 kb, 2 kb, 1.5 kb, 1 kb, 750 bp, 500 bp. 250bp; lane2 in Fig. 5O is the Sac II restriction of pOCTS3019t: the bands are: 11652bp, 887bp from top to bottom; the lane1 in Fig. 5P is the electrophoresis result of the 1kb DNA ladder Marker; the lane2 in Fig. 5P is Sac II digestion electrophoresis results of pOCTS3019t;
  • Example 6 is a schematic diagram showing the results of titer detection of the recombinant lentiviral vector in Example 1 of the present invention.
  • FIG. 7 is a flow chart showing the steps of constructing OCTS-CAR-T cells according to Example 1 of the present invention, comprising the steps of isolation culture, activation, gene transduction, and OCTS-CAR-T cell identification;
  • lane1 is DL2000marker, and the strips from top to bottom are from top to bottom: 2 kb, 1 kb, 750 bp, 500 bp, 250bp, 100bp;
  • lane2 is a positive control;
  • lane3 is a negative control;
  • lane4 is PBS;
  • lane5 is lysate;
  • lane6 is OCTS12319s-CAR-T cells;
  • lane7 is OCTS2219s-CAR-T cells;
  • lane8 is OCTS2019s-CAR-T cells;
  • Lane9 is OCTS3019s-CAR-T cells;
  • lane10 is OCTS12319t-CAR-T cells;
  • lane11 is OCTS2219t-CAR-T cells;
  • lane12 is OCTS2019t-CAR-T cells;
  • lane13 is OCTS3019t-CAR-T cells;
  • Figure 9 is a schematic diagram showing the transduction efficiency and immunological typing results of flow detection OCTS-CAR-T cells in Example 2 of the present invention; wherein, Figure 9A shows the transduction efficiency results of OCTS12319s-CAR-T cells; and Figure 9B shows OCTS12319s- Immunophenotyping results of CAR-T cells; Figure 9C shows the results of transduction efficiency of OCTS2219s-CAR-T cells; Figure 9D shows the results of immunophenotyping of OCTS2219s-CAR-T cells; Figure 9E shows the results of OCTS2019s-CAR-T cells Transduction efficiency results; Figure 9F shows the immunophenotyping results of OCTS2019s-CAR-T cells; Figure 9G shows the transduction efficiency results of OCTS3019s-CAR-T cells; Figure 9H shows the immunophenotyping results of OCTS3019s-CAR-T cells; Figure 9I shows the results of transduction efficiency of OCTS12319t-CAR
  • Figure 10 is a bar graph showing the killing efficiency of OCTS-CAR-T cells against target cells under different effect ratio conditions in Example 3 of the present invention; wherein, Figure 10A shows that OCTS12319s-CAR-T cells and OCTS12319t-CAR-T cells are different. Killing results of target cells; Figure 10B shows OCTS2219s-CAR-T cells and OCTS2219t-CAR-T cells for different target sizes Cell killing results; Figure 10C shows the killing results of OCTS2019s-CAR-T cells and OCTS2019t-CAR-T cells against different target cells; Figure 10D shows OCTS3019s-CAR-T cells and OCTS3019t-CAR-T cells for different target cells Killing results.
  • Lentiviral backbone plasmid pLenti-3G basic lentiviral packaging plasmid pPac-GP, pPac-R and membrane protein granule pEnv-G, HEK293T/17 cells, homologous recombinase, Oligo Annealing Buffer, Mycoplasma detection kit, Toxin detection kit, CD19 + K562, CD20 + K562, CD22 + K562, CD30 + K562, CD123 + K562, CD19 + CD123 + K562, CD19 + CD20 + K562, CD19 + CD22 + K562, CD19 + CD30 + K562, K562
  • the cell was purchased from Shiyan (Shanghai) Biomedical Technology Co., Ltd.; the specific preparation method of lentiviral backbone plasmid pLenti-3G basic has been disclosed in the invention entitled "A replication-defective recombinant lentivirus-based CAR-T transgenic vector and its Construction method and application
  • Human fresh peripheral blood is provided by a healthy donor
  • D-PBS (-), 0.4% trypan blue, mesh, various types of cell culture dishes, culture bags, culture plates are purchased from Corning;
  • Opti-MEM, Pen-Srep, Hepes, FBS, AIM-V, RPMI 1640, DMEM, lipofectamine 3000 were purchased from Invitrogen;
  • Biotinylated protein L was purchased from GeneScript;
  • the LDH test kit was purchased from Promega;
  • Ficoll lymphocyte separation solution was purchased from GE;
  • cryoPremium cryopreservation solution and sorting buffer are from Shanghai Youkadi Company;
  • rIL-2, rIL-7,, rIL-15, rIL-21 were purchased from peprotech;
  • CD3 monoclonal antibody CD28 monoclonal antibody, CD3/CD28 magnetic beads CD4/CD8 magnetic beads purchased from Germany Miltenyi company;
  • FACS flow cytometer was purchased from Thermo Corporation;
  • a fluorescent inverted microscope was purchased from Olympus;
  • CD4-FITC and CD8-APC were purchased from BioLegend;
  • ProteinL Magnetic Beads was purchased from BioVision;
  • RetroNectin purchased from Takara company
  • phycoerythrin (PE)-conjugated streptavidin was purchased from BD Bioscience;
  • the plasmid extraction kit and the agarose gel recovery kit were purchased from MN Corporation;
  • Competent cells TOP10 were purchased from tiangen;
  • DNeasy kit was purchased from Shanghai Jierui Company;
  • SA-HRP was purchased from Shanghai Shengsheng Company
  • EF1 ⁇ -F 5'-ATTCAAAATTTTATCGATGCTCCGGTGCCCGTCAGT-3' (SEQ ID NO. 34)
  • 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)
  • IL6Rscab-F AAAACACGATGATAATGCCACCATGAACTCCTTCTCCACAAGCG (SEQ ID NO. 40)
  • IL6Rscab-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)
  • the construction method of the recombinant lentiviral vector of the present invention is as follows:
  • Human EF1 ⁇ promoter (SEQ ID NO. 14), OCTS structure [CD8 leader chimeric receptor signal peptide as shown in SEQ ID NO. 15, two sets of single chain antibodies: the first group is selected from the following four groups Any one of the single-chain antibodies: a CD20 single-chain antibody light chain VL as shown in SEQ ID NO. 18, a CD20 single-chain antibody heavy chain VH as set forth in SEQ ID NO. 19; as shown in SEQ ID NO. CD22 single-chain antibody light chain VL, CD22 single-chain antibody heavy chain VH as set forth in SEQ ID NO. 21; CD30 single-chain antibody light chain VL as set forth in SEQ ID NO. 22, as set forth in SEQ ID NO.
  • the CD30 single-chain antibody heavy chain VH is shown; the CD123 single-chain antibody light chain VL as shown in SEQ ID NO. 24, the CD123 single-chain antibody heavy chain VH as shown in SEQ ID NO. 25; the second group is SEQ.
  • CD28 chimeric receptor co-stimulatory factor shown in 30 the CD134 chimeric receptor co-stimulatory factor as set forth in SEQ ID NO. 31
  • TCR chimeric receptor T cell activation domain as set forth in SEQ ID NO.
  • the lentiviral backbone plasmid pLenti-3G basic was digested with Cla I and EcoR I restriction enzymes, and the product was subjected to 1.5% agarose gel electrophoresis to confirm the 5623 bp fragment V1, and the gel was recovered and placed. In the Eppendorf tube, the corresponding fragment was recovered using MN's agarose gel recovery kit (see Table 1), and the purity and concentration of the product were determined;
  • sol The sol solution was added in a ratio of 200 ⁇ l NTI/100 mg gel, and placed in a water bath at 50 ° C for 5-10 minutes. 2, combined with DNA Centrifuge at 11,000 g for 30 seconds and discard the filtrate. 3, wash the film 700 ⁇ l of NT3 was added and centrifuged at 11,000 g for 30 seconds, and the filtrate was discarded. 4, wash the film Repeat the third step once 5, dry Centrifuge at 11000g for 1 minute, replace with a new collection tube and leave it at room temperature for 1 minute. 6, eluting DNA 15-30 ⁇ l of NE was added, and the mixture was allowed to stand at room temperature for 1 minute, centrifuged at 11,000 g for 1 minute, and the filtrate was collected.
  • the PCR cycle conditions were: 98 ° C for 3 min, (98 ° C for 10 sec, 55 ° C for 15 sec, 72 ° C for 30 sec) * 35 cycle , 72 ° C 5 min.
  • the product was subjected to 1.5% agarose gel electrophoresis, and the 2381 bp fragment d was confirmed, and the gel was recovered and placed in an Eppendorf tube, and the corresponding fragment was recovered by MN's agarose gel recovery kit (see Table 1), and the product was determined. Purity and concentration;
  • the PCR cycle conditions were: 98 ° C for 3 min, (98 ° C for 10 sec, 55 ° C for 15 sec, 72 ° C for 30 sec) * 35 cycle , 72 ° C 5 min.
  • the product was subjected to 1.5% agarose gel electrophoresis, and the 2399 bp fragment g was confirmed, and the gel was collected and placed in an Eppendorf tube, and the corresponding fragment was recovered by MN's agarose gel recovery kit (see Table 1), and the product was determined. Purity and concentration;
  • Recombinant lentiviral plasmid Fragment combination pOCTS12319s a, b, j, k pOCTS2219s a, c, j, k pOCTS2019s a, d, j, k pOCTS3019s a, e, j, k pOCTS12319t a, f, j, k pOCTS2219t a, g, j, k pOCTS2019t a, h, j, k pOCTS3019t a, i, j, k
  • the clones were picked for colony PCR identification.
  • the correct clones were identified as recombinant lentiviral plasmids pOCTS12319s, pOCTS2219s, pOCTS2019s, pOCTS3019s, pOCTS12319t, pOCTS2219t, pOCTS2019t, pOCTS3019t, and the correct clones were identified by enzyme digestion (see Figure 5). Sequencing review results.
  • Trypsin solution Weigh Trypsin 2.5g, EDTA 0.19729g in 1000ml beaker, add 900ml 1XPBS to dissolve, dissolve it, use 1000ml measuring cylinder to make up to 1000ml, 0.22 ⁇ M filter sterilization, long-term use can be saved to -20 ° C refrigerator;
  • a DNA/CaCl 2 solution was prepared in accordance with N + 0.5.
  • the amount of HEK293T/17 cell transfection plasmid per dish was used in the following ratios: recombinant lentiviral plasmid (20 ⁇ g), pPac-GP (15 ⁇ g), pPac-R (10 ⁇ g), pEnv-G (7.5 ⁇ g).
  • the supernatant collected at this time contained the recombinant lentiviral vectors lvOCTS12319s, lvOCTS2219s, lvOCTS2019s. , lvOCTS3019s, lvOCTS12319t, lvOCTS2219t, lvOCTS2019t, lvOCTS3019t.
  • the eluate is divided into 25 to 50 ⁇ l tubes, frozen and stored in a -80 ° C refrigerator for long-term storage;
  • a 24-well plate was used to inoculate 293T cells.
  • the cell volume per well is 5 ⁇ 10 4
  • the volume of the added medium is 500 ul
  • the growth rate of different kinds of cells is different
  • the cell fusion rate when the virus is infected is 40%-60%;
  • the cells were digested with 0.2 ml of 0.25% trypsin-EDTA solution and allowed to stand at 37 ° C for 1 minute. The entire cell surface was washed with a medium, and the cells were collected by centrifugation. Genomic DNA was extracted according to the instructions of the DNeasy kit. 200 ⁇ l of eluate was added to each sample tube to wash the DNA and quantify;
  • the total number of reactions is 40, and 1 ml of 2 ⁇ TaqMan Universal PCR Master Mix, 4 ⁇ l of forward primer, 4 ⁇ l of reverse primer, 4 ⁇ l of probe and 788 ⁇ l of H 2 O are mixed. Put on the ice after the shock;
  • the total number of reactions is 40, and 1 ml of 2 ⁇ TaqMan Universal PCR Master Mix, 100 ⁇ l of 10 ⁇ RNaseP primer/probe mix and 700 ⁇ l of H 2 O are mixed. Put on the ice after the shock;
  • the quantitative PCR instrument used was the ABI PRISM 7500 quantitative system.
