RESUMO
Ectoenzyme CD38 is increased on lymphocytes in response to an antigenic challenge and it is hypothesized that targeting these activated lymphocytes could ameliorate pathologic activities in autoimmune diseases. The cynomolgus monkey is an appropriate model for assessing potential effects of targeting CD38 in humans because these species exhibit similar expression profiles. TAK-079 is a human monoclonal antibody (IgG1 λ ) that binds to CD38 and lyses bound cells by complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity. TAK-079 binds to monkey CD38 with an affinity at EC50 4.5 nM, and the potential activity of TAK-079 was investigated in a monkey collagen-induced arthritis model of autoimmune disease. Prophylactic administration of TAK-079 (3 mg/kg i.v. weekly) was well tolerated and prevented arthritis development compared with vehicle-treated control animals, which exhibited progressive disease with radiographic damage and worsening clinical scores over the study course. Therapeutic treatment of arthritic monkeys with TAK-079 (3 mg/kg i.v. weekly) was also well tolerated and reduced disease progression and symptoms. Arthritis scores and joint swelling were significantly lower than the vehicle control, accompanied by decreases in blood levels of C-reactive protein, alkaline phosphatase, and natural killer, B, and T cells. Histopathology, morphometry, and radiology revealed significantly less joint damage in animals exposed prophylactically to TAK-079 treatment compared with vehicle-treated animals and significantly less damage in animals treated therapeutically with TAK-079 or dexamethasone (0.1 mg/kg oral gavage daily), illustrating potential disease-modifying activity. In conclusion, these data indicate that depletion of CD38-expressing cells could be a therapeutic mechanism for treating autoimmune diseases. SIGNIFICANCE STATEMENT: This study demonstrates that targeting CD38-expressing leukocytes with a cytolytic antibody can ameliorate autoimmune disease in cynomolgus monkeys. The study gives a unique perspective into this therapeutic strategy because the three other anti-CD38 cytolytic antibodies in clinical development (daratumumab, isatuximab, and MOR202) cannot be tested in similar models because they do not crossreact with CD38 expressed by new world primates.
Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Anticorpos Monoclonais/imunologia , Artrite Experimental/imunologia , Linfócitos B/metabolismo , Regulação da Expressão Gênica/imunologia , Células Matadoras Naturais/metabolismo , Linfócitos T/metabolismo , ADP-Ribosil Ciclase 1/imunologia , Animais , Artrite Experimental/metabolismo , Artrite Experimental/patologia , Linfócitos B/imunologia , Células CHO , Cricetulus , Progressão da Doença , Células Matadoras Naturais/imunologia , Macaca fascicularis , Linfócitos T/imunologiaRESUMO
The observed structure-activity relationship of three distinct ATP noncompetitive With-No-Lysine (WNK) kinase inhibitor series, together with a crystal structure of a previously disclosed allosteric inhibitor bound to WNK1, led to an overlay hypothesis defining core and side-chain relationships across the different series. This in turn enabled an efficient optimization through scaffold morphing, resulting in compounds with a good balance of selectivity, cellular potency, and pharmacokinetic profile, which were suitable for in vivo proof-of-concept studies. When dosed orally, the optimized compound reduced blood pressure in mice overexpressing human WNK1, and induced diuresis, natriuresis and kaliuresis in spontaneously hypertensive rats (SHR), confirming that this mechanism of inhibition of WNK kinase activity is effective at regulating cardiovascular homeostasis.
Assuntos
Anti-Hipertensivos/farmacologia , Hipertensão/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Tiazóis/farmacologia , Regulação Alostérica , Animais , Anti-Hipertensivos/síntese química , Anti-Hipertensivos/farmacocinética , Células HEK293 , Humanos , Masculino , Camundongos Transgênicos , Antígenos de Histocompatibilidade Menor , Simulação de Acoplamento Molecular , Piperazinas/síntese química , Piperazinas/farmacocinética , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Serina-Treonina Quinases/metabolismo , Ratos Sprague-Dawley , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/farmacocinética , Proteína Quinase 1 Deficiente de Lisina WNKRESUMO
Protein kinases are known for their highly conserved adenosine triphosphate (ATP)-binding site, rendering the discovery of selective inhibitors a major challenge. In theory, allosteric inhibitors can achieve high selectivity by targeting less conserved regions of the kinases, often with an added benefit of retaining efficacy under high physiological ATP concentration. Although often overlooked in favor of ATP-site directed approaches, performing a screen at high ATP concentration or stringent hit triaging with high ATP concentration offers conceptually simple methods of identifying inhibitors that bind outside the ATP pocket. Here, we applied the latter approach to the With-No-Lysine (K) (WNK) kinases to discover lead molecules for a next-generation antihypertensive that requires a stringent safety profile. This strategy yielded several ATP noncompetitive WNK1-4 kinase inhibitors, the optimization of which enabled cocrystallization with WNK1, revealing an allosteric binding mode consistent with the observed exquisite specificity for WNK1-4 kinases. The optimized compound inhibited rubidium uptake by sodium chloride cotransporter 1 (NKCC1) in HT29 cells, consistent with the reported physiology of WNK kinases in renal electrolyte handling.
