TW201638108A - 抗c-Met抗體和抗c-Met抗體-細胞毒性藥物偶聯物及其醫藥用途 - Google Patents
抗c-Met抗體和抗c-Met抗體-細胞毒性藥物偶聯物及其醫藥用途 Download PDFInfo
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
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- A61K31/4738—Quinolines; Isoquinolines ortho- or peri-condensed with heterocyclic ring systems
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- A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
- A61K31/537—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines spiro-condensed or forming part of bridged ring systems
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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Abstract
本發明涉及抗c-Met抗體和抗c-Met抗體-細胞毒性藥物偶聯物及其醫藥用途。具體地,本發明涉及一種抗c-Met抗體,c-Met的抗原結合片段,包含該抗c-Met抗體CDR區的嵌合抗體、人源化抗體,及其抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物,以及包含人源化抗c-Met抗體和抗原結合片段及其抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物的醫藥組成物,以及其作為抗癌藥物的用途,尤其在製備用於治療c-Met介導的疾病或病症的藥物中的用途。
Description
本發明公開了一種抗c-Met抗體或其抗原結合片段,包含所述抗c-Met抗體CDR區的嵌合抗體、人源化抗體,及抗c-Met抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物,以及包含人c-Met抗體或其抗原結合片段及其抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物的醫藥組成物,以及其作為抗癌藥物的用途。尤其涉及一種人源化的抗c-Met抗體及抗c-Met抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物,在製備用於治療c-Met介導的疾病或病症的藥物中的用途。
近年來分子生物學和腫瘤藥理學研究表明,酪胺酸激酶(Protein Tyrosine Kinases,PTKs)相關的細胞信號轉導通路在腫瘤的形成和發展中發揮了極其重要的作用,超過50%的原癌基因和癌基因產物都具有酪胺酸激酶活性。c-Met原癌基因屬於PTKs家族中Ron亞族,其編碼的c-Met
蛋白是肝細胞生長因子/離散因子(Hepatocyte Growth Factor/Scatter Factor,HGF/SF)的高親和性受體。HGF/c-Met信號通路與血管新生和腫瘤生長過程密切相關,其持續啟動是組織細胞癌變或癌細胞增殖亢進的重要原因,抑制該通路已成為腫瘤靶向治療的新手段。
c-Met原癌基因位於人類第7號染色體長臂(7q31),大小超過120kb,編碼分子量約150kD的c-Met蛋白前體,經局部糖基化生成一個170kD的糖蛋白,該糖蛋白進一步剪切為α亞基(50kDa)和β亞基(140kDa),以二硫鍵相連,形成成熟的c-Met蛋白受體。該異二聚體包含兩條鏈,β鏈有胞外區、跨膜區(也稱膜伸展片段)和胞內區(包含細胞內酪胺酸激酶結合位點)。α鏈只有胞外部分,但它是高度糖基化,藉由二硫鍵附著於β鏈上。兩個亞基的胞外區域是相應配體的識別部位,胞內區域具有酪胺酸激酶活性。
c-Met啟動的機制分為三種:一是依賴HGF的啟動機制,二是不依賴HGF啟動機制,三是經過其他膜途徑,例如藉由透明質酸膜表面受體的CD44、黏附素以及RON信號傳導途徑等等。其中最常見的是依賴HGF的啟動機制。HGF的N末端與c-Met結合,促進β鏈上Tyr1234和Tyr1235二聚化和自磷酸化,C-末端附近的Tyr1349和Tyr1356磷酸化產生多個接頭蛋白的結合位點,這些接頭蛋白誘導了P13K/Akt、Ras/Mapk、c-Src和STAT3/5介導的下游信號的啟動,引發不同細胞反應,如細胞生存和活動(與P13K/Akt通路密切相關),腫瘤轉移和細胞增殖(主要由Ras/Mapk介
導)。此外,c-Met與其它膜受體存在交聯(cross-talk),現已確知這種交聯可促進腫瘤形成及轉移,由於c-Met是導致腫瘤形成及轉移的許多通路的交叉點,以c-Met為靶標可相對較容易地實現對許多通路的同時干擾,c-Met成為抗腫瘤生成和轉移治療的一個有希望的靶點。
抗體藥物偶聯物(antibody drug conjugate,ADC)把單克隆抗體或者抗體片段藉由穩定的化學接頭化合物與具有生物活性的細胞毒素相連,充分利用了抗體對腫瘤細胞特異或高表現抗原結合的特異性和細胞毒素的高效性,避免對正常細胞的毒副作用。這也就意味著,與以往傳統的化療藥物相比,抗體藥物偶聯物能精准地結合腫瘤細胞並降低將對正常細胞的影響。
ADC藥物由抗體(靶向),接頭和毒素三部分組成。其中,好的靶點(抗體部分)決定了ADC藥物的特異性,這不僅包括特異靶向結合,還包括有效的內吞。
目前針對c-Met激酶靶點抑制劑主要有三類:HGF和c-Met生物拮抗劑、HGF和c-Met抗體,以及小分子c-Met抑制劑。現有的臨床結果表明,直接針對HGF,c-Met的抗體,或c-Met小分子抑制療效不甚理想。針對c-Met的ADC藥物可能是該靶點最有效的方法治療腫瘤。目前,尚沒有c-Met ADC藥物臨床研究。
本發明首創抗c-Met抗體ADC藥物,不僅保留了本發明抗c-Met抗體的抗體依賴性的細胞增殖抑制作用,同時又增加了潛在的細胞毒素藥物的效應。而且因為其毒素靶
向在腫瘤細胞內釋放,藥物毒副作用並沒有隨著療效的增加而同步增大。本發明提供特異性結合人c-Met的人源化抗體和嵌合抗體,並且該人源化抗體和嵌合抗體特徵在於具有高親和力,高藥效,有內吞功效,穩定性好和無c-Met激動活性等特點。在這些優良性能基礎上,本發明同時還提供特異性結合人c-Met的抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物,不僅保留了本發明抗c-Met抗體的抗體依賴性的細胞增殖抑制作用,同時又增加了潛在的細胞毒素藥物的效應和治療疾病的廣譜性。而且因為其毒素靶向在腫瘤細胞內釋放(本發明抗c-Met抗體的內吞作用),藥物毒副作用並沒有隨著療效的增加而同步增大。
本發明提供一種特異性結合c-Met受體的抗體或其抗原結合片段,其包含至少1個選自以下CDR區的序列或其突變序列:抗體重鏈可變區HCDR區序列:SEQ ID NO:6,SEQ ID NO:7或SEQ ID NO:8;和抗體輕鏈可變區LCDR區序列:SEQ ID NO:9,SEQ ID NO:10或SEQ ID NO:11。
在本發明一個較佳的實施方案中,提供一種如上所述的c-Met抗體或其抗原結合片段,其中該抗體重鏈可變區包含至少1個選自如下的HCDR區序列或其突變序列:SEQ ID NO:6,SEQ ID NO:7或SEQ ID NO:8。
在本發明一個較佳的實施方案中,提供一種如上所述
的c-Met抗體或其抗原結合片段,其中該抗體輕鏈可變區包含至少1個選自如下的LCDR區序列或其突變序列:SEQ ID NO:9,SEQ ID NO:10或SEQ ID NO:11。
在本發明一個較佳的實施方案中,提供一種如上所述的c-Met抗體或其抗原結合片段,其中該抗體包含重鏈可變區序列SEQ ID NO:6(HCDR1),SEQ ID NO:7(HCDR2)和SEQ ID NO:8(HCDR3),或其突變序列,和輕鏈可變區序列SEQ ID NO:9(LCDR1),SEQ ID NO:10(LCDR2)和SEQ ID NO:11(LCDR3)或其突變序列。
在本發明一個較佳的實施方案中,提供一種如上所述的c-Met抗體或其抗原結合片段,其中該CDR區突變序列為CDR區發生1至3個優化抗體活性的胺基酸突變,其中該HCDR2區突變序列較佳為SEQ ID NO:12。
在本發明一個較佳的實施方案中,提供一種如上所述的c-Met抗體或其抗原結合片段,其中該c-Met抗體或其抗原結合片段為鼠源抗體或其片段。
在本發明一個較佳的實施方案中,提供一種如上所述的c-Met抗體或其抗原結合片段,其中該鼠源抗體重鏈可變區序列為:SEQ ID NO:4。
在本發明一個較佳的實施方案中,提供一種如上所述的c-Met抗體或其抗原結合片段,其中該鼠源抗體輕鏈可變區序列為:SEQ ID NO:5。
在本發明一個較佳的實施方案中,提供一種如上所述的c-Met抗體或其抗原結合片段,其中該鼠源抗體的-重鏈
可變區為:SEQ ID NO:4,輕鏈可變區為:SEQ ID NO:5。
在本發明一個較佳的實施方案中,提供一種如上所述的鼠源抗體或其片段,其抗體重鏈可變區進一步包含源自鼠源的IgG1、IgG2、IgG3或IgG4或其變體的重鏈FR區。
在本發明一個較佳的實施方案中,提供一種如上所述的鼠源抗體或其片段,其進一步包含源自鼠源IgG1、IgG2、IgG3或IgG4、或其變體的重鏈恆定區。
在本發明一個較佳的實施方案中,提供一種如上所述的鼠源抗體或其片段,其抗體輕鏈可變區進一步包含選自鼠源κ或λ鏈、或其變體的輕鏈FR區。
在本發明一個較佳的實施方案中,提供一種如上所述的鼠源抗體或其片段,其進一步包含選自鼠源κ或λ鏈、或其變體的輕鏈恆定區。
在本發明一個較佳的實施方案中,提供一種如上所述的c-Met抗體或其抗原結合片段,其為嵌合抗體或人源化抗體或其片段。
在本發明一個較佳的實施方案中,提供一種如上所述的c-Met抗體或其抗原結合片段,其中該人源化抗體重鏈可變區進一步包含人源IgG1、IgG2、IgG3、或IgG4或其變體的重鏈FR區。
在本發明一個較佳的實施方案中,提供一種如上所述的c-Met抗體或其抗原結合片段,其中該人源化抗體重鏈可變區上的重鏈FR區序列,來源於人種系重鏈序列,較佳人種系重鏈IGHV 3-33*01;包含人種系重鏈IGHV
3-33*01的FR1,FR2,FR3區和FR4區的框架序列或其突變序列,較佳該突變序列為在前述框架序列上發生0至10個胺基酸的回復突變。
在本發明一個較佳的實施方案中,提供一種如上所述的c-Met抗體或其抗原結合片段,其中該人源化抗體包含SEQ ID NO:13至15所示的重鏈可變區序列或其變體。
在本發明一個較佳的實施方案中,提供一種如上所述的c-Met抗體或其抗原結合片段,其中該人源化抗體輕鏈可變區上的輕鏈FR區序列,選自人種系輕鏈序列,較佳人種系輕鏈IGKV085或IGKV 4-1*01,包含人種系輕鏈IGKV085和IGKV 4-1*01的FR1,FR2,FR3區和FR4區的框架序列或其突變序列,較佳該突變序列為在前述框架序列上發生0至10個胺基酸的回復突變。
在本發明一個較佳的實施方案中,提供一種如上所述的c-Met抗體或其抗原結合片段,其中該人源化抗體包含選自SEQ ID NO:16至18所示的輕鏈可變區序列或其變體。
在本發明一個較佳的實施方案中,提供一種如上所述的c-Met抗體或其抗原結合片段,該人源化抗體包含選自SEQ ID NO:13至15的重鏈可變區和選自SEQ ID NO:16至18的輕鏈可變區。
在本發明一個較佳的實施方案中,提供一種如上所述的c-Met抗體或其抗原結合片段,其包含選自(a)至(c)任一的重鏈可變區序列和輕鏈可變區序列的组合:
(a)SEQ ID NO:13的重鏈可變區序列和SEQ ID NO:16的輕鏈可變區序列;(b)SEQ ID NO:14的重鏈可變區序列和SEQ ID NO:17的輕鏈可變區序列;或(c)SEQ ID NO:15的重鏈可變區序列和SEQ ID NO:18的輕鏈可變區序列。
在本發明一個較佳的實施方案中,提供一種如上所述的c-Met抗體或其抗原結合片段,其中該人源化抗體的重鏈恆定區包含源自人源IgG1或其變體、人源IgG2或其變體、人源IgG3或其變體或人源IgG4或其變體的恆定區,較佳包含人源IgG1或其變體或人源IgG2或其變體或人源IgG4或其變體的恆定區,更佳包含人源IgG2或其變體的恆定區。
在本發明一個較佳的實施方案中,提供一種如上所述的c-Met抗體或其抗原結合片段,其包含選自SEQ ID NO:23-25或與其具有至少90%同源性的全長重鏈序列。
在本發明一個較佳的實施方案中,提供一種如上所述的c-Met抗體或其抗原結合片段,其中該人源化抗體輕鏈可變區進一步包含任選自人源κ或λ鏈或其變體的輕鏈FR區。
在本發明一個較佳的實施方案中,提供一種如上所述的c-Met抗體或其抗原結合片段,其中該人源化抗體的輕鏈恆定區包含選自人源κ或λ鏈或其變體的恆定區。
在本發明一個較佳的實施方案中,提供一種如上所述
的c-Met抗體或其抗原結合片段,其包含選自SEQ ID NO:26至28或與其具有至少90%序列同源性的全長輕鏈序列。
在本發明一個較佳的實施方案中,提供一種如上所述的c-Met抗體或其抗原結合片段,其中該人源化抗體包含選自SEQ ID NO:23至25的全長重鏈序列和SEQ ID NO:26至28的全長輕鏈序列。
在本發明一個較佳的實施方案中,提供一種如上所述的c-Met抗體或其抗原結合片段,其中該人源化抗體選自(a)至(c)任一的全長輕鏈序列和全長重鏈序列的组合:Ab-9:SEQ ID NO:23的重鏈序列和SEQ ID NO:26的輕鏈序列;Ab-10:SEQ ID NO:24的重鏈序列和SEQ ID NO:27的輕鏈序列;或Ab-11:SEQ ID NO:25的重鏈序列和SEQ ID NO:28的輕鏈序列。
在本發明一個較佳的實施方案中,提供一種如上所述的c-Met抗體或其抗原結合片段,其中該抗原結合片段包含Fab、Fv、sFv或F(ab’)2。
本發明進一步提供一種編碼如上所述的c-Met抗體或其抗原結合片段的表現前體產物的DNA分子。本發明進一步提供一種含有如上所述的DNA分子的表現載體。
本發明進一步提供一種用如上所述的表現載體轉化的宿主細胞。
在本發明一個較佳的實施方案中,提供一種如上所述的宿主細胞,其中該宿主細胞較佳為哺乳動物細胞,更佳為CHO細胞。
本發明進一步提供一種藥物组合物,其包含含有如上所述的c-Met抗體或其抗原結合片段,和一種或多種可藥用的賦形劑、稀釋劑或載體。
本發明進一步提供一種如上所述的c-Met抗體或其抗原結合片段、或包含其的醫藥組成物,在製備用於治療c-Met介導的疾病或病症的藥物中的用途,其中該疾病或病症較佳為癌症;更佳為表現c-Met的癌症;最佳為胃癌、胰腺癌、肺癌、腸癌、腎癌、黑色素瘤、非小細胞肺癌;最佳為胃癌和非小細胞肺癌。
本發明進一步提供一種治療和預防c-Met介導的疾病或病症的方法,該方法包括给予所需患者治療有效量的如上所述的c-Met抗體或其抗原結合片段、或包含其的藥物组合物;其中該疾病或病症較佳為癌症;更佳為表現c-Met的癌症;最佳為胃癌、胰腺癌、肺癌、腸癌、腎癌、黑色素瘤、非小細胞肺癌;最佳為胃癌和非小細胞肺癌。
本發明進一步提供一種通式(I)所示的抗體-細胞毒性藥物偶聯物或其藥學上可接受的鹽或溶劑化合物:Ab-[(L2)t-L1-D)]y (I)
其中:D為藥物模組;L1,L2是接頭單元;
t為0或1,較佳為1;y為1至8,較佳為2至5;y可以為小數;Ab為如上所述的特異性結合c-Met受體的抗體或其抗原結合片段。
在本發明一個較佳的實施方案中,提供一種通式(I)所示的抗體-細胞毒性藥物偶聯物或其藥學上可接受的鹽或溶劑化合物,其中-L2-為以下通式(-L2-)所示的化合物:
其中X1選自自氫原子、鹵素、羥基、氰基、烷基、烷氧基和環烷基;X2選自-烷基-、-環烷基-和-雜環基-;m為0至5,較佳1至3;S為硫原子。
在本發明一個較佳的實施方案中,提供一種通式(I)所示的抗體-細胞毒性藥物偶聯物或其藥學上可接受的鹽或溶劑化合物,其中該藥物模組D為選自毒素、化療劑、抗生素、放射性同位素和核溶酶的細胞毒劑。
在本發明一個較佳的實施方案中,提供一種通式(I)所示的抗體-細胞毒性藥物偶聯物或其藥學上可接受的鹽或溶劑化合物,其中D為以下通式(D)所示的化合物:
或其互變異構體、內消旋體、外消旋體、對映異構體、非對映異構體、或其混合物形式,或其可藥用的鹽,其中:R1-R7選自氫原子、鹵素、羥基、氰基、烷基、烷氧基和環烷基;R8-R11任選自氫原子、鹵素、烯基、烷基、烷氧基和環烷基;R8-R11較佳至少其中一個選自鹵素、烯基、烷基和環烷基,其餘為氫原子;或者R8-R11之中的任意兩個形成環烷基,餘下的兩個基團任選自氫原子、烷基和環烷基;R12-R13選自氫原子、烷基或鹵素;R14選自芳基或雜芳基,該芳基或雜芳基任選進一步被選自氫原子、鹵素、羥基、烷基、烷氧基和環烷基的取代基所取代;R15任選自鹵素、烯基、烷基、環烷基和COOR17;R16選自氫原子、鹵素、羥基、氰基、烷基、烷氧基和環烷基;R17選自氫原子、烷基和烷氧基。
在本發明一個較佳的實施方案中,提供一種通式(I)所示的抗體-細胞毒性藥物偶聯物或其藥學上可接受的鹽或溶劑化合物,其中L2包含選自Val-Cit,MC,PAB和MC-PAB
的接頭,較佳為MC。
在本發明一個較佳的實施方案中,提供一種通式(I)所示的抗體-細胞毒性藥物偶聯物或其藥學上可接受的鹽或溶劑化合物,其中D是美登木素生物鹼;較佳自DM1、DM3和DM4,更較佳為DM1。
在本發明一個較佳的實施方案中,提供一種通式(I)所示的抗體-細胞毒性藥物偶聯物或其藥學上可接受的鹽或溶劑化合物,其中該L2選自N-琥珀醯亞胺基4-(2-吡啶基硫代)戊酸酯(SPP)、N-琥珀醯亞胺基4-(N-馬來醯亞胺基甲基)-環己烷-1-羧酸酯(SMCC)、和N-琥珀醯亞胺基(4-碘-乙醯基)胺基苯甲酸酯(SIAB);較佳為SPP或SMCC。
在本發明一個較佳的實施方案中,提供一種通式(I)所示的抗體-細胞毒性藥物偶聯物或其藥學上可接受的鹽或溶劑化合物,其中D是喜樹鹼類生物鹼;較佳自CPT、10-羥基-CPT、CPT-11(伊立替康)、SN-38和托泊替康,更較佳SN-38。
在本發明一個較佳的實施方案中,提供一種通式(I)所示的抗體-細胞毒性藥物偶聯物或其藥學上可接受的鹽或溶劑化合物,其中該接頭L2包含任選自Val-Cit,MC,PAB或MC-PAB的結構,較佳為MC或MC-vc-PAB。
