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Publication numberCN101932606 A
Publication typeApplication
Application numberCN 200980102891
PCT numberPCT/IB2009/000124
Publication date29 Dec 2010
Filing date22 Jan 2009
Priority date23 Jan 2008
Also published asCA2712221A1, EP2245069A1, EP2245069B1, US8236315, US20090232804, WO2009093138A1
Publication number200980102891.9, CN 101932606 A, CN 101932606A, CN 200980102891, CN-A-101932606, CN101932606 A, CN101932606A, CN200980102891, CN200980102891.9, PCT/2009/124, PCT/IB/2009/000124, PCT/IB/2009/00124, PCT/IB/9/000124, PCT/IB/9/00124, PCT/IB2009/000124, PCT/IB2009/00124, PCT/IB2009000124, PCT/IB200900124, PCT/IB9/000124, PCT/IB9/00124, PCT/IB9000124, PCT/IB900124
InventorsC伍兹, E拉扎里德斯, H莫特尔, M博特施格尔, S侯, S布莱恩, X范
Applicant格兰马克药品股份有限公司
Export CitationBiBTeX, EndNote, RefMan
External Links: SIPO, Espacenet
Humanized antibodies specific for von willebrand factor
CN 101932606 A
Abstract
The present disclosure relates to humanized antibodies or binding fragments thereof specific for human von Willebrand factor (vWF), methods for their preparation and use, including methods for treating vWF mediated diseases or disorders.
Claims(118)  translated from Chinese
  1. 一种具有冯维勒布兰德氏因子(vWF)特异性的人源化抗体或其结合片段,包含:(a)如SEQ ID NO:19中所述的重链可变区序列;及(b)如SEQ ID NO:28中所述的轻链可变区序列。 People having a von Willebrand factor (vWF) specific humanized antibody or binding fragment thereof comprising: (a) as SEQ ID NO: heavy chain variable region sequence described in 19; and ( b) as SEQ ID NO: 28 the light chain variable region sequence described.
  2. 2. 一种具有vWF特异性的人源化抗体或其结合片段,该人源化抗体包含:(a)如SEQ ID NO :237中所述的重链可变区序列;及(b)如SEQ ID NO :238中所述的轻链可变区序列。 2. A person with a specific humanized vWF antibody or binding fragment thereof, the humanized antibody comprises: (a) as SEQ ID NO: heavy chain variable region sequence in the 237; and (b) such as SEQ ID NO: 238 in the light chain variable region sequence.
  3. 3. 一种具有vWF特异性的人源化抗体或其结合片段,该人源化抗体包含:(a)重链及轻链互补决定区(CDR),其对应于存在于鼠类抗体NMC-4的重链及轻链可变区(分别为SEQ ID NO :1及2)中的⑶R ;以及(b)重链框架区和/或轻链框架区,前者对应于存在于人类抗体AAC18165. 1 (SEQ ID NO :4)的可变区中的框架区,后者对应于存在于人类抗体AAK94808 (SEQ ID NO :6)的可变区中的框架区。 3. A person having specificity for vWF humanized antibody or binding fragment thereof, the humanized antibody comprises: (a) the heavy chain and light chain complementarity determining region (CDR), which corresponds to the murine antibody present in NMC- 4 heavy and light chain variable regions (SEQ ID NO: 1 and 2) ⑶R; and (b) a heavy chain framework region and / or light chain framework regions, the former corresponds to the presence of human antibodies AAC18165. 1 (SEQ ID NO: 4) of the variable region framework region, which corresponds to the presence of human antibodies AAK94808 (SEQ ID NO: 6) of the variable region framework regions.
  4. 4. 一种具有vWF特异性的人源化抗体或其结合片段,该人源化抗体包含:选自于以下重链CDR其中一种或多种:HCDRl :GFSLTDYGVD(SEQ ID NO :7), HCDR2 : MIffGDGSTDYNSALKS(SEQ ID NO :8),及HCDR 3 :DPADYGNYDYALDY(SEQ ID NO :9);和/ 或选自于以下轻链CDR 其中一种或多种=LCDRl :SASQDINKYLN(SEQ ID NO :10), LCDR2 : YTSSLHS(SEQ ID NO :11),及LCDR3 :QQYEKLPWT(SEQ ID NO: 12)。 4. A person having vWF-specific humanized antibody or binding fragment thereof, the humanized antibody comprises: a heavy chain CDR selected from the following wherein one or more of: HCDRl: GFSLTDYGVD (SEQ ID NO: 7), HCDR2: MIffGDGSTDYNSALKS (SEQ ID NO: 8), and HCDR 3: DPADYGNYDYALDY (SEQ ID NO: 9); and / or light chain CDR selected from the following wherein one or more = LCDRl: SASQDINKYLN (SEQ ID NO: 10 ), LCDR2: YTSSLHS (SEQ ID NO: 11), and LCDR3: QQYEKLPWT (SEQ ID NO: 12).
  5. 5. 一种具有vWF特异性的人源化抗体或其结合片段,该人源化抗体包含:重链CDR =HCDRl :GFSLTDYGVD(SEQ ID NO :7),HCDR2 =MIffGDGSTDYNSALKS (SEQ ID NO : 8),及HCDR3 :DPADYGNYDYALDY(SEQ IDNO :9);禾口/ 或轻链CDR=LCDRl :SASQDINKYLN(SEQ ID NO :10),LCDR2 :YTSSLHS(SEQID NO :11),及LCDR3 =QQYEKLPffT(SEQ ID NO :12)。 A person having vWF-specific humanized antibody or binding fragment thereof, the humanized antibody comprises: a heavy chain CDR = HCDRl: GFSLTDYGVD (SEQ ID NO: 7), HCDR2 = MIffGDGSTDYNSALKS (SEQ ID NO: 8) and HCDR3: DPADYGNYDYALDY (SEQ IDNO: 9); Hekou / or light chain CDR = LCDRl: SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQID NO: 11), and LCDR3 = QQYEKLPffT (SEQ ID NO: 12).
  6. 6.权利要求第4或5项的人源化抗体或其结合片段,还包含:来自人类抗体AAC18165. KSEQ ID NO :4)的重链框架区;和/或来自人类抗体AAK94808 (SEQ ID NO :6) 的轻链框架区。 According to claim 4 or 5 of the first humanized antibody or binding fragment thereof, further comprising: KSEQ ID NO from a human antibody AAC18165: 4) of the heavy chain framework regions; and / or from a human antibody AAK94808 (SEQ ID NO. : 6) light chain framework region.
  7. 7.权利要求第6项的人源化抗体或其结合片段,其中该重链框架区还包含一个或多个鼠类残基。 The first six people claim humanized antibody or binding fragment thereof, wherein the heavy chain framework region further comprises one or more murine residues.
  8. 8.权利要求第6项的人源化抗体或其结合片段,其中该重链框架区不包含一个或多个鼠类残基。 The first six people claim humanized antibody or binding fragment thereof, wherein the heavy chain framework region does not contain one or more murine residues.