  • the cycle conditions were set to: 50 ° C for 2 minutes, 95 ° C for 10 minutes, then 95 ° C for 15 seconds, 60 ° C for 1 minute of 40 cycles.
  • N number of cells at the time of infection (approximately 1 ⁇ 10 5 )
  • V volume of diluted virus added
  • the construction method of the OCTS-CAR-T cells of the present invention is as follows:
  • the upper layer of plasma was transferred to a new 50 ml centrifuge tube, the plasma was inactivated at 56 ° C for 30 min, returned to room temperature, 2000 g, centrifuged for 30 min, and the supernatant was taken to a 50 ml centrifuge tube for use.
  • the liquid in the centrifuge tube is divided into four layers, from top to bottom: a yellow plasma layer (recovered for use), a white film layer, a colorless transparent Ficoll layer, and a red-black mixed cell layer.
  • the obtained PBMC count was added to the sorting buffer at a ratio of 80 ul/10 7 cells, and the cell pellet was resuspended.
  • the LS separation column was separated from the magnetic frame.
  • the cell suspension was connected with a B tube, 5 ml of buffer solution was added, and the column was stoppered with a little force to collect CD4+/CD8+ cells, and the samples were counted.
  • the cell pellet was resuspended in AIM-V medium at a cell density of 1 x 10 6 /ml - 4 x 10 6 /ml, and 2 ⁇ 10 5 - 1 ⁇ 10 6 U / L IFN- ⁇ factor was added.
  • RetroNectin 1 ⁇ 10 3 ug/L to 1 ⁇ 10 4 ug/L RetroNectin was coated in a 24-well plate one day in advance, and the sealing film was sealed and coated overnight at 4°C.
  • OCTS-CAR-T cells were expanded in vitro.
  • the endotoxin working standard is 15EU/piece;
  • Step 4 is repeated once;
  • PCR reaction system was: ddH20 6.5 ⁇ l, Myco Mix 1 ⁇ l, 2 ⁇ Taq Plus Mix Master (Dye Plus) 12.5 ⁇ l, template 5 ⁇ l; PCR cycle conditions were: 95 °C30sec, (95°C30sec, 56°C30sec, 72°C30sec)*30cycle, 72°C for 5min.
  • the virus-transduced T cells were collected, and the cells were resuspended in a D-PBS(-) solution containing 1 to 4% human albumin and adjusted to 1 ⁇ 10 6 /ml.
  • Target cells were separately cultured [CD19 + K562, CD20 + K562, CD22 + K562, CD30 + K562, CD123 + K562, CD19 + CD123 + K562, CD19 + CD20 + K562, CD19 + CD22 + K562, CD19 + CD30 + K562, K562 cells] and effector cells [OCTS-CAR-T cells];
  • OCTS-CAR-T has a good killing effect on each of the single target cells and the dual target cells.
  • the killing efficiency of the Turn OCTS-structured CAR-T cells to the target cells is slightly higher than that of the Series OCTS structure.
  • Killing efficiency (experimental well - effector cell hole - target cell well) / (target cell maximum pore - target cell well) X100%

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Abstract

提供了一种基于OCTS技术的淋系白血病CAR-T治疗载体，包括慢病毒骨架质粒、人EF1α启动子、OCTS嵌合受体结构域和IL6R单链抗体；OCTS嵌合受体结构域包括：CD8 leader嵌合受体信号肽、两组单链抗体：第一组选自以下四组单链抗体的任意一组：CD20单链抗体轻链VL、CD20单链抗体重链VH；CD22单链抗体轻链VL、CD22单链抗体重链VH；CD30单链抗体轻链VL、CD30单链抗体重链VH；CD123单链抗体轻链VL、CD123单链抗体重链VH；第二组为CD19单链抗体轻链VL以及CD19单链抗体重链VH；抗体内铰链Inner-Linker、单链抗体间铰链Inter-Linker、CD8 Hinge嵌合受体铰链、CD8 Transmembrane嵌合受体跨膜区、TCR嵌合受体T细胞激活域以及嵌合受体共刺激因子区域。还提供了该载体的构建方法及其在制备治疗淋系白血病的药物中的应用。

Description

一种基于OCTS技术的淋系白血病CAR-T治疗载体及其构建方法和应用 技术领域
本发明属于医学生物领域，具体涉及一种载体，尤其涉及一种基于OCTS技术的淋系白血病CAR-T治疗载体。此外，本发明还涉及该载体的构建方法和应用。
背景技术
肿瘤免疫治疗的理论基础是免疫系统具有识别肿瘤相关抗原、调控机体攻击肿瘤细胞(高度特异性的细胞溶解)的能力。1950年代，Burnet和Thomas提出了“免疫监视”理论，认为机体中经常会出现的突变的肿瘤细胞可被免疫系统所识别而清除，为肿瘤免疫治疗奠定了理论基础[Burnet FM.Immunological aspects of malignant disease.Lancet,1967；1:1171-4]。随后，各种肿瘤免疫疗法包括细胞因子疗法、单克隆抗体疗法、过继免疫疗法、疫苗疗法等相继应用于临床。
2013年一种更先进的肿瘤免疫疗法---CAR-T疗法成功用于临床，并表现了前所未有的临床疗效。CAR-T，全称是Chimeric Antigen Receptor T-Cell Immunotherapy，嵌合抗原受体T细胞免疫疗法。该疗法是通过转基因的手段，将启动子、抗原识别区、共刺激因子、效应区等共同组成的嵌合分子，导入T细胞基因组内，从而使T细胞对靶细胞的识别、信号转导、杀伤等功能融为一体，实现了对靶细胞的特异性杀伤[Eleanor J.Cheadle,et al.CAR T cells:driving the road from the laboratory to the clinic.Immunological Reviews 2014.Vol.257:91–106]。CAR-T疗法在临床上最领先的有诺华的CLT019，采用CLT019治疗复发难治急性淋巴细胞白血病患者，六个月的肿瘤无进展生存率达到67％，其中最长的应答时间达到两年多。总部位于中国上海的上海优卡迪生物医药科技有限公司与医院合作，截止到2017年2月，共治疗复发难治急性淋巴细胞白血病患者36例，其中完全24例，缓解比例达到66.6％。这是抗癌研究的颠覆性突破。CAR-T细胞疗法可能是最有可能治愈癌症的手段之一，并被《Science》杂志评为2013年度十大科技突破之首。
CAR-T目前在治疗B-淋巴细胞白血病等几种类型的血液肿瘤方面疗效显著，但是也存在一些局限性，目前一个嵌合抗原受体只能识别一种抗原靶标，肿瘤细胞是个复杂的群体，含有相应抗原的肿瘤细胞被清除以后，不含相应抗原的肿瘤细胞会迅速增殖，一段时间后导致肿瘤复发。那么要使CAR-T识别能同时识别两种抗原，就有两个方案可选：一是将两组嵌合抗原受体构建进入一个慢病毒转基因载体，一次性将两组嵌合抗原受体转导进入原代T淋巴细胞；二是用两个慢病毒转基因载体分两次转导，将两组嵌合抗原受体分别转导进入原代T淋巴细胞。
方案一的缺点在于占用慢病毒转基因载体的宝贵容量，不利于装载其它功能元件；转基因载体包装效率低；基因转导效率非常低，很难转导进入原代T淋巴细胞内。
方案二的缺点在于需要经过两次转导，两次转导的综合效率较低，转导周期时间长，原代细胞容易衰老，导致增殖能力衰退，杀伤功能下降，影响肿瘤清除疗效。
人B-lymphocyte antigen CD19，又称为CD19，是一种由人CD19基因编码的蛋白质。CD19主要表达在滤泡树突状细胞和B细胞表面。CD19在造血干细胞表面不表达，它存在于Pro-Bcell到Memory B cell的发育阶段，消失于浆细胞(Plasma cell)表面。CD19在大多数B细胞恶性肿瘤表面表达，包括慢性淋巴细胞白血病(CLL)，B急性淋巴细胞白血病(B-ALL)，弥漫大B细胞淋巴瘤(DLBCL)，滤泡性淋巴瘤(FL)套细胞淋巴瘤(MCL)。因此，CD19是一个良好的肿瘤细胞免疫治疗的靶点[Scheuermann RH,Racila E.CD19 antigen in leukemia and lymphoma diagnosis and immunotherapy.Leukemia & Lymphoma.1995,18(5-6):385–97.]。
CD22是主要在成熟B淋巴细胞表达的Ⅰ型跨膜蛋白，在B细胞信号传导中起重要作用。 CD22作为B细胞受体(BCR)的一个共受体，通过抗原引起CD22与BCR交联，触发CD22磷酸化，主要使下游信号蛋白去磷酸化和失活，抑制BCR信号传导。CD22普遍存在于正常B细胞和B细胞恶性肿瘤中。
CD20作为一种信号转导复合物的成分对B淋巴细胞的生长有调节作用，并且CD20特异性表达在pre B cell到mature B cell的分化阶段，尤其是大多数淋巴瘤细胞表面(Mohamed-Rachid Boulassel and Ahmed Galal.Immunotherapy for B-Cell Neoplasms using T Cells expressing Chimeric Antigen Receptors.Sultan Qaboos University Med J,August 2012,Vol.12,Iss.3,pp.273-285.)。
CD30属于肿瘤坏死因子受体家族，在淋巴细胞的凋亡和增殖中起重要作用，几乎在所有的霍奇金淋巴瘤和部分非霍奇金淋巴瘤表面都有表达，目前在临床作为霍奇金淋巴瘤和间变性大细胞淋巴瘤诊断的重要标志(Carlos A.Ramos,et al.Chimeric T-Cells for Therapy of CD30+Hodgkin and Non-Hodgkin Lymphomas(HL & NHL).Biology of Blood and MarrowTransplantation,2016,22(3):S145-S146.)。
