Next Article in Journal
Pectin Nanoparticle-Loaded Soft Coral Nephthea sp. Extract as In Situ Gel Enhances Chronic Wound Healing: In Vitro, In Vivo, and In Silico Studies
Previous Article in Journal
Reactive Palladium–Ligand Complexes for 11C-Carbonylation at Ambient Pressure: A Breakthrough in Carbon-11 Chemistry
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Pharmaceuticals
Volume 16
Issue 7
10.3390/ph16070956
pharmaceuticals-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

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

by
Linnea Charlotta Hjelm
,
Hanna Lindberg
,
Stefan Ståhl
and
John Löfblom
*
Department of Protein Science, School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, 106 91 Stockholm, Sweden
*
Author to whom correspondence should be addressed.
Pharmaceuticals 2023, 16(7), 956; https://doi.org/10.3390/ph16070956
Submission received: 29 May 2023 / Revised: 27 June 2023 / Accepted: 30 June 2023 / Published: 3 July 2023
(This article belongs to the Section Biopharmaceuticals)
Browse Figures
Versions Notes

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.

1. Introduction

Antibodies and other biological drugs have had a profound impact on the treatment of many different diseases, such as different forms of cancers and auto-immune disorders [1,2]. However, for diseases of the central nervous system (CNS), the restricted uptake over the blood–brain barrier (BBB) [3,4] makes development of therapeutics and diagnostic tools more challenging.
The BBB protects the sensitive neuronal environment by substantially decreasing the paracellular diffusion of substances from blood. Various receptors and transporters such as the transferrin receptor-1, the insulin-like growth factor 1 receptor, the L-type amino acid transporter, and the glucose transporter isoform 1 control the transcytosis of molecules in and out between the brain and blood compartments [3,5,6]. Transfer can either be by adsorption [7,8,9,10], passive or active transportation [5], or receptor-mediated transcytosis (RMT) [5]. It has been shown that about 0.1–0.2% of systemically administered antibodies pass into the CSF [11], which is potentially too low to achieve efficient disease-modifying effects [12]. Therefore, efforts have been made to find a transportation system across the BBB for biological drugs [5,13,14,15,16,17]. One of the more well-studied approaches is RMT via the transferrin receptor-1 (TfR), which naturally transports transferrin (Tf) across the BBB to the neuronal environment [7,18,19]. An example is the anti-murine TfR-specific 8D3 shuttle antibody [20], which has shown up to 80-fold increased uptake to the brain in rodent models [21]. For antibodies targeting TfR, several parameters have to be considered, such as affinity [22], valency [21,23], and antibody effector functions to minimize potential adverse effects [24].
In addition to antibodies and antibody derivatives [17,24,25], shuttles based on modified fragment crystallizable (Fc) from antibodies [16] and alternative scaffolds are emerging. The alternative scaffolds include a variety of protein structures, such as VNARs [26] and cysteine-dense peptides [27]. With a monovalent format and lack of Fc, they might potentially circumvent some limitations and safety liabilities previously seen for bivalent antibodies, such as harmful immune responses caused by effector function and TfR receptor depletion [21,28,29,30]. Moreover, the small size of alternative scaffolds might benefit biodistribution and targeting due to more rapid diffusion in the brain parenchyma, as has been shown for single-chain variable fragments (scFv) when compared to full-length antibodies [31].
Affibody molecules are small (6.5 kDa) affinity proteins that fold into a three-helical structure and typically demonstrate high thermal stability, high solubility, and efficient and complete refolding after denaturation [32]. Production is efficient in prokaryotic Escherichia coli, and the small size allows for production by solid-phase chemical peptide synthesis, enabling the straightforward incorporation of unnatural amino acids [32,33]. Affibodies with new specificities are generated by directed evolution (e.g., phage and bacterial display [32,34,35]), and binders to over 60 different targets have been reported in the literature [32]. The most advanced affibody molecule in clinical testing is izokibep, with a femtomolar affinity for interleukin 17-A (IL17-A) [32,36]. Results from phase I and II clinical trials show high efficacy in various IL17-driven auto-immune disorders and a good safety profile [36,37]. Selections from affibody libraries against intrinsically disordered neurodegenerative peptides have resulted in dimeric variants of the affibody scaffold with an unusual three-dimensional structure and mode of binding. The atypical dimeric binders are denoted sequestrins [38], and an example is a sequestrin (ZSYM73) targeting amyloid beta with subnanomolar affinity [39]. In (APP)/PS1 transgenic AD animal models, ZSYM73 restored cognitive function, inhibited amyloid beta aggregation, and eliminated neurotoxic effects [39]. Compared to antibodies, the small size of affibodies and sequestrins results in improved tissue penetration [19], but transportation across the BBB is still limited, corresponding to bioavailability of around 0.1–0.2% in CSF [39]. In a follow-up study, ZSYM73 was therefore fused to an scFv(8D3) brain shuttle, resulting in a 9-fold increase in CSF bioavailability [40].
Encouraged by the results, affibody molecules were here selected against TfR using phage display technology. Two of the binders demonstrated cross-reactivity to both human and murine TfR and displayed non-competitive binding with human Tf. The two candidates were further refined by single amino acid mutagenesis and evaluated in terms of different characteristics, such as thermal stability and cell binding. Finally, the top candidates were investigated for their ability of RMT using TfR-expressing brain endothelial cell lines in a recombinant silk-based in vitro assay where an increased uptake to the basal side for the TfR-specific affibodies was observed.

2. Results

2.1. Phage Display Selections

Affibody molecules were selected against the extracellular domain of recombinant human and murine transferrin receptors from an M13 filamentous phage library containing 3 × 1010 affibody molecules. The library was designed to include 14 surface-located randomized positions distributed over helices 1 and 2 (Figure 1A). Five rounds of panning of the library were performed in different tracks against human TfR, murine TfR, or a cross-selection strategy with alternating human and murine TfR (Figure S1). Target concentrations were gradually decreased from 100 to 12.5 nM (or 40 nM in cross-selection tracks) in the final rounds (Figure S1). Candidate binders were analyzed by phage ELISA for binding to the receptors, and 174 colonies from the last or second last cycle of panning were subjected to DNA sequencing.

2.2. Production of His6-Tagged and ABD-Fused Affibody Molecules

Nineteen different affibody molecules from the selections (Figure S2) were produced in both a His6-ZTfR-ABD and a ZTfR-His6 format. Affibody molecules can be fused to an albumin-binding domain (ABD) to prolong their half-life in circulation by interaction with human serum albumin (HSA) [36,41,42]. The production indicated a higher yield from the smaller format compared to the ABD-fusions (Tables S1 and S2). Molecular weight was verified by MS and correlated well with expected values (Tables S1 and S2). The purity was verified by SDS-PAGE (Figures S3 and S4), and all constructs showed the expected alpha-helical secondary structure content as measured using circular dichroism (CD) spectroscopy (Figure S5).

2.3. Flow Cytometry Analysis of ZTfR Candidates Aimed to Bind to Murine and Human Cells

The affibody candidates in ZTfR-ABD format were analyzed using flow cytometry for binding to human SK-OV-3 and murine brain endothelial cells (bEnd.3) expressing human and mouse TfR (Figure 2). Two of the affibody molecules (ZTfR#14 and ZTfR#18) demonstrated binding to both human SK-OV-3 and murine bEnd.3 (Figure 2, Table S3). The two binders originated from the selection track with alternating mTfR and hTfR as targets (Figure S1). ZTfR#14 and ZTfR#18 showed low sequence homology, which is typically an indication of non-overlapping epitopes (Figure 1B).
Next, TfR-positive human SK-OV-3 and brain endothelial hCMEC/D3 cells were incubated with a dilution series of ZTfR#14 and ZTfR#18, and binding was analyzed by flow cytometry, showing concentration-dependent signal (Figure S6 and Figure 3).
Cell-specific binding was investigated by co-incubating the His6-ZTfR-ABD construct with ZTfR-His6 at different molar excess ratios, showing a substantial decrease in signal in comparison to no blocking (Table S4). Moreover, flow cytometry was also used for epitope binning by co-incubating the His6-ZTfR#14-ABD construct with ZTfR#18-His6 and vice versa, showing a negligible decrease in binding and thus confirming non-overlapping epitopes (Table S4).
The high concentration of Tf in blood and results from previous studies demonstrating negative effects on reticulocyte count for transferrin-blocking agents in animal models [29] suggest that competitive binding with transferrin (Tf) is probably not optimal. Another epitope-binning experiment was thus performed, where the ZTfR candidates #2, #4, #10, #14, and #18 in ZTfR-ABD format were pre-incubated with fluorescently labeled transferrin (Tf-488) prior to analysis of cell-binding. For clones #2, #4, and #10, the Tf-488 signal decreased after co-incubation, whereas no shift in signal was observed for co-incubation with ZTfR#14 and ZTfR#18 (Figure S7). Furthermore, the signal from ZTfR#14 and ZTfR#18 was maintained at a 5-fold molar excess of Tf (Figure 4). A non-TfR binding control affibody (Ztaq) [43] was included in the experiment, showing no effect on TfR binding (Table S5) and supports the indication that the TfR epitope is not shared between the ZTfR#14, ZTfR#18, and Tf.

