N-glycosylation and Immunotherapy for cancer

项目摘要 几十年来，癌症的治疗一直依靠手术切除、化疗和/或放射治疗。 最近，一些基于免疫的治疗方法为癌症的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(如抗PD-1)，双特异性抗体 将T细胞与癌细胞(如双特异性T细胞结合蛋白-BITE)和T细胞进行基因工程表达 针对癌细胞的抗原受体(如嵌合抗原受体T细胞或CAR T)。三个都是 方法诱导T细胞介导的对癌细胞的杀伤。然而，双特定的广泛发展 抗体和CAR T细胞受到少量已知细胞表面蛋白的限制 对癌症的特异性，以安全地允许通过抗体靶向。对于实体癌尤其如此，不同于 在恶性血液病中，健康细胞的丧失不能由干细胞前体细胞轻易补充。一个 这个问题的解决方案是针对癌症特异性糖链抗原，而不是蛋白质抗原。事实上，改变了 糖基化是癌症的一个几乎普遍的特征，代表着最丰富和最广泛表达的细胞 表面癌抗原，同时在正常组织中也有有限的表达或没有表达。然而，新一代 针对复杂碳水化合物的单抗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效性。在这里，我们建议解决这些问题，并 开发一种新型免疫疗法，靶向一种N-连接的碳水化合物抗原，该抗原与 绝大多数实体癌和造血癌。我们将这些分子命名为葡聚糖- 依赖T细胞招募器(GlyTR)技术。关键的是，GlyTR技术不利用抗体来 靶向碳水化合物癌症抗原。初步数据表明，GlyTR双特异性蛋白1) 特异性结合人CD3及其N-糖链靶标，2)只有在存在的情况下才能强效激活T细胞 在癌细胞中，3)以低至5 PM的EC50诱导T细胞依赖性杀伤癌细胞，4)抑制 人源化NSG小鼠体内已建立的结肠癌异种移植瘤的体内生长和5)不触发产生 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 针对N-糖链的人和鼠反应性GlyTR双特异性蛋白的特征。目标2将 利用人源化NSG小鼠检测针对N-糖链的GlyTR蛋白的有效性和安全性。 目的3将检测针对N-糖链的GlyTR蛋白的有效性和安全性。 正常的免疫系统。如果成功，这些实验将为整个新的 一种可靶向多种实体和造血细胞肿瘤的抗癌免疫治疗剂 毒性最小。

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.