lag-3 affinity peptide n13, preparation method and application thereof

A LAG-3, affinity technology, applied in the field of anti-tumor application, preparation, LAG-3 affinity peptide N13, can solve problems such as poor efficacy

Active Publication Date: 2019-01-04
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The biggest challenge encountered in the current process of tumor immunotherapy is poor efficacy due to tumor immune tolerance and escape.

Method used

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  • lag-3 affinity peptide n13, preparation method and application thereof
  • lag-3 affinity peptide n13, preparation method and application thereof
  • lag-3 affinity peptide n13, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] This example mainly briefly introduces the process of screening and obtaining LAG-3 affinity peptide N13.

[0038] The LAG-3 affinity peptide N13 in the present invention is mainly obtained by liquid-phase and solid-phase reverse panning methods for phage display dodecapeptide library screening, and its main technical process is: binding to the target protein→amplification→binding to the target protein → Elution. After 3-5 rounds of screening, phage monoclonals with affinity to the extracellular segment of the target protein LAG-3 were enriched round by round, and the recovery rate was greatly improved. Then, the blue spots were selected from the third round and the fifth round for sequencing, and a plurality of different inserted dodecapeptide sequences, ie, affinity peptide sequences, were obtained respectively, among which the N13 peptide was shared by the two screening methods. The specific process of screening is described below.

[0039] The first thing that nee...

Embodiment 2

[0089] This example mainly briefly introduces the artificial synthesis preparation method of LAG-3 affinity peptide N13 as follows.

[0090] The preparation method of LAG-3 affinity peptide N13 is prepared by Fmoc solid-phase peptide synthesis method. The technical principle is: select Rink resin to connect with the first Fmoc-amino acid carboxyl group at the C-terminal of the peptide to be synthesized in the form of a covalent bond, Then use the N-terminal of the amino acid as the starting point for the synthesis of the polypeptide, and allow it to undergo a dehydration condensation reaction with the carboxy-terminal of the next amino acid to form a peptide bond; then, deprotect the protecting group of the Fmoc-amino acid at the N-terminal, and then Let the N-terminal of the second amino acid react with the carboxyl group of the following amino acid; repeat this process until the synthesis of the polypeptide is completed; finally, the synthetic polypeptide is cut off from the ...

Embodiment 3

[0120] Taking the LAG-3 affinity peptide N13 prepared in Example 2 as an example, this example mainly conducted an in vitro blocking experiment on the affinity effect of the LAG-3 affinity peptide N13 and LAG-3, and the results showed that it can block LAG3 / MHC-II signaling pathway, related experiments are introduced as follows.

[0121] (1) THP-1 cells were stimulated with 20ng / mL IFN-r for 12h, and then incubated on ice with Anti-Human HLA-DR APC flow antibody, and then the ligand HLA-DR molecules on the surface were detected by flow cytometry The expression level reaches the highest level, and the cells are in good condition;

[0122] (2) Incubate the above stimulated THP-1 cells with different concentrations of LAG3-Fc fusion protein, then incubate Anti-human IgG1-Fc PE antibody on ice, and use flow cytometry to detect the surface of the above stimulated THP-1 cells The binding amount of LAG3-Fc fusion protein is about 400ng;

[0123] (3 Dissolve the affinity peptide N13...

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Abstract

The invention belongs to the technical field of biopharmaceuticals, and in particular relates to a LAG‑3 affinity peptide N13, a preparation method and an antitumor application thereof. The affinity peptide includes 12 amino acids, the molecular weight is 1634.8 Da, and its sequence is DDFRVWWPNFPR. The peptide can be prepared by the Fmoc solid-phase polypeptide synthesis method, and at the same time, the peptide can be used as a drug in tumor treatment. In this application, the eukaryotic protein rhLAG3-Fc is used as the target molecule, hIgG1-Fc is used as the tag protein, and the affinity peptide N13 of the extracellular segment of LAG-3 is screened out through the high-throughput technology of phage display peptide library. For this peptide, further in vitro signal pathway blocking experiments and mouse tumor-bearing experiments showed that this peptide has good application prospects in tumor treatment and prolonging the survival of organisms, and provides a new method for the immunotherapy of organism tumors. possible.

Description

technical field [0001] The invention belongs to the technical field of biopharmaceuticals, and specifically relates to a LAG-3 affinity peptide N13, a preparation method and an antitumor application thereof. Background technique [0002] Among the existing tumor treatment technologies, immunotherapy against tumors is considered to be the ultimate means of curing tumors. Existing studies generally believe that compared with traditional treatment methods, tumor immunotherapy can activate or induce tumor patients to establish a specific immune response to tumor antigens, eliminate primary tumor cells, establish immune memory, and prevent tumor recurrence and transfer. However, due to the diversity and complexity of tumor types, tumor immunotherapy is not only a hot spot but also a difficult point in current tumor research. [0003] In the existing tumor immunotherapy, CD8 + Cytotoxic T lymphocytes (CTLs) are the main effector cells, CD8 + CTL needs to be activated through t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K7/08A61K38/10A61P35/00
CPCA61K38/00C07K7/08C07K2319/00
Inventor 高艳锋张璐宁李国栋祁元明翁海波
Owner ZHENGZHOU UNIV
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