WO2020062794A1 - 一种mrfft1细胞 - Google Patents

一种mrfft1细胞 Download PDF

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WO2020062794A1
WO2020062794A1 PCT/CN2019/077924 CN2019077924W WO2020062794A1 WO 2020062794 A1 WO2020062794 A1 WO 2020062794A1 CN 2019077924 W CN2019077924 W CN 2019077924W WO 2020062794 A1 WO2020062794 A1 WO 2020062794A1
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cells
cell
mrfft1
antigen
tcr
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PCT/CN2019/077924
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French (fr)
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焦顺昌
张嵘
周子珊
宋玉洁
解佳森
张天赋
王海燕
袁翰
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北京鼎成肽源生物技术有限公司
焦顺昌
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Priority to US16/630,794 priority Critical patent/US20210071142A1/en
Priority to EP19856461.9A priority patent/EP3858998A1/en
Publication of WO2020062794A1 publication Critical patent/WO2020062794A1/zh

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  • the invention belongs to the field of biotechnology, and particularly relates to an MRFFT1 cell and a preparation method thereof.
  • CAR- T cells also have deficiencies in safety and treatment of solid tumors.
  • DC cells are specifically modified to produce specific killing by DC-presenting T cells.
  • Some laboratories are trying to use viruses as vectors to transfect and present T cells, and induce specific killing of T cells.
  • Some laboratories use TCR-T technology to target and present MAGE A3 antigen.
  • the target is single (MAGE-3), which is effective only for individual cancer species such as non-small cell lung cancer.
  • MAGE-3 single
  • the safety and convenience are not as good as the peptide method.
  • the direct stimulation of simple mixed peptides although simple and convenient, is less efficient.
  • the secondary stimulation of specific precise peptides is not as direct as tumor-specific antigens transduced by T cell receptors.
  • Existing TCR-T solutions for hematological and solid tumors lack accurate TCRs that cover more tumor types.
  • the present invention uses human peripheral blood for ctDNA sequencing or tumor tissue for whole exon sequencing, screens out mutation sites for antigen epitope prediction, connects and synthesizes mutant polypeptide expression gene sequences; simultaneously constructs MVA virus vectors to package MVA viruses , Transfect APC cells, complete the transformation of specific MV cells, co-culture with PBMC isolated from peripheral blood in vitro, screen out effective peptides, and transform ordinary T cells into The precise killing ability of RFF cells was modified by using the TCR-T technology principle. The modified T cells were then knocked out of the immunosuppressive targets by gene editing technology to precisely protect the specific killing T cells from Intrareceptor inhibition increases the lethality of T cells to tumor cells.
  • the MRFFT1 cell provided by the present invention can be widely used for individualized and precise treatment of solid tumors.
  • MRFFT1 cells that is, cells obtained through the above technical solutions or technical means of M, R, FF, T, and 1.
  • RNA mutations 1) Use human peripheral blood for ctDNA sequencing or commercially available engineered cell lines (such as H1299, H226, H358, H1563, H2228, A549, Renca, LLC mouse Lewis lung cancer cells, CRL-6323B16F1, CRL-2539 4T1, U14 small Mouse cervical cancer cells, BV-2 mouse microglioma cells, G422 mouse glioma cells, etc.) MHC type detection and full exon sequencing to detect RNA mutations;
  • the ligated polypeptide is reduced to a nucleic acid sequence and codon optimized
  • the gene sequence of the epitope peptide is synthesized by solid-phase synthesis method; or synthesized by a technical service company;
  • T cells obtained by the above scheme are collected by centrifugation, and the polypeptide is used as an antigen to directly stimulate the T cells to screen for accurate peptides:
  • the experimental group that is significantly larger than the negative control group is an effective precise peptide
  • CD8 CD137, IFN- ⁇ staining of T cells after stimulation, and sorting by flow cytometry;
  • Cell surface inhibitory signal molecules include: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, 2B4 (CD244);
  • Tumor antigen is a mutant antigen, which is different from other tissues, has strong target specificity, is not prone to off-target effects, and has high safety;
  • the ratio of specific cells obtained is high, and it is usually able to identify tumor antigen-specific cells.
  • the distribution in PBMC is less than 0.5%.
  • Cells transformed by the MRFFT1 protocol have a tumor antigen-specific T cell (TCR +) ratio of 70. %the above;
  • MRFFT1 cells knock out immunosuppressive targets such as PD1, CTLA4, TIM3, and LAG3, the killing ability of tumors is not limited, and the killing efficiency is higher.
  • Figure 1 APC efficiency test of MVA virus transfection; where 1A: control group, 1B: transfection group.
  • FIG. 1 MFF cell typing.
  • Figure 4 Flow cytometric detection of specific T cell proportions; 4A: control group, 4B: MRFF protocol.
  • FIG. 6 Knockout of inhibitory targets; 6A: after knockout, 6B: before knockout.
