Neoantigen-specific TCR sequences are identified for a newly characterized CLL neoantigen. (A) Summary of filtering process used to identify CLL neoantigens arising from indel mutations. (B) A frameshift deletion in a putative CLL driver MGA (4227 del T) generates a neoantigen, with predicted binding affinity of IC₅₀ = 106.5 nM to HLA-A*02:01. Other mutations identified in 16 patients with CLL shown here were previously described (* denotes nonsense mutation).¹⁶  (C) IFNγ ELISPOT confirmed the immunogenicity of mut-MGA_106 peptide in HLA-A*02:01⁺ healthy donor PBMCs cultured with mut-MGA_106 peptide (P = .0029, compared with control peptide with 1-sided 2-sample t-test). Abrogation of response was observed with class I blocking antibody, W6/32 (P = .0005). (D) Single-cell TCR sequencing identified enriched clones (red for downstream cloning and expression; open triangle for matched mut-MGA_106-specific TCR). (E) Dominant TCRs were cloned and expressed on Jurkat∆αβ reporter cells, and cocultured with autologous APCs pulsed with mut-MGA_106 peptide. One mut-MGA-specific TCR was identified by IL-2 ELISA. (F) Functional avidity of mut-MGA-specific TCR is determined.