2.4. pH-Dependent Binding to TfR Expressing Cells

During transcytosis across the brain endothelial cells in the BBB, the pH of the endosomes containing TfR decreases to around 5.5 [18,19]. Thus, the dissociation of the affibodies from cells at different pH was investigated by employing a flow-cytometric assay similar to previously described studies [44] (Figure 5A). The analysis was performed at 4 °C to decrease internalization, and fluorescently labeled Tf (Tf-488) was included for comparison (Figure 5B). After incubation of binders with SK-OV-3 cells, cells were washed with buffers of different pH, followed by an analysis of cell-binding using flow cytometry. The pH-dependent binding of Tf–TfR was verified, shown as a decrease in signal for pH below 6.0 (Figure 5B). Interestingly, ZTfR#14 showed a pH-independent profile, and ZTfR#18 had opposite dependence compared to Tf, where the dissociation appeared slower at lower pH (Figure 5B). CD spectroscopy was used to verify secondary structure content, thermal stability, and refolding in the pH range (5.5–7.4) (Figure S8). The spectra measured at different pH were overlapping, indicating that the structure was similar and independent of pH in the given range (Figure 5C,D). The thermal melting point (Tm) was estimated to be 56 °C for ZTfR#14 and 58 °C for ZTfR#18, and spectra before and after the variable temperature measurements were overlapping, indicating full refolding (Figure S9).

2.5. Surface Plasmon Resonance Analysis of Binding between ZTfR and TfR

The affinity of the ZTfR#14-ABD and ZTfR#18-ABD to recombinant TfR was estimated by surface plasmon resonance (SPR) using an immobilized extracellular domain of human and murine his-tagged TfR. Both affibody molecules showed a higher affinity for murine TfR with a KD of 90 nM for ZTfR#14 and 170 nM for ZTfR#18. For human TfR, the KD was 150 nM for ZTfR#14 and 710 nM for ZTfR#18 (Figure S9 and Table S6).

2.6. Single Amino Acid Mutagenesis of ZTfR#14 and ZTfR#18

Single amino acid mutagenesis was performed on ZTfR#14 and ZTfR#18 to investigate the contribution of individual residues in binding to TfR. The previously 14 randomized positions of respective affibodies were mutated to either histidine or the wildtype amino acid from the original IgG-binding Z domain [45]. The genes for the mutated affibody molecules were subcloned in fusion to a gene encoding an engineered albumin-binding domain (ABD) [41] into an expression vector for Adhesin Involved in Diffuse Adherence (AIDA) 1-mediated display on the surface of E. coli [34]. E. coli expressing the different mutants on the surface were analyzed by flow cytometry for assessment of TfR-binding, and the original binders (ZTfR#14 and ZTfR#18) were included for comparison. In addition to the analysis of TfR-binding, cells were also incubated with saturating concentrations of fluorescently labeled HSA to assess surface expression levels.
For both affibody molecules, most mutations had a negative impact on the binding to TfR (Figure 6). However, two mutations with improved binding to TfR, for respective binder and with the introduction of histidine, were found, corresponding to A9H and L27H for ZTfR#14, and M14H and I11N for ZTfR#18, although M14H seemed to have a negative effect on the cell surface expression (Figure 6 and Figure S10).

2.7. Characterization of Second-Generation Affibody Molecules Binding TfR

The four mutants (ZTfR#14_A9H, ZTfR#14_L27H, ZTfR#18_I11N, and ZTfR#18_M14H) that showed improved binding in the mutagenesis study were subcloned for expression of soluble affibody molecules in a (HE)3-ZTfR-cys format. In addition, two double mutants were generated and included in the study, corresponding to ZTfR#14_A9H_L27H and ZTfR#18_I11N_M14H, as well as a control affibody with an affinity for the HER2 receptor [46] (Table S7, Figure S11). After purification, the C-terminal cysteine in the proteins was conjugated with biotin and FITC followed by verification of secondary structure content, molecular mass, thermal stability, and binding to TfR-positive cells (Figure S12, Tables S7 and S8).
The secondary structure content of the six mutants was similar to ZTfR#14 and ZTfR#18, and thermal stability was either improved or similar (Table 1). However, ZTfR#14_L27H and ZTfR#18_I11N showed non-complete refolding after heat-induced denaturation. All six mutants demonstrated binding to both SK-OV-3 and brain endothelial bEnd.3 cells and the highest signals were observed for ZTfR#14_L27H and ZTfR#18_M14H (Figure S12). The pH-dependency of cell binding was similar for the mutants compared to ZTfR#14 and ZTfR#18 (Figure S13).

2.8. Flow Cytometric Endocytosis Assay

To study the potential endocytosis of the affibody constructs, a previously described flow cytometry-based assay was used [47]. In the assay, the pH sensitivity of FITC is exploited. One emission maximum (FL1 525 nm) of FITC is pH-dependent, whereas the other maximum (FL2 575 nm) is not. Dextran-FITC was added to bEnd.3 cells and incubated at different pH, followed by flow-cytometric analysis of the fluorescence in FL1 and FL2. The pH was plotted against the FL1/FL2 ratio for a standard curve (Figure S14). The FITC-labeled affibody molecules were thereafter incubated with bEnd.3 cells, followed by washing with acidic buffer (pH 5.0) and neutral (pH 7.0) to release remaining membrane-bound affibodies. After neutralization with buffer (pH 7.4), FL1 and FL2 fluorescence were analyzed using flow cytometry.
The TfR-specific BBB shuttle antibody 8D3 has previously been analyzed using the assay, revealing a pH of around 6, which corresponds to recycling TfR endosomes [18,19,47]. The obtained FL1/FL2 ratios for the affibodies corresponded to a pH of around 5–6 for ZTfR#18-derived variants and to a pH of around 6–7 for the ZTfR#14-derived variants (Table 2).

2.9. Transcytosis across a Murine In Vitro BBB Model

To study potential transcytosis across the BBB, a previously described in vitro assay was employed [47]. The assay is based on transwells with recombinant spider silk nanomembranes supporting confluent monolayers of brain endothelial cells. The FITC-labeled affibody molecules were added to the apical side of bEnd.3 cell membranes together with a fluorescently labeled antibody (IgG-AF647) that is used as an internal control for the barrier integrity of the membranes. The HER2-specific affibody (ZHER2) was included as a control. After 90 min, the media on both sides were collected, and the AF647 and FITC fluorescence were measured using a fluorescence spectrophotometer. The fluorescence was finally used to calculate the apparent permeability (papp) [47].
All six TfR-specific affibody molecules except for ZTfR#14_A9H showed higher apparent permeability than the internal control antibody (Table S9, Figure 7). Three of the variants also showed higher permeability compared to ZHER2, indicating transcytosis over TfR-positive bEnd.3 cells.