在本發明一個較佳的實施方案中,提供一種通式(I)所示的抗體-細胞毒性藥物偶聯物或其藥學上可接受的鹽或溶劑化合物,其包括通式(II)所示的偶聯藥物或其藥學上可接受的鹽或溶劑化合物:
其中:R2-R16如通式(D)中所定義;Ab,t,y,L1,L2如通式(I)中所定義。
在本發明一個較佳的實施方案中,提供一種通式(I)所示的抗體-細胞毒性藥物偶聯物或其藥學上可接受的鹽或溶劑化合物,其包括通式(UI)所示的偶聯藥物或其藥學上可接受的鹽或溶劑化合物:
其中:R2-R16如通式(D)中所定義;Ab,t,y,L1,L2如通式(I)中所定義;n為3至6,較佳為5。
在本發明一個較佳的實施方案中,提供一種通式(I)所示的抗體-細胞毒性藥物偶聯物或其藥學上可接受的鹽或溶劑化合物,其包括通式(IV)所示的偶聯藥物或其藥學上可接受的鹽或溶劑化合物:
其中:R2-R16如通式(D)中所定義;Ab,y如通式(I)中所定義;n如通式(III)中所定義;X1,X2,m如通式L2中所定義。
在本發明一個較佳的實施方案中,提供一種通式(I)所示的抗體-細胞毒性藥物偶聯物或其藥學上可接受的鹽或溶劑化合物,其包括通式(V)所示的偶聯藥物或其藥學上可接受的鹽或溶劑化合物:
其中:Ab,D,y如通式(I)中所定義;n如通式(III)中所定義;X1,X2,m如通式L2中所定義。
本發明所述的抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物包括但不限於:
本發明進一步提供一種製備通式(V)所示的抗體-細胞毒性藥物偶聯物的方法,該方法包括:
通式(Ab-L2)化合物與通式(L1-D)化合物在有機溶劑中反應,得到通式(V)化合物;該有機溶劑較佳為乙腈或乙醇;其中:Ab如前所述的特異性結合c-Met受體的抗體或其抗原結合片段;X1選自自氫原子、鹵素、羥基、氰基、烷基、烷氧基和環烷基;X2選自烷基、環烷基和雜環基;X為0至5,較佳為1至3;m為0至5,較佳為1至3。
在本發明一個較佳的實施方案中,提供一種如上所述的抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物,其具有體外或體內細胞殺傷活性。
本發明進一步提供一種醫藥組成物,其含有如上所述
的c-Met抗體或其抗原結合片段、或抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物和可藥用的賦形劑、稀釋劑或載體。
本發明進一步提供一種如上所述的c-Met抗體或其抗原結合片段、或抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物、或包含其的醫藥組成物,在製備用於治療c-Met介導的疾病或病症的藥物中的用途,其中該疾病或病症較佳為癌症;更佳為表現c-Met的癌症;最佳為胃癌、胰腺癌、肺癌、腸癌、腎癌、黑色素瘤、非小細胞肺癌;最佳為胃癌、胰腺癌、非小細胞肺癌和腎癌。
本發明進一步提供一種治療和預防c-Met介導的疾病或病症的方法,該方法包括給予所需患者治療有效量的如上所述的c-Met抗體或其抗原結合片段、或抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物、或包含其的醫藥組成物;其中該疾病或病症較佳為癌症;更佳為表現c-Met的癌症;最佳為胃癌、胰腺癌、肺癌、腸癌、腎癌、黑色素瘤、非小細胞肺癌;最佳為胃癌、胰腺癌、非小細胞肺癌和腎癌。
發明詳述
一、術語
為了更容易理解本發明,以下具體定義了某些技術和科學術語。除顯而易見在本檔中的它處另有明確定義,否則本文使用的所有其它技術和科學術語都具有本發明所屬領域的一般技術人員通常理解的含義。
本發明所用胺基酸三字母代碼和單字母代碼如J.biol.chem,243,p3558(1968)中所述。
術語“c-Met”或“c-Met多肽”或“c-Met受體”是指結合細胞生長因子(HGF)的受體酪胺酸激酶。本發明中如非特指,比如鼠c-Met(m-c-Met)或猴c-Met(cyno-c-Met),均指人的c-Met(h-c-Met)。本發明中所用的人、鼠、食蟹猴c-Met均藉由GenBank提供的核苷酸序列或多肽序列進行編碼,例如GenBank登錄號NM_000245中提供的核苷酸序列編碼的人多肽,或由GenBank登錄號NP_000236中提供的多肽序列編碼的人蛋白質或其細胞外結構域。原始的單鏈前體蛋白質在轉譯後被剪切以產生α和β亞基,其藉由二硫鍵連接以形成成熟受體。受體酪胺酸激酶c-Met參與細胞過程包括,例如伴隨胚胎發生的組織再生的遷移、侵入和形態發生的過程。
術語“c-Met相關病症或狀況”指任何源自c-Met的不利表現或缺乏表現,不利調控或缺乏調控,或有害活性或缺乏活性,或可以藉由調節c-Met表現或活性來進行調節、治療或治癒的疾病、病症或狀況。例如在大部分癌症
患者中,或在其疾病確實由與c-Met途徑相關的變化驅動的患者中,可以預期HGF/c-Met途徑的啟動。例如上調歸因於不同的機制,像HGF和/或c-Met的過表現,或藉由c-Met突變的組成型啟動。c-Met相關病症或狀況包括但不限於,例如增生性疾病和紊亂和炎性疾病和紊亂。增生性疾病包括但不限於,例如癌症,其包括例如,胃癌、食道癌、腎癌包括乳突狀腎細胞癌、肺癌、神經膠質瘤、頭頸癌、上皮癌、皮膚癌、白血病、淋巴癌,骨髓瘤、腦癌、胰腺癌,結直腸癌、胃腸癌、腸癌、生殖器癌症、泌尿器癌症、黑色素瘤、前列腺癌以及本領域技術人員已知的其它腫瘤。炎性疾病包括但不限於,例如細菌感染,包括李斯特菌屬細菌引起的感染。
本發明所述的抗體指免疫球蛋白,是由兩條相同的重鏈和兩條相同的輕鏈藉由鏈間二硫鍵連接而成的四肽鏈結構。免疫球蛋白重鏈恆定區的胺基酸組成和排列順序不同,故其抗原性也不同。據此,可將免疫球蛋白分為五類,或稱為免疫球蛋白的同種型,即IgM,IgD,IgG,IgA和IgE,其相應的重鏈分別為μ鏈,δ鏈,γ鏈,α鏈,ε鏈。同一類Ig根據其鉸鏈區胺基酸組成和重鏈二硫鍵的數目和位置的差別,又可分為不同的亞類,如IgG可分為IgG1,IgG2,IgG3,IgG4。輕鏈藉由恆定區的不同分為κ鏈或λ鏈。五類Ig中第每類Ig都可以有κ鏈或λ鏈。
抗體重鏈和輕鏈靠近N端的約110個胺基酸的序列變化很大,為可變區(V區);靠近C端的其餘胺基酸序列相
對穩定,為恆定區(C區)。可變區包括3個高變區(HVR)和4個序列相對保守的骨架區(FR)。3個高變區決定抗體的特異性,又稱為互補性決定區(CDR)。每條輕鏈可變區(LCVR)和重鏈可變區(HCVR)由3個CDR區4個FR區組成,從胺基端到羧基端依次排列的順序為:FR1,CDR1,FR2,CDR2,FR3,CDR3,FR4。輕鏈的3個CDR區指LCDR1,LCDR2,和LCDR3;重鏈的3個CDR區指HCDR1,HCDR2和HCDR3。發明所述的抗體或抗原結合片段的LCVR區和HCVR區的CDR胺基酸殘基在數量和位置符合已知的Kabat編號規則(LCDR1-3,HCDE2-3),或者符合kabat和chothia的編號規則(HCDR1)。
術語“鼠源抗體”在本發明中為根據本领域知识和技能用小鼠製備的抗人c-Met的單克隆抗體。製備時用c-Met抗原注射試驗對象,然後分離表現具有所需序列或功能特性的抗體的雜交瘤。在本發明一個較佳的實施方案中,該鼠源c-Met抗體或其抗原結合片段,可進一步包含鼠源κ、λ鏈或其變體的輕鏈恆定區,或進一步包含鼠源IgG1,IgG2,IgG3或IgG4或其變體的重鏈恆定區。
術語“嵌合抗體(chimeric antibody)”,是將鼠源性抗體的可變區與人抗體的恆定區融合而成的抗體,可以減輕鼠源性抗體誘發的免疫應答反應。建立嵌合抗體,要先建立分泌鼠源性特異性單抗的雜交瘤,然後從小鼠雜交瘤細胞中克隆可變區基因,再克隆到人抗體的恆定區基因,進行重组表現。
術語“人源化抗體(humanized antibody)”,也稱為CDR移植抗體(CDR-grafted antibody)人源化,是指將小鼠的CDR序列移植到人的抗體可變區框架,即不同類型的人種系抗體構架序列中產生的抗體。可以克服嵌合抗體由於攜带大量小鼠蛋白成分,從而誘導的强烈的抗體可變抗體反應。此類構架序列可以從包括種系抗體基因序列的公共DNA數據庫或公開的参考文獻獲得。如人重鏈和輕鏈可變區基因的種系DNA序列可以在“VBase”人種系序列数據庫(在因特網www.mrccpe.com.ac.uk/vbase可獲得),以及在Kabat,E.A.等人,1991 Sequences of Proteins of Immunological Interest,第5版中找到。在本發明一個較佳的實施方案中,該c-Met人源化抗體小鼠的CDR序列選自SEQ ID NO:6,7,8,9,10,11(please check the #s,in case just copy from sost draft)。人的抗體可變區框架經過設計選擇,其中該抗體輕鏈可變區上的輕鏈FR區序列,選自人種系輕鏈序列,較佳人種系輕鏈IGKV085或IGKV 4-1*01,包含人種系輕鏈IGKV085和IGKV 4-1*01的FR1,FR2,FR3區和FR4區;其中該抗體重鏈可變區上的重鏈FR區序列,來源於人種系重鏈序列,較佳人種系重鏈IGHV 3-33*01;包含人種系重鏈IGHV 3-33*01的FR1,FR2,FR3區和FR4區。為避免免疫原性下降的同时,引起的活性下降,可對該人抗體可變區可進行最少反向突變,以保持活性。
本領域中有可獲得的多種方法來產生人源化抗體。例如,可藉由獲得抗c-Met特異抗體(例如,鼠類抗體或由
雜交瘤產生的抗體)HCVR和LCVR序列,將其移植到所選的人抗體框架編碼序列上來產生人源化抗體。任選地,可藉由隨機誘變或在特定位置誘變來優化CDR區,以在將CDR區移植到框架區中之前用不同的胺基酸置換CDR中的一個或更多個胺基酸。或者,可使用本領域技術人員可獲得的方法在將CDR區插入人框架區後對其進行優化。較佳地,“人源化抗體”具有起源於或來自親代抗體(即非人抗體,較佳為小鼠單株抗體)的CDR,而在其存在的程度上框架區和恆定區(或其主要部分或基本部分,即至少約90%、92%、94%、95%、96%、97%、98%或99%)序列同人類種系免疫球蛋白區(參見,例如the,International,ImMunoGeneTics,Database)或其重組或突變形式中,無論該抗體是否在人類細胞中產生。較佳地,從人源化抗體來源的非人親代抗體的CDR優化人源化抗體的至少2、3、4、5或6個CDR,以產生期望的性質,例如改善的特異性、親和力或中和作用,其可以藉由篩選測定,例如ELISA測定來進行鑒定。較佳地,本發明抗體中優化的CDR當與親本抗體中存在的CDR相比時,包含至少一個胺基酸置換。與親本抗體的CDRs相比,本發明人源化抗體的CDR中某些胺基酸置換(參見本文的實施例6)降低了抗體不穩定性的可能性(例如除去CDR中Asn殘基)或當對人受試者施用時降低了抗體的免疫原性(例如,由IMMUNOFILTERTM,Technology預測的)。
在CDR編碼序列移植到所選人框架編碼序列上之
後,然後表現編碼人源化可變重鏈和可變輕鏈序列的所得DNA序列,以產生結合c-Met的人源化抗體。可將人源化HCVR和LCVR表現為整個抗硬骨素抗體分子的部分,即表現為與人恆定域序列的融合蛋白。然而,HCVR和LCVR序列也可在不存在恆定序列的情況下進行表現,以產生人源化抗c-Met scFv。
進一步描述參與人源化可使用小鼠抗體的方法的文獻包括,例如Queen等,Proc.,Natl.Acad.Sci.USA,88,2869,1991和Winter及其同事的方法[Jones等,Nature,321,522(1986),Riechmann,等,Nature,332,323-327(1988),Verhoeyen,等,Science,239,1534(1988)]。
本發明中所述的“抗原結合片段”,指具有抗原結合活性的Fab片段,Fab’片段,F(ab’)片段,以及與人c-Met結合的Fv片段scFv片段;包含本發明所述抗體的選自SEQ ID NO:3至SEQ ID NO:8中的一個或多個CDR區。Fv片段含有抗體重鏈可變區和輕鏈可變區,但沒有恆定區,並具有全部抗原結合位元點的最小抗體片段。一般地,Fv抗體還包含在VH和VL結構域之間的多肽接頭,且能夠形成抗原結合所需的結構。也可以用不同的連接物將兩個抗體可變區連接成一條多肽鏈,稱為單鏈抗體(single chain antibody)或單鏈Fv(scFv)。scFv還可以和其它抗體,例如抗EGFR抗體構建雙特異抗體(bispecific antibody)本發明的術語“與c-Met結合”,指能與人c-Met相互作用。本發明的術語“抗原結合位點”指抗原上不連續的,由本發明
抗體或抗原結合片段識別的三維空間位點。本發明中所述的“ADCC”,即antibody-dependent cell-mediated cytotoxicity,抗體依賴的細胞介導的細胞毒作用,是指表現Fc受體的細胞藉由識別抗體的Fc段直接殺傷被抗體包被的靶細胞。可藉由對IgG上Fc段的修飾,降低或消除抗體的ADCC效應功能。該修飾指在抗體的重鏈恆定區進行突變,如選自IgG1的N297A,L234A,L235A;IgG2/4chimera,IgG4的F235E,或L234A/E235A突變。
本發明中所述的融合蛋白是一種藉由DNA重組,得到的兩個基因共表現的蛋白產物。重組c-Met胞外區Fc融合蛋白藉由DNA重組,把c-Met胞外區和人抗體Fc片段共表現的融合蛋白。該c-Met胞外區,是指c-Met蛋白表現在細胞膜以外的部分。
本發明工程化的抗體或抗原結合片段可用常規方法製備和純化。比如,編碼重鏈(SEQ ID NO:4)和輕鏈(SEQ ID NO:5)的cDNA序列,可以選殖並重組至表現載體pEE6.4((Lonza Biologics)。重組的免疫球蛋白表現載體可以穩定地轉染CHO細胞。作為一種更推薦的現有技術,哺乳動物類表現系統會導致抗體的糖基化,特別是在FC區的高度保守N端。藉由表現與人c-Met特異性結合的抗體得到穩定的克隆。陽性的純株在生物反應器的無血清培養基中擴大培養以生產抗體。分泌了抗體的培養液可以用常規技術純化。比如,用含調整過的緩衝液的A或G Sepharose FF管柱進行過柱。洗脫非特異性結合的組分。再用PH梯度
法洗脫結合的抗體,用SDS-PAGE檢測抗體片段,收集。抗體可用常規方法進行過濾濃縮。可溶的混合物和多聚體,也可以用常規方法去除,比如分子篩,離子交換。得到的產物需立即冷凍,如-70℃,或者凍乾。
本發明的抗體指單株抗體。本發明所述的單株抗體或mAb,指由單一的純株細胞株得到的抗體,該細胞株不限於真核的,原核的或噬菌體的純株細胞株。單株抗體或抗原結合片段可以用如雜交瘤技術、重組技術、噬菌體展示技術,合成技術(如CDR-grafting),或其它現有技術進行重組得到。
“給予”和“處理”當應用於動物、人、實驗受試者、細胞、組織、器官或生物流體時,是指外源性藥物、治療劑、診斷劑或組合物與動物、人、受試者、細胞、組織、器官或生物流體的接觸。“給予”和“處理”可以指例如治療、藥物代謝動力學、診斷、研究和實驗方法。細胞的處理包括試劑與細胞的接觸,以及試劑與流體的接觸,其中該流體與細胞接觸。
“治療”意指給予患者內用或外用治療劑,諸如包含本發明的任一種結合化合物的組合物,該患者具有一種或多種疾病症狀,而已知該治療劑對這些症狀具有治療作用。通常,在受治療患者或群體中以有效緩解一種或多種疾病症狀的量給予治療劑,無論是藉由誘導這類症狀退化還是抑制這類症狀發展到任何臨床右測量的程度。有效緩解任何具體疾病症狀的治療劑的量(也稱作“治療有效
量”)可根據多種因素變化,例如患者的疾病狀態、年齡和體重,以及藥物在患者產生需要療效的能力。藉由醫生或其它專業衛生保健人士通常用於評價該症狀的嚴重性或進展狀況的任何臨床檢測方法,可評價疾病症狀是否已被減輕。盡本發明的實施方案(例如治療方法或製品)在緩解每個患都有的目標疾病症狀方面可能無效,但是根據本領域已知的任何統計學檢驗方法如Student t檢驗、卡方檢驗、依據Mann和Whitney的U檢驗、Kruskal-Wallis檢驗(H檢驗)、Jonckheere-Terpstra核對總和Wilcoxon檢驗確定,其在統計學顯著數目的患者中應當減輕目標疾病症狀。
“保守修飾”或“保守置換或取代”是指具有類似特徵(例如電荷、側鏈大小、疏水性/親水性、主鏈構象和剛性等)的其它胺基酸置換蛋白中的胺基酸,使得可頻繁進行改變而不改變蛋白的生物學活性。本領域技術人員知曉,一般而言,多肽的非必需區域中的單個胺基酸置換基本上不改變生物學活性(參見例如Watson等(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224頁,(第4版))。另外,結構或功能類似的胺基酸的置換不大可能破環生物學活性。
整個說明書和申請專利範圍中使用的術語“基本上由……組成”或其變形表示包括所有所述元件或元件組,並且任選包括與所述元件類似或不同性質的其它元件,該其它元件非顯著改變指定給藥方案、方法或組合物的基本性質或新性質。作為非限制性例子,基本上由所提及的胺
基酸序列組成的結合化合物還可以包括一種或多種胺基酸,其不顯著影響結合化合物的性質。
“有效量”包含足以改善或預防醫學病症的症狀或病症的量。有效量還意指足以允許或促進診斷的量。用於特定患者或獸醫學受試者的有效量可依據以下因素而變化:如待治療的病症、患者的總體健康情況、給藥的方法途徑和劑量以及副作用嚴重性。有效量可以是避免顯著副作用或毒性作用的最大劑量或給藥方案。
“外源性”指根據背景在生物、細胞或人體外產生的物質。“內源性”指根據背景在細胞、生物或人體內產生的物質。
“同源性”是指兩個多核苷酸序列之間或兩個多肽之間的序列相似性。當兩個比較序列中的位置均被相同鹼基或胺基酸單體亞基佔據時,例如如果兩個DNA分子的每一個位置都被腺嘌呤佔據時,那麼該分子在該位置是同源的。兩個序列之間的同源懷百分率是兩個序列共有的匹配或同源位置數除以比較的位置數×100的函數。例如,在序列最佳比對時,如果兩個序列中的10個位置有6個匹配或同源,那麼兩個序列為60%同源。一般而言,當比對兩個序列而得到最大的同源性百分率時進行比較。
“任選”或“任選地”意味著隨後所描述地事件或環境可以但不必發生,該說明包括該事件或環境發生或不發生地場合。例如,“任選包含1至3個抗體重鏈可變區”意味著特定序列的抗體重鏈可變區可以但不必須存在。
“醫藥組成物”表示含有一種或多種本文所述化合物或其生理學上/可藥用的鹽或前體藥物與其他化學組分的混合物,以及其他組分例如生理學/可藥用的載體和賦形劑。醫藥組成物的目的是促進對生物體的給藥,利於活性成分的吸收進而發揮生物活性。
常規的醫藥組成物的製備見中國藥典。
