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Experimental Therapeutics, Preclinical Pharmacology |
1 Department of Immunology and 2 Second Department of Internal Medicine, Kurume University, Fukuoka, Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental Design: Seven peptides possessing the ability to induce HLA-A31-restricted and tumor-reactive CTLs were examined for their ability to induce HLA-A3-, -A11-, and -A33-restricted and tumor-reactive CTLs from peripheral blood mononuclear cells (PBMCs) of 18 epithelial cancer patients. The five reference peptides all have the ability to induce CTL activity restricted with one of the HLA-A3 supertypes, and, thus, were also examined as positive controls.
Results: Three peptides (2 from ß-tublin5- and 1 from CGI37-derived peptides) induced tumor-reactive CTLs in PBMCs of HLA-A3+, -A11+, and -A33+ cancer patients with various frequencies (17–50%). One RLI- or KIAA0036-derived peptide induced tumor-reactive CTLs in PBMCs of HLA-A3+ and -A11+ or HLA-A11+ and -A33+ cancer patients also with various frequencies (22–67%), respectively, whereas the other peptide induced CTL activity in only HLA-A33+ patients. Among the five reference peptides tested, one peptide, TRP2–197, induced CTL activity in both HLA-A11+- and -A33+-restricted manners.
Conclusions: We identified new peptide vaccine candidates for HLA-A3, -A11, -A31, and -A33 positive cancer patients. This study may facilitate the development of both basic and clinical studies of peptide-based immunotherapy for cancer patients with other alleles of HLA-A2 and -A24.
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The following tumor cell lines were used in this study, esophageal cancer cell line KE5 (A1101, B1501/5504, Cw0303/0401); lung adenocarcinoma cell lines 1–87 (A0207/1101, B4601/5401, Cw0102), LC1 (A3101/3303, B1511/44031, Cw0303/1403), 11–18 (A0201/2402, B5201/5401, Cw0102/1202), and QG56 (A2601, B4601, Cw0102); gastric adenocarcinoma cell line MKN28 (A3101, B5101, Cw0304); colon adenocarcinoma cell line SW620 (A0201/2402, B0702/1518, Cw0702/0704); cervical cancer cell line RKN (A3303, B44/51, Cw1403); and astrocytoma cell line Becher (A0301/6802). All of the cell lines were maintained in RPMI 1640 (Invitrogen, Carlsbad, CA) with 10% FCS. Phytohemagglutinin-activated normal T cells were also used as a negative control. To generate HLA-A11- and -A33-transfected C1R cell lines, an HLA-A1101 or -A3303 cDNA clone was inserted into the eukaryotic expression vector pCR3.1 (Invitrogen) by a method reported previously (8) . Electroporation was performed using a Gene Pulser (Bio RAD, Richmond, CA). G418-resistant single clones were selected by limiting dilution. To generate HLA-A3 transfected COS-7 cells, 100 ng of HLA-A0301 cDNA, which was inserted into pCR3.1, were mixed in 100 µl of Opti-MEM (Invitrogen) with 0.3 µl of Fugene 6 (Invitrogen) and incubated for 30 min. The mixture was then added to the COS-7 cells (1 x 103 cells), which were then incubated for 3 h. One hundred µl of RPMI 1640 containing 20% FCS was added, and COS-7 cells were cultured for 2 days. The surface expression of HLA-A3, -A11, or -A33 molecules was examined by anti-HLA-A3 (IgM; One Lambda, Canoga Park, CA), anti-HLA-A11 (A11.1M, IgG3), and anti-HLA-A33 mAb (IgM; One Lambda), respectively. Surface phenotypes of the CTLs were analyzed by fluorescence-activated cell sorter with FITC-conjugated anti-CD3, anti-CD4, anti-CD8, and anti-CD14 monoclonal antibody (mAb; Nichirei, Tokyo, Japan).
Peptides.
