WO2017031863A1 - 双嵌合抗原受体修饰的t淋巴细胞及其制备方法 - Google Patents

双嵌合抗原受体修饰的t淋巴细胞及其制备方法 Download PDF

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WO2017031863A1
WO2017031863A1 PCT/CN2015/096861 CN2015096861W WO2017031863A1 WO 2017031863 A1 WO2017031863 A1 WO 2017031863A1 CN 2015096861 W CN2015096861 W CN 2015096861W WO 2017031863 A1 WO2017031863 A1 WO 2017031863A1
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chimeric antigen
antigen receptor
car
modified
lymphocyte
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姜舒
纪惜銮
张芸
罗朝霞
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深圳市茵冠生物科技有限公司
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464403—Receptors for growth factors
    • A61K39/464406—Her-2/neu/ErbB2, Her-3/ErbB3 or Her 4/ ErbB4
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    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
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    • A61K39/4631—Chimeric Antigen Receptors [CAR]
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    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
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    • A61K2239/27—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterized by targeting or presenting multiple antigens
    • A61K2239/29—Multispecific CARs
    • A—HUMAN NECESSITIES
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    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
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    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K2239/46—Indexing codes associated with cellular immunotherapy of group A61K39/46 characterised by the cancer treated
    • A61K2239/47—Brain; Nervous system

Definitions

  • the invention belongs to the field of biotechnology, and relates to a double chimeric antigen receptor modified T lymphocyte and a preparation method thereof.
  • Adoptive cellular immunotherapy refers to the infusion of autologous or allogeneic immune effector cells activated in vitro to patients to kill tumor cells in patients. It is one of the most important methods for the treatment of malignant tumors. Good results have been achieved in the clinical treatment of various solid tumors and hematological tumors.
  • chimeric antigen receptor (CAR)-modified T lymphocyte technology is a newly developed cell therapy technology, which can modify the effector T lymphocytes by genetic engineering technology to overcome the local immunosuppressive microenvironment and host of tumor. The immune tolerance state enhances anti-tumor targeting, killing activity and persistence.
  • CARs consist of an extracellular antigen binding region, a transmembrane region, and an intracellular signal transduction region.
  • the extracellular antigen binding region consists of a variable region derived from the light chain (VL) and heavy chain (VH) of the monoclonal antibody, and is joined by a hinge region with a toughness to form a single chain fragment variable (scFv).
  • CAR modifies the patient's T lymphocyte by gene transfection by genetically recombining the scFv and the intracellular signal domain "immunoreceptor tyrosine-based activation motifs (ITAM)" that recognize the tumor antigen in vitro.
  • ITAM immunoimmunoreceptor tyrosine-based activation motifs
  • the cells which allow the patient's T lymphocytes to express tumor antigen receptors, are purified and large-scale amplified modified T lymphocytes, called chimeric antigen receptor-modified T lymphocytes (CAR-T).
  • CAR-T lymphocyte technology has developed to the third generation.
  • the first generation of CAR is linked to the intracellular signaling region (ITAM) by a single-chain antibody through a transmembrane region, ITAM is usually CD3 ⁇ or Fc ⁇ RI ⁇ ;
  • ITAM intracellular signaling region
  • CM costimulatory molecule
  • CM1 and CM2 dual costimulatory molecules
  • CD134 or CD137 CD137.
  • the first generation of CAR-T lymphocytes were more studied, but most of the experiments were still insufficient in cell expansion, in vivo survival time, cytokine secretion, etc., and did not achieve the expected clinical results.
  • the first signal is a specific signal, and the TCR recognizes the antigen-peptide-MHC complex on the surface of the antigen-presenting cell.
  • the second signal is a costimulatory signal, which promotes IL-2 synthesis through important co-stimulatory molecules such as CD28/B7, and fully activates T lymphocytes and protects against apoptosis. Even if T lymphocytes are in contact with an antigen, it is difficult for cells to function normally if there is no costimulatory signal.
  • chimeric antigen receptors containing only the CD3 ⁇ sequence are also difficult to efficiently activate CAR-T lymphocytes. Therefore, according to the dual-signal theory of T lymphocyte activation, the second and third generation CARs add co-stimulatory molecules such as CD28 and CD137 to the chimeric antigen receptor to enhance the cytotoxicity and proliferative activity of T lymphocytes. T lymphocyte response, prolonging the survival time of T lymphocytes, and the like. Studies have confirmed that the second generation of CAR-T lymphocytes are superior to the first generation in both tumoricidal activity and survival time in vivo. At present, the clinical application of the third generation of CAR-T lymphocytes is still relatively small, and the structure, safety and effectiveness of the structure need further observation and optimization.
  • CAR-T lymphocytes The primary risk of clinical application of CAR-T lymphocytes is off-target effects, which can lead to autoimmune responses to normal tissues, mainly due to the fact that there are currently few known tumor-specific antigens, and most CARs are directed at important tissues that do not express or express. Fewer tumor-associated antigens. Therefore, how to improve the targeting of CAR-T lymphocytes is the primary problem facing clinical application.
  • the present invention constructs a low affinity chimeric antigen receptor (CAR-L) and a high affinity chimeric antigen receptor (CAR-H), respectively recognizes two tumor-associated antigens, and respectively contains a CD3 ⁇ sequence and a CM sequence, They are simultaneously transfected into T lymphocytes, and the modified T lymphocytes can be effectively activated only by simultaneously identifying two tumor-associated antigens, which enhances the targeting of CAR-T cells to kill tumors and reduces damage to normal tissues.
  • CAR-L low affinity chimeric antigen receptor
  • CAR-H high affinity chimeric antigen receptor
  • a first aspect of the invention provides a dual chimeric antigen receptor modified T lymphocyte (BiCAR-T) which expresses two chimeric antigen receptors on the surface.
