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. 2014 Aug 19;22(10):1768–1778. doi: 10.1038/mt.2014.132

Chaperone Nanobodies Protect Gelsolin Against MT1-MMP Degradation and Alleviate Amyloid Burden in the Gelsolin Amyloidosis Mouse Model

Wouter Van Overbeke 1, Adriaan Verhelle 1, Inge Everaert 2, Olivier Zwaenepoel 1, Joël Vandekerckhove 1, Claude Cuvelier 3, Wim Derave 2, Jan Gettemans 1,*
PMCID: PMC4428403  PMID: 25023329

Abstract

Gelsolin amyloidosis is an autosomal dominant incurable disease caused by a point mutation in the GSN gene (G654A/T), specifically affecting secreted plasma gelsolin. Incorrect folding of the mutant (D187N/Y) second gelsolin domain leads to a pathological proteolytic cascade. D187N/Y gelsolin is first cleaved by furin in the trans-Golgi network, generating a 68 kDa fragment (C68). Upon secretion, C68 is cleaved by MT1-MMP-like proteases in the extracellular matrix, releasing 8 kDa and 5 kDa amyloidogenic peptides which aggregate in multiple tissues and cause disease-associated symptoms. We developed nanobodies that recognize the C68 fragment, but not native wild type gelsolin, and used these as molecular chaperones to mitigate gelsolin amyloid buildup in a mouse model that recapitulates the proteolytic cascade. We identified gelsolin nanobodies that potently reduce C68 proteolysis by MT1-MMP in vitro. Converting these nanobodies into an albumin-binding format drastically increased their serum half-life in mice, rendering them suitable for intraperitoneal injection. A 12-week treatment schedule of heterozygote D187N gelsolin transgenic mice with recombinant bispecific gelsolin-albumin nanobody significantly decreased gelsolin buildup in the endomysium and concomitantly improved muscle contractile properties. These findings demonstrate that nanobodies may be of considerable value in the treatment of gelsolin amyloidosis and related diseases.

Introduction

Amyloid disease arises when proteins or peptides aggregate and accumulate in tissues. The amyloid protein precursor, the site of deposition and clinical features differ greatly amongst different amyloidoses but cross-β sheet formation is a common denominator.1 Amyloid fibril deposition and insoluble protein fibrils cause cellular damage and disease although a growing body of evidence also attributes cellular toxicity to various intermediates prior to fiber formation.2 Over 30 amyloid based degenerative disorders are known, including Alzheimer's disease, maturity-onset diabetes and prion disease.3 Familial amyloidosis–Finnish type (FAF) or gelsolin amyloidosis is a rare autosomal dominant disorder in which mutant plasma gelsolin (PG*) is the indirect amyloid precursor.4,5 Gelsolin is a calcium-sensitive actin binding protein and founding member of the gelsolin family of actin associated proteins.6 Alternative splicing of the gelsolin gene results in a cytoplasmic and a secreted variant.7 Intracellular (81 kDa) gelsolin is involved in remodeling of the actin cytoskeleton during cell migration.8 Plasma gelsolin (PG, 83 kDa) acts as an actin scavenger in the circulation to prevent increasing blood viscosity following tissue damage.9 In gelsolin amyloidosis patients, a D187N/Y (aspartate to asparagine or tyrosine) mutation compromises calcium binding by gelsolin domain 2 which results in disturbed folding of plasma gelsolin and subsequent aberrant proteolysis.10,11 A 68 kDa secreted gelsolin fragment (C68) arises after a first cleavage by furin in the trans-Golgi network. Amyloidogenic peptides (8 and 5 kDa) are released in the extracellular matrix upon cleavage of C68 by MT1-MMP-like proteases12,13 and these cause systemic amyloid deposition which results in cardiac, renal, muscular and dermatological problems. Furthermore, corneal lattice dystrophy is very typical, accompanied by cranial neuropathy.14 Therapy is currently restricted to symptomatic treatment such as eyedrops, management of intraocular pressure and plastic surgery.15,16 A mouse model of gelsolin amyloidosis recapitulates the endoproteolytic cascade and associated amyloid deposition.17

Llama VHH antibodies or nanobodies correspond to the variable part of heavy chain antibodies. These single domain antibodies were found in Camelidae and represent the smallest, intact antigen-binding fragment (15 kDa).18 Nanobodies are endowed with unique features of solubility and stability which make them the instrument of choice in a broad range of biotechnological applications.19 Our recent work has shown that they can be instrumental in preventing breast cancer metastasis in vivo20 or perturb selected functions of proteins when used as intrabody (protein domain knockout).21,22,23,24

In this study, we used nanobodies against the 8 kDa gelsolin peptide to unsettle MT1-MMP proteolysis, which plays an essential role in the gelsolin amyloidosis pathological cascade. MT1-MMP is a membrane bound protease, member of the matrix-metalloprotease (MMP) family. This family plays a key role in pericellular protein degradation processes.25 MT1-MMP degrades several ECM components and its activity is critical during development.26 It seems reasonable to assume that inhibition of MT1-MMP as a therapeutic approach in gelsolin amyloidosis may result in severe side effects, particularly given the chronic nature of the disease. For this reason, we aimed to perturb C68 degradation by MT1-MMP using chaperone nanobodies. We show here that nanobodies raised against the 8 kDa gelsolin amyloidogenic peptide bind to the C68 amyloid precursor and drastically reduce cleavage of C68 by MT1-MMP in vitro. We further provide evidence that these nanobodies interact with the C68 amyloid precursor in plasma and muscle tissue of gelsolin amyloidosis transgenic mice expressing D187N human gelsolin. We demonstrate that intraperitoneal injection of FAF nanobodies in heterozygote gelsolin amyloidosis mice reduces gelsolin deposits in the endomysium and has a beneficial effect on muscle contractile properties. This new approach could be useful in related diseases.

