JP2007145715A - Hla-a24- or hla-a2-binding peptide of parathyroid hormone-related protein - Google Patents

Hla-a24- or hla-a2-binding peptide of parathyroid hormone-related protein Download PDF

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JP2007145715A
JP2007145715A JP2004156460A JP2004156460A JP2007145715A JP 2007145715 A JP2007145715 A JP 2007145715A JP 2004156460 A JP2004156460 A JP 2004156460A JP 2004156460 A JP2004156460 A JP 2004156460A JP 2007145715 A JP2007145715 A JP 2007145715A
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hla
peptide
pthrp
prostate cancer
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JP4579581B2 (en
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Kyogo Ito
恭悟 伊東
Mamoru Harada
守 原田
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Green Peptide Co Ltd
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Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel treating method of prostate cancers. <P>SOLUTION: A cancer antigen peptide that is a partial peptide derived from a parathyroid hormone-related protein and can bind to an HLA-A24 or HLA-A2 antigen and be recognized by cytotoxic T lymphocytes, or a derivative thereof having the functionally equivalent properties, can be useful for the treatment and prevention of prostate cancers. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

本発明は、ヒト主要組織適合抗原HLA-A24またはHLA-A2陽性前立腺癌患者において免疫原を有する副甲状腺ホルモン関連タンパク質由来抗原ペプチドの同定に関し、詳細には、前立腺癌を処置または予防するための特異的癌抗原、それを含む癌ワクチン、およびそのための治療学的方法に関する。   The present invention relates to the identification of antigenic peptides derived from parathyroid hormone-related proteins having immunogens in human major histocompatibility antigen HLA-A24 or HLA-A2 positive prostate cancer patients, and in particular for treating or preventing prostate cancer It relates to specific cancer antigens, cancer vaccines containing them, and therapeutic methods therefor.

前立腺癌は、老齢男性において頻度の高い癌の１つである[1]。前立腺癌は骨に転移することが多く、骨転移を有する患者にはアンドロゲン遮断療法が適用されている。ホルモン療法はこのような患者における疾患進行を一時的に抑制できるが、ほとんどの場合、ホルモン不応性の前立腺癌への進展することが避けられない。従って、新たな治療方法の開発が求められている。   Prostate cancer is one of the most common cancers in older men [1]. Prostate cancer often metastasizes to bone, and androgen blocking therapy is applied to patients with bone metastasis. Hormone therapy can temporarily suppress disease progression in such patients, but in most cases, progression to hormone refractory prostate cancer is inevitable. Therefore, development of a new treatment method is demanded.

腫瘍免疫学の最近の進歩により、種々のタイプの癌患者においてヒト癌関連抗原をコードする遺伝子、および細胞傷害性Tリンパ球（CTL）によって認識されるそれらの抗原エピトープが同定されている[2 - 4]。これらの同定された癌抗原やそのペプチドは特異的免疫療法に応用されている[5 - 7]。前立腺癌の場合、正常な前立腺に発現している組織特異的な抗原も前立腺癌患者に対する特異的免疫療法のための標的分子となりうる。前立腺特異的抗原（PSA）または前立腺特異膜抗原（PSMA）を標的とする免疫療法はすでに実施されており、抗腫瘍効果が限られた症例であるが確認されている[8 - 12]。   Recent advances in tumor immunology have identified genes encoding human cancer-associated antigens and their antigenic epitopes recognized by cytotoxic T lymphocytes (CTLs) in patients with various types of cancer [2 - Four]. These identified cancer antigens and their peptides have been applied to specific immunotherapy [5-7]. In the case of prostate cancer, tissue-specific antigens expressed in normal prostate can also be a target molecule for specific immunotherapy for prostate cancer patients. Immunotherapy targeting prostate specific antigen (PSA) or prostate specific membrane antigen (PSMA) has already been performed and has been identified in cases with limited antitumor effects [8-12].

副甲状腺ホルモン関連タンパク質(PTHrP)は、骨芽細胞の受容体に結合するオートクリンまたはパラクリン因子であり、骨の形成と再吸収を刺激する。PTHrPはN端側で副甲状腺ホルモン(PTH)と相同性を有しており、PTHと同じ受容体に結合できるため、PTHと同様の活性をもたらす[13]。PTHrPは、カルシウム輸送、ケラチン形成細胞の分化、平滑筋の弛緩や軟骨の発育など種々の生理学的役割を担っている[14]。副甲状腺細胞の場合は、高い細胞外カルシウム濃度が負のフィードバック調節を生じ、PTHの分泌と副甲状腺細胞の増殖とを抑制するが、PTHrPの場合は、分泌が持続し、骨再吸収の増悪を促進する[15]。従って、PTHrPは悪性腫瘍に付随する高カルシウム血症に関与すると考えられている[16]。さらに、前立腺癌はPTHrPを産生すると報告されている[17]。これらは、PTHrPが骨転移を伴う前立腺癌患者の免疫療法における格好の標的分子である可能性を示している。PTHrP 59-68 および PTHrP 165-173 ペプチドはHLA-A2陽性前立腺癌患者を特異的に免疫治療するための候補ペプチドであると報告されている[16, 17]。
本研究では、HLA-A24またはHLA-A2陽性前立腺癌患者において免疫原性を有する新たなPTHrP由来ペプチドの同定を試みた。 Parathyroid hormone-related protein (PTHrP) is an autocrine or paracrine factor that binds to osteoblast receptors and stimulates bone formation and resorption. PTHrP is homologous to parathyroid hormone (PTH) on the N-terminal side and can bind to the same receptor as PTH, resulting in activity similar to PTH [13]. PTHrP plays various physiological roles such as calcium transport, keratinocyte differentiation, smooth muscle relaxation and cartilage development [14]. In the case of parathyroid cells, high extracellular calcium concentration results in negative feedback regulation and suppresses PTH secretion and parathyroid cell proliferation, but in the case of PTHrP, secretion persists and bone resorption is exacerbated. Promote [15]. Therefore, PTHrP is thought to be involved in hypercalcemia associated with malignant tumors [16]. In addition, prostate cancer has been reported to produce PTHrP [17]. These indicate that PTHrP may be a good target molecule in immunotherapy of prostate cancer patients with bone metastases. PTHrP 59-68 and PTHrP 165-173 peptides have been reported to be candidate peptides for the specific immunotherapy of patients with HLA-A2 positive prostate cancer [16, 17].
In this study, we attempted to identify a new PTHrP-derived peptide that is immunogenic in patients with HLA-A24 or HLA-A2 positive prostate cancer.

前立腺癌は特異的免疫療法を開発するうえで格好な標的と考えられる[25]。近年、本発明者らは、前立腺癌患者から前立腺癌反応性CTLを生じさせることのできる前立腺関連抗原由来のエピトープペプチドの同定を試みている[22, 23, 26]。しかし、前立腺癌患者を治療する際の主な障害は、骨組織に転移した前立腺癌に対する治療である。従って、本発明者らは、転移を有するHLA-A24またはHLA-A2陽性前立腺癌患者を特異的免疫療法により処置するのに適している可能性ある抗原エピトープを同定するため実験を行った。   Prostate cancer is considered a good target for developing specific immunotherapy [25]. In recent years, the inventors have attempted to identify epitope peptides derived from prostate-related antigens that can generate prostate cancer-reactive CTLs from prostate cancer patients [22, 23, 26]. However, a major obstacle in treating prostate cancer patients is treatment for prostate cancer that has metastasized to bone tissue. Therefore, the inventors conducted experiments to identify antigenic epitopes that may be suitable for treating HLA-A24 or HLA-A2 positive prostate cancer patients with metastases with specific immunotherapy.

本実験では、ヒト主要組織適合抗原(HLA)-A24またはHLA-A2陽性前立腺癌患者において免疫原性を有するPTHrP-誘導ペプチドの同定を試みた。HLA-A24結合モチーフを有する４つの異なるPTHrPペプチドのうちPTHrP 36-44 および PTHrP 102-111ペプチドが、HLA-A2結合モチーフを有する７つの異なるPTHrPペプチドのうちPTHrP 42-51 および PTHrP 59-67ペプチドが、それぞれのHLA陽性前立腺癌患者の末梢血単核細胞（PBMC）からペプチド特異的な細胞傷害性Tリンパ球を効率的に誘導した。ペプチドで刺激したPBMCは前立腺癌細胞に対してHLA-A24またはHLA-A2拘束性に細胞傷害活性を示した。抗体およびコールド標的細胞を用いた実験により、それらの細胞傷害活性はPTHrPペプチド特異的なCD8陽性T細胞に依存していることが判明した。HLA-A24分子に結合するPTHrP 102-111 またはPTHrP 110-119ペプチド、およびHLA-A2分子に結合するPTHrP 42-51ペプチドに反応する免疫グロブリンG(IgG)は前立腺癌患者の血漿中において高頻度に検出でき、このことは、PTHrP 102-111およびPTHrP 42-51ペプチドが癌患者の細胞性および液性の両方の免疫応答に認識されることを示している。これらの結果は、本発明のペプチドが、転移を伴うHLA-A24またはHLA-A2陽性前立腺癌患者に対する特異的な免疫療法のための有望な標的分子でありうることを示す。   In this experiment, we attempted to identify PTHrP-derived peptides that are immunogenic in patients with human major histocompatibility antigen (HLA) -A24 or HLA-A2 positive prostate cancer. PTHrP 36-44 and PTHrP 102-111 peptides out of 4 different PTHrP peptides with HLA-A24 binding motifs, PTHrP 42-51 and PTHrP 59-67 peptides out of 7 different PTHrP peptides with HLA-A2 binding motifs However, peptide-specific cytotoxic T lymphocytes were efficiently induced from peripheral blood mononuclear cells (PBMC) of each HLA-positive prostate cancer patient. PBMC stimulated with peptide showed HLA-A24 or HLA-A2 restricted cytotoxic activity against prostate cancer cells. Experiments with antibodies and cold target cells revealed that their cytotoxic activity was dependent on PTHrP peptide-specific CD8 positive T cells. Immunoglobulin G (IgG) reacts with PTHrP 102-111 or PTHrP 110-119 peptide that binds to HLA-A24 molecule and PTHrP 42-51 peptide that binds to HLA-A2 molecule in plasma of prostate cancer patients This indicates that the PTHrP 102-111 and PTHrP 42-51 peptides are recognized by both cellular and humoral immune responses in cancer patients. These results indicate that the peptides of the present invention may be promising target molecules for specific immunotherapy against HLA-A24 or HLA-A2 positive prostate cancer patients with metastasis.

