RESUMO
Natural killer (NK) cells are an important effector cell type for adoptive cancer immunotherapy. Similar to T cells, NK cells can be modified to express chimeric antigen receptors (CARs) to enhance antitumor activity, but experience with CAR-engineered NK cells and their clinical development is still limited. Here, we redirected continuously expanding and clinically usable established human NK-92 cells to the tumor-associated ErbB2 (HER2) antigen. Following GMP-compliant procedures, we generated a stable clonal cell line expressing a humanized CAR based on ErbB2-specific antibody FRP5 harboring CD28 and CD3ζ signaling domains (CAR 5.28.z). These NK-92/5.28.z cells efficiently lysed ErbB2-expressing tumor cells in vitro and exhibited serial target cell killing. Specific recognition of tumor cells and antitumor activity were retained in vivo, resulting in selective enrichment of NK-92/5.28.z cells in orthotopic breast carcinoma xenografts, and reduction of pulmonary metastasis in a renal cell carcinoma model, respectively. γ-irradiation as a potential safety measure for clinical application prevented NK cell replication, while antitumor activity was preserved. Our data demonstrate that it is feasible to engineer CAR-expressing NK cells as a clonal, molecularly and functionally well-defined and continuously expandable cell therapeutic agent, and suggest NK-92/5.28.z cells as a promising candidate for use in adoptive cancer immunotherapy.
Assuntos
Expressão Gênica , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Receptor ErbB-2/imunologia , Receptores Imunológicos/genética , Receptores Imunológicos/imunologia , Proteínas Recombinantes de Fusão/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Neoplasias da Mama/terapia , Linhagem Celular Transformada , Linhagem Celular Tumoral , Evolução Clonal , Citotoxicidade Imunológica , Modelos Animais de Doenças , Epitopos de Linfócito T/imunologia , Feminino , Vetores Genéticos/genética , Humanos , Imunofenotipagem , Imunoterapia , Lentivirus/genética , Teste de Cultura Mista de Linfócitos , Neoplasias/genética , Neoplasias/imunologia , Neoplasias/patologia , Neoplasias/terapia , Fenótipo , Transdução Genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Reactivation of latent viruses such as human cytomegalovirus (HCMV) after allogeneic hematopoietic stem cell transplantation (HSCT) results in high morbidity and mortality. Effective immunization against HCMV shortly after allo-HSCT is an unmet clinical need due to delayed adaptive T cell development. Donor-derived dendritic cells (DCs) have a critical participation in stimulation of naïve T cells and immune reconstitution, and therefore adoptive DC therapy could be used to protect patients after HSCT. However, previous methods for ex vivo generation of adoptive donor-derived DCs were complex and inconsistent, particularly regarding cell viability and potency after thawing. We have previously demonstrated in humanized mouse models of HSCT the proof-of-concept of a novel modality of lentivirus-induced DCs ("SmyleDCpp65") that accelerated antigen-specific T cell development. METHODS: Here we demonstrate the feasibility of good manufacturing practices (GMP) for production of donor-derived DCs consisting of monocytes from peripheral blood transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-α and the cytomegalovirus antigen pp65) that were cryopreserved and thawed. RESULTS: Upscaling and standardized production of one lot of IDLV and three lots of SmyleDCpp65 under GMP-compliant conditions were feasible. Analytical parameters for quality control of SmyleDCpp65 identity after thawing and potency after culture were defined. Cell recovery, uniformity, efficacy of gene transfer, purity and viability were high and consistent. SmyleDCpp65 showed only residual and polyclonal IDLV integration, unbiased to proto-oncogenic hot-spots. Stimulation of autologous T cells by GMP-grade SmyleDCpp65 was validated. CONCLUSION: These results underscore further developments of this individualized donor-derived cell vaccine to accelerate immune reconstitution against HCMV after HSCT in clinical trials.
