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.

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.

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.

Antibodies can be successfully engineered and isolated by yeast or phage display of combinatorial libraries. Still, generation of libraries comprising heavy chain as well as light chain diversities is a cumbersome process involving multiple steps. Within this study, we set out to compare the output of yeast display screening of antibody Fab libraries from immunized rodents that were generated by Golden Gate Cloning with the conventional three step method of individual heavy- and light chain sub-library construction followed by chain combination via yeast mating. We demonstrate that the Golden Gate Cloning-based one-step process delivers libraries and antibodies from heavy- and light chain diversities with similar quality to the traditional method while being significantly less complex and faster. Additionally, we show that this method can also be used to successfully screen and isolate chimeric chicken/human antibodies following avian immunization.

Recent years have shown a tremendous increase and diversification in antibody-based therapeutics with advances in production techniques and formats. The plethora of currently investigated bi- to multi-specific antibody architectures can be harnessed to elicit a broad variety of specific modes of actions in oncology and immunology, spanning from enhanced selectivity to effector cell recruitment, all of which cannot be addressed by monospecific antibodies. Despite continuously growing efforts and methodologies, the identification of an optimal bispecific antibody as the best possible combination of two parental monospecific binders, however, remains challenging, due to tedious cloning and production, often resulting in undesired extended development times and increased expenses. Although automated high throughput screening approaches have matured for pharmaceutical small molecule development, it was only recently that protein bioconjugation technologies have been developed for the facile generation of bispecific antibodies in a ‘plug and play’ manner. In this review, we provide an overview of the most relevant methodologies for bispecific screening purposes—the DuoBody concept, paired light chain single cell production approaches, Sortase A and Transglutaminase, the SpyTag/SpyCatcher system, and inteins—and elaborate on the benefits as well as drawbacks of the different technologies.