Affibody Molecules Intended for Receptor-Mediated Transcytosis via the Transferrin Receptor

Abstract

The development of biologics for diseases affecting the central nervous system has been less successful compared to other disease areas, in part due to the challenge of delivering drugs to the brain. The most well-investigated and successful strategy for increasing brain uptake of biological drugs is using receptor-mediated transcytosis over the blood-brain barrier and, in particular, targeting the transferrin receptor-1 (TfR). Here, affibody molecules are selected for TfR using phage display technology. The two most interesting candidates demonstrated binding to human TfR, cross-reactivity to the murine orthologue, non-competitive binding with human transferrin, and binding to TfR-expressing brain endothelial cell lines. Single amino acid mutagenesis of the affibody molecules revealed the binding contribution of individual residues and was used to develop second-generation variants with improved properties. The second-generation variants were further analyzed and showed an ability for transcytosis in an in vitro transwell assay. The new TfR-specific affibody molecules have the potential for the development of small brain shuttles for increasing the uptake of various compounds to the central nervous system and thus warrant further investigations.

Keywords: affibody molecules; blood–brain barrier; directed evolution; phage display; receptor-mediated transcytosis; transferrin receptor-1.

Figure 1

(A) Affibody structure with 14 randomized positions (beige) in helices 1 and 2 (PDB:2B89). (B) Z library sequence with randomized positions marked with “X” aligned to sequences for Z_TfR#14 and Z_TfR#18.

Figure 2

Analysis by flow cytometry of Z_TfR-ABD constructs at 1 μM on (A) murine hTfR expressing bEnd.3 cells and (B) human TfR-expressing SK-OV-3 cells. The bar chart shows the MFI (mean fluorescent intensity) of TfR-binding, and signals are normalized to blank cells for respective cell lines. The first bar in both groups is negative control with cells incubated only with a secondary reagent (*H). The dashed line represents the signal from the negative control affibody molecule (Ztaq-ABD) (*C). A construct scFv8D3-Z_SYM73-ABD (*P) with an scFv fragment of the murine TfR-specific antibody 8D3 is included as a positive control. Values are given as mean ± s.d. based on n = 3 samples.

Figure 3

Binding to human brain endothelial cells. Flow cytometry showing binding to hCMEC/D3 cells for 300 nM of Z_TfR#14-ABD (purple) and Z_TfR#18-ABD (blue) with detection via HSA-647 at 640 nm excitation and 660 nm BP filter. In total, 20,000 cells were analyzed per sample.

Figure 4

Flow cytometry of SK-OV-3 cells. (A,B) Fluorescence from cells corresponding to affibody-binding. Z_TfR#14-ABD or Z_TfR#18-ABD co-incubated with a different molar excess of transferrin-AF488 (Tf-488). Cells incubated with only affibody are shown in purple. Cells incubated with affibody and Tf-488 are shown in blue (higher molar excess in a darker shade). The Z_TfR-ABD is detected by HSA-647 at 640/660 nm laser and filter. (C,D) Fluorescence from cells corresponding to Tf-488 binding. Z_TfR#14-ABD or Z_TfR#18-ABD co-incubated with a different molar excess of Tf-488. Cells incubated with only Tf-488 are shown in purple. Cells incubated with affibody and Tf-488 are shown in green (higher molar excess in a darker shade). Tf-488 is detected at 488/525 nm laser and filter. All samples are measured for 20,000 cells.

Figure 5

Evaluation for pH-dependent binding of the affibody constructs. (A) Schematic illustration of the experimental flow cytometric procedure. The sample is subjected to cells at physiological pH before washing and divided into different samples for dissociation at different pH for 30 min. The samples are finally washed at pH 7.4 and analyzed in the flow cytometer. Values are given as mean ± s.d. based on n = 3 samples. (B) The signal was obtained after incubation at different pH for Tf, Z_TfR#14, and Z_TfR#18. (C,D) Measurement of secondary structure by circular dichroism spectroscopy for Z_TfR#14 and Z_TfR#18 at pH 5.5, 6.5, and 7.4.

Figure 6

Mean fluorescence intensity (MFI) from flow cytometry on E. coli cells displaying single amino acid mutants of Z_TfR#14 or Z_TfR#18 in fusion to an albumin-binding domain. A negative control Z_wt [45] was included for the binding of hTfR to E. coli. Cells were incubated with labeled human TfR and fluorescently labeled HSA. MFI values are normalized by hTfR binding to expression levels (measured by HSA) and normalized with the signal for the original binder (Z_TfR#14 or Z_TfR#18). (A) Mutants of Z_TfR#14 incubated with 100 nM hTfR, and (B) Mutants of Z_TfR#18 incubated with 75 nM hTfR. Values are given as mean ± s.d. based on n = 2 samples.

Figure 7

Apparent permeability (p_app) for transcytosis of FITC labeled affibodies over a bEnd.3 cell barrier formed on recombinant silk membranes. Each sample is analyzed in at least triplicate and compared with the simultaneously added internal control in a two-sided students t-test (* p-value < 0.05, ** p-value < 0.01, *** p-value < 0.005). The Z_HER2 control targets the HER2 receptor. Values are given as mean ± s.d. based on n = 3 samples.