Method for improving efficacy of antibody drug and antibody drug
An antibody drug and antibody technology, applied in the field of methods and antibody drugs to improve the efficacy of antibody drugs, can solve problems such as limitations in the laboratory level, unclear and complex glycosylation modification functions, etc.
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Embodiment 1
[0051] Example 1 Effect of sialic acid modification at Asn162 site of CH1 domain of tumor SIA-IgG on tumor growth
[0052] first part:
[0053] RP215 is a monoclonal antibody produced by one of the hybridoma cells obtained by immunizing mice with proteins extracted from ovarian cancer cell lines, and can specifically recognize IgG molecules.
[0054] Please refer to patents: CN201510776518.9, CN201810330585.1, SIA-IgG is a type of IgG with unique sialic acid modification, mainly expressed on tumor stem cells. Cancer stem cells can rely on the unique sialylation modification of SIA-IgG to promote the maintenance of stemness of cancer stem cells and the immune escape of tumors, respectively.
[0055] The inventors previously overexpressed wild-type SIA-IgG, recombinant SIA-IgG with 162-site mutations in the CH1 domain (S160A and N162C), and recombinant SIA-IgG with 297-site mutations in the CH2 domain (N297Q) in 293 cells , and the expressed protein product was detected by Wes...
Embodiment 2
[0071] Example 2 The asialylation modification at the Asn162 site of the commercialized antibody CH1 domain has better drug efficacy
[0072]In this example, two monoclonal antibodies, RP215 and recombinant antibody BD11, were used as tools to analyze the sialylation modification of the CH1 domain of the 10 commercial antibodies in Table 1. BD11 is a humanized chimeric antibody composed of CDR (complementarity determining region) of RP215 and human FR (framework region), which can specifically recognize the sialylation modification at Asn162 site of CH1 domain of IgG heavy chain. For analysis results, see Figure 4 and Table 1.
[0073] Since the sialylation modification occurs at position 162 of CH1 of the IgG heavy chain, reduction electrophoresis can separate the IgG heavy chain and light chain of the antibody drug, so that when using RP215 or BD11 for detection, the IgG heavy chain (55kD ) whether there is sialylation modification. Figure 4 The results of reduction ele...
Embodiment 3
[0082] Example 3 Removing the sialylation modification at the Asn162 site of the CH1 domain of a commercial antibody can improve the efficacy of the drug
[0083] Herceptin was incubated with neuraminidase at 37°C for an appropriate time to remove the sialylation modification at the Asn162 site of the CH1 domain of Herceptin to obtain Herceptin without sialic acid modification.
[0084] Next, a comparative experiment of injection treatment was carried out on tumor-forming nude mice.
[0085] Tumor-forming nude mice were injected with Herceptin, sialic acid-removed modified Herceptin (Herceptin+Neuraminidase), pertuzumab and PBS buffer, respectively, with 8 nude mice for each group. The experimental period was 22 days, and the injection dose was 50mg / kg, once every three days. The tumor size in the mice of each group was measured in vivo on different days during the experiment, and the mice were sacrificed after the experiment, and the tumors in the mice were taken out to meas...
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