  • BiCAR-T dual chimeric antigen receptor modified T lymphocyte
  • the chimeric antigen receptor CAR-L is a single-chain antibody targeting the EGBB2, a CD8 alpha hinge region and a transmembrane region, and a CD3 ⁇ sequence by splicing the amino terminus to the carboxy terminus.
  • the structure of the obtained chimeric antigen receptor CAR-L is ERBB2(scFv)-CD8 ⁇ -CD3 ⁇ , and the amino acid sequence thereof is shown in SEQ ID NO: 1, and the nucleotide sequence thereof is SEQ ID NO: :2 is shown.
  • the chimeric antigen receptor CAR-H is a single-chain antibody targeting EGFR, a CD8 alpha hinge region, a CD28 transmembrane region, and the like, which are sequentially spliced from the amino terminus to the carboxy terminus.
  • Intracellular region, and intracellular region of inducible co-stimulator (ICOS) The structure of the chimeric antigen receptor CAR-H is EGFR (scFv)-CD8 ⁇ -CD28-ICOS, and its amino acid sequence is shown in SEQ ID NO: 3, and its coding nucleotide sequence is shown in SEQ ID NO: 4.
  • the single-chain antibody targeting EGBB2 has low affinity for a targeting protein with a K D value > 2 nM; the single-chain antibody targeting EGFR has high affinity for a targeting protein with a K D value of ⁇ 0.5 nM.
  • a second aspect of the invention is the use of the dual chimeric antigen receptor-modified T lymphocytes and their associated chimeric antigen receptors for the preparation of anti-tumor drugs.
  • a third aspect of the present invention provides a method for preparing the double chimeric antigen receptor-modified T lymphocytes, and the specific steps are as follows:
  • steps (1) and (2) were sequentially loaded into the vector pCDH-CMV-MCS; thereby obtaining a lentiviral transfer vector containing the chimeric antigen receptor CAR-L gene fragment, designated as pCDH- ERBB2-CD3 ⁇ (CAR-L);
  • the lentiviral transfer vectors pCDH-EGFR-CD28-ICOS (CAR-H) and pCDH-ERBB2-CD3 ⁇ (CAR-L) were transfected into 293FT cells; the virus particles were obtained by packaging, and concentrated by centrifugation to obtain high titer. Lentiviral suspension;
  • the lentivirus suspension infects T lymphocytes to obtain a double chimeric antigen receptor-modified T lymphocyte (BiCAR-T).
  • the nucleotide sequence of the ERBB2scFv antibody with high specificity and low affinity for the extracellular ligand binding region RLD protein of ERBB2 was designed according to genetic engineering technology (nucleotide sequence is shown as SEQ ID No: 5); The restriction endonuclease EcoRI site was added to the end. Expression of the antibody by expression system ERBB2scFv, K D measured by ELISA on protein affinity value RLD 2.14nM.
  • the Hinge-TM-CD3 ⁇ expression cassette of the chimeric antigen receptor CAR-L was designed according to the CD8 ⁇ and CD3 ⁇ gene sequences in Genebank (NCBI), including the CD8 ⁇ hinge region and transmembrane region (aa135-205) and the CD3 ⁇ chain intracellular region ( Aa52-163), synthetic gene sequence. Primers were designed, and restriction endonucleases EcoRI and BamHI sites were added to both ends of the primers, and the above gene sequences were amplified by PCR. EcoRI and BamHI double-transgested lentiviral transfer plasmid pCDH-CMV-MCS were placed in a 37 ° C water bath for 4 h.
  • the PCR product purification kit was used to recover the column.
  • the Hinge-TM-CD3 ⁇ fragment was inserted into the vector gene fragment using Solution I ligase. All the ligation systems were added to DH 5 ⁇ competent cells, and coated on ampicillin-resistant LB plates. The cells were allowed to be absorbed for about half an hour, and placed in a 37 ° C incubator for inversion culture overnight. A single clone screened by Amp on the plate was picked and the ligation product was amplified according to the plasmid DNA mini-extraction kit protocol. The plasmid was extracted and identified by double digestion with EcoRI and BamHI, and the name was pCDH-CD3 ⁇ .
  • the plasmid pCDH-CD3 was digested with EcoRI and placed in a 37 ° C water bath for 4 h. After the reaction, the PCR product purification kit was used to purify and recover. 1 ⁇ L of FastAP was added to the digested product (dephosphorization of the cut vector end), and the reaction was carried out for 10 min in a 37 ° C water bath, and then the enzyme was inactivated in a 70 ° C electric heating dryer for 10 min, and the carrier fragment was purified by column purification. The ERBB2scFv fragment of interest was inserted into the vector gene fragment using Solution I ligase and ligated overnight at 4 °C.
  • the ligation system was placed in a 70 ° C water bath to inactivate the enzyme for 10 min, and cooled on ice.
  • the ligation product was transformed into DH5 ⁇ competent cells, which were subjected to ice bath for 30 min, heat shock at 42 ° C for 90 s, and immediately placed on ice for 5 min.
  • the bacteria solution was applied to ampicillin (Amp + ) antibiotic.
  • Amp + ampicillin
  • On a LB plate incubate at 37 ° C in an incubator overnight. The colony growth on the plate was observed the next day.
  • a single clone screened by Amp on the plate was picked and identified by colony PCR, and analyzed by 1% agarose gel electrophoresis to screen out the amplified bacteria with the expected band.
  • the filtered remaining aqueous lysate was added to 2.5 mL of LB liquid medium containing 2.5 ⁇ L of ampicillin, and shaken overnight to expand the culture. On the next day, a part of the bacterial liquid was stored as a 15% glycerol in a -100 ° C refrigerator. The remaining bacterial solution was extracted with a small amount of plasmid DNA extraction kit. The extracted plasmid was identified by DNA sequencing.
