PML is a fatal disease of the central nervous system caused by JCV. Survival is dependent on early diagnosis and ability to establish T cell immunity. Adoptive transfer of polyomavirus-specific T cells is possible; however, current delays in creating HLA-matched anti-viral T cells can be fatal.

PBMCs from healthy donors with specific HLA class I subtypes were stimulated with peptide libraries tiled across the JCV VP1 protein. Multiple rounds of stimulation were performed to identify the peptides that induced the largest CD8⁺ T cell activation (i.e., INFg, TNFa, CD137, and CD69 expression). For the immunogenic peptides antigens were narrowed and high-affinity antigen-specific CD8⁺ T cells were isolated using tetramers. Single-cell T cell receptor (TCR) sequencing identified high-affinity, VP1-specific TCR-a/b chain pairs. Candidate TCRs were then precisely introduced into the genome of T cells via CRISPR-Cas9 for further characterization.

A highly conserved region of the JCV VP1 protein (VP1_100-108) was identified as an antigen for HLA-A2 CD8⁺ T cells with little potential for inducing autoimmunity. Fourteen high avidity TCRs specific for VP1_100-108 were isolated, and ten demonstrated specific binding to VP1_100-108 HLA-A2 tetramers. The top candidates have been integrated into T cells via CRISPR-Cas9 and are being further characterized. Other immunogenic regions of VP1 have been similarly identified for HLA-A1 and A3 alleles.   

We identified immunogenic epitopes from the JCV VP1 protein that bind HLA-A1, A2, and A3 and isolated HLA-A2-restricted TCRs that demonstrated recognition and activation to the epitope of interest. While additional in vitro and in vivo validation is in progress, we believe these cells will offer a rapid, “off-the-shelf”, allogeneic anti-JCV T cell therapy for PML patients who are unable to immune reconstitute.