RESUMO
X-linked chronic granulomatous disease (X-CGD) is a primary immunodeficiency caused by mutations in the CYBB gene, resulting in the inability of phagocytic cells to eliminate infections. To design a lentiviral vector (LV) capable of recapitulating the endogenous regulation and expression of CYBB, a bioinformatics-guided approach was used to elucidate the cognate enhancer elements regulating the native CYBB gene. Using this approach, we analyzed a 600-kilobase topologically associated domain of the CYBB gene and identified endogenous enhancer elements to supplement the CYBB promoter to develop MyeloVec, a physiologically regulated LV for the treatment of X-CGD. When compared with an LV currently in clinical trials for X-CGD, MyeloVec showed improved expression, superior gene transfer to hematopoietic stem and progenitor cells (HSPCs), corrected an X-CGD mouse model leading to complete protection against Burkholderia cepacia infection, and restored healthy donor levels of antimicrobial oxidase activity in neutrophils derived from HSPCs from patients with X-CGD. Our findings validate the bioinformatics-guided design approach and have yielded a novel LV with clinical promise for the treatment of X-CGD.
Assuntos
Doença Granulomatosa Crônica , Animais , Camundongos , Doença Granulomatosa Crônica/genética , Doença Granulomatosa Crônica/terapia , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , NADPH Oxidase 2/genética , Terapia Genética/métodos , MutaçãoRESUMO
Bispecific antibodies are an important tool for the management and treatment of acute leukemias. As a next step toward clinical translation of engineered plasma cells, we describe approaches for secretion of bispecific antibodies by human plasma cells. We show that human plasma cells expressing either fragment crystallizable domain-deficient anti-CD19 × anti-CD3 (blinatumomab) or anti-CD33 × anti-CD3 bispecific antibodies mediate T cell activation and direct T cell killing of B acute lymphoblastic leukemia or acute myeloid leukemia cell lines in vitro. We demonstrate that knockout of the self-expressed antigen, CD19, boosts anti-CD19-bispecific secretion by plasma cells and prevents self-targeting. Plasma cells secreting anti-CD19-bispecific antibodies elicited in vivo control of acute lymphoblastic leukemia patient-derived xenografts in immunodeficient mice co-engrafted with autologous T cells. In these studies, we found that leukemic control elicited by engineered plasma cells was similar to CD19-targeted chimeric antigen receptor-expressing T cells. Finally, the steady-state concentration of anti-CD19 bispecifics in serum 1 month after cell delivery and tumor eradication was comparable with that observed in patients treated with a steady-state infusion of blinatumomab. These findings support further development of ePCs for use as a durable delivery system for the treatment of acute leukemias, and potentially other cancers.
Assuntos
Anticorpos Biespecíficos , Antígenos CD19 , Plasmócitos , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Anticorpos Biespecíficos/farmacologia , Animais , Camundongos , Antígenos CD19/imunologia , Antígenos CD19/genética , Antígenos CD19/metabolismo , Plasmócitos/metabolismo , Plasmócitos/imunologia , Linhagem Celular Tumoral , Linfócitos T/imunologia , Linfócitos T/metabolismo , Complexo CD3/imunologia , Complexo CD3/metabolismo , Complexo CD3/genética , Ativação Linfocitária/imunologia , Citotoxicidade ImunológicaRESUMO
BACKGROUND: Preclinical studies suggest that bb2121, a chimeric antigen receptor (CAR) T-cell therapy that targets B-cell maturation antigen (BCMA), has potential for the treatment of multiple myeloma. METHODS: In this phase 1 study involving patients with relapsed or refractory multiple myeloma, we administered bb2121 as a single infusion at doses of 50×106, 150×106, 450×106, or 800×106 CAR-positive (CAR+) T cells in the dose-escalation phase and 150×106 to 450×106 CAR+ T cells in the expansion phase. Patients had received at least three previous lines of therapy, including a proteasome inhibitor and an immunomodulatory agent, or were refractory to both drug classes. The primary end point was safety. RESULTS: Results for the first 33 consecutive patients who received a bb2121 infusion are reported. The data-cutoff date was 6.2 months after the last infusion date. Hematologic toxic effects were the most common events of grade 3 or higher, including neutropenia (in 85% of the patients), leukopenia (in 58%), anemia (in 45%), and thrombocytopenia (in 45%). A total of 25 patients (76%) had cytokine release syndrome, which was of grade 1 or 2 in 23 patients (70%) and grade 3 in 2 patients (6%). Neurologic toxic effects occurred in 14 patients (42%) and were of grade 1 or 2 in 13 patients (39%). One patient (3%) had a reversible grade 4 neurologic toxic effect. The objective response rate was 85%, including 15 patients (45%) with complete responses. Six of the 15 patients who had a complete response have had a relapse. The median progression-free survival was 11.8 months (95% confidence interval, 6.2 to 17.8). All 16 patients who had a response (partial response or better) and who could be evaluated for minimal residual disease (MRD) had MRD-negative status (≤10-4 nucleated cells). CAR T-cell expansion was associated with responses, and CAR T cells persisted up to 1 year after the infusion. CONCLUSIONS: We report the initial toxicity profile of a BCMA-directed cellular immunotherapy for patients with relapsed or refractory multiple myeloma. Antitumor activity was documented. (Funded by Bluebird Bio and Celgene; CRB-401 ClinicalTrials.gov number, NCT02658929.).
