Ubiquitin-derived artificial binding proteins targeting oncofetal fibronectin reveal scaffold plasticity by ß-strand slippage.

Affilin proteins, artificial binding proteins based on the ubiquitin scaffold, have been generated by directed protein evolution to yield de-novo variants that bind the extra-domain B (EDB) of oncofetal fibronectin, an established marker of tumor neovasculature. The crystal structures of two EDB-specific Affilin variants reveal a striking structural plasticity of the ubiquitin scaffold, characterised by ß-strand slippage, leading to different negative register shifts of the ß5 strands. This process recruits amino acid residues from ß5 towards the N-terminus to an adjacent loop region and subsequent residues into ß5, respectively, remodeling the binding interface and leading to target specificity and affinity. Protein backbone alterations resulting from ß-strand register shifts, as seen in the ubiquitin fold, can pose additional challenges to protein engineering as structural evidence of these events is still limited and they are difficult to predict. However, they can surface under the selection pressure of directed evolution and suggest that backbone plasticity allowing ß-strand slippages can increase structural diversity, enhancing the evolutionary potential of a protein scaffold.

Evaluation of mild pH elution protein A resins for antibodies and Fc-fusion proteins.

Protein A affinity chromatography is widely used as a capture step for monoclonal antibodies (mAb) and molecules that possess an Fc-domain, such as fusion proteins and bispecific antibodies. However, the use of low pH (3.0-4.0) to elute the molecule and achieve acceptable yield (>85 %) can lead to product degradation (e.g. fragmentation, aggregation) for molecules sensitive to low pH. In this paper, we describe a comprehensive evaluation of two protein A resins with ligands designed to elute at a milder pH as a result of modified sequences in their Fc and VH3 binding regions. One of the evaluated resins has been made commercially available by Purolite and named Praesto Jetted A50 HipH. Results demonstrated that Jetted A50 HipH could elute the Fc-fusion protein and most mAbs evaluated with an elution pH at or above 4.6. Elution and wash optimization determined run conditions for high recovery (>90 % monomer yield), reduction of high molecular weight (HMW) species (>50 %), and significant host cell protein (HCP) clearance at the mildest elution pH possible. For a pH-stable mAb and a pH-sensitive fusion protein, cell culture material was purified with optimized conditions and demonstrated the mild elution pH resins' ability to purify product with acceptable yield, comparable or better impurity clearance, and significantly milder native eluate pH compared to traditional resins. The benefits of the mild elution pH resins were clearly exemplified for the pH-sensitive protein, where a milder elution buffer and native eluate pH resulted in only 2 % HMW in the eluate that remained stable over 48 h. In contrast, a traditional protein A resin requiring low pH elution led to eluate HMW levels of 8 %, which increased to 16 % over the same hold time. Additionally, these resins have high dynamic binding capacity and allow the use of traditional HCP washes. Therefore, Jetted A50 HipH is an ideal candidate for a platform protein A resin and provides flexibility for pH-sensitive proteins and stable mAbs, while preserving product quality, recovery, and seamless integration into a downstream process.

Affilin-based retargeting of adenoviral vectors to the epidermal growth factor receptor.

INTRODUCTION: Treatment of head and neck squamous cell carcinomas (HNSCC) by oncolytic adenoviral vectors holds promise as an efficient anti-cancer therapy. The epidermal growth factor receptor (EGFR) represents an attractive target receptor since it is frequently overexpressed in many types of HNSCC. METHODS: To achieve EGFR-specific targeting by human adenovirus type 5 (HAdV-5) based vectors, the EGFR affinity ligand Affilin was covalently attached in a position specific manner either to the fiber or the hexon protein of the vector capsid. In vitro and in vivo studies investigated EGFR-specific cancer cell transduction, susceptibility to natural sequestration mechanisms, pharmacokinetics and biodistribution profiles of Affilin-decorated vectors. RESULTS: Affilin-decorated vectors showed strongly enhanced and EGFR-specific cancer cell transduction in vitro and less susceptibility to known sequestration mechanisms of HAdV-5 particles. However, in vivo neither systemic nor intratumoral vector administration resulted in an improved transduction of EGFR-positive tumors. Comprehensive analyses indicated hampered EGFR-targeting by Affilin-decorated vectors was caused by rapid vector particle consumption due to binding to the murine EGFR, insufficient tumor vascularization and poor target accessibility for Affilin in the solid tumor caused by a pronounced tumor stroma. CONCLUSION: In vitro studies yielded proof-of-concept results demonstrating that covalent attachment of a receptor-specific Affilin to the adenoviral capsid provides an effective and versatile tool to address cancer-specific target receptors by adenoviral vectors. Regarding EGFR as the vector target, off-target tissue transduction and low receptor accessibility within the tumor tissue prevented efficient tumor transduction by Affilin-decorated vectors, rendering EGFR a difficult-to-target receptor for adenoviral vectors.

