JP5960064B2 - Immunogenic compositions against human progastrin peptides - Google Patents
Immunogenic compositions against human progastrin peptides Download PDFInfo
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- JP5960064B2 JP5960064B2 JP2012556067A JP2012556067A JP5960064B2 JP 5960064 B2 JP5960064 B2 JP 5960064B2 JP 2012556067 A JP2012556067 A JP 2012556067A JP 2012556067 A JP2012556067 A JP 2012556067A JP 5960064 B2 JP5960064 B2 JP 5960064B2
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- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
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- A61K47/646—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the entire peptide or protein drug conjugate elicits an immune response, e.g. conjugate vaccines
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Description
本発明はヒトプロガストリンペプチドに対する免疫原性組成物に関する。 The present invention relates to immunogenic compositions against human progastrin peptides.
胃腸(GI)癌は、世界的にヒトにおける最も一般的な形態の癌であり、多くの国で死亡原因の第一位である。いくつかの増殖因子が、GI悪性腫瘍の増殖を行うことが知られており、特にそれらは一般にプレプロガストリンのプロセッシングから生成されたペプチドホルモンのファミリーである。正常な組織におけるプレプロガストリンの成熟は、中間体プロガストリンの多段階プロセッシングを含み、循環中に認められる主要なアミド化形態のガストリン；それぞれ、「大」および「小」ガストリンとして知られるガストリン-34(G34)およびガストリン-17(G17)を誘導する。これらのホルモンは、粘膜の細胞増殖および特に、食品の摂取後の胃酸の(食後)分泌を調節する。プロガストリンおよびガストリン、特に、G17およびグリシン伸長型のG17(gly-G17)は、in vitroおよびin vivoの双方において、GI悪性腫瘍における栄養的な役割を果たすことが報告されており、いくつかの観察が、治療目的のためのこれらのガストリンの標的化された阻害に関する事例を支持する。 Gastrointestinal (GI) cancer is the most common form of cancer in humans worldwide and is the leading cause of death in many countries. Several growth factors are known to effect the growth of GI malignancies, in particular they are generally a family of peptide hormones generated from preprogastrin processing. Maturation of preprogastrin in normal tissues involves multi-step processing of the intermediate progastrin, the major amidated form of gastrin found in the circulation; gastrin-34, known as “large” and “small” gastrins, respectively. Induces (G34) and gastrin-17 (G17). These hormones regulate mucosal cell proliferation and, in particular, (post-prandial) secretion of gastric acid after food intake. Progastrin and gastrin, in particular G17 and glycine-extended G17 (gly-G17), have been reported to play a nutritional role in GI malignancies both in vitro and in vivo, and several Observations support the case for targeted inhibition of these gastrins for therapeutic purposes.
特定の癌促進性の増殖因子およびホルモンに対する免疫化は、特定の癌、特に、乳癌、肺癌、および特定の型のGI癌の治療および予防に有用であることが公知である。さらに、胃-食道および胃-十二指腸潰瘍性疾患の治療および予防に対する免疫学的手法も、これらの慢性症状の治療に有効であり得る。 Immunization against certain cancer-promoting growth factors and hormones is known to be useful for the treatment and prevention of certain cancers, particularly breast cancer, lung cancer, and certain types of GI cancer. In addition, immunological approaches to the treatment and prevention of gastro-esophageal and gastro-duodenal ulcer disease may be effective in treating these chronic conditions.
いくつかの治療手法が成功裏に用いられてきたが、特にその一つは標的化されたヒトまたはヒト化モノクローナル抗体(huMAb)を用いるものである。しかしながら、商業的なhuMAbの製造および送達を設定する費用および困難性を考えると、特に、発展途上国のための、さほど高価ではない代替戦略が必要である。 Several therapeutic approaches have been used successfully, particularly one using targeted human or humanized monoclonal antibodies (huMAbs). However, given the cost and difficulty of setting up commercial huMAb production and delivery, there is a need for less expensive alternative strategies, especially for developing countries.
GI癌および疾患について、これらの免疫学的手法は、疾患を促進する胃腸ペプチドホルモンの生物活性を中和する特異的抗体の生成を必要とする。必要とされる抗体は、特定の増殖因子もしくはホルモン、またはホルモン前駆体に対して特異的でなければならない。1種以上の因子またはホルモンを選択的に標的化して、特定の疾患を治療することができる。 For GI cancers and diseases, these immunological approaches require the generation of specific antibodies that neutralize the biological activity of gastrointestinal peptide hormones that promote the disease. The antibody required must be specific for a particular growth factor or hormone, or hormone precursor. One or more factors or hormones can be selectively targeted to treat a particular disease.
例えば、ヒト胃腸ホルモンガストリン17(「huG17」)は、酸を刺激し、従って胃および十二指腸の潰瘍を引き起こすその能力のため、胃食道逆流疾患などの胃腸疾患プロセスに関与する。さらに、huG17は、いくつかのGI癌の増殖を刺激することが示されている。 For example, the human gastrointestinal hormone gastrin 17 ("huG17") is involved in gastrointestinal disease processes such as gastroesophageal reflux disease because of its ability to stimulate acid and thus cause gastric and duodenal ulcers. Furthermore, huG17 has been shown to stimulate the growth of several GI cancers.
従って、huG17の作用を中和することができる特異的抗huG17抗体が、huG17が関与する疾患を治療するための臨床試験において用いられてきた。抗huG17抗体を、例えば、受動免疫によって患者に投与するか、またはそれらを能動免疫により患者中で誘導することができる。 Therefore, specific anti-huG17 antibodies that can neutralize the action of huG17 have been used in clinical trials to treat diseases involving huG17. Anti-huG17 antibodies can be administered to a patient, for example, by passive immunization or they can be induced in a patient by active immunization.
同様に、アミド化ガストリンは循環中で唯一の生物学的に活性な形態のガストリンであると考えられていたが、現在では、ヒトGI癌患者および誘導された癌細胞系を用いる研究に基づいて、プロガストリン形態のプレプロガストリンが増殖能力を有することの相当の証拠が存在する。実際、GI癌細胞は一般に、プレプロガストリンおよびプロガストリンのようなガストリン前駆体をその通常のアミド化形態に十分にプロセッシングすることができない；従って、癌患者における血清レベルは、通常のアミド化形態のレベルよりも非常に高いこれらの前駆体形態のガストリンのレベルを示す。アミド化ガストリンまたはグリシン伸長ガストリンではなく、プロガストリンの血漿レベルは、結腸直腸ポリープを有する患者または対照と比較して、結腸直腸癌を有する患者において有意に上昇することが報告されている(Siddheshwarら、Gut 48: 47-52, 2001)。また、プロガストリン、アミド化ガストリン、総ガストリン、およびグリシン伸長ガストリンは、それぞれ、結腸直腸癌(CRC)患者の100％、69％、56％および44％において検出され(Ciccotostoら、Gastroenterology 109: 1142-53, 1995)、これは癌が実際にはガストリンを完全にプロセッシングできなかったことを示唆している。 Similarly, amidated gastrin was thought to be the only biologically active form of gastrin in the circulation, but is now based on research using human GI cancer patients and derived cancer cell lines. There is considerable evidence that the progastrin form of preprogastrin has proliferative capacity. In fact, GI cancer cells are generally unable to fully process gastrin precursors such as preprogastrin and progastrin to their normal amidated forms; thus, serum levels in cancer patients are in the normal amidated form. The levels of gastrin in these precursor forms are much higher than the levels. Plasma levels of progastrin, but not amidated gastrin or glycine-extended gastrin, have been reported to be significantly elevated in patients with colorectal cancer compared to patients with colorectal polyps or controls (Siddheshwar et al. Gut 48: 47-52, 2001). Progastrin, amidated gastrin, total gastrin, and glycine-extended gastrin are also detected in 100%, 69%, 56%, and 44% of colorectal cancer (CRC) patients, respectively (Ciccotosto et al., Gastroenterology 109: 1142 -53, 1995), suggesting that the cancer was in fact unable to fully process gastrin.