  9. 9.权利要求第6项的人源化抗体或其结合片段,其中该轻链框架区还包含一个或多个鼠类残基。 Article 6 of people claim humanized antibody or binding fragment thereof, wherein the light chain framework region further comprises one or more murine residues.
  10. 10.权利要求第6项的人源化抗体或其结合片段,其中该轻链框架区不包含一个或多个鼠类残基。 Article 6 of people claim humanized antibody or binding fragment thereof, wherein the light chain framework region does not contain one or more murine residues.
  11. 11.权利要求第4或5项的人源化抗体或其结合片段,其中该人源化抗体包含选自以下的重链可变区:H2(SEQ ID NO: 13),H4 (SEQ ID NO: 14),H5 (SEQ ID NO: 15),H6 (SEQ ID NO : 16),H7 (SEQ ID NO : 17),H8 (SEQID NO : 18),H9 (SEQ ID NO : 19),H12 (SEQ ID NO :20), H13(SEQ ID NO :21),H14(SEQ ID NO :22),H15 (SEQ ID NO : 145),或H16 (SEQ ID NO: 146)。 11. The article of claim 4 or 5 of the humanized antibody or binding fragment thereof, wherein the humanized antibody comprises a heavy chain selected from the variable region: H2 (SEQ ID NO: 13), H4 (SEQ ID NO : 14), H5 (SEQ ID NO: 15), H6 (SEQ ID NO: 16), H7 (SEQ ID NO: 17), H8 (SEQID NO: 18), H9 (SEQ ID NO: 19), H12 ( SEQ ID NO: 20), H13 (SEQ ID NO: 21), H14 (SEQ ID NO: 22), H15 (SEQ ID NO: 145), or H16 (SEQ ID NO: 146).
  12. 12.权利要求第4或5项的人源化抗体或其结合片段,其中该人源化抗体包含选自以下的轻链可变区:L5 (SEQ ID NO :23),L4(SEQ ID NO :24),L6 (SEQ ID NO :25),L7 (SEQ ID NO:26), L8 (SEQ ID NO :27), L9 (SEQID NO :28), LlO (SEQ ID NO :29),或Lll (SEQ ID NO :30)。 12. The article of claim 4 or 5 of the humanized antibody or binding fragment thereof, wherein the humanized antibody comprises a light chain selected from the variable region: L5 (SEQ ID NO: 23), L4 (SEQ ID NO : 24), L6 (SEQ ID NO: 25), L7 (SEQ ID NO: 26), L8 (SEQ ID NO: 27), L9 (SEQID NO: 28), LlO (SEQ ID NO: 29), or Lll (SEQ ID NO: 30).
  13. 13.权利要求第4或5项的人源化抗体或其结合片段,其中该人源化抗体包含:选自以下的重链可变区:H2 (SEQ ID N0:13),H4(SEQ ID NO : 14), H5 (SEQ ID NO: 15), H6 (SEQ ID NO :16),H7(SEQ ID NO :17),H8(SEQID NO : 18),H9(SEQ ID NO : 19),H12(SEQ ID NO :20), H13(SEQ ID NO :21),H14 (SEQ ID NO :22),H15 (SEQ ID NO :145),或H16 (SEQ ID NO :146);以及选自以下的轻链可变区:L5 (SEQ ID NO :23), L4(SEQ ID NO :24),L6 (SEQ ID NO -.25), L7 (SEQ ID NO :26),L8(SEQ ID NO :27),L9(SEQID NO :28),LlO (SEQ ID NO :29),或Lll(SEQ ID NO :30)。 13. The article of claim 4 or 5 of the humanized antibody or binding fragment thereof, wherein the humanized antibody comprises: a heavy chain selected from the variable region: H2 (SEQ ID N0: 13), H4 (SEQ ID NO: 14), H5 (SEQ ID NO: 15), H6 (SEQ ID NO: 16), H7 (SEQ ID NO: 17), H8 (SEQID NO: 18), H9 (SEQ ID NO: 19), H12 (SEQ ID NO: 20), H13 (SEQ ID NO: 21), H14 (SEQ ID NO: 22), H15 (SEQ ID NO: 145), or H16 (SEQ ID NO: 146); and selected from light chain variable region: L5 (SEQ ID NO: 23), L4 (SEQ ID NO: 24), L6 (SEQ ID NO -.25), L7 (SEQ ID NO: 26), L8 (SEQ ID NO: 27 ), L9 (SEQID NO: 28), LlO (SEQ ID NO: 29), or Lll (SEQ ID NO: 30).
  14. 14.权利要求第5项的人源化抗体或其结合片段,其中该人源化抗体包含:重链可变区序列,其含有至少80%与SEQ ID NO :19的框架区相同的框架区;和/或轻链可变区序列, 其含有至少80%与SEQ ID NO :28的框架区相同的框架区。 14. The first person to claim 5 of a humanized antibody or binding fragment thereof, wherein the humanized antibody comprises: a heavy chain variable region sequence, containing at least 80% with SEQ ID NO: 19 of the same framework region framework regions ; and / or light chain variable region sequence, containing at least 80% with SEQ ID NO: 28 of the same framework region framework regions.
  15. 15.权利要求第5项的人源化抗体或其结合片段,还包含:对应于人类抗体家族VH4中的框架区的重链框架区;以及对应于人类抗体家族VKl中的框架区的轻链框架区。 Corresponding to the light chain and a human antibody VKl family of framework regions; heavy chain framework region correspond to the VH4 family of human antibody framework region: one of the first five of claim humanized antibody or binding fragment thereof, further comprising framework regions.
  16. 16.权利要求第5项的人源化抗体或其结合片段,还包含:对应于人类抗体重链种系序列4-59中的框架区的重链框架区1、2和3,其中重链框架区1 为QVQLQESGPGLVKPSETLSLTCTVS (SEQ ID NO :171),重链框架区2 为WIRQPPGKGLEffIG (SEQ ID NO : 172),且重链框架区3 为RVTISVDTSKNQFSLKLSSVTAADTAVYYCA R (SEQ ID NO :173);以及对应于存在于人类抗体轻链种系序列018中的框架区的轻链框架区1、2和3, 其中轻链框架区1为DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO : 186),轻链框架区2为WYQQKPGKAPKLLIY (SEQ ID NO :187),且轻链框架区3 为GVPSRFSGSGSGTDFTFTISSLQPEDIATY YC(SEQ ID NO :188)。 People claim 16. Section 5 of the humanized antibody or binding fragment thereof, further comprising: corresponding to the human antibody heavy chain germline sequences of the heavy chain framework regions 4-59 framework regions 1, 2 and 3, wherein the heavy chain framework region 1 QVQLQESGPGLVKPSETLSLTCTVS (SEQ ID NO: 171), heavy chain framework region 2 WIRQPPGKGLEffIG (SEQ ID NO: 172), and a heavy chain framework region 3 RVTISVDTSKNQFSLKLSSVTAADTAVYYCA R (SEQ ID NO: 173); and corresponds to the presence in the light chain framework region of a human antibody light chain germline sequence 018 in the framework regions 1, 2 and 3, wherein the light chain framework region 1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 186), the light chain framework region 2 is WYQQKPGKAPKLLIY (SEQ ID NO : 187), and the light chain framework region 3 GVPSRFSGSGSGTDFTFTISSLQPEDIATY YC (SEQ ID NO: 188).
