Affinity recovery of eight HER2-binding affibody variants using an anti-idiotypic affibody molecule as capture ligand

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Abstract

Affibody molecules generated by combinatorial protein engineering to bind the human epidermal growth factor receptor 2 (HER2) have in earlier studies proven to be promising tracers for HER2-mediated molecular imaging of cancer. Amino acid extensions either at the N- or C-terminus of these ZHER2 affibody molecules, have been successfully employed for site-specific radiolabeling of the tracer candidates. Hexahistidyls or other tags, which would be convenient for recovery purposes, should be avoided since they could negatively influence the tumor targeting efficacy and biodistribution properties of the tracer. Using a new ß-lactamase-based protein fragment complementation assay (PCA), an affibody molecule was isolated which bound a ZHER2 affibody molecule with sub-micromolar affinity, but not unrelated affibody molecules. This suggests that the interacting area include the HER2-binding surface of ZHER2. This novel anti-idiotypic affibody molecule ZE01 was produced in Escherichia coli, purified, and chemically coupled to a chromatography resin in order to generate an affibody-based affinity column, suitable for recovery of different variants of ZHER2 affibody molecules, having a common binding surface for HER2. Eight such ZHER2 affibody molecules, designed for future radioimaging investigations, having different C-terminal peptide extensions aimed for radioisotope (99mTc)-chelation, were successfully produced and recovered in a single step to high purity using the anti-idiotypic affibody ligand for the affinity purification. These results clearly suggest a potential for the development of anti-idiotypic affibody-based resins for efficient recovery of related variants of a target protein that might have altered biochemical properties, thus avoiding the cumbersome design of specific recovery schemes for each variant of a target protein.

Introduction

Affibody molecules are a class of small so called scaffold protein domains that have been exploited in a number of biotechnological applications and also proved potential for diagnostic and therapeutic use [1], [2], [3]. The proteins are based on a 58 amino acid three helical bundle domain Z, derived from staphylococcal protein A [4]. By randomizing 13 solvent exposed residues on helix one and two, a combinatorial library was created at the binding surface [5]. By displaying the library on bacteriophage, and subjecting the phage to a biopanning procedure against target proteins of choice, specific binders can be obtained with a typical affinity range of 0.1 to 100 nM. The binding strength can be improved in an affinity maturation procedure, in such cases when higher affinity is desired [6]. Phage display technology has been the preferred format for selection of new or improved affibody molecules. However, today there are alternative technologies available, e.g., microbead display [7], a protein fragment complementation assay [8], [9] (PCA) and bacterial display [10].

Affibody molecules are considered to have properties making them suitable as tracers in molecular imaging applications [11]; i.e., high affinity and specificity in their binding, small size (6.5 kDa) and high structural stability. HER2-specific affibody molecules have earlier been generated by phage display selection [12]. After directed evolution based on the first generation affibody molecule, second generation binders with high affinities were isolated [13]. One of these variants, the ZHER2:342, displayed properties, including a very high affinity of 22 pM, good solubility, stability and tumor uptake, making it a promising candidate for further studies [13]. In fact, the ZHER2:342 was the first affibody molecule to be administered to humans, when studied in a limited number of patients with recurrent breast cancer [14]. Administration of a microdose (less than 100 μg) of 111In-labeled ZHER2:342 resulted in high-quality SPECT images enabling the detection even of small lesions (12–14 mm) as early as 2 h post injection. This was also shown to be applicable on patients that were on Herceptin treatment [14].

In the initial imaging studies, affibody molecules were radiolabeled by non-site-specific iodination or conjugation of chelators to the ε-NH2-group of lysines using amine chemistry and the results have been summarized in several reviews [15], [16]. Nevertheless, site-specific labeling of tracers is indeed desired and of utmost importance to allow production of well-defined and homogenous products for clinical use. Labeling either by site-specific coupling of a chelator during peptide synthesis or by incorporation of a unique cysteine and using thiol-directed chemistry, have been evaluated [15], [16]. More recently, significant effort has been devoted to develop and optimize methods for 99mTc labeling of affibody molecules taking advantage of amino acid-based chelators placed either at the N- or C-terminus of the tracer. More than 20 different such constructs for ZHER2:342 have been investigated [17]. Taken together, the results using N- or C-terminal amino acid extensions as chelators for radiolabeling of candidate tracers for molecular imaging seem indeed promising. However, small differences in the amino acid sequences can significantly influence biodistribution and a number of variants might need to be investigated in order to find the optimal tracer. Such differences in properties e.g., in terms of hydrophilicity and charge, would argue for a general recovery scheme. Unfortunately, hexahistidyl tags, which would offer straightforward recovery using immobilized metal ion affinity chromatography (IMAC) are not optimal in this case. His6-tagging of ZHER2:342 site-specifically labeled with 99mTc or 111In has demonstrated to significantly increase liver radioactivity, as compared to when an untagged ZHER2:342 affibody tracer labeled in the same way was used in tumor targeting studies in mice [18], [19]. Thus, the use of His6-tags on affibody-based imaging tracers should be avoided. Instead, a general recovery scheme of different HER2-binding affibody variants to be evaluated as tracer candidates and not dependent on any affinity tag, would be appreciated.

