Multiple chimeric antigen receptors successfully target chondroitin sulfate proteoglycan 4 in several different cancer histologies and cancer stem cells

Abstract

BackgroundThe 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.MethodsCSPG4 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.ResultsImmunohistochemistry 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.ConclusionsThe 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.

Mesothelin-Targeted CARs: Driving T Cells to Solid Tumors

“…EGFRvIII and chondroitin sulfate proteoglycan-4 (CSPG-4) have been suggested to be examples for such favorable scenarios. (14, 15)…”

Current modalities in cancer immunotherapy: Immunomodulatory antibodies, CARs and vaccines

“…For hematological cancers these antigens include CD22 and CD20 (Haso et al, 2013; Till et al, 2012). For solid tumors they include abnormally glycosylated MUC1 and CSPG4 proteins and the abnormal splice variant of EGFR, EGFRvIII (Beard et al, 2014; Johnson et al, 2015; Lohmueller et al, 2016; Wilkie et al, 2008). Search for other abnormally glycosylated or abnormally spliced targets could yield exciting new antigen targets for CAR therapy.…”

Bispecific antibodies and CARs: generalized immunotherapeutics harnessing T cell redirection

“…Some investigators have designed CAR T cells specifically to target the microenvironment, such as CAR T cells direct to fibroblast activation protein [90] or chondroitin sulfate [91]. However, it is not clear that targeting the tumor stroma will have an anti-tumor effect.…”