RESUMO
The inability of people living with HIV (PLWH) to eradicate human immunodeficiency virus (HIV) infection is due in part to the inadequate HIV-specific cellular immune response. The antiviral function of cytotoxic CD8+ T cells, which are crucial for HIV control, is impaired during chronic viral infection because of viral escape mutations, immune exhaustion, HIV antigen downregulation, inflammation, and apoptosis. In addition, some HIV-infected cells either localize to tissue sanctuaries inaccessible to CD8+ T cells or are intrinsically resistant to CD8+ T cell killing. The novel design of synthetic chimeric antigen receptors (CARs) that enable T cells to target specific antigens has led to the development of potent and effective CAR-T cell therapies. While initial clinical trials using anti-HIV CAR-T cells performed over 20 years ago showed limited anti-HIV effects, the improved CAR-T cell design, which enabled its success in treating cancer, has reinstated CAR-T cell therapy as a strategy for HIV cure with notable progress being made in the recent decade.Effective CAR-T cell therapy against HIV infection requires the generation of anti-HIV CAR-T cells with potent in vivo activity against HIV-infected cells. Preclinical evaluation of anti-HIV efficacy of CAR-T cells and their safety is fundamental for supporting the initiation of subsequent clinical trials in PLWH. For these preclinical studies, we developed a novel humanized mouse model supporting in vivo HIV infection, the development of viremia, and the evaluation of novel HIV therapeutics. Preclinical assessment of anti-HIV CAR-T cells using this mouse model involves a multistep process including peripheral blood mononuclear cells (PBMCs) harvested from human donors, T cell purification, ex vivo T cell activation, transduction with lentiviral vectors encoding an anti-HIV CAR, CAR-T cell expansion and infusion in mice intrasplenically injected with autologous PBMCs followed by the determination of CAR-T cell capacity for HIV suppression. Each of the steps described in the following protocol were optimized in the lab to maximize the quantity and quality of the final anti-HIV CAR-T cell products.
Assuntos
Infecções por HIV , Imunoterapia Adotiva , Receptores de Antígenos Quiméricos , Humanos , Animais , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Camundongos , Infecções por HIV/imunologia , Infecções por HIV/terapia , Infecções por HIV/virologia , Imunoterapia Adotiva/métodos , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T CD8-Positivos/imunologia , HIV-1/imunologia , Linfócitos T/imunologia , Transdução GenéticaRESUMO
Pediatric patients with relapsed or refractory rhabdomyosarcoma (RMS) have dismal cure rates, and effective therapy is urgently needed. The oncogenic receptor tyrosine kinase fibroblast growth factor receptor 4 (FGFR4) is highly expressed in RMS and lowly expressed in healthy tissues. Here, we describe a second-generation FGFR4-targeting chimeric antigen receptor (CAR), based on an anti-human FGFR4-specific murine monoclonal antibody 3A11, as an adoptive T cell treatment for RMS. The 3A11 CAR T cells induced robust cytokine production and cytotoxicity against RMS cell lines in vitro. In contrast, a panel of healthy human primary cells failed to activate 3A11 CAR T cells, confirming the selectivity of 3A11 CAR T cells against tumors with high FGFR4 expression. Finally, we demonstrate that 3A11 CAR T cells are persistent in vivo and can effectively eliminate RMS tumors in two metastatic and two orthotopic models. Therefore, our study credentials CAR T cell therapy targeting FGFR4 to treat patients with RMS.