CD123是人白介素3受体的alpha链，在大多数的急性髓系白血病(AML)细胞和很多造血细胞表面均有表达，CD123是一个非常好的免疫治疗靶点，因为即使在CD123表达很少的细胞里，它的表达会随时间增长逐渐增强，并且急性髓系白血病很可能是由于处于白血病前期的造血干细胞经过克隆演化而来，以CD123为靶点，则可以起到清髓的作用(Saar Gill,Carl H.June et al.Preclinical targeting of human acute myeloid leukemia and myeloablation using chimeric antigen receptor-modified T cells.Blood.2014；123(15):2343-2354.)。
IL-6受体(IL-6R)由一个IL6受体亚单位和两个gp130信号转导亚单位组成，主要分布于肝细胞、中性粒细胞、单核、巨噬细胞以及淋巴细胞表面；另外还有一种可溶性受体(sIL-6R)，这种受体缺乏跨膜成分和胞质成分，在信号转导中，活化的sIL-6R与膜结合gp130亚单位结合，发挥其作用[RoseJS,SchellerJ,ElsonG,et al.Interleukin-6biology is coordinated by membrane-bound and soluble receptors:role in in-flammation and cancer.J Leukoc Biol,2006,80:227-236]。CAR-T细胞治疗中的细胞因子风暴(CRS)就与IL-6信号通路的过度活化相关，阻断封闭IL-6R，有利于阻断IL-6信号通路的过度激活，从而控制CRS反应。
目前，尚未有克服上述缺点针对CD19以及选自CD22、CD20、CD30、CD123中的任意一种抗原组成的双抗原的CAR-T治疗的相关报道。
发明内容
本发明要解决的技术问题之一是提供一种基于OCTS技术的淋系白血病CAR-T治疗载体。首先，其仅需要一次转导，转导效率高，不影响CAR-T治疗的疗效；其次，其不占用慢病毒转基因载体的宝贵容量，利于装载其它功能元件。第三，能有效封闭IL6R，阻断IL6信号通路，防止炎症因子风暴(CRS)升级。
本发明要解决的技术问题之二是提供该载体的构建方法。
本发明要解决的技术问题之三是提供该载体的应用。
为解决上述技术问题，本发明采用如下技术方案：
在本发明的一方面，提供一种基于OCTS技术的淋系白血病CAR-T治疗载体，包括慢病毒骨架质粒、人EF1α启动子、OCTS嵌合受体结构域和IL6R单链抗体；
所述慢病毒骨架质粒包括：用于目的菌株大量扩增的含氨苄青霉素抗性基因AmpR序列，如SEQ ID NO.1所示；用于质粒复制的原核复制子pUC Ori序列，如SEQ ID NO.2所示；用于增强真核细胞内的复制的病毒复制子SV40Ori序列，如SEQ ID NO.3所示；用于慢病毒包装的慢病毒包装顺式元件；ZsGreen1绿色荧光蛋白，如SEQ ID NO.11所示；IRES核糖体结合序列，如SEQ ID NO.12所示；用于增强转基因的表达效率的eWPRE增强型土拨鼠乙肝病毒转录后调控元件，如SEQ ID NO.13所示；
所述人EF1α启动子的序列如SEQ ID NO.14所示；
所述OCTS嵌合受体结构域包括：如SEQ ID NO.15所示的CD8 leader嵌合受体信号肽、两组单链抗体：第一组选自以下四组单链抗体的任意一组：如SEQ ID NO.18所示的CD20单链抗体轻链VL、如SEQ ID NO.19所示的CD20单链抗体重链VH；如SEQ ID NO.20所示的CD22单链抗体轻链VL、如SEQ ID NO.21所示的CD22单链抗体重链VH；如SEQ ID NO.22所示的CD30单链抗体轻链VL、如SEQ ID NO.23所示的CD30单链抗体重链VH；如SEQ ID NO.24所示的CD123单链抗体轻链VL、如SEQ ID NO.25所示的CD123单链抗体重链VH；第二组为如SEQ ID NO.16所示的CD19单链抗体轻链VL以及如SEQ ID NO.17所示的CD19单链抗体重链VH；如SEQ ID NO.26所示的抗体内铰链Inner-Linker、如SEQ ID NO.27所示的单链抗体间铰链Inter-Linker、如SEQ ID NO.28所示的CD8 Hinge嵌合受体铰链、如SEQ ID NO.29所示的CD8 Transmembrane嵌合受体跨膜区、如SEQ ID NO.32所示的TCR嵌合受体T细胞激活域以及嵌合受体共刺激因子区域；所述嵌合受体共刺激因子区域选自4-1BB、ICOS、CD27、OX40、CD28、MYD88、IL1R1、CD70、TNFRSF19L、TNFRSF27、TNFRSF1OD、TNFRSF13B、TNFRSF18、CD134等肿瘤坏死因子超家族(tumor necrosis factor receptor superfamily，TNFRSF)中的任意一种或多种的组合。
所述慢病毒包装顺式元件可以采用第二代慢病毒载体，也可以采用第三代慢病毒载体。所述慢病毒包装顺式元件采用第二代慢病毒载体包括：如SEQ ID NO.5所示的慢病毒5terminal LTR、如SEQ ID NO.6所示的慢病毒3terminal Self-Inactivating LTR、如SEQ ID NO.7所示的Gag顺式元件、如SEQ ID NO.8所示的RRE顺式元件、如SEQ ID NO.9所示的env顺式元件、如SEQ ID NO.10所示的cPPT顺式元件。所述慢病毒包装顺式元件采用第三代慢病毒载体包括：如SEQ ID NO.5所示的慢病毒5terminal LTR、如SEQ ID NO.6所示的慢病毒3terminal Self-Inactivating LTR、如SEQ ID NO.7所示的Gag顺式元件、如SEQ ID NO.8所示的RRE顺式元件、如SEQ ID NO.9所示的env顺式元件、如SEQ ID NO.10所示的cPPT顺式元件，以及如SEQ ID NO.4所示的RSV启动子。本发明优选采用第三代慢病毒载体。
优选的，所述两组单链抗体采用串联连接方式或者转角连接方式；如图4所示：
当两组单链抗体为CD20单链抗体和CD19单链抗体时，所述串联连接方式具体为：CD20单链抗体轻链VL与CD19单链抗体轻链VL采用单链抗体间铰链Inter-Linker连接，CD20单链抗体轻链VL与CD20单链抗体重链VH采用抗体内铰链Inner-Linker连接，CD19单链抗体轻链VL与CD19单链抗体重链VH采用抗体内铰链Inner-Linker连接，即pOCTS2019s(见图4A、图4C)；所述转角连接方式具体为：CD19单链抗体轻链VL与CD19单链抗体重链VH采用抗体内铰链Inner-Linker连接，CD20单链抗体轻链VL与CD19单链抗体重链VH采用单链抗体间铰链Inter-Linker连接，CD20单链抗体重链VH与CD19单链抗体轻链VL采用单链抗体间铰链Inter-Linker连接，即pOCTS2019t(见图4B、图4C)；
当两组单链抗体为CD30单链抗体和CD19单链抗体时，所述串联连接方式具体为：CD30单链抗体轻链VL与CD19单链抗体轻链VL采用单链抗体间铰链Inter-Linker连接，CD30单链抗体轻链VL与CD30单链抗体重链VH采用抗体内铰链Inner-Linker连接，CD19单链抗体轻链VL与CD19单链抗体重链VH采用抗体内铰链Inner-Linker连接，即pOCTS3019s(见图4A、图4C)；所述转角连接方式具体为：CD19单链抗体轻链VL与CD19单链抗体重链VH采用抗体内铰链Inner-Linker连接，CD30单链抗体轻链VL与CD19单链抗体重链VH采用单链抗体间铰链Inter-Linker连接，CD30单链抗体重链VH与CD19单链抗体轻链VL采用单链抗体间铰链Inter-Linker连接，即pOCTS3019t(见图4B、图4C)；
当两组单链抗体为CD22单链抗体和CD19单链抗体时，所述串联连接方式具体为：CD22单链抗体轻链VL与CD19单链抗体轻链VL采用单链抗体间铰链Inter-Linker连接，CD22单链抗体轻链VL与CD22单链抗体重链VH采用抗体内铰链Inner-Linker连接，CD19单链抗体轻链VL与CD19单链抗体重链VH采用抗体内铰链Inner-Linker连接，即pOCTS2219s(见图4A、图4C)；所述转角连接方式具体为：CD19单链抗体轻链VL与CD19单链抗体重链VH采用抗体内铰链Inner-Linker连接，CD22单链抗体轻链VL与CD19单链抗体重链VH采用单链抗体间铰链Inter-Linker连接，CD22单链抗体重链VH与CD19单链抗体轻链VL采用单链抗体间铰链Inter-Linker连接，即pOCTS2219t(见图4B、图4C)；
当两组单链抗体为CD123单链抗体和CD19单链抗体时，所述串联连接方式具体为：CD123单链抗体轻链VL与CD19单链抗体轻链VL采用单链抗体间铰链Inter-Linker连接，CD123单链抗体轻链VL与CD123单链抗体重链VH采用抗体内铰链Inner-Linker连接，CD19单链抗体轻链VL与CD19单链抗体重链VH采用抗体内铰链Inner-Linker连接，即pOCTS12319s(见图4A、图4C)；所述转角连接方式具体为：CD19单链抗体轻链VL与CD19单链抗体重链VH采用抗体内铰链Inner-Linker连接，CD123单链抗体轻链VL与CD19单链抗体重链VH采用单链抗体间铰链Inter-Linker连接，CD123单链抗体重链VH与CD19单链抗体轻链VL采用单链抗体间铰链Inter-Linker连接，即pOCTS12319t(见图4B、图4C)。
优选的，所述IL6R单链抗体的序列如SEQ ID NO.33所示。
优选的，所述eWPRE增强型土拨鼠乙肝病毒转录后调控元件有6个核苷酸的增强突变，具体为：g.396G>A、g.397C>T、g.398T>C、g.399G>A、g.400A>T、g.411A>T。
优选的，由所述人EF1α启动子启动整个OCTS结构基因表达，所述CD8 leader嵌合受体信号肽位于OCTS编码序列的N端，用于引导OCTS蛋白定位于细胞膜；所述两组单链抗体组合成双抗原识别区，用于识别相应靶抗原；所述CD8 Hinge嵌合受体铰链用于将scFv锚定于细胞膜外侧；所述CD8 Transmembrane嵌合受体跨膜区用于将整个嵌合受体固定于细胞膜上；所述CD28嵌合受体共刺激因子用于刺激T淋巴细胞体外激活和体内肿瘤细胞杀伤作用；所述CD134嵌合受体共刺激因子用于促进T淋巴细胞增殖和因子分泌，增强肿瘤免疫，有利于记忆T细胞的长期存活；所述TCR嵌合受体T细胞激活域用于激活下游信号通路的表达；所述IL6R单链抗体分泌到细胞外，封闭IL6R，阻断IL6信号通路，防止炎症因子风暴升级；当抗原识别区域与靶抗原结合时，信号通过嵌合受体传递至细胞内，从而产生T细胞增殖、细胞因子分泌增加、抗细胞凋亡蛋白分泌增加、细胞死亡延迟、裂解靶细胞等一系列生物学效应。
优选的，所述嵌合受体共刺激因子区域采用如SEQ ID NO.30所示的CD28嵌合受体共刺激因子以及如SEQ ID NO.31所示的CD134嵌合受体共刺激因子组合。
优选的，所述CD19单链抗体轻链VL、CD19单链抗体重链VH、CD20单链抗体轻链VL、CD20单链抗体重链VH、CD30单链抗体轻链VL、CD30单链抗体重链VH、CD123单链抗体轻链VL、CD123单链抗体重链VH、IL6R单链抗体均经过人源化改造。
在本发明的第二方面，提供一种上述基于OCTS技术的淋系白血病CAR-T治疗载体的构建方法，包括以下步骤：
(1)将如SEQ ID NO.1所示的含氨苄青霉素抗性基因AmpR序列、如SEQ ID NO.2所示的原核复制子pUC Ori序列、如SEQ ID NO.3所示的病毒复制子SV40Ori序列、用于慢病毒包装的慢病毒包装顺式元件、如SEQ ID NO.11所示的ZsGreen1绿色荧光蛋白、如SEQ ID NO.12所示的IRES核糖体结合序列、如SEQ ID NO.13所示的eWPRE增强型土拨鼠乙肝病毒转录后调控元件存储于慢病毒骨架质粒上；
(2)将如SEQ ID NO.14所示的人EF1α启动子、所述OCTS嵌合受体结构域以及如SEQ ID NO.33所示的IL6R单链抗体组合成OCTS嵌合受体设计方案，经过酶切、连接、重组反应克隆至慢病毒骨架质粒中，得到第三代OCTS设计的重组慢病毒质粒；
(3)将得到的重组慢病毒质粒分别与慢病毒包装质粒pPac-GP、pPac-R以及膜蛋白质粒pEnv-G共同转染HEK293T/17细胞，在HEK293T/17细胞中进行基因转录表达后，包装成功重组慢病毒载体会释放到细胞培养上清中，收集包含的重组慢病毒载体的上清液；
