JP2024019894A - Pharmaceutical composition for treatment of galectin-4 positive gastric cancer - Google Patents
Pharmaceutical composition for treatment of galectin-4 positive gastric cancer Download PDFInfo
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Abstract
Description
本発明は、ガレクチン-4陽性胃がん治療用医薬組成物に関する。 The present invention relates to a pharmaceutical composition for treating galectin-4-positive gastric cancer.
日本において、胃がんは年間罹患数が約13万人、そして年間死亡者数が約5万人であり、癌の中で第3位と頻度が高い。特に、転移や再発をきたし切除することができない場合は、非常に予後が悪い癌である。胃がんの予後に大きく関わる転移として、腹膜播種、リンパ節転移、血行性転移という3つの経路が存在する。現在、転移を有する胃がんは癌診療ガイドライン上でも「ステージIV胃がん」として扱われている。例えば腹膜播種を起こす胃がんにおいて、ガレクチン-4との関連が報告されている(非特許文献1~3)。しかしながら、それぞれの転移経路に特異的な診断マーカーや分子標的治療薬は存在しない。従って、化学療法と外科的な癌組織切除とを組み合わせた治療が行われている。転移が無い限局性の癌では5年生存率が95%あるが、活発に転移し全身に拡散する遠隔転移性の胃がんでは5年生存率は3%と言われている。 In Japan, stomach cancer has an annual incidence of about 130,000 people and an annual death rate of about 50,000 people, ranking third among cancers. In particular, cancer that has metastasized or recurred and cannot be removed is a cancer with a very poor prognosis. There are three routes of metastasis that greatly affect the prognosis of gastric cancer: peritoneal dissemination, lymph node metastasis, and hematogenous metastasis. Currently, gastric cancer that has metastasized is treated as "Stage IV gastric cancer" in cancer treatment guidelines. For example, a relationship with galectin-4 has been reported in gastric cancer that causes peritoneal dissemination (Non-patent Documents 1 to 3). However, there are no diagnostic markers or molecular target therapeutics specific to each metastatic pathway. Therefore, treatment that combines chemotherapy and surgical removal of cancerous tissue is being performed. Localized cancer that has not metastasized has a 5-year survival rate of 95%, but distant metastatic gastric cancer that has actively metastasized and spread throughout the body is said to have a 5-year survival rate of 3%.
本発明の目的は、胃がんに有効な治療薬を提供することである。 An object of the present invention is to provide an effective therapeutic agent for gastric cancer.
本発明者は、胃がんに有効な治療薬について、鋭意研究した結果、驚くべきことに、ガレクチン-4の阻害剤によって、胃がんを効率的に治療できることを見出した。
本発明は、こうした知見に基づくものである。
従って、本発明は、
[1]ガレクチン-4阻害剤を有効成分として含む、ガレクチン-4陽性胃がん治療用医薬組成物、
[2]前記ガレクチン-4阻害剤が、遺伝子に対してRNAi効果を有する二本鎖核酸である、[1]に記載の胃がん治療用医薬組成物、及び
[3]前記胃がんが、腹膜播種能を有する胃がんである、[1]又は[2]に記載の胃がん治療用医薬組成物、
に関する。
As a result of intensive research into therapeutic agents effective for gastric cancer, the present inventor surprisingly discovered that gastric cancer can be efficiently treated with a galectin-4 inhibitor.
The present invention is based on these findings.
Therefore, the present invention
[1] A galectin-4-positive pharmaceutical composition for treating gastric cancer containing a galectin-4 inhibitor as an active ingredient,
[2] The pharmaceutical composition for gastric cancer treatment according to [1], wherein the galectin-4 inhibitor is a double-stranded nucleic acid that has an RNAi effect on genes, and [3] the gastric cancer has peritoneal dissemination ability. The pharmaceutical composition for gastric cancer treatment according to [1] or [2], which is gastric cancer having
Regarding.
本発明の医薬組成物によれば、ガレクチン-4陽性胃がんを効率的に治療することができる。 According to the pharmaceutical composition of the present invention, galectin-4-positive gastric cancer can be efficiently treated.
《医薬組成物》
本発明のガレクチン-4陽性胃がん治療用医薬組成物は、ガレクチン-4阻害剤を有効成分として含む。ガレクチン-4阻害剤としては、特に限定されるものではないが、ガレクチン-4タンパク質の機能を抑制する成分、又はガレクチン-4遺伝子の機能を抑制する成分が挙げられる。
《Pharmaceutical composition》
The galectin-4-positive pharmaceutical composition for gastric cancer treatment of the present invention contains a galectin-4 inhibitor as an active ingredient. Galectin-4 inhibitors include, but are not particularly limited to, components that suppress the function of galectin-4 protein or components that suppress the function of galectin-4 gene.
(ガレクチン-4)
ガレクチンは、β-ガラクトシドを認識するレクチンであり、15種類のガレクチンファミリーが知られている。ガレクチン-4は、2つの糖結合領域を有するタンデムリピート型のガレクチンである。ガレクチン-4は、小腸及び大腸で強く発現していることが報告されているが、大腸がんにおいて、ガレクチン-4の発現が低下することが報告されている。
(Galectin-4)
Galectin is a lectin that recognizes β-galactoside, and 15 types of galectin families are known. Galectin-4 is a tandem repeat type galectin that has two sugar-binding regions. It has been reported that galectin-4 is strongly expressed in the small intestine and large intestine, but it has been reported that the expression of galectin-4 is decreased in colon cancer.
本発明の医薬組成物の対象となる胃がんは、ガレクチン-4陽性の胃がんである。ガレクチン-4が陽性であることによって、ガレクチン-4阻害剤によって、癌細胞の増殖を抑制することができる。ガレクチン-4陽性の胃がん細胞は、胃に限局した胃がん細胞、リンパ節転移を示す胃がん細胞、又は血行性転移を示す胃がん細胞、が挙げられる。限定されるものではないが、ガレクチン-4陽性の胃がんとしては、腹膜播種能を有する胃がんが多い。 Gastric cancer targeted by the pharmaceutical composition of the present invention is galectin-4 positive gastric cancer. By being positive for galectin-4, the proliferation of cancer cells can be suppressed by a galectin-4 inhibitor. Examples of galectin-4-positive gastric cancer cells include gastric cancer cells localized to the stomach, gastric cancer cells showing lymph node metastasis, and gastric cancer cells showing hematogenous metastasis. Although not limited to, most galectin-4-positive gastric cancers are gastric cancers that have the ability to disseminate into the peritoneum.
(ガレクチン-4タンパク質の機能を抑制する成分)
ガレクチン-4タンパク質の機能を抑制する成分としては、例えばガレクチン-4タンパク質と結合して、ガレクチン-4タンパク質の機能を抑制する成分が挙げられる。このような成分としては、限定されるものではないが、硫酸デキストラン、ヘパリン、硫酸化糖脂質、又はコレステロール硫酸が挙げられる。
(component that suppresses the function of galectin-4 protein)
Examples of the component that suppresses the function of galectin-4 protein include a component that binds to galectin-4 protein and suppresses the function of galectin-4 protein. Such ingredients include, but are not limited to, dextran sulfate, heparin, sulfated glycolipids, or cholesterol sulfate.
(ガレクチン-4遺伝子の機能を抑制する成分)
ガレクチン-4遺伝子(配列番号1)の機能を抑制する成分としては、限定されるものではないが、ガレクチン-4遺伝子に対してRNAi効果を有する阻害性核酸としてdsRNA、siRNA、shRNA、mi-RNA、および人工miRNA等の干渉RNAが挙げられる。
前記二本鎖核酸は、ガレクチン-4遺伝子に対してRNAi効果を有する二本鎖核酸であり、所望のセンス鎖とアンチセンス鎖とがハイブリダイズしてなる二本鎖核酸領域を含む核酸分子を意味し、siRNA(small interfering RNA)であることが好ましい。
(component that suppresses galectin-4 gene function)
Components that suppress the function of the galectin-4 gene (SEQ ID NO: 1) include, but are not limited to, inhibitory nucleic acids that have an RNAi effect on the galectin-4 gene such as dsRNA, siRNA, shRNA, and mi-RNA. , and interfering RNAs such as artificial miRNAs.
The double-stranded nucleic acid is a double-stranded nucleic acid that has an RNAi effect on the galectin-4 gene, and is a nucleic acid molecule containing a double-stranded nucleic acid region formed by hybridizing a desired sense strand and an antisense strand. It is preferably siRNA (small interfering RNA).
(センス鎖及びアンチセンス鎖)
前記二本鎖核酸は、23塩基からなる配列番号1のいずれかの標的配列に対応する塩基配列を含むセンス鎖と、前記センス鎖に相補的な塩基配列を含むアンチセンス鎖とを含む。
ここで、「標的配列に対応する塩基配列」とは、標的配列と同一の塩基配列であるか、あるいは、前記標的配列において1若しくは数個(例えば、2~3個)の塩基が置換された塩基配列を意味する。二本鎖核酸がsiRNAである場合、1~数塩基のミスマッチを含んでいても、RNAi効果が得られることが知られている。本発明では、標的配列と同一の塩基配列だけでなく、RNAi効果が得られる限り、ミスマッチを含む塩基配列であってもよい。
(sense strand and antisense strand)
The double-stranded nucleic acid includes a sense strand containing a base sequence corresponding to any target sequence of SEQ ID NO: 1 consisting of 23 bases, and an antisense strand containing a base sequence complementary to the sense strand.
