Research ArticleIMMUNOLOGY

Imaging CAR T cell therapy with PSMA-targeted positron emission tomography

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Science Advances  03 Jul 2019:
Vol. 5, no. 7, eaaw5096
DOI: 10.1126/sciadv.aaw5096
  • Fig. 1 Engineered expression of PSMA variants on CD19 CAR T cells.

    (A) CAR construct illustration demonstrating the CD19-targeting domain, costimulatory domain, and the location of the PSMA expression tag. Three different molecular constructs of PSMA were produced: WT, W2G, and N9del. VH, heavy chain variable domain; VL, light chain variable domain; GST, glutathione S-transferase; IgG4, immunoglobulin G4. (B) CD19-EGFRt or CD19-PSMA CAR T cells were generated, and flow cytometry was performed to identify CAR+ and PSMA+ CD4 or CD8 T cells. Gating was based on the mock transduction control. (C) Surface expression of PSMA was assessed by flow cytometry. Geometric mean fluorescent intensity (gMFI) is reported after gating on PSMA+ cells. (D) Mock, CD19-EGFRt, or CD19-PSMA CAR T cells were incubated with K562-CD19-NLR target cells at a ratio of 4:1. Target cells were analyzed every 2 hours using an IncuCyte live cell imaging system. TM, transmembrane; NLR, NucLight Red.

  • Fig. 2 CD19 CAR T cells expressing tPSMA maintain function in vitro and in vivo.

    (A) Mock or CD19-tPSMA CAR T cells were incubated with K562-CD19-NLR target cells at a ratio of 4:1. Target cells were analyzed every 2 hours using an IncuCyte live cell imaging system. (B) A kill index [1/area under the curve (AUC)] was calculated at the indicated effector:target ratio. (C and D) Nalm6-GFP-fLuc tumor cells were injected intravenously into female NOD scid (nonobese diabetic severe combined immunodeficient) gamma (NSG) mice (n = 8 per group) on day 0, and 1 × 106 mock or CAR T cells were injected on day 4. Whole-body bioluminescence imaging (BLI) was performed to determine tumor burden, with data displayed as average radiance for each group (C), and survival events were recorded (D).

  • Fig. 3 PET detects CD19-tPSMA CAR T cells with high sensitivity in vitro and in vivo.

    (A) Standard curve demonstrating a linear relationship between the PET signal and the number of CD19-tPSMA(N9Del) CAR T cells. Inset: In vitro phantom from which the standard curve was derived. The in vitro phantom used varying numbers of CD19-tPSMA(N9del) CAR T cells incubated with 37 MBq of [18F]DCFPyL, a high affinity, positron-emitting ligand targeting PSMA, in a 384-well plate. Plates were scanned using the SuperArgus small-animal PET/CT device at 1 hour after beginning the incubation. There were three rows and eight columns of wells presented in a checkerboard pattern: (top row) 40,000, 20,000, 10,000, and 8000 cells; (middle row) 6000, 4000, 2000, and 1000 cells; and (bottom row) 800, 600, 400, and 200 cells. Detection limit was at or near 2000 cells. Images from one of eight separate plates are shown. Error bars, SD; n = 8. (B) Representative images of NSG mice injected with the indicated number (K = 1000; M = 1 × 106) of CD19-tPSMA(N9del) CAR T cells in 50 μl (50% Matrigel) in the shoulders (white arrows); n = 5. Mice were imaged on the SuperArgus small-animal PET/CT at 1 hour after injection of 14.8 MBq of [18F]DCFPyL. PET data are expressed in percentage of injected dose per cubic centimeter of tissue imaged (%ID/cc). To improve the display contrast of the in vivo images, relatively high renal radiotracer uptake was masked using a thresholding method.

  • Fig. 4 PSMA PET/CT enables visualization of CD19-tPSMA(N9del) CAR T cell infiltration into local and metastatic tumors.

    Tumors were derived from Nalm6-eGFP-fLuc cells. (A and B) Mice were infused with 2 × 106 CD19-tPSMA(N9del) CAR T cells; n = 5. (C) Untreated (left mouse) and treated (right mouse) with infusion of 2 × 106 mock T cells. Mice were imaged on the SuperArgus small-animal PET/CT at 1 hour after injection of 14.8 MBq of [18F]DCFPyL and were evaluated at various times (in days) after infusion of the CAR T cells, as indicated. Images alternate between fLuc-tagged bioluminescence (BLI, radiance) for visualization of tumor cells and PET/CT for T cells, with each mouse undergoing both imaging studies; n = 2. (D) Tumor was dissected and stained with anti-PSMA antibody for CD19-tPSMA(N9del) CAR T cells and anti–enhanced green fluorescent protein (eGFP) antibody for tumor cells. CAR T cells infiltrated into the center of the tumor (magnification boxes). Regions where CD19-tPSMA(N9del) CAR T cells infiltrated stained negative with anti-eGFP antibody, indicating tumor cell death. This is a representative example of n = 8. PET data are expressed in percentage of injected dose per cubic centimeter of tissue imaged (%ID/cc). To improve the display contrast of the in vivo images, the relatively high renal radiotracer uptake was masked using a thresholding method. Arrows demonstrate tumor, where indicated. Images are scaled to the same maximum value within each modality.

  • Fig. 5 CD19-tPSMA(N9del) CAR T cell numbers in mouse and human.

    Values were obtained from tumor biopsies, with the human samples derived from the TRANSCEND NHL-001 trial. TRANSCEND uses the same CAR construct but an EGFRt rather than tPSMA tag. Note that CD19-tPSMA(N9del) CAR T cell numbers in mouse versus human biopsy samples are on the same order of magnitude.

  • Fig. 6 The number of CD19-tPSMA(N9del) cells in the peripheral blood and the bone marrow does not correlate with the total number of the CD19-tPSMA(N9del) cells localized to the tumors.

    (A) PET/CT and BLI images of five mice. Days are marked from the day of infusion of CD19-tPSMA(N9del) CAR T cells. Mice were imaged on the SuperArgus small-animal PET/CT at 1 hour after injection of 14.8 MBq of [18F]DCFPyL and were scanned at various times after injection, as indicated. Images alternate between fLuc-tagged bioluminescence (BLI, radiance) for visualization of tumor cells and PET/CT for CAR T cells, with each mouse undergoing both imaging studies. PET data are expressed in percentage of injected dose per cubic centimeter of tissue imaged (%ID/cc), with arrows designating accumulation of CAR T cells. To improve the display contrast of the in vivo images, the relatively high renal radiotracer uptake was masked using a thresholding method. Images are scaled to the same maximum value within each modality. (B) Quantified numbers of the CD19-tPSMA(N9del) cells in the region of interest drawn to cover the entire tumor area were plotted with the percentage number of PSMA+/CAR+ cell populations in the peripheral blood (PPB) and the bone marrow (BM). Each data point (M) represents each mouse; n = 5.

Supplementary Materials

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    • Fig. S1. DCFPyL does not affect CD19-tPSMA(N9del) CAR T cells in vitro.

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