Calreticulin mutant myeloproliferative neoplasms induce MHC-I skewing, which can be overcome by an optimized peptide cancer vaccine

Abstract

The majority of JAK2 V617F -negative myeloproliferative neoplasms (MPNs) have disease-initiating frameshift mutations in calreticulin ( CALR ), resulting in a common carboxyl-terminal mutant fragment (CALR MUT ), representing an attractive source of neoantigens for cancer vaccines. However, studies have shown that CALR MUT -specific T cells are rare in patients with CALR MUT MPN for unknown reasons. We examined class I major histocompatibility complex (MHC-I) allele frequencies in patients with CALR MUT MPN from two independent cohorts. We observed that MHC-I alleles that present CALR MUT neoepitopes with high affinity are underrepresented in patients with CALR MUT MPN. We speculated that this was due to an increased chance of immune-mediated tumor rejection by individuals expressing one of these MHC-I alleles such that the disease never clinically manifested. As a consequence of this MHC-I allele restriction, we reasoned that patients with CALR MUT MPN would not efficiently respond to a CALR MUT fragment cancer vaccine but would when immunized with a modified CALR MUT heteroclitic peptide vaccine approach. We found that heteroclitic CALR MUT peptides specifically designed for the MHC-I alleles of patients with CALR MUT MPN efficiently elicited a CALR MUT cross-reactive CD8 + T cell response in human peripheral blood samples but not to the matched weakly immunogenic CALR MUT native peptides. We corroborated this effect in vivo in mice and observed that C57BL/6J mice can mount a CD8 + T cell response to the CALR MUT fragment upon immunization with a CALR MUT heteroclitic, but not native, peptide. Together, our data emphasize the therapeutic potential of heteroclitic peptide–based cancer vaccines in patients with CALR MUT MPN.

Biology and therapeutic targeting of molecular mechanisms in MPNs

“…This suggests that immune-mediated clearance by individuals expressing a mutant CALR neo-epitope high-affinity binding MHC-I allele occurs such that CALR -mutant MPN may not manifest clinically in these individuals. 140 In the first vaccine trial with a mutant CALR peptide, none of the 10 patients had any clinical response and only 2 had a CD8 + T-cell response, consistent with impaired MHC processing and/or presentation of neoepitope antigens. 145 Alternative approaches to enhance the mutant CALR-directed T-cell immune response include the use of a modified vaccine approach, such as heteroclitic peptides, which optimize MHC-I binding affinity, 140 or combination approaches with an MPN-directed neoantigen vaccine plus an immune checkpoint inhibitor, which is the focus of a recently opened phase 1 clinical trial (# NCT05444530 ).…”

“…Using in silico approaches, it has been shown that the mutant-specific C-terminus of mutant CALR is predicted to generate multiple neoepitopes that bind to major histocompatibility class I (MHC-I) with high affinity. 140 This has led to interest in developing T-cell receptor–mediated immune therapy, including vaccination approaches ( Figure 1 D). However, peptide-stimulated T-cell responses against mutant CALR epitopes are decreased in patients with MPN compared with those in healthy control subjects, suggesting defects in immune response and/or antigen presentation in MPN.…”

Effects of calreticulin mutations on cell transformation and immunity

“…On the other hand, six MHC class I alleles, HLA‐A*11:01, HLA‐B*08:01, HLA‐B*44:02, HLA‐C*07:01, HLA‐C*07:02 and HLA‐C*06:02, predicted as strong binders of mutant CRT‐derived peptides were underrepresented in both cohorts. 88 MHC class I skewing was specifically observed in CRT‐mutated MPNs. Pre‐stimulation of healthy donor PBMCs or pre‐immunization of mice with modified peptide sequences (heteroclitic peptides) could induce immune activation to an otherwise weak binding peptide.…”

“…MHC class I allele representation in MPN patients from US and Danish cohorts was biased towards allotypes exhibiting weak predicted binding affinities to mutant CRT‐derived peptide sequences. 88 HLA‐B*51:01, predicted to bind poorly to mutant CRT peptides, was found to be overrepresented. On the other hand, six MHC class I alleles, HLA‐A*11:01, HLA‐B*08:01, HLA‐B*44:02, HLA‐C*07:01, HLA‐C*07:02 and HLA‐C*06:02, predicted as strong binders of mutant CRT‐derived peptides were underrepresented in both cohorts.…”

“…MHC class I skewing was specifically observed in CRT‐mutated MPNs. Pre‐stimulation of healthy donor PBMCs or pre‐immunization of mice with modified peptide sequences (heteroclitic peptides) could induce immune activation to an otherwise weak binding peptide 88 . Thus, optimization of peptide sequences to allow presentation by overrepresented MHC alleles might provide an opportunity to activate mutant CRT‐specific T cell responses in MPN patients.…”