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Vegf and PDGFRβ bispecific fusion protein and use thereof

A fusion protein and bispecific technology, applied in the direction of hybrid immunoglobulin, fusion polypeptide, immunoglobulin, etc., can solve problems such as limited effect of means

Active Publication Date: 2018-10-19
SHANGHAI HENGRUI PHARM CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0013] At present, it is believed that the effect of neutralizing VEGF is limited, mainly because the pericytes (vascularmural cells) attached to blood vessels protect blood vessels in the absence of VEGF

Method used

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  • Vegf and PDGFRβ bispecific fusion protein and use thereof
  • Vegf and PDGFRβ bispecific fusion protein and use thereof
  • Vegf and PDGFRβ bispecific fusion protein and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0103] Example 1. Bispecific fusion proteins

[0104] The present invention provides a bispecific fusion protein comprising a VEGF-binding peptide and a PDGFRβ-binding peptide; more specifically, a modified extracellular domain of VEGFR (Flt-1, Flk-1, Flt-4), A novel fusion protein formed by the chimerism of PDGFRβ-binding peptide and IgG Fc region.

[0105] In the embodiment of the present invention, the amino acid sequence of the VEGF-binding peptide of the present invention is called VTE; the second component, PDGFR antibody, is called PDX; the third component, the Fc region of IGg, is called Fc. There are three combinations of the three components:

[0106] (1) The amino acid sequence of VTE is upstream of the amino acid sequence of Fc, and the amino acid sequence of Fc is upstream of the amino acid sequence of PDX, such as VTE---Fc---PDX, referred to as VFP;

[0107] (2) The amino acid sequence of VTE is upstream of the amino acid sequence of PDX, and the amino acid seq...

Embodiment 2

[0162] Embodiment 2 vector construction

[0163] All PCR and cloning-related operations were carried out in accordance with molecular cloning standard operations. For details, refer to "Molecular Cloning" (Sambrook et al., Cold Spring Harbor Laboratory).

[0164] Experimental Materials:

[0165] Eukaryotic expression vector pcDNA3.1(+) (Life technologies, Cat. No. V790-20);

[0166] Nucleotide sequence encoding VTE protein (SEQ ID NO: 1): synthesized by Gene Synthesis Company (Jinweizhi, Suzhou);

[0167] Nucleotide sequence encoding Fc protein fragment (SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9): Fc fragment of human antibody heavy chain γ1 constant region, synthesized by Gene Synthesis Company (Jinweizhi, Suzhou );

[0168] Nucleotide sequence of PDX protein fragment (SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6): synthesized by Gene Synthesis Company (Jinweizhi, Suzhou).

[0169] experimental method:

[0170] 1. Fragment splicing

[0171] According to the...

Embodiment 3

[0176] Example 3. Protein expression

[0177] experimental method:

[0178] FreeStyle 293 cells (GIBCO, Cat#R79007) were used to transiently transfect and express the expression vector constructed in Example 2.

[0179] FreeStyle 293 cells were cultured in suspension in a culture medium (Freestyle 293expression medium, GIBCO, Ca#12338018), and ultra-low immunoglobulin fetal bovine serum (Ultra Low IgGetal Bovine Serum, GIBCO, Cat#16250078) was added with a final concentration of 1%.

[0180] Prepare the expression vector constructed in Example 2 and the transfection reagent PEI (Polysciences, Ca#239662), the amount of the expression vector is 100 μg / 100 ml cells, and the mass ratio of the expression vector to PEI is 1:2. On the day of transfection, the cell density was 1×10 6 / ml. For 1L of FreeStyle 293 cells to be transfected, take 50ml of Opti-MEM medium (GIBCO, Cat#11058021) and mix with the expression vector, let stand for 5min, and filter; take another 50ml of Opti-ME...

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Abstract

Provided are VEGF and PDGFRβ bispecific fusion protein and application thereof. More specifically, a bifunctional fusion protein comprising VEGFR extracellular ligand binding domain and PDGFRβ antibody fragment is provided, which can be used to treat wet age-related macular degeneration (AMD) and any other diseases that can be improved by inhibiting VEGF and PDGF signaling pathways , such as tumors and diabetic retinopathy.

Description

technical field [0001] The invention relates to a bispecific fusion protein of VEGF and PDGFRβ, including its pharmaceutical composition and application. Background technique [0002] Age-related macular degeneration (AMD) is a disabling and blinding disease. It primarily affects the macula, or the central area of ​​the retina responsible for high acuity vision, and is a cause of irreversible vision loss in older adults. According to statistics, more than 30 million people around the world suffer from this disease. With the gradual increase of the aging population in China, the disease has seriously endangered the life and health of the people of our country. [0003] There are two forms of AMD, dry AMD and wet AMD. Although dry AMD is more common, it generally causes milder, slower vision loss. New blood vessels form under the retina in people with wet AMD. As photoreceptor cells and retinal pigment epithelial (RPE) cells slowly degenerate, blood vessels tend to develo...

Claims

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Application Information

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IPC IPC(8): C07K19/00C07K16/46C07K14/475C07K16/28C12N15/62C12N15/63C12N5/10A61K38/00A61K39/395A61P35/00A61P27/02
CPCA61K38/00A61P27/02A61P35/00C07K14/475C07K14/49C07K16/2863C07K2319/00
Inventor 屈向东曹国庆叶鑫陈侃张蕾许志宾袁纪军张连山潘琴管雁宾
Owner SHANGHAI HENGRUI PHARM CO LTD
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