Fixed-point conjugate of anti-CD24 antibody and diethylamine diazeniumdiolate molecule and application of fixed-point conjugate

A technology of diethylamine azonium diolide, CD24, applied in anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, medical preparations of inactive ingredients, anti-animal/human immunoglobulin It can solve the problems of product inhomogeneity and poor druggability, and achieve the effect of expanding application prospects, reducing side effects, and improving product inhomogeneity.

Pending Publication Date: 2021-04-02
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The design of HN-02 is to connect the chimeric TG7 antibody with site-specific active cysteine ​​and the nitric oxide donor molecule through a Linker containing a maleimide ring and a disulfide bond, and play a nitric oxide molecule The anti-tumor activity reduces the side effects caused by its non-targeted release, improves the shortcomings of the first generation of ANC molecules, such as inhomogeneous products and poor druggability, and expands the application prospects of nitric oxide donor drugs. Rectal cancer treatment offers new options

Method used

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  • Fixed-point conjugate of anti-CD24 antibody and diethylamine diazeniumdiolate molecule and application of fixed-point conjugate
  • Fixed-point conjugate of anti-CD24 antibody and diethylamine diazeniumdiolate molecule and application of fixed-point conjugate
  • Fixed-point conjugate of anti-CD24 antibody and diethylamine diazeniumdiolate molecule and application of fixed-point conjugate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Synthetic method and identification of embodiment 1 HL-2:

[0068]

[0069] 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;

[0070] 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;

[0071] 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;

[0072] Step d: Dissolve 20...

Embodiment 2

[0087] Example 2 Construction and preparation of site-directed mutagenesis antibody TG7

[0088] Firstly, according to the results of MOE simulation, the light chain nucleic acid sequence of the chimeric antibody cG7 was mutated from valine (GTC) at position 211 to cysteine ​​(TGC) by PCR to obtain products at the upper and lower ends of the mutation site, and then overlap- PCR splicing, enzyme ligation with PTT5 vector, transformation into DH5α Escherichia coli to obtain cloning vector sequencing identification. For the correctly sequenced TG7 recombinant plasmid transfected into HEK 293 cells for expression, the protein A column was purified and its molecular weight was identified by SDS-PAGE. The results are shown in figure 1 .

Embodiment 3

[0089] Example 3 Identification of site-directed mutagenesis antibody TG7 binding ability to rhCD24 antigen

[0090] The TG7 antibody we obtained needs to determine its ability to bind to rhCD24 molecules. We used western blot and ELISA to verify the specific binding ability of TG7 antibody to rhCD24 molecule.

[0091] (1) Western blot detection of TG7 antibody assembly

[0092] ① Select the appropriate separation gel concentration (12%) according to the molecular weight of the target protein (45kDa), and run SDS-PAGE electrophoresis with rhCD24 antigen at four concentrations of 40, 20, 10, and 5nM respectively.

[0093] ②After electrophoresis, carefully pry open the glass plate, cut off the separation gel containing the target band, measure the length and width of the separation gel containing the target band, and place it in the transfer buffer. Cut out six pieces of PVDF membrane that are 2 mm larger than the length and width of the separation gel and six pieces that are ...

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Abstract

The invention belongs to the field of bioengineering, and particularly relates to a fixed-point conjugate of an anti-CD24 antibody and a diethylamine diazeniumdiolate molecule and application of the fixed-point conjugate. The fixed-point conjugate is formed by coupling a chimeric antibody TG7 with fixed-point active cysteine and nitric oxide donor diethylamine diazeniumdiolate molecules through amaleimide-disulfide bond Linker. Diethylamine diethylamine molecules are enriched to the surface of CD24 positive tumor cells by utilizing the CD24 antigen targeting effect of the TG7 antibody and areinternalized into the cells in the form of an antigen-antibody compound, so that the treatment index of the nitric oxide donor molecules is improved, the toxic-side effect on normal tissue cells dueto the non-targeting property during the single use of the nitric oxide donor is avoided, the targeting delivery problem of the nitric oxide donor is solved, a new choice is provided for the colorectal cancer treatment, and the nitric oxide donor can be popularized to other CD24 molecule overexpressed malignant tumor treatment.

Description

Background technique [0001] At present, the morbidity and mortality of malignant tumors are showing a significant upward trend in the world. Specific to colorectal cancer, according to the statistics of the World Health Organization in 2018, its incidence rate ranks third, and its mortality rate ranks second. Drugs for the treatment of colorectal cancer in clinical use are mainly divided into chemotherapy drugs and biological targeted drugs. Chemotherapy drugs directly rely on small molecules with high toxicity to kill colorectal cancer cells, such as 5-FU / LV, irinotecan, oxaliplatin, capecitabine, etc. Biological targeted drugs block tumor-related signaling pathways by specifically targeting colorectal cancer cell surface membrane receptors. These two types of drugs all act unilaterally, and even if they are administered in combination, it is difficult to exert complementary effects, such as bevacizumab and cetuximab. Therefore, the development of an antibody small molecul...

Claims

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Application Information

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IPC IPC(8): C07K16/28C07K1/107C07K1/34A61K47/68A61P35/00
CPCC07K16/2896A61K47/6849A61P35/00C07K2317/24
Inventor 张娟黄张建郭敏姬黄林华
Owner CHINA PHARM UNIV
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