Björn Hock

Generation and analysis of the improved human HAL9/10 antibody phage display libraries.

BackgroundAntibody phage display is a proven key technology that allows the generation of human antibodies for diagnostics and therapy. From naive antibody gene libraries - in theory - antibodies against any target can be selected. Here we describe the design, construction and characterization of an optimized antibody phage display library.ResultsThe naive antibody gene libraries HAL9 and HAL10, with a combined theoretical diversity of 1.5×1010 independent clones, were constructed from 98 healthy donors using improved phage display vectors. In detail, most common phagemids employed for antibody phage display are using a combined His/Myc tag for detection and purification. We show that changing the tag order to Myc/His improved the production of soluble antibodies, but did not affect antibody phage display. For several published antibody libraries, the selected number of kappa scFvs were lower compared to lambda scFvs, probably due to a lower kappa scFv or Fab expression rate. Deletion of a phenylalanine at the end of the CL linker sequence in our new phagemid design increased scFv production rate and frequency of selected kappa antibodies significantly. The HAL libraries and 834 antibodies selected against 121 targets were analyzed regarding the used germline V-genes, used V-gene combinations and CDR-H3/-L3 length and composition. The amino acid diversity and distribution in the CDR-H3 of the initial library was retrieved in the CDR-H3 of selected antibodies showing that all CDR-H3 amino acids occurring in the human antibody repertoire can be functionally used and is not biased by E. coli expression or phage selection. Further, the data underline the importance of CDR length variations.ConclusionThe highly diverse universal antibody gene libraries HAL9/10 were constructed using an optimized scFv phagemid vector design. Analysis of selected antibodies revealed that the complete amino acid diversity in the CDR-H3 was also found in selected scFvs showing the functionality of the naive CDR-H3 diversity.

A generic approach to engineer antibody pH-switches using combinatorial histidine scanning libraries and yeast display

There is growing interest in the fast and robust engineering of protein pH-sensitivity that aims to reduce binding at acidic pH, compared to neutral pH. Here, we describe a novel strategy for the incorporation of pH-sensitive antigen binding functions into antibody variable domains using combinatorial histidine scanning libraries and yeast surface display. The strategy allows simultaneous screening for both, high affinity binding at pH 7.4 and pH-sensitivity, and excludes conventional negative selection steps. As proof of concept, we applied this strategy to incorporate pH-dependent antigen binding into the complementary-determining regions of adalimumab. After 3 consecutive rounds of separate heavy and light chain library screening, pH-sensitive variants could be isolated. Heavy and light chain mutations were combined, resulting in 3 full-length antibody variants that revealed sharp, reversible pH-dependent binding profiles. Dissociation rate constants at pH 6.0 increased 230- to 780-fold, while high affinity binding at pH 7.4 in the sub-nanomolar range was retained. Furthermore, binding to huFcRn and thermal stability were not affected by histidine substitutions. Overall, this study emphasizes a generalizable strategy for engineering pH-switch functions potentially applicable to a variety of antibodies and further proteins-based therapeutics.

An online monitoring system based on a synthetic sigma32-dependent tandem promoter for visualization of insoluble proteins in the cytoplasm of Escherichia coli

The expression of heterologous proteins in the cytoplasm of Escherichia coli is often accompanied by limitations resulting in uncontrollable fermentation processes, increased rates of cell lysis, and thus limited yields of target protein. To deal with these problems, reporter tools are required to improve the folding properties of recombinant protein. In this work, the well-known σ32-dependent promoters ibpAB and fxsA were linked in a tandem promoter (ibpfxs), fused with the luciferase reporter gene lucA to allow enhanced monitoring of the formation of misfolded proteins and their aggregates in E. coli cells. Overexpression of MalE31, a folding-defective variant of the maltose-binding protein, and other partially insoluble heterologous proteins showed that the lucA reporter gene was activated in the presence of these misfolded proteins. Contrary to this, the absence of damaged proteins or overexpression of mostly soluble proteins led to a reduced level of luciferase induction. Through performing expression of aggregation-prone proteins, we were able to demonstrate that the ibpfxs::lucA reporter unit is 2.5–4.5 times stronger than the single reporter units ibp::lucA and fxs::lucA. Data of misfolding studies showed that this reporter system provides an adequate tool for in vivo folding studies in E. coli from microtiter up to fermentation scales.

