Noncompetitive allosteric antagonism of death receptor 5 by a synthetic affibody ligand

Abstract

Fatty acid-induced upregulation of death receptor 5 (DR5) and its cognate ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), promotes hepatocyte lipoapoptosis, which is a key mechanism in the progression of fatty liver disease. Accordingly, inhibition of DR5 signaling represents an attractive strategy for treating fatty liver disease. Ligand competition strategies are prevalent in tumor necrosis factor (TNF) receptor antagonism, but recent studies suggest that non-competitive inhibition through perturbation of receptor conformation may be a compelling alternative. To this end, we used yeast display and a designed combinatorial library to identify a synthetic 58-amino acid affibody ligand that specifically binds the DR5. Biophysical and biochemical studies show that the affibody neither blocks TRAIL binding nor prevents the receptor-receptor interaction. Live-cell fluorescence lifetime measurements indicate that the affibody induces a conformational change in transmembrane dimers of DR5 and favors an inactive state of the receptor. The affibody inhibits apoptosis in TRAIL-treated Huh-7 cells, an in vitro model of fatty liver disease. Thus, this lead affibody serves as a potential drug candidate, with a unique mechanism of action, for fatty liver disease.

Graphical Abstract

INTRODUCTION

Tumor necrosis factor (TNF) ligands and TNF receptors (TNFR) are essential modulators of the immune response and are critically involved in organ development and tissue homeostasis^{1, 2}. Activation of the TNFR members via their cognate ligands effects cell proliferation, survival, and apoptosis. Excessive or impaired cell death is associated with pathophysiology of several acute and chronic diseases, including developmental, autoimmune, neurodegenerative diseases, and cancer^{3, 4}. Neutralization of TNF ligand with monoclonal antibodies has significant clinical benefit in patients with rheumatoid arthritis, inflammatory bowel disease, and psoriasis^4–7. However, the global blockade of TNF ligand causes several undesirable side effects, such as tuberculosis, pneumonia, sepsis, increased risk of congestive heart failure, as well as a potentially increased risk for specific malignancies, such as lymphomas^8–14. Recent breakthroughs regarding the structure and biophysics of TNF receptors have shifted the current therapeutic paradigm of global TNF ligand inhibition to selective targeting of the receptor itself^15–20. In particular, blocking oligomerization of TNF receptors has been considered a potential therapeutic target^21–28. In our previous work, we showed that it is possible to inhibit TNFR signaling by specifically targeting the receptor dimer, without interrupting ligand binding^29–31. However, despite progress, these approaches and others like them have failed to discover even nanomolar potency small molecule inhibitors. These studies have been limited to molecules that competitively target the receptor-receptor or receptor-ligand interfaces. Non-competitive agents offer unique potential for greater efficacy. Also, protein ligands may provide larger surface area for elevated affinity³² and target a wider array of epitopes.

Recently, non-antibody protein scaffolds with picomolar affinities have been discovered from affibody libraries to numerous targets, including tumor necrosis factor α^{33, 34}, human epidermal growth factor receptor 2³⁵ and amyloid-β peptide³⁶. Affibody molecules are small ligands based on the three helical bundle Z domain of the Ig-binding region of protein A^{37, 38}. Combinatorial libraries containing different affibody molecules have been generated by diversifying 17 solvent-exposed amino acids located in helices 1 and 2 of the Z domain^39–41. The small size, robust structure, high affinity binding ability to protein targets, efficient conjugation, and relatively easy production procedures – either by chemical synthesis or by recombinant production in bacteria – makes them attractive targeting agents for therapeutics and diagnostics^41–43.

In the current study, we used an affibody library composed of 2×10⁹ variants along with directed evolution to discover a high affinity functional modulator of death receptor 5 (DR5; also known as TRAIL-R2), a member of TNF receptor superfamily. Upon binding to its cognate ligand, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), DR5 recruits the adaptor molecule Fas-associated death domain (FADD) and caspase-8 to form the death inducing signaling complex, which activates caspase-8, subsequently leading to apoptosis^{44, 45}. Recent studies show that liver expression of DR5 is increased in both human and experimental nonalcoholic steatohepatitis (NASH)⁴⁶. Hepatocyte apoptosis by free fatty acids is considered to be a key histological feature of NASH and plays a critical role in pathogenesis of nonalcoholic fatty liver disease (NAFLD)^{47, 48}. In vitro studies of NAFLD showed that hepatocyte treatment with free fatty acid palmitate leads to DR5 clustering in the plasma membrane, which triggers ligand-independent receptor activation and hepatocyte cytotoxicity⁴⁹. In hepatocellular carcinoma cells (Huh-7), knockdown of DR5 expression attenuates fatty acid mediated apoptosis⁴⁹. Likewise, in a dietary mouse model of NASH, DR5 deletion suppresses hepatocyte apoptosis and fibrosis⁵⁰. Furthermore, inhibitors of apoptosis have been developed as drugs for the treatment of NASH^{51, 52}. Therefore, inhibition of TRAIL-induced and/or fatty acid-induced activation of DR5 signaling is an attractive strategy for NASH therapy.

Utilizing yeast surface display^{53, 54} and directed evolution, we identified an affibody variant that specifically binds to human DR5 with high affinity. Cell-based functional assays showed that the affibody inhibits TRAIL-induced apoptosis in Huh-7 cells, an in vitro model for NAFLD and NASH. Using a combination of biophysical and biochemical studies, we showed that the affibody does not block TRAIL binding or disrupt DR5–DR5 interactions. These results suggest that the affibody allosterically inhibits DR5 signaling. In summary, we have discovered a non-immunoglobulin and non-competitive DR5 antagonist that can serve as a potential drug candidate for NASH.

