A humanized anti-her2 monoclonal antibody
A monoclonal antibody and humanized technology, applied in the direction of antibodies, anti-tumor drugs, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the problem of randomness, variable region with certain Uncertainty, the influence of protein advanced structure thermal stability is difficult to predict accurately, etc., to achieve good thermal stability, long product shelf life, and long storage time
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Embodiment 1
[0037] Example 1. Preparation and screening of mouse-derived anti-HER2 monoclonal antibody
[0038] The extracellular region of recombinant human HER2 protein (named rHER2-ECD) was prepared according to the method of "Molecular architecture of the ErbB2 extracellular domainhomodimer" (Oncotarget. 2015 Jan 30;6(3):1695-706.) for immunization Balb / c mice were injected intraperitoneally with 50 μg / mouse / time, mixed with complete Freund's adjuvant for the first immunization, and mixed with incomplete Freund's adjuvant for 2-4 times. Two weeks later, the blood was taken from the orbit, and the antibody titer was detected by ELISA method. Animals with high titer were selected and injected intravenously with 50 μg per animal. Three days later, the spleen was taken, and the polyethylene glycol method was used for cell fusion, and the HAT medium (Invitrogen Company) was pressurized for screening. The supernatant of the fused cell culture was detected by ELISA to screen for positive clo...
Embodiment 2
[0042] Example 2. Cloning of antibody variable region genes (5'RACE)
[0043] Trizol method (Trizol reagent was purchased from Thermofisher Company, product number 15596026) was used to extract the total RNA of the hybridoma cell No. m14C5B screened in Example 1. Select the appropriate positions of the heavy chain and light chain constant regions of the antibody (IgG1, κ) to design three gene-specific primers: GSP1-H~GSP3-H and GSP1-L~GSP3-L. The primer sequences are as follows:
[0044] GSP1-H:GTAGAGGTCAGACTGCAGGAC;
[0045] GSP2-H: CTCAGGGAAATAGCCCTTGAC;
[0046] GSP3-H: AGATCCAGGGGCCAGTGGATAGAC;
[0047] GSP1-L:TTGCTGTCCTGATCAGTCCAACT;
[0048] GSP2-L: TGTCGTTCACTGCCATCAATCTT;
[0049] GSP3-L: TTGTTCAAGAAGCACACGACTGA.
[0050] Among them, GSP1 is farthest from the variable region gene and is used for reverse transcription reaction, GSP2 is used for the first round of PCR amplification, and GSP3 is used for nested amplification. AAP primers and AUAP primers in the 5' R...
Embodiment 3
[0052] Example 3, humanized design of murine monoclonal antibody
[0053] The consensus sequences of human immunoglobulin heavy chain subgroup III and Igκ chain subgroup I were respectively selected as humanized templates for antibody light and heavy chains, and the amino acid sequences of the variable regions were as follows:
[0054] Heavy chain III subgroup:
[0055] EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSVISGDGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGFDYWGQGTLVTVS
[0056] Kappa chain subgroup I:
[0057] DIQMTQSPSSLSASSVGDRVTITCRASQSISNYLAWYQQKPGKAPKLLIYAASSLESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYNSLPWTFGQGTKVEIKR
[0058] Then, the heavy chain and light chain CDR regions of the murine antibody m14C5B were transplanted into the human template heavy chain subgroup III and Igκ chain I subgroup respectively, that is, the CDR1, CDR2, and CDR3 of the light chain replaced the Ig chain I subgroup respectively. Amino acids at positions 24-34, 50-56, and 89-97 are re...
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