A conjugate of monoclonal antibody targeting CD24 and diethylaminoazonium dialkoxide and its application

A technology of diethylaminoazonium dialkoxide and monoclonal antibody, applied in the field of bioengineering, can solve problems such as unclear direct action mechanism and affecting DNA replication of tumor cells

Active Publication Date: 2020-04-17
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The direct action mechanism of NO on tumor cells is still unclear, and there are mainly three aspects reported so far: (1) NO reacts with intracellular superoxide anion to form peroxynitrite, which is protonated and decomposed into NO 2 And hydroxyl free radicals, hydroxyl free radicals can combine with various molecules of tumor cells, thereby causing tumor cell damage, such as lipid peroxidation, protein, amino acid cross-linking reaction; (2) NO is very easy to combine with Fe-S center-containing Protein Fe forms Fe-NO, which causes the degradation of Fe-S prosthetic group and aconitase on the mitochondrial respiratory chain, thus preventing the synthesis of intracellular energy and inducing apoptosis; (3) NO can directly act on the reduction of ribonucleic acid Enzymes that affect the replication of tumor cell DNA, and can also nitrate DNA to inhibit tumor cell proliferation

Method used

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  • A conjugate of monoclonal antibody targeting CD24 and diethylaminoazonium dialkoxide and its application
  • A conjugate of monoclonal antibody targeting CD24 and diethylaminoazonium dialkoxide and its application
  • A conjugate of monoclonal antibody targeting CD24 and diethylaminoazonium dialkoxide and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Synthetic method and identification of embodiment 1 HL-2:

[0046] (1)

[0047] (2)

[0048]Step a: Slowly add 435 mL of 0.1 M sodium bicarbonate aqueous solution into 80 mL of compound 1 aqueous solution with a concentration of 0.55 mol / L through the dropping funnel. React at room temperature for 20 minutes; pull dry with an oil pump, and then perform final drying on a freeze-drying device to obtain compound 2;

[0049] Step b: Add 12.4g of compound 3 to a 250mL reaction flask, add 100mL of benzene, stir, slowly add 26.6g of NBS, react at room temperature for 5h, TLC detects that the reaction is complete, and compound 4 is obtained;

[0050] Step c: add 0.82g of azobisisobutyronitrile to the reaction flask of compound 4, and add 26.6g of NBS, and reflux at 80°C for 20h; spin off the solvent, then extract with ethyl acetate, wash with saturated brine, no Water Na 2 SO 4 Drying, concentration and column chromatography gave compound 5 as a white solid;

[0051] ...

Embodiment 2

[0066] Example 2 Preparation of Antibody Protein Conjugate HL-01

[0067] 1. Anti-CD24 monoclonal antibody G7mAb (prepared according to literature: Ma Z et al., Selective targeted

[0068] delivery of doxorubicin via conjugating to anti-CD24antibody results in enhanced antitumor potency for hepatocellular carcinoma both in vitro and in vivo.J Cancer Res Clin Oncol.2017,143(10):1929-1940) Purified by Protein A column affinity chromatography and glucose After replacing the antibody solution system with sugar gel G25FF desalting column molecular sieve chromatography, BCA method was used to measure the antibody concentration, and the reducing agent TCEP was mixed with the antibody at a molar ratio of 2.5:1. After adding, stir slowly and evenly, react at 4°C for 1h, 4000rpm, low temperature Residual TCEP was removed by ultrafiltration, and replaced with a PBS (pH 7.0) system containing 1M antioxidant diethylenetriaminepentaacetic acid to prepare for coupling reaction.

[0069] 2. ...

Embodiment 3

[0072]Example 3 Non-reducing SDS-PAGE electrophoresis identification of antibody protein conjugate HN-01

[0073] 1. Sample preparation: Take several EP tubes, add 6 μL of 5×Loading Buffer containing 250 mM Tris-HCl (pH 6.8), 10% SDS, 0.5% bromophenol blue, and 50% glycerol, and add 24 μL of samples to each tube in turn, Mix the solution evenly, put it in a boiling water bath for 5 minutes, centrifuge at 8000 rpm for 3 minutes, and set aside.

[0074] 2. Glue dispensing: Fix the double-layer glass plate to the glue dispensing mold, check for leaks with single distilled water, and configure the lower layer separation glue and the upper layer lamination glue respectively. First add the separating gel to the double-layer glass plate, and immediately flatten it with 1mL absolute ethanol. The separation gel in the lower layer was solidified in about 25 minutes, then the stacking gel was added, and the comb teeth were inserted. After the stacking gel is completely solidified, disa...

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Abstract

Disclosed are a method for preparing an anti-CD24 antibody-drug conjugate in the field of bioengineering and the use thereof. The diethylamine azo onium diol salt molecules, as novel nitric oxide donors, are conjugated with the heavy chain constant regions of the anti-CD24 monoclonal antibody G7mAb via maleimide-disulfide bonds by means of chemical coupling technology, and accordingly, the antibody-drug conjugate HN-01 can be prepared; and nitric oxide donor molecules are enriched on the surfaces of tumor cells by means of the specific targeting effects of antibodies and are internalized into the cells for directionally releasing nitric oxide, increasing intratumor therapeutic indexes and, at the same time, reducing the toxic and side effects on normal tissues. As proved by the results of in vivo and in vitro experiments, the antibody-drug conjugate HN-01 sufficiently utilizes the targeting of the antibodies and the antitumor specificity of the nitric oxide, solves the problem of tumor targeting delivery of the nitric oxide donors, and has a good clinical application value.

Description

[0001] The invention belongs to the field of bioengineering, and specifically relates to a novel antibody-drug conjugate HN-01 that can specifically bind to the leukocyte differentiation antigen CD24 molecule overexpressed on the surface of tumor cells, which consists of monoclonal antibody G7mAb and a new Nitrogen donor diethylamine azonium dialkoxide molecules were coupled via maleimide-disulfide bonds. It utilizes the specific targeting effect of G7mAb to enrich the diethylaminoazonium dialkoxide molecule on the surface of tumor cells, which improves the therapeutic index of the nitric oxide donor molecule and avoids the low targeting effect when it is used alone. Toxic and side effects on normal tissue cells caused by sex, solves the problem of targeted release of nitric oxide donors, provides a new option for the treatment of liver cancer, and can also be extended to the treatment of other malignant tumors with overexpression of CD24 molecules middle. Background technique...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K47/68A61K31/655A61K33/00C07K16/28C07K1/06C07K1/36C07K1/22C07K1/34A61P35/00C07D207/448
CPCA61K31/655A61K33/00A61K47/6803A61P35/00C07D207/448C07K16/2896A61K47/6849A61K47/6851C07D207/452
Inventor 张娟黄张建张鑫荣王旻马招兄徐瑶
Owner CHINA PHARM UNIV
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