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A kind of antibody-coupled drug and its application

An antibody and coupling technology, applied in the field of biopharmaceuticals, can solve the problems of weak immune effect and weak CDC effect, and achieve the effects of good uniformity, broadening research ideas, and good tumor killing ability

Active Publication Date: 2022-04-29
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Anti-EGFR antibody drugs currently on the market, such as cetuximab and panitumumab, panitumumab itself is an IgG2 antibody, its immune effect itself is very weak, almost no CDC effect, and IgG1 cetuximab, The CDC effect in vivo and in vitro is also weak

Method used

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  • A kind of antibody-coupled drug and its application
  • A kind of antibody-coupled drug and its application
  • A kind of antibody-coupled drug and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Preparation of 97m-vcMMAE conjugates.

[0040] Nanobody Fc fusion protein targeting EGFR, 7D12 and 9G8 nanobody fusion, the sequence of the nanobody is from the literature (Karl R.Schmitz et al., Structure.2013; 21(7):1214–1224), the following It is called 97m for short. The Fc segment sequence of the fusion protein is preserved in the laboratory. In this embodiment, 7D12 and 9G8 were passed through the flexible linker peptide (GGGGS) by PCR method 3 The C-terminus is connected to the IgG1 heavy chain constant region, and finally the DNA sequence is obtained.

[0041] The sequence is as follows:

[0042] 9G8: the amino acid sequence is shown in SEQ ID No.1, and the gene sequence is shown in SEQ ID No.2.

[0043] 7D12: the amino acid sequence is shown in SEQ ID No.3, and the gene sequence is shown in SEQ ID No.4.

[0044] Flexible connecting peptide: the amino acid sequence is shown in SEQ ID No.5, and the gene sequence is shown in SEQ ID No.6.

[0045] IgG1 type h...

Embodiment 2

[0051] Drug-antibody conjugation ratio determination of conjugates.

[0052] In order to quantitatively detect the DAR value after coupling toxic small molecules, the coupled products were analyzed by hydrophobic interaction chromatography. The ADC concentration after coupling was diluted to 1 mg / mL before injection. Due to the strong hydrophobicity of vcMMAE small molecules, the changes in hydrophobicity after coupling small molecules can be used to distinguish the components of different drug small molecule coupling ratios in the final coupling product. The specific analysis conditions are as follows:

[0053] Chromatographic column: TSKgel Butyl-NPR hydrophobic chromatographic column (2.5μm, 4.6mm×3.5cm); mobile phase A: 2M (NH 4 ) 2 SO 4 , 150mM Na 3 PO 4 , pH 7.0; mobile phase B: 150mM Na 3 PO 4 , pH 7.0; mobile phase C: isopropanol; flow rate: 0.8mL / min; column temperature: 25°C; injection volume: 10μL;

[0054] Table 1

[0055] time (min) A％ B％ C％ ...

Embodiment 3

[0059] Flow cytometry affinity assay.

[0060] Cells expressing EGFR, such as A431, were cultured with DMEM medium containing double antibodies. After the cells grew to the logarithmic growth phase, the cells were divided into 5×10 5 The number of cells was co-incubated with a series of serially diluted antibodies or ADCs in 1% BSA in PBS for 30 min on ice.

[0061] After the incubation, the cells were washed twice with pre-cooled PBS to remove proteins non-specifically adsorbed to the cell surface. Finally, a 1:200 dilution of FITC-labeled goat anti-human IgG secondary antibody was added in a dark environment, and the incubation was continued on ice for 30 min in the dark. After the incubation, the cells were washed with pre-cooled PBS, and the cell suspension resuspended in PBS was loaded on a flow cytometer to detect the mean fluorescence intensity (MFI) of the cells.

[0062] The affinity of the fusion protein to EGFR-positive cells before and after coupling was detected...

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Abstract

The invention discloses an antibody-coupled drug and its application. The antibody-coupled drug is that the antibody is coupled with the drug dolastatin, and the antibody includes the Fc of 9G8 nanobody, 7D12 nanobody and IgG1 antibody, and the two chains of the Fc of the double-chain structure are fused to one molecule of 9G8 nanobody. Antibodies and 7D12 nanobodies, the amino acid sequence of 9G8 nanobodies is that the 7th and / or 71st serines are mutated to cysteines in the sequence shown in SEQ ID No.1 for coupling dolastatin, so The DAR value of the antibody-coupled drug is 1-2. In the present invention, the fusion protein can be easily connected with the small molecule toxin through the site-directed cysteine ​​mutation at the conserved site of the nanobody, the coupling product has good uniformity, and exhibits good tumor killing ability in in vivo and in vitro experiments. Compared with the currently marketed cetuximab, the fusion protein conjugate has a stronger CDC effect and can overcome a series of drug resistance mutations in the extracellular region of EGFR.

Description

technical field [0001] The invention relates to the technical field of biopharmaceuticals, in particular to an antibody-coupled drug and its application. Background technique [0002] Antibody-drug conjugates are a class of antibody-modified drugs that combine antibodies with cytotoxic small-molecule drugs. They have attracted people's attention because they combine the targeting of antibodies and the lethality of chemotherapy drugs. If the antibody part is regarded as a targeted guidance system, then the high-efficiency cytotoxic small-molecule drug attached to it is the drug carrier responsible for killing, which can theoretically achieve the effect of targeted killing of the target. By coupling chemical small molecules to antibodies, the therapeutic window of small molecule drugs can be broadened, their side effects can be reduced, and the long half-life of antibody drugs and various immune effects mediated by antibodies can be exerted. [0003] Epidermal growth factor r...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/07A61K47/68A61P35/00
CPCA61K38/07A61K47/6817A61P35/00
Inventor 樊建声陈枢青
Owner ZHEJIANG UNIV
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