JP4233608B2 - Autoantibody measurement method - Google Patents
Autoantibody measurement method Download PDFInfo
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- JP4233608B2 JP4233608B2 JP51816398A JP51816398A JP4233608B2 JP 4233608 B2 JP4233608 B2 JP 4233608B2 JP 51816398 A JP51816398 A JP 51816398A JP 51816398 A JP51816398 A JP 51816398A JP 4233608 B2 JP4233608 B2 JP 4233608B2
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Description
技術分野
本発明はCD38ペプチド断片を用いた抗CD38自己抗体の測定方法、CD38ペプチド、その断片およびその変異体を含有する自己免疫疾患治療用医薬組成物に関する。
背景技術
インスリン依存型糖尿病（IDDM）は、膵島に自己免疫的炎症（膵島炎）が起こってインスリンを産生・分泌する細胞（β細胞）が破壊され、インスリンが極度に欠乏するために発病する。従って、IDDMおよび膵島炎の根本的治療としては発病前治療、早期治療、即ち膵β細胞が破壊される前に治療を行う必要があり、その早期診断方法が重要である。しかし、現在行われているのは病理解剖または生体検査による発病後の診断方法のみであり、診断された時点では既にインスリン生産細胞が破壊されている。発病前の病理解剖等の上記方法による診断は倫理上不可能であることから、インスリン依存型糖尿病および膵島炎患者を発病前に診断する方法が種々試みられているが、いずれも不十分であった。
CD38は分子量46kDaの糖タンパク質であり、主としてリンパ球表面に認められ、Ｔ細胞、Ｂ細胞の分化および活性化のマーカーとして、さらに、Ｔ細胞、Ｂ細胞の悪性度（幼若度）の指標として用いられている。最近、CD38分子のcDNAをプローブにしたノーザンブロット解析や、CD38分子に対する特異抗体を用いた免疫組織染色により、CD38は白血球、免疫細胞に限らず、ラット脳、十二指腸、心臓、脳下垂体、膵島にも発現していることが確認された。
CD38はさらに、細胞内に環状ADPリボース（cyclic ADP-ribose；cADPR）を生成させ、このcADPRを介して細胞内カルシウムの移動を制御していることも明らかとなった。1993年Okamotoらは、膵島細胞においてcADPRが細胞内カルシウムの増加を介してインスリン分泌を促進するという新たな仮説を提唱した。
発明の開示
CD38は膜結合型蛋白で可溶性部分が血中流出し、機能発現すると考えられる。そこで、CD38のアミノ酸配列から細胞膜外にあり、機能調節に関与していると推定されるペプチド断片３種類を用い、抗CD38抗体陽性血清を測定したところ、３種類のペプチド断片のうちＣ末端部分（配列番号１）のペプチドが最も強い反応を示した。
本発明者は膵島細胞においてCD38免疫活性が細胞外周（おそらく細胞表面）に局在し、さらに市販の抗CD38モノクローナル抗体処理によってインスリン分泌が有意に抑制されることを見出した。
糖尿病患者において抗CD38抗体の存在を検討したところ、IDDM患者血清の約68％に検出された。さらに、IDDMの最適モデル動物として評価の確立しているNODマウスにおいては、膵島における自己免疫的炎症が起こっている第７週齢以後、50-70%に抗CD38抗体が検出された。このように、ヒトおよびモデル動物の血中にCD38に対する自己抗体が出現していることを見出した。
この抗体はそれ自体がインスリン分泌を抑制していると同時に、膵島において膵島炎が起こりβ細胞の破壊が進行していることを示すマーカーとしての意義を有すると考えられる。従って、抗CD38抗体はインスリン分泌機構に深く関与していることから、抗CD38抗体を測定することはIDDMおよび膵島炎の発病予知に寄与し得ると考えられる。
一方、抗CD38抗体によりインスリン分泌が抑制されることからCD38はインスリン分泌の調節・制御に重要な役割を果たしている可能性がある。
本発明はCD38のＣ末端部分のペプチドを用い、糖尿病患者血清中の抗CD38自己抗体の測定系を確立し、抗CD38自己抗体と糖尿病発生機構との関連性について解析を行った結果を基礎とする。
