WO2008007711A1

WO2008007711A1 - Sart3-derived peptide useful in cancer vaccine therapy for hla-a3 supertype allele-positive prostate cancer patient

Info

Publication number: WO2008007711A1
Application number: PCT/JP2007/063838
Authority: WO
Inventors: Kyogo Itoh; Mamoru Harada
Original assignee: Kurume University
Priority date: 2006-07-11
Filing date: 2007-07-11
Publication date: 2008-01-17
Also published as: JPWO2008007711A1; JP5114403B2

Abstract

Disclosed is a SART3-derived peptide which can bind to an HLA-A3 supertype allele molecule and can be recognized by cellular immunity. The peptide is particularly useful for the treatment of a prostate cancer patient who is negative with respect to a HLA-A2 or HLA-A24 molecule for which many cancer vaccine candidate peptides have been identified previously.

Description

Specification

SART3-derived peptides useful for cancer vaccine therapy for HLA-A3 supertype allele positive prostate cancer patients

Technical field

[0001] The present invention relates to a SART3-derived peptide useful for the treatment or prevention of HLA-A3 supertype allele positive prostate cancer patients.

Background art

[0002] Prostate cancer is a cancer that is common in elderly men (Non-patent Document 1). For prostate cancer, the ability of androgon-removal therapy to be transiently successful S, effective treatment for relapse as hormone-resistant or bone-metastatic prostate cancer. Specific immunotherapy can be a promising option for such patients. This is because prostate cancer reactive cytotoxic τ cells can specifically find metastases. Many cancer antigen peptides that can be used for specific immunotherapy for prostate cancer patients have been identified so far (Non-patent Document 2). However, because of the high frequency of HLA-A2 and -A24 alleles worldwide, most of the conventional cancer antigen peptides were for patients positive for HLA-A2 or -A24 alleles (Non-Patent Document 3). ).

[0003] HLA-A2, -A3, _B7 and -B44 supertype alleles have been proposed for HLA class I alleles based on structural homology and peptide binding motif analysis (Non-Patent Document 4). Among them, the HLA-A3 supertype allele is found in 38% of Caucasians, 53% of Chinese, 46% of Japanese, and 43% of North American African Americans and Hispanics (Non-patent Document 4). Nonetheless, cancer antigen peptides that can be used to treat HLA-A3 supertype allele positive prostate cancer patients are limited (Non-patent Documents 5-7) Non-patent Document 1: Greenlee RT, Murray T, Bolden S, Wingo PA. Cancer statistics, 2000. CA Cancer J Clin 2000; 50: 7-33.

Non-patent literature 2: Renkvist N, Castelli C, Robbins PF, Parmiani GA listing of human tu mor antigens recognized by T cells. Cancer Immunol Immunother 2001; 50: 3-15. Non-patent literature 3: Imanishi T, Akazawa Satoshi, Kimura A. Allele and haplotype frequencies fo r HLA and complement loci in various ethnic groups. In Tsuji, Aizawa M, Sasazuki T. editors. HLA 1991.Vol. l Oxford: Oxford Scientific Publications; 1992 1065- 1220 P- Non-patent literature 4: Sette A, Sidney J Ninemajor HLA class I supertypes account for the vast preponderance of HLA- A and B polymorphism.Immunogenetics 1999; 50: 201-2 12.

Non-Patent Document 5: Wang RF, Johnston SL, Southwood S, Sette A, Rosenberg SA.Recognition of an antigenic peptide derived from tyrosinase-related protein-2 by CTL in t he context of HLA-A31 and A33. J Immunol 1998 ; 160: 890-897.

Non-Patent Document 6: Takedatsu H, Shichijo S, atagiri, Sawamizu H, Sata M, Itoh. Identification of peptide vaccine candidates sharing among HLA-A3 +, -A11 +, -A31 +, and nd -A33 + cancer patients. Clin Cancer Res 2004 ; 10: 1112-1120.

Non-Patent Document 7: Matsueda S, Takedatsu H, Yao A, Tanaka M, Noguchi M, Itoh, Harada M. identification of peptide vaccine candidates for prostate cancer patients with

HLA- A3 supertype alleles.Clin Cancer Res. 2005; 11: 6933-6943.

Disclosure of the invention

Problems to be solved by the invention

[0004] An object of the present invention is to provide a cancer antigen peptide useful for cancer vaccine therapy for HLA-A3 supertype allele positive prostate cancer patients.

