Overexpression of B7-H3 in prostate cancer cell lines and tumor tissues
To assess whether B7-H3 could be a target for PCa, we examined the expression of this molecule in PCa tissues and cell lines. Immunohistochemistry of tumor tissue from three clinical prostate cancer patients revealed high expression of B7-H3 (Figure 1A). The result of flow cytometry (FCM) showed that B7-H3 was highly expressed on the surface of prostate cancer cell lines PC3, DU145 and LNCaP (Figure 1B). These results indicate that B7-H3 is a potential PCa target for the development of novel therapeutic strategies.
Construction of CAR-T cells targeting B7-H3
After confirming that B7-H3 is highly expressed in prostate cancer, we synthesized the B7-H3 scFv sequence based on 8H9 clone, and constructed the second-generation B7-H3 CAR containing CD8α transmembrane region, CD28 intracellular costimulatory domain and CD3ζ intracellular signaling domain (Figure 2A). B7-H3 CAR-T cells were constructed by infecting CD3/CD28-activated T cells with a retroviral vector encoding B7-H3 CAR, and the activated T cells were used as control T cells. The positive rate of CAR expression on the surface of CAR-T cells reached 85% (Figure 2B, C). On the sixth day after transfection, CAR-T cells expanded approximately 60 fold, which was comparable to control T cells, indicating that the expression of CAR did not affect the proliferation of T cells (Figure 2D).
B7-H3 redirected CAR-T cells efficiently kill PCa cells
We tested the function of B7-H3 specific T cells in vitro using FCM and RTCA techniques. Three prostate cancer cells were co-incubated with control T and B7-H3 CAR-T cells at effector-to-target ratio (E:T) of 1:1, respectively. RTCA was used to record the cell index in real time to reflect the adherence and killing of tumor cells. After 4 days of co-culture, the percentage of surviving CAR-T cells and tumor cells was analyzed by flow cytometry to calculate the killing rate. The results of RTCA showed that, tumor cells alone as blank group, control T cells had no obvious effect on three B7-H3 positive tumor cells. Compared with blank and control T groups, B7-H3 CAR-T cells induced almost complete elimination of tumor cells (Figure 3A, B, C). FCM results demonstrated that B7-H3 CAR-T cell group had less residual tumor cells at higher target ratio, that is, CAR-T cells had stronger killing effects on PC3, DU145 and LNCaP cells. The killing effect was dose-dependent and enhanced with the increase of effector-to-target ratio (Figure 3D, E, F). The above results suggest that B7-H3 CAR-T cells have significant anti-tumor effect on B7-H3 positive PCa cells.
Control T and CAR-T cells were stained with CFSE, then incubated with PC3 or DU145 cells at E:T of 1:1. After 48 h, the CFSE fluorescence intensity of T cells was detected by flow cytometry, and the fluorescence intensity decreased with the increase of T cell proliferation. As shown in Figure 3G and 3H, the fluorescence intensity of control T cells co-cultured with the two tumor cells was not different from that of T cells alone, while the CFSE intensity of B7-H3 CAR-T cells was significantly weaker after co-culture, demonstrating that tumor cells enhanced the proliferation of CAR-T cells. The above shows that B7-H3 CAR-T cells could eradicate tumor cells, and tumor cells stimulate CAR-T cells to proliferate efficiently. CAR-T cells activate, proliferate, and release cytokines IFN-γ, TNF-α, Granzyme A, and Granzyme B in the presence of target antigen. Hence, we collected T cells co-cultured with tumor cells at E:T of 1:5 for 48 hours, then detected the release of IFN-γ and TNF-α from CAR-T cells by flow cytometry. The detection of the cytokines illustrated PC3, DU145 and LNCaP cells expressing B7-H3 significantly increased the release of IFN-γ (Fig 3I) and TNF-α (Fig 3J) from CAR-T cells compared with control T cells.
