Humanized CD47 antibody or antigen binding fragment thereof and application

A combination of fragments and humanized technology, applied in the field of biomedicine, can solve problems such as red blood cell and platelet reduction, side effects, and greatly reduced therapeutic effect

Active Publication Date: 2021-04-20
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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  • Humanized CD47 antibody or antigen binding fragment thereof and application
  • Humanized CD47 antibody or antigen binding fragment thereof and application
  • Humanized CD47 antibody or antigen binding fragment thereof and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1: Production and purification of CD47 humanized antibody and control antibody

[0084] The amino acid sequence and CDR sequence of the humanized CD47 antibody of the present invention are shown in Table 1 and Table 2, and the expression vector of the humanized CD47 antibody was constructed in vitro.

[0085] Specifically, the light chain variable region (VL) sequence in Table 1 was constructed to the human antibody light chain κ chain constant region (SEQ ID NO: 58), and the heavy chain variable region (VH) sequence was constructed to the human antibody A constant region of a heavy chain, preferably a human IgG4 (S228P) heavy chain constant region (SEQ ID NO: 60). Construct the expression vectors of the following humanized antibodies: h99B5-IgG4-1, h99B5-IgG4-2, h99B5-IgG4-3, h99B5-IgG4-4, h99B5-IgG4-5, h81C1-IgG4-1, h81C1-IgG4- 2, h81C1-IgG4-3, h81C1-IgG4-4, h81C1-IgG4-5, h100-IgG4-1, h100-IgG4-2, h100-IgG4-3, h100-IgG4-4, h100-IgG4-5, h100-IgG4-6, h100-IgG4...

Embodiment 2

[0090] Example 2: The dose effect (ELISA) of the binding of the humanized CD47 antibody of the present invention to the human CD47 recombinant fusion protein

[0091] Coat hCD47-his (Cat#CD7-H5227, Lot#C56P1-737F1-FA) at 1 μg / m, 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 ...

Embodiment 3

[0094] Example 3: The humanized CD47 antibody of the present invention and the cell binding activity (FACS) of various cancer cell lines expressing human CD47 / cynomolgus monkey were measured by flow cytometry (BD FACS Celesta Cell Analyzer) of the CD47 antibody of the present invention Binding activity to CHO-K1 stable cell lines expressing human or cynomolgus monkey CD47, Jurkat, Raji, MDA-MB-231, SHP77, U-87 cells and other cancer cell lines.

[0095]In this experiment, two stable cell lines expressing CD47 (CHO-K1-Cyno-CD47 / CHO-K1-Human-CD47) and five cancer cell lines, CHO-K1-Cyno-CD47 / CHO-K1-Human -CD47 was derived from Nanjing GenScript Biotechnology Co., Ltd., Jurkat, Raji, MDA-MB-231, SHP77, U-87 (Cell Bank, Chinese Academy of Sciences, Shanghai). After digesting the listed cells (suspension cells do not need to be digested), centrifuge at room temperature, 1000rpm, 5mins, discard the supernatant, wash with PBS, resuspend in PBS in the cell flow tube, adjust the cell c...

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PUM

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Abstract

The invention provides a humanized CD47 antibody or an antigen binding fragment thereof and application, the humanized CD47 antibody or the antigen binding fragment thereof is low in toxicity and efficient, erythrocyte agglutination does not occur in vitro, and erythrocyte removal cannot be caused. In addition, the humanized CD47 antibody provided by the invention shows extremely weak level of low binding or non-binding with platelets and red blood cells, and shows more specific targeting specificity on CD47+ tumor cells. The humanized CD47 antibody or the antigen binding fragment thereof provided by the invention can effectively block the binding of CD47 and SIRP alpha in function, and activate and mediate the phagocytic activity of macrophages to tumor cells.

Description

technical field [0001] The present invention relates to the field of biomedicine, specifically to the field of antibody technology and the field of immunity, and more specifically to the humanized CD47 antibody or its antigen-binding fragment and application. Background technique [0002] CD47 is also known as Integrin Associated Protein (IntegrinAssociated Protein, IAP), which is widely expressed on the surface of cells, and can bind to signal regulatory protein α (Signalreg μlatory protein α, SIRPα), thrombospondin-1 (TSP1) and integrin ( integrins) to 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) an...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13C07K16/46A61K39/395A61P35/00A61P35/02
Inventor 张崇骞李虹侠宗超张楠汤春张晓霞J·彭D·张
Owner BETA PHARM SUZHOU LTD
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