Results

Properties and specificity of 8 kDa FAF peptide-specific nanobodies

Consecutive cleavage of D187N/Y plasma gelsolin by furin and MT1-MMP results in formation of peptides which act as amyloidogenic precursors underlying gelsolin amyloidosis pathology (Figure 1). We reasoned that chaperone FAF gelsolin nanobodies might interfere with MT1-MMP proteolysis by sterical hindrance, and hence indirectly reduce amyloid buildup. To test this hypothesis, a dromedary was immunized with biotinylated and chemically synthesized human gelsolin 8 kDa FAF peptide (173A-243M) containing an asparagine residue at position 187 (D187N). The same animal was simultaneously immunized with recombinant GST-tagged human gelsolin domain 2, harbouring the same mutation (G2D187N).

Figure 1.

Figure 1

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Schematic representation of the pathological proteolytic cascade in gelsolin amyloidosis. Mutant plasma gelsolin (right) is proteolytically processed, in contrast to wild type plasma gelsolin (left). D187N/Y in the second domain of mutant plasma gelsolin (white asterisk) perturbs folding, rendering the protein susceptible to aberrant cleavage by furin in the trans-Golgi network (TGN). The resulting 68 kDa secreted fragment (C68) is further proteolysed by MT1-MMP-like proteases in the extracellular matrix (ECM), generating 8 kDa and 5 kDa amyloidogenic peptides that give rise to amyloid deposition. Gelsolin domains (squares) are numbered 1–6.

After several rounds of phage panning with biotinylated peptide or His6-tagged gelsolin G2D187N, three different nanobodies were isolated (FAF Nb1-3). The binding characteristics of purified FAF Nb1-3 were investigated by ELISA, demonstrating dose-dependent interaction between nanobodies and the biotinylated peptide or C68 (Figure 2a–c). No significant binding was observed with GST-CapG, a negative control. CapG (~40 kDa) is an actin associated protein displaying 49% similarity with gelsolin27 that binds reversibly to the barbed end of actin filaments (F-actin capping) in a calcium-dependent manner. Binding characteristics were more thoroughly investigated by ITC (isothermal titration calorimetry), indicating a Kd of FAF nanobodies for the 8 kDa peptide in the range of 4–8 × 10−7 mol/l (Supplementary Table S1a).

Figure 2.

Figure 2

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Familial amyloidosis–Finnish type (FAF) Nb1-3 bind to C68 and the 8 kDa amyloidogenic peptide in ELISA. FAF Nb1-3 were tested for interaction with recombinant C68 or 8 kDa peptide in an ELISA assay. A tenfold dilution series of FAF Nb was used, starting from 1 µg (=1) up to 10–5 µg. FAF Nb1-3 (shown in a, b, and c, respectively) displayed concentration dependent absorbance (measured at 450 nm) reflecting interaction with the 8 kDa peptide (black bars) or with C68 (gray bars). GST-CapG (white bars) was used as a negative control. Signals are represented relative to the highest value, normalized to 1.

We next investigated the specificity of FAF Nb1-3 by western blot analysis. Protein lysates from E. coli cells expressing C68, PG, or PG* were used for this purpose. A lysate of GST-CapG expressing E. coli cells was included as a negative control. As shown in Figure 3a,c, all three FAF nanobodies recognized C68, PG, and PG* as well as the 8 kDa FAF peptide but did not cross-react with CapG, attesting to their specificity. GST-CapG expression in the E. coli lysate was verified as shown in Figure 3b.

Figure 3.

Figure 3

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Familial amyloidosis–Finnish type (FAF) Nb1-3 bind specifically to gelsolin (fragments). (a) 5 µg bacterial protein extract containing recombinant GST-CapG (negative control), C68, PG, or PG* were fractionated by SDS-PAGE and western blot analysis was performed using V5-tagged FAF Nb1-3 as primary antibody. Monoclonal anti-gelsolin antibody was used as a positive control. (b) To confirm GST-CapG expression in the negative control lysate, a polyclonal anti-CapG antibody was used. (c) The same procedure was repeated for the 8 kDa peptide. Polyclonal anti-FAF peptide antibody was used as a positive control. Monomeric 8 kDa peptide and peptide oligomers are visualized, the latter particularly by the nanobodies.

We then ascertained the ability of recombinant FAF nanobodies to recognize their native epitope by co-immunoprecipitation experiments using E. coli lysates containing recombinant C68, PG, or PG*. FAF Nb1-3 bound to C68, PG, and PG*, albeit to varying degrees (Supplementary Figure S1a–c). Indeed, C68 was efficiently retrieved by the FAF nanobodies whereas PG* and particularly PG was much less efficiently precipitated. GST-CapG (negative control) was not precipitated from the bacterial extract, further indicating that FAF Nb1-3 recognize an epitope that is unique to mutant gelsolin and more accessible in the C68 degradation product. This is in agreement with a previous report where the D187N mutation was shown to prevent normal folding of the second domain of plasma gelsolin.28 C68, PG, PG*, or GST-CapG were not retained by anti-V5 agarose in the absence of a FAF nanobody (Supplementary Figure S1d).

Mono- and bispecific FAF nanobodies reduce MT1-MMP proteolysis of C68 in vitro

We hypothesized that the C68-FAF Nb interaction might interfere with MT1-MMP catalyzed proteolysis of C68 through sterical hindrance of the interaction between MT1-MMP and C68 or by shielding of the scissile peptide bond that is cleaved by MT1-MMP at 243M-244L.13 We therefore investigated if FAF nanobodies counteract MT1-MMP mediated proteolysis of C68. This would constitute a therapeutic effect since peptide aggregation and amyloid fibril formation involve a nucleation phase requiring a critical peptide monomer concentration before polymerization is initiated.29 Preventing formation of the 8 kDa peptide could thus slow down an early and rate-limiting step in the amyloid aggregation process. FAF Nb1-3 were pre-incubated at increasing concentrations with C68, up to a twofold molar excess, before addition of the MT1-MMP catalytic domain. Formation of the 8 kDa peptide was assessed by western blotting. All three nanobodies significantly reduced amyloid peptide formation when used in equimolar ratios relative to C68 (nanobodies bind in a 1:1 ratio) (Figure 4a,b). By contrast, a previously characterized gelsolin nanobody (GSN Nb13) that binds in a calcium-dependent manner with high affinity (5 nmol/l) to gelsolin domains 4–5 in the C-terminal half of the protein,30 showed no effect whatsoever. An additional in vitro cleavage assay was performed to investigate if FAF Nb1-3 could affect gelsolin proteolysis by furin. This proprotein convertase generates C68 from mutant plasma gelsolin and hydrolyzes the scissile peptide bond at residues 172R-173A (273M-274L is cleaved by MT1-MMP). Yet, the FAF nanobodies had no effect on proteolysis of mutant plasma gelsolin by furin (Figure 4c), further emphasizing their specific impact on MT1-MMP dependent C68 degradation. To confirm substrate specificity, we checked for a specific interaction of the FAF nanobodies with a physiological MT1-MMP substrate. We performed an in vitro gelatin degradation assay and first checked in vitro breakdown of the FAF nanobodies to discriminate nanobody breakdown from gelatin degradation (Supplementary Figure S2a). FAF Nb1 nor FAF Nb2-MSA21 disturbed gelatin degradation (Supplementary Figure S2b, lanes 3–4). On the other hand, TIMP-2 (an MT1-MMP inhibitor) inhibited proteolysis and protected the full length protein (Supplementary Figure S2b, lane 5). Taken together, these findings demonstrate that FAF Nb1-3 significantly and specifically reduce degradation of C68 in vitro.

