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1.
Mol Cancer ; 23(1): 236, 2024 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-39443955

RESUMO

Enhancing the efficacy of CD19 CAR-T cell therapy can significantly improve patient outcomes by reducing relapse rates in CD19 + B cell malignancies. Exogenous or transgenic cytokines are often used to boost the expansion and durability of CAR-T cells but pose risks of severe toxicities. A promising approach to address these limitations is to immobilize cytokines on the surface of CAR-T cells using transmembrane (TM) anchor domains. Given IL-7 can enhance T-cell proliferation and antitumor activity, our study developed membrane-bound IL-7 constructs using different TM anchor domains (CD8, CD28 and B7-1). We primarily found that the CD8 TM provided superior anchoring for IL-7 compared to CD28 and B7-1. Moreover, the IL-7 construct with a CD8 TM (IL7/CD8) enhanced naïve T cell proliferation and effector functions, and improved the in vitro and in vivo antitumor activity of CD19 CAR-T cells. Importantly, although IL7/CD8 could promote T-cell proliferation, it did not sustain long-term autonomous expansion, which could ensure the safety of CD19 CAR-T cells expressing IL7/CD8 in clinical applications. Collectively, the IL7/CD8 construct represents a promising strategy for enhancing the therapeutic potential of CD19 CAR-T cell therapy.


Assuntos
Antígenos CD19 , Imunoterapia Adotiva , Interleucina-7 , Humanos , Interleucina-7/metabolismo , Antígenos CD19/imunologia , Animais , Camundongos , Imunoterapia Adotiva/métodos , Antígenos CD8/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Proliferação de Células , Receptores de Antígenos Quiméricos/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/genética , Linhagem Celular Tumoral , Linfócitos T/imunologia , Linfócitos T/metabolismo
2.
Ann Hematol ; 2024 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-39304585

RESUMO

Chronic myeloid leukemia presenting de novo in the blast phase (CML-BP) is a rare diagnosis among pediatric malignancies. We report on a 16-year-old male who presented with CML-BP lymphoid at diagnosis. He was treated with shortened acute lymphoblastic leukemia induction plus the tyrosine kinase inhibitor (TKI) imatinib followed by dasatinib. After achieving molecular remission (MR), hematopoietic stem cell transplantation (HSCT) was performed early after diagnosis. Despite prophylactic dasatinib, he relapsed 3 months later with the kinase domain mutation T315I. Multiple therapeutic approaches including ponatinib, blinatumomab, a 2nd HSCT from a different donor, donor lymphocyte infusions, and high-dose asciminib all resulted in subsequent relapse. Another molecular response was achieved by combining ponatinib plus asciminib with chemotherapy. In this situation, CD19-directed CAR-T cells (Kymriah®) were administered for compassionate use and tolerated without adverse events. Compared to all prior therapies, CAR T-cells maintained remission. After 12 months of follow-up, complete B-cell aplasia and low numbers of CAR-T cells are detectable in the peripheral blood, potentially mediating long-term disease control.

3.
Biotechnol Bioeng ; 121(10): 3252-3268, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-38963234

RESUMO

Bringing effective cancer therapy in the form of chimeric antigen receptor technology to untapped markets faces numerous challenges, including a global shortage of therapeutic lentiviral or retroviral vectors on which all current clinical therapies using genetically modified T cells are based. Production of these lentiviral vectors in academic settings in principle opens the way to local production of therapeutic cells, which is the only economically viable approach to make this therapy available to patients in developing countries. The conditions for obtaining and concentrating lentiviral vectors have been optimized and described. The calcium phosphate precipitation method was found to be suitable for transfecting high cell-density cultures, a prerequisite for high titers. We describe protocols for gradually increasing production from 6-well plates to P100 plates, T-175 flasks, and 5-layer stacks while maintaining high titers, >108 transducing units. Concentration experiments using ultracentrifugation revealed the advantage of lower centrifugation speeds compared to competing protocols. The resulting batches of lentiviral vectors had a titer of 1010 infectious particles and were used to transduce primary human T lymphocytes generating chimeric antigen receptor T cells, the quality of which was checked and found potential applicability for treatment.


