Project description:Chimeric antigen receptor (CAR) T cells mediate durable complete responses in patients with certain hematologic malignancies, but antigen downregulation is a common mechanism of resistance. While the native TCR can respond to very low levels of peptide presented in MHC, engineered CARs are incapable of responding to antigen-low targets, likely due to a disorganized immune synapse and poor recruitment of proximal signaling molecules. We developed a platform that endows CARs with the ability to kill antigen-low cancer cells, consisting of a membrane tethered version of the signaling molecule SLP-76 (MT-SLP-76). MT-SLP-76 can be expressed alongside any CAR to lower its threshold for activation and overcomes antigen low escape in multiple xenograft models. While SLP-76 is natively in the cytosol, only the engineered membrane tethered version can adequately amplify CAR signaling, a process that is mediated through recruitment of ITK and PLC1. MT-SLP-76 was designed based on biologic principles to render CAR T cell therapies less susceptible to antigen downregulation and is poised for clinical development to overcome this common mechanism of resistance.
Project description:While CAR T cells have altered the treatment landscape for B cell malignancies, the risk of on-target, off-tumor toxicity has hampered their development for solid tumors because most target antigens are shared with normal cells. Researchers have attempted to apply Boolean logic gating to CAR T cells to prevent on-target, off-tumor toxicity; however, a truly safe and effective logic-gated CAR has remained elusive. Here, we describe a novel approach to CAR engineering in which we replace traditional ITAM-containing CD3ζ domains with intracellular proximal T cell signaling molecules. We demonstrate that certain proximal signaling CARs, such as a ZAP-70 CAR, can activate T cells and eradicate tumors in vivo while bypassing upstream signaling proteins such as CD3ζ. The primary role of ZAP-70 is to phosphorylate LAT and SLP-76, which form a scaffold for the propagation of T cell signaling. We leveraged the cooperative role of LAT and SLP-76 to engineer Logic-gated Intracellular NetworK (LINK) CAR, a rapid and reversible Boolean-logic AND-gated CAR T cell platform that outperforms other systems in both efficacy and the prevention of on-target, off-tumor toxicity. LINK CAR will dramatically expand the number and types of molecules that can be targeted with CAR T cells, enabling the deployment of these powerful therapeutics for solid tumors and diverse diseases such as autoimmunity9 and fibrosis10. In addition, this work demonstrates that the internal signaling machinery of cells can be repurposed into surface receptors, a finding that could have broad implications for new avenues of cellular engineering.
Project description:Regulatable CAR platforms could circumvent toxicities associated with CAR-T therapy, but existing systems have shortcomings including leakiness and attenuated activity. Here, we present SNIP-CARs, a novel protease-based platform for regulating CAR activity using an FDA-approved small molecule. Design iterations yielded CAR-T cells that manifest full functional capacity with drug and no leaky activity in the absence of drug. In numerous models, SNIP CAR-T cells were more potent than constitutive CAR-T cells and showed diminished T cell exhaustion and greater stemness. In a ROR1-based CAR lethality model, drug cessation following toxicity onset reversed toxicity, thereby credentialling the platform as a safety switch. In the same model, reduced drug dosing opened a therapeutic window that resulted in tumor eradication in the absence of toxicity. SNIP-CARs enable remote tuning of CAR activity, which provides solutions to safety and efficacy barriers currently limiting progress in using CAR T cells to treat solid tumors.
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:To characterize transfer of molecules from target cells into CAR T cells via trogocytosis we cultured NALM-6 leukemia cell line expressing a CD19-mCherry fusion protein with CAR T cells. NALM6-CD19-mCherry were loaded with heavy amino acid and cocultured with CAR T cells for 1 hour. CAR T cells were next sorted into two fractions, mCherry-positive (TrogPos), and -negative (TrogNeg). Proteomics analysis revealed the presence of targeted antigen (CD19) in the TrogPos only.
