Eukaryotic expression, protein purification and biological effects research of human CS1-Fc fusion protein
【Abstract】Signaling lymphocyte activation family 7 (SLAMF7/CS1) is a cell surface glycoprotein that is highly expressed in multiple myeloma cells. CS1 is a sensitive and specific biomarker for multiple myeloma. CAR-T cell immunotherapy is a new method for the treatment of multiple myeloma. CS1 CAR-T cell immunotherapy has good effect on relapsed refractory multiple myeloma. To detect the expression efficiency of CS1 CAR on CS1 CAR-T cells and to find an auxiliary means to CAR-T cell immunotherapy, we prepared a CS1-Fc fusion protein. First, the extracellular domain of CS1 was amplified from the existing plasmid by PCR and ligated with human IgG1-Fc fragment by overlap extension PCR. The recombinant fragment was ligated into pMH3 eukaryotic expression vector. After restriction enzyme digestion and DNA sequencing, the pMH3-CS1-Fc-his recombinant plasmid was successfully constructed. The recombinant plasmid was transfected into Chinese hamster ovary cell (CHO-S) by liposome. The expression of the CS1-Fc fusion protein in CHO-S cells was identified by flow cytometry after G418 pressure screening. Next, the CS1-Fc fusion protein was purified by nickel column. Western-blot analysis showed that molecular weight of the fusion protein was about 70 kDa. The CS1-Fc fusion protein could effectively detect the expression rate of CS1 CAR and promote the activation, proliferation and cytokine secretion of the CS1 CAR-T cells. The results will lay the experimental foundation for the in vitro detection and potentiation of CAR-T cells in multiple myeloma treated with CS1 CAR-T cell.
【Keywords】 human signaling lymphocyte activation family 7 (SLAMF7/CS1); CS1-Fc fusion protein; eukaryotic expression; CAR-T; multiple myeloma;
(Translated by HE Z)
 Al-Hujaily EM, Oldham RAA, Hari P, et al. Development of novel immunotherapies for multiple myeloma. Int J Mol Sci, 2016, 17 (9): 1506.
 Palumbo A, Bringhen S, Ludwig H, et al. Personalized therapy in multiple myeloma according to patient age and vulnerability: a report of the European Myeloma Network (EMN). Blood, 2011, 118 (17): 4519–4529.
 Kumar SK, Dispenzieri A, Lacy MQ, et al. Continued improvement in survival in multiple myeloma: changes in early mortality and outcomes in older patients. Leukemia, 2014, 28 (5): 1122–1128.
 June CH, O’Connor RS, Kawalekar OU, et al. CART cell immunotherapy for human cancer. Science, 2018, 359 (6382): 1361–1365.
 Hudecek M, Einsele H. Myeloma CARs are rolling into the clinical arena. Blood, 2016, 128 (13): 1667–1668.
 Mikkilineni L, Kochenderfer JN. Chimeric antigen receptor T-cell therapies for multiple myeloma. Blood, 2017, 130 (24): 2594–2602.
 Morgan RA, Yang JC, Kitano M, et al. Case report of a serious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2. Mol Ther, 2010, 18 (4): 843–851.
 Brentjens RJ, Rivière I, Park JH, et al. Safety and persistence of adoptively transferred autologous CD19-targeted T cells in patients with relapsed or chemotherapy refractory B-cell leukemias. Blood, 2011, 118 (18): 4817–4828.
 Di Bernardo A, Macor P, Guarnotta C, et al. Humoral immunotherapy of multiple myeloma: perspectives and perplexities. Exp Opin Biol Ther, 2010, 10 (6): 863–873.
 Malaer JD, Mathew PA. CS1 (SLAMF7, CD319) is an effective immunotherapeutic target for multiple myeloma. Am J Cancer Res, 2017, 7 (8): 1637–1641.
 Wang XL, Walter M, Urak R, et al. Lenalidomide enhances the function of CS1 chimeric antigen receptor-redirected T cells against multiple myeloma. Clin Cancer Res, 2018, 24 (1): 106–119.
 Gogishvili T, Danhof S, Prommersberger S, et al. SLAMF7-CAR T cells eliminate myeloma and confer selective fratricide of SLAMF7+normal lymphocytes. Blood, 2017, 130 (26): 2838–2847.
 Jafari R, Zolbanin NM, Rafatpanah H, et al. Fc-fusion proteins in therapy: an updated view. Curr Med Chem, 2017, 24 (12): 1228–1237.
 Ghose S, Hubbard B, Cramer SM. Binding capacity differences for antibodies and Fc-fusion proteins on protein A chromatographic materials. Biotechnol Bioeng, 2007, 96 (4): 768–779.
 Ma QZ, De Marte L, Wang YW, et al. Carcinoembryonic antigen-immunoglobulin Fc fusion protein (CEA-Fc)for identification and activation of anti-CEA immunoglobulin-T-cell receptor-modified T cells, representative of a new class of Ig fusion proteins. Cancer Gene Ther, 2004, 11 (4): 297–306.
 Structural Genomics Consortium, Architecture et Fonction des Macromolécules Biologiques, Berkeley Structural Genomics Center, et al. Protein production and purification. Nat Methods, 2008, 5 (2): 135–146.
 Chang ZL, Lorenzini MH, Chen XM, et al. Rewiring T-cell responses to soluble factors with chimeric antigen receptors. Nat Chem Biol, 2018, 14 (3): 317–324.
 Irving BA, Weiss A. The cytoplasmic domain of the T cell receptorζchain is sufficient to couple to receptor-associated signal transduction pathways. Cell, 1991, 64 (5): 891–901.
 Letourneur F, Klausner RD. T-cell and basophil activation through the cytoplasmic tail of T-cell-receptor zeta family proteins. Proc Natl Acad Sci USA, 1991, 88 (20): 8905–8909.
 Romeo C, Seed B. Cellular immunity to HIVactivated by CD4 fused to T cell or Fc receptor polypeptides. Cell, 1991, 64 (5): 1037–1046.
 Marzio R, Mauël J, Betz-Corradin S. CD69 and regulatiof the immune function. Immunopharmacol Immunotoxicol, 1999, 21 (3): 565–582.
 Schwartzentruber DJ, Hom SS, Dadmarz R, et al. In vitro predictors of therapeutic response in melanoma patients receiving tumor-infiltrating lymphocytes and interleukin-2. J Clin Oncol, 1994, 12 (7): 1475–1483.
 Beecham EJ, Ma QZ, Ripley R, et al. Coupling CD28 co-stimulation to immunoglobulin T-cell receptor molecules: the dynamics of T-cell proliferation and death. J Immunother, 2000, 23 (6): 631–642.
 Iezzi G, Karjalainen K, Lanzavecchia A. The duration of antigenic stimulation determines the fate of naive and effector T cells. Immunity, 1998, 8 (1): 89–95.