| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Immunology |
Cancer Vaccine Development Division, Kurume University Research Center for Innovative Cancer Therapy [A. Y., K. K., M. K., M. N., K. I.], Department of Immunology [A. Y., K. I.], and Department of Surgery [S. T.], Kurume University School of Medicine, Kurume 830-0011, Japan
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
We attempted to clone new tumor antigen genes from lung cancer cells to obtain new candidate peptides in use for vaccination and, here, report three new tumor antigen genes and their derived four peptides recognized by HLA-A2402-restricted CTLs.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Cloning and Identification of New Tumor Antigen Genes.
The expression-gene cloning method was used to identify genes that encode tumor antigens recognized by the CTL line, GK-CTL, as reported elsewhere (4)
. The precise methods for preparation of a cDNA library of the 11-18 cells, screening of cDNA clones, and DNA sequencing have been described elsewhere (5)
. Briefly, mRNA of the 11-18 lung adenocarcinoma cells was converted to cDNA, ligated to SalI adapter, and inserted into the expression vector pCMV-SPORT2.0 (Life Technologies, Inc., Gaithersburg, MD). A total of 1 x 105 clones from the cDNA library of the 11-18 cells were divided into 1000 wells (the expected number of clones/well was 100) and subjected to the first screening. Purified DNA from the divided pools and 100 ng of HLA-A2402 cDNA were cotransfected into VA13 cells (1 x 104 cells/well) and analyzed for their activity to stimulate IFN-
production by the GK-CTLs. Assay protocol is described in the "Peptides and Assays" section. Full-length HBP3
cDNA was cloned into pCR3.1 (Invitrogen, Carlsbad, CA) by TA-cloning method. A BTBD2 clone (KAIA4184) was purchased from Takara (Tokyo, Japan).
Northern Blot Analysis.
Total RNA (5 µg/lane) extracted from various cells or tissue specimens (six lung cancers, two renal cell carcinomas, one colon cancer, four gastric cancers, two ovarian cancers, one esophageal cancer, and one oral cancer) using RNA zol B (TEL-TEST, Friendswood, TX) was separated on formaldehyde-agarose gel and transferred to nylon membranes (Hybond-N+; Amersham, Buckinghamshire, United Kingdom). Total RNA preparations of normal tissues were purchased from Sawady Technology (Tokyo, Japan). The membranes were additionally hybridized with the following probes: MluI cut 0.7-kb fragments of original clone 50; PstI cut 1.2-kb fragments of clone 83; MluI cut 1.2-kb fragments of original clone 96; MluI cut 0.9-kb fragments of original clone 111; and MluI cut 1.6-kb fragments of original clone 114. Human ß-actin cDNA (Clontech, Tokyo, Japan) was also used as a control probe. Hybridization method has been described previously (5)
.
Peptides and Assays.
Synthetic peptides (purity >70%) derived from the deduced amino acid sequence of each of the clones with binding motifs for HLA-A2402 molecules, as described in the literature (20)
, including motifs of tyrosine or phenylalanine at position 2 and isoleucine, leucine, phenylalanine, or tryptophan at position 9, searched using BIMAS software (BioInformatics and Molecular Analysis Section, Center for Information Technology, NIH, Bethesda, MD; Ref. 21
), were obtained from Sawady Technology. An HIV nef-derived peptide (RYPLTFGWCF) and an EBV membrane-protein-derived peptide (TYGPVFMCL) capable of binding to HLA-A2402 molecules were used as negative and positive controls, respectively. Peptides of >95% purity were used for experiments regarding dose dependency and CTL induction. The estimated half-time of the dissociation scores of each peptide for HLA-A24 molecules was calculated using BIMAS software. For detection of antigenic peptides recognized by the GK-CTLs, the peptides were loaded onto C1R-A2402 cells by incubation at a concentration of 10 µg/ml, unless stated otherwise. Two h later, the supernatant was removed, and the GK-CTLs (1 x 105) were added to the culture, incubated for an additional 18 h, and the concentration of IFN- in the culture supernatants was measured by ELISA (limit of sensitivity: 10 pg/ml) in triplicate assays. Student’s t test was used for the statistical analyses.