本発明において、HLA-A24またはHLA-A2陽性前立腺癌患者において前立腺癌特異的なCTLを産生させる可能性ある新たなPTHrPペプチドが同定され、それによりPTHrPを標的とするペプチドワクチンを作成できる可能性が広がった。本発明者らは、PTHrP 36-44およびPTHrP 102-111ペプチドの両者がHLA-A24陽性前立腺癌患者において、また、PTHrP 42-51 および PTHrP 59-67ペプチドがHLA-A2陽性前立腺癌患者において、それぞれ前立腺癌反応性CTLを誘導する可能性があることを示した。HLA-A24陽性前立腺癌患者由来のPBMCは、PTHrP 36-44ペプチドまたはPTHrP 102-110ペプチドで刺激するとそれぞれ、10人の患者のうち７または6人においてペプチド特異的なIFN-γ産生を示した。HLA-A2結合性PTHrP 42-51ペプチドおよびPTHrP 59-67ペプチドは、10人の患者のうち５または４人においてペプチド特異的なIFN-γ産生を誘導した。さらに重要なことは、これらのPTHrPペプチドで刺激されたPBMCはHLA-A24またはHLA-A2拘束性に前立腺癌細胞に対して細胞傷害活性を示したことである。これらの結果は、上記のPTHrPペプチドが免疫原性を有しており、転移を伴うHLA-A24またはHLA-A2陽性前立腺癌患者の特異的な免疫療法に有用である可能性を示している。   In the present invention, a new PTHrP peptide capable of producing prostate cancer-specific CTLs in HLA-A24 or HLA-A2-positive prostate cancer patients has been identified, and thereby the possibility of creating a peptide vaccine targeting PTHrP Spread. We have found that both PTHrP 36-44 and PTHrP 102-111 peptides are HLA-A24 positive prostate cancer patients, and PTHrP 42-51 and PTHrP 59-67 peptides are HLA-A2 positive prostate cancer patients, It was shown that each could induce prostate cancer reactive CTL. PBMC from HLA-A24 positive prostate cancer patients showed peptide-specific IFN-γ production in 7 or 6 out of 10 patients when stimulated with PTHrP 36-44 peptide or PTHrP 102-110 peptide, respectively . HLA-A2 binding PTHrP 42-51 peptide and PTHrP 59-67 peptide induced peptide-specific IFN-γ production in 5 or 4 out of 10 patients. More importantly, PBMCs stimulated with these PTHrP peptides showed cytotoxic activity against prostate cancer cells in a HLA-A24 or HLA-A2 restricted manner. These results indicate that the above PTHrP peptide is immunogenic and may be useful for specific immunotherapy of HLA-A24 or HLA-A2 positive prostate cancer patients with metastasis.

これらペプチドはさらに、HLA-A24またはHLA-A2陽性健常ドナーのPBMCからペプチド特異的かつ腫瘍反応性のCTLを誘導した。この結果は、HLA-A2陽性健常ドナーのPBMCからPTHrPペプチド特異的CTLが誘導されることを示した既報[17]と整合している。これらペプチドとPTHとは相同性が低いことから、PTHrP ペプチドとPTHとの間の交差反応の可能性は除外できると考えられる。PTHrPはケラチン生成細胞、子宮および授乳期の乳腺において散発的に低レベルで検出される[27]。腫瘍免疫の最近の進歩により、ヒト癌細胞における自己抗原が免疫系に認識される主な腫瘍抗原であることが示された[2-4]。変異していない自己抗原に反応するCTL前駆体が特定の健常ドナーおよび癌患者の両者の末梢血に循環している可能性がある。   These peptides further induced peptide-specific and tumor-reactive CTLs from PBMC of HLA-A24 or HLA-A2 positive healthy donors. This result is consistent with a previous report [17] showing that PTHrP peptide-specific CTLs are induced from PBMC of HLA-A2-positive healthy donors. Since these peptides and PTH have low homology, the possibility of cross-reaction between PTHrP peptide and PTH can be excluded. PTHrP is detected sporadically at low levels in keratinocytes, uterus and lactating mammary glands [27]. Recent advances in tumor immunity have shown that autoantigens in human cancer cells are the main tumor antigens recognized by the immune system [2-4]. CTL precursors that react to unmutated autoantigens may be circulating in the peripheral blood of both certain healthy donors and cancer patients.

ここでは、本発明者らは、クラスI結合性腫瘍ペプチドに対する抗体が特定の癌患者や健常ドナーに既に観察されていることから[20, 21]、PTHrPペプチドに対するIgGがHLA-A24またはHLA-A2陽性前立腺癌患者由来の血漿中において検出可能か否かを調べた。本発明者らは以前、前立腺関連抗原から誘導されたペプチドに反応するIgGが健常ドナーと前立腺癌患者に頻繁に検出されることを報告している[22-24]。本発明では、HLA-A24分子結合性ペプチドではPTHrP 102-111ペプチドまたはPTHrP 110-119ペプチドのいずれかに反応するIgGが、HLA-A2分子結合性ペプチドではPTHrP 42-51ペプチドに対するIgGが、健常ドナーおよび前立腺癌患者に頻繁に検出された。このことは、PTHrP 102-111およびPTHrP 42-51ペプチドが細胞性および液性免疫系の両者に認識されていることを意味する。抗腫瘍免疫応答においてペプチド特異的IgGが果たす役割は明確ではないが、本発明者らの臨床試験では、ペプチドワクチンにより投与したペプチドに反応するIgGを頻繁に誘導させることを観察している[28, 29]。さらに、投与したペプチドに反応するIgGの誘導は、進行性肺癌の患者の生存率の改善と正に相関していた[30]。胃癌の患者にペプチドワクチンを投与すると、投与ペプチドに対する細胞性免疫応答のみならず液性応答を示す患者では生存率が改善することが観察された[31]。さらに、投与したペプチドに反応するIgGの誘導は、再発性婦人科癌の患者における臨床効果と相関していた[32]。また、進行HRPCの患者におけるPTHrP 102-111ペプチドに特異的なIgGレベルは、非進行前立腺癌患者におけるそれよりも低いことを観察している（未掲載）。PTHrP 102-111ペプチドをこのような患者にワクチン投与すると、ペプチド特異的なIgGが誘導され、臨床効果を導くと考えられる。さらなる研究により、抗腫瘍免疫応答におけるペプチド特異的なIgGの役割が明らかになるであろう。   Here, the inventors have already observed antibodies against class I binding tumor peptides in certain cancer patients and healthy donors [20, 21], so IgG against PTHrP peptides is HLA-A24 or HLA- Whether it was detectable in plasma from A2-positive prostate cancer patients was examined. We have previously reported that IgG that responds to peptides derived from prostate-related antigens is frequently detected in healthy donors and prostate cancer patients [22-24]. In the present invention, IgG that reacts with either PTHrP 102-111 peptide or PTHrP 110-119 peptide is used in HLA-A24 molecule-binding peptide, and IgG for PTHrP 42-51 peptide is healthy in HLA-A2 molecule-binding peptide. Frequently detected in donors and prostate cancer patients. This means that PTHrP 102-111 and PTHrP 42-51 peptides are recognized by both the cellular and humoral immune systems. Although the role played by peptide-specific IgG in the anti-tumor immune response is unclear, our clinical trials have observed frequent induction of IgG in response to peptides administered by peptide vaccines [28 , 29]. Furthermore, the induction of IgG in response to administered peptides was positively correlated with improved survival in patients with advanced lung cancer [30]. It was observed that administration of peptide vaccines to patients with gastric cancer improved survival in patients with humoral responses as well as cellular immune responses to the administered peptides [31]. In addition, the induction of IgG in response to administered peptides was correlated with clinical efficacy in patients with recurrent gynecologic cancer [32]. We have also observed that IgG levels specific for the PTHrP 102-111 peptide in patients with advanced HRPC are lower than those in patients with non-progressive prostate cancer (not shown). Vaccination of such patients with the PTHrP 102-111 peptide is thought to induce peptide-specific IgG and lead to clinical effects. Further studies will reveal the role of peptide-specific IgGs in anti-tumor immune responses.

