PROJECT SUMMARY
Over 37 million individuals in the U.S. have chronic kidney disease (CKD) and are at high risk to die from
cardiovascular complications. While great strides have been made to improve CKD care and dialysis access,
minimal advances have been made in drug development to stall or reverse kidney damage and associated
pathologies. Currently, therapeutic options to prevent cardiovascular damage in CKD do not exist, and the only
cure for CKD is kidney transplantation. Elevations in serum levels of phosphate and fibroblast growth factor
(FGF) 23 are a hallmark of CKD and associated with an increased risk of cardiovascular death. Expression levels
of klotho, a regulator of phosphate metabolism in the kidney, are reduced in CKD. Klotho can be released from
the kidney as soluble klotho (sKL) that circulates in the blood and acts as a binding partner for FGF receptors
(FGFR) on various tissues. Reductions in serum sKL levels have been shown to contribute to CKD-associated
pathologies. sKL seems to protect tissues by substituting for renal klotho thereby promoting FGF23/FGFR1-
induced renal phosphate excretion and lowering systemic phosphate levels, as well blocking the direct pathologic
actions of FGF23 and of paracrine FGFs. While elevating klotho expression has shown therapeutic potential in
animal models of CKD, further advances have been stymied by sKL’s short half-life and technical difficulties to
produce the recombinant sKL protein in sufficient amounts, along with a lack of tools to measure sKL activity.
Alpha Young LLC has developed a novel method to produce the recombinant sKL protein as well as a novel
assays to determine the bioactivity of sKL based on its ability to bind FGF23 and FGFR1. We have generated
an early-stage mimetic protein, and here we will introduce additional point mutations to increase sKL’s stability
and bioactivity. In Phase 1, we will modify sKL’s glycosylation sites and heparin binding domain, and we will
screen for mutant variants with increased binding affinities for FGF23 and FGFR1 to improve bioactivity, and
decreased heparin binding affinity to increase half-life. In Phase 2, we will optimize our identified sKL variants
by utilizing a phage display-based approach to introduce mutations into sKL’s FGFR binding domain with the
goal to increase FGFR1 binding affinity. Candidates with the desired changes in binding properties will be tested
for their biological activity using cell culture models that can determine the effect of sKL on FGF23-regulated
signaling, renal phosphate uptake, cardiac hypertrophy and on fibroblast activation induced by paracrine FGFs.
The half-life of the most promising candidates will be tested by injection studies in in rats. Finally, the most active
and stable sKL variant will be injected into mouse models of CKD, followed by the analysis of renal phosphate
excretion and cardiovascular damage. We propose that the administration of our sKL mimetic can serve as a
novel therapeutic approach in CKD to lower serum phosphate levels and to protect from the damaging actions
of FGFs. A successful completion of our project would provide us with a potent drug candidate and the
opportunity to pursue early-stage partners for advancing and validating its potential for future clinical trials.