Assuntos
Antígeno de Maturação de Linfócitos B/antagonistas & inibidores , Imunoterapia Adotiva , Mieloma Múltiplo/terapia , Receptores de Antígenos Quiméricos/uso terapêutico , Adulto , Idoso , Relação CD4-CD8 , Feminino , Humanos , Imunoterapia Adotiva/efeitos adversos , Infusões Intravenosas , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/imunologia , Intervalo Livre de Progressão , Linfócitos T/metabolismoRESUMO
ß-globin lentiviral vectors (ß-LV) have faced challenges in clinical translation for gene therapy of sickle cell disease (SCD) due to low titer and sub-optimal gene transfer to hematopoietic stem and progenitor cells (HSPCs). To overcome the challenge of preserving efficacious expression while increasing vector performance, we used published genomic and epigenomic data available through ENCODE to redefine enhancer element boundaries of the ß-globin locus control region (LCR) to construct novel ENCODE core sequences. These novel LCR elements were used to design a ß-LV of reduced proviral length, termed CoreGA-AS3-FB, produced at higher titers and possessing superior gene transfer to HSPCs when compared to the full-length parental ß-LV at equal MOI. At low vector copy number, vectors containing the ENCODE core sequences were capable of reversing the sickle phenotype in a mouse model of SCD. These studies provide a ß-LV that will be beneficial for gene therapy of SCD by significantly reducing the cost of vector production and extending the vector supply.
Assuntos
Anemia Falciforme/terapia , Terapia Genética/métodos , Vetores Genéticos , Lentivirus/genética , Região de Controle de Locus Gênico/genética , Transdução Genética/métodos , Globinas beta/genética , Animais , Células da Medula Óssea/metabolismo , Modelos Animais de Doenças , Células HEK293 , Voluntários Saudáveis , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Fenótipo , TransfecçãoRESUMO
Emerging gene-editing technologies are nearing a revolutionary phase in genetic medicine: precisely modifying or repairing causal genetic defects. This may include any number of DNA sequence manipulations, such as knocking out a deleterious gene, introducing a particular mutation, or directly repairing a defective sequence by site-specific recombination. All of these edits can currently be achieved via programmable rare-cutting endonucleases to create targeted DNA breaks that can engage and exploit endogenous DNA repair pathways to impart site-specific genetic changes. Over the past decade, several distinct technologies for introducing site-specific DNA breaks have been developed, yet the different biological origins of these gene-editing technologies bring along inherent differences in parameters that impact clinical implementation. This review aims to provide an accessible overview of the various endonuclease-based gene-editing platforms, highlighting the strengths and weakness of each with respect to therapeutic applications.
