WO2019242338A1 - 一种包含共刺激受体的嵌合抗原受体及应用 - Google Patents

一种包含共刺激受体的嵌合抗原受体及应用 Download PDF

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WO2019242338A1
WO2019242338A1 PCT/CN2019/077922 CN2019077922W WO2019242338A1 WO 2019242338 A1 WO2019242338 A1 WO 2019242338A1 CN 2019077922 W CN2019077922 W CN 2019077922W WO 2019242338 A1 WO2019242338 A1 WO 2019242338A1
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car
cells
scfv
seq
cd3zeta
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PCT/CN2019/077922
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French (fr)
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杨选明
傅阳心
汪鑫
叶圣勤
李范林
张会会
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上海隆耀生物科技有限公司
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Priority to KR1020217001093A priority Critical patent/KR20210023990A/ko
Priority to CN201980041270.8A priority patent/CN112673024B/zh
Priority to EP19823745.5A priority patent/EP3816190A4/en
Priority to JP2020571595A priority patent/JP7427255B2/ja
Priority to CN202210391005.6A priority patent/CN114805604A/zh
Publication of WO2019242338A1 publication Critical patent/WO2019242338A1/zh
Priority to US17/126,966 priority patent/US11590168B2/en
Priority to US18/159,887 priority patent/US20230277666A1/en

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Definitions

  • the invention relates to the technical field of cellular immunotherapy, in particular to a chimeric antigen receptor containing a co-stimulatory receptor and application thereof.
  • chimeric antigen receptor T cells were first proposed by Gross, Waks, and Eshhar in 1989. They expressed antibodies that recognize TNP on T cells, achieving the activation and effects of antigen-specific, non-MHC-restricted T cells. Enhancement and put forward the concept of the application of CAR-T technology in tumor treatment. According to this principle, embedding tumor-specific antibodies into T cells will give T cells new tumor-killing capabilities. Later, CAR-T technology was introduced into anti-tumor clinical trials. However, early CAR-T cells contained only the first signal because of their intracellular signalling domain, and the selected tumor type was solid tumor. The final clinical results were not Too ideal.
  • the structure of CAR consists of an extracellular antigen recognition domain, an extracellular hinge region, a transmembrane region, and an intracellular signaling domain.
  • the extracellular antigen recognition domain is usually composed of a single chain antibody, which specifically recognizes the surface molecules of tumor cell membranes, and may also be a ligand or receptor for certain tumor-specific antigens.
  • the extracellular hinge region is a space structure that separates the antigen recognition domain from the transmembrane region. The purpose is to provide a suitable spatial location so that the extracellular antigen recognition domain can maintain the correct structure before and after recognition of the antigen and conduct intracellularly. signal.
  • the transmembrane region is a domain that ensures the localization of CAR molecules on the membrane surface.
  • the intracellular signaling domain is a key part of mediating CAR signaling, usually one or several first signals (recognition of TCR and MHC-I-peptide complexes), and second signals (co-stimulatory receptors and co-stimulatory receptors). Body recognition).
  • the first generation CAR contains only the first signal
  • the second generation CAR has a first signal and a second signal
  • the third generation CAR has a first signal and two second signal domains.
  • CAR-T for B cell surface targeting molecules CD19 and CD20 prepared from patient's own blood cells has been relatively mature in the treatment of B cell leukemia, but although the response rate is high, there are a large number of recurrences. Tumor treatment efficiency is relatively low, which is related to the immunosuppressive microenvironment in solid tumors.
  • tumor microenvironment In solid tumors, there are a variety of immune cells, tumor cells and stromal cells, which together constitute the tumor microenvironment.
  • the tumor microenvironment is usually immunosuppressive. It can inhibit endogenous anti-tumor T cell responses or adoptive T cells (such as CAR-T) at multiple levels. For example, T cells are depleted and tumor-killing cells are lost. Function, eventually T cells are cleared. How to enhance the activation capacity of CAR-T in solid tumors so that it can fight against immunosuppression in the tumor microenvironment is an important idea and direction for the expansion of CAR-T to solid tumors.
  • CAR-T domains use novel regulatory molecules such as IL-12, 4 -1BBL, etc. In addition to affecting CAR-T, these molecules will also have non-specific activation of other non-CAR-T cells, potentially causing immune side effects.
  • the purpose of the present invention is to overcome the defects in the prior art, provide a chimeric antigen receptor containing a co-stimulatory receptor and use thereof, and provide a CAR constructed from a recombinant expression vector such as the chimeric antigen receptor.
  • -T cells such as OX40, are an important co-stimulatory receptor, mainly expressed in activated CD4 and CD8 T cells, and exhibit multiple functions during T cell activation, can promote T cell activation and express more effector molecules As well as reducing apoptosis-related gene expression, integrating the co-stimulatory receptor signal in CAR-T has the potential to enhance the effect.
  • the present invention adopts the following technical solutions:
  • the first object of the present invention is to provide a chimeric antigen receptor comprising a co-stimulatory receptor, and the structure of the chimeric antigen receptor is scFv (X)-(Y) CD3zeta-2A- (Z); wherein , X includes a tumor-targeting antibody or a ligand and a receptor capable of specifically binding to the tumor; Y is an intracellular region of a co-stimulatory receptor selected from ICOS, CD28, CD27, HVEM, LIGHT, and CD40L , 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, CD226; Z is a co-stimulatory receptor selected from ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4- 1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, CD226.
  • the technical measures taken by the present invention also include:
  • the X is selected from the group consisting of an anti-CD19 antibody, an anti-CD20 antibody, an EGFR antibody, a HER2 antibody, an EGFRVIII antibody, an anti-PSMA antibody, an anti-BCMA antibody, an anti-CD22 antibody, and an anti-CD30 antibody. It can be understood that X may also be another protein capable of specifically binding to a tumor.
  • the X is an anti-CD20 antibody
  • the Y is 4-1BB
  • the Z is selected from one of OX40, HVEM, ICOS, CD27, and 4-1BB.
  • the scFv (X)-(Y) CD3zeta is scFv-antihCD20-20BBZ, and its sequence is shown in SEQ ID No. 1; the sequence of the OX40 is shown in SEQ ID No. 2; the HVEM The sequence is shown in SEQ ID No. 3; the sequence of the ICOS is shown in SEQ ID No. 4; the sequence of the CD27 is shown in SEQ ID No. 5; the sequence of the 4-1BB is shown in SEQ ID No. 6; the sequence of 2A is shown in SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9 or SEQ ID No. 10.
  • SEQ ID No. 7 GSGATNFSLLKQAGDVEENPGP;
  • SEQ ID No. 8 GSGEGRGSLLTCGDVEENPGP;
  • SEQ ID No. 9 GSGQCTNYALLKLAGDVESNPGP
  • SEQ ID No. 10 GSGVKQTLNFDLLKLAGDVESNPGP.
  • the extracellular hinge region of the chimeric antigen receptor is selected from a region of CD8a or IgG; the transmembrane region of the chimeric antigen receptor is selected from one of CD8a, CD28, CD137, or CD3.
  • a second object of the present invention is to provide a recombinant expression vector of any of the aforementioned chimeric antigen receptors.
  • a third object of the present invention is to provide a CAR-T cell constructed from a recombinant expression vector of any of the aforementioned chimeric antigen receptors.
