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.

A Generic Strategy to Generate Bifunctional Two-in-One Antibodies by Chicken Immunization

“…Currently, various monoclonal antibodies targeting either EGFR, including the therapeutic antibodies panitumumab ( 37 ), necitumumab ( 38 ), nimotuzumab ( 39 ) and cetuximab ( 40 ), or PD-L1, among them durvalumab ( 41 ), avelumab ( 42 ) and atezolizumab ( 43 ) are approved for tumor treatment in multiple countries. Recently, our group isolated a chicken-derived anti‐PD‐L1 antibody called ICI2 ( 44 ). Since ICI2 exhibited a common light chain as it was utilized in a multispecific setup, we assumed that the heavy chain CDRs were mainly responsible for antigen recognition and could tolerate various light chains, as reported for other antibodies ( 45 , 46 ).…”

“…In order to isolate a Two-in-One antibody targeting both PD-L1 and EGFR, the ICI2 heavy chain was paired with a diversity of anti‐EGFR light chains ( Figure 1 ). The light chain library was generated by amplification of VL genes from cDNA derived from a chicken immunized with EGFR-ECD and subsequent insertion into a pYD 1 ‐derived vector encoding a human lambda CL by homologous recombination in BJ5464 yeast as previously described ( 44 ). The light chain diversity of 2.9x10 8 transformants was combined with EBY100 yeast cells encoding the ICI2 VH-CH 1 fragment by yeast mating ( Figure 1 ), resulting in adequate oversampling of the estimated light chain diversity, which was estimated to be about 5x10 8 unique variants.…”

Challenges and Recent Advances in NK Cell-Targeted Immunotherapies in Solid Tumors

“…Compared with CAR-NKs, BiKEs and TriKEs are more easily manufactured and cost-effective. However, BiKEs and TriKEs suffer from short half-life and structural instability due to the lack of Fc region [ 169 ]. Similarly, ICIs also have relatively short half-life.…”

“…Moreover, Bogen et al [ 169 ] proposed an innovative approach to simultaneously block EGFR and PD-1/PD-L1 signalings, and meanwhile to potentiate NK-mediated ADCC against EGFR + PD-L1 + cancer cells, with a tri-specific anti-EGFR×CD16a×PD-L1 antibody. In this study, they chose an Fc-based format to elongate the half-life of this tri-specific antibody, thus overcoming the limitation of short half-life of BiKEs and TriKEs.…”

Development of Bispecific Antibody for Cancer Immunotherapy: Focus on T Cell Engaging Antibody

“…TriKE is a BiKE molecule, which is crosslinked with IL-15. IL-15 moiety improves the proliferation and survival of NK cells by inducing superior NK cell cytotoxicity and degranulation ( 37 ). The 161519 (CD16×IL-15×CD19) TriKE recruits CD19 + tumor cells to NK cells, promoting tumor cell killing.…”