  17. 17.权利要求第5项的人源化抗体或其结合片段,还包含:对应于人类抗体重链种系序列4-34中的框架区的重链框架区1、2和3,其中重链框架区1 为QVQLQQWGAGLLKPSETLSLTCAVY (SEQ ID NO :165),重链框架区2 为WIRQPPGKGLEffIG (SEQ ID NO :166),且重链框架区3 为RVTISVDTSKNQFSLKLSSVTAADTAVYYCA R (SEQ ID NO :167);以及对应于存在于人类抗体轻链种系序列018中的框架区的轻链框架区1、2和3, 其中轻链框架区1为DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO : 186),轻链框架区2为WYQQKPGKAPKLLIY (SEQ ID NO :187),且轻链框架区3 为GVPSRFSGSGSGTDFTFTISSLQPEDIATY YC(SEQ ID NO :188)。 The first five people to claim 17. The humanized antibody or binding fragment thereof, further comprising: corresponding to the human antibody heavy chain germline sequences of the heavy chain framework region of 4-34 framework regions 2 and 3, wherein the heavy chain framework region 1 QVQLQQWGAGLLKPSETLSLTCAVY (SEQ ID NO: 165), heavy chain framework region 2 WIRQPPGKGLEffIG (SEQ ID NO: 166), and a heavy chain framework region 3 RVTISVDTSKNQFSLKLSSVTAADTAVYYCA R (SEQ ID NO: 167); and corresponds to the presence in the light chain framework region of a human antibody light chain germline sequence 018 in the framework regions 1, 2 and 3, wherein the light chain framework region 1 DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 186), the light chain framework region 2 is WYQQKPGKAPKLLIY (SEQ ID NO : 187), and the light chain framework region 3 GVPSRFSGSGSGTDFTFTISSLQPEDIATY YC (SEQ ID NO: 188).
  18. 18.权利要求第1-17项中任一项的人源化抗体或其结合片段,其中该人源化抗体以IOnM或更小的亲和力(Kd)结合至vWF。 People of any one of claims 1-17 Item 18. The humanized antibody or binding fragment thereof, wherein the humanized antibody IOnM or less affinity (Kd) binding to vWF.
  19. 19.权利要求第1-17项中任一项的人源化抗体或其结合片段,其中该人源化抗体用IOOnM或更小的亲和力(Ki)竞争结合至vWF。 People of any one of claims 1-17 Item 19. The humanized antibody or binding fragment thereof, wherein the humanized antibody or less by IOOnM affinity (Ki) compete for binding to vWF.
  20. 20.权利要求第1-17项中任一项的人源化抗体或其结合片段,其中该人源化抗体用IOnM或更小的亲和力(Kd)结合至vWF的Al结构域。 People of any one of claims 1-17 Item 20. The humanized antibody or binding fragment thereof, wherein the humanized antibody or less by IOnM affinity (Kd) of binding to the vWF Al domain.
  21. 21.权利要求第1-17项中任一项的人源化抗体或其结合片段,其中该人源化抗体用IOOnM或更小的亲和力(Ki)竞争结合至vWF的Al结构域。 People of any one of claims 1-17 Item 21. The humanized antibody or binding fragment thereof, wherein the humanized antibody with IOOnM or less affinity (Ki) to compete for binding of vWF Al domain.
  22. 22.权利要求第1-17项中任一项的人源化抗体或其结合片段,其中该人源化抗体具有大于65C的FAB片段热稳定性温度。 People of any one of claims 1-17 Item 22. The humanized antibody or binding fragment thereof, wherein the humanized antibody FAB fragments having thermal stability temperature of greater than 65 C.
  23. 23.权利要求第1-17项中任一项的人源化抗体或其结合片段,其中该人源化抗体具有高于亲本非人源化抗体的FAB片段热稳定性温度。 People of any one of claims 1-17 Item 23. The humanized antibody or binding fragment thereof, wherein the humanized antibody has a higher affinity of the non-humanized antibody fragment FAB temperature thermal stability.
  24. 24. 一种具有vWF特异性的人源化抗体或其结合片段,其中该人源化抗体包含:重链CDR1,为GFSLTDYGVD(SEQ ID NO :7);重链CDR2,为MIWGDGSTDYNSALKS (SEQ IDNO:8);重链CDR 3,为DPADYGNYDYALDY(SEQID NO:9);轻链CDRl ;轻链CDR2 ;及轻链CDR3, % QQYEKLPffT (SEQ ID NO :12),条件为轻链CDRl 非SASQDINKYLN(SEQ ID NO :10)和/ 或轻链CDR2 非YTSSLHS (SEQ ID NO: 11)。 24. A person having vWF-specific humanized antibody or binding fragment thereof, wherein the humanized antibody comprises: a heavy chain CDR1, a GFSLTDYGVD (SEQ ID NO: 7); heavy chain CDR2, a MIWGDGSTDYNSALKS (SEQ IDNO: 8); the heavy chain CDR 3, a DPADYGNYDYALDY (SEQID NO: 9); a light chain CDRl; light chain CDR2; and the light chain CDR3,% QQYEKLPffT (SEQ ID NO: 12), the condition for the light chain CDRl non SASQDINKYLN (SEQ ID NO: 10) and / or the light chain CDR2 non YTSSLHS (SEQ ID NO: 11).
  25. 25.权利要求第24项的人源化抗体或其结合片段,还包含人类抗体重链框架区和/或人类抗体轻链框架区。 The first 24 people to claim 25. The humanized antibody or binding fragment thereof, further comprising a human antibody heavy chain framework regions and / or human antibody light chain framework region.
  26. 26.权利要求第25项的人源化抗体或其结合片段,其中该重链框架区对应于存在于4-59衍生人类抗体中的重链框架区。 The first 25 people to claim 26. The humanized antibody or binding fragment thereof, wherein the heavy chain framework region corresponding to the presence in the 4-59-derived human antibody heavy chain framework region.
  27. 27.权利要求第26项的人源化抗体或其结合片段,其中存在于4-59衍生人类抗体中的该重链框架区还包含一个或多个鼠类残基。 27. The first 26 people to claim a humanized antibody or binding fragment present in the 4-59 human antibodies derived heavy chain framework region further comprises one or more murine residues.
  28. 28.权利要求第26项的人源化抗体或其结合片段,其中存在于4-59衍生人类抗体中的该重链框架区不包含一个或多个鼠类残基。 28. The first 26 people to claim a humanized antibody or binding fragment present in the 4-59 human antibodies derived heavy chain framework region does not contain one or more murine residues.