Assuntos
Receptores de Antígenos Quiméricos , Rabdomiossarcoma , Animais , Criança , Humanos , Camundongos , Linhagem Celular Tumoral , Imunoterapia Adotiva , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/metabolismo , Receptores de Antígenos Quiméricos/genética , Rabdomiossarcoma/tratamento farmacológicoRESUMO
With greater accessibility and an increased number of patients being treated with CAR T cell therapy, real-world toxicity continues to remain a significant challenge to its widespread adoption. We have previously shown that allogeneic umbilical cord blood-derived (UCB) regulatory T cells (Tregs) can resolve inflammation and treat acute and immune-mediated lung injuries. Allogeneic, cryopreserved UCB Tregs have shown a clinical benefit in patients suffering from COVID-19 acute respiratory distress syndrome. The unique properties of UCB Treg cells include a lack of plasticity under inflammatory micro-environments, no requirement for HLA matching, a long shelf life of cryopreserved cells, and immediate product availability, which makes them attractive for treating acute inflammatory syndromes. Therefore, we hypothesized that adjunct therapy with UCB Tregs may resolve the undesirable inflammation responsible for CAR T cell therapy-associated toxicity. In in vitro analysis, no interference from the addition of UCB Tregs was observed on CD19 CAR T cells' ability to kill CD19 Raji cells at different CAR T: Raji cell ratios of 8:1 (80.4% vs. 81.5%); 4:1 (62.0% vs. 66.2%); 2:1 (50.1% vs. 54.7%); and 1:1 (35.4% vs. 44.1%). In the xenogeneic B-cell lymphoma model, multiple injections of UCB Tregs were administered 3 days after CD19 CAR T cell injection, and no detrimental effect of add-on Tregs was noted on the circulating CD8+ T effector cells. The distribution of CAR T cells in multiple organs remained unaffected by the addition of the UCB Tregs. Specifically, no difference in the overall tumor burden was detected between the UCB Treg + CAR T vs. CAR T alone recipients. No tumor was detected in the liver or bone marrow in CAR T cells + UCB Tregs recipients, with a notable corresponding decrease in multiple circulating inflammatory cytokines when compared to CART alone recipients. Here we show the proof of concept for adjunct therapy with UCB Tregs to mitigate the hyper-inflammatory state induced by CAR T cells without any interference in their on-target anti-tumor activity. Administration of UCB Tregs after CAR T cells allows sufficient time for their synapse formation with tumor cells and exerts cytotoxicity, such that the UCB Tregs are diverted to interact with the antigen-presenting cells at the site of inflammation. Such a differential distribution of cells would allow for a two-pronged strategy of a UCB Treg "cooling blanket" effect and lay the groundwork for clinical study.
Assuntos
COVID-19 , Neoplasias , Receptores de Antígenos Quiméricos , Humanos , Linfócitos T Reguladores , COVID-19/terapia , Inflamação , Microambiente TumoralRESUMO
The landscape of therapeutic options for B cell malignancies has fundamentally changed with regulatory and marketing approval of chimeric antigen receptor (CAR)-engineered T cell products. The cell types used for CAR-T production, the length of time of manufacture, the stimulation matrix, and the nature of the gene vector used to transduce human T cells all are significant variables that require adequate quality control before infusion. Having approved products available to clinicians using a centralized production paradigm has not stopped innovation in investigator-initiated trials. Moreover, the high costs of the commercial products have been a significant wake-up call to those concerned about rising costs in health care, and the ability of developing nations, and nations with managed care systems to support these costs. Place-of-care manufacturing is a clear alternative to the approved products created in a centralized manufacturing approach. It is supported by continued technological innovation and the willingness of clinicians to develop new ways to decrease costs and make these curative therapies equitably available.
Assuntos
Neoplasias , Receptores de Antígenos Quiméricos , Humanos , Imunoterapia Adotiva , Linfócitos TRESUMO
Chimeric Antigen Receptor (CAR) T cell therapy is an accepted standard of care for relapsed/refractory B cell malignancies. However, the high cost of existing industry-driven centralized production makes this therapy unaffordable in low and middle-income countries. Decentralized or point of care manufacturing has the potential to overcome some of these challenges. Here we demonstrate a decentralized manufacturing process for anti-CD19-CAR-T cells using a fully automated closed system (Miltenyi CliniMACS Prodigy®) is feasible in a developing country setting. Validation run data, as part of a pre-clinical trial safety evaluation, demonstrates the successful and robust manufacturing of anti-CD19 CAR-T cells with T cell expansion of 25 to 47-fold. The median transduction efficiency was 48.8%, with a median viability of 98% and fulfillment of all standard release criteria assays for clinical application. Evaluation of production costs in an academic, not for profit setting in India provide a benchmark for low and middle-income pricing which could greatly increase access to this therapy. Based on our analysis, the cost per product would be approximately $35,107 US dollars. Our data highlights the safety, efficacy, and reproducibility of the process for use in planned future clinical trials.
