Construction and binding analysis of recombinant single-chain TCR derived from tumor-infiltrating lymphocytes and a cytotoxic T lymphocyte clone directed against MAGE-1

The TCR is responsible for the specificity of cytotoxic T lymphocytes (CTL) by recognizing peptides presented in the context of MHC. By producing recombinant soluble TCR, it is possible to study this interaction at the molecular level. We generated single-chain TCR (scTCR) from tumor infiltrating lymphocytes (TIL) and one CTL clone directed against melanoma-associated antigen (MAGE)-1. Sixty-eight day anti-MAGE-1 TIL and one cloned anti-MAGE-1 CTL were analyzed by PCR for their V(α) and V(β) gene usage. The TIL population showed a restriction in V(α) and V(β) usage with only V(α)4 and V(α)9 and V(β)2 and V(β)7 expressed. The anti-MAGE-1 CTL clone demonstrated absolute restriction with only V(α)12 and V(β)1 expressed. DNA sequence analysis was performed on all V regions, For the TIL, each possible V(α)-V(β) combination (i.e. V(α)4-V(β)2, V(α)9-V(β)2, V(α)4-V(β)7 and V(α)9-V(β)7) was constructed as a distinct scTCR and the recombinant proteins expressed in bacteria. From the anti-MAGE-1 TIL, V(α)4-V(β)2 scTCR demonstrated binding activity to HLA-A1⁺ cells pulsed with MAGE-1 peptide. Results obtained from screening a panel of our scTCR constructs on HLA-A1⁺ cells pulsed with MAGE-1 peptide or irrelevant peptide demonstrated that V(β)2 plays a significant role in binding to the MAGE-1 peptide. Amino acid alignment analysis showed that each V(β) sequence is distinctly different from the others. These findings demonstrate that soluble TCR in single-chain format have binding activity. Furthermore, the results indicate that in TCR, like antibodies, one chain may contribute a dominant portion of the binding activity.