JP6516235B2 - Chimeric protein and microglial activity inhibitor using the same - Google Patents
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Description
本発明は、脳内の免疫反応を司るミクログリアの活性化を局所的に必要なときにのみ不活性化させ、炎症反応を鎮静化させる抗炎症性サイトカインに関する。 The present invention relates to an anti-inflammatory cytokine which inactivates the activation of microglia responsible for the immune response in the brain only when necessary locally, and suppresses the inflammatory response.
例えば、パーキンソン病治療等を目的に神経幹/前駆細胞等の幹細胞移植治療を検討した場合に、移植後初期に惹起される初期炎症反応が問題になる。
脳内における炎症反応および免疫反応を担うのがミクログリアであるが、移植直後に活性化され異物となる移植細胞の排除に向けて機能する。
移植後初期に惹起される炎症反応では、ミクログリアが活性化され炎症性サイトカインIL−1β,IL−6,TNFa等を放出し、移植幹細胞を攻撃することが知られている。
そこでミクログリアを不活性化させるための、抗炎症性サイトカインを用いた炎症反応の抑制が必要になると考えられる。
しかしながら、免疫抑制剤の投与等、過剰な免疫抑制は患者自身の生体防御能を低下させることになるため、できることならば避けなければならない。
本発明は患者自身の免疫応答を妨害することなく、局所的に移植幹細胞を炎症反応から保護できないか研究した結果得られたものである。
For example, when stem cell transplantation treatments such as neural stem / progenitor cells are considered for the purpose of Parkinson's disease treatment etc., the initial inflammatory reaction that is triggered early after transplantation becomes a problem.
Although microglia are responsible for inflammatory and immune responses in the brain, they function to eliminate transplanted cells that are activated immediately after transplantation and become foreign.
It is known that, in the inflammatory reaction which is triggered early after transplantation, microglia are activated to release inflammatory cytokines IL-1β, IL-6, TNFa and the like to attack transplanted stem cells.
Therefore, it is considered that suppression of the inflammatory reaction using anti-inflammatory cytokines is required to inactivate microglia.
However, excessive immunosuppression, such as administration of an immunosuppressant, reduces the ability of the patient to protect itself and should therefore be avoided if at all possible.
The present invention has been obtained as a result of studying whether or not transplant stem cells can be protected from an inflammatory response locally without interfering with the patient's own immune response.
特許文献1には、多官能化学成分を含む水溶性ポリマーセグメントに酵素的に切断可能なリンカーを介して接合された生物学的に活性な物質を含む重合薬物接合体を開示する。
しかし、同公報に開示する重合薬物接合体は、生物学的に活性な物質を化学的に修飾する技法にて接合しており、この生物学的に活性な物質であるタンパク質の活性が化学的接合により維持されなくなる恐れが高い。
また、同公報には水溶性ポリマーセグメントとしてポリエチレングリコールを開示しているが、この種のポリマーはタンパク質を放出した後も生体内に残留し、生体に悪い影響を与える毒素となる恐れが高い。
U.S. Pat. No. 5,956,095 discloses polymeric drug conjugates comprising a biologically active substance conjugated via an enzymatically cleavable linker to a water soluble polymer segment comprising a multifunctional chemical moiety.
However, the polymerized drug conjugate disclosed in the publication is conjugated by a technique for chemically modifying a biologically active substance, and the activity of the protein which is the biologically active substance is chemically There is a high risk that it will not be maintained by bonding.
In addition, although polyethylene glycol is disclosed as the water-soluble polymer segment in the same publication, this type of polymer remains in the living body even after releasing the protein and is likely to be a toxin which adversely affects the living body.
本発明は、必要とする時に且つ局所的に作用する抗炎症性サイトカインを放出するキメラタンパク質及びこれを用いたミクログリア活性阻害剤の提供を目的とする。 An object of the present invention is to provide a chimeric protein that releases an anti-inflammatory cytokine that acts locally and when necessary, and a microglial activity inhibitor using the same.
