TW202241951A - 抗fgfr2抗體及其用途 - Google Patents
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Abstract
本申請提供了抗FGFR2抗體以及所述抗體的編碼核酸,用於其表現和生產的載體和宿主細胞,抗體藥物偶聯物和包含所述抗體的醫藥組合物。本發明進一步涉及所述抗體分子在製備用於診斷FGFR2通路相關失調的癌症中的用途,以及所述抗體或抗體-藥物偶聯物在製備用於治療FGFR2通路相關失調的癌症特別是胃癌的藥物中的用途。
Description
本發明是有關於一種抗體分子,具體而言是人抗FGFR2抗體分子,尤其是抗FGFR2IIIb的抗體分子。
成纖維細胞生長因子受體2(fibroblast growth factor receptor 2,FGFR2) 是一種酪胺酸激酶受體,其胞外區有2個免疫球蛋白類似結構域(β異構體)(分別是D2和D3結構域)或3個免疫球蛋白類似結構域(α異構體)(分別是D1、D2和D3結構域),與胞外區結構域相連的是跨膜區和胞內雙酪胺酸激酶亞結構域。根據外顯子來源不同,FGFR2可分為IIIb及IIIc亞型,他們的區別主要體現在D3結構域。IIIb亞型根據胞外區免疫球蛋白類似結構的數目不同而分成FGFR2αIIIb(3個免疫球蛋白類似結構域D1、D2和D3)以及FGFR2βIIIb(2個免疫球蛋白類似結構域D2和D3)。另外,IIIb亞型主要表現在上皮組織,IIIc亞型主要表現在間充質組織,這兩種受體的某些FGF配體有相反的表現模式例如與FGFR2IIIb結合的FGF7、FGF10 和FGF22在間充質組織表現,而與FGFR2IIIc結合的FGF4、FGF5和FGF6 在上皮細胞表現,因此推測FGFR2在上皮和間充質轉化中起到重要作用。
根據序列的同源性,與FGFR2同一家族的還有FGFR1、FGFR3以及FGFR4,該家族的信號通路的啟動需要成纖維生長因子(fibroblast growth factors,FGFs)作為配體,FGF與受體FGFR2的結合主要通過受體中的D2和D3區域,同時結合硫酸類肝素糖蛋白,誘發FGFR二聚體化,進而自身磷酸化,通過FGFR底物2(FGFR substrate 2,FRS2)和鈣調蛋白信號通路PLCA轉導RAS-ERK和PI3K-AKT信號級聯反應,並且還涉及 DAG-PKC和IP3信號級聯反應。FGFR信號的異常啟動與多種惡性腫瘤相關。
正常細胞中,FGFR2位於染色體10q26,主要參與組織修復和發育過程中的細胞分化、增殖和凋亡。研究顯示小鼠敲除FGFR2IIIb基因會導致胚胎致死。同時,大量證據表明,FGFR2或FGFs的過表現以及基因上的變化,如基因擴增、基因融合和重排、基因點突變及染色體易位,會導致FGFR2信號通路失調,失調的FGFR/FGF信號通路與細胞癌變密切相關。潛在的FGFR2的過表現、錯義突變活化或異常蛋白融合已經在多種癌症類型中報導,其中包括子宮內膜癌、卵巢癌、乳腺癌、肺癌、胃癌、食道癌、膀胱癌和膽管癌。例如FGFR2IIIb在40%的胃癌組織樣本中有高表現,FGFR2IIIb基因擴增在胃癌患者群體中有最高15%的突變頻率。相比無FGFR2IIIb基因擴增的病人,FGFR2IIIb基因基因擴增導致FGFR2IIIb過表現的胃癌病人伴有顯著的淋巴結轉移,與低分化胃腺癌顯著相關,生存率更低,是胃癌患者一個極差的預後指標。約在9%~14%的膽管癌患者中發現了FGFR2基因融合和重排。
綜上所述,FGFR2可成為腫瘤治療的潛在靶點,在治療中使用阻斷劑如抗體以阻斷FGF與FGFR2的結合,從而抑制FGFR/FGF信號通路作用,該治療思路已在其他酪胺酸激酶,如HER2、EGFR等陽性腫瘤中證實有效。
本發明提供了一組針對FGFR2IIIb的新抗體。本發明還提供了含有本發明所述的抗FGFR2IIIb抗體的醫藥組合物,以及所述抗體在製備用於診斷或治療胃癌特別是FGFR2IIIb基因擴增導致FGFR2IIIb過表現的胃癌的藥物中的用途。
具體地,本發明提供了抗FGFR2IIIb抗體。本發明的優選實施例為一種特異性結合FGFR2IIIb的抗體或抗原結合片段,其包含重鏈CDR1、CDR2和CDR3以及輕鏈CDR1、CDR2和CDR3,所述重鏈CDR1、CDR2和CDR3以及輕鏈CDR1、CDR2和CDR3分別與選自下組的抗體的重鏈CDR1、CDR2和CDR3以及輕鏈CDR1、CDR2和CDR3序列具有至少80%,優選至少90%,更優選至少95%的同一性:FWB1904、FWB1905、FWB1906、FWB1907、FWB1908、FWB1910、FWB1911、FWB1912、FWB1913、FWB1914、FWB1915、FWB1916、FWB1918、FWB1919、FWB1920、FWB1921、FWB1922、FWB1923、FWB1924和FWB1925。更優選地,本發明涉及一種特異性結合FGFR2IIIb的抗體或抗原結合片段,其包含選自下組的抗體的重鏈CDR1、CDR2和CDR3以及輕鏈CDR1、CDR2和CDR3:FWB1904、FWB1905、FWB1906、FWB1907、FWB1908、FWB1910、FWB1911、FWB1912、FWB1913、FWB1914、FWB1915、FWB1916、FWB1918、FWB1919、FWB1920、FWB1921、FWB1922、FWB1923、FWB1924和FWB1925。
本發明的更優選實施例為特異性結合FGFR2IIIb的抗體或抗原結合片段,其包含重鏈可變區和輕鏈可變區,其中所述重鏈可變區和輕鏈可變區與選自下組的抗體的重鏈可變區和輕鏈可變區序列具有至少85%同一性,優選至少90%同一性,更優選95%同一性,最優選98%同一性:FWB1904、FWB1905、FWB1906、FWB1907、FWB1908、FWB1910、FWB1911、FWB1912、FWB1913、FWB1914、FWB1915、FWB1916、FWB1918、FWB1919、FWB1920、FWB1921、FWB1922、FWB1923、FWB1924和FWB1925。進一步更優選地,本發明涉及一種特異性結合FGFR2IIIb的抗體或其抗原結合片段,其包含選自下組的抗體的重鏈可變區和輕鏈可變區序列:FWB1904、FWB1905、FWB1906、FWB1907、FWB1908、FWB1910、FWB1911、FWB1912、FWB1913、FWB1914、FWB1915、FWB1916、FWB1918、FWB1919、FWB1920、FWB1921、FWB1922、FWB1923、FWB1924和FWB1925。
本發明的尤其優選的實施例是包含在本文中被稱為FWB1913、FWB1914和FWB1925的抗體分子的三個重鏈CDR和三個輕鏈CDR的抗體。
本發明還提供了一種分離的核酸,其編碼本發明所述的抗體或抗原結合片段。
本發明還提供了一種表現載體,其包含本發明所述的分離的核酸。本發明還提供了一種宿主細胞,其包含本發明所述的核酸或表現載體,優選地所述宿主細胞是真核宿主細胞,更優選所述宿主細胞是哺乳動物宿主細胞。
本發明還提供了一種組合物,其包含第一核酸和第二核酸,其中所述第一核酸編碼本發明所述的抗體的重鏈,所述第二核酸編碼本發明所述的抗體的輕鏈。
本發明提供了一種醫藥組合物,其用於治療FGFR2IIIb相關疾病或失調,其包含本發明所述的抗體或其抗原結合片段、分離的核酸、表現載體、或宿主細胞,所述醫藥組合物還包含醫藥上可接受的載劑。優選地,所述FGFR2IIIb相關疾病或失調是胃癌,特別是FGFR2IIIb基因擴增導致FGFR2IIIb過表現的胃癌。
本發明還涉及抗體-藥物偶聯物(antibody-drug conjugate,ADC)。本發明還涉及包含抗體-藥物偶聯物和醫藥上可接受的載劑的醫藥組合物。所述抗體-藥物偶聯物包含本發明所述的抗體或抗原結合片段以及藥物。
本發明還涉及本發明所述的抗體或其抗原結合片段或抗體-藥物偶聯物在製備用於治療FGFR2通路相關失調的癌症的藥物中的用途,所述癌症優選為胃癌,最優選為FGFR2IIIb基因擴增導致FGFR2IIIb過表現的胃癌。
如本說明書和所附申請專利範圍中使用的,單數形式“一個”,“一種”和“該/所述”包括複數提及物,除非上下文明確另有規定。如此,例如,提及“一個/種分子”任選包括兩個/種或更多個/種此類分子的組合,諸如此類。
如本文中使用的,術語“約”指業內熟習此項技術者容易知道的相應數值的常規誤差範圍。本文中提及“約”某個數值或參數包括(並描述)涉及該數值或參數本身的實施例。
除非另有說明,本文中使用的術語具有本領域公知的含義。
除非另外指明,本文涉及的FGFR2IIIb通常指人FGFR2IIIb,在本文的若干處還稱為“抗原”。本發明提供了抗人FGFR2IIIb的抗體。FGFR2IIIb抗體可以是與FGFR2IIIb,特別是哺乳動物FGFR2IIIb (如人)特異性結合的抗體。抗體分子可以是分離的抗體分子。
在本申請描述的任何實施例中,所述抗體可結合FGFR2IIIb而不結合FGFR2IIIc。
在一個實施例中,抗FGFR2IIIb抗體分子包含重鏈和輕鏈。抗FGFR2IIIb的抗原結合片段為Fab片段、F (ab')片段、Fv片段、F(ab')2片段、單鏈抗體(scFV)和雙抗體。
