Targeting molecule for reversing treatment drug resistance of anti-PD-1 antibody
A technology targeting molecules and drugs, applied in the fields of molecular immunology and tumor therapy
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Embodiment 1
[0092] Embodiment 1 Construction of anti-TIM-3 antibody expression vector
[0093] In the early stage, several antibodies with strong binding ability to TIM antigen were obtained through the screening of phage antibody library. This application is one of the antibodies, numbered 37#.
[0094] (1) Synthesize antibody heavy chain sequence (wherein, the heavy chain nucleic acid sequence of the 37# antibody is shown in SEQ ID NO: 19) and light chain sequence (wherein, the 37# antibody light chain nucleic acid sequence is shown in SEQ ID NO: :20), and it utilizes the molecular cloning method to clone the fragment into the ZY-CDMO carrier, and the ZY-CDMO carrier map is as follows figure 1 shown.
[0095] The specific operation is as follows: introduce an EcoRI restriction site at the 5' end of the light chain by conventional PCR, introduce an XbaI restriction site at the 3' end, and insert it between EcoRI and XbaI of the ZY-CDMO vector; A HindIII restriction site was introduced ...
Embodiment 2
[0096] Example 2 Expression and purification of anti-TIM-3 antibody
[0097] Inoculate 30ml in a 125ml cell culture shaker flask with a density of 0.5×10 6 cells / ml of CHO cells, when the cell density grows to 2×10 6 cells / ml or more, the cell viability is more than 95%, and the cells are 1×10 7 The cells / ml density was inoculated in the electrotransfer buffer, 40 μg plasmid was added to the electric shock cup, and then 0.7ml of cell suspension was added, and finally the electrotransfer buffer was added to 0.8ml, and mixed gently to avoid bubbles. Electric shock once under the conditions of 300V, 900μF, place the electric shock cup in the ice box for 5min, and dilute it into 6ml of culture medium at 37°C CO 2 After recovering in the incubator for 48 hours, add 75 μM MSX to the seed medium to inoculate the 96-well plate and perform clone screening, and divide the monoclonal cells into the 96-well plate by the limited dilution method, 37 ° C, 5% CO 2 Cultivate in an incubator...
Embodiment 3
[0099] Example 3 Anti-TIM-3 Antibody Affinity Activity Detection to TIM-3 Antigen
[0100] Use ELISA to detect affinity, the specific steps are as follows:
[0101] Coating: Take the ELISA plate and dilute the target protein human TIM-3 (Sino Biological, 10390-H08H) to 0.5 μg / ml with pH 9.6 coating solution, and coat 100 μl / well in a humid chamber overnight at 4°C.
[0102] Discard the liquid in the well, invert the plate and pat dry the residual liquid on a paper towel, add washing solution (0.1% PBST) 300 μl / well and wash 3 times.
[0103] Blocking: adding 300 μl / well of blocking solution (2% M-PBS) and standing at 37° C. for 1.0 h.
[0104] Discard the liquid in the well, invert the plate and pat dry the residual liquid on a paper towel, add washing solution (0.1% PBST) 300 μl / well and wash 3 times.
[0105] Add the primary antibody: Dilute the anti-TIM-3 antibody (37#, 67#, 75#) to 1mg / ml with DPBS, and then perform 10-fold dilutions in sequence, a total of 7 concentrati...
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