The Anti-tumor Effect Of A Bispecific Antibody On Gastric Cancer And Its Mechanism Of Action

Worldwide,more than 70,000 patients die from gastric cancer every year,which makes it the second leading cause of cancer death.Besides,the burden of gastric cancer is particularly high in most developing countries,including China.Gastric cancer is most often diagnosed at an advanced stage which makes most patients with gastric cancer having a somber prognosis.Expression of human epidermal growth factor receptor-2(ErbB2)oncogene is found in 12%-20% of human gastric cancers and the ErbB2-positive status is supposed to be associated with even worsen prognosis.In recent years,more and more evidence proves that targeted therapies may bring clinical benefit to patients with advanced gastric cancer.Trastuzumab(a humanized monoclonal antibody)which targets the extracellular domain IV of ErbB2 has become a standard treatment and has been shown to prolong the overall survival of patients with ErbB2-positive advanced gastric cancer.Pertuzumab,which binds to the extracellular domain II of ErbB2,is another humanized monoclonal antibody targeting ErbB2 and its development in therapy of ErbB2-positive advanced gastric cancer has shown encouraging results.Meanwhile,clinical trials investigating the effects of Pertuzumab plus Trastuzumab in advanced gastric cancer have also shown promising results.Despite the effectiveness of ErbB2-targeted therapies,the efficacy of Trastuzumab and Pertuzumab is still modest and variable.Thus,it is imperative to improve the efficacy of ErbB2-targeted therapies.During the last decade,genetic engineering has provided us with a variety of methods to develop and optimize antibody structure and function.In our previous study,we developed a genetically engineered bispecific anti-ErbB2 antibody(TPL)using Trastuzumab and Pertuzumab.In the present study,we investigated the antitumor effect of this bispecific antibody on gastric cancer and its mechanisms of action.First,we examined the antiproliferative activity of Trastuzumab,Pertuzumab,Trastuzumab plus Pertuzumab,and TPL in human ErbB2-overexpressing gastric cancer cell line N87.It is obvious that Trastuzumab and Pertuzumab in combination is more potent in antiproliferative activity than Trastuzumab or Pertuzumab alone.Notably,the most potent in vitro antiproliferative effect was observed in TPL.Next,we investigated the therapeutic efficacy of Trastuzumab,Pertuzumab,Trastuzumab plus Pertuzumab,and TPL in nude mice bearing established N87 xenograft tumors.Our data showed that both Trastuzumab and Pertuzumab could effectively inhibit tumor growth.The combination of Trastuzumab plus Pertuzumab was more potent in inhibition of tumors than each antibody alone.Remarkably,TPL showed even stronger inhibitory effects than Trastuzumab plus Pertuzumab.Previous studies by other researchers have shown that the combination of two anti-ErbB2 antibodies that did not compete with each other for the binding to ErbB2 can enhance ErbB2 internalization which may eventually improve the therapeutic activity of antireceptor antibodies.Here we also evaluated the ability of Trastuzumab,Pertuzumab,Trastuzumab plus Pertuzumab,and TPL to induce internalization in N87 cells.Results from confocal and flow cytometry demonstrated that 8 hour treatment with Pertuzumab or Trastuzumab could not induce internalization effectively in N87 cells.8h treatment with Trastuzumab plus Pertuzumab or 2h treatment with TPL can induce slight internalization.However,significant internalization was observed when treated with TPL for 8h in N87 cells.In order to further investigate the relationship between ErbB2 internalization and non-competitive anti-ErbB2 mAb pairs,we developed 5 new mouse anti-ErbB2 mAbs through standard hybridoma technique.We created 21 anti-ErbB2 mAbs pairs using Pertuzumab,Trastuzumab,and the 5 new antibodies.Then we identified 18 non-competitive mAbs pairs by competitive binding assay out of the 21 pairs.Interestingly,the results of confocal shows that only 4 of 18 non-competitive mAbs pairs effectively induce internalization.In summary,our present study showed that the bispecific antibody TPL can significantly accelerate receptor internalization and display effective antitumor activity in ErbB2-overexpressing gastric cancer cells,suggesting that it might have the potential to improve the effectiveness of targeted therapies for ErbB2-overexpressing gastric cancer.We have also demonstrated that only certain non-competitive anti-ErbB2 mAb pairs could effectively induce ErbB2 receptor internalization.