Antibody targeting of Cathepsin S induces antibody-dependent cellular cytotoxicity

<p>Abstract</p> <p>Background</p> <p>Proteolytic enzymes have been implicated in driving tumor progression by means of their cancer cell microenvironment activity where they promote proliferation, differentiation, apoptosis, migration, and invasion. Therapeutic strategies have focused on attenuating their activity using small molecule inhibitors, but the association of proteases with the cell surface during cancer progression opens up the possibility of targeting these using antibody dependent cellular cytotoxicity (ADCC). Cathepsin S is a lysosomal cysteine protease that promotes the growth and invasion of tumour and endothelial cells during cancer progression. Our analysis of colorectal cancer patient biopsies shows that cathepsin S associates with the cell membrane indicating a potential for ADCC targeting.</p> <p>Results</p> <p>Here we report the cell surface characterization of cathepsin S and the development of a humanized antibody (Fsn0503h) with immune effector function and a stable <it>in vivo </it>half-life of 274 hours. Cathepsin S is expressed on the surface of tumor cells representative of colorectal and pancreatic cancer (23%-79% positive expression). Furthermore the binding of Fsn0503h to surface associated cathepsin S results in natural killer (NK) cell targeted tumor killing. In a colorectal cancer model Fsn0503h elicits a 22% cytotoxic effect.</p> <p>Conclusions</p> <p>This data highlights the potential to target cell surface associated enzymes, such as cathepsin S, as therapeutic targets using antibodies capable of elicitingADCC in tumor cells.</p

IMAC purification of His6-HwTx-IV(acid).

<p>A) Slow gradient elution IMAC. Lane M, Seeblue pre-stained molecular weight marker (Invitrogen); Lanes 1 to 26, fractions collected during imidazole gradient elution. Elution of monomers starts at 60mM imidazole. At 105mM imidazole, only monomers elute. B) Capture IMAC with step elution at 105mM Imidazole. Lane M, Seeblue pre-stained molecular weight marker (Invitrogen); Lanes 1 to 12, fractions collected during the 105mM imidazole elution; Lanes 13 and 14, fractions collected during the second elution step at 500mM imidazole. </p

Cleavage and purification of HwTx-IV(acid).

<p>A) SDS-PAGE analysis of the cleavage reaction and depletion IMAC. Lane 1, Combined capture IMAC fractions; lane 2, Concentrated IMAC fractions in cleavage buffer; lane 3, Concentrated IMAC fractions after overnight SUMO protease cleavage; lane M, Seeblue protein MW marker; lanes 4 to 13, Depletion IMAC fractions. B) Polishing by preparative RP-HPLC. Chromatogram of the RP-HPLC polishing step showing that one main peak is observed. The corresponding fractions were combined and lyophilised.</p

Representation of HwTx-IV.

<p>A) Amino acid sequence of native HwTx-IV (C-terminal amide) with the correct disulfide pairing. B) Structure of HwTx-IV (1mb6). The three disulfide bonds are shown in yellow. C-D) Interpolated charge solvent surfaces of HwTx-IV(amide) (C) and HwTx-IV(acid) (D) shows an important charge difference in the C-terminal region (Discovery Studio 3.5, Accelrys). </p

Recombinant Expression and<i> In Vitro</i> Characterisation of Active Huwentoxin-IV

