AAV-mediated intra-tumoral immunotherapy for the treatment of immunologically “cold” tumors

重组腺相关病毒载体(AAV)是一种安全有效的体内基因治疗工具。病毒衣壳的修饰使得能够产生适合于体内肿瘤内基因转移的新型AAV变体，针对肿瘤细胞和来自肿瘤微环境的细胞。这些载体为肿瘤的原位免疫治疗提供了很有前景的工具。该项目的目的是操纵肿瘤及其微环境，以提高肿瘤的免疫原性。为此，我们将利用工程AAV来刺激先天免疫和获得性免疫，并诱导两种类型的免疫原性死亡(ICD)，即焦下垂和坏死性下垂。在第一种策略中，我们将利用AAV表达分泌型或细胞质鞭毛蛋白。鞭毛蛋白是一种有效的细菌衍生蛋白疫苗佐剂，可被Toll样受体-5(TLR5)感知，例如在巨噬细胞/树突状细胞(DC)上。感知通过核因子-kappa B途径激活先天免疫系统。鞭毛蛋白还通过形成NLRC4炎性小体诱导下垂。上睑下垂被定义为细胞死亡的一种高度炎症性变体，其关键分子执行物最近被确定为Gasdermin D(GSDMD)，它是炎症体途径的下游靶标。被激活的caspase-1裂解GSDMD导致促炎症细胞因子IL-1β和IL-18的活性形式的孔形成和释放，代表着一种灾难性的细胞死亡形式，有利于危险相关分子模式(DAMP)和肿瘤抗原的释放。ICD的诱导正在成为抗肿瘤免疫反应的有效触发因素，不仅可以由GSDMD或Gasdermin E(GSDME)等焦下垂执行者的激活诱导，也可以通过混合谱系激酶结构域样蛋白(MLKL)的激活而诱导，后者同样可以寡聚，也可以直接破坏膜的完整性而导致坏死症。因此，在我们的第二个互补策略中，我们将使用AAV通过表达GSDMD、GSDME或MLKL的活性截断/突变形式来诱导肿瘤内的ICD，从而绕过肿瘤对诱导下垂或坏死性下垂的抵抗。在急性炎症的背景下诱导肿瘤细胞死亡以及结合肿瘤抗原释放DAMP有望增强肿瘤抗原通过DC向T细胞递送，最终诱导强大、持久的适应性抗肿瘤免疫。重要的是，我们两种互补方法的结合有很强的科学依据，因为协同效应是可以预期的。此外，我们的策略可以很容易地与已有的免疫治疗方法相结合(例如，免疫检查点封锁)。AAV良好的安全性强调了我们的策略在临床上的高潜力。最后，我们的策略提供了相当大的潜力来提高肿瘤的免疫原性，即使在免疫“冷”的肿瘤，仍然是一个临床挑战。

Recombinant adeno-associated virus vectors (AAV) represent a safe and efficient tool for in vivo gene therapy. Modification of the viral capsid enables generation of novel AAV variants suitable for in vivo intra-tumoral gene transfer, targeting both tumor cells and cells from the tumor microenvironment. Such vectors represent promising tools for in situ immunotherapy of tumors. The aim of the project is to manipulate the tumor and its microenvironment to increase tumor immunogenicity. For that, we will make use of engineered AAV to stimulate innate and adaptive immunity and to induce two types of immunogenic death (ICD), namely pyroptosis or necroptosis.In the first strategy, we will make use of AAV to express secreted or cytoplasmic flagellin. Flagellin is a potent bacterial-derived protein vaccine adjuvant sensed by Toll-like receptor-5 (TLR5) for example on macrophages/dendritic cells (DC). Sensing activates the innate immune system via the NF-kappa B pathway. Flagellin also induces pyroptosis through formation of the NLRC4 inflammasome. Pyroptosis is defined as a highly inflammatory variant of cell death and its key molecular executioner has recently been identified as gasdermin D (GSDMD), a downstream target of the inflammasome pathway. GSDMD cleavage by activated caspase-1 leads to pore formation and release of the active forms of the proinflammatory cytokines IL-1beta and IL-18, representing a catastrophic form of cell death, favoring the release of danger associated molecular patterns (DAMPs) as well as tumor antigens.Induction of ICD is emerging as potent trigger of anti-tumor immune responses and can be induced not only by the activation of pyroptosis executioners like GSDMD or gasdermin E (GSDME), but also by the activation of Mixed Lineage Kinase Domain-like Protein (MLKL), which can similarly oligomerize and also directly disrupts the membrane integrity to cause necroptosis. Therefore, in our second, complementary strategy, we will use AAV to induce ICD intra-tumorally by expressing active truncated/mutated forms of GSDMD, GSDME or MLKL, thus bypassing tumor resistance to the induction of pyroptosis or necroptosis. Induction of tumor cell death in the context of acute inflammation and release of DAMPs in conjunction with tumor antigens is expected to enhance presentation of tumor antigens via DC to T cells and finally induce a robust, long lasting adaptive anti-tumor immunity.Importantly, there is a strong scientific rationale for the combination of our two complementary approaches, since synergistic effects can be expected. In addition, our strategy can be combined easily with established immunotherapeutic approaches (e.g. immune checkpoints blockade). The favorable safety profile of AAV underscores the high potential for clinical translation of our strategy. Finally, our strategy offers the considerable potential to increase tumor immunogenicity even in immunologically “cold” tumors that still represent a clinical challenge.