本發明的抗FGFR2IIIb抗體分子可以是人有效的、人的、人類化的、CDR移植的、嵌合的、突變的、親和成熟的、去免疫的、合成的或者是體外產生的抗體分子。在一個實施例中,FGFR2IIIb抗體為人類化抗體。在另一實施例中,抗體分子具有選自以下的重鏈恒定區如IgG1、IgG2、IgG3、IgG4、IgM、IgA1、IgA2、IgD和IgE的重鏈恒定區、特別是IgG1、IgG2、IgG3和IgG4的重鏈恒定區,更特別是IgG1 (如人IgG1)的重鏈恒定區。重鏈恒定區一般是人的或者是人恒定區的修飾形式。在另一實施例中,抗體分子具有選自如Lambda或Kappa(優選Lambda,如人Lambda)輕鏈恒定區的輕鏈恒定區。在一個實施例中,改變(如突變)了恒定區,以修飾抗體分子的特性(如提高或降低以下一種或多種Fc受體結合、抗體糖基化、半胱氨酸殘基數、效應細胞的功能或補體的功能)。
本發明提供了本發明所述的抗FGFR2IIIb抗體在製備用於治療胃癌的藥物中的用途。在一些實施例中,所述胃癌包含FGFR2基因擴增。在一些實施例中,FGFR2擴增包括>3的FGFR2:CEN10 (染色體10著絲粒)比。在一些實施例中,所述癌症過表現FGFR2。在一些實施例中,包含FGFR2擴增包括>3的FGFR2IIIb:CEN10 (染色體10著絲粒)比過表現的癌症過表現FGFR2IIIb的程度高於FGFR2IIIc。在一些實施例中,包含FGFR2擴增的癌症表現FGFR2IIIb的歸一化水準超過FGFR2IIIc表現的歸一化水準的2倍,3倍,5倍或10倍。在一些實施例中,所述表現水準對GUSB歸一化。在一些實施例中,所述癌症過表現FGFR2IIIb但不包含FGFR2基因擴增。在一些實施例中,FGFR2IIIb的表現或過表現通過IHC確定。在一些實施例中,IHC對腫瘤細胞的1+、2+或3+染色表明FGFR2IIIb過表現。在一些實施例中,腫瘤細胞中IHC的2+或3+染色表明FGFR2IIIb過表現。
抗體藥物偶聯物(antibody-drug conjugate,ADC)是通過化學連接將具有生物活性的小分子藥物連接到單株抗體上,單株抗體作為載體將小分子藥物靶向運輸到目標細胞中。本發明的抗體藥物偶聯物為本發明的抗FGFR2IIIb抗體與藥物通過化學連接而製成。
FGF/FGFR信號通路與細胞增殖、分化、凋亡和遷移有關,腫瘤細胞的FGFR啟動突變或配體/受體過表現導致其信號通路持續啟動,不僅與多種惡性腫瘤的發生、增殖、不良預後等密切相關,且在腫瘤新生血管生成、腫瘤的侵襲與轉移等過程中發揮重要作用。本發明的抗 FGFR2IIIb 抗體可以通過阻斷FGFR2IIIb與其配體FGF(這些配體涵蓋了FGF1、FGF7(KGF)和FGF7亞家族的其它成員FGF3、FGF10 和 FGF22)的結合,從而抑制腫瘤細胞FGFR/FGF信號通路的異常啟動,進而抑制腫瘤細胞增殖,及腫瘤血管內皮細胞的新生、分化和遷移。
表1和表2中列出了本發明所述的抗體的6個CDR的序列。
表1. 本發明的抗體的重鏈CDR的序列
抗體編號 FWB | HCDR1 | HCDR1 SEQ ID NO: | HCDR2 | HCDR2 SEQ ID NO: | HCDR3 | HCDR3 SEQ ID NO: |
FWB1904 | SYNVH | 1 | SIYPDNGDTSYNQNFRG | 2 | GDFAY | 3 |
FWB1905 | SYNVH | 7 | SIYPDNGDSSYNQNYKG | 8 | GDFAY | 9 |
FWB1906 | SYNVH | 13 | SIYPDNGDSSYNQNYRG | 14 | GDFAY | 15 |
FWB1907 | SYNVN | 19 | SIYPDNGDSSYNNNYKG | 20 | GDFAY | 21 |
FWB1908 | TYNVH | 25 | SIYPDNGDSTYNQNFKG | 26 | GDFAY | 27 |
FWB1910 | TYNVH | 31 | SIYPDNGDTSYDEDFKG | 32 | GDFAY | 33 |
FWB1911 | SYNVH | 37 | SIYPDNGDSSYNQNYKG | 38 | GDFAY | 39 |
FWB1912 | SYNVH | 43 | SIYPDNGDSSYNQNYKG | 44 | GDYAY | 45 |
FWB1913 | SYNVH | 49 | SIYPDNGDSSYNQNYKG | 50 | GDYAY | 51 |
FWB1914 | SYNVH | 55 | SIYPDNGDSSYNNNYKG | 56 | GDFAY | 57 |
FWB1915 | SYNVH | 61 | SIYPDNGDSSYDEDYKG | 62 | GDFAY | 63 |
FWB1916 | SYNVH | 67 | SIYPDNGDSSYNQNYKG | 68 | GDFAY | 69 |
FWB1918 | SYNVH | 73 | SIYPDNGDSSYNQNFRG | 74 | GDFAY | 75 |
FWB1919 | SYNVH | 79 | SIYPDNGDSSYNQNYRG | 80 | GDFAY | 81 |
FWB1920 | SYNIH | 85 | SIYPDNGDSSYNQNYRG | 86 | GDFAY | 87 |
FWB1921 | SYNVH | 91 | SIYPDNGDSTYNQNYRG | 92 | GDFAY | 93 |
FWB1922 | SYNVH | 97 | SIYPDNGDSTYNQNYRG | 98 | GDFAY | 99 |
FWB1923 | SYNVH | 103 | SIYPDNGDSTYDEDFKG | 104 | GDFAY | 105 |
FWB1924 | SYNVH | 109 | SLYPDNGDTSYDEDYKG | 110 | GDFAY | 111 |
FWB1925 | SYNVH | 115 | SIYPDNGDSTYDEDYRG | 116 | GDFAY | 117 |
表2. 本發明的抗體的輕鏈CDR的序列
抗體編號 FWB | LCDR1 | LCDR1 SEQ ID NO: | LCDR2 | LCDR2 SEQ ID NO: | LCDR3 | LCDR3 SEQ ID NO: |
FWB1904 | KASNGISNDIA | 4 | SASYRYS | 5 | QQHSTTPYT | 6 |
FWB1905 | KASNGVSNDIA | 10 | SASYRYS | 11 | QQHSTTPYT | 12 |
FWB1906 | KASNGISNDIA | 16 | SASYRYS | 17 | QQHSTTPYT | 18 |
FWB1907 | RASNGISNDIA | 22 | SASYRYS | 23 | QQHSTTPYT | 24 |
FWB1908 | KGSQGVSNDVA | 28 | SASYRYT | 29 | QQHSTTPYT | 30 |
FWB1910 | KVSQGVSNDAV | 34 | SASYRYT | 35 | QQHSTTPYT | 36 |
FWB1911 | KASNGVSNDIA | 40 | SASYRYS | 41 | QQHSTTPYS | 42 |
FWB1912 | KASNGVSNDIA | 46 | SASYRYS | 47 | QQHSTTPYT | 48 |
FWB1913 | KASNGVSNDIA | 52 | SASYRYS | 53 | QQHSTTPYS | 54 |
FWB1914 | KASNGVSNDIA | 58 | SASYRYS | 59 | QQHSTTPYT | 60 |
FWB1915 | KASNGVSNDIA | 64 | SASYRYS | 65 | QQHSTTPYT | 66 |
FWB1916 | KASNGISNDIA | 70 | SASYRYS | 71 | QQHSTTPYT | 72 |
FWB1918 | KGSNGISNDIA | 76 | SASYRYS | 77 | QQHSTTPYT | 78 |
FWB1919 | RGSNGISNDIA | 82 | SASYRYS | 83 | QQHSTTPYT | 84 |
FWB1920 | KGSNGVSNDIA | 88 | SASYRYS | 89 | QQHSTTPYT | 90 |
FWB1921 | KGSNGVSNDIA | 94 | SASYRYS | 95 | QQHSTTPYT | 96 |
FWB1922 | KGSNGISNDIA | 100 | SASYRYS | 101 | QQHSTTPYT | 102 |
FWB1923 | KVSQGVSNDAV | 106 | SASYRYS | 107 | QQHSTTPYT | 108 |
FWB1924 | KVSQGVSNDAV | 112 | SASYRYS | 113 | QQHSTTPYT | 114 |
FWB1925 | KGSNGISNDIA | 118 | SASYRYS | 119 | QQHSTTPYT | 120 |
表3. 本發明的抗體的重鏈可變區和輕鏈可變區的序列
抗體編號 FWB | VH序列 | VH編號 SEQ ID NO: | VL序列 | VL編號 SEQ ID NO: |