CTL Induction by Peptides.
PBMCs (1 x 105/well) obtained from HLA-A24+ healthy donors or cancer patients were incubated with 10 µg/ml (
10 µM) of the peptide in 96-well plates in the presence of 100 units/ml interleukin-2 (Shionogi, Osaka, Japan) at day 0, as reported previously (5)
. At culture days 3, 6, and 9, the cells were restimulated by 10 µg/ml of the peptide. The culture supernatant of each well was removed, resuspended in fresh medium, and separated into two wells at day 10. Each of the two wells was stimulated with the corresponding peptide or control HIV-peptide. After 16 h of culture, the concentration of IFN- in the culture supernatants was measured by ELISA in triplicate assays. Tumor-specific response was similarly measured; 11-18, Sq-1, or QG56 cells (1 x 104 cells/well) were overnight culture in flat-bottomed 96-well plates, and the peptide-stimulated PBMCs were added to the culture. The concentration of IFN- in the supernatant of the assay culture stimulated by control HIV-peptide-loaded C1R-A2402 cells or HLA-A24- QG56 cells was subtracted from that of the specific peptide-loaded C1R-A2402 cells or HLA-A24+ tumor cells (11-18, Sq-1), respectively. In this culture system, antigenic peptides are presented by the initially contained antigen-presenting cells in the PBMCs, and no more addition of exogenous antigen-presenting cells is necessary during entire culture periods (22)
. For cytotoxicity tests, the cells were additionally cultured in 96-well U-bottomed microculture plates in the presence of interleukin 2 without peptide stimulation to obtain a sufficient number of cells for the assay. The cells were harvested at culture days 28–42, and the cytotoxic activity was measured using a standard 6-h 51Cr-release assay at different E:T ratios, as reported previously (5)
.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
production by GK-CTLs after cotransfection with HLA-A2402 cDNA into the VA13 cells. After repeated screening cycles, three clones, i.e., clones 50, 83, and 96, were confirmed to encode tumor antigens recognized by the GK-CTLs after cotransfection with HLA-A2402 but not with control HLA-A2601 (Fig. 1)
. Different reactivity of GK-CTL-derived sublines to each transfectant suggested that the presence of specific T cell clones to the above three gene products (data not shown).
|
Expression of the Genes at the mRNA Level.
The expression of the three newly identified genes in lung and other cancer cell lines, cancer tissues, and normal tissues at the mRNA level was analyzed by Northern blotting (Fig. 2)
. BTBD2/clone 50, clone 83, and HBP/clone 96 were expressed in almost all of the lung and other cancer cell lines tested to different degrees (Fig. 2A)
. It should be noted that the expression of these genes in VA13 cells was detectable, but expression levels were low.
|
. The expression of BTBD2/clone 50 was rarely observed in these cancer tissues, and only weak expression was detected in five lung cancer tissue specimens. In contrast, clone 83 and HBP/clone 96 were more frequently expressed in the cancer tissues. Expression of the three genes in normal tissues was also examined (Fig. 2C)
. BTBD2/clone 50 was ubiquitously expressed in the normal tissue specimens at different levels, i.e., relatively strong to intermediate expression was detected in the brain, colon, kidney, and skeletal muscle, and weak expression was observed in the PBMC, heart, lung, stomach, small intestine, liver, spleen, placenta, and testis. In contrast to the ubiquitous expression of BTBD2/clone 50, clone 83 was expressed only in the colon, and expression levels in the other normal tissues were almost same as the back ground level. Expression of HBP/clone 96 was not detected in the normal tissue specimens (data not shown).
Identification of Antigenic Peptides Recognized by the CTL.
