Conjugate of an IgG Binding Domain with Botulinum Neurotoxin A

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Conjugate of an IgG binding domain with botulinum neurotoxin A lacking the acceptor moiety targets its SNARE protease into TrkA-expressing cells when coupled to anti-TrkA IgG or Fc-#NGF Marc Nugent, Jiafu Wang, Gary W. Lawrence, Tomas Zuraswki, Joan A. Geoghegan, and J Oliver Dolly Bioconjugate Chem., Just Accepted Manuscript • Publication Date (Web): 10 May 2017 Downloaded from http://pubs.acs.org on May 11, 2017

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Conjugate of an IgG binding domain with botulinum neurotoxin A lacking the acceptor moiety targets its SNARE protease into TrkA-expressing cells when coupled to anti-TrkA IgG or FcβNGF Marc Nugent*, Jiafu Wang*, Gary Lawrence*, Tomas Zurawski*, Joan A. Geoghegan‡ and J. Oliver Dolly* *

International Centre for Neurotherapeutics, Dublin City University, Glasnevin, Dublin 9, and ‡ Department of

Microbiology, Moyne Institute of Preventive Medicine, School of Genetics and Microbiology, Trinity College Dublin, Dublin 2, Ireland.

Corresponding Author: J. Oliver Dolly email address: [email protected]

ABSTRACT Numerous naturally-occurring toxins can perturb biological systems when they invade susceptible cells. Coupling of pertinent targeting ligands to the active domains of such proteins provides a strategy for directing these to particular cellular populations implicated in disease. A novel approach described herein involved fusion of one mutated immunoglobulin G (IgG) binding moiety of staphylococcal protein A to the SNARE protease and translocation domain of botulinum neurotoxin A (BoNT/A). This chimera could be monovalently coupled to IgG or via its Fc region to recombinant targeting ligands. The utility of the resulting conjugates is demonstrated by the delivery of a SNARE protease into a cell line expressing tropomyosin receptor kinase A (TrkA) through coupling to anti-TrkA IgG or a fusion of Fc and nerve-growth factor. Thus, this is a versitile and innovative technology for conjugating toxins to diverse ligands for retargeted cell delivery of potential therapeutics.

INTRODUCTION An emerging therapeutic strategy for treating a range of debilitating and even life-threatening disorders involves conjugating toxins, devoid of their natural binding domains, to antibodies or recombinant ligands which selectively target defined cellular populations1, 2. The generation of such chimerae is largely achieved by sequential chemical or enzymatic linkage 1 ACS Paragon Plus Environment

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of the relevant components via multifarious protocols3, 4, or their recombinant expression as a single protein after genetic fusion5. These conjugation approaches exhibit their own respective benefits, and disadvantages such as efficiency6, solubility7, reproducibility8, applicability to diverse ligands and the biological activity of the products9. Therefore, an unmet need exists for a simplified and flexible protein-based method for coupling biotherapeutics to a broad range of putative targeting ligands. Moreover, achieving intracellular delivery would greatly extend their research and medical applications. One useful building block of such a vehicle is provided by the virulence factor protein A (SpA) of Staphylococcus aureus. It consists of 5 highly-modular immunoglobulin G (IgG) interactive domains (A-E)10 (Figure 1A), that bind to IgG with high efficiency and stability11. These binding domains consist of thermodynamically autonomous triple-helical bundles12, with each capable of binding to the IgG fragment crystallisable (Fc) and antigenbinding (Fab) regions of IgG, using separate sites (Figure 1B). A coupling strategy developed herein utilizes one copy of IgG-binding domain B of SpA (SpA-B) in which residues essential for binding IgG Fab (D36, D37) have been substituted with alanine (SpABmut)13, to preclude the potential hindrance of target binding. The fusion of SpA-Bmut to pertinent toxin derivatives should afford their selective monovalent coupling to IgG Fc without any chemical modification of components. Thus, this strategy supports cellular targeting of these conjugates through coupled IgG which can be raised against any cellsurface antigen. However, intracellular delivery of toxins’ enzymic moieties usually requires additional constituents that stimulate internalization of the membrane-bound complex. Hence, an ideal conjugation strategy should also be applicable to suitable recombinant ligands which provoke receptor uptake. Tagging of these proteins with a minimal portion of the Fc of rabbit IgG (rFc) and coupling via SpA-Bmut could fulfil these criteria. As a functionally important protein for targeting, tropomyosin kinase A (TrkA) was chosen because of being expressed on the surface of neurons and neuronal cell lines. TrkA signalling mediates neurotrophic effects during development, promoting neuronal survival and differentiation, which are indispensable for the maturation of peripheral sensory neurons14. However, upon reaching adulthood expression of TrkA is restricted to smalldiameter peptidergic C-fibre nociceptors15, where the principle function of TrkA signalling transitions from a role in neuronal survival to mediation of nociception16. As such, TrkA has emerged as a potential therapeutic target for treating pain conditions arising from nociceptor hyper-sensitivity17,