Means for solving the problem

[0005] The present invention provides a SART3-derived peptide that can bind to an HLA-A3 supertype allele molecule and is recognized by cellular immunity. Specifically, the present invention relates to a peptide consisting of the amino acid sequence shown in SEQ ID NO: 16 or 20, and substitution, deletion and / or addition of 1 or 2 amino acids in the amino acid sequence shown in SEQ ID NO: 16 or 20 And a derivative thereof having a property that is functionally equivalent to the peptide consisting of the amino acid sequence introduced with and having the amino acid sequence shown in SEQ ID NO: 16 or 20.

[0006] The present invention also provides a nucleic acid molecule encoding the peptide or derivative of the present invention and a vector comprising the nucleic acid molecule. [0007] The present invention also provides a pharmaceutical composition for treating or preventing prostate cancer, particularly the pharmaceutical composition which is a cancer vaccine, comprising the peptide, derivative or vector of the present invention.

[0008] Furthermore, the present invention relates to prostate cancer reactive cytotoxicity, which comprises contacting peripheral blood mononuclear cells collected from HLA-A3 supertype allele positive prostate cancer patients with the peptide or derivative of the present invention. Methods of inducing sex T cells are provided.

[0009] The present invention also includes introducing a peptide or derivative of the present invention into a cell having an antigen-presenting ability derived from an HLA-A3 supertype allele-positive prostate cancer patient, or introducing the vector of the present invention. And a method for preparing an antigen-presenting cell that presents a complex of a SART3-derived peptide or a derivative thereof and a HLA-A3 supertype allele molecule on the cell surface.

The invention's effect

[0010] The present invention has expanded the options for peptide-based immunotherapy, particularly cancer vaccine therapy, for HLA-A3 supertype allele positive prostate cancer patients. The present invention is particularly useful for the treatment of prostate cancer patients who are negative for HLA-A2 or HLA-A24 molecules for which many cancer vaccine candidate peptides have been identified so far.

Brief Description of Drawings

[0011] [FIG. 1] A: SART3 mRNA expression in esophageal cancer cell line KE4, normal PBMC, and three prostate cancer cell lines (PC3, PC93, and LNCaP). B: Expression of HLA-A11, _A31, and A33 molecules in three types of LNCaP transfectants established in the present invention. The dotted line shows the result of staining without the primary antibody.

[Fig. 2A] Cytotoxic activity of peptide-stimulated PBMC from HLA-A3 supertype allele positive prostate cancer patients. * p <0.05.

[FIG. 2B] Cytotoxic activity of peptide-stimulated PBMC derived from HLA-A3 supertype allele positive prostate cancer patients. * p <0.05.

[FIG. 3A] Cytotoxic activity against prostate cancer cells depending on peptide-specific CD8-positive T cells. * ρ <0 · 05.

[Fig. 3Β] Peptide-specific CD8-positive cell damage on prostate cancer cells Activity. * ρ <0 · 05.

BEST MODE FOR CARRYING OUT THE INVENTION

[0012] The peptide of the present invention is a peptide comprising a part of the amino acid sequence of SART3 identified as a cancer antigen. The amino acid sequence of SART3 is disclosed in Genebank at ΑΒ020880.

[0013] The phrase "can bind to an HLA-A3 supertype allele molecule" means that the peptide can form a complex with the HLA-A3 supertype allele molecule and be displayed on the cell surface. In general, the amino acid sequence of a peptide that binds to an HLA molecule has regularity that depends on the type of HLA. The amino acid sequence according to the regularity is called a binding motif. HLA-A3 supertype allele molecules include HLA-A11, -A31, _A33, -A0301 and —A6801 molecules, which share the binding motif (Sette, A., and Sidney, J. Ninemajor, Immunogenetics, 50: 201-212, 1999.) Peptides with binding motifs for HLA-A3 supertype allele molecules should be determined by computer analysis such as Bioinformatics and Molecular Analysis Section (NIH, Bethesd a, MD). Can do.

In the present invention, the phrase “recognized by cellular immunity” means that the peptide has the ability to induce peptide-specific CTL. Peptide-specific CTL inducibility can be achieved, for example, by stimulating peripheral blood mononuclear cells (PBMC) with a peptide and reacting with antigen-presenting cells in which the peptide-stimulated PB MC is pulsed with the corresponding peptide (eg, IFN-γ). ) Can be measured by ELISA or the like. In addition, the cytotoxic activity of the induced CTL can be confirmed by a ⁵¹ Cr release measurement method or the like. In consideration of the recognition by CTL, the number of amino acid residues of the peptide of the present invention should be in the range of 8-14. S is preferable, more preferably 8 to; 11, particularly preferably 9 or 10. is there.

[0015] The phrase "recognized by humoral immunity" means that IgG specific to the peptide exists in the living body, that is, peptide-specific IgG is detected from plasma. Peptides recognized by both cellular immunity and humoral immunity are preferred as the peptides of the present invention because they are expected to have high immunogenicity and excellent CTL inducing ability. Specific IgG in plasma can be measured by a conventional ELISA method or the like.