術語“載體”用於本發明的藥物,是指能改變藥物進入人體的方式和在體內的分佈、控制藥物的釋放速度並將藥物輸送到靶向器官的體系。藥物載體釋放和靶向系統能夠減少藥物降解及損失,降低副作用,提高生物利用度。如可作為載體的高分子表面活性劑由於其獨特的兩親性結構,可以進行自組裝,形成各種形式的聚集體,較佳的實例如膠束、微乳液、凝膠、液晶、囊泡等。這些聚集體具有包載藥物分子的能力,同時又對膜有良好的滲透性,可以作為優良的藥物載體。術語“稀釋劑”又稱填充劑,其主要用途是增加片劑的重量和體積。稀釋劑的加入不僅保證一定的體積大小,而且減少主要成分的劑量偏差,改善藥物的壓縮成型性等。當片劑的藥物含有油性組分時,需加入吸收劑吸收油性物,使保持“乾燥”狀態,以利於製成片劑。
術語“可藥用鹽”是指本發明配體-細胞毒性藥物偶聯物的鹽,這類鹽用於哺乳動物體內時具有安全性和有效性,且具有應有的生物活性,本發明抗體-抗體藥物偶聯化合物至少含有一個胺基,因此可以與酸形成鹽。
術語“溶劑化合物”指本發明的配體-藥物偶聯化合物與一種或多種溶劑分子形成可藥用的溶劑化合物。
術語“配體”是能識別和結合目標細胞相關的抗原或受體的大分子化合物。配體的作用是將藥物呈遞給與配體結合的目標細胞群,這些配體包括但不限於蛋白類激素、凝集素、生長因子、抗體或其他能與細胞結合的分子。
治療劑是與結合部分如抗體或抗體片段、或其亞片段分別、同時或相繼地給藥的分子或原子,並且可用於疾病的治療。治療劑的實例包括但不限於抗體、抗體片段、共軛物、藥物、細胞毒性劑、促細胞凋亡劑、毒素、核酸酶(包括DNA酶和RNA酶)、激素、免疫調節劑、螯合劑、硼化合物、光敏劑或染料、放射性同位素或放射性核素、寡核苷酸、干擾RNA、肽、抗血管發生劑、化療劑、細胞因子、趨化因子、前藥、酶、結合蛋白或肽、或其組合。
偶聯物是與如上所述的治療劑偶聯的抗體組分或其他靶向部分。本文所用的術語“偶聯物”和“免疫偶聯物”可交換地使用。
術語“細胞毒劑”在用於本文時指抑制或防止細胞的功能和/或引起細胞死亡或破壞的物質。
“毒素”指能夠對細胞的生長或增殖產生有害效果的任何物質。
“化療劑”指可用於治療癌症的化學化合物。該定義還包括發揮調節、降低、阻斷或抑制可促進癌生長的激素效果作用的抗激素劑,且常常是系統或全身治療的形式。
它們自身可以是激素。
澳瑞他汀是全合成藥物,化學結構式相對容易改造,以便優化其物理性質和成藥特性。用於和抗體偶聯的澳瑞他汀衍生物主要包括單甲基澳瑞他汀E(MMAE)和單甲基澳瑞他汀F(MMAF),前者是又天然微管蛋白聚合酶抑制劑尾海兔素-10(dolastatin-10)衍生出的合成五肽,在C-端加上一個2-胺基-1-苯基丙基-1-醇而合成。MMAE對多種人類腫瘤細胞株的抑制活性小於一奈米莫耳。為了降低MMAE自身細胞毒活性,MMAF在尾海兔素-10的C-端加上一個苯丙胺酸,因為在結構上引入一個羧基,MMAF的細胞膜通透性較差,因此對細胞的生物活性顯著降低,但是和抗體偶聯後對細胞的抑制活性大幅度提高(US7750116)。
術語“微管蛋白抑制劑”是指藉由抑制微管蛋白的聚合或促進微管蛋白的聚合而干擾細胞的有絲分裂過程,從而發揮抗腫瘤作用的一類化合物。其非限制性實例包括:美登素類、卡利奇黴素、紫杉烷類、長春新鹼、秋水仙鹼、尾海兔素/澳瑞他汀,較佳選自美登素類或尾海兔素/澳瑞他汀;更佳選自通式D1或DM所示的化合物。
CPT是喜樹鹼的縮寫,並且在本申請中CPT用於表示喜樹鹼本身或喜樹鹼的類似物或衍生物。具有所示的編號和用字母A-E標記的環的喜樹鹼和一些其類似物的結構在以下式提供。
CPT:R1=R2=R3=H
10-羥基-CPT:R1=OH;R2=R3=H
伊立替康(CPT-11):R1=;R2=乙基;R3=H
SN-38:R1=OH;R2=乙基;R3=H
托泊替康:R1=OH;R2=H;R3=CH-N(CH3)2
術語“胞內代謝物”指由細胞內對抗體-藥物偶聯物(ADC)的代謝過程或反應產生的化合物。該代謝過程或反應可以是酶促過程,諸如ADC的肽接頭的蛋白水解切割、或官能團諸如腙、酯或醯胺的水解。胞內代謝物包括但不限於在進入、擴散、攝取或轉運進入細胞後經歷胞內切割的抗體和游離藥物。
術語“胞內切割的”和“胞內切割”指細胞內對抗體-藥物偶聯物(ADC)的代謝過程或反應,由此藥物模組(D)與抗體(Ab)之間的共價附著,即接頭被打斷,導致細胞內游離藥物與抗體解離。ADC被切割的模組因而是胞內代謝物。
術語“生物利用度”指施用於患者的給定量的藥物的
系統利用度(即血液/血漿水準)。生物利用度是表明藥物從所施用的劑量形式到達大循環的時間(速率)和總量(程度)二者度量的絕對項。
術語“細胞毒活性”指抗體-藥物偶聯物或抗體-藥物偶聯物的胞內代謝物的細胞殺傷、細胞抑制、或生長抑制效果。細胞毒活性可以表述為IC50值,即半數細胞存活時每單位體積的濃度(莫耳或品質)。
術語“烷基”指飽和脂肪族烴基團,其為包含1至20個碳原子的直鏈或支鏈基團,較佳含有1至12個碳原子的烷基,更佳含有1至10個碳原子的烷基,最佳含有1至6個碳原子的烷基。非限制性實例包括甲基、乙基、正丙基、異丙基、正丁基、異丁基、第三丁基、第二丁基、正戊基、1,1-二甲基丙基、1,2-二甲基丙基、2,2-二甲基丙基、1-乙基丙基、2-甲基丁基、3-甲基丁基、正己基、2,2-二乙基己基,及其各種支鏈異構體等。烷基可以是取代的或非取代的,當被取代時,取代基可以在任何可使用的連接點上被取代,該取代基較佳為一個或多個以下基團,其獨立地選自烷基、烯基、炔基、烷氧基、烷硫基、烷基胺基、鹵素、巰基、羥基、硝基、氰基、環烷基、雜環烷基、芳基、雜芳基、環烷氧基、雜環烷氧基、環烷硫基、雜環烷硫基、側氧基。
術語“環烷基”指飽和或部分不飽和單環或多環環狀烴取代基,環烷基環包含3至20個碳原子,較佳包含3至12個碳原子,更佳包含3至10個碳原子,最佳包含3
至8個碳原子。單環環烷基的非限制性實例包括環丙基、環丁基、環戊基、環戊烯基、環己基、環己烯基、環己二烯基、環庚基、環庚三烯基、環辛基等;多環環烷基包括螺環、稠環和橋環的環烷基。
術語“雜環基”指飽和或部分不飽和單環或多環環狀烴取代基,其包含3至20個環原子,其中一個或多個環原子為選自氮、氧或S(O)m(其中m是整數0至2)的雜原子,但不包括-O-O-、-O-S-或-S-S-的環部分,其餘環原子為碳。較佳包含3至12個環原子,其中1至4個是雜原子;更佳環烷基環包含3至10個環原子。單環雜環基的非限制性實例包括吡咯烷基、哌啶基、哌嗪基、嗎啉基、硫代嗎啉基、高哌嗪基等。多環雜環基包括螺環、稠環和橋環的雜環基。
該雜環基環可以稠合於芳基、雜芳基或環烷基環上,其中與母體結構連接在一起的環為雜環基,其非限制性實例包括:和等。
雜環基可以是任選取代的或非取代的,當被取代時,取代基較佳為一個或多個以下基團,其獨立地選自烷基、烯基、炔基、烷氧基、烷硫基、烷基胺基、鹵素、巰基、羥基、硝基、氰基、環烷基、雜環烷基、芳基、雜芳基、環烷氧基、雜環烷氧基、環烷硫基、雜環烷硫基、側氧基。
術語“芳基”指具有共軛的π電子體系的6至14員全碳單環或稠合多環(也就是共用毗鄰碳原子對的環)基團,
較佳為6至10員,例如苯基和萘基,最較佳苯基。該芳基環可以稠合於雜芳基、雜環基或環烷基環上,其中與母體結構連接在一起的環為芳基環,其非限制性實例包括:
芳基可以是取代的或非取代的,當被取代時,取代基較佳為一個或多個以下基團,其獨立地選自烷基、烯基、炔基、烷氧基、烷硫基、烷基胺基、鹵素、巰基、羥基、硝基、氰基、環烷基、雜環烷基、芳基、雜芳基、環烷氧基、雜環烷氧基、環烷硫基、雜環烷硫基。
術語“雜芳基”指包含1至4個雜原子、5至14個環原子的雜芳族體系,其中雜原子選自氧、硫和氮。雜芳基較佳為5至10員,更佳為5員或6員,例如呋喃基、噻吩基、吡啶基、吡咯基、N-烷基吡咯基、嘧啶基、吡嗪基、咪唑基、四唑基等。該雜芳基環可以稠合於芳基、雜環基或環烷基環上,其中與母體結構連接在一起的環為雜芳基環,其非限制性實例包括:
雜芳基可以是任選取代的或非取代的,當被取代時,
取代基較佳為一個或多個以下基團,其獨立地選自烷基、烯基、炔基、烷氧基、烷硫基、烷基胺基、鹵素、巰基、羥基、硝基、氰基、環烷基、雜環烷基、芳基、雜芳基、環烷氧基、雜環烷氧基、環烷硫基、雜環烷硫基。
術語“烷氧基”指-O-(烷基)和-O-(非取代的環烷基),其中烷基的定義如上所述。烷氧基的非限制性實例包括:甲氧基、乙氧基、丙氧基、丁氧基、環丙氧基、環丁氧基、環戊氧基、環己氧基。烷氧基可以是任選取代的或非取代的,當被取代時,取代基較佳為一個或多個以下基團,其獨立地選自烷基、烯基、炔基、烷氧基、烷硫基、烷基胺基、鹵素、巰基、羥基、硝基、氰基、環烷基、雜環烷基、芳基、雜芳基、環烷氧基、雜環烷氧基、環烷硫基、雜環烷硫基。
術語“鍵”指用“-”表示的共價鍵。
術語“羥基”指-OH基團。
術語“鹵素”指氟、氯、溴或碘。
術語“胺基”指-NH2。
術語“氰基”指-CN。
術語“硝基”指-NO2。
術語“側氧基”指=O。
“任選”或“任選地”意味著隨後所描述的事件或環境可以但不必發生,該說明包括該事件或環境發生或不發生地場合。例如,“任選被烷基取代的雜環基團”意味著烷基可以但不必須存在,該說明包括雜環基團被烷基取代
的情形和雜環基團不被烷基取代的情形。
“取代的”指基團中的一個或多個氫原子,較佳為最多5個,更佳為1~3個氫原子彼此獨立地被相應數目的取代基取代。不言而喻,取代基僅處在它們的可能的化學位置,本領域技術人員能夠在不付出過多努力的情況下確定(藉由實驗或理論)可能或不可能的取代。例如,具有游離氫的胺基或羥基與具有不飽和(如烯屬)鍵的碳原子結合時可能是不穩定的。
“接頭”指包含使抗體共價附著於藥物模組的共價鍵或原子鏈的化學模組。在各個實施方案中,接頭包括:二價基,諸如亞烴基(alkyldiyl)、亞芳基、亞雜芳基,諸如-(CR2)nO(CR2)n-、烴氧基重複單元(例如聚亞乙基氧基(polyethyleneoxy)、PEG、聚亞甲基氧基(polymethyleneoxy)和烴胺基(例如聚乙烯胺基,JeffamineTM)等模組;及二酸酯和醯胺類,包括琥珀酸酯、琥珀醯胺、二乙醇酸酯、丙二酸酯和己醯胺。
縮寫
接頭組件
MC=6-馬來醯亞胺基己醯基
Val-Cit或“vc”=纈胺酸-瓜胺酸(蛋白酶可切割接頭中的例示二肽)
瓜胺酸=2-胺基-5-脲基戊酸
PAB=對胺基苄氧羰基(“自我犧牲”接頭組件的例示)
Me-Val-Cit=N-甲基-纈胺酸-瓜胺酸(其中接頭肽鍵已
經修飾以防止其受到組織蛋白酶B的切割)
MC(PEG)6-OH=馬來醯亞胺基己醯基-聚乙二醇(可附著於抗體半胱胺酸)
SPP=N-琥珀醯亞胺基4-(2-吡啶基硫代)戊酸酯
SPDP=N-琥珀醯亞胺基3-(2-吡啶基二硫代)丙酸酯
SMCC=琥珀醯亞胺基-4-(N-馬來醯亞胺基甲基)環己烷-1-羧酸酯
IT=亞胺基硫烷
細胞毒性藥物:
MMAE=單甲基澳瑞他汀E(MW 718)
MMAF=澳瑞他汀E(MMAE)的變體,其在藥物的C-末端處有苯丙胺酸(MW731.5)
MMAF-DMAEA=有DMAEA(二甲基胺基乙胺)以醯胺連接至C-末端苯丙胺酸的MMAF(MW 801.5)
MMAF-TEG=有四乙二醇酯化至苯丙胺酸的MMAF
MMAF-NtBu=N-第三丁基作為醯胺附著於MMAF的C-末端
DM1=N(2’)-脫乙醯基-N(2’)-(3-巰基-1-氧丙基)-美登素
DM3=N(2’)-脫乙醯基-N2-(4-巰基-1-氧戊基)-美登素
DM4=N(2’)-脫乙醯基-N2-(4-巰基-4-甲基-1-氧戊基)-美登素
本發明還提供了包含偶聯有一種或多種細胞毒劑的本發明任何抗c-Met抗體或其它具有內吞活性的c-Met抗體(如,LY-2875358)的抗體-細胞毒性藥物偶聯物或其可藥用
鹽或溶劑化合物(可互換的稱為“抗體-藥物偶聯物”或“ADC”),該細胞毒劑諸如化療劑、藥物、生長抑制劑、毒素(例如細菌、真菌、植物或動物起源的酶活性毒素或其片段)或放射性同位素(即放射偶聯物)。
在某些實施方案中,抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物包含抗c-Met抗體和化療劑或其它毒素。本文中(上文))描述了可用於生成抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物的化療劑。也可使用酶活性毒素及其片段,這些酶活性毒素及其片段已在說明書中描述。
在某些實施方案中,抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物包含抗c-Met抗體和一種或多種小分子毒素,包括但不限於小分子藥物,諸如喜樹鹼衍生物、加利車黴素(calicheamicin)、美登木素生物鹼(maytansinoids)、朵拉司他汀(dolastatin)、澳瑞他汀、單端孢黴素(trichothecene)和CC1065及這些藥物具有細胞毒活性的片段。
例示性的接頭L2包括6-馬來醯亞胺基己醯基(“MC”)、馬來醯亞胺基丙醯基(“MP”)、纈胺酸-瓜胺酸(“val-cit”或“vc”)、丙胺酸-苯丙胺酸(“ala-phe”)、對胺基苄氧羰基(“PAB”)、N-琥珀醯亞胺基4-(2-吡啶基硫代)戊酸酯(“SPP”)、N-琥珀醯亞胺基4-(N-馬來醯亞胺基甲基)環己烷-1羧酸酯(“SMCC”)、和N-琥珀醯亞胺基(4-碘-乙醯基)胺基苯甲酸酯(“SIAB”)。本領域知道多種接
頭,下文也描述了一些。
接頭可以是便於在細胞中釋放藥物的“可切割接頭”。例如,可使用酸不穩定接頭(例如腙)、蛋白酶敏感(例如肽酶敏感)接頭、光不穩定接頭、二甲基接頭、或含二硫化物接頭(Chari等,Cancer Research 52:127-131(1992);美國專利No.5,208,020)。
在有些實施方案中,接頭構件可以是將抗體連接至另一接頭構件或藥物模組的“延伸物單元”(stretcher unit)。例示性的延伸物單元顯示於下文(其中波形線指示共價附著至抗體的位點):
在有些實施方案中,接頭單元可以是胺基酸單元。在一個這樣的實施方案中,胺基酸單元容許蛋白酶切割接頭,由此便於在暴露於胞內蛋白酶(諸如溶酶體酶)後從抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物釋放
藥物。參見例如Doronina等(2003)Nat.Biotechhol.21:778-784。例示性的胺基酸單元包括但不限於二肽、三肽、四肽、和五肽。例示性的二肽包括:纈胺酸-瓜胺酸(VC或val-cit);丙胺酸-苯丙胺酸(AF或ala-phe);苯丙胺酸-賴胺酸(FK或phe-lys);或N-甲基-纈胺酸-瓜胺酸(Me-val-cit)。例示性的三肽包括:甘胺酸-纈胺酸-瓜胺酸(gly-val-cit)和甘胺酸-甘胺酸-甘胺酸(gly-gly-gly)。胺基酸單元可以包含天然存在的胺基酸殘基,以及次要胺基酸和非天然存在胺基酸類似物,諸如瓜胺酸。胺基酸單元可以在它們對特定酶(例如腫瘤相關蛋白酶,組織蛋白酶B、C和D,或血漿蛋白酶)的酶促切割的選擇性方面進行設計和優化。
在有些實施方案中,接頭構件可以是將抗體連接(或是直接的或是藉由延伸物單元和/或胺基酸單元)至藥物模組的“間隔物”單元。間隔物單元可以是“自我犧牲的”(self-immolative)或“非自我犧牲的”。“非自我犧牲的”間隔物單元指間隔物單元的部分或整體在ADC的酶促(蛋白水解)切割後保持結合於藥物模組的間隔物單元。非自我犧牲的間隔物單元的例子包括但不限於甘胺酸間隔物單元和甘胺酸-甘胺酸間隔物單元。還涵蓋對序列特異性酶促切割易感的肽間隔物的其它組合。例如,腫瘤細胞相關蛋白酶對含甘胺酸-甘胺酸間隔物單元的ADC的酶促切割將導致甘胺酸-甘胺酸-藥物模組從ADC的剩餘部分釋放。在一個這樣的實施方案中,甘胺酸-甘胺酸-藥物模組然後在腫瘤細胞中進行分開的水解步驟,如此從藥物模組切割甘胺
酸-甘胺酸間隔物單元。
“自我犧牲的”間隔物單元容許釋放藥物模組而沒有分開的水解步驟。在某些實施方案中,接頭的間隔物單元包含對胺基苄基單元。在一個這樣的實施方案中,將對胺基苯甲醇經醯胺鍵附著至胺基酸單元,並且在苯甲醇與細胞毒劑之間生成胺基甲酸酯、甲基胺基甲酸酯、或碳酸酯。參見例如Hamann等(2005)Expert Opin.Ther.Patents(2005)15:1087-1103。在一個實施方案中,間隔物單元是對胺基苄氧羰基(PAB)。
本發明中的示例性接頭如下:
例示性的藥物模組
美登素和美登木素生物鹼
在有些實施方案中,抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物包含偶聯有一個或多個美登木素生物鹼分子的本發明抗體。美登木素生物鹼是藉由抑制微管蛋白多聚化來發揮作用的有絲分裂抑制劑。美登素最初從東
非灌木齒葉美登木(Maytenus serrata)分離得到(美國專利No.3,896,111)。隨後發現某些微生物也生成美登木素生物鹼,諸如美登醇和C-3美登醇酯(美國專利No.4,151,042)。
美登木素生物鹼藥物模組在抗體-藥物偶聯物中是有吸引力的藥物模組,因為它們:(i)相對易於藉由發酵或發酵產物的化學修飾或衍生化來製備;(ii)易於用適於藉由非二硫化物接頭偶聯至抗體的官能團衍生化;(iii)在血漿中穩定;且(iv)有效針對多種腫瘤細胞系。
適於用作美登木素生物鹼藥物模組的美登素化合物是本領域公知的,而且可以依照已知方法從天然來源分離,或是使用遺傳工程技術生產(參見Yu等(2002)PNAS 99:7968-7973)。美登醇和美登醇類似物也可以依照已知方法合成製備。
美登木素生物鹼藥物模組的例示性實施方案包括:DM1;DM3;和DM4,正如本文中所公開的。
澳瑞他汀和朵拉司他汀
在有些實施方案中,抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物包含與朵拉司他汀(dolastatin)或朵拉司他汀肽類似物或衍生物(例如澳瑞他汀)(美國專利No.5,635,483;5,780,588)偶聯的本發明抗體。朵拉司他汀和澳瑞他汀已經顯示出干擾微管動力學、GTP水解、及核和細胞分裂(Woyke等(2001)Antimicrob.Agents and Chemother.45(12):3580-3584)且具有抗癌(美國專利No.5663149)和抗真菌活性(Pettit等(1998)Antimicrob.Agents Chemother.42:
2961-2965)。朵拉司他汀或澳瑞他汀藥物模組可經由肽藥物模組的N(胺基)末端或C(羧基)末端附著於抗體(WO02/088172)。
例示性的澳瑞他汀實施方案包括N-末端連接的單甲基澳瑞他汀藥物模組DE和DF,披露於Senter等,Proceedings of the American Association for CancerResearch,卷45,摘要號623,2004年3月28日,明確將其公開內容完整收入本文作為參考。肽藥物模組可以選自下文通式DE和DF:
其中DE和DF的波形線指示抗體或抗體-接頭的共價附著位點,且每個位置是獨立的;R2選自H和C1-C8烴基;R3選自H、C1-C8烴基、C3-C8碳環、芳基、C1-C8烴基-芳基、C1-C8烴基-(C3-C8碳環)、C3-C8雜環和C1-C8烴基-(C3-C8雜環);R4選自H、C1-C8烴基、C3-C8碳環、芳基、C1-C8烴基-芳基、C1-C8烴基-(C3-C8碳環)、C3-C8雜環和C1-C8烴基-(C3-C8雜環);