Figure 4.

Figure 4

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Familial amyloidosis–Finnish type (FAF) Nb1-3 reduce C68 proteolysis by MT1-MMP in vitro. (a) FAF Nb1-3 were incubated with C68 (molar ratio nanobody:C68 indicated on top) prior to addition of MT1-MMP. The 8 kDa peptide was detected with anti-His HRP coupled antibody. Lane 1: negative control without MT1-MMP; lane 2: positive control with MT1-MMP but without addition of a nanobody. Increasing concentrations of nanobody (lanes 3–5, molar ratios indicated on top) progressively reduce 8 kDa peptide formation. Control GSN Nb13 has no effect on MT1-MMP cleavage of C68. (b) Quantification of data shown in a. Data are presented relative to the positive control. In a 1:1 ratio, FAF nanobodies reduced peptide formation by 74 ± 5% (P < 0.001, FAF Nb1), 56 ± 8% (P < 0.01, FAF Nb2) and 83 ± 5% (P < 0.001, FAF Nb3). Data shown as mean of triplicates + SE. **P < 0.01; ***P < 0.001 (two sided unpaired t-test). (c) FAF nanobodies do not affect proteolysis of PG* by furin. PG* was incubated with FAF Nb1-3 (lanes 3–5, molar ratios indicated on top) prior to furin addition. C68 formation was detected with anti-8 kDa peptide antiserum and was not affected by FAF nanobodies or by GSN Nb13. Negative (lane 1) and positive (lane 2) controls were included as in a.

We hypothesized that FAF nanobodies may be endowed with the ability to reduce amyloid formation in vivo since MT1-MMP is a membrane-bound protease that cleaves C68 upon secretion. For this reason, FAF nanobodies do not require intracellular expression. Hence, with a view to applying a FAF Nb in the mouse model that recapitulates the endoproteolytic cascade,17 we set out to increase nanobody half-life by converting them into a bulkier format. Nanobodies are rapidly cleared via the kidneys,31 rendering them inapt for medium or long term therapeutic purposes. Linkage with albumin via an albumin binding peptide has been reported to increase serum half-life.32 We therefore coupled FAF Nb1-3 to MSA21, an albumin binding nanobody (Kd = 12 nmol/l for mouse serum albumin), separated by a GGGSGGG spacer (Figure 5a). The ability of MSA21 to bind mouse albumin was verified by gelfiltration and SDS-PAGE (Figure 5b). ELISA experiments showed that the bispecific construct retained its ability to bind C68 gelsolin (Supplementary Figure S3a–c) while calorimetry demonstrated that FAF Nb2-MSA21 displayed an affinity for the 8 kDa peptide that was in the same range as compared to the monospecific FAF Nb2 format (Supplementary Table S1a,b). In the following experiments, monospecific FAF Nb1 and bispecific FAF Nb2-MSA21 were used because they displayed the highest affinity. Affinity determination was also performed for the FAF Nb2-MSA21/C68 interaction, which was in the same range as the affinity for the 8 kDa peptide (Supplementary Table S1a,b). The effect of MSA21 linkage on the serum half-life was subsequently investigated by intraperitoneal injection of 100 µg recombinant nanobody into wild type C57BL/6 mice. Western blot analysis on blood samples taken at different time points revealed that the monospecific nanobody was nearly completely cleared from the circulation after four hours whereas the bispecific FAF Nb2-MSA21 remained stable in plasma up to 1 week after injection, and was not degraded (Figure 5c). Importantly, modifying the FAF nanobodies from a monospecific into a bispecific format did not affect their ability to reduce MT1-MMP catalyzed degradation of C68 (Figure 5d).

Figure 5.

Figure 5

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Bispecific Familial amyloidosis–Finnish type (FAF) Nb2-MSA21 displays a prolonged half-life in mice and retains its effect on MT1-MMP proteolysis of C68 in vitro. (a) Schematic representation of the bispecific FAF Nb-MSA21, bound to its respective targets, C68 and albumin. (b) Upper panel: binding of FAF Nb2-MSA21 to albumin. Superdex 200 gel filtration chromatography of FAF Nb2-MSA21 in the absence of albumin (dashed line); peak fractions eluted at 15 ml. In the presence of mouse serum albumin peak fractions eluted at 12 ml (solid line), indicating that FAF Nb2-MSA21 and albumin had formed a complex. Lower panel: SDS-PAGE shows co-elution of FAF Nb2-MSA21 and albumin in the same peak fraction. (c) Wild type C57BL/6 mice were injected intraperitoneally with 100 µg V5-tagged FAF Nb1 or FAF Nb2-MSA21. Blood samples were taken at the indicated time points. Plasma was fractionated by SDS-PAGE followed by western blotting. Nanobody was detected with polyclonal anti-V5 antibody. (d) MT1-MMP assay with bispecific FAF Nb-MSA21 nanobodies. Lane 1; negative control without MT1-MMP; lane 2: positive control with MT1-MMP but without addition of a nanobody. Lanes 3–4 represent additional controls in which an equimolar amount of either albumin or nanobody was added. Lanes 5–7 represent samples where an increasing amount of albumin and nanobody was added (ratio of albumin and Nb in proportion to C68 indicated on top).

