RESUMEN
Through a whole-cell panning approach, we previously identified a panel of antibodies that bound to prostate cancer cell surface antigens. One such antigen, CUB domain-containing protein 1 (CDCP1), was recognized by monoclonal antibody 25A11 and is a single transmembrane molecule highly expressed in several metastatic cancers as well as on CD34(+)CD133(+) myeloid leukemic blast cells. We show CDCP1 expression on prostate cancer cell lines by real-time quantitative PCR (RT-qPCR), flow cytometry, and immunohistochemistry and on prostate cancer patient samples by RT-qPCR and immunohistochemical staining. In cell-based assays, antibody 25A11 inhibited prostate cancer cell migration and invasion in vitro. Further characterization showed that CDCP1 is internalized on antibody binding. When 25A11 was coupled to the cytotoxin saporin either directly or via a secondary antibody, both resulted in prostate cancer cell killing in vitro. In vivo targeting studies with an anti-CDCP1 immunotoxin showed significant inhibition of primary tumor growth as well as metastasis in a mouse xenograft model. These data provide support for continued evaluation of anti-CDCP1 therapy for potential use in cancer in primary and metastatic disease.
Asunto(s)
Anticuerpos Monoclonales/química , Antígenos CD/química , Antígenos de Neoplasias/química , Moléculas de Adhesión Celular/química , Proteínas de Neoplasias/química , Neoplasias de la Próstata/patología , Antígeno AC133 , Animales , Antígenos CD/biosíntesis , Antígenos CD/fisiología , Antígenos CD34/biosíntesis , Antígenos de Neoplasias/fisiología , Moléculas de Adhesión Celular/fisiología , Movimiento Celular , Glicoproteínas/biosíntesis , Humanos , Masculino , Glicoproteínas de Membrana , Ratones , Invasividad Neoplásica , Metástasis de la Neoplasia , Proteínas de Neoplasias/fisiología , Trasplante de Neoplasias , Péptidos , Neoplasias de la Próstata/metabolismo , Estructura Terciaria de ProteínaRESUMEN
Although the immune system is capable of mounting a response against many cancers, that response is insufficient for tumor eradication in most patients due to factors in the tumor microenvironment that defeat tumor immunity. We previously identified the immune-suppressive molecule CD200 as up-regulated on primary B cell chronic lymphocytic leukemia (B-CLL) cells and demonstrated negative immune regulation by B-CLL and other tumor cells overexpressing CD200 in vitro. In this study we developed a novel animal model that incorporates human immune cells and human tumor cells to address the effects of CD200 overexpression on tumor cells in vivo and to assess the effect of targeting Abs in the presence of human immune cells. Although human mononuclear cells prevented tumor growth when tumor cells did not express CD200, tumor-expressed CD200 inhibited the ability of lymphocytes to eradicate tumor cells. Anti-CD200 Ab administration to mice bearing CD200-expressing tumors resulted in nearly complete tumor growth inhibition even in the context of established receptor-ligand interactions. Evaluation of an anti-CD200 Ab with abrogated effector function provided evidence that blocking of the receptor-ligand interaction was sufficient for control of CD200-mediated immune modulation and tumor growth inhibition in this model. Our data indicate that CD200 expression by tumor cells suppresses antitumor responses and suggest that anti-CD200 treatment might be therapeutically beneficial for treating CD200-expressing cancers.
Asunto(s)
Anticuerpos Bloqueadores/uso terapéutico , Antígenos CD/efectos de los fármacos , Inmunoterapia/métodos , Neoplasias/terapia , Proteínas Recombinantes/uso terapéutico , Animales , Anticuerpos Bloqueadores/genética , Anticuerpos Bloqueadores/inmunología , Antígenos CD/análisis , Antígenos CD/inmunología , Línea Celular Tumoral , Membrana Celular/química , Humanos , Leucocitos Mononucleares/inmunología , Ratones , Ratones Endogámicos , Neoplasias/tratamiento farmacológico , Biblioteca de Péptidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
ALXN4100, a fully human antibody that binds to the protective antigen of anthrax toxin, was generated from a Fab isolated from a phage display library and was analyzed for its pharmacokinetic properties in rabbits and then used to protect rabbits from challenge with a lethal aerosol dose of Bacillus anthracis spores (approximately 322X LD(50)). All rabbits receiving 15 or 40 mg/kg of antibody 24 hours before challenge survived; survival of rabbits receiving 4 mg/kg either subcutaneously or intravenously was 80 or 90%, respectively. Susceptibility to anthrax disease appeared to be correlated with serum antibody concentration. This study indicates that ALXN4100 has the potential to be useful for prophylaxis of anthrax disease in humans.
Asunto(s)
Carbunco/prevención & control , Anticuerpos Antibacterianos/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/inmunología , Aerosoles , Animales , Anticuerpos Monoclonales/farmacocinética , Ensayo de Inmunoadsorción Enzimática , Humanos , Dosificación Letal Mediana , Conejos , Esporas BacterianasRESUMEN
The C-type lectin L-SIGN is expressed on liver and lymph node endothelial cells, where it serves as a receptor for a variety of carbohydrate ligands, including ICAM-3, Ebola, and HIV. To consider targeting liver/lymph node-specific ICAM-3-grabbing nonintegrin (L-SIGN) for therapeutic purposes in autoimmunity and infectious disease, we isolated and characterized Fabs that bind strongly to L-SIGN, but to a lesser degree or not at all to dendritic cell-specific ICAM-grabbing nonintegrin (DC-SIGN). Six Fabs with distinct relative affinities and epitope specificities were characterized. The Fabs and those selected for conversion to IgG were tested for their ability to block ligand (HIV gp120, Ebola gp, and ICAM-3) binding. Receptor internalization upon Fab binding was evaluated on primary human liver sinusoidal endothelial cells by flow cytometry and confirmed by confocal microscopy. Although all six Fabs internalized, three Fabs that showed the most complete blocking of HIVgp120 and ICAM-3 binding to L-SIGN also internalized most efficiently. Differences among the Fab panel in the ability to efficiently block Ebola gp compared with HIVgp120 suggested distinct binding sites. As a first step to consider the potential of these Abs for Ab-mediated Ag delivery, we evaluated specific peptide delivery to human dendritic cells. A durable human T cell response was induced when a tetanus toxide epitope embedded into a L-SIGN/DC-SIGN-cross-reactive Ab was targeted to dendritic cells. We believe that the isolated Abs may be useful for selective delivery of Ags to DC-SIGN- or L-SIGN-bearing APCs for the modulation of immune responses and for blocking viral infections.
Asunto(s)
Moléculas de Adhesión Celular/inmunología , Células Dendríticas/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Lectinas Tipo C/inmunología , Activación de Linfocitos/inmunología , Receptores de Superficie Celular/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Afinidad de Anticuerpos , Células Presentadoras de Antígenos/inmunología , Antígenos Virales/inmunología , Western Blotting , Células Cultivadas , Endocitosis , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Fragmentos Fab de Inmunoglobulinas/genética , Microscopía Confocal , Datos de Secuencia Molecular , Biblioteca de Péptidos , Proteínas Virales/inmunologíaRESUMEN
A panel of Fabs that neutralize anthrax toxin in vitro was selected from libraries generated from human donors vaccinated against anthrax. At least two of these antibodies protect rats from anthrax intoxication in vivo. Fabs 83K7C and 63L1D bind with subnanomolar affinity to protective antigen (PA) 63, and Fab 63L1D neutralizes toxin substoichiometrically, inhibits lethal factor (LF) interaction with PA63 and binds to a conformational epitope formed by PA63.