Johan Nilvebrant

Engineering Bispecificity into a Single Albumin-Binding Domain

Bispecific antibodies as well as non-immunoglobulin based bispecific affinity proteins are considered to have a very high potential in future biotherapeutic applications. In this study, we report on a novel approach for generation of extremely small bispecific proteins comprised of only a single structural domain. Binding to tumor necrosis factor-α (TNF-α) was engineered into an albumin-binding domain while still retaining the original affinity for albumin, resulting in a bispecific protein composed of merely 46 amino acids. By diversification of the non albumin-binding side of the three-helix bundle domain, followed by display of the resulting library on phage particles, bispecific single-domain proteins were isolated using selections with TNF-α as target. Moreover, based on the obtained sequences from the phage selection, a second-generation library was designed in order to further increase the affinity of the bispecific candidates. Staphylococcal surface display was employed for the affinity maturation, enabling efficient isolation of improved binders as well as multiparameter-based sortings with both TNF-α and albumin as targets in the same selection cycle. Isolated variants were sequenced and the binding to albumin and TNF-α was analyzed. This analysis revealed an affinity for TNF-α below 5 nM for the strongest binders. From the multiparameter sorting that simultaneously targeted TNF-α and albumin, several bispecific candidates were isolated with high affinity to both antigens, suggesting that cell display in combination with fluorescence activated cell sorting is a suitable technology for engineering of bispecificity. To our knowledge, the new binders represent the smallest engineered bispecific proteins reported so far. Possibilities and challenges as well as potential future applications of this novel strategy are discussed.

A small bispecific protein selected for orthogonal affinity purification

A novel protein domain with dual affinity has been created by randomization and selection. The small alkali‐stabilized albumin‐binding domain (ABD*), used as scaffold to construct the library, has affinity to human serum albumin (HSA) and is constituted of 46 amino acids of which 11 were randomized. To achieve a dual binder, the binding site of the inherent HSA affinity was untouched and the randomization was made on the opposite side of the molecule. Despite its small size and randomization of almost a quarter of its amino acids, a bifunctional molecule, ABDz1, with ability to bind to both HSA and the Z2 domain/protein A was successfully selected using phage display. Moreover, the newly selected variant showed improved affinity for HSA compared to the parental molecule. This novel protein domain has been characterized regarding secondary structure and affinity to the two different ligands. The possibility for affinity purification on two different matrices has been investigated using the two ligands, the HSA matrix and the protein A‐based, MabSelect SuRe matrix, and the new protein domain was purified to homogeneity. Furthermore, gene fusions between the new domain and three different target proteins with different characteristics were made. To take advantage of both affinities, a purification strategy referred to as orthogonal affinity purification using two different matrices was created. Successful purification of all three versions was efficiently carried out using this strategy.

The albumin-binding domain as a scaffold for protein engineering

The albumin-binding domain is a small, three-helical protein domain found in various surface proteins expressed by gram-positive bacteria. Albumin binding is important in bacterial pathogenesis and several homologous domains have been identified. Such albumin-binding regions have been used for protein purification or immobilization. Moreover, improvement of the pharmacokinetics, through the non-covalent association to albumin, by fusing such domains to therapeutic proteins has been shown to be successful. Domains derived from streptococcal protein G and protein PAB from Finegoldia magna, which share a common origin and therefore represent an interesting evolutionary system, have been thoroughly studied structurally and functionally. Their albumin-binding sites have been mapped and these domains form the basis for a wide range of protein engineering approaches. By substitution-mutagenesis they have been engineered to achieve a broader specificity, an increased stability or an improved binding affinity, respectively. Furthermore, novel binding sites have been incorporated either by replacing the original albumin-binding surface, or by complementing it with a novel interaction interface. Combinatorial protein libraries, where several residues have been randomized simultaneously, have generated a large number of new variants with desired binding characteristics. The albumin-binding domain has also been utilized to explore the relationship between three-dimensional structure and amino acid sequence. Proteins with latent structural information built into their sequence, where a single amino acid substitution shifts the equilibrium in favor of a different fold with a new function, have been designed. Altogether, these examples illustrate the versatility of the albumin-binding domain as a scaffold for protein engineering.

