Novel functional anti-HER3 monoclonal antibodies with potent anti-cancer effects on various human epithelial cancers

Resistance of progressive cancers against chemotherapy is a serious clinical problem. In this context, human epidermal growth factor receptor 3 (HER3) can play important roles in drug resistance to HER1- and HER2- targeted therapies. Since clinical testing of anti-HER3 monoclonal antibodies (mAbs) such as patritumab could not show remarkable effect compared with existing drugs, we generated novel mAbs against anti-HER3. Novel rat mAbs reacted with HEK293 cells expressing HER3, but not with cells expressing HER1, HER2 or HER4. Specificity of mAbs was substantiated by the loss of mAb binding with knockdown by siRNA and knockout of CRISPR/Cas9-based genome-editing. Analyses of CDR sequence and germline segment have revealed that seven mAbs are classified to four groups, and the binding of patritumab was inhibited by one of seven mAbs. Seven mAbs have shown reactivity with various human epithelial cancer cells, strong internalization activity of cell-surface HER3, and inhibition of NRG1 binding, NRG1-dependent HER3 phosphorylation and cell growth. Anti-HER3 mAbs were also reactive with in vivo tumor tissues and cancer tissue-originated spheroid. Ab4 inhibited in vivo tumor growth of human colon cancer cells in nude mice. Present mAbs may be superior to existing anti-HER3 mAbs and support existing anti-cancer therapeutic mAbs

スケールフリーネットワーク上で観察されるEffectiveness model 適用Vaccination Gameにおける感染率へのadditive ノイズがもたらす確率共鳴現象

修士（工学）Master of Engineerin

Dual‐targeting therapy against HER3/MET in human colorectal cancers

Abstract Background Colorectal cancer (CRC) is the most common malignancy in the world, and novel molecular targeted therapies for CRC have been vigorously pursued. We searched for novel combination therapies based on the expression patterns of membrane proteins in CRC cell lines. Results A positive correlation was observed between the expression of human pidermal growth factor receptor (HER) 3 and mesenchymal‐to‐epithelial transition factor (MET) on the cell surface of CRC cell lines. The brief stimulation of HER3/MET‐high SW1116 CRC cells with both neuregulin‐1 (NRG1) and hepatocyte growth factor enhanced ERK phosphorylation and cell proliferation more than each stimulation alone. In addition, a prolonged NRG1 stimulation resulted in the tyrosine phosphorylation of MET. In this context, the Forkhead Box protein M1 (FOXM1)‐regulated tyrosine phosphorylation of MET by NRG1 was demonstrated, suggesting the existence of a signaling pathway mediated by FOXM1 upon the NRG1 stimulation. Since the co‐expression of HER3 and MET was also demonstrated in in vivo CRC tissues by immunohistochemistry, we investigated whether the co‐inhibition of HER3 and MET could be an effective therapy for CRC. We established HER3‐and/or MET‐KO SW1116 cell lines, and HER3/MET‐double KO resulted in the inhibition of in vitro cell proliferation and in vivo tumor growth in nude mice by SW1116 cells. Furthermore, the combination of patritumab, an anti‐HER3 fully human mAb, and PHA665752, a MET inhibitor, markedly inhibited in vitro cell proliferation, 3D‐colony formation, and in vivo tumor growth in nude mice by SW1116 cells Conclusion The dual targeting of HER3/MET has potential as CRC therapy

Immuno-PET Imaging of HER3 in a Model in which HER3 Signaling Plays a Critical Role.

HER3 is overexpressed in various carcinomas including colorectal cancer (CRC), which is associated with poor prognosis, and is involved in the development of therapy resistance. Thus, an in vivo imaging technique is needed to evaluate the expression of HER3, an important therapeutic and diagnostic target. Here, we report successful HER3 PET imaging using a newly generated anti-human HER3 monoclonal antibody, Mab#58, and a mouse model of a HER3-overexpressing xenograft tumor. Furthermore, we assessed the role of HER3 signaling in CRC cancer tissue-originated spheroid (CTOS) and applied HER3 imaging to detect endogenous HER3 in CTOS-derived xenografts. Cell binding assays of 89Zr-labeled Mab#58 using the HER3-overexpressing cell line HER3/RH7777 demonstrated that [89Zr]Mab#58 specifically bound to HER3/RH7777 cells (Kd = 2.7 nM). In vivo biodistribution study in mice bearing HER3/RH7777 and its parent cell xenografts showed that tumor accumulation of [89Zr]Mab#58 in HER3/RH7777 xenografts was significantly higher than that in the control from day 1 to day 4, tending to increase from day 1 to day 4 and reaching 12.2 ± 4.5%ID/g. Radioactivity in other tissues, including the control xenograft, decreased or remained unchanged from day 1 to day 6. Positron emission tomography (PET) in the same model enabled clear visualization of HER3/RH7777 xenografts but not of RH7777 xenografts. CTOS growth assay and signaling assay revealed that CRC CTOS were dependent on HER3 signaling for their growth. In PET studies of mice bearing a CRC CTOS xenograft, the tumor was clearly visualized with [89Zr]Mab#58 but not with the 89Zr-labeled control antibody. Thus, tumor expression of HER3 was successfully visualized by PET with 89Zr-labeled anti-HER3 antibody in CTOS xenograft-bearing mice, a model that retains the properties of the patient tumor. Non-invasive targeting of HER3 by antibodies is feasible, and it is expected to be useful for cancer diagnosis and treatment

