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

1University Hospital Würzburg, Würzburg, Bayern, Germany, 2 University Hospital and Rudolf Virchow Center, University of Würzburg, Würzburg, Bayern, Germany, 3University of Birmingham, Birmingham, UK, Birmingham, England, United Kingdom, 4Institute of Experimental Biomedicine, University Hospital Würzburg and Rudolf Virchow Center for Integrative and Translational Bioimaging, University of Würzburg, Würzburg, Bayern, Germany, 5University Hospital Würzburg and Rudolf Virchow Center for Integrative and Translational Biomaging University of Würzburg, Würzburg, Bayern, Germany

Abstract Number: OC 10.2

Meeting: ISTH 2022 Congress

Theme: Platelets and Megakaryocytes » Platelet Receptors

Background: Platelet C-type lectin-like receptor 2 (CLEC-2) is a unique platelet activation receptor signaling through a hemITAM motif. Due to its roles in thrombosis, tumor metastasis and inflammation CLEC-2 has been suggested as therapeutic target. However, there are currently no methods to test potential human therapeutics targeting CLEC-2, such as antibodies, in vivo.

Aims: We have therefore generated and characterized a humanized CLEC-2 mouse (hCLEC-2KI) and developed a novel monoclonal anti-human CLEC-2 antibody, HEL1, for in vivo testing.

Methods: Murine CLEC-2 was replaced with the human variant and the mice, as well as their platelets characterized in vitro and in vivo. HEL1 was generated by hybridoma technology following immunization of rats with immunoprecipitated human CLEC-2. HEL1 was characterized using human and hCLEC-2KI platelets. CLEC-2 regulation was assessed using the anti-hCLEC-2 monoclonal antibodies AYP1 and HEL1.

Results: hCLEC-2KI mice were viable, fertile and born at Mendelian ratio, without evidence of blood-lymphatic mixing. hCLEC-2KI platelets displayed glycoprotein receptor expression, activation and aggregation comparable to wildtype controls. Likewise, tail bleeding times of hCLEC-2KI mice were like those of WT mice. The novel antibody HEL1 activates platelets upon binding to CLEC-2. However, HEL1-fab fragments neither activate platelets nor affect rhodocytin-induced platelet activation. Challenging hCLEC-2KI mice with HEL1- or AYP1-IgG, which bind different epitopes, resulted in a transient thrombocytopenia as well as CLEC-2 depletion for more than 2 weeks, but did not affect hemostasis.

Conclusion(s): Here, we provide proof of principle that the hCLEC-2KI mouse can be used to test anti-hCLEC-2 agents in vivo. In addition, we demonstrated that hCLEC-2 can be immunodepleted.

Image

Antibody-mediated depletion of human CLEC-2 in a novel humanized mouse model. The novel anti-human CLEC-2 antibody HEL1 binds a different epitope than established AYP1 antibody, however, both antibodies activate platelets. In vivo, injection of either anti-CLEC-2 antibody in hCLEC-2KI mice, that express human instead of murine CLEC-2, results in CLEC-2 depletion from the platelet surface for more than 2 weeks -parts of the figure were created in BioRender-.

To cite this abstract in AMA style:

Brown H, Beck S, Navarro S, Watson S, Nieswandt B, Stegner D. Antibody-mediated depletion of human CLEC-2 in a novel humanized mouse model [abstract]. https://abstracts.isth.org/abstract/antibody-mediated-depletion-of-human-clec-2-in-a-novel-humanized-mouse-model/. Accessed September 22, 2023.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/antibody-mediated-depletion-of-human-clec-2-in-a-novel-humanized-mouse-model/