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CD8+ T cell-mediated recognition of peptide-major histocompatibility complex class I (pMHCI) molecules involves cooperative binding of the T cell receptor (TCR), which confers antigen specificity, and the CD8 coreceptor, which stabilizes the TCR/pMHCI complex. Earlier work has shown that the sensitivity of antigen recognition can be regulated in vitro by altering the strength of the pMHCI/CD8 interaction. Here, we characterized two CD8 variants with moderately enhanced affinities for pMHCI, aiming to boost antigen sensitivity without inducing non-specific activation. Expression of these CD8 variants in model systems preferentially enhanced pMHCI antigen recognition in the context of low-affinity TCRs. A similar effect was observed using primary CD4+ T cells transduced with cancer-targeting TCRs. The introduction of highaffinity CD8 variants also enhanced the functional sensitivity of primary CD8+ T cells expressing cancer-targeting TCRs, but comparable results were obtained using exogenous wild-type CD8. Specificity was retained in every case, with no evidence of reactivity in the absence of cognate antigen. Collectively, these findings highlight a generically applicable mechanism to enhance the sensitivity of low-affinity pMHCI antigen recognition, which could augment the therapeutic efficacy of clinically relevant TCRs. [ABSTRACT FROM AUTHOR]

Simple Summary: Acute myeloid leukemia (AML) is an aggressive hematological malignancy with poor prognosis. For AML relapses after chemotherapy, new and effective therapies are needed. In 30–35% of AMLs, a frameshift mutation in the nucleophosmin 1 gene (dNPM1) creates potential neoantigens that are attractive targets for immunotherapy. We previously isolated a T-cell receptor (TCR) that targets an HLA-A*02:01-binding dNPM1 neoantigen on primary AML. Here, we investigated whether AVEEVSLRK is another dNPM1 neoantigen that can be targeted by TCR gene transfer. We isolated various T-cells, cloned the HLA-A*11:01-restricted TCR from one T-cell clone and, upon transfer to CD8 cells, demonstrated targeting of dNPM1 primary AMLs in vitro. However, the TCR failed to mediate an anti-tumor effect in immunodeficient mice engrafted with dNPM1 OCI-AML3 cells. Our results demonstrate that AVEEVSLRK is an HLA-A*11:01-binding neoantigen on dNPM1 AML. Whether the isolated TCR is of sufficient affinity to treat patients remains uncertain. 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. [ABSTRACT FROM AUTHOR]

Background: Cellular immunotherapy using modified T cells offers new avenues for cancer treatment. T-cell receptor (TCR) engineering of CD8 T cells enables these cells to recognize tumor-associated antigens and tumor-specific neoantigens. Improving TCR T-cell therapy through increased potency and in vivo persistence will be critical for clinical success., Methods: We evaluated a novel drug combination to enhance TCR therapy in mouse models for acute myeloid leukemia (AML) and multiple myeloma (MM)., Results: Combining TCR therapy with the SUMO E1 inhibitor TAK981 and the DNA methylation inhibitor 5-Aza-2' deoxycytidine resulted in strong antitumor activity in a persistent manner against two in vivo tumor models of established AML and MM. We uncovered that the drug combination caused strong T-cell proliferation, increased cytokine signaling in T cells, improved persistence of T cells, and reduced differentiation towards exhausted phenotype. Simultaneously the drug combination enhanced immunogenicity of the tumor by increasing HLA and co-stimulation and surprisingly reducing inhibitory ligand expression., Conclusion: Combining T-cell therapy with TAK981 and 5-Aza-2' deoxycytidine may be an important step towards improved clinical outcome., Competing Interests: Competing interests: LUMC has applied for a patent on the triple therapy with MHMH and ACOV as inventors., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The CD3/T cell receptor (TCR) complex is responsible for antigen-specific pathogen recognition by T cells, and initiates the signaling cascade necessary for activation of effector functions. CD3 agonistic antibodies are commonly used to expand T lymphocytes in a wide range of clinical applications, including in adoptive T cell therapy for cancer patients. A major drawback of expanding T cell populations ex vivo using CD3 agonistic antibodies is that they expand and activate T cells independent of their TCR antigen specificity. Therapeutic agents that facilitate expansion of T cells in an antigen-specific manner and reduce their threshold of T cell activation are therefore of great interest for adoptive T cell therapy protocols. To identify CD3-specific T cell agonists, several RNA aptamers were selected against CD3 using Systematic Evolution of Ligands by EXponential enrichment combined with high-throughput sequencing. The extent and specificity of aptamer binding to target CD3 were assessed through surface plasma resonance, P32 double-filter assays, and flow cytometry. Aptamer-mediated modulation of the threshold of T cell activation was observed in vitro and in preclinical transgenic TCR mouse models. The aptamers improved efficacy and persistence of adoptive T cell therapy by low-affinity TCR-reactive T lymphocytes in melanoma-bearing mice. Thus, CD3-specific aptamers can be applied as therapeutic agents which facilitate the expansion of tumor-reactive T lymphocytes while conserving their tumor specificity. Furthermore, selected CD3 aptamers also exhibit cross-reactivity to human CD3, expanding their potential for clinical translation and application in the future., Competing Interests: F.P. is associate editor in this journal., (© 2024 The Authors.)

To increase the number of cancer patients that can be treated with T cell receptor (TCR) gene therapy, we aimed to identify a set of high-affinity cancer-specific TCRs targeting different melanoma-associated antigens (MAGEs). In this study, peptides derived from MAGE genes with tumor-specific expression pattern were identified by human leukocyte antigen (HLA) peptidomics. Next, peptide-HLA tetramers were generated, and used to sort MAGE-specific CD8 + T cell clones from the allogeneic (allo) HLA repertoire of healthy donors. To evaluate the clinical potential, most potent TCRs were sequenced, transferred into peripheral blood-derived CD8 + T cells, and tested for antitumor efficacy. In total we identified, seven MAGE-specific TCRs that effectively target MAGE-A1, MAGE-A3, MAGE-A6, and MAGE-A9 in the context of HLA-A∗01:01, -A∗02:01, -A∗03:01, -B∗07:02, -B∗35:01, or -C∗07:02. TCR gene transfer into CD8⁺ T cells resulted in efficient reactivity against a variety of different tumor types, while no cross-reactivity was detected. In addition, major in vivo antitumor effects of MAGE-A1 specific TCR engineered CD8⁺ T cells were observed in the orthotopic xenograft model for established multiple myeloma. The identification of seven MAGE-specific TCRs expands the pool of cancer patients eligible for TCR gene therapy and increases possibilities for personalized TCR gene therapy., Competing Interests: The authors report no competing interests., (© 2022 The Author(s).)