RESUMO
Chimeric antigen receptor (CAR T) therapy produced excellent activity in patients with relapsed/refractory B-lineage malignancies. However, extending these therapies to T cell cancers requires overcoming unique challenges. In the recent years, multiple approaches have been developed in preclinical models and some were tested in clinical trials in patients with treatment-refractory T-cell malignanices with promising early results. Here, we review main hurdles impeding the success of CAR T therapy in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/LBL), discuss potential solutions, and summarize recent progress in both preclinical and clinical development of CAR T therapy for these diseases.
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Chimeric antigen receptor T cells (CART) targeting lymphocyte antigens can induce T cell fratricide and require additional engineering to mitigate self-damage. We demonstrate that the expression of a chimeric antigen receptor (CAR) targeting CD5, a prominent pan-T cell antigen, induces rapid internalization and complete loss of the CD5 protein on T cells, protecting them from self-targeting. Notably, exposure of healthy and malignant T cells to CD5.CART cells induces similar internalization of CD5 on target cells, transiently shielding them from cytotoxicity. However, this protection is short-lived, as sustained activity of CD5.CART cells in patients with T cell malignancies results in full ablation of CD5+ T cells while sparing healthy T cells naturally lacking CD5. These results indicate that continuous downmodulation of the target antigen in CD5.CART cells produces effective fratricide resistance without undermining their on-target cytotoxicity.
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Antígenos CD5 , Receptores de Antígenos Quiméricos , Antígenos CD5/metabolismo , Humanos , Receptores de Antígenos Quiméricos/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Citotoxicidade Imunológica , Linfócitos T/imunologia , Linfócitos T/metabolismo , Imunoterapia Adotiva/métodos , Animais , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
BACKGROUND AIMS: Vγ9Vδ2 T cells are an attractive cell platform for the off-the-shelf cancer immunotherapy as the result of their lack of alloreactivity and inherent multi-pronged cytotoxicity, which could be further amplified with chimeric antigen receptors (CARs). In this study, we sought to enhance the in vivo longevity of CAR-Vδ2 T cells by modulating ex vivo manufacturing conditions and selecting an optimal CAR costimulatory domain. METHODS: Specifically, we compared the anti-tumor activity of Vδ2 T cells expressing anti-CD19 CARs with costimulatory endodomains derived from CD28, 4-1BB or CD27 and generated in either standard fetal bovine serum (FBS)- or human platelet lysate (HPL)-supplemented medium. RESULTS: We found that HPL supported greater expansion of CAR-Vδ2 T cells with comparable in vitro cytotoxicity and cytokine secretion to FBS-expanded CAR-Vδ2 T cells. HPL-expanded CAR-Vδ2 T cells showed enhanced in vivo anti-tumor activity with longer T-cell persistence compared with FBS counterparts, with 4-1BB costimulated CAR showing the greatest activity. Mechanistically, HPL-expanded CAR Vδ2 T cells exhibited reduced apoptosis and senescence transcriptional pathways compared to FBS-expanded CAR-Vδ2 T cells and increased telomerase activity. CONCLUSIONS: This study supports enhancement of therapeutic potency of CAR-Vδ2 T cells through a manufacturing improvement.
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Apoptose , Plaquetas , Senescência Celular , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos , Humanos , Receptores de Antígenos Quiméricos/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Animais , Imunoterapia Adotiva/métodos , Camundongos , Plaquetas/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Antígenos CD19/imunologia , Antígenos CD19/metabolismo , Linhagem Celular Tumoral , Antígenos CD28/metabolismo , Antígenos CD28/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
ABSTRACT: For patients with high-risk or relapsed/refractory acute myeloid leukemia (AML), allogeneic stem cell transplantation (allo-HSCT) and the graft-versus-leukemia effect mediated by donor T cells, offer the best chance of long-term remission. However, the concurrent transfer of alloreactive T cells can lead to graft-versus-host disease that is associated with transplant-related morbidity and mortality. Furthermore, â¼60% of patients will ultimately relapse after allo-HSCT, thus, underscoring the need for novel therapeutic strategies that are safe and effective. In this study, we explored the feasibility of immunotherapeutically targeting neoantigens, which arise from recurrent nonsynonymous mutations in AML and thus represent attractive targets because they are exclusively present on the tumor. Focusing on 14 recurrent driver mutations across 8 genes found in AML, we investigated their immunogenicity in 23 individuals with diverse HLA profiles. We demonstrate the immunogenicity of AML neoantigens, with 17 of 23 (74%) reactive donors screened mounting a response. The most immunodominant neoantigens were IDH2R140Q (n = 11 of 17 responders), IDH1R132H (n = 7 of 17), and FLT3D835Y (n = 6 of 17). In-depth studies of IDH2R140Q-specific T cells revealed the presence of reactive CD4+ and CD8+ T cells capable of recognizing distinct mutant-specific epitopes restricted to different HLA alleles. These neo-T cells could selectively recognize and kill HLA-matched AML targets endogenously expressing IDH2R140Q both in vitro and in vivo. Overall, our findings support the clinical translation of neoantigen-specific T cells to treat relapsed/refractory AML.
