Facile generation of biepitopic antibodies with intrinsic agonism for activating tumor necrosis factor receptors.

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.

Unlocking the potential of agonist antibodies for treating cancer using antibody engineering.

Agonist antibodies that target immune checkpoints, such as those in the tumor necrosis factor receptor (TNFR) superfamily, are an important class of emerging therapeutics due to their ability to regulate immune cell activity, especially for treating cancer. Despite their potential, to date, they have shown limited clinical utility and further antibody optimization is urgently needed to improve their therapeutic potential. Here, we discuss key antibody engineering approaches for improving the activity of antibody agonists by optimizing their valency, specificity for different receptors (e.g., bispecific antibodies) and epitopes (e.g., biepitopic or biparatopic antibodies), and Fc affinity for Fcγ receptors (FcγRs). These powerful approaches are being used to develop the next generation of cancer immunotherapeutics with improved efficacy and safety.

Facile generation of biepitopic antibodies with intrinsic agonism for activating receptors in the tumor necrosis factor superfamily.

Agonist antibodies that activate cellular receptors 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 potent activation. This can be achieved using antibodies that recognize two unique epitopes on the same receptor and mediate receptor superclustering. However, identifying compatible pairs of antibodies to generate biepitopic antibodies (also known as biparatopic antibodies) for activating TNF receptors typically requires animal immunization and is a laborious and unpredictable process. Here, we report a simple method for systematically identifying biepitopic antibodies that potently activate TNF receptors without the need for additional animal immunization. Our approach uses off-the-shelf, receptor-specific IgG antibodies, which lack intrinsic (Fc-gamma receptor-independent) agonist activity, to first block their corresponding epitopes. Next, we perform selections for single-chain antibodies from human nonimmune libraries that bind accessible epitopes on the same ectodomains using yeast surface display and fluorescence-activated cell sorting. The selected single-chain antibodies are finally fused to the light chains of IgGs to generate human tetravalent antibodies that engage two different receptor epitopes and mediate potent receptor activation. We highlight the broad utility of this approach by converting several existing clinical-stage antibodies against TNF receptors, including ivuxolimab and pogalizumab against OX40 and utomilumab against CD137, into biepitopic antibodies with highly potent agonist activity. We expect that this widely accessible methodology can be used to systematically generate biepitopic antibodies for activating other receptors in the TNF receptor superfamily and many other receptors whose activation is dependent on strong receptor clustering.

Agonist antibody discovery: Experimental, computational, and rational engineering approaches.

Agonist antibodies that activate cellular signaling have emerged as promising therapeutics for treating myriad pathologies. Unfortunately, the discovery of rare antibodies with the desired agonist functions is a major bottleneck during drug development. Nevertheless, there has been important recent progress in discovering and optimizing agonist antibodies against a variety of therapeutic targets that are activated by diverse signaling mechanisms. Herein, we review emerging high-throughput experimental and computational methods for agonist antibody discovery as well as rational molecular engineering methods for optimizing their agonist activity.