The Mechanism Of An EGFRvⅢ Targeting Monoclonal Antibody CH12 Induced Species Specific Thrombocytopenia In Cynomolgus Monkeys

Epidermal growth factor receptor(EGFR)plays a key role in the regulation of cell proliferation,survival and differentiation.Overexpression and mutation can lead to cell proliferation,angiogenesis,increased local tissue invasion,and anti-apoptotic Thus,it is considered to be a proto-oncogene and promotes tumor cell proliferation and development.The epidermal growth factor receptor variant III(EGFRvIII)is the most common EGFR mutants and EGFRvIII expression has been detected in a variety of malignancies.In addition,since EGFRvIII is not expressed in normal tissues,it is also an ideal target for cancer treatmentCH12 is a novel humanized monoclonal antibody against EGFRvIII for cancer treatment.Previous pharmacodynamic studies have shown that CH12 can inhibit the growth of EGFRvIII-positive cells and xenografts,and the efficacy is better than the commercial drug EGFR-targeted cetuximab.However,in the preclinical safety evaluation of CH12,single(≥ 25 mg/kg)and repeated(>5 mg/kg)intravenous infusion of CH12 resulted in a significant reduction in platelet counts in cynomolgus monkeys,accompanied by purpura such as ecchymosis,i.e.unexpected thrombocytopenia occurred.On the contrary,it has no effect on rats(400 mg/kg)Further in vitro experiments found that CH12 can bind and activate platelets from cynomolgus monkey peripheral blood samples,but not from human.Cynomolgus monkey-specific thrombocytopenia has been reported previously,but the underlying mechanism remains unclear,such as omalizumab,AMG X,and MAbY.1.Clarify the toxicity mechanism can also provide a reference for this type of toxicity induced by other monoclonal antibodies,and clarify species differences can also help the assessment and prediction of clinical adverse reactionsHere,we first showed that CH12 induced thrombocytopenia in cynomolgus monkeys through off-target platelet binding and activation,resulting in platelet destruction.We subsequently found that integrin αIIbβ3(which is expressed on platelets)contributed to this off-target toxicity.Furthermore,three-dimensional structural modeling of the αIIbβ3 molecules in cynomolgus monkey,human,and rat suggested that an additional unique loop exists in the ligand-binding pocket of theαIIb subunit in cynomolgus monkeys,which may explain why CH12 binds to platelets only in cynomolgus monkeys.Amino-acid sequence alignment of αIIb subunit revealed that the unique loop identified in cynomolgus monkeys was located in the ligand-binding pocket,the corresponding sequence was "DKR",and two amino-acid deletions were identified before "DKR" sequence in the cynomolgus monkeys,which may the reason caused this species limited toxicity.Moreover,this study supported the hypothesis that the minor differences between cynomolgus monkeys and humans can confuse human risk assessments and clarify the corresponding species differences can help the prediction of human risks and avoid losses in drug development.