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ABSTRACTInvasive aspergillosis (IA) is a common and deadly mold infection in immunocompromised patients. As morbidity and mortality of IA are primarily driven by poor immune defense, adjunct immunotherapies, such as chimeric antigen receptor (CAR) T cells, are direly needed. Here, we propose a novel approach to generate Aspergillus fumigatus (AF)-CAR T cells using the single-chain variable fragment domain of monoclonal antibody AF-269-5 and a lentiviral vector system. These cells successfully targeted mature hyphal filaments of representative clinical and reference AF isolates and elicited a potent release of cytotoxic effectors and type 1 T cell cytokines. Furthermore, AF-CAR T cells generated from peripheral blood mononuclear cells of four healthy human donors and expanded with either of three cytokine stimulation regimens (IL-2, IL-2 + IL-21, or IL-7 + IL-15) significantly suppressed mycelial growth of AF-293 after 18 hours of co-culture and synergized with the immunomodulatory antifungal agent caspofungin to control hyphal growth for 36 hours. Moreover, cyclophosphamide-immunosuppressed NSG mice with invasive pulmonary aspergillosis that received two doses of 5 million AF-CAR T cells (6 and 48 hours after AF infection) showed significantly reduced morbidity on day 4 post-infection (P < 0.001) and significantly improved 7-day survival (P = 0.049) compared with mice receiving non-targeting control T cells, even without concomitant antifungal chemotherapy. In conclusion, we developed a novel lentiviral strategy to obtain AF-CAR T cells with high targeting efficacy, yielding significant anti-AF activity in vitro and short-term protection in vivo. Our approach could serve as an important steppingstone for future clinical translation of antifungal CAR T-cell therapy after further refinement and thorough preclinical evaluation.IMPORTANCEInvasive aspergillosis (IA) remains a formidable cause of morbidity and mortality in patients with hematologic malignancies and those undergoing hematopoietic stem cell transplantation. Despite the introduction of several new Aspergillus-active antifungals over the last 30 years, the persisting high mortality of IA in the setting of continuous and profound immunosuppression is a painful reminder of the major unmet need of effective antifungal immune enhancement therapies. The success of chimeric antigen receptor (CAR) T-cell therapy in cancer medicine has inspired researchers to translate this approach to opportunistic infections, including IA. Aiming to refine anti-Aspergillus CAR T-cell therapy and improve its feasibility for future clinical translation, we herein developed and validated a novel antibody-based CAR construct and lentiviral transduction method to accelerate the production of CAR T cells with high targeting efficacy against Aspergillus fumigatus. Our unique approach could provide a promising platform for future clinical translation of CAR T-cell-based antifungal immunotherapy.

Bladder cancer has a 60%–70% recurrence rate most likely due to any residual tumour left behind after a transurethral resection (TUR). Failure to completely resect the cancer can lead to recurrence and progression into higher grade tumours with metastatic potential. We present here a novel therapy to treat superficial tumours with the potential to decrease recurrence. The therapy is a heat-based approach in which bladder tumour specific single-walled carbon nanotubes (SWCNTs) are delivered intravesically at a very low dose (0.1 mg SWCNT per kg body weight) followed 24 h later by a short 30 s treatment with a 360° near-infrared light that heats only the bound nanotubes. The energy density of the treatment was 50 J cm−2, and the power density that this treatment corresponds to is 1.7 W cm−2, which is relatively low. Nanotubes are specifically targeted to the tumour via the interaction of annexin V (AV) and phosphatidylserine, which is normally internalised on healthy tissue but externalised on tumours and the tumour vasculature. SWCNTs are conjugated to AV, which binds specifically to bladder cancer cells as confirmed in vitro and in vivo. Due to this specific localisation, NIR light can be used to heat the tumour while conserving the healthy bladder wall. In a short-term efficacy study in mice with orthotopic MB49 murine bladder tumours treated with the SWCNT-AV conjugate and NIR light, no tumours were visible on the bladder wall 24 h after NIR light treatment, and there was no damage to the bladder. In a separate survival study in mice with the same type of orthotopic tumours, there was a 50% cure rate at 116 days when the study was ended. At 116 days, no treatment toxicity was observed, and no nanotubes were detected in the clearance organs or bladder. [ABSTRACT FROM AUTHOR]