Assuntos
Regulação Alostérica/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Descoberta de Drogas , Células HEK293 , Células HT29 , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Antígenos de Histocompatibilidade Menor/química , Antígenos de Histocompatibilidade Menor/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Proteína Quinase 1 Deficiente de Lisina WNKRESUMO
The With-No-Lysine (K) (WNK) kinases play a critical role in blood pressure regulation and body fluid and electrolyte homeostasis. Herein, we introduce the first orally bioavailable pan-WNK-kinase inhibitor, WNK463, that exploits unique structural features of the WNK kinases for both affinity and kinase selectivity. In rodent models of hypertension, WNK463 affects blood pressure and body fluid and electro-lyte homeostasis, consistent with WNK-kinase-associated physiology and pathophysiology.
Assuntos
Sistema Cardiovascular/efeitos dos fármacos , Imidazóis/farmacologia , Rim/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirrolidinas/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Animais , Sistema Cardiovascular/metabolismo , Humanos , Imidazóis/química , Rim/metabolismo , Testes de Função Renal , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Pirrolidinas/química , Ratos , Ratos Sprague-Dawley , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Chimeric antigen receptors (CARs) are synthetic molecules designed to redirect T cells to specific antigens. CAR-modified T cells can mediate long-term durable remissions in B cell malignancies, but expanding this platform to solid tumors requires the discovery of surface targets with limited expression in normal tissues. The variant III mutation of the epidermal growth factor receptor (EGFRvIII) results from an in-frame deletion of a portion of the extracellular domain, creating a neoepitope. We chose a vector backbone encoding a second-generation CAR based on efficacy of a murine scFv-based CAR in a xenograft model of glioblastoma. Next, we generated a panel of humanized scFvs and tested their specificity and function as soluble proteins and in the form of CAR-transduced T cells; a low-affinity scFv was selected on the basis of its specificity for EGFRvIII over wild-type EGFR. The lead candidate scFv was tested in vitro for its ability to direct CAR-transduced T cells to specifically lyse, proliferate, and secrete cytokines in response to antigen-bearing targets. We further evaluated the specificity of the lead CAR candidate in vitro against EGFR-expressing keratinocytes and in vivo in a model of mice grafted with normal human skin. EGFRvIII-directed CAR T cells were also able to control tumor growth in xenogeneic subcutaneous and orthotopic models of human EGFRvIII(+) glioblastoma. On the basis of these results, we have designed a phase 1 clinical study of CAR T cells transduced with humanized scFv directed to EGFRvIII in patients with either residual or recurrent glioblastoma (NCT02209376).
