IGF1R- and ROR1-Specific Chimeric Antigen Receptor (CAR) T Cell Immunotherapy for Poor Risk Sarcomas

Background:  Patients with metastatic or recurrent/refractory sarcomas have a dismal prognosis with 5-year survival of 10-20%. IGF1R (insulin-like growth factor-1 receptor) is highly expressed on the majority of sarcomas. Clinical trials with anti-IGF1R antibody in patients with refractory Ewing sarcoma (EWS) have produced low response rates (10-14%), which exemplify the need to develop alternative new therapies toward this target.

Objective:  We hypothesize that IGF1R chimeric antigen receptor (CAR) T cells will be more effective than antibody alone against IGF-1R⁺ sarcomas even at low levels of expression.

Methods:  To test our hypothesis, we transfected peripheral blood lymphocytes from 6 healthy donors and 3 sarcoma patients with Sleeping Beauty (SB) transposons encoding IGF1R CAR with a 4-1BB signaling domain and GFP (IG) or GFP:Zeocin fusion gene (IGZ) and SB100X transposase-expressing plasmids and selected for GFP or drug resistance.

Results:  ⁵¹Cr-release assays demonstrated the cytotoxicity of IGF-1R CAR T cells against a panel of IGF-1R⁺ sarcoma cell lines including 2 osteosarcoma (OS), 3 alveolar rhabdomyosarcoma (ARMS), 2 embryonial rhabdomyosarcoma (ERMS), 1 fibrosarcoma (FS), and 1 EWS. CAR⁺ T cells were not cytotoxic for IGF1R^- K562 erythroleukemia and Daudi lymphoma cells and unmodified T cells did not kill IGF1R⁺ target cells (Fig. 1A). CAR⁺ T cells also killed human IGF1R cDNA transfected mouse fibroblast cells derived from IGF1R knockout mice but not parental cells. In addition, we also demonstrated expression of the alternative target ROR1(receptor tyrosine kinase-like orphan receptor 1) in 13 of 17 sarcoma cell lines tested as it has been shown that ROR1 is highly expressed in certain types of cancer but not in normal adult tissues. Multiplex cytokine assays showed that both IGF1R and ROR1 CAR T cells produced high levels of IFN-g, TNF-a and IL-13 in an antigen-specific manner. Furthermore, both IGF1R and ROR1 CAR T cells were generated from 3 sarcoma patients and were shown to release significant amounts of IFN-g in response to stimulation with cultured allogeneic sarcoma lines. The in vivo adoptive transfer of both IGF1R and ROR1 CAR T cells derived from a sarcoma patient significantly (p<0.02) reduced tumor growth and prolonged the survival of animals in both pre-established, systemic (i.v.) and local (i.p.) osteosarcoma xenograft models (Fig. 1B and 1C).

Conclusion: These data indicate that both IGF1R and ROR1 can be effectively targeted by CAR T cells and that such CAR T cells may be useful in the treatment of metastatic or refractory sarcomas.