FAF Nb2-MSA21 binds human C68 from plasma and muscle tissue of gelsolin amyloidosis mice

When administered to mice, the therapeutic efficacy of the bispecific FAF nanobody may be “diluted” by interaction with mouse plasma gelsolin since mouse and human gelsolin show a very high sequence similarity (96%). For this reason, we performed different co-immunoprecipitation experiments prior to injection of nanobodies into gelsolin amyloidosis mice. Importantly, mouse plasma gelsolin does not co-immunoprecipitate with FAF Nb1 or FAF Nb2-MSA21 (Figure 6a, lanes 3 and 4). Therefore, binding of FAF nanobody to mouse plasma gelsolin, when injected into gelsolin amyloidosis mice, will likely be negligible. Next, we investigated in a co-immunoprecipitation experiment if FAF Nb1 and FAF Nb2-MSA21 bind to C68 that is present in plasma of gelsolin amyloidosis transgenic animals. For this purpose, plasma was collected from a 3 weeks old homozygous animal, reported to contain relatively high levels of circulating C68.17 The input C68 signal is distorted due to the high concentration of albumin (Figure 6b, lane 1). FAF Nb1 and FAF Nb2-MSA21 (Figure 6b, lanes 3 and 4, respectively) specifically retrieved the C68 fragment from mouse plasma and did not co-immunoprecipitate PG*, emphasizing their specificity. Hence, “dilution” of the therapeutic efficacy by non-specific interaction with full length PG* is not expected to be a disturbing factor.

Figure 6.

Figure 6

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Familial amyloidosis–Finnish type (FAF) Nb1 and FAF Nb2-MSA21 bind human C68 from gelsolin amyloidosis transgenic mouse plasma and muscle lysate. (a) Albumin-cleared plasma from a wild type C57BL/6 mouse was used in a co-immunoprecipitation assay with V5 tagged FAF nanobodies (detection with polyclonal anti-gelsolin antibody). Lane 1: detection of mouse gelsolin in albumin-cleared plasma (5 µg); lane 2: mouse gelsolin does not bind in a non-specific manner to anti-V5 agarose (negative control); lanes 3 and 4: mouse gelsolin is not precipitated by FAF Nb1 or FAF Nb2-MSA21. (b) FAF nanobodies bind to C68 that is present in plasma from a 3 weeks old homozygous mouse (detection with polyclonal anti-FAF antibody). Lane 1: detection of PG* and C68 in 5 µg of plasma; lane 2: negative control; lanes 3–4: FAF Nb1 and FAF Nb2-MSA21 immunoprecipitate C68, but not PG*. (c) FAF nanobodies interact with C68 in muscle lysate (detection with polyclonal anti-FAF antibody). Lanes 1–2: detection of human gelsolin in muscle lysate (10 µg) from a WT animal or a 4.5 months old heterozygous mouse, demonstrating the specificity of the anti-FAF antibody for ectopic human mutant gelsolin and C68. Lane 3: negative control; lanes 4–5: immunoprecipitation of C68 by FAF Nb1 or FAF Nb2-MSA21.

The C68 fragment is not only present in the circulation but also resides in skeletal muscle tissue of transgenic mice. Mere sequestration of circulating C68 by a nanobody is unlikely to trigger major therapeutic effects since C68 cleavage by membrane bound MT1-MMP occurs immediately upon secretion of the 68 kDa fragment from cells. Hence, once the fragment has “escaped” MT1-MMP proteolysis and ends up in the circulation, it is less likely to give rise to amyloidogenic peptides in the short term. We therefore investigated if FAF nanobodies interact with C68 in muscle tissue. This is important since MT1-MMP cleaves C68 locally, so any potential inhibitory effects by the nanobody strongly depend on its ability to bind C68 in skeletal muscle. To this end, a musculus gastrocnemius extract was obtained from a 4.5 months old heterozygous gelsolin amyloidosis mouse and this extract was used in a co-immunoprecipitation assay with FAF Nb1 or FAF Nb2-MSA21. PG* (weak) as well as the C68 gelsolin fragment were readily detected in the heterozygous muscle extract (Figure 6c, lane 2) whereas they were not detected in WT muscle extract (Figure 6c, lane 1). A fraction of the detected transgenic proteins may originate from blood vessels that irrigate the muscle tissue. C68 was enriched by immunoprecipitation with the FAF nanobodies (Figure 6c, lanes 4–5). C68 gelsolin showed a weak tendency to interact non-specifically with the agarose beads (Figure 6c, lane 3) which may be caused by its propensity to oligomerize.29

FAF nanobodies accumulate around muscle fibers upon injection in a heterozygous gelsolin amyloidosis mouse

To verify if FAF Nb2-MSA21 binds C68 in vivo, we performed immunohistochemistry on muscle tissue. A FAF heterozygous mouse (5.5 months of age) was injected with FAF Nb1 or FAF Nb2-MSA21 (both V5-tagged) and 1 hour later a musculus gastrocnemius tissue sample was obtained and stained for gelsolin (anti-8 kDa antiserum) or nanobody (anti-V5). The specificity of this antiserum was verified by western blot (Supplementary Figure S4). The circumferential myofiber staining pattern observed for gelsolin is very similar to what has been reported earlier17 (Figure 7a,b). Importantly, the V5 nanobody staining pattern was identical to the gelsolin pattern for both the mono- and bispecific nanobody, (Figure 7c,d) indicating that both nanobody formats penetrate into muscle tissue and colocalize with gelsolin deposits or truncated gelsolin (Figure 7e,f). Thus, the injected nanobodies interact with their targets after intraperitoneal injection. Gelsolin staining in wild type mouse muscle tissue did not result in any significant background staining, as expected (Figure 7g).

Figure 7.