Each of the 18 different synthetic peptides derived from clone83, clone 50, and clone 96, with binding motifs to HLA-A2402 molecules, was loaded onto an HLA-A2402 stable transformant, C1R-A2402 cells, and the ability of each peptide to induce IFN- production by GK-CTL was tested. A total of 7 of 18 peptides derived from the genes induced significant levels of IFN- production (Fig. 3A)
, and four (clone 50-1-767, clone 83-3-297, clone 83-3-301, and clone 96-3-380) of these peptides expressed the effect in a dose-dependent manner (Fig. 3B)
. However, remaining 3 of 7 peptides did not express such dose dependency. Therefore, we selected the former 4 peptides for additional analysis. The optimal concentration of the 4 peptides for loading C1R-A2402 cells varied, ranging from 0.1 to 10 µg/ml (compatible to 0.1–10 µM). The binding affinity of the 4 peptides to the HLA-A2402 molecules varied; the estimated half-time score of dissociation of each peptide ranged between 20 and 660 (Fig. 3A)
. Therefore, the binding affinity shows no relationship to the stimulatory property. This type of discordance has been observed in our previous study (2, 3, 4, 5, 6, 7, 8)
.
|
. For example, clone 83-3-297 peptide induced significant levels of IFN- in PBMCs of patients 2 and 8 (left panel of patients). Control HIV peptide did not induce the specific T cells after being stimulated four times in any of the cases (data not shown). The induction rate of the peptides for peptide-specific T cells was as follows: 5 of 8 for clone 50-1-767; 3 of 8 for clones 83-3-301 and 96-3-380; and 2 of 8 for clone 83-3-297. Reactivity of the peptide-stimulated T cells to HLA-A24+ lung cancer cells (11, 12, 13, 14, 15, 16, 17, 18)
was also examined, and a similar tendency was observed, except for in the case of EBV (Fig. 4A
, right panel of patients). It is noted that the magnitude of T-cell response to peptide-loaded cells and that to tumor cells cannot compare each other because the set of experiments were performed at different times and different culture conditions. The peptide-stimulated T cells also recognized HLA-A24+ other origin of cancer cells such as ovarian cancer (KOC-3S) and renal cell carcinoma (TUHR-10TKB; Fig. 4B
). In contrast to the observed effects of peptides in the PBMCs of cancer patients, these peptides rarely induced the peptide-specific T cells in the PBMC cultures of healthy donors (Fig. 4A)
.
|
. Control EBV- or HIV-peptide-induced T cells did not show such cytotoxicity against either 11-18 or QG56 cells (data not shown). The cytotoxic activities of the CTLs against autologous lung cancer cells were also confirmed (Fig. 5B)
. Peptide specificity of the CTLs was additionally analyzed by a cold target inhibition assay (Fig. 5C)
. SS-EBB cells or peptide-loaded SS-EBB cells were used as cold targets, but not hot targets, because spontaneous release of 51Cr from the cells was markedly increased after peptide loading. The cytotoxicity of each of the peptide-induced CTLs against 11-18 cells was not inhibited by addition of a 20-fold excess of cold SS-EBB cells or irrelevant control peptide-loaded SS-EBB cells. In contrast, corresponding peptide-loaded SS-EBB cells inhibited the cytotoxicity. Effects of mAbs on the IFN- production and cytotoxicity of the peptide-induced T cells were additionally examined. Representative results are shown in Fig. 5, D and E
. IFN- production in response to the 11-18 cells (Fig. 5D)
, or the cytotoxicity against 11-18 cells (Fig. 5E)
was inhibited by an addition of anti-CD8, anti-HLA-class I (W6/32), or anti-HLA-A24 mAb but not by that of anti-CD4 or anti-HLA-DR mAb. These results suggest that the four peptide-induced CD8+ T cells specifically react to the HLA-A24+ cancer cells, but not to nontumorous cells, through the recognition of the original peptide used for their induction; moreover, this reaction results in lysis of the tumor cells in a HLA-A24-restricted manner.
