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Hybridoma cell strain 105D11, antibody and application thereof

A hybridoma cell line, monoclonal antibody technology, applied in application, antibody, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the problems of no product, market, etc., and achieve affinity High, promoting phagocytosis, good specificity

Active Publication Date: 2020-01-24
ZHEJIANG BLUE SHIELD PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the number of units developing CD47 antibody drugs at home and abroad is gradually increasing, so far no products have been launched, and Hu5F9-G4 from FORTY SEVEN is at the forefront of clinical phase II.

Method used

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  • Hybridoma cell strain 105D11, antibody and application thereof
  • Hybridoma cell strain 105D11, antibody and application thereof
  • Hybridoma cell strain 105D11, antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028]Example 1: Construction of CHO-K1 stable cell lines overexpressing human CD47 and cynomolgus monkey CD47

[0029] 1) Shuttle carrier construction

[0030] Human CD47 protein sequence

[0031] NP_001768leukocyte surface antigen CD47 isoform 1precursor[Homosapiens]

[0032] MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE

[0033] 35,214Da, 323aa, 1-18 are signal peptides, 19-141,198-207,257-268 are extracellular domains, 142-162,177-197,208-228,236-256,269-289 are transmembrane helices, 163-176,229-235,290-323 are intracellular domains .

[0034] Cynomolgus monkey CD47 protein sequence

[0035] XP_005548289PREDICTED:leukocyte surface antigen CD47 isoform X1[Macaca fascicularis]

[0036] MWPLVAALLL...

Embodiment 2

[0059] Example 2: Animal immunity and antiserum ELISA titer and flow cytometry assay

[0060] Five 6- to 8-week-old Balb / c mice (Beijing Speifu) were selected and housed in the same cage, numbered #1, #2, #3, #4, and #5 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 immunogen of recombinant human CD47-mFc fusion protein (Acro Biosystems CD7-H52A5) 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. 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. The diff...

Embodiment 3

[0064] The preparation of embodiment 3 hybridoma monoclonal cell lines

[0065] 1. Cell Fusion

[0066] Cell fusion was performed using the polyethylene glycol method. The specific operation is as follows:

[0067]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.

[0068] 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.

[0069] 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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Abstract

The invention discloses a hybridoma cell strain 105D11 and an anti-human CD47 monoclonal antibody generated from the hybridoma cell strain 105D11. A high-affinity anti-human CD47 specific monoclonal antibody which can block the interaction of CD47-SIRPalpha is prepared by using CD47 recombinant protein with bioactivity as an antigen through screening of a hybridoma technique. Meanwhile, an in-vitro experiment proves that the anti-human CD47 specific monoclonal antibody generated from the hybridoma cell strain 105D11 can promote macrophages to phagocytize tumor cells and cannot cause agglutination of erythrocytes. Therefore, the antibody has a potential value in tumor immunotherapy.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a hybridoma cell line 105D11, a monoclonal antibody capable of binding to human CD47 with specificity and high affinity produced by it, a preparation method thereof, a variable region sequence thereof, and a pharmaceutical application thereof. 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 tumo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/28C12N15/13A61K39/395A61P35/00G01N33/577G01N33/574
CPCA61K2039/505A61P35/00C07K16/2803C07K2317/76C07K2317/92G01N33/57492G01N33/577G01N2333/70503
Inventor 郭志刚黄子祥邬婷李露露
Owner ZHEJIANG BLUE SHIELD PHARM CO LTD
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