Chimeric antigen receptor-modified T cells (CAR-T cells) have shown good effects in the treatment of hematologic cancers; however, they may cause on-target off-tumor toxicity because of minimal expression of tumor-associated antigens (TAAs) on normal tissues, particularly in the context of treating solid tumors. Hypoxia is a common hallmark of solid tumors because of the Warburg effect. To minimize side effects, we designed a hypoxia-inducible CAR (HiCAR), which is driven by a hypoxia response element (HRE), and consists of a conventional CAR and an oxygen-dependent degradation domain (ODD) that is actively degraded under normoxia but stabilized under hypoxia. HiCAR-T cells showed enhanced cytotoxicity against tumor cells under hypoxia compared to normoxia in vitro and antitumor efficacy comparable to that of conventional CAR-T cells in vivo. Overall, our study demonstrates the potential of the HiCAR for improving the safety of CAR-T cells to promote the clinical application of CAR-T immunotherapy.

Background On-target off-tumor toxicity impedes the clinical application of chimeric antigen receptor-modified T cells (CAR-T cells) in the treatment of solid tumors. Previous reports proved that the combinatorial antigen recognition strategy could improve the safety profile of CAR-T cells by targeting two different tumor-associated antigens (TAAs), one as a CAR-T targeted antigen and the other as a chimeric costimulatory receptor (CCR) ligand. The programmed death-ligand 1 (PD-L1, also known as B7-H1) is preferentially overexpressed on multiple tumors, it will be highly interesting to explore the potential of PD-L1 as a universal target for designing CCR. Methods A novel dual-targeted CAR, which is composed of first-generation CD19/HER2 CAR with CD3ζ signaling domain and PD-L1 CCR containing the CD28 costimulatory domain, was constructed and delivered into T cells by pseudotyped lentivirus. The cytokine release, cytotoxicity and proliferation of dual-targeted CAR-T cells were tested in vitro, and their safety and therapeutic efficacy were evaluated using a human tumor xenograft mouse model in vivo. Results The dual-targeted CAR-T cells exerted a similar cytotoxic activity against CD19/HER2+ tumor cells with or without PD-L1 in vitro, however, enhanced cytokine releases and improved proliferative capacity were only observed in the presence of both CD19/HER2 and PD-L1. Importantly, the dual-targeted CAR-T cells displayed no cytotoxicity against PD-L1+ cells alone in the absence of tumor antigen CD19/HER2. In addition, the dual-targeted CAR-T cells preferably destroyed tumor xenografts bearing both CD19/HER2 and PD-L1, but spared only antigen-positive tumor xenografts without PD-L1 in vivo. Furthermore, PD-L1 CCR also improved the antitumor efficacy of the low-affinity HER2 CAR-T cells against PD-L1+ tumors expressing high levels of HER2. Conclusion Our observations demonstrated that PD-L1 could be used as a universal target antigen for designing CCR, and the dual-targeted CAR-T cells equipped with PD-L1 CCR could be used to reduce the risk of on-target off-tumor toxicity while retaining their potent antitumor efficacy in the treatment of PD-L1+ solid tumors.

Despite the success of antiretroviral therapy in suppressing HIV to an undetectable level in the blood and improving patients' quality of life, HIV persists in antiretroviral therapy-treated patients and threatens their lives. Anti-HIV chimeric antigen receptor (CAR) T cells could offer a cure by recognizing and killing virus-producing cells in an Env-specific manner. In this review, the authors summarize several important aspects of the development of anti-HIV CAR T cells, with a special focus on the evolution of CAR design for enhanced potency and targeting specificity, and also outline the challenges that still need to be addressed to take anti-HIV CAR T cells from a hopeful approach to a real HIV cure.