本発明に係るキメラタンパク質(融合タンパク質)は、抗炎症性サイトカインに酵素分解性ペプチド及び基材結合性ペプチドを、その順に融合したキメラタンパク質であって、前記酵素分解性ペプチドは脳内に有するミクログリアの活性化により産生されるマトリックスメタロプロテアーゼ類により切断されるものであり、前記基材結合性ペプチドは天然ハイドロゲルからなる基材と分子間相互作用により結合可能であり、前記抗炎症性サイトカインは、前記ミクログリアの活性化を抑制するものであることを特徴とする。
本発明に係るキメラタンパク質は、基材結合性ペプチドを介して天然ハイドロゲルに分子間相互作用にて結合して使用できるものである。
ここで分子間相互作用は、天然ハイドロゲルと基材結合性ペプチドが108〜1010/Mの会合定数を有する特異的な結合をいう。
このようにキメラタンパク質を天然ハイドロゲルからなる基材に結合させる方法を採用すると、結合によりキメラタンパク質に有する生物学的活性が失われることはない。
The chimeric protein (fusion protein) according to the present invention is a chimeric protein in which an enzyme-degradable peptide and a substrate-binding peptide are fused in that order to an anti-inflammatory cytokine, and said enzyme-degradable peptide is a microglia possessed in the brain. And the substrate binding peptide is capable of binding to a substrate composed of a natural hydrogel by intermolecular interaction, and the anti-inflammatory cytokine is The present invention is characterized by suppressing the activation of the microglia.
The chimeric protein according to the present invention can be used by binding to a natural hydrogel through intermolecular interaction via a substrate binding peptide.
Here, the intermolecular interaction refers to a specific binding between a natural hydrogel and a substrate binding peptide having an association constant of 10 8 to 10 10 / M.
As such, when the method for binding a chimeric protein to a substrate comprising a natural hydrogel is adopted, the biological activity possessed by the chimeric protein is not lost by the binding.
本発明において、前記マトリックスメタロプロテアーゼ類はMMP−3又はMMP−9であるのが好ましい。
MMP−3はストロメライシン−1と称され、MMP−9はゼラチナーゼ−B(92kDa)と称される。
これらは、ペプチドを特異選択的に切断する作用を有する。
例えば他のMMP−2等は複数のプロテアーゼにて切断される点で明確に相違する。
In the present invention, the matrix metalloproteinases are preferably MMP-3 or MMP-9.
MMP-3 is referred to as stromelysin-1 and MMP-9 is referred to as gelatinase-B (92 kDa).
These have the effect of specifically selectively cleaving the peptide.
For example, other MMP-2 and the like are distinctly different in that they are cleaved by a plurality of proteases.
本発明において、前記抗炎症性サイトカインは、インターロイキン−10であるのが好ましい。
例えば、抗炎症性サイトカインの一つであるインターロイキン−4では、本開発方法で構築したキメラタンパク質では、抗炎症活性が低下してしまう。
また、前記天然ハイドロゲルはコラーゲン又はヒアルロン酸であり、当該基材に前記キメラタンパク質を担持させることでミクログリア活性阻害剤となる。
In the present invention, the anti-inflammatory cytokine is preferably interleukin-10.
For example, in interleukin-4 which is one of the anti-inflammatory cytokines, the chimeric protein constructed by the present development method has a reduced anti-inflammatory activity.
In addition, the natural hydrogel is collagen or hyaluronic acid, and when the chimeric protein is supported on the base material, it becomes a microglial activity inhibitor.
本発明者は、これまでパーキンソン病治療を目指した神経幹/前駆細胞移植医療における低生着率を改善する目的で、細胞移植時に天然ハイドロゲルを用いた。
この天然ハイドロゲルが移植幹細胞を物理的に保護し、その生着率が約40%まで向上できている。
本発明により、脳内の免疫反応を司るミクログリアの活性化を抑制し、移植部位周辺のみの免疫反応を抑制できるので生物学的にも保護可能になる。
このように本発明は、物理的な保護と生物学的な保護の両方が可能になり、移植幹細胞の生着率はさらに向上することができる(約55%に向上)。
それに伴い、中枢神経系における細胞移植による再生医療が実現に向けて大きく前進すると考えられる。
The present inventors used natural hydrogel at the time of cell transplantation in order to improve the low survival rate in neural stem / progenitor cell transplantation medical treatment aiming at treatment of Parkinson's disease until now.