  • the lentiviral transfer vector containing the chimeric antigen receptor gene fragment was identified as pCDH-ERBB2-CD3 ⁇ (CAR-L); sequencing revealed that each gene fragment encoding CAR-L ERBB2scFv, hinge region, transmembrane The region and CD3 ⁇ sequence and linkage are correct, and the nucleotide sequence thereof is shown in SEQ ID NO: 2, and the encoded amino acid sequence is shown in SEQ ID NO: 1.
  • the nucleotide sequence of the EGFR scFv antibody with high specificity and high affinity for the PEP3-KLH antigen was designed according to genetic engineering technology (the nucleotide sequence is shown in SEQ ID No: 6); Endonuclease EcoRI site. Expression of EGFR scFv antibody by expression system, measured by ELISA on K D PEP3-KLH antigen affinity value of 0.47nM.
  • the CD8 ⁇ hinge region, the CD28 transmembrane region and the intracellular region, and the ICOS intracellular region gene sequence were designed and synthesized according to the CD8 ⁇ , CD28 and ICOS gene sequences in Genebank (NCBI). Primers were designed, and restriction endonucleases EcoRI and BamHI sites were added to both ends of the primers, and the above gene sequences were amplified by PCR.
  • the target fragment after the recovery of the gel was double-digested and placed in a 37 ° C water bath for 4 h to make the ends end with a sticky end. After the reaction, 4 ⁇ L of the sample was applied and analyzed by 1% agarose gel electrophoresis. Hinge-TM-CD28-ICOS target fragment with sticky ends.
  • the EcoRI and BamHI double-transgested lentiviral transfer plasmid pCDH-CMV-MCS was recovered by PCR using a PCR product purification kit.
  • the Hinge-TM-CD28-ICOS fragment was inserted into the vector gene fragment using Solution I ligase and ligated overnight at 4 °C.
  • the ligation system was added to an EP tube containing DH5 ⁇ competent cells, mixed and placed on ice for 30 min, heat shocked at 42 ° C for 90 s, quickly placed on ice for 5 min, and 600 ⁇ L of LB liquid medium (without antibiotics) was added.
  • the plasmid pCDH-CD28-ICOS was digested with EcoRI and purified by PCR using a PCR product purification kit. 1 ⁇ L of FastAP was added to the digested product (dephosphorization of the cut vector end), and the reaction was carried out for 10 min in a 37 ° C water bath, and then the enzyme was inactivated in a 70 ° C electric heating dryer for 10 min, and the carrier fragment was purified by column purification.
  • the EGFR scFv fragment of interest was inserted into the vector gene fragment using Solution I ligase and ligated overnight at 4 °C. The ligation system was placed in a 70 ° C water bath to inactivate the enzyme for 10 min, and cooled on ice.
  • the ligation product was transformed into DH 5 ⁇ competent cells, and subjected to ice bath for 30 min, heat shock at 42 ° C for 90 s, and immediately placed on ice for 5 min. After adding LB liquid medium, the bacteria solution was applied to ampicillin (Amp + ). The resistant LB plates were incubated overnight at 37 ° C in an incubator. The colony growth on the plate was observed the next day. A single clone screened by Amp on the plate was picked and identified by colony PCR, and analyzed by 1% agarose gel electrophoresis to screen out the amplified bacteria with the expected band.
  • the filtered remaining water-soluble bacterial solution was added to 2.5 mL of an LB liquid medium containing 2.5 ⁇ L of ampicillin, and shaken overnight to expand the culture. The next day, a portion of the bacterial liquid was stored as a 15% glycerol broth in a -100 ° C refrigerator. The remaining bacterial solution was extracted with a small amount of plasmid DNA extraction kit. The extracted plasmid was identified by DNA sequencing.
  • 293FT cells in good growth state were selected for subculture.
  • the medium was discarded and washed in serum-free DMEM medium, and 20 ⁇ L of liposome Lipofectamine TM 2000 was added to 500 ⁇ L of serum-free DMEM medium.
  • two recombinant plasmids CAR-L/CAR-H 15 ⁇ g, ⁇ 8.210 ⁇ g, and VSV-G 5 ⁇ g were quickly added, and the cells were incubated in a 293FT cell culture flask after standing for 20 minutes. After 6 hours, complete medium culture was replaced, and 48h fluorescence microscope was used. Under the observation, the virus supernatant was collected after 72 hours, and centrifuged at 5000 rpm for 10 min to remove floating cells and cell debris. The virus titer was detected by ELISA and stored at -80 °C.
  • PBMC peripheral blood mononuclear cells
  • the cells were washed with an appropriate amount of MACS buffer (according to 1.5 mL/10 7 PBMC), centrifuged at 800 r/min for 10 min to precipitate the cells, the supernatant was discarded, and the cells were resuspended in 500 ⁇ L of MACS buffer.
  • the MS separation column was placed on a MiniMACS separator, and the separation column was washed once with 500 ⁇ L of MACS buffer.
  • the cell suspension was added to the MS separation column, and the first flow of the cells was CD3-cells that were not labeled with magnetic beads.
  • the separation column was washed three times with 500 ⁇ L of MACS buffer, and the MS separation column was removed from the MiniMACS separator and placed on a 15 mL centrifuge tube.
  • T lymphocytes were activated according to the human T lymphocyte CD3/CD28 immune activation magnetic beads instructions.
  • the isolated CD3+ T lymphocytes were plated on a 24-well plate at 1 ⁇ 10 6 per well. 25 ⁇ L of pre-washed magnetic beads were added to each well, and recombinant human IL-2 was added to give a final concentration of 30 U/mL.
  • the 24-well plates were incubated in a 37 ° C, 5% CO 2 incubator. The medium containing recombinant human IL-2 was replaced every 2-3 days. Passage was performed according to cell growth density.
  • CD3+ T lymphocytes grow well and have a density of about 70%, infection can occur.
  • Polybrene was added to a 24-well plate to a final concentration of 4 ⁇ g/mL, while a lentivirus suspension CAR-L/CAR-H was added, and the fourth day was changed to continue culture and cryopreservation.
