Pancreatic cancer is the seventh leading cause of cancer-associated mortality in both sexes and has an extremely poor prognosis, with a 5-year overall survival (OS) rate below 10% (Schizas et al., 2020). The poor prognosis is mainly due to the untimely diagnosis, limited efficacy of available drugs and rapid tumour progression (Mizrahi et al., 2020). Traditional treatment modalities for pancreatic cancer, such as surgery, chemotherapy, radiotherapy and other locoregional treatments, have poor therapeutic effects (Manji et al., 2017). Tumour immunotherapy, such as immune checkpoint inhibitors, has shown encouraging results for pancreatic cancer (Torphy et al., 2018), but the overall response rate is still low (Timmer et al., 2021). Due to the major challenges above, CAR T cell therapy may offer a new approach for the treatment of pancreatic cancer.
The effective treatment of CAR T cells depends on the specific expression of the recognized target antigens on the surface of tumour cells, while solid tumours usually express tumour-associated antigens rather than tumour-specific antigens. Moreover, the expression of antigens in tumour tissues is highly heterogeneous. Therefore, it is extremely important to find a tumour-related antigen with an excellent expression profile, which will enhance the antitumour activity of CAR T cell therapy against solid tumours.
Trop2, also known as tumour-associated calcium signal transducer 2, is overexpressed in epithelial cancer (Bignotti et al., 2011) and plays a critical role in tumour growth. Specifically, Trop2 can increase the intracellular calcium concentration, acts as a calcium signal transducer (Ripani et al., 1998), and is closely related to the JAK/STAT (Hou et al., 2019), MAPK/ERK (Cubas et al., 2010), PI3K/AKT (Guerra et al., 2016) and EPK/JNK (Guan et al., 2017) signalling pathways. As reported, knockdown or knockout of Trop2 on the cell surface significantly inhibits the proliferation of tumour cells (Zhang et al., 2018). Since Trop2 is indispensable in the proliferation and migration of tumour cells, it may be a potential and attractive target for epithelial carcinomas (Cubas et al., 2009), and Trop2-specific chimeric antigen T cells may offer a new therapeutic approach for pancreatic cancer.
Despite the efficacy of Trop2 CAR T cells in gastric cancer (Zhao et al., 2019) and triple-negative breast cancer (Chen et al., 2021) in vitro and in vivo, the efficacy in pancreatic cancer has not been reported thus far. Herein, we employed the human scFv phage library to isolate a human single-chain fragment variable that specifically recognizes EMD of Trop2. Then, we identified its binding specificity and affinity to native Trop2 as well as Trop2-positive cancer cells. Furthermore, we designed Trop2-targeted CAR T cells with this novel scFv and evaluated the antitumour effects of CAR T cells in vitro and in vivo.
Surprisingly, Trop2-specific CAR T cells displayed strong antitumour effects in vivo and completely eliminated BxPC-3 pancreatic cancer xenograft tumours 3 weeks after infusion. Compared with several ADC drugs targeting Trop2 (Cardillo et al., 2015; Mao et al., 2016; Strop et al., 2016), which could effectively inhibit tumour growth but not eliminate tumours in a similar BxPC-3 xenograft model, Trop2-specific CAR T cells exhibited a potent tumour elimination capacity. The functional difference between Trop2-targeted ADC drugs and CAR T cell therapy may be ascribed to CAR T cells being “living drugs” with the capacity of proliferation. Interestingly, we observed that Trop2-specific CAR T cells proliferated when co-cultured with Trop2-positive cells but not with the negative cells (Fig. S3). In addition, CAR T cells targeting Trop2 failed to eliminate tumours in previous studies of gastric cancer (Zhao et al., 2019) and triple-negative breast cancer (Chen et al., 2021), which was probably related to the affinity of scFv for the CAR structure. Our 2F11-scFv bound to Trop2 with high affinity (Kd=0.17 nM), which can be one of the reasons for the thorough tumour clearance.
One of the main hurdles of CAR T cell therapy for solid tumours is the poor infiltration of CAR T cells into tumour tissue (Martinez and Moon, 2019). At present, two kinds of infusion methods, intravenous or intratumoural injection, are mainly used in solid tumour-related in vivo studies. Compared with intratumoural injection, intravenous infusion increases the difficulty of CAR T cells infiltrating solid tumours because of the abundant deposition of extracellular matrix. CAR T cells were injected intratumourally in several CAR T cell-related studies to enhance infiltration (Golubovskaya et al., 2017; Zhao et al., 2019). It has been reported that CD47 CAR T cells injected intratumourally could inhibit pancreatic tumour growth in the same BxPC-3 xenograft model but did not completely remove tumours (Golubovskaya et al., 2017). Thus, intratumoural injection may have better short-term infiltration but not long-term maintenance to eliminate tumours. In the in vivo study, Trop2-targeted CAR T cells were injected only intravenously and then eliminated pancreatic tumours in 3 weeks. We performed immunohistochemical staining with an anti-CD3 antibody on the removed tumours and the tissues at the inoculation site. Unexpectedly, T cell infiltration in the BxPC-3 tumours treated with Trop2 CAR T cells was more abundant than that in the tumours treated with CD19 CAR T cells (Fig. S4), which should partially be ascribed to 2F11 having a high binding capacity for the Trop2 antigen.
The poor persistence of CAR T cells in vivo is another difficulty of CAR T cell treatment. Even though the existing CAR T therapy has greatly improved the complete remission rate of leukaemia and lymphoma, up to one-third of patients eventually relapse (Park et al., 2018), which may be related to the persistence of CAR T cells in vivo (Heng et al., 2020). Surprisingly, no relapse was observed in the pancreatic tumour-bearing mice treated with Trop2 CAR T cells after tumour disappearance, and all mice remained disease-free. We have three hypotheses that may explain this surprising disease-free survival. First, the tumour was completely cleared. Second, no deleterious toxicities were observed. Third, CAR T cells persistently circulated in vivo for 50 days after infusion (Fig. 5c).
Furthermore, one of the factors affecting the persistence of CAR T cells in the clinic is T cell-mediated anti-transgene rejection responses, which will lead to a reduction in CAR-positive T cells. CAR-positive T cells could be detected in the blood of two patients with vesicular lymphoma 24 hours after the first infusion of CD19 CAR T cells but were not detected after 1 week. Even if higher doses of CAR T cells were infused, they could still not survive in vivo. It is speculated that the antitransgene rejection responses may be caused by the use of mouse anti-CD19 scFv on CAR (Jensen et al., 2010). Additionally, Jennifer N et al. reported that the human scFv segment can eliminate antitransgene rejection responses and reduce the risk for off-tumour toxicity (Song et al., 2015) and neurological toxicity (Brudno et al., 2020). Therefore, Trop2 CAR T cells equipped with the fully human 2F11 scFv may have superior persistence and safety in future clinical applications.
Nevertheless, the cell line xenograft model cannot accurately simulate the complex tumour microenvironment of solid tumours in humans. One limitation of our study was the absence of a Trop2-positive pancreatic tumour patient-derived xenograft (PDX) model in the Trop2 CAR T cell efficacy studies. It is unknown whether Trop2 CAR T cells exert the same antitumour activity and persistence against patient-derived pancreatic tumours. In a future study, we intend to use patient-derived tumour xenograft (PDX) models to evaluate the in vivo antitumour activities of CAR T cells.