RESUMEN
Agonist antibodies are being pursued for therapeutic applications ranging from neurodegenerative diseases to cancer. For the tumor necrosis factor (TNF) receptor superfamily, higher-order clustering of three or more receptors is key to their activation, which can be achieved using antibodies that recognize two unique epitopes. However, the generation of biepitopic (i.e., biparatopic) antibodies typically requires animal immunization and is laborious and unpredictable. Here, we report a simple method for identifying biepitopic antibodies that potently activate TNF receptors without the need for additional animal immunization. Our approach uses existing, receptor-specific IgGs, which lack intrinsic agonist activity, to block their corresponding epitopes, then selects single-chain antibodies that bind accessible epitopes. The selected antibodies are fused to the light chains of IgGs to generate human tetravalent antibodies. We highlight the broad utility of this approach by converting several clinical-stage antibodies against OX40 and CD137 (4-1BB) into biepitopic antibodies with potent agonist activity.
Asunto(s)
Epítopos , Humanos , Epítopos/inmunología , Epítopos/química , Animales , Receptores del Factor de Necrosis Tumoral/agonistas , Receptores del Factor de Necrosis Tumoral/inmunología , Receptores del Factor de Necrosis Tumoral/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/agonistas , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/inmunología , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismo , Miembro 9 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/antagonistas & inhibidores , Receptores OX40/agonistas , Receptores OX40/inmunología , Receptores OX40/metabolismo , Receptores OX40/antagonistas & inhibidores , Anticuerpos/inmunología , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/farmacología , RatonesRESUMEN
Immune checkpoint inhibitors (ICI) have transformed cancer treatment. However, only a minority of patients achieve a profound response. Many patients are innately resistant while others acquire resistance to ICIs. Furthermore, hepatotoxicity and suboptimal efficacy have hampered the clinical development of agonists of 4-1BB, a promising immune-stimulating target. To effectively target 4-1BB and treat diseases resistant to ICIs, we engineered ATG-101, a tetravalent "2+2â³ PD-L1×4-1BB bispecific antibody. ATG-101 bound PD-L1 and 4-1BB concurrently, with a greater affinity for PD-L1, and potently activated 4-1BB+ T cells when cross-linked with PD-L1-positive cells. ATG-101 activated exhausted T cells upon PD-L1 binding, indicating a possible role in reversing T-cell dysfunction. ATG-101 displayed potent antitumor activity in numerous in vivo tumor models, including those resistant or refractory to ICIs. ATG-101 greatly increased the proliferation of CD8+ T cells, the infiltration of effector memory T cells, and the ratio of CD8+ T/regulatory T cells in the tumor microenvironment (TME), rendering an immunologically "cold" tumor "hot." Comprehensive characterization of the TME after ATG-101 treatment using single-cell RNA sequencing further revealed an altered immune landscape that reflected increased antitumor immunity. ATG-101 was well tolerated and did not induce hepatotoxicity in non-human primates. According to computational semimechanistic pharmacology modeling, 4-1BB/ATG-101/PD-L1 trimer formation and PD-L1 receptor occupancy were both maximized at around 2 mg/kg of ATG-101, providing guidance regarding the optimal biological dose for clinical trials. In summary, by localizing to PD-L1-rich microenvironments and activating 4-1BB+ immune cells in a PD-L1 cross-linking-dependent manner, ATG-101 safely inhibits growth of ICI resistant and refractory tumors. SIGNIFICANCE: The tetravalent PD-L1×4-1BB bispecific antibody ATG-101 activates 4-1BB+ T cells in a PD-L1 cross-linking-dependent manner, minimizing the hepatotoxicity of existing 4-1BB agonists and suppressing growth of ICI-resistant tumors. See related commentary by Ha et al., p. 1546.