Assuntos
Endonucleases/metabolismo , Edição de Genes , Engenharia Genética , Terapia Genética , Genoma , Animais , Edição de Genes/história , Edição de Genes/métodos , Técnicas de Transferência de Genes , Engenharia Genética/história , Engenharia Genética/métodos , Terapia Genética/métodos , Vetores Genéticos/genética , História do Século XX , Humanos , Transdução GenéticaRESUMO
UNLABELLED: Adoptive transfer of CD8 T cells genetically engineered to express "chimeric antigen receptors" (CARs) represents a potential approach toward an HIV infection "functional cure" whereby durable virologic suppression is sustained after discontinuation of antiretroviral therapy. We describe a novel bispecific CAR in which a CD4 segment is linked to a single-chain variable fragment of the 17b human monoclonal antibody recognizing a highly conserved CD4-induced epitope on gp120 involved in coreceptor binding. We compared a standard CD4 CAR with CD4-17b CARs where the polypeptide linker between the CD4 and 17b moieties is sufficiently long (CD4-35-17b CAR) versus too short (CD4-10-17b) to permit simultaneous binding of the two moieties to a single gp120 subunit. When transduced into a peripheral blood mononuclear cell (PBMC) or T cells thereof, all three CD4-based CARs displayed specific functional activities against HIV-1 Env-expressing target cells, including stimulation of gamma interferon (IFN-γ) release, specific target cell killing, and suppression of HIV-1 pseudovirus production. In assays of spreading infection of PBMCs with genetically diverse HIV-1 primary isolates, the CD4-10-17b CAR displayed enhanced potency compared to the CD4 CAR whereas the CD4-35-17b CAR displayed diminished potency. Importantly, both CD4-17b CARs were devoid of a major undesired activity observed with the CD4 CAR, namely, rendering the transduced CD8(+) T cells susceptible to HIV-1 infection. Likely mechanisms for the superior potency of the CD4-10-17b CAR over the CD4-35-17b CAR include the greater potential of the former to engage in the serial antigen binding required for efficient T cell activation and the ability of two CD4-10-17b molecules to simultaneously bind a single gp120 subunit. IMPORTANCE: HIV research has been energized by prospects for a cure for HIV infection or, at least, for a "functional cure" whereby antiretroviral therapy can be discontinued without virus rebound. This report describes a novel CD4-based "chimeric antigen receptor" (CAR) which, when genetically engineered into T cells, gives them the capability to selectively respond to and kill HIV-infected cells. This CAR displays enhanced features compared to previously described CD4-based CARs, namely, increased potency and avoidance of the undesired rendering of the genetically modified CD8 T cells susceptible to HIV infection. When adoptively transferred back to the individual, the genetically modified T cells will hopefully provide durable killing of infected cells and sustained virus suppression without continued antiretroviral therapy, i.e., a functional cure.
Assuntos
Fármacos Anti-HIV/metabolismo , HIV-1/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , Receptores de Antígenos/metabolismo , Receptores de HIV/metabolismo , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Antígenos CD4/genética , Antígenos CD4/metabolismo , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Ligação Proteica , Receptores de Antígenos/genética , Receptores de HIV/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/metabolismo , Transdução GenéticaRESUMO
Programmed cell death-1 (PD-1) is expressed on activated T cells and represents an attractive target for gene-editing of tumor targeted T cells prior to adoptive cell transfer (ACT). We used zinc finger nucleases (ZFNs) directed against the gene encoding human PD-1 (PDCD-1) to gene-edit melanoma tumor infiltrating lymphocytes (TIL). We show that our clinical scale TIL production process yielded efficient modification of the PD-1 gene locus, with an average modification frequency of 74.8% (n = 3, range 69.9-84.1%) of the alleles in a bulk TIL population, which resulted in a 76% reduction in PD-1 surface-expression. Forty to 48% of PD-1 gene-edited cells had biallelic PD-1 modification. Importantly, the PD-1 gene-edited TIL product showed improved in vitro effector function and a significantly increased polyfunctional cytokine profile (TNFα, GM-CSF, and IFNγ) compared to unmodified TIL in two of the three donors tested. In addition, all donor cells displayed an effector memory phenotype and expanded approximately 500-2,000-fold in vitro. Thus, further study to determine the efficiency and safety of adoptive cell transfer using PD-1 gene-edited TIL for the treatment of metastatic melanoma is warranted.
Assuntos
Endorribonucleases/genética , Regulação Neoplásica da Expressão Gênica , Linfócitos do Interstício Tumoral/imunologia , Melanoma/terapia , Receptor de Morte Celular Programada 1/genética , Dedos de Zinco , Alelos , Animais , Separação Celular , Citocinas/metabolismo , Feminino , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Humanos , Memória Imunológica , Imunoterapia Adotiva , Interferon gama/metabolismo , Ativação Linfocitária/imunologia , Camundongos , Metástase Neoplásica , Transplante de Neoplasias , Fenótipo , Receptor de Morte Celular Programada 1/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
In this issue of Blood, Casucci et al present an elegant study that describes a potential new target for adoptive cell transfer (ACT), in this case CD44 splice variant 6 (CD44v6), and detail why it may be a good target for ACT and how to manage expected off-tumor/on-target toxicities.
