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CD20-resisting all humanized monoclonal antibodies and application thereof

A monoclonal antibody, antibody technology, applied in applications, antibodies, anti-animal/human immunoglobulin, etc., can solve problems such as failure, multiple infusion reactions, and inability to continue medication

Active Publication Date: 2015-10-21
BEIJING ANBAOKANG BIO PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But due to its 25% murine portion, it is more prone to murine immunogenicity, i.e. human anti-mouse antibody response
This will cause more infusion reactions, and it will not allow long-term medication
Because the human anti-mouse antibody circulating in the patient's serum will neutralize Rituxan and reduce its efficacy or even fail
In addition, there is still room for improvement in the tumor-killing efficacy of Rituxan itself. For example, its current clinical effective rate (CR+PR) is about 50%, continuous medication cannot be used, and the remission period is only one year. Therefore, it is necessary to invent new antibody molecules to improve The efficacy of these drugs [29]

Method used

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  • CD20-resisting all humanized monoclonal antibodies and application thereof
  • CD20-resisting all humanized monoclonal antibodies and application thereof
  • CD20-resisting all humanized monoclonal antibodies and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Embodiment 1: the preparation of hybridoma and carrier

[0057] 1. Antigen and lymphocyte preparation and immunization

[0058] 1.1 Antigen preparation:

[0059] 1.1.1 CD20 protein

[0060] Human CD20 (Novusbio) antigen was purchased, diluted with PBS, and filtered with a 0.22 μm needle sterile filter.

[0061] 1.1.2 Preparation of 293F cells expressing CD20 membrane protein

[0062] Purchase CD20 cDNA (Origene, SC101205), and design and synthesize PCR amplification primers:

[0063] CD20 forward primer: 5'-TCAGGAGTTTTGAGAGCAAAATG-3'

[0064] CD20 reverse primer: 5'-AACAGAAGAATCACTTAAGGAG-3'

[0065] Then use the cDNA of CD20 as a template to amplify and purify, and clone into pCR3.1 vector (Invitrogen, K300001). TM After 48 hours, 293-F cells (Invitrogen, R790-07) were cultured with 1 mg / ml G418 (Invitrogen, 10131035) for 2 weeks. Stable G418-resistant clones were screened out and frozen.

[0066] 1.1.3 Extracted CD20 membrane protein

[0067] CD20 membrane p...

Embodiment 2

[0098] Example 2: Preparation and Identification of Monoclonal Antibody

[0099] 1. Preparation of antibodies by in vitro culture method

[0100] The 1.1.81, 1.4.68, 1.6.14, 1.36.2, 1.72.108, 1.89.45, 1.105.3, 1.134.42, 1.146.78, 1.176.109 cells, gradually reduce serum to serum-free or serum-free medium containing only a small amount of serum (Invitrogen, 12338-026), put in 225cm 2 Square bottle, according to inoculation 1×10 5 cells / ml, inoculate 100ml, and inoculate 4 bottles of each strain of cells.

[0101] 2. Antibody purification

[0102] Take out at 225cm 2The supernatant of the cells cultured in the square flask for about 10 days was centrifuged to remove the cell debris, and the antibody was purified by rProtein A affinity chromatography. Sample loading: the cell culture supernatant at pH 7.4 was filtered through a 0.22um filter membrane and loaded directly; washing: washing with PBS at pH 7.4, 10 times the volume of the column bed; elution: glycine solution ...

Embodiment 3

[0103] Example 3: Complement-dependent cytotoxicity assay (CDC)

[0104] Ramos, Raji and Daudi cells were purchased from ATCC in DMEM buffered with 10% FBS, 1% sodium pyruvate and 1% HEPES. CellTiterGlo kit was purchased from Promega (Madison, WI). Normal human serum was obtained from whole blood of healthy blood donors.

[0105] Press 1×10 5 cells / well, spread Ramos, Raji and Daudi cells into 96-well plates. At 37°C, 5% CO 2 The cells in the cell incubator were incubated with different concentrations of the antibody to be tested for 10 minutes, wherein the antibody to be tested was the cell supernatant antibody obtained in Example 2 or the affinity matured antibody obtained in Example 6, the positive control was Rituxan, and the negative The control was purified human IgG (purified from healthy human serum). Then normal human serum was added to make the final concentration in the culture solution 10%. At 37°C, 5% CO 2 In a cell culture incubator, cells, different conce...

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Abstract

The invention belongs to the technical field of antibody drugs and provides a completely anti-CD20 human monoclonal antibody and an application thereof. The antibodies come from human hybridomas by virtue of cellular poison functional test screening and CD20 affinity screening, and the antibodies have excellent CD20 affinity and specificity and an obvious cellular poison effect on lymphoma cells; in a pharmacodynamic experiment of a Raji cell infiltrating tumor model for treating human B cell lymphoma, lifetime of an experimental animal is obviously prolonged; in a Raji cell orthotopic transplantation tumor inhibiting experiment for treating human B cell lymphoma, growth of transplant subcutaneous sarcoma of Raji cells is obviously inhibited; curative effect not lower than or better than that of rituximab is respectively shown in lifetime and tumor growth inhibiting; and the completely anti-CD20 human monoclonal antibody has potential diagnosis and treatment values on diseases such as non-hodgkin lymphomas, B cell lymphoma, rheumatic and rheumatoid diseases, systemic lupus erythematosus, immune thrombocytopenic purpura and multiple sclerosis.

Description

[0001] This application is a divisional application, the original application number is 201210082298.6, the application date is March 26, 2012, and the title of the invention is "anti-CD20 fully human monoclonal antibody and its application". technical field [0002] The invention relates to the technical field of antibody drugs. Specifically, it relates to a group of new molecules of fully human monoclonal antibodies, which can be used in the diagnosis and treatment of lymphoma. Background technique [0003] 1. CD20 [0004] CD20 is a non-glycosylated phosphoprotein with a molecular weight of 33-37 kDa. It has 4 transmembrane regions, the amino-terminal and carboxy-terminal are located inside the plasma membrane, between the third transmembrane region and the fourth transmembrane region, There is a loop region consisting of 43 amino acid residues, which constitutes its main antigenic epitope. As a differentiation antigen on the surface of B lymphocytes, it is initially ex...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/63C07K17/00A61K39/395A61K51/10A61K39/44A61P35/00A61P29/00A61P7/04A61P37/02G01N33/577
Inventor 冯晓王涛李新灵
Owner BEIJING ANBAOKANG BIO PHARMA CO LTD
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