ABSTRACT
Anti-angiogenic therapies using biological molecules that neutralize vascular endothelial growth factor-A (VEGF-A) have revolutionized treatment of retinal vascular diseases including age-related macular degeneration (AMD). This study reports preclinical assessment of a strategy to enhance anti-VEGF-A monotherapy efficacy by targeting both VEGF-A and angiopoietin-2 (ANG-2), a factor strongly upregulated in vitreous fluids of patients with retinal vascular disease and exerting some of its activities in concert with VEGF-A. Simultaneous VEGF-A and ANG-2 inhibition was found to reduce vessel lesion number, permeability, retinal edema, and neuron loss more effectively than either agent alone in a spontaneous choroidal neovascularization (CNV) model. We describe the generation of a bispecific domain-exchanged (crossed) monoclonal antibody (CrossMAb; RG7716) capable of binding, neutralizing, and depleting VEGF-A and ANG-2. RG7716 showed greater efficacy than anti-VEGF-A alone in a non-human primate laser-induced CNV model after intravitreal delivery. Modification of RG7716's FcRn and FcγR binding sites disabled the antibodies' Fc-mediated effector functions. This resulted in increased systemic, but not ocular, clearance. These properties make RG7716 a potential next-generation therapy for neovascular indications of the eye.
Subject(s)
Angiopoietin-2/antagonists & inhibitors , Antibodies, Monoclonal/administration & dosage , Eye Diseases/drug therapy , Immunologic Factors/administration & dosage , Neovascularization, Pathologic/drug therapy , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Animals , Antibodies, Monoclonal/isolation & purification , Antibodies, Monoclonal/pharmacology , Disease Models, Animal , Drug Evaluation, Preclinical , Eye Diseases/pathology , Immunologic Factors/isolation & purification , Immunologic Factors/pharmacology , Macaca fascicularis , Treatment OutcomeABSTRACT
Inhibition of the vascular endothelial growth factor (VEGF) pathway has failed to improve overall survival of patients with glioblastoma (GBM). We previously showed that angiopoietin-2 (Ang-2) overexpression compromised the benefit from anti-VEGF therapy in a preclinical GBM model. Here we investigated whether dual Ang-2/VEGF inhibition could overcome resistance to anti-VEGF treatment. We treated mice bearing orthotopic syngeneic (Gl261) GBMs or human (MGG8) GBM xenografts with antibodies inhibiting VEGF (B20), or Ang-2/VEGF (CrossMab, A2V). We examined the effects of treatment on the tumor vasculature, immune cell populations, tumor growth, and survival in both the Gl261 and MGG8 tumor models. We found that in the Gl261 model, which displays a highly abnormal tumor vasculature, A2V decreased vessel density, delayed tumor growth, and prolonged survival compared with B20. In the MGG8 model, which displays a low degree of vessel abnormality, A2V induced no significant changes in the tumor vasculature but still prolonged survival. In both the Gl261 and MGG8 models A2V reprogrammed protumor M2 macrophages toward the antitumor M1 phenotype. Our findings indicate that A2V may prolong survival in mice with GBM by reprogramming the tumor immune microenvironment and delaying tumor growth.
Subject(s)
Antibodies, Bispecific/pharmacology , Antibodies, Neoplasm/pharmacology , Antineoplastic Agents/pharmacology , Glioblastoma/drug therapy , Macrophages/metabolism , Neoplasm Proteins/antagonists & inhibitors , Neoplasms, Experimental/drug therapy , Ribonuclease, Pancreatic/antagonists & inhibitors , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vesicular Transport Proteins/antagonists & inhibitors , Animals , Cell Line, Tumor , Glioblastoma/metabolism , Glioblastoma/pathology , Humans , Macrophages/pathology , Mice , Neoplasm Proteins/metabolism , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Ribonuclease, Pancreatic/metabolism , Vascular Endothelial Growth Factor A/metabolism , Vesicular Transport Proteins/metabolism , Xenograft Model Antitumor AssaysABSTRACT
We describe a generic approach to assemble correctly two heavy and two light chains, derived from two existing antibodies, to form human bivalent bispecific IgG antibodies without use of artificial linkers. Based on the knobs-into-holes technology that enables heterodimerization of the heavy chains, correct association of the light chains and their cognate heavy chains is achieved by exchange of heavy-chain and light-chain domains within the antigen binding fragment (Fab) of one half of the bispecific antibody. This "crossover" retains the antigen-binding affinity but makes the two arms so different that light-chain mispairing can no longer occur. Applying the three possible "CrossMab" formats, we generated bispecific antibodies against angiopoietin-2 (Ang-2) and vascular endothelial growth factor A (VEGF-A) and show that they can be produced by standard techniques, exhibit stabilities comparable to natural antibodies, and bind both targets simultaneously with unaltered affinity. Because of its superior side-product profile, the CrossMab(CH1-CL) was selected for in vivo profiling and showed potent antiangiogenic and antitumoral activity.
