Isolation of T cell receptors targeting recurrent neoantigens in hematological malignancies

Abstract

Mutation-derived neoantigens represent an important class of tumour-specific, tumour rejection antigens, and are attractive targets for TCR gene therapy of cancer. The majority of such mutations are patient-specific and targeting these requires a fully personalized approach. However, some mutations are found recurrently among cancer patients, and represent potential targets for neoantigen-specific TCR gene therapy that is more widely applicable. Therefore, we have investigated if some cancer mutations found recurrently in hematological malignancies encode immunogenic neoantigens presented by common European Caucasoid HLA class I alleles and can form targets for TCR gene therapy. We initially focused on identifying HLA class I neoepitopes derived from calreticulin (CALR) exon 9 mutations, found in ~ 80% of JAK2wt myeloproliferative neoplasms (MPN). Based on MHC class I peptide predictions, a number of peptides derived from mutant CALR (mCALR) were predicted to bind to HLA-A*03:01 and HLA-B*07:02. However, using mass spectrometry and ex vivo pMHC multimer staining of PBMC from MPN patients with CALR exon 9 mutations, we found no evidence that these peptides were naturally processed and presented on the surface of mCALR-expressing target cells. We next developed a protocol utilizing pMHC multimers to isolate CD8+ T cells from healthy human donor PBMC that are specific for mCALR and additional putative neoepitopes found recurrently in hematological malignancies. Using this approach, CD8+ T cells specific for HLA-A*03:01- and HLA-B*07:02-presented mCALR peptides and an HLA-A*11:01-presented mutant FBXW7 (mFBXW7) peptide were successfully isolated. TCRs isolated from mCALR-specific CD8+ T cell populations were not able to recognize target cells engineered to express mCALR. In contrast, mFBXW7-specific CD8+ T cells were able to recognize target cells engineered to express mFBXW7. In conclusion, while we found no evidence for mCALR derived neoepitope presentation in the context of the HLA class I alleles studied, our data suggests that the recurrent pR465H mutation in FBXW7 may encode an HLA-A*11:01 presented neoepitope, and warrants further investigation as a target for T cell based immunotherapy of cancer.Electronic supplementary materialThe online version of this article (10.1186/s40425-018-0386-y) contains supplementary material, which is available to authorized users.

Immune Checkpoint Blockade Enhances Shared Neoantigen-Induced T-cell Immunity Directed against Mutated Calreticulin in Myeloproliferative Neoplasms

“…Tubb and colleagues focused on identifying mut-CALR epitopes recognized by CD8 + T cells based on predicted epitope-MHC information. However, mut-CALR reactive CD8 + T cells were elicited only in healthy donors but not in patients with MPN (38). In either case, the majority of predicted peptides failed to elicit T-cell responses and the observed mut-CALR-specific T-cell responses were weak.…”

“…Recent studies from Holmstörm and colleagues and Tubb and colleagues also evaluated mut-CALR-specific T-cell immunity (36)(37)(38). Holmstörm and colleagues reported that .…”

Selection of Tumor-Specific Cytotoxic T Lymphocytes in Acute Myeloid Leukemia Patients Through the Identification of T-Cells Capable to Establish Stable Interactions With the Leukemic Cells: “Doublet Technology”

“…One of the main treatment modalities in cancer immunotherapy is based on adoptive transfer of tumor-specific cytotoxic T cells into cancer patients with the goal of recognizing, targeting, and destroying tumor cells ( 2 , 34 ). The conventional methods to identify and isolate tumor-specific CTLs are based on cytokine production assays and soluble pMHC multimers ( 16 , 21 , 35 ). The first one is based on selecting those CTLs that highly release INF-γ upon exposure to a specific antigen.…”

Neoantigens in Hematologic Malignancies

“…Another group described cytokine production, primarily by CD4 + T cells, in response to ex vivo stimulation of peripheral blood mononuclear cells from patients with MPNs with long (31mer) CALR mut peptides (133). CD8 + T cells specific for CALR mut peptides presented on HLA-A * 03:01 and -B * 07:02 were identified by another group, but the low avidity of the T cells prevented them from assessing whether the epitopes were naturally processed and presented on CALR mut cells (115). Additionally, a 9mer peptide spanning the JAK2 V617F mutation (VLNYGVCFC) was identified as a ligand of HLA-A * 02:01 by HLA-binding prediction; epitope-specific CD8 + T cells lysed target cells either pulsed with the mutant peptide or naturally expressing JAK2 V617F, but also recognized targets pulsed with wildtype JAK2 peptide with lower efficiency (116).…”