  • a fourth object of the present invention is to provide a method for preparing the above CAR-T cells, which includes the following steps:
  • Step 1 Construction of lentiviral vector and virus production
  • ScFv (X)-(Y) CD3zeta, Z was added with 2A to form a fusion protein, lentiviral vectors were added at both ends, and co-transfection with the lentiviral packaging plasmid was performed to obtain scFv (X)-(Y) CD3zeta-2A- (Z) virus;
  • Step 2 Preparation of scFv (X)-(Y) CD3zeta-2A- (Z) CAR-T cells;
  • the isolated and purified human PBMCs were cultured and infected with scFv (X)-(Y) CD3zeta-2A- (Z) virus obtained in step 1.
  • Cell expansion was performed under appropriate conditions to prepare scFv (X)-(Y ) CD3zeta-2A- (Z) CAR-T cells.
  • the technical measures adopted by the present invention further include:
  • the specific steps of constructing the lentiviral vector and virus production include: adding scAv (X)-(Y) CD3zeta, Z by overlapping PCR to add 2A in the middle to form a fusion protein, and adding restriction sites to clone at both ends Lentiviral vector; clone the endotoxin-free clone of the correctly sequenced clone and transfection with the lentivirus packaging plasmid, collect the supernatant at a predetermined time, filter, and centrifuge to concentrate the virus to obtain scFv (X)-(Y) CD3zeta-2A- ( Z) Viruses.
  • the specific steps for the construction of the lentiviral vector and virus production are: adding scAv (X)-(Y) CD3zeta and OX40 to the 2A sequence by overlap PCR, and adding EcoRI and SalI digestion sites at both ends
  • the pCDH-MSCVEF vector was cloned, and the clone that was sequenced correctly was endotoxin-free, and transfected with 293X and lentiviral packaging plasmid. The supernatant was collected after 48 and 72 hours. After filtering at 0.45 uM, the virus was concentrated by centrifugation at 25,000 RPM for 2 hours to obtain scFv (X)-(Y) CD3zeta-2A- (Z) virus.
  • the specific steps for preparing the scFv (X)-(Y) CD3zeta-2A- (Z) CAR-T cells include: after separating and purifying human PBMC, inoculating it into a culture plate with appropriate stimulation conditions, and culturing After a predetermined time, the scFv (X)-(Y) CD3zeta-2A- (Z) virus produced in step 1 is infected, and the cells are expanded according to appropriate stimulation conditions. After 2 rounds of stimulation and expansion, the cells obtained are scFv (X)-(Y) CD3zeta-2A- (Z) CAR-T cells.
  • the stimulating conditions of cultured, isolated and purified human PBMC are anti-hCD3 and anti-hCD28, and the stimulating conditions of expanded cells are stimulated with artificial antigen presenting cells or anti-hCD3 / 28 every 6 days.
  • the X is selected from an anti-CD19 antibody, an anti-CD20 antibody, an EGFR antibody, a HER2 antibody, and an EGFRVIII antibody.
  • the X is an anti-CD20 antibody
  • the Y is 4-1BB
  • the Z is selected from one of OX40, HVEM, ICOS, CD27, and 4-1BB.
  • the scFv (X)-(Y) CD3zeta is scFv-antihCD20-20BBZ, whose sequence is shown in SEQ ID No. 1; the sequence of the OX40 is shown in SEQ ID No. 2; and the sequence of the HVEM is shown in FIG. SEQ ID No. 3; the sequence of the ICOS is shown in SEQ ID No. 4; the sequence of the CD27 is shown in SEQ ID No. 5; the sequence of the 4-1BB is shown in SEQ ID No. 6; The sequence of 2A is shown in SEQ ID No.7.
  • the lentiviral packaging plasmid in step 1 includes VSV-g, pMD, Gag / Pol, RSV-REV, and a Beckman ultracentrifuge and a SW28 rotor are used for centrifugation.
  • a fifth object of the present invention is to provide a preparation containing the above CAR-T cells or a CAR-T cell prepared by the above-mentioned preparation method; further, the preparation further includes a pharmaceutically acceptable diluent or excipient. Agent.
  • a sixth object of the present invention is to provide an application of the above-mentioned chimeric antigen receptor, the above-mentioned CAR-T cell, or the CAR-T cell prepared by the above-mentioned preparation method in preparing a medicine for treating or preventing tumor.
  • the tumor is a solid tumor
  • examples of the solid tumor include, but are not limited to, lymphoma, kidney tumor, neuroblastoma, germ cell tumor, osteosarcoma, chondrosarcoma, soft tissue sarcoma, liver tumor, thymoma , Pulmonary blastoma, pancreatoblastoma, hemangioma and so on.
  • the present invention has the following beneficial effects:
  • CAR-T cells of the present invention significantly improve tumor killing ability and expansion ability, and significantly improve the killing ability of solid tumors / metastatic tumors;
  • CAR-T cells according to the present invention include costimulatory receptors (ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, CD226, etc.), instead of conventionally used ligands or secreted factors, only affect CAR-T cells , Reduces the risk of causing immune side effects.
  • costimulatory receptors ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, CD226, etc.
  • the present invention uses a co-stimulatory receptor for the construction of CAR-T for the first time. Compared with the currently used CAR-T technology, it significantly improves the activation and survival ability of CAR-T cells in tumors and the ability to control solid / metastatic tumors. Therefore, the efficacy of CAR-T cells can be improved to have more excellent anti-tumor efficacy.
  • FIG. 1 is a schematic diagram illustrating a molecular structure of a chimeric antigen receptor (CAR) containing a third signal receptor in each embodiment of the present invention
  • FIG. 2 is a schematic diagram of a virus titer measured after BBZ-2A-OX40 virus infected 293 cells in an embodiment of the present invention
  • FIG. 3 is a schematic diagram of a virus titer measured after BBZ-2A-HVEM virus infected 293 cells in an embodiment of the present invention
  • FIG. 4 is a schematic diagram of a virus titer measured after BBZ-2A-ICOS virus infects 293 cells in an embodiment of the present invention
  • FIG. 5 is a schematic diagram of a virus titer measured after 293 cells were infected by BBZ-2A-CD27 virus according to an embodiment of the present invention
  • FIG. 6 is a schematic diagram of a virus titer measured after BBZ-2A-4-1BB virus infects 293 cells according to an embodiment of the present invention
  • FIG. 7 is a schematic diagram of the results of phenotypic analysis of BBZ CAR-T cells and BBZ-2A-OX40 CAR-T cells in an embodiment of the present invention
  • FIG. 8 is a schematic diagram of the results of phenotypic analysis of BBZ CAR-T cells and BBZ-2A-HVEM CAR-T cells in an embodiment of the present invention
  • FIG. 9 is a schematic diagram of the results of phenotypic analysis of BBZ CAR-T cells and BBZ-2A-ICOS CAR-T cells in an embodiment of the present invention.
  • FIG. 10 is a schematic diagram of the results of phenotypic analysis of BBZ CAR-T cells and BBZ-2A-CD27 CAR-T cells in an embodiment of the present invention
  • FIG. 11 is a schematic diagram of the results of phenotypic analysis of BBZ CAR-T cells and BBZ-2A-4-1BB CAR-T cells in an embodiment of the present invention
  • FIG. 12 is a schematic diagram of the expansion capacity of BBZ CAR-T cells and BBZ-2A-OX40 CAR-T cells in an embodiment of the present invention
  • FIG. 13 is a schematic diagram of tumor killing ability of BBZ CAR-T cells and BBZ-2A-OX40 CAR-T cells according to an embodiment of the present invention
  • FIG. 14 is a schematic diagram of the anti-tumor ability of BBZ CAR-T cells and BBZ-2A-OX40 CAR-T cells in an embodiment of the present invention
  • FIG. 15 is a schematic diagram of the in vivo viability of BBZ CAR-T cells and BBZ-2A-OX40 CAR-T cells in an embodiment of the present invention.
