Therefore, novel anti-HER2 mAbs and modalities have been desired

Therefore, novel anti-HER2 mAbs and modalities have been desired. defucosylated mouse IgG2a type of mAb against HER2 (H2Mab-139-mG2a-f) to enhance antibody-dependent cellular cytotoxicity (ADCC)-mediated antitumor activity. H2Mab-139-mG2a-f exhibits a high binding affinity in circulation cytometry with the dissociation constant (= 3). Fluorescence data were recognized using the SA3800 Cell Analyzer. The apparent dissociation constant (= 8) or control mouse IgG (mIgG, Wako) (= 8) in 100 L PBS were intraperitoneally injected. On days 14 and 22, additional antibody injections were performed. The tumor volume was measured on days 8, 12, 14, 19, 22, and 26 after the inoculation of cells. We injected BT-474 and MDA-MB-468 (5 106 cells) subcutaneously into the remaining flank of BALB/c nude mice, as indicated above. On day time 7 post-inoculation, 100 g of H2Mab-139-mG2a-f (= 8) or control mIgG (= 8) in 100 L PBS was intraperitoneally injected. On days 14 and 21, additional antibody injections were performed. The tumor volume was measured on days 7, 10, 14, 16, 21, 24, and 28 after the inoculation of cells. 2.11. Statistical Analyses All data are demonstrated SGL5213 as mean standard error of the mean (SEM). Welchs < 0.05 was considered to indicate a statistically significant difference. 3. Results 3.1. Detection of HER2 Using H2Mab-139-mG2a-f by Flow Cytometry We previously founded an anti-HER2 mAb (H2Mab-139, IgG1, kappa) from the immunization of the HER2 ectodomain produced by glioblastoma LN229 cells [23]. H2Mab-139 was advantageous for western blotting, circulation cytometry, and IHC [23]. Here, we manufactured a class-switched and defucosylated H2Mab-139 (H2Mab-139-mG2a-f) by fusing the Vchains of H2Mab-139 with the Cchains of mouse IgG2a (Number 1A). We confirmed that H2Mab-139-mG2a-f was selectively identified by anti-IgG2a, but not anti-IgG1 secondary antibodies in circulation cytometry (Number 1B). H2Mab-139-mG2a-f recognized CHO/HER2 cells, but not parental CHO-K1 cells (Number 1C). The reactivity did not change compared to the unique H2Mab-139 (Number 1C). Furthermore, H2Mab-139-mG2a-f reacted with HEK-293T cells, but not with HER2-KO HEK293T (BINDS-23) cells (Number 1D). We next investigated the reactivity of H2Mab-139-mG2a-f against breast tumor cell lines. As demonstrated in Number 1E, H2Mab-139-mG2a-f reacted HER2-positive breast cancer cell collection, BT-474, but not the triple-negative breast tumor (TNBC) cell collection, MDA-MB-468. Open in a separate window Number 1 Circulation cytometry using H2Mab-139-mG2a-f. (A) A core-fucose-deficient mouse IgG2a mAb, H2Mab-139-mG2a-f was produced from H2Mab-139 (mouse IgG1). (B) LN229/HER2 cells were treated with 1 g/mL of H2Mab-139-mG2a-f (reddish) or buffer control (packed gray), followed by Alexa Fluor 488-conjugated anti-mouse IgG or Fluorescein-conjugated anti-mouse weighty chains (IgG1 and IgG2a). (C) CHO-K1 and CHO/HER2 cells were treated with 10 g/mL of H2Mab-139-mG2a-f (reddish), H2Mab-139 (reddish), or buffer control (packed gray), followed by Alexa Fluor 488-conjugated anti-mouse IgG. (D) HEK293T and HER2-KO HEK293T (BINDS-23) cells were treated with 10 g/mL of H2Mab-139-mG2a-f (reddish) or buffer control (packed gray), followed by Alexa Fluor 488-conjugated anti-mouse IgG. (E) Breast tumor cell lines, BT-474 and MDA-MB-468 cells were treated with 10 g/mL of H2Mab-139-mG2a-f (reddish) or buffer control (packed gray), followed by Alexa SGL5213 Fluor 488-conjugated anti-mouse IgG. A kinetic analysis of the relationships of H2Mab-139-mG2a-f with CHO/HER2 and BT-474 was performed by circulation cytometry. The apparent < 0.05) (Figure 3A). No difference was observed between H2Mab-139-mG2a-f and control mIgG2a about ADCC against CHO-K1 (Number 3B). Open in a separate windowpane Number 3 H2Mab-139-mG2a-f-mediated ADCC and CDC activities in CHO/HER2 and CHO-K1 cells. (A,B) ADCC induced by H2Mab-139-mG2a-f or control mouse IgG2a (mIgG2a) against CHO/HER2 (A) and CHO-K1 (B) cells. (C,D) CDC induced by H2Mab-139-mG2a-f or control mIgG2a against CHO/HER2 (C) and CHO-K1 (D) cells. Ideals are demonstrated as mean SEM. Asterisks show statistical significance (* < 0.05; Welchs < 0.05). There was no difference between H2Mab-139-mG2a-f and control mIgG2a in CDC for CHO-K1 (Number 3D). These results showed that H2Mab-139-mG2a-f exerted significantly high levels of ADCC/CDC against CHO/HER2 cells. 3.4. Antitumor Effects of H2Mab-139-mG2a-f in the Mouse Xenografts of CHO/HER2 Cells Following a inoculation of CHO/HER2, we injected H2Mab-139-mG2a-f and control mIgG intraperitoneally into CHO/HER2 xenograft tumor-bearing mice on days 8, 14, and 22. On days 8, 12, 14, 19, 22, and 26 after the tumor inoculation, we measured the tumor volume. The H2Mab-139-mG2a-f administration reduced the tumor volume on days 19 (< 0.01), 22 (< 0.01), and 26 (< 0.01) compared with that SGL5213 of mIgG (Number 4A). The H2Mab-139-mG2a-f administration led to a 52% reduction of the tumor volume compared with that of the control mIgG on day time 26 post-injection. Open in a separate window Number 4 Antitumor activity of H2Mab-139-mG2a-f against CHO/HER2 xenograft. (A) CHO/HER2 cells were subcutaneously injected into BALB/c nude Lypd1 mice (day time 0). On day time 8, 100 g of H2Mab-139-mG2a-f or control normal mouse IgG (mIgG) was injected intraperitoneally into mice. Additional antibodies were injected on days 14 and 22. We measured the tumor.