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Fully human monoclonal antibody against CD20 and its application

A monoclonal antibody, fully human technology, applied in the direction of antibodies, applications, anti-animal/human immunoglobulin, etc., can solve the problems of continuous drug use, long-term drug use, multiple infusion reactions, etc.

Active Publication Date: 2020-05-22
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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  • Fully human monoclonal antibody against CD20 and its application
  • Fully human monoclonal antibody against CD20 and its application
  • Fully human monoclonal antibody against CD20 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1: the preparation of hybridoma and carrier

[0064] 1. Antigen and lymphocyte preparation and immunization

[0065] 1.1 Antigen preparation:

[0066] 1.1.1 CD20 protein

[0067] Human CD20 (Novusbio) antigen was purchased, diluted with PBS, and filtered with a 0.22 μm needle sterile filter.

[0068] 1.1.2 Preparation of 293F cells expressing CD20 membrane protein

[0069] Purchase CD20 cDNA (Origene, SC101205), and design and synthesize PCR amplification primers:

[0070] CD20 forward primer: 5'-TCAGGAGTTTTGAGAGCAAAATG-3'

[0071] CD20 reverse primer: 5'-AACAGAAGAATCACTTAAGGAG-3'

[0072] Then use the cDNA of CD20 as a template to amplify and purify, and clone into the pCR3.1 vector (Invitrogen, K300001). TMAfter 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.

[0073] 1.1.3 Extracted CD20 membrane protein

[0074] CD20 membran...

Embodiment 2

[0105] Example 2: Preparation and Identification of Monoclonal Antibody

[0106] 1. Preparation of antibodies by in vitro culture method

[0107] 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.

[0108] 2. Antibody purification

[0109] Take out at 225cm 2 The cell supernatant of the square flask cultured for about 10 days was centrifuged to remove cell debris, and the antibody was purified by rProteinA affinity chromatography. 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 column bed volume; elution: glycine solution at pH 3.0, each section C...

Embodiment 3

[0110] Example 3: Complement-dependent cytotoxicity assay (CDC)

[0111] 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.

[0112] 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

Provided is a fully humanized anti-CD20 monoclonal antibody, which is prepared by human hybridoma technology. The antibody has excellent affinity and specificity, and a significant cytotoxic effect on lymphoma cells, and so can be used for the preparation of pharmaceutical compositions for treating or diagnosing diseases such as non-Hodgkin lymphoma, B cell lymphoma, rheumatic and rheumatoid diseases, systemic lupus erythematosus, immune thrombocytopenic purpura and multiple sclerosis.

Description

technical field [0001] 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 [0002] 1. CD20 [0003] 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 expressed at the pre-B cell stage and ends when B cells terminally differentiate into plasma cells. It has always been considered a unique marker on the surface of B lineage cells. It is mainly involved in regulating the proliferation and differe...

Claims

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Application Information

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IPC IPC(8): C07K16/30C12N15/13C12N15/63A61K39/395A61P35/00A61P37/00
CPCA61P35/00A61P37/00C07K16/2887C07K16/3061C07K2317/21C07K2317/73C07K2317/92
Inventor 冯晓
Owner BEIJING ANBAOKANG BIO PHARMA CO LTD
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