Figure 7

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Intraperitoneally injected Familial amyloidosis–Finnish type (FAF) Nb1 and FAF Nb2-MSA21 co-localize with gelsolin deposits in the endomysium of heterozygous gelsolin amyloidosis mice. Confocal microscopy images of heterozygous 5.5 months old gelsolin amyloidosis mice injected with 100 µg V5-tagged FAF Nb1 or FAF Nb2-MSA21. Musculus gastrocnemius tissue was dissected 1 hour post-injection and cryosections were stained for gelsolin and nanobody. (a,b) Gelsolin staining reveals a pattern that surrounds the myofibers. (c,d) V5 staining indicates presence of the nanobodies between the muscle fibers. (e,f) Merged images indicate co-localization between injected nanobody and gelsolin deposits in gelsolin amyloidosis mice. (g) Musculus gastrocnemius tissue from a wild type mouse stained for gelsolin (scale bar = 50 µm).

FAF Nb2-MSA21 reduces gelsolin buildup in endomysium and improves muscle contractile properties

To assess if FAF Nb1 and FAF Nb2-MSA21 trigger a therapeutic response in gelsolin amyloidosis mice, we set up an experiment in which heterozygous mice were injected weekly (IP) with 100 µg of recombinant FAF Nb1 (n = 5) or FAF Nb2-MSA21 (n = 6). A control group of heterozygous mice (n = 11) was injected with 200 µl PBS at the same time intervals. Injections started at the age of 4 weeks and the mice were injected 12 times. Potential adverse effects of systemic nanobody administration were assessed by measuring liver transaminase values after 3 months of injection. AST (aspartate transaminase) and ALT (alanine transaminase) levels did not significantly differ between the PBS and FAF Nb2-MSA21 injected mice (Supplementary Figure S5). To investigate the effectiveness of FAF Nb1 and FAF Nb2-MSA21 at single muscle level, in vitro contractile function of two different hind leg muscles (extensor digitorum longus (EDL) and soleus) was evaluated by repeated electrically stimulated tetanic contractions of the intact incubated muscles. Typical features of contractile fatigue, such as the decrease in relaxation rate (Figure 8a) and contraction speed (Supplementary Figure S6a) in the beginning (30 and 60 seconds) of the fatigue protocol, were attenuated in EDL of FAF Nb2-MSA21 injected mice, compared to PBS (not for FAF Nb1 group). Relaxation and contraction speed was not affected by the interventions in soleus (Figure 8b and Supplementary Figure S6b). In EDL, the force-frequency relationship was not affected by nanobody treatment (Supplementary Figure S6c). In soleus, a left-ward shift of the force-frequency was observed with FAF Nb2-MSA21 compared to PBS (75 and 100 Hz) and FAF Nb1 (1, 35, and 50 Hz) treated mice (Supplementary Figure S6d).

Figure 8.

Figure 8

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Familial amyloidosis–Finnish type (FAF) Nb2-MSA21 injection in gelsolin amyloidosis heterozygote mice significantly improves muscle contractile properties and reduces pathological gelsolin buildup in the endomysium. (a,b) Repeated in vitro muscle contractions in FAF Nb1 (gray circles) and FAF Nb2-MSA21 (white circles) injected mice compared to PBS controls (black circles). Injections with FAF Nb2-MSA21, but not with FAF Nb1, resulted in an attenuation of the slowing of the relaxation rate during fatigue in EDL (a) but not in soleus (b), *P < 0.05 FAF Nb2-MSA21 versus PBS (two sided unpaired t-test). (c) Upper panels: confocal microscopy images show sections of dissected musculus gastrocnemius, taken from gelsolin amyloidosis mice injected with PBS (left), FAF Nb1 (middle) or FAF Nb2-MSA21 (right). Lower panels: co-staining of the same tissues for laminin, a basement membrane marker. (d) Quantification of the gelsolin staining surface percentage. A statistically significant decrease of 15 ± 3% was observed for FAF Nb1 (P < 0.05) and 30 ± 2% for FAF Nb2-MSA21 (*P < 0.001) injected mice, in comparison to the PBS group. The additional 15% effect of FAF Nb2-MSA21, in comparison to FAF Nb1 is statistically significant (*P < 0.01). Scale bar = 50 µm. *P < 0.05; **P < 0.01; ***P < 0.001 (two sided unpaired t-test).

The effect of FAF nanobody administration was further corroborated by immunohistochemistry. Musculus gastrocnemius was dissected and staining was performed with anti-8 kDa antiserum (Figure 8c) followed by confocal microscopy. The staining pattern was more homogenous around the muscle fibers in PBS injected mice (Figure 8c, upper left panel). By contrast, the pattern was more focal and less intense in nanobody injected mice (Figure 8c, upper middle and right panels). Moreover, we co-stained laminin in the same sections (Figure 8c, lower panels) showing that the pattern for this basement membrane marker remains constant in all three groups, which validates the reducing effect of FAF nanobody on gelsolin staining. To quantify this effect, Image J analysis was performed to calculate the gelsolin staining surface percentage (Figure 8d). Quantification revealed a significant decrease for both FAF Nb1 (P < 0.05) and FAF Nb2-MSA21 (P < 0.001) injected mice, compared to PBS controls. The additional effect triggered by MSA21 linkage is statistically significant (P < 0.01) when comparing the FAF Nb2-MSA21 versus FAF Nb1 group.

Discussion

Nanobodies have proven to be a potent tool for functional perturbation of extracellular33 or intracellular proteins.20,21,22,23,30 In this study, detailed insight into the proteolytic gelsolin amyloidosis cascade12,13 enabled us to target the C68 precursor that gives rise to amyloidogenic peptides. Preventing C68 proteolysis by MT1-MMP may embody a therapeutically preferred strategy since metalloproteases participate in many diverse physiological processes.25 Moreover, in vitro experiments revealed that other MT1-MMP-like proteases are also capable of degrading C68.13 By using FAF nanobodies as a molecular chaperone protecting 243M-244L cleavage, the implementation of multiple inhibitors targeting various metalloproteases may prove to be redundant. Co-immunoprecipitation experiments demonstrated a binding preference of FAF Nb1-3 to C68 as compared to full length PG or PG*. This discriminative property of FAF nanobodies is therapeutically advantageous because it prevents “dilution” of the FAF nanobody effect through potential interaction with gelsolin configurations other than C68. Potentially, a fraction of the circulating nanobody may be taken up in cells through albumin mediated internalization34 and will be temporarily exposed to intracellular epitopes before degradation through the lysosomal pathway.