EXPERIMENTAL SECTION

Identification and evolution of DR5 specific ABY binders using yeast surface display

The naïve affibody library containing 2×10⁹ variants was previously generated by designed diversification of 17 solvent-exposed amino acids located in helices 1 and 2 of the Z domain of the Ig-binding region of protein A followed by introduction into a yeast display system⁴⁰. The affibody yeast library was grown in SD-CAA selection media (16.8 g/L sodium citrate dihydrate, 3.9 g/L citric acid, 20.0 g/L dextrose, 6.7 g/L yeast nitrogen base, 5.0 g/L casamino acids) at 30 °C with shaking. After ~20 hours of incubation, yeast cells were centrifuged and resuspended in SG-CAA induction media (10.2 g/L sodium phosphate dibasic heptahydrate, 8.6 g/L sodium phosphate monobasic monohydrate, 19.0 g/L galactose, 1.0 g/L dextrose, 6.7 g/L yeast nitrogen base, 5.0 g/L casamino acids) and grown overnight to induce affibody display on the yeast surface.

To discover affibody molecules that specifically bind to DR5, we performed magnetic activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS). DR5 is expressed as two functional isoforms, long and short, that differ in their transmembrane domains. In our previous studies we show that the oligomeric structure of two functional isoforms of DR5 is indistinguishable²⁰. To target conformationally accurate DR5, sorts used cell lysates from HEK293 cells with a stable expression of long isoform of death receptor 5 (DR5) in which the cytoplasmic domain is replaced by GFP (DRL5ΔCD-GFP). Magnetic sorts on the naïve library were performed at 4 °C and with two washes during positive selections. For magnetic sorting, target and control proteins were conjugated to GFP-trap magnetic beads (40 μm magnetic beads coated in anti-GFP antibody, [ChromoTek, gtma-20]). To coat the beads with proteins, HEK293 cells with stable expression of DR5ΔCD-GFP or transient expression of non-target protein (lymphocyte activation gene 3 (LAG3)-GFP, [OriGene, RG220269]) were detached, washed thrice with phosphate-buffered saline (PBS), and then lysed (2 mM ethylenediaminetetraacetic acid 1% Triton X-100, 1x protease inhibitor in PBS) on ice for 30 minutes. After incubation, cell lysates were centrifuged at 13000 rpm for 15 minutes at 4 °C to remove insoluble debris. Soluble supernatants were mixed with GFP-trap magnetic beads and incubated for 1–2 hours at 4 °C. After the conjugation, beads were washed three times with PBS. Yeast displaying the ABY library was exposed to bare beads and then beads with immobilized LAG3-GFP to remove any non-specific binding interactions. The remaining yeast were incubated with DR5ΔCD-GFP coated beads, and bound yeast were selected. These DR5-bound yeast populations were grown, induced, and sorted more stringently with another round of MACS with depletion on control beads (bare beads and LAG3-GFP coated beads) and enrichment on DR5ΔCD-GFP coated beads with three washes. For FACS selection, DR5-bound yeast populations were induced, labeled with anti-c-Myc antibody (9E10, BioLegend), followed by AlexaFluor647-conjugated anti-mouse antibody. Next, these labeled yeast populations were incubated with DR5ΔCD-GFP lysate. All GFP⁺/AlexaFluor647⁺ yeast cells were collected via BD FACS Aria II. Sorted yeast was grown and plasmid DNA was extracted using Zymoprep Yeast Plasmid Miniprep Kit II (Zymo Research Corp.).

Generation of randomly mutagenized ABY library

Sorted DR5 binders were further engineered to increase binding affinity using random mutagenesis to the full gene and the helices of ABY in parallel by error-prone PCR using nucleotide analogues 2’-deoxy-P-nucleoside-5’-triphosphate and 8-oxo-2’-deoxyguanosine-5’- triphosphate as described⁴⁰. The mutagenized gene fragments were transformed into yeast with homologous recombination with linearized pCT vector. The resultant mutant ABY population underwent two rounds of MACS and a FACS against mammalian cell lysates expressing DR5ΔCD-GFP (or LAG3-GFP for comparative control). Finally, FACS sorted ABY populations were labeled with mouse c-Myc antibody, followed by AlexaFluor647-conjugated anti-mouse antibody and incubated with DR5ΔCD-GFP lysate (or LAG3 GFP lysate for comparative control) for two hours at room temperature. Binding of ABY to DR5ΔCD-GFP was detected by flow cytometry. Yeast clones that showed double positive fluorescence signals (AlexaFluor647⁺/GFP⁺) were collected. These cells were grown and zymoprepped to isolate plasmid DNA. Clonal plasmid was obtained by transforming extracted DNA into One Shot TOP10 Escherichia coli (Invitrogen). Individual colonies were grown in LB medium and plasmids were extracted from bacterial culture using the QIAGEN Miniprep Kit. Purified DNA was sequenced by ACGT, Inc.

Cell cultures and reagents

HEK293 cells were cultured in phenol red-free Dulbecco’s modified Eagle’s medium (Gibco, 31053–028), and Jurkat cells were cultured in RPMI 1640 medium (ATCC, 30–2001). All media were supplemented with 2 mM L-glutamine (Gibco, 25030149), heat-inactivated 10% fetal bovine serum (Sigma, F0926), 100 U/mL penicillin, and 100 μg/mL streptomycin (Gibco, 15140122). Human hepatocellular carcinoma cells (Huh-7) were cultured in Dulbecco’s modified Eagle’s medium containing glucose (4.5 g/L), penicillin (100 units/mL), streptomycin (100 mg/mL) and 10% fetal bovine serum (all Gibco, Carlsbad, CA). The N-terminal FLAG-tagged soluble TRAIL (residues 114–281) was overexpressed using the pT7-FLAG-1 inducible expression system in Escherichia coli and purified by anti-FLAG-affinity column (Sigma-Aldrich, A2220).

Molecular biology

EGFP and TagRFP vectors were a kind gift from David D. Thomas. cDNA encoding DR5ΔCD (1–240) was inserted at the N-terminus of the EGFP and TagRFP vectors using standard cloning techniques. To prevent the dimerization and aggregation of EGFP, we mutated alanine 206 to lysine (A206K)⁵⁵. All mutations were introduced by Quikchange mutagenesis and sequenced for confirmation.