以下で詳細に考察されるように、従来技術における欠点に対処するため、本発明は、その一態様に従って、(A)(i)プロガストリンまたはN末端および／もしくはC末端プロセッシングされたプロガストリンの種のアミノ酸配列と、それに連結された(ii)7アミノ酸のスペーサーから構成される模倣ペプチドならびに(B)該模倣ペプチドにカップリングした免疫原性担体を含むポリペプチド免疫原を提供する。本発明の一実施形態によれば、前記ポリペプチド免疫原は、アミノ酸配列：Cys-pro-Pro-Pro-Pro-Ser-Ser-Gly-Trp-Met-Asp-nPhe-Gly-Arg-Arg-Ser-Ala-Glu-Asp-Glu-Asn(配列番号1)を有する模倣ペプチドを含む。別の実施形態によれば、模倣ペプチドは、pGlu-Gly-Pro-Trp-β-イソVal-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (配列番号2);
pGlu-Gly-Pro-Trp-Ile-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (配列番号3);
pGlu-Gly-Pro-Trp-Val-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (配列番号4);
Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-Gly (配列番号5); および
Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-nPhe-Gly-Trp-Met-Asp-Phe-Gly (配列番号6)
からなる群より選択されるアミノ酸配列を有する。本発明に従えば、免疫原性担体を、特に、破傷風トキソイド、ジフテリアトキソイド、百日咳トキソイド、およびツベルクリン純粋タンパク質誘導体から選択することができる。
As will be discussed in detail below, in order to address the shortcomings in the prior art, the present invention, according to one aspect thereof, comprises (A) (i) progastrin or N-terminal and / or C-terminal processed progastrin. Provided is a polypeptide immunogen comprising an amino acid sequence of a species and (ii) a mimetic peptide composed of a 7 amino acid spacer linked thereto, and (B) an immunogenic carrier coupled to the mimetic peptide. According to one embodiment of the invention, the polypeptide immunogen comprises the amino acid sequence: Cys-pro-Pro-Pro-Pro-Ser-Ser-Gly-Trp-Met-Asp-nPhe-Gly-Arg-Arg- A mimetic peptide having Ser-Ala-Glu-Asp-Glu-Asn (SEQ ID NO: 1) is included. According to another embodiment, the mimetic peptide is pGlu-Gly-Pro-Trp-β-isoVal -Glu-Glu-Glu-Glu-Glu- Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 2);
pGlu-Gly-Pro-Trp-Ile-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 3);
pGlu-Gly-Pro-Trp-Val-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 4);
Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-Gly (SEQ ID NO: 5); and
Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-nPhe-Gly-Trp-Met-Asp-Phe-Gly (SEQ ID NO: 6)
Having an amino acid sequence selected from the group consisting of According to the invention, the immunogenic carrier can be selected in particular from tetanus toxoid, diphtheria toxoid, pertussis toxoid and tuberculin pure protein derivatives.
本発明の別の態様によれば、有効量の上記ポリペプチド免疫原、および免疫原のための製薬上許容し得るビヒクルを含む免疫原性組成物が提供される。好ましい実施形態においては、製薬上許容し得る担体は、免疫原が存在する水相、および油相のエマルジョンを含む。この油相は、スクアレン、スクアラン、モノオレイン酸ソルビタン、ポリソルベート(登録商標)40および／またはポリソルベート80などの少なくとも1種の生分解性油を含む。油相はまた、個別の乳化剤を含んでもよい。さらに、油相または水相は1種以上のアジュバントを含んでもよい。
According to another aspect of the present invention, there is provided an immunogenic composition comprising an effective amount of the above polypeptide immunogen and a pharmaceutically acceptable vehicle for the immunogen. In a preferred embodiment, the pharmaceutically acceptable carrier comprises an aqueous phase in which the immunogen is present and an emulsion of the oil phase. The oil phase comprises squalene, squalane, sorbitan monooleate, at least one biodegradable oils such as Polysorbate ® 40 and / or
さらに別の態様によれば、本発明は、上記の模倣ペプチドとの連結において特に好適である7アミノ酸のスペーサーペプチドを提供する。そのようなスペーサーペプチドの例は、Ser-Ser-Pro-Pro-Pro-pro-Cys (配列番号7); Ser-Ser-pro-pro-pro-pro-Cys (配列番号8); Thr-Thr-Pro-Pro-Pro-pro-Cys (配列番号9); およびThr-Thr-pro-pro-pro-pro-Cys (配列番号10)からなる群より選択されるアミノ酸配列を有するものである。 According to yet another aspect, the present invention provides a 7 amino acid spacer peptide that is particularly suitable for linking with the above mimetic peptides. Examples of such spacer peptides are Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 7); Ser-Ser-pro-pro-pro-pro-Cys (SEQ ID NO: 8); Thr-Thr It has an amino acid sequence selected from the group consisting of -Pro-Pro-Pro-pro-Cys (SEQ ID NO: 9); and Thr-Thr-pro-pro-pro-pro-Cys (SEQ ID NO: 10).
増殖因子または胃腸ペプチドホルモンに対する能動免疫は、標的化された因子またはホルモンに結合する抗体を誘導する化学構造を含む免疫原を患者に投与することにより達成される。そのような化学構造は、標的化された因子またはホルモンの免疫学的ペプチド模倣体として構築され、それらは標的またはその標的のエピトープと免疫学的に交叉反応する任意の分子から構成させることができる。これらの免疫原は、抗体を誘導する固有の能力を有し得る免疫学的ペプチド模倣体(「免疫模倣体」)であり、例えば、それらは免疫原性であってよい。さらに、免疫模倣体は、本質的に免疫原性ではないことも多く、従って、強力な免疫原性特性を有する担体(「免疫原性担体」)に連結し、それによって、複合体を免疫原性にしなければならない。 Active immunization against growth factors or gastrointestinal peptide hormones is achieved by administering to the patient an immunogen comprising a chemical structure that induces antibodies that bind to the targeted factors or hormones. Such chemical structures are constructed as immunological peptidomimetics of targeted factors or hormones, which can be composed of any molecule that immunologically cross-reacts with the target or epitope of that target . These immunogens are immunological peptidomimetics (“immunomimetics”) that may have an inherent ability to induce antibodies, for example, they may be immunogenic. In addition, immunomimetics are often not immunogenic in nature and are therefore linked to carriers with strong immunogenic properties (“immunogenic carriers”), thereby linking the conjugate to the immunogen. Must be sex.
本発明に従えば、任意の化学構造が、例えば、Watsonら、Expert Opin. Biol. 1: 309-17 (2001)およびWatsonら、Cancer Res. 56: 880-85 (1996)により例示されたようなhuG17のエピトープと免疫学的に交叉反応する免疫模倣体として働くことができる。本発明の好ましい実施形態においては、ペプチド模倣体は、ヒト小ガストリンならびにグリシン伸長ガストリンに結合する交叉反応性抗体を刺激するように設計された、huG17のアミノ末端エピトープをその中に含むhuG17の一部である。免疫原のスペーサーエレメントは、免疫模倣体(ペプチド模倣体 + スペーサー)が担体に結合する連結点として働く。スペーサーはまた、免疫原のエピトープ部分に対する免疫応答に影響し得る。 In accordance with the present invention, any chemical structure is as exemplified by, for example, Watson et al., Expert Opin. Biol. 1: 309-17 (2001) and Watson et al., Cancer Res. 56: 880-85 (1996). It can act as an immunomimetic that immunologically cross-reacts with the epitope of huG17. In a preferred embodiment of the invention, the peptidomimetic is a single huG17 amino-terminal epitope of huG17 that is designed to stimulate human small gastrin as well as cross-reactive antibodies that bind to glycine-extended gastrin. Part. The spacer element of the immunogen serves as a junction point where the immunomimetic (peptidomimetic + spacer) binds to the carrier. Spacers can also affect the immune response to the epitope portion of the immunogen.
スペーサーを介してジフテリアトキソイドにカップリングしたhuG17のN末端ノナペプチド部分を用いる免疫化は、ラットにおける実験的潰瘍および免疫不全マウスにおけるGIヒト癌異種移植片の両方を阻害することが報告されている。GIを有する患者において、「G17DT」として知られるこの免疫原を用いるいくつかの臨床試験が実施され、有意な結果を出しているが(Watsonら(2001)を参照)、この免疫原を用いる最新の米国での第III相臨床試験は、後期段階の膵臓癌の治療において統計的有意性に到達できなかった。 Immunization with the N-terminal nonapeptide portion of huG17 coupled to diphtheria toxoid via a spacer has been reported to inhibit both experimental ulcers in rats and GI human cancer xenografts in immunodeficient mice. Several clinical trials using this immunogen known as `` G17DT '' have been performed in patients with GI and have yielded significant results (see Watson et al. (2001)), but the latest using this immunogen US Phase III clinical trials failed to reach statistical significance in the treatment of late-stage pancreatic cancer.
初期の臨床試験においてはいくらかの成功を示しているが、G17DT手法は、特に、GI悪性疾患におけるガストリン遺伝子発現の顕著な不均一性を考慮に入れていなかった。正常な胃の細胞および組織中では、最も豊富な分泌／循環ガストリン種はG17およびG34である。しかしながら、多くの科学的報告書は、これらの後者の種が実際には、循環中に50％未満、特定の事例では、10％〜20％ほどの少なさのガストリン形態を構成することがあることを示している。 Although showing some success in early clinical trials, the G17DT approach did not take into account significant heterogeneity of gastrin gene expression, particularly in GI malignancies. In normal gastric cells and tissues, the most abundant secreted / circulating gastrin species are G17 and G34. However, many scientific reports indicate that these latter species may actually constitute less than 50% gastrin forms in the circulation, and in certain cases as little as 10% to 20%. It is shown that.
主要な種は、例えば、Rehfeldら、Regulatory Peptides 120: 177-83(2004)に記載された「プロガストリン」として知られるプロセッシングされた中間体である。プロガストリンは、伝統的なガストリン受容体とは無関係である実質的な増殖促進活性を有することが示されている。かくして、上記のG17DT免疫原により生じた抗体は同様に、GI癌患者の循環中に豊富であるプロセッシングされていない／部分的にプロセッシングされた形態のガストリン(プロガストリン)の主要な循環種ではなく、G17およびC末端伸長された形態のみを捕捉する。 The major species is a processed intermediate known as “progastrin” as described, for example, in Rehfeld et al., Regulatory Peptides 120: 177-83 (2004). Progastrin has been shown to have substantial growth promoting activity that is unrelated to traditional gastrin receptors. Thus, the antibodies generated by the G17DT immunogen described above are similarly not the primary circulating species of unprocessed / partially processed forms of gastrin (progastrin) that are abundant in the circulation of patients with GI cancer Only the G17 and C-terminal extended forms are captured.
