Immunotherapeutic Interventions of AIDS and Cancer

我们对mRNA表达基本机制的理解有助于鉴定介导基因表达转录后控制的分子机制，这导致了RNA优化（也称为密码子优化）的发展，作为改善基因表达的关键策略，这种方法目前是许多基因治疗应用的关键技术，包括治疗性HIV疫苗和细胞因子dna，在这个项目中使用。使用细胞因子pDNAs （IL-12, IL-15）作为分子疫苗佐剂可以改善免疫应答的数量和质量。为了优化这些细胞因子，我们发现IL-15/IL-15Ra （hetIL-15）和IL-12细胞因子家族使用类似的高度调控步骤，转录后和翻译后调控策略。我们报道了来自同一细胞的IL-15和IL-15Ra的共表达对于hetIL-15的产生至关重要，并分析了其复杂的糖基化模式。hetIL-15作为一种淋巴细胞生长因子，在艾滋病和癌症的免疫治疗中具有重要的应用前景。HetIL-15具有利用患者自身免疫系统，诱导NK细胞和T细胞增殖，诱导CTL减少或清除HIV库的潜力，同样，作为癌症免疫治疗的一部分，诱导抗肿瘤CTL。因此，艾滋病毒和癌症免疫治疗具有共同的潜在免疫特征，我们使用细胞因子治疗方法的研究可能会为两者的治愈提供线索。为了测试hetIL-15在癌症和艾滋病免疫治疗中的应用，我们从哺乳动物细胞中制备了hetIL-15。HetIL-15目前正处于转移性癌症免疫治疗的临床试验中。我们进一步表明，hetIL-15能够在过继细胞移植（ACT）治疗癌症之前取代淋巴细胞清除的需要。hetIL-15诱导淋巴细胞进入肿瘤部位并激活肿瘤内CD8+ T细胞。与其他方案相比，hetIL-15随着时间的推移诱导肿瘤中抗原特异性淋巴细胞的富集，并大大增加CD8+/Treg细胞比率。hetIL-15在ACT中的应用将为癌症免疫治疗方案提供新的工具和技术。消除对淋巴细胞清除的需求将使更多的患者有资格接受细胞移植方案。此外，IL-15治疗可以作为将T细胞植入肿瘤的一般方法，提高其他免疫治疗干预的成功率。在另一项研究中，我们生产并开发了细胞外囊泡（EV）技术，将hetIL-15作为免疫疗法输送到肿瘤部位。我们的方法有可能很容易地应用于产生用于研究和开发的工程EV的纯化制剂。为了充分利用IL-12 DNA的潜力，我们研究了这种糖基化的70 kDa异二聚体细胞因子的生物学特性，以最大化细胞因子的产生。虽然每个亚基的产生都是独立调节的，但这两个分子在同一细胞中的共表达对于形成具有生物活性的异二聚体是必不可少的。基于我们对IL-15/IL15Ra交叉稳定的关键细胞内调控步骤的发现，我们研究了p35和p40亚基的转录后调控和相互作用，从而导致IL-12p70的最佳产生。研究控制IL-12p70生物合成的分子步骤，我们发现rna优化基因序列的组合，以及重要的是，在细胞内对两个亚基的相对表达水平进行微调，导致IL-12p70异源二聚体的产量大大增加。重要的是，我们发现p40增强p35的稳定性并促进其细胞内运输，从而形成稳定、有效分泌的IL-12p70复合物。设计IL-12p70的双表达质粒，以获得有利的两个亚基的相对水平和最佳的IL-12表达，用于我们的DNA疫苗研究的佐剂。与IL-12一样，相关的IL-23和IL-27也同样受到调控，细胞因子的产生分别由p40和EBI3链控制。这些细胞因子pDNAs提供了重要的分子工具，可作为疫苗和癌症免疫治疗中的分子佐剂进行测试，具有很好的未来转化应用前景。为此，我们进一步开发了在规定的无血清条件下生长的高表达哺乳动物细胞系。我们报道了一种不带亲和标签的高效一步纯化IL-12蛋白。使用纯化的IL-12细胞因子，我们现在首次证明肝素作为一种辅助受体，能够在细胞表面维持IL-12的储存库，从而增强其活性。我们对这些细胞因子分子生物学的理解为有效的细胞因子生产提供了必要的关键信息。尽管在了解艾滋病毒感染的免疫生物学和艾滋病发病机制方面取得了很大进展，但治疗性艾滋病毒疫苗的开发尚未实现。由于病毒的变异、病毒抗原的性质、免疫优势和疫苗在粘膜部位诱导的体液免疫的短暂持续，HIV疫苗的开发变得复杂。如项目1所述，我们开发了基于pdna的疫苗，以诱导对HIV-1 Gag保守元件（CE）的强大细胞毒性T细胞应答，选择严格保守的区域，功能重要性，独立于与病毒控制相关的“保护性”单倍型，以及与hiv感染LTNP的免疫控制相关的区域。我们证明了一种包含HIV-1 p24gag保守元件（CE）作为引物，然后结合CE+gag pDNA作为增强剂的疫苗，可以引发针对这些保守区域的广泛、有效和持久的T细胞反应，有效地克服gag可变区域施加的优势。与HIV CE DNA疫苗类似，我们通过类比的方法开发了SIV CE DNA，并发现与p57gag pDNA疫苗相比，p27CE pDNA疫苗对于有效诱导和增加具有细胞毒性潜能（颗粒酶B+ CD107a+）靶向亚显性CE表位的ag特异性T细胞的频率也至关重要。在p27CE pDNA引物之后，CE+gag pDNA增强疫苗接种引起了更广泛的CE表位识别，从而更深刻地改变了免疫优势结构。因此，含有CE pDNA引物和CE+gag pDNA加强疫苗的SIV/HIV疫苗方案显著增加了细胞毒性T细胞对亚显性高度保守的gag表位的反应，并最大化了反应广度。我们在接受抗逆转录病毒治疗的猕猴感染SIV时使用CE DNA疫苗方案，发现它诱导了强大的细胞毒性T细胞反应。目前正在评估HIV CE疫苗与hetIL-15治疗相结合作为有效的SIV免疫治疗方法。作为一项合作努力，我们还在HAART （A5369）下的HIV感染者中开发了一项临床试验，以测试HIV CE疫苗方案的免疫原性（2017年底开放）。HVTN 119和A5369这两项试验将在同一实验室使用该技术进行评估。这将使我们能够直接比较和对比不同情况下CE的免疫原性。

Our understanding of the basic mechanisms of mRNA expression has been instrumental in the identification of molecular mechanisms mediating posttranscriptional control of gene expression, which led to the development of RNA optimization (also referred to as codon optimization) as a key strategy to improve gene expression, a method which is presently a key technology for many gene therapy applications including therapeutic HIV vaccines and cytokine DNAs which are used in this project. The use of cytokine pDNAs (IL-12, IL-15) as molecular vaccine adjuvants was found to improve the quantity and alter the quality of the immune responses. To optimally use these cytokines, we found that IL-15/IL-15Ra (hetIL-15) as well as the IL-12 cytokine family use similar highly regulated steps posttranscriptional