In preclinical studies, Affibody scaffolds have shown promising prospects in numerous HER2
-specific targeting applications. Moreover, the recent first application of 68
Ga- and 111
(ABY-002) in patients with metastatic breast cancer highlights the great potential of Affibody molecules to localize themselves to HER2
-positive metastatic lesions in patients (30
). However, the use of Affibody molecules for radiotherapeutic applications is questionable, mainly due to their relatively fast clearance (compared to antibody) and extremely high kidney uptakes (generally > 100% ID/g for radiometal-labeled Affibody scaffolds) (8
). The high renal accumulation of the radiometal-labeled Affibody molecules could result in very high radiation doses to the radiation-sensitive kidneys. Therefore this represents a critical concern for using Affibody molecules for radionuclide therapy. Thus, we aimed to develop a generalizable strategy to design Affibody bioconjugates which possess high tumor targeting specificity as well as low kidney accumulations.
As shown in earlier studies, the fusion of an Affibody protein to a small albumin binding domain (ABD) results in a 20 kDa Affibody-ABD fusion protein that shows reduced kidney uptake and that can be efficient in radionuclide therapy approaches (31
). Inspired by these results, we explored a simple and generalizable method to chemically conjugate several Affibody molecules to HSA for in vivo
applications. The resulting bioconjugate is a multimeric ligand with a size much below conventional antibodies (< 100 kDa). The chemical conjugation of Affibody molecules to HSA has several distinctive advantages compared to Affibody-ABD fusion proteins. First, our method avoids time-consuming techniques, such as molecular cloning and protein purification. Moreover, compared to the Affibody-ABD fusion proteins, the conjugation used in this paper is more versatile, and allows for a quick and efficient conjugation of many other peptides or proteins in a similar fashion. Second, the HSA modification not only improves the pharmacokinetics of the Affibody (e.g. reduced kidney uptake) due to a higher molecular weight, it also allows the attachment of multiple monomeric ligands to one HSA molecule. This may allow multiple and simultaneous binding to HER2
receptors and lead to an improved tumor targeting efficacy and retention. The advantages of using multivalent binders have been well documented and reviewed before (33
). Third, covalent conjugation of the Affibody molecule to HSA seems to be a more accessible approach than a non-covalent binding of the Affibody-fusion protein to the patients’ own serum albumin in vivo
The synthesis of chemically conjugated DOTA-HSA-ZHER2:342
was performed in three steps. First, HSA was modified with the metal chelator DOTA. It was found that two DOTA were coupled with one HSA molecule. The conjugation of DOTA to the backbone of HSA as the first step is advantageous and avoids a potential unfavorable modification of the Affibody protein. On the contrary, for radiolabeling of many Affibody proteins or preparations of Affibody-ABD fusion proteins, the lysine residues in the Affibody molecules were used for sites of chemical modification (6
). These approaches are not ideal and may generate products with reduced targeting capacity because multiple lysine residues presented in the Affibody are responsible for receptor recognition. Second, the bifunctional crosslinker Sulfo-SMCC was conjugated to DOTA-HSA through lysine residues. As shown by MALDI-TOF-MS analyses, 6 to 8 reactive groups were found on one HSA molecule. In the third step, the HER2
Affibody analog Ac-Cys-ZHER2:342
was covalently conjugated with DOTA-HSA-SMCC. It was found that up to five Affibody molecules were covalently conjugated onto one HSA molecule. We also tested different Affibody-to-HSA reaction ratios and found that higher ratios of Affibody molecules did not result in a higher number of attached Affibody molecules (data not shown).
Evaluation of the radiolabeled conjugates in mice demonstrated that 64Cu-DOTA-HSA-ZHER2:342 and 111In-DOTA-HSA-ZHER2:342 are promising agents for HER2 imaging via PET and SPECT/CT. For small-animal PET, excellent tumor-to- background contrast was obtained at 4 h, 24 h, and 48 h after injection of 64Cu-DOTA-HSA-ZHER2:342.Whereas the SKOV3 tumors in 64Cu-DOTA-HSA injected mice could be barely seen. Quantitative analysis of PET images suggested a significant difference in tumor uptake of 64Cu-DOTA-HSA-ZHER2:342 and 64Cu-DOTA-HSA. This is a good reflection of the specificity of 64Cu-DOTA-HSA-ZHER2:342 in HER2 positive tumors. Micro SPECT/CT with 111In-DOTA-HSA-ZHER2:342 clearly showed uptake into the tumors of mice at 24 h p.i. and even at 96 h p.i. For both PET and SPECT imaging the kidneys showed low uptakes. These imaging studies clearly indicate that HSA modification does not compromise the tumor targeting ability of the Affibody molecules, while significantly reducing the kidney uptakes of the protein.
The biodistribution studies of 111In-DOTA-HSA-ZHER2:342 showed the capacity of 111In-DOTA-HSA-ZHER2:342 to accumulate in HER2 expressing tumors. Such findings were in good agreement with the PET studies of 64Cu-DOTA-HSA-ZHER2:342 in xenograft-bearing mice. Due to the relatively high molecular weight of the compound (~ 75.4 to 96.2 kDa), the probe exhibited mainly a hepatic accumulation.
Additionally, spleen uptake was found to be high probably due to uptake through scavenger receptors in sessile macrophages within the spleen. Consistent with our expectation, the kidney retention (11.50% ID/g at 1 h and 5.37% ID/g at 48 h) of the conjugate was very low in comparison to the radiometal-labeled Affibody monomer itself (16
), trastuzumab (35
), anti-HER2 diabodies (37
) or minibodies (38
). Furthermore, the reduction of the renal clearance could be decreased to even lower levels compared to an 177
Lu-labelled Affibody-ABD fusion protein (15% vs
6.1% ID/g) as shown in biodistribution experiments in SKOV3 tumor bearing mice (31
). The specificities of 111
were also confirmed by the use of an excess of nonradiolabeled Ac-Cys-ZHER2:342 in vitro
and in vivo
. Biodistribution studies revealed a specific binding of 111
protein as shown by a reduced SKOV3 tumor uptake after co-injection with Ac-Cys-Z HER2:342