JP2018508227A - 新型抗egfrモノクローナル抗体、その製造方法およびその応用 - Google Patents
新型抗egfrモノクローナル抗体、その製造方法およびその応用 Download PDFInfo
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Abstract
Description
a)新規抗EGFRモノクローナル抗体に、配列番号2で示されるアミノ酸配列を有する軽鎖と、配列番号4で示されるアミノ酸配列を有する重鎖とを含有させる;
b)配列番号1と3で示される核酸断片を用いて組換えプラスミドを構築し、宿主細胞にトランスフェクトし、高発現クローンをスクリーニングする;
c)細胞培養条件を最適化し、大規模培養し、新規抗EGFRモノクローナル抗体を得、分離精製する。
Gatatccttctgacacagtctccagtgatactgtcagtttctccaggggagcgcgtctca 60
Tttagttgtcgggccagtcagagtatcggcacaaacatccattggtaccagcagcggaca 120
Aacggctccccccggttgctcattaagtacgcaagcgagtctatctctgggataccaagt 180
Cgcttctcgggtagtggtagcggaacagattttactctgagtatcaatagcgtcgaatcc 240
Gaagatattgccgattactactgtcagcagaataacaactggccaaccacattcggcgcc 300
Ggtaccaagctggaactcaagcgcacagttgccgcacctagtgtcttcatcttcccacca 360
Tctgacgagcaactaaagagtggcactgcaagtgtcgtatgtctgctgaacaacttttac 420
Ccacgggaggctaaagtgcagtggaaggtagacaacgcccttcagagcggaaattctcag 480
Gaaagcgtcaccgaacaagattccaaggatagcacatactccctgtcctctaccctgaca 540
Ctgtcaaaagctgactacgaaaagcataaagtgtatgcttgcgaggtgactcatcagggg 600
Ctcagctcgcccgtcaccaagtccttcaaccgtggagaatgt (配列番号:1)
RFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPP 120
SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLT 180
LSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (配列番号:2)
RFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKR (配列番号:5;軽鎖の可変領域であり、CDRは黄色を付ける)
Acttgtactgtgagtgggttctcgttgacgaactacggggtgcattgggtgcgccagagt 120
Cccggtaaagggctggagtggttaggcgtgatttggagcggcggtaacactgactataat 180
Acccctttcaccagtcgcttgagtatcaataaggataattcaaagtctcaagtgtttttt 240
Aagatgaactccctacagagcaacgatacggctatctactactgtgcccgcgcccttaca 300
Tactacgactatgagttcgcttattggggccaggggaccttggtcactgtgtctgcagct 360
Tctacaaaagggccatccgtgttcccactggcccccagttccaagagcactagtggtggc 420
Acagcagccctcgggtgcctcgtgaaggattacttcccggagccagtgaccgtcagttgg 480
Aactccggcgctctaacaagcggagtacatacttttccagccgtgctgcagtcttcaggg 540
Ctttacagtctttcctccgttgtgacagtgcccagcagcagcctgggcacccagacttat 600
Atttgtaatgtgaaccataagccttctaatactaaggtggacaagagagttgagccaaag 660
tcctgtgacaaaactcacacatgccccccttgcccagctcctgagttgttgggcggccct 720
tccgtcttcctgtttcccccgaaacctaaggataccctgatgatatctcggacaccagaa 780