即ち、本発明はCD38ペプチド断片を用いた抗CD38自己抗体の測定方法、CD38ペプチド断片を用いた自己免疫疾患治療用医薬組成物、および配列番号１記載のアミノ酸配列を有するペプチドに関する。
本発明はCD38ペプチド断片、好ましくはCD38ペプチドの細胞外領域における10−30アミノ酸残基からなるペプチド断片、より好ましくは配列番号１記載のアミノ酸配列を含むペプチドまたはそのアミノ酸配列に１または数個のアミノ酸残基の置換、欠失、挿入および付加の中から選ばれる少なくとも１つの変異を有するペプチドを用いた抗CD38自己抗体の測定方法に関する。
抗CD38自己抗体の測定は競合法、サンドウィッチ法等を用いることができる。例えば、CD38ペプチド断片をマイクロタイタープレートにアビジン−ビオチン法などにより固相し、患者より入手した検体、例えば血清、血漿を加え、ペルオキシダーゼまたはアルカリホスファターゼなどで標識した抗CD38抗体に対する抗体を反応させ、対応する基質、例えばオルトフェニレンジアミンまたはテトラメチルベンチジン、あるいは4-ニトロフェニルホスフェートを加え、呈色反応を行った後、吸光度を測定する。
また、本発明のペプチド断片またはその変異体はヒトCD38のアミノ酸配列（Jackson DG．，Bell JI．，J Immonol．144，2811-2815，1990）に基づいてメリーフィールド固相合成方法（Merrifield，R．B．，J．Am．Chem．Soc．，85，2149-2156（1963））により化学合成することができる。
本発明はさらに、化学的に修飾されていてもよい、CD38ペプチド、そのペプチド断片またはその変異体を含有する自己免疫疾患治療用医薬組成物、特に自己免疫疾患が膵島炎または糖尿病である該組成物に関する。
「化学的修飾」とは治療薬としてペプチドを投与する場合、生体中に存在する様々なプロテアーゼによる分解から保護するために行われるあらゆる修飾を意味し、典型的にはポリエチレングリコール（PEG）などでペプチドを修飾することを意味する。例えば、治療薬としてペプチドを投与すると生体中に存在する様々なプロテアーゼによる分解を受ける場合がある。そのためペプチドを治療薬として投与する場合、大量投与しなければその効果は期待できないが、生体への影響から大量投与は避けなければならない。そのためプロテアーゼ等の影響を受けにくい形態で投与すれば少量でかつ有効濃度をより長く維持することが可能となる。インターロイキン（IL）等をPEG等で修飾することにより、生体中での分解が抑制され有効濃度が長時間維持されることが報告されている。そこで、それらのペプチド修飾方法をCD38ペプチド、そのペプチド断片およびその変異体に応用することができる。
CD38ペプチドのペプチド断片とは配列番号２記載のアミノ酸配列から任意に選ばれる５以上のアミノ酸残基を有するペプチドであり、好ましくは細胞外領域を含むペプチド、より好ましくは自己抗体の抗原部位を含むペプチドを意味する。
CD38ペプチドまたはそのペプチド断片の変異体とはCD38ペプチドまたはそのペプチド断片のアミノ酸配列に１または数個のアミノ酸の置換、欠失、挿入および付加等の中から選ばれる少なくとも１つの変異を有するペプチドを意味する。このようなペプチドおよびそれらの変異体の作製方法はMolecular Cloning A Laboratory Manual 第２版（Maniatis，T．et al，Cold Spring Harbor Laboratories（New York），（1989））に記載されている。
インスリン依存型糖尿病は膵臓が自己免疫反応によりβ細胞が破壊され、インスリン分泌機能が低下することに起因する自己免疫疾患である。一般に、自己免疫疾患に対して自己抗原投与により免疫応答を起こさなくする状態、つまり「免疫寛容」を誘導することによって抗原特異的に免疫抑制し、発病を防ぐ治療方法がある。従って、本発明のCD38ペプチド、その断片およびそれらの変異体は自己免疫疾患であるIDDMの治療に用い得る。これらは膵島に対する自己抗体の攻撃を中和する効果も期待できる。
一般に、自己抗原タンパク質、抗原ペプチドなどの投与方法は抗原量、投与経路および抗原形態などの様々な要素が関係する。このうち特に治療への応用が検討されているのは経口投与による「免疫寛容」の誘導である。抗原が経口的に摂取された時に生じる免疫寛容を「経口トレランス」といい、原因抗原が明確な自己免疫疾患の治療方法としては経口投与が有用と考えられている。自己免疫疾患である慢性関節リュウマチにおいて原因抗原であるコラーゲンの経口投与により関節炎の発生が抑制されたことが報告されており、臨床への応用も試みられている。そこで、慢性関節リュウマチでのコラーゲン療法を応用し、IDDMおよび膵島炎においても発病前もしくは発病早期にCD38ペプチド、そのペプチド断片またはそれらの変異体を経口投与し、経口トレランスによる抗原特異的免疫抑制の有用性が示されれば患者の負担が大きく軽減され、画期的な治療薬となることが期待できる。
