JPWO2017061602A1 - Antibody specifically binding to CD147 molecule and use thereof - Google Patents
Antibody specifically binding to CD147 molecule and use thereof Download PDFInfo
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- JPWO2017061602A1 JPWO2017061602A1 JP2017544242A JP2017544242A JPWO2017061602A1 JP WO2017061602 A1 JPWO2017061602 A1 JP WO2017061602A1 JP 2017544242 A JP2017544242 A JP 2017544242A JP 2017544242 A JP2017544242 A JP 2017544242A JP WO2017061602 A1 JPWO2017061602 A1 JP WO2017061602A1
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Abstract
ヒトCD147に特異的に結合するモノクローナル抗体の提供、CD147発現を特徴づけられる多彩な癌種に対する治療への利用。次のa〜cの性質を有する、ヒトCD147に対するモノクローナル抗体またはその抗原結合領域を含む断片。a.ヒトCD147発現がん細胞株に対して反応する、b.カニクイザルCD147発現細胞株に対して反応する、c.ヒトCD147発現がん細胞に対してin vivoで抗腫瘍活性を示す。Providing monoclonal antibodies that specifically bind to human CD147, and therapeutic use for various cancer types characterized by CD147 expression. A fragment comprising a monoclonal antibody against human CD147 or an antigen-binding region thereof having the following properties a to c: a. Reacts against a human CD147 expressing cancer cell line, b. Reacts against a cynomolgus monkey CD147 expressing cell line, c. It exhibits antitumor activity in vivo against human CD147-expressing cancer cells.
Description
本発明は、ヒトCD147に特異的に結合する抗体およびその利用に関する。 The present invention relates to antibodies that specifically bind to human CD147 and uses thereof.
CD147はタイプI膜型の糖タンパク質であり、免疫グロブリン様スーパーファミリーに分類される。多彩な生理的機能を持ち、例えば精子形成や神経ネットワークなどで重要な役割を果たすと共に、動脈硬化やリウマチ、ウィルス感染などへの関連が報告されている。また、多くのがん種において発現が認められており、がんの転移や浸潤、化学薬剤耐性にもCD147の関連が示唆されている(非特許文献1)。CD147はインテグリンやmonocarboxylate transporters(MCTs)やシクロフィリンA、カベオリン−1などと相互作用することが知られており(非特許文献2)、さまざまな状況下で役割を果たしていることが伺える。 CD147 is a type I membrane glycoprotein and is classified into an immunoglobulin-like superfamily. It has various physiological functions and plays an important role in, for example, spermatogenesis and neural networks, and has been reported to be associated with arteriosclerosis, rheumatism, and viral infection. In addition, expression has been observed in many cancer types, and it has been suggested that CD147 is related to cancer metastasis, invasion, and chemical drug resistance (Non-patent Document 1). CD147 is known to interact with integrins, monocarboxylate transporters (MCTs), cyclophilin A, caveolin-1 and the like (Non-Patent Document 2), and it can be seen that it plays a role in various situations.
CD147の機能を調節する抗体が報告されている。例えば、シクロフィリンAとの結合によるT細胞の活性化を阻害する抗体(ABX−CBL)やVEFG産生刺激を抑制する抗体(UM−8D6)などが知られている。その中でも、中国で開発された抗CD147マウス抗体HAb18がよく知られている。HAb18のF(ab’)フラグメントに放射性元素ヨード131(I−131)を標識したLicartin(商標)は肝細胞癌に対する治療薬として中国国内でのみ承認されており、さらにHAb18のキメラ化およびFcにフコースの付加を欠損させた抗体は、肺細胞株への細胞障害活性が増強された事が報告されている(非特許文献3、4)。 Antibodies that regulate the function of CD147 have been reported. For example, an antibody (ABX-CBL) that inhibits activation of T cells by binding to cyclophilin A, an antibody (UM-8D6) that suppresses VEGF production stimulation, and the like are known. Among them, anti-CD147 mouse antibody HAb18 developed in China is well known. Licartin ™, which is labeled with the radioactive element iodine-131 (I-131) on the F (ab ′) fragment of HAb18, has been approved only in China as a therapeutic agent for hepatocellular carcinoma. It has been reported that antibodies lacking fucose addition have enhanced cytotoxic activity against lung cell lines (Non-Patent Documents 3 and 4).
急性骨髄性白血病の患者での予後(非特許文献5)や、大腸がんのリンパ節への転移(非特許文献6)にCD147が大きく関与しているという報告があり、治療のターゲットになりうると推測される。しかし、実際にこれらのがん細胞株を用いた実験でin vivoにおける抗腫瘍効果を示す抗CD147抗体は報告されていない。 There are reports that CD147 is significantly involved in the prognosis of patients with acute myeloid leukemia (Non-patent Document 5) and metastasis of colon cancer to lymph nodes (Non-patent Document 6). Presumed to be possible. However, no anti-CD147 antibody exhibiting an antitumor effect in vivo has been reported in actual experiments using these cancer cell lines.
本発明は、ヒトCD147に特異的に結合するモノクローナル抗体の提供、CD147発現を特徴づけられる多彩な癌種に対する治療への利用を課題とする。 An object of the present invention is to provide a monoclonal antibody that specifically binds to human CD147, and to use it for the treatment of various cancer types characterized by CD147 expression.
本発明者らは上記課題を解決するために鋭意検討を行ったところ、CD147発現癌細胞に対してin vivoで優れた抗腫瘍活性を持つ新規抗CD147モノクローナル抗体、及びそのヒト化抗体の取得に成功し、当該抗体が、既存の抗CD147抗体では試みられていない大腸がんや白血病の細胞株を用いたゼノグラフトマウスモデルにおいて抗腫瘍活性を有することを見出し、本発明を完成した。 The present inventors have conducted extensive studies to solve the above-mentioned problems. As a result, in order to obtain a novel anti-CD147 monoclonal antibody having excellent antitumor activity in vivo against CD147-expressing cancer cells, and a humanized antibody thereof. The present inventors have succeeded and found that the antibody has antitumor activity in a xenograft mouse model using colon cancer and leukemia cell lines that have not been attempted with existing anti-CD147 antibodies, and have completed the present invention.
本発明抗ヒトCD147モノクローナル抗体は、カニクイザルCD147にも反応し、CD147抗原の発現を特徴付けるがん細胞株に対して強い抗腫瘍効果を示すことが判明した。 It was found that the anti-human CD147 monoclonal antibody of the present invention also reacts with cynomolgus CD147 and exhibits a strong antitumor effect against cancer cell lines that characterize the expression of CD147 antigen.
すなわち、本発明は、次の〔1〕〜〔16〕を提供するものである。 That is, the present invention provides the following [1] to [16].
〔1〕次のa〜cの性質を有する、ヒトCD147に対するモノクローナル抗体またはその抗原結合領域を含む断片。
a.ヒトCD147発現がん細胞株に対して反応する、
b.カニクイザルCD147発現細胞株に対して反応する、
c.ヒトCD147発現がん細胞に対してin vivoで抗腫瘍活性を示す。
〔2〕重鎖の可変領域にCDR1として配列SYGIS(配列番号17)、CDR2として配列WINPNSGGTNYAQKFQG(配列番号18)、CDR3として配列GRGSYYAFDI(配列番号19)を有し、軽鎖の可変領域にCDR1として配列KSSQSVLYSSNNKNYLA(配列番号20)、CDR2として配列WASTRES(配列番号21)、CDR3として配列QQYYSTPT(配列番号22)を有する〔1〕に記載のモノクローナル抗体またはその抗原結合領域を含む断片。
〔3〕重鎖の可変領域に配列番号4のアミノ酸配列と、軽鎖の可変領域に配列番号8のアミノ酸配列を有する〔1〕に記載のモノクローナル抗体またはその抗原結合領域を含む断片。
〔4〕重鎖の可変領域にCDR1として配列SYGMS(配列番号35)、CDR2として配列TISSGGSYTYYQDSIKG(配列番号36)、CDR3として配列GDWADY(配列番号37)を有し、軽鎖の可変領域にCDR1として配列KASQDINSYLS(配列番号38)、CDR2として配列RANRLVA(配列番号39)、CDR3として配列LQYDEFPLT(配列番号40)を有する〔1〕に記載のモノクローナル抗体またはその抗原結合領域を含む断片。
〔5〕重鎖の可変領域に配列番号6のアミノ酸配列と、軽鎖の可変領域に配列番号10のアミノ酸配列を有する〔1〕に記載のモノクローナル抗体またはその抗原結合領域を含む断片。
〔6〕重鎖の可変領域にCDR1として配列TYWIE(配列番号29)、CDR2として配列EFLPGSGSTNFNEKFKG(配列番号30)、CDR3として配列SGGNFGARFAS(配列番号31)を有し、軽鎖の可変領域にCDR1として配列RSSKSLLSNNGNTYLY(配列番号32)、CDR2として配列RMSSLAS(配列番号33)、CDR3として配列MQHLEYPFT(配列番号34)を有する〔1〕に記載のモノクローナル抗体またはその抗原結合領域を含む断片。
〔7〕重鎖の可変領域に配列番号5のアミノ酸配列と、軽鎖の可変領域に配列番号9のアミノ酸配列を有する〔1〕に記載のモノクローナル抗体またはその抗原結合領域を含む断片。
〔8〕〔2〕〜〔7〕のいずれかに記載の抗CD147モノクローナル抗体の可変領域のCDR周辺を含むアミノ酸配列と、ヒトイムノグロブリンのアミノ酸配列とからなる、ヒト化された抗CD147モノクローナル抗体またはその抗原結合領域を含む断片。
〔9〕〔8〕に記載の抗体であって、重鎖可変領域および軽鎖可変領域の組み合わせが、1)配列番号13および14で示されるアミノ酸配列、または、2)配列番号15および16で示されるアミノ酸配列を有する、ヒト化重鎖およびヒト化軽鎖抗CD147モノクローナル抗体またはその抗原結合領域を含む断片。
〔10〕〔2〕〜〔9〕いずれかに記載の抗体のアミノ酸配列において1または複数のアミノ酸が置換、欠失および付加または挿入され、同一性のレベルが少なくとも90%以上である〔1〕〜〔9〕のいずれかに記載の抗CD147モノクローナル抗体またはその抗原結合領域を含む断片。
〔11〕〔1〕〜〔10〕いずれかに記載の抗CD147モノクローナル抗体またはその抗原結合領域を含む断片に、細胞傷害性薬物を結合させたコンジュゲート抗体。
〔12〕〔1〕〜〔10〕のいずれかに記載の抗CD147モノクローナル抗体またはその抗原結合領域を含む断片に、放射性同位元素を標識させたコンジュゲート抗体。
〔13〕〔1〕〜〔12〕のいずれかに記載の抗CD147モノクローナル抗体、その抗原結合領域を含む断片またはコンジュゲート抗体を有効成分として含む医薬組成物。
〔14〕〔1〕〜〔12〕のいずれかに記載の抗CD147モノクローナル抗体、その抗原結合領域を含む断片またはコンジュゲート抗体を有効成分として含むヒトCD147抗原の発現を特徴づける腫瘍に対する抗腫瘍剤。
〔15〕ヒトCD147抗原の発現を特徴づける腫瘍を治療するための、〔1〕〜〔12〕のいずれかに記載の抗CD147モノクローナル抗体、その抗原結合領域を含む断片またはコンジュゲート抗体。
〔16〕ヒトCD147抗原の発現を特徴づける腫瘍に対する抗腫瘍剤製造のための、〔1〕〜〔12〕のいずれかに記載の抗CD147モノクローナル抗体、その抗原結合領域を含む断片またはコンジュゲート抗体の使用。
〔17〕ヒトCD147抗原の発現を特徴づける細胞を殺傷するための方法であって、該細胞にヒト細胞表面CD147抗原と特異的に結合する〔1〕〜〔12〕のいずれかに記載の抗CD147モノクローナル抗体、その抗原結合領域を含む断片またはコンジュゲート抗体を投与することを特徴とする、該抗体またはその抗原結合領域を含む断片がCD147に結合することで該細胞を殺傷させる、細胞傷害方法。
〔18〕ヒトCD147抗原の発現により特徴づけられる腫瘍を有する対象者を治療するための方法であって、該対象者に、ヒトCD147と特異的に結合する〔1〕〜〔12〕のいずれかに記載の抗CD147モノクローナル抗体、その抗原結合領域を含む断片またはコンジュゲート抗体を投与することを特徴とする、該抗体又はその抗原結合領域を含む断片のCD147への結合により該腫瘍を殺傷させる、腫瘍の治療方法。[1] A fragment comprising a monoclonal antibody against human CD147 or an antigen-binding region thereof having the following properties a to c:
a. Reacts against a human CD147 expressing cancer cell line,
b. Reacts against a cynomolgus monkey CD147 expressing cell line,
c. It exhibits antitumor activity in vivo against human CD147-expressing cancer cells.
[2] The heavy chain variable region has the sequence SYGIS (SEQ ID NO: 17) as CDR1, the sequence WINPNSGGTNYAQKFQG (SEQ ID NO: 18) as CDR2, the sequence GRGSYYAAFDI (SEQ ID NO: 19) as CDR3, and the CDR1 in the light chain variable region A fragment comprising the monoclonal antibody according to [1], which has the sequence KSSQSVLYSSNNNKNYLA (SEQ ID NO: 20), the sequence WASTRES (SEQ ID NO: 21) as CDR2, and the sequence QQYYSTPT (SEQ ID NO: 22) as CDR3, or an antigen-binding region thereof.
[3] The monoclonal antibody or the antigen-binding region thereof according to [1], which has the amino acid sequence of SEQ ID NO: 4 in the variable region of the heavy chain and the amino acid sequence of SEQ ID NO: 8 in the variable region of the light chain.
[4] The heavy chain variable region has the sequence SYGMS (SEQ ID NO: 35) as CDR1, the sequence TISSGGSYTYYQDSIGG (SEQ ID NO: 36) as CDR2, the sequence GDWADY (SEQ ID NO: 37) as CDR3, and the CDR1 in the variable region of the light chain A fragment comprising the monoclonal antibody according to [1], which has the sequence KASQDINSYLS (SEQ ID NO: 38), the sequence RANRLVA (SEQ ID NO: 39) as CDR2, and the sequence LQYDEFPLT (SEQ ID NO: 40) as CDR3.
[5] The monoclonal antibody or the antigen-binding region thereof according to [1], which has the amino acid sequence of SEQ ID NO: 6 in the variable region of the heavy chain and the amino acid sequence of SEQ ID NO: 10 in the variable region of the light chain.
[6] The heavy chain variable region has the sequence TYWIE (SEQ ID NO: 29) as CDR1, the sequence EFLPGSGSTNFNEKFKG (SEQ ID NO: 30) as CDR2, the sequence SGGNFGARFAS (SEQ ID NO: 31) as CDR3, and the CDR1 in the variable region of the light chain A fragment comprising the monoclonal antibody or the antigen-binding region thereof according to [1], which has the sequence RSSSKSLSNNGNTLYLY (SEQ ID NO: 32), the sequence RMSSLAS (SEQ ID NO: 33) as CDR2, and the sequence MQHLEYPFT (SEQ ID NO: 34) as CDR3.
[7] The fragment comprising the monoclonal antibody or the antigen-binding region thereof according to [1] having the amino acid sequence of SEQ ID NO: 5 in the variable region of the heavy chain and the amino acid sequence of SEQ ID NO: 9 in the variable region of the light chain.
[8] A humanized anti-CD147 monoclonal antibody comprising an amino acid sequence including the CDR periphery of the variable region of the anti-CD147 monoclonal antibody according to any one of [2] to [7], and a human immunoglobulin amino acid sequence Or a fragment containing the antigen-binding region thereof.
[9] The antibody according to [8], wherein the combination of the heavy chain variable region and the light chain variable region is 1) an amino acid sequence represented by SEQ ID NOs: 13 and 14, or 2) SEQ ID NOs: 15 and 16 A fragment comprising a humanized heavy chain and a humanized light chain anti-CD147 monoclonal antibody or antigen binding region thereof having the amino acid sequence shown.
[10] One or more amino acids are substituted, deleted and added or inserted in the amino acid sequence of the antibody according to any one of [2] to [9], and the level of identity is at least 90% or more [1] A fragment containing the anti-CD147 monoclonal antibody or the antigen-binding region thereof according to any one of to [9].
[11] A conjugated antibody in which a cytotoxic drug is bound to the anti-CD147 monoclonal antibody according to any one of [1] to [10] or a fragment containing the antigen-binding region thereof.
[12] A conjugate antibody in which a radioisotope is labeled on the anti-CD147 monoclonal antibody according to any one of [1] to [10] or a fragment containing the antigen-binding region thereof.
[13] A pharmaceutical composition comprising as an active ingredient the anti-CD147 monoclonal antibody according to any one of [1] to [12], a fragment containing the antigen-binding region thereof, or a conjugate antibody.
[14] Antitumor agent against tumor characterized by expression of human CD147 antigen comprising as an active ingredient the anti-CD147 monoclonal antibody according to any one of [1] to [12], a fragment containing the antigen-binding region thereof, or a conjugate antibody .
[15] The anti-CD147 monoclonal antibody according to any one of [1] to [12], a fragment containing the antigen-binding region thereof, or a conjugate antibody for treating a tumor characterized by the expression of human CD147 antigen.
[16] The anti-CD147 monoclonal antibody according to any one of [1] to [12], a fragment containing the antigen-binding region thereof, or a conjugate antibody for the production of an antitumor agent against a tumor characterized by the expression of human CD147 antigen Use of.
[17] A method for killing a cell that characterizes the expression of a human CD147 antigen, which specifically binds to the human cell surface CD147 antigen on the cell, according to any one of [1] to [12] A cytotoxic method comprising killing the cells by binding to CD147, comprising administering a CD147 monoclonal antibody, a fragment containing the antigen-binding region thereof, or a conjugated antibody. .
[18] A method for treating a subject having a tumor characterized by the expression of a human CD147 antigen, wherein the subject specifically binds to human CD147, according to any one of [1] to [12] An anti-CD147 monoclonal antibody, a fragment comprising the antigen-binding region thereof, or a conjugate antibody, wherein the tumor is killed by binding of the antibody or a fragment comprising the antigen-binding region thereof to CD147, How to treat a tumor.
本発明の抗ヒトCD147モノクローナル抗体またはその抗原結合領域を含む断片は、白血病やバーキットリンパ腫、Bリンパ腫、肺がん、肝癌、膵臓がん、前立腺がん、大腸がんなどのCD147抗原の発現を特徴づけられる腫瘍に対するin vivoで強い抗腫瘍効果を示し、多様な癌種への抗がん剤として有用である。 The anti-human CD147 monoclonal antibody of the present invention or a fragment containing the antigen-binding region thereof is characterized by the expression of CD147 antigen such as leukemia, Burkitt lymphoma, B lymphoma, lung cancer, liver cancer, pancreatic cancer, prostate cancer, and colon cancer. It exhibits a strong antitumor effect in vivo against tumors that are attached, and is useful as an anticancer agent for various cancer types.
