Oligospecific Human Antibodies with Therapeutic Potential Derived from Immunized Chickens

In the last decades, monoclonal antibodies (mAbs) emerged as a powerful biologic entity for diagnostic and therapeutic applications and is today the most successful class of biologics. Even though rodent immunization combined with subsequent humanization is conventionally used to produce monoclonal antibodies for therapeutic use, this technology comes with some limitations. Human-derived antigens utilized for immunization are often sequence homologous to their murine counterpart and therefore less immunogenic, restricting the number of potential addressed epitopes. Therefore, in recent years avian-immunization has emerged as a potential new approach for the generation of monoclonal antibodies. Due to the phylogenetic distance of chickens to humans, additional epitopes can be addressed that could not be targeted utilizing rodents, enabling additional mode of actions for therapeutic applications. Next generation antibodies, like antibody-drug-conjugates (ADCs) or bispecific antibodies (bsAb), were excessively investigated in recent years. However, the generation of bsAb in an IgG-like design remains challenging due to the need of four polypeptide chains to assemble in the correct way to facilitate bispecific binding. To circumvent the light chain pairing problem, the easiest way is to utilize common light chains (cLC). Such light chains complement both heavy chains and allow an IgG-like architecture while facilitating bispecific binding. The first investigation in the frame of the present cumulative study was focused on the establishment of yeast surface display (YSD)-based panning in a fluorescence-activated cell sorting (FACS)-assisted high throughput manner. Furthermore, to verify whether this technology is suitable for the discovery of bispecific and biparatopic antibodies, a common light chain approach was conducted. Panning of a cLC-based chicken-derived anti-epidermal growth factor receptor (EGFR) immune library was performed against Calcein-AM stained EGFR+++ A431 cells. By utilizing a viability staining approach, only living cells were investigated without the need do genetically modify the target cell, as mandatory for prior published studies. Via FACS, cell-cell complexes, consisting of immuno-stained yeast cells expressing a Fab fragment bound to Calcein-AM-stained mammalian cells, were sorted over multiple selection rounds. Antibodies exhibiting diverse sequences were isolated that specifically bound A431 cells but not EGFR- Jurkat cells. Reformatted antibodies reveled favorable stability, aggregation and specificity while exhibiting notable affinity. Furthermore, a broad epitope space was covered, including the therapeutically interesting epitope of Cetuximab, an Food and Drug Administration (FDA)-approved anti-EGFR mAb. This was the first report of yeast panning utilizing an immune library, and furthermore the first study investigating cLC-based antibodies derived from chickens. These results paved the way for panning against other membrane proteins that could not be produced as a soluble antigen, such as G-protein coupled receptors (GPCRs). Additionally, it expanded the methodology used for the generation of cLC-based biparatopic and bispecific antibodies. The second investigation elucidated the generation of biparatopic anti-EGFR chicken antibodies, derived from a common light chain YSD library. At first, an EGF-blocking antibody targeting domain III on EGFR was isolated from a chicken-derived cLC-library. This antibody was then utilized as a detection antibody to screen the same library again for EGFR-binding. As the newly isolated antibody must target an orthogonal epitope to the first antibody, a second mAb targeting the domain II was isolated. By utilizing the knob-into-hole (KiH) technology, a bispecific antibody was generated. Besides improved affinity, it was able to cluster the soluble extracellular domains (ECD) of EGFR as shown by size-exclusion chromatography (SEC) experiments. Furthermore, cell-bound EGFR was clustered as shown by improved antibody-dependent cell-mediated cytotoxicity (ADCC). This mode of action was mediated by the mix-site binding profile of the biparatopic antibody. Mixed-site binding referred to the ability of a biparatopic antibody to bind two different EGFR molecules at the same time or the inability to bind both epitopes on one EGFR molecule simultaneously. This allows for the additional binding of two biparatopic antibodies to one antigen, resulting in clustering of the antigen. Effective clustering of the target was demonstrated using Biolayer interferometry (BLI)- and enzyme-linked immunosorbent assay (ELISA)-based experiments. The methodology to generate biparatopic antibodies