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HLA-E molecules can present self- and pathogen-derived peptides to both natural killer (NK) cells and T cells. T cells that recognize HLA-E peptides via their T cell receptor (TCR) are termed donor-unrestricted T cells due to restricted allelic variation of HLA-E. The composition and repertoire of HLA-E TCRs is not known so far. We performed TCR sequencing on CD8+ T cells from 21 individuals recognizing HLA-E tetramers (TMs) folded with two Mtb-HLA-E-restricted peptides. We sorted HLA-E Mtb TM+ and TM- CD8+ T cells directly ex vivo and performed bulk RNA-sequencing and single-cell TCR sequencing. The identified TCR repertoire was diverse and showed no conservation between and within individuals. TCRs selected from our single-cell TCR sequencing data could be activated upon HLA-E/peptide stimulation, although not robust, reflecting potentially weak interactions between HLA-E peptide complexes and TCRs. Thus, HLA-E-Mtb-specific T cells have a highly diverse TCR repertoire.
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Antibody-mediated delivery of immunogenic epitopes to redirect virus-specific CD8+ T-cells towards cancer cells is an emerging and promising new therapeutic strategy. These so-called antibody-epitope conjugates (AECs) rely on the proteolytic release of the epitopes close to the tumor surface for presentation by HLA class I molecules to eventually redirect and activate virus-specific CD8+ T-cells towards tumor cells. We fused the immunogenic EBV-BRLF1 epitope preceded by a protease cleavage site to the C-terminus of the heavy and/or light chains of cetuximab and trastuzumab. We evaluated these AECs and found that, even though all AECs were able to redirect the EBV-specific T-cells, AECs with an epitope fused to the C-terminus of the heavy chain resulted in higher levels of T-cell activation compared to AECs with the same epitope fused to the light chain of an antibody. We observed that all AECs were depending on the presence of the antibody target, that the level of T-cell activation correlated with expression levels of the antibody target, and that our AECs could efficiently deliver the BRLF1 epitope to cancer cell lines from different origins (breast, ovarian, lung, and cervical cancer and a multiple myeloma). Moreover, in vivo, the AECs efficiently reduced tumor burden and increased the overall survival, which was prolonged even further in combination with immune checkpoint blockade. We demonstrate the potential of these genetically fused AECs to redirect the potent EBV-specific T-cells towards cancer in vitro and in vivo.
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Inmunoconjugados , Neoplasias , Humanos , Linfocitos T CD8-positivos , Epítopos , Herpesvirus Humano 4/genética , Neoplasias/terapia , Epítopos de Linfocito TRESUMEN
[This corrects the article DOI: 10.1016/j.omto.2022.11.007.].
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HLA-sensitized patients on the transplant waiting list harbor antibodies and memory B cells directed against allogeneic HLA molecules, which decreases the chance to receive a compatible donor organ. Current desensitization strategies non-specifically target circulating antibodies and B cells, warranting the development of therapies that specifically affect HLA-directed humoral immune responses. We developed Chimeric HLA Antibody Receptor (CHAR) constructs comprising the extracellular part of HLA-A2 or HLA-A3 coupled to CD28-CD3ζ domains. CHAR-transduced cells expressing reporter constructs encoding T-cell activation markers, and CHAR-transduced CD8+ T cells from healthy donors were stimulated with HLA-specific monoclonal antibody-coated microbeads, and HLA-specific B cell hybridomas. CHAR T cell activation was measured by upregulation of T cell activation markers and IFNγ secretion, whereas CHAR T cell killing of B cell hybridomas was assessed in chromium release assays and by IgG ELISpot. HLA-A2- and HLA-A3-CHAR expressing cells were specifically activated by HLA-A2- and HLA-A3-specific monoclonal antibodies, either soluble or coated on microbeads, as shown by CHAR-induced transcription factors. HLA-A2 and HLA-A3 CHAR T cells efficiently produced IFNγ with exquisite specificity and were capable of specifically lysing hybridoma cells expressing HLA-A2- or HLA-A3-specific B-cell receptors, respectively. Finally, we mutated the α3 domain of the CHAR molecules to minimize any alloreactive T-cell reactivity against CHAR T cells, while retaining CHAR activity. These data show proof of principle for CHAR T cells to serve as precision immunotherapy to specifically desensitize (highly) sensitized solid organ transplant candidates and to treat antibody-mediated rejection after solid organ transplantation.