  • the present invention provides a chimeric antigen receptor comprising a co-stimulatory receptor, and the structure of the chimeric antigen receptor is scFv (X)-(Y) CD3zeta-2A- (Z); wherein X is tumor targeting Antibody or other protein; Y is the intracellular region of a co-stimulatory receptor selected from the group consisting of ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2, CD226; Z is a costimulatory receptor selected from ICOS, CD28, CD27, HVEM, LIGHT, CD40L, 4-1BB, OX40, DR3, GITR, CD30, TIM1, SLAM, CD2 , CD226.
  • X tumor targeting Antibody or other protein
  • Y is the intracellular region of a co-stimulatory receptor selected from the group consisting of ICOS, CD28,
  • the invention also relates to a CAR-T cell constructed from any of the above-mentioned chimeric antigen receptor recombinant expression vectors and a preparation method thereof, a preparation containing the CAR-T cell, and an application of the CAR-T cell.
  • the chimeric antigen receptor (CAR) molecules containing costimulatory receptors involved in the following examples of the present invention are BBZ-2A-OX40, BBZ-2A-HVEM, BBZ-2A-ICOS, BBZ-2A-CD27, BBZ-2A-4-1BB, its structure is shown in Figure 1.
  • the preparation of the 20BBZ-2A-OX40 CAR-T cells described in this example includes the following steps:
  • the scFv-antihCD20-20BBZ (SEQ ID No. 1) and OX40 (SEQ ID No. 2) were added to the 2A (SEQ ID No. 7) sequence in the middle of the overlap PCR, and EcoRI and SalI digestion sites were added at both ends to clone pCDH.
  • -MSCVEF vector Endotoxin-free clones that were sequenced correctly were cloned with lentiviral packaging plasmids (VSV-g, pMD, Gag / Pol, RSV-REV) to transfect 293X. Supernatants were collected after 48 and 72 hours.
  • the Beckman ultracentrifuge and SW28 rotor were centrifuged at 25,000 RPM for 2 hours to concentrate the virus, namely pCDH-MSCVEF-20BBZ-2A-OX40 virus (referred to as 20BBZ-2A-OX40 virus) for subsequent CAR-T cell production.
  • pCDH-MSCVEF-20BBZ virus 20BBZ virus for short
  • the obtained virus was used to infect 293 cells, and the virus titer was measured, as shown in FIG. 2.
  • the preparation of the 20BBZ-2A-HVEM CAR-T cells described in this example includes the following steps:
  • the scFv-antihCD20-20BBZ (SEQ ID No. 1) and HVEM (SEQ ID No. 3) were added to the 2A (SEQ ID No. 8) sequence in the middle of the overlap PCR, and EcoRI and SalI restriction sites were added at both ends to clone pCDH.
  • -MSCVEF vector Endotoxin-free clones that were sequenced correctly were cloned with lentiviral packaging plasmids (VSV-g, pMD, Gag / Pol, RSV-REV) to transfect 293X. Supernatants were collected after 48 and 72 hours.
  • pCDH-MSCVEF-20BBZ-2A-HVEM virus referred to as 20BBZ-2A-HVEM virus
  • 20BBZ-2A-HVEM virus pCDH-MSCVEF-20BBZ virus
  • the preparation of the 20BBZ-2A-ICOS CAR-T cells described in this example includes the following steps:
  • the scFv-antihCD20-20BBZ (SEQ ID No. 1) and ICOS (SEQ ID No. 4) were added to the 2A (SEQ ID No. 9) sequence through the middle of the overlap PCR, and EcoRI and SalI restriction sites were added at both ends to clone pCDH.
  • -MSCVEF vector Endotoxin-free clones that were sequenced correctly were cloned with lentiviral packaging plasmids (VSV-g, pMD, Gag / Pol, RSV-REV) to transfect 293X. Supernatants were collected after 48 and 72 hours.
  • the Beckman ultracentrifuge and SW28 rotor were centrifuged at 25,000 RPM for 2 hours to concentrate the virus, which was pCDH-MSCVEF-20BBZ-2A-ICOS virus (referred to as 20BBZ-2A-ICOS virus) for subsequent CAR-T cell production.
  • pCDH-MSCVEF-20BBZ virus 20BBZ virus for short
  • the obtained virus was used to infect 293 cells, and the virus titer was measured, as shown in FIG. 4.
  • the preparation of the 20BBZ-2A-CD27 CAR-T cells described in this example includes the following steps:
  • the scFv-antihCD20-20BBZ (SEQ ID No. 1) and CD27 (SEQ ID No. 5) were added to the 2A (SEQ ID No. 10) sequence in the middle of the overlap PCR, and EcoRI and SalI restriction sites were added at both ends to clone pCDH.
  • -MSCVEF vector Endotoxin-free clones that were sequenced correctly were cloned with lentiviral packaging plasmids (VSV-g, pMD, Gag / Pol, RSV-REV) to transfect 293X. Supernatants were collected after 48 and 72 hours.
  • the Beckman ultracentrifuge and SW28 rotor were centrifuged at 25,000 RPM for 2 hours to concentrate the virus, which was pCDH-MSCVEF-20BBZ-2A-CD27 virus (referred to as 20BBZ-2A-CD27 virus) for subsequent CAR-T cell production.
  • 20BBZ-2A-CD27 virus pCDH-MSCVEF-20BBZ-2A-CD27 virus
  • a control pCDH-MSCVEF-20BBZ virus (20BBZ virus for short) was produced, and the obtained virus was used to infect 293 cells, and the virus titer was measured, as shown in FIG. 5.
  • the preparation of the 20BBZ-2A-4-1BB CAR-T cells described in this example includes the following steps:
  • ScFv-antihCD20-20BBZ (SEQ ID No. 1) and 4-1BB (SEQ ID No. 6) are added in the middle of the overlap PCR 2A (SEQ ID No. 7) sequence, EcoRI and SalI restriction sites are added at both ends
  • the pCDH-MSCVEF vector was cloned. Endotoxin-free clones that were sequenced correctly were cloned with lentiviral packaging plasmids (VSV-g, pMD, Gag / Pol, RSV-REV) to transfect 293X. Supernatants were collected after 48 and 72 hours.
  • pCDH-MSCVEF-20BBZ-2A-4-1BB virus referred to as 20BBZ-2A-4-1BB virus
  • 20BBZ-2A-4-1BB virus pCDH-MSCVEF-20BBZ-2A-4-1BB virus
  • a control pCDH-MSCVEF-20BBZ virus (20BBZ virus for short) was produced, and the obtained virus was used to infect 293 cells, and the virus titer was measured, as shown in FIG. 6.
  • the 20BBZ CAR-T cells and 20BBZ-2A-OX40 CAR-T cells prepared in step 2 in Example 1 were continuously cultured for 14 days, stimulated with artificial antigen-presenting cells every 6 days, and the cells were counted. The results are shown in FIG. 12 shown. It can be seen from the figure that the 20BBZ-2A-OX40 CAR-T cells have a stronger proliferation capacity than the 20BBZ CAR-T cells.