3. Discussion

The limited efficacy of many CNS-targeted biological drugs could potentially be improved by increased transportation across the BBB. Utilizing small alternative scaffolds (e.g., affibodies) as brain shuttles would potentially have an advantage over antibodies due to the faster biodistribution in the brain parenchyma after transcytosis. The smaller mass of the complex might additionally decrease potential systemic toxic effects from long serum half-life in the periphery [31,48]. RMT via the transferrin receptor-1 (TfR), which shuttles transferrin (Tf) and iron over the BBB to the brain [7,18,19], is one of the more well-investigated strategies. However, targeting the TfR receptor is still problematic as the receptor is abundantly present in other organs and cells in the body, resulting in a sink effect and potential toxicity issues, which have been observed in previous studies [40].
Herein, we selected TfR-specific affibody molecules using phage display technology. Nineteen candidates were selected from the output, and the two most promising binders were further characterized in vitro. Affinity, binding kinetics, epitope, valency, and pH-dependent binding are examples of properties that have been suggested in the literature to be important for RMT via TfR [23,29,49,50]. Moreover, cross-reactivity to TfR orthologues from model animals would facilitate future preclinical evaluation and clinical translation. When analyzing the two top candidates in flow cytometry on TfR-positive SKOV-3 cells, it was noted that the cell-binding signal decreased when labeling and analysis were performed at room temperature (data not shown), perhaps indicating internalization of the binders prior to the addition of secondary detection reagents.
The two candidates, ZTfR#14 and ZTfR#18, were cross reactive for murine and human TfR and bound to an epitope that was non-overlapping with the site for Tf. Avoiding the binding site of Tf is probably important as the high concentration of Tf in blood would otherwise greatly limit available receptors for RMT. Furthermore, blocking the interaction between Tf and TfR has previously been connected to toxicity in mice [29]. Using multi-color flow cytometry, we could show the simultaneous binding of Tf and respective affibody to cells at molar excess of either Tf or affibody. Thus, these affibodies will likely not be toxic due to decreased uptake of Tf.
It has been hypothesized and demonstrated for some TfR-specific antibodies in in vitro models that pH-dependent binding is important for transcytosis [49,51]. The idea is, in principle, to mimic the natural pH dependency in the range between pH 5.0 to 7.4, which is part of the iron transport mechanism via Tf and TfR. However, it should be noted that several TfR-specific brain shuttles without pH-dependent binding have been reported, and it is speculated that the importance of pH dependency is linked to TfR epitope and affinity, where both fast on rate and off rate are considered important [15,28,50,52]. The pH-dependent binding can, however, be of importance since certain epitopes, in combination with bivalent binders, are believed to cross-link the receptor during endocytosis, directing it to lysosomal degradation. Still, release by pH or by fast off rates after endocytosis probably has the potential to improve transcytosis [49,51]. The two candidates, ZTfR#14 and ZTfR#18, displayed different pH-dependent behavior, where ZTfR#14 was not affected by pH in the given range, and ZTfR#18 had a slower off rate from the receptor with lower pH.
Single amino acid mutagenesis was performed on the two candidates, where histidine or the Zwt [45] amino acid was incorporated in the 14 previously randomized positions. The results from the mutagenesis study indicated the contribution of individual residues to the interaction with TfR. Moreover, a few mutants showed increased binding to TfR, and two single amino acid mutants for each affibody were analyzed further. The two mutations were also combined in double mutants to evaluate potential additive effects.
The new variants (ZTfR#14_A9H, ZTfR#14_L27H, ZTfR#14_A9H_L27H, ZTfR#18_I11N, ZTfR#18_M14H, and ZTfR#18I_11N_M14H) were site-specifically conjugated to FITC and showed binding to both SK-OV-3 and brain endothelial bEnd.3 cells in flow cytometry. Internalization was analyzed using a fluorescence assay, exploiting the pH-dependency of FITC and indicating that the binders were located in a cell compartment with a pH of around 5–6 for the ZTfR#18-derived variants and around 6–7 for the ZTfR#18-derived variants. The results suggest that the ZTfR#18-derived variants are more efficiently internalized, although a pH of around five might indicate that it is partly directed to lysosomes, which is undesired.
Finally, the six affibodies were studied with a method where nanofibrillar membranes of recombinant silk seeded with brain endothelial cells are used as a simplified BBB model to assess active transcytosis. The results showed higher apparent permeability for three of the TfR-binders compared to negative control. As epitope, affinity, and internalization behavior only give indications about the capability of transcytosis over BBB, this assay is important as a predictor for the in vivo functionality.
The results are promising, and further optimization of the binders is warranted before studies on brain uptake in animal models.

4. Materials and Methods

4.1. Protein Labeling

Biotinylation of recombinant human and mouse transferrin receptor-1 (TfR: Sino Biological Inc., Bejing, China) was performed using a Biotin-XX Microscale Protein Labeling Kit (Invitrogen, Waltham, MA, USA) according to supplier’s recommendations. The concentration of the proteins was determined using absorbance at 280 nm.

4.2. Phage Display Selections

A combinatorial phage library of the Z domain with randomization in 14 positions was essentially prepared as described previously [53] (Figure 1). Selection and amplification were performed in phosphate-buffered saline with tween (PBST: 0.1% Tween-20) with bovine serum albumin (BSA: Saveen and Werner, Limhamn, Sweden) (PBSTB: 3 w/v% BSA) at room temperature, as described previously [35]. Prior to the first cycle of bio-panning, a negative selection of the library was conducted by incubating the library with streptavidin-coated magnetic beads. The remaining phage particles were panned against recombinant TfR for 1 h in five cycles with an increasing number of washes over the cycles. Different strategies for panning of binders were implemented based on recombinant human TfR, recombinant murine TfR, or cross-selection strategies. In the tracks where the target was kept constant, the concentration of the receptor was decreased from 100 nM in the first panning cycle to 12.5 nM in cycle five. In the cross-selection strategies, human and mouse receptor targets were altered in the five panning cycles, with target concentration lowered from 100 nM in the first two cycles to 40 nM in the last cycle (Figure S1). Selections were followed by DNA sequencing (Microsynth AG, Balgach, Switzerland) of 174 randomly picked colonies.

4.3. Production of Recombinant Affibody Molecules in E. coli

The affibody molecules selected for further characterization were produced with either a C-terminal hexahistidine (His6) tag or an N-terminal hexahistidine (His6) in combination with a C-terminal albumin binding domain (ABD) [41] for purification and characterization strategies. For His6-tagged proteins, the genetic sequences were subcloned into the pET26b(+) vector, introducing a C-terminal His6-tag and yielding the protein constructs [ZTfR]-His6. For ABD-fused proteins, the affibody genes were cloned into a pT7 vector introducing a C-terminal ABD035 molecule [41], yielding the final proteins His6-[ZTfR]-(G4S)3-ABD035. Transformation to Escherichia coli BL21STAR (DE3) cells (Thermo Scientific, Waltham, MA, USA) was performed by heat shock with the expression vectors, and colonies were sequence-verified by the Sanger sequencing (Microsynth AG, Balgach, Switzerland). For protein expression, cells were cultivated in tryptic soy broth with yeast extract (TSBY) media (Merck KGaA, Darmstadt, Germany) with 50 µg × mL−1 kanamycin at 37 °C with 150 rpm shaking. At OD600 of approximately 0.7 AU, protein expression was induced by the addition of Isopropyl β-D-1-thiogalactopyranoside (IPTG: Chemtronica, Stockholm, Sweden) to a final concentration of 1 mM. The cultures were incubated for approximately 18 h at 25 °C prior to harvest, and cells were lyzed by sonication with a Vibra-Cell VCX 130 sonicator (Sonics, Newtown, CT, USA). The affibody molecules were purified by immobilized metal affinity chromatography (IMAC) using a HisPurTM Cobalt resin (Thermo Fisher Scientific, Waltham, MA, USA) with running buffer (47 mM Na2HPO4, 3 mM NaH2PO4, 300 mM NaCl, 15 mM imidazole, pH 7.4), and elution buffer supplemented with 150 mM imidazole. All eluted proteins were buffer exchanged on PD-10 columns (Cytiva, Marlborough, MA, USA) to phosphate-buffered saline (PBS). The protein concentrations were determined by a Pierce™ BCA Protein Assay Kit as per the manufacturer’s instructions (Thermo Scientific, Waltham, MA, USA). The molecular weight and purity of the proteins were confirmed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (NuPAGE Bis-Tris 4–12%, Invitrogen, Waltham, MA, USA) and MALDI mass spectrometry (MS) analysis using SCIEX 4200 MALDI-TOF instrument (SCIEX, Framingham, MA, USA).