and posttranslational regulation strategies. We reported that coexpression of IL-15 and IL-15Ra from the same cell is critical for the production of hetIL-15 and analyzed its complex glycosylation pattern. hetIL-15, as a lymphocyte growth factor, represents a promising candidate in HIV immunotherapies against HIV and cancer. HetIL-15 has the potential that harness the patient's own immune system, inducing proliferation of NK and T cells, and to induce CTL to reduce or clear the HIV reservoir and, similarly, as part of cancer immunotherapy to induce anti-tumor CTL. Thus, HIV and cancer immunotherapy share underlying immunologic features and our studies using cytokine therapy approach may shed light towards the cure of both. To test applications of hetIL-15 in immunotherapy against cancer and HIV, we produced hetIL-15 from mammalian cells. HetIL-15 is presently in clinical trials for immunotherapy of metastatic cancers. We have further shown that hetIL-15 is able to replace the need for lymphodepletion prior to Adoptive Cell Transfer (ACT) for cancer therapy. hetIL-15 induces lymphocyte entry into tumor sites and activation of intratumoral CD8+ T cells. In contrast to other protocols, hetIL-15 induces an enrichment of antigen specific lymphocytes in the tumor over time and greatly increases CD8+/Treg cell ratio. Application of hetIL-15 to ACT will provide new tools and techniques for cancer immunotherapy protocols. Elimination of the need for lymphodepletion will make more patients eligible for cell transfer protocols. In addition, IL-15 treatment could be a general method to place T cells into tumors, increasing the success rate of other immunotherapy interventions. In another line of research, we produced and developed the extracellular vesicle (EV) technology to deliver hetIL-15 as immunotherapy to tumor sites. Our procedure have the potential to be be readily applied to generate purified preparations of engineered EV for research and development. To use IL-12 DNA to its full potential, we studied the biology of this glycosylated 70 kDa heterodimeric cytokine to maximize cytokine production. Although the production of each subunit is independently regulated, coexpression of both molecules in the same cell is essential to form biologically active heterodimer. Prompted by our findings on the critical intracellular regulatory step of IL-15/IL15Ra cross-stabilization, we investigated the posttranscriptional regulation and interaction of the p35 and p40 subunits leading to optimal IL-12p70 production. Investigating molecular steps controlling IL-12p70 biosynthesis, we found that the combination of RNA-optimized gene sequences, and importantly, fine-tuning of the relative expression levels of the two subunits within a cell resulted in greatly increased production of the IL-12p70 heterodimer. Importantly, we discovered that p40 enhances the p35 stability and promotes its intracellular trafficking, resulting in formation of a stable, efficiently secreted IL-12p70 complex. Dual expression plasmids for IL-12p70 were designed to obtain favorable relative levels of the two subunits and optimal IL-12 expression used as adjuvant for our DNA vaccine studies. Like IL-12, the related IL-23 and IL-27 are similarly regulated