Site-Specific Radiometal Labeling and Improved Biodistribution Using ABY-027, A Novel HER2-Targeting Affibody Molecule–Albumin-Binding Domain Fusion Protein

Abstract

Because of their better penetration, smaller targeting proteins may be superior to antibodies for radioimmunotherapy of solid tumors. Therefore, Affibody molecules (6.5 kDa) have a potential for being suitable as targeted moiety for radiolabeled therapeutic proteins. Previous studies have demonstrated that a fusion of an Affibody molecule with an albumin-binding domain (ABD) provides a strong noncovalent binding to albumin in vivo. This strong noncovalent binding can be used for reduction of the renal uptake of the Affibody molecule while maintaining a size smaller than that of an antibody, which is important when using residualizing radionuclide labels conjugated to Affibody molecules. The goal of this study was to design and evaluate a new targeting Affibody-ABD fusion protein with improved biodistribution properties for radionuclide therapy. Methods: A novel Affibody-based construct, Z HER2:2891 -ABD 035 -DOTA (ABY-027), was created by fusion of the reengineered HER2-binding Affibody molecule Z HER2:2891 to the N terminus of the high-affinity ABD 035 , and a maleimido-derivative of DOTA was conjugated at the C terminus of the construct. Binding and processing of 177 Lu-ABY-027 by HER2-expressing cells were evaluated in vitro. Targeting of HER2-expressing SKOV-3 xenografts was evaluated in BALB/C nu/nu mice and compared with targeting of previously reported ABD-(Z HER2:342 ) 2 . Results: The binding affinity (dissociation constant) of ABY-027 to HER2 (74 pM) was the same as for the parental Z HER2:2891 (76 pM). ABY-027 was stably labeled with 177 Lu and 111 In with preserved specific binding to HER2-expressing cells in vitro. In vivo receptor saturation experiments demonstrated that targeting of SKOV-3 xenografts in BALB/C nu/nu mice was HER2-specific. 177 Lu-ABY-027 demonstrated substantially (2-to 3-fold) lower renal and hepatic uptake than previously assessed HER2-specific Affibody-based albumin-binding agents. Tumor uptake of radiolabeled ABY-027 at 48 h after injection was 2-fold higher than that for previously reported ABD-(Z HER2:342 ) 2 . Conclusion: An optimized molecular design of an ABD fusion protein resulted in an Affibody molecule construct with better properties for therapy. Fully preserved in vivo targeting of the fusion protein was shown in xenografted mice. Site-specific coupling of DOTA provides a uniform conjugate and creates the potential for labeling with a broad range of therapeutic radionuclides. The biodistribution of 177 Lu-ABY-027 in a murine model suggests it is more suitable for therapy than alternative approaches.

A truncated and dimeric format of an Affibody library on bacteria enables FACS‐mediated isolation of amyloid‐beta aggregation inhibitors with subnanomolar affinity

“…Alternative scaffold-proteins are in general much smaller than full-length antibodies and can usually be engineered into multivalent as well as multispecific units with an overall size that remain smaller than the conventional antibody [17,18]. Such features can potentially improve in vivo biodistribution of the agent [19][20][21][22]. One type alternative binding-protein that has been investigated for various diagnostic and therapeutic applications is the small three-helical bundle Affibody molecule (6.5 kDa) [17,23].…”

Diabody Pretargeting with Click Chemistry In Vivo

“…Interestingly, approaches focused on increasing the apparent size of the targeting agents did reduce the glomerular filtration rate and thereby decreased renal reabsorption and strongly improved radionuclide tumor-to-kidney ratios (15,21). For example, both the PEGylation of diabodies and the modification of Affibody molecules with albumin-binding domains resulted in a strong reduction in glomerular filtration and tumor uptake that was 7-fold higher than kidney uptake (in %ID/g) (15,21).…”

“…In recent years, several strategies that reduce the renal uptake of radiometal-labeled low-molecular-weight agents have been developed (6,(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). One example is the conjugation of polyethylene glycol (PEG) to antibody fragments to increase the molecular weight beyond the renal molecular weight cutoff of 60 kDa, resulting in an impressive reduction in kidney uptake (21,22).…”

Positron Emission Tomography Imaging with 18F-Labeled ZHER2:2891 Affibody for Detection of HER2 Expression and Pharmacodynamic Response to HER2-Modulating Therapies

“…In this context previous Affibodies labeled with 68 Ga or 111 In showed approximately 10-fold higher renal localization than that seen in tumor (34,35,38), precluding imaging of tumors in the region around the kidney, as well as having an impact on dosimetry. Bioconjugation of the Affibody molecule with albumin, histidine containing tags or 18 F radiolabels have been proposed as alternative approaches to avoid tubular reabsorption and permitting rapid glomerular filtration (26,(39)(40)(41). Using a radiohalogen strategy with 18 F, we demonstrated rapid renal clearance of […”