Dual-payload antibody-drug conjugate for chemo-immunotherapy of triple-negative breast cancers

总结 三阴性乳腺癌（TNBC）具有高度侵袭性，并且通常在标准细胞毒性化疗后复发。 化疗免疫疗法如靶向PD-1、PD-L1或PD-L2的免疫检查点阻断剂（ICB）， CTLA-4代表了癌症治疗的重大突破，但75-90%的TNBC患者由于缺乏足够的免疫力而未能应答。 原发性和获得性抵抗Sacituzumab-SN 38和Pembrolizumab-化疗已经被证实是有效的。 开发并应用于治疗难治性转移性TNBC，显示出联合靶向治疗的巨大潜力。 目前，TNBC的治疗方法还不成熟，但迫切需要新的有效的TNBC治疗策略。我们最近发现 跨膜CD 276（B7-H3）与大多数TNBC中的血管生成、转移和免疫耐受相关 人源化抗CD 276单克隆抗体（mAb），能够靶向CD 276 + TNBC和上调肿瘤免疫。此外，我们还建立了创新平台， 在一个抗体中偶联高细胞毒性的emtansine和免疫调节toll样受体（TLR）激动剂， 药物偶联物（ADC）。我们的初步评估表明，CD 276靶向的双有效载荷ADC（276- 278） DualADC）有效杀死多种TNBC亚型，显著增强免疫功能，克服了 ICB对PD-1 mAb的耐药性，并在三种动物模型中将肿瘤负荷降低90-100%和转移。 这些结果表明276-DualADC是治疗TNBC的有希望的治疗剂。我们的目标是发展和 检查组合人源化CD 276 mAb-导向的Hu 276-DualADC的有效性，其靶向 提供有效的化疗和免疫治疗TLR 7/8激动剂和PD-1靶向ADC，以消除 异种和转移性TNBC细胞。据推测，这种新的组合策略，命名为 作为Hu 276/PD-1-DualADC，协同多种化疗和免疫介导的抗癌机制，即， 直接杀死癌细胞、肿瘤免疫、肿瘤细胞因子和免疫检查点阻断，以增强 TNBC治疗效果。提出了三个目标来检验这一假设。目标1将产生大规模的 Hu 276-携带mertansine和咪唑并喹啉的DualADC，并表征其亲和力，TNBC特异性， 生物分布和毒性。最佳治疗策略将以最大耐受剂量确定， 药代动力学研究。最后，将在原发性异种移植物模型和远处移植物模型中评价抗TNBC功效。 转移模型目的2将评估HuCD 276/PD-1-DualADC在三种肿瘤中的协同作用。 免疫活性模型。潜在的机制（增殖、凋亡、免疫细胞浸润和 活化、肿瘤细胞因子和ICB恢复）也将被描绘。目标3将全面评估长期 在手术切除后的转移性同基因TNBC异种移植物模型中的治疗功效和患者来源的 异种移植物（PDX）模型。还将研究IND前毒理学，以收集临床前数据，用于未来的临床研究。 试发射。该项目的成功完成将为治疗侵袭性TNBC提供新的策略。

SUMMARY Triple-negative breast cancers (TNBCs) are highly aggressive and often relapse post standard cytotoxic chemotherapies. The immunotherapies such as immune checkpoint blockers (ICBs) that target PD-1, PD-L1 or CTLA-4 represent a major breakthrough in cancer treatment, but 75-90% of TNBC patients failed to respond due to primary and acquired resistance. The Sacituzumab-SN38 and Pembrolizumab-chemotherapy have been developed and applied to treat refractory metastatic TNBC, showing the great potential of combined and targeted therapies, but novel effective treatment strategies for TNBC are urgently needed. We recently detected transmembrane CD276 (B7-H3) associated with angiogenesis, metastasis and immune tolerance in most TNBC patients, and developed a humanized anti-CD276 monoclonal antibody (mAb) capable of targeting CD276+ TNBC and upregulating tumoral immunity. Furthermore, we established innovative platforms for concurrent conjugation of highly cytotoxic emtansine and immunoregulating toll-like receptor (TLR) agonist in one antibody- drug conjugate (ADC). Our preliminary evaluations showed that the CD276-targeted dual-payload ADC (276- DualADC) effectively killed multiple TNBC subtypes, significantly enhanced immune functions and overcame ICB resistance to PD-1 mAb, and reduced tumor burden by 90-100% and metastasis in three animal models. These results indicate 276-DualADC is a promising therapeutic to treat TNBCs. Our goal is to develop and examine the effectiveness of combining humanized CD276 mAb-directed Hu276-DualADC, which targeting delivers a potent chemotherapy and immunotherapy TLR 7/8 agonist, and PD-1-targeting ADC to eliminate heterogenous and metastatic TNBC cells in vivo. It is hypothesized that this novel combinatory strategy, named as Hu276/PD-1-DualADC, synergizes multiple chemo- and immuno-mediated anti-cancer mechanisms, i.e., direct cancer cell killing, tumoral immunity, tumoral cytokine, and immune checkpoint blockade, to enhance TNBC treatment efficacy. Three aims were proposed to test the hypothesis. Aim 1 will produce large-scale Hu276-DualADC carrying mertansine and imidazoquinoline, and characterize its affinity, TNBC-specificity, biodistribution and toxicity. Optimal treatment strategy will be determined in maximal tolerated dose and pharmacokinetics studies. Finally, anti-TNBC efficacy will be evaluated in primary xenograft models and distant metastatic models. Aim 2 will assess the synergistic effects of HuCD276/PD-1-DualADC in three immunocompetent models. The underlying mechanisms (proliferation, apoptosis, immune cell infiltration and activation, tumoral cytokine, and ICB restoration) will also be delineated. Aim 3 will fully evaluate the long-term therapeutic efficacy in metastatic syngeneic TNBC xenograft models post surgical resection and patient-derived xenograft (PDX) models. Pre-IND toxicology will also be investigated to collect preclinical data for future clinical trial launching. Successful completion of this project will provide a new strategy to treat aggressive TNBCs.