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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 high-affinity 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.
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Antígenos CD8 , Linfocitos T CD8-positivos , Antígenos de Histocompatibilidad Clase I , Activación de Linfocitos , Antígenos de Histocompatibilidad Clase I/metabolismo , Péptidos/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , HumanosRESUMEN
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.
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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.
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Decitabina , Epigénesis Genética , Receptores de Antígenos de Linfocitos T , Animales , Decitabina/farmacología , Decitabina/uso terapéutico , Ratones , Humanos , Epigénesis Genética/efectos de los fármacos , Receptores de Antígenos de Linfocitos T/metabolismo , Procesamiento Proteico-Postraduccional , Línea Celular Tumoral , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/inmunología , Mieloma Múltiple/metabolismoRESUMEN
[This corrects the article DOI: 10.1016/j.omto.2022.11.007.].
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BACKGROUND: The immunoglobulin J chain (Jchain) is highly expressed in the majority of multiple myeloma (MM), and Jchain-derived peptides presented in HLA molecules may be suitable antigens for T-cell therapy of MM. METHODS: Using immunopeptidomics, we identified Jchain-derived epitopes presented by MM cells, and pHLA tetramer technology was used to isolate Jchain-specific T-cell clones. RESULTS: We identified T cells specific for Jchain peptides presented in HLA-A1, -A24, -A3, and -A11 that recognized and lysed JCHAIN-positive MM cells. TCRs of the most promising T-cell clones were sequenced, cloned into retroviral vectors, and transferred to CD8 T cells. Jchain TCR T cells recognized target cells when JCHAIN and the appropriate HLA restriction alleles were expressed, while JCHAIN or HLA-negative cells, including healthy subsets, were not recognized. Patient-derived JCHAIN-positive MM samples were also lysed by Jchain TCR T cells. In a preclinical in vivo model for established MM, Jchain-A1, -A24, -A3, and -A11 TCR T cells strongly eradicated MM cells, which resulted in 100-fold lower tumor burden in Jchain TCR versus control-treated mice. CONCLUSIONS: We identified TCRs targeting Jchain-derived peptides presented in four common HLA alleles. All four TCRs demonstrated potent preclinical anti-myeloma activity, encouraging further preclinical testing and ultimately clinical development.
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Cadenas J de Inmunoglobulina , Mieloma Múltiple , Animales , Ratones , Mieloma Múltiple/terapia , Receptores de Antígenos de Linfocitos T/genética , Alelos , Linfocitos T CD8-positivosRESUMEN
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⺠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⺠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.
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Recurrent disease emerges in the majority of patients with ovarian cancer (OVCA). Adoptive T-cell therapies with T-cell receptors (TCRs) targeting tumor-associated antigens (TAAs) are considered promising solutions for less-immunogenic 'cold' ovarian tumors. In order to treat a broader patient population, more TCRs targeting peptides derived from different TAAs binding in various HLA class I molecules are essential. By performing a differential gene expression analysis using mRNA-seq datasets, PRAME, CTCFL and CLDN6 were selected as strictly tumor-specific TAAs, with high expression in ovarian cancer and at least 20-fold lower expression in all healthy tissues of risk. In primary OVCA patient samples and cell lines we confirmed expression and identified naturally expressed TAA-derived peptides in the HLA class I ligandome. Subsequently, high-avidity T-cell clones recognizing these peptides were isolated from the allo-HLA T-cell repertoire of healthy individuals. Three PRAME TCRs and one CTCFL TCR of the most promising T-cell clones were sequenced, and transferred to CD8+ T cells. The PRAME TCR-T cells demonstrated potent and specific antitumor reactivity in vitro and in vivo. The CTCFL TCR-T cells efficiently recognized primary patient-derived OVCA cells, and OVCA cell lines treated with demethylating agent 5-aza-2'-deoxycytidine (DAC). The identified PRAME and CTCFL TCRs are promising candidates for the treatment of patients with ovarian cancer, and are an essential addition to the currently used HLA-A*02:01 restricted PRAME TCRs. Our selection of differentially expressed genes, naturally expressed TAA peptides and potent TCRs can improve and broaden the use of T-cell therapies for patients with ovarian cancer or other PRAME or CTCFL expressing cancers.