  • Figure 7 Detection of knockout efficiency of the original TCR; 7A: after knockout, 7B: before knockout.
  • FIG. 8 Expression efficiency of specific TCRs; 8A: 7 days after transfection, 8B: before transfection.
  • Figure 10 ELISA for cytokine IFN- ⁇ release.
  • Figure 11 Survival curves of animal tumor-bearing models.
  • IC50 ⁇ 1000nM is considered weak immunogenic and can be connected; IC50 ⁇ 1000nM is considered strong immunogenic and cannot be connected (here should be Considering the IC50 calculation results of 3 prediction softwares, it can be regarded as weak immunogenicity when the IC50 calculated by ⁇ 2 softwares is ⁇ 1000nM, and it can be considered as strong immunogenicity when the IC50 calculated by ⁇ 2 softwares is ⁇ 1000nM);
  • the antigenic epitopes are linked together, and the IC50 at the linker is higher than the IC50 of the antigenic epitopes on both sides (that is, try to avoid strong binding antigens at the linker); if necessary, use weak immunogenic peptides as Linker peptides space strong immunogenic peptides; or add patient's own amino acids to the linker to reduce the possibility of producing strong antigens.
  • the ligated polypeptide is reduced to a nucleic acid sequence and codon optimized
  • the amino acid sequence can be appropriately repeated. However, when reverting to the gene sequence, care should be taken to avoid the occurrence of reverse, direct and mirror repeats in the gene sequence.
  • CEF cells dry embryo fibroblasts
  • BHK-21 cells hamster kidney fibroblasts
  • CEF cells dry embryo fibroblasts
  • BHK-21 cells hamster kidney fibroblasts
  • MVA transfected with shuttle plasmid
  • Monolayer cells the monolayer cells were frozen and thawed to obtain rMVA solution
  • the rMVA solution was seeded into RK-13 cells (rabbit kidney cells) and cultured to obtain rMVA-infected RK-13 aggregation points, and the aggregation points were selected for screening
  • Purify to obtain the required rMVA remove the wtMVA in the rMVA, culture the rMVA in the monolayer of CEF or BHK-21 cells, select the rMVA that does not contain the selection gene K1L, and purify to obtain the rMVA-antigen epitope peptide.
  • rMVA-epitope peptide the required rMVA
  • the culture medium is OKM200 + 5% FBS, and cultured for 10 to 14 days can obtain T cells obtained by the secondary impact of precise peptides, that is, MRFF cells;
  • CD8 + CD137 +, or CD8 + IFN- ⁇ + cells CD8 + CD137 +, or CD8 + IFN- ⁇ + cells
  • Cell surface inhibitory signal molecules include: PD-1, Tim-3, LAG3, CTLA-4, BTLA, VISTA, CD160, 2B4 (CD244);
  • step 10 to complete the construction of the TCR knockout vector and virus packaging
  • the CD8 + T cells are cultured in the culture medium for 0-5 days, preferably 3 days, and then transferred into the constructed TCR expression vector;
  • the cells were transferred from the 175cm 2 to 75cm 2 flasks to large bottles OKM-200 + 5% FBS;
  • TCR-T which is a knockout immunosuppressive signal molecule, can be harvested, that is, MRFFT1 cells.
  • NGS mice were inoculated with tumor cell lines stably overexpressing specific antigen peptides to make ectopic tumor-bearing animal models.
  • 5 ⁇ 10 5 tumor cells expressing specific antigens were suspended in 100 ⁇ l of physiological saline and injected subcutaneously into the right flank of 30 NSG mice, respectively, and the mice were numbered at the same time.
  • the cells are retransfused in groups.
  • the animal model is randomly divided into three groups, each group has 5-6 mice, one group is given a placebo saline, and the other is given a group.
  • T cells (control group) without any genetic manipulation 1 ⁇ 10 7 one group was given MRFFT1 cells 1 ⁇ 10 7 , and the second injection was performed 7 days after the first injection of cells, and the cells were injected for the third time after 7 days. Days, statistics of survival data, drawing survival curves.
  • Table 1 shows the prediction results of the mutation sites and epitopes detected by sequencing.
  • CD8 + T cells were 68% and CD4 + T cells were 9.45%.
  • the selected peptide 3 was used to stimulate MFF protocol cells, and the proportion of T cells specific to the precise peptide was detected by flow cytometry.
  • the results are shown in Figure 4.
  • the black boxes are specific T cells: MRFF protocol cells.
  • the proportion of IFN- ⁇ -releasing cells was significantly higher than that of non-stimulated cells (control), indicating that the MRFF protocol can obtain specific T cells for precise polypeptides; meanwhile, CD8 + IFN- ⁇ + cells were performed by flow cytometry (In black box) sorting;
  • TCR lentivirus genome extraction and TCR sequencing.
  • the distribution of TCR is shown in Figure 5 (top 22 of the high-frequency distribution).
