Project description:Single-cell RNA-seq analysis of bioengineered human bone marrow leukemia chip established with human B-ALL cells (REH, ATCC), Human umbilical vein cells (HUVECs, Lonza), Human mesenchymal stem cells (hMSC, Lonza), Human Bone marrow mononuclear cells (STEMCELL Technologies), and Human MSC osteoblasts (Lonza) using the reported Leukemia-on-a-Chip method treated with Healthy donor derived CAR Tcells, Patient derived CAR T cells, Mock T cells, and or left non-treated.
Project description:Background: Although most patients with newly diagnosed high-risk neuroblastoma (NB) achieve remission after initial therapy, more than 50% experience late relapses caused by minimal residual disease (MRD) and succumb to their cancer. Therapy strategies to target MRD may benefit these children. We developed a new chimeric antigen receptor (CAR) targeting glypican (GPC)2 and conducted iterative preclinical engineering of the CAR structure to maximize its anti-tumor efficacy before clinical translation. Methods: We evaluated different GPC2-CAR constructs by measuring the CAR activity against several NB cell lines in vitro. NOD-SCID mice engrafted with human NB cell lines or orthotopic patient-derived xenograft (PDX) and treated with human CAR T cells served as in vivo models. Mechanistic studies were performed using single-cell RNA-sequencing. Results: Applying stringent in vitro assays and orthotopic in vivo NB models, we demonstrated that our single-chain variable fragment, CT3, integrated into a CAR backbone with a CD28 hinge, CD28 transmembrane, and 4-1BB co-stimulatory domain elicits the best preclinical anti-NB activity compared to other tested CAR constructs. This enhanced activity was associated with an enrichment of CD8+ effector T cells in the tumor-microenvironment and upregulation of several effector molecules such as GNLY, GZMB, ZNF683, and HMGN2. Finally, we also showed that the CT3.28H.BBζ CAR was more potent than a recently clinically tested GD2-targeted CAR to control NB in vivo. Conclusion: Given the robust preclinical activity of CT3.28H.BBζ, these promising results warrant further clinical testing in children with NB.
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:Spatial transcriptomic profiling of two replicates of Patient and two replicates of Patient-corrected bioengineered vascular grafts harvested at week 8 post-implantation.
Project description:Allogeneic chimeric antigen receptor (CAR)-T cell therapies hold the potential to overcome many of the challenges associated with patient-derived (autologous) CAR-T cells. Key considerations in the development of allogeneic CAR-T cell therapies include prevention of GvHD and suppression of allograft rejection. Here we describe preclinical data supporting the ongoing first-in-human clinical trial (CaMMouflage) in relapsed/refractory multiple myeloma patients evaluating CB-011, a hypoimmunogenic, allogeneic anti–B cell maturation antigen (BCMA) CAR-T cell therapy candidate. CB-011 cells feature 4 genomic alterations and were engineered from healthy donor-derived T cells using a Cas12a CRISPR hybrid RNA-DNA (chRDNA) genome-editing technology platform. To address allograft rejection, CAR-T cells were engineered to prevent endogenous human leukocyte antigen (HLA) class I complex expression and overexpress a single-chain polyprotein complex composed of beta-2 microglobulin (B2M) tethered to HLA-E. Additionally, T cell receptor expression was disrupted at the T cell receptor alpha constant locus in combination with the site-specific insertion of a humanized BCMA-specific CAR. CB-011 cells exhibited robust plasmablast cytotoxicity in vitro in a mixed lymphocyte reaction in cell co-cultures derived from patients with multiple myeloma. Additionally, CB-011 cells demonstrated suppressed recognition by and cytotoxicity from HLA-mismatched T cells. CB-011 cells were protected from natural killer (NK) cell–mediated cytotoxicity in vitro and in vivo due to endogenous promoter-driven expression of B2M–HLA-E. Potent antitumor efficacy, when combined with an immune-cloaking armoring strategy to dampen allograft rejection, offers optimized therapeutic potential in multiple myeloma.
Project description:This is a preclinical study investigating the role of CREM transcription factor in CAR NK cells. We found thata CREM is a regulatory checkpoint induced by CAR CD3z signaling and IL-15 stimulation that leads to suppression of NK cell function. CREM KO enhances the anti-tumor efficacy of CAR NK cells.