Assuntos
Vetores Genéticos , Lentivirus , Receptores de Antígenos Quiméricos , Linfócitos T , Lentivirus/genética , Humanos , Vetores Genéticos/genética , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Imunoterapia Adotiva/métodos , Transdução Genética
4.
Pediatr Nephrol ; 39(8): 2495-2503, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38507119

RESUMO

BACKGROUND: CD19-specific chimeric antigen receptor (CAR) T-cell therapy has shown promising disease responses in patients with high-risk B-cell malignancies. However, its use may be related to complications such as immune-mediated complications, infections, and end-organ dysfunction. The incidence of post-CAR T-cell therapy acute kidney injury (AKI) in the children, adolescent, and young adult (CAYA) patient population is largely unreported. METHODS: The objectives of this study were to determine the incidence of AKI in CAYA patients with high-risk B-cell malignancies treated with CD19-CAR T-cell therapy, evaluate potential risk factors for developing AKI, and determine patterns of kidney function recovery. We conducted a retrospective analysis of 34 CAYA patients treated with CD19-CAR T-cell at a single institution. RESULTS: There was a cumulative incidence of any grade AKI by day 30 post-infusion of 20% (n = 7), with four cases being severe AKI (stages 2-3) and one patient requiring kidney replacement therapy. All episodes of AKI developed within the first 14 days after receiving CAR T-cell therapy and 50% of patients with AKI recovered kidney function to baseline within 30 days post-infusion. No evaluated pre-treatment risk factors were associated with the development of subsequent AKI; there was an association between AKI and cytokine release syndrome and neurotoxicity. We conclude that the risk of developing AKI following CD19-CAR T-cell therapy is highest early post-infusion, with most cases of AKI being severe. CONCLUSIONS: Frequent monitoring to facilitate early recognition and subsequent management of kidney complications after CD19-CAR T-cell therapy may reduce the severity of AKI in the CAYA patient population.


Assuntos
Injúria Renal Aguda , Antígenos CD19 , Imunoterapia Adotiva , Humanos , Injúria Renal Aguda/terapia , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/imunologia , Injúria Renal Aguda/epidemiologia , Adolescente , Masculino , Feminino , Estudos Retrospectivos , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Criança , Adulto Jovem , Incidência , Pré-Escolar , Antígenos CD19/imunologia , Fatores de Risco , Adulto , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/complicações , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos Quiméricos/imunologia
5.
Mol Ther ; 31(7): 2089-2104, 2023 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-36945773

RESUMO

CAR T cells recognizing CD19 effectively treat relapsed and refractory B-ALL and DLBCL. However, CD19 loss is a frequent cause of relapse. Simultaneously targeting a second antigen, CD22, may decrease antigen escape, but is challenging: its density is approximately 10-fold less than CD19, and its large structure may hamper immune synapse formation. The characteristics of the optimal CD22 CAR are underexplored. We generated 12 distinct CD22 antibodies and tested CARs derived from them to identify a CAR based on the novel 9A8 antibody, which was sensitive to low CD22 density and lacked tonic signaling. We found no correlation between affinity or membrane proximity of recognition epitope within Ig domains 3-6 of CD22 with CART function. The optimal strategy for CD19/CD22 CART co-targeting is undetermined. Co-administration of CD19 and CD22 CARs is costly; single CARs targeting CD19 and CD22 are challenging to construct. The co-expression of two CARs has previously been achieved using bicistronic vectors. Here, we generated a dual CART product by co-transduction with 9A8-41BBζ and CAT-41BBζ (obe-cel), the previously described CD19 CAR. CAT/9A8 CART eliminated single- and double-positive target cells in vitro and eliminated CD19- tumors in vivo. CAT/9A8 CART is being tested in a phase I clinical study (NCT02443831).


Assuntos
Linfoma de Burkitt , Receptores de Antígenos Quiméricos , Humanos , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T , Recidiva Local de Neoplasia , Imunoterapia Adotiva , Proteínas Adaptadoras de Transdução de Sinal , Antígenos CD19 , Anticorpos , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico
6.
Int J Mol Sci ; 25(1)2024 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-38203814

RESUMO

Cluster of differentiation 19 (CD19) chimeric antigen receptor (CAR) T cells are a highly effective immunotherapy for relapsed and refractory B-cell malignancies, but their utility can be limited by the development of immune effector cell-associated neurotoxicity syndrome (ICANS). The recent discovery of CD19 expression on the pericytes in the blood-brain barrier (BBB) suggests an important off-target mechanism for ICANS development. In addition, the release of systemic cytokines stimulated by the engagement of CD19 with the CAR T cells can cause endothelial activation and decreased expression of tight junction molecules, further damaging the integrity of the BBB. Once within the brain microenvironment, cytokines trigger a cytokine-specific cascade of neuroinflammatory responses, which manifest clinically as a spectrum of neurological changes. Brain imaging is frequently negative or nonspecific, and treatment involves close neurologic monitoring, supportive care, interleukin antagonists, and steroids. The goal of this review is to inform readers about the normal development and microstructure of the BBB, its unique susceptibility to CD19 CAR T cells, the role of individual cytokines on specific elements of the brain's microstructural environment, and the clinical and imaging manifestations of ICANS. Our review will link cellular pathophysiology with the clinical and radiological manifestations of a complex clinical entity.