  • the prepared 20BBZ-2A-CD27 CAR-T cells were seeded into a 96-well plate, and Raji tumor cells were added according to CAR-T: tumor cell ratio 1: 1, 1: 2, 1: 4, and tumors were compared after 24 and 48 hours.
  • the survival ratio of the cells is shown in Fig. 13.
  • 20BBZ-2A-OX40 / ICOS / CD27 CAR-T cells have similar tumor-killing ability compared to 20BBZ CAR-T cells, and even CAR-T containing some co-stimulatory receptors has stronger tumor-killing ability.
  • mice 10 6 Nalm-6 tumor cells were inoculated intravenously into B-NDG mice, and 6 7 days later were treated with 10 7 20BBZ CAR-T cells and 20BBZ-2A-OX40 CAR-T cells, and the survival rate of the mice was observed.
  • Some mice The content of tumor cells and CAR-T cells in the bone marrow was measured on the seventh day, and the results are shown in Figs. As can be seen from the figure, compared with 20BBZ CAR-T cells, 20BBZ-2A-OX40 CAR-T cells significantly prolonged the survival of mice and expanded more in vivo.
  • the present invention enables CAR-T cells to activate, survive, and expand CAR-T cells in tumors compared to the currently used CAR-T technology in clinical practice.
  • the ability is significantly enhanced, and it has more excellent anti-tumor efficacy.

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Abstract

本发明提供了一种包含共刺激受体的嵌合抗原受体，该嵌合抗原受体的结构为scFv(X)-(Y)CD3zeta-2A-(Z)；其中，X包括肿瘤靶向抗体或能与肿瘤特异结合的配体、受体；Y为共刺激受体的胞内区，Z为共刺激受体，所述共刺激受体选自ICOS、CD28、CD27、HVEM、LIGHT、CD40L、4-1BB、OX40、DR3、GITR、CD30、TIM1、SLAM、CD2、CD226。本发明还提供一种由上述嵌合抗原受体的重组表达载体构建的CAR-T细胞及其制备方法和应用。本发明所述的CAR-T细胞显著提高了肿瘤杀伤能力和扩增能力。

Description

一种包含共刺激受体的嵌合抗原受体及应用 技术领域
本发明涉及细胞免疫治疗技术领域，尤其涉及一种包含共刺激受体的嵌合抗原受体及其应用。
背景技术
利用免疫学治疗手段来攻克肿瘤，一直是免疫学在转化医学方面应用的重要方向。随着各种组学(基因组学，蛋白组学等)的发展，肿瘤细胞由于突变产生的免疫原性得到了广泛的认可，这为肿瘤免疫治疗奠定了理论的基础。同时，随着肿瘤免疫学研究自身的积累，肿瘤免疫治疗近期取得了巨大的进步，一系列新的免疫治疗手段逐步进入临床。当前的肿瘤免疫学研究，奠定了T细胞杀伤在肿瘤免疫治疗中的中心地位，而嵌和抗原受体T细胞(CAR-T细胞)就是结合了抗体的靶向识别和T细胞的肿瘤杀伤功能，人工修饰生成的肿瘤杀伤细胞。
嵌和抗原受体T细胞的概念最早在1989年被Gross，Waks和Eshhar提出，他们将识别TNP的抗体表达在T细胞上，实现了抗原特异性的、非MHC限制的T细胞的活化和效应的增强，并提出了CAR-T技术在肿瘤治疗中的应用的概念。根据这一原理，将具有肿瘤特异性的抗体嵌入T细胞，将赋予T细胞新的杀瘤能力。之后，CAR-T技术被引入到抗肿瘤临床试验中，但早期的CAR-T细胞由于其胞内信号传递域仅含有第一信号，而且选择的肿瘤类型为实体瘤，最终的临床结果都不太理想。在2008年，Fred Hutchison肿瘤研究所等机构使用CAR-T对B细胞淋巴瘤来进行治疗，虽然治疗结果不太理想，但这一临床实验的关键在于证实了以表达CD20的B细胞作为靶点的CAR-T治疗是相对安全的。随后，在2010年NCI报道了一例B细胞淋巴瘤治疗成功的病例，利用针对CD19的CAR-T，病人的淋巴瘤得到控制，正常B细胞也被清除，血清Ig显著降低，为CAR-T治疗B细胞来源的淋巴瘤的有效性提供了理论和实际的支持。在2011年，美国宾夕法尼亚大学的的Carl June博士领导的团队将特异性识别CD19的CAR-T用于B细胞来源的慢性淋巴细胞白血病的治疗，显示了“治愈” 的疗效，随后在复发难治性急性淋巴细胞白血病展开临床实验，也取得了良好的疗效。由于这一突破性的进展以及其他免疫调控手段的发展，Science杂志将肿瘤免疫治疗评为了2013年科技突破进展的第一名。这一成功在世界各国引起了广泛的影响，各国开始大量开展基于CAR-T的科学研究和肿瘤治疗的临床试验。
CAR的结构由胞外抗原识别结构域，胞外铰链区，跨膜区和胞内的信号传导结构域组成。胞外的抗原识别域通常是由单链抗体组成，特异性识别肿瘤细胞膜表面分子，也可以是某些肿瘤特异抗原的配体或受体等。胞外铰链区是一段用于隔开抗原识别域和跨膜区的空间结构，其目的是为了提供合适的空间位置，使得胞外抗原识别域能够在识别抗原前后维持正确的结构并传导胞内信号。跨膜区是为了保证CAR分子在膜表面定位的结构域。胞内信号传导结构域是介导CAR信号传导的关键部分，通常是一个或几个第一信号(TCR和MHC-I-peptide复合物识别)，第二信号(共刺激受体和共刺激配体识别)的组合。第一代CAR只含有第一信号，第二代CAR有一个第一信号和一个第二信号，第三代CAR有一个第一信号和两个第二信号结构域。虽然CAR-T在针对B细胞来源的白血病治疗中获得了巨大的成功，但是其相对较高的复发率以及对实体瘤的低有效率是当前的重要挑战。因此发展新一代的高效CAR-T是目前临床所急需的。除了第三代CAR-T，目前还有其他新的CAR-T设计策略，即在第二代CAR-T的基础上引入了新的独立于CAR的调节分子，以进一步增强CAR-T的功能。
