Abstract
We previously described (Fuentes, J Clin Invest 2016) a chimeric protein, GPIIb/uPA-T, that provided effective thromboprophylaxis composed of an N-terminal scFv specifically to human (h) GPIIb linked to a C-terminal variant of low molecular weight urokinase containing a thrombin cleavable activating site. uPA-T prevented FeCl3-induced carotid arterial thrombosis in immunocompromized NOD/SCID, IL2 receptor γ-chain deficient (NSG) mice that had been pre-infused with human platelets (hPlts) without causing rebleeding from pre-existing tail-clips clots. However, this construct and subsequent hGPIIb-targeting scFv uPA-T chimeric constructs caused significant thrombocytopenia in hPlt-infused NSG (hPlt/NSG) mice and in hGPIIb+ mice. Therefore, we developed a new chimeric uPA-T targeting hGPIbα based on 5G6, a monoclonal antibody that binds to the hGPIbα juxtamembrane ADAM17 cleavage site and is known to not cause thrombocytopenia. The chimera 5G6/uPA-T binds to the surface of hPlts and hGPIbα-expressing mouse platelets at levels ~30% of those seen with hGPIIb/uPA-T, consistent with the difference in expression of GPIIb vs. GPIbα. 5G6/uPA-T did not reduce platelet counts in hPlt/NSG or hGPIα+ mice at doses that caused significant thrombocytopenia after infusion of GPIIb/uPA-T (1 mg/kg each, bolus + 30-minute infusion). We then tested the efficacy of 5G6/uPA-T in hPlt/NSG mice that that also carried a single mouse-to-human arginine (R) to histidine (H) substitution at position 1326 in von Willebrand factor, VWFH, created using CRISPR/Cas9 technology in NSG mice. This substitution, described by T. Diacovo (Megalion, Circulation 2011), markedly impairs binding of mouse Plts that express murine GPIbα, but increases the affinity for hPlt expressing hGPIbα. We hypothesized that such mice would allow studies of hPlt-enriched thrombi in mice. To test this hypothesis, hPlts were infused into NSG mice to achieve a circulating level of 10-20% of the total platelet pool. Following FeCl3-induced carotid arterial injury, similar numbers of hPlts were incorporated into the thrombi in wildtype VWFR/R NSG and heterozygous VWFR/H littermate NSG mice, whereas 1.4-1.8-fold more hPlts were incorporated in VWFH/H NSG littermates over the 3-minute observation period (n = 12-17, p<0.04 between VWFH/H NSG mice vs. VWFR/R and VWFR/H mice.
Infusion of 5G6/uPA-T (1 mg/kg bolus+infusion) into hPlts-infused VWFH/H NSG mice prevented carotid artery thrombosis induced by FeCl3 and observed for 30 minutes as follows: In the absence of infused hPlts, 2/5 injured carotid arteries in the VWFH/H NSG mice completely occluded with a total flow volume in all vessels measured by Doppler of 1158±377 ml/min.s area under the curve (AUC). In contrast, 5/5 vessels occluded hPlts-infused VWFH/H NSG mice with an average time to occlusion of 13.0±3.4 mins and an AUC reduced to 692±75 ml/min.s (p<0.04 compared to no infused hPlts). In contrast, only 1/5 vessels occluded in hPlts-infused VWFH/H NSG mice given 5G6/uPA-T prior to injury, with an AUC of 1487±190 ml/min.s (p<0.006 compared to hPlt-infused VWFh/h NSG mice). None of the mice given 5G6/uPA-T exhibited untoward bleeding at the surgical site. 5G6/uPA-T did not induce thrombocytopenia, visible bleeding elsewhere, rebleeding in hPlt+/VWFH/H NSG mice when given 10 minutes following tail vein clipping, nor were other overt toxicities observed.
In summary, we believe that 5G6/uPA-T combines a number of desirable features for a drug designed to prevent untoward platelet-mediated thrombosis: i. 5G6/uPA-T binds specifically to a hPlt-specific receptor, allowing targeted delivery to evolving thrombi while sparing established clots where it likely poorly penetrates into the thrombi. ii. The drug is effective even while binding to the base of the GPIbα chain near the platelet surface, where it also likely maintains GPIb/IX biology by limiting GPIbα cleavage, and does not cause thrombocytopenia. iii. Finally, 5G6/uPA-T delivers a form of urokinase that needs the co-presence of thrombin to become activated further limiting its activity to sites of evolving thrombi. These properties were demonstrated in an in vivo model that selectively incorporates hPlts. We believe that 5G6 may have clinical application in settings where there is a high risk for thrombosis, such as maintaining stent patency, while avoiding untoward lysis of previously established clots.
Disclosures
Li: Neoletix: Consultancy, Equity Ownership. Poncz:Incyte Corporation: Consultancy, Research Funding.
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