本発明の治療用医薬組成物に用いるCD38ペプチド、そのペプチド断片またはそれらの変異体は遺伝子工学的に作製されたものでもよい。
本発明は、さらに、経口、経鼻、静脈内、腹腔内および胸腺内投与可能な剤形から選ばれる治療用医薬組成物に関する。
本発明のペプチドを含有する治療用医薬組成物を投与する場合は通常の剤形、例えば、錠剤、散剤、顆粒剤、カプセル剤等の固形製剤；水剤；油性懸濁剤；シロップ剤またはエリキシル剤等の液剤；および水性または油製懸濁注射剤等を用いることができる。いずれの場合も、その調製に際しては、慣用の賦形剤、結合剤、水溶剤、油性溶剤、乳化剤、懸濁化剤等の担体を必要に応じて用いることができ、また、他の添加剤、例えば保存剤、安定化剤等を含む物であっても良い。
【図面の簡単な説明】
図１はインスリン放出作用に対する抗CD38抗体の作用を示すグラフである。
実施例
次に実施例によって本発明をさらに詳細に説明するが、本発明の範囲はこれらのみに限定されるものではない。
実施例１
CD38抗原のインスリン分泌機構における役割−CD38ペプチド断片
CD38抗原に対する抗体がインスリン分泌を抑制することから、この抗原はインスリン分泌機序において重要な役割を果たしていると推定される。一方、本抗原は様々な酵素活性などの多彩な機能を有することが知られている。今回は、CD38抗原の断片に対する抗体を作成してそれらのインスリン分泌に及ぼす効果について検討し、本抗原の構造−機能連関と、インスリン分泌機構に果たす意義について追求した。
方法
（１）Min 6細胞（膵臓β細胞由来インスリン分泌細胞株）：Min 6細胞は10％ FCS DMEM（Glucose 20mM）にて培養した。
（２）抗CD38抗体：抗CD38モノクローナル抗体（T10；マウスIgG1），および二種類のCD38ペプチド断片287-297（N-Cys Val Lys Asn Pro Glu Asp Ser Ser Cys Thr-C；287-Ag）、241-255（N-Cys Ser Asn Asn Pro Val Ser Val Phe Trp Lys Thr Val Ser Arg-C；241-Ag）をそれぞれKeyhole Limpet Hemocyanineと抱合し、家兎に免疫して作成したポリクローナル抗体を用いた。
（３）インスリン分泌：Min 6細胞を48穴プレートに培養し、分泌実験時には１％FCS添加KRB緩衝液を培地とした。これに抗CD38抗体、D-グルコース（5,20mM）、および種々のインスリン分泌刺激物質を加え、30分間で培養液に分泌されたインスリンをRIAによって測定した。
（４）［Ca2+］iの測定：1-［2-（5’カルボキシオキサゾル-2’-イル）-6-アミノベンゾフラン-5-オキシ］-2-（2’-アミノ-5’-メチルフェノキシ）エタン-N,N,N’,N’-四酢酸五カリウム塩（C29H22K5N30O14；Fura-2）AM（終濃度２μM）を負荷したMin 6細胞を、流速0.3ml/minで灌流しながらARGUS l00（浜松PHOTONICS社製）によって蛍光340nm/380nm比を測光し、［Ca2+］iを経時的に測定した。
成績
（１）抗CD38モノクローナル抗体を前処理することによって、D-glucose（20mM）、D-mannose（20mM）、L-arginine（10mM）、α-KIC（10mM）、glucagon（１μM）、forskolin（10μM）、Carbamylcholine（100μM）、などによるインスリン分泌が強く抑制された。得られた結果を図１に示す。
（２）287-Abを用いた場合には、抗CD38モノクローナル抗体とほぼ同様の効果が見られた。なお、CD38抗原の断片67-81（N-Cys Val Lys Tyr Thr Glu Ile His Pro Glu Met Arg His Val Asp-C；67-Ag）を免疫抗原として得られた抗体67-Abを用いた場合287-Abのような強いインスリン分泌抑制効果は得られなかった。
（３）241-Abはインスリン分泌に明らかな効果を及ぼさなかった。
（４）抗CD38モノクローナル抗体および287-Abの処理はD-glucoseによる［Ca2+］i反応を遅延、低下させた。
結論
