Patients with progressive metastatic melanoma (n = 11) or synovial cell sarcoma (n = 6) expressing high levels of NY-ESO-1 received nonmyeloablative chemotherapy followed by a median of 5 × 1010 T cells transduced with an anti–NY-ESO-1 TCR (range, 1.6 to 130 × 109) plus systemic IL-2 (). Patients presented with metastases in a wide variety of anatomic sites and were refractory to extensive prior treatment. All of the patients with melanoma had received prior treatment with high-dose IL-2, and all of the patients with synovial cell sarcoma experienced progression after extensive treatment with multiple courses of chemotherapy, all of which included doxorubicin and alkylating agents such as ifosfamide or cyclophosphamide. Many patients had also received second-line agents such as epirubicin, etoposide, or irinotecan and experimental monoclonal antibodies (). With the exception of patient 15, CD8+ T cells composed more than two thirds of the administered T cells (). A median of 78% of the transferred CD8+ T cells (range, 63% to 87%) and 65% of CD4+ T cells (range, 57% to 79%) bound the NY-ESO-1 tetramer, and a median of 92% of total CD3+ cells (range, 85% to 96%) bound an anti-Vβ13.1 antibody, which was reactive with the β chain of the transduced TCR (). The transduced T cells were highly reactive with an HLA-A*0201–positive, NY-ESO-1–positive tumor cell line (), and peptide titrations revealed that T cells from each of the 16 treatment samples that were tested recognized HLA-A*0201–positive target cells pulsed with a minimum concentration of between 0.1 and 0.01 nmol/L of the NY-ESO-1:157-165 peptide (Appendix Fig. A1, online only). In response to the NY-ESO-1–positive and HLA-A*0201–positive tumor target 624 mel, between 9,400 and 20,600 IFN-γ ELISPOTs/105 T cells were detected in the five samples of infused T cells that were tested, and responses against peptide pulsed targets ranged between 20,300 and 30,700 IFN-γ ELISPOTs/105 T cells in the four samples of infused T cells that were tested.
Characteristics of Patients and Administered T Cells
Prior Systemic Therapies in Patients Treated With Anti–NY-ESO-1TCR Transduced T Cells
Five of the 11 patients with metastatic melanoma experienced an objective response by Response Evaluation Criteria in Solid Tumors (RECIST), including two complete responses, ongoing at 22 and 20 months, and one partial response, ongoing at 9 months. Four of the six patients with synovial cell sarcoma exhibited objective partial responses, with one lasting 18 months. Examples of these responses are shown in
Fig 1. Computed tomography scans demonstrating tumor regression. Radiologic studies were obtained before therapy and after adoptive transfer of NY-ESO-1 T-cell receptor (TCR) –transduced T cells. Tumors indicated by arrows. (A) Regression of multiple (more ...)
Significant anti–NY-ESO-1 antibody titers were observed in pretreatment serum samples from six of 11 patients with melanoma and one of six patients with synovial cell sarcoma (). In all patients, there was no association between anti–NY-ESO-1 antibody titers in serum obtained from responders and nonresponders before treatment (P = .70) when analyzed using the nonparametric Mann-Whitney U test; however, it is difficult to draw any firm conclusions regarding the association between antibody titers and clinical response, given the relatively small number of patients treated in this trial.
Fig 2. Serum anti–NY-ESO-1 antibody titers. The titers of anti–NY-ESO-1 antibodies in pretreatment sera were evaluated by enzyme-linked immunosorbent assay. Clinically responding patients are designated with square symbols, and nonresponding (more ...)
