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 Valpha and Vbeta gene usage. The TIL population showed a restriction in Valpha and Vbeta usage with only Valpha4 and Valpha9 and Vbeta2 and Vbeta7 expressed. The anti-MAGE-1 CTL clone demonstrated absolute restriction with only Valpha12 and Vbeta1 expressed. DNA sequence analysis was performed on all V regions. For the TIL, each possible Valpha-Vbeta combination (i.e. Valpha4-Vbeta2, Valpha9-Vbeta2, Valpha4-Vbeta7 and Valpha9-Vbeta7) was constructed as a distinct scTCR and the recombinant proteins expressed in bacteria. From the anti-MAGE-1 TIL, Valpha4-Vbeta2 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 Vbeta2 plays a significant role in binding to the MAGE-1 peptide. Amino acid alignment analysis showed that each Vbeta 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.
D000595:Amino Acid Sequence; D000951:Antigens, Neoplasm; D015788:HLA-A1 Antigen / Q000502:physiology; D006801:Humans; D016246:Lymphocytes, Tumor-Infiltrating / Q000378:metabolism*; D058950:Melanoma-Specific Antigens; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000276:immunology / Q000378:metabolism*; D017510:Protein Folding; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000378:metabolism*