以下、この発明の実施の形態について詳細に説明する。
1.定義
本明細書で使用する用語の定義は以下のとおりである。Hereinafter, embodiments of the present invention will be described in detail.
1. Definitions The terms used in this specification are defined as follows.
ヒトCD147は4つのアイソフォームが同定されている。第1のアイソフォーム(「長いアイソフォーム」または「アイソフォーム1」としてもまた公知である)は、385個のアミノ酸の前駆ポリペプチドとして発現され、これは最初の21残基によって表されるシグナル配列を有する。第2のアイソフォーム(「アイソフォーム2」としてもまた公知である)は、第1のアイソフォームの23〜139番目のアミノ酸配列が欠損した269個のアミノ酸の前駆ポリペプチドとして発現され、これも最初の21残基によって表されるシグナル配列を有する。第2アイソフォームはいくつかの正常組織で発現が認められ、多くのがん種で発現の亢進が報告されている。第3のアイソフォームは第1のアイソフォームの1〜209番目のアミノ酸配列が欠損した176個のアミノ酸から成り、第4のアイソフォームは第1のアイソフォームの1〜11番目のアミノ酸配列が異なり、12〜191番目のアミノ酸が欠損した205個のアミノ酸から形成される。第3と4のアイソフォームの発現はまれに正常細胞や腫瘍細胞で見られる程度である。第1のアイソフォームは網膜に特異的に発現が確認されている。
本発明では、「CD147抗原」または「CD147」は、主に第2のアイソフォームを包含する。本発明の抗CD147抗体は、本明細書中で以下に示されるようにヒトCD147のエピトープに結合し、このヒトCD147は、この細胞表面抗原の第1のアイソフォームまたは第2のアイソフォームのいずれかのうちの同じ位置に存在する。また、「CD147」の前に付したものがなければ、「ヒトCD147」を意味する。Four isoforms of human CD147 have been identified. The first isoform (also known as “long isoform” or “isoform 1”) is expressed as a precursor polypeptide of 385 amino acids, which is a signal represented by the first 21 residues. Has an array. The second isoform (also known as “isoform 2”) is expressed as a 269 amino acid precursor polypeptide lacking the 23rd to 139th amino acid sequence of the first isoform, which is also It has a signal sequence represented by the first 21 residues. The second isoform is expressed in several normal tissues, and increased expression has been reported in many cancer types. The third isoform consists of 176 amino acids from which the 1st to 209th amino acid sequences of the first isoform are deleted, and the 4th isoform differs in the 1st to 11th amino acid sequences of the first isoform. , Amino acids 12 to 191 are formed from 205 amino acids deleted. The expression of the third and fourth isoforms is rarely seen in normal cells and tumor cells. The first isoform is confirmed to be specifically expressed in the retina.
In the present invention, “CD147 antigen” or “CD147” mainly includes the second isoform. The anti-CD147 antibodies of the invention bind to an epitope of human CD147 as set forth herein below, which human CD147 is either the first or second isoform of the cell surface antigen. Exist in the same position. If there is nothing before “CD147”, it means “human CD147”.
CD147抗原は、種々の細胞型の表面に提示される。「発現を特徴付ける」および「発現する」により、CD147抗原のうちのすべてまたは一部が、細胞の外側に曝露されることが意図される。提示または発現されたCD147抗原は、完全にまたは部分的にグリコシル化され得る。 CD147 antigen is presented on the surface of various cell types. By “characterize expression” and “express” is intended that all or part of the CD147 antigen is exposed outside the cell. The presented or expressed CD147 antigen can be fully or partially glycosylated.
本明細書中で用いる「CD147エピトープ」とは、抗CD147抗体と免疫反応することができる分子(例えば、ペプチド)または該分子の断片を意味し、例えば、モノクローナル抗体により認識されるCD147抗原決定基が含まれる。CD147抗原エピトープはタンパク質、タンパク質断片、ペプチドなどに含まれていてよい。エピトープは最も一般的には、タンパク質、短いオリゴペプチド、オリゴペプチド模倣物(すなわち、CD147抗原の抗体結合特性を模倣する有機化合物)、またはそれらの組合せである。 As used herein, “CD147 epitope” means a molecule (eg, a peptide) or a fragment of the molecule that can immunoreact with an anti-CD147 antibody, eg, a CD147 antigenic determinant recognized by a monoclonal antibody. Is included. CD147 antigen epitopes may be included in proteins, protein fragments, peptides and the like. An epitope is most commonly a protein, a short oligopeptide, an oligopeptide mimetic (ie, an organic compound that mimics the antibody binding properties of the CD147 antigen), or a combination thereof.
「抗体」とは、本明細書では、2本の同一の軽(L)鎖と2本の同一の重(H)鎖からなる、典型的には約150,000ダルトンの、ヘテロ四量体糖タンパク質として定義される。各軽鎖は1つのジスルフィド結合によって重鎖と共有結合で連結されて、ヘテロ二量体を形成している。そのようなヘテロ二量体の2本の同一の重鎖間のジスルフィド共有結合によってヘテロ四量体が形成される。軽鎖と重鎖は1つのジスルフィド結合によって一緒に連結されるが、2本の重鎖間のジスルフィド結合の数は免疫グロブリンのアイソタイプにより変化する。それぞれの重鎖および軽鎖はまた、規則的に間隔をあけて配置された鎖内ジスルフィド架橋を有する。各重鎖は、アミノ酸の末端に、可変ドメイン(VH)、その後に3または4つの定常ドメイン(CH1、CH2、CH3、およびCH4)を有し、CH1とCH2の間にヒンジ領域が存在する。各軽鎖は2つのドメイン、すなわち、アミノ末端の可変ドメイン(VL)とカルボキシ末端の定常ドメイン(CL)を有する。VLドメインはVHドメインと非共有結合で会合しており、一方CLドメインは通常はジスルフィド結合を介してCH1ドメインと共有結合で連結されている。特定のアミノ酸残基が軽鎖可変ドメインと重鎖可変ドメイン間の界面を形成していると考えられている(Chothia et al.,J MoI Biol 1985;186:651−663)。“Antibody” as used herein is a heterotetramer, typically about 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Defined as glycoprotein. Each light chain is covalently linked to the heavy chain by one disulfide bond to form a heterodimer. A heterotetramer is formed by a disulfide covalent bond between two identical heavy chains of such a heterodimer. The light and heavy chains are linked together by a single disulfide bond, but the number of disulfide bonds between the two heavy chains varies with the immunoglobulin isotype. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (V H ) at the end of the amino acid followed by 3 or 4 constant domains (C H 1, C H 2, C H 3, and C H 4), and C H 1 And a hinge region exists between C H 2. Each light chain has two domains: an amino-terminal variable domain (V L ) and a carboxy-terminal constant domain (C L ). V L domains are associated non-covalently with a V H domain, whereas the C L domain usually are covalently linked with C H 1 domain via a disulfide bond. It is believed that certain amino acid residues form an interface between the light chain variable domain and the heavy chain variable domain (Chothia et al., J MoI Biol 1985; 186: 651-663).
「可変」とは、可変ドメインの特定の部分が抗体間で広範囲にわたって配列が異なり、その特定の抗原に対する各々の特定の抗体の結合および特異性に使用される事実をいう。しかし、その可変性は、抗体の可変ドメインの全体にわたって均一に分布しない。可変性は、軽鎖可変ドメインおよび重鎖可変ドメインの両方で相補性決定領域(CDR;complementarity determining region)と呼ばれる3つのセグメントに集中する。可変ドメインのうち、より高度に保存される部分は、フレームワーク領域(framework region;FR)と呼ばれる。 “Variable” refers to the fact that a particular portion of a variable domain varies widely in sequence between antibodies and is used for the binding and specificity of each particular antibody for that particular antigen. However, the variability is not evenly distributed throughout the variable domain of the antibody. Variability is concentrated in three segments called complementarity determining regions (CDRs) in both the light chain variable domain and the heavy chain variable domain. The more highly conserved portion of the variable domain is called the framework region (FR).
「相補性決定領域(CDR)」とは、可変ドメイン内のある種の配列が抗体間で配列が大きく異なり、その特異的抗原決定基に対する特定の各抗体の結合および特異性に直接関与するある種の残基を含むということを示す。軽鎖可変ドメインでも重鎖可変ドメインでも、3つのセグメントに集中している。CDRはKabat et al,Sequences of Proteins of Immunological Interest,5thEd.Public Health Service,National Institutes of Health,1993:Bethesda,MD.の配列比較により定義される。Kabatにより定義されているように、CDR−L1は軽鎖可変ドメインの24〜34残基付近に、CDR−L2は50〜56残基付近に、そしてCDR−L3は89〜97残基付近に位置し、CDR−H1は重鎖可変ドメインの31〜35付近、CDR−H2は50〜65付近、そしてCDR−H3は95〜102付近に位置する。A “complementarity determining region (CDR)” is a sequence in which certain sequences within a variable domain vary greatly between antibodies and are directly involved in the binding and specificity of each particular antibody to its specific antigenic determinant Indicates that it contains species residues. Both light and heavy chain variable domains are concentrated in three segments. CDR is Kabat et al, Sequences of Proteins of Immunological Interest, 5 th Ed. Public Health Service, National Institutes of Health, 1993: Bethesda, MD. Defined by sequence comparison. As defined by Kabat, CDR-L1 is around residues 24-34 of the light chain variable domain, CDR-L2 is around residues 50-56, and CDR-L3 is around residues 89-97. CDR-H1 is located near 31-35 of the heavy chain variable domain, CDR-H2 is located near 50-65, and CDR-H3 is located near 95-102.
重鎖および軽鎖の各々の3つのCDRは、変動が少ない傾向にある配列を含むフレームワーク領域(FR)によって隔てられている。重鎖可変ドメインおよび軽鎖可変ドメインのアミノ末端〜カルボキシ末端までは、FRとCDRは、FR1、CDR1、FR2、CDR2、FR3、CDR3およびFR4の順で並んでいる。FRの大まかなβ−シート配置は各鎖内のCDRを互いと近接させるととともに、互いのCDRと近接させる。この結果生じた配置は抗原結合部位に寄与するが、全てのCDR残基が必ずしも直接抗原結合に関与するわけではない。 The three CDRs of each heavy and light chain are separated by a framework region (FR) containing sequences that tend to be less variable. From the amino terminus to the carboxy terminus of the heavy chain variable domain and the light chain variable domain, FR and CDR are arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The rough β-sheet arrangement of FRs brings the CDRs in each chain close to each other and close to each other CDRs. The resulting configuration contributes to the antigen binding site, but not all CDR residues are directly involved in antigen binding.
FR残基およびIg定常ドメインは抗原結合には直接関与しないが、抗原結合に寄与し、かつ/または抗体エフェクター機能を媒介する。いくつかのFR残基は、エピトープと直接非共有結合すること、1以上のCDR残基と相互作用すること、また、重鎖と軽鎖の間の境界に影響を及ぼすことによる、少なくとも3つの方法で抗原結合に有意な作用を持ち得る。定常ドメインは抗原結合には直接関与しないが、抗体依存性細胞傷害性(ADCC)、補体依存性細胞傷害性(CDC)および抗体依存性細胞食作用(ADCP)における抗体の関与など、種々のIgエフェクター機能を媒介する。 FR residues and Ig constant domains are not directly involved in antigen binding, but contribute to antigen binding and / or mediate antibody effector functions. Some FR residues are at least 3 by directly non-covalently binding to the epitope, interacting with one or more CDR residues, and affecting the boundary between the heavy and light chains. The method can have a significant effect on antigen binding. Constant domains are not directly involved in antigen binding, but include a variety of antibodies, including antibody-dependent cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody involvement in antibody-dependent cellular phagocytosis (ADCP). Mediates Ig effector function.
「抗原結合領域を含む断片」は、インタクトな抗体の一部、好ましくは、インタクトな抗体の抗原結合フラグメントまたは可変領域を含む。抗原結合フラグメントの例としては、Fab、Fab’、F(ab’)2およびFvフラグメント;二重特異性抗体;直鎖状抗体(Zapata et.Al.,Protein Eng 1995;8(10):1057−1062);単鎖抗体分子;ならびに抗原結合フラグメントから形成された多重特異性抗体が挙げられる。抗体のパパイン消化により、各々単一の抗原結合部位を有する2つの同一の抗原結合フラグメント(「Fab」フラグメントと呼ばれる)、および残りの「Fc」フラグメント(この名称は、容易に結晶化する能力を反映する)が生成される。ペプシン処理により、F(ab’)2フラグメントが得られ、これは、2つの抗原結合部位を有し、依然として抗原と架橋結合し得る。 A “fragment comprising an antigen binding region” comprises a portion of an intact antibody, preferably an antigen binding fragment or variable region of an intact antibody. Examples of antigen-binding fragments include Fab, Fab ′, F (ab ′) 2 and Fv fragments; bispecific antibodies; linear antibodies (Zapata et. Al., Protein Eng 1995; 8 (10): 1057 -1062); single chain antibody molecules; as well as multispecific antibodies formed from antigen binding fragments. Upon papain digestion of the antibody, two identical antigen-binding fragments (referred to as “Fab” fragments) each having a single antigen-binding site, and the remaining “Fc” fragment (this name has the ability to easily crystallize). Is reflected). Pepsin treatment yields an F (ab ') 2 fragment that has two antigen binding sites and can still crosslink with antigen.
「Fv」は、完全な抗原認識部位および抗原結合部位を含む最小の抗原結合フラグメントである。2本の鎖(two−chain)のFv種では、この領域は、固く非共有結合した1つの重鎖可変ドメインと1つの軽鎖可変ドメインとのダイマーからなる。単鎖のFv種では、1つの重鎖可変ドメインと1つの軽鎖可変ドメインとが、その軽鎖と重鎖とが2本の鎖のFv種における構造に類似する「ダイマーの」構造に会合し得るように、フレキシブルなペプチドリンカーによって共有結合され得る。この構成において、各々の可変ドメインの3つのCDRが相互作用して、VH−VLダイマーの表面における抗原結合部位を規定する。合わせて、6つのCDRが抗体に対する抗原結合特異性を与える。しかし、単一の可変ドメイン(または抗原に対して特異的な3つのCDRのみを含むFvの半分)でも、抗原を認識し結合する能力を有するが、完全な結合部位よりは親和性は低い。“Fv” is the minimum antigen-binding fragment that contains a complete antigen recognition and binding site. In the two-chain Fv species, this region consists of a dimer of one heavy chain variable domain and one light chain variable domain that are tightly non-covalently linked. In a single chain Fv species, one heavy chain variable domain and one light chain variable domain associate in a “dimeric” structure whose light and heavy chains are similar to the structure in a two chain Fv species. As such, it can be covalently linked by a flexible peptide linker. In this configuration, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the V H -V L dimer. Together, the six CDRs provide antigen binding specificity for the antibody. However, even a single variable domain (or half of an Fv that contains only three CDRs specific for the antigen) has the ability to recognize and bind the antigen, but has a lower affinity than the complete binding site.
任意の脊椎動物種由来の抗体(免疫グロブリン)の「軽鎖」は、それらの定常ドメインのアミノ酸配列に基づいて、2つの明らかに異なる型(カッパ(κ)およびラムダ(λ)と呼ばれる)の一方に割り当てられ得る。 “Light chains” of antibodies (immunoglobulins) from any vertebrate species are of two distinct types (called kappa (κ) and lambda (λ)), based on the amino acid sequences of their constant domains. Can be assigned to one.
それらの重鎖の定常ドメインのアミノ酸配列に依存して、免疫グロブリンが異なるクラスに割り当てられ得る。ヒト免疫グロブリンの5つの主要なクラスが存在する:IgA、IgD、IgE、IgGおよびIgM、そしてこれらの1または数個か、さらにサブクラス(アイソタイプ)(例えば、IgG1、IgG2、IgG3、IgG4、IgAおよびIgA2)に分けられ得る。免疫グロブリンの異なるクラスに対応する重鎖定常ドメインは、それぞれ、アルファ、デルタ、イプシロン、ガンマおよびミューと呼ばれる。免疫グロブリンの異なるクラスのサブユニット構造および三次元配置は周知である。異なるアイソタイプは異なるエフェクター機能を有する。例えば、ヒトIgG1アイソタイプおよびIgG3アイソタイプは、抗体依存性細胞傷害性(ADCC)の活性を媒介する。 Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to different classes. There are five major classes of human immunoglobulins: IgA, IgD, IgE, IgG and IgM, and one or several of these, plus subclasses (isotypes) (eg, IgG1, IgG2, IgG3, IgG4, IgA and IgA2). The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known. Different isotypes have different effector functions. For example, the human IgG1 and IgG3 isotypes mediate antibody-dependent cytotoxicity (ADCC) activity.
「抗腫瘍効果」は、腫瘍細胞の増殖または生存を阻害する活性を意味する。この腫瘍細胞の増殖または生存の阻害は、in vitroで、また、in vivoでもよい。例えば、in vitroで腫瘍細胞の数を減少させる活性、腫瘍細胞の数の増加を阻害する活性、腫瘍細胞の細胞死を引き起こす活性、ADCC活性、CDC活性などが挙げられる。in vivoでは腫瘍の重量または体積を減少させる活性、腫瘍の重量または体積の増加を抑制させる活性、他薬剤による腫瘍の重量または体積の減少を促進させる活性、腫瘍細胞による個体死亡を抑制する活性等が挙げられる。溶媒と比較して抗体による効果が、30%以上の活性、好ましくは50%以上の活性、より好ましくは80%以上の活性、さらに好ましくは90%以上の活性、特に好ましくは95%以上の活性を有する抗体を挙げることができる。 “Anti-tumor effect” means an activity that inhibits the growth or survival of tumor cells. This inhibition of tumor cell growth or survival may be in vitro or in vivo. For example, the activity of reducing the number of tumor cells in vitro, the activity of inhibiting the increase of the number of tumor cells, the activity of causing cell death of tumor cells, the ADCC activity, the CDC activity and the like can be mentioned. In vivo activity to reduce tumor weight or volume, activity to suppress tumor weight or volume increase, activity to promote tumor weight or volume decrease by other drugs, activity to suppress individual death due to tumor cells, etc. Is mentioned. 30% or more activity, preferably 50% or more activity, more preferably 80% or more activity, more preferably 90% or more activity, particularly preferably 95% or more activity compared to the solvent. The antibody which has can be mentioned.