R5選自H和甲基;或者R4與R5一起形成碳環且具有通式-(CRaRb)n-,其中Ra和Rb獨立地選自H、C1-C8烴基和C3-C8碳環,而n選自2、3、4、5和6;R6選自H和C1-C8烴基;R7選自H、C1-C8烴基、C3-C8碳環、芳基、C1-C8烴基-芳基、C1-C8烴基-(C3-C8碳環)、C3-C8雜環和C1-C8烴基-(C3-C8雜環);每個R8獨立地選自H、OH、C1-C8烴基、C3-C8碳環和O-(C1-C8烴基);R9選自H和C1-C8烴基;R10選自芳基或C3-C8雜環;Z為O、S、NH或NR12,其中R12為C1-C8烴基;R11選自H、C1-C20烴基、芳基、C3-C8雜環、-(R13O)m-R14和-(R13O)m-CH(R15)2;m是選自1-1000的整數;R13為C2-C8烴基;R14為H或C1-C8烴基;R15每次出現獨立為H、COOH、-(CH2)n-N(R16)2、-(CH2)n-SO3H或-(CH2)n-SO3-C1-C8烴基;R16每次出現獨立為H、C1-C8烴基或-(CH2)n-COOH;R18選自-C(R8)2-C(R8)2-芳基、-C(R8)2-C(R8)2-(C3-C8雜環)和-C(R8)2-C(R8)2-(C3-C8碳環);且n是為選自0到6的整數。
通式DE的一種例示性澳瑞他汀,實施方案是MMAE,其中波形線指示共價附著至抗體-藥物偶聯物的接頭(L):
通式DF的一種例示性澳瑞他汀,實施方案是MMAF,其中波形線指示共價附著至抗體-藥物偶聯物的接頭(L)(參見US2005/0238649及Doronina等(2006)Bioconjugate Chem.17:114-124):
其它藥物模組包括選自以下的MMAF衍生物,其中波形線指示共價附著至抗體-藥物偶聯物的接頭(L):
一方面,可以將親水性基團在R11處附著於藥物模組,該親水性基團包括但不限於三乙二醇酯(triethylene glycol ester,TEG),如上所述。不限於任何特定理論,該親水性基團有助於藥物模組的內在化和不聚集(non-agglomeration)。包含澳瑞他汀/朵拉司他汀或其衍生物的通式I ADC的例示性實施方案記載於US2005-0238649A1及Doronina等(2006)Bioconjugate Chem.17:114-124,明確收入本文作為參考。包含MMAE或MMAF及各種接頭的通式I ADC的例示性實施方案具有如下結構和縮寫(其中“Ab”是抗體;p是1到約8;“Val-Cit”是纈胺酸-瓜胺酸二肽;而“S”是硫原子):
典型的是,基於肽的藥物模組可藉由在兩個或更多胺基酸和/或肽片段之間形成肽鍵來製備。此類肽鍵可依照例如肽化學領域眾所周知的液相合成法來製備(參見E.Schroder和K.Lübke,“The Peptides”,卷1,pp 76-136,1965,Academic Press)。澳瑞他汀/朵拉司他汀藥物模組可依照以下文獻中的方法來製備)US2005-0238649A1;美國專利No.5635483;美國專利No.5780588;Pettit等(1989)J.Am.Chem.Soc.111:5463-5465;Pettit等(1998)Anti-Cancer Drug Design 13:243-277;Pettit,G.R.等,Synthesis,1996,719-725;Pettit等(1996)J.Chem.Soc.Perkin Trans.15:859-863;及Doronina(2003)Nat.Biotechnol.21(7):778-784。
具體而言,通式DF的澳瑞他汀/朵拉司他汀藥物模組諸如MMAF及其衍生物可以使用US2005-0238649A1及Doronina等(2006)Bioconjugate Chem.17:114-124中記載的方法來製備。通式DE的澳瑞他汀/朵拉司他汀藥物模組諸如MMAE及其衍生物可以使用Doronina等(2003)Nat.
Biotech.21:778-784中記載的方法來製備。可以藉由常規方法方便地合成藥物-接頭模組MC-MMAF、MC-MMAE、MC-vc-PAB-MMAF和MC-vc-PAB-MMAE,例如Doronina等(2003)Nat.Biotech.21:778-784及美國專利申請公開號US2005/0238649A1中所記載的,然後將它們偶聯至感興趣的抗體。
藥物載荷
藥物載荷(loading)由y表示,即通式I的分子中每個抗體的平均藥物模組數。藥物載荷的範圍可以為每個抗體1至20個藥物模組(D)。通式I的ADC包括偶聯有一定範圍(1至20個)藥物模組的抗體的集合。來自偶聯反應的ADC製備物中每個抗體的平均藥物模組數可以藉由常規手段來表徵,諸如質譜、ELISA測定法、和HPLC。還可以測定ADC在y方面的定量分佈。在有些情況中,將p為某數值的同質ADC從具有其它藥物載荷的ADC中分離、純化、和表徵可以藉由諸如反相HPLC或電泳的手段來實現。
對於有些抗體-藥物偶聯物,y可能受到抗體上附著位元點數目的限制。例如,若附著是半胱胺酸硫醇,正如上文例示性實施方案中的那樣,則抗體可能只有一個或數個半胱胺酸硫醇基,或者可能只有一個或數個有足夠反應性的硫醇基,可附著接頭。在某些實施方案中,較高的藥物載荷,例如y>5,可引起某些抗體-藥物偶聯物的聚集、不溶性、毒性、或喪失細胞通透性。在某些實施方案中,本發明ADC的藥物載荷的範圍為1到約8;約2到約6;約
3到約5;約3到約4;約3.1到約3.9;約3.2到約3.8;約3.2到約3.7;約3.2到約3.6;約3.3到約3.8;或約3.3到約3.7。事實上,對於某些ADC已經顯示了每個抗體藥物模組的最佳比率可以為小於8,可以為約2到約5。參見US2005-0238649A1(完整收入本文作為參考)。
在某些實施方案中,在偶聯反應中將少於理論最大值的藥物模組偶聯至抗體。抗體可包含例如賴胺酸殘基,其不與藥物-接頭中間物或接頭試劑起反應,如下文所討論的。只有最具反應性的賴胺酸基團可以與胺反應性接頭試劑起反應。一般而言,抗體不包含許多游離的和反應性的半胱胺酸硫醇基,其可連接藥物模組;事實上,抗體中的大多數半胱胺酸硫醇基以二硫橋形式存在。在某些實施方案中,可以在部分或完全還原性條件下用還原劑諸如二硫蘇糖醇(DTT)或三羰基乙基膦(TCEP)還原抗體以產生反應性半胱胺酸硫醇基。在某些實施方案中,將抗體置於變性條件以暴露反應性親核基團,諸如賴胺酸或半胱胺酸。
ADC的載荷(藥物/抗體比率DAR)可以以不同方式來控制,例如藉由:(i)限制藥物-接頭中間物或接頭試劑相對於抗體的莫耳過量,(ii)限制偶聯反應的時間或溫度,(iii)半胱胺酸硫醇修飾的部分或限制還原性條件,(iv)藉由重組技術對抗體的胺基酸序列進行工程改造,使得半胱胺酸殘基的數目和位置為了控制接頭-藥物附著的數目和/或位置而進行改變(諸如如本文中和WO2006/034488(完整收入本文作為參考)中所述而製備的thioMab或thioFab)。
應當理解,若超過一個親核基團與藥物-接頭中間物或者與接頭試劑和接下來的藥物模組試劑起反應,則所得產物是具有一個或多個藥物模組附著於抗體之分佈的ADC化合物混合物。可以藉由對抗體特異性的和對藥物特異性的雙重ELISA抗體測定法自混合物計算每個抗體的平均藥物數。混合物中的各種ADC分子可以藉由質譜來鑒定,並藉由HPLC來分離,例如疏水相互作用層析。在某些實施方案中,可以藉由電泳或層析從偶聯混合物中分離具有單一載荷值的同質ADC。
製備抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物的某些方法
可以採用本領域技術人員知道的有機化學反應、條件和試劑藉由數種路徑來製備通式I的ADC,包括:(1)抗體的親核基團經共價鍵與二價接頭試劑反應形成Ab-L,接著與藥物模組D反應;和(2)藥物模組的親核基團經共價鍵與二價接頭試劑反應形成D-L,接著與抗體的親核基團反應。經後一種路徑製備通式I的ADC的例示性方法記載於US2005-0238649A1,明確收入本文作為參考。
抗體的親核基團包括但不限於:(i)N末端胺基;(ii)側鏈胺基,例如賴胺酸;(iii)側鏈硫醇基,例如半胱胺酸;和(iv)糖基化抗體中糖的羥基或胺基。胺、硫醇、和羥基是親核的,能夠與接頭模組上的親電子基團反應而形成共價鍵,而接頭試劑包括:(i)活性酯類,諸如NHS酯、HOBt酯、鹵代甲酸酯、和酸性鹵化物;(ii)烴基和苄基鹵化物,
諸如鹵代乙醯胺;(iii)醛、酮、羧基和馬來醯亞胺基團。某些抗體具有可還原的鏈間二硫化物,即半胱胺酸橋。可藉由還原劑諸如DTT(二硫蘇糖醇)或三羰基乙基膦(TCEP)處理使抗體完全或部分還原,從而具有與接頭試劑偶聯的反應性。每個半胱胺酸橋理論上將形成兩個反應性硫醇親核體。或者,可經由賴胺酸殘基的修飾將硫氫基引入抗體,例如藉由使賴胺酸殘基與2-亞胺基硫烷(Traut氏試劑)起反應,導致胺轉變為硫醇。
還可藉由抗體上的親電子基團(諸如醛或酮羰基)與接頭試劑或藥物上的親核基團之間的反應來生成本發明的抗體-藥物偶聯物。接頭試劑上的有用親核基團包括但不限於醯肼(hydrazide)、肟(oxime)、胺基(amino)、肼(hydrazine)、縮胺基硫脲(thiosemicarbazone)、肼羧酸酯(hydrazine carboxylate)、和芳基醯肼(arylhydrazide)。在一個實施方案中,可以用例如高碘酸鹽氧化劑氧化糖基化抗體的糖,從而形成可與接頭試劑或藥物模組的胺基團反應的醛或酮基團。所得亞胺Schiff鹼基可形成穩定的連接,或者可以用例如硼氫化物試劑還原而形成穩定的胺連接。在一個實施方案中,糖基化抗體的碳水化合物部分與半乳糖氧化酶或偏高碘酸鈉的反應可以在抗體中生成羰基(醛基和酮基),它可與藥物上的適宜基團反應(Hermanson,Bioconjugate Techniques)。在另一個實施方案中,包含N-末端絲胺酸或蘇胺酸殘基的抗體可以與偏高碘酸鈉反應,導致在第一個胺基酸處生成醛(Geoghegan和Stroh,(1992)Bioconjugate
Chem.3:138-146;US5362852)。這樣的醛可與藥物模組或接頭親核體反應。
藥物模組上的親核基團包括但不限於:胺、硫醇、羥基、醯肼、肟、肼、縮胺基硫脲、肼羧酸酯、和芳基醯肼基團,它們能夠與接頭模組上的親電子基團反應而形成共價鍵,而接頭試劑包括:(i)活性酯類,諸如NHS酯、HOBt酯、鹵代甲酸酯、和酸性鹵化物;(ii)烴基和苄基鹵化物,諸如鹵代乙醯胺;(iii)醛、酮、羧基、和馬來醯亞胺基團。
本發明的化合物明確涵蓋但不限於用如下交聯試劑製備的ADC:BMPS、EMCS、GMBS、HBVS、LC-SMCC、MBS、MPBH、SBAP、SIA、SIAB、SMCC、SMPB、SMPH、sulfo-EMCS、sulfo-GMBS、sulfo-KMUS、sulfo-MBS、sulfo-SIAB、sulfo-SMCC和sulfo-SMPB、及SVSB(琥珀醯亞胺基-(4-乙烯基碸)苯甲酸酯)它們可藉由商業途徑獲得(例如Pierce Biotechnology,Inc.,Rockford,IL.,U.S.A參見2003-2004年度應用手冊和產品目錄(2003-2004Applications Handbook and Catalog)第467-498頁)。
還可使用多種雙功能蛋白質偶聯劑來製備包含抗體和細胞毒劑的抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物,諸如N-琥珀醯亞胺基3-(2-吡啶基二硫代)丙酸酯(SPDP),琥珀醯亞胺基-4-(N-馬來醯亞胺基甲基)環己烷-1-羧酸酯(SMCC),亞胺基硫烷(IT),亞胺酸酯(諸如鹽酸己二醯亞胺酸二甲酯)、活性酯類(諸如辛二酸二琥珀醯亞胺基酯)、醛類(諸如戊二醛)、雙疊氮化合物(諸如雙(對-疊氮
苯甲醯基)己二胺)、雙重氮衍生物(諸如雙(對-重氮苯甲醯基)-乙二胺)、二異硫氰酸酯(諸如甲苯2,6-二異氰酸酯)、和雙活性氟化合物(諸如1,5-二氟-2,4-二硝基苯)的雙功能衍生物。例如,可以如Vitetta等,Science 238:1098(1987)中所述製備蓖麻毒蛋白免疫毒素。碳-14標記的1-異硫氰酸苄基-3-甲基二亞乙基三胺五乙酸(MX-DTPA)是用於將放射性核苷酸與抗體偶聯的例示性螯合劑。參見WO94/11026。
或者,可藉由例如重組技術或肽合成來製備包含抗體和細胞毒劑的融合蛋白。重組DNA分子可以包含各自編碼偶聯物的抗體和細胞毒部分的區域,彼此或是毗鄰或是由編碼接頭肽的區域分開,該接頭肽不破壞偶聯物的期望特性。
在又一個實施方案中,可以將抗體與“受體”(諸如鏈黴親合素)偶聯從而用於腫瘤預先靶向,其中對患者施用抗體-受體偶聯物,接著使用清除劑由迴圈中清除未結合的偶聯物,然後施用與細胞毒劑(例如放射性核苷酸)偶聯的“配體”(例如親合素)。提供以下實施例僅為說明性目的,並不旨在限制本發明的範圍。
第1圖顯示本發明抗c-Met抗體和ADC分子抑制腫瘤作用,結果表明ADC新分子藉由帶入的毒素能達到對腫瘤的完全抑制作用,而抗體單獨無法達到。結果還表明,本發明ADC分子沒有因為毒素的偶聯而影響到T1/2,本發明ADC藥物在小鼠體內未見毒性。
以下結合實施例用於進一步描述本發明,但這些實施例並非限制本發明的範圍。
本發明實施例或測試例中未註明具體條件的實驗方法,通常按照常規條件,或按照原料或商品製造廠商所建
議的條件。參見Sambrook等,分子克隆,實驗室手冊,冷泉港實驗室;當代分子生物學方法,Ausubel等著,Greene出版協會,Wiley Interscience,NY。未註明具體來源的試劑,為市場購買的常規試劑。
實施例1. 抗原抗體克隆表現
本發明所用抗體(輕、重鏈),抗原用領域周知的重疊延伸PCR方法構建,將重疊延伸PCR得到的DNA片段用HindIII/BstBI這兩個酶切位點插入到表現載體pEE6.4((Lonza Biologics),在293F細胞(Invitrogen,Cat# R790-07)中表現得到。所得重組蛋白用於免疫或篩選。c-Met基因範本來源於origene公司(貨號RC217003)。所選殖表現的DNA序列如下。
人c-Met細胞外區域(ECD)和鼠Fc區域融合蛋白(human c-Met ECD-mFc)DNA序列:
(SEQ ID NO:1)人c-Met細胞外Sema區域和Flag-His標籤(Human c-Met Sema-Flis)DNA序列:
(SEQ ID NO:2)人c-Met ECD his標籤(Human c-Met ECD-His)重組蛋白DNA序列:
(SEQ ID NO:3)
實施例2. 抗原抗體結合實驗(ELISA)
本實驗是利用酶聯免疫吸附的方法檢驗抗c-Met抗體(包括雜交瘤上清或重組表現的單株抗體等)在體外對c-Met抗原的親和力。
實驗步驟為:用包被液(PBS)(Hyclone,Cat No.:SH30256.01B)稀釋抗原(human c-Met-His,實施例1)至2μg/ml,加入100μl/孔於96孔酶標板(Costar 9018,Cat No.:03113024),4℃孵育過夜。次日將包被有抗原的96孔酶標板恢復至室溫,洗滌液(PBS+0.05%Tween 20(Sigma,Cat No.:P1379)洗滌三次。隨後加入200μl/孔封閉液(PBS+1%BSA(Roche,Cat No.:738328),37℃孵育1小時。洗滌液洗滌三次。加入待測抗c-Met抗體於96孔酶標板,室溫孵育1小時。洗滌液洗滌三次。加入封閉液10000倍稀釋二抗(Goat anti-Mouse IgG(H+L)(HRP)(Thermo,貨號:31432)100μl/孔至96孔酶標板,室溫孵育1小時。洗滌液洗滌三次。加入100μl/孔顯色液TMB(eBioscience REF:00-4201-56)至96孔酶標板。加入100μl/孔終止液(2N H2SO4)至96孔酶標板。使用讀板儀450nm讀板。
實施例3. 抗人c-Met的小鼠單株抗體細胞株產生
本發明藉由免疫小鼠,脾細胞融合、融合瘤篩選方法得到小鼠來源抗人c-Met單株細胞株。該方法為本領域熟知。即將重組表現抗原(human c-Met ECD-mFc,human c-Met Sema-flis,見實施例1)用PBS(Hyclone,Cat No.:SH30256.01B)稀釋至1mg/ml,與弗氏佐劑(首次免疫用完全
弗氏佐劑,加強免疫用不完全弗氏佐劑)乳化後,皮下注射接種Balb/C小鼠(每組5隻),每隻小鼠接種100μg抗原,間隔兩週加強免疫。從第一次加強免疫開始,每次加強免疫後的7至10天內採集小鼠血清用ELISA測定(方法見實施例2)效價。
選擇免疫後血清效價高於1:105的小鼠進行細胞融合。分別無菌製備小鼠B細胞和骨髓瘤細胞(SP2/0,ATCC number:CRL-1581TM)並計數,將兩種細胞按照B細胞:SP2/0=1:4的比例進行混合後離心(1500r/min,7min),棄上清加入1ml 50%聚乙二醇(Supplier:SIGMA,Catalogue# RNBB306),之後用1ml無血清RPMI1640(Supplier:GIBCO,Catalogue#C22400)終止,離心10min,棄上清,用含融合瘤細胞生長因子(Supplier:Roche,Catalogue# 1363735001),血清(Supplier:GIBCO,Catalogue#C20270)和HAT(Supplier:Invitrogen,Catalogue# 21060-017)的RPMI1640重懸沉澱,按照每孔105個B細胞的標準鋪板,每孔100μl,置37℃細胞培養箱中培養,3天後加入每孔100μl含雜交瘤細胞生長因子,血清和HT(Supplier:Invitrogen,Catalogue# 11067-030)的RPMI1640,2至4天后全換液每孔加入150μl含融合瘤細胞生長因子,血清和HT的RPMI1640,次日ELISA(方法見實施例2)檢測陽性純株。結果見表1。
實施例4 抗人c-Met單株體對胃癌細胞MKN45增值的抑制作用
選擇上述純株,進一步培養得到單株,ELISA驗證了結合活性後,挑選單株培養上清進行細胞活性檢測。該實驗原理是利用本發明抗人c-Met抗體能抑制人胃癌細胞(MKN45)表面c-Met發生磷酸化,而抑制MNK45細胞的增殖。
人胃癌細胞(MKN45,JCRB,JCRB0254,P11)1×105個細胞/mL,50μl/孔加至96孔細胞培養板(costar,#3799),培養基為RPMI 1640 medium:(GIBCO,cat#11835)+10%胎牛血清(FBS)(GIBCO-10099141)。隨後加入50μl/孔抗人c-Met待測抗體,37度培養箱(廠商:SANYO設備編號