gtgacatgcgtcgtggtcgatgtgtcacacgaagaccctgaggtgaaatttaactggtac 840
gtagacggtgtagaagttcacaacgctaagacaaagcctcgggaagagcagtacaactca 900
acctaccgagtagtgtccgtgcttactgttctgcaccaggactggctgaatggaaaggaa 960
tataagtgtaaagtgtccaataaggcactgcctgctccaatcgagaagacgatttctaaa 1020
gccaagggacaaccaagagaacctcaggtgtataccttgcccccatctagagaagagatg 1080
accaaaaaccaggtgtcacttacatgcctcgtgaaaggcttctatccttctgacattgcc 1140
gtcgaatgggagagtaacggacagcccgagaacaactacaagaccacacctccagtgctg 1200
gattcggatggctctttcttcctttatagtaagctcactgtggacaagtcccgatggcag 1260
caggggaacgtgttctcttgcagcgtgatgcacgaggcattgcataatcactacacccag 1320
aagtctctctcattatcccctggcaag (配列番号:3)
TPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAA 120
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG 180
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGP 240
SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS 300
TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM 360
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQ 420
QGNVFSCSVMHEALHNHYTQKSLSLSPGK (配列番号:4)
TPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA (配列番号:9;軽鎖の可変領域であり、CDRは黄色を付ける)
チャイニーズハムスター卵巣細胞発現システムでより高効率の発現が得られるように、ハムスター好適コドンを選択して真核高効率発現ベクターを構築した。ハムスター好適コドンは表1に示す。
MATMVPSSVPCHWLLFLLLLFSGSS (配列番号: 13), ATG GCC ACC ATG GTG CCC TCT TCT GTG CCC TGC CAC TGG CTG CTG TTC CTG CTG CTG CTG TTC TCT GGC TCT TCT (配列番号: 14)。
生物製薬分野において、宿主細胞の選択には、以下のいくつかの重要な面に注目する必要がある:グリコシル化および他の翻訳後修飾のタイプは免疫原性をもたらすことを避けられること;宿主細胞はバイオリアクターでの大規模培養に適し、かつ、動物由来成分フリーの化学成分限定的(chemically defined and animal component free、ACDF)培地中で高密度まで増殖できること;ウイルス安全性;ACDF中で行われるクローニングおよびストレススクリーニングに適すること。
リポソーム法でCHO−CR−GS-/-をコトランスフェクトし、CSスクリーニングシステムでストレススクリーニングし、抗EGFRモノクローナル抗体を高効率で発現する安定な細胞クローンを得た。複数回のトランスフェクションおよびスクリーニングにより、発現量が20pg/cell.dayを超える細胞クローンを得た。
CHO−CR−G5-/-用の汎用基本培地を開発した。それは合成型培地(Chemical Defined、CD)、即ち細胞成長の需要に応じてアミノ酸、ビタミン、無機塩、グルコースおよび微量元素などを一定の比率で組み合わせてなる基本培地である。基本培地はスクリーニングで得られる操作細胞の成長の需要を基本的に満たすことができる。操作細胞から所望の抗体収率をより改善するために、ホルモン、遺伝子改変の組換え成長因子の添加、アミノ酸量の調節を含む基礎培地の最適化を行った。