In vivoの動物モデルとしては、ヌードマウス等の免疫不全マウスにヒト癌組織由来の培養細胞株を移植した異種移植モデル、培養マウス癌細胞株を正常な免疫系を有する野生型マウスへ移植した同系移植モデルなどがあげられる。 Examples of in vivo animal models include xenograft models in which cultured cancer cell lines derived from human cancer tissue are transplanted into immunodeficient mice such as nude mice, and syngeneic strains in which cultured mouse cancer cell lines are transplanted into wild-type mice having a normal immune system. Examples include transplantation models.
異種移植モデルはヌードマウス等の免疫不全マウスの皮下、皮内、腹腔内、静脈内等様々な部位にヒト癌細胞株を移植することにより作製することができる。 A xenograft model can be prepared by transplanting human cancer cell lines to various sites such as subcutaneous, intradermal, intraperitoneal, intravenous, etc. of immunodeficient mice such as nude mice.
「細胞傷害性薬物」とは、細胞の機能を阻害するまたは妨げ、かつ/または細胞の破壊を生じる物質を指す。この用語は化学療法薬ならびに毒素(細菌、真菌、植物または動物起源の酵素的に活性な毒素)およびそのフラグメントを含むものとする。このような細胞傷害性薬物は、既知の標準的手順を用いて例えばヒト化CD147抗体などの抗体とカップリングさせ、例えば抗体による療法が指示される患者の治療に使用することができる。 “Cytotoxic drug” refers to a substance that inhibits or prevents the function of cells and / or causes destruction of cells. The term is intended to include chemotherapeutic agents as well as toxins (enzymatically active toxins of bacterial, fungal, plant or animal origin) and fragments thereof. Such cytotoxic drugs can be coupled to an antibody, such as a humanized CD147 antibody, using known standard procedures and used, for example, to treat a patient for whom therapy with the antibody is indicated.
「放射性同位元素」とは、I131、1125、Y90およびRe186などの放射性核種であり、既知の標準的手順を用いて例えばヒト化CD147抗体などの抗体とカップリングさせ、例えば抗体による療法が指示される患者の治療に使用することができる。A “radioisotope” is a radionuclide such as I 131 , 1 125 , Y 90 and Re 186 and is coupled to an antibody, such as a humanized CD147 antibody, using known standard procedures, eg, by an antibody It can be used to treat patients for whom therapy is indicated.
「CD147の発現をするがん細胞」とは、急性リンパ球性白血病、慢性白血病、リンパ腫(例えば、ホジキン病または非ホジキン病)、多発性骨髄腫、癌腫(例えば、大腸癌、膵臓癌、肝癌、乳癌、卵巣癌、前立腺癌、扁平上皮癌、腎癌、肺癌、または食道癌)などが挙げられるが、これらに限定されない。 “A cancer cell expressing CD147” refers to acute lymphocytic leukemia, chronic leukemia, lymphoma (eg, Hodgkin's disease or non-Hodgkin's disease), multiple myeloma, carcinoma (eg, colon cancer, pancreatic cancer, liver cancer) Breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, kidney cancer, lung cancer, or esophageal cancer), and the like.
「CD147関連疾患」とは、喘息、過敏性肺炎、関節リウマチ、乾癬性関節炎、潰瘍性大腸炎、全身性エリテマトーデス、移植片対宿主病、全身性炎症性応答症候群、子宮内膜症、心筋梗塞、動脈硬化症、多発性硬化症、アルツハイマー病、肝臓線維症、神経線維症、HIV感染症、髄膜炎、肝炎などが挙げられるが、これらに限定されない。 “CD147-related disease” means asthma, hypersensitivity pneumonia, rheumatoid arthritis, psoriatic arthritis, ulcerative colitis, systemic lupus erythematosus, graft-versus-host disease, systemic inflammatory response syndrome, endometriosis, myocardial infarction , Arteriosclerosis, multiple sclerosis, Alzheimer's disease, liver fibrosis, neurofibrosis, HIV infection, meningitis, hepatitis and the like.
2.抗体の作製方法
本発明において、ヒトCD147に結合する抗体は当業者に公知の方法により作成することができる。例えば、モノクローナル抗体産生ハイブリドーマは、基本的には公知技術を使用し、以下のようにして作製できる。すなわち、CD147タンパク質またはCD147発現細胞を感作抗原として使用して、これを通常の免疫方法にしたがって哺乳動物を免疫する。得られる免疫細胞を通常の細胞融合法によって公知の親細胞と融合させ、通常のスクリーニング法により、モノクローナルな抗体産生細胞を選択することができる。 2. Antibody Production Method In the present invention, an antibody that binds to human CD147 can be produced by methods known to those skilled in the art. For example, a monoclonal antibody-producing hybridoma can be basically produced using a known technique as follows. That is, a CD147 protein or a CD147-expressing cell is used as a sensitizing antigen, and this is used to immunize a mammal according to a normal immunization method. The obtained immune cells can be fused with a known parent cell by an ordinary cell fusion method, and monoclonal antibody-producing cells can be selected by an ordinary screening method.
具体的には、モノクローナル抗体を作製するには次のようにすればよい。まず、配列番号1または2に示されるCD147のアミノ酸配列にしたがってCD147タンパク質を取得し、これを抗体取得の感作抗原として使用する。すなわち、ヒトCD147をコードする遺伝子配列を公知の発現ベクター系に挿入して適当な宿主細胞を形質転換させた後、その宿主細胞の表面上のCD147安定発現を作製する、または培養上清中から目的のCD147タンパク質を公知の方法で精製する。 Specifically, the monoclonal antibody can be produced as follows. First, a CD147 protein is obtained according to the amino acid sequence of CD147 shown in SEQ ID NO: 1 or 2, and this is used as a sensitizing antigen for antibody acquisition. That is, a gene sequence encoding human CD147 is inserted into a known expression vector system to transform an appropriate host cell, and then stable expression of CD147 on the surface of the host cell is produced, or from the culture supernatant The target CD147 protein is purified by a known method.
次に、この精製CD147タンパク質やCD147安定発現細胞を感作抗原として用いる。あるいは、CD147の部分ペプチドを感作抗原として使用することもできる。この際、部分ペプチドはヒトCD147のアミノ酸配列にしたがって化学合成により得ることも可能である。 Next, this purified CD147 protein or CD147 stably expressing cells is used as a sensitizing antigen. Alternatively, a partial peptide of CD147 can be used as a sensitizing antigen. In this case, the partial peptide can also be obtained by chemical synthesis according to the amino acid sequence of human CD147.
感作抗原で免疫される哺乳動物としては、特に限定されるものではないが、細胞融合に使用する親細胞との適合性を考慮して選択するのが好ましく、一般的にはげっ歯類の動物、例えば、マウス、ラット、ハムスター、あるいはウサギ、サル等が使用される。 The mammal to be immunized with the sensitizing antigen is not particularly limited, but is preferably selected in consideration of compatibility with the parent cell used for cell fusion. Animals such as mice, rats, hamsters, rabbits, monkeys and the like are used.
感作抗原を動物に免疫するには、公知の方法にしたがって行われる。例えば、一般的方法として、感作抗原を哺乳動物の腹腔内または皮下に注射することにより行われる。具体的には、感作抗原をPBS(Phosphate−Buffered Saline)や生理食塩水等で適当量に希釈、懸濁したものに所望により通常のアジュバント、例えばフロイント完全アジュバントを適量混合し、乳化後、哺乳動物に4〜21日毎に数回投与する。また、感作抗原免疫時に適当な担体を使用することもできる。 In order to immunize an animal with a sensitizing antigen, a known method is performed. For example, as a general method, a sensitizing antigen is injected into a mammal intraperitoneally or subcutaneously. Specifically, the sensitizing antigen is diluted to an appropriate amount with PBS (Phosphate-Buffered Saline), physiological saline or the like, mixed with an appropriate amount of an ordinary adjuvant, for example, Freund's complete adjuvant, if necessary, and emulsified. The mammal is dosed several times every 4-21 days. In addition, an appropriate carrier can be used during immunization with the sensitizing antigen.
このように哺乳動物を免疫し、血清中に所望の抗体レベルが上昇するのを確認した後に、哺乳動物から免疫細胞を採取し、細胞融合に付される。好ましい免疫細胞としては、特に脾細胞が挙げられる。 Thus, after immunizing a mammal and confirming that a desired antibody level rises in serum, immune cells are collected from the mammal and subjected to cell fusion. Preferable immune cells include spleen cells.
前記免疫細胞と融合すべき親細胞として、哺乳動物のミエローマ細胞を用いる。このミエローマ細胞は、公知の種々の細胞株、例えば、P3(P3x63Ag8.653)(Kearney et al.,J Immnol 1979;123:1548−1550)、NS−1(Kohler.G. and Milstein,C Eur J Immunol 1976;6:511−519)、SP2/0(Shulman,M.et al.,Nature 1978;276:269−270)等が好適に使用される。 Mammalian myeloma cells are used as parent cells to be fused with the immune cells. The myeloma cells are known from various known cell lines such as P3 (P3x63Ag8.653) (Kearney et al., J Immunol 1979; 123: 1548-1550), NS-1 (Kohler. G. and Milstein, C Eur. J Immunol 1976; 6: 511-519), SP2 / 0 (Shulman, M. et al., Nature 1978; 276: 269-270) and the like are preferably used.
前記免疫細胞とミエローマ細胞との細胞融合は、基本的には公知の方法、たとえば、ケーラーとミルステインらの方法(Kohler.G.and Milstein,C.,Methods Enzymol 1981;73:3−46)等に準じて行うことができる。 The cell fusion between the immune cells and myeloma cells is basically performed by a known method, for example, the method of Kohler and Milstein et al. (Kohler. G. and Milstein, C., Methods Enzymol 1981; 73: 3-46). It can carry out according to etc.
より具体的には、前記細胞融合は、例えば細胞融合促進剤の存在下に通常の栄養培養液中で実施される。融合促進剤としては、例えばポリエチレングリコール(PEG)、センダイウイルス(HVJ)等が使用され、更に所望により融合効率を高めるためにジメチルスルホキシド等の補助剤を添加使用することもできる。 More specifically, the cell fusion is performed in a normal nutrient culture medium in the presence of a cell fusion promoter, for example. For example, polyethylene glycol (PEG), Sendai virus (HVJ), or the like is used as the fusion promoter, and an auxiliary agent such as dimethyl sulfoxide can be added and used to increase the fusion efficiency as desired.
免疫細胞とミエローマ細胞との使用割合は任意に設定することができる。例えば、ミエローマ細胞に対して免疫細胞を1〜10倍とするのが好ましい。前記細胞融合に用いる培養液としては、例えば、前記ミエローマ細胞株の増殖に好適なRPMI−1640培養液、MEM培養液、その他、この種の細胞培養に用いられる通常の培養液が使用可能であり、さらに、牛胎児血清(FBS)等の血清補液を併用することもできる。 The use ratio of immune cells and myeloma cells can be arbitrarily set. For example, the number of immune cells is preferably 1 to 10 times that of myeloma cells. As the culture solution used for the cell fusion, for example, RPMI-1640 culture solution suitable for growth of the myeloma cell line, MEM culture solution, and other normal culture solutions used for this kind of cell culture can be used. Furthermore, serum replacement fluid such as fetal bovine serum (FBS) can be used in combination.
細胞融合は、前記免疫細胞とミエローマ細胞との所定量を前記培養液中でよく混合し、予め37℃程度に加温したPEG溶液(例えば平均分子量1000〜6000程度)を通常30〜60%(w/v)の濃度で添加し、混合することによって目的とする融合細胞(ハイブリドーマ)を形成する。続いて、適当な培養液を逐次添加し、遠心して上清を除去する操作を繰り返すことによりハイブリドーマの生育に好ましくない細胞融合剤等を除去する。 In cell fusion, a predetermined amount of the immune cells and myeloma cells are mixed well in the culture medium, and a PEG solution (for example, an average molecular weight of about 1000 to 6000) preheated to about 37 ° C. is usually 30 to 60% ( The target fusion cell (hybridoma) is formed by adding at a concentration of w / v) and mixing. Subsequently, cell fusion agents and the like that are undesirable for the growth of the hybridoma are removed by sequentially adding an appropriate culture medium and centrifuging to remove the supernatant.
このようにして得られたハイブリドーマは、通常の選択培養液、例えばHAT培養液(ヒポキサンチン、アミノプテリンおよびチミジンを含む培養液)で培養することにより選択される。上記HAT培養液での培養は、目的とするハイブリドーマ以外の細胞(非融合細胞)が死滅するのに十分な時間(通常、数日〜数週間)継続する。ついで、通常の限界希釈法を実施し、目的とする抗体を産生するハイブリドーマのスクリーニングおよび単一クローニングを行う。 The hybridoma thus obtained is selected by culturing in a normal selective culture solution, for example, a HAT culture solution (a culture solution containing hypoxanthine, aminopterin and thymidine). The culture in the HAT culture solution is continued for a sufficient time (usually several days to several weeks) for cells other than the target hybridoma (non-fused cells) to die. Subsequently, a normal limiting dilution method is performed, and screening and single cloning of a hybridoma that produces the target antibody are performed.
標的とするタンパク質に対する抗体を作製する別の方法として、特許4870348号に記載されているように、1000億個もの独立したクローンからなる巨大なヒト抗体ライブラリーを作製し、それを利用した癌細胞及び組織の細胞膜表面に存在するタンパク質(細胞表面抗原)に対する抗体の網羅的取得法がある。スクリーニングの抗原として、精製した細胞外領域を有する可溶性の膜タンパク質を使用することができる。 As another method for producing an antibody against a target protein, as described in Japanese Patent No. 4870348, a huge human antibody library consisting of 100 billion independent clones was prepared, and cancer cells using the library There is also a comprehensive method for obtaining antibodies against proteins (cell surface antigens) present on the cell membrane surface of tissues. A soluble membrane protein having a purified extracellular region can be used as an antigen for screening.
スクリーニングは、感作抗原として用いたCD147安定発現細胞やCD147の発現を特徴付けるがん細胞を用いたFACS解析により、CD147に反応する抗体を選抜することができる。また精製CD147タンパク質を用いたELISAによっても、CD147に反応する抗体を選抜することができる。本発明における抗体の好ましい態様として治療に用いる抗体の選抜として有望な方法は、CD147の発現を特徴付けるがん細胞を用いたFACS解析である。 In the screening, an antibody that reacts with CD147 can be selected by FACS analysis using CD147 stably expressing cells used as a sensitizing antigen or cancer cells characterizing the expression of CD147. An antibody that reacts with CD147 can also be selected by ELISA using purified CD147 protein. As a preferred embodiment of the antibody of the present invention, a promising method for selecting an antibody to be used for treatment is FACS analysis using cancer cells that characterize the expression of CD147.
このようにして作製されるモノクローナル抗体を産生するハイブリドーマは、通常の培養液中で継代培養することが可能であり、また、液体窒素中で長期保存することが可能である。 The hybridoma producing the monoclonal antibody thus produced can be subcultured in a normal culture solution, and can be stored for a long time in liquid nitrogen.
当該ハイブリドーマからモノクローナル抗体を取得するには、当該ハイブリドーマを通常の方法に従って培養し、その培養上清として得る方法、あるいはハイブリドーマをこれと適合性がある哺乳動物に投与して増殖させ、その腹水として得る方法などが採用される。前者の方法は、高純度の抗体を得るのに適しており、一方、後者の方法は、抗体の大量生産に適している。 In order to obtain a monoclonal antibody from the hybridoma, the hybridoma is cultured according to a usual method and obtained as a culture supernatant, or the hybridoma is administered to a mammal compatible therewith to proliferate, and as ascites The method of obtaining is adopted. The former method is suitable for obtaining highly pure antibodies, while the latter method is suitable for mass production of antibodies.
3.本発明モノクローナル抗体の性質
本発明の抗ヒトCD147モノクローナル抗体またはその抗原結合領域は、次のa〜cの性質を有する。
a.ヒトCD147発現がん細胞株に対して反応する。
b.カニクイザルCD147発現細胞株に対して反応する。
c.ヒトCD147発現がん細胞に対してin vivoで抗腫瘍活性を示す。 3. Properties of the monoclonal antibody of the present invention The anti-human CD147 monoclonal antibody of the present invention or an antigen-binding region thereof has the following properties a to c.
a. Reacts against a human CD147 expressing cancer cell line.
b. Reacts against cynomolgus monkey CD147 expressing cell lines.
c. It exhibits antitumor activity in vivo against human CD147-expressing cancer cells.
このようなa〜cの性質を有する抗ヒトCD147モノクローナル抗体は、従来報告されていない。
ここで、ヒトCD147発現がん細胞株は、前述のCD147の発現をするがん細胞と同義である。また、iv vivoで抗腫瘍活性を示すとは、ヒトCD147発現がん細胞を有する動物(ヒトを含む)に対して抗ヒトCD147モノクローナル抗体を投与することにより、抗腫瘍活性、例えば腫瘍重量または腫瘍体積を減少させる作用が得られることを意味する。No anti-human CD147 monoclonal antibody having such properties a to c has been reported so far.
Here, the human CD147-expressing cancer cell line is synonymous with the above-described cancer cell expressing CD147. In addition, iv vivo exhibits antitumor activity means that antitumor activity, for example, tumor weight or tumor, can be obtained by administering anti-human CD147 monoclonal antibody to animals (including humans) having human CD147-expressing cancer cells. It means that the effect | action which reduces a volume is acquired.
4.抗ヒトCD147抗体の可変領域のクローニング
本発明におけるヒトCD147に結合する抗体について、可変領域のクローニングは当業者に公知の方法により作成することができる。 4). Cloning of variable region of anti-human CD147 antibody Regarding the antibody that binds to human CD147 in the present invention , cloning of the variable region can be prepared by methods known to those skilled in the art.
具体的には、抗ヒトCD147抗体を産生するハイブリドーマから、抗ヒトCD147抗体の可変領域をコードするmRNAを単離する。mRNAの単離は、公知の方法、例えば、グアニジン超遠心法(Chirgwin,J.M.et al.,Biochemistry 1979;18:5294−5299)、AGPC法(Chomczynski,P.et al.,Anal Biochem 1987;162:156−159)等により行ってtotal RNAを調製し、mRNA Purification Kit(Pharmacia社)等を使用して目的のmRNAを調製する。また、QuickPrep mRNA Purification Kit(Pharmacia社)を用いることによりmRNAを直接調製することもできる。 Specifically, mRNA encoding the variable region of anti-human CD147 antibody is isolated from the hybridoma producing anti-human CD147 antibody. Isolation of mRNA is performed by a known method such as guanidine ultracentrifugation (Chirgwin, JM et al., Biochemistry 1979; 18: 5294-5299), AGPC method (Chomczynski, P. et al., Anal Biochem). 1987; 162: 156-159) and the like to prepare total RNA, and mRNA of interest is prepared using mRNA Purification Kit (Pharmacia) and the like. Alternatively, mRNA can be directly prepared by using QuickPrep mRNA Purification Kit (Pharmacia).