Assuntos
Neoplasias Encefálicas/terapia , Receptores ErbB/imunologia , Glioblastoma/terapia , Imunoterapia , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Modelos Animais de Doenças , Xenoenxertos , Humanos , CamundongosRESUMO
BACKGROUND: Seneca Valley virus (SVV-001) is a nonpathogenic oncolytic virus that can be systemically administered and can pass through the blood-brain barrier. We examined its therapeutic efficacy and the mechanism of tumor cell infection in pediatric malignant gliomas. METHODS: In vitro antitumor activities were examined in primary cultures, preformed neurospheres, and self-renewing glioma cells derived from 6 patient tumor orthotopic xenograft mouse models (1 anaplastic astrocytoma and 5 GBM). In vivo therapeutic efficacy was examined by systemic treatment of preformed xenografts in 3 permissive and 2 resistant models. The functional role of sialic acid in mediating SVV-001 infection was investigated using neuraminidase and lectins that cleave or competitively bind to linkage-specific sialic acids. RESULTS: SVV-001 at a multiplicity of infection of 0.5 to 25 replicated in and effectively killed primary cultures, preformed neurospheres, and self-renewing stemlike single glioma cells derived from 4 of the 6 glioma models in vitro. A single i.v. injection of SVV-001 (5 × 10(12) viral particles/kg) led to the infection of orthotopic xenografts without harming normal mouse brain cells, resulting in significantly prolonged survival in all 3 permissive and 1 resistant mouse models (P < .05). Treatment with neuraminidase and competitive binding using lectins specific for α2,3-linked and/or α2,6-linked sialic acid significantly suppressed SVV-001 infectivity (P < .01). CONCLUSION: SVV-001 possesses strong antitumor activity against pediatric malignant gliomas and utilizes α2,3-linked and α2,6-linked sialic acids as mediators of tumor cell infection. Our findings support the consideration of SVV-001 for clinical trials in children with malignant glioma.
Assuntos
Neoplasias Encefálicas/terapia , Glioma/terapia , Terapia Viral Oncolítica , Vírus Oncolíticos , Picornaviridae , Animais , Neoplasias Encefálicas/metabolismo , Modelos Animais de Doenças , Glioma/metabolismo , Injeções Intravenosas , Camundongos , Camundongos SCID , Ácido N-Acetilneuramínico/metabolismo , Análise de Sobrevida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Seneca Valley virus (SVV-001) is an oncolytic picornavirus with selective tropism for a subset of human cancers with neuroendocrine differentiation. To characterize further the specificity of SVV-001 and its patterns and kinetics of intratumoral spread, bacterial plasmids encoding a cDNA clone of the full-length wild-type virus and a derivative virus expressing GFP were generated. The full-length cDNA of the SVV-001 RNA genome was cloned into a bacterial plasmid under the control of the T7 core promoter sequence to create an infectious cDNA clone, pNTX-09. A GFP reporter virus cDNA clone, pNTX-11, was then generated by cloning a fusion protein of GFP and the 2A protein from foot-and-mouth disease virus immediately following the native SVV-001 2A sequence. Recombinant GFP-expressing reporter virus, SVV-GFP, was rescued from cells transfected with in vitro RNA transcripts from pNTX-11 and propagated in cell culture. The proliferation kinetics of SVV-001 and SVV-GFP were indistinguishable. The SVV-GFP reporter virus was used to determine that a subpopulation of permissive cells is present in small-cell lung cancer cell lines previously thought to lack permissivity to SVV-001. Finally, it was shown that SVV-GFP administered to tumour-bearing animals homes in to and infects tumours whilst having no detectable tropism for normal mouse tissues at 1×10(11) viral particles kg(-1), a dose equivalent to that administered in ongoing clinical trials. These infectious clones will be of substantial value in further characterizing the biology of this virus and as a backbone for the generation of additional oncolytic derivatives.
Assuntos
Vírus Oncolíticos/genética , Picornaviridae/genética , Sequência de Aminoácidos , Animais , Carcinoma de Células Pequenas/terapia , Carcinoma de Células Pequenas/virologia , Linhagem Celular Tumoral , Clonagem Molecular , Efeito Citopatogênico Viral/genética , DNA Complementar/genética , DNA Viral/genética , Feminino , Genes Reporter , Proteínas de Fluorescência Verde/genética , Humanos , Neoplasias Pulmonares/terapia , Neoplasias Pulmonares/virologia , Camundongos , Camundongos Nus , Dados de Sequência Molecular , Terapia Viral OncolíticaRESUMO
The RNA genome of Seneca Valley virus (SVV), a recently identified picornavirus, contains an internal ribosome entry site (IRES) element which has structural and functional similarity to that from classical swine fever virus (CSFV) and hepatitis C virus, members of the Flaviviridae. The SVV IRES has an absolute requirement for the presence of a short region of virus-coding sequence to allow it to function either in cells or in rabbit reticulocyte lysate. The IRES activity does not require the translation initiation factor eIF4A or intact eIF4G. The predicted secondary structure indicates that the SVV IRES is more closely related to the CSFV IRES, including the presence of a bipartite IIId domain. Mutagenesis of the SVV IRES, coupled to functional assays, support the core elements of the IRES structure model, but surprisingly, deletion of the conserved IIId(2) domain had no effect on IRES activity, including 40S and eIF3 binding. This is the first example of a picornavirus IRES that is most closely related to the CSFV IRES and suggests the possibility of multiple, independent recombination events between the genomes of the Picornaviridae and Flaviviridae to give rise to similar IRES elements.