[0016] The amino acid represented by SEQ ID NO: 16 or 20 is particularly preferred as the peptide of the present invention. A peptide consisting of a sequence.

[0017] In the derivative of the peptide consisting of the amino acid sequence shown in SEQ ID NO: 16 or 20 in the present invention, substitution, deletion and / or addition of 1 or 2 amino acids in the amino acid sequence shown in SEQ ID NO: 16 or 20 It has the functionally equivalent properties of a peptide consisting of the introduced amino acid sequence and consisting of the amino acid sequence shown in SEQ ID NO: 16 or 20. “Has functionally equivalent properties to the peptide consisting of the amino acid sequence shown in SEQ ID NO: 16 or 20” means that it can bind to the HLA-A3 supertype allele molecule and is recognized by cellular immunity. To do. Whether or not they have functionally equivalent properties is determined by the ability S to investigate according to the method described above.

[0018] Substitution of amino acids from the viewpoint of not changing the properties of peptides, such as homologous amino acids (polar amino acids, nonpolar amino acids, hydrophobic amino acids, hydrophilic amino acids, positively charged amino acids, negatively charged amino acids, aromatic amino acids, etc.) ) Is preferable. Deletion and attachment of amino acids are preferably performed so that the derivative has 8 to 11 amino acid residues.

[0019] In addition, amino acid substitutions, deletions and / or additions are preferably allowed on the HLA molecule-binding motif. That is, amino acid substitution, deletion and / or addition is preferably performed so that the peptide terminal amino acid force S-lysine or arginine of the peptide derivative is obtained. For example, substitution, deletion and / or addition of other amino acids with the C-terminal amino acid of SEQ ID NO: 16 or 20 preserved in arginine, substitution of its C-terminal amino acid with lysine, or its C-terminal The amino acid is replaced with lysine, and other amino acid substitutions, deletions and / or additions are performed. Of these, derivatives comprising an amino acid sequence in which the C-terminal amino acid of the amino acid sequence shown in SEQ ID NO: 16 or 20 is substituted with lysine are particularly suitable for the present invention.

[0020] The amino acids constituting the peptides and derivatives of the present invention may be natural amino acids or amino acid analogs. Examples of amino acid analogs include N-acylated, 0-acylated, esterified, and acid amino acids. Examples include amidated products and alkylated products. The peptides and derivatives of the present invention may be modified in their constituent amino acids or carboxyl groups as long as the functions are not significantly impaired. Modifications include binding of formyl, acetyl, and t-butoxycarbonyl groups to the N-terminus and free amino groups, and C-terminus and free amino groups. And those having a methyl group, an ethyl group, a t-butyl group, a benzyl group or the like bonded to the carboxyl group.

[0021] The peptides and derivatives of the present invention can be produced by ordinary peptide synthesis. For example, Peptide Synthesis, Interscience, New York, 1966; The Proteins, Vol2, Academic Press Inc., New York, 1976; peptide synthesis, Maruzen Co., Ltd., 1975; Experiment, Maruzen Co., Ltd., 1985; Development of pharmaceuticals, Vol. 14-Peptide synthesis, Hirokawa Shoten, 1991).

[0022] The peptide and derivative of the present invention may be produced by fragmenting a polypeptide containing the amino acid sequence IJ of the peptide or derivative of the present invention in a cell and provided as a complex with an HLA molecule. . It is also within the scope of the present invention to utilize the peptides or derivatives of the present invention in such embodiments. As long as the peptide or derivative of the present invention can be provided, the number of amino acid residues and the amino acid sequence of the polypeptide are arbitrary.

[0023] The peptides and derivatives of the present invention can efficiently induce and proliferate CTLs that damage HLA-A3 supertype allele-positive prostate cancer cells. That is, the peptides and derivatives of the present invention can be used for inducing prostate cancer-reactive CTLs and for producing pharmaceutical compositions for prostate cancer, etc., in the treatment or prevention of prostate cancer. Useful.

[0024] The pharmaceutical composition of the present invention contains one or more of the peptides or derivatives of the present invention, and has a therapeutic effect by inducing prostate cancer reactive CTL specific for the peptides or derivatives. Demonstrate. The pharmaceutical composition of the present invention can be used as a cancer vaccine. Since CTL of cancer patients is a collection of cells that recognize different cancer antigen peptides, it is more effective to use a combination of multiple types of peptides and / or derivatives. You may combine with cancer antigen peptides other than the peptide of this invention. The pharmaceutical composition of the present invention can be administered together with an adjuvant conventionally known to be used for vaccine administration so that an immune response is effectively established. Further, a liposomal preparation, a particulate preparation bound to beads having a diameter of several meters, or a preparation bound to lipid may be used.