Assuntos
Antígenos de Neoplasias/imunologia , Receptores de Hialuronatos/imunologia , Imunoterapia Adotiva , Leucemia Mieloide Aguda/terapia , Terapia de Alvo Molecular , Mieloma Múltiplo/terapia , Subpopulações de Linfócitos T/imunologia , Animais , HumanosRESUMO
Despite significant progress in the development of adoptive cell-transfer therapies (ACTs) using gene-engineered T cells, little is known about the fate of cells following infusion. To address that, we performed a comparative analysis of gene expression between T-cell receptor-engineered lymphocytes persisting in the circulation 1 month after administration and the product that was infused. We observed that 156 genes related to immune function were differentially expressed, including underexpression of stimulators of lymphocyte function and overexpression of inhibitory genes in postinfusion cells. Of genes overexpressed postinfusion, the product of programmed cell death 1 (PDCD1), coinhibitory receptor PD-1, was expressed at a higher percentage in postinfusion lymphocytes than in the infusion product. This was associated with a higher sensitivity to inhibition of cytokine production by interaction with its ligand PD-L1. Coinhibitory receptor CD160 was also overexpressed in persisting cells, and its expression was associated with decreased reactivity, which surprisingly was found to be ligand-independent. These results contribute to a deeper understanding of the properties of transgenic lymphocytes used to treat human malignancies and may provide a rationale for the development of combination therapies as a method to improve ACT.
Assuntos
Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Transferência Adotiva , Adulto , Animais , Antígenos CD/metabolismo , Antígenos de Neoplasias/metabolismo , Feminino , Citometria de Fluxo , Proteínas Ligadas por GPI/metabolismo , Engenharia Genética , Humanos , Ligantes , Masculino , Melanoma/sangue , Melanoma/tratamento farmacológico , Camundongos , Pessoa de Meia-Idade , Receptor de Morte Celular Programada 1/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores Imunológicos/metabolismo , Adulto JovemRESUMO
Immune targeting of B-cell malignancies using chimeric antigen receptors (CARs) is a promising new approach, but critical factors impacting CAR efficacy remain unclear. To test the suitability of targeting CD22 on precursor B-cell acute lymphoblastic leukemia (BCP-ALL), lymphoblasts from 111 patients with BCP-ALL were assayed for CD22 expression and all were found to be CD22-positive, with median CD22 expression levels of 3500 sites/cell. Three distinct binding domains targeting CD22 were fused to various TCR signaling domains ± an IgG heavy chain constant domain (CH2CH3) to create a series of vector constructs suitable to delineate optimal CAR configuration. CARs derived from the m971 anti-CD22 mAb, which targets a proximal CD22 epitope demonstrated superior antileukemic activity compared with those incorporating other binding domains, and addition of a 4-1BB signaling domain to CD28.CD3 constructs diminished potency, whereas increasing affinity of the anti-CD22 binding motif, and extending the CD22 binding domain away from the membrane via CH2CH3 had no effect. We conclude that second-generation m971 mAb-derived anti-CD22 CARs are promising novel therapeutics that should be tested in BCP-ALL.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras B/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras B/terapia , Receptores de Antígenos de Linfócitos T/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T/imunologia , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/antagonistas & inibidores , Lectina 2 Semelhante a Ig de Ligação ao Ácido Siálico/imunologia , Animais , Linhagem Celular Tumoral , Citocinas/metabolismo , Citotoxicidade Imunológica , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Recombinantes de Fusão/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Like the p16, SMAD4, and RB1 genes, FAM190A (alias CCSER1) lies at a consensus site of homogeneous genomic deletions in human cancer. FAM190A transcripts in 40% of cancers also contain in-frame deletions of evolutionarily conserved exons. Its gene function was unknown. We found an internal deletion of the FAM190A gene in a pancreatic cancer having prominent focal multinuclearity. The experimental knockdown of FAM190A expression by shRNA caused focal cytokinesis defects, multipolar mitosis, and multinuclearity as observed in time-lapse microscopy. FAM190A was localized to the γ-tubulin ring complex of early mitosis and to the midbody in late cytokinesis by immunofluorescence assay and was present in the nuclear fraction of unsynchronized cells by immunoblot. FAM190A interacted with EXOC1 and Ndel1, which function in cytoskeletal organization and the cell division cycle. Levels of FAM190A protein peaked 12 hours after release from thymidine block, corresponding to M-phase. Slower-migrating phosphorylated forms accumulated toward M-phase and disappeared after release from a mitotic block and before cytokinesis. Studies of FAM190A alterations may provide mechanistic insights into mitotic dysregulation and multinuclearity in cancer. We propose that FAM190A is a regulator or structural component required for normal mitosis and that both the rare truncating mutations and common in-frame deletion alteration of FAM190A may contribute to the chromosomal instability of cancer.