This natural hydrogel physically protects the transplanted stem cells, and its engraftment rate can be improved to about 40%.
According to the present invention, the activation of microglia responsible for the immune response in the brain can be suppressed, and the immune response only around the transplantation site can be suppressed, so that biological protection is also possible.
Thus, the present invention enables both physical protection and biological protection, and the survival rate of transplanted stem cells can be further improved (improved to about 55%).
Along with this, it is thought that regenerative medicine by cell transplantation in the central nervous system will make great progress toward realization.
まず、発明の機序を説明する。
本材料は、炎症反応が起こっているときのみ放出され局所で作用し、炎症反応が鎮静化されたあとは、放出がストップする機能を有している。
例えば図1に示した模式図で説明すると、幹細胞の移植後に惹起される炎症反応でミクログリアが活性化されると、マトリックスメタロプロテアーゼ(MMP)が放出される。
この放出されたMMPにより基材に担持されたIL−10等の抗炎症性サイトカインが切断されて、放出される。
この放出された抗炎症性サイトカインがミクログリアを不活性化し、炎症反応が鎮静化される。
これにより、移植幹細胞を保護しつつも、通常生体内で起こっている生体防御反応を妨害しないシステムが構築できる。
本発明に係る阻害剤は、移植幹細胞を保護して、且つ、不必要な免疫抑制が起こらないようにすることができ、患者にとって負担軽減になると期待できる。
前述したように、我々はこれまでに、移植幹細胞を保護するための天然ハイドロゲルの設計を進めてきた。
その天然ハイドロゲルにキメラタンパク質を安定に固定する。
これにより、移植後でも炎症反応が起こらない限り拡散しないため、不必要に免疫抑制することはない。
移植後に惹起される炎症反応に伴い、ミクログリアが活性化され、移植部位に遊走してくるが、その際に、マトリックスメタロプロテアーゼ類(MMP−3やMMP−9)を大量に産生する。
その現象に着目し、抗炎症性サイトカインIL−10を天然ハイドロゲルに安定に固定し、IL−10と天然ハイロドゲル結合部位との間に、MMP−9で特異的に切断されるペプチド配列を導入した(図2)。
これにより、炎症反応誘発時のMMP−9の大量放出時にのみ、IL−10が放出され、炎症反応を鎮静化させるシステムを構築した。
First, the mechanism of the invention will be described.
This material is released only when an inflammatory response is taking place and acts locally, and has the function of stopping the release after the inflammatory response has subsided.
For example, as described in the schematic diagram shown in FIG. 1, when microglia are activated in an inflammatory reaction elicited after transplantation of stem cells, matrix metalloproteinases (MMPs) are released.
The released MMP cleaves the anti-inflammatory cytokine such as IL-10 supported on the substrate and releases it.
This released anti-inflammatory cytokine inactivates microglia and the inflammatory response is suppressed.
This makes it possible to construct a system that protects the transplanted stem cells but does not interfere with the biodefense reaction that normally occurs in vivo.
The inhibitor according to the present invention can protect transplanted stem cells, prevent unnecessary immunosuppression from occurring, and can be expected to reduce the burden on patients.
As mentioned above, we have previously designed natural hydrogels to protect transplanted stem cells.
The chimeric protein is stably fixed to the natural hydrogel.
As a result, even after transplantation, there is no unnecessary immune suppression, since the disease does not spread unless an inflammatory reaction occurs.
The microglia are activated and migrate to the transplantation site in response to the inflammatory reaction elicited after transplantation, at which time a large amount of matrix metalloproteinases (MMP-3 and MMP-9) is produced.
Focusing on the phenomenon, the anti-inflammatory cytokine IL-10 is stably fixed to the natural hydrogel, and a peptide sequence specifically cleaved by MMP-9 is introduced between the IL-10 and the natural hydrogel gel binding site (Figure 2).
Thus, IL-10 was released only at the time of massive release of MMP-9 at the time of induction of the inflammatory reaction, and a system was established to calm the inflammatory reaction.