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Anticuerpos , Linfocitos B , Desensibilización Inmunológica , Trasplante de Riñón , Anticuerpos/genética , Anticuerpos/inmunología , Aloinjertos/inmunología , Linfocitos T , Antígeno HLA-A2/metabolismo , Antígeno HLA-A3/metabolismo , Interferón gamma/inmunología , Citotoxicidad Inmunológica , Linfocitos B/inmunología , Desensibilización Inmunológica/métodos , Prueba de Estudio Conceptual , Línea Celular , Donantes de Sangre , HumanosRESUMEN
Therapeutic antibody-epitope conjugates (AECs) are promising new modalities to deliver immunogenic epitopes and redirect virus-specific T-cell activity to cancer cells. Nevertheless, many aspects of these antibody conjugates require optimization to increase their efficacy. Here we evaluated different strategies to conjugate an EBV epitope (YVL/A2) preceded by a protease cleavage site to the antibodies cetuximab and trastuzumab. Three approaches were taken: chemical conjugation (i.e. a thiol-maleimide reaction) to reduced cysteine side chains, heavy chain C-terminal enzymatic conjugation using sortase A, and genetic fusions, to the heavy chain (HC) C-terminus. All three conjugates were capable of T-cell activation and target-cell killing via proteolytic release of the EBV epitope and expression of the antibody target was a requirement for T-cell activation. Moreover, AECs generated with a second immunogenic epitope derived from CMV (NLV/A2) were able to deliver and redirect CMV specific T-cells, in which the amino sequence of the attached peptide appeared to influence the efficiency of epitope delivery. Therefore, screening of multiple protease cleavage sites and epitopes attached to the antibody is necessary. Taken together, our data demonstrated that multiple AECs could sensitize cancer cells to virus-specific T cells.
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Infecciones por Citomegalovirus , Inmunoconjugados , Neoplasias , Humanos , Epítopos , Péptidos , Anticuerpos , Péptido Hidrolasas , Neoplasias/terapiaRESUMEN
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
The balance between safety and efficacy of T cell therapies remains challenging and T cell mediated toxicities have occurred. The stringent selection of tumor-specific targets and careful selection of tumor-specific T cells using T cell toxicity screenings are essential. In vitro screening options against vital organs or specialized cell subsets would be preferably included in preclinical pipelines, but options remain limited. Here, we set up preclinical models with human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes, epicardial cells, and kidney organoids to investigate toxicity risks of tumor-specific T cells more thoroughly. CD8+T cells reactive against PRAME, HA-1H, CD20, or WT1, currently used or planned to be used in phase I/II clinical studies, were included. Using these hiPSC-derived preclinical models, we demonstrated that WT1-specific T cells caused on-target toxicity that correlated with target gene expression. Multiple measures of T cell reactivity demonstrated this toxicity on the level of T cells and hiPSC-derived target cells. In addition, phenotypic analysis illustrated interaction and crosstalk between infiltrated T cells and kidney organoids. In summary, we demonstrated the benefit of hiPSC-derived models in determining toxicity risks of tumor-specific T cells. Furthermore, our data emphasizes the additional value of other measures of T cell reactivity on top of the commonly used cytokine levels.
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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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Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) specific CD4+ and CD8+ T cells in SARS-CoV-2-unexposed donors has been explained by the presence of T cells primed by other coronaviruses. However, based on the relatively high frequency and prevalence of cross-reactive T cells, we hypothesized cytomegalovirus (CMV) may induce these cross-reactive T cells. Stimulation of pre-pandemic cryo-preserved peripheral blood mononuclear cells (PBMCs) with SARS-CoV-2 peptides revealed that frequencies of SARS-CoV-2-specific T cells were higher in CMV-seropositive donors. Characterization of these T cells demonstrated that membrane-specific CD4+ and spike-specific CD8+ T cells originate from cross-reactive CMV-specific T cells. Spike-specific CD8+ T cells recognize SARS-CoV-2 spike peptide FVSNGTHWF (FVS) and dissimilar CMV pp65 peptide IPSINVHHY (IPS) presented by HLA-B*35:01. These dual IPS/FVS-reactive CD8+ T cells were found in multiple donors as well as severe COVID-19 patients and shared a common T cell receptor (TCR), illustrating that IPS/FVS-cross-reactivity is caused by a public TCR. In conclusion, CMV-specific T cells cross-react with SARS-CoV-2, despite low sequence homology between the two viruses, and may contribute to the pre-existing immunity against SARS-CoV-2.