得られたmRNAから抗体可変領域をコードする遺伝子を増幅させる。抗体可変領域の遺伝子の増幅は、マウスIgGまたはマウスκ鎖の定常領域配列に相補的なオリゴヌクレオチドをプライマーとして、SMART RACE cDNA Amplication Kit(Clontech社)等を用いて行うことができる。または、得られたmRNAから逆転写酵素 Kit(Invitrogen社)等を用いてcDNAを合成し、作製したcDNAを鋳型として、degenerate primer等を用いたPCR法により可変領域をコードする遺伝子を増幅することができる。 A gene encoding the antibody variable region is amplified from the obtained mRNA. Amplification of the antibody variable region gene can be performed using SMART RACE cDNA Amplification Kit (Clontech) or the like using an oligonucleotide complementary to the constant region sequence of mouse IgG or mouse κ chain as a primer. Alternatively, cDNA is synthesized from the obtained mRNA using reverse transcriptase Kit (Invitrogen) or the like, and the gene encoding the variable region is amplified by PCR using degenerate primer using the prepared cDNA as a template. Can do.
得られたPCR増幅物から目的とするDNA断片を精製し、ベクターDNAと連結する。さらに、これより組換えベクターを作製し、大腸菌等に導入してコロニーを選択して所望の組換えベクターを調製する。そして、目的とするDNAの塩基配列を公知の方法、例えば、ジデオキシヌクレオチドチェインターミネーション法等により確認する。 The target DNA fragment is purified from the obtained PCR amplification product and ligated with vector DNA. Further, a recombinant vector is prepared from this, introduced into Escherichia coli, etc., and colonies are selected to prepare a desired recombinant vector. Then, the base sequence of the target DNA is confirmed by a known method such as the dideoxynucleotide chain termination method.
DNAの塩基配列は公開されているKabat DatabaseやIMGT(ImMunoGeneTics)Database等により、軽鎖可変領域および重鎖可変領域のアミノ酸残基とCDR等を同定することができる。 The nucleotide sequence of DNA can identify the amino acid residues and CDRs of the light chain variable region and the heavy chain variable region by using publicly available Kabat Database or IMGT (ImmunoGeneTics) Database.
本発明の抗ヒトCD147モノクローナル抗体の例としては、次のPPAT−082−01、PPAT−082−02及びPPAT−082−03が挙げられ、その可変領域は、次のとおりである。
(1)PPAT−082−01
重鎖の可変領域にCDR1として配列TYWIE(配列番号29)、CDR2として配列EFLPGSGSTNFNEKFKG(配列番号30)、CDR3として配列SGGNFGARFAS(配列番号31)を有し、軽鎖の可変領域にCDR1として配列RSSKSLLSNNGNTYLY(配列番号32)、CDR2として配列RMSSLAS(配列番号33)、CDR3として配列MQHLEYPFT(配列番号34)を有するモノクローナル抗体である。好ましくは、重鎖の可変領域に配列番号5のアミノ酸配列と、軽鎖の可変領域に配列番号9のアミノ酸配列を有するヒト由来のモノクローナル抗体である。Examples of the anti-human CD147 monoclonal antibody of the present invention include the following PPAT-082-01, PPAT-082-02, and PPAT-082-03, and the variable regions thereof are as follows.
(1) PPAT-082-01
The variable region of the heavy chain has the sequence TYWIE (SEQ ID NO: 29) as the CDR1, the sequence EFLPGSGSTNFNEKFKG (SEQ ID NO: 30) as the CDR2, the sequence SGGNFGARFAS (SEQ ID NO: 31) as the CDR3, and the sequence RSSKSLLSNNGTYLY (as the CDR1 in the light chain variable region) SEQ ID NO: 32), a monoclonal antibody having the sequence RMSSLAS (SEQ ID NO: 33) as CDR2 and the sequence MQHLYPFT (SEQ ID NO: 34) as CDR3. Preferably, it is a human-derived monoclonal antibody having the amino acid sequence of SEQ ID NO: 5 in the variable region of the heavy chain and the amino acid sequence of SEQ ID NO: 9 in the variable region of the light chain.
(2)PPAT−082−02
重鎖の可変領域にCDR1として配列SYGMS(配列番号35)、CDR2として配列TISSGGSYTYYQDSIKG(配列番号36)、CDR3として配列GDWADY(配列番号37)を有し、軽鎖の可変領域にCDR1として配列KASQDINSYLS(配列番号38)、CDR2として配列RANRLVA(配列番号39)、CDR3として配列LQYDEFPLT(配列番号40)を有するモノクローナル抗体である。好ましくは、重鎖の可変領域に配列番号6のアミノ酸と、軽鎖の可変領域に配列番号10のアミノ酸配列を有するモノクローナル抗体である。(2) PPAT-082-02
The variable region of the heavy chain has the sequence SYGMS (SEQ ID NO: 35) as the CDR1, the sequence TISSGSYTYYQDSIGG (SEQ ID NO: 36) as the CDR2, the sequence GDWADY (SEQ ID NO: 37) as the CDR3, and the sequence KASQDINSYLS (as the CDR1 in the light chain variable region) SEQ ID NO: 38), a monoclonal antibody having the sequence RANRLVA (SEQ ID NO: 39) as CDR2 and the sequence LQYDEFPLT (SEQ ID NO: 40) as CDR3. A monoclonal antibody having the amino acid sequence of SEQ ID NO: 6 in the variable region of the heavy chain and the amino acid sequence of SEQ ID NO: 10 in the variable region of the light chain is preferable.
(3)PPAT−082−03
重鎖の可変領域にCDR1として配列SYGIS(配列番号17)、CDR2として配列WINPNSGGTNYAQKFQG(配列番号18)、CDR3として配列GRGSYYAFDI(配列番号19)を有し、軽鎖の可変領域にCDR1として配列KSSQSVLYSSNNKNYLA(配列番号20)、CDR2として配列WASTRES(配列番号21)、CDR3として配列QQYYSTPT(配列番号22)を有するモノクローナル抗体である。好ましくは、重鎖の可変領域に配列番号4のアミノ酸配列と、軽鎖の可変領域に配列番号8のアミノ酸配列を有するモノクローナル抗体である。(3) PPAT-082-03
The variable region of the heavy chain has the sequence SYGIS (SEQ ID NO: 17) as CDR1, the sequence WINPNSGGTNYAQKQQG (SEQ ID NO: 18) as CDR2, the sequence GRGSYYAFDI (SEQ ID NO: 19) as CDR3, and the sequence KSSQSVLYSSSNNKNYLA as the CDR1 in the variable region of the light chain ( SEQ ID NO: 20), monoclonal antibody having the sequence WASTRES (SEQ ID NO: 21) as CDR2 and the sequence QQYYSTPT (SEQ ID NO: 22) as CDR3. A monoclonal antibody having the amino acid sequence of SEQ ID NO: 4 in the variable region of the heavy chain and the amino acid sequence of SEQ ID NO: 8 in the variable region of the light chain is preferable.
本発明のモノクローナル抗体またはその抗原結合領域を含む断片には、前記のアミノ酸配列において1または複数のアミノ酸が置換、欠失および付加または挿入され、同一性のレベルが少なくとも90%以上であるモノクローナル抗体またはその抗原結合領域を含む断片が含まれる。 The monoclonal antibody of the present invention or a fragment containing the antigen-binding region thereof has one or more amino acid substitutions, deletions, additions or insertions in the amino acid sequence, and the monoclonal antibody having a level of identity of at least 90% or more Or a fragment containing the antigen-binding region thereof is included.
5.抗CD147抗体のヒト化作製
本発明において抗体の好ましい態様の一つとして、ヒトCD147に結合するヒト抗体やヒト化抗体を挙げることができる。ヒト化抗体は既知の方法を用いて製造することができる。 5. Preparation of humanized anti-CD147 antibody One preferred embodiment of the antibody in the present invention includes a human antibody or a humanized antibody that binds to human CD147. Humanized antibodies can be produced using known methods.
ヒト化抗体は、再構成(reshaped)ヒト抗体とも称され、これは、ヒト以外の哺乳動物、例えばマウス抗体の相補性決定領域(CDR)をヒト抗体のCDRへ移植したものであり、その一般的な遺伝子組換え手法も知られている(WO 96/02576号公報)。 A humanized antibody is also called a reshaped human antibody, which is a non-human mammal such as a mouse antibody complementarity-determining region (CDR) grafted to a human antibody CDR. A known genetic recombination technique is also known (WO 96/02576).
具体的には、例えばCDRがマウス抗体由来である場合には、マウス抗体のCDRとヒト抗体のフレームワーク領域(FR)とを連結するように設計したDNA配列を、CDRおよびFR両方の末端領域にオーバーラップする部分を有するように作製した数個のオリゴヌクレオチドをプライマーとして用いてPCR法により合成する、または人工的に軽鎖可変領域および重鎖可変領域の遺伝子をコードするDNAを合成することも可能である。 Specifically, for example, when the CDR is derived from a mouse antibody, a DNA sequence designed to link the CDR of the mouse antibody and the framework region (FR) of the human antibody is used as a terminal region of both the CDR and FR. Synthesized by PCR using several oligonucleotides prepared to have overlapping portions as primers, or artificially synthesizing DNA encoding light chain variable region and heavy chain variable region genes Is also possible.
CDRと連結されるヒト抗体のフレームワーク領域は、相補性決定領域が良好な抗原結合部位を形成するもの等が選択される。必要に応じ、ヒト化抗体の相補性決定領域が適切な抗原結合部位を形成するように、抗体の可変領域におけるフレームワーク領域のアミノ酸を置換してもよい(Sato K et al.,Cancer Res 1993;53:851−856)。また、CDRの一部のアミノ酸をヒト抗体のアミノ酸に置換してもよい。 The framework region of the human antibody to be linked to the CDR is selected such that the complementarity determining region forms a favorable antigen binding site. If necessary, the amino acid of the framework region in the variable region of the antibody may be substituted so that the complementarity determining region of the humanized antibody forms an appropriate antigen binding site (Sato K et al., Cancer Res 1993). 53: 851-856). In addition, some amino acids of CDR may be substituted with amino acids of human antibodies.
ヒト化抗体の定常領域には、ヒト抗体のものが使用され、例えば重鎖では、Cγ1、Cγ2、Cγ3、Cγ4を、軽鎖ではCκ、Cλを使用することができる。また、抗体またはその産生の安定性を改善するために、ヒト抗体の定常領域を修飾してもよい。ヒト化の際に用いられるヒト抗体は、IgG、IgM、IgA、IgE、IgDなど如何なるアイソタイプのヒト抗体でもよいが、本発明においてはIgGを用いることが好ましく、さらにIgG1又はIgG3が好ましく、特にIgG1が好ましい。IgG1は高い細胞傷害活性を有している点で抗体を抗癌剤として利用する場合に有効である(Clark MR,Chemical immunology 1997;65:88−110)。 The constant region of a humanized antibody is that of a human antibody. For example, Cγ1, Cγ2, Cγ3, Cγ4 can be used for the heavy chain, and Cκ, Cλ can be used for the light chain. In addition, the constant region of a human antibody may be modified to improve the stability of the antibody or its production. Human antibodies used for humanization may be human antibodies of any isotype such as IgG, IgM, IgA, IgE, IgD. In the present invention, it is preferable to use IgG, more preferably IgG1 or IgG3, particularly IgG1. Is preferred. IgG1 is effective when an antibody is used as an anticancer agent because it has high cytotoxic activity (Clark MR, Chemical immunology 1997; 65: 88-110).
なお、ヒト化抗体を作製した後に、可変領域や定常領域中のアミノ酸を他のアミノ酸で置換等してもよい。 In addition, after producing a humanized antibody, amino acids in the variable region or constant region may be substituted with other amino acids.
さらに、本発明の抗体は細胞傷害活性が増強された抗体でもよい。細胞傷害活性が増強された抗体としては、例えば、フコースが欠損した抗体、糖鎖にバイセクティング(bise cting)N−アセチルグルコサミン(GlcNAc)が付加した抗体、Fc領域のアミノ酸を置換することによりFcγ受容体との結合活性を変化させた抗体などを挙げることができる。これら細胞傷害活性が増強された抗体は当業者に公知の方法で作製することができる。 Furthermore, the antibody of the present invention may be an antibody with enhanced cytotoxic activity. Examples of antibodies with enhanced cytotoxic activity include antibodies lacking fucose, antibodies in which bisecting N-acetylglucosamine (GlcNAc) is added to the sugar chain, and Fcγ by substituting amino acids in the Fc region. Examples thereof include an antibody whose binding activity to a receptor is changed. These antibodies with enhanced cytotoxic activity can be prepared by methods known to those skilled in the art.
ヒト化抗体におけるCDRの由来は特に限定されず、どのような動物由来でもよい。例えば、マウス抗体、ラット抗体、ウサギ抗体、ラクダ抗体などの配列を用いることが可能であるが、好ましくはマウス抗体のCDR配列である。 The origin of the CDR in the humanized antibody is not particularly limited, and may be derived from any animal. For example, sequences of mouse antibody, rat antibody, rabbit antibody, camel antibody and the like can be used, but the CDR sequence of mouse antibody is preferable.
目的とする抗CD147のヒト化抗体の可変領域をコードするDNAを得たのち、これを、所望のヒト抗体定常領域をコードするDNAを含有する発現ベクターへ組み込む。 After obtaining the DNA encoding the variable region of the anti-CD147 humanized antibody of interest, this is incorporated into an expression vector containing DNA encoding the desired human antibody constant region.
本発明で使用される抗CD147抗体を製造するには、抗体遺伝子を発現制御領域、例えば、エンハンサー、プロモーターの制御のもとで発現するよう発現ベクターに組み込む。次に、この発現ベクターにより、宿主細胞を形質転換し、抗体を発現させる。 In order to produce the anti-CD147 antibody used in the present invention, an antibody gene is incorporated into an expression vector so as to be expressed under the control of an expression control region such as an enhancer or promoter. Next, host cells are transformed with this expression vector to express the antibody.
抗体遺伝子の発現は、軽鎖または重鎖をコードするポリヌクレオチドを別々に発現ベクターに組み込んで宿主細胞を同時形質転換させてもよいし、あるいは軽鎖および重鎖をコードするポリヌクレオチドを単一の発現ベクターに組み込んで宿主細胞を形質転換させてもよい(WO 94/11523 号公報)。 The expression of the antibody gene may be achieved by separately incorporating polynucleotides encoding light or heavy chains into an expression vector and co-transforming host cells, or by using a single polynucleotide encoding light and heavy chains. The host cell may be transformed by incorporating it into an expression vector (WO 94/11523).
抗体のヒト化において、通常、由来となった抗体の抗原結合活性を維持したままヒト化を行うことは困難であるが、本発明においては、由来となったマウス抗体と同様に抗原結合活性を有するヒト化抗体の取得に成功した。ヒト化抗体はヒト体内における抗原性が低下しているため、治療目的などでヒトに投与する場合に有用である。 In humanization of antibodies, it is usually difficult to perform humanization while maintaining the antigen-binding activity of the derived antibody. However, in the present invention, the antigen-binding activity is demonstrated in the same manner as the derived mouse antibody. We succeeded in obtaining humanized antibodies. Since humanized antibodies have reduced antigenicity in the human body, they are useful when administered to humans for therapeutic purposes.
本発明のヒト化された抗CD147モノクローナル抗体またはその抗原結合領域を含む断片としては、前記の抗CD147モノクローナル抗体(例えば、PPAT−082−01、PPAT−082−02)の可変領域のCDR周辺を含むアミノ酸配列とヒトイムノグロブリンのアミノ酸配列とからなるヒト化された抗CD147モノクローナル抗体またはその抗原結合領域を含む断片が好ましい。 Examples of the humanized anti-CD147 monoclonal antibody of the present invention or a fragment containing the antigen-binding region thereof include the CDR region of the variable region of the above-mentioned anti-CD147 monoclonal antibodies (for example, PPAT-082-01 and PPAT-082-02). A humanized anti-CD147 monoclonal antibody consisting of an amino acid sequence containing it and an amino acid sequence of human immunoglobulin, or a fragment containing the antigen-binding region thereof is preferred.
より具体的には、重鎖可変領域および軽鎖可変領域の組み合わせが、1)配列番号13および14で示されるアミノ酸配列、または2)配列番号15および16で示されるアミノ酸配列を有する、ヒト化重鎖およびヒト化軽鎖抗CD147モノクローナル抗体またはその抗原結合領域を含む断片がより好ましい。 More specifically, the combination of heavy chain variable region and light chain variable region has 1) the amino acid sequence shown by SEQ ID NO: 13 and 14, or 2) the amino acid sequence shown by SEQ ID NO: 15 and 16 More preferred are heavy chain and humanized light chain anti-CD147 monoclonal antibodies or fragments comprising the antigen binding region thereof.
なお、これらのヒト化抗CD147モノクローナル抗体またはその抗原結合領域を含む断片においても、前記のアミノ酸配列において1または複数のアミノ酸配列が置換、欠失および付加され、同一性のレベルが少なくとも90%以上であるヒト化抗CD147モノクローナル抗体またはその抗原結合領域を含む断片が含まれる。 In these humanized anti-CD147 monoclonal antibodies or fragments containing the antigen-binding region thereof, one or more amino acid sequences are substituted, deleted, and added in the amino acid sequence, and the level of identity is at least 90% or more. And a fragment containing the humanized anti-CD147 monoclonal antibody or antigen binding region thereof.
6.抗CD147抗体によるADCC活性及びCDC活性測定
本発明の抗CD147抗体の好ましい態様としては、CD147を発現する細胞に対して高いADCC活性を有する抗体または高いCDC活性を有する抗体を挙げることができる。6). ADCC activity and CDC activity measurement by anti-CD147 antibody As a preferable aspect of the anti-CD147 antibody of the present invention, an antibody having a high ADCC activity or an antibody having a high CDC activity against cells expressing CD147 can be mentioned.
ヒトCD147を発現する細胞としては、例えば、HepG2(ATCC HB-8065)やPC−3(ATCC CRL−1435)などの腫瘍細胞、ヒトCD147をコードする遺伝子を組み込まれた細胞などを挙げることができる。 Examples of cells that express human CD147 include tumor cells such as HepG2 (ATCC HB-8065) and PC-3 (ATCC CRL-1435), and cells into which a gene encoding human CD147 has been incorporated. .
