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Anti-CD47/VEGF bispecific antibody and application thereof

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

Active Publication Date: 2020-03-17
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/VEGF bispecific antibody and application thereof
  • Anti-CD47/VEGF bispecific antibody and application thereof
  • Anti-CD47/VEGF bispecific antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0204] Example 1 Molecular Sequence Design of Anti-CD47 / VEGF Bispecific Antibody

[0205] The structure of the bispecific antibody that can simultaneously bind CD47 and VEGF extracellular domain provided by the present invention is as follows: figure 1 shown. The CD47 single domain antibody sequence is derived from the patent 201810151752.6; then the above CD47 single domain antibody is fused with the heavy chain of the Bevacizumab monoclonal antibody, and the heavy chain is fused by Linker ((GGGGS)4) to the Bevacizia antibody heavy chain (LALA) and CD47 monoclonal antibody Domain antibody connection, the amino acid sequence is as follows (the underlined part is the Linker sequence):

[0206] A: The amino acid sequence is shown in SEQ ID NO.: 13, and the CD47 single domain antibody is connected to the C-terminal of the heavy chain of the bevacizumab through a linker (GGGGS) 4;

[0207] EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAED...

Embodiment 2

[0212] Example 2 Expression and purification of anti-CD47 / VEGF bispecific antibody

[0213]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 oth...

Embodiment 3

[0216] Example 3 Candidate Double Antibody A Purity Analysis

[0217] The purified sample bis-antibody A was subjected to SEC analysis: (1) Sample preparation: Dilute the sample to 1.0 mg / mL with mobile phase as the test sample. Use a 0.22um syringe filter to filter into a sample vial and put it into an HPLC autosampler for sample injection. (2) Mobile phase: 200mM phosphate aqueous solution with pH 7.0, chromatographic column: X bridge BEH200SEC 3.5μm 7.8x300mm, the parameters are set as follows: detection wavelength 280nm, column temperature 25℃, flow rate 0.5mL / min, injection volume 10uL. (3) Test results such as figure 2 As shown, the purity of double antibody A reached 95.06%, and the purity of double antibody B reached 97.67%, which can be used in subsequent experiments.

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Abstract

The invention provides an anti-CD47 / VEGF bispecific antibody and an application thereof. The anti-CD47 / VEGF bispecific antibody comprises: (a) the anti-VEGF antibody bevacizumab; and (b) the anti-CD47monodomain antibody in a monovalent form linked to the anti-VEGF antibody. The bifunctional antibody disclosed by the invention not only can specifically target CD47 and VEGF and effectively block the interaction between a target and a ligand thereof, but also can effectively inhibit tumor cell proliferation, reserves the anti-tumor functions of a CD47 single-domain antibody and bevacizumab, andhas a good application prospect.

Description

technical field [0001] The invention belongs to the field of tumor immunology, and in particular relates to anti-CD47 / VEGF bispecific antibody and application thereof. Background technique [0002] Members of the vascular epidermal growth factor family—A (VEGF), B, C, D, and placental growth factor all belong to the cysteine ​​knot growth factor superfamily proteins, which play a role in normal conditions (such as development, injury repair) and pathological conditions Angiogenesis, angiogenesis and lymphangiogenesis play an important role in conditions such as tumor growth and intraocular neovascularization. VEGFA plays an important role in functions important to angiogenesis such as endothelial cell proliferation and survival, increased vascular permeability, and chemotaxis and homing of hematopoietic cells to the bone marrow. VEGFA exists in multiple subtypes: 121 (VEGFA), 165, 189 and 206, among which VEGFA165 is the most common subtype expressed in normal tissues and t...

Claims

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Application Information

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IPC IPC(8): C07K16/46C12N15/13A61K39/395A61P35/00A61P35/02
CPCA61K2039/505A61P35/00C07K16/22C07K16/2803C07K2317/31C07K2317/569C07K2317/76C07K2317/92
Inventor 万亚坤朱敏沈晓宁
Owner SHANGHAI NOVAMAB BIOPHARM CO LTD
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