Assuntos
Picornaviridae/genética , Biossíntese de Proteínas , RNA Viral/genética , Ribossomos/metabolismo , Animais , Extratos Celulares , Linhagem Celular , Vírus da Febre Suína Clássica/genética , Análise Mutacional de DNA , Humanos , Mutação , Conformação de Ácido Nucleico , Pestivirus/genética , Picornaviridae/química , RNA Viral/química , RNA Viral/metabolismo , Coelhos , Deleção de SequênciaRESUMO
Difficulties of drug delivery across the blood-brain barrier (BBB) and failure to eliminate cancer stem cells (CSCs) are believed to be the major causes of tumor recurrences in children with medulloblastoma (MB). Seneca Valley virus-001 (SVV-001) is a naturally occurring oncolytic picornavirus that can be systemically administered. Here, we report its antitumor activities against MB cells in a panel of 10 primary tumor-based orthotopic xenograft mouse models. We found that SVV-001 killed the primary cultured xenograft cells, infected and replicated in tumor cells expressing CSC surface marker CD133, and eliminated tumor cells capable of forming neurospheres in vitro in 5 of the 10 xenograft models. We confirmed that SVV-001 could pass through BBB in vivo. A single i.v. injection of SVV-001 in 2 anaplastic MB models led to widespread infection of the preformed intracerebellar (ICb) xenografts, resulting in significant increase in survival (2.2-5.9-fold) in both models and complete elimination of ICb xenografts in 8 of the 10 long-term survivors. Mechanistically, we showed that the intracellular replication of SVV-001 is mediated through a subverted autophagy that is different from the bona fide autophagic process induced by rapamycin. Our data suggest that SVV-001 is well suited for MB treatment. This work expands the current views in the oncolytic therapy field regarding the utility of oncolytic viruses in simultaneous targeting of stem and nonstem tumor cells.
Assuntos
Barreira Hematoencefálica/virologia , Neoplasias Encefálicas/terapia , Meduloblastoma/terapia , Células-Tronco Neoplásicas/patologia , Terapia Viral Oncolítica , Vírus Oncolíticos/fisiologia , Animais , Apoptose , Western Blotting , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Criança , Humanos , Técnicas Imunoenzimáticas , Injeções Intravenosas , Meduloblastoma/patologia , Camundongos , Camundongos SCID , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de Sobrevida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The crystal structure of Seneca Valley Virus-001 (SVV-001), the representative member of a new genus, Senecavirus, is reported at 2.3A resolution. SVV-001 is the first naturally occurring nonpathogenic picornavirus shown to mediate selective cytotoxicity towards tumor cells with neuroendocrine cancer features. The nonsegmented (+) ssRNA genome of SVV-001 shares closest sequence similarity with the genomes of the members of Cardiovirus. The overall tertiary structure of VP1-VP4 subunits is conserved with the exception of loops, especially those of VP1 that show large deviations relative to the members of the cardioviruses. The surface loops of VP1 and VP2 are predicted to mediate cell tropism of SVV-001. In addition, the organization of the packaged nucleic acid density indicates that certain regions of VP2 and VP4 interact closely with the packaged nucleic acid.
