WO2023033049A1 - Antibody and antibody fragment having binding properties to mutant sod1 protein causing amyotrophic lateral sclerosis - Google Patents
Antibody and antibody fragment having binding properties to mutant sod1 protein causing amyotrophic lateral sclerosis Download PDFInfo
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Abstract
Disclosed is an antibody or antibody fragment having binding properties to the mutant SOD1 protein, including: a heavy chain variable region including heavy chain CDR1 composed of an amino acid sequence of GFSLNTSGMG (SEQ ID NO: 1), heavy chain CDR2 composed of an amino acid sequence of IWWDDDK (SEQ ID NO: 2), and heavy chain CDR3 composed of an amino acid sequence of ARLGYAMDY (SEQ ID NO: 3), which may have an amino acid substitution of three or less; and/or a light chain variable region including light chain CDR1 composed of an amino acid sequence of QNVGGTN (SEQ ID NO: 4), light chain CDR2 composed of an amino acid sequence of SAS, and light chain CDR3 composed of an amino acid sequence of QQYYIYPYT (SEQ ID NO: 5), which may have an amino acid substitution of three or less. Also disclosed are: a nucleic acid for encoding the antibody or antibody fragment; a vector containing the nucleic acid; a diagnostic agent and diagnostic kit containing the antibody or antibody fragment; and a pharmaceutical composition containing the antibody or antibody fragment and the nucleic acid or the vector.
Description
本発明は、変異型SOD1タンパク質に結合性を有する抗体又は抗体断片に関する。さらに、本発明は、該抗体又は抗体断片をコードする核酸、該核酸を含むベクター、該抗体又は抗体断片を含む診断薬及び診断用キット、並びに該抗体若しくは抗体断片、該核酸又は該ベクターを含む医薬組成物に関する。
The present invention relates to antibodies or antibody fragments that have binding properties to mutant SOD1 proteins. Furthermore, the present invention includes nucleic acids encoding said antibodies or antibody fragments, vectors containing said nucleic acids, diagnostic agents and diagnostic kits containing said antibodies or antibody fragments, and said antibodies or antibody fragments, said nucleic acids or said vectors. It relates to pharmaceutical compositions.
筋萎縮性側索硬化症(Amyotrophic lateral sclerosis: ALS)は、60～70歳で発症する全身の筋萎縮と筋力低下とを主症状とする神経変性疾患であり、発症後4年以内に致死性の嚥下障害と呼吸不全とを呈する。ALS患者の20%は家族性で、その30%はCu/Zn superoxide dismutase I (SOD1)遺伝子に突然変異を有し、その突然変異の数は200種類にも及ぶ。そして、ALSでは、変異型SOD1タンパク質が、異常蓄積することが知られている。
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by generalized muscle atrophy and weakness that develops between the ages of 60 and 70, and is fatal within 4 years after onset. dysphagia and respiratory failure. Twenty percent of ALS patients are familial, and 30% of them have mutations in the Cu/Zn superoxide dismutase I (SOD1) gene, with as many as 200 mutations. In ALS, it is known that the mutant SOD1 protein is abnormally accumulated.
SOD1はスーパーオキシドという活性酸素を除去する酵素であるが、変異型SOD1による神経毒性は抗活性酸素活性とは無関係であり、変異タンパク質の除去が創薬開発の主要な戦略となっている。そして近年SOD1に対するアンチセンスオリゴヌクレオチド治療が実用化され、いよいよ難治性ALSも発症阻止、進行停止に向けた先進医療の恩恵に浴する時代を迎えており、早期診断がますます重要となっている。
SOD1 is an enzyme that removes superoxide, a reactive oxygen species, but the neurotoxicity of mutant SOD1 is unrelated to its anti-reactive oxygen activity, and removal of mutant proteins has become a major strategy for drug development. In recent years, antisense oligonucleotide therapy against SOD1 has been put to practical use, and we are entering an era in which we can benefit from advanced medical treatment aimed at preventing the onset and progression of refractory ALS, and early diagnosis is becoming more and more important. .
このようなALSの診断及び治療に使用するための抗体について、各種の抗体が報告されている(例えば、特許文献１－４及び非特許文献１)。
Various antibodies have been reported for use in diagnosing and treating such ALS (eg, Patent Documents 1-4 and Non-Patent Document 1).
本発明は、幅広い種類の変異型SOD1タンパク質に結合性を有する抗体又は抗体断片を提供することを目的とする。
The purpose of the present invention is to provide antibodies or antibody fragments that have binding properties to a wide variety of mutant SOD1 proteins.
さらに、本発明は、該抗体又は抗体断片をコードする核酸、該核酸を含むベクター、該抗体又は抗体断片を含む診断薬及び診断用キット、並びに該抗体若しくは抗体断片、該核酸又は該ベクターを含む医薬組成物を提供することを目的とする。
Furthermore, the present invention includes nucleic acids encoding said antibodies or antibody fragments, vectors containing said nucleic acids, diagnostic agents and diagnostic kits containing said antibodies or antibody fragments, and said antibodies or antibody fragments, said nucleic acids or said vectors. The object is to provide a pharmaceutical composition.
本発明者らは、上記目的を達成すべく鋭意研究を重ねた結果、本発明者らが取得した抗体について試験したところ、アミノ酸置換の部位を問わず、多くの変異型SOD1タンパク質を認識し、異なる変異型SOD1遺伝子を有するヒト組織での染色性を確認し、ALSモデルラットに抗体を投与することでALSの進行抑制と寿命延長効果とが見られるという知見を得た。
The present inventors have conducted intensive studies to achieve the above object, and as a result of testing the antibodies obtained by the present inventors, they recognize many mutant SOD1 proteins regardless of the site of amino acid substitution, We confirmed staining in human tissues with different mutant SOD1 genes, and found that administration of the antibody to ALS model rats inhibited the progression of ALS and extended lifespan.
本発明は、これら知見に基づき、更に検討を重ねて完成されたものであり、次の抗体又は抗体断片、核酸、ベクター、診断薬、診断用キット、及び医薬組成物を提供するものである。
The present invention was completed through further studies based on these findings, and provides the following antibodies or antibody fragments, nucleic acids, vectors, diagnostic agents, diagnostic kits, and pharmaceutical compositions.
項１．変異型SOD1タンパク質に結合性を有し、
3以下のアミノ酸置換をしてもよい、GFSLNTSGMG (配列番号１)のアミノ酸配列からなる重鎖CDR1、IWWDDDK (配列番号２)のアミノ酸配列からなる重鎖CDR2、及びARLGYAMDY (配列番号３)のアミノ酸配列からなる重鎖CDR3を含む重鎖可変領域、並びに／又は
3以下のアミノ酸置換をしてもよい、QNVGGTN (配列番号４)のアミノ酸配列からなる軽鎖CDR1、SASのアミノ酸配列からなる軽鎖CDR2、及びQQYYIYPYT (配列番号５)のアミノ酸配列からなる軽鎖CDR3を含む軽鎖可変領域
を含む、抗体又は抗体断片。
項２．前記抗体断片がFab、Fab'、F(ab')2、Fv、scFv、dsFv、ds-scFv、ダイアボディ又はミニボディである、項１に記載の抗体又は抗体断片。
項３．前記抗体断片がscFvである、項１又は２に記載の抗体又は抗体断片。
項４．前記抗体又は抗体断片がヒト化抗体又は抗体断片である、項１～３のいずれか一項に記載の抗体又は抗体断片。
項５．項１～４のいずれか一項に記載の抗体又は抗体断片をコードする核酸。
項６．項５に記載の核酸を含むベクター。
項７．項１～４のいずれか一項に記載の抗体又は抗体断片を含む診断薬。
項８．筋萎縮性側索硬化症の診断用である、項７に記載の診断薬。
項９．項１～４のいずれか一項に記載の抗体又は抗体断片を含む診断用キット。
項１０．項１～４のいずれか一項に記載の抗体若しくは抗体断片、項５に記載の核酸、又は項６に記載のベクターを含む医薬組成物。
項１１．筋萎縮性側索硬化症の治療及び／又は予防用である、項１０に記載の医薬組成物。
項１２．筋萎縮性側索硬化症等の変異型SOD1が蓄積する疾患の診断方法であって、
(1) 被検体から採取された生体試料について項１～４のいずれか一項に記載の抗体又は抗体断片を用いて変異型SOD1を検出する工程を含む、方法。
項１３．さらに、(2) 前記工程(1)で測定された前記変異型SOD1の量又は濃度の値が、予め設定されたカットオフ値以上である場合に、前記被検体が筋萎縮性側索硬化症等の変異型SOD1が蓄積する疾患の発症の可能性が高いと判定する工程を含む、項１２に記載の方法。
項１４．項１～４のいずれか一項に記載の抗体若しくは抗体断片、項５に記載の核酸、又は項６に記載のベクターの有効量を哺乳動物に投与する工程を含む、筋萎縮性側索硬化症等の変異型SOD1が蓄積する疾患の治療方法。
項１５．筋萎縮性側索硬化症等の変異型SOD1が蓄積する疾患の診断用の診断薬の製造における、項１～４のいずれか一項に記載の抗体又は抗体断片の使用。
項１６．筋萎縮性側索硬化症等の変異型SOD1が蓄積する疾患の治療及び／又は予防用医薬組成物の製造における、項１～４のいずれか一項に記載の抗体若しくは抗体断片、項５に記載の核酸、又は項６に記載のベクターの使用。 Section 1. have binding properties to mutant SOD1 proteins,
A heavy chain CDR1 consisting of the amino acid sequence of GFSLNTSGMG (SEQ ID NO: 1), a heavy chain CDR2 consisting of the amino acid sequence of IWWDDDK (SEQ ID NO: 2), and the amino acids of ARLGYAMDY (SEQ ID NO: 3), which may have up to 3 amino acid substitutions A heavy chain variable region comprising a heavy chain CDR3 consisting of a sequence, and/or a light chain consisting of a light chain CDR1 consisting of the amino acid sequence of QNVGGTN (SEQ ID NO: 4), which may have 3 or less amino acid substitutions, and a light chain consisting of the SAS amino acid sequence. An antibody or antibody fragment comprising a light chain variable region comprising a CDR2 and a light chain CDR3 consisting of the amino acid sequence of QQYYIYPYT (SEQ ID NO: 5).