4.4. Cultivation of TfR-Positive Cell Lines

Three cell lines were used to test for binding to TfR. The murine TfR-positive BBB cell line bEnd.3 (ATCC, United States) was cultivated in DMEM high glucose media (Thermo Fisher, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS: Sigma-Aldrich/Merck KGaA, Darmstadt, Germany) at 37 °C with 5% CO2 until 80% confluency. The human TfR-positive SK-OV-3 (ATCC, Manassas, VA, USA) cell line was cultivated in McCoy media (Thermo Fisher, Waltham, MA, USA) supplemented with 10% FBS (Sigma-Aldrich/Merck KGaA, Darmstadt, Germany) under the same conditions. The human TfR-positive hCMEC/D3 (#SCC066, Merck KGaA, Darmstadt, Germany) endothelial brain cell line was cultivated in EndoGROTM-MV Complete Media Kit (#SCME004, Merck KGaA, Darmstadt, Germany) supplemented with 1 ng × mL−1 fibroblast growth factor-2 (FGF-2) (#GF003, Merck KGaA, Darmstadt, Germany) in T75 flasks pre-coated with Collagen Type I, Rat Tail (#08-115, Merck KGaA, Darmstadt, Germany) in an atmosphere of 5% CO2 at 37 °C as by manufacturer’s instructions. To detach cells, TrypLE Express GibcoTM (Thermo Fisher, Waltham, MA, USA) was added to cells in an incubator for 3–5 min. Recovered and washed cells were resuspended in ice-cold PBS supplemented with 1 w/v% BSA (PBSB) (Saveen and Werner, Limhamn, Sweden).

4.5. Flow Cytometry Analysis of ZTfR-Binding to Murine and Human Cells

A dilution series of each affibody in His6-[ZTfR]-ABD format was incubated with 100,000 human TfR-positive SK-OV-3 cells for 45 min, 15 rpm, 4 °C before washing twice with PBSB (1 w/v%). Fluorescently in-house labeled HSA (Sigma-Aldrich, St. Louis, MO, USA) with AF-647 (Thermo Scientific, Waltham, MA, USA) was used as a secondary reagent to detect binding and incubated with cells for 15 min on ice before washing away non-bound reagent. TfR expression on the cell lines was verified with fluorescently labeled human transferrin (Tf-488, Thermo Scientific, Waltham, MA, USA) and with ab18242 (Abcam, Cambridge, UK), as by manufacturer’s instructions. A GalliosTM flow cytometer (Beckman Coulter, Brea, CA, USA) was used to analyze 20,000 events of each sample, and data were further analyzed in Kaluza (Version 2.1, Beckman Coulter, Brea, CA, USA). All experiments were performed in duplicates, and samples were normalized to blank cells for comparison between batches.
To analyze pH-dependent binding, SK-OV-3 cells were treated and analyzed as above, except for the following differences. His6-[ZTfR]-ABD and HSA-647 were preincubated for 20 min and then added to SK-OV-3 cells. After incubation, cells were washed in PBSB (1 w/v%) with different pHs in the range 5.0–7.4 for 30 min at 4 °C. All samples were analyzed in triplicate. Experimental set-up with inspiration from Neiveyans et al. [44].
To verify TfR-specific binding, a blocking experiment was performed by co-incubating the ZTfR-ABD construct with 5–10× molar excess of ZTfR-His6. The SK-OV-3 cells were treated and analyzed as above.
To analyze competitive binding with human transferrin, 25 μg × mL−1 (313 nM) fluorescently labeled transferrin (AF488) (#T13342, Invitrogen, Waltham, MA, USA) was pre-incubated with 100–600 nM of His6-[ZTfR]-ABD constructs prior to SK-OV-3 cell labeling and flow-cytometric analysis at 488/525 nm and 640/660 nm in triplicate. Additionally, 200 nM of respective His6-[ZTfR]-ABD construct was pre-incubated with 15–85 μg × mL−1 (186–1063 nM) of Tf-488 prior to SK-OV-3 cell labeling and flow-cytometric analysis at 488/525 nm and 640/660 nm in triplicate. Values were calculated as mean ± s.d.

4.6. Circular Dichroism Analysis for Secondary Structure

Circular dichroism (CD) spectroscopy was used to verify the secondary structure content of the affibody molecules in (HE)3-[ZTfR]-bio or His6-[ZTfR]-ABD format. CD spectra between 195–260 nm at 20 °C were recorded with a Chirascan system (Applied Photophysics, Leatherhead, UK) using a 1 mm High precision cell (110-1P-40 cuvettes, Hellma Analytics, Munich, Germany). Five scans were recorded for each protein sample at a concentration of 0.2 mg × mL−1 in PBS.
The thermal melting point for the affibody molecules was determined using a variable temperature measurement (VTM) at 221 nm with a temperature gradient of 5 °C or 1 °C per minute (depending on samples as indicated in results) with five readings at each temperature point. After cooling down to 20 °C, refolding was accessed by recording spectra and compared to the spectra before denaturation.

4.7. Biosensor Analysis of the ZTfR and TfR Interaction of Both Murine and Human TfR

The affinity and kinetics of the interaction between the His6-[ZTfR]-ABD constructs and TfR of human and murine origin were analyzed using surface plasmon resonance (SPR) on a Biacore T200 system (Cytiva, Marlborough, MA, USA) at 25 °C with PBST (0.05% Tween-20) as running buffer. Approximately 1100 response units (RU) of hTfR-His6 and mTfR-His6 (Sino Biological Inc., Bejing, China) were immobilized on Series S CM5 chips (Cytiva, Marlborough, MA, USA) via amine coupling as per the manufacturer’s recommendations. The ZTfR constructs were injected for 300 s at 30 μL × min−1 in a dilution series between 582–19.4 nM, in duplicate. Dissociation was monitored for 1000 s before regeneration with 10 mM HCl for 30 s, followed by a stabilization period of 60 s. Sensorgrams were double-referenced with the blank surface and a buffer injection of PBST and analyzed with Biacore evaluation software using 1:1 binding model.

4.8. Single Amino Acid Mutagenesis and E. coli Surface Display Binding Analysis

The ZTfR#14 and ZTfR#18 affibodies were mutated at 14 positions by introducing codons for histidine and the wildtype amino acid of the Z domain [45] at each position. Oligos encoding the mutated affibody genes (HT genparts, GenScript, Piscataway, NJ, USA) were subcloned to the E. coli display vector pBad2.2 [34] using Gibson assembly according to the supplier’s recommendations. Four fragments were assembled simultaneously in the Gibson assembly method (New England Biolabs, Ipswich, MA, USA) with a two times molar excess of the insert to pBad2.2 vector and transformed to BL21STAR (DE3) E. coli (Thermo Scientific, Waltham, MA, USA) by standard heat shock protocol. Colonies were sequence-verified by the Sanger sequencing (Eurofins Genomics, Ebersberg, Germany) and further transformed into electrocompetent E. coli JK321 [54].

4.9. Analysis of TfR-Binding Using E. coli Surface Display and Flow Cytometry

Transformed E. coli were grown in LB media at 37 °C until OD600 reached 0.5 AU before inducing with arabinose (0.6%) and incubation at 25 °C for 16 h protein expression. Cells expressing affibody molecules on the surface were incubated in 50 nM, 100 nM, and 200 nM of biotinylated hTfR (#H82E5, Acro Biosystems, Newark, DE, USA) for 45 min. After washing with PBS with 0.01% Pluronic F108 NF surfactant (PBSP), cells were incubated for 15 min on ice, with streptavidin phycoerythrin conjugate (SA-PE: Thermo Scientific, Waltham, MA, USA) and HSA-AF647 (in-house labeled). After washing, cells were analyzed on a Gallios™ Flow Cytometer system (Beckman Coulter, Brea, CA, USA), and data were analyzed in Kaluza (Version 2.1, Beckman Coulter, Brea, CA, USA) for forward/side scatter and relevant fluorophore signals (488/575 nm and 640/660 nm). Values were calculated as mean ± s.d based on duplicate readings.