Assuntos
Vírus Oncolíticos/química , Vírus Oncolíticos/classificação , Picornaviridae/química , Picornaviridae/classificação , Modelos Moleculares , Conformação de Ácido Nucleico , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , RNA Viral/química , Receptores Virais/química , Receptores Virais/metabolismo , Vírion/químicaRESUMO
Seneca Valley Virus-001 (SVV-001) is a newly found species in the Picornaviridae family. SVV-001 is the first naturally occurring nonpathogenic picornavirus observed to mediate selective cytotoxicity towards tumor cells with neuroendocrine cancer features. The nonsegmented (+)ssRNA genome of SVV-001 shares closest sequence similarity to the genomes of the members of the Cardiovirus genus. However, based on the distinct characteristics of the genome organization and other biochemical properties, it has been suggested that SVV-001 represents a new genus, namely 'Senecavirus', in the Picornaviridae family. In order to understand the oncolytic properties of SVV-001, the native virus was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to space group R3, with unit-cell parameters (in the hexagonal setting) a = b = 311.5, c = 1526.4 A. Although the SVV crystals diffracted to better than 2.3 A resolution, the data quality is acceptable [I/sigma(I) > 2.0] to 2.6 A resolution. The unit-cell volume and the locked rotation-function analysis suggest that six particles could be accommodated in the unit cell, with two distinct sets of one third of a particle, each containing 20 protomers, occupying the crystallographic asymmetric unit. (ClinicalTrials.gov identifier NCT00314925)
Assuntos
Proteínas do Capsídeo/química , Picornaviridae/química , Difração de Raios X , Proteínas do Capsídeo/isolamento & purificação , Cardiovirus/química , Cristalização , Picornaviridae/isolamento & purificaçãoRESUMO
BACKGROUND: Numerous clinical trials have demonstrated that oncolytic viruses can elicit antitumor responses when they are administered directly into localized cancers. However, the treatment of metastatic disease with oncolytic viruses has been challenging due to the inactivation of viruses by components of human blood and/or to inadequate tumor selectivity. METHODS: We determined the cytolytic potential and selectivity of Seneca Valley Virus-001 (SVV-001), a newly discovered native picornavirus, in neuroendocrine and pediatric tumor cell lines and normal cells. Suitability of the virus for intravenous delivery in humans was assessed by blood inactivation assays. Safety was evaluated in vivo using an immune-competent mouse model, and efficacy was evaluated in vivo in athymic mice bearing tumors derived from human small-cell lung cancer and retinoblastoma cell lines. RESULTS: Cell lines derived from small-cell lung cancers and solid pediatric cancers were at least 10,000-fold more sensitive to the cytolytic activity of SVV-001 than were any of the adult normal human cells tested. Viral infectivity was not inhibited by human blood components. Intravenous doses up to 1 x 10(14) virus particles (vp) per kg were well tolerated, and no dose-limiting toxicity was observed in immune-competent mice. A single intravenous dose of 1 x 10(8) vp per kg into athymic mice bearing preestablished small-cell lung or retinoblastoma tumors resulted in complete, durable responses in ten of ten and five of eight mice, respectively. CONCLUSIONS: SVV-001 has potent cytolytic activity and high selectivity for tumor cell lines having neuroendocrine properties versus adult normal cells. Systemically administered SVV-001 has potential for the treatment of metastatic neuroendocrine cancers.
Assuntos
Antineoplásicos/uso terapêutico , Carcinoma Neuroendócrino/terapia , Terapia Viral Oncolítica/métodos , Vírus Oncolíticos , Picornaviridae , Animais , Carcinoma Neuroendócrino/tratamento farmacológico , Carcinoma de Células Pequenas/terapia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Testes de Hemaglutinação , Humanos , Imuno-Histoquímica , Injeções Intravenosas , Neoplasias Pulmonares/terapia , Camundongos , Camundongos Nus , Vírus Oncolíticos/classificação , Vírus Oncolíticos/patogenicidade , Picornaviridae/classificação , Picornaviridae/patogenicidade , Projetos de Pesquisa , Retinoblastoma/terapia , Transplante HeterólogoRESUMO
Selective replication of oncolytic viruses in tumor cells provides a promising approach for the treatment of human cancers. One of the limitations observed with oncolytic viruses currently used in the treatment of solid tumors is the inefficient spread of virus throughout the tumor mass following intratumoral injection. Data are presented showing that oncolytic adenoviruses expressing the relaxin gene and containing an Ad5/Ad35 chimeric fiber showed significantly enhanced transduction and increased virus spread throughout the tumor when compared with non-relaxin-expressing, Ad5-based viruses. The increased spread of such viruses throughout tumors correlated well with improved antitumor efficacy and overall survival in two highly metastatic tumor models. Furthermore, nonreplicating viruses expressing relaxin did not increase metastases, suggesting that high level expression of relaxin will not enhance metastatic spread of tumors. In summary, the data show that relaxin may play a role in rearranging matrix components within tumors, which helps recombinant oncolytic adenoviruses to spread effectively throughout the tumor mass and thereby increase the extent of viral replication within the tumor. Expressing relaxin from Ad5/Ad35 fiber chimeric adenoviruses may prove a potent and novel approach to treating patients with cancer.