Assuntos
Biomarcadores Tumorais/deficiência , Proteínas de Ciclo Celular/deficiência , Divisão Celular/fisiologia , Neoplasias/metabolismo , Biomarcadores Tumorais/genética , Proteínas de Ciclo Celular/genética , Divisão Celular/genética , Linhagem Celular Tumoral , Instabilidade Cromossômica , Citometria de Fluxo , Imunofluorescência , Humanos , Immunoblotting , Mutação , Neoplasias/genética , Neoplasias/patologiaRESUMO
We conducted a clinical trial to assess adoptive transfer of T cells genetically modified to express an anti-CD19 chimeric Ag receptor (CAR). Our clinical protocol consisted of chemotherapy followed by an infusion of anti-CD19-CAR-transduced T cells and a course of IL-2. Six of the 8 patients treated on our protocol obtained remissions of their advanced, progressive B-cell malignancies. Four of the 8 patients treated on the protocol had long-term depletion of normal polyclonal CD19(+) B-lineage cells. Cells containing the anti-CD19 CAR gene were detected in the blood of all patients. Four of the 8 treated patients had prominent elevations in serum levels of the inflammatory cytokines IFNγ and TNF. The severity of acute toxicities experienced by the patients correlated with serum IFNγ and TNF levels. The infused anti-CD19-CAR-transduced T cells were a possible source of these inflammatory cytokines because we demonstrated peripheral blood T cells that produced TNF and IFNγ ex vivo in a CD19-specific manner after anti-CD19-CAR-transduced T-cell infusions. Anti-CD19-CAR-transduced T cells have great promise to improve the treatment of B-cell malignancies because of a potent ability to eradicate CD19(+) cells in vivo; however, reversible cytokine-associated toxicities occurred after CAR-transduced T-cell infusions.
Assuntos
Linfócitos B/imunologia , Citocinas/efeitos adversos , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Leucemia Linfocítica Crônica de Células B/terapia , Linfoma de Células B/terapia , Linfócitos T/imunologia , Antígenos CD19/imunologia , Citocinas/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Leucemia Linfocítica Crônica de Células B/imunologia , Linfoma de Células B/imunologia , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/uso terapêutico , Indução de Remissão , Linfócitos T/transplante , Transdução GenéticaRESUMO
Engineering CD8⺠T cells to deliver interleukin 12 (IL-12) to the tumor site can lead to striking improvements in the ability of adoptively transferred T cells to induce the regression of established murine cancers. We have recently shown that IL-12 triggers an acute inflammatory environment that reverses dysfunctional antigen presentation by myeloid-derived cells within tumors and leads to an increase in the infiltration of adoptively transferred antigen-specific CD8⺠T cells. Here, we find that local delivery of IL-12 increased the expression of Fas within tumor-infiltrating macrophages, dendritic cells, and myeloid-derived suppressor cells (MDSC), and that these changes were abrogated in mice deficient in IL-12-receptor signaling. Importantly, upregulation of Fas in host mice played a critical role in the proliferation and antitumor activity of adoptively transferred IL-12-modified CD8⺠T cells. We also observed higher percentages of myeloid-derived cell populations within tumors in Fas-deficient mice, indicating that tumor stromal destruction was dependent on the Fas death receptor. Taken together, these results describe the likely requirement for costimulatory reverse signaling through Fasl on T cells that successfully infiltrate tumors, a mechanism triggered by the induction of Fas expression on myeloid-derived cells by IL-12 and the subsequent collapse of the tumor stroma.
