T-Cell Therapy Using Interleukin-21-Primed Cytotoxic T-Cell Lymphocytes Combined With Cytotoxic T-Cell Lymphocyte Antigen-4 Blockade Results in Long-Term Cell Persistence and Durable Tumor Regression

Abstract

Purpose Peripheral blood-derived antigen-specific cytotoxic T cells (CTLs) provide a readily available source of effector cells that can be administered with minimal toxicity in an outpatient setting. In metastatic melanoma, this approach results in measurable albeit modest clinical responses in patients resistant to conventional therapy. We reasoned that concurrent cytotoxic T-cell lymphocyte antigen-4 (CTLA-4) checkpoint blockade might enhance the antitumor activity of adoptively transferred CTLs. Patients and Methods Autologous MART1-specific CTLs were generated by priming with peptide-pulsed dendritic cells in the presence of interleukin-21 and enriched by peptide-major histocompatibility complex multimer-guided cell sorting. This expeditiously yielded polyclonal CTL lines uniformly expressing markers associated with an enhanced survival potential. In this first-in-human strategy, 10 patients with stage IV melanoma received the MART1-specific CTLs followed by a standard course of anti-CTLA-4 (ipilimumab). Results The toxicity profile of the combined treatment was comparable to that of ipilimumab monotherapy. Evaluation of best responses at 12 weeks yielded two continuous complete remissions, one partial response (PR) using RECIST criteria (two PRs using immune-related response criteria), and three instances of stable disease. Infused CTLs persisted with frequencies up to 2.9% of CD8+ T cells for as long as the patients were monitored (up to 40 weeks). In patients who experienced complete remissions, PRs, or stable disease, the persisting CTLs acquired phenotypic and functional characteristics of long-lived memory cells. Moreover, these patients also developed responses to nontargeted tumor antigens (epitope spreading). Conclusion We demonstrate that combining antigen-specific CTLs with CTLA-4 blockade is safe and produces durable clinical responses, likely reflecting both enhanced activity of transferred cells and improved recruitment of new responses, highlighting the promise of this strategy.

Trial registration: ClinicalTrials.gov NCT00871481.

Conflict of interest statement

Authors’ disclosures of potential conflicts of interest are found in the article online at www.jco.org. Author contributions are found at the end of this article.

Figures

Fig 1.

Tumor regressions after melanoma-reactive polyclonal cytotoxic T cells (CTLs) combined with anti–cytotoxic T-cell lymphocyte antigen-4. (A) Spider plot of all treated patients showing changes from baseline in the tumor burden (y-axis), measured as the products of the perpendicular diameters of all target lesions, assessed weeks after the CTL infusion (x-axis). The dashed line above the solid line indicates 25% progression (modified WHO progressive disease [PD]), and the dashed line below the solid line indicates 50% reduction (modified WHO partial response [PR]). Red lines indicate patients with PD, purple lines indicate patients with stable disease, blue lines indicate patients with PRs, and green lines indicate patients with complete remissions. Red squares indicate the occurrence of new lesions, asterisks indicate the start of an alternate treatment, pound signs indicate disease progression sufficient to transition to comfort care, and blue horizontal arrows indicate continued monitoring. (B) Serial images of computed tomography scans performed before infusion (left) and 64 and 55 weeks after treatment (right) for patients 1 (top) and 7 (bottom), respectively. Arrows indicate the location of the largest index lesions for each patient.

Fig 2.

Kinetics of in vivo persistence of melanoma-reactive polyclonal cytotoxic T cells (CTLs). (A) Percent multimer-positive CD8+ T cells (y-axis) in peripheral-blood mononuclear cells (solid circles) collected 7 days (± 2 days) before and at defined time points after infusions is shown for patients who achieved complete remissions, partial responses, or stable disease after treatment. Green arrows indicate CTL infusions, black vertical arrows indicate anti–cytotoxic T-cell lymphocyte antigen-4 infusions, asterisks indicate the start of an alternate treatment, pound signs indicate comfort care, orange arrows indicate concurrent corticosteroid therapy, and blue horizontal arrows indicate ongoing monitoring. (B) The same analysis performed for patients who experienced disease progression after treatment. IL-2, interleukin-2.

Fig 3.

Phenotypic and functional characteristics of transferred melanoma-reactive cytotoxic T cells (CTLs). (A) Expression of CD27, CD28, CD127, CD62L, and CCR7 (y-axis) on gated multimer-positive cells for CD8+ CTL products immediately before infusion and after 3, 6, 9, and 23 weeks in vivo for patients who achieved complete remissions (CRs), partial responses (PRs), or stable disease (SD). (B) The same analysis in panel A for expression of CD57 and programmed death-1 (PD1; y-axis) performed for patients who experienced progressive disease (PD). (C) Open symbols indicate patients who achieved CRs, PRs, or SD (left column); solid symbols indicate patients who experienced PD (right column). Left plots: percentage of cells within the infusion products producing interferon gamma (IFN-γ) in response to MART1 peptide and in peripheral-blood mononuclear cells (PBMCs) before and 6 and 12 weeks after the CTL infusion; right plots: respective percentages of tumor necrosis factor alpha (TNF-α) and interleukin-2 (IL-2) cells among IFN-γ–positive cells. (D) Mean intranuclear Ki67 expression of endogenous CD8+ multimer-negative cells (black columns) and preinfusion CTL products and multimer-positive CD8+ T cells (gray columns) at indicated time points for all patients combined. Two-tailed paired t tests were used for statistical analysis. NS, not significant. (*)P < .05. (†) P < .005. (‡) P < .001.

Fig A1.

Reactivity to nontargeted epitopes. Interferon gamma (IFN-γ) spots per 105 peripheral-blood mononuclear cells (PBMCs;y-axis) at indicated time points (x-axis) before and after the infusion of polyclonal cytotoxic T cells (CTLs) generated in the presence of interleukin-21 for patients who (A) achieved complete remissions, partial responses, or stable disease or (B) experienced disease progression after receiving treatment. Pound signs indicate patients who had received prior ipilimumab, green arrows indicate CTL infusions, and horizontal lines indicate ipilimumab administration with the total number of doses received indicated immediately above. The scales of they-axis for graphs for patients 1 (maximum, 700 spots/105 PBMCs) and 4 (maximum, 3,000 spots/105 PBMCs) are different from all others (maximum, 400 spots/105 PBMCs). Two-tailed paired ttests were used for statistical analysis. NS, not significant. (*)P < .05. (†) P < .005.