Strategy for specific delivery of antisense oligonucleotides to T cells

项目摘要 反义寡核苷酸（ASO）是有前途的药物，因为它们具有改变疾病表达的潜力。 相关基因，包括那些以前被认为是“坚不可摧”的基因。尽管他们的临床潜力，他们的成功 由于输送ASO的局限性， 除了肝脏、肌肉和中枢神经系统（CNS）以外的组织，这些组织是富含 ASO的分布或特定的交付方法。不幸的是，应用于免疫条件或 可受益于免疫调节的病症（例如，癌症，疫苗）由于缺乏 将ASO递送至体内免疫细胞，特别是T细胞的有效工具。因此， 可以增强阿索递送至T细胞的效率和特异性仍然是一个关键的未满足的需求。 为了满足这一关键需求，ABS正在开发一种模块化交付平台，专门针对T细胞的ASO 利用针对T细胞特异性细胞表面受体的缀合的单特异性单克隆抗体（mmAb）。 mmAb是高度特异性的抗体，其已经针对人蛋白质组进行了筛选，并被选择用于 它们与靶蛋白的唯一结合。这种选择性筛选通常不用于开发 单克隆抗体，导致抗体结合到其他蛋白质，除了他们的预期目标，从而 增强了由于脱靶结合引起的潜在毒性作用。因此，本文提出的mmAb具有 增强ASO向T细胞的递送同时降低抗体缀合物的潜在毒性作用的潜力。 ABS的T细胞特异性递送平台利用了T细胞主导的程序化表达。 细胞死亡蛋白1（PDCD 1、PD 1、CD 279）和白细胞介素2受体亚基β（IL 2 RB、CD 122）。除了 它们的T细胞主导表达，它们在T细胞中的生物学作用使它们成为有吸引力的候选物。PDCD 1是 在肿瘤浸润性T细胞中高度表达，并介导这些肿瘤反应性T细胞的抑制，从而 阻碍了它们杀死癌细胞的功效。因此，PDCD 1抗体在增强免疫应答中可能具有双重作用。 通过将免疫调节性ASO导向这些关键细胞，同时也减轻PDCD 1- 介导的肿瘤反应性T细胞的抑制。IL 2 RB促进T细胞中受体介导的内吞作用，和 在此，我们利用该功能靶向并驱动特异性结合的阿索在T细胞中的内化。 该I期提案的目标是测试PDCD 1和IL 2 RB mmAbs对免疫应答的效率和特异性。 将缀合的ASO离体和在小鼠中递送至T细胞。成功完成这一目标将验证 ABS的T细胞特异性递送平台在体内将ASO递送至T细胞的效用。这个模块化的交付平台 将对人类健康产生广泛的影响，因为它广泛适用于治疗癌症，免疫缺陷， 自身免疫性疾病和感染性疾病，以及在疫苗开发中的应用。在随后的第二阶段提案中， 我们将创建最佳mmAb的单特异性纳米抗体，以进一步提高其治疗指数，并测试 其在小鼠和非人灵长类动物中递送ASO至T细胞的功效和安全性。

PROJECT SUMMARY Antisense oligonucleotides (ASOs) are promising drugs given their potential to modify expression of disease- related genes, including those previously considered ‘undruggable’. Despite their clinical potential, their success has been limited to hepatic, muscular and neurodegenerative conditions due to limitations with delivering ASOs to tissues other than liver, muscle and the central nervous system (CNS), which are tissues with either rich distribution of ASOs or specific delivery methods. Unfortunately, applications to immunological conditions or conditions that could benefit from immuno-modulation (e.g., cancer, vaccines) have been limited due to the lack of effective tools to deliver ASOs to immune cells in vivo, in particular to T cells. Accordingly, delivery tools that could enhance the efficiency and specificity of ASO delivery to T cells remain a critical unmet need. To address this critical need, ABS is developing a modular delivery platform to target ASOs specifically to T cells utilizing conjugated mono-specific monoclonal antibodies (mmAbs) against T cell-specific cell surface receptors. mmAbs are highly specific antibodies that have been screened against the human proteome and selected for their exclusive binding to the target protein. Such selectivity screen is not usually employed in the development of monoclonal antibodies, resulting in antibodies that bind to other proteins besides their intended target, thereby enhancing potentially toxic effects due to off-target binding. Accordingly, the mmAbs proposed here have the potential to enhance delivery of ASOs to T cells while reducing potential toxic effects of the antibody conjugate. ABS’ T cell-specific delivery platform takes advantage of the T cell-predominant expression of the Programmed Cell Death Protein 1 (PDCD1, PD1, CD279) and Interleukin 2 Receptor Subunit Beta (IL2RB, CD122). Besides their T cell-predominant expression, their biological roles in T cells make them attractive candidates. PDCD1 is highly expressed in tumor-infiltrating T cells and mediates suppression of these tumor-reactive T cells, thereby hindering their efficacy to kill cancer cells. Accordingly, -PDCD1 antibodies could have a dual role in enhancing tumor reactivity by directing immuno-modulatory ASOs to these critical cells, while also relieving the PDCD1- mediated suppression of tumor-reactive T cells. IL2RB promotes receptor-mediated endocytosis in T cells, and here we leverage this function to target and drive internalization of the conjugated ASO specifically in T cells. The goal of this Phase I proposal is to test the efficiency and specificity of -PDCD1 and -IL2RB mmAbs to deliver the conjugated ASOs to T cells ex vivo and in mice. Successful completion of this goal will validate the utility of ABS’ T cell-specific delivery platform to deliver ASOs to T cells in vivo. This modular delivery platform will have broad impact on human health given its wide applicability for treatment of cancers, immunodeficiencies, autoimmune and infectious diseases, and applications in vaccine development. In the ensuing Phase II proposal, we will create a mono-specific nanobody of the optimal mmAb to further improve its therapeutic index, and test its efficacy and safety to deliver ASOs to T cells in mice and nonhuman primates.