Eleven different peptides, each with the ability to induce peptide-specific and tumor-reactive CTL activity with an HLA-A3-, -A11-, -A31-, or -A33-restricted manner, all belonging to the HLA-A3 supertype, were used in this study (3
, 12
, 13)
. We recently reported 7 of these peptides as being capable of inducing HLA-A31-restricted and tumor-reactive CTLs (10)
. The remaining 5 peptides that were used as reference peptides in this study were reported from the other laboratories as CTL epitope peptides with the ability to induce CTLs restricted to one of the HLA-A3 supertypes (3
, 12
, 13)
. EBV- and Influenza virus-derived peptides with an HLA-A11-binding motif were used as positive controls, whereas an HIV-derived peptide with an HLA-A31-binding motif was used as a negative control (14
, 15)
. The peptide binding affinities for these alleles of the HLA-A3 supertype were calculated based on a predicted half-time of dissociation to HLA class I molecules, as reported previously (16)
; the binding scores obtained from the bioinformatics and molecular analysis section website3
are given in Table 1
. All of the peptides (>90% purity) used in this study were purchased from Biosynthesis (Lewisville, TX) and were dissolved with DMSO at a dose of 10 mg/ml.
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in response to HLA-A3, -A11, or -A33 expressing COS-7 cells or C1R cells, designated as COS-7-A3, C1R-A11, and -A33 respectively; the cells were preloaded with either a corresponding peptide or an HIV peptide, used as a negative control. Four wells were prepared for each peptide, and the assays were performed in duplicate. The background IFN- production (<50 pg/ml) in response to the HIV peptide was subtracted from the values given in the data. The concentration of IFN- was assessed by ELISA as reported previously (16)
. For the inhibition assay, the peptide-reactive CTLs were positively purified using a CD8 Isolation kit (DYNAL, Oslo, Norway), and their peptide-specific IFN- production was measured in the presence of 20 µg/ml of anti-HLA class I (W6/32, IgG2a), anti-CD8 (Nu-Ts/c, IgG2a), or anti-HLA class II (H-DR-1, IgG2a) mAbs, as reported previously (17)
. The background IFN- production (<100 pg/ml) was subtracted from the data. A two-tailed Student’s t test was used for the statistical analysis throughout the study.
Cytotoxicity Assay.
The peptide-stimulated PBMCs were incubated for >21 days, and cytotoxicity was tested by a standard 6-h 51Cr release assay (17)
. A two-tailed Student’s t test was used for the statistical analysis. For the cold target inhibition assays, unlabeled C1R-A11 or C1R-A33 cells were incubated with the corresponding peptide or with an HIV-peptide for 2 h, and then the cells were added to the 51Cr-labeled targets at a cold:hot target cell ratio of 10:1.
Recognition of MHC/Peptide Complexes.
One hundred ng each of HLA-A cDNA, which were inserted into pCR3.1, or 100 ng of HLA-A cDNA with 100 ng of each clones (RLI, ß-tublin5, CGI37, and KIAA0036) were mixed in 100 µl of Opti-MEM (Invitrogen) with 0.3 or 0.6 µl of Fugene 6 (Invitrogen), respectively, and incubated for 30 min. The mixture was then added to the COS-7 cells (1 x 103 cells), which were then incubated for 3 h. One hundred µl of RPMI 1640 containing 20% FCS was added and COS-7 cells were cultured for 2 days. The peptide-stimulated PBMCs were then added and incubated for 12 h. Then, their ability to produce IFN- was measured by ELISA.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 3
, and 4
show the representative results of CTL induction by IFN- production in response to peptide-loaded C1R-A11, C1R-A33 cells, and HLA-A3-transfected COS-7 cells in HLA-A11, -A33, and -A3 cancer patients, respectively. RLI-522, ß-tublin5–154, ß-tublin5–232, ß-tublin5–309, CGI37–72, KIAA0036–241, and KIAA0036–356 peptides were shown to have the ability to induce peptide-reactive CTL activity in PBMCs from 2, 4, 1, 2, 2, 0, and 4 of 9 HLA-A11+ cancer patients tested, respectively (Table 2)
. Similarly, these peptides possessed the ability to induce peptide-reactive CTL activity in PBMCs from 0, 2, 1, 1, 1, 4, and 4 of 6 HLA-A33+ cancer patients tested, respectively (Table 3)
. RLI-522, ß-tublin5–154, ß-tublin5–232, ß-tublin5–309, and CGI37–72, but not KIAA0036–241 and KIAA0036–356 peptides, were shown to have the ability to induce peptide-reactive CTL activity in a PBMCs from at least 1 of the 3 HLA-A3+ cancer patients tested (Table 4)
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IFN- production by these peptide-stimulated PBMCs in response to C1R-A11 or -A33 cells pulsed with a corresponding peptide was inhibited by both the anti-HLA class I mAb and the anti-CD8 mAb, but not by the anti-HLA class II mAb in all of the tested cases. Representative data for each of HLA-A11 and -A33 from patients 8 and 11, respectively, are shown in Fig. 1
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for HLA-A11 patients, Fig. 2B
for HLA-A33 patients, and Fig. 2C
for HLA-A3 patients). None of them were cytotoxic against HLA-A allele-matched phytohemagglutinin blastoid T cells (Fig. 2A
for HLA-A11 patients and Fig. 2B
for HLA-A33 patients). In contrast, PBMCs cultured with HIV peptide (Fig. 2, A and B)
or flu peptide (data not shown), both of which were used as negative controls for an antitumor response, failed to display HLA-A11- or -A33-restricted cytotoxicity against cancer cells, respectively.
