Validation of Siglec-6 as a novel target for cancer immunotherapy

项目摘要 为了响应PAR-16-176的资助机会公告，Christoph Rader博士的这份联合提案 来自斯克里普斯研究所（木星，佛罗里达州）和Natarajan Muthusamy来自俄亥俄州州立大学 （哥伦布，OH）提出验证用于慢性淋巴细胞白血病（CLL）的免疫疗法的新靶点。 作为所提出的研究的一部分，我们将开发新型T细胞接合双特异性抗体（biAb）和一种新的免疫抑制剂。 转基因小鼠模型，以评估和启动进一步的临床前开发。我们的研究团队将产生， 验证和递送新的biAb，其被专门设计用于募集和激活T细胞以进行选择性和特异性免疫应答。 有效根除CLL患者中的恶性B细胞。CLL是美国最常见的白血病， 终身风险为0.6%，年死亡率约为5000人。尽管免疫化疗的出现和小 分子激酶抑制剂，对于新诊断的， 难治性和复发性CLL患者。事实上，目前没有食品和药物管理局（FDA）- 在CLL中允许选择性靶向恶性B细胞而不损害 健康的细胞和组织。值得注意的是，异基因造血干细胞移植（alloHSCT）仍然存在。 CLL的唯一潜在治愈性治疗，强调了免疫疗法无与伦比的能力。 为了寻找强大的移植物抗白血病（GVL）反应的潜在贡献者，我们设计了一个协调一致的 抗体药物和靶点发现策略，通过从治愈的HSCT患者产生第一个alloHSCT后抗体文库， CLL患者并通过针对原代CLL细胞的噬菌体展示来选择它。我们发现了一组完全的人类 在一个实施方案中，所述方法包括在原代CLL细胞上使用具有共同抗原的单克隆抗体（mAb）。在尚未公开的工作中， 在该提案中，我们首次将新靶标确定为Siglec-6。在健康个体中， Siglec-6的表达高度限于胎盘、肥大细胞和某些B细胞亚群。其表达 对慢性淋巴细胞白血病细胞的作用尚未见报道。我们提出的研究，建立在我们的综合 在抗体工程和转基因小鼠模型的优势，从这两个临床样品的可用性 未治疗和治疗的CLL患者进行离体和体内临床前研究，以及我们广泛的 支持数据，目的是严格检验Siglec-6可以作为T细胞免疫调节的靶点的假设。 招募和激活biAb以介导CLL患者中白血病细胞的有效和安全根除。的 这一目标的实验性执行将基于两个具体目标。在目标1中，我们将构建和测试一个 Siglec-6 x CD 3 biAb组，用于通过自体T细胞离体和体内杀死原代CLL细胞。在目标2中， 将开发表达转基因人Siglec-6的新型CLL小鼠模型，并将其用于体内 替代Siglec-6 x CD 3 biAb在天然和活性免疫系统中的评价。总的来说，我们的研究 探索了CLL靶向治疗的独特途径，通过用新药物和新药物询问新靶点， 小鼠模型，以评估和启动进一步的临床前开发。

PROJECT SUMMARY In response to funding opportunity announcement PAR-16-176, this joint proposal by Drs. Christoph Rader from The Scripps Research Institute (Jupiter, FL) and Natarajan Muthusamy from The Ohio State University (Columbus, OH) proposes to validate a novel target for immunotherapy of chronic lymphocytic leukemia (CLL). As part of the proposed study, we will develop novel T-cell engaging bispecific antibodies (biAbs) and a novel transgenic mouse model to assess and initiate further preclinical development. Our research team will generate, validate, and deliver novel biAbs that are specifically designed to recruit and activate T cells for selective and potent eradication of malignant B cells in CLL patients. CLL is the most common leukemia in the U.S. with a lifetime risk of 0.6% and an annual mortality of ~5,000. Despite the advent of immunochemotherapy and small molecule kinase inhibitors, there remains a great need for potent and safe treatment options for newly diagnosed, refractory, and relapsed CLL patients. In fact, there are currently no Food and Drug Administration (FDA)- approved therapeutic modalities in CLL that allow for selective targeting of malignant B cells without harming healthy cells and tissues. Notably, allogeneic hematopoietic stem cell transplantation (alloHSCT) has remained the only potentially curative treatment of CLL, underscoring the unmatched capability of immunotherapy. Pursuing potential contributors of the powerful graft-versus-leukemia (GVL) response, we devised a concerted antibody drug and target discovery strategy by generating the first post-alloHSCT antibody library from a cured CLL patient and selecting it by phage display against primary CLL cells. We identified a panel of fully human monoclonal antibodies (mAbs) with a common antigen on primary CLL cells. In still undisclosed work presented for the first time in this proposal, we identified the new target as Siglec-6. In healthy individuals, the expression of Siglec-6 is highly restricted to the placenta, to mast cells, and to certain B-cell subpopulations. Its expression on leukemia cells in CLL has not been reported previously. Our proposed study, which builds on our combined strengths in antibody engineering and transgenic mouse models, on the availability of clinical samples from both untreated and treated CLL patients for ex vivo and in vivo preclinical studies, as well as on our extensive supporting data, has the objective to rigorously test the hypothesis that Siglec-6 can serve as a target for T-cell recruiting and activating biAbs to mediate potent and safe eradication of leukemia cells in CLL patients. The experimental execution of this objective will be based on two Specific Aims. In Aim 1, we will build and test a panel of Siglec-6 x CD3 biAbs for primary CLL cell killing by autologous T-cells ex vivo and in vivo. In Aim 2, we will develop a novel CLL mouse model expressing transgenic human Siglec-6 and employ it for the in vivo evaluation of surrogate Siglec-6 x CD3 biAbs in a native and competent immune system. Collectively, our study explores a unique route for targeted therapy of CLL by interrogating a novel target with novel agents and a novel mouse model to assess and initiate further preclinical development.