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COVID-19 , Infecciones por Citomegalovirus , Humanos , SARS-CoV-2 , Linfocitos T CD8-positivos , Citomegalovirus , Leucocitos Mononucleares , Receptores de Antígenos de Linfocitos T , Linfocitos T CD4-PositivosRESUMEN
BACKGROUND: Transcription factor Wilms' tumor gene 1 (WT1) is an ideal tumor target based on its expression in a wide range of tumors, low-level expression in normal tissues and promoting role in cancer progression. In clinical trials, WT1 is targeted using peptide-based or dendritic cell-based vaccines and T-cell receptor (TCR)-based therapies. Antitumor reactivities were reported, but T-cell reactivity is hampered by self-tolerance to WT1 and limited number of WT1 peptides, which were thus far selected based on HLA peptide binding algorithms. METHODS: In this study, we have overcome both limitations by searching in the allogeneic T-cell repertoire of healthy donors for high-avidity WT1-specific T cells, specific for WT1 peptides derived from the HLA class I associated ligandome of primary leukemia and ovarian carcinoma samples. RESULTS: Using broad panels of malignant cells and healthy cell subsets, T-cell clones were selected that demonstrated potent and specific anti-WT1 T-cell reactivity against five of the eight newly identified WT1 peptides. Notably, T-cell clones for WT1 peptides previously used in clinical trials lacked reactivity against tumor cells, suggesting limited processing and presentation of these peptides. The TCR sequences of four T-cell clones were analyzed and TCR gene transfer into CD8+ T cells installed antitumor reactivity against WT1-expressing solid tumor cell lines, primary acute myeloid leukemia (AML) blasts, and ovarian carcinoma patient samples. CONCLUSIONS: Our approach resulted in a set of naturally expressed WT1 peptides and four TCRs that are promising candidates for TCR gene transfer strategies in patients with WT1-expressing tumors, including AML and ovarian carcinoma.
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Leucemia Mieloide Aguda , Neoplasias Ováricas , Receptores de Antígenos de Linfocitos T , Proteínas WT1 , Linfocitos T CD8-positivos/inmunología , Carcinoma Epitelial de Ovario/inmunología , Carcinoma Epitelial de Ovario/terapia , Femenino , Humanos , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/terapia , Neoplasias Ováricas/inmunología , Neoplasias Ováricas/terapia , Péptidos/inmunología , Péptidos/farmacología , Receptores de Antígenos de Linfocitos T/inmunología , Proteínas WT1/inmunologíaRESUMEN
Unconventional HLA class I-restricted CD8+ T cell epitopes, longer than 10 aa, have been implicated to play a role in human immunity against viruses and cancer. T cell recognition of long peptides, centrally bulging from the HLA cleft, has been described previously. Alternatively, long peptides can contain a linear HLA-bound core peptide, with a N- or C-terminal peptide "tail" extending from the HLA peptide binding groove. The role of such a peptide "tail" in CD8+ T cell recognition remains unclear. In this study, we identified a 20mer peptide (FLPTPEELGLLGPPRPQVLA [FLP]) derived from the IL-27R subunit α gene restricted to HLA-A*02:01, for which we solved the crystal structure and demonstrated a long C-terminal "tail" extension. FLP-specific T cell clones demonstrated various recognition modes, some T cells recognized the FLP core peptide, while for other T cells the peptide tail was essential for recognition. These results demonstrate a crucial role for a C-terminal peptide tail in immunogenicity.