|
production of the GK-CTLs (Table 1)
. It makes sense because the CTL-epitope peptide encoded by clone 96 is included in the whole HBP protein. The cotransfectants of HLA-A2402 and full-length BTBD2 (KAIA4184) also stimulated IFN- production of clone 50-1-767-induced CTLs (Table 1)
. This result suggested that the first ORF-encoding clone 50-1-767 peptide was naturally translated from the full-length BTBD2 cDNA.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
There are two putative ORFs in BTBD2/clone 50, and the first ORF (nt position 2038–2431) is present in both the BTBD2 and original clone 50. The first ORF possesses a Kozak motif at the initiation codon, and Reinhardt’s method suggested that the putative protein of the first ORF was a nuclear protein. The second ORF (nt position 758-1372) encodes BTBD2 protein, which has been identified as a topoisomerase I-interacting zinc finger protein (23)
. The second ORF is only present in BTBD2 but not in the original clone 50, and it does not possess a Kozak motif at the initiation codon. The antigenic epitope peptide, clone 50-1-767, is encoded by the first ORF. Whether the first ORF was translated from the full-length BTBD2 cDNA, reactivity of clone 50-1-767-induced CTLs to a transfectant, which had been cotransfected with full-length BTBD2 and HLA-A2401 cDNA, was additionally examined, and the recognition of the transfectant by the CTLs was confirmed (Table 1)
. This result suggested that the first ORF-encoding clone 50-1-767 peptide was naturally translated from the full-length BTBD2 cDNA. Although the second ORF was not included in the original clone 50, we additionally attempted to find new epitope peptides encoded by the second ORF and identified another HLA-A2402-restricted epitope peptide (clone 50-2-383: MFKEPVEVL) in the BTBD2 protein (data not shown). These results suggest that both of the ORFs are translated to protein in these lung cancer cells.
HBP encodes HBP/stem-loop (histon) binding protein (25)
. The putative ORF of clone 96 encodes a partial HBP/stem-loop (histon) binding protein containing a COOH-terminal; thus, the epitope peptide, clone 96-3-380, is encoded by both the HBP and clone 96. Indeed, cells cotransfected with full-length HBP and HLA-A2402 cDNA were able to stimulate IFN- production of the GK-CTLs (Table 1)
. The HBP binds the stem-loop structure of replication-dependent histone pre-mRNAs and contributes to efficient 3' end processing by stabilizing the complex between pre-mRNA and U7 small nuclear ribonucleoprotein (25)
. Therefore, all of the putative proteins encoded by clone 83 and the first ORF of BTBD2/clone 50 and the HBP/stem-loop (histon) binding protein are localized in the nucleus. We previously reported that some of the transcription-related proteins located in the nucleus could be recognized by the CTLs as tumor antigens (6
, 8)
.
Clone83 and HBP/clone 96 were preferentially expressed in the cancer cells. In contrast, BTBD2/clone 50 was ubiquitously expressed in both normal tissues and cancer cells. These expressions in normal tissues could possibly induce adverse effects such as tissue distraction when the newly defined gene product-derived antigenic peptides are used in treatment vaccines for patients with lung and other cancers. Therefore, clone83 and HBP/clone 96 are more suitable target molecules for cancer vaccines. However, it should be noted that no severe adverse effects in normal tissues or organs have been reported in the clinical trials of cancer vaccines specific to MAGE-1, MAGE-3, Melan-A, gp100, tyrosinase, and NY-ESO-1 in melanoma patients, although these molecules are expressed in the normal testis, retina, and/or melanocytes at both mRNA and protein levels (12, 13, 14, 15, 16, 17) . Similarly, no severe adverse effects on the function of normal organs have been observed in our clinical trials of peptide cancer vaccines, although some of the target molecules are ubiquitously expressed in normal organs (11 , 29) . Subcellular traffic of antigenic molecules and/or subsequent processing of the antigenic peptides in the proteasomes of normal cells may differ from that of tumor cells in these cases. Alternatively, some molecules in normal cells, including a family of serpins (a group of serine-protease inhibitors), might be involved in normal cell resistance to CTL-mediated lysis (30) . Therefore, BTBD2/clone 50 is also a possible candidate for the cancer vaccine. Study of CTL induction by the BTBD2/clone 50-derived peptide also suggests such a possibility, i.e., peptide clone 50-1-767-induced peptide-specific and tumor-specific CTLs in 5 of 8 PBMC cultures from lung cancer patients, whereas no such induction was observed in the PBMCs of healthy donors.
