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The versatility of antibodies in modern life science or medicine is based not only on their ability to bind antigens with very high affinity but also on intrinsic stability, which allows for multiple protein engineering approaches to generate antibodies with tailor-made characteristics. This work focused on expanding the applicability of antibodies with responsive binding elements for in vitro and in vivo experiments while streamlining the screening process by optimizing the library preparation. The first project presented in this work focuses on pH-responsive sweeping bispecific antibodies (bsAbs). bsAbs comprise – in contrast to conventional antibodies – two antigen specificities that can be utilized to generate immunological synapses and subsequent target degradation. However, if solid tumor-associated antigens are targeted, bsAbs can suffer from antibody buffering if the antigen is also present in soluble form in the bloodstream, just as monospecific antibodies do. Recent publications describe the generation of recycling and sweeping antibodies which comprise pH-responsive or Calcium-responsive antigen binding in order to circumvent antibody buffering. This mechanism is based on the internalization of the antigen-antibody complex by endothelial cells and subsequent calcium concentration and pH change during vesicle maturation causing the antigen to dissociate. While the antibody is secreted and again able to bind antigen, the antigen is degraded in the forming lysosome. Our approach describes the first implementation of a recycling function into a bsAb equipped with common light chains. To this end, a histidine-doped common light chain library was generated based on the common light chain of a CEACAM5/CEACAM6 bsAb. This bsAb was isolated from an Omniflic immunization with the antigens CEACAM5 and CEACAM6, respectively, both relevant markers for colorectal cancer. As an additional goal, we aimed at introducing the pH-responsive binding solely for CEACAM5. The his-doped cLC library was screened for antigen-binding at pH 7.4 and antigen release at pH 5.0. Isolated candidates were characterized towards melting temperature and antigen affinity, only showing minor differences compared to the parental common light chain. More importantly, it was examined and confirmed that the generated common light chain remains a common light chain which can be used for a CEACAM5/CEACAM6 specific bsAb. This work mediates more profound insights into the engineering of common light chains for tailor-made binding characteristics resulting in a molecule where one antibody arm gains pH-responsive behavior while one arm remains pH-independent. The second investigation focuses on the generation of single chain Fragment variable molecules (scFvs) generated by chicken immunization as affinity ligands for affinity chromatography, expanding the utilization of chicken immunization derived antibody fragments. Affinity chromatography is a powerful tool to purify protein from complex solutions. Especially in the context of antibodies, naturally occurring ligands such as Protein A enable simplifying and accelerating the purification process. But the very high affinity of Protein A also comes with the drawback of requiring harsh elution conditions such as low pH or high ionic strength, resulting in antibody degradation during purification. Therefore, newly generated ligands for antibody purification are often engineered to include responsive binding elements allowing mild elution conditions during purification. We aimed at creating affinity ligands based on chicken antibodies, which are known for easy library generation, intrinsic thermal stability, and can be generated by a simple immunization process. Additionally, chickens are not mammals increasing the chance of provoking an immune reaction upon immunization with a mammal protein, unlike rodent hosts. Our chosen format – scFv – should also allow easy and fast production in bacterial hosts to ensure favorable production. The generated chicken scFv library could be utilized to isolate a plethora of different scFvs against our model protein, a human IgG scaffold. The resulting variants showed high affinity and comprised pH- and/or Magnesium-responsive binding behavior, which was an additional sorting criterion to enable mild elution conditions in a DSP setup in the end. After coupling the scFvs to a solid support, chromatographies were performed confirming the specificity as well as the pH-/Magnesium-responsive binding behavior in a DSP setup. Our experimental design proved to be a very time-efficient procedure to isolate potent affinity ligands enabling a mild elution purification of human proteins. The third part of this work focuses on the method of yeast display itself and the refinement thereof. The online monitoring during the sorting of yeast display libraries is enabled by immunofluorescent staining. Due to the staining, time has to be spent to prepare the library prior sorting. We developed an expression system where the surface presentation is entangled with intracellular tGFP expression to circumvent this procedure. To this end, a ribosomal skipping sequence (also known as 2A peptide) was genetically fused to the 3’ of the presented protein and 5’ of tGFP. Upon translation, the polypeptide chain is elongated until the last codon of the 2A peptide gene. A strand break occurs without causing the ribosome to stop but rather to continue translation with a second polypeptide chain (tGFP in this work) until a stop codon is recognized. This setup allows the detection of full-length surface protein constructs without utilizing immunological staining resulting in a more time- and cost-friendly alternative to immunological staining.

Affinity chromatography provides an excellent platform for protein purification, which is a key step in the large scale downstream processing of therapeutic monoclonal antibodies (Mabs). Protein A chromatography constitutes the gold standard for Mab purification. However, the required acidic conditions (2.8–3.5) for elution from the affinity matrix limit their applicability, particularly for next generation antibodies and antibody fusion proteins, since denaturation and irreversible aggregation can occur due to the acidic buffer conditions. Here we describe a generic procedure for the generation of antigen-specific chromatography ligands with tailor-made elution conditions. To this end, we generated a scFv-library based on mRNA from a chicken immunized with human Fc. The antibody repertoire was displayed on yeast Saccharomyces cerevisiae screened via FACS toward pH- and magnesium-responsive scFvs which specifically recognize human IgG antibodies. Isolated scFvs were reformatted, produced in Escherichia coli and immobilized on NHS-agarose columns. Several scFvs were identified that mediated antibody binding at neutral pH and antibody recovery at pH values of 4.5 and higher or even at neutral pH upon MgCl2 exposure. The iterative screening methodology established here is generally amenable to the straightforward isolation of stimulus-responsive antibodies that may become valuable tools for a variety of applications.