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Anti-cd47/cd20 bispecific antibody and use thereof

A bifunctional antibody and antibody technology, applied in the field of tumor immunology

Active Publication Date: 2020-10-09
SHANGHAI NOVAMAB BIOPHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, many technologies can only solve one or two problems, which makes the development of bispecific antibody products always face technical bottlenecks

Method used

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  • Anti-cd47/cd20 bispecific antibody and use thereof
  • Anti-cd47/cd20 bispecific antibody and use thereof
  • Anti-cd47/cd20 bispecific antibody and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0200] Example 1 Molecular Sequence Design of Anti-CD47 / CD20 Bispecific Antibody

[0201] The structure of the bispecific antibody that can simultaneously bind to the extracellular domains of CD47 and CD20 provided by the present invention is as follows: figure 1 shown. The CD47 nanobody sequence is derived from patent 201810151752.6; then the above CD47 nanobody is fused with the heavy chain of rituximab, and the heavy chain is produced by Linker ((GGGGS) 4 ) to link the rituximab heavy chain (LALA) and the CD47 Nanobody, the amino acid sequence is as follows (the underlined part is the Linker sequence):

[0202] A: The amino acid sequence is shown in SEQ ID NO.: 14, and the CD47 Nanobody is connected to the N-terminal of the heavy chain of the rituximab through a linker (GGGGS) 4;

[0203] QVQLQESGGGLVQPGGSLRLSCAASGYAYTSDCMGWFRQTPGKGLEGVALIYTPGNSTNYADSVKGRFTISQDNSKSTVYLQMNSLRAEDTAMYYCAARRGACSLRLPFFYWGQGTLVTVSS GGGGSGGGGSGGGGSGGGGS QVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQT...

Embodiment 2

[0214] Example 2 Expression and purification of anti-CD47 / CD20 bispecific antibody

[0215] Transiently transfer the two plasmids containing the synthetic gene into HEK293F cells. The specific method is as follows: (1) Use the OMEGA plasmid answering kit to prepare a large number of plasmids containing the heavy chain and the plasmid containing the light chain respectively. Filter and sterilize in the stage for standby; (2) Cultivate HEK293F cells to 2.0×10 6 each / mL; (3) Mix the heavy chain plasmid and the light chain plasmid according to the mass ratio of 2:3, and mix the mixed plasmid with the transfection reagent PEI at a ratio of 1:3 to the transfection medium F17 (Gibco) Stand in the middle for 20min, then add to HEK293F cells, at 37℃, 6%CO 2 Cultivate in a shaker incubator for 6 days; (4) Centrifuge to obtain the supernatant, combine the supernatant with protein A beads at room temperature for 1 hour; (5) Use pH 7.0 phosphate buffer to wash away foreign proteins and ot...

Embodiment 3

[0218] Embodiment 3 FACS detects the binding situation of antibody and cell surface antigen

[0219] Binding of anti-CD47 / CD20 bispecific antibody (A-F) to CD20 on cell surface: (1) 3×10 5 Raji (CD20+) cells (each sample) were incubated with 100uL of CD47 nanobody Nb1902 (expressed in E.coli) at a concentration of 80ug / mL for 20min at 4°C; (2) Antibody dilution: each group of antibodies were serially diluted ( 100ug / mL, 50ug / mL, 25ug / mL, 12.5ug / mL, 6.25ug / mL, 3.125ug / mL, 1.56ug / mL, 0.78ug / mL, 0.39ug / mL, 0.195ug / mL, 0.0976ug / mL mL, 0.0488ug / mL), incubate 100uL of the diluted antibody with each well of Raji cells for 20min at 4°C, and set a positive control (rituximab) at the same time; (2) Wash the cells twice with PBS, add eBioscience’s Anti-hFc-FITC (diluted at 1:200) was incubated at 4°C for 20 min, cells were washed twice with PBS and detected by flow cytometry (BD FACS Calibur), and data processing was performed using graphpad prism 6 software. The result is as figure ...

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PUM

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Abstract

The invention relates to an anti-CD47 / CD20 bispecific antibody and an application thereof. Specifically, the invention provides the anti-CD47 / CD20 bispecific antibody, which comprises: (a) the anti-CD20 antibody (such as rituximab); and (b) the anti-CD47 nanobody in a monovalent form linked to the anti-CD20 antibody. The bifunctional antibody disclosed by the invention has good stability, the bifunctional antibody has the advantages that CD47 and CD20 can be specifically targeted, the interaction between CD47 and its ligand SIRPa is effectively blocked, CD20 on the cell surface is effectivelycombined, tumor cell proliferation can be remarkably inhibited, the synergistic effect of the bifunctional antibody is achieved, and the bifunctional antibody has a good application prospect.

Description

technical field [0001] The invention belongs to the field of tumor immunology, and in particular relates to anti-CD47 / CD20 bispecific antibodies and uses thereof. Background technique [0002] Leukocyte differentiation antigen 47 (CD47), also known as integrin associated protein (IAP), is a member of the immunoglobulin superfamily. As a highly glycosylated transmembrane protein of about 50kD, CD47 molecule consists of an extracellular N-terminal immunoglobulin variable domain, five transmembrane segments with highly hydrophobic extensions and a short alternatively spliced ​​cytoplasmic region . CD47 is widely distributed and expressed on the surface of myeloid cells such as macrophages, granulocytes, dendritic cells, mast cells and hematopoietic stem cells, especially highly expressed in stem cells. CD47 is a multipotent molecule, and its main functions include ① binding to CD47 ligand signal-regulatory protein (SIRP) α and interacting with integrin to regulate intercellul...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/46C12N15/13A61K39/395A61P35/00
CPCA61K2039/505A61P35/00C07K16/2803C07K16/2887C07K2317/31C07K2317/569C07K2317/76C07K2317/92C07K2317/94
Inventor 万亚坤朱敏沈晓宁
Owner SHANGHAI NOVAMAB BIOPHARM CO LTD
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