Adriaan Verhelle

An ER-directed gelsolin nanobody targets the first step in amyloid formation in a gelsolin amyloidosis mouse model

Hereditary gelsolin amyloidosis is an autosomal dominantly inherited amyloid disorder. A point mutation in the GSN gene (G654A being the most common one) results in disturbed calcium binding by the second gelsolin domain (G2). As a result, the folding of G2 is hampered, rendering the mutant plasma gelsolin susceptible to a proteolytic cascade. Consecutive cleavage by furin and MT1-MMP-like proteases generates 8 and 5 kDa amyloidogenic peptides that cause neurological, ophthalmological and dermatological findings. To this day, no specific treatment is available to counter the pathogenesis. Using GSN nanobody 11 as a molecular chaperone, we aimed to protect mutant plasma gelsolin from furin proteolysis in the trans-Golgi network. We report a transgenic, GSN nanobody 11 secreting mouse that was used for crossbreeding with gelsolin amyloidosis mice. Insertion of the therapeutic nanobody gene into the gelsolin amyloidosis mouse genome resulted in improved muscle contractility. X-ray crystal structure determination of the gelsolin G2:Nb11 complex revealed that Nb11 does not directly block the furin cleavage site. We conclude that nanobodies can be used to shield substrates from aberrant proteolysis and this approach might establish a novel therapeutic strategy in amyloid diseases.

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

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.

A nanobody modulates the p53 transcriptional program without perturbing its functional architecture

The p53 transcription factor plays an important role in genome integrity. To perform this task, p53 regulates the transcription of genes promoting various cellular outcomes including cell cycle arrest, apoptosis or senescence. The precise regulation of this activity remains elusive as numerous mechanisms, e.g. posttranslational modifications of p53 and (non-)covalent p53 binding partners, influence the p53 transcriptional program. We developed a novel, non-invasive tool to manipulate endogenous p53. Nanobodies (Nb), raised against the DNA-binding domain of p53, allow us to distinctively target both wild type and mutant p53 with great specificity. Nb3 preferentially binds ‘structural’ mutant p53, i.e. R175H and R282W, while a second but distinct nanobody, Nb139, binds both mutant and wild type p53. The co-crystal structure of the p53 DNA-binding domain in complex with Nb139 (1.9 Å resolution) reveals that Nb139 binds opposite the DNA-binding surface. Furthermore, we demonstrate that Nb139 does not disturb the functional architecture of the p53 DNA-binding domain using conformation-specific p53 antibody immunoprecipitations, glutaraldehyde crosslinking assays and chromatin immunoprecipitation. Functionally, the binding of Nb139 to p53 allows us to perturb the transactivation of p53 target genes. We propose that reduced recruitment of transcriptional co-activators or modulation of selected post-transcriptional modifications account for these observations.

A new survivin tracer tracks, delocalizes and captures endogenous survivin at different subcellular locations and in distinct organelles

Survivin, the smallest member of the inhibitor of apoptosis protein family, plays a central role during mitosis and exerts a cytoprotective function. Survivin is highly expressed in most cancer types and contributes to multiple facets of carcinogenesis. The molecular mechanisms underlying its highly diverse functions need to be extensively explored, which is crucial for rational design of future personalized therapeutics. In this study, we have generated an alpaca survivin nanobody (SVVNb8) that binds with low nanomolar affinity to its target. When expressed as an intrabody in HeLa cells, SVVNb8 faithfully tracks survivin during different phases of mitosis without interfering with survivin function. Furthermore, coupling SVVNb8 with a subcellular delocalization tag efficiently redirects endogenous survivin towards the nucleus, the cytoplasm, peroxisomes and even to the intermembrane space of mitochondria where it presumably interacts with resident mitochondrial survivin. Based on our findings, we believe that SVVNb8 is an excellent instrument to further elucidate survivin biology and topography, and can serve as a model system to investigate mitochondrial and peroxisomal (survivin) protein import.

AAV9 delivered bispecific nanobody attenuates amyloid burden in the gelsolin amyloidosis mouse model

Gelsolin amyloidosis is a dominantly inherited, incurable type of amyloidosis. A single point mutation in the gelsolin gene (G654A is most common) results in the loss of a Ca2+  binding site in the second gelsolin domain. Consequently, this domain partly unfolds and exposes an otherwise buried furin cleavage site at the surface. During secretion of mutant plasma gelsolin consecutive cleavage by furin and MT1-MMP results in the production of 8 and 5 kDa amyloidogenic peptides. Nanobodies that are able to (partly) inhibit furin or MT1-MMP proteolysis have previously been reported. In this study, the nanobodies have been combined into a single bispecific format able to simultaneously shield mutant plasma gelsolin from intracellular furin and extracellular MT1-MMP activity. We report the successful in vivo expression of this bispecific nanobody following adeno-associated virus serotype 9 gene therapy in gelsolin amyloidosis mice. Using SPECT/CT and immunohistochemistry, a reduction in gelsolin amyloid burden was detected which translated into improved muscle contractile properties. We conclude that a nanobody-based gene therapy using adeno-associated viruses shows great potential as a novel strategy in gelsolin amyloidosis and potentially other amyloid diseases.

