WO2020125277A1 - 基因工程修饰的双靶点嵌合抗原受体及其用途 - Google Patents

基因工程修饰的双靶点嵌合抗原受体及其用途 Download PDF

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WO2020125277A1
WO2020125277A1 PCT/CN2019/117601 CN2019117601W WO2020125277A1 WO 2020125277 A1 WO2020125277 A1 WO 2020125277A1 CN 2019117601 W CN2019117601 W CN 2019117601W WO 2020125277 A1 WO2020125277 A1 WO 2020125277A1
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chimeric antigen
antigen receptor
dual
target
tumor
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French (fr)
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王永生
李丹
杨骐毓
马启智
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四川大学华西医院
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Priority to EP19900565.3A priority Critical patent/EP3950718A4/en
Priority to US17/442,720 priority patent/US20220185893A1/en
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    • C12N2510/00—Genetically modified cells

Definitions

  • the invention belongs to the field of genetic engineering, and in particular relates to a genetically modified dual-target chimeric antigen receptor.
  • the invention also relates to an immune response cell expressing the chimeric antigen receptor, as well as the antigen receptor and immune response Use of cells in the preparation of drugs for diagnosis and treatment of malignant tumors or solid tumors.
  • CAR-T cell therapy and CTLA-4 and PD-1/PD-L1 antibody therapy were considered the three major advances in tumor immunotherapy.
  • the use of antigen-antibody scFv fragments, combined with intracellular activation and proliferation signals of T cells, to construct chimeric antigen receptor (CAR)-modified T cells (CAR-T) allows T cells to directly obtain antibody-specific recognition Ability to become an effector T cell independent of HLA (human leukocyte antigen) limitation.
  • the killing activity of the obtained CAR-T cells mainly depends on the single-chain receptor recognition antigen on the surface of the CAR-T cells, and has specific killing activity.
  • CAR-T can be expanded in vivo, survive for a long time and form an immune memory, and it shows high anti-tumor activity even for refractory hematological tumors.
  • CAR-T faces huge challenges in the treatment of solid tumors, and its clinical application has been slow to date.
  • CAR-T is used in the systemic treatment of solid tumors, there are several major challenges: (1) Most solid tumor treatment targets are tumor-associated antigens (Tumor-associated antigens, TAA). Because TAA is expressed in normal tissues, there is potential toxicity for targeting TAA. CAR-T cells targeting Her2, MART1, and CAIX are used to observe "on-target” toxicity in clinical treatment, suggesting that systemic CAR-T treatment for TAA needs to be cautious Design; (2) CAR-T cells are used to treat solid tumors.
  • TAA tumor-associated antigens
  • CAR-T cells enter the solid tumor through the blood vessels is an important step to exert the therapeutic effect, but CAR-T is often difficult to penetrate the vascular basement membrane, affecting the therapeutic effect; (3) Inside the solid tumor is a suppressive immunosuppressive microenvironment that can secrete TGF- ⁇ , activate PD-1/PD-L1 inhibitory signals, and suppress effector cell activity through immunosuppressive cells MDSC and Treg. It can be seen that there are still many difficulties in using CAR-T for systemic CAR-T treatment.
  • Malignant tumors such as lung cancer, colon cancer, ovarian cancer, breast cancer, gastric cancer, lymphoma and other tumors are prone to the serous cavity metastasis of the thoracic cavity, abdominal cavity and pericardial cavity, often causing the chest cavity, abdominal cavity to spread or further merge with malignant serous cavity effusion , Great harm.
  • Thoracic and abdominal cavity metastases and pleural or peritoneal effusions in patients with tumors are often difficult to tolerate systemic therapy. Even if drainage + perfusion chemotherapy is used for clinical treatment, the efficacy is still limited. Complications such as dyspnea and intestinal obstruction seriously affect the patient's physiological function and life Quality, median survival time is often only 3-6 months.
  • PC Peritoneal Carcinomatosis
  • EMT epithelial-mesenchymal transition
  • CSC cancer stem cells
  • CAR-T chimeric antigen receptor
  • CAR-T serosal cavity infusion therapy has two major advantages: First, it is safe and has low systemic toxicity. CAR-T has specific killing activity on tumor cells, completely depends on the targeted tumor antigen, and will also kill normal tissue expressing the antigen. CAR-T plays a killing role in the serosal cavity, even if it multiplies on a large scale, it is not easy to enter the blood circulation in large quantities, and it is easy to local treatment, which can eliminate potential toxicity. Second, the therapeutic targets of CAR-T cells have been extended, and more TAA can be used as therapeutic targets.
  • CAR-T plays a role and depends on the antigen. Independent of the source of the tumor, CAR-T therefore has a broad-spectrum tumoricidal activity (killing activity against a variety of tumors expressing this antigen), and therefore clinically can treat serous cavity metastasis as an independent indication for treatment.
  • tumor cells and immune cells both up-regulate PD-L1 to escape immune attack.
  • VEGFR1 or HER2
  • PD-L1 dual targeting CAR-T
  • VEGFR1 or HER2
  • PD-L1 dual targeting CAR-T dual CAT-T
  • the dual-target chimeric antigen receptor containing the PD-L1 target can eliminate the immune escape of tumor cells ,Remove the immune suppression of immune cells, prevent and treat malignant tumors/solid tumors, and be used for clinical related prevention and treatment.
  • the present invention provides a genetically modified dual-target chimeric antigen receptor and its host cell .
  • the first technical problem to be solved by the present invention is to provide a genetically modified dual-target chimeric antigen receptor.
  • the dual-target chimeric antigen receptor can bind two different targets and deliver two Species signal.
  • the genetically modified dual-target chimeric antigen receptor of the present invention wherein the dual-target chimeric antigen receptor is connected by a chimeric antigen receptor 1 and a chimeric antigen receptor 2 capable of recognizing PD-L1 through a connecting peptide Made.
  • the chimeric antigen receptor 2 includes: a single-chain antibody of PD-L1, a transmembrane region, and an intracellular domain.
  • the single-chain antibody of PD-L1 refers to a single-chain antibody of PD-L1 capable of binding to PD-L1 molecules on the surface of tumor cells or immune cells.
  • transmembrane region is a CD8 transmembrane region.
  • the intracellular domain is 4-1BB intracellular domain.
  • the chimeric antigen receptor 2 is composed of: scFv of human PD-L1, CD8 transmembrane region, and 4-1BB costimulatory molecular peptide.
  • amino acid sequence of the chimeric antigen receptor 2 is shown in SEQ ID NO: 1.
  • coding nucleotide sequence of the chimeric antigen receptor 2 is shown in SEQ ID NO: 2:
  • the chimeric antigen receptor 1 includes: a single-chain antibody capable of binding tumor-specific antigen or tumor-associated antigen, a transmembrane region and an intracellular immune receptor tyrosine activation motif.
  • the tumor-specific antigen or tumor-associated antigen is at least one of CD19, CD20, MUC1, EGFR, EGFRvIII, HER2, ERBB3, ERBB4, VEGFR1, VEGFR2, EpCAM, CD44 or IGFR.
  • the single-chain antibody capable of binding tumor-specific antigen or tumor-associated antigen refers to a protein capable of binding EGFR family, including EGFR, HER2, ERBB3, ERBB4 or EGFRvIII, VEGFR1, VEGFR2, EpCAM, CD19, CD20, CD44 Single chain antibody.
  • the single-chain antibody is VEGFR1 scFv or HER2scFv.