本発明の新規免疫原組成物が、癌患者において循環中に認められるガストリンおよびプロガストリンの形態を標的化することにより対処することは、特に従来技術におけるこの欠点である。これに関して、本発明者らは、好適なスペーサーペプチドにカップリングさせた場合、huG17の特定のペプチド模倣体により、従来の免疫原と比較して、予想外に改善された免疫応答をもたらす免疫原が得られることを発見した。 It is this deficiency, particularly in the prior art, that the novel immunogenic compositions of the present invention address by targeting the gastrin and progastrin forms found in the circulation in cancer patients. In this regard, the inventors have shown that immunogens that, when coupled to a suitable spacer peptide, result in an unexpectedly improved immune response due to certain peptidomimetics of huG17 compared to conventional immunogens. I found out that
本発明者らはまた、本発明に従えば、改変アミノ酸を用いて、しばしば、天然アミノ酸を含有するペプチドよりも高レベルで交叉反応性抗体の惹起を許容しながらでも、隣接するペプチドの「外来」の固有の特性を増強することができることを発見した。より具体的には、侵入する微生物および「外来」抗原のマクロファージおよび単球によるプロセッシングが、ハロゲン化反応および酸化およびニトロシル化反応をもたらすことが公知である。かくして、得られた化学的改変は、タンパク質およびペプチドの抗原性およびその後の免疫原性を増加させると理解された。従って、本発明は、ペプチド免疫原の免疫原性を増加させるための、パラ-クロロまたはパラ-ニトロ-フェニルアラニン残基などの1個以上のハロゲン化またはニトロシル化アミノ酸残基の含有を包含する。これが、例えば、パラ-NO2-フェニルアラニン(nPhe)が以下に詳述される特定の本発明の免疫原(配列番号1および6)に出現する理由である。 The inventors have also found that, according to the present invention, modified amino acids are often used to allow for the “foreign” of neighboring peptides, while allowing for the induction of cross-reactive antibodies at higher levels than peptides containing natural amino acids. Has been found to be able to enhance the inherent properties of More specifically, it is known that processing of invading microorganisms and “foreign” antigens by macrophages and monocytes results in halogenation and oxidation and nitrosylation reactions. Thus, the resulting chemical modifications were understood to increase the antigenicity and subsequent immunogenicity of proteins and peptides. Thus, the present invention includes the inclusion of one or more halogenated or nitrosylated amino acid residues, such as para-chloro or para-nitro-phenylalanine residues, to increase the immunogenicity of the peptide immunogen. This is why, for example, para-NO 2 -phenylalanine (nPhe) appears in certain inventive immunogens (SEQ ID NOs: 1 and 6) detailed below.
従って、本発明の一態様によれば、改良された免疫原は、それぞれ、プロガストリンならびにプロガストリンのN末端および／またはC末端プロセッシングされた種に対するポリクローナル抗体を生成する。そのような免疫原の例は、(i)アミノ酸残基：Cys-pro-Pro-Pro-Pro-Ser-Ser-Gly-Trp-Met-Asp-nPhe-Gly-Arg-Arg-Ser-Ala-Glu-Asp-Glu-Asn(配列番号1)のペプチド、それにカップリングした(ii)免疫原性担体を含むものである(本明細書中の配列表において適用された慣例については以下を参照されたい)。従って、この実施形態は、プロガストリン成分(プレプロガストリンの残基88〜101)と、7アミノ酸のスペーサーを包含し、21アミノ酸(G21)のペプチドである免疫模倣体を構成する。 Thus, according to one aspect of the invention, the improved immunogens generate polyclonal antibodies to progastrin and N-terminal and / or C-terminal processed species of progastrin, respectively. Examples of such immunogens are (i) amino acid residues: Cys-pro-Pro-Pro-Pro-Ser-Ser-Gly-Trp-Met-Asp-nPhe-Gly-Arg-Arg-Ser-Ala- Glu-Asp-Glu-Asn (SEQ ID NO: 1) peptide, including (ii) an immunogenic carrier coupled thereto (see below for conventions applied in the sequence listing herein) . Thus, this embodiment comprises a progastrin component (residues 88-101 of preprogastrin) and a 7 amino acid spacer and constitutes an immunomimic that is a 21 amino acid (G21) peptide.
本明細書の意味において、免疫原性担体は、任意の好適な高分子量担体、典型的には、タンパク質またはそれに共有連結されたハプテンもしくはペプチド配列に対する免疫応答を生じることができる十分な分子的複雑性を有する大分子(すなわち、一般に6000 kDを超える)であってよい。好適な免疫原性担体のカテゴリーは、限定されるものではないが、ジフテリアトキソイド(DT)、破傷風トキソイド(TT)、百日咳トキソイド、およびツベルクリン純粋タンパク質誘導体(PPD)により例示される。これらのうち、破傷風トキソイドが好ましい免疫原性担体である。このカテゴリーはまた、Fifisら、J. Immunol. 173: 3148-54 (2004)により記載されたナノビーズなどの粒子状担体ならびに市販のデンドリマー、例えば、PAMAMデンドリマーおよびMAPデンドリマーも包含する。Aguilarら、J. Pept. Sci. 15: 78-88 (2009)を参照されたい。 In the sense of the present specification, an immunogenic carrier is of sufficient molecular complexity that is capable of producing an immune response against any suitable high molecular weight carrier, typically a protein or a hapten or peptide sequence covalently linked thereto. It can be a large molecule (ie, generally greater than 6000 kD). Suitable categories of immunogenic carriers are, but are not limited to, diphtheria toxoid (DT), tetanus toxoid (TT), pertussis toxoid, and tuberculin pure protein derivative (PPD). Of these, tetanus toxoid is a preferred immunogenic carrier. This category also includes particulate carriers such as nanobeads described by Fifis et al., J. Immunol. 173: 3148-54 (2004) and commercially available dendrimers, such as PAMAM dendrimers and MAP dendrimers. See Aguilar et al., J. Pept. Sci. 15: 78-88 (2009).
本明細書の意味において、語句「製薬上許容し得るビヒクル」は、免疫原の免疫原性効果を減じることなく免疫原を運搬する医学的に安全な非毒性物質を指す。好適なビヒクルは、以下でさらに説明されるような液体エマルジョンであってよく、またはそれは安定な粒子状物質、例えば、製薬上安全な凍結乾燥粉末もしくは製薬上許容し得るシリカゲルもしくは合成の非感染性ウイルス様粒子(VLP)であってよい。Fields Virology, Vol. 1, D.M. Knipe & P. Howley (編), Lippincott Williams & Wilkins (2007)を参照されたい。 In the sense of the present specification, the phrase “pharmaceutically acceptable vehicle” refers to a medically safe non-toxic substance that carries the immunogen without diminishing the immunogenic effect of the immunogen. A suitable vehicle may be a liquid emulsion as further described below, or it may be a stable particulate material, such as a pharmaceutically safe lyophilized powder or a pharmaceutically acceptable silica gel or synthetic non-infectious. It may be a virus-like particle (VLP). See Fields Virology, Vol. 1, D.M. Knipe & P. Howley (ed.), Lippincott Williams & Wilkins (2007).
本発明にとって、好ましい形態の製薬上許容し得るビヒクルは、ポリペプチド免疫原を含有する水相、および油相のエマルジョンである。油相は、意図される投与の用量範囲において非毒性的である、水相と混和しない少なくとも1種の生分解性油を含む。油は天然のものまたは合成のものであってもよく、治療的使用のための国際規制基準を満たすと一般に認識される様々な利用可能なそのような油が存在する。そのような好適な油の例は、スクアレン、スクアラン、モノオレイン酸ソルビタン、ポリソルベート40およびポリソルベート80である。好ましい油相は、これらの5種の油の全部を含む。
For the present invention, a preferred form of a pharmaceutically acceptable vehicle is an aqueous phase containing a polypeptide immunogen and an oil phase emulsion. The oil phase contains at least one biodegradable oil that is non-toxic in the intended dosage range of administration and is immiscible with the aqueous phase. Oils may be natural or synthetic, and there are a variety of such oils that are generally recognized to meet international regulatory standards for therapeutic use. Examples of such suitable oils are squalene, squalane, sorbitan monooleate,
さらに、油相は、モノステアリン酸アルミニウムまたはアジュバント活性オレイン酸サッカリドもしくはステアリン酸サッカリドエステルなどの個別の乳化剤を含有してもよい。 In addition, the oil phase may contain individual emulsifiers such as aluminum monostearate or adjuvant-active oleic acid saccharides or stearic acid saccharide esters.