Section 2. 2. The antibody or antibody fragment of paragraph 1, wherein said antibody fragment is Fab, Fab', F(ab') 2 , Fv, scFv, dsFv, ds-scFv, diabodies or minibodies.
Item 3. Item 3. The antibody or antibody fragment according to Item 1 or 2, wherein the antibody fragment is scFv.
Section 4. Item 4. The antibody or antibody fragment according to any one of Items 1 to 3, wherein the antibody or antibody fragment is a humanized antibody or antibody fragment.
Item 5. A nucleic acid encoding the antibody or antibody fragment of any one of Items 1-4.
Item 6. Item 6. A vector comprising the nucleic acid of Item 5.
Item 7. A diagnostic agent comprising the antibody or antibody fragment according to any one of Items 1 to 4.
Item 8. Item 8. The diagnostic agent according toitem 7, which is used for diagnosing amyotrophic lateral sclerosis.
Item 9. A diagnostic kit comprising the antibody or antibody fragment according to any one of Items 1 to 4.
Item 10. A pharmaceutical composition comprising the antibody or antibody fragment of any one of Items 1 to 4, the nucleic acid of Item 5, or the vector of Item 6.
Item 11. Item 11. The pharmaceutical composition according to Item 10, which is for treatment and/or prevention of amyotrophic lateral sclerosis.
Item 12. A method for diagnosing a disease in which mutant SOD1 accumulates, such as amyotrophic lateral sclerosis, comprising:
(1) A method comprising the step of detecting mutant SOD1 in a biological sample collected from a subject using the antibody or antibody fragment according to any one of items 1 to 4.
Item 13. Furthermore, (2) the subject has amyotrophic lateral sclerosis when the amount or concentration of the mutant SOD1 measured in step (1) is equal to or greater than a preset cutoff value. Item 13. The method according to Item 12, comprising the step of determining that there is a high possibility of developing a disease in which mutant SOD1 such as SOD1 accumulates.
Item 14. Amyotrophic lateral sclerosis, comprising the step of administering to a mammal an effective amount of the antibody or antibody fragment of any one of items 1 to 4, the nucleic acid of item 5, or the vector of item 6. A method for treating a disease in which mutant SOD1 accumulates, such as disease.
Item 15. Item 5. Use of the antibody or antibody fragment according to any one of Items 1 to 4 in the production of a diagnostic agent for diagnosing diseases in which mutant SOD1 accumulates, such as amyotrophic lateral sclerosis.
Item 16. The antibody or antibody fragment according to any one of Items 1 to 4 in the production of a pharmaceutical composition for the treatment and/or prevention of diseases in which mutant SOD1 accumulates, such as amyotrophic lateral sclerosis; Use of the nucleic acid described or the vector of item 6.
3以下のアミノ酸置換をしてもよい、GFSLNTSGMG (配列番号１)のアミノ酸配列からなる重鎖CDR1、IWWDDDK (配列番号２)のアミノ酸配列からなる重鎖CDR2、及びARLGYAMDY (配列番号３)のアミノ酸配列からなる重鎖CDR3を含む重鎖可変領域、並びに／又は
3以下のアミノ酸置換をしてもよい、QNVGGTN (配列番号４)のアミノ酸配列からなる軽鎖CDR1、SASのアミノ酸配列からなる軽鎖CDR2、及びQQYYIYPYT (配列番号５)のアミノ酸配列からなる軽鎖CDR3を含む軽鎖可変領域
を含む、抗体又は抗体断片。
項２．前記抗体断片がFab、Fab'、F(ab')2、Fv、scFv、dsFv、ds-scFv、ダイアボディ又はミニボディである、項１に記載の抗体又は抗体断片。
項３．前記抗体断片がscFvである、項１又は２に記載の抗体又は抗体断片。
項４．前記抗体又は抗体断片がヒト化抗体又は抗体断片である、項１～３のいずれか一項に記載の抗体又は抗体断片。
項５．項１～４のいずれか一項に記載の抗体又は抗体断片をコードする核酸。
項６．項５に記載の核酸を含むベクター。
項７．項１～４のいずれか一項に記載の抗体又は抗体断片を含む診断薬。
項８．筋萎縮性側索硬化症の診断用である、項７に記載の診断薬。
項９．項１～４のいずれか一項に記載の抗体又は抗体断片を含む診断用キット。
項１０．項１～４のいずれか一項に記載の抗体若しくは抗体断片、項５に記載の核酸、又は項６に記載のベクターを含む医薬組成物。
項１１．筋萎縮性側索硬化症の治療及び／又は予防用である、項１０に記載の医薬組成物。
項１２．筋萎縮性側索硬化症等の変異型SOD1が蓄積する疾患の診断方法であって、
(1) 被検体から採取された生体試料について項１～４のいずれか一項に記載の抗体又は抗体断片を用いて変異型SOD1を検出する工程を含む、方法。
項１３．さらに、(2) 前記工程(1)で測定された前記変異型SOD1の量又は濃度の値が、予め設定されたカットオフ値以上である場合に、前記被検体が筋萎縮性側索硬化症等の変異型SOD1が蓄積する疾患の発症の可能性が高いと判定する工程を含む、項１２に記載の方法。
項１４．項１～４のいずれか一項に記載の抗体若しくは抗体断片、項５に記載の核酸、又は項６に記載のベクターの有効量を哺乳動物に投与する工程を含む、筋萎縮性側索硬化症等の変異型SOD1が蓄積する疾患の治療方法。
項１５．筋萎縮性側索硬化症等の変異型SOD1が蓄積する疾患の診断用の診断薬の製造における、項１～４のいずれか一項に記載の抗体又は抗体断片の使用。
項１６．筋萎縮性側索硬化症等の変異型SOD1が蓄積する疾患の治療及び／又は予防用医薬組成物の製造における、項１～４のいずれか一項に記載の抗体若しくは抗体断片、項５に記載の核酸、又は項６に記載のベクターの使用。 Section 1. have binding properties to mutant SOD1 proteins,
A heavy chain CDR1 consisting of the amino acid sequence of GFSLNTSGMG (SEQ ID NO: 1), a heavy chain CDR2 consisting of the amino acid sequence of IWWDDDK (SEQ ID NO: 2), and the amino acids of ARLGYAMDY (SEQ ID NO: 3), which may have up to 3 amino acid substitutions A heavy chain variable region comprising a heavy chain CDR3 consisting of a sequence, and/or a light chain consisting of a light chain CDR1 consisting of the amino acid sequence of QNVGGTN (SEQ ID NO: 4), which may have 3 or less amino acid substitutions, and a light chain consisting of the SAS amino acid sequence. An antibody or antibody fragment comprising a light chain variable region comprising a CDR2 and a light chain CDR3 consisting of the amino acid sequence of QQYYIYPYT (SEQ ID NO: 5).
Section 2. 2. The antibody or antibody fragment of paragraph 1, wherein said antibody fragment is Fab, Fab', F(ab') 2 , Fv, scFv, dsFv, ds-scFv, diabodies or minibodies.
Item 3. Item 3. The antibody or antibody fragment according to Item 1 or 2, wherein the antibody fragment is scFv.
Section 4. Item 4. The antibody or antibody fragment according to any one of Items 1 to 3, wherein the antibody or antibody fragment is a humanized antibody or antibody fragment.
Item 5. A nucleic acid encoding the antibody or antibody fragment of any one of Items 1-4.
Item 6. Item 6. A vector comprising the nucleic acid of Item 5.
Item 8. Item 8. The diagnostic agent according to
Item 9. A diagnostic kit comprising the antibody or antibody fragment according to any one of Items 1 to 4.
Item 10. A pharmaceutical composition comprising the antibody or antibody fragment of any one of Items 1 to 4, the nucleic acid of Item 5, or the vector of Item 6.
Item 11. Item 11. The pharmaceutical composition according to Item 10, which is for treatment and/or prevention of amyotrophic lateral sclerosis.
Item 12. A method for diagnosing a disease in which mutant SOD1 accumulates, such as amyotrophic lateral sclerosis, comprising:
(1) A method comprising the step of detecting mutant SOD1 in a biological sample collected from a subject using the antibody or antibody fragment according to any one of items 1 to 4.
Item 13. Furthermore, (2) the subject has amyotrophic lateral sclerosis when the amount or concentration of the mutant SOD1 measured in step (1) is equal to or greater than a preset cutoff value. Item 13. The method according to Item 12, comprising the step of determining that there is a high possibility of developing a disease in which mutant SOD1 such as SOD1 accumulates.
Item 14. Amyotrophic lateral sclerosis, comprising the step of administering to a mammal an effective amount of the antibody or antibody fragment of any one of items 1 to 4, the nucleic acid of item 5, or the vector of item 6. A method for treating a disease in which mutant SOD1 accumulates, such as disease.
Item 15. Item 5. Use of the antibody or antibody fragment according to any one of Items 1 to 4 in the production of a diagnostic agent for diagnosing diseases in which mutant SOD1 accumulates, such as amyotrophic lateral sclerosis.
本発明の抗体及び抗体断片は、アミノ酸置換の部位を問わず、幅広い種類の変異型SOD1タンパク質を認識することが可能である。そのため、本発明の抗体及び抗体断片により、SOD1遺伝子に様々な変異を有するALSの診断、治療及び予防が可能となる。
The antibodies and antibody fragments of the present invention are capable of recognizing a wide variety of mutant SOD1 proteins, regardless of the site of amino acid substitution. Therefore, the antibodies and antibody fragments of the present invention enable diagnosis, treatment and prevention of ALS having various mutations in the SOD1 gene.
