RESUMO
Inhibition of specific matrix metalloproteinases (MMP) is an attractive noncytotoxic approach to cancer therapy. MMP-14, a membrane-bound zinc endopeptidase, has been proposed to play a central role in tumor growth, invasion, and neovascularization. Besides cleaving matrix proteins, MMP-14 activates proMMP-2 leading to an amplification of pericellular proteolytic activity. To examine the contribution of MMP-14 to tumor growth and angiogenesis, we used DX-2400, a highly selective fully human MMP-14 inhibitory antibody discovered using phage display technology. DX-2400 blocked proMMP-2 processing on tumor and endothelial cells, inhibited angiogenesis, and slowed tumor progression and formation of metastatic lesions. The combination of potency, selectivity, and robust in vivo activity shows the potential of a selective MMP-14 inhibitor for the treatment of solid tumors.
Assuntos
Anticorpos Monoclonais , Antineoplásicos/uso terapêutico , Divisão Celular/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Inibidores de Metaloproteinases de Matriz , Neovascularização Patológica/prevenção & controle , Animais , Anticorpos Monoclonais Humanizados , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Feminino , Genes Reporter , Humanos , Imuno-Histoquímica , Camundongos , Invasividade Neoplásica/patologia , Transfecção , Transplante Heterólogo , Veias Umbilicais/citologia , Veias Umbilicais/efeitos dos fármacosRESUMO
A number of small charged carbohydrate moieties have been associated with inflammation and cancer. However, the development of therapeutic Abs targeting these moieties has been hampered by their low immunogenicity and their structural relationship to self-Ag. We report the design of an Ab repertoire enriched in Abs binding to small charged carbohydrates and the construction of a human Fab phagemid library, "FAB-CCHO." This library combines L chain Ig sequences from human donors and H chain synthetic diversity constructed in key Ag contact sites in CDRs 1, 2, and 3 of the human framework V(H)3-23. The H chain CDR3 has been engineered to enrich the library in Abs that bind charged carbohydrates by the introduction of basic residues at specific amino acid locations. These residues were selected on the basis of anti-carbohydrate Ab sequence alignment. The success of this design is demonstrated by the isolation of phage Abs against charged carbohydrate therapeutic target Ags such as sulfated sialyl-Lewis X glycan and heparan sulfate.
Assuntos
Bacteriófago M13/genética , Regiões Determinantes de Complementaridade/genética , Fragmentos Fab das Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/genética , Oligossacarídeos/genética , Oligossacarídeos/imunologia , Biblioteca de Peptídeos , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Animais , Diversidade de Anticorpos , Bacteriófago M13/química , Bacteriófago M13/imunologia , Sítios de Ligação de Anticorpos , Sequência de Carboidratos , Regiões Determinantes de Complementaridade/química , Desenho de Fármacos , Humanos , Fragmentos Fab das Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/química , Antígenos do Grupo Sanguíneo de Lewis , Camundongos , Dados de Sequência Molecular , Oligossacarídeos/química , Eletricidade EstáticaRESUMO
The use of oligonucleotide-assisted cleavage and ligation (ONCL), a novel approach to the capture of gene repertoires, in the construction of a phage-display immune antibody library is described. ONCL begins with rapid amplification of cDNA ends to amplify all members equally. A single, specific cut near 5' and/or 3' end of each gene fragment (in single stranded form) is facilitated by hybridization with an appropriate oligonucleotide adapter. Directional cloning of targeted DNA is accomplished by ligation of a partially duplex DNA molecule (containing suitable restriction sites) and amplification with primers in constant regions. To demonstrate utility and reliability of ONCL, a human antibody repertoire was cloned from IgG mRNA extracted from human B-lymphocytes engrafted in Trimera mice. These mice were transplanted with peripheral blood lymphocytes from Candida albicans infected individuals and subsequently immunized with C.albicans glyceraldehyde-3-phosphate dehydrogenase (GAPDH). DNA sequencing showed that ONCL resulted in efficient capture of gene repertoires. Indeed, full representation of all V(H) families/segments was observed showing that ONCL did not introduce cloning biases for or against any V(H) family. We validated the efficiency of ONCL by creating a functional Fab phage-display library with a size of 3.3 x 10(10) and by selecting five unique Fabs against GAPDH antigen.
Assuntos
Clonagem Molecular/métodos , DNA Complementar , Genes de Imunoglobulinas , Oligonucleotídeos/química , Biblioteca de Peptídeos , Adolescente , Adulto , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Biotecnologia/métodos , Candida albicans/enzimologia , Candida albicans/imunologia , Feminino , Gliceraldeído-3-Fosfato Desidrogenases/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/genética , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Oligonucleotídeos/metabolismo , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
Combinatorial libraries of rearranged hypervariable V(H) and V(L) sequences from nonimmunized human donors contain antigen specificities, including anti-self reactivities, created by random pairing of V(H)s and V(L)s. Somatic hypermutation of immunoglobulin genes, however, is critical in the generation of high-affinity antibodies in vivo and occurs only after immunization. Thus, in combinatorial phage display libraries from nonimmunized donors, high-affinity antibodies are rarely found. Lengthy in vitro affinity maturation is often needed to improve antibodies from such libraries. We report the construction of human Fab libraries having a unique combination of immunoglobulin sequences captured from human donors and synthetic diversity in key antigen contact sites in heavy-chain complementarity-determining regions 1 and 2. The success of this strategy is demonstrated by identifying many monovalent Fabs against multiple therapeutic targets that show higher affinities than approved therapeutic antibodies. This very often circumvents the need for affinity maturation, accelerating discovery of antibody drug candidates.