RESUMO
Antibodies that block interaction of immune checkpoint receptors with its ligands have revolutionized the treatment of several cancers. Despite the success of this approach, the high cost has been restricted the use of this class of drugs. In this context, the development of biosimilar can be an important strategy for reducing prices and expanding access after patent has been dropped. Here, we evaluated the use of HEK293 cells for transient expression of an immune checkpoint-blocking antibody as a first step for biosimilar development. Antibody light and heavy chain genes were cloned into pCI-neo vector and transiently expressed in HEK293 cells. The culture supernatant was then subjected to protein A affinity chromatography, which allowed to obtain the antibody with high homogeneity. For physicochemical comparability, biosimilar antibody and reference drug were analyzed by SDS-PAGE, isoelectric focusing, circular dichroism and fluorescence spectroscopy. The results indicated that the both antibodies have a high degree of structural similarity. Lastly, the biosimilar antibody binding capacity to target receptor was shown to be similar to reference product in ELISA and flow cytometry assays. These data demonstrate that the HEK293 system can be used as an important tool for candidate selection and early development of biosimilar antibodies.
Assuntos
Anticorpos Monoclonais/farmacologia , Medicamentos Biossimilares/farmacologia , Inibidores de Checkpoint Imunológico/farmacologia , Proteínas de Checkpoint Imunológico/genética , Cadeias Pesadas de Imunoglobulinas/farmacologia , Cadeias Leves de Imunoglobulina/farmacologia , Anticorpos Monoclonais/biossíntese , Afinidade de Anticorpos , Especificidade de Anticorpos , Medicamentos Biossimilares/metabolismo , Cromatografia de Afinidade , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Inibidores de Checkpoint Imunológico/imunologia , Proteínas de Checkpoint Imunológico/imunologia , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Leves de Imunoglobulina/biossíntese , Focalização IsoelétricaRESUMO
Complementarity-determining regions (CDRs) from monoclonal antibodies tested as synthetic peptides display anti-infective and antitumor activities, independent of the specificity of the native antibody. Previously, we have shown that the synthetic peptide C7H2, based on the heavy chain CDR 2 from monoclonal antibody C7, a mAb directed to a mannoprotein of Candida albicans, significantly reduced B16F10 melanoma growth and lung colony formation by triggering tumor apoptosis. The mechanism, however, by which C7H2 induced apoptosis in tumor cells remained unknown. Here, we demonstrate that C7H2 interacts with components of the tumor cells cytoskeleton, being rapidly internalized after binding to the tumor cell surface. Mass spectrometry analysis and in vitro validation revealed that ß-actin is the receptor of C7H2 in the tumor cells. C7H2 induces ß-actin polymerization and F-actin stabilization, linked with abundant generation of superoxide anions and apoptosis. Major phenotypes following peptide binding were chromatin condensation, DNA fragmentation, annexin V binding, lamin disruption, caspase 8 and 3 activation, and organelle alterations. Finally, we evaluated the cytotoxic efficacy of C7H2 in a panel of human tumor cell lines. All tumor cell lines studied were equally susceptible to C7H2 in vitro. The C7H2 amide without further derivatization significantly reduced lung metastasis of mice endovenously challenged with B16F10-Nex2 melanoma cells. No significant cytotoxicity was observed toward nontumorigenic cell lines on short incubation in vitro or in naïve mice injected with a high dose of the peptide. We believe that C7H2 is a promising peptide to be developed as an anticancer drug.
Assuntos
Actinas/imunologia , Anticorpos Monoclonais Murinos/farmacologia , Anticorpos Antineoplásicos/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Cadeias Pesadas de Imunoglobulinas/farmacologia , Região Variável de Imunoglobulina/farmacologia , Melanoma/prevenção & controle , Proteínas de Neoplasias/imunologia , Animais , Anticorpos Monoclonais Murinos/imunologia , Antineoplásicos/imunologia , Candida albicans/imunologia , Caspase 3/imunologia , Caspase 8/imunologia , Linhagem Celular Tumoral , Fragmentação do DNA/efeitos dos fármacos , DNA de Neoplasias/imunologia , Proteínas Fúngicas/imunologia , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Região Variável de Imunoglobulina/imunologia , Masculino , Melanoma/imunologia , Melanoma/patologia , Glicoproteínas de Membrana/imunologia , Camundongos , Metástase NeoplásicaRESUMO
Binding to a specific receptor is an essential step for most enteropathogens to initiate an intestinal infection. We analyzed the inhibitory effect of human milk and its protein components on adhesion of two diarrheagenic Escherichia coli strains, diffusely adherent E. coli (DAEC) and enteroaggregative E. coli (EAEC), to HeLa cells. Defatted milk, whey proteins, immunoglobulin and non-immunoglobulin fractions, in concentrations lower than usually found in whole milk, inhibited both DAEC and EAEC adhesion, indicating that human milk components may contribute to the defense of the infants against enteropathogens.