(CANCER RESEARCH 58. 5144-5150. November 15, 1998] The Peptide Recognized by HLA-A68.2-restricted, Squamous Cell Carcinoma of the Lung-specific Cytotoxic T Lymphocytes Is Derived from a Mutated Elongation Factor 2 Gene1 Kevin T. Hogan,2 Dominic P. Eisinger, Samuel B. Cupp HI, Kristen J. Lekstrom, Donna D. Deacon, Jeffrey Shabanowitz, Donald F. Hunt, Victor H. Engelhard, Craig L. Slingluff, Jr., and Mark M. Ross Argonex Pharmaceuticals, Charlollesville, Virginia 22903 ¡K.T. H., K. J. L, S. B. C.. M. M. K.I and Lake Placid. New York ID. P. E.¡,and the Departments of Surgery ¡D.D. D.. C. L. S.¡.Chemistry ¡J.S.. D. F. H.¡.Pathology ¡D.F. H.I, and Microbiology ¡V.H. £./, University of Virginia. Charlotte'sviile, Virginia 2290« ABSTRACT The identification of naturally processed tumor peptides that can stim ulate a tumor-specific, (II response is crucial to the development of a vaccine-based, immunotherapeutic approach to cancer treatment. One type of cancer in which a tumor-specific, (II response has been observed is squamous cell carcinoma of the lung. In the system investigated here, the tumor-specific (Ils are HLA-A68.2 restricted. Immunoaffinity chro- matography »asused to isolate the HLA-A68.2 molecules from the tumor cell line, and peptide «aseluted with acid from the HLA-A68.2 molecules and subjected to three rounds of separation by reversed phase-high performance liquid chromatography (RP-HPLC). To determine which fractions contained the peptide recognized by the tumor-specific <"l'I,s. an aliquot of each RP-HPLC fraction was added to the autologous, B- lymphoblastoid cell line, and the cells were then tested as targets for tumor-specific CTLs. After the third round of RP-HPLC, mass spectro- metry was used to sequence individual peptide candidates, and a peptide with a in/:, of 497 was identified as the active peptide. Collision-activated dissociation of HI/- 497 allowed identification of the peptide sequence as ETVSEQSNV. With the exception of a single amino acid difference (glu- tamic acid versus glutamine as the sixth position in the peptide), this peptide is identical to residues 581 to 589 of elongation factor 2. The PCR was used to amplify the elongation factor 2 gene in both the tumor cells and the autologous B cell line, and DNA sequencing of the products revealed the presence of a heterozygous mutation in the tumor cells that accounts for the difference between the two peptide sequences. Although a similar analysis did not reveal the presence of the mutation in three additional lung cell carcinomas, this does not rule out the possibility that a survey of a larger population of tumor cells would reveal the presence of the mutation at a low frequency. These results demonstrate the utility of this approach for identifying tumor-specific antigens that are the targets of a CTL response. INTRODUCTION Lung cancer is expected to account for 178,100 (13%) of all new cancer cases in 1997.3 Because the disease typically metastasizes to distant locations before diagnosis, a combination of radiation therapy, chemotherapy, and surgery must frequently be used in an attempt to eradicate the disease. In spite of advances in these treatment areas, the combined 5-year survival rate for all stages of the disease is only 14%. The development of a new therapeutic approach to lung cancer that would be effective against both the primary lesion and distant métas tases would, therefore, have a significant impact on disease mortality. One such potential approach to lung cancer treatment is CTL-medi- ated immunotherapy. CTL-mediated immunotherapy is based on the observation that the Received 6/4/98: accepted 9/18/98. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported in pan by SBIR Grant 1R43CA768I8 from the National Cancer Institute (to M. M. R.I. ~ To whom requests for reprints should be addressed, at Argonex Pharmaceuticals, 706 Forrest St.. Suite 1. Charlottesville, VA 22903. ' American Cancer Society Website (www.cancer.org). immune system can make an effective response to specific antigens expressed on autologous tumor cells. It has been shown that human CTLs recognize sarcomas (1), renal cell carcinomas (2), colorectal carcinomas (3), ovarian carcinomas (4, 5), pancreatic carcinomas (6, 7), squamous tumors of the head and neck (8), and squamous carci nomas of the lung (9, 10). The largest number of reports of human tumor-reactive CTLs, however, concern melanoma (reviewed in Ref. 11). The ability of tumor-reactive CTLs to mediate tumor regression, both in humans (12) and in animal models (13-15), suggests that efforts directed at increasing CTL activity would likely have a bene ficial effect with respect to cancer treatment. Although CTLs can be directly stimulated with tumor cells, vacci nation of a patient population would be greatly expedited if the antigen that is recognized by the CTLs can be isolated and identified. Unlike the antigens that are recognized by antibodies, the antigens recognized by the T-cell receptor on a CTL are small peptides, 8-10 amino acids in length, that are bound to class I MHC4-encoded molecules (16). In humans, both tumor-specific and tumor-associated T-cell epitopes have been identified in melanoma (17-28), ovarian carcinoma (29-32), breast carcinoma (32), non-small cell lung carci noma (10), and pancreatic carcinoma (7). Because initial studies of peptide immunization in human melanoma patients have yielded promising results (33, 34), the identification of additional peptides that act as T-cell epitopes for these and other cancers is of critical importance for the development of a vaccine-based, immunotherapeu tic approach to cancer treatment. Toward this end, we have developed previously a CTL line that recognizes an autologous squamous cell cancer of the lung (9). Antibody-blocking experiments demonstrated that the CTLs recognized antigen in association with HLA-A68, and reconstitution experiments with peptide derived from HLA-A68 and separated by RP-HPLC demonstrated that at least one peptide fraction could reconstitute the epitope recognized by the CTLs. The objective of the work described was to determine: (a) the identity of the peptide that was recognized by the CTL; (b) the protein origin of the antigenic peptide; and (c) whether this antigen is expressed in additional lung cell carcinomas. MATERIALS AND METHODS Cell Lines. VBT2, a squamous cell carcinoma of the lung, was established previously in culture from a metastatic lesion in a 45-year-old African- American male (9). All of the material from this patient was obtained follow ing informed, written consent. The lung cell cancer lines CALU-1 (epider- moid), Sk-Mes-1 (squamous), and Sk-Lu-1 (adenocarcinoma) were obtained from the American Type Culture Collection. The cells were maintained in RPMI 1640 containing 5% FBS and 2 mM L-glutamine. VBT2-EBV, a B- lymphoblastoid cell line obtained from the same patient as were the VBT2 tumor cells (9), was maintained in RPMI 1640 containing 10% FBS and 2 mM 4 The abbreviations used are: MHC. major histocompatibility complex; RP-HPLC. reversed phase-high performance liquid chromatography; FBS, fetal bovine serum; HFBA. heptafluorobutyric acid; TFA. trifluoroacetic acid; i.d.. inside diameter; o.d., outside diameter: CAD, collision-activated dissociation: EF2, elongation factor 2. 5144 Research. on August 23, 2015. © 1998 American Association for Cancer cancerres.aacrjournals.org Downloaded from