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.
Adoptive transfer of T cell receptor (TCR)-transduced T cells may be an attractive strategy to target both hematological malignancies and solid tumors. By introducing a TCR, large numbers of T cells ...with defined antigen (Ag) specificity can be obtained. However, by introduction of a TCR, mixed TCR dimers can be formed. Besides the decrease in TCR expression of the introduced and endogenous TCR, these mixed TCR dimers could harbor potentially harmful specificities. In this study, we demonstrate that introduction of TCRs resulted in formation of neoreactive mixed TCR dimers, composed of the introduced TCR chains pairing with either the endogenous TCR α or β chain. Neoreactivities observed were HLA class I or class II restricted. Most neoreactive mixed TCR dimers were allo-HLA reactive; however, neoreactive mixed TCR dimers with autoreactive activity were also observed. We demonstrate that inclusion of an extra disulfide bond between the constant domains of the introduced TCR markedly reduced neoreactivity, whereas enhanced effectiveness of the introduced TCR was observed. In conclusion, TCR transfer results in the formation of neoreactive mixed TCR dimers with the potential to generate off-target effects, underlining the importance of searching for techniques to facilitate preferential pairing.
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.
Adoptive transfer of T cells engineered with a cancer-specific T cell receptor (TCR) has demonstrated clinical benefit. However, the risk for off-target toxicity of TCRs remains a concern. Here, we ...examined the cross-reactive profile of T cell clone (7B5) with a high functional sensitivity for the hematopoietic-restricted minor histocompatibility antigen HA-2 in the context of HLA-A*02:01. HA-2pos Epstein-Barr virus-transformed B lymphoblastic cell lines (EBV-LCLs) and primary acute myeloid leukemia samples, but not hematopoietic HA-2neg samples, are effectively recognized. However, we found unexpected off-target recognition of human fibroblasts and keratinocytes not expressing the HA-2 antigen. To uncover the origin of this off-target recognition, we performed an alanine scanning approach, identifying six out of nine positions to be important for peptide recognition. This indicates a low risk for broad cross-reactivity. However, using a combinatorial peptide library scanning approach, we identified a CDH13-derived peptide activating the 7B5 T cell clone. This was confirmed by recognition of CDH13-transduced EBV-LCLs and cell subsets endogenously expressing CDH13, such as proximal tubular epithelial cells. As such, we recommend the use of a combinatorial peptide library scan followed by screening against additional cell subsets to validate TCR specificity and detect off-target toxicity due to cross-reactivity directed against unrelated peptides before selecting candidate TCRs for clinical testing.
Adoptive transfer of T cells engineered with a cancer-specific T cell receptor (TCR) has demonstrated clinical benefit. To validate TCR specificity and detect off-target toxicity before selecting candidate TCRs for clinical testing, Bijen et al. recommend the use of a combinatorial peptide library scan followed by screening against additional cell subsets.
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) replicates
. 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)-DR
monocytes, CD38
PD1
CD4
T effector (T
) 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 (T
). 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.
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.
Medulloblastoma is the most frequent malignant childhood brain tumor with a high morbidity. Identification of new therapeutic targets would be instrumental in improving patient outcomes. We evaluated ...the expression of the tumor-associated antigen PRAME in biopsies from 60 patients with medulloblastoma. PRAME expression was detectable in 82% of tissues independent of molecular and histopathologic subgroups. High PRAME expression also correlated with worse overall survival. We next investigated the relevance of PRAME as a target for immunotherapy. Medulloblastoma cells were targeted using genetically modified T cells with a PRAME-specific TCR (SLL TCR T cells). SLL TCR T cells efficiently killed medulloblastoma HLA-A*02
DAOY cells as well as primary HLA-A*02
medulloblastoma cells. Moreover, SLL TCR T cells controlled tumor growth in an orthotopic mouse model of medulloblastoma. To prevent unexpected T-cell-related toxicity, an inducible caspase-9 (iC9) gene was introduced in frame with the SLL TCR; this safety switch triggered prompt elimination of genetically modified T cells. Altogether, these data indicate that T cells genetically modified with a high-affinity, PRAME-specific TCR and iC9 may represent a promising innovative approach for treating patients with HLA-A*02
medulloblastoma.
