Hersintuzumab: A novel humanized anti-HER2 monoclonal antibody induces potent tumor growth inhibition

Abstract

Humanized monoclonal antibodies (mAbs) against HER2 including trastuzumab and pertuzumab are widely used to treat HER2 overexpressing metastatic breast cancers. These two mAbs recognize distinct epitopes on HER2 and their combination induces a more potent blockade of HER2 signaling than trastuzumab alone. Recently, we have reported characterization of a new chimeric mAb (c-1T0) which binds to an epitope different from that recognized by trastuzumab and significantly inhibits proliferation of HER2 overexpressing tumor cells. Here, we describe humanization of this mAb by grafting all six complementarity determining regions (CDRs) onto human variable germline genes. Humanized VH and VL sequences were synthesized and ligated to human γ1 and κ constant region genes using splice overlap extension (SOE) PCR. Subsequently, the humanized antibody designated hersintuzumab was expressed and characterized by ELISA, Western blot and flow cytometry. The purified humanized mAb binds to recombinant HER2 and HER2-overexpressing tumor cells with an affinity comparable with the chimeric and parental mouse mAbs. It recognizes an epitope distinct from those recognized by trastuzumab and pertuzumab. Binding of hersintuzumab to HER2 overexpressing tumor cells induces G1 cell cycle arrest, inhibition of ERK and AKT signaling pathways and growth inhibition. Moreover, hersintuzumab could induce antibody-dependent cell cytotoxicity (ADCC) on BT-474 cells. This new humanized mAb is a potentially valuable tool for single or combination breast cancer therapy.

Inhibitory Effect of Polyclonal Antibodies Against HER3 Extracellular Subdomains on Breast Cancer Cell Lines

“…Approval of Pertuzumab by FDA as a combination therapy together with Trastuzumab in HER2 overexpressing metastatic breast cancer patients is an example of these mAbs (Scheuer et al, 2009;D'Souza et al, 2014;Yonesaka et al, 2016). We have recently produced a novel humanized mAb (Hersintuzumab) which displays superior tumor inhibitory activity in combination with Trastuzumab as compared to the combination of Pertuzumab and Trastuzumab (Amiri et al, 2018).The same principle may be applicable for HER3 specific mAbs.…”

A Novel Anti-HER2 Bispecific Antibody With Potent Tumor Inhibitory Effects In Vitro and In Vivo

“…Two forms of anti-HER2 DVD-Igs, based on the proximity of variable domains of trastuzumab or hersintuzumab to N-terminal of the heavy and light chains of DVD-Ig, were designed as described previously ( 31 ). Briefly, the VL and VH sequences of hersintuzumab ( 12 ) and VL (GenBank: GM685466.1) and VH (GenBank: GM685464.1) sequences of trastuzumab were attached with two linkers ASTKGPSVFPLAP and TVAAPSVFIFPP, respectively, and then joined to human IgG1 heavy and Cκ light chain constant domains. These linkers are basically the same as the natural linkers present between the constant and variable domains of the heavy and light chains of an IgG molecule, providing a flexible conformation to both domains.…”

“…Binding affinity of DVD-Igs was measured with ELISA, as described previously ( 12 ). Briefly, serial concentrations (1200–75 ng/mL) of recombinant subdomains I+II, III+IV, or full HER2-ECD were coated in ELISA plate for 1.5 hours at 37°C.…”

“…Recently, we produced a humanized mAb, hersintuzumab, which targets domains I–II of human HER2, a distinct epitope from those recognized by trastuzumab and pertuzumab ( 12 ). Hersintuzumab induced growth inhibition, G1 cell cycle arrest, and cell signaling inhibition in HER2 expressing tumor cell lines ( 12 ). This mAb displayed significant synergistic inhibitory effects in combination with trastuzumab.…”

Antitumor Effects of a Recombinant Baculovirus Displaying Anti-HER2 Scfv Expressing Apoptin in HER2 Positive SK-BR-3 Breast Cancer Cells

“…Then 50 μl of recombinant HER2-ECD antigen solution at 2 μg/ml in PBS added to each well and incubated for 1 hr at 37 ˚C. After washing the wells, 50 μl of HRP conjugated anti-HER2 monoclonal antibody (1T0 mAb), which recognizes different epitopes to trastuzumab [26], was added to each well and incubated for 1 hr at 37 ˚C. Following a final wash, TMB substrate was added to each well and absorbance read at 450 nm.…”