  • the transmembrane region is at least one of CD28, CD8, CD3 ⁇ , CD134, CD137, ICOS, DAP10 or CD27 transmembrane region.
  • the transmembrane regions of the chimeric antigen receptors 1 and 2 are selected from different transmembrane regions. More preferably, the transmembrane region of the chimeric antigen receptor 1 is a CD28 transmembrane region, and the transmembrane region of the chimeric antigen receptor 2 is a CD8 transmembrane region.
  • the intracellular immunoreceptor tyrosine activation motif includes an immunoreceptor tyrosine activation motif signal chain selected from CD3 ⁇ or Fc ⁇ RI.
  • the above-mentioned chimeric antigen receptor 1 is composed of: a signal peptide, a scFv of human VEGFR1, a CD28 transmembrane region, and a CD3 ⁇ binding domain.
  • amino acid sequence of the signal peptide is shown in SEQ ID NO: 11:
  • nucleotide sequence of the signal peptide is shown in SEQ ID NO: 12:
  • amino acid sequence of the chimeric antigen receptor 1 is shown in SEQ ID NO: 3:
  • nucleotide sequence of the chimeric antigen receptor 1 is shown in SEQ ID NO: 4:
  • the above-mentioned chimeric antigen receptor 1 is composed of: a signal peptide, a scFv of HER2 of the human body, a CD28 transmembrane region, and a CD3 ⁇ binding domain.
  • amino acid sequence of the signal peptide is shown in SEQ ID NO: 11.
  • the nucleotide sequence of the signal peptide is shown in SEQ ID NO: 12.
  • amino acid sequence of the chimeric antigen receptor 1 is shown in SEQ ID NO: 5:
  • coding nucleotide sequence of the chimeric antigen receptor 1 is shown in SEQ ID NO: 6:
  • the connecting peptide is at least one of Furin or P2A.
  • the amino acid sequence of the connecting peptide P2A is shown in SEQ ID NO: 7, and the nucleotide sequence encoding the connecting peptide P2A is shown in SEQ ID NO: 8.
  • the amino acid sequence of the connecting peptide Furin is shown in SEQ ID NO: 9, and the nucleotide sequence encoding the connecting peptide Furin is shown in SEQ ID NO: 10.
  • SEQ ID NO: 8 The nucleotide sequence of the connecting peptide P2A:
  • the chimeric antigen receptor 1 and the chimeric antigen receptor 2 of the present invention are co-expressed by one vector.
  • the present invention also provides an expression vector.
  • the expression vector is an expression vector that simultaneously expresses the above chimeric antigen receptor 1 and chimeric antigen receptor 2.
  • the expression vector is a eukaryotic or prokaryotic expression vector, and the eukaryotic expression vector is a plasmid;
  • the prokaryotic expression vector is a viral vector, and the viral vector includes retrovirus, recombinant lentivirus, Recombinant adenovirus; further, the viral vector is pWPXLd.
  • the present invention also provides a host cell containing the above expression vector.
  • the host cell is an immune response cell.
  • they are T cells, monocytes, natural killer cells, and neutrophils. More preferably, they are T cells or natural killer cells.
  • the present invention also provides the use of the above-mentioned dual-target chimeric antigen receptor, recombinant vector containing the above-mentioned chimeric antigen receptor, and host cells containing the above-mentioned recombinant vector in the preparation of drugs for preventing or treating malignant tumor serous cavity metastasis .
  • the malignant tumor is a solid tumor, and may be lung cancer, hepatocellular carcinoma, colon cancer, rectal cancer, breast cancer, ovarian cancer, gastric cancer, cholangiocarcinoma, gallbladder cancer, esophageal cancer, and kidney cancer. , At least one of pancreatic cancer or prostate cancer.
  • the present invention can simultaneously express two antigen receptors in a host cell, one of which is a receptor that binds to a tumor-specific antigen or a tumor-associated antigen. It can produce a specific targeting effect.
  • the other antigen receptor is PD-L1 receptor, and it can also have a specific binding effect.
  • the dual-target specific binding form of the method of the present invention can be used to prepare drugs for the prevention and treatment of malignant tumor serous cavity metastasis, which provides a basis for the prevention and treatment of malignant tumor serous cavity metastasis.
  • FIG. 1 is a schematic diagram of the framework of the pro-antigen receptor of the present invention (wherein the variable region can be replaced with any single-chain antibody fragment);
  • FIG. 2 is a schematic diagram of a specific implementation manner of binding HER2 and binding PD-L1 dual-target CAR;
  • Figure 3 is a flow chart for measuring the expression of CAR molecules on the surface of 293T cells
  • Figure 4 is a flow chart for measuring the expression of CAR molecules on the surface of T cells
  • Figure 5 shows a flow diagram for the construction of stable cell lines
  • FIG. 6 shows the results of IFN- ⁇ killing of two CAR-T cells and control T cells in vitro
  • Figure 7 shows the killing effect of genetically modified T cells in vitro, and the ratio of viable negative cells to positive cells (T includes: T, HER2 CAR-T and HER2/PD-L1 CAR-T three cell types; k is short for k562, herk is short for k562-her2, and hlk is short for k562-her2-pdl1);
  • Figure 8 shows the results of the peritoneal implantation model of HER2 CAR-T and HER2/PD-L1 CAR-T treated SKOV3 ovarian cancer mice.
  • Recombinant vectors for constructing dual-target chimeric antigen receptors The expression framework is: 5 to 3 ends in order: HER2scFv-CD28 transmembrane region-CD3 ⁇ -Furin-P2A-PD-L1scFv-CD8 transmembrane region-4-1BB .
  • the amino acid sequence of the signal peptide of HER2 is shown in SEQ ID NO: 11, and the nucleotide sequence of the signal peptide of HER2 is shown in SEQ ID NO: 12.
  • amino acid sequence of HER2scFv is shown in SEQ ID NO: 13:
  • the coding nucleotide sequence of HER2 scFv is shown in SEQ ID NO: 14:
  • the amino acid sequence of the CD28 transmembrane region is shown in SEQ ID NO: 15:
  • the nucleotide sequence of the CD28 transmembrane region is shown in SEQ ID NO: 16:
  • the amino acid sequence of CD3 ⁇ is shown in SEQ ID NO: 17:
  • the nucleotide sequence of CD3 ⁇ is shown in SEQ ID NO: 18:
  • the coding nucleotide sequence of Furin-P2A is shown in SEQ ID NO: 20:
  • the amino acid sequence of the signal peptide of PD-L1 is shown in SEQ ID NO: 11: The nucleotide sequence of the signal peptide of PD-L1 is shown in SEQ ID NO: 12.
  • amino acid sequence of PD-L1scFv is shown in SEQ ID NO: 21:
  • the coding nucleotide sequence of PD-L1scFv is shown in SEQ ID NO: 22:
  • the amino acid sequence of the CD8 transmembrane region is shown in SEQ ID NO: 23:
  • the nucleotide sequence of the CD8 transmembrane region is shown in SEQ ID NO: 24:
  • amino acid sequence of 4-1BB is shown in SEQ ID NO: 25:
  • the nucleotide sequence of 4-1BB is shown in SEQ ID NO: 26:
  • Day 1 Paving. Digestion of 293T cells with a good density of 90%, passage 1:5, about 1.0x107cells/20ml/15cm plate, 5% CO2, 37°C overnight. 24h cell density is about 50-70% (not more than 70%).
  • Day 2 Transfection. Two hours before transfection, the cells were exchanged with pre-warmed 10% DMEM high glucose medium 20ml/dish. All reagents are equilibrated to room temperature. Transfection: a.
