Combined preparation for treating prostatic cancer and medical application thereof
An inhibitor and peptide-binding technology, applied in the field of biomedicine, can solve the problems of preparing single-chain antibody sequences, low specific binding efficiency, and inability to target antigens, etc., and achieve the effect of enhancing anti-tumor effect, good clinical application value and prospect
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Embodiment 2
[0127] Example 2 Verification of WTN polypeptide specificity
[0128] 1. Experimental method
[0129] The WTN polypeptide was detected by immunofluorescence with the Lncap cell line with high PSMA expression and the PC3 cell line with low PSMA expression, and the fluorescent marker used was FITC (ie, FITC-labeled WTN polypeptide).
[0130] 2. Experimental results
[0131] Fluorescence detection of WTN polypeptide and Lncap cell line such as figure 1 As shown, the fluorescence detection of WTN polypeptide and PC3 cell line is as follows figure 2 shown; figure 1 The center of the dot is the nucleus, and the light color around the dot is the fluorescence displayed by the combination of WTN and the cell surface antigen PSMA; figure 2 Only the nuclei were stained, and it can be seen that the cell surface fluorescence marked by WTN was only in figure 1 Appeared in , proving that the combination of WTN and cell surface antigen PSMA has high specificity.
Embodiment 3
[0132] Embodiment 3 The cell line involved in the present invention, its culture method, construction method
[0133] 1. Construction method of TABP-EIC-WTN
[0134] (1) Preparation of TABP-EIC-WTN cells
[0135] The NK cells used in the experiments involved in this patent are obtained from the expansion of peripheral blood mononuclear cells (PBMC).
[0136] (2) Construction of tumor antigen-binding peptide expression vector
[0137] The tumor antigen binding peptide structure (the complete structure is: WTN polypeptide region-CD8α hinge region-2B4 transmembrane domain-2B4 co-stimulatory domain-NKG2D primary signal transduction domain, the nucleic acid sequence is shown in SEQ ID NO:12) sequence Obtained for gene synthesis (General Biology), the expression vector is pLenti-EF1a-Backbone(NN) (addgene #27961). The tumor antigen-binding peptide structure is inserted into the restriction sites BsiWI and EcoRI (that is, the sequence shown in SEQ ID NO: 12 is replaced with the se...
Embodiment 4
[0151] Example 4 Upregulation of PD-L1 expression on C4-2 cells co-cultured with TABP-EIC-WTN cells depends on IFN-γ
[0152] C4-2 GFP cells were co-cultured with TABP-EIC-WTN cells, and flow cytometry was performed at 2, 6, 12, and 24 hours, and the results were as follows image 3 Shown: And detect the PD-L1 expressed in C4-2 cells, the results are as follows image 3 Shown: The mean fluorescence intensity (MFI) of PD-L1 in C4-2 cells and the percentage of C4-2 cells expressing PD-L1 were significantly up-regulated over time.
[0153] TABP-EIC-WTN cells were cultured, and in the presence or absence of C4-2 GFP co-cultivation, the IFN-γ secretion level in the medium supernatant was measured by ELISA at the time points of 2, 6, 12, and 24 hours, The result is as Figure 4 shown.
[0154] Add IFN-γ to C4-2 cells, and the expression of PD-L1 on C4-2 cells under different concentrations of IFN-γ, the results are as follows Figure 5 Shown: The mean fluorescence intensity (MFI...
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