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LAG-3 affinity peptide N13, preparing method and application thereof

A LAG-3, affinity technology, applied in chemical instruments and methods, peptides, fusion peptides, etc., can solve problems such as poor efficacy

Active Publication Date: 2016-04-20
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, preparing method and application thereof
  • LAG-3 affinity peptide N13, preparing method and application thereof
  • LAG-3 affinity peptide N13, preparing 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] first What needs to be...

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 for Anti-HumanHLA-DRAPC flow antibody, and then the expression of ligand HLA-DR molecules on the surface was detected by flow cytometry reached the highest, and the cells are in good condition;

[0122] (2) Incubate the stimulated THP-1 cells with different concentrations of LAG3-Fc fusion protein, then incubate Anti-humanIgG1-FcPE antibody on ice, and use flow cytometry to detect the LAG3-Fc fusion protein on the surface of the stimulated THP-1 cells. The binding amount of Fc fusion protein is about 400ng;

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

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Abstract

The invention belongs to the technical field of biological pharmacy and particularly relates to LAG-3 affinity peptide N13, a preparing method and application thereof to the anti-tumor aspect. The affinity peptide comprises twelve amino acids, the molecular weight is 1634.8 Da, and the sequence is DDFRVWWPNFPR specifically. An Fmoc solid-phase polypeptide synthesis method is adopted for preparing the peptide, and the peptide can be applied to drugs for treating tumor. According to the affinity peptide N13, eukaryon protein rhLAG3-Fc serves as target molecules, hIgG1-Fc serves as tag protein, and the phage display peptide library high-throughput technology is used for screening the affinity peptide N13 of an LAG-3 extracellular domain. For the peptide, further in-vitro signal channel blocking tests and mouse tumor-bearing tests show that the peptide has good application prospects in the aspects of treating tumor and prolonging the survival period of a living body, and new possibility is provided for immune therapy for tumor of the living body.

Description

Technical field [0001] The invention is a biopharmaceutical technology field, which involves a LAG-3 affinity peptide N13, preparation methods and its application in anti-tumor. Background technique [0002] In the existing tumor treatment technology, immunotherapy for tumors is considered to be the ultimate means to cure tumors.It is generally believed that compared with traditional treatment methods, tumor immunotherapy can activate or induce tumor patients to establish a specific immune response to tumor antigen, remove primary tumor cells, and establish immune memory to prevent the recurrence of tumors from recurrence of tumor recurrenceAnd transfer.However, due to the diversity and complexity of tumor types, the immunotherapy of tumors is not only a hot spot in existing tumor research, but also the difficulty of research. [0003] In the existing tumor immunotherapy, CD8 + Cytotoxic T lymphocyte (CTL) is the main effect cell, CD8 + CTL needs to be activated through MHC-I mol...

Claims

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Application Information

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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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