RESUMO
CD33 and CD123 are expressed on the surface of human acute myeloid leukemia blasts and other noncancerous tissues such as hematopoietic stem cells. On-target off-tumor toxicities may limit chimeric antigen receptor T cell therapies that target both CD33 and CD123. To overcome this limitation, we developed bispecific human CD33/CD123 chimeric antigen receptor (CAR) T cells with an "AND" logic gate. We produced novel CD33 and CD123 scFvs from monoclonal antibodies that bound CD33 and CD123 and activated T cells. Screening of CD33 and CD123 CAR T cells for cytotoxicity, cytokine production, and proliferation was performed, and we selected scFvs for CD33/CD123 bispecific CARs. The bispecific CARs split 4-1BB co-stimulation on one scFv and CD3ζ on the other. In vitro testing of cytokine secretion and cytotoxicity resulted in selecting bispecific CAR 1 construct for in vivo analysis. The CD33/CD123 bispecific CAR T cells were able to control acute myeloid leukemia (AML) in a xenograft AML mouse model similar to monospecific CD33 and CD123 CAR T cells while showing no on-target off-tumor effects. Based on our findings, human CD33/CD123 bispecific CAR T cells are a promising cell-based approach to prevent AML and support clinical investigation.
RESUMO
Higher γδ T cell counts in patients with malignancies are associated with better survival. However, γδ T cells are rare in the blood and functionally impaired in patients with malignancies. Promising results are reported on the treatment of various malignancies with in vivo expansion of autologous γδ T cells using zoledronic acid (zol) and interleukin-2 (IL-2). Here we demonstrated that zol and IL-2, in combination with a novel genetically engineered K-562 CD3scFv/CD137L/CD28scFv/IL15RA quadruplet artificial antigen-presenting cell (aAPC), efficiently expand allogeneic donor-derived γδ T cells using a Good Manufacturing Practice (GMP) compliant protocol sufficient to achieve cell doses for future clinical use. We achieved a 633-fold expansion of γδ T cells after day 10 of coculture with aAPC, which exhibited central (47%) and effector (43%) memory phenotypes. In addition, >90% of the expanded γδ T cells expressed NKG2D, although they have low cell surface expression of PD1 and LAG3 inhibitory checkpoint receptors. In vitro real-time cytotoxicity analysis showed that expanded γδ T cells were effective in killing target cells. Our results demonstrate that large-scale ex vivo expansion of donor-derived γδ T cells in a GMP-like setting can be achieved with the use of quadruplet aAPC and zol/IL-2 for clinical application.
Assuntos
Células Apresentadoras de Antígenos , Interleucina-2 , Interleucina-2/farmacologia , Linfócitos TRESUMO
An obstacle to the development of chimeric antigen receptor (CAR) T cells is the limited understanding of CAR T-cell biology and the mechanisms behind their antitumor activity. We and others have shown that CARs with a CD28 costimulatory domain drive high T-cell activation, which leads to exhaustion and shortened persistence. This work led us to hypothesize that by incorporating null mutations of CD28 subdomains (YMNM, PRRP, or PYAP), we could optimize CAR T-cell costimulation and enhance function. In vivo, we found that mice given CAR T cells with only a PYAP CD28 endodomain had a significant survival advantage, with 100% of mice alive after 62 days compared with 50% for mice with an unmutated endodomain. We observed that mutant CAR T cells remained more sensitive to antigen after ex vivo antigen and PD-L1 stimulation, as demonstrated by increased cytokine production. The mutant CAR T cells also had a reduction of exhaustion-related transcription factors and genes such as Nfatc1, Nr42a, and Pdcd1 Our results demonstrated that CAR T cells with a mutant CD28 endodomain have better survival and function. This work allows for the development of enhanced CAR T-cell therapies by optimizing CAR T-cell costimulation.