  • Centrifugation The centrifugation conditions are: 20°C, 70000g, 2h. Wait until the speed of the centrifuge rises to 7000g and then leave. After centrifugation, the culture medium is discarded, and the centrifuge tube is placed upside down on sterilized filter paper to suck up the remaining culture medium. Use the purchased PBS to resuspend the virus pellet. The amount of PBS in each centrifuge tube is determined according to your needs. Generally, each centrifuge tube is resuspended with 100ul. Finally, 100ul PBS is used to wash each centrifuge tube and suck it out again. Dispense the resuspended virus into small EP tubes and store in -80 degree refrigerator for use.
  • the green head covers the blood vessels for anticoagulation, usually 15-20ml of blood can be drawn at a time.
  • FICOLL lymphocyte separation liquid is slowly added dropwise to the drawn blood, and the ratio of lymphocyte separation liquid to blood is 1:1.
  • the mixture of lymphocyte separation liquid and blood is centrifuged. The conditions are 1000g, 45min, 32 degrees, and the acceleration/deceleration rate is 3. After centrifugation, you can see that the blood is divided into three layers, and the layer where the lymphocytes are located is the middle white transparent layer.
  • the tip of the gun slowly aspirates the lymphocytes in the middle white transparent layer. Do not aspirate the remaining two layers of liquid.
  • the absorbed lymphocytes were added to 20mL serum-free and antibiotic-free X-VIVO medium and centrifuged at 500g for 10 minutes. Discard the supernatant and resuspend the precipitated lymphocytes with 10 mL of sterile lysate. The lysing time should not exceed 5 minutes, 2-3 minutes. Centrifuge at 500g for 10min. Discard the supernatant and use 4 mL of 5% human fetal bovine serum, 2.5% IL-2 X-vivo medium, and X-VIVO medium with serum and IL-2. Resuspend the T lymphocytes, and then count the cells. Determine the amount of medium to be added to each well of the 6-well plate according to the number of cells. The volume of each well is generally 3 ⁇ 10 6 lymphocytes. However, the specific addition amount should be calculated according to the virus titer and the amount used to kill the experimental cells.
  • BSA bovine serum albumin
  • Target cell screening screening for HER2 and/or PDL1-positive tumor cell lines (see Figure 5) by flow cytometry antibody staining to construct stable target cells/cell lines.
  • the K562 cell line is a HER2-negative and PDL1-negative double-negative cell line.
  • the HER2 and/or PDL1 molecules are transferred to construct a K562 cell line that stably expresses the HER2 and/or PDL1 molecules (see Figure 5).
  • Fillco lymphocyte separation fluid
  • PBMC peripheral blood mononuclear cells
  • X-VIVO X-VIVO
  • concentration of PBMC should be controlled at 1-2 ⁇ 10 6 /mL as much as possible, and pipetted to resuspend.
  • Commercial human AB serum (sigma) was added at 5% of the total medium volume. Control the density of T cells to 1-2 ⁇ 10 6 /mL.
  • One well of a six-well plate can culture up to 3 ⁇ 10 6 T cells. If it exceeds, it will be distributed to 2 wells, and so on. Change the medium at least once every 48 hours. If the medium becomes obviously yellow, you can change the medium 24 hours and readjust the cell density.
  • T cells In the cultivation of T cells, observe every 24h to observe the morphology of clones, to understand the morphological changes of T cells, the tendency of apoptosis and senescence and whether there is fungal bacterial contamination. At the same time estimate the total number of T cells. Each time the medium is changed, serum and IL-2 need to be dissolved and matched immediately. For centrifugation of T cells, it is recommended to centrifuge a 5mL BD tube as the centrifuge tube at 1300rpm/min at room temperature for 3min.
  • Effector cells and target cells were stained with Cell Trace TM CFSE Cell Proliferation Kit (Thermo) and Cell Trace TM Far Red Cell Proliferation Kit (Thermo), respectively.
  • Add effector cells such as T cells and CAR-T cells
  • target cells such as SKOV3 and 293T cells
  • the number of cells per well is 1*10 6 cells, and a control well with only effector cells or target cells is added.
  • SKOV3 is the target cell
  • 293T is the negative cell of the control.
  • the death or proliferation of the target cell reflects the in vitro killing ability of CAR-T (see Figure 7).
  • Fig. 7 show that the in vitro killing ability of dual-targeted CAR-T cells of ERBB2 and PDL1 against tumor cells is significantly better than the in vitro killing ability of ERBB2's single-targeted CAR-T cells against the same tumor cells. The ability of simple T cells to kill the same tumor cells in vitro.
  • the amount of dilution is large, please add the same amount of sample and sample analysis buffer, and make up to 100ul with the standard dilution. Wash the plate 5 times, and pat it dry on thick absorbent paper for the last time. Add biotinylated antibody working solution (100ul/well). Seal the reaction well with sealing tape and incubate at room temperature for 60 minutes. Wash the plate 5 times, and pat it dry on thick absorbent paper for the last time. Add enzyme conjugate working solution (100ul/well). Seal the reaction well with sealing tape and incubate at room temperature for 20 minutes in the dark. Wash the plate 5 times, and pat it dry on thick absorbent paper for the last time. Add the developer TMB100ul/well and incubate at room temperature for 20 minutes in the dark.
  • the results of re-welling are only valid within 20% of the difference.
  • the average value of re-welling can be used as the measurement value; the OD value of each standard or specimen should be subtracted from the OD value of the background correction hole; manual Draw a standard curve.
  • the standard concentration is used as the abscissa, and the OD value is used as the ordinate, and the coordinate points of each standard are connected with a smooth line.
  • the concentration can be found on the standard curve; if the OD value of the sample is higher than the upper limit of the standard curve, it should be diluted and re-tested, and the concentration should be multiplied by the dilution factor (see Figure 6).
  • the results in FIG. 6 indicate that the in vitro killing ability of dual-targeted CAR-T cells of ERBB2 and PDL1 against tumor cells is significantly better than that of simple T cells against the same tumor cells in vitro.
  • HER2 CAR-T and HER2/PD-L1 CAR-T treatment mice SKOV3 ovarian cancer intraperitoneal implantation model.
  • Mouse SKOV3 ovarian cancer peritoneal model Female 8-12 week old NOD.Cg-PrkdcscidIl2rgtmWjl/SzJ (NSG) mice were selected. 5 ⁇ 10 5 FFLuc-GFP SKOV3 cells were injected intraperitoneally. After 10 days, 5 ⁇ 10 6 different types of CAR T cells were intraperitoneally injected; 7 days later, the second CAR-T intraperitoneal injection was performed. Tumor burden was measured by bioluminescence imaging using Xenogen IVIS imaging system (Xenogen) every 7 days after the first CAR-T injection. The obtained bioluminescence data was analyzed using Living Image software (Xenogen). It can be clearly seen from the results in FIG. 8 that the HER2/PDL1 dual-targeted CAR-T cells have a significantly better therapeutic effect on the mouse SKOV3 peritoneal model than the HER2CAR-T cells.
  • the present invention constructs a dual-target chimeric antigen receptor containing HER2 and PD-L1.
  • the experiment proves that the dual-target chimeric antigen receptor has a certain anti-tumor effect in vitro, and the in vitro test It has been verified to be highly effective and aimed to provide more effective treatments for malignant tumors.
  • the HER2 and PDL1 double-targeted CAR-T cells of the present invention can kill tumor cells in vitro, which is obviously superior to the HER2 single-targeted CAR-T cells in killing the same tumor cells in vitro, and even better than simple T cells. The ability to kill the same tumor cells in vitro.