Here, the "base sequence corresponding to the target sequence" is the same base sequence as the target sequence, or one or several (for example, 2 to 3) bases have been substituted in the target sequence. means a base sequence. It is known that when the double-stranded nucleic acid is siRNA, an RNAi effect can be obtained even if it contains one to several base mismatches. In the present invention, not only a base sequence that is the same as the target sequence but also a base sequence containing a mismatch may be used as long as an RNAi effect can be obtained.
また、アンチセンス鎖における「センス鎖に相補的な塩基配列」とは、センス鎖とハイブリダイズすることができる程度に相補的な塩基配列であればよく、センス鎖に完全に相補的な塩基配列であるか、あるいは、前記センス鎖に完全に相補的な塩基配列において1若しくは数個(例えば、2~3個)の塩基が置換された塩基配列であることができる。 In addition, the "nucleotide sequence complementary to the sense strand" in the antisense strand may be any nucleotide sequence that is complementary to the extent that it can hybridize with the sense strand, and is a nucleotide sequence that is completely complementary to the sense strand. Alternatively, it may be a base sequence in which one or several (for example, 2 to 3) bases are substituted in a base sequence that is completely complementary to the sense strand.
(核酸の種類と修飾)
二本鎖核酸を構成する核酸の種類は、特に限定されるものではなく、適宜選択することができ、例えば、二本鎖RNA、DNA-RNAキメラ型二本鎖核酸を挙げることができる。キメラ型はRNAi効果を有する二本鎖RNAの一部をDNAに換えたものであり、血清中での安定性が高く、免疫応答誘導性が低いことが知られている。
また、二本鎖核酸は、例えば、2’-OH基の修飾、バックボーンのホスホロチオエートによる置換やボラノホスフェート基による修飾、リボースの2位と4位が架橋されたLNA(locked nucleic acid)の導入などによって、ヌクレアーゼに対する耐性や安定化を高めることもできる。あるいは、細胞への導入効率を高める等の目的から、二本鎖核酸のセンス鎖の5’端、或いは3’端に、例えば、ナノ粒子、コレステロール、細胞膜通過ペプチド等の修飾を施すこともできる。
(Nucleic acid types and modifications)
The type of nucleic acid constituting the double-stranded nucleic acid is not particularly limited and can be selected as appropriate, and includes, for example, double-stranded RNA and DNA-RNA chimera type double-stranded nucleic acid. The chimeric type is a double-stranded RNA that has an RNAi effect, with part of it replaced with DNA, and is known to have high stability in serum and low immune response induction.
In addition, double-stranded nucleic acids can be produced by, for example, modification of the 2'-OH group, substitution of the backbone with phosphorothioate, modification with boranophosphate group, introduction of LNA (locked nucleic acid) in which the 2- and 4-positions of ribose are cross-linked. It is also possible to increase resistance to nucleases and stability. Alternatively, for the purpose of increasing the efficiency of introduction into cells, the 5' or 3' end of the sense strand of the double-stranded nucleic acid can be modified with nanoparticles, cholesterol, cell membrane-transmitting peptides, etc. .
(siRNA)
二本鎖RNAは、siRNA(キメラ型を含む)であることが好ましい。ここで、「siRNA」とは、18塩基長~29塩基長の小分子二本鎖RNAであり、前記siRNAのアンチセンス鎖(ガイド鎖)と相補的な配列をもつ標的遺伝子のmRNAを切断し、標的遺伝子の発現を抑制する機能を有する。
前記siRNAは、先述したようなセンス鎖及びアンチセンス鎖を含み、かつ所望のRNAi効果を示すものであれば、その末端構造に特に制限はなく、適宜選択することができ、例えば、前記siRNAは、平滑末端を有するものであってもよいし、突出末端(オーバーハング)を有するものであってもよい。中でも、前記siRNAは、各鎖の3’末端が2塩基~6塩基突出した構造を有することが好ましく、各鎖の3’末端が2塩基突出した構造を有することがより好ましい。
(siRNA)
The double-stranded RNA is preferably siRNA (including chimeric type). Here, "siRNA" is a small double-stranded RNA with a length of 18 to 29 bases, which cleaves the mRNA of a target gene that has a complementary sequence to the antisense strand (guide strand) of the siRNA. , has the function of suppressing the expression of target genes.
The terminal structure of the siRNA is not particularly limited as long as it contains the sense strand and antisense strand as described above and exhibits the desired RNAi effect, and can be appropriately selected. For example, the siRNA may be , may have blunt ends, or may have protruding ends (overhangs). Among these, the siRNA preferably has a structure in which the 3' end of each strand protrudes by 2 to 6 bases, and more preferably has a structure in which the 3' end of each strand protrudes by 2 bases.
本発明のsiRNAとしては、例えば、配列番号1を標的配列とする配列番号3(5’-CCGGACAUUGCCAUCAACAGCUGAA-3’)のセンス鎖と配列番号4(5’-UUCAGCUGUUGAUGGCAAUGUCCGG-3’)のアンチセンス鎖とからなるsiRNAを挙げることができる。 The siRNA of the present invention includes, for example, the sense strand of SEQ ID NO: 3 (5'-CCGGACAUUGCCAUCAACAGCUGAA-3') whose target sequence is SEQ ID NO: 1, and the antisense strand of SEQ ID NO: 4 (5'-UUCAGCUGUUGAUGGCAAUGUCCGG-3'). Examples include siRNA consisting of
(製造方法)
前記二本鎖RNA(特にはsiRNA)は、従来公知の手法に基づき作製することができる。
例えば、所望のセンス鎖とアンチセンス鎖とに相当する18塩基長~29塩基長の一本鎖RNAを、それぞれ既存のDNA/RNA自動合成装置等を利用して化学的に合成し、それらをアニーリングすることにより作製することができる。また、後述する本発明のベクターのような、所望のsiRNA発現ベクターを構築し、前記発現ベクターを細胞内に導入することにより、細胞内の反応を利用してsiRNAを作製することもできる。
(Production method)
The double-stranded RNA (particularly siRNA) can be produced based on conventionally known techniques.
For example, single-stranded RNAs of 18 to 29 bases in length corresponding to the desired sense strand and antisense strand are chemically synthesized using existing automatic DNA/RNA synthesizers, respectively, and It can be produced by annealing. Furthermore, siRNA can also be produced by utilizing intracellular reactions by constructing a desired siRNA expression vector, such as the vector of the present invention described below, and introducing the expression vector into cells.
(DNA、発現ベクター)
DNAは、先述した、前記二本鎖核酸(特にはsiRNA)をコードする塩基配列を含むことを特徴とするDNAであり、発現ベクターは、前記DNAを含むことを特徴とする発現ベクターである。
(DNA, expression vector)
The DNA is a DNA characterized by containing the aforementioned double-stranded nucleic acid (particularly siRNA) encoding base sequence, and the expression vector is an expression vector characterized by containing the aforementioned DNA.
(DNA)
前記DNAとしては、先述した、前記二本鎖核酸をコードする塩基配列を含むDNAであれば特に制限はなく、目的に応じて適宜選択することができるが、前記二本鎖核酸をコードする塩基配列の上流(5’側)に、前記二本鎖核酸の転写を制御するためのプロモーター配列が連結されていることが好ましい。前記プロモーター配列としては、特に制限はなく、適宜選択することができ、例えば、CMVプロモーター等のpol II系プロモーター、H1プロモーター、U6プロモーター等のpol III系プロモーターなどが挙げられる。
(DNA)
The DNA is not particularly limited as long as it includes the base sequence encoding the double-stranded nucleic acid mentioned above, and can be appropriately selected depending on the purpose; It is preferable that a promoter sequence for controlling transcription of the double-stranded nucleic acid is linked upstream (5' side) of the sequence. The promoter sequence is not particularly limited and can be selected as appropriate, and includes, for example, pol II promoters such as CMV promoter, pol III promoters such as H1 promoter, and U6 promoter.
また、更に、前記二本鎖核酸をコードする塩基配列の下流(3’側)に、前記二本鎖核酸の転写を終結させるためのターミネーター配列が連結されていることがより好ましい。前記ターミネーター配列としても、特に制限はなく、目的に応じて適宜選択することができる。 Furthermore, it is more preferable that a terminator sequence for terminating the transcription of the double-stranded nucleic acid is linked downstream (3' side) of the base sequence encoding the double-stranded nucleic acid. The terminator arrangement is not particularly limited and can be appropriately selected depending on the purpose.
前記プロモーター配列、前記二本鎖核酸をコードするヌクレオチド配列、及び前記ターミネーター配列を含む転写ユニットは、前記DNAにおける好ましい一態様である。なお、前記転写ユニットは、従来公知の手法を用いて構築することができる。 A transcription unit including the promoter sequence, the nucleotide sequence encoding the double-stranded nucleic acid, and the terminator sequence is a preferred embodiment of the DNA. Note that the transfer unit can be constructed using a conventionally known method.