Assuntos
Infecções por Citomegalovirus/imunologia , Células Dendríticas/citologia , Lentivirus , Transplante de Células-Tronco/métodos , Animais , Técnicas de Cultura de Células , Sobrevivência Celular , Criopreservação , Citomegalovirus , Infecções por Citomegalovirus/prevenção & controle , Células Dendríticas/virologia , Citometria de Fluxo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Células HEK293 , Transplante de Células-Tronco Hematopoéticas , Humanos , Interferon-alfa/metabolismo , Leucócitos Mononucleares/citologia , Fosfoproteínas/metabolismo , Plasmídeos/metabolismo , Transgenes , Proteínas da Matriz Viral/metabolismoRESUMO
The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive primary immunodeficiency disorder associated with thrombocytopenia, eczema, and autoimmunity. We treated two patients who had this disorder with a transfusion of autologous, genetically modified hematopoietic stem cells (HSC). We found sustained expression of WAS protein expression in HSC, lymphoid and myeloid cells, and platelets after gene therapy. T and B cells, natural killer (NK) cells, and monocytes were functionally corrected. After treatment, the patients' clinical condition markedly improved, with resolution of hemorrhagic diathesis, eczema, autoimmunity, and predisposition to severe infection. Comprehensive insertion-site analysis showed vector integration that targeted multiple genes controlling growth and immunologic responses in a persistently polyclonal hematopoiesis. (Funded by Deutsche Forschungsgemeinschaft and others; German Clinical Trials Register number, DRKS00000330.).
Assuntos
Terapia Genética , Transplante de Células-Tronco Hematopoéticas , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Técnicas de Transferência de Genes , Terapia Genética/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Lactente , Masculino , Mutagênese Insercional , Transplante Autólogo , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/imunologiaRESUMO
Gene transfer into hematopoietic stem cells has been used successfully for correcting lymphoid but not myeloid immunodeficiencies. Here we report on two adults who received gene therapy after nonmyeloablative bone marrow conditioning for the treatment of X-linked chronic granulomatous disease (X-CGD), a primary immunodeficiency caused by a defect in the oxidative antimicrobial activity of phagocytes resulting from mutations in gp91(phox). We detected substantial gene transfer in both individuals' neutrophils that lead to a large number of functionally corrected phagocytes and notable clinical improvement. Large-scale retroviral integration site-distribution analysis showed activating insertions in MDS1-EVI1, PRDM16 or SETBP1 that had influenced regulation of long-term hematopoiesis by expanding gene-corrected myelopoiesis three- to four-fold in both individuals. Although insertional influences have probably reinforced the therapeutic efficacy in this trial, our results suggest that gene therapy in combination with bone marrow conditioning can be successfully used to treat inherited diseases affecting the myeloid compartment such as CGD.
Assuntos
Proteínas de Transporte/genética , Proteínas de Ligação a DNA/genética , Terapia Genética/métodos , Doença Granulomatosa Crônica/terapia , Células-Tronco Hematopoéticas/fisiologia , Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Adulto , Cromossomos Humanos X , Ensaios Clínicos como Assunto , Técnicas de Transferência de Genes , Ligação Genética , Marcadores Genéticos , Vetores Genéticos , Doença Granulomatosa Crônica/sangue , Doença Granulomatosa Crônica/etiologia , Doença Granulomatosa Crônica/genética , Humanos , Proteína do Locus do Complexo MDS1 e EVI1 , Mutagênese Insercional , Neutrófilos/fisiologia , Proto-Oncogenes , RNA Mensageiro/análise , Retroviridae/genética , Resultado do TratamentoRESUMO
Drug toxicity and viral resistance limit the long-term efficacy of antiviral drug treatment for human immunodeficiency virus (HIV) infection. Thus, alternative therapies need to be explored. We tested the infusion of T lymphocytes transduced with a retroviral vector (M87o) that expresses an HIV entry-inhibitory peptide (maC46). Gene-modified autologous T cells were infused into ten HIV-infected patients with advanced disease and multidrug-resistant virus during anti-retroviral combination therapy. T-cell infusions were tolerated well, with no severe side effects. A significant increase of CD4 counts was observed after infusion. At the end of the 1-year follow-up, the CD4 counts of all patients were still around or above baseline. Gene-modified cells could be detected in peripheral blood, lymph nodes, and bone marrow throughout the 1-year follow-up, and marking levels correlated with the cell dose. No significant changes of viral load were observed during the first 4 months. Four of the seven patients who changed their antiviral drug regimen thereafter responded with a significant decline in plasma viral load. In conclusion, the transfer of gene-modified cells was safe, led to sustained levels of gene marking, and may improve immune competence in HIV-infected patients with advanced disease and multidrug-resistant virus.
