Pre-clinical assessment of chimeric antigen receptor t cell therapy targeting CD19+ B cell malignancy

Abstract

Background: Autologous chimeric antigen receptor (CAR) T cell therapy is a promising therapeutic strategy for treating hematologic malignancies. A spectrum of serious complications caused by CAR-T cells has caught great attention. We developed a novel CAR against CD19 namely UWC19, consisting anti-CD19 single-chain variable fragment (scFv) hinged with 4-1BB and CD3z signaling domains. In this study, preclinical assessments of UWC19 were conducted to evaluate the safety and efficacy in vitro and in vivo. Methods: To evaluate the binding activity of UWC19 cells to CD19, we measured the saturation degree of CAR with human CD19 molecules using flow cytometry in vitro. The antitumor efficacy of UWC19 cells was determined by in vitro cytotoxicity assay against CD19 positive cells and in vivo using a xenograft mouse model. Cross tissue reactivity of UWC19 cells was examined by co-culturing with cell lines from difference human tissues. Tumorigenicity was determined by subcutaneously injecting UWC19 in immunodeficient mice. Persistence was analyzed using quantitative PCR. Results: We showed that UWC19 CAR T cells exerted highly specific binding affinity and cytotoxicity against CD19+ cells in vitro. In vivo, UWC19 CAR T cells are able to fully control disease progression in a Raji-xenografted immunodeficient mouse model. UWC19 exerted no obvious effects on the mean body mass and graft versus host disease were observed in surviving mice. We showed that UWC19 cells specifically recognized and eliminated CD19 positive cells, whereas CD19 negative cells were much less affected. No tumorigenicity of UWC19 in immunodeficient mice was observed. Conclusions: UWC19 treatment effectively eliminated CD19 positive tumor cells with favorable toxicity profile. The findings suggest encouraging clinical prospects for its use in patients with CD19 positive B cell malignancies. Our study presented an alternative evaluation strategy for CAR-T cell products.

Comparison of the efficacy of second and third generation lentiviral vector transduced CAR CD19 T cells for use in the treatment of acute lymphoblastic leukemia both in vitro and in vivo models

“…The amounts of CAR-CD19 T cells were chosen based on the clinical doses tested in the clinical trial for treating patients with ALL. This study is consistent with the previous findings that showed CARs increased the potency of the engineered T cells in a pre-clinical setting [ 36 – 40 ]. Nevertheless, further pre-clinical testing is required, including experiments in NOD SCID gamma mouse (NSG) animals, to ensure the dose and the in vivo activity of modified CAR T cells.…”

“…Therefore, a single dose of tumor cell line (Raji LUC/GFP) was injected into immunodeficient mice (C.B.17/Icr-scid/scidJcl) to create a model as close to the human disease as possible. The dose of the Raji cancer cell line (500,000 cells/mouse) was based on previously published data [ 36 ]. Administration of both the Raji cell line and CAR-CD19 T cells did not affect the body weight of the treated mice throughout the study period.…”

Homology mediated end joining enables efficient non-viral targeted integration of large DNA templates in primary human T cells

“…T cells transfected with HMEJ template, gRNAs and Cas9 were more efficiently engineered (52.4% ±5.16) than those transfected with HR template, gRNA and Cas9 (42.8% ±4. 43), but at a cost of a slightly lower viability (79.1% ±2.07 vs 73.1% ±4.25, respectively) ( Figure 2C&D ). We also compared HMEJ mediated integration to another previously reported HR-based integration strategy that uses a linear double stranded DNA template generated through PCR 12,14 .…”

“…Specific pathogen-free female NODscid IL2Rgamma null (NSG) mice were purchased from Jackson Labs. Tumor challenge studies were performed using the Raji-luc cell line, a well-defined model of Burkitt Lymphoma used for testing CAR-T function 42,43 . Specifically, mice were implanted with 1x10 5 Raji-luc cells resuspended in 50% Matrigel (Corning) through a 100 µL intraperitoneal (IP) injection.…”

CAR T-Cells for CNS Lymphoma: Driving into New Terrain?

“…Anti-tumor effects of CD19-directed CAR T-cells against PCNSL have not only been demonstrated in vitro, but also in murine in vivo models [16,17]. Mulazzani et al designed an orthotopic PCNSL model by combining a chronic cranial window with two-photon intravital microscopy, allowing the repetitive visualization of brain tumor growth [16].…”