(発現ベクター)
前記ベクターとしては、前記DNAを含むものであれば特に制限はなく、目的に応じて適宜選択することができ、例えば、プラスミドベクター、ウイルスベクターなどが挙げられる。前記ベクターは、前記二本鎖核酸(特にsiRNA)を発現可能な発現ベクターであることが好ましい。
前記二本鎖核酸の発現様式としては、特に制限はなく、目的に応じて適宜選択することができ、例えば二本鎖核酸としてsiRNAを発現させる方法として、短い一本鎖RNAを二本発現させる方法(タンデム型)、shRNA(short hairpin RNA)として一本鎖RNAを発現させる方法(ヘアピン型)等が挙げられる。ここで、shRNAとは、18塩基~29塩基程度のdsRNA領域と3塩基~9塩基程度のloop領域を含む一本鎖RNAであるが、shRNAは、生体内で発現されることにより、塩基対を形成してヘアピン状の二本鎖RNAとなる。その後、shRNAはDicer(RNase III酵素)により切断されてsiRNAとなり、標的遺伝子の発現抑制に機能することができる。
(expression vector)
The vector is not particularly limited as long as it contains the DNA, and can be appropriately selected depending on the purpose. Examples include plasmid vectors, virus vectors, and the like. The vector is preferably an expression vector capable of expressing the double-stranded nucleic acid (particularly siRNA).
The expression mode of the double-stranded nucleic acid is not particularly limited and can be appropriately selected depending on the purpose. For example, as a method for expressing siRNA as a double-stranded nucleic acid, two short single-stranded RNAs are expressed. Examples include a method (tandem type), a method of expressing single-stranded RNA as shRNA (short hairpin RNA) (hairpin type), and the like. Here, shRNA is a single-stranded RNA that includes a dsRNA region of about 18 to 29 bases and a loop region of about 3 to 9 bases. to form a hairpin-shaped double-stranded RNA. Thereafter, the shRNA is cleaved by Dicer (RNase III enzyme) to become siRNA, which can function to suppress the expression of the target gene.
前記タンデム型siRNA発現ベクターは、前記siRNAを構成するセンス鎖をコードするDNA配列と、アンチセンス鎖をコードするDNA配列とを含み、かつ、各鎖をコードするDNA配列の上流(5’側)にプロモーター配列がそれぞれ連結され、また、各鎖をコードするDNA配列の下流(3’側)にターミネーター配列がそれぞれ連結されたDNAを含む。 The tandem siRNA expression vector includes a DNA sequence encoding a sense strand and a DNA sequence encoding an antisense strand constituting the siRNA, and upstream (5' side) of the DNA sequence encoding each strand. A promoter sequence is linked to each chain, and a terminator sequence is linked to the downstream (3' side) of the DNA sequence encoding each chain.
また、前記ヘアピン型siRNA発現ベクターは、前記siRNAを構成するセンス鎖をコードするDNA配列と、アンチセンス鎖をコードするDNA配列とが逆向きに配置され、前記センス鎖DNA配列とアンチセンス鎖DNA配列とがループ配列を介して接続されており、かつ、それらの上流(5’側)にプロモーター配列が、また、下流(3’側)にターミネーター配列が連結されたDNAを含む。
前記各ベクターは、従来公知の手法を用いて構築することができ、例えば、前記DNAを、予め制限酵素で切断したベクターの切断部位に連結(ライゲーション)することにより構築することができる。
Further, in the hairpin-type siRNA expression vector, the DNA sequence encoding the sense strand and the DNA sequence encoding the antisense strand constituting the siRNA are arranged in opposite directions, and the sense strand DNA sequence and the antisense strand DNA are arranged in opposite directions. sequences are connected via a loop sequence, and a promoter sequence is connected upstream (5' side) of these sequences, and a terminator sequence is connected downstream (3' side) thereof.
Each of the vectors can be constructed using a conventionally known method, for example, by ligating the DNA to a cleavage site of a vector that has been previously cleaved with a restriction enzyme.
前記DNA又は前記ベクターを細胞に導入(トランスフェクト)することにより、プロモーターが活性化され、前記二本鎖核酸を生成することができる。例えば、前記タンデム型ベクターにおいては、前記DNAが細胞内で転写されることにより、センス鎖及びアンチセンス鎖が生成され、それらがハイブリダイズすることによりsiRNAが生成される。前記ヘアピン型ベクターにおいては、前記DNAが細胞内で転写されることにより、まずヘアピン型RNA(shRNA)が生成され、次いで、ダイサーによるプロセシングにより、siRNAが生成される。 By introducing (transfecting) the DNA or the vector into cells, the promoter is activated and the double-stranded nucleic acid can be produced. For example, in the tandem vector, the DNA is transcribed within cells to generate a sense strand and an antisense strand, and siRNA is generated by hybridization of these strands. In the hairpin vector, hairpin RNA (shRNA) is first generated by intracellular transcription of the DNA, and then siRNA is generated by processing by Dicer.
(miRNA)
本発明の医薬組成物は、miRNAを含むことができる。miRNA(microRNA)は、20~25塩基長の1本鎖RNA(成熟miRNA)であり、遺伝子の転写後発現調節に関与する。miRNAの産生過程では、初めにRNAポリメラーゼIIによる転写産物(Primary-miRNA;Pri-miRNA)が生じ、次にPri-miRNAは切断されステムループ配列を有するmiRNA前駆体(Pre-miRNA)を生じ、さらにDicerで切断されることにより成熟miRNAが生じる。このmiRNAを補充する方法として、内在性microRNAの機能を効果的に模倣し機能増加するようにデザインされた化学合成したmiRNA mimicを使用する方法、miRNA発現プラスミドを使用する方法等を用いることができる。
(miRNA)
Pharmaceutical compositions of the invention can include miRNA. miRNA (microRNA) is a single-stranded RNA (mature miRNA) with a length of 20 to 25 bases, and is involved in post-transcriptional expression regulation of genes. In the miRNA production process, a transcription product (Primary-miRNA; Pri-miRNA) is first produced by RNA polymerase II, and then Pri-miRNA is cleaved to produce a miRNA precursor (Pre-miRNA) having a stem-loop sequence. Further cleavage with Dicer produces mature miRNA. As a method for replenishing this miRNA, a method using a chemically synthesized miRNA mimic designed to effectively mimic and increase the function of endogenous microRNA, a method using a miRNA expression plasmid, etc. can be used. .
(アンチセンスオリゴ)
本発明の医薬組成物は、アンチセンスオリゴを含むことができる。アンチセンスオリゴは標的と相補的な配列を有する一本鎖DNAやRNAで、タンパク質への発現を抑制するはたらきを持つ。核酸に様々な化学修飾を導入することで、安定性や機能などを追加することができる。その一つの例としてモルフォリノアンチセンスオリゴは、モルフォリノ環を有するオリゴDNAであり、RNAに対する親和性が高く、そしてDNA分解酵素に対して抵抗性を有する。ガレクチン-4の5′非翻訳領域、またはエキソンとイントロンとの境界領域に相同性をもつように設計することによって、ガレクチン-4のmRNAからの翻訳やスプライシングを抑制することができる。
(antisense oligo)
Pharmaceutical compositions of the invention can include antisense oligos. Antisense oligos are single-stranded DNA or RNA that have a sequence complementary to the target, and have the function of suppressing protein expression. By introducing various chemical modifications to nucleic acids, stability and functionality can be added. As one example, a morpholino antisense oligo is an oligo DNA having a morpholino ring, has a high affinity for RNA, and is resistant to DNA degrading enzymes. By designing homology to the 5' untranslated region of galectin-4 or the exon-intron boundary region, translation and splicing from galectin-4 mRNA can be inhibited.
本発明のガレクチン-4陽性胃がん治療用医薬組成物は、ガレクチン-4タンパク質の機能を抑制する成分(例えば、硫酸デキストラン、ヘパリン、硫酸化糖脂質、又はコレステロール硫酸)、又はガレクチン-4遺伝子の機能を抑制する成分(例えば、前記二本鎖核酸、DNA、又はベクター)を有効成分として含み、更に必要に応じてその他の成分を含むことができる。 The galectin-4-positive pharmaceutical composition for gastric cancer treatment of the present invention contains a component that suppresses the function of galectin-4 protein (for example, dextran sulfate, heparin, sulfated glycolipid, or cholesterol sulfate) or the function of galectin-4 gene. (for example, the double-stranded nucleic acid, DNA, or vector) as an active ingredient, and may further contain other components as necessary.
