Antibody binding to programmed death receptor 1 (PD-1) and application thereof
A programmed death and PD-1 technology, which is applied in the direction of antibody, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, application, etc., can solve the problem that the affinity needs to be improved, and avoid causing immunogenicity Response, significant clinical value, high expression effect
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Embodiment 1
[0103] Example 1. Natural phage library screening for anti-PD-1 antibodies
[0104] 1.1. Phage panning
[0105] The PD-1 antigen (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., product number: 10377-H08H) was coated in an immune tube according to 10 μg, and the large-capacity human natural library prepared by our company was put into 5 × 10E11 per round. Phage, a total of 3 rounds of screening and enrichment were carried out, and the output after each round of screening was calculated. Calculate the input / output ratio of phage input ratio for each round, and the specific results are as follows figure 1 shown. The results showed that clones specific for PD-1 antigen were significantly enriched.
[0106] 1.2. Phage binding and sequencing
[0107] 1 mL / well of 96-well deep-well plate was added with 2-YT medium containing resistance (the medium contained 100 μg / mL ampicillin and 2% glucose). Pick monoclonal colonies from the plates enriched by the 3rd round ...
Embodiment 2
[0108] Example 2. Analysis of physicochemical properties of antibodies
[0109] 2.1 Detecting the integrity of antibody light and heavy chains by reducing SDS-PAGE
[0110] The clone number E2, H6 antibody light and heavy chain antibody sequences were constructed into IgG4 full anti-eukaryotic expression vector, and the use of ExpiCHO TM Expression system kit (purchased from Thermo Fisher Scientific, Cat. No. A29133) to express and prepare the whole antibody molecule, and set the commercial antibody sequence As a control (http: / / www.imgt.org / 3Dstructure-DB / cgi / details.cgi?pdbcode=9798), double-sandwich ELISA was used to detect the expression of the antibody after expression, and the differences in the expression of different groups were compared. The results are as follows Figure 10 It can be seen from the figure that the expression level of F6 is significantly higher than that of commercial antibodies At the same time, protein A was used for protein purification of E2 and...
Embodiment 3
[0119] Example 3. ELISA assay for the binding activity of antibodies to human PD-1
[0120]The binding activity of the antibody to human PD-1 was determined by protease-linked immunosorbent assay (ELISA). The PD-1 antigen (purchased from Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., product number: 10377-H08H) was immobilized on a 96-well plate and placed at 4°C overnight. 4% milk (configured in PBS buffer) was incubated at 37°C for 1 hour as a blocking solution to block non-specific binding sites, and the ELISA plate was washed 3 times with PBST (2% Tween 20 in PBS). Antibody samples (clone F6, Keytruda) were prepared in PBS buffer, and the antibodies were uniformly diluted to 10 μg / mL and diluted twice to obtain a total of 12 concentration points. The gradient antibody was added to the corresponding well plate, placed at 37°C for 1 hour, washed the enzyme-linked plate 3 times with PBST, diluted at 1:8000 and added goat anti-human IgG-HRP (purchased from Thermo Fisher Sci...
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