Mechanisms of action of a TCR-like antibody to WT1

描述（由申请人提供）：本提案建立在R 01发现用于治疗白血病的裸和放射性标记抗体并将其转化为临床的20年基础上。在这里，我们已经把我们的目标推进到一个更困难和更有趣的目标， a mAb：细胞内癌蛋白Wilms' tumor 1（WT 1），其选择性地在大多数白血病和许多其它癌症类型的肿瘤细胞中表达。我们和其他人先前已经鉴定了来自WT 1蛋白的肽，其诱导HLA-A0201限制性细胞毒性CD 8 T细胞，能够通过TCR识别杀死肿瘤细胞。我们假设特异性针对WT 1肽/HLA-A2复合物（模拟TCR）的mAb将是研究WT 1免疫生物学的一种新的有用工具，并可能是一种有效的治疗剂。我们发现了一种针对细胞表面HLA-A0201上呈递的WT 1肽的人细胞毒性mAb（ESK 1）。我们已经提出了一种新的方法来治疗癌症患者，通过靶向一个否则“非药物”的细胞内癌蛋白。我们发现的mAb的临床前性能超过了我们作为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/细胞抗性，以利用其在人体中的活性。这种方法还将为其他肿瘤特异性细胞内靶点提供概念验证。在这里，在街对面的两个互补实验室（Scheinberg和Ravetch）的合作中，我们试图详细了解这种新试剂的作用机制，并使用抗体作为高度特异性的工具来探测抗原呈递和效应器功能之间关系的某些方面。目的1：确定目前人类白血病和实体瘤小鼠模型对埃斯克作用的可能耐药机制。ESK 1在体外和体内的治疗活性是Fc依赖性的，并且ADCC主导该活性。我们将探讨治疗活性是如何被逃避导致复发的，包括可能的靶细胞、药物和宿主药代动力学以及宿主效应细胞原因。目的2：利用WT 1肽-生物素标记探针，了解表位数目对ESK 1体内外功能的重要性。我们问：如何用如此少的表位实现治疗（我们的靶细胞显示0.1-1.0%的典型mAb表位）？这个表位数有下限吗？基于我们的初步数据，我们假设募集免疫效应细胞用于ADCC（目前未知）所需的mAb结合事件的数量将远低于（<100个/细胞）先前的预期。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。我们还将探索用α发射形式治疗所需的表位位点的最小下限（其仅需要1次击中即可杀死）。目的3：创造一个新的，人源化的Fc？R小鼠作为癌症模型，并在其中表征ESK 1的治疗活性。我们能否建立一个更相关的小鼠模型来测试人类mAb？该工具对于许多研究人员进行裸人mAb疗法的临床前设计和研究至关重要。在该模型中，将确定携带关键FcR的效应细胞。

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.