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Abstract

一种基因工程修饰的双靶点嵌合抗原受体，表达上述双靶点抗原受体的宿主细胞，以及它们在预防或治疗恶性肿瘤/实体瘤中的用途。针对肿瘤细胞暴露于浆膜腔积液后出现的肿瘤细胞和免疫细胞的PD-L1表达上调这一现象，提供一种基因工程修饰的双靶点嵌合抗原受体及其宿主细胞。所述双靶点嵌合抗原受体会竞争结合到PD-L1，将PD-L1的抑制信号转化为激活信号，增强T细胞杀伤活性，同时在其下游引入的4-1BB能促进T细胞增殖、存活。

Description

基因工程修饰的双靶点嵌合抗原受体及其用途 技术领域
本发明属于基因工程领域，具体涉及一种基因工程修饰的双靶点嵌合抗原受体，本发明还涉及表达所述嵌合抗原受体的免疫反应细胞，以及所述抗原受体、免疫反应细胞在制备诊断治疗恶性肿瘤或实体瘤的药物中的用途。
背景技术
2013年，Science杂志将肿瘤的免疫治疗列为年度十大科技进展之首，其中CAR-T细胞治疗与CTLA-4、PD-1/PD-L1抗体治疗被认为肿瘤免疫治疗三大进展。利用抗原抗体scFv片段，联合T细胞胞内激活、增殖信号，构建嵌合型抗原受体(Chimeric antigen receptor，CAR)修饰的T细胞(CAR-T)，可使T细胞直接获得抗体的特异识别能力、成为不依赖HLA(人类白细胞抗原)限制的效应T细胞。获得的CAR-T细胞的杀伤活性主要依赖CAR-T细胞表面的单链受体识别抗原，具有特异的杀伤活性。临床研究证实这类CAR-T能够在体内扩增，长期存活并形成免疫记忆，即使对难治性血液肿瘤也显示出高效的抗肿瘤活性。遗憾的是，CAR-T在实体瘤的治疗中面临巨大挑战，至今在临床应用上进展缓慢。
CAR-T用于实体瘤的全身治疗，存在几大挑战：(1)多数实体瘤治疗靶点都是肿瘤相关抗原(Tumor-associated antigens,TAA)。由于TAA表达在正常组织，靶向TAA存在潜在毒性，采用CAR-T细胞靶向Her2、MART1、CAIX在临床治疗中都观察“on-target”毒性，提示针对TAA的全身CAR-T治疗需要慎重设计；(2)CAR-T细胞治疗实体肿瘤，CAR-T细胞通过血管进入实体瘤内部是发挥治疗效应的重要步骤，但是CAR-T常常难以穿过血管基底膜，影响治疗疗效；(3)实体瘤内部为抑制性的免疫抑制微环境，可分泌TGF-β，激活PD-1/PD-L1抑制信号，通过免疫抑制细胞MDSC和Treg等来抑制效应细胞活性。由此可知，将CAR-T用于全身CAR-T治疗目前还存在诸多困难。
我们认为，将CAR-T用于实体瘤的局部治疗在一定程度上可以回避上述挑战，在特定实体瘤患者的特定情况中发挥积极作用。在本研究中，我们拟将CAR-T用于浆膜腔输注(Serous cavity infusion)来预防和治疗恶性肿瘤的浆膜腔转移，探讨CAR-T在浆膜腔的抗肿瘤效应及其免疫机制。
恶性肿瘤，如肺癌、结肠癌、卵巢癌、乳腺癌、胃癌、淋巴瘤等肿瘤易发生胸腔、腹腔以及心包腔的浆膜腔转移，常常造成胸腔、腹腔扩散或者进一步合并恶性浆膜腔积液，危害极大。肿瘤患者胸腹腔发生转移合并胸腔或腹腔积液，常常难以耐受全身治疗，临床上即使采用引流+灌注化疗进行治疗，疗效仍然有限，呼吸困难、肠梗阻等并发症严重影响患者生理功能及生活质量，中位生存期常常只有3-6个月。此外，部分肿瘤患者术后常常仅仅表现为浆膜腔播散，如腹膜癌扩散(Peritoneal carcinomatosis，PC)，这些患者有条件通过接受肿瘤减灭术联合腹膜内热灌注化疗来显著延长生存率。但遗憾的是，即使接受减瘤术联合腹膜内热灌注化疗的患者，治疗后三年内约80％的患者会出现腹膜内复发，其原因在于不能有效清除腹腔内的肿瘤细胞。因此，对于这些浆膜腔转移患者，尽管局部治疗是目前主要治疗手段，但疗效有限。我们的前期研究发现：肿瘤细胞一旦暴露在胸腹腔的恶性积液中，就会发生上皮间质转化(Epithelial-mesenchymal transition,EMT)，并进一步产生高频率肿瘤干细胞(Cancer stem cell,CSC)，这群细胞高表达耐药蛋白ABCB1及ABCG2，产生治疗抵抗。
研究表明，CAR-T细胞对CSC也具有有效的杀伤活性。目前已有研究探讨CAR-T的局部应用，如在靶向Mesothelin(间皮素)的MSLNCAR-T在动物实验中采用胸腔局部输注治疗肿瘤证实了局部使用的有效性和安全性。因此，采用嵌合抗原受体(CAR-T)有望成为预防和治疗恶性肿瘤/实体瘤的浆膜腔转移的有效手段，但目前国内外对浆膜腔环境下CAR-T的应用研究极为有限。
采用CAR-T浆膜腔输注治疗，具备两大优势：一是安全，全身毒性小。CAR-T对肿瘤细胞具有特异杀伤活性，完全依赖于靶向的肿瘤抗原，也会杀伤表达抗原的正常组织。CAR-T在浆膜腔发挥杀伤作用，即使大规模增殖，不易大量进入血液循环，且易于局部处理，可消除潜在毒性。二是CAR-T细胞的治疗靶点得到延伸，更多的TAA可以作为治疗靶点。由于浆膜腔主要为结缔组织包裹，与上皮来源的肿瘤细胞表达谱差异较大，大量上皮源性的抗原可能成为潜在治疗靶点；更重要的是，CAR-T发挥作用，关键依赖抗原，不依赖肿瘤来源，CAR-T因而具有广谱杀瘤活性(对表达该抗原的多种肿瘤具备杀伤活性)，临床上也因此可以将浆膜腔转移作为独立适应症来进行治疗。
在浆膜腔环境中，肿瘤细胞和免疫细胞都会上调PD-L1来逃脱免疫攻击。研究发现，阻断PD-1/PD-L1信号能够增加CAR-T治疗疗效，但临床治疗成本昂贵，肿瘤细胞经过恶性积液处理，仍然会高表达VEGFR1或HER2。
针对肿瘤细胞在浆膜腔积液暴露后出现肿瘤细胞和免疫细胞的PD-L1表达上调这 一挑战，我们在前期基础上设计构建了同时靶向VEGFR1(或HER2)和PD-L1的双靶点CAR病毒载体。该发明以VEGFR1(或HER2)及PD-L1双靶向CAR-T(dual CAT-T)为例，说明包含PD-L1靶点的双靶点嵌合抗原受体能消除肿瘤细胞的免疫逃逸，解除免疫细胞的免疫抑制，预防和治疗恶性肿瘤/实体瘤，并用于临床相关预防与治疗。
发明内容
针对上述肿瘤细胞暴露于浆膜腔积液后出现的肿瘤细胞和免疫细胞的PD-L1表达上调这一现象，本发明提供一种基因工程修饰的双靶点嵌合抗原受体及其宿主细胞。
本发明要解决的第一个技术问题为：提供一种基因工程修饰的双靶点嵌合抗原受体，所述的双靶点嵌合抗原受体可结合两个不同的靶点，传递两种信号。
本发明基因工程修饰的双靶点嵌合抗原受体，所述的双靶点嵌合抗原受体由嵌合抗原受体1和能够识别PD-L1的嵌合抗原受体2通过连接肽连接而成。
其中，上述基因工程修饰的双靶点嵌合抗原受体中，所述的嵌合抗原受体2包括：PD-L1的单链抗体、跨膜区和胞内结构域。