好ましい態様の説明Description of preferred embodiments

即ち、本発明は、
（１）副甲状腺ホルモン関連タンパク質由来の部分ペプチドであり、HLA-A24またはHLA-A2抗原に結合でき、細胞傷害性Tリンパ球に認識されうる癌抗原ペプチド、または機能的に同等の性質を有するその誘導体；好ましくは液性免疫系にさらに認識される癌抗原ペプチド；具体的には、配列番号１または２、もしくは配列番号３または４に示すアミノ酸配列を含む癌抗原ペプチド、または機能的に同等の性質を有するその誘導体；
（２）上記（１）記載の癌抗原ペプチドおよび機能的に同等の性質を有するその誘導体を少なくとも１つ活性成分として含む、前立腺癌を処置または予防するための医薬組成物、好ましくは前立腺癌が骨転移を伴う医薬組成物；
（３）上記（１）記載の癌抗原ペプチドおよび機能的に同等の性質を有するその誘導体を少なくとも１つ活性成分として含む、転移性骨病変を処置または予防するための医薬組成物；好ましくは転移性骨病変が前立腺癌に付随する医薬組成物；
（４）HLA-A24またはHLA-A2抗原と上記（１）記載の癌抗原ペプチドまたは機能的に同等の性質を有するその誘導体との複合体を特異的に認識する細胞傷害性Tリンパ球；および
（５）上記（４）記載の細胞傷害性Tリンパ球を活性成分として含む、前立腺癌を処置するための医薬組成物；好ましくは前立腺癌が骨転移を伴う医薬組成物；に関する。 That is, the present invention
(1) A partial peptide derived from a parathyroid hormone-related protein, which can bind to HLA-A24 or HLA-A2 antigen and can be recognized by cytotoxic T lymphocytes, or has a functionally equivalent property. A derivative thereof; preferably a cancer antigen peptide further recognized by the humoral immune system; specifically, a cancer antigen peptide comprising the amino acid sequence shown in SEQ ID NO: 1 or 2, or SEQ ID NO: 3 or 4, or functionally equivalent Derivatives thereof having the following properties:
(2) A pharmaceutical composition for treating or preventing prostate cancer comprising at least one of the cancer antigen peptide of (1) above and a derivative thereof having functionally equivalent properties as an active ingredient, preferably prostate cancer A pharmaceutical composition with bone metastases;
(3) A pharmaceutical composition for treating or preventing metastatic bone lesions comprising at least one of the cancer antigen peptide of (1) above and a derivative thereof having functionally equivalent properties as an active ingredient; preferably metastasis A pharmaceutical composition in which a sexual bone lesion is associated with prostate cancer;
(4) cytotoxic T lymphocytes that specifically recognize a complex of the HLA-A24 or HLA-A2 antigen and the cancer antigen peptide of (1) above or a derivative thereof having functionally equivalent properties; and (5) A pharmaceutical composition for treating prostate cancer comprising the cytotoxic T lymphocyte according to (4) as an active ingredient; preferably a pharmaceutical composition in which prostate cancer is accompanied by bone metastasis.

本発明において、「癌抗原ペプチド」なる用語は、副甲状腺ホルモン関連タンパク質の一部を含む部分ペプチドであって、HLA-A24またはHLA-A2抗原に結合でき、CTLに認識されうる部分ペプチドを意味する。「副甲状腺ホルモン関連タンパク質」なる用語はPTHrPと略称され、生体のすべての組織ではないがほとんどの組織から産生されているタンパク質ホルモンのファミリーである。PTHrPは種間において高度に保存されている単一遺伝子にコードされており、PTHrPのアミノ酸配列は文献(35)に記載されている。本発明の癌抗原ペプチドは、PTHrPの一部を含むペプチド候補物質を合成し、そのペプチド候補物質とHLA分子との複合体がCTLによって認識されるか否かを決定する検定を行うことで、同定できる。   In the present invention, the term “cancer antigen peptide” means a partial peptide containing a part of a parathyroid hormone-related protein, which can bind to HLA-A24 or HLA-A2 antigen and can be recognized by CTL. To do. The term “parathyroid hormone-related protein” is abbreviated as PTHrP and is a family of protein hormones produced from most but not all tissues of the body. PTHrP is encoded by a single gene that is highly conserved among species, and the amino acid sequence of PTHrP is described in Reference (35). The cancer antigen peptide of the present invention synthesizes a peptide candidate substance containing a part of PTHrP, and performs an assay to determine whether or not a complex of the peptide candidate substance and an HLA molecule is recognized by CTL. Can be identified.

ペプチドの合成は、ペプチド化学において通常使用される方法によって行うことができる。既知の方法としては、“Peptide Synthesis”, Interscience, New York, 1966; “The Proteins”, vol. 2, Academic Press Inc., New York, 1976; “Pepuchido-Gosei”, Maruzen Co. Ltd., 1975などの文献に記載されている方法が挙げられる。
本発明の癌抗原ペプチドは、以下の実施例に示す手法により同定できる。
本発明において、「機能的に同等の性質を有する癌抗原ペプチドの誘導体」とは、本発明の癌抗原ペプチドのアミノ酸配列において１つまたは数個の置換、欠失および／または付加を含むアミノ酸配列を有し、かつ癌抗原ペプチドとしての性質、すなわちHLA-A24分子またはHLA-A2分子に結合できCTLに認識されうる性質を備えた改変ペプチドを意味する。 Peptide synthesis can be performed by methods commonly used in peptide chemistry. Known methods include “Peptide Synthesis”, Interscience, New York, 1966; “The Proteins”, vol. 2, Academic Press Inc., New York, 1976; “Pepuchido-Gosei”, Maruzen Co. Ltd., 1975. The method described in literatures, such as these, is mentioned.
The cancer antigen peptide of the present invention can be identified by the technique shown in the following examples.
In the present invention, “a derivative of a cancer antigen peptide having functionally equivalent properties” means an amino acid sequence containing one or several substitutions, deletions and / or additions in the amino acid sequence of the cancer antigen peptide of the present invention. And a modified peptide having properties as a cancer antigen peptide, that is, a property capable of binding to HLA-A24 molecule or HLA-A2 molecule and being recognized by CTL.

本発明においては、HLA-A24またはHLA-A2陽性前立腺癌患者において液性および細胞性免疫系の両者に効率的に認識されるPTHrPペプチドが好ましい。
本発明の癌抗原ペプチドまたはその誘導体は、前立腺癌を処置または予防するための医薬組成物に使用でき、好ましくは前立腺癌は骨転移を伴っており、さらに転移性骨病変を処置または予防するための医薬組成物に使用でき、好ましくは転移性骨病変は前立腺癌に付随する。これら発明は、前立腺癌が非常に骨転移しやすく、骨破壊や骨吸収に関わるPTHrPを産生するという事実、および本発明のPTHrPペプチドが前立腺癌反応性CTLを誘導できるという結果に基づく。 In the present invention, PTHrP peptides that are efficiently recognized by both humoral and cellular immune systems in HLA-A24 or HLA-A2 positive prostate cancer patients are preferred.
The cancer antigen peptide or derivative thereof of the present invention can be used in a pharmaceutical composition for treating or preventing prostate cancer. Preferably, prostate cancer is accompanied by bone metastasis, and further for treating or preventing metastatic bone lesions. Preferably, the metastatic bone lesion is associated with prostate cancer. These inventions are based on the fact that prostate cancer is very prone to bone metastasis and produces PTHrP involved in bone destruction and bone resorption, and the results that the PTHrP peptide of the present invention can induce prostate cancer reactive CTL.

本発明の癌抗原ペプチドまたはその誘導体は少なくとも１つ、またはその２つもしくはそれ以上を患者に投与し、要すれば免疫調整物質や化学療法物質などの他の抗癌剤とともに投与する。癌抗原ペプチドまたはその誘導体を投与すれば、それらは抗原提示細胞のHLA-A24またはHLA-A2とともに提示され、提示されたHLA抗原複合体に対するCTLが増殖し、腫瘍細胞を破壊する。その結果、患者の癌が治療され、あるいは腫瘍の増殖または転移が予防される。   The cancer antigen peptide of the present invention or a derivative thereof is administered to a patient at least one, or two or more thereof, if necessary, together with other anticancer agents such as immunomodulators and chemotherapeutics. When cancer antigen peptides or derivatives thereof are administered, they are presented together with HLA-A24 or HLA-A2 of antigen presenting cells, and CTL against the presented HLA antigen complex proliferates and destroys tumor cells. As a result, the patient's cancer is treated or tumor growth or metastasis is prevented.

本発明の癌抗原ペプチドまたはその誘導体を活性成分として含む組成物は、細胞性および／または液性免疫を効率的に確立するためにアジュバントとともに投与することができる。投与方法としては、皮内投与、皮下投与または静脈注射が挙げられる。製剤中の本発明の腫瘍抗原ペプチドまたはその誘導体の投与量は、例えば処置目的の疾患状態、個々の患者の年齢、体重等により適宜調整することができるが、通常0.1mg〜10.0mg、好ましくは 0.5mg〜5.0mg、より好ましくは1.0mg〜3.0mgであり、これを数日ごとに投与する。   The composition containing the cancer antigen peptide of the present invention or a derivative thereof as an active ingredient can be administered together with an adjuvant in order to efficiently establish cellular and / or humoral immunity. Administration methods include intradermal administration, subcutaneous administration or intravenous injection. The dose of the tumor antigen peptide of the present invention or a derivative thereof in the preparation can be adjusted as appropriate depending on, for example, the disease state to be treated, the age, weight, etc. of each patient, but is usually 0.1 mg to 10.0 mg, preferably 0.5 mg to 5.0 mg, more preferably 1.0 mg to 3.0 mg, which is administered every few days.

本発明は、HLA-A24またはHLA-A2抗原と癌抗原ペプチドまたはその誘導体との複合体を特異的に認識するCTLを、さらに、そのCTLを活性成分として含む前立腺癌を処置するための医薬組成物をも提供する。本発明の組成物は好ましくは生理食塩水またはリン酸緩衝化食塩水（PBS）を含む。これらは例えば静脈内、皮下または皮内に投与することができる。   The present invention relates to a CTL that specifically recognizes a complex of HLA-A24 or HLA-A2 antigen and a cancer antigen peptide or a derivative thereof, and further a pharmaceutical composition for treating prostate cancer containing the CTL as an active ingredient Also provide things. The composition of the present invention preferably comprises physiological saline or phosphate buffered saline (PBS). They can be administered, for example, intravenously, subcutaneously or intradermally.

