Construction and application of a bispecific antibody epcam×cd3
A bispecific antibody, -CD3 technology, applied in the direction of antibody, anti-animal/human immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the limited curative effect of solid tumors, etc. problems, to achieve the effect of increasing immunotherapy and improving efficacy
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Embodiment 1
[0067] Example 1: Construction of expression vectors for bispecific antibodies (EpCAM×CD3 SMBODY, M702)
[0068] 1. Bispecific antibody sequence design
[0069] The bispecific antibody targeting EpCAM and CD3 was named M702 (EpCAM×CD3 SMBODY), as figure 2 The anti-EpCAM side is in ScFv-Fc form, including anti-EpCAM VH, VL, and Fc domains; the anti-CD3 side is in IgG form, including anti-CD3 heavy chain and light chain, including Fab and Fc domains. Among them, ScFv-Fc undergoes KKW transformation on one side of Fc, and one side of IgG form undergoes LDY transformation on Fc. For the specific Fc transformation process, see PCT / CN2012 / 084982, so that they are not easy to form homodimers, but easy to form hybrid dimers, that is EpCAM×CD3 SMBODY bispecific antibody. At the same time, in order to express the diabody in CHO cells and secrete it into the culture medium, the leader peptide sequence of the mouse kappa chain was selected as the secretion signal peptide. The amino ac...
Embodiment 2
[0092] Example 2: Expression and purification of bispecific antibodies
[0093] 1. Expression of bispecific antibodies
[0094] The endotoxin-free large-scale extraction kit (Qiagen, 12391) was used for large-scale extraction of plasmids, and the specific operation was performed according to the instructions provided by the manufacturer. CHO-S cells were cultured in CD CHO medium (Gibco, 10743-029) at 37°C in 5% CO according to the instructions provided by the manufacturer. 2 Cultured in a cell culture incubator, after the cells were prepared, the plasmid pCHO1.0-anti-CD3-HL-LDY and pCHO1.0-hygromycin-anti- EpCAM-ScFv-Fc-KKW were co-transfected together into CHO-S cells, and the co-transfection of these two plasmids was designed to express the bispecific antibody against EpCAM×CD3.
[0095] On the second day after transfection, the culture temperature was lowered to 32° C., and 3.5% FeedA was added every day. After 14 days of culture, the expression supernatant was harvested...
Embodiment 3
[0098] Example 3: Determination of the binding activity of bispecific antibodies to cells (FACS)
[0099] The bispecific antibody of the invention binds to the target antigen on the corresponding cell. In the present invention, HCT116 (purchased from ATCC, CCL-247) is used as EpCAM-positive cells, Jurkat (Jurkat, TIB-152) is used as CD3-positive cells, and the cell binding activity is determined by the diabody prepared in the present invention.
[0100] 1. Detection of binding activity of bispecific antibody to HCT116 cells by flow cytometry
[0101] Sufficient HCT116 cells were cultured, digested with 0.25% trypsin, and collected by centrifugation. Dilute the bispecific antibody at the same time, the concentration starts from 500nmol, and 3-fold serial dilution, to obtain 12 concentration gradients, set aside. Wash the collected cells twice with PBS+1%FBS, then resuspend the cells in PBS+1%FBS to 4×10 6 cells / ml, the cells were plated in a 96-well plate, 50ul per well (2×1...
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