  • the frequency of TCR1 and TCR17 is higher, indicating that this TCR is closely related to the mutant antigen.
  • TCR was amplified to construct a lentivirus expression vector.
  • the horizontal line is the CDR3 sequence, which needs to be replaced
  • the horizontal line is the replacement CDR3 sequence
  • sgRNA1 has the highest knockout efficiency and is effective.
  • the original TCR on PBMC was knocked out, and the result is shown in Figure 7:
  • the original TCR expression can be effectively reduced.
  • expression-specific TCR lentivirus transfection can be performed.
  • PBMCs were transfected with lentivirus specific for TCR packaging. On day 7, the expression efficiency of TCR was detected by flow cytometry. The results are shown in Figure 8. The constructed TCR can be expressed normally, and the TCR + cell ratio is 25.1%.
  • Control cells and MRFFT1 cells were used to detect the killing efficiency of target cells derived from mutant epitopes, and untreated cells were used as controls (Mock). The results are shown in FIG. The cells have a strong killing effect.
  • IFN- ⁇ is one of the most important cytokines in antitumor effects.
  • Figure 10 shows The detection of IFN- ⁇ released when MRFFT1 cells were co-cultured with tumor cells showed that compared with IFN- ⁇ produced by effector cells (T cells), after co-culture with tumor cells, MRFFT1 cells can produce a large number of This result of IFN- ⁇ is consistent with the results of the killing experiment. It shows that T cells expressing specific TCR, combined with the knockout of inhibitory targets, can more effectively improve the anti-tumor ability.
  • the first course of treatment MRFFT1 cells once a month, the number of 1 ⁇ 10 9 cells, a total of 2 times;
  • the second course of treatment MRFFT1 cells every six months, the number of 1 ⁇ 10 9 cells, a total of 2 times;

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Abstract

本发明涉及一种MRFFT1细胞及其制备方法，通过抗原表位预测筛选出突变多肽，连接并合成突变多肽表达基因序列；同时构建MVA病毒载体，包装MVA病毒，转染APC细胞，完成特异性MV细胞的改造，体外与从外周血中分离的PBMC共培养，筛选出有效多肽，通过精准有效多肽刺激的第二次冲击，将普通T细胞改造成为具有更精准杀伤能力的RFF细胞，再利用TCR－T技术原理进行了改造，改造后的T细胞再用基因编缉技术对其进行免疫抑制靶点的敲除，精准的保护了特异性杀伤T细胞免受体内抑制，提高了T细胞对肿瘤细胞的杀伤力。