进一步的，所述的PD-L1的单链抗体是指能够结合肿瘤细胞或免疫细胞表面PD-L1分子的PD-L1的单链抗体。
进一步的，所述跨膜区为CD8跨膜区。
进一步的，所述的胞内结构域为4-1BB胞内结构域。
其中，所述的嵌合抗原受体2组成为：人体的PD-L1的scFv、CD8跨膜区、4-1BB共刺激分子肽段。
具体的，所述的嵌合抗原受体2的氨基酸序列为SEQ ID NO：1所示。
Figure PCTCN2019117601-appb-000001
进一步的，所述的嵌合抗原受体2的编码核苷酸序列为SEQ ID NO：2所示：
Figure PCTCN2019117601-appb-000002
Figure PCTCN2019117601-appb-000003
其中，所述的嵌合抗原受体1包括：能够结合肿瘤特异性抗原或肿瘤相关抗原的单链抗体、跨膜区和胞内免疫受体酪氨酸活化基序。
其中，所述的肿瘤特异性抗原或肿瘤相关抗原为CD19、CD20、MUC1、EGFR、EGFRvIII、HER2、ERBB3、ERBB4、VEGFR1、VEGFR2、EpCAM、CD44或IGFR中的至少一种。
其中，所述的能够结合肿瘤特异性抗原或肿瘤相关抗原的单链抗体是指能够结合EGFR家族蛋白，包括EGFR、HER2、ERBB3、ERBB4或EGFRvIII、VEGFR1、VEGFR2、EpCAM、CD19、CD20、CD44的单链抗体。优选地，所述单链抗体为VEGFR1 scFv或HER2scFv。
其中，所述的跨膜区为CD28、CD8、CD3δ、CD134、CD137、ICOS、DAP10或CD27跨膜区中的至少一种。优选的，所述嵌合抗原受体1和2的跨膜区选择不不同的 跨膜区。更优选的，所述嵌合抗原受体1的跨膜区为CD28跨膜区，所述嵌合抗原受体2的跨膜区为CD8跨膜区。
其中，上述胞内免疫受体酪氨酸活化基序包含选自CD3δ或FcεRI的免疫受体酪氨酸活化基序信号链。
其中，上述嵌合抗原受体1组成为：信号肽、人体的VEGFR1的scFv、CD28跨膜区、CD3δ结合域。
所述信号肽的氨基酸序列为SEQ ID NO：11所示：
Figure PCTCN2019117601-appb-000004
所述信号肽的核苷酸序列为SEQ ID NO：12所示：
Figure PCTCN2019117601-appb-000005
进一步的，所述的嵌合抗原受体1的氨基酸序列为SEQ ID NO：3所示：
Figure PCTCN2019117601-appb-000006
进一步的，所述的嵌合抗原受体1的核苷酸序列为SEQ ID NO：4所示：
Figure PCTCN2019117601-appb-000007
Figure PCTCN2019117601-appb-000008
另一方面，上述嵌合抗原受体1组成为：信号肽、人体的HER2的scFv、CD28跨膜区、CD3δ结合域。
所述的信号肽的氨基酸序列为SEQ ID NO：11所示。
信号肽的核苷酸序列为SEQ ID NO：12所示。
进一步的，所述的嵌合抗原受体1的氨基酸序列为SEQ ID NO：5所示：
Figure PCTCN2019117601-appb-000009
Figure PCTCN2019117601-appb-000010
进一步的，所述的嵌合抗原受体1的编码核苷酸序列为SEQ ID NO：6所示：
Figure PCTCN2019117601-appb-000011
其中，所述的连接肽为Furin或P2A的至少一种。
连接肽P2A的氨基酸序列如SEQ ID NO:7所示，编码连接肽P2A的核苷酸序列如SEQ ID NO:8所示。连接肽Furin的氨基酸序列如SEQ ID NO:9所示，编码连接肽Furin的核苷酸序列如SEQ ID NO:10所示
SEQ ID NO:7 连接肽P2A的氨基酸序列：
Figure PCTCN2019117601-appb-000012
SEQ ID NO:8 连接肽P2A的核苷酸序列：
Figure PCTCN2019117601-appb-000013
SEQ ID NO：9 连接肽Furin的氨基酸序列：
Figure PCTCN2019117601-appb-000014
SEQ ID NO：10 连接肽Furin的核苷酸序列：
Figure PCTCN2019117601-appb-000015
本发明的上述嵌合抗原受体1和嵌合抗原受体2，由一个载体共同表达。
本发明还提供了一种表达载体，所述的表达载体为同时表达上述嵌合抗原受体1和嵌合抗原受体2的表达载体。进一步的，所述的表达载体为真核或原核表达载体，所述的真核表达载体为质粒；所述的原核表达载体为病毒载体，所述的病毒载体包括逆转录病毒、重组慢病毒、重组腺病毒；进一步的，所述的病毒载体为pWPXLd。
本发明还提供了一种含有上述表达载体的宿主细胞。优选的，所述宿主细胞为免疫反应细胞。优选为T细胞、单核细胞、自然杀伤细胞、中性粒细胞。更优选为T细胞或自然杀伤细胞。
本发明还提供了一种上述双靶点嵌合抗原受体、含有上述嵌合抗原受体的重组载体、含有上述重组载体的宿主细胞在制备预防或治疗恶性肿瘤浆膜腔转移药物中的用途。
进一步的，上述用途中，所述的恶性肿瘤为实体瘤，尤其可以为肺癌、肝细胞癌、结肠癌、直肠癌、乳腺癌、卵巢癌、胃癌、胆管癌、胆囊癌、食管癌、肾癌、胰腺癌或前列腺癌中的至少一种。
与现有技术相比，本发明的有益效果为：
本发明通过构建含有双靶点嵌合抗原受体表达单元的重组载体，能够在宿主细胞中同时表达两种抗原受体，其中一种抗原受体为结合肿瘤特异性抗原或肿瘤相关抗原的受 体，能够产生特异性靶向的作用，另一种抗原受体为PD-L1受体，也能够有特异性结合的作用，当两者共同存在于宿主细胞时，能够达到双靶点同时结合的效果，本发明方法的双靶点特异性结合形式，能够用于制备预防和治疗恶性肿瘤浆膜腔转移的药物，为预防和治疗恶性肿瘤浆膜腔转移提供了基础。
附图说明
图1所示为本发明前合抗原受体的构架简图(其中可变区可替换为任意一单链抗体片段)；
图2所示为结合HER2与结合PD-L1双靶点CAR的具体实施方式模式图；
图3所示为测293T细胞表面CAR分子表达的流式图；
图4所示为测T细胞表面CAR分子表达的流式图；
图5所示为构建稳定细胞株的流式图；
图6所示为两种CAR-T细胞与对照组T细胞体外杀伤的IFN-γ结果；
图7所示为基因工程修饰的T细胞体外杀伤作用结果，存活的阴性细胞与阳性细胞的比例图(T包括：T、HER2 CAR-T和HER2/PD-L1 CAR-T三种细胞类型；k是k562的简写，herk是k562-her2的简写，hlk是k562-her2-pdl1的简写)；
图8所示为HER2 CAR-T和HER2/PD-L1 CAR-T治疗小鼠SKOV3卵巢癌腹腔种植模型结果。