スクリーニングされた高発現クローンを無血清培地で増幅培養し、上澄を収集し、9000rpm*20min、4℃で遠心し、沈殿した細胞と破片を捨てた;Millipore社の50KD限外濾過カセットで限外濾過濃縮を行った後、さらに9000rpm*30min、4℃で遠心し、細胞破片を除去した;0.45μm濾過膜で濾過し、rProtein A(組換えタンパク質A)アフィニティークロマトグラフィーで予備精製を行った;同所洗浄緩衝液は6M GuClで、カラム用結合緩衝液は20mM PB+150mM NaCl pH7.0であった;カラム体積の3〜5倍量で平衡化した後、カラム体積の3〜5倍量の溶出緩衝液20mM Citric Acid(クエン酸緩衝液) pH3.0で溶出した。平衡化および洗浄の後、カラムを20%EtOH中に保存した。rProtein Aから溶出された目的タンパク質に対して、Hitrap G25(GE Healthcare)で脱塩、緩衝液交換を行い、カラム溶出緩衝液をPBS(20mMPB+150mMNaCl pH7.0)とし、同所洗浄液を0.5M NaOHとした。以上の精製ステップは全て氷上で行われ、精製で得られた抗体を50KD限外濾過遠心チューブ(Merck Millipore)で濃縮し、CMAB009モノクローナル抗体を得た。
CMAB009モノクローナル抗体とセツキシマブ(Erbitux(登録商標)、C225モノクローナル抗体)の糖鎖の比較分析には、LC/MS、MS/MS技術を使用した。
CMAB009 mAb臨床的耐性の初期評価
最初の研究では、合計18名の被験者を、単回静脈内投与の研究において、100mg/m2用量、250mg/m2用量および400mg/m2用量の用量群それぞれに、3、6、6名の被験者を割り当てた。単回投与試験を行った被験者のうち、3名が疾患の進行により撤回した;研究デザインによれば、残りの15名の被験者は複数回投与の組入れ基準により複数回投与群に参加し、3名の追加被験者は複数回投与された(表1)。
CMAB009モノクローナル抗体臨床安全性:ほとんどの有害事象は薬物関連発疹であり、臨床的に有意な新たな毒性は観察されず、73名の被験者の間で重篤な過敏症は観察されなかった。
第2/3相の研究では、転移性結腸直腸癌を有する患者にCMAB009を投与して、CMAB009の有効性および免疫原性を確認した。以下に記載するように、研究の結果は、Erbitux(登録商標)(セツキシマブ)を用いて実施された類似の研究と比較した。驚くべきことに、CMAB009は、Erbitux(登録商標)の既知の効力を超える有効性を有することが判明した。例えば、CMAB009は、患者の全生存期間および疾患進行までの時間を延長させることができる。以下の研究は、Alberto F. Sobrero et al.、Clin Oncol、2008、26:2311−2319に記載されたErbitux(登録商標)(セツキシマブ)研究に相当する。
Claims (36)
- 下記工程を備えることを特徴とする、新規抗EGFRモノクローナル抗体の製造方法。
a)新規抗EGFRモノクローナル抗体に、配列番号2で示されるアミノ酸配列を有する軽鎖と、配列番号4で示されるアミノ酸配列を有する重鎖とを含有させる;
b)a)の核酸断片を用いて組換えプラスミドを構築し、宿主細胞にトランスフェクトし、高発現クローンをスクリーニングする;
c)細胞培養条件を最適化し、大規模培養することで、新規抗EGFRモノクローナル抗体を得、分離精製する。 - 主にハムスターに最適化したコドンに基づいて、新規抗EGFRモノクローナル抗体の軽鎖と重鎖のコード配列をデザインおよび合成することを特徴とする請求項1に記載された方法。
- 宿主細胞は、真核哺乳動物CHO細胞であることを特徴とする請求項1に記載された方法。
- 細胞培養の温度は33℃〜36℃、好ましくは34℃であることを特徴とする請求項1に記載された方法。
- 細胞培養の成長培地のpH値は6.5〜6.9、好ましくはpH6.6であることを特徴とする請求項1に記載された方法。
- 細胞培養の成長培地における浸透圧は290mOsm/kg〜350mOsm/kg、好ましくは340mOsm/kgであることを特徴とする請求項1に記載された方法。
- 細胞培養成長培地は無血清培地であって、無血清条件で前記宿主細胞を培養することを特徴とする請求項4〜6のいずれかの一つに記載された方法。
- 従来の方法で製造された抗体よりも低い免疫原性を有することを特徴とする請求項1の新規抗体。
- 請求項1に記載された抗体と薬学的に許容される担体とを含む組成物。
- 請求項1に記載された新規抗EGFRモノクローナル抗体を含む、上皮成長因子受容体(EGFR)を発現する腫瘍を治療する薬物の製造方法。
- 薬物で上皮成長因子受容体(EGFR)を発現する腫瘍を治療する方法であって、前記薬物は、請求項9に記載された組成物を含むことを特徴とする方法。
- さらに、上皮成長因子受容体(EGFR)を発現する腫瘍を治療する他の薬物と併用することを含む、請求項10または11に記載された方法。
- 水と抗EGFR抗体を含む液体薬物組成物であって、前記抗EGFR抗体は、配列番号2で示されるアミノ酸配列を有する軽鎖と配列番号4で示されるアミノ酸配列を有する重鎖とを含有し、
前記抗体は、動的光散乱(DLS)分析で測定されたz−平均(z−avg)が約10〜25nmであり、かつ
