JP6531262B2 - Anti-fluorescent dye monoclonal antibody - Google Patents
Anti-fluorescent dye monoclonal antibody Download PDFInfo
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- JP6531262B2 JP6531262B2 JP2015550572A JP2015550572A JP6531262B2 JP 6531262 B2 JP6531262 B2 JP 6531262B2 JP 2015550572 A JP2015550572 A JP 2015550572A JP 2015550572 A JP2015550572 A JP 2015550572A JP 6531262 B2 JP6531262 B2 JP 6531262B2
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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- Immunology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Pathology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- General Physics & Mathematics (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Description
本発明は、アレクサフルオール（Alexa Fluor；登録商標、インビトロジェン社製）６４７のエピトープに結合することができる抗蛍光色素モノクローナル抗体や、該モノクローナル抗体を構成するポリペプチドや、該ポリペプチドをコードするＤＮＡに関する。 The present invention encodes an anti-fluorescent dye monoclonal antibody capable of binding to an epitope of Alexa Fluor (registered trademark, Invitrogen Corp.) 647, a polypeptide constituting the monoclonal antibody, and the polypeptide It relates to DNA.
抗体は研究用試薬、診断用試薬、各種物質モニター用試薬、抗体医薬等として、膨大な種類が開発・販売されている。抗体は、一般的に抗原で免疫した動物の血清から調製されるが、抗原は複数のエピトープ（抗原決定基）をもつことが多いため、単に血清から調製されたままでは、各々のエピトープに対する抗体が混ざっており、これをポリクローナル抗体という。一方、モノクローナル抗体は、免疫グロブリン分子種自体が均一であり、一つのエピトープに対する単一の分子種となるため、抗原特異性が全く同一の抗体となる。 A large number of antibodies have been developed and marketed as research reagents, diagnostic reagents, reagents for monitoring various substances, antibody drugs, and the like. Antibodies are generally prepared from the sera of animals immunized with the antigen, but since the antigens often have multiple epitopes (antigenic determinants), as prepared from serum, antibodies against each of the epitopes may be used. Are mixed, and this is called a polyclonal antibody. On the other hand, a monoclonal antibody is an antibody having identical antigen specificity since the immunoglobulin molecular species itself is uniform and becomes a single molecular species for one epitope.
従来、モノクローナル抗体の作製には、抗原で免疫した動物の脾臓やリンパ節から取り出した抗体産生リンパ球のＢ細胞と、無限に増殖して免疫グロブリンを産生する形質細胞腫瘍の骨髄腫細胞を融合した、ハイブリドーマ細胞を用い、目的の抗体を産生する１個のハイブリドーマ細胞から増殖した細胞集団を、限界希釈法により分離し、該細胞集団が分泌する抗体を精製する方法がとられてきた。 Conventionally, for the production of monoclonal antibodies, B cells of antibody-producing lymphocytes removed from the spleen or lymph node of an animal immunized with an antigen are fused with myeloma cells of plasma cell tumor that proliferates indefinitely to produce immunoglobulin. A method has been taken, using the hybridoma cells, separating a cell population grown from one hybridoma cell producing an antibody of interest by limiting dilution, and purifying the antibody secreted by the cell population.
その他のモノクローナル抗体の作製法としては、目的の抗体が認識する抗原を標識化してなる標識化抗原を、前記抗体を産生するターゲット細胞を含む細胞集団に接触させ、前記標識化抗原を前記ターゲット細胞に結合させ、得られる標識化ターゲット細胞を分離し、分離した標識化ターゲット細胞を用いて、それが保有する抗体遺伝子を調製し、調製した抗体遺伝子を、発現ベクターを用いて発現させる抗体作製方法［特許文献１］等が知られる。 As another method for producing a monoclonal antibody, a labeled antigen obtained by labeling an antigen recognized by a target antibody is brought into contact with a cell population containing target cells producing the antibody, and the labeled antigen is used as the target cell. A method for producing an antibody gene comprising the antibody gene of the present invention prepared using the expression vector, the isolated labeled target cell is isolated, and the isolated labeled target cell is used. [Patent Document 1] etc. are known.
また、本発明者らは、既に確立した抗原特異的なヒト抗体を産生するＢ細胞を１細胞レベルで検出・同定し、ＰＣＲクローニング法により、直接抗体遺伝子を取得する技術［特許文献２］により、感染症やがん関連抗原に対する特異的なヒト抗体を産生するＢ細胞の検出が可能であることを示してきた。 In addition, the present inventors detect and identify B cells producing an antigen-specific human antibody that has already been established on a single cell level, and obtain a direct antibody gene by PCR cloning method [Patent Document 2] It has been shown that it is possible to detect B cells producing specific human antibodies against infectious diseases and cancer-related antigens.
アレクサフルオール６４７は、以下の構造式を有し、その極大吸収波長が６５０ｎｍの蛍光物質である。また、蛍光物質を認識するモノクローナル抗体の応用としては、免疫組織化学におけるアルカリフォスファターゼ標識抗ＦＩＴＣ（フルオレセインイソチオシアネート）抗体の二次抗体としての使用［特許文献３］や、抗Ｃｙ（登録商標）５モノクローナル抗体を結合させたマイクロビーズによる、Ｃｙ５、ＰＥ−Ｃｙ５又はアレクサフルオール６４７標識の一次抗体で染色した細胞の間接磁気標識分離法［非特許文献１］等が知られている。 Alexafluor 647 has the following structural formula, and is a fluorescent substance whose maximum absorption wavelength is 650 nm. Moreover, as an application of a monoclonal antibody that recognizes a fluorescent substance, use of an alkaline phosphatase-labeled anti-FITC (fluorescein isothiocyanate) antibody as a secondary antibody in immunohistochemistry [Patent Document 3], and anti-Cy (registered trademark) 5 Indirect magnetic labeling separation method [Non-patent document 1] etc. of cells stained with Cy5, PE-Cy5 or Alexafluor 647-labeled primary antibody by microbeads to which a monoclonal antibody is bound are known.
本発明の課題は、アレクサフルオール６４７を認識する新規なモノクローナル抗体を提供することにある。 An object of the present invention is to provide a novel monoclonal antibody that recognizes Alexafluor 647.
本発明者らは、悪性グリオーマ患者１９症例において数種類のがん関連抗原タンパク（ＥＧＦＲ，ＶＥＧＦＲ２，ＶＥＧＦＲ３，ＣＤＨ１１等）に対する血液中のがん関連抗原に対する自家抗体価を測定していた。ＶＥＧＦＲ２に対する抗体価が最も高かった症例（患者）の末梢血Ｂ細胞を用いて、アレクサフルオール６４７標識ＶＥＧＦＲ２タンパク（細胞外ドメイン）とＰＥ標識マウス抗ヒトＩｇＧ抗体による染色にて、ＶＥＧＦＲ２特異的な抗体をもつＢ細胞を選別し、更に、Single cell sorter (FACSAria, BD Bioscience)を用いて１細胞ごとのｍＲＮＡを回収し、ｃＤＮＡクローニングを行った。クローニングされた抗体遺伝子を発現ベクターに組み込んで、バキュロウイルス発現系にて完全長抗体タンパクを発現・分泌させ、抗体を精製した。ＥＬＩＳＡ法にてＶＥＧＦＲ２−アレクサフルオール６４７及びＶＥＧＦＲ２タンパクに対する、精製抗体の結合活性を評価したところ、蛍光色素であるアレクサフルオール６４７特異的に結合活性を示すヒトモノクローナル抗体が、偶発的に同定された。本発明はこれらの知見に基づき完成に至ったものである。 The present inventors measured autoantibody titers to cancer-associated antigens in blood against several types of cancer-associated antigen proteins (EGFR, VEGFR2, VEGFR3, CDH11, etc.) in 19 cases of malignant glioma patients. VEGFR2 specific by staining with Alexafluor 647 labeled VEGFR2 protein (extracellular domain) and PE labeled mouse anti-human IgG antibody using peripheral blood B cells of the case (patient) with the highest antibody titer against VEGFR2 Antibody-bearing B cells were sorted, and mRNA for each cell was recovered using Single cell sorter (FACSAria, BD Bioscience), and cDNA cloning was performed. The cloned antibody gene was incorporated into an expression vector, and full-length antibody protein was expressed and secreted in a baculovirus expression system to purify the antibody. When the binding activity of the purified antibody to VEGFR2-Alexafluor 647 and VEGFR2 protein was evaluated by ELISA method, a human monoclonal antibody showing a binding activity specifically to Alexafluor 647, which is a fluorescent dye, was accidentally identified. The The present invention has been completed based on these findings.
