118P London, UK
Pharmacology 2016

 

 

Novel shark antibodies engineered to characterise and improve delivery of therapeutics across the blood-brain barrier

A. L. Fleckney1, R. C. Brown1, S. Drndarski1, K. B. Wicher2, L. Rutkowski3, S. A. Thomas1. 1King\'s College London, Institute of Pharmaceutical Science, London, UNITED KINGDOM, 2Ossianix Inc., Stevenage, UNITED KINGDOM, 3Ossianix Inc., Philadelphia, UNITED STATES.

Introduction: The blood-brain barrier prevents >98% of therapeutics from reaching the brain, posing a major problem for treatment of CNS afflictions, such as Parkinson’s and Alzheimer’s disease. Novel shark antigen receptor variable domains - termed VNARs - can be used as molecular Trojan Horses to deliver therapeutics to the brain. This study aimed to investigate brain uptake and biodistribution of transferrin receptor 1 (TfR1) targeted VNARs in vivo.

Methods: All procedures were performed within the Animal Scientific Procedures Act (1986) and Amendment Regulations 2012. VNARs were tritiated and approx. 0.3µCi 8D3 antibody, 2.2µCi VNAR1-Fc or 1.7µCi VNAR2-Fc were injected (IV) into adult male Balb/c mice. After 60min, mice were anaesthetised, vasculature was flushed, organs harvested and radioactivity determined. VNAR tissue uptake was expressed as percentage of injected dose (DPMs in tissue/total DPMs injected) per gram of tissue - termed %ID/g (±SEM; n=5 per treatment). TfR1 and 2 expression was investigated using Western Blots. ELISAs investigated binding of each VNAR-Fc to mouse TfR2.

Results: There was no significant difference in brain uptake between the 2 VNAR-Fcs (Mann-Whitney Rank Sum Test (p=0.548)) or between each VNAR-Fc and 8D3 (Student’s t-test, p=0.7 VNAR1 and p=0.378 VNAR2) (Table 1). However, there were differences in distribution to the liver. In all cases, uptake in liver was higher than that achieved in brain: 8D3 two-fold, VNAR1-Fc 1.5-fold and VNAR2-Fc ~20-fold. Preliminary Western Blots show that both TfRs are expressed in mouse liver, but only TfR1 is expressed in brain. ELISA data shows that binding of both VNARs to mouse TfR2 is negligible, equal to non-specific binding to human serum albumin. However, both VNARs have a high affinity for human and mouse TfR1 (Table 2).

Table 1: Mean concentrations of VNAR in plasma, VNAR brain uptake and VNAR liver uptake
8D3 mAb VNAR1-Fc VNAR2-Fc
Plasma (DPM/μl) 27 154 166
Plasma (ng/μl) 1.1 0.9 0.6
Brain Uptake (%ID/g) 0.32 ± 0.08 0.27 ± 0.10 0.21 ± 0.09
Liver Uptake (%ID/g) 0.66 ± 0.23 0.45 ± 0.18 4.39 ± 0.34
 
Table 2: VNAR EC50\'s for binding to TfR1 (nM)
Mouse TfR1 Human TfR1
VNAR1-Fc 1.67 0.43
VNAR2-Fc 0.57 0.13

 

Conclusions: Both VNAR-Fc molecules exhibit similar brain uptake, but VNAR2-Fc shows greatest uptake in liver - suggesting VNAR2-Fc may be cross-reactive with TfR2 in liver, which is reportedly expressed at higher levels than TfR1 (1, 2). However, our preliminary data shows TfR1 is expressed ~2.4 times more than TfR2. VNARs showed high affinity for TfR1 and did not significantly cross-react with TfR2. It may be that the epitope VNAR2-Fc binds is more exposed in liver than brain, or simply that the vasculature was not flushed effectively, leaving residual VNAR in liver tissue.

Funded by a BBSRC-CASE PhD studentship

References: 1. Fleming, R. E. et al. (2000). Proc. Natl. Acad. Sci. U.S.A., 97(5), 2214-9. 2. Wallace, D. F., et al. (2007). Gastroenterol., 132(1), 301-10.