Mechanisms of action of a TCR-like antibody to WT1

描述(申请人提供)：这项建议建立在这个R01发现用于治疗白血病的裸露和放射性标记抗体并将其转化为临床的20年的基础上。在这里，我们将我们的目标推进到一个更困难、更耐人寻味的目标 单抗：细胞内癌蛋白Wilms‘s Tumor 1(WT1)，在大多数白血病和许多其他癌症类型的肿瘤细胞中选择性表达。我们和其他人之前已经发现了来自WT1蛋白的多肽，这些多肽可以诱导HLA-A0201限制性的细胞毒性CD8 T细胞，能够通过TCR识别杀死肿瘤细胞。我们推测，针对WT1多肽/人类白细胞抗原A2复合体的单抗(模拟TCR)将是研究WT1免疫生物学的一种新的有用工具，并可能成为一种有效的治疗药物。我们发现了一种针对细胞表面的WT1多肽的人细胞毒性单抗(ESK1)。我们已经提出了一种新的治疗癌症患者的方法，通过靶向一种原本“无法下药”的细胞内癌蛋白。我们发现的单抗的临床前表现，超过了我们作为一种可能的治疗先导药物的预期，但我们需要充分了解其作用机制和宿主/细胞耐药，以便利用其在人类中的活性。这一方法也将为其他肿瘤特异性细胞内靶点提供概念证明。在这里，在街对面两个互补的实验室(Scheinberg和Ravetch)的合作下，我们试图更详细地了解这种新制剂的作用机制，并将抗体作为一种高度特异的工具来探索抗原呈递和效应器功能之间关系的某些方面。目的1：在现有的人白血病和实体瘤小鼠模型中，确定对Esk作用产生抵抗的可能机制。ESK1在体外和体内的治疗活性是FC依赖的，ADCC主导这一活性。我们将探讨如何避免治疗活性导致复发，包括可能的靶细胞、制剂和宿主的药代动力学以及宿主效应细胞的原因。目的：通过使用标记的WT1多肽-生物素探针，了解表位数目对ESK1功能的影响。我们问：如何用这么少的表位(我们的靶细胞显示0.1-1.0%的典型单抗表位)实现治疗？这个表位编号有下限吗？基于我们的初步数据，我们假设为ADCC(目前未知)招募免疫效应细胞所需的mAb结合事件的数量将远远低于先前预期的数量(&lt；100/cell)。如果这是真的，它将对抗体特异性、活性和治疗产生深远的影响。我们还将探索使用阿尔法发射形式(只需点击一次即可杀死)治疗所需的表位位置的最低限度。目的3：建立一种新的人源化Fc？R小鼠作为肿瘤模型，并在其中鉴定ESK1的治疗活性。我们能创造一个更相关的小鼠模型来测试人类单抗吗？这一工具将对许多研究人员在临床前设计和研究裸露的人类mAb疗法至关重要。在模型中，将确定关键的FCR承载效应细胞。

DESCRIPTION (provided by applicant): This proposal builds on the 20 years of this R01 discovering naked and radiolabeled antibodies for the treatment of leukemia and translating them into the clinic. Here we have advanced our goals to a more difficult and intriguing target for a mAb: the intracellular oncoprotein Wilms' tumor 1 (WT1), which is selectively expressed in neoplastic cells of most leukemias, and many other cancer types. We, and others, had previously identified peptides derived from the WT1 protein that induce HLA-A0201-restricted cytotoxic CD8 T cells, capable of killing tumor cells via TCR recognition. We hypothesized that a mAb specific for the WT1 peptide/HLA-A2 complex (mimicking a TCR) would be a novel and useful tool to study the immunobiology of WT1 and possibly an effective therapeutic agent. We discovered a human, cytotoxic mAb (ESK1) directed to a WT1 peptide that is presented on cell surface HLA-A0201. We have proposed a new approach to the treatment of patients with cancers by targeting an otherwise "un-druggable" intracellular oncoprotein. The preclinical performance of the mAb we discovered, more than exceeded our expectations as a possible therapeutic lead drug, but we need to fully understand its mechanism of action and host/cellular resistance in order to exploit its activity in humans. This approach would also provide a proof of concept for other tumor-specific, intracellular targets. Here, in a collaboration of two complementary labs across the street from each other (Scheinberg and Ravetch), we seek to understand in great detail the mechanisms of action of this new agent and to use the antibody as a highly specific tool to probe some aspects of the relationship between antigen presentation and effector function. AIM 1: To determine the mechanisms of possible resistance to ESK action in current mouse models of human leukemia and solid tumors. ESK1 therapeutic activity in vitro and in vivo is Fc-dependent, and ADCC dominates this activity. We will explore how the therapeutic activity is evaded causing relapse including possible target cell, agent and host pharmacokinetic, and host effector cell causes. AIM 2: By use of a tagged WT1 peptide- biotin probe, to understand the importance of epitope site number on the function of ESK1 in vivo and in vitro. We ask: How can therapy be achieved with so few epitopes (our target cells display 0.1-1.0% of typical mAb epitopes)? Is there a floor to this epitope number? Based on our preliminary data, we hypothesize that the number of mAb binding events required to recruit immune effector cells for ADCC (now unknown) will be far lower (<100/cell) than previously expected. If this is true, it would have profound implications for antibody specificity, activity ad therapy. We will also explore the minimal floor of epitope sites needed for therapy with an alpha emitting form (which requires only 1 hit to kill). AIM 3: To create a new, humanized Fc?R mouse as a model of cancer and characterize within it, ESK1 therapeutic activity. Can we create a more relevant mouse model to test human mAb? This tool will be critical to many investigators for the preclinical design and study of naked human mAb therapies. Within the model, the key FcR bearing effector cells will be determined.