本発明の別の態様によれば、上記のエマルジョンの油相または水相は、ポリペプチド免疫原の免疫原性担体成分とは異なる少なくとも1種のアジュバントを含有する。様々な公知のアジュバントが存在し、その任意の1種以上を本発明における使用のために考慮することができる。そのような公知のアジュバントの例は、Nor-MDP、イミキモッド、環状ジグアニル酸、トレオニル-N-アセチル-ムラミル-L-アラニル-D-イソグルタミン、イソプリノシン、トレハロースジミコール酸、QS-21、α-ガラクトシルセラミド(α-GalCer)、およびα-グルコシルセラミド(α-GluCer)である。さらに、このアジュバント規則に関して、本発明は、典型的には、アジュバント自体と見なされない場合に、それにもかかわらず免疫刺激的である材料の使用を包含する。これらの材料の例は、Ergamisol(登録商標)（レバミソール塩酸塩、すなわち(S)-(-)-6-フェニル-2,3,5,6-テトラヒドロイミダゾ[2,1-b]チアゾール塩酸塩）、Cimetidine（1-シアノ-2-メチル-3-(2-[[5-メチル-1H-イミダゾール-4-イル)メチル]スルファニル]エチル)グアニジン）、Praziquantel、尿酸、マンナンおよびマンナンの誘導体、ならびにビタミンEである。
According to another aspect of the invention, the oil phase or aqueous phase of the above emulsion contains at least one adjuvant that is different from the immunogenic carrier component of the polypeptide immunogen. There are a variety of known adjuvants, any one or more of which can be considered for use in the present invention. Examples of such known adjuvants include Nor-MDP, imiquimod, cyclic diguanylic acid, threonyl-N-acetyl-muramyl-L-alanyl-D-isoglutamine, isoprinosine, trehalose dimycolic acid, QS-21, α- Galactosylceramide (α-GalCer) and α-glucosylceramide (α-GluCer). Furthermore, with respect to this adjuvant rule, the present invention typically encompasses the use of materials that are nevertheless immunostimulatory when not considered the adjuvant itself. Examples of these materials are Ergamisol® (levamisole hydrochloride, ie (S)-(−)-6-phenyl-2,3,5,6-tetrahydroimidazo [2,1-b] thiazole hydrochloride ) , Cimetidine (1-cyano-2-methyl-3- (2-[[5-methyl-1H-imidazol-4-yl) methyl] sulfanyl] ethyl) guanidine) , Praziquantel, uric acid, mannan and mannan derivatives, As well as vitamin E.
本発明のさらなる態様によれば、改良された免疫原は、huG17およびgly-huG17のアミノ末端エピトープに対するポリクローナル抗体を生成する。これらの免疫原の例は、(i)配列：pGlu-Gly-Pro-Trp-isoVal-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys(配列番号2)のペプチド、それにカップリングした(ii)上記の免疫原性担体を含むものである。ここで、本明細書中の他の場所と同様、「イソVal」は、Leuのβアミノ酸模倣体を指し、一般にLeuに置換することができ、「pGlu」は、huG17ホルモンの生物活性N末端に見出されるアミノ酸誘導体であるピログルタミン酸である。再び、破傷風トキソイドは、この実施形態のための好ましい免疫原性担体であり、そこでN末端huG17の改変物(プレプロガストリンの残基76〜86) + 7アミノ酸のスペーサーがG-18ペプチドである免疫模倣体を構成する。 According to a further aspect of the invention, the improved immunogen generates polyclonal antibodies against the amino terminal epitopes of huG17 and gly-huG17. Examples of these immunogens are: (i) sequence: pGlu-Gly-Pro-Trp-isoVal-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (sequence The peptide of No. 2) and (ii) the above-mentioned immunogenic carrier coupled thereto. Here, as elsewhere in this specification, “isoVal” refers to a beta amino acid mimic of Leu and can generally be replaced by Leu, and “pGlu” is the biologically active N-terminus of the huG17 hormone. Pyroglutamic acid, an amino acid derivative found in Again, tetanus toxoid is a preferred immunogenic carrier for this embodiment, where an N-terminal huG17 variant (residues 76-86 of preprogastrin) +7 amino acid spacer is a G-18 peptide. Construct a mimic.
本発明のさらなる実施形態は、(i)配列pGlu-Gly-Pro-Trp-Val-Glu-Glu-Glu-Glu-Glu-Ala-Thr-Thr-Pro-Pro-Pro-pro-Cys(配列番号3)のペプチド、それにカップリングした(ii)上記の免疫原性担体を含む免疫模倣体である。同様に、破傷風トキソイドが、この実施形態において好ましい免疫原性担体であり、そこでN末端huG17の改変物(プレプロガストリンの改変された残基76〜86) + 7アミノ酸のスペーサーがG-18 LVペプチド、huG17の免疫模倣体を構成する。代替として、この実施形態によれば、「G18 LI」と命名された配列pGlu-Gly-Pro-Trp-Ile-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cysのペプチドが、huG17の相同的免疫模倣体を構成するペプチド(i)として用いられる。 A further embodiment of the present invention provides: (i) the sequence pGlu-Gly-Pro-Trp-Val-Glu-Glu-Glu-Glu-Glu-Ala-Thr-Thr-Pro-Pro-Pro-pro-Cys (SEQ ID NO: An immunomimetic comprising the peptide of 3) and (ii) the above immunogenic carrier coupled thereto. Similarly, tetanus toxoid is a preferred immunogenic carrier in this embodiment, where a modification of the N-terminal huG17 (modified residues 76-86 of preprogastrin) + a 7 amino acid spacer is a G-18 LV peptide Constitutes an immunomimic of huG17. Alternatively, according to this embodiment, the sequence pGlu-Gly-Pro-Trp-Ile-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro named “G18 LI” The peptide of -pro-Cys is used as peptide (i) that constitutes a homologous immune mimic of huG17.
本発明のさらに別の実施形態は、(i)配列：Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-Gly(配列番号5)のペプチド、それにカップリングした(ii)上記の免疫原性担体を含む免疫模倣体である。かくして、この実施形態は、プレプロガストリンの残基79〜93 + 7アミノ酸のスペーサーがG-20ペプチドを構成する、huG17のグリシン伸長免疫模倣体である。 Yet another embodiment of the present invention provides: (i) the sequence: Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp A peptide of -Phe-Gly (SEQ ID NO: 5), coupled to it (ii) an immunomimetic comprising the above immunogenic carrier. Thus, this embodiment is a glycine-elongated immunomimetic of huG17 in which the spacer of residues 79-93 + 7 amino acids of preprogastrin constitutes the G-20 peptide.
また、本発明の一実施形態は、(i)配列：Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-nPhe-Gly-Trp-Met-Asp-Phe-Gly(配列番号6)のペプチド、それにカップリングした(ii)上記の免疫原性担体を含む免疫模倣体である。従って、この実施形態は、プレプロガストリンの残基79〜93 + 7アミノ酸のスペーサーがG-20 YFペプチドを構成する、huG17の改変グリシン伸長免疫模倣体である。 One embodiment of the present invention also provides: (i) the sequence: Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-nPhe-Gly-Trp-Met-Asp A peptide of -Phe-Gly (SEQ ID NO: 6), coupled to it (ii) an immunomimetic comprising the above immunogenic carrier. Thus, this embodiment is a modified glycine extension immunomimic of huG17 in which the spacer of residues 79-93 + 7 amino acids of preprogastrin constitutes the G-20 YF peptide.
以下に列挙される残りのペプチド配列(配列番号7〜10)は、1個以上のD異性体プロリルアミノ酸を含むスペーサーであり、本発明者らが発見した特徴は、担体上への隣接する免疫原ペプチドの適切な位置的提示にとって重要である。この意味において、Dアミノ酸異性体は好適な配置を可能にし、ならびにAPC提示のための免疫原の持続性を増強し、より高力価の抗体が得られる。 The remaining peptide sequences listed below (SEQ ID NOs: 7-10) are spacers containing one or more D-isomer prolyl amino acids, and the features we have found are adjacent to the carrier It is important for proper positional presentation of immunogenic peptides. In this sense, the D amino acid isomers allow for a favorable configuration as well as enhance the persistence of the immunogen for APC presentation, resulting in higher titer antibodies.
以下の一覧において、配列番号7として同定されたペプチドは、スペーサーペプチド：Ser-Ser-Pro-Pro-Pro-pro-Cysである。さらに、配列番号8は、スペーサーペプチド：Ser-Ser-pro-pro-pro-pro-Cysであり、配列番号9は、スペーサーペプチド：Thr-Thr-Pro-Pro-Pro-pro-Cysであり、および配列番号10は、スペーサーペプチド：Thr-Thr-pro-pro-pro-pro-Cysである。 In the following list, the peptide identified as SEQ ID NO: 7 is the spacer peptide: Ser-Ser-Pro-Pro-Pro-pro-Cys. Furthermore, SEQ ID NO: 8 is a spacer peptide: Ser-Ser-pro-pro-pro-pro-Cys, SEQ ID NO: 9 is a spacer peptide: Thr-Thr-Pro-Pro-Pro-pro-Cys, And SEQ ID NO: 10 is the spacer peptide: Thr-Thr-pro-pro-pro-pro-Cys.
