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A polypeptide specifically binding to CD3 and its application

A specific and reactive technology, applied in the field of non-antibody binding peptides, can solve the problems of mitogenicity, limited promotion and long-term medication

Active Publication Date: 2022-04-15
HOSPITAL OF STOMATOLOGY SUN YAT SEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The immunosuppressive effect of the OKT3 antibody has achieved good curative effect and wide recognition, but its mouse-derived immune response in the human body limits its promotion and long-term medication
Moreover, this CD3 monoclonal antibody also has a defect of mitogenicity, which is related to the Fc segment of the OKT3 antibody. The Fc segment of the OKT3 antibody activates lymphocytes by binding to the Fc receptors on the surface of lymphocytes, releasing a large number of cytokines

Method used

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  • A polypeptide specifically binding to CD3 and its application
  • A polypeptide specifically binding to CD3 and its application
  • A polypeptide specifically binding to CD3 and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Screening and analysis of physicochemical properties of novel CD3 ligands

[0045] 1. Materials and methods

[0046] 1.1. Human liver cancer T7 phage cDNA library screening for novel CD3 ligands

[0047] According to the conventional peptide screening method, CD3ε recombinant protein was coated on a 96-well plate, and T7 phage was added to incubate at room temperature for 30 min the next day. After washing with TBST, T7 phage eluent was incubated at room temperature for 10-20 min to elute the phage bound to CD3ε recombinant protein. It was added to 6 ml of shaken BLT5403 host bacteria (Novagen Company), and the bacteria were shaken at 37° C. until lysed. The phage screening process was repeated four times, and the binding ability of the phage monoclonal to the CD3ε recombinant protein was detected by ELISA.

[0048] 1.2. Identification of phage insertion sequence by PCR and sequencing

[0049] After the above four rounds of phage screening, 46 phage plaque...

Embodiment 2

[0069] Example 2 Study on cell binding specificity of Limhotin

[0070] 1. Materials and methods

[0071] Jurkat cells: Human acute T lymphocytic leukemia cells (Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences);

[0072] Cal27 cells (or called CAL27 cells): human tongue squamous cell carcinoma cell line (Cell Bank of Type Culture Collection Committee, Chinese Academy of Sciences);

[0073]CTSC-2 cells: human tongue squamous cell carcinoma cell line (constructed in our laboratory)

[0074] PBMC cells: human peripheral blood mononuclear cells;

[0075] 1.2 Immunofluorescence analysis of Limhotin binding to Jurkat cells, PBMC cells, and CAL27 cells

[0076] Culture Jurkat cells, PBMC cells, and CAL27 cells in 6-well plates. When the cell density grows to about 70%, add 5 μM Limhotin polypeptide respectively. The negative control group is the same amount of polypeptide dissolution reagent. After incubation for 30 minutes, 4% PFA Fix with r...

Embodiment 3

[0093] Example 3 Research on Cell Regulatory Function of Limhotin

[0094] 1. Experimental reagents

[0095]

[0096] 2. Experimental method

[0097] 2.1. Cell Viability Detection

[0098] Add 10 to 96-well culture plate 4 For Jurkat, PBMC and CAL27 cells, the treatment group was added with gradient concentration of Limhotin. After culturing for 24h, 48h and 72h, 10 μl of CCK-8 solution was added to each well of Jurkat and CAL27 cells, and the absorbance at 450nm was measured by a multifunctional microplate reader. Calculation of relative cell activity: relative cell activity=(OD value of cells in experimental group-OD value of cells in blank group) / (OD value of cells in negative control group-OD value of cells in blank group)×100%.

[0099] Add 100 μl of cell titer glo reagent to PBMC cells, transfer the liquid to an opaque black 96-well plate, measure the fluorescence value CPS with a multi-functional microplate reader, and calculate the relative cell activity: Relativ...

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Abstract

The invention provides a polypeptide specifically binding to CD3 and its application. The polypeptide is the polypeptide Limhotin, and the amino acid sequence of the polypeptide Limhotin is shown in Sequence 1. Limphotin can inhibit the proliferation of CD3-positive T lymphocytes and Jurkat tumor cells, intraperitoneal injection of Limphotin can significantly reduce the infiltration of T lymphocytes in the skin of psoriasis mice, and reduce the skin damage of psoriasis mice. Therefore, Limphotin can be used in the diagnosis and targeted therapy of CD3-positive T lymphocyte-related diseases and CD3-positive tumors.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to non-antibody-binding polypeptides capable of specifically binding to CD3 molecules and applications thereof. Background technique [0002] CD3 (cluster of differentiation 3) is mainly expressed on the membrane of T lymphocytes (referred to as T cells), and usually forms a TCR-CD3 complex with T cell surface antigen receptor (TCR), which is the key to T cell differentiation and maturation. It is an important regulatory molecule and a target molecule for the treatment of immune-related diseases. TCR recognizes the major histocompatibility complex (MHC) on the surface of antigen-presenting cells (APC) and the antigenic polypeptides presented, and transmits signals to T cells through CD3 molecules, causing a series of T cell growth and function Changes, including T cell selection, differentiation, proliferation, and cytokine production. [0003] T cells are an important part of the body'...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/47C12N5/09C12N5/0783C12N15/11A61K47/64A61K38/16A61P35/00A61P35/02A61P37/02A61P17/06
CPCC07K14/4702C12N5/0694C12N5/0636A61K47/64A61P35/00A61P35/02A61P37/02A61P17/06A61K38/00
Inventor 张雁黄鸿兴汪华
Owner HOSPITAL OF STOMATOLOGY SUN YAT SEN UNIV
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