Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Antibody for resisting human CD79a extracellular terminal protein, coding gene and application

An antibody and coding technology, applied in anti-animal/human immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, application, etc., can solve loss of CD20, recurrence, non-expression of CD20, etc. problem, to achieve the effect of high internalization rate

Active Publication Date: 2017-12-19
ZHEJIANG UNIV
View PDF1 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Problems existing in the current antibody-targeted therapy for B lymphocyte-related diseases: In summary, many B-NHL and B lymphocytic leukemias have been successfully treated clinically through the combination chemotherapy of CD20 chimeric antibody rituximab. However, some B-cell tumors do not express CD20, and some will lose CD20 surface markers during the treatment of MabThera, which leads to problems of drug resistance and recurrence. In the immunotherapy of these tumor patients, it is necessary to find other effective drugs. Target
Therefore, it is possible that CD79a molecule is more suitable for the therapeutic target of lymphoma and autoimmune disease than CD79b molecule. It is because there is currently no effective monoclonal antibody that responds to the CD79a molecule in living cells, so far there is no literature report on the use of live cells to immunize to obtain a monoclonal antibody against the extracellular part of the CD79a molecule
Although reported in the literature (Mason DY, Cordell JL, Brown MH, Borst J, Jones M, Pulford K, Jaffe E, Ralfkiaer E, Dallenbach F, Stein H.CD79a: a novel marker for B-cell neoplasms in routinely processedtissue samples.Blood .1995; 15; 86(4):1453-9.) Using the immunogen as the computer-simulated 15-amino acid synthetic peptide-coupled thyroglobulin of the primary structure of the protein at the extracellular end of the CD79a molecule to obtain an antibody (clone number JCB117 ), but it still does not react with living B cells as detected by flow cytometry (probably because the synthetic peptide immunogen has lost the glycosylation structure of the peptide chain folding machine), and cannot carry out the immune signal transduction function of CD79a molecule and the disease Targeted therapy research, so there is no literature report on this aspect
Literature reports (Tribolet L, Cantacessi C, Pickering DA, Navarro S, Doolan DL, Trieu A, Fei H, Chao Y, Hofmann A, Gasser RB, Giacomin PR. Loukas A.Probing of a human proteome microarray with a recombinantpathogen protein reveals a novel mechanism by which hookworms suppress B-cell receptor signaling. J Infect Dis.2015; 211(3):416-25.) The recombinant vaccine Na-ASP-2 protein used in hookworms binds to CD79a, leading to the activation of lyn and pi3k in the BCR signaling pathway Transcriptional downregulation controls BCR signaling processes, leading to B cell immunodeficiency

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Antibody for resisting human CD79a extracellular terminal protein, coding gene and application
  • Antibody for resisting human CD79a extracellular terminal protein, coding gene and application
  • Antibody for resisting human CD79a extracellular terminal protein, coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Development of ZUCH-S3 monoclonal antibody.

[0044] According to the classic method of mouse-mouse hybridoma reported by Koller and Milstein (Lin Xueyan, Zhang Ling editors. Modern Cell and Molecular Immunology. First Edition. Beijing: Science and Technology Press. 2000, p489-521.), with B cell lymphoma cell line Raji cells as immunogen, 10 7 Raji cells were injected intraperitoneally into 8-week-old female Balb / C mice, once a week, for a total of 4 times. On the 3rd day after the fourth injection, the mice were killed by dislocation, and the splenocytes were aseptically collected and kept in logarithmic The mouse myeloma cell line NS-1 cells (products of American ATCC) in the growth phase were mixed at a ratio of 6:1, and 50% polyethylene glycol (PEG, American Sigma Company, molecular weight 3350 Daltons) solution was used as the fusion medium. After fusion, selective culture was carried out in a 96-well plate (Falcon, USA). On the 9th to 20th day after fusion, the ...

Embodiment 2

[0046] Purification and subtyping of monoclonal antibodies.