(4)将得到的重组慢病毒上清采用抽滤、吸附、洗脱的柱纯化方式进行纯化，分别得到重组慢病毒载体。
优选的，步骤(4)中，所述抽滤步骤要控制上清体积在200ml～2000ml，控制真空度在-0.5MPA～-0.9MPA，防止由于堵孔带来的载体损失；所述吸附步骤要控制溶液的PH值在6～8，防止PH的变化导致载体失活；所述洗脱步骤要控制洗脱液的离子强度在0.5M～1.0M，防止离子强度的变化导致洗脱不完全或者载体失活。
在本发明的第三方面，提供所述的载体在制备治疗淋系白血病的药物中的应用。
与现有技术相比，本发明具有如下有益效果：
本发明所采用的OCTS-CAR-T技术，是在目前传统CAR-T细胞治疗的基础上，通过对嵌合抗原受体(CAR)结构的优化改造，使得嵌合抗原受体能够识别两种抗原，大大拓展了CAR-T细胞的识别范围，针对肿瘤群体的清除更彻底，疗效更持久；避免分批培养CAR-T细胞，大大节约成本；避免患者多次回输不同靶向CAR-T细胞，节约了患者的经济支出，降低复发的几率，间接提高患者生存质量。仅需要一次转导，转导效率高，不影响CAR-T治疗的疗效；不占用慢病毒转基因载体的宝贵容量，利于装载其它功能元件，转基因载体包装效率高，基因转导效率高。
OCTS的全称是One CAR with Two ScFvs，通过串联OCTS(Series OCTS)或者转角OCTS(Turn OCTS)的连接方式，将两段scFv与整合成一个嵌合分子(如图1所示)，赋予T淋巴细胞HLA非依赖的方式识别两种肿瘤抗原的能力，相对于传统的CAR-T细胞能够识别更广泛的目标，进一步扩大了肿瘤细胞的清除范围。OCTS的基础设计中包括两个肿瘤相关抗原(tumor-associated antigen，TAA)结合区(通常来源于单克隆抗体抗原结合区域的scFv段)，一个胞外铰链区，一个跨膜区，两个胞内信号转导区和一个效应元件区。scFv区域对于OCTS的特异性、有效性以及基因改造T细胞自身的安全性来说是关键的决定因素。随着OCTS-CAR-T的即将进入临床研究阶段标志着CAR-T细胞治疗即将进入2.0时代。
本发明所采用的载体骨架可以应用于第三代慢病毒载体结构上，也可以应用于第二代慢病毒载体结构上。第二代和第三代慢病毒载体在结构上的区别如图2B所示。本发明优选第三代慢病毒载体(如图2A所示)，3’SIN LTR去除了U3区域，消除了慢病毒载体自我复制的可能性，大大提高了安全性；增加了cPPT和WPRE元件，提高了转导效率和转基因的表达效率；采用RSV启动子保证了慢病毒载体包装时核心RNA的持续高效转录；采用人自身的EF1α启动子，使CAR基因能够在人体内长时间持续表达。
本发明所述的CD19单链抗体轻链VL、CD19单链抗体重链VH、CD20单链抗体轻链VL、CD20单链抗体重链VH、CD22单链抗体轻链VL、CD22单链抗体重链VH、CD30单链抗体轻链VL、CD30单链抗体重链VH、CD123单链抗体轻链VL、CD123单链抗体重链VH、IL6R单链抗体均经过人源化改造，能有有效减少体内人抗鼠抗(Human anti-mouse antibodies，HAMA)的产生，延长scFv的半衰期和作用效果，增加OCTS-CAR-T细胞的存在时间。
本发明中使用的共刺激因子的一种或若干种组合，能够增加转导后细胞的增殖速率、存活时间、杀伤效率、免疫记忆等特性。
本发明采用的OCTS-CAR-T细胞由GMP级别的车间生产后，可用于人体临床实验。
本发明的重组慢病毒载体可以实现在人T淋巴细胞上表达CD19、CD20、CD22、CD30、CD123等组合的双靶向嵌合抗原受体，引导并激活T淋巴细胞对CD19、CD20、CD22、CD30、CD123等阳性细胞的杀伤作用，在临床上可用于治疗B淋巴细胞白血病、B淋巴瘤的治疗。
本发明通过重组慢病毒载体骨架、OCTS结构域、白介素-6受体(IL6R)的single chain antibody(单链抗体)构建形成重组慢病毒载体，该方式得到的重组慢病毒载体可以实现在人T淋巴细胞内表达IL-6R的单链抗体，能有效封闭IL6R，阻断IL-6信号通路，临床上可用于缓解细胞因子释放综合症(Cytokine Release Syndrome，CRS)，保障细胞治疗过程中患者的生命安全。
可见，本发明所述的OCTS-CAR-T细胞将给肿瘤细胞治疗提供可靠的保障。
附图说明
图1是本发明所述的OCTS嵌合受体的示意图，包含了串联OCTS(Series OCTS)和转角OCTS(Turn OCTS)示意图；
图2本发明所述的慢病毒载体结构示意图；其中图2A是本发明采用的第三代慢病毒载体结构示意图，图2B是第二代和第三代慢病毒载体结构比较示意图；
图3为本发明实施例1中构建本发明所述的重组慢病毒载体的构建流程图。其中，(A)图是慢病毒骨架质粒pLenti-3G basic的结构示意图；(B)图是8个OCTS质粒的示意图；(C)图是pPac-GP质粒的结构示意图；(D)图是pPac-R质粒的结构示意图；(E)图是pEnv-G包装质粒的结构示意图；
图4是本发明实施例1中OCTS结构的元件顺序示意图，其中，A图是串联OCTS(SeriesOCTS)的结构示意图，B图是转角OCTS(Turn OCTS)的结构示意图，C图是OCTS结构的质粒编号(OCTS Symbol)的列表示意图；
图5是本发明实施例1中重组慢病毒质粒pOCTS12319s、pOCTS2219s、pOCTS2019s、pOCTS3019s、pOCTS12319t、pOCTS2219t、pOCTS2019t、pOCTS3019t的酶切预测及酶切琼脂糖凝胶电泳图；其中图5A是pOCTS12319s的酶切预测示意图，图5B是pOCTS12319s的酶切琼脂糖凝胶电泳图；图5C是pOCTS2219s的酶切预测示意图，图5D是pOCTS2219s的酶切琼脂糖凝胶电泳图；图5E是pOCTS2019s的酶切预测示意图，图5F是pOCTS2019s的酶切琼脂糖凝胶电泳图；图5G是pOCTS3019s的酶切预测示意图，图5H是pOCTS3019s的酶切琼脂糖凝胶电泳图；图5I是pOCTS12319t的酶切预测示意图，图5J是pOCTS12319t的酶切琼脂糖凝胶电泳图；图5K是pOCTS2219t的酶切预测示意图，图5L是pOCTS2219t的酶切琼脂糖凝胶电泳图；图5M是pOCTS2019t的酶切预测示意图，图5N是pOCTS2019t的酶切琼脂糖凝胶电泳图；图5O是pOCTS3019t的酶切预测示意图，图5P是pOCTS3019t的酶切琼脂糖凝胶电泳图；图5A中的lane1是1kb DNA ladder Marker：条带从上到下依次为：10kb、8kb、6kb、5kb、4kb、3.5kb、3kb、2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp；图5A中的lane2是pOCTS12319s的BamH I酶切预测：条带从上到下依次为：11384bp、818bp；图5B中的lane1是1kb DNA ladder Marker的电泳结果；图5B中的lane2是pOCTS12319s的BamH I酶切电泳结果；图5C中的lane1是1kb DNA ladder Marker：条带从上到下依次为：10kb、8kb、6kb、5kb、4kb、3.5kb、3kb、2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp；图5C中的lane2是pOCTS2219s的Kpn I酶切预测：条带从上到下依次为：7824bp、4322bp；图5D中的lane1是1kb DNA ladder Marker的电泳结果；图5D中的lane2是pOCTS2219s的Kpn I酶切电泳结果；图5E中的lane1是1kb DNA ladder Marker：条带从上到下依次为：10kb、8kb、6kb、5kb、4kb、3.5kb、3kb、2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp；图5E中的lane2是pOCTS2019s的ApaL I酶切预测：条带从上到下依次为：4883bp、3931bp、1726bp、1246bp、497bp；图5F lane1是1kb DNA ladder Marker的电泳结果；图5F lane2是pOCTS2019s的ApaL I酶切电泳结果；图5G中的lane1是1kb DNA  ladder Marker：条带从上到下依次为：10kb、8kb、6kb、5kb、4kb、3.5kb、3kb、2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp；图5G中的lane2是pOCTS3019s的BamH I酶切预测：条带从上到下依次为：10268bp、1197bp、818bp；图5H中的lane1是1kb DNA ladder Marker的电泳结果；图5H中的lane2是pOCTS3019s的BamH I酶切电泳结果；图5I中的lane1是1kb DNA ladder Marker：条带从上到下依次为：10kb、8kb、6kb、5kb、4kb、3.5kb、3kb、2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp；图5I中的lane2是pOCTS12319t的Sal I酶切预测：条带从上到下依次为：6934bp、5389bp；图5J中的lane1是1kb DNA ladder Marker的电泳结果；图5J中的lane2是pOCTS12319t的Sal I酶切电泳结果；图5K中的lane1是1kb DNA ladder Marker：条带从上到下依次为：10kb、8kb、6kb、5kb、4kb、3.5kb、3kb、2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp；图5K中的lane2是pOCTS2219t的EcoR I酶切预测：条带从上到下依次为：10626bp、1399bp、311bp；图5L中的lane1是1kb DNA ladder Marker的电泳结果；图5L中的lane2是pOCTS2219t的EcoR I酶切电泳结果；图5M中的lane1是1kb DNA ladder Marker：条带从上到下依次为：10kb、8kb、6kb、5kb、4kb、3.5kb、3kb、2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp；图5M中的lane2是pOCTS2019t的Pst I酶切预测：条带从上到下依次为：9231bp、2554bp、617bp；图5N中的lane1是1kb DNA ladder Marker的电泳结果；图5N中的lane2是pOCTS2019t的Pst I酶切电泳结果；图5O中的lane1是1kb DNA ladder Marker：条带从上到下依次为：10kb、8kb、6kb、5kb、4kb、3.5kb、3kb、2.5kb、2kb、1.5kb、1kb、750bp、500bp、250bp；图5O中的lane2是pOCTS3019t的Sac II酶切预测：条带从上到下依次为：11652bp、887bp；图5P中的lane1是1kb DNA ladder Marker的电泳结果；图5P中的lane2是pOCTS3019t的Sac II酶切电泳结果；
图6是本发明实施例1中重组慢病毒载体的滴度检测结果示意图；
图7为本发明实施例1中所述的OCTS-CAR-T细胞构建的步骤流程图，包含分离培养、激活、基因转导、OCTS-CAR-T细胞鉴定等阶段；
图8是本发明实施例2中OCTS-CAR-T细胞的支原体检测结果示意图，其中，lane1为DL2000marker，从上到下条带条带从上到下依次为：2kb、1kb、750bp、500bp、250bp、100bp；lane2为阳性对照；lane3为阴性对照；lane4为PBS；lane5为裂解液；lane6为OCTS12319s-CAR-T细胞；lane7为OCTS2219s-CAR-T细胞；lane8为OCTS2019s-CAR-T细胞；lane9为OCTS3019s-CAR-T细胞；lane10为OCTS12319t-CAR-T细胞；lane11为OCTS2219t-CAR-T细胞；lane12为OCTS2019t-CAR-T细胞；lane13为OCTS3019t-CAR-T细胞；