  • Lentivirus infection of the target cells by limiting dilution method, 96-well plates (80 cells/plate), 37 ° C, 5% CO 2 to grow into cell clones, select monoclonal cells to identify positive clones, ie obtain dual chimeric antigen receptors Modified T lymphocytes (BiCAR-T).
  • the experiment was divided into four experimental groups according to different target cells: blank control group (4T1 cells), ERBB2 group (4T1 cells transfected with ERBB2), EGFR group (4T1 cells transfected with EGFR), and ERBB2-EGFR group ( The 4T1 cells transfected with ERBB2 and EGFR were placed, and the effector cell control group and the target cell control group were set.
  • the effector cells BiCAR-T (prepared in Example 3) and the corresponding target cell densities of each group were adjusted to be 1 ⁇ 10 6 /mL and 1 ⁇ 10 7 /mL, respectively, and the target ratio was 5:1 in each experimental group.
  • the experimental grouping was the same as in Example 4, and each group was provided with 3 duplicate holes.
  • the antibody-labeled plate was taken out, and the TMB blank color-developing well was set, and 0.1 mL of the standard diluted with a certain number and the sample diluted with the sample diluent were sequentially added.
  • the plate was capped and reacted at 37 ° C for 90 min. After the reaction, the liquid in the microplate was removed by an automatic washer.
  • the biotin anti-human IFN- ⁇ antibody working solution was sequentially added in 0.1 mL per well (except TMB blank coloring well), reacted at 37 ° C for 60 min, and washed three times with 0.01 M PBS.
  • the ABC working solution was sequentially added in 0.1 mL per well (except for the TMB blank coloring hole), reacted at 37 ° C for 30 min, and washed 5 times with 0.01 M PBS.
  • TMB coloring solution was added in order of 90 ⁇ L per well, and the reaction was stopped at 20 ° C for 20-25 min at 37 ° C.
  • TMB stop solution was added in order of 0.1 mL per well.
  • the blue color turned yellow, and the OD value was measured at 450 nm with a microplate reader.
  • the OD value of the sample was subtracted from the OD value of the blank well, and the OD value and the standard concentration were plotted on the XY axis.
  • the IFN- ⁇ concentration was found on the standard curve and multiplied by the dilution factor to calculate the IFN- ⁇ concentration in the sample; the specific results are as follows:
  • Control group BiCAR-T cell group Comparative example 1 Comparative example 2 Tumor volume (mm 3 ) 1214 ⁇ 120 376 ⁇ 36 745 ⁇ 65 1094 ⁇ 99 Survival rate (%) 0 76.5% 37.8% 5.3%