Overexpression and purification of DR5 binders

Evolved DR5 binding ABY variant sequences were transferred from pCT vector into a pET expression vector with a C-terminal His₆ tag using NheI and BamHI restriction enzymes. Recombinant soluble DR5 binders were overexpressed using pET expression system in Escherichia coli and purified by immobilized cobalt affinity chromatography. Purity of proteins was analyzed by 4–20% SDS-PAGE gels (Bio-Rad) under reducing conditions followed by Coomassie staining.

Binding affinity measurements of ABY variants to DR5ΔCD-GFP on HEK293 cells

HEK293 cells with stable expression of DR5ΔCD-GFP, TNFR1ΔCD-GFP or transient expression of LAG3-GFP or DR4ΔCD-GFP were detached using trypsin, washed with PBS, and then incubated with soluble ABY variants at increasing concentrations for 2–4 hours at room temperature until equilibrated. After incubation, cells were washed with PBS with 0.1% bovine serum albumin to remove unbound affibody and labeled with AlexaFluor647-conjugated anti-His₅ antibody (Qiagen, 35370) for 1–2 hours at 4 °C. Fluorescence was analyzed on a BD Accuri C6 flow cytometer and quantified using FlowJo software.

ABY_DR5–6 pull-down assays

Binding specificity of ABY_DR5–6 was determined by a pull-down assay. Anti-His₆ magnetic beads were mixed with purified His₆-tagged ABY_DR5–6 (200 nM), incubated at 4 °C for 2 hours by end over end rotation, and washed three times with PBS to remove any unbound ABY_DR5–6. Next, recombinant DR4-Fc or DR5-Fc (100 nM) was added to ABY_DR5–6-coated magnetic beads and rotated at 4 °C for 2–4 hours. Beads were washed thrice with PBS, resuspended in 2x SDS sample buffer, and heated at 95 °C for 10 minutes to elute pulled-down proteins. Samples were resolved using 4–20% SDS-PAGE gels and subjected to Western blotting using anti-DR4 and DR5 antibodies.

Functional assays

The effects of binders on biological functions of DR5 were assessed using cell viability assays: MTT, caspase-8 glow, and Fas-associated protein with death domain (FADD) recruitment.

MTT

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to determine viability of cells treated with DR5 binder. MTT assay is a colorimetric assay, which measures the ability of viable cells to reduce a soluble tetrazolium salt to an insoluble purple formazan precipitate. Jurkat cells were seeded in 96-well plates at 5,000 cells/well and incubated for 24 hours at 37 °C, 5% CO₂. After 24 hours of incubation, cells were treated with soluble affibodies for 2 – 4 hours and then treated with TRAIL (0.1 μg/mL) for 16 hours at 37 °C. Cell viability was assessed with a Cytation 3 Cell Imaging Multi-Mode Reader luminometer (BioTek).

Caspase-8 assays

Jurkat cells were seeded in 96-well plates at 5,000 cells/well and incubated for 24 h at 37 °C. Cells were treated with soluble affibody, incubated for 1 – 2 h, and then treated with TRAIL (0.1 μg/mL), followed by 16 hours of incubation at 37 °C. An equal volume of Caspase-Glo 8 reagent (Promega) was added to each well, and the luminescence was measured after 30 min using a Cytation 3 Cell Imaging Multi-Mode Reader luminometer (BioTek).

Analysis of TRAIL-induced recruitment of FADD to DR5

Jurkat cells were treated with affibody (200 nM), incubated for 2 – 4 hours at 37 °C, and then stimulated with FLAG-tagged TRAIL (0.1 μg/mL) for 4 – 6 hours. Post-stimulation cells were washed with cold PBS and lysed in RIPA buffer supplemented with protease inhibitors (ProteoGuard™ Protease Inhibitor Cocktail, Takara). Supernatants were transferred to a tube containing anti-FLAG antibody-coated magnetic beads and rotated overnight at 4 °C, followed by three washes with wash buffer (50 mM Tris–HCl [pH 7.5], 150 mM NaCl, 0.5% sodium deoxycholate, 1% NP-40). Immunoprecipitation samples were resolved using SDS-PAGE gels and subjected to Western blotting using anti-DR5 (Biolegend, 307302) and FADD (BD Biosciences, 610399) antibodies.

Cell death assay in Huh-7 cells

Huh-7 cells grown in 96-well plates were treated with recombinant human TRAIL (20 ng/mL, R&D Systems, 375-TL-010) for 16 h in the presence or absence of increasing concentrations of ABY_DR5–6 (78 nM −10 μM). Palmitate was dissolved in isopropyl alcohol and conjugated to bovine serum albumin resuspended in DMEM media (1% bovine serum albumin) as previously described⁴⁹. Following the incubation period, cells were stained with cell permeable Hoechst 33342 (Invitrogen, H1399) and live cell-impermeant SYTOX Green (Invitrogen, S7020) to label all and dead cells, respectively. Stained cells were visualized, analyzed, and quantified using a plate-based image cytometer Celigo (Nexcelom Bioscience).

Pull-down assay

The effect of affibody on the TRAIL-DR5 interaction was determined by a pull-down assay with anti-GFP magnetic beads. DR5ΔCD-GFP lysate (from HEK293 cells with a stable expression of DR5ΔCD-GFP) was mixed with anti-GFP beads and incubated at 4 °C for 2 hours. The beads were then washed thrice to remove the unbound proteins. Soluble TRAIL (1 μg/mL) or a mixture of TRAIL and affibody (200 μM) were added to DR5-GFP coated magnetic beads and rotated at 4 °C for 2 – 4 hours. Then beads were washed thrice. Pulled down proteins were resolved by SDS-PAGE and then immunoblotted with anti-GFP (Abcam, ab290), -TRAIL (Abcam, ab9959) and –His₅ antibodies (Qiagen, 35370).

