Fig. 4. Retroviral transduction of lymphocytes with four TCRs and assessment of their HLA restriction elements and specificity.

(A) Design of gammaretroviral TCR construct used for transduction of peripheral blood mononuclear cells (PBMC) with four TCRs derived from patient 3 and 5 cytolytic TIL clones. The human constant chains were replaced with mouse constant chains (mTCRα and mTCRβ) to reduce mispairing of transduced alpha and beta chains with endogenous TCR chains. A ribosomal skipping motif (2A) is inserted between the alpha and beta chains for separate transcription and expression of the two TCR chains.

(B) Determination of the restriction elements by which TCR react to autologous cancers (See Table S1 for HLA listing). For TCRs in patient 3, TRBV6-1 TCR recognizes its epitope by HLA-C*0701, whereas TRBV9 is restricted by HLA-B*4901, blocked by Bw4 and not by Bw6, the latter having the potential to block HLA-B*0801. The reactivity of both TCRs in patient 5 are not blocked by the anti-BC antibody, and are restricted by HLA-A*0201. The specificity of the Class I and Class II blocking antibodies was demonstrated in the same experiment by the selective block of known Class I (F5 mel) and Class II (Tyr 450) lymphocyte lines.

(C) Reactivity of native TIL and PBMC transduced with TCRs from patient 3 (upper), TCRs from patient 5 (middle), TCR that recognize MART1, MAGE A3 and CEA presented by HLA-A*020101, to 17 targets (see Table S2 for all target tested): 1. GALMa, 2. GALMb, 3. GALMc, 4. GALMd, 5. BAOMa, 6. BAOMb, 7. BAAM, 8. CALM, 9. CALM transduced to express HLA-A*020101, 10. CAPM, 11. CAAWMa, 12. NCI H508 colon cancer (Ca.), 13. SK-CO-1 colon Ca., 14. 624 melanoma TC, 15. SW1463 rectal Ca, 16. KATO III gastric Ca, 17. HCT15 colon Ca. Patients 3 and 5 TCRs reactivity are restricted to the autologous setting.