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125 Co-opting IL-8 to enhance efficacy of B7H3 CAR T cells against pediatric sarcoma
  1. Jessica Lake,
  2. Kevin Winkler2,
  3. Alexander Harrant2,
  4. Ashley Yingst2,
  5. Kristin Schaller2,
  6. Eric Hoffmeyer2,
  7. Madeline Larson2,
  8. Dejene Tufa2,
  9. Laura Cobb2,
  10. Dallas Jones2 and
  11. Michael Verneris2
  1. 1University of Colorado Anschutz Medical Campus, Aurora, CO, USA
  2. 2University of Colorado, Aurora, CO, USA


Background The 5-year disease-free survival for children and young adults with metastatic sarcoma at diagnosis or recurrent disease after front-line therapy is 20–30%.1 2 Cellular immunotherapy using chimeric antigen receptor (CAR) T cells has shown dramatic benefits in leukemia, but only limited success in solid tumors.3 4 One limitation of CAR T cell therapy has been poor trafficking into solid tumors.5–7 Chemokines are small, secreted, cytokine-like molecules that mediate lymphocyte homing and migration.8 In this study, we discovered that both osteosarcoma (OS) and rhabdomyosarcoma (RMS) cells significantly increase expression of the chemokine IL-8 after clinically achievable doses of radiation, but not at rest. Given that CAR T cells do not express the receptor for IL-8, we created a construct with an IL-8 receptor (CXCR2) and a B7H3 CAR in T cells to improve CAR T homing and to create an effective new immunotherapy for patients with sarcoma.

Methods Multiple OS and RMS cell lines were irradiated at 10 Gy and IL-8 was measured by ELISA. We created retroviral constructs, B7H3 CAR-T2a-CXCR2 and B7H3 CAR. Peripheral blood T lymphocytes were stimulated with IL-2 and anti-CD3/28 antibodies for 48 hours prior to transduction with the retroviral vectors. Surface expression of the scFv (by L protein) and CXCR2 (mAb) were assessed using flow cytometry. In vitro cytotoxicity assays using sarcoma tumor spheroids were conducted using Incucyte. INF-γ and IL-2 production were measured by ELISA. NSG mice injected orthotopically with an IL-8 overexpressing RMS cell line were treated 4–7 days later with the B7H3 CAR-CXCR2 T cells or B7H3 T cells (control) and followed weekly with bioluminescent imaging.

Results Irradiated (10 Gy) sarcoma cells express 2-9x higher IL-8 than non-irradiated sarcoma. T cells were transduced with efficiencies of 60–90%. INF-γ production was equivalent between the B7H3 CAR-T2a-CXCR2 T cells and B7H3 CAR T cells, but IL-2 production was significantly higher in the dual expressing CAR T cells. In vitro cytotoxicity with sarcoma spheroids was measured by Incucyte and showed faster and greater killing by B7H3 CAR-T2a-CXCR2 T cells than B7H3 CAR T cells. Furthermore, when sarcoma tumor bearing mice were treated with B7H3 CAR-T2a-CXCR2 T cells, tumors resolved completely by 4–5 weeks and had long-lasting remission.

Conclusions Chemokine receptor expressing CAR T cells showed superior cytokine production and T cell activation/cytotoxicity compared to a CAR T construct alone. These finding lead to better efficacy in animal models and suggest a promising approach for pediatric sarcoma.


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Ethics Approval The animal experiments discussed in the abstract were approved by the University of Colorado IACUC, protocol #00251.

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