従来技術においては、免疫模倣体-担体複合体を用いる免疫化による有効な抗体応答の誘導には、典型的には免疫原の2回以上の投与を必要とし、抗体力価が所望のレベルまで上昇するには数週間または数ヶ月かかる。対照的に、本発明の改良された免疫原は、免疫原の初期過程の投与の直後に有効なレベルの抗体を誘導する。かくして、惹起された抗体のレベルは数ヶ月間にわたって上昇したままであり、免疫原の単回注射によるその後の追加免疫の際により高レベルまで容易に上昇する。 In the prior art, induction of an effective antibody response by immunization with an immunomimetic-carrier complex typically requires two or more doses of the immunogen, with antibody titers reaching the desired level. It takes weeks or months to rise. In contrast, the improved immunogens of the present invention induce effective levels of antibodies immediately following administration of the initial course of immunogen. Thus, the level of elicited antibody remains elevated over several months and easily rises to higher levels during subsequent boosts with a single injection of immunogen.
本発明を以下の実施例を参照することによりさらに説明するが、これは例示に過ぎず、本発明を限定するものではない。 The invention is further illustrated by reference to the following examples, which are illustrative only and not limiting.
標準的な固体状態の合成方法により、ペプチドを調製した。それぞれのペプチドを、アミノ酸含量および純度に関して特性評価した。 Peptides were prepared by standard solid state synthesis methods. Each peptide was characterized for amino acid content and purity.
かくして、以下に列挙されるアミノ酸配列を有するペプチドを合成した。これらの配列において、本明細書の他の箇所におけると同様、大文字で始まるアミノ酸はL異性体アミノ酸であるが、小文字で始まるものはD異性体である。 Thus, peptides having the amino acid sequences listed below were synthesized. In these sequences, as elsewhere in the specification, amino acids starting with a capital letter are L-isomer amino acids, but those starting with a lowercase letter are D-isomers.
(1)Cys-pro-Pro-Pro-Pro-Ser-Ser-Gly-Trp-Met-Asp-nPhe-Gly-Arg-Arg-Ser-Ala-Glu-Asp-Glu-Asn (配列番号1)、「G-21」と命名、
(2)pGlu-Gly-Pro-Trp-イソVal-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (配列番号2)、「G-18」と命名、
(3)pGlu-Gly-Pro-Trp-イソVal-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (配列番号3)、「G-18 LV」と命名(ヒトG17相同体)、
(4)pGlu-Gly-Pro-Trp-Ile- Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (配列番号4)、「G18 LI」と命名、
(5)Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-Gly (配列番号5)、「G-20」と命名、
(6)Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-nPhe-Gly-Trp-Met-Asp-Phe-Gly (配列番号6)、「G-20」と命名、
(7)Ser-Ser-Pro-Pro-Pro-pro-Cys (配列番号7)、7アミノ酸のペプチドであるセリル-プロリルスペーサー、
(8)Ser-Ser-pro-pro-pro-pro-Cys (配列番号8)、7アミノ酸のペプチドであるセリル-全部D-プロリルのスペーサー、
(9)Thr-Thr-Pro-Pro-Pro-pro-Cys (配列番号9)、7アミノ酸のペプチドであるトレオニル-プロリルスペーサー、
(10)Thr-Thr-pro-pro-pro-pro-Cys (配列番号10)、7アミノ酸のペプチドであるトレオニル-全部D-プロリルのスペーサー。
(1) Cys-pro-Pro-Pro-Pro-Ser-Ser-Gly-Trp-Met-Asp-nPhe-Gly-Arg-Arg-Ser-Ala-Glu-Asp-Glu-Asn (SEQ ID NO: 1), Named "G-21",
(2) pGlu-Gly-Pro-Trp-isoVal-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 2), `` G-18 '' Named,
(3) pGlu-Gly-Pro-Trp-isoVal-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 3), `` G-18 LV '' (Human G17 homologue),
(4) pGlu-Gly-Pro-Trp-Ile- Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 4), named `` G18 LI '' ,
(5) Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-Gly (SEQ ID NO: 5), `` G -20 ",
(6) Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-nPhe-Gly-Trp-Met-Asp-Phe-Gly (SEQ ID NO: 6), `` G -20 ",
(7) Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 7), a seryl-prolyl spacer which is a 7 amino acid peptide,
(8) Ser-Ser-pro-pro-pro-pro-Cys (SEQ ID NO: 8), a 7 amino acid peptide seryl-all-D-prolyl spacer,
(9) Thr-Thr-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 9), a 7-amino acid peptide, a threonyl-prolyl spacer,
(10) Thr-Thr-pro-pro-pro-pro-Cys (SEQ ID NO: 10), a 7-amino acid peptide threonyl-all-D-prolyl spacer.
ペプチド1(配列番号1)は、プレプロガストリンのN末端側の残基88〜101に結合したカルボキシ末端スペーサー-Ser-Ser-Pro-Pro-Pro-pro-Cys(配列番号7)が先行するプロガストリンのパラ-ニトロフェニルアラニン改変アミノ末端免疫模倣体(-Gly-Trp-Met-Asp-nPhe-Gly-Arg-Arg-Ser-Ala-Glu-Asp-Glu-Asn)を含む。ペプチド2(配列番号2)は、イソValがLeuに置換されたhuG17の相同体であるhuG17の11アミノ酸の免疫模倣体(pGlu-Gly-Pro-Trp-Leu-isoVal-Glu-Glu-Glu-Glu-Ala-)、次いで、huG17免疫模倣体のプレプロガストリン残基のアミノ酸番号86に結合したスペーサー-Ser-Ser-Pro-Pro-Pro-D-Pro-Cys(配列番号7)を含む。ペプチド3(配列番号3)は、上記のプレプロガストリンのアミノ酸番号86に結合した、ValがイソValに置換されている以外はペプチド2と同じである、11アミノ酸の免疫模倣相同体、-Ser-Ser-pro-pro-pro-pro-Cys(配列番号8)を含む。ペプチド4(配列番号4)は、IleがイソValに置換されている以外はペプチド2と同様に、huG17の11アミノ酸の免疫模倣体、pGlu-Gly-Pro-Trp-Ile-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-Gly、次いで、huG17の免疫模倣体のプレプロガストリンのC末端の残基番号79に結合したスペーサー-Ser-Ser-Pro-Pro-Pro-pro-Cys(配列番号7)を含む。ペプチド5(配列番号5)は、huG17のグリシン伸長免疫模倣体のプレプロガストリンN末端の残基番号79に結合したスペーサー-Ser-Ser-Pro-Pro-Pro-pro-Cys(配列番号7)が先行する、gly-huG17の15アミノ酸の免疫模倣体、Trp-Ile-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-Glyを含む。ペプチド6(配列番号6)は、huG17のグリシン伸長免疫模倣体のプレプロガストリンN末端の残基番号79に結合したスペーサー-Ser-Ser-Pro-Pro-Pro-pro-Cys(配列番号7)が先行する、gly-huG17のパラ-ニトロフェニルアラニン改変された15アミノ酸の免疫模倣体、Trp-Ile-Glu-Glu-Glu-Glu-Glu-Ala-nPhe-Gly-Trp-Met-Asp-Phe-Glyを含む。ペプチド9(配列番号1)は、プレプロガストリンのN末端の残基88〜101のペプチドに結合した、カルボキシ末端スペーサー-Ser-Ser-pro-pro-pro-pro-Cys(配列番号8)が先行する、プロガストリンのアミノ末端免疫模倣体、-Gly-Trp-Met-Asp-Phe-Gly-Arg-Arg-Ser-Ala-Glu-Asp-Glu-Asnを含み、プロガストリンのヒトペプチド模倣体を形成する。 Peptide 1 (SEQ ID NO: 1) is a pro-peptide preceded by a carboxy-terminal spacer-Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 7) attached to the N-terminal residues 88-101 of preprogastrin. Contains a para-nitrophenylalanine modified amino-terminal immunomimetic of gastrin (-Gly-Trp-Met-Asp-nPhe-Gly-Arg-Arg-Ser-Ala-Glu-Asp-Glu-Asn). Peptide 2 (SEQ ID NO: 2) is an 11 mimetic immunomimic of huG17 (pGlu-Gly-Pro-Trp-Leu-isoVal-Glu-Glu-Glu-Glu- Glu-Ala-) followed by a spacer-Ser-Ser-Pro-Pro-Pro-D-Pro-Cys (SEQ ID NO: 7) linked to amino acid number 86 of the preprogastrin residue of the huG17 immunomimetic. Peptide 3 (SEQ ID NO: 3) is the same as peptide 2, except that Val is replaced with isoVal, linked to amino acid number 86 of the above preprogastrin, -Ser- Contains Ser-pro-pro-pro-pro-Cys (SEQ ID NO: 8). Peptide 4 (SEQ ID NO: 4) is similar to peptide 2 except that Ile is replaced with isoVal, an 11 amino acid immunomimetic of huG17, pGlu-Gly-Pro-Trp-Ile-Glu-Glu-Glu -Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-Gly, then spacer-Ser-Ser-Pro- linked to residue number 79 at the C-terminus of huG17 immunomimetic preprogastrin Includes Pro-Pro-pro-Cys (SEQ ID NO: 7). Peptide 5 (SEQ ID NO: 5) has spacer-Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 7) bound to residue number 79 at the N-terminal preprogastrin of the glycine-elongated immunomimetic of huG17. The preceding, 15-amino acid immunomimic of gly-huG17, including Trp-Ile-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-Gly. Peptide 6 (SEQ ID NO: 6) has spacer-Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 7) linked to residue number 79 at the N-terminal preprogastrin of the glycine-extended immunomimetic of huG17. Preceding, gly-huG17 para-nitrophenylalanine modified 15 amino acid immunomimetic, Trp-Ile-Glu-Glu-Glu-Glu-Glu-Ala-nPhe-Gly-Trp-Met-Asp-Phe-Gly including. Peptide 9 (SEQ ID NO: 1) is preceded by a carboxy-terminal spacer-Ser-Ser-pro-pro-pro-pro-Cys (SEQ ID NO: 8) linked to the peptide at residues 88-101 at the N-terminus of preprogastrin Including an amino-terminal immunomimetic of progastrin, -Gly-Trp-Met-Asp-Phe-Gly-Arg-Arg-Ser-Ala-Glu-Asp-Glu-Asn, and a human peptidomimetic of progastrin Form.
