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Sleeping Beauty (SB) transposon, a type of nonviral integrative vectors, provides an alternative to modify primary T cells. Creative biolabs has developed SB transposon CAR vector pSBCAR1 CD20 (Rituximab) h(28ζ), which is constructed for the engineering of T cells to target human CD20. The T cells are genetically modified through transduction with a nonviral vector expressing scFv of anti-CD20 antibody linked to CD28 and CD3ζ signaling domains. And the vector product was designed for the treatment of Acute lymphoblastic leukemia (ALL).
CAR Construction : Rituximab-CD28-CD3ζ Fig.1 Tumor progression after the CD20-targeting CAR-T cells treatment. NSG mice were injected intravenously (i.v.) with 0.5 × 10^6 firefly-luciferase-expressing Raji cells 6 days prior to treatment with 5 × 10^6 CD8+ CD20-targeting CAR-T cells delivered i.v. Tumor progression was monitored by bioluminescence imaging. Chen, X., Khericha, M., Lakhani, A., Meng, X., Salvestrini, E., Chen, L. C., ... & Chen, Y. Y. (2020). Rational tuning of CAR tonic signaling yields superior T-cell therapy for cancer. bioRxiv. |
CAR Construction : Rituximab-CD28-CD3ζ Fig.2 Evualution of tumor activation and exhaustion. Activation and exhaustion maker expression on CAR+ T cells were evaluated 11 days post Dynabead removal, without CD20 antigen stimulation. Chen, X., Khericha, M., Lakhani, A., Meng, X., Salvestrini, E., Chen, L. C., ... & Chen, Y. Y. (2020). Rational tuning of CAR tonic signaling yields superior T-cell therapy for cancer. bioRxiv. |
CAR Construction : Rituximab-CD28-CD3ζ Fig.3 T-cell proliferation assay A 4-day T-cell proliferation assay on CAR+T cells with CellTrace Violet (CTV) dye in the absence or presence of target cells (on-target, CD19+/CD20+K562 cells; off-target, parental K562 cells) at 2:1 effector-to-target (E:T) ratio. Chen, X., Khericha, M., Lakhani, A., Meng, X., Salvestrini, E., Chen, L. C., ... & Chen, Y. Y. (2020). Rational tuning of CAR tonic signaling yields superior T-cell therapy for cancer. bioRxiv. |
CAR Construction : Fig.4 Binding of rituximab to mutant CD20 variants on intact cells. Point-mutated CD20 variants were transiently transfected in Freestyle 293-F cells. By gating on live cells with high transfection efficiency, FACS-binding curves were determined for the indicated antibodies. Niederfellner, G., Lammens, A., Mundigl, O., Georges, G. J., Schaefer, W., Schwaiger, M., ... & Klein, C. (2011). Epitope characterization and crystal structure of GA101 provide insights into the molecular basis for type I/II distinction of CD20 antibodies. Blood, The Journal of the American Society of Hematology, 118(2), 358-367. |
CAR Construction : Fig.5 Rituximab binding to puried CD20. Size exclusion chromatography of CD20/ rituximab complex Top: Gel fltration chromatograms of CD20/ rituximab assembly and rituximab alone are shown in orange and red, respectively; Bottom: SDS-PAGE and stain free detection was used to analyze the gel fltration fractions. Agez, M., Mandon, E. D., Iwema, T., Gianotti, R., Limani, F., Herter, S., ... & Jawhari, A. (2019). Biochemical and biophysical characterization of purified native CD20 alone and in complex with rituximab and obinutuzumab. Scientific reports, 9(1), 1-13. |
CAR Construction : Fig.6 CD20/antibodies binding by SPR. Sensorgram overlays showing the binding of anti-CD20 rituximab (blue), obinutuzumab (red) obinutuzumab wt (green) to purified CD20 captured by anti-His antibody. Agez, M., Mandon, E. D., Iwema, T., Gianotti, R., Limani, F., Herter, S., ... & Jawhari, A. (2019). Biochemical and biophysical characterization of purified native CD20 alone and in complex with rituximab and obinutuzumab. Scientific reports, 9(1), 1-13. |
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