Heterodimeric IL-15 in Cancer Immunotherapy

我们对IL-15的生物学进行了探索，发现只有与所谓的IL-15受体-α在同一细胞中共表达，才有可能有效地产生IL-15。我们还发现，以前在人类和啮齿动物中被鉴定为细胞内或核内的第二种形式的IL-15(SSP IL-15)，当与IL-15受体α共表达时，也可以有效地从细胞中分泌出来。这些结果为IL-15的生物学和调控提供了新的线索，并为该细胞因子的高效生产和临床应用提供了方法。我们已经构建了大量产生可溶性生物活性IL-15/IL-15受体α异二聚体的细胞系，并将其用于生产体内发现的生物活性形式的IL-15，以及用于临床试验的cGMP材料的开发。IL-15因其刺激包括CD8和NK细胞在内的淋巴细胞的生长、激活和存活的能力而备受关注。因此，IL-15已被考虑用于癌症免疫治疗和过继转移后支持细胞毒细胞克隆的生长。IL-15的其他拟议用途是用于淋巴细胞减少症、支持NK细胞在NK转移后的生长和激活，以及作为疫苗佐剂。我们已经证明，IL-15注射可以加速细胞毒药物治疗后淋巴细胞减少的小鼠淋巴细胞的恢复。我们还发现hetIL-15可以替代过继细胞转移(ACT)中淋巴耗竭的需要，因为在hetIL-15治疗后，被转移的细胞可以存活、增殖和进入肿瘤。这可能对ACT方案有重要的临床意义。我们已经使用以前开发的RNA优化技术来优化IL-15细胞因子的表达，并已经证明我们可以在小鼠和猕猴体内传递DNA后过量产生生物活性细胞因子。异源二聚体IL-15表达载体的DNA传递导致系统活跃的细胞因子水平，并促进NK和T细胞的增殖。我们已经证明hetIL-15在小鼠模型和猕猴的几种肿瘤中极大地增加了淋巴细胞的渗透，提出了一种普遍的方法来增加淋巴细胞的渗透，这与抗肿瘤活性有关。在这些结果的基础上，我们启动了hetIL-15在转移性癌症中的首个人类临床试验，并与抗PD-1检查点抑制剂(NCT02452268；与诺华公司合作)联合使用。除了癌症免疫治疗外，IL-15还引起了人们对临床用于治疗艾滋病毒感染的浓厚兴趣，特别是在针对病毒根除或功能性治愈的方案中。使用IL-15作为抗HIV感染的免疫治疗剂是基于其作为一种生长因子和免疫系统的天然(NK细胞)和适应性(CTL)臂介导的细胞毒反应的关键调节因子的作用。在HIV感染的RM模型中使用hetIL-15治疗，我们已经证明：(I)增加hetIL-15剂量(阶梯式剂量)的方案对不同的递送途径具有良好的耐受性，并增加了外周血、粘膜部位和LN中颗粒酶含量较高的T淋巴细胞(CD8、CD4和Gamma/Delta)和NK细胞。(Ii)重要的是，hetIL-15治疗促进了细胞毒性(GrzB+)CD8+T细胞进入B细胞滤泡，在LN内通常排除CTL的区域以及SIV/HIV感染的滤泡辅助CD4+T细胞驻留的区域。HetIL-15治疗可显著降低SIV感染猕猴LN内的细胞相关病毒RNA和血浆病毒血症。(Iii)我们还发现hetIL-15可以增强LN中的ADCC，这提供了一种额外的清除感染细胞的机制。我们还利用优化的载体在动物体内表达了IL-12细胞因子。高效的表达产生生物活性水平，从而提高DNA疫苗后的免疫应答，从而成为我们疫苗的重要分子佐剂。我们还开发了大规模分离和纯化外切体的方法。我们已经证明hetIL-15被整合到外切体中，并产生了足够的量用于动物实验。我们认为，外切体的多功能性可用于将免疫治疗外切体输送到疾病部位(肿瘤或淋巴组织)。我们开发了有效地将外切体运送到肿瘤部位的方法。在小鼠肿瘤模型上的其他实验已经证明了淋巴系和髓系细胞网络的快速相互作用，导致不同细胞群的数量和迁移的变化。广泛的转录组学和蛋白质组学分析揭示了hetIL-15在药理学上影响的其他途径。值得注意的是，我们研究了hetIL-15对肿瘤中树突状细胞(DC)数量和性质的影响。我们已经证明，hetIL-15治疗后，肿瘤中的DC增加。DC参与hetIL-15治疗期间诱导的细胞网络，进而支持更多的效应细胞在肿瘤部位的招募。在小鼠原位模型上的实验表明，DC群体与肿瘤的消退相关，并揭示了hetIL-15在诱导或增强针对肿瘤的长期保护性免疫反应方面的额外有益作用。

We explored the biology of IL-15 and showed that efficient production of IL-15 is possible only by co-expression in the same cell with the so-called IL-15 Receptor-alpha. We also showed that a second form of IL-15 (SSP IL-15) previously identified in humans and rodents as intracellular or nuclear IL-15 is also efficiently secreted from the cells when co-expressed with the IL-15 Receptor alpha. These results shed new light in the biology and regulation of IL-15 and provide methods for the efficient production and clinical application of this cytokine. Cell lines overproducing soluble bioactive IL-15/IL-15 Receptor alpha heterodimers have been constructed and were used for the production of the bioactive form of IL-15 found in the body and for the development of cGMP material for clinical trials. IL-15 is of interest due to its ability to stimulate the growth, activation and survival of lymphocytes, including CD8 and NK cells. Thus, IL-15 has been considered for cancer immunotherapy and for support of the growth of cytotoxic cell clones after adoptive transfer. Other proposed uses of IL-15 are in lymphopenia, in supporting NK cell growth and activation after NK transfer, and as vaccine adjuvant. We have shown that IL-15 injection accelerates the recovery of lymphocytes in mice rendered lymphopenic after treatment with cytotoxic drugs. We also showed that hetIL-15 can replace the need for lymphodepletion in Adoptive Cell Transfer (ACT), since the transferred cells can survive, proliferate and enter the tumors after hetIL-15 treatment. This may have important clinical implications for ACT protocols. We have used the previously developed technologies of RNA optimization to optimize expression of IL-15 cytokine, and have