CD38抗原のなかでも特に、フラグメント287-297近傍がインスリン分泌、［Ca2+］i反応、すなわち膵β細胞内情報伝達系に深く関与しており、この膜外蛋白の構造−機能連関の重要な意義が確認された。
実施例２
抗CD38抗体の測定
方法
配列番号１に記載のペプチド287-Agを抗原とし、EIA用96穴プレートへ吸着・固相化を行った後、BSAで一昼夜ブロッキングを行い測定に用いた。サンプルとしてヒト、ラット、マウス等の血清を用いた。上記で作製した抗原固相96穴マイクロプレートに血清サンプルを添加し、室温で約60分間緩やかに振盪で十分に反応させる。血清サンプルを捨て、洗浄操作を十分に行った後、ビオチン化二次抗体液を加える。
ビオチン化二次抗体は血清サンプルがヒトの場合にはビオチン化抗ヒトIgGを、ラット、マウスの場合にはそれぞれビオチン化抗ラットIgG、ビオチン化抗マウスIgGを用いる。室温で約60分間緩やかに振盪した後、洗浄操作を十分に行う。次いでアビジン−DHおよび、ビオチン化ペルオキシダーゼまたはビオチン化アルカリフォスファターゼを加えて、室温で約30分間緩やかに振盪する。ABC試薬を捨て、PBSを100ml／wellずつ加えて約５分間緩やかに振盪した後、洗浄操作を十分に行う。基質（オルトフェニレンジアミン（OP）、テトラメチルベンチジン（TMB）、4-ニトロフェニルフォスフェートまたは2,2’-アジノ-ビス（1-エチルベンゾチアゾリン-6-スルホン酸）（ABTS）等）溶液を加え、室温、暗所で呈色反応を起こさせ、吸光度を計測する。
成績
ヒトにおいては150例の糖尿病患者血清を用いて、抗CD38抗体を測定した。その結果、IDDMにおいては32例中22例（68％）が陽性であり、これに対してインスリン非依存型糖尿病（NIDDM）においては128例中16例（12.5％）のみが陽性であった。
IDDMのモデル動物であるNODマウスにおいては、膵島における自己免疫的炎症（膵島炎）が起こっている第７週齢以降では84％の陽性率であった。本マウスの系では第11週齢以降に糖尿病が発病する。なお、膵島炎が未だ起こっていない第５週齢では全例が陰性であった。
結論
抗CD38抗体は糖尿病発病前、膵島炎出現と同時期に検出されることから、糖尿病発病の予知に有用であり、インスリン依存型糖尿病の早期治療、発病予防に寄与しうるものと考えられる。
配列表
配列番号：１
配列の長さ：１１
配列の型：ペプチド
配列：
配列の長さ：３００
配列の型：アミノ酸
トポロジー：直鎖状
配列の種類：ペプチド
配列：
Background Art Insulin-dependent diabetes mellitus (IDDM) is caused by autoimmune inflammation (islet inflammation) in pancreatic islets, destroying cells that produce and secrete insulin (β cells), and insulin is extremely deficient. Therefore, as the fundamental treatment of IDDM and pancreatic isletitis, it is necessary to carry out pretreatment, early treatment, that is, treatment before pancreatic β cells are destroyed, and the early diagnosis method is important. However, currently only a diagnostic method after pathogenesis by pathological anatomy or biopsy is performed, and insulin-producing cells have already been destroyed at the time of diagnosis. Since diagnosing by the above methods such as pathological anatomy before the onset of disease is ethically impossible, various methods for diagnosing patients with insulin-dependent diabetes and pancreatic insulitis before the onset of disease have been attempted. It was.