1904 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDTSYNQNFRGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSS | 121 | DIQMTQSPSSLSASVGDRVTITCKASNGISNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIK | 122 |
1905 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSSYNQNYKGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSS | 123 | DIQMTQSPSSLSASVGDRVTITCKASNGVSNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIK | 124 |
1906 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSSYNQNYRGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSS | 125 | DIQMTQSPSSLSASVGDRVTITCKASNGISNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIK | 126 |
1907 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVNWVRQAPGQGLEWIGSIYPDNGDSSYNNNYKGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSS | 127 | DIQMTQSPSSLSASVGDRVTITCRASNGISNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIK | 128 |
1908 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTTYNVHWVRQAPGQGLEWIGSIYPDNGDSTYNQNFKGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSS | 129 | DIQMTQSPSSLSASVGDRVTITCKGSQGVSNDVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIK | 130 |
1910 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTTYNVHWVRQAPGQGLEWIGSIYPDNGDTSYDEDFKGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSS | 131 | DIQMTQSPSSLSASVGDRVTITCKVSQGVSNDAVWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIK | 132 |
1911 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSSYNQNYKGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSS | 133 | DIQMTQSPSSLSASVGDRVTITCKASNGVSNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYSFGQGTKLEIK | 134 |
1912 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSSYNQNYKGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDYAYWGQGTLVTVSS | 135 | DIQMTQSPSSLSASVGDRVTITCKASNGVSNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIK | 136 |
1913 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSSYNQNYKGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDYAYWGQGTLVTVSS | 137 | DIQMTQSPSSLSASVGDRVTITCKASNGVSNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYSFGQGTKLEIK | 138 |
1914 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSSYNNNYKGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSS | 139 | DIQMTQSPSSLSASVGDRVTITCKASNGVSNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIK | 140 |
1915 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSSYDEDYKGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSS | 141 | DIQMTQSPSSLSASVGDRVTITCKASNGVSNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIK | 142 |
1916 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSSYNQNYKGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSS | 143 | DIQMTQSPSSLSASVGDRVTITCKASNGISNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIK | 144 |
1918 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSSYNQNFRGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSS | 145 | DIQMTQSPSSLSASVGDRVTITCKGSNGISNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIK | 146 |
1919 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSSYNQNYRGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSS | 147 | DIQMTQSPSSLSASVGDRVTITCRGSNGISNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIK | 148 |
1920 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNIHWVRQAPGQGLEWIGSIYPDNGDSSYNQNYRGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSS | 149 | DIQMTQSPSSLSASVGDRVTITCKGSNGVSNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIK | 150 |
1921 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSTYNQNYRGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSS | 151 | DIQMTQSPSSLSASVGDRVTITCKGSNGVSNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIK | 152 |
1922 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSTYNQNYRGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSS | 153 | DIQMTQSPSSLSASVGDRVTITCKGSNGISNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIK | 154 |