by an epitope binning-based screening might accelerate the discovery of therapeutic mAbs exhibiting biparatopic binding behaviors in the future. Furthermore, this study represents the first bispecific chicken-derived antibody as well as the first cLC-based biparatopic molecule. The third investigation evaluated the usage of a new next generation sequencing (NGS)-assisted method for the humanization of chicken-derived single-chain fragment variable (scFvs) using YSD and FACS. To this end, the complementary determining regions (CDRs) of two chicken-derived anti-EGFR scFvs were grafted onto the most homologous human germline sequences encoding for VH and VL, respectively. Additionally, six Vernier residues in the VH and four in the VL sequences were partially randomized to encode either the human or the chicken germline residue at this position. Since the Vernier residues are crucial for the orientation of the CDRs and therefore for antigen binding, the most human antibody with the highest affinity can be identified. Humanization was performed in a library-based approach, both in the scFv and the Fab format. Next generation sequencing after two FACS sorting rounds revealed enrichment of certain Vernier combinations. The humanized antibodies were produced as scFv-Fc fusions or full-length IgG antibodies, respectively. Biophysical analysis showed improved stability and aggregation behavior while nearly maintaining the wild-type affinity. Additionally, germline identity was comparable to those of therapeutically approved humanized antibodies. This technology therefore enables the fast and convenient humanization of chicken-derived antibodies through a generic process, further expanding the usage of avian immunization for therapeutic applications. In the fourth study, the previously generated EGF-blocking mAb was affinity maturated by a novel type of light chain shuffling approach based on the disruption of the binding by mutating the heavy chain CDRs and the selection of a novel VL domain, able to restore binding. This new light chain was chosen as a cLC for the screening of an anti-CD16a and an anti-PD-L1 library. The resulting CD16a binder bound with high affinity to both human CD16a isotypes and blocked the interaction to human IgG1 Fc. The PD-L1 binder exhibited an overlapping epitope with Durvalumab, an FDA-approved anti-PD-L1 antibody, and effectively blocked the interaction of PD-1 and PD-L1. The three Fab fragments were subsequently humanized and subcloned into a trispecific 2+1 antibody, with one heavy chain comprising the anti-EGFR-Fab arm and one heavy chain encoding the anti-PD-L1 Fab as a head-to-tail fusion to the anti-CD16a Fab. The trispecific molecule was able to bind all three antigens with high affinity and in a simultaneous manner. By co-targeting EGFR and PD-L1, the trispecific antibody facilitated an enhanced CD16a-mediated ADCC effect compared to the EGFR×CD16a bispecific. The utilization of a common light chain eases the production and engineering of 2+1 bi- and trispecifics and might contribute to the generation of avian-derived oligospecific therapeutic antibodies in the future. The last part of this work elucidated the possibilities to engineer pH-dependent binding behavior into a common light chain of a bispecific CEACAM5×CEACAM6 antibody. To this end, two histidines were incorporated into the CDR-L1 or the CDR-L3 or both, respectively, of the VL germline IGKV3-15. A YSD library was screened for antibodies binding to CEACAM5 at pH 7.4 and non-binding at pH 6.0. Two light chain variants with pH-dependent binding properties were isolated. By permutation of respective histidines in a rational manner, three new variants were generated, exhibiting elevated pH-dependence as shown by BLI-experiments. The light chain mediating the strongest pH-responsive binding was furthermore paired with the CEACAM6-binding heavy chain. Subsequent BLI experiments revealed no pH-dependence, indicating that pH-responsive binding is only mediated in combination with the VH domain the light chain was originally screened with. A bispecific antibody was generated, which bound CEACAM5 pH-dependently and CEACAM6 pH-independently. Furthermore, upon pH-dependent dissociation, the antibody was able to rebind to CEACAM5 for multiple cycles. This methodology could be used to engineer additional modes of action into already existing common light chain bi- or multispecifics antibodies as well as to swiftly generate pH-responsive mAbs from scratch. In summary, these investigations illuminated a straightforward methodology to generate humanized multispecific chicken-derived antibodies. Different engineering strategies were elucidated to optimize affinities and incorporate pH-dependent specificity, paving the way for tailor-made therapeutics