Antibody to ras Proteins in Patients with Colon Cancer1

The current study examined sera from 160 colon cancer patients and 60 normal individuals to determine whether antibody to mutated p21 ran protein was present. Studies focused on the aspartic acid substitution at amino acid position 12 (denoted D12), one of the most common mutations in colon adenocarcinoma. IgA antibodies directed against mutated p2! ras-D12 protein were detected in 51 (32%) of 160 colon cancer patients, but only in 1 (2.5%) of 40 normal individuals. The greater incidence of antibody in cancer patients provides presumptive evidence that immunization to the ras proteins occurred as a result of the malignancy. Examination of sera for antibody reactivity to wild-type p21 ras protein (denoted p2! ras-G12) as well as p2! ran proteins bearing the D12, V12, S12, or L61 mutations showed that antibody detected was largely to normal segments of the p21 ras protein. Epitope mapping, using peptide neutralization assays with mutated or normal ras peptides as competitors, demonstrated that in 10 (67%) of 15 sera examined the antibody reactivity to p2! ras-G12 protein was neutralized by peptides near the carboxyl terminus of p21 ras protein, but not by peptides spanning the specific point mutation region. Antibody reactivities correlated with peripheral blood lymphocyte count, but did not correlate with patient age, sex, histology, stage, tumor locus, lymph node metastasis, or serum carcinoembryonic antigen. Introduction The ras oncogenes are cancer-related genes that become activated by specific point mutations and can be identified in a Received 5/4/95; revised 7/10/95; accepted 7/13/95. This work was supported by Grant CA54561 from the National Cancer Institute, Department of Health and Human Services. 2 Present address: Department of Surgery, Yokohama City University School of Medicine, 3-9 Fukuura Kanazawa-ku, Yokohama 236, Japan. To whom requests for reprints should be addressed, at Division of Oncology, University of Washington, Box 356527, Seattle, WA 981956527. wide variety of human malignancies (I, 2). Three ras genes have been defined in the human genome, H-ras-1, K-ras-2, and N-ras (3). These genes encode a highly conserved group of Mr 21,000 proteins, denoted as p21 ras. Activation of the ras proto-oncogene occurs most commonly at codon 12 or codon 61 and results in corresponding single amino acid substitutions within the p2l protein. The activating amino acid substitutions impair the GTPase activity of the ras protein and generate constitutively activated signaling complexes with transforming activity (4, 5). Mutated ras proteins are implicated in malignant transformation of many human cancers including approximately 45% of colon adenocarcinomas. The most common specific amino acid substitutions replacing glycine at amino acid position 12 of the wild-type p21 ras protein in colon adenocarcinoma are aspartic acid (13%), valine (15%), cysteine (6%), or serine (4%; Ref. 6). Only a few cases with leucine replacing glutamine at amino acid position 61 have been reported. Mutated p2l ras proteins are not expressed by normal tissue and thus represent cancer-specific proteins. Mice immunized to whole mutated ras proteins are able to mount a T-cell response that is specific for the mutated segment of the molecule (7, 8). Conversely, immunizing animals with peptides that span the mutated segment can result in the generation of both helper/ inducer and cytolytic T-cell responses that recognize mutated protein (9, 10). Recently, it has been demonstrated that T cells from normal individuals can recognize peptides that span the mutated segment of the molecule (11-13). Furthermore, memory T cells, isolated from a patient with a follicular thyroid carcinoma, were found to specifically recognize a ras peptide with a leucine substitution at residue 61 (14), a common mutation in that tumor. Studies in our laboratory examining peripheral blood lymphocytes from patients with pancreatic