Mutated nucleophosmin 1 as immunotherapy target in acute myeloid leukemia

Abstract

The most frequent subtype of acute myeloid leukemia (AML) is defined by mutations in the nucleophosmin 1 (NPM1) gene. Mutated NPM1 (ΔNPM1) is an attractive target for immunotherapy, since it is an essential driver gene and 4 bp frameshift insertions occur in the same hotspot in 30%-35% of AMLs, resulting in a C-terminal alternative reading frame of 11 aa. By searching the HLA class I ligandome of primary AMLs, we identified multiple ΔNPM1-derived peptides. For one of these peptides, HLA-A*02:01-binding CLAVEEVSL, we searched for specific T cells in healthy individuals using peptide-HLA tetramers. Tetramer-positive CD8+ T cells were isolated and analyzed for reactivity against primary AMLs. From one clone with superior antitumor reactivity, we isolated the T cell receptor (TCR) and demonstrated specific recognition and lysis of HLA-A*02:01-positive ΔNPM1 AML after retroviral transfer to CD8+ and CD4+ T cells. Antitumor efficacy of TCR-transduced T cells was confirmed in immunodeficient mice engrafted with a human AML cell line expressing ΔNPM1. In conclusion, the data show that ΔNPM1-derived peptides are presented on AML and that CLAVEEVSL is a neoantigen that can be efficiently targeted on AML by ΔNPM1 TCR gene transfer. Immunotherapy targeting ΔNPM1 may therefore contribute to treatment of AML.

Keywords: Cancer immunotherapy; Hematology; Leukemias; T-cell receptor.

Figure 1. DNA and protein sequences for WTNPM1 and ΔNPM1.

Nucleic and amino acid sequences are shown for WTNPM1 (upper) and ΔNPM1 (lower). Indicated is a TCTG insertion between c.863_864 in the coding sequence of NPM1, which is the most frequent frameshift mutation in the hotspot region. Translated aa sequences are shown until the termination sequence (stop, indicated by asterisks). As a consequence of the TCTG insertion, the C-terminal aa CLAVEEVSLRK of ΔNPM1 are translated in an alternative reading frame. WT and mutant aa are indicated in gray and red, respectively.

Figure 2. Validation of C*LAVEEVSL as peptide eluted from HLA-A*02:01–positive AML with ΔNPM1.

C*LAVEEVSL was validated as peptide eluted from HLA-A*02:01–positive AML with ΔNPM1 by comparing MS/MS between eluted peptides and synthetic peptide C*LAVEEVSL. Matching MS/MS are shown for an eluted peptide from AML10197 (upper panel) and synthetic peptide C*LAVEEVSL (lower panel). C*, cysteinylation of the Cys residue.

Figure 3. CLAVEEVSL as ΔNPM1-derived neoantigen.

CD8⁺ T cells specific for ΔNPM1 were single cell isolated from PBMCs from HLA-A*02:01–positive healthy individuals using a mix of ΔNPM1-CLA and ΔNPM1-C*LA pHLA tetramers. (A) T cell clones were stained with pHLA tetramers. T cell clones 1A2 (left) and 4A8 (right) were both positive for the ΔNPM1-CLA tetramer (red), whereas only clone 1A2 stained with ΔNPM1-C*LA (orange). (B) T cell clones 1A2 (upper panel) and 4A8 (lower panel) were tested for reactivity against HLA-A*02:01–positive T2 cells exogenously loaded with titrated concentrations of ΔNPM1 peptide CLAVEEVSL (red circles), the cysteinylated variant C*LAVEEVSL (orange squares), or an irrelevant HLA-A*02:01–binding CMV peptide NLVPMVATV (blue triangles) by IFN-γ ELISA. Both T cell clones showed recognition of CLAVEEVSL, and clone 1A2 also showed recognition of C*LAVEEVSL. Release of IFN-γ (ng/ml) in duplicate wells is shown. (C) Clones 1A2 (upper panel) and 4A8 (middle panel) were tested for reactivity against 5 HLA-A*02:01–positive primary AMLs by IFN-γ ELISA. The panel included 3 AMLs with ΔNPM1 and 2 AMLs with WTNPM1. T cell clone 1A2 reacted against all 3 AMLs with ΔNPM1, whereas clone 4A8 recognized 2 AMLs. An HLA-A*02:01–specific alloreactive T cell clone (Allo-A2 clone) was included as a positive control. Release of IFN-γ (ng/ml) in duplicate wells is shown; bars represent mean IFN-γ release.

Figure 4. Targeting ΔNPM1 by TCR gene transfer.

