Stability of the recombinant anti‑erbB2 scFv‑Fc‑interleukin‑2 fusion protein and its inhibition of HER2‑overexpressing tumor cells

Du,Yu‑Jia; Lin,Ze‑Min; Zhao,Ying‑Hua; Feng,Xiu‑Ping; Wang,Chang‑Qing; Wang,Gang; Wang,Chun‑Di; Shi,Wei; Zuo,Jian‑Ping; Li,Fan; Wang,Cheng‑Zhong

doi:10.3892/ijo.2012.1747

Stability of the recombinant anti‑erbB2 scFv‑Fc‑interleukin‑2 fusion protein and its inhibition of HER2‑overexpressing tumor cells

Authors:
- Yu‑Jia Du
- Ze‑Min Lin
- Ying‑Hua Zhao
- Xiu‑Ping Feng
- Chang‑Qing Wang
- Gang Wang
- Chun‑Di Wang
- Wei Shi
- Jian‑Ping Zuo
- Fan Li
- Cheng‑Zhong Wang
View Affiliations

Affiliations: Norman Bethune College of Medicine, Jilin University, Changchun 130021, P.R. China, Laboratory of Immunology and Virology, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, P.R. China, Key Laboratory for Molecular Enzymology and Engineering, The Ministry of Education, College of Life Science, Jilin University, Changchun, P.R. China, Pharmaceutical R&D Center, Gene Science Pharmaceuticals Co. Ltd., Changchun 130012, P.R. China
Published online on: December 20, 2012 https://doi.org/10.3892/ijo.2012.1747
Pages: 507-516

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

The anti‑erbB2 scFv‑Fc‑IL‑2 fusion protein (HFI) is the basis for development of a novel targeted anticancer drug, in particular for the treatment of HER2‑positive cancer patients. HFI was fused with the anti‑erbB2 antibody and human IL‑2 by genetic engineering technology and by antibody targeting characteristics of HFI. IL‑2 was recruited to target cells to block HER2 signaling, inhibit or kill tumor cells, improve the immune capacity, reduce the dose of antibody and IL‑2 synergy. In order to analyse HFI drug ability, HFI plasmid stability was verified by HFI expression of the trend of volume changes. Additionally, HFI could easily precipitate and had progressive characteristics and thus, the buffer system of the additive phosphate‑citric acid buffer, arginine, Triton X‑100 or Tween‑80, the establishment of a microfiltration, ion exchange, affinity chromatography and gel filtration chromatography‑based purification process were explored. HFI samples were obtained according to the requirements of purity, activity and homogeneity. In vivo, HFI significantly delayed HER2 overexpression of non‑small cell lung cancer (Calu‑3) in human non‑small cell lung cancer xenografts in nude mice, and the inhibition rate was more than 60% (P<0.05) in the group treated with 1 mg/kg the HFI dose; HFI significantly inhibited HER2 expression of breast cancer (FVB/neu) transgenic mouse tumor growth in 1 mg/kg of the HFI dose group, and in the following treatment the 400 mm3 tumors disappeared completely. Combined with other HFI test data analysis, HFI not only has good prospects, but also laid the foundation for the development of antibody‑cytokine fusion protein‑like drugs.

