Separate antigen PSMA conjugated protein and use thereof
A protein-binding and chimeric antigen receptor technology, applied in the field of biomedicine, can solve the problems of unknown epitope information, lack of cross-species cross-reactivity, etc., and achieve the effect of alleviating and/or treating tumors
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Embodiment 1
[0162] Example 1. Generation and screening of monoclonal hybridoma cells.
[0163] 1.1 Preparation of CHO-K1 / cyno PSMA cell stable transfection line.
[0164] Lentiviral particles packaged with nucleic acid sequences encoding human PSMA (GeneChem, cat#LVCON335) infected CHO-K1 at a ratio of M.O.I.=100 ((M.O.I. = (lentiviral particle titer*volume) / number of infected cells) Cells (ATCC, cat# CCL-61). Cells 48 hours post-infection were passaged at a ratio of 1:10 in F2K medium containing 8 µg / ml of selection resistance and 10% (w / v) fetal bovine serum1 About one week until the uninfected control group CHO-K1 was killed by screening resistance. Perform limiting dilution to 0.5 cells per well in a 96-well plate, continue to add medium containing screening resistance and culture for about 5 days to observe whether it is a monoclonal , Marked. Continue to culture in a 37-degree carbon dioxide incubator for about 1 week to 50% full, and expand the monoclonal to 6 wells. Use Tab antib...
Embodiment 2
[0171] Example 2. Sequencing, expression and purification of monoclonal PSMA antibodies.
[0172] The monoclonal PSMA antibody screened in Example 1 was sequenced, and the amino acid sequence is shown in Table 1.
[0173] Table 1. Sequencing results of monoclonal PSMA antibodies
[0174] sequence name SEQ ID NO: HCDR1 1 HCDR2 2 HCDR3 3 LCDR1 4 LCDR2 5 LCDR3 6 V H
15 V L
16
[0175] The heavy chain variable region sequence of the above monoclonal PSMA antibody was subcloned into pTT5 expression vector containing signal peptide and human heavy chain IgG1 constant region (SEQ ID NO: 19). The light chain variable region sequence of the monoclonal PSMA antibody was subcloned into an expression vector containing the signal peptide and the human antibody light chain kappa constant region (SEQ ID NO: 20). After the recombinant plasmid was confirmed by sequencing, the plasmid was extracted with a large extraction kit...
Embodiment 3
[0182] Example 3. Binding ability of monoclonal PSMA antibodies to PSMA on the cell surface.
[0183] HEK293 / hPSMA cells or CHO-K1 / cyno PSMA cells or LNCAP cells were cultured and expanded in T-75 culture flasks. After reaching 90% confluency, the culture medium was aspirated and the cells were washed twice with PBS. Cells were treated with trypsin (Invitrogen, cat#15050065) for about 1 minute, and the trypsin was neutralized with culture medium. Then the cells were washed twice with PBS, the cell count was determined, and the cells were resuspended in PBS to 2×10 6 cells / ml. Add 100 µl of cell suspension to each well of a 96-well V-plate. Incubate with different concentrations of purified PSMA antibody or each isotype control antibody on ice for 1 hour. Cells were washed twice with PBS and incubated with goat anti-human (H+L)-Alexa Fluor 647 (Life technology, cat#A21445) at 4°C for 30-45 minutes. After washing with PBS again, the median fluorescence intensity (MFI) of the...
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