Novel antibody-drug conjugates targeting T-cell lymphoma

翻译后摘要：T细胞淋巴瘤仍然是治疗的挑战，尤其是因为他们的显着 异质性Robert Orlovsky（Asylia的联合创始人）的实验室分析了外周血淋巴细胞的膜蛋白质组， 和皮肤T细胞淋巴瘤，并发现表面热休克蛋白（csHSP）-70， 一直过度表达。虽然HSP 70已被公认为细胞内应激反应伴侣，但其 在癌症中定位于细胞表面，正获得越来越多的关注。考虑到最近的临床成功 抗体-药物偶联物和细胞表面HSP 70的异常高和选择性过表达， T细胞淋巴瘤，我们总结了csHSP 70作为一个非常有吸引力的抗体药物靶点 因此，本发明提供了用于治疗这种恶性肿瘤的药物共辄物（ADC）。我们已经产生了高亲和力的单克隆抗体 （克隆239-87）特异性识别细胞表面HSP 70。在初步数据中，我们已经生成了 mAb 239-87与MMAE，其特异性结合T细胞NHL细胞系上的csHSP 70，但不结合正常 外周血来源的细胞。与MMAE连接，239-87-MMAE ADC显示出对两种细胞的细胞毒活性。 外周和皮肤T细胞淋巴瘤细胞系具有可比的，或更大的效力比领先的 临床ADC，维布妥昔单抗。 我们现在提出了一系列关键实验，以加速239-87 ADC的临床转化， T细胞淋巴瘤的治疗在目标1中，我们将人源化mAb 239-87，并优化药物样 当前小鼠克隆的特征。我们将研究引线与可溶性HSP 70和csHSP 70的结合 在野生型和基因组编辑的细胞模型中，以验证它们保留了csHSP 70亲和力和特异性。在特定 目的2，我们将克隆239-87 mAb转化为ADC。我们将首先综合一组ADC， 与MMAE、美登素、喜树碱和吡咯并苯并二氮杂卓连接的最佳先导抗体分子 与组织蛋白酶和酸可切割的接头，并比较它们对T细胞淋巴瘤细胞系的功效。接下来， 我们将对csHSP 70进行遗传调节，并进行内化和定量流动研究，以确定 最小csHSP 70表达需要看到抗癌功效。最后，我们将验证协同增效的潜力 我们的ADC和增强csHSP 70的药物之间的关系，如组氨酸脱乙酰酶抑制剂。在目标3中，我们 使用体内肿瘤细胞系衍生的模型验证克隆239-87 ADC的活性。接下来，我们将验证 在更生理相关的患者来源的异种移植物（PDX）模型中选择的最佳表现者的活性。 最后，我们将在这些PDX中进行药代动力学研究，以开始了解潜在的药物分布 和毒性参数。 综上所述，这些研究将提供一个概念验证，即HSP 70靶向ADC具有 作为有吸引力的候选药物开发所必需的功效和药理学特性，并使我们能够 推进新药毒理学研究，为I期临床试验做准备。

Abstract: T-cell lymphomas have remained treatment-challenging, not least because of their remarkable heterogeneity. The lab of Robert Orlovsky (co-founder of Asylia) analyzed the membrane proteome of peripheral and cutaneous T-cell lymphoma and discovered that surface Heat shock protein (csHSP)-70 as being consistently overexpressed. While HSP70 is well established as an intracellular stress-response chaperone, its localization to the cell surface in cancer, is gaining increased attention. Considering the recent clinical successes of antibody-drug conjugates and the exceptionally high and selective overexpression of cell surface HSP70 in T-cell lymphoma, we summarize that csHSP70 presents as an exceptionally attractive target for antibody drug conjugates (ADC) for the treatment of this malignancy. We have generated a high affinity monoclonal antibody (clone 239-87) specifically recognizing cell-surface HSP70. In preliminary data, we have generated an ADC of mAb 239-87 with MMAE, which specifically bound csHSP70 on T-cell NHL cell lines but did not bind normal peripheral blood-derived cells. Linked to MMAE, the 239-87-MMAE ADC showed cytotoxic activity against both peripheral and cutaneous T-cell lymphoma lines cell lines with comparable, or greater potency than the leading clinical ADC, brentuximab vedotin. We now propose a series of critical experiments to accelerate the clinical translation of 239-87 ADCs for the treatment of T-cell lymphoma. In aim 1, we will humanize mAb 239-87 and optimize the drug-like characteristics of the current murine clone. We will study binding of the leads to soluble HSP70, and to csHSP70 in wild-type and genome edited cell models to verify that they retain csHSP70 affinity and specificity. In specific aim 2, we will convert clone 239-87 mAb into an ADC. We will first synthesize a panel of ADCs based on the optimal lead antibody molecule linked to MMAE, a maytansine, a camptothecin, and a pyrrolobenzodiazepine with cathepsin- and acid-cleavable linkers and compare their efficacy against T-cell lymphoma cell lines. Next, we will genetically modulate csHSP70 and perform internalization and quantitative flow studies to determine the minimum csHSP70 expression needed to see anti-cancer efficacy. Finally, we will verify the potential for synergy between our ADCs and agents that enhance csHSP70, such as histione deacetylase inhibitors. In Aim 3, we will validate the activity of clone 239-87 ADCs using in vivo tumor cell line-derived models. Next, we will validate the activity of selected best performers in more physiologically relevant patient-derived xenograft (PDX) models. Finally, we will perform pharmacokinetic studies in these PDXs to begin to understand potential drug distribution and toxicity parameters. Taken together, these studies will provide a proof-of-concept that HSP70-targeted ADCs have the necessary efficacy and pharmacologic properties for development as attractive drug candidates and allow us to move forward with investigational new drug-enabling toxicology studies in preparation for phase I clinical testing.