TINC035)中培養5天。使用細胞增殖檢測試劑盒(CellTiter-Glo® Luminescent Cell Viability Assay)(Promega,G7573),按試劑盒說明書方法檢測細胞增殖情況。讀板儀(PerkinElmer,TREA001-RDA-IBA100)讀板。使用如下公式計算細胞的增值百分率:細胞增殖率%=(1-實驗組細胞讀數/不作處理組細胞讀數)×100%。結果見表2。
實施例5. 抗c-Met抗體序列選殖
將實施例4中得到的活性好的單細胞株Ab-5進行cDNA序列選殖,然後重組表現出單株抗體並進行各項活性檢測。本發明用逆轉錄PCR擴增抗體基因的重鏈和輕鏈可變區,連接到載體測序得到單株抗體輕重鏈序列。首先採用RNA純化試劑盒(Qiagen公司,貨號74134,步驟見此
說明書)提取實施例4中活性好的單細胞株的細胞總RNA。然後使用Invitrogen公司的貨號為18080-051 cDNA合成試劑盒製備cDNA單鏈,即Oligo-dT primers cDNA反轉錄。以此為模版,採用PCR方法合成抗體輕重鏈可變區序列,PCR產物選殖到TA載體pMD-18T,然後送去測定序列。將得到的抗體輕重鏈序列分別選殖到表現載體(見實施例1),表現重組單株抗體,驗證活性(見實施例2,4)後,進行人源化工作。
本發明小鼠融合瘤細胞單株抗體Ab-5的序列:
重鏈可變區:
(SEQ ID NO:4)
輕鏈可變區:
(SEQ ID NO:5)
抗人c-Met抗體的VH/VL CDR的胺基酸殘基由Kabat編號系統確定並注釋。
本發明中鼠源的CDR序列如表3所述:
實施例6. 抗c-Met抗體人源化
將實施例5得到鼠源抗c-Met單株抗體輕重鏈序列在抗體資料庫裡進行同源性比較後,建立人源化抗體模型,根據模型選擇回復突變篩選最優的人源化抗c-Met單株抗體為本發明較佳分子。該方法從已經發表的小鼠Fab晶體結構模型資料庫(比如PDB資料庫)中查找與所得鼠源候選分子同源性相似的晶體結構開始,挑取高解析度(比如<2.5Å)的Fab晶體結構,建立小鼠Fab模型。將本發明鼠源抗體輕重鏈序列和模型中的序列比對,保留和模型中和鼠源抗體序列一致的序列,得到本發明鼠抗體的結構模型,其中不一致胺基酸為可能的回復突變位點。用Swiss-pdb viewer軟體運行鼠抗體結構模型,優化能量(最小化)。將模型中除CDR外的不同胺基酸位點進行回復突變,將所得的突變抗體(人源化)和人源化之前抗體對比進行活性檢測。保留活性好的人源化抗體。之後,對CDR區
域優化,包括避免糖基化,脫醯胺化,氧化位點等。優化後的人源化抗c-Met抗體的CDR區如表4所示:
人源化以後的輕重鏈可變區如以下所示:
Ab-9
(SEQ ID NO:13)
Ab-10
(SEQ ID NO:14)
Ab-11
(SEQ ID NO:15)
Ab-9
(SEQ ID NO:16)
Ab-10
(SEQ ID NO:17)
Ab-11
(SEQ ID NO:18)
人源化以後的輕重鏈和IgG Fc區段重組,得到本發明人源化抗c-Met單株抗體。所用的Fc序列任選自以下序列:
重鏈恆定區:
(SEQ ID NO:19)
(SEQ ID NO:20)
(SEQ ID NO:21)
輕鏈恆定區:
(SEQ ID NO:22)
用基因克隆、重組表現的方法分別選殖、表現、純化上述抗體,經ELISA(實施例2)和體外結合活性檢測(實施例7),最終選出活性保持最好的人源化抗體Ab-9,Ab-10,Ab-11,序列如下:
Ab-9人源化抗體:
重鏈:
(SEQ ID NO:23)
輕鏈:
(SEQ ID NO:26)
Ab-10人源化抗體:
重鏈:
(SEQ ID NO:24)
輕鏈:
(SEQ ID NO:27)
Ab-11人源化抗體:
重鏈:
(SEQ ID NO:25)
輕鏈:
(SEQ ID NO:28)
實施例7. 抗c-Met人源化抗體體結合外活性檢測
本發明人源化抗體體外活性除了用ELISA(實施例2)檢測之外,還檢測了其與c-Met高表現細胞株MKN45的結合,及其與c-Met抗原的親和力(BIACore測定)。結果見表5和表6。
用FACS方法檢測本發明抗c-Met人源化體同c-Met高表現細胞株MKN45的結合活性。
在具有10%(v/v)胎牛血清(FBS)(GIBCO,Cat No.:10099-141)和青黴素/鏈黴素(GIBCO,Cat No.:15070-063)的RPMI 1640培養基(GIBCO,Cat No.:11835-030)中重懸浮MKN45細胞(JCRB,Cat No.:JCRB0254)至10000,000細胞/mL。將2mL重懸的MKN45細胞以150,000細胞/孔加入96孔微滴定板(Corning,Cat No.:3799)中,加入8個濃度(20μg/ml起5倍濃度梯度稀釋)的c-Met抗體至對應的孔中,最終體積為100μl,在4℃孵育1小時。加入FACS緩衝液(具有2.5%(v/v)胎牛血清(FBS)的磷酸鹽緩衝液(PBS)(Hyclone,Cat:SH30256.01B),4℃,1300rpm,4分鐘,離心棄上清,重複三次。每孔加入100μl二抗溶液(螢光標記羊抗鼠二抗:1:200稀釋,Biolegend,Cat#405307;螢光標記抗人二抗:1:30稀釋,Biolegend,Cat#409304),在4℃孵育1小時。加入FACS緩衝液,4℃,1300rpm,4分鐘,
離心棄上清,重複三次。加入200μl FACS緩衝液,重懸細胞,準備好樣品,使用流式細胞儀(BD FACS Array)檢測。
本發明採用表面等離子共振技術(surface plasmon resonance,SPR)檢測c-Met抗體與c-Met抗原Sema-His之間的親和力。
Anti-mouse IgG(GE Life Sciences catalog # BR-1008-38)或anti-human IgG(GE Life Sciences catalog # BR-1008-39)抗體用pH5.0乙酸鈉溶液(GE Healthcare,Cat#BR-1003-51)分別稀釋至30μg/ml和50μg/ml,用胺基偶聯試劑盒(GE Life Sciences,Cat#BR100050)固定化至CM5晶片(GE Life Sciences catalog # BR-1000-12)的試驗及對比通道,偶聯水準設定在15000RU。運行緩衝液PBS(Hyclone,Cat#SH30256.01B)+0.05% P20(GE Life Sciences,Cat#BR-1000-54))稀釋c-Met抗體至1.5μg/ml,運行緩衝液稀釋抗原sema-his至200nM,之後用同樣緩衝液1:2倍稀釋直至0.78nM。將稀釋好的抗體在30μl/min的流速下流過實驗通道一分鐘,抗原在同樣的流速下流經實驗通道和對比通道3分鐘,解離10分鐘後將流速調至10μl/min,在實驗通道和對比通道流過再生緩衝液3分鐘。資料經雙重扣減後用BiaEvaluation 4.1擬合,擬合模型用1:1(Langmuir)模型。
上述結果表明,本發明人源化抗體和抗原的結合活性在0.13至8nM,因檢測方法的不同,檢測結果有所不同。結果表明了人源化後的抗c-Met抗體保留了人源化之前抗體的結合活性。
實施例8. 抗c-Met人源化抗體體外功能,細胞活性評價
為了檢測本發明抗體的功能,用阻斷c-Met ligand(肝細胞生長因子HGF)和c-Met的結合實驗,以及抑制細胞增值實驗(實施例4)對實施例7中的抗體進行了評估。
HGF與c-Met的結合造成c-Met分子的酪胺酸磷酸化以及c-Met信號通路的活化。本實驗藉由ELISA方法檢測本發明抗c-Met抗體阻斷HGF結合在受體c-Met蛋白上的活力,即IC50。
c-Met ECD-mFc(實施例子1)稀釋在PBS(Hyclone,Cat #SH30256.01B)中,終濃度為2μg/ml,包被在96孔ELISA板(Costar,cat#2592)常溫過夜。使用洗板機(Suppler:BioTex;Model:ELX405;S/N:251504)用PBST(PBS+0.05%Tween 20(Simga,Cat#P1379))洗滌3次,300μl封閉液PBS+1%BSA(Roche,Cat #738328)加入到96孔板37℃孵育60分鐘。用PBST洗滌3次後,封閉液稀釋好的抗體50μl加入到96孔中,37℃孵育90分鐘。添加封閉液稀釋好的終濃度為20ng/ml的人HGF(Sino Biological,#10463-HNAS)50μl到含有c-Met抗體的96孔板中,在室溫孵育120分鐘。用PBST洗滌三次後,添加100μl封閉液稀釋好的終濃度為100ng/ml生物素標記的抗HGF抗體(R&D,Cat #BAF294)到96孔板中孵育90分鐘。PBST洗滌後,添加100μl封閉液稀釋好的辣根過氧化物酶(ebioscience,#18-4100-51)到96孔板中孵育30分鐘。PBST洗滌後,加入100μl受質(ebioscience,cat#00-4201-56)室溫孵育10分鐘,加入100μl終止液(2N H2SO4),用450nM酶標儀(Supplier:Moleculer Devices;Model:MNR0643;Equip ID:TMRP001)讀數。使用SoftMax Pro v5進行資料分析。結果見表7。
上述結果表明,本發明人源化抗體不僅保留了和抗原的結合活性,而且能夠阻止抗原和配體(ligand)的結合,同時顯示了抑制腫瘤細胞的生長活性。
實施例9 本發明抗c-Met人源化抗體的激動劑(agonist)活性評估
抗c-Met抗體阻止HGF/c-Met結合,也有可能啟動c-Met信號,即具有激動劑活性。抗c-Met激動劑活性不是本發明所需要的。為了檢測本發明抗體是否有激動劑活性,用c-Met磷酸化,人腎透明細胞癌皮膚轉移細胞(caki-1)增值,人肺癌H441細胞遷移等三項實驗進行檢測評估。
HGF與c-Met的結合啟動c-Met分子的酪胺酸磷酸化以及活化c-Met信號通路。因此,HGF啟動c-Met用於激動劑實驗的正對照,用人肺癌細胞株A459來評估誘導c-Met酪胺酸殘基1349處磷酸化的作用強弱。
將A549 cells懸浮在Ham’s F12K+2mM穀胺醯胺(Invitrogen,# 21127-022)+10%(v/v)胎牛血清(FBS)(GIBCO,
#10099141),取0.2mL細胞懸液加入到96孔板(Corning,# 3599),細胞濃度是60,000細胞/孔。37℃,5% CO2條件下孵育24小時。24小時後棄96孔的培養基,加入100μL的低血清培養基(Ham’s F12K+2mM穀胺醯胺+0.5% FBS)37℃,5% CO2條件下饑餓6小時。抗體用上面的低血清培養基進行稀釋(終濃度為20μg/ml)。陽性對照HGF的濃度是200ng/ml。37℃孵育15分鐘。棄掉培養基後,加入50μl細胞裂解液(10mM Tris,150mM NaCl,2mM EDTA),50mM NaF,1%(v/v)TRITON-X 100,蛋白酶抑制劑(Roche cat # 05892791001),磷酸酶抑制劑cocktail II(Sigma #P5726)及磷酸酶抑制劑cocktail III(Sigma #P0044)。細胞裂解後用ELISA方法檢測c-Met酪胺酸磷酸化。c-Metcapture antibody(CST,cat#3148s)用PBS 1:1000稀釋後加入到96孔ELISA板(costar,cat#9018),每個孔100μl,在4℃孵育過夜。用TBS-T洗滌3次後加入300μl封閉液(TBS-T plus 2%(w/v)BSA)孵育1小時。用TBS-T洗滌3次後加入75μL細胞封閉液,加入25μl細胞裂解物,4℃孵育過夜。用TBS-T洗滌3次,用封閉液1:1000稀釋pY1349 c-Met抗體(cell signal,#3133),每個孔100μl。室溫孵育2小時後TBST洗滌4次,用封閉液1:12000稀釋HRP標記的羊抗兔多株抗體(cell signaling,cat#7074),每個孔100μl室溫孵育1小時。TBS-T洗滌5次,加入100μL TMB(ebioscience#TMB,004201)到每個孔,再加入100μl終止液(2N H2SO4),用450nM酶標儀(Supplier:Moleculer Devices;
Model:MNR0643;Equip ID:TMRP001)讀數。使用SoftMax Pro v5進行資料分析。結果見表8。
人腎透明細胞癌皮膚(Caki-1)細胞表現肝細胞生長因子受體(c-Met),HGF能結合c-Met刺激Caki-1細胞增殖。因此,本發明的人源化抗c-Met抗體和HGF平行出來Caki-1細胞,可評價抗c-Met抗體的激動劑活性。
Caki-1(上海中科院,TCHu135,P12)細胞1000個/孔加至96孔細胞培養板(costar,#3799),培養基為McCoy’s 5A(invitrogen,#16600)+10%胎牛血清(FBS)(GIBCO-10099141),37℃,24小時。隨後細胞饑餓24小時(細胞饑餓培養基為McCoy’s 5A+0.5%胎牛血清)。饑餓後,細胞用梯度稀釋的抗c-Met抗體(最高濃度為20μg/ml)及陽性對照處理5天後,用細胞增殖檢測試劑盒(CellTiter-Glo® Luminescent Cell Viability Assay(Promega,G7573)檢測細胞增殖情況。讀板儀(廠商:PerkinElmer設備編號:TREA001-RDA-IBA100)讀板。計算細胞的增值百分率:細胞增殖%=實驗組細胞讀數/不作處理組細胞讀數×100%。結果表明,本發明抗體均對Caki-1細胞沒有增殖作用。見表8。
抗c-Met抗體如果有激動劑活性,能影響細胞的遷移能力。本發明用表現c-Met的H441細胞株來評估本發明c-Met抗體影響細胞遷移能力。
在具有10%(v/v)胎牛血清(FBS)(GIBCO,Cat No.:10099-141)和青黴素/鏈黴素(GIBCO,Cat No.:15070-063)
的RPMI 1640培養基(GIBCO,Cat No.:11835-030)中重懸浮H441細胞(ATCC,Cat No.:HTB-174)至500,000細胞/ml。將重懸的H441細胞以1ml/孔加入12孔培養板(Costar,Cat No.:3513)中,在37℃,5% CO2下培養3天,用磷酸鹽緩衝液(PBS)洗兩遍細胞,加入含低濃度胎牛血清(0.5% FBS)的RPMI 1640培養基,37℃,5% CO2下培養16小時。用5ml槍頭在每個孔的底部劃痕,並用含低濃度胎牛血清培養基清洗一遍,加入1ml低濃度血清培養基RPMI 164(),在4倍倒置顯微鏡下隨機選取劃痕區域照相保存並在培養板做好標記,此時的記為時間零點,細胞用10μg/ml的c-Met抗體或HGF對照(200ng/ml)處理(37℃,5% CO2)16小時後,在4倍倒置顯微鏡下將已標記的劃痕區域照相保存,此時的記為遷移後時間。影響細胞遷移百分比為相對於零點時的遷移距離除以培養基組相對於零點時的遷移距離乘以100。結果見表8。
上述結果表明,本發明人源化抗體激動劑活性比較弱(c-Met磷酸化,H441遷移實驗結果)或沒有(20μg/ml抗體未能看到刺激Caki-1增殖)。
實施例10 抗c-Met抗體體內藥效評價
為了評價本發明抗體的抗腫瘤活性,用BALB/c裸小鼠皮下移植人源胃癌MKN45細胞模型進行檢測。
MKN45細胞用RPMI-1640培養基(10%胎牛血清)單層培養,培養條件為37℃,5% CO2。在對數生長期計數並且收集細胞。將細胞用PBS重懸至合適濃度,在小鼠(BALB/c Nude小鼠,雌性,10週,體重22至28g。購自上海斯萊克實驗動物有限公司,動物合格證編號:2007000548777。飼養環境:SPF級。)右翼皮下接種0.1m13×106細胞。腫瘤平均體積達到114mm3時,稱量體重,測量腫瘤體積,分組,開始給藥。對照組給PBS,抗體治療組給本發明抗體5mg/kg,每週一次,給藥兩次。每週2次,測量腫瘤體積和體重,第25天終止試驗。腫瘤大小計算公式:腫瘤體積(mm3)-0.5×(腫瘤長徑×腫瘤短徑2)。抑瘤率計算公式:抑瘤率=(V0-VT)/V0×100%,其中V0、VT分別為實驗開始時及實驗結束時的腫瘤體積。
結果表明本發明抗體Ab-9,Ab-10,的腫瘤抑制率分別為56%,和64%。小鼠體重在試驗過程中無明顯變化(22至24g)。該結果表明,本發明人源化抗c-Met抗體在體內有抑制腫瘤生長的活性。
實施例11 抗c-Met抗體的內吞作用
本發明抗體結合人c-Met,具有非常好的體外活性,體內抑制腫瘤活性。此外,該抗體沒有或有很弱的激動劑活性。為了檢測本發明抗體結合人c-Met後,是否能夠和人c-Met一起內吞到細胞內,用表現c-Met的人胃癌細胞MKN45(JCRB,Cat No.:JCRB0254)進行了評估。
在具有10%(v/v)胎牛血清(FBS)(GIBCO,Cat No.:10099-141)和青黴素/鏈黴素(GIBCO,Cat No.:15070-063)的RPMI 1640培養基(GIBCO,Cat No.:11835-030)中重懸浮MKN45細胞至10000,000細胞/mL。將2mL重懸的MKN45細胞以250,000細胞/孔加入96孔微滴定板中,加入10μg/ml的c-Met抗體至對應的孔中,最終體積為100μl,在4℃孵育1小時。加入FACS緩衝液(具有2.5%胎牛血清的磷酸鹽緩衝液(Hyclone,Cat:SH30256.01B),4℃,1300rpm,4分鐘,離心棄上清,重複三次。每孔加入100ul二抗溶液(螢光標記羊抗鼠二次抗體:1:200稀釋,Biolegend,Cat#405307;螢光標記抗人二次抗體:1:30稀釋,Biolegend,Cat#409304),在4℃孵育1小時。加入FACS緩衝液,4℃,1300rpm,4分鐘,離心棄上清,重複三次。加入細胞完全培養基(具有10%胎牛血清的RPMI 1640培養基),在37℃,5% CO2下孵育0小時,0.5小時,1小時,2小時,4小時。每孔加入5ul 7-AAD(Biolegend,Cat:420403)至100μl FACS緩衝液中,在4℃孵育30分鐘,加入FACS緩衝液,4℃,1300rpm,4分鐘,離心棄上清,重複三次。加入200μl Stripping buffer(0.05M甘胺酸,pH 3.0;
0.1M氯化鈉,按照1:1(v/v)混勻)至每孔細胞中,重懸細胞,室溫孵育7分鐘。室溫,1300rpm,4分鐘,離心棄上清。加入200μl中和洗液(0.15M三羥甲基胺基甲烷,pH 7.4)至每孔細胞中,重懸細胞,室溫,1300rpm,4分鐘,離心棄上清。加入200μl FACS緩衝液,重懸細胞,準備好樣品,使用流式細胞儀(BD FACS Calibur)檢測。結果見表9。
