EpoxySEAKER and onSEAKER: CAR T-Cell Systems for Targeted Local Biosynthesis of Therapeutic Warheads

EPOXYSEAKER和ONSEAKER：用于靶向局部生物合成的CAR T细胞系统 治疗性弹头 我们的实验室已经开发出新型的表达酶的SEAKER(合成酶-武装杀手)CAR T细胞 这将遮盖组与系统管理的无毒前药分开。Seaker的本土化 细胞转化为肿瘤允许在感兴趣的部位将前药特异性地转化为抗癌剂。这些 Seaker细胞克服了CAR T细胞治疗的局限性，如T细胞耗竭、免疫抑制和 通过T细胞的增殖、酶的结构性分泌而产生级联效应的抗原变异 活性药物的催化生成。这些酶继续在肿瘤中产生细胞毒性化合物。 即使站点后的T细胞功能衰竭。我们将对这个系统进行改造，以产生新的 表达细菌生物合成酶并将非生物合成酶转化为 通过酶介导的治疗性弹头将毒性前药底物转化为细胞毒性化合物 安装。通过消除对遮盖部分的需要来限制前药的体积，我们预计会有更大的 前药的药理性质。将弹头部分安装到前药上，而不是切割 掩蔽组将消除细胞毒剂的非特异性激活，并减少靶点上、场外 与目标化合物相关的毒性。 我们计划使用我们的两个新的Seaker系统来靶向肿瘤环境，引发免疫反应， 随后分泌EpnF或TsnB9，其将酶激活FDA批准的前药物变体 就地用药。我们将为两个Seaker系统生成多个前药，并评估它们的转换为 这些活性化合物在体外使用重组酶。卡非佐米和帕诺比妥等化合物 在治疗多发性骨髓瘤时，与靶点外毒性有关。肿瘤 靶向“Epoxy Seaker”或“On Seaker”细胞将合成酶的分泌定位到 肿瘤部位，允许全身给药无毒前药，随后的原位化合物 合成并降低了目标化合物的异地毒性。相加疗法 CAR-T细胞和小分子的作用将增加治疗的有效性，并可能扩大 CAR-T细胞治疗的靶点范围。

EPOXYSEAKER AND ONSEAKER: CAR T-CELL SYSTEMS FOR TARGETED LOCAL BIOSYNTHESIS OF THERAPEUTIC WARHEADS Our labs have developed novel SEAKER (Synthetic Enzyme-Armed KillER) CAR T-cells that express enzymes that cleave masking groups from systemically administered non-toxic prodrugs. The localization of SEAKER cells to tumors allows for specific conversion of the prodrug to anticancer agent at the site of interest. These SEAKER cells overcome limitations of CAR T-cell therapy such as T-cell exhaustion, immunosuppression and antigen variance by creating a cascade effect through proliferation of T-cells, constitutive secretion of enzymes and catalytic generation of the active drugs. The enzymes continue to produce cytotoxic compounds at the tumor site even after the T-cells functionally exhaust. We will engineer this system to produce novel “epoxySEAKER” and “onSEAKER” cells that express bacterial biosynthetic enzymes and convert non- toxic prodrug substrates into cytotoxic compounds through an enzyme-mediated therapeutic warhead installation. Limiting the bulkiness of the prodrug by eliminating the need for masking moieties we expect greater pharmacological properties of the prodrugs. Installing the warhead moiety onto the prodrug rather than cleaving a masking group will eliminate the non-specific activation of cytotoxic agents and reduce the on-target, off-site toxicity associated with the target compounds. We plan to use our two novel SEAKER systems to target the tumor environment, elicit immunologic responses, and subsequently secrete EpnF or TsnB9 which will enzymatically activate prodrug variants of FDA approved drugs in situ. We will generate multiple prodrugs for the two SEAKER systems and evaluate their conversion to the active compounds using recombinant enzymes in vitro. Compounds such as carfilzomib and panobinostat are associated with on-target off-site toxicity when administered for the treatment of multiple myeloma. Tumor targeting “epoxySEAKER” or “onSEAKER” cells will localize the secretion of synthetic enzyme to the tumor site, allowing systemic administration of non-toxic prodrug, subsequent in situ compound synthesis and reduced off-site toxicity demonstrated by the target compounds. The additive therapeutic effect of the CAR-T cells and small-molecules will increase the effectiveness of treatment and may expand the target scope of CAR-T cell therapy.