すなわち、本発明は以下のとおりである。
（１）アレクサフルオール６４７を認識するモノクローナル抗体。
（２）アレクサフルオール６４７を認識する抗体の重鎖可変領域を構成するポリペプチドであって、該重鎖可変領域が配列番号２に示すアミノ酸配列からなる超可変領域を含むことを特徴とするポリペプチド。
（３）アレクサフルオール６４７を認識する抗体の重鎖可変領域を構成するポリペプチドであって、該重鎖可変領域が配列番号４に示すアミノ酸配列、又は配列番号４と少なくとも９７％同一であるアミノ酸配列からなることを特徴とするポリペプチド。
（４）アレクサフルオール６４７を認識する抗体の重鎖を構成するポリペプチドであって、該重鎖が配列番号６に示すアミノ酸配列、又は配列番号６と少なくとも９５％同一であるアミノ酸配列からなることを特徴とするポリペプチド。
（５）アレクサフルオール６４７を認識する抗体の軽鎖可変領域を構成するポリペプチドであって、該軽鎖可変領域が、配列番号８に示すアミノ酸配列からなる超可変領域を含むことを特徴とするポリペプチド。
（６）アレクサフルオール６４７を認識する抗体の軽鎖可変領域を構成するポリペプチドであって、該軽鎖可変領域が、配列番号１０に示すアミノ酸配列、又は配列番号１０と少なくとも９７％同一であるアミノ酸配列からなることを特徴とするポリペプチド。
（７）アレクサフルオール６４７を認識する抗体の軽鎖を構成するポリペプチドであって、該軽鎖が、配列番号１２に示すアミノ酸配列、又は配列番号１２と少なくとも９５％同一であるアミノ酸配列からなることを特徴とするポリペプチド。
（８）上記（２）〜（７）のいずれかに記載のポリペプチドをコードすることを特徴とするＤＮＡ。
（９）上記（２）〜（４）のいずれかに記載のポリペプチド、及び上記（５）〜（７）のいずれかに記載のポリペプチドを含むことを特徴とする抗アレクサフルオール６４７モノクローナル抗体。
（１０）上記（２）又は（３）に記載のポリペプチド、及び上記（５）又は（６）に記載のポリペプチドを含むことを特徴とする上記（９）記載の抗アレクサフルオール６４７モノクローナル抗体。
（１１）上記（４）に記載のポリペプチド、及び上記（７）に記載のポリペプチドを含むことを特徴とする上記（９）記載の抗アレクサフルオール６４７モノクローナル抗体。That is, the present invention is as follows.
(1) A monoclonal antibody that recognizes Alexaful 647.
(2) A polypeptide comprising a heavy chain variable region of an antibody that recognizes Alexafluor 647, wherein the heavy chain variable region comprises a hypervariable region consisting of the amino acid sequence shown in SEQ ID NO: 2 Polypeptide.
(3) A polypeptide constituting a heavy chain variable region of an antibody that recognizes alexafluor 647, wherein the heavy chain variable region is at least 97% identical to the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 4 A polypeptide characterized by consisting of an amino acid sequence.
(4) A polypeptide constituting a heavy chain of an antibody that recognizes Alexafluor 647, wherein the heavy chain consists of the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence at least 95% identical to SEQ ID NO: 6 A polypeptide characterized by
(5) A polypeptide constituting a light chain variable region of an antibody that recognizes Alexafluor 647, wherein the light chain variable region comprises a hypervariable region consisting of the amino acid sequence shown in SEQ ID NO: 8 Polypeptide.
(6) A polypeptide constituting a light chain variable region of an antibody that recognizes alexafluor 647, wherein the light chain variable region is at least 97% identical to the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 10 A polypeptide characterized by consisting of a certain amino acid sequence.
(7) A polypeptide constituting the light chain of an antibody that recognizes alexafluor 647, wherein the light chain comprises the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence at least 95% identical to SEQ ID NO: 12 The polypeptide characterized by becoming.
(8) A DNA encoding the polypeptide according to any one of the above (2) to (7).
(9) An anti-alexafulol 647 monoclonal antibody characterized by comprising the polypeptide according to any of (2) to (4) above, and the polypeptide according to any of (5) to (7) above antibody.
(10) The anti-alexafluol 647 monoclonal antibody according to the above (9), which comprises the polypeptide according to the above (2) or (3), and the polypeptide according to the above (5) or (6) antibody.
(11) The anti-alexafluor 647 monoclonal antibody according to the above (9), which comprises the polypeptide according to the above (4) and the polypeptide according to the above (7).
本発明によると、アレクサフルオール６４７と特異的に結合するモノクローナル抗体を用いることから、アレクサフルオール６４７で標識された物質をモニタリングすることや、アレクサフルオール６４７で標識された物質を定量することが可能となる。 According to the present invention, since a monoclonal antibody specifically binding to alexafluor 647 is used, monitoring of a substance labeled with alexafluor 647, and quantification of a substance labeled with alexafluor 647 Is possible.
本発明のポリペプチドとしては、アレクサフルオール６４７を認識する抗体の重鎖可変領域や重鎖、及び軽鎖可変領域や軽鎖を構成するポリペプチドであって、１）配列番号２に示すアミノ酸配列からなる超可変領域（相補性決定領域：ＣＤＲ３）を含む重鎖可変領域を構成するポリペプチド［Ｐ１］、２）配列番号４に示すアミノ酸配列、若しくは配列番号４と少なくとも９７％同一、好ましくは９８％以上同一、より好ましくは９９％以上同一であるアミノ酸配列からなる重鎖可変領域を構成するポリペプチド［Ｐ２］、３）配列番号６に示すアミノ酸配列、又は配列番号６と少なくとも９５％同一、好ましくは９８％以上同一、より好ましくは９９％以上同一であるアミノ酸配列からなる重鎖を構成するポリペプチド［Ｐ３］、又は、４）配列番号８に示すアミノ酸配列からなる超可変領域（相補性決定領域：ＣＤＲ３）を含む軽鎖可変領域を構成するポリペプチド［Ｐ４］、５）配列番号１０に示すアミノ酸配列、若しくは配列番号１０と少なくとも９７％同一、好ましくは９８％以上同一、より好ましくは９９％以上同一であるアミノ酸配列からなる軽鎖可変領域を構成するポリペプチド［Ｐ５］、６）配列番号１２に示すアミノ酸配列、若しくは配列番号１２と少なくとも９５％同一、好ましくは９８％以上同一、より好ましくは９９％以上同一であるアミノ酸配列からなる軽鎖を構成するポリペプチド［Ｐ６］を挙げることができる。 The polypeptide of the present invention is a heavy chain variable region or a heavy chain of an antibody that recognizes Alexafluor 647, and a light chain variable region or a polypeptide constituting a light chain, and the amino acid shown in 1) SEQ ID NO: 2 Polypeptide [P1] comprising a heavy chain variable region including a hypervariable region (complementarity determining region: CDR3) consisting of a sequence 2) The amino acid sequence shown in SEQ ID NO: 4 or at least 97% identical to SEQ ID NO: 4 Is a polypeptide comprising a heavy chain variable region consisting of an amino acid sequence that is 98% or more identical, more preferably 99% or more identical, [3] amino acid sequence shown in SEQ ID NO: 6 or at least 95% with SEQ ID NO: 6 Polypeptide [P3] constituting heavy chain consisting of amino acid sequences identical, preferably 98% or more identical, more preferably 99% or more identical, or ) A polypeptide [P4] constituting a light chain variable region comprising a hypervariable region (complementarity determining region: CDR3) consisting of the amino acid sequence shown in SEQ ID NO: 8, 5) the amino acid sequence shown in SEQ ID NO: 10, or SEQ ID NO: 10 A light chain variable region consisting of an amino acid sequence which is at least 97% identical, preferably 98% or more, more preferably 99% or more identical to [P5], 6) the amino acid sequence shown in SEQ ID NO: 12 Polypeptide [P6] which comprises the light chain which consists of an amino acid sequence which is at least 95% identical, preferably 98% or more, more preferably 99% or more identical to SEQ ID NO: 12 can be mentioned.