Project description:Chimeric antigen receptor (CAR) T cell therapy is a promising immunotherapy against cancer. Although there is a growing interest in other cell types, a comparison of CAR immune effector cells in challenging solid tumor contexts is lacking. Here, we compare mouse and human NKG2D-CAR expressing T cells, NK cells and macrophages against glioblastoma, the most aggressive primary brain tumor. In vitro we show that T cell cancer killing is CAR-dependent, whereas intrinsic cytotoxicity overrules CAR-dependence for NK cells and CAR macrophages reduce glioma cells in co-culture assays. In orthotopic immunocompetent glioma mouse models, systemically administered CAR T cells demonstrate superior accumulation in the tumor and each immune cell type induces distinct changes in the tumor microenvironment. An otherwise low therapeutic efficacy is significantly enhanced by co-expression of pro-inflammatory cytokines in all CAR immune effector cells, underscoring the necessity for multifaceted cell engineering strategies to overcome the immunosuppressive solid tumor microenvironment.
Project description:Immunotherapies with CAR-T cells achieve remarkable success, especially against B-cell malignancies. However, their activity against non-hematopoietic solid tumors is very limited. One central cause of this failure may be that a pro-oxidative tumor microenvironment can downmodulate the activation and migration of T-cells. In contrast to tumor cells, primary T cells have very low levels of antioxidants. This makes them prone to pro-oxidative effects. Oxidation-induced dysregulation of actin cytoskeletal dynamics hinders T-cell migration which allows only low tumor infiltration rates. In addition, the proliferation and the effector functions of T-cells (tumor cell killing) are disrupted. In order to strengthen human CAR-T cells against a pro-oxidative tumor microenvironment, we have increased increase their antioxidant capacity, e.g. by expressing antioxidants. We aimed to characterize the importance of antioxidant empowerment at functional level and at the molecular level. Among the antioxidant systems, thioredoxin1 (TRX1)-empowerment, allowed CAR T cells to retain their capacities for cytolytic immune synapse formation, cytokine release, proliferation, and cytotoxicity under pro-oxidative conditions. At the molecular level, gene expression analysis revealed that a pro-oxidative micromilieu caused a downmodulation of costimulatory and cytokine signals of T cells. One unique focus of this study was also to investigate global influence of reactive oxygen species on protein oxidation in T cells. Therefore, using TRX1 kinetic trapping and consequently mass spectrometry was employed to get insight into global oxidation levels in T cells. This analysis, namely redoxosome analysis, unveiled 196 oxidized proteins which were annotated to regulate several antitumor T cell functions. Taken together, our results provide evidence that TRX1 empowerment can increase CAR T cell efficacy against solid tumors.
Project description:Chimeric antigen receptor (CAR)-T cell therapies have shown great success in treating hematologic malignancies. Nonetheless, their therapeutic effect on solid tumors remains to be improved. Recently, macrophages have attracted great attention, given their ability to infiltrate solid tumors, phagocytize tumor cells as well as their immunomodulatory capacities. The first generation of CD3ζ-based CAR-macrophages demonstrated that the CAR could stimulate macrophage phagocytosis in a tumor antigen-dependent way. Here, we genetically engineered induced pluripotent stem cell (iPSC)-derived macrophages (iMACs) with TLR4 intracellular TIR domain-containing CARs against EGFRvIII and GPC3, which yielded markedly enhanced antitumor effect in two different solid tumor models including glioblastoma, and hepatocellular carcinoma in which complete remission was achieved with CAR-iMACs alone or in combination with CD47 antibody. Moreover, the tandem CD3ζ-TIR-CAR, or the “second-generation” design of TIR-based dual signaling CAR, endowed iMACs with both target engulfment/efferocytosis capacity against antigen-expressing solid tumor cells, and potency of antigen-dependent M1 state polarization and M2 state resistance in an NF-κB dependent manner. We also illustrated a surprising mechanism of tumor cell elimination by CAR-induced efferocytosis against tumor cell apoptotic bodies. Taken together, we established the next generation CAR-iMACs equipped with orthogonal phagocytosis and polarization capacity for better antitumor functions in treating solid tumors.
Project description:To investigate the activation of CAR-macrophages by the antigen CEA, we treated CAR-macrophages with CEA and detected them by RNA-Seq.