View Figures

View References

1.	Jallal B, Schlessinger J and Ullrich A: Tyrosine phosphatase inhibition permits analysis of signal transduction complexes in p185HER2/neu-overexpressing human tumor cells. J Biol Chem. 267:4357–4363. 1992.
2.	Rusnak DW, Lackey K, Affleck K, et al: The effects of the novel, reversible epidermal growth factor receptor/ErbB-2 tyrosine kinase inhibitor, GW2016, on the growth of human normal and tumor-derived cell lines in vitro and in vivo. Mol Cancer Ther. 1:85–94. 2001.
3.	Agus DB, Akita RW, Fox WD, et al: Targeting ligand-activated ErbB2 signaling inhibits breast and prostate tumor growth. Cancer Cell. 2:127–137. 2002. View Article : Google Scholar : PubMed/NCBI
4.	Konopleva M, Zhang W, Shi YX, et al: Synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid induces growth arrest in HER2-overexpressing breast cancer cells. Mol Cancer Ther. 5:317–328. 2006. View Article : Google Scholar : PubMed/NCBI
5.	Bergman I, Barmada MA, Griffin JA and Slamon DJ: Treatment of meningeal breast cancer xenografts in the rat using an anti-p185/HER2 antibody. Clin Cancer Res. 7:2050–2056. 2001.PubMed/NCBI
6.	Spiridon CI, Ghetie MA, Uhr J, et al: Targeting multiple Her-2 epitopes with monoclonal antibodies results in improved anti-growth activity of a human breast cancer cell line in vitro and in vivo. Clin Cancer Res. 8:1720–1730. 2002.PubMed/NCBI
7.	Meng S, Tripathy D, Shete S, et al: HER-2 gene amplification can be acquired as breast cancer progresses. Proc Natl Acad Sci USA. 101:9393–9398. 2004. View Article : Google Scholar : PubMed/NCBI
8.	Jerome L, Alami N, Belanger S, et al: Recombinant human insulin-like growth factor binding protein 3 inhibits growth of human epidermal growth factor receptor-2-overexpressing breast tumors and potentiates herceptin activity in vivo. Cancer Res. 66:7245–7252. 2006. View Article : Google Scholar
9.	Merlin JL, Barberi-Heyob M and Bachmann N: In vitro comparative evaluation of trastuzumab (Herceptin) combined with paclitaxel (Taxol) or docetaxel (Taxotere) in HER2-expressing human breast cancer cell lines. Ann Oncol. 13:1743–1748. 2002. View Article : Google Scholar
10.	Nahta R, Hung MC and Esteva FJ: The HER-2-targeting antibodies trastuzumab and pertuzumab synergistically inhibit the survival of breast cancer cells. Cancer Res. 64:2343–2346. 2004. View Article : Google Scholar
11.	Moulder SL, Yakes FM, Muthuswamy SK, Bianco R, Simpson JF and Arteaga CL: Epidermal growth factor receptor (HER1) tyrosine kinase inhibitor ZD1839 (Iressa) inhibits HER2/neu (erbB2)-overexpressing breast cancer cells in vitro and in vivo. Cancer Res. 61:8887–8895. 2001.PubMed/NCBI
12.	Liang K, Lu Y, Jin W, Ang KK, Milas L and Fan Z: Sensitization of breast cancer cells to radiation by trastuzumab. Mol Cancer Ther. 2:1113–1120. 2003.PubMed/NCBI
13.	Tseng PH, Wang YC, Weng SC, et al: Overcoming trastuzumab resistance in HER2-overexpressing breast cancer cells by using a novel celecoxib-derived phosphoinositide-dependent kinase-1 inhibitor. Mol Pharmacol. 70:1534–1541. 2006. View Article : Google Scholar
14.	Arpino G, Gutierrez C, Weiss H, et al: Treatment of human epidermal growth factor receptor 2-overexpressing breast cancer xenografts with multiagent HER-targeted therapy. J Natl Cancer Inst. 99:694–705. 2007. View Article : Google Scholar : PubMed/NCBI
15.	Talmadge JE, Phillips H, Schindler J, Tribble H and Pennington R: Systematic preclinical study on the therapeutic properties of recombinant human interleukin 2 for the treatment of metastatic disease. Cancer Res. 47:5725–5732. 1987.PubMed/NCBI
16.	Kovacs JA, Vogel S, Albert JM, et al: Controlled trial of interleukin-2 infusions in patients infected with the human immunodeficiency virus. N Engl J Med. 335:1350–1356. 1996. View Article : Google Scholar : PubMed/NCBI
17.	Kozlowski T, Sablinski T, Basker M, et al: Decreased graft-versus-host disease after haplotype mismatched bone marrow allografts in miniature swine following interleukin-2 treatment. Bone Marrow Transplant. 25:47–52. 2000. View Article : Google Scholar
