Establishment of a simple and efficient platform for car-t cell generation and expansion: from lentiviral production to in vivo studies

ABSTRACT Introduction: Adoptive transfer of T cells expressing a CD19-specific chimeric antigen receptor (CAR) has shown impressive response rates for the treatment of CD19 + B-cell malignancies in numerous clinical trials. The CAR molecule, which recognizes cell-surface tumor-associated antigen independently of human leukocyte antigen (HLA), is composed by one or more signaling molecules to activate genetically modified T cells for killing, proliferation, and cytokine production. Objectives: In order to make this treatment available for a larger number of patients, we developed a simple and efficient platform to generate and expand CAR-T cells. Methods: Our approach is based on a lentiviral vector composed by a second-generation CAR that signals through a 41BB and CD3-ζ endodomain. Conclusions: In this work, we show a high-level production of the lentiviral vector, which was successfully used to generate CAR-T cells. The CAR-T cells produced were highly cytotoxic and specific against CD19+ cells in vitro and in vivo, being able to fully control disease progression in a xenograft B-cell lymphoma mouse model. Our work demonstrates the feasibility of producing CAR-T cells in an academic context and can serve as a paradigm for similar institutions. Nevertheless, the results presented may contribute favoring the translation of the research to the clinical practice.

Humoral immune response in yersinia enterocolitica 0: 3 - infected balb/c mice

Balb/c mice were experimentally infected by oral or intravenous route with Yersinia enterocolitica 0:3 for evaluation of their humaral immune response. Agroup of normal mice was used as control. Five mice from each group were bled by heart puncture and their spleens were removed on the 3rd, 7th, 14th, 21st and 28th day after infection. Immunoglobulin-secreting spleen cells were detected by the isotype-specific protein A plaque assay. The presence of specific anti-Yersinia antibodies was determined in mouse serum by ELISA. In the group infected by the intragastric route there was a reduction of immunoglobuling-secreting spleen cells compared to control during the first week after infection; the animals did not develop anti-Yersinia antibodies. In the group infected by the intravenous route there was ans increase in the cells secreting immunoglobulins of all isotypes, with the following peaks of maximum activation: IgM on the 3rd day, IgG2a and IgG3 on the 7th day and IgA, IgG1 and IgG2b on the 14th day after infection. Specific antibodies of the IgG and IgM isotype were detected in the sera of these animals. Previous Balb/c mouse infection with Y. enterocolitica by the oral route did not change the patterns of humoral response to sheep red blood cells by these animals. We conclude that the immune humoral response differs between the animals inoculated intravenously or orally, with a short-term immunosupression being observed only in the latter group.