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Antígenos de Neoplasias , Isocitrato Desidrogenase , Leucemia Mieloide Aguda , Humanos , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/genética , Transplante de Células-Tronco Hematopoéticas , Imunoterapia/métodos , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/imunologia , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , MutaçãoRESUMO
ABSTRACT: Despite newer targeted therapies, patients with primary refractory or relapsed (r/r) T-cell lymphoma have a poor prognosis. The development of chimeric antigen receptor (CAR) T-cell platforms to treat T-cell malignancies often requires additional gene modifications to overcome fratricide because of shared T-cell antigens on normal and malignant T cells. We developed a CD5-directed CAR that produces minimal fratricide by downmodulating CD5 protein levels in transduced T cells while retaining strong cytotoxicity against CD5+ malignant cells. In our first-in-human phase 1 study (NCT0308190), second-generation autologous CD5.CAR T cells were manufactured from patients with r/r T-cell malignancies. Here, we report safety and efficacy data from a cohort of patients with mature T-cell lymphoma (TCL). Among the 17 patients with TCL enrolled, CD5 CAR T cells were successfully manufactured for 13 out of 14 attempted lines (93%) and administered to 9 (69%) patients. The overall response rate (complete remission or partial response) was 44%, with complete responses observed in 2 patients. The most common grade 3 or higher adverse events were cytopenias. No grade 3 or higher cytokine release syndrome or neurologic events occurred. Two patients died during the immediate toxicity evaluation period due to rapidly progressive disease. These results demonstrated that CD5.CAR T cells are safe and can induce clinical responses in patients with r/r CD5-expressing TCLs without eliminating endogenous T cells or increasing infectious complications. More patients and longer follow-up are needed for validation. This trial was registered at www.clinicaltrials.gov as #NCT0308190.
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Imunoterapia Adotiva , Linfoma de Células T , Humanos , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Recidiva Local de Neoplasia/tratamento farmacológico , Linfócitos T , Doença Crônica , Linfoma de Células T/tratamento farmacológico , Antígenos CD19RESUMO
Chimeric antigen receptor (CAR) T cell (CAR-T) therapy represents a revolutionary treatment for patients with relapsed/refractory hematologic malignancies. However, its use can result in significant toxicities, including cytokine release syndrome (CRS), a potentially life-threatening clinical syndrome resulting from the release of proinflammatory cytokines upon T cell activation. In addition, patients who develop CRS often experience prolonged cytopenias, and those with the most severe CRS also have the longest delays in full marrow recovery. Although an association between CRS and delayed bone marrow recovery has been established, the precise mechanism underlying this phenomenon remains unknown. This study was conducted to test our hypothesis that delayed bone marrow recovery following CAR-T therapy is caused by elevation of proinflammatory cytokines, leading to apoptosis and depletion of hematopoietic stem and progenitor cells (HSPCs). SCID-beige mice bearing intraperitoneal CD19+ Raji cell tumors were treated with injection of human CD19.28z CAR T cells. Bone marrow was then harvested for analysis by flow cytometry, and HSPCs were isolated for whole-transcriptome analysis by RNA sequencing. Complete blood counts and serum cytokine levels were measured as well. A second model was developed in which SCID-beige mice were treated with murine IFN-γ (mIFN-γ), murine IL-6 (mIL-6), or both. Bone marrow was harvested, and flow cytometry assays were conducted to evaluate the degree of apoptosis and proliferation on specific HSPC populations. SCID-beige mice bearing intraperitoneal Raji cell tumors that were treated with CAR-T therapy developed CRS, with elevations of several proinflammatory cytokines, including profound elevation of human IFN-γ. Gene set enrichment analysis of RNA sequencing data revealed that genes associated with apoptosis were significantly upregulated in HSPCs from mice that developed CRS. Endothelial protein C receptor (EPCR)-negative HSCs, a subset of HSCs that is poised for terminal differentiation, was found to be specifically decreased in mice that were treated with CAR T cells. Furthermore, HSPCs were found to have increased levels of apoptosis upon treatment with mIFN-γ and mIL-6, whereas short-term HSCs and multipotent progenitors exhibited increases in proliferation with mIFN-γ treatment alone. The results from this study provide evidence that the elevation of proinflammatory cytokines following CAR-T therapy impacts the bone marrow through a combined mechanism: pluripotent HSCs that are exposed to elevated levels of IFN-γ and IL-6 undergo increased cell death, while more committed progenitor cells become more proliferative in response to elevated IFN-γ. These combined effects lead to depleted stores of repopulating HSCs and ultimately cytopenias. © 2023 American Society for Transplantation and Cellular Therapy. Published by Elsevier Inc.