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, A-1). The similar results were obtained with RLI-522, ß-tublin5–154, and CGI37–72 peptides (Fig. 3
, A-2). In the inhibition test, the cytotoxicity against the HLA-A33+ LC1 tumor cell line mediated by each of the 5 peptides (2 from ß-tublin5, CGI37–72, and 2 from KIAA0036) was blocked by unlabeled C1R-A33 cells loaded with the corresponding peptide, but not by unlabeled C1R-A33 cells loaded with the HIV peptide (Fig. 3B)
. The cytotoxicity against the HLA-A11+ lung cancer 1–87, HLA-A33+ lung cancer LC1, or HLA-A3+ astrocytoma cell lines mediated by each of the peptide reactive-PBMCs was blocked by anti-CD8 and anti-HLA class I but not by the other mAbs (Fig. 2C
and Fig. 3C
). All of these results indicate that CD8+ T cells from the peptide-stimulated PBMCs, except for those with ß-tublin5–232, exhibited peptide-specific CTL activity reactive to tumor cells with an HLA class I-restricted manner.
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by recognition of COS-7 cells that were cotransfected with both HLA-A11 and the corresponding cDNA clone (Fig. 4A)
. In contrast, these PBMCs failed to produce IFN- either by recognition of COS-7 cells that were cotransfected with any of the irrelevant HLA class I (A33, A31, or A2) and corresponding clones (ß-tublin5, CGI37, RLI, or KIAA0036), or by recognition of COS-7 cells that were transfected with any of the HLA class I (A33, A31, A11, or A2) clones with an irrelevant (ppMAPkkk) cDNA clone (Fig. 4A)
. ß-tublin5–309 peptide was not examined by transfection study because of the sample limitation.
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, B-1). Namely, the HLA-A33+ PBMCs stimulated with each of ß-tublin5–154, ß-tublin5–309, or CGI37–72 produced significant levels of IFN- by recognition of COS-7 cells that were cotransfected with both HLA-A33 and the corresponding cDNA clone. In contrast, these PBMCs failed to produce IFN- either by recognition of COS-7 cells that were cotransfected with any of the irrelevant HLA class I (A31, A11, or A2) and ß-tublin5 or CG137 cDNA clones or by recognition of COS-7 cells that were transfected with any of the HLA class I (A33, A31, A11, or A2) clones and with an irrelevant (ppMAPkkk) cDNA clone (Fig. 4
, B-1).
Secondly, we tested the six peptides. Namely, HLA-A33+ PBMCs stimulated with each of the six peptides tested (ß-tublin5–154 or CGI37–72, 2 from KIAA0036, and MAGE1–95 and MAGE1–73 as reference peptides) produced significant levels of IFN- by recognition of COS-7 cells that were transfected with the HLA-A33 cDNA clone, followed by loading of the corresponding peptide (Fig. 4
, B-2). In contrast, these PBMCs failed to produce IFN- by either of the two following experiments, by recognition of COS-7 cells that were transfected with any of the irrelevant HLA class I cDNA clones, followed by loading of the corresponding peptide or by recognition of COS-7 cells that were transfected with any of the HLA class I (A33, A31, A11, or A2) clones, followed by loading of an irrelevant peptide (HIV).