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Linfocitos T CD8-positivos , Epítopos de Linfocito T , Antígeno HLA-A2 , Linfocitos T CD8-positivos/inmunología , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/inmunología , Genes MHC Clase I/genética , Genes MHC Clase I/inmunología , Antígenos HLA-A/genética , Antígenos HLA-A/inmunología , Antígeno HLA-A2/genética , Antígeno HLA-A2/inmunología , Humanos , Péptidos/genética , Péptidos/inmunologíaRESUMEN
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
Systemic immune cell dynamics during coronavirus disease 2019 (COVID-19) are extensively documented, but these are less well studied in the (upper) respiratory tract, where severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replicates1-6. Here, we characterized nasal and systemic immune cells in individuals with COVID-19 who were hospitalized or convalescent and compared the immune cells to those seen in healthy donors. We observed increased nasal granulocytes, monocytes, CD11c+ natural killer (NK) cells and CD4+ T effector cells during acute COVID-19. The mucosal proinflammatory populations positively associated with peripheral blood human leukocyte antigen (HLA)-DRlow monocytes, CD38+PD1+CD4+ T effector (Teff) cells and plasmablasts. However, there was no general lymphopenia in nasal mucosa, unlike in peripheral blood. Moreover, nasal neutrophils negatively associated with oxygen saturation levels in blood. Following convalescence, nasal immune cells mostly normalized, except for CD127+ granulocytes and CD38+CD8+ tissue-resident memory T cells (TRM). SARS-CoV-2-specific CD8+ T cells persisted at least 2 months after viral clearance in the nasal mucosa, indicating that COVID-19 has both transient and long-term effects on upper respiratory tract immune responses.
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Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Nasofaringe/inmunología , Nariz/citología , Mucosa Respiratoria/inmunología , SARS-CoV-2/inmunología , Anticuerpos Antivirales/sangre , COVID-19/inmunología , COVID-19/patología , Granulocitos/inmunología , Antígenos HLA-DR/metabolismo , Humanos , Células Asesinas Naturales/inmunología , Células T de Memoria/inmunología , Monocitos/inmunología , Nasofaringe/citología , Nasofaringe/virología , Neutrófilos/inmunología , Nariz/inmunología , Nariz/virología , Estudios Prospectivos , Mucosa Respiratoria/citología , Mucosa Respiratoria/virologíaRESUMEN
CAR T cell therapy has shown great promise for the treatment of B cell malignancies. However, antigen-negative escape variants often cause disease relapse, necessitating the development of multi-antigen-targeting approaches. We propose that a T cell receptor (TCR)-based strategy would increase the number of potential antigenic targets, as peptides from both intracellular and extracellular proteins can be recognized. Here, we aimed to isolate a broad range of promising TCRs targeting multiple antigens for treatment of B cell malignancies. As a first step, 28 target genes for B cell malignancies were selected based on gene expression profiles. Twenty target peptides presented in human leukocyte antigen (HLA)-A∗01:01, -A∗24:02, -B∗08:01, or -B∗35:01 were identified from the immunopeptidome of B cell malignancies and used to form peptide-HLA (pHLA)-tetramers for T cell isolation. Target-peptide-specific CD8 T cells were isolated from HLA-mismatched healthy donors and subjected to a stringent stepwise selection procedure to ensure potency and eliminate cross-reactivity. In total, five T cell clones specific for FCRL5 in HLA-A∗01:01, VPREB3 in HLA-A∗24:02, and BOB1 in HLA-B∗35:01 recognized B cell malignancies. For all three specificities, TCR gene transfer into CD8 T cells resulted in cytokine production and efficient killing of multiple B cell malignancies. In conclusion, using this systematic approach we successfully identified three promising TCRs for T cell therapy against B cell malignancies.
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Neoplasias , Receptores de Antígenos de Linfocitos T , Linfocitos T CD8-positivos , Tratamiento Basado en Trasplante de Células y Tejidos , Humanos , Inmunoterapia Adoptiva/métodos , Neoplasias/terapia , Receptores de Antígenos de Linfocitos T/metabolismoRESUMEN
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.