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Doenças da Medula Óssea , Imunoterapia Adotiva , Neoplasias , Receptores de Antígenos Quiméricos , Animais , Humanos , Camundongos , Apoptose , Medula Óssea/patologia , Doenças da Medula Óssea/metabolismo , Doenças da Medula Óssea/patologia , Citocinas/metabolismo , Células-Tronco Hematopoéticas , Interleucina-6/metabolismo , Camundongos SCID , Imunoterapia Adotiva/efeitos adversosRESUMO
Chimeric antigen receptor (CAR)-mediated targeting of T lineage antigens for the therapy of blood malignancies is frequently complicated by self-targeting of CAR T cells or their excessive differentiation driven by constant CAR signaling. Expression of CARs targeting CD7, a pan-T cell antigen highly expressed in T cell malignancies and some myeloid leukemias, produces robust fratricide and often requires additional mitigation strategies, such as CD7 gene editing. In this study, we show fratricide of CD7 CAR T cells can be fully prevented using ibrutinib and dasatinib, the pharmacologic inhibitors of key CAR/CD3ζ signaling kinases. Supplementation with ibrutinib and dasatinib rescued the ex vivo expansion of unedited CD7 CAR T cells and allowed regaining full CAR-mediated cytotoxicity in vitro and in vivo on withdrawal of the inhibitors. The unedited CD7 CAR T cells persisted long term and mediated sustained anti-leukemic activity in two mouse xenograft models of human T cell acute lymphoblastic leukemia (T-ALL) by self-selecting for CD7-, fratricide-resistant CD7 CAR T cells that were transcriptionally similar to control CD7-edited CD7 CAR T cells. Finally, we showed feasibility of cGMP manufacturing of unedited autologous CD7 CAR T cells for patients with CD7+ malignancies and initiated a phase I clinical trial (ClinicalTrials.gov: NCT03690011) using this approach. These results indicate pharmacologic inhibition of CAR signaling enables generating functional CD7 CAR T cells without additional engineering.
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Leucemia-Linfoma Linfoblástico de Células T Precursoras , Receptores de Antígenos Quiméricos , Camundongos , Animais , Humanos , Linfócitos T , Imunoterapia Adotiva/métodos , Dasatinibe/metabolismo , Estudos de Viabilidade , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismoRESUMO
Acute graft-versus-host disease (aGVHD) limits the therapeutic benefit of allogeneic hematopoietic stem cell transplantation (allo-HSCT) and requires immunosuppressive prophylaxis that compromises antitumor and antipathogen immunity. OX40 is a costimulatory receptor upregulated on circulating T cells in aGVHD and plays a central role in driving the expansion of alloreactive T cells. Here, we show that OX40 is also upregulated on T cells infiltrating GVHD target organs in a rhesus macaque model, supporting the hypothesis that targeted ablation of OX40+ T cells will mitigate GVHD pathogenesis. We thus created an OX40-specific cytotoxic receptor that, when expressed on human T cells, enables selective elimination of OX40+ T cells. Because OX40 is primarily upregulated on CD4+ T cells upon activation, engineered OX40-specific T cells mediated potent cytotoxicity against activated CD4+ T cells and suppressed alloreactive T-cell expansion in a mixed lymphocyte reaction model. OX40 targeting did not inhibit antiviral activity of memory T cells specific to Epstein-Barr virus, cytomegalovirus, and adenoviral antigens. Systemic administration of OX40-targeting T cells fully protected mice from fatal xenogeneic GVHD mediated by human peripheral blood mononuclear cells. Furthermore, combining OX40 targeting with a leukemia-specific chimeric antigen receptor in a single T cell product provides simultaneous protection against leukemia and aGVHD in a mouse xenograft model of residual disease posttransplant. These results underscore the central role of OX40+ T cells in mediating aGVHD pathogenesis and support the feasibility of a bifunctional engineered T-cell product derived from the stem cell donor to suppress both disease relapse and aGVHD following allo-HSCT.