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Abstract

提供了双嵌合抗原受体修饰的T淋巴细胞及其制备方法。构建了低亲和力嵌合抗原受体（chimeric antigen receptor, CAR-L）和高亲和力的嵌合抗原受体（CAR-H），分别识别两种肿瘤相关抗原，并且分别含有CD3ζ序列和共刺激分子信号序列（costimulatory molecule, CM）序列，将它们同时转染至T淋巴细胞中，修饰后的T淋巴细胞只有同时识别两种肿瘤相关抗原才能被有效激活，增强了CAR-T细胞杀伤肿瘤的靶向性，降低对正常组织的损伤。

Description

双嵌合抗原受体修饰的T淋巴细胞及其制备方法 技术领域
本发明属于生物技术领域，本发明涉及双嵌合抗原受体修饰的T淋巴细胞及其制备方法。
背景技术
过继性细胞免疫治疗(adoptive cellular immunotherapy,ACI)是指将体外激活的自体或异体免疫效应细胞输注给患者,以杀伤患者体内的肿瘤细胞，是目前治疗恶性肿瘤的重要手段之一，已在多种实体瘤和血液肿瘤的临床治疗中取得较好疗效。其中，嵌合抗原受体(chimeric antigen receptor，CAR)修饰的T淋巴细胞技术是新近迅速发展的一种细胞治疗技术，通过基因工程技术修饰效应T淋巴细胞，克服肿瘤局部免疫抑制微环境和宿主免疫耐受状态，提高了抗肿瘤的靶向性、杀伤活性和持久性。
目前，大多数CAR由胞外抗原结合区、跨膜区和胞内信号转导区组成。胞外抗原结合区由来源于单克隆抗体的轻链(VL)和重链(VH)的可变区组成，中间由带韧性的铰链区连接形成单链抗体(single chain fragment variable,scFv)。CAR通过将识别肿瘤抗原的scFv和胞内信号域“免疫受体酪氨酸活化基序(immunoreceptor tyrosine-based activation motifs，ITAM)”在体外进行基因重组，通过基因转染技术修饰患者的T淋巴细胞，使患者T淋巴细胞表达肿瘤抗原受体，经过纯化和大规模扩增修饰后的T淋巴细胞，称为嵌合抗原受体修饰的T淋巴细胞(CAR-T)。
目前CAR-T淋巴细胞技术发展到了第三代。第一代CAR由单链抗体通过跨膜区域与胞内信号传导区(ITAM)相连，ITAM通常为CD3ζ或FcεRIγ；第二代CAR的胞内信号转导区引入了共刺激分子(costimulatory molecule，CM)，主要为CD28分子；第三代CAR引入了双共刺激分子(CM1和CM2)，主要为CD28分子加上CD134或CD137等。第一代CAR-T淋巴细胞研究较多，但是大多数试验在细胞扩增、体内存活时间、细胞因子分泌等方面还存在不足,没有达到预期的临床效果。研究表明，T淋巴细胞的完全活化有赖于双信号和细胞因子的作用。其中第一信号为特异性信号，由TCR识别抗原递呈细胞表面的抗原肽-MHC复合物所 启动；第二信号为协同刺激信号，通过CD28/B7等重要的共刺激分子，促进IL-2合成，并使T淋巴细胞充分活化及免于凋亡。即使T淋巴细胞与抗原接触，如果没有协同刺激信号，细胞难以发挥正常功能。相应的，仅含有CD3ζ序列的嵌合抗原受体，如没有协同刺激信号2，也难以高效激活CAR-T淋巴细胞。因此，依照T淋巴细胞活化的双信号学说，第二和第三代CAR在嵌合抗原受体上加上如CD28、CD137等共刺激分子，以提高T淋巴细胞的细胞毒性、增殖活性，维持T淋巴细胞应答，延长T淋巴细胞存活时间等。研究证实第二代的CAR-T淋巴细胞在杀瘤活性和体内存活时间均优于第一代。目前第三代CAR-T淋巴细胞临床应用还比较少，其结构的构建、安全性和有效性还需进一步观察和优化。
CAR-T淋巴细胞临床应用的首要风险是脱靶效应，可导致针对正常组织的自身免疫反应，主要是由于目前已知的肿瘤特异性抗原较少，大多数CAR针对的是重要组织不表达或表达较少的肿瘤相关抗原。因此，如何提高CAR-T淋巴细胞的靶向性是目前临床应用面临的首要问题。
发明内容
本发明构建低亲和力嵌合抗原受体(chimeric antigen receptor，CAR-L)和高亲和力嵌合抗原受体(CAR-H)，分别识别两种肿瘤相关抗原，并且分别含有CD3ζ序列和CM序列，将它们同时转染至T淋巴细胞中，修饰后的T淋巴细胞只有同时识别两种肿瘤相关抗原才能被有效激活，增强了CAR-T细胞杀伤肿瘤的靶向性，降低对正常组织的损伤。
本发明的第一方面是提供一种双嵌合抗原受体修饰的T淋巴细胞(BiCAR-T)，所述T淋巴细胞表面表达两种嵌合抗原受体。
在本发明的一个实施方案中，所述嵌合抗原受体CAR-L是通过氨基端到羧基端顺次拼接所述的靶向EGBB2的单链抗体、CD8α铰链区及跨膜区、和CD3ζ链胞内区，所得到的嵌合抗原受体CAR-L的结构为ERBB2(scFv)-CD8α-CD3ζ，其氨基酸序列如SEQ ID NO：1所示，其编码核苷酸序列如SEQ ID NO：2所示。
在本发明的另一个实施方案中，所述嵌合抗原受体CAR-H是通过氨基端到羧基端顺次拼接所述的靶向EGFR的单链抗体、CD8α铰链区、CD28跨膜区及胞内区、和可诱导协同刺激分子(inducibleco-stimulator,ICOS)胞内区，所得到的 嵌合抗原受体CAR-H的结构为EGFR(scFv)-CD8α-CD28-ICOS，其氨基酸序列如SEQ ID NO：3所示，其编码核苷酸序列如SEQ ID NO：4所示。
在本发明中，所述靶向EGBB2的单链抗体对靶向蛋白具有低亲和力，KD值>2nM；所述靶向EGFR的单链抗体对靶向蛋白具有高亲和力，KD值<0.5nM。
本发明的第二方面是所述双嵌合抗原受体修饰的T淋巴细胞及其相关嵌合抗原受体在制备抗肿瘤药物中的应用。
本发明的第三方面是提供所述双嵌合抗原受体修饰的T淋巴细胞的制备方法，具体步骤如下：
(1)通过PCR方法获得编码人源ERBB2单链抗体的核苷酸片段；