本発明のＤＮＡとしては、上記本発明のアレクサフルオール６４７を認識する抗体の重鎖可変領域を構成するポリペプチド［Ｐ１］及び［Ｐ２］、重鎖を構成するポリペプチド［Ｐ３］、軽鎖可変領域を構成するポリペプチド［Ｐ４］及び［Ｐ５］、並びに、軽鎖を構成するポリペプチド［Ｐ６］をそれぞれコードするＤＮＡであれば特に制限されず、より具体的には、１）重鎖超可変領域をコードする配列番号１に示す塩基配列を含む重鎖可変領域をコードするＤＮＡ［Ｄ１］、２）重鎖可変領域をコードする配列番号３に示す塩基配列、若しくは配列番号３と少なくとも９５％同一、好ましくは９８％以上同一、より好ましくは９９％以上同一である塩基配列を含むＤＮＡ［Ｄ２］、３）重鎖をコードする配列番号５に示す塩基配列、若しくは配列番号５と少なくとも９０％同一、好ましくは９５％以上同一、より好ましくは９８％以上同一である塩基配列からなるＤＮＡ［Ｄ３］、４）軽鎖超可変領域をコードする配列番号７に示す塩基配列を含む軽鎖可変領域をコードするＤＮＡ［Ｄ４］、５）軽鎖可変領域をコードする配列番号９に示す塩基配列、若しくは配列番号９と少なくとも９５％同一、好ましくは９８％以上同一、より好ましくは９９％以上同一である塩基配列を含むＤＮＡ［Ｄ５］、６）軽鎖をコードする配列番号１１に示す塩基配列、若しくは配列番号１１と少なくとも９０％同一、好ましくは９５％以上同一、より好ましくは９８％以上同一である塩基配列からなるＤＮＡ［Ｄ６］を挙げることができる。 The DNA of the present invention includes the polypeptides [P1] and [P2] constituting the heavy chain variable region of the antibody recognizing the above-mentioned Alexafluor 647 of the present invention, the polypeptide [P3] constituting the heavy chain, the light chain It is not particularly limited as long as it is a DNA encoding each of the polypeptides [P4] and [P5] constituting the variable region and the polypeptide [P6] constituting the light chain, more specifically, 1) heavy chain DNA encoding heavy chain variable region including base sequence shown in SEQ ID NO: 1 encoding hypervariable region [D1] 2) Base sequence shown in SEQ ID NO: 3 encoding heavy chain variable region or SEQ ID NO: 3 and at least DNA [D2] containing a base sequence that is 95% identical, preferably 98% or more identical, more preferably 99% or more identical, 3) a base sequence shown in SEQ ID NO: 5 encoding a heavy chain, Or a DNA [D3] consisting of a nucleotide sequence at least 90% identical, preferably 95% or more identical, more preferably 98% or more identical to SEQ ID NO: 5 to SEQ ID NO: 7 encoding the light chain hypervariable region DNA encoding a light chain variable region containing the nucleotide sequence shown [5], 5) The nucleotide sequence shown in SEQ ID NO: 9 encoding the light chain variable region, or at least 95% identical, preferably 98% or more identical to SEQ ID NO: 9 More preferably, the DNA [D5] containing a nucleotide sequence identical to 99% or more, 6) The nucleotide sequence shown in SEQ ID NO: 11 encoding light chain, or at least 90% identical to SEQ ID NO: 11, preferably 95% or more More preferably, DNA [D6] which consists of a base sequence which is 98% or more identical can be mentioned.
また、上記アミノ酸配列や塩基配列において、例えば、少なくとも９５％同一とは、問題の配列が、アミノ酸やヌクレオチドの各挿入、欠失、置換等をただ１つの違いとして数えたとき、比較しようとしている配列と５％未満の異なるアミノ酸やヌクレオチドを含むことを意味する。 In the above amino acid sequence or base sequence, for example, at least 95% identical means that the sequence in question is to be compared when each insertion, deletion, substitution, etc. of amino acids or nucleotides is counted as one difference. By sequence it is meant to contain less than 5% different amino acids and nucleotides.
本発明のモノクローナル抗体としては、アレクサフルオール６４７に特異的に結合する抗アレクサフルオール６４７モノクローナル抗体であれば特に制限されず、抗アレクサフルオール６４７モノクローナル抗体の重鎖可変領域を構成するポリペプチド［Ｐ１］及び［Ｐ２］、重鎖を構成するポリペプチド［Ｐ３］のいずれかと、軽鎖可変領域を構成するポリペプチド［Ｐ４］及び［Ｐ５］、軽鎖を構成するポリペプチド［Ｐ６］のいずれかを含むモノクローナル抗体、例えば、［Ｐ１］と［Ｐ４］、［Ｐ２］と［Ｐ５］、［Ｐ３］と［Ｐ６］、［Ｐ１］と［Ｐ５］、［Ｐ２］と［Ｐ４］等をそれぞれ含むモノクローナル抗体を具体的に挙げることができる。 The monoclonal antibody according to the present invention is not particularly limited as long as it is an anti-alexafluor 647 monoclonal antibody that specifically binds to alexafluor 647, and a polypeptide constituting the heavy chain variable region of the anti-alexafluor 647 monoclonal antibody [P1] and [P2], any of the heavy chain polypeptide [P3] and the light chain variable region polypeptide [P4] and [P5], the light chain polypeptide [P6] A monoclonal antibody containing any of them, for example, [P1] and [P4], [P2] and [P5], [P3] and [P6], [P1] and [P5], [P2] and [P4], etc. Specific examples include monoclonal antibodies that contain each.
本発明の抗アレクサフルオール６４７モノクローナル抗体には、ＩｇＧ、ＩｇＡ、ＩｇＭ、ＩｇＤ、ＩｇＥ等のいずれのアイソタイプを有するモノクローナル抗体が含まれ、また、本発明の抗アレクサフルオール６４７モノクローナル抗体には、完全長（Full body）モノクローナル抗体の他、モノクローナル抗体の活性フラグメント、例えば、Ｆ（ａｂ′）２、Ｆａｂ′、Ｆａｂ、Ｆｖ（variable fragment of antibody）、ｓＦｖ、ｄｓＦｖ（disulphide stabilised Fv）、ｄＡｂ（single domain antibody）等も含まれる。The anti-alexafluor 647 monoclonal antibodies of the present invention include monoclonal antibodies having any isotype such as IgG, IgA, IgM, IgD, IgE, etc. The anti-alexafluor 647 monoclonal antibodies of the present invention include In addition to full-body monoclonal antibodies, active fragments of monoclonal antibodies, such as F (ab ') 2 , Fab', Fab, Fv (variable fragment of antibody), sFv, dsFv (disulphide stabilized Fv), dAb (d Also included are single domain antibody) and the like.
