Genome-Wide Integration Site Profiling of Multi-Component CAR T-Cell Engineering Systems with ADA1/CD26 Co-Transduction

Background: Retroviral vectors remain widely used for CAR T-cell manufacturing, including emerging multi-component engineering strategies that incorporate additional functional modules to enhance therapeutic performance. However, genome-wide profiling of integration patterns in CAR T cells subjected to multiplex retroviral engineering has not previously been reported. Methods: We performed genome-wide profiling of retroviral integration sites in CAR T-cell products derived from two targeting platforms (IL15-GPC3 and HER2). Each platform was evaluated under conditions with or without co-transduction of a retroviral vector encoding ADA1 and CD26. Experiments were conducted using cells obtained from a single donor with three technical replicates. Vector copy number (VCN), chromosomal distribution, genomic feature annotation, and integration hotspot analyses were conducted. Results: VCN levels were comparable across all CAR T-cell products and were not significantly affected by ADA1/CD26 co-expression. Integration events were broadly distributed across the genome with enrichment in gene-dense chromosomal regions. Chromosome 19 showed a prominent integration preference, with a secondary enrichment observed on chromosome 17. The majority of integration sites were located within intronic regions, with no enrichment observed in promoter regions. Recurrent integration hotspots were identified; however, no evidence of dominant clonal expansion was observed across conditions. Comparative analyses between ADA1/CD26 and control groups showed minimal differences in integration patterns. Conclusions: To our knowledge, this study provides the first comprehensive, genome-wide characterization of retroviral integration site profiles in CAR T-cell products manufactured by multi-vector co-transduction with an ADA1/CD26 module. By employing harmonized comparison with external reference datasets, we offer a directly benchmarked integration site characterization of multi-component CAR T-cell manufacturing. These findings establish a reference framework for integration site monitoring in next-generation CAR T-cell engineering systems and inform risk evaluation for emerging combinatorial vector strategies.