(2)通过PCR方法获得编码CD8α铰链区及跨膜区和CD3ζ链胞内区的核苷酸片段；
(3)将步骤(1)和(2)获得核苷酸片段依次装入载体pCDH-CMV-MCS；从而获得含有嵌合抗原受体CAR-L基因片段的慢病毒转移载体，命名为pCDH-ERBB2-CD3ζ(CAR-L)；
(4)合成已知编码人源EGFR单链抗体的核苷酸片段；
(5)通过PCR方法获得编码CD8α铰链区、CD28跨膜区及胞内区、和ICOS胞内区的核苷酸片段；
(6)将步骤(4)和(5)获得核苷酸片段依次装入载体pCDH-CMV-MCS；从而获得含有嵌合抗原受体CAR-H基因片段的慢病毒转移载体，命名为pCDH-EGFR-CD28-ICOS(CAR-H)；
(7)将慢病毒转移载体pCDH-EGFR-CD28-ICOS(CAR-H)和pCDH-ERBB2-CD3ζ(CAR-L)转染至293FT细胞；包装获得病毒颗粒，经离心浓缩后获得高滴度的慢病毒悬液；
(8)慢病毒悬液感染T淋巴细胞从而获得双嵌合抗原受体修饰的T淋巴细胞(BiCAR-T)。
具体实施方式
下面将进一步地详细说明本发明。需要指出的是，以下说明仅仅是对本发 明要求保护的技术方案的举例说明，并非对这些技术方案的任何限制。本发明的保护范围以所附权利要求书记载的内容为准。
实施例1 CAR-L表达质粒载体的构建
根据基因工程技术设计对ERBB2的胞外配体结合区RLD蛋白高特异性和低亲和性的ERBB2scFv抗体的核苷酸序列(核苷酸序列如SEQ ID No：5所示)；且在两端加上限制性内切酶EcoRI位点。通过表达系统表达ERBB2scFv抗体，经ELISA测定其对RLD蛋白亲和力的KD值为2.14nM。
按照Genebank(NCBI)中CD8α及CD3ζ基因序列设计合成嵌合抗原受体CAR-L的Hinge-TM-CD3ζ表达框，包括CD8α铰链区和跨膜区(aa135-205)及CD3ζ链胞内区(aa52-163)，合成基因序列。设计引物，在引物两端分别加上限制性内切酶EcoRI、BamHI位点，PCR扩增上述基因序列。EcoRI、BamHI双酶切慢病毒转移质粒pCDH-CMV-MCS，置于37℃水浴中反应4h，反应结束后用PCR产物纯化试剂盒过柱回收。用Solution I连接酶将Hinge-TM-CD3ζ片段插入到载体基因片段中。将连接体系全部加入装有DH 5α感受态细胞，涂在氨苄青霉素抗性的LB平板上，正置约半小时待菌液基本被吸收，放入37℃培养箱倒置培养过夜。挑取平板上Amp筛选的单个克隆，按质粒DNA少量提取试剂盒操作说明扩增连接产物。提取质粒，用EcoRI、BamHI双酶切来鉴定，符合要求的命名为pCDH-CD3ζ。
EcoRI酶切质粒pCDH-CD3ζ，置于37℃水浴中反应4h，反应结束后用PCR产物纯化试剂盒过柱纯化回收。向酶切产物中加入1μL FastAP(使切开的载体末端去磷酸化)，37℃水浴中反应10min，然后置于70℃电热烘干机中灭活酶10min，过柱纯化回收载体片段。用Solution I连接酶将ERBB2scFv目的片段插入到载体基因片段中，4℃连接过夜。将连接体系放入70℃水浴灭活酶10min，置冰上冷却。将连接产物转化DH5α感受态细胞，经冰浴30min，42℃水浴热休克90s，再次立即置于冰上5min，加入LB液体培养基摇菌后，将菌液涂在氨苄青霉素(Amp+)抗性的LB平板上，37℃培养箱倒置培养过夜。次日观察平板上菌落生长情况。挑取平板上Amp筛选的单个克隆，进行菌落PCR鉴定，用1％琼脂糖凝胶电泳分析，筛选出扩增条带与预期相符的单克隆菌。将筛选出的单克隆余下的水溶菌液加到2.5mL含2.5μL氨苄青霉素的LB液体培养基中， 过夜摇菌，扩大培养。次日，保存一部分菌液为15％的甘油菌于-100℃冰箱中。剩余菌液用质粒DNA少量提取试剂盒提取质粒。将提取的质粒进行DNA测序鉴定。经鉴定符合要求的含有嵌合抗原受体基因片段的慢病毒转移载体，命名为pCDH-ERBB2-CD3ζ(CAR-L)；测序表明，编码CAR-L的各基因片段ERBB2scFv、铰链区、跨膜区和CD3ζ序列和连接正确，其核苷酸序列如SEQ ID NO：2所示，其编码氨基酸序列如SEQ ID NO：1所示。
实施例2 CAR-H表达质粒载体的构建
根据基因工程技术设计对PEP3-KLH抗原高特异性和高亲和性的EGFR scFv抗体的核苷酸序列(核苷酸序列如SEQ ID No：6所示)；且在两端加上限制性内切酶EcoRI位点。通过表达系统表达EGFR scFv抗体，经ELISA测定其对PEP3-KLH抗原亲和力的KD值为0.47nM。
根据Genebank(NCBI)中CD8α、CD28及ICOS基因序列设计合成CD8α铰链区、CD28跨膜区及胞内区、和ICOS胞内区基因序列。设计引物，在引物两端分别加上限制性内切酶EcoRI、BamHI位点，PCR扩增上述基因序列。将胶回收后的目的片段双酶切，置于37℃水浴中反应4h，使两端带有粘性末端，反应结束后取4μL上样经1％琼脂糖凝胶电泳分析，纯化获取两端带有粘性末端的Hinge-TM-CD28-ICOS目的片段。
EcoRI、BamHI双酶切慢病毒转移质粒pCDH-CMV-MCS，用PCR产物纯化试剂盒过柱回收。用Solution I连接酶将Hinge-TM-CD28-ICOS片段插入到载体基因片段中，4℃连接过夜。将连接体系加入装有DH5α感受态细胞的EP管中，混匀后置于冰上30min，42℃水浴热休克90s，迅速置于冰上5min，加入600μL LB液体培养基(不含抗生素)放入37℃水平摇床(180r/min)摇菌1h，6000r/min离心2min，弃去上清450μL，剩余的轻轻吹打混匀后涂在氨苄青霉素(Amp+)抗性的LB平板上，正置约半小时，放入37℃培养箱倒置培养过夜。挑取平板上Amp筛选的单个克隆扩增，提取质粒，用EcoRI、BamHI双酶切来鉴定，符合要求的命名为pCDH-CD28-ICOS。
