Antibody-mediated depletion of human CLEC-2 in a novel humanised mouse model

Abstract

Platelet C-type lectin-like receptor 2 (CLEC-2) has been proposed as a potential anti-thrombotic target as genetic or antibody-mediated receptor deficiency prevents occlusive thrombus formation in mice. This occurs through interaction with an unknown ligand as the endogenous ligand podoplanin is not present in the vasculature. However, the CLEC-2-podoplanin interaction does have an important role in tumour metastasis. There are currently no methods to test potential human therapeutics targeting CLEC-2, such as antibodies, in vivo. We have therefore generated and characterised a humanised CLEC-2 mouse (hCLEC-2KI) and developed a novel monoclonal anti-human CLEC-2 antibody, HEL1, for in vivo testing. hCLEC-2KI mice were phenotypically normal and had comparable platelet glycoprotein receptor expression, activation and aggregation to wildtype platelets. hCLEC-2KI mice had both comparable bleeding and vessel occlusion times to WT mice. Challenging hCLEC-2KI mice with HEL1 or a second monoclonal anti-hCLEC-2 antibody, AYP1, resulted in transient thrombocytopenia as well as CLEC-2 depletion for more than 2 weeks but had no effect on haemostasis. This illustrates the power of the humanised CLEC-2 mouse model in evaluating novel therapeutics in vivo, including antibodies that target CLEC-2, as well as the limited effect on haemostasis when targeting CLEC-2.

Divalent nanobodies to platelet CLEC-2 can serve as agonists or antagonists

“…These data demonstrate that a divalent ligand to CLEC-2 is an antagonist in human platelets but an agonist in mouse platelets expressing the human CLEC-2 receptor. A similar result is also seen with the mAb AYP1 which recognises human CLEC-2 29 . Thus, this differential action is not due to a difference between human and mouse CLEC-2, indicating an inherent difference between human and mouse.…”

“…These results indicate a species difference in the ability of divalent ligands to stimulate aggregation. To address whether this is due to a difference between mouse and human CLEC-2 or between mouse and human platelets, we used platelets from a humanised CLEC-2 knock-in transgenic mouse (hCLEC1b KI ) 29 .…”

CLEC-2 Supports Platelet Aggregation in Mouse but not Human Blood at Arterial Shear

“…Haining et al demonstrated that CLEC-2 did not contribute to thrombus occlusion in a mechanical injury of the aorta, which was recently confirmed using a humanized CLEC-2 mouse model. 15,50 This illustrates a varied contribution of vascular beds to thrombus formation in mice.…”

Rac Inhibition Causes Impaired GPVI Signalling in Human Platelets through GPVI Shedding and Reduction in PLCγ2 Phosphorylation

“…The following antibodies were used: for flow cytometry, PE-conjugated anti-human GPVI (clone: HY101; BD Biosciences, San Diego, CA, USA); for FRET, the F (ab’) fragment of the anti-human GPVI (clone: 313A10) [ 17 ]; for super-resolution microscopy, the 1G5 F (ab’) fragment [ 62 ] and for GPVI shedding analysis, the affinity-purified anti-GPVI cytoplasmic tail IgG [ 63 ] (both gifts from Dr E. Gardiner, Australia) were used. Anti-GPIX (clone: p0p6) [ 64 ], anti-integrin αIIb (clone: MWReg30) [ 65 ], anti-integrin β3-subunit (clone: EDL1) [ 66 ], anti-GPV (clone: 10C10) and anti-CLEC-2 (clone: HEL1) [ 67 ] antibodies were all purified and fluorophore-conjugated in house. Anti-PLCγ2 pY1217 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-SYK Py525/526, anti-GAPDH and anti-LAT antibodies were from Cell Signalling (Danvers, MA, USA); anti-LAT pY200 was from Abcam (Cambridge, UK); Alexa Fluor ® 488-Phalloidin was from Invitrogen (Carlsbad, CA, USA); Alexa Fluor ® 647 labelled anti-human CD41/CD61 antibody (clone: PAC-1) was from BioLegend (San Diego, CA, USA); APC-labelled anti-human CD42b (clone: HIP1) and FITC-labelled anti-human P-selectin (clone: AK-4) antibodies were both from BD Biosciences (San Diego, CA, USA).…”