Developing Off-the-Shelf T-cell Receptor Therapy Targeting Resistant Pathogens Using CRISPR Genome-Editing

Introduction
Iatrogenic immunosuppression caused by chemotherapy, allogeneic hematopoietic stem cell transplantation, and solid organ transplantation places patients at significant risk of opportunistic infections and viral reactivations. Pathogens such as adenovirus (Adv), BK virus (BKV), and cytomegalovirus (CMV) frequently lack effective treatments, progress rapidly, and result in increased morbidity, mortality, and poor transplant outcomes. To address this critical clinical challenge, we are developing HLA-restricted, allogeneic "off-the-shelf" T-cell therapies targeting these viral pathogens.

Materials and Methods
Virus-specific T-cell receptors (TCRs) were identified and isolated from healthy donors using peptide libraries and functional stimulation assays. The TCRs were transduced into allogeneic T-cells via gammaretroviral vectors. CRISPR/Cas9 technology was used to disrupt the endogenous TCRα (TRAC) gene to eliminate graft-versus-host disease risk. Engineered T-cells were evaluated for antigen-specific responses via OX40 and 4-1BB activation marker upregulation and IFNγ secretion.

Additionally, a CRISPR-based non-viral method is being developed to integrate TCR genes directly into the TRAC locus under the control of its endogenous promoter. This approach ensures precise, uniform TCR expression while avoiding the risks of random vector integration, enhancing both safety and therapeutic consistency.

Results and Discussion
Four TCRs targeting CMV, BKV, and Adv were isolated and characterized. T-cells expressing these TCRs demonstrated robust antigen-specific responses. The CMV-specific TCR showed 50-70% upregulation of OX40 and 4-1BB activation markers in response to the pp65 peptide, along with strong IFNγ. Similarly, TCRs targeting BKV and Adv showed significant T-cell activation and cytokine release upon stimulation with peptide libraries. CRISPR-mediated TRAC knockout successfully prevented alloreactivity, as shown by basal IFNγ secretion levels in mixed lymphocyte reaction assays with different donor leukocytes.

Conclusions
This research demonstrates the feasibility of developing virus-specific TCR-T cells that are effective, safe, and clinically applicable for immunocompromised patients. Conducted in collaboration with the Advanced Biotherapy Center at Sheba Medical Center, this project integrates cutting-edge genome-editing technologies with advanced clinical infrastructure. By addressing the unmet need for treatments for Adv, BKV, and CMV infections, this platform offers a transformative therapeutic approach to improve patient outcomes.