Affibody molecules, being based on the well-characterized protein A, which has been extensively used as affinity ligand for immunoglobulins, were for obvious reasons early explored for bioseparation purposes. A number of affibody molecules coupled to affinity resins have demonstrated to very specifically capture their target in complex samples such as fermentation media, cell lysates, and human body fluids. This includes affibody molecules directed to human apolipoprotein A [20], Taq DNA polymerase [20], human factor VIII [21], the G protein of human respiratory syncytial virus [22], human IgA [23], human amyloid beta peptides [24], and human transferrin [25]. Efficient bioseparation methods can be of great value in industrial processes but also of significant benefit for purification purposes in research. Furthermore, selection of anti-idiotypic affibody molecules has previously been achieved on a few occasions, with the parental protein A domain Zwt or Taq DNA polymerase-binding ZTaq as target affibody molecules, respectively [26], [27]. In those cases, where phage display has been used as selection principle, the affinities have been in the low or sub-micromolar range, and the binding sites have been found to predominantly overlap with the target binding site of the original affibody molecule [26], [27], i.e., anti-idiotypic sites. The three-dimensional structures for these affibody:affibody interactions have been described [28], [29]. The anti-idiotypic affibody molecule binding the Zwt protein A domain (denoted ZSPA-1) has furthermore been successfully used as an affinity tag for affinity separation purposes [30], [31]. These results encouraged our effort to generate an anti-idiotypic affibody molecule binding the ZHER2:342 which could be evaluated as an affinity ligand of related affibody molecules.

In this study we employed a recently developed ß-lactamase-based protein fragment complementation assay (PCA) [8], [9] to generate affibody molecules binding the ZHER2:342 affibody molecule. The most promising candidate was characterized, expressed in a head-to-tail dimeric form, purified and used to generate a ZHER2-specific affinity chromatography column. Eight variants of a ZHER2 affibody molecule, all with different C-terminal amino acid extensions aimed to act as 99mTc-chelating peptides, were designed, expressed in Escherichia coli, and recovered using the generated tailor-made affinity resin.

Section snippets

Vectors, bacterial strains, and library construction

The target vector pPCA-TARGET and the library vector pPCA-LIB used in the selection system have been described in detail elsewhere [8], [32]. A gene fragment encoding a HER2-binding affibody molecule denoted ZHER2:342 [13] was inserted into the target vector utilizing the AscI and NotI restriction sites. The affibody library used in selection procedures, PCA-lib, consisting of 1.3 × 109 members [8], was designed by NN(G/T) codon variation as earlier described by Nord et al. [5]. Selected clones

PCA selection of anti-ZHER2:342 affibody molecules

Anti-ZHER2:342 affibody molecules were selected by the recently described β-lactamase protein complementation assay [8], [9]. Members of a naive affibody library (1.3 × 109 members), were individually co-expressed in E. coli cells as fused to a C-terminal fragment of the ß-lactamase reporter, together with a fusion protein comprising the N-terminal fragment of the reporter and the ZHER2:342 affibody molecule as the target protein (Fig. 1A and B). Clone-survival selections were performed in two

Conclusions

The use of various affinity tags, such as hexahistidyl-tags, is a convenient way to recover proteins in purification strategies. However, in some cases such affinity tags must be avoided, e.g., when the tag itself has a negative influence on the properties of the protein. We here report on the production and evaluation of an affibody-based affinity chromatography resin based on an anti-idiotypic affibody molecule selected to bind to HER2-binding affibody molecules. An anti-ZHER2 affibody

Acknowledgments

This research was supported by grants from VINNOVA (P35722-1) and the Swedish Foundation for Strategic Research (RBa08-0067) (Stiftelsen för Strategisk Forskning). The authors wish to express their gratitude to Prof. Sophia Hober for productive discussions and to Dr. Tove Alm, Johanna Steen, and Joel Lindgren, KTH, for technical assistance.

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