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Hybridoma cell strain 6F9, antibody and application of hybridoma cell strain 6F9

A hybridoma cell line, monoclonal antibody technology, applied in application, antibody, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., to achieve the effect of good specificity and high affinity

Active Publication Date: 2020-01-17
ZHEJIANG BLUE SHIELD PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no anti-CD70 antibody drug on the market, basically all of them are in different clinical stages, and mainly ADC

Method used

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  • Hybridoma cell strain 6F9, antibody and application of hybridoma cell strain 6F9
  • Hybridoma cell strain 6F9, antibody and application of hybridoma cell strain 6F9
  • Hybridoma cell strain 6F9, antibody and application of hybridoma cell strain 6F9

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Construction of CHO-K1 stable cell lines overexpressing human CD70 and cynomolgus monkey CD70

[0027] 1) Shuttle carrier construction

[0028] Human CD70 protein sequence

[0029] NP_001243 CD70 antigen isoform 1[Homo sapiens]

[0030] MPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLPL ESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIYMVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTCTNLTGTLLPSRNTDETFFGVQWVRP

[0031] 21,118Da, 193aa, 1-17 is the intracellular domain, 18-38 is the transmembrane helix, 39-193 is the extracellular domain.

[0032] Cynomolgus monkey CD70 protein sequence

[0033] XP_005587773PREDICTED:CD70 antigen[Macaca fascicularis]

[0034] MNGPRKNEVEREIGRSGGEGLGTGNSVAHPRPLPGPSGNHLHPLCELQTGSSWREFPLANRSSPSPRPAGHPQRGAGWSPDKLRQVDAQEPREGAAVAFLPFPAALCAPLAPPALAEVIAAAMPEEGSGCAVRRRPYACVLRAAVVPLVAGLAICLVVCVQRLSRAQQQLPLESLGWDIAELQLNHTGPQQDPRLYWQGGPALGRSFLRGPELDKGQLRIRRDGIYMVHIQVTLAICSSTSTSRHHHPTTLAVGICSPASRSISLLRLSFHQGCTIASQRLTPLARGDTLCT...

Embodiment 2

[0057] Example 2: Animal immunity and antiserum ELISA titer and flow cytometry assay

[0058] Three 6- to 8-week-old Balb / c mice (Beijing Speyford) were selected and housed in the same cage, numbered #1, #2, and #3 respectively. Four days before the first immunization, about 0.05ml of blood was collected from each mouse through the tail vein, placed at 4°C for half an hour, then centrifuged at 10,000rpm at 4°C for 10min, and the serum was separated as negative control serum for subsequent experiments. For the first immunization, the recombinant human CD70-mFc fusion protein immunogen (Acro Biosystems CDL-H525a) was fully mixed with an equal volume of Freund's complete adjuvant (SIGMA), and the phacoemulsification was complete, and two subcutaneous and two intraperitoneal injections were taken. The immunization dose For 50 μg of immunogen per mouse, the injection volume was 0.2 ml. The second and third immunizations were carried out at intervals of 14 days and 35 days respecti...

Embodiment 3

[0062] The preparation of embodiment 3 hybridoma monoclonal cell lines

[0063] 1. Cell Fusion

[0064] Cell fusion was performed using the polyethylene glycol method. The specific operation is as follows:

[0065] 1) One week before fusion, revive SP2 / 0-Ag14 myeloma cells (Beijing Beina). Two days before cell fusion, expand the culture of SP2 / 0-Ag14 so that it is in logarithmic growth phase on the day of fusion.

[0066] 2) Half an hour before cell fusion, pre-treat SP2 / 0-Ag14 cells, resuspend SP2 / 0-Ag14 cells and count, take 2-3×10 7 The cells were placed in a 37°C water bath for later use.

[0067] 3) Collect blood from the heart of the mouse to be fused, collect serum in the same manner as above, and store it at -20°C, which can be used as a positive control for screening after fusion. The mice were killed by neck dislocation, soaked in 75% alcohol, and transferred to the cell room. The spleen was taken for grinding, and filtered through a 70 μm sieve to make a singl...

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PUM

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Abstract

The invention belongs to the technical field of biology, and particularly discloses a hybridoma cell strain 6F9, an anti-human CD70 monoclonal antibody generated by the hybridoma cell strain 6F9 and an application of the hybridoma cell strain 6F9. The the hybridoma cell strain 6F9 uses a biologically-active human CD70 recombinant protein as an antigen, and obtains a specific monoclonal antibody capable of binding human CD70 with high affinity at the cell level through hybridoma technology screening. The antibody has potential value in tumor immunotherapy.

Description

technical field [0001] The invention belongs to biogenetic engineering antibody technology, in particular to a hybridoma cell line 6F9, the anti-human CD70 monoclonal antibody produced by it and its application. [0002] technical background [0003] CD70 is a type II transmembrane glycoprotein and also belongs to the TNF family. Human CD70 is a trimer composed of 193 amino acids, of which 155 amino acid sequences outside the cell membrane have certain homology with other members of the TNF ligand superfamily. The cytokine receptor CD27 is a member of the tumor necrosis factor receptor (TFNR) superfamily, which plays a role in cell growth and differentiation as well as apoptosis. The ligand for CD27 is CD70, which belongs to the tumor necrosis factor family of ligands. [0004] Under normal circumstances, CD70 is only transiently expressed on the surface of activated T and B lymphocytes and mature DC cells, and is generally not expressed on other non-lymphoid normal tissues...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/28C12N15/13G01N33/68G01N33/577A61K39/395A61P35/00C12R1/91
CPCA61K2039/505A61P35/00C07K16/2875C07K2317/92G01N33/577G01N33/68G01N2333/70575
Inventor 郭志刚黄子祥杨洋李梦晗
Owner ZHEJIANG BLUE SHIELD PHARM CO LTD
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