4.10. Production and Labeling of Second-Generation Affibody Molecules

Selected clones from the mutagenesis study and a HER2-specific affibody ZHER2:0342 [46] as control were subcloned into the pET21a+-vector (Thermo Scientific, Waltham, MA, USA) modified to contain an N-terminal (HE)3-tag and C-terminal cystine using infusion cloning according to supplier’s recommendations (Takara, Kusatsu, Japan). The protein constructs were expressed in E. coli BL21STAR (DE3) as described above, with the change of carbenicillin (100 μg × mL−1). After sonication, the samples were treated at 70 °C for 10 min to precipitate host proteins. After incubation on ice for 20 min, samples were centrifuged for 20 min at 25,000× g. Samples were treated with 5 mM tris(2-carboxyethyl)phosphine (TCEP, Sigma-Aldrich/Merck KGaA, Darmstadt, Germany) for 30 min and thereafter purified using an ÄKTA start system (Cytiva, Marlborough, MA, USA) with a HisTrap crude column (Cytiva, Marlborough, MA, USA) using running buffer as described above (without imidazole) and elution buffer with 300 mM imidazole in a gradient elution (0–80%). Eluate fractions containing protein were buffer exchanged to PBS (pH 7.0) and concentrated to 2 mg × mL−1 before reducing with TCEP again for 30 min. The reduced proteins were thereafter conjugated to biotin (EZ-Link™ Maleimide-PEG2-Biotin, Thermo Scientific, Waltham, MA, USA) and Fluorescein-5-Maleimide (FITC) (Invitrogen, Waltham, MA, USA) as by manufacturer’s instructions. The secondary structure was verified by CD spectroscopy. Labeling efficacy and concentration after labeling was estimated by SCIEX 4200 MALDI-TOF instrument (SCIEX, Framingham, MA, USA), concertation by Pierce™ BCA Protein Assay Kit (Thermo Scientific, Waltham, MA, USA), and degree of labeling was estimated by using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). Purity was analyzed by SDS-PAGE (NuPAGE™ 4 to 12%, Bis-Tris, Invitrogen, Waltham, MA, USA).

4.11. Analysis of Endocytosis Using Flow Cytometry

Endocytosis was studied using a previously described flow cytometry method [47]. Briefly, bEnd.3 cells were resuspended in 0.1 mg × mL−1 Dextran-FITC (40 kDa Mw, Merck KGaA, Darmstadt, Germany) in ice-cold buffers ranging between pH 4.0–5.0 (25 mM Sodium Acetate, 25 mM NaCl, 125 mM KCl, 10 μM nigericin), pH 5.5–6.5 (25 mM MES, 25 mM NaCl, 125 mM KCl, 10 μM nigericin), or pH 7.0 (25 mM HEPES, 25 mM NaCl, 125 mM KCl, 10 μM nigericin), where nigericin was added just before use. Labeled cells were analyzed using flow cytometry for FL1 (488/525 nm) and FL2 (488/575 nm) to obtain a standard curve for pH. Each sample was analyzed in triplicate and normalized to blank cells without dextran. Values were calculated as mean ± s.d.
Next, cells were resuspended in ice-cold buffer pH 7.4 (25 mM HEPES, 25 mM NaCl, 125 mM KCl) buffer without nigericin and with 250 nM of respective FITC-labeled affibody molecule and incubated for 15 min, 15 rpm, at room temperature before spun down and washed once in a buffer of pH 5.0 and once at pH 7.0 (no nigericin) for 5 min at 15 rpm. The sample was resuspended in a pH 7.4 buffer and analyzed in the flow cytometer with the same settings as for the standard curve.

4.12. Transcytosis In Vitro Murine BBB Model

A previously described method based on recombinant spider silk nanomembranes with confluent monolayers of murine brain endothelial cells (bEnd.3) was used for the assessment of TfR-mediated transcytosis [47]. 500 nM of respective FITC-labeled affibody molecule together with an AF647-labeled internal-control IgG2a antibody was added in complete pre-warmed cell media to the silk membrane apical side with (n = 3) or without confluent bEnd.3 cell layer (n = 3) and placed into a well. Cell media was added to the basal side and incubated for 90 min (37 °C, 5% CO2). The apical and basal samples were aspired, and together with the starting sample, the fluorescent intensity was measured at 483-14/530-30 nm and 575-20/621-10 nm with 1200/3000 gain at 25 °C in a CLARIOStar Plus (BMG Labtech, Ortenberg, Germany). Values were averaged by technical and biological replicates, and signals from complete cell media were subtracted. Statistical analysis was performed in Microsoft Excel using a two-sided Student’s t-test with equal variance for the apparent permeability (papp, cm × s−1) calculated as by Equation ()1, where A is the surface area (cm2), dQ/dt the steady-state flux (mmol × s−1), VR is the volume of the receiver chamber (cm3) and C0 the initial concentration of the affibody (mM).
p a p p = d Q d t × V R A × C 0

5. Conclusions

To conclude, we describe the development of affibodies targeting TfR by phage display technology. Two top candidates were evaluated in vitro and further improved by single amino acid substitutions, yielding a total of six new variants. The six affibodies were studied in terms of cell binding and transcytosis, demonstrating higher apparent permeability for three of the TfR-binders compared to a negative control. Although the affibody constructs evaluated herein show promising results, further optimization of the binders is likely warranted prior to studies on brain uptake in animal models.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ph16070956/s1, Figure S1: Phage selection procedure; Figure S2: Sequence alignment of clones, Table S1: Production of His6-ZTfR-ABD, Figure S3: Sodium Dodecyl Sulphate–Poly Acrylamide Gel (SDS-PAGE) of constructs His6-ZTfR-ABD, Table S2: Production of ZTfR-His6, Figure S4: SDS-PAGE of constructs ZTfR-His6, Figure S5: Circular dichroism (CD) of His6-ZTfR-ABD constructs, Table S3: Cellular binding of His6-ZTfR-ABD, Figure S6: Concentration dependent human cell binding, Table S4: Self-block of His6-ZTfR-ABD construct at cell binding, Figure S7: Histograms of competitive transferrin binding of His6-ZTfR-ABD constructs, Table S5: Transferrin competitive binding to His6-ZTfR-ABD, Figure S8: Secondary structure pH dependency, Figure S9: Surface plasmon resonance (SPR) sensorgram for TfR binding, Table S6: Kinetic data of SPR sensorgram, Figure S10: Single amino acid mutagenesis binding analysis, Table S7: Producibility of (HE)3-ZTfR-cys constructs, Figure S11: SDS-PAGE of production from (HE)3-ZTfR-cys constructs, Table S8: Biotinylation efficacy of (HE)3-ZTfR-cys constructs and thermal melting point, Figure S12: Cellular binding verification of (HE)3-ZTfR-FITC constructs, Figure S13: Cellular pH dependent binding verification of (HE)3-ZTfR-FITC constructs, Figure S14: Standard curve for pH dependent FITC signal, Table S9: Apparent permeability of (HE)3-ZTfR-FITC constructs.

Author Contributions

Conceptualization, L.C.H., H.L., S.S. and J.L.; methodology, L.C.H., H.L., S.S. and J.L.; software, L.C.H.; validation, L.C.H. and H.L.; formal analysis, L.C.H. and H.L.; investigation, L.C.H. and H.L; resources, S.S. and J.L.; data curation, L.C.H., H.L. and J.L.; writing—original draft preparation, L.C.H.; writing—review and editing, L.C.H., H.L., S.S. and J.L.; visualization, L.C.H.; supervision, H.L., S.S. and J.L.; project administration, L.C.H., H.L., S.S. and J.L.; funding acquisition, S.S. and J.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Swedish Brain Foundation (grant FO2018-0094, FO2021-0407, FO2022-0253, FO2023-0400, FO2023-0141), the Knut and Alice Wallenberg Foundation (grants KAW 2019.0341 and KAW 2021.0197), the Swedish Agency for Innovation VINNOVA (2019/00104 and CellNova Center; 2017/02105), StratNeuro, the Tussilago foundation (FL-0002.025.551-7), and the Schörling Family foundation via the Swedish FTD Initiative.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article and supplementary material.

Acknowledgments

The authors thank Eva Baum for the production of the ZTfR-ABD constructs and Siri Flemming Svedmark for help with CD measurements. Spiber AB for providing silk for the transcytosis assay and GUTVIBRATIONS for the 3D inserts. JK321 cells were a gift from Arne Skerra with permission from Joachim Jose.