Approximately 1 month after transfer, between 2% and 60% of the CD8+ T cells present in PBMCs obtained from 14 of the 17 treated patients bound the NY-ESO-1 tetramer (). In PBMCs obtained at the same time from patients 6, 10, and 17, as well PBMCs obtained from each of the patients before transfer, less than 1% of the CD8+ T cells bound to the NY-ESO-1 tetramer. Between 4% and 45% of peripheral CD4+ T cells from 11 of 17 patients stained with the NY-ESO-1 tetramer, whereas significant tetramer staining was not seen in samples obtained from the remaining six patients (). The percentage of T cells detected in PBMCs at 1 month that expressed Vβ13.1 ranged between 3% and 85% of CD8+ T cells and between 3% and 63% of CD4+ T cells. The expression of Vβ13.1 was highly correlated with NY-ESO-1 tetramer expression (A). The disparity between NY-ESO-1 tetramer staining and Vβ13.1 staining ( and ) presumably reflects higher affinity of the anti-Vβ13.1 antibody for TCR-transduced T cells, the detection of the Vβ13.1-positive transgene paired with endogenous α chains that results in nonfunctional TCRs, and the population of native Vβ13.1-positive T cells in the recovering peripheral repertoire after adoptive transfer.
Characteristics of Patient PBMCs Analyzed Approximately 1 Month After Adoptive Transfer
Fig 3. Correlations between T-cell persistence and enzyme-linked immunosorbent spot (ELISPOT) responses. Highly significant correlations were found (A) between NY-ESO-1 tetramer and Vβ13.1 expression (r2 = 0.82; P < .001), (B) between NY-ESO-1 (more ...)
Peptide-specific and tumor-specific IFN-γ ELISPOT responses were detected in PBMCs obtained from all of the patients 1 month after therapy with the exception of patients 6, 10, and 17, whose peripheral CD8+ T cells at 1 month also failed to demonstrate significant binding of the NY-ESO-1 tetramer (). Before transfer of 1G4-α95:LY TCR-transduced T cells, less than 10 IFN-γ ELISPOTs/105 PBMCs were detected in response to NY-ESO-1 peptide pulsed target cells in PBMC samples obtained from 16 of the 17 patients treated in this trial, whereas 55 ELISPOTs/105 PBMCs were detected in patient 3. After adoptive transfer, more than 1,000 ELISPOTs/105 PBMCs were detected in response to NY-ESO-1 peptide pulsed target cells in samples from 13 of 17 patients, whereas less than 100 ELISPOTs/105 PBMCs were detected in samples from patients 3, 6, 10, and 17. The number of NY-ESO-1 peptide–specific IFN-γ ELISPOTs detected in PBMCs 1 month after treatment correlated with NY-ESO-1 tetramer and anti-Vβ13.1 antibody binding (B and C). Tumor regression was not correlated with persistence of the transferred T cells as measured by NY-ESO-1 tetramer staining, Vβ13.1 staining, or ELISPOT responses.
The levels of NY-ESO-1–specific T cells detected using IFN-γ ELISPOT assays were significantly lower than the number of NY-ESO-1 tetramer–positive T cells detected 1 month after transfer in the 14 patients who demonstrated significant T-cell persistence. Several factors may influence these findings, including the fact that only approximately 10% to 30% of the in vitro cultured T cells assayed before infusion were detected in the ELISPOT assay, as well as the fact that whole PBMCs rather than purified T cells were used to evaluate these responses. The analysis of clinical adoptive immunotherapy trials carried out with MART-1– and gp100-reactive TCRs revealed that the levels of expression of multiple genes in PBMCs containing persistent T cells 1 month after adoptive transfer were low relative to those observed in in vitro cultured T cells.14
The IFN-γ ELISPOT assay results also demonstrated that responses of PBMCs obtained 1 month after transfer to an HLA-A*0201–positive and NY-ESO-1–positive tumor target were lower than responses to peptide pulsed target cells. This may have resulted from the insensitivity of quiescent T cells present in peripheral blood at this time to the relatively low levels of endogenously presented antigen on the tumor cell surface.
No toxicities were attributed to the transferred cells, although all patients experienced the transient neutropenia and thrombocytopenia induced by the preparative regimen and the transient toxicities associated with IL-2. All patients recovered well after the completion of treatment.