DataSheet1_Development and therapeutic evaluation of 5D3(CC-MLN8237)3.2 antibody-theranostic conjugates for PSMA-positive prostate cancer therapy.DOCX

Prostate cancer (PC) is an aggressive cancer that can progress rapidly and eventually become castrate-resistant prostate cancer (CRPC). Stage IV metastatic castrate-resistant prostate cancer (mCRPC) is an incurable late-stage cancer type with a low 5-year overall survival rate. Targeted therapeutics such as antibody-drug conjugates (ADCs) based on high-affinity monoclonal antibodies and potent drugs conjugated via smart linkers are being developed for PC management. Conjugating further with in vitro or in vivo imaging agents, ADCs can be used as antibody-theranostic conjugates (ATCs) for diagnostic and image-guided drug delivery. In this study, we have developed a novel ATC for PSMA (+) PC therapy utilizing (a) anti-PSMA 5D3 mAb, (b) Aurora A kinase inhibitor, MLN8237, and (c) for the first time using tetrazine (Tz) and trans-cyclooctene (TCO) click chemistry-based conjugation linker (CC linker) in ADC development. The resulting 5D3(CC-MLN8237)3.2 was labeled with suitable fluorophores for in vitro and in vivo imaging. The products were characterized by SDS-PAGE, MALDI-TOF, and DLS and evaluated in vitro by optical imaging, flow cytometry, and WST-8 assay for cytotoxicity in PSMA (+/−) cells. Therapeutic efficacy was determined in human PC xenograft mouse models following a designed treatment schedule. After the treatment study animals were euthanized, and toxicological studies, complete blood count (CBC), blood clinical chemistry analysis, and H&E staining of vital organs were conducted to determine side effects and systemic toxicities. The IC50 values of 5D3(CC-MLN8237)3.2-AF488 in PSMA (+) PC3-PIP and PMSA (−) PC3-Flu cells are 8.17 nM and 161.9 nM, respectively. Pure MLN8237 shows 736.9 nM and 873.4 nM IC50 values for PC3-PIP and PC3-Flu cells, respectively. In vivo study in human xenograft mouse models confirmed high therapeutic efficacy of 5D3(CC-MLN8237)3.2-CF750 with significant control of PSMA (+) tumor growth with minimal systemic toxicity in the treated group compared to PSMA (−) treated and untreated groups. Approximately 70% of PSMA (+) PC3-PIP tumors did not exceed the threshold of the tumor size in the surrogate Kaplan-Meyer analysis. The novel ATC successfully controlled the growth of PSMA (+) tumors in preclinical settings with minimal systemic toxicities. The therapeutic efficacy and favorable safety profile of novel 5D3(CC-MLN8237)3.2 ATC demonstrates their potential use as a theranostic against aggressive PC.

前列腺癌（Prostate cancer, PC）是一种侵袭性较强的癌症，可快速进展并最终发展为去势抵抗性前列腺癌（castrate-resistant prostate cancer, CRPC）。Ⅳ期转移性去势抵抗性前列腺癌（metastatic castrate-resistant prostate cancer, mCRPC）是一种无法治愈的晚期癌症类型，5年总生存率较低。针对前列腺癌的治疗，目前正在开发基于高亲和力单克隆抗体与通过智能连接子偶联的强效药物的靶向治疗手段，如抗体药物偶联物（antibody-drug conjugates, ADCs）。若将抗体药物偶联物进一步与体外或体内显像剂偶联，则可作为抗体治疗诊断偶联物（antibody-theranostic conjugates, ATCs），用于诊断及影像引导下的药物递送。本研究开发了一种针对前列腺特异性膜抗原阳性（PSMA(+)）前列腺癌的新型治疗诊断偶联物，具体采用了（a）抗PSMA 5D3单克隆抗体、（b）极光激酶A抑制剂MLN8237，且首次在抗体药物偶联物开发中使用了基于四嗪（tetrazine, Tz）与反式环辛烯（trans-cyclooctene, TCO）点击化学的偶联连接子（CC linker）。将所得产物5D3(CC-MLN8237)3.2用合适的荧光团标记，用于体外及体内成像。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、基质辅助激光解吸电离飞行时间质谱（MALDI-TOF）及动态光散射（DLS）对产物进行表征，并通过光学成像、流式细胞术及WST-8细胞毒性检测，在PSMA(+/−)细胞中开展体外评价。按照预设的治疗方案，在人前列腺癌异种移植小鼠模型中评估其治疗效果。治疗结束后对实验动物实施安乐死，并开展毒理学研究、全血细胞计数（complete blood count, CBC）、血液生化分析及重要脏器的苏木精-伊红染色，以确定其不良反应及全身毒性。5D3(CC-MLN8237)3.2-AF488在PSMA(+) PC3-PIP细胞与PSMA(−) PC3-Flu细胞中的半抑制浓度（IC50）分别为8.17 nM与161.9 nM；游离MLN8237在PC3-PIP与PC3-Flu细胞中的IC50值分别为736.9 nM与873.4 nM。人异种移植小鼠模型的体内研究证实，5D3(CC-MLN8237)3.2-CF750具有较高的治疗效果，与PSMA(−)治疗组及未治疗组相比，治疗组可显著控制PSMA(+)肿瘤的生长，且全身毒性极低。在替代卡普兰-迈耶（Kaplan-Meyer）分析中，约70%的PSMA(+) PC3-PIP肿瘤未超过肿瘤体积阈值。该新型治疗诊断偶联物可在临床前研究中成功控制PSMA(+)肿瘤的生长，且全身毒性极低。新型5D3(CC-MLN8237)3.2 ATC的治疗效果及良好的安全性特征，证明其有望作为针对侵袭性前列腺癌的治疗诊断手段。