Engineering T cells with chimeric antigen receptors (CARs) is an effective method for directing T cells to attack tumors, but may cause adverse side effects such as the potentially lethal cytokine release syndrome. Here the authors show that the T cell antigen coupler (TAC), a chimeric receptor that co-opts the endogenous TCR, induces more efficient anti-tumor responses and reduced toxicity when compared with past-generation CARs. TAC-engineered T cells induce robust and antigen-specific cytokine production and cytotoxicity in vitro, and strong anti-tumor activity in a variety of xenograft models including solid and liquid tumors. In a solid tumor model, TAC-T cells outperform CD28-based CAR-T cells with increased anti-tumor efficacy, reduced toxicity, and faster tumor infiltration. Intratumoral TAC-T cells are enriched for Ki-67+ CD8+ T cells, demonstrating local expansion. These results indicate that TAC-T cells may have a superior therapeutic index relative to CAR-T cells.

Natural killer (NK) cells are useful for cancer immunotherapy and have proven clinically effective against hematologic malignancies. However, immunotherapies for poor prognosis solid malignancies, including ovarian cancer, have not been as successful due to immunosuppression by solid tumors. Although rearming patients' own NK cells to treat cancer is an attractive option, success of that strategy is limited by the impaired function of NK cells from cancer patients and by inhibition by self-MHC. In this study, we show that expansion converts healthy donor and immunosuppressed ovarian cancer patient NK cells to a cytotoxic CD56CD16 subset with activation state and antitumor functions that increase with CD56 brightness. We investigated whether these expanded NK cells may overcome the limitations of autologous NK cell therapy against solid tumors. Peripheral blood- and ascites-derived NK cells from ovarian cancer patients were expanded and then adoptively transferred into cell-line and autologous patient-derived xenograft models of human ovarian cancer. Expanded ovarian cancer patient NK cells reduced the burden of established tumors and prolonged survival. These results suggest that CD56 NK cells harbor superior antitumor function compared with CD56 cells. Thus, NK cell expansion may overcome limitations on autologous NK cell therapy by converting the patient's NK cells to a cytotoxic subset that exerts a therapeutic effect against autologous tumor. These findings suggest that the value of expanded autologous NK cell therapy for ovarian cancer and other solid malignancies should be clinically assessed. .

BackgroundAdoptive cell transfer of tumor-specific T lymphocytes (T cells) is proving to be an effective strategy for treating established tumors in cancer patients. One method of generating these cells is accomplished through engineering bulk T cell populations to express chimeric antigen receptors (CARs), which are specific for tumor antigens. Traditionally, these CARs are targeted against tumor antigens using single-chain antibodies (scFv). Here we describe the use of a designed ankyrin repeat protein (DARPin) as the tumor-antigen targeting domain.MethodsWe prepared second generation anti-HER2 CARs that were targeted to the tumor antigen by either a DARPin or scFv. The CARs were engineered into human and murine T cells. We then compared the ability of CARs to trigger cytokine production, degranulation and cytotoxicity.ResultsThe DARPin CARs displayed reduced surface expression relative to scFv CARs in murine cells but both CARs were expressed equally well on human T cells, suggesting that there may be a processing issue with the murine variants. In both the murine and human systems, the DARPin CARs were found to be highly functional, triggering cytokine and cytotoxic responses that were similar to those triggered by the scFv CARs.ConclusionsThese findings demonstrate the utility of DARPins as CAR-targeting agents and open up an avenue for the generation of CARs with novel antigen binding attributes.

Ligands for the NKG2D receptor are overexpressed on tumors, making them interesting immunotherapy targets. To assess the tumoricidal properties of T cells directed to attack NKG2D ligands, we engineered murine T cells with two distinct NKG2D-based chimeric antigen receptors (CARs): (i) a fusion between the NKG2D receptor and the CD3ζ chain and (ii) a conventional second-generation CAR, where the extracellular domain of NKG2D was fused to CD28 and CD3ζ. To enhance the CAR surface expression, we also engineered T cells to coexpress DAP10. In vitro functionality and surface expression levels of all three CARs was greater in BALB/c T cells than C57BL/6 T cells, indicating strain-specific differences. Upon adoptive transfer of NKG2D-CAR-T cells into syngeneic animals, we observed significant clinical toxicity resulting in morbidity and mortality. The severity of these toxicities varied between the CAR configurations and paralleled their in vitro NKG2D surface expression. BALB/c mice were more sensitive to these toxicities than C57BL/6 mice, consistent with the higher in vitro functionality of BALB/c T cells. Treatment with cyclophosphamide prior to adoptive transfer exacerbated the toxicity. We conclude that while NKG2D ligands may be useful targets for immunotherapy, the pursuit of NKG2D-based CAR-T cell therapies should be undertaken with caution.