[0025] The administration method is, for example, intradermal administration or subcutaneous administration. Dosage, disease state The amount of the peptide or derivative of the present invention in the pharmaceutical composition is usually 0.0001 mg to 1000 mg, preferably 0.0001 mg to 100 mg, more preferably 0.001 mg. ~ 10mg. It is preferable to administer this once every several days, weeks or months for 1 to 3 years.

[0026] The nucleic acid molecule of the present invention can provide the peptide or derivative of the present invention. When a vector containing the nucleic acid molecule of the present invention is introduced into an antigen-presenting cell and expressed, the peptide or derivative of the present invention is produced and forms a complex with the HLA molecule and presented on the cell surface. This antigen-presenting cell is capable of efficiently proliferating peptide-specific prostate cancer reactive CTL.

[0027] The vector of the present invention can be used for administration to a patient to express the peptide or derivative of the present invention in the patient. Further, after the vector of the present invention is introduced into an appropriate cell outside the body, for example, a dendritic cell derived from a patient, the cell may be returned to the patient. These methods are well known in the art (Hrouda D, Dalgleish AG. Gene therapy for prostate cancer. Gene Ther 3: 845-52, 1996).

[0028] Examples of the vector of the present invention include various plasmids and viral vectors such as adenovirus, adeno-associated virus, retrovirus, vaccinia virus and the like (Liu M, Acres B, Balloul jM, Bizouarne N, Paul S, MOS). P, ^ quioan P. Gene-based vaccines and immunotherapeutics. Proc Natl Acad Sci USA 101 Suppl, 14567-71, 2004). Vector preparation methods are well known in the art (Molecular Cloning: A laboraroy manual, 2nd edn. New York, Cold Spring Harbor Laboratory).

[0029] When the vector of the present invention is administered to a patient as a pharmaceutical composition for treating or preventing prostate cancer, the dosage varies depending on the disease state, individual patient age, weight, etc. The DNA content is 0.1 μg to 100 mg, preferably 1 μg to 50 mg. Examples of the administration method include intravenous injection, subcutaneous administration, intradermal administration and the like.

[0030] The CTL induction method of the present invention provides a CTL that damages HLA-A3 supertype allele-positive prostate cancer cells. In the present invention, “prostate cancer reactivity” means that it has a property of recognizing a complex of a cancer antigen peptide on a prostate cancer cell and an HLA molecule and damaging the cell. Induction of CTL is, for example, HLA-A3 supertype array PBMC collected from a patient with le-positive prostate cancer is cultured in vitro in the presence of the peptide or derivative of the present invention. The CTL induced by the method of the present invention is useful for adoptive immunotherapy, that is, a cancer treatment method in which CTL induced in a patient's body from which PBMCs are collected and cancer cells are damaged. That is, CTL induced by the method of the present invention can be used as a medicament for treating or preventing prostate cancer.

[0031] The CTL induction kit of the present invention is used for carrying out the CTL induction method. The kit of the present invention contains one or more of the peptides or derivatives of the present invention, and further includes an appropriate buffer or medium.

[0032] The antigen-presenting cell preparation method of the present invention provides an antigen-presenting cell for inducing CTL that damages HLA-A3 supertype allele-positive prostate cancer cells. Antigen-presenting cells can be prepared, for example, by the ability of the peptide or derivative of the present invention to be incorporated into cells having antigen-presenting ability derived from HLA-A3 supertype allele-positive prostate cancer patients, or such This is carried out by introducing and expressing the vector of the present invention in cells by a well-known method. Cells with antigen-presenting ability are, for example, dendritic cells, and are prepared by separating cultured plate adherent cells from PBMC collected from patients and culturing them in the presence of IL-4 and GM-CSF for about 1 week. be able to. Antigen-presenting cells prepared by the method of the present invention can induce CTLs that specifically recognize complexes of peptides or derivatives presented on the cell surface and HLA molecules, and when administered to patients, It can promote the induction of prostate cancer reactive CTL in the body. That is, the antigen-presenting cells prepared by the method of the present invention can be used as a medicament for treating or preventing prostate cancer.

[0033] The antigen-presenting cell preparation kit of the present invention is used for performing the antigen-presenting cell preparation method. The kit of the present invention contains one or more kinds of the peptide or derivative of the present invention, and can further contain an appropriate buffer or medium.

[0034] Power to explain the present invention in more detail by the following examples The present invention is not limited by these examples in any way.

