MO074TILVESTAMAB, A FUNCTION-BLOCKING MONOCLONAL ANTIBODY INHIBITOR OF AXL RTK SIGNALLING, LIMITS THE ONSET OF RENAL FIBROTIC CHANGES IN HUMAN KIDNEYS EX VIVO

Abstract

Abstract Background and Aims Interstitial fibrosis, characterised by the accumulation of extracellular matrix in the cortical interstitium, is directly correlated with progressive chronic kidney disease secondary to inflammatory, immunologic, obstructive or metabolic causes. An invariant histologic marker of this progression is the accumulation of fibroblasts, with the phenotypic appearance of activated myofibroblasts expressing alpha smooth muscle actin (αSMA) within intracellular contractile stress fibres. Once present, these myofibroblasts are prognostic indicators of expansion of fibrotic matrix and progressive tubular atrophy, leading towards end-stage disease. The Receptor Tyrosine Kinase AXL is involved in a range of kidney pathologies, with increased activity associated with Epithelial to Mesenchymal Transition (EMT) and tubular proliferation following podocyte loss. In mice treated with an angiotensin-converting enzyme (ACE) inhibitor, enhancement of AXL expression is localised to tubular segments within the medulla and there is evidence of parallel regulatory control of ACE and AXL. We have demonstrated enhanced expression of AXL and the mesenchymal marker, vimentin in diseased human kidney tissue secondary to diabetes or hypertension. Targeting AXL with a small-molecule inhibitor has previously been reported to attenuate fibrosis and reduce inflammation in the unilateral ureteric-outflow obstruction (UUO) model of kidney fibrosis in mice (Landolt et al., 2019). Tilvestamab is a novel function blocking humanized anti-AXL antibody. Tilvestamab blocks GAS6-mediated AXL receptor activation in fibroblasts and renal tubule epithelial cells and mediates AXL receptor internalization and degradation. In this study we aimed to further characterise AXL as a target in CKD and to investigate anti-fibrotic efficacy of tilvestamab. Method Eight weeks old male C57BL/6 mice underwent UUO operation. After 15 days, kidneys were dissociated and stained with a high dimensional single cell mass cytometry 33 markers antibody panel. Data were analysed using JMP Genomics (v.8.2). Precision Cut Kidney Slices (PCKSs) from explanted human kidney tissue were propagated in a bioreactor (Paish et al., 2019, FibroFind, UK). PCKS were incubated for 72hrs in the presence of investigational drugs. Secreted collagen1a1 were quantified by ELISA. RNA was reverse transcribed to cDNA and used in qPCRs to measure Col1a1 and αSMA. FFPE sections were stained for αSMA. High magnification images were taken of each slide and analysed for surface area covered by the stain. Results Expression pattern of AXL during development of kidney fibrosis in the UUO model was investigated using a mass cytometry antibody panel designed for identifying subpopulations of immune cells as well as cell populations of the fibrotic stroma. Two predominant cell populations were affected by ligation; the mesenchymal and the immune island. AXL was a marker characterising several of the key populations that expanded upon ligation supporting a role for AXL in kidney fibrosis pathogenesis. In an ex vivo model of human PCKS, tilvestamab dose-dependently reduced the levels of αSMA. When combined with the lower of two doses of the ACE inhibitor enalapril, the lowest dose of tilvestamab synergized to reduce αSMA levels further as well as reducing secreted Collagen 1a1. Conclusion AXL expression is induced in key cell populations during development of kidney fibrosis supporting AXL as a novel target in CKD. Tilvestamab represents a promising strategy for the pharmacologic intervention of kidney fibrosis, and the potential synergy with current reno-protective therapies warrants further exploration.