Conflicts of Interest

The authors declare no conflict of interest, and the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

References

  1. Chan, A.C.; Carter, P.J. Therapeutic antibodies for autoimmunity and inflammation. Nat. Rev. Immunol. 2010, 10, 301–316. [Google Scholar] [CrossRef]
  2. Zahavi, D.; Weiner, L. Monoclonal Antibodies in Cancer Therapy. Antibodies 2020, 9, 34. [Google Scholar] [CrossRef]
  3. McConnell, H.L.; Kersch, C.N.; Woltjer, R.L.; Neuwelt, E.A. The translational significance of the neurovascular unit. J. Biol. Chem. 2017, 292, 762–770. [Google Scholar] [CrossRef] [Green Version]
  4. Zlokovic, B.V. Neurovascular mechanisms of Alzheimer’s neurodegeneration. Trends Neurosci. 2005, 28, 202–208. [Google Scholar] [CrossRef]
  5. Barar, J.; Rafi, M.A.; Pourseif, M.M.; Omidi, Y. Blood-brain barrier transport machineries and targeted therapy of brain diseases. BioImpacts 2016, 6, 225–248. [Google Scholar] [CrossRef] [Green Version]
  6. Saunders, N.R.; Habgood, M.D.; Møllgård, K.; Dziegielewska, K.M. The biological significance of brain barrier mechanisms: Help or hindrance in drug delivery to the central nervous system? F1000Research 2016, 5, 313. [Google Scholar] [CrossRef] [Green Version]
  7. Pardridge, W.M. Blood-Brain Barrier and Delivery of Protein and Gene Therapeutics to Brain. Front. Aging Neurosci. 2020, 11, 373. [Google Scholar] [CrossRef]
  8. Hawkins, R.A.; Mokashi, A.; Simpson, I.A. An active transport system in the blood-brain barrier may reduce levodopa availability. Exp. Neurol. 2005, 195, 267–271. [Google Scholar] [CrossRef]
  9. Walter, F.R.; Santa-Maria, A.R.; Mészáros, M.; Veszelka, S.; Dér, A.; Deli, M.A. Surface charge, glycocalyx, and blood-brain barrier function. Tissue Barriers 2021, 9, 1904773. [Google Scholar] [CrossRef]
  10. Lajoie, J.M.; Katt, M.E.; Waters, E.A.; Herrin, B.R.; Shusta, E.V. Identification of lamprey variable lymphocyte receptors that target the brain vasculature. Sci. Rep. 2022, 12, 6044. [Google Scholar] [CrossRef]
  11. Atwal, J.K.; Chen, Y.; Chiu, C.; Mortensen, D.L.; Meilandt, W.J.; Liu, Y.; Heise, C.E.; Hoyte, K.; Luk, W.; Lu, Y.; et al. A Therapeutic Antibody Targeting BACE1 Inhibits Amyloid-β Production In Vivo. Sci. Transl. Med. 2011, 3, 4ra43. [Google Scholar] [CrossRef]
  12. Yadav, D.B.; Maloney, J.A.; Wildsmith, K.R.; Fuji, R.N.; Meilandt, W.J.; Solanoy, H.; Lu, Y.; Peng, K.; Wilson, B.; Chan, P.; et al. Widespread brain distribution and activity following i.c.v. infusion of anti-β-secretase (BACE1) in nonhuman primates. Br. J. Pharmacol. 2017, 174, 4173–4185. [Google Scholar] [CrossRef] [Green Version]
  13. Alata, W.; Yogi, A.; Brunette, E.; Delaney, C.E.; Faassen, H.; Hussack, G.; Iqbal, U.; Kemmerich, K.; Haqqani, A.S.; Moreno, M.J.; et al. Targeting insulin-like growth factor-1 receptor (IGF1R) for brain delivery of biologics. FASEB J. 2022, 36, e22208. [Google Scholar] [CrossRef]
  14. Boado, R.J.; Zhang, Y.; Zhang, Y.; Xia, C.F.; Wang, Y.; Pardridge, W.M. Genetic engineering of a lysosomal enzyme fusion protein for targeted delivery across the human blood-brain barrier. Biotechnol. Bioeng. 2008, 99, 475–484. [Google Scholar] [CrossRef]
  15. Stocki, P.; Szary, J.; Rasmussen, C.L.M.; Demydchuk, M.; Northall, L.; Logan, D.B.; Gauhar, A.; Thei, L.; Moos, T.; Walsh, F.S.; et al. Blood-brain barrier transport using a high affinity, brain-selective VNAR antibody targeting transferrin receptor 1. FASEB J. 2021, 35, e21172. [Google Scholar] [CrossRef]
  16. Kariolis, M.S.; Wells, R.C.; Getz, J.A.; Kwan, W.; Mahon, C.S.; Tong, R.; Kim, J.; Srivastava, A.; Bedard, C.; Henne, K.R.; et al. Brain delivery of therapeutic proteins using an Fc fragment blood-brain barrier transport vehicle in mice and monkeys. Sci. Transl. Med. 2020, 12, eaay1359. [Google Scholar] [CrossRef]
  17. Wouters, Y.; Jaspers, T.; Rué, L.; Serneels, L.; De Strooper, B.; Dewilde, M. VHHs as tools for therapeutic protein delivery to the central nervous system. Fluids Barriers CNS 2022, 19, 79. [Google Scholar] [CrossRef]
  18. Mayle, K.M.; Le, A.M.; Kamei, D.T. The intracellular trafficking pathway of transferrin. Biochim. Biophys. Acta-Gen. Subj. 2012, 1820, 264–281. [Google Scholar] [CrossRef] [Green Version]
  19. Lamb, J.E.; Ray, F.; Ward, J.H.; Kushner, J.P.; Kaplan, J. Internalization and subcellular localization of transferrin and transferrin receptors in HeLa cells. J. Biol. Chem. 1983, 258, 8751–8758. [Google Scholar] [CrossRef]
  20. Kissel, K.; Hamm, S.; Schulz, M.; Vecchi, A.; Garlanda, C.; Engelhardt, B. Immunohistochemical localization of the murine transferrin receptor (TfR) on blood–tissue barriers using a novel anti-TfR monoclonal antibody. Histochem. Cell Biol. 1998, 110, 63–72. [Google Scholar] [CrossRef]
  21. Hultqvist, G.; Syvänen, S.; Fang, X.T.; Lannfelt, L.; Sehlin, D. Bivalent brain shuttle increases antibody uptake by monovalent binding to the transferrin receptor. Theranostics 2017, 7, 308–318. [Google Scholar] [CrossRef] [PubMed]
  22. Yu, Y.J.; Zhang, Y.; Kenrick, M.; Hoyte, K.; Luk, W.; Lu, Y.; Atwal, J.; Elliott, J.M.; Prabhu, S.; Watts, R.J.; et al. Boosting Brain Uptake of a Therapeutic Antibody by Reducing Its Affinity for a Transcytosis Target. Sci. Transl. Med. 2011, 3, 84ra44. [Google Scholar] [CrossRef] [PubMed]
  23. Niewoehner, J.; Bohrmann, B.; Collin, L.; Urich, E.; Sade, H.; Maier, P.; Rueger, P.; Stracke, J.O.; Lau, W.; Tissot, A.C.; et al. Increased Brain Penetration and Potency of a Therapeutic Antibody Using a Monovalent Molecular Shuttle. Neuron 2014, 81, 49–60. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Weber, F.; Bohrmann, B.; Niewoehner, J.; Fischer, J.A.A.; Rueger, P.; Tiefenthaler, G.; Moelleken, J.; Bujotzek, A.; Brady, K.; Singer, T.; et al. Brain Shuttle Antibody for Alzheimer’s Disease with Attenuated Peripheral Effector Function due to an Inverted Binding Mode. Cell Rep. 2018, 22, 149–162. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. Lessard, E.; Rennie, K.; Haqqani, A.; Ling, B.; Whitfield, J.; Paradis, A.; Araujo, J.; Yoganathan, N.; Gillard, J.; Stanimirovic, D.; et al. Pharmacokinetics and Pharmacodynamic Effect of a Blood-Brain Barrier-Crossing Fusion Protein Therapeutic for Alzheimer’s Disease in Rat and Dog. Pharm. Res. 2022, 39, 1497–1507. [Google Scholar] [CrossRef]
  26. Stocki, P.; Szary, J.M.; Jacobsen, C.L.M.; Demydchuk, M.; Northall, L.; Moos, T.; Walsh, F.S.; Lynn Rutkowski, J. High efficiency blood-brain barrier transport using a VNAR targeting the Transferrin Receptor 1 (TfR1). bioRxiv 2019. [Google Scholar] [CrossRef] [Green Version]