(剤形、投与方法)
前記医薬組成物の剤型としては、特に制限はなく、所望の投与方法に応じて適宜選択することができ、例えば、経口固形剤(錠剤、被覆錠剤、顆粒剤、散剤、カプセル剤等)、経口液剤(内服液剤、シロップ剤、エリキシル剤等)、注射剤(溶液、懸濁液、用事溶解用固形剤等)、軟膏剤、貼付剤、ゲル剤、クリーム剤、外用散剤、スプレー剤、吸入散剤などが挙げられる。
また、有効成分以外のその他の成分として、所望の医薬添加物、例えば、賦形剤、結合剤、崩壊剤、滑沢剤、着色剤、矯味・矯臭剤等を含むことができる。
(Dosage form, administration method)
The dosage form of the pharmaceutical composition is not particularly limited and can be appropriately selected depending on the desired administration method, such as oral solid dosage forms (tablets, coated tablets, granules, powders, capsules, etc.), Oral solutions (oral solutions, syrups, elixirs, etc.), injections (solutions, suspensions, solid preparations for dissolution, etc.), ointments, patches, gels, creams, external powders, sprays, inhalers Examples include powders.
In addition, other ingredients other than the active ingredient may include desired pharmaceutical additives, such as excipients, binders, disintegrants, lubricants, coloring agents, flavoring agents, and the like.
前記医薬組成物の投与方法としては、特に制限はなく、例えば、前記医薬組成物の剤型、疾患の種類、患者の状態等に応じて、局所投与、全身投与のいずれかを選択することができる。例えば、局所投与においては、前記医薬を、所望の部位(例えば、腫瘍部位)に直接注入することにより投与することができる。前記注入には、注射等の従来公知の手法を適宜利用することができる。また、全身投与(例えば、経口投与、腹腔内投与、血液中への投与等)においては、前記医薬の有効成分が所望の部位(例えば、腫瘍部位)まで安定に、かつ効率良く送達されるよう、従来公知の薬剤送達技術を適宜応用することが好ましい。 There are no particular restrictions on the method of administering the pharmaceutical composition; for example, local administration or systemic administration may be selected depending on the dosage form of the pharmaceutical composition, the type of disease, the patient's condition, etc. can. For example, for local administration, the medicament can be administered by injection directly into the desired site (eg, tumor site). For the injection, conventionally known techniques such as injection can be used as appropriate. In addition, in systemic administration (e.g., oral administration, intraperitoneal administration, administration into the blood, etc.), the active ingredient of the drug is stably and efficiently delivered to the desired site (e.g., tumor site). It is preferable to appropriately apply conventionally known drug delivery techniques.
前記医薬組成物の投与量としては、特に制限はなく、投与対象である患者の年齢、体重、所望の効果の程度等に応じて適宜選択することができるが、例えば、成人への1日の投与あたり、例えば二本鎖核酸の量として、1mg~100mgが好ましい。
また、前記医薬組成物の投与回数としても、特に制限はなく、投与対象である患者の年齢、体重、所望の効果の程度等に応じて、適宜選択することができる。
The dosage of the pharmaceutical composition is not particularly limited and can be appropriately selected depending on the age, body weight, degree of desired effect, etc. of the patient to whom it is administered. For example, the amount of double-stranded nucleic acid per administration is preferably 1 mg to 100 mg.
Furthermore, the number of times the pharmaceutical composition is administered is not particularly limited, and can be appropriately selected depending on the age, body weight, degree of desired effect, etc. of the patient to be administered.
《ガレクチン-4陽性胃がんの治療方法》
本発明は、ガレクチン-4陽性胃がんの治療の必要がある対象に、その有効量でガレクチン-4タンパク質の機能を抑制する成分(例えば、硫酸デキストラン、ヘパリン、硫酸化糖脂質、又はコレステロール硫酸)、又はガレクチン-4遺伝子の機能を抑制する成分(例えば、前記二本鎖核酸、DNA、又はベクター)を投与することを含む、ガレクチン-4陽性胃がんの治療または予防方法を提供する。
《Treatment method for galectin-4 positive gastric cancer》
The present invention provides effective doses of ingredients that suppress the function of galectin-4 proteins (for example, dextran sulfate, heparin, sulfated glycolipids, or cholesterol sulfate) to subjects in need of treatment for galectin-4-positive gastric cancer. Alternatively, the present invention provides a method for treating or preventing galectin-4-positive gastric cancer, which comprises administering a component (eg, the double-stranded nucleic acid, DNA, or vector) that suppresses the function of the galectin-4 gene.
《ガレクチン-4陽性胃がん用医薬組成物の製造への使用》
本発明は、ガレクチン-4タンパク質の機能を抑制する成分(例えば、硫酸デキストラン、ヘパリン、硫酸化糖脂質、又はコレステロール硫酸)、又はガレクチン-4遺伝子の機能を抑制する成分(例えば、前記二本鎖核酸、DNA、又はベクター)の、ガレクチン-4陽性胃がんの治療用医薬組成物の製造に使用することができる。
《Use for manufacturing galectin-4 positive pharmaceutical composition for gastric cancer》
The present invention provides a component that suppresses the function of galectin-4 protein (for example, dextran sulfate, heparin, sulfated glycolipid, or cholesterol sulfate), or a component that suppresses the function of galectin-4 gene (for example, the double-stranded (nucleic acid, DNA, or vector) can be used to produce a pharmaceutical composition for treating galectin-4-positive gastric cancer.
《ガレクチン-4陽性胃がん治療用のガレクチン-4阻害剤》
本発明は、ガレクチン-4陽性胃がん治療用のガレクチン-4阻害剤に関する。ガレクチン-4阻害剤としては、前記の通りガレクチン-4タンパク質の機能を抑制する成分(例えば、硫酸デキストラン、ヘパリン、硫酸化糖脂質、又はコレステロール硫酸)、又はガレクチン-4遺伝子の機能を抑制する成分(例えば、前記二本鎖核酸、DNA、又はベクター)が挙げられる。
《Galectin-4 inhibitor for treatment of galectin-4 positive gastric cancer》
The present invention relates to a galectin-4 inhibitor for the treatment of galectin-4-positive gastric cancer. As described above, galectin-4 inhibitors include components that suppress the function of galectin-4 protein (for example, dextran sulfate, heparin, sulfated glycolipids, or cholesterol sulfate), or components that suppress the function of galectin-4 gene. (For example, the double-stranded nucleic acid, DNA, or vector).
《作用》
本発明の医薬組成物が、ガレクチン-4陽性胃がんの治療、特に増殖を抑制できる理由は、詳細には検討されていない。しかしながら、以下のように推定することができる。但し、本発明は以下の推定によって、限定されるものではない。
ガレクチン-4遺伝子の機能を抑制する成分は、ガレクチン-4のmRNAの発現を抑制することによって、癌細胞の増殖を抑制すると考えられる。ガレクチン-4陽性胃がん細胞の増殖には、ガレクチン-4の発現が関与していると考えられ、ガレクチン-4の発現をmRNAのレベルで抑制することによって、ガレクチン-4陽性胃がん細胞の増殖を抑制できると推定される。
一方、ガレクチン-4タンパク質の機能を抑制する成分は、ガレクチン-4タンパク質に結合することによって、ガレクチン-4陽性胃がん細胞の増殖を抑制できると考えられる。ガレクチン-4は癌細胞の悪性度に関係する分子群(例えばcMET、CD44等)に糖結合ドメインを通じて結合することによって、細胞表面への局在を安定化し、ガレクチン-4が-複合糖鎖(糖タンパク質または糖脂質)格子を形成し、格子内に存在する受容体の側方拡散を拘束することにより、受容体の凝集や情報伝達を起こす閾値を高めることが推定される。ガレクチン-4タンパク質の機能を抑制する成分は、ガレクチン-4に結合することでガレクチン-4によって発現や局在が制御されている分子への結合を阻害することができる。具体的には、ガレクチン-4の結合阻害によってcMETの活性化を阻害する(すなわちpMETの抑制)ことにより増殖を抑制していると考えられる。
《Effect》
The reason why the pharmaceutical composition of the present invention is able to treat galectin-4-positive gastric cancer, particularly suppress its proliferation, has not been investigated in detail. However, it can be estimated as follows. However, the present invention is not limited by the following estimation.
A component that suppresses the function of the galectin-4 gene is thought to suppress the proliferation of cancer cells by suppressing the expression of galectin-4 mRNA. Galectin-4 expression is thought to be involved in the proliferation of galectin-4-positive gastric cancer cells, and by suppressing galectin-4 expression at the mRNA level, the proliferation of galectin-4-positive gastric cancer cells is suppressed. It is estimated that it can be done.
On the other hand, a component that suppresses the function of galectin-4 protein is thought to be able to suppress the proliferation of galectin-4-positive gastric cancer cells by binding to galectin-4 protein. Galectin-4 stabilizes its localization on the cell surface by binding to molecules related to the malignancy of cancer cells (e.g., cMET, CD44, etc.) through the sugar-binding domain, It is presumed that by forming a lattice (glycoprotein or glycolipid) and restricting the lateral diffusion of receptors present within the lattice, the threshold for receptor aggregation and signal transmission is increased. A component that suppresses the function of galectin-4 protein can inhibit binding to molecules whose expression and localization are controlled by galectin-4 by binding to galectin-4. Specifically, proliferation is thought to be suppressed by inhibiting cMET activation (ie, pMET suppression) by inhibiting galectin-4 binding.
