RESUMO
CC49 is a clinically validated antibody with specificity for TAG-72, a carbohydrate epitope that is overexpressed and exposed on the cell surface in a large fraction of solid malignancies. We constructed a single-chain fragment (scFv) based on CC49 and fused it to beta-lactamase (BLA). Following optimization of the scFv domain by combinatorial consensus mutagenesis (CCM) for increased expression and stability, we characterized the protein variant for binding, in vivo pharmacokinetics (PK), and antitumor efficacy. The fusion protein TAB2.5 possessed a similar binding specificity relative to the parent antibody CC49. TAB2.5 also showed prolonged retention (T(1/2) = 36.9 h) in tumor-bearing mice with tumor/plasma ratios of up to 1000. Preliminary evaluation of TAB2.5, in combination with a novel prodrug, GC-Mel, resulted in significant efficacy in a colorectal xenograft tumor model and supports the utility of the protein as an agent for tumor-selective prodrug activation.
Assuntos
Anticorpos Antineoplásicos , Cefalosporinas/química , Cefalosporinas/metabolismo , Cefalosporinas/uso terapêutico , Compostos de Mostarda Nitrogenada/química , Compostos de Mostarda Nitrogenada/metabolismo , Compostos de Mostarda Nitrogenada/uso terapêutico , Pró-Fármacos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/uso terapêutico , beta-Lactamases , Animais , Antibióticos Antineoplásicos/uso terapêutico , Anticorpos Antineoplásicos/química , Anticorpos Antineoplásicos/genética , Anticorpos Antineoplásicos/metabolismo , Anticorpos Antineoplásicos/uso terapêutico , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/metabolismo , Antineoplásicos Alquilantes/uso terapêutico , Antineoplásicos Fitogênicos/uso terapêutico , Camptotecina/análogos & derivados , Camptotecina/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Doxorrubicina/uso terapêutico , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Sistemas de Liberação de Medicamentos , Feminino , Humanos , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/metabolismo , Região Variável de Imunoglobulina/genética , Região Variável de Imunoglobulina/metabolismo , Irinotecano , Melfalan/química , Melfalan/metabolismo , Melfalan/uso terapêutico , Camundongos , Camundongos Nus , Estrutura Molecular , Transplante de Neoplasias , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Pró-Fármacos/uso terapêutico , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , beta-Lactamases/química , beta-Lactamases/genética , beta-Lactamases/metabolismo , beta-Lactamases/uso terapêuticoRESUMO
CC49 is a clinically validated antibody with specificity for TAG-72, a carbohydrate epitope that is over-expressed and exposed on a large fraction of solid malignancies. We constructed a single chain fragment (scFv) based on CC49 and fused it to beta-lactamase. The first generation fusion protein, TAB2.4, was expressed at low levels in Escherichia coli and significant degradation was observed during production. We optimized the scFv domain of TAB2.4 by Combinatorial Consensus Mutagenesis (CCM). An improved variant TAB2.5 was identified that resulted in an almost 4-fold improved expression and 2.5 degrees higher thermostability relative to its parent molecule. Soluble TAB2.5 can be manufactured in low-density E.coli cultures at 120 mg/l. Our studies suggest that CCM is a rapid and efficient method to generate antibody fragments with improved stability and expression. The fusion protein TAB2.5 can be used for antibody directed enzyme prodrug therapy (ADEPT).
