RESUMO
The macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine that recently emerged as an attractive therapeutic target for a variety of diseases. A diverse panel of fully human anti-MIF antibodies was generated by selection from a phage display library and extensively analyzed in vitro. Epitope mapping studies identified antibodies specific for linear as well as structural epitopes. Experimental animal studies revealed that only those antibodies binding epitopes within amino acids 50-68 or 86-102 of the MIF molecule exerted protective effects in models of sepsis or contact hypersensitivity. Within the MIF protein, these two binding regions form a ß-sheet structure that includes the MIF oxidoreductase motif. We therefore conclude that this ß-sheet structure is a crucial region for MIF activity and a promising target for anti-MIF antibody therapy.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Neutralizantes/química , Oxirredutases Intramoleculares/química , Fatores Inibidores da Migração de Macrófagos/química , Motivos de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/uso terapêutico , Dermatite de Contato/tratamento farmacológico , Dermatite de Contato/imunologia , Modelos Animais de Doenças , Humanos , Oxirredutases Intramoleculares/imunologia , Fatores Inibidores da Migração de Macrófagos/imunologia , Camundongos , Sepse/tratamento farmacológico , Sepse/imunologiaRESUMO
Background: Activated cardiac fibroblasts (CF) play a central role in cardiac fibrosis, a condition associated with most cardiovascular diseases. Conversion of quiescent into activated CF sustains heart integrity upon injury. However, permanence of CF in active state inflicts deleterious heart function effects. Mechanisms underlying this cell state conversion are still not fully disclosed, contributing to a limited target space and lack of effective anti-fibrotic therapies. Materials and methods: To prioritize targets for drug development, we studied CF remodeling upon activation at transcriptomic and proteomic levels, using three different cell sources: primary adult CF (aHCF), primary fetal CF (fHCF), and induced pluripotent stem cells derived CF (hiPSC-CF). Results: All cell sources showed a convergent response upon activation, with clear morphological and molecular remodeling associated with cell-cell and cell-matrix interactions. Quantitative proteomic analysis identified known cardiac fibrosis markers, such as FN1, CCN2, and Serpine1, but also revealed targets not previously associated with this condition, including MRC2, IGFBP7, and NT5DC2. Conclusion: Exploring such targets to modulate CF phenotype represents a valuable opportunity for development of anti-fibrotic therapies. Also, we demonstrate that hiPSC-CF is a suitable cell source for preclinical research, displaying significantly lower basal activation level relative to primary cells, while being able to elicit a convergent response upon stimuli.
RESUMO
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
Antibody phage display, coupled with automated screening, facilitates and potentiates the mining of complex combinatorial libraries and the identification of potent drug leads. In managing phage screening data, the behavior of individual phage isolates in binding assays must be linked to their antibody identities as deduced from DNA sequencing. Reviewed here are recently reported approaches for high-throughput screening of clones isolated from phage antibody libraries after selection on a defined antigen. Specific information management challenges, and possible solutions, are described for organizing screening data to enable rapid lead discovery using these antibody libraries.
Assuntos
Anticorpos/química , Biblioteca de Peptídeos , Animais , Anticorpos/isolamento & purificação , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Análise Serial de Proteínas , Proteínas/imunologiaRESUMO
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.