B7-H3 CAR-T cells do not kill B7-H3 negative PCa cells
In order to clarify the specificity of B7-H3 CAR-T cells, we used CRISPR-Cas9 technology to knock out B7-H3 in PC3 and DU145 cells, and constructed PC3 (B7H3-) and DU145 (B7H3-) cells by puromycin selection and flow sorting. Flow cytometry was used to detect the expression of B7-H3 on cell clones (Figure 4A, B), and western blot was used to verify the successful construct of PC3(B7H3-) and DU145(B7H3-) cells (Figure 4C, D). The effect of B7-H3 CAR-T cells on B7-H3 negative cells was detected by RTCA and flow cytometry. RTCA curve showed that control T cells did not kill PC3(B7H3-) and DU145(B7H3-) cells, and the curve of B7-H3 CAR-T group was similar to blank group and control T group, suggesting that B7-H3 directed CAR-T cells had no cytotoxicity against B7-H3 negative tumor cells (Figure 4E, F). The results of FCM were consistent with RTCA (Figure 4G, H).
As shown in Figure 4I and 4J, the CFSE intensity of B7-H3 CAR-T cells co-incubated with PC3(B7H3-) or DU145(B7H3-) cells had no discernible difference from that of CAR-T cells alone, but higher than co-incubation with PC3/DU145 cells, denoting that B7-H3 negative PC3 and DU145 cells had no effect on the proliferation of B7-H3 CAR-T cells. Cytokine assays revealed that PC3(B7H3-) and DU145(B7H3-) cells did not promote the release of IFN-γ (Figure 4K) and TNF-α (Figure 4L) from B7-H3 CAR-T cells. The above results imply that B7-H3 CAR-T cells specifically target and kill B7-H3 positive tumor cells and release cytokines.
B7-H3 CAR-T cells inhibit the growth of prostate cancer in vivo
Since B7-H3 specific CAR-T cells could effectively eliminate B7-H3 positive PCa cells in vitro, we evaluated anti-tumor activity of B7-H3 CAR-T cells in vivo. We infused immunodeficient NCG mice with DU145 or DU145(B7H3-) cells to construct subcutaneous xenograft models of prostate cancer (Figure 5A). On day 29 and 65, 5×106 control T or B7-H3 CAR-T cells were infused via tail vein. After treatment, blood was collected from the tail vein every week to detect the content of T cells and CAR-T cells in peripheral blood to evaluate the survival of T cells in mice. As show in Figure 5B, tumors in PBS group and control T group of DU145 mice continued to grow, while B7-H3 CAR-T cells significantly inhibited the tumor growth (p < 0.05). Tumors in the B7-H3 CAR-T group of DU145(B7H3-) mice were slightly smaller than PBS and control T groups, but not statistically significant (Figure 5C). Treatment with control T and B7-H3 CAR-T cells in both models did not lead to weight loss in mice (Figure 5D, E), demonstrating that B7-H3 CAR-T cells had no obvious toxic or side effects on mice.
As for the content of T cells in the peripheral blood of DU145 mice, there were fewer T cells in control group than in B7-H3 CAR-T group (p < 0.05), while T cells in both groups of DU145(B7H3-) mice were lower. The contents of T cells and CAR-T cells in the peripheral blood of CAR-T group of DU145 mice were higher than those of CAR-T group of DU145(B7H3-) mice (p < 0.01) (Figure 5F、G). At the end of experiment, mouse spleen was taken and ground to detect the ratio of T cells (Figure 5H) and CAR-T cells (Figure 5I). There was no difference in the proportion of spleen T cells between control T group and B7-H3 CAR-T group of the two mice, and the ratio of T cells and CAR-T cells in the spleen of DU145(B7H3-) mice was obvious lower than that of DU145 mice. The tumors of mice were ground to measure the content of T cells and CAR-T cells, and excised tumor tissues for CD3 immunohistochemical staining to evaluate the infiltration of T cells. The numbers of T cells in the tumors of control T group and B7-H3 CAR-T group in the two mouse models were comparable, and the numbers of T cells and CAR-T cells in the tumors of B7-H3 knockout mice were less than that of the DU145 mice ( p < 0.05) (Figure 5J, K). There were obvious T cell infiltration in the tumor tissues of control T group and B7-H3 CAR-T group (Figure 5L).
Overall, the above results verified that B7-H3 specific CAR-T cells inhibited the tumor growth of B7-H3 positive DU145 mice, and effectively expanded and infiltrated in mice, but had no obvious side effects.