CTL Induction by the Reference Peptides.
The following five reference peptides of the HLA-A3 supertype were also tested in this study, MAGE1-, MAGE2-, and PAP-derived peptides capable of inducing HLA-A11-restricted CTLs (14)
; a gp100-derived peptide capable of inducing HLA-A3 and -A11-restricted CTLs (18)
; and a TRP2-derived peptide capable of inducing HLA-A31 and -A33-restricted CTLs (3)
. In addition, EBV- and flu-derived peptides with an HLA-A11-restricted type of immunogenicity were used as the other reference peptides in this study (14)
. HIV-derived peptide was used as a negative control (15)
. The representative results regarding CTL induction in PBMCs from HLA-A11 and -A33 cancer patients are shown in Tables 2
and 3
, respectively. The MAGE1–95, MAGE2–73, PAP-274, gp100–87, TRP2–197, EBV, flu, and HIV-derived peptides had the ability to induce peptide-reactive CTL activity in PBMCs from 2, 5, 3, 3, 2, 5, 5, and 0 of 9 HLA-A11+ cancer patients tested, respectively. Similarly, these peptides had the ability to induce peptide-reactive CTL activity from 1, 2, 2, 1, 3, 3, 2, and 0 of 5 HLA-A33+ cancer patients tested, respectively. Although most of these results are consistent with those of previous studies (3
, 14
, 18)
, it is of note that the TRP2–197 peptide, which can induce HLA-A31- and-A33-restricted CTLs, also induced HLA-A11-restricted CTL activity in 2 of 9 HLA-A11+ and 1 of 3 cancer patients tested.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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30%, 35%, 44%, 52%, and 35% among Caucasians, North American African-Americans, Japanese, Chinese, and Hispanics, respectively (11)
. Although HLA-A3303 is not included in the A3 supertype classically, it was reported that HLA-A3303 binding peptides shared the same anchor residues for HLA-A3101 binding peptides (19
, 20)
. We used HLA-A3303+ PBMCs in this study, instead of HLA-A3301 for the experiments, because it is a predominant allele in Asian population. Therefore, ß-tublin5–154, ß-tublin5–309, and CGI37–72 peptides, along with the TRP2–197 reference peptide, might be appropriate for use in a cancer vaccine for HLA-A3 supertype+ cancer patients except with HLA-A6801+ patients. Among them, the ß-tublin5–154 is the most widely shared peptide possessing the ability to induce CTLs among HLA-A3 supertype under the present conditions. On the other hand, RLI-522 or KIAA0036–356 peptide induced tumor-reactive CTLs in HLA-A3+ and -A11+ or HLA-A11+ and -A33+ cancer patients with various frequencies (22–67%), respectively. Thus, these two peptides might also be useful for relatively large numbers of cancer patients. However, the PBMCs of only three HLA-A3+ patients were tested, and -A68.1+ patients were not tested, because these allele frequencies are extremely low in Japan (1.6 and 0.5%). Therefore, additional studies with more subjects with HLA-A0301 and -A6801 alleles are needed to confirm the results of this small-scale study. Although the estimated binding affinities of the majority of peptides tested were relatively low in each of the HLA-A3 supertypes, these peptides, including KIAA0036–356 and ß-tublin5–154, induced HLA-A3-supertype-restricted and tumor-reactive CTL activity in PBMCs from cancer patients tested with relatively high frequencies. No correlation between binding affinity and the ability to induce CTLs was obtained in this study; this result is consistent with those from previous studies (3 , 21) . These results suggest that the binding affinity for MHC class I molecules is not the only factor that is determinative of immunogenicity.
