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

在开发中的许多免疫治疗方法中，使用双特异性抗体或免疫球蛋白的能力是非常重要的。 嵌合抗原受体（汽车）指导T细胞选择性地杀死肿瘤细胞已经证明了显著的 早期的成功通常，双特异性抗体或双特异性T细胞增殖物（即，BiTes）交联T细胞， 通常通过单价相互作用结合CD 3和靶癌细胞表面抗原。一个 另一种方法是对癌症患者的T细胞进行基因工程改造，使其表达单链抗体 （scFv）-CD 3+融合蛋白，其可以靶向癌细胞表面抗原。在重新引入患者体内后， 表达CAR的T细胞已经能够选择性地消除靶癌细胞。虽然成功， 细胞表面的基因工程是耗时且不可逆的， 持续输液此外，已经观察到由于抗原损失而对两种方法的抗性。 我们的研究小组已经表明，两个二氢叶酸还原酶分子（DHFR 2）融合到α CD 3单链上， 抗体（scFv）可以被工程化以在添加化学物质时自发地自组装 二聚剂，双甲氨蝶呤（BisMTX），转化为高度稳定的八价化学自组装纳米环 （CSAN）。已经用含有第三臂的BisMTX制备了CSAN，从而使其能够与 寡核苷酸、荧光团、放射性标记和药物。最近，我们制备了αEpCAM/α CD 3 CSAN， 和α CD 133/α CD 3-CSAN。双特异性CSAN快速（分钟）和稳定（天）结合T细胞上的CD 3。 膜，从而形成化学自组装的人工抗原受体（PAR）T细胞。后 将PAR T细胞与EpCAM+和/或CD 133+癌细胞孵育，快速和选择性杀伤原发性 观察肿瘤起始癌干细胞（CSC）。我们还用原位的 αEpCAM和α CD 133 PAR T细胞是无毒的，并且能够联合 在体内根除肿瘤。我们的方法的一个独特的安全功能是能够从 通过以临床相关浓度给药FDA批准的无毒抗生素甲氧苄啶， 从而使我们能够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方案，我们确定了T细胞诱导的杀伤的一般性。 以及用αEpCAM/α-CD 3-CSANS和α CD 133/α CD 3-CSANS根除TNBC肿瘤。此外，我们将 开发tripspecific αEpCAM/α CD 133/α CD 3 CSAN，可同时消除TNBC 原发肿瘤细胞和CSC。项目里程碑的成功完成应导致 阐明控制基于化学生物学的非遗传和可逆方法的关键特征， T细胞靶向，以及CD 133对TNBC增殖的重要性。这些规则将适用于 具有更高选择性、更低毒性和更低毒性的抗肿瘤免疫疗法的临床开发 发展抵抗力的能力。

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.