1923 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSTYDEDFKGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSS | 155 | DIQMTQSPSSLSASVGDRVTITCKVSQGVSNDAVWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIK | 156 |
1924 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSLYPDNGDTSYDEDYKGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSS | 157 | DIQMTQSPSSLSASVGDRVTITCKVSQGVSNDAVWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIK | 158 |
1925 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSTYDEDYRGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSS | 159 | DIQMTQSPSSLSASVGDRVTITCKGSNGISNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIK | 160 |
表4. 本發明的抗體的重鏈和輕鏈的序列
抗體編號 FWB | HC | VH編號 SEQ ID NO: | VC | VL編號 SEQ ID NO: |
1904 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDTSYNQNFRGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK | 161 | DIQMTQSPSSLSASVGDRVTITCKASNGISNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC | 162 |
1905 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSSYNQNYKGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK | 163 | DIQMTQSPSSLSASVGDRVTITCKASNGVSNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC | 164 |
1906 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSSYNQNYRGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK | 165 | DIQMTQSPSSLSASVGDRVTITCKASNGISNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC | 166 |
1907 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVNWVRQAPGQGLEWIGSIYPDNGDSSYNNNYKGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK | 167 | DIQMTQSPSSLSASVGDRVTITCRASNGISNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC | 168 |
1908 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTTYNVHWVRQAPGQGLEWIGSIYPDNGDSTYNQNFKGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK | 169 | DIQMTQSPSSLSASVGDRVTITCKGSQGVSNDVAWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC | 170 |
1910 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTTYNVHWVRQAPGQGLEWIGSIYPDNGDTSYDEDFKGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK | 171 | DIQMTQSPSSLSASVGDRVTITCKVSQGVSNDAVWYQQKPGKAPKLLIYSASYRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC | 172 |
1911 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSSYNQNYKGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK | 173 | DIQMTQSPSSLSASVGDRVTITCKASNGVSNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYSFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC | 174 |
1912 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSSYNQNYKGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDYAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK | 175 | DIQMTQSPSSLSASVGDRVTITCKASNGVSNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC | 176 |
1913 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSSYNQNYKGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDYAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK | 177 | DIQMTQSPSSLSASVGDRVTITCKASNGVSNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYSFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC | 178 |
1914 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSSYNNNYKGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK | 179 | DIQMTQSPSSLSASVGDRVTITCKASNGVSNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC | 180 |
1915 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSSYDEDYKGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK | 181 | DIQMTQSPSSLSASVGDRVTITCKASNGVSNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC | 182 |
1916 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSSYNQNYKGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK | 183 | DIQMTQSPSSLSASVGDRVTITCKASNGISNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC | 184 |
1918 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSSYNQNFRGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK | 185 | DIQMTQSPSSLSASVGDRVTITCKGSNGISNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC | 186 |