At the time of muscle dissection in the nanobody treated heterozygous mice, the animals were 4 months of age. Congo red staining, a dye commonly used for amyloid detection35 is not that pronounced at this stage.17 For this reason, we used the anti-8 kDa antiserum which recognizes all gelsolin configurations present (PG*, C68, and 8 kDa peptide). While the anti-8 kDa antiserum does not allow us to discriminate between different gelsolin fragments, we are confident that this antiserum is specific for detecting pathological accumulation of C68 or amyloid peptide between muscle fibers because staining of wild type mice muscle sections was negative.

In accordance with the decrease in gelsolin staining in FAF Nb2-MSA21 treated mice, the in vitro muscle contractile function was improved in this group. Furthermore, an improved relaxation speed at the onset of fatigue was observed in the EDL of FAF Nb2-MSA21 injected mice. A slowing of the relaxation rate of repeated tetanic contractions is a well-known feature of fatigue. Homeostatic control is considerably strained during fatiguing muscle contractions. It can therefore be hypothesized that extracellular amyloid accumulation will negatively affect energy supply and metabolic waste elimination. These pathological features seem to be reversed, at least in part, by the FAF Nb2 MSA21 treatment.

Nanobody technology is maturing into clinical trials. ALX-0061 (an IL-6R nanobody,36 has successfully completed a phase IIa trial and is en route of becoming a clinical therapeutic for rheumatoid arthritis. To further increase the potential of FAF Nb2-MSA21 in human gelsolin amyloidosis patients, certain modifications would be required. For instance, the MSA21 albumin binding nanobody should be replaced by a human serum albumin binder. Human serum albumin has a longer serum half-life (±19 days,)37 which would positively affect the administration frequency schedule. In addition, an undesirable immune response triggered by a nanobody can be tackled by a humanization strategy involving mutation of camelid-specific residues 49 and 50 located in framework 2. These mutations are neutral to typical advantageous nanobody properties (affinity, expression, stability) and have lead the authors to propose a universal humanized nanobody scaffold onto which antigen-binding loops from other nanobodies can be grafted.38

The concept of shielding an amyloid precursor protein from proteolysis may be of use in the research field of Alzheimer's disease and related afflictions. More specifically, extracellular cleavage of APP (amyloid precursor protein) by BACE (β-secretase 1) constitutes the first of two consecutive cleavages which ultimately result in formation of Aβ amyloidogenic peptide.39 A bispecific monoclonal antibody directed against the transferrin receptor and BACE was recently shown to penetrate the blood-brain-barrier and reduce Aβ40 levels in the brain of mice.40 Nanobodies can be produced at very low cost and have also been demonstrated to penetrate the blood-brain barrier41,42,43 without requiring specific targeting to a receptor. Therefore, side effects elicited by receptor function disruption can be avoided. Hypothetically, nanobodies directed against the BACE cleavage site in APP could indirectly counter BACE activity by shielding APP, thus moderating subsequent Aβ formation. Hence, the use of nanobodies as a chaperone to protect precursor proteins from aberrant proteolysis can be instrumental in mounting therapeutic initiatives against gelsolin amyloidosis and related diseases.

Materials and Methods

Antibodies and reagents. See Supplementary Materials and Methods for a detailed description on the antibodies and reagents used in this study.

Generation of FAF gelsolin-specific nanobodies. See Supplementary Materials and Methods for details on the generation of FAF gelsolin-specific nanobodies.

cDNA cloning. All reactions were performed using the Cold Fusion Kit (System Biosciences, Mountain View, CA). Cloning of the FAF nanobodies into the pHEN6-V5-His was performed using following primers: 5′ CCA GGT GCA GCT GCA GGA GTC TGG GGG AGG CTC 3′ (forward) and 5′ TGA GGA GAC GGT GAC CTG GGT CCC CTG GCC CCA 3′ (reverse). The albumin binding nanobody (MSA21, Ablynx nv), was introduced C-terminally to the FAF nanobodies in pHEN6-V5-His using the following primers: 5′ GGC CAG GGG ACC CAG GTC ACC GTC TCC TCA GGT GGT GGT AGC GGT GGT GGT CAA GTC CAA CTG CAG GAA TCG GG 3′ (forward) and 5′ GCT TGA GAC GGT GAC CTG GGT GCC TTG ACC 3′ (reverse). The resulting bispecific FAF Nb-MSA21 construct was subcloned into the pTYB1 vector (New England Biolabs) using the primers 5′ CTT TAA GAA GGA GAT ATA CAT ATG GCC CAG GTG CAG CTG CAG GAG TCT GG 3′ (forward) and 5′ CGC CAT TAA AAC ATT GGT ACC CTT GGC AAA GCA ATG GTG ATG GTG ATG ATG ACC 3′ (reverse).

