CBFB-MYH11 fusion neoantigen enables T cell recognition and killing of acute myeloid leukemia

Abstract

Proteins created from recurrent fusion genes like CBFB-MYH11 are prevalent in acute myeloid leukemia (AML), often necessary for leukemogenesis, persistent throughout the disease course, and highly leukemia specific, making them attractive neoantigen targets for immunotherapy. A nonameric peptide derived from a prevalent CBFB-MYH11 fusion protein was found to be immunogenic in HLA-B*40:01+ donors. High-avidity CD8+ T cell clones isolated from healthy donors killed CBFB-MYH11+ HLA-B*40:01+ AML cell lines and primary human AML samples in vitro. CBFB-MYH11-specific T cells also controlled CBFB-MYH11+ HLA-B*40:01+ AML in vivo in a patient-derived murine xenograft model. High-avidity CBFB-MYH11 epitope-specific T cell receptors (TCRs) transduced into CD8+ T cells conferred antileukemic activity in vitro. Our data indicate that the CBFB-MYH11 fusion neoantigen is naturally presented on AML blasts and enables T cell recognition and killing of AML. We provide proof of principle for immunologically targeting AML-initiating fusions and demonstrate that targeting neoantigens has clinical relevance even in low-mutational frequency cancers like fusion-driven AML. This work also represents a first critical step toward the development of TCR T cell immunotherapy targeting fusion gene-driven AML.

Keywords: Cancer immunotherapy; Immunology; Leukemias; Oncology; T cells.

Figure 1. Schematic of the type A CBFB-MYH11 fusion resulting from an inversion in chromosome 16.

In the type A fusion, exon 5 of CBFB is fused to exon 34 of MYH11, creating a fusion protein with the junctional amino acid sequence depicted.

Figure 2. Highly avid CD8⁺ T cells recognizing an HLA-B*40:01–restricted epitope of CBFB-MYH11 can be isolated from healthy donors.

(A) Percentage specific lysis of peptide-pulsed targets for individual wells of CD8⁺ T cell lines after in vitro peptide stimulation of 2 HLA-B*40:01⁺ donors. Percent specific lysis was calculated as percent lysis of peptide-pulsed targets minus percent lysis of no-peptide targets to remove nonspecific reactivity to autologous target cells alone. Gray circles indicate wells with no peptide-specific reactivity. The single-peptide specificity of T cells in wells with peptide-specific lysis was subsequently determined: red triangles, REEMEVHEL-specific T cells; black triangles, T cells specific for control (known immunogenic, non–CBFB-MYH11) epitopes. Each plot depicts a single experiment. (B) Percentage lysis of HLA-B*40:01⁺ LCL targets with REEMEVHEL peptide (circles), pooled additional predicted HLA-B*40:01–binding peptides (squares), or no exogenous peptide (triangles) by REEMEVHEL-specific CD8⁺ T cell clones isolated from 3 healthy donors in 4-hour CRA. (C) Percentage lysis of HLA-B*40:01⁺ LCL targets with either REEMEVHEL peptide (circles), known HLA-B*40:01–binding peptide KECVLHDDL (diamonds), or no exogenous peptide (triangles) by REEMEVHEL-specific CD8⁺ T cell clones in 4-hour CRA. For B and C, peptides were used at 1000 ng/mL each; error bars are SD of 3 biological replicates. (D) Percentage lysis of targets pulsed with various concentrations of REEMEVHEL peptide by REEMEVHEL-specific CD8⁺ T cell clones. Mean and SEM of 3 technical replicates are shown. (E) Representative flow plots (from 3 experiments) of CBFB-MYH11/HLA-B*40:01 pHLA tetramer staining of REEMEVHEL-specific T cell clones and an irrelevant clone specific for an epitope (IPRAHNRLV) presented on HLA-B*07:02 (negative control). Cells are gated on live single CD4^–CD8⁺ cells. (F) Percentage lysis of REEMEVHEL-pulsed LCLs (1000 ng/mL) with varying HLA types (including all class I HLA types of the 3 donors) by 6 REEMEVHEL-specific T cell clones.