The use of engineered T cells in adoptive transfer therapies has shown significant promise in treating hematological cancers. However, successes treating solid tumors are much less prevalent. Oncolytic viruses (OVs) have the capacity to induce specific lysis of tumor cells and indirectly impact tumor growth via vascular shutdown. These viruses bear natural abilities to associate with lymphocytes upon systemic administration, but therapeutic doses must be very high in order to evade antibodies and other components of the immune system. As T cells readily circulate through the body, using these cells to deliver OVs directly to tumors may provide an ideal combination. Our studies demonstrate that loading chimeric antigen receptor–engineered T cells with low doses of virus does not impact receptor expression or function in either murine or human T cells. Engineered T cells can deposit virus onto a variety of tumor targets, which can enhance the tumoricidal activity of the combination treatment. This concept appears to be broadly applicable, as we observed similar results using murine or human T cells, loaded with either RNA or DNA viruses. Overall, loading of engineered T cells with OVs represents a novel combination therapy that may increase the efficacy of both treatments.

, the pathogen causing pulmonary tuberculosis (TB) in humans, has evolved to delay Th1 immunity in the lung. Although conventional dendritic cells (cDCs) are known to be critical to the initiation of T cell immunity, the differential roles and molecular mechanisms of migratory CD11b and CD103 cDC subsets in anti- Th1 activation remain unclear. Using a murine model of pulmonary infection, we found that slow arrival of-bearing migratory CD11b and CD103 cDCs at the draining lymph nodes preceded the much-delayed Th1 immunity and protection in the lung. Contrary to their previously described general roles in Th polarization, CD11b cDCs, but not CD103 cDCs, were critically required for Th1 activation in draining lymph nodes following infection. CD103 cDCs counterregulated CD11b cDC-mediated Th1 activation directly by producing the immune-suppressive cytokine IL-10. Thus, our study provides new mechanistic insights into differential Th immune regulation by migratory cDC subsets and helps to develop novel vaccines and therapies.

Despite the remarkable success of chimeric antigen receptor (CAR)-T cells against hematologic malignancies, severe off-tumor effects have constrained their use against solid tumors. Recently, CAR-engineered natural killer (NK) cells have emerged as an effective and safe alternative. Here, we demonstrate that HER2 CAR-expression in NK cells from healthy donors and patients with breast cancer potently enhances their anti-tumor functions against various HER2-expressing cancer cells, regardless of MHC class I expression. Moreover, HER2 CAR-NK cells exert higher cytotoxicity than donor-matched HER2 CAR-T cells against tumor targets. Importantly, unlike CAR-T cells, HER2 CAR-NK cells do not elicit enhanced cytotoxicity or inflammatory cytokine production against non-malignant human lung epithelial cells with basal HER2 expression. Further, HER2 CAR-NK cells maintain high cytotoxic function in the presence of immunosuppressive factors enriched in solid tumors. These results show that CAR-NK cells may be a highly potent and safe source of immunotherapy in the context of solid tumors.

The AMP‐activated protein kinase (AMPK) is a heterotrimeric protein complex that is an important sensor of cellular energy status. Reduced expression of the AMPK β1 isoform has been linked to reduced survival in different cancers, but whether this accelerates tumor progression and the potential mechanism mediating these effects are not known. Furthermore, it is unknown whether AMPK β1 is implicated in tumorigenesis, and if so, what tissues may be most sensitive. In the current study, we find that in the absence of the tumor suppressor p53, germline genetic deletion of AMPK β1 accelerates the appearance of a T‐cell lymphoma that reduces lifespan compared to p53 deficiency alone. This increased tumorigenesis is linked to increases in interleukin‐1β (IL1β), reductions in acetyl‐CoA carboxylase (ACC) phosphorylation, and elevated lipogenesis. Collectively, these data indicate that reductions in the AMPK β1 subunit accelerate the development of T‐cell lymphoma, suggesting that therapies targeting this AMPK subunit or inhibiting lipogenesis may be effective for limiting the proliferation of p53‐mutant tumors.