<抗炎症性サイトカイン(IL−10)キメラタンパク質の合成>
抗炎症サイトカイン(IL−10)キメラタンパク質は、遺伝子工学技術を応用し、大腸菌・酵母・動物細胞などの発現系を利用して合成する。
まず、タンパク質のアミノ酸配列をコードする遺伝子をインターロイキン10(IL10)、MMP9切断サイト(M9CS)、基材結合ペプチド(SBP)、それぞれポリメラーゼ連鎖反応(PCR)を利用して得る。
それぞれのDNAをオーバーラップ伸長PCR法や制限酵素法など様々な方法を駆使することによって、IL10−M9CS−SBPの順に連結させる。
そのDNAを生物発現系で一般的に用いられるプラスミドベクターに挿入する。作製したプラスミドベクターを宿主(大腸菌・酵母・動物細胞など)に導入し、強制発現させ、一般的なタンパク質精製法を利用して目的となるタンパク質を得た。
<Synthesis of anti-inflammatory cytokine (IL-10) chimeric protein>
An anti-inflammatory cytokine (IL-10) chimeric protein is synthesized by applying genetic engineering technology and utilizing expression systems such as E. coli, yeast, animal cells and the like.
First, a gene encoding an amino acid sequence of a protein is obtained using interleukin 10 (IL10), MMP9 cleavage site (M9CS), substrate binding peptide (SBP), and polymerase chain reaction (PCR), respectively.
The respective DNAs are ligated in the order of IL10-M9CS-SBP by using various methods such as overlap extension PCR method and restriction enzyme method.
The DNA is inserted into a plasmid vector commonly used in biological expression systems. The prepared plasmid vector was introduced into a host (E. coli, yeast, animal cell, etc.), forced expression, and a target protein was obtained using a general protein purification method.
その例を以下具体的に説明する。
まず、ヒトIL10コード遺伝子よりPCRによりクローニングし、IL10遺伝子を得た。
その塩基配列を配列番号1に示す。
遺伝子増幅に用いたフォワードプライマーは下記のとおりであり、
5-catgcatatgagcccaggccagggcacccag-3(配列番号4)
リバースプライマーは下記のとおりである。
5-gtttcgtatcttcattgtcatgtagg-3(配列番号5)
次にIL10遺伝子にオーバーラップエクステンションPCR法により、MMP−9により切断される切断サイト(M9CS)のペプチド及び天然ハイドゲルからなる基材に結合させるための基材結合ペプチド(SBP)を、この順にて連結させたIL10−M9CS−SBP遺伝子を作成した。
オーバーラップエクステンションPCRに用いたプライマーは下記のとおりである。
フォワードプライマー :5-ctacatgacaatgaagatacgaaacggtggcgggggaccacctggtgtagtgggagaacaaggggagcagggaccaccgcc-3(配列番号6)
リバースプライマー :5-gctcgagggtgatattggtatcagcaatgcggatgtaggaaccgcccccacccccgccacctggcggtggtccctgctcccct-3(配列番号7)
上記プライマーにて得られた遺伝子の増幅には下記のプライマーを用いた。
フォワードプライマー :5-ctacatgacaatgaagatac-3(配列番号8)
リバースプライマー :5-gctcgagggtgatattggta-3(配列番号9)
また、IL10−M9CS−SBPの増幅には配列番号4のフォワードプライマーと下記のリバースプライマーを用いた。
リバースプライマー :5-gctcgagggtgatattggtatcagc-3(配列番号10)
得られたIL10−M9CS−SBP遺伝子の塩基配列を図8に示し、そのアミノ配列を配列番号2,3に示す。
図8にて説明すると、□の部分がIL10のコード配列,「 」の部分がMMP−9により切断されるペプチドコード配列(M9CS),『 』の部分がコラーゲン結合性ペプチドコード配列,一重下線の部分は柔軟リンカーペプチドコード配列,二重下線の部分は制限酵素切断配列である。
なお、図8に示したタンパク質には、高純度精製を可能とするアフィニティーカラムクロマトグラフィーを用いるため、ヒスチジンタグを含ませた例になっている。