A dual expression platform to optimize the soluble production of heterologous proteins in the periplasm of Escherichia coli

The functional analysis of individual proteins or of multiprotein complexes—since the completion of several genome sequencing projects—is in focus of current scientific work. Many heterologous proteins contain disulfide-bonds, required for their correct folding and activity, and therefore, need to be transported to the periplasm. The production of soluble and functional protein in the periplasm often needs target-specific regulatory genetic elements, leader peptides, and folding regimes. Usually, the optimization of periplasmic expression is a step-wise and time-consuming procedure. To overcome this problem we developed a dual expression system, containing a degP-promoter-based reporter system and a highly versatile plasmid set. This combines the differential protein expression with the selection of a target-specific expression plasmid. For the validation of this expression tool, two different molecular formats of a recombinant antibody directed to the human epidermal growth factor receptor and human 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) were used. By application of this expression system we demonstrated that the amount of functional protein is inversely proportional to the on-line luciferase signal. We showed that this technology offers a simple tool to evaluate and improve the yield of functionally expressed proteins in the periplasm, which depends on the used regulatory elements and folding strategies.

Comparison of two expression platforms in respect to protein yield and quality: Pichia pastoris versus Pichia angusta

The methylotrophic yeasts Pichia pastoris and Pichia angusta (Hansenula polymorpha) were used for the comparative heterologous production of two model mammalian proteins of pharmaceutical interest, the NK1-fragment (22 kDa) of human hepatocyte growth factor and the extracellular domain (28 kDa) of mouse tissue factor (MTF). Both recombinant proteins were engineered to contain an N-terminal Strep- (WSHPQFEK) and a C-terminal His(6)-tag. In addition, both proteins contained the pre-pro-sequence of Saccharomyces cerevisiae mating factor alpha to allow secretion. Following vector construction, transformation and zeocin amplification, the best Pichia producers were identified in a screening procedure using Western blot and a Luminex xMAP based high-throughput method. Recombinant NK1-fragment and MTF were purified from culture supernatants of the best producers by affinity chromatography (Ni-nitrilotriacetic acid columns). Using P. pastoris as a host for the synthesis of NK1-fragment a protein yield of 5.7 mg/l was achieved. In comparable expression experiments P.angusta yielded 1.6 mg/l of NK1-fragment. NK1-fragment apparently was not glycosylated in either system. For the production of MTF, P. pastoris was also the superior host yielding 1.2mg/l glycosylated recombinant protein whereas P. angusta was clearly less efficient (<0.2mg/l MTF). For both expression systems no correlation between the amount of recombinant protein and the copy number of the chromosomally integrated heterologous genes was found. In P. pastoris strains less degradation of the two model recombinant proteins was observed. Altogether, this paper provides a structured protocol for rapidly identifying productive Pichia strains for the synthesis of full-length recombinant proteins.

Spontaneous Isopeptide Bond Formation as a Powerful Tool for Engineering Site-Specific Antibody-Drug Conjugates

Spontaneous isopeptide bond formation, a stabilizing posttranslational modification that can be found in gram-positive bacterial cell surface proteins, has previously been used to develop a peptide-peptide ligation technology that enables the polymerization of tagged-proteins catalyzed by SpyLigase. Here we adapted this technology to establish a novel modular antibody labeling approach which is based on isopeptide bond formation between two recognition peptides, SpyTag and KTag. Our labeling strategy allows the attachment of a reporting cargo of interest to an antibody scaffold by fusing it chemically to KTag, available via semi-automated solid-phase peptide synthesis (SPPS), while equipping the antibody with SpyTag. This strategy was successfully used to engineer site-specific antibody-drug conjugates (ADCs) that exhibit cytotoxicities in the subnanomolar range. Our approach may lead to a new class of antibody conjugates based on peptide-tags that have minimal effects on protein structure and function, thus expanding the toolbox of site-specific antibody conjugation.

