Hotspot DNA Methyltransferase 3A (DNMT3A) and Isocitrate Dehydrogenase 1 and 2 (IDH1/2) Mutations in Acute Myeloid Leukemia and Their Relevance as Targets for Immunotherapy.

DNA methyltransferase 3A (DNMT3A) and isocitrate dehydrogenase 1 and 2 (IDH1/2) are genes involved in epigenetic regulation, each mutated in 7-23% of patients with acute myeloid leukemia. Here, we investigated whether hotspot mutations in these genes encode neoantigens that can be targeted by immunotherapy. Five human B-lymphoblastoid cell lines expressing common HLA class I alleles were transduced with a minigene construct containing mutations that often occur in DNMT3A or IDH1/2. From these minigene-transduced cell lines, peptides were eluted from HLA class I alleles and analyzed using tandem mass spectrometry. The resulting data are available via ProteomeXchange under the identifier PXD050560. Mass spectrometry revealed an HLA-A*01:01-binding DNMT3AR882H peptide and an HLA-B*07:02-binding IDH2R140Q peptide as potential neoantigens. For these neopeptides, peptide-HLA tetramers were produced to search for specific T-cells in healthy individuals. Various T-cell clones were isolated showing specific reactivity against cell lines transduced with full-length DNMT3AR882H or IDH2R140Q genes, while cell lines transduced with wildtype genes were not recognized. One T-cell clone for DNMT3AR882H also reacted against patient-derived acute myeloid leukemia cells with the mutation, while patient samples without the mutation were not recognized, thereby validating the surface presentation of a DNMT3AR882H neoantigen that can potentially be targeted in acute myeloid leukemia via immunotherapy.

Expanding the repertoire reveals recurrent, cryptic, and hematopoietic HLA class I minor histocompatibility antigens.

ABSTRACT: Allogeneic stem cell transplantation (alloSCT) is a curative treatment for hematological malignancies. After HLA-matched alloSCT, antitumor immunity is caused by donor T cells recognizing polymorphic peptides, designated minor histocompatibility antigens (MiHAs), that are presented by HLA on malignant patient cells. However, T cells often target MiHAs on healthy nonhematopoietic tissues of patients, thereby inducing side effects known as graft-versus-host disease. Here, we aimed to identify the dominant repertoire of HLA-I-restricted MiHAs to enable strategies to predict, monitor or modulate immune responses after alloSCT. To systematically identify novel MiHAs by genome-wide association screening, T-cell clones were isolated from 39 transplanted patients and tested for reactivity against 191 Epstein-Barr virus transformed B cell lines of the 1000 Genomes Project. By discovering 81 new MiHAs, we more than doubled the antigen repertoire to 159 MiHAs and demonstrated that, despite many genetic differences between patients and donors, often the same MiHAs are targeted in multiple patients. Furthermore, we showed that one quarter of the antigens are cryptic, that is translated from unconventional open reading frames, for example long noncoding RNAs, showing that these antigen types are relevant targets in natural immune responses. Finally, using single cell RNA-seq data, we analyzed tissue expression of MiHA-encoding genes to explore their potential role in clinical outcome, and characterized 11 new hematopoietic-restricted MiHAs as potential targets for immunotherapy. In conclusion, we expanded the repertoire of HLA-I-restricted MiHAs and identified recurrent, cryptic and hematopoietic-restricted antigens, which are fundamental to predict, follow or manipulate immune responses to improve clinical outcome after alloSCT.

Automated manufacture of ΔNPM1 TCR-engineered T cells for AML therapy.

Acute myeloid leukemia (AML) is a heterogeneous malignancy that requires further therapeutic improvement, especially for the elderly and for subgroups with poor prognosis. A recently discovered T cell receptor (TCR) targeting mutant nucleophosmin 1 (ΔNPM1) presents an attractive option for the development of a cancer antigen-targeted cellular therapy. Manufacturing of TCR-modified T cells, however, is still limited by a complex, time-consuming, and laborious procedure. Therefore, this study specifically addressed the requirements for a scaled manufacture of ΔNPM1-specific T cells in an automated, closed, and good manufacturing practice-compliant process. Starting from cryopreserved leukapheresis, 2E8 CD8-positive T cells were enriched, activated, lentivirally transduced, expanded, and finally formulated. By adjusting and optimizing culture conditions, we additionally reduced the manufacturing time from 12 to 8 days while still achieving a clinically relevant yield of up to 5.5E9 ΔNPM1 TCR-engineered T cells. The cellular product mainly consisted of highly viable CD8-positive T cells with an early memory phenotype. ΔNPM1 TCR CD8 T cells manufactured with the optimized process showed specific killing of AML in vitro and in vivo. The process has been implemented in an upcoming phase 1/2 clinical trial for the treatment of NPM1-mutated AML.

An HLA-A*11:01-Binding Neoantigen from Mutated NPM1 as Target for TCR Gene Therapy in AML.