Assuntos
Adenoviridae/fisiologia , Terapia Viral Oncolítica/métodos , Neoplasias da Próstata/terapia , Relaxina/biossíntese , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Linhagem Celular Tumoral , Colágeno/metabolismo , Feminino , Vetores Genéticos/genética , Humanos , Masculino , Camundongos , Metástase Neoplásica , Neoplasias da Próstata/genética , Neoplasias da Próstata/virologia , Relaxina/genética , Replicação Viral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
One of the most time-consuming steps in the generation of adenoviral vectors is the construction of recombinant plasmids. This chapter describes a detailed method for the rapid construction of adenoviral vectors. The method described here uses homologous recombination machinery of Escherichia coli BJ5183 to construct plasmids used in generation of adenoviral vectors. With this method, no ligation steps are involved in generating the plasmids, and any region of the adenoviral genome can be easily modified. Briefly, the full-length adenoviral genome flanked by unique restriction enzyme sites is first cloned into a bacterial plasmid. Next, the region of the viral genome to be modified is subcloned into a bacterial shuttle plasmid, and the desired changes are introduced by molecular biology techniques. The modified viral DNA fragment is gel-purified and cotransformed with the full-length plasmid, linearized in the targeted region, into BJ5183 cells. Homologous recombination in E. coli generates plasmids containing the modified adenoviral genome. Recombinant virus is generated following release of the viral DNA sequences from the plasmid backbone and transfection into a producer cell line. With this method, homogeneous recombinant adenoviruses can be obtained without plaque purification.
Assuntos
Adenoviridae/genética , Escherichia coli/genética , Recombinação Genética , DNA Viral/genética , Vetores Genéticos , Genoma Viral , Indicadores e Reagentes , Plasmídeos , Transfecção/métodosRESUMO
PURPOSE: Transcriptionally controlled oncolytic adenovirus CG5757 is engineered with two tumor-specific promoters from E2F-1 and human telomerase reverse transcriptase genes. This virus has broad anticancer spectrum and higher specificity. The objective of the current study is to show its antitumor selectivity and therapeutic potential. EXPERIMENTAL DESIGN: The antitumor specificity of E2F-1 and human telomerase reverse transcriptase promoters was evaluated in a panel of tumor and normal cells. Under the control of these promoters, the tumor-selective expression of E1a and E1b genes was evaluated. Further in vitro antitumor specificity and potency of this virus were characterized by viral replication and cytotoxicity assays followed by a newly developed ex vivo tumor culture assay. Subsequently, in vivo antitumor efficacy and toxicology studies were carried out to assess the therapeutic potential of this oncolytic agent. RESULTS: In a broad panel of cells, E2F-1 and human telomerase reverse transcriptase promoters were activated in a tumor-selective manner. Under the control of these promoters, expression of E1a and E1b genes appears only in tumor cells. This specificity is extended to viral replication and hence the cytotoxicity in a broad range of cancer cells. Furthermore, CG5757 only replicates in cancer tissues but not in normal tissues that are derived from clinical biopsies. The safety profile was further confirmed in in vivo toxicology studies, and strong efficacy was documented in several tumor xenograft models after CG5757 was given via different routes and regimens. CONCLUSIONS: CG5757 has strong antitumor selectivity and potency. It has low toxicity and has great potential as a therapeutic agent for different types of cancers.
Assuntos
Adenoviridae/genética , Proteínas de Ligação a DNA/genética , Neoplasias/terapia , Regiões Promotoras Genéticas/genética , Telomerase/genética , Animais , Linhagem Celular Tumoral , Expressão Gênica , Genes Virais/genética , Terapia Genética , Humanos , Masculino , Camundongos , Camundongos Nus , Replicação Viral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Adenovirus serotype 5 (Ad5)-based vectors can bind at least three separate cell surface receptors for efficient cell entry: the coxsackie-adenovirus receptor (CAR), alpha nu integrins, and heparan sulfate glycosaminoglycans (HSG). To address the role of each receptor involved in adenoviral cell entry, we mutated critical amino acids in fiber or penton to inhibit receptor interaction. A series of five adenoviral vectors was prepared and the biodistribution of each was previously characterized in mice. To evaluate possible species differences in Ad vector tropism, we characterized the effects of each detargeting mutation in non-human primates after systemic delivery to confirm our conclusions made in mice. In non-human primates, CAR was found to have minimal effects on vector delivery to all organs examined including liver and spleen. Cell-surface alpha nu integrins played a significant role in delivery of vector to the spleen, lung and kidney. The fiber shaft mutation S*, which presumably inhibits HSG binding, was found to significantly decrease delivery to all organs examined. The ability to detarget the liver corresponded with decreased elevations in liver serum enzymes (aspartate transferase [AST] and alanine transferase [ALT]) 24 hr after vector administration and also in serum interleukin (IL)-6 levels 6 hr after vector administration. The biodistribution data generated in cynomolgus monkeys correspond with those data derived from mice, demonstrating that CAR binding is not the major determinant of viral tropism in vivo. Vectors containing the fiber shaft modification may provide for a detargeted adenoviral vector on which to introduce new tropisms for the development of targeted, systemically deliverable adenoviral vectors for human clinical application.