Figure 3. T cells specific for the HLA-B*40:01–restricted CBFB-MYH11 epitope kill AML cell lines.

(A) Absolute cell number of viable NB-4 cells either CBFB-MYH11–transduced (TD) (solid line) or mock-TD (dashed line) after coculture with high-avidity REEMEVHEL-specific clone D2.C24 from a representative experiment. (B) Representative flow plots for experiments shown in A and C, depicting viable single cells at time points after coculture. (C) Percentage survival of NB-4 cells, either WT (left) or transduced to express the full-length CBFB-MYH11 type A fusion (right), at time points after coculture with the D2.C24 T cell clone. (D) Absolute cell number of viable ME-1 cells either HLA-B*40:01–TD (solid line) or mock-TD (dashed line) and cocultured with clone D2.C24 from a representative experiment. (E) Representative flow plots for experiments shown in D and F, depicting viable single cells at time points after coculture. (F) Percentage survival of ME-1 cells, either WT (left) or transduced to express HLA-B*40:01 (right), at time points after coculture with the D2.C24 T cell clone. Viable cell numbers were assessed at varying time points by flow cytometry and percent survival calculated as described in Supplemental Methods. For C and F, colored bars indicate mean and error bars SD for 3–10 technical replicate samples. Two-sample unpaired 2-tailed t tests were performed to compare 0 hours with each subsequent time point.

Figure 4. The HLA-B*40:01–restricted CBFB-MYH11 epitope is a bona fide AML antigen.

Four high-avidity T cell clones were tested for recognition of CBFB-MYH11⁺ HLA-B*40:01⁺ primary AML, as well as primary AML lacking either the fusion or restricting HLA genotype. (A) Degranulation of T cell clones in response to primary AML was determined by measurement of T cell CD107a presentation in response to stimulation with primary AML (blue bars, CBFB-MYH11⁺ HLA-B*40:01⁺, n = 3 different primary AML samples; orange bars, CBFB-MYH11⁺ HLA-B*40:01^–, n = 7; green bars, CBFB-MYH11^– HLA-B*40:01⁺, n = 6). (B) Representative flow plots from one CD107a degranulation experiment are shown from left to right: CBFB-MYH11⁺ HLA-B*40:01⁺ AML (blue background), negative control stimulators CBFB-MYH11⁺ HLA-B*40:01^– AML (orange background), and CBFB-MYH11^– HLA-B*40:01⁺ AML (green background). (C) Lysis of primary AML by T cell clones was tested in a 4-hour CRA with effector/target (E:T) ratio of 20:1 (blue bars, CBFB-MYH11⁺ HLA-B*40:01⁺, n = 3; orange bars, CBFB-MYH11⁺ HLA-B*40:01^–, n = 6; green bars, CBFB-MYH11^– HLA-B*40:01⁺, n = 5). For A and C, mean and SD for each clone are shown are from a single experiment with 3–7 biological replicates for each group of AML samples. Statistics were calculated using unpaired 2-tailed t tests with Welch’s correction.

Figure 5. CBFB-MYH11/B*40:01–specific T cells control AML in vivo in a PDX murine model.