以下に本発明を実施例によりさらに詳細に説明するが、本発明はいかなる意味においてもこれら実施例により制限されるものではない。データの統計学的有意差は両側スチューデントｔ検定により求めた。０．０５以下のｐ値を統計学的に有意であると判断した。
実施例１
HLA-A24陽性前立腺癌患者からペプチド特異的CTLを惹起させる候補物質の同定
1.1 患者
すべてのHLA-A24陽性前立腺癌患者およびHLA-A24陽性健常ドナーはインフォームドコンセントを得た後に本試験に供した。前立腺癌患者＃2、＃3、＃5、および＃9が骨転移を伴っていた。すべての被験者にはHIVに非感染であった。末梢血２０ｍｌを採取し、フィコール・コンレイ密度勾配遠心によりPBMCを調製した。癌患者および健常ドナーのPBMCにおけるHLA-A24分子の発現はフローサイトメトリーにより測定した。 The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples in any way. Statistical significance of the data was determined by a two-sided student t test. A p value of 0.05 or less was judged to be statistically significant.
Example 1
Identification of candidate substances that induce peptide-specific CTL from HLA-A24 positive prostate cancer patients
1.1 Patients All HLA-A24 positive prostate cancer patients and HLA-A24 positive healthy donors were submitted to the study after obtaining informed consent. Prostate cancer patients # 2, # 3, # 5, and # 9 were associated with bone metastases. All subjects were uninfected with HIV. 20 ml of peripheral blood was collected and PBMC was prepared by Ficoll-Conlay density gradient centrifugation. The expression of HLA-A24 molecule in PBMC of cancer patients and healthy donors was measured by flow cytometry.

1.2 細胞株
C1R-A24は、C1Rリンパ腫由来でHLA-A*2402分子を発現する亜細胞株である(Oiso M, Eura M, Katsura F, Takiguchi M, Sobao Y, Masuyama K, Nakashima M, Itoh K, Ishikawa T. A newly identified MAGE-3-derived epitope recognized by HLA-A24-restricted cytotoxici T lymphocytes. Int. J. Cancer 81: 387-394, 1999)。すべての細胞株は、10％ FCSを添加したRPMI-1640培地(Gibco BRL, Grand Island, NY)に維持させた。 1.2 Cell lines
C1R-A24 is a subcell line derived from C1R lymphoma and expressing HLA-A * 2402 molecule (Oiso M, Eura M, Katsura F, Takiguchi M, Sobao Y, Masuyama K, Nakashima M, Itoh K, Ishikawa T A newly identified MAGE-3-derived epitope recognized by HLA-A24-restricted cytotoxici T lymphocytes. Int. J. Cancer 81: 387-394, 1999). All cell lines were maintained in RPMI-1640 medium (Gibco BRL, Grand Island, NY) supplemented with 10% FCS.

1.3 ペプチド
４つのPTHrP由来ペプチド（以下の表１に列記）を、HLA-A24分子に対する結合親和性、すなわちHLA-A24結合モチーフに基づき準備した[18, 19]。すべてのペプチドは>90%純度であり、Biologica Co.（名古屋、日本）から入手した。HLA-A24結合モチーフを有する、インフルエンザ（Flu）ウイルス由来ペプチド（RFYIQMCYEL）、EBV由来ペプチド（(TYGPVFMCL）およびHIV由来ペプチド（RYLRQQLLGI）を対照として用いた。すべてのペプチドをDMSOに溶解し、用量10 mg/mlとした。
PTHrP 1-36ペプチドはプロペプチドであるが[35]、PTHrP 25-34ペプチドを含めた。PTHrP 36-44とPTHとでは３つアミノ酸が相違し、他の３つのPTHrPペプチドとPTHとではすべてのアミノ酸が相違していた。 1.3 Peptides Four PTHrP derived peptides (listed in Table 1 below) were prepared based on the binding affinity for the HLA-A24 molecule, ie the HLA-A24 binding motif [18, 19]. All peptides were> 90% pure and were obtained from Biologica Co. (Nagoya, Japan). Influenza (Flu) virus-derived peptide (RFYIQMCYEL), EBV-derived peptide ((TYGPVFMCL) and HIV-derived peptide (RYLRQQLLGI) with HLA-A24 binding motif were used as controls.All peptides were dissolved in DMSO and dose 10 mg / ml.
The PTHrP 1-36 peptide is a propeptide [35], but the PTHrP 25-34 peptide was included. Three amino acids differed between PTHrP 36-44 and PTH, and all amino acids differed between the other three PTHrP peptides and PTH.

1.4 PBMCにおけるペプチド特異的CTLの検定
PBMCにおけるペプチド特異的CTLを検定し、４つのPTHrPペプチドの免疫原性を調べるため、HLA-A24陽性健常ドナー10名およびHLA-A24陽性前立腺癌患者10名のPBMCを４つのPTHrPペプチドそれぞれを用いて刺激し、次いで対応するPTHrPペプチドまたはHIVペプチドいずれかで前もってパルスしておいたC1R-A24細胞に応答して産生されるIFN-γを調べた。Flu-およびEBV-由来ペプチドを対照として用いた。 1.4 Peptide-specific CTL assay in PBMC
To test peptide-specific CTL in PBMC and examine the immunogenicity of four PTHrP peptides, PBMCs from 10 HLA-A24 positive healthy donors and 10 HLA-A24 positive prostate cancer patients were used with each of the 4 PTHrP peptides. IFN-γ produced in response to C1R-A24 cells that had been stimulated and then pre-pulsed with either the corresponding PTHrP peptide or HIV peptide was examined. Flu- and EBV-derived peptides were used as controls.

PBMCにおけるペプチド特異的CTLを検出するための上記検定は、既報の方法に従って行った[34]。簡単に説明すると、U底型96ウエルマイクロカルチャープレート（Nunc, Roskilde, Denmark）において、培養培地200μｌの容量にてPBMCs (1 X 105 細胞/ウエル)を10 μg/mlの各ペプチドとインキュベートした。この培養培地には45% RPMI-1640、45% AIM-V 培地 (Gibco BRL)、10% FCS、100 U/ml IL-2および0.1 mM MEM非必須アミノ酸溶液 (Gibco, BRL)が含まれている。3日毎に、培養培地の半分を取り出し、対応するペプチドを含む新たな培地(20 μg/ml)と置き換えた。培養の15日目に、培養細胞を４つのウエルに分け、２つのウエルにはPTHrPペプチド-パルスC1R-A24細胞を、他の２つのウエルにはHIV-パルスC1R-A24細胞を用いた。18時間インキュベートした後、上清を採取し、ELISA（限界感度：10 pg/ml）によりIFN-γレベルを測定した。
検定は個々のペプチドで各4ウエル回行い、２つの結果の平均を表１に示す。 The above assay for detecting peptide-specific CTLs in PBMC was performed according to previously reported methods [34]. Briefly, PBMCs (1 × 10 5 cells / well) were incubated with 10 μg / ml of each peptide in a volume of 200 μl of culture medium in a U-bottom 96-well microculture plate (Nunc, Roskilde, Denmark). . This culture medium contains 45% RPMI-1640, 45% AIM-V medium (Gibco BRL), 10% FCS, 100 U / ml IL-2 and 0.1 mM MEM non-essential amino acid solution (Gibco, BRL) Yes. Every 3 days, half of the culture medium was removed and replaced with fresh medium (20 μg / ml) containing the corresponding peptide. On the 15th day of culture, the cultured cells were divided into 4 wells, PTHrP peptide-pulsed C1R-A24 cells were used in 2 wells, and HIV-pulsed C1R-A24 cells were used in the other 2 wells. After 18 hours of incubation, the supernatant was collected and the IFN-γ level was measured by ELISA (limit sensitivity: 10 pg / ml).
The assay was performed 4 times for each peptide and the average of the two results is shown in Table 1.

Figure 2007145715
Figure 2007145715

有意な値（両側スチューデントｔ検定によりp<0.05）が得られた場合、ペプチド特異的CTLの誘導の成功を陽性と判断した。結果、PTHrP 36-44、PTHrP 102-111、PTHrP 25-34およびPTHrP 110-119ペプチドがHLA-A24陽性健常ドナー１０のうちそれぞれ6、5、2および3名においてペプチド特異的CTLを誘導した。これらのPTHrPペプチドはさらに、HLA-A24陽性前立腺癌患者10名のうちそれぞれ7、6、3および1名においてペプチド特異的CTLを誘導した。これらは、PTHrP 36-44およびPTHrP 102-111がともに、HLA-A24陽性前立腺癌患者においてペプチド特異的CTLを産生させるための候補ペプチドであることを示している。   If a significant value (p <0.05 by two-sided student t-test) was obtained, the successful induction of peptide-specific CTL was considered positive. As a result, PTHrP 36-44, PTHrP 102-111, PTHrP 25-34 and PTHrP 110-119 peptides induced peptide-specific CTLs in 6, 5, 2 and 3 of HLA-A24 positive healthy donors 10, respectively. These PTHrP peptides further induced peptide-specific CTLs in 7, 6, 3 and 1 out of 10 HLA-A24 positive prostate cancer patients, respectively. These indicate that both PTHrP 36-44 and PTHrP 102-111 are candidate peptides for producing peptide-specific CTL in HLA-A24 positive prostate cancer patients.