利用病人自体血液细胞制备的针对B细胞表面靶向分子CD19、CD20的CAR-T在B细胞白血病治疗中的应用已相对成熟，但虽然响应率高，但是存在大量的复发现象，此外对实体淋巴瘤的治疗效率相对较低，这与实体肿瘤中的免疫抑制微环境有关。
在实体肿瘤中，存在多种免疫细胞、肿瘤细胞和基质细胞，共同组成肿瘤微环境。肿瘤微环境通常是免疫抑制的，能够对内源的抗肿瘤T细胞反应或过继性的T细胞(譬如CAR-T)在多种层次进行抑制，譬如，导致T细胞发生耗竭，失去杀伤肿瘤的功能，最终T细胞被清除。如何增强CAR-T在实体瘤中的活化能力，使其能够对抗肿瘤微环境中的免疫抑制，是扩增CAR-T到实体瘤治疗的重要思路和方向。
而当前临床使用的CAR-T结构域，在肿瘤杀伤能力和扩增能力上还存在不 足，在控制实体肿瘤/转移肿瘤上疗效较差，部分CAR-T采用新型调控分子如IL-12，4-1BBL等，这些分子除了影响CAR-T，也会对其他非CAR-T细胞产生非特异的活化作用，有造成免疫副作用的潜在可能。
发明内容
本发明的目的在于克服现有技术中的缺陷，提供一种包含共刺激受体的嵌合抗原受体及其应用，并提供一种由如该嵌合抗原受体的重组表达载体构建的CAR-T细胞，例如OX40是一个重要的共刺激受体，主要表达在活化的CD4和CD8T细胞，在T细胞活化过程中表现出多种功能，能够促进T细胞的活化，表达更多的效应分子以及减少细胞凋亡相关的基因表达，将共刺激受体信号整合在CAR-T中，具有潜在的增强效应的功能。
为实现上述目的，本发明采用如下技术方案：
本发明的第一个目的是提供一种包含共刺激受体的嵌合抗原受体，所述嵌合抗原受体的结构为scFv(X)-(Y)CD3zeta-2A-(Z)；其中，X包括肿瘤靶向抗体或能与肿瘤特异结合的配体、受体；Y为共刺激受体的胞内区，所述共刺激受体选自ICOS、CD28、CD27、HVEM、LIGHT、CD40L、4-1BB、OX40、DR3、GITR、CD30、TIM1、SLAM、CD2、CD226；Z为共刺激受体，所述共刺激受体选自ICOS、CD28、CD27、HVEM、LIGHT、CD40L、4-1BB、OX40、DR3、GITR、CD30、TIM1、SLAM、CD2、CD226。
为了进一步优化上述嵌合抗原受体，本发明采取的技术措施还包括：
进一步地，所述X选自anti-CD19抗体、anti-CD20抗体、EGFR抗体、HER2抗体、EGFRVIII抗体、anti-PSMA抗体、anti-BCMA抗体、anti-CD22抗体、anti-CD30抗体。可理解的是，X还可为能与肿瘤特异结合的其他蛋白。
进一步地，所述X为anti-CD20抗体，所述Y为4-1BB，所述Z选自OX40、HVEM、ICOS、CD27、4-1BB中的一种。
进一步地，所述scFv(X)-(Y)CD3zeta为scFv-antihCD20-20BBZ，其序列如SEQ ID No.1所示；所述OX40的序列如SEQ ID No.2所示；所述HVEM的序列如SEQ ID No.3所示；所述ICOS的序列如SEQ ID No.4所示；所述CD27的序列如SEQ ID No.5所示；所述4-1BB的序列如SEQ ID No.6所示；所述2A的序 列如SEQ ID No.7、SEQ ID No.8、SEQ ID No.9或SEQ ID No.10所示。
其中上述各序列具体如下：
SEQ ID No.1：
Figure PCTCN2019077922-appb-000001
SEQ ID No.2：
Figure PCTCN2019077922-appb-000002
SEQ ID No.3：
Figure PCTCN2019077922-appb-000003
SEQ ID No.4：
Figure PCTCN2019077922-appb-000004
Figure PCTCN2019077922-appb-000005
SEQ ID No.5：
Figure PCTCN2019077922-appb-000006
SEQ ID No.6：
Figure PCTCN2019077922-appb-000007
SEQ ID No.7：GSGATNFSLLKQAGDVEENPGP；
SEQ ID No.8：GSGEGRGSLLTCGDVEENPGP；
SEQ ID No.9：GSGQCTNYALLKLAGDVESNPGP；
SEQ ID No.10：GSGVKQTLNFDLLKLAGDVESNPGP。
进一步地，所述嵌合抗原受体的胞外铰链区选自CD8a或IgG的一段区域；所述嵌合抗原受体的跨膜区选自CD8a、CD28、CD137或CD3中的一个。
本发明的第二个目的是提供一种任一上述的嵌合抗原受体的重组表达载体。
本发明的第三个目的是提供一种由任一上述的嵌合抗原受体的重组表达载体构建的CAR-T细胞。
本发明的第四个目的是提供一种上述CAR-T细胞的制备方法，其包括以下步骤：
步骤一、慢病毒载体的构建及病毒生产；
将scFv(X)-(Y)CD3zeta，Z的中间加入2A形成融合蛋白，两端加入慢病毒载体，并和慢病毒包装质粒共同转染，获得scFv(X)-(Y)CD3zeta-2A-(Z)病毒；
步骤二、scFv(X)-(Y)CD3zeta-2A-(Z)CAR-T细胞的制备；
分离纯化后的人PBMC经过培养后，感染步骤一获得的scFv(X)-(Y)CD3zeta-2A-(Z)病毒，在合适的条件下进行细胞扩增，制备scFv(X)-(Y)CD3zeta-2A-(Z)CAR-T细胞。
为了进一步优化上述CAR-T细胞的制备方法，本发明所采用的技术措施还包括：
进一步地，所述慢病毒载体的构建及病毒生产的具体步骤包括：将scFv(X)-(Y)CD3zeta，Z通过overlap PCR在其中间加入2A形成融合蛋白，两端加入酶切位点克隆慢病毒载体；将测序正确的克隆无内毒素大提，和慢病毒包装质粒共同转染，预定时间收取上清，过滤、离心浓缩病毒，获得scFv(X)-(Y)CD3zeta-2A-(Z)病毒。
更进一步地，所述慢病毒载体的构建及病毒生产的具体步骤为：将scFv(X)-(Y)CD3zeta和OX40通过overlap PCR在其加入2A序列，两端加入EcoRI和SalI酶切位点克隆pCDH-MSCVEF载体，将测序正确的克隆无内毒素大提，和慢病毒包装质粒共同转染293X，48和72小时后收取上清，0.45uM过滤后，25000RPM离心2小时浓缩病毒，获得scFv(X)-(Y)CD3zeta-2A-(Z)病毒。
进一步地，所述scFv(X)-(Y)CD3zeta-2A-(Z)CAR-T细胞的制备的具体步骤包括：将人PBMC经过分离纯化后，接种到具有合适刺激条件的培养板，培养预定时间后，感染步骤一生产的scFv(X)-(Y)CD3zeta-2A-(Z)病毒，按照合适的刺激条件进行细胞扩增，经过2轮刺激扩增后，获得的细胞即为scFv(X)-(Y)CD3zeta-2A-(Z)CAR-T细胞。
进一步地，培养分离纯化后的人PBMC的刺激条件为anti-hCD3和anti-hCD28，扩增细胞的刺激条件为每6天使用人工抗原呈递细胞或anti-hCD3/28刺激。
