RESUMEN
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.
Asunto(s)
Anticuerpos Antineoplásicos , Cefalosporinas/química , Cefalosporinas/metabolismo , Cefalosporinas/uso terapéutico , Compuestos de Mostaza Nitrogenada/química , Compuestos de Mostaza Nitrogenada/metabolismo , Compuestos de Mostaza Nitrogenada/uso terapéutico , Profármacos , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/uso terapéutico , beta-Lactamasas , Animales , Antibióticos Antineoplásicos/uso terapéutico , Anticuerpos Antineoplásicos/química , Anticuerpos Antineoplásicos/genética , Anticuerpos Antineoplásicos/metabolismo , Anticuerpos Antineoplásicos/uso terapéutico , Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/metabolismo , Antineoplásicos Alquilantes/uso terapéutico , Antineoplásicos Fitogénicos/uso terapéutico , Camptotecina/análogos & derivados , Camptotecina/uso terapéutico , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Doxorrubicina/uso terapéutico , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/metabolismo , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/metabolismo , Irinotecán , Melfalán/química , Melfalán/metabolismo , Melfalán/uso terapéutico , Ratones , Ratones Desnudos , Estructura Molecular , Trasplante de Neoplasias , Profármacos/química , Profármacos/metabolismo , Profármacos/uso terapéutico , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , beta-Lactamasas/química , beta-Lactamasas/genética , beta-Lactamasas/metabolismo , beta-Lactamasas/uso terapéuticoRESUMEN
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).