18

. Upon the IgG-like domain of this receptor binding IgG its

internalization is induced19; hopefully, this can result in the intracellular delivery of 2 ACS Paragon Plus Environment


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prospective therapeutics. Furthermore, TrkA serves as the high-affinity receptor for nervegrowth factor homodimer (βNGF)20, and its binding also results in internalization of the complex21. Botulinum neurotoxin type A (BoNT/A) has shown significant efficacy in treating an extensive number of clinical conditions resulting from neuronal over-activity including alleviation of pain22-24 and, thus, serves as an appropriate toxin candidate for targeting to TrkA-expressing cells. BoNT/A is a modular di-chain protein comprised of a protease lightchain (LC/A) that is attached by a disulfide and non-covalent bond to a heavy chain (HC). The latter has distinct domains responsible for neuronal surface binding (HC/A) and translocation to the cytosol (HN/A) of the attached protease, respectively. Delivery of LC/A into the neuronal cytoplasm results in cleavage of synaptosomal-associated protein with Mr of

25k (SNAP-25).

As

this

is

an

essential

soluble

N-ethylmaleimide-sensitive 2+

factor attachment protein receptor (SNARE) involved in Ca -dependent exocytosis of neurotransmitters25, its truncation prevents neuronal exocytosis. Advantageously, the LCHN/A domains are amenable to genetic manipulation26-28 and BoNT/A derivatives devoid of HC/A have been targeted into various cell types following conjugation to appropriate ligands29. Conveniently, the delivery of LC/A into susceptible cells can be detected by the production of cleaved SNAP-25 and, thus, is easy to assay. Herein, a novel chimeric fusion of LC.HN/A and SpA-Bmut (Figure 1C,D) was generated for evaluation of SpA-Bmut-mediated coupling of this core-therapeutic to either anti-TrkA IgG or rFc-βNGF. Both ligands were found to deliver the SNARE-cleaving protease into TrkA-expressing cells, unlike the nonliganded control protein.

RESULTS Design and cloning of LC.HN/A-SpA-Bmut. Site-directed mutagenesis of nucleotides (D36A, D37A), encoding SpA-B residues previously reported as essential for IgG Fab interaction13, created a gene encoding SpA-Bmut (Figure 1A,B). This was subsequently fused to the 3ꞌ end of LC.HN/A with an intervening short nucleotide sequence encoding a serineglycine flexible linker. The resultant chimeric gene was inserted into the pGEX-KG bacterial vector to encode the single-chain protein (GST-LC.HN/A-SpA-Bmut) depicted in Figure 1C. The N-terminal glutathione-S-transferase (GST) tag (Mr of 25k) enabled affinity purification on glutathione (GSH) agarose. The two thrombin protease cleavage sites inserted at the Ctermini of GST and LC/A, respectively, facilitated thrombin-mediated release of LC.HN/A-

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SpA-Bmut from GST (depicted in Figure 1D) and conversion of LC.HN/A to fully-active disulfide linked di-chain form.

Figure 1. Schematic of the generation of the LC.HN/A-SpA-Bmut. (A) Representation of the SpA protein, indicating the relative stability of 5 IgG binding domains, region X and the LPXTG sortase signal which are both responsible for attachment to the bacterial cell wall. (B) Amino acid sequence of SpA-B domain, residues involved in binding of Fc (Q9, 10) and IgG Fab (D36, 37) are highlighted in green and red, respectively. (C) LC.HN/A-SpA-Bmut DNA cloned into pGEX-KG expression vector, indicating the thrombin protease sites, and inter-chain disulfide bond. (D) Hypothetical structure of the resultant purified protein. [Adapted and combined from the crystal structure of BoNT/A30 and SpA-B31].

Recombinantly created LC.HN/A-SpA-Bmut was successfully expressed in E. coli. This entailed induction of transformed E. coli BL21 with isopropyl β-D-1-thiogalactopyranoside (IPTG). Analysis of both the soluble and insoluble fractions of the resultant bacterial lysate, by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), confirmed appreciable expression of soluble product with the predicted Mr ~135k for single-chain GSTLC.HN/A-SpA-Bmut (Figure 2A). Subsequent affinity-chromatography on GSH-agarose immobilized a significant quantity of the latter, although a residual amount could be detected in the flow-through and washes (Figure 2B). Incubation with thrombin protease released LC.HN/A-SpA-Bmut from the agarose-associated GST tag, yielding the predicted decrease in size of Mr ~25K corresponding to the removal of GST. Analysis of the separated eluate determined LC.HN/A-SpA-Bmut had been isolated to reasonable purity, although a number of contaminating bands were identified. The subsequent addition of reductant, confirmed that 4 ACS Paragon Plus Environment


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LC.HN/A-SpA-Bmut had been predominantly converted to the disulfide linked di-chain. A final yield of ~1mg/L of culture was determined by bicinchoninic acid protein assay (BCA) protein assay.

