Antibody-dual drug conjugates for eradicating triple-negative breast cancer with heterogeneity

摘要 三阴性乳腺癌（TNBC）是乳腺癌的侵袭性和难治性亚型， 目前临床选择有限。虽然最初对铂类新辅助治疗有反应， 化疗，转移性TNBC患者很少在诊断后存活5年。大多数TNBC肿瘤 由具有不同基因表达谱的异质细胞群体组成。这种异质性 导致耐药性、早期复发和治疗失败。尽管广泛的研究工作 提供了一些临床选择，开发了可以有效根除各种疾病的治疗方法， TNBC细胞群仍然是一个挑战。基于抗体的药物，例如抗体-药物缀合物（ADC） 是治疗TNBC的最有前途的靶向治疗方式。滋养层细胞表面抗原2 TROP2是一种在超过80%的TNBC中过表达的蛋白质，代表了一种有前途的分子靶标。 最近，Sacituzumab Govitecan（Trodelvy），一种靶向TROP2的抗体-药物偶联物（ADC），被授予 加速批准转移性TNBC的三线或更高线治疗。然而，TNBC经常复发 持续治疗后，TROP2在肿瘤内的异质性表达和耐药诱导 SN-38的有效载荷可能导致临床结果不佳。此外，接受Trodelvy治疗的患者通常 患有包括中性粒细胞减少症在内的副作用。从而提高疗效，扩大靶向范围， 确保这类药物的安全性仍然是开发安全有效疗法的关键临床需求 对于异质性TNBC患者。我们开发了新的ADC技术，包括：1）肽 用于使ADC治疗指数最大化的连接子，和2）双功能化学间隔子和位点特异性连接子。 在一些实施方案中，药物缀合用于产生含有两种不同有效载荷的均质ADC（称为双药物ADC）。 具体而言，我们的含有两种不同抗有丝分裂剂的双药ADC显示出极大的安全性改善， 与FDA批准的ADC相比， 卡德西拉和恩赫图基于这一成功和我们的初步数据，我们假设， 抗TR0P2双药ADC有效地根除广泛的实体和转移性TNBC肿瘤。在aim中 1，我们将通过基于细胞的测定研究我们的新型接头如何在癌症和正常细胞中加工， 蛋白质组学分析和体内生物分布分析。目的二是合成新型的DNA烷基化剂 具有不同化学性质的有效载荷和增效剂。然后我们将准备一系列抗TROP2双- 药物ADC的组合和与抗体缀合的药物的数量不同。在目标3中， 评估双药ADC在TNBC小鼠模型中的毒性功效。成功完成该项目 将为这种新型药物奠定基础，有可能克服异质性和耐药性。 TNBC，这可能最终彻底改变目前这种毁灭性疾病的治疗策略。

ABSTRACT Triple-negative breast cancer (TNBC) is an aggressive and refractory subtype of breast cancer for which limited clinical options are currently available. Although initially responsive to platinum-based neoadjuvant chemotherapy, patients with metastatic TNBC rarely survive 5 years after diagnosis. Most TNBC tumors consist of heterogeneous cell populations with different gene expression profiles. This heterogeneity contributes to drug resistance, early relapse, and therapeutic failure. Although extensive research efforts have made a few clinical options available, developing therapeutics that can effectively eradicate a broad range of TNBC cell populations remains a challenge. Antibody-based drugs such as antibody-drug conjugates (ADCs) are the most promising targeted therapeutic modality for treating TNBC. Trophoblast cell-surface antigen 2 (TROP2) is a protein overexpressed in more than 80% of TNBC, representing a promising molecular target. Recently, Sacituzumab Govitecan (Trodelvy), a TROP2-targeted antibody-drug conjugate (ADC), was granted accelerated approval for the third or later line of treatment of metastatic TNBC. However, TNBC often relapses after continuous treatment; the intratumor heterogeneous expression of TROP2 and drug resistance induced by its payload SN-38 likely contribute to poor clinical outcomes. In addition, patients treated with Trodelvy often suffer from adverse effects, including neutropenia. Thus, enhancing the efficacy, expanding the target scope, and ensuring the safety of this drug class remain critical clinical needs to develop safe and effective therapies for patients with heterogeneous TNBC. We have developed novel ADC technologies, including: 1) peptide linkers for maximizing ADC therapeutic index, and 2) bifunctional chemical spacers and a site-specific conjugation for generating homogeneous ADCs containing two distinct payloads (termed dual-drug ADCs). Specifically, our dual-drug ADCs containing two different antimitotic agents showed greatly improved safety and efficacy in mouse models of HER2-low refractory breast cancer compared with the FDA-approved ADCs Kadcyla and Enhertu. Based on this success and our preliminary data, we hypothesize that optimally designed anti-TROP2 dual-drug ADCs effectively eradicate a broad range of solid and metastatic TNBC tumors. In Aim 1, we will investigate how our novel linkers are processed in cancer and normal cells by cell-based assays, proteomic analysis, and in vivo biodistribution analysis. In Aim 2, we will synthesize novel DNA-alkylator payloads and potentiators with varying chemical properties. We will then prepare a series of anti-TROP2 dual- drug ADCs varying in the combination and the number of drugs conjugated to the antibody. In Aim 3, we evaluate the dual-drug ADCs for toxicity efficacy in TNBC mouse models. Successful completion of this project will lay the foundations for this novel drug class with the potential to overcome heterogeneity and resistance of TNBC, which may eventually revolutionize current therapeutic strategies for this devastating disease.