Evaluation of prime/boost regimens using recombinant poxvirus/tyrosinase vaccines for the treatment of patients with metastatic melanoma

Abstract

Purpose: Two clinical trials were conducted to evaluate the clinical efficacy and immunologic impact of vaccination against the tyrosinase protein plus systemic interleukin 2 (IL-2) administration in patients with advanced metastatic melanoma.

Experimental design: Full-length tyrosinase was employed as an immunogen to induce diverse immunologic responses against a commonly expressed melanoma antigen. Heterologous prime/boost vaccination with recombinant vaccinia and fowlpox vectors encoding tyrosinase was first explored in a randomized three-arm phase II trial, in which vaccines were administered alone or concurrently with low-dose or high-dose IL-2. In a subsequent single cohort phase II trial, all patients received the same vaccines and high-dose IL-2 sequentially rather than concurrently.

Results: Among a total of 64 patients treated on these trials, 8 objective partial responses (12.5%) were observed, all in patients receiving high-dose IL-2. Additional patients showed evidence of lesional regression (mixed tumor response) or overall regression that did not achieve partial response status (minor response). In vitro evidence of enhanced immunity against tyrosinase following protocol treatments was documented in 3 of 49 (6%) patients tested serologically, 3 of 23 (13%) patients tested for T-cell recognition of individual tyrosinase peptides, and 4 of 16 (25%) patients tested for T-cell recognition of full-length tyrosinase protein with real-time reverse transcription-PCR techniques.

Conclusions: Whereas prime/boost immunization with recombinant vaccinia and fowlpox viruses enhanced antityrosinase immunity in some patients with metastatic melanoma, it was ineffective alone in mediating clinical benefit, and in combination with IL-2 did not mediate clinical benefit significantly different from that expected from treatment with IL-2 alone.

Fig. 1

Protocol schemas for the randomized phase II trial that accrued patients from 1999 to 2002 (A) and the single cohort phase II trial that accrued patients from 2003 to 2004 (B). Vaccines were administered i.m. at 2 × 10⁹ plaque-forming units per dose.”Response” refers to complete or partial clinical response.

Fig. 2

Tumor regression associated with intratumoral lymphoid infiltrates in patient 42, who manifested a mixed response following treatment with poxvirus/tyrosinase vaccines plus high-dose IL-2 (arm 3 on the randomized trial). S100 and tyrosinase are expressed in normal/atypical melanocytes as well as melanoma cells. Whereas tyrosinase protein is expressed in both a regressing s.c. (SQ) lesion and a progressing gall bladder (GB) metastasis, CD8+ lymphoid infiltrates are seen only in the regressing lesion. Staining with anti-CD4 gave similar results (not shown). Both metastatic lesions expressed MHC I uniformly and MHC II poorly (not shown). Concomitant infiltration of immune cells into an atypical nevus correlated clinically with the development of cutaneous depigmentation surrounding benign or atypical nevi and suggested the development of immunity against a shared melanoma-melanocyte lineage antigen.

Fig. 3

Antiviral IgG titers induced by vaccination. Patients on the randomized clinical trial received rF-TYR (FP) and rV-TYR (VV) on an alternating schedule every 4 weeks, starting with rF-TYR. Sera collected before treatment or 4 weeks following each inoculation were tested by ELISA for antiviral IgG. A, following one round of heterologous prime/boost inoculation in 12 patients, there was no evidence for serologic cross-reactivity between the two poxviruses. Naïve serum titers in the same assays were <50 for vaccinia virus and <50 to 229 for fowlpox virus. B, repeat immunizations with the same recombinant viruses in 11 patients showed maximal antivaccinia titers after the first rV-TYR and maximal anti-fowlpox titers after the second rF-TYR.

Fig. 4

Antityrosinase IgG response developing in patient 18 after vaccination plus low-dose IL-2 (arm 2 on the randomized trial). Western blots containing lysates of COS-7 cells transfected with pcDNA3.1/tyrosinase (1.2 × 10⁵ cell equivalents/lane) or infected with the nonrecombinant vaccinia vector TBC-Wy (0.7 × 10⁵ cell equivalents/lane) were probed simultaneously with sera (1:50) collected before treatment, after the first treatment cycle (rF-TYR + IL-2), and after the second cycle (rV-TYR + IL-2). Lane 1, untransfected COS-7 cells; lane 2, COS-7 transfected with pcDNA3.1/tyrosinase; lane 3, uninfected COS-7; lane 4, COS-7 infected with TBC-Wy. The murine antityrosinase monoclonal antibody T311 provides a positive control for tyrosinase protein expression; arrows, monomeric and multimeric tyrosinase bands. Tyrosinase protein was visualized only with serum collected after the second treatment cycle. Antivaccinia IgG was evident pretreatment but increased significantly after the rV-TYR inoculation.

Fig. 5

Antityrosinase T-cell responses generated in patients undergoing treatment. A, PBL were collected from patient 35 before treatment, after four cycles (course 1), and after six cycles (mid-course 2) of vaccines plus high-dose IL-2 (arm 3 on the randomized trial). After repetitive in vitro sensitization for 4 weeks with the HLA-A1-restricted Ty 244S peptide, PBL obtained posttreatment, but not before treatment, could specifically recognize Ty 244S. Results of an IFNγ ELISA following an overnight stimulation of cultured PBL with Ty 244S or other HLA-A1-restricted peptides are shown. Simultaneous in vitro sensitization with Ty 146-156 or Ty 243-251 failed to raise reactive T cells (not shown). Clinically, this patient had a mixed tumor regression. B, in real-time RT-PCR assays quantifying IFNγ mRNA, PBL from patient 3 (HLA-A1 and -A24) failed to recognize known tyrosinase epitopes but showed specific reactivity against tyrosinase protein expressed in autologous dendritic cells. Reactivity increased following sequential treatments with recombinant vaccines and high-dose IL-2 (arms 1 and 4 on the randomized trial). Clinically, this patient had a partial response after IL-2 therapy. Stimulation index for peptide reactivity, copies IFNγ mRNA/copies CD8 mRNA in PBL stimulated with peptide, compared with no peptide; for protein reactivity, PBL response to dendritic cells infected with Ad2/Ty or Ad2/gp100, compared with Ad2/green fluorescent protein control. Indices ≥2 are considered positive. Modified from ref. .