ADCC活性またはCDC活性の測定方法は、当業者に公知の方法により行うことが可能であり、例えば、溶解した細胞から遊離されるラクトースデヒドゲナーゼを測定するCytoTox96 Non−Radioactive Cytotoxicity Assay(Promega社)を用いることにより行うことが可能である。ADCC活性を測定する際の具体的な条件としては、特に限定されないが、例えば、実施例記載の条件を用いて測定することができる。 ADCC activity or CDC activity can be measured by methods known to those skilled in the art. For example, CytoTox96 Non-Radioactive Cytotoxicity Assay (Promega) for measuring lactose dehydrogenase released from lysed cells. It is possible to carry out by using. Although it does not specifically limit as specific conditions at the time of measuring ADCC activity, For example, it can measure using the conditions of an Example description.
さらに、本発明の抗体は細胞障害活性が増強された抗体でもよい。細胞障害活性が増強された抗体としては、例えば、フコースが欠損した抗体、糖鎖にバイセクティング(bise cting)N−アセチルグルコサミン(GlcNAc)が付加した抗体、Fc領域のアミノ酸を置換することによりFcγ受容体との結合活性を変化させた抗体などを挙げることができる。これら細胞障害活性が増強された抗体は当業者に公知の方法で作製することができる。 Furthermore, the antibody of the present invention may be an antibody with enhanced cytotoxic activity. Examples of antibodies with enhanced cytotoxic activity include antibodies lacking fucose, antibodies in which bisecting N-acetylglucosamine (GlcNAc) is added to the sugar chain, and Fcγ by substituting amino acids in the Fc region. Examples thereof include an antibody whose binding activity to a receptor is changed. These antibodies with enhanced cytotoxic activity can be prepared by methods known to those skilled in the art.
7.細胞傷害性薬物をコンジュゲートした抗CD147抗体
本発明の別の実施態様として、抗CD147抗体に細胞傷害性薬物などの各種分子と結合させた、コンジュゲート抗体を挙げることができる。 7). Anti-CD147 Antibody Conjugated with Cytotoxic Drug Another embodiment of the present invention includes a conjugated antibody in which various molecules such as a cytotoxic drug are bound to anti-CD147 antibody.
本発明で用いられる細胞傷害性薬物の例としては、デュオカルマイシン、デュオカルマイシンのアナログ及び誘導剤、CC−1065、CBIを主成分とするデュオカルマイシンアナログ、MCBIを主成分とするデュオカルマイシンアナログ、CCBIを主成分とするデュオカルマイシンアナログ、ドキソルビシン、ドラスタチン、メイタンシン、メイタンシンアナログ、DM1,DM2,DM3,DM4、DMI、アウリスタチンE、アウリスタチンEB(AEB)、アウリスタチンEFP(AEFP)、モノメチルアウリスタチンE(MMAE)、モノメチルアウリスタチンF(MMAF)、メトトレキサート、メトプテリン、ジクロロメトトレキサート、5−フルオロウラシル、マイトマイシンC、マイトマイシンA、カルミノマイシン、アミノプテリン、ビンクリスチン、タキソール、タキソテールレチノイン酸、酪酸、N8−アセチルスペルミジン並びにカンプトセシン等を挙げることができるが、これだけに限定されるわけではない。 Examples of cytotoxic drugs used in the present invention include duocarmycin, duocarmycin analogs and inducers, CC-1065, duocarmycin analogs based on CBI, and duocarls based on MCBI. Mycin analogs, duocarmycin analogs based on CCBI, doxorubicin, dolastatin, maytansine, maytansine analogs, DM1, DM2, DM3, DM4, DMI, auristatin E, auristatin EB (AEB), auristatin EFP (AEFP) ), Monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), methotrexate, methopterin, dichloromethotrexate, 5-fluorouracil, mitomycin C, mitomycin A, carminomy Emissions, aminopterin, vincristine, taxol, taxotere retinoic acid, butyric acid, there may be mentioned N8- acetyl spermidine and camptothecin like, but is not limited thereto.
本発明における細胞傷害性薬物をコンジュゲートした抗CD147抗体は、前記の薬物と抗CD147抗体とを結合し、公知の方法により作製できる。抗体と薬物は、それら自身が有する連結基などを介して直接結合されてもよいし、また、リンカーや他の物質を介して間接的に結合されてもよい。 The anti-CD147 antibody conjugated with the cytotoxic drug in the present invention can be prepared by binding the above-mentioned drug and the anti-CD147 antibody and using a known method. The antibody and the drug may be directly bonded via a linking group or the like possessed by themselves, or may be indirectly bonded via a linker or other substance.
薬物が直接結合される場合の連結基は、例えばSH基を用いたジスルフィド結合やマレイミドを介する結合が挙げられる。例えば、抗体のFc領域の分子内ジスルフィド結合と、薬物のジスルフィド結合を還元して、両者をジスルフィド結合にて結合する。また、マレイミドを介する方法もある。また別の方法として、抗体内にシステインを遺伝子工学的に導入する方法もある。 Examples of the linking group when the drug is directly bonded include a disulfide bond using an SH group and a bond via maleimide. For example, the intramolecular disulfide bond in the Fc region of the antibody and the disulfide bond of the drug are reduced, and both are bonded by a disulfide bond. There is also a method via maleimide. Another method is to introduce cysteine into the antibody by genetic engineering.
抗体と薬物を、他の物質(リンカー)を介して間接的に結合することも可能である。リンカーには、抗体または薬剤または両方と反応する官能基を1または2種類以上有することが望ましい。官能基の例としてはアミノ基、カルボキシル基、メルカプト基、マレイミド基、ピリジニル基等を挙げることができる。 It is also possible to bind an antibody and a drug indirectly through another substance (linker). The linker preferably has one or more functional groups that react with the antibody or drug or both. Examples of functional groups include amino groups, carboxyl groups, mercapto groups, maleimide groups, pyridinyl groups, and the like.
リンカーの例としては、スルフォスクシイミジル−4−(N−マレイミドメチル)シクロヘキサン−1−カルボキシレート(Sulfo−SMCC)、N−スクシンイミジル4−(マレイミドメチル)シクロヘキサンカルボキシレート(SMCC)、γ−マレイミド酪酸N−スクシンイミジルエステル(GMBS)、ε−マレイミドカプロン酸N−ヒドロキシスクシンイミドエステル(EMCS)、m−マレイミドベンゾイル−N−ヒドロキシスクシンイミドエステル(MBS)、N−(α−マレイミドアセトキシ)−スクシンイミドエステル(AMAS)、N−スクシンイミジル4−(p−マレイミドフェニル)−ブチレート(SMPB)、およびN−(p−マレイミドフェニル)イソシアネート(PMPI)、p−アミノベンジルオキシカルボンイル(PAB)、N−スクシンイミジル4(2−ピリジルチオ)ペンタノエート(SPP)及びN−スクシンイミジル(4−イオド−アセチル)アミノ安息香酸エステル(SIAB)等が挙げられるが、これらに限定されるものではない。また、このリンカーは例えば、バリン−シトルリン(Val−Cit)、アラニン−フェニルアラニン(Ala−Phe)のようなペプチドリンカーであってもよいし、上記にあげたリンカーをそれぞれ適宜組み合わせて使用しても良い。 Examples of linkers include sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC), N-succinimidyl 4- (maleimidomethyl) cyclohexanecarboxylate (SMCC), γ- Maleimidobutyric acid N-succinimidyl ester (GMBS), ε-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N- (α-maleimidoacetoxy) -succinimide Ester (AMAS), N-succinimidyl 4- (p-maleimidophenyl) -butyrate (SMPB), and N- (p-maleimidophenyl) isocyanate (PMPI), p-aminobenzyloxyca Bonyl (PAB), N-succinimidyl 4 (2-pyridylthio) pentanoate (SPP), N-succinimidyl (4-iodo-acetyl) aminobenzoic acid ester (SIAB) and the like are exemplified, but not limited thereto. . The linker may be a peptide linker such as valine-citrulline (Val-Cit) or alanine-phenylalanine (Ala-Phe), or the linkers listed above may be used in appropriate combinations. good.
薬物と抗体との結合方法に関しては、例えば、Cancer Research;68(22)9280(2008)、Nature Biotechnology;26(8)925(2008)、Bio Conjugate Chemistry;19、1673(2008)、Cancer Research;68(15)6300(2008)、又は特表2008−516896号公報などに記載の方法に準じて行うことができる。 Regarding the method for binding a drug to an antibody, for example, Cancer Research; 68 (22) 9280 (2008), Nature Biotechnology; 26 (8) 925 (2008), Bio Conjugate Chemistry; 19, 1673 (2008), Cancer Research; 68 (15) 6300 (2008), or Japanese National Patent Publication No. 2008-516896.
8.放射性同位元素をコンジュゲートした抗CD147抗体
本発明の別の実施態様として、抗CD147抗体に放射性同位元素を標識させたコンジュゲート抗体を挙げることができる。抗腫瘍剤として用いる場合には、細胞傷害性放射性金属元素を標識するのが好ましい。このような細胞傷害性放射性金属元素としては、例えばイットリウム90(90Y)、レニウム186(186Re)、レニウム188(188Re)、銅67(67Cu)、鉄59(59Fe)、ストロンチウム89(89Sr)、金198(198Au)、水銀203(203Hg)、鉛212(212Pb)、ジスプロシウム165(165Dy)、ルテニウム103(103Ru)、ビスマス212(212Bi)、ビスマス213(213Bi)、ホルミウム166(166Ho)、サマリウム153(153Sm)、ルテチウム177(177Lu)などを挙げることができる。これらの放射性金属元素の中でも、90Y、153Sm、177Luが、半減期、放射線エネルギー、容易な標識反応、標識率、錯体の安定性等の点から好ましい。 8). Anti-CD147 antibody conjugated with a radioisotope Another embodiment of the present invention includes a conjugated antibody in which a radioisotope is labeled with an anti-CD147 antibody. When used as an antitumor agent, it is preferable to label a cytotoxic radioactive metal element. Examples of such cytotoxic radioactive metal elements include yttrium 90 (90Y), rhenium 186 (186Re), rhenium 188 (188Re), copper 67 (67Cu), iron 59 (59Fe), strontium 89 (89Sr), gold 198 (198Au), mercury 203 (203Hg), lead 212 (212Pb), dysprosium 165 (165Dy), ruthenium 103 (103Ru), bismuth 212 (212Bi), bismuth 213 (213Bi), holmium 166 (166Ho), samarium 153 ( 153Sm) and lutetium 177 (177Lu). Among these radioactive metal elements, 90Y, 153Sm, and 177Lu are preferable from the viewpoints of half-life, radiation energy, easy labeling reaction, labeling rate, and complex stability.
これらの放射性金属元素を抗CD147抗体に結合させるには、該抗体に金属キレート試薬を反応させ、これに放射性金属元素を反応させて錯体とするのが好ましい。このようにして得られたコンジュゲート抗体は、放射性金属元素が金属キレート試薬を介して抗CD147抗体に結合している。 In order to bind these radioactive metal elements to the anti-CD147 antibody, it is preferable to react the antibody with a metal chelating reagent and react with the radioactive metal element to form a complex. In the conjugate antibody thus obtained, the radioactive metal element is bound to the anti-CD147 antibody via a metal chelating reagent.
このような錯体形成に用いられる金属キレート試薬の例としては、例えば(1)8−ヒドロキシキノリン、8−アセトキシキノリン、8−ヒドロキシキナルジン、キノリン骨格を有するキノロン系化合物であるノルフロキサシン、オフロキサシン、スパルフロキサシン等のキノリン誘導体;(2)クロラニル酸、アルミノン、チオ尿素、テトラフェニルアルソニウムクロライド等の化合物;(3)エチレンジアミン四酢酸(EDTA)、ジエチレントリアミン五酢酸(DTPA)およびこれらに類似した骨格を有するジヒドロキシエチルグリシン、エチレンジアミン二酢酸、エチレンジアミン二プロピオン酸塩酸塩、イソチオシアノベンジルEDTA、イソチオシアノベンジルDTPA、メチルイソチオシアノベンジルDTPA、シクロヘキシルイソチオシアノベンジルDTPA、マレイミドプロピルアミドベンジルEDTA、マレイミドデシルアミドベンジルDTPA;(4)1,4,7,10−テトラアザシクロドデカン−1,4,7,10−四酢酸(DOTA)、イソチオシアノベンジルDOTA等を挙げることができるが、これだけに限定されるわけではない。 Examples of metal chelating reagents used for such complex formation include (1) 8-hydroxyquinoline, 8-acetoxyquinoline, 8-hydroxyquinaldine, norfloxacin, which is a quinolone compound having a quinoline skeleton, ofloxacin, and spall. Quinoline derivatives such as floxacin; (2) compounds such as chloranilic acid, aluminone, thiourea, tetraphenylarsonium chloride; (3) ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA) and similar skeletons Dihydroxyethyl glycine, ethylenediaminediacetic acid, ethylenediaminedipropionate hydrochloride, isothiocyanobenzyl EDTA, isothiocyanobenzyl DTPA, methylisothiocyanobenzyl DTPA, cyclohexane Ruisothiocyanobenzyl DTPA, maleimidopropylamidobenzyl EDTA, maleimidodecylamidobenzyl DTPA; (4) 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), iso Although thiocyanobenzyl DOTA etc. can be mentioned, it is not necessarily limited to this.
これらの金属キレート試薬のうち、イソチオシアノベンジルDOTA、メチルイソチオシアノベンジルDTPA、シクロヘキシルイソチオシアノベンジルDTPAが金属キレートの容易な抗体への導入反応、標識率、錯体の安定性等の点で好ましい。 Among these metal chelate reagents, isothiocyanobenzyl DOTA, methylisothiocyanobenzyl DTPA, and cyclohexylisothiocyanobenzyl DTPA are easy to introduce metal chelates into antibodies, labeling rate, complex stability, etc. preferable.
抗CD147抗体への放射性金属元素の結合は、常法に従って行うことができる。例えば抗CD147抗体に金属キレート試薬を反応させ、予め標識前駆体を調製しておき、次いで放射性金属元素を反応させることにより行うことができる。 The binding of the radioactive metal element to the anti-CD147 antibody can be performed according to a conventional method. For example, the reaction can be performed by reacting an anti-CD147 antibody with a metal chelate reagent, preparing a labeling precursor in advance, and then reacting with a radioactive metal element.
9.医薬組成物
前記の抗CD147モノクローナル抗体、コンジュゲート抗体またはその抗原結合領域を含む断片は、CD147の発現を特徴づける腫瘍や疾患に対する医薬組成物の有効成分として有用である。
このような医薬組成物は、好ましくは、前記抗体等に加えて、生理学的に許容され得る希釈剤またはキャリアを含んでおり、他の抗体または抗生物質のような他の薬剤との混合物であってもよい。適切なキャリアには、生理的食塩水、リン酸緩衝生理食塩水、リン酸緩衝生理食塩水グルコース液、および緩衝生理食塩水が含まれるが、これらに限定されるものではない。投与経路は、経口ルート、並びに静脈内、筋肉内、皮下および腹腔内の注射または配薬を含む非経腸的ルートである。 9. Pharmaceutical Composition The anti-CD147 monoclonal antibody, conjugated antibody or fragment containing the antigen-binding region thereof is useful as an active ingredient of a pharmaceutical composition for tumors and diseases characterized by CD147 expression.
Such a pharmaceutical composition preferably contains a physiologically acceptable diluent or carrier in addition to the antibody or the like, and is a mixture with other drugs or other drugs such as antibiotics. May be. Suitable carriers include, but are not limited to, physiological saline, phosphate buffered saline, phosphate buffered saline glucose solution, and buffered saline. The routes of administration are oral routes and parenteral routes including intravenous, intramuscular, subcutaneous and intraperitoneal injection or delivery.
この場合、本発明の抗体の有効量と適切な希釈剤及び薬理学的に使用し得るキャリアとの組合せとして投与される有効量は、1回につき体重1kgあたり0.1mg〜100mgであり、2日から8週間間隔で投与される。 In this case, the effective amount administered as a combination of an effective amount of the antibody of the present invention and an appropriate diluent and a pharmacologically usable carrier is 0.1 mg to 100 mg per kg body weight at a time. It is administered at intervals of 8 weeks from the day.
以下、実施例により本発明をさらに具体的に説明する。但し、本発明はこれら実施例に技術的範囲が限定されるものではない。 Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.
実施例1
実施例1に係る抗原の作製を説明する。ヒトCD147の第2のアイソフォームであるアミノ酸情報(Genbankアクセッション番号NP_940991.1)のアミノ酸残基22−207番目までの細胞外ドメインのCD147のアミノ酸配列(配列番号1)をコードする遺伝子とそれに続く8xHisタグをコードする遺伝子を設計し、人工的に合成した。カニクイザルCD147のアミノ酸情報(Genbankアクセッション番号 EHH59001.1)を参照にして、ヒトCD147のアミノ酸残基22−207番目に相当する細胞外ドメインのカニクイザルCD147アミノ酸配列(配列番号2)をコードする遺伝子+8xHisを設計し、人工的に合成した。これらの合成遺伝子をそれぞれpCXN3ベクターに挿入して、ヒトまたはカニクイザルCD147発現ベクターを作製した。発現ベクターをFreeStyle−293F細胞(in vitrogen社)に一過性トランスフェクションした。その後1週間のフラスコ培養をして、その培養上清をサンプルとしHis−Trap HPカラム(GE社)に通すことにより精製し、各種の細胞外ドメインCD147−His蛋白質はPBSに置換した。SDS−PAGEおよび抗His tag抗体(キアゲン社)を使ったウエスタンブロティング法により、細胞外ドメインCD147−Hisが精製されたことを確認した。Example 1
Production of the antigen according to Example 1 will be described. A gene encoding the amino acid sequence of CD147 (SEQ ID NO: 1) of the extracellular domain from amino acid residues 22 to 207 of amino acid information (Genbank accession number NP_940991.1), which is the second isoform of human CD147, and The gene encoding the subsequent 8xHis tag was designed and synthesized artificially. Gene encoding cynomolgus monkey CD147 amino acid sequence (SEQ ID NO: 2) of extracellular domain corresponding to amino acid residues 22-207 of human CD147 with reference to amino acid information of cynomolgus monkey CD147 (Genbank accession number EHH59001.1) + 8xHis Was designed and synthesized artificially. Each of these synthetic genes was inserted into a pCXN3 vector to prepare a human or cynomolgus CD147 expression vector. The expression vector was transiently transfected into FreeStyle-293F cells (Invitrogen). Thereafter, flask culture was carried out for 1 week, and the culture supernatant was used as a sample for purification by passing through a His-Trap HP column (GE), and various extracellular domain CD147-His proteins were replaced with PBS. It was confirmed that the extracellular domain CD147-His was purified by Western blotting using SDS-PAGE and anti-His tag antibody (Qiagen).