PCRにより増幅したIL10−M9CS−SBP遺伝子をプラスミドに導入し、その導入プラスミドを、ヒートショック法により大腸菌に形質導入した。
なお、エレクトロポレーションにより動物細胞に形質導入してもよい。
培養増殖により発現したタンパク質を宿主から抽出し、アフィニティーカラムクロマトグラフィーにより分画精製した。
なお、コラーゲンゲル等の天然ハイドロゲルからなる基材に担持するには、低温にてコラーゲンゲル前駆溶液と上記タンパク質溶液を混合し、その溶液を37℃×15〜30min加温する。
このように特異的な相互作用を利用して、特定の方向でタンパク質を基材に担持できる。
これにより、従来の化学的結合と異なりタンパク質の活性が失われることはない。
<IL−10キメラタンパク質のキャラクタリゼーション>
発現し精製したタンパク質は、ドデシル硫酸ナトリウム−ポリアクリルアミド電気泳動法(SDS−PAGE)による分析、ウエスタンブロッティング(WB)法によるIL10キメラタンパク質の証明、円偏光二色性(CD)測定によるタンパク質の構造評価、基材への担持能力の評価および基材に担持されたIL10キメラタンパク質の選択放出評価、活性化ミクログリアを用いた活性評価を行った。
<SDS−PAGEおよびWB分析>
本実験では、IL10キメラタンパク質と対照実験として天然のIL10を用い、SDS−PAGE分析、WB分析を行った(図3)。
本開発タンパク質の理論的な分子量(21.8 kDa)にほぼ一致するバンドが得られている。
また、WB分析によって、本発明に係るキメラタンパク質がIL10ドメインを含むタンパク質であることがこの結果より証明された。
<CD測定>
本開発キメラタンパク質の構造評価を行った(図4)。
対照として天然のIL10のCDスペクトルと比較した。
本開発キメラタンパク質のCDスペクトルは、197〜260nmの領域において、天然IL10とほぼ一致するスペクトルであった。
このことから、得られたキメラタンパク質が天然IL10と同様の二次構造を有することが明らかとなった。
また、190〜197nmにおいて、天然IL10のスペクトルと異なるスペクトルが得られた。
これは、本開発キメラタンパク質がIL10のC末端側に存在するM9CSおよびSBPのオリゴペプチド配列(28 amino acids)のランダムコイル構造に由来するものと考えられる。
An example is specifically described below.
First, cloning was performed by PCR from a human IL10 coding gene to obtain an IL10 gene.
The nucleotide sequence is shown in SEQ ID NO: 1.
The forward primers used for gene amplification are as follows:
5-catgcatatgagccccaggcagggcacccag-3 (SEQ ID NO: 4)
The reverse primer is as follows.
5-gtttcgtatcttcattgtcatgtagg-3 (SEQ ID NO: 5)
Next, a peptide binding site (M9CS) cleaved by MMP-9 by an overlap extension PCR method to the IL10 gene and a substrate binding peptide (SBP) for binding to a substrate consisting of a natural hydrogel are sequentially arranged in this order The ligated IL10-M9CS-SBP gene was generated.
The primers used for overlap extension PCR are as follows.
Forward primer: 5-ctacatgacaatgaagatacacaa cggtggcgggggaccctgtgtagtgggagaacaaggggagcagggaccaccgcc-3 (SEQ ID NO: 6)
Reverse primer: 5-gctcgagggtgatatttgtatcagcaatgcggatgtaggaaccg ccgaccgccccccccccg ccacctggcggtgct ccctgctccct-3 (SEQ ID NO: 7)
The following primers were used for amplification of the gene obtained by the said primer.
Forward primer: 5-ctacatgacaatgaagatac-3 (SEQ ID NO: 8)
Reverse primer: 5-gctcgagggtgatattggta-3 (SEQ ID NO: 9)
In addition, the forward primer of SEQ ID NO: 4 and the following reverse primer were used for amplification of IL10-M9CS-SBP.