  27. Crook, Z.R.; Girard, E.; Sevilla, G.P.; Merrill, M.; Friend, D.; Rupert, P.B.; Pakiam, F.; Nguyen, E.; Yin, C.; Ruff, R.O.; et al. A TfR-Binding Cystine-Dense Peptide Promotes Blood–Brain Barrier Penetration of Bioactive Molecules. J. Mol. Biol. 2020, 432, 3989–4009. [Google Scholar] [CrossRef]
  28. Bien-Ly, N.; Yu, Y.J.; Bumbaca, D.; Elstrott, J.; Boswell, C.A.; Zhang, Y.; Luk, W.; Lu, Y.; Dennis, M.S.; Weimer, R.M.; et al. Transferrin receptor (TfR) trafficking determines brain uptake of TfR antibody affinity variants. J. Exp. Med. 2014, 211, 233–244. [Google Scholar] [CrossRef] [Green Version]
  29. Couch, J.A.; Yu, Y.J.; Zhang, Y.; Tarrant, J.M.; Fuji, R.N.; Meilandt, W.J.; Solanoy, H.; Tong, R.K.; Hoyte, K.; Luk, W.; et al. Addressing safety liabilities of TfR bispecific antibodies that cross the blood-brain barrier. Sci. Transl. Med. 2013, 5, 183ra57. [Google Scholar] [CrossRef]
  30. Villaseñor, R.; Schilling, M.; Sundaresan, J.; Lutz, Y.; Collin, L. Sorting Tubules Regulate Blood-Brain Barrier Transcytosis. Cell Rep. 2017, 21, 3256–3270. [Google Scholar] [CrossRef] [Green Version]
  31. Faresjö, R.; Bonvicini, G.; Fang, X.T.; Aguilar, X.; Sehlin, D.; Syvänen, S. Brain pharmacokinetics of two BBB penetrating bispecific antibodies of different size. Fluids Barriers CNS 2021, 18, 26. [Google Scholar] [CrossRef] [PubMed]
  32. Ståhl, S.; Gräslund, T.; Eriksson Karlström, A.; Frejd, F.Y.; Nygren, P.Å.; Löfblom, J. Affibody Molecules in Biotechnological and Medical Applications. Trends Biotechnol. 2017, 35, 691–712. [Google Scholar] [CrossRef] [PubMed]
  33. Engfeldt, T.; Renberg, B.; Brumer, H.; Nygren, P.Å.; Karlström, A.E. Chemical synthesis of triple-labelled three-helix bundle binding proteins for specific fluorescent detection of unlabelled protein. ChemBioChem 2005, 6, 1043–1050. [Google Scholar] [CrossRef]
  34. Andersson, K.G.; Persson, J.; Ståhl, S.; Löfblom, J. Autotransporter-Mediated Display of a Naïve Affibody Library on the Outer Membrane of Escherichia coli. Biotechnol. J. 2019, 14, e1800359. [Google Scholar] [CrossRef]
  35. Grönwall, C.; Jonsson, A.; Lindström, S.; Gunneriusson, E.; Ståhl, S.; Herne, N. Selection and characterization of Affibody ligands binding to Alzheimer amyloid β peptides. J. Biotechnol. 2007, 128, 162–183. [Google Scholar] [CrossRef]
  36. Klint, S.; Feldwisch, J.; Gudmundsdotter, L.; Dillner Bergstedt, K.; Gunneriusson, E.; Höidén Guthenberg, I.; Wennborg, A.; Nyborg, A.C.; Kamboj, A.P.; Peloso, P.M.; et al. Izokibep: Preclinical development and first-in-human study of a novel IL-17A neutralizing Affibody molecule in patients with plaque psoriasis. mAbs 2023, 15, 2209920. [Google Scholar] [CrossRef]
  37. Affibody, A.B. Izokibep, a Bispecific Molecule Targeting Interleukin-17A (IL-17). 2022. Available online: https://www.affibody.se/ (accessed on 25 January 2023).
  38. Hjelm, L.C.; Lindberg, H.; Ståhl, S.; Löfblom, J. Construction and Validation of a New Naïve Sequestrin Library for Directed Evolution of Binders against Aggregation-Prone Peptides. Int. J. Mol. Sci. 2023, 24, 836. [Google Scholar] [CrossRef] [PubMed]
  39. Boutajangout, A.; Lindberg, H.; Awwad, A.; Paul, A.; Baitalmal, R.; Almokyad, I.; Höidén-Guthenberg, I.; Gunneriusson, E.; Frejd, F.Y.; Härd, T.; et al. Affibody-Mediated Sequestration of Amyloid β Demonstrates Preventive Efficacy in a Transgenic Alzheimer’s Disease Mouse Model. Front. Aging Neurosci. 2019, 11, 64. [Google Scholar] [CrossRef]
  40. Meister, S.W.; Hjelm, L.C.; Dannemeyer, M.; Tegel, H.; Lindberg, H.; Ståhl, S.; Löfblom, J. An affibody molecule is actively transported into the cerebrospinal fluid via binding to the transferrin receptor. Int. J. Mol. Sci. 2020, 21, 2999. [Google Scholar] [CrossRef] [Green Version]
  41. Jonsson, A.; Dogan, J.; Herne, N.; Abrahmsén, L.; Nygren, P.Å. Engineering of a femtomolar affinity binding protein to human serum albumin. Protein Eng. Des. Sel. 2008, 21, 515–527. [Google Scholar] [CrossRef]
  42. Tolmachev, V.; Orlova, A.; Pehrson, R.; Galli, J.; Baastrup, B.; Andersson, K.; Sandström, M.; Rosik, D.; Carlsson, J.; Lundqvist, H.; et al. Radionuclide therapy of HER2-positive microxenografts using a 177Lu-labeled HER2-specific affibody molecule. Cancer Res. 2007, 67, 2773–2782. [Google Scholar] [CrossRef] [Green Version]
  43. Eklund, M.; Axelsson, L.; Uhlén, M.; Nygren, P.Å. Anti-idiotypic protein domains selected from protein A-based affibody libraries. Proteins Struct. Funct. Genet. 2002, 48, 454–462. [Google Scholar] [CrossRef] [PubMed]
  44. Neiveyans, M.; Melhem, R.; Arnoult, C.; Bourquard, T.; Jarlier, M.; Busson, M.; Laroche, A.; Cerutti, M.; Pugnière, M.; Ternant, D.; et al. A recycling anti transferrin receptor 1 monoclonal antibody as an efficient therapy for erythroleukemia through target up regulation and antibody. mAbs 2019, 11, 359–605. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  45. Nilsson, B.; Moks, T.; Jansson, B.; Abrahmsén, L.; Elmblad, A.; Holmgren, E.; Henrichson, C.; Jones, T.A.; Uhlén, M. A synthetic IgG-binding domain based on staphylococcal protein A. Protein Eng. Des. Sel. 1987, 1, 107–113. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  46. Orlova, A.; Magnusson, M.; Eriksson, T.L.J.; Nilsson, M.; Larsson, B.; Höiden-Guthenberg, I.; Widström, C.; Carlsson, J.; Tolmachev, V.; Ståhl, S.; et al. Tumor imaging using a picomolar affinity HER2 binding Affibody molecule. Cancer Res. 2006, 66, 4339–4348. [Google Scholar] [CrossRef] [Green Version]
  47. Hjelm, L.C.; Hedhammar, M.; Löfblom, J. In vitro Blood–Brain barrier model based on recombinant spider silk protein nanomembranes for evaluation of transcytosis capability of biomolecules. Biochem. Biophys. Res. Commun. 2023, 669, 77–84. [Google Scholar] [CrossRef]
  48. Morrison, J.I.; Metzendorf, N.G.; Rofo, F.; Petrovic, A.; Hultqvist, G. A single-chain fragment constant design enables easy production of a monovalent blood-brain barrier transporter and provides an improved brain uptake at elevated doses. J. Neurochem. 2023, 165, 413–425. [Google Scholar] [CrossRef]
  49. Sade, H.; Baumgartner, C.; Hugenmatter, A.; Moessner, E.; Freskgård, P.O.; Niewoehner, J. A human blood-brain barrier transcytosis assay reveals antibody transcytosis influenced by pH-dependent receptor binding. PLoS ONE 2014, 9, e96340. [Google Scholar] [CrossRef] [Green Version]
  50. Yu, Y.J.; Atwal, J.K.; Zhang, Y.; Tong, R.K.; Wildsmith, K.R.; Tan, C.; Bien-Ly, N.; Hersom, M.; Maloney, J.A.; Meilandt, W.J.; et al. Therapeutic bispecific antibodies cross the blood-brain barrier in nonhuman primates. Sci. Transl. Med. 2014, 6, 261ra154. [Google Scholar] [CrossRef]