c-Met抗體內吞百分比=(各個時間點螢光強度值-零點時的平均螢光強度值)/零點時的平均螢光強度值。
表9結果表明,本發明抗體除了沒有激動劑活性外,還有很好的內吞作用。結合靶細胞後,抗體和受體一起迅速內吞到靶細胞內,2-4小時內吞達到最大值。
實施例12 抗c-Met抗體生物物理穩定性特性分析
為了評估本發明抗體的生物物理穩定性,比如糖基化,脫胺基化位點是否存在,以及其穩定性等,用質譜(LC-MS)分析方法對本發明抗c-Met抗體進行了綜合評價。
樣品直接LC-MS檢測輕重鏈分子量分析糖基化。在4℃長時間(3個月以上),或40℃,21天加速條件下,LC-MS分析脫胺基化。不同條件處理樣品後,取出樣品,用pH7.2Tris-HCl稀釋樣品至2mg/ml,加入終濃度為10mM TCEP和6M尿素(AMRESCO,Cat# 0378)3 7℃孵育20min.加入終濃度為20mM IAA(Sigma-Aldrich,Cat#I1149)室溫避光孵育15min保護巰基,用pH 7.2 Tris-HCl稀釋樣品以調節pH,按照蛋白:酶=10:1(重量比)的比例添加蛋白酶(Sigma-Aldrich,Cat# T6567),37℃孵育25min後添加終濃度為0.1%的甲酸(Fluca,Cat#94318)終止反應,離心上LC-MS分析。
用BiopharmaLynx來分析有無脫醯胺基作用。所得到的質譜資料藉由找到包含脫醯胺化位點的原生肽(native peptide)和修飾產物,提取母離子得到EIC(Extracted Ion Chromatogram)圖,積分得到峰面積並計算脫醯胺化和氧化產物所占比例。結果見表10。
上述結果表明,本發明的抗體穩定,物理性能好。
實施例13、抗c-Met抗體Ab-10偶聯毒素MC-MMAF
本發明抗c-Met抗體具有受體結合阻止活性、無激動劑活性、具有靶細胞內吞活性和物理穩定性等特性,這些特性使得本發明抗體特別適合和毒素偶聯成ADC藥物用於c-Met表現癌症治療。偶聯過程見下圖:
第一步將硫代乙酸S-(3-羰基丙基)酯(0.7mg,5.3μmol)溶解於0.9mL乙腈溶液,備用。向Ab-10單抗pH=4.3的乙酸/乙酸鈉緩衝液(10.35mg/ml,9.0mL,0.97mmol)加入上述預製的硫代乙酸S-(3-羰基丙基)酯的乙腈溶液,然後滴加1.0mL的氰基硼氫化鈉(14.1mg,224μmol)的水溶液,於25℃下振盪反應2小時。反應結束後,用Sephadex G25凝膠管柱脫鹽純化(洗脫相:pH為6.5的0.05M的PBS溶液)後,得產物1b溶液,濃縮到約10mg/ml後直接進行下一步反應。
第二步,向1b溶液(11.0mL)中加入0.35mL的2.0M鹽酸羥胺溶液,於25℃下振盪反應30分鐘後,將反應液用Sephadex G25凝膠管柱脫鹽純化(洗脫相:pH為6.5的0.05M的PBS溶液)後,得標題產物Ab-10單抗-丙硫醇1c溶液(濃度6.17mg/ml,14.7mL)。
第三步,將化合物MC-MMAF(1.1mg,1.2μmol,採用
PCT專利WO2005081711公開的方法製備而得)溶解於0.3mL乙腈中,加入Ab-10單抗-丙硫醇溶液1c(6.17mg/mL,3.0mL)中,於25℃下振盪反應4小時後,將反應液用Sephadex G25凝膠管柱脫鹽純化(洗脫相:pH為6.5的含0.05M的PBS溶液),在無菌條件下藉由0.2μm濾器過濾後得標題產物ADC-1的PBS緩衝液(3.7mg/mL,4.7mL),於4℃冷凍儲存。
Q-TOF LC/MS:特徵峰:148119.2(MAb+0D)、149278.1(MAb+1D)、150308.1(MAb+2D)、151314.1(MAb+3D)。分析得每個抗體分子連接毒素量(DAR)平均值:y=1.7。
實施例14、抗c-Met抗體Ab-10偶聯毒素MC-VC-PAB-MMAE
將化合物MC-VC-PAB-MMAE(1.6mg,1.2μmol,採用PCT專利WO2004010957公開的方法製備而得)溶解於0.3mL乙腈中,加入Ab-10單抗-丙硫醇溶液1c(6.17mg/mL,3.0mL)中,於25℃下振盪反應4小時後,將反應液用Sephadex G25凝膠管柱脫鹽純化(洗脫相:pH為6.5的含0.05M的PBS溶液),在無菌條件下藉由0.2μm濾器過濾後得標題產物ADC-2的PBS緩衝液(3.6mg/mL,4.8mL),於4℃冷凍儲存。
Q-TOF LC/MS:特徵峰:148118.4(MAb+0D)、149509.2
(MAb+1D)、150903.1(MAb+2D)、152290.4(MAb+3D)、153680.7(MAb+4D)。分析得每個抗體分子連接毒素量(DAR)平均值:y=1.8。
實施例15、抗c-Met抗體Ab-10偶聯毒素MC-VC-PAB-MMAF
將化合物MC-VC-PAB-MMAF(1.6mg,1.2μmol,採用PCT專利WO2005081711公開的方法製備而得)溶解於0.3mL乙腈中,加入Ab-10單抗-丙硫醇溶液1c(6.17mg/mL,3.0mL)中,於25℃下振盪反應4小時後,將反應液用Sephadex G25凝膠管柱脫鹽純化(洗脫相:pH為6.5的含0.05M的PBS溶液),在無菌條件下藉由0.2μm濾器過濾後得標題產物ADC-3的PBS緩衝液(3.5mg/mL,4.9mL),於4℃冷凍儲存。
Q-TOF LC/MS:特徵峰:148119.1(MAb+0D)、149525.3(MAb+1D)、150930.7(MAb+2D)、152335.2(MAb+3D)、153739.8(MAb+4D)。分析得每個抗體分子連接毒素量(DAR)平均值:y=1.6。
實施例16、抗c-Met抗體Ab-10偶聯毒素MC-MMAE
化合物MC-MMAE(1.2mg,1.2μmol,採用專利申請”US7/750/116 B1”公開的方法製備而得)溶解於0.3mL乙腈中,加入Ab-10單抗-丙硫醇溶液1c(6.17mg/mL,3.0mL)中,於25℃下振盪反應4小時後,將反應液用Sephadex G25凝膠管柱脫鹽純化(洗脫相:pH為6.5的含0.05M的PBS溶液),在無菌條件下藉由0.2μm濾器過濾後得標題產物ADC-4的PBS緩衝液(3.4mg/mL,5.0mL),於4℃冷凍儲存。
Q-TOF LC/MS:特徵峰:148118.6(MAb+0D)、149104.3(MAb+1D)、150090.1(MAb+2D)、151075.8(MAb+3D)。分析得每個抗體分子連接毒素量(DAR)平均值:y=1.6。
實施例17、抗c-Met抗體Ab-9偶聯毒素MC-MMAE
第一步,將硫代乙酸S-(3-羰基丙基)酯(0.7mg,5.3μmol),溶解於0.9mL乙腈溶液,備用;向Ab-9單抗pH=4.3的乙酸/乙酸鈉緩衝液(10.85mg/ml,9.0mL,0.976mmol)加入上述預製的硫代乙酸S-(3-羰基丙基)酯的乙腈溶液,
然後滴加1.0mL的氰基硼氫化鈉(14.1mg,224μmol)的水溶液,於25℃下振盪反應2小時。反應結束後,用Sephadex G25凝膠管柱脫鹽純化(洗脫相:pH為6.5的0.05M的PBS溶液)後,得標題產物5b溶液,濃縮到約10mg/ml後直接進行下一步反應。
第二步,向5b溶液(11.0mL)中加入0.35mL的2.0M鹽酸羥胺溶液,於25℃下振盪反應30分鐘後,將反應液用Sephadex G25凝膠管柱脫鹽純化(洗脫相:pH為6.5的0.05M的PBS溶液)後,得標題產物Ab-9單抗-丙硫醇5c溶液(濃度6.2mg/ml,15.0mL)。
第三步,將化合物MC-MMAE(1.1mg,1.2μmol)溶解於0.3mL乙腈中,加入Ab-9單抗-丙硫醇溶液5c(6.2mg/mL,3.0mL)中,於25℃下振盪反應4小時後,將反應液用Sephadex G25凝膠管柱脫鹽純化(洗脫相:pH為6.5的含0.05M的PBS溶液),在無菌條件下藉由0.2μm濾器過濾後得標題產物ADC-5的PBS緩衝液(3.8mg/mL,4.6mL),於4℃冷凍儲存。
Q-TOF LC/MS:特徵峰:150530.9(MAb+0D)、151915.7(MAb+1D)、153333.6(MAb+2D)、154763.4(MAb+3D)、156271.9(MAb+4D)。分析得每個抗體分子連接毒素量(DAR)平均值:y=1.5。
實施例18、抗c-Met抗體Ab-9偶聯毒素MC-MMAF
將化合物MC-MMAF(1.1mg,1.2μmol)溶解於0.3mL乙腈中,加入Ab-9單抗-丙硫醇溶液5c(6.2mg/mL,3.0mL)中,於25℃下振盪反應4小時後,將反應液用Sephadex G25凝膠管柱脫鹽純化(洗脫相:pH為6.5的含0.05M的PBS溶液),在無菌條件下藉由0.2μm濾器過濾後得標題產物ADC-6的PBS緩衝液(3.8mg/mL,4.6mL),於4℃冷凍儲存。
Q-TOF LC/MS:特徵峰:150537.8(MAb+0D)、152087.9(MAb+1D)、153486.5(MAb+2D)、154911.7(MAb+3D)、156499.9(MAb+4D)。分析得每個抗體分子連接毒素量(DAR)平均值:y=1.7。
實施例19、抗c-Met抗體Ab-9偶聯毒素MC-VC-PAB-MMAF
將化合物MC-VC-PAB-MMAF(1.6mg,1.2μmol)溶解於0.3mL乙腈中,加入Ab-9單抗-丙硫醇溶液5c(6.2mg/mL,3.0mL)中,於25℃下振盪反應4小時後,將反應液用Sephadex G25凝膠管柱脫鹽純化(洗脫相:pH為6.5的含
0.05M的PBS溶液),在無菌條件下藉由0.2μm濾器過濾後得標題產物ADC-7的PBS緩衝液(3.8mg/mL,4.6mL),於4℃冷凍儲存。
Q-TOF LC/MS:特徵峰:150537.8(MAb+0D)、152087.9(MAb+1D)、153486.5(MAb+2D)、154911.7(MAb+3D)、156499.9(MAb+4D)。分析得每個抗體分子連接毒素量(DAR)平均值:y=1.8。
實施例20、抗c-Met抗體Ab-9偶聯毒素MC-VC-PAB-MMAE
將化合物MC-VC-PAB-MMAE(1.6mg,1.2μmol)溶解於0.3mL乙腈中,加入Ab-9單抗-丙硫醇溶液5c(6.2mg/mL,3.0mL)中,於25℃下振盪反應4小時後,將反應液用Sephadex G25凝膠管柱脫鹽純化(洗脫相:pH為6.5的含0.05M的PBS溶液),在無菌條件下藉由0.2μm濾器過濾後,得標題產物ADC-8的PBS緩衝液(3.8mg/mL,4.6mL),於4℃冷凍儲存。
Q-TOF LC/MS:特徵峰:150508.6(MAb+0D)、151903.6(MAb+1D)、153314.5(MAb+2D)、154747.8(MAb+3D)、156039.5(MAb+4D)。分析得每個抗體分子連接毒素量(DAR)平均值:y=1.6。
實施例21、抗c-Met抗體Ab-10偶聯毒素SMCC-DM1
將SMCC(4-(N-馬來醯亞胺基甲基)環己烷-1-羧酸琥珀醯亞胺酯(1.65mg,4.94μmol,購自上海瀚鴻化工科技有限公司,批號BH-4857-111203),溶解於0.9mL乙腈溶液,備用;向Ab-10單抗pH=6.5的PBS緩衝液(10.15mg/ml,9.0mL,0.62μmol)加入上述預製的4-(N-馬來醯亞胺基甲基)環己烷-1-羧酸琥珀醯亞胺酯的乙腈溶液,於25℃下振盪反應2小時。反應結束後用Sephadex G25凝膠管柱脫鹽純化(洗脫相:pH為6.5的0.05M的PBS溶液)後得標題產物9b
溶液,濃縮到約10mg/ml(8.3mg/ml,11ml)後直接進行下一步反應。
向9b溶液(11.0mL)中加入3.0mg的L-DM1(採用公知的方法文獻“Journal of Medicinal Chemistry.2006,49,4392-4408”製備而得)乙醇溶液(3.0mgL-DM1/1.1ml乙醇),於25℃下振盪反應約4.0小時後,將反應液用Sephadex G25凝膠管柱脫鹽純化(洗脫相:pH為6.5的0.05M的PBS溶液)後,得標題產物ADC-9溶液(濃度6.3mg/ml,14mL),於4℃冷凍儲存。
Q-TOF LC/MS:特徵峰:148119.6(MAb+0D)、149078.1(MAb+1D)、149836.4(MAb+2D)、150593.7(MAb+3D)、151552.5(MAb+4D)。
平均值:y=2.3。
實施例22、抗c-Met抗體Ab-9偶聯毒素SMCC-DM1
將SMCC(4-(N-馬來醯亞胺基甲基)環己烷-1-羧酸琥珀醯亞胺酯(1.65mg,4.94μmol),溶解於0.9mL乙腈溶液,備用;向Ab-9單抗pH=6.5的PBS緩衝液(10.15mg/ml,9.0mL,0.62umol)加入上述預製的4-(N-馬來醯亞胺基甲基)環己烷-1-羧酸琥珀醯亞胺酯的乙腈溶液,於25℃下振盪反應2小時。反應結束後用Sephadex G25凝膠管柱脫鹽純化(洗脫相:pH為6.5的0.05M的PBS溶液)後,得標題產物10b溶液,濃縮到約10mg/ml(8.3mg/ml,11ml)後直接進行下一步反應。
向9b溶液(11.0mL)中加入3.0mg的L-DM1(3.0mgL-DM1/1.1ml乙醇)乙醇溶液,於25℃下振盪反應約4.0小時後,將反應液用Sephadex G25凝膠管柱脫鹽純化(洗脫相:pH為6.5的0.05M的PBS溶液)後得標題產物
ADC-10溶液(濃度6.3mg/ml,14mL),於4℃冷凍儲存。
Q-TOF LC/MS:特徵峰:150534.2(MAb+0D)、151492.6(MAb+1D)、152451.7(MAb+2D)、153409.7(MAb+3D)、154368.1(MAb+4D)。
平均值:y=2.2。
實施例23、抗c-Met抗體Ab-9偶聯毒素-SN-38
將化合物MC-VC-PAB-SN-38(1.3mg,1.2μmol)溶解於0.3mL乙腈中,加入Ab-9單抗-丙硫醇溶液5c(6.2mg/mL,3.0mL)中,於25℃下振盪反應4小時後,將反應液用Sephadex G25凝膠管柱脫鹽純化(洗脫相:pH為6.5的含0.05M的PBS溶液),在無菌條件下藉由0.2μm濾器過濾後,得標題產物ADC-11的PBS緩衝液(3.7mg/mL,4.5mL),
於4℃冷凍儲存。
Q-TOF LC/MS:特徵峰:150537.1(MAb+0D)、151786.6(MAb+1D)、152948.6(MAb+2D)、154161.7(MAb+3D)、155365.9(MAb+4D)、156477.8(MAb+5D)。
平均值:y=2.6。
實施例24、抗c-Met抗體Ab-10偶聯毒素
將原料((S)-2-胺基-3-(2-氟苯基)丙酸12a(400mg,2.18mmol,採用公知的方法“Advanced Synthesis & Catalysis,2012,354(17),3327-3332”製備而得)溶於10ml乙酸第三丁酯,加入高氯酸(300mg(70%),3.3mmol),於室溫下攪拌16小時。反應完畢後加入6Ml水,分液,有機相用飽和碳酸氫鈉溶液(5Ml)洗滌。水相用飽和碳酸氫鈉溶液調節至Ph=8,二氯甲烷(5Ml×3)萃取,合併有機相,依次用
水(3Ml),飽和氯化鈉溶液(5Ml)洗滌,無水硫酸鈉乾燥,過濾,濾液減壓濃縮得粗品標題產物(S)-第三丁酯2-胺基-3-(2-氟苯基)丙酸12b(390mg,黃色油狀物),產品不經純化直接進行下一步反應。
將原料(2R,3R)-3-((1S,3S,5S)-2-(第三丁氧羰基)-2-氮雜雙環[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙酸12e(100mg,0.334mmol)溶於6Ml二氯甲烷和二甲基甲醯胺(V/V=5:1)混合溶劑中,加入反應物粗品(S)-第三丁酯2-胺基-3-(2-氟苯基)丙酸12b(80mg,0.334mmol)。再加入N,N-二異丙基乙基胺(0.29Ml,1.67mmol)和2-(7-偶氮苯並三氮唑)-N,N,N’,N’-四甲基脲六氟磷酸酯(152.3mg,0.40mmol)。反應體系在氬氣氛下,於室溫攪拌1小時。反應結束後,加10Ml水攪拌,分層,二氯甲烷層用飽和氯化鈉溶液(10Ml)洗滌,無水硫酸鈉乾燥,過濾,濾液減壓濃縮。用矽膠管柱色譜法以洗脫劑體系B純化所得殘餘物,得到標題產物(1S,3S,5S)-第三丁酯3-((1R,2R)-3-(((S)-1-(第三丁氧基)-3-(2-氟苯基)-1-羰基丙基-2-基)胺基)-1-甲氧基-2-甲基-3-羰基丙基)-2-氮雜雙環[3.1.0]己烷-2-羧酸12c(173mg,無色液體),收率99.5%。
MS m/z(ESI):521.2[M+1]
將原料(1S,3S,5S)-第三丁酯3-((1R,2R)-3-(((S)-1-(第三丁氧基)-3-(2-氟苯基)-1-羰基丙基-2-基)胺基)-1-甲氧基-2-甲基-3-羰基丙基)-2-氮雜雙環[3.1.0]己烷-2-羧酸12c(173mg,0.33mmol)溶於2Ml二噁烷中,加入5.6M的氯化氫二噁烷溶液(0.21Ml,1.16mmol),氬氣氛下,於室溫攪拌1小時,置於0℃冰箱內12小時。反應結束後,將反應液減壓濃縮,加入5Ml二氯甲烷稀釋,加入10Ml飽和碳酸氫鈉溶液,攪拌10分鐘。體系分層,水層用二氯甲烷萃取(5Ml×3)。合併二氯甲烷層,用飽和氯化鈉溶液(10Ml)洗滌,無水硫酸鈉乾燥。過濾,濾液減壓濃縮,得到粗品標題產品(S)-第三丁酯2-((2R,3R)-3-((1S,3S,5S)-2-氮雜雙環[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙醯胺)-3-(2-氟苯基)丙酸12d(77mg,黃色液體),產品不經純化直接進行下一步反應。
MS m/z(ESI):421.2[M+1]
將粗品(S)-第三丁酯2-((2R,3R)-3-((1S,3S,5S)-2-氮雜雙環[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙醯胺)-3-(2-氟苯基)丙酸12d(77mg,0.183mmol),(5S,8S,11S,12R)-11-((S)-第二丁基)-1-(9H-芴-9-基)-5,8-二異丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧雜-4,7,10-三氮雜十四烷-14-羧酸12i(116.8mg,0.183mmol,採用專利申請“WO 2013072813”公開的方法製備而得)溶於6Ml二氯甲烷和二甲基甲醯胺(V/V=5:1)混合溶劑中,加入N,N-二異丙基乙基胺(0.16Ml,0.915mmol)和2-(7-偶氮苯並三氮唑)-N,N,N’,N’-四甲基脲六氟磷酸酯(84mg,0.22mmol)。反應體系在氬氣氛下,於室溫下攪拌1小時。反應結束後,加入10Ml水攪拌,分層。二氯甲烷層用飽和氯化鈉溶液(10Ml)洗滌,無水硫酸鈉乾燥。過濾,濾液減壓濃縮。用矽膠管柱色譜法以洗脫劑體系B純化殘留物,得到標題產品(S)-第三丁酯2-((2R,3R)-3-((1S,3S,5S)-2-((5S,8S,11S,12R)-11-((S)-第二丁基)-1-(9H-芴-9-基)-5,8-二異丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧雜-4,7,10-三氮雜十四烷基-14-醯基)-2-氮雜雙環[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙醯胺)-3-(2-氟苯基)丙酸12e(190.5mg,黃色黏稠物),收率100%。