EcoRI酶切质粒pCDH-CD28-ICOS，用PCR产物纯化试剂盒过柱纯化回收。向酶切产物中加入1μL FastAP(使切开的载体末端去磷酸化)，37℃水浴中反应 10min，然后置于70℃电热烘干机中灭活酶10min，过柱纯化回收载体片段。用Solution I连接酶将EGFR scFv目的片段插入到载体基因片段中，4℃连接过夜。将连接体系放入70℃水浴灭活酶10min，置冰上冷却。将连接产物转化DH 5α感受态细胞，经冰浴30min，42℃水浴热休克90s，再次立即置于冰上5min，加入LB液体培养基摇菌后，将菌液涂在氨苄青霉素(Amp+)抗性的LB平板上，37℃培养箱倒置培养过夜。次日观察平板上菌落生长情况。挑取平板上Amp筛选的单个克隆，进行菌落PCR鉴定，用1％琼脂糖凝胶电泳分析，筛选出扩增条带与预期相符的单克隆菌。将筛选出的单克隆余下的水溶菌液加到2.5mL含2.5μL氨苄青霉素的LB液体培养基中，过夜摇菌，扩大培养。次日，保存一部分菌液为15％的甘油菌液于-100℃冰箱中。剩余菌液用质粒DNA少量提取试剂盒提取质粒。将提取的质粒进行DNA测序鉴定。经鉴定符合要求的含有嵌合共刺激受体基因片段的慢病毒转移载体，命名为pCDH-EGFR-CD28-ICOS(CAR-H)，其核苷酸序列如SEQ ID NO：4所示，其编码氨基酸序列如SEQ ID NO：3所示。
实施例3T淋巴细胞转染及表达检测
选择生长状态良好的293FT细胞进行传代培养，当细胞生长达70％融合时，弃培养基并以无血清DMEM培养基冲洗，将20μL脂质体LipofectamineTM 2000加至500μL的无血清DMEM培养基，充分振荡后快速加入两种重组质粒CAR-L/CAR-H 15μg、△8.210μg、VSV-G 5μg，静置20min移至293FT细胞培养瓶中培养；6h后更换完全培养基培养，48h荧光显微镜下观察，72h后收集病毒上清，5000rpm低温离心10min，去除漂浮细胞及细胞碎片。ELISA法检测病毒滴度后置于-80℃保存。
抽取50mL健康志愿者的新鲜外周血，通过常规方法获得人外周血单个核细胞(PBMC)。用适量的MACS缓冲液(按1.5mL/107个PBMC)洗涤细胞一次，800r/min离心10min沉淀细胞，弃上清。用适量的MACS缓冲液重悬细胞(按80μL/107个PBMC)后，加入适量的anti human-CD3免疫磁珠(按20μL/107个PBMC)混匀，4℃孵育15min。再加入适量的MACS缓冲液洗涤细胞(按1.5mL/107个PBMC)，800r/min离心10min沉淀细胞，弃上清，用500μL MACS缓冲液 重悬细胞。将MS分离柱放入MiniMACS分离器上，用500μL MACS缓冲液洗涤分离柱一次。加入细胞悬液于MS分离柱内，先流出来的细胞是未被磁珠标记的CD3-细胞。用500μL MACS缓冲液洗涤分离柱三次，从MiniMACS分离器上取下MS分离柱，放到15mL离心管上。加入1mL MACS缓冲液至分离柱上，迅速将滞留的细胞洗脱下来，洗脱液即为分离的CD3+ T淋巴细胞。加入适量的MACS缓冲液，充分混匀后计数。1000r/min离心10min，弃上清。用含10％FBS的RPMI 1640培养基重悬，调整细胞浓度至1×106/mL于6孔板中。将培养板置于37℃、5％CO2培养箱中培养。
按照人T淋巴细胞CD3/CD28免疫激活磁珠说明书激活T淋巴细胞。将分离出的CD3+ T淋巴细胞以每孔1×106个铺于24孔板上。每孔加入25μL已预洗的磁珠，加入重组人IL-2使终浓度为30U/mL。将24孔板置于37℃、5％CO2培养箱中培养。每2-3d更换含有重组人IL-2的培养基。根据细胞生长密度进行传代。
当CD3+ T淋巴细胞生长状态良好，密度为70％左右时可进行感染。将Polybrene加入24孔板中至终浓度为4μg/mL，同时加入慢病毒悬液CAR-L/CAR-H，第四天换液继续培养并冻存。慢病毒感染目的细胞，经有限稀释法，铺96孔板(80cells/plate)，37℃、5％CO2至细胞克隆长出，挑选单克隆细胞鉴定阳性克隆，即获得双嵌合抗原受体修饰的T淋巴细胞(BiCAR-T)。
实施例4BiCAR-T体外杀瘤活性检测
实验根据不同的靶细胞分为四个实验组：空白对照组(4T1细胞)、ERBB2组(转染了ERBB2的4T1细胞)、EGFR组(转染了EGFR的4T1细胞)、ERBB2-EGFR组(转染了ERBB2和EGFR的4T1细胞)，设置效应细胞对照组和靶细胞对照组。调整效应细胞BiCAR-T(实施例3制备)和各组相应的靶细胞密度，分别为1×106/mL和1×107/mL，在各实验组中按效靶比5：1、10：1、20：1、40：1往96孔板加入效应细胞悬液和靶细胞悬液，总体积为200μL，放入37℃，5％C02培养箱中培养48h后每孔加入20uL CCK-8,继续孵育2h后上酶标仪检测，于450nm读取OD值，杀伤率＝【1-(实验组OD值-效应细胞对照组OD值)/靶 细胞对照组OD值】×100％。
具体结果如下：
杀伤率％ 空白对照组 ERBB2组 EGFR组 ERBB2-EGFR组
效靶比5：1 0.7±0.1 2.4±0.1 1.7±0.1 9.7±0.6
效靶比10：1 1.1±0.1 4.8±0.3 2.2±0.2 18.8±0.7
效靶比20：1 1.6±0.1 7.3±0.6 2.4±0.2 29.1±1.7
效靶比40：1 1.9±0.1 8.9±0.7 3.2±0.3 45.2±2.5
实施例5 BiCAR-T分泌IFN-γ的检测
实验分组同实施例4，每组设3个复孔。取出已包被抗体的酶标板，设置TMB空白显色孔，依次加入0.1mL按一定倍数稀释的标准品和用样品稀释液稀释的样品。酶标板加上盖，37℃反应90min。反应后用自动洗板机吸去酶标板内的液体。将生物素抗人IFN-γ抗体工作液按每孔0.1mL依次加入(TMB空白显色孔除外)，37℃反应60min，0.01M PBS洗涤3次。将ABC工作液按每孔0.1mL依次加入(TMB空白显色孔除外)，37℃反应30min，0.01M PBS洗涤5次。按每孔90μL依次加入TMB显色液，37℃避光反应20-25min，按每孔0.1mL依次加入TMB终止液，此时蓝色立转黄色，用酶标仪在450nm测定OD值。样品OD值减去空白孔OD值后以OD值及标准品浓度为XY轴绘图，在标准曲线上查找IFN-γ浓度后乘以稀释倍数，计算样本中IFN-γ浓度；具体结果如下：