Description

一种MRFFT1细胞 技术领域
本发明属于生物技术领域，具体涉及一种MRFFT1细胞及其制备方法。
背景技术
目前，在肿瘤的特异性免疫治疗方面，现有的LAK、DC、CIK、DC-CIK细胞和方法基本被证明是无效的，而NK、CAR-NK、TIL等细胞技术还有待成熟，CAR－T细胞在安全性和实体瘤治疗中还有缺陷。现有技术一般通过改造DC细胞，由DC递呈T细胞产生特异杀伤。有些实验室在尝试用病毒做为载体的方法进行转染递呈T细胞，诱导T细胞的特异性杀伤。我们也曾用突变混合多肽直接刺激PBMC，诱导T细胞。还有实验室利用TCR-T技术，靶向递呈MAGE A3抗原。
以上治疗方法并不成熟，尤其是体外诱导DC细胞及DC细胞负载肿瘤抗原技术理论上研究较多，但在具体实施过程中还有许多问题，缺乏明确的、肿瘤细胞发生发展关键的信号传导通路相关分子作为诱导抗原，因为肿瘤抗原不明及肿瘤微环境免疫抑制的障碍，使实现特异性细胞靶向免疫治疗难以顺利实施。另外，有的虽然进行了抗原体外冲击，但没有进行体外共培育和体外扩增，让较为单薄的特异性细胞直接面对复杂的肿瘤免疫微环境，因此，很难起到预期的效果。也有的虽然也可以体外递呈和共培育，但靶点单一(MAGE-3)，仅对非小细胞肺癌等个别癌种起效。虽然也有尝试慢毒为载体的方法进行转染递呈，但安全性、方便性不如多肽方式。而简单混合多肽的直接刺激，虽然简单方便，但效率较低。特异性精准多肽的二次刺激不如T细胞受体转导的肿瘤特异性抗原更直接。现有的TCR-T在治疗血液肿瘤和实体肿瘤的解决方案中，缺乏覆盖更多瘤种的精准的TCR。
上述方案均没有考虑T细胞的自我防护技术，使得数量不多的特异性T细胞直接面对强大的肿瘤免疫微环境。
发明内容
本发明通过使用人源外周血进行ctDNA测序或肿瘤组织进行全外显子测序，筛选出突变位点进行抗原表位预测，连接并合成突变多肽表达基因序列；同时构建MVA病毒载体，包装MVA病毒，转染APC细胞，完成特异性MV细胞的改造，体外与从外周血中分离的PBMC共培养，筛选出有效多肽，通过精准有效多肽刺激的第二次冲击，将普通T细胞改造成为具有更精准杀伤能力的RFF细胞，再利用TCR－T技术原理进行了改造，改造后的T细胞再用 基因编缉技术对其进行免疫抑制靶点的敲除，精准的保护了特异性杀伤T细胞免受体内抑制，提高了T细胞对肿瘤细胞的杀伤力。
本发明提供的MRFFT1细胞可广泛应用于个体化精准治疗实体肿瘤。
对于专用名词的解释：
M：MVA病毒转染技术
R：精准多肽二次冲击技术
FF：混合多肽技术
T：TCR-T技术
1：靶点敲除防护技术
例如：MRFFT1细胞，即经由上述M、R、FF、T、1各项技术方案或技术手段改造而获得的细胞。
MRFFT1细胞改造方案：
1、抗原表位预测
1)使用人源外周血进行ctDNA测序或市售工程细胞系(如H1299、H226、H358、H1563、H2228、A549、Renca、LLC小鼠Lewis肺癌细胞、CRL-6323B16F1、CRL-2539 4T1、U14小鼠子宫颈癌细胞、BV-2小鼠小胶质瘤细胞、G422小鼠神经胶质瘤细胞等)进行MHC类型检测和全外显子测序检测RNA突变；
2)利用MHC类型和基因突变信息预测抗原表位：以突变的氨基酸位点为中心，向两侧各延伸8个氨基酸，将这段17个氨基酸的多肽作为潜在抗原表位；
3)使用预测软件分析潜在抗原表位的IC50，如IC50＜1000nM则认为此潜在抗原表位为抗原表位；
2、多肽连接
1)使用前述软件分析任意抗原表位两两相连后接头处的IC50，IC50≥1000nM时认为是弱免疫原性，可以连接；IC50＜1000nM时认为是强免疫原性，不能连接；
2)根据上述结果，将弱免疫原性的抗原表位连接在一起，接头处IC50要高于两侧抗原表位的IC50(也就是接头处尽量避免产生强结合抗原)；
3、合成多肽
1)将连接后的多肽还原为核酸序列，并进行密码子优化；
2)使用固相合成法合成抗原表位肽的基因序列；或由技术服务公司进行合成；
4、构建表达抗原表位肽的MVA病毒
将上步合成的基因序列构建表达抗原表位肽的MVA病毒表达质粒，进行病毒包装；
5、转染抗原递呈细胞(APC)并与PBMC共培养
1)使用表达抗原表位肽的rMVA病毒转染抗原递呈细胞(包括但不限于：外周血单个核细胞、树突状细胞、中性粒细胞、B淋巴细胞、巨噬细胞)；
2)收集处理完成的APC，以APC：PBMC＝1：5-20的比例混合共培养，得到效应细胞；
6、筛选有效的精准多肽，并使用精准多肽再次刺激T细胞
1)离心收集以上方案获得的T细胞，多肽作为抗原直接刺激T细胞筛选精准多肽：