Figure 2. Recombinant expression and purification of tag-free di-chain LC.HN/A-SpA-Bmut. (A) The bacterial lysate containing recombinantly expressed GST-LC.HN/A-SpA-Bmut was fractionated by centrifugation and the supernatant subjected to SDS-PAGE followed by Coomassie staining, this revealed a band (numbered arrow) at the predicted molecular size of GST-LC.HN/A-SpA-Bmut, as specified below the image. (B) Samples from the affinity-chromatography were subjected to SDS-PAGE (+/- 2.5% reductant) followed by Coomassie staining. Cleavage with thrombin (+Thr) released LC.HN/A-SpA-Bmut from GST-tag, and largely achieved conversion to the disulfide linked di-chain. Numbered arrows indicate the predicted molecular sizes of the expected proteins

IgG efficiently couples to LC.HN/A-SpA-Bmut. Immobilization of GST-LC.HN/A-SpA-Bmut on GSH-agarose proved convenient for the subsequent coupling to IgG. The latter can be incubated in excess to saturate the SpA-Bmut binding sites, prior to removal by washing of both surplus unbound IgG and any contaminants present. Commercial rabbit IgG, containing bovine serum albumin (BSA) as stabilizer (included by the supplier), was incubated with GSH-agarose bound GST-LC.HN/A-SpA-Bmut at a ~3-fold molar excess. Ensuing analysis of the washes and the agarose resin by SDS-PAGE and Coomassie staining demonstrated a substantial amount of IgG remained coupled to GST-LC.HN/A-SpA-Bmut (Figure 3A); both proteins gave similar staining intensity when the gels were subjected to densitometric analysis. Specificity of this interaction was unveiled by the contrasting presence of BSA only in the washes. Parallel analysis of all the above noted samples by Western blotting, using an anti-rabbit IgG, substantiated the results from SDS-PAGE and confirmed the identity of the rabbit IgG (Figure 3B). Moreover, no disassociation of coupled IgG was observed following overnight incubation at 37°C in cell culture medium. Subsequent release of GST-LC.HN/ASpA-Bmut-IgG from the GSH-associated GST tag, by thrombin, was reflected by the reduced 5 ACS Paragon Plus Environment

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size on SDS-PAGE (Figure 3C). Analysis of the eluate by non-denaturing polyacrylamide gel electrophoresis (native-PAGE) confirmed the integrity of the coupled complex (Figure 3D). Furthermore, LC.HN/A-SpA-Bmut-IgG released from GSH was found to retain protease activity comparable to that of wild-type BoNT/A26, using an established cleavage assay and a SNAP-25 fragment substrate26 (Figure 3E,F). Thus, this was not impeded by either replacing HC/A with SpA-Bmut or the subsequent coupling to IgG.

Figure 3. Coupling of LC.HN/A-SpA-Bmut to IgG and confirmation of its protease activity. (A) GSH-agarose immobilized GST-LC.HN/A-SpA-Bmut was incubated with ~3-fold molar excess of rabbit IgG, followed by extensive washing with PBS. Coupling of IgG was demonstrated by SDS-PAGE followed by Coomassie staining of the washes and the resin. (B) Samples from (A) were subjected to Western blotting with HRPconjugated secondary antibodies to rabbit IgG (1:5,000). (C) GST-LC.HN/A-SpA-Bmut-IgG coupled complex was cleaved by thrombin, releasing LC.HN/A-SpA-Bmut-IgG from the GSH-agarose, shown by SDS-PAGE followed with Coomassie staining. (D) Native-PAGE followed by Coomassie staining demonstrated a shift in mobility of released LC.HN/A-SpA-Bmut due to binding of IgG. (E) Assay of SNAP-25 cleavage was performed with 1.25 – 20 nM of LC.HN/A-SpA-Bmut either uncoupled, or coupled to rabbit IgG. Cleaved and intact SNAP25 were separated by SDS-PAGE and visualized with Coomassie staining. (F) Densitometric analysis of data from (E) was performed to determine % cleavage. Data plotted are means ± s.e.m; from 3 independent experiments.

