Using gene expression cloning, we have identified Npm as the antigen recognized by CTL007 on CRC cells. This is the first identification of Npm as a T cell antigen. The cDNA clone we have isolated from CRC cells encodes full-length non-mutated Npm. In contrast, genomic translocation of Npm and expression of a fusion protein (Npm –ALK) are common in several haematological malignancies 24, 25
. Npm mRNA and protein analysis in WC007 CRC cells recognized by CTL007 excluded the presence of an Npm fusion protein in these cells. Npm protein levels were increased in CRC cells compared to normal colon tissue (), in agreement with the demonstrated increase in Npm mRNA levels in CRC 8, 22
. Increased tumor expression of Npm, which has been related to decreased cell doubling time 26
, was correlated with unfavorable prognosis in bladder cancer patients 10
. A possible role of Npm as an oncogene has been suggested (reviewed in 5, 6
). Antigens encoded by oncogenes that drive proliferation of tumor cells are unlikely to be lost from the cells and hence may make more stable and consistent target antigens. Furthermore, immunological targeting of such antigens may be beneficial for patients as their expression by tumors may be associated with poor prognostic outcome of the disease. A growth stimulatory role has been suggested for the antigens ABCE1 27
, TYMS and PGK1 28
which are recognized by CRC patients’ CTL, and therefore these antigens, in addition to Npm, have potential as immunotherapeutic targets for CRC patients.
CTL007 recognized 2 of 3 Npm peptides tested (). Recognition of two peptides with quite distinct amino acid sequences by the CTL may be explained by the polyclonality of the CTL. However, Npm may be the only antigen recognized by the CTL line as treatment of the target cells with shRNA specific for Npm totally abolished target cell lysis by the CTL (). Peptide 2, which induced highest sensitization of target cells for lysis by CTL007 (), not only induced CTL in PBMC of patient 007, but also in the PBMC of three of five additional HLA-A1 positive CRC patients available to us for study (). The CTL induced by the peptide in three patients not only lysed peptide-coated target cells, but also, albeit to a lesser extent, CRC cells endogenously expressing Npm (). Unfortunately autologous tumor cells were not available in any of the patients. It needs to be emphasized that CTL007 which was induced by stimulating 007PBMC with autologous WC007 CRC cells showed significantly higher lysis [up to 54%; 4
] of the CRC cells than the CTL induced in the same PBMC by peptide 2 (up to 20% lysis in various experiments; shows one such experiment). Tumor cells presenting multiple Npm epitopes, including both CTL and T helper epitopes, may be superior T cell stimulators than a peptide representing only a single CTL epitope. High peptide concentrations (12.5µM) were needed to generate CTLs from PBMC (data not shown), suggesting that the CTLs are of low avidity.
CTL007 lysed 2 (WC007, HT29) of 4 HLA-A1 expressing CRC cell lines tested 4
(). Target cell lysis was not associated with Npm total protein expression levels, as all 4 CRC cell lines tested and one non-lysed HLA-A1 positive melanoma cell line (WM793) expressed the protein by Western blot analysis ( and ). These data suggest that differences in the susceptibility of target cells to lysis by CTL007 cannot be explained by differences in Npm protein expression levels by the target cells. Npm gene sequence analysis of a few cell lines (WC007, WC013) indictated no difference in the Npm gene sequence from cells lysed by CTL007 and cells not lysed (data not shown). There was also no difference in the amount or size of nuclear and cytoplasmic Npm protein in cells lysed or not lysed by CTL007 (data not shown). The reason for differences in lysis of different target cells is unknown and needs further investigation. Npm may provide a vaccine for HLA-A1 positive CRC patients and patients with tumors of other tissue origins expressing Npm, such as melanoma (29
and ), breast carcinoma (), hepatocellular carcinoma 9
, and squamous cell carcinoma 21
. Furthermore, in addition to HLA-A1 binding epitopes, Npm expresses multiple other epitopes potentially binding to each of the HLA listed at http://www.syfpeithi.de
and therefore also has vaccine potential for patients with these HLA types.
Absence of correlation of Npm protein expression and lysis of cells by CTL007
In conclusion, overexpression of Npm by tumors of various histological types, recognition of the antigen by T cells derived from different CRC patients, expression of anchors for all known HLA types on the protein, and association of the antigen with poor prognostic outcome make it a promising target for immunotherapeutic intervention in cancer patients.