Expression Enhancement in Trastuzumab Therapeutic Monoclonal Antibody Production using Genomic Amplification with Methotrexate

Abstract

Background: Trastuzumab (Herceptin) is a humanized monoclonal antibody (mAb) which is used for specific treatment of metastatic breast cancer in patients with overexpression of HER2/neu receptor. In this study, we have attempted to develop a biosimilar version of trastuzumab mAb.

Methods: According to in silico studies, the heavy and light chains of trastuzumab mAb were designed and constructed. The recombinant constructs were co-transfected in CHO DG44 cell line. Stable transformants were selected on a semi solid medium. Genomic amplification with methotrexate was achieved for heavy chain gene amplification. Biological activity of produced antibody in comparison with Herceptin was tested by flow cytometry method.

Results: Three folds of amplification were obtained after seven rounds of methotrexate treatments. The results indicated the equal expression level of heavy and light chains. The yield of purified mAb was between 50 to 60 mg/l /day. According to the results, the produced mAb had similar affinity to HER2(+) tumor cells to that of Herceptin.

Conclusion: High-level recombinant protein expression can be achieved by amplification of the recombinant gene with a selectable marker, such as Dihydrofolate Reductase (DHFR). It is usually accepted that DHFR gene can be amplified in DHFR(－) CHO cells, which consequently leads to amplification of the co-linked target gene, and finally amplification of recombinant protein. In this research, with the aim of producing a biosimilar version of herceptin, the effect of genomic amplification was investigated on the increasing the gene copy number using quantitative real-time PCR.

Keywords: Dihydrofolate reductase (DHFR); Monoclonal antibody; Recombinant proteins; Trastuzumab.

Figure 1

A clonal cell line that was derived from a single cell on semi-solid cloning matrix

Figure 2

Multiplex PCR. Lanes 1-11: Multiplex PCR products of STD 3-6 and 8-14 genomic DNA. Lane 12: untransfected CHO DG44 cell line as negative control. Lane 13: GeneRuler™ 100bp DNA Ladder (Fermentas). Lane 14: pSLO7 plasmid as positive control for the light chain. Lane 15: pSHC12 plasmid as positive control for the heavy chain

Figure 3

Western blotting using anti-human IgG1 antibody on PVDF membrane. Lanes 1-9: concentrated supernatant of STD 12-18 and 20-21 transformants. Lane 10: PageRuler™ plus pre-stained protein ladder (Fermentas). Lane 11: concentrated supernatant of untransfected CHO DG44 cell line as negative control

Figure 4

Genomic amplification with methotrexate. Lanes 1 and 2: concentrated supernatant of STD7 transformant in reducing and non-reducing conditions. Lanes 3 and 4: concentrated supernatant of STD72G transformant in reducing and non-reducing condition. Lanes 5 and 6: concentrated supernatant of STD76G transformant in reducing and non-reducing condition. Lanes 7 and 8: concentrated supernatant of STD77G transformant in reducing and non-reducing condition. Lane 9: PageRuler™ unstained broad range protein ladder (Fermentas). Lanes 10 and 11: Herceptin in reducing and non-reducing condition as positive control. Lane 12: culture supernatant from untransfected CHO DG44 cell line as negative control. Lane 13: cell lysate of STD77G construct

Figure 5

Flow cytometry. Cell lines staining; A, A’) CHO DG44; B, B’) MDA-MB-361; C, C’) MCF7; D, D’) MDA-MB-468; E, E’) SK-BR-3; F, F’) SK-OV-3; G, G’) T-47D and H, H’) MDA-MB-453) using the anti-human IgG FITC conjugate antibody. The treatment were performed using produced purified Trastuzumab antibody (A-H figs) as experiment (blue-dash lines), Herceptin antibody (A’-H’ figs) as positive control (blue-dash lines) and anti-human IgG1 (A-H and A’-H’ figs) as isotype-matched control antibody (black lines)