Coupling of LC.HN/A-SpA-Bmut to anti-TrkA IgG increases the binding and delivery of the SNARE protease into TrkA-expressing PC-12 cells. Having successfully conjugated IgG to LC.HN/A-SpA-Bmut, the next stage was to ascertain if attaching anti-TrkA IgG could deliver the SNARE protease into cells expressing TrkA. A commercial polyclonal IgG against TrkA was selected based on its antigenic epitope region (residues 342-356), coinciding with a receptor region to which antibody binding was known to induce crosslinking and consequent internalization19. PC-12 cells, a clonal pheochromocytoma line which express both TrkA and SNAP-2532, 33 were cultured and differentiated by treatment with 2.5S 6 ACS Paragon Plus Environment


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NGF, reaffirming the presence of TrkA. Binding of the anti-TrkA IgG to both differentiated and undifferentiated PC-12 cells was demonstrated by immuno-cytochemistry (Figure 4A), confirming the functionality of the IgG. Dot-blotting showed that LC.HN/A-SpA-Bmut does not bind proteins in PC-12 cell lysate unless conjugated to anti-TrkA IgG (Figure 4B). This elevated binding correlated with a substantial improvement in the cytosolic delivery of LC into treated PC-12 cells via targeted LC.HN/A-SpA-Bmut. Analysis by Western blotting demonstrated a dose-dependent increase in SNAP-25 cleavage (Figure 4C,D), in contrast to the non-IgG targeted control.

Figure 4. Anti-TrkA IgG coupled to LC.HN/A-SpA-Bmut delivers the protease of BoNT/A into PC-12 cells. (A) Undifferentiated and differentiated PC-12 cells were fixed and incubated with anti-TrkA IgG (1:50). Cells were imaged by bright-field microscopy and binding of anti-TrkA IgG was detected with a goat anti-rabbit secondary (Alexa Fluor 488, 1:500) and visualized by fluorescence microscopy. (B) PC-12 cell lysate dot blotted onto nitrocellulose membrane was incubated with LC.HN/A-SpA-Bmut, with or without coupled anti-TrkA IgG. The level of bound protein was determined using an anti-LC/A antibody (1: 1,000) followed by a second antibody conjugated to HRP for detection of signal. Densitometric analysis was performed with ImageJ software; data plotted are means ± s.e.m; statistical analysis by unpaired t-test: *** p < 0.001, from 3 independent experiments. (C) PC-12 cells were treated with the indicated concentration of LC.HN/A-SpA-Bmut with and without coupled anti-TrkA IgG for 48 hours prior to lysis. Western-blotting was performed with antibodies against α-tubulin (1:3,000) and SNAP-25A (1: 3,000). (D) Densitometric analysis of data from (C), normalized against the α-tubulin loading control. Data plotted are means ± s.e.m; statistical analysis performed using unpaired t-test: * p < 0.05, ** p < 0.01, *** p < 0.001, from 3 independent experiments.


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Design, expression and purification of biologically-active rFC-βNGF-(His)6. The versatility of the SpA-Bmut coupling was assessed from its ability to deliver the SNARE protease into TrkA-expressing cells by targeting mediated via IgG or a recombinant ligand. For the latter, a novel fusion of βNGF tagged with a fragment of rFc was designed. NGF is expressed as an immature monomeric precursor containing an N-terminal pre-pro peptide, which directs the nascent protein into organelles of the secretory pathway and orchestrates extensive post-translational modifications34. As recombinant expression of functional NGF is difficult to achieve in bacterial systems35, but baculovirus mediated expression has been shown to produce functional βNGF in insect cells36, a baculovirus was generated for recombinant expression of rFc-βNGF-(His)6 in Sf9 insect cells (Figure 5A). The C-terminal (His)6 tag was incorporated to facilitate purification by immobilized metal affinity chromatography (IMAC). As the expressed fusion protein is expected to be secreted, the cell supernatant from Sf9 insect cells infected with the aforementioned baculovirus was subjected to IMAC, with both the Sf9 supernatant and IMAC elution fractions analysed by Western blotting using an antibody against (His)6. The latter visualized a protein at the predicted size for the rFc-βNGF-(His)6 homodimer (Mr ~80 k), in both the supernatant prior to IMAC, and the eluate from IMAC (Figure 5B). Due to the low purification yield the product was visualized by immuno-blotting. The functional activity of purified rFc-βNGF-(His)6 was measured by an established PC-12 survival assay37, which uses a cell-permeable fluorescent metabolites producing compound (AlamarBLUE™) as a means of determining cell viability. This revealed that 10 µl of the purified rFc-βNGF-(His)6 sample exhibited functional activity comparable to 10 ng/ml of commercial 2.5S NGF (Figure 5C). Further neurotrophic activity of rFc-βNGF(His)6 was reflected by its ability to induce differentiation and neurite outgrowth in PC-12 cells, consistent with its expected direct interaction with TrkA (Figure 5D).



