The study on newly developed McAb NJ001 specific to non-small cell lung cancer and its biological characteristics

Abstract

Monoclonal antibody (McAb) is the key tool for cancer immunodiagnosis and immunotherapy. McAb-based immunotherapy that targets tumor antigens has had great achivement. In this study, a cell clone which kept secreting high-titer IgG1-type McAb named NJ001 against human non-small cell lung cancer (NSCLC) cells was obtained. The titer of purified NJ001 was 2×10(6). The antigen named SP70 of NSCLC specifically identified by NJ001 was proved to be a protein with the relative molecular mass (Mr) of 70 kDa. The results of immunohistochemical staining indicated that NJ001 could positively react to NSCLC, but weak positively or negatively react to human small-cell lung cancer (SCLC), pulmonary pseudotumor and other epithelial tumors. In soft agar assay, the colony formation efficiency in NJ001 groups decreased in a dose-dependent manner. For the concentration of 100 µg/ml, 200 µg/ml and 400 µg/ml, the inhibition ratio of colony formation was 23.4%, 62.5% and 100% respectively. Meanwhile, NJ001 caused significant reduction in tumor volume and tumor weight compared to control mice in lung cancer xenograft model. The tumor growth inhibition ratio in 200 µg, 400 µg and 800 µg NJ001 groups was 10.44%, 37.29% and 44.04%, respectively. NJ001 also led to cytomorphological changes and induced the apoptosis of human lung adenocarcinoma cell line SPC-A1 significantly. The newly developed NJ001 selectively reacted to NSCLC and exhibited anti-tumor activity both in vitro and in vivo. NJ001 is of great value concerning immunodiagnostics and immunotherapy for NSCLC and holds promise for further research regarding the mechanism underlying tumor progression of NSCLC.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Production and characterization of NJ001.

(A) Karyotype of NM001 hybridoma cell line (×400). (B) Binding activity of McAbs to human maligant and nonmaligant cells in culture. Undiluted supernatants from hybridomas NM001, NM004, NM005 were tested in triplicate with indirect cell ELISA as described in “Materials and Methods”. Each number represents the average absorbance of the substrate end product at 450 nm. Controls without McAbs exhibited an average absorbance of 0.02. NM001 was selected for further study as it exhibited the greatest binding ratio with lung tumor cells and the greatest specificity for the lung cancer cell lines compared with the other tumor cell lines. (C) Western blot analysis for the reaction of NJ001 produced from hybridoma NM001 with different cells (human maligant and nonmaligant cells) in culture. (Lane 1, SPC-A1; lane 2, A549; lane 3, NCI-H520; lane 4, NCI-H460; lane 5, HepG2; lane 6, ZR-75-30; lane 7, COLO 205; lane 8, WI-38; lane 9, PBMC; lane 10, Marker). GAPDH was used as a loading control. Results indicated that the expression of protein was specific to antibody NJ001 in non-small cell lung cancer cell lines, not in other cancer cell lines and normal cells. (D) Representative positive and negative results obtained from IIF analysis of reaction of NJ001 with different cells observed by fluorescence and confocal microscopy analysis (×400). (a, SPC-A1; b, ZR-75-30; c, HepG2; d, WI-38). Presence of NJ001 can be visualized in cellular cytoplasm of SPC-A1 cells, but other cell lines showed no fluorescence. Localization of the antigen specific to NJ001 may be in cellular cytoplasm of SPC-A1 cells.

Figure 2. Photomicrographs of immunohistochemistry staining with NJ001 (×200).

Representative areas of tumor sections from (A) NSCLC lung adenocarcinoma; (B) NSCLC squamous lung cancer; (C) SCLC; (D) Breast carcinoma; (E) Gastric cancer; (F) Colon cancer; (G) Ovarian cancer; (H) Liver cancer; (I) Pulmonary pseudotumor; (J) Adjacent nontumourous lung tissues.

Figure 3. Inhibitory effect of NJ001 on SPC-A1 cell proliferation.

Compared with the control treated cells, the level of proliferation of NJ001 treated SPC-A1 cells significantly decreased after 48 h and 72 h (** P<0.001).

Figure 4. Inhibition of colony formation of SPC-A1 cells by NJ001 in soft agar. (×100).

The cell suspensions (2×104 cells) mixed with 0.3% agarose and different concentrations of NJ001 or MCA2849 were layered on the top of culture media in 6-well culture plates and allowed to grow for 2 weeks before colonies were counted. Representative contrast images were shown. (A) 0 µg/mL NJ001, (B) 100 µg/mL NJ001, (C) 200 µg/mL NJ001, (D) 400 µg/mL NJ001, (E) 0 µg/mL MCA2849, (F) 100 µg/mL MCA2849, (G) 200 µg/mL MCA2849, (H) 400 µg/mL MCA2849.

Figure 5. Photomicrographs of H&E staining in the preliminary study (×200).

Representative areas of tissue sections from inoculation sites in NJ001 group (A) and the excised tumors in control group (B).

Figure 6. Inhibition of tumor growth in vivo by NJ001 in the SPC-A1 xenograft model.

(A) Tumor growth curve. Animals were subcutaneously injected with 2×106 SPC-A1 cells and intraperitoneally injected with normal saline, 200 µg, 400 µg, or 800 µg NJ001. Tumor volumes were measured at 4-day intervals. The error bars represent standard deviation. (B) Average tumor weight in the antibody and control groups. After 3 weeks of treatment, tumors were excised and weighed. The error bars represent standard deviation. * P<0.05 compared to the control group. ∧P<0.05 compared to the 200 µg NJ001 group.

Figure 7. NJ001 induced apoptosis of SPC-A1 cells.

SPC-A1 cells were cultured with or without 200 µg/mL NJ001 or MCA2849 for 24 h and 48 h. (A) Morphological changes in SPC-A1 cells were observed under inverted microscope (×100). a, 24 h NJ001; b, 24 h MCA2849; c, 24 h McAb free; d, 48 h NJ001; e, 48 h MCA2849; f, 48 h McAb free. (B) Apoptosis was analyzed by flow cytometry. (C) Each column and error bar represents the mean ± SD of three independent experiments (**P<0.001). The amount of late apoptosis was determined as the percentage of Annexin V+/PI+ cells.