Purification recombinant C68, PG* and nanobodies. pET11a-C68 (or pTrc-His-TOPO-PG*) cDNA was transformed into chemically competent E. coli cells and the mixture was incubated overnight at 37 °C on LB-agar plates containing 100 µg/ml ampicilline. A single colony was picked and grown overnight at 37 °C in 5 ml LB medium containing 100 µg/ml ampicillin. The preculture was diluted in 500 ml of LB/ampicillin (100 µg/ml) and induced for expression with 0.5 mmol/l IPTG at OD600 of 0.6–0.9 (4 hours incubation at 37 °C). The cells were collected by centrifugation (~11,000g) for 15 minutes at 4 °C. Cells were resuspended in phosphate buffered saline (PBS) with 0.2 mg/ml lysozyme and lysed during 30 minutes rotation at room temperature. This suspension was finally sonicated (Vibracell, Sonics and Materials, Newtown, CT) and centrifuged again (~29,000g) for 30 minutes at 4 °C to obtain the bacterial protein lysate. His6-tagged C68/PG* was further purified by IMAC (immobilized metal affinity chromatography). Briefly, the lysate was incubated with TALON metal affinity resin (Clontech, Mountain View, CA) for 2 hours at 4 °C in the presence of 10 mmol/l imidazole (with end over end rotation). The beads were washed with buffer A (50 mmol/l NaH2PO4 pH 8.0, 500 mmol/l NaCl, 20 mmol/l imidazole, 1 mmol/l PMSF (phenylmethanesulfonylfluoride) and protease inhibitor cocktail (200 µg/ml of benzamidin, leupeptin, and aprotinin). His6-tagged C68/PG* was eluted with buffer B (50 mmol/l NaH2PO4 pH 8.0, 500 mmol/l NaCl, 250 mmol/l imidazole, 1 mmol/l PMSF, and 200 µg/ml protease inhibitor cocktail). Further purification was done by ion exchange chromatography on a MONO Q HR 10/10 column (GE Healthcare, Diegem, Belgium). The column was equilibrated in buffer A (20 mmol/l Tris pH 8.0, 50 mmol/l NaCl, 1 mmol/l DTT, 1 mmol/l EGTA). After loading the sample, a linear concentration gradient of NaCl was established with buffer B (20 mmol/l Tris pH 8.0, 500 mmol/l NaCl, 1 mmol/l DTT, 1 mmol/l EGTA) to elute the proteins at a flow rate of 0.5–1 ml/min. Final purity was confirmed by SDS-PAGE and Coomassie staining. Nanobodies in the pHEN6-V5-His6 vector were expressed and purified as described.30 Mono- and bispecific FAF Nb1-3 in the pTYB1 vector were transformed in E. coli BL21 cells, which were grown until OD600 = 2 and subsequently induced with 0.5 mmol/l IPTG (overnight incubation at 20 °C). Nanobodies were purified with chitin beads according to the manufacturer's instructions and cleaved from the intein moiety with 50 mmol/l DTT. Final purification was performed with a size exclusion chromatography Superdex 75 16/60 column (GE Healthcare, Diegem, Belgium).

Size exclusion chromatography. Interaction between recombinant FAF Nb2-MSA21 and mouse serum albumin (Sigma-Aldrich, Diegem, Belgium) was investigated by incubating equimolar quantities of the nanobody and albumin (4 µmol/l) in 500 µl PBS and analyzing the complex on a Superdex 200 HR 10/30 column (GE Healthcare).

ELISA. Enzyme-linked immunosorbent assay (ELISA) experiments were performed in Nunc 96-well plates (Roskilde, Denmark). 0.2 µg protein per well was coated in coating buffer (100 mmol/l bicarbonate/carbonate, pH 9.6). PBS + 0.5% Tween was used as washing buffer; 1% BSA in PBS as blocking buffer. V5-tagged nanobodies were added to the wells in a tenfold dilution series, ranging from 1 µg to 10−5 µg. Anti-V5 antibody and anti-mouse HRP served as secondary and tertiary antibody. Readout of the reaction was performed with a 3,3′,5,5′ tetramethylbenzidine (TMB) substrate kit (Thermo Scientific, Erembodegem, Belgium).

In vitro MT1-MMP assay. The in vitro cleavage reaction13 was performed in a total volume of 20 µl. 3 µmol/l purified recombinant C68 was incubated with FAF nanobody (or negative control GSN Nb13) in reaction buffer (50 mmol/l Tris pH 7.5, 5 mmol/l CaCl2, 200 mmol/l NaCl, 20 μmol/l ZnSO4, and 0.05% Brij-35) for 1 hour at 4 °C. In case of bispecific nanobodies, 3 µmol/l mouse serum albumin was added and the sample was incubated for another hour at 4 °C. The MT1-MMP cleavage reaction was initiated by addition of the catalytic domain of MT1-MMP (50 ng) and further incubation for 20 minutes at 37 °C. The reaction was terminated by adding 5 µl Laemmli sample buffer supplemented with 12.5 mmol/l EDTA. Samples were immediately boiled for 5 minutes, fractionated by tricine SDS-PAGE followed by western blotting, performed as described.44 Penta-His6 HRP was used for peptide detection. Quantification was done with Image J software.

Gelatin degradation assay. The experiment was performed essentially as described by Woskowicz and coworkers.45 5 µg FAF Nb1 or FAF Nb2-MSA21 was used. 100 ng of MT1-MMP was added and TIMP-2 was used at a concentration of 1 µmol/l.

In vitro furin assay. The in vitro cleavage reaction46 was performed in a total volume of 20 µl. 2 µmol/l purified recombinant PG* was incubated with FAF nanobody (or negative control GSN Nb13) in reaction buffer (25 mmol/l Tris pH 7.0, 2 mmol/l CaCl2, 1 mmol/l 2-mercaptoethanol) for 1 hour at 4 °C. To initiate cleavage, 1 unit of furin was added and the mixture was further incubated at 37 °C for 1 hour. The reaction was terminated by adding 5 µl Laemmli sample buffer and samples were immediately boiled for 5 minutes and fractionated by 10% SDS-PAGE followed by western blotting. The anti-8 kDa antiserum was used as primary antibody.

Co-immunoprecipitation assay. Bacterial pellets containing GST-CapG, C68, PG, or PG* constructs were resuspended in lysis buffer containing 0.5% NP40 and further processed as described above. 1 mg bacterial protein lysate was incubated with 5 µg V5-tagged nanobody in binding buffer (PBS + 0.5% NP-40, 1 mmol/l PMSF and 200 µg/ml protease inhibitor cocktail) at 4 °C for 1 hour. Anti-V5-agarose (12.5 µl settled beads) was added and the sample was further incubated at 4 °C for 2 hours. The suspension was washed 4 times with binding buffer and once with PBS. Proteins bound to anti-V5 agarose were eluted with Laemmli sample buffer, boiled for 5 minutes and fractionated by SDS-PAGE. Mouse muscle lysates were obtained by homogenization of 50 mg tissue in PBS with a glass rod and subsequent centrifugation (~29,000g). Albumin-cleared mouse plasma was obtained using the ProMax Albumin Removal Kit (Thermo Scientific, Erembodegem, Belgium), according to the manufacturer's instructions. Co-immunoprecipitation experiments on mouse muscle lysates and albumin-cleared plasma were performed as described for bacterial lysates using 100 µg of muscle lysate or 20 µg of albumin-cleared plasma.