Assuntos
Imunoterapia Adotiva , Neoplasias , Humanos , Reprodutibilidade dos Testes , Linfócitos T , Custos e Análise de Custo , Antígenos CD19RESUMO
The gene and cell therapy field saw its first approved treatments in Europe in 2012 and the United States in 2017 and is projected to be at least a $10B USD industry by 2025. Despite this success, a massive gap exists between the companies, clinics, and researchers developing these therapeutic approaches, and their availability to the patients who need them. The unacceptable reality is a geographic exclusion of low-and middle-income countries (LMIC) in gene therapy development and ultimately the provision of gene therapies to patients in LMIC. This is particularly relevant for gene therapies to treat human immunodeficiency virus infection and hemoglobinopathies, global health crises impacting tens of millions of people primarily located in LMIC. Bridging this divide will require research, clinical and regulatory infrastructural development, capacity-building, training, an approval pathway and community adoption for success and sustainable affordability. In 2020, the Global Gene Therapy Initiative was formed to tackle the barriers to LMIC inclusion in gene therapy development. This working group includes diverse stakeholders from all sectors and has set a goal of introducing two gene therapy Phase I clinical trials in two LMIC, Uganda and India, by 2024. Here we report on progress to date for this initiative.
Assuntos
Países em Desenvolvimento , Infecções por HIV , Humanos , Estados UnidosRESUMO
HIV-specific chimeric antigen receptor-T cell (CAR T cell) therapies are candidates to functionally cure HIV infection in people with HIV (PWH) by eliminating reactivated HIV-infected cells derived from latently infected cells within the HIV reservoir. Paramount to translating such therapeutic candidates successfully into the clinic will require anti-HIV CAR T cells to localize to lymphoid tissues in the body and eliminate reactivated HIV-infected cells such as CD4+ T cells and monocytes/macrophages. Here we show that i.v. injected anti-HIV duoCAR T cells, generated using a clinical-grade anti-HIV duoCAR lentiviral vector, localized to the site of active HIV infection in the spleen of humanized mice and eliminated HIV-infected PBMCs. CyTOF analysis of preinfusion duoCAR T cells revealed an early memory phenotype composed predominantly of CCR7+ stem cell-like/central memory T cells (TSCM/TCM) with expression of some effector-like molecules. In addition, we show that anti-HIV duoCAR T cells effectively sense and kill HIV-infected CD4+ T cells and monocytes/macrophages. Furthermore, we demonstrate efficient genetic modification of T cells from PWH on suppressive ART into anti-HIV duoCAR T cells that subsequently kill autologous PBMCs superinfected with HIV. These studies support the safety and efficacy of anti-HIV duoCAR T cell therapy in our presently open phase I/IIa clinical trial (NCT04648046).
Assuntos
Infecções por HIV , HIV-1 , Receptores de Antígenos Quiméricos , Animais , Camundongos , Linfócitos T CD4-Positivos , Infecções por HIV/tratamento farmacológico , Leucócitos Mononucleares , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como AssuntoRESUMO
Background: With the rising number of chimeric antigen receptor (CAR) T cell treated patients, it is increasingly important to understand the treatment's impact on patient-reported outcomes (PROs) and, ideally, identify biomarkers of central nervous system (CNS) adverse effects. Methods: The purpose of this exploratory study was to assess short-term PROs and serum kynurenine metabolites for associated neurotoxicity among patients treated in an anti-CD20, anti-CD19 (LV20.19) CAR T cell phase I clinical trial (NCT03019055). Fifteen CAR T treated patients from the parent trial provided serum samples and self-report surveys 15 days before and 14, 28, and 90 days after treatment. Results: Blood kynurenine concentrations increased over time in patients with evidence of neurotoxicity (p = 0.004) and were increased in self-reported depression (r = 0.52, p = 0.002). Depression improved after CAR T infusion (p = 0.035). Elevated 3-hydroxyanthranilic acid (3HAA) concentrations prior to cell infusion were also predictive of neurotoxicity onset (p = 0.031), suggesting it is a biomarker of neurotoxicity following CAR T cell therapy. Conclusions: Elevated levels of kynurenine pathway metabolites among CAR T cell recipients are associated with depressed mood and neurotoxicity. Findings from this exploratory study are preliminary and warrant validation in a larger cohort.
