Identification and characterization of the MAGEA1-specific TCR

For priming of CD8⁺ T cells from healthy donors, streptavidin-coated microspheres served as artificial antigen-presenting cells and were loaded with anti-CD28 antibody (clone 9.3, purified from mouse hybridoma supernatant, University of Tübingen, Germany) and MAGEA1-derived target peptide (KVLEYVIKV)-HLA (pHLA) monomers.²³ After 3 weeks of culture with repeated stimulation and medium exchange, cells were analyzed for primed populations using specific MAGEA1-target HLA-A*02:01 tetramers (MAGEA1-Brilliant Violet 650 and PE-Cy7), viability and anti-CD8 staining. Single cells of 2D target tetramer-binding populations were sorted on a BD ARIAIII FACS device into lysis buffer (64.9 mmol/L Tris, 810.8 mmol/L LiCl, 6.5 mmol/L EDTA, pH 7.5) for single cell rapid amplification of cDNA 5’ ends (5’RACE).

After cell lysis, mRNA was captured by paramagnetic oligo-deoxythymine-beads and cDNA was synthesized using TCR gene-specific primers. TCR transcripts were amplified via nested multiplex PCR.²⁴ The resulting PCR products were analyzed by Sanger sequencing. The sequencing data were used to assemble full length coding DNA sequences in silico using BLAST, CDR3 determination and final chain assembly. The TCRs were resynthesized via gene synthesis at GenScript (Rijswijk, Netherlands).

For transient re-expression, TCR mRNA was in vitro transcribed with the help of mMESSAGE mMACHINE T7 Transcription Kit according to the manufacturer’s instructions. As a template for in vitro transcription, individual TCR chains were PCR amplified with T7 and Kozak sequences at the 5’ end and a 64-adenine 3’ tail. Primary CD8⁺ T cells were isolated from leukaphereses from HLA-A*02-positive donors via CD8⁺ magnetic-activated cell sorting (Miltenyi Biotec, Bergisch Gladbach). After 1 day resting, the CD8⁺ cells were prestimulated for 3–5 days with plate-bound anti-CD3 (10 µg/mL coating concentration) and soluble anti-CD28 (0.1 µg/mL) antibodies. Cells were harvested, electroporated with TCR mRNA in ECM830 electroporator (BTX, Holliston, USA) at 500 V for 3 ms and rested for 20 hours. TCR re-expression was evaluated via tetramer staining along with anti-CD3 and viability staining.

For stable TCR expression, PBMC-derived T cells were activated overnight using immobilized anti-CD3 and anti-CD28 antibodies (0.5 µg/mL each), followed by transduction with a lentiviral vector encoding the MAGEA1-specific TCR. Cells were cultured in the presence of cytokines for additional 6–9 days and harvested for monitoring transduction efficiency, transgene expression, and functional assessment.

TCR mRNA electroporated T cells were used for activation assay by interferon (IFN)-γ release ELISA after co-culture with target cells loaded with peptides or target-expressing tumor cell lines as well as primary cells from healthy tissues. Released IFN-γ levels were determined after 20 hours of co-culture with the help of BD OptEIA Human IFN-γ ELISA or Biolegend Human IFN-γ-ELISA MAX Deluxe Kits. Primary cells from healthy tissues were obtained from PromoCell (Heidelberg, Germany) or induced pluripotent stem cell-derived cell types were obtained from FUJIFILM Cellular Dynamics (Madison, USA). Tumor cell lines were obtained from ATCC (Virginia, USA) or DSMZ (Braunschweig, Germany). All cells were cultured according to the manufacturer’s instructions and genotyped for HLA-A*02. Culture periods were kept short to maintain cellular characteristics. The T cell activation assays were performed in T cell medium to enable optimal activity of the effector cells. T cell medium consists of RPMI 1640 GlutaMAX supplemented with 10% heat-inactivated human serum, 1% penicillin/streptomycin, 0.2% gentamycin and 1% sodium pyruvate. The EC₅₀ of the MAGEA1-specific TCR was determined from two donors using GraphPad Prism V.6 via nonlinear fit (sigmoidal, 4PL) of log-dose (loaded peptide concentration) versus response (IFN-γ release).

For TCR specificity testing, 10 similar peptides were selected based on their sequence similarity to the target peptide. For this purpose, an in-house database was searched for peptides that share at least five identical amino acids with the target and have been detected at least once on an HLA-A*02-positive healthy tissue by LC-MS/MS. For the selection of a representative set of similar peptides, parameters such as prediction of binding to HLA-A*02:01 (NetMHCpan≤0.5), the number of identical amino acids (with or without anchoring positions), similarity to the target peptide (based on PMBEC-score), display of a unique similarity motif and number of detections on healthy tissues were considered.

Cytotoxic response of MAGEA1 TCR-positive transduced and non-transduced (NT) T cells was measured against red fluorescent protein (RFP)-labeled U2OS and UACC-257 MAGEA1-positive (HLA-A*02-positive) tumor cells using IncuCyte live imaging. The assay was performed at various effector-to-target (E:T) ratios and fold-tumor growth monitored for at least 72 hours based on RFP fluorescence signal. Results are presented as mean±SD of three replicates at each imaging time point.

The MAGEA1-specific TCR was expressed as soluble protein and refolded according to a published protocol in Escherichia coli.²⁵ Refolded TCRs were purified via anion exchange and size exclusion chromatography. The protein concentration was determined using Bradford assays and refolding was determined via native and denaturing polyacrylamide gel electrophoresis. Biolayer interferometry technology was used to determine the affinity of the refolded TCR toward target pHLA compared with unrelated pHLA.