cancer showed that CD4 T-celI immunity to the mutated segment of ras protein was present in some patients with pancreatic cancer. Studies focused on the aspartic acid substitution (denoted D12) as the most common mutation. Patients were found in whom T cells responded to both ras D12 peptides and p21 ras-D12 protein (15). Existent specific T-celI reactivity to mutated ras peptides and proteins imply that the patient’s own tumor acts as a source of immunizing protein and that mutated ras proteins can elicit immune responses in humans. Often, helper/inducer T cells and antibodies can respond to the same protein. The current study examined whether antibodies specific to ras proteins exist in sera of patients with colon cancer. Sera from 160 patients with colon cancer and 60 normal donors were examined to evaluate antibody reactivity to p21 ras proteins using ELISA. Studies focused on the aspartic acid substitution (D12). IgA antibodies directed against p21 ras-Dl2 protein could be detected in colon cancer patients to a substantially greater extent than in normal individuals. However, the majority of antibody detected was to the normal but not the mutated portion of p21 ras proteins. The greater incidence of antibody in cancer patients provides evidence that immunization to the ras proteins occurred as a result of the malignancy. Antibody correlated to peripheral blood lymphocyte counts. 1072 Antibody to ras Proteins in Colon Adenocarcinoma The abbreviations used are: HRP, horseradish peroxidase; CEA, carcinoembryonic antigen. Whether or not the presence of antibody correlates with outcome has not yet been evaluated. Materials and Methods Sera. Sera were collected from 155 patients with newly diagnosed colon cancer, 5 patients with recurrent colon cancer, and 60 healthy normal individuals. One hundred sixteen patients from Yokohama City University Hospital (Yokohama, Japan) and Kinki University Hospital (Osaka, Japan), and 44 patients from the University of Washington Medical Center (Seattle, WA), Loyola University Medical Center (Chicago, IL), and Mayo Clinic (Rochester, MN) were studied. Blood (10 ml) from each colon cancer patient or normal individual was obtained with informed consent. Sera were stored at -20#{176}C prior to testing. p2! ras Proteins. Purified wild-type p21 K-ras-G12 protein and mutated K-ras-D12 protein were purchased from Oncogene Science (Manhasset, NY). These were purified without SDS and BSA. Endotoxin was removed using the endotoxin removal device (END-X B15; Cape Cod, Woods Hole, MA). Mutated H-ras-V12 protein, H-ras-S12 protein, and H-ras-L61 protein were expressed in Escherichia coli and purified in our laboratory. In brief, plasmids capable of expressing p21 ras protein in a eukaryotic host were constructed by cloning a synthetic C-Ha-ras gene into the pGH-L9 plasmid (16, 17). The synthetic gene was altered by cassette mutagenesis to encode various activated forms of p21 ras including valine 12 (V-12), serine 12 (5-12), and leucine 61 (L-61). E. coli (strain HB 101) was transfected with plasmid containing coding sequences for one of the three activated p21 ras proteins (18). The transfected bacteria were expanded, and the detergent lysates of transfected bacteria were purified by size on a Sephadex 075 (SG75) column. The fractions containing M 21,000 ras protein were concentrated by Amicon filtration (PM1O). The ras protein was further purified by HPLC (Waters 625 LC System; Millipore, Milford, MA) on a DEAE ion exchange column (Bio-Rad Laboratories, Richmond, CA). Fractions containing ras protein were analyzed using SDS-PAGE (Pharmacia PhastSystem; Pharmacia LKB Biotechnology, Uppsala, Sweden) and Western blotting. The primary antibody was a monoclonal mouse IgG pan-ras antibody (pan-ras Ab3) specific for both mutated and wild-type proteins (Oncogene Science). The fractions containing p2 1 ras were pooled, dialyzed, sterile filtered, and dried, and endotoxin was removed as previously described. Protein