2)设置阳性对照：T细胞+100ng/mL OKT3；阴性对照：T细胞+1640+10％FBS+200U/mL IL2；
3)精准多肽评判标准：
a.阳性对照和阴性对照正常，则说明此数据可信；
b.实验组显著大于阴性对照组的为有效的精准多肽；
4)以筛选出的精准多肽二次冲击T细胞；
7、构建TCR-T细胞
1)对刺激后的T细胞进行CD8、CD137、IFN-γ的染色，并以流式细胞仪进行分选；
2)分选出能够识别精准多肽的特异性细胞，测序确定高频TCR序列并扩增；
3)构建TCR基因表达载体，包装病毒；
4)敲除所述外周血T细胞中原有的TCR基因，转入上步构建的TCR基因，培养即得TCR-T细胞；
8、敲除细胞表面免疫抑制性信号分子，即得MRFFT1细胞
细胞表面抑制性信号分子包括：PD-1、Tim-3、LAG3、CTLA-4、BTLA、VISTA、CD160、2B4(CD244)；
9、构建特异性抗原表达靶细胞及肿瘤模型生存实验。
本发明的有益效果：
1.肿瘤抗原为突变抗原，与其它组织不同，靶点专一性强，不易发生脱靶效应，安全性高；
2.获得的特异性细胞比例高，通常能够识别肿瘤抗原的特异性细胞，在PBMC的分布为0.5％以下，经过MRFFT1方案改造的细胞，识别肿瘤抗原的特异性T细胞(TCR+)比例为70％以上；
3.MRFFT1细胞由于对PD1、CTLA4、TIM3、LAG3等免疫抑制性靶点进行敲除，因 此，对肿瘤的杀伤能力不受限制，杀伤效率更高。
附图说明
图1：MVA病毒转染APC效率检测；其中，1A：对照组，1B：转染组。
图2：MFF细胞分型检测。
图3：精准多肽的筛选。
图4：流式检测特异性T细胞比例；其中，4A：对照组，4B：MRFF方案。
图5：TCR分布频率。
图6：抑制性靶点的敲除情况；其中，6A：敲除后，6B：敲除前。
图7：原有TCR的敲除效率检测；其中，7A：敲除后，7B：敲除前。
图8：特异性TCR的表达效率；其中，8A：转染7天后，8B：转染前。
图9：LDH释放检测杀伤效率。
图10：ELISA检测细胞因子IFN-γ的释放。
图11：动物荷瘤模型生存曲线。
具体实施方式
下面通过具体的实施方案叙述本发明。除非特别说明，本发明中所用的技术手段均为本领域技术人员所公知的方法。另外，实施方案应理解为说明性的，而非限制本发明的范围，本发明的实质和范围仅由权利要求书所限定。对于本领域技术人员而言，在不背离本发明实质和范围的前提下，对这些实施方案中的物料成分和用量进行的各种改变或改动也属于本发明的保护范围。
技术方案详述如下：
1、抗原表位预测
1)使用肺癌患者外周血进行ctDNA测序和HLA分型检测；
2)利用软件对测序信息进行分析：将ctDNA测序结果与正常细胞的基因组相比，筛选出突变位点；
3)以突变的氨基酸位点为中心，向两侧各延伸8个氨基酸，将这段17个氨基酸的多肽作为潜在抗原表位；
4)使用预测软件分析潜在抗原表位的IC50(推荐软件：NetMHCpan 3.0、PickPocket、artificial neural networks(ANN))，如IC50＜1000nM则认为此潜在抗原表位为抗原表位。
2、多肽连接
1)使用前述软件分析任意抗原表位两两相连后接头处的IC50，IC50≥1000nM时认为是弱免疫原性，可以连接；IC50＜1000nM时认为是强免疫原性，不能连接(此处应考虑3个预测软件的IC50计算结果，当≥2个软件计算的IC50≥1000nM时才能认为是弱免疫原性，当≥2个软件计算的IC50＜1000nM时才能认为是强免疫原性)；
2)根据上述结果，将抗原表位连接在一起，接头处IC50要高于两侧抗原表位的IC50(也就是接头处尽量避免产生强结合抗原)；必要时将弱免疫原性肽做为连接肽将强免疫原性肽间隔；或者添加患者自身氨基酸到接头处，用来降低产生强抗原的可能性。
3、合成编码多肽的基因序列
1)将连接后的多肽还原为核酸序列，并进行密码子优化；
连接完成后的核酸序列如果较短(＜100bp)可以适当把氨基酸序列进行重复，但是，需要注意还原成基因序列时，应尽量避免基因序列中反向重复、直接重复和镜像重复序列的出现
2)使用固相合成法合成抗原表位肽的基因序列(由技术服务公司进行合成)。
4、构建表达抗原表位肽的MVA病毒
1)构建穿梭质粒：将合成的抗原表位肽基因序列克隆到pIIIdHR-P7.5质粒中；
2)重组MVA病毒构建：CEF细胞(鸡胚成纤维细胞)或者BHK-21细胞(仓鼠肾成纤维细胞)单层生长到70％～90％覆盖率依次感染MVA、转染穿梭质粒，培养获得单层细胞；所述单层细胞通过冻融、破碎获得rMVA溶液；所述rMVA溶液接种到RK-13细胞(兔肾细胞)培养获取rMVA感染RK-13聚集点，挑取所述聚集点筛选纯化得到所需rMVA；清除掉所述rMVA中wtMVA，将rMVA培养于CEF或者BHK-21细胞单层中，筛选不含选择基因K1L的rMVA，纯化得到rMVA-抗原表位肽，扩增分离得到rMVA-抗原表位肽；