These findings identify PRAME as a medulloblastoma tumor-associated antigen that can be targeted using genetically modified T cells.
.
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 × 10
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
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.
IMPORTANCE: Uveal melanoma (UM) is an intraocular primary malignant neoplasm that often gives rise to metastatic disease for which there are no effective therapies. A substantial proportion of UMs ...express the cancer-testis antigen PRAME (preferentially expressed antigen in melanoma), which can potentially be targeted by adoptive T-cell therapy. OBJECTIVE: To determine whether there may be a rationale for PRAME-directed T-cell therapy for metastatic UM. DESIGN, SETTING, AND PARTICIPANTS: An experimental study using a retrospective cohort of 64 patients with UM (median follow-up, 62 months) was conducted from January 8, 2015, to November 20, 2016, at the Leiden University Medical Center. Clinical, histopathologic, and genetic parameters were compared between 64 PRAME-positive and PRAME-negative UMs. HLA class I restricted, PRAME-specific T cells were stimulated with UM cell lines to measure their antigen-specific reactivity against these cell lines, which were analyzed for PRAME expression by real-time quantitative polymerase chain reaction. Uveal melanoma metastases from 16 unrelated patients were assessed for PRAME expression by messenger RNA fluorescence in situ hybridization and for HLA class I expression by immunofluorescence staining. MAIN OUTCOMES AND MEASURES: Interferon γ production for antigen-specific reactivity and detection of PRAME and HLA class I expression in primary and metastatic UM. RESULTS: Of the 64 patients in the study (31 women and 33 men; mean SD age at the time of enucleation, 60.6 15.6 years), PRAME expression was negative in 35 primary UMs and positive in 29 primary UMs. Positive PRAME expression was associated with a high largest basal diameter (15.0 vs 12.0 mm; P = .005), ciliary body involvement (59% vs 26%; P = .008), and amplification of chromosome 8q (66% vs 23%; P = .002). PRAME-specific T cells reacted against 4 of 7 UM cell lines, demonstrating that T-cell reactivity correlated with PRAME expression. Metastatic UM samples were positive for PRAME messenger RNA in 11 of 16 patients and for HLA class I in 10 of 16 patients, with 8 of 16 patients demonstrating coexpression of both PRAME and HLA class I. CONCLUSIONS AND RELEVANCE: PRAME is expressed in many primary and metastatic UMs, and about half of the metastatic UMs coexpress PRAME and HLA class I. The finding that PRAME-specific T cells in this study reacted against PRAME-positive UM cell lines suggests a potential role for PRAME-directed immunotherapy for selected patients with metastatic UM.
Adoptive T cell therapy using TCR transgenic autologous T cells has shown great potential for the treatment of tumor patients. Thorough characterization of genetically reprogrammed T cells is ...necessary to optimize treatment success. Here, we describe the generation of triple parameter reporter T cells based on the Jurkat 76 T cell line for the evaluation of TCR and chimeric antigen receptor functions as well as adoptive T cell strategies. This Jurkat subline is devoid of endogenous TCR alpha and TCR beta chains, thereby circumventing the problem of TCR miss-pairing and unexpected specificities. The resultant reporter cells allow simultaneous determination of the activity of the transcription factors NF-κB, NFAT and AP-1 that play key roles in T cell activation. Human TCRs directed against tumor and virus antigens were introduced and reporter responses were determined using tumor cell lines endogenously expressing the antigens of interest or via addition of antigenic peptides. Finally, we demonstrate that coexpression of adhesion molecules like CD2 and CD226 as well as CD28 chimeric receptors represents an effective strategy to augment the response of TCR-transgenic reporters to cells presenting cognate antigens.
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