Anti-cd40 antibody and use thereof
An antibody and variant technology, applied in the direction of antibodies, antibody medical components, anti-tumor drugs, etc., can solve the problems of lack of targeted cancer cells, excess selectivity, etc.
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example 1
[0179] Example 1: Production of mouse anti-human CD40 antibody
[0180] Monoclonal antibodies are produced by immunizing Balb / C mice or SJL mice intraperitoneally with mouse 3T12 cells overexpressing human CD40. Spleens were harvested and splenocytes were fused with the multiple myeloma cell line NS0. Hybridomas were selected using aminopterin. Selected hybridomas expressing anti-CD40 antibodies with agonistic activity were screened and subcloned to isolate individual clones.
[0181] To screen for antibodies with agonistic activity, a panel of functional assays was developed that included NFκB pathway stimulation, monocyte activation, dendritic cell (DC) activation, and CD40 ligand (CD40L) competition. In these analyses, anti-human CD40G28-5 (mouse IgGl) (Biolegend) was included as a positive control and an isotype-matched mouse antibody (mIgGl ) was included as a negative control.
[0182] 8.1.1. Analysis of HEK293 blue CD40 NFκB reporter
[0183] HEK293 blue CD40 cell l...
example 2
[0192] Example 2: Epitope classification of mouse anti-human CD40 antibodies
[0193] BIAcore analysis and ELISA methods were used to classify mouse anti-human CD40 agonist antibodies according to their ability to compete with each other or CD40 ligand (CD40L) for binding to CD40.
[0194] BIAcore analysis was performed at 12°C using a BIAcore T100 instrument. A goat anti-mouse Fc antibody (Pierce, cat. no. 31170) was first immobilized on the CM5 sensor chip, followed by capture of the first test antibody on the surface. After blocking with a mouse isotype antibody cocktail at 50 μg / mL, flow cells were injected with a soluble form of the extracellular domain of human CD40 (Creative BioMart, catalog # CD402221H). Subsequently, a second test antibody or CD40L (PeproTech, cat. no. 0308145) was injected to measure its binding to the complex of CD40 and the first test antibody. As shown in Table 2-1, the anti-CD40 antibodies of the present invention were classified into three epi...
example 3
[0201] Example 3: Humanization of mouse anti-human CD40 antibody
[0202] Humanization of antibody V regions was performed as outlined by Queen, C. et al. (Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America], 1989; 86: 10029-10033). Typical structures of CDRs were determined according to Huang et al. (Methods, 2005; 36:35-42). Identify human alternative germline sequences with the same or most similar CDR canonical structures, and select appropriate human V H , V L and J segment sequences to provide the framework for the anti-CD40 variable region. At framework positions where the computer model indicated significant contacts with the CDRs, the original human framework amino acids were substituted with amino acids from the murine anti-CD40 V region (back mutation). Unless otherwise specified, human IgG with natural variants D356E and L358M in the heavy chain was used 1 The constant region and the κ light chain. V of ...
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