MS m/z(ESI):1040.6[M+1]
將原料(S)-第三丁酯2-((2R,3R)-3-((1S,3S,5S)-2-((5S,8S,11S,12R)-11-((S)-第二丁基)-1-(9H-芴-9-基)-5,8-二異丙基-12-甲氧基-4,10-二甲基-3,6,9-三羰基-2-氧雜-4,7,10-三氮雜十四烷基-14-醯基)-2-氮雜雙環[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙醯胺基)-3-(2-氟苯基)丙酸12e(190.5mg,0.183mmol)溶於1.5Ml二氯甲烷中,加入2Ml二乙胺。反應體系在氬氣氛下,於室溫攪拌3小時。反應結束後,將反應液減壓濃縮,得到粗品標題產品(S)-第三丁酯2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基胺基)丁醯胺基)丁醯胺基)-3-甲氧基-5-甲基庚醯基)-2-氮雜雙環[3,1.0]己烷-3-基)-3-甲氧基-2-甲基丙醯胺)-3-(2-氟苯基)丙酸12f(150mg,黃色黏稠物),產品不經純化直接進行下一步反應。
MS m/z(ESI):818.5[M+1]
將粗品(S)-第三丁酯2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基胺基)丁醯胺基)丁醯胺基)-3-甲氧基-5-甲基庚醯基)-2-氮雜雙環
[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙醯胺基)-3-(2-氟苯基)丙酸12f(150mg,0.183mmol)溶於1Ml二噁烷中,加入5.6M的氯化氫二噁烷溶液3Ml,氬氣氛下,於室溫攪拌12小時。反應結束後,將反應液減壓濃縮,用乙醚帶旋溶劑。所得殘餘物用高效液相色譜法純化得標題產品(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基胺基)丁醯胺基)丁醯胺基)-3-甲氧基-5-甲基庚醯基)-2-氮雜雙環[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙醯胺基)-3-(2-氟苯基)丙酸12g(28mg,白色粉末固體),收率20%。
MS m/z(ESI):762.7[M+1]
1H NMR(400MHz,CD3OD):δ 7.38-7.18(m,2H),7.13-7.01(m,2H),4.80-4.67(m,2H),4.30-4.15(m,1H),4.13-4.01(m,1H),3.96-3.83(m,2H),3.75-3.60(m,2H),3.42-3.11(m,9H),3.06-2.95(m,1H),2.70-2.58(m,4H),2.28-2.01(m,4H),1.88-1.70(m,3H),1.57-1.25(m,4H),1.22-0.95(m,18H),0.92-0.80(m,4H),0.78-0.65(m,1H).
將原料(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-N,3-二甲基-2-((S)-3-甲基-2-(甲基胺基)丁醯胺基)丁醯胺基)-3-甲氧基-5-甲基庚醯基)-2-氮雜雙環[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙醯胺基)-3-(2-氟苯基)丙酸12g(25mg,0.033mmol)溶於3mL二氯甲烷中,加入N,N-二異丙基乙基胺(0.029mL,0.164mmol),反應體系在氬氣氛下,冰浴下滴加預製的6-(2,5-二羰基-2,5-二氫-1H-吡咯-1-基)己醯氯4b(11.3mg,0.049mmol)的二氯甲烷溶液,於室溫反應3小時。反應結束後,加入5mL水,攪拌20分鐘,分液,有機層用無水硫酸鈉乾燥,過濾,濾液減壓濃縮,殘留物用高效液相色譜法純化得標題產物(S)-2-((2R,3R)-3-((1S,3S,5S)-2-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(2,5-二羰基-2,5-二氫-1H-吡咯-1-基)-N-甲基己醯胺基)-3-甲基丁醯胺基)-N,3-二甲基丁醯胺基)-3-甲氧基-5-甲基庚醯基)-2-氮雜雙環[3.1.0]己烷-3-基)-3-甲氧基-2-甲基丙醯胺基)-3-(2-氟苯基)丙酸12h(7mg,黃色黏稠物),收率22.4%。
MS m/z(ESI):955.4[M+1]
1H NMR(400MHz,CD3OD):δ 7.36-7.30(m,1H),7.29-7.21(m,1H),7.17-7.02(m,2H),6.83-6.79(m,2H),4.81-4.71(m,2H),4.69-4.55(m,2H),4.25-4.15(m,1H),4.13-4.04(m,1H),3.96-3.85(m,2H),3.70-3.61(m,1H),3.55-3.46(m,3H),3.40-3.21(m,4H),3.18-3.10(m,2H),3.07-2.96(m,4H),2.67-2.56(m,2H),2.54-2.34(m,3H),2.29-2.17(m,2H),2.10-1.99(m,1H),1.89-1.57(m,7H),1.52-1.28(m,6H),1.21-1.11(m,4H),1.07-0.96(m,6H),0.95-0.81(m,12H),0.80-0.69(m,1H).
將化合物12h(1.2mg,1.2μmol)溶解於0.3mL乙腈中,加入Ab-10單抗-丙硫醇1c溶液(6.17mg/mL,3.0mL)中,於
25℃下振盪反應4小時後將反應液用Sephadex G25凝膠柱脫鹽純化(洗脫相:pH為6.5的含0.05M的PBS溶液),在無菌條件下藉由0.2μm濾器過濾後得標題產物ADC-12的PBS緩衝液(3.3mg/mL,5.0mL),於4℃冷凍儲存。
Q-TOF LC/MS:特徵峰:148119.6(MAb+0D)、149150.5(MAb+1D)、150221.1(MAb+2D)、151265.1(MAb+3D)、152314.3(MAb+4D)。
平均值:y=1.6。
參照實施例13-24,製備實施例25-27的ADC化合物。
測試例1、抗c-Met抗體毒素偶聯(ADC)分子穩定性評價
本發明ADC分子或其它具有內吞作用的c-Met抗體-毒素偶聯物(如,LY-2875358-ADC)的毒素中間體和毒素,在PBS、人和猴血漿中進行了游離毒素的穩定性評價。
將實施例化合物ADC-1和ADC-12的毒素中間體和毒素用PBS、人或猴血漿(蘇州西山中科藥物研究開發有限公司,動物生產許可證號:SCXK(蘇)2012-0009)稀釋至500μg/mL,37℃孵育7天,於0、3、7天取樣測定樣品中游離毒素和毒素中間體的濃度。50μL含藥人、猴血漿或PBS樣品,加入20uL內對照(喜樹鹼,上海融禾醫藥科技發展有限公司,批號090107,100ng/ml),加入150μL乙腈,渦旋混勻3min,15000rpm,離心10min,取上清液80μL與80μL 0.2%甲酸混勻後,10μL進樣。標準曲線分析方法為50μL空白人、猴血漿或PBS樣品,加入50μL系列工作溶液,加入20μL內對照(喜樹鹼,100ng/ml),加入100μL乙腈,渦旋混勻3min,15000rpm,離心10min,取上清液80μL與80μL 0.2%甲酸混勻後,10μL進樣。
使用Shimadzu LC-30AD超高效液相色譜系統(日本島津公司),UPLC-MS/MS質譜儀為API4000三重四極杆串聯質譜儀(美國AB SCIEX公司),設定色譜條件(色譜管柱:Waters XBridgeTM BEH300 C18(100mm×4.6mm i.d.,3.5mm),流動相為0.2%甲酸-乙腈(梯度洗脫)。結果見表11、表12。
上述結果表明,本發明ADC-1,ADC-12的毒素中間體和毒素在各種溶劑(PBS,人血漿,猴血漿等)中穩定。37℃孵育0、3和7天均未檢測到降解產物游離毒素和毒素中間體(毒素-linker)。
測試例2、抗c-Met抗體毒素偶聯(ADC)分子體外活性
評價
本發明ADC-1,ADC-12的體外活性用FACS(檢測和c-Met陽性細胞的結合力)和內吞作用(方法見實施例11)評價。結果見表13。
上述結果表明,本發明抗體和毒素偶聯後,保留了抗體的結合活性和內吞活性。
測試例3、抗c-Met抗體毒素偶聯(ADC)分子細胞毒性實驗
為了評估本發明ADC分子對細胞的毒性作用,用細胞ATP毒性實驗進行了評估。ATP是活細胞新陳代謝的一個指標,檢測ATP可以反應分子對細胞的毒性大小。
HepG2細胞(中科院細胞庫,Cat# TCHu 72)培養在含10% FBS的EMEM完全培養基中,MKN45細胞培養在含10% FBS的RPMI1640完全培養基中,實驗時加入2至3ml胰蛋白酶消化2至3min,待細胞消化完全,加入10至15ml完全培養基將經過消化的細胞洗脫下來,1000rpm離心
3min,棄上清,接著加入10至20mk完全培養基將細胞重懸,製成單細胞懸液,調整細胞密度為4×104cells/ml。在96孔細胞培養板各孔中加0.1ml上述細胞懸液,37℃ 5% CO2的培養箱中培養,24小時後去掉培養基,每孔加入90μl含2%FBS的EMEM培養基或含2%FBS的RPMI1640培養基,將待測樣品(實施例13化合物和毒素)用PBS稀釋成不同濃度梯度,每孔加入10μl,37℃ 5% CO2的培養箱中孵育72小時。用CellTiter-Glo®Luminescent Cell Viability Assay試劑盒(Promega,Cat#G7571)按說明書檢測。用酶標儀(VICTOR 3,PerkinElmer公司)檢測化學發光,GraphPad Prism(version 5.0)軟體進行資料分析。結果將表14。
討論:上述結果表明,本發明ADC-1,ADC-12對c-Met陽性細胞MKN45的細胞毒性作用相同(IC50分別為0.51nM,和0.59nM)。但是各自的毒素部分對c-Met陽性細胞MKN45
的細胞毒性作用不同,兩者相差93倍(79.4/0.85)。
本發明ADC-1,ADC-12對c-Met陰性性細胞HepG2均沒有細胞毒性作用,表明ADC化合物具有特異靶向作用。但是各自的毒素部分對c-Met陰性細胞HepG2的細胞毒性作用不同,兩者相差82倍(400.8/4.88)。
這些結果表明,本發明ADC-1,ADC-12具有特異靶向作用,能抑制c-Met陽性細胞增值,但對非特異(正常細胞)沒有毒性作用。ADC-1,ADC-12不同之處在於,各自的游離毒素對靶向細胞和非靶向細胞的毒性不同。ADC-12的毒素部分對c-Met陽性細胞和陰性細胞HepG2的細胞毒性要比ADC-1的毒素部分弱93,82倍。因此,該分子到達靶細胞過程中,如果有游離的毒素被釋放,其非特異的毒性作用要比ADC-1弱。因而,毒副作用要小,安全性好。
測試例4、抗c-Met抗體毒素偶聯(ADC)分子對腫瘤細胞的增殖抑制作用
上述結果表明,ADC-1(實施例13)能夠特異殺死c-Met表現的腫瘤靶細胞。為了檢測該毒性作用對腫瘤細胞的增殖抑制所用。用本發明分子檢測了多種腫瘤細胞,採用CCK法測試樣品對細胞增殖的抑制作用,根據IC50大小評價本發明ADC分子體外細胞活性。
所用細胞及相應的培養基見下表15,用Cell Counting Kit(東仁化學科技有限公司,Cat#CK04)檢測細胞增值(按說明書進行操作)。
實驗時加入2至3ml胰蛋白酶消化2至3min,待細胞
消化完全,加入10至15ml完全培養基將經過消化的細胞洗脫下來,1000rpm離心3min,棄上清。接著加入10至20ml培養基將細胞重懸,製成單細胞懸液,調整細胞密度為4×104cells/ml。在96孔細胞培養板各孔中加0.1ml上述細胞懸液,37℃ 5% CO2的培養箱中培養,24小時後去掉培養基,每孔加入90μl含2%FBS的培養基,將待測樣品用PBS稀釋成不同濃度梯度,每孔加入10μl,37℃ 5% CO2的培養箱中孵育72小時。每孔加入10μl CCK8,培養箱中繼續孵育2小時,酶標儀(VICTOR 3,PerkinElmer公司)檢測OD450,採用GraphPad Prism(version 5.0)軟體進行資料分析。結果見表16。
表16結果表明本發明抗c-Met抗體在胃癌細胞系MKN45,SUN上有比較好的活性,而在其它c-Met表現低或不表現的腫瘤細胞,例如肺癌細胞上活性很弱,或者沒有。而本發明的ADC-1因為帶有額外的毒素,對c-Met表現的腫瘤細胞,包括胃癌細胞系MKN45,SUN,特別抗c-Met抗體沒有作用或作用非常微弱的是肺癌、胰腺癌和腎細胞癌細胞上顯示出很好的活性。
測試例5、抗c-Met抗體毒素偶聯(ADC)分子體內藥效評價
為了更好評價本發明抗c-Met抗體和ADC分子的抗腫瘤藥效活性,用實施例10方法對抗體Ab-10和ADC-1進
行了平行比較實驗。和實施例10不同的是,本測試例是單次給藥,觀察到腫瘤抑制作用直到有回復後趨勢停止試驗。
Ab-10(5mg/kg),原液(2.18mg/ml)用PBS配成終濃度0.5mg/ml;Ab-10(10mg/kg),原液(2.18mg/ml)用PBS配成終濃度1mg/ml;Ab-10(30mg/kg),原液(2.18mg/ml)用PBS配成終濃度3mg/ml;ADC-1(2.5mg/kg),原液(10mg/ml)用PBS配成終濃度0.25mg/ml;ADC-1(5mg/kg),原液(10mg/ml)用PBS配成終濃度0.5mg/ml;ADC-1(10mg/kg),原液(10mg/ml)用PBS配成終濃度1mg/ml;所有動物給藥方式均為尾靜脈注射,給藥體積0.2ml/隻。
裸小鼠右肋部皮下接種MKN-45細胞(1×106/隻),腫瘤生長至平均體積(150.19+8.44)mm3,隨機分組給藥,每組8隻。具體給藥方案見表17。
每週測2次瘤體積,稱體重,記錄資料。
使用Excel統計軟體:平均值以avg計算;SD值以STDEV計算;SEM值以STDEV/SQRT計算;組間差異P值
以TTEST計算。
腫瘤體積(V)計算公式為:V=1/2×L長×L短 2
抑瘤率=(V0-VT)/V0*100%
其中V0、VT分別為實驗開始時及實驗結束時的腫瘤體積。
結論:本發明抗體及ADC化合物對MKN-45裸小鼠移植瘤有明顯的療效。
為了評價本發明ADC-12的體內藥效,用上述同樣的試驗方法,平行比較了ADC-1和ADC-12。同樣劑量3mg/kg,單次給藥後,結果見表18。
上述結果表明ADC-1和ADC-12在11天抑瘤率相近,但是從15天之後,ADC-1的藥效減弱(21天為27.1%),而ADC-12的抑瘤作用仍然維持在第11天的水準(50.4%)。
測試例6、ADC-12對人肺癌NCI-H1993裸小鼠皮下移植瘤的療效
評價並比較ADC-12、Ab-10抗體原液對人肺癌NCI-H1993裸小鼠皮下移植瘤的療效。
ADC-12用注射用水溶解成20mg/ml溶液,分裝保存-80℃冰箱,臨用時用0.1% BSA生理鹽水稀釋成相應濃度;Ab-10抗體原液濃度16.3mg/ml,用0.1% BSA生理鹽水稀釋後,分裝保存於-80℃冰箱。
BALB/cA-nude裸小鼠,6-7週,♀,購自上海靈暢生物科技有限公司。生產許可證號:SCXK(滬)2013-0018;動物合格證號2013001814303。飼養環境:SPF級。
裸小鼠皮下接種人肺癌NCI-H1993細胞,待腫瘤生長
至100至150mm3後,將動物隨機分組(D0)。給藥劑量和給藥方案見表19。每週測2至3次瘤體積,稱鼠重,記錄資料。腫瘤體積(V)計算公式為:V=1/2×a×b2其中a、b分別表示長、寬。T/C(%)=(T-T0)/(C-C0)×100其中T、C為實驗結束時的腫瘤體積;T0、C0為實驗開始時的腫瘤體積。
ADC-12是抗c-Met抗體-毒素偶聯物。ADC-12(1、3、10mg/kg,IV,D0)劑量依賴性地抑制高表現c-Met人肺癌NCI-H1993裸小鼠皮下移植瘤的生長,抑瘤率分別為45%、63%、124%,10mg/kg劑量組有7/10腫瘤部分消退(D21);Ab-10抗體原液為製備ADC-12的裸抗體,Ab-10抗體原液(30mg/kg,IV,每週2次,共6次)對NCI-H1993的抑瘤率為42%;荷瘤小鼠對以上藥物均能很好耐受,沒有體重減輕等症狀發生。相比較,ADC-12對NCI-H1993的療效明顯強於Ab-10抗體原液。
討論:ADC-12(1、3、10mg/kg,IV,D0)劑量依賴性地抑制高表現c-Met人肺癌NCI-H1993裸小鼠皮下移植瘤的生長,引起腫瘤部分消退;Ab-10抗體原液(30mg/kg,IV,每週2次,共6次)對NCI-H1993也有效;ADC-12對NCI-H1993的療效明顯強於Ab-10抗體原液。荷瘤小鼠對以上藥物均能很好耐受。
<110> 江蘇恆瑞醫藥股份有限公司、上海恆瑞醫藥有限公司
<120> 抗c-Met抗體和抗c-Met抗體-細胞毒性藥物偶聯物及其醫藥用途
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Claims (66)
- 一種特異性結合c-Met受體的抗體或其抗原結合片段,其包含至少1個選自以下的CDR區序列或其突變序列:抗體重鏈可變區HCDR區序列:SEQ ID NO:6,SEQ ID NO:7或SEQ ID NO:8;和抗體輕鏈可變區LCDR區序列:SEQ ID NO:9,SEQ ID NO:10或SEQ ID NO:11。
- 如申請專利範圍第1項所述的特異性結合c-Met受體的抗體或其抗原結合片段,其中該抗體重鏈可變區包含至少1個選自如下的HCDR區序列或其突變序列:SEQ ID NO:6,SEQ ID NO:7或SEQ ID NO:8。
- 如申請專利範圍第1項所述的特異性結合c-Met受體的抗體或其抗原結合片段,其中該抗體輕鏈可變區包含至少1個選自如下的LCDR區序列或其突變序列:SEQ ID NO:9,SEQ ID NO:10或SEQ ID NO:11。
