title: Merge Two into One: Multispecific Symmetric Antibodies for Cancer Immunotherapy
Creators: name: Harwardt, Julia
Publisher: name: UNSPECIFIED
Year: 2024

description: This work is focused on the generation and characterization of multispecific symmetric antibodies for cancer immunotherapy. In contrast to asymmetric antibodies, where protein engineering technologies need to be applied to ensure the correct pairing of the different polypeptide chains, symmetric antibodies consist of two identical heavy and light chains. Consequently, these molecules exhibit monoclonal antibody (mAb)-like characteristics in terms of established manufacturing and a conventional approval process. Two-in-One antibodies are symmetrical tetravalent IgG-like bispecific antibodies (bsAbs) in which each fragment antigen binding (Fab) addresses two distinct antigens. Simultaneous targeting of two tumor-associated antigens (TAAs) on the same malignant cell offers advantages like increased specificity and reduced immune escape. Affinity optimization can further improve the efficacy of drug candidates and limit adverse effects. 

The first investigation within this cumulative thesis was dedicated on the generation of a symmetrical Two-in-One antibody targeting epidermal growth factor receptor (EGFR) and programmed cell death ligand-1 (PD-L1), two therapeutic targets upregulated in many solid tumors. To this end, the heavy chain of a chicken-derived anti-PD-L1 common light chain (cLC) antibody was combined with a chicken-derived anti-EGFR light chain yeast surface display (YSD) library, followed by subsequent screening for binding properties towards both antigens. The isolated Two-in-One antibody HCP-LCE simultaneously targeted EGFR and PD-L1 at the same Fab fragment and exhibited favorable biophysical characteristics. BLI measurements and cell-based assays revealed that HCP-LCE inhibited EGFR signaling by binding to EGFR dimerization domain II and blocked the PD-1/PD-L1 interaction. Remarkably, both antigens were addressed with comparatively low binding affinities in the double- to triple-digit nanomolar range, but specific and high-affinity cellular binding properties were demonstrated on EGFR and PD-L1 double positive tumor cells. HCP-LCE represented the first Two-in-One antibody without complementarity-determining region (CDR) engineering, targeting two antigens simultaneously with a single Fab fragment. This approach of library generation paves the way for the further development of Two-in-One antibodies derived from avian immunization with tailor-made binding properties. 

In a second project, a symmetrical trispecific natural killer (NK) cell engager (NKCE) was generated based on the previously isolated Two-in-One antibody. By the simultaneous targeting of a TAA and a specific marker on the surface of NK cells, the immune function of NK cells to kill tumor cells is harnessed for tumor therapy. For the generation of such an antibody, the cLC technology was applied, an established method to circumvent light chain mispairing in multispecific antibodies. To this end, the light chain of the Two-in-One antibody HCP-LCE was used as cLC for the generation of a chicken-derived anti-CD16a YSD library. The isolated CD16a engaging cLC Fab fragment was fused in a head-to-tail setup with the parental Two-in-One antibody, resulting in a symmetrical trispecific 2+2 antibody that simultaneously bound EGFR, PD-L1 and CD16a with six independent paratopes on four Fabs. The antibody exhibited specific cellular binding on EGFR and PD-L1 double positive tumor cells and induced NK cell-mediated tumor cell killing (ADCC) already at low concentrations. This study pioneers the straightforward generation of trispecific cLC immune cell engager molecules in a 2+2 design, which facilitates subsequent process development due to the symmetrical architecture. 

The third part of this work focused on the affinity maturation of the Two-in-One antibody for EGFR binding by site-directed mutagenesis and YSD in combination with fluorescence-activated cell sorting (FACS). Individual amino acids of the light chain CDR1 and CDR3 were randomized and the resulting YSD library provided a Two-in-One variant that exhibited a 60-fold improvement in EGFR binding affinity due to the replacement of a single amino acid at position three of the light chain CDR3, while PD-L1 binding was not impaired. AlphaFold2-based modeling predicted that the exchange of the neutral amino acid tyrosine to the acidic amino acid glutamic acid causes the formation of an additional salt bridge between the introduced glutamic acid and an arginine at EGFR position 165. The increase in affinity was demonstrated by BLI measurements, real-time antigen binding measurements on surfaces with a mixture of both recombinant proteins and cellular binding studies using flow cytometry and real-time interaction cytometry. This easily adaptable approach provides a generic strategy for the affinity maturation of Two-in-One antibodies.