Mechanisms of Action of a TCR-like mAb Directed Against WT1

 描述(申请人提供)：Wilms Tumor 1(WT1)蛋白在多种癌症类型中广泛表达，但在成人组织中很少表达。WT1参与了肿瘤形成过程和癌症干细胞。然而，由于WT1是一种细胞内转录因子，针对WT1蛋白的治疗仍然难以捉摸。我们实验室已经发现并开发了一种T细胞受体(TCR)样单抗(MAb)，它能在癌细胞表面表达的主要组织相容性复合体(MHC)I类单倍型HLA-A02的背景下与WT1衍生的多肽特异性结合。这一单抗ESK1是一种与该表位具有高亲和力的人IgG1，并在体内显示了显著的抗癌活性。ESK1的疗效促使其从实验室发展到临床使用。因此，了解这种抗体在体内的作用机制，以便选择从该疗法中受益最大的患者，确定扩大疗效的方法，并预测耐药和/或毒性机制，对我们来说是很重要的。体内外研究表明，Fc-γ受体(Fc-γ-Rs)介导的抗体依赖的细胞毒作用在ESK1活性中起主导作用，并排除了Fc非依赖的机制。商业上可用的单抗针对的是癌细胞表面的数万到数十万个表位，而ESK1只针对数百到几千个表位。尽管这些数字很低，ESK1在体内显示出惊人的清除肿瘤负担的能力，这使我们相信ADCC比之前认为的更强大。因此，在这个项目的第一个目标中，我们将量化 为促进ADCC效应细胞的招募和激活，ESK1结合所需的最少表位数量。识别这个结合位点号，我们预测它是 每个细胞少于100个，不仅将使我们了解表位数量在患者选择和预测毒性方面的潜在影响，而且还将支持进一步开发针对其他低密度癌症特异性表位的治疗性单抗。在人类中，NK细胞是ADCC的主要介质，而小鼠NK细胞几乎不表达低亲和力激活的Fcγ受体，这使得这些细胞对于小鼠成功的抗体治疗几乎是必不可少的。由于单抗治疗在进入人体试验之前在小鼠模型中得到验证，这可能会导致观察到的疗效、毒性和已确定的治疗机制存在很大差异。因此，该项目还将首次开发一种新的小鼠模型，以适当地评价人单抗：一种免疫缺陷的、表达人FcγR的小鼠。该小鼠模型将用于验证ESK1的治疗活性，以及确定哪些人Fcγ受体和免疫效应细胞参与其抗肿瘤活性。这些信息对于确定如何最好地设计ESK1的Fc区域以促进最有效的抗肿瘤反应以及适当地选择患者(例如根据他们治疗后的免疫细胞含量或根据疾病类型)以增加临床试验的成功机会至关重要。

 DESCRIPTION (provided by applicant): The Wilms Tumor 1 (WT1) protein is widely expressed across a variety of cancer types, but is rarely expressed in adult tissues. WT1 is involved in the oncogenic process and in cancer stem cells. However, because WT1 is an intracellular transcription factor, therapies that target the WT1 protein have remained elusive. Our lab has discovered and developed a T-cell receptor (TCR)-like monoclonal antibody (mAb) that specifically binds to a WT1-derived peptide in the context of the major histocompatibility complex (MHC) class I haplotype HLA-A02 expressed on cancer cell surfaces. This mAb, ESK1, is a human IgG1 that has high affinity for this epitope and has demonstrated significant anti-cancer potency in vivo. The efficacy of ESK1 has prompted its movement from the laboratory to development for clinical use. Therefore, it is important for us to understand the mechanism of this antibody in vivo in order to select patients that will benefit most from this therapy, to determine ways to amplify therapeutic efficacy, and to predict mechanisms of resistance and/or toxicities. Studies in vitro and in vivo have demonstrated the dominant role of antibody-dependent cellular cytotoxicity (ADCC) mediated by Fcγ receptors (FcγRs) in ESK1 activity and have ruled out Fc-independent mechanisms. While commercially available mAbs target tens of thousands to hundreds of thousands of epitopes on a cancer cell surface, ESK1 only targets hundreds to a few thousand epitopes. Despite these low numbers, ESK1 exhibits strikingly potent clearance of tumor burden in vivo, which led us to believe that ADCC is more powerful than previously thought. Therefore, in the first aim of this project, we will quantify the minimum number of epitopes required for ESK1 to bind in order to promote the recruitment and activation of effector cells for ADCC. Identifying this binding site number, which we predict to be less than one hundred per cell, will not only inform us on the potential impact of epitope site number in patient selection and in predicting toxicity, but will also support the further development of therapeutic mAbs against other low density cancer specific epitopes. NK cells are the main mediators of ADCC in humans, yet murine NK cells express few low-affinity activating FcγRs, making these cells virtually dispensable for successful antibody therapy in a mouse. As mAb therapies are validated in mouse models before moving to human trials, this may lead to large discrepancies in the observed efficacy, toxicities, and mechanisms of therapy identified. Therefore, this project will also develop a novel mouse model for appropriately evaluating human mAbs for the first time: an immunodeficient, human FcγR-expressing mouse. This mouse model will be used to validate the therapeutic activity of ESK1, as well as to determine which human FcγRs and immune effector cells are involved in its anti-tumor activity. This information will be essential to determine ways to best engineer the Fc region of ESK1 to promote the most effective anti-tumor response, as well as to properly select patients (e.g. based on their immune cell content post therapy or according to disease type) for increasing the chance of success in clinical trials.