采用新型抗Her2嵌合抗原受体T细胞联用免疫抑制通路阻遏来克服乳腺癌赫赛汀耐药的研究

The de novo and acquired resistance to Herceptin therapy is a serious problem in treating Her2-positive breast cancer patients. Herceptin can not kill all of the Her2-positive breast cancer cells even in patients who are sensitive to Herceptin treatment. Current strategies to overcome Herceptin resistance are focusing mainly on inhibiting different cell signaling pathways activated in different patients with Herceptin resistance. Those strategies certainly can not be applied to patients with unknown mechanism of Herceptin resistance. In this study, we will test the novel therapeutic concept, combining the new-generation anti-Her2 CAR-T cells with immunosuppression blockade (anti-PD1 antibody) to combat the problem of Herceptin resistance of Her2-positive breast cancer. The anti-Her2 CAR-T cells can target Her2 present on the surface of breast cancer cells, and can be activated efficiently. The anti-PD1 antibody can block immunosuppression induced during T cell activation. Thus the combination therapy, through the ways of humoral and cellular immunity together with immunosuppression blockade, can kill all the Her2-positive breast cancer cells regardless of different Herceptin resistance mechanisms. The principal investigator started using CAR-T cells for the therapy of Her2-positive tumors 10 years ago. In this proposal, first, mouse tumor model with the normal immune system will be used to determine the enhanced activity, efficacy and consistency of the new-generation anti-Her2 CAR-T cells in combination with immunosuppression blockade. Then, Herceptin-resistant breast cancer cell model and xenograft model will be used to examine the efficacy and mechanism of this combination therapy for overcoming Herceptin resistance of breast cancer.

Her2+乳腺癌对赫赛汀的原发和继发耐药问题严重。赫赛汀不足以彻底杀灭所有Her2+乳腺癌细胞。已报道的任何一种克服赫赛汀耐药策略是通过调控各个耐药相关细胞信号通路的各个环节，不能同时克服所有原因已知的耐药，更不能克服原因未明的耐药。本研究假设新型抗Her2嵌合抗原受体（CAR）T细胞联用PD1免疫抑制通路阻遏，能通过靶向Her2的体液免疫、激活T细胞的细胞免疫、阻遏因T细胞激活而诱发的免疫抑制，三管齐下而彻底杀灭所有Her2+乳腺癌细胞，因耐药相关细胞信号通路随细胞死亡而不复存在，也就消除了任何原因的赫赛汀耐药问题。本课题负责人十年前开始Her2+肿瘤CAR-T细胞治疗研究。本研究首先采用正常免疫小鼠肿瘤模型，以确证新型抗Her2 CAR-T细胞联用PD1阻遏是否能进一步加强抗Her2 CAR-T细胞的活化和杀伤效能及持续性。然后采用耐药肿瘤模型，研究其针对赫赛汀耐药乳腺癌的作用及机制。

1、抗HER2 CAR重组慢病毒制备方案的优化.需要优化抗HER2嵌合抗原受体(CAR)T细胞的制备以使其成为可靠的治疗。研究表明:共转染时，悬浮包装细胞优于常用贴壁培养条件，包装系统pspax 2/pmd2.g优于pcmv-dr8.91/pvsv-g。最佳离心浓缩速度为20000 g，而不是一般采用的超高速。重要的是，加入瞬时离心纯化可显著提高人外周血T细胞的存活率(从13.25%提高到62.80%)，这是CAR-T细胞制备技术上的一大突破。转染人外周血T细胞的最佳MOI值为40。plvx-ef1a-car-eris-zsGreen 1在人外周血T细胞中表达率最高，细胞毒性达63.56%。我们优化了在T细胞中表达第三代抗HER2 CAR的重组慢病毒的制备，这应该为提高CAR-T细胞对于杀死靶细胞的效力奠定基础。.2、抗HER2 CAR-人T细胞联合PD1阻遏克服人HER2+乳腺癌曲妥珠单抗耐药问题.试图通过检测第三代抗HER2 CAR-T细胞单独和联合PD1阻遏对HER2阳性和曲妥珠单抗耐药乳腺癌细胞的体外和异种移植模型的治疗效果，来克服曲妥珠单抗耐药问题。抗HER2 CAR-T细胞特异性靶向HER2阳性的BT474细胞和曲妥珠单抗耐药的HCC1954细胞。抗HER2 CAR-T细胞与HCC1954细胞共培养后，IL-2和IFN-γ的分泌均增加，并随共培养体系中抗PD1抗体的加入而进一步增加。另外，抗HER2 CAR-T细胞对HCC1954和BT474细胞具有较强的细胞毒作用。抗PD1抗体的加入进一步增强了抗HER2CAR-T细胞对HCC1954细胞的杀伤作用。最后，注射抗HER2 CAR-T细胞可显著抑制HCC1954移植瘤的生长。联合抗HER2 CAR-T细胞和抗PD1抗体可进一步抑制HCC1954肿瘤的生长。因此，第三代抗HER2 CAR-T细胞联合PD1阻遏是克服乳腺癌曲妥珠单抗耐药的潜在疗法。.3、抗HER2 CAR-小鼠T细胞联合PD1阻遏对免疫正常小鼠HER2+乳腺癌模型的作用.在免疫正常小鼠模型中，发现CAR-T细胞能明显浸润肿瘤，抑制肿瘤生长，增加肿瘤细胞凋亡。加入抗PD1抗体可增强CAR-T细胞的抗肿瘤活性。结果表明，抗PD1抗体能显著提高抗HER2CAR-T对HER2阳性实体瘤的疗效。

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