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Infecções por Vírus Epstein-Barr , Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas , Leucemia , Humanos , Animais , Camundongos , Leucócitos Mononucleares/patologia , Infecções por Vírus Epstein-Barr/complicações , Macaca mulatta , Herpesvirus Humano 4 , Doença Enxerto-Hospedeiro/etiologia , Leucemia/complicações , Doença Crônica , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , RecidivaRESUMO
Chimeric antigen receptor (CAR) T-cell therapy targeting T-cell acute lymphoblastic leukemia (T-ALL) faces limitations such as antigen selection and limited T-cell persistence. CD7 is an attractive antigen for targeting T-ALL, but overlapping expression on healthy T cells leads to fratricide of CD7-CAR T cells, requiring additional genetic modification. We took advantage of naturally occurring CD7- T cells to generate CD7-CAR (CD7-CARCD7-) T cells. CD7-CARCD7- T cells exhibited a predominantly CD4+ memory phenotype and had significant antitumor activity upon chronic antigen exposure in vitro and in xenograft mouse models. Based on these encouraging results, we next explored the utility of CD7- T cells for the immunotherapy of CD19+ hematological malignancies. Direct comparison of nonselected (bulk) CD19-CAR and CD19-CARCD7- T cells revealed that CD19-CARCD7- T cells had enhanced antitumor activity compared with their bulk counterparts in vitro and in vivo. Lastly, to gain insight into the behavior of CD19-CAR T cells with low levels of CD7 gene expression (CD7lo) in humans, we mined single-cell gene and T-cell receptor (TCR) expression data sets from our institutional CD19-CAR T-cell clinical study. CD19-CARCD7lo T cells were present in the initial CD19-CAR T-cell product and could be detected postinfusion. Intriguingly, the only functional CD4+ CD19-CAR T-cell cluster observed postinfusion exhibited CD7lo expression. Additionally, samples from patients responsive to therapy had a higher proportion of CD7lo T cells than nonresponders (NCT03573700). Thus, CARCD7- T cells have favorable biological characteristics and may present a promising T-cell subset for adoptive cell therapy of T-ALL and other hematological malignancies.
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Neoplasias Hematológicas , Leucemia-Linfoma Linfoblástico de Células T Precursoras , Humanos , Camundongos , Animais , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Receptores de Antígenos de Linfócitos T , Imunoterapia Adotiva , Neoplasias Hematológicas/terapia , Imunoterapia , Antígenos CD19RESUMO
Subsequent malignancies are well-documented complications in long-term follow-up of cancer patients. Recently, genetically modified immune effector (IE) cells have shown benefit in hematologic malignancies and are being evaluated in clinical trials for solid tumors. Although the short-term complications of IE cells are well described, there is limited literature summarizing long-term follow-up, including subsequent malignancies. We retrospectively reviewed data from 340 patients treated across 27 investigator-initiated pediatric and adult clinical trials at our center. All patients received IE cells genetically modified with γ-retroviral vectors to treat relapsed and/or refractory hematologic or solid malignancies. In a cumulative 1027 years of long-term follow-up, 13 patients (3.8%) developed another cancer with a total of 16 events (4 hematologic malignancies and 12 solid tumors). The 5-year cumulative incidence of a first subsequent malignancy in the recipients of genetically modified IE cells was 3.6% (95% confidence interval, 1.8% to 6.4%). For 11 of the 16 subsequent tumors, biopsies were available, and no sample was transgene positive by polymerase chain reaction. Replication-competent retrovirus testing of peripheral blood mononuclear cells was negative in the 13 patients with subsequent malignancies tested. Rates of subsequent malignancy were low and comparable to standard chemotherapy. These results suggest that the administration of IE cells genetically modified with γ retroviral vectors does not increase the risk for subsequent malignancy.
