Targeting Effector Immune cells to Cancer with Chemically Self-Assembled Nanorings (CSANs)

在许多正在开发的免疫治疗方法中，使用双特异性抗体或 嵌合抗原受体(CARS)指导T细胞选择性杀伤肿瘤细胞已显示出重要意义 早期的成功。通常，双特异性抗体或双特异性T细胞结合体(即BITE)通过以下方式使T细胞交联 与CD3和靶肿瘤细胞表面抗原结合，通常通过单价相互作用。一个 另一种方法是通过基因工程使癌症患者的T细胞表达单链抗体 (ScFv)-CD3ζ融合蛋白，可靶向肿瘤细胞表面抗原。在重新介绍给病人后， 表达CAR的T细胞已经能够选择性地清除靶癌细胞。虽然成功，但 细胞表面的基因工程是耗时且不可逆转的，而咬合的使用需要 持续输液。此外，由于抗原丢失，已经观察到对这两种方法的抵抗力。 我们的团队已经证明了两个二氢叶酸还原酶分子(DHFR2)融合到一个αCD3单链上 抗体(ScFv)可以被设计成在添加化学物质后自发自组装 二聚体，双甲氨蝶呤(BisMTX)，制成高度稳定的八价化学自组装纳米环 (CSANS)。CSAN已经用含有第三个臂的BisMTX制备，从而使其能够偶联到 寡核苷酸、荧光团、放射性标记和药物。最近，我们制备了αEPCAM/αCD3CSAN 和αCD133/αCD3-CSANS。双特异性CSAN快速(分钟)和稳定(天数)与T细胞表面CD3结合 膜，从而形成化学自组装的人工抗原受体(PAR)T细胞。vt.在.的基础上 PAR T细胞与EPCAM和/或CD133癌细胞孵育后对原代细胞的快速选择性杀伤 观察肿瘤起始肿瘤干细胞(CSC)的表达。我们还演示了一种直立式 αEPCAM和αCD133PAR T细胞无毒并能联合应用的小鼠肿瘤模型 在体内根除肿瘤。我们方法的一个独特的安全功能是能够从 通过给T细胞注射FDA批准的临床相关浓度的无毒抗生素甲氧苄啶， 从而使我们能够从药物上使细胞失活，并减少细胞因子的释放。 因此，作为当前方法的替代，我们确定了T细胞诱导的杀伤的一般性 αEPCAM/α-CD3-CSANS和αCD133/αCD3-CSANS根治肿瘤。此外，我们还将 开发一种Trip特异性αEPCAM/αCD133/αCD3CSAN，使其能够同时消除TNBC 原代肿瘤细胞和CSC。项目里程碑的成功完成应该会导致 阐明一种基于化学生物的非遗传和可逆方法的关键特征 T细胞靶向，以及CD133在TNBC增殖中的重要性。本规则适用于 高选择性、低毒、低剂量抗肿瘤免疫治疗的临床研究进展 抗病能力的发展。

Of the many immunotherapy approaches under development, the ability to use bispecific antibodies or chimeric antigen receptors (CARs) to direct T-cells to selectively kill tumor cells has demonstrated significant early success. Typically, bispecific antibodies or bispecific T-cell engagers (i.e., BiTes) cross-link T-cells by binding to CD3 and to a target cancer cell surface antigen, usually through a monovalent interaction. An alternative approach is to genetically engineer a cancer patient’s T-cells to express a single chain antibody (scFv)-CD3ζ fusion protein that can target the cancer cell surface antigen. After re-introduction into the patient, CAR-expressing T-cells have been able to selectively eliminate the target cancer cells. While successful, the genetic engineering of cell surfaces is time consuming and irreversible and the use of BiTes requires continuous infusion. Furthermore, the resistance to both approaches due to antigen loss has been observed. Our group has shown that two dihydrofolate reductase molecules (DHFR2) fused to an αCD3 single chain antibody (scFv) can be engineered to spontaneously self-assemble upon the addition of the chemical dimerizer, bis-methotrexate (BisMTX), into either highly stable octavalent chemically self-assembled nanorings (CSANs). CSANs have been prepared with BisMTX containing a third arm, thus enabling it to be conjugated to oligonucleotides, fluorophores, radiolabels and drugs. Recently, we have prepared αEpCAM/αCD3 CSANs and αCD133/αCD3-CSANs. The bispecific CSANs rapidly (min) and stably (days) bind to CD3 on T-cell membranes, thus forming chemically self assembled prosthetic antigen receptor (PAR) T-cells. Upon incubation of the PAR T-cells with EpCAM+ and/or CD133+ cancer cells, rapid and selective killing of primary and tumor initiating cancer stem cells (CSC) was observed. We have also demonstrated with an orthotopic murine cancer model that αEpCAM and αCD133 PAR T-cells are non-toxic and able in combination to eradicate tumors in vivo. A unique safety feature of our approach is the ability to remove the CSANs from the T-cells by dosing with the FDA-approved non-toxic antibiotic trimethoprim at clinically relevant concentrations, thus allowing us to deactivate the cells pharmacologically and reduce cytokine release. Consequently, as an alternative to current approaches, we determine the generality T-cell induced killing and eradication of TNBC tumors with αEpCAM/α-CD3-CSANS and αCD133/αCD3-CSANS. In addition, we will develop a tripspecific αEpCAM/αCD133/αCD3 CSANs that will allow the simultaneous elimination of TNBC primary tumor cells and CSC. The successful completion of the project milestones should result in the elucidation of the key features governing a chemical biologically based non-genetic and reversible method for T-cell targeting, as well as the importance of CD133 on TNBC proliferation. These rules will be applicable to the clinical development of anti-cancer immunotherapy with greater selectivity, lower toxicity and a reduced ability for the development of resistance.