本発明の抗アレクサフルオール６４７モノクローナル抗体の完全長抗体の作製方法としては、抗アレクサフルオール６４７モノクローナル抗体の重鎖をコードするＤＮＡ［Ｄ３］と軽鎖をコードするＤＮＡ［Ｄ６］を組み込んだ発現ベクターにより宿主細胞を形質転換し、形質転換細胞を適切な培地で培養して発現させることにより得ることができる。Ｆａｂ抗体の作製には、精製された上記完全長抗体をパパイン消化して、Ｆａｂ断片のみを再度精製する従来法が挙げられる。また、一本鎖Ｆｖフラグメント（ｓｃＦｖ）抗体の作製には、抗アレクサフルオール６４７モノクローナル抗体の重鎖可変領域をコードするＤＮＡ［Ｄ１］又は［Ｄ２］と軽鎖可変領域をコードするＤＮＡ［Ｄ４］又は［Ｄ５］とをリンカーで連結して宿主細胞内で発現させる方法等を挙げることができる。 As a method for producing a full-length antibody of the anti-alexafluor 647 monoclonal antibody of the present invention, a DNA encoding the heavy chain of the anti-alexafluor 647 monoclonal antibody [D3] and a DNA encoding the light chain [D6] were incorporated. It can be obtained by transforming host cells with an expression vector and culturing transformed cells in an appropriate medium for expression. Preparation of a Fab antibody includes conventional methods in which papain is digested with the purified full-length antibody and only Fab fragment is purified again. In addition, for the production of a single chain Fv fragment (scFv) antibody, a DNA [D1] or [D2] encoding the heavy chain variable region of the anti-alexafluor 647 monoclonal antibody and a DNA [D4 encoding the light chain variable region] ] Or [D5] with a linker, and the method of making it express in a host cell etc. can be mentioned.
本発明の抗アレクサフルオール６４７モノクローナル抗体には、抗体可変領域（Ｆｖ）と、様々な動物種由来の抗体定常領域（Ｆｃ）を組み合わせたキメラ抗体やヒト化抗体も含まれる。かかるキメラ抗体やヒト化抗体は、Ｆｖ領域をコードする遺伝子とＦｃ領域をコードする遺伝子と結合させて発現させることにより、調製することができる。かかる動物種としては、一般的な抗体の動物種であるマウス、ラット、ハムスター、モルモット、ウサギ、ヤギ、ヒツジ、ロバ、ブタ、ウシ、ウマ、イヌ、ネコ、ニワトリ、サル、ヒト等を例示することができる。定常領域は、可変領域の由来するモノクローナル抗体と同一のものであっても、あるいは、異なるモノクローナル抗体に由来するものであってもよい。例えば、Ｆｃ領域として、公知のヒトのＩｇＧ１、マウスのＩｇＧ１のＦｃ領域が使用可能である。 The anti-alexafluor 647 monoclonal antibodies of the present invention also include chimeric antibodies and humanized antibodies in which antibody variable regions (Fv) and antibody constant regions (Fc) derived from various animal species are combined. Such a chimeric antibody or a humanized antibody can be prepared by linking and expressing a gene encoding an Fv region and a gene encoding an Fc region. As such animal species, mice, rats, hamsters, guinea pigs, rabbits, goats, sheep, donkeys, pigs, cattle, horses, dogs, cats, chickens, monkeys, humans etc. which are animal species of common antibodies are exemplified. be able to. The constant region may be identical to the monoclonal antibody from which the variable region is derived or may be derived from a different monoclonal antibody. For example, as the Fc region, known human IgG1, Fc region of mouse IgG1 can be used.
なお、サンドイッチＥＬＩＳＡ法において本発明のヒト抗アレクサフルオール６４７モノクローナル抗体を捕獲抗体として用いる場合には、かかる捕獲抗体の定常領域が検出抗体の定常領域と異なる動物種由来とすることで、捕獲抗体と検出抗体との交差反応をより防ぐことが可能となる。例えば、図１に示すように、検出抗体として酵素標識抗ヒトＩｇＧ抗体等の抗ヒト抗体を用いる場合には、捕獲抗体としてヒト−マウスキメラ抗体（マウス化抗体）を用いればよい。前記ヒト−マウスキメラ抗体は、例えば、ヒト由来の配列番号６に示すアミノ酸配列からなるポリペプチドコードする遺伝子のうちＦｃ領域配列を人工合成されたマウス由来のＦｃ領域配列と入れ替えた、配列番号１３に示すアミノ酸配列からなるポリペプチドをコードする遺伝子を発現する発現ベクターを作製し、ヒト由来の配列番号１２に示すアミノ酸配列をコードする遺伝子と共にｅｘｐｉ２９３細胞のような哺乳類細胞に導入して培養上清中に産生させることにより得ることができる。 When the human anti-alexafluor 647 monoclonal antibody of the present invention is used as a capture antibody in a sandwich ELISA method, the capture antibody has a constant region derived from an animal species different from the constant region of the detection antibody. It is possible to further prevent cross-reaction between the antibody and the detection antibody. For example, as shown in FIG. 1, when using an anti-human antibody such as an enzyme-labeled anti-human IgG antibody as a detection antibody, a human-mouse chimeric antibody (mouse-ized antibody) may be used as a capture antibody. The human-mouse chimeric antibody is, for example, a gene encoding a polypeptide consisting of an amino acid sequence represented by SEQ ID NO: 6 derived from human, wherein the Fc domain sequence is replaced with an Fc domain sequence derived from artificially synthesized mouse, An expression vector expressing a gene encoding a polypeptide consisting of the amino acid sequence shown in Table 1 is prepared, and introduced into a mammalian cell such as expi 293 cell together with a gene encoding the amino acid sequence shown in SEQ ID NO: 12 derived from human and culture supernatant It can be obtained by being produced therein.
本発明のモノクローナル抗体の製造に用いることができる発現ベクターとしては、本発明のポリペプチドをコードするＤＮＡを、大腸菌、枯草金、酵母、昆虫細胞、植物細胞、動物細胞等の宿主細胞において発現しうるものが好ましく、バキュロウイルスのAutographa californica nuclear polyhedrosis virus（AcNPV）ベクター及びバキュロウイルス用のシャトルベクター（pFastBac Dual vector）を好適に挙げることができる。また、宿主細胞がカイコ（Bombix mori）細胞のＢｍＮ４では、バキュロウイルスの核多角体病ウイルスBombix mori nuclear polyhedrosis virus（BmNPV）ベクターを好適に挙げることができる。また、上記発現ベクターには、プロモーター、ターミネーター、マーカー遺伝子としての薬剤耐性遺伝子を含んでいてもよい。 As an expression vector that can be used for producing the monoclonal antibody of the present invention, a DNA encoding the polypeptide of the present invention is expressed in host cells such as E. coli, Bacillus subtilis, yeast, insect cells, plant cells, animal cells and the like. Preferred are baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) vectors and shuttle vectors for baculovirus (pFastBac Dual vector). In addition, as BmN4 in which the host cell is Bombyx mori cells, Baculovirus nucleopolyhedrosis virus Bombix mori nuclear polyhedrosis virus (BmNPV) vector can be preferably mentioned. In addition, the expression vector may contain a promoter, a terminator, and a drug resistance gene as a marker gene.
また、宿主細胞へのベクターの導入法としては、In-Fusionクローニングシステム（Clontech社）、リポソーム法、リポフェクション法、マイクロインジェクション法、ＤＥＡＥデキストラン法、リン酸カルシウム法、エレクトロポレーション法等を挙げることができ、Lipofectin Reagent（登録商標）、Lipofectamine（登録商標）、Lipofectamine（登録商標）2000 Reagent（インビトロジェン社製）や、SuperFect（登録商標）Transfection Reagent（キアゲン社製）、FuGENE（登録商標）HD Transfection Reagent（ロシュ・ダイアグノスティックス社製）、FuGENE（登録商標）6 Transfection Reagent（ロシュ・ダイアグノスティックス社製）等の市販のトランスフェクション試薬を用いる当技術分野で広く用いられている手法を挙げることができる。 Moreover, examples of methods for introducing a vector into host cells include In-Fusion cloning system (Clontech), liposome method, lipofection method, microinjection method, DEAE dextran method, calcium phosphate method, electroporation method and the like. , Lipofectin Reagent (registered trademark), Lipofectamine (registered trademark), Lipofectamine (registered trademark) 2000 Reagent (manufactured by Invitrogen Corporation), SuperFect (registered trademark) Transfection Reagent (manufactured by Qiagen), FuGENE (registered trademark) HD Transfection Reagent (manufactured by Qiagen) List widely used techniques in the art using commercially available transfection reagents such as Roche Diagnostics, FuGENE® 6 Transfection Reagent (Roche Diagnostics), etc. Can.