CD38 is a glycoprotein with a molecular weight of 46 kDa and is found mainly on the surface of lymphocytes, as a marker for differentiation and activation of T cells and B cells, and as an index of malignancy (juvenileity) of T cells and B cells. It is used. Recently, CD38 is not limited to leukocytes or immune cells, but also from rat brain, duodenum, heart, pituitary gland, and pancreatic islets by Northern blot analysis using cDNA of CD38 molecule as a probe and immunohistochemical staining using a specific antibody against CD38 molecule. It was also confirmed that
It was also revealed that CD38 generates cyclic ADP-ribose (cADPR) in the cell and regulates the movement of intracellular calcium via this cADPR. In 1993, Okamoto et al. Proposed a new hypothesis that cADPR promotes insulin secretion through increased intracellular calcium in islet cells.
Disclosure of the invention
CD38 is a membrane-bound protein and the soluble part is thought to be expressed in the blood. Thus, anti-CD38 antibody-positive sera were measured using three types of peptide fragments that were estimated to be involved in functional regulation from the amino acid sequence of CD38. The C-terminal portion of the three types of peptide fragments was measured. The peptide of (SEQ ID NO: 1) showed the strongest reaction.
The present inventor has found that CD38 immunoreactivity is localized around the cell periphery (probably the cell surface) in islet cells, and that insulin secretion is significantly suppressed by treatment with a commercially available anti-CD38 monoclonal antibody.
The presence of anti-CD38 antibody in diabetic patients was detected in about 68% of IDDM patient sera. Furthermore, in the NOD mouse, which has been evaluated as an optimal model animal for IDDM, anti-CD38 antibody was detected in 50-70% after the seventh week when autoimmune inflammation occurred in the islets. Thus, it was found that autoantibodies against CD38 appeared in the blood of humans and model animals.
This antibody is considered to have significance as a marker that itself suppresses insulin secretion and at the same time indicates that pancreatic islet inflammation has occurred and β-cell destruction is in progress. Therefore, since anti-CD38 antibody is deeply involved in the insulin secretion mechanism, it is considered that measuring anti-CD38 antibody can contribute to the onset prediction of IDDM and isletitis.
On the other hand, since insulin secretion is suppressed by the anti-CD38 antibody, CD38 may play an important role in regulation and control of insulin secretion.
The present invention uses a peptide at the C-terminal part of CD38, establishes a measurement system for anti-CD38 autoantibodies in the serum of diabetic patients, and analyzes the relationship between anti-CD38 autoantibodies and the mechanism of diabetes development. To do.
That is, the present invention relates to a method for measuring an anti-CD38 autoantibody using a CD38 peptide fragment, a pharmaceutical composition for treating an autoimmune disease using a CD38 peptide fragment, and a peptide having the amino acid sequence set forth in SEQ ID NO: 1.
The present invention relates to a CD38 peptide fragment, preferably a peptide fragment consisting of 10-30 amino acid residues in the extracellular region of the CD38 peptide, more preferably a peptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or one or several amino acid sequences thereof. The present invention relates to a method for measuring an anti-CD38 autoantibody using a peptide having at least one mutation selected from amino acid residue substitution, deletion, insertion and addition.
For the measurement of the anti-CD38 autoantibody, a competitive method, a sandwich method or the like can be used. For example, a CD38 peptide fragment is solid-phased on a microtiter plate by an avidin-biotin method, a sample obtained from a patient, such as serum or plasma, is added, and an antibody against an anti-CD38 antibody labeled with peroxidase or alkaline phosphatase is reacted. Corresponding substrates such as orthophenylenediamine or tetramethylbenzidine, or 4-nitrophenyl phosphate are added, and after performing a color reaction, the absorbance is measured.
In addition, the peptide fragment of the present invention or a variant thereof is based on the amino acid sequence of human CD38 (Jackson DG., Bell JI., J Immonol. 144, 2811-2815, 1990). B., J. Am. Chem. Soc., 85, 2149-2156 (1963)).
The present invention further relates to a pharmaceutical composition for treating an autoimmune disease containing a CD38 peptide, a peptide fragment thereof or a variant thereof, which may be chemically modified, particularly the composition wherein the autoimmune disease is pancreatic insulitis or diabetes. Related to things.
