Therapeutic combinations and methods of treating melanoma
A melanoma and toxin technology, applied in the field of melanoma treatment, can solve problems such as disease recurrence
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Embodiment 1
[0383] Example 1: In vitro evaluation of specific cell killing by anti-ETBR ADC
[0384] In melanomas expressing a relatively low copy number of ETBR (in the case of cell line A2058 (from the American Type Culture Collection)) or a higher copy number of ETBR (in the case of cell line UACC-257X2.2) The anti-ETBR antibody-ADC candidate Hu5E9v1-ADC was evaluated in vitro on the cell line. The UACC-257X2.2 cell line is a derivative of the parental UACC-257 cell line (NCI-Frederick Cancer DCT Tumor Repository), optimized for in vivo growth. The parental UACC-257 cells were injected subcutaneously into the right side of female NCr nude mice, and a tumor was harvested and dissociated, and cultured in vitro to generate the UACC-257X1.2 cell line. The UACC-257X1.2 line was injected subcutaneously into the right side of female NCr nude mice again to try to improve the growth of the cell line. A tumor was collected from this study and adapted again to grow in vitro to generate the UACC-2...
Embodiment 2
[0388] Example 2: In vivo evaluation of specific tumor killing by anti-ETBR ADC
[0389] Based on the research described in Example 1 above, the melanoma cell lines A2058 and UACC-257X2.2 were selected as suitable models for in vivo anti-tumor activity studies representing broad ETBR expression. The UACC-257X2.2 melanoma cell line is a derivative of the parental UACC-257 melanoma cell line (National Cancer Institute (NCI)), optimized for in vivo growth. Specifically, parental UACC-257 cells were injected subcutaneously into the right side of female NCr nude mice, and a tumor was harvested and cultured in vitro to generate UACC-257X1.2 cell line. The UACC-257X1.2 line was injected subcutaneously into the right side of female NCr nude mice again to try to improve the growth of the cell line. A tumor was collected from this study and adapted again to grow in vitro to generate the UACC-257X2.2 cell line. This cell line and tumors derived therefrom express ETBR equivalent to the pa...
Embodiment 3
[0394] Example 3: Effect of BRAF inhibitor drugs on the expression level of ETBR
[0395] Evaluation of the effects of BRAF inhibitor drugs on ETBR transcripts and proteins in a variety of melanoma cells representing various genetic backgrounds of melanoma (such as BRAF mutant (V600E), BRAF wild type and RAS mutant (Q61L)). Total protein and cell surface protein) expression levels.
[0396] By adding the appropriate drug volume to the cultured cells on a 4-well plate for 24 hours, the melanoma cell line UACC-257X2.2 was treated with different concentrations of BRAF inhibitor drugs ("BRAFi") (specifically RG7204), A2058, COLO 829, IPC-298 (ATCC).
[0397] In order to determine the effect of BRAFi on ETBR and control ribosomal protein L19 (RPL19) transcript levels, the following experiment was performed. Cells treated with RG7204 for 24 hours were harvested from the plate by scraping and processed for total RNA using Qiashredder and RNeasy mini kit (79654, 74104, Qiagen, Valencia, C...
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