Colocalization of Affibody and DR5

To test the colocalization of soluble affibody with DR5, HEK293 cells with a stable expression of DR5ΔCD-GFP were grown on 35-mm glass bottom MatTek culture dishes (MatTek Corporation) and treated with soluble affibody and incubated for 2 – 4 h at 37 °C. Cells were gently washed twice with PBS to remove the unbound soluble proteins and labeled with mouse His₆-antibody (Biolegend, 652502), followed by AlexaFluor555-conjugated anti-mouse secondary antibody (Invitrogen, A32727). Colocalization was evaluated using fluorescence microscopy.

Fluorescence lifetime measurements

HEK293 cells with stable expression of DR5ΔCD-GFP and DR5ΔCD-GFP-RFP were grown to 80–90% confluence in a 10 cm plate. For lifetime measurements, stable cells were detached with trypsin, washed thrice with PBS, treated with soluble DR5 binder (0 −10 μM), and incubated for 1–2 hours. After incubation, cells were dispensed (50 μL/well) into a 96 well glass-bottom plate. Donor lifetime in the presence and absence of acceptor was measured by using a fluorescence lifetime plate reader (Fluorescence Innovations, Inc., Minneapolis, MN). GFP fluorescence was excited with a 473-nm microchip laser, and emission was filtered with 488-nm long-pass and 517/20-nm band-pass filters. Time-resolved fluorescence waveforms for each well were fitted to single-exponential decays using least-squares minimization global analysis software (Fluorescence Innovations, Inc.) to give donor lifetime (τ_D) and donor–acceptor lifetime (τ_DA). FRET efficiency (E) was then calculated based on Eq. (1)

$$\text{E}=1-\left(\frac{{\tau }_{\text{DA}}}{{\tau }_{\text{D}}}\right)$$ (1)

RESULTS

Discovery and evolution of DR5-specific ABY binders using yeast surface display

Yeast surface display, with magnetic and flow cytometric selection methods, was used to isolate affibody molecules with specific binding to the extracellular domain of DR5. The previously described⁴⁰ naïve yeast surface display affibody library comprises 2×10⁹ unique affibody variants with designed diversity at 17 sites on the surface of two helices (Figure 1A). To enrich specific binders with a high likelihood to bind DR5 in its cellular conformation⁵⁶ the DR5 molecular target was isolated from the lysate of mammalian cells expressing the long isoform of DR5 with a deleted cytoplasmic domain replaced by green fluorescent protein (DR5ΔCD-GFP). The GFP enables purified immobilization on magnetic beads via anti-GFP antibody and fluorescent detection via FACS. An analogous construct with lymphocyte-activation gene 3 (LAG3)-GFP served as a negative control (Figure 1B). LAG3 is an immune checkpoint receptor protein found on the cell surface of effector T cells that has a very low sequence homology (15%) with DR5. The ABY library underwent three magnetic sorts to enrich DR5-specific binders and one FACS sort for the presence of the C-terminal peptide epitope to isolate full-length affibodies. Sorted DR5-binding ABY sequences were mutated through error-prone PCR targeting the helical paratope and the genes of enriched ABY variants⁵⁷. The mutagenized ABY population was further enriched with two magnetic sorts with DR5ΔCD-GFP coated beads and one FACS with DR5ΔCD-GFP expressing cell lysates. After these six sorts for binders and one round of mutagenesis, the enriched population exhibited substantial binding to DR5ΔCD-GFP (Figure 1C) whereas significant binding was not observed to LAG3-GFP cell lysate (Figure 1D), which suggests that the population evolved specifically with DR5-ABY interactions rather than non-specific interactions in the selection process. From this evolved population, we sequenced six randomly selected clones, which revealed high sequence diversity in the functional population with amino acid variations at the initially diversified helical sites (Figure 2A). The six DR5 binders were produced in E. coli using a pET expression system with a C-terminal His₆-tag and purified by metal affinity chromatography. Electrophoresis revealed good yields (~1 mg/L), high purity, and proper molecular weight of all soluble proteins (Figure 2B).

Figure 1.

(A) Schematic of the affibody scaffold displayed on the surface of yeast. (B) Flow chart for the discovery and evolution of DR5 binders from the naïve ABY library. Yeast displaying the affibody population evolved for binding the extracellular domain of DR5 were labeled with mouse c-Myc antibody, followed by AF647-conjugated anti-mouse antibody as well as cellular lysate with DR5ΔCD-GFP (C) or LAG3-GFP (D) for two hours at room temperature. Affibody display and target binding were detected by flow cytometry. DR5-specific GFP signal indicates DR5-specific binding. Absence of GFP signal for AF647- cells indicates absence of non-specific DR5 binding to yeast as well as absence of truncated ABY binders.

Figure 2.

(A) Amino acid sequences of six clones from the evolved population against DR5 were aligned with the wild-type affibody to demonstrate amino acid sequence variances of the six clones. Horizontal bars: amino acids that are identical with the wild-type (B) Coomassie blue staining of soluble ABY protein. ABY variants were purified using affinity chromatography and analyzed by 4–20% SDS-PAGE gels. Open circle represents soluble affibody. (C) Binding of ABY_DR5 variants to DR5ΔCD-GFP expressing stable cells. HEK293 cells with stable expression of DR5ΔCD-GFP were incubated with 1 μM soluble ABY_DR5 variants. Binding was detected with AF647 conjugated anti-His₅ antibody by flow cytometry. Black: HEK293 stable cells; blue, cyan, green, magenta, purple and red: HEK293 stable cells treated with ABY_DR5 1–6, respectively. (D) Affinity titration of ABY_DR5–6. HEK293 cells with a stable expression of DR5ΔCD-GFP or TNFR1ΔCD-GFP or transient expression of LAG3-GFP were incubated with increasing concentrations of soluble ABY_DR5–6 Binding was detected by AF647-conjugated anti-His₅ antibody via flow cytometry. Data are presented as mean ± standard deviation of three independent experiments. The line represents the minimization of the sum of squared errors for a 1:1 binding model, which indicates K_d = 94 ± 5 nM.

