Tumor Targeting Using Affibody Molecules: Interplay of Affinity, Target Expression Level, and Binding Site Composition

Abstract

Radionuclide imaging of cancer-associated molecular alterations may contribute to patient stratification for targeting therapy. Scaffold high-affinity proteins, such as Affibody molecules, are a new, promising class of probes for in vivo imaging. Methods. The effects of human epidermal growth factor receptor 2 (HER2) affinity and binding site composition of HER2-binding Affibody molecules, and of the HER2 density on the tumor targeting, were studied in vivo. The tumor uptake and tumor-to-organ ratios of Affibody molecules with moderate (dissociation constant [K D ] 5 10 29 M) or high (K D 5 10 210 M) affinity were compared between tumor xenografts with a high (SKOV-3) and low (LS174T) HER2 expression level in BALB/C nu/nu mice. Two Affibody molecules with similar affinity (K D 5 10 210 M) but having alternative amino acids in the binding site were compared. Results. In SKOV-3 xenografts, uptake was independent of affinity at 4 h after injection, but high-affinity binders provided 2-fold-higher tumor radioactivity retention at 24 h. In LS174T xenografts, uptake of high-affinity probes was already severalfold higher at 4 h after injection, and the difference was increased at 24 h. The clearance rate and tumor-to-organ ratios were influenced by the amino acid composition of the binding surface of the tracer protein. Conclusion. The optimal affinity of HER2-binding Affibody molecules depends on the expression of a molecular target. At a high expression level (.10 6 receptors per cell), an affinity in the low-nanomolar range is sufficient. At moderate expression, subnanomolar affinity is desirable. The binding site composition can influence the imaging contrast. This information may be useful for development of imaging agents based on scaffold affinity proteins.

Imaging of HER3-expressing xenografts in mice using a 99mTc(CO)3-HEHEHE-ZHER3:08699 affibody molecule

“…Taking into account that the in vivo specificity study demonstrated that radioactivity uptake in these organs was saturable, we can conclude that this uptake was receptor specific. Previously, an affibody molecule with an unrelated specificity for Taq polymerase (ZTaq), which has the same 3-helix structure and that only differs in the few amino acids in the binding region, was tested for biodistribution and tumor uptake in LS174T xenograft model [20,22]. The level of radioactivity concentration in tumors due to unspecific uptake was lower or on the level of muscles and blood.…”

“…Together with very high affinity, this permits high-contrast imaging of target expression within a few hours after injection. Multiple studies with different xenograft models have demonstrated that there is no unspecific uptake of affibody molecules in tumors, which is important to exclude false-positive findings [20,21,22]. Two clinical studies have confirmed the potential of affibody-based agents for imaging of HER2-expressing metastases in patients with disseminated breast cancer [23,24].…”

“…One of the challenges of imaging from HER2-targeting affibody molecules is that imaging of such expression levels requires low picomolar affinity [22]. To meet these requirements, additional affinity maturation was performed using bacterial display [27] and fluorescence-activated cell sorting (FACS), resulting in the selection of the Z 08698 and Z 08699 affibody molecules with affinities of around 50 and 21 pM, respectively [28].…”

Evaluation of backbone-cyclized HER2-binding 2-helix Affibody molecule for In Vivo molecular imaging

“…The affinity toward its target (18 nM, as measured using Biacore) (Figure 3) was substantially decreased in comparison with the parental 3-helix Z HER2:342 Affibody molecule (22 pM) [4]. However, previous studies have demonstrated that low nanomolar affinity of Affibody molecules is suitable for imaging of abundantly expressed HER2 [4,[15][16][17][18]31].…”

“…Further optimization of the sequence and the use of homocysteine for disulfide bridge formation allowed obtaining a 2-helix Affibody variant with affinity of 5 nM [14]. Although this affinity is appreciably lower than the affinity of parental Affibody molecules Z HER2:342 (22 pM, [4]), it is still sufficient for imaging applications [15]. A DOTA-conjugated 2-helix variant, DOTA-MUT-DS (other designation PEP09239), has previously been labeled with 68 Ga, 64 Cu and 111 In, and successfully used for imaging of HER2-expressing tumor xenografts in mice [16][17][18].…”