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Immunocompetent cell that expresses a cell surface molecule specifically recognizing human mesothelin, il-7 and ccl19

An immune activity and cell surface technology, applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, receptor/cell surface antigen/cell surface determinant, anti-animal/human immunoglobulin, etc. , can solve the problems of partial loss of function, side effects, inability to treat recurrence or metastasis, etc.

Active Publication Date: 2020-08-07
NOILE IMMUNE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are various problems such as side effects, partial loss of function, and inability to treat recurrence or metastasis

Method used

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  • Immunocompetent cell that expresses a cell surface molecule specifically recognizing human mesothelin, il-7 and ccl19
  • Immunocompetent cell that expresses a cell surface molecule specifically recognizing human mesothelin, il-7 and ccl19
  • Immunocompetent cell that expresses a cell surface molecule specifically recognizing human mesothelin, il-7 and ccl19

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0263] [Example 1] Production of anti-human mesothelin CAR

[0264] (synthesis of scFv sequence and DNA fragment of anti-human mesothelin CAR)

[0265] In order to compare the sequences, sequences and types of appropriate signal peptides of VL and VH, a figure 1 Sequences of nine anti-human mesothelin scFvs shown.

[0266] VH07(15)VL07 consists of the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 1, the amino acid sequence of the peptide linker shown in SEQ ID NO: 26, and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 2.

[0267] VH36(15) VL36 consists of the amino acid sequence of the heavy chain variable region shown in SEQ ID NO: 3, the amino acid sequence of the peptide linker shown in SEQ ID NO: 26, and the amino acid sequence of the light chain variable region shown in SEQ ID NO: 4.

[0268] VL07(15)VH07 consists of the amino acid sequence of the light chain variable region shown in SEQ ID NO: 2, the amino ac...

Embodiment 2

[0285] [Example 2] CAR expression measurement by flow cytometry

[0286] (Flow Cytometry Analysis)

[0287] Analysis of the expression level of CAR that recognizes mesothelin as an antigen was performed by flow cytometry analysis. The prepared T cells expressing anti-human mesothelin CAR-IL-7 / CCL19 were mixed with recombinant human mesothelin (containing 6-His at the C-terminus) (manufactured by BioLegend), and phycoerythrin (PE)-labeled anti-6-His monoclonal The cloned antibody (manufactured by Abcam) was reacted with an allophycocyanin (APC)-labeled anti-CD8 monoclonal antibody (manufactured by Affymetrix) for staining. EC800 (manufactured by Sony Corporation) was used for the flow cytometer, and FlowJo software (manufactured by Tree Star Corporation) was used for data analysis.

[0288] (result)

[0289] First, anti-human mesothelin CAR-IL-7 / CCL19 with VH07(15)VL07 (expressed by signal peptide T), VH07(15)VL07 (expressed by signal peptide P) or VH36(15)VL36 as the scFv r...

Embodiment 3

[0291] [Example 3] Mesothelin expression of each tumor cell

[0292] (Flow Cytometry Analysis)

[0293] In order to identify tumor cell lines expressing mesothelin, the expression level of mesothelin in each tumor cell line was confirmed. The malignant mesothelioma cell lines ACC-MESO-1, Y-MESO8A, NCI-H2052, NCI-H226, MSTO211H, and kidney cancer cell line A498 were labeled with PE-labeled commercially available mesothelin antibody (catalogue number FAB32652P; R&D Systems, Inc.) was stained, and the expression of mesothelin in each tumor cell was measured by flow cytometry analysis. It should be noted that the PE-labeled anti-human mesothelin antibody was used for staining at 3 μg / sample. EC800 (manufactured by Sony Corporation) was used for the flow cytometer, and FlowJo software (manufactured by Tree Star Corporation) was used for data analysis.

[0294] (result)

[0295] show the result in Figure 4 . Expression of mesothelin was confirmed in malignant mesothelioma cel...

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PUM

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Abstract

The purpose of this invention is to provide an immunocompetent cell that targets mesothelin. Another purpose is to produce an immunocompetent cell that expresses a cell surface molecule specifically recognizing human mesothelin, interleukin-7 (IL-7), and chemokine (C-C motif) ligand 19 (CCL19). It is preferable that: the cell surface molecule that specifically recognizes human mesothelin is a chimera antigen receptor (CAR) provided with a single-chain antibody, a membrane-spanning domain and a signal transmission domain which induces the activation of the immunocompetent cell; and that the heavy chain variable domain and the light chain variable domain are linked via a peptide linker comprising a sequence of 2-30 amino acids.

Description

technical field [0001] The present invention relates to the expression of cell surface molecules specifically recognizing human mesothelin (Human Mesothelin), interleukin 7 (Interleukin-7: IL-7) and chemokine (C-C motif) ligand 19 (chemokine (C-C motif) )ligand 19: CCL19) immunocompetent cells; a pharmaceutical composition containing the immunocompetent cells; comprising the expression of a nucleic acid encoding a cell surface molecule that specifically recognizes mesothelin, a nucleic acid encoding IL-7, and a nucleic acid encoding CCL19 Carrier; a method for making immunocompetent cells expressing cell surface molecules that specifically recognize human mesothelin, IL-7 and CCL19, characterized in that the nucleic acid encoding the cell surface molecules that specifically recognize human mesothelin, Nucleic acid encoding IL-7 and nucleic acid encoding CCL19 are introduced into immunocompetent cells. Background technique [0002] Malignant tumors are diseases that affect m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A61K35/17A61P35/00C07K14/52C07K14/54C07K14/705C07K19/00C12N5/0783
CPCA61K35/17A61P35/00C07K14/52C07K14/5418C07K2319/03C07K14/7051A61K39/001168A61K2039/5158A61K2039/5156A61K2039/55522A61K2039/55527A61K2039/86A61K2039/852C12N5/0636C07K16/28C12N2510/00A61K38/00C07K14/521C07K14/70521C07K14/70575C12N15/63
Inventor 玉田耕治佐古田幸美安达圭志
Owner NOILE IMMUNE BIOTECH
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