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International Immunology, Vol. 11, No. 5, 745-751, May 1999
© 1999 Japanese Society for Immunology

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

D. F. Lake, M. L. Salgaller1, P. van der Bruggen2, R. M. Bernstein3 and J. J. Marchalonis

Department of Microbiology and Immunology, and Arizona Cancer Center, University of Arizona, Tucson, AZ 85724, USA
1 Northwest Biotherapeutics LLC, Seattle, WA, USA
2 Ludwig Institute for Cancer Research, Brussels, Belgium
3 FDA, Rockville, MD, USA

Correspondence to: D. F. Lake

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{alpha} and Vß gene usage. The TIL population showed a restriction in V{alpha} and Vß usage with only V{alpha}4 and V{alpha}9 and Vß2 and Vß7 expressed. The anti-MAGE-1 CTL clone demonstrated absolute restriction with only V{alpha}12 and Vß1 expressed. DNA sequence analysis was performed on all V regions. For the TIL, each possible V{alpha}–Vß combination (i.e. V{alpha}4–Vß2, V{alpha}9–Vß2, V{alpha}4–Vß7 and V{alpha}9–Vß7) was constructed as a distinct scTCR and the recombinant proteins expressed in bacteria. From the anti-MAGE-1 TIL, V{alpha}4–Vß2 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 Vß2 plays a significant role in binding to the MAGE-1 peptide. Amino acid alignment analysis showed that each Vß 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.

Keywords: single chain TCR

Transmitting editor: G. Klein


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