1919 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSSYNQNYRGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK | 187 | DIQMTQSPSSLSASVGDRVTITCRGSNGISNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC | 188 |
1920 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNIHWVRQAPGQGLEWIGSIYPDNGDSSYNQNYRGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK | 189 | DIQMTQSPSSLSASVGDRVTITCKGSNGVSNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC | 190 |
1921 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSTYNQNYRGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK | 191 | DIQMTQSPSSLSASVGDRVTITCKGSNGVSNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC | 192 |
1922 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSTYNQNYRGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK | 193 | DIQMTQSPSSLSASVGDRVTITCKGSNGISNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC | 194 |
1923 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSTYDEDFKGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK | 195 | DIQMTQSPSSLSASVGDRVTITCKVSQGVSNDAVWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC | 196 |
1924 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSLYPDNGDTSYDEDYKGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK | 197 | DIQMTQSPSSLSASVGDRVTITCKVSQGVSNDAVWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC | 198 |
1925 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFTSYNVHWVRQAPGQGLEWIGSIYPDNGDSTYDEDYRGRATITADKSTSTAYMELSSLRSEDTAVYYCARGDFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK | 199 | DIQMTQSPSSLSASVGDRVTITCKGSNGISNDIAWYQQKPGKAPKLLIYSASYRYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHSTTPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC | 200 |
實例
實例
1
使用基因工程方法製備抗體
分別根據以下表5和6所示的重鏈和輕鏈胺基酸序列合成基因序列,並選殖至表現載體pcDNA3.4(Invitrogen),雙質體通過電轉方法共轉染至HEK293細胞,使轉化的HEK293細胞表現抗體,37℃搖床培養一周後,收集上清,純化抗體。使用Protein A 親和層析柱純化,純化後分別使用SDS-PAGE 和SEC-HPLC 檢測方法對抗體純度進行檢測,所有抗體純度均達到95%以上。對25個候選抗體通過ELISA篩選得到與FGFR2IIIb結合活性高的候選抗體。
表5:實例1的抗體的重鏈CDR和FR的序列
FWB編號 | HFR1 | HCDR1 | HFR2 | HCDR2 | HFR3 | HCDR3 | HFR4 |
FWB1901 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFT | TYDVH | WVRQAPGQGLEWIG | SIYPNDGDTSYNQNFKG | RATITADKSTSTAYMELSSLRSEDTAVYYCAR | GDFAY | WGQGTLVTVSS |
FWB1902 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFT | TYDVH | WVRQAPGQGLEWIG | SIYPNDGDTSYNQNFKG | RATITADKSTSTAYMELSSLRSEDTAVYYCAR | GNFAY | WGQGTLVTVSS |
FWB1903 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFT | TYDVH | WVRQAPGQGLEWIG | SIYPNNGDTSYNQNFKG | RATITADKSTSTAYMELSSLRSEDTAVYYCAR | GNFAY | WGQGTLVTVSS |
FWB1904 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFT | SYNVH | WVRQAPGQGLEWIG | SIYPDNGDTSYNQNFRG | RATITADKSTSTAYMELSSLRSEDTAVYYCAR | GDFAY | WGQGTLVTVSS |
FWB1905 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFT | SYNVH | WVRQAPGQGLEWIG | SIYPDNGDSSYNQNYKG | RATITADKSTSTAYMELSSLRSEDTAVYYCAR | GDFAY | WGQGTLVTVSS |
FWB1906 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFT | SYNVH | WVRQAPGQGLEWIG | SIYPDNGDSSYNQNYRG | RATITADKSTSTAYMELSSLRSEDTAVYYCAR | GDFAY | WGQGTLVTVSS |
FWB1907 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFT | SYNVN | WVRQAPGQGLEWIG | SIYPDNGDSSYNNNYKG | RATITADKSTSTAYMELSSLRSEDTAVYYCAR | GDFAY | WGQGTLVTVSS |
FWB1908 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFT | TYNVH | WVRQAPGQGLEWIG | SIYPDNGDSTYNQNFKG | RATITADKSTSTAYMELSSLRSEDTAVYYCAR | GDFAY | WGQGTLVTVSS |
FWB1909 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFT | TYNVH | WVRQAPGQGLEWIG | SIYPNDGDTSYNQNFKG | RATITADKSTSTAYMELSSLRSEDTAVYYCAR | GDFAY | WGQGTLVTVSS |
FWB1910 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFT | TYNVH | WVRQAPGQGLEWIG | SIYPDNGDTSYDEDFKG | RATITADKSTSTAYMELSSLRSEDTAVYYCAR | GDFAY | WGQGTLVTVSS |
FWB1911 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFT | SYNVH | WVRQAPGQGLEWIG | SIYPDNGDSSYNQNYKG | RATITADKSTSTAYMELSSLRSEDTAVYYCAR | GDFAY | WGQGTLVTVSS |
FWB1912 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFT | SYNVH | WVRQAPGQGLEWIG | SIYPDNGDSSYNQNYKG | RATITADKSTSTAYMELSSLRSEDTAVYYCAR | GDYAY | WGQGTLVTVSS |
FWB1913 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFT | SYNVH | WVRQAPGQGLEWIG | SIYPDNGDSSYNQNYKG | RATITADKSTSTAYMELSSLRSEDTAVYYCAR | GDYAY | WGQGTLVTVSS |
FWB1914 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFT | SYNVH | WVRQAPGQGLEWIG | SIYPDNGDSSYNNNYKG | RATITADKSTSTAYMELSSLRSEDTAVYYCAR | GDFAY | WGQGTLVTVSS |
FWB1915 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFT | SYNVH | WVRQAPGQGLEWIG | SIYPDNGDSSYDEDYKG | RATITADKSTSTAYMELSSLRSEDTAVYYCAR | GDFAY | WGQGTLVTVSS |
FWB1916 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFT | SYNVH | WVRQAPGQGLEWIG | SIYPDNGDSSYNQNYKG | RATITADKSTSTAYMELSSLRSEDTAVYYCAR | GDFAY | WGQGTLVTVSS |
FWB1917 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFT | SYNVH | WVRQAPGQGLEWIG | SIYPDNGDSSYNQNYRG | RATITADKSTSTAYMELSSLRSEDTAVYYCAR | GDFAY | WGQGTLVTVSS |
FWB1918 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFT | SYNVH | WVRQAPGQGLEWIG | SIYPDNGDSSYNQNFRG | RATITADKSTSTAYMELSSLRSEDTAVYYCAR | GDFAY | WGQGTLVTVSS |
FWB1919 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFT | SYNVH | WVRQAPGQGLEWIG | SIYPDNGDSSYNQNYRG | RATITADKSTSTAYMELSSLRSEDTAVYYCAR | GDFAY | WGQGTLVTVSS |
FWB1920 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFT | SYNIH | WVRQAPGQGLEWIG | SIYPDNGDSSYNQNYRG | RATITADKSTSTAYMELSSLRSEDTAVYYCAR | GDFAY | WGQGTLVTVSS |
FWB1921 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFT | SYNVH | WVRQAPGQGLEWIG | SIYPDNGDSTYNQNYRG | RATITADKSTSTAYMELSSLRSEDTAVYYCAR | GDFAY | WGQGTLVTVSS |
FWB1922 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFT | SYNVH | WVRQAPGQGLEWIG | SIYPDNGDSTYNQNYRG | RATITADKSTSTAYMELSSLRSEDTAVYYCAR | GDFAY | WGQGTLVTVSS |
FWB1923 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFT | SYNVH | WVRQAPGQGLEWIG | SIYPDNGDSTYDEDFKG | RATITADKSTSTAYMELSSLRSEDTAVYYCAR | GDFAY | WGQGTLVTVSS |
FWB1924 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFT | SYNVH | WVRQAPGQGLEWIG | SLYPDNGDTSYDEDYKG | RATITADKSTSTAYMELSSLRSEDTAVYYCAR | GDFAY | WGQGTLVTVSS |
FWB1925 | QVQLVQSGAEVKKPGSSVKVSCKASGYIFT | SYNVH | WVRQAPGQGLEWIG | SIYPDNGDSTYDEDYRG | RATITADKSTSTAYMELSSLRSEDTAVYYCAR | GDFAY | WGQGTLVTVSS |
所有抗體的重鏈恒定區均為:
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
表6:實例1的抗體的輕鏈CDR和FR的序列
FWB編號 | LFR1 | CDR1 | LFR2 | CDR2 |
FWB1901 | DIQMTQSPSSLSASVGDRVTITC | KASQGVSDDVA | WYQQKPGKAPKLLIY | SASYRYV |
FWB1902 | DIQMTQSPSSLSASVGDRVTITC | KASQGVSDDVA | WYQQKPGKAPKLLIY | SASYRYV |
FWB1903 | DIQMTQSPSSLSASVGDRVTITC | KASQGLSNDVA | WYQQKPGKAPKLLIY | SASYRYV |
FWB1904 | DIQMTQSPSSLSASVGDRVTITC | KASNGISNDIA | WYQQKPGKAPKLLIY | SASYRYS |
FWB1905 | DIQMTQSPSSLSASVGDRVTITC | KASNGVSNDIA | WYQQKPGKAPKLLIY | SASYRYS |
FWB1906 | DIQMTQSPSSLSASVGDRVTITC | KASNGISNDIA | WYQQKPGKAPKLLIY | SASYRYS |
FWB1907 | DIQMTQSPSSLSASVGDRVTITC | RASNGISNDIA | WYQQKPGKAPKLLIY | SASYRYS |
FWB1908 | DIQMTQSPSSLSASVGDRVTITC | KGSQGVSNDVA | WYQQKPGKAPKLLIY | SASYRYT |
FWB1909 | DIQMTQSPSSLSASVGDRVTITC | KATQGVSNDAV | WYQQKPGKAPKLLIY | SASYRYT |
FWB1910 | DIQMTQSPSSLSASVGDRVTITC | KVSQGVSNDAV | WYQQKPGKAPKLLIY | SASYRYT |
FWB1911 | DIQMTQSPSSLSASVGDRVTITC | KASNGVSNDIA | WYQQKPGKAPKLLIY | SASYRYS |
FWB1912 | DIQMTQSPSSLSASVGDRVTITC | KASNGVSNDIA | WYQQKPGKAPKLLIY | SASYRYS |
FWB1913 | DIQMTQSPSSLSASVGDRVTITC | KASNGVSNDIA | WYQQKPGKAPKLLIY | SASYRYS |
FWB1914 | DIQMTQSPSSLSASVGDRVTITC | KASNGVSNDIA | WYQQKPGKAPKLLIY | SASYRYS |
FWB1915 | DIQMTQSPSSLSASVGDRVTITC | KASNGVSNDIA | WYQQKPGKAPKLLIY | SASYRYS |
FWB1916 | DIQMTQSPSSLSASVGDRVTITC | KASNGISNDIA | WYQQKPGKAPKLLIY | SASYRYS |
FWB1917 | DIQMTQSPSSLSASVGDRVTITC | KGSNGISNDIA | WYQQKPGKAPKLLIY | SASYRYS |
FWB1918 | DIQMTQSPSSLSASVGDRVTITC | KGSNGISNDIA | WYQQKPGKAPKLLIY | SASYRYS |
FWB1919 | DIQMTQSPSSLSASVGDRVTITC | RGSNGISNDIA | WYQQKPGKAPKLLIY | SASYRYS |
FWB1920 | DIQMTQSPSSLSASVGDRVTITC | KGSNGVSNDIA | WYQQKPGKAPKLLIY | SASYRYS |
FWB1921 | DIQMTQSPSSLSASVGDRVTITC | KGSNGVSNDIA | WYQQKPGKAPKLLIY | SASYRYS |
FWB1922 | DIQMTQSPSSLSASVGDRVTITC | KGSNGISNDIA | WYQQKPGKAPKLLIY | SASYRYS |
FWB1923 | DIQMTQSPSSLSASVGDRVTITC | KVSQGVSNDAV | WYQQKPGKAPKLLIY | SASYRYS |
