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
Patients chronically infected with Trypanosoma cruzi develop chronic Chagas' heart disease (cChHD). Their Ab response is suspected to be involved in the cardiac pathogenesis. Reactivity of serum Abs from these patients has been extensively studied but little is known about the diversity of the in vivo IgG repertoire. We analyzed 125 variable H chain (VH) genes and compared it to repertoires from healthy individuals, and patients with autoimmune processes and other infections. VH were from plasma cells isolated from heart tissue of three cChHD patients and from a Fab combinatorial library derived from bone marrow of another cChHD patient. The role of the parasite in shaping the Ab repertoire was assessed analyzing VH genes before and after panning against T. cruzi Ag. Among recovered VH genes, a significantly increased representation of VH4 was observed. Plasma cells at the site of cardiac infiltration showed an increased VH1 usage. CDR3 lengths were similar to the ones found in the healthy repertoire and significantly shorter than in other infections. VH derived from anti-T. cruzi Fab and plasma cells showed a higher proportion of hypermutated genes, 46.9% and 43.75%, respectively, vs 30.9% of the cChHD patient repertoire, pointing to the role of parasite Ags in the shaping of the humoral response in Chagas' disease. No histological evidence of germinal center-like structures was observed in heart tissue. In accordance, VH analysis of heart plasmocytes revealed no evidence of clonal B cell expansion, suggesting that they migrated into heart tissue from secondary lymphoid organs.
Assuntos
Anticorpos Antiprotozoários/genética , Cardiomiopatia Chagásica/imunologia , Rearranjo Gênico do Linfócito B/genética , Cadeias Pesadas de Imunoglobulinas/biossíntese , Cadeias Pesadas de Imunoglobulinas/genética , Região Variável de Imunoglobulina/biossíntese , Região Variável de Imunoglobulina/genética , Adulto , Sequência de Aminoácidos , Anticorpos Antiprotozoários/biossíntese , Antígenos de Protozoários/imunologia , Linfócitos B/imunologia , Linfócitos B/parasitologia , Linfócitos B/patologia , Cardiomiopatia Chagásica/parasitologia , Cardiomiopatia Chagásica/patologia , Doença Crônica , Regiões Determinantes de Complementaridade/biossíntese , Regiões Determinantes de Complementaridade/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Hipermutação Somática de Imunoglobulina/genética , Trypanosoma cruzi/imunologiaRESUMO
Cruzipain, the major proteinase of Trypanosoma cruzi, plays an important role in the biology of this parasite. This study reports the development of a recombinant Fab antibody, using RNA isolated from the anti-Ag163B6 hybridoma against cruzipain. This procedure involves the use of cDNAs obtained with the aid of a specific set of primers complementary to the complete light kappa chain (L kappa) and the first two domains of the IgG1 heavy chain (VH/CH1). These products were subsequently cloned in the pComb3 system, from which the gIII gene had been removed, and expressed in Escherichia coli cells. The recombinant Fab molecule recognized cruzipain by ELISA, in a fashion similar to the original mAb anti-Ag163B6. Nucleotide sequence analysis of the recombinant molecule, together with its immunological recognition by specific anti-mouse IgG (Fab)2, indicated the immunoglobulin nature of the recombinant product. Moreover, both the mAb anti-Ag163B6 and the soluble Fab fragment described here react similarly with the intact parasite surface, as observed in an indirect immunofluorescence assay. In conclusion, our recombinant Fab anti-Ag 163B6 allows the possible use of this molecule for diagnosis, antigen purification, and eventually treatment of Chagas-afflicted individuals.