Novel ubiquitin-derived high affinity binding proteins with tumor targeting properties.

Targeting effector molecules to tumor cells is a promising mode of action for cancer therapy and diagnostics. Binding proteins with high affinity and specificity for a tumor target that carry effector molecules such as toxins, cytokines, or radiolabels to their intended site of action are required for these applications. In order to yield high tumor accumulation while maintaining low levels in healthy tissues and blood, the half-life of such conjugates needs to be in an optimal range. Scaffold-based binding molecules are small proteins with high affinity and short systemic circulation. Due to their low molecular complexity, they are well suited for combination with effector molecules as well as half-life extension technologies yielding therapeutics with half-lives adapted to the specific therapy. We have identified ubiquitin as an ideal scaffold protein due to its outstanding biophysical and biochemical properties. Based on a dimeric ubiquitin library, high affinity and specific binding molecules, so-called Affilin® molecules, have been selected against the extradomain B of fibronectin, a target almost exclusively expressed in tumor tissues. Extradomain B-binding molecules feature high thermal and serum stability as well as strong in vitro target binding and in vivo tumor accumulation. Application of several half-life extension technologies results in molecules of largely unaffected affinity but significantly prolonged in vivo half-life and tumor retention. Our results demonstrate the utility of ubiquitin as a scaffold for the generation of high affinity binders in a modular fashion, which can be combined with effector molecules and half-life extension technologies.

NH exchange in point mutants of human ubiquitin.

Several point mutants of human ubiquitin (Ub(T9V), Ub(F45W), Ub(F45G), and Ub(A46S)) were prepared by recombinant techniques. The NH exchange rate constants were measured by the NMR diffusion and the MEXICO methods and compared with those in the wild type to examine the influence of structural changes and to improve the understanding of this important reaction in studies of protein folding and denaturation. The observed changes follow qualitatively the polarity and steric alterations caused by the introduced amino acids. Attempts to reproduce quantitatively the observed changes by modeling studies and molecular dynamics simulations were not satisfactory.

Affilin-novel binding molecules based on human gamma-B-crystallin, an all beta-sheet protein.

The concept of novel binding proteins as an alternative to antibodies has undergone rapid development and is now ready for practical use in a wide range of applications. Alternative binding proteins, based on suitable scaffolds with desirable properties, are selected from combinatorial libraries in vitro. Here, we describe an approach using a beta-sheet of human gamma-B-crystallin to generate a universal binding site through randomization of eight solvent-exposed amino acid residues selected according to structural and sequence analyses. Specific variants, so-called Affilin, have been isolated from a phage display library against a variety of targets that differ considerably in size and structure. The isolated Affilin variants can be produced in Escherichia coli as soluble proteins and have a high level of thermodynamic stability. The crystal structures of the human wild-type gamma-B-crystallin and a selected Affilin variant have been determined to 1.7 A and 2.0 A resolution, respectively. Comparison of the two molecules indicates that the human gamma-B-crystallin tolerates amino acid exchanges with no major structural change. We conclude that the intrinsically stable and easily expressed gamma-B-crystallin provides a suitable framework for the generation of novel binding molecules.

Artificial, non-antibody binding proteins for pharmaceutical and industrial applications.