以下、実施例によって本発明を具体的に説明するが、これらは本発明の範囲を限定するものではない。 EXAMPLES Hereinafter, the present invention will be specifically explained with reference to Examples, but these are not intended to limit the scope of the present invention.
《実施例1》
本実施例では、ガレクチン-4陽性胃がん細胞(NUGC4細胞)のガレクチン-4をノックアウトした細胞を取得した。
ガレクチン-4遺伝子のノックアウトは、ホライゾンディスカバリー社のDharmacon Edit-R CRISPR-Cas9ゲノム編集試薬システムによるゲノム編集ツールを利用して行った。Puromycin耐性遺伝子を含んだEdit-R hCMV-PuroR-Cas9 Expression Plasmid、Edit-R Human LGALS4(3960) crRNA(CM-012232-05)とEdit-R CRISPR-Cas9 Synthetic tracrRNAをDharmaFECT Duo Transfection Reagentを用いてNUGC4細胞に共導入した。Puromycinを培地に添加することによりノックアウト細胞を濃縮し、さらにコロニーをピックアップして、ガレクチン-4の発現細胞が混入していないクローンを蛍光免疫染色法で選別した。ガレクチン-4の発現が認められないノックアウト株クローン、KO-1a、KO-1b、及びKO-2を選択した。ノンコーディング遺伝子に対してデザインされたEdit-R crRNA Non-targeting Control #1をガレクチン-4用にデザインされたcrRNAの代わりに用いて同様の操作を行い、対照株C1を樹立した。
《Example 1》
In this example, galectin-4-positive gastric cancer cells (NUGC4 cells) in which galectin-4 was knocked out were obtained.
The galectin-4 gene was knocked out using a genome editing tool using Horizon Discovery's Dharmacon Edit-R CRISPR-Cas9 genome editing reagent system. Edit-R hCMV-PuroR-Cas9 Expression Plasmid containing Puromycin resistance gene, Edit-R Human LGALS4 (3960) crRNA (CM-012232-05) and Edit-R CRISPR-Cas9 Sy nthetic tracrRNA using DharmaFECT Duo Transfection Reagent Cotransfected into NUGC4 cells. Knockout cells were concentrated by adding Puromycin to the medium, colonies were picked up, and clones that were not contaminated with cells expressing galectin-4 were selected by fluorescent immunostaining. Knockout strain clones, KO-1a, KO-1b, and KO-2, in which galectin-4 expression was not observed were selected. A control strain C1 was established by performing the same operation using Edit-R crRNA Non-targeting Control #1 designed for a non-coding gene instead of the crRNA designed for galectin-4.
ノックアウト細胞各クローンからRNAを抽出し逆転写してcDNAを取得後、塩基配列を調べた結果を図1に示す。
ノックアウト細胞株ではターゲットとしたcrRNA部位近傍に塩基対の欠失が確認された。ノックアウト株KO-1a及びKO-1bは同じ遺伝子配列を持ち、ノックアウト株KO-2の欠失範囲はKO-1aやKO-1bよりも狭かった。
図1aのように遺伝子配列から推定されるアミノ酸配列の欠失はそれぞれ45アミノ酸及び12アミノ酸である。ウェスタンブロット法で、ガレクチン-4の発現を調べたところ、それぞれのクローンは、ガレクチン-4タンパク質の発現は認められなかった(図1b)。
After RNA was extracted from each knockout cell clone and reverse transcribed to obtain cDNA, the base sequence was examined and the results are shown in FIG.
In the knockout cell line, deletion of base pairs was confirmed near the targeted crRNA site. Knockout strains KO-1a and KO-1b had the same gene sequence, and the deletion range of knockout strain KO-2 was narrower than that of KO-1a and KO-1b.
As shown in Figure 1a, the deletions in the amino acid sequence deduced from the gene sequence are 45 amino acids and 12 amino acids, respectively. When the expression of galectin-4 was examined by Western blotting, no expression of galectin-4 protein was observed in each clone (Fig. 1b).
(ノックアウト細胞株の増殖能)
前記ノックアウト細胞株の増殖能を、細胞倍化時間によって測定した。
(Proliferation ability of knockout cell line)
The proliferation ability of the knockout cell line was measured by cell doubling time.
表1に示すように、ノックアウト株の細胞倍化時間は親株、対照株に比べ長く、増殖能が低下していた。また、親株(Wild)とKO-2株の増殖カーブを図2に示す。KO-2株の増殖カーブは、親株と比較して、有意に低下していた。図中の**はp<0.01を示し、**は2つのグループの間で統計学的に著しく有意な差があることを示している。 As shown in Table 1, the cell doubling time of the knockout strain was longer than that of the parent strain and the control strain, and the proliferation ability was decreased. Furthermore, the growth curves of the parent strain (Wild) and the KO-2 strain are shown in FIG. The growth curve of the KO-2 strain was significantly lower than that of the parent strain. ** in the figure indicates p<0.01, and ** indicates a statistically significant difference between the two groups.
(ノックアウト細胞株の接着非依存性増殖能)
ノックアウト細胞株の接着非依存性増殖能をGILAアッセイで測定した。接着非依存性増殖能とは細胞外基質に接着しなくても生存し増殖できる細胞の性質である。
GILAアッセイは、細胞培養用96ウェルプレート(No.3704、コーニング社)及び超低吸着96ウェルプレート(No.3474、コーニング社)に1000個/wellで細胞を播種し、5日間培養後、それぞれのプレートの生細胞に由来するATP量を測定した。ATP量は、CellTiter-Glo(登録商標)2.0試薬(プロメガ社)を100μL/wellで添加後、反応液を96ウェル白プレート(No.3362、コーニング社)に移し、発光量をTristar2 LB942プレートリーダー(ベルトールド社)で測定した。超低吸着プレート上の細胞のATP量と細胞培養用プレート上の細胞のATP量の比をLow/High比とした。
図3に示すように、対照株C1に比較して、ノックアウト細胞株ではLow/High比が低下し、接着非依存性増殖能が低下していることが示された。
(Adhesion-independent proliferation ability of knockout cell line)
The adhesion-independent proliferation ability of the knockout cell line was measured by GILA assay. Adhesion-independent growth capacity is the property of cells that can survive and proliferate without adhesion to extracellular matrix.
In the GILA assay, cells were seeded at 1000 cells/well in a 96-well plate for cell culture (No. 3704, Corning Inc.) and an ultra-low adsorption 96-well plate (No. 3474, Corning Inc.), and after culturing for 5 days, each The amount of ATP derived from living cells of the plate was measured. The amount of ATP was determined by adding CellTiter-Glo (registered trademark) 2.0 reagent (Promega) at 100 μL/well, then transferring the reaction solution to a 96-well white plate (No. 3362, Corning), and measuring the amount of luminescence using Tristar2 LB942. Measurement was performed using a plate reader (Berthold). The ratio of the ATP amount of the cells on the ultra-low adsorption plate to the ATP amount of the cells on the cell culture plate was defined as the Low/High ratio.
As shown in FIG. 3, the knockout cell line had a lower Low/High ratio, indicating a lower adhesion-independent growth ability, compared to the control line C1.
(マウス腹膜播種モデルを用いた解析)
ノックアウト細胞株で腹膜播種を、マウスモデルを用いて解析した。免疫不全マウス(BALBc nu/nu、オス、6週齢)に、1×106個のヒト胃癌細胞株NUGC4細胞を腹腔内に移植し、39日間飼育後開腹し腫瘍の数及び重量を計測した。
NUGC4細胞親株投与群は2匹、NUGC4対照株C1投与群は2匹、ノックアウト株KO-1a投与群は3匹、ノックアウト株KO-2投与群は3匹を使用し、各細胞を個体当たり1×106個/100~200μL RPMI1640 mediumで腹腔内に移植した。39日後に剖検を行った結果と、剖検時に腫瘍重量を測定した。
(Analysis using mouse peritoneal dissemination model)
Peritoneal dissemination with knockout cell lines was analyzed using a mouse model. Immunodeficient mice (BALBc nu/nu, male, 6 weeks old) were intraperitoneally transplanted with 1 x 10 6 human gastric cancer cell line NUGC4 cells, kept for 39 days, and then subjected to laparotomy to measure the number and weight of tumors. .
Two animals were used for the NUGC4 cell parent line administration group, two animals were used for the NUGC4 control line C1 administration group, three animals were used for the knockout strain KO-1a administration group, and three animals were used for the knockout strain KO-2 administration group. ×10 6 cells/100 to 200 μL were implanted intraperitoneally in RPMI1640 medium. The results of autopsy performed 39 days later and the weight of the tumor at the time of autopsy were measured.
表2及び図4に示すように、NUGC4胃癌細胞親株投与群及び対照株C1投与群はどちらも腹水の貯留が認められ、腹腔内に多数の腫瘍が観察されたが、ノックアウト株投与群のマウスにはどちらも腹腔内に腫瘍は認められなかった。図中の*はp<0.05を示し、*は2つのグループの間で腫瘍重量に統計学的に有意な差があることを示している。 As shown in Table 2 and Figure 4, ascites was observed in both the NUGC4 gastric cancer cell parent line administration group and the control cell C1 administration group, and numerous tumors were observed in the peritoneal cavity; No tumor was found within the peritoneal cavity in either case. * in the figure indicates p<0.05, and * indicates a statistically significant difference in tumor weight between the two groups.
