Separated antigen PSMA binding protein and purpose 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
[0181] Example 1. Generation and screening of monoclonal hybridoma cells
[0182] 1.1 Preparation of CHO-K1 / cyno PSMA cell stable transfection line
[0183] Lentiviral particles (Jikai Gene, cat#LVCON335) packaged with a nucleic acid sequence encoding cynomolgus PSMA (the Genbank accession number of the corresponding cynomolgus PSMA amino acid sequence is XP_005579379) with M.O.I.=100 ((M.O.I.=(lentivirus Particle titer*volume) / number of infected cells) to infect CHO-K1 cells (ATCC, cat#CCL-61). Cells after 48 hours of infection were passaged at a ratio of 1:10 with a screening antibody containing 8 μg / ml and 10% (w / v) fetal bovine serum F2K medium for about 1 week until the uninfected control group CHO-K1 was screened for resistance and killed, while the infected group CHO-K1 cells still survived. The lentivirus CHO-K1 cells in the infection group were digested and limitedly diluted to 0.5 cells per well in a 96-well plate, and then cultured with medium containing selection ...
Embodiment 2
[0190] Example 2. Sequencing, expression and purification of monoclonal PSMA antibodies
[0191] The monoclonal PSMA antibody screened in Example 1 was sequenced, and the amino acid sequence is shown in Table 1.
[0192] Table 1. Sequencing results of monoclonal PSMA antibodies
[0193] sequence name SEQ ID NO: HCDR1 1 HCDR2 2 HCDR3 3 LCDR1 4 LCDR2 5 LCDR3 6 V H
15 V L
16
[0194] The heavy chain variable region sequence of the above monoclonal PSMA antibody was subcloned into the pTT5 expression vector containing the 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, a large pumping kit (Macherey-Nagel, Xtra M...
Embodiment 3
[0200] Example 3. Binding Ability of Monoclonal PSMA Antibody to Cell Surface PSMA
[0201] HEK293T cells overexpressing human PSMA (HEK293T / human PSMA, Kangyuan Bochuang, cat#KC-1005) or CHO-K1 cells overexpressing cynomolgus PSMA (CHO-K1 / cyno PSMA) or overexpressing human PSMA Tumor cells (LNCAP) were cultured and expanded in T-75 culture flasks, and the medium was aspirated after reaching 90% confluency, and the cells were washed twice with PBS. The cells were treated with trypsin (Invitrogen, cat#15050065) for about 1 minute, and the trypsin was neutralized with 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 isotype control antibody for 1 hour on ice. Cells were washed twice with PBS and incubated with goat anti-human (H+L)-Alexa Fluor 647 (Life t...
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