  29. 29.权利要求第25项的人源化抗体或其结合片段,其中该轻链框架区对应于存在于018衍生人类抗体中的轻链框架区。 The first 25 people to claim 29. The humanized antibody or binding fragment thereof, wherein the light chain framework region corresponding to the presence in the 018-derived human antibody light chain framework region.
  30. 30.权利要求第29项的人源化抗体或其结合片段,其中存在于018衍生人类抗体中的该轻链框架区还包含一个或多个鼠类残基。 30. The first 29 people to claim a humanized antibody or binding fragment present in the 018-derived human antibody in the light chain framework region further comprises one or more murine residues.
  31. 31.权利要求第29项的人源化抗体或其结合片段,其中存在于018衍生人类抗体中的该轻链框架区不包含一个或多个鼠类残基。 31. The first 29 people to claim a humanized antibody or binding fragment present in the 018-derived human antibody light chain framework in the region does not contain one or more murine residues.
  32. 32.权利要求第26项的人源化抗体或其结合片段,其中该4-59衍生人类抗体为AAC18165. 1。 The first 26 people to claim 32. The humanized antibody or binding fragment thereof, wherein the human antibody derived 4-59 AAC18165. 1.
  33. 33.权利要求第29项的人源化抗体或其结合片段,其中该018衍生人类抗体为AAK94808。 The first 29 people to claim 33. The humanized antibody or binding fragment thereof, wherein the human antibody is derivatized 018 AAK94808.
  34. 34.权利要求第24项的人源化抗体或其结合片段,其中LCDRl和/或LCDR2是来自于人类抗体。 The first 24 people to claim 34. The humanized antibody or binding fragment thereof, wherein LCDRl and / or LCDR2 derived from a human antibody.
  35. 35.权利要求第34项的人源化抗体或其结合片段,其中IXDR2为DASNLET(SEQ ID NO : 118)。 The first 34 people to claim 35. The humanized antibody or binding fragment thereof, wherein IXDR2 is DASNLET (SEQ ID NO: 118).
  36. 36.权利要求第1-17及24项中任一项的人源化抗体或其结合片段,其中HCDRl包含一个或多个选自F27G,L29I,T30S及V34W的氨基酸取代。 People of any one of 1-17 and 24 to claim 36. The humanized antibody or binding fragment thereof, which contain one or more groups selected from HCDRl F27G, amino acid substitutions L29I, T30S and V34W of.
  37. 37.权利要求第1-17及24项中任一项的人源化抗体或其结合片段,其中HCDR2包含一个或多个选自S61P及A62S的氨基酸取代。 1-17 and as in any one of claims 24 37. A humanized antibody or binding fragment thereof, wherein HCDR2 comprises one or more amino acid substitutions selected from the S61P and A62S.
  38. 38.权利要求第1-17及24项中任一项的人源化抗体或其结合片段,其中IXDRl包含一个或多个选自S24Q,N30S及K31N的氨基酸取代。 People of any one of 1-17 and 24 to claim 38. The humanized antibody or binding fragment thereof, wherein IXDRl contain one or more substituents selected from S24Q, N30S and K31N amino acids.
  39. 39.权利要求第1-17及24项中任一项的人源化抗体或其结合片段,其中HCDRl包含一个或多个选自F27G,L29I,T30S及V34W的氨基酸取代,HCDR2包含一个或多个选自S61P 及A62S的氨基酸取代,且IXDRl包含一个或多个选自S24Q,N30S及K31N的氨基酸取代。 People of any one of 1-17 and claim 24 39. A humanized antibody or binding fragment thereof, which comprises one or more selected HCDRl F27G, substituted amino L29I, T30S and the V34W, HCDR2 contains one or more amino acid selected from S61P and A62S substitution, and IXDRl contain one or more substituents selected from S24Q, N30S and K31N amino acids.
  40. 40.权利要求第4或5项的人源化抗体或其结合片段,还包含来自人类抗体的重链框架区,其中该人类重链框架区不包含一个或多个鼠类残基。 40. Claim 4 or 5 of the first humanized antibody or binding fragment thereof, further comprising a heavy chain framework region from a human antibody, wherein the human heavy chain framework region does not contain one or more murine residues.
  41. 41.权利要求第4或5项的人源化抗体或其结合片段,还包含来自人类抗体的轻链框架区,其中该人类轻链框架区不包含一个或多个鼠类残基。 41. Claim 4 or 5 of the first humanized antibody or binding fragment thereof, further comprises a light chain framework region from a human antibody, wherein the human light chain framework region does not contain one or more murine residues.
  42. 42.权利要求第15-17项中任一项的人源化抗体或其结合片段,其中该重链框架区不包含一个或多个鼠类残基。 People of any one of claims 15-17 Item 42. The humanized antibody or binding fragment thereof, wherein the heavy chain framework region does not contain one or more murine residues.
  43. 43.权利要求第15-17项中任一项的人源化抗体或其结合片段,其中该轻链框架区不包含一个或多个鼠类残基。 People of any one of claims 15-17 Item 43. The humanized antibody or binding fragment thereof, wherein the light chain framework region does not contain one or more murine residues.
  44. 44.权利要求第1-43项中任一项的人源化抗体或其结合片段,其中该人源化抗体保留与亲本非人源化抗体、或包含来自亲本非人源化抗体的可变区及人类Fc区的嵌合体相同的活性。 People of any one of the first to claim 1-43 Item 44. The humanized antibody or binding fragment thereof, wherein the humanized antibodies retain parental non-humanized antibody, or a humanized antibody comprising non-human variable from parent chimera same active region and the human Fc region.
  45. 45.权利要求第44项的人源化抗体或其结合片段,其中该活性用瑞斯特霉素诱导血小板凝集活性加以测量。 The first 44 people to claim 45. The humanized antibody or binding fragment thereof, wherein the active neomycin-induced platelet aggregation by Rist activity is measured.
  46. 46.权利要求第1-43项中任一项的人源化抗体或其结合片段,其中该人源化抗体缺乏效应子功能。 People of any one of the first to claim 1-43 Item 46. The humanized antibody or binding fragment thereof, wherein the humanized antibody lacks effector function.
  47. 47.权利要求第1-43项中任一项的人源化抗体或其结合片段,其中该人源化抗体包含源自于IgG4的Fc区。 People of any one of claim s 1-43 Item 47. The humanized antibody or binding fragment thereof, wherein the humanized antibody comprises an Fc region derived from IgG4.
  48. 48.权利要求第1-43项中任一项的人源化抗体或其结合片段,其中该人源化抗体具有对人类vWF的Al结构域的特异性。 People of any one of the first to claim 1-43 Item 48. The humanized antibody or binding fragment thereof, wherein the humanized antibody having human vWF Al domain of specificity.
  49. 49.权利要求第1-43项中任一项的人源化抗体,其中该人源化抗体为全长抗体。 1-43 49. The first item in person to any of claims humanized antibody, wherein the humanized antibody is a full-length antibody.