shown that we can over-produce bioactive cytokine after DNA delivery in mice and macaques. DNA delivery of vectors expressing heterodimeric IL-15 leads to systemically active levels of cytokine and the increased proliferation of NK and T cells. We have shown that hetIL-15 greatly increases lymphocyte infiltration in several tumors in mouse models and in macaques, suggesting a general method to increase lymphocyte infiltration, which is associated with anti-tumor activity. On the basis of these results, we have initiated first-in-human clinical trials of hetIL-15 in metastatic cancers and also in combination with anti-PD-1 check point inhibitor (NCT02452268; collaboration with Novartis). In addition to cancer immunotherapy, IL-15 has generated strong interest for clinical use to treat HIV infection, especially in protocols targeting viral eradication or a functional cure. The use of IL-15 as an immune therapeutic agent against HIV infection is based on its effects as a growth factor and key regulator of cytotoxic responses mediated by both the innate (NK cells) and the adaptive (CTL) arms of the immune system. Using hetIL-15 treatment in the RM model for HIV infection, we have demonstrated that: (i) a regimen with increasing dosing (step-dose) of hetIL-15 is well tolerated by different routes of delivery and increases T lymphocytes (CD8, CD4 and gamma/delta) and NK cells with high granzyme content in peripheral blood, mucosal sites and LN. (ii) Importantly, hetIL-15 treatment promotes the entrance of cytotoxic (GrzB+) CD8+ T cells in the B cell follicles, areas within the LN where CTL are typically excluded and where SIV/HIV infected follicular helper CD4+ T cells reside. hetIL-15 treatment led to significant decrease in cell-associated viral RNA within the LN as well as in plasma viremia in SHIV infected macaques. (iii) We also showed that hetIL-15 can enhance ADCC in LN, which provides an additional mechanism of elimination of infected cells. We have also used optimized vectors to express IL-12 cytokine in animals. Efficient expression results in bioactive levels, which increase immune response after DNA vaccination, thus becoming important molecular adjuvant for our vaccines. We have also developed methods for large-scale isolation and purification of exosomes. We have shown that hetIL-15 is incorporated in exosomes and have produced sufficient quantities for animal experiments. We propose that the versatility of exosomes can be used for the delivery of immunotherapy exosomes to disease sites (tumor or lymphoid tissue). We developed methods for the efficient delivery of exosomes to tumor sites. Additional experiments in mouse tumor models have demonstrated the rapid interaction of lymphoid and myeloid cell networks resulting in changes in numbers and migration of different cell populations. Extensive transcriptomics and proteomics analysis has revealed additional pathways affected by hetIL-15 pharmacologically. Of note, we have studied the effects of hetIL-15 in the number and properties of Dendritic Cells (DC) in tumors. We have demonstrated that DC in tumors increase upon hetIL-15 treatment. DC participate in a network of cells that are induced during hetIL-15 treatment and in turn support more recruitment of effector cells in tumor sites. Experiments in mouse orthotopic models show that DC populations correlate with tumor regression and reveal additional beneficial effects of hetIL-15 in inducing or enhancing long term protective immune response against tumors.