本発明の好ましい態様によれば、これらのペプチドはそれぞれ、破傷風トキソイド(TT)免疫原性担体上に存在するアミノ基にコンジュゲートされた。この連結は、連結剤の一方の末端にスクシンイミジルエステルを含有し、他方の末端にマレイミドを含有するヘテロ二官能性連結剤を用いる、末端ペプチドのシステイン残基を介するものであった。上記のペプチド1および2のいずれかと、担体との連結を達成するために、ペプチドのシステインを最初に還元した。乾燥ペプチドを0.1Mのリン酸ナトリウムバッファー、pH 7〜9に、5〜50モル過剰量のジチオトレイトールと共に溶解した。このペプチドを凍結乾燥し、用いるまで減圧下で保存した。 According to a preferred embodiment of the present invention, each of these peptides was conjugated to an amino group present on a tetanus toxoid (TT) immunogenic carrier. This linkage was through the cysteine residue of the terminal peptide using a heterobifunctional linking agent containing a succinimidyl ester at one end of the linking agent and a maleimide at the other end. To achieve ligation of either of peptides 1 and 2 above with the carrier, the peptide cysteine was first reduced. The dried peptide was dissolved in 0.1 M sodium phosphate buffer, pH 7-9, with a 5-50 molar excess of dithiothreitol. The peptide was lyophilized and stored under reduced pressure until used.
105分子量のTTあたり約25の遊離アミノ基の活性化を達成するのに十分な比率で、ヘテロ二官能性連結剤ε-マレイミドカプロン酸N-ヒドロキシスクシンイミドエステル(EMCS)で処理することにより、TTを活性化した。 Treatment with the heterobifunctional linker ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS) at a ratio sufficient to achieve activation of about 25 free amino groups per 105 molecular weight TT Activated.
精製破傷風トキソイドの調製：TTを限外濾過により精製した。回収された精製TTの最終濃度は、5〜40 mg/mlであると予想された。クロマトグラフィー(SEC HPLC)、タンパク質濃度(Lowry)、および遊離アミノ基(ニンヒドリン)により、純度を決定した。 Preparation of purified tetanus toxoid: TT was purified by ultrafiltration. The final concentration of purified TT recovered was expected to be 5-40 mg / ml. Purity was determined by chromatography (SEC HPLC), protein concentration (Lowry), and free amino groups (ninhydrin).
ペプチドを商業的に入手し(Biosyn Corp, USA)、既知の純度および含量を有する還元ペプチドをコンジュゲーションに用いた。ペプチドをトリス(2-カルボキシエチル)-ホスフィン-HCl(TCEP)を用いて還元し、混合物をコンジュゲーションにおいて用いた。Ellmansアッセイを用いて、遊離スルフヒドラル(sulfhydral)基を決定した。 Peptides were obtained commercially (Biosyn Corp, USA) and reduced peptides with known purity and content were used for conjugation. The peptide was reduced using tris (2-carboxyethyl) -phosphine-HCl (TCEP) and the mixture was used in conjugation. The Ellmans assay was used to determine free sulfhydral groups.
破傷風トキソイドの活性化：精製TTを活性化バッファー中に5〜50 mg/mlに希釈した。所望の量のTTを、テフロン(登録商標)コートされた撹拌棒を含むガラスバイアルに移し、EMCS(50〜90 mg/mlのDMF)をTT溶液に添加した。EMCS/DTのモル比は活性化レベルを決定する。最後の濃縮工程において、総量を減少させて5〜50 mg TT/mlを得た。SEC HPLC、Lowryによるタンパク質濃度、Ellman'sによる活性化レベルにより、TT溶液を決定した。 Tetanus toxoid activation: Purified TT was diluted to 5-50 mg / ml in activation buffer. The desired amount of TT was transferred to a glass vial containing a Teflon-coated stir bar and EMCS (50-90 mg / ml DMF) was added to the TT solution. The molar ratio of EMCS / DT determines the activation level. In the final concentration step, the total amount was reduced to obtain 5-50 mg TT / ml. The TT solution was determined by SEC HPLC, protein concentration by Lowry, and activation level by Ellman's.
ペプチド-TTのコンジュゲーション：マレイミド-TTと反応するペプチドの量を算出した後、ペプチドをM-TT溶液に添加した。ペプチド-TTコンジュゲートを限外濾過により精製した。 Peptide-TT conjugation: After calculating the amount of peptide that reacts with maleimide-TT, the peptide was added to the M-TT solution. The peptide-TT conjugate was purified by ultrafiltration.
ペプチドG18およびG21のコンジュゲートをEMCSによりTTに連結し、0.1〜0.5M重炭酸アンモニウムで平衡化させたG50 Sephadexカラム上、4℃での低圧クロマトグラフィーにより混合物の他の成分から分離した。それぞれの場合、コンジュゲートをカラム空隙容量中で溶出し、凍結乾燥および保存し、使用するまで4〜0℃で乾燥した。 Peptides G18 and G21 conjugates were linked to TT by EMCS and separated from the other components of the mixture by low pressure chromatography at 4 ° C. on a G50 Sephadex column equilibrated with 0.1-0.5 M ammonium bicarbonate. In each case, the conjugate was eluted in the column void volume, lyophilized and stored, and dried at 4-0 ° C. until use.
重量増加、アミノ酸分析などの当業者には公知のいくつかの方法により、免疫模倣ペプチド含量に関してコンジュゲートを特性評価することができる。これらの方法により作製されたペプチドG18およびG21のTTとのコンジュゲートを、アミノ酸分析により決定したところ、104〜106 MWのTTあたり10〜30モルのペプチドを有し、全てが試験動物の免疫化にとって免疫原として好適であると考えられた。同様に、G18およびG21のDTコンジュゲートを同じ様式で調製して、抗体結合のアッセイのための基質としてhuG17を用いてELISA力価を決定した。 The conjugate can be characterized for immunomimetic peptide content by several methods known to those skilled in the art, such as weight gain, amino acid analysis, and the like. Conjugates of peptides G18 and G21 made by these methods with TT were determined by amino acid analysis and had 10-30 moles of peptide per 104-106 MW TT, all immunizing test animals It was considered suitable as an immunogen. Similarly, G18 and G21 DT conjugates were prepared in the same manner and ELISA titers were determined using huG17 as a substrate for antibody binding assays.
実施例1のペプチド-TTコンジュゲートを、以下のように調製された水相および油相成分のエマルジョン中で投与した。コンジュゲートおよびアジュバントをリン酸緩衝生理食塩水(PBS)中に溶解して、水相を作製した。コンジュゲートの濃度が、これらの成分が最終的なエマルジョン中に有する濃度の2倍となるように水相を調製する。以下の実施例4で用いられる免疫原を調製するために、コンジュゲートをPBS、pH 6.5〜8.0中に、5〜12 mg/mlの濃度で溶解した(勿論この広い範囲を超えてもよく、本発明者らはコンジュゲートを欲する場合、担体の程度を制御するためにそれを用いる)。 The peptide-TT conjugate of Example 1 was administered in an emulsion of aqueous and oil phase components prepared as follows. The conjugate and adjuvant were dissolved in phosphate buffered saline (PBS) to create an aqueous phase. The aqueous phase is prepared so that the concentration of the conjugate is twice the concentration that these components have in the final emulsion. To prepare the immunogen used in Example 4 below, the conjugate was dissolved in PBS, pH 6.5-8.0 at a concentration of 5-12 mg / ml (of course this wide range may be exceeded, If we want a conjugate, we use it to control the extent of the carrier).