具体实施方式
以下通过具体实施方式结合附图对本发明进行详细说明。下述实施例中，凡未注明具体实验条件的，均为按照本领域技术人员熟知的常规条件，例如Sambrook J,Russell D.W.,2001,Molecular Cloning:A laboratory manual(3 rd ed),Spring Harbor Laboratory Press中所述的条件，或按照制造厂商所建议的条件。
实施例1 双靶点嵌合抗原受体的重组慢病毒载体的构建
构建双靶点嵌合抗原受体的重组载体，表达框架为：5端到3端依次为：HER2scFv-CD28跨膜区-CD3δ-Furin-P2A-PD-L1scFv-CD8跨膜区-4-1BB。
HER2的信号肽的氨基酸序列为SEQ ID NO：11所示，HER2的信号肽的核苷酸序列为为SEQ ID NO：12所示。
HER2scFv的氨基酸序列为SEQ ID NO：13所示：
Figure PCTCN2019117601-appb-000016
Figure PCTCN2019117601-appb-000017
HER2 scFv的编码核苷酸序列为SEQ ID NO：14所示：
Figure PCTCN2019117601-appb-000018
CD28跨膜区的氨基酸序列为SEQ ID NO：15所示：
Figure PCTCN2019117601-appb-000019
CD28跨膜区的核苷酸序列为SEQ ID NO：16所示：
Figure PCTCN2019117601-appb-000020
CD3δ的氨基酸序列为为SEQ ID NO：17所示：
Figure PCTCN2019117601-appb-000021
Figure PCTCN2019117601-appb-000022
CD3δ的核苷酸序列为SEQ ID NO：18所示：
Figure PCTCN2019117601-appb-000023
Furin-P2A的氨基酸为SEQ ID NO：19所示：
Figure PCTCN2019117601-appb-000024
Furin-P2A的编码核苷酸序列为为SEQ ID NO：20所示：
Figure PCTCN2019117601-appb-000025
PD-L1的信号肽的氨基酸序列为SEQ ID NO：11所示：PD-L1的信号肽的核苷酸序列为SEQ ID NO：12所示。
PD-L1scFv的氨基酸序列为SEQ ID NO：21所示：
Figure PCTCN2019117601-appb-000026
PD-L1scFv的编码核苷酸序列为SEQ ID NO：22所示：
Figure PCTCN2019117601-appb-000027
Figure PCTCN2019117601-appb-000028
CD8跨膜区的氨基酸序列为SEQ ID NO：23所示：
Figure PCTCN2019117601-appb-000029
CD8跨膜区的核苷酸序列为SEQ ID NO：24所示：
Figure PCTCN2019117601-appb-000030
4-1BB的氨基酸序列为为SEQ ID NO：25所示：
Figure PCTCN2019117601-appb-000031
4-1BB的核苷酸序列为SEQ ID NO：26所示：
Figure PCTCN2019117601-appb-000032
按上述序列合成双靶点嵌合抗原受体并插入到慢病毒pWPXLd载体(Invitrogen)BamH1-NdeI位点(见图2)，转化到大肠杆菌感受态细胞，经测序正确后，使用Qiagen公司的质粒纯化试剂盒提取并纯化质粒，纯化步骤参照试剂盒说明书，获得重组表达载体的高品质质粒，插入的目的片段结果见图1。
实施例2 重组载体转化细胞
1、293T细胞的培养和传代：
打开生物安全柜，用75％的酒精棉擦拭台面，并将移液器、移液枪、枪尖盒、15ml离心管、离心管架、10cm 2新的细胞培养皿放于生物安全柜中，关闭柜门，开启生物安全柜的紫外开关，照射半小时以消毒灭菌。将含10％的胎牛血清和100U/ml青霉素链霉 素的DMEM及胰酶放于37℃水浴锅中预热。打开生物安全柜，开启通风开关，将已长到80％-90％的293T细胞培养皿从37℃、5％的CO 2的培养箱中取出，放于生物安全柜。用75％的酒精消毒双手、培养基瓶瓶口、移液管筒口等。用无菌移液管吸尽培养皿中的培养基，弃于废液缸中。加入1ml胰酶简略冲洗掉平皿中残存的培养基，以中和胰酶抑制物，随后吸尽并移除。向培养皿中滴入1-2ml胰酶，镜下观察细胞，直到细胞变圆分离，吸除胰酶。向培养皿中加入6-8ml新鲜完全培养基，轻柔吹打下细胞。将细胞悬液分于其他培养皿，并添加培养基以达到每皿10ml。十字式晃动培养皿数次，摇匀细胞，镜下观察后放入37℃培养箱。24小时后观察细胞状态，待细胞长到80％-90％时进行下一次传代培养。
2、慢病毒原液的获取：
第一天:铺板。90％密度状态良好的293T细胞消化，1:5传代,约1.0x107cells/20ml/15cm平皿，5％CO2、37℃培养过夜。24h细胞密度约50-70％(不超过70％)。第二天：转染。转染前2小时，细胞换液--预热的10％DMEM高糖培养基20ml/皿。所有试剂平衡至室温。转染：a.在50ml BD管中配制以下DNA混合物(每15cm平皿)，psPAX2(packaging plasmid(包装质粒))22.5ug；pMD.2G(envelope plasmid(包膜质粒))11.25ug；pWPXLd(lentivirus vector(慢病毒载体))22.5ug。b.加水定容至1125ul。c.2.5M CaCl2 125ul滴入DNA溶液,涡旋5s。d、将BD管置于涡旋仪上(4档)，2×BBS(1250ul)溶液逐滴加入DNA-CaCl2混合液，震荡5s。e.室温静置15分钟。将2.25ml转染混合物滴入平皿，十字交叉(各10次)轻轻摇晃混匀，3％CO2，37°培养(12-16h)。吸去培养基，10ml PBS洗一次。换液--预热的5％DMEM培养基15ml，5％CO2，37°培养至48h。第四天：转染后48hr。收细胞上清，加预热的5％FBS新鲜DMEM培养基15ml，5％CO2，37°培养；病毒上清0.45μm滤器过滤，4℃保存(最多1周)。第五天：转染后72hr，收病毒上清，0.45μm滤器过滤，4℃保存。
3、慢病毒的浓缩：
