A fully humanized anti-vegfr-2 monoclonal antibody and its preparation method
A VEGFR-2, monoclonal antibody technology, applied in the field of bioengineering, can solve the problems of difficult assembly, small molecules, high tumor penetration, achieve good biochemical and biological activities, effective anti-angiogenesis effect.
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Embodiment 1
[0061] Example 1 Construction and Screening and Separation of Antibody Library
[0062] 1. Construction of antibody library
[0063] The single-chain antibody library used in the present invention is a human natural antibody library, and its main construction process is:
[0064] (1) Isolate B lymphocytes from human peripheral blood or spleen, lymph nodes and other tissues, Trizol extracts total mRNA and reverse transcribes it into cDNA;
[0065] (2) According to the human immunoglobulin gene sequence library (such as Kabat database, V-base, IMGT, etc.), design a set of primers for antibody heavy chain and light chain with degenerate sequences to amplify different VH by PCR technology and VL gene fragments, spliced into a full-length scFv single-chain antibody through the linker region;
[0066] The base sequences of the primers used are as follows:
[0067] V H back (amplification heavy chain variable region VH primer)
[0068] HuVH1B / 7A-BACK 5'-CAG RTG CAG CTG GTG CAR...
Embodiment 2
[0112] Example 2 Construction and expression of high-expression engineered cell lines
[0113] 1. Antibody cloning and transfection
[0114] (1) The screened variable region sequence is composed of SEQ ID NO: 1-6, using conventional gene cloning methods, cloned into the FC fusion eukaryotic expression vector Pcantab-5E or pFUSEIgG1-FC to express the full-length antibody in the carrier. The correct insertion of the antibody gene was identified by enzyme digestion and sequencing, followed by transfection and antibody secretory expression.
[0115] (2) 24 hours before transfection, digest 1×10 5 / mL cell density HEK293T / 17 cells were seeded in 6-well plates and cultured overnight. Take two test tubes containing 200ul serum-free medium, add 1ugDNA working solution, mix well, incubate at room temperature for 5 minutes, quickly add 2ul PEI (polyethyleneimine polymer) dilution solution to the DNA dilution solution, and immediately vortex for 15 seconds , incubate at room temperat...
Embodiment 3
[0132] Identification of Biochemical and Biological Functions of Embodiment 3 Antibodies
[0133] 1. Antibody affinity detection
[0134] The affinity constants of the target antibody samples of the three clones (A139, A140, A141) were detected by using Biacore T100 (Biacore AB, Uppsala, Sweden). The reference product VEGF165 (product of R&D Company) was covalently bonded to the CM5 biosensor chip (GE Healthcare) through the amino group, and the RU response value of the antibody to be tested was determined, and the appropriate antibody concentration was determined according to the result, usually at 100-200RU. should. Then test the concentration of antigen VEGFR2, determine the range of antigen concentration and exposure time. Finally, the kinetic parameters and affinity of the test antibody were determined using the kinetics / affinity method. The results are shown in Table 1.
[0135]
[0136] Table 1 Affinity test results
[0137] It can be seen from the table that di...
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