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Fusion protein capable of inducing and activating cancer-targeted T cells, preparation method and use thereof

A fusion protein and cancer cell technology, applied in the field of biomedicine, to achieve the effect of inhibiting tumor growth

Active Publication Date: 2013-08-14
孙嘉琳 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using these T cells without cancer cell specificity to target cancer cells is a new and powerful method in the field of anti-cancer. Such drugs are different from antibodies and have not been reported yet.

Method used

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  • Fusion protein capable of inducing and activating cancer-targeted T cells, preparation method and use thereof
  • Fusion protein capable of inducing and activating cancer-targeted T cells, preparation method and use thereof
  • Fusion protein capable of inducing and activating cancer-targeted T cells, preparation method and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031]Example 1 Construction of B7.2 (CD86) expression vector

[0032] According to the information of the human-derived B7.2 gene (aka CD86) in the GenBank database (NM_175862 and L25259), TAKARA was commissioned to synthesize a nucleic acid sequence fragment including a linker peptide and a B7.2 gene (222 encoding the extracellular part) amino acid) and a few bases of the restriction enzyme cleavage point HindIII in front of the whole fragment and XhoI at the back, insert this synthesized nucleic acid fragment into the T vector and carry out DNA sequencing identification, and then use the double digestion method to use HindIII and XhoI After treatment, this fragment was inserted into the pET22b plasmid, thus producing the expression vector pET22b-B7.2, which expresses the B7.2 protein. The sequence listing (SEQ ID NO. 2) is only the B7.2 protein, and another sequence listing (SEQ ID NO. 14) can be found in the preceding linker peptide.

Embodiment 2

[0033] Example 2 Construction of TGF-α-B7.2 expression vector

[0034] According to the information of human-derived TGF-α gene (NM_003236) in the GenBank database, TAKARA was commissioned to synthesize a nucleic acid sequence fragment including TGF-α gene and several restriction endonuclease sites BamHI and EcoRI added in front of TGF-α base, containing restriction endonuclease sites for SalI and HindIII at the end of the fragment. The synthesized nucleic acid fragment was inserted into the T vector and identified by DNA sequencing. Then, after treatment with BamHI and HindIII by double digestion method, the fragment was inserted into the pET22b-B7.2 plasmid, thus producing the expression vector pET22b. -TGF-α-B7.2, which can express a TGF-α-B7.2 fusion protein (see SEQ ID NO. 4 of the Sequence Listing).

Embodiment 3

[0035] Example 3 Construction of EGF-B7.2 expression vector

[0036] According to the human-derived EGF gene information (NM_001963 and NM_001178130) in the GenBank database, TAKARA was commissioned to synthesize a nucleic acid sequence fragment including the EGF gene and a few bases of restriction endonuclease sites BamHI and EcoRI in front of EGF. Following the fragment contains restriction enzyme sites for SalI and HindIII. The synthesized nucleic acid fragment was inserted into the T vector and identified by DNA sequencing. Then, after treatment with BamHI and HindIII by double digestion method, the fragment was inserted into the pET22b-B7.2 plasmid, thus producing the expression vector pET22b. -EGF-B7.2, which can express the EGF-B7.2 fusion protein (see SEQ ID NO. 6 of the sequence listing).

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Abstract

The present invention discloses a fusion protein capable of inducing and activating cancer-targeted T cells, a preparation method and use thereof. The protein comprises peptides which function on the cancer cells and costimulatory molecules B7.2. The peptides which function on the cancer cells are selected from transforming growth factor-alpha, epidernal growth factor, vascular endothelial growthfactor, gonadotropin-releasing hormone or gastrin-releasing peptide. The fusion protein of the invention has a cancer targeting function. On one hand, the fusion protein can respectively function with the VEGFR, EGFR, GnRH-R or GRP-R; and on the other hand, the fusion protein interacts with corresponding receptors CD28 and CTLA-4 which are expressed on the T cell. Thus, the T cell is targetedly positioned to the periphery of cancer cells which express VEGFR, EGFR, GnRH-R or GRP-R in great numbers. Experiments prove that the fusion protein of the invention can restrain tumor growth and causes cancer cell apoptosis.

Description

technical field [0001] The invention relates to a fusion protein, a preparation method and application thereof, and belongs to the field of biomedicine. Background technique [0002] Epidermal growth factor (EGF), vascular endothelial cell growth factor (VEGF), gonadotropin-releasing hormone (GnRH) and gastrin-releasing peptide (GRP) ) and other receptors are abundantly expressed in various tumor tissues, for example, the EGF receptor in intestinal mucosal tumors is 300 times higher than that in normal tissues (Gastroenterology, 98, 961-967, 1990). Therefore, abnormally highly expressed EGF receptors, VEGF receptors, GnRH receptors, and GRP receptors have become attractive targets for cancer therapy. The receptor of transforming growth factor-α (TGF-α) is the same as that of EGF. [0003] The above cytokines, hormones and polypeptides can interact with the corresponding receptors (Receptors) on cancer cells. Therefore, a fusion protein is formed by connecting an effector ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/15C12N1/19C12N1/21C12N5/10C12N5/0783
Inventor 孙嘉琳
Owner 孙嘉琳
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