PRX302 is a highly potent, mutant bacterial pore-forming biologic protoxin engineered for selective activation by PSA, a serine protease expressed by benign and malignant prostate epithelial cells. Although being developed as a local therapy for benign prostatic hyperplasia and localized prostate cancer, PRX302 cannot be administered systemically as a treatment for metastatic disease due to binding to ubiquitously expressed glycosylphosphatidylinositol (GPI)-anchored proteins, which leads to poor accumulation within the tumor microenvironment. To overcome this limitation, poly-lactic-co-glycolic acid (PLGA) microparticles encapsulating the protoxin were developed, which are known to accumulate in the liver, a major site of metastasis for prostate cancer and other solid tumors. A highly sensitive and reproducible sandwich ELISA to quantify PRX302 released from microparticles was developed. Utilizing this assay, PRX302 release from different microparticle formulations was assessed over multiple days. Hemolysis assays documented PSA-dependent pore formation and lytic potential (i.e., function) of the released protoxin. MTT assays demonstrated that conditioned supernatant from PRX302-loaded, but not blank (i.e., unloaded), PLGA microparticles was highly cytotoxic to PC3 and DU145 human prostate cancer cells in the presence of exogenous PSA. Microparticle encapsulation prevented PRX302 from immediately interacting with GPI-anchored proteins as demonstrated in a competition assay, which resulted in an increased therapeutic index and significant antitumor efficacy following a single dose of PRX302-loaded microparticles in a preclinical model of prostate cancer liver metastasis with no obvious toxicity. These results document that PRX302 released from PLGA microparticles demonstrate in vivo antitumor efficacy in a clinically relevant preclinical model of metastatic prostate cancer.

中文翻译：

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前列腺靶向生物制剂的微粒封装治疗去势抵抗性前列腺癌临床前模型中的肝转移

PRX302 是一种高效、突变的细菌成孔生物原毒素，经过工程改造，可被 PSA 选择性激活，PSA 是一种由良性和恶性前列腺上皮细胞表达的丝氨酸蛋白酶。尽管 PRX302 被开发为治疗良性前列腺增生和局限性前列腺癌的局部疗法，但由于与普遍表达的糖基磷脂酰肌醇 (GPI) 锚定蛋白结合，因此不能全身性给药作为转移性疾病的治疗，这会导致在肿瘤微环境中积聚不良. 为了克服这一限制，开发了包封原毒素的聚乳酸-乙醇酸 (PLGA) 微粒，已知其在肝脏中积聚，肝脏是前列腺癌和其他实体瘤的主要转移部位。开发了一种高度灵敏且可重复的夹心 ELISA，用于量化从微粒释放的 PRX302。利用该测定法，在多日内评估了 PRX302 从不同微粒制剂的释放。溶血测定记录了释放的原毒素的PSA依赖性孔形成和溶解潜力（即功能）。MTT 测定表明，在外源性 PSA 存在下，来自负载 PRX302 而非空白（即未负载）PLGA 微粒的条件上清液对 PC3 和 DU145 人前列腺癌细胞具有高度细胞毒性。如竞争测定所示，微粒封装阻止 PRX302 立即与 GPI 锚定蛋白相互作用，在前列腺癌肝转移的临床前模型中，单剂量负载 PRX302 的微粒可提高治疗指数和显着的抗肿瘤功效，且无明显毒性。这些结果表明，从 PLGA 微粒释放的 PRX302 在临床相关的转移性前列腺癌临床前模型中显示出体内抗肿瘤功效。