  51. Toth, A.E.; Nielsen, S.S.E.; Tomaka, W.; Abbott, N.J.; Nielsen, M.S. The endo-lysosomal system of bEnd.3 and hCMEC/D3 brain endothelial cells. Fluids Barriers CNS 2019, 16, 14. [Google Scholar] [CrossRef] [Green Version]
  52. Stocki, P.; Szary, J.; Demydchuk, M.; Northall, L.; Rasmussen, C.L.M.; Logan, D.B.; Gauhar, A.; Thei, L.; Coker, S.F.; Moos, T.; et al. CDR3 Variants of the TXB2 Shuttle with Increased TfR1 Association Rate and Enhanced Brain Penetration. Pharmaceutics 2023, 15, 739. [Google Scholar] [CrossRef] [PubMed]
  53. Yu, F.; Gudmundsdotter, L.; Akal, A.; Gunneriusson, E.; Frejd, F.; Nygren, P.Å. An affibody-adalimumab hybrid blocks combined IL-6 and tnf-triggered serum amyloid a secretion in vivo. mAbs 2014, 6, 1598–1607. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  54. Maurer, J.; Jose, J.; Meyer, T.F.; Molekulare Biologie, A. Autodisplay: One-Component System for Efficient Surface Display and Release of Soluble Recombinant Proteins from Escherichia coli. J. Bacteriol. 1997, 179, 794–804. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Pharmaceuticals 16 00956 g001 550
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 ZTfR#14 and ZTfR#18.
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 ZTfR#14 and ZTfR#18.
Pharmaceuticals 16 00956 g001
Pharmaceuticals 16 00956 g002 550
Figure 2. Analysis by flow cytometry of ZTfR-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-ZSYM73-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 2. Analysis by flow cytometry of ZTfR-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-ZSYM73-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.
Pharmaceuticals 16 00956 g002
Pharmaceuticals 16 00956 g003 550
Figure 3. Binding to human brain endothelial cells. Flow cytometry showing binding to hCMEC/D3 cells for 300 nM of ZTfR#14-ABD (purple) and ZTfR#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 3. Binding to human brain endothelial cells. Flow cytometry showing binding to hCMEC/D3 cells for 300 nM of ZTfR#14-ABD (purple) and ZTfR#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.
Pharmaceuticals 16 00956 g003
Pharmaceuticals 16 00956 g004 550
Figure 4. Flow cytometry of SK-OV-3 cells. (A,B) Fluorescence from cells corresponding to affibody-binding. ZTfR#14-ABD or ZTfR#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 ZTfR-ABD is detected by HSA-647 at 640/660 nm laser and filter. (C,D) Fluorescence from cells corresponding to Tf-488 binding. ZTfR#14-ABD or ZTfR#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 4. Flow cytometry of SK-OV-3 cells. (A,B) Fluorescence from cells corresponding to affibody-binding. ZTfR#14-ABD or ZTfR#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 ZTfR-ABD is detected by HSA-647 at 640/660 nm laser and filter. (C,D) Fluorescence from cells corresponding to Tf-488 binding. ZTfR#14-ABD or ZTfR#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.
Pharmaceuticals 16 00956 g004
Pharmaceuticals 16 00956 g005 550
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, ZTfR#14, and ZTfR#18. (C,D) Measurement of secondary structure by circular dichroism spectroscopy for ZTfR#14 and ZTfR#18 at pH 5.5, 6.5, and 7.4.
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, ZTfR#14, and ZTfR#18. (C,D) Measurement of secondary structure by circular dichroism spectroscopy for ZTfR#14 and ZTfR#18 at pH 5.5, 6.5, and 7.4.
Pharmaceuticals 16 00956 g005
Pharmaceuticals 16 00956 g006 550
Figure 6. Mean fluorescence intensity (MFI) from flow cytometry on E. coli cells displaying single amino acid mutants of ZTfR#14 or ZTfR#18 in fusion to an albumin-binding domain. A negative control Zwt [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 (ZTfR#14 or ZTfR#18). (A) Mutants of ZTfR#14 incubated with 100 nM hTfR, and (B) Mutants of ZTfR#18 incubated with 75 nM hTfR. Values are given as mean ± s.d. based on n = 2 samples.
Figure 6. Mean fluorescence intensity (MFI) from flow cytometry on E. coli cells displaying single amino acid mutants of ZTfR#14 or ZTfR#18 in fusion to an albumin-binding domain. A negative control Zwt [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 (ZTfR#14 or ZTfR#18). (A) Mutants of ZTfR#14 incubated with 100 nM hTfR, and (B) Mutants of ZTfR#18 incubated with 75 nM hTfR. Values are given as mean ± s.d. based on n = 2 samples.
Pharmaceuticals 16 00956 g006
Pharmaceuticals 16 00956 g007 550
Figure 7. Apparent permeability (papp) 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 ZHER2 control targets the HER2 receptor. Values are given as mean ± s.d. based on n = 3 samples.
Figure 7. Apparent permeability (papp) 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 ZHER2 control targets the HER2 receptor. Values are given as mean ± s.d. based on n = 3 samples.
Pharmaceuticals 16 00956 g007
Table
Table 1. Thermal melting point (Tm) and refolding capability for biotinylated (HE)3-ZTfR-cys constructs.
Table 1. Thermal melting point (Tm) and refolding capability for biotinylated (HE)3-ZTfR-cys constructs.
CloneTm [°C]Refolding Capability
(HE)3-ZTfR#14_A9H-biotin64Yes
(HE)3-ZTfR#14_L27H-biotin66Partly
(HE)3-ZTfR#14_A9H_L27H-biotin62Partly
(HE)3-ZTfR#18_I11N-biotin52Partly
(HE)3-ZTfR#18_M14H--biotin62Yes
(HE)3-ZTfR#18_I11N_M14H-biotin63Yes
(HE)3-ZHER2-biotin62Yes
Table
Table 2. Estimated pH environment for ZTfR-FITC constructs incubated with bEnd.3 cells. Values are given as mean ± s.d. based on n = 3 samples.
Table 2. Estimated pH environment for ZTfR-FITC constructs incubated with bEnd.3 cells. Values are given as mean ± s.d. based on n = 3 samples.
SampleCalculated pH Environment by Standard CurveEndocytosis Classification
(HE)3-ZHER2-FITC5.75 ± 0.02Late/lysosomal
(HE)3-ZTfR#14_A9H-FITC7.15 ± 0.31Partial cell surface or vesicle
(HE)3-ZTfR#14_L27H-FITC6.94 ± 0.02Early
(HE)3-ZTfR#14_A9H_L27H-FITC6.18 ± 0.04Recycling
(HE)3-ZTfR#18_I11N-FITC5.13 ± 0.02Lysosome
(HE)3-ZTfR#18_M14H-FITC6.56 ± 0.02Early
(HE)3-ZTfR#18_I11N_M14H-FITC5.16 ± 0.01Lysosome
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Hjelm, L.C.; Lindberg, H.; Ståhl, S.; Löfblom, J. Affibody Molecules Intended for Receptor-Mediated Transcytosis via the Transferrin Receptor. Pharmaceuticals 2023, 16, 956. https://doi.org/10.3390/ph16070956

AMA Style

Hjelm LC, Lindberg H, Ståhl S, Löfblom J. Affibody Molecules Intended for Receptor-Mediated Transcytosis via the Transferrin Receptor. Pharmaceuticals. 2023; 16(7):956. https://doi.org/10.3390/ph16070956

Chicago/Turabian Style

Hjelm, Linnea Charlotta, Hanna Lindberg, Stefan Ståhl, and John Löfblom. 2023. "Affibody Molecules Intended for Receptor-Mediated Transcytosis via the Transferrin Receptor" Pharmaceuticals 16, no. 7: 956. https://doi.org/10.3390/ph16070956

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop