Research paperT cell activation upon exposure to patient-derived tumor tissue: A functional assay to select patients for adoptive T cell therapy
Introduction
Localized cancers are often treated by surgical removal of the diseased tissue or organ. Metastasized cancers, for example prostate cancer (PC), harbor difficulties of treatment when they are spread throughout the patients' body, at which stage most often only palliative treatments are available (Hanks and Dawson, 1986, Henry and O'Mahony, 1999, Paulson et al., 1990, Sinha et al., 1977). The development of novel therapeutic regimens such as therapy with T cells or gene-engineered T cells in order to provide anti-tumor immunity may represent an attractive option to treat metastasized cancers (Pinthus et al., 2003, Willemsen et al., 2003). In fact, receptor-engineered T lymphocytes have already shown clinical feasibility. In example, T lymphocytes engineered to express single-chain variable fragment scFv (defined as chimeric antibody-based receptor, CAR) directed against carboxy anhydrase-IX (CAIX) and alpha-foliate receptor have been applied towards renal cell carcinoma (RCC) and ovarian carcinoma, respectively, but as of yet without clear objective clinical responses (Kershaw et al., 2006, Lamers et al., 2006a). Clinical response rates following adoptive therapy with CAR-engineered T cells are expected to improve by careful assessment of the safety and efficacy of the target antigen of choice as well as the ligand-binding affinity and molecular design of CAR receptors to engineer the T cells.
Tumors may escape the host's immune response because of lack or compromised expression of major histocompatibility complex (MHC) and/or co-stimulatory ligands (Marincola et al., 2003). Specific CARs for a surface tumor associated antigen (TAA), in combination with the anti-tumor potential of T lymphocytes, may bypass immunological escape of tumors that have down-regulated or lost MHC molecules. Furthermore, in case of CAR that incorporate co-stimulatory molecules, such as CAR:CD28-CD3ζ, tumor recognition does not depend on co-stimulatory ligands nor induces loss of T cells by activation-induced cell death and results in more durable T cell responses (Eshhar et al., 1993, Hombach et al., 2001, Emtage et al., 2008). The design and validation of CARs that permit optimal antigen-specific T lymphocyte activation are of critical importance to the clinical implementation of receptor genes in the treatment of cancers.
Currently, the cloning and functional validations of CAR receptors are laborious and time-consuming, and clinical adoptive therapies with CAR-modified T cells depend on GMP-grade and therefore expensive gene transductions, expansions and infusions of patient's T cells. Pre-selection of patients based on antigen expression in patient's tumor and non-tumor tissue may improve the therapeutic efficacy, safety and cost effectiveness of adoptive T cell therapy, since only those patients who are anticipated to benefit from CAR T cell therapy will be treated. Screening of patient's tumor tissue by antibody staining does provide information on the presence of the target antigen but not necessarily on the tumor's ability to induce a T cell response. A functional screening assay based on CAR-engineered T cells is expected to better facilitate the selection of patients for adoptive therapy with CAR-modified T cells.
In this manuscript, we have set up and validated activation of CAR-directed T cells upon exposure to freshly prepared and patient-derived tumor tissue. First, we explored the ability of CAR-engineered primary T cells to respond in an antigen-dependent manner upon exposure to tumor tissue. Second, we translated the tumor tissue driven response into a sensitive and donor-independent NFAT activation assay in CAR-transduced Jurkat T cells. The assays in both primary human T cells and the Jurkat T cell line were set up with a CAIX-specific CAR, and the reporter gene assay was validated for two independent prostate-specific membrane antigen (PSMA) specific CARs.
Section snippets
CAIX CAR-transduced human T cells produce cytokines in response to antigen-positive RCC patient-derive tumor tissue
Earlier studies have shown that the CAIX CAR used in the current paper (i.e., a CAIX-specific CAR:CD4-Fc(ε)RIγ) enables primary human T cells to respond to antigen-positive target cells in vitro (Weijtens et al., 1998, Weijtens et al., 1996). Moreover, this receptor is implemented in an ongoing clinical immune gene therapy trial to treat metastatic RCC patients with autologous gene-modified T cells (Lamers et al., 2006a). Our preclinical and clinical experience with the CAIX CAR made this
Discussion
We introduced an assay to measure functional antigen expression on patient-derived tumor tissue using T cells gene-modified with a therapeutic CAR receptor of interest. First, we report the ability of primary human T cells transduced with a CAIX CAR, currently implemented in a clinical trial to treat RCC patients, to respond towards CAIX-positive but not CAIX-negative tumor tissue by increased production of IFNγ, TNFα, IL-10 and IL-4. Next, we set up a donor-independent and sensitive NFAT
Cells and culture reagents
The Jurkat T cell cl. E6.1, the CAIX-negative renal cell carcinoma (RCC)-derived SKRC-17 cl.1 and the CAIX cDNA-transfected SKRC-17 cl.4 (both kindly provided by dr. Egbert Oosterwijk, Nijmegen, The Netherlands) were cultured with RPMI 1640 medium (Cambrex Bio Sciences, Verviers, Belgium) supplemented with 200 nM l-glutamine, 10% bovine calf serum (BCS, Greiner Bio-one, Alphen a/d Rijn, The Netherlands), and the antibiotics streptomycin (100 μg/ml) and penicillin (100 U/ml) (both from Cambrex Bio
Acknowledgements
This work is in part financed by an Erasmus MC grant for translational research. Authors would like to thank Dr. Egbert Oosterwijk (Nijmegen, The Netherlands) for providing us with both the renal cell lines SKRC-17 cl.1 and SKRC-17 cl.4.
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