Assuntos
Adenoviridae/genética , Técnicas de Transferência de Genes , Animais , Proteínas do Capsídeo/química , Membrana Celular/metabolismo , Vetores Genéticos , Humanos , Imuno-Histoquímica , Integrina alfaV/biossíntese , Interleucina-6/biossíntese , Interleucina-6/metabolismo , Fígado/metabolismo , Macaca fascicularis , Masculino , Camundongos , Mutação , Ligação Proteica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Distribuição Tecidual , Transdução GenéticaRESUMO
Adenoviral vectors used in gene therapy are predominantly derived from adenovirus serotype 5 (Ad5), which infects a broad range of cells. Ad5 cell entry involves interactions with the coxsackie-adenovirus receptor (CAR) and integrins. To assess these receptors in vivo, we mutated amino acid residues in fiber and penton that are involved in receptor interaction and showed that CAR and integrins play a minor role in hepatic transduction but that integrins can influence gene delivery to other tissues. These data suggest that an alternative entry pathway exists for hepatocyte transduction in vivo that is more important than CAR or integrins. In vitro data suggest a role for heparan sulfate glycosaminoglycans (HSG) in adenovirus transduction. The role of the fiber shaft in liver uptake was examined by introducing specific amino acid changes into a putative HSG-binding motif contained within the shaft or by preparing fiber shaft chimeras between Ad5 and Ad35 fibers. Results were obtained that demonstrate that the Ad5 fiber shaft can influence gene transfer both in vitro and to the liver in vivo. These observations indicate that the currently accepted two-step entry pathway, which involves CAR and integrins, described for adenoviral infection in vitro, is not used for hepatic gene transfer in vivo. In contrast, alpha(v) integrins influence gene delivery to the lung, spleen, heart, and kidney. The detargeted vector constructs described here may provide a foundation for the development of targeted adenoviral vectors.
Assuntos
Adenoviridae/genética , Proteínas do Capsídeo/genética , Vetores Genéticos/farmacocinética , Transdução Genética , Motivos de Aminoácidos , Animais , Western Blotting , Proteínas do Capsídeo/metabolismo , Linhagem Celular , Células Cultivadas , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Vetores Genéticos/administração & dosagem , Vetores Genéticos/sangue , Glicosaminoglicanos/fisiologia , Hepatócitos/metabolismo , Humanos , Integrinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Receptores Virais/metabolismo , Vírion/metabolismo , beta-Galactosidase/genéticaRESUMO
Systemic administration of adenoviral vectors leads to a widespread distribution of vector. Therefore, targeting of adenoviral vectors to specific tissues or cell types will require methods to ablate the normal tropism of the vector simultaneously with the introduction of new receptor specificities. To inhibit native receptor binding, we mutated residues in the AB loop of the adenovirus type 5 (Ad5) fiber. We genetically incorporated the S408E-P409A mutation, referred to as KO1, into the adenoviral genome alone or in combination with an RGD-targeting ligand in the HI loop of fiber. Transduction experiments confirmed that the KO1 mutation results in a significant reduction in fiber-dependent gene transfer on A549 and primary fibroblast cells that could be restored via the RGD-targeting ligand. Competition transduction experiments verified the receptor-binding properties of each vector on A549 and hepatocytes in vitro. Unexpectedly, in mice systemic delivery of the vector containing the KO1 mutation resulted in efficient liver transduction that was localized specifically to hepatocytes. We confirmed these results in three different mouse strains, indicating that hepatic adenoviral gene transfer may be independent of the coxsackievirus-adenovirus receptor and that in vivo retargeting will require further viral capsid modifications to generate a fully detargeted adenoviral vector upon which to introduce new tropisms.