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Figure 5. Production of biologically-active rFc-βNGF-(His)6. (A) Illustration of the rFc-NGF recombinant fusion protein. Pre-ProNGF-rFc-(His)6, when expressed in Sf9 insect cells is undergoes intracellular processing, entering the secretion pathway, removing the pre-pro signal and resulting in secretion of homo-dimerized rFcβNGF into the Sf9 supernatant. A C-terminal (His)6 enables IMAC purification, with later removal of the tag by thrombin cleavage. (B) Western blotting of aliquots from the IMAC purification of rFc-βNGF-(His)6 was carried out using anti-(His)6 antibody (1 µg/ml). Arrow indicates predicted size of rFc-βNGF-(His)6. (C) PC-12 cells were incubated with indicated dose of either purified rFc-βNGF-(His)6 or 2.5S NGF in a final volume of 500 µl for 48 hours at 37°C, 5% CO2. AlamarBLUE™ (50 µl) was added to each well, incubated for 24 hours and fluorescence measured using Tecan Safire II plate reader (excitation 570 nm, emission 585 nm). Data plotted are means ± s.e.m, n=3. (D) PC-12 cells were treated with either 100 ng/ml 2.5S NGF or 10 µl of purified rFc-βNGF-(His)6 (protein concentration could not be accurately determined by standard protein assays) in a final volume of 1 ml for 96 hours at 37°C, 5% C02, prior to image acquisition.

Coupling of rFC-βNGF to LC.HN/A-SpA-Bmut greatly increases delivery of the SNAP25 protease into PC-12 cells. IMAC purified rFc-βNGF-(His)6 coupled successfully following incubation with GSH immobilized GST-LC.HN/A-SpA-Bmut, and the resultant complex (LC.HN/A-SpA-Bmut-rFc-βNGF) was released from the GSH-agarose by thrombin cleavage, as determined by Western blotting with an anti-βNGF antibody (Figure 6A). GSTLC.HN/A-SpA-Bmut immobilized on GSH agarose, and the eluted protein without GST, could be detected along with rFc-βNGF due to binding of the SpA-Bmut domain to secondary antibodies raised in rabbit38. Strikingly, treatment of PC-12 cells with LC.HN/A-SpA-Bmut-rFc-βNGF produced significantly greater SNAP-25 cleavage than equivalent concentrations of uncoupled control (Fig. 6B,C). Furthermore, this revealed comparable levels of cleaved SNAP-25 as attained with anti-TrkA IgG (cf. Figure 4D). When consideration is made for the proportion of 9 ACS Paragon Plus Environment

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LC.HN/A-SpA-Bmut coupled to IgG (cf. Figure 3C), relative to that coupled to rFc-βNGF (cf. Figure 6A), it can be surmised that rFc-βNGF is considerably more efficient at delivering the SNAP-25 protease into PC-12 cells.

Figure 6. rFc-βNGF coupled to LC.HN/A-SpA-Bmut is delivered into PC-12 cells as reflected by SNAP-25 cleavage. (A) IMAC purified rFc-βNGF-(His)6 was coupled to GSH immobilized GST-LC.HN/A-SpA-Bmut, washed extensively and released from the GSH-agarose as described in Figure 3C. Coupling was analysed by Western blotting using anti-βNGF antibody (1:200). (B) Comparison of SNAP-25 cleavage levels in PC-12 cells treated with LC.HN/A-SpA-Bmut, with or without coupling to rFc-βNGF. Experiment was performed as in Figure 4C, with indicated concentrations of LC.HN/A-SpA-Bmut, with or without coupling to rFc-βNGF. (C) Densitometric analysis of data from (B). Data plotted are means ± s.e.m, normalized against α-tubulin as a loading control. Statistical analysis carried out by unpaired t-test: * p < 0.05, ** p < 0.01, *** p < 0.001, from 3 independent experiments.

DISCUSSION The development of targeted therapeutics that can exploit the exquisite potency and selectivity of enzymes in naturally-occurring toxins is often constrained by the availability and suitability of relevant targeting moieties. Consequently, this frequently necessitates extensive screening and optimization of ligands39; furthermore, their identification, 10 ACS Paragon Plus Environment


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expression and conjugation is often a rate-limiting step40. Our SpA-Bmut strategy overcomes some of these hurdles by independent expression of core-therapeutic proteins (typified by LC.HN/A-SpA-Bmut) which can be rapidly and efficiently coupled to commercial IgG or rFcfusions. Previous attempts to exploit the avidity of bacterial proteins for IgG have shown their potential for conjugating diphtheria toxin and pseudomonas exotoxin A to IgG41,

42

.