以下、本発明の実施の形態について詳細に説明する。
Hereinafter, embodiments of the present invention will be described in detail.
なお、本明細書において「含む(comprise)」とは、「本質的にからなる(essentially consist of)」という意味と、「のみからなる(consist of)」という意味をも包含する。
In this specification, the term "comprise" includes the meaning of "essentially consist of" and the meaning of "consist of".
また、本発明において「核酸」とはRNA又はDNAを意味する。本発明において「遺伝子」とは、特に言及しない限り、2本鎖DNA、1本鎖DNA(センス鎖又はアンチセンス鎖)、及びそれらの断片が含まれる。また、本発明において「遺伝子」とは、特に言及しない限り、調節領域、コード領域、エクソン、及びイントロンを区別することなく示すものとする。
In addition, in the present invention, "nucleic acid" means RNA or DNA. In the present invention, the term "gene" includes double-stranded DNA, single-stranded DNA (sense strand or antisense strand), and fragments thereof, unless otherwise specified. In the present invention, the term "gene" refers to regulatory regions, coding regions, exons, and introns without distinction, unless otherwise specified.
本発明において、「核酸」、「ヌクレオチド」及び「ポリヌクレオチド」は同義であって、これらはDNA及びRNAの両方を含み、2本鎖であっても1本鎖であってもよい。
In the present invention, "nucleic acid", "nucleotide" and "polynucleotide" are synonymous, and include both DNA and RNA, and may be double-stranded or single-stranded.
本明細書において、「SOD1」は特に明示が無い限りタンパク質を意味するものとするが、遺伝子として解釈することが適切な場合は遺伝子を意味するものとする。
In this specification, "SOD1" shall mean protein unless otherwise specified, but shall mean gene when it is appropriate to interpret it as a gene.
なお、以下に示すRefSeq IDはNCBIのweb siteに登録されているものである。
The RefSeq IDs shown below are those registered on the NCBI website.
本発明の抗体又は抗体断片は、変異型SOD1タンパク質に結合性を有し、
3以下のアミノ酸置換をしてもよい、GFSLNTSGMG (配列番号１)のアミノ酸配列からなる重鎖CDR1、IWWDDDK (配列番号２)のアミノ酸配列からなる重鎖CDR2、及びARLGYAMDY (配列番号３)のアミノ酸配列からなる重鎖CDR3を含む重鎖可変領域、並びに／又は
3以下のアミノ酸置換をしてもよい、QNVGGTN (配列番号４)のアミノ酸配列からなる軽鎖CDR1、SASのアミノ酸配列からなる軽鎖CDR2、及びQQYYIYPYT (配列番号５)のアミノ酸配列からなる軽鎖CDR3を含む軽鎖可変領域
を含むことを特徴とする。 The antibody or antibody fragment of the present invention has a binding property to mutant SOD1 protein,
A heavy chain CDR1 consisting of the amino acid sequence of GFSLNTSGMG (SEQ ID NO: 1), a heavy chain CDR2 consisting of the amino acid sequence of IWWDDDK (SEQ ID NO: 2), and the amino acids of ARLGYAMDY (SEQ ID NO: 3), which may have up to 3 amino acid substitutions A heavy chain variable region comprising a heavy chain CDR3 consisting of a sequence, and/or a light chain consisting of a light chain CDR1 consisting of the amino acid sequence of QNVGGTN (SEQ ID NO: 4), which may have 3 or less amino acid substitutions, and a light chain consisting of the SAS amino acid sequence. It is characterized by comprising a light chain variable region including CDR2 and light chain CDR3 consisting of the amino acid sequence of QQYYIYPYT (SEQ ID NO: 5).
3以下のアミノ酸置換をしてもよい、GFSLNTSGMG (配列番号１)のアミノ酸配列からなる重鎖CDR1、IWWDDDK (配列番号２)のアミノ酸配列からなる重鎖CDR2、及びARLGYAMDY (配列番号３)のアミノ酸配列からなる重鎖CDR3を含む重鎖可変領域、並びに／又は
3以下のアミノ酸置換をしてもよい、QNVGGTN (配列番号４)のアミノ酸配列からなる軽鎖CDR1、SASのアミノ酸配列からなる軽鎖CDR2、及びQQYYIYPYT (配列番号５)のアミノ酸配列からなる軽鎖CDR3を含む軽鎖可変領域
を含むことを特徴とする。 The antibody or antibody fragment of the present invention has a binding property to mutant SOD1 protein,
A heavy chain CDR1 consisting of the amino acid sequence of GFSLNTSGMG (SEQ ID NO: 1), a heavy chain CDR2 consisting of the amino acid sequence of IWWDDDK (SEQ ID NO: 2), and the amino acids of ARLGYAMDY (SEQ ID NO: 3), which may have up to 3 amino acid substitutions A heavy chain variable region comprising a heavy chain CDR3 consisting of a sequence, and/or a light chain consisting of a light chain CDR1 consisting of the amino acid sequence of QNVGGTN (SEQ ID NO: 4), which may have 3 or less amino acid substitutions, and a light chain consisting of the SAS amino acid sequence. It is characterized by comprising a light chain variable region including CDR2 and light chain CDR3 consisting of the amino acid sequence of QQYYIYPYT (SEQ ID NO: 5).
スーパーオキシドディスムターゼ(SOD)は、スーパオキシドアニオンを酸素と過酸化水素に不均化する反応を触媒する酵素である(EC 1.15.1.1)。ヒトでは、SODは3種の形態が存在し、SOD1は細胞質に存在し通常2量体である。SOD2はミトコンドリアに存在し、SOD3は細胞外に存在し、これらは通常4量体である。SOD1とSOD3は反応中心に銅と亜鉛とを含み、SOD2は反応中心にマンガンを含む。
Superoxide dismutase (SOD) is an enzyme that catalyzes the dismutation of superoxide anions into oxygen and hydrogen peroxide (EC 1.15.1.1). In humans, SOD exists in three forms, SOD1 is present in the cytoplasm and is usually a dimer. SOD2 is present in mitochondria, SOD3 is present extracellularly, and they are usually tetramers. SOD1 and SOD3 contain copper and zinc in the reaction center, and SOD2 contains manganese in the reaction center.
本発明におけるSOD1は、通常、動物由来であり、好ましくは哺乳動物由来、特に好ましくはヒト由来である。
SOD1 in the present invention is usually animal-derived, preferably mammalian-derived, and particularly preferably human-derived.
ヒト由来のSOD1タンパク質のアミノ酸配列は、RefSeq Accession No.NP_000445として登録され、配列番号６に示されている。また、ヒト由来のSOD1タンパク質をコードする遺伝子は、RefSeq Accession No.NM_000454として登録され、その塩基配列は配列番号７に示されている。
The amino acid sequence of the human-derived SOD1 protein has been registered as RefSeq Accession No.NP_000445 and is shown in SEQ ID NO:6. A gene encoding a human-derived SOD1 protein has been registered as RefSeq Accession No. NM_000454, and its nucleotide sequence is shown in SEQ ID NO:7.
本発明における変異型SOD1とは、アミノ酸が置換、欠失、挿入又は付加されることなどによりタンパク質に変異が生じることで、天然構造とは異なる構造にフォールディングしたSOD1のことであり、特にALSと関連する病原構造を有するSOD1のことである。変異型SOD1はタンパク質配列の変異によるものであってもよいし、又は他の修飾によるものであってもよい。また、変異型SOD1とは、孤発性変異又は家族性変異のいずれの変異を有するものであってもよく、特に家族性変異を有するSOD1である。
Mutant SOD1 in the present invention refers to SOD1 that is folded into a structure different from the native structure due to mutations in the protein caused by substitution, deletion, insertion or addition of amino acids, particularly ALS. SOD1 with related pathogenic structures. Mutant SOD1 may result from mutations in the protein sequence or may result from other modifications. Mutant SOD1 may have either a sporadic mutation or a familial mutation, particularly SOD1 having a familial mutation.
本発明の抗体又は抗体断片に含まれる上記CDR (相補性決定領域)は、変異型SOD1に結合性を有することを特徴とする。本発明の抗体又は抗体断片は、変異型SOD1に特異的に結合し、正常構造を有する野生型SOD1には結合しないことが望ましい。
The CDRs (complementarity determining regions) contained in the antibody or antibody fragment of the present invention are characterized by having binding properties to mutant SOD1. Desirably, the antibody or antibody fragment of the present invention specifically binds to mutant SOD1 and does not bind to wild-type SOD1 having a normal structure.
本発明の抗体又は抗体断片は、好ましくは、3以下のアミノ酸置換をしてもよい、GFSLNTSGMG (配列番号１)のアミノ酸配列からなる重鎖CDR1、IWWDDDK (配列番号２)のアミノ酸配列からなる重鎖CDR2、及びARLGYAMDY (配列番号３)のアミノ酸配列からなる重鎖CDR3を含む重鎖可変領域、並びに3以下のアミノ酸置換をしてもよい、QNVGGTN (配列番号４)のアミノ酸配列からなる軽鎖CDR1、SASのアミノ酸配列からなる軽鎖CDR2、及びQQYYIYPYT (配列番号５)のアミノ酸配列からなる軽鎖CDR3を含む軽鎖可変領域を含む。
The antibody or antibody fragment of the present invention preferably has a heavy chain CDR1 consisting of the amino acid sequence of GFSLNTSGMG (SEQ ID NO: 1), which may have up to 3 amino acid substitutions, and a heavy chain consisting of the amino acid sequence of IWWDDDK (SEQ ID NO: 2). A heavy chain variable region comprising chain CDR2 and a heavy chain CDR3 consisting of the amino acid sequence of ARLGYAMDY (SEQ ID NO: 3), and a light chain consisting of the amino acid sequence of QNVGGTN (SEQ ID NO: 4), optionally with up to 3 amino acid substitutions It comprises a light chain variable region comprising CDR1, light chain CDR2 consisting of the amino acid sequence of SAS, and light chain CDR3 consisting of the amino acid sequence of QQYYIYPYT (SEQ ID NO: 5).