水相を1:1(vol:vol)で油性ビヒクル相と混合して、最終免疫原製剤を含むエマルジョンを作製した。一つのそのようなビヒクルは、20〜60部のスクアレン、70〜30部のスクアラン、2〜12部のモノオレイン酸ソルビタン、0.6〜2.0部のモノステアリン酸アルミニウム、0.1〜1部のポリソルベート80および0.2〜1.2部のポリソルベート40の混合物である。水相および油相ビヒクルは、安定化乳化混合物を形成させるために任意の公知の方法により混合してもよい。エマルジョンは保存時に安定でなければならない、すなわち、それは数週間から数ヶ月の最少保存時間にわたって水相およびビヒクル相への有意な程度の分離を受けるべきではない。エマルジョンはまた、許容可能なサイズの皮下注射針により容易に注入することができる稠度のものでなければならない。
The aqueous phase was mixed 1: 1 (vol: vol) with the oily vehicle phase to make an emulsion containing the final immunogenic formulation. One such vehicle is 20-60 parts squalene, 70-30 parts squalane, 2-12 parts sorbitan monooleate, 0.6-2.0 parts aluminum monostearate, 0.1-1
免疫原を含有する水相を、2つのガラスシリンジ間の18ゲージの二本連結した針により、1:1(vol:vol)で2つの溶液の油性ビヒクル混合物を用いて乳化した。混合物を針を介して50回押した。次いで、乳化した混合物を動物への注射のために使い捨てシリンジ中に取り出した。実施例4中のための、エマルジョン中の最終免疫原濃度は、約1〜約5 mg/mlの濃度範囲のコンジュゲート：hG18TTであった。 The aqueous phase containing the immunogen was emulsified with an oil vehicle mixture of the two solutions at 1: 1 (vol: vol) with two 18 gauge needles between two glass syringes. The mixture was pushed 50 times through the needle. The emulsified mixture was then removed into a disposable syringe for injection into animals. The final immunogen concentration in the emulsion for in Example 4 was conjugate: hG18TT in a concentration range of about 1 to about 5 mg / ml.
本発明者らは、実施例1および2に記載のように、TTおよびDTに連結された実施例1に記載のG18およびG21ペプチドの各々を含むコンジュゲートを構築した。次いで、ペプチドG18免疫原(図1A、B)を用いて6匹のマウスを免疫し、ペプチドG21免疫原(図1C、D)を用いて6匹のマウスを免疫した。 We constructed a conjugate comprising each of the G18 and G21 peptides described in Example 1 linked to TT and DT as described in Examples 1 and 2. Peptide G18 immunogen (FIGS. 1A, B) was then used to immunize 6 mice, and peptide G21 immunogen (FIGS. 1C, D) was used to immunize 6 mice.
本発明者らは、実施例1および2に記載のように、TTおよびDTに連結された、上記のG18およびG21ペプチドの各々を含むコンジュゲートを構築した。次いで、G18免疫原(図2A)を用いて4匹のウサギを免疫し、G21免疫原(図2B)を用いて4匹のウサギを免疫した。 We constructed conjugates containing each of the above G18 and G21 peptides linked to TT and DT as described in Examples 1 and 2. Then, 4 rabbits were immunized with G18 immunogen (FIG. 2A) and 4 rabbits were immunized with G21 immunogen (FIG. 2B).
図1および2に提示されるように、これらのELISA試験の結果は、免疫原1および2(実施例1および2の)が、いくつかの動物種において、ならびに誘導された抗体応答の期間において、その効力および抗体の惹起の両方に関して有効であったことを示している。 As presented in FIGS. 1 and 2, the results of these ELISA tests show that immunogens 1 and 2 (of Examples 1 and 2) are in some animal species and in the period of the induced antibody response. , Indicating that it was effective both in terms of its potency and eliciting antibodies.
本発明者らは、上記に記載のように、TTおよびDTに連結されたG18およびG21ペプチドの各々を含むコンジュゲートを構築した。次いで、G18TT免疫原およびG21TT免疫原を用いて6匹のマウスを免疫した。ピーク力価(42日目)において、全てのマウスは、50,000個のヒト胃癌細胞／1〜2 cmのチューブ(BCG-823)を含有する滅菌された腹腔内中空ファイバーインプラントを5日間受けた。中空ファイバー管はCTLまたはNK細胞ではなく500 KD未満の分子の浸透を許容し、免疫応答性マウス中でのヒト癌細胞の生存を可能にする。5日間の終わりに、それぞれのマウスのインプラントを除去し、生細胞をMTTアッセイにより計数し、それらを非免疫マウス中の対照インプラントと比較した。示されるように、いくらかの動物は10 mg/kgのシスプラチン(CP)または20 mg/kgの5-フルオロウラシル(5-FU)または10 mg/kgのCP + 5-FUの各々の組合せ(FUP)の単回投与でも処理した。 We constructed a conjugate comprising each of the G18 and G21 peptides linked to TT and DT as described above. Six mice were then immunized with G18TT and G21TT immunogens. At peak titer (day 42), all mice received a sterile intraperitoneal hollow fiber implant containing 50,000 human gastric cancer cells / 1-2 cm tube (BCG-823) for 5 days. Hollow fiber tubes allow the penetration of molecules less than 500 KD rather than CTL or NK cells, allowing survival of human cancer cells in immunocompetent mice. At the end of 5 days, each mouse implant was removed and viable cells were counted by MTT assay and compared to control implants in non-immunized mice. As indicated, some animals have 10 mg / kg cisplatin (CP) or 20 mg / kg 5-fluorouracil (5-FU) or 10 mg / kg CP + 5-FU each combination (FUP) A single dose of was also processed.
この試験の結果を図3に提示する。免疫原1および2(実施例1および2の)は、いずれかの抗ガストリン／プロガストリン(G18およびG21免疫原)を用いる場合、ヒト胃癌細胞の増殖の阻害、ならびに従来の胃癌化学療法剤の存在下で有効であった十分な抗体応答の誘導に関して有効であることを見てとることができる。 The results of this test are presented in FIG. Immunogens 1 and 2 (of Examples 1 and 2) inhibit the growth of human gastric cancer cells when using either anti-gastrin / progastrin (G18 and G21 immunogens), as well as conventional gastric cancer chemotherapeutic agents It can be seen that it is effective in inducing a sufficient antibody response that was effective in the presence.
本発明者らは、上記のG18-a、G-18-b、G20およびG20-aペプチド(それぞれ、配列番号3〜6)の各々を含むコンジュゲートを構築し、それらをG21(配列番号1)と比較した。それらは、実施例1および2に記載のように、TTに全て連結された。次いで、本発明者らは、これらの免疫原(図4A、B、CおよびDを参照)を用いて6匹のマウスを免疫し、比較のために、G21免疫原を用いて6匹のマウスを免疫した。 We constructed a conjugate comprising each of the G18-a, G-18-b, G20 and G20-a peptides (SEQ ID NOs: 3-6, respectively) described above, and combined them with G21 (SEQ ID NO: 1 ). They were all linked to TT as described in Examples 1 and 2. We then immunized 6 mice with these immunogens (see FIGS. 4A, B, C and D) and, for comparison, 6 mice with the G21 immunogen. Immunized.
かくして示された改良は、本発明による免疫原ペプチドの免疫模倣体およびユニークなスペーサー領域中で具体化された改変物から生じる。本発明のペプチド免疫原を、上記の免疫原性模倣体およびスペーサーをいずれも含まない免疫原と比較して試験したところ、後者はさほど有効でないことがわかった。従って、従来の免疫原は、本発明者らが本発明に従ってその免疫模倣体および／またはそのスペーサーを改変することにより改良された。
[１] (A)(i)プロガストリンのアミノ酸配列またはプロガストリンのN末端および／もしくはC末端プロセッシングされた種、それに連結された(ii)7アミノ酸のスペーサーから構成される模倣ペプチドならびに(B)該模倣ペプチドにカップリングされた免疫原性担体を含むポリペプチド免疫原。
[２] 模倣ペプチドが、アミノ酸配列：Cys-pro-Pro-Pro-Pro-Ser-Ser-Gly-Trp-Met-Asp-nPhe-Gly-Arg-Arg-Ser-Ala-Glu-Asp-Glu-Asn(配列番号1)を有する、１に記載のポリペプチド免疫原。
[３] 模倣ペプチドが、
pGlu-Gly-Pro-Trp-β-isoVal-Glu-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (配列番号2);
pGlu-Gly-Pro-Trp-Ile-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (配列番号3);
pGlu-Gly-Pro-Trp-Val-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (配列番号4);
Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-Gly (配列番号5); および
Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-nPhe-Gly-Trp-Met-Asp-Phe-Gly (配列番号6)
からなる群より選択されるアミノ酸配列を有する、１に記載のポリペプチド免疫原。
[４] 免疫原性担体が、破傷風トキソイド、ジフテリアトキソイド、百日咳トキソイド、およびツベルクリン純粋タンパク質誘導体からなる群より選択される、１に記載のポリペプチド免疫原組成物。
[５] 免疫原性担体が破傷風トキソイドである、４に記載のポリペプチド免疫原性組成物。
[６] 有効量の１に記載のポリペプチド免疫原および該免疫原のための製薬上許容し得るビヒクルを含む免疫原性組成物。
[７] 製薬上許容し得る担体が、前記免疫原が存在する水相、および油相のエマルジョンを含む、６に記載の免疫原性組成物。
[８] 油相がスクアレン、スクアラン、モノオレイン酸ソルビタン、ポリソルベート40およびポリソルベート80のうちの少なくとも1種を含む、７に記載の免疫原性組成物。
[９] 油相が乳化剤を含む、７に記載の免疫原性組成物。
[１０] 油相または水相のいずれかが少なくとも1種のアジュバントを含有する、７に記載の免疫原性組成物。
[１１] アジュバントが、Nor-MDP、イミキモッド、環状ジグアニル酸、トレオニル-N-アセチル-ムラミル-L-アラニル-D-イソグルタミン、イソプリノシン、トレハロースジミコール酸、QS-21、α-ガラクトシルセラミド、およびα-グルコシルセラミドからなる群より選択される、１０に記載の免疫原性組成物。
[１２] アジュバントが、Ergamisol(登録商標)、Cimetidine、Praziquantel、尿酸、マンナンおよびマンナンの誘導体、ならびにビタミンEからなる群より選択される、１０に記載の免疫原性組成物。
[１３] Ser-Ser-Pro-Pro-Pro-pro-Cys (配列番号7);
Ser-Ser-pro-pro-pro-pro-Cys (配列番号8);
Thr-Thr-Pro-Pro-Pro-pro-Cys (配列番号9); および
Thr-Thr-pro-pro-pro-pro-Cys (配列番号10)
からなる群より選択されるスペーサーペプチド。
The improvements thus shown result from the immunomimetics of the immunogenic peptides according to the invention and the modifications embodied in the unique spacer region. When the peptide immunogens of the present invention were tested in comparison to immunogens that did not contain any of the above immunogenic mimetics and spacers, the latter was found to be less effective. Thus, conventional immunogens have been improved by the inventors by modifying their immunomimetics and / or their spacers in accordance with the present invention .