Background: On-target off-tumor toxicity impedes the clinical application of chimeric antigen receptor-modified T cells (CAR-T cells) in the treatment of solid tumors. The combinatorial antigen recognition strategy can improve the therapeutic safety of CAR-T cells by targeting two different tumor-associated antigens (TAAs) using a CAR and a chimeric costimulatory receptor (CCR). Although programmed death-ligand 1 (PD-L1, also known as B7-H1) is expressed on multiple tumors, the potential of PD-L1 as a universal target for designing CCR remains unknown.Methods: A first-generation CD19 or HER2 CAR and a PD-L1 CCR containing the CD28 signaling domain were constructed and delivered into Jurkat T cells or primary T cells by a pseudotyped lentivirus. The release of cytokines, including IL-2, IFN-γ and TNF-α, was quantified using enzyme-linked immunosorbent assay (ELISA) kits or a cytometric bead array (CBA). The in vitro cytotoxicity of CAR-T cells was detected with a luciferase-based killing assay. The in vitro proliferation of CAR-T cells was assessed by flow cytometry. The therapeutic safety and efficacy of CAR-T cells was evaluated using a subcutaneous dual-tumor model in vivo.Results: Jurkat T cells or primary T cells expressing both the CD19/HER2 CAR and PD-L1 CCR produced higher levels of cytokines in the presence of CD19/HER2 and PD-L1 than in the presence of HER2/CD19. Compared to HER2-z-engineered T cells, HER2-z-PD-L1-28-engineered T cells had higher in vitro cytotoxicity potential against PD-L1-positive tumor cells. CD19/HER2-z-PD-L1-28-engineered T cells showed higher proliferation potential in the presence of CD19/HER2 and PD-L1 than in the absence of PD-L1. CD19/HER2-z-PD-L1-28-engineered T cells preferably destroyed xenograft tumors expressing CD19/HER2 and PD-L1 in vivo and did not significantly affect CD19/HER2-expressing tumors. The PD-L1 CCR improved the antitumor efficacy of low-affinity HER2 CAR-T cells against PD-L1-positive tumors expressing high levels of HER2.Conclusion: Our findings confirmed that PD-L1 can be used as a universal target antigen for designing CCR, improving the efficacy of CAR-T cells in the treatment of PD-L1-positive solid tumors but reducing toxicity within PD-L1-negative normal tissues expressing low levels of TAA in vivo.

Background: On-target off-tumor toxicity impedes the clinical application of chimeric antigen receptor-modified T cells (CAR-T cells) in the treatment of solid tumors. Previous reports proved that the combinatorial antigen recognition strategy could improve the safety profile of CAR-T cells by targeting two different tumor-associated antigens (TAAs), one as a CAR-T targeted antigen and the other as a chimeric costimulatory receptor (CCR) ligand. The programmed death-ligand 1 (PD-L1, also known as B7-H1) is preferentially overexpressed on multiple tumors, it will be highly interesting to explore the potential of PD-L1 as a universal target for designing CCR.Methods: A novel dual-targeted CAR, which is composed of first-generation CD19/HER2 CAR with CD3ζ signaling domain and PD-L1 CCR containing the CD28 costimulatory domain, was constructed and delivered into T cells by pseudotyped lentivirus. The cytokine release, cytotoxicity and proliferation of dual-targeted CAR-T cells were tested in vitro, and their safety and therapeutic efficacy were evaluated using a human tumor xenograft mouse model in vivo.Results: The dual-targeted CAR-T cells exerted a similar cytotoxic activity against CD19/HER2+ tumor cells with or without PD-L1 in vitro, however, enhanced cytokine releases and improved proliferative capacity were only observed in the presence of both CD19/HER2 and PD-L1. Importantly, the dual-targeted CAR-T cells displayed no cytotoxicity against PD-L1+ cells alone in the absence of tumor antigen CD19/HER2. In addition, the dual-targeted CAR-T cells preferably destroyed tumor xenografts bearing both CD19/HER2 and PD-L1, but spared only antigen-positive tumor xenografts without PD-L1 in vivo. Furthermore, PD-L1 CCR also improved the antitumor efficacy of the low-affinity HER2 CAR-T cells against PD-L1+ tumors expressing high levels of HER2.Conclusion: Our observations demonstrated that PD-L1 could be used as a universal target antigen for designing CCR, and the dual-targeted CAR-T cells equipped with PD-L1 CCR could be used to reduce the risk of on-target off-tumor toxicity while retaining their potent antitumor efficacy in the treatment of PD-L1+ solid tumors.