Persistence of multiple tumor-specific T-cell clones is associated with complete tumor regression in a melanoma patient receiving adoptive cell transfer therapy

Abstract

The authors recently reported that adoptive immunotherapy with autologous tumor-reactive tumor infiltrating lymphocytes (TILs) immediately following a conditioning nonmyeloablative chemotherapy regimen resulted in an enhanced clinical response rate in patients with metastatic melanoma. These observations led to the current studies, which are focused on a detailed analysis of the T-cell antigen reactivity as well as the in vivo persistence of T cells in melanoma patient 2098, who experienced a complete regression of all metastatic lesions in lungs and soft tissues following therapy. Screening of an autologous tumor cell cDNA library using transferred TILs resulted in the identification of novel mutated growth arrest-specific gene 7 (GAS7) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene transcripts. Direct sequence analysis of the expressed T-cell receptor beta chain variable regions showed that the transferred TILs contained multiple T-cell clonotypes, at least six of which persisted in peripheral blood for a month or more following transfer. The persistent T cells recognized both the mutated GAS7 and GAPDH. These persistent tumor-reactive T-cell clones were detected in tumor cell samples obtained from the patient following adoptive cell transfer and appeared to be represented at higher levels in the tumor sample obtained 1 month following transfer than in the peripheral blood obtained at the same time. Overall, these results indicate that multiple tumor-reactive T cells can persist in the peripheral blood and at the tumor site for prolonged times following adoptive transfer and thus may be responsible for the complete tumor regression in this patient.

FIGURE 1

Clinical response in melanoma patient 2098 after adoptive cell transfer. Computed axial tomography scans showed the disease status prior to adoptive cell transfer (A) and 2 months after treatment (B) in the lung and prior to adoptive cell transfer (C) and 2 months after treatment (D) in the subcutaneous site. Arrows show sites of metastases.

FIGURE 2

Diversity of TRBV sequences expressed in the in vitro expanded and transferred TILs from melanoma patient 2098 and T-cell persistence in the peripheral blood. TRBV sequences amplified from the resected tumor sample (2098Tu) used in TIL 2098 generation, TIL 2098, and PBMC-d6 and PBMC-d27 samples obtained at 6 days and 27 days respectively following transfer were aligned with each other as well as to germline TRBV sequences, and the percentage corresponding to each of the individual clonotypes was determined. Following TRBV family names, lowercase letters are used to distinguish unique sequences that are derived from the same TRBV family but that contain distinct CDR3 junctional region sequences. A minimum of 96 clones were sequenced from each sample.

FIGURE 3

Comparison of T-cell persistence between the peripheral blood and tumor sites of melanoma patient 2098 after adoptive cell transfer. The TRBV sequences expressed in TIL 2098 as well as PBMC-6d, PBMC-27d, and PBMC-63d samples obtained at 6, 27, and 63 days following adoptive TIL 2098 transfer were compared with the TRBV sequences expressed in tumor samples obtained at 8, 29, and 64 days following adoptive TIL 2098 transfer, designated TU-d8, TU-d29, and TU-d64.

FIGURE 4

Tumor reactivity of TIL 2098. Tumor reactivity of TIL 2098 as well as a PBMC sample obtained at 8 weeks following adoptive cell transfer (PBMC8w) was evaluated in an IFN-γ release assay. PBMC8w was cultured in 500 CU/mL IL-2 for 16 hours before assay. The tumor reactivity of TIL 2098 and PBMC8w cells was tested with autologous 2098 tumor cells, allogeneic HLA-A2 positive 624mel, 1088mel, 1907mel, 1909mel, and 2023mel tumor cells, and HLA-A2 negative 397mel and 888mel tumor cells.

FIGURE 5

Upregulation of CD107a cell surface expression in response to tumor stimulation in TIL 2098. 1 × 10⁵ TIL 2098 T cells were co-cultured with 1 × 10⁵ allogeneic 888mel tumor cells (A) or 1 × 10⁵ autologous 2098mel tumor cells (B) for 16 hours. CD107a expression as well as TRBV2 and TRBV27 expression detected by corresponding TCR VB22 antibody and VB12 antibody respectively in TIL 2098 T cells was analyzed by FACS.

FIGURE 6

Titration of GAS7 and GAPDH antigenic epitopes recognized by TIL 2098. 1 × 10⁵ 1978EBV-B cells were pulsed with different concentrations of GAS7 (A) or GAPDH (B) peptides for 2 hours, washed with T-cell assay medium, and co-cultured with 1 × 10⁵ TIL 2098 T cells for 16 hours. The concentration of IFN-γ in the culture medium released by T cells was measured by ELISA assay. The sequences of antigenic epitopes are shown in Table 1.

FIGURE 7

Determination of tumor antigen reactive T-cell clones and persistence of 2098BV12-4a T-cell clone in melanoma patient 2098. A, 1 × 10⁵ cloned 2098BV2a, 2098BV7-9a, 2098BV12-4a, or 2098BV27 T cells were co-cultured for 16 hours with 1 × 10⁵ 2098mel tumor cells or 1978EBV-B cells pulsed with no peptide, 1 μM GAS7-10merMut, or 1 μM GAPDH-10merMut for 2 hours. The concentration of IFN-γ in the culture medium released by T cells was measured by ELISA assay. B, TRBV12-4 sequences were amplified from TIL 2098 as well as PBMC1w, PBMC8w, and PBMC32w samples obtained at 1, 8, and 32 weeks respectively following adoptive TIL 2098 transfer by RT-PCR using specific TRBV12-4 gene family primers. The percentage of TRBV12-4a sequences was normalized by FACS analysis in the total populations.