実施例２
HLA-A24拘束性でありPTHrP 36-44 または PTHrP 102-111ペプチドに特異的な前立腺癌反応性CTLの誘導
2.1 HLA-A24発現前立腺癌細胞株
ペプチド刺激PBMCのHLA-A24拘束性および前立腺癌に対する細胞傷害活性を調べるため、LNCaP-A24と命名したHLA-A24分子を恒常的に発現したLNCaP細胞系株を作製した。LNCaPはHLA-A24陰性の前立腺癌細胞株である。HLA-A24分子を恒常的に発現するLNCaP細胞（LNCaP-A24と命名）を樹立させるため、HLA-A*2402 遺伝子を挿入したpcDNA3.1/Hygro ベクター (Invitrogen, CA) をLNCaP細胞株 (ATCC 番号: CRL-1740)に電気的穿孔法で導入し、用量170μg/ml (Invitrogen)のハイグロマイシンBを用いて選別を行った。すべての細胞株は、10% FCSを添加したRPMI-1640培地 (Gibco BRL, Grand Island, NY)にて維持させた。LNCaPはPTHrPを産生することが既に報告されている(17)。親LNCaP細胞株はHLA-A24分子の細胞表面発現に関して陰性であるが、LNCaP-A24細胞株はその細胞表面にHLA-A24分子を発現している。LNCaP および LNCaP-A24に関するフローサイトメトリー分析の結果を図１に示す。フローサイトメトリー分析はLNCaP および LNCaP-A24細胞に対して行った。これらの細胞を抗-HLA-A24モノクローナル抗体、次いでFITC結合抗-マウスIgGモノクローナル抗体を用いて染色した。点線は第一モノクローナル抗体を用いずに染色した結果である。 Example 2
Induction of prostate cancer reactive CTL specific for HLA-A24 and specific for PTHrP 36-44 or PTHrP 102-111 peptide
2.1 HLA-A24-expressing prostate cancer cell line In order to investigate the peptide-stimulated PBMC HLA-A24 restriction and cytotoxic activity against prostate cancer, we used an LNCaP cell line that constitutively expressed the HLA-A24 molecule, named LNCaP-A24. Produced. LNCaP is an HLA-A24 negative prostate cancer cell line. In order to establish LNCaP cells constitutively expressing HLA-A24 molecules (named LNCaP-A24), the pcDNA3.1 / Hygro vector (Invitrogen, CA) with the HLA-A * 2402 gene inserted was used as the LNCaP cell line (ATCC No .: CRL-1740) was introduced by electroporation, and selection was performed using hygromycin B at a dose of 170 μg / ml (Invitrogen). All cell lines were maintained in RPMI-1640 medium (Gibco BRL, Grand Island, NY) supplemented with 10% FCS. LNCaP has already been reported to produce PTHrP (17). The parent LNCaP cell line is negative for cell surface expression of HLA-A24 molecules, whereas the LNCaP-A24 cell line expresses HLA-A24 molecules on its cell surface. The results of flow cytometry analysis for LNCaP and LNCaP-A24 are shown in FIG. Flow cytometry analysis was performed on LNCaP and LNCaP-A24 cells. These cells were stained with anti-HLA-A24 monoclonal antibody followed by FITC-conjugated anti-mouse IgG monoclonal antibody. The dotted line is the result of staining without using the first monoclonal antibody.

2.2 指示したペプチドによる前立腺癌反応性CTLの誘導
次いで、PTHrP 36-44 またはPTHrP 102-111ペプチドいずれかで刺激したPBMCがHLA-A24陽性健常ドナーおよび前立腺癌患者由来の癌患者反応性CTLを誘導できるか否かを調べた。HLA-A24陽性健常ドナーおよび前立腺癌患者由来のPBMCをこれらのPTHrPペプチドで繰り返し刺激した。その結果、健常ドナー#2、患者#1および患者#2由来のPTHrPペプチド刺激PBMCが、HIV ペプチド-パルスC1R-A24細胞に対するよりも、刺激に用いたPTHrPペプチドをパルスしたC1R-A24細胞に応答してIFN-γを高いレベルで産生することが示された(図2A)。詳細には、15日目に4つのウエルから培養細胞の半分を採取し、プールし、それをHIVペプチド (白抜き記号) および上記PTHrPペプチド (黒色記号)で前もって18時間パルスしておいたC1R-A24細胞とともに培養した。次いで、上清におけるIFN-γのレベルをELISAにより測定した。 2.2 Induction of prostate cancer reactive CTL by the indicated peptide PBMC stimulated with either PTHrP 36-44 or PTHrP 102-111 peptide then induced cancer patient reactive CTL from HLA-A24 positive healthy donors and prostate cancer patients We investigated whether it was possible. PBMCs from HLA-A24 positive healthy donors and prostate cancer patients were repeatedly stimulated with these PTHrP peptides. As a result, PTHrP peptide-stimulated PBMC from healthy donor # 2, patient # 1, and patient # 2 responded to C1R-A24 cells pulsed with the PTHrP peptide used for stimulation rather than to HIV peptide-pulsed C1R-A24 cells It was shown that IFN-γ was produced at a high level (FIG. 2A). Specifically, on day 15, half of the cultured cells were collected from 4 wells, pooled, and C1R previously pulsed with HIV peptide (open symbol) and the above PTHrP peptide (black symbol) for 18 hours -Cultured with A24 cells. The level of IFN-γ in the supernatant was then measured by ELISA.

2.3 ３つの標的に対するペプチド刺激PBMCの細胞傷害活性
上記ペプチド刺激細胞がPTHrPペプチド-パルスC1R-A24細胞に応答してIFN-γを産生できることを確認した後、これらペプチド刺激PBMCを３つの標的であるLNCaP細胞 (HLA-A24陰性)、LNCaP-A24細胞 (HLA-A24陽性)およびPHA-芽球様T細胞 (HLA-A24陽性)に対する細胞傷害活性について試験した。詳細には、PTHrPペプチドによりインビトロ刺激した後、ペプチド刺激PBMCを100 U/ml IL-2とともにさらに約10日間培養し、細胞傷害活性検定を行うために充分な数の細胞を得た。次いで、得られた細胞を、6時間 51Cr放出検定によりLNCaP細胞、LNCaP-A24細胞およびPHA-芽球様T細胞に対する細胞傷害活性について試験した。96丸底ウエルプレートにおいて指示したエフェクター/標的比率において１ウエル当たり2000個の51Cr標識細胞をエフェクター細胞とともに培養した。この結果も図２Aに示す。健常ドナー#2、患者#1および患者#2由来のPTHrPペプチド刺激PBMCが、LNCaPおよびHLA-A24陽性PHA-誘導T芽球様細胞に対するよりもLNCaP-A24細胞株に対して高い細胞傷害活性レベルを有することが示された。 2.3 Cytotoxic activity of peptide-stimulated PBMC against three targets After confirming that the peptide-stimulated cells can produce IFN-γ in response to PTHrP peptide-pulsed C1R-A24 cells, these peptide-stimulated PBMC are three targets Cytotoxic activity against LNCaP cells (HLA-A24 negative), LNCaP-A24 cells (HLA-A24 positive) and PHA-blast-like T cells (HLA-A24 positive) was tested. Specifically, after in vitro stimulation with PTHrP peptide, the peptide-stimulated PBMC was further cultured with 100 U / ml IL-2 for about 10 days to obtain a sufficient number of cells to perform the cytotoxic activity assay. The resulting cells were then tested for cytotoxic activity against LNCaP cells, LNCaP-A24 cells and PHA-blast-like T cells by a 6 hour 51 Cr release assay. 2000 51 Cr-labeled cells per well were cultured with effector cells at the effector / target ratios indicated in 96 round bottom well plates. This result is also shown in FIG. 2A. PTHrP peptide-stimulated PBMC from healthy donor # 2, patient # 1, and patient # 2 have higher levels of cytotoxic activity against the LNCaP-A24 cell line than against LNCaP and HLA-A24 positive PHA-induced T blast-like cells It was shown to have

2.4 PTHrPペプチド刺激PBMCの細胞傷害活性の抗体による阻害
次いで、いくつかの実験として、抗-HLAクラスI (W6/32: マウスIgG2a)、抗-HLA-DR (L243: マウスIgG2a)、抗-CD4 (NU-TH/I: マウスIgG1)、抗-CD8 (NU-TS/C: マウスIgG2a)、または抗-CD14 (H14: マウスIgG2a)抗体のいずれかを20 μg/mlで、検定の開始時にウエルに加えた。その結果、LNCaP-A24に対する細胞傷害活性が抗-HLAクラスIおよび抗-CD8モノクローナル抗体の添加により有意に阻害されたが、他の抗-HLAクラスII、抗-CD4または抗-CD14モノクローナル抗体の添加では阻害されないことが分かった（図２B）。 2.4 Inhibition of cytotoxic activity of PTHrP peptide-stimulated PBMC by antibodies Next, in some experiments, anti-HLA class I (W6 / 32: mouse IgG2a), anti-HLA-DR (L243: mouse IgG2a), anti-CD4 (NU-TH / I: mouse IgG1), anti-CD8 (NU-TS / C: mouse IgG2a), or anti-CD14 (H14: mouse IgG2a) antibody at 20 μg / ml at the start of the assay Added to the well. As a result, cytotoxic activity against LNCaP-A24 was significantly inhibited by the addition of anti-HLA class I and anti-CD8 monoclonal antibodies, but other anti-HLA class II, anti-CD4 or anti-CD14 monoclonal antibodies It was found that the addition was not inhibited (FIG. 2B).

2.5 PTHrPペプチド刺激PBMCの特異性
さらに、PTHrPペプチド刺激PBMCの特異性をコールド標的細胞を用いた実験により確かめた。簡単に説明すると、2 X 104 コールド標的細胞を含有する96丸底ウエルプレートにおいて、51Cr-標識標的細胞 (2 X 103 細胞/ウエル)をペプチド刺激PBMC (4 X 104 細胞/ウエル)とともに培養した。HIVペプチドまたは対応するPTHrPペプチドのいずれかで前もってパルスしておいたC1R-A24細胞をコールド標的として使用した。LNCaP-A24細胞株に対する細胞傷害活性は、対応するPTHrPペプチド-パルスC1R-A24細胞をコールド標的として添加することにより有意に抑制されたが、この抑制は、HIVペプチド-パルスC1R-A24細胞を添加しても認められなかった(図2C)。
これらの結果は、PTHrP 36-44 および PTHrP 102-111ペプチドの両者がHLA-A24陽性前立腺癌患者において前立腺癌反応性CTLを誘導する可能性があることを、さらに、それらの前立腺癌に対する細胞傷害活性がPTHrPペプチド特異的なCD8陽性T細胞に依存していることを示している。 2.5 Specificity of PTHrP peptide-stimulated PBMC The specificity of PTHrP peptide-stimulated PBMC was confirmed by experiments using cold target cells. Briefly, in 96 round bottom well plates containing 2 x 10 4 cold target cells, 51 Cr-labeled target cells (2 x 10 3 cells / well) were peptide-stimulated PBMC (4 x 10 4 cells / well) Incubated with. C1R-A24 cells previously pulsed with either the HIV peptide or the corresponding PTHrP peptide were used as cold targets. Cytotoxic activity against the LNCaP-A24 cell line was significantly suppressed by adding the corresponding PTHrP peptide-pulsed C1R-A24 cell as a cold target, but this suppression was achieved by adding HIV peptide-pulsed C1R-A24 cells. Even this was not observed (FIG. 2C).
These results indicate that both PTHrP 36-44 and PTHrP 102-111 peptides can induce prostate cancer-reactive CTL in HLA-A24 positive prostate cancer patients, and in addition, their cytotoxicity against prostate cancer The activity is dependent on PTHrP peptide-specific CD8 positive T cells.

