Fusion protein capable of inducing and activating cancer targeting T-cells as well as preparation method and application of the fusion protein

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

Active Publication Date: 2012-03-28
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 targeting T-cells as well as preparation method and application of the fusion protein
  • Fusion protein capable of inducing and activating cancer targeting T-cells as well as preparation method and application of the fusion protein
  • Fusion protein capable of inducing and activating cancer targeting T-cells as well as preparation method and application of the fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031]Example 1 Construction of B7.1(CD80) expression vector

[0032] According to the information (NM_005191) of the B7.1 gene (also known as CD80) of human origin in the GenBank database, TAKARA was commissioned to synthesize a nucleic acid sequence fragment including a connecting peptide and a B7.1 gene (208 amino acids encoding the extracellular part) and a few bases of HindIII in front of the entire fragment and a few bases of the XhoI restriction enzyme cut point in the back, insert this synthesized nucleic acid fragment into a T vector and perform DNA sequencing identification, and then use the double enzyme digestion method to treat it with HindIII and XhoI , Insert this fragment into the pET22b plasmid, thus producing the expression vector pET22b-B7.1, which can express the B7.1 protein. The sequence listing (SEQ ID NO.2) is only the B7.1 protein, and the previous connecting peptide can be found in another sequence listing (SEQ ID NO.14).

Embodiment 2

[0033] Embodiment 2 Construction of TGF-α-B7.1 expression vector

[0034] According to the TGF-α gene information (NM_003236) of human origin in the GenBank database, TAKARA was commissioned to synthesize a nucleic acid sequence fragment including the TGF-α gene and several restriction endonuclease sites BamHI and EcoRI in front of TGF-α. bases, the end of the fragment contains the restriction endonuclease sites of SalI and HindIII. The synthesized nucleic acid fragment was inserted into the T vector and identified by DNA sequencing, and then treated with BamHI and HindIII by double enzyme digestion, and then inserted into the pET22b-B7.1 plasmid, thus producing the expression vector pET22b - TGF-α-B7.1, capable of expressing TGF-α-B7.1 fusion protein (see SEQ ID NO.4 in the sequence listing).

Embodiment 3

[0035] The construction of embodiment 3EGF-B7.1 expression vector

[0036] According to the information of the EGF gene (NM_001963 and NM_001178130) of the human origin of GenBank database, entrust TAKARA company to synthesize a nucleic acid sequence fragment and comprise EGF gene and several bases of restriction endonuclease site BamHI and EcoRI in front of EGF again, in The rear of the fragment contains restriction endonuclease sites for SalI and HindIII. The synthesized nucleic acid fragment was inserted into the T vector and identified by DNA sequencing, and then treated with BamHI and HindIII by double enzyme digestion, and then inserted into the pET22b-B7.1 plasmid, thus producing the expression vector pET22b - EGF-B7.1, capable of expressing EGF-B7.1 fusion protein (see SEQ ID NO.6 in the sequence listing).

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Abstract

The invention discloses a fusion protein capable of inducing and activating cancer targeting T-cells as well as a preparation method and application of the fusion protein. The fusion protein contains peptide acting with cancer cells as well as costimulatory molecules B7.1, wherein the peptide acting with the cancer cells is selected from a transforming growth factor-alpha, an epidermal growth factor, a vascular endothelial growth factor, gonadotropinreleasing hormone or gastrin-releasing peptide. The fusion protein has a targeting function; the fusion protein can act with VEGFR, EGFR, GnRH-R, or GRP-R respectively on one hand and interact with corresponding receptors CD28 and CTLA-4 expressed on the T-cells, therefore the targeting of the T-cells is positioned at the periphery of the cancer cells greatly expressing VEGFR, EGFR, GnRH-R, or GRP-R. Proven by experiments, the fusion protein can restrain the growth of tumors and cause cancer cells to apoptosis.

Description

technical field [0001] The invention relates to a fusion protein, its preparation method and application, 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, EGF receptors in intestinal mucosal tumors are 300 times higher than those in normal tissues (Gastroenterology, 98, 961-967, 1990). Therefore, abnormally high-expressed EGF receptors, VEGF receptors, GnRH receptors, GRP receptors, etc. have become attractive targets for cancer therapy. The receptor of transforming growth factor-α (Transforming growth factor-α, TGF-α) is the same as that of EGF. [0003] The above cytokines, hormones and peptides can interact with the corresponding receptors (Receptor) on cancer cells. Therefore, an effector such as a toxin protein ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/21C12P21/02A61K38/25A61K38/17A61K38/18A61K38/09A61K47/48A61P35/00
Inventor 孙嘉琳
Owner 孙嘉琳
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