图9为本发明实施例2中流式检测OCTS-CAR-T细胞的转导效率以及免疫分型结果示意图；其中，图9A表示OCTS12319s-CAR-T细胞的转导效率结果；图9B表示OCTS12319s-CAR-T细胞的免疫分型结果；图9C表示OCTS2219s-CAR-T细胞的转导效率结果；图9D表示OCTS2219s-CAR-T细胞的免疫分型结果；图9E表示OCTS2019s-CAR-T细胞的转导效率结果；图9F表示OCTS2019s-CAR-T细胞的免疫分型结果；图9G表示OCTS3019s-CAR-T细胞的转导效率结果；图9H表示OCTS3019s-CAR-T细胞的免疫分型结果；图9I表示OCTS12319t-CAR-T细胞的转导效率结果；图9J表示OCTS12319t-CAR-T细胞的免疫分型结果；图9K表示OCTS2219t-CAR-T细胞的转导效率结果；图9L表示OCTS2219t-CAR-T细胞的免疫分型结果；图9M表示OCTS2019t-CAR-T细胞的转导效率结果；图9N表示OCTS2019t-CAR-T细胞的免疫分型结果；图9O表示OCTS3019t-CAR-T细胞的转导效率结果；图9P表示OCTS3019t-CAR-T细胞的免疫分型结果；
图10为本发明实施例3中不同效靶比条件下，OCTS-CAR-T细胞对靶细胞杀伤效率柱状图；其中，图10A表示OCTS12319s-CAR-T细胞和OCTS12319t-CAR-T细胞对不同靶细胞的杀伤结果；图10B表示OCTS2219s-CAR-T细胞和OCTS2219t-CAR-T细胞对不同靶细 胞的杀伤结果；图10C表示OCTS2019s-CAR-T细胞和OCTS2019t-CAR-T细胞对不同靶细胞的杀伤结果；图10D表示OCTS3019s-CAR-T细胞和OCTS3019t-CAR-T细胞对不同靶细胞的杀伤结果。
具体实施方式
下面结合具体实施例进一步阐述此发明。应理解的是，在此描述的特定实施方式通过举例的方式来表示，并不作为对本发明的限制。在不偏离本发明范围的情况下，本发明的主要特征可以用于各种实施方式。
材料
1、慢病毒骨架质粒pLenti-3G basic，慢病毒包装质粒pPac-GP、pPac-R以及膜蛋白质粒pEnv-G，HEK293T/17细胞，同源重组酶，Oligo Annealing Buffer，支原体检测试剂盒，内毒素检测试剂盒，CD19+K562、CD20+K562、CD22+K562、CD30+K562、CD123+K562、CD19+CD123+K562、CD19+CD20+K562、CD19+CD22+K562、CD19+CD30+K562、K562细胞购自世翱(上海)生物医药科技有限公司；慢病毒骨架质粒pLenti-3G basic的具体制备方法已经公开在发明名称为“一种基于复制缺陷性重组慢病毒的CAR-T转基因载体及其构建方法和应用”，专利申请号为201610008360.5的专利申请说明书中；
2、人新鲜外周血由健康供者提供；
3、OCTS12319s、OCTS2219s、OCTS2019s、OCTS3019s、OCTS12319t、OCTS2219t、OCTS2019t、OCTS3019t DNA序列组合由上海优卡迪公司设计(参见图4C)，交给上海捷瑞生物工程有限公司合成，并以寡核苷酸干粉或者质粒形式保存；
4、工具酶Cla I、Pst I、Sac II、Sal I、EcoR I、BamH I、ApaL I、Kpn I、T4 DNA连接酶均购自NEB公司；
5、0.22μm-0.8μm PES滤器购自millipore公司；
6、D-PBS(-)、0.4％台盼蓝、筛网、各类型细胞培养皿、培养袋、培养板均购自corning公司；
7、Opti-MEM、Pen-Srep、Hepes、FBS、AIM-V、RPMI 1640、DMEM、lipofectamine 3000购自invitrogen公司；
8、Biotinylated protein L购自GeneScript公司；
9、LDH检测试剂盒购自promega公司；
10、Ficoll淋巴细胞分离液购自GE公司；
11、20％人血白蛋白注射液购自杰特贝林公司；
12、CryoPremium冻存液、分选缓冲液来自上海优卡迪公司；
13、rIL-2，rIL-7，，rIL-15，rIL-21购自peprotech公司；
14、CD3单克隆抗体，CD28单克隆抗体，CD3/CD28磁珠CD4/CD8磁珠购自德国Miltenyi公司；
15、冷冻离心机(美国ThermoScientific公司；
16、FACS流式细胞仪购自Thermo公司；
17、荧光倒置显微镜购自Olympus公司；
18、CD4-FITC、CD8-APC购自BioLegend公司；
19、0.9％生理盐水购自今迈公司；
20、ProteinL Magnetic Beads购自BioVision公司；
21、PrimeSTAR、RetroNectin购自Takara公司；
22、phycoerythrin(PE)-conjugated streptavidin购自BD Bioscience公司；
23、质粒抽提试剂盒、琼脂糖凝胶回收试剂盒均购自MN公司；
24、感受态细胞TOP10购自tiangen公司；
25、NaCl、KCl、Na2HPO4.12H2O、KH2PO4、Trypsin、EDTA、CaCl2、NaOH、PEG6000均购自上海生工；
26、DNeasy试剂盒购自上海捷瑞公司；
27、SA-HRP购自上海翊圣公司；
28、引物：根据引物设计原则设计扩增DNA片段和靶位点所需的引物，该引物由上海生物公司合成，具体为：
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)
IL6Rscab-F：AAAACACGATGATAATGCCACCATGAACTCCTTCTCCACAAGCG(SEQ ID NO.40)
IL6Rscab-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)
29、本发明中，所述SEQ ID NO.1，SEQ ID NO.2，SEQ ID NO.3，SEQ ID NO.4，SEQ ID NO.5，SEQ ID NO.6，SEQ ID NO.7，SEQ ID NO.8，SEQ ID NO.9，SEQ ID NO.10，SEQ ID NO.11，SEQ ID NO12，SEQ ID NO.13，SEQ ID NO.14，SEQ ID NO.15，SEQ ID NO.16，SEQ ID NO.17，SEQ ID NO.18，SEQ ID NO.19，SEQ ID NO.20，SEQ ID NO.21，SEQ ID NO.22，SEQ ID NO.23，SEQ ID NO.24，SEQ ID NO.25，SEQ ID NO.26，SEQ ID NO.27，SEQ ID NO.28，SEQ ID NO.29，SEQ ID NO.30，SEQ ID NO.31，SEQ ID NO.32，SEQ ID NO.33所示DNA片段由上海捷瑞生物工程有限公司根据本发明人提供的序列合成。
实施例1OCTS-CAR-T细胞构建
一、重组慢病毒载体lvOCTS12319s、lvOCTS2219s、lvOCTS2019s、lvOCTS3019s、lvOCTS12319t、lvOCTS2219t、lvOCTS2019t、lvOCTS3019t的构建、纯化、检测方法。
参见图3，本发明所述重组慢病毒载体的构建方法如下：
1、将人EF1α启动子(SEQ ID NO.14)、OCTS结构【如SEQ ID NO.15所示的CD8 leader嵌合受体信号肽、两组单链抗体：第一组选自以下四组单链抗体的任意一组：如SEQ ID NO.18所示的CD20单链抗体轻链VL、如SEQ ID NO.19所示的CD20单链抗体重链VH；如SEQ ID NO.20所示的CD22单链抗体轻链VL、如SEQ ID NO.21所示的CD22单链抗体重链VH；如SEQ ID NO.22所示的CD30单链抗体轻链VL、如SEQ ID NO.23所示的CD30单链抗体重链VH；如SEQ ID NO.24所示的CD123单链抗体轻链VL、如SEQ ID NO.25所示的CD123单链抗体重链VH；第二组为如SEQ ID NO.16所示的CD19单链抗体轻链VL以及如SEQ ID NO.17所示的CD19单链抗体重链VH；如SEQ ID NO.26所示的抗体内铰链Inner-Linker、如SEQ ID NO.27所示的单链抗体间铰链Inter-Linker、如SEQ ID NO.28所示的CD8 Hinge嵌合受体铰链、如SEQ ID NO.29所示的CD8 Transmembrane嵌合受体跨膜区、如SEQ ID NO.30所示的CD28嵌合受体共刺激因子、如SEQ ID NO.31所示的CD134嵌合受体共刺激因子、如SEQ ID NO.32所示的TCR嵌合受体T细胞激活域。
OCTS12319s、OCTS2219s、OCTS2019s、OCTS3019s、OCTS12319t、OCTS2219t、OCTS2019t、OCTS3019t，结构详见图4】、IL6R单链抗体(SEQ ID NO.33)组合成OCTS嵌合受体设计方案，经过酶切、连接、重组反应克隆至慢病毒骨架质粒pLenti-3G basic中，分别得到重组慢病毒质粒pOCTS12319s、pOCTS2219s、pOCTS2019s、pOCTS3019s、pOCTS12319t、pOCTS2219t、pOCTS2019t、pOCTS3019t，元件顺序和编号如图4C所示。
(1)将慢病毒骨架质粒pLenti-3G basic使用Cla I和EcoR I限制性内切酶进行双酶切，产物经过1.5％的琼脂糖凝胶电泳，确认5823bp的片段V1，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
1、溶胶 按200μl NTI/100mg gel比例加入溶胶液，50℃水浴放置5-10分钟。
2、结合DNA 11000g离心30秒，弃去滤液。
3、洗膜 加入700μl NT3，11000g离心30秒，弃去滤液。
4、洗膜 重复第三步一次
5、晾干 11000g离心1分钟，换新的收集管，室温放置1分钟。
6、洗脱DNA 加入15-30μl NE，室温放置1分钟，11000g离心1分钟，收集滤液。
表1 琼脂糖凝胶回收步骤
(2)用引物EF1α-F和EF1α-R以合成的人EF1α启动子(SEQ ID NO.14)为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃2min)*35cycle，72℃10min。产物经过1.5％的琼脂糖凝胶电泳，确认1208bp的片段a，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
试剂 体积(μl)
H2O 32.5
5×Buffer(with Mg2+) 10
dNTP(各2.5mM) 4
Primer1(+)(10μM) 1
Primer2(－)(10μM) 1
Template 1
PrimeSTAR 0.5
表2 50μl PCR反应体系
(3)用引物OCTS-F和OCTS-R以合成的OCTS12319s为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃30sec)*35cycle，72℃5min。产物经过1.5％的琼脂糖凝胶电泳，确认2363bp的片段b，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(4)用引物OCTS-F和OCTS-R以合成的OCTS2219s为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃30sec)*35cycle，72℃5min。产物经过1.5％的琼脂糖凝胶电泳，确认2369bp的片段c，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(5)用引物OCTS-F和OCTS-R以合成的OCTS2019s为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃30sec)*35cycle，72℃5min。产物经过1.5％的琼脂糖凝胶电泳，确认2381bp的片段d，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(6)用引物OCTS-F和OCTS-R以合成的OCTS3019s为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃30sec)*35cycle，72℃5min。产物经过1.5％的琼脂糖凝胶电泳，确认2381bp的片段e，并割胶回收置于Eppendorf管内， 用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(7)用引物OCTS-F和OCTS-R以合成的OCTS12319t为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃30sec)*35cycle，72℃5min。