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Neoplasias Hematológicas , Neoplasias , Adulto , Criança , Seguimentos , Neoplasias Hematológicas/genética , Neoplasias Hematológicas/terapia , Humanos , Leucócitos Mononucleares , Neoplasias/genética , Neoplasias/terapia , Estudos RetrospectivosRESUMO
The seventh multi-stakeholder Paediatric Strategy Forum focused on chimeric antigen receptor (CAR) T-cells for children and adolescents with cancer. The development of CAR T-cells for patients with haematological malignancies, especially B-cell precursor acute lymphoblastic leukaemia (BCP-ALL), has been spectacular. However, currently, there are scientific, clinical and logistical challenges for use of CAR T-cells in BCP-ALL and other paediatric malignancies, particularly in acute myeloid leukaemia (AML), lymphomas and solid tumours. The aims of the Forum were to summarise the current landscape of CAR T-cell therapy development in paediatrics, too identify current challenges and future directions, with consideration of other immune effector modalities and ascertain the best strategies to accelerate their development and availability to children. Although the effect is of limited duration in about half of the patients, anti-CD19 CAR T-cells produce high response rates in relapsed/refractory BCP-ALL and this has highlighted previously unknown mechanisms of relapse. CAR T-cell treatment as first- or second-line therapy could also potentially benefit patients whose disease has high-risk features associated with relapse and failure of conventional therapies. Identifying patients with very early and early relapse in whom CAR T-cell therapy may replace haematopoietic stem cell transplantation and be definitive therapy versus those in whom it provides a more effective bridge to haematopoietic stem cell transplantation is a very high priority. Development of approaches to improve persistence, either by improving T cell fitness or using more humanised/fully humanised products and co-targeting of multiple antigens to prevent antigen escape, could potentially further optimise therapy. Many differences exist between paediatric B-cell non-Hodgkin lymphomas (B-NHL) and BCP-ALL. In view of the very small patient numbers with relapsed lymphoma, careful prioritisation is needed to evaluate CAR T-cells in children with Burkitt lymphoma, primary mediastinal B cell lymphoma and other NHL subtypes. Combination trials of alternative targets to CD19 (CD20 or CD22) should also be explored as a priority to improve efficacy in this population. Development of CD30 CAR T-cell immunotherapy strategies in patients with relapsed/refractory Hodgkin lymphoma will likely be most efficiently accomplished by joint paediatric and adult trials. CAR T-cell approaches are early in development for AML and T-ALL, given the unique challenges of successful immunotherapy actualisation in these diseases. At this time, CD33 and CD123 appear to be the most universal targets in AML and CD7 in T-ALL. The results of ongoing or planned first-in-human studies are required to facilitate further understanding. There are promising early results in solid tumours, particularly with GD2 targeting cell therapies in neuroblastoma and central nervous system gliomas that represent significant unmet clinical needs. Further understanding of biology is critical to success. The comparative benefits of autologous versus allogeneic CAR T-cells, T-cells engineered with T cell receptors T-cells engineered with T cell receptor fusion constructs, CAR Natural Killer (NK)-cell products, bispecific T-cell engager antibodies and antibody-drug conjugates require evaluation in paediatric malignancies. Early and proactive academia and multi-company engagement are mandatory to advance cellular immunotherapies in paediatric oncology. Regulatory advice should be sought very early in the design and preparation of clinical trials of innovative medicines, for which regulatory approval may ultimately be sought. Aligning strategic, scientific, regulatory, health technology and funding requirements from the inception of a clinical trial is especially important as these are very expensive therapies. The model for drug development for cell therapy in paediatric oncology could also involve a 'later stage handoff' to industry after early development in academic hands. Finally, and very importantly, strategies must evolve to ensure appropriate ease of access for children who need and could potentially benefit from these therapies.