本発明の抗アレクサフルオール６４７モノクローナル抗体は、被検非ヒト動物対象に投与された、アレクサフルオール６４７で標識された物質のモニタリングに有利に使用することができる。より詳細には、特定疾患の生物学的製剤（医薬品）の開発に向け実験動物に使用する場合を例として、医薬品への感受性の確認や投与間隔及び投与量の調整等を目的とした、生物学的製剤濃度による有効性、及びin vivo／in situにおける生物学的製剤の局在に対するモニタリングに有利に使用することができる。具体的には、アレクサフルオール６４７で標識された開発候補医薬品を実験動物に投与したのち、適宜観察対象の臓器を取り出し、本発明の抗体を用いた免疫組織化学法、免疫沈降法、ＥＬＩＳＡ法、ウェスタンブロッティング法等により、対象の臓器に局在する該医薬品を同定、可視化、定量する手法を例示することができる。 The anti-alexafluor 647 monoclonal antibody of the present invention can be advantageously used for monitoring a substance labeled with alexafluor 647, which has been administered to a subject non-human animal subject. More specifically, the organism is used for the development of a biological preparation (drug) for a specific disease, for example, to check the sensitivity to a drug and adjust the administration interval and dose, using as an example a laboratory animal. It can be advantageously used for monitoring the efficacy by chemical concentration and the localization of the biologic in vivo / in situ. Specifically, after administering a development candidate drug labeled with Alexafluor 647 to an experimental animal, the organ to be observed is taken out as appropriate, and immunohistochemistry, immunoprecipitation, ELISA using the antibody of the present invention The method of identifying, visualizing and quantifying the medicine localized in the target organ can be exemplified by Western blotting and the like.
本発明の抗アレクサフルオール６４７モノクローナル抗体は、磁気細胞分離法に基づく細胞分離・濃縮を目的とした利用を例示することができ、ＭＡＣＳ（登録商標）細胞分離システム（Miltenyi Biotec社）や、Dynabeads（登録商標）細胞分離システム（インビトロジェン社）、ＢＤ ＩＭａｇ（商標）細胞分離システム等好適に挙げることができる。（ａ）アレクサフルオール６４７で標識された任意の抗体と特異的に結合している濃縮対象細胞を含む、細胞懸濁液試料を調製し；（ｂ）前記試料に、本発明の抗アレクサフルオール６４７モノクローナル抗体が結合した磁気ビーズを添加し、濃縮対象細胞をビーズに結合させ；（ｃ）前記試料を磁場の存在下で強磁性マトリックスに通過させ、強磁性マトリックスに結合しない濃縮非対象細胞を回収し；（ｄ）濃縮対象細胞を提供するために、実質的に磁場の非存在下で該マトリックスから結合細胞を溶出し、濃縮対象細胞を回収する方法を挙げることができる。磁気細胞分離法に基づく細胞分離・濃縮方法の概略を図２に示す。 The anti-alexafluor 647 monoclonal antibody of the present invention can be exemplified for the purpose of cell separation / concentration based on magnetic cell separation method, and MACS (R) Cell Separation System (Miltenyi Biotec), Dynabeads (Registered trademark) cell separation system (Invitrogen), BD IMagTM cell separation system, and the like can be suitably mentioned. (A) preparing a cell suspension sample comprising cells to be concentrated which are specifically bound to any antibody labeled with Alexafluor 647; (b) an anti-alexaflu of the invention to said sample Adding magnetic beads bound by the all 647 monoclonal antibody to bind the cells to be concentrated to the beads; (c) passing the sample through the ferromagnetic matrix in the presence of a magnetic field and not binding to the ferromagnetic matrix And (d) eluting the bound cells from the matrix substantially in the absence of a magnetic field to provide cells to be concentrated, and recovering the cells to be concentrated. An outline of the cell separation / concentration method based on the magnetic cell separation method is shown in FIG.
以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの例示に限定されるものではない。また、実施例において使用した患者検体（細胞および血漿）は、静岡県立静岡がんセンターにて治療中の患者の同意のもと、施設内の臨床研究倫理審査委員会の承認を受けた研究の範囲内で使用された。 Hereinafter, the present invention will be more specifically described by way of examples, but the technical scope of the present invention is not limited to these examples. In addition, the patient samples (cells and plasma) used in the examples are of the research approved by the Clinical Research Ethics Review Board in the institution with the consent of the patient being treated at the Shizuoka Prefectural Shizuoka Cancer Center. Used within range.
１．悪性グリオーマ患者血液中の自家抗体測定
悪性グリオーマ１９症例においてがん関連抗原タンパクの上皮成長因子受容体（ＥＧＦＲ）、血管内皮細胞増殖因子受容体（ＶＥＧＦＲ２及びＶＥＧＦＲ３）、カドヘリン（ＣＤＨ１１）、血小板凝集因子（Ｐｏｄｏｐｌａｎｉｎ）及び陰性コントロールのグルタチオンＳ−トランスフェラーゼ（ＧＳＴ）に対する、患者血液中の自家抗体の測定を、特開２０１０−２３７１２６号公報に開示される、本発明者が開発した「ＥＬＩＳＡ法を用いたヒト血清中の抗原特異的なＩｇＧ抗体力価測定の標準化法」にて実施した（図３）。
各組換えタンパク２０ｎｇを９６ウェルプレートに固相化し、４℃で一晩インキュベートした後、３％ＢＳＡ溶液にてブロッキングを行った。次に希釈した患者由来の血漿１００ｕｌを添加し、２時間室温にてインキュベートした。洗浄後、１０００倍希釈のＨＲＰ標識ヒツジ抗ヒトＩｇＧ抗体（GEヘルスケア社製）、その後発色基質を添加し、イムノリーダー（Immuno Mini NJ-2300、ナルジェヌンク社製）にて吸光度の測定を行った。
その結果、ＧＢ−ＳＣＣ００８症例の血液中には、ＥＧＦＲ、ＶＥＧＦＲ２及びＶＥＧＦＲ３について自家抗体が顕著に存在することが示された。また、ＧＢ−ＳＣＣ００８症例の血液中の各種がん関連抗原タンパクに対する自家抗体は、ウェスタンブロッティングによっても確認できた（図４）。1. Autologous antibody measurement in blood of malignant glioma patients Epidermal growth factor receptor (EGFR) of cancer related antigen protein, vascular endothelial growth factor receptor (VEGFR2 and VEGFR3), cadherin (CDH11), platelet aggregation factor in 19 cases of malignant glioma Measurement of autoantibodies in patient blood against (Podoplanin) and glutathione S-transferase (GST) as a negative control was disclosed in Japanese Patent Laid-Open No. 2010-237126. It carried out by the "standardized method of antigen specific IgG antibody titer measurement in human serum" (FIG. 3).
After 20 ng of each recombinant protein was immobilized on a 96-well plate and incubated overnight at 4 ° C., blocking was performed with a 3% BSA solution. Next, 100 ul of diluted patient plasma was added and incubated for 2 hours at room temperature. After washing, HRP-labeled sheep anti-human IgG antibody (manufactured by GE Healthcare Co., Ltd.) diluted 1000-fold and then a chromogenic substrate were added, and the absorbance was measured with an immunoreader (Immuno Mini NJ-2300, manufactured by Nargenunc). .
As a result, it was shown that in the blood of GB-SCC 008 cases, autoantibodies were prominently present for EGFR, VEGFR2 and VEGFR3. In addition, autoantibodies against various cancer-related antigen proteins in the blood of GB-SCC 008 cases were also confirmed by Western blotting (FIG. 4).
