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:Medulloblastoma (MB) is the most common malignant brain tumor in children. Patients with high-risk features, such as MYCN aberrations, and relapsed/refractory disease have poor clinical outcomes. This underscores the urgent need for new therapies. Glypican-2 (GPC2) is a MYCN-regulated and recently discovered oncofetal antigen. Given that GPC2 is also expressed in brain tumors, we evaluated the preclinical activity of our GPC2-CAR (CT3-CD28HTM-BBζ) against MB and compared it to two existing CARs targeting GD2 and B7-H3. A newly generated patient-derived xenograft (PDX) MAF1433, with MYCN aberrations, were used to test CAR T-cells in vivo. Single-cell RNA-sequencing were used for mechanistic studies. MB patient samples express intermediate to high levels of GPC2 and can be targeted with a GPC2-CAR, leading to significant in vivo tumor regression in orthotopic tumor models. GPC2-CAR T-cells had equivalent activity to the B7-H3-CAR and enhanced activity compared to the GD2-CAR in vivo. T-cell kinetic studies revealed that GPC2-CAR T-cells home to the TME, expand, and upregulate genes critical for cytotoxicity and T-cell homing to induce MB cell death. CT3-CD28HTM-BBζ GPC2-CAR can regress GPC2+ MB with MYCN aberrations in preclinical studies, providing a preclinical rationale for including children with GPC2+ MB in our upcoming clinical GPC2-CAR T-cell trial.
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:Poor tumor trafficking and the immunosuppressive tumor microenvironment (TME) limit chimeric antigen receptor (CAR) T cell efficacy in solid tumors, such as neuroblastoma. We previously optimized GPC2 CARs in human neuroblastoma xenografts leading to clinical translation, however, there have not been preclinical studies using immunocompetent models. Thus, here we generated murine GPC2 CAR T cells using the D3-GPC2-targeting single-chain variable fragment being utilized clinically (NCT05650749) and tested them in neuroblastoma syngeneic allografts. Immune profiling of GPC2 CAR T cell-treated tumors revealed significant reprogramming of the TME, most notably poor intra-tumor CAR T cell persistence being associated with increased recruitment of myeloid-derived suppressor cells (MDSCs), along with MDSC-recruiting CXCL1/2 chemokines. These tumor-infiltrating MDSCs directly inhibited GPC2 CAR T cell activation and proliferation ex vivo. To both capitalize on this chemokine gradient and mitigate MDSC-tumor trafficking, we engineered GPC2 CAR T cells to express the CXCL1/2 receptor, CXCR2. CXCR2-armored GPC2 CAR T cells migrated towards CXCL1/2 gradients, enhanced anti-neuroblastoma efficacy, and reduced the level of MDSCs in the TME. Together, these findings suggest CAR T cell studies in immunocompetent models are imperative to define mechanisms of solid tumor immune escape and rationally design armoring strategies that will lead to durable clinical efficacy.
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.