RESUMO
The ability to isolate antibodies against any antigen of interest has become increasingly important as antibodies have proved their utility both in antigen detection, quantification and as specific in vivo targeting agents. To this end, we have constructed a large antibody phage library in the single chain Fv (scFv) phagemid format based on the naive human variable (V) gene repertoire dictated by IgD and IgM. Optimizing each step of the library construction has resulted in a highly diverse and functional library, as assessed by sequencing analysis, large-scale automated expression analysis and antigen screening. Furthermore, the versatile format of the library, which comprises 14 separate sub-libraries, adds considerably flexibility with respect to which part of the antibody repertoire that is to be probed. This versatility has been further exploited to generate a refined antibody library, which exhibits one of the highest prokaryotic expression levels reported to date for a naive repertoire. The construction of the refined library was based on the functional purification of expressed V genes in the context of the protein L interaction with correctly folded V genes of the kappa light chain family. Antigen screening of this library indicated that the functional purification improved the ability to retrieve antigen specific antibodies, but at the cost of potential loss of diversity in the isolated repertoire.
Assuntos
Anticorpos/isolamento & purificação , Imunoglobulina D/genética , Imunoglobulina M/genética , Região Variável de Imunoglobulina/genética , Biblioteca de Peptídeos , Sequência de Aminoácidos , Anticorpos/genética , Antígenos/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Leves de Imunoglobulina/genética , Dados de Sequência MolecularRESUMO
The factor VII genes of five unrelated Finnish female patients, F1-F5, with moderate bleeding tendency, were screened for mutations using single strand conformational polymorphisms and DNA sequencing. Heterozygous shifts were detected in exons 5 and 8 for patient F1, and sequencing confirmed the presence of the silent dimorphism H115H, the polymorphism R353Q and the mutation A294V. The patient F1 was also heterozygous for a novel -59T/G transversion mutation in the Hepatocyte nuclear factor 4-binding site. The remaining four patients carried a -32A/C transversion mutation located in a footprint (-51 to -32) covering the major transcription initiation start site -51). There was also a consensus sequence match to an initiator response-like binding element covering -51. Two patients were homozygous and two heterozygous for this mutation. Plasma FVII:Ag and FVII:C levels were reduced in parallel. A strong reduction in binding affinity of a specific nuclear protein to the -32C-containing oligonucleotide was found by electrophoretic mobility shift assays on nuclear extracts from HepG2 cells. EDTA caused no reduced binding. A minimal promoter (-191 to +15) containing the wild-type sequence or the -32A/C or -59T/G mutations was cloned in front of the firefly luciferase reporter gene and transiently transfected into Hep3B cells. Reduced activities [23.0 +/- 3.1% (-32C), 55.4 +/- 6.3% (-59G), 100% (wild-type construct)] were found for the mutated promoters. Southwestern blotting and UV crosslinking analysis showed binding of three proteins (20, 20 and 50 kDa) to the putative initiator response element. The -32A/C mutant oligonucleotide bound two proteins.
Assuntos
Proteínas de Ligação a DNA , Deficiência do Fator VII/genética , Fator VII/genética , Mutação , Regiões Promotoras Genéticas/genética , Adulto , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Sítios de Ligação/genética , Linhagem Celular , Quelantes/farmacologia , DNA/genética , Ácido Edético/farmacologia , Eletroforese , Feminino , Fator 4 Nuclear de Hepatócito , Heterozigoto , Homozigoto , Humanos , Pessoa de Meia-Idade , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Polimorfismo Conformacional de Fita Simples , Titulometria , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , TransfecçãoRESUMO
A novel mutation in the factor VII gene resulting in procoagulant activity of 7.5% and antigen levels of 23% is presented. Single-stranded conformational polymorphism and DNA sequencing analysis revealed heterozygous shifts, and mutations were detected in exons 5, 7 and 8. The mutant L204P in exon 7 was novel, while the common polymorphisms, H115H and R353Q, were located in exons 5 and 8, respectively. The molecular effect of the L204P mutation was characterized using recombinant mammalian expression in Chinese hamster ovary cells. Low levels (4 ng/ml) of secreted mutant protein were found in transiently transfected cells compared to wild-type factor VII (83 ng/ml). Metabolic labeling demonstrated that the rate of mutant protein synthesis was similar to that of wild-type FVII, and the mutant protein accumulated intracellularly with no signs of increased degradation during a four-hour chase. No interaction between secreted P204 protein and immobilized soluble tissue factor was detected using surface plasmon reso-nance. The activation rate of recombinant mutant FVII protein was strongly reduced compared to wild-type FVII. A 9-fold reduction in the rate of FX activation was detected whereas Km was nearly the same for wild-type and the mutant. This slow rate was caused by a correspondingly lowered rate of P204 activation. A synthetic peptide sequence comprising amino acids 177-206 blocked binding of FVIIa to the TF-chip, and the subsequent factor X activation with an IC(50) value of 0.5 micro M in a chromogenic factor Xa assay. Additionally, evaluation of the peptide by surface plasmon resonance analysis resulted in inhibition of complex formation with an apparent K(I) of 7 micro M.
