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Anti-VEGF and anti-PD1 bispecific antibody with novel structure

A bispecific antibody and a new structure technology, applied in the field of molecular immunology, can solve the problems of not being able to adapt to complex diseases and single antibody effect, achieve the best experimental results and inhibit tumor growth

Active Publication Date: 2019-11-26
ANHUI RUBIOX VISION BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a stable and brand-new anti-VEGF-anti-PD1 bispecific antibody Ps3Vm, which has high affinity and high specificity, can specifically recognize two targets of VEGF and PD1, and solves the problem of existing antibodies Single effect, unable to adapt to defects such as complex diseases

Method used

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  • Anti-VEGF and anti-PD1 bispecific antibody with novel structure
  • Anti-VEGF and anti-PD1 bispecific antibody with novel structure
  • Anti-VEGF and anti-PD1 bispecific antibody with novel structure

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Preparation of PD1 and VEGF antigens and antibodies

[0041] 1. Construction of expression vector for PD-1 antigen

[0042] The cDNA of human PD-1 was synthesized from Nanjing GenScript Company, the GeneID is 5133, and the cDNAID is NM_005018.2. After the PD-1 gene in the extracellular region was synthesized, the Fc purification tag was added to obtain PD-1-mFc, and the PD-1-mFc was obtained in two The Xba I and Bam H I restriction sites were introduced into the pTT5 expression plasmid, which was verified to be correct by sequencing. The sequenced plasmid was transfected into Trans10 (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), and a single clone was picked and inoculated into 1 liter of LB liquid medium until OD 600 When the temperature is 1, the bacteria are collected by centrifugation, and the plasmid is extracted with a large plasmid extraction kit (purchased from Qiagen).

[0043] 2. Construction of expression vector for VEGF antigen

...

Embodiment 2

[0068] Example 2 Preparation of Candidate Bispecific Antibodies

[0069] 1. Preparation of scFv-VEGF-linker-PD1-H chain structure bispecific antibody (A3P4):

[0070] On the basis of the existing anti-VEGF humanized antibody, the heavy chain and light chain variable region genes were extracted and connected with peptides to form a single-chain antibody scFv-VEGF. The scFv-VEGF was cloned into the N-terminal of the heavy chain of the anti-PD1 antibody to construct a bispecific antibody with the structure of scFv-VEGF-linker-PD1-Hchain. The heavy chain expression vector and the light chain expression vector of anti-PD1 antibody were co-transformed into 293F cells, the supernatant was collected, purified, and the molecular weight and purity were identified by SDS-PAGE ( Figure 4 ), SEC detected that there were more antibody dimers in this sequence ( Figure 5 ).

[0071] 2. Preparation of bispecific antibody (Vs3P4) with dsFv-VEGF-linker-PD1-H chain structure:

[0072] On th...

Embodiment 3

[0075] Example 3 Bispecific Antibody Affinity Determination

[0076] 1. Affinity of bispecific antibody to PD-1

[0077] Coat the microtiter plate with PD-1-mFc, block with 1% BSA, add different concentrations of antibodies PDAB, A3P4, Vs3P4, and Ps3Vm to the microtiter plate, incubate at 37°C, and then add the enzyme-labeled secondary antibody and incubate at 37°C for 30 minutes. The absorbance at 450 nm was detected on a microplate reader. Antibody PDAB, A3P4, Vs3P4, Ps3Vm combined with antigen PD-1 The results show that the antibody PDAB, A3P4, Vs3P4, Ps3Vm can effectively bind to PD-1 protein, and the binding efficiency is dose-dependent, the results are shown in Figure 12 and Table 4.

[0078] Table 4 Binding efficiency of antibodies PDAB, A3P4, Vs3P4, Ps3Vm to PD-1 protein

[0079]

[0080] 2. Affinity of bispecific antibody to VEGF

[0081] Coat the microtiter plate with VEGF-mFc, block with 1% BSA, add different concentrations of antibodies Avastin, A3P4, Vs3P4...

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Abstract

The invention relates to an anti-vascular endothelial growth factor (VEGF) and anti-PD-1 bispecific antibody with a novel structure, and belongs to the technical field of molecular immunology. In a heavy-chain variable region of the antibody, CDR-H1 is an amino acid sequence shown as SEQ ID NO: 1, CDR-H2 is an amino acid sequence shown as SEQ ID NO: 2, and CDR-H3 is an amino acid sequence shown asSEQ ID NO: 3; and in a light-chain variable region of the antibody, CDR-L is an amino acid sequence shown as SEQ ID NO: 4. The bispecific antibody Ps3Vm can be effectively combined with PD-1 proteinand VEGF protein, can effectively compete with PDL-1 to be combined with PD-1 protein and compete with VEGF-A to be combined with VEGF protein, and can effectively stimulate T cell function and secrete cytokines IL-2 and IFN-gamma at the same time. An isotype control antibody cannot promote T cell proliferation and IL-2 and IFN-gamma secretion. In addition, the bispecific antibody Ps3Vm can also obviously inhibit tumor growth of mice.

Description

technical field [0001] The invention relates to a novel structure anti-VEGF-anti-PD-1 bispecific antibody Ps3Vm, which belongs to the technical field of molecular immunology. Background technique [0002] Vascular endothelial growth factor (vascular endothelial growth factor, VEGF), also known as vascular permeability factor (vascular permeability factor, VPF), is a highly specific factor that promotes the growth of vascular endothelial cells. Extracellular matrix degeneration, vascular endothelial cell migration, proliferation and angiogenesis. Vascular endothelial growth factors are a family that includes VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental growth factor (PGF). Usually VEGF is VEGF-A. VEGF-A can promote the formation of new blood vessels and increase the permeability of blood vessels, VEGF-B plays a role in non-neovascular tumors, VEGF-C and VEGF-D play a role in the formation of new blood vessels and new lymphatic vessels in cancer tissues VEGF-E is a...

Claims

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Application Information

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IPC IPC(8): C07K16/46C07K16/28A61K39/395A61P35/00
CPCC07K16/468C07K16/2863C07K16/2803A61P35/00C07K2317/56C07K2317/565C07K2317/31A61K2039/505
Inventor 汪国兴程联胜胡思怡袁红武婷樊丽
Owner ANHUI RUBIOX VISION BIOTECH
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