Nevertheless, efficient conjugation could not be achieved, or required chemical crosslinking to stabilize the resultant complex. Furthermore, binding to IgG Fab has not been addressed, and multiple IgG binding domains were employed, thereby, running the risk of steric hindrance between coupled IgG. Thus, SpA-Bmut provides a novel, effective platform in which IgG can be coupled without a requirement for chemical conjugation or cross-linking. Moreover, IgG-mediated cellular internalization of the well-characterised protease from BoNT/A was achieved, as demonstrated by Western blotting of cleaved SNAP-25. Previous evaluation of such whole antibody-mediated targeting of toxin moieties have involved microscopy43, radiolabelling44, thymidine incorporation41 or cell viability assays42. Therefore, LC.HN/A-SpA-Bmut represents an advance over preceding techniques in terms of simplicity and reproducibility of IgG coupling, no compromise of cell-viability and a straightforward detection method. Likewise, this strategy permits more expeditious screening of

recombinantly-expressed

ligands,

allowing

their

functional

optimization

and

characterization prior to conjugation and assessment of targeting. However, generation of such suitable ligands can be time-consuming and challenging. As exemplified by rFc-βNGF(His)6, obtaining adequate expression of functional proteins which contain disulphide bonds or require extensive post-translational modification can prove difficult45, 46. In this regard, the SpA-Bmut conjugation affords a unique advantage whereby the initial utilization of appropriate IgG against a candidate receptor can be rapidly examined, and its suitability as a therapeutic target established before justifying the investment in recombinant ligand development. Hence, it is preferable to employ this unified approach which enables screening and assessment of IgG and ligand targeting, as well as the actual receptor targets themselves. Significantly, LC.HN/A-SpA-Bmut coupled to rFc-βNGF delivered protease into PC12 cells with efficiency comparable to considerably higher concentrations of coupled antiTrkA IgG. As targeted therapeutics exhibit different internalization pathways, dependent upon their targeting ligand5, 47, it can be assumed that the substantial biological activity of rFc-βNGF makes up for the low quantity of coupled complex. This highlights that high11 ACS Paragon Plus Environment

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affinity agonistic ligands may serve as more efficient delivery agents than their IgG counterparts. PC-12 cells were treated with up to 1.2 nM of LC.HN/A-SpA-Bmut, of which only a small proportion was coupled to rFc-βNGF, likely corresponding to low pM concentrations. In spite of this, the considerable SNAP-25 cleavage observed demonstrates the significant targeting efficiency of rFc-βNGF. The development of retargeted BoNT therapeutics (commonly termed targetedsecretion inhibitors), to expand the toxins clinical utility, is the focus of intense academic and commercial investigation [reviewed by

40, 48

]. Chronic pain, a disorder which involves the

sensitization of sub-populations of sensory neurons responsible for the perception of noxious stimuli, represents one such condition for which the development of novel therapeutics is an economic and social imperative49. Notably, peripheral expression of TrkA is restricted to such neurons15, where it influences the surface expression and activation thresholds of receptors and channels concerned with pain sensation16,

50

. Their increased membrane

insertion during chronic sensitization is mediated by Ca2+-dependent SNARE complex formation51, which is also required for the stimulated exocytosis of pain-signalling peptides52. Consequently, inhibition of such mechanisms by a retargeted BoNT-derived protease has considerable potential as an innovative therapeutic for chronic pain. To this end, the demonstrated ability of rFC-βNGF to target LC.HN/A-SpA-Bmut into PC-12 cells indicates its potential for delivering this SNARE inactivating enzyme into TrkA-expressing cells. However, further work is required to generate sufficient quantities of material for a comprehensive assessment of its targeting efficiency in vitro and, potentially, of its antinociceptive efficacy in vivo.

EXPERIMENTAL PROCEDURES Materials. Baculodirect™ insect cell expression system, Sf-900II™ medium and Bolt® SDS-PAGE gels were bought from Biosciences (Dublin, Ireland), a distributor of Life Technologies. Restriction enzymes were purchased from Brennan and Company (Dublin, Ireland), a distributor for New England Biolabs. Oligonucleotides for polymerase chain reaction manipulation of DNA were ordered from Eurofins MWG Operon (Ebersberg, Germany). Anti-TrkA IgG was purchased from Alomone Labs (Jerusalem, Israel) and primary antibodies used for Western blotting were from Sigma Aldrich (Wicklow, Ireland) or Abcam (Cambridge, UK). Secondary antibodies for Western blotting and immunocytochemistry were supplied by Jackson ImmunoResearch (Suffolk, UK) and Life