The HLA-A24 allele is found in 60% of Japanese (95% of these cases are genotypically A2402), in 30% of Chinese, and in 20% of Caucasians (31) . The four peptides derived from the three genes were able to induce HLA-A24-restricted and tumor-specific CTLs in the PBMCs of lung cancer patients. The four peptides might therefore be appropriate vaccine candidates for use in specific immunotherapy for HLA-A24+ patients with lung cancer.
|
FOOTNOTES |
|---|
1 Supported, in part, by Ministry of Education, Culture, Sports, Science, and Technology of Japan Grant-in-Aids 12670583 (to A. Y.) and 12213134 (to K. I.) and Second Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labor and Welfare, Japan Grant-in-Aids H12-cancer-025 (to A. Y.) and H12-11-16 (to K. I.). 
2 To whom requests for reprints should be addressed, at Cancer Vaccine Development Division, Kurume University Research Center for Innovative Cancer Therapy, Asahi-machi 67, Kurume 830-0011, Japan. Phone: 81-942-31-7744; Fax: 81-942-31-7745; E-mail: akiymd{at}med.kurume-u.ac.jp
3 The abbreviations used are: HBP, hairpin-binding protein; BTBD2, BTB domain containing 2; PBMC, peripheral blood mononuclear cell; nt, nucleotide; ORF, open-reading frame; mAb, monoclonal antibody.
Received 4/ 2/02. Accepted 3/27/03.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  J. Nemunaitis, R. O. Dillman, P. O. Schwarzenberger, N. Senzer, C. Cunningham, J. Cutler, A. Tong, P. Kumar, B. Pappen, C. Hamilton, et al. Phase II Study of Belagenpumatucel-L, a Transforming Growth Factor Beta-2 Antisense Gene-Modified Allogeneic Tumor Cell Vaccine in Non-Small-Cell Lung Cancer J. Clin. Oncol., October 10, 2006; 24(29): 4721 - 4730. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. H. Gelman and L. Gao SSeCKS/Gravin/AKAP12 Metastasis Suppressor Inhibits Podosome Formation via RhoA- and Cdc42-Dependent Pathways Mol. Cancer Res., March 1, 2006; 4(3): 151 - 158. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Motomura, S. Senju, T. Nakatsura, H. Matsuyoshi, S. Hirata, M. Monji, H. Komori, D. Fukuma, H. Baba, and Y. Nishimura Embryonic Stem Cell-Derived Dendritic Cells Expressing Glypican-3, a Recently Identified Oncofetal Antigen, Induce Protective Immunity against Highly Metastatic Mouse Melanoma, B16-F10 Cancer Res., February 15, 2006; 66(4): 2414 - 2422. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. E. Raez, P. A. Cassileth, J. J. Schlesselman, K. Sridhar, S. Padmanabhan, E. Z. Fisher, P. A. Baldie, and E. R. Podack Allogeneic Vaccination With a B7.1 HLA-A Gene-Modified Adenocarcinoma Cell Line in Patients With Advanced Non-Small-Cell Lung Cancer J. Clin. Oncol., July 15, 2004; 22(14): 2800 - 2807. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Cancer Research | Clinical Cancer Research |
| Cancer Epidemiology Biomarkers & Prevention | Molecular Cancer Therapeutics |
| Molecular Cancer Research | Cancer Prevention Research |
| Cancer Prevention Journals Portal | Cancer Reviews Online |
| Annual Meeting Education Book | Meeting Abstracts Online |
) and 11-18 cells (
) at E:T ratio 20. Values represent the means of triplicate assays and background values against QG56 cells were subtracted. *, P < 0.05. C, cold target inhibition of the cytotoxicity. Indicated peptides (10 µg/ml) were loaded on SS-EBB cells and used as cold target cells for the cytotoxicity assay against 11-18 cells. Cold target cells/hot target