Effective Suppression Of Breast Tumor Growth By A Bispeciflc Antibody Targeting Distinct ErbB2Epitopes

Overexpression of human epidermal growth factor receptor-2(HER2or ErbB2), amember of the ErbB family of receptor tyrosine kinases, is found in20-30%of humanbreast cancers. Trastuzumab, a humanized monoclonal antibody (mAb) directed againstErbB2, is the first anti-ErbB2treatment approved for clinical use for patients withhigh-ErbB2-expressing breast cancer. However, even in ErbB2-overexpressing breasttumors, Trastuzumab has antitumor activity in only up to one-third of patients, and it doesnot have any activity against tumors expressing lower levels of ErbB2. Thus, there is anurgent need to improve ErbB2-directed therapy in Trastuzumab-resistant breast cancer.Heterodimerization between ErbB2and other ErbBs may redundantly trigger cellproliferation signals and confer Trastuzumab resistance. Although no specific ligand forErbB2has been identified, it is the preferred heterodimerization partner of the ErbB family.ErbB2forms heterodimers with both ligand-free and ligand-bound forms of the other ErbBfamily members (EGFR, ErbB3and ErbB4), which activates ErbB receptors anddownstream MAPK and AKT signaling pathways, thereby promoting cell proliferation andsurvival. Previous studies have demonstrated that Trastuzumab only partially inhibitsErbB2-containing heterodimer formation. ErbB2heterodimerization may still initiatesignaling events that confer resistance when ErbB2is inhibited by Trastuzumab. Here wedeveloped a bispecific anti-ErbB2antibody using Trastuzumab and Pertuzumab, anotherErbB2-specific humanized antibody that binds to a distinct epitope from Trastuzumab.Competitive binding assay results indicated that this bispecific antibody, denoted as TPL,effectively compete with both Trastuzumab and Pertuzumab for binding toErbB2-overexpressing BT-474cells. The affinity constant (Kd) of TPLfor the extracellulardomain of HER2was determined by an enzyme-linked immunosorbent assay (ELISA).The results showed that TPLhad an ErbB2-bindng affinity comparable to that ofTrastuzumab and much higher than that of Pertuzumab. Pharmacokinetic analysis furthershowed that TPLhad pharmacokinetic properties similar to those of a conventional IgGmolecule, suggesting that it is highly stable in vivo.Next, we examined the capability of TPLto disrupt ErbB2heterodimerization inbreast cancer cell lines. TPLwas far more potent in blocking both ligand-independent andligand-induced ErbB2heterodimerization than Trastuzumab plus Pertuzumab, possiblythrough steric hindrance and/or inducing ErbB2conformational change. We also examinedthe inhibitory effects of anti-ErbB2mAb treatment on the activation of ErbB2and downstream MAPK and AKT signaling pathways. Our results indicated that the ability ofanti-ErbB2mAbs to inhibit ErbB2signaling corresponded with their capacity to blockErbB2heterodimerization. Accordingly, TPLshowed the most potent inhibitory effect.We evaluated the ability of Trastuzumab, Pertuzumab, Trastuzumab plus Pertuzumab,and TPLto inhibit the in vitro proliferation of high-ErbB2-expressing (BT-474andSK-BR-3) and low-ErbB2-expressing (MCF-7, MDA-MB-468and MDA-MB-231) breastcancer cell lines. The results clearly indicate that the antiproliferative activity of theseanti-ErbB2mAbs is directly related to their ability to block ErbB2heterodimerization andactivation. TPLdemonstrated far greater antiproliferative activity than that of any of theothers both in the absence and presence of ErbB ligand. Next, the therapeutic efficacy ofanti-ErbB2mAbs was examined in nude mice bearing established BT-474, MDA-MB-231,or MCF-7breast cancer xenograft tumors. Our data showed that TPLwas far moreeffective in suppressing the in vivo growth of these three breast cancer cell lines than allthe other anti-ErbB2mAbs tested, suggesting that it has the great potential to be translatedto the clinic.