RESUMO
We have demonstrated that a trypsin sensitive enzyme such as L-asparaginase can be rendered trypsin resistant by genetically fusing its gene with that of a single-chain antibody derived from a preselected monoclonal antibody capable of providing protection against trypsin. The chimeric L-asparaginase retained 75% of its original activity upon exposure to trypsin, whereas the native unprotected L-asparaginase control was totally inactivated.
Assuntos
Asparaginase/genética , Engenharia de Proteínas , Tripsina/farmacologia , Anticorpos Monoclonais/genética , Asparaginase/efeitos dos fármacos , Sequência de Bases , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Tripsina/imunologiaAssuntos
Anticorpos Monoclonais/química , Anticorpos , Proteínas/química , Proteínas/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Estabilidade Enzimática , Enzimas/química , Enzimas/imunologia , Enzimas/metabolismo , Humanos , Proteínas/imunologiaRESUMO
We show that a non-inhibitory monoclonal antibody (MAB) can be selected that provides substantial and sustained protection against proteolytic inactivation of L-asparaginase by trypsin. Of six non-inhibitory, high affinity, monoclonal antibodies to L-asparaginase, one afforded approximately 70% protection. Inactivation of L-asparaginase is associated with a single cleavage adjacent to lysine-29 that results in loss of an N-terminal fragment with a calculated MW of 2,647. The protective MAB prevented this trypsin cleavage. The products of gene fusions of "humanized" fragments of such antibodies and L-asparaginase could have increased clinical utility.
Assuntos
Anticorpos Monoclonais/imunologia , Asparaginase/imunologia , Tripsina , Sequência de Aminoácidos , Western Blotting , Eletroforese em Gel de Poliacrilamida , ImmunoblottingRESUMO
In nature, increased stability of enzymes has often been found to be associated with noncovalent protein-protein interactions. Specific antibodies should be suitable for this purpose. To test this hypothesis, we used a number of model enzymes, complexed them with their specific antibodies, and exposed them and the free enzymes to low and high temperature, lyophilization, oxidation, and alcohol. The retained activity of the antibody-complexed enzymes was substantially, and in some cases dramatically, higher. In general mechanistic terms, stabilization may have been accomplished either by noncovalent antibody crosslinking of discontinuous oligopeptide chains on the surface of the enzyme, thereby increasing resistance to unfolding of the enzyme, or by physical shielding by the antibodies of vulnerable sites on the surface of the enzyme.
Assuntos
Anticorpos , Estabilidade Enzimática , Glucana 1,4-alfa-Glucosidase/imunologia , alfa-Amilases/imunologia , Animais , Complexo Antígeno-Anticorpo , Glucana 1,4-alfa-Glucosidase/metabolismo , Humanos , Cinética , Camundongos/imunologia , Coelhos/imunologia , Termodinâmica , alfa-Amilases/metabolismoRESUMO
The effectiveness of a methodology designed to protect the antigen binding capacity of monoclonal antibodies undergoing labelling with a number of reagents was examined. The antigen binding sites of monoclonal antibodies were protected by complexing them with their antigen. Chemical modification with 6 mM of the water soluble Bolton-Hunter reagent of site protected monoclonal antibodies to glucoamylase resulted in antibodies that could tolerate a four-fold increase in reagent incorporation, without any loss of antigen binding capacity. Iodination of these antibodies (modified under site protected conditions) yielded over 70% increase in radioactivity incorporated in the active antibody fraction, compared with the incorporation into unprotected antibodies. Site protected labeling was found to be effective in retaining the antigen binding capacity of monoclonal antibodies modified with all reagents tested with the exception of chloramine-T.