Assuntos
Acetilcisteína/análogos & derivados , Fator VII/química , Fator VII/genética , Mutação , Acetilcisteína/química , Animais , Anticorpos Monoclonais/química , Encéfalo/metabolismo , Brefeldina A/química , Células CHO , Dicroísmo Circular , Cricetinae , Inibidores de Cisteína Proteinase/farmacologia , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Precursores Enzimáticos/química , Éxons , Fator X/química , Fator Xa/química , Heterozigoto , Humanos , Concentração Inibidora 50 , Cinética , Modelos Moleculares , Peptídeos/química , Reação em Cadeia da Polimerase , Polimorfismo Genético , Polimorfismo Conformacional de Fita Simples , Testes de Precipitina , Ligação Proteica , Estrutura Terciária de Proteína , Inibidores da Síntese de Proteínas/farmacologia , Proteínas Recombinantes/química , Mapeamento por Restrição , Análise de Sequência de DNA , Serina Endopeptidases/química , Ressonância de Plasmônio de Superfície , Fatores de Tempo , TransfecçãoRESUMO
There is increasing experimental evidence for an important role of Angiopoietin-2 (Ang-2) in tumor angiogenesis and progression. In addition, Ang-2 is up-regulated in many cancer types and correlated with poor prognosis. To investigate the functional role of Ang-2 inhibition in tumor development and progression, we generated novel fully human antibodies that neutralize specifically the binding of Ang-2 to its receptor Tie2. The selected antibodies LC06 and LC08 recognize both rodent and human Ang-2 with high affinity, but LC06 shows a higher selectivity for Ang-2 over Ang-1 compared to LC08 which can be considered an Ang-2/Ang-1 cross-reactive antibody. Our data demonstrate that Ang-2 blockade results in potent tumor growth inhibition and pronounced tumor necrosis in subcutaneous and orthotopic tumor models. These effects are attended with a reduction of intratumoral microvessel density and tumor vessels characterized by fewer branches and increased pericyte coverage. Furthermore, anti-Ang-2 treatment strongly inhibits the dissemination of tumor cells to the lungs. Interestingly, in contrast to the Ang-2/Ang-1 cross-reactive antibody LC08 that leads to a regression of physiological vessels in the mouse trachea, the inhibition with the selective anti-Ang-2 antibody LC06 appears to be largely restricted to tumor vasculature without obvious effects on normal vasculature. Taken together, these data provide strong evidence for the selective Ang-2 antibody LC06 as promising new therapeutic agent for the treatment of various cancers.
Assuntos
Angiopoietina-1/antagonistas & inibidores , Angiopoietina-2/antagonistas & inibidores , Angiopoietina-2/imunologia , Anticorpos Neutralizantes/farmacologia , Antineoplásicos/farmacologia , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/tratamento farmacológico , Angiopoietina-1/imunologia , Animais , Anticorpos Neutralizantes/imunologia , Especificidade de Anticorpos , Antineoplásicos/imunologia , Linhagem Celular Tumoral , Neoplasias do Colo/imunologia , Córnea/efeitos dos fármacos , Córnea/imunologia , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Microvasos/efeitos dos fármacos , Microvasos/imunologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/imunologia , Fosforilação , Distribuição Aleatória , Receptor TIE-2/antagonistas & inibidores , Receptor TIE-2/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Vascular endothelial growth factor (VEGF) is critical for physiological and pathological angiogenesis. Within the tumor microenvironment, VEGF functions as an endothelial cell survival factor, permeability factor, mitogen, and chemotactic agent. The majority of these functions are mediated by VEGF-induced activation of VEGF receptor 2 (VEGFR2), a high affinity receptor tyrosine kinase expressed by endothelial cells and other cell types in the tumor microenvironment. VEGF can also ligate other cell surface receptors including VEGFR1 and neuropilin-1 and -2. However, the importance of VEGF-induced activation of these receptors in tumorigenesis is still unclear. We report the development and characterization of r84, a fully human monoclonal antibody that binds human and mouse VEGF and selectively blocks VEGF from interacting with VEGFR2 but does not interfere with VEGF:VEGFR1 interaction. Selective blockade of VEGF binding to VEGFR2 by r84 is shown through ELISA, receptor binding assays, receptor activation assays, and cell-based functional assays. Furthermore, we show that r84 has potent anti-tumor activity and does not alter tissue histology or blood and urine chemistry after chronic high dose therapy in mice. In addition, chronic r84 therapy does not induce elevated blood pressure levels in some models. The ability of r84 to specifically block VEGF:VEGFR2 binding provides a valuable tool for the characterization of VEGF receptor pathway activation during tumor progression and highlights the utility and safety of selective blockade of VEGF-induced VEGFR2 signaling in tumors.
Assuntos
Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/toxicidade , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Fator A de Crescimento do Endotélio Vascular/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/imunologia , Antineoplásicos/uso terapêutico , Ligação Competitiva , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Feminino , Humanos , Vasos Linfáticos/efeitos dos fármacos , Vasos Linfáticos/metabolismo , Camundongos , Microvasos/efeitos dos fármacos , Microvasos/metabolismo , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Especificidade por Substrato , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Traditional antibody-mediated neutralization of HIV-1 infection is thought to result from the binding of antibodies to virions, thus preventing virus entry. However, antibodies that broadly neutralize HIV-1 are rare and are not induced by current vaccines. We report that four human anti-phospholipid monoclonal antibodies (mAbs) (PGN632, P1, IS4, and CL1) inhibit HIV-1 CCR5-tropic (R5) primary isolate infection of peripheral blood mononuclear cells (PBMCs) with 80% inhibitory concentrations of <0.02 to approximately 10 microg/ml. Anti-phospholipid mAbs inhibited PBMC HIV-1 infection in vitro by mechanisms involving binding to monocytes and triggering the release of MIP-1alpha and MIP-1beta. The release of these beta-chemokines explains both the specificity for R5 HIV-1 and the activity of these mAbs in PBMC cultures containing both primary lymphocytes and monocytes.