Example

[0035] 1. Method 1.1 Patients

PBMCs were collected with prior written consent from HLA-A3 supertype allele positive prostate cancer patients and healthy volunteers (HD). Patients included HLA-A11 positive, -A31 positive, and -A33 positive patients. HLA-A3 positive or -A68.1 positive patients are extremely infrequent in Japanese (1.6% and 0.5%) (Aizawa M. The Proceedings of the 3ra Asia-Oceania Histocompaatioility Workshop Conference, pp. 1090-1103 uxror d: Oxford University Press, 1986 ·), PBMCs derived from them were not available. None of the patients who collected PBM C were HIV-infected. 20 ml of peripheral blood was collected, and PBMCs were prepared by the Huycol 'Conley specific gravity centrifugation method. All samples were stored at low temperatures until used in experiments. Expression of HLA-A11, -A31 and -A33 molecules on PBMC was confirmed by flow cytometry using the following antibody: anti-HLA-A11 monoclonal antibody (mAb) (C at # 0284HA; One Lambda Inc., Canoga, CA, USA); anti-HLA-A31 mAb (Cat # 0273 HA; One Lambda); anti-HLA- A33 mAb (Cat # 0612HA; One Lambda); and FITC-conjugated anti-mouse immunoglobulin G (IgG) mAb . The start of this study was approved by the Kurume University Medical Ethics Committee.

1.2 Detection of peptide-specific IgG

The level of SART3-derived peptide-specific IgG in plasma was measured by the Luminex (trademark) method as previously reported (Komatsu N, Shichijo S, Nakagawa M, Itoh Κ, Scand J Clin Invest 2004; 64: 1-11 ·). Specifically, diluted serum (100 L) was added to SART3-derived peptide-coated color-coded beads (Luminex Corp (Austin, TX, USA) (5 μ 1)) and 96-well filter plate (MABVN1250, Millipore Corp., Incubated for 2 hours at room temperature on a plate shaker in Bedford, MA, USA After 2 hours, the plate was washed with T-PBS and biotinylated anti-human IgG (BA-3080 (VECTOR LAB, (CA, USA) (100 1) for 1 hour at room temperature, wash the plate, add streptavidin-labeled PE (100) (S-866, Invitrogen Detection Technologies, Eugene, Oregon, USA), 30 The bound beads were washed 4 times, followed by addition of Tween-PBS dOO l) to each well 50 1 samples were used for measurements by the Luminex ™ method SART3-derived peptides In order to confirm the specificity of IgG for Control The cells were cultured on plates coated with HIV-derived peptides. Thereafter, the level of IgG specific for the corresponding SART3-derived peptide in the obtained supernatant was measured by the Luminex (trademark) method.

[0037] 1.3 Cell lines and flow cytometry

C1R_A11, -A31, and -A33 are C1R subcell lines that stably express the HLA-A1101, -A3101, and -A3303 genes, respectively. Expression of HLA-A11, -A31 and -A33 molecules on these subcell lines has been reported previously (Takedatsu H, Shichijo S, Katagiri K, Saw amizu H, Sata M, Itoh Κ, Clin Cancer Res 2004; 10: 1112-1120 ·). PC3, PC-93, and LNCaP are prostate cancer cell lines, and KE4 is an esophageal cancer cell line. To produce LNCaP subcell lines expressing HLA_A11, -A 31 and -A33 molecules, respectively, the HLA-A11 01, -A3101 or -A3303 plasmid cDNA was transformed into the eukaryotic expression vector pCR3.1 (Invitrogen, Carlsbad , CA, USA) and electoral positioning was performed using Gene Panoresa (Bio RAD, Richmond, CA, USA). LNCaP_All, -A31 and -A33 are subcell lines that stably express the HLA-A1101, -A3101 and -A3303 genes, respectively (Fig. 1). All these cell lines were cultured in RPMI 1640 (Invitrogen) containing 10% FCS.

[0038] 1.4 RT-PCR

Total RNA of cancer cell lines was isolated using RNAzol ™ B (Tel-Test Inc., Friendswood, TX, USA). cDNA was prepared using the Superscript ™ Preamplification System for First-Strand cDNA Synthesis (Invitrogen) and amplified using the following primers: SART3: 5′-AAGTACGCCAACATGTGGC-3 ′ (sense, SEQ ID NO: 34) , 5,-CTCTG CTCATTGACACGAGC-3 '(antisense, SEQ ID NO: 35)); β-actin: 5, -CTTCG CGGGCGATGC-3' (sense, SEQ ID NO: 36), 5, -CGTACATGGCTGGGGTGTTG-3 '(antisense, sequence Number 37). PCR is 95 in a DNA thermorecycler (ICycler, Bio-Rad laboratories, Hercules, CA, USA) using Taq DNA polymerase. C1 min, 60 ° C 1 min, 72 ° C 1 min, 30 cycles. PCR products were separated by electrophoresis on a 2% agarose gel.