Reverse primer: 5-gctcgagggtgatattggtatcagc-3 (SEQ ID NO: 10)
The nucleotide sequence of the obtained IL10-M9CS-SBP gene is shown in FIG. 8, and the amino sequences thereof are shown in SEQ ID NOS: 2 and 3.
Referring to FIG. 8, the part of □ represents the coding sequence of IL10, the part of “” represents the peptide coding sequence (M9CS) cleaved by MMP-9, the part of “” represents the collagen-binding peptide coding sequence, single-underlined The part is a soft linker peptide coding sequence, and the double underlined part is a restriction enzyme cleavage sequence.
The protein shown in FIG. 8 is an example in which a histidine tag is included in order to use affinity column chromatography that enables high purity purification.
The IL10-M9CS-SBP gene amplified by PCR was introduced into a plasmid, and the introduced plasmid was transduced into E. coli by the heat shock method.
Alternatively, animal cells may be transduced by electroporation.
The protein expressed by culture growth was extracted from the host and fractionated and purified by affinity column chromatography.
In addition, in order to carry | support to the base material which consists of natural hydrogels, such as a collagen gel, a collagen gel precursor solution and the said protein solution are mixed at low temperature, and 37 degreeC * 15 to 30 min.
Such specific interaction can be used to support proteins on a substrate in a specific direction.
This, unlike conventional chemical binding, does not result in loss of protein activity.
Characterization of IL-10 Chimeric Protein
The expressed and purified protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), verification of IL10 chimeric protein by Western blotting (WB) method, structure of protein by circular dichroism (CD) measurement Evaluation, evaluation of the ability to support the substrate and evaluation of selective release of IL10 chimeric protein supported on the substrate, and activity evaluation using activated microglia were performed.
<SDS-PAGE and WB analysis>
In this experiment, SDS-PAGE analysis and WB analysis were performed using an IL10 chimeric protein and natural IL10 as a control experiment (FIG. 3).
A band almost corresponding to the theoretical molecular weight (21.8 kDa) of the developed protein is obtained.
Moreover, it was proved from this result that the chimeric protein according to the present invention is a protein containing an IL10 domain by WB analysis.
<CD measurement>
Structural evaluation of this developed chimeric protein was performed (FIG. 4).
As a control, it was compared to the CD spectrum of native IL10.
The CD spectrum of this developed chimeric protein was a spectrum in the region of 197 to 260 nm, which was almost identical to native IL10.
From this, it became clear that the obtained chimeric protein has the same secondary structure as native IL10.
In addition, a spectrum different from that of natural IL10 was obtained at 190 to 197 nm.
This is considered to be derived from the random coil structure of the M9CS and SBP oligopeptide sequences (28 amino acids) present in the C-terminal side of IL10 in this developed chimeric protein.
<基材への固定能力評価および基材固定されたIL10の選択放出評価>
本開発タンパク質は、様々な基材に固定することのできるように基材結合ペプチド(SBP)を導入している。
本実験では、IL10キメラタンパク質にオリゴヒスチジン(His)を導入したタンパク質を使用した。
ガラス基板上に2価の金属配位子(Ni(II)またはZn(II))を有する置換基を導入し、IL10キメラタンパク質を固定させた。
ガラス基材上に固定されたIL10キメラタンパク質の表面密度は、0.685 μg/cm2であった。
