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A kind of preparation method of antibody-conjugated drug, antibody-conjugated drug and application

A technology of antibody-drug coupling and monoclonal antibody, which is applied in the biological field, can solve problems such as instability of thiol-maleimide linkers, uncommon screening of mutation sites, and low coupling efficiency, so as to save cost and input , good anti-tumor effect, and improved coupling efficiency

Active Publication Date: 2021-03-23
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] The present invention aims at the heterogeneity problem caused by traditional antibody-conjugated drugs using lysine residues on antibodies or cysteine ​​residues produced by the reduction of interchain disulfide bonds; The imide linker is unstable in the circulatory system and the screening of mutation sites is not universal; the direct Sortase A enzyme transpeptide reaction requires a large amount of dolastatin (Monomethyl auristatin E, MMAE) and coupling due to steric hindrance In order to solve the problems of low efficiency, a simple, flexible, and general-purpose fixed-point coupling method is provided. The uniformity and coupling efficiency of the anti-CD20 antibody-dolastatin conjugate obtained by this preparation method are greatly improved, and the target CD20 Positive tumor cells have good anti-tumor effects in vivo and in vitro, and are more suitable for large-scale production

Method used

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  • A kind of preparation method of antibody-conjugated drug, antibody-conjugated drug and application
  • A kind of preparation method of antibody-conjugated drug, antibody-conjugated drug and application
  • A kind of preparation method of antibody-conjugated drug, antibody-conjugated drug and application

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Production of anti-CD20 antibody variants.

[0044] 1. Construct the expression vector of the anti-CD20 antibody with LPETG tag on the light and heavy chains.

[0045] Ofatumumab (OFA) is a fully humanized anti-CD20 monoclonal antibody, and the gene sequences encoding its heavy chain and light chain are shown in SEQ ID No.3 and SEQ ID No.4, respectively.

[0046] The plasmids containing ofatumumab heavy chain and light chain encoding genes (heavy chain expression vector H and light chain expression vector L) were respectively obtained by PCR technology from the inventor's previous experiments. Invention patent.) amplifies the sequences of the heavy chain coding gene and the light chain coding gene, and then adds a signal peptide sequence to the N-terminals of the heavy chain coding gene and the light chain coding gene by the PCR method (the nucleotide sequence is as follows: shown in SEQ ID No.5), C-terminal plus coding (GGGGS) n LPETG (GHHHHHH) m (n = 0, 1, 2; m = 0...

Embodiment 2

[0061] Enzyme-chemical preparation of anti-CD20 antibody conjugate drug.

[0062] 1. Expression and purification of SortaseA enzyme

[0063] Specific steps reference Pan, L.Q., et al. (2017) Sortase A-generated highlypotent anti-CD20-MMAE conjugates for efficient elimination of B-lineagelymphomas.Small.13(6).

[0064] 2. Transpeptide reaction catalyzed by SortaseA enzyme

[0065] Bifunctional small molecule GGG-PEG-N 3 (GPN for short) was purchased from Nanjing Lianning Biopharmaceutical Co., Ltd., and its chemical structure is as follows: figure 2shown. Add respectively to 50mM Tris, 150mM NaCl (pH7.4) reaction system: 2μM recombinant anti-CD20 antibody variant (prepared in Example 1), 50μM Sortase A enzyme, 200μM bifunctional small molecule, 5μM CaCl 2 . Sortase A has a binding site for calcium ions, and the presence of calcium ions will increase the catalytic efficiency of the enzyme. React at 37°C for 12-24 hours. The antibody was coupled with the bifunctional smal...

Embodiment 3

[0072] Analysis of properties of anti-CD20 antibody drug conjugate prepared by enzyme-chemical method.

[0073] 1. RP-HPLC analysis

[0074] Column: Varian PLRP-S column (8 μm, 150×25 mm); column temperature: 80° C.; mobile phase A: 0.1% trifluoroacetic acid aqueous solution; mobile phase B: acetonitrile. Gradient elution conditions: 25% B (3 min); 25-50% B (25 min); 50-95% B (2 min); 95-25% B (1 min); 25% B (2 min). Flow rate: 0.6mL / min; the sample was reduced with DTT at 37°C for 30min.

[0075] The result is as Image 6 As shown, the anti-CD20 antibody OFA still has an unreduced peak under DTT reducing conditions, and the anti-CD20 antibody conjugated drug OFA-GPN-vcMMAE is connected to the light and heavy chains of sea rabbit toxin. The peak time is similar, resulting in a combined peak ( L1+H1), the drug-antibody conjugation ratio (DAR) cannot be accurately judged.

[0076] 2. HIC analysis

[0077] The anti-CD20 antibody conjugated drug OFA-GPN-vcMMAE was further a...

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Abstract

The invention discloses a preparation method of an antibody-conjugated medicine, the antibody-conjugated medicine and applications. The preparation method comprises the following steps: firstly, underthe catalysis of Sortase A enzyme, leading LPXTG sequence and oligoglycine linker to generate transpeptidation reaction, so that the anti-CD20 monoclonal antibody is connected with a first linker arm; and then leading an azide group linker and alkynyl linker to generate cycloaddition reaction, so that the first linker arm is connected with a second linker arm. The antibody-conjugated medicine isprepared by utilizing an enzyme-chemical method, so that the usage amount of expensive medicines can be reduced, and the cost can be saved. By adopting difunctional small molecules, the steric hindrance of Sortase A enzyme in transpeptidation reaction can be reduced, and the flexibility is good. The conjugation sites and quantity on the antibody medicines can be efficiently controlled, the uniformity is enhanced, and the in-vivo and in-vitro anti-tumor activity can be improved on the basis of keeping the original antibody affinity.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a preparation method of an antibody-coupled drug, an antibody-coupled drug and its application. Background technique [0002] As one of the most serious diseases, cancer threatens the life and health of human beings. Due to the poor targeting selectivity of traditional chemotherapy and high toxicity and side effects; and the common monoclonal antibody therapy is often ineffective for tumor patients due to its weak tumor killing ability. good. As an innovative therapeutic option, antibody-drug conjugates (ADCs) are composed of antibodies or their fragments and cytotoxic drugs, which have the advantages of both biological and chemical drugs, that is, the unique target of antibodies The tropism and strong cytotoxicity of small molecule chemical drugs have shown unique advantages in cancer treatment. [0003] As early as a century ago, German immunologist Paul Ehrlich proposed the conc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K47/68A61K47/65A61K38/07A61P35/00A61P35/02
CPCA61K38/07
Inventor 徐瑛赵文彬陈枢青
Owner ZHEJIANG UNIV
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