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Neoplasias Ováricas , Receptores de Antígenos de Linfocitos T , Humanos , Femenino , Antígenos de Neoplasias , Linfocitos T CD8-positivos , Neoplasias Ováricas/terapia , Neoplasias Ováricas/metabolismo , Péptidos/metabolismo , Proteínas de Unión al ADN/metabolismoRESUMEN
BACKGROUND: T cell receptor (TCR)-engineered cells can be powerful tools in the treatment of malignancies. However, tumor resistance by Human Leukocyte antigen (HLA) class I downregulation can negatively impact the success of any TCR-mediated cell therapy. Allogeneic natural killer (NK) cells have demonstrated efficacy and safety against malignancies without inducing graft-versus-host-disease, highlighting the feasibility for an 'off the shelf' cellular therapeutic. Furthermore, primary NK cells can target tumors using a broad array of intrinsic activation mechanisms. In this study, we combined the antitumor effector functions of NK cells with TCR engineering (NK-TCR), creating a novel therapeutic strategy to avoid TCR-associated immune resistance. METHODS: BOB1, is a transcription factor highly expressed in all healthy and malignant B cell lineages, including multiple myeloma (MM). Expression of an HLA-B*07:02 restricted BOB1-specifc TCR in peripheral blood-derived NK cells was achieved following a two-step retroviral transduction protocol. NK-TCR was then compared with TCR-negative NK cells and CD8-T cells expressing the same TCR for effector function against HLA-B*07:02+ B-cell derived lymphoblastoid cell lines (B-LCL), B-cell acute lymphoblastic leukemia and MM cell lines in vitro and in vivo. RESULTS: Firstly, TCR could be reproducibly expressed in NK cells isolated from the peripheral blood of multiple healthy donors generating pure NK-TCR cell products. Secondly, NK-TCR demonstrated antigen-specific effector functions against malignancies which were previously resistant to NK-mediated lysis and enhanced NK efficacy in vivo using a preclinical xenograft model of MM. Moreover, antigen-specific cytotoxicity and cytokine production of NK-TCR was comparable to CD8 T cells expressing the same TCR. Finally, in a model of HLA-class I loss, tumor cells with B2M KO were lysed by NK-TCR in an NK-mediated manner but were resistant to T-cell based killing. CONCLUSION: NK-TCR cell therapy enhances NK cell efficacy against tumors through additional TCR-mediated lysis. Furthermore, the dual efficacy of NK-TCR permits the specific targeting of tumors and the associated TCR-associated immune resistance, making NK-TCR a unique cellular therapeutic.
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Mieloma Múltiple , Escape del Tumor , Antígenos de Histocompatibilidad Clase I , Humanos , Células Asesinas Naturales , Receptores de Antígenos de Linfocitos T/genéticaRESUMEN
Chimeric antigen receptor (CAR) T cell therapies have resulted in profound clinical responses in the treatment of CD19-positive hematological malignancies, but a significant proportion of patients do not respond or relapse eventually. As an alternative to CAR T cells, T cells can be engineered to express a tumor-targeting T cell receptor (TCR). Due to HLA restriction of TCRs, CARs have emerged as a preferred treatment moiety when targeting surface antigens, despite the fact that functional differences between engineered TCR (eTCR) T and CAR T cells remain ill-defined. Here, we compared the activity of CAR T cells versus engineered TCR T cells in targeting the B cell malignancy-associated antigen CD20 as a function of antigen exposure. We found CAR T cells to be more potent effector cells, producing higher levels of cytokines and killing more efficiently than eTCR T cells in a short time frame. However, we revealed that the increase of antigen exposure significantly impaired CAR T cell expansion, a phenotype defined by high expression of coinhibitory molecules and effector differentiation. In contrast, eTCR T cells expanded better than CAR T cells under high antigenic pressure, with lower expression of coinhibitory molecules and maintenance of an early differentiation phenotype, and comparable clearance of tumor cells.
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Recurrencia Local de Neoplasia , Linfocitos T , Antígenos CD20/metabolismo , Humanos , Inmunoterapia Adoptiva/métodos , Receptores de Antígenos de Linfocitos T/genéticaRESUMEN
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.