２．シングルセルソーターによるＶＥＧＦＲ２−アレクサフルオール６４７特異的抗体産生Ｂ細胞の分取
ＧＢ−ＳＣＣ００８症例の全血からFicoll-PaqueTM plus（GEヘルスケア社製）処理による密度勾配遠心分離を行い、末梢血単核球を分離した後、CD19マイクロビーズ（Miltenyi社製）を用いてAutoMACS (Miltenyi社製）装置にてCD19陽性細胞を選別した。その後、アレクサフルオール６４７標識ＶＥＧＦＲ２（細胞外ドメイン）タンパク(当研究室にて作製)、ＰＥ標識マウス抗ヒトＩｇＧ抗体（BD Bioscience社製）、ＰｅｒＣＰ標識マウス抗ヒトＣＤ１４モノクローナル抗体（クローン：MfP9）（BD Bioscience社製）及びＰＩ（propidium iodide）を用いてＶＥＧＦＲ２抗体を産生する細胞分取用の染色を行った。また抗CEACAM5マウスハイブリドーマ細胞（クローン：T84.66A3.1A.1F2, ATCC, cat. HB-8747）を使用した実験では、アレクサフルオール６４７標識ヒトCEA-related cell adhesion molecules（ＣＥＡＣＡＭ５）タンパク(当研究室にて作製)、ＰＥ標識ラット抗マウスＣＤ１３８モノクローナル抗体（クローン：２８１−２）（BD Bioscience社製）を使用して細胞の染色を行った。
シングルセルソーター(FACSAria，BD Bioscience社製)を用いて、ＰＩ陰性生細胞のＣＤ１４陰性かつＣＤ１９陽性Ｂ細胞のうち、ＶＥＧＦＲ２（細胞外ドメイン）タンパクに結合する免疫グロブリンＧ（ＩｇＧ）が細胞膜に結合しているＢ細胞にゲートを設定したところ、ＣＤ１９陽性細胞の０．０６％がＶＥＧＦＲ２特異的抗体産生Ｂ細胞であった（図５Ｂ）。セルソーターの検出感度は、抗ＣＥＡＣＡＭ５マウスハイブリドーマ細胞を含む患者末梢血単核球を用いて検討した。含まれる抗ＣＥＡＣＡＭ５マウスハイブリドーマ細胞は、アレクサフルオール６４７標識ＣＥＡＣＡＭ５抗原と抗マウスＣＤ１３８抗体を用いて検出した。セルソーターの検出限界は、１０細胞／１０６末梢血単核球であり、０．００１％であった（図５Ａ）。2. By single cell sorter VEGFR2- Alexa (manufactured by GE Healthcare) full-ol 647-specific antibody-producing B cells of the preparative GB-SCC008 cases whole blood from Ficoll-Paque TM plus perform density gradient centrifugation by treatment, peripheral blood After separating the nuclear cells, CD19-positive cells were sorted by an AutoMACS (manufactured by Miltenyi) apparatus using CD19 microbeads (manufactured by Miltenyi). After that, Alexafluor 647-labeled VEGFR2 (extracellular domain) protein (made in our laboratory), PE-labeled mouse anti-human IgG antibody (BD Bioscience), PerCP-labeled mouse anti-human CD14 monoclonal antibody (clone: MfP9) Staining for cell separation for producing VEGFR2 antibody was performed using (BD Bioscience) and PI (propidium iodide). In the experiment using anti-CEACAM5 mouse hybridoma cells (clone: T84.66A3.1A.1F2, ATCC, cat. HB-8747), Alexaful 647 labeled human CEA-related cell adhesion molecules (CEACAM5) protein (this study) Cells were stained using a PE-labeled rat anti-mouse CD138 monoclonal antibody (clone: 281-2) (manufactured by BD Bioscience).
Immunoglobulin G (IgG) binding to VEGFR2 (extracellular domain) protein is bound to the cell membrane among CD14 negative and CD19 positive B cells of PI negative living cells using a single cell sorter (FACSAria, BD Bioscience) When the gate was set to B cells, 0.06% of CD19 positive cells were VEGFR2 specific antibody producing B cells (FIG. 5B). The detection sensitivity of the cell sorter was examined using patient peripheral blood mononuclear cells containing anti-CEACAM5 mouse hybridoma cells. The included anti-CEACAM5 mouse hybridoma cells were detected using Alexafluor 647 labeled CEACAM5 antigen and anti-mouse CD138 antibody. The detection limit of the cell sorter was 0.001 / 10 6 peripheral blood mononuclear cells (FIG. 5A).
３．１細胞レベルでの抗体遺伝子の解析及び同定
上記により、セルソーターで１細胞ごとに分取したＶＥＧＦＲ２−アレクサフルオール６４７特異的抗体産生Ｂ細胞の、１細胞レベルでの抗体遺伝子の解析を、特許番号第５２０５５９７号公報に開示される、本発明者が開発した「１細胞レベルでの抗体遺伝子の解析・同定方法」に準拠して実施した。まずセルソーターにて１細胞ごとに９６ウェルプレートに分取した後、細胞を破砕させ、直接１細胞ごとにｍＲＮＡを回収した。その後ＲＴ−ＰＣＲを行ったところ、採取した６０個のＢ細胞のうち４０個でＩｇＨ遺伝子の増幅に成功した（図６ 最上段）。また、分取したウェルごとの細胞の有無を確認するために実施した、βアクチンのＲＴ−ＰＣＲでは、３８個で成功した（図６ 最下段）。最終的に同一細胞でＩｇＨ及びＩｇＬ遺伝子がともに増幅された細胞は、２２個であった。これらのすべての細胞からＨ鎖及びＬ（kappa）鎖の抗体遺伝子のｃＤＮＡがクローニングされた。3.1 Analysis and Identification of Antibody Genes at the Cell Level By the above, analysis of antibody genes at the 1 cell level of VEGFR2-Alexafluor 647 specific antibody-producing B cells sorted for each cell by the cell sorter, It implemented based on "the analysis and identification method of the antibody gene in 1 cell level" which this inventor disclosed to the patent No. 5205 597 disclosed. First, each cell was sorted into 96 well plates by a cell sorter, then the cells were disrupted and mRNA was collected directly per cell. Thereafter, RT-PCR was performed, and the IgH gene was successfully amplified in 40 out of 60 collected B cells (FIG. 6, top row). In addition, RT-PCR of β-actin, which was performed to confirm the presence or absence of cells in each well separated, was successful in 38 cells (FIG. 6, bottom row). Finally, there were 22 cells in which IgH and IgL genes were amplified together in the same cell. The cDNAs of antibody genes of H chain and L (kappa) chain were cloned from all these cells.
４．バキュロウイルス発現系による完全長抗体タンパクの作製
２２クローンの抗体遺伝子を発現ベクターに組み込んで、バキュロウイルス発現系にて完全長抗体タンパクを発現・分泌させ、精製を行った。
抗体遺伝子（Ｈ鎖及びＬ鎖）をバキュロウイルス用のシャトルベクター（pFastBac Dual vector）に組み込んで、大腸菌DH10Bacに導入し、相同組換えにより組換えバクミドの作製を行った。バクミドＤＮＡは、昆虫由来のＳｆ９細胞に導入され、組換えバキュロウイルスの作製を行った。増幅されたウイルスは、タンパクの高産生株である昆虫由来のHigh Five細胞に感染させ、２７℃で６４時間無血清培地にて培養した。培養上清中に産生された抗体タンパクは、プロテインＡプレパックカラム（GEヘルスケア社製）を用いて精製を行った。取得できた１５クローンについて、アレクサフルオール６４７標識ＶＥＧＦＲ２及びＶＥＧＦＲ２タンパクに対する結合活性をＥＬＩＳＡ法にて評価した（図７）。アレクサフルオール６４７標識ＶＥＧＦＲ２タンパクに対して強く結合したクローンが５種類認められた（＃４８，５１，５４，５５，５６）。このうちＶＥＧＦＲ２にも結合したものが、クローン＃４８，＃５１であった。従ってこの結果から、＃５４，＃５５，＃５６のクローンは、ＶＥＧＦＲ２タンパクではなく、アレクサフルオール６４７標識特異的に結合活性のある抗体である可能性が示された。ＶＥＧＦＲ２タンパクに対する抗体クローンの＃４８，＃５１，＃５５精製抗体を、ＳＤＳ−ＰＡＧＥ法にて電気泳動し、泳動後のゲルをクマシーブリリアントブルー染色したところ、Ｈ鎖、Ｌ鎖に分かれた明瞭なバンドを確認できた（図８）。4. Preparation of Full-Length Antibody Protein by Baculovirus Expression System The antibody gene of 22 clones was incorporated into an expression vector, and the full-length antibody protein was expressed and secreted in the baculovirus expression system, and purification was performed.