仪器：超高速离心仪，配套的转子与套筒，超速离心管，配平用天平。将套筒及天平至生物安全柜紫外仪下消毒。确保每个套筒内无液滴后，将合适的离心管放至套筒里。将用0.45um的滤器过滤的病毒悬液加至离心管内。每个装了病毒悬液的离心管严格配平，使用精度为0.001g及以上的天平,盖上套筒盖后再次用天平验证是否完全配平。将配平好的各套筒装至离心仪转子里，准备离心。离心：离心条件为：20℃，70000g,2h.待离心仪速度上升至7000g再离开。离心完毕后，将培养基倒掉，离心管倒置在灭菌滤 纸上吸干剩余培养基。使用购买的PBS重悬病毒沉淀，每个离心管PBS量依据自己需要而定，一般每个离心管用100ul重悬，最后再用100ulPBS将各离心管洗一遍吸出。将重悬的病毒分装至小EP管里，-80度冰箱保存，待使用。
4、浓缩病毒液感染293T细胞后，293T细胞表面CAR分子表达的检测。
HER2-PDL1双CAR病毒上清液感染293Td细胞，6孔板铺293td细胞，收取48h病毒上清液，上清液：10％培养基＝1：1，各1ml，感染细胞，24h换2ml10％培养基，48h做流式，检测CAR的表达量(见图3)。
5、人外周血T淋巴细胞的分离：
抽取血液，绿头盖抗凝取血管，一般一次抽取15-20ml血液即可。FICOLL淋巴细胞分离液缓慢逐滴加入抽取血液中，淋巴细胞分离液与血液比例为1：1。淋巴细胞分离液与血液的混合液离心。条件是1000g,45min,32度，加/减速度为3。离心过后可以看到血液分为3层，而淋巴细胞所在层，为中间白色透明层。枪尖缓慢吸取中间白色透明层淋巴细胞，切勿吸取其余2层液体。吸取的淋巴细胞加入到20mL无血清无抗生素X-VIVO培养基中离心，500g，10min。倒掉上清，用10mL无菌裂红液重悬沉淀的淋巴细胞，裂红时间不超过5min，2-3min即可。离心500g，10min。倒掉上清，用4mL5％人胎牛血清，2.5％IL-2的X-vivo培养基，带血清和IL-2的X-VIVO培养基最好现配现用。重悬T淋巴细胞，之后进行细胞计数，根据细胞数量决定6孔板每孔加入培养基量，一般每孔的体积为3×10 6个淋巴细胞。但具体的加入量要根据病毒的滴度以及杀伤实验细胞所用量计算得到。
病毒感染T细胞前一天，用纤维连接蛋白(RetroNectin)稀释液包被病毒感染实验中将要使用的六孔板(Retronectin使用PBS稀释，浓度为50ug/ml)，稀释好后每2ml的retronectin包被六孔板的一个孔。4℃密封过夜备用。感染当天，吸去RetroNectin稀释液，使用2％BSA(牛血清白蛋白)溶液(PBS配制)封闭六孔板30min。吸去BSA，使用PBS润洗数次。(此步骤后，该六孔板可于4℃保存一周)。每孔预备1ml慢病毒悬液，混匀，加入六孔板中，32℃，1000g，2h离心。取出六孔板，吸去上清。PBS润洗一次。各孔加入2mL浓度为1.5×10 6cell/mL的PBMC细胞悬液，32℃，1000g，10min离心。37℃，5％CO 2细胞培养箱培养。48h后换液。慢病毒感染时，MOI值控制在4～40之间效果最佳。
6、浓缩病毒感染T细胞后，T细胞表面CAR分子表达的检测：
HER2-PDL1双CAR病毒上清液感染T细胞，6孔板铺293td细胞，收取48h病毒 上清液，上清液：10％培养基＝1：1，各1mL，感染细胞，24h换2mL10％培养基，48h做流式，检测CAR的表达量(见图4)。
靶细胞筛选：通过流式抗体分子染色，筛选HER2和/或PDL1阳性的肿瘤细胞系(见图5)构建稳定的靶细胞/细胞株。
K562细胞系为HER2阴性和PDL1阴性的双阴细胞系，通过慢病毒感染方式，转入HER2和/或PDL1分子，构建稳定表达HER2和/或PDL1分子的K562细胞系(见图5)。
CAR-T细胞的培养：
淋巴细胞分离液(Fillco)密度梯度离心分离出的外周血单核细胞，细胞技术板对其进行计数，获得细胞数总量，然后以1：1比率加入同数量相同的CD3/CD28磁珠(Gibco)。在有磁力架的情况下，在50mL的BD管里加入10mL左右X-VIVO培养基，然后加入经计数计算后的实际磁珠体积，轻吹重悬后，把BD管放入磁力架中静置3-4分钟，仅剩下纯磁珠吸附在BD管壁上。向BD管中加入用X-VIVO重悬了的PBMC(外周血单核细胞)。PBMC的浓度尽量控制在1-2×10 6/mL，用移液器吹打重悬。加入5％总培养基体积的商品化人AB血清(sigma)。控制T细胞的密度为1-2×10 6/mL，一个六孔板的一个孔可培养最高至3×10 6个T细胞，超过则分配到2孔中，依次类推。至少48小时换液一次，若培养基出现明显变黄的现象，可以24h换液，重新调整细胞密度。在T细胞的培养中，每24h观察一次，观察克隆形态，清楚T细胞的形态变化，凋亡衰老倾向以及是否存在真菌细菌污染。同时估算T细胞总量。每次换液的时候血清和IL-2都需要即时溶解即时配。T细胞的离心换液，离心推荐为5mLBD管作为离心管，室温下1300rpm/min，离心3min。
实施例3 HER2和PDL1的双靶向CAR-T细胞的性能测定
1、CAR-T细胞体外杀伤能力的测定。
用Cell Trace TM CFSE Cell Proliferation Kit(Thermo)和Cell Trace TM Far Red Cell Proliferation Kit(Thermo)分别对效应细胞和靶细胞进行染色。将效应细胞(如T细胞和CAR-T细胞)和靶细胞(如SKOV3和293T细胞)按效靶比1:1，2:1，4:1，8:1加入12孔版中，靶细胞在每孔的细胞数为1*10 6个，增设仅有效应细胞或靶细胞的对照孔。其中，SKOV3为靶细胞，293T为对照的阴性细胞，观察流式结果，靶细胞的死亡或增殖情况反应CAR-T的体外杀伤能力(见图7)。图7的结果，说明，ERBB2和PDL1的双靶向CAR-T细胞对肿瘤细胞的体外杀伤能力，明显优于ERBB2的单靶向CAR-T细胞 对相同肿瘤细胞的体外杀伤能力,更优于单纯T细胞对相同肿瘤细胞的体外杀伤能力。
2、CAR-T细胞体外杀伤能力，IFN-γ分泌量的测定。
使用IFN gamma Human ELISA Kit(Thermo)，通过计算并确定一次性实验所需的板条数，取出所需板条放置在框架内，暂时用不到板条请放回铝箔袋密封，保存于4℃。建议设置本底较正孔，即空白孔,设置方法为该孔只加TMB显色液和中止液。每次实验均需做标准品对照并画出标准曲线。分别将标本或不同浓度标准品(100ul/孔)加入相应孔中，用封板胶纸封住反应孔，室温孵育120分钟。对于血清或血浆标本，请加入50ul样本分析缓冲液后加50ul标本,如稀释量大，请将样本与样本分析缓冲液等量加入，不足部分用标准品稀释液补充至100ul。洗板5次，且最后一次置厚吸水纸上拍干。加入生物素化抗体工作液(100ul/孔)。用封板胶纸封住反应孔，室温孵育60分钟。洗板5次，且最后一次置厚吸水纸上拍干。加入酶结合物工作液(100ul/孔)。用封板胶纸封住反应孔，避光室温孵育20分钟。洗板5次，且最后一次置厚吸水纸上拍干。加入显色剂TMB100ul/孔，避光室温孵育20分钟。加入终止液50ul/孔,混匀后即刻测量OD450值。结果判断:复孔的值在20％的差异范围内结果才有效，复孔的值平均后可作为测量值；每个标准品或标本的OD值应减去本底校正孔的OD值；手工绘制标准曲线。以标准品浓度作横坐标，OD值作纵坐标，以平滑线连接各标准品的坐标点。通过标本的OD值可在标准曲线上查出其浓度；若标本OD值高于标准曲线上限，应适当稀释后重测，计算浓度时应乘以稀释倍数(见图6)。图6的结果，说明，ERBB2和PDL1的双靶向CAR-T细胞对肿瘤细胞的体外杀伤能力，明显优于单纯T细胞对相同肿瘤细胞的体外杀伤能力。