“Chemical modification” means any modification made to protect against degradation by various proteases present in the body when a peptide is administered as a therapeutic agent, typically with polyethylene glycol (PEG), etc. It means modifying the peptide. For example, when a peptide is administered as a therapeutic agent, it may be degraded by various proteases present in the living body. Therefore, when a peptide is administered as a therapeutic agent, its effect cannot be expected unless it is administered in a large amount, but a large amount of administration must be avoided because of its influence on the living body. Therefore, if it is administered in a form that is not easily affected by protease or the like, a small amount and an effective concentration can be maintained longer. It has been reported that by modifying interleukin (IL) or the like with PEG or the like, degradation in the living body is suppressed and the effective concentration is maintained for a long time. Therefore, these peptide modification methods can be applied to the CD38 peptide, its peptide fragment and its variants.
The peptide fragment of CD38 peptide is a peptide having 5 or more amino acid residues arbitrarily selected from the amino acid sequence shown in SEQ ID NO: 2, preferably a peptide containing an extracellular region, more preferably an antigen site of an autoantibody. Means peptide.
A variant of CD38 peptide or peptide fragment thereof is a peptide having at least one mutation selected from substitution, deletion, insertion and addition of one or several amino acids in the amino acid sequence of CD38 peptide or peptide fragment thereof. means. Methods for producing such peptides and their variants are described in Molecular Cloning A Laboratory Manual Second Edition (Maniatis, T. et al, Cold Spring Harbor Laboratories (New York), (1989)).
Insulin-dependent diabetes mellitus is an autoimmune disease caused by the β-cells being destroyed by the autoimmune reaction in the pancreas and the insulin secretion function being lowered. In general, there is a therapeutic method for preventing autoimmune diseases by causing immunosuppression in an antigen-specific manner by inducing a state in which an immune response is not caused by autoantigen administration, that is, “immune tolerance”. Therefore, the CD38 peptide of the present invention, fragments thereof and variants thereof can be used for the treatment of IDDM, which is an autoimmune disease. They can also be expected to neutralize autoantibody attack on the islets.
In general, administration methods such as self-antigen proteins and antigen peptides involve various factors such as antigen amount, administration route, and antigen form. Of these, the application to treatment is being investigated especially for induction of “immune tolerance” by oral administration. Immune tolerance that occurs when an antigen is taken orally is called "oral tolerance", and oral administration is considered useful as a method for treating autoimmune diseases with a clear cause antigen. In rheumatoid arthritis, which is an autoimmune disease, it has been reported that the occurrence of arthritis was suppressed by oral administration of collagen, which is the causative antigen, and clinical application has also been attempted. Therefore, by applying collagen therapy in rheumatoid arthritis, CD38 peptide, its peptide fragments or their variants are orally administered before or early in IDDM and insulitis, and antigen-specific immunosuppression by oral tolerance is achieved. If usefulness is shown, it can be expected that the burden on patients will be greatly reduced and that it will be an epoch-making therapeutic agent.
The CD38 peptide, peptide fragment thereof or variant thereof used in the therapeutic pharmaceutical composition of the present invention may be produced by genetic engineering.
The present invention further relates to a therapeutic pharmaceutical composition selected from dosage forms that can be administered orally, nasally, intravenously, intraperitoneally and intrathymically.
When a therapeutic pharmaceutical composition containing the peptide of the present invention is administered, a usual dosage form, for example, solid preparations such as tablets, powders, granules, capsules, etc .; liquids; oily suspensions; syrups or elixirs Liquid agents such as agents; and aqueous or oil suspension injections can be used. In any case, carriers such as conventional excipients, binders, aqueous solvents, oily solvents, emulsifiers, and suspending agents can be used as necessary in the preparation, and other additives can be used. For example, a substance containing a preservative, a stabilizer and the like may be used.
[Brief description of the drawings]
FIG. 1 is a graph showing the action of an anti-CD38 antibody on the insulin releasing action.
EXAMPLES Next, the present invention will be described in more detail by way of examples. However, the scope of the present invention is not limited to these examples.
Example 1
Role of CD38 antigen in insulin secretion mechanism-CD38 peptide fragment
Since an antibody against CD38 antigen suppresses insulin secretion, it is presumed that this antigen plays an important role in the mechanism of insulin secretion. On the other hand, this antigen is known to have various functions such as various enzyme activities. In this study, antibodies against CD38 antigen fragments were prepared and their effects on insulin secretion were examined, and the structure-function relationship of this antigen and the significance of this antigen in the mechanism of insulin secretion were pursued.
