Heterodimeric IL-15 in Cancer Immunotherapy

我们探索了IL-15的生物学，并表明IL-15的有效生产只有通过与所谓的IL-15受体-α在同一细胞中共表达才有可能。我们还表明，先前在人类和啮齿动物中鉴定为细胞内或细胞核IL-15的第二种形式的IL-15（SSP IL-15）在与IL-15受体α共表达时也从细胞中有效分泌。这些结果为IL-15的生物学和调控提供了新的线索，并为这种细胞因子的有效生产和临床应用提供了方法。已经构建了过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，并将其用于产生体内发现的IL-15的生物活性形式和用于开发用于临床试验的cGMP材料。IL-15由于其刺激淋巴细胞（包括CD 8和NK细胞）的生长、活化和存活的能力而受到关注。因此，IL-15已被考虑用于癌症免疫治疗和用于支持过继转移后细胞毒性细胞克隆的生长。IL-15的其他建议用途是在淋巴细胞减少症中，在NK转移后支持NK细胞生长和活化，以及作为疫苗佐剂。我们已经表明，IL-15注射加速淋巴细胞在用细胞毒性药物治疗后呈现淋巴细胞减少的小鼠中的恢复。我们还表明，hetIL-15可以取代免疫细胞转移（ACT）中淋巴细胞清除的需要，因为转移的细胞可以在hetIL-15治疗后存活，增殖并进入肿瘤。这可能对ACT协议具有重要的临床意义。我们已经使用先前开发的RNA优化技术来优化IL-15细胞因子的表达，并且已经表明我们可以在小鼠和猕猴中在DNA递送后过量产生生物活性细胞因子。表达异源二聚体IL-15的载体的DNA递送导致细胞因子的全身活性水平以及NK和T细胞的增殖增加。我们已经表明，hetIL-15大大增加了小鼠模型和猕猴中几种肿瘤中的淋巴细胞浸润，这表明了增加淋巴细胞浸润的一般方法，其与抗肿瘤活性相关。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次人体临床试验，并与抗PD-1检查点抑制剂联合使用（NCT 02452268;与诺华公司合作）。除了癌症免疫疗法外，IL-15还引起了人们对临床用于治疗HIV感染的强烈兴趣，特别是在针对病毒根除或功能性治愈的方案中。IL-15作为抗HIV感染的免疫治疗剂的用途是基于其作为生长因子和由免疫系统的先天性（NK细胞）和适应性（CTL）臂介导的细胞毒性应答的关键调节剂的作用。在用于HIV感染的RM模型中使用hetIL-15治疗，我们已经证明：（i）hetIL-15的剂量增加（逐步剂量）的方案通过不同的递送途径耐受良好，并且增加外周血、粘膜部位和LN中具有高颗粒酶含量的T淋巴细胞（CD 8、CD 4和γ/δ）和NK细胞。(ii)重要的是，hetIL-15处理促进细胞毒性（Grz B+）CD 8 + T细胞进入B细胞滤泡，即LN内CTL通常被排除的区域和SIV/HIV感染的滤泡辅助CD 4 + T细胞所在的区域。hetIL-15处理导致LN内细胞相关病毒RNA以及SHIV感染的猕猴中血浆病毒血症的显著减少。(iii)我们还表明，hetIL-15可以增强LN中的ADCC，这提供了消除感染细胞的另一种机制。我们还使用优化的载体在动物中表达IL-12细胞因子。高效表达导致生物活性水平，其增加DNA疫苗接种后的免疫应答，从而成为我们的疫苗的重要分子佐剂。我们还开发了大规模分离和纯化外来体的方法。我们已经表明hetIL-15被掺入外泌体中，并且已经产生了足够的数量用于动物实验。我们提出外泌体的多功能性可用于将免疫治疗外泌体递送至疾病部位（肿瘤或淋巴组织）。我们开发了将外来体有效递送至肿瘤部位的方法。在小鼠肿瘤模型中的其他实验已经证明了淋巴和骨髓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受hetIL-15 β影响的其他途径。值得注意的是，我们已经研究了hetIL-15对肿瘤中树突状细胞（DC）的数量和性质的影响。我们已经证明，肿瘤中的DC在hetIL-15治疗后增加。DC参与在hetIL-15治疗期间诱导的细胞网络，并反过来支持效应细胞在肿瘤部位的更多募集。小鼠原位模型中的实验显示DC群体与肿瘤消退相关，并揭示了hetIL-15在诱导或增强针对肿瘤的长期保护性免疫应答中的额外有益作用。

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.