CD8⁺ and CD4⁺ T cells isolated from HLA-A*02:01–positive healthy individuals were transduced with the TCR for ΔNPM1 from clone 1A2 and a TCR for HLA-A*02:01–binding CMV peptide NLVPMVATV. TCR-transduced T cells were purified by MACS. (A) TCR-transduced T cells were analyzed by flow cytometry using ΔNPM1-CLA (red) and CMV-NLV (blue) pHLA tetramers. CD8⁺ (CD8ØNPM1) and CD4⁺ (CD4ØNPM1) T cells transduced with the TCR for ΔNPM1 stained with the ΔNPM1-CLA tetramer, whereas CD8⁺ (CD8ØCMV) and CD4⁺ (CD4ØCMV) T cells transduced with the CMV-specific TCR showed binding to the CMV-NLV tetramer. Results are shown for donor 1. (B) TCR-transduced T cells were analyzed for reactivity against T2 cells exogenously loaded with titrated concentrations of CLAVEEVSL (red circles) or NLVPMVATV (blue squares) by IFN-γ ELISA. CD8ØNPM1 (upper left panel) and CD4ØNPM1 (lower left panel) showed half-maximum recognition of CLAVEEVSL at concentrations of 30–100 nM (dotted lines), but no recognition of NLVPMVATV. Conversely, CD8ØCMV (upper right panel) and CD4ØCMV (lower right panel) reacted against NLVPMVATV, but not CLAVEEVSL. Release of IFN-γ (ng/ml) in duplicate wells is shown for donor 1. (C) TCR-transduced T cells were tested for recognition of HLA-A*02:01–positive AML cell lines with ΔNPM1 (OCI-AML3) or WTNPM1 (OCI-AML2) in the absence or presence of blocking antibodies against HLA class I (αHLA-I) or HLA class II (αHLA-II) by IFN-γ ELISA. Recognition of OCI-AML3 by CD8⁺ and CD4⁺ T cells transduced with the TCR for ΔNPM1 is mediated by HLA class I. Release of IFN-γ (ng/ml) in duplicate wells is shown for donor 2. Bars represent mean IFN-γ release.

Figure 5. Recognition of primary AMLs after ΔNPM1 TCR gene transfer.

TCR-transduced CD8⁺ and CD4⁺ T cells were tested for reactivity against a panel of 13 HLA-A*02:01–positive primary AMLs, including 9 samples with ΔNPM1 and 4 samples with WTNPM1 by IFN-γ ELISA (Supplemental Table 4). CD8⁺ (CD8ØNPM1; red circles) and CD4⁺ (CD4ØNPM1; red squares) T cells transduced with the TCR for ΔNPM1 reacted against all 9 AMLs with ΔNPM1, whereas none of the AMLs were specifically recognized by CD8⁺ (CD8ØCMV; blue circles) or CD4⁺ (CD4ØCMV; blue squares) T cells after transfer of the CMV-specific TCR. The allo-A2 clone (gray triangles) was included as a positive control. Release of IFN-γ (ng/ml) in duplicate wells is shown for donor 1.

Figure 6. Lysis of primary AMLs after ΔNPM1 TCR gene transfer.

TCR-transduced CD8⁺ and CD4⁺ T cells were tested for cytolytic capacity by a 9-hour ⁵¹Cr-release assay on 6 HLA-A*02:01–positive primary AMLs, including 4 samples with ΔNPM1 and 2 samples with WTNPM1. CD8⁺ (CD8ØNPM1; red circles) and CD4⁺ (CD4ØNPM1; red squares) T cells transduced with the TCR for ΔNPM1 showed specific lysis of all 4 AMLs with ΔNPM1, whereas none of the AMLs were specifically lysed by CD8⁺ (CD8ØCMV; blue circles) or CD4⁺ (CD4ØCMV; blue squares) T cells after transfer of the CMV-specific TCR. The allo-A2 clone (gray triangles) was included as a positive control. Data represent mean percentage of specific lysis in triplicate wells ± SD for donor 2.

Figure 7. In vivo antitumor efficacy after ΔNPM1 TCR gene transfer.

Male NSG mice were engrafted with 1 × 10⁶ HLA-A*02:01–positive OCI-AML3 cells with ΔNPM1. OCI-AML3 cells were transduced with luciferase to follow in vivo tumor growth by bioluminescence imaging. Two weeks after tumor inoculation, mice were either left untreated or injected i.v. with 4 × 10⁶ CD8⁺/CD4⁺ T cells (2:3 ratio) transduced with the TCR for ΔNPM1 or with the CMV-specific control TCR. TCR-transduced T cells were produced from HLA-A*02:01–positive healthy donor 3. (A and B) After T cell injection, tumor burden was followed twice per week for 21 days. In mice receiving CD8⁺/CD4⁺ T cells transduced with the TCR for ΔNPM1 (ΔNPM1 T cells; n = 5; red), tumor load was significantly reduced within 2 weeks of T cell transfer, resulting in delayed tumor outgrowth as compared with that in untreated mice (no T cells; n = 4; black) and mice treated with CD8⁺/CD4⁺ T cells transduced with the CMV-specific control TCR (CMV T cells; n = 2; blue). (C) Kaplan-Meier curve showing better overall survival of mice treated with the ΔNPM1-specific TCR (n = 5; red line) as compared with untreated mice (n = 4; black line) or mice treated with the CMV-specific control TCR (n = 2; blue line). P = 0.0206, log-rank (Mantel-Cox) test.