RESUMO
For more than 2 centuries active immunotherapy has been at the forefront of efforts to prevent infectious disease [Waldmann TA (2003) Nat Med 9:269-277]. However, the decreased ability of the immune system to mount a robust immune response to self-antigens has made it more difficult to generate therapeutic vaccines against cancer or chronic degenerative diseases. Recently, we showed that the site-specific incorporation of an immunogenic unnatural amino acid into an autologous protein offers a simple and effective approach to overcome self-tolerance. Here, we characterize the nature and durability of the polyclonal IgG antibody response and begin to establish the generality of p-nitrophenylalanine (pNO(2)Phe)-induced loss of self-tolerance. Mutation of several surface residues of murine tumor necrosis factor-alpha (mTNF-alpha) independently to pNO(2)Phe leads to a T cell-dependent polyclonal and sustainable anti-mTNF-alpha IgG autoantibody response that lasts for at least 40 weeks. The antibodies bind multiple epitopes on mTNF-alpha and protect mice from severe endotoxemia induced by lipopolysaccharide (LPS) challenge. Immunization of mice with a pNO(2)Phe(43) mutant of murine retinol-binding protein (RBP4) also elicited a high titer IgG antibody response, which was cross-reactive with wild-type mRBP4. These findings suggest that this may be a relatively general approach to generate effective immunotherapeutics against cancer-associated or other weakly immunogenic antigens.
Assuntos
Aminoácidos/genética , Imunoterapia/métodos , Engenharia de Proteínas/métodos , Tolerância a Antígenos Próprios/imunologia , Aminoácidos/imunologia , Animais , Formação de Anticorpos , Autoanticorpos , Autoantígenos/genética , Imunoglobulina G , Camundongos , Fenilalanina/análogos & derivados , Fenilalanina/genética , Linfócitos T/imunologia , Fator de Necrose Tumoral alfa/genéticaRESUMO
We have devised a phage display system in which an expanded genetic code is available for directed evolution. This system allows selection to yield proteins containing unnatural amino acids should such sequences functionally outperform ones containing only the 20 canonical amino acids. We have optimized this system for use with several unnatural amino acids and provide a demonstration of its utility through the selection of anti-gp120 antibodies. One such phage-displayed antibody, selected from a naïve germline scFv antibody library in which six residues in V(H) CDR3 were randomized, contains sulfotyrosine and binds gp120 more effectively than a similarly displayed known sulfated antibody isolated from human serum. These experiments suggest that an expanded "synthetic" genetic code can confer a selective advantage in the directed evolution of proteins with specific properties.
Assuntos
Evolução Molecular Direcionada , Código Genético , Proteínas/genética , Aminoácidos/genética , Anticorpos Antivirais/imunologia , Ensaio de Imunoadsorção Enzimática , Escherichia coli , Vetores Genéticos , Células Germinativas/imunologia , HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Humanos , Biblioteca de Peptídeos , Análise de Sequência de Proteína , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
The ability to selectively induce a strong immune response against self-proteins, or increase the immunogenicity of specific epitopes in foreign antigens, would have a significant impact on the production of vaccines for cancer, protein-misfolding diseases, and infectious diseases. Here, we show that site-specific incorporation of an immunogenic unnatural amino acid into a protein of interest produces high-titer antibodies that cross-react with WT protein. Specifically, mutation of a single tyrosine residue (Tyr(86)) of murine tumor necrosis factor-alpha (mTNF-alpha) to p-nitrophenylalanine (pNO(2)Phe) induced a high-titer antibody response in mice, whereas no significant antibody response was observed for a Tyr(86) --> Phe mutant. The antibodies generated against the pNO(2)Phe are highly cross-reactive with native mTNF-alpha and protect mice against lipopolysaccharide (LPS)-induced death. This approach may provide a general method for inducing an antibody response to specific epitopes of self- and foreign antigens that lead to a neutralizing immune response.
Assuntos
Substituição de Aminoácidos , Formação de Anticorpos/efeitos dos fármacos , Mutação de Sentido Incorreto , Tolerância a Antígenos Próprios/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Animais , Formação de Anticorpos/genética , Formação de Anticorpos/imunologia , Doenças Transmissíveis/genética , Doenças Transmissíveis/imunologia , Endotoxemia/induzido quimicamente , Endotoxemia/tratamento farmacológico , Endotoxemia/genética , Endotoxemia/imunologia , Epitopos/genética , Epitopos/imunologia , Epitopos/farmacologia , Imunoquímica , Lipopolissacarídeos/toxicidade , Masculino , Doenças Metabólicas/genética , Doenças Metabólicas/imunologia , Camundongos , Neoplasias/genética , Neoplasias/imunologia , Nitrofenóis/imunologia , Nitrofenóis/farmacologia , Tolerância a Antígenos Próprios/genética , Tolerância a Antígenos Próprios/imunologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologia , Vacinas/genética , Vacinas/imunologiaRESUMO
We report a general method to display peptide-containing unnatural amino acids on filamentous M13 phage. Five distinct unnatural amino acids were site-specifically incorporated at the N-terminal of the M13 phage minor coat protein pIII. Phages that contain p-azidophenylalanine can undergo a highly specific azide-alkyne [3 + 2] cycloaddition reaction with an alkyne-derivatized fluorophore. The generalization of phage display to include unnatural amino acids should significantly increase the scope of phage display technology.
Assuntos
Aminoácidos/genética , Aminoácidos/metabolismo , Biossíntese Peptídica/genética , Biblioteca de Peptídeos , Proteínas do Capsídeo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Mathanococcus/genética , Mathanococcus/metabolismo , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/metabolismoRESUMO
The structures of halogen atom-benzene complexes were investigated by modern DFT and ab initio computational methods. The spectroscopic properties of the complexes are also predicted and are in good agreement with experiment where such data have been reported. The fluorine atom-benzene complex is predicted to be a sigma complex due to the strength of a C-F bond. The chlorine atom-benzene complex is predicted to have an eta(1) pi complex structure, which is only slightly more favorable (1.1 kcal/mol with the BH&HLYP/6-311++G method including the ZPE correction) than a sigma complex but is significantly more stable (4.4 kcal/mol with the BH&HLYP/6-311++G method including the ZPE correction) than the eta(6) pi complex. The bromine and iodine benzene complexes are also predicted to prefer an eta(1) pi complex structure.