実施例2
実施例2に係るマウス免疫による抗CD147モノクローナル抗体の作製を説明する。ヒトとカニクイザルまたはマウスの細胞外ドメインCD147(Genbankアクセッション番号 NP_001070652.1参照)との相同性は図1と図2に示すようにヒトとカニクイザル間では78%と高く、ヒトとマウス間では47%であり高くない。従って、ヒトまたはカニクイザルの細胞外ドメインCD147−Hisをマウスに免疫することでヒトまたはカニクニザル、または両種のCD147に対する抗体が得られると考えられる。ヒトとカニクイザルCD147の両方を認識するモノクローナル抗体を取得するために、Balb/cマウスを免疫動物として、6〜8週齢より免疫を開始した。Example 2
Production of anti-CD147 monoclonal antibody by mouse immunization according to Example 2 will be described. The homology between human and cynomolgus monkey or mouse extracellular domain CD147 (see Genbank accession number NP_0010706652.1) is as high as 78% between human and cynomolgus monkey as shown in FIGS. 1 and 2, and 47 between human and mouse. % And not high. Therefore, it is considered that antibodies against human or cynomolgus monkey, or both types of CD147 can be obtained by immunizing mice with human or cynomolgus monkey extracellular domain CD147-His. In order to obtain a monoclonal antibody that recognizes both human and cynomolgus monkey CD147, immunization was started from 6 to 8 weeks of age using Balb / c mice as immunized animals.
初回免疫では、細胞外ドメインCD147−Hisは50μg/headと百日咳毒素 100ng/headおよびフロイト完全アジュバントまたはTiter Maxでエマルジョン化したものを腹腔内に投与した。2週間後に、CD147−His 25μg/headをフロイト不完全アジュバントまたはTiter Maxでエマルジョン化したもので追加免疫した。細胞融合を行う3日前に、PBS中の細胞外ドメインCD147−His 25μg/headで最終免疫を施した。 In the first immunization, extracellular domain CD147-His was intraperitoneally administered with 50 μg / head and pertussis toxin 100 ng / head and emulsified with Freud's complete adjuvant or Titer Max. Two weeks later, booster was performed with CD147-His 25 μg / head emulsified with Freud's incomplete adjuvant or Titer Max. Three days prior to cell fusion, final immunization was performed with extracellular domain CD147-His 25 μg / head in PBS.
細胞融合は脾臓細胞を摘出し、マウスミエローマP3−X63Ag8U1を混合し、PEG1500(ロッシュ・ダイアグノティック社)を用いて使用説明書に従って実施した。RPMI1640培地を加えPEG1500を希釈し、遠心操作によりPEG1500を除去した。細胞ペレットを慎重に10%FBS/RPMI1640培地にて懸濁したものを100μL/wellで96wellプレートに播種し、一晩培養した。翌日、10%FBS/1x HAT media supplement/1x BM−condimed H1 Hybridoma cloning supplementを含むRPMI1640培地(HAT培地)を100μL/wellで添加した。融合日から2、4日後に培養液の半分を新しいHAT培地に交換し、8日後にその培養上清を用いて、スクリーニングを実施した。 Cell fusion was performed by removing spleen cells, mixing mouse myeloma P3-X63Ag8U1, and using PEG1500 (Roche Diagnostics) according to the instruction manual. RPMI1640 medium was added to dilute PEG1500, and PEG1500 was removed by centrifugation. A cell pellet carefully suspended in 10% FBS / RPMI1640 medium was seeded on a 96-well plate at 100 μL / well and cultured overnight. The next day, RPMI 1640 medium (HAT medium) containing 10% FBS / 1x HAT media supplement / 1x BM-consolidated H1 Hybridoma cloning supplement was added at 100 μL / well. Two to four days after the fusion day, half of the culture medium was replaced with fresh HAT medium, and screening was performed using the culture supernatant after 8 days.
スクリーニングは細胞外ドメインCD147−Hisを固相したELISAを行った。ヒトまたはサルの細胞外ドメインCD147−HisをPBSで2.0μg/mLになるようにそれぞれ調製し、Costar Plate(CORNING)50μL/wellに添加し、4℃で通夜静置した。翌朝、抗原液を捨て、ブロッキング液(40% Block Ace/TBS)100μL/wellで添加し、室温で1時間ブロッキングを行った。ブロッキング溶液を除き、PBS/0.05% Tween−20により洗浄し、ブロッキング液で5倍または50倍希釈したハイブリドーマwellの培養上清を50μL/well添加し、室温で1時間反応させた。PBS/0.05% Tween−20で5回洗浄後、10% Block Ace/TBSで5000倍希釈したHRP標識 anti−mouse IgG(H+L)抗体を50μL/well添加し、室温で1時間反応させた。PBS/0.05% Tween−20で5回洗浄後、TMB試薬を50μL/well添加し室温で5分間反応させた。2NH2SO4を50μL/well加え、発色反応を停止した。その後、Plate readerにて450nm(referenceとして620nm)の吸光度を測定した。ELISAまたはFACSで検討した結果、多数の陽性ハイブリドーマwellを得た。Screening was performed by ELISA using extracellular domain CD147-His as a solid phase. Human or monkey extracellular domain CD147-His was prepared with PBS to 2.0 μg / mL, added to Costar Plate (CORNING) 50 μL / well, and allowed to stand at 4 ° C. overnight. The next morning, the antigen solution was discarded, and 100 μL / well of blocking solution (40% Block Ace / TBS) was added, followed by blocking at room temperature for 1 hour. The blocking solution was removed, the plate was washed with PBS / 0.05% Tween-20, 50 μL / well of a hybridoma well culture supernatant diluted 5 or 50 times with a blocking solution, and reacted at room temperature for 1 hour. After washing 5 times with PBS / 0.05% Tween-20, 50 μL / well of HRP-labeled anti-mouse IgG (H + L) antibody diluted 5000 times with 10% Block Ace / TBS was added and allowed to react at room temperature for 1 hour. . After washing 5 times with PBS / 0.05% Tween-20, 50 μL / well of TMB reagent was added and reacted at room temperature for 5 minutes. 2 NH 2 SO 4 was added at 50 μL / well to stop the color reaction. Thereafter, the absorbance at 450 nm (620 nm as a reference) was measured with a plate reader. As a result of examination by ELISA or FACS, a large number of positive hybridoma wells were obtained.
陽性クローンについては限界希釈法によりモノクローン化した。最終的に細胞表面上のCD147との反応できるモノクローナル抗体を選定するために、ヒトCD147に関しては前立腺がんの細胞株であるPC−3(ATCC CRL−1435)とバーキットリンパ腫の細胞株であるRaji(ATCC CCL−86)を、カニクイザルCD147に関してはHSV不死化T細胞であるHSC−F(JCRB細胞バンクJCRB1164)によるFACS解析を行った。その結果、両CD147に強く反応する2つの陽性クローンPPAT−082−01とPPAT−082−02を取得した。 Positive clones were monocloned by the limiting dilution method. In order to finally select a monoclonal antibody capable of reacting with CD147 on the cell surface, human CD147 is a prostate cancer cell line, PC-3 (ATCC CRL-1435), and a Burkitt lymphoma cell line. Raji (ATCC CCL-86) was analyzed for cynomolgus monkey CD147 by HSC-F (JCRB cell bank JCRB1164), an HSV immortalized T cell. As a result, two positive clones PPAT-082-01 and PPAT-082-02 that strongly react to both CD147 were obtained.
なお、本実施例を含め以下のいずれの実施例中、並びに実施例における試験結果として示した表または図中においては、各々の本発明のヒト抗ヒトCD147モノクローナル抗体を産生するハイブリドーマクローンは記号を用いて命名した。また、当該記号の次に「抗体」を付したものは、それぞれのハイブリドーマにより産生される抗体、または当該ハイブリドーマから単離された抗体遺伝子を保持する宿主細胞により生産された組換え抗体を意味する。また文脈上明らかな範囲において、ハイブリドーマクローンの名称が抗体の名称をあらわす場合がある。 In any of the following examples including this example, and in the tables or figures shown as test results in the examples, each of the hybridoma clones producing the human anti-human CD147 monoclonal antibody of the present invention has a symbol. Named. In addition, the term “antibody” after the symbol means an antibody produced by each hybridoma, or a recombinant antibody produced by a host cell carrying an antibody gene isolated from the hybridoma. . In addition, the name of the hybridoma clone may represent the name of an antibody within a range that is clear from the context.
実施例3
実施例3に係る抗ヒトCD147抗体の可変領域のクローニングを説明する。培養した2つの陽性ハイブリドーマから調製したtotal RNAを鋳型として、のSuperScriptIII First Strand Synthesis System(invitrogen社)を用いて添付説明書に従ってcDNAを合成した。重鎖可変領域の増幅は、重鎖シグナル配列をコードする核酸の縮重配列を含むフォワードプライマーと重鎖定常領域特異的なリバースプライマーを用いてExTag polymerase(タカラバイオ)によりcDNAを鋳型としてPCRした。軽鎖可変領域の増幅は、軽鎖シグナル配列をコードする核酸の縮重配列を含むフォワードプライマーと軽鎖定常領域特異的なリバースプライマーを用いてExTag polymeraseによりcDNAを鋳型としてPCRした。増幅したPCR産物をQIAquick Gel Extraction Kit(キアゲン社)を用いて、アガロースゲル電気泳動のシングルバンドから精製した後、pGEM−T easyベクター(プロメガ社)へクローニングして塩基配列の決定を行い、重鎖可変領域と軽鎖可変領域のアミノ酸残基およびCDRを同定した。抗体の重鎖および軽鎖の可変領域のアミノ酸残基と各CDR配列を、表1(重鎖)と表2(軽鎖)に示す。Example 3
The cloning of the variable region of the anti-human CD147 antibody according to Example 3 will be described. Using total RNA prepared from two cultured positive hybridomas as a template, cDNA was synthesized using SuperScriptIII First Strand Synthesis System (Invitrogen) according to the attached instructions. Amplification of the heavy chain variable region was performed by PCR using Extag polymerase (Takara Bio) as a template using a forward primer containing a degenerate sequence of a nucleic acid encoding a heavy chain signal sequence and a reverse primer specific to the heavy chain constant region. . Amplification of the light chain variable region was performed by PCR using Extag polymerase as a template using a forward primer containing a degenerate sequence of a nucleic acid encoding a light chain signal sequence and a reverse primer specific to the light chain constant region. The amplified PCR product was purified from a single band of agarose gel electrophoresis using QIAquick Gel Extraction Kit (Qiagen), and then cloned into pGEM-T easy vector (Promega) to determine the base sequence. The amino acid residues and CDRs of the chain variable region and the light chain variable region were identified. Table 1 (heavy chain) and 2 (light chain) show the amino acid residues and the respective CDR sequences of the variable regions of the heavy and light chains of the antibody.
実施例4
実施例4に係るヒト抗体ファージライブラリーによる抗CD147モノクローナル抗体の作製を説明する。まずはCD147を強く発現しているがん細胞を利用して、CD147に結合するファージ抗体の1stスクリーニングを実施した。CD147強発現である膵臓癌細胞株であるMIApaca−2(ATCC CRL−1420)を任意の方法で培養した。その後細胞を回収し、冷却したPBSで洗浄した後、1×1013cfuのヒト抗体ファージライブラリー(特開2005−185281号公報、WO2008/007648号公報、WO2006/090750号公報を参照)を混ぜ、最終容積1.6mLになるように反応液(1%BSA、0.1%NaN3、MEM)を添加し、4℃にて4時間ゆっくり回転させて反応させた。反応終了後、反応液を二つに分け、それぞれ事前に用意した0.6mLの有機溶液(dibutyl phthalate cycloheximide9:1)に重層し、マイクロ遠心機により2分間遠心(3000rpm)した。上清を捨て、チューブの底に沈降した細胞を0.7mLの1%BSA/MEMで懸濁し、更に0.7mLの有機溶媒に重層した。同じように遠心により上清を捨て、細胞を0.3mLのPBSで懸濁し、液体窒素で凍結した。Example 4
The production of anti-CD147 monoclonal antibody using the human antibody phage library according to Example 4 will be described. First, the 1st screening of the phage antibody couple | bonded with CD147 was implemented using the cancer cell which is expressing CD147 strongly. MIApaca-2 (ATCC CRL-1420), which is a pancreatic cancer cell line highly expressing CD147, was cultured by an arbitrary method. Thereafter, the cells are collected, washed with chilled PBS, and mixed with a 1 × 10 13 cfu human antibody phage library (see Japanese Patent Application Laid-Open Nos. 2005-185281, WO2008 / 007648, and WO2006 / 090750). Then, a reaction solution (1% BSA, 0.1% NaN 3 , MEM) was added to a final volume of 1.6 mL, and the reaction was performed by slowly rotating at 4 ° C. for 4 hours. After completion of the reaction, the reaction solution was divided into two, and each layer was layered on a 0.6 mL organic solution (dibutyl phthalate cyclohexane 9: 1) prepared in advance, and centrifuged for 2 minutes (3000 rpm) with a microcentrifuge. The supernatant was discarded, and the cells that had settled to the bottom of the tube were suspended in 0.7 mL of 1% BSA / MEM and overlaid with 0.7 mL of an organic solvent. Similarly, the supernatant was discarded by centrifugation, and the cells were suspended in 0.3 mL of PBS and frozen in liquid nitrogen.
凍結した細胞を37℃で融解し、これをOD 0.5の大腸菌DH12S 20mLに1時間感染させ、その一部をアンピシリンプレートに蒔いて回収されたファージのtiterを算出した。ファージ感染大腸菌は600mLの2xYTGA培地(2xYT,200μg/mL ampicillin sulfate,1% glucose)にて30℃で通夜培養した。この通夜培養10mLを2xYTA培地(2xYT,200μg/mL ampicillin sulfate)200mLと混ぜ、37℃にて1.5時間培養後ヘルパーファージKO7を1x1011入れ、37℃にて1時間培養したのち、800mLの2xYTGAK(2xYT,200μg/mL ampicillin sulfate,0.05% glucose, 50μg/mL kanamycin)を入れて30℃にて通夜培養した。これを8000rpmにて10分間遠心して上清1Lを調製、それに200mLのPEG液(20% polyetyleneglycol 6000,2.5M NaCl)を混ぜてよくかきまぜたのち、8000rpm 10分間の遠心を行うことでファージを沈殿させた。これを10mLのPBSに懸濁し、その一部を使用して大腸菌感染数を調べた。これが1stスクリーニングのファージである。The frozen cells were thawed at 37 ° C., infected with 20 mL of E. coli DH12S having an OD of 0.5 for 1 hour, and a portion thereof was spread on an ampicillin plate to calculate the titer of the recovered phage. Phage-infected Escherichia coli was cultured overnight at 30 ° C. in 600 mL of 2 × YTGA medium (2 × YT, 200 μg / mL ampicillin sulfate, 1% glucose). 10 mL of this overnight culture was mixed with 200 mL of 2 × YTA medium (2 × YT, 200 μg / mL ampicillin sulfate), cultured at 37 ° C. for 1.5 hours, then added with 1 × 10 11 helper phage KO7, cultured at 37 ° C. for 1 hour, and then 800 mL of 2 × YTGAK (2 × YT, 200 μg / mL ampicillin sulfate, 0.05% glucose, 50 μg / mL kanmycin) was added and cultured overnight at 30 ° C. This was centrifuged at 8000 rpm for 10 minutes to prepare 1 L of the supernatant, and 200 mL of PEG solution (20% polyethyleneglycol 6000, 2.5 M NaCl) was mixed and stirred well, followed by centrifugation at 8000 rpm for 10 minutes. Precipitated. This was suspended in 10 mL of PBS, and a portion thereof was used to examine the number of E. coli infections. This is the 1st screening phage.
次に、ヒトCD147の細胞外ドメイン抗原を用いて2ndスクリーニングを行った。
PBSで50μg/mLになるように抗原を調製し、Immuno Module/Strip Plates(NUNK)に100μL/well添加し、4℃で通夜静置した。その後、抗原液を捨て、ブロッキング液(5% スキムミルク/ PBS)200μL/wellを添加し、37℃で2時間ブロッキングを行った。
次にブロッキング液を除いたImmuno Module/Strip Plates に5% スキムミルク/ PBS で調製した1×1010の1stスクリーニングファージを混合し、2時間室温で反応させた。
反応終了後、PBSにて10回洗浄し、0.2M Glycine HCl(pH3.0)を100μL/wellに添加してファージを回収した。回収したファージはOD0.5の大腸菌DH12S 20mLに1時間感染させ1stスクリーニング時と同様にファージの調製を行い、これを2ndスクリーニングファージとした。Next, 2nd screening was performed using the extracellular domain antigen of human CD147.
Antigen was prepared to 50 μg / mL with PBS, 100 μL / well was added to Immuno Module / Stripe Plates (NUNK), and allowed to stand at 4 ° C. overnight. Thereafter, the antigen solution was discarded, 200 μL / well of a blocking solution (5% skim milk / PBS) was added, and blocking was performed at 37 ° C. for 2 hours.
Next, 1 × 10 10 1st screening phage prepared with 5% skim milk / PBS was mixed with Immuno Module / Stripe Plates excluding the blocking solution, and reacted at room temperature for 2 hours.
After completion of the reaction, the plate was washed 10 times with PBS, and 0.2 M Glycine HCl (pH 3.0) was added to 100 μL / well to collect phages. The recovered phage was infected with 20 mL of E. coli DH12S having an OD of 0.5 for 1 hour, and the phage was prepared in the same manner as in the 1st screening, and this was used as a 2nd screening phage.
3rdスクリーニングでは、カニクイザルCD147細胞外ドメイン抗原と2ndスクリーニングのファージを使用し、上記2ndスクリーニングと同じ工程を行った。さらにもう一度カニクイザルCD147細胞外ドメイン抗原を用いた4thスクリーニングとして、一連の操作を繰り返した。 In the 3rd screening, cynomolgus monkey CD147 extracellular domain antigen and 2nd screening phage were used, and the same steps as the 2nd screening were performed. Further, a series of operations was repeated as a 4th screening using the cynomolgus monkey CD147 extracellular domain antigen again.