実施例３
HLA-A24分子結合性PTHrPペプチドに対するIgG反応性の検出
本発明者らはすでに、CTLエピトープペプチドに反応するIgGが健常ドナーと種々の型の癌患者において検出されることを報告した[20, 21]。前立腺関連抗原に反応するIgGも健常ドナーおよび前立腺癌患者において検出されている[22-24]。従って、ここにおいて、上記４つのPTHrP-由来ペプチドに反応するIgGが癌患者および健常ドナーの血漿中に検出されるか否かを調べた。 Example 3
Detection of IgG reactivity to HLA-A24 molecule binding PTHrP peptide We have already reported that IgG reactive to CTL epitope peptide is detected in healthy donors and various types of cancer patients [20, 21 ]. IgG responding to prostate-related antigen has also been detected in healthy donors and prostate cancer patients [22-24]. Therefore, it was examined here whether IgG reacting with the four PTHrP-derived peptides was detected in the plasma of cancer patients and healthy donors.

既報[20, 21]のようにして、血漿中のペプチド特異的IgGレベルをELISAにより測定した。簡単に説明すると、ペプチド（20 μｇ/ウエル）固定化プレートをBlock Ace (雪印, 東京, 日本)によりブロックし、0.05% Tween20-PBSで洗浄し、次いで0.05% Tween20-Block Aceで希釈した血漿試料を100μｌ/ウエルでプレートに加えた。３７℃で2時間インキュベートした後、プレートを洗浄し、1:1000に希釈したウサギ抗-ヒトIgG (γ-鎖特異的) (DAKO, Glostrup, デンマーク)とともにさらに2時間インキュベートした。得られたプレートを洗浄し後、1:100に希釈したヤギ抗-ウサギIgG-結合ペルオキシダーゼ(EnVision, DAKO)１００μｌを各ウエルに加え、次いで室温にて40分間インキュベートした。プレートを1回洗浄し、100μｌ/ウエルでテトラメチルベンジジン基質溶液(KPL, Guildford, UK)を加えた後、1Mリン酸を加えて反応を停止させた。値は吸光度(OD)単位/mlで示し、HIVペプチドに対する応答を差し引いた。対応するPTHrPペプチドに反応するIgGは、1:100希釈血漿におけるODの差異が0.05を超えた場合に陽性を判断した。PTHrP102-111 または PTHrP 109-119ペプチドのいずれかに反応するIgGは10人の健常ドナーのうち8人に、そして10人の前立腺癌患者のうち7人に検出される結果が以下の表２に示すように得られた。結果は図３Aにも示している。   Peptide-specific IgG levels in plasma were measured by ELISA as described previously [20, 21]. Briefly, a peptide sample (20 μg / well) immobilized plate was blocked with Block Ace (Snow Brand, Tokyo, Japan), washed with 0.05% Tween20-PBS, and then diluted with 0.05% Tween20-Block Ace. Was added to the plate at 100 μl / well. After 2 hours incubation at 37 ° C., the plates were washed and incubated for an additional 2 hours with rabbit anti-human IgG (γ-chain specific) diluted 1: 1000 (DAKO, Glostrup, Denmark). After washing the resulting plates, 100 μl of 1: 100 diluted goat anti-rabbit IgG-conjugated peroxidase (EnVision, DAKO) was added to each well and then incubated for 40 minutes at room temperature. The plate was washed once, and tetramethylbenzidine substrate solution (KPL, Guildford, UK) was added at 100 μl / well, and then 1M phosphoric acid was added to stop the reaction. Values are given in absorbance (OD) units / ml and the response to HIV peptide was subtracted. IgG responding to the corresponding PTHrP peptide was judged positive when the OD difference in 1: 100 diluted plasma exceeded 0.05. Table 2 below shows the results of IgG detected in 8 of 10 healthy donors and 7 of 10 prostate cancer patients responding to either PTHrP102-111 or PTHrP 109-119 peptide. Obtained as shown. The results are also shown in FIG. 3A.

示したPTHrPペプチドに対するIgGの特異性を確認するため、健常ドナー#1および患者#6のいずれか由来の血漿試料100μｌを、対応するPTHrPペプチドまたは無関係のPTHrPペプチドのいずれかで前もってコートしておいたプレート中で培養した。次いで、得られた上清におけるPTHrP 102-111ペプチドまたはPTHrP 110-119ペプチドに反応するIgGレベルをELISAにより測定した。   To confirm the specificity of IgG for the indicated PTHrP peptides, 100 μl plasma samples from either healthy donor # 1 and patient # 6 were pre-coated with either the corresponding PTHrP peptide or an irrelevant PTHrP peptide. In the plate. Subsequently, the IgG level in response to the PTHrP 102-111 peptide or PTHrP 110-119 peptide in the obtained supernatant was measured by ELISA.

他方、PTHrP 36-44ペプチドに反応するIgGが10人の健常ドナーのうち３人に、そして10人の前立腺癌患者のうち１人にそれぞれ検出された。PTHrP 25-34に反応するIgGは健常ドナー、癌患者いずれにも検出されなかった。PTHrPペプチド特異的IgGのレベルは、対応するPTHrPペプチドコートウエル中で血漿を培養することにより減少した（図３B）。このペプチド特異的な吸収は、本検定システムが信頼できることを示している。   On the other hand, IgG reactive to the PTHrP 36-44 peptide was detected in 3 of 10 healthy donors and in 1 of 10 prostate cancer patients, respectively. IgG reactive to PTHrP 25-34 was not detected in either healthy donors or cancer patients. The level of PTHrP peptide-specific IgG was reduced by culturing plasma in the corresponding PTHrP peptide-coated well (FIG. 3B). This peptide-specific absorption indicates that the assay system is reliable.

Figure 2007145715
Figure 2007145715

実施例４
HLA-A2陽性前立腺癌患者からペプチド特異的CTLを誘導できる候補物質の同定
4.1 患者、細胞株、およびペプチド
実施例１と同様にして、HLA-A2陽性前立腺癌患者においてペプチド特異的CTLを誘導できるPTHrP由来ペプチドを同定した。癌患者＃1、＃3および＃10が骨転移を伴っていた。標的細胞としてHLA-A0201*分子を発現するリンパ腫由来細胞株であるT2細胞（ATCC寄託番号：CRL-1992）を用いた。HLA-A2結合モチーフを有する７つのPTHrP由来ペプチドを準備した（表３に列記）。PTHrP 59-68およびPTHrP 165-173はHLA-A0201*健常ドナーから特異的CTLを誘導することが以前に報告されているが[17]、本発明においては推定結合スコアーが１未満であった（表３）。PTHと共有するアミノ酸の数を以下に示す：PTHrP 42-51（5）、PTHrP 143-51（4）、PTHrP 51-60（2）、PTHrP 59-67（1）、PTHrP 103-111（0）。 Example 4
Identification of candidate substances capable of inducing peptide-specific CTL from HLA-A2-positive prostate cancer patients
4.1 Patients, cell lines, and peptides In the same manner as in Example 1, PTHrP-derived peptides capable of inducing peptide-specific CTLs in HLA-A2-positive prostate cancer patients were identified. Cancer patients # 1, # 3, and # 10 were associated with bone metastases. T2 cells (ATCC deposit number: CRL-1992), a lymphoma-derived cell line that expresses HLA-A0201 * molecule, were used as target cells. Seven PTHrP-derived peptides having an HLA-A2 binding motif were prepared (listed in Table 3). PTHrP 59-68 and PTHrP 165-173 have been previously reported to induce specific CTL from HLA-A0201 * healthy donors [17], but in the present invention the estimated binding score was less than 1 ( Table 3). The number of amino acids shared with PTH is shown below: PTHrP 42-51 (5), PTHrP 143-51 (4), PTHrP 51-60 (2), PTHrP 59-67 (1), PTHrP 103-111 (0 ).

4.2 PBMCにおけるペプチド特異的CTLの検定
HLA-A2陽性健常ドナー10名およびHLA-A2陽性前立腺癌患者10名において、これらペプチドの特異的CTL誘導能を検討した結果を表３に示す。 4.2 Peptide-specific CTL assay in PBMC
Table 3 shows the results of examining the specific CTL inducing ability of these peptides in 10 HLA-A2-positive healthy donors and 10 HLA-A2-positive prostate cancer patients.