5、转染抗原递呈细胞(APC)并与PBMC共培养
1)使用表达抗原表位肽的rMVA病毒转染抗原递呈细胞(包括但不限于：外周血单个核细胞、树突状细胞、中性粒细胞、B淋巴细胞、巨噬细胞)；
2)将抗原递呈细胞(APC)以1-10×10 6/mL铺入6-24孔板中；
3)将表达抗原多肽的rMVA病毒以MOI＝0.1-2感染APC，感染2-24h后，每孔加入0.5-2mL含100-2000IU/mL IL-2、10-100ng/ml hTNF-α、1000-5000IU/mL IL-6和10-100ng/mL IL-1β的1-20％FBS AIM V培养基，继续培养2-48h；
4)收集处理完成的APC，以APC：PBMC＝1：5-20的比例混合，PBMC约为5×10 7，加入50mL OKM100培养基到T75细胞培养瓶中，放入30-37℃细胞培养箱中培养14天，即获得MFF方案效应细胞。
6、筛选有效的精准多肽
多肽作为抗原直接刺激效应细胞筛选精准多肽：
1)离心收集以上MFF方案细胞，1500rpm离心5min收集T细胞，加入10mL PBS重悬细胞并计数，1500rpm离心5min，收集T细胞以1640+10％FBS+200U/mL IL2重悬，计数调整至1×10 6cells/mL；
2)以排枪将T细胞分至96孔平底板中，200μL/孔，细胞数为2×10 5cells；再分别加入10μL 1mg/mL的突变多肽，终浓度为50μg/mL，每条多肽设置3复孔；
3)设置阳性对照：T细胞+100ng/mL OKT3；阴性对照：T细胞+1640+10％FBS+200U/mL IL2；
4)37℃、5％CO 2刺激24h后，1500rpm离心10min，转移140μL上清至新的96孔板中；
5)再将96孔板进行离心，1500rpm 10min，取样品进行ELISA检测(或将样品至于-80℃保存)；
检测IFN-γ的ELISA系统：
1)目前测试过可用于检测IFN-γ的ELISA试剂盒有Biolegend：LEGEND MAX Human IFN-γELISA Kit with Pre-coated Plates(货号：430107)和达科为：Human IFN-γELISA Kit(货号：DKW12-1000-096)，请严格按照厂家说明书进行操作；
2)ELISA手动包板系统(15块板)：Human IFN-gamma DuoSet 15plate(货号：DY285B)×1，DuoSet ELISA Ancillary Reagent Kit 2(货号：DY008)×3；
精准多肽评判标准：
1)阳性对照和阴性对照正常，则说明此数据可信；
2)实验组显著大于阴性对照时，说明多肽为有效的精准多肽。
7、以筛选出的精准多肽二次冲击T细胞
1)PBMC以步骤5培养至第2～14天时，取2×10 7的效应细胞，加入终浓度为10μg/mL～100μg/mL的精准多肽，冲击1-4h；
2)冲击4h后，转入OKM25预包板的6孔板中或T25cm 2培养瓶，补OKM100+12％FBS，37℃5％CO 2培养，根据细胞生长情况，转移至T75培养瓶中，尽量保持细胞密度在1×10 6cells/mL；
3)进入T175培养瓶中时，培养基为OKM200+5％FBS，培养10～14天即可获得精准多肽二次冲击得到的T细胞，即MRFF细胞；
8、突变抗原特异性杀伤T细胞的培养及分离
1)以筛选出的精准多肽直接作为抗原刺激，对步骤7获得的MRFF细胞进行刺激，刺激12～72h后，备用；
2)对刺激后的T细胞进行CD8、CD137、IFN-γ的染色，并以流式细胞仪进行分选，选择CD8+CD137+、或者CD8+IFN-γ+细胞；
9、CD8+T细胞TCR频率检测及高频TCR的克隆
1)提取分选的CD8+CD137+、或者CD8+IFN-γ+细胞的基因组，进行TCR频率的检测，确定高频的TCR序列；
2)提取分选细胞的mRNA，反转录成从DNA，根据高频TCR的序列，设计引物，扩增得到TCR基因；
3)构建TCR基因表达载体，包装病毒；
10、构建免疫抑制性信号分子敲除的CRISPR载体
1)细胞表面抑制性信号分子包括：PD-1、Tim-3、LAG3、CTLA-4、BTLA、VISTA、CD160、2B4(CD244)；
2)分析抑制性信号分子的外显子，在pubmed上找到基因的mRNA的CDS区，分别将每个外显子进行敲除靶点的预测；
3)设计合成sgRNA所需的正向引物和反向引物，将正向引物和反向引物1：1混合后，95℃处理5～60min，再进行缓慢降温，形成sgRNA的DNA序列；
4)将CRISPR慢病毒表达载体进行双酶切，并与sgRNA对应的双链DNA进行连接，转入克隆感受态细胞，12h后，挑取单克隆进行测序，保留测序正确的克隆；
