Isolated T cell receptor, modified cell thereof, encoded nucleic acid and application thereof

Abstract

The invention provides an isolated T cell receptor, a modified cell thereof, an encoded nucleic acid and an application thereof. The isolated T cell receptor (TCR) comprises at least one of an alpha chain and a beta chain. Both the alpha chain and the beta chain comprise a variable region and a constant region. The invention is characterized in that the T cell receptor can specifically recognize the antigen Her2 / neu expressed by tumor cells, the amino acid sequence of the variable region of the alpha chain has at least 98% consistency with the amino acid sequence as shown in SEQ ID NO: 1, andthe amino acid sequence of the variable region of the beta chain has at least 98% consistency with the amino acid sequence as shown in SEQ ID NO: 2. The TCR can specifically recognize tumor antigens while avoiding possible off-target side effects. Immune cells modified with the TCR have significant anti-tumor effects.

Description

technical field

[0001] The invention belongs to the field of biotechnology, in particular, relates to isolated T cell receptors, modified cells, encoding nucleic acids, expression vectors, preparation methods, pharmaceutical compositions and applications. Background technique

[0002] Her2 / neu (ERBB2) is a transmembrane protein belonging to the EGFR family, which forms a dimer with other proteins in the family and regulates cell proliferation, differentiation and carcinogenesis through a series of intracellular signal transduction pathways (see the literature "Growth Factors, 2008;26:263", "Oncol Biol. Phys, 2004;58:903"). Her2 / neu protein is overexpressed in a variety of epithelial cancer cells, such as breast cancer, gastric cancer, colorectal cancer, ovarian cancer, pancreatic cancer, lung cancer, esophageal cancer, bladder cancer, kidney cancer, etc. (see the literature "Trends in Molecular Med , 2013; 19:677"), and the expression in primary and metastatic cancer cells ...

Examples

preparation example Construction

[0146] Preparation of recombinant TCR lentiviral expression vector: the viral vector used to express TCR is a replication-defective lentiviral vector, including: the lentiviral vector pCDH-EF1α-MCS-(PGK-GFP) expressing GFP, which can be purchased from System Biosciences (Cat#CD811A-1); and the vector pCDH-EF1α-MCS that does not express GFP, obtained by removing the PGK promoter and GFP gene on the pCDH-EF1α-MCS-(PGK-GFP) vector by using conventional techniques in the art. According to the obtained TCR gene sequence, synthesize the whole gene sequence of TCR β chain and α chain and the cleavable F2A sequence and Furin restriction fragment between them, and link to the multiple cloning site downstream of the EF-1α promoter of the vector , the sequence of transcription inserted into TCR is TCRβ chain (without stop codon), Furin fragment, F2A fragment, and TCRα chain (see the literature "Gene Ther. 2008 Nov; 15(21): 1411-1423"). The vector expressing GFP is driven by the inverted ...

Embodiment 1

[0149] Example 1: Induction of Her2 / neu 369-377 polypeptide (Her2-E75 epitope polypeptide) specific killer T cells from HLA-A2 positive normal donor peripheral blood

[0150] In this example, a low-concentration Her2 / neu 369-377 polypeptide of 1 μg / ml was used to induce polypeptide-specific killer T cells from HLA-A2-positive normal PBMC (#2) after two rounds of in vitro stimulation, and flow cytometric analysis was performed and single cell isolation. The specific method is as described above. The result is as follows:

[0151] figure 1 A The right figure shows that 0.024% of the lymphocytes are CD8-positive killer T cells that can bind to the Her2 / neu 369-377 / HLA-A2 pentamer (ie Her2-E75 pentamer), and the left figure does not pass through the Her2 polypeptide Stimulated control cells did not show CD8-positive pentamer-positive cells. The results showed that in the natural T cell pool, the number of specific T cells recognizing Her2 / neu 369-377 antigen polypeptide was ve...