アミノ酸置換は、抗体又は抗体断片の変異型SOD1に対する結合性が維持されるように行われる。上記「3以下のアミノ酸置換をしてもよい」とは、重鎖可変領域又は軽鎖可変領域において合計3個以下のアミノ酸を置換してもよいことを意味し、アミノ酸置換はCDRの部分で行われるのが望ましい。
Amino acid substitutions are made so that the binding of the antibody or antibody fragment to mutant SOD1 is maintained. The above-mentioned "may have 3 or less amino acid substitutions" means that a total of 3 or less amino acids may be substituted in the heavy chain variable region or the light chain variable region, and the amino acid substitutions are in the CDR portion. preferably done.
アミノ酸置換の数は、好ましくは2以下、より好ましくは1以下であり、更に好ましくは0である。アミノ酸を置換する場合、性質の似たアミノ酸に置換すれば、元の抗体断片の活性が維持されやすいと考えられる。特定のアミノ酸配列において、アミノ酸を置換させる技術は公知である。
The number of amino acid substitutions is preferably 2 or less, more preferably 1 or less, and still more preferably 0. When amino acids are substituted, substitution with amino acids with similar properties is considered to facilitate maintenance of the activity of the original antibody fragment. Techniques for substituting amino acids in a specific amino acid sequence are known.
抗体断片とは、その抗体断片が由来する抗体の結合活性の一部又は全部を保持する抗体の一部を示すものとする。抗体断片としては、例えば、Fab、Fab'、F(ab')2、Fv、scFv、dsFv、ds-scFv、ダイアボディ又はミニボディなどが挙げられる。
An antibody fragment refers to a portion of an antibody that retains all or part of the binding activity of the antibody from which the antibody fragment is derived. Antibody fragments include, for example, Fab, Fab', F(ab') 2 , Fv, scFv, dsFv, ds-scFv, diabodies or minibodies.
本発明の抗体断片としては、scFvであることが望ましい。scFvとは、重鎖可変領域と軽鎖可変領域とからなるFvを、適当なペプチドリンカーで連絡した抗体断片のことである。scFvとしては、重鎖可変領域(VH)と軽鎖可変領域(VL)とがいずれの順序で結合しているものも使用することができる。
The antibody fragment of the present invention is preferably scFv. scFv is an antibody fragment in which Fv consisting of a heavy chain variable region and a light chain variable region are linked by an appropriate peptide linker. A scFv in which a heavy chain variable region (VH) and a light chain variable region (VL) are linked in any order can also be used.
本発明の抗体又は抗体断片の可変領域におけるフレームワーク領域(FR領域)の配列は、抗体断片が変異型SOD1に結合性を有する限り特に制限されず、いずれの配列も使用することができる。本発明の抗体又は抗体断片は、フレームワーク領域がヒト由来であるヒト化抗体又は抗体断片であることが望ましい。このようなヒト化抗体又は抗体断片は公知の方法により作製することができる。
The sequence of the framework region (FR region) in the variable region of the antibody or antibody fragment of the present invention is not particularly limited as long as the antibody fragment has binding properties to mutant SOD1, and any sequence can be used. The antibody or antibody fragment of the present invention is desirably a humanized antibody or antibody fragment whose framework region is of human origin. Such humanized antibodies or antibody fragments can be produced by known methods.
本発明の抗体としては、マウス、ラット、モルモット、ウサギ、ウシ、ヤギ、ヒツジなどいずれの動物由来のものであってもよく、特に限定されない。また、本発明の抗体又は抗体断片のアイソタイプも特に限定されず、IgG (IgG1、IgG2、IgG3、IgG4)、IgA、IgD、IgE及びIgMのいずれであってもよい。
The antibody of the present invention may be derived from any animal such as mouse, rat, guinea pig, rabbit, bovine, goat, and sheep, and is not particularly limited. Also, the isotype of the antibody or antibody fragment of the present invention is not particularly limited, and may be any of IgG (IgG1, IgG2, IgG3, IgG4), IgA, IgD, IgE and IgM.
本発明の抗体又は抗体断片は、酵素、蛍光物質、発光物質、色素、放射性化合物、ビオチン等により標識化されたものであってもよい。
The antibody or antibody fragment of the present invention may be labeled with an enzyme, fluorescent substance, luminescent substance, dye, radioactive compound, biotin, or the like.
本発明の核酸は、上記抗体又は抗体断片をコードすることを特徴とする。当該核酸は、抗体又は抗体断片は、PCR法、化学合成、生化学的切断／再結合などの常法で作製することができる。
The nucleic acid of the present invention is characterized by encoding the antibody or antibody fragment described above. The nucleic acid, antibody or antibody fragment can be produced by conventional methods such as PCR, chemical synthesis, biochemical cleavage/recombination, and the like.
本発明のベクター(特に発現ベクター)は、上記抗体又は抗体断片を産生させるためのものであって、上記核酸を含むことを特徴とする。当該ベクターとしては、特に制限されず、公知のベクターを広く使用することができる。上記核酸を導入する細胞の種類等を考慮し、適切なベクターを適宜選択すればよい。ベクターは、上記核酸以外にも、プロモーター、エンハンサー、ターミネーター、ポリアデニル化シグナル、選択マーカー、複製起点などを含有し得る。ベクターは、自立的に複製するベクター、及び宿主細胞に導入された際に宿主細胞のゲノムに組み込まれ、組み込まれた染色体と共に複製されるもののいずれも使用することができる。上記核酸は、公知の方法によりベクターに挿入することができる。
The vector (especially the expression vector) of the present invention is for producing the above antibody or antibody fragment, and is characterized by comprising the above nucleic acid. The vector is not particularly limited, and a wide range of known vectors can be used. An appropriate vector may be appropriately selected in consideration of the type of cell into which the nucleic acid is to be introduced. Vectors may contain promoters, enhancers, terminators, polyadenylation signals, selectable markers, origins of replication, etc., in addition to the above nucleic acids. Both autonomously replicating vectors and vectors that, when introduced into the host cell, integrate into the genome of the host cell and replicate with the integrated chromosome can be used. The above nucleic acids can be inserted into vectors by known methods.
ベクターの構築、及び当該ベクターの細胞への導入法は周知であり、例えば、Sambrook and Russell, Molecular Cloning, A Laboratory Manual 3rd edition, Cold Spring Harbor Laboratory Press (2001)等の記載を参考にして実施することができる。
Construction of vectors and methods for introducing the vectors into cells are well known, and are carried out with reference to descriptions such as Sambrook and Russell, Molecular Cloning, A Laboratory Manual 3rd edition, Cold Spring Harbor Laboratory Press (2001), etc. be able to.
ベクターを公知の方法により宿主細胞に導入し、次に形質転換された細胞を培養することにより上記抗体又は抗体断片を製造することができる。宿主細胞へのベクターの導入は、コンピテントセル法、プロトプラスト法、エレクトロポーレーション法、リン酸カルシウム法、リポフェクション法、リポソーム法、マイクロインジェクション法、酢酸リチウム法、パーティクルガン法、ウイルス法等の周知の方法により行うことができる。上記抗体又は抗体断片を製造するために、種々の宿主－発現ベクター系を利用することができる。形質転換体を作製するための宿主としては、例えば、大腸菌、酵母、哺乳動物細胞、植物細胞、昆虫細胞などが挙げられる。
The above antibody or antibody fragment can be produced by introducing the vector into host cells by a known method and then culturing the transformed cells. Introduction of vectors into host cells can be performed by well-known methods such as competent cell method, protoplast method, electroporation method, calcium phosphate method, lipofection method, liposome method, microinjection method, lithium acetate method, particle gun method, virus method, etc. It can be done by A variety of host-expression vector systems are available to produce the antibodies or antibody fragments described above. Hosts for producing transformants include, for example, E. coli, yeast, mammalian cells, plant cells, insect cells and the like.
形質転換体によって生産された上記抗体又は抗体断片の精製は、例えば、イオン交換クロマトグラフィー、ゲル濾過クロマトグラフィー、遠心分離、塩析等によって行うことができる。また、上記抗体又は抗体断片にポリヒスチジンタグ、HAタグ、mycタグなどを付加しておき、これらのタグを利用して精製することもできる。
Purification of the antibody or antibody fragment produced by the transformant can be performed, for example, by ion exchange chromatography, gel filtration chromatography, centrifugation, salting out, and the like. Alternatively, a polyhistidine tag, HA tag, myc tag, or the like may be added to the antibody or antibody fragment described above, and these tags may be used for purification.
本発明の診断薬及び診断用キットは、上記抗体又は抗体断片を含むことを特徴とし、変異型SOD1を検出することが可能である。そのため、本発明の診断薬及び診断用キットは、変異型SOD1が蓄積する疾患、特にALSの診断が可能となる。
The diagnostic agent and diagnostic kit of the present invention are characterized by containing the antibody or antibody fragment described above, and are capable of detecting mutant SOD1. Therefore, the diagnostic agent and diagnostic kit of the present invention enable diagnosis of diseases in which mutant SOD1 accumulates, particularly ALS.