Selection and in vitro characterization of human CD44v6-binding antibody fragments

The cluster of differentiation (CD) 44v6 antigen has been suggested to be involved in tumor formation, invasion, and metastasis formation, and has been observed in a majority of primary and metastatic squamous cell carcinomas of the head and neck. Probes specifically binding to this region may be utilized as tools for the challenging tasks of early detection and targeted treatments of small residual disease. In this project, an epitope‐guided phage display selection of human fragment antigen‐binding (Fab) fragments with affinity to the v6 sequence was performed. A selected set of Fab fragments was shown to specifically recognize increasingly complex forms of the target sequence, both in the form of a short synthetic or recombinant peptide and in the context of a purified extracellular domain of CD44. The binding was independent of known v6‐sequence variation and posttranslational modifications that are common in the CD44 protein family. Furthermore, real‐time interaction measurements on antibody fragments labeled with 125I showed specific and high‐affinity binding to the antigen present on cultured head and neck squamous cell carcinoma cells. There was no cross‐reactivity toward cells that lack the target protein. As hypothesized, characterization of the interaction between Fab fragments and the targets using the mathematical tool Interaction Map revealed more heterogeneous interactions on cells than with pure proteins analyzed by surface plasmon resonance. One main candidate Fab fragment with optimal affinity for all forms of the target sequence was identified. The flexible recombinant source of the Fab fragments might aid the development of tailored molecules adapted for therapeutic or diagnostic applications in the future.

Engineering of bispecific affinity proteins with high affinity for ERBB2 and adaptable binding to albumin.

The epidermal growth factor receptor 2, ERBB2, is a well-validated target for cancer diagnostics and therapy. Recent studies suggest that the over-expression of this receptor in various cancers might also be exploited for antibody-based payload delivery, e.g. antibody drug conjugates. In such strategies, the full-length antibody format is probably not required for therapeutic effect and smaller tumor-specific affinity proteins might be an alternative. However, small proteins and peptides generally suffer from fast excretion through the kidneys, and thereby require frequent administration in order to maintain a therapeutic concentration. In an attempt aimed at combining ERBB2-targeting with antibody-like pharmacokinetic properties in a small protein format, we have engineered bispecific ERBB2-binding proteins that are based on a small albumin-binding domain. Phage display selection against ERBB2 was used for identification of a lead candidate, followed by affinity maturation using second-generation libraries. Cell surface display and flow-cytometric sorting allowed stringent selection of top candidates from pools pre-enriched by phage display. Several affinity-matured molecules were shown to bind human ERBB2 with sub-nanomolar affinity while retaining the interaction with human serum albumin. Moreover, parallel selections against ERBB2 in the presence of human serum albumin identified several amino acid substitutions that dramatically modulate the albumin affinity, which could provide a convenient means to control the pharmacokinetics. The new affinity proteins competed for ERBB2-binding with the monoclonal antibody trastuzumab and recognized the native receptor on a human cancer cell line. Hence, high affinity tumor targeting and tunable albumin binding were combined in one small adaptable protein.

Development and characterization of small bispecific albumin-binding domains with high affinity for ErbB3.

Affinity proteins based on small scaffolds are currently emerging as alternatives to antibodies for therapy. Similarly to antibodies, they can be engineered to have high affinity for specific proteins. A potential problem with small proteins and peptides is their short in vivo circulation time, which might limit the therapeutic efficacy. To circumvent this issue, we have engineered bispecificity into an albumin-binding domain (ABD) derived from streptococcal Protein G. The inherent albumin binding was preserved while the opposite side of the molecule was randomized for selection of high-affinity binders. Here we present novel ABD variants with the ability to bind to the epidermal growth factor receptor 3 (ErbB3). Isolated candidates were shown to have an extraordinary thermal stability and affinity for ErbB3 in the nanomolar range. Importantly, they were also shown to retain their affinity to albumin, hence demonstrating that the intended strategy to engineer bispecific single-domain proteins against a tumor-associated receptor was successful. Moreover, competition assays revealed that the new binders could block the natural ligand Neuregulin-1 from binding to ErbB3, indicating a potential anti-proliferative effect. These new binders thus represent promising candidates for further development into ErbB3-signaling inhibitors, where the albumin interaction could result in prolonged in vivo half-life.

In vivo characterization of the novel CD44v6-targeting Fab fragment AbD15179 for molecular imaging of squamous cell carcinoma: a dual-isotope study

BackgroundPatients with squamous cell carcinoma in the head and neck region (HNSCC) offer a diagnostic challenge due to difficulties to detect small tumours and metastases. Imaging methods available are not sufficient, and radio-immunodiagnostics could increase specificity and sensitivity of diagnostics. The objective of this study was to evaluate, for the first time, the in vivo properties of the radiolabelled CD44v6-targeting fragment AbD15179 and to assess its utility as a targeting agent for radio-immunodiagnostics of CD44v6-expressing tumours.MethodsThe fully human CD44v6-targeting Fab fragment AbD15179 was labelled with 111In or 125I, as models for radionuclides suitable for imaging with SPECT or PET. Species specificity, antigen specificity and internalization properties were first assessed in vitro. In vivo specificity and biodistribution were then evaluated in tumour-bearing mice using a dual-tumour and dual-isotope setup.ResultsBoth species-specific and antigen-specific binding of the conjugates were demonstrated in vitro, with no detectable internalization. The in vivo studies demonstrated specific tumour binding and favourable tumour targeting properties for both conjugates, albeit with higher tumour uptake, slower tumour dissociation, higher tumour-to-blood ratio and higher CD44v6 sensitivity for the 111In-labelled fragment. In contrast, the 125I-Fab demonstrated more favourable tumour-to-organ ratios for liver, spleen and kidneys.ConclusionsWe conclude that AbD15179 efficiently targets CD44v6-expressing squamous cell carcinoma xenografts, and particularly, the 111In-Fab displayed high and specific tumour uptake. CD44v6 emerges as a suitable target for radio-immunodiagnostics, and a fully human antibody fragment such as AbD15179 can enable further clinical imaging studies.