产物经过1.5％的琼脂糖凝胶电泳，确认2390bp的片段f，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(8)用引物OCTS-F和OCTS-R以合成的OCTS2219t为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃30sec)*35cycle，72℃5min。产物经过1.5％的琼脂糖凝胶电泳，确认2399bp的片段g，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(9)用引物OCTS-F和OCTS-R以合成的OCTS2019t为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃30sec)*35cycle，72℃5min。产物经过1.5％的琼脂糖凝胶电泳，确认2411bp的片段h，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(10)用引物OCTS-F和OCTS-R以合成的OCTS3019t为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃30sec)*35cycle，72℃5min。产物经过1.5％的琼脂糖凝胶电泳，确认2424bp的片段i，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(11)用引物IRES-F和IRES-R以合成的IRES核糖体结合序列(SEQ ID NO.12)为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃30sec)*35cycle，72℃5min。产物经过1.5％的琼脂糖凝胶电泳，确认575bp的片段j，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(12)用引物IL6Rscab-F和IL6Rscab-R以合成的IL6R单链抗体(SEQ ID NO.33)为模板，使用表2中的体系，PCR循环条件为：98℃3min，(98℃10sec，55℃15sec，72℃30sec)*35cycle，72℃5min。产物经过1.5％的琼脂糖凝胶电泳，确认1569bp的片段k，并割胶回收置于Eppendorf管内，用MN公司的琼脂糖凝胶回收试剂盒回收相应的片段(见表1)，并测定产物的纯度和浓度；
(16)将重组慢病毒质粒DNA片段组合(见表3)以5μl总体积且摩尔比1:1:1:1的比例加入Eppendorf管内，加入同源重组酶反应液15μl，混匀后在42℃孵育30分钟，转移至冰上放置2-3分钟，将反应液加入50μl TOP10中，轻轻旋转以混匀内容物，在冰中放置30分钟，将管放到预加温到42℃的恒温水浴锅中热激90秒，快速将管转移到冰浴中，使细胞冷却2-3分钟，每管加900μl LB培养液，然后将管转移到37℃摇床上，温育1小时使细菌复苏，取100μl的转化菌液涂布于Amp LB琼脂平板上，倒置平皿，于恒温培养箱中37℃培养，16小时。
重组慢病毒质粒 片段组合
pOCTS12319s a、b、j、k
pOCTS2219s a、c、j、k
pOCTS2019s a、d、j、k
pOCTS3019s a、e、j、k
pOCTS12319t a、f、j、k
pOCTS2219t a、g、j、k
pOCTS2019t a、h、j、k
pOCTS3019t a、i、j、k
表3 重组慢病毒质粒DNA片段组合
挑取克隆进行菌落PCR鉴定，鉴定正确的克隆即为重组慢病毒质粒pOCTS12319s、pOCTS2219s、pOCTS2019s、pOCTS3019s、pOCTS12319t、pOCTS2219t、pOCTS2019t、pOCTS3019t，对正确的克隆进行酶切鉴定(见图5)，并送测序复核结果。
2、重组慢病毒载体lvOCTS12319s、lvOCTS2219s、lvOCTS2019s、lvOCTS3019s、lvOCTS12319t、lvOCTS2219t、lvOCTS2019t、lvOCTS3019t的包装。
(1)完全培养基：取出预热好的新鲜培养基，加入10％FBS+5ml Pen-Srep，上下颠倒混匀即可；
(2)1XPBS溶液：称量NaCl 8g，KCl 0.2，Na2HPO4.12H2O 3.58g，KH2PO4 0.24g置于1000ml烧杯中，加入900ml Milli-Q grade超纯水溶解，溶解完成后，使用1000ml量筒定容至1000ml，121℃高温湿热灭菌20min；
(3)0.25％Trypsin溶液:称量Trypsin 2.5g，EDTA 0.19729g置于1000ml烧杯中，加入900ml1XPBS溶解，溶解完成后，使用1000ml量筒定容至1000ml，0.22μM过滤除菌，长期使用可保存至-20℃冰箱；
(4)0.5M CaCl2溶液：称量36.75g CaCl2用400ml Milli-Q grade超纯水溶解；用Milli-Q grade超纯水将总体积定容至500ml，混匀；0.22μm过滤除菌，分装保存到50ml离心管中，每管45ml左右，4℃保存。
(5)2XHBS溶液：称量4.09g NaCl，0.269g Na2HPO4，5.96g Hepes，用400ml Milli-Q grade超纯水溶解；校准pH仪后，用2M NaOH溶液将HBS溶液的pH调到7.05。调整每瓶HBS的PH消耗2M NaOH为3ml左右；
(6)从液氮罐中取出冻存的HEK293T/17细胞，迅速转移到37℃水浴中，1～2min后转移到超净台中，无菌操作将冻存管中的液体全部转移至10cm2培养皿中，补足含10％FBS的DMEM至8mL/10cm2dish，24h后显微镜观察细胞，细胞汇合的程度大于80％进行传代；
(7)选择细胞状态良好、无污染的HEK293T/17细胞，每2-6个培养皿为一组，将细胞胰酶消化后，用电动移液器吸取4-12ml完全培养基，向每个消化后的培养皿中加2ml，避免培养皿变干；使用1ml移液器将所有细胞吹打成单细胞悬液，转移到培养基瓶中；
(8)将上述2-6个培养皿中的剩余细胞转移到培养基瓶中，并用培养基再冲洗一便培养皿；
(9)盖紧培养基瓶盖，上下颠倒10次左右充分混匀细胞悬液，将细胞传到8-24个10cm2培养皿中，每皿的细胞密度应当约4×106个/10ml完全培养基左右。如果细胞密度和预期的相差较大，则需要对细胞进行计数，然后按照4×106个/皿的量接种；
(10)每6个培养皿整理为一摞，注意保持上下皿之间的配合。将培养皿左右，前后晃动数次，使细胞充分铺开，然后放入5％CO2培养箱。剩余细胞做同样处理；
(11)检查所传代细胞，细胞汇合度应当为70-80％，轮廓饱满，贴壁良好，在细胞培养皿中均匀分布；
(12)为细胞换液，将培养基替换为新鲜完全培养基,每皿9ml，并将培养箱的CO2浓度设定值提高到8％；
(13)按照N+0.5配DNA/CaCl2溶液。每皿HEK293T/17细胞转染质粒量按照下列比例使用：重组慢病毒质粒(20μg),pPac-GP(15μg)，pPac-R(10μg),pEnv-G(7.5μg)。取一个新的5ml离心管，加入0.5M CaCl2：0.25ml，重组慢病毒质粒20μg：pPac-GP 15μg：pPac-R 10μg：pEnv-G 7.5μg，补充超纯水至0.5ml盖上盖子，充分混匀；
(14)另取一支5ml离心管，加入0.5ml DNA/CaCl2溶液。打开涡旋振荡器，一只手拿住5ml离心管的上端，使管底接触振荡头，使液体在管壁上散开流动，另一只手拿一把1mL移液枪，吸取0.5mL 2×HBS溶液，缓慢滴加进入离心管，控制流速，以半分钟滴完为宜。2×HBS加入后，继续振荡5秒钟，停止振荡，可直接加入需要转染的细胞中；
(15)取一皿细胞，将离心管中的1mL钙转液滴加进去，尽可能使钙转试剂分布到整个培 养皿中；
(16)钙转液加入后，在皿盖上做好标记，将培养皿放还到另一个5％CO2培养箱中。确保培养皿水平放置，每摞培养皿不要超过6个。在5％CO2培养箱中放置(6–8h)；
(17)将第一个培养箱的CO2浓度设定值调回到5％；
(18)24小时后，检查细胞状态。细胞汇合度应当为80–85％左右，状态良好。将培养基吸走，更换10ml新鲜的DMEM完全培养基；
(19)48小时后，观察转染效率。绝大多数细胞仍然是贴壁的。可以看到超过95％细胞都会带有绿色荧光。将同一个病毒包装上清液收集到一起，并向培养皿中继续添加10mL新鲜培养基；
(20)72小时后，再次将同一个病毒上清液收集到一起，两次收集的病毒可以放在一起，丢弃培养皿；此时收集的上清里包含了重组慢病毒载体lvOCTS12319s、lvOCTS2219s、lvOCTS2019s、lvOCTS3019s、lvOCTS12319t、lvOCTS2219t、lvOCTS2019t、lvOCTS3019t。
3、离子交换色谱法纯化重组慢病毒载体；
(1)将收集的上清液使用Thermo真空泵，经0.22μm-0.8μm的PES滤器抽滤，除去杂质；
(2)按1:1～1:10的比例往上清中加入1.5M NaCl 250mM Tris-HCl(pH 6-8)；
(3)将2个离子交换柱串联放置，用4ml 1M NaOH、4ml 1M NaCl、5ml 0.15M NaCl 25mM Tris-HCl(pH 6-8)溶液依次过柱；
(4)将步骤2中获得的溶液通过蠕动泵以1-10ml/min的速度给离子交换柱上样；
(5)全部上清液过柱后，使用10ml 0.15M NaCl 25mM Tris-HCl(pH 6-8)溶液清洗一遍；
(6)根据上样量使用1-5ml 1.5M NaCl 25mM Tris-HCl(pH 6-8)进行洗脱，收集洗脱液；
(7)将洗脱液分成25到50μl一管，冻存到-80℃冰箱，进行长期保存；
4、重组慢病毒载体滴度测定；
(1)取24孔板接种293T细胞。每孔细胞为5×104个，所加培养基体积为500ul,不同种类的细胞生长速度有所差异，进行病毒感染时的细胞融合率为40％-60％；
(2)准备3个无菌EP管，在每个管中加入90ul的新鲜完全培养基(高糖DMEM+10％FBS)接种细胞24小时后，取两个孔的细胞用血球计数板计数，确定感染时细胞的实际数目，记为N；
(3)取待测定的病毒原液10ul加入到第一个管中，轻轻混匀后，取10ul加入到第二个管中，然后依次操作直到最后一管；在每管中加入410ul完全培养基(高糖DMEM+10％FBS),终体积为500ul；
(4)感染开始后20小时，除去培养上清，更换为500μl完全培养基(高糖DMEM+10％FBS)，5％CO2继续培养48小时；
(5)72小时后，观察荧光表达情况，正常情况下，荧光细胞数随稀释倍数增加而相应减少,并拍照；
(6)用0.2ml 0.25％胰酶-EDTA溶液消化细胞，在37℃放置1分钟。用培养基吹洗整个细胞面，离心收集细胞。按照DNeasy试剂盒的说明抽提基因组DNA。每个样品管中加入200μl洗脱液洗下DNA并定量；
(7)准备目的DNA检测qPCRmix总管Ⅰ(QPCR引物序列为SEQ ID NO.42---SEQ ID NO.43)：
Figure PCTCN2017110667-appb-000001
n＝number of reactions.例如：总反应数为40，将1ml 2×TaqMan Universal PCR Master Mix，4μl forward primer，4μl reverse primer，4μl probe和788μl H2O混和。震荡后放在冰上；
(8)准备内参DNA检测qPCRmix管Ⅱ(QPCR引物序列为SEQ ID NO.44---SEQ ID NO.45)：
2×TaqMan Master Mix                      25μl×n
10×RNaseP primer/probe mix               2.5μl×n
H2O                                       17.5μl×n
n＝number of reactions.例如：总反应数为40，将1ml 2×TaqMan Universal PCR Master Mix，100μl 10×RNaseP primer/probe mix和700μl H2O混和。震荡后放在冰上；
(9)在预冷的96孔PCR板上完成PCR体系建立。从总管Ⅰ中各取45μl加入到A-D各行的孔中，从总管Ⅱ中各取45μl加入到E-G各行的孔中。
(10)分别取5μl质粒标准品和待测样品基因组DNA加入到A-D行中，每个样品重复1次。另留1个孔加入5μl的水做为无模板对照(no-template control)。
(11)分别取5μl基因组标准品和待测样品基因组DNA加入到E-G行中，每个样品重复1次。另留1个孔加入5μl的水做为无模板对照(no-template control)。