This study examined the impact of chimeric antigen receptor (CAR) T cell therapya therapy that gets immune cells to fight cancer by changing them in the lab to find and destroy cancer cellson blood markers associated with depression, anxiety, pain, fatigue, and poor sleep. Fifteen CAR T cell patients provided blood samples and completed surveys before and three timepoints after treatment. We found that the amount of kynurenine, a normal blood constituent, and related molecules was higher in patients who experienced significant CAR T cell side effects on the brain and in patients reporting more depression. These results identify the excessive elevation of blood constituents related to the mood that may also be associated with depression and brain dysfunction following CAR T. These blood constituents could potentially be used as markers and targeted with interventions to prevent brain dysfunction.
RESUMO
Chimeric antigen receptor T-cell (CAR-T cell) therapy directed at CD19 produces durable remissions in the treatment of relapsed/refractory non-Hodgkin lymphoma (NHL). Nonetheless, many patients receiving CD19 CAR-T cells fail to respond for unknown reasons. To reveal changes in 4-1BB-based CD19 CAR-T cells and identify biomarkers of response, we used single-cell RNA sequencing and protein surface marker profiling of patient CAR-T cells pre- and postinfusion into patients with NHL. At the transcriptional and protein levels, we note the evolution of CAR-T cells toward a nonproliferative, highly differentiated, and exhausted state, with an enriched exhaustion profile in CAR-T cells of patients with poor response marked by TIGIT expression. Utilizing in vitro and in vivo studies, we demonstrate that TIGIT blockade alone improves the antitumor function of CAR-T cells. Altogether, we provide evidence of CAR-T cell dysfunction marked by TIGIT expression driving a poor response in patients with NHL. SIGNIFICANCE: This is the first study investigating the mechanisms linked to CAR-T patient responses based on the sequential analysis of manufactured and infused CAR-T cells using single-cell RNA and protein expression data. Furthermore, our findings are the first to demonstrate an improvement of CAR-T cell efficacy with TIGIT inhibition alone. This article is highlighted in the In This Issue feature, p. 1825.
Assuntos
Linfoma não Hodgkin , Receptores de Antígenos Quiméricos , Receptores Imunológicos , Linfócitos T , Antígenos CD19 , Humanos , Imunoterapia Adotiva , Linfoma não Hodgkin/genética , Receptores de Antígenos de Linfócitos T , Receptores de Antígenos Quiméricos/genética , Receptores Imunológicos/genética , Linfócitos T/patologiaRESUMO
BACKGROUND: HIV cure research involving cell and gene therapy has intensified in recent years. There is a growing need to identify ethical standards and safeguards to ensure cell and gene therapy (CGT) HIV cure research remains valued and acceptable to as many stakeholders as possible as it advances on a global scale. METHODS: To elicit preliminary ethical and practical considerations to guide CGT HIV cure research, we implemented a qualitative, in-depth interview study with three key stakeholder groups in the United States: (1) biomedical HIV cure researchers, (2) bioethicists, and (3) community stakeholders. Interviews permitted evaluation of informants' perspectives on how CGT HIV cure research should ethically occur, and were transcribed verbatim. We applied conventional content analysis focused on inductive reasoning to analyze the rich qualitative data and derive key ethical and practical considerations related to CGT towards an HIV cure. RESULTS: We interviewed 13 biomedical researchers, 5 community members, and 1 bioethicist. Informants generated considerations related to: perceived benefits of CGT towards an HIV cure, perceived risks, considerations necessary to ensure an acceptable benefit/risk balance, CGT strategies considered unacceptable, additional ethical considerations, and considerations for first-in-human CGT HIV cure trials. Informants also proposed important safeguards to developing CGT approaches towards an HIV cure, such as the importance of mitigating off-target effects, mitigating risks associated with long-term duration of CGT interventions, and mitigating risks of immune overreactions. CONCLUSION: Our study identified preliminary considerations for CGT-based HIV cure across three key stakeholder groups. Respondents identified an ideal cure strategy as one which would durably control HIV infection, protect the individual from re-acquisition, and eliminate transmission to others. Known and unknown risks should be anticipated and perceived as learning opportunities to preserve and honor the altruism of participants. Preclinical studies should support these considerations and be transparently reviewed by regulatory experts and peers prior to first-in-human studies. To protect the public trust in CGT HIV cure research, ethical and practical considerations should be periodically revisited and updated as the science continues to evolve. Additional ethics studies are required to expand stakeholder participation to include traditionally marginalized groups and clinical care providers.