ng/ml 空白对照组 ERBB2组 EGFR组 ERBB2-EGFR组
效靶比5：1 0.10±0.01 0.19±0.01 0.13±0.01 0.97±0.04
效靶比10：1 0.11±0.01 0.32±0.02 0.15±0.01 2.65±0.16
效靶比20：1 0.14±0.01 0.56±0.03 0.17±0.01 3.69±0.28
效靶比40：1 0.18±0.01 0.67±0.05 0.23±0.02 4.51±0.33
实施例6 BiCAR-T体内杀瘤活性检测
收集对数生长期的U251肿瘤细胞，计数细胞数量后调整细胞浓度，每只小鼠右侧背部皮下接种1×107个细胞。待肿瘤体积达到300-400mm3时，计数BiCAR-T (实施例3制备)浓度调整至2×108个/mL，尾静脉注射小鼠100μL/只，隔一天注射一次，共三次；对照组注射未进行双嵌合抗原受体修饰的T淋巴细胞。接种肿瘤30天后，用游标卡尺测量肿瘤的长与宽，计算肿瘤体积并统计生存率。
另外，设计以下对比例用于对比试验，具体如下：
对比例1：仅包含CAR-L(实施例1制备)的T细胞，给药量同实施例3制备的BiCAR-T细胞；
对比例2：仅包含CAR-H(实施例2制备)的T细胞，给药量同实施例3制备的BiCAR-T细胞；
具体结果如下：
  对照组 BiCAR-T细胞组 对比例1 对比例2
肿瘤体积(mm3) 1214±120 376±36 745±65 1094±99
生存率(％) 0 76.5％ 37.8％ 5.3％
本发明内容仅仅举例说明了要求保护的一些具体实施方案，其中一个或更多个技术方案中所记载的技术特征可以与任意的一个或多个技术方案相组合，这些经组合而得到的技术方案也在本申请保护范围内，就像这些经组合而得到的技术方案已经在本发明公开内容中具体记载一样。
Figure PCTCN2015096861-appb-000001
Figure PCTCN2015096861-appb-000002
Figure PCTCN2015096861-appb-000003
Figure PCTCN2015096861-appb-000004
Figure PCTCN2015096861-appb-000005
Figure PCTCN2015096861-appb-000006
Figure PCTCN2015096861-appb-000007
Figure PCTCN2015096861-appb-000008
Figure PCTCN2015096861-appb-000009
Figure PCTCN2015096861-appb-000010

Claims (9)

  1. 一种双嵌合抗原受体修饰的T淋巴细胞，所述T淋巴细胞表面表达双嵌合抗原受体。
  2. 根据权利要求1所述嵌合抗原受体修饰的T淋巴细胞，其特征在于，所述嵌合抗原受体CAR-L是通过氨基端到羧基端顺次拼接所述的靶向ERBB2的单链抗体、CD8α铰链区及跨膜区、CD3ζ链胞内区，所得到的嵌合抗原受体的结构为ERBB2(scFv)-CD8α-CD3ζ。
  3. 根据权利要求2所述嵌合抗原受体修饰的T淋巴细胞，其特征在于，所述嵌合抗原受体CAR-L的氨基酸序列如SEQ ID NO：1所示。
  4. 根据权利要求3所述嵌合抗原受体修饰的T淋巴细胞，其特征在于，所述嵌合抗原受体CAR-L的编码核苷酸序列如SEQ ID NO：2所示。
  5. 根据权利要求1所述嵌合抗原受体修饰的T淋巴细胞，其特征在于，所述嵌合抗原受体CAR-H是通过氨基端到羧基端顺次拼接所述的靶向EGFR的单链抗体、CD8α铰链区、CD28跨膜区及胞内区及ICOS胞内区，所得到的嵌合抗原受体的结构为EGFR(scFv)-CD8α-CD28-ICOS。
  6. 根据权利要求5所述嵌合抗原受体修饰的T淋巴细胞，其特征在于，所述嵌合抗原受体CAR-H的氨基酸序列如SEQ ID NO：3所示。
  7. 根据权利要求6所述嵌合抗原受体修饰的T淋巴细胞，其特征在于，所述嵌合抗原受体CAR-H的编码核苷酸序列如SEQ ID NO：4所示。
  8. 权利要求1-7任一项所述嵌合抗原受体修饰的T淋巴细胞在制备抗肿瘤药物中的应用。
  9. 一种制备权利要求1-7任一项所述嵌合抗原受体修饰的T淋巴细胞的方法，具体步骤如下：
    (1)通过PCR方法获得人源编码低亲和力ERBB2单链抗体的核苷酸片段；
    (2)通过PCR方法获得编码CD8α铰链区和跨膜区以及CD3ζ链胞内区的核苷酸片段；
    (3)将步骤(1)和(2)获得核苷酸片段依次装入载体pCDH-CMV-MCS；从而获得含有嵌合抗原受体CAR-L基因片段的慢病毒转移载体，命名为 pCDH-ERBB2-CD3ζ(CAR-L)；
    (4)通过PCR方法获得已知人源编码高亲和力EGFR单链抗体的核苷酸片段；
    (5)通过PCR方法获得编码CD8α铰链区、CD28跨膜区和胞内区以及ICOS胞内区的核苷酸片段；
    (6)将步骤(4)和(5)获得核苷酸片段依次装入载体pCDH-CMV-MCS；从而获得含有嵌合抗原受体CAR-H基因片段的慢病毒转移载体，命名为pCDH-EGFR-CD28-ICOS(CAR-H)；
    (7)将慢病毒转移载体pCDH-EGFR-CD28-ICOS和pCDH-ERBB2-CD3ζ转染至293FT细胞；包装获得病毒颗粒，经离心浓缩后获得高滴度的慢病毒悬液；
    (8)慢病毒悬液感染T淋巴细胞从而获得双嵌合抗原受体修饰的T淋巴细胞。
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