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Updated: Nov 4, 2025
Assessment of Human Natural Killer Cell Events Driven by FcγRIIIa Engagement in the Presence of Therapeutic Antibodies
Published on: May 22, 2020
Design of a Trispecific Checkpoint Inhibitor and Natural Killer Cell Engager Based on a 2 + 1 Common Light Chain
Jan P Bogen1,2, Stefania C Carrara1,2, David Fiebig1,2
1Institute for Organic Chemistry and Biochemistry, Technical University of Darmstadt, Darmstadt, Germany.
Abstract:
Natural killer cell engagers gained enormous interest in recent years due to their potent anti-tumor activity and favorable safety profile. Simultaneously, chicken-derived antibodies entered clinical studies paving the way for avian-derived therapeutics. In this study, we describe the affinity maturation of a common light chain (cLC)-based, chicken-derived antibody targeting EGFR, followed by utilization of the same light chain for the isolation of CD16a- and PD-L1-specific monoclonal antibodies. The resulting binders target their respective antigen with single-digit nanomolar affinity while blocking the ligand binding of all three respective receptors. Following library-based humanization, bispecific and trispecific variants in a standard 1 + 1 or a 2 + 1 common light chain format were generated, simultaneously targeting EGFR, CD16a, and PD-L1. The trispecific antibody mediated an elevated antibody-dependent cellular cytotoxicity (ADCC) in comparison to the EGFR×CD16a bispecific variant by effectively bridging EGFR/PD-L1 double-positive cancer cells with CD16a-positive effector cells. These findings represent, to our knowledge, the first detailed report on the generation of a trispecific 2 + 1 antibodies exhibiting a common light chain and illustrate synergistic effects of trispecific antigen binding. Overall, this generic procedure paves the way for the engineering of tri- and oligospecific therapeutic antibodies derived from avian immunizations.
Insights
This study engineered chicken-derived antibodies to target cancer cells, creating a novel trispecific antibody. This avian-derived therapeutic demonstrated enhanced anti-tumor activity by engaging natural killer cells.
Area of Science:
- Immunology
- Antibody Engineering
- Oncology
Background:
- Natural killer cell engagers show potent anti-tumor activity and safety.
- Avian-derived therapeutics, specifically chicken antibodies, are emerging in clinical studies.
- Targeting Epidermal Growth Factor Receptor (EGFR), CD16a, and Programmed Death-Ligand 1 (PD-L1) is crucial in cancer therapy.
Purpose of the Study:
- To develop novel avian-derived antibody formats for cancer immunotherapy.
- To engineer a common light chain (cLC)-based trispecific antibody targeting EGFR, CD16a, and PD-L1.
- To evaluate the efficacy of the trispecific antibody in mediating antibody-dependent cellular cytotoxicity (ADCC).
Main Methods:
- Affinity maturation of a chicken-derived antibody targeting EGFR using a cLC platform.
- Isolation of CD16a- and PD-L1-specific monoclonal antibodies utilizing the same cLC.
- Generation of bispecific and trispecific antibodies in a 2 + 1 cLC format targeting EGFR, CD16a, and PD-L1.
- Library-based humanization and assessment of antibody-dependent cellular cytotoxicity (ADCC).
Main Results:
- Chicken-derived binders targeting EGFR, CD16a, and PD-L1 achieved single-digit nanomolar affinity.
- The generated trispecific antibody effectively blocked ligand binding for all three targeted receptors.
- The trispecific antibody demonstrated enhanced ADCC compared to a bispecific variant by bridging cancer cells and effector cells.
Conclusions:
- This study presents the first detailed report of a trispecific 2 + 1 antibody with a common light chain derived from avian immunization.
- The findings illustrate synergistic effects of trispecific antigen binding in enhancing anti-tumor immune responses.
- The developed generic procedure supports the engineering of multi-specific therapeutic antibodies from avian sources.