Mouse intraperitoneal injection of FAF nanobodies. All animal work was approved by the Animal Experimental Ethics Committee of Ghent University Hospital (ECD 10/32). The animals were kept under environmentally controlled conditions (12 h normal light/dark cycles, 20–23 °C and 50% relative humidity) with food and water ad libitum. Heterozygous gelsolin amyloidosis mice were injected weekly intraperitoneally (IP) with 100 µg of FAF Nb1 (n = 5) or FAF Nb2-MSA21 (n = 6) (dissolved in 200 µl of PBS). IP injections were initiated at the age of 4 weeks and the mice were injected 12 times. A control group of heterozygous mice (n = 11) was injected with 200 µl PBS, at the same time intervals. Wild type C57BL/6 mice for breeding were purchased at Charles River (L'Arbresle Cedex, France).

Evaluation of in vitro contractile function in mice. Evaluation of muscle contractile function was performed as described before.47 In brief, mice were anaesthetized by an intraperitoneal infusion of 80% Ketalar / 20% Rompun (5 µl/g body weight). Following dissection of the slow-twitch soleus and fast-twitch EDL (extensor digitorum longus) muscles, wires were attached to the tendons and muscles were mounted vertically in an incubation bath with one tendon attached to a force transducer (PowerLab, ADInstruments, Spechbach, Germany) and stimulated with capacitor discharges between platinum electrodes. The incubation medium (10 ml) was a Krebs–Henseleit solution, which was continuously gassed with a mixture of 95% O2 and 5% CO2 and maintained at 30 °C. After mounting, a 15 minutes stabilization period was allowed and optimal muscle length (L0) was determined by tetanic contractions. Next, the force–frequency relation was determined by stimulating at 10, 20, 35, 50, 75, 100, and 125 Hz with 1 minute rest interval for soleus and stimulating at 25 (1 minute rest), 40 (1 minute rest), 55 (1 minute rest), 70, 100, 125, 150, and 175 Hz with 2 minutes rest interval, unless stated otherwise, for EDL. Furthermore, fatigability was evaluated as the percentage decrease in tetanic force, contraction speed (maximum slope) and relaxation rate (minimum slope) values during 8 minutes of repeated tetanic contractions (train duration, 350 microseconds; soleus, 50 Hz every 5 seconds; EDL, 100 Hz every 10 seconds).

AST/ALT measurements. AST/ALT level measurements were performed at Ghent University Hospital by using the Cobas c701 (a+b) method (Roche, Basel, Switzerland) in the UV-Tris buffer changed IFCC method without addition of pyridoxal phosphate.

Immunostaining. Musculus gastrocnemius was dissected from the hind limb and snap frozen in liquid nitrogen. Cryosections were made and thawed for 15 minutes. Subsequently, the sections were incubated in aceton for 20 minutes at −20 °C, followed by a quick wash with PBS and 10 minutes incubation in PBS. Next, sections were incubated in 50 mmol/l NH4Cl/PBS for 10 minutes and washed again in PBS. Endogenous peroxidase activity was blocked by incubating in 0.3% H2O2 for 20 minutes followed by washing in PBS. Sections were incubated in 1% BSA/PBS for 20 minutes and incubated overnight with primary antibody (1:500) at 4 °C (anti-8 kDa antiserum). Slides were washed with PBS and monoclonal anti-V5 antibody (1:800) or rat anti-laminin (1:500) was incubated at room temperature for 1 hour. Next, a PBS wash was performed and secondary antibody (Alexa fluor 594 goat anti-rabbit, 488 goat anti-mouse or 594 anti-rat) was incubated (1/500) for 1 hour. Sections were rinsed in PBS and stained with DAPI (1/500) for 2 minutes. Finally, sections were mounted with VectaShield and imaged at room temperature using an Olympus IX81 FluoView 1000 confocal laser scanning microscope (UplanSApo 20×/0.75 objective) with FluoView FV1000 software. Quantification of the confocal images was performed with ImageJ software. The images were converted to a 3-slice RGB stack (red, green, and blue) and Li's Minimum Cross Entropy thresholding method was used to measure the surface percentage of gelsolin staining in the entire image.

Statistical analysis. For statistical analysis, two sided unpaired t-tests were performed using SPSS software. Data are represented as mean + SE. For quantification of gelsolin staining in nanobody injected mice, six cryosections were made for each mouse to reduce technical variance. The average of the six technical replicates was used to perform the t-test (*P < 0.05; **P < 0.01; ***P < 0.001).

SUPPLEMENTARY MATERIAL Figure S1. Establishing FAF Nb1-3 specificity. Figure S2. FAF nanobodies do not prevent gelatin degradation by MT1-MMP. Figure S3. MSA21 linked FAF Nb1-3 retain the ability to bind C68 in ELISA. Figure S4. Specificity of the anti-8 kDa antiserum. Figure S5. AST/ALT levels are not significantly altered in nanobody treated gelsolin amyloidosis mice. Figure S6. FAF Nb2-MSA21 injection has a benefical effect on contraction speed during fatigue in EDL and force-frequency relationship in soleus. Table S1. Isothermal titration calorimetry (ITC) parameters for (a) FAF Nb1-3 and (b) bispecific MSA21-FAF Nb1-3. Supplementary Materials and Methods.

Acknowledgments

We thank JW Kelly, LJ Page, and A Guerrero (Scripps Research Institute) for sharing the gelsolin amyloidosis mouse model, L Supply (Ghent University) for help with immunohistochemistry, B Vanloo (Ghent University) for initial ITC measurements, M Goethals (Ghent University) for peptide synthesis, D Tondeleir (Ghent University) for inital help with mice work, L Van Troys (Ghent University) for help with confocal microscopy, B Vanheel (Ghent University) for provinding lab space and Ablynx NV (Zwijnaarde) for sharing the MSA21 nanobody. This work was supported by the Foundation for Alzheimer Research (SAO-FRA), the G.S.K.E. (Geneeskundige Stichting Koningin Elisabeth), the Amyloidosis Foundation (USA) and the Interuniversity Attraction Poles Programme of the Belgian State, Federal Office for Scientific, Technical and Cultural Affairs (IUAP P7/13). W.V.O. and A.V. are supported by the Agency for Innovation by Science and Technology in Flanders (IWT-Vlaanderen). The authors declare no conflict of interest.

Supplementary Material

Supplementary Information
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