Assuntos
Barreira Hematoencefálica , Síndromes Neurotóxicas , Humanos , Encéfalo/diagnóstico por imagem , Antígenos CD19 , Citocinas , Permeabilidade , Linfócitos T
7.
Rinsho Ketsueki ; 65(9): 1164-1173, 2024.
Artigo em Japonês | MEDLINE | ID: mdl-39358274

RESUMO

Tisagenlecleucel, a commercially available CD19-targeted CAR-T cell product, has dramatically changed the treatment of relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). Tisagenlecleucel infusion has been linked to distinct acute adverse events, including cytokine release syndrome, neurotoxicity, hemophagocytic lymphohistiocytosis and prolonged pancytopenia, which are rare with cytocidal chemotherapy. In addition, recent retrospective studies have revealed pre-infusion prognostic factors including high tumor burden (bone marrow leukemia cell fraction ≥5%) and non-response to blinatumomab, another CD19-targeting agent. Not only physicians providing CAR-T cell therapy but also those referring patients for this therapy should thoroughly understand the indications and limitations, characteristic acute complications, pre-treatment factors affecting prognosis, and late complications. This article outlines the current understanding regarding the use of tisagenlecleucel in children and adolescents with B-ALL.


Assuntos
Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Criança , Adolescente , Imunoterapia Adotiva , Receptores de Antígenos de Linfócitos T
8.
Br J Haematol ; 202(1): 65-73, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37082780

RESUMO

Large B-cell lymphoma (LBCL) patients with comorbidities and/or advanced age are increasingly considered for treatment with CD19 CAR T, but data on the clinical benefit of CAR T in the less fit patient population are still limited. We analysed outcomes of consecutive patients approved for treatment with axicabtagene ciloleucel (axi-cel) or tisagenlecleucel (tisa-cel) by the UK National CAR T Clinical Panel, according to fitness for autologous stem cell transplant (ASCT). 81/404 (20%) of approved patients were deemed unfit for ASCT. Unfit patients were more likely to receive tisa-cel versus axi-cel (52% vs. 48%) compared to 20% versus 80% in ASCT-fit patients; p < 0.0001. The drop-out rate from approval to infusion was significantly higher in the ASCT-unfit group (34.6% vs. 23.5%; p = 0.042). Among infused patients, response rate, progression-free and overall survival were similar in both cohorts. CAR T was well-tolerated in ASCT-unfit patients with an incidence of grade ≥3 cytokine release syndrome and neurotoxicity of 2% and 11%, respectively. Results from this multicentre real-world cohort demonstrate that CD19 CAR T can be safely delivered in carefully selected older patients and patients with comorbidities who are not deemed suitable for transplant.


Assuntos
Linfoma Difuso de Grandes Células B , Receptores de Antígenos Quiméricos , Transplantes , Humanos , Autoenxertos , Transplante Autólogo , Proteínas Adaptadoras de Transdução de Sinal , Antígenos CD19 , Síndrome da Liberação de Citocina , Linfoma Difuso de Grandes Células B/terapia , Imunoterapia Adotiva/efeitos adversos
9.
Ann Hematol ; 102(1): 155-165, 2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36394582