Figure 2007145715
Figure 2007145715

PTHrP 42-51およびPTHrP 59-67ペプチドが、HLA-A2陽性健常ドナー10名のうちそれぞれ7名および4名においてペプチド特異的CTLを誘導した。既報のPTHrP 59-68ぺプチドはPTHrP 59-67ペプチドと１アミノ酸しか相違しないが、PTHrP 59-68ぺプチドはHLA-A2陽性健常ドナー由来のPBMCにおいてペプチド特異的CTLを誘導しなかった。既報のPTHrP 165-173ぺプチドは、HLA-A2陽性健常ドナー10名のうち4名においてペプチド特異的CTLを誘導した。PTHrP 42-51およびPTHrP 59-67ペプチドは、HLA-A2陽性前立腺癌患者10名のうちそれぞれ5名および4名においてペプチド特異的CTLを誘導した。既報のPTHrP 59-68およびPTHrP 165-173ぺプチドは、HLA-A2陽性前立腺癌患者10名のうちそれぞれ5名および3名においてペプチド特異的CTLを誘導した。   PTHrP 42-51 and PTHrP 59-67 peptides induced peptide-specific CTL in 7 and 4 out of 10 HLA-A2-positive healthy donors, respectively. The reported PTHrP 59-68 peptide differs from the PTHrP 59-67 peptide by only one amino acid, whereas the PTHrP 59-68 peptide did not induce peptide-specific CTL in PBMC from HLA-A2-positive healthy donors. The previously reported PTHrP 165-173 peptide induced peptide-specific CTL in 4 out of 10 HLA-A2-positive healthy donors. PTHrP 42-51 and PTHrP 59-67 peptides induced peptide-specific CTL in 5 and 4 out of 10 HLA-A2-positive prostate cancer patients, respectively. Previously reported PTHrP 59-68 and PTHrP 165-173 peptides induced peptide-specific CTLs in 5 and 3 out of 10 HLA-A2 positive prostate cancer patients, respectively.

PTHrP 59-68ペプチドとPTHrP 59-67ペプチドでは、ペプチド特異的CTLが誘導される患者がほとんど異なっている（表３）。これらの結果は、本発明のPTHrP 42-51およびPTHrP 59-67ぺプチドが、HLA-A2陽性前立腺癌患者においてペプチド特異的CTLを誘導するのに有用な新規の候補ペプチドであることを示している。   PTHrP 59-68 peptide and PTHrP 59-67 peptide are almost different in patients in which peptide-specific CTLs are induced (Table 3). These results indicate that the PTHrP 42-51 and PTHrP 59-67 peptides of the present invention are novel candidate peptides useful for inducing peptide-specific CTLs in HLA-A2-positive prostate cancer patients. Yes.

実施例５
HLA-A2拘束性でありPTHrP 42-51 または PTHrP 59-67ペプチドに特異的な前立腺癌反応性CTLの誘導
実施例２と同様にして、HLA-A2陽性健常ドナーおよびHLA-A2陽性前立腺癌患者由来PBMCにおいて、PTHrP 42-51およびPTHrP 59-67ぺプチドが癌細胞反応性CTLを誘導できるかについて調べた。HLA-A2発現前立腺細胞株は、HLA-A2陰性前立腺癌細胞株であるPC93細胞（Dr. K. Ohnishi, Department of Urology, Kyoto University, Japanにより樹立）にHLA-A*0201分子を恒常的に発現させることにより作製した（PC93-A2細胞）。本細胞株のHLA-A*0201分子の発現は以前に報告されている[24]。またPC93細胞は、2.2pmol/l（1×106 細胞/ml、24時間）のレベルのPTHrPを産生した。 Example 5
Induction of HLA-A2-restricted prostate cancer-reactive CTL specific for PTHrP 42-51 or PTHrP 59-67 peptide In the same manner as in Example 2, HLA-A2-positive healthy donors and HLA-A2-positive prostate cancer patients It was investigated whether PTHrP 42-51 and PTHrP 59-67 peptides can induce cancer cell-reactive CTL in the derived PBMC. HLA-A2-expressing prostate cell line is a HLA-A2-negative prostate cancer cell line, PC93 cells (established by Dr. K. Ohnishi, Department of Urology, Kyoto University, Japan). It was produced by expression (PC93-A2 cells). The expression of HLA-A * 0201 molecules in this cell line has been reported previously [24]. PC93 cells also produced PTHrP at a level of 2.2 pmol / l (1 × 10 6 cells / ml, 24 hours).

PTHrP 42-51およびPTHrP 59-67ぺプチドで刺激したPBMCが、対応ペプチドでパルスしたT2細胞に反応してIFN-γを産生できることを確認した（図４A）。これらペプチド刺激PBMCの、PC93細胞、PC93-A2細胞、およびPHA芽球様T細胞に対する細胞傷害活性を図4Bに示す。図５には、これらのペプチド刺激PBMCの細胞傷害活性を（A）ブロッキング抗体、および（B）コールド阻害アッセイを用いて検討した結果を示す。これらの結果は、PTHrP 42-51およびPTHrP 59-67ぺプチドが、HLA-A2陽性前立腺癌患者において前立腺癌反応性CTLを誘導し得ること、およびその細胞傷害活性がペプチド特異的なCD8陽性細胞に依存することを示唆している。   It was confirmed that PBMC stimulated with PTHrP 42-51 and PTHrP 59-67 peptides can produce IFN-γ in response to T2 cells pulsed with the corresponding peptide (FIG. 4A). FIG. 4B shows the cytotoxic activity of these peptide-stimulated PBMCs against PC93 cells, PC93-A2 cells, and PHA blast-like T cells. FIG. 5 shows the results of examining the cytotoxic activity of these peptide-stimulated PBMCs using (A) blocking antibodies and (B) cold inhibition assays. These results indicate that PTHrP 42-51 and PTHrP 59-67 peptides can induce prostate cancer reactive CTL in HLA-A2 positive prostate cancer patients and that their cytotoxic activity is peptide-specific CD8 positive cells It is suggested that it depends on.

実施例６
HLA-A2結合性PTHrPペプチドに反応するIgGの検出
実施例３と同様にして、健常ドナー10人および癌患者10人の血漿中にPTHrP 42-51およびPTHrP 59-67ぺプチドに特異的なIgGが存在するかについて検討した。1：100希釈血清でのカットオフ値はOD0.054とした。結果は表4に示す。

Figure 2007145715
Example 6
Detection of IgG in response to HLA-A2 binding PTHrP peptide As in Example 3, IgG specific for PTHrP 42-51 and PTHrP 59-67 peptides in the plasma of 10 healthy donors and 10 cancer patients Was examined. The cut-off value with 1: 100 diluted serum was OD0.054. The results are shown in Table 4.
Figure 2007145715

PTHrP 42-51ペプチドに反応するIgGは、健常ドナー10人のうち8人において、および前立腺癌患者10人のうち7人において検出された。PTHrP 59-67ぺプチドに反応するIgGは、健常ドナー10人のうち2人において、および前立腺癌患者10人のうち2人において検出された。健常ドナー2人（HD＃１およびHD＃6）および癌患者2人（Pt＃1およびPt＃2）の代表的結果を図６Aに示す。IgGのペプチド特異性を対応ペプチドでの吸収によって検討した結果を図６Bに示す。以上の結果は、これらペプチド、特にPTHrP 42-51ペプチドが、癌患者において細胞性および液性免疫応答の両者を誘導することを示唆している。   IgG in response to PTHrP 42-51 peptide was detected in 8 of 10 healthy donors and in 7 of 10 prostate cancer patients. IgG in response to PTHrP 59-67 peptide was detected in 2 of 10 healthy donors and in 2 of 10 prostate cancer patients. Representative results for two healthy donors (HD # 1 and HD # 6) and two cancer patients (Pt # 1 and Pt # 2) are shown in FIG. 6A. The result of examining the peptide specificity of IgG by absorption with the corresponding peptide is shown in FIG. 6B. The above results suggest that these peptides, especially PTHrP 42-51 peptide, induce both cellular and humoral immune responses in cancer patients.

本発明者らは、HLA-A24または-HLAA2陽性前立腺癌患者において免疫原性である新たな４つのPTHrP由来ペプチドを同定した。HLA-A24アレルの頻度は全世界を通じて比較的高い[33]。また、白人の多くがHLA-A*0201陽性である。一方日本人においては、HLA-A2サブタイプは非常に変化に富んでいる。本発明のHLA-A2分子結合性PTHrP由来ペプチドはHLA-A*0201分子結合モチーフに基づいて調製し、かつT2細胞およびPC93-A2細胞もHLA-A*0201分子を発現していた。しかしながら本発明のペプチドは、表３に示されるように、HLA-A*0206、HLA-A*0207およびHLA-A*0210を含む他のHLA-A2サブタイプを有する患者においてもペプチド特異的CTLを誘導した。本発明の情報は、転移を伴うHLA-A24またはHLA-A2陽性前立腺癌患者に対するペプチドに基づく免疫療法により処置する可能性を開くものである。   The inventors have identified four new PTHrP derived peptides that are immunogenic in patients with HLA-A24 or -HLAA2 positive prostate cancer. The frequency of HLA-A24 alleles is relatively high throughout the world [33]. Many whites are HLA-A * 0201 positive. On the other hand, in Japanese, the HLA-A2 subtype is very varied. The HLA-A2 molecule-binding PTHrP-derived peptide of the present invention was prepared based on the HLA-A * 0201 molecule-binding motif, and T2 cells and PC93-A2 cells also expressed HLA-A * 0201 molecules. However, the peptides of the present invention, as shown in Table 3, are also peptide specific CTLs in patients with other HLA-A2 subtypes including HLA-A * 0206, HLA-A * 0207 and HLA-A * 0210. Induced. The information of the present invention opens up the possibility of treatment with peptide-based immunotherapy for HLA-A24 or HLA-A2 positive prostate cancer patients with metastasis.

