Bispecific antibody for multiple myeloma (MM) and application of bispecific antibody
A bispecific antibody, antibody technology, applied in the direction of antibody, application, fermentation, etc., can solve the problem of complex structure of bispecific antibody, and achieve the effect of ensuring biological activity, avoiding heterologous mismatch, and maintaining affinity
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Embodiment 1
[0035] Example 1 Construction of bispecific antibody DF00.
[0036] Firstly, the BCMA-targeting single-chain antibody 2A9 and the PD-1-targeting antibody D00, which were screened by the laboratory through phage display technology and optimized by the company, were used as templates to design primers to transfer the heavy and light chain variable region genes. The IgG4 constant region (the Fc segment is mutated by knobs-into-holes, and the Fab segment is transformed by Crossover) gene is used as a template to design primers to transfer the heavy and light chain constant region genes, and the variable and constant regions of the heavy and light chains are separated by overlapping PCR technology The heavy and light chain genes of the bispecific antibody DF00 were constructed by ligation; EcoRI and NotI double-enzyme digestion was performed on the obtained PCR product and pCMV3 vector respectively, and after recovering the target fragment and double-enzyme-digested vector, T4 DNA l...
Embodiment 2
[0037] Example 2 Expression, purification and identification of bispecific antibody DF00.
[0038] First, the four recombinant plasmids pCMV3-F00-H / L-Chain (knob end) and pCMV3-D00-H / L-Chain (hole end) were transfected into HEK293 cells by PEI transfection reagent in a certain ratio, and then Change the medium daily and passage the cells, gradually expand the scale of cell fermentation and culture, collect the cell culture medium, filter the sample with a 0.22 μm filter membrane, and then perform Protein A column affinity chromatography purification, and finally obtain a large amount of the target protein. 8% non-reducing and 12% reducing SDS-PAGE electrophoresis were performed respectively, and Western blotting was used to identify its molecular weight and assembly status, and to conduct preliminary verification of the target protein. see results figure 2 , F00 and DF00 were successfully expressed and assembled correctly.
Embodiment 3
[0039] Example 3 The binding affinity of DF00 to BCMA and PD-1 was verified by ELISA analysis.
[0040]First, the antigens BCMA and PD-1 were treated with 50mM NAHCO 3 The buffer was diluted to 1 μg / ml, and coated on the activated microplate at 4°C overnight; after washing with PBS to remove the antigen not bound to the plate, block with 5% skimmed milk at 37°C for 2 hours; wash the plate again with PBST Then start to incubate a series of diluted antibodies (500nM-0.244nM / 250nM-0.122nM) at 37°C for 2h; then wash the plate again with PBST to wash away the antibodies not bound to the antigen, and use goat antibody with HRP tag Incubate with human IgG (H+L) at 37°C for 2 hours; finally wash the plate with PBST and use TMB chromogenic solution to develop color. When a clear color gradient appears, use 2M dilute sulfuric acid to terminate the reaction and detect OD with a microplate reader 450 -OD 630 value as the final result. see results image 3 , the double antibody DF00 ha...
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