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Desenvolvimento de Medicamentos/organização & administração , Oncologia/organização & administração , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Adolescente , Criança , Europa (Continente) , Humanos , Pediatria , Estados Unidos , United States Food and Drug AdministrationRESUMO
BACKGROUND AIMS: Efforts to safely and effectively treat acute myeloid leukemia (AML) by targeting a single leukemia-associated antigen with chimeric antigen receptor (CAR) T cells have met with limited success, due in part to heterogeneous expression of myeloid antigens. The authors hypothesized that T cells expressing CARs directed toward two different AML-associated antigens would eradicate tumors and prevent relapse. METHODS: For co-transduction with the authors' previously optimized CLL-1 CAR currently in clinical study (NCT04219163), the authors generated two CARs targeting either CD123 or CD33. The authors then tested the anti-tumor activity of T cells expressing each of the three CARs either alone or after co-transduction. The authors analyzed CAR T-cell phenotype, expansion and transduction efficacy and assessed function by in vitro and in vivo activity against AML cell lines expressing high (MOLM-13: CD123 high, CD33 high, CLL-1 intermediate), intermediate (HL-60: CD123 low, CD33 intermediate, CLL-1 intermediate/high) or low (KG-1a: CD123 low, CD33 low, CLL-1 low) levels of the target antigens. RESULTS: The in vitro benefit of dual expression was most evident when the target cell line expressed low antigen levels (KG-1a). Mechanistically, dual expression was associated with higher pCD3z levels in T cells compared with single CAR T cells on exposure to KG-1a (P < 0.0001). In vivo, combinatorial targeting with CD123 or CD33 and CLL-1 CAR T cells improved tumor control and animal survival for all lines (KG-1a, MOLM-13 and HL-60); no antigen escape was detected in residual tumors. CONCLUSIONS: Overall, these findings demonstrate that combinatorial targeting of CD33 or CD123 and CLL-1 with CAR T cells can control growth of heterogeneous AML tumors.
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Leucemia Linfocítica Crônica de Células B , Leucemia Mieloide Aguda , Animais , Linhagem Celular Tumoral , Imunoterapia Adotiva , Subunidade alfa de Receptor de Interleucina-3 , Leucemia Mieloide Aguda/terapia , Linfócitos TRESUMO
ABSTRACT: Banked chimeric antigen receptor (CAR) T cells immediately available for off-the-shelf (OTS) application can solve key limitations of patient-specific CAR T-cell products while retaining their potency. The allogeneic nature of OTS cell therapies requires additional measures to minimize graft-versus-host disease and host-versus-graft immune rejection in immunocompetent recipients. In this review, we discuss engineering and manufacturing strategies aimed at minimizing unwanted interactions between allogeneic CAR T cells and the host. Overcoming these limitations will improve safety and antitumor potency of OTS CAR T cells and facilitate their wider use in cancer therapy.
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Doença Enxerto-Hospedeiro , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos , Terapia Baseada em Transplante de Células e Tecidos , Humanos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Linfócitos TRESUMO
Engineered T cells are effective therapies against a range of malignancies, but current approaches rely on autologous T cells, which are difficult and expensive to manufacture. Efforts to develop potent allogeneic T cells that are not rejected by the recipient's immune system require abrogating both T- and natural killer (NK)-cell responses, which eliminate foreign cells through various mechanisms. In the present study, we engineered a receptor that mediates deletion of activated host T and NK cells, preventing rejection of allogeneic T cells. Our alloimmune defense receptor (ADR) selectively recognizes 4-1BB, a cell surface receptor temporarily upregulated by activated lymphocytes. ADR-expressing T cells resist cellular rejection by targeting alloreactive lymphocytes in vitro and in vivo, while sparing resting lymphocytes. Cells co-expressing chimeric antigen receptors and ADRs persisted in mice and produced sustained tumor eradication in two mouse models of allogeneic T-cell therapy of hematopoietic and solid cancers. This approach enables generation of rejection-resistant, 'off-the-shelf', allogeneic T-cell products to produce long-term therapeutic benefit in immunocompetent recipients.
