RESUMO
UNLABELLED: During antiviral therapy, specific delivery of interferon-α (IFNα) to infected cells may increase its antiviral efficacy, trigger a localized immune reaction, and reduce the side effects caused by systemic administration. Two T-cell receptor-like antibodies (TCR-L) able to selectively bind hepatitis B virus (HBV)-infected hepatocytes of chronic hepatitis B patients and recognize core (HBc18-27) and surface (HBs183-91) HBV epitopes associated with different human leukocyte antigen (HLA)-A*02 alleles (A*02:01, A*02:02, A*02:07, A*02:11) were generated. Each antibody was genetically linked to two IFNα molecules to produce TCR-L/IFNα fusion proteins. We demonstrate that the fusion proteins triggered an IFNα response preferentially on the hepatocytes presenting the correct HBV-peptide HLA-complex and that the mechanism of the targeted IFNα response was dependent on the specific binding of the fusion proteins to the HLA/HBV peptide complexes through the TCR-like variable regions of the antibodies. CONCLUSION: TCR-L antibodies can be used to target cytokines to HBV-infected hepatocytes in vitro. Fusion of IFNα to TCR-L decreased the intrinsic biological activity of IFNα but preserved the overall specificity of the protein for the cognate HBV peptide/HLA complexes. This induction of an effective IFNα response selectively in HBV-infected cells might have a therapeutic advantage in comparison to the currently used native or pegylated IFNα.
Assuntos
Anticorpos Antivirais/farmacologia , Antivirais/farmacologia , Antígenos HLA-A/imunologia , Vírus da Hepatite B/imunologia , Hepatite B/imunologia , Interferon-alfa/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Animais , Anticorpos Antivirais/imunologia , Fusão Gênica Artificial , Linfócitos T CD8-Positivos/efeitos dos fármacos , Quimiocinas/metabolismo , Portadores de Fármacos/farmacologia , Células Hep G2 , Hepatite B/tratamento farmacológico , Hepatite B/virologia , Vírus da Hepatite B/genética , Humanos , Ativação Linfocitária/efeitos dos fármacos , CamundongosRESUMO
B7-H4 protein is expressed on the surface of a variety of immune cells and functions as a negative regulator of T cell responses. We independently identified B7-H4 (DD-O110) through a genomic effort to discover genes upregulated in tumors and here we describe a new functional role for B7-H4 protein in cancer. We show that B7-H4 mRNA and protein are overexpressed in human serous ovarian cancers and breast cancers with relatively little or no expression in normal tissues. B7-H4 protein is extensively glycosylated and displayed on the surface of tumor cells and we provide the first demonstration of a direct role for B7-H4 in promoting malignant transformation of epithelial cells. Overexpression of B7-H4 in a human ovarian cancer cell line with little endogenous B7-H4 expression increased tumor formation in SCID mice. Whereas overexpression of B7-H4 protected epithelial cells from anoikis, siRNA-mediated knockdown of B7-H4 mRNA and protein expression in a breast cancer cell line increased caspase activity and apoptosis. The restricted normal tissue distribution of B7-H4, its overexpression in a majority of breast and ovarian cancers and functional activity in transformation validate this cell surface protein as a new target for therapeutic intervention. A therapeutic antibody strategy aimed at B7-H4 could offer an exciting opportunity to inhibit the growth and progression of human ovarian and breast cancers.
Assuntos
Antígeno B7-1/genética , Neoplasias da Mama/patologia , Células Epiteliais/patologia , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias Ovarianas/patologia , Animais , Apoptose/genética , Apoptose/fisiologia , Antígeno B7-1/metabolismo , Western Blotting , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Membrana Celular/química , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , DNA Complementar/genética , Células Epiteliais/metabolismo , Feminino , Glicosilação , Humanos , Imuno-Histoquímica , Glicoproteínas de Membrana/análise , Camundongos , Camundongos SCID , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Inibidor 1 da Ativação de Células T com Domínio V-Set , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Human testisin, a serine protease, is highly expressed in ovarian cancer and premeiotic spermatocytes with relatively little expression in other normal tissues. We first showed that testisin was localized on the surface of cultured tumor cells as a glycosyl-phosphatidylinositol-linked protein. We next explored the biological function of testisin in malignant transformation through manipulation of testisin expression in cell culture model systems. Small interfering RNA-mediated knockdown of endogenous testisin mRNA and protein expression in tumor cell lines led to increased apoptosis and diminished growth in soft agar. Conversely, overexpression of testisin in an epithelial cell line induced colony formation in soft agar as well as s.c. tumor growth in severe combined immunodeficient mice. A catalytic domain mutant was unable to induce soft-agar growth indicating that testisin protease activity is required for transformation. Ectopic expression of testisin in a human ovarian cancer cell line without endogenous testisin expression, led to the formation of larger tumors in severe combined immunodeficient mice. Data presented here provide the first demonstration that testisin can promote cellular processes that drive malignant transformation. Our functional data coupled with the restricted normal tissue distribution of testisin and its overexpression in a majority of ovarian cancers validates this cell surface protein as a target for therapeutic intervention.
