Adoptive T cell therapy using antigen-specific CD8+ T cell clones for the treatment of patients with metastatic melanoma: in vivo persistence, migration, and antitumor effect of transferred T cells

Abstract

Adoptive T cell therapy, involving the ex vivo selection and expansion of antigen-specific T cell clones, provides a means of augmenting antigen-specific immunity without the in vivo constraints that can accompany vaccine-based strategies. A phase I study was performed to evaluate the safety, in vivo persistence, and efficacy of adoptively transferred CD8+ T cell clones targeting the tumor-associated antigens, MART1MelanA and gp100 for the treatment of patients with metastatic melanoma. Four infusions of autologous T cell clones were administered, the first without IL-2 and subsequent infusions with low-dose IL-2 (at 0.25, 0.50, and 1.0 x 10(6) unitsm(2) twice daily for the second, third, and fourth infusions, respectively). Forty-three infusions of MART1MelanA-specific or gp100-specific CD8+ T cell clones were administered to 10 patients. No serious toxicity was observed. We demonstrate that the adoptively transferred T cell clones persist in vivo in response to low-dose IL-2, preferentially localize to tumor sites and mediate an antigen-specific immune response characterized by the elimination of antigen-positive tumor cells, regression of individual metastases, and minor, mixed or stable responses in 8 of 10 patients with refractory, metastatic disease for up to 21 mo.

Fig 1.

Treatment schedule. Four infusions of antigen-specific CD8⁺ CTL clones are scheduled at 2-wk intervals between the first and second infusion, and at 3-wk intervals for subsequent infusions given with a 14-day course of low-dose IL-2. The IL-2 is administered s.c., twice daily for 14 days at 0.50, 1.0, and 2.0 million units/m²/day for the second, third, and fourth infusions, respectively.

Fig 2.

Analysis of T cell frequency in PBMCs after infusion of MART1-specific CD8⁺ T cell clones without IL-2. Two methods were used: semiquantitative PCR analysis of a clone-specific CDR3 region (gel bands digitized and quantified based on dilution standards 10⁻² to 10⁻⁵) and tetramer analysis by flow cytometry. The T cell frequency in both cases was characterized by a rapid rise on day +1 after the infusion followed by a steady decline to nearly undetectable levels by day +14 and a median T cell survival in each case of ≈7 days.

Fig 3.

Analysis of T cell persistence in vivo after infusion of MART1-specific CD8⁺ T cell clones without IL-2 (INFUSION #1) and with IL-2 (INFUSION #2). Peripheral blood samples from patient 1017-6 were analyzed pre-T cell infusion (day 0), days 1, 7, 14, and 21 after T cell infusion without IL-2 (INFUSION #1) and with low-dose IL-2 (INFUSION #2). Percentage of tetramer⁺, CD8⁺ T cells over total CD8⁺ T cells is displayed in the right upper quadrant. Sustained elevation in the frequency of MART1-tetramer⁺ T cells in the peripheral blood to at least day 14 (1.02 vs. <0.05%) is observed when low-dose IL-2 is given (INFUSION #2) compared with infusion of T cells when no IL-2 is given (INFUSION #1).

Fig 4.

Analysis of T cell infiltrate from tumor nodule harvested from patient 1017-2 after infusion of MART1-specific CD8⁺ T cell clones. Percentage of infused clone (tetramer⁺, CD8⁺ T cells) among all CD8⁺ T cells is displayed in the upper right quadrant. Significant infiltrate comprised of MART1-specific CD8⁺ T cells is seen in postinfusion tumor nodule (37.7%; A) compared with preinfusion nodule (0.0%; B) and postinfusion peripheral blood (0.8%; C) demonstrating preferential localization of infused clones to tumor site. Less than 0.2% of CD8 T cells in tumor nodule stain with an irrelevant tetramer (tyrosinase-peptide tetramer; D).

Fig 5.

Radiographic response in metastatic lesions after adoptive T cell therapy. Two peripheral pulmonary nodules in the left lower lobe present pre-T cell therapy in patient 1017-6 are absent after the fourth infusion of MART1-specific T cell clones (A). However, a new 1+ cm nodule appears more in the lobe during this interval, demonstrating a mixed response to therapy. In patient 1017-2 (B), partial resolution of paratracheal adenopathy is observed after the fourth infusion of MART1-specific T cells.

Fig 6.

Immunohistochemical analysis of antigen expression in preinfusion tumor and recurrent postinfusion tumor nodule appearing after MART1-specific T cell therapy. Gp100, tyrosinase, and MART1 expression are seen in the tumor sample preinfusion. Selective loss of MART1 expression is seen in postinfusion tumor cells, demonstrating the outgrowth of antigen-loss tumor variant.