Development of antisense oligonucleotides that enhance sIL7R as novel cancer immunotherapy

项目摘要 免疫疗法旨在释放免疫系统攻击和杀死癌细胞， 治疗癌症的希望。这一承诺得到了2018年诺贝尔医学奖或 生理学奖授予James P. Allison博士（UT MD安德森癌症中心）和Tasuku Honjo博士（京都 发现通过抑制负性免疫调节来治疗癌症。免疫检查点 抑制剂，如靶向负性免疫调节剂CTLA-4和PD-1的单克隆抗体， 获得了食品药品监督管理局对几种癌症的批准。举例说明它们的潜力，抗CTLA-4单克隆抗体 2011年批准抗体伊匹木单抗（Yerlimumab，布里斯托Myers Squibb）用于治疗黑色素瘤， 随后是其他类型的癌症。同样，抗PD 1单克隆抗体派姆单抗（Keytruda， Merck）和Nivolumab（Opdivo，布里斯托Myers Squibb）也被批准用于黑色素瘤，后来用于其他癌症。 癌症的类型。这些生物制剂的一些结果非常令人印象深刻。然而，承诺 由于许多患者没有反应，免疫疗法受到限制。 具有提高对免疫疗法的应答率的潜力的潜在促免疫调节剂已经出现， 我们在自身免疫方面的工作这项工作使我们发现可溶性白细胞介素-7受体（sIL-7 R）是一种驱动因子， 自身免疫，这通常不是一个理想的结果，但在免疫肿瘤学的背景下，它可以 单独或与免疫检查点抑制剂协同增强抗癌免疫力。在这一提议中， 我们检验了这样一个假设，即增加自身免疫的驱动因子sIL 7 R的水平将导致增强的免疫- 反应性，从而提高抗癌免疫力。由于sIL 7 R是通过选择性剪接产生的， IL 7 R前体mRNA的第六外显子，特别是通过排除该外显子，其表达可以使用 剪接调节反义寡核苷酸（ASO）。为了增加sIL 7 R水平，我们产生了两种ASO，IL 7 R- 001和IL 7 R-004，其增加IL 7 R外显子6的排除和sIL 7 R的产生（pro-sIL 7 R ASO）。IL7R 表达主要限于T细胞，因此这种细胞类型是sIL 7 R的主要生产者。我们优化了 将这些ASO体外递送到人原代T细胞中，这将有助于本提案中的体外测试， 对于随后的体内功效研究至关重要。作为概念验证，我们将在体外测试pro-sIL 7 R ASO可以调节T细胞功能并增强T细胞介导的对癌细胞的杀伤。 如果pro-sIL 7 R ASO在体外增强抗癌免疫力，在II期，我们将测试其抗癌治疗效果。 在狒狒（Papio anubis）中高度相关的遗传诱导的肝细胞癌模型中的潜力。 这个模型是由我们在西南国家灵长类动物研究中心（圣安东尼奥， TX），是非人灵长类动物中第一个实验上易处理的癌症模型之一，并且是研究癌症的理想模型。 免疫治疗候选药物如pro-sIL 7 R ASO的体内测试。

PROJECT SUMMARY Immunotherapy, which is designed to unleash the immune system to attack and kill cancer cells, holds enormous promise for the treatment of cancer. Such promise was recognized by the 2018 Nobel Prize in Medicine or Physiology awarded to Drs. James P. Allison (UT MD Anderson Cancer Center) and Tasuku Honjo (Kyoto University) for their discovery of cancer therapy by inhibition of negative immune regulation. Immune check point inhibitors, such as monoclonal antibodies that target the negative immune regulators CTLA-4 and PD-1 have gained FDA approval for several types of cancer. Exemplifying their potential, the anti-CTLA-4 monoclonal antibody Ipilimumab (Yervoy, Bristol Myers Squibb) was approved in 2011 for the treatment of melanoma and subsequently for other types of cancer. Likewise, anti-PD1 monoclonal antibodies Pembrolizumab (Keytruda, Merck) and Nivolumab (Opdivo, Bristol Myers Squibb) were also approved for melanoma and later for other types of cancers. Some results with these biologics have been very impressive. However, the promise of immunotherapy has been limited by the fact that in many patients there is no response. Potential pro-immune modulators with potential to boost response rate to immunotherapy have emerged from our work on autoimmunity. This work has led us to discover that soluble interleukin-7 receptor (sIL7R) is a driver of autoimmunity, which is usually not a desirable outcome, but in the context of immuno-oncology it could enhance anti-cancer immunity either alone or synergistically with immune checkpoint inhibitors. In this proposal, we test the hypothesis that increasing levels of sIL7R, a driver of autoimmunity, will lead to enhanced immune- reactivity and thus improved anti-cancer immunity. Given that sIL7R is produced by alternative splicing of the sixth exon of IL7R pre-mRNAs, specifically by exclusion of this exon, its expression can be controlled using splicing-modulating antisense oligonucleotides (ASOs). To increase sIL7R levels we generated two ASOs, IL7R- 001 and IL7R-004, that increase exclusion of IL7R exon 6 and production of sIL7R (pro-sIL7R ASOs). IL7R expression is largely restricted to T cells, and thus this cell type is the main producer of sIL7R. We have optimized delivery of these ASOs into human primary T cells in vitro, which will facilitate in vitro testing in this proposal and is critical for subsequent in vivo efficacy studies. As proof-of-concept, here we will test in vitro whether pro-sIL7R ASOs can modulate T cell function and enhance T cell-mediated killing of cancer cells. If pro-sIL7R ASOs enhance anti-cancer immunity in vitro, in Phase II we will test their anti-cancer therapeutic potential in a highly relevant, genetically-induced hepatocellular carcinoma model in baboons (Papio anubis). This model developed by our collaborators at the Southwest National Primate Research Center (San Antonio, TX), is one of the first experimentally tractable models of cancer in nonhuman primates and an ideal model for in vivo testing of immunotherapy candidate drugs, such as pro-sIL7R ASOs.