br When culturing T cells in vitro
When culturing T CH 223191 in vitro, diﬀerent expansion methods result in diﬀerent ratios of naive T and eﬀector T cells. IL-2 promotes the expansion of more eﬀector T cells, despite their reduced memory function, whereas IL-7 and IL-15 have the opposite eﬀect . As, to our knowledge, this is the first study to report the eﬀects of ATF-CAR
Fig. 6. Cytokines secretion as well as granzyme B release of ATF-CAR T cells against target cells. (A) The concentration of IFN-γ, TNF, IL-2, IL-4, IL-5 and IL-6 in the cocultured supernatants were determined by a CBA assay. Generally, Th1 cytokines had higher levels than Th2 cytokines in each cocultured group. In uPAR-positive cancer cells, ATF-CAR T cells secreted drastically higher levels of Th1 cytokines than CT cells especially IFN-γ. (B) In uPAR-positive cancer cells (uPAR-A2780 and vector ES-2), levels of granzyme B released from ATF-CAR T cells were higher than CT cells which demonstrated the specific killing against antigen uPAR, while, the result was not in antigen-deficient cancer cells. Data are presented as mean ± SEM. Statistical significance was calculated by the two-tailed Student’s t-test between the two groups. *P < 0.05, **P < 0.01, and ***P < 0.001.
against uPAR cancer cells, a relatively high ratio of eﬀector T cells is acceptable; however, in further studies, memory function and eﬀector capability must be balanced by optimizing the protocol. Most clinical trials rely on second- or third-generation CARs; our study used the third-generation CAR framework to produce ATF-CAR. Third-genera-tion CARs include two intracellular costimulatory molecules and have been shown to enhance T cell activation, sustain proliferation and tumor-lytic activity, and reduce activation-induced cell death . Epithelial ovarian cancer comprises 90% of all ovarian cancers . CAR T clinical trials for patients with epithelial ovarian cancer have involved many target antigens, including FRα, MUC16, mesothelin, etc. [23,24]. As uPAR is expressed not only in cancer cells but also in stromal cells in the tumour microenvironment, including tumor fibroblasts, macro-phages, and endothelial cells, targeting uPAR for CAR T cell therapy could help overcome the physical obstacles of cancer tissue masses [4,25]. However, relying only on the spatio-temporal restriction of uPAR expression in normal tissues would be a negative coping strategy. Strategies such as, co-expression of an inhibitory CAR (iCAR) that re-cognizes another antigen expressed on non-tumor tissues, the combi-nation of two specific antigens, and the modulation of antigen re-sponsiveness by modifying CAR construction will be investigated for anti-uPAR ATF-CAR in the future [13,26,27].
The elevated expression of uPAR is relatively restricted within pa-tients with ovarian cancer and human ovarian cancer cells making uPAR a promising biomarker for prognostic, therapeutic, and predictive applications in ovarian cancer [28,29]. Our prospective study demon-strated the elevated expression of uPA and soluble uPAR in patients with ovarian cancer. Although the sample size was relatively small, the results were consistent with previous studies [30,31]. The circulating form of uPAR, which is soluble uPAR (suPAR) released from the membrane-bound form due to GPI flexibility, can be detected by ELISA in serum samples and suPAR could represent the intermediary products of uPAR metabolism [32,33]. UPAR is a uPA receptor; however, its function is not restricted to plasminogen activation as it cooperates with co-receptors, such as integrins, G-protein-coupled receptors
(GPCRs), and growth factor receptors to participate in tumor develop-ment, particularly in invasion and metastasis . Interestingly, uPAR is not only expressed in ovarian cancer cells but also in non-neoplastic stromal cells in cancer tissue masses and targeting cancer stroma is a promising therapeutic aspect of oncotherapy . Although uPAR has not been a target antigen for any CAR T cell therapy before, uPAR has been targeted in multiple therapeutic strategies to treat cancer and has served as a diagnostic probe in the field of nuclear medicine [10,36]. To the best of our knowledge, this is the first study to target uPAR to treat cancer using CAR T cells.
ATF is used to replace scFv in the CAR framework, but not due to a lack of humanization antibodies for uPAR. The antibodies ATN-615 and ATN-658 targeting uPAR and many complex structures based on those antibodies have exhibited anti-cancer activities [37,38]. ATF is a nat-ural ligand for uPAR with a stable aﬃnity (Kd = 1 nm) and less po-tential toxicity in terms of immunogenicity. Optimal extracellular an-tigen-binding domain selection should be based on minimum immunogenicity; the scFv used for CAR design is usually either murine scFv, humanized scFv, or fully human scFv, in order of decreasing immunogenicity . Engineering the immunogenicity of therapeutic proteins is hotly debated, with predictions varying for each individual case . The ATF segment used in our study is derived from the natural binding part of uPA, reducing immunogenicity to some extent. The disadvantages of immunogenicity and the benefits of therapeutic eﬀects still need to be examined by further studies. ATF is also a competitive antagonist of uPA at the same uPAR binding site, inhibiting the tumor-promoting action of uPA. In addition, ATF has 157 amino acids, making it easier to insert in lentiviral vectors and leaving more space for other CAR components, such as additional costimulatory do-mains. Nowadays, instead of using scFv as an extra recognition domain, antibody mimetic proteins are used as antigen-binding domains, such as the DARPins G3 and 4D5 which target Her-2-positive cancer cells . The only natural ligand-receptor binding partner involved in CAR de-sign is NKG2D-CAR . The ligand NKG2D, which is widely expressed by cancer cells and some NK cells and NKG2D-CAR T therapy has made