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Preparation method and application of multiple target complex antigen-loaded CD8<+> cytotoxic T lymphocyte

A technology of lymphocytes and compound antigens, applied in the fields of biology and medicine, can solve the problem of tumor heterogeneity

Active Publication Date: 2015-09-23
BEIJING BIOHEALTHCARE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

And tumor antigen peptides are restricted by MHC, only patients with the same MHC class I molecules can use the same peptide, and due to tumor heterogeneity, some tumor antigen peptides may induce immune tolerance instead of activating immune response

Method used

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  • Preparation method and application of multiple target complex antigen-loaded CD8&lt;+&gt; cytotoxic T lymphocyte
  • Preparation method and application of multiple target complex antigen-loaded CD8&lt;+&gt; cytotoxic T lymphocyte
  • Preparation method and application of multiple target complex antigen-loaded CD8&lt;+&gt; cytotoxic T lymphocyte

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Example 1 Construction of lentiviral LentihGM-CSF vector

[0070] PCR primers were provided for the CDS region sequence of human peripheral blood hGM-CSF gene (GeneID: NM_000758.3), and restriction sites were added to the 5' and 3' ends of the above gene by PCR amplification, and the plasmid pORFhGM-CSF (purchased from Invivogen, article number: porf-hgmcsf) as a template, the hGM-CSF gene fragment was amplified by the PCR method; the hGM-CSF gene was directional cloned into the lentiviral ligation vector system ( figure 1 Shown) to construct the recombinant vector FattbC31UGW-hGM-CSF. The recombinant vector FattbC31UGW-hGM-CSF and envelope plasmid VSVG ( figure 2 shown) and the packaging structure plasmid CMVΔ8.9-D64N / D116N ( image 3 Shown) mixed, the mixing ratio is 1.5-10:1-5:1 to form a lentiviral vector system, use liposome LipofectamineTM to transfect on 293T cells, observe under a fluorescence microscope after 24-48 hours, and a large amount of fluorescence ...

Embodiment 2

[0077] Example 2 Synthesis of Multi-target Compound Antigen Peptides

[0078] The polypeptide shown in SEQ ID NO: 5 was entrusted to Shanghai Qiangyao Biotechnology Co., Ltd. to synthesize it using a standard Fmoc protocol, and to use high performance liquid chromatography for purification and purity analysis, and mass spectrometry for identification and molecular weight determination. The result shows that the purity of the polypeptide is higher than 95%, and the molecular weight is consistent with the theoretical value.

[0079] Specific synthesis steps: 1. Fmoc-protected columns and monomers must be protected by an alkaline solvent (such as piperidine) to remove the amino protection group; 2. Use an activator to activate and dissolve the carboxyl end of the amino acid that needs to be cross-linked, and convert the activated mono The body and the free amino group are cross-linked under the action of a cross-linking agent to form a peptide bond; 3. The first two steps are rep...

Embodiment 3

[0080] Example 3 Synthesis of Multi-target Compound Antigen Peptides

[0081] The peptide shown as SEQ ID NO: 10 was entrusted to Shanghai Qiangyao Biotechnology Co., Ltd. to synthesize it using a standard Fmoc protocol, and to use high performance liquid chromatography for purification and purity analysis, and mass spectrometry for identification and molecular weight determination. The result shows that the purity of the polypeptide is higher than 95%, and the molecular weight is consistent with the theoretical value. Specific synthesis steps: 1. Fmoc-protected columns and monomers must be protected by an alkaline solvent (such as piperidine) to remove the amino protection group; 2. Use an activator to activate and dissolve the carboxyl end of the amino acid that needs to be cross-linked, and convert the activated mono The body and the free amino group are cross-linked under the action of a cross-linking agent to form a peptide bond; 3. The first two steps are repeated until ...

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Abstract

The invention provides a multiple target complex antigen, namely a combined cytotoxic T lymphocyte (CTL) antigen epitope peptide, a polyvalent vaccine with multiple target positions for a tumor cell surface, a multiple target complex antigen-loaded CD8<+> CTL, a preparation method of the CTL, and an application of the lymphocyte to preparation of a drug for treating a cancer. The invention also provides an epitope-induced special CTL effect cell.

Description

[0001] Technical field: the present invention relates to the fields of biology and medicine, and more specifically relates to multi-target composite antigen-loaded CD8 + Preparation method and application of cytotoxic T lymphocytes. Background technique: [0002] Malignant tumors are one of the major diseases that threaten human health. In recent years, the important role of the immune system in tumor prevention and treatment has been widely recognized. Immunotherapy based on anti-tumor specific immune reconstitution is currently recognized internationally following surgery, After radiotherapy and chemotherapy, the most promising means of completely eradicating tumor cells in the body to cure tumors will become a major direction of future tumor treatment. Immunotherapy mainly activates and mobilizes cytotoxic and biologically active cells and cytokines by supplementing, inducing and activating the body's inherent biological response regulation system in vitro, improves the fun...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N5/0783C12N5/10C12N15/867A61K39/00A61P35/00
Inventor 刘静维卢戌王跃杨照敏
Owner BEIJING BIOHEALTHCARE BIOTECH
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