最終的な陽性ファージクローンは、CD147抗原を用いたELISAによる抗原抗体の反応性から選定した。すなわち、ヒトまたはカニクイザルの細胞外ドメインCD147−HisをPBSで10μg/mLになるようにそれぞれ調製し、Immuno Module/Strip Plates(NUNK)に50μL/wellに添加し、37℃で2時間静置した。その後、抗原液を捨て、ブロッキング液(5% スキムミルク/PBS) 200μL/wellで添加し、37℃で2時間ブロッキングを行った。ブロッキング溶液を除き、PBSにより洗浄し、上記4thスクリーニングより調製した培養上清を100μL/well添加し、37℃で1時間反応させた。PBSで5回洗浄後、PBS/0.05%Tween20で希釈した1μg/mL Rabbit anti−cp3抗体を100μL/well添加し、37℃で1時間反応させた。PBSで5回洗浄後、更にPBS/0.05%Tween20で2000倍希釈したHRP標識anti−Rabbit IgG(H+L)抗体を100μL/well添加し、37℃で1時間反応させた。PBSで5回洗浄後、OPD in 0.1Mクエン酸リン酸バッファー(pH5.1)+0.01%H2O2 を100μL/well添加し室温で5分間反応させた。2NH2SO4を100μL/well加え、発色反応を停止した。その後、SPECTRAmax340PC(Molecular Devices)にて492nmの吸光度を測定した。その結果からヒトおよびカニクイザル細胞外ドメインCD147抗原の両タンパク質に陽性反応を示すファージクローンを選別し、そのDNA配列を解析した。それぞれのCDR配列を確認し、独立配列を有するものを分類した結果、ヒト抗体1つ PPAT−082−03を取得できた。また特許5382692号に記載の抗CD147モノクローナルヒト抗体の配列(配列番号177および181)を参考にしてPPAT−082−04を作製した。その2つのヒト抗体の重鎖および軽鎖の可変領域のアミノ酸残基と各CDR配列を、表1(重鎖)と表2(軽鎖)に示す。但し、PPAT−082−04の軽鎖はλ鎖のため、その定常領域も含めて表2に示す。The final positive phage clone was selected from the reactivity of the antigen antibody by ELISA using the CD147 antigen. Specifically, human or cynomolgus monkey extracellular domain CD147-His was prepared with PBS to 10 μg / mL, added to Immuno Module / Stripe Plates (NUNK) at 50 μL / well, and allowed to stand at 37 ° C. for 2 hours. . Thereafter, the antigen solution was discarded, and 200 μL / well of blocking solution (5% skim milk / PBS) was added, followed by blocking at 37 ° C. for 2 hours. The blocking solution was removed, the plate was washed with PBS, 100 μL / well of the culture supernatant prepared from the 4th screening was added, and the mixture was reacted at 37 ° C. for 1 hour. After washing with PBS 5 times, 100 μL / well of 1 μg / mL Rabbit anti-cp3 antibody diluted with PBS / 0.05% Tween 20 was added and reacted at 37 ° C. for 1 hour. After washing with PBS 5 times, 100 μL / well of HRP-labeled anti-Rabbit IgG (H + L) antibody diluted 2000 times with PBS / 0.05% Tween 20 was further added and reacted at 37 ° C. for 1 hour. After washing 5 times with PBS, 100 μL / well of OPD in 0.1 M citrate phosphate buffer (pH 5.1) + 0.01% H 2 O 2 was added and allowed to react at room temperature for 5 minutes. 2 NH 2 SO 4 was added at 100 μL / well to stop the color reaction. Thereafter, the absorbance at 492 nm was measured with SPECTRAmax340PC (Molecular Devices). From the results, phage clones positive for both human and cynomolgus monkey extracellular domain CD147 antigen proteins were selected and their DNA sequences were analyzed. As a result of confirming each CDR sequence and classifying those having independent sequences, one human antibody PPAT-082-03 was obtained. In addition, PPAT-082-04 was prepared with reference to the sequence of the anti-CD147 monoclonal human antibody (SEQ ID NOs: 177 and 181) described in Japanese Patent No. 5382629. The amino acid residues and the respective CDR sequences of the variable regions of the heavy and light chains of the two human antibodies are shown in Table 1 (heavy chain) and Table 2 (light chain). However, since the light chain of PPAT-082-04 is a λ chain, it is shown in Table 2 including its constant region.
実施例5
実施例5に係る抗CD147ヒト化抗体の作製を説明する。IMGTより公開されているヒト抗体の配列データを入手し、軽鎖可変領域、重鎖可変領域に分けてコンセンサス配列を同定した。その結果の重鎖可変領域のアミノ酸配列は配列番号11、軽鎖可変領域のアミノ酸配列は配列番号12に示す。これらのヒト抗体コンセンサス配列のフレームワーク領域のアミノ酸残基を参照として、抗体PPAT−082−01と02は、重鎖および軽鎖の各CDRを中心としてアミノ酸残基を移植し、下記の1)と2)に示すヒト化抗体を設計した。
1)hPPAT−082−01
2)hPPAT−082−02
設計したそれぞれのヒト化抗体について、それぞれの重鎖可変領域および軽鎖可変領域のアミノ酸配列を表3に示す。Example 5
The production of the anti-CD147 humanized antibody according to Example 5 will be described. Human antibody sequence data published by IMGT was obtained, and consensus sequences were identified by dividing into light chain variable regions and heavy chain variable regions. The resulting heavy chain variable region amino acid sequence is shown in SEQ ID NO: 11, and the light chain variable region amino acid sequence is shown in SEQ ID NO: 12. With reference to the amino acid residues in the framework regions of these human antibody consensus sequences, antibodies PPAT-082-01 and 02 transplanted amino acid residues around the CDRs of the heavy chain and the light chain, and the following 1) And the humanized antibody shown in 2) was designed.
1) hPPAT-082-01
2) hPPAT-082-02
Table 3 shows the amino acid sequences of each heavy chain variable region and light chain variable region for each designed humanized antibody.
実施例6
実施例6に係る抗ヒトCD147抗体のキメラ抗体とヒト抗体の調製方法を説明する。マウス免疫から取得した抗体PPAT−082−01とPPAT−082−02、ファージからの抗体PPAT−082−03とPPAT−082−04のそれぞれの軽鎖と重鎖の可変領域のアミノ酸配列をコードする遺伝子を人工的に合成した。ヒトIgG1由来の定常領域の遺伝子が組み込まれたpCXN3ベクターに重鎖可変領域の遺伝子を、ヒトκ鎖由来の定常領域の遺伝子が組み込まれたpCXN3ベクターに軽鎖可変領域の遺伝子をそれぞれ挿入した発現ベクターを作製した。PPAT−082−04の軽鎖のアミノ酸配列をコードする遺伝子に関しては、可変領域の遺伝子の後にヒトλ鎖由来の定常領域の遺伝子を人工的に合成した遺伝子をpCXN3ベクターに挿入した発現ベクターを作製した。重鎖可変領域と軽鎖可変領域の遺伝子をそれぞれ組み込んだ発現ベクターを同時にFreeStyle−293F細胞(invitrogen社)に導入して、マウス−ヒトのキメラ抗体とヒト抗体を産生させた。マウス抗体であるPPAT−082−01とPPAT−082−02の可変領域を含むマウス−ヒトのキメラ抗体は、cPPAT−082−01とcPPAT−082−02とそれぞれ命名する。産生したそれぞれの抗体は、培養上清中に分泌されているのでProtein Gカラムで精製し、さらに陰イオンカラムまたはゲル濾過カラムにより精製し、PBSに置換した。精製した2つのキメラ抗体と2つヒト抗体は、実施例2のように、FACS解析よりヒトとカニクイザルCD147と反応性を確認した。その結果を示す図3のように、cPPAT−082−01とcPPAT−082−02のマウス−ヒトのキメラ抗体とPPAT−082−03のヒト抗体はHSC−FとRaji細胞に濃度依存的に結合したが、PPAT−082−04のヒト抗体はHSC−Fとは反応しなかった。従って、作製したすべての抗体は元の抗体と同じ特性、少なくともCD147抗原との反応を継承している事が分かった。Example 6
A method for preparing a chimeric antibody and a human antibody of the anti-human CD147 antibody according to Example 6 will be described. Encodes the amino acid sequences of the variable regions of the light and heavy chains of antibodies PPAT-082-01 and PPAT-082-02 obtained from mouse immunity, and antibodies PPAT-082-03 and PPAT-082-04 from phage, respectively. Genes were synthesized artificially. Expression in which a heavy chain variable region gene is inserted into a pCXN3 vector into which a constant region gene derived from human IgG1 is incorporated, and a light chain variable region gene is inserted into a pCXN3 vector into which a constant region gene from human κ chain is incorporated A vector was prepared. Regarding the gene encoding the amino acid sequence of the light chain of PPAT-082-04, an expression vector is prepared by inserting a gene obtained by artificially synthesizing a constant region gene derived from human λ chain after the variable region gene into the pCXN3 vector did. Expression vectors incorporating the heavy chain variable region gene and the light chain variable region gene were simultaneously introduced into FreeStyle-293F cells (Invitrogen) to produce mouse-human chimeric antibodies and human antibodies. The mouse-human chimeric antibodies comprising the variable regions of the mouse antibodies PPAT-082-01 and PPAT-082-02 are named cPPAT-082-01 and cPPAT-082-02, respectively. Since each produced antibody was secreted into the culture supernatant, it was purified with a Protein G column, further purified with an anion column or gel filtration column, and replaced with PBS. The purified two chimeric antibodies and two human antibodies were confirmed to react with human and cynomolgus monkey CD147 by FACS analysis as in Example 2. As shown in FIG. 3 showing the results, cPPAT-082-01 and cPPAT-082-02 mouse-human chimeric antibody and PPAT-082-03 human antibody bound to HSC-F and Raji cells in a concentration-dependent manner. However, the human antibody of PPAT-082-04 did not react with HSC-F. Therefore, it was found that all the antibodies produced inherited the same characteristics as the original antibody, at least the reaction with the CD147 antigen.
実施例7
実施例7に係る抗CD147抗体による慢性骨髄性白血病の細胞株であるK562移植モデルにおける抗腫瘍効果を説明する。生後6週齢のC.B.17/Icr−scidJc1マウス(日本クレア)に、K562細胞(ATCC CCL−432)をマウス1匹当たり5×106個で、MatriGel(BD社)とRPMI1640(比率1対1)の溶液中で腹側部皮下へ注射した。腫瘍体積が平均230mm3に達した日に、PBSに溶解したcPPAT−082−01、cPPAT−082−02、PPAT−082−03、またはPPAT−082−04を静脈注射した。投与量は抗CD147抗体をマウス1匹当たり15mg/kgをそれぞれ5個体のマウスに週2回で5回投与した。比較として、抗体を含まないPBSを静脈注射した。経過日時と腫瘍体積(平均値)関係を図4に示す。PBS投与群は日を追って腫瘍体積が増加したが、抗CD147抗体15mg/kgを投与された群すべては5匹すべて腫瘍体積は初回抗体投与時からあまり変化していない、もしくは減少を示した。従って、抗CD147抗体投与において抗腫瘍効果が確認された。Example 7
The antitumor effect in the K562 transplantation model which is a cell line of chronic myelogenous leukemia by the anti-CD147 antibody according to Example 7 will be described. 6 weeks old C.I. B. 17 / Icr-scidJc1 mice (CLEA, Japan) and K562 cells (ATCC CCL-432) at 5 × 10 6 per mouse in a solution of MatriGel (BD) and RPMI 1640 (ratio 1: 1). Injection into the subcutaneous side. On the day when the tumor volume reached an average of 230 mm 3 , cPPAT-082-01, cPPAT-082-02, PPAT-082-03, or PPAT-082-04 dissolved in PBS was injected intravenously. The dose was 15 mg / kg of anti-CD147 antibody per mouse, and 5 mice were administered twice a week for 5 times. As a comparison, PBS without antibody was injected intravenously. The relationship between the elapsed date and time and the tumor volume (average value) is shown in FIG. In the PBS-administered group, the tumor volume increased day by day, but in all of the groups administered with 15 mg / kg of the anti-CD147 antibody, the tumor volume did not change much from the initial antibody administration or showed a decrease. Therefore, the anti-tumor effect was confirmed by anti-CD147 antibody administration.
実施例8
実施例8に係る抗CD147抗体による大腸がんの細胞株であるSW480移植モデルにおける抗腫瘍効果を説明する。生後6週齢のC.B.17/Icr−scidJc1マウス(日本クレア)に、SW480細胞(ATCC CCL−280)をマウス1匹当たり5×106個で、RPMI1640の溶液中で腹側部皮下へ注射した。腫瘍体積が平均270mm3に達した日に、cPPAT−082−01、cPPAT−082−02、PPAT−082−03、またはPPAT−082−04の抗CD147抗体を静脈注射した。投与量は抗CD147抗体をマウス1匹当たり15mg/kgをそれぞれ5個体のマウスに週2回で5回投与した。比較として、抗体を含まないPBSを静脈注射した。経過日時と腫瘍体積(平均値)関係を図5に示す。抗CD147抗体15mg/kgを投与された群では5匹すべてにおいて、PBS投与群より腫瘍体積の増加度合は遅延しており、抗CD147抗体は抗腫瘍効果があることが確認された。Example 8
The antitumor effect in the SW480 transplantation model which is a colon cancer cell line by the anti-CD147 antibody according to Example 8 will be described. 6 weeks old C.I. B. 17 / Icr-scidJc1 mice (CLEA Japan) were injected subcutaneously in the ventral region with 5 × 10 6 SW480 cells (ATCC CCL-280) per mouse in a solution of RPMI1640. On the day tumor volumes reached an average of 270 mm 3 , cPPAT-082-01, cPPAT-082-02, PPAT-082-03, or PPAT-082-04 anti-CD147 antibodies were injected intravenously. The dose was 15 mg / kg of anti-CD147 antibody per mouse, and 5 mice were administered twice a week for 5 times. As a comparison, PBS without antibody was injected intravenously. The relationship between elapsed date and time and tumor volume (average value) is shown in FIG. In all 5 animals in the group administered with the anti-CD147 antibody 15 mg / kg, the degree of increase in tumor volume was delayed as compared with the PBS administration group, and it was confirmed that the anti-CD147 antibody has an antitumor effect.
実施例9
実施例9に係る対象抗体HAb18の作製およびCD147との反応性の確認を説明する。HAb18の名称としてCD147抗体が知られており、その抗体のF(ab’)2フラグメントに放射性I−131標識した肝がんの治療薬(Licartin)が中国国内のみで承認されている。未標識でのHAb18との薬効を比較するために、特表2006−525946に記載されている遺伝子配列情報から、実施例6と同様に重鎖と軽鎖の可変領域のアミノ酸配列をコードする遺伝子を人工的に合成し、抗体を作製し、精製した。その重鎖(配列番号41)と軽鎖(配列番号42)の可変領域のアミノ酸配列を表4に示す。また精製したHAb18は、実施例2で記載したように細胞外ドメインのヒトCD147-HisまたはカニクイザルCD147-Hisを固相したELISAにてCD147との反応性を確認した。その結果の図6に示したように、HAb18はヒトCD147との反応が認められたが、カニクイザルCD147との反応は認められなかった。このことから、HAb18はPPAT−082−04以外の抗体とはエピトープが異なる事が示唆される。Example 9
The production of the target antibody HAb18 according to Example 9 and confirmation of the reactivity with CD147 will be described. The CD147 antibody is known as the name of HAb18, and a therapeutic agent for liver cancer (Licartin) labeled with radioactive I-131 on the F (ab ′) 2 fragment of the antibody is approved only in China. In order to compare the drug efficacy with unlabeled HAb18, from the gene sequence information described in JP-T-2006-525946, a gene encoding the amino acid sequences of the variable regions of the heavy chain and the light chain as in Example 6. Were artificially synthesized to produce antibodies and purified. Table 4 shows the amino acid sequences of the variable regions of the heavy chain (SEQ ID NO: 41) and light chain (SEQ ID NO: 42). Further, as described in Example 2, the purified HAb18 was confirmed to be reactive with CD147 in an ELISA in which extracellular domain human CD147-His or cynomolgus monkey CD147-His was solid-phased. As shown in FIG. 6 of the result, HAb18 was found to react with human CD147 but not with cynomolgus CD147. This suggests that HAb18 has an epitope different from that of antibodies other than PPAT-082-04.
実施例10
実施例10に係る抗CD147抗体によるBリンパ腫の細胞株であるNCI−H929移植モデルにおける抗腫瘍効果を説明する。生後6週齢のC.B.17/Icr−scidJc1マウスに、NCI−H929細胞(ATCC CRL−9068)をマウス1匹当たり5×106個で、MatriGel(BD社)とRPMI1640(比率1対1)の溶液中で腹側部皮下へ注射した。腫瘍体積が平均141mm3に達した日に、HAb-18、またはPPAT−082−03の抗CD147抗体を静脈注射した。投与量は抗CD147抗体をマウス1匹当たり15mg/kgをそれぞれ3個体のマウスに週1回で4回投与した。比較として、抗体を含まないPBSを静脈注射した。経過日時と腫瘍体積(平均値)関係を図7に示す。2つの抗CD147抗体15mg/kgを投与された群では3匹すべてにおいて、PBS投与群より腫瘍体積の増加度合は遅延しており、抗CD147抗体は抗腫瘍効果があることが確認された。PPAT−082−03とHAb18との抗腫瘍効果は同等程度であった。Example 10
The antitumor effect in the NCI-H929 transplantation model which is a cell line of B lymphoma by the anti-CD147 antibody according to Example 10 will be described. 6 weeks old C.I. B. 17 / Icr-scidJc1 mice with 5 × 10 6 NCI-H929 cells (ATCC CRL-9068) per mouse and ventral in a solution of MatriGel (BD) and RPMI 1640 (ratio 1: 1) It was injected subcutaneously. On the day when the tumor volume reached an average of 141 mm 3 , HAb-18, or PPAT082-03 anti-CD147 antibody was injected intravenously. The dose was 15 mg / kg of anti-CD147 antibody per mouse, and each mouse was administered to 3 mice, 4 times a week. As a comparison, PBS without antibody was injected intravenously. The relationship between the elapsed date and time and the tumor volume (average value) is shown in FIG. In all three mice administered with 15 mg / kg of the two anti-CD147 antibodies, the increase in tumor volume was delayed as compared with the PBS-administered group, confirming that the anti-CD147 antibody has an antitumor effect. The anti-tumor effect of PPAT-082-03 and HAb18 was comparable.
