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

摘要 三阴性乳腺癌是一种侵袭性和难治性的乳腺癌亚型。 目前可用的临床选择有限。尽管最初对铂基新佐剂有反应 化疗后，转移性TNBC患者很少能在确诊后存活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。尽管广泛的研究努力已经 提供了一些临床选择，开发了可以有效根除广泛的 TNBC细胞种群仍然是一个挑战。抗体为基础的药物，如抗体-药物结合物(ADC) 是治疗TNBC最有前景的靶向治疗方法。滋养层细胞表面抗原2 TROP2是一种在超过80%的TNBC中高表达的蛋白质，是一个有前途的分子靶点。 最近，一种TROP2靶向抗体-药物结合物(ADC)Sacituzumab Govitecan(Trodelvy)获得批准 加速批准转移性TNBC的第三线或更晚的治疗。然而，TNBC经常复发。 TROP2在肿瘤内的异质性表达与肿瘤耐药 根据其有效载荷，SN-38可能会导致较差的临床结果。此外，接受特罗维治疗的患者通常 有不良反应，包括中性粒细胞减少症。从而增强疗效，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要 适用于具有异质性TNBC的患者。我们开发了新的ADC技术，包括：1)多肽 用于最大化ADC治疗指数的连接物，以及2)双功能化学间隔物和特定部位的 用于产生包含两个不同有效载荷的均质ADC的共轭(称为双药ADC)。 具体地说，我们的双药adc含有两种不同的抗有丝分裂药物，显示出极大的安全性。 在HER2低度难治性乳腺癌小鼠模型中的疗效与FDA批准的ADC的比较 卡西拉和恩赫图。基于这一成功和我们的初步数据，我们假设优化设计 抗TROP2双药ADC可有效根除多种实体和转移性TNBC肿瘤。在AIM 1，我们将通过基于细胞的分析来研究我们的新连接子是如何在癌细胞和正常细胞中处理的， 蛋白质组学分析和体内生物分布分析。在目标2中，我们将合成新型的dna-烷化剂。 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双重- 药物ADCs在结合和与抗体结合的药物数量上各不相同。在目标3中，我们 评估双药ADCs在TNBC小鼠模型中的毒性效果。本项目圆满完成 将为这种新的药物类别奠定基础，具有克服异质性和抗药性的潜力 TNBC，这可能最终彻底改变目前对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.