本発明の診断薬及び診断用キットによる罹患の可能性の判定は、被検体から採取された生体試料における変異型SOD1を検出することにより行うことができる。被検体としては、特に限定されず、例えば、ヒト、サル、マウス、ラット、イヌ、ネコ、ウサギなどの哺乳動物が挙げられ、好ましくはヒトである。生体試料としては、変異型SOD1を含有し得るものであれば特に限定されず、例えば、全血、血清、血漿、髄液、唾液、鼻腔粘膜、関節液、尿、組織液、汗、涙、唾液などの体液、細胞、組織(例えば、脳脊髄、末梢神経、筋肉の生検及び剖検組織)などが挙げられる。
The possibility of morbidity can be determined by using the diagnostic agent and diagnostic kit of the present invention by detecting mutant SOD1 in a biological sample collected from a subject. Subjects are not particularly limited, and examples include mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits, preferably humans. The biological sample is not particularly limited as long as it can contain mutant SOD1. body fluids, cells, tissues (eg, cerebrospinal cord, peripheral nerve, muscle biopsy and autopsy tissue), and the like.
変異型SOD1を検出する方法としては、例えば、酵素免疫測定法(ELISA)、ラジオイムノアッセイ(RIA)、エンザイムイムノアッセイ(EIA)、蛍光イムノアッセイ、ウェスタンブロット法、イムノクロマト法、免疫沈降、免疫組織染色法などが挙げられる。上記抗体又は抗体断片に結合する変異型SOD1を検出するために用いられる抗体又は抗体断片の標識については特に限定されず、例えば、酵素、蛍光物質、発光物質、色素、放射性化合物、ビオチンなどが挙げられる。
Examples of methods for detecting mutant SOD1 include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence immunoassay, western blotting, immunochromatography, immunoprecipitation, and immunohistochemical staining. is mentioned. The label of the antibody or antibody fragment used for detecting the mutant SOD1 that binds to the above antibody or antibody fragment is not particularly limited, and examples thereof include enzymes, fluorescent substances, luminescent substances, dyes, radioactive compounds, biotin, and the like. be done.
本発明の診断薬及び診断用キットにより、上記抗体又は抗体断片に結合する変異型SOD1が検出された場合、あるいは検出された変異型SOD1の量が予め設定されたカットオフ値以上である場合に、変異型SOD1が蓄積する疾患の発症リスクが高いと判定することができる。カットオフ値とは、変異型SOD1が蓄積する疾患に罹患していない被検体から採取された生体試料における上記抗体又は抗体断片により検出される変異型SOD1の検出量である。カットオフ値は、過去に得られたデータから標準化されたものであってもよい。
When a mutant SOD1 that binds to the above antibody or antibody fragment is detected by the diagnostic agent and diagnostic kit of the present invention, or when the amount of the detected mutant SOD1 is equal to or greater than a preset cutoff value , it can be determined that the risk of developing a disease in which mutant SOD1 accumulates is high. The cut-off value is the amount of mutant SOD1 detected by the antibody or antibody fragment in a biological sample collected from a subject not suffering from a disease in which mutant SOD1 accumulates. The cutoff value may be normalized from previously obtained data.
上記抗体又は抗体断片を診断薬及び診断用キットとして使用する場合の使用量は、ヒトの変異型SOD1の存在についての判別が可能になる程度にまで結合量が十分となる量であればよく、生体試料の種類等の条件により適宜選択される。また、本発明の診断薬及び診断用キットは、上記抗体又は抗体断片の機能を阻害しない程度で他の添加剤を含むことができる。
When the above antibody or antibody fragment is used as a diagnostic agent or diagnostic kit, the amount used may be any amount as long as the binding amount is sufficient to the extent that the presence of human mutant SOD1 can be determined. It is appropriately selected according to conditions such as the type of biological sample. In addition, the diagnostic agent and diagnostic kit of the present invention can contain other additives to the extent that they do not inhibit the functions of the antibody or antibody fragment.
本発明の診断用キットには、上記抗体又は抗体断片以外にも、被検体から採取された生体試料における変異型SOD1を検出するための器具、試薬などが含まれていてもよい。
The diagnostic kit of the present invention may contain, in addition to the above antibodies or antibody fragments, instruments, reagents, etc. for detecting mutant SOD1 in biological samples collected from subjects.
本発明の医薬組成物は、ヒトを含む哺乳動物に投与されるものであって、上記抗体又は抗体断片、核酸又はベクターを含むことを特徴とし、変異型SOD1が蓄積する疾患、特に筋萎縮性側索硬化症の治療及び／又は予防の効果を有する。
The pharmaceutical composition of the present invention, which is administered to mammals including humans, is characterized by comprising the above-mentioned antibody or antibody fragment, nucleic acid or vector, and is used for diseases in which mutant SOD1 accumulates, particularly muscle atrophy. It has the effect of treating and/or preventing lateral sclerosis.
上記核酸又はベクターを含む本発明の医薬組成物により上記核酸を細胞内で発現させることで、上記抗体又は抗体断片を細胞内抗体(イントラボディ)として使用することができる。
The antibody or antibody fragment can be used as an intracellular antibody (intrabody) by intracellularly expressing the nucleic acid using the pharmaceutical composition of the present invention containing the nucleic acid or vector.
上記核酸を細胞内で発現させるために、例えば、非ウイルスベクター又はウイルスベクターを使用することができる。非ウイルスベクターとしては、リポソーム、高分子ミセル、カチオン性キャリアなどが挙げられる。また、ウイルスベクターとしては、レンチウイルス、レトロウイルス、アデノウイルス、アデノ随伴ウイルス、センダイウイルスなどが挙げられ、上記核酸を含むベクターを無毒化したウイルスに導入し、細胞又は組織にこのウイルスを感染させることにより細胞又は組織内に核酸を導入することができる。
For example, non-viral vectors or viral vectors can be used to express the above nucleic acids in cells. Non-viral vectors include liposomes, polymeric micelles, cationic carriers, and the like. Examples of viral vectors include lentivirus, retrovirus, adenovirus, adeno-associated virus, Sendai virus, etc. A vector containing the nucleic acid is introduced into a detoxified virus to infect cells or tissues with this virus. Nucleic acids can be introduced into cells or tissues.
本発明の医薬組成物は、その使用形態に応じて、生物学的に許容される担体、賦形剤等を任意に含有できる。本発明の医薬組成物は、常套手段に従って製造することができる。例えば、必要に応じて糖衣又は腸溶性被膜を施した錠剤、カプセル剤、エリキシル剤、マイクロカプセル剤等として経口的に、軟膏、硬膏等の外用剤、噴霧剤、吸入剤等として経皮的、経鼻的又は経気管的に、水又はそれ以外の薬学的に許容し得る液との無菌性溶液、懸濁液剤等の注射剤の形で非経口的に使用できる。
The pharmaceutical composition of the present invention can optionally contain biologically acceptable carriers, excipients, etc., depending on its usage form. The pharmaceutical composition of the present invention can be manufactured according to conventional procedures. For example, if necessary, it may be administered orally as sugar-coated or enteric-coated tablets, capsules, elixirs, microcapsules, etc.; It can be used parenterally in the form of injections such as sterile solutions or suspensions with water or other pharmaceutically acceptable liquids, nasally or tracheally.
本発明の医薬組成物における有効成分である抗体若しくは抗体断片、核酸又はベクターの配合量は、剤型、投与経路等に応じて適宜選択され、通常、製剤全量中0.0001～90質量%程度、好ましくは0.001～70質量%程度である。
The amount of the antibody or antibody fragment, nucleic acid, or vector, which are active ingredients in the pharmaceutical composition of the present invention, is appropriately selected according to the dosage form, administration route, etc., and is usually about 0.0001 to 90% by mass, preferably about 0.0001 to 90% by mass of the total amount of the formulation. is about 0.001 to 70% by mass.
本発明の医薬組成物の投与は、局所的であってもよく、全身的であってもよい。投与方法には特に制限はなく、経口的又は非経口的に投与される。非経口的投与経路としては、皮下、腹腔内、髄腔内、静脈、動脈又は脊髄液への注射又は点滴、経皮的投与等が挙げられる。本発明の医薬組成物の投与量は、剤型の種類、投与方法、患者の年齢及び体重、患者の症状等を考慮して、最終的には医師の判断により適宜決定できる。
The administration of the pharmaceutical composition of the present invention may be local or systemic. The administration method is not particularly limited, and is administered orally or parenterally. Parenteral routes of administration include subcutaneous, intraperitoneal, intrathecal, intravenous, arterial or spinal fluid injection or infusion, transdermal administration and the like. The dosage of the pharmaceutical composition of the present invention can be finally determined as appropriate by the doctor's judgment, taking into account the type of dosage form, administration method, age and weight of the patient, symptoms of the patient, and the like.
本発明の医薬組成物は、細胞内で抗体又は抗体断片を発現させてミスフォールドしたSOD1を分解除去できるため、変異型SOD1が蓄積する疾患の治療に使用することができる。変異型SOD1が蓄積する疾患としては、特に限定されず、例えば、筋萎縮性側索硬化症等が挙げられる。本発明の診断薬又は診断用キットにより、被検体が変異型SOD1が蓄積する疾患に罹患している可能性が高いと判定された場合に、本発明の医薬組成物を投与し被検体の変異型SOD1が蓄積する疾患を治療することができる。
The pharmaceutical composition of the present invention can be used to treat diseases in which mutant SOD1 accumulates, because it can express antibodies or antibody fragments intracellularly to degrade and remove misfolded SOD1. Diseases in which mutant SOD1 accumulates are not particularly limited, and examples thereof include amyotrophic lateral sclerosis. When it is determined by the diagnostic agent or diagnostic kit of the present invention that there is a high possibility that the subject is suffering from a disease in which mutant SOD1 accumulates, the pharmaceutical composition of the present invention is administered to the subject for mutation. Diseases in which type SOD1 accumulates can be treated.