[0039] 1.5 peptide

All SART3-derived peptides shown in Table 1 were prepared based on the binding motif for the HLA-A3 supertype allele molecule (Parker C, Bednarek MA, Coligan JE., J Immu nol 1994; 152: 163-175.) ₀ Influenza (Flu) virus-derived peptide, Epstein's virus (EBV) -derived peptide, tyrosinase-related protein 2 (TRP2) -derived peptide, and HIV-derived peptide, HLA- Used as a binding control for the A3 supertype allele. All peptides were purchased from Biologica Co. (Nagoya, Japan) and dissolved in dimethyl sulfoxide at a dose of 10 Hg / ml.

[table 1]

? ? ? f ,.

Table 1 H¾ of SART3-derived peptides that bind to ΗΙΛ-Α3 supertype allele molecules

33-41 TRR VLSR Q.O

-\ n RLEGELTKV 2.7

127-135 ELTKVRMA 27.0

158-166 S AQDGLDR 15.0

G .ALWEAYR 9.0 LEKVHSL R 2

301-309 ALLQAEAPR 9.0

CLVPDLW! 9.0

364-273 VI, SVHNRAiR 9.0

396-405 HQV1SVTFF. Q.O

QVISVTi 'K 0.0

455-463 YL QEVF.ER y.o

472-! VIMQNWA. Q.O

4,4-493 RLCNNM <) A 2.7

EL.WDSIMTR 27.0 WLEYY LEi 9.0 IU.ARVNEpR 2.7 AL K I 9.0 SMQEPDT LR 15.0

734-742 QiRPIPSNR 15.0

759-769 ALQALE DR 9.0

(EMDRKSVEGR 45.0 RLVT RAOK 0.1)

872- SSI VAISNPPQR 45

KVPEKFET 4.5 M. QTYGAR 9.0

910-91 TQLSLLP Q.O

914-922 SLL AI.Q 9.0

M ADFA D.O

1VTDFSVIK 0.5

NVKNLYE V 0.5

T.P2 LLGPORPYR 9.0a: The binding score was calculated based on the expected half-life of dissociation from the HLA class molecule.

('No 31 Busite; Bioinfomiaics and M! Ecular Analysts Section, Cotwtttalsonal Bioscience m <5 Er

Division of Con¾> uti ^ eseach & eehno! Ov _T NIH)

HIV-derived nucleotides are not calculated because they are composed of 11 amino acids.

b: The HLA class that was previously reported that the peptide is immunogenic ¾! _U allele

1.6 Induction of peptide-specific CTL from PBMC

Peptide-reactive CTL was detected with some modifications to the previously reported method (Hida N, Maeda Y, Katagiri Κ, TaKasu Η, Harada Μ, Itoh Κ, Cancer Immunol Immunother 2002; 51: 219-28 U bottom 96 PBMC (1 X 10 ⁵ cells / well) was added to each peptide (10 μ 1 / ml) and 4 μL in a culture medium of 200 μΐ in a well-microphone culture plate (Nunc, Roskilde, Denmark). Incubated in eru set. This culture is 45% RPMI 1640, 45% AIM-V medium (Gib co-BRL, Gaithersburg, MD), 10% FCS, 100 U / ml interleukin-2 (IL-2) and 0. ImM MEM not required It consisted of an amino acid solution (Gibco_BRL). Every 3 or 4 days, half of the culture was removed and replaced with a new culture containing the corresponding peptide (20 g / ml) and IL-2 (100 U / ml). On the 15th day of culture, the cultured cells were divided into 4 wells. Two wells were used for C1R_A11, -A31 or -A33 cells pulsed with the corresponding peptide, and the other two wells were cultured with C1R_A11, -A31 or -A33 cells pulsed with HIV peptide. After 18 hours of incubation, the supernatant was collected, and the level of IFN-γ was measured by enzyme linked immunoassay (ELISA). Peptide-reactive CTL induction is ρ <0 · 05 by two-sided student t-test and more than 100 pg / ml interferon in response to cells pulsed with the corresponding peptide compared to cells pulsed with HIV peptide ( IFN) _-y was judged positive when produced.

