Figure 1.
Generation of retroviral vectors for the expression of SSX2-specific TCRs.
The coding region of each TCR alpha-chain was amplified by PCR using primers flanked by an NcoI restriction site in the 5′ end and an overhanging sequencing containing the element in the 3′ end. In parallel, each TCR beta-chain was amplified by PCR using a forward primer containing a 5′ overhang that overlaps with the 3′ overhang present in the primer used for the amplification of the alpha-chain. The reverse primer used for amplification of the beta-chain contained a stop codon and an EcoRI restriction site. In a second round of PCR, the products of the alpha- and beta-chain amplifications were pooled, and ligation of both cDNA fragments through the overlapping overhangs was achieved by PCR using external primers. The resulting PCR products were cloned in pMSGV1 vector for retrovirus production. LTR: long terminal repeat, sd: splice donor, sa: splice acceptor, ψ: retrovirus encapsidation signal.
Table 1.
TCRs isolated from SSX2-reactive lymphocyte clones.
Figure 2.
Expression and biological function of SSX2 TCR-5, -8, -9 and -11.
A) Analysis of surface expression of SSX241-49-specific TCRs in CD8 and CD4 T cell populations by flow cytometry. Human peripheral blood mononuclear cells were stimulated with OKT3 and transduced with the indicated retroviral expression vectors. One week later, cells were stained with anti-human CD3, CD8 antibodies and with a fluorescently labeled tetramer containing the SSX241-49 peptide. Results from one representative donor of at least four independent experiments. Events gated on lymphoid, single, viable, CD3+ cells. B) – D) Concentration of IFNg in supernatants of TCR-transduced T cells cultured overnight with the indicated targets (1×105 effectors vs 1×105 targets). Results shown as average of duplicates for two representative donors out of four. UT: untranduced.
Figure 3.
Analysis of recognition of other genes by TCR-5.
Peripheral blood T cells expressing TCR-5 were cocultured overnight with T2 cells previously pulsed with the serial dilutions of the indicated peptides. Results of IFNg concentration in culture supernatants are expressed as average of duplicates in a representative experiment. Sequence alignment of the tested peptides is shown in the figure legend for A) SSX-family genes and B) non-SSX genes with overlapping sequences. IGSF22: immunoglobulin superfamily member 22, ARHGAP1: Rho GTPase-activating protein 1, GPR82: Probable G-protein coupled receptor 82, PHF8: histone lysine demethylase PHF8, LIPM: lipase member M, SYT14: synaptotagmin-14, TCOF1: treacle protein, RBL2: retinoblastoma-like protein 2, FRAS1: extracellular matrix protein FRAS1. Prediction of binding affinity to HLA-A2*0201 is shown for each peptide, expressed as dissociation constant (KD, nM).
Figure 4.
Codon optimization and replacement of TCR constant regions with murine sequences.
A) Schematic representation of the three constructs generated for the expression of TCR-5 and derivatives. LTR: long terminal repeat, sd: splice donor, sa: splice acceptor, ψ: retrovirus encapsidation signal, MC: mouse TCR constant region, 2A: linker peptide. B) Analysis of expression of TCR-5 variants by tetramer staining. OKT3-stimulated lymphocytes were transduced twice with the corresponding TCR-expressing vector and stained with anti-CD3, anti-CD8 and SSX241-49 tetramers one week after transduction. Representative results from three independent experiments. Values in parentheses represent the mean fluorescence intensity of tetramer staining within the CD8 T cell population. C) 51Cr-release assay for the evaluation of antigen-specific cytolysis induced by TCR-5-transduced lymphocytes after four-hour coculture with the indicated target cells. Percentage of lysis depicted for each target cell line at different effector:target ratios is the average of duplicates in a representative experiment of three independent experiments. UT: untransduced T cells used as negative control of unspecific lysis, WT: Wild-type TCR, Co Op: copon-optimized TCR, MCR: codon-optimized TCR with mouse constant region.
Table 2.
IFNg secretion (pg/mL) by TCR-5 transduced lymphocytes upon coculture with indicated targets.
Figure 5.
Analysis of expression and biological function of codon-optimized TCR-5 in human lymphocytes transduced with clinical grade retroviral vector supernatants.
A) Flow cytometry analysis of tetramer staining of T cells transduced with vector supernatants produced by stable packaging line clones A8, A10, C3, D8, F2 and H2. Clones were previously selected as those displaying the highest expression of viral RNA using dot plot analysis. Results of tetramer and CD8 staining of T cells are shown for three patients. B) IFNg ELISA of supernatants from cocultures of T cells transduced with vectors derived from clones A8, A10, C3, D8, F2 and H2. HLA-A*0201+ SSX2+ melanoma cells 624, HLA-A*0201- SSX2+ melanoma cells 938 and HLA-A*0201+ SSX2- cells CosA2 were used as targets.