Assuntos
Infecções por HIV , Eticistas , Terapia Genética , Infecções por HIV/prevenção & controle , Humanos , Pesquisa Qualitativa , Pesquisadores , Estados UnidosRESUMO
CAR T-cell therapies targeting the B-cell maturation antigen eliminate tumors in relapsed/refractory multiple myeloma patients, however durable remissions remain difficult to attain. Transforming growth factor beta (TGF-ß) is a multifunctional cytokine abundantly expressed in the multiple myeloma bone marrow niche, where it promotes an immunosuppressive tumor microenvironment. We hypothesized that BCMA CAR T-cells armored to resist the suppressive effects of TGF-ß will provide an advantage in treating multiple myeloma. The armored B2ARM CAR T cells, co-expressing BCMA targeting CAR with TGF-ß dominant-negative receptor II, were generated by lentiviral transduction of primary human CD4+ and CD8+ T cells. The B2ARM CAR T cells eliminated MM.1S multiple myeloma targets in long-term cytotoxicity assays, even under TGF-ß-high conditions, whereas cytotoxic function of the non-armored B2 CAR -T cells was inhibited by TGF-ß. Concordantly, after long-term exposure to targets in the presence of TGF-ß, the B2ARM CAR T cells were enriched for Granzyme B, CD107a, Ki67 and polyfunctional cells T-cells (double or triple-positive for IFN-γ, IL-2 and/or TNF-α), as determined by flow cytometry. In addition, the B2ARM CAR T-cells, but not the conventional B2 CAR T-cells, resisted the TGF-ß-mediated suppression of activation (CD25), exhaustion (PD-1, LAG3), and differentiation to T effectors (CD45RA+ CD45RO-CD62L-). In NSG mice bearing RPMI-8226 tumors overexpressing TGF-ß, the B2ARM CAR mediated 100% tumor rejection and survival, superior infiltration of tumors on day 7 post CAR T treatment (%CD3+CAR+), and greater expression of IFN-γ, TNF-α, Ki67, Granzyme B, and PD-1, as compared to tumor-infiltrating non-armored B2 CAR T-cells. In NSG RPMI-8226 xenograft model in which tumors were additionally supplemented with TGF-ß injections on days -1 through 11 of CAR T treatment, the B2ARM CAR T cells rejected tumors faster than the non-armored B2 CARs, and showed greater numbers of CD3+ and CD3+CAR+, central memory (CD45RO+CD62L+) and effector memory (CD45RO+CD62L-) T cells in the peripheral blood 18 days after treatment. In summary, the armored B2ARM CAR T cells mediate superior persistence, proliferation, multi-functionality, effector differentiation and anti-tumor function in pre-clinical models of multiple myeloma, while abrogating TGF-ß-mediated suppression.