RESUMO

The complete remission (CR) rate and overall survival (OS) of relapsed/refractory (R/R) B-cell acute lymphoblastic leukaemia (B-ALL) are not satisfactory. The available salvage regimens include standard chemotherapy, inotuzumab ozogamicin, blinatumomab and cluster of differentiation (CD)19 chimeric antigen receptor T cells (CAR T), and the NCCN guidelines recommend all of these therapies with no preference. Dual CD19/CD22 CAR T-cells have emerged as new treatments and have shown some efficacy, with high CR rates and preventing CD19-negative relapse. However, direct comparisons of the CR rate and long-term survival among the different salvage therapies are lacking. Databases including PubMed, Embase, Web of Science and Cochrane were searched from inception to January 31, 2022, for relevant studies. The outcomes of interest were complete remission/complete remission with incomplete haematologic recovery (CR/CRi) rates and 1-year overall survival (OS) rates. Odds ratios (ORs) were generated for binary outcomes, and the mean difference (MD) was generated for consecutive outcomes by network meta-analysis. CD19 CAR T-cells demonstrated a significantly better effect in improving the CR/CRi rate than blinatumomab (OR = 8.32, 95% CI: 1.18 to 58.44) and chemotherapy (OR = 16.4, 95% CI: 2.76 to 97.45). In terms of OS, CD19 CAR T-cells and dual CD19/CD22 CAR T-cells both had a higher 1-year OS rate than blinatumomab, inotuzumab ozogamicin and chemotherapy. There was no significant difference between CD19 CAR T-cells and dual CD19/CD22 CAR T-cells in terms of 1-year OS and CR/CRi rates. CD19 CAR T-cells are effective in inducing CR, and CD19 CAR T-cells and dual CD19/CD22 CAR T-cells show benefits for overall survival. More high-quality randomized controlled trials and longer follow-ups are needed to confirm and update the results of this analysis in the future.


Assuntos
Recidiva Local de Neoplasia , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Inotuzumab Ozogamicina/uso terapêutico , Metanálise em Rede , Recidiva Local de Neoplasia/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Indução de Remissão , Antígenos CD19
10.
Transpl Infect Dis ; 25 Suppl 1: e14191, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37987114

RESUMO

BACKGROUND: CD19 chimeric antigen receptor (CAR)-T cell therapy has emerged as an effective treatment in those with refractory or relapsed lymphoma. CD19 CAR-T cell therapy can cause direct and indirect toxic adverse effects and increased risk for infection. Infectious complications and optimal antimicrobial prophylaxis strategies are an ongoing area of investigation. METHODS: A single-center retrospective cohort study was conducted to review recipients of CD19 CAR-T cell therapy between April 2018 and December 2020. Patient characteristics and clinical outcomes were extracted from the electronic health records. RESULTS: Infectious complications were identified in 18/50 (36%) recipients with 31 episodes of infection. The median time to infection was 225 days (range 0-614). Bacterial infections were most common with bloodstream infection followed by sinusitis and skin and soft tissue infection. Eight viral infections were identified, most being respiratory viral illnesses. Two fungal infections were identified: Pneumocystis jirovecii pneumonia (PJP) and disseminated fusariosis. Seventeen infections (54.8%) were classified as severe: leading to death, requiring hospitalization, need for empiric intravenous antibiotics, or significant alteration in hospital course. No characteristics were found to be statistically significant risks for infection, although a trend toward significance was seen in prior autologous stem cell transplant recipients (p = .12) and those with recurrent neutropenia (p = .14). Three patients (6%) died from infection. CONCLUSION: Infections were common after CD19 CAR-T cell therapy and occurred beyond the first year. Further multicenter studies are needed to define infectious risks and optimize antimicrobial prophylaxis recommendations in recipients of CD19 CAR-T cell therapy.


Assuntos
Receptores de Antígenos Quiméricos , Humanos , Anti-Infecciosos , Antígenos CD19 , Terapia Baseada em Transplante de Células e Tecidos , Imunoterapia Adotiva/efeitos adversos , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Estudos Retrospectivos
11.
Mol Ther ; 30(2): 534-549, 2022 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-34628050

RESUMO

We generated dual-antigen receptor (DR) T cells from induced pluripotent stem cells (iPSCs) to mitigate tumor antigen escape. These cells were engineered to express a chimeric antigen receptor (CAR) for the antigen cell surface latent membrane protein 1 (LMP1; LMP1-CAR) and a T cell receptor directed to cell surface latent membrane protein 2 (LMP2), in association with human leucocyte antigen A24, to treat therapy-refractory Epstein-Barr virus-associated lymphomas. We introduced LMP1-CAR into iPSCs derived from LMP2-specific cytotoxic T lymphocytes (CTLs) to generate rejuvenated CTLs (rejTs) active against LMP1 and LMP2, or DRrejTs. All DRrejT-treated mice survived >100 days. Furthermore, DRrejTs rejected follow-up inocula of lymphoma cells, demonstrating that DRrejTs persisted long-term. We also demonstrated that DRrejTs targeting CD19 and LMP2 antigens exhibited a robust tumor suppressive effect and conferred a clear survival advantage. Co-operative antitumor effect and in vivo persistence, with unlimited availability of DRrejT therapy, will provide powerful and sustainable T cell immunotherapy.