concentration was determined by spectrophotometer (Bio-Rad Protein Assay; Bio-Rad Laboratories). Recombinant myoglobin (Mr 19,000; Sigma) was used as negative controls. Synthetic ran Peptides. Five peptides derived from the amino acid sequence of the wild-type p21 K-ras-G12 protein were constructed and used in the current studies. All peptides are 18 amino acids in length. Two ras peptides, YKLVVVGAGGVGKSALTI (p4-21) and GETCLLDILDTAGQEEYS (p4861), were constructed to span the specific point mutation region at codon 12 or 61. The other three peptides, DTKQAQDLARSYGIPFIE (p125-142), KTRQRVEDAFYTLVREIR (plzV7-l64), and EIRQYRLKKISKEEKTPG (p162-179) with an a-helix constructing protein molecule surface, are from the carboxyl terminus amino acid sequence of the wild-type p21 K-ras-G12 protein. Peptides were synthesized by Dr. Patrick S. H. Chou (Biopolymer Facility, Department of Immunology, University of Washington) using 9-fluorenylmethoxycarbonyl chemistry in an automated peptide synthesizer (Model 433A; Applied Biosystems, Inc., Foster City, CA) and purified by HPLC. Serum Antibody Detection Using ELISA. The purified ras proteins were diluted in carbonate buffer (pH 9.6), and 50 pi of a 0.02-p.g protein solution were added to Immulon II polystyrene ELISA plate wells (Dynatech Laboratories Inc., Chantilly, VA) and incubated for 18 h at 4#{176}C. Human sera were diluted to 1:25 in 0.2% casein buffer (1.58 g/liter Tris-HC1, 9 g/liter NaCl, 3.72 g/liter EDTA, 0.2 g/liter Thimerisol, and 0.05% NP4O) and rocked for 18 h at 4#{176}C. The monoclonal mouse IgG anti-ras antibody (pan-ras Ab3; Oncogene Science) specific for both mutated and wild-type ras proteins was diluted 1:500 in 0.2% casein buffer as a positive control and incubated in the same manner as the human sera. Excess ras protein was washed six times from each well. Two hundred p l of 0.2% casein buffer without NP4O were added to each well and incubated for 3 h at room temperature for blocking. After washing, 100 pA of the diluted human sera, diluted pan-ras antibody, or buffer alone were added to the appropriate wells and incubated overnight at 4#{176}C. The next day the plates were washed six times. As the secondary antibody, antihuman IgA HRP3 (Accurate Chemicals, Westbury, NY) diluted 1:500 in 0.2% casein buffer with 0.05% NP4O and 1% mouse sera was added. Antimouse immunoglobulin HRP (Amersham, Arlington Heights, IL) diluted in 0.2% casein buffer with 0.05% NP4O was used for positive control wells. The plates were incubated for 2 h at room temperature. After the incubation the plates were washed six times and developed with equal parts tetramethylbenzidine peroxidase substrate and peroxidase Solution B (Kirkegaard and Perry Laboratory, Inc., Gaithersburg, MD). After 40 mm, the development reaction was stopped by the addition of I N HCI. Absorbency was read at a wavelength of 450 nm. All evaluations were performed in quadruplicate. The positive antibody control was a monoclonal mouse IgG pan-ras antibody specific for both mutated and wild-type proteins. Peptide Neutralization ELISA. Sera from 15 patients with detectable antibody to p21 ras-G12 in the previous ELISA study were selected by the level of absorbency, between 0.4 and 1.0, for this study. Patient sera as primary antibody were preincubated for 1 h with or without ras peptides (100 i.g/ml) or p21 ras (1 ji ml) as competitors. Other procedures for the ELISA were as follows as detailed above. The percentage of inhibition of the absorbency by the competitor was calculated. Antibody Detection Using Western Blotting. Immunoblotting with chemoluminescence was used to verify antibody reactivity. Depending on the stock concentration of the recombinant protein, between 80 and 100 ng purified protein were analyzed in each lane. Briefly, protein was transferred to nitrocellulose (Hybond-C; Amersham) and then blocked in 5% BSA0.05% NP4O in PBS for 1 h. The blot was incubated with human sera diluted 1:200-1:1000 in 1% BSA-0.1% NP4O in PBS