IGF1R- and ROR1-Specific CAR T Cells as a Potential Therapy for High Risk Sarcomas

Authors:
Xin Huang, Haein Park, Joseph Greene, James Pao, Erin Mulvey, Sophia X. Zhou, Catherine M. Albert, Fred Moy, Deepali Sachdev, Douglas Yee, Christoph Rader, Carl V. Hamby, David M. Loeb, Mitchell S. Cairo, Xianzheng Zhou
In:
Source: PLoS ONE
Publication Date: (2015)
Issue: 10(7): e0133152
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Gene Expression
Cells used in publication:
PBMC, human
Species: human
Tissue Origin: blood
Platform:
Nucleofector™ I/II/2b
Experiment
Human T cell gene transfer was carried out using a Nucleofector device with the human T cell Nucleofector kit (Lonza) as previously described (Huang, et al. Molecular therapy, 2008). 5E+6 PBMCs (from at least 8 healthy donors and 3 pediatric sarcoma patients) were Nucleofected with Sleeping Beauty (SB) CAR transposons (10 µg) mixed with SB100X transposase plasmid (10 µg). The cells were activated by anti-CD3/CD28 microbeads, and maintained in human T-cell medium consisting of RPMI-1640, 10% FBS, 10 mM HEPES, 2 mM L-glutamine, 50 µM ß-mercaptoethanol, 50 U/ml penicillin, and 50 µg/ml streptomycin supplemented with IL-2 (50 IU/ml) and IL-7 (10 µg/ml). The activated T cells were sorted or selected for GFP+ cells by flow cytometry or zeocin (0.2 mg/ml) selection and expanded every 10–14 days with anti-CD3/28 beads or OKT3. T cells cultured for 1–3 months were used for assays and were phenotypically effector memory T cells with variable ratios of CD4/CD8 subsets and stable expression of transgenes due to SB-mediated integration. Fig.1 shows that greater than 58% of nucleofected T cells from patient 1 expressed CARs and GFP in both IGF1R CAR (IGZ) and ROR1 CAR (RGZ) T cell populations. Mock T cells from the same patient were double negative for CARs and GFP. Lonza summary: The authors generated successfully chimeric antigen receptor (CAR) human T cells targeting the type I insulin-like growth factor receptor (IGF1R) or tyrosine kinase-like orphan receptor 1 (ROR1) selected for their therapeutic potential against sarcomas. To do so, PBMC from normal and pediatric sarcoma donors were transfected using the Nucleofector Technology with Sleeping Beauty (SB) transposons harboring the CAR constructs, GFP tagged in presence or absence of a selection marker. The engineered CAR T cells showed to be highly effective, in comparison to negative controls, for the treatments of human sarcomas mouse models validating IGF1R and ROR1 molecular targets for anti-sarcomas adoptive T-cell therapy (ACT).
Abstract
Patients with metastatic or recurrent and refractory sarcomas have a dismal prognosis. Therefore, new targeted therapies are urgently needed. This study was designed to evaluate chimeric antigen receptor (CAR) T cells targeting the type I insulin-like growth factor receptor (IGF1R) or tyrosine kinase-like orphan receptor 1 (ROR1) molecules for their therapeutic potential against sarcomas. Here, we report that IGF1R (15/15) and ROR1 (11/15) were highly expressed in sarcoma cell lines including Ewing sarcoma, osteosarcoma, alveolar or embryonal rhabdomyosarcoma, and fibrosarcoma. IGF1R and ROR1 CAR T cells derived from eight healthy donors using the Sleeping Beauty (SB) transposon system were cytotoxic against sarcoma cells and produced high levels of IFN-?, TNF-a and IL-13 in an antigen-specific manner. IGF1R and ROR1 CAR T cells generated from three sarcoma patients released significant amounts of IFN-? in response to sarcoma stimulation. The adoptive transfer of IGF1R and ROR1 CAR T cells derived from a sarcoma patient significantly reduced tumor growth in pre-established, systemically disseminated and localized osteosarcoma xenograft models in NSG mice. Infusion of IGF1R and ROR1 CAR T cells also prolonged animal survival in a localized sarcoma model using NOD/scid mice. Our data indicate that both IGF1R and ROR1 can be effectively targeted by SB modified CAR T cells and that such CAR T cells may be useful in the treatment of high risk sarcoma patients.