- 如申請專利範圍第1至3項中任一項所述的特異性結合c-Met受體的抗體或其抗原結合片段,其中該抗體包含重鏈可變區序列SEQ ID NO:6,SEQ ID NO:7和SEQ ID NO:8,或其突變序列,和輕鏈可變區序列SEQ ID NO:9,SEQ ID NO:10和SEQ ID NO:11或其突變序列。
- 如申請專利範圍第1至4項中任一項所述的特異性結合c-Met受體的抗體或其抗原結合片段,其中該突變序 列為CDR區發生1至3個優化抗體活性的胺基酸突變:。
- 如申請專利範圍第5項所述的特異性結合c-Met受體的抗體或其抗原結合片段,其中HCDR2區突變序列為SEQ ID NO:12。
- 如申請專利範圍第1至6項中任一項所述的特異性結合c-Met受體的抗體或其抗原結合片段,其中該特異性結合c-Met受體的抗體或其抗原結合片段為鼠源抗體或其片段。
- 如申請專利範圍第7項所述的特異性結合c-Met受體的抗體或其抗原結合片段,其中該鼠源抗體重鏈可變區序列為:SEQ ID NO:4。
- 如申請專利範圍第7項所述的特異性結合c-Met受體的抗體或其抗原結合片段,其中該鼠源抗體輕鏈可變區序列為:SEQ ID NO:5。
- 如申請專利範圍第7至9項中任一項所述的特異性結合c-Met受體的抗體或其抗原結合片段,其中該鼠源抗體的重鏈可變區為:SEQ ID NO:4,輕鏈可變區為:SEQ ID NO:5。
- 如申請專利範圍第1至5項中任一項所述的特異性結合c-Met受體的抗體或其抗原結合片段,其為嵌合抗體或人源化抗體或其片段。
- 如申請專利範圍第11項所述的特異性結合c-Met受體的抗體或其抗原結合片段,其中該人源化抗體重鏈可 變區上的重鏈FR區序列,來源於人種系重鏈序列;其中該重鏈FR區序列包含人種系重鏈IGHV 3-33*01的FR1,FR2,FR3區和FR4區的框架序列或其突變序列。
- 如申請專利範圍第12項所述的特異性結合c-Met受體的抗體或其抗原結合片段,其中人源化抗體重鏈可變區上的重鏈FR區序列,來源於人種系重鏈IGHV 3-33*01;其中該突變序列為在框架序列上發生0至10個胺基酸的回復突變。
- 如申請專利範圍第12項所述的特異性結合c-Met受體的抗體或其抗原結合片段,其中該人源化抗體包含選自SEQ ID NO:13-15所示的重鏈可變區序列或其變體。
- 如申請專利範圍第11項所述的特異性結合c-Met受體的抗體或其抗原結合片段,其中該人源化抗體輕鏈可變區上的輕鏈FR區序列,選自人種系輕鏈序列,其中該輕鏈FR區序列包含人種系輕鏈IGKV085和IGKV 4-1*01的FR1,FR2,FR3區和FR4區的框架序列或其突變序列。
- 如申請專利範圍第15項所述的特異性結合c-Met受體的抗體或其抗原結合片段,其中該人源化抗體輕鏈可變區上的輕鏈FR區序列,選自人種系輕鏈IGKV085或IGKV 4-1*01,其中該突變序列為在框架序列上發生0至10個胺基酸的回復突變。
- 如申請專利範圍第15項所述的特異性結合c-Met受體的抗體或其抗原結合片段,其中該人源化抗體包含選 自SEQ ID NO:16-18所示的輕鏈可變區序列或其變體。
- 如申請專利範圍第11至17項中任一項所述的特異性結合c-Met受體的抗體或其抗原結合片段,其中該人源化抗體包含選自SEQ ID NO:13-15的重鏈可變區序列和選自SEQ ID NO:16-18的輕鏈可變區序列。
- 如申請專利範圍第1至5項和11至18項中任一項所述的特異性結合c-Met受體的抗體或其抗原結合片段,其包含選自a)至c)任一的重鏈可變區序列和輕鏈可變區序列的组合:a)SEQ ID NO:13的重鏈可變區序列和SEQ ID NO:16的輕鏈可變區序列;b)SEQ ID NO:14的重鏈可變區序列和SEQ ID NO:17的輕鏈可變區序列;或c)SEQ ID NO:15的重鏈可變區序列和SEQ ID NO:18的輕鏈可變區序列。
- 如申請專利範圍第11至19項中任一項所述的特異性結合c-Met受體的抗體或其抗原結合片段,其中該人源化抗體的重鏈恒定區包含源自人源IgG1或其變體、人源IgG2或其變體、人源IgG3或其變體或人源IgG4或其變體的恒定區。
- 如申請專利範圍第20項中所述的特異性結合c-Met受體的抗體或其抗原結合片段,其中該人源化抗體的重鏈恒定區包含人源IgG1或其變體或人源IgG2或其變體或人源IgG4或其變體的恒定區。
- 如申請專利範圍第20項中所述的特異性結合c-Met受體的抗體或其抗原結合片段,其中該人源化抗體的重鏈恆定區包含人源IgG2或其變體的恆定區。
- 如申請專利範圍第20項所述的特異性結合c-Met受體的抗體或其抗原結合片段,其包含選自SEQ ID NO:23-25或與其具有至少90%同源性的全長重鏈序列。
- 如申請專利範圍第11至19項中任一項所述的特異性結合c-Met受體的抗體或其抗原結合片段,其中該人源化抗體的輕鏈恆定區包含選自人源κ或λ鏈或其變體的恆定區。
- 如申請專利範圍第24項所述的特異性結合c-Met受體的抗體或其抗原結合片段,其包含選自SEQ ID NO:26-28或與其具有至少90%序列同源性的全長輕鏈序列。
- 如申請專利範圍第11至25項中任一項所述的特異性結合c-Met受體的抗體或其抗原結合片段,其中該人源化抗體包含選自以下全長輕鏈序列和全長重鏈序列的组合;Ab-9:SEQ ID NO:23的重鏈序列和SEQ ID NO:26的輕鏈序列;Ab-10:SEQ ID NO:24的重鏈序列和SEQ ID NO:27的輕鏈序列;或Ab-11:SEQ ID NO:25的重鏈序列和SEQ ID NO:28的輕鏈序列。
- 一種DNA分子,其編碼如申請專利範圍第1至26項中任一項所述的特異性結合c-Met受體的抗體或其抗原結合片段。
- 一種表現載體,其含有如申請專利範圍第27項所述的DNA分子。
- 一種用如申請專利範圍第28項所述的表現載體轉化的宿主細胞。
- 如申請專利範圍第29項所述的宿主細胞,其中該宿主細胞為哺乳動物細胞。
- 如申請專利範圍第29項所述的宿主細胞,其中該宿主細胞為CHO細胞。
- 一種醫藥組成物,其包含如申請專利範圍第1至26項中任一項所述的特異性結合c-Met受體的抗體或其抗原結合片段,和一種或多種可藥用的赋形劑、稀释劑或載體。
- 一種通式(I)所示的抗體-細胞毒性藥物偶聯物或其藥學上可接受的鹽或溶劑化合物:Ab-[(L2)t-L1-D)]y (I)其中:D為藥物模組;L1,L2是接頭單元;t為0或1;y為1至8;Ab如申請專利範圍第1至26項中任一項所述的特 異性結合c-Met受體的抗體或其抗原結合片段。
- 如申請專利範圍第33項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其藥學上可接受的鹽或溶劑化合物,其中t為1。
- 如申請專利範圍第33項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其藥學上可接受的鹽或溶劑化合物,其中y為2至5。
- 如申請專利範圍第33項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其藥學上可接受的鹽或溶劑化合物,其中-L2-為以下通式(-L2-)所示的化合物:
- 如申請專利範圍第36項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其藥學上可接受的鹽或溶劑化合物,其中m為1至3。
- 如申請專利範圍第33項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物,其中該藥物模組D為選自毒素、化療劑、抗生素、放射性同位素和核溶酶的細胞毒劑。
- 如申請專利範圍第33項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其藥學上可接受的鹽或溶劑化合物,其中D為以下通式(D)所示的化合物:
- 如申請專利範圍第39項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其藥學上可接受的鹽或溶劑化合物,其中R8至R11至少其中一個選自鹵素、烯基、烷基和環烷基,其餘為氫原子。
- 如申請專利範圍第39項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物,其中L2包含選自Val-Cit,MC,PAB和MC-PAB的接頭。
- 如申請專利範圍第41項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物,其中L2包含MC。
- 如申請專利範圍第33項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物,其中D是美登木素生物鹼。
- 如申請專利範圍第43項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物,其中D是DM1、DM3和DM4。
- 如申請專利範圍第44項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物,其中D是DM1。
- 如申請專利範圍第43項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物,其中該L2選自N-琥珀醯亞胺基4-(2-吡啶基硫代)戊酸酯(SPP)、N-琥珀醯亞胺基4-(N-馬來醯亞胺基甲基)-環己烷-1-羧酸酯(SMCC)、和N-琥珀醯亞胺基(4-碘-乙醯基) 胺基苯甲酸酯(SIAB)。
- 如申請專利範圍第46項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物,其中該L2選自SPP或SMCC。
- 如申請專利範圍第33項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物,其中D是喜樹鹼類生物鹼。
- 如申請專利範圍第48項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物,其中D選自CPT、10-羥基-CPT、CPT-11(伊立替康)、SN-38和托泊替康。
- 如申請專利範圍第49項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物,其中D是SN-38。
- 如申請專利範圍第48項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物,其中該接頭L2包含任選自Val-Cit,MC,PAB或MC-PAB的結構。
- 如申請專利範圍第51項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物,其中該接頭L2包含MC或MC-vc-PAB。
- 如申請專利範圍第33項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其藥學上可接受的鹽或溶劑化合物,其為通式(II)所示的偶聯藥物或其藥學上可接受的 鹽或溶劑化合物:
- 如申請專利範圍第33項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其藥學上可接受的鹽或溶劑化合物,其為通式(III)所示的偶聯藥物或其藥學上可接受的鹽或溶劑化合物:
- 如申請專利範圍第54項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其藥學上可接受的鹽或溶劑化合 物,其中n為5。
- 如申請專利範圍第33項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其藥學上可接受的鹽或溶劑化合物,其為通式(IV)所示的偶聯藥物或其藥學上可接受的鹽或溶劑化合物:
- 如申請專利範圍第33項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其藥學上可接受的鹽或溶劑化合物,其為通式(V)所示的偶聯藥物或其藥學上可接受的鹽或溶劑化合物:
- 如申請專利範圍第33至57項中任一項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物,其中該抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物選自:
- 如申請專利範圍第58項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物,其中y的範圍為2至5。
- 一種製備如申請專利範圍第57項中所述的通式(V)所示的偶聯藥物或其藥學上可接受的鹽或溶劑化合物的方法,該方法包括:
- 如申請專利範圍第60項所述的方法,其中X為1至3;m為1至3。
- 一種醫藥組成物,其含有如申請專利範圍第33至58項中任一項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物和可藥用的賦形劑、稀釋劑或載體。
- 一種申請專利範圍第1至26項中任一項所述的特異性結合c-Met受體的抗體或其抗原結合片段、或申請專 利範圍第32項中所述的醫藥組成物、或如申請專利範圍第33至58項中任一項所述的通式(I)所示的抗體-細胞毒性藥物偶聯物或其可藥用鹽或溶劑化合物、或如申請專利範圍第62項所述的醫藥組成物的用途,其用在製備用於治療c-Met介導的疾病或病症的藥物,其中該疾病或病症為癌症。
- 如申請專利範圍第63項所述的用途,其中該癌症為表現c-Met的癌症。
- 如申請專利範圍第64項所述的用途,其中該癌症為胃癌、胰腺癌、肺癌、腸癌、腎癌、黑色素瘤、非小細胞肺癌。
- 如申請專利範圍第65項所述的用途,其中該癌症為胃癌、胰腺癌、非小細胞肺癌和腎癌。
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TW202430557A (zh) * | 2022-12-23 | 2024-08-01 | 大陸商蘇州宜聯生物醫藥有限公司 | Anti-Met抗體、抗體藥物偶聯物及其製備方法和用途 |
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US7659241B2 (en) * | 2002-07-31 | 2010-02-09 | Seattle Genetics, Inc. | Drug conjugates and their use for treating cancer, an autoimmune disease or an infectious disease |
EP3120861B1 (en) * | 2003-11-06 | 2018-08-15 | Seattle Genetics, Inc. | Intermediate for conjugate preparation comprising auristatin derivatives and a linker |
JP5068651B2 (ja) * | 2004-08-05 | 2012-11-07 | ジェネンテック, インコーポレイテッド | ヒト化抗cmet抗体 |
US7750116B1 (en) * | 2006-02-18 | 2010-07-06 | Seattle Genetics, Inc. | Antibody drug conjugate metabolites |
UY32914A (es) * | 2009-10-02 | 2011-04-29 | Sanofi Aventis | Anticuerpos que se usan específicamente al receptor epha2 |
KR20130037153A (ko) * | 2011-10-05 | 2013-04-15 | 삼성전자주식회사 | 항 c-Met 항체 및 그의 용도 |
AR090549A1 (es) * | 2012-03-30 | 2014-11-19 | Genentech Inc | Anticuerpos anti-lgr5 e inmunoconjugados |
CN105452297B (zh) * | 2013-04-30 | 2019-10-11 | 新加坡科技研究局 | mAb 2抗-Met抗体 |
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2015
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- 2016-04-07 EP EP16779555.8A patent/EP3284751A4/en not_active Withdrawn
- 2016-04-15 TW TW105111792A patent/TWI731856B/zh active
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WO2018129029A1 (en) * | 2017-01-04 | 2018-07-12 | Immunogen, Inc. | Met antibodies and immunoconjugates and uses thereof |
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RU2017135257A (ru) | 2019-05-17 |
KR20170138451A (ko) | 2017-12-15 |
US10543284B2 (en) | 2020-01-28 |
MX2017012965A (es) | 2018-06-06 |
CA2982777A1 (en) | 2016-10-20 |
RU2017135257A3 (zh) | 2019-09-24 |
JP2018516539A (ja) | 2018-06-28 |
TWI731856B (zh) | 2021-07-01 |
CN106687480A (zh) | 2017-05-17 |
CN106687480B (zh) | 2019-04-19 |
CN106188293A (zh) | 2016-12-07 |
EP3284751A1 (en) | 2018-02-21 |
BR112017021245A2 (zh) | 2018-06-26 |
US20180110875A1 (en) | 2018-04-26 |
AU2016248357A1 (en) | 2017-10-26 |
CN111196853A (zh) | 2020-05-26 |
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