Assuntos
Interleucina-12/uso terapêutico , Melanoma Experimental/metabolismo , Receptor fas/metabolismo , Animais , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Células Dendríticas/metabolismo , Feminino , Citometria de Fluxo , Interleucina-12/administração & dosagem , Melanoma Experimental/terapia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Receptor fas/genéticaRESUMO
B cells are an attractive platform for engineering to produce protein-based biologics absent in genetic disorders, and potentially for the treatment of metabolic diseases and cancer. As part of pre-clinical development of B cell medicines, we demonstrate a method to collect, ex vivo expand, differentiate, radioactively label, and track adoptively transferred non-human primate (NHP) B cells. These cells underwent 10- to 15-fold expansion, initiated IgG class switching, and differentiated into antibody secreting cells. Zirconium-89-oxine labeled cells were infused into autologous donors without any preconditioning and tracked by PET/CT imaging. Within 24 hours of infusion, 20% of the initial dose homed to the bone marrow and spleen and distributed stably and equally between the two. Interestingly, approximately half of the dose homed to the liver. Image analysis of the bone marrow demonstrated inhomogeneous distribution of the cells. The subjects experienced no clinically significant side effects or laboratory abnormalities. A second infusion of B cells into one of the subjects resulted in an almost identical distribution of cells, suggesting a non-limiting engraftment niche and feasibility of repeated infusions. This work supports the NHP as a valuable model to assess the potential of B cell medicines as potential treatment for human diseases.
RESUMO
The genetic modification of CD8+ T cells using anti-tumor T-cell receptors (TCR) or chimeric antigen receptors is a promising approach for the adoptive cell therapy of patients with cancer. We previously developed a simplified method for the clinical-scale generation of central memory-like (Tcm) CD8+ T cells following transduction with lentivirus encoding anti-tumor TCR and culture in the presence of IL-2. In this study, we compared different cytokines or combinations of IL-2, IL-7, IL-12, IL-15, and IL-21 to expand genetically engineered CD8+ T cells. We demonstrated that specific cytokine combinations IL-12 plus IL-7 or IL-21 for 3 days followed by withdrawal of IL-12 yielded the phenotype of CD62L(high)CD28(high) CD127(high)CD27(high)CCR7(high), which is associated with less-differentiated T cells. Genes associated with stem cells (SOX2, NANOG, OCT4, and LIN28A), were also up-regulated by this cytokine cocktail. Moreover, the use of IL-12 plus IL-7 or IL-21 yielded CD8 T cells showing enhanced persistence in the NOD/SCID/γc-/- mouse model. This defined cytokine combination could also alter highly differentiated TIL from melanoma patients into cells with a less-differentiated phenotype. The methodology that we developed for generating a less-differentiated anti-tumor CD8+ T cells ex vivo may be ideal for the adoptive immunotherapy of cancer.
Assuntos
Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Citocinas/imunologia , Citocinas/farmacologia , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Linfócitos do Interstício Tumoral/imunologia , Animais , Linfócitos T CD8-Positivos/citologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/imunologia , Humanos , Interleucinas/imunologia , Interleucinas/farmacologia , Linfócitos do Interstício Tumoral/citologia , Melanoma/imunologia , Melanoma/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , FenótipoRESUMO
Cluster of differentiation (CD)8(+) T cells exist as naive, central memory, and effector memory subsets, and any of these populations can be genetically engineered into tumor-reactive effector cells for adoptive immunotherapy. However, the optimal subset from which to derive effector CD8(+) T cells for patient treatments is controversial and understudied. We investigated human CD8(+) T cells and found that naive cells were not only the most abundant subset but also the population most capable of in vitro expansion and T-cell receptor transgene expression. Despite increased expansion, naive-derived cells displayed minimal effector differentiation, a quality associated with greater efficacy after cell infusion. Similarly, the markers of terminal differentiation, killer cell lectin-like receptor G1 and CD57, were expressed at lower levels in cells of naive origin. Finally, naive-derived effector cells expressed higher CD27 and retained longer telomeres, characteristics that suggest greater proliferative potential and that have been linked to greater efficacy in clinical trials. Thus, these data suggest that naive cells resist terminal differentiation, or "exhaustion," maintain high replicative potential, and therefore may be the superior subset for use in adoptive immunotherapy.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Memória Imunológica/imunologia , Imunoterapia Adotiva/métodos , Subpopulações de Linfócitos T/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/transplante , Diferenciação Celular/imunologia , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Citocinas/metabolismo , Citometria de Fluxo , Expressão Gênica , Vetores Genéticos/genética , Humanos , Retroviridae/genética , Subpopulações de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/transplante , Telômero/genética , Transdução Genética , Membro 7 da Superfamília de Receptores de Fatores de Necrose Tumoral/metabolismoRESUMO
INTRODUCTION: Preclinical studies have demonstrated that tumor-reactive T cells expressing the interleukin (IL)-15 transgene had enhanced activity. Gene therapy strategies using IL-15 should include a safety mechanism in anticipation of possible adverse effects because IL-15 overexpression has been implicated in autoimmune disorders and may be involved in the pathogenesis of some leukemias. We developed a retroviral vector carrying both IL-15 and the herpes simplex virus-thymidine kinase (HSV-TK) suicide gene and characterized its application in the transduction of human T lymphocytes. METHODS: A retroviral vector carrying IL-15 and HSV-TK genes was optimized for the transduction of human T lymphocytes. IL-15 production was measured by enzyme-linked immunosorbent assay. Thymidine incorporation and cell viability assays were used to assess the efficacy of the HSV-TK suicide gene. Genetically modified tumor-infiltrating lymphocytes (TILs) were assayed for survival after withdrawal from exogenous IL-2. The activity and specificity of retrovirally transduced TILs were assessed using tumor coculture assays. RESULTS: Human T cells transduced with the IL-15 HSV-TK vector exhibited thymidine uptake in the absence of exogenous cytokine support and survived in culture for up to 80 d without IL-2. IL-15 HSV-TK-transduced T cells were efficiently killed by ganciclovir at concentrations as low as 0.1 µM. TILs transduced with the IL-15 HSV-TK vector retained specific recognition of HLA-A2+, MART1+ melanomas, even after withdrawal of IL-2. CONCLUSIONS: Human T lymphocytes genetically modified with the IL-15 HSV-TK retroviral vector retained the ability to recognize tumor antigen while gaining the ability to secrete IL-15 and prolong their own survival. IL-15 HSV-TK-transduced T cells expressed HSV-TK and could be efficiently eliminated by ganciclovir.
Assuntos
Engenharia Genética/métodos , Imunoterapia Adotiva/métodos , Interleucina-15/genética , Simplexvirus/genética , Linfócitos T/fisiologia , Timidina Quinase/genética , Animais , Doenças Autoimunes/terapia , Linhagem Celular Tumoral , Expressão Gênica/imunologia , Humanos , Interleucina-2/farmacologia , Leucemia/terapia , Melanoma/terapia , Camundongos , Células NIH 3T3 , Retroviridae/genética , Neoplasias Cutâneas/terapia , Linfócitos T/citologia , Transdução Genética , Transgenes/genéticaRESUMO
Adoptive immunotherapy using TCR-engineered PBLs against melanocyte differentiation Ags mediates objective tumor regression but is associated with on-target toxicity. To avoid toxicity to normal tissues, we targeted cancer testis Ag (CTA) MAGE-A3, which is widely expressed in a range of epithelial malignancies but is not expressed in most normal tissues. To generate high-avidity TCRs against MAGE-A3, we employed a transgenic mouse model that expresses the human HLA-A*0201 molecule. Mice were immunized with two HLA-A*0201-restricted peptides of MAGE-A3: 112-120 (KVAELVHFL) or MAGE-A3: 271-279 (FLWGPRALV), and T cell clones were generated. MAGE-A3-specific TCR α- and ß-chains were isolated and cloned into a retroviral vector. Expression of both TCRs in human PBLs demonstrated Ag-specific reactivity against a range of melanoma and nonmelanoma tumor cells. The TCR against MAGE-A3: 112-120 was selected for further development based on superior reactivity against tumor target cells. Interestingly, peptide epitopes from MAGE-A3 and MAGE-A12 (and to a lesser extent, peptides from MAGE-A2 and MAGE-A6) were recognized by PBLs engineered to express this TCR. To further improve TCR function, single amino acid variants of the CDR3 α-chain were generated. Substitution of alanine to threonine at position 118 of the α-chain in the CDR3 region of the TCR improved its functional avidity in CD4 and CD8 cells. On the basis of these results, a clinical trial is planned in which patients bearing a variety of tumor histologies will receive autologous PBLs that have been transduced with this optimized anti-MAGE-A3 TCR.
