Chimeric antigen receptor and immune effector cell expressing chimeric antigen receptor
A chimeric antigen receptor and antibody technology, applied to genetically modified cells, cells modified by introducing foreign genetic material, animal cells, etc., can solve the problems of lack of specificity and not ideal, and achieve the effect of prolonging the survival time
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Embodiment 1
[0116] Example 1: Detection of B7-H3 expression in tumors
[0117] In order to determine the expression of B7-H3 antigen in tumors, we first selected different tumor cell lines to test. At the same time, in order to further verify the expression of B7-H3 in primary tumors, we detected the expression of B7-H3 in slices containing multiple patient glioma tissues and normal tissues.
[0118] Method: Collect tumor cells and wash them three times with 1xPBS, then stain with B7-H3 antibody 15C11 on ice for 30 minutes, add APC-coupled goat anti-mouse secondary antibody after washing with 1xPBS, incubate at room temperature for 20 minutes, wash with 1xPBS and wash with The expression of B7-H3 on tumor cells was detected by flow cytometry.
[0119] Immunohistochemical staining: Sections of glioma tissues and normal tissues of multiple patients were used to detect the expression of B7-H3 in primary tumors with 15C11 antibody by immunohistochemical method.
[0120] Paraffin sections we...
Embodiment 2
[0122] Example 2: Cell Culture
[0123] The cell lines used in the present invention include 293T cells, which are cultured in IMDM medium containing 10% FBS, 1% glutaMAX, and 1% double antibody.
[0124] WM-266-4, SK-OV-3, LN-229, LN-18, U-87MG cell culture medium is in RPMI640 or DMEM with 10% FBS.
[0125] The retrovirus encoding GFP was prepared by transient transfection of 293T cells using a three-plasmid system (GFS-GFP, MMLV-gag-pol and pVSV-G. GFP-positive cells were prepared by infecting GFP-encoding retrovirus. Encoding Luciferase (Luc ) lentivirus was prepared by transiently transfecting 293T cells with Lenti-Luciferase, pspAX2 and pVSV-G, while the retrovirus encoding GFP-firefly luciferase (GFP-FFLuc) was prepared by GFS-GFP-FFLuc, MMLV-gag-pol and pVSV -G is prepared by transiently transfecting 293T cells. The tumor cell line used in mice is infected by the lentivirus encoding Luc or the retrovirus of GFP-FFLuc.
Embodiment 3
[0126] Example 3: Construction of Chimeric Antigen Receptor
[0127] A chimeric antigen receptor consists of a single-chain antibody region, a CD8α hinge region, a CD8α transmembrane region, a CD28 or 4-1BB co-stimulatory factor region, and a CD3ζ signal transduction region (see attached image 3 shown). The single-chain antibody region (scFV) of the chimeric antigen receptor is derived from the antibody against B7-H3 produced by each monoclonal hybridoma cell, and the amino acid sequence of the scFV is as shown in SEQ ID NO: 2, 14, 26, 38, 50, 62, 74, 86, 98 or 110. Chimeric antigen receptors are produced by gene synthesis and then cloned into retroviral vectors. After cloning, the correctness of the sequence was confirmed by sequencing.
[0128] Table 1. Sequence listing:
[0129] SEQ ID NO.1: Nucleic acid sequence of 15C11 single-chain antibody region
[0130] ATGGAGTTTGGGCTGAGCTGGCTTTTTCTTGTGGCTATTTTAAAAGGTGTCCAGTGCTCTAGAGACATCCAGATGACACAGAGCCCTAGCAGCCTGTCTGCCAGCGTGGG...
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