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Antibody coupling medicine and application thereof

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

Active Publication Date: 2021-09-17
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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  • Antibody coupling medicine and application thereof
  • Antibody coupling medicine and application thereof
  • Antibody coupling medicine and application thereof

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 coupling medicine and application thereof. According to the antibody coupling medicine, medicine aplysiatoxin is coupled to an antibody, the antibody comprises a 9G8 nano antibody, a 7D12 nano antibody and an Fc of an IgG1 type antibody, two chains of the Fc of a double-chain structure are fused with the 9G8 nano antibody and the 7D12 nano antibody of a molecule respectively, an amino acid sequence of the 9G8 nano antibody is characterized in that serine at the 7th site and / or 71th site is mutated into cysteine to be used for coupling aplysiatoxin in a sequence shown as SEQ ID No.1, and the DAR value of the antibody coupling medicine is 1-2. The fusion protein can be easily connected with small molecule toxins through site-specific cysteine mutation of a conserved site of the nano antibody, the homogeneity of a coupling product is good, and the good tumor killing ability is shown in in-vivo and in-vitro experiments. Compared with cetuximab on the market at present, the fusion protein conjugate has a stronger CDC effect and can overcome drug-resistant mutation of a series of EGFR extracellular regions.

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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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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