(12)所使用定量PCR仪为ABI PRISM 7500定量系统。循环条件设定为：50℃2分钟，95℃10分钟，然后是95℃15秒，60℃1分钟的40个循环。
数据分析：测得的DNA样品中整合的慢病毒载体拷贝数用基因组数加以标定，得到每基因组整合的病毒拷贝数。
滴度(integration units per ml，IU ml-1)的计算公式如下：
IU ml-1＝(C×N×D×1000)/V
其中：C＝平均每基因组整合的病毒拷贝数
N＝感染时细胞的数目(约为1×105)
D＝病毒载体的稀释倍数
V＝加入的稀释病毒的体积数
(13)重组慢病毒载体lvOCTS12319s、lvOCTS2219s、lvOCTS2019s、lvOCTS3019s、lvOCTS12319t、lvOCTS2219t、lvOCTS2019t、lvOCTS3019t的滴度结果(如图6所示)；
二、OCTS-CAR-T细胞构建
参见图7，本发明所述OCTS-CAR-T细胞的构建方法如下：
1、分离PBMC。
(1)抽取健康供者新鲜外周血50ml；
(2)将采血袋喷拭酒精两遍，并擦干。
(3)用50ml注射器将袋中的血细胞吸出来移至新50ml管中。
(4)400g，20℃离心10min。
(5)将上层血浆移到新的50ml离心管中，56℃，30min灭活血浆，恢复至室温，2000g，离心30min，取上清到50ml离心管中待用。
(6)用D-PBS(-)补至50ml，拧紧盖子，颠倒混匀。
(7)取2个新50ml离心管，每管加入15ml Ficoll淋巴细胞分离液。
(8)向每管Ficoll上小心加入血细胞稀释液25ml。800g，20℃离心20min。
(9)离心管中液体分为四层，从上至下分别为：黄色的血浆层(回收待用)、白膜层、无色透明的Ficoll层、红黑色的混合细胞层。
(10)小心吸取白膜层到新50ml离心管中，补加D-PBS(-)至50ml，颠倒混匀后500g，20℃离心10min。
(11)加入25ml 5％人血白蛋白并重悬细胞，400g，20℃离心10min。
(12)弃上清，加入25ml 5％人血白蛋白重悬细胞沉淀，并过70um筛网，计数。
(13)取1份含1.25x108cells用于激活；剩余细胞悬液400g，20℃离心10min，加CryoPremium并冻存。
2、CD4/CD8阳性T细胞分选。
(1)将获得的PBMC计数，以80ul/107cells的比例加入分选缓冲液，重悬细胞沉淀。
(2)再以20ul/107cells的比例加入CD4/CD8磁珠，吹打混匀后放入4℃中孵育15min。
(3)取出磁珠-细胞混合液，以2ml/107cells的比例加入分选缓冲液，颠倒混匀后，250g，4℃离心10min。
(4)以500ul/108cells的比例加入分选缓冲液，重悬细胞沉淀。
(5)用镊子夹取LS分离柱到磁力架上。
(6)同时准备2个15ml离心管，分别标记：CD4-/CD8-细胞液(A管)、CD4+/CD8+细胞液(B管)。
(7)用3ml分离缓冲液润洗LS,并用A管接缓冲液。
(8)加入细胞-磁珠混合液，滴完后加入3ml缓冲液冲洗柱子(每次无液体残留时再加入新的液体)，总共三次，收集得到CD4/CD8-细胞。
(9)LS分离柱与磁力架分离，用B管接细胞悬液，加入5ml缓冲液，将并用柱子内塞稍用力冲洗，收集为CD4+/CD8+细胞，取样计数。
(10)按1x106/ml-4x106/ml的细胞密度用AIM-V培养基重悬细胞沉淀，并加入2×105～1×106U/L IFN-γ因子。
3、T细胞激活。
(1)提前一天将1×103ug/L～1×104ug/L CD3单克隆抗体和1×103ug/L～1×104ug/L CD28单克隆抗体加入24孔板，封口膜封口，4℃过夜包被。
(2)取出包被的T75瓶，倒掉包被液，用D-PBS(-)洗涤一次，并将分选得到的细胞悬液接种到T75瓶中，摇匀，放入37℃、5％CO2培养箱中培养。
4、CAR基因转导及OCTS-CAR-T细胞诱导培养。
(1)提前一天包被1×103ug/L～1×104ug/L RetroNectin于24孔板内，封口膜封口，4℃过夜包被。
(2)往24孔板中，根据每孔5×105细胞量，按MOI＝5～20的量，分别加入lvOCTS12319s、lvOCTS2219s、lvOCTS2019s、lvOCTS3019s、lvOCTS12319t、lvOCTS2219t、lvOCTS2019t、lvOCTS3019t慢病毒转基因载体，同时添加含2×105～5×105U/L rIL-2，5×103ng/L～1×104ng/LrIL-7，5×103ng/L～1×104ng/L rIL-15，5×103ng/L～1×104ng/L rIL-21和含10％自体血清的AIM-V培养基37℃、5％CO2继续培养。
5、OCTS-CAR-T细胞体外扩增。
(1)每2天等量补加含2×105～5×105U/L rIL-2，5×103ng/L～1×104ng/L rIL-7，5×103ng/L～1×104ng/L rIL-15，5×103ng/L～1×104ng/L rIL-21和含10％自体血清的AIM-V培养基，使PH值维持在6.5～7.5之间，细胞密度维持在5×105～2×106/ml之间，37℃、5％CO2继续培养10-14天。
(2)第7天左右，冻存培养的OCTS-CAR-T细胞用于后续检测。
实施例2
OCTS-CAR-T细胞病原检测和表达检测。
一、内毒素检测；
(1)、内毒素工作标准品为15EU/支；
(2)、鲎试剂灵敏度λ＝0.25EU/ml,0.5ml/管
(3)、内毒素标准品稀释：取内毒素标准品一支，分别用BET水按比例稀释成4λ和2λ的溶 解，封口膜封口，震荡溶解15min；稀释时每稀释一步均应在漩涡混合器上混匀30s；
(4)、加样：取鲎试剂若干支，每支加入BET水0.5ml溶解，分装至若干支无内毒素试管中，每管0.1ml。其中2支为阴性对照管，加入BET水0.1ml；
2支为阳性对照管，加入2λ浓度的内毒素工作标准品溶液0.1ml；
2支为样品阳性对照管，加入0.1ml含2λ内毒素标准品的样品溶液(稀释20倍的待测样品1ml+4λ的内毒素标准品溶液1ml＝2ml含2λ内毒素标准品的稀释40倍样品)。
样品管中加入0.1ml样品，稀释比例见表4，37±1℃水浴(或培养箱)保温60±1min；
稀释倍数 原液 5 10 20 40 80 160
对应EU/ml 0.25 1.25 2.5 5 10 20 40
结果              
表4 内毒素稀释比例及对应内毒素含量
(5)、OCTS-CAR-T细胞的内毒素检测结果(如表5所示)，所有细胞的内毒素含量均小于2.5EU/ml，符合《中华人民共和国药典》中小于10EU/ml的标准；
表5
稀释倍数 原液 5 10 20 40 80 160
对应EU/ml 0.25 1.25 2.5 5 10 20 40
OCTS12319s-CAR-T (+) (-) (-) (-) (-) (-) (-)
OCTS2219s-CAR-T (+) (+) (-) (-) (-) (-) (-)
OCTS2019s-CAR-T (+) (-) (-) (-) (-) (-) (-)
OCTS3019s-CAR-T (+) (-) (-) (-) (-) (-) (-)
OCTS12319t-CAT-T (+) (-) (-) (-) (-) (-) (-)
OCTS2219t-CAR-T (+) (+) (-) (-) (-) (-) (-)
OCTS2019t-CAR-T (+) (+) (-) (-) (-) (-) (-)
OCTS3019t-CAR-T (+) (-) (-) (-) (-) (-) (-)
二、支原体检测；
(1)在实验前三日，细胞样品用无抗生素培养基进行培养；
(2)收集1ml细胞悬浮液(细胞数大于1*105)，置于1.5ml离心管中；
(3)13000g离心1min，收集沉淀，弃去培养基；
(4)加入500ul PBS用枪头吹吸或涡旋振荡，重悬沉淀。13000g离心5min；
(5)步骤4重复一次；
(6)加入50μl Cell Lysis Buffer，用枪头吹吸，充分混匀后,在55℃水浴中孵育20min；
(7)将样品置于95℃中加热5min；
(8)13000g离心5min后，取5μl上清作为模板，25μl PCR反应体系为：ddH20 6.5μl、Myco Mix 1μl、2x Taq Plus Mix Master(Dye Plus)12.5μl、模板5μl；PCR循环条件为：95℃30sec，(95℃30sec，56℃30sec，72℃30sec)*30cycle，72℃5min。
(9)支原体检测结果显示(如图8所示)，OCTS-CAR-T细胞中均不含支原体。
三、OCTS基因转导效率检测及免疫分型检测；
(1)收集经病毒转导后的T细胞，用含1～4％人血白蛋白的D-PBS(-)溶液重悬细胞并调整为1×106/ml。
(2)向离心管中加入含1～4％人血白蛋白的D-PBS(-)溶液1ml并混匀，350g离心5min，弃上清。
(3)重复步骤2一次。
(4)用0.2ml的含1～4％人血白蛋白的D-PBS(-)溶液重悬细胞，并向离心管中加入1ul的1mg/ul protein L，5ul CD4-FITC，5ul CD8-APC，混匀，4℃孵育45min。
(5)向离心管中加入1ml含1～4％人血白蛋白的D-PBS(-)溶液并混匀，350g离心5min，弃上清。
(6)重复步骤5两次。
(7)用0.2ml含1～4％人血白蛋白的D-PBS(-)溶液重悬细胞，并向离心管中加入0.2ul PE-SA，混匀，37℃避光孵育15min。
(8)向离心管中加入1ml含1～4％人血白蛋白的D-PBS(-)溶液重并混匀，350g离心5min，弃上清。
(9)用1ml D-PBS(-)溶液重悬细胞沉淀，350g离心5min，弃上清。
(10)重复步骤9两次。
(11)用0.4ml D-PBS(-)溶液重悬细胞沉淀，流式细胞仪进行检测。
(12)OCTS基因转导效率及免疫分型检测的检测结果如图9所示，制备的OCTS-CAR-T细胞的感染效率大多数位于30％～40％之间，CD4阳性细胞和CD8阳性细胞的比例位于1:3～3:1之间，可以进行后续功能检测。
实施例3 OCTS-CAR-T细胞的功能检测。
一、靶细胞杀伤效果评估。
(1)分别培养靶细胞[CD19+K562、CD20+K562、CD22+K562、CD30+K562、CD123+K562、CD19+CD123+K562、CD19+CD20+K562、CD19+CD22+K562、CD19+CD30+K562、K562细胞]和效应细胞[OCTS-CAR-T细胞]；
(2)收集靶细胞4x105cells和OCTS-CAR-T细胞2.8x106cells，800g，6min离心，弃上清；
(3)用1ml D-PBS(-)溶液分别重悬靶细胞和效应细胞，800g，6min离心，弃上清；
(4)重复步骤3一次；
(5)用700ul培养基(AIM-V培养基+1～10％FBS)重悬效应细胞，用2ml培养基(AIM-V培养基+1～10％FBS)重悬靶细胞；
(6)设置效靶比为1:1、5:1、10:1的实验孔，效应细胞分别与单靶细胞和双靶细胞共孵育的分组情况如表6所示，并设置对照组(K562细胞)，每组3个复孔；
表6
效应细胞 靶细胞1 靶细胞2 靶细胞3
OCTS12319s-CAR-T CD123+K562 CD19+K562 CD19+CD123+K562
OCTS2219s-CAR-T CD22+K562 CD19+K562 CD19+CD22+K562
OCTS2019s-CAR-T CD20+K562 CD19+K562 CD19+CD20+K562
OCTS3019s-CAR-T CD30+K562 CD19+K562 CD19+CD30+K562
OCTS12319t-CAR-T CD123+K562 CD19+K562 CD19+CD123+K562
OCTS2219t-CAR-T CD22+K562 CD19+K562 CD19+CD22+K562
OCTS2019t-CAR-T CD20+K562 CD19+K562 CD19+CD20+K562
OCTS3019t-CAR-T CD30+K562 CD19+K562 CD19+CD30+K562
(7)250g，5min平板离心；
(8)37℃，5％CO2培养箱中培养4小时；
(9)250g，5min平板离心；
(10)取每个孔的50ul上清到新96孔板中，并且每孔加入50ul底物溶液(避光操作)；
(11)避光孵育25min；
(12)每孔加入50ul终止液；
(13)酶标仪检测490nm吸光度；
(14)将3个复孔取平均值；将所有实验孔、靶细胞孔和效应细胞孔的吸光值减去培养基背景吸光值的均值；将靶细胞最大值的吸光值减去体积校正对照吸光值的均值。