前記抗EGFR抗体は、N−グリコリルノイラミン酸(NGNA)を含有しない、Gal−α(1,3)−Galグリカンを含有しない、および/または、Gal−α(2,3/6)−Galグリカンを含有する
ことを特徴とする組成物。 - 抗体のz−平均は、15〜20nmであることを特徴とする請求項13に記載された組成物。
- がんを有するヒト被験者を治療する方法であって、請求項13または14に記載された組成物を被験者に投与することにより、前記がんを治療することを特徴とする方法。
- ヒト被験者のがんの進行を抑制する方法であって、請求項13または14に記載された組成物を被験者に投与することにより、当該進行を抑制することを特徴とする方法。
- がんは頭頸部扁平上皮がん(SCCHN)または結腸直腸がんであることを特徴とする請求項15または16に記載された方法。
- 結腸直腸がんは、K−Ras野生型、EGFR−発現の結腸直腸がんであることを特徴とする請求項17に記載された方法。
- 抗体は、FOLFIRI(イリノテカン、5−フルオロウラシル、フォリン酸)と併用して投与されることを特徴とする請求項18に記載された方法。
- 抗体は、イリノテカンと併用して投与されることを特徴とする請求項18に記載された方法。
- 被験者は、再発性または転移性頭頸部扁平上皮がんを有し、かつ、先行のプラチナベースの治療に失敗したことを特徴とする請求項15または16に記載された方法。
- 被験者は、局所的または局部的に進行した頭頸部扁平上皮がんを有することを特徴とする請求項15または16に記載された方法。
- 抗体は、放射性治療と併用し、がんの初期治療に用いられることを特徴とする請求項22に記載された方法。
- 被験者は、再発性の局所疾患または転移性頭頸部扁平上皮がんを有することを特徴とする請求項15または16に記載された方法。
- 抗体は、5−FUを用いるプラチナベースの治療と併用されることを特徴とする請求項21に記載された方法。
- 抗体は、追加の治療薬と併用されることを特徴とする請求項15または16に記載された方法。
- 追加の治療薬は、化学療法剤であることを特徴とする請求項26に記載された方法。
- 被験者は、オキサリプラチンおよびイリノテカンベースの化学療法に失敗したことを特徴とする請求項15または16に記載された方法。
- 被験者は、イリノテカンに対して不耐症であることを特徴とする請求項15または16に記載された方法。
- 結腸直腸がんを有する被験者の結腸直腸がんの進行を治療または抑制する方法であって、
前記方法は、抗EGFR抗体とイリノテカンを投与することにより、結腸直腸がんを治療することを含み、前記抗体は、配列番号2で示されるアミノ酸配列を有する軽鎖と配列番号4で示されるアミノ酸配列を有する重鎖とを含有し、かつ、
Gal−α(2,3/6)−Galグリカンを含有することを特徴とする方法。 - 結腸直腸がんを有する被験者の結腸直腸がんの進行を治療または抑制する方法であって、
前記方法は、抗EGFR抗体とイリノテカンを投与することにより、結腸直腸がんを治療することを含み、前記抗体は、配列番号2で示されるアミノ酸配列を有する軽鎖と配列番号4で示されるアミノ酸配列を有する重鎖とを含有し、かつ、
N−グリコリルノイラミン酸(NGNA)グリカンおよびGal−α(1,3)−Galグリカンを含有しない
ことを特徴とする方法。 - 前記結腸直腸がんは、進行した結腸直腸がんであることを特徴とする請求項31または32に記載された方法。
- 前記抗体は、400mg/m2の初期投与量、続いて250mg/m2の週投与量で被験者に点滴によって投与されることを特徴とする請求項31〜33のいずれかの一つに記載された方法。
- 前記抗体は、チャイニーズハムスターの卵巣(CHO)細胞において製造されることを特徴とする請求項31〜34のいずれかの一つに記載された方法。
- 水と抗EGFR抗体とを含む液体薬物組成物であって、
前記抗EGFR抗体は、配列番号2で示されるアミノ酸配列を有する軽鎖と配列番号4で示されるアミノ酸配列を有する重鎖とを含有し、
前記抗EGFR抗体は、チャイニーズハムスターの卵巣(CHO)細胞において製造され、
前記組成物は、ポリソルベートおよび/またはサッカロースを含有しない
ことを特徴とする組成物。 - 基本的に、水、抗EGFR抗体、塩化ナトリウム、リン酸二水素ナトリウム二水和物、および、リン酸二ナトリウム二水和物を含む液体医薬組成物であって、
前記抗EGFR抗体は、配列番号2で示されるアミノ酸配列を有する軽鎖と配列番号4で示されるアミノ酸配列を有する重鎖とを含有し、
前記抗EGFR抗体は、N−グリコリルノイラミン酸(NGNA)グリカンを含有しない、Gal−α(1,3)−Galグリカンを含有しない、および/または、Gal−α(2,3/6)−Galグリカンを含有する
ことを特徴とする組成物。
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