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Technologies, respectively. All other reagents and chemicals were purchased from Sigma Aldrich. Cloning of pGEX-KG-LC.HN/A-SpA-Bmut. cDNA encoding only residues 1-872 of BoNT/A, encompassing LC/A and HN/A with an intervening thrombin cleavage site 27, was inserted between the BamHI and XbaI restriction sites of the pGEX-KG vector. Nucleotides encoding the two aspartic acids at position 36 and 37 of SpA-B cDNA were mutated to alanine by site-directed mutagenesis using the Quikchange™ method to generate SpA-Bmut. A 5’ linker sequence encoding 10 alternating glycines/serines was inserted at the 5’ end of SpA-Bmut by polymerase chain reaction; the resultant cDNA was inserted between XbaI and XhoI sites of pGEX-KG already containing LC.HN/A, to generate pGEX-KG-LC.HN/A-SpABmut. At all stages the fidelity of the cDNA was confirmed by DNA sequencing. Cloning was performed using E. coli TOP-10™ cells; prior to transformation, these cells were made chemically competent using a previously published protocol53. Expression and purification of GST- LC.HN/A-SpA-Bmut. The pGEX-KGLC.HN/A-SpA-Bmut vector was transformed into BL21 (DE3) E. coli expression strain and grown in Luria broth at 37°C until an Abs600

nm

0.6 - 0.8 was reached. Expression was

induced by adding 0.1 mM IPTG, lowering of the temperature to 22°C and incubating overnight. The cells were centrifuged at 5,000 x g for 30 minutes, the pellet resuspended in “bacteria lysis buffer” (150 mM NaCl, 20 mM HEPES, pH 8, containing lysozyme at a final concentration of 2 mg/ml) and shaken at room temperature for 40 minutes followed by three freeze-thaw cycles. The resulant clarified lysate was subjected to affinity chromatography using GSH-agarose. The pGEX-KG vector incorporates a thrombin cleavage site between the GST affinity tag and the recombinant protein, enabling release by the protease of LC.HN/A-SpA-Bmut from the GSH-agarose. IgG coupling reaction. Typically, GST-LC.HN/A-SpA-Bmut (10 µg) immobilized on 10 µl of GSH-agarose was incubated with ~3-fold molar excess of either rabbit anti-goat IgG or rabbit anti-TrkA IgG, with agitation for 1 hour at 4°C. The agarose was washed extensively with PBS (137 mM NaCl, 10 mM phosphate, 2.7 mM KCl, pH 8.3) to remove excess IgG. The protein complexes were released from GSH-agarose by incubation with thrombin (1 unit/mg of protein) for 40 minutes at room temperature, before the eluate was separated on filtration spin columns. Samples were then run on either 10% SDS-PAGE or 10% native-PAGE, prior to Coomassie staining. Assay of SNAP-25 cleavage in vitro. Proteolytic activity of LC.HN/A-SpA-Bmut with and without coupling to IgG, was assessed using a SNAP-25 cleavage assay in vitro26. 13 ACS Paragon Plus Environment

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Concentrations of protein samples were determined by BCA protein assay, and diluted appropriately into the assay buffer (20 mM HEPES, 100 mM NaCl, 10 µM ZnCl2, 5 mM dithiothreitol, 10 µg/ml BSA, pH 7.4). GFP-SNAP-25(134-206)26 was incubated with the indicated toxin concentrations at 37°C for 30 minutes, before resolving samples on 12% SDS-PAGE, Coomassie staining and densitometric analysis with ImageJ software. Immuno-cytochemistry. PC-12 cells were seeded at 3x104 per well in a collagencoated 12-well plate either undifferentiated, or differentiated for 96 hours with 100 ng/ml 2.5S NGF. Samples were fixed with ice-cold methanol, blocked with 10% goat serum in PBS and incubated with anti-TrkA IgG (1:50) overnight at 4°C. Binding of TrkA IgG was visualized by labelling with Alexa Fluor 488-conjugated anti-rabbit secondary (1:500), and imaged using a fluorescence microscope (Olympus, IX71). Binding of PC-12 lysate by LC.HN/A-SpA-Bmut coupled to TrkA IgG. Lysate (2 µg) was blotted onto nitrocellulose membrane and blocked with 5% BSA to prevent nonspecific binding of other proteins to the membrane, before applying 5 ng of LC.HN/A-SpABmut with and without coupled TrkA IgG, and incubating for 10 minutes at room temperature. The membrane was washed extensively to remove unbound protein and dot-blot analysis performed with an anti-LC/A antibody (1: 1,000), followed by a secondary antibody conjugated to horseradish peroxidase for 1 hour at room temperature. Chemiluminescent signals were imaged using G BOX CHEMI HR-16. Analysis of SNAP-25 cleavage in PC-12 cells. After seeding at 2x105 per well in a collagen-coated 24-well plate, the cells were incubated for 48 hours at 37°C, 5% C02, washed three times with chilled PBS, lysed in 100 µl ice-cold SDS sample-buffer, heated to 95°C for 5 minutes and run on 12% precast Bolt® SDS-PAGE gels. Proteins were transferred onto PVDF membrane. Incubation with anti-α-tubulin (1:3,000) and anti-SNAP-25A (1: 3,000) was performed overnight at 4°C. This was followed by exposure to either anti-mouse or rabbit secondary antibodies conjugated to horseradish peroxidase for 1 hour at room temperature. Chemiluminescent signals were imaged using G BOX CHEMI HR-16. Generation of baculovirus for recombinant expression of rFc-NGF-(His)6 in insect cells. DNA encoding full-length human pre-pro-NGF was designed with a direct 3’ fusion of nucleotides encoding residues 102-324 of rabbit Ig gamma chain C region, with a subsequent 3’ sequence encoding a thrombin cleavage site and a polyhistidine purification tag. The resultant gene (Pre-pro-NGF-rFc-thrombin-His6) was synthesised with codon optimization for insect cell expression. Recombinant baculovirus for expressing rFc-βNGF-



