Goal-oriented transgenic TCR-T cell design for efficient cancer immunotherapy 2266120

Introduction Adoptive cell transfer (ACT) therapy is a promising approach for cancer treatment facing several limitations. Tumor-infiltrating lymphocyte (TILs) therapy is limited by patient condition and tumor accessibility. Peripheral lymphocyte redirection strategies by low transduction efficiency and potency. We previously showed that transgenic TCR-T cells suffer inconsistent expression of transgenic receptor and surface dilution by endogenous TCR, both limiting effective avidity for antigen. In this work we compare 3 general strategies for transgenic TCR-T cell engineering: Viral transduction, viral transduction coupled with endogenous TCR knock-out and Crispr-cas9 knock-in under endogenous TCR promoter. Thus, we identify their use cases for goal oriented therapeutic T-cells design. Methods We use pMIGII retroviral vector for viral transduction. For endogenous TCR knock-out we deliver CRISPR-CAS9-gRNA RNP targeting TRAC and TRBC loci, using NEON system. For CRISPRS-CAS9 knock-in we use the above method followed by AAV6 transduction to deliver HDR template targeting TRAC. For Cytolytic Assay we use Chromium 51 release. Results Regular viral transduction resulted in transgenic TCR-T cells with low cytolytic activity, that were rescued by CRISPR-CAS9 knock-out of endogenous TCR. Both these approaches, however, resulted in loss of cytolytic activity and receptor expression with subsequent restimulations, due to semi-random gene insertion by viral vector. CRISPR-CAS9 HDR knock-in under endogenous TCR promoter produced consistent near-physiological transgenic TCR expression level, and cytolytic activity, maintained over multiple restimulations. Conclusion Viral Transduction couples with endogenous TCR knock-out is sufficient to produce highly potent transgenic TCR -T cells for short-term use, with repeated infusions or in the context of in vivo transduction. Precision knock-in under endogenous TCR locus produces TCR-T cells more suitable for off-the-shelf reagent with long lasting cytolytic activity. Funding Source MRIE at SKCC, DOD CDMP HT9425-25-1-0902 Topic Categories Technological Innovations in Immunology (TECH)