本発明の抗体又は抗体断片は、アミノ酸置換の部位を問わず、幅広い種類の変異型SOD1タンパク質を認識することが可能であり、SOD1遺伝子に突然変異を有するALSの診断、予防、治療、研究のための抗体又は抗体断片として用途が広い。アンチセンスオリゴヌクレオチドは変異遺伝子特異的な治療が必要となるが、本発明の抗体又は抗体断片は変異の種類を問わず診断、予防及び治療に利用可能である。
The antibody or antibody fragment of the present invention can recognize a wide variety of mutant SOD1 proteins regardless of the site of amino acid substitution, and is useful for diagnosis, prevention, treatment and research of ALS having mutations in the SOD1 gene. It is versatile as an antibody or antibody fragment for Antisense oligonucleotides require treatment specific to mutant genes, but the antibody or antibody fragment of the present invention can be used for diagnosis, prevention and treatment regardless of the type of mutation.
以下、本発明を更に詳しく説明するため実施例を挙げる。しかし、本発明はこれら実施例等になんら限定されるものではない。
Examples are given below to describe the present invention in more detail. However, the present invention is by no means limited to these examples.
＜実験方法＞
以下の試験例で使用した変異型SOD1タンパク質に結合性を有する抗SOD1抗体のVH及びVLのアミノ酸配列及び塩基配列を以下に示す。下線部分は、順にCDR1、CDR2、CDR3である。
VH (アミノ酸配列)
QVTLKESGPGILQPSQTLSLTCSFSGFSLNTSGMGVGWIRQPSGRVLEWLAHIWWDDDKRYNPALKSRLTISKDTSTNQVFLKIASVDTTDTATYYCARLGYAMDYWGHGTSVTVS (配列番号８)
VL (アミノ酸配列)
DIVMTQSQKFMSTSLGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYYIYPYTFGGGTKLEI (配列番号９)
VH (塩基配列)
CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAACACTTCTGGTATGGGTGTAGGCTGGATTCGTCAGCCTTCAGGGAGGGTTCTGGAGTGGCTGGCACACATTTGGTGGGATGATGACAAGCGCTATAACCCAGCCCTGAAGAGCCGACTGACAATCTCCAAGGATACCTCCACCAACCAGGTATTCCTCAAGATCGCCAGTGTGGACACTACAGATACTGCCACATACTACTGTGCTCGACTTGGCTATGCTATGGACTACTGGGGTCACGGAACCTCAGTCACCGTCTCC (配列番号１０)
VL (塩基配列)
GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCACTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATTACATCTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATA (配列番号１１) <Experimental method>
The amino acid sequences and nucleotide sequences of the VH and VL of the anti-SOD1 antibody that binds to the mutant SOD1 protein used in the following test examples are shown below. Underlined parts are CDR1, CDR2 and CDR3 in order.
VH (amino acid sequence)
QVTLKESGPGILQPSQTLSLTCSFS GFSLNTSGMG VGWIRQPSGRVLEWLAH IWWDDDK RYNPALKSRLTISKDTSTNQVFLKIASVDTTDTATYYC ARLGYAMDY WGHGTSVTVS (SEQ ID NO: 8)
VL (amino acid sequence)
DIVMTQSQKFMSTSLGDRVSVTCKAS QNVGTN VAWYQQKPGQSPKALIY SAS YRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFC QQYYIYPYT FGGGTKLEI (SEQ ID NO: 9)
VH (nucleotide sequence)
CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAACACTTCTGGTATGGGTGTAGGCTGGATTCGTCAGCCTTCAGGGAGGGTTCTGGAGTGGCTGGCACACATTTGGTGGGATGATGACAAGCGCTATAACCCAGCCCTGAAGAGCCGACTGACAATCTCCAAGGATACCTCCACCAACCAGGTATTCCTCAAGATCGCCAGTGTGGACACTACAGATACTGCCACATACTACTGTGCTCGACTTGGCTATGCTATGGACTACTGGGGTCACGGAACCTCAGTCACCGTCTCC (配列番号１０)
VL (nucleotide sequence)
GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCACTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATTACATCTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATA (配列番号１１)
以下の試験例で使用した変異型SOD1タンパク質に結合性を有する抗SOD1抗体のVH及びVLのアミノ酸配列及び塩基配列を以下に示す。下線部分は、順にCDR1、CDR2、CDR3である。
VH (アミノ酸配列)
QVTLKESGPGILQPSQTLSLTCSFSGFSLNTSGMGVGWIRQPSGRVLEWLAHIWWDDDKRYNPALKSRLTISKDTSTNQVFLKIASVDTTDTATYYCARLGYAMDYWGHGTSVTVS (配列番号８)
VL (アミノ酸配列)
DIVMTQSQKFMSTSLGDRVSVTCKASQNVGTNVAWYQQKPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFCQQYYIYPYTFGGGTKLEI (配列番号９)
VH (塩基配列)
CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAACACTTCTGGTATGGGTGTAGGCTGGATTCGTCAGCCTTCAGGGAGGGTTCTGGAGTGGCTGGCACACATTTGGTGGGATGATGACAAGCGCTATAACCCAGCCCTGAAGAGCCGACTGACAATCTCCAAGGATACCTCCACCAACCAGGTATTCCTCAAGATCGCCAGTGTGGACACTACAGATACTGCCACATACTACTGTGCTCGACTTGGCTATGCTATGGACTACTGGGGTCACGGAACCTCAGTCACCGTCTCC (配列番号１０)
VL (塩基配列)
GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCACTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATTACATCTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATA (配列番号１１) <Experimental method>
The amino acid sequences and nucleotide sequences of the VH and VL of the anti-SOD1 antibody that binds to the mutant SOD1 protein used in the following test examples are shown below. Underlined parts are CDR1, CDR2 and CDR3 in order.
VH (amino acid sequence)
QVTLKESGPGILQPSQTLSLTCSFS GFSLNTSGMG VGWIRQPSGRVLEWLAH IWWDDDK RYNPALKSRLTISKDTSTNQVFLKIASVDTTDTATYYC ARLGYAMDY WGHGTSVTVS (SEQ ID NO: 8)
VL (amino acid sequence)
DIVMTQSQKFMSTSLGDRVSVTCKAS QNVGTN VAWYQQKPGQSPKALIY SAS YRYSGVPDRFTGSGSGTDFTLTISNVQSEDLAEYFC QQYYIYPYT FGGGTKLEI (SEQ ID NO: 9)
VH (nucleotide sequence)
CAGGTTACTCTGAAAGAGTCTGGCCCTGGGATATTGCAGCCCTCCCAGACCCTCAGTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAACACTTCTGGTATGGGTGTAGGCTGGATTCGTCAGCCTTCAGGGAGGGTTCTGGAGTGGCTGGCACACATTTGGTGGGATGATGACAAGCGCTATAACCCAGCCCTGAAGAGCCGACTGACAATCTCCAAGGATACCTCCACCAACCAGGTATTCCTCAAGATCGCCAGTGTGGACACTACAGATACTGCCACATACTACTGTGCTCGACTTGGCTATGCTATGGACTACTGGGGTCACGGAACCTCAGTCACCGTCTCC (配列番号１０)
VL (nucleotide sequence)
GACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCACTAGGAGACAGGGTCAGCGTCACCTGCAAGGCCAGTCAGAATGTGGGTACTAATGTAGCCTGGTATCAACAGAAACCAGGGCAATCTCCTAAAGCACTGATTTACTCGGCATCCTACCGGTACAGTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAATGTGCAGTCTGAAGACTTGGCAGAGTATTTCTGTCAGCAATATTACATCTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATA (配列番号１１)
試験例１：培養細胞における本発明の抗体による変異型SOD1特異的染色
HeLa 細胞にSOD1-FLAG (野生型、A4V、H46R、G85R、G93A)を発現するプラスミドを遺伝子導入させ、4%パラホルムアルデヒド(PFA)に常温30分反応させた後、4%正常ヤギ血清で30分間ブロッキングを行った。その後、抗SOD1抗体(緑)とウサギ抗FLAG抗体(ともにPBS-Tバッファーにて1:500希釈)に常温30分反応させ、同バッファーで3回洗浄した後、抗SOD1抗体に対して抗マウスIgG (CF48; 緑)、FLAGに対して抗ウサギIgG (CF568; 赤)を同バッファーに1:500に希釈し常温30分反応させた。蛍光退色防止薬を含む包埋薬を加え、共焦点顕微鏡で観察した。 Test Example 1: Mutant SOD1-specific staining of cultured cells with the antibody of the present invention HeLa cells were transfected with a plasmid expressing SOD1-FLAG (wild type, A4V, H46R, G85R, G93A), and treated with 4% paraformaldehyde ( PFA) at room temperature for 30 minutes, followed by blocking with 4% normal goat serum for 30 minutes. Then, react with anti-SOD1 antibody (green) and rabbit anti-FLAG antibody (both diluted 1:500 in PBS-T buffer) for 30 minutes at room temperature. IgG (CF48; green) and anti-rabbit IgG (CF568; red) against FLAG were diluted 1:500 in the same buffer and allowed to react at room temperature for 30 minutes. An embedding agent containing a fluorescent anti-fading agent was added and observed with a confocal microscope.
HeLa 細胞にSOD1-FLAG (野生型、A4V、H46R、G85R、G93A)を発現するプラスミドを遺伝子導入させ、4%パラホルムアルデヒド(PFA)に常温30分反応させた後、4%正常ヤギ血清で30分間ブロッキングを行った。その後、抗SOD1抗体(緑)とウサギ抗FLAG抗体(ともにPBS-Tバッファーにて1:500希釈)に常温30分反応させ、同バッファーで3回洗浄した後、抗SOD1抗体に対して抗マウスIgG (CF48; 緑)、FLAGに対して抗ウサギIgG (CF568; 赤)を同バッファーに1:500に希釈し常温30分反応させた。蛍光退色防止薬を含む包埋薬を加え、共焦点顕微鏡で観察した。 Test Example 1: Mutant SOD1-specific staining of cultured cells with the antibody of the present invention HeLa cells were transfected with a plasmid expressing SOD1-FLAG (wild type, A4V, H46R, G85R, G93A), and treated with 4% paraformaldehyde ( PFA) at room temperature for 30 minutes, followed by blocking with 4% normal goat serum for 30 minutes. Then, react with anti-SOD1 antibody (green) and rabbit anti-FLAG antibody (both diluted 1:500 in PBS-T buffer) for 30 minutes at room temperature. IgG (CF48; green) and anti-rabbit IgG (CF568; red) against FLAG were diluted 1:500 in the same buffer and allowed to react at room temperature for 30 minutes. An embedding agent containing a fluorescent anti-fading agent was added and observed with a confocal microscope.