Embodiment 2

[0152] Example 2: Obtaining the full sequence of Her2 / neu 369-377 polypeptide-specific TCR

[0153] This embodiment directly purifies total RNA from a certain number of Her2 CTL 6A5 cells obtained in Example 1, and obtains paired TCR α chain and β chain gene sequences by the method of 5'-RACE RT-PCR (that is, the two chains can be combined Consists of a functional TCR that recognizes antigenic polypeptides), and the encoded TCR is called "Her2 TCR-6A5". The amino acid sequence of the α chain of the TCR is shown in SEQ ID NO: 4, the coding sequence is shown in SEQ ID NO: 12, and the amino acid sequence of the β chain of the TCR is shown in SEQ ID NO: 7, and the coding sequence is shown in SEQ ID NO:15 shown. This TCR exists in the peripheral T cell pool of HLA-A2 positive normal people, and will not cross-react with normal cells that express Her2 / neu protein in a small amount and cause autoimmune reactions. To test the antigen specificity of the obtained TCR and its function,...

technical field

[0001] The invention belongs to the field of biotechnology, in particular, relates to isolated T cell receptors, modified cells, encoding nucleic acids, expression vectors, preparation methods, pharmaceutical compositions and applications. Background technique

[0002] Her2 / neu (ERBB2) is a transmembrane protein belonging to the EGFR family, which forms a dimer with other proteins in the family and regulates cell proliferation, differentiation and carcinogenesis through a series of intracellular signal transduction pathways (see the literature "Growth Factors, 2008;26:263", "Oncol Biol. Phys, 2004;58:903"). Her2 / neu protein is overexpressed in a variety of epithelial cancer cells, such as breast cancer, gastric cancer, colorectal cancer, ovarian cancer, pancreatic cancer, lung cancer, esophageal cancer, bladder cancer, kidney cancer, etc. (see the literature "Trends in Molecular Med , 2013; 19:677"), and the expression in primary and metastatic cancer cells ...

Examples

preparation example Construction

[0146] Preparation of recombinant TCR lentiviral expression vector: the viral vector used to express TCR is a replication-defective lentiviral vector, including: the lentiviral vector pCDH-EF1α-MCS-(PGK-GFP) expressing GFP, which can be purchased from System Biosciences (Cat#CD811A-1); and the vector pCDH-EF1α-MCS that does not express GFP, obtained by removing the PGK promoter and GFP gene on the pCDH-EF1α-MCS-(PGK-GFP) vector by using conventional techniques in the art. According to the obtained TCR gene sequence, synthesize the whole gene sequence of TCR β chain and α chain and the cleavable F2A sequence and Furin restriction fragment between them, and link to the multiple cloning site downstream of the EF-1α promoter of the vector , the sequence of transcription inserted into TCR is TCRβ chain (without stop codon), Furin fragment, F2A fragment, and TCRα chain (see the literature "Gene Ther. 2008 Nov; 15(21): 1411-1423"). The vector expressing GFP is driven by the inverted ...

Embodiment 1

[0149] Example 1: Induction of Her2 / neu 369-377 polypeptide (Her2-E75 epitope polypeptide) specific killer T cells from HLA-A2 positive normal donor peripheral blood

[0150] In this example, a low-concentration Her2 / neu 369-377 polypeptide of 1 μg / ml was used to induce polypeptide-specific killer T cells from HLA-A2-positive normal PBMC (#2) after two rounds of in vitro stimulation, and flow cytometric analysis was performed and single cell isolation. The specific method is as described above. The result is as follows:

[0151] figure 1 A The right figure shows that 0.024% of the lymphocytes are CD8-positive killer T cells that can bind to the Her2 / neu 369-377 / HLA-A2 pentamer (ie Her2-E75 pentamer), and the left figure does not pass through the Her2 polypeptide Stimulated control cells did not show CD8-positive pentamer-positive cells. The results showed that in the natural T cell pool, the number of specific T cells recognizing Her2 / neu 369-377 antigen polypeptide was ve...

Embodiment 2

[0152] Example 2: Obtaining the full sequence of Her2 / neu 369-377 polypeptide-specific TCR

[0153] This embodiment directly purifies total RNA from a certain number of Her2 CTL 6A5 cells obtained in Example 1, and obtains paired TCR α chain and β chain gene sequences by the method of 5'-RACE RT-PCR (that is, the two chains can be combined Consists of a functional TCR that recognizes antigenic polypeptides), and the encoded TCR is called "Her2 TCR-6A5". The amino acid sequence of the α chain of the TCR is shown in SEQ ID NO: 4, the coding sequence is shown in SEQ ID NO: 12, and the amino acid sequence of the β chain of the TCR is shown in SEQ ID NO: 7, and the coding sequence is shown in SEQ ID NO:15 shown. This TCR exists in the peripheral T cell pool of HLA-A2 positive normal people, and will not cross-react with normal cells that express Her2 / neu protein in a small amount and cause autoimmune reactions. To test the antigen specificity of the obtained TCR and its function,...

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