18.	Gaffen SL and Liu KD: Overview of interleukin-2 function, production and clinical applications. Cytokine. 28:109–123. 2004. View Article : Google Scholar : PubMed/NCBI
19.	Malek TR and Bayer AL: Tolerance, not immunity, crucially depends on IL-2. Nat Rev Immunol. 4:665–674. 2004. View Article : Google Scholar : PubMed/NCBI
20.	Cesana GC, DeRaffele G, Cohen S, et al: Characterization of CD4⁺CD25⁺ regulatory T cells in patients treated with high-dose interleukin-2 for metastatic melanoma or renal cell carcinoma. J Clin Oncol. 24:1169–1177. 2006.PubMed/NCBI
21.	Goldsmith MA, Lai SY, Xu W, et al: Growth signal transduction by the human interleukin-2 receptor requires cytoplasmic tyrosines of the beta chain and non-tyrosine residues of the gamma c chain. J Biol Chem. 270:21729–21737. 1995. View Article : Google Scholar
22.	Hughes-Fulford M, Sugano E, Schopper T, Li CF, Boonyaratanakornkit JB and Cogoli A: Early immune response and regulation of IL-2 receptor subunits. Cell Signal. 17:1111–1124. 2005. View Article : Google Scholar : PubMed/NCBI
23.	Dybkaer K, Iqbal J, Zhou G, et al: Genome wide transcriptional analysis of resting and IL2 activated human natural killer cells: gene expression signatures indicative of novel molecular signaling pathways. BMC Genomics. 8:2302007. View Article : Google Scholar
24.	Harvill ET and Morrison SL: An IgG3-IL2 fusion protein activates complement, binds Fc gamma RI, generates LAK activity and shows enhanced binding to the high affinity IL-2R. Immunotechnology. 1:95–105. 1995. View Article : Google Scholar : PubMed/NCBI
25.	Budagian V, Nanni P, Lollini PL, et al: Enhanced inhibition of tumour growth and metastasis, and induction of antitumour immunity by IL-2-IgG2b fusion protein. Scand J Immunol. 55:484–492. 2002. View Article : Google Scholar : PubMed/NCBI
26.	Heuser C, Ganser M, Hombach A, et al: An anti-MUC1-antibody-interleukin-2 fusion protein that activates resting NK cells to lysis of MUC1-positive tumour cells. Br J Cancer. 89:1130–1139. 2003. View Article : Google Scholar : PubMed/NCBI
27.	Neal ZC, Yang JC, Rakhmilevich AL, et al: Enhanced activity of hu14.18-IL2 immunocytokine against murine NXS2 neuroblastoma when combined with interleukin 2 therapy. Clin Cancer Res. 10:4839–4847. 2004. View Article : Google Scholar : PubMed/NCBI
28.	Gillies SD, Lan Y, Williams S, et al: An anti-CD20-IL-2 immunocytokine is highly efficacious in a SCID mouse model of established human B lymphoma. Blood. 105:3972–3978. 2005. View Article : Google Scholar : PubMed/NCBI
29.	Wagner K, Schulz P, Scholz A, Wiedenmann B and Menrad A: The targeted immunocytokine L19-IL2 efficiently inhibits the growth of orthotopic pancreatic cancer. Clin Cancer Res. 14:4951–4960. 2008. View Article : Google Scholar : PubMed/NCBI
30.	Marlind J, Kaspar M, Trachsel E, et al: Antibody-mediated delivery of interleukin-2 to the stroma of breast cancer strongly enhances the potency of chemotherapy. Clin Cancer Res. 14:6515–6524. 2008. View Article : Google Scholar : PubMed/NCBI
31.	Shi M, Xie Z, Feng J, et al: A recombinant anti-erbB2, scFv-Fc-IL-2 fusion protein retains antigen specificity and cytokine function. Biotechnol Lett. 25:815–819. 2003. View Article : Google Scholar : PubMed/NCBI
32.	Shi M, Xie Z, Yu M, Shen B and Guo N: Controlled growth of Chinese hamster ovary cells and high expression of antibody-IL-2 fusion proteins by temperature manipulation. Biotechnol Lett. 27:1879–1884. 2005. View Article : Google Scholar : PubMed/NCBI
33.	Shi M, Zhang L, Gu HT, et al: Efficient growth inhibition of ErbB2-overexpressing tumor cells by anti-ErbB2 ScFv-Fc-IL-2 fusion protein in vitro and in vivo. Acta Pharmacol Sin. 28:1611–1620. 2007. View Article : Google Scholar : PubMed/NCBI
34.	Arakawa T, Prestrelski SJ, Kenney WC and Carpenter JF: Factors affecting short-term and long-term stabilities of proteins. Adv Drug Deliv Rev. 46:307–326. 2001. View Article : Google Scholar : PubMed/NCBI
