Cancer Immunotherapy Targeting Tn Antigen

摘要 肿瘤免疫治疗双特异性蛋白与工程化嵌合抗原受体T细胞 T)表现出显著的临床活动性，对B细胞恶性肿瘤的完全缓解率高达90%。 然而，将这两种治疗方法应用于绝大多数癌症类型是有限的。 受到多种因素的影响。首先，只有一小部分已知的细胞表面蛋白足以 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 恶性血液病；健康细胞的丧失不能轻易由干细胞前体细胞来补充。第二, 因为每个单独的双特异性蛋白和/或CAR T细胞只针对一种癌症类型、不同的双特异性 和/或CAR T细胞将需要针对每种癌症类型进行培养。这极大地增加了开发时间和 成本。第三，两种疗法都不能有效地针对最丰富和最广泛表达的细胞表面 已知的肿瘤抗原，即肿瘤相关糖类抗原(TACA‘s)。许多癌症特异性的 抗原不是蛋白质，而是在正常组织中有限表达或不表达的复杂碳水化合物。 事实上，糖基化改变几乎是癌症的普遍特征。虽然TACA已经被知道了几十年， 针对复杂碳水化合物的有效单抗的产生已被证明是非常 具有挑战性，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 这些问题并开发了一类针对TN抗原的新型免疫疗法，TN抗原是一种异常的 O-连接碳水化合物常见于许多实体和造血系肿瘤，但在正常人群中不存在 组织。我们将这项技术称为糖依赖T细胞招募器(Glytr，发音为 《闪闪发光》)。我们已经产生并优化了一种GlyTR双特异性蛋白，它可以1)与两种TN特异性结合 抗原和CD3，2)在TN+癌细胞存在而不是不存在的情况下激活T细胞，以及3)诱导T细胞 体外和体内对多种固体和液体癌细胞的依赖杀伤作用。而血清半衰期为~2小时， 这类似于FDA批准的需要持续静脉输注的双特异性蛋白质Blincyto (通过泵)超过28天(前9天住院)。为了避免GlyTR的这种繁琐的治疗方案， 在这里，我们建议通过增加人血清白蛋白(HSA)结构域来延长半衰期。人血清白蛋白的半衰期 是~3周，并已成功地与治疗性蛋白融合，显著延长半衰期，包括两个 FDA批准的治疗方法。在这里，我们建议将HSA基因融合到Glytr(HSA-Glytr)，并确认 结合TN抗原，杀癌活性和改善半衰期。具体地说，我们提出了以下两点 目标。目的1优化人血清白蛋白-甘氨酸受体双特异性蛋白的活性和药物开发。《目标2》探索 优化的人血清白蛋白-甘氨酸双特异性蛋白的有效性和安全性。如果成功，这些实验将允许 随后的IND使研究能够开发出一种全新的抗癌免疫疗法 能够通过一次治疗针对多个实体和造血系癌症。

Abstract Cancer immunotherapeutic bi-specific proteins and engineered Chimeric Antigen Receptor T cells (CAR T) have shown remarkable clinical activity, with complete response rates as high as ~90% for B cell malignancies. However, applying these two therapeutic approaches to the vast majority of cancer types is restricted by multiple factors. First, there are only a 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. Second, as each individual bi-specific protein and/or CAR T cell only target a single cancer type, different bi-specific and/or CAR T cells will need to be developed for each cancer type. This greatly increases development time and costs. Third, neither therapy is able to effectively target the most abundant and widely expressed cell surface cancer antigens known, namely Tumor associated carbohydrate antigens (TACA’s). Many cancer specific antigens are not proteins, but rather complex carbohydrates that have limited or no expression in normal tissues. Indeed, altered glycosylation is a near universal feature of cancer. While TACA’s have been known for decades, generation of effective 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 the Tn antigen, an abnormal O-linked carbohydrate common on many solid and hematopoietic cancers but not present on normal tissue. We have termed this technology as Glycan-dependent T cell Recruiter (GlyTR, pronounced ‘glitter’). We have generated and optimized a GlyTR bi-specific protein that 1) specifically binds to both Tn antigen and CD3, 2) activated T cells in the presence but not absence of Tn+ cancer cells and 3) induced T cell dependent killing of diverse solid and liquid cancer cells in vitro and in vivo. However, serum half-life was ~2 hrs, which is similar to the FDA approved bi-specific protein Blincyto that requires continuous intravenous infusion (via a pump) over 28-days (first 9 days in hospital). To avoid this cumbersome treatment regimen for GlyTR, here we propose to extend the half-life by adding a human-serum albumin (HSA) domain. The half-life of HSA is ~3 weeks and has been successfully fused to therapeutic proteins to markedly increase half-life, including two FDA approved therapeutics. Here we propose to genetically fuse HSA to GlyTR (HSA-GlyTR) and confirm binding to Tn antigen, cancer-killing activity and improved half-life. Specifically, we propose the following two Aims. Aim 1 optimizes the HSA-GlyTR bi-specific protein for activity and drug development. Aim 2 explores the efficacy and safety of the optimized HSA-GlyTR bi-specific protein. If successful, these experiments will allow subsequent IND enabling studies to develop an entire new class of cancer killing immunotherapeutic’s uniquely capable of targeting multiple solid and hematopoietic cancers with a single therapeutic.