Binding affinity measurements of ABY variants to DR5ΔCD-GFP on HEK293 cells

To test the binding of soluble ABY variants to DR5, HEK293 cells with stable expression of DR5ΔCD-GFP were incubated, separately, with each of the six soluble ABY variants (ABY_DR5). Among these six variants, five clones showed weak binding and one clone (ABY_DR5–6) showed significant binding to DR5ΔCD-GFP cells (Figure 2C). Subsequently, we measured the binding affinity of ABY_DR5–6 to DR5 in HEK293 cells with stable expression of DR5ΔCD-GFP. Titration exhibited an affinity of K_d = 94 ± 5 nM to DR5ΔCD-GFP, while no significant binding was observed to cells expressing TNFR1ΔCD-GFP or LAG3-GFP (Figure 2D). To further verify ABY_DR5–6 specificity, we tested binding to death receptor 4 (DR4), which is a homologous member of the TNF-receptor superfamily (51% identity and 68% similarity to DR5) that binds to TRAIL and triggers apoptosis^{58, 59}. No significant binding was observed to cells expressing DR4ΔCD-GFP (Figure 3A). Moreover, a pull-down assay was performed in which ABY_DR5–6 was immobilized on anti-His₆ beads and incubated with DR5 and DR4. Western blot analysis of pull-down samples showed that ABY_DR5–6 does not bind to DR4; only DR5 appeared in the pulled-down samples (Figure 3B). These results confirm that the ABY variant specifically binds DR5 rather than non-specifically binding other proteins expressed on the surface of HEK293 cells.

Figure 3.

Lack of binding of ABY variants to DR4. (A) Affinity titration of ABY_DR5–6. HEK293 cells with a stable expression of DR5ΔCD-GFP or transient expression of DR4ΔCD-GFP were incubated with increasing concentrations of soluble ABY_DR5–6 Binding was detected by AF647-conjugated anti-His₅ antibody via flow cytometry. Data are presented as mean ± standard deviation of three independent experiments. (B) ABY_DR5–6-DR4 binding was determined by a pull-down assay with anti-His magnetic beads. Soluble ABY_DR5–6 (200 nM) was mixed with anti-His beads and incubated at 4 °C for 2 hours. The beads were then washed thrice to remove the unbound proteins. Soluble-DR4-Fc (100 nM) or Soluble-DR5-Fc (100 nM) was added to ABY_DR5–6 coated magnetic beads and rotated at 4 °C for 2–4 hours. Beads were washed thrice, and pulled-down proteins were resolved by SDS-PAGE and immunoblotted with anti-DR4 and anti-DR5 antibodies.

Colocalization of membrane bound DR5ΔCD-GFP and ABY_DR5–6

Next, we sought to directly observe the binding of the ABY_DR5–6 molecule to DR5ΔCD-GFP expressed on the cell surface. For this assay, we incubated DR5ΔCD-GFP stably expressing cells with ABY_DR5–6 binder and observed colocalization with fluorescence microscopy (Figure 4). However, some cells were only stained with AlexaFluor-labeled affibody, which might be originating from binding of ABY_DR5–6 to endogenous DR5 (very low levels) in HEK293 cells. These results confirm the direct interaction between affibody and DR5. Next, we sought to study the effect of ABY_DR5–6 binder on biological function of DR5.

Figure 4:

Colocalization of membrane-bound DR5ΔCD-GFP and ABY_DR5–6 on HEK293 cell surface. The yellow color in the overlay of the red and green signals indicates colocalization of ABY_DR5–6 and DR5ΔCD-GFP. Scale bars correspond to 100 μm.

Effect of ABY_DR5–6 on TRAIL-induced apoptosis

It is well documented that TRAIL selectively induces apoptosis in several different cancer cells without harming normal cells, and DR5 is implicated as the primary TRAIL target^{44, 60–63}. To test the biological effect of ABY binding to DR5 on TRAIL-induced apoptosis, functional assays were performed with the human lymphoma Jurkat cell line, which is a well-established model for the study of the apoptotic death pathways^{64, 65}. We first examined the surface expression of DR5 in Jurkat cells using flow cytometry, which showed significant expression of DR5 on the surface of Jurkat cells (Figure 5A). Next, the functional effect of ABY_DR5–6 on TRAIL-induced apoptosis was determined by MTT assay. ABY_DR5–6 inhibited TRAIL-induced apoptosis in a dose-dependent manner (IC₅₀ of 15 ± 1 nM), while nonbinding ABY_NB control caused no effect (Figure 5B).

Figure 5:

ABY_DR5–6 inhibits TRAIL-induced apoptosis. (A) FACS data demonstrates surface expression of DR5 on Jurkat cells. Jurkat cells were incubated with anti-DR5 antibody, followed by AF647-conjugated mouse secondary antibody, and analyzed by flow cytometry. Red: fully labeled cells; green: cells lacking primary DR5 antibody; black: unlabeled cells. (B) ABY_DR5–6 inhibits TRAIL-induced cell death as determined by MTT assay. Jurkat cells were treated with increasing concentrations of soluble ABY_DR5–6 (1 pM - 10 μM) then stimulated with TRAIL (0.1 μg/mL; 3 nM) for 16 hours. The line represents the minimization of the sum of squared errors for a 1:1 inhibition model, which indicates IC₅₀ = 15 ± 1 nM. Data are presented as mean ± standard deviation of three independent experiments. (C) Effect of ABY_DR5–6 on TRAIL-induced FADD recruitment to DR5. Jurkat cells were incubated with 200 nM affibody then stimulated with FLAG-tagged TRAIL. TRAIL and associated molecules were immunoprecipitated on anti-FLAG-conjugated magnetic beads, resolved using SDS-PAGE gels, and subjected to western blotting using anti-DR5 and FADD antibodies. (D) Caspase-8 activity was measured in Jurkat cells treated with increasing concentrations of soluble ABY_DR5–6 (1 pM - 10 μM) and TRAIL (0.1 μg/mL). The line represents the minimization of the sum of squared errors for a 1:1 inhibition model, which indicates IC₅₀ = 160 ± 20 nM. Data are presented as mean ± standard deviation of three independent experiments.