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Graft-vs.-leukemia (GVL) reactivity after HLA-matched allogeneic stem cell transplantation (alloSCT) is mainly mediated by donor T cells recognizing minor histocompatibility antigens (MiHA). If MiHA are targeted that are exclusively expressed on hematopoietic cells of recipient origin, selective GVL reactivity without severe graft-vs.-host-disease (GVHD) may occur. In this phase I study we explored HA-1H TCR gene transfer into T cells harvested from the HA-1H negative stem-cell donor to treat HA-1H positive HLA-A*02:01 positive patients with high-risk leukemia after alloSCT. HA-1H is a hematopoiesis-restricted MiHA presented in HLA-A*02:01. Since we previously demonstrated that donor-derived virus-specific T-cell infusions did not result in GVHD, we used donor-derived EBV and/or CMV-specific T-cells to be redirected by HA-1H TCR. EBV and/or CMV-specific T-cells were purified, retrovirally transduced with HA-1H TCR, and expanded. Validation experiments illustrated dual recognition of viral antigens and HA-1H by HA-1H TCR-engineered virus-specific T-cells. Release criteria included products containing more than 60% antigen-specific T-cells. Patients with high risk leukemia following T-cell depleted alloSCT in complete or partial remission were eligible. HA-1H TCR T-cells were infused 8 and 14 weeks after alloSCT without additional pre-conditioning chemotherapy. For 4/9 included patients no appropriate products could be made. Their donors were all CMV-negative, thereby restricting the production process to EBV-specific T-cells. For 5 patients a total of 10 products could be made meeting the release criteria containing 3-280 × 106 virus and/or HA-1H TCR T-cells. No infusion-related toxicity, delayed toxicity or GVHD occurred. One patient with relapsed AML at time of infusions died due to rapidly progressing disease. Four patients were in remission at time of infusion. Two patients died of infections during follow-up, not likely related to the infusion. Two patients are alive and well without GVHD. In 2 patients persistence of HA-1H TCR T-cells could be illustrated correlating with viral reactivation, but no overt in-vivo expansion of infused T-cells was observed. In conclusion, HA-1H TCR-redirected virus-specific T-cells could be made and safely infused in 5 patients with high-risk AML, but overall feasibility and efficacy was too low to warrant further clinical development using this strategy. New strategies will be explored using patient-derived donor T-cells isolated after transplantation transduced with HA-1H-specific TCR to be infused following immune conditioning.
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Enfermedad Injerto contra Huésped/terapia , Efecto Injerto vs Leucemia , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Herpesvirus Humano 4/inmunología , Inmunoterapia Adoptiva , Leucemia/cirugía , Antígenos de Histocompatibilidad Menor/inmunología , Oligopéptidos/inmunología , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/trasplante , Adulto , Anciano , Femenino , Enfermedad Injerto contra Huésped/genética , Enfermedad Injerto contra Huésped/inmunología , Enfermedad Injerto contra Huésped/metabolismo , Trasplante de Células Madre Hematopoyéticas/mortalidad , Humanos , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/mortalidad , Leucemia/genética , Leucemia/inmunología , Leucemia/metabolismo , Masculino , Persona de Mediana Edad , Antígenos de Histocompatibilidad Menor/genética , Antígenos de Histocompatibilidad Menor/metabolismo , Países Bajos , Oligopéptidos/genética , Oligopéptidos/metabolismo , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Linfocitos T/virología , Factores de Tiempo , Trasplante Homólogo , Resultado del TratamientoRESUMEN
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.
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Traslado Adoptivo , Leucemia Mieloide Aguda , Mutación , Proteínas de Neoplasias , Proteínas Nucleares , Péptidos , Animales , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/patología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/patología , Femenino , Antígeno HLA-A2/genética , Antígeno HLA-A2/inmunología , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/terapia , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/inmunología , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Nucleofosmina , Péptidos/genética , Péptidos/inmunología , Transducción Genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Synovial sarcoma expresses multiple cancer testis antigens that could potentially be targeted by T-cell receptor (TCR) gene therapy. In this study we investigated whether PRAME-TCR-gene therapy could be an effective treatment for synovial sarcoma by investigating the potential of PRAME-specific T-cells to recognize sarcoma cells and by evaluating the expression patterns of PRAME and HLA class I (HLA-I) in synovial sarcoma tumor samples. All PRAME expressing sarcoma cell lines, including 2 primary synovial sarcoma cell cultures (passage < 3), were efficiently recognized by PRAME-specific T-cells. mRNA FISH demonstrated that PRAME was expressed in all synovial sarcoma samples, mostly in an homogeneous pattern. Immunohistochemistry demonstrated low HLA-I baseline expression in synovial sarcoma, but its expression was elevated in specific areas of the tumors, especially in biphasic components of biphasic synovial sarcoma. In 5/11 biphasic synovial sarcoma patients and in 1/17 monophasic synovial sarcoma patients, elevated HLA-I on tumor cells was correlated with infiltration of T-cells in these specific areas. In conclusion, low-baseline expression of HLA-I in synovial sarcoma is elevated in biphasic areas and in areas with densely infiltrating T-cells, which, in combination with homogeneous and high PRAME expression, makes synovial sarcoma potentially a suitable candidate for PRAME-specific TCR-gene therapy.