更进一步地，所述scFv(X)-(Y)CD3zeta-2A-(Z)CAR-T细胞的制备的具体步骤为：将人PBMC经过Stemcell T细胞分离试剂盒纯化后，接种到anti-hCD3和anti-hCD28包被的96孔培养板，2天后，按照MOI＝10-20感染scFv(X)-(Y)CD3zeta-2A-(Z)病毒，1天后换液继续细胞培养，按照每6天使用人工抗原呈递细胞或anti-hCD3/28刺激，经过2轮刺激后，所得细胞即为scFv(X)-(Y)CD3zeta-2A-(Z)CAR-T细胞。
进一步地，所述X选自anti-CD19抗体、anti-CD20抗体、EGFR抗体、HER2抗体、EGFRVIII抗体。
进一步地，所述X为anti-CD20抗体，所述Y为4-1BB，所述Z选自OX40、HVEM、ICOS、CD27、4-1BB中的一种。
进一步地，所述scFv(X)-(Y)CD3zeta为scFv-antihCD20-20BBZ，其序列如SEQID No.1所示；所述OX40的序列如SEQID No.2所示；所述HVEM的序列如SEQID No.3所示；所述ICOS的序列如SEQID No.4所示；所述CD27的序列如SEQID No.5所示；所述4-1BB的序列如SEQID No.6所示；所述2A的序列如SEQID No.7所示。
进一步地，所述步骤一中慢病毒包装质粒包括VSV-g，pMD Gag/Pol，RSV-REV，离心使用贝克曼超速离心机和SW28转头。
本发明的第五个目的是提供一种含有上述CAR-T细胞或含有由上述制备方法制得的的CAR-T细胞的制剂；进一步地，所述制剂还包括药用的稀释剂或赋形剂。
本发明的第六个目的是提供一种上述嵌合抗原受体、上述CAR-T细胞或由上述制备方法制得的的CAR-T细胞在制备治疗或预防肿瘤药物中的应用。
进一步地，所述肿瘤为实体肿瘤，所述实体肿瘤的例子包括但不限于，淋巴瘤、肾脏肿瘤、神经母细胞瘤、生殖细胞瘤、骨肉瘤、软骨肉瘤、软组织肉瘤、肝脏肿瘤、胸腺瘤、肺母细胞瘤、胰母细胞瘤、血管瘤等。
与现有技术相比，本发明具有以下有益效果：
本发明所述的CAR-T细胞显著提高了肿瘤杀伤能力和扩增能力，显著提高的实体肿瘤/转移肿瘤的杀伤能力；本发明所述的CAR-T细胞，包含共刺激受体(ICOS、CD28、CD27、HVEM、LIGHT、CD40L、4-1BB、OX40、DR3、GITR、CD30、TIM1、SLAM、CD2、CD226等)而不是常规所用的配体或分泌因子，只对CAR-T细胞有作用，降低了造成免疫副作用的风险。
本发明首次将共刺激受体用于CAR-T的构建，相对目前临床使用的CAR-T技术，其显著提高CAR-T细胞在肿瘤中的活化能力和存活能力，控制实体/转移肿瘤的能力，从而提高CAR-T细胞疗效，以具有更优异的抗肿瘤疗效。
附图说明
图1为本发明各实施例中含有第三信号受体的嵌合抗原受体(CAR)分子结构的说明示意图；
图2为本发明一实施例中BBZ-2A-OX40病毒感染293细胞后测得的病毒滴度的示意图；
图3为本发明一实施例中BBZ-2A-HVEM病毒感染293细胞后测得的病毒滴度的示意图；
图4为本发明一实施例中BBZ-2A-ICOS病毒感染293细胞后测得的病毒滴度的示意图；
图5为本发明一实施例中BBZ-2A-CD27病毒感染293细胞后测得的病毒滴度的示意图；
图6为本发明一实施例中BBZ-2A-4-1BB病毒感染293细胞后测得的病毒滴度的示意图；
图7为本发明一实施例中BBZ CAR-T细胞和BBZ-2A-OX40 CAR-T细胞表型分析的结果示意图；
图8为本发明一实施例中BBZ CAR-T细胞和BBZ-2A-HVEM CAR-T细胞表型分析的结果示意图；
图9为本发明一实施例中BBZ CAR-T细胞和BBZ-2A-ICOS CAR-T细胞表型分析的结果示意图；
图10为本发明一实施例中BBZ CAR-T细胞和BBZ-2A-CD27 CAR-T细胞表型分析的结果示意图；
图11为本发明一实施例中BBZ CAR-T细胞和BBZ-2A-4-1BB CAR-T细胞表型分析的结果示意图；
图12为本发明一实施例中BBZ CAR-T细胞和BBZ-2A-OX40 CAR-T细胞的扩增能力的示意图；
图13为本发明一实施例BBZ CAR-T细胞和BBZ-2A-OX40 CAR-T细胞的肿瘤杀伤能力的示意图；
图14为本发明一实施例中BBZ CAR-T细胞和BBZ-2A-OX40 CAR-T细胞的抗肿瘤能力的示意图；
图15为本发明一实施例中BBZ CAR-T细胞和BBZ-2A-OX40 CAR-T细胞的体内存活能力的示意图。
具体实施方式
本发明提供一种包含共刺激受体的嵌合抗原受体，所述嵌合抗原受体的结构为scFv(X)-(Y)CD3zeta-2A-(Z)；其中，X为肿瘤靶向抗体或其他蛋白；Y为共刺激受体的胞内区，所述共刺激受体选自ICOS、CD28、CD27、HVEM、LIGHT、CD40L、4-1BB、OX40、DR3、GITR、CD30、TIM1、SLAM、CD2、CD226；Z为共刺激受体，所述共刺激受体选自ICOS、CD28、CD27、HVEM、LIGHT、CD40L、4-1BB、OX40、DR3、GITR、CD30、TIM1、SLAM、CD2、CD226。本发明还涉及一种由任一上述的嵌合抗原受体的重组表达载体构建的CAR-T细胞及其制备方法，含有该CAR-T细胞的制剂以及该CAR-T细胞的应用。
下面结合附图和实施例，对本发明的具体实施方式作进一步描述。以下实施例仅用于更加清楚地说明本发明的技术方案，而不能以此来限制本发明的保护范围。
本发明下述实施例所涉及的含有共刺激受体的嵌合抗原受体(CAR)分子分别为BBZ-2A-OX40、BBZ-2A-HVEM、BBZ-2A-ICOS、BBZ-2A-CD27、BBZ-2A-4-1BB，其结构如图1所示。
实施例1-20BBZ-2A-OX40 CAR-T细胞的制备
本实施例所述的20BBZ-2A-OX40 CAR-T细胞的制备包括以下步骤：
1.慢病毒载体pCDH-MSCVEF-20BBZ-2A-OX40的构建及病毒生产
将scFv-antihCD20-20BBZ(SEQ ID No.1)和OX40(SEQ ID No.2)通过overlap PCR的中间加入2A(SEQ ID No.7)序列，两端加入EcoRI和SalI酶切位点克隆pCDH-MSCVEF载体。将测序正确的克隆无内毒素大提，和慢病毒包装质粒(VSV-g，pMD Gag/Pol，RSV-REV)共同转染293X，48和72小时后收取上清，0.45uM过滤后，使用贝克曼超速离心机和SW28转头，25000RPM离心2小时浓缩病毒，即为pCDH-MSCVEF-20BBZ-2A-OX40病毒(简称为20BBZ-2A-OX40病毒)，用于后续CAR-T细胞生产。同时生产对照pCDH-MSCVEF-20BBZ病毒(简称20BBZ病毒)，将所得病毒感染293细胞，测得病毒滴度，如图2所 示。
2. 20BBZ-2A-OX40 CAR-T细胞和20BBZ CAR-T细胞的制备