IL10の大きさから概算すると、ある程度密にIL10キメラタンパク質が固定されていることが分かった(表面占有率79.4%)。
IL10キメラタンパク質固定基材を、500mMイミダゾールを含むリン酸緩衝液に浸漬させ、IL10キメラタンパク質の脱離について評価した。
イミダゾールは、Hisと金属配位子間の相互作用を阻害する。
これにより、キメラタンパク質がSBPによって固定されているかどうかを評価した。
イミダゾール溶液に浸漬後のIL10キメラタンパク質の表面密度は、0.101μg/cm2であり、約85%がSBPによりアンカーリングされていることが分かった。
IL10キメラタンパク質を固定した表面に、種々の濃度MMP−9を3時間接触させ、IL10ドメインの放出を観察した(図5A)。
また、2.5μg/mL MMP−9を所定時間接触させ、経時的なタンパク質放出を観察した(図5B)。
MMP−9の切断によって溶液中に放出されたタンパク質をSDS−PAGE分析により検出し定量した。
その結果、1μg/mL以上のMMP−9を3時間反応させると、固定されたIL10のうち90%放出されることが分かった。
また、2.5μg/mL MMP−9を反応させた場合、接触後15分で固定されたIL10の約70%が放出されたことが分かった。
また、MMP−9接触後1時間でほぼ放出量は一定となり、2.5μg/mLのMMP−9では、約1時間の反応でほとんどのIL10が放出されることが明らかとなった。
これらの結果から、基材に安定固定させたIL10キメラタンパク質を、MMP−9の切断により、選択的に放出させることができることが分かり、本システムが目的通りに機能することが証明された。
<活性化ミクログリアを用いた活性評価>
合成したIL10キメラタンパク質の活性を、活性化ミクログリアの不活性化により評価した。
ミクログリアは、リポポリサッカライド(LPS)およびインターフェロンγにより活性化し、炎症反応を司るマクロファージなどと同等の機能を果たす。
活性化ミクログリアは、炎症性サイトカインを産生するが、その産生量をIL10キメラタンパク質により、抑制できるかで、その活性をリコンビナントIL10との比較により判断した。
その結果、リコンビナントIL10と同等の活性を有していることが明らかになった。
<IL−10キメラタンパク質の有効性評価(in vitro)>
IL10キメラタンパク質をコラーゲンゲルに担持させることにより、ゲル中の細胞を炎症反応から保護できるかについて、in vitroにより評価した。
この結果、IL10キメラタンパク質を担持させた天然ハイドロゲル中では、活性化ミクログリアが存在しても、細胞は、死滅しないことが分かった。
また、その理由として、Figure 7Dに示すように、炎症性サイトカインの産生量が、大幅に減少しているためと考えられる。
これらの結果から、IL10キメラタンパク質により、炎症反応から細胞を保護することが出来ると強く示唆される。
これは、細胞移植による治療において非常に有用なツールになると思われる。
<Evaluation of ability to fix to substrate and selective release of IL10 immobilized on substrate>
The present developed protein introduces a substrate-binding peptide (SBP) so that it can be immobilized on various substrates.
In this experiment, a protein in which oligohistidine (His) was introduced to IL10 chimeric protein was used.
A substituent having a divalent metal ligand (Ni (II) or Zn (II)) was introduced onto a glass substrate to immobilize the IL10 chimeric protein.
The surface density of the IL10 chimeric protein immobilized on the glass substrate was 0.685 μg / cm 2 .
As estimated from the size of IL10, it was found that the IL10 chimeric protein was immobilized to a certain degree (surface occupancy rate 79.4%).
The IL10 chimeric protein immobilization substrate was immersed in a phosphate buffer containing 500 mM imidazole and evaluated for elimination of the IL10 chimeric protein.
The imidazole inhibits the interaction between His and the metal ligand.
This assessed whether the chimeric protein was immobilized by SBP.
The surface density of the IL10 chimeric protein after immersion in the imidazole solution was 0.101 μg / cm 2 and was found to be approximately 85% anchored by SBP.
Various concentrations of MMP-9 were contacted for 3 hours on the surface on which the IL10 chimeric protein was immobilized, and the release of the IL10 domain was observed (FIG. 5A).
In addition, 2.5 μg / mL MMP-9 was contacted for a predetermined time, and protein release over time was observed (FIG. 5B).
The proteins released into solution by cleavage of MMP-9 were detected and quantified by SDS-PAGE analysis.
As a result, it was found that when 1 μg / mL or more of MMP-9 was reacted for 3 hours, 90% of the immobilized IL10 was released.
In addition, it was found that when the 2.5 μg / mL MMP-9 was reacted, about 70% of the fixed IL10 was released 15 minutes after the contact.
In addition, it was revealed that the release amount was almost constant in 1 hour after MMP-9 contact, and in the case of 2.5 μg / mL of MMP-9, most of IL10 was released in about 1 hour of reaction.
