Kaneko MK, Suzuki H, Kato Y.
Establishment of a novel cancer-specific anti-HER2 monoclonal antibody H2Mab-250/H2CasMab-2 for breast cancers
Monoclon. Antib. Immunodiagn. Immunother., https://doi.org/10.20944/preprints202309.0906.v3, 2023 (preprint)
Figure 1. Flow cytometry using anti-HER2 mAbs. (A) Flow cytometry using H2Mab-250 (10 μg/mL; Red line) and H2Mab-119 (10 μg/mL; Blue line) against CHO-K1, CHO/HER2, HER2-positive breast cancers (BT-474 and SK-BR-3), a triple-negative breast cancer (MDA-MB-468), and non-transformed normal epithelial cells (HaCaT and MCF 10A). (B) Flow cytometry using H2Mab-250 (10 μg/mL; Red line) and H2Mab-119 (10 μg/mL; Blue line) against immortalized normal epithelial cells including hTERT TIGKs (gingiva), HBEC3-KT (lung bronchus), hTERT-HME1 (mammary gland), hTCEpi (corneal), hTEC/SVTERT24-B (thymus), RPTEC/TERT1 (kidney proximal tubule), and HCEC-1CT (colon). The black line represents the negative control (blocking buffer).
Figure 2. ADCC reporter assay by H2Mab-250-mG2a-f and trastuzumab in the presence of BT-474 and HaCaT cells. Target cells such as BT-474 (A) or HaCaT (B) were cultured in a 96-well white solid plate. H2Mab-250-mG2a-f and trastuzumab were serially diluted and added to the target cells. The engineered Jurkat cells were then added and co-cultured with antibody-treated target cells. Luminescence using the Bio-Glo Luciferase Assay System was measured using a GloMax luminometer. N.D., not determined. Error bars represent means ± SDs.
Figure 3. Immunohistochemical analysis of H2Mab-250 in breast cancer and normal epithelium. (A) The HER2-positive breast cancer tissue sections were treated with H2Mab-250 or H2Mab-119 (0.1 or 0.5 µg/mL). (B) A normal heart section was treated with H2Mab-250 (1 µg/mL). (C) Sections of normal breast, stomach, lung, colon, kidney, and esophagus were treated with H2Mab-250 (0.1 µg/mL). The sections were then treated with the Envision+ kit. The chromogenic reaction was performed using DAB, and the sections were counterstained with hematoxylin. Scale bar = 100 µm.
Figure 4. Epitope identification for H2Mab-250. (A) Epitope determination of H2Mab-250 and H2Mab-119 using flow cytometry. The schematic representation of HER2 and the deletion mutants (left). Flow cytometry using H2Mab-250 (10 μg/mL; Red line) and H2Mab-119 (10 μg/mL; Blue line) against CHO/HER2 (WT and deletion mutants). The cell surface expression was confirmed by an anti-PA tag mAb, NZ-1 (10 μg/mL; Green). The cells were treated with Alexa Fluor 488-conjugated anti-mouse IgG for H2Mab-250 and H2Mab-119 or Alexa Fluor 488-conjugated anti-rat IgG for NZ-1. The black line represents the negative control (blocking buffer). (B and C) Determination of H2Mab-250 epitope by ELISA. Five synthesized peptides that cover the HER2 domain IV (B), alanine-substituted peptides of HER2 domain IV (603–622) (C), HER2ec, or buffer control (NC) were immobilized on immunoplates. The plates were incubated with H2Mab-250 (10 μg/mL), followed by incubation with peroxidase-conjugated anti-mouse immunoglobulins. Optical density was measured at 655 nm. Error bars represent means ± SDs. (D) Flow cytometry using H2Mab-250 (10 μg/mL; Red line) against CHO/HER2 (WT and W614A). The cell surface expression was confirmed by an anti-PA tag mAb, NZ-1 (10 μg/mL; Red line). The black line represents the negative control (blocking buffer). (E) Surface plasmon resonance analysis between H2Mab-250 and HER2 domain IV (603–622) peptides. The affinity constant (KA) at equilibrium was calculated as 1/KD.
Supplementary Figure 1. Flow cytometry using anti-HER2 mAbs. The cells were treated with H2Mab-77 (10 μg/mL), H2Mab-139 (10 μg/mL), H2Mab-193 (10 μg/mL), H2Mab-215 (10 μg/mL), H2Mab-19 (10 μg/mL), H2Mab-181 (10 μg/mL), H2Mab-41 (10 μg/mL), and trastuzumab (10 μg/mL) against CHO-K1, CHO/HER2, HER2-positive breast cancers (BT-474 and SK-BR-3), a triple-negative breast cancer (MDA-MB-468), and non-transformed normal epithelial cells (HaCaT and MCF 10A). The cells were treated with Alexa Fluor 488-conjugated anti-mouse IgG for mouse anti-HER2 mAbs or FITC-conjugated anti-human IgG for trastuzumab. The black line represents the negative control (blocking buffer).
Supplementary Figure 2. Flow cytometry using anti-HER2 mAbs. The cells were treated with H2Mab-77 (10 μg/mL), H2Mab-139 (10 μg/mL), H2Mab-193 (10 μg/mL), H2Mab-215 (10 μg/mL), H2Mab-19 (10 μg/mL), H2Mab-181 (10 μg/mL), H2Mab-41 (10 μg/mL), and trastuzumab (10 μg/mL) against immortalized normal epithelial cells, including hTERT TIGKs (gingiva), HBEC3-KT (lung bronchus), hTERT-HME1 (mammary gland), hTCEpi (corneal), hTEC/SVTERT24-B (thymus), RPTEC/TERT1 (kidney proximal tubule), and HCEC-1CT (colon). The cells were treated with Alexa Fluor 488-conjugated anti-mouse IgG for mouse anti-HER2 mAbs or FITC-conjugated anti-human IgG for trastuzumab. The black line represents the negative control (blocking buffer).
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