Identification and Characterization of HLA Class I-Restricted MYD88 L265P-Derived Peptides As Tumor-Specific Targets for Immunotherapy

Non-Hodgkin lymphomas (NHL) are frequent malignancies with considerable mortality. A recurrent somatic and oncogenic driver mutation in the Toll-like receptor adaptor gene MYD88, Leu265Pro (L265P) has been identified in up to 90% of certain NHL subtypes. Genetic alterations affecting a protein-coding region have the potential to generate mutation-derived peptides that are presented by HLA class I proteins and might be recognized by cytotoxic T cells. Because MYD88^L265P is a widely occurring and tumor-specific mutation, we investigated the potential of MYD88^L265P-containing peptides for CD8⁺ T cell mediated immunotherapy as a new therapeutic approach for MYD88^L265P+ NHL.

Based on in silico prediction we identified potential HLA ligands encompassing the MYD88^L265P mutation for several HLA class I allotypes. Functional characterization of the candidate HLA class I MYD88^L265P-derived HLA class I ligands with regard to induction of T cell responses identified a set of immunogenic peptides for HLA-B*07 and ‑B*15. In one MYD88^L265P-mutated NHL patient, memory T cell responses targeting three different MYD88^L265P-derived HLA class I ligands were detected by IFN-γ ELISPOT. Efficient T cell priming was demonstrated in vitro using naïve T cells of healthy volunteers (HVs). In detail, three HLA‑B*07 peptides (P1-3_B*07) and one HLA‑B*15-restricted peptide (P4_B*15) were analyzed using artificial antigen-presenting cell-based (aAPC) in vitro priming experiments in three to six HVs, respectively. For all tested peptides proliferation of peptide-specific CD8⁺ T cells could be detected after in vitro priming. For the HLA-B*07-restricted ligands, peptide-specific CD8⁺ T cells could be induced in 6/6 (P1_B*15), 1/3 (P2_B*07) and 3/4 (P3_B*07) HVs, respectively, with a maximum frequency of 14.1% peptide-specific CD8⁺ T cells. For the HLA-B*15-restricted ligand (P4_B*15), peptide-specific CD8⁺ T cells could be induced in 2/3 HVs with a maximum frequency of 9.5% tetramer-positive CD8⁺ T cells. The functionality and specificity of peptide-specific CD8⁺ T cells after aAPC-based in vitro priming was validated by intracellular cytokine staining for IFN-γ and TNF-α as well as for the expression of the degranulation marker CD107a. In 3/3 HVs primed with P1_B*07 (RPIPIKYKAM) as well as in 1/2 HVs primed with P4_B*15 (HQKRPIPIKY), we detected specific and functional CD8⁺ T cell populations after stimulation with the mutated peptides, but not after stimulation with the corresponding wild type peptides (P1_WT: RLIPIKYKAM, P4_WT: HQKRLIPIKY). Furthermore, the peptide-specific cytotoxic activity of specific CD8⁺ T cells was demonstrated in a VITAL assay. The polyclonal P1_B*07- and P4_B*15-specific CD8⁺ T cells (0.12% and 0.76% peptide-specific T cells, respectively) lysed autologous peripheral blood mononuclear cells loaded with the mutated peptides, but not cells presenting the wild type peptides. P4_B*15-specific CD8⁺ T cells showed 17.9% (±1.2%) MYD88^L265P-peptide-specific cell killing at an E/T ratio of 1:1 compared to 2.6% (±1.2%) of non-specific cell lysis of unspecific effector cells against the same targets in three independent replicates, respectively. The specific lysis showed an E/T ratio-dependent manner as the specific lysis decreases with reducing E/T ratios. P1_B*07-specific CD8⁺ T cells specifically killed 11.4% (±1.7%) of MYD88^L265P loaded targets at an E/T ratio of 0.7:1 in comparison to 2.1% unspecific lysis of unspecific effector cells.

In this study, we identified and characterized MYD88^L265P mutation-derived HLA class I ligands for T cell mediated immunotherapy. The strong immunogenicity of the HLA-B*07 and HLA-B*15-restricted mutation-derived peptides as well as the functionality and specificity of peptide-specific CD8⁺ T cells, demonstrated by cytotoxicity assays, underline the potential of the MYD88^L265P mutation as tumor-specific target. These data highlight the potential of MYD88^L265P mutation-specific immunotherapy as a novel broadly applicable and tumor-specific treatment approach for patients with MYD88^L265P+ NHL.