5)提取携带sgRNA对应DNA序列的CRISPR慢病毒载体质粒，进行病毒包装；
11、构建敲除TCR的CRISPR载体
1)在pubmed上找到TCR基因的mRNA的CDS区，并分析TCR的保守区，将保守区进行敲除靶点的预测；
2)按照步骤10中的步骤3)～5)完成TCR敲除载体的构建及病毒包装；
12、构建敲除免疫抑制性信号分子的TCR-T：
1)以步骤10和11中获得的病毒，感染步骤8获得的CD8+T细胞，同时进行原有TCR的敲除，以及免疫抑制性信号分子的敲除；
2)敲除后，CD8+T细胞在培养基中培养0-5天后，优选3天，再转入构建的TCR表达载体；
3)以OKM100+12％FBS将感染后的CD8+T细胞重悬，并置于刺激因子OKM-25的预铺板上，记为第0天；
4)观察细胞情况和细胞密度，在第5天，将共培养细胞转移至大培养瓶中，补新鲜的培养液OKM-100+12％FBS；
5)将细胞从75cm 2瓶中转移至175cm 2大瓶后培养液为OKM-200+5％FBS；
6)培养至14-21天时，即可收获敲除免疫抑制性信号分子的TCR-T，即MRFFT1细胞。
13、构建特异性抗原表达靶细胞及肿瘤模型生存实验
1)构建可以表达筛选的精准多肽(特异性抗原)的慢病毒载体。
2)将特异性抗原表达慢病毒载体包装成慢病毒颗粒，感染HLA配型合适的肿瘤细胞，稳定过表达特异性抗原，流式检测表达水平及表达强度。
3)稳定过表达特异性抗原肽的肿瘤细胞系接种NGS小鼠，做异位荷瘤动物模型。将5×10 5表达特异性抗原的肿瘤细胞悬于100μl生理盐水中，分别皮下注射至30只NSG小鼠的右侧胁肋部皮下，同时对小鼠进行编号。
4)在肿瘤生长至100-120mm 3左右时分组回输细胞，根据肿瘤体积大小，将动物模型随机分成三组，每组5-6只小鼠，一组给予安慰剂生理盐水，一组给予没有进行任何遗传操作的T细胞(对照组)1×10 7，一组给予MRFFT1细胞1×10 7，首次注射细胞7天后进行第二次注射，7天之后第三次注射细胞，连续观察60天，统计存活数据，绘制生存曲线。
试验结果：
1、突变位点及抗原表位预测
表1为测序检测到的突变位点及抗原表位预测结果。
表1 抗原表位预测
Figure PCTCN2019077924-appb-000001
Figure PCTCN2019077924-appb-000002
2、MVA病毒转染APC效率检测
1)使用表达抗原表位肽的rMVA病毒转染抗原递呈细胞(包括但不限于：外周血单个核细胞、树突状细胞、中性粒细胞、B淋巴细胞、巨噬细胞)；
2)将抗原递呈细胞(APC)以1-10×10 6/mL铺入6-24孔板中；
3)将表达抗原多肽的rMVA病毒以MOI＝0.1-2感染APC，感染2-24h后，每孔加入0.5-2mL含100-2000IU/mL IL-2、10-100ng/ml hTNF-α、1000-5000IU/mL IL-6和10-100ng/mL IL-1β的1-20％FBS AIM V培养基，继续培养2-48h；
4)使用流式细胞仪检测APC中GFP阳性比例(如图1所示)。
3、MFF细胞分型检测
MFF方案细胞培养结束后，进行CD4+和CD8+细胞的分型检测，结果如图2所示：CD8+T细胞为68％，CD4+T细胞为9.45％。
4、以MFF细胞筛选精准多肽
以12条多肽分别刺激培养的T细胞，通过检测IFN-γ的分泌，检测有效的多肽，结果如图3所示：3号的多肽引起的IFN-γ的释放量>阴性对照的释放量，属于有效精准多肽。
5、对精准多肽特异性的T细胞的鉴定及分选
以筛选的3号多肽，刺激MFF方案细胞，以流式检测对精准多肽特异性的T细胞比例，结果如图4所示，黑色框内为特异性T细胞：MRFF方案细胞，3号多肽引起的释放IFN-γ的细胞比例，明显高于没有刺激的细胞(对照)，说明，MRFF方案，可以获得对精准多肽的特异性T细胞；同时以流式细胞仪进行CD8+IFN-γ+细胞(黑色框内)的分选；
6、高频TCR的鉴定及克隆
将分选得到细胞进行基因组的提取，及TCR的测序，TCR的分布情况如图5所示(高频分布的前22)，TCR1和TCR17分布频率较高，说明，此TCR与突变抗原密切相关，根据TCR17序列，对TCR进行扩增，构建慢病毒表达载体。
表2 TCRβ链CDR3的序列情况
Figure PCTCN2019077924-appb-000003
Figure PCTCN2019077924-appb-000004
已知的TCR-α：
氨基酸序列：
Figure PCTCN2019077924-appb-000005
碱基序列：
Figure PCTCN2019077924-appb-000006
已知TCR-β：
氨基酸：
Figure PCTCN2019077924-appb-000007
横线为CDR3序列，需要被替换的序列
