Screening of multiple myeloma by polyclonal rabbit anti-human plasmacytoma cell immunoglobulin

Abstract

Antibody-based immunotherapy has been effectively used for tumor treatment. However, to date, only a few tumor-associated antigens (TAAs) or therapeutic targets have been identified. Identification of more immunogenic antigens is essential for improvements in multiple myeloma (MM) diagnosis and therapy. In this study, we synthesized a polyclonal antibody (PAb) by immunizing rabbits with whole human plasmacytoma ARH-77 cells and identified MM-associated antigens, including enlonase, adipophilin, and HSP90s, among others, via proteomic technologies. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that 200 µg/mL PAb inhibits the proliferation of ARH-77 cells by over 50% within 48 h. Flow cytometric assay indicated that PAb treatment significantly increases the number of apoptotic cells compared with other treatments (52.1% vs. NS, 7.3% or control rabbit IgG, 9.9%). In vivo, PAb delayed tumor growth and prolonged the lifespan of mice. Terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that PAb also induces statistically significant changes in apoptosis compared with other treatments (P<0.05). We therefore conclude that PAb could be used for the effective screening and identification of TAA. PAb may have certain anti-tumor functions in vitro and in vivo. As such, its combination with proteomic technologies could be a promising approach for sieving TAA for the diagnosis and therapy of MM.

Figure 1. Production and characterization of PAb.

(A) ELISA of PAb on ARH-77. Control rabbit IgG and PAb were incubated with ARH-77 at dilutions from 1∶2,000 to 1∶20,000. After addition of an alkaline phosphatase-conjugated secondary antibody, the absorbance was measured at 450 nm. Represented here is the mean of 4 wells to 6 wells ± standard deviation for every dilution. (B) Western blot showed the multiple protein bands recognized by PAb but not by control IgG. (C) Indirect immunofluorescence assay of PAb on myeloma and non-myeloma cell line by flow cytometry. Gray line represents 1∶2,000 PAb dilutions reacted with ARH-77 (left panel), U266 (upper part, middle panel), and Raji (upper part, right panel), human hepatocellular carcinoma cell line HepG2 (lower part, middle panel) and human pancreatic carcinoma cell line Panc-1(lower part, right panel). Black line represents control IgG diluted to 1∶2,000 used as a negative control. (D) Indirect immunofluorescence assay of antigens on ARH-77 by fluorescence microscopy with FITC-goat anti-rabbit IgG (left, green fluorescence) and with hoechst33258 (middle, blue fluorescence). Up line represents the treatment group with PAb and down line represents the treatment group with control IgG. Merged images (right) show localization of antigens on ARH-77 cells (400×).

Figure 2. 2-D PAGE and Western blot analysis of ARH-77 cell proteins.

(A) Western blot detection of the targeted-protein spot recognized by PAb. (B) 2-D protein pattern of ARH-77 cells after Commassie Blue staining. (C) MALDI-MS spectrum obtained from spot A1 after trypsin digestion and peptide sequences from ENO1 matching peaks obtained from MALDI-MS spectra. (D) The peptide of 703.6864 selected from the PMF of the A1 spot was sequenced by nano-ESI-MS/MS.

Figure 3. Inhibition of myeloma cells growth in vitro determined by MTT.

(A)The growth of PAb-treated cells was significantly inhibited compared with the control IgG and NS groups, and the inhibitory rates on different concentrations on ARH-77 cells after 48 h were 16.7%, 23.98%, 28.47%, and 56.84%. (B)The similar results were shown in U266 cell line. (C) The PAb did not effect growth of HepG2 cell line.

Figure 4. PAb-induced apoptosis in myeloma cell lines.

Flow cytometric analysis revealed the proportion of sub-G1 phase cells (apoptotic cells) to be 7.3% (NS), 9.9% (control), and 52.1% (PAb). The experiments were repeated at least three times.

Figure 5. Inhibitory effect of PAb on tumor growth in xenograft SCID mouse models.

(A) A significant difference in tumor volume (P<0.05) was observed between PAb-treated mice and other treatment groups. The mean ± standard error of the mean of tumor growth of five mice is shown. (B) Representative picture for tumor volume different groups. (C) A significant increase in survival was observed in PAb-treated mice compared with other treatment groups (P<0.05).

Figure 6. PAb-induced tumor cells apoptosis in vivo by TUNEL assay.

(A) Sections from the tumor-bearing mice treated with NS (left panel), control IgG (middle panel), or PAb (right panel) were stained with FITC-dUTP as described in the Materials and Methods section (200×). (B) An apparent increase in the number of apoptotic cells and apoptotic index was observed within residual tumors treated with PAb compared with other treatment groups in the ARH-77 subcutaneous injection tumor models. * represents the PAb group showing significant difference compared with NS and control IgG group mice (P<0.05).