Assuntos
Infecções por Vírus Epstein-Barr , Células-Tronco Pluripotentes Induzidas , Linfoma , Receptores de Antígenos Quiméricos , Animais , Terapia Baseada em Transplante de Células e Tecidos , Herpesvirus Humano 4/genética , Imunoterapia Adotiva , Células-Tronco Pluripotentes Induzidas/metabolismo , Linfoma/genética , Linfoma/terapia , Camundongos , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T Citotóxicos , Proteínas da Matriz Viral/genética
12.
Immunopharmacol Immunotoxicol ; 45(6): 709-718, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37382435

RESUMO

BACKGROUND: Multiple targets of chimeric antigen receptor T cells (CAR-T cells) are shared expressed by tumor cells and T cells, these self-antigens may stimulate CAR-T cells continuously during the expansion. Persistent exposure to antigens is considered to cause metabolic reprogramming of T cells and the metabolic profiling is critical in determining the cell fate and effector function of CAR-T cells. However, whether the stimulation of self-antigens during CAR-T cell generation could remodel the metabolic profiling is unclear. In this study, we aim to investigate the metabolic characteristics of CD26 CAR-T cells, which expressed CD26 antigens themselves. METHODS: The mitochondrial biogenesis of CD26 and CD19 CAR-T cells during expansion was evaluated by the mitochondrial content, mitochondrial DNA copy numbers and genes involved in mitochondrial regulation. The metabolic profiling was investigated by the ATP production, mitochondrial quality and the expression of metabolism-related genes. Furthermore, we assessed the phenotypes of CAR-T cells through memory-related markers. RESULTS: We reported that CD26 CAR-T cells had elevated mitochondrial biogenesis, ATP production and oxidative phosphorylation at early expansion stage. However, the mitochondrial biogenesis, mitochondrial quality, oxidative phosphorylation and glycolytic activity were all weakened at later expansion stage. On the contrary, CD19 CAR-T cells did not exhibit such characteristics. CONCLUSION: CD26 CAR-T cells showed distinctive metabolic profiling during expansion that was extremely unfavorable to cell persistence and function. These findings may provide new insights for the optimization of CD26 CAR-T cells in terms of metabolism.


Assuntos
Glicólise , Mitocôndrias , Biogênese de Organelas , Linfócitos T , Linfócitos T/citologia , Linfócitos T/metabolismo , Dipeptidil Peptidase 4 , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Metaboloma , Humanos , Células Cultivadas , Espécies Reativas de Oxigênio/metabolismo , Metaloproteinases da Matriz/metabolismo , Receptores de Antígenos de Linfócitos T
13.
J Allergy Clin Immunol ; 149(5): 1525-1560, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35176351

RESUMO

Secondary hypogammaglobulinemia (SHG) is characterized by reduced immunoglobulin levels due to acquired causes of decreased antibody production or increased antibody loss. Clarification regarding whether the hypogammaglobulinemia is secondary or primary is important because this has implications for evaluation and management. Prior receipt of immunosuppressive medications and/or presence of conditions associated with SHG development, including protein loss syndromes, are histories that raise suspicion for SHG. In patients with these histories, a thorough investigation of potential etiologies of SHG reviewed in this report is needed to devise an effective treatment plan focused on removal of iatrogenic causes (eg, discontinuation of an offending drug) or treatment of the underlying condition (eg, management of nephrotic syndrome). When iatrogenic causes cannot be removed or underlying conditions cannot be reversed, therapeutic options are not clearly delineated but include heightened monitoring for clinical infections, supportive antimicrobials, and in some cases, immunoglobulin replacement therapy. This report serves to summarize the existing literature regarding immunosuppressive medications and populations (autoimmune, neurologic, hematologic/oncologic, pulmonary, posttransplant, protein-losing) associated with SHG and highlights key areas for future investigation.


