Novel knock-in mouse model for the evaluation of the therapeutic efficacy and toxicity of human podoplanin-targeting agents

Cancer Sci. 2021 Jun;112(6):2299-2313. doi: 10.1111/cas.14891. Epub 2021 Apr 5.


Podoplanin is a key molecule for enhancing tumor-induced platelet aggregation. Podoplanin interacts with CLEC-2 on platelets via PLatelet Aggregation-inducing domains (PLAGs). Among our generated antibodies, those targeting the fourth PLAG domain (PLAG4) strongly suppress podoplanin-CLEC-2 binding and podoplanin-expressing tumor growth and metastasis. We previously performed a single-dose toxicity study of PLAG4-targeting anti-podoplanin-neutralizing antibodies and found no acute toxicity in cynomolgus monkeys. To confirm the therapeutic efficacy and toxicity of podoplanin-targeting antibodies, a syngeneic mouse model that enables repeated dose toxicity tests is needed. Replacement of mouse PLAG1-PLAG4 domains with human homologous domains drastically decreased the platelet-aggregating activity. Therefore, we searched the critical domain of the platelet-aggregating activity in mouse podoplanin and found that the mouse PLAG4 domain played a critical role in platelet aggregation, similar to the human PLAG4 domain. Human/mouse chimeric podoplanin, in which a limited region containing mouse PLAG4 was replaced with human homologous region, exhibited a similar platelet-aggregating activity to wild-type mouse podoplanin. Thus, we generated knock-in mice with human/mouse chimeric podoplanin expression (PdpnKI/KI mice). Our previously established PLAG4-targeting antibodies could suppress human/mouse chimeric podoplanin-mediated platelet aggregation and tumor growth in PdpnKI/KI mice. Repeated treatment of PdpnKI/KI mice with antibody-dependent cell-mediated cytotoxicity activity-possessing PG4D2 antibody did not result in toxicity or changes in hematological and biochemical parameters. Our results suggest that anti-podoplanin-neutralizing antibodies could be used safely as novel anti-tumor agents. Our generated PdpnKI/KI mice are useful for investigating the efficacy and toxicity of human podoplanin-targeting drugs.

Keywords: CLEC-2; knock-in mice; platelet aggregation; podoplanin; toxicity.

MeSH terms

  • Animals
  • Antibodies, Monoclonal / pharmacology
  • Antibodies, Monoclonal / therapeutic use*
  • Antibodies, Neutralizing / pharmacology
  • Antibodies, Neutralizing / therapeutic use*
  • Antineoplastic Agents / pharmacology
  • Antineoplastic Agents / therapeutic use*
  • Cell Line, Tumor
  • Humans
  • Membrane Glycoproteins / chemistry
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / immunology
  • Membrane Glycoproteins / metabolism*
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Platelet Activation / drug effects
  • Platelet Aggregation / drug effects
  • Protein Domains
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Xenograft Model Antitumor Assays


  • Antibodies, Monoclonal
  • Antibodies, Neutralizing
  • Antineoplastic Agents
  • Gp38 protein, mouse
  • Membrane Glycoproteins
  • PDPN protein, human
  • Recombinant Fusion Proteins