Design of a Trispecific Checkpoint Inhibitor and Natural Killer Cell Engager Based on a 2 + 1 Common Light Chain Antibody Architecture

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.

Keywords: NK cell engager; bispecific antibody; checkpoint inhibitor; common light chain; trispecific antibody.

Figure 1

Possible light chain pairing combinations and structural model of trispecific antibodies. (A) In 2 + 1 trispecifics, 27 different light chain pairing combinations are possible, resulting in only ~3.7% correctly paired antibodies. The correctly paired variant is highlighted. (B) Structural model of a 2 + 1 trispecific antibody exhibiting common light chains to circumvent light chain mispairing. Intended functionalities are depicted.

Figure 2

Affinity maturation of EGFR-binding FEB4 (A) Schematic representation of light chain shuffling approach. The mutations in the CDR-H2 and CDR-H3 disrupt the binding to EGFR. By shuffling with a light chain immune repertoire derived from EGFR-immunized chicken by yeast mating and FACS sorting, an affinity maturated variant was isolated. (B) BLI affinity measurements of FEB4-derived antibodies at different maturation steps.

Figure 3

Ligand-receptor blockage of isolated cLC-mAbs. (A) BLI-assisted EGF-inhibition assay. Immobilized dFEB4-1 binds to EGFR at different EGF concentrations, revealing a dose-dependent binding. (B) Western blot analysis of inhibition of EGF-induced phosphorylation of AKT in A549 cells. 50 µg/mL dFEB1-4 and 10 ng/mL EGF were utilized. The western blot was performed three times, yielding reproducible results. (C) Epitope determination of NKE14. Immobilized NKE14 binds to CD16a at different scFv-Fc concentrations. CD16a:Fc-binding led to diminished binding of NKE14 to CD16a in a dose-dependent manner. (D) PD-1:PD-L1 interaction inhibition assay. Immobilized ICI2, ICI12, ICI13, or durvalumab binding to PD-L1 at different amPD-1-Fc concentrations, revealing dose-dependent binding.

Figure 4

BLI-measurements of humanized mono-, bi-, and trispecific antibodies against EGFR, CD16a, and PD-L1. While all antibodies bind exclusively to their respective target with distinguished affinity, the trispecific antibody binds all three antigens.

Figure 5

Characterization of bi- and trispecific humanized antibodies. BLI-assisted simultaneous binding assay. The bi- or trispecific antibodies were loaded onto biosensors, and antigens are added step-wise, revealing EGFR×CD16a bispecific or EGFR×CD16a×PD-L1 trispecific binding, respectively.

Figure 6

PD-1/PD-L1 blockage and ADCC cell-based reporter assays. (A) PD-1/PD-L1 blockage assay. hICI2-3 (pink), durvalumab (gray) were tested in comparison to the trispecific construct (blue) and the bispecific construct (green). EC₅₀ values: durvalumab, 586 pM; hICI2-3, 728 pM; trispecific, 5.2 nM. (B) ADCC reporter bioassay. Bivalent hdFEB4-1-4 with a wildtype IgG1 Fc (red) and the bispecific construct with the LALA mutation were tested in comparison to the trispecific antibody. As controls, one-armed hdFEB4-1-4 (orange), one-armed hNKE14-8 (black), and hICI2-3 were tested. EC₅₀ values: hdFEB4-1-4 (wtFc), 271 pM, bispecific antibody, 362 pM; trispecific antibody, 7.0 pM. A&B) Luciferase activity is plotted against the logarithmic antibody concentration. All measurements were performed in duplicates, and the experiments were repeated at least three times, yielding similar results.

Figure 7

Schematic representation of the intended mode of action of the trispecific checkpoint inhibitor and natural killer cell engager. As EGF-binding is blocked, downstream signaling is inhibited. Simultaneously, PD-L1 is blocked, inhibiting interaction with PD-1 on T cells and NK cells. PD-L1 inhibition facilitates T cell-mediated cytotoxicity against the tumor. While the simultaneous binding of EGFR and PD-L1 mediates an elevated tumor specificity, the recruitment of cytotoxic NK cells via CD16a engagement, paired with checkpoint inhibition, leads to an effective ADCC reaction.