  50. 50.权利要求第1-43项中任一项的结合片段,其中该结合片段为选自Fab,Fab', Fab,-SH, Fv, scFv,F(ab' ) 2及双链抗体的抗体片段。 Binding fragment of any one of the first claim 50. Item 1-43, wherein the binding fragment is selected from Fab, Fab ', Fab, -SH, Fv, scFv, F (ab') 2 antibodies and diabodies fragments.
  51. 51.权利要求第50项的结合片段,其中该抗体片段不为Fab。 Binding fragment 51. Section 50 wherein the antibody fragment is not Fab.
  52. 52. 一种分离核酸,编码权利要求第1-43项中任一项的抗体或其结合片段。 52. An isolated nucleic acid, antibody or binding fragment of any one of claim s 1-43 item coded.
  53. 53. 一种分离核酸,其包含载体GS264的编码轻链的核酸序列,该载体GS264在DSMZ保藏于微生物中,其登记号为DSM 21059。 53. An isolated nucleic acid vector comprising a nucleic acid sequence encoding the light chain of GS264, GS264 the vector deposited at DSMZ microorganisms, which registration number DSM 21059.
  54. 54. 一种分离核酸,其包含载体GS265的编码重链的核酸序列,该载体GS265在DSMZ保藏于微生物中,其登记号为DSM 21060。 54. An isolated nucleic acid vector comprising a nucleic acid sequence encoding the heavy chain of GS265, GS265 the vector deposited at DSMZ microorganisms, which registration number DSM 21060.
  55. 55. 一种分离核酸,编码具有vWF特异性的人源化抗体或其结合片段,该人源化抗体或其结合片段包含如SEQ ID NO: 19中所述的重链可变区序列及如SEQ ID N0:28中所述的轻链可变区序列。 55. An isolated nucleic acid encoding a vWF-specific humanized antibody or binding fragment thereof, the humanized antibody or binding fragment thereof comprising as SEQ ID NO: heavy chain variable region sequence and 19 as described SEQ ID N0: 28 the light chain variable region sequence described.
  56. 56. 一种分离核酸,编码具有vWF特异性的人源化抗体或其结合片段,该人源化抗体或其结合片段包含如SEQ ID NO :237中所述的重链序列及如SEQ ID NO :238中所述的轻链序列。 56. An isolated nucleic acid encoding a vWF-specific humanized antibody or binding fragment thereof, the humanized antibody or binding fragment thereof comprising as SEQ ID NO: heavy chain sequence described in SEQ ID NO 237 and e.g. : 238 light chain sequence described.
  57. 57. 一种载体,其包含权利要求第52-56项中任一项的分离核酸。 57. A vector comprising an isolated nucleic acid of claim any one of claims 52-56 in the first item.
  58. 58. 一种宿主细胞,其包含权利要求第52-56项中任一项的分离核酸或权利要求第57项的载体。 58. A host cell comprising an isolated nucleic acid or rights to claim any one of the first carrier 52-56 requirement of Article 57.
  59. 59. 一种人源化抗体或其结合片段的生产方法,包含:培养权利要求第58项的宿主细胞,使得核酸被表达并产生抗体。 59. A humanized antibody or binding fragment production process, comprising: culturing a host cell as claimed in claim 58 first, so that the nucleic acid is expressed and the antibody produced.
  60. 60.权利要求第59项的方法,还包含从宿主细胞培养物回收该抗体。 60. The method of claim 59, further comprising recovering the antibody from the host cell culture.
  61. 61.权利要求第59项的方法,其中该抗体是回收自宿主细胞培养基。 61. The method of claim 59, wherein the antibody is recovered from the host cell culture medium.
  62. 62.权利要求第59项的方法,其中,于该培养步骤之前,该宿主细胞用包含编码重链可变区的核酸的载体及包含编码轻链可变区的核酸的载体共转染。 62. The method of claim 59, wherein, before the incubation step, with the host cell comprises a heavy chain variable region encoding nucleic acid vector and a vector comprising a nucleic acid encoding the light chain variable region co-transfection.
  63. 63. 一种组合物,包含:权利要求第1-43项中任一项的人源化抗体或其结合片段、及可药用的载体。 63. A composition, comprising: a first one of any one of claims 1-43 humanized antibody or binding fragment thereof, and a pharmaceutically acceptable carrier.
  64. 64. 一种组合物,包含:权利要求第1-43项中任一项的第一人源化抗体或其结合片段、 及结合至vWF的Al结构域的第二抗体。 64. A composition, comprising: a first one of any one of claims 1-43 first humanized antibody or binding fragment thereof, and a second antibody binds to the Al domain of vWF.
  65. 65.权利要求第64项的组合物,其中该第二抗体为AJW200。 Section 64 of the composition of claim 65., wherein the second antibody is AJW200.
  66. 66. 一种受试者中的vWF介导的疾病或紊乱的治疗方法,该方法包含施用给该受试者治疗有效量的权利要求第1-43项中任一项的人源化抗体或结合片段。 66. A method of treating a disease or disorder in a subject mediated by vWF, which method comprises administering to the subject a therapeutically effective amount of people in any one of claims 1-43 items first humanized antibody or binding fragment thereof.
  67. 67.权利要求第66项的方法,其中该受试者为人类。 67. The method of claim 66, wherein the subject is a human.
  68. 68.权利要求第66项的方法,其中该vWF介导的疾病为血栓性疾病或紊乱 68. The method of claim 66, wherein the vWF-mediated disease is thrombotic disease or disorder
  69. 69.权利要求第68项的方法,其中该血栓性疾病或紊乱为心血管疾病或脑血管疾病如缺血性卒中。 69. The method of claim 68, wherein the thrombotic disease or disorder is a cardiovascular disease or cerebrovascular diseases such as ischemic stroke.
  70. 70.权利要求第69项的方法,其中该心血管疾病为动脉粥状硬化症、再狭窄、心绞痛、 急性心肌梗塞、急性冠状动脉综合症、或与糖尿病相关联的心血管疾病。 70. The method of claim 69, wherein the cardiovascular disease is atherosclerotic sclerosis, restenosis, angina pectoris, acute myocardial infarction, acute coronary syndrome, or diabetes associated with cardiovascular disease.
  71. 71.权利要求第68项的方法,其中该血栓性疾病或紊乱为血管发炎、静脉血栓、镰刀形细胞病、异种移植排斥、外周血管病、血栓性血小板减少性紫癜症、囊性纤维化、血管性痴呆、雷诺病、类风湿性关节炎、或糖尿病。 71. The method of claim 68, wherein the thrombotic disease or disorder is inflammation of blood vessels, venous thrombosis, sickle cell disease, xenograft rejection, peripheral vascular disease, thrombotic thrombocytopenic purpura disease, cystic fibrosis, vascular dementia, Raynaud's disease, rheumatoid arthritis, or diabetes.
