Anti-PD-L1/TGF-beta bifunctional antibodies and uses thereof
A bifunctional antibody, PD-L1 technology, applied in the field of tumor immunology, can solve the problems of poor stability, complex process and large differences in quality control
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Embodiment 1
[0224] Example 1 Construction of expression vector
[0225] Entrusted Suzhou Jinweizhi Biotechnology Co., Ltd. (Jinweizhi for short) to synthesize amino acids 24-159 (ECD) with human TGF-βRII extracellular domain (accession number: P37173). 24-159 ) of the N-fusion and C-fusion genes, N-fusion and C-fusion indicate that the TGF-βRII ECD is fused to the N- and C-terminus of the heavy chain of the humanized PD-L1 antibody through a GS flexible linker, respectively. During gene synthesis, a HindIII endonuclease recognition site was added to the 5' end of N-fusion, and the heavy chain variable region and part C of the PD-L1 antibody (HB0023) were connected downstream of the receptor ECD. H 1 gene sequence, add the NheI endonuclease recognition site at the 3' end. The 5' end of C-fusion is derived from the heavy chain constant region C of the PD-L1 antibody (HB0023) H 3 begins with the recognition site for the SexAI endonuclease and contains part C H 3 and the receptor ECD gene,...
Embodiment 2
[0244] Example 2 Expression and purification of fusion protein
[0245] The expression of the protein in the present invention is divided into two modes: transient transfection expression and stable transfection expression. For homeopathic transfection expression, the constructed heavy chain expression vectors 500054 and 500055 are respectively mixed with the light chain vector 400085 in a ratio of 1:1 , and pre-incubated with PEI (Polyetherimide), then co-transfected into CHO-S (Thermo Fisher, R80007) cells at 32°C, 5% CO 2 , After culturing at 125rpm / min for 7 days, the supernatant was collected by centrifugation and purified for use. For stable transfection expression, the constructed heavy chain expression vectors 500054 and 500055 were mixed with the light chain vector 400085 in a ratio of 1:2 and added to blank CHO-K1 cells, mixed with medium, and carried out with a pulse voltage of 250-300V. Click transfection, MSX pressurized screening of stably transfected cell clone...
Embodiment 3
[0246] Example 3 Binding activity of fusion protein and target
[0247] 3.1 ELISA method to detect the binding activity of fusion protein to human TGF-β
[0248]Dilute TGF-β1 (ACRO, TG1-H4212) or TGF-β3 (R&D, 8420-B3-025) to 0.5 μg / ml with PBS, add 100 μl / well to a 96-well microtiter plate, and coat overnight at 4°C. After washing with PBST, the plate was blocked with blocking solution for 1 h. The sample to be tested starts from 30μg / ml, 12 gradients are diluted by 3 times, and TGF-βRII-Fc (ACRO, TG2-H5252) and control drug 900544 (according to the patent PD-L1 / TGF-β double antibody M7824 issued by Merck) Sequence synthesis gene, self-expressed by Huabo Biotechnology) as a positive control, 900201 (900201 is a human IgG1 isotype control antibody targeting non-target antigens, used for multiple detections, as a negative control) as a negative control, 100μl / well was added, The reaction was carried out at room temperature for 2h. After washing the plate with PBST, add HRP-la...
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