Serpin B9-Insensitive Granzyme B Mutant Delivered by Engineered Capsid AAV Vectors Demonstrates Selective Killing of EGFR-Positive Cancer Cells

Dennis Makafui Dogbey, Krupa Naran, Stefan Barth
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Abstract

Cellular immunotherapy strategies that harness the cytotoxic properties of T and NK cells by releasing granzyme B have emerged as a crucial approach in cancer therapy for both hematological and solid tumors. However, the effective release and antitumor activities of granzyme B are significantly influenced by protease inhibitors including serpin B9. Gene delivery mechanisms that can counteract these limitations of cell-based immunotherapies are highly desired. In this regard, genetic modification of adeno-associated virus (AAV) capsids as vehicles for targeted delivery of therapeutic genes is an emerging area of research in the field of gene therapy of both monogenic and polygenic diseases. The insertion of antigen-specific antibody fragments and peptides into capsid sequences has been shown to redirect capsid tropism from native antigens to specific targets. Herein, we report on genetically engineered AAV2 capsid modified by inserting two antibody fragments: single-chain variable and single-domain antibodies and GE11 peptide, all specific for targeting epidermal growth factor receptor, at the N terminus of VP2. We observed an inverse correlation between the size of the inserted anti-EGFR moiety, capsid structure conservation, and vector yield. After demonstrating the proof of concept by confirming antigen-dependent transduction of cancer cells using AAV-GFP vectors, we further demonstrated that targeted AAV vectors encoding a protease-insensitive granzyme B mutant DNA cargo selectively eliminated cancer cells expressing serpin B9 in comparison to vectors encoding wild-type granzyme B in vitro. Our findings suggest that the challenges posed by intracellular expression of serpin B9 to antitumor T(NK) cell–dependent immunotherapies can be curtailed by utilizing the R201K granzyme mutant in immune-based therapy development.

Abstract Image

由工程衣壳AAV载体传递的Serpin b9不敏感颗粒酶B突变体显示选择性杀死egfr阳性癌细胞
通过释放颗粒酶B来利用T细胞和NK细胞的细胞毒性的细胞免疫治疗策略已经成为血液病和实体瘤癌症治疗的重要方法。然而,颗粒酶B的有效释放和抗肿瘤活性受到蛋白酶抑制剂包括丝氨酸蛋白酶B9的显著影响。人们非常需要能够抵消细胞免疫疗法这些局限性的基因传递机制。在这方面,腺相关病毒(AAV)衣壳的遗传修饰作为靶向递送治疗基因的载体是单基因和多基因疾病基因治疗领域的一个新兴研究领域。在衣壳序列中插入抗原特异性抗体片段和肽已被证明可以将衣壳从天然抗原转向特异性靶标。在此,我们报道了通过在VP2的N端插入两个抗体片段修饰的基因工程AAV2衣壳:单链可变和单域抗体和GE11肽,它们都是针对表皮生长因子受体的特异性抗体。我们观察到插入的抗egfr片段的大小、衣壳结构保守性和载体产量之间呈负相关。在利用AAV- gfp载体证实癌细胞抗原依赖性转导后,我们进一步证明,与编码野生型颗粒酶B的载体相比,编码蛋白酶不敏感颗粒酶B突变DNA货物的靶向AAV载体在体外选择性地消除了表达serpin B9的癌细胞。我们的研究结果表明,利用R201K颗粒酶突变体在免疫治疗开发中可以减少serpin B9细胞内表达对抗肿瘤T(NK)细胞依赖性免疫疗法的挑战。
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