《実施例2》
本実施例では、ガレクチン-4の発現と相関する分子を探索した。具体的には、親株、ノックアウト細胞株(KO-1a株、KO-2株)、及び再発現株R3のcMETの発現をウェスタンブロット法で調べた。再発原株R3は、KO-2株にFLAGガレクチン-4発現プラスミドを導入し、ガレクチン-4を再発現させた細胞株である。
図5に示すように、cMETの発現はノックアウト株KO-1a及びKO-2両方で低下していた。一方、再発現株R3では、対照株及び親株とほぼ同じ程度まで回復しており、ガレクチン-4の発現がcMETの発現に影響を与えている可能性が示唆された。
《Example 2》
In this example, we searched for molecules that correlate with the expression of galectin-4. Specifically, the expression of cMET in the parent line, knockout cell lines (KO-1a line, KO-2 line), and re-expression line R3 was examined by Western blotting. The re-originating strain R3 is a cell line obtained by introducing a FLAG galectin-4 expression plasmid into the KO-2 strain to re-express galectin-4.
As shown in FIG. 5, cMET expression was decreased in both knockout strains KO-1a and KO-2. On the other hand, the re-expression strain R3 recovered to almost the same level as the control strain and the parent strain, suggesting that the expression of galectin-4 may influence the expression of cMET.
(ガレクチン-4の発現の変化とpMETの発現)
ガレクチン-4の発現に相関してcMETの発現が変化していることから、cMETと結合して活性化させる分子HGFの有無で、cMETの活性化体であるpMETの発現を調べた。
前日に12ウェルプレートに4×105個/wellで撒いた各細胞(対照株C1、ノックアウト株KO-1a及びKO-2)を、PBS洗浄後0.1%FCSを含むRPMI1640培地に置換し、4時間後HGFを含まない培地(-)あるいは50ng/mLの濃度でHGFを含む培地(+)を添加して30分反応させた。PBSで洗浄後、SDSサンプルバッファーを加えて回収した。12%SDS-PAGEゲルに添加、泳動後PVDF膜に転写し、実施例2で使用したガレクチン-4抗体、cMET抗体、β-アクチン抗体、pMET抗体(Y1234/1235)(D26)XP(登録商標) Rabbit mAb(Cell Signaling Technology社)で染色を行った。結果を図6に示す。
ノックアウト細胞株は、両クローン共にHGFの存在の有無に関わらず、親株や再発現株R3に比べてpMETのレベルは極めて低かった。このことから、ガレクチン-4の発現がcMETの活性化とも相関することが明らかになった。
(Changes in galectin-4 expression and pMET expression)
Since the expression of cMET changes in correlation with the expression of galectin-4, the expression of pMET, which is an activated form of cMET, was investigated based on the presence or absence of HGF, a molecule that binds to and activates cMET.
Each cell (control strain C1, knockout strain KO-1a and KO-2) seeded at 4 x 10 cells/well in a 12-well plate the previous day was washed with PBS and replaced with RPMI 1640 medium containing 0.1% FCS. After 4 hours, a medium without HGF (-) or a medium containing HGF at a concentration of 50 ng/mL (+) was added and allowed to react for 30 minutes. After washing with PBS, SDS sample buffer was added and collected. Galectin-4 antibody, cMET antibody, β-actin antibody, pMET antibody (Y1234/1235) (D26) XP (registered trademark) used in Example 2 were added to a 12% SDS-PAGE gel and transferred to a PVDF membrane after electrophoresis ) Staining was performed with Rabbit mAb (Cell Signaling Technology). The results are shown in FIG.
Both clones had extremely low pMET levels in the knockout cell lines compared to the parent line and the re-expression line R3, regardless of the presence or absence of HGF. This revealed that the expression of galectin-4 also correlates with the activation of cMET.
(ガレクチン-4の発現の変化とCD44の発現)
次に、親株、ノックアウト細胞株(KO-1a株、KO-2株)、再発現株R3でのCD44の発現をウェスタンブロット法で調べた。
図7に示すように、CD44の発現はノックアウト株KO-1a及びKO-2の両方で低下していた。一方、再発現株R3では、対照株及び親株とほぼ同じ程度まで回復しており、ガレクチン-4の発現がCD44の発現に影響を与えている可能性が示唆された。
(Changes in galectin-4 expression and CD44 expression)
Next, the expression of CD44 in the parent strain, knockout cell lines (KO-1a strain, KO-2 strain), and re-expression strain R3 was examined by Western blotting.
As shown in FIG. 7, the expression of CD44 was decreased in both knockout strains KO-1a and KO-2. On the other hand, the re-expression strain R3 recovered to almost the same level as the control strain and the parent strain, suggesting that the expression of galectin-4 may influence the expression of CD44.
《実施例3》
本実施例では、pMETの発現とガレクチン-4の発現とを、近接ライゲーションアッセイ(PLA)によって解析した。
PLAはDuolink(登録商標) PLA試薬(シグマアルドリッチ社)の蛍光法のマニュアルに従って行った。NUGC4細胞の親株及びノックアウト株KO-2を室温で15分間4%パラホルムアルデヒドを用いて固定し、PBSを用いて洗浄し、浸透処理バッファー(0.5%Triton X-100、PBS)中で5分間浸透処理した後、37℃で30分間PLA用ブロッキングバッファーを用いてブロッキングした。1次抗体として抗pMET抗体及び抗ガレクチン-4抗体(AF1227、R&D Systems社)と4℃で終夜インキュベートした。
洗浄後、希釈PLA二次抗体-PLAプローブ溶液[抗ウサギ(+)および抗ヤギ(-)]を加え、37℃で1時間加湿チャンバー内にてスライドをインキュベートした。洗浄後、ライゲーション反応を実施し、グリーンキットを用いて増幅反応を実施した。反応後の細胞をDuolink(登録商標) In Situ Mounting Medium with DAPIを用いて封入し、透明なネイルポリッシュ硬化剤で周囲を密封し、蛍光顕微鏡で観察した。
PLAでは各抗体の結合する分子が40nm以内に近接して位置する時は、ライゲーション反応によって環状DNA鋳型が形成し、それに続く増幅反応によってハイブリダイズする蛍光オリゴプローブを使用して、局在増幅DNAを視覚化する。
図8で示されるように親株では細胞膜の近傍付近にPLAシグナルが観察され、ガレクチン-4とpMETとの近接が観察された。一方、ノックアウト細胞株KO-2ではPLAシグナルが観察されないことから、親株で認められたPLAシグナルが特異的であることが確かめられた。
《Example 3》
In this example, pMET expression and galectin-4 expression were analyzed by proximity ligation assay (PLA).
PLA was performed according to the fluorescence method manual of Duolink (registered trademark) PLA reagent (Sigma-Aldrich). The parental and knockout strain KO-2 of NUGC4 cells were fixed with 4% paraformaldehyde for 15 min at room temperature, washed with PBS, and incubated in permeabilization buffer (0.5% Triton X-100, PBS) for 5 min. After permeation treatment for a minute, blocking was performed using a blocking buffer for PLA at 37°C for 30 minutes. The cells were incubated overnight at 4°C with anti-pMET antibody and anti-galectin-4 antibody (AF1227, R&D Systems) as primary antibodies.
After washing, diluted PLA secondary antibody-PLA probe solutions [anti-rabbit (+) and anti-goat (-)] were added and the slides were incubated at 37°C for 1 hour in a humidified chamber. After washing, a ligation reaction was performed, and an amplification reaction was performed using a green kit. The cells after the reaction were encapsulated using Duolink (registered trademark) In Situ Mounting Medium with DAPI, the surrounding area was sealed with a transparent nail polish hardening agent, and the cells were observed using a fluorescence microscope.
In PLA, when the molecules bound by each antibody are located close to each other within 40 nm, a circular DNA template is formed by a ligation reaction, and a fluorescent oligo probe that hybridizes in a subsequent amplification reaction is used to locally amplify the DNA. Visualize.
As shown in FIG. 8, in the parent strain, PLA signals were observed near the cell membrane, and galectin-4 and pMET were observed to be in close proximity. On the other hand, since no PLA signal was observed in the knockout cell line KO-2, it was confirmed that the PLA signal observed in the parent cell line was specific.