替换后的TCR-β：
Figure PCTCN2019077924-appb-000008
横线为替换的CDR3序列
7、抑制性靶点敲除效率的检测
利用CRISPR技术，将PBMC上的抑制性靶点PD-1进行敲除，sgRNA序列见表3，抑制性靶点的敲除效率结果如图6所示：sgRNA1的敲除效率最高，可有效的阻断抑制性信号分子PD-1的表达；sgRNA优选sgRNA1、sgRNA2；也可利用此方法，对Tim-3、LAG3、CTLA-4、BTLA、VISTA、CD160、2B4(CD244)等抑制性信号分子进行敲除；可有效的阻断抑制性信号分子的表达。
表3 抑制性靶点sgRNA序列
Figure PCTCN2019077924-appb-000009
8、原有TCR敲除效率的检测
利用CRISPR技术，将PBMC上的原有的TCR进行敲除，结果如图7所示：可有效的减少原有TCR的表达，此时，可进行表达特异性TCR慢病毒的转染。
9、特异性TCR表达情况的检测
以包装特异性TCR的的慢病毒转染PBMC，在第7天时，以流式检测TCR的表达效率，结果如图8所示：构建的TCR可以正常表达，TCR+的细胞比例为25.1％
10、MRFFT1细胞对靶细胞的杀伤作用
分别以对照细胞和MRFFT1细胞对突变抗原表位来源的靶细胞进行杀伤效率的检测，以无处理的细胞作为对照(Mock)，结果如图9所示，与对照组相比，MRFFT1细胞对靶细胞有较强的杀伤效果。
11、MRFFT1细胞释放细胞因子的检测
肿瘤细胞与效应细胞共培养时，由于效应细胞，可以识别肿瘤细胞上突变抗原，因此，会产生一系列的细胞因子，IFN-γ是抗肿瘤作用中最主要的细胞因子之一，图10为MRFFT1细胞与肿瘤细胞共培养时，释放的IFN-γ的检测，结果表明：与效应细胞自身产生的IFN-γ相比(T cells only)，与肿瘤细胞共培养后，MRFFT1细胞可产生大量的IFN-γ此结果与杀伤实验结果一致说明：表达特异性TCR的T细胞，结合抑制性靶点的敲除可以更有效的提高抗 肿瘤能力。
13、构建特异性抗原表达靶细胞及肿瘤模型生存实验
成功构建了特异性抗原表达肿瘤靶细胞系，建立荷瘤动物模型，结果显示(图11)，MRFFT1细胞对肿瘤荷瘤小鼠的生存改善具有显著影响作用。
14、临床案例：
某男：61岁
疾病诊断：双肺低分化腺癌；左肺结核
第一疗程：每个月一次MRFFT1细胞，数量1×10 9个细胞，共2次；
第二疗程：每半年一次MRFFT1细胞，数量1×10 9个细胞，共2次；
给药结束后，22个月无进展生存；
其它案例：
患者编号 疾病诊断 无进展生存时间
1 胃腺癌肝转移 2016.3-至今
2 胃癌 2016.3-至今
3 肺癌 2016.4-至今
4 肺腺癌 2016.4-至今
5 肺腺癌 2016.6-至今
6 食管癌 2016.6-至今
注：“至今”的含意为“申请日前一天”

Claims (6)

  1. 一种MRFFT1细胞，其特征在于，所述MRFFT1细胞由以下步骤制备：1)使用人源外周血进行ctDNA测序或肿瘤组织进行全外显子测序，筛选出突变位点；2)根据突变位点进行抗原表位预测，合成突变多肽的基因序列；3)构建表达突变多肽的MVA病毒载体，包装MVA病毒；4)转染抗原递呈细胞并与PBMC共培养，获得MFF细胞；4)所述突变多肽作为抗原刺激所述MFF细胞，筛选出有效的精准多肽；5)以所述精准多肽作为抗原刺激所述MFF细胞，筛选出能够识别所述精准多肽的特异性细胞，测序并得到特异性细胞的高频TCR基因；6)敲除外周血T细胞中原有的TCR基因，转入上步获得的能够与精准多肽特异性结合的TCR基因，获得TCR-T细胞；7)敲除细胞表面免疫抑制性信号分子，即得MRFFT1细胞。
  2. 如权利要求1所述的MRFFT1细胞，其特征在于，所述人源外周血也可以是市售工程细胞系。
  3. 如权利要求1所述的MRFFT1细胞，其特征在于，所述抗原表位预测是以突变的氨基酸位点为中心，向两侧各延伸8个氨基酸，将这段17个氨基酸的多肽作为潜在抗原表位；使用预测软件分析潜在抗原表位的IC50，如IC50＜1000nM则认为此潜在抗原表位为抗原表位。
  4. 如权利要求1所述的MRFFT1细胞，其特征在于，所述敲除外周血T细胞中原有的TCR基因和/或细胞表面免疫抑制性信号分子的方法为CRISPR技术。
  5. 如权利要求1所述的MRFFT1细胞，其特征在于，所述细胞表面抑制性信号分子包括：PD-1、Tim-3、LAG3、CTLA-4、BTLA、VISTA、CD160、2B4(CD244)。
  6. 如权利要求1所述的MRFFT1细胞，其特征在于，所述抗原递呈细胞包括：外周血单个核细胞、树突状细胞、中性粒细胞、B淋巴细胞、巨噬细胞。
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