結果を図１に示す。図１から本発明の抗体により全ての種類の変異型SOD1タンパク質を認識できていることが分かる。
The results are shown in Figure 1. From FIG. 1, it can be seen that the antibodies of the present invention can recognize all kinds of mutant SOD1 proteins.
試験例２：ALSモデルマウス、SOD1に遺伝子変異を有する家族性ALS患者脊髄組織における本発明の抗体による染色
生後91日齢の変異SOD1(G93A)トランスジェニックマウスと同腹の野生型マウスとをケタミン麻酔後にリン酸バッファーと4%パラホルムアルデヒドにて灌流固定したのち、脊髄をパラフィン包埋した。SOD1遺伝子に突然変異を有する家族性ALS患者の剖検脊髄をホルマリン固定後、同様にパラフィン包埋した。7μmの切片を、ヒストファイン脱パラフィンバッファーにpH6、120℃で20分間反応させて抗原賦活し、抗SOD1抗体を3% BSA入りPBSで500倍希釈し、4℃条件で一昼夜反応させた。抗体反応液をPBSで3回洗浄した後、2次抗体キット反応液(株式会社ニチレイ)で37℃、30分反応させた。PBSで3回洗浄した後、切片をペルオキシダーゼDAB染色キット(ナカライテスク株式会社)で発色させた。 Test Example 2: Staining with the antibody of the present invention in spinal cord tissues of ALS model mice and familial ALS patients with genetic mutations in SOD1. After perfusion fixation with phosphate buffer and 4% paraformaldehyde, the spinal cord was embedded in paraffin. Autopsy spinal cords of familial ALS patients with mutations in the SOD1 gene were fixed in formalin and then embedded in paraffin. The 7 μm section was reacted with Histofine deparaffinization buffer at pH 6 and 120° C. for 20 minutes to activate the antigen, and the anti-SOD1 antibody was diluted 500-fold with PBS containing 3% BSA and reacted at 4° C. overnight. After the antibody reaction solution was washed with PBS three times, it was reacted with a secondary antibody kit reaction solution (Nichirei Corporation) at 37°C for 30 minutes. After washing with PBS three times, the sections were developed with a peroxidase DAB staining kit (Nacalai Tesque, Inc.).
生後91日齢の変異SOD1(G93A)トランスジェニックマウスと同腹の野生型マウスとをケタミン麻酔後にリン酸バッファーと4%パラホルムアルデヒドにて灌流固定したのち、脊髄をパラフィン包埋した。SOD1遺伝子に突然変異を有する家族性ALS患者の剖検脊髄をホルマリン固定後、同様にパラフィン包埋した。7μmの切片を、ヒストファイン脱パラフィンバッファーにpH6、120℃で20分間反応させて抗原賦活し、抗SOD1抗体を3% BSA入りPBSで500倍希釈し、4℃条件で一昼夜反応させた。抗体反応液をPBSで3回洗浄した後、2次抗体キット反応液(株式会社ニチレイ)で37℃、30分反応させた。PBSで3回洗浄した後、切片をペルオキシダーゼDAB染色キット(ナカライテスク株式会社)で発色させた。 Test Example 2: Staining with the antibody of the present invention in spinal cord tissues of ALS model mice and familial ALS patients with genetic mutations in SOD1. After perfusion fixation with phosphate buffer and 4% paraformaldehyde, the spinal cord was embedded in paraffin. Autopsy spinal cords of familial ALS patients with mutations in the SOD1 gene were fixed in formalin and then embedded in paraffin. The 7 μm section was reacted with Histofine deparaffinization buffer at pH 6 and 120° C. for 20 minutes to activate the antigen, and the anti-SOD1 antibody was diluted 500-fold with PBS containing 3% BSA and reacted at 4° C. overnight. After the antibody reaction solution was washed with PBS three times, it was reacted with a secondary antibody kit reaction solution (Nichirei Corporation) at 37°C for 30 minutes. After washing with PBS three times, the sections were developed with a peroxidase DAB staining kit (Nacalai Tesque, Inc.).
結果を図２に示す。ALSモデルマウス、SOD1に遺伝子変異を有する家族性ALS患者の脊髄組織において、本発明の抗体により全ての種類の変異型SOD1タンパク質を特異的に認識できていることが分かる。
The results are shown in Figure 2. It can be seen that the antibodies of the present invention can specifically recognize all kinds of mutant SOD1 proteins in ALS model mice and spinal cord tissues of familial ALS patients with SOD1 gene mutations.
試験例３：変異型SOD1に対する抗SOD1抗体(scFv)の特異的結合性の免疫沈降による検証
6ウェルのマルチプレートに播種したHEK293A細胞に対して、FuGene HDをウェルあたり6μl、Opti-MEM 100μl、DNA (pcDNA3-SOD1-FLAG (野生型(W)、A4V、H46R、G85R、G93A))とpcDNA3-scFv-Mycとを1.5μgずつ混合し20分静置した後、細胞に投与した。48時間後に細胞を回収し100μl RIPAバッファーに溶解し、そのうち90μlを免疫沈降に用い、10μlをインプットとして用いた。前者に410μlのRIPAバッファーと20μlの抗FLAG抗体アガロースビーズ、あるいは抗Myc抗体アガロースビーズと常温60分間、回転させながら撹拌反応させ、その後RIPAバッファーで4回ビーズを洗浄した。洗浄液を除去した後、各々のビーズに２メルカプトエタノール(2ME)を含む2%SDSサンプリングバッファー30μlを加え、撹拌後95℃で5分間反応させ、プロテインG結合タンパク質を溶出させた。 Test Example 3: Verification by Immunoprecipitation of Specific Binding of Anti-SOD1 Antibody (scFv) to Mutant SOD1 For HEK293A cells seeded in a 6-well multiplate, 6 μl of FuGene HD per well, 100 μl of Opti-MEM, DNA (pcDNA3-SOD1-FLAG (wild-type (W), A4V, H46R, G85R, G93A)) and pcDNA3-scFv-Myc (1.5 µg each) were mixed, allowed to stand for 20 minutes, and administered to cells. After 48 hours, cells were harvested and lysed in 100 μl RIPA buffer, 90 μl of which was used for immunoprecipitation and 10 μl as input. The former was reacted with 410 µl of RIPA buffer and 20 µl of anti-FLAG antibody agarose beads or anti-Myc antibody agarose beads at room temperature for 60 minutes while rotating and stirring, and then the beads were washed four times with RIPA buffer. After removing the washing solution, 30 μl of 2% SDS sampling buffer containing 2-mercaptoethanol (2ME) was added to each bead, and after stirring, the beads were allowed to react at 95° C. for 5 minutes to elute the protein G binding protein.
6ウェルのマルチプレートに播種したHEK293A細胞に対して、FuGene HDをウェルあたり6μl、Opti-MEM 100μl、DNA (pcDNA3-SOD1-FLAG (野生型(W)、A4V、H46R、G85R、G93A))とpcDNA3-scFv-Mycとを1.5μgずつ混合し20分静置した後、細胞に投与した。48時間後に細胞を回収し100μl RIPAバッファーに溶解し、そのうち90μlを免疫沈降に用い、10μlをインプットとして用いた。前者に410μlのRIPAバッファーと20μlの抗FLAG抗体アガロースビーズ、あるいは抗Myc抗体アガロースビーズと常温60分間、回転させながら撹拌反応させ、その後RIPAバッファーで4回ビーズを洗浄した。洗浄液を除去した後、各々のビーズに２メルカプトエタノール(2ME)を含む2%SDSサンプリングバッファー30μlを加え、撹拌後95℃で5分間反応させ、プロテインG結合タンパク質を溶出させた。 Test Example 3: Verification by Immunoprecipitation of Specific Binding of Anti-SOD1 Antibody (scFv) to Mutant SOD1 For HEK293A cells seeded in a 6-well multiplate, 6 μl of FuGene HD per well, 100 μl of Opti-MEM, DNA (pcDNA3-SOD1-FLAG (wild-type (W), A4V, H46R, G85R, G93A)) and pcDNA3-scFv-Myc (1.5 µg each) were mixed, allowed to stand for 20 minutes, and administered to cells. After 48 hours, cells were harvested and lysed in 100 μl RIPA buffer, 90 μl of which was used for immunoprecipitation and 10 μl as input. The former was reacted with 410 µl of RIPA buffer and 20 µl of anti-FLAG antibody agarose beads or anti-Myc antibody agarose beads at room temperature for 60 minutes while rotating and stirring, and then the beads were washed four times with RIPA buffer. After removing the washing solution, 30 μl of 2% SDS sampling buffer containing 2-mercaptoethanol (2ME) was added to each bead, and after stirring, the beads were allowed to react at 95° C. for 5 minutes to elute the protein G binding protein.