(MKN45細胞のPLA)
本実施例では、NUGC4細胞に代えて、MKN45細胞を用いて、近接ライゲーションアッセイ(PLA)を行った。MKN45細胞もガレクチン-4を発現している胃がん細胞である。
図9で示されるように無処理(-)の細胞では細胞膜の近傍付近にガレクチン-4とpMETとの近接を示すPLAシグナルが観察された。また、N-結合型糖鎖合成阻害剤(NGI-1)を10μMの濃度で培地に3日間添加した場合、PLAシグナルは大幅に減弱したことから、ガレクチン-4とpMETとの近接に糖鎖が関係している可能性が示唆された。抗pMETウサギ抗体の代わりに非免疫ウサギIgGを使用した場合では、極少量のPLAシグナルしか観察されなかった。
また、ガレクチン-4ウサギ抗体とCD44マウス抗体を使用し、PLA二次抗体-PLAプローブ溶液[抗ウサギ(+)および抗マウス(-)]を使用し同様の近接ライゲーションアッセイを行ったところ、膜近傍にPLAシグナルが観察された。このことから、CD44とガレクチン-4との近接も示唆された。
(PLA of MKN45 cells)
In this example, proximity ligation assay (PLA) was performed using MKN45 cells instead of NUGC4 cells. MKN45 cells are also gastric cancer cells that express galectin-4.
As shown in FIG. 9, in untreated (-) cells, a PLA signal indicating the proximity of galectin-4 and pMET was observed near the cell membrane. Furthermore, when N-linked glycan synthesis inhibitor (NGI-1) was added to the medium at a concentration of 10 μM for 3 days, the PLA signal was significantly attenuated, indicating that glycan chains are present in the vicinity of galectin-4 and pMET. It was suggested that they may be related. When non-immune rabbit IgG was used instead of anti-pMET rabbit antibody, only a minimal amount of PLA signal was observed.
In addition, when a similar proximity ligation assay was performed using a galectin-4 rabbit antibody and a CD44 mouse antibody and a PLA secondary antibody-PLA probe solution [anti-rabbit (+) and anti-mouse (-)], the membrane A PLA signal was observed nearby. This suggested the close proximity of CD44 and galectin-4.
《実施例4》
本実施例では、近接標識法の1つであるSPPLAT(Selective Proteomic Proximity Labeling Assay using Tyramide)法を用いて、MKN45及びNUGC4細胞表面のガレクチン-4の近接分子の同定を行った。
《Example 4》
In this example, proximal molecules of galectin-4 on the surface of MKN45 and NUGC4 cells were identified using the SPPLAT (Selective Proteomic Proximity Labeling Assay using Tyramide) method, which is one of the proximity labeling methods.
(ビオチン-PEG4-チラミド調製]
チラミン塩酸塩(ナカライ社)を0.1Mホウ酸バッファー(pH8.8)で1.55mg/mLの濃度に調製し、2mgのEZLink NHS-PEG4-biotin No-Weigh(登録商標) Format(A39259、サーモフィッシャー社)を0.34mLのDMSOに溶解した。調製したチラミン塩酸溶液とNHS-PEG4-ビオチン溶液を0.34mLずつ混和し、遮光して16時間反応させた。その後、0.22μmフィルターでろ過した後、分注し、使用時まで-30℃フリーザーで凍結保存した。
(Biotin-PEG4-tyramide preparation)
Tyramine hydrochloride (Nacalai) was prepared at a concentration of 1.55 mg/mL with 0.1 M boric acid buffer (pH 8.8), and 2 mg of EZLink NHS-PEG4-biotin No-Weight (registered trademark) Format (A39259, Thermo Fisher) was dissolved in 0.34 mL of DMSO. 0.34 mL each of the prepared tyramine hydrochloride solution and NHS-PEG4-biotin solution were mixed and reacted for 16 hours in the dark. Thereafter, the mixture was filtered through a 0.22 μm filter, aliquoted, and stored frozen in a -30°C freezer until use.
(SPPLAT法による細胞表面標識)
培養した細胞をPBSで3回洗浄し、HRP標識した抗体をPBSに溶解した1%BSA溶液に加え、細胞と4℃で1時間反応させた。反応後、細胞をPBSで3回洗浄し、6cm培養ディッシュの場合は1mLの50mMトリス塩酸あたり6.25μLのビオチン-PEG4-チラミド溶液、0.03%の過酸化水素を加えて調製した溶液と5分反応させた。反応後、1mLのカタラーゼ溶液(200U/mL)を加え反応を停止させた。細胞をPBSで3回洗浄し、cOmplete(登録商標) Protease Inhibitor Cocktail(ロシュ社)、PhosSTOP(登録商標)(ロシュ社)及び4mM 1,10-フェナントロリン(ナカライ社)を含む0.5mLのEzRipa buffer(Atto社)を添加しスクレーパーで回収し、-30℃で凍結保存した。
(Cell surface labeling by SPPLAT method)
The cultured cells were washed three times with PBS, and the HRP-labeled antibody was added to a 1% BSA solution dissolved in PBS, and reacted with the cells at 4° C. for 1 hour. After the reaction, the cells were washed three times with PBS, and in the case of a 6 cm culture dish, a solution prepared by adding 6.25 μL of biotin-PEG4-tyramide solution and 0.03% hydrogen peroxide per 1 mL of 50 mM Tris-HCl. The reaction was allowed to proceed for 5 minutes. After the reaction, 1 mL of catalase solution (200 U/mL) was added to stop the reaction. Cells were washed three times with PBS and treated with 0.5 mL of EzRipa buffer containing cOmplete® Protease Inhibitor Cocktail (Roche), PhosSTOP® (Roche), and 4 mM 1,10-phenanthroline (Nacalai). (Atto) was added, collected with a scraper, and stored frozen at -30°C.
(ビオチン標識分子の回収)
凍結保存した細胞溶解液を解凍後、超音波処理を行い、4000Gで5分遠心した。上清にHigh Capacity Magne(登録商標) ストレプトアビジンビーズ(V7820、プロメガ社)を添加し、4℃、16時間ローテーションしながら反応させた。遠心及び磁気ビーズ分離ラックでストレプトアビジンビーズを分離しRipa bufferで3回洗浄後、SDSサンプルバッファーでビーズから標識分子を回収した。
(Recovery of biotin-labeled molecules)
After thawing the cryopreserved cell lysate, it was treated with ultrasound and centrifuged at 4000G for 5 minutes. High Capacity Magne (registered trademark) streptavidin beads (V7820, Promega) were added to the supernatant, and the mixture was allowed to react at 4°C for 16 hours with rotation. Streptavidin beads were separated using centrifugation and a magnetic bead separation rack, washed three times with Ripa buffer, and labeled molecules were recovered from the beads using SDS sample buffer.
(ガレクチン-4抗体による標識分子)
SPPLAT法でHRP標識ガレクチン-4抗体(aG4)及びHRP標識ウサギIgG(C)で細胞表面を標識した結果を図10aに示す。ガレクチン-4抗体(aG4)の代わりにウサギIgG(C)を反応させた細胞の溶解液にみられるいくつかのバンド(*)は細胞に存在するアビジン結合分子である。ガレクチン-4抗体(aG4)特異的に多くの分子がビオチン標識された。
ストレプトアビジンビーズで回収されたビオチン標識分子の中にpMET及びCD44が染色されたことから、これらの分子がガレクチン-4に近接して存在することがSPPLAT法でも明らかになった。
HRP標識ガレクチン-4抗体の代わりにHRP標識ガレクチン-4を用い、標識時、50mMの濃度でスクロース(Suc)またはラクトース(Lac)、あるいは1mg/mLの濃度でアシアロフェチュイン(AF)を共存させてSPPLAT法で細胞表面を標識した結果を図10bに示す。ガレクチン-4でビオチン標識される分子は、ガレクチン-4の結合阻害活性の無いスクロースに比べ、ガレクチン-4の結合阻害活性を有するラクトースやアシアロフェチュインの共存で大幅に減少した。
ストレプトアビジンビーズで回収したビオチン標識分子の中にウェスタンブロットでpMET及びCD44が染色されたことから、これらの分子がガレクチン-4に近接して存在することが明らかになった。pMET及びCD44の染色も阻害糖の存在で大幅に減少したことから、これらの分子とガレクチン-4の近接に糖鎖が関与していることが明らかになった。
(Labeled molecule by galectin-4 antibody)
Figure 10a shows the results of labeling the cell surface with HRP-labeled galectin-4 antibody (aG4) and HRP-labeled rabbit IgG (C) using the SPPLAT method. Several bands (*) seen in the lysate of cells reacted with rabbit IgG (C) instead of galectin-4 antibody (aG4) are avidin-binding molecules present in the cells. Many molecules were biotin-labeled specifically with galectin-4 antibody (aG4).
Since pMET and CD44 were stained in the biotin-labeled molecules recovered with streptavidin beads, it was also revealed by the SPPLAT method that these molecules were present in close proximity to galectin-4.
HRP-labeled galectin-4 was used instead of HRP-labeled galectin-4 antibody, and during labeling, sucrose (Suc) or lactose (Lac) at a concentration of 50 mM, or asialofetuin (AF) at a concentration of 1 mg/mL was coexisting. The results of cell surface labeling using the SPPLAT method are shown in FIG. 10b. Molecules biotin-labeled with galectin-4 were significantly reduced by the coexistence of lactose and asialofetuin, which have galectin-4 binding inhibitory activity, compared to sucrose, which does not have galectin-4 binding inhibitory activity.