Assuntos
Agamaglobulinemia , Imunodeficiência de Variável Comum , Síndromes de Imunodeficiência , Agamaglobulinemia/diagnóstico , Agamaglobulinemia/etiologia , Agamaglobulinemia/terapia , Imunodeficiência de Variável Comum/complicações , Humanos , Doença Iatrogênica , Imunidade , Imunoglobulinas , Síndromes de Imunodeficiência/complicações , Síndromes de Imunodeficiência/diagnóstico , Síndromes de Imunodeficiência/terapia
14.
Int J Mol Sci ; 24(13)2023 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-37445684

RESUMO

Natural killer (NK) cells are a part of innate immunity that can be activated rapidly in response to malignant transformed cells without prior sensitization. Engineering NK cells to express chimeric antigen receptors (CARs) allows them to be directed against corresponding target tumor antigens. CAR-NK cells are regarded as a promising candidate for cellular immunotherapy alternatives to conventional CAR-T cells, due to the relatively low risk of graft-versus-host disease and safer clinical profile. Human induced pluripotent stem cells (iPSCs) are a promising renewable cell source of clinical NK cells. In the present study, we successfully introduced a third-generation CAR targeting CD19, which was validated to have effective signaling domains suitable for NK cells, into umbilical cord blood NK-derived iPSCs, followed by a single-cell clone selection and thorough iPSC characterization. The established single-cell clone of CAR19-NK/iPSCs, which is highly desirable for clinical application, can be differentiated using serum- and feeder-free protocols into functional CAR19-iNK-like cells with improved anti-tumor activity against CD19-positive hematologic cancer cells when compared with wild-type (WT)-iNK-like cells. With the feasibility of being an alternative source for off-the-shelf CAR-NK cells, a library of single-cell clones of CAR-engineered NK/iPSCs targeting different tumor antigens may be created for future clinical application.


Assuntos
Células-Tronco Pluripotentes Induzidas , Receptores de Antígenos Quiméricos , Humanos , Receptores de Antígenos Quiméricos/genética , Imunoterapia Adotiva , Células Matadoras Naturais , Antígenos de Neoplasias
15.
Mol Ther ; 29(9): 2677-2690, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-33940156

RESUMO

Adoptively transferred CD19 chimeric antigen receptor (CAR) T cells have led to impressive clinical outcomes in B cell malignancies. Beyond induction of remission, the persistence of CAR-T cells is required to prevent relapse and provide long-term disease control. To improve CAR-T cell function and persistence, we developed a composite co-stimulatory domain of a B cell signaling moiety, CD79A/CD40, to induce a nuclear translocating signal, NF-κB, to synergize with other T cell signals and improve CAR-T cell function. CD79A/CD40 incorporating CD19CAR-T cells (CD19.79a.40z) exhibited higher NF-κB and p38 activity upon CD19 antigen exposure compared with the CD28 or 4-1BB incorporating CD19CAR-T cells (CD19.28z and CD19.BBz). Notably, we found that CD19.79a.40z CAR-T cells continued to suppress CD19+ target cells throughout the co-culture assay, whereas a tendency for tumor growth was observed with CD19.28z CAR-T cells. Moreover, CD19.79a.40z CAR-T cells exhibited robust T cell proliferation after culturing with CD19+ target cells, regardless of exogenous interleukin-2. In terms of in vivo efficiency, CD19.79a.40z demonstrated superior anti-tumor activity and in vivo CAR-T cell proliferation compared with CD19.28z and CD19.BBz CD19CAR-T cells in Raji-inoculated mice. Our data demonstrate that the CD79A/CD40 co-stimulatory domain endows CAR-T cells with enhanced proliferative capacity and improved anti-tumor efficacy in a murine model.


Assuntos
Antígenos CD19/imunologia , Antígenos CD40/metabolismo , Antígenos CD79/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Receptores de Antígenos Quiméricos/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Técnicas de Cocultura , Humanos , Imunoterapia Adotiva , Células K562 , Ativação Linfocitária , Camundongos , NF-kappa B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Int J Mol Sci ; 23(21)2022 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-36361619