Method (1) Min 6 cells (insulin-secreting cell line derived from pancreatic β cells): Min 6 cells were cultured in 10% FCS DMEM (
(2) Anti-CD38 antibody: anti-CD38 monoclonal antibody (T10; mouse IgG1), and two types of CD38 peptide fragments 287-297 (N-Cys Val Lys Asn Pro Glu Asp Ser Ser Cys Thr-C; 287-Ag), 241-255 (N-Cys Ser Asn Asn Pro Val Ser Val Phe Trp Lys Thr Val Ser Arg-C; 241-Ag) was conjugated with Keyhole Limpet Hemocyanine, and a polyclonal antibody prepared by immunizing rabbits was used. .
(3) Insulin secretion: Min 6 cells were cultured in a 48-well plate, and 1% FCS-added KRB buffer was used as the medium during the secretion experiment. To this, an anti-CD38 antibody, D-glucose (5,20 mM), and various insulin secretagogues were added, and insulin secreted into the culture medium for 30 minutes was measured by RIA.
(4) Measurement of [Ca 2+ ] i: 1- [2- (5′carboxyoxazol-2′-yl) -6-aminobenzofuran-5-oxy] -2- (2′-amino-5 ′ Min 6 cells loaded with 5 -methylphenoxy) ethane-N, N, N ′, N′-tetraacetic acid pentapotassium salt (C 29 H 22 K 5 N 30 O 14 ; Fura-2) AM (final concentration 2 μM) While perfusing at a flow rate of 0.3 ml / min, the fluorescence 340 nm / 380 nm ratio was measured with ARGUS 100 (manufactured by Hamamatsu Photonics), and [Ca 2+ ] i was measured over time.
Results (1) By pre-treatment with anti-CD38 monoclonal antibody, D-glucose (20 mM), D-mannose (20 mM), L-arginine (10 mM), α-KIC (10 mM), glucagon (1 μM), forskolin ( 10 μM), Carbamylcholine (100 μM), etc. strongly inhibited insulin secretion. The obtained results are shown in FIG.
(2) When 287-Ab was used, almost the same effect as the anti-CD38 monoclonal antibody was observed. In the case of using antibody 67-Ab obtained by using CD38 antigen fragment 67-81 (N-Cys Val Lys Tyr Thr Glu Ile His Pro Glu Met Arg His Val Asp-C; 67-Ag) as an immunizing antigen 287 The strong insulin secretion inhibitory effect like -Ab was not obtained.
(3) 241-Ab had no obvious effect on insulin secretion.
(4) Treatment with anti-CD38 monoclonal antibody and 287-Ab delayed and reduced the [Ca 2+ ] i reaction by D-glucose.
Conclusion
Among CD38 antigens, in particular, the vicinity of fragment 287-297 is deeply involved in insulin secretion, [Ca 2+ ] i reaction, ie, pancreatic β-cell signal transduction system. The significance was confirmed.
Example 2
Measurement method of anti-CD38 antibody Peptide 287-Ag described in SEQ ID NO: 1 was used as an antigen, adsorbed and immobilized on a 96-well plate for EIA, and then blocked for 24 hours with BSA and used for measurement. Serum from human, rat, mouse, etc. was used as a sample. A serum sample is added to the antigen solid phase 96-well microplate prepared above and allowed to react sufficiently by gentle shaking at room temperature for about 60 minutes. After discarding the serum sample and thoroughly washing, add the biotinylated secondary antibody solution.
As the biotinylated secondary antibody, biotinylated anti-human IgG is used when the serum sample is human, and biotinylated anti-rat IgG and biotinylated anti-mouse IgG are used when the serum sample is rat or mouse, respectively. After gently shaking at room temperature for about 60 minutes, the washing operation is performed sufficiently. Avidin-DH and biotinylated peroxidase or biotinylated alkaline phosphatase are then added and gently shaken for about 30 minutes at room temperature. Discard the ABC reagent, add 100 ml / well of PBS and shake gently for about 5 minutes, and then wash thoroughly. Substrate (orthophenylenediamine (OP), tetramethylbenzidine (TMB), 4-nitrophenyl phosphate or 2,2'-azino-bis (1-ethylbenzothiazoline-6-sulfonic acid) (ABTS) solution) To cause a color reaction in the dark at room temperature and measure the absorbance.
