Defucosylated mouse‑dog chimeric anti‑HER2 monoclonal antibody exerts antitumor activities in mouse xenograft models of canine tumors

Abstract

Human epidermal growth factor receptor 2 (HER2) overexpression has been reported in various types of cancer, including breast, gastric, lung, colorectal and pancreatic cancer. A humanized anti-HER2 monoclonal antibody (mAb), trastuzumab, has been shown to improve survival of patients in HER2-positive breast and gastric cancer. An anti-HER2 mAb, H 2 Mab-77 (mouse IgG 1 , kappa) was previously developed. In the present study, a defucosylated version of mouse-dog chimeric anti-HER2 mAb (H77Bf) was generated. H77Bf possesses a high binding-affinity [a dissociation constant (K D ): 7.5x10 -10 M, as determined by flow cytometric analysis] for dog HER2-overexpressed CHO-K1 (CHO/dHER2) cells. H77Bf highly exerted antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) for CHO/dHER2 cells by canine mononuclear cells and complement, respectively. Moreover, administration of H77Bf significantly suppressed the development of CHO/dHER2 xenograft tumor in mice compared with the control dog IgG. H77Bf also possesses a high binding-affinity (K D : 7.2x10 -10 M) for a canine mammary gland tumor cell line (SNP), and showed high ADCC and CDC activities for SNP cells. Intraperitoneal administration of H77Bf in mouse xenograft models of SNP significantly suppressed the development of SNP xenograft tumors compared with the control dog IgG. These results indicated that H77Bf exerts antitumor activities against dHER2-positive canine cancers, and could be valuable treatment regimen for canine cancers.

Antitumor Activity of an anti-EGFR/HER2 Bispecific Antibody in a Mouse Xenograft Model of Canine Osteosarcoma

“…CHO-K1 and a canine OS cell line D-17 were purchased from the American Type Culture Collection. Stable dog EGFR-overexpressed CHO-K1 (CHO/dEGFR) [26] and dog HER2overexpressed CHO-K1 (CHO/dHER2) [27] were established as described previously. CHO-K1, CHO/dEGFR, and CHO/dHER2 were cultured in RPMI-1640 medium (Nacalai Tesque, Inc.), supplemented with 10% heat-inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, Inc.), 100 units/mL of penicillin, 100 μg/mL streptomycin, and 0.25 μg/mL amphotericin B (Nacalai Tesque, Inc.).…”

“…Previously, we have established mouse-dog chimeric anti-EGFR mAb (E134B) [26] and anti-HER2 mAb (H77B) [27]. In this study, to generate E134Bf-H77scFv, we first constructed a single chain Fv of H77B (H77scFv) by connecting the VH and VL cDNA of H77B with a linker sequence (GGGGSGGGGSGGGGS).…”

“…The H77scFv cDNA was further fused at the 3' end of the light chain cDNA of E134B. The cDNA of the E134B heavy chain and E134B light chain-H77scFv were transduced into BINDS-09 (FUT8 knockout ExpiCHO-S) cells using the ExpiCHO Expression System (Thermo Fisher Scientific, Inc.) to produce the defucosylated form [25][26][27][28][29][30][31][32]. A defucosylated and bispecific antibody, E134Bf-H77scFv, was purified using Ab-Capcher (ProteNova Co., Ltd.).…”

“…Canine mononuclear cells (MNCs) obtained from Yamaguchi University were resuspended in DMEM (Nacalai Tesque, Inc.) with 10% FBS and were used as effector cells. Target D-17 cells were labeled with 10 μg/mL Calcein AM (Thermo Fisher Scientific, Inc.) [25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42]. The target cells (2 × 10 4 cells) were plated in 96-well plates and mixed with effector canine MNCs (effector/target cells ratio, 50), 100 μg/mL of control normal dog IgG, E134Bf, H77Bf, and E134Bf-H77scFv.…”

“…Subsequently, we produced mouse-dog chimeric anti-EGFR mAb (E134B) and anti-HER2 mAb (H77B) from the information of variable regions of EMab-134 and H2Mab-77, respectively. Furthermore, we evaluated the antitumor effect of their respective defucosylated forms, E134Bf [25,26] and H77Bf [27] against canine tumors in mouse xenograft models. In this study, we produced a defucosylated bispecific Ab against dEGFR and dHER2, and evaluated the ADCC, CDC, and antitumor activities in canine osteosarcoma D-17 xenograft.…”