Assuntos
Mieloma Múltiplo , Receptores de Antígenos Quiméricos , Animais , Antígeno de Maturação de Linfócitos B , Granzimas , Humanos , Antígeno Ki-67 , Camundongos , Receptor de Morte Celular Programada 1 , Receptores de Antígenos Quiméricos/genética , Fator de Crescimento Transformador beta , Microambiente Tumoral , Fator de Necrose Tumoral alfaRESUMO
Chimeric antigen receptor (CAR) T cells targeting the CD19 antigen are effective in treating adults and children with B-cell malignancies. Place-of-care manufacturing may improve performance and accessibility by obviating the need to cryopreserve and transport cells to centralized facilities. Here we develop an anti-CD19 CAR (CAR19) comprised of the 4-1BB co-stimulatory and TNFRSF19 transmembrane domains, showing anti-tumor efficacy in an in vivo xenograft lymphoma model. CAR19 T cells are manufactured under current good manufacturing practices (cGMP) at two disparate clinical sites, Moscow (Russia) and Cleveland (USA). The CAR19 T-cells is used to treat patients with relapsed/refractory pediatric B-cell Acute Lymphocytic Leukemia (ALL; n = 31) or adult B-cell Lymphoma (NHL; n = 23) in two independently conducted phase I clinical trials with safety as the primary outcome (NCT03467256 and NCT03434769, respectively). Probability of measurable residual disease-negative remission was also a primary outcome in the ALL study. Secondary outcomes include complete remission (CR) rates, overall survival and median duration of response. CR rates are 89% (ALL) and 73% (NHL). After a median follow-up of 17 months, one-year survival rate of ALL complete responders is 79.2% (95%CI 64.5â97.2%) and median duration of response is 10.2 months. For NHL complete responders one-year survival is 92.9%, and median duration of response has not been reached. Place-of-care manufacturing produces consistent CAR-T cell products at multiple sites that are effective for the treatment of patients with B-cell malignancies.
Assuntos
Antígenos CD19/imunologia , Linfócitos B/imunologia , Linfoma de Células B/imunologia , Receptores de Antígenos Quiméricos/imunologia , Linfócitos T/imunologia , Adolescente , Adulto , Idoso , Animais , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Masculino , Camundongos , Camundongos Endogâmicos NOD , Pessoa de Meia-Idade , Neoplasia Residual , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Intervalo Livre de Progressão , Receptores de Antígenos de Linfócitos T , Receptores do Fator de Necrose Tumoral/química , Federação Russa , Estados Unidos , Adulto JovemRESUMO
Anti-CD19 chimeric antigen receptor T (CAR-T) cells have demonstrated activity against relapsed/refractory lymphomas. Cytokine release syndrome (CRS) and immune effector cell - associated neurotoxicity syndrome (ICANS) are well-known complications. Tocilizumab, a monoclonal antibody targeting the interleukin-6 (IL-6) receptor was administered 1 hour prior to infusion of anti-CD19 CAR-T cells with CD3ζ/4-1BB costimulatory signaling used to treat non-Hodgkin lymphoma patients. Relapsed/refractory lymphoma patients treated with anti-CD19 CAR-T cells were included in this analysis. Cytokine plasma levels were measured by electrochemiluminescence before lymphodepleting chemotherapy, prior to infusion and then on days 2, 4,6, and 14 days after treatment. Twenty patients were treated. Cell products included locally manufactured anti-CD19 CAR-T (n=18) and tisagenlecleucel (n=2). There were no adverse events attributed to tocilizumab. Ten patients had grade 1-2 CRS at a median of 4 (range 3-7) days. There were no cases of grade ≥3 CRS. Five patients had ICANS, grade 1 (n=4) and grade 4 (n=1). Laboratory studies obtained prior to lymphodepleting chemotherapy were comparable between patients with and without CRS, except for interleukin (IL)-15 plasma concentrations. patients with CRS had higher post-infusion ferritin and C reactive protein, with more marked increases in inflammatory cytokines, including IL-6, IL-15, IFN-γ, fractalkine and MCP-1. Fifteen patients (75%) achieved CR and 2 (10%), PR. One-year OS and PFS estimates were 83% and 73%. Prophylactic tocilizumab was associated with low CRS incidence and severity. There were no adverse events associated with tocilizumab, no increase in frequency or severity of ICANS and excellent disease control and overall survival.
