Project description:Analysis of TH17 cells redirected with chimeric antigen receptors (CAR) expressing various signaling domains (including CD28, 4-1BB and ICOS) after surrogate antigen stimulation. Our results showed that T cells redirected with an ICOS-based CAR specifically retained a genotype of TH17 cells with expression of Il17a, Il17f, Il1r1, Ccl20, Rorc, and in the absence of Foxp3 CAR-redirected TH17 cells from three different human normal donors were stimulated with immobilized recombinant mesothelin. Gene expression levels were determined prior to stimulation (day 0) and 4, 8, 24 and 96 hours upon antigen recognition.
Project description:Adoptive cell therapy, a subset of cancer immunotherapy, is collection of therapeutic approaches which aim to redirect the immune system by reprogramming patient T-cells to target antigenic molecules differentially and specifically expressed in certain cancers. One promising immunotherapy technique is CAR T-cell therapy, where cancer cells are targeted through the expression a chimeric antigen receptor (CAR), a synthetic trans- membrane receptor that functionally compensates for the T-cell receptor (TCR) but targets a tumor associated antigen on the cancer cell surface. While CAR T-cell therapy is promising with two clinically approved second-generation CARs (Kymriah and Yescarta), few studies have investigated the mechanism of signal propagation in T-cells and no studies have investigated the potential signaling response in the target cells. To gain further insight to CAR-based signaling, we stimulated third generation CD19 CAR-expressing Jurkat T-cells by co-culture with SILAC labeled CD19HI Raji B-cells and used two phosphoenrichment strategies coupled with liquid chromatography-tandem mass spec- trometry (LC-MS/MS) to detect and analyze global phosphorylation changes in both cell populations. Analysis of the phosphopeptides originating from the CD19-CAR T cells revealed an increase in many phosphorylation events necessary for canonical TCR signaling. We also observed for the first time a significant decrease in B-cell receptor- related phosphopeptide abundance in CD19HI Raji B-cells after co-culture with CD19-targetted CAR T-cells.
Project description:Chimeric antigen receptor (CAR)-T cell therapy is a promising therapeutic approach for relapsed or refractory acute myeloid leukemia (AML). However, optimal target antigens have yet to be established. A subset of AML cells express TNF, which is initially expressed on the cell surface and subsequently secreted by proteolytic cleavage of the extracellular portion. We generated CAR-T cells targeting the N-terminal fragment of TNF (TNF-NTF) that remains on the cell surface after shedding. We ablated TNF gene to avoid fratricide and coexpressed chimeric cytokine receptor (G6/7R) that we had previously developed in CAR-T cells to provide constitutive IL-7 signaling. The G6/7R-expressing TNF-TNF CAR-T cells exhibited potent and durable therapeutic efficacy in vivo. Our data suggest that TNF-TNF may be a promising target antigen for CAR-T cells applicable to AML.
Project description:Chimeric antigen receptor (CAR) T-cells induce responses in patients with relapsed/refractory leukemia; however, long-term efficacy is frequently limited by post-CAR relapses. The inability to target antigen-low cells is an intrinsic vulnerability of second-generation CAR T-cells and underlies the majority of relapses following CD22BBz CAR T-cell therapy. We interrogated CD22BBz CAR signaling in response to low antigen and found inefficient phosphorylation of LAT, limiting downstream signaling. To overcome this, we designed the Adjunctive LAT-Activating CAR T-cell (ALA-CART) platform, pairing a second-generation CAR with a LAT-CAR incorporating the intracellular domain of LAT. ALA-CART cells demonstrated reduced differentiation during manufacturing and increased LAT phosphorylation, MAPK signaling and AP-1 activity. Consequently, ALA-CART cells showed improved cytotoxicity, proliferation, persistence and efficacy against antigen-low leukemias that were refractory to clinically-active CD22BBz CAR T-cells. These data suggest restoration of LAT signaling through the ALA-CART platform represents a promising strategy for overcoming multiple mechanisms of CAR T-cell failure.
Project description:The main purpose of this research is to verify the safety of CEA targeted chimeric antigen receptor T cells and to determine the proper dosage of CAR T cells infused.
Project description:The main purpose of this research is to verify the safety of CEA targeted chimeric antigen receptor T cells and to determine the proper dosage of CAR T cells infused.
Project description:This is a single arm, open-label, dose escalation clinical study to evaluate the safety and tolerability of autologous mesothelin (MSLN)-targeted chimeric antigen receptor (MSLN-CAR) T cells secreting PD-1 nanobodies (αPD1-MSLN-CAR T cells) in patients with solid tumors.
Project description:This is a single arm, open-label, dose escalation clinical study to evaluate the safety and tolerability of autologous mesothelin (MSLN)-targeted chimeric antigen receptor (MSLN-CAR) T cells secreting PD-1 nanobodies (αPD1-MSLN-CAR T cells) in patients with solid tumors.