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(His)6 was generated using the Baculodirect™ system according to the manufacturer’s protocol. Expression, purification and coupling of rFc-βNGF-(His)6. Suspension cultures of Sf9 insect cells were maintained at 27.5°C, with shaking at 130 revolutions per minute in Sf900II™ medium. Conditions for recombinant expression of rFc-βNGF-(His)6 were extensively screened; the optimal protocol involved seeding Sf9 cells in log-phase growth at 2.5 x 106/ml in a total of 500 ml. Cells were infected with low-passage recombinant baculovirus at a multiplicity of infection of 0.05, and incubated for 96 hours prior to harvesting. rFc-βNGF-(His)6 was purified by IMAC from Sf9 supernatant, using an ÄKTAexplorer FPLC with a 1 ml HisTrap Excel™ column. Protein was eluted with 500 mM imidazole, 100 mM sodium phosphate, pH 7.4 in 1 ml fractions and subsequently precipitated by ammonium sulfate (80% saturation) to further concentrate the sample. The resultant protein pellet was then reconstituted into 200 µl of protein storage buffer (100 mM NaCl, 20 mM HEPES, pH 7.4). Coupling of rFc-βNGF-(His)6 to GST-LC.HN/A-SpA-Bmut was performed as described for IgG, with the exception that 10 µg of GSH-agarose immobilized GST-LC.HN/A-SpA-Bmut was incubated with 200 µl of purified rFc-βNGF(His)6. Assay of the survival of PC-12 cells. This was performed as previously described 37; briefly, PC-12 cells were seeded at 4x104 per well in a 24-well plate in serum-free medium, and incubated with the indicated quantity of either 2.5S NGF or purified rFc-βNGF-(His)6 for 48 hours at 37°C, 5% C02. AlamarBLUE™, an indicator of cellular metabolism, was added to each well and incubated for a further 24 hours; fluorescence was measured using a Tecan Safire II plate reader (excitation 570 nm, emission 585 nm).

ABBREVIATIONS USED BoNT/A, Botulinum neurotoxin serotype A; BCA, bicinchoninic acid; BSA, bovine serum albumin; Fab, fragment antigen-binding; Fc, fragment crystallizable; GSH, glutathione; GST, glutathione-S-transferase; (His)6, polyhistidine purification tag; IgG, immunoglobulin G; IMAC, immobilized metal affinity chromatography; native-PAGE, non-denaturing polyacrylamide gel electrophoresis; βNGF, nerve-growth factor homodimer; PBS, phosphate buffered saline; SpA, Staphylococcus aureus Protein A; rFC, rabbit immunoglobulin G Fc domain; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; s.e.m, standard error of the mean; SNAP-25, synaptosomal-associated protein of Mr 25k; SNARE, 15 ACS Paragon Plus Environment

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soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TrkA, tropomyosinrelated kinase A.

AUTHOR INFORMATION Corresponding Author J.O.D. email: [email protected] Author contributions MN devised the study, performed the experiments, prepared the figures and drafted the manuscript; JG provided reagents and expert advice; MN, JW, TZ, JG and JOD reviewed the manuscript; GL, JW and JOD supervised the study. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the Irish Research Council and an IvP award (to JOD) from Science Foundation Ireland.

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Conjugate of an IgG Binding Domain with Botulinum Neurotoxin A

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