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CD47 antibody or immunocompetence fragment thereof and application

An immune activity, 1. CD47 technology, applied in the field of biomedicine, can solve the problems of greatly reduced therapeutic effect, reduction of red blood cells and platelets, and side effects

Active Publication Date: 2020-07-28
BETA PHARM SUZHOU LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, these CD47 monoclonal antibodies have high target binding to red blood cells and platelets, which not only greatly reduces the therapeutic effect of the corresponding antibodies, but also introduces drug side effects, and clinically occurs red blood cells and platelets and other related side effects

Method used

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  • CD47 antibody or immunocompetence fragment thereof and application
  • CD47 antibody or immunocompetence fragment thereof and application
  • CD47 antibody or immunocompetence fragment thereof and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1 Preparation of CD47 antibody of the present invention

[0068] (1) Mouse immunization and fusion

[0069] Ten mice (5 balb / c, 5 C57bl / 6) will be immunized according to Table 3, using CD47-mFc (Acro, CD7-H52A5, mFc tag) as the immunogen.

[0070] Table 3. Immunization Schedule

[0071]

[0072] Select 4 mice with the best response for fusion (ELISA serum titer is greater than 256K, FACS serum titer is greater than 1 log shift degree.) as Figure 1A , Figure 1B , AD335, AD336, AD355, and AD356 were selected for fusion of spleen B cells and mouse myeloma cells, and hybridoma screening was carried out one week later, among which AD335, AD336 belonged to the balb / c strain, and AD355, AD356 belonged to the C57bl / 6 strain.

[0073] The antibody screening method involves double screening at the protein level and the cell level. In the protein level screening, the ELISA detection method of human CD47 protein binding is used; in the cell level screening, the FACS ...

experiment example 1

[0079] Experimental example 1 The dose effect (ELISA) of the combination of CD47 antibody of the present invention and human CD47 recombinant fusion protein

[0080] Coat hCD47-his (Cat#CD7-H5227, Lot#C56P1-737F1-FA) at 1 μg / ml, and select PBS (HyClone Lot: AC13298279) as the coating buffer, 100 μl / well, at room temperature (25°C) After 16-18h, wash the plate twice with TBST, block with PBS+3%BSA, 200μl / well, block at room temperature (25°C) for 16-18h, wash the plate once with TBST, tap dry, and dry at 37°C for 2 hours. Prepare the CD47 antibody of the present invention and the control antibody according to 330 μl 100 μg / ml, 10 μg / ml is the first gradient, and perform a 4-fold gradient dilution. For example, the second gradient is to add 80 μl of the first gradient to 240 μl PBS, and so on, a total of 11 a gradient concentration. Incubate at 37°C for 1 hour. After the plate was washed 3 times with PBST by an automatic plate washer, 100 μl of 1:20000 diluted goat anti-human ...

experiment example 2

[0082] Experimental Example 2 CD47 Antibody of the Present Invention Binding Activity to Cells Expressing Human CD47 (FACS)

[0083] The binding activity of the CD47 antibody of the present invention to CHO-K1 stable cell lines, Jurkat, CCRF-CEM, Raji and other cancer cell lines expressing human or cynomolgus CD47 was determined by flow cytometry (BD FACSCelesta Cell Analyzer).

[0084] In this experiment, two stable cell lines expressing CD47 (CHO-K1-Cyno-CD47 / CHO-K1-Human-CD47) and three cancer cell lines, CHO-K1-Cyno-CD47 / CHO-K1-Human -CD47 was derived from Nanjing GenScript Biotechnology Co., Ltd., Jurkat ( TIB-152 TM ) belongs to T lymphocytic leukemia cells, CCRF-CEM (Cell Bank, Chinese Academy of Sciences, Shanghai) belongs to human acute lymphoblastic leukemia T lymphocytes, and Raji (Cell Bank, Chinese Academy of Sciences, Shanghai) belongs to human lymphoma cells. After digesting the listed cells (suspension cells do not need to be digested), centrifuge at room te...

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Abstract

The invention provides a novel CD47 antibody or an immunocompetence fragment thereof, or a composition including the antibody / immunocompetence fragment. The CD47 antibody cannot cause red cell agglutination, and shows low combination at an extremely weak level or non-combination with red cells and blood platelets. The invention provides nucleic acid coding the CD47 antibody or the immunocompetencefragment, an expression vector and a host cell. In addition, the invention provides the purpose of the CD47 antibody or a medical composition containing the CD47 antibody / immunocompetence fragment for preparation of medicines for treating CD47 mediated diseases.

Description

technical field [0001] The present invention relates to the field of biomedicine, specifically to the field of antibody technology and immunity, and more specifically to the CD47 antibody or its immune active fragment and application. Background technique [0002] CD47, also known as Integrin Associated Protein (IAP), is widely expressed on the surface of cells and can interact with signal regulatory protein α (Signalreg μlatory protein α, SIRPα), thrombospondin-1 (TSP1) and integrin (integrins) interact and mediate a series of responses such as apoptosis, proliferation, and immunity. CD47 was first identified as a tumor antigen of human ovarian cancer in the 1980s, and then CD47 was found to be expressed in various types of human tumors, including acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic Cellular leukemia (ALL), non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), bladder cancer (BC) and other solid tumors. Its expression and activ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13C12N15/85C12N5/10A61K39/395A61P35/00
CPCA61K39/395A61K2039/505A61P35/00C07K16/2803C07K2317/31C07K2317/54C07K2317/55C07K2317/56C07K2317/622C12N15/85A61K2300/00
Inventor 王雷黄苏萍张楠张崇骞赵永浩汤春张晓霞J·彭D·张
Owner BETA PHARM SUZHOU LTD
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