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于体内肿瘤内基因转移的新型AAV变体，其靶向肿瘤细胞和来自肿瘤微环境的细胞。这样的载体代表了肿瘤原位免疫治疗的有前途的工具。该项目的目的是操纵肿瘤及其微环境，以增加肿瘤免疫原性。为此，我们将利用工程化的AAV来刺激先天性和适应性免疫，并诱导两种类型的免疫原性死亡（ICD），即焦亡或坏死性凋亡。鞭毛蛋白是由例如巨噬细胞/树突细胞（DC）上的Toll样受体-5（TLR 5）感测的有效细菌衍生蛋白疫苗佐剂。感应通过NF-κ B途径激活先天免疫系统。鞭毛蛋白还通过NLRC 4炎性体的形成诱导焦亡。焦亡被定义为细胞死亡的高度炎性变体，其关键分子执行器最近被鉴定为gasdermin D（GSDMD），其是炎性体途径的下游靶标。活化的半胱天冬酶-1切割GSDMD导致孔形成和促炎细胞因子IL-1 β和IL-18活性形式的释放，代表细胞死亡的灾难性形式，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-肿瘤免疫应答，并且不仅可以通过激活焦亡执行因子如GSDMD或gasdermin E（GSDME）来诱导，而且可以通过激活混合谱系激酶结构域样蛋白（MLKL）来诱导，其可以类似地寡聚化，并且还直接破坏膜完整性以引起坏死性凋亡。因此，在我们的第二种互补策略中，我们将使用AAV通过表达活性截短/突变形式的GSDMD、GSDME或MLKL来在肿瘤内诱导ICD，从而绕过肿瘤对诱导细胞凋亡或坏死凋亡的抗性。在急性炎症的背景下诱导肿瘤细胞死亡并释放DAMPs与肿瘤抗原结合，预计将增强肿瘤抗原通过DC向T细胞的呈递，并最终诱导强大的、持久的适应性抗肿瘤免疫。重要的是，我们的两种互补方法的组合有很强的科学依据，因为可以预期协同效应。此外，我们的策略可以很容易地与已建立的免疫抑制方法（例如免疫检查点阻断）相结合。AAV的有利安全性特征强调了我们策略的临床转化的高潜力。最后，我们的策略提供了相当大的潜力，增加肿瘤免疫原性，即使在免疫学上的“冷”肿瘤，仍然代表着临床挑战。

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.