将人PBMC经过Stemcell T细胞分离试剂盒纯化后，接种到anti-hCD3和anti-hCD28包被的96孔培养板，2天后，按照MOI＝10-20感染20BBZ病毒和20BBZ-2A-OX40病毒，1天后换液继续细胞培养，按照每6天使用人工抗原呈递细胞或anti-hCD3/28刺激，经过2轮刺激后，所得细胞即为20BBZ CAR-T细胞和20BBZ-2A-OX40 CAR-T细胞，用于后续实验和表型分析，其结果如图7所示。由图可知，所得细胞为CAR阳性。
实施例2-20BBZ-2A-HVEM CAR-T细胞的制备
本实施例所述的20BBZ-2A-HVEM CAR-T细胞的制备包括以下步骤：
1.慢病毒载体pCDH-MSCVEF-20BBZ-2A-HVEM的构建及病毒生产
将scFv-antihCD20-20BBZ(SEQ ID No.1)和HVEM(SEQ ID No.3)通过overlap PCR的中间加入2A(SEQ ID No.8)序列，两端加入EcoRI和SalI酶切位点克隆pCDH-MSCVEF载体。将测序正确的克隆无内毒素大提，和慢病毒包装质粒(VSV-g，pMD Gag/Pol，RSV-REV)共同转染293X，48和72小时后收取上清，0.45uM过滤后，使用贝克曼超速离心机和SW28转头，25000RPM离心2小时浓缩病毒，即为pCDH-MSCVEF-20BBZ-2A-HVEM病毒(简称为20BBZ-2A-HVEM病毒)，用于后续CAR-T细胞生产。同时生产对照pCDH-MSCVEF-20BBZ病毒(简称20BBZ病毒)，将所得病毒感染293细胞，测得病毒滴度，如图3所示。
2. 20BBZ-2A-HVEM CAR-T细胞和20BBZ CAR-T细胞的制备
将人PBMC经过Stemcell T细胞分离试剂盒纯化后，接种到anti-hCD3和anti-hCD28包被的96孔培养板，2天后，按照MOI＝10-20感染20BBZ病毒和20BBZ-2A-HVEM病毒，1天后换液继续细胞培养，按照每6天使用人工抗原呈递细胞或anti-hCD3/28刺激，经过2轮刺激后，所得细胞即为20BBZ CAR-T细胞和20BBZ-2A-HVEM CAR-T细胞，用于后续实验和表型分析，其结果如图8所示。由图可知，所得细胞为CAR阳性。
实施例3-20BBZ-2A-ICOS CAR-T细胞的制备
本实施例所述的20BBZ-2A-ICOS CAR-T细胞的制备包括以下步骤：
1.慢病毒载体pCDH-MSCVEF-20BBZ-2A-ICOS的构建及病毒生产
将scFv-antihCD20-20BBZ(SEQ ID No.1)和ICOS(SEQ ID No.4)通过overlap PCR的中间加入2A(SEQ ID No.9)序列，两端加入EcoRI和SalI酶切位点克隆pCDH-MSCVEF载体。将测序正确的克隆无内毒素大提，和慢病毒包装质粒(VSV-g，pMD Gag/Pol，RSV-REV)共同转染293X，48和72小时后收取上清，0.45uM过滤后，使用贝克曼超速离心机和SW28转头，25000RPM离心2小时浓缩病毒，即为pCDH-MSCVEF-20BBZ-2A-ICOS病毒(简称为20BBZ-2A-ICOS病毒)，用于后续CAR-T细胞生产。同时生产对照pCDH-MSCVEF-20BBZ病毒(简称20BBZ病毒)，将所得病毒感染293细胞，测得病毒滴度，如图4所示。
2. 20BBZ-2A-ICOS CAR-T细胞和20BBZ CAR-T细胞的制备
将人PBMC经过Stemcell T细胞分离试剂盒纯化后，接种到anti-hCD3和anti-hCD28包被的96孔培养板，2天后，按照MOI＝10-20感染20BBZ病毒和20BBZ-2A-ICOS病毒，1天后换液继续细胞培养，按照每6天使用人工抗原呈递细胞或anti-hCD3/28刺激，经过2轮刺激后，所得细胞即为20BBZ CAR-T细胞和20BBZ-2A-ICOS CAR-T细胞，用于后续实验和表型分析，其结果如图9所示。由图可知，所得细胞为CAR阳性。
实施例4-20BBZ-2A-CD27 CAR-T细胞的制备
本实施例所述的20BBZ-2A-CD27 CAR-T细胞的制备包括以下步骤：
1.慢病毒载体pCDH-MSCVEF-20BBZ-2A-CD27的构建及病毒生产
将scFv-antihCD20-20BBZ(SEQ ID No.1)和CD27(SEQ ID No.5)通过overlap PCR的中间加入2A(SEQ ID No.10)序列，两端加入EcoRI和SalI酶切位点克隆pCDH-MSCVEF载体。将测序正确的克隆无内毒素大提，和慢病毒包装质粒(VSV-g，pMD Gag/Pol，RSV-REV)共同转染293X，48和72小时后收取上清，0.45uM过滤后，使用贝克曼超速离心机和SW28转头，25000RPM离心2小时浓缩病毒，即为pCDH-MSCVEF-20BBZ-2A-CD27病毒(简称为20BBZ-2A-CD27病毒)，用于后续CAR-T细胞生产。同时生产对照pCDH-MSCVEF-20BBZ病毒(简称20BBZ病毒)，将所得病毒感染293细胞，测得病毒滴度，如图5所示。
2. 20BBZ-2A-CD27 CAR-T细胞和20BBZ CAR-T细胞的制备
将人PBMC经过Stemcell T细胞分离试剂盒纯化后，接种到anti-hCD3和anti-hCD28包被的96孔培养板，2天后，按照MOI＝10-20感染20BBZ病毒和20BBZ-2A-CD27病毒，1天后换液继续细胞培养，按照每6天使用人工抗原呈递细胞或anti-hCD3/28刺激，经过2轮刺激后，所得细胞即为20BBZ CAR-T细胞和20BBZ-2A-CD27 CAR-T细胞，用于后续实验和表型分析，其结果如图10所示。由图可知，所得细胞为CAR阳性。
实施例5-20BBZ-2A-4-1BB CAR-T细胞的制备
本实施例所述的20BBZ-2A-4-1BB CAR-T细胞的制备包括以下步骤：
1.慢病毒载体pCDH-MSCVEF-20BBZ-2A-4-1BB的构建及病毒生产
将scFv-antihCD20-20BBZ(SEQ ID No.1)和4-1BB(SEQ ID No.6)通过overlap PCR的中间加入2A(SEQ ID No.7)序列，两端加入EcoRI和SalI酶切位点克隆pCDH-MSCVEF载体。将测序正确的克隆无内毒素大提，和慢病毒包装质粒(VSV-g，pMD Gag/Pol，RSV-REV)共同转染293X，48和72小时后收取上清，0.45uM过滤后，使用贝克曼超速离心机和SW28转头，25000RPM离心2小时浓缩病毒，即为pCDH-MSCVEF-20BBZ-2A-4-1BB病毒(简称为20BBZ-2A-4-1BB病毒)，用于后续CAR-T细胞生产。同时生产对照pCDH-MSCVEF-20BBZ病毒(简称20BBZ病毒)，将所得病毒感染293细胞，测得病毒滴度，如图6所示。
2. 20BBZ-2A-4-1BB CAR-T细胞和20BBZ CAR-T细胞的制备