RESUMO

Acute myeloid leukemia (AML) and B-cell acute lymphocytic leukemia (B-ALL) are severe blood malignancies affecting both adults and children. Chimeric antigen receptor (CAR)-based immunotherapies have proven highly efficacious in the treatment of leukemia. However, the challenge of the immune escape of cancer cells remains. The development of more affordable and ready-to-use therapies is essential in view of the costly and time-consuming preparation of primary cell-based treatments. In order to promote the antitumor function against AML and B-ALL, we transduced NK-92 cells with CD276-CAR or CD19-CAR constructs. We also attempted to enhance cytotoxicity by a gene knockout of three different inhibitory checkpoints in NK cell function (CBLB, NKG2A, TIGIT) with CRISPR-Cas9 technology. The antileukemic activity of the generated cell lines was tested with calcein and luciferase-based cytotoxicity assays in various leukemia cell lines. Both CAR-NK-92 exhibited targeted cytotoxicity and a significant boost in antileukemic function in comparison to parental NK-92. CRISPR-Cas9 knock-outs did not improve B-ALL cytotoxicity. However, triple knock-out CD276-CAR-NK-92 cells, as well as CBLB or TIGIT knock-out NK-92 cells, showed significantly enhanced cytotoxicity against U-937 or U-937 CD19/tag AML cell lines. These results indicate that the CD19-CAR and CD276-CAR-NK-92 cell lines' cytotoxic performance is suitable for leukemia killing, making them promising off-the-shelf therapeutic candidates. The knock-out of CBLB and TIGIT in NK-92 and CD276-CAR-NK-92 should be further investigated for the treatment of AML.


Assuntos
Leucemia Mieloide Aguda , Linfoma de Células B , Leucemia-Linfoma Linfoblástico de Células Precursoras , Receptores de Antígenos Quiméricos , Humanos , Antígenos CD19 , Antígenos B7/metabolismo , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Imunoterapia Adotiva/métodos , Células Matadoras Naturais , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/terapia , Leucemia Mieloide Aguda/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo
17.
Chin J Cancer Res ; 34(1): 53-62, 2022 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-35355931

RESUMO

Objective: Previous studies reported that 4-1BB-based CD19 chimeric antigen receptor (CAR)-T cells were more beneficial for the clinical outcomes than CD28-based CAR-T cells, especially the lower incidence rate of severe adverse events. However, the median progression-free survival (mPFS) of 4-1BB-based product Kymriah was shorter than that of CD28-based Yescarta (2.9 monthsvs. 5.9 months), suggesting that Kymriah was limited in the long-term efficacy. Thus, a safe and durable 4-1BB-based CD19 CAR-T needs to be developed. Methods: We designed a CD19-targeted CAR-T (named as IM19) which consisted of an FMC63 scFv, 4-1BB and CD3ζ intracellular domain and was manufactured into a memory T-enriched formulation. A phase I/II clinical trial was launched to evaluate the clinical outcomes of IM19 in relapsed or refractory (r/r) B cell non-Hodgkin lymphoma (B-NHL). Dose-escalation investigation (at a dose of 5×105/kg, 1×106/kg and 3×106/kg) was performed in 22 r/r B-NHL patients. All patients received a single infusion of IM19 after 3-day conditional regimen. Results: At month 3, the overall response rate (ORR) was 59.1%, the complete response rate (CRR) was 50.0%. The mPFS was 6 months and the 1-year overall survival rate was 77.8%. Cytokine release syndrome (CRS) occurred in 13 patients (59.1%), with 54.5% of grade 1-2 CRS. Only one patient (4.5%) experienced grade 3 CRS and grade 3 neurotoxicity. Conclusions: These results demonstrated the safety and durable efficacy of a 4-1BB-based CD19 CAR-T, IM19, which is promising for further development and clinical investigation.

18.
J Cell Mol Med ; 25(15): 7451-7461, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34245101

RESUMO

In recent years, chimeric antigen receptor T (CAR T)-cell therapy has shown great potential in treating haematologic disease, but no breakthrough has been achieved in solid tumours. In order to clarify the antitumour mechanism of CAR T cell in solid tumours, the pharmacokinetic (PK) and pharmacodynamic (PD) investigations of CD19 CAR T cell were performed in human leukaemic xenograft mouse models. For PK investigation, we radiolabelled CD19 CAR T cell with 89 Zr and used PET imaging in the CD19-positive and the CD19-negative K562-luc animal models. For PD evaluation, optical imaging, tumour volume measurement and DNA copy-number detection were performed. Unfortunately, the qPCR results of the DNA copy number in the blood were below the detection limit. The tumour-specific uptake was higher in the CD19-positive model than in the CD19-negative model, and this was consistent with the PD results. The preliminary PK and PD studies of CD19 CAR T cell in solid tumours are instructive. Considering the less efficiency of CAR T-cell therapy of solid tumours with the limited number of CAR T cells entering the interior of solid tumours, this study is suggestive for the subsequent CAR T-cell design and evaluation of solid tumour therapy.