REAL-Select: Full-Length Antibody Display and Library Screening by Surface Capture on Yeast Cells

We describe a novel approach named REAL-Select for the non-covalent display of IgG-molecules on the surface of yeast cells for the purpose of antibody engineering and selection. It relies on the capture of secreted native full-length antibodies on the cell surface via binding to an externally immobilized ZZ domain, which tightly binds antibody Fc. It is beneficial for high-throughput screening of yeast-displayed IgG-libraries during antibody discovery and development. In a model experiment, antibody-displaying yeast cells were isolated from a 1∶1,000,000 mixture with control cells confirming the maintenance of genotype-phenotype linkage. Antibodies with improved binding characteristics were obtained by affinity maturation using REAL-Select, demonstrating the ability of this system to display antibodies in their native form and to detect subtle changes in affinity by flow cytometry. The biotinylation of the cell surface followed by functionalization with a streptavidin-ZZ fusion protein is an approach that is independent of the genetic background of the antibody-producing host and therefore can be expected to be compatible with other eukaryotic expression hosts such as P. pastoris or mammalian cells.

Generation of human bispecific common light chain antibodies by combining animal immunization and yeast display

Bispecific antibodies (bsAbs) pave the way for novel therapeutic modes of action along with potential benefits in several clinical applications. However, their generation remains challenging due to the necessity of correct pairings of two different heavy and light chains and related manufacturability issues. We describe a generic approach for the generation of fully human IgG-like bsAbs. For this, heavy chain repertoires from immunized transgenic rats were combined with either a randomly chosen common light chain or a light chain of an existing therapeutic antibody and screened for binders against tumor-related targets CEACAM5 and CEACAM6 by yeast surface display. bsAbs with subnanomolar affinities were identified, wherein each separate binding arm mediated specific binding to the respective antigen. Altogether, the described strategy represents a combination of in vivo immunization with an in vitro selection method, which allows for the integration of existing therapeutic antibodies into a bispecific format.

Engineering bispecific antibodies with defined chain pairing

Bispecific IgG-like antibodies can simultaneously interact with two epitopes on the same or on different antigens. Therefore, these molecules facilitate novel modes of action, which cannot be addressed by conventional monospecific IgGs. However, the generation of such antibodies still appears to be demanding due to their specific architecture comprising four different polypeptide chains that need to assemble correctly. This review focusses on different strategies to circumvent this issue or to enforce a correct chain association with a focus on common-chain bispecific antibodies.

A high-throughput microtiter plate-based screening method for the detection of full-length recombinant proteins

The Gram-negative bacterium Escherichia coli is an important host for the (heterologous) production of recombinant proteins. The development and optimization of a protocol to overproduce a desired protein in E. coli is often tedious. A novel high-throughput screening method based on the Luminex xMAP bead technology was developed allowing a rapid evaluation of a certain expression strategy. A variant of green fluorescent protein (GFPuv) from Aequorea victoria was used as a reporter to establish the methodology. The N-terminus and the C-terminus of GFPuv were engineered to contain a His(6)- and an HA-tag (YPYDVPDYA), respectively. The double-tagged protein was loaded onto Luminex-microspheres via its His(6)-tag, the presence of the HA-tag was verified using an anti-HA antibody. High-throughput detection of full-length proteins (containing both tags) on the beads was performed using an automated Luminex 100IS analyzer. The results were compared to results obtained by classical Western blot analysis. Comparison of the two methods revealed that the Luminex-based method is faster and more economical in detecting full-length (intact) soluble recombinant protein, allowing one to routinely screen a high number of parameters in gene expression experiments. As proof of concept, different protocols to overproduce double-tagged model eucaryotic proteins (human protein S6 kinase 1 and human tankyrase) in E. coli were monitored using the new approach. Relevant parameters for optimizing gene expression of the corresponding genes were rapidly identified using the novel high-throughput method.