RESUMO
We report on a series of sequential events leading to long-term survival and cure of pediatric X-linked chronic granulomatous disease (X-CGD) patients after gamma-retroviral gene therapy (GT) and rescue HSCT. Due to therapyrefractory life-threatening infections requiring hematopoietic stem cell transplantation (HSCT) but absence of HLAidentical donors, we treated 2 boys with X-CGD by GT. Following GT both children completely resolved invasive Aspergillus nidulans infections. However, one child developed dual insertional activation of ecotropic viral integration site 1 (EVI1) and signal transducer and activator of transcription 3 (STAT3) genes, leading to myelodysplastic syndrome (MDS) with monosomy 7. Despite resistance to mismatched allo-HSCT with standard myeloablative conditioning, secondary intensified rescue allo-HSCT resulted in 100 % donor chimerism and disappearance of MDS. The other child did not develop MDS despite expansion of a clone with a single insertion in the myelodysplasia syndrome 1 (MDS1) gene and was cured by early standard allo-HSCT. The slowly developing dominance of clones harboring integrations in MDS1-EVI1 may guide clinical intervention strategies, i.e. early rescue allo-HSCT, prior to malignant transformation. GT was essential for both children to survive and to clear therapy-refractory infections, and future GT with safer lentiviral self-inactivated (SIN) vectors may offer a therapeutic alternative for X-CGD patients suffering from life-threatening infections and lacking HLA-identical HSC donors.
Assuntos
Terapia Genética/métodos , Doença Granulomatosa Crônica/terapia , Transplante de Células-Tronco Hematopoéticas/métodos , Aspergilose/terapia , Aspergillus nidulans/patogenicidade , Criança , Deleção Cromossômica , Cromossomos Humanos Par 7 , Proteínas de Ligação a DNA/genética , Gammaretrovirus/genética , Terapia Genética/efeitos adversos , Humanos , Proteína do Locus do Complexo MDS1 e EVI1 , Masculino , Glicoproteínas de Membrana/genética , Síndromes Mielodisplásicas/etiologia , NADPH Oxidase 2 , NADPH Oxidases/genética , Proto-Oncogenes/genética , Fator de Transcrição STAT3/genética , Fatores de Transcrição/genéticaRESUMO
Wiskott-Aldrich syndrome (WAS) is characterized by microthrombocytopenia, immunodeficiency, autoimmunity, and susceptibility to malignancies. In our hematopoietic stem cell gene therapy (GT) trial using a γ-retroviral vector, 9 of 10 patients showed sustained engraftment and correction of WAS protein (WASP) expression in lymphoid and myeloid cells and platelets. GT resulted in partial or complete resolution of immunodeficiency, autoimmunity, and bleeding diathesis. Analysis of retroviral insertion sites revealed >140,000 unambiguous integration sites and a polyclonal pattern of hematopoiesis in all patients early after GT. Seven patients developed acute leukemia [one acute myeloid leukemia (AML), four T cell acute lymphoblastic leukemia (T-ALL), and two primary T-ALL with secondary AML associated with a dominant clone with vector integration at the LMO2 (six T-ALL), MDS1 (two AML), or MN1 (one AML) locus]. Cytogenetic analysis revealed additional genetic alterations such as chromosomal translocations. This study shows that hematopoietic stem cell GT for WAS is feasible and effective, but the use of γ-retroviral vectors is associated with a substantial risk of leukemogenesis.
Assuntos
Terapia Genética/efeitos adversos , Mutagênicos/efeitos adversos , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/uso terapêutico , Síndrome de Wiskott-Aldrich/terapia , Adolescente , Animais , Plaquetas/metabolismo , Criança , Pré-Escolar , Células Clonais , Colite/etiologia , Progressão da Doença , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Humanos , Leucócitos Mononucleares/metabolismo , Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Leucemia-Linfoma Linfoblástico de Células T Precursoras/etiologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Trombocitopenia/terapia , Transplante Autólogo , Resultado do Tratamento , Síndrome de Wiskott-Aldrich/patologia , Proteína da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by impaired antimicrobial activity in phagocytic cells. As a monogenic disease affecting the hematopoietic system, CGD is amenable to gene therapy. Indeed in a phase I/II clinical trial, we demonstrated a transient resolution of bacterial and fungal infections. However, the therapeutic benefit was compromised by the occurrence of clonal dominance and malignant transformation demanding alternative vectors with equal efficacy but safety-improved features. In this work we have developed and tested a self-inactivating (SIN) gammaretroviral vector (SINfes.gp91s) containing a codon-optimized transgene (gp91(phox)) under the transcriptional control of a myeloid promoter for the gene therapy of the X-linked form of CGD (X-CGD). Gene-corrected cells protected X-CGD mice from Aspergillus fumigatus challenge at low vector copy numbers. Moreover, the SINfes.gp91s vector generates substantial amounts of superoxide in human cells transplanted into immunodeficient mice. In vitro genotoxicity assays and longitudinal high-throughput integration site analysis in transplanted mice comprising primary and secondary animals for 11 months revealed a safe integration site profile with no signs of clonal dominance.
