Transduction and analysis of TCR-engineered cells. (A) CD8⁺human lymphocytes were electroporated with RNA encoding control [green fluorescent protein (GFP)] or cloned TCRs reactive with HLA-A2 restricted epitopes from the human TAAs MART-1, gp100, NY-ESO-1, and p53. Effector T cells were cocultured with T2 cells pulsed with 1 μM of the indicated peptide (values are expressed as IFN-γ in pg/ml). Values demonstrating the specific release of cytokine are in bold. (B) Diagram of the recombinant retroviral vector MSGV1AIB used to engineer human lymphocytes. LTR, long terminal repeat;Ψ, extended packaging signal; sd, splice donor; sa, splice acceptor; Alpha, alpha chain; IRES, internal ribosomal entry site; Beta, beta chain. (C) Transduced (Td) lymphocytes were analyzed 5 days after transduction for the expression of Vβ12 and MART-1 tetramer [Ala²⁷ →Leu²⁷ (A27L)] in CD8⁺cells in comparison with untransduced (UnTd) cells. Numbers in the upper-right corners indicate the percentage of positive cells in that quadrant. (D) TCR vector-engineered cells from patient 6 (TCR) were cocultured with MART-1 peptide-pulsed T2 cells, HLA-A2⁻ melanoma line (Mel 888), or HLA-A2⁺ melanoma line (Mel 526), and the amount of IFN-γ produced was determined. Control effectors were untransduced cells (PBL) and the MART-1–reactive TIL JKF6 (JKF6). (E) Anti-melanoma properties of genetically engineered lymphocytes were determined for all patients before infusion. The production of IFN-γ (pg/ml) after coculture with peptide-pulsed T2 cells (Peptide Reactivity) and anti-melanoma activity (Tumor Reactivity) for HLA-A2⁺ lines (526 and 624) and HLA-A2⁻ lines (888 and 938).

Persistence of gene-marked cells. DNA extracted from peripheral blood mononuclear cells (PBMCs) was subjected to real-time quantitative PCR to determine the percentage of vector-transduced cells in patient circulation at various times after infusion. Each line represents data from a separate patient. (A) Cohort 1; (B) Cohort 2; (C) Cohort 3. (D) Mean value of the percentage of gene-marked cells for all patients in each cohort at the given time interval after treatment. (E) The percentage of CD8⁺/Vβ12⁺ cells in the intermediate gate (13) for patients in cohorts 2 and 3 is shown. (F) The percentage of CD8⁺/MART-1⁺ tetramer cells was determined for patients in cohorts 2 and 3 at the times shown. Pretreatment values for each patient are plotted as day 0 after infusion.

Cancer regression in two patients. (A) Computed tomography (CT) images of liver metastasis in patient 4 taken at pretreatment, 1 month, and 10 months after treatment with TCR-engineered T cells. (B) Size of liver and axillary tumors and tempo of regression of tumor sites in patient 4. Day 0, beginning of treatment. L Axill LN, left axillary lymph node. (C) CT images of hilar lymph node metastasis in patient 14; pretreatment, day 0, and 2 months and 12 months after treatment. The white arrow indicates the mass in the lung hilum. (D) Size of tumor and tempo of regression in patient 14. (E) Quantitation of gene-marked cells in the PBMCs of patients 4 and 14 was determined by real-time quantitative PCR. Pt, patient. Day of infusion (Inf.) indicated by arrow. (F) The percentage of CD8⁺/Vβ12⁺ cells in the intermediate gate (13) in the circulation of patients 4 and 14.