Fusion protein combined with CD40L and application thereof

A fusion protein and protein technology, which is applied in the field of fusion protein binding to CD40L, can solve the problems of platelet aggregation, decreased activity, uncertain connection sites, etc., and achieve the goal of promoting maturation and antigen presentation, reducing therapeutic side effects, and reducing production costs Effect

Active Publication Date: 2022-03-08
SUZHOU PRO HEAL PHARM TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, anti-CD40L antibody drugs containing human antibody Fc have experienced severe thromboembolism in clinical trials, leading to the failure of drug development.
On the other hand, PEGylation is considered to eliminate this risk, but the immunogenicity of the PEGylated protein will increase, and PEGylation will also affect the functional domain of the protein, resulting in a decrease in activity. In addition, PEGylation has uncertain connection sites Increased difficulty in process quality control
[0005] At present, there is no therapeutic antibody drug against CD40L on the market, and it is urgent to develop fusion protein molecules that bind to CD40L for the treatment of immune system diseases while further reducing its side effects

Method used

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  • Fusion protein combined with CD40L and application thereof
  • Fusion protein combined with CD40L and application thereof
  • Fusion protein combined with CD40L and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0127] Example 1. Expression and purification of fusion protein

[0128] In certain embodiments, the antigen binding protein provided by the invention comprises the following heavy chain HC and light chain LC:

[0129] (1) fusion protein 1, HC as shown in SEQ ID NO:21, and / or LC as shown in SEQ ID NO:9; or,

[0130] (2) Fusion protein 2, HC as shown in SEQ ID NO:31, and / or LC as shown in SEQ ID NO:9; or,

[0131] (3) Fusion protein 3, HC as shown in SEQ ID NO:23, and / or LC as shown in SEQ ID NO:9;

[0132](4) Fusion protein 4, HC as shown in SEQ ID NO:32, and / or LC as shown in SEQ ID NO:9.

[0133] The light chain sequence and the heavy chain fragment sequence of the fusion protein were optimized for amino acid codons according to the human host cell expression system, then the signal peptide nucleotide sequence for protein expression was added and gene synthesis was carried out, and then the synthesized target gene was passed through 5 'EcoRI and 3'HindIII were cloned into...

Embodiment 2

[0140] Example 2. Binding detection of fusion protein to human CD40L and HSA antigen

[0141] The fusion protein 1 and CD40L protein (manufacturer: Beijing Baipusaisi Biotechnology Co., Ltd., article number: CDL-H5248) and HSA (manufacturer: Beijing Baipusaisi Biotechnology Co., Ltd.) purified in Example 1 were analyzed using a Gator non-labeled bioanalyzer. Adams Biotechnology Co., Ltd., Cat. No.: HSA-H5220) for determination of affinity. Anti-His tag biosensors were selected to capture CD40L and HSA respectively (loading concentration 10 μg / mL), and then the kinetic detection of binding and dissociation between the captured samples and the fusion protein was performed. Kinetics were analyzed by fit using a 1:1 binding model. The brief action steps are: protein loading for 200s, binding for 180s, dissociation for 300s, and regeneration for 30s. The determined affinities are shown in Table 1 below.

[0142] Table 1

[0143]

[0144]

[0145] As shown in the table abo...

Embodiment 3

[0146] Example 3. Determination of fusion protein blocking CD40 binding to CD40L

[0147] Coat CD40 protein (manufacturer: Beijing Baipusaisi Biotechnology Co., Ltd., article number: CD0-H5228) on an ELISA microtiter plate at 4°C overnight. / mL initial concentration was serially diluted and then mixed with biotinylated CD40L protein (manufacturer: Beijing Biopsy Biotechnology Co., Ltd., product number: CDL-H82F1). Add 100 μL of the mixed solution to each well of the ELISA plate, incubate at 37°C, wash the plate 6 times, add the enzyme-labeled secondary antibody, incubate at 37°C, wash the plate 6 times, then develop the color and read the results. Test results such as Figure 5 As shown, its IC50 value is 0.1182 μg / mL.

[0148] The result is as Figure 5 As shown, fusion protein 1 can effectively block the binding of CD40 and CD40L at a lower concentration, further indicating that the fusion protein has the ability to block CD40 and CD40L signal transduction.

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Abstract

The invention provides a fusion protein combined with CD40L and application of the fusion protein, and belongs to the field of biological medicine. The fusion protein combined with CD40L provided by the invention does not contain a human antibody Fc structural domain, and research finds that the fusion protein does not cause activation of any blood platelets while being combined with CD40L, so that the safety of the fusion protein is greatly improved and the treatment side effects are reduced while the biological activity of the fusion protein is ensured; and a new thought and a new method are provided for treatment of immune system diseases. In addition, the fusion protein shows extremely high expression quantity in the construction and screening process of a stably transfected cell line, and is beneficial to large-scale development and production.

Description

technical field [0001] The invention belongs to the field of biomedicine, and in particular relates to a fusion protein binding to CD40L and an application thereof. Background technique [0002] CD40L (SEQ ID NO: 24), also known as CD154, is a member of the tumor necrosis factor (TNF) family. CD40L is a type II transmembrane glycoprotein, mainly expressed in mature activated CD4 positive T lymphocytes and platelets, and partially expressed in natural killer cells, dendritic cells, mononuclear macrophages, B lymphocytes, and non-hematopoietic origin endothelial cells, epithelial cells and vascular smooth muscle cells, etc. 62.; Antoniades C, et al. "The CD40 / CD40 ligand system: linking inflammation with atherothrombosis." Journal of the American College of Cardiology, 2009, 54(8):669-677.). CD40L usually exists in two forms: membrane-bound CD40 ligand (mCD40L) and soluble CD40L (sCD40L). Both mCD40L and sCD40L bind to the corresponding receptor CD40 (SEQ ID NO: 25) in a tr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/46C07K19/00A61K39/395A61K47/64A61K47/65A61K47/68A61K48/00A61P37/02A61P37/06
CPCC07K16/2875C07K16/18C07K14/765A61K47/643A61K47/65A61K47/6811A61K47/6889A61K48/005A61K48/0008A61P37/02A61P37/06C07K2317/565C07K2317/56C07K2317/51C07K2317/515C07K2317/31C07K2317/569C07K2319/31A61K2039/505Y02A50/30
Inventor 张鹏郭树华马琳
Owner SUZHOU PRO HEAL PHARM TECH CO LTD
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