C57BL/6 mice were s.c. tumor tissue and significantly inhibited tumor growth in 3 mouse models of cancer (Rip-Tag2, mPDAC, and Lewis lung carcinoma). Reduced tumor burden also correlated with significant loss of CLEC14A expression and reduced vascular density within malignant tissues. These data suggest the tumor vasculature can be safely and effectively targeted with CLEC14A-specific CAR T cells, offering a potent and widely applicable therapy for cancer. values shown were calculated using a Wilcoxon matched-pairs signed rank test. Human T cells were then transduced with these retroviral constructs and analyzed by flow cytometry. As illustrated in Figure 1B, CD34 expression was readily detected in T cells transduced with vectors encoding CARs based on either of the 2 CLEC14A-specific antibodies. Using recombinant CLEC14A protein, it was also possible to stain directly for surface CAR expression (Figure 1C). In vitro functions of CLEC14A-specific CAR engineered T cells. In vitro tests were used to assess the function of these engineered T cells. Using an ELISA to detect IFN- release, T cells expressing the CARs were diluted with mock T cells to equalize the proportion of transduced cells, and they were then compared for their ability to respond to human CLEC14A. The target antigen was expressed either as a recombinant Fc-fusion protein immobilized on a plate, overexpressed on the surface of engineered CHO cells, or naturally expressed at physiological levels on the surface of HUVECs grown under static culture conditions. As shown in Figure 1, DCF, in all cases, there was a specific response to CLEC14A above control targets. Note that these CAR T cells also produced the cytokines TNF- and IL-2 in response to CLEC14A (Supplemental Figure 2). Using (-)-Borneol a chromium release assay, we assessed the cytotoxic function of the CAR T cells. CHO cells expressing human CLEC14A (or CHO cells plus vector only control) were cocultured with CAR T cells or mock T cells. Again CAR T cell preparations were diluted with mock T cells to equalize for transduction efficiencies. Both CAR constructs tested mediated specific lysis of CLEC14A+ targets (Figure 2A). Open in a separate window Figure 2 Further characterization of functional responses in CAR-transduced T cells.(A) Human T cells expressing CLEC14A-specific CARs (or mock T cell controls) were tested for cytotoxicity against CHO cells engineered to express full-length human CLEC14A (or control CHO cells transduced with vector alone). Results show data from 8 repeat experiments (effector/target ratio = 9:1). (B) Such T cells were also tested for proliferation, measured by CFSE staining of CD34+ T cells (solid line) and CD34C T cells (dotted line) when cocultured with HUVECs or medium alone (unstimulated). Results show a histogram of T cells expressing CAR5.28z, and the 2 graphs below show data from 2 repeat experiments giving the percentage of CD34+ cells that proliferated for each of the CARs indicated (having subtracted the percentage of CD34+ T cells that proliferated in medium alone). (C) CLEC14A-specific (-)-Borneol CAR T cells (or mock T cell controls) were also tested for IFN- release in response to plate-bound (-)-Borneol recombinant human or mouse CLEC14A (both expressed (-)-Borneol as Fc-fusion proteins) or to Agt Fc alone. Results show data from 6 repeat experiments. All values shown were calculated using a Wilcoxon matched-pairs signed rank test. CFSE labeling of (-)-Borneol CAR T cells demonstrated that they can also proliferate when cultured with HUVECs. This proliferation was induced only in CD34+ T cells and not in the nontransduced (CD34C) subset within the T cell preparation, indicating that it is in response to CLEC14A (Figure 2B). Next, we sought to compare responses of our CAR T cells with human and mouse versions of CLEC14A. CAR T cell preparations.