Simultaneous humoral and cellular immune response against cancer-testis antigen NY-ESO-1: definition of human histocompatibility leukocyte antigen (HLA)-A2-binding peptide epitopes

Abstract

A growing number of human tumor antigens have been described that can be recognized by cytotoxic T lymphocytes (CTLs) in a major histocompatibility complex (MHC) class I-restricted fashion. Serological screening of cDNA expression libraries, SEREX, has recently been shown to provide another route for defining immunogenic human tumor antigens. The detection of antibody responses against known CTL-defined tumor antigens, e.g., MAGE-1 and tyrosinase, raised the question whether antibody and CTL responses against a defined tumor antigen can occur simultaneously in a single patient. In this paper, we report on a melanoma patient with a high-titer antibody response against the "cancer-testis" antigen NY-ESO-1. Concurrently, a strong MHC class I-restricted CTL reactivity against the autologous NY-ESO-1-positive tumor cell line was found. A stable CTL line (NW38-IVS-1) was established from this patient that reacted with autologous melanoma cells and with allogeneic human histocompatibility leukocyte antigen (HLA)-A2(-), NY-ESO-1-positive, but not NY-ESO-1-negative, melanoma cells. Screening of NY-ESO-1 transfectants with NW38-IVS-1 revealed NY-ESO-1 as the relevant CTL target presented by HLA-A2. Computer calculation identified 26 peptides with HLA-A2-binding motifs encoded by NY-ESO-1. Of these, three peptides were efficiently recognized by NW38-IVS-1. Thus, we show that antigen-specific humoral and cellular immune responses against human tumor antigens may occur simultaneously. In addition, our analysis provides a general strategy for identifying the CTL-recognizing peptides of tumor antigens initially defined by autologous antibody.

Figure 1

Western blot analysis of NW38 serum antibody reactivity. Lanes 1–5 contained 1 μg of recombinant NY-ESO-1 protein, tested against serum from a healthy donor (lane 1), NW38 serum (lanes 2–4, serum dilutions 1:1,000, 1:10,000, and 1:100,000), or an anti–NY-ESO-1 mouse monoclonal antibody (lane 5, hybridoma supernatant 1:50 dilution). NW38 serum was also tested, at 1:1,000 dilution, against lysates of 5 × 10³ autologous NW-MEL-38 tumor cells (lane 6), of 2 × 10⁴ untransfected COS-7 cells (lane 7), and of 2 × 10⁴ NY-ESO-1–transfected COS-7 cells (lane 8).

Figure 2

Cytotoxicity of NW38-IVS-1 against NY-ESO-1⁺ and NY-ESO-1⁻ cell lines. Specific lysis of the autologous NY-ESO-1⁺ melanoma cell line NW-MEL-38, and the NY-ESO-1⁺/HLA-A2⁺ allogeneic melanoma cell lines SK-MEL-37 and MZ-MEL-19 was observed. The NY-ESO-1⁺ MHC class I^- melanoma cell line SK-MEL-19, the NY-ESO-1⁻ HLA-A2⁺ melanoma cell line NW-MEL-145, K562, and NW38 PHA blasts used as controls were not lysed. Values represent the specific lysis of ⁵¹Cr-labeled target cells (E/T ratios: 90 [solid], 30 [checkered], 10 [striped], and 1:1 [open], as assessed by a standard ⁵¹Cr-release assay.

Figure 3

TNF-α–release assays after stimulation of the CTL line NW38-IVS-1 by COS-7 cell transfectants. TNF release was detected against COS-7 cells cotransfected with the expression vector pcDNA3.1 (−) containing NY-ESO-1 cDNA, and pcDNA1Amp containing HLA-A2 cDNA. No TNF release was detected against COS-7 transfected with vector alone, vector/HLA-A1, vector/HLA-A2, vector/NY-ESO-1, and vector/NY-ESO-1/HLA-A1.

Figure 4

TNF-α–release assays after stimulation of CTL line NW38-IVS-1 by HLA-A2–transfected COS-7 cells pulsed with HLA-A2–binding peptides encoded by NY-ESO-1-at 10 μg/ml. Cotransfectants of NY-ESO-1 and HLA-A2 were used as a positive control.