The chimeric antigen receptor (CAR) T-cell therapy has been effective for patients with CD19 B-cell malignancies. Most studies have investigated the second-generation CARs with either CD28 or 4-1BB costimulatory domains in the CAR receptor. Here, we describe the first clinical phase I/IIa trial using third-generation CAR T cells targeting CD19 to evaluate safety and efficacy. Fifteen patients with B-cell lymphoma or leukemia were treated with CAR T cells. The patients with lymphoma received chemotherapy during CAR manufacture and 11 of 15 were given low-dose cyclophosphamide and fludarabine conditioning prior to CAR infusion. Peripheral blood was sampled before and at multiple time points after CAR infusion to evaluate the persistence of CAR T cells and for immune profiling, using quantitative PCR, flow cytometry, and a proteomic array. Treatment with third-generation CAR T cells was generally safe with 4 patients requiring hospitalization due to adverse reactions. Six of the 15 patients had initial complete responses [4/11 lymphoma and 2/4 acute lymphoblastic leukemia (ALL)], and 3 of the patients with lymphoma were in remission at 3 months. Two patients are still alive. Best predictor of response was a good immune status prior to CAR infusion with high IL12, DC-Lamp, Fas ligand, and TRAIL. Responding patients had low monocytic myeloid-derived suppressor cells (MDSCs; CD14CD33HLADR) and low levels of IL6, IL8, NAP3, sPDL1, and sPDL2. Third-generation CARs may be efficient in patients with advanced B-cell lymphoproliferative malignancy with only modest toxicity. Immune profiling pre- and posttreatment can be used to find response biomarkers.

BackgroundCancer immunotherapy can be potentiated by conditioning regimens such as cyclophosphamide, which reduces the level of regulatory T cells (tregs). However, myeloid suppressive cells are still remaining. Accordingly to previous reports, gemcitabine improves immune status of cancer patients. In this study, the role of gemcitabine was further explored to map its immunological target cells and molecules in patients with pancreatic cancer.MethodsPatient blood was investigated by flow cytometry and cytokine arrays at different time points during gemcitabine treatment.ResultsThe patients had elevated myeloid-derived suppressor cells (MDSCs), and Tregs at diagnosis. Myeloid cells were in general decreased by gemcitabine. The granulocytic MDSCs were significantly reduced while monocytic MDSCs were not affected. In vitro, monocytes responding to IL-6 by STAT3 phosphorylation were prevented to respond in gemcitabine medium. However, gemcitabine could not prevent STAT3 phosphorylation in IL-6-treated tumor cell lines. TGFβ-1 was significantly reduced after only one treatment and continued to decrease. At the same time, the effector T cell:Treg ratio was increased and the effector T cells had full proliferative capacity during the gemcitabine cycle. However, after a resting period, the level of suppressor cells and TGFβ-1 had been restored showing the importance of continuous conditioning.ConclusionsGemcitabine regulates the immune system in patients with pancreatic cancer including MDSCs, Tregs and molecules such as TGFβ-1 but does not hamper the ability of effector lymphocytes to expand to stimuli. Hence, it may be of high interest to use gemcitabine as a conditioning strategy together with immunotherapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-016-1037-z) contains supplementary material, which is available to authorized users.

Pancreatic cancer is a severe indication with short expected survival despite surgery and/or combination chemotherapeutics. Checkpoint blockade antibodies are approved for several cancer indications, but pancreatic cancer has remained refractory. However, there are clinical data suggesting that stimulation of the CD40 pathway may be of interest for these patients. Oncolytic viruses armed with immunostimulatory genes represent an interesting approach. Herein, we present LOAd703, a designed adenovirus armed with trimerized and that activates the CD40 and 4-1BB pathways, respectively. As many cells in the tumor stroma, including stellate cells and the infiltrating immune cells, express CD40 and some 4-1BB, we hypothesize that LOAd703 activates immunity and simultaneously modulates the biology of the tumor stroma. Tumor, stellate, endothelial, and immune cells were infected by LOAd703 and investigated by flow cytometry, proteomics, and functional analyses. LOAd703-infected pancreatic cell lines were killed by oncolysis, and the virus was more effective than standard-of-care gemcitabine. In xenograft models, LOAd703 efficiently reduced established tumors and could be combined with gemcitabine for additional effect. Infected stellate and tumor cells reduced factors that promote tumor growth (Spp-1, Gal-3, HGF, TGFβ and collagen type I), while chemokines were increased. Molecules involved in lymphocyte migration were upregulated on infected endothelial cells. Dendritic cells were robustly stimulated by LOAd703 to produce costimulators, cytokines and chemokines, and such DCs potently expanded both antigen-specific T cells and NK cells. LOAd703 is a potent immune activator that modulates the stroma to support antitumor responses. .