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Engenharia Celular/métodos , Rejeição de Enxerto/imunologia , Receptores de Antígenos de Linfócitos T , Receptores de Antígenos Quiméricos , Linfócitos T , Animais , Linhagem Celular , Terapia Baseada em Transplante de Células e Tecidos , Células Cultivadas , Rejeição de Enxerto/prevenção & controle , Humanos , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Camundongos , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
BACKGROUND: C-type lectin-like molecule 1 (CLL-1) is highly expressed in acute myeloid leukemia (AML) but is absent in primitive hematopoietic progenitors, making it an attractive target for a chimeric antigen receptor (CAR) T-cell therapy. Here, we optimized our CLL-1 CAR for anti-leukemic activity in mouse xenograft models of aggressive AML. METHODS: First, we optimized the CLL-1 CAR using different spacer, transmembrane and costimulatory sequences. We used a second retroviral vector to coexpress transgenic IL15. We measured the effects of each construct on T cell phenotype and sequential (recursive) co culture assays with tumor cell targets to determine the durability of the anti tumor activity by flow cytometry. We administered CAR T cells to mice engrafted with patient derived xenografts (PDX) and AML cell line and determined anti tumor activity by bioluminescence imaging and weekly bleeding, measured serum cytokines by multiplex analysis. After euthanasia, we examined formalin-fixed/paraffin embedded sections. Unpaired two-tailed Student's t-tests were used and values of p<0.05 were considered significant. Survival was calculated using Mantel-Cox log-rank test. RESULTS: In vitro, CLL-1 CAR T cells with interleukin-15 (IL15) were less terminally differentiated (p<0.0001) and had superior expansion compared with CD28z-CD8 CAR T cells without IL15 (p<0.001). In both AML PDX and AML cell line animal models, CLL-1 CAR T coexpressing transgenic IL15 initially expanded better than CD28z-CD8 CAR T without IL15 (p<0.0001), but produced severe acute toxicity associated with high level production of human tumor necrosis factor α (TNFα), IL15 and IL2. Histopathology showed marked inflammatory changes with tissue damage in lung and liver. This acute toxicity could be managed by two strategies, individually or in combination. The excessive TNF alpha secretion could be blocked with anti-TNF alpha antibody, while excessive T cell expansion could be arrested by activation of an inducible caspase nine safety switch by administration of dimerizing drug. Both strategies successfully prolonged tumor-free survival. CONCLUSION: Combinatorial treatment with a TNFα blocking antibody and subsequent activation of the caspase-9 control switch increased the expansion, survival and antileukemic potency of CLL-1 CAR T-cells expressing transgenic IL15 while avoiding the toxicities associated with excessive cytokine production and long-term accumulation of activated T-cells.
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Imunoterapia Adotiva/métodos , Interleucina-15/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Humanos , Leucemia Linfocítica Crônica de Células B , Camundongos , Camundongos Endogâmicos NODRESUMO
Human cytomegalovirus (HCMV) reactivations are associated with lower overall survival after transplantations. Adoptive transfer of HCMV-reactive expanded or selected T cells can be applied as a compassionate use, but requires that the human leukocyte antigen-matched donor provides memory cells against HCMV. To overcome this, we developed engineered T cells expressing chimeric antigen receptors (CARs) targeted against the HCMV glycoprotein B (gB) expressed upon viral reactivation. Single-chain variable fragments (scFvs) derived from a human high-affinity gB-specific neutralizing monoclonal antibody (SM5-1) were fused to CARs with 4-1BB (BBL) or CD28 (28S) costimulatory domains and subcloned into retroviral vectors. CD4+ and CD8+ T cells obtained from HCMV-seronegative adult blood or cord blood (CB) transduced with the vectors efficiently expressed the gB-CARs. The specificity and potency of gB-CAR-T cells were demonstrated and compared in vitro using the following: 293T cells expressing gB, and with mesenchymal stem cells infected with a HCMV TB40 strain expressing Gaussia luciferase (HCMV/GLuc). BBL-gB-CAR-T cells generated with adult or CB demonstrated significantly higher in vitro activation and cytotoxicity performance than 28-gB-CAR-T cells. Nod.Rag.Gamma (NRG) mice transplanted with human CB CD34+ cells with long-term human immune reconstitution were used to model HCMV/GLuc infection in vivo by optical imaging analyses. One week after administration, response to BBL-gB-CAR-T cell therapy was observed for 5/8 mice, defined by significant reduction of the bioluminescent signal in relation to untreated controls. Response to therapy was sporadically associated with CAR detection in spleen. Thus, exploring scFv derived from the high-affinity gB-antibody SM5-1 and the 4-1BB signaling domain for CAR design enabled an in vitro high on-target effect and cytotoxicity and encouraging results in vivo. Therefore, gB-CAR-T cells can be a future clinical option for treatment of HCMV reactivations, particularly when memory T cells from the donors are not available.