将人PBMC经过Stemcell T细胞分离试剂盒纯化后，接种到anti-hCD3和anti-hCD28包被的96孔培养板，2天后，按照MOI＝10-20感染20BBZ病毒和20BBZ-2A-4-1BB病毒，1天后换液继续细胞培养，按照每6天使用人工抗原呈递细胞或anti-hCD3/28刺激，经过2轮刺激后，所得细胞即为20BBZCAR-T细胞和20BBZ-2A-4-1BB CAR-T细胞，用于后续实验和表型分析，其结果如图11所示。由图可知，所得细胞为CAR阳性。
实施例6-比较20BBZ CAR-T细胞和20BBZ-2A-OX40 CAR-T细胞的扩增能力
将实施例1中步骤2制备所得的20BBZ CAR-T细胞和20BBZ-2A-OX40  CAR-T细胞，连续培养14天，每隔6天用人工抗原呈递细胞刺激一次，细胞计数，其结果如图12所示。由图可知，20BBZ-2A-OX40 CAR-T细胞相对20BBZ CAR-T细胞有更强的增殖能力。
实施例7-比较20BBZ CAR-T细胞和20BBZ-2A-OX40 CAR-T细胞的肿瘤杀伤能力
将实施例1中步骤2制备所得的20BBZ CAR-T细胞和20BBZ-2A-OX40 CAR-T细胞，实施例3步骤2制备所得的20BBZ-2A-ICOS CAR-T细胞，和实施例4步骤2制备所得的20BBZ-2A-CD27 CAR-T细胞，接种到96孔板，按照CAR-T：肿瘤细胞比例1:1、1:2、1:4加入Raji肿瘤细胞，24、48小时后比较肿瘤细胞的存活比例，其结果如图13所示。由图可知，20BBZ-2A-OX40/ICOS/CD27 CAR-T细胞相对20BBZ CAR-T细胞有相似的肿瘤杀伤能力，甚至部分含共刺激受体的CAR-T有更强的肿瘤杀伤能力。
实施例8-比较20BBZ CAR-T细胞和20BBZ-2A-OX40 CAR-T细胞的抗肿瘤能力和体内存活能力
将10 6Nalm-6肿瘤细胞通过静脉接种到B-NDG小鼠，6天后给予10 7 20BBZ CAR-T细胞和20BBZ-2A-OX40 CAR-T细胞治疗，观察小鼠的存活率，部分小鼠在第7天检测其骨髓中的肿瘤细胞和CAR-T细胞的含量，其结果分别如图14和图15所示。由图可知，20BBZ-2A-OX40 CAR-T细胞相对20BBZ CAR-T细胞显著延长了小鼠的生存，并在体内扩增更多。
由上述实施例可知，本发明通过构建新型含共刺激受体的CAR-T细胞，相对目前临床使用的CAR-T技术，其使CAR-T细胞在肿瘤中的活化能力、存活能力、扩增能力显著增强，具有更优异的抗肿瘤疗效。
以上对本发明的具体实施例进行了详细描述，但其只作为范例，本发明并不限制于以上描述的具体实施例。对于本领域技术人员而言，任何对本发明进行的等同修改和替代也都在本发明的范畴之中。因此，在不脱离本发明的精神和范围下所作的均等变换和修改，都应涵盖在本发明的范围内。

Claims (10)

  1. 一种包含共刺激受体的嵌合抗原受体，其特征在于，所述嵌合抗原受体的结构为scFv(X)-(Y)CD3zeta-2A-(Z)；
    其中，X包括肿瘤靶向抗体或能与肿瘤特异结合的配体、受体；
    Y为共刺激受体的胞内区，所述共刺激受体选自ICOS、CD28、CD27、HVEM、LIGHT、CD40L、4-1BB、OX40、DR3、GITR、CD30、TIM1、SLAM、CD2、CD226；
    Z为共刺激受体，所述共刺激受体选自ICOS、CD28、CD27、HVEM、LIGHT、CD40L、4-1BB、OX40、DR3、GITR、CD30、TIM1、SLAM、CD2、CD226。
  2. 根据权利要求1所述的一种包含共刺激受体的嵌合抗原受体，其特征在于，所述X选自anti-CD19抗体、anti-CD20抗体、EGFR抗体、HER2抗体、EGFRVIII抗体、anti-PSMA抗体、anti-BCMA抗体、anti-CD22抗体、anti-CD30抗体。
  3. 根据权利要求1所述的一种包含共刺激受体的嵌合抗原受体，其特征在于，所述X为anti-CD20抗体，所述Y为4-1BB，所述Z选自OX40、HVEM、ICOS、CD27、4-1BB中的一种。
  4. 根据权利要求3所述的一种包含共刺激受体的嵌合抗原受体，其特征在于，所述scFv(X)-(Y)CD3zeta为scFv-antihCD20-20BBZ，其序列如SEQ ID No.1所示；所述OX40的序列如SEQ ID No.2所示；所述HVEM的序列如SEQ ID No.3所示；所述ICOS的序列如SEQ ID No.4所示；所述CD27的序列如SEQ ID No.5所示；所述4-1BB的序列如SEQ ID No.6所示；所述2A的序列如SEQ ID No.7、SEQ ID No.8、SEQ ID No.9或SEQ ID No.10所示。
  5. 一种由如权利要求1～4中任一项所述的嵌合抗原受体的重组表达载体构建的CAR-T细胞
  6. 根据权利要求5所述的CAR-T细胞的制备方法，其特征在于，包括如下步骤：
    步骤一、慢病毒载体的构建及病毒生产；
    将scFv(X)-(Y)CD3zeta，Z的中间加入2A形成融合蛋白，两端加入慢病毒载体，并和慢病毒包装质粒共同转染，获得scFv(X)-(Y)CD3zeta-2A-(Z)病毒；
    步骤二、scFv(X)-(Y)CD3zeta-2A-(Z)CAR-T细胞的制备；
    分离纯化后的人PBMC经过培养后，感染步骤一获得的 scFv(X)-(Y)CD3zeta-2A-(Z)病毒，在合适的条件下进行细胞扩增，制备scFv(X)-(Y)CD3zeta-2A-(Z)CAR-T细胞。
  7. 根据权利要求6所述的CAR-T细胞的制备方法，其特征在于，所述慢病毒载体的构建及病毒生产的具体步骤包括：
    将scFv(X)-(Y)CD3zeta，Z通过overlap PCR在其中间加入2A形成融合蛋白，两端加入酶切位点克隆慢病毒载体；将测序正确的克隆无内毒素大提，和慢病毒包装质粒共同转染，预定时间收取上清，过滤、离心浓缩病毒，获得scFv(X)-(Y)CD3zeta-2A-(Z)病毒。
  8. 根据权利要求6所述的CAR-T细胞的制备方法，其特征在于，所述scFv(X)-(Y)CD3zeta-2A-(Z)CAR-T细胞的制备的具体步骤包括：将人PBMC经过分离纯化后，接种到具有合适刺激条件的培养板，培养预定时间后，感染步骤一生产的scFv(X)-(Y)CD3zeta-2A-(Z)病毒，按照合适的刺激条件进行细胞扩增，经过2轮刺激扩增后，获得的细胞即为scFv(X)-(Y)CD3zeta-2A-(Z)CAR-T细胞。
  9. 一种含有如权利要求5所述的CAR-T细胞的制剂。
  10. 如权利要求1～4中任一项所述的嵌合抗原受体，如权利要求5所述的CAR-T细胞在制备治疗或预防肿瘤药物中的应用。
PCT/CN2019/077922 2018-06-20 2019-03-13 一种包含共刺激受体的嵌合抗原受体及应用 WO2019242338A1 (zh)

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