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

Antibody Targeting of Cathepsin S Inhibits Angiogenesis and Synergistically Enhances Anti-VEGF

Angiogenesis is a key hallmark of tumourigenesis and its inhibition is a proven strategy for the development of novel anti-cancer therapeutics. An important aspect of early angiogenesis is the co-ordinated migration and invasion of endothelial cells through the hypoxic tumour tissue. Cathepsin S has been shown to play an important role in angiogenesis as has vascular endothelial growth factor (VEGF). We sought to assess the anti-angiogenic effect of Fsn0503, a novel cathepsin S inhibitory antibody, when combined with anti-VEGF on vascular development. where it significantly retarded the development of vasculature in human xenograft models. Furthermore, when Fsn0503 was combined with an anti-VEGF antibody, a synergistic inhibition of microvascular development was observed.Taken together, this data demonstrates that the antibody-mediated targeting of cathepsin S represents a novel method of inhibiting angiogenesis. Furthermore, when used in combination with anti-VEGF therapies, Fsn0503 has the potential to significantly enhance current treatments of tumour neovascularisation and may also be of use in the treatment of other conditions associated with inappropriate angiogenesis

Fsn0503 demonstrates anti-angiogenic effects <i>in vitro</i>.

<p>(<b>A</b>) Fsn0503 attenuates HUVEC invasion using a modified Boyden chamber assay at 500 nM. (<b>B</b>) Fsn0503 (500 nM) inhibits HUVEC degradation of quenched fluorescent substrate DQ gelatin. Cells treated with Fsn0503 or controls were analysed for the presence of fluorescent degradation products by confocal microscopy (representative images shown). (<b>C</b>) A reduction of 70% of DQ gelatin degradation was observed when quantified by mean energy per cell (p = 0.03) (5 fields per assay). (<b>D</b>) Fsn0503 (400 nM) inhibits HUVEC tube formation compared to isotype control (representative images shown). (<b>E</b>) Tube formation was assessed by counting nodes with 1, 2, 3, or more branches and the average calculated per field of view. The number of nodes with 3 or more branches, which are indicative of normal tube formation, were significantly reduced in Fsn0503 treated samples (p<0.01).</p

Fsn0503 inhibits angiogenesis <i>in vivo</i>; CD34 Immunohistochemical analysis of Fsn0503 treated tumors.

<p>(<b>A</b>) Pattern of small vessel (white arrows) and large vessel (black arrows) distribution in isotype control and Fsn0503 treated tumors (10 mg/kg 5 times a week for 4 weeks) (20×). (<b>B</b>) Analysis of total vessel number as characterized by CD34 staining shows that Fsn0503 caused an increase in small vessel number and a significant decrease in the number of large vessels (p<0.001). (<b>C</b>) A significant reduction is observed in the mean vessel area of tumours treated with Fsn0503 (p<0.001).</p

Cathepsin S expression and activity is up-regulated by pro-angiogenic stimuli.

<p>(<b>A</b>) Total RNA was isolated from endothelial cell lines (HUVEC and HMEC-1) and used to assess Cathepsin S mRNA by RT-PCR. GAPDH serves as a control. (<b>B,C</b>) Human recombinant VEGF (10 ng/ml) up-regulates cathepsin S mRNA and protein in HUVECs when assessed by RT-PCR and western blot using GAPDH and tubulin as respective controls (<b>D</b>) Human recombinant VEGF (10 ng/ml) stimulates Cathepsin S-like activity, as assessed by the cleavage of fluorigenic substrate, Cbz-Val-Val-Arg-AMC, by 37% in HUVEC cell lysates. (<b>E,F</b>) To demonstrate the effect of hypoxia on Cathepsin S up-regulation total RNA and protein was isolated from HUVEC endothelial cells (grown in normal or hypoxic conditions) and examined by RT-PCR and western blot. GAPDH and tubulin serves as a control.</p