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Preparation method of antibody-conjugated medicine, antibody-conjugated medicine and applications

A technology of antibody-conjugated drug and monoclonal antibody, which is applied in the preparation of antibody-conjugated drug, antibody-conjugated drug and its application field, and can solve the problem of instability of thiol-maleimide linker and the incompatibility of screening mutation sites , low coupling efficiency and other issues, to achieve the effects of cost savings, good anti-tumor effect, and improved coupling efficiency

Active Publication Date: 2018-04-06
ZHEJIANG UNIV
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  • Abstract
  • Description
  • Claims
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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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  • Preparation method of antibody-conjugated medicine, antibody-conjugated medicine and applications
  • Preparation method of antibody-conjugated medicine, antibody-conjugated medicine and applications
  • Preparation method of antibody-conjugated medicine, antibody-conjugated medicine and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Preparation of anti-CD20 antibody variants.

[0044] 1. Construct the expression vector of anti-CD20 antibody with LPETG label for light and heavy chain.

[0045] Ofatumumab (ofatumumab, OFA) is a fully humanized targeting anti-CD20 monoclonal antibody. 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 the heavy chain and light chain encoding genes of ofatumumab (heavy chain expression vector H and light chain expression vector L) were obtained by the inventor’s preliminary experiments by PCR technology. For details, please refer to the Chinese application number ZL201310046396.9 Invention patent.) amplified the heavy chain coding gene and light chain coding gene sequence, and then by PCR method to add a signal peptide sequence (nucleotide sequence such as SEQ ID No.5), the C-terminal plus the code (GGGGS) n LPETG(GHHHHHH) m (n = 0, 1, 2; m = 0, 1, a total of 6 combinations) t...

Embodiment 2

[0061] Preparation of anti-CD20 antibody conjugated drugs by enzyme-chemical method.

[0062] 1. Expression and purification of SortaseA enzyme

[0063] For specific steps, refer to 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 (Referred to as GPN) purchased from Nanjing Lianning Biopharmaceutical Co., Ltd., with chemical structure such as figure 2 Shown. In the 50mM Tris, 150mM NaCl (pH7.4) reaction system, respectively add: 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 is catalyzed by SortaseA and the bifu...

Embodiment 3

[0072] The property analysis of anti-CD20 antibody conjugated drugs prepared by enzyme-chemical method.

[0073] 1. RP-HPLC analysis

[0074] Column: Varian PLRP-S column (8μm, 150×25mm); 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 37°C for 30min.

[0075] The result is Image 6 As shown, the anti-CD20 antibody OFA still has an unreduced peak under DTT reduction conditions. The anti-CD20 antibody conjugated drug OFA-GPN-vcMMAE and Shanghai Latoxin have similar light and heavy chain peak times, resulting in a combined peak ( L1+H1), the drug-antibody coupling ratio (DAR) cannot be accurately determined.

[0076] 2. HIC analysis

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

[0078] Chromatographic column...

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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 present invention relates to the field of biotechnology, in particular to a method for preparing antibody-conjugated drugs, antibody-conjugated drugs and applications. 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, the toxic and side effects are large; and the common monoclonal antibody therapy has weak tumor killing ability, and the efficacy of tumor patients is often not good. good. Antibody-drug conjugates (ADCs), as an innovative treatment option, are formed by linking antibodies or their fragments with cytotoxic drugs. They have the advantages of both biological drugs and chemical drugs, that is, they simultaneously utilize the unique targets of antibodies The strong cytotoxicity of tropism and small molecule chemical drugs shows unique advantages in cancer treatment. [0003] As early as a century ago, German...

Claims

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Application Information

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Patent Type & Authority Applications(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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