Assuntos
Anticorpos Monoclonais Humanizados/uso terapêutico , Síndrome da Liberação de Citocina/prevenção & controle , Imunoterapia Adotiva/efeitos adversos , Linfoma não Hodgkin/terapia , Síndromes Neurotóxicas/prevenção & controle , Corticosteroides/uso terapêutico , Adulto , Idoso , Anticorpos Monoclonais Humanizados/administração & dosagem , Proteína C-Reativa/análise , Síndrome da Liberação de Citocina/sangue , Citocinas/sangue , Esquema de Medicação , Feminino , Ferritinas/sangue , Humanos , Proteína Antagonista do Receptor de Interleucina 1/uso terapêutico , Estimativa de Kaplan-Meier , Linfoma Difuso de Grandes Células B/sangue , Linfoma Difuso de Grandes Células B/terapia , Linfoma não Hodgkin/sangue , Masculino , Pessoa de Meia-Idade , Síndromes Neurotóxicas/etiologia , Pré-Medicação , Intervalo Livre de Progressão , Receptores de Interleucina-6/antagonistas & inibidores , Terapia de Salvação , Índice de Gravidade de Doença , Resultado do TratamentoRESUMO
Chimeric antigen receptor (CAR) T cells typically use a strong constitutive promoter to ensure maximal long-term CAR expression. However, recent evidence suggests that restricting the timing and magnitude of CAR expression is functionally beneficial, whereas constitutive CAR activation may lead to exhaustion and loss of function. We created a self-driving CD19-targeting CAR, which regulates its own function based on the presence of a CD19 antigen engaged by the CAR itself, by placing self-driving CAR19 constructs under transcriptional control of synthetic activator protein 1 (AP1)-nuclear factor κB (NF-κB) or signal transducer and activator of transcription (STAT)5 promoters. CD19 antigen-regulated expression was observed for self-driving AP1-NFκB-CAR19, with CAR19 upregulation within 18 h after exposure to target CD19, and corresponded to the level of tumor burden. Self-driving CAR-T cells showed enhanced tumor-dependent activation, expansion, and low exhaustion in vitro as compared to constitutively expressed EF1α and murine stem cell virus (MSCV) CARs and mediated tumor regression and survival in Raji-bearing NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Long-term CAR function correlated with upregulated CAR expression within 24 h of exposure to tumor antigen. The self-driving AP1-NFκB-CAR19 circuit was also used to inducibly express dominant-negative transforming growth factor ß receptor II (TGFBRIIdn), which effectively countered the negative effects of TGF-ß on CAR-T activation. Thus, a self-driving CAR approach may offer a new modality to express CAR and auxiliary proteins by enhancing CAR-T functional activity and limiting exhaustion.
Assuntos
Linfoma de Burkitt/terapia , Imunoterapia Adotiva/métodos , NF-kappa B/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Fator de Transcrição STAT5/genética , Fator de Transcrição AP-1/genética , Animais , Linfoma de Burkitt/genética , Linfoma de Burkitt/imunologia , Linfoma de Burkitt/patologia , Linhagem Celular Tumoral , Células HEK293 , Humanos , Células K562 , Camundongos , Camundongos Endogâmicos NOD , Regiões Promotoras Genéticas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A substantial number of patients with leukemia and lymphoma treated with anti-CD19 or anti-CD22 monoCAR-T cell therapy relapse because of antigen loss or down-regulation. We hypothesized that B cell tumor antigen escape may be overcome by a chimeric antigen receptor (CAR) design that simultaneously targets three B cell leukemia antigens. We engineered trispecific duoCAR-T cells with lentiviral vectors encoding two CAR open reading frames that target CD19, CD20, and CD22. The duoCARs were composed of a CAR with a tandem CD19- and CD20-targeting binder, linked by the P2A self-cleaving peptide to a second CAR targeting CD22. Multiple combinations of intracellular T cell signaling motifs were evaluated. The most potent duoCAR architectures included those with ICOS, OX40, or CD27 signaling domains rather than those from CD28 or 4-1BB. We identified four optimal binder and signaling combinations that potently rejected xenografted leukemia and lymphoma tumors in vivo. Moreover, in mice bearing a mixture of B cell lymphoma lines composed of parental triple-positive cells, CD19-negative, CD20-negative, and CD22-negative variants, only the trispecific duoCAR-T cells rapidly and efficiently rejected the tumors. Each of the monoCAR-T cells failed to prevent tumor progression. Analysis of intracellular signaling profiles demonstrates that the distinct signaling of the intracellular domains used may contribute to these differential effects. Multispecific duoCAR-T cells are a promising strategy to prevent antigen loss-mediated relapse or the down-regulation of target antigen in patients with B cell malignancies.