The antibody gene (H chain and L chain) was incorporated into a shuttle vector (pFastBac Dual vector) for baculovirus, introduced into E. coli DH10Bac, and recombinant bacmid was prepared by homologous recombination. The bacmid DNA was introduced into insect-derived Sf9 cells to produce a recombinant baculovirus. The amplified virus was used to infect High Five cells derived from insect, which is a high protein-producing strain, and cultured in serum-free medium at 27 ° C. for 64 hours. The antibody protein produced in the culture supernatant was purified using a protein A pre-packed column (manufactured by GE Healthcare). The binding activity to Alexafluor 647 labeled VEGFR2 and VEGFR2 protein was evaluated by ELISA for 15 clones obtained (FIG. 7). Five types of clones strongly bound to Alexafluor 647-labeled VEGFR2 protein were observed (# 48, 51, 54, 55, 56). Among them, those bound to VEGFR2 were clones # 48 and # 51. Therefore, from this result, it is indicated that the # 54, # 55, and # 56 clones may not be the VEGFR2 protein, but may be an antibody having Alexafluor 647 label-specific binding activity. The # 48, # 51, and # 55 purified antibodies of the antibody clone against VEGFR2 protein were electrophoresed by SDS-PAGE method, and the gel after electrophoresis was stained with Coomassie Brilliant Blue, and it was clearly separated into H chain and L chain. I could confirm the band (Figure 8).
５．抗体クローンのＳＰＲ法を用いた親和性測定
抗体クローンの＃４８，＃５１，＃５５精製抗体と、ＶＥＧＦＲ２タンパク及びアレクサフルオール６４７標識ＶＥＧＦＲ２タンパクとの親和性を、BIAcore X100（GEヘルスケア社製）の機器を用いた表面プラズモン共鳴法（ＳＰＲ解析）にて測定した。チップへのリガンド（各抗体クローン）固定化量は１０００〜１００００レスポンスユニット（ＲＵ）の間で行われた。抗体はＨＢＳ緩衝液（０．１５Ｍ ＮａＣｌ、３ｍＭ ＥＤＴＡ、０．０５％ Ｔｗｅｅｎ２０）に溶解し、流速１０μｌ／ｍｉｎ又は３０μｌ／ｍｉｎ、２５℃、最大１５０μＭの濃度で測定した。チップの再生を行う際には３molar塩化マグネシウムを用いた。BIAcore X100エバリュエーションソフトウェアを用いて測定データから動態定数を算出した。5. Affinity measurement of antibody clones using SPR method Affinity of the # 48, # 51, and # 55 purified antibodies of antibody clones with the VEGFR2 protein and Alexafluor 647 labeled VEGFR2 protein was determined by BIAcore X100 (GE Healthcare) It measured by the surface plasmon resonance method (SPR analysis) using the instrument of 2.). The amount of ligand (each antibody clone) immobilized on the chip was between 1000 and 10000 response units (RU). The antibody was dissolved in HBS buffer (0.15 M NaCl, 3 mM EDTA, 0.05% Tween 20), and measured at a flow rate of 10 μl / min or 30 μl / min at 25 ° C. and a maximum concentration of 150 μM. When regenerating the chip, 3 molar magnesium chloride was used. Kinetic constants were calculated from the measured data using BIAcore X100 evaluation software.
抗体クローン＃４８及び＃５１については、１：１結合モデルにて結合速度定数（Ｋａ）、解離速度定数（Ｋｄ）及び解離定数（ＫＤ）を求め、抗体クローン＃５５については、１：１結合反応の解離速度の差を利用したTwo-state reactionモデルにて解離定数を求めた。結果を表１に示す。ＳＰＲ解析の結果から、クローン＃４８及び＃５１抗体は、ＶＥＧＦＲ２に比較的強く結合活性を示すが、一方クローン＃５５抗体は、ＶＥＧＦＲ２タンパクには親和性を示さず、アレクサフルオール６４７標識ＶＥＧＦＲ２タンパクのみに結合することが明らかとなった。これよりクローン＃５５抗体は、蛍光色素であるアレクサフルオール６４７を特異的に認識するヒトモノクローナル抗体であることがわかった（図９）。 For antibody clones # 48 and # 51, association rate constant (Ka), dissociation rate constant (Kd) and dissociation constant (KD) are determined in 1: 1 binding model, and for antibody clone # 55, 1: 1 binding The dissociation constant was determined by a two-state reaction model using the difference in the dissociation rate of the reaction. The results are shown in Table 1. From the results of SPR analysis, the clone # 48 and # 51 antibodies show relatively strong binding activity to VEGFR2, while the clone # 55 antibody does not show affinity for the VEGFR2 protein, and Alexafluor 647 labeled VEGFR2 protein It became clear to bind only. From this, it was found that the clone # 55 antibody is a human monoclonal antibody that specifically recognizes the fluorescent dye Alexafluor 647 (FIG. 9).
６．サンドイッチＥＬＩＳＡ法を用いたアレクサフルオール６４７標識抗体濃度の測定
アレクサフルオール６４７標識抗体として、アレクサフルオール６４７標識トラスツズマブを次の方法で作製した。まずトラスツズマブ（抗Ｈｅｒ２抗体）の重鎖を構成するポリペプチド（配列番号１４）と軽鎖を構成するポリペプチド（配列番号１５）をコードする遺伝子をそれぞれpcDNA3.3ベクター（サーモフィッシャーサイエンティフィック社製）に組み込み、Expi293細胞に遺伝子導入し、５〜７日間培養した後に培養上清からプロテインＡを用いて回収精製することでトラスツズマブを得た。次に、得られたトラスツズマブに対して、Alexa Fluor 647 Protein Labeling Kit(サーモフィッシャーサイエンティフィック社製）を用い、製品付属のマニュアルに従ってアレクサフルオール６４７標識操作を行い、アレクサフルオール６４７標識トラスツズマブを得た。6. Measurement of Alexafluor 647 Labeled Antibody Concentration Using Sandwich ELISA Method Alexafluor 647 labeled trastuzumab was prepared by the following method as alexafluor 647 labeled antibody. First of all, a gene encoding a polypeptide (SEQ ID NO: 14) constituting the heavy chain of trastuzumab (anti-Her2 antibody) and a polypeptide (SEQ ID NO: 15) constituting the light chain are pcDNA3.3 vectors (Thermo Fisher Scientific Co., Ltd.) And the gene was introduced into Expi 293 cells, cultured for 5 to 7 days, and then recovered and purified from the culture supernatant using protein A to obtain trastuzumab. Next, using the Alexa Fluor 647 Protein Labeling Kit (manufactured by Thermo Fisher Scientific Co., Ltd.), the obtained trastuzumab is labeled with Alexafulol 647 according to the manual attached to the product, and Alexafulol 647 labeled trastuzumab Obtained.
