Identification of tyrosinase-related protein 2 as a tumor rejection antigen for the B16 melanoma

Abstract

Recently, major advances have been made in the identification of antigens from human melanoma which are recognized by T cells. In spite of this, little is known about the optimal ways to use these antigens to treat patients with cancer. Progress in this area is likely to require accurate preclinical animal models, but the availability of such models has lagged behind developments in human tumor immunology. Whereas many of the identified human melanoma antigens are normal tissue differentiation proteins, analogous murine tumor antigens have not yet been identified. In this paper we identify a normal tissue differentiation antigen, tyrosinase-related protein 2 (TRP-2), expressed by the murine B16 melanoma which was found by screening a cDNA library from B16 with tumor-reactive cytotoxic T lymphocytes (CTL). A peptide conforming to the predicted MHC class I H2-Kb binding motif, TRP-2181-188, was identified as the major reactive epitope within TRP-2 recognized by these anti-B16 CTLs. By site-directed mutagenesis, it was shown that alteration of this epitope eliminated recognition of TRP-2. It was further demonstrated that a CTL line raised from splenocytes by repeated stimulation in vitro with this peptide could recognize B16 tumor and was therapeutic against 3-d-old established pulmonary metastases. The use of TRP-2 in a preclinical model of tumor immunotherapy may be helpful in suggesting optimal vaccination strategies for cancer therapy in patients.

Figure 1

Stimulation of mINF-γ release from line A T cells by various murine tumor lines. 10⁵ line A T cells and 10⁵ tumor cells were coincubated in 2 ml of CM for 24 h. Aliquots of supernatant were assayed for mINF-γ. All targets were MHC type H2^b except P815, 9873, and CT26 (H2^d). Line A CTLs demonstrated relative specificity for the B16 tumor.

Figure 2

Alignment of TRP-2 inserts found by cDNA expression library screening with anti-B16 CTLs. The top line indicates the composite TRP-2 cDNA sequence described by Jackson et al. (8). The next line (shaded) represents the coding region for the TRP-2 protein. The location of the five TRP-2 cDNA clones identified are shown below (striped).

Figure 3

Release of mIFN-γ by line A when incubated with MC-38 (H2^b) and varying amounts of TRP-2_181–188 peptide (circles). No significant mIFN-γ release was seen when MC-38 was incubated with peptide without line A (square) or with line A without peptide (triangle).

Figure 4

Treatment of 3-d-old pulmonary metastases from B16 by the intravenous administration of T cells reactive with TRP-2_181–188 generated by in vitro peptide stimulation of splenocytes from normal or B16immunized mice. Low-dose systemic IL-2 was given with adoptive cell transfer. Therapeutic effects were seen from anti–TRP-2_181–188 CTLs from normal mice (P = 0.0004 vs. IL-2 alone, and P = 0.014 vs. ovalbumin_257–264 stimulated cultures), as well as from anti-B16 immunized mice (P = 0.0003 vs. IL-2 alone, and P = 0.010 vs. ovalbumin_257–264 stimulated cultures).