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Preparation method of muramyl dipeptide-anti-CD20 immune conjugate and application thereof

A technology of immunoconjugates and muramyl dipeptide, which is applied in the direction of anti-animal/human immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, dipeptide components, etc., and can solve the problem of Difficulty in identifying or eliminating tumor cells, immune function impairment, etc.

Inactive Publication Date: 2013-05-08
THE AFFILIATED HOSPITAL OF QINGDAO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, with the prolongation of chemotherapy in children with lymphoma, the immune function of the body is further damaged, and it is difficult to identify or remove residual tumor cells.

Method used

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  • Preparation method of muramyl dipeptide-anti-CD20 immune conjugate and application thereof
  • Preparation method of muramyl dipeptide-anti-CD20 immune conjugate and application thereof
  • Preparation method of muramyl dipeptide-anti-CD20 immune conjugate and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] [Example 1] Preparation of Compounds 1-5

[0013] Preparation of Compound 1

[0014] Take, for example, a linker arm containing three carbon atoms. Under argon protection, in a 10ml flask, add Boc-protected linker N-Boc-1,3-propanediamine hydrochloride (20mg, 0.12mmol, CAS number: 127346-48-9, Sigma-Aldrich company) , dissolved with 3ml dimethylformamide, added excess triethylamine, and then added N-succinimide-3-(2-pyridylthio)propionate (SPDP, 74mg, 0.24mmol, CAS No.: 68181 -17-9, Sigma-Aldrich Company), stirred at room temperature for 1 h, and the spot plate detection reaction was completed, then separated through a silica gel column (ethyl acetate: petroleum ether = 1: 1), and evaporated to dryness in vacuo to obtain the product compound 1 (19.8 mg). ESI-MS m / z 372.1[M+H] + .

[0015] Preparation of Compound 2

[0016] Under the protection of argon, in a 10ml reaction flask, add compound 1 (10mg, 0.02mmol), add 1.5ml of dichloromethane, stir in ice bath for 10...

Embodiment 2

[0029] [Example 2] Test of immune activity

[0030] 1. In vitro immune activity

[0031] 1.1 Test method

[0032] Preparation of the test sample solution: the test sample is the compound 5 synthesized in the above-mentioned Example 1 and unconjugated anti-CD20 monoclonal antibody, MDP, lipopolysaccharide (LPS, Escherichia coli 0128: B12, Sigma-Aldrich Company). Accurately weigh an appropriate amount of sample, and use RPMI1640 complete culture medium to prepare a solution of the required concentration for activity testing.

[0033] The present invention adopts the method of indirect flow cytometry combined with antibody-antigen specific binding to test and evaluate the binding activity of the tested sample to CD20+ lymphoma cells (Raji cells, ≥90%). The anti-CD20 monoclonal antibody specifically binds to the CD20 antigen of lymphoma cells, FITC-labeled non-specific secondary antibody is added, incubated in the dark, and the fluorescence intensity is detected by flow cytometr...

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Abstract

By taking guide functions of specific cellular immunity and monoclonal antibody immunity generated in vaccine inoculation as theoretical basis, the effective component of bacillus calmette Guerin (BCG), namely muramyl dipeptide (MDP), and a monoclonal antibody Rituximab of lymphoma cell membrane resistant antigen CD20 are combined to prepare a muramyl dipeptide-anti-CD20 immune conjugate, namely MDP-Rituximab, wherein in-vivo and in-vitro anti-tumour effects of immunocompetent cells subjected to BCG immunity under mediation of MDP-Rituximab are known. The conjugate disclosed by the invention keeps the immunogenicity of MDP, stimulates organisms to generate specific T cell immune response, and brings MDP to be around residual lymphoma cells with the help of immune guide function that a Rituximab antibody is combined with lymphoma cell membrane CD20 molecules; the final purpose of eliminating residual tumours is achieved through immune response mediated by MDP induced T cells; therefore, due to the method, the drug resistance caused by exclusive use of Rituximab is overcome; furthermore, the use amount of the antibody is reduced; and the application prospect of the muramyl dipeptide-anti-CD20 immune conjugate in malignant lymphoma is predicted.

Description

Technical field: [0001] The present invention relates to a muramyl dipeptide-anti-CD20 immunoconjugate (MDP-Rituximab) with targeted antigen binding and anti-tumor effect by stimulating immunocompetent cells. The present invention discloses its preparation method, and Role in lymphoma therapy. Background technique: [0002] According to the statistics of the World Health Organization, the incidence rate of lymphoma increases by 7.5% every year, and it is one of the nauseating tumors with the fastest increasing incidence rate. The incidence of lymphoma in my country is about 2 / 100,000 population, with 25,000 new cases and 20,000 deaths every year, and children account for 30% of the national population. Therefore, there are nearly 10,000 new cases of children every year. Its mortality rate ranks first among childhood malignancies. Improving the level of diagnosis and treatment of lymphoma in children is an urgent problem for the majority of blood workers. More than 90% of ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K16/28C07K5/072A61K47/48A61K38/05A61P35/02A61P35/00A61P37/02A61P19/04A61P19/02A61P29/00
Inventor 孙立荣王玲珍
Owner THE AFFILIATED HOSPITAL OF QINGDAO UNIV
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