35.	Chen L, Xie Z, Teng Y, et al: Highly efficient selection of the stable clones expressing antibody-IL-2 fusion protein by a dicistronic expression vector containing a mutant neo gene. J Immunol Methods. 295:49–56. 2004. View Article : Google Scholar
36.	Lin JX and Leonard WJ: Signaling from the IL-2 receptor to the nucleus. Cytokine Growth Factor Rev. 8:313–332. 1997. View Article : Google Scholar : PubMed/NCBI
37.	Lacosta S, Merali Z and Anisman H: Central monoamine activity following acute and repeated systemic interleukin-2 administration. Neuroimmunomodulation. 8:83–90. 2000. View Article : Google Scholar : PubMed/NCBI
38.	Sereti I, Anthony KB, Martinez-Wilson H, et al: IL-2-induced CD4⁺ T-cell expansion in HIV-infected patients is associated with long-term decreases in T-cell proliferation. Blood. 104:775–780. 2004.PubMed/NCBI
39.	Jo D, Liu D, Yao S, Collins RD and Hawiger J: Intracellular protein therapy with SOCS3 inhibits inflammation and apoptosis. Nat Med. 11:892–898. 2005. View Article : Google Scholar : PubMed/NCBI
40.	Lewis GD, Figari I, Fendly B, et al: Differential responses of human tumor cell lines to anti-p185HER2 monoclonal antibodies. Cancer Immunol Immunother. 37:255–263. 1993. View Article : Google Scholar : PubMed/NCBI
41.	Smith KA and Griffin JD: Following the cytokine signaling pathway to leukemogenesis: a chronology. J Clin Invest. 118:3564–3573. 2008. View Article : Google Scholar : PubMed/NCBI
42.	Friess T, Scheuer W and Hasmann M: Combination treatment with erlotinib and pertuzumab against human tumor xenografts is superior to monotherapy. Clin Cancer Res. 11:5300–5309. 2005. View Article : Google Scholar : PubMed/NCBI
43.	Johnson BE and Janne PA: Rationale for a phase II trial of pertuzumab, a HER-2 dimerization inhibitor, in patients with non-small cell lung cancer. Clin Cancer Res. 12:4436s–4440s. 2006. View Article : Google Scholar : PubMed/NCBI
44.	Konecny GE, Pegram MD, Venkatesan N, et al: Activity of the dual kinase inhibitor lapatinib (GW572016) against HER-2-overexpressing and trastuzumab-treated breast cancer cells. Cancer Res. 66:1630–1639. 2006. View Article : Google Scholar : PubMed/NCBI
45.	King DM, Albertini MR, Schalch H, et al: Phase I clinical trial of the immunocytokine EMD 273063 in melanoma patients. J Clin Oncol. 22:4463–4473. 2004. View Article : Google Scholar : PubMed/NCBI
46.	Zhang X, Feng J, Ye X, Yao Y, Zhou P and Chen X: Development of an immunocytokine, IL-2-183B2scFv, for targeted immunotherapy of ovarian cancer. Gynecol Oncol. 103:848–852. 2006. View Article : Google Scholar : PubMed/NCBI
47.	Johnson EE, Lum HD, Rakhmilevich AL, et al: Intratumoral immunocytokine treatment results in enhanced antitumor effects. Cancer Immunol Immunother. 57:1891–1902. 2008. View Article : Google Scholar : PubMed/NCBI
48.	Singh H, Serrano LM, Pfeiffer T, et al: Combining adoptive cellular and immunocytokine therapies to improve treatment of B-lineage malignancy. Cancer Res. 67:2872–2880. 2007. View Article : Google Scholar : PubMed/NCBI
49.	Spiridon CI, Guinn S and Vitetta ES: A comparison of the in vitro and in vivo activities of IgG and F(ab’)2 fragments of a mixture of three monoclonal anti-Her-2 antibodies. Clin Cancer Res. 10:3542–3551. 2004.PubMed/NCBI
50.	Xie H, Lin L, Tong L, et al: AST1306, a novel irreversible inhibitor of the epidermal growth factor receptor 1 and 2, exhibits antitumor activity both in vitro and in vivo. PLoS One. 6:e214872011. View Article : Google Scholar
51.	Zou W: Regulatory T cells, tumour immunity and immunotherapy. Nat Rev Immunol. 6:295–307. 2006. View Article : Google Scholar : PubMed/NCBI
52.	Koreth J, Matsuoka K, Kim HT, et al: Interleukin-2 and regulatory T cells in graft-versus-host disease. N Engl J Med. 365:2055–2066. 2011. View Article : Google Scholar : PubMed/NCBI
53.	Vesely MD, Kershaw MH, Schreiber RD and Smyth MJ: Natural innate and adaptive immunity to cancer. Annu Rev Immunol. 29:235–271. 2011. View Article : Google Scholar : PubMed/NCBI