15%ポリアクリルアミドゲル(富士フイルム和光純薬株式会社)で10μlのインプット液、免疫沈降液を電気泳動した後、PVDFメンブレンに転写した。同メンブレンをPBS-Tバッファーで洗浄した後5%スキムミルクを加えたPBS-Tバッファーで常温30分反応させブロッキングし、その後、抗Myc抗体、あるいは抗FLAG抗体(1:1000倍、PBS-T＋5%スキムミルク)に常温1時間振盪させながら反応させ、PBS-Tで3回洗浄した後、抗マウスIgG-ペルオキシダーゼ2次抗体に反応させた。PBS-Tで3回洗浄した後、ECL発行キット(ナカライテスク株式会社)を用いて発光反応をさせ、CCDカメラにて撮像した(ウェスタンブロット法)。
After electrophoresis of 10 μl of the input solution and the immunoprecipitate on a 15% polyacrylamide gel (Fujifilm Wako Pure Chemical Industries, Ltd.), they were transferred to a PVDF membrane. After washing the same membrane with PBS-T buffer, react with PBS-T buffer containing 5% skimmed milk for 30 minutes at room temperature for blocking. Skimmed milk) was allowed to react with shaking at room temperature for 1 hour, washed with PBS-T three times, and then reacted with an anti-mouse IgG-peroxidase secondary antibody. After washing with PBS-T three times, an ECL publishing kit (Nacalai Tesque, Inc.) was used to cause a luminescence reaction, followed by imaging with a CCD camera (Western blotting method).
結果を図３に示す。図３から本発明の抗体断片scFvは変異型SOD1タンパク質を特異的に認識することが分かる。
The results are shown in Figure 3. It can be seen from FIG. 3 that the antibody fragment scFv of the present invention specifically recognizes the mutant SOD1 protein.
試験例４：ALSモデルラットを用いた抗SOD1抗体の髄腔内持続投与による進行抑制の検証
東北大学の作製したH46Rトランスジェニックラットに対して生後19週目にALZET(商標)ポンプ2ML4 (室町機械株式会社)を用いて抗SOD1抗体の4週間持続投与を行った。2ML4は2 mlの液体を2.5μl/hで4週間に渡って持続投与が可能である。1 mg/mlの抗SOD1抗体が溶解したPBS 2 ml若しくは抗SOD1抗体が溶解していないPBS 2 mlをALZETポンプに充填し、先端に取り付けたカテーテルをL5腰椎の椎弓より髄腔内に挿入し、ポンプは皮下に留置した。治療効果判定として生後17週目より週に1回、体重と前足の握力を測定し、後肢反射(Hind foot reflex)試験と傾斜板試験(Inclined plate test)とを行った。 Test Example 4: Verification of inhibition of progression by continuous intrathecal administration of anti-SOD1 antibody using ALS model rats. Co., Ltd.), the anti-SOD1 antibody was continuously administered for 4 weeks. 2ML4 can be continuously administered in 2 ml liquid at 2.5 μl/h for 4 weeks. Fill the ALZET pump with 2 ml of PBS containing 1 mg/ml anti-SOD1 antibody or 2 ml of PBS without anti-SOD1 antibody, and insert the catheter attached to the tip into the medullary canal from the L5 lumbar vertebral arch. The pump was placed subcutaneously. To evaluate the therapeutic effect, body weight and grip strength of the forelimbs were measured once a week from 17 weeks after birth, and a hind foot reflex test and an inclined plate test were performed.
東北大学の作製したH46Rトランスジェニックラットに対して生後19週目にALZET(商標)ポンプ2ML4 (室町機械株式会社)を用いて抗SOD1抗体の4週間持続投与を行った。2ML4は2 mlの液体を2.5μl/hで4週間に渡って持続投与が可能である。1 mg/mlの抗SOD1抗体が溶解したPBS 2 ml若しくは抗SOD1抗体が溶解していないPBS 2 mlをALZETポンプに充填し、先端に取り付けたカテーテルをL5腰椎の椎弓より髄腔内に挿入し、ポンプは皮下に留置した。治療効果判定として生後17週目より週に1回、体重と前足の握力を測定し、後肢反射(Hind foot reflex)試験と傾斜板試験(Inclined plate test)とを行った。 Test Example 4: Verification of inhibition of progression by continuous intrathecal administration of anti-SOD1 antibody using ALS model rats. Co., Ltd.), the anti-SOD1 antibody was continuously administered for 4 weeks. 2ML4 can be continuously administered in 2 ml liquid at 2.5 μl/h for 4 weeks. Fill the ALZET pump with 2 ml of PBS containing 1 mg/ml anti-SOD1 antibody or 2 ml of PBS without anti-SOD1 antibody, and insert the catheter attached to the tip into the medullary canal from the L5 lumbar vertebral arch. The pump was placed subcutaneously. To evaluate the therapeutic effect, body weight and grip strength of the forelimbs were measured once a week from 17 weeks after birth, and a hind foot reflex test and an inclined plate test were performed.
結果を図４に示す。全ての項目で末期である29週目には抗SOD1抗体投与群で有意に改善がみられた上(図４b-e)、生存期間の延長がみられた(図４a)。
The results are shown in Figure 4. At week 29, which is the terminal stage, significant improvement was observed in all items in the anti-SOD1 antibody administration group (Fig. 4b-e), and the survival period was prolonged (Fig. 4a).
Claims (11)
- 変異型SOD1タンパク質に結合性を有し、
3以下のアミノ酸置換をしてもよい、GFSLNTSGMG (配列番号１)のアミノ酸配列からなる重鎖CDR1、IWWDDDK (配列番号２)のアミノ酸配列からなる重鎖CDR2、及びARLGYAMDY (配列番号３)のアミノ酸配列からなる重鎖CDR3を含む重鎖可変領域、並びに／又は
3以下のアミノ酸置換をしてもよい、QNVGGTN (配列番号４)のアミノ酸配列からなる軽鎖CDR1、SASのアミノ酸配列からなる軽鎖CDR2、及びQQYYIYPYT (配列番号５)のアミノ酸配列からなる軽鎖CDR3を含む軽鎖可変領域
を含む、抗体又は抗体断片。 have binding properties to mutant SOD1 proteins,
A heavy chain CDR1 consisting of the amino acid sequence of GFSLNTSGMG (SEQ ID NO: 1), a heavy chain CDR2 consisting of the amino acid sequence of IWWDDDK (SEQ ID NO: 2), and the amino acids of ARLGYAMDY (SEQ ID NO: 3), which may have up to 3 amino acid substitutions A heavy chain variable region comprising a heavy chain CDR3 consisting of a sequence, and/or a light chain consisting of a light chain CDR1 consisting of the amino acid sequence of QNVGGTN (SEQ ID NO: 4), which may have 3 or less amino acid substitutions, and a light chain consisting of the SAS amino acid sequence. An antibody or antibody fragment comprising a light chain variable region comprising a CDR2 and a light chain CDR3 consisting of the amino acid sequence of QQYYIYPYT (SEQ ID NO: 5). - 前記抗体断片がFab、Fab'、F(ab')2、Fv、scFv、dsFv、ds-scFv、ダイアボディ又はミニボディである、請求項１に記載の抗体又は抗体断片。 2. The antibody or antibody fragment of claim 1, wherein said antibody fragment is Fab, Fab', F(ab') 2 , Fv, scFv, dsFv, ds-scFv, diabodies or minibodies.
- 前記抗体断片がscFvである、請求項１又は２に記載の抗体又は抗体断片。 The antibody or antibody fragment according to claim 1 or 2, wherein the antibody fragment is scFv.
- 前記抗体又は抗体断片がヒト化抗体又は抗体断片である、請求項１又は２に記載の抗体又は抗体断片。 The antibody or antibody fragment according to claim 1 or 2, wherein the antibody or antibody fragment is a humanized antibody or antibody fragment.
- 請求項１又は２に記載の抗体又は抗体断片をコードする核酸。 A nucleic acid encoding the antibody or antibody fragment according to claim 1 or 2.
- 請求項５に記載の核酸を含むベクター。 A vector comprising the nucleic acid according to claim 5.
- 請求項１又は２に記載の抗体又は抗体断片を含む診断薬。 A diagnostic agent comprising the antibody or antibody fragment according to claim 1 or 2.
- 筋萎縮性側索硬化症の診断用である、請求項７に記載の診断薬。 The diagnostic agent according to claim 7, which is used for diagnosing amyotrophic lateral sclerosis.
- 請求項１又は２に記載の抗体又は抗体断片を含む診断用キット。 A diagnostic kit containing the antibody or antibody fragment according to claim 1 or 2.
- 請求項１又は２に記載の抗体若しくは抗体断片、請求項５に記載の核酸、又は請求項６に記載のベクターを含む医薬組成物。 A pharmaceutical composition comprising the antibody or antibody fragment of claim 1 or 2, the nucleic acid of claim 5, or the vector of claim 6.
- 筋萎縮性側索硬化症の治療及び／又は予防用である、請求項１０に記載の医薬組成物。 The pharmaceutical composition according to claim 10, which is for treatment and/or prevention of amyotrophic lateral sclerosis.
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| JP2009507475A (en) * | 2005-08-31 | 2009-02-26 | ユニバルシテ ラバル | Antibodies and use of said antibodies in the treatment, prevention and diagnosis of diseases associated with SOD1 abnormalities |
| WO2011142461A1 (en) * | 2010-05-13 | 2011-11-17 | 国立大学法人 東京大学 | Antibody for diagnosing amyotrophic lateral sclerosis (als) |
| JP2017046686A (en) * | 2010-12-17 | 2017-03-09 | ニューリミューン ホールディング エイジー | Human anti-SOD1 antibody |
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| JP2009507475A (en) * | 2005-08-31 | 2009-02-26 | ユニバルシテ ラバル | Antibodies and use of said antibodies in the treatment, prevention and diagnosis of diseases associated with SOD1 abnormalities |
| WO2011142461A1 (en) * | 2010-05-13 | 2011-11-17 | 国立大学法人 東京大学 | Antibody for diagnosing amyotrophic lateral sclerosis (als) |
| JP2017046686A (en) * | 2010-12-17 | 2017-03-09 | ニューリミューン ホールディング エイジー | Human anti-SOD1 antibody |
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