  72. 72.权利要求第69项的方法,其中该脑血管疾病为血管性痴呆、缺血性卒中、或再发性中风。 72. The method of claim 69, wherein the cerebrovascular disease is vascular dementia, ischemic stroke, or recurrent stroke.
  73. 73.权利要求第66-72项中任一项的方法,其中该治疗有效量从约0. 001至约IOOmg/kg ο The method according to any one of claims 66-72 Item 73., wherein the therapeutically effective amount of from about 0.001 to about IOOmg / kg ο
  74. 74.权利要求第73项的方法,其中该治疗有效量从约0. 002至约20mg/kg。 74. The method of claim 73, wherein the therapeutically effective amount of from about 0.002 to about 20mg / kg.
  75. 75.权利要求第73项的方法,其中该治疗有效量从约0. 002至约10mg/kg。 75. The method of claim 73, wherein the therapeutically effective amount of from about 0.002 to about 10mg / kg.
  76. 76.权利要求第66-75项中任一项的方法,其中是将单一或多次小剂量的该治疗有效量的该人源化抗体或其结合片段施用给受试者。 The method according to any one of the first items 66-75 76. wherein is a single or multiple small doses of the therapeutically effective amount of the humanized antibody or binding fragment thereof is administered to a subject.
  77. 77.权利要求第66-75项中任一项的方法,其中该治疗有效量足以抑制血小板凝集,但不足以引发明显的临床出血迹象。 The method according to any one of claim s 66-75 Item 77., wherein the therapeutically effective amount sufficient to inhibit platelet aggregation, but not enough to lead to significant clinical signs of bleeding.
  78. 78.权利要求第66-72项中任一项的方法,其中该治疗有效量从EDiqq的约1至约250 倍,而不致引发明显的临床出血迹象。 The method according to any one of claims 66-72 Item 78., wherein the therapeutically effective amount of from about 1 to about 250 times EDiqq, and does not cause any significant clinical signs of bleeding.
  79. 79. —种权利要求第1-43项中任一项的人源化抗体或其结合片段作为药物的用途,包含以治疗有效量施用该人源化抗体或其结合片段。 79. - The first human in any one of claims 1-43 seed item humanized antibody or binding fragment for use as a medicament, comprising administering a therapeutically effective amount of the humanized antibody or binding fragment thereof.
  80. 80. 一种权利要求第1-43项中任一项的人源化抗体或其结合片段的用途,其是用于制备vWF介导的疾病或紊乱的治疗药物,包含以治疗有效量施用该人源化抗体或其结合片段。 People of any one of the first claim 1-43 Item 80. A humanized antibody or binding fragment purposes, which is the preparation of a medicament for the treatment of vWF mediated disease or disorder, comprising administering a therapeutically effective amount of the humanized antibody or binding fragment thereof.
  81. 81.权利要求第79项的用途,其中该药物被用来治疗vWF介导的疾病或紊乱。 Article 81. The use of claim 79, wherein the drug is used to treat vWF mediated disease or disorder.
  82. 82.权利要求第80或81项的用途,其中该vWF介导的疾病或紊乱为血栓性疾病或紊乱。 Use section 80 or 81 of 82. wherein the vWF-mediated disease or disorder is thrombotic disease or disorder.
  83. 83.权利要求第82项的用途,其中该血栓性疾病为心血管疾病或脑血管疾病如缺血性卒中。 Article 83. The use of claim 82, wherein the thrombotic disease is cardiovascular disease or cerebrovascular diseases such as ischemic stroke.
  84. 84.权利要求第83项的用途,其中该心血管疾病为动脉粥状硬化症、再狭窄、心绞痛、 急性心肌梗塞、急性冠状动脉综合症、或与糖尿病相关联的心血管疾病。 Article 84. The use of claim 83, wherein the cardiovascular disease is atherosclerotic sclerosis, restenosis, angina, acute myocardial infarction, acute coronary syndrome, or diabetes associated with cardiovascular disease.
  85. 85.权利要求第82项的用途,其中该血栓性疾病包含:血管发炎、静脉血栓、镰刀形细胞病、异种移植排斥、外周血管病、血栓性血小板减少性紫癜症、囊性纤维化、血管性痴呆、 雷诺病、类风湿性关节炎、或糖尿病。 Article 85. The use of claim 82, wherein the thrombotic disorders comprising: inflammation of blood vessels, venous thrombosis, sickle cell disease, xenograft rejection, peripheral vascular disease, thrombotic thrombocytopenic purpura disease, cystic fibrosis, vascular dementia, Raynaud's disease, rheumatoid arthritis, or diabetes.
  86. 86.权利要求第83项的用途,其中该脑血管疾病为血管性痴呆、缺血性卒中、或再发性中风。 86. The use of the first 83 wherein the cerebrovascular disease is vascular dementia, ischemic stroke, or recurrent stroke.
  87. 87.权利要求第79-86项中任一项的用途,其中该治疗有效量从约0. 001至约IOOmg/kg ο The use of any one of the first items 87. 79-86 wherein the therapeutically effective amount of from about 0.001 to about IOOmg / kg ο
  88. 88.权利要求第87项的用途,其中该治疗有效量从约0. 002至约20mg/kg。 Article 88. The use of claim 87, wherein the therapeutically effective amount of from about 0.002 to about 20mg / kg.
  89. 89.权利要求第87项的用途,其中该治疗有效量从约0. 002至约10mg/kg。 Article 89. The use of claim 87, wherein the therapeutically effective amount of from about 0.002 to about 10mg / kg.
  90. 90.权利要求第79-89项中任一项的用途,其中是将单一或多次小剂量的该治疗有效量的该人源化抗体或其结合片段施用给受试者。 The use of any one of the first 79-89 Item 90. The claim is a single or multiple small doses of the therapeutically effective amount of the humanized antibody or binding fragment thereof is administered to a subject.
  91. 91.权利要求第79-89项中任一项的用途,其中该治疗有效量足以抑制血小板凝集,但不足以引发明显的临床出血迹象。 The use of any one of claim s 79-89 Item 91., wherein the therapeutically effective amount sufficient to inhibit platelet aggregation, but not enough to lead to significant clinical signs of bleeding.
  92. 92.权利要求第79-86项中任一项的用途,其中该治疗有效量从EDiqq的约1至约250 倍,而不致引发明显的临床出血迹象。 The use of any one of the first items 92. 79-86 wherein the therapeutically effective amount of from about 1 to about 250 times EDiqq, and does not cause any significant clinical signs of bleeding.
  93. 93.权利要求第1-43项中任一项的人源化抗体或其结合片段,用作药物时,包含以一治疗有效量施用该人源化抗体或其结合片段。 People of any one of claim s 1-43 Item 93. The humanized antibody or binding fragment thereof, when used as a drug, comprising administering to a therapeutically effective amount of the humanized antibody or binding fragment thereof.