ADAPT, a Novel Scaffold Protein-Based Probe for Radionuclide Imaging of Molecular Targets That Are Expressed in Disseminated Cancers.

Small engineered scaffold proteins have attracted attention as probes for radionuclide-based molecular imaging. One class of these imaging probes, termed ABD-Derived Affinity Proteins (ADAPT), has been created using the albumin-binding domain (ABD) of streptococcal protein G as a stable protein scaffold. In this study, we report the development of a clinical lead probe termed ADAPT6 that binds HER2, an oncoprotein overexpressed in many breast cancers that serves as a theranostic biomarker for several approved targeting therapies. Surface-exposed amino acids of ABD were randomized to create a combinatorial library enabling selection of high-affinity binders to various proteins. Furthermore, ABD was engineered to enable rapid purification, to eradicate its binding to albumin, and to enable rapid blood clearance. Incorporation of a unique cysteine allowed site-specific conjugation to a maleimido derivative of a DOTA chelator, enabling radionuclide labeling, ¹¹¹In for SPECT imaging and ⁶⁸Ga for PET imaging. Pharmacologic studies in mice demonstrated that the fully engineered molecule (111)In/⁶⁸Ga-DOTA-(HE)3-ADAPT6 was specifically bound and taken up by HER2-expressing tumors, with a high tumor-to-normal tissue ratio in xenograft models of human cancer. Unbound tracer underwent rapid renal clearance followed by high renal reabsorption. HER2-expressing xenografts were visualized by gamma-camera or PET at 1 hour after infusion. PET experiments demonstrated feasibility for discrimination of xenografts with high or low HER2 expression. Our results offer a preclinical proof of concept for the use of ADAPT probes for noninvasive in vivo imaging.

Engineered Autonomous Human Variable Domains.

BACKGROUND The complex multi-chain architecture of antibodies has spurred interest in smaller derivatives that retain specificity but can be more easily produced in bacteria. Domain antibodies consisting of single variable domains are the smallest antibody fragments and have been shown to possess enhanced ability to target epitopes that are difficult to access using multidomain antibodies. However, in contrast to natural camelid antibody domains, human variable domains typically suffer from low stability and high propensity to aggregate. METHODS This review summarizes strategies to improve the biophysical properties of heavy chain variable domains from human antibodies with an emphasis on aggregation resistance. Several protein engineering approaches have targeted antibody frameworks and complementarity determining regions to stabilize the native state and prevent aggregation of the denatured state. CONCLUSION Recent findings enable the construction of highly diverse libraries enriched in aggregation-resistant variants that are expected to provide binders to diverse antigens. Engineered domain antibodies possess unique advantages in expression, epitope preference and flexibility of formatting over conventional immunoreagents and are a promising class of antibody fragments for biomedical development.

Purification Systems Based on Bacterial Surface Proteins

Affinity purification is based on the selective and reversible interaction between two binding partners, of which one is bound to a chromatography matrix and the other may be either a native target protein or a recombinant protein fused with an affinity tag (Cuatrecasas et al. 1968). Recombinant DNA-technology allows straightforward construction of gene fusions to provide fusion proteins with two or more functions. The main intention is to facilitate downstream purification; however gene fusions may also improve solubility and proteolytic stability and assist in refolding (Waugh 2005). There are many fusion partners for which commercially available purification systems exist, ranging in size from a few amino acids to whole proteins (Flaschel & Friehs 1993; Terpe 2003). A commonly used purification handle is the poly-histidine (His) tag, enabling purification of the recombinant protein on a column with immobilized metal ions (Hochuli et al. 1988). Other commonly used tags include the FLAG peptide (binding to anti-FLAG monoclonal antibodies), the strep-tag (binding to streptavidin), glutathione S-transferase (binding to glutathione) and maltose binding protein (binding to amylose) (Terpe 2003). Many affinity chromatography strategies also exist for the purification of native proteins, however these are slightly less specific and generally purify classes of proteins, as individual proteins each need a specific ligand. Today, many different ligands are available that can separate specific groups of proteins, for example phosphorylated, glycosylated or ubiquitinylated proteins (Azarkan et al. 2007).