Western blot staining of pMET and CD44 in the biotin-labeled molecules recovered with streptavidin beads revealed that these molecules were present in close proximity to galectin-4. Since the staining of pMET and CD44 was also significantly reduced in the presence of the inhibitory sugar, it became clear that sugar chains were involved in the proximity of these molecules and galectin-4.
《実施例5》
本実施例では、siRNAを用いて、ガレクチン-4陽性細胞のガレクチン-4の発現を阻害した。すなわち、ガレクチン-4高発現胃癌細胞株(NUGC4、MKN45)を用いてガレクチン-4遺伝子の発現阻害(ノックダウン)実験を行い、ノックアウトと同様のフェノタイプが見られ、腹膜播種が阻害されるか調べた。
ノックダウン実験には、インビトロジェン社のsiRNAを用いた。ガレクチン-4用のデザイン済みStealth RNAi(登録商標) siRNA(Stealth siRNA)#3(5’-CCGGACAUUGCCAUCAACAGCUGAA-3’)、及びコントロールとしてNegative control High GC(12935400)のStealth siRNAを用いた。
細胞へのsiRNAの導入にはLipofectamine RNAiMax(インビトロジェン社)を用い、同社のプロトコールに従って行った。前日に撒き替えた細胞に、一定の濃度になるようにOptiMEM培地で希釈したsiRNAとRNAiMaxのコンプレックスを加え数日間培養後に試験に用いた。
《Example 5》
In this example, siRNA was used to inhibit the expression of galectin-4 in galectin-4 positive cells. That is, we performed an expression inhibition (knockdown) experiment of the galectin-4 gene using gastric cancer cell lines that highly express galectin-4 (NUGC4, MKN45), and found that the same phenotype as knockout was observed, and whether peritoneal dissemination was inhibited. Examined.
Invitrogen's siRNA was used in the knockdown experiment. Pre-designed Stealth RNAi (registered trademark) siRNA (Stealth siRNA) #3 (5'-CCGGACAUUGCCAUCAACAGCUGAA-3') for galectin-4 and Stealth siRNA of Negative control High GC (12935400) were used as a control. .
siRNA was introduced into cells using Lipofectamine RNAiMax (Invitrogen) according to the company's protocol. A complex of siRNA and RNAiMax diluted in OptiMEM medium to a constant concentration was added to the cells seeded the previous day, and after culturing for several days, the cells were used in the test.
(ガレクチン-4標的siRNAによる関連分子の発現への影響)
siRNAによって、cMET及びpMETタンパク質の発現が低下するか調べた。NUGC4細胞へは10nM、MKN45細胞へは25nMの濃度でsiRNAを導入し、SDSサンプルバッファーで回収し、両分子の発現をウェスタンブロットで調べた。
図11に示すように、48時間後に回収した細胞で、ガレクチン-4をノックダウンした細胞(Gi)ではコントロールsiRNAを導入した細胞(Ci)に比べ、NUGC4細胞及びMKN45細胞の両方でガレクチン-4タンパク質の発現低下が認められ、ノックアウトと同様にノックダウンでも特にpMETタンパク質の低下が認められた。
(Influence of galectin-4 targeting siRNA on expression of related molecules)
It was investigated whether the expression of cMET and pMET proteins was reduced by siRNA. siRNA was introduced into NUGC4 cells at a concentration of 10 nM and into MKN45 cells at a concentration of 25 nM, collected with SDS sample buffer, and the expression of both molecules was examined by Western blotting.
As shown in Figure 11, among the cells collected after 48 hours, galectin-4 was knocked down in cells with galectin-4 knocked down (Gi) compared to cells into which control siRNA was introduced (Ci). A decrease in protein expression was observed, and in knockdown as well as knockdown, a decrease in pMET protein in particular was observed.
(ガレクチン-4標的siRNA導入による増殖能への影響)
siRNAの導入によって、胃がん細胞の増殖能に与える影響を調べた。前日に細胞を96ウェルプレートに播種し、siRNA添加から72時間後の生細胞をWST-8法で調べ、コントロールsiRNAを導入した細胞と比較した。
図12に示すように、ガレクチン-4をノックダウンした細胞(Gi)では、無処理の細胞及びコントロールsiRNAを導入した細胞(Ci)に比べ、増殖能の低下が認められた。MKN45細胞の方が、増殖抑制効果が高いのは、MKN45細胞の方がよりその増殖にcMET経路の関与する割合が高い為と考えられた。
(Influence on proliferation ability by introducing siRNA targeting galectin-4)
The effect of introducing siRNA on the proliferation ability of gastric cancer cells was investigated. Cells were seeded in a 96-well plate the previous day, and 72 hours after addition of siRNA, living cells were examined by the WST-8 method and compared with cells introduced with control siRNA.
As shown in FIG. 12, in cells in which galectin-4 was knocked down (Gi), a decrease in proliferation ability was observed compared to untreated cells and cells introduced with control siRNA (Ci). The reason why MKN45 cells had a higher proliferation inhibitory effect was thought to be because the cMET pathway was involved in the proliferation of MKN45 cells at a higher rate.
(ガレクチン-4標的siRNA導入細胞のPLA)
ガレクチン-4標的siRNA導入細胞でpMETとガレクチン-4のPLAを行った。図13で示されるように無処理の細胞及びコントロールsiRNAを導入した細胞(Ci)で、膜の近傍付近にPLAシグナルが観察され、ガレクチン-4とpMETとの近接が観察された。一方、ガレクチン-4標的siRNA導入細胞(Gi)ではPLAが減弱し、膜の近傍でのシグナルが観察されなかった。このことから、ガレクチン-4のノックダウンによって、膜近傍でのガレクチン-4とpMETとの近接が大きく抑制されることが示唆された。
(PLA of cells introduced with galectin-4 target siRNA)
PLA of pMET and galectin-4 was performed in cells introduced with galectin-4 target siRNA. As shown in FIG. 13, in untreated cells and cells introduced with control siRNA (Ci), PLA signals were observed near the membrane, and proximity of galectin-4 and pMET was observed. On the other hand, in galectin-4 target siRNA-introduced cells (Gi), PLA was attenuated and no signal was observed near the membrane. This suggested that knockdown of galectin-4 greatly suppresses the proximity of galectin-4 and pMET near the membrane.
《実施例6》
本実施例では、siRNAの投与によって腹膜播種が抑制されるかを、マウスモデルを用いて解析した。
免疫不全マウス(BALBc nu/nu、オス、6週齢)に、ヒト胃癌細胞株MKN45細胞3×106個を腹腔内に移植し、腹膜播種モデルとした。80μgのsiRNA溶解液、80μLのin vivo実験用トランスフェクション試薬(LEO-10、北海道システム・サイエンス株式会社)、5%ブドウ糖液の計250μLを腫瘍細胞投与後、1、4、7、11、14、18、21日目に計7回腹腔内に投与した。
動物試験に用いたガレクチン-4標的siRNA及びコントロールsiRNAは、実施例5と同じ配列で、かつin vivo用に調製されたもの(インビトロジェン社)を用いた。両siRNA腹腔内投与群とも、4匹ずつのマウスで実験を行った。また、2匹のマウスは腫瘍細胞のみを投与し、無処置群とした。
28日後に開腹、剖検し、腫瘍重量を測定した結果を図14に示す。無処置群+対照siRNA投与群(Ci)とガレクチン-4標的siRNA投与群(Gi)で統計処理を行うとp=0.017となり、有意水準5%でガレクチン-4標的siRNAによる腫瘍重量の抑制が認められ、ガレクチン-4標的siRNAによって腹膜播種を抑制できると考えられる。
《Example 6》
In this example, a mouse model was used to analyze whether administration of siRNA suppressed peritoneal dissemination.
3×10 6 human gastric cancer cell line MKN45 cells were intraperitoneally transplanted into an immunodeficient mouse (BALBc nu/nu, male, 6 weeks old) to serve as a peritoneal dissemination model. After administering a total of 250 μL of 80 μg of siRNA solution, 80 μL of in vivo experimental transfection reagent (LEO-10, Hokkaido System Science Co., Ltd.), and 5% glucose solution to tumor cells, 1, 4, 7, 11, 14 It was administered intraperitoneally a total of 7 times on the 18th and 21st days.
The galectin-4 target siRNA and control siRNA used in the animal test had the same sequence as in Example 5 and was prepared for in vivo use (Invitrogen). For both siRNA intraperitoneal administration groups, experiments were conducted with four mice each. In addition, two mice received only tumor cells and were set as an untreated group.
After 28 days, the tumor was subjected to laparotomy and autopsy, and the weight of the tumor was measured. The results are shown in FIG. When performing statistical processing on the untreated group + control siRNA administration group (Ci) and the galectin-4 target siRNA administration group (Gi), p = 0.017, and the tumor weight was suppressed by galectin-4 target siRNA at a significance level of 5%. It is thought that peritoneal dissemination can be suppressed by siRNA targeting galectin-4.
本発明の医薬組成物は、ガレクチン-4陽性胃がん細胞の増殖を抑制し、胃がんの抗がん剤として用いることができる。 The pharmaceutical composition of the present invention suppresses the proliferation of galectin-4-positive gastric cancer cells and can be used as an anticancer agent for gastric cancer.
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