文献
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LNCaPおよびHLA-A24-発現LNCaP細胞株LNCaP-A24についてのフローサイトメトリー分析の結果である。点線は第1のモノクローナル抗体不存在下での染色である。FIG. 5 is the result of flow cytometry analysis for LNCaP and HLA-A24-expressing LNCaP cell line LNCaP-A24. The dotted line is staining in the absence of the first monoclonal antibody. 健常ドナー（HD）および癌患者（Pt）のPBMCからのHLA-A24拘束性前立腺癌反応性CTLの誘導を示す。(A)：図示したペプチドによる前立腺癌反応性CTLの誘導、およびLNCaP 細胞 (HLA-A24陰性)、LNCaP-A24 細胞 (HLA-A24陽性)およびPHA-芽球様T細胞 (HLA-A24陽性)に対するそれらの細胞傷害活性を示すグラフである。値は3回の検定の平均である。* P<0.05は統計学的に有意と考えられた。(B)：PTHrPペプチド刺激PBMCの細胞傷害活性に対する抗体の阻害を示すグラフである。値は3回の検定の平均である。* P<0.05は統計学的に有意と考えられた。(C)：PTHrPペプチド刺激CTLの細胞傷害活性の阻害を示すグラフである。値は3回の検定の平均である。* P<0.05は統計学的に有意と考えられた。FIG. 5 shows the induction of HLA-A24-restricted prostate cancer reactive CTL from PBMC of healthy donors (HD) and cancer patients (Pt). (A): Prostate cancer-reactive CTL induction with the indicated peptides, and LNCaP cells (HLA-A24 negative), LNCaP-A24 cells (HLA-A24 positive) and PHA-blast-like T cells (HLA-A24 positive) FIG. The value is the average of 3 tests. * P <0.05 was considered statistically significant. (B) is a graph showing antibody inhibition against the cytotoxic activity of PTHrP peptide-stimulated PBMC. The value is the average of 3 tests. * P <0.05 was considered statistically significant. (C) is a graph showing inhibition of the cytotoxic activity of PTHrP peptide-stimulated CTL. The value is the average of 3 tests. * P <0.05 was considered statistically significant. 健常ドナーおよび前立腺癌患者由来の血漿におけるPTHrPペプチドに対するIgG反応性を示す。(A)：6人の健常ドナーおよび６人の前立腺癌患者由来のペプチド特異的IgGの結果を吸光度 (OD)として示すグラフである。(B)：図示したPTHrPペプチドに対するIgGのペプチド特異的吸収を示すグラフである。1 shows IgG reactivity to PTHrP peptide in plasma from healthy donors and prostate cancer patients. (A): It is a graph which shows the result of the peptide specific IgG derived from 6 healthy donors and 6 prostate cancer patients as absorbance (OD). (B) is a graph showing peptide-specific absorption of IgG against the illustrated PTHrP peptide. 健常ドナーおよび癌患者のPBMCからのHLA-A2拘束性前立腺癌反応性CTLの誘導を示す。 （A）：図示したペプチドによるペプチド特異的CTLの誘導、および（B）：PC93細胞 (HLA-A2陰性)、PC93-A2 細胞 (HLA-A2陽性)およびPHA-芽球様T細胞 (HLA-A2陽性)に対するそれらの細胞傷害活性を示すグラフである。値は3回の検定の平均である。* P<0.05は統計学的に有意と考えられた。FIG. 5 shows the induction of HLA-A2 restricted prostate cancer reactive CTL from PBMC of healthy donors and cancer patients. (A): Peptide-specific CTL induction by the indicated peptides, and (B): PC93 cells (HLA-A2 negative), PC93-A2 cells (HLA-A2 positive) and PHA-blast-like T cells (HLA- It is a graph which shows those cytotoxic activity with respect to (A2 positive). The value is the average of 3 tests. * P <0.05 was considered statistically significant. (A)：PTHrPペプチド刺激PBMCの細胞傷害活性に対する抗体の阻害を示すグラフである。値は3回の検定の平均である。* P<0.05は統計学的に有意と考えられた。(B)： PTHrPペプチド刺激CTLの細胞傷害活性の阻害を示すグラフである。コールド標的細胞としてペプチド-パルス非標識T2細胞を用いた。値は3回の検定の平均である。* P<0.05は統計学的に有意と考えられた。(A): It is a graph which shows the inhibition of the antibody with respect to the cytotoxic activity of PTHrP peptide stimulation PBMC. The value is the average of 3 tests. * P <0.05 was considered statistically significant. (B) is a graph showing inhibition of the cytotoxic activity of PTHrP peptide-stimulated CTL. Peptide-pulse unlabeled T2 cells were used as cold target cells. The value is the average of 3 tests. * P <0.05 was considered statistically significant. 健常ドナーおよび前立腺癌患者由来の血漿におけるPTHrPペプチドに対するIgG反応性を示す。(A)：健常ドナー2人および前立腺癌患者2人由来のペプチド特異的IgGの結果を吸光度 (OD)として示すグラフである。(B)：図示したPTHrPペプチドに対するIgGのペプチド特異的吸収を示すグラフである。1 shows IgG reactivity to PTHrP peptide in plasma from healthy donors and prostate cancer patients. (A): It is a graph which shows the result of the peptide specific IgG derived from two healthy donors and two prostate cancer patients as absorbance (OD). (B) is a graph showing peptide-specific absorption of IgG against the illustrated PTHrP peptide.

Claims (18)

副甲状腺ホルモン関連タンパク質由来の部分ペプチドであり、HLA-A24抗原に結合でき、細胞傷害性Tリンパ球に認識されうる癌抗原ペプチド、または機能的に同等の性質を有するその誘導体。   A partial peptide derived from a parathyroid hormone-related protein, a cancer antigen peptide that can bind to HLA-A24 antigen and can be recognized by cytotoxic T lymphocytes, or a derivative having functionally equivalent properties. 液性免疫系に認識される、請求項１記載の癌抗原ペプチド、または機能的に同等の性質を有するその誘導体。   The cancer antigen peptide according to claim 1, which is recognized by the humoral immune system, or a derivative having functionally equivalent properties. 配列番号１または２に示すアミノ酸配列を含む請求項１または２記載の癌抗原ペプチド、または機能的に同等の性質を有するその誘導体。   The cancer antigen peptide according to claim 1 or 2, comprising the amino acid sequence shown in SEQ ID NO: 1 or 2, or a derivative thereof having functionally equivalent properties. 請求項１から３までのいずれか記載の癌抗原ペプチドおよび機能的に同等の性質を有するその誘導体を少なくとも１つ活性成分として含む、前立腺癌を処置または予防するための医薬組成物。   A pharmaceutical composition for treating or preventing prostate cancer comprising the cancer antigen peptide according to any one of claims 1 to 3 and a derivative thereof having functionally equivalent properties as an active ingredient. 前立腺癌が骨転移を伴う、請求項４記載の医薬組成物。   The pharmaceutical composition according to claim 4, wherein the prostate cancer is accompanied by bone metastasis. 請求項１から３までのいずれか記載の癌抗原ペプチドおよび機能的に同等の性質を有するその誘導体を少なくとも１つ活性成分として含む、転移性骨病変を処置または予防するための医薬組成物。   A pharmaceutical composition for treating or preventing metastatic bone lesions, comprising as an active ingredient at least one of the cancer antigen peptides according to any one of claims 1 to 3 and derivatives thereof having functionally equivalent properties. 転移性骨病変が前立腺癌に付随する、請求項６記載の医薬組成物。   The pharmaceutical composition of claim 6, wherein the metastatic bone lesion is associated with prostate cancer. HLA-A24抗原と請求項１から３までのいずれか記載の癌抗原ペプチドまたは機能的に同等の性質を有するその誘導体との複合体を特異的に認識する細胞傷害性Tリンパ球。   A cytotoxic T lymphocyte that specifically recognizes a complex of the HLA-A24 antigen and the cancer antigen peptide according to any one of claims 1 to 3 or a derivative thereof having functionally equivalent properties. 請求項８記載の細胞傷害性Tリンパ球を活性成分として含む、前立腺癌を処置するための医薬組成物。   A pharmaceutical composition for treating prostate cancer comprising the cytotoxic T lymphocyte according to claim 8 as an active ingredient. 前立腺癌が骨転移を伴う、請求項９記載の医薬組成物。   The pharmaceutical composition according to claim 9, wherein the prostate cancer is accompanied by bone metastasis. 配列番号３または４に示すアミノ酸配列を含む癌抗原ペプチド、またはHLA-A2抗原に結合でき、細胞傷害性Tリンパ球に認識されうる機能的に同等の性質を有するその誘導体。   A cancer antigen peptide comprising the amino acid sequence shown in SEQ ID NO: 3 or 4, or a derivative thereof capable of binding to HLA-A2 antigen and having functionally equivalent properties that can be recognized by cytotoxic T lymphocytes. 請求項１１記載の癌抗原ペプチドおよび機能的に同等の性質を有するその誘導体を少なくとも１つ活性成分として含む、前立腺癌を処置または予防するための医薬組成物。   A pharmaceutical composition for treating or preventing prostate cancer comprising the cancer antigen peptide according to claim 11 and a derivative thereof having functionally equivalent properties as an active ingredient. 前立腺癌が骨転移を伴う、請求項１２記載の医薬組成物。   The pharmaceutical composition according to claim 12, wherein the prostate cancer is accompanied by bone metastasis. 請求項１１記載の癌抗原ペプチドおよび機能的に同等の性質を有するその誘導体を少なくとも１つ活性成分として含む、転移性骨病変を処置または予防するための医薬組成物。   A pharmaceutical composition for treating or preventing metastatic bone lesions, comprising as an active ingredient the cancer antigen peptide according to claim 11 and a derivative thereof having functionally equivalent properties. 転移性骨病変が前立腺癌に付随する、請求項１４記載の医薬組成物。   15. The pharmaceutical composition of claim 14, wherein the metastatic bone lesion is associated with prostate cancer. HLA-A2抗原と請求項１１記載の癌抗原ペプチドまたは機能的に同等の性質を有するその誘導体との複合体を特異的に認識する細胞傷害性Tリンパ球。   A cytotoxic T lymphocyte that specifically recognizes a complex of the HLA-A2 antigen and the cancer antigen peptide of claim 11 or a derivative thereof having functionally equivalent properties. 請求項１６記載の細胞傷害性Tリンパ球を活性成分として含む、前立腺癌を処置するための医薬組成物。   A pharmaceutical composition for treating prostate cancer comprising the cytotoxic T lymphocyte according to claim 16 as an active ingredient. 前立腺癌が骨転移を伴う、請求項１７記載の医薬組成物。
The pharmaceutical composition of claim 17, wherein the prostate cancer is associated with bone metastasis.
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