Structural and functional characterization of MBS301, an afucosylated bispecific anti-HER2 antibody

Abstract

MBS301, a glyco-engineered bispecific anti-human epidermal growth factor receptor 2 (HER2) antibody with a typical IgG1 monoclonal antibody structure, was developed through dual-cell expression and in vitro assembling process. MBS301 consists of two half antibodies engineered from trastuzumab and pertuzumab, respectively. Integrity and purity profiles of MB301 indicated that the heterodimerization of the two half antibodies was successful. The high and similar melting temperatures (Tm1,72.0°C and Tm2, 84.8°C) of MBS301 compared with those of its parental monoclonal antibodies trastuzumab and pertuzumab (in-house made T-mab and P-mab, respectively) revealed its structural compactness. With computer-modeling experiments and Biacore binding and competition kinetics studies, the binding stoichiometry between MBS301 and HER2-ECD was determined to be 1:1 and the two arms of MBS301 were shown to bind to domains II and IV of HER2-ECD antigen simultaneously. MBS301 displayed synergistic bioactivities as the combination of T-mab and P-mab in vitro in multiple cancer cell lines and in vivo in xenograft mouse model studies, and showed more effective activity than T-mab or P-mab used individually. Moreover, fucose-knockout dramatically increased MBS301’s binding affinity to low affinity FcγRIIIa allotype 158F (KD = 2.35 × 10−7M) to near the high affinity level of allotype V158 (KD = 1.17 × 10−7M). This resulted in far more effective ADCC activity of MBS301 than the combination of T-mab and P-mab in killing HER2-positive cancer cells. Hence, a novel fully afucosylated anti-HER2 bispecific antibody with improved antitumor activities was generated and shown to have the potential to be used for treating HER2-positive but trastuzumab-resistant solid tumors.

Cryo-EM Structure of HER2-trastuzumab-pertuzumab complex

“…[19] The second approach was the knob-into-hole Fc technique, in which half antibodies of trastuzumab and pertuzumab were assembled together. [20, 21] Strong inhibition of HER2-positive breast cancer cell proliferation was also observed in vitro and in vivo for these bispecific antibodies KN026 and MBS301. [20, 21] However, the anti-tumor activities of all the bispecific antibodies from both approaches were comparable to or slightly better than the combination of trastuzumab and pertuzumab.…”

“…[20, 21] Strong inhibition of HER2-positive breast cancer cell proliferation was also observed in vitro and in vivo for these bispecific antibodies KN026 and MBS301. [20, 21] However, the anti-tumor activities of all the bispecific antibodies from both approaches were comparable to or slightly better than the combination of trastuzumab and pertuzumab. It appears from the cryo-EM structure of HER2-trastuzumab-pertuzumab that, even with the flexibility of the hinge region and the flexibility between the variable and the constant regions, the two Fab arms of one bispecific antibody cannot bind to both domains II and IV of one HER2 molecule simultaneously (Figs 4D and 5A), considering all the various conformations of IgG1.…”

Anti-ErbB2 immunotherapeutics: struggling to make better antibodies for cancer therapy

“…MBS301 54 and BCD-147 (Phase 1 study NCT03912441) are also bispecific antibodies that include binding sites derived from trastuzumab and pertuzumab. MBS301 was genetically modified to knock out fucosylation, thus improving its binding to low affinity FcγR.…”

“…MBS301 was genetically modified to knock out fucosylation, thus improving its binding to low affinity FcγR. 54 MBS301 is being evaluated in a Phase 1 study (NCT03842085) of patients with ErbB2-positive recurrent or metastatic malignant solid tumor, while the Phase 1 study of BCD-147 (NCT03912441) includes healthy volunteers.…”

Bp-Bs, a Novel T-cell Engaging Bispecific Antibody with Biparatopic Her2 Binding, Has Potent Anti-tumor Activities

“…Trastuzumab combined with pertuzumab plus docetaxel has already been approved for the first-line treatment of patients with Her2-positive metastatic breast cancer 17 . Bispecific antibodies targeting both Her2 and Her3, 18 targeting two different epitopes of Her2,19, 20 or engaging T cells to Her2 cancer cells by targeting Her2 and CD3 21 have also exhibited encouraging results in phase I or pre-clinical studies.…”