[0041] 1.7 Measurement of cytotoxic activity

The cytotoxic activity of peptide-stimulated PBM C against LNCaP, LNCaP_All, LNCaP_A31 or LNCaP_A33 was measured by standard 6 hour ⁵¹ Cr release assay. Phyto-Magnolechun (PHA) activated T cells were used as negative control cells. In a round-bottom 96-well plate, 2000 ⁵¹ Cr-labeled cells were cultured with effector cells at the indicated effector cell / target cell ratio. Specific ⁵¹ Cr release was calculated by the following formula: Specific lysis (%) = (Test sample release spontaneous release) / (Maximum release spontaneous release). When ⁵¹ Cr-labeled cells were incubated without effector cells, the amount of ⁵¹ Cr in the supernatant was spontaneous release, and when ⁵¹ Cr-labeled cells were incubated with 1% Triton X (Wako Pure Chemical Industries, Osaka, Japan) The amount of ⁵¹ Cr in the supernatant is the maximum release.

[0042] 1.8 Cold target cell inhibition experiment

The specificity of peptide-reactive CTLs was confirmed by inhibition experiments with cold target cells. Immediately before the experiment, CD8 positive T cells were isolated using a CD8 Positive Isolation Kit (Dynal, Oslo, Norway). Specifically, purified CD8 positive T cells (2 x 10 ⁴ cells / well) were placed in a round bottom 96-well plate in the presence of cold target cells (4 x 10 ⁴ cells) with ⁵¹ Cr labeled target cells ( 2 × 10 ³ cells / well). Corresponding SART3 peptide or HIV peptide C1R_A11, -A31, and -A33 pre-pulsed with either of these were used as cold target cells.

[0043] 1.9 statistics

Statistical significance of the data was determined by a two-sided student t test. P values less than 0.05 were considered statistically significant.

[0044] 2. Results

2.1 Measurement of SART3-derived peptide-reactive IgG in the plasma of prostate cancer patients

First, 29 SART-3-derived peptides were prepared based on the binding motif for the HLA-A3 supertype allele (Table 1). HLA-A3 and HLA-A68 molecules also belong to the HLA-A3 supertype allele (Sette A, Sidney J., Immunogenetics 1999; 50: 201-21 2.), but they are extremely rare in Japanese. The binding ability to HLA-A11, -A31, and -A33 molecules was preferentially examined. The purpose of this study is to identify peptides that have the ability to induce cancer-reactive CTL in HLA-A3 supertype positive prostate cancer patients. Has been observed in the plasma of patients with various cancers (Nakatsura T, Senju S, Ito M, Nishimu ra Y, Itoh Satoshi, Eur J Immunol 2002; 32: 826-836 .; Ohkouchi S , Yamada A, Imai N, Minet, Harada, Shichijo S, Maeda Y, Saijo Y, Nukiwa T, Itoh., Tissue Antigens 2 002; 59: 259-272.) _{O In} addition, PBMC obtained from prostate cancer patients Because of the limited number, 29 peptides were too numerous to test individual peptides for their ability to induce peptide-specific CTL from PBMC. Therefore, these peptide candidates were first screened based on their ability to be recognized by IgG from prostate cancer patients.

[0045] As a result, SART3 (SEQ ID NO: 6), SART3 (SEQ ID NO: 16), SART3 (

243-252 511-519 734-742 SEQ ID NO: 20), SART3 (SEQ ID NO: 23) or SART3 (SEQ ID NO: 27)

831-839, 910-918

IgG response to chido was detected in plasma of 5, 7, 13, 5, and 5 of 20 prostate cancer patients, respectively (Table 2). IgG that reacts with these five SART3-derived peptides was also detected in plasma of 2, 2, 8, 1, and 4 of 20 HDs (data not shown). SART3-derived peptide-reactive IgG for these five types was detected more frequently in the plasma of prostate cancer patients than in HD. Against 24 other SART3-derived peptides 0064

We investigated whether it was possible. In vitro, PBMCs were stimulated with each SART3-derived peptide or control peptide, and the stimulated cells responded to C1R_A11, C1R-A31, or C1R-A33 cells with the corresponding peptide nors in response to IFN-γ. The production was examined.

Table 3 shows representative results of 15 HLA-A3 supertype allele positive prostate cancer patients (5 per allele). Induction of peptide-specific CTL was considered successful when p <0.05 and the difference from IFN-γ production by the control HIV peptide was greater than 100 pg / ml. As a result, SART3 (SEQ ID NO: 6), SART3 (SEQ ID NO: 16), SART3 (SEQ ID NO: 16)

243-252 511-519 734-742 number 20), SART3 (SEQ ID NO: 23) and SART3 (SEQ ID NO: 27) peptides

831-839, 910-918

Induced corresponding peptide-reactive CTLs from 0, 11, 6, 3, and 5 PBMCs of 15 HLA-A3 supertype allele positive prostate cancer patients, respectively. These five SART3-derived peptides also induced peptide-reactive CTLs from 1, 6, 3, 1, and 3 PBMCs, respectively, of 15 HLA-A3 supertype allele positive HDs (de) Data not shown).