VCA nanobodies target N-WASp to reduce invadopodium formation and functioning

Invasive cancer cells develop small actin-based protrusions called invadopodia, which perform a primordial role in metastasis and extracellular matrix remodelling. Neural Wiskott-Aldrich syndrome protein (N-WASp) is a scaffold protein which can directly bind to actin monomers and Arp2/3 and is a crucial player in the formation of an invadopodium precursor. Expression modulation has pointed to an important role for N-WASp in invadopodium formation but the role of its C-terminal VCA domain in this process remains unknown. In this study, we generated alpaca nanobodies against the N-WASp VCA domain and investigated if these nanobodies affect invadopodium formation. By using this approach, we were able to study functions of a selected functional/structural N-WASp protein domain in living cells, without requiring overexpression, dominant negative mutants or siRNAs which target the gene, and hence the entire protein. When expressed as intrabodies, the VCA nanobodies significantly reduced invadopodium formation in both MDA-MB-231 breast cancer and HNSCC61 head and neck squamous cancer cells. Furthermore, expression of distinct VCA Nbs (VCA Nb7 and VCA Nb14) in PC-3 prostate cancer cells resulted in reduced overall matrix degradation without affecting MMP9 secretion/activation or MT1-MMP localisation at invadopodial membranes. From these results, we conclude that we have generated nanobodies targeting N-WASp which reduce invadopodium formation and functioning, most likely via regulation of N-WASp—Arp2/3 complex interaction, indicating that this region of N-WASp plays an important role in these processes.

Intracellular displacement of p53 using transactivation domain (p53 TAD) specific nanobodies

ABSTRACT The tumor suppressor p53 is of crucial importance in the prevention of cellular transformation. In the presence of cellular stress signals, the negative feedback loop between p53 and Mdm2, its main negative regulator, is disrupted, which results in the activation and stabilization of p53. Via a complex interplay between both transcription-dependent and – independent functions of p53, the cell will go through transient cell cycle arrest, cellular senescence or apoptosis. However, it remains difficult to completely fathom the mechanisms behind p53 regulation and its responses, considering the presence of multiple layers involved in fine-tuning them. In order to take the next step forward, novel research tools are urgently needed. We have developed single-domain antibodies, also known as nanobodies, that specifically bind with the N-terminal transactivation domain of wild type p53, but that leave the function of p53 as a transcriptional transactivator intact. When the nanobodies are equipped with a mitochondrial-outer-membrane (MOM)-tag, we can capture p53 at the mitochondria. This nanobody-induced mitochondrial delocalization of p53 is, in specific cases, associated with a decrease in cell viability and with morphological changes in the mitochondria. These findings underpin the potential of nanobodies as bona fide research tools to explore protein function and to unravel their biochemical pathways.

Nanobodies targeting cortactin proline rich, helical and actin binding regions downregulate invadopodium formation and matrix degradation in SCC-61 cancer cells

Cortactin is a multidomain actin binding protein that activates Arp2/3 mediated branched actin polymerization. This is essential for the formation of protrusive structures during cancer cell invasion. Invadopodia are cancer cell-specific membrane protrusions, specialized at extracellular matrix degradation and essential for invasion and tumor metastasis. Given the unequivocal role of cortactin at every stage of invadopodium formation, it is considered an invadopodium marker and potential drug target. We used cortactin nanobodies to examine the role of cortactin domain-specific function at endogenous protein level. Two cortactin nanobodies target the central region of cortactin with high specificity. One nanobody interacts with the actin binding repeats whereas the other targets the proline rich region and was found to reduce EGF-induced cortactin phosphorylation. After intracellular expression as an intrabody, they are both capable of tracing their target in the complex environment of the cytoplasm, and disturb cortactin functions during invadopodia formation and extracellular matrix degradation. These data illustrate the use of nanobodies as a research tool to dissect the role of cortactin in cancer cell motility. This information can contribute to the development of novel therapeutics for tumor cell migration and metastasis.

A Nanobody‐Based Approach to Amyloid Diseases, the Gelsolin Case Study

Gelsolin amyloidosis (AGel) is an autosomal‐dominant inherited disease caused by point mutations in the gelsolin gene. At the protein level, these mutations result in the loss of a Ca2+‐binding site, crucial for the correct folding and function. In the trans‐Golgi network, this mutant plasma gelsolin is cleaved by furin, giving rise to a 68 kDa Cterminal fragment. When secreted in the extracellular matrix, this fragment under‐ goes proteolysis by MT1‐MMP–like proteases, resulting in the production of 8 and 5 kDa amyloidogenic peptides. Nanobodies, the variable part of the heavy chain of heavy‐ chain antibodies, have been used as molecular chaperones for mutant plasma gelsolin and the 68 kDa C‐terminal fragment in an attempt to inhibit their pathogenic proteoly‐ sis. Furthermore, these nanobodies have also been tested and applied as a 99mTc‐based imaging agent in the gelsolin amyloidosis mouse model.