Acute myeloid leukemia (AML) is a hematological malignancy caused by clonal expansion of myeloid progenitor cells. Most patients with AML respond to chemotherapy, but relapses often occur and infer a very poor prognosis. Thirty to thirty-five percent of AMLs carry a four base pair insertion in the nucleophosmin 1 gene (NPM1) with a C-terminal alternative reading frame of 11 amino acids. We previously identified various neopeptides from the alternative reading frame of mutant NPM1 (dNPM1) on primary AML and isolated an HLA-A*02:01-restricted T-cell receptor (TCR) that enables human T-cells to kill AML cells upon retroviral gene transfer. Here, we isolated T-cells recognizing the dNPM1 peptide AVEEVSLRK presented in HLA-A*11:01. The TCR cloned from a T-cell clone recognizing HLA-A*11:01+ primary AML cells conferred in vitro recognition and lysis of AML upon transfer to CD8 cells, but failed to induce an anti-tumor effect in immunodeficient NSG mice engrafted with dNPM1 OCI-AML3 cells. In conclusion, our data show that AVEEVSLRK is a dNPM1 neoantigen on HLA-A*11:01+ primary AMLs. CD8 cells transduced with an HLA-A*11:01-restricted TCR for dNPM1 were reactive against AML in vitro. The absence of reactivity in a preclinical mouse model requires further preclinical testing to predict the potential efficacy of this TCR in clinical development.

Alphavirus-based hepatitis C virus therapeutic vaccines: can universal helper epitopes enhance HCV-specific cytotoxic T lymphocyte responses?

BACKGROUND: Antigen-specific T cell immune responses play a pivotal role in resolving acute and chronic hepatitis C virus (HCV) infections. Currently, no prophylactic or therapeutic vaccines against HCV are available. We previously demonstrated the preclinical potency of therapeutic HCV vaccines based on recombinant Semliki Forest virus (SFV) replicon particles. However, clinical trials do not always meet the high expectations of preclinical studies, thus, optimization of vaccine strategies is crucial. In efforts to further increase the frequency of HCV-specific immune responses in the candidate SFV-based vaccines, the authors assessed whether inclusion of three strong, so-called universal helper T cell epitopes, and an endoplasmic reticulum localization, and retention signal (collectively termed sigHELP-KDEL cassette) could enhance HCV-specific immune responses. METHODS: We included the sigHELP-KDEL cassette in two of the candidate SFV-based HCV vaccines, targeting NS3/4A and NS5A/B proteins. We characterized the new constructs in vitro for the expression and stability of the transgene-encoded proteins. Their immune efficacy with respect to HCV-specific immune responses in vivo was compared with the parental SFV vaccine expressing the corresponding HCV antigen. Further characterization of the functionality of the HCV-specific CD8+ T cells was assessed by surface and intracellular cytokine staining and flow cytometry analysis. RESULTS: Moderate, but significantly, enhanced frequencies of antigen-specific immune responses were achieved upon lower/suboptimal dosage immunization. In optimal dosage immunization, the inclusion of the cassette did not further increase the frequencies of HCV-specific CD8+ T cells when compared with the parental vaccines and the frequencies of effector and memory populations were identical. CONCLUSION: We hypothesize that the additional effect of the sigHELP-KDEL cassette in SFV-based vaccines depends on the immunogenicity, nature, and stability of the target antigen expressed by the vaccine.

An alphavirus-based therapeutic cancer vaccine: from design to clinical trial.

Cancer immunotherapy has greatly advanced in recent years. Most immunotherapeutic strategies are based on the use of immune checkpoint blockade to unleash antitumor immune responses or on the induction or adoptive transfer of immune effector cells. We aim to develop therapeutic vaccines based on recombinant Semliki Forest virus vectors to induce tumor-specific effector immune cells. In this review, we describe our ongoing work on SFV-based vaccines targeted against human papillomavirus- and hepatitis C virus-related infections and malignancies, focusing on design, delivery, combination strategies, preclinical efficacy and product development for a first-in-man clinical trial with an HPV-specific vaccine.

Synthesis and study of 2-(pyrrolesulfonylmethyl)-N-arylimines: a new class of inhibitors for human glutathione transferase A1-1.

Overexpression of human GSTA1-1 in tumor cells is part of MDR mechanisms. We report on the synthesis of 11 pyrrole derivatives as hGSTA1-1 inhibitors starting from 1-methyl-2-[(2-nitrobenzylsulfanyl]-1H-pyrrole. Molecular modeling revealed two locations in the enzyme H binding site: the catalytic primary one accommodating shorter and longer derivatives and the secondary one, where shorter derivatives can occupy. Derivative 9, displaying the highest inhibition and bearing a p-nitroarylimino moiety, and derivative 4, lacking this moiety, were studied kinetically. Derivative 9 binds (K(i(9)) = 71 ± 4 µM) at the primary site competitively vs CDNB. Derivative 4 binds (K(i(4)) = 135 ± 27 µM) at the primary and secondary sites, allowing the binding of a second molecule (4 or CDNB) leading to formation of unreactive and reactive complexes, respectively. The arylmethylsulfonylpyrrole core structure is a new pharmacophore for hGSTA1-1, whereas its derivative 9 may serve as a lead structure.