Immuno-PET Imaging of HER3 in a Model in which HER3 Signaling Plays a Critical Role.

HER3 is overexpressed in various carcinomas including colorectal cancer (CRC), which is associated with poor prognosis, and is involved in the development of therapy resistance. Thus, an in vivo imaging technique is needed to evaluate the expression of HER3, an important therapeutic and diagnostic target. Here, we report successful HER3 PET imaging using a newly generated anti-human HER3 monoclonal antibody, Mab#58, and a mouse model of a HER3-overexpressing xenograft tumor. Furthermore, we assessed the role of HER3 signaling in CRC cancer tissue-originated spheroid (CTOS) and applied HER3 imaging to detect endogenous HER3 in CTOS-derived xenografts. Cell binding assays of 89Zr-labeled Mab#58 using the HER3-overexpressing cell line HER3/RH7777 demonstrated that [89Zr]Mab#58 specifically bound to HER3/RH7777 cells (Kd = 2.7 nM). In vivo biodistribution study in mice bearing HER3/RH7777 and its parent cell xenografts showed that tumor accumulation of [89Zr]Mab#58 in HER3/RH7777 xenografts was significantly higher than that in the control from day 1 to day 4, tending to increase from day 1 to day 4 and reaching 12.2 ± 4.5%ID/g. Radioactivity in other tissues, including the control xenograft, decreased or remained unchanged from day 1 to day 6. Positron emission tomography (PET) in the same model enabled clear visualization of HER3/RH7777 xenografts but not of RH7777 xenografts. CTOS growth assay and signaling assay revealed that CRC CTOS were dependent on HER3 signaling for their growth. In PET studies of mice bearing a CRC CTOS xenograft, the tumor was clearly visualized with [89Zr]Mab#58 but not with the 89Zr-labeled control antibody. Thus, tumor expression of HER3 was successfully visualized by PET with 89Zr-labeled anti-HER3 antibody in CTOS xenograft-bearing mice, a model that retains the properties of the patient tumor. Non-invasive targeting of HER3 by antibodies is feasible, and it is expected to be useful for cancer diagnosis and treatment

<i>In vitro</i> assays of radiolabeled anti-HER3 antibody Mab#58.

<p>(A) Cell binding assay for [⁸⁹Zr]Mab#58 comparing HER3 overexpressing HER3/RH7777 cells (closed triangles) and parent RH7777 cells (closed circles). (B) Competitive inhibition assay for DF-conjugated (closed circles) and unconjugated Mab#58 (closed squares). (C) Internalization assay for [⁸⁹Zr]Mab#58 at 37°C. Changes in the percentage of total radioactivity for each fraction are plotted against incubation time at 37°C (closed circles, internalized fraction; open triangles, membrane-bound fraction; open circles, protein-bound fraction in the culture medium; closed triangles, non-protein-bound fraction in the culture medium). These assays were conducted in duplicate. Data represent the mean.</p

CTOS growth in various culture conditions.

<p>(A) Growth curve of CTOS cultured in StemPro hESC (Invitrogen) or basal medium containing one of the growth factors as indicated. (B) Representative images of CTOS at day 14 in (A).</p

Representative PET images of mice bearing CTOS C45 xenografts.

<p>Serial PET images of mice bearing C45 xenograft (white arrow) injected with [⁸⁹Zr]Mab#58 (upper panels) or similarly ⁸⁹Zr-labeled control IgG.</p

Representative immunohistochemical staining of HER3/RH7777 (A) and RH7777 (B) xenografts for HER3 expression.

<p>Representative immunohistochemical staining of HER3/RH7777 (A) and RH7777 (B) xenografts for HER3 expression.</p