High-stability humanized anti-VEGF (vascular endothelial growth factor) antibody and application thereof
An antibody and amino acid technology, applied in the direction of antibodies, anti-growth factor immunoglobulin, anti-animal/human immunoglobulin, etc., can solve problems such as poor structural stability
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Embodiment 1
[0059] Example 1 Stability study of anti-VEGF antibody Amv6
[0060] 1. Materials and methods
[0061] 1. Materials: Mammalian HEK293T cells were purchased from Invitrogen, serum-free medium Freestyle TM 293 expression medium (Cat. No.: 12338-026) and transfection medium Opti-MEMI (Cat. No.: 31985-070) are products of Gibco, and the transfection reagent 293fectin TM reagent (product number: 12347-019) is also a product of Invitrogen. High-fidelity DNA polymerase primeStar DNA polymerase (product of Takara Company), primer synthesis was completed by Shanghai Sangong. HRP-goat anti-human IgG antibody was purchased from Zhongshan Jinqiao Biological Co., Ltd. The 96-well ELISA is the product of Cornning Company. Purification medium ProteinA 1ml prepacked column is the product of Invitrogen Company. The pure recombinant VEGF165 protein is obtained by HEK293T transient expression and purification. Amv6 antibody light and heavy chain expression vectors pABκ-Amv6L and pABG1-Amv...
Embodiment 2
[0081] Example 2 Effect of Amv6 light chain mutation on thermostability
[0082] 1. Method
[0083] 1. Amv6 variable region structure modeling
[0084] Antibodies are a class of functional proteins that have been widely studied. By summarizing and analyzing the structural information of antibodies, a set of effective structure prediction and screening schemes have been summarized, which can accurately model the variable regions of antibodies. . In the present invention, some codes in Rosetta Antibody are modified and processed in parallel to make it suitable for the computing environment. In actual work, localized Rosetta Antibody software was used to model the variable region of Amv6, and a total of 5,000 models were generated. Score and sort these models, select the top 10 models for detailed analysis, and select the model with the best comprehensive properties as the final model. Using this method, all CDR regions except the heavy chain CDR3 can be modeled very accurate...
Embodiment 3
[0100] Example 3 Effect of Amv6 heavy chain mutation on thermostability
[0101] Taking the Amv6 heavy chain variable region sequence (SEQ ID NO.4) as the object, a total of 17 heavy chain single point mutations were designed. After expressing, purifying and quantifying the protein of each single point mutant, Fortebio protein interaction system was used for analysis. For the determination of relative affinity, the difference between its affinity and the parental antibody Amv6 was evaluated, and the statistical results are shown in Table 2. For the further thermal stability evaluation of the mutant antibody whose affinity remains basically unchanged, the method refers to Example 2, and the statistical results are shown in Table 2. Among them, there are 6 single-point mutants with improved thermal stability compared with the parental antibody, and some results are as follows Figure 8 shown.
[0102] Table 2 Effect of single-point directed mutation of Amv6 heavy chain on affi...
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