Assuntos
Antígenos CD28/antagonistas & inibidores , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Citocinas/biossíntese , Feminino , Humanos , Imunoterapia Adotiva , Ativação Linfocitária/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fatores de Transcrição NFATC/genética , Células NIH 3T3 , Membro 2 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Receptor de Morte Celular Programada 1/genética , Receptores de Antígenos Quiméricos/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Graft-versus-host disease (GVHD) remains an important cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (allo-HCT). For decades, GVHD prophylaxis has included calcineurin inhibitors, despite their incomplete efficacy and impairment of graft-versus-leukemia (GVL). Distinct from pharmacologic immune suppression, we have developed what we believe is a novel, human CD83-targeted chimeric antigen receptor (CAR) T cell for GVHD prevention. CD83 is expressed on allo-activated conventional CD4+ T cells (Tconvs) and proinflammatory dendritic cells (DCs), which are both implicated in GVHD pathogenesis. Human CD83 CAR T cells eradicate pathogenic CD83+ target cells, substantially increase the ratio of regulatory T cells (Tregs) to allo-activated Tconvs, and provide durable prevention of xenogeneic GVHD. CD83 CAR T cells are also capable of treating xenogeneic GVHD. We show that human acute myeloid leukemia (AML) expresses CD83 and that myeloid leukemia cell lines are readily killed by CD83 CAR T cells. Human CD83 CAR T cells are a promising cell-based approach to preventing 2 critical complications of allo-HCT - GVHD and relapse. Thus, the use of human CD83 CAR T cells for GVHD prevention and treatment, as well as for targeting CD83+ AML, warrants clinical investigation.
Assuntos
Transferência Adotiva , Antígenos CD/imunologia , Linfócitos T CD4-Positivos/imunologia , Doença Enxerto-Hospedeiro/prevenção & controle , Transplante de Células-Tronco Hematopoéticas , Imunoglobulinas/imunologia , Leucemia Mieloide Aguda/terapia , Glicoproteínas de Membrana/imunologia , Proteínas de Neoplasias/imunologia , Receptores de Antígenos Quiméricos/imunologia , Aloenxertos , Animais , Linfócitos T CD4-Positivos/transplante , Linhagem Celular Tumoral , Doença Enxerto-Hospedeiro/imunologia , Doença Enxerto-Hospedeiro/patologia , Humanos , Leucemia Mieloide Aguda/imunologia , Leucemia Mieloide Aguda/patologia , Camundongos , Antígeno CD83RESUMO
Adoptive cell therapy with ex vivo expanded tumor infiltrating lymphocytes or gene engineering T cells expressing chimeric antigen receptors (CAR) is a promising treatment for cancer patients. This production utilizes T-cell activation and transduction with activation beads and RetroNectin, respectively. However, the high cost of production is an obstacle for the broad clinical application of novel immunotherapeutic cell products. To facilitate production we refined our approach by using artificial antigen presenting cells (aAPCs) with receptors that ligate CD3, CD28, and the CD137 ligand (CD137L or 41BBL), as well as express the heparin binding domain (HBD), which binds virus for gene-transfer. We have used these aAPC for ex vivo gene engineering and expansion of tumor infiltrating lymphocytes and CAR T cells. We found that aAPCs can support efficacious T-cell expansion and transduction. Moreover, aAPCs expanded T cells exhibit higher production of IFN-γ and lower traits of T-cell exhaustion compared with bead expanded T cells. Our results suggest that aAPC provide a more physiological stimulus for T-cell activation than beads that persistently ligate T cells. The use of a renewable cell line to replace 2 critical reagents (beads and retronectin) for CAR T-cell production can significantly reduce the cost of production and make these therapies more accessible to patients.
Assuntos
Células Apresentadoras de Antígenos/imunologia , Células Apresentadoras de Antígenos/metabolismo , Imunoterapia Adotiva , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Biomarcadores , Linhagem Celular , Terapia Baseada em Transplante de Células e Tecidos , Citotoxicidade Imunológica , Técnicas de Transferência de Genes , Humanos , Imunofenotipagem , Imunoterapia Adotiva/métodos , Células K562 , Ativação Linfocitária/imunologia , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Camundongos , Neoplasias/etiologia , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/terapiaRESUMO
Chimeric antigen receptors (CARs) have an antigen-binding domain fused to transmembrane, costimulatory, and CD3ζ domains. Two CARs with regulatory approval include a CD28 or 4-1BB costimulatory domain. While both CARs achieve similar clinical outcomes, biologic differences have become apparent but not completely understood. Therefore, in this study we aimed to identify mechanistic differences between 4-1BB and CD28 costimulation that contribute to the biologic differences between the 2 CARs and could be exploited to enhance CAR T cell function. Using CD19-targeted CAR T cells with 4-1BB we determined that enhancement of T cell function is driven by NF-κB. Comparison to CAR T cells with CD28 also revealed that 4-1BB is associated with more antiapoptotic proteins and dependence on persistence for B cell killing. While TNF receptor-associated factor 2 (TRAF2) has been presupposed to be required for 4-1BB costimulation in CAR T cells, we determined that TRAF1 and TRAF3 are also critical. We observed that TRAFs impacted CAR T viability and proliferation, as well as cytotoxicity and/or cytokines, in part by regulating NF-κB. Our study demonstrates how 4-1BB costimulation in CAR T cells impacts antitumor eradication and clinical outcomes and has implications for enhanced CAR design.