[Table 3]

# i * ¾ 16ffl b VHHffi c¾ lΘ 01 e Ί10--,

[0048] Of these five types of SART3-derived peptides, SART3 (SEQ ID NO: 16)

511-519

And SART3 (SEQ ID NO: 20) peptide, especially the HLA-A3 supertype allele

734-742

It shows that it is suitable as a peptide for inducing peptide-specific CTL in PBMC of patients with positive prostate cancer.

[0049] 2.3 Prostate cancer response from PBMC in HLA-A3 supertype allele positive prostate cancer patients Induction of sex CTL

Next, SART3 (SEQ ID NO: 16) and SART3 (SEQ ID NO: 20) peptides were used.

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We investigated whether CTL induced by in vitro stimulation showed cytotoxic activity against prostate cancer cells. Prior to the cytotoxic activity test, expression of the SART3 gene in prostate cancer cell lines was examined by an RT-PCR based method (FIG. 1A). SART3 mRNA expression was observed in the KE4 esophageal cancer cell line (cells in which SART3 was identified by tumor-infiltrating T cells (Yang D, Nakao M, ^ nicmjo Sasatomi Ί, Takasu H, Matsumoto H, Mori, Hayanshi A, Yama na H , Shirouzu K, Itoh Κ, Cancer Res 1999; 59: 4056-4063.))! Prostate cancer cell lines PC3, PC93, and LNCaP also expressed SART3 mRNA. Furthermore, in order to clarify the ability of HLA-A3 supertype allele-restricted cytotoxic activity, LNCaP transfectants expressing each of the HLA-A11, -A31, and -A33 molecules were prepared (Fig. 1B).

[0050] In vitro, PBMC from HLA-A3 supertype allele-positive prostate cancer patients were stimulated with either SART3 or SART3 peptide and induced thereby

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It was investigated whether the obtained peptide-reactive CTL could show cytotoxic activity against LNCaP transfectant (Fig. 2A, Fig. 2B). In vitro SART3 (SEQ ID NO: 16) and

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HLA-A11 positive patients (Pt. 1, 2, 5) stimulated with each of SART3 (SEQ ID NO: 20)

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And PBMCs derived from 17) showed higher levels of cytotoxic activity against LNCaP-All cells than against LNCaP cells and HLA-A11-positive immunized T cells. Similarly, these peptides had the ability to induce LNCaP transfectant-reactive CTLs from PBMCs of HLA-A31 positive patients and HLA-A33 positive patients. That is, these peptide-reactive CTLs showed higher levels of cytotoxic activity against LNCaP_A31 cells and LNCaP_A33 cells than to LNCaP cells or immunized T cells.

[0051] These results indicate that SART3 (SEQ ID NO: 16) or SART3 (

511-519 734-742 SEQ ID NO: 20) PBMC stimulated with the peptide, HLA-A11 against prostate cancer cells,

It shows that -A31 or -A33 exerts cytotoxic activity in a restricted manner.

2.4 Cytotoxic activity against prostate cancer cells dependent on peptide-specific CD8 + T cells In addition, we attempted to identify the types of cells involved in the cytotoxic activity of peptide-stimulated PBMC. Purified CD8 positive T cells were used for the following experiments.

[0053] As shown in FIGS. 3A and 3B, SART3 (SEQ ID NO: 16) and SART3 (

511-519 734-742 SEQ ID NO: 20) Cytotoxic activity of PBMC from HLA-A11, HLA-A31, and HLA-A33 positive patients stimulated with peptide against LNCaP_All, LNCaP_A31, and LNCaP_A33, pulse corresponding peptide Significantly suppressed by addition of unlabeled C1R_A11, C1R_A31, and C1R-A33 cells Unlabeled C1R-All, C1R_A31, and C1R-A33 cells pulsed with HIV peptide were not suppressed.

[0054] The results of this cold inhibition experiment indicate that the peptide-specific CD8-positive T cells are likely to have cytotoxic activity against peptide-stimulated PBMC against LNCaP transfectants.

[0055] 3. Summary

The above results indicate that the SART3-derived peptide of the present invention can induce prostate cancer-reactive CTL in HLA-A3 supertype allele-positive prostate cancer patients and is useful for specific immunotherapy, particularly cancer vaccine therapy.

Claims

The scope of the claims

[1] A peptide consisting of the amino acid sequence shown in SEQ ID NO: 16 or 20.

[2] A pharmaceutical composition comprising a peptide consisting of the amino acid sequence shown in SEQ ID NO: 16 or 20.

[3] The pharmaceutical composition according to claim 2, wherein the pharmaceutical composition is a cancer vaccine.