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

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.