  94. 94.权利要求第1-43项中任一项的人源化抗体或其结合片段,当其被用于治疗vWF介导的疾病或紊乱时,包含以一治疗有效量施用该人源化抗体或其结合片段。 People of any one of the first to claim 1-43 Item 94. The humanized antibody or binding fragment thereof, when it is used to treat vWF mediated disease or disorder comprises administering a therapeutically effective amount of the humanized antibody or binding fragment thereof.
  95. 95.权利要求第93项的人源化抗体或其结合片段,其中该药物被用来治疗vWF介导的疾病或紊乱。 95. The first person to claim 93 humanized antibody or binding fragment thereof, wherein the drug is used to treat vWF mediated disease or disorder.
  96. 96.权利要求第94或95项的人源化抗体或其结合片段,其中该vWF介导的疾病或紊乱为血栓性疾病或紊乱。 96. The first person to claim 94 or 95 of the humanized antibody or binding fragment thereof, wherein the vWF-mediated disease or disorder is thrombotic disease or disorder.
  97. 97.权利要求第96项的人源化抗体或其结合片段,其中该血栓性疾病为心血管疾病或脑血管疾病如缺血性卒中。 97. The first person to claim 96 humanized antibody or binding fragment thereof, wherein the thrombotic disease is cardiovascular disease or cerebrovascular diseases such as ischemic stroke.
  98. 98.权利要求第97项的人源化抗体或其结合片段,其中该心血管疾病为动脉粥状硬化症、再狭窄、心绞痛、急性心肌梗塞、急性冠状动脉综合症、或与糖尿病相关联的心血管疾病。 98. The article of claim 97 humanized antibody or binding fragment thereof, wherein the cardiovascular disease is atherosclerotic sclerosis, restenosis, angina, acute myocardial infarction, acute coronary syndrome, or associated with diabetes linked cardiovascular disease.
  99. 99.权利要求第96项的人源化抗体或其结合片段,其中该血栓性疾病包含:血管发炎、 静脉血栓、镰刀形细胞病、异种移植排斥、外周血管病、血栓性血小板减少性紫癜症、囊性纤维化、血管性痴呆、雷诺病、类风湿性关节炎、或糖尿病。 The first 96 people to claim 99. The humanized antibody or binding fragment thereof, wherein the thrombotic disease comprising: inflammation of blood vessels, venous thrombosis, sickle cell disease, xenograft rejection, peripheral vascular disease, thrombotic thrombocytopenic purpura disease , cystic fibrosis, vascular dementia, Raynaud's disease, rheumatoid arthritis, or diabetes.
  100. 100.权利要求第97项的人源化抗体或其结合片段,其中该脑血管疾病为血管性痴呆、 缺血性卒中、或再发性中风。 The first 97 people to claim 100. The humanized antibody or binding fragment thereof, wherein the cerebrovascular disease is vascular dementia, ischemic stroke, or recurrent stroke.
  101. 101.权利要求第93-100项中任一项的人源化抗体或其结合片段,其中该治疗有效量从约0. 001 至约100mg/kg。 Any one of the first people 93-100 item 101. Claim humanized antibody or binding fragment thereof, wherein the therapeutically effective amount of from about 0.001 to about 100mg / kg.
  102. 102.权利要求第101项的人源化抗体或其结合片段,其中该治疗有效量从约0. 002至约20mg/kg。 102. Section 101 people claim humanized antibody or binding fragment thereof, wherein the therapeutically effective amount of from about 0.002 to about 20mg / kg.
  103. 103.权利要求第101项的人源化抗体或其结合片段,其中该治疗有效量从约0. 002至约10mg/kg。 The first 101 people to claim 103. The humanized antibody or binding fragment thereof, wherein the therapeutically effective amount of from about 0.002 to about 10mg / kg.
  104. 104.权利要求第93-103项中任一项的人源化抗体或其结合片段,其中是将单一或多次小剂量的该治疗有效量的该人源化抗体或其结合片段施用该受试者。 People of any one of the first items 104. Claim 93-103 humanized antibody or binding fragment is a single or multiple small doses of the therapeutically effective amount of the humanized antibody or binding fragment thereof is administered to the subject Test person.
  105. 105.权利要求第93-103项中任一项的人源化抗体或其结合片段,其中该治疗有效量足以抑制血小板凝集,但不足以引发明显的临床出血迹象。 People of any one of the first item 105. 93-103 claim humanized antibody or binding fragment thereof, wherein the therapeutically effective amount sufficient to inhibit platelet aggregation, but not enough to lead to significant clinical signs of bleeding.
  106. 106.权利要求第93-100项中任一项的人源化抗体或其结合片段,其中该治疗有效量从EDiqq的约1至约250倍,而不致引发明显的临床出血迹象。 Any one of the first people 93-100 106. The claim item humanized antibody or binding fragment thereof, wherein the therapeutically effective amount is from about 1 to about 250 times EDiqq, and does not cause any significant clinical signs of bleeding.
  107. 107. 一种具有冯维勒布兰德氏因子(vWF)特异性的人类抗体或其结合片段,其用范围自EDiqq的约1至约250倍的治疗有效量加以投药,而不致引发明显的临床出血迹象。 107. having von Willebrand factor (vWF) specific for human antibody or binding fragment thereof, with a range from EDiqq from about 1 to about 250 times the therapeutically effective amount to be administered, and does not cause any significant Clinical signs of bleeding.
  108. 108.权利要求第107项的人类抗体或其结合片段,其中该抗体或其结合片段具有人类vWF的Al结构域的特异性。 The first human antibody or binding fragment of claim 108. 107, wherein the antibody or binding fragment thereof having specificity for human vWF Al domain.
  109. 109. 一种将权利要求第1-43项中任一项的人源化抗体或其结合片段施用有此需要的受试者的方法,包含:施用一治疗有效量的该人源化抗体或其结合片段,其足以抑制血小板凝集而不致引发明显的临床出血迹象。 109. A man would in any one of claims 1-43 of the first humanized antibody or binding fragment method of administering a subject in need thereof, comprising: administering a therapeutically effective amount of the humanized antibody or binding fragment thereof, which is sufficient to inhibit platelet aggregation triggered without significant clinical signs of bleeding.
  110. 110.权利要求第66或109项的方法,其中该人源化抗体或其结合片段是经由皮下方式加以施用。 Method 109 or 110. Claim 66, wherein the humanized antibody or binding fragment thereof is to be administered by subcutaneous way.
  111. 111.权利要求第66或109项的方法,其中该人源化抗体或其结合片段是经由静脉方式加以施用。 Method 109 or 111. Claim 66, wherein the humanized antibody or binding fragment thereof is to be administered by intravenous way.
  112. 112.权利要求第66或109项的方法,其中该人源化抗体或其结合片段是经由静脉方式结合放射性治疗法加以施用。 Method 109 or 112. Claim 66, wherein the humanized antibody or binding fragment thereof is combined with radiation therapy to be administered via intravenous way.