and cytokine production is controlled by the p40 and EBI3 chains, respectively. These cytokine pDNAs provide important molecular tools to be tested as molecular adjuvants in vaccine and in cancer immunotherapy, with promising future translational applications. Towards this, we further developed high expresser mammalian cell lines grown under defined serum-free conditions. We reported an efficient one-step purification of IL-12 protein without affinity tag. Using the purified IL-12 cytokine, we now demonstrated for the first time that heparin serves as a co-receptor capable of maintaining a reservoir of IL-12 at the cell surface and, thus, enhancing its activity. Our understanding of the molecular biology of these cytokines provided the critical information necessary for efficient cytokine production. Despite great progress in understanding the immunobiology of HIV infection and AIDS pathogenesis, the development of a therapeutic HIV vaccine has not been achieved. HIV vaccine development is complicated by virus variability, the nature of the viral antigens, immune dominance and the short persistence of vaccine-induced humoral immunity at mucosal sites. As described in project 1, we developed pDNA-based vaccines to induce robust cytotoxic T cell responses to the conserved elements (CE) of HIV-1 Gag, selecting regions by stringent conservation, functional importance, independent of 'protective' haplotypes associated with virus control, and association with immune control in HIV-infected LTNP. We demonstrated that a vaccine comprising conserved elements (CE) of HIV-1 p24gag as prime followed by a combination of CE+gag pDNA as boost elicits broad, potent and durable T cell responses targeting these conserved regions, efficiently overcoming the dominance imposed by the Gag variable regions. Similar to the HIV CE DNA vaccine, we have developed SIV CE DNA by analogy and found that vaccination with p27CE pDNA was also critical for the efficient induction and increased the frequency of Ag-specific T cells with cytotoxic potential (granzyme B+ CD107a+) targeting subdominant CE epitopes, compared with the responses elicited by the p57gag pDNA vaccine. Following p27CE pDNA priming, the CE+gag pDNA booster vaccination elicited significantly broader CE epitope recognition, and thus, a more profound alteration of the immunodominance hierarchy. Hence, SIV/HIV vaccine regimens comprising CE pDNA prime and CE+gag pDNA booster vaccination significantly increased cytotoxic T cell responses to subdominant highly conserved Gag epitopes and maximized response breadth. We are using the CE DNA vaccine regimen in SIV infected in ART-treated macaques and found that it induced robust cytotoxic T cell response. Combination of the HIV CE vaccine with hetIL-15 treatment is currently under evaluation as potent immunotherapeutic approach against SIV. As a collaborative effort, we have also developed and clinical trial in HIV-infected persons under HAART (A5369) to test the immunogenicity of the HIV CE vaccine regimen (opens end 2017). Both trials, HVTN 119 in HIV naive persons and A5369 will be evaluated using the techniques in the same lab. This will allow us to directly compare and contrast the CE immunogenicity under different circumstances.