(15)将步骤14中获得的经过校正的值带入下面公式，计算每个效靶比所产生的细胞毒性百分比。结果如图10所示，OCTS-CAR-T对各自的单靶细胞和双靶细胞均有较好的杀伤效果，Turn OCTS结构的CAR-T细胞对靶细胞的杀伤效率略高于Series OCTS结构的CAR-T细胞；
杀伤效率＝(实验孔-效应细胞孔-靶细胞孔)/(靶细胞最大孔-靶细胞孔)X100％
(16)上述实验结果表明，通过对传统CAR结构中抗原识别区的改造形成的OCTS结构，能够显著提高OCTS-CAR-T细胞识别并杀伤靶细胞的范围，因此OCTS-CAR-T细胞将在未来的CD19阳性/CD22阳性/CD20阳性/CD123阳性/CD30阳性/CD19、CD22双阳性/CD19、CD20双阳性/CD19、CD123双阳性/CD19、CD30双阳性的B淋巴细胞白血病、B淋巴瘤等恶性肿瘤的细胞治疗中发挥巨大的作用。
Figure PCTCN2017110667-appb-000002
Figure PCTCN2017110667-appb-000003
Figure PCTCN2017110667-appb-000004
Figure PCTCN2017110667-appb-000005
Figure PCTCN2017110667-appb-000006
Figure PCTCN2017110667-appb-000007
Figure PCTCN2017110667-appb-000008
Figure PCTCN2017110667-appb-000009
Figure PCTCN2017110667-appb-000010
Figure PCTCN2017110667-appb-000011
Figure PCTCN2017110667-appb-000012
Figure PCTCN2017110667-appb-000013
Figure PCTCN2017110667-appb-000014

Claims (11)

  1. 一种基于OCTS技术的淋系白血病CAR-T治疗载体，其特征在于，包括慢病毒骨架质粒、人EF1α启动子、OCTS嵌合受体结构域和IL6R单链抗体；
    所述慢病毒骨架质粒包括：用于目的菌株大量扩增的含氨苄青霉素抗性基因AmpR序列，如SEQ ID NO.1所示；用于质粒复制的原核复制子pUC Ori序列，如SEQ ID NO.2所示；用于增强真核细胞内的复制的病毒复制子SV40 Ori序列，如SEQ ID NO.3所示；用于慢病毒包装的慢病毒包装顺式元件；ZsGreen1绿色荧光蛋白，如SEQ ID NO.11所示；IRES核糖体结合序列，如SEQ ID NO.12所示；用于增强转基因的表达效率的eWPRE增强型土拨鼠乙肝病毒转录后调控元件，如SEQ ID NO.13所示；
    所述人EF1α启动子的序列如SEQ ID NO.14所示；
    所述OCTS嵌合受体结构域包括：如SEQ ID NO.15所示的CD8 leader嵌合受体信号肽、两组单链抗体：第一组选自以下四组单链抗体的任意一组：如SEQ ID NO.18所示的CD20单链抗体轻链VL、如SEQ ID NO.19所示的CD20单链抗体重链VH；如SEQ ID NO.20所示的CD22单链抗体轻链VL、如SEQ ID NO.21所示的CD22单链抗体重链VH；如SEQ ID NO.22所示的CD30单链抗体轻链VL、如SEQ ID NO.23所示的CD30单链抗体重链VH；如SEQ ID NO.24所示的CD123单链抗体轻链VL、如SEQ ID NO.25所示的CD123单链抗体重链VH；第二组为如SEQ ID NO.16所示的CD19单链抗体轻链VL以及如SEQ ID NO.17所示的CD19单链抗体重链VH；如SEQ ID NO.26所示的抗体内铰链Inner-Linker、如SEQ ID NO.27所示的单链抗体间铰链Inter-Linker、如SEQ ID NO.28所示的CD8 Hinge嵌合受体铰链、如SEQ ID NO.29所示的CD8 Transmembrane嵌合受体跨膜区、如SEQ ID NO.32所示的TCR嵌合受体T细胞激活域以及嵌合受体共刺激因子区域；所述嵌合受体共刺激因子区域选自4-1BB、ICOS、CD27、OX40、CD28、MYD88、IL1R1、CD70、TNFRSF19L、TNFRSF27、TNFRSF1OD、TNFRSF13B、TNFRSF18、CD134等肿瘤坏死因子超家族中的任意一种或多种的组合。
  2. 如权利要求1所述的载体，其特征在于，所述慢病毒包装顺式元件采用第二代慢病毒载体或第三代慢病毒载体；所述第二代慢病毒载体包括：如SEQ ID NO.5所示的慢病毒5 terminal LTR、如SEQ ID NO.6所示的慢病毒3 terminal Self-Inactivating LTR、如SEQ ID NO.7所示的Gag顺式元件、如SEQ ID NO.8所示的RRE顺式元件、如SEQ ID NO.9所示的env顺式元件、如SEQ ID NO.10所示的cPPT顺式元件；所述第三代慢病毒载体包括：如SEQ ID NO.5所示的慢病毒5 terminal LTR、如SEQ ID NO.6所示的慢病毒3 terminal Self-Inactivating LTR、如SEQ ID NO.7所示的Gag顺式元件、如SEQ ID NO.8所示的RRE顺式元件、如SEQ ID NO.9所示的env顺式元件、如SEQ ID NO.10所示的cPPT顺式元件，以及如SEQ ID NO.4所示的RSV启动子。
  3. 如权利要求1所述的载体，其特征在于，所述两组单链抗体采用串联连接方式或者转角连接方式；
    当两组单链抗体为CD20单链抗体和CD19单链抗体时，所述串联连接方式具体为：CD20单链抗体轻链VL与CD19单链抗体轻链VL采用单链抗体间铰链Inter-Linker连接，CD20单链抗体轻链VL与CD20单链抗体重链VH采用抗体内铰链Inner-Linker连接，CD19单链抗体轻链VL与CD19单链抗体重链VH采用抗体内铰链Inner-Linker连接；所述转角连接方式具体为：CD19单链抗体轻链VL与CD19单链抗体重链VH采用抗体内铰链Inner-Linker连接，CD20单链抗体轻链VL与CD19单链抗体重链VH采用单链抗体间铰链Inter-Linker连接，CD20单链抗体重链VH与CD19单链抗体轻链VL采用单链抗体间铰链Inter-Linker连接；
    当两组单链抗体为CD30单链抗体和CD19单链抗体时，所述串联连接方式具体为：CD30单链抗体轻链VL与CD19单链抗体轻链VL采用单链抗体间铰链Inter-Linker连接，CD30单链抗体轻链VL与CD30单链抗体重链VH采用抗体内铰链Inner-Linker连接，CD19单链抗体轻链VL与CD19单链抗体重链VH采用抗体内铰链Inner-Linker连接；所述转角连接方式具体为：CD19单链抗体轻链VL与CD19单链抗体重链VH采用抗体内铰链Inner-Linker连接，CD30单链抗体轻链VL与CD19单链抗体重链VH采用单链抗体间铰链Inter-Linker连接，CD30单链抗体重链VH与CD19单链抗体轻链VL采用单链抗体间铰链Inter-Linker连接；
    当两组单链抗体为CD22单链抗体和CD19单链抗体时，所述串联连接方式具体为：CD22单链抗体轻链VL与CD19单链抗体轻链VL采用单链抗体间铰链Inter-Linker连接，CD22单链抗体轻链VL与CD22单链抗体重链VH采用抗体内铰链Inner-Linker连接，CD19单链抗体轻链VL与CD19单链抗体重链VH采用抗体内铰链Inner-Linker连接；所述转角连接方式具体为：CD19单链抗体轻链VL与CD19单链抗体重链VH采用抗体内铰链Inner-Linker连接，CD22单链抗体轻链VL与CD19单链抗体重链VH采用单链抗体间铰链Inter-Linker连接，CD22单链抗体重链VH与CD19单链抗体轻链VL采用单链抗体间铰链Inter-Linker连接；
    当两组单链抗体为CD123单链抗体和CD19单链抗体时，所述串联连接方式具体为：CD123单链抗体轻链VL与CD19单链抗体轻链VL采用单链抗体间铰链Inter-Linker连接，CD123单链抗体轻链VL与CD123单链抗体重链VH采用抗体内铰链Inner-Linker连接，CD19单链抗体轻链VL与CD19单链抗体重链VH采用抗体内铰链Inner-Linker连接；所述转角连接方式具体为：CD19单链抗体轻链VL与CD19单链抗体重链VH采用抗体内铰链Inner-Linker连接，CD123单链抗体轻链VL与CD19单链抗体重链VH采用单链抗体间铰链Inter-Linker连接，CD123单链抗体重链VH与CD19单链抗体轻链VL采用单链抗体间铰链Inter-Linker连接。
  4. 如权利要求1所述的载体，其特征在于，所述IL6R单链抗体的序列如SEQ ID NO.33所示。
  5. 如权利要求1所述的载体，其特征在于，所述eWPRE增强型土拨鼠乙肝病毒转录后调控元件有6个核苷酸的增强突变，具体为：g.396G>A、g.397C>T、g.398T>C、g.399G>A、g.400A>T、g.411A>T。
  6. 如权利要求1所述的载体，其特征在于，由所述人EF1α启动子启动整个OCTS结构基因表达，所述CD8 leader嵌合受体信号肽位于OCTS编码序列的N端，用于引导OCTS蛋白定位于细胞膜；所述两组单链抗体组合成双抗原识别区，用于识别相应靶抗原；所述CD8 Hinge嵌合受体铰链用于将scFv锚定于细胞膜外侧；所述CD8 Transmembrane嵌合受体跨膜区用于将整个嵌合受体固定于细胞膜上；所述CD28嵌合受体共刺激因子用于刺激T淋巴细胞体外激活和体内肿瘤细胞杀伤作用；所述CD134嵌合受体共刺激因子用于促进T淋巴细胞增殖和因子分泌，增强肿瘤免疫，有利于记忆T细胞的长期存活；所述TCR嵌合受体T细胞激活域用于激活下游信号通路的表达；所述IL6R单链抗体分泌到细胞外，封闭IL6R，阻断IL6信号通路，防止炎症因子风暴升级；当抗原识别区域与靶抗原结合时，信号通过嵌合受体传递至细胞内，从而产生T细胞增殖、细胞因子分泌增加、抗细胞凋亡蛋白分泌增加、细胞死亡延迟、裂解靶细胞一系列生物学效应。
  7. 如权利要求1所述的载体，其特征在于，所述嵌合受体共刺激因子区域采用如SEQ ID NO.30所示的CD28嵌合受体共刺激因子以及如SEQ ID NO.31所示的CD134嵌合受体共刺激因子组合。
  8. 如权利要求1所述的载体，其特征在于，所述CD19单链抗体轻链VL、CD19单链抗体重链VH、CD20单链抗体轻链VL、CD20单链抗体重链VH、CD30单链抗体轻链VL、CD30单链抗体重链VH、CD123单链抗体轻链VL、CD123单链抗体重链VH、IL6R单链抗体均经过人源化改造。
  9. 一种如权利要求1-8任一项所述的基于OCTS技术的淋系白血病CAR-T治疗载体的构建方法，其特征在于，包括以下步骤：
    (1)将如SEQ ID NO.1所示的含氨苄青霉素抗性基因AmpR序列、如SEQ ID NO.2所示的原核复制子pUC Ori序列、如SEQ ID NO.3所示的病毒复制子SV40 Ori序列、用于慢病毒包装的慢病毒包装顺式元件、如SEQ ID NO.11所示的ZsGreen1绿色荧光蛋白、如SEQ ID NO.12所示的IRES核糖体结合序列、如SEQ ID NO.13所示的eWPRE增强型土拨鼠乙肝病毒转录后调控元件存储于慢病毒骨架质粒上；
    (2)将如SEQ ID NO.14所示的人EF1α启动子、所述OCTS嵌合受体结构域以及如SEQ ID NO.33所示的IL6R单链抗体组合成OCTS嵌合受体设计方案，经过酶切、连接、重组反应克隆至慢病毒骨架质粒中，得到第三代OCTS设计的重组慢病毒质粒；
    (3)将得到的重组慢病毒质粒分别与慢病毒包装质粒pPac-GP、pPac-R以及膜蛋白质粒pEnv-G共同转染HEK293T/17细胞，在HEK293T/17细胞中进行基因转录表达后，包装成功重组慢病毒载体会释放到细胞培养上清中，收集包含的重组慢病毒载体的上清液；
    (4)将得到的重组慢病毒上清采用抽滤、吸附、洗脱的柱纯化方式进行纯化，分别得到重组慢病毒载体。
  10. 如权利要求9所述的方法，其特征在于，步骤(4)中，所述抽滤步骤要控制上清体积在200ml～2000ml，控制真空度在-0.5MPA～-0.9MPA，防止由于堵孔带来的载体损失；所述吸附步骤要控制溶液的PH值在6～8，防止PH的变化导致载体失活；所述洗脱步骤要控制洗脱液的离子强度在0.5M～1.0M，防止离子强度的变化导致洗脱不完全或者载体失活。
  11. 如权利要求1-8任一项所述的载体在制备治疗淋系白血病的药物中的应用。
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