FWB1924 | DIQMTQSPSSLSASVGDRVTITC | KVSQGVSNDAV | WYQQKPGKAPKLLIY | SASYRYS |
FWB1925 | DIQMTQSPSSLSASVGDRVTITC | KGSNGISNDIA | WYQQKPGKAPKLLIY | SASYRYS |
FWB編號 | LFR3 | CDR3 | LFR4 | |
FWB1901 | WYQQKPGKAPKLLIY | QQHSTTPYT | FGQGTKLEIK | |
FWB1902 | WYQQKPGKAPKLLIY | QQHSTTPYV | FGQGTKLEIK | |
FWB1903 | WYQQKPGKAPKLLIY | QQHSTTPYV | FGQGTKLEIK | |
FWB1904 | WYQQKPGKAPKLLIY | QQHSTTPYT | FGQGTKLEIK | |
FWB1905 | WYQQKPGKAPKLLIY | QQHSTTPYT | FGQGTKLEIK | |
FWB1906 | WYQQKPGKAPKLLIY | QQHSTTPYT | FGQGTKLEIK | |
FWB1907 | WYQQKPGKAPKLLIY | QQHSTTPYT | FGQGTKLEIK | |
FWB1908 | WYQQKPGKAPKLLIY | QQHSTTPYT | FGQGTKLEIK | |
FWB1909 | WYQQKPGKAPKLLIY | QQHSTTPYT | FGQGTKLEIK | |
FWB1910 | WYQQKPGKAPKLLIY | QQHSTTPYT | FGQGTKLEIK | |
FWB1911 | WYQQKPGKAPKLLIY | QQHSTTPYS | FGQGTKLEIK | |
FWB1912 | WYQQKPGKAPKLLIY | QQHSTTPYT | FGQGTKLEIK | |
FWB1913 | WYQQKPGKAPKLLIY | QQHSTTPYS | FGQGTKLEIK | |
FWB1914 | WYQQKPGKAPKLLIY | QQHSTTPYT | FGQGTKLEIK | |
FWB1915 | WYQQKPGKAPKLLIY | QQHSTTPYT | FGQGTKLEIK | |
FWB1916 | WYQQKPGKAPKLLIY | QQHSTTPYT | FGQGTKLEIK | |
FWB1917 | WYQQKPGKAPKLLIY | QQHSTTPYT | FGQGTKLEIK | |
FWB1918 | WYQQKPGKAPKLLIY | QQHSTTPYT | FGQGTKLEIK | |
FWB1919 | WYQQKPGKAPKLLIY | QQHSTTPYT | FGQGTKLEIK | |
FWB1920 | WYQQKPGKAPKLLIY | QQHSTTPYT | FGQGTKLEIK | |
FWB1921 | WYQQKPGKAPKLLIY | QQHSTTPYT | FGQGTKLEIK | |
FWB1922 | WYQQKPGKAPKLLIY | QQHSTTPYT | FGQGTKLEIK | |
FWB1923 | WYQQKPGKAPKLLIY | QQHSTTPYT | FGQGTKLEIK | |
FWB1924 | WYQQKPGKAPKLLIY | QQHSTTPYT | FGQGTKLEIK | |
FWB1925 | WYQQKPGKAPKLLIY | QQHSTTPYT | FGQGTKLEIK |
所有抗體的輕鏈恒定區均為:
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
實例
2
通過
ELISA
方法測定抗
FGFR2
抗體的結合活性
首先將96孔ELISA板用100 μL 的 2 μg/mL rhFGFR2IIIb-Fc(recombinant humanFGFR2IIIb-Fc)在4°C過夜,並用250 μL的封閉液(在PBST中的3%BSA)在37°C下孵育2小時,之後用PBST洗板3次。將待測抗體從10 μg/mL進行10倍系列稀釋,共8個濃度點(包括空白對照),並將系列稀釋的抗體每孔100 μL加入到ELISA孔中,在37°C下孵育1小時,隨後用PBST洗板3遍。加入二抗抗Fab HRP偶聯物並在37°C下孵育1小時,最後用PBST洗板3次並加入100 μL TMB反應15分鐘。顯色反應用1N鹽酸(50 μL)進行終止,並在讀板機(M5)上檢測450 nm的吸光度值,以此來計算各個抗體對人FGFR2IIIb的結合活性(EC
50)。結果如下表7所示,最終篩選出與人FGFR2IIIb、人FGFR2βIIIb和鼠FGFR2IIIb結合活性強,但與人FGFR2IIIc、FGFR3IIIb、FGFR3IIIc、FGFR4、FGFR1IIIb和FGFR1IIIc無結合的FWB1904、FWB1905、FWB1906、FWB1907、FWB1908、FWB1910、FWB1911、FWB1912、FWB1913、FWB1914、FWB1915、FWB1916、FWB1918、FWB1919、FWB1920、FWB1921、FWB1922、FWB1925繼續進行實驗。
表7:抗體與多種FGFR的結合
“--”是指無結合信號
抗體編號 | ELISA EC 50(nM) | ||||||||
人FGFR2IIIb | 人FGFR2βIIIb | 鼠FGFR2IIIb | 人FGFR2IIIc | 人FGFR3IIIb | 人FGFR3IIIc | 人 FGFR4 | 人FGFR1IIIb | 人FGFR1IIIc | |
FWB1904 | 0.3515 | 0.1625 | 0.3084 | -- | -- | -- | -- | -- | -- |
FWB1905 | 0.2984 | 0.1732 | 0.2617 | -- | -- | -- | -- | -- | -- |
FWB1906 | 0.3236 | 0.1674 | 0.2891 | -- | -- | -- | -- | -- | -- |
FWB1907 | 1.059 | 0.2618 | 5.898 | -- | -- | -- | -- | -- | -- |
FWB1908 | 0.2813 | 0.1258 | 0.2254 | -- | -- | -- | -- | -- | -- |
FWB1910 | 0.2791 | 0.2099 | 0.2955 | -- | -- | -- | -- | -- | -- |
FWB1911 | 0.6968 | 0.2464 | 0.3383 | -- | -- | -- | -- | -- | -- |
FWB1912 | 0.7241 | 0.2069 | 0.2928 | -- | -- | -- | -- | -- | -- |
FWB1913 | 0.9029 | 0.1962 | 0.2961 | -- | -- | -- | -- | -- | -- |
FWB1914 | 1.006 | 0.2026 | 0.2497 | -- | -- | -- | -- | -- | -- |
FWB1915 | 0.8769 | 0.185 | 0.2727 | -- | -- | -- | -- | -- | -- |
FWB1916 | 0.5823 | 0.1429 | 0.2904 | -- | -- | -- | -- | -- | -- |
FWB1918 | 0.6259 | 0.1353 | 0.2698 | -- | -- | -- | -- | -- | -- |
FWB1919 | 0.8791 | 0.1491 | 0.3135 | -- | -- | -- | -- | -- | -- |
FWB1920 | 1.209 | 0.1527 | 0.274 | -- | -- | -- | -- | -- | -- |
FWB1921 | 0.6205 | 0.1762 | 0.2605 | -- | -- | -- | -- | -- | -- |
FWB1922 | 0.6564 | 0.2153 | 0.139 | -- | -- | -- | -- | -- | -- |
FWB1923 | 1.684 | 未檢測 | 未檢測 | 未檢測 | 未檢測 | 未檢測 | 未檢測 | 未檢測 | 未檢測 |
FWB1924 | 1.885 | 未檢測 | 未檢測 | 未檢測 | 未檢測 | 未檢測 | 未檢測 | 未檢測 | 未檢測 |
FWB1925 | 0.6434 | 0.1733 | 0.0908 | -- | -- | -- | -- | -- | -- |
實例
3
通過
FACS
測定抗
FGFR2
抗體的結合活性