Assuntos
Ligante 4-1BB/metabolismo , NF-kappa B/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/metabolismo , Ligante 4-1BB/genética , Animais , Antígenos CD19 , Linfócitos B , Antígenos CD28 , Linhagem Celular , Receptores Coestimuladores e Inibidores de Linfócitos T , Citocinas/metabolismo , Feminino , Terapia Genética , Proteínas de Homeodomínio/genética , Humanos , Imunoterapia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Linfócitos T/imunologia , Fator 1 Associado a Receptor de TNF/metabolismo , Fator 2 Associado a Receptor de TNF/metabolismo , Fator 3 Associado a Receptor de TNF/metabolismo , Fator de Transcrição RelA/metabolismo , Transcriptoma , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Respiratory syncytial virus (RSV) infection is the most frequent cause of hospitalization in infants and young children worldwide. Although mucosal RSV vaccines can reduce RSV disease burden, little is known about mucosal immune response capabilities in children. Neonatal or adult mice were infected with RSV; a subset of neonatal mice received interferon alpha (IFN-α) (intranasal) prior to RSV infection. B cells, B cell activating factor (BAFF) and IgA were measured by flow cytometry. RSV specific IgA was measured in nasal washes. Nasal associated lymphoid tissue (NALT) and lungs were stained for BAFF and IgA. Herein, we show in a mouse model of RSV infection that IFN-α plays a dual role as an antiviral and immune modulator and age-related differences in IgA production upon RSV infection can be overcome by IFN-α administration. IFN-α administration before RSV infection in neonatal mice increased RSV-specific IgA production in the nasal mucosa and induced expression of the B-cell activating factor BAFF in NALT. These findings are important, as mucosal antibodies at the infection site, and not serum antibodies, have been shown to protect human adults from experimental RSV infection.
Assuntos
Imunoglobulina A/imunologia , Imunoglobulina A/metabolismo , Interferon Tipo I/metabolismo , Infecções por Vírus Respiratório Sincicial/imunologia , Infecções por Vírus Respiratório Sincicial/metabolismo , Animais , Fator Ativador de Células B/metabolismo , Citometria de Fluxo , Camundongos , Camundongos Endogâmicos BALB C , Palivizumab/uso terapêutico , Reação em Cadeia da Polimerase em Tempo Real , Infecções por Vírus Respiratório Sincicial/tratamento farmacológicoRESUMO
BACKGROUND: Exposures to elevated levels of particulate matter (PM) enhance severity of influenza virus infection in infants. The biological mechanism responsible for this phenomenon is unknown. The recent identification of environmentally persistent free radicals (EPFRs) associated with PM from a variety of combustion sources suggests its role in the enhancement of influenza disease severity. METHODS: Neonatal mice (< seven days of age) were exposed to DCB230 (combustion derived PM with a chemisorbed EPFR), DCB50 (non-EPFR PM sample), or air for 30 minutes/day for seven consecutive days. Four days post-exposure, neonates were infected with influenza intranasally at 1.25 TCID50/neonate. Neonates were assessed for morbidity (% weight gain, peak pulmonary viral load, and viral clearance) and percent survival. Lungs were isolated and assessed for oxidative stress (8-isoprostanes and glutathione levels), adaptive immune response to influenza, and regulatory T cells (Tregs). The role of the EPFR was also assessed by use of transgenic mice expressing human superoxide dismutase 2. RESULTS: Neonates exposed to EPFRs had significantly enhanced morbidity and decreased survival following influenza infection. Increased oxidative stress was also observed in EPFR exposed neonates. This correlated with increased pulmonary Tregs and dampened protective T cell responses to influenza infection. Reduction of EPFR-induced oxidative stress attenuated these effects. CONCLUSIONS: Neonatal exposure to EPFR containing PM resulted in pulmonary oxidative stress and enhanced influenza disease severity. EPFR-induced oxidative stress resulted in increased presence of Tregs in the lungs and subsequent suppression of adaptive immune response to influenza.