Identification of a colorectal tumor-associated antigen (COA-1) recognized by CD4(+) T lymphocytes

Abstract

Only a limited number of target molecules have been shown to be recognized by colon tumor-reactive T cells, limiting the options for the development of immunotherapies for patients with colon cancer. The current studies were undertaken in an attempt to generate tumor-reactive T cells that could be used to identify and characterize novel colon tumor-associated antigens. Multiple CD4(+) T-cell clones isolated either from tumor-infiltrating lymphocytes or peripheral blood mononuclear cells that were sensitized in vitro with autologous tumor cells from a colon cancer patient, 1869, recognized autologous tumor cells in a class II HLA-DR-restricted manner. One of the peripheral blood mononuclear cell clones, clone C111, was used to screen pools of clones that were generated from an autologous colon tumor cell line cDNA library. A cDNA clone that was isolated encoded a protein that was termed colorectal tumor-associated antigen-1 (COA-1). This product was recognized in the context of the two autologous HLA-DRbeta1 alleles, HLA-DRbeta1*0402 and DRbeta1*1301. The nucleotide sequence of the COA-1 transcript was nearly identical to multiple expressed sequence tag sequences that encode variants of Socius, a protein that was found recently to bind to members of the Rnd family of GTPases. The COA-1 gene was expressed at relatively comparable levels in colorectal and melanoma tumor cells, EBV-infected B cells, normal B cells, and cultured fibroblast cell lines. However, the gene that was isolated from normal cell types contained a single nucleotide substitution, resulting in an amino acid change near the COOH terminus of the protein. Although the minimal epitope recognized by CD4(+) cells was encoded by sequences that were upstream from this substitution, C111 T cells did not appear to recognize the normal gene product. Therefore, this alteration may either affect the localization or the processing of this gene product, which may at least in part be responsible for the differential recognition of tumor and normal cells.

Fig. 1

Phenotypic characterization of the colorectal cancer line 1869 col. A, antibodies directed against MHC class I (W6/32) and class II (L243) molecules, an epithelium marker (Ber-EP4), and the β subunit of prolyl-4-hydroxylase (5B5), a protein expressed exclusively in fibroblasts, were used to stain the 1869 col cell line. B, intracellular staining was carried out using three cytokeratin reactive mAbs, CK18, which reacts with cytokeratin 18, LP34, which reacts with multiple cytokeratins, and MNF116, which reacts with cytokeratins 5, 6, 8, 17, and probably 19. C, staining of 1869 col cells at passage 6 (P6) and passage 20 (P20) was carried out with the anti-CEA mAb Col-1.

Fig. 2

A cDNA clone isolated from the 1869 cDNA library encodes an antigen recognized by C111 T cells. The 293 cells expressing the MHC DRβ1*0402 or 1301 molecules were transfected with the 1D8 cDNA clone, or COA-1a, which corresponds to nucleotides 209-1318 of the COA-1 gene (Fig. 3). Target cells were either transfected with the COA-1a product alone or were cotransfected with a mixture of COA-1a and the full-length HLA class II Ii. Additional targets were transfected with a control plasmid encoding green fluorescent protein. Eighteen h after the addition of 5 × 10⁴ C111 T cells to the transfectants, supernatants were collected and IFN-γ release was measured by ELISA.

Fig. 3

Sequence of the COA-1 gene isolated from the mRNA of the tumor line 1869 col. The COA-1 gene was isolated by RT-PCR from the 1869 col tumor cell line. The sequence of the 1D8 cDNA clone is indicated by bold letters, the amino acid sequence corresponding to the T-cell epitope is underlined, and the single nucleotide difference between the normal and tumor transcripts at position 1280 is noted. The position of the primers used to generate the COA-1a product is indicated by the arrows, and the amino acid sequence of the longest open reading frame in this transcript, which is similar to the Socius gene product (20), is noted beneath the nucleotide sequence.

Fig. 4

The COA-1 transcript derived from normal B cells is not recognized by the clone C111 T cells. 293 cells expressing the indicated MHC DRβ1 molecules were transfected with COA-1a cDNAs isolated by RT-PCR from either the 1869 col cell line or from 1869 CD40L-stimulated B cells. The green fluorescent protein and Ii-1D8 constructs were used as negative and positive controls, respectively. Eighteen h after the addition of 5 × 10⁴ C111 T cells to the transfectants, supernatants were collected, and IFN-γ release was measured by ELISA.