ABSTRACT
OX40 is a costimulatory molecule that belongs to the tumor necrosis factor receptor (TNFR) superfamily. OX40 agonist-based combinations are emerging as promising candidates for novel cancer immunotherapy. Clinical trials have shown that OX40 agonist antibodies could lead to better results in cancer patients. Using a hybridoma platform and three different types of immunization strategies, namely recombinant protein, DNA, and overexpressing cells, we identified a chimeric anti-OX40 antibody (mAb035-hIgG1 from DNA immunization) that shows excellent binding specificity, and slightly stronger activation of human memory CD4+ T cells and similar potent antitumor activity compared with BMS 986178, an anti-OX40 antibody currently being evaluated for the treatment of solid tumors. This paper further systematically investigates the antigen-specific immune response, the number of binders, epitope bins, and functional activities of antibodies among different immunization strategies. Interestingly, we found that different immunization strategies affect the biological activity of monoclonal antibodies.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Agents, Immunological/pharmacology , CD4-Positive T-Lymphocytes/drug effects , Immunization/methods , Receptors, OX40/immunology , Recombinant Fusion Proteins/pharmacology , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/isolation & purification , Antibody Affinity , Antibody Specificity , Antineoplastic Agents, Immunological/isolation & purification , Antineoplastic Agents, Immunological/metabolism , Biological Assay , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , CHO Cells , Cricetulus , Female , Freund's Adjuvant/administration & dosage , Gene Expression , Genes, Reporter , HEK293 Cells , Humans , Hybridomas/chemistry , Hybridomas/immunology , Immunoglobulin Fc Fragments/biosynthesis , Immunoglobulin Fc Fragments/isolation & purification , Immunoglobulin Fc Fragments/pharmacology , Jurkat Cells , Luciferases/genetics , Luciferases/metabolism , Lymphocyte Activation/drug effects , Mice , Mice, Inbred BALB C , NF-kappa B/genetics , NF-kappa B/immunology , Receptors, OX40/antagonists & inhibitors , Receptors, OX40/genetics , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/isolation & purificationABSTRACT
Roxarsone, an organoarsenic feed additive, is widely used worldwide to promote animal growth. It has been found to exhibit a higher angiogenic index than As(III) at lower concentrations and to promote angiogenic phenotype in human endothelial cell in vitro. Little research has focused on the potential angiogenic effect of roxarsone in vitro or in vivo. Here, we investigated the pro-angiogenic effect of roxarsone in vivo. The effects of 0.1-10.0 µM roxarsone were tested in the rat endothelial cell Matrigel plug assay, chicken chorioallantoic membrane (CAM) model and MCF-7 cell xenograft tumour model; 10 ng/mL vascular endothelial growth factor (VEGF) was used as a positive control and PBS as a negative control. Roxarsone significantly increased the volume, weight and haemoglobin content of the Matrigel plugs compared to PBS group (p < 0.05); 1.0 µM roxarsone exerted the most significant effects. H&E staining and CD31 immunochemistry revealed obviously more new vessels or capillary-like structures in the plugs of the roxarsone and VEGF groups. Roxarsone significantly increased the numbers of primary/secondary vessels and area of vessels in the CAM assay and obviously increased tumour weight and volume in the xenograft model compared to PBS (p < 0.05). Histochemistry indicated local necrosis was observed at the centre of the xenograft tumours in the PBS and roxarsone groups, with less necrosis apparent in the VEGF-treated tumours. The growth of endothelial cells and VEGF level was obviously affected at blockade of VEGF and its receptor Flt-1/Flk-1 by SU5416 or its antibody in vitro. This study demonstrates roxarsone promotes angiogenesis in vivo, and a VEGF/VEGFR mechanism may be involved.