サンドイッチＥＬＩＳＡ法を用いて、アレクサフルオール６４７標識トラスツズマブを次の方法で測定した。９６穴イモビライザーアミノプレート（サーモフィッシャーサイエンティフィック社製）に、ＰＢＳ中に希釈した１０μｇ／ｍｌの抗アレクサフルオール６４７抗体（抗体クローン＃５５）を捕獲抗体として５０μｌ／ウェルずつ分注し室温で２時間反応固定化した後、洗浄液（０．０５％Ｔｗｅｅｎ−２０含有ＰＢＳ）を用いて３回洗浄し、３％ＢＳＡを含むＰＢＳを用いて４℃で一晩反応させブロッキングを行った。上記プレートを洗浄液で３回洗浄した後、０、１５．６２５、３１．２５、６２．５、１２５、２５０、５００及び１０００ｎｇ／ｍｌのアレクサフルオール６４７標識トラスツズマブを各ウェルに加え、室温で２時間反応させた。次に上記プレートを、洗浄液で３回洗浄後、検出抗体として５μｇ／ｍｌのビオチン標識した抗アレクサフルオール６４７抗体（抗体クローン＃５５）を５０μｌ／ウェルずつ加えて３０分間反応させ、洗浄液で３回洗浄した。さらに、０．２μｇ／ｍｌのホースラディッシュペルオキシダーゼ標識ストレプトアビジン（PN21130、サーモフィッシャーサイエンティフィック社製）を５０μｌ／ウェルずつ加えて３０分間反応させ、洗浄液で７回洗浄した後、基質液（TMB substrate reagent set, BDバイオサイエンス社製）を１００μｌ／ウェルずつ加え、室温で３０分間インキュベートし、１Ｍの硫酸水溶液を５０μｌ／ウェルずつ添加し発色反応を停止させた。各ウェルの吸光度（４５０ｎｍ）をプレートリーダー（ImmunoMini NJ-2300、バイオテック社製)を用いて測定した。 Alexafluor 647 labeled trastuzumab was measured by the following method using a sandwich ELISA method. Dispense 50 μl / well of 10 μg / ml anti-alexafluor 647 antibody (antibody clone # 55) diluted in PBS as a capture antibody into a 96-well immobilizer amino plate (manufactured by Thermo Fisher Scientific Co., Ltd.) at room temperature After 2 hours of reaction immobilization, the cells were washed 3 times with a washing solution (PBS containing 0.05% Tween-20) and reacted overnight at 4 ° C. to perform blocking using PBS containing 3% BSA. After washing the plate three times with wash solution, add 0, 15.625, 31.25, 62.5, 125, 250, 500 and 1000 ng / ml of Alexafluor 647-labeled trastuzumab to each well and It was made to react for time. Next, the plate is washed 3 times with a washing solution, then 50 μl / well of 5 μg / ml of a biotin-labeled anti-alexafluor 647 antibody (antibody clone # 55) is added as a detection antibody, and reacted for 30 minutes. Washed several times. Furthermore, 50 μl / well of 0.2 μg / ml horseradish peroxidase-labeled streptavidin (PN 21130, manufactured by Thermo Fisher Scientific Co., Ltd.) is added and reacted for 30 minutes, washed seven times with a washing solution, and then a substrate solution (TMB substrate) 100 μl / well of reagent set (BD Biosciences) was added and incubated for 30 minutes at room temperature, and 50 μl / well of 1 M aqueous sulfuric acid solution was added to stop the color reaction. The absorbance (450 nm) of each well was measured using a plate reader (ImmunoMini NJ-2300, manufactured by Biotech).
吸光度（４５０ｎｍ）をＸ軸に、アレクサフルオール６４７標識トラスツズマブの濃度（ｎｇ／ｍｌ）をＹ軸にしてプロットしたグラフを作成した（図１０）。その結果、吸光度とアレクサフルオール６４７標識トラスツズマブの濃度から相関係数が高い検量線を作成できることが確認できた。したがって、抗体クローン＃５５を用いたサンドイッチＥＬＩＳＡ法により、アレクサフルオール６４７標識抗体の検量線を作成し、被検物質を上記と同様に処理して吸光度（４５０ｎｍ）を測定することで、被検物質中のアレクサフルオール６４７標識抗体の濃度を定量することが可能であることが明らかとなった。 A graph was created in which the absorbance (450 nm) was plotted on the X axis and the concentration (ng / ml) of Alexafluor 647 labeled trastuzumab on the Y axis (FIG. 10). As a result, it was confirmed that a calibration curve having a high correlation coefficient could be created from the absorbance and the concentration of Alexafluor 647-labeled trastuzumab. Therefore, a calibration curve of Alexafluor 647-labeled antibody is prepared by sandwich ELISA using antibody clone # 55, and the test substance is treated in the same manner as described above to measure absorbance (450 nm). It turned out that it is possible to quantify the concentration of Alexafluor 647 labeled antibody in the substance.
サンドイッチＥＬＩＳＡ法によりレクサフルオール６４７標識抗体の濃度を定量することは、抗体医薬の開発に応用できる。例えば、正常な動物と腫瘍細胞を移植した動物に対して、アレクサフルオール６４７で標識した、前記腫瘍細胞を特異的に認識する抗体（以下、「アレクサ標識−腫瘍細胞認識抗体」ともいう）を投与した後、所定期間後にそれぞれの動物から血液を採取する。次に、本発明の抗体を用いたサンドイッチＥＬＩＳＡ法により、それぞれの血液中に含まれるアレクサ標識−腫瘍細胞認識抗体の濃度を定量して比較することで、動物体内の腫瘍細胞に結合するアレクサ標識−腫瘍細胞認識抗体量を測定することができる。かかる腫瘍細胞に結合するアレクサ標識−腫瘍細胞認識抗体量の測定により、抗体医薬として用いた場合の抗体の投与量、投与時期等を判断することが可能となる。 Determining the concentration of Lexafluor 647 labeled antibody by sandwich ELISA can be applied to the development of antibody drugs. For example, an antibody that specifically recognizes the tumor cells (hereinafter also referred to as "Alexa label-a tumor cell recognition antibody") labeled with Alexafluor 647 against normal animals and animals into which tumor cells have been transplanted After administration, blood is collected from each animal after a predetermined period of time. Then, the concentration of Alexa-labeled tumor cell-recognizing antibody contained in each blood is quantified and compared by a sandwich ELISA method using the antibody of the present invention, thereby binding Alexa-labeled to tumor cells in the animal body. The amount of tumor cell recognition antibody can be measured. By measuring the amount of Alexa label-tumor cell recognition antibody that binds to such tumor cells, it becomes possible to determine the dosage, administration timing, etc. of the antibody when used as an antibody drug.
本発明は、アレクサフルオール６４７標識抗体に対する２次抗体として標的細胞の選別や、アレクサフルオール６４７標識タンパクを定量しうるＥＬＩＳＡ系の構築が可能である。特にアレクサフルオール６４７標識抗体のin vivoでの血中濃度のモニタリング等に対し好適に利用することができ、抗体医薬の代謝や薬物動態の解析に有用性を発揮しうると期待される。 The present invention makes it possible to select target cells as secondary antibodies to Alexafluor 647 labeled antibody, and to construct an ELISA system capable of quantifying Alexaful 647 labeled protein. In particular, it can be suitably used for monitoring in vivo blood concentration of Alexafluor 647 labeled antibody, etc., and is expected to be useful for analysis of metabolism and pharmacokinetics of antibody drug.
Claims (3)
アレクサフルオール６４７を認識する抗体の軽鎖可変領域を構成するポリペプチドであって、該軽鎖可変領域が、配列番号１０に示すアミノ酸配列又は配列番号１０と少なくとも９７％同一であるアミノ酸配列からなる、前記軽鎖可変領域を構成するポリペプチド
を含むことを特徴とする抗アレクサフルオール６４７モノクローナル抗体。 A polypeptide constituting the heavy chain variable region of an antibody that recognizes Alexafluor (registered trademark) 647, wherein the heavy chain variable region is at least 97% identical to the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 4 A polypeptide comprising the heavy chain variable region consisting of an amino acid sequence , and
A polypeptide comprising the light chain variable region of an antibody that recognizes Alexafluor 647, wherein the light chain variable region is the amino acid sequence shown in SEQ ID NO: 10 or an amino acid sequence at least 97% identical to SEQ ID NO: 10 An anti-alexafluor 647 monoclonal antibody, comprising: a polypeptide that constitutes the light chain variable region .
アレクサフルオール６４７を認識する抗体の軽鎖を構成するポリペプチドであって、該軽鎖が、配列番号１２に示すアミノ酸配列又は配列番号１２と少なくとも９５％同一であるアミノ酸配列からなる、前記軽鎖を構成するポリペプチド
を含むことを特徴とする請求項１記載の抗アレクサフルオール６４７モノクローナル抗体。 A polypeptide constituting the heavy chain of an antibody that recognizes Alexafluor 647, wherein said heavy chain consists of the amino acid sequence shown in SEQ ID NO: 6 or an amino acid sequence at least 95% identical to SEQ ID NO: 6 A polypeptide constituting a chain , and
A polypeptide constituting the light chain of an antibody that recognizes Alexafluor 647, wherein said light chain consists of the amino acid sequence shown in SEQ ID NO: 12 or an amino acid sequence at least 95% identical to SEQ ID NO: 12 anti Alexa full-ol 647 monoclonal antibody according to claim 1, characterized in that it comprises a polypeptide constituting the chain.
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