  113. 113.权利要求第66或109项的方法,其中该人源化抗体或其结合片段的该治疗有效量从EDltltl的约1至约250倍。 Method 109 or 113. Claim 66, wherein the humanized antibody or binding fragment of such treatment an effective amount of from about 1 to about 250 times EDltltl of.
  114. 114.权利要求第79项的用途,其中该人源化抗体或其结合片段用自EDltltl的约1至约250倍的治疗有效量加以施用。 Article 114. The use of claim 79, wherein the humanized antibody or binding fragment to be administered from about 1 to about 250 times the therapeutically effective amount EDltltl of.
  115. 115. 一种制品,其是用以治疗vWF介导的疾病或紊乱,该制品包含权利要求第1-43项中任一项的人源化抗体或其结合片段。 115. An article, which is for the treatment of vWF mediated disease or disorder, comprising the article of claim any one of the first human 1-43 item humanized antibody or binding fragment thereof.
  116. 116. 一种试剂盒,其是用以治疗vWF介导的疾病或紊乱,该试剂盒包含权利要求第1-43项中任一项的人源化抗体或其结合片段。 116. A kit, which is for the treatment of vWF mediated disease or disorder, the kit comprising one of claims 1-43 according to any one of the first item humanized antibody or binding fragment thereof.
  117. 117. —种权利要求第1-43项中任一项的人源化抗体或其结合片段的用途,其是用于非治疗性应用上。 117. - people in any one of the first kinds of claims 1-43 items humanized antibody or binding fragment of purpose, which is used for non-therapeutic applications.
  118. 118.权利要求第117项的用途,其中该非治疗性应用为诊断性测定。 118. Section 117 of use claim, wherein the non-therapeutic applications of diagnostic assays.
Description  translated from Chinese

对冯维勒布兰德氏因子特异的人源化抗体 Von Willebrand factor specific humanized antibody

技术领域 Technical Field

[0001] 本公开基本涉及具有冯维勒布兰德氏(von ffillebrand)因子特异性的人源化抗体或其结合片段。 Al. [0001] The present disclosure relates to a basic Von Willebrand (von ffillebrand) factor specific humanized antibody or binding fragment thereof. 更特别地,本公开基本涉及包含CDR的具有冯维勒布兰德氏因子特异性的人源化抗体或其结合片段,该CDR对应于存在于鼠类抗体NMC-4中的CDR。 More particularly, the present disclosure relates to the basic comprising CDR having specificity for human von Willebrand factor humanized antibody or binding fragment thereof, which corresponds to the CDR present in the murine antibody NMC-4 in the CDR.

背景技术 Background

[0002] 血小板附着至血管创伤部位的调节涉及若干蛋白质的协调良好的相互作用,且其在止血及血栓形成两方面均扮演重要角色。 [0002] platelet adhesion to the vascular wound site coordination of several proteins involved in the regulation of good interactions, and which both have played an important role in hemostasis and thrombosis. 一种促成血小板附着的此类蛋白质为冯维勒布兰德氏因子(vWF),其为存在于血浆中的大型多体(multimeric)糖蛋白。 Such is an enabling platelet adhesion protein von Willebrand factor (vWF), which is present in the plasma of large multi-body (multimeric) glycoprotein. 假说认为:vWF 可经由其Al结构域而与血小板受体GPIb-α产生相互作用,从而促进血小板滚动及附着(Moake等,(1986) J. Clin. Invest. 78 : 1456-61)。 Hypothesis that: vWF Al domain via its receptor and platelet GPIb-α interact, thereby facilitating platelet adhesion and rolling (Moake et, (1986) J. Clin Invest 78:.. 1456-61). 在血小板滚动及附着之后,可形成导致出血停止的血小板/纤维蛋白栓(plug);然而,过度的血小板及/或凝血反应可能导致病理性血栓状态。 After rolling and adhesion of platelets can cause bleeding to stop the formation of platelet / fibrin plug (plug); however, excessive platelets and / or clotting reaction may lead to pathologic thrombosis state.

[0003] 由于现今针对抑制血小板活化(例如GPIIbIIla、ADP受体、环加氧酶(cyclo-oxygenase)或磷酸二酉旨酶拮抗齐Ll (phosphodiesteraseantagonist))或凝血(例如凝血酶及Xa因子抑制剂)的治疗方式与出血并发症相关联,有需要开发出在不严重削弱止血反应的情况下而能够实质上抑制血栓形成的物质。 [0003] Since the current for the inhibition of platelet activation (e.g. GPIIbIIla, ADP receptor, cyclooxygenases (cyclo-oxygenase) or phosphodiesterase enzyme antagonistic homogeneous unitary purpose Ll (phosphodiesteraseantagonist)) or coagulation (e.g. thrombin and factor Xa inhibitor ) The treatment is associated with bleeding complications, there is a need to develop without serious bleeding reactions and can weaken substantially inhibiting substance thrombosis.

[0004] 发明_既述 [0004] The invention, both said _

[0005] 本发明基本涉及具有人类冯维勒布兰德氏因子(vWF)特异性的人源化抗体或其结合片段、其制备及使用方法,包含vWF介导的疾病或紊乱的治疗方法。 [0005] The present invention relates to a basic human von Willebrand factor (vWF) specific humanized antibody or binding fragment thereof, their preparation and use, including the treatment of vWF-mediated disease or disorder. 具有人类vWF特异性的人源化抗体或其结合片段可包含来自非人类抗体的互补决定区(CDR)(例如小鼠CDR) 及人类框架区。 People having specificity for human vWF humanized antibody or binding fragment can comprise a complementarity determining region derived from a non-human antibody (CDR) (e.g., mouse CDR) and human framework regions.

[0006] 本发明提供具有vWF特异性的人源化抗体或其结合片段,包含如SEQ IDNO :19中所示的重链可变区序列及如SEQ ID NO :28中所示的轻链可变区序列。 [0006] The present invention provides human vWF having specificity for the humanized antibody or binding fragment thereof, comprising as SEQ IDNO: 19 heavy chain variable region sequence as shown in and SEQ ID NO: 28 the light chain can be shown variable region sequences.

[0007] 本发明提供具有vWF特异性的人源化抗体或其结合片段,包含如SEQ IDNO :237中所示的重链序列及如SEQ ID NO :238中所示的轻链序列。 [0007] The present invention provides human vWF having specificity for the humanized antibody or binding fragment thereof, comprising as SEQ IDNO: heavy chain sequence and 237 as shown in SEQ ID NO: light chain sequence shown in 238.

[0008] 本发明提供具有vWF特异性的人源化抗体或其结合片段,包含:(a)重链及轻链互补决定区(⑶R),其对应于存在于鼠类抗体NMC-4重链及轻链可变区(分别为SEQ ID N0: 1及2)中的CDR;(b)重链框架区和/或轻链框架区,前者对应于存在于VH 4-59衍生人类抗体(例如抗体AAC18165. 1(SEQ ID NO :4))的可变区中的框架区,后者对应于存在于人类抗体AAK94808 (VL 018) (SEQ ID NO :6