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CD24 monoclonal antibody and diethylamine azo onium diol salt targeting conjugate and application thereof

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

Active Publication Date: 2018-08-17
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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  • CD24 monoclonal antibody and diethylamine azo onium diol salt targeting conjugate and application thereof
  • CD24 monoclonal antibody and diethylamine azo onium diol salt targeting conjugate and application thereof
  • CD24 monoclonal antibody and diethylamine azo onium diol salt targeting conjugate and application thereof

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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

The invention belongs to the technical field of bioengineering antibodies, and particularly relates to a method for preparing a CD24 antibody resistant medicine conjugate and application thereof. Themethod and the application have the advantages that diethylamine azo onium diol salt molecules which are novel nitric oxide donors are coupled to CD24 monoclonal antibody resistant G7mAb heavy chain constant regions via maleimide-disulfide bonds by the aid of chemical coupling technologies, and accordingly the antibody medicine conjugate HN-01 can be prepared; nitric oxide donor molecules are enriched on the surfaces of tumor cells by the aid of specific targeting effects of antibodies and are internalized into the cells, nitric oxide can be directionally released, accordingly, intratumor therapeutic indexes can be increased, and toxic and side effects on normal tissues can be deteriorated; as proved by in-vivo and in-vitro experiment results, the targeting of the antibodies and the antitumor specificity of the nitric oxide can be sufficiently utilized by the antibody medicine conjugate HN-01, the problems in the aspect of tumor targeting delivery of existing nitric oxide donors can besolved, and the method and the CD24 antibody resistant medicine conjugate have excellent 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 Applications(China)
IPC IPC(8): A61K47/68A61K31/655A61K33/00C07K16/28C07K1/06C07K1/36C07K1/22C07K1/34A61P35/00C07D207/448
CPCA61K47/6803A61P35/00A61K31/655A61K33/00C07D207/448C07K16/2896C07D207/452A61K47/6849A61K47/6851
Inventor 张娟黄张建张鑫荣王旻马招兄徐瑶
Owner CHINA PHARM UNIV
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