To further understand the effect of ABY_DR5–6 on downstream signaling of the DR5 apoptotic pathway, we examined the TRAIL-induced recruitment of FADD to DR5 and activation of caspase-8 in the presence or absence of ABY_DR5–6. It has been shown previously that TRAIL binding to DR5 triggers the formation of the death-inducing signaling complex by the recruitment of the adaptor FADD to DR5 and initiates caspase-8 activation⁴⁵. Co-immunoprecipitation showed that cells treated with ABY_DR5–6 significantly inhibited TRAIL-induced recruitment of FADD to DR5 compared with untreated cells (Figure 5C). These results demonstrate that the DR5 binder effectively inhibits the TRAIL-induced downstream signaling of DR5. Subsequently, we examined the activation of caspase-8, a further downstream cell death signaling protein, in response to affibody treatment. ABY_DR5–6 inhibited TRAIL-induced caspase-8 activation in a dose-dependent manner, with an IC₅₀ of 160 ± 20 nM (Figure 5D). Taken together, these results confirm that ABY_DR5–6 is a functional inhibitor of TRAIL-induced apoptosis.

Effect of ABY_DR5–6 on TRAIL–DR5 interactions

It has been shown previously that blocking TRAIL binding to DR5 inhibits apoptosis^{20, 66, 67}. We therefore investigated if the inhibitory function of ABY_DR5–6 on TRAIL-induced apoptosis resulted from blocking TRAIL binding. We tested the effect of ABY_DR5–6 on TRAIL-DR5 interactions in live cells using flow cytometry (Figure 6A). No significant difference was observed in TRAIL binding to DR5ΔCD-GFP expressing cells in the presence versus absence of ABY_DR5–6. To further support this observation, a pull-down assay was used to assess the effect of ABY_DR5–6 on TRAIL-DR5 interactions. Western blot analysis of pull-down samples showed that ABY_DR5–6 does not affect TRAIL binding: DR5 and TRAIL interacted similarly in the presence or absence of ABY_DR5–6 (Figure 6B). Moreover, ABY_DR5–6 also appeared in the pulled-down sample, which suggests that TRAIL and ABY_DR5–6 are binding at different sites in DR5. These results confirm that DR5 binder does not block TRAIL binding.

Figure 6:

ABY_DR5–6 binds DR5 without competing TRAIL-DR5 complex formation. (A) HEK293 cells with stable expression of DR5ΔCD–GFP were incubated with TRAIL (50 nM) and TRAIL + soluble ABY_DR5–6 (200 nM). TRAIL binding was detected with rabbit anti-TRAIL antibody, followed by AF647-conjugated anti-rabbit antibody, as measured by flow cytometry. Black: HEK293 stable cells; blue: HEK293 stable cells treated with TRAIL only; and red: HEK293 stable cells treated with TRAIL and ABY_DR5–6. (B) TRAIL-DR5 binding was determined by a pull-down assay with anti-GFP magnetic beads. DR5ΔCD-GFP lysate was mixed with anti-GFP beads and incubated at 4 °C for 2 hours. The beads were then washed thrice to remove the unbound proteins. Soluble-TRAIL (50 nM) and TRAIL+ ABY_DR5–6 (200 nM) was added to DR5-GFP coated magnetic beads and rotated at 4 °C for 2–4 hours. Beads were washed thrice, and pulled-down proteins were resolved by SDS-PAGE and immunoblotted with anti-GFP, TRAIL and His₅ antibodies.

Effect of ABY_DR5–6 on ligand-independent DR5–DR5 interactions

Next, we evaluated the effect of soluble ABY_DR5–6 on ligand-independent DR5-DR5 interactions. It has been shown previously that several members of the TNF receptor superfamily, including DR5, TNFR1 and FAS, exist as ligand-independent oligomers, which are important for TNFR signaling pathways^{21, 22, 66, 68–71}. In our previous work, we established that it is possible to inhibit TRAIL-induced apoptosis by disrupting the DR5-DR5 interaction^29–31. Thus, we hypothesized that the anti-apoptotic effect of ABY_DR5–6 might be due to disruption of pre-ligand assembly of DR5 receptors. We therefore investigated the effect of ABY_DR5–6 on DR5–DR5 interactions using live-cell time-resolved Förster resonance energy transfer (TR-FRET). We previously showed that TR-FRET could directly report on receptor oligomerization (increase in FRET) and disruption of receptor-receptor interactions (decrease in FRET)^{30, 72}. Experiments were carried out in HEK293 cells stably expressing the long isoform of DR5 without a cytoplasmic domain (DR5ΔCD) fused to GFP and co-expressing DR5ΔCD fused to RFP just downstream of the transmembrane domain of the receptors. DR5ΔCD-GFP (donor) lifetime in the presence and absence of acceptor (DR5ΔCD-RFP) was measured and then used to calculate FRET efficiency using Equation 1. Measurements showed a substantial decrease in the fluorescence lifetime of the donor in the presence of the acceptor compared with the donor only, which confirms efficient energy transfer between the FRET pairs. These results confirm that DR5 exists as ligand-independent oligomers. We then evaluated the effect of ABY_DR5–6 on DR5–DR5 interactions. Interestingly, cells treated with ABY_DR5–6 showed higher FRET compared with untreated cells (Figure 7). Increase in FRET suggests that upon ABY_DR5–6 binding DR5 undergoes conformational rearrangements that lead to a decrease in distance between the cytoplasmic ends of pre-assembled DR5 receptors. Cells treated with soluble bovine serum albumin or non-binder (negative controls) caused no significant FRET change compared with untreated cells (Figure 7). These results suggest that ABY_DR5–6 does not block the DR5-DR5 interaction but rather modulates the local conformation. This local conformational change reduces apoptosis by inhibiting the recruitment of FADD to DR5 as shown in Figure 5C.