Assuntos
Antígenos de Neoplasias/imunologia , Testes Imunológicos de Citotoxicidade , Epitopos de Linfócito T/metabolismo , Antígenos HLA-A/metabolismo , Proteínas de Neoplasias/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/administração & dosagem , Antígenos de Neoplasias/biossíntese , Sequência de Bases , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/terapia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/terapia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Técnicas de Cocultura , Testes Imunológicos de Citotoxicidade/métodos , Epitopos de Linfócito T/administração & dosagem , Epitopos de Linfócito T/imunologia , Feminino , Antígenos HLA-A/biossíntese , Antígenos HLA-A/imunologia , Antígeno HLA-A2 , Humanos , Imunoterapia Adotiva/métodos , Interferon gama/metabolismo , Melanoma/imunologia , Melanoma/metabolismo , Melanoma/terapia , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Família Multigênica/imunologia , Proteínas de Neoplasias/administração & dosagem , Proteínas de Neoplasias/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T/transplanteRESUMO
The aim of this review is to acquaint the reader with the concept of T cell receptor (TCR) gene therapy for metastatic melanoma. We first review antigen choice, followed by gene delivery technology and, finally, we discuss selected clinical applications. To be successful, TCR gene therapy must combine multiple elements and research disciplines. First, there is a need for an understanding of tumor immunology because this is essential in proper antigen choice. Second, gene therapy is a technology-driven field and cutting edge knowledge of protein engineering and gene delivery methods are indispensable. Finally, a dedicated team of physician/scientists and healthcare providers is fundamental to clinical success. TCR gene therapy is now a realistic treatment option for metastatic melanoma. Both tumor regression and on-target/off-tumor toxicities have been observed, which emphasizes the experimental nature of this approach. As with any new medical procedure, future large-scale randomized trials will be necessary to validate this approach, and these are within reason in the next few years. This article is a US Government work and is in the public domain in the USA.
Assuntos
Terapia Genética , Melanoma/imunologia , Melanoma/terapia , Receptores de Antígenos de Linfócitos T/genética , Linfócitos T/imunologia , Ensaios Clínicos como Assunto , Técnicas de Transferência de Genes , Genes Codificadores dos Receptores de Linfócitos T , Engenharia Genética , Humanos , Melanoma/secundário , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais , Pele/imunologiaRESUMO
BACKGROUND: There has been significant progress in the last two decades on the design of chimeric antigen receptors (CAR) for adoptive immunotherapy targeting tumor-associated antigens. Structurally CARs consist of a single chain antibody fragment directed against a tumor-associated antigen fused to an extracellular spacer and transmembrane domain followed by T cell cytoplasmic signaling moieties. Currently several clinical trials are underway using gene modified peripheral blood lymphocytes (PBL) with CARs directed against a variety of tumor associated antigens. Despite the improvements in the design of CARs and expansion of the number of target antigens, there is no universal flow cytometric method available to detect the expression of CARs on the surface of transduced lymphocytes. METHODS: Currently anti-fragment antigen binding (Fab) conjugates are most widely used to determine the expression of CARs on gene-modified lymphocytes by flow cytometry. The limitations of these reagents are that many of them are not commercially available, generally they are polyclonal antibodies and often the results are inconsistent. In an effort to develop a simple universal flow cytometric method to detect the expression of CARs, we employed protein L to determine the expression of CARs on transduced lymphocytes. Protein L is an immunoglobulin (Ig)-binding protein that binds to the variable light chains (kappa chain) of Ig without interfering with antigen binding site. Protein L binds to most classes of Ig and also binds to single-chain antibody fragments (scFv) and Fab fragments. RESULTS: We used CARs derived from both human and murine antibodies to validate this novel protein L based flow cytometric method and the results correlated well with other established methods. Activated human PBLs were transduced with retroviral vectors expressing two human antibody based CARs (anti-EGFRvIII, and anti-VEGFR2), two murine antibody derived CARs (anti-CSPG4, and anti-CD19), and two humanized mouse antibody based CARs (anti-ERBB2, and anti-PSCA). Transduced cells were stained first with biotin labeled protein L followed by phycoerythrin (PE)-conjugated streptavidin (SA) and analyzed by flow cytometry. For comparison, cells were stained in parallel with biotin conjugated goat-anti-mouse Fab or CAR specific fusion proteins. Using protein L, all CAR transduced lymphocytes exhibited specific staining pattern ranging from 40 to 80% of positive cells (compared to untransduced cells) and staining was comparable to the pattern observed with anti-Fab antibodies. CONCLUSION: Our data demonstrate the feasibility of employing Protein L as a general reagent for the detection of CAR expression on transduced lymphocytes by flow cytometry.