Assuntos
Gammaretrovirus/genética , Vetores Genéticos/metabolismo , Doença Granulomatosa Crônica/terapia , Animais , Aspergillus fumigatus/patogenicidade , Células Cultivadas , Metilação de DNA , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Terapia Genética , Vetores Genéticos/genética , Humanos , Pneumopatias/microbiologia , Pneumopatias/patologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , NADPH Oxidase 2 , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Fenótipo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-fes/genética , Superóxidos/metabolismoRESUMO
Drug toxicity and viral resistance limit the long-term efficacy of antiviral drug treatment for human immunodeficiency virus (HIV) infection. Thus, alternative therapies need to be explored. We tested the infusion of T lymphocytes transduced with a retroviral vector (M87o) that expresses an HIV entry-inhibitory peptide (maC46). Gene-modified autologous T cells were infused into ten HIV-infected patients with advanced disease and multidrug-resistant virus during anti-retroviral combination therapy. T-cell infusions were tolerated well, with no severe side effects. A significant increase of CD4 counts was observed after infusion. At the end of the 1-year follow-up, the CD4 counts of all patients were still around or above baseline. Gene-modified cells could be detected in peripheral blood, lymph nodes, and bone marrow throughout the 1-year follow-up, and marking levels correlated with the cell dose. No significant changes of viral load were observed during the first 4 months. Four of the seven patients who changed their antiviral drug regimen thereafter responded with a significant decline in plasma viral load. In conclusion, the transfer of gene-modified cells was safe, led to sustained levels of gene marking, and may improve immune competence in HIV-infected patients with advanced disease and multidrug-resistant virus.
Assuntos
Transferência Adotiva/métodos , Infecções por HIV/terapia , HIV/imunologia , Linfócitos T/imunologia , Adulto , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Farmacorresistência Viral/imunologia , Citometria de Fluxo , Vetores Genéticos/genética , Infecções por HIV/imunologia , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Retroviridae/genética , Linfócitos T/citologia , Linfócitos T/metabolismo , Resultado do Tratamento , Carga ViralRESUMO
Increasing use of hematopoietic stem cells for retroviral vector-mediated gene therapy and recent reports on insertional mutagenesis in mice and humans have created intense interest to characterize vector integrations on a genomic level. We studied retrovirally transduced human peripheral blood progenitor cells with bone marrow-repopulating ability in immune-deficient mice. By using a highly sensitive and specific ligation-mediated polymerase chain reaction (PCR) followed by sequencing of vector integration sites, we found a multitude of simultaneously active human stem cell clones 8 weeks after transplantation. Vector integrations occurred with significantly increased frequency into chromosomes 17 and 19 and into specific regions of chromosomes 6, 13, and 16, although most of the chromosomes were targeted. Preferred genomic target sites have previously only been reported for wild-type retroviruses. Our findings reveal for the first time that retroviral vector integration into human marrow-repopulating cells can be nonrandom (P =.000 37).
Assuntos
Células da Medula Óssea/citologia , Vetores Genéticos , Retroviridae/genética , Animais , Cromossomos Humanos Par 13 , Cromossomos Humanos Par 16 , Cromossomos Humanos Par 17 , Cromossomos Humanos Par 19 , Cromossomos Humanos Par 6 , Clonagem Molecular , Feminino , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Hibridização in Situ Fluorescente , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Reação em Cadeia da Polimerase/métodos , Transfecção , Integração ViralRESUMO
Methods to analyze the clonality of an adverse event in preclinical or clinical retroviral stem cell gene therapy protocols are needed. We analyzed the progeny of retrovirally transduced human peripheral blood progenitor cells (PBPCs) after transplantation and engraftment in immune-deficient mice. The integration site of the provirus serves as a unique tag of the individual transduced PBPC. A plasmid library of junctions between proviral and human genomic DNA was generated. We were able to detect individual transduced cell clones that amounted to 0.14%-0.0001% of chimeric bone marrow cells. This is the first report in which the contribution of individual marrow-repopulating cells to human hematopoiesis is directly quantified.