Using combinatorial chemistry to generate novel binding molecules based on protein frameworks ('scaffolds') is a concept that has been strongly promoted during the past five years in both academia and industry. Non-antibody recognition proteins derive from different structural families and mimic the binding principle of immunoglobulins to varying degrees. In addition to the specific binding of a pre-defined target, these proteins provide favourable characteristics such as robustness, ease of modification and cost-efficient production. The broad spectrum of potential applications, including research tools, separomics, diagnostics and therapy, has led to the commercial exploitation of this technology by various small- and medium-sized companies. It is predicted that scaffold-based affinity reagents will broaden and complement applications that are presently covered by natural or recombinant antibodies. Here, we provide an overview on current approaches in the biotech industry, considering both scientific and commercial aspects.

Effect of coenzyme modification on the structural and catalytic properties of wild-type transketolase and of the variant E418A from Saccharomyces cerevisiae.

Transketolase from baker's yeast is a thiamin diphosphate-dependent enzyme in sugar metabolism that reconstitutes with various analogues of the coenzyme. The methylated analogues (4'-methylamino-thiamin diphosphate and N1'-methylated thiamin diphosphate) of the native cofactor were used to investigate the function of the aminopyrimidine moiety of the coenzyme in transketolase catalysis. For the wild-type transketolase complex with the 4'-methylamino analogue, no electron density was found for the methyl group in the X-ray structure, whereas in the complex with the N1'-methylated coenzyme the entire aminopyrimidine ring was disordered. This indicates a high flexibility of the respective parts of the enzyme-bound thiamin diphosphate analogues. In the E418A variant of transketolase reconstituted with N1'-methylated thiamin diphosphate, the electron density of the analogue was well defined and showed the typical V-conformation found in the wild-type holoenzyme [Lindqvist Y, Schneider G, Ermler U, Sundstrom M (1992) EMBO J11, 2373-2379]. The near-UV CD spectrum of the variant E418A reconstituted with N1'-methylated thiamin diphosphate was identical to that of the wild-type holoenzyme, while the CD spectrum of the variant combined with the unmodified cofactor did not overlap with that of the native protein. The activation of the analogues was measured by the H/D-exchange at C2. Methylation at the N1' position of the cofactor activated the enzyme-bound cofactor analogue (as shown by a fast H/D-exchange rate constant). The absorbance changes in the course of substrate turnover of the different complexes investigated (transient kinetics) revealed the stability of the alpha-carbanion/enamine as the key intermediate in cofactor action to be dependent on the functionality of the 4-aminopyrimidine moiety of thiamin diphosphate.

Snapshot of a key intermediate in enzymatic thiamin catalysis: crystal structure of the alpha-carbanion of (alpha,beta-dihydroxyethyl)-thiamin diphosphate in the active site of transketolase from Saccharomyces cerevisiae.

Kinetic and spectroscopic data indicated that addition of the donor substrate hydroxypyruvate to the thiamin diphosphate (ThDP)-dependent enzyme transketolase (TK) led to the accumulation of the alpha-carbanion/enamine of (alpha,beta-dihydroxyethyl) ThDP, the key reaction intermediate in enzymatic thiamin catalysis. The three-dimensional structure of this intermediate trapped in the active site of yeast TK was determined to 1.9-A resolution by using cryocrystallography. The electron density suggests a planar alpha-carbanion/enamine intermediate having the E-configuration. The reaction intermediate is firmly held in place through direct hydrogen bonds to His-103 and His-481 and an indirect hydrogen bond via a water molecule to His-69. The 4-NH(2) group of the amino-pyrimidine ring of ThDP is within 3 A distance to the alpha-hydroxy oxygen atom of the dihydroxyethyl moiety but at an angle unfavorable for a strong hydrogen bond. No structural changes occur in TK on formation of the reaction intermediate, suggesting that the active site is poised for catalysis and conformational changes during the enzyme reaction are not very likely. The intermediate is present with high occupancy in both active sites, arguing against previous proposals of half-of-the-sites reactivity in yeast TK.