3、HER2 CAR-T和HER2/PD-L1 CAR-T治疗小鼠SKOV3卵巢癌腹腔种植模型。
小鼠SKOV3卵巢癌腹腔模型。选择雌性8-12周龄NOD.Cg-PrkdcscidIl2rgtmWjl/SzJ(NSG)小鼠。通过腹腔注射5×10 5FFLuc-GFP SKOV3细胞。10天后，腹腔注射5×10 6不同种类的CAR T细胞；7天后进行第2次CAR-T腹腔注射。从第1次CAR-T注射后每隔7天通过生物发光成像使用Xenogen IVIS成像系统(Xenogen)测量肿瘤负荷。使用Living Image软件(Xenogen)分析获取的生物发光数据。从图8的结果可以明显看出，HER2/PDL1双靶向CAR-T细胞对小鼠SKOV3腹腔模型的疗效明显优于HER2CAR-T细胞。
由上述试验结果可知：本发明构建了一种含有HER2和PD-L1的双靶点嵌合抗原受体，实验证明该双靶点嵌合抗原受体具有一定的体外抗肿瘤效应，且体外试验已经验证其有高效性，旨在为恶性肿瘤提供更有效的治疗方法。说明本发明的HER2和PDL1的 双靶向CAR-T细胞对肿瘤细胞的体外杀伤能力，明显优于HER2的单靶向CAR-T细胞对相同肿瘤细胞的体外杀伤能力，更优于单纯T细胞对相同肿瘤细胞的体外杀伤能力。

Claims (26)

  1. 基因工程修饰的双靶点嵌合抗原受体，其特征在于：所述的双靶点嵌合抗原受体由嵌合抗原受体1和能够识别PD-L1的嵌合抗原受体2通过连接肽连接而成。
  2. 根据权利要求1所述的双靶点嵌合抗原受体，其特征在于：所述的嵌合抗原受体2包括：PD-L1的单链抗体、跨膜区和胞内结构域。
  3. 根据权利要求2所述的双靶点嵌合抗原受体，其特征在于：满足下述至少一项；
    所述的PD-L1的单链抗体是指能够结合肿瘤细胞或免疫细胞表面PD-L1分子的PD-L1的单链抗体；
    所述跨膜区为CD8跨膜区；
    所述的胞内结构域为4-1BB胞内结构域。
  4. 根据权利要求3所述的双靶点嵌合抗原受体，其特征在于：所述的嵌合抗原受体2组成为：人体的PD-L1的scFv、CD8跨膜区、4-1BB共刺激分子肽段。
  5. 根据权利要求4所述的双靶点嵌合抗原受体，其特征在于：所述的嵌合抗原受体2的氨基酸序列为SEQ ID NO：1所示。
  6. 根据权利要求4所述的双靶点嵌合抗原受体，其特征在于：所述的嵌合抗原受体2的编码核苷酸序列为SEQ ID NO：2所示。
  7. 根据权利要求1所述的双靶点嵌合抗原受体，其特征在于：所述的嵌合抗原受体1包括：能够结合肿瘤特异性抗原或肿瘤相关抗原的单链抗体、跨膜区和胞内免疫受体酪氨酸活化基序。
  8. 根据权利要求7所述的双靶点嵌合抗原受体，其特征在于：所述的肿瘤特异性抗原或肿瘤相关抗原为CD19、CD20、MUC1、EGFR、EGFRvIII、HER2、ERBB3、ERBB4、VEGFR1、VEGFR2、EpCAM、CD44或IGFR中的至少一种。
  9. 根据权利要求7所述的双靶点嵌合抗原受体，其特征在于：所述的能够结合肿瘤特异性抗原或肿瘤相关抗原的单链抗体是指能够结合EGFR家族蛋白，包括EGFR、HER2、ERBB3、ERBB4或EGFRvIII、VEGFR1、VEGFR2、EpCAM、CD19、CD20、CD44的单链抗体。
  10. 根据权利要求9所述的双靶点嵌合抗原受体，其特征在于：所述的能够结合肿瘤特异性抗原或肿瘤相关抗原的单链抗体为VEGFR1scFv或HER2scFv。
  11. 根据权利要求7所述的双靶点嵌合抗原受体，其特征在于：所述的跨膜区为CD28、CD8、CD3ζ、CD134、CD137、ICOS、DAP10或CD27跨膜区中的至少一种。
  12. 根据权利要求1所述的双靶点嵌合抗原受体，其特征在于：所述嵌合抗原受体1和2的跨膜区不同。
  13. 根据权利要求12所述的双靶点嵌合抗原受体，其特征在于：所述嵌合抗原受体1的跨膜区为CD28跨膜区，所述嵌合抗原受体2的跨膜区为CD8跨膜区。
  14. 根据权利要求7所述的双靶点嵌合抗原受体，其特征在于：所述胞内免疫受体酪氨酸活化基序包含选自CD3ζ或FcεRI的免疫受体酪氨酸活化基序信号链。
  15. 根据权利要求7所述的双靶点嵌合抗原受体，其特征在于：所述嵌合抗原受体1为：人体的VEGFR1的scFv、CD28跨膜区和CD3ζ结合域。
  16. 根据权利要求15所述的双靶点嵌合抗原受体，其特征在于：所述嵌合抗原受体1的氨基酸序列为SEQ ID NO：3所示。
  17. 根据权利要求15所述的双靶点嵌合抗原受体，其特征在于：所述嵌合抗原受体1的编码核苷酸序列为SEQ ID NO：4所示。
  18. 根据权利要求7所述的双靶点嵌合抗原受体，其特征在于：所述的嵌合抗原受体1为：人体的HER2的scFv、CD28跨膜区和CD3ζ结合域。
  19. 根据权利要求18所述的双靶点嵌合抗原受体，其特征在于：所述嵌合抗原受体1的氨基酸序列为SEQ ID NO：5所示。
  20. 根据权利要求18所述的双靶点嵌合抗原受体，其特征在于：所述嵌合抗原受体1的编码核苷酸序列为SEQ ID NO：6所示。
  21. 根据权利要求1所述的双靶点嵌合抗原受体，其特征在于：所述的连接肽为Furin或P2A的至少一种。
  22. 根据权利要求1所述的双靶点嵌合抗原受体，其特征在于：所述的嵌合抗原受体1和嵌合抗原受体2，由一个载体共同表达。
  23. 同时表达权利要求1-22任一项所述的双靶点嵌合抗原受体的表达载体。
  24. 含有权利要求23所述的表达载体的宿主细胞。
  25. 权利要求1-22任一项所述的嵌合抗原受体、权利要求23所述的表达载体、权利要求24所述的宿主细胞在制备预防或治疗恶性肿瘤浆膜腔转移药物中的用途。
  26. 根据权利要求25所述的用途，其特征在于：所述的恶性肿瘤为实体瘤，为肺癌、肝细胞癌、结肠癌、直肠癌、乳腺癌、卵巢癌、胃癌、胆管癌、胆囊癌、食管癌、肾癌、胰腺癌或前列腺癌中的至少一种。
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