Assuntos
Antígenos CD19/imunologia , Imunoterapia Adotiva/métodos , Leucemia Experimental/terapia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Feminino , Humanos , Células K562 , Leucemia Experimental/diagnóstico por imagem , Camundongos , Camundongos Endogâmicos NOD , Imagem Multimodal/métodos , Imagem Óptica/métodos , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Zircônio/química
19.
Cancer Immunol Immunother ; 70(7): 1979-1993, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33416942

RESUMO

INTRODUCTION: Although recent clinical trials have demonstrated the efficacy of CD19-directed chimeric antigen receptor (CAR) T-cell therapy for refractory or relapsed B acute lymphoblastic leukemia (r/r B-ALL), most trials exclude patients with high-burden CNS leukemia (CNSL) to avoid the risk of severe neurotoxicity. There were only sparse cases describing the effect of CAR T cells on low-burden CNSL, and the safety and effectiveness of CAR T cells in high-burden CNSL remains unknown. METHODS: Here, we retrospectively analyzed the results of CD19 CAR T-cell therapy in 12 pediatric patients that had low (Blasts < 20/µL in CSF) or high-burdens (Blasts or intracranial solid mass) of CNS B-ALL, that are enrolled in three clinical trials and one pilot study at Beijing Boren Hospital RESULTS: Eleven patients (91.7%) achieved complete remission (CR) on day 30, and one patient got CR on day 90 after infusion. Most patient experienced mild cytokine-release syndrome. However, of the five patients who retained > 5/µL blasts in CSF or a solid mass before CAR T-cell expansion, four developed severe (grade 3-4) neurotoxicity featured by persistent cerebral edema and seizure, and they fully recovered after intensive managements. Sustained remission was achieved in 9 of the 12 patients, resulted in a 6-month leukemia-free survival rate of 81.8% (95% CI 59.0-100). Only one patient has CNS relapse again. CONCLUSION: Our study demonstrates that CAR T cells are effective in clearing both low- and high-burden CNSL, but a high CNSL burden before CAR T-cell expansion may cause severe neurotoxicity requiring intense intervention.


Assuntos
Antígenos CD19/imunologia , Neoplasias do Sistema Nervoso Central/tratamento farmacológico , Síndrome da Liberação de Citocina/patologia , Imunoterapia Adotiva/efeitos adversos , Síndromes Neurotóxicas/patologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Adolescente , Neoplasias do Sistema Nervoso Central/imunologia , Neoplasias do Sistema Nervoso Central/patologia , Criança , Pré-Escolar , Síndrome da Liberação de Citocina/etiologia , Feminino , Seguimentos , Humanos , Lactente , Masculino , Síndromes Neurotóxicas/etiologia , Projetos Piloto , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida
20.
Cytotherapy ; 23(9): 852-860, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-33941482

RESUMO

BACKGROUND AIMS: Next-generation immune cell therapy products will require complex modifications using engineering technologies that can maintain high levels of cell functionality. Non-viral engineering methods have the potential to address limitations associated with viral vectors. However, while electroporation is the most widely used non-viral modality, concerns about its effects on cell functionality have led to the exploration of alternative approaches. Here the authors have examined the suitability of the Solupore non-viral delivery system for engineering primary human T cells for cell therapy applications. METHODS: The Solupore system was used to deliver messenger RNA (mRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) guide RNA ribonucleoprotein (RNP) cargos to T cells, and efficiency was measured by flow cytometry. Cell perturbation was assessed by immune gene expression profiling, including an electroporation comparator. In vitro and in vivo cytotoxicity of chimeric antigen receptor (CAR) T cells generated using the Solupore system was evaluated using a real-time cellular impedance assay and a Raji-luciferase mouse tumor model, respectively. RESULTS: Efficient transfection was demonstrated through delivery of mRNA and CRISPR CAS9 RNP cargos individually, simultaneously and sequentially using the Solupore system while consistently maintaining high levels of cell viability. Gene expression profiling revealed minimal alteration in immune gene expression, demonstrating the low level of perturbation experienced by the cells during this transfection process. By contrast, electroporation resulted in substantial changes in immune gene expression in T cells. CAR T cells generated using the Solupore system exhibited efficient cytotoxicity against target cancer cells in vitro and in vivo. CONCLUSIONS: The Solupore system is a non-viral means of simply, rapidly and efficiently delivering cargos to primary human immune cells with retention of high cell viability and functionality.


Assuntos
Vetores Genéticos , Linfócitos T , Animais , Terapia Baseada em Transplante de Células e Tecidos , Eletroporação , Humanos , Camundongos , Transfecção
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