title: Novel technologies for antibody hit discovery and engineering of antibody-like proteins with therapeutic relevance
Creators: name: Pekar, Lukas
Publisher: name: UNSPECIFIED
Year: 2021

description: In 1975, scientists Köhler and Milstein successfully fused B-lymphocytes from immunized mice with myeloma cells and, through this procedure entitled hybridoma technology, were able to combine the advantages of both precursor cells. These immortalized cells are culturable and able to secrete antigen-specific antibodies. The scientists' invention, revolutionary for the production of monoclonal antibodies, was awarded with a Nobel Prize in 1984. It became apparent, however, that the hybridoma technology is a lengthy process with low efficiency. In addition, the murine origin of the antibodies causes a potential problem with immunogenicity when applied to humans. Several new technologies have been developed in the last decades in order to overcome these limitations. These technologies include the use of human naïve, semi-synthetic or synthetic antibody diversities (to avoid the immunogenicity issue) in combination with cellular or non-cellular in vitro selection systems for the presentation and isolation of antibodies, such as Phage or Yeast Display. By the invention of these methods, large repertoires could be examined for predefined characteristics, thus enabling the isolation of specific molecules, which advanced today's successful use of monoclonal antibodies in biotechnological and medical applications. Despite all improvements in the finding process of antibodies, the generation of initial display libraries is still complex and laborious. It is often a multistage process which, as with Yeast Display, encompasses multiple cloning steps, the generation of separated libraries for heavy and light chains as well as, ultimately, their mating. This process can be simplified and condensed to only one reaction through the application of Golden Gate Cloning (GGC). The cloning reaction within GGC is based on the use of Type IIs restriction enzymes, which digest DNA in a defined distance from their recognition sequences and, thereby, allow for the incorporation of overhangs with requested signatures. Type IIs restriction enzymes enable a targeted, single-stage cloning process in which all recognition sequences are removed during the reaction, thereby causing an equilibrium shift towards the specific cloning product. As part of this work, the feasibility of GGC for the generation of Yeast and Phage Display libraries could be demonstrated for three different selection campaigns. Specific Fab antibodies against CEACAM6, EGFR and hCG were isolated from the immune repertoires of transgenic rats and wild type chickens (Yeast Display), and EGFR-specific scFv and VHH antibodies were isolated from the immune repertoires of chickens and camelids (Phage Display). In comparison to the traditional generation of antibody libraries, GGC could be validated with regards to gene repertoire distribution, the overall size of libraries as well as to the biophysical characteristics of isolated antibodies. The developed single-stage GGC process is able to generate antibody libraries based on combinatorial heavy and light chain diversities with the same quality as the traditional method. In addition, the process is a faster and less laborious method, which parallels the multiple steps of the original approach. Besides classical monoclonal antibodies and antibody fragments, new antibody formats can broaden the therapeutic space of new biological entities. Among these are molecules such as bispecific antibodies (bsAbs) and immunoligands, as wells as other antibody fragments and derivates - such as camelid single domain antibody Fc-fusions. In theory, four different plasmids have to be used during the expression of IgG-based bispecific antibodies in cell culture, as they consist of two different heavy chains and two different light chains. This would lead to a statistical yield of only 12.5 % of the desired assembled protein. The Strand Exchange Engineered Domain (SEED) technology is one possibility to avoid heterogeneity during the expression of several similar polypeptide chains. Through the introduction of alternating IgA and IgG β sheet structures in the CH3 domains, antiparallel immunoglobulin structures occur, which promote a heterodimerization of the heavy chains. In order to avoid heavy chain homodimerization, additional technologies, such as "knob-into-holes", controlled Fab arm exchange or targeted amino acid exchanges used to introduce contrary electrostatic charges into both heavy chains have been developed. Moreover, mispairing of antibodies light chains can be prevented by technologies such as CrossMab or the use of a Common Light Chain. Another possibility is the utilization of camelid VHH domains. VHHs, compared to canonical antibodies, provide specific antigen binding exclusively via the variable domain of the heavy chain. In this context, it was one of the goals of the present work to combine the SEED Technology with a bispecific Fab-like VHH antibody which was conceptionally described by Baty and coworkers in 2013. This combination enabled the generation of a novel bi- and trispecific, IgG-like VHH-based antibody platform with related variable valences. The results of the characterization of this antibody format with regards to its biophysical and biochemical characteristics - such as the specific NK cell-mediated ADCC - verify the versatility of the generic platform in expressing fully functional mono-, bi- and trispecific antibodies with different valences as a "plug and play" application. Furthermore, the use of bispecific molecules for a targeted NK cell recruitment is of substantial interest nowadays. The specific NK cell recruitment towards tumorous or infected cells enables a specific cytolysis of target cells and a simultaneous immune modulation through NK cell-mediated cytokine release. One possibility to address the recruitment of NK cells are the Natural Cytotoxicity Receptors (NCRs), comprehensively expressed by NK cells. For this work, and through the use of Yeast Display, the N-terminal IgV-like domain of B7 H6 (natural ligand of NKp30), which is relevant for receptor binding, could be affinity matured. Via Fluorescence Activated Cell Sorting (FACS)-based selection, B7 H6 variants with significantly increased affinity to NKp30 were achieved. Additionally, these variants showed significantly increased NK cell mediated cytotoxicities and cytokine release when used in the format of bispecific immunoligands, compared to the parental B7-H6 molecule. These results verify the underlying hypothesis that an increased affinity of B7 H6 to NKp30 results in an increased NK cell-based tumor cell cytolysis as well as in increased NK cell-mediated secretion of proinflammatory cytokines. An increased affinity of activating NK cell receptors natural ligands could, therefore, be used in the development of potential immunotherapies for the treatment of patients with various cancerous diseases.