Assuntos
Adenovírus Humanos/genética , Vetores Genéticos , Receptores Virais/genética , Transdução Genética , Adenovírus Humanos/metabolismo , Animais , Western Blotting , Proteínas do Capsídeo/genética , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Feminino , Terapia Genética , Células HeLa , Hepatócitos/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mutação , Oligopeptídeos/genética , Receptores Virais/metabolismo , Especificidade da Espécie , Tropismo/genética , Células Tumorais CultivadasRESUMO
Gutless adenoviral vectors are devoid of all viral coding regions and display reduced cytotoxicity, diminished immunogenicity, and an increased coding capacity compared with early generation vectors. Using hemophilia A, a deficiency in clotting factor VIII (FVIII), as a model disease, we generated and evaluated a gutless vector encoding human FVIII. The FVIII gutless vector grew to high titer and was reproducibly scaled-up from vector seed lots. Extensive viral DNA analyses revealed no rearrangements of the vector genome. A quantitative PCR assay demonstrated helper virus contamination levels of <2%, with the best preparation containing 0.3% helper virus. We compared the gutless vector with an E1/E2a/E3-deficient (Av3) early generation vector encoding an identical FVIII expression cassette following intravenous administration to hemophilia A mice. Gutless vector-treated mice displayed 10-fold higher FVIII expression levels that were sustained for at least 9 months. In contrast, mice treated with the Av3 vector displayed FVIII levels below the limit of sensitivity of the assay at 3 months. Assessment of hepatotoxicity by measuring the serum levels of liver enzymes demonstrated that the gutless vector was significantly less toxic than the Av3 vector at time points later than 7 days. At the highest dose used, both vectors caused a transient 10-fold increase in liver enzymes 1 day after vector administration, suggesting that this increase was caused by direct toxicity of the input capsid proteins. These data demonstrate that the gutless vector displayed increased duration and levels of FVIII expression, and was significantly less toxic than an analogous early generation vector.
Assuntos
Adenoviridae/genética , Fator VIII/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Hemofilia A/genética , Animais , Sequência de Bases , Primers do DNA , Fator VIII/administração & dosagem , Feminino , Terapia Genética , Hemofilia A/terapia , Humanos , Camundongos , Reação em Cadeia da Polimerase , Transdução GenéticaRESUMO
Adenovirus binds to mammalian cells via interaction of fiber with the coxsackie-adenovirus receptor (CAR). Redirecting adenoviral vectors to enter target cells via new receptors has the advantage of increasing the efficiency of gene delivery and reducing nonspecific transduction of untargeted tissues. In an attempt to reach this goal, we have produced bifunctional molecules with soluble CAR (sCAR), which is the extracellular domain of CAR fused to peptide-targeting ligands. Two peptide-targeting ligands have been evaluated: a cyclic RGD peptide (cRGD) and the receptor-binding domain of apolipoprotein E (ApoE). Human diploid fibroblasts (HDF) are poorly transduced by adenovirus due to a lack of CAR on the surface. Addition of the sCAR-cRGD or sCAR-ApoE targeting protein to adenovirus redirected binding to the appropriate receptor on HDF. However, a large excess of the monomeric protein was needed for maximal transduction, indicating a suboptimal interaction. To improve interaction of sCAR with the fiber knob, an isoleucine GCN4 trimerization domain was introduced, and trimerization was verified by cross-linking analysis. Trimerized sCAR proteins were significantly better at interacting with fiber and inhibiting binding to HeLa cells. Trimeric sCAR proteins containing cRGD and ApoE were more efficient at transducing HDF in vitro than the monomeric proteins. In addition, the trimerized sCAR protein without targeting ligands efficiently blocked liver gene transfer in normal C57BL/6 mice. However, addition of either ligand failed to retarget the liver in vivo. One explanation may be the large complex size, which serves to decrease the bioavailability of the trimeric sCAR-adenovirus complexes. In summary, we have demonstrated that trimerization of sCAR proteins can significantly improve the potency of this targeting approach in altering vector tropism in vitro and allow the efficient blocking of liver gene transfer in vivo.