Assuntos
Imunoterapia Adotiva , Linfoma de Células B , Animais , Antígenos CD19 , Linfócitos B , Humanos , Linfoma de Células B/terapia , Camundongos , Receptores de Antígenos de Linfócitos T , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico , Linfócitos TRESUMO
Lentiviral vectors (LV) have emerged as a robust technology for therapeutic gene delivery into human cells as advanced medicinal products. As these products are increasingly commercialized, there are concomitant demands for their characterization to ensure safety, efficacy and consistency. Standards are essential for accurately measuring parameters for such product characterization. A critical parameter is the vector copy number (VCN) which measures the genetic dose of a transgene present in gene-modified cells. Here we describe a set of clonal Jurkat cell lines with defined copy numbers of a reference lentiviral vector integrated into their genomes. Genomic DNA was characterized for copy number, genomic integrity and integration coordinates and showed uniform performance across independent quantitative PCR assays. Stability studies during continuous long-term culture demonstrated sustained renewability of the reference standard source material. DNA from the Jurkat VCN standards would be useful for control of quantitative PCR assays for VCN determination in LV gene-modified cellular products and clinical samples.
Assuntos
Dosagem de Genes , Lentivirus/genética , Transdução Genética , Calibragem/normas , Técnicas de Transferência de Genes/normas , Vetores Genéticos/genética , Humanos , Células Jurkat , Mutagênese Insercional/genética , Padrões de Referência , Reprodutibilidade dos Testes , Transdução Genética/métodos , Transdução Genética/normas , Transfecção/métodos , Transfecção/normas , Estudos de Validação como Assunto , Integração Viral/genéticaRESUMO
Chimeric antigen receptor (CAR) T cells targeting CD19 are a breakthrough treatment for relapsed, refractory B cell malignancies1-5. Despite impressive outcomes, relapse with CD19- disease remains a challenge. We address this limitation through a first-in-human trial of bispecific anti-CD20, anti-CD19 (LV20.19) CAR T cells for relapsed, refractory B cell malignancies. Adult patients with B cell non-Hodgkin lymphoma or chronic lymphocytic leukemia were treated on a phase 1 dose escalation and expansion trial (NCT03019055) to evaluate the safety of 4-1BB-CD3ζ LV20.19 CAR T cells and the feasibility of on-site manufacturing using the CliniMACS Prodigy system. CAR T cell doses ranged from 2.5 × 105-2.5 × 106 cells per kg. Cell manufacturing was set at 14 d with the goal of infusing non-cryopreserved LV20.19 CAR T cells. The target dose of LV20.19 CAR T cells was met in all CAR-naive patients, and 22 patients received LV20.19 CAR T cells on protocol. In the absence of dose-limiting toxicity, a dose of 2.5 × 106 cells per kg was chosen for expansion. Grade 3-4 cytokine release syndrome occurred in one (5%) patient, and grade 3-4 neurotoxicity occurred in three (14%) patients. Eighteen (82%) patients achieved an overall response at day 28, 14 (64%) had a complete response, and 4 (18%) had a partial response. The overall response rate to the dose of 2.5 × 106 cells per kg with non-cryopreserved infusion (n = 12) was 100% (complete response, 92%; partial response, 8%). Notably, loss of the CD19 antigen was not seen in patients who relapsed or experienced treatment failure. In conclusion, on-site manufacturing and infusion of non-cryopreserved LV20.19 CAR T cells were feasible and therapeutically safe, showing low toxicity and high efficacy. Bispecific CARs may improve clinical responses by mitigating target antigen downregulation as a mechanism of relapse.