<div><p>Huwentoxin-IV (HwTx-IV) is a 35-residue neurotoxin peptide with potential application as a novel analgesic. It is a member of the inhibitory cystine knot (ICK) peptide family, characterised by a compact globular structure maintained by three intramolecular disulfide bonds. Here we describe a novel strategy for producing non-tagged, fully folded ICK-toxin in a bacterial system. HwTx-IV was expressed as a cleavable fusion to small ubiquitin-related modifier (SUMO) in the cytoplasm of the SHuffle T7 Express <i>lysY Escherichia coli</i> strain, which allows cytosolic disulfide bond formation. Purification by IMAC with selective elution of monomeric SUMO fusion followed by proteolytic cleavage and polishing chromatographic steps yielded pure homogeneous toxin. Recombinant HwTx-IV is produced with a C-terminal acid, whereas the native peptide is C-terminally amidated. HwTx-IV(acid) inhibited Na_v1.7 in a dose dependent manner (IC₅₀ = 463-727 nM). In comparison to HwTx-IV(amide) (IC₅₀ = 11 ± 3 nM), the carboxylate was ~50 fold less potent on Na_v1.7, which highlights the impact of the C-terminus. As the amide bond of an additional amino acid may mimic the carboxamide, we expressed the glycine-extended analogue HwTx-IV^G36(acid) in the SUMO/SHuffle system. The peptide was approximately three fold more potent on Na_v1.7 in comparison to HwTx-IV(acid) (IC₅₀ = 190 nM). In conclusion, we have established a novel system for expression and purification of fully folded and active HwTx-IV(acid) in bacteria, which could be applicable to other structurally complex and cysteine rich peptides. Furthermore, we discovered that glycine extension of HwTx-IV(acid) restores some of the potency of the native carboxamide. This finding may also apply to other C-terminally amidated peptides produced recombinantly.</p> </div

Effect of C-terminal glycine addition on potency.

<p>Na_v1.7 currents were measured from HEK cells stably expressing the hNa_v1.7 alpha subunit using whole cell voltage clamp. Peak sodium current in the presence of increasing doses of HwTx-IV (10 minutes incubation per dose) is plotted as a fraction of basal current (I/Ibasal). Statistics indicate significant difference in IC₅₀ between selected groups (one-way ANOVA with Tukey post-test). * = vs recombinant HwTx-IV(acid), *** = p<0.001; † = vs. synthetic HwTx-IV^G36(acid), ††† = p<0.001; $= vs synthetic HwTx-IV(amide),$ = p<0.05, $$$ = p<0.001. No significant difference was observed between the recombinant and synthetic HwTx-IV^G36(acid).</p

Characterisation of recombinant HwTx-IV(acid).

<p>A) Analytical RP-HPLC. The recombinant and synthetic HwTx-IV(acid) were analysed separately and combined (1:1). B) LC-MS analysis of the recombinant and synthetic HwTx-IV(acid). The peptides are identical. C) MALDI-TOF analysis of recombinant HwTx-IV(acid). The calculated and observed isotope distributions and m/z ions match. A minor cleaved form was detected: HwTx-IV^E1-V23. The theoretical m/z ions are: [M+H]⁺ = 4105.928 m/z, [M+2H]²⁺ = 2053.464 m/z, [M+3H]³⁺ = 1369.981 m/z and [M+4H]⁴⁺ = 1027.232 m/z. </p

Purification and characterisation of recombinant HwTx-IV^G36(acid).

<p>A) Preparative RP-HPLC. The main peak was collected and lyophilised. B) LC-MS of recombinant and synthetic HwTx-IV^G36(acid). Synthetic and recombinant HwTx-IV^G36(acid) are identical. E) MALDI-TOF analysis of recombinant HwTx-IV^G36(acid). The calculated and observed isotope distributions and m/z ions match. The minor cleaved form HwTx-IV^E1-V23 was detected again. The theoretical m/z ions are: [M+H]⁺ = 4162.950 m/z, [M+2H]²⁺ = 2081.975 m/z, [M+3H]³⁺ = 1388.317 m/z and [M+4H]⁴⁺ = 1041.488 m/z.</p

Electrophysiological characterisation of HwTx-IV(acid/amide) potency at hNa_v1.7.

<p>Na_v1.7 currents were measured from HEK cells stably expressing the hNa_v1.7 alpha subunit using whole cell voltage clamp. Peak sodium current in the presence of increasing doses of HwTx-IV (10 minutes incubation per dose) is plotted as a fraction of basal current (I/Ibasal). Statistics indicate significant difference in IC₅₀ between selected groups and synthetic HwTx-IV(amide), (one-way ANOVA with Tukey post-test; p<0.001, ***). No significant difference was observed between the recombinant and synthetic HwTx-IV(acid).</p