(A) Experiment overview: Newborn, preconditioned MISTRG mice were injected intrahepatically with 1 × 10⁶ PBMCs (OKT3-pretreated to prevent xenogeneic graft-versus-host disease) from HLA-B*40:01⁺ patients with active CBFB-MYH11⁺ AML. After 12 weeks of AML engraftment, mice received 10 × 10⁶ CD8⁺ T cells i.v., either D2.C24 clone (high-avidity, CBFB-MYH11/B*40:01–specific) or a control clone (specific for a candidate neoantigen epitope, IPRAHNRLV, presented by HLA-B*07:02, for which this AML was genotypically negative), then were monitored by weekly PB sampling. (B) Primary AML PBMCs (AML1, 81% blasts) before OKT3 treatment and injection into mice were stained for myeloid markers using AML tracking in mice. (C) Representative flow plots of mice PB pretreatment (left) and 7 days after injection (right) with either CBFB-MYH11/B40:01–specific (red box) or control (blue box) T cell clones. (D) Summary of PB disease burden by flow cytometry after CBFB-MYH11/B40:01–specific (red circles) or control (blue squares) T cell treatment. Statistics were calculated using repeated-measures 2-way ANOVA. (E) Human CBFB-MYH11 type A transcript expression, normalized to murine CD45 (Ptprc) as 2^–ΔCq, was assessed before and 7 days after administration of CBFB-MYH11/B40:01–specific or control T cells. (F–H) AML burden in terminal bone marrow as percentage (F) or absolute number (G) of human CD33⁺ cells, and relative CBFB-MYH11 type A transcript expression (H). (I) Correlation between marrow disease burden as measured by flow cytometry and real-time qPCR was determined by calculation of Pearson correlation coefficient. For all groups and time points, n = 5, except for control T cell–treated mice on day 7 (n = 4) owing to poor RNA yield from 1 sample. Except where noted, statistics were calculated using unpaired 2-tailed parametric t tests. Mean and SD are shown.

Figure 6. CBFB-MYH11/B*40:01–specific CD8⁺ T cells recognize a unique epitope and have a naive phenotype in healthy donors.

(A) Percentage lysis by REEMEVHEL-specific T cell clones of HLA-B*40:01⁺ LCLs pulsed with individual peptides (at 1000 ng/mL) with alanine substitution at each position. Each bar represents percentage lysis of targets by one T cell clone from 3 technical replicate experiments with mean and SD shown. (B) Sequence logo of critical residues of the CBFB-MYH11/B*40:01 epitope for T cell clones compared with residues shared among known HLA-B*40:01–restricted microbial epitopes from IEDB. (C) Phenotypic evaluation of epitope-specific T cells isolated in healthy donors by CBFB-MYH11/B*40:01 tetramer enrichment of PBMCs from healthy donors and surface marker staining. Representative flow plots of viable CD8⁺ single cells from tetramer enrichment of donor D1 are shown (of 3 biological replicate experiments summarized in D). (D) Summary of phenotypic evaluation from C across 3 biological replicates (donors D1–D3); mean and SD are shown.

Figure 7. TCRs specific for the CBFB-MYH11/B*40:01 epitope confer antileukemic activity.

(A) Expression of transgenic TCRs in CD8⁺ T cells transduced (TD) with D1.C6 B1A1, D2.C8 B1A1, D2.C24 B1A1, and D3.C5 B1A2 TCR constructs or mock-TD is shown by staining with CBFB-MYH11/B*40:01 tetramer from a single experiment. (B) HLA-B*40:01⁺ LCLs pulsed with REEMEVHEL peptide at various concentrations were lysed in CRA by D2.C24 TCR-TD CD8⁺ T cells (dashed line) and D2.C24 parental clone (solid line). Mean and SEM for 3 technical replicates are shown. (C) Degranulation of D2.C24 TCR-TD CD8⁺ T cells and D2.C24 parental clone in response to primary AML was determined by measurement of T cell CD107a presentation in response to stimulation with primary AML (blue bars, CBFB-MYH11⁺ HLA-B*40:01⁺, n = 3 different primary AML samples; orange bars, CBFB-MYH11⁺ HLA-B*40:01^–, n = 3; green bars, CBFB-MYH11^– HLA-B*40:01⁺, n = 4). (D) Lysis of primary AML by D2.C24 TCR-TD CD8⁺ T cells, D2.C24 parental clone, and mock-TD CD8⁺ T cells was evaluated in a standard 4-hour CRA with E:T of 20:1 (CBFB-MYH11⁺ HLA-B*40:01⁺, n = 3 different AML samples; CBFB-MYH11⁺ HLA-B*40:01^–, n = 6; CBFB-MYH11^– HLA-B*40:01⁺, n = 5). For C and D, mean and SD are shown. Statistics were calculated using unpaired 2-tailed t tests with Welch’s correction.