N-glycosylation and Immunotherapy for cancer

项目摘要 几十年来，癌症的治疗一直依赖于手术切除、化疗和/或放疗。 最近，许多基于免疫的疗法为癌症治疗提供了有前途的新方法。 这些包括阻断T细胞抑制的检查点抑制剂（例如抗PD-1）， 将T细胞与癌细胞交联（例如双特异性T细胞嵌合体- BiTE）和T细胞工程化以表达 对癌细胞特异性的抗原受体（例如嵌合抗原受体T细胞或CAR T）。所有三 方法诱导T细胞介导的癌细胞杀伤。然而，双特异性的广泛发展 抗体和CAR T细胞的结合受到少量已知细胞表面蛋白的限制，这些蛋白足以 特异于癌症，以安全地允许抗体靶向。这对于实体癌尤其如此， 在造血系统恶性肿瘤中，健康细胞的损失不能容易地由干细胞祖细胞补充。一 该问题的解决方案是靶向癌症特异性聚糖抗原而不是蛋白质抗原。事实上， 糖基化几乎是癌症的普遍特征 表面癌抗原，同时在正常组织中也具有有限的表达或不表达。然而，一代 对复合碳水化合物具有特异性的单克隆抗体已被证明是非常具有挑战性的，极大地限制了 它们作为癌症免疫治疗靶点的有用性。我们在此建议解决这些问题， 开发了一类新型免疫治疗剂，其靶向N-连接的糖抗原， 绝大多数实体和造血系统癌症。我们将这些分子称为聚糖- 依赖性T细胞募集（GlyTR）技术。关键的是，GlyTR技术不利用抗体来 靶向碳水化合物癌抗原。初步数据表明，GlyTR双特异性蛋白1） 特异性结合人CD 3及其N-聚糖靶标，2）仅在存在下稳健活化的T细胞， 3）诱导T细胞依赖性杀伤癌细胞，EC 50低至5 pM，4）抑制癌细胞增殖， 在人源化NSG小鼠中建立的结肠癌异种移植物的体内生长，和5）不触发 小鼠体内的抗体为了进一步扩展这一方法，提出了以下目标。目标1将 表征靶向N-聚糖的人和小鼠反应性GlyTR双特异性蛋白。目标2将 使用人源化NSG小鼠检查靶向N-聚糖的GlyTR蛋白的功效和安全性。 目的3将使用具有以下缺陷的小鼠来检查靶向N-聚糖的GlyTR蛋白的功效和安全性： 正常的免疫系统如果成功的话，这些实验将为一个全新的 一类能够靶向多种实体癌和造血系统癌的癌症杀伤免疫球蛋白， 毒性极小。

Project Summary For decades, the treatment of cancer has relied on surgical resection, chemotherapy and/or radiotherapy. Recently, a number of immune based therapies have provided promising new approaches for cancer treatment. These include checkpoint inhibitors that block T cell suppression (eg anti-PD-1), bi-specific antibodies that cross-link T cells to cancer cells (eg Bi-specific T cell engagers – BiTE) and T cells engineered to express antigen receptors specific to cancer cells (eg Chimeric Antigen Receptor T cells or CAR T). All three approaches induce T cell mediated killing of cancer cells. However, widespread development of bi-specific antibodies and CAR T cells is limited by the small number of known cell-surface proteins that are sufficiently specific to cancer to safely allow targeting by antibodies. This is particularly true for solid cancers, where unlike hematopoietic malignancies, loss of healthy cells cannot be readily replenished by stem cell progenitors. A solution to this issue is to target cancer specific glycan antigens rather than protein antigens. Indeed, altered glycosylation is a near universal feature of cancer and represent the most abundant and widely expressed cell surface cancer antigens, while also having limited or no expression in normal tissue. However, generation of monoclonal antibodies specific to complex carbohydrates has proven to be very challenging, greatly limiting their usefulness as targets for cancer immunotherapy. Here we propose to address these issues and develop a novel class of immunotherapeutics that target an N-linked carbohydrate antigen common to the vast majority of solid and hematopoietic cancers. We have termed these molecules as Glycan- dependent T cell Recruiter (GlyTR) technology. Critically, GlyTR technology does not utilize antibodies to target carbohydrate cancer antigens. Preliminary data demonstrates that the GlyTR bi-specific protein 1) specifically bound to both human CD3 and its N-glycan target, 2) robustly activated T cells only in the presence of cancer cells, 3) induced T cell dependent killing of cancer cells with an EC50 as low as 5pM, 4) inhibited in vivo growth of established colon cancer xenografts in humanized NSG mice and 5) did not trigger production of antibodies in mice. To further expand on this approach, the following Aims are proposed. Aim 1 will characterize human and mouse reactive GlyTR bi-specific proteins targeting N-glycans. Aim 2 will examine the efficacy and safety of GlyTR proteins targeting N-glycans using humanized NSG mice. Aim 3 will examine the efficacy and safety of GlyTR proteins targeting N-glycans using mice with a normal immune system. If successful, these experiments will provide proof of principal data for an entire new class of cancer killing immunotherapeutic's capable of targeting multiple solid and hematopoietic cancers with minimal toxicity.