From these results, it was found that the IL10 chimeric protein stably immobilized on the substrate can be selectively released by cleavage of MMP-9, demonstrating that the system functions as intended.
<Evaluation of activity using activated microglia>
The activity of the synthesized IL10 chimeric protein was assessed by inactivation of activated microglia.
Microglia are activated by lipopolysaccharide (LPS) and interferon γ, and function in the same manner as macrophages and the like responsible for the inflammatory response.
Activated microglia produce inflammatory cytokines, but their activity could be judged by comparison with recombinant IL10, depending on whether their production could be suppressed by the IL10 chimeric protein.
As a result, it became clear that it has activity equivalent to recombinant IL10.
<Evaluation of efficacy of IL-10 chimeric protein (in vitro)>
It was evaluated in vitro whether the cells in the gel could be protected from the inflammatory reaction by loading the IL10 chimeric protein on a collagen gel.
As a result, it was found that in the natural hydrogel loaded with IL10 chimeric protein, the cells were not killed even in the presence of activated microglia.
Moreover, as the reason, as shown in FIG. 7D, it is considered that the production amount of inflammatory cytokines is significantly reduced.
These results strongly suggest that IL10 chimeric protein can protect cells from the inflammatory response.
This seems to be a very useful tool in the treatment by cell transplantation.
本発明は抗炎症性サイトカインを、移植材料に複合化させ、移植直後の初期炎症・免疫反応が起こったときのみ抗炎症性サイトカインが放出され、移植部位周辺のみに局所的に作用するシステムとなっている。
このシステムにより、移植に伴い惹起された炎症反応のみを抑制することとなり、生体防御のために常に起こっている免疫反応・炎症反応を抑制することなく、レシピエントへの負担が最大限軽減されることが期待される。
また、一般的に用いられる免疫抑制剤の前身投与も必要がなくなると考えられ、この点からもレシピエントへの負担軽減につながると期待される。
The present invention is a system in which an anti-inflammatory cytokine is complexed to a graft material, and the anti-inflammatory cytokine is released only when the initial inflammation / immune reaction immediately after transplantation occurs, acting locally only around the graft site. ing.
With this system, only the inflammatory reaction elicited by transplantation is suppressed, and the burden on the recipient is alleviated maximally without suppressing the immune reaction / inflammatory reaction that is constantly occurring for biological protection. It is expected.
In addition, it is thought that the prior administration of a commonly used immunosuppressant is also unnecessary, and it is expected that this will lead to a reduction in the burden on the recipient.
Claims (1)
前記酵素分解性ペプチドは脳内に有するミクログリアの活性化により産生されるマトリックスメタロプロテアーゼ類により切断されるものであり、
前記基材結合性ペプチドは天然ハイドロゲルからなる基材と分子間相互作用により結合可能であり、
前記抗炎症性サイトカインは、前記ミクログリアの活性化を抑制するものであり、
前記マトリックスメタロプロテアーゼ類はMMP−3又はMMP−9であり、
前記抗炎症性サイトカインはインターロイキン−10である、キメラタンパク質を含んでなり、
前記キメラタンパク質をコラーゲン又はヒアルロン酸からなる前記天然ハイドロゲルの基材に担持するとともに、移植細胞と複合化して用いることを特徴とする、前記移植細胞の移植に伴い惹起された炎症反応を抑制する炎症抑制剤。 A chimeric protein in which an enzyme-degradable peptide and a substrate-binding peptide are fused in that order to an anti-inflammatory cytokine,
The enzyme-degradable peptide is cleaved by matrix metalloproteinases produced by activation of microglia contained in the brain,
The substrate binding peptide is capable of binding to a substrate comprising a natural hydrogel by intermolecular interaction,
The anti-inflammatory cytokine suppresses the activation of the microglia.
The matrix metalloproteinases are MMP-3 or MMP-9,
The anti-inflammatory cytokine comprises interleukin-10, a chimeric protein ,
Inhibiting to be supported on the substrate of the natural hydrogel comprising the chimeric protein of collagen or hyaluronic acid, and transplanted cells, characterized by using in combination of, the inflammatory response to with raised transplantation of transplanted cells Antiinflammatory agent.
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