Results In humans, anti-CD38 antibody was measured using 150 diabetic sera. As a result, 22 of 32 cases (68%) were positive in IDDM, whereas only 16 out of 128 cases (12.5%) were positive in non-insulin dependent diabetes mellitus (NIDDM).
The NOD mouse, which is a model animal of IDDM, had a positive rate of 84% after 7 weeks of age when autoimmune inflammation (islet inflammation) occurred in the islets. In this mouse system, diabetes develops after 11 weeks of age. In addition, all the cases were negative at the 5th week age when pancreatic insulitis has not yet occurred.
Conclusion Since anti-CD38 antibody is detected before the onset of diabetes and at the same time as the onset of pancreatic insulitis, it is useful for the prediction of the onset of diabetes and may contribute to the early treatment and prevention of the onset of insulin-dependent diabetes.
Sequence Listing SEQ ID NO: 1
Sequence length: 11
Sequence type: Peptide sequence:
Sequence length: 300
Sequence type: Amino acid Topology: Linear sequence type: Peptide sequence:
Claims (7)
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| JP27253796 | 1996-10-15 | ||
| PCT/JP1997/001259 WO1998016245A1 (en) | 1996-10-15 | 1997-04-11 | Method of autoantibody assay |
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| US6586008B1 (en) * | 1999-08-25 | 2003-07-01 | Advanced Inhalation Research, Inc. | Use of simple amino acids to form porous particles during spray drying |
| MX2007011064A (en) | 2005-03-23 | 2008-02-19 | Genmab As | Antibodies against cd38 for treatment of multiple myeloma. |
| CN103554259B (en) * | 2005-10-12 | 2016-05-18 | 莫佛塞斯公司 | Specificity is for generation and the qualification of the derivative treatment of the complete people HuCAL GOLD-antibody of people CD38 |
| TR201910145T4 (en) | 2006-09-26 | 2019-08-21 | Genmab As | Anti-CD38 plus corticosteroids plus a corticosteroid non-chemotherapeutic for treating tumors. |
| RS59769B1 (en) | 2010-06-09 | 2020-02-28 | Genmab As | Antibodies against human cd38 |
| US9732154B2 (en) | 2014-02-28 | 2017-08-15 | Janssen Biotech, Inc. | Anti-CD38 antibodies for treatment of acute lymphoblastic leukemia |
| US9603927B2 (en) | 2014-02-28 | 2017-03-28 | Janssen Biotech, Inc. | Combination therapies with anti-CD38 antibodies |
| WO2016040294A2 (en) | 2014-09-09 | 2016-03-17 | Janssen Biotech, Inc. | Combination therapies with anti-cd38 antibodies |
| CA2969717A1 (en) | 2014-12-04 | 2016-06-09 | Janssen Biotech, Inc. | Anti-cd38 antibodies for treatment of acute myeloid leukemia |
| BR112017024877A2 (en) | 2015-05-20 | 2019-09-17 | Janssen Biotech, Inc. | anti-cd38 antibody and its use in the treatment of light chain amyloidosis and other cd38 positive haematological malignancies |
| MX2018000265A (en) | 2015-06-22 | 2018-05-23 | Janssen Biotech Inc | Combination therapies for heme malignancies with anti-cd38 antibodies and survivin inhibitors. |
| US20170044265A1 (en) | 2015-06-24 | 2017-02-16 | Janssen Biotech, Inc. | Immune Modulation and Treatment of Solid Tumors with Antibodies that Specifically Bind CD38 |
| US10781261B2 (en) | 2015-11-03 | 2020-09-22 | Janssen Biotech, Inc. | Subcutaneous formulations of anti-CD38 antibodies and their uses |
| PE20181365A1 (en) | 2015-11-03 | 2018-08-27 | Janssen Biotech Inc | SUBCUTANEOUS FORMULATIONS OF ANTI-CD38 ANTIBODIES AND THEIR USES |
| WO2019089832A1 (en) | 2017-10-31 | 2019-05-09 | Janssen Biotech, Inc. | Methods of treating high risk multiple myeloma |
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