Figure 7:

Effect of ABY_DR5–6 on ligand-independent DR5-DR5 interactions was determined using live-cell TR-FRET measurements. For lifetime measurements, HEK293 cells with a stable expression of DR5ΔCD-GFP and DR5ΔCD-GFP-RFP were lifted with trypsin, washed thrice with PBS, and resuspended in PBS at a concentration of 1 million cells/mL. Then cells were treated with soluble ABY_DR5–6 (0.1 – 10 μM), BSA and non-binder, and incubated for 1 – 2 hours. After incubation cells were washed with PBS and dispensed (50 μL/well) into a 96 well glass-bottom plate. Donor lifetime was measured using a fluorescence lifetime plate reader. Note: *, **, and *** indicates statistically significant increase relative to no affibody control with p < 0.05, 0.01, and 0.001.

Effect of ABY_DR5–6 on TRAIL-induced apoptosis in Huh-7 hepatocytes

It has been shown that TRAIL-induced apoptosis contributes to hepatocyte apoptosis in fatty liver disease^{46, 47, 49}. Therefore, we examined the effect of ABY_DR5–6 on TRAIL-induced apoptosis in Huh-7 cells, a hepatocyte-derived cell line. Huh-7 cells are well established and validated as an in vitro model for studying hepatocyte lipoapoptosis in NAFLD^{46, 73}. Huh-7 cells were treated with TRAIL in the presence and absence of ABY_DR5–6, and cell death was assessed using MTT assay, caspase-8 assay and plate-based image cytometric analysis of Hoechst 33342 and SYTOX Green staining. ABY_DR5–6 consistently inhibited cell death as assessed by the maintenance of MTT activity, even at sub-nanomolar concentrations, whereas the non-binder control yielded no protection (Figure 8A). ABY_DR5–6 inhibits caspase-8 activation in a dose-dependent manner with 50% inhibition at low nanomolar concentrations whereas the non-binder control has no effect (Figure 8B). The Hoechst/SYTOX assay yields a more nuanced result (Figure 8C). Moderate inhibition is observed at mid-nanomolar concentrations with more complete inhibition at low micromolar levels. Collectively, these results confirm that soluble ABY_DR5–6 protects Huh-7 cells from TRAIL-induced apoptosis.

Figure 8:

Effect of ABY_DR5–6 on TRAIL-induced apoptosis in Huh-7 hepatocytes. Huh-7 cells grown in 96-well plates were treated with recombinant human TRAIL (0.1 μg/mL) for 16 h in the presence or absence of increasing concentrations of ABY_DR5–6 (0.001 nM - 10 μM). TRAIL-induced cell death was determined by MTT assay (A), Caspase-8 assay (B) and a mixture of cell-permeable Hoechst 33342 and impermeable Sytox Green DNA fluorescent dyes (C). Triangle and dotted lines represent TRAIL-induced cell death in the absence of affibody treatment and square represents cell death in untreated cells. Data are means ± SD of three independent experiments. All ABY_DR5–6 samples have lower death (A) and caspase-8 activity (B) than non-binder by two-tailed unpaired t test (P < 0.0001).

DISCUSSION

Emerging evidence suggests that ligand-dependent and/or ligand-independent activation of DR5 signaling contributes to hepatocyte apoptosis, which is a key mechanism for disease progression in patients with fatty liver disease^{46, 47, 49, 74, 75}. Recently, several inhibitors of hepatic cell death have been suggested as potential treatment for NASH^{76, 77}. Thus, blocking the DR5-apoptosis signaling pathway is a highly promising approach for nonalcoholic fatty liver disease therapy. In this way, we believe that with further optimization ABY_DR5–6 and its variants can become a viable therapy. Improvements can include further enhancement in the already strong potency, which can be achieved through additional rounds of directed evolution (affinity maturation). Epitope mapping^{54, 78, 79}, —an experimental strategy through which the binding interface of the affibody and DR5 is identified—can provide increased insight into mechanism and guide ligand engineering. Ultimately, favorable pharmacokinetics must be achieved as well, which is facilitated by the modularity of the affibody scaffold⁴¹. Finally, increased testing for activity in more advanced cellular models and animal studies will be necessary.

In order to most effectively treat fatty liver diseases, therapeutics should ultimately ameliorate the effects of cellular stresses, such as lipotoxicity or unmitigated endoplasmic reticulum stress, which induce apoptotic cell death via ligand-independent DR5 activation^{46, 47, 49}. We attempted to assess the effect of ABY_DR5–6 on ligand-independent induction of apoptosis with a model of hepatocyte lipotoxicity in which fatty acid-induced cell death is largely mediated by DR5⁴⁹. Huh-7 cells were treated with the fatty acid palmitate in the presence or absence of ABY_DR5–6, and cell death was quantified. While we found that ABY_DR5–6 inhibited palmitate-induced apoptosis in Huh-7 cells at higher concentrations, recapitulating the Jurkat results, the data (not shown) was confounding at mid-range concentration. Thus, full elucidation of these findings will be the subject of future studies. Nonetheless, the collective results highlight the power of the technology platform we introduce here, which can be readily modified and improved to accommodate cellular models like this one with increased complexity and translational relevance. For example, in the context of palmitate-induced apoptosis, we note that in our original study of DR5-induced lipoapoptosis we showed that DR5 clusters into lipid rafts upon treatment with palmitate⁴⁹. In another of our previous studies, we likewise showed that removal of membrane cholesterol—a key component of lipid rafts—reduces the dimerization of DR5 (ablating function) in response to TRAIL stimulation¹⁸. Thus, in both cases (palmitate and TRAIL), lipid raft-induced changes in DR5 structure are important to function. This points to a rational future optimization of our affibody engineering strategy, which could include evolving binders specifically against raft-associated DR5 in the presence of palmitate and/or TRAIL. Nevertheless, in the absence of direct selection, we have discovered and evolved an affibody with a unique mode of conformation-based activity.