[1] (A) (i) the amino acid sequence of progastrin or the N-terminal and / or C-terminal processed species of progastrin, (ii) a mimetic peptide composed of a 7 amino acid spacer linked thereto and (B A polypeptide immunogen comprising an immunogenic carrier coupled to the mimetic peptide.
[2] The mimetic peptide has the amino acid sequence: Cys-pro-Pro-Pro-Pro-Ser-Ser-Gly-Trp-Met-Asp-nPhe-Gly-Arg-Arg-Ser-Ala-Glu-Asp-Glu- 2. The polypeptide immunogen according to 1, having Asn (SEQ ID NO: 1).
[3] The mimetic peptide is
pGlu-Gly-Pro-Trp-β-isoVal-Glu-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 2);
pGlu-Gly-Pro-Trp-Ile-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 3);
pGlu-Gly-Pro-Trp-Val-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 4);
Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-Gly (SEQ ID NO: 5); and
Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-nPhe-Gly-Trp-Met-Asp-Phe-Gly (SEQ ID NO: 6)
The polypeptide immunogen according to 1, having an amino acid sequence selected from the group consisting of:
[4] The polypeptide immunogenic composition according to 1, wherein the immunogenic carrier is selected from the group consisting of tetanus toxoid, diphtheria toxoid, pertussis toxoid, and tuberculin pure protein derivative.
[5] The polypeptide immunogenic composition according to 4, wherein the immunogenic carrier is tetanus toxoid.
[6] An immunogenic composition comprising an effective amount of the polypeptide immunogen according to 1 and a pharmaceutically acceptable vehicle for the immunogen.
[7] The immunogenic composition according to 6, wherein the pharmaceutically acceptable carrier comprises an aqueous phase in which the immunogen is present and an oil phase emulsion.
[8] The immunogenic composition according to 7, wherein the oil phase comprises at least one of squalene, squalane, sorbitan monooleate,
[9] The immunogenic composition according to 7, wherein the oil phase contains an emulsifier.
[10] The immunogenic composition according to 7, wherein either the oil phase or the aqueous phase contains at least one adjuvant.
[11] The adjuvant is Nor-MDP, imiquimod, cyclic diguanylic acid, threonyl-N-acetyl-muramyl-L-alanyl-D-isoglutamine, isoprinosine, trehalose dimycolic acid, QS-21, α-galactosylceramide, and 11. The immunogenic composition according to 10, selected from the group consisting of α-glucosylceramide.
[12] The immunogenic composition according to 10, wherein the adjuvant is selected from the group consisting of Ergamisol (registered trademark), Cimetidine, Praziquantel, uric acid, mannan and mannan derivatives, and vitamin E.
[13] Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 7);
Ser-Ser-pro-pro-pro-pro-Cys (SEQ ID NO: 8);
Thr-Thr-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 9); and
Thr-Thr-pro-pro-pro-pro-Cys (SEQ ID NO: 10)
A spacer peptide selected from the group consisting of
Claims (12)
pGlu-Gly-Pro-Trp-β-isoVal-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (配列番号2);
pGlu-Gly-Pro-Trp-Ile-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (配列番号3);
pGlu-Gly-Pro-Trp-Val-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (配列番号4);
Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-Gly (配列番号5); および
Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-nPhe-Gly-Trp-Met-Asp-Phe-Gly (配列番号6)
からなる群より選択されるアミノ酸配列の少なくとも１つから構成される模倣ペプチドならびに(B)該模倣ペプチドにカップリングされた免疫原性担体を含むポリペプチド免疫原性組成物、ここでProはプロリンのL異性体であり、proはプロリンのD異性体である、前記ポリペプチド免疫原性組成物。 (A ) Cys-pro-Pro-Pro-Pro-Ser-Ser-Gly-Trp-Met-Asp-nPhe-Gly-Arg-Arg-Ser-Ala-Glu-Asp-Glu-Asn (SEQ ID NO: 1) ;
pGlu-Gly-Pro-Trp-β-isoVal-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 2);
pGlu-Gly-Pro-Trp-Ile-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 3);
pGlu-Gly-Pro-Trp-Val-Glu-Glu-Glu-Glu-Glu-Ala-Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 4);
Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-Gly (SEQ ID NO: 5); and
Cys-pro-Pro-Pro-Pro-Trp-Leu-Glu-Glu-Glu-Glu-Glu-Ala-nPhe-Gly-Trp-Met-Asp-Phe-Gly (SEQ ID NO: 6)
At least one or that consists of mimetic peptide of an amino acid sequence selected from the group consisting of and (B) a polypeptide immunogenic compositions comprising the coupled immunogenic carrier to the mimetic peptide, wherein Pro is proline The polypeptide immunogenic composition, wherein L is an L isomer and pro is the D isomer of proline.
Ser-Ser-pro-pro-pro-pro-Cys (配列番号8);
Thr-Thr-Pro-Pro-Pro-pro-Cys (配列番号9); および
Thr-Thr-pro-pro-pro-pro-Cys (配列番号10)
からなる群より選択されるスペーサーペプチド。
Ser-Ser-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 7);
Ser-Ser-pro-pro-pro-pro-Cys (SEQ ID NO: 8);
Thr-Thr-Pro-Pro-Pro-pro-Cys (SEQ ID NO: 9); and
Thr-Thr-pro-pro-pro-pro-Cys (SEQ ID NO: 10)
A spacer peptide selected from the group consisting of
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|---|---|---|---|
| CN201010116229.3 | 2010-03-03 | ||
| CN2010101162293A CN101797382B (en) | 2010-03-03 | 2010-03-03 | Anti-human progastrin polypeptide immune composition |
| PCT/US2011/000413 WO2011109106A2 (en) | 2010-03-03 | 2011-03-03 | Immunogenic compositions against human progastrin peptides |
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| JP (1) | JP5960064B2 (en) |
| KR (2) | KR101847224B1 (en) |
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| EA037027B1 (en) * | 2015-12-31 | 2021-01-27 | Прогастрин Э Кансер С.А Р.Л. | Use and method for preventing or treating gastric cancer using a composition comprising a monoclonal progastrin-binding antibody |
| WO2019038671A1 (en) * | 2017-08-22 | 2019-02-28 | Onkologix Ltd | Composition of tumor-associated proliferative peptides and related anti-cancer immunogen for the treatment of lung cancers and other cancers |
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| US2005102A (en) * | 1931-05-21 | 1935-06-18 | Barrett Co | Distillation of tar |
| US5023077A (en) * | 1989-01-24 | 1991-06-11 | Aphton Corporation | Immunogenic compositions and methods for the treatment and prevention of gastric and duodenal ulcer disease |
| US6258550B1 (en) * | 1993-04-23 | 2001-07-10 | Virginia Commonwealth University | Polypeptides that include conformation-constraining groups which flank a protein-protein interaction site |
| US6084066A (en) * | 1993-10-29 | 2000-07-04 | Virginia Commonwealth University | Polypetides that include conformation-constraining groups which flank a protein-protein interaction site |
| US5468494A (en) * | 1993-11-12 | 1995-11-21 | Aphton Corp. | Immunogenic compositions against human gastrin 17 |
| DK0889735T3 (en) | 1996-02-08 | 2011-08-08 | Cancer Advances Inc | Immunogenic methods for the treatment of gastrointestinal cancer |
| CA2327439C (en) * | 1998-05-15 | 2005-07-05 | Aphton Corporation | Combination therapy for the treatment of tumors |
| CA2580965C (en) * | 2004-09-22 | 2014-04-08 | Receptor Biologix, Inc. | Monoclonal antibodies to progastrin |
| CN100558747C (en) * | 2006-04-06 | 2009-11-11 | 海南天源康泽医药科技有限公司 | With the diphtheria toxin muton CRM 197 immunogen and preparation method thereof and application of carrier |
| US7854932B2 (en) * | 2006-12-19 | 2010-12-21 | The Board Of Regents Of The University Of Texas System | Immunogenic compositions comprising progastrin and uses thereof |
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| CN101797382A (en) | 2010-08-11 |
| WO2011109106A3 (en) | 2012-01-19 |
| WO2011109106A2 (en) | 2011-09-09 |
| KR20170142214A (en) | 2017-12-27 |
| CN101797382B (en) | 2013-11-20 |
| KR101847224B1 (en) | 2018-04-10 |
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