Preclinical Evaluation of CAR T Cells

All in vivo studies were conducted in compliance with Institutional Animal Care and Use Committee (IACUC)–approved protocols [Memorial Sloan Kettering Cancer Center (MSKCC) 00-05-065 or Juno 15–06]. Six- to 12-week-old NOD.Cg-Prkdc^scidIl2rg^tm1Wjl/SzJ (NSG) mice (The Jackson Laboratory) were injected subcutaneously with RPMI-8226 cells or systemically via tail vein with OPM2 cells (30 ) stably transduced with ffLuc. Injection of D-luciferin substrate (Millipore-Sigma) allowed for longitudinal in vivo BLI. A single dose of human genetically modified CAR T cells was administered at the indicated time points. In some cases, T cells were modified with a bicistronic construct including a CAR and membrane-tethered external Gaussia luciferase (31 (link)), which could be imaged after injection of coelenterazine substrate (NanoLight Technology). BLI was conducted using an IVIS Spectrum, and images were analyzed using Living Image software (PerkinElmer). Survival was graphically represented as Kaplan-Meier curves. The log-rank (Mantel-Cox) test was used to test statistical significance, with P values adjusted for multiple comparisons via Benjamini-Hochberg correction.

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Smith E.L., Harrington K., Staehr M., Masakayan R., Jones J., Long T.J., Ng K.Y., Ghoddusi M., Purdon T.J., Wang X., Do T., Pham M.T., Brown J.M., De Larrea C.F., Olson E., Peguero E., Wang P., Liu H., Xu Y., Garrett-Thomson S.C., Almo S.C., Wendel H.G., Riviere I., Liu C., Sather B, & Brentjens R.J. (2019). GPRC5D is a target for the immunotherapy of multiple myeloma with rationally designed CAR T cells. Science translational medicine, 11(485), eaau7746.

Publication 2019

NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice D-luciferin substrate coelenterazine substrate IVIS Spectrum Living Image software

Cancer Cells Coelenterazine Human Institutional animal care and use committee Luciferase Luciferin Membrane Mice T cells Tail Vein

Corresponding Organization : Memorial Sloan Kettering Cancer Center

Other organizations : Kettering University, Universitat de Barcelona, Eureka Therapeutics (United States), Albert Einstein College of Medicine

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Variable analysis

independent variables

Injection of human genetically modified CAR T cells at indicated time points

dependent variables

Survival of mice as represented by Kaplan-Meier curves
In vivo bioluminescence imaging (BLI) of ffLuc or membrane-tethered external Gaussia luciferase expressing cells

control variables

NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice (6- to 12-week-old)
RPMI-8226 cells injected subcutaneously or OPM2 cells injected systemically via tail vein
Injection of D-luciferin or coelenterazine substrates for BLI

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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