We have shown here unambiguously that the synthetic affibody ligand approach is capable of protecting liver cells from DR5-induced apoptosis with substantial potency and specificity. Among six ABY_DR5 variants we tested, one affibody molecule (ABY_DR5–6) strongly and selectively binds to the extracellular domain of DR5 and showed biological activity in two different cell lines with three different cell-based assays. In all these cell-based assays, ABY_DR5–6 produced similar efficacy, albeit at different potencies (IC₅₀). The variance in IC₅₀ could be attributed to the fundamental difference between the cell-based assays and the different molecules that elicit outcomes in their systems: the MTT assay monitors metabolic activity of cells; caspase-8 assay monitors extrinsic/receptor-mediated caspase-8 activity; and Hoechst/Sytox Green DNA fluorescent dyes measure the number of live and dead cells. Metabolic activity and cell viability represent two different aspects of cellular function, and both are required for the estimation of the physiological state of a cell after exposure to various types of stress. We note that ABY_DR5–6 elicits effects (cell binding, Figure 4; apoptotic inhibition, Figures 5 and 8; conformational modulation, Figure 7) at concentrations below the binding K_d. As the K_d represents the concentration for half-maximal binding, these results are expectedly consistent with the ability for ABY_DR5–6 to yield biological outcomes with a small fraction of receptors bound. We also note that the apoptotic inhibition in Huh-7 hepatocytes was surprisingly sensitive but nevertheless reproducible in controlled experiments. Future studies would be needed to evaluate if this sensitivity results from single ABY_DR5–6 molecules inhibiting numerous receptors within coupled oligomeric complexes^17,20.

There are currently three therapeutic strategies that have been developed to ameliorate TNF receptor related diseases: sequestration of ligand^80–82; competitive inhibition of ligand-receptor interactions^83–85; and competitive inhibition of receptor-receptor interactions^{29, 30}. While competitively targeting receptor assembly is a valid approach, we have shown with small molecule studies of TNFR1 inhibition that an even more effective approach is to develop protein antagonists that stabilize an inactive receptor conformation without competing with ligand binding or receptor-receptor interactions^{86, 87}. In the current study we have discovered a novel DR5 antagonist that acts in this non-competitive, allosteric manner. The discovery of a ligand with this unique mechanism of action via sampling only six lead affibodies after enrichment of binders without epitope- or mechanism-driven selections was perhaps fortuitous. While it is plausible to expect synthetic ligands to preferentially bind at hot spots affiliated with natural binding interfaces, sequencing and mechanistic characterization of many more clones would be needed to elucidate the relative frequency of binders that compete with TRAIL, compete with DR5 dimerization, are passive, or induce unique conformations (such as ABY_DR5–6). Our biophysical and biochemical data show that ABY_DR5–6 binds to DR5 without blocking the ligand-receptor or the receptor-receptor interactions and triggers conformational changes in DR5 transmembrane (TM) domain-dimer. We have shown previously that TM-dimer of DR5 exists in open-active and closed-inactive conformations⁸⁸. Using TR-FRET, we also previously demonstrated a conformational change in the DR5 TM-dimers upon ligand binding⁷² or soluble protein binding³⁰. This phenomenon was also observed in other families of receptors^{89, 90}. Here, an increase in FRET efficiency upon ABY_DR5–6 binding suggests that the monomers within the DR5 TM-dimer moved closer to each other. Importantly, this FRET data confirms that the affibody is in fact binding the dimeric form of DR5, despite the engineering process taking place absent ligand and raft association (as discussed above). These findings also confirm that binding of ABY_DR5–6 induces conformational changes in DR5 and favors the closed-inactive state of TM domain. These biophysical results correlate remarkably well with the functional inhibition by ABY_DR5–6, lending further support to the efficacy and potency of targeting conformational changes in TNF receptors.

CONCLUSION

Members of the TNFR superfamily are implicated in numerous diseases, are heavily targeted in ongoing clinical trials and are inhibited by drugs actively used in the clinic. For the past two decades, treatments have relied on sequestering the receptor’s ligand as the main mode of action. Unfortunately, currently available anti-TNF ligand therapeutics induce dangerous side effects. The reason for these well-known problems is that TNF ligands also signal via other receptors; thus depleting them inhibits other useful cellular pathways. Recent breakthroughs regarding TNF receptor structural biophysics have shifted the current therapeutic paradigm of global TNF ligand inhibition to selective targeting of the receptor itself. Moreover, recent studies suggest that non-competitive inhibition through perturbation of receptor conformation may be a compelling alternative to competitive inhibition. In the current study, using yeast surface display and directed evolution, we identified a synthetic affibody ligand that specifically binds to human DR5 with high affinity and inhibits TRAIL-induced apoptosis in Huh-7 cells, an in vitro model of fatty liver disease. Downstream inhibition is achieved without blocking TRAIL binding or preventing receptor-receptor interaction. Thus, our results validate the proposed non-competitive, allosteric mechanism for TNF receptor antagonism, which provides an intriguing opportunity for modulation of this important receptor family.

ABBREVIATIONS

DR5

death receptor 5

DR4

death receptor 4

FACS

fluorescence-activated cell sorting

FADD

Fas-associated death domain

FRET

Förster resonance energy transfer

GFP

green fluorescent protein

HEK293

human embryonic kidney cells

Huh-7

hepatocellular carcinoma cells

LAG3

lymphocyte activation gene 3

NAFLD

nonalcoholic fatty liver disease

NASH

nonalcoholic steatohepatitis

MACS

magnetically activated cells sorting

MTT

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

TNF

tumor necrosis factor

TNFR1

tumor necrosis factor receptor 1

TRAIL

tumor necrosis factor-related apoptosis-inducing ligand