Hybridoma cell strain 12G6, antibody and application of antibody

A hybridoma cell line and monoclonal antibody technology, applied in the biological field, to achieve the effect of good specificity and high affinity

Pending Publication Date: 2020-01-31
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, the only antibody drug targeting SIRPα reported at home and abroad is OSE-172 developed by Boehringer Ingelheim, which is still in the preclinical stage

Method used

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  • Hybridoma cell strain 12G6, antibody and application of antibody
  • Hybridoma cell strain 12G6, antibody and application of antibody
  • Hybridoma cell strain 12G6, antibody and application of antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

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

[0027] 1) Shuttle carrier construction

[0028] Human SIRPα protein sequence

[0029] NP_542970tyrosine-protein phosphatase non-receptor type substrate1isoform 1precursor[Homo sapiens]

[0030]MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAAENTGSNERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTRLHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLTYADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK

[0031] 54,967Da, 504aa, 1-30 is the signal peptide, 31-373 is the extracellular domain, 374-394 is the transmembrane helix, and 395-504 is the intracellular domain.

[0032]...

Embodiment 2

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

[0058] Two 6- to 8-week-old Balb / c mice (Beijing Speifu) were selected and housed in the same cage, numbered #1 and #2 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 SIRPα-hFc fusion protein immunogen (Acro Biosystems SIA-H5251) was fully mixed with an equal volume of Freund's complete adjuvant (SIGMA), and the phacoemulsification was complete. Two subcutaneous and two intraperitoneal injections were taken, and the immunization dose was 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 respectively...

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 single-...

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PUM

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Abstract

The invention discloses a hybridoma cell strain 12G6 and an anti-human SIRP alpha monoclonal antibody generated by the hybridoma cell strain 12G6. The high-affinity anti-human SIRP alpha specific monoclonal antibody capable of blocking CD47-SIRP alpha interaction is screened and prepared by taking SIRP alpha recombinant protein with biological activity as an antigen through a hybridoma technology.Therefore, the antibody has potential value in tumor immunotherapy.

Description

technical field [0001] The present invention relates to the field of biotechnology, in particular to a hybridoma cell line 12G6, a monoclonal antibody capable of binding to human SIRPα with specificity and high affinity produced by it, its preparation method, its variable region sequence, and its application. Background technique [0002] Under normal circumstances, the immune system can recognize and eliminate tumor cells in the tumor microenvironment. However, in order to survive and grow, tumor cells can adopt different strategies to suppress the immune system of the human body and cannot kill tumor cells normally. All stages of the immune response were survived. The tumor-immune cycle is divided into the following seven steps: 1. Tumor antigen release; 2. Tumor antigen presentation; 3. Initiation and activation of effector T cells; 4. T cell migration to tumor tissue; 5. T cell infiltration of tumor tissue; 6. T cells recognize tumor cells; 7. Clear tumor cells. Abnorm...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/28C12N15/13A61K39/395A61P35/00G01N33/577G01N33/574G01N33/68C12R1/91
CPCC07K16/2896A61P35/00G01N33/577G01N33/57484G01N33/6872C07K2317/92C07K2317/73C07K2317/56C07K2317/565G01N2333/70596A61K2039/505
Inventor 郭志刚黄子祥赵行琦
Owner ZHEJIANG BLUE SHIELD PHARM CO LTD
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