Recognition of NY-ESO-1+ tumor cells by engineered lymphocytes is enhanced by improved vector design and epigenetic modulation of tumor antigen expression

Abstract

The therapeutic use of T cell receptor (TCR)-transduced peripheral blood lymphocytes (PBL) targeting tumor-associated antigens is emerging as a promising investigational treatment for patients with cancer. Initial response rates to therapy were low, suggesting the need to improve the function of TCR-transduced PBL. We constructed standard bicistronic retroviral vectors using an internal promoter or internal ribosomal entry site element as well as vectors incorporating coding sequences for 2A linker peptides between coding sequences for α and β chains targeting the cancer-testis (CT) antigen, NY-ESO-1. Incorporation of coding sequences for 2A linker peptides in the bicistronic TCR expression cassette resulted in up to a fourfold increase in TCR expression and a significant improvement in effector function as measured by interferon-gamma release following co-culture with peptide-pulsed targets and NY-ESO-1+ tumors. We also sought to enhance reactivity of TCRtransduced PBL against tumor targets by modulation of tumor antigen expression on target cells. Induction of NY-ESO-1 expression on tumor targets using the demethylating agent 5-aza-2′-deoxycytidine (alone or in combination with the histone deacetylase inhibitor depsipeptide) resulted in enhanced interferon-gamma secretion by the TCR-transduced PBL on culture with treated targets. Taken together, these results indicate that design of TCR-based vectors incorporating 2A linker peptides improves TCR expression and effector function of transduced PBL. Furthermore, induction of CT antigen expression through treatment of tumor targets with chromatin-remodeling agents may augment TCR-based immunotherapy targeting these antigens. These results have relevance for TCRbased gene therapies targeting common epithelial malignancies.

Incorporation of Transmembrane Hydrophobic Mutations in the TCR Enhance Its Surface Expression and T Cell Functional Avidity

“…T2 (ATCC/CRL-1992) is a lymphoblastoid cell line deficient in TAP function whose HLA/A2 protein can be easily loaded with exogenous peptides. Viral packaging line 293GP, which stably expresses GAG and POL proteins, has been previously described (26). Adherent cells were cultured in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated FBS (Biological Industries, Beth Haemek, Israel) and were maintained in a 37˚C and 5% CO 2 incubator.…”

“…The sequences of the peptides used in this study are as follows: MART-1 [26][27][28][29][30][31][32][33][34][35] (ELAGIGILTV), gp100 209M (IMDQVPFSV), and p53 264-272 (LLGRNSFEV).…”

“…The a-and b-chains from the previously characterized TCRs specific for MART-1 [26][27][28][29][30][31][32][33][34][35] termed F4 (or DMF4) (7,27), for p53 (4), and for gp100 (28) were subcloned into the pGEM-4Z/64A vector as described previously (11). In addition, we created mutant TCRs in which we mutated residues in the TM region (20,29) of the TCRa-and TCRb-chains.…”

“…Freshly isolated PBLs were stimulated for 48 h in the presence of 50 ng/ml OKT3 before transduction. After stimulation, lymphocytes were transduced with retroviral vectors by transfer to nontreated tissue culture dishes (Nunc, Rochester, NY) that had been precoated with RetroNectin (Takara, Otsu, Japan) and retroviral vectors as previously described (26).…”

Engineered Drug Resistant Cell-Mediated Immunotherapy

“…These genetically engineered CTLs harboring a transgenic αβ TCR, in addition to their native αβ TCR, acquire the same antigen specificity as the high affinity T cells from which the TCR was cloned. Several CTLs harboring transgenic TCRs have now been developed that are directed specifically to tumor antigens, such as MART-1, gp100, NY-ESO-1 and CEA, resulting in tumor elimination in animal models (Abad, et al, 2008;Morgan, et al, 2003;Kessels et al, 2001;Stanislawski et al, 2001;Wargo et al, 2009;Xue et al, 2005). Bicistronic viral vectors encoding cDNA sequences for both α and β chains have been successfully incorporated into retroviral based strategies to transfer αβ transgenic TCR into T lymphocytes (Yang et al, 2008).…”