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.
Highlights
With the FDA approval of blinatumomab in 2014, bispecific antibodies, those bringing immune cells in spatial proximity to malignant tumor cells, raised further interest for therapeutic application [1, 2]
We recently showed that immunization of chickens enables the isolation of common light chain antibodies targeting a broad epitope space [53], and that these antibodies can be assembled in a heterodimerized manner resulting in biparatopic antibodies comprising cLCs [54]
The respective antibody showed affinity in the lower-double-digit nanomolar range, targeted a conformational epitope on epidermal growth factor receptor (EGFR) domain III, exhibited an overlapping binding site with matuzumab, and inhibited epidermal growth factor (EGF) binding to its receptor [54]
Summary
With the FDA approval of blinatumomab in 2014, bispecific antibodies (bsAb), those bringing immune cells in spatial proximity to malignant tumor cells, raised further interest for therapeutic application [1, 2]. Their unique mechanism is based on the simultaneous binding of a cancer-specific antigen on the surface of a tumor cell and a specific marker on the surface of immune cells, activating the latter, leading to efficient killing of the malignant cells. To overcome the toxic effects of T cell engagers, the concept of natural killer (NK) cell engagers was created, based on their anti-tumor activity [10, 11]. Engagement of CD16a is less demanding compared to CD3 engagement due to lower steric hindrances and facilitated by the lack of accessory molecules [14]
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