待測抗體為FWB1904、FWB1905、FWB1906、FWB1907、FWB1908、FWB1910、FWB1911、FWB1912、FWB1913、FWB1914、FWB1915、FWB1916、FWB1918、FWB1919、FWB1920、FWB1921、FWB1922、FWB1925。微孔板中每孔收集2×105個細胞(KATO III 以及SNU16,購買自ATCC,其細胞表面均有高表現的FGFR2IIIb受體),離心,用含2%FBS的PBS重懸細胞,抗體以30μg/mL為初始濃度,5倍梯度稀釋,在每孔細胞加入相應濃度的抗體或空白對照100μL,放置4℃孵育1小時,用含2%FBS的PBS清洗細胞2次後,加入Alexa 488羊抗人IgG, 放置4℃避光孵育1小時,用含2%FBS的PBS清洗細胞2次,然後用100 μL含2%FBS的PBS重懸細胞,最後用流式細胞儀檢測細胞表面的螢光信號。最終篩選出與KATO III和SNU16結合活性強的FWB1904、FWB1905、FWB1912、FWB1914、FWB1915、FWB1916、FWB1919、FWB1921、FWB1925繼續進行實驗。
表8:抗體與KATO III和SNU16細胞上的FGFR2IIIb受體結合的能力
抗體編號 | ELISA EC 50(nM) | |
KATOIII | SNU16 | |
FWB1904 | 1.621 | 1.133 |
FWB1905 | 1.092 | 1.331 |
FWB1906 | 1.849 | 2.278 |
FWB1908 | 2.172 | 1.604 |
FWB1910 | 4.156 | 2.301 |
FWB1911 | 1.958 | 3.012 |
FWB1912 | 1.901 | 2.747 |
FWB1913 | 1.846 | 2.169 |
FWB1914 | 2.555 | 2.851 |
FWB1915 | 2.188 | 2.445 |
FWB1916 | 1.974 | 3.024 |
FWB1918 | 1.604 | 2.959 |
FWB1919 | 1.283 | 2.842 |
FWB1920 | 1.83 | 4.328 |
FWB1921 | 1.774 | 3.857 |
FWB1922 | 2.047 | 4.418 |
FWB1925 | 1.958 | 4.193 |
實例 4 通過 FACS 測定抗體阻斷細胞表面受體FGFR2IIIb
與其配體 FGF7 結合的活性
待測抗體為FWB1904、FWB1905、FWB1912、FWB1914、FWB1915、FWB1916、FWB1919、FWB1921、FWB1925。微孔板中每孔收集3×10
5個細胞(KATO III 以及SNU16),離心,用含2%FBS的PBS重懸細胞,抗體以30μg/mL為初始濃度,5倍梯度稀釋,在每孔細胞加入的相應濃度的抗體或空白對照100 μL,放置4℃孵育0.5小時後,再加入100uμL 0.32μg/mL的生物素偶聯的FGF7,放置4℃孵育1小時用含2%FBS的PBS清洗細胞2次後,加入Alexa 488鏈黴親和素,放置4℃避光孵育0.5小時,用含2%FBS的PBS清洗細胞2次,然後用100 uL含2%FBS的PBS重懸細胞,最後用流式細胞儀檢測細胞表面的螢光信號,檢測時每孔吸取細胞2×10
4個。
表9:通過FACS測定抗體阻斷細胞表面受體FGFR2IIIb與其配體FGF7結合的活性的結果
抗體編號 | ELISA EC 50(nM) | 最大抑制率(%) | ||
KATO III | SNU16 | KATO III | SNU16 | |
FWB1904 | 9.73 | 3.438 | 94.5 | 98.6 |
FWB1905 | 9.95 | 3.097 | 93.2 | 98.3 |
FWB1912 | 10.2 | 4.171 | 82.9 | 87.2 |
FWB1914 | 8.75 | 3.452 | 93.2 | 98.3 |
FWB1915 | 9.95 | 3.824 | 91.3 | 97.2 |
FWB1916 | 20.2 | 6.932 | 95.1 | 97.1 |
FWB1919 | 12 | 4.997 | 86.3 | 87.5 |
FWB1921 | 10.95 | 5.202 | 85.7 | 87.8 |
FWB1925 | 9.84 | 7.855 | 77.5 | 83.0 |
無
無
Claims (16)
- 一種特異性結合FGFR2IIIb的抗體或其抗原結合片段,其包含重鏈CDR1、CDR2和CDR3以及輕鏈CDR1、CDR2和CDR3,所述重鏈CDR1、CDR2和CDR3以及輕鏈CDR1、CDR2和CDR3分別與選自下組的抗體的重鏈CDR1、CDR2和CDR3以及輕鏈CDR1、CDR2和CDR3序列具有至少80%,優選至少90%同一性:FWB1904、FWB1905、FWB1906、FWB1907、FWB1908、FWB1910、FWB1911、FWB1912、FWB1913、FWB1914、FWB1915、FWB1916、FWB1918、FWB1919、FWB1920、FWB1921、FWB1922、FWB1923、FWB1924和FWB1925。
- 如請求項1所述的特異性結合FGFR2IIIb的抗體或其抗原結合片段,其包含選自下組的抗體的重鏈CDR1、CDR2和CDR3以及輕鏈CDR1、CDR2和CDR3:FWB1904、FWB1905、FWB1906、FWB1907、FWB1908、FWB1910、FWB1911、FWB1912、FWB1913、FWB1914、FWB1915、FWB1916、FWB1918、FWB1919、FWB1920、FWB1921、FWB1922、FWB1923、FWB1924和FWB1925。
- 如請求項1或請求項2所述的抗體或其抗原結合片段,其包含重鏈可變區和輕鏈可變區,其中所述重鏈可變區和輕鏈可變區與選自下組的抗體的重鏈可變區和輕鏈可變區序列具有至少85%同一性,優選至少90%同一性,更優選95%同一性,最優選98%同一性:FWB1904、FWB1905、FWB1906、FWB1907、FWB1908、FWB1910、FWB1911、FWB1912、FWB1913、FWB1914、FWB1915、FWB1916、FWB1918、FWB1919、FWB1920、FWB1921、FWB1922、FWB1923、FWB1924和FWB1925。
- 如請求項3所述的抗體或其抗原結合片段,其包含選自下組的抗體的重鏈可變區和輕鏈可變區序列:FWB1904、FWB1905、FWB1906、FWB1907、FWB1908、FWB1910、FWB1911、FWB1912、FWB1913、FWB1914、FWB1915、FWB1916、FWB1918、FWB1919、FWB1920、FWB1921、FWB1922、FWB1923、FWB1924和FWB1925。
- 如請求項1或請求項2所述的抗體或其抗原結合片段,其包含重鏈和輕鏈,其中所述重鏈和輕鏈分別與選自下組的抗體的重鏈和輕鏈序列具有至少80%,優選至少85%、更優選90%,最優選至少95%,98%,99%同一性:FWB1904、FWB1905、FWB1906、FWB1907、FWB1908、FWB1910、FWB1911、FWB1912、FWB1913、FWB1914、FWB1915、FWB1916、FWB1918、FWB1919、FWB1920、FWB1921、FWB1922、FWB1923、FWB1924和FWB1925。
- 如請求項5所述的抗體或其抗原結合片段,其包含選自下組的抗體的重鏈和輕鏈序列:FWB1904、FWB1905、FWB1906、FWB1907、FWB1908、FWB1910、FWB1911、FWB1912、FWB1913、FWB1914、FWB1915、FWB1916、FWB1918、FWB1919、FWB1920、FWB1921、FWB1922、FWB1923、FWB1924和FWB1925。
- 如請求項1至請求項6中任一請求項的抗體,其中所述抗體是人抗體、人類化抗體或嵌合抗體。
- 如請求項1至請求項6中任一請求項的抗體,其中所述抗體是IgA、IgG和IgD。
- 如請求項1至請求項6中任一請求項的抗原結合片段,其中所述抗原結合片段選自Fab片段、F (ab')片段、Fv片段、F(ab')2片段、單鏈抗體(scFV)和雙抗體。
- 一種分離的核酸,其編碼如請求項1至請求項9中任一項所述的抗體或其抗原結合片段。
- 一種表現載體,其包含如請求項10所述的分離的核酸。
- 一種宿主細胞,其包含如請求項10所述的分離的核酸或如請求項11所述的表現載體,優選地所述宿主細胞是真核宿主細胞,更優選所述宿主細胞是哺乳動物宿主細胞。
- 一種醫藥組合物,其用於治療FGFR2相關疾病或失調,其包含如請求項1至請求項9中任一請求項所述的抗體或其抗原結合片段、如請求項10所述的分離的核酸、如請求項11所述的表現載體、或如請求項12所述的宿主細胞,所述醫藥組合物還包含醫藥上可接受的載劑。
- 一種組合物,其包含第一核酸和第二核酸,其中所述第一核酸編碼如請求項1至請求項9中任一請求項所述的抗體的重鏈,所述第二核酸編碼如請求項1至請求項9中任一請求項所述的抗體的輕鏈。
- 一種抗體-藥物偶聯物,所述抗體-藥物偶聯物包含如請求項1至請求項9中任一請求項的抗體或抗原結合片段以及藥物。
- 如請求項1至請求項9中任一請求項所述的抗體或其抗原結合片段或如請求項15所述的抗體-藥物偶聯物在製備用於治療FGFR2通路相關失調的癌症的藥物中的用途,所述癌症優選為胃癌,更優選為FGFR2IIIb基因擴增導致FGFR2IIIb過表現的胃癌。
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