実施例11
実施例11に係るK562移植モデルにおける抗腫瘍効果の抗CD147抗体の用量依存性を説明する。生後8週齢のC.B.17/Icr−scidJc1マウスに、K562細胞をマウス1匹当たり5×106個で、MatriGelとRPMI1640(比率1対1)の溶液中で腹側部皮下へ注射した。腫瘍体積が平均117mm3に達した日に、PPAT−082−03の抗CD147抗体を静脈注射した。投与量は抗CD147抗体をマウス1匹当たり0.12、0.6、3mg/kgの3種の濃度でそれぞれ5個体のマウスに週1回で4回投与した。比較として、HAb-18を0.6mg/kgと、抗体を含まないPBSを静脈注射した。経過日時と腫瘍体積(平均値)関係を図8に示す。PPAT−082−03の3mg/kgを投与された群では腫瘍がほぼ消失し、その後の経過観察でも再発は認められなかった。0.6mg/kgを投与されたPPAT−082−03とHAb18の抗腫瘍効果は同等程度であり、腫瘍体積の減少が見られ、その後の経過観察では再発の兆しが認められた。0.12mg/kgを投与されたPPAT−082−03では、PBS投与群とほぼ変わらず、抗腫瘍効果を認める事ができなかった。Example 11
The dose dependence of the anti-CD147 antibody of the antitumor effect in the K562 transplantation model according to Example 11 will be described. 8 weeks old C.I. B. 17 / Icr-scidJc1 mice were injected subcutaneously in the ventral region with 5 × 10 6 K562 cells per mouse in a solution of MatriGel and RPMI 1640 (ratio 1: 1). On the day when the tumor volume reached an average of 117 mm 3 , PPAT-082-03 anti-CD147 antibody was injected intravenously. The doses of anti-CD147 antibody were administered four times once a week to five mice each at three concentrations of 0.12, 0.6, and 3 mg / kg per mouse. As a comparison, HAb-18 was intravenously injected at 0.6 mg / kg and PBS containing no antibody. The relationship between the elapsed date and time and the tumor volume (average value) is shown in FIG. In the group to which 3 mg / kg of PPAT-082-03 was administered, the tumor almost disappeared, and no recurrence was observed in the subsequent follow-up. The antitumor effects of PPAT-082-03 and HAb18 administered at 0.6 mg / kg were comparable, showing a decrease in tumor volume, and subsequent follow-up showed signs of recurrence. PPAT-082-03 administered with 0.12 mg / kg was almost the same as the PBS administration group, and no antitumor effect could be observed.
実施例12
実施例12に係る肝癌の細胞株であるHepG2移植モデルにおける抗腫瘍効果の抗CD147抗体の用量依存性を説明する。生後8週齢のC.B.17/Icr−scidJc1マウスに、HepG2細胞(ATCC HB−8065)をマウス1匹当たり8×106個で、RPMI1640の溶液中で腹側部皮下へ注射した。腫瘍体積が平均110mm3に達した日に、PPAT−082−03の抗CD147抗体を静脈注射した。投与量は抗CD147抗体をマウス1匹当たり0.2、1、5mg/kgをそれぞれ5個体のマウスに週1回で4回投与した。比較として、HAb-18を1mg/kgと、抗体を含まないPBSを静脈注射した。経過日時と腫瘍体積(平均値)関係を図9に示す。抗CD147抗体のPPAT−082−03を5mg/kgと1mg/kgをそれぞれ投与された群、およびHAb18 1mg/kgを投与された群において、腫瘍体積が早期に減少しており、投与3回目(34日目)時にはほぼ消失しており、その後の長期の経過観察でも再発が認められなかった。抗CD147抗体は抗腫瘍効果があることが確認された。1mg/kg投与のPPAT−082−03とHAb18との抗腫瘍効果は同等程度であった。PPAT−082−03 0.2mg/kg投与群は、PBS投与群よりも腫瘍体積の増加度合は遅延しており、この濃度においてもPPAT−082−03の抗CD147抗体は抗腫瘍効果があることが確認された。Example 12
The dose dependence of the anti-CD147 antibody of the antitumor effect in the HepG2 transplantation model, which is a liver cancer cell line according to Example 12, will be described. 8 weeks old C.I. B. 17 / Icr-scidJc1 mice were injected subcutaneously in the ventral region with 8 × 10 6 HepG2 cells (ATCC HB-8065) per mouse in a solution of RPMI 1640. On the day when the tumor volume reached an average of 110 mm 3 , PPAT-082-03 anti-CD147 antibody was injected intravenously. The doses were 0.2, 1 and 5 mg / kg of anti-CD147 antibody per mouse for 5 mice each once a week for 4 times. As a comparison, HAb-18 was intravenously injected at 1 mg / kg and PBS containing no antibody. The relationship between the elapsed date and time and the tumor volume (average value) is shown in FIG. In the group administered with 5 mg / kg and 1 mg / kg of the anti-CD147 antibody PPAT-082-03, and the group administered with HAb18 1 mg / kg, respectively, the tumor volume decreased early, and the third administration ( On day 34), it almost disappeared, and no recurrence was observed in the subsequent long-term follow-up. It was confirmed that the anti-CD147 antibody has an antitumor effect. The antitumor effect of PPAT-082-03 and HAb18 administered at 1 mg / kg was comparable. The PPAT-082-03 0.2 mg / kg administration group is delayed in the degree of increase in tumor volume compared to the PBS administration group, and the anti-CD147 antibody of PPAT-082-03 has an antitumor effect even at this concentration. Was confirmed.
実施例13
実施例13に係るADCC活性の測定を説明する。
組み換えヒトCD16安定発現KHYG−1の調製
IL−2存在下RPMI1640培地中でナチュラルキラー様細胞株であるKHYG-1(JCRB細胞バンク JCRB0156)にヒトCD16を安定発現した細胞株をエフェクター細胞として培養した。使用直前に細胞を回収し、2.5%FBS/RPMI1640(フェノールレッドなし)培地に懸濁した。Example 13
The measurement of ADCC activity according to Example 13 will be described.
Preparation of recombinant human CD16 stably expressing KHYG-1 A cell line stably expressing human CD16 was cultured as an effector cell in KHYG-1 (JCRB cell bank JCRB0156), a natural killer-like cell line, in RPMI1640 medium in the presence of IL-2. . The cells were collected immediately before use and suspended in 2.5% FBS / RPMI1640 (no phenol red) medium.
マウス骨髄細胞溶液の調製
BALB/cマウスの大腿骨を無菌的に回収し大腿骨の中に存在する骨髄細胞を、25G注射針を用いてRPMI1640培地で採取した。これを40μmフィルターに通した後に遠心(500xG、5分間、20℃)して、細胞を2.0x106細胞/mLにIL−2とGM−CSFを含む10%FBS/RPMI1640培地中に調製し、6日間培養することでeffector cellに分化させた。使用直前にセルクレーパーで細胞を剥がし回収し、2.5%FBS/RPMI1640(フェノールレッドなし)培地に懸濁した。Preparation of mouse bone marrow cell solution The femur of BALB / c mice was aseptically collected, and the bone marrow cells present in the femur were collected in RPMI 1640 medium using a 25G injection needle. This was passed through a 40 μm filter and then centrifuged (500 × G, 5 minutes, 20 ° C.) to prepare cells in 10% FBS / RPMI1640 medium containing IL-2 and GM-CSF at 2.0 × 10 6 cells / mL. The cells were differentiated into effector cells by culturing for 6 days. Immediately before use, the cells were peeled off with a cell scraper and collected, and suspended in 2.5% FBS / RPMI1640 (no phenol red) medium.
標的細胞の調製
培養したHepG2、SW480、PC−3細胞を、トリプシン-EDTA(Life Tech社)を用いてディッシュから剥離し、表5のように96ウェルU字底プレート(Thermo社)の各ウェルに分注し、1日間培養した。測定する翌日に、2.5%FBS/RPMI1640(フェノールレッドなし)培地 150uL/wellで洗浄した後、40uL/wellを加えた。Preparation of target cells Cultured HepG2, SW480 and PC-3 cells were detached from the dish using trypsin-EDTA (Life Tech), and each well of a 96-well U-bottom plate (Thermo) as shown in Table 5 The aliquots were cultured for 1 day. On the next day of measurement, after washing with 2.5% FBS / RPMI1640 (without phenol red) medium 150 uL / well, 40 uL / well was added.
ADCC測定(LDH法)
ADCC活性のkit試薬は、CytoTox96 Non−Radioactive Cytotoxicity Assay(Promega社)を用いて、添付手順書に従って実施した。具体的には、各細胞株にCD147抗体cPPAT−082−01,−02,PPAT−082−03またはHAb18を最終濃度が 0.004、0.016,0.25,1,4ug/mLになるように、それぞれ25uL/wellで加え、その上にヒトCD16安定発現KHYG−1細胞株のeffector cellを表5のように 25uL/wellで加え、5%CO2中、37℃で4時間培養した。
また、マウス移植モデルでの抗腫瘍効果の機序としてのADCC活性を調査するために、表5のように、HepG2細胞に対してマウス骨髄細胞由来のeffector cell 25uL/wellを加え、さらにCD147抗体cPPAT−082−01,−02,PPAT−082−03またはHAb18を最終濃度が0.03、0.3、3ug/mLになるように、それぞれ25uL/wellで加えて同様に培養した。
培養終了予定の30分前に、maximum wellにlysis bufferを添加した。培養4時間後、250xg 4分間の遠心で上清50uLのみ回収し新しいプレートを移し、基質液 50uL/wellを添加して室温 30分間遮光して放置した。ストップ液 50uL/wellを添加し、A492を測定し(A620はレファレンスとして測定)、%細胞毒性を求めた。
%細胞毒性=(A−B−C)/(D−C)x100
Aは抗体とeffector cellの両方を添加したwellのA492値、Bは標的細胞なしで抗体とeffector cellの両方を添加したwellのA492値(effector cellのバックグラウンド)、Cは抗体とeffector cellの両方を添加しなかったwellのA492値(標的細胞のバックグラウンド)、Dは100%LDHの放出を表すmaximum wellのA492値を示す。またA,B,C,DのA492値は、細胞なしの培地のみのバックグランドA492値を差し引いている。試験は二重に行い、ADCC活性(%)について平均値を算出した。
その結果を図10に示す。どのCD147抗体もHepG2やSW480、PC−3に対してADCC活性を示したが、cPPAT−082−01は、他の3つの抗体より活性は低かった。マウス骨髄細胞をエフェクター細胞とした時もすべての抗体はHepG2に対してADCC活性を認めた。このことからマウスHepG2移植モデルでの抗腫瘍効果には、ADCC活性が関与している事が分かった。ADCC measurement (LDH method)
The kit reagent for ADCC activity was carried out using CytoTox96 Non-Radioactive Cytotoxicity Assay (Promega) according to the attached procedure. Specifically, the CD147 antibody cPPAT-082-01, -02, PPAT-082-03 or HAb18 is added to each cell line to a final concentration of 0.004, 0.016, 0.25, 1, 4 ug / mL. As shown in Table 5, an effector cell of KHYG-1 cell line stably expressing human CD16 was added at 25 uL / well as shown in Table 5 and cultured at 37 ° C. in 5% CO 2 for 4 hours.
In addition, in order to investigate ADCC activity as a mechanism of antitumor effect in a mouse transplantation model, as shown in Table 5, mouse bone marrow cell-derived effector cell 25 uL / well was added to HepG2 cells, and further CD147 antibody cPPAT-082-01, -02, PPAT-082-03, or HAb18 was added at 25 uL / well to a final concentration of 0.03, 0.3, 3 ug / mL, and cultured in the same manner.
Lysis buffer was added to the maximum well 30 minutes before the end of the culture. After 4 hours of culture, only 50 uL of the supernatant was recovered by centrifugation at 250 × g for 4 minutes, and a new plate was transferred. Stop solution 50 uL / well was added, A492 was measured (A620 was measured as a reference), and% cytotoxicity was determined.
% Cytotoxicity = (ABC) / (DC) × 100
A is the A492 value of the well to which both the antibody and the effector cell were added, B is the A492 value of the well to which both the antibody and the effector cell were added without the target cell (background of the effector cell), and C was the antibody and the effector cell Well A492 value (target cell background) without both added, D indicates maximum well A492 value representing 100% LDH release. The A492 values of A, B, C, and D are subtracted from the background A492 value of only the medium without cells. The test was performed in duplicate, and an average value was calculated for ADCC activity (%).
The result is shown in FIG. All CD147 antibodies showed ADCC activity against HepG2, SW480, and PC-3, but cPPAT-082-01 was less active than the other three antibodies. All antibodies also showed ADCC activity against HepG2 when mouse bone marrow cells were used as effector cells. From this, it was found that ADCC activity is involved in the antitumor effect in the mouse HepG2 transplantation model.
実施例14
実施例14に係るCDC活性の測定を説明する。
標的細胞の調製
培養したHepG2、肺がんの細胞株であるPC-9(RIKEN BRC RCB4455)、PC−3を、トリプシン-EDTA(Life Tech社)を用いてディッシュから剥離し、表6のように96ウェルU字底プレート(Thermo社)の各ウェルに分注し、1日間培養した。測定する翌日に、2.5%FBS/RPMI1640(フェノールレッドなし)培地 150uL/wellで洗浄した後、50uL/wellを加えた。K562細胞は測定する当日に、表6のcell数でwellに加えて、2.5%FBS/RPMI1640(フェノールレッドなし)培地 150uL/wellで洗浄した後、50uL/wellを加えた。Example 14
The measurement of CDC activity according to Example 14 will be described.
Preparation of target cells Cultured HepG2, lung cancer cell lines PC-9 (RIKEN BRC RCB4455) and PC-3 were detached from the dish using trypsin-EDTA (Life Tech), and 96 as shown in Table 6 The solution was dispensed into each well of a well U-bottom plate (Thermo) and cultured for 1 day. On the next day of measurement, the cells were washed with 2.5% FBS / RPMI1640 (no phenol red) medium 150 uL / well, and 50 uL / well was added. On the day of measurement, K562 cells were washed with 2.5% FBS / RPMI1640 (without phenol red) medium 150 uL / well in addition to the number of cells shown in Table 6 and then added with 50 uL / well.
CDC測定(LDH法)
CDC活性のkit試薬は、ADCC活性と同様にCytoTox96 Non−Radioactive Cytotoxicity Assay(Promega社)を用いて、添付手順書に従って実施した。具体的には、各細胞株にCD147抗体cPPAT−082−01,−02,PPAT−082−03またはHAb18を最終濃度が 0.1、1,10ug/mLになるように、それぞれ25uL/wellで加え、その上に補体(Baby rabbit serum、Cedarlane社)を最終希釈倍率が表6のようになるように調製した溶液を 25uL/wellで加え、5%CO2中、37℃で2時間培養した。
培養終了予定の30分前に、maximum wellにlysis bufferを添加した。培養4時間後、250xg 4分間の遠心で上清50uLのみ回収し新しいプレートを移し、基質液 50uL/wellを添加して室温 30分間遮光して放置した。ストップ液 50uL/wellを添加し、A492を測定し(A620はレファレンスとして測定)、%細胞毒性を実施例115と同じ計算式で求めた。
その結果を図11に示す。CD147抗体cPPAT−082−01は、どの細胞株に対してもCDC活性は認められなかった。HepG2やPC−3に対して、cPPAT−082−02やHAb18Gは弱いCDC活性を、PPAT−082−03はそれ以上のCDC活性を示した。K562とPC−9に対しては、PPAT−082−03のみCDC活性を認めた。以上の事から、PPAT−082−03はHAb18Gや他の抗体よりも優れたCDC活性を有している事が分かった。CDC measurement (LDH method)
The kit reagent for CDC activity was carried out using CytoTox96 Non-Radioactive Cytotoxicity Assay (Promega) in the same manner as ADCC activity according to the attached procedure manual. Specifically, the CD147 antibody cPPAT-082-01, -02, PPAT-082-03 or HAb18 was added to each cell line at a final concentration of 0.1, 1, 10 ug / mL at 25 uL / well, respectively. In addition, a solution prepared by adding complement (Babbit rabbit serum, Cedarlane) so that the final dilution ratio was as shown in Table 6 was added at 25 uL / well and incubated at 37 ° C. in 5% CO 2 for 2 hours. .
Lysis buffer was added to the maximum well 30 minutes before the end of the culture. After 4 hours of culture, only 50 uL of the supernatant was collected by centrifugation at 250 × g for 4 minutes, and a new plate was transferred. The substrate solution was added 50 uL / well, and the mixture was allowed to stand at room temperature for 30 minutes. Stop solution 50 uL / well was added, A492 was measured (A620 was measured as a reference), and% cytotoxicity was determined by the same formula as in Example 115.
The result is shown in FIG. CD147 antibody cPPAT-082-01 showed no CDC activity against any cell line. In contrast to HepG2 and PC-3, cPPAT-082-02 and HAb18G showed weak CDC activity, and PPAT-082-03 showed higher CDC activity. For K562 and PC-9, only PPAT-082-03 showed CDC activity. From the above, it was found that PPAT-082-03 has a CDC activity superior to HAb18G and other antibodies.
実施例15
実施例15に係る抗CD147抗体のPPAT−082−03によるHepG2移植モデルにおける腫瘍の病理所見を説明する。生後8週齢のC.B.17/Icr−scidJc1マウスに、HepG2細胞をマウス1匹当たり8×106個で、RPMI1640の溶液中で腹側部皮下へ注射した。腫瘍体積が平均147mm3に達した日に、PPAT−082−03の抗CD147抗体5mg/kgで静脈注射した。抗体投与前、投与後6、18、30、48、72、96時間後のHepG2の腫瘍を採取して、HE染色を実施した。その結果の一部を図12に示し、まとめを表7に示す。PPAT−082−03の投与によって、分葉状形態のHepG2の腫瘍は増殖が抑えられて、かつアポトーシスや好中球やマクロファージの浸潤が認められた。その後96時間後には腫瘍体積自体の減少が確認された。Example 15
The pathological findings of the tumor in the HepG2 transplantation model by PPAT-082-03 of the anti-CD147 antibody according to Example 15 will be described. 8 weeks old C.I. B. 17 / Icr-scidJc1 mice were injected subcutaneously in the ventral region in RPMI 1640 solution with 8 × 10 6 HepG2 cells per mouse. On the day the tumor volume reached an average of 147 mm 3 , it was intravenously injected with 5 mg / kg of anti-CD147 antibody of PPAT-082-03. HepG2 tumors were collected before antibody administration and 6, 18, 30, 48, 72, and 96 hours after administration, and HE staining was performed. A part of the result is shown in FIG. By administration of PPAT-082-03, the growth of the lobulated HepG2 tumor was suppressed, and apoptosis, neutrophil and macrophage infiltration were observed. Thereafter, a decrease in the tumor volume itself was confirmed 96 hours later.
本発明の抗体は、抗腫瘍剤として利用することができる。 The antibody of the present invention can be used as an antitumor agent.
Claims (18)
a.ヒトCD147発現がん細胞株に対して反応する、
b.カニクイザルCD147発現細胞株に対して反応する、
c.ヒトCD147発現がん細胞に対してin vivoで抗腫瘍活性を示す。A fragment comprising a monoclonal antibody against human CD147 or an antigen-binding region thereof having the following properties a to c:
a. Reacts against a human CD147 expressing cancer cell line,
b. Reacts against a cynomolgus monkey CD147 expressing cell line,
c. It exhibits antitumor activity in vivo against human CD147-expressing cancer cells.
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Title |
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KOCH, CHRISTIAN ET AL.: "T cell activation-associated epitopes of CD147 in regulation of the T cell response, and their defin", INTERNATIONAL IMMUNOLOGY, vol. 11, no. 5, JPN6016042365, 1999, pages 777 - 786, XP055373859, ISSN: 0004408513, DOI: 10.1093/intimm/11.5.777 * |
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