In VitroCharacterization of a Bivalent Anti-HER-2 Affibody with Potential for Radionuclide-Based Diagnostics

Abstract

The 185 kDa transmembrane glycoprotein human epidermal growth factor receptor 2 (HER-2) (p185/neu, c-ErbB-2) is overexpressed in breast and ovarian cancers. Overexpression in breast cancer correlates with poor patient prognosis, and visualization of HER-2 expression might provide valuable diagnostic information influencing patient management. We have previously described the generation of a new type of affinity ligand, a 58-amino-acid affibody (Z(HER2:4)) with specific binding to HER-2. In order to benefit from avidity effects, we have created a bivalent form of the affibody ligand, (Z(HER2:4))2. The monovalent and bivalent ligands were compared in various assays. The new bivalent affibody has a molecular weight of 15.6 kDa and an apparent affinity (K(D)) against HER-2 of 3 nM. After radioiodination, using the linker molecule N-succinimidyl p-(trimethylstannyl) benzoate (SPMB), in vitro binding assays showed specific binding to HER-2 overexpressing cells. Internalization of 125I was shown after delivery with both the monovalent and the bivalent affibody. The cellular retention of 125I was longer after delivery with the bivalent affibody when compared to delivery with the monovalent affibody. With approximately the same affinity as the monoclonal antibody trastuzumab (Herceptin) but only one tenth of the size, this new bivalent molecule is a promising candidate for radionuclide-based detection of HER-2 expression in tumors. 125I was used in this study as a surrogate marker for the diagnostically relevant radioisotopes 123I for single photon emission computed tomography (SPECT)/gamma-camera imaging and 124I for positron emission tomography (PET).

Engineering and characterization of a bispecific HER2 × EGFR‐binding affibody molecule

“…The affinities (equilibrium dissociation constant, K D ) of the monomeric affibody molecules were previously determined to be 22 pM for Z HER2:342 [30] and 5.4 nM for Z EGFR:1907 [31]. The dimer format of each affibody molecule in the bs affibody protein will most likely give an additional increase in functional affinity (avidity), as demonstrated previously [38,39]. Furthermore, high selectivity of the Z HER2:342 and Z EGFR:1907 affibody molecules to their respective target protein has been shown in previous studies [30,39].…”

Adenovirus 5 vector genetically re-targeted by an Affibody molecule with specificity for tumor antigen HER2/neu

“…54 No studies have been made to distinguish between internalization and natural receptor recycling pathways for the ZH Affibody. It is most likely that, the binding to Her2/neu promotes receptor internalization as it has been shown that about 30% of the monovalent ZH and 50% of the bivalent ZHZH are internalized after binding to SKBR-3 cells at all measured time points between 0-24 h. 32 It can also be assumed that internalization into endosomes can be stimulated if the virus, with its multivalent ZH ligands, pulls together several Her2/neu antigens, as multi-valency or bi-valency is often needed for internalization of antibodies. A somewhat contradicting result has been published by us earlier, when targeting by binding of the Herceptin antibody to the Ad5/R7-C2C2 virus needed RGD in the penton base to be able to infect HER2/neuexpressing cells.…”

“…The tandem repeat also has a higher avidity for HER2/neu than the monomer. 32 However, the affinity of the monomeric ZH is already in the nanomolar range, 30 which is comparable to the affinity of the WT knob for CAR (3 nM). 55 By trying to allow for correct folding of the knob sequence and by choosing a ligand that can fold correctly in the cytoplasm of infected cells, we have been able to produce a re-targeted Ad with improved phenotype and a new specificity with clinical relevance.…”

“…The reactivity of ZH ligands genetically introduced into Ad fibers was analyzed by a cell-binding assay with or without blocking by affinity-purified His6-(ZH) 2 Affibody molecules. 32 Baculovirus-infected Sf9 cells were harvested 48 h post-infection (pi), lysed in hypotonic buffer (10 mM Tris-HCl buffer, pH 7.5) at 01C, adjusted to isotonic conditions (150 mM NaCl in10 mM Tris-HCl buffer, pH 7.5) and centrifuged at 10 000 g, 41C, for 10 min. For the cell-binding assay the fiber containing supernatants were applied to cells in suspension (293 and SKOV-3) prewashed in phosphate-buffered saline (PBS) (0.01 M phosphate, 0.15 M NaCl).…”

HER2/neu: an increasingly important therapeutic target. Part 1: basic biology & therapeutic armamentarium

“…Part 1 Clinical Trial Outcomes PET agent (zr89-labeled trastuzumab) as a predictor of response to trastuzumab emtansine (ZEPHIR) has recently been initiated in Europe (NCT01565200). Additionally, bispecific antibodies targeting HER2/neu have been developed [123][124][125][126][127] taking advantage of the intrinsic immunologic function of antibodies recognizing HER2/neu. Finally, aptamers selected for recognition and binding to the HER2/neu molecule can deliver cytotoxic agents to HER2/neu-positive cells as an alternative to antibody-based delivery [128].…”