title: Multiple chimeric antigen receptors successfully target chondroitin sulfate proteoglycan 4 in several different cancer histologies and cancer stem cells.
abstract: BACKGROUND
  The development of immunotherapy has led to significant progress in the treatment of metastatic cancer, including the development of genetic engineering technologies that redirect lymphocytes to recognize and target a wide variety of tumor antigens. Chimeric antigen receptors (CARs) are hybrid proteins combining antibody recognition domains linked to T cell signaling elements. Clinical trials of CAR-transduced peripheral blood lymphocytes (PBL) have induced remission of both solid organ and hematologic malignancies. Chondroitin sulfate proteoglycan 4 (CSPG4) is a promising target antigen that is overexpressed in multiple cancer histologies including melanoma, triple-negative breast cancer, glioblastoma, mesothelioma and sarcoma.
  
  
  METHODS
  CSPG4 expression in cancer cell lines was assayed using flow cytometry (FACS) and reverse-transcription PCR (RT-PCR). Immunohistochemistry was utilized to assay resected melanomas and normal human tissues (n = 30) for CSPG4 expression and a reverse-phase protein array comprising 94 normal tissue samples was also interrogated for CSPG4 expression. CARs were successfully constructed from multiple murine antibodies (225.28S, TP41.2, 149.53) using second generation (CD28.CD3ζ) signaling domains. CAR sequences were cloned into a gamma-retroviral vector with subsequent successful production of retroviral supernatant and PBL transduction. CAR efficacy was assayed by cytokine release and cytolysis following coculture with target cell lines. Additionally, glioblastoma stem cells were generated from resected human tumors, and CSPG4 expression was determined by RT-PCR and FACS.
  
  
  RESULTS
  Immunohistochemistry demonstrated prominent CSPG4 expression in melanoma tumors, but failed to demonstrate expression in any of the 30 normal human tissues studied. Two of 94 normal tissue protein lysates were positive by protein array. CAR constructs demonstrated cytokine secretion and cytolytic function after co-culture with tumor cell lines from multiple different histologies, including melanoma, breast cancer, mesothelioma, glioblastoma and osteosarcoma. Furthermore, we report for the first time that CSPG4 is expressed on glioblastoma cancer stem cells (GSC) and demonstrate that anti-CSPG4 CAR-transduced T cells recognize and kill these GSC.
  
  
  CONCLUSIONS
  The functionality of multiple different CARs, with the widespread expression of CSPG4 on multiple malignancies, suggests that CSPG4 may be an attractive candidate tumor antigen for CAR-based immunotherapies using appropriate technology to limit possible off-tumor toxicity.
authors.affiliation: Surgery Branch, Center for Cancer Research, National Cancer Institute, 10 Center Drive, Building 10 Hatfield CRC, Rm 3-5930, 20892-1201 Bethesda, MD, USA.
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Beard
authors.affiliation: Surgery Branch, Center for Cancer Research, National Cancer Institute, 10 Center Drive, Building 10 Hatfield CRC, Rm 3-5930, 20892-1201 Bethesda, MD, USA.
authors.forename: Zhili
authors.initials: Z
authors.lastname: Zheng
authors.affiliation: Surgery Branch, Center for Cancer Research, National Cancer Institute, 10 Center Drive, Building 10 Hatfield CRC, Rm 3-5930, 20892-1201 Bethesda, MD, USA.
authors.forename: Kiran H
authors.initials: KH
authors.lastname: Lagisetty
authors.affiliation: Surgery Branch, Center for Cancer Research, National Cancer Institute, 10 Center Drive, Building 10 Hatfield CRC, Rm 3-5930, 20892-1201 Bethesda, MD, USA.
authors.forename: William R
authors.initials: WR
authors.lastname: Burns
authors.affiliation: Surgery Branch, Center for Cancer Research, National Cancer Institute, 10 Center Drive, Building 10 Hatfield CRC, Rm 3-5930, 20892-1201 Bethesda, MD, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Tran
authors.affiliation: Laboratory of Pathology, Center for Cancer Research, National Cancer Institute, 10 Center Drive, Building 10 Hatfield CRC, MSC 4605, 20892-1201 Bethesda, MD, USA.
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Hewitt
authors.affiliation: Surgery Branch, Center for Cancer Research, National Cancer Institute, 10 Center Drive, Building 10 Hatfield CRC, Rm 3-5930, 20892-1201 Bethesda, MD, USA.
authors.forename: Daniel
authors.initials: D
authors.lastname: Abate-Daga
authors.affiliation: Surgery Branch, Center for Cancer Research, National Cancer Institute, 10 Center Drive, Building 10 Hatfield CRC, Rm 3-5930, 20892-1201 Bethesda, MD, USA.
authors.forename: Shannon F
authors.initials: SF
authors.lastname: Rosati
authors.affiliation: Division of Hematology and Medical Oncology, New York University Langone Medical Center, New York, New York, USA.
authors.forename: Howard A
authors.initials: HA
authors.lastname: Fine
authors.affiliation: Department of Surgery, Massachusetts General Hospital, 55 Fruit Street, Boston, MA 02114, USA.
authors.forename: Soldano
authors.initials: S
authors.lastname: Ferrone
authors.affiliation: Surgery Branch, Center for Cancer Research, National Cancer Institute, 10 Center Drive, Building 10 Hatfield CRC, Rm 3-5930, 20892-1201 Bethesda, MD, USA.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Rosenberg
authors.affiliation: Surgery Branch, Center for Cancer Research, National Cancer Institute, 10 Center Drive, Building 10 Hatfield CRC, Rm 3-5930, 20892-1201 Bethesda, MD, USA ; Current address: Bluebird bio, 150 Second St, Cambridge, MA 02141, USA.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Morgan
keywords: CSPG4; Cancer stem cells; Chimeric antigen receptor; Glioblastoma; Immunotherapy; Melanoma