Cancer Immunotherapy Targeting Tn Antigen

摘要 癌症免疫双特异性蛋白和工程化嵌合抗原受体T细胞（CAR T）显示出显著的临床活性，对于B细胞恶性肿瘤的完全反应率高达~90%。 然而，将这两种治疗方法应用于绝大多数癌症类型受到限制 受到多种因素的影响。首先，只有少数已知的细胞表面蛋白质能够充分地 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， 造血系统恶性肿瘤;健康细胞的损失不能容易地由干细胞祖细胞补充。第二、 由于每种单独的双特异性蛋白和/或CAR T细胞仅靶向单一癌症类型，因此不同的双特异性蛋白和/或CAR T细胞可以靶向不同的癌症类型。 和/或CAR T细胞将需要为每种癌症类型开发。这大大增加了开发时间， 成本第三，这两种疗法都不能有效地靶向最丰富和最广泛表达的细胞表面 已知的癌症抗原，即肿瘤相关碳水化合物抗原（TACA）。许多癌症特异性 抗原不是蛋白质，而是复杂的碳水化合物，在正常组织中表达有限或不表达。 事实上，糖基化改变是癌症的一个几乎普遍的特征。虽然TACA已经知道了几十年， 产生对复合碳水化合物具有特异性的有效单克隆抗体已被证明是非常 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用性。在这里，我们建议解决 这些问题，并开发了一种新的免疫治疗药物，靶向Tn抗原，一种异常的 O-连接的碳水化合物常见于许多实体和造血系统癌症，但不存在于正常 组织.我们将这种技术称为聚糖依赖性T细胞募集（GlyTR，发音为 'glitter'）。我们已经产生并优化了GlyTR双特异性蛋白，其1）特异性结合两种Tn 抗原和CD 3，2）在Tn+癌细胞存在但不缺乏的情况下活化T细胞，和3）诱导T细胞 在体外和体内依赖性杀死不同的固体和液体癌细胞。然而，血清半衰期约为2小时， 它类似于FDA批准的需要持续静脉输注的双特异性蛋白Blincyto (via泵）超过28天（住院前9天）。为了避免这种繁琐的GlyTR治疗方案， 在此，我们提出通过加入人血清白蛋白（HSA）结构域来延长半衰期。HSA的半衰期 约3周，并已成功融合到治疗性蛋白质，以显着增加半衰期，包括两个 FDA批准的治疗方法。在这里，我们提出将HSA与GlyTR基因融合（HSA-GlyTR），并证实 与Tn抗原结合，具有抗癌活性和改善的半衰期。具体而言，我们提出以下两点 目标。目的1优化HSA-GlyTR双特异性蛋白的活性和药物开发。目标2探索 优化的HSA-GlyTR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癌症杀伤免疫系统， 能够用单一治疗剂靶向多种实体和造血系统癌症。

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.