In regard to biological function of genes coding the peptides studied herein, RLI is a member of the superfamily of ATP-binding cassette transporters and blocks the activity of protein synthesis in the 2–5A/RNase L system, the central pathway for viral IFN action (22
, 23)
. RLI regulates mitochondrial mRNA stability by inhibiting antiproliferative action after IFN-
treatment (22)
. Thus, RLI appears to become activated under cytokine influence near tumor sites, along with enhancing intrinsic antitumor immune responses. It has been shown that ß-tubulin is an integral component of microtubules, and that it plays important roles in mitosis, cell signaling, and motility (24)
. The elevated expression of ß-tubulin is reported more often in cancer cells than in normal cells (25
, 26)
. Thus, ß-tubulin is one of the major targets of anticancer therapies (24)
. The function of CGI37 or KIAA0036 has not yet been determined. However, the mRNA expression levels of these genes are higher in cancer cells than in normal tissues except for testis (10)
.
As mentioned in the introduction, clinical trials of peptide vaccination are currently underway, although most are limited to use in either HLA-A2+ or -A24+ cancer patients (2 , 9) . This limitation is largely due to the relative infrequency of HLA-A alleles other than HLA-A2 and -A24 (<20%). The present results suggest potential vaccine candidates for HLA-A3 supertype+ cancer patients, and, thus, may facilitate the development of both basic and clinical studies of peptide-based immunotherapy for cancer patients with alleles other than HLA-A2 and -A24.
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FOOTNOTES |
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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.
Requests for reprints: Shigeki Shichijo, Department Immunology, Kurume University, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan. Phone: 81-942-31-7551; Fax: 81-942-31-7699; E-mail: shichijo{at}med.kurume-u.ac.jp
3 Internet address: http://bimas.dcrt.nih.gov/. 
Received 5/27/03; revised 10/15/03; accepted 10/15/03.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B crystallin in C6 glioma cells is induced by agents that promote the disassembly of microtubules. J. Biol. Chem., 271: 26989-26994, 1996.This article has been cited by other articles:
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  A. Jalili, S. Ozaki, T. Hara, H. Shibata, T. Hashimoto, M. Abe, Y. Nishioka, and T. Matsumoto Induction of HM1.24 peptide-specific cytotoxic T lymphocytes by using peripheral-blood stem-cell harvests in patients with multiple myeloma Blood, November 15, 2005; 106(10): 3538 - 3545. [Abstract] [Full Text] [PDF]
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 S. Matsueda, H. Takedatsu, A. Yao, M. Tanaka, M. Noguchi, K. Itoh, and M. Harada Identification of Peptide Vaccine Candidates for Prostate Cancer Patients with HLA-A3 Supertype Alleles Clin. Cancer Res., October 1, 2005; 11(19): 6933 - 6943. [Abstract] [Full Text] [PDF]
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N. Yajima, R. Yamanaka, T. Mine, N. Tsuchiya, J. Homma, M. Sano, T. Kuramoto, Y. Obata, N. Komatsu, Y. Arima, et al. Immunologic Evaluation of Personalized Peptide Vaccination for Patients with Advanced Malignant Glioma Clin. Cancer Res., August 15, 2005; 11(16): 5900 - 5911. [Abstract] [Full Text] [PDF]
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K. Azuma, T. Sasada, H. Takedatsu, H. Shomura, M. Koga, Y. Maeda, A. Yao, T. Hirai, A. Takabayashi, S. Shichijo, et al. Ran, a Small GTPase Gene, Encodes Cytotoxic T Lymphocyte (CTL) Epitopes Capable of Inducing HLA-A33-restricted and Tumor-Reactive CTLs in Cancer Patients Clin. Cancer Res., October 1, 2004; 10(19): 6695 - 6702. [Abstract] [Full Text] [PDF]
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 T. Sasada, H. Takedatsu, K. Azuma, M. Koga, Y. Maeda, S. Shichijo, H. Shoumura, T. Hirai, A. Takabayashi, and K. Itoh Immediate Early Response Gene X-1, a Stress-Inducible Antiapoptotic Gene, Encodes Cytotoxic T-Lymphocyte (CTL) Epitopes Capable of Inducing Human Leukocyte Antigen-A33-Restricted and Tumor-Reactive CTLs in Gastric Cancer Patients Cancer Res., April 15, 2004; 64(8): 2882 - 2888. [Abstract] [Full Text] [PDF]
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