Antibody display technologies enable the successful isolation of antigen-specific antibodies with therapeutic potential. The key feature that facilitates the selection of an antibody with prescribed properties is the coupling of the protein variant to its genetic information and is referred to as genotype phenotype coupling. There are several different platform technologies based on prokaryotic organisms as well as strategies employing higher eukaryotes. Among those, phage display is the most established system with more than a dozen of therapeutic antibodies approved for therapy that have been discovered or engineered using this approach. In recent years several other technologies gained a certain level of maturity, most strikingly mammalian display. In this review, we delineate the most important selection systems with respect to antibody generation with an emphasis on recent developments.

Monoclonal antibody therapeutics have proven to be successful treatment options for patients in various indications. Particularly in oncology, therapeutic concepts involving antibodies often rely on the so-called effector functions, such as antibody dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC), which are programed in the antibody Fc region. However, Fc-mediated effector mechanisms often seem to be insufficient in properly activating the immune system to act against tumor cells. Furthermore, long term treatments can lead to resistance against the applied drug, which is monospecific by nature. There is promise in using specific antibodies to overcome such issues due to their capability of recruiting and activating T-cells directly at the tumor site, for instance. During the last decade, two of these entities, which are referred to as Blinatumomab and Catumaxomab, have been approved to treat patients with acute lymphoblastic leukemia and malignant ascites. In addition, Emicizumab, which is a bispecific antibody targeting clotting factors IXa and X, was recently granted market approval by the FDA in 2017 for the treatment of hemophilia A. However, the generation of these next generation therapeutics is challenging and requires tremendous engineering efforts as two distinct paratopes need to be combined from two different heavy and light chains. This mini review summarizes technologies, which enable the generation of antibodies with dual specificities.

Activating NK cell receptors represent promising target structures to elicit potent antitumor immune responses. In this study, novel immunoligands were generated that bridge the activating NK cell receptor NKp30 on NK cells with epidermal growth factor receptor (EGFR) on tumor cells in a bispecific IgG-like format based on affinity-optimized versions of B7-H6 and the Fab arm derived from cetuximab. To enhance NKp30 binding, the solitary N-terminal IgV domain of B7-H6 (ΔB7-H6) was affinity matured by an evolutionary library approach combined with yeast surface display. Biochemical and functional characterization of 36 of these novel ΔB7-H6–derived NK cell engagers revealed an up to 45-fold–enhanced affinity for NKp30 and significantly improved NK cell–mediated, EGFR-dependent killing of tumor cells compared with the NK cell engager based on the wild-type ΔB7-H6 domain. In this regard, potencies (EC₅₀ killing) of the best immunoligands were substantially improved by up to 87-fold. Moreover, release of IFN-γ and TNF-α was significantly increased. Importantly, equipment of the ΔB7-H6–based NK cell engagers with a human IgG1 Fc part competent in Fc receptor binding resulted in an almost 10-fold superior killing of EGFR-overexpressing tumor cells compared with molecules either triggering FcγRIIIa or NKp30. Additionally, INF-γ and TNF-α release was increased compared with molecules solely triggering FcγRIIIa, including the clinically approved Ab cetuximab. Thus, incorporating affinity-matured ligands for NK cell–activating receptors might represent an effective strategy for the generation of potent novel therapeutic agents with unique effector functions in cancer immunotherapy. Key Points B7-H6 was affinity matured by yeast surface display. NK cell engagers harboring optimized B7-H6 have improved cytotoxic capacities. NK cell engagers based on improved B7-H6 display a differentiated cytokine profile.

Here, we report the characterization of a VHH-derived IgG-like bi- and trispecific antibody platform that essentially relies on the replacement of the VH and VL regions of a conventional antibody by two independently functioning VHH domains. Consequently, a VHH is engrafted onto constant region CH1 while the other VHH-based paratope is engrafted on the constant region of the light chain, Cκ or Cλ, resulting in a tetravalent bispecific IgG-like molecule. Combined with a heavy chain heterodimerization technique, this platform allows facile engineering of bi- and trispecific antibodies with flexible valencies. We demonstrate the general applicability of this generic platform approach and elaborate on the limitations of specific formats.

Yeast surface display (YSD) has proven to be a versatile platform technology for antibody discovery. However, the construction of antibody Fab libraries typically is a tedious three-step process that involves the generation of heavy chain as well as light chain display plasmids in different haploid yeast strains followed by yeast mating. Within this study, we aimed at implementing a focused Golden Gate Cloning approach for the generation of YSD libraries. For this, antibodies heavy and light chains were encoded on one single plasmid. Fab display on yeast cells was either mediated by a two-directional promoter system (2dir) or by ribosomal skipping (bicis). The general applicability of this methodology was proven by the functional display of a therapeutic antibody. Subsequently, we constructed large antibody libraries with heavy chain diversities derived from CEACAM5 immunized animals in combination with a common light chain. Target-specific antibodies from both display systems were readily obtained after three rounds of fluorescence activated cell sorting. Isolated variants exhibited high affinities in the nanomolar and subnanomolar range as well as appropriate biophysical properties. We demonstrated that Golden Gate Cloning appears to be a valid tool for the generation of large yeast surface display antibody Fab libraries. This procedure simplifies the hit discovery process of antibodies from immune repertoires.

Controlled Fab arm exchange (cFAE) has proven to be a generic and versatile technology for the efficient generation of IgG-like bispecific antibodies (DuoBodies or DBs), with several in clinical development and one product, amivantamab, approved by the Food and Drug Administration. In this study, we expand the cFAE-toolbox by incorporating VHH-modules at the C-termini of DB-IgGs, termed DB-VHHs. This approach enables the combinatorial generation of tri- and tetraspecific molecules with flexible valencies in a straightforward fashion. Using cFAE, a variety of multispecific molecules was produced and assessed for manufacturability and physicochemical characteristics. In addition, we were able to generate DB-VHHs that efficiently triggered natural killer cell mediated lysis of tumor cells, demonstrating the utility of this format for potential therapeutic applications.