[0047] 1. Purification of monoclonal antibodies: In order to induce ascites, BALB / C mice were intraperitoneally injected with 0.5 0.5 per mouse of pristane 1 week before inoculation with hybridoma cells (hybridoma cell line ZUCH-S3 obtained in Example 1). ml, then inoculate 5.0 × 10 each 6 After 10 days, the ascites fluid was collected and centrifuged, and the antibody titer was determined. Monoclonal antibodies in ascites were purified by affinity purification (Sepharose cross-linked with Protein G). ①Ascites fluid was diluted 3 times with cold Binding Buffer, then centrifuged at 10,000 rpm for 15 minutes at 4°C to remove the precipitate. ② Thoroughly wash the affinity purification column preloaded with Sepharose-Protein G with Binding Buffer of 10 times the column bed volume. ③Put the diluted ascites into a column, and control the flow rate to 8-10 drops / min. ④ Repeat the flow of ascites on the column once. ⑤...

Embodiment 3

[0050] The ability of S3 monoclonal antibody to recognize antigen was detected by flow cytometry and fluorescence microscope.

[0051] The steps are as follows: ① Count Raji cells, Nmlm-6 cells, normal peripheral blood cells, B-cell lymphoma patient cells and B-cell acute leukemia cells (B-ALL), block Fc receptors with bovine serum albumin, wash with PBS, and adjust them respectively. Concentration to 1×10 7 / ml, take 100μl of cell suspension from each tube; ②Add 50μl of S3 monoclonal antibody culture supernatant or 10μl of purified antibody to each tube, incubate at 4°C for 30min; ③Add PBS and centrifuge at 1000g for 10 minutes to wash away unbound S3; ④Add 5 μl of FITC-labeled goat anti-mouse monoclonal antibody (GAM-FITC) respectively, incubate at 4°C for 30min; ⑤Add PBS respectively, centrifuge at 1000g for 10min, remove the supernatant; ⑥Detect and analyze by flow cytometry or fluorescence microscope. ⑦ The results are shown in Figure 2 to Figure 6 :

[0052] like f...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

PropertyMeasurementUnit
molecular weightaaaaaaaaaa
molecular weightaaaaaaaaaa
Login to View More

Abstract

The invention discloses an antibody for resisting human CD79a extracellular terminal protein, a coding gene and application. The antibody is characterized in that a human B cell lymphoma Raji cell line is used as an immunogen for immunizing mice; splenocyte is obtained and then is fused with a mouse myeloma cell to obtain a hybridoma cell; a monoclonal antibody for specifically aiming at the human CD79a extracellular terminal protein is obtained via screening; an amino acid sequence of the monoclonal antibody is obtained by sequence analysis; variable region sequence with a heavy chain and a light chain and CDR (Complementarity-Determining Region) sequences with light chains and heavy chains are obtained via analyzing; an chimeric antibody expressed via cloning can be used for effectively and specifically recognizing the human CD79a extracellular terminal protein; in addition, when the antibody acts on human B lymphocyte, the internalization rate is high; the antibody has a good prospect for preparing a targeted drug of targeted B lymphocyte and can be used for treating diseases associated with the B lymphocyte.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an antibody against human CD79a extracellular terminal protein, an encoding gene and an application. Background technique [0002] B cell antigen recognition receptor (BCR) is the most important molecule on the surface of B cells. It consists of a mIg that recognizes and binds antigen and two CD79a / CD79b (also known as Igα / Igβ) heterodimers that transmit antigen-stimulating signals constitute. The B cell molecule CD79a / CD79b is a member of the immunoglobulin superfamily with molecular weights of 45kDa, 33kDa and 37kDa, respectively, which have extracellular, transmembrane and cytoplasmic regions. The structure of the extracellular domain belongs to IgSF, with a C2-like domain and a V-like domain, respectively, and a glutamic acid in the transmembrane domain. The negatively charged glutamate or glutamine in the transmembrane region of CD79a and CD79b and the positively charged amino...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13A61K47/68A61K31/537A61K38/07A61P35/02A61P37/02
CPCA61K31/537A61K38/07C07K16/2803C07K2317/56C07K2317/77A61K2300/00
Inventor 沈红强舒强
Owner ZHEJIANG UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com