CD40 targeted binding protein, coding nucleic acid and application thereof

A technology of targeted binding, 1. CD40, applied in the field of CD40-targeted binding proteins, can solve the problems of poor tumor penetration, high production cost, large molecular weight, etc., and achieve good tissue penetration, high yield, and simple production process Effect

Active Publication Date: 2022-02-22
NANJING DRUM TOWER HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the current antibody drugs targeting CD40 have disadvantages such as large molecular weight, poor tumor penetration, complicated process, and high production costs. Therefore, it is of great significance to develop low-molecular-weight antibody drugs with high affinity and high permeability

Method used

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  • CD40 targeted binding protein, coding nucleic acid and application thereof
  • CD40 targeted binding protein, coding nucleic acid and application thereof
  • CD40 targeted binding protein, coding nucleic acid and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1: ELISA detects the affinity of target binding protein to CD40 molecule:

[0030] 1. Use the E. coli expression system to synthesize the following targeting binding proteins targeting binding proteins #1, 2, 3, 6, 7, and express the negative control targeting binding protein #10, use nickel column purification, use GenScript Endotoxin removal kits remove endotoxins to <0.1EU / μg:

[0031] Targeted binding protein #1 (SEQ ID No. 1 with a histidine tag at the N-terminus)

[0032] Targeted binding protein #2 (SEQ ID No. 2 with a histidine tag at the N-terminus)

[0033] Targeted binding protein #3 (SEQ ID No. 3 with a histidine tag at the N-terminus)

[0034] Targeted binding protein #6 (SEQ ID No. 6 with a histidine tag at the N-terminus)

[0035] Targeted binding protein #7 (SEQ ID No. 7 with a histidine tag at the N-terminus)

[0036] Targeted binding protein #10 (SEQ ID No. 10 with a histidine tag at the N-terminus);

[0037]2. Coat the flat bottom 96-well...

Embodiment 2

[0040] Example 2: Flow detection of the binding of targeted binding proteins to CD40 on the surface of tumor cells and antigen-presenting cells:

[0041] 1. Binding to CD40 on the surface of tumor cells:

[0042] (1) The CD40+ B lymphoma cell line Raji was cultured. When the number of cells reaches 10 6 Cells were collected at 24 hours, resuspended in PBS containing 2% fetal bovine serum, centrifuged, the supernatant was discarded, and resuspended with PBS containing 2% fetal bovine serum 100 μL / tube.

[0043] (2) On the basis of Example 2, target binding proteins #1, 3, 6, 10 were added to each group respectively, incubated at 4°C for 1 h, centrifuged, discarded the supernatant, and used 2% fetal bovine serum. Washed once with PBS.

[0044] (3) Add anti-His secondary antibody labeled with phycoerythrin (PE) fluorescent dye, incubate at 4°C for 1 h, centrifuge, discard the supernatant, and wash once with PBS containing 2% fetal bovine serum.

[0045] (4) After resuspending...

Embodiment 3

[0052] Example 3: Tandem multivalent CD40-specific targeting binding proteins effectively activate dendritic cells:

[0053] 1. Use the E. coli expression system to synthesize a trivalent tandem CD40-specific targeting binding protein targeting binding protein #14 (SEQ ID No. 14 with a histidine tag at the N-terminus). A trivalent tandem non-targeted targeting binding protein targeting binding protein #16 (SEQ ID No. 16 with a histidine tag at the N-terminus) was used as a control. Purified using a nickel column, endotoxin was removed to <0.1 EU / μg using the GenScript Endotoxin Removal Kit.

[0054] 2. Flow cytometry detection of dendritic cell surface activation markers:

[0055] (1) According to the method described in Example 2, PBMCs were isolated, adhered to obtain peripheral blood mononuclear cells, and were cultured with GM-CSF and IL-4 for 5 days to promote dendritic cell differentiation.

[0056] (2) On the 6th day, different concentrations of target binding protein...

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Abstract

The invention discloses a CD40 targeted binding protein, which is characterized in that the amino acid sequence of the protein is shown as SEQ ID No.8, Xaa at the 30th site, the 32th site, the 33th site, the 35th site, the 43rd site, the 44th site, the 63rd site, the 65th site, the 66th site, the 68th site, the 76th site, the 77th site, the 96th site, the 98th site, the 99th site, the 101th site, the 109th site and the 110th site are respectively any amino acid. The multivalent CD40 targeted binding protein is formed by carrying out multivalent series connection on the CD40 targeted binding protein through a linker. According to the invention, the molecular weight of the antibody is only about 18kDa and is about one tenth of that of the antibody, and the antibody has good tissue penetrability, and is simple in production process and high in yield; and the CD40 specific targeted binding protein also has a good immune activation effect after being connected in series in a multivalent manner by using a proper linker.

Description

technical field [0001] The present invention belongs to the technical field of biology and new medicine, in particular, the present invention relates to a CD40 targeting binding protein, its encoding nucleic acid and use. Background technique [0002] In recent years, immunotherapy has become one of the important means of tumor treatment. Immunotherapy refers to a treatment method that suppresses tumor growth and causes tumor regression by mobilizing the body's anti-tumor immune response. The function of immune cells is co-regulated by co-inhibitory and co-stimulatory molecules. In the face of foreign antigens, co-stimulatory molecules are responsible for activating the immune response, while co-inhibitory molecules are responsible for limiting the strength of the immune response to prevent damage to the own tissue. These immune checkpoints are all critical for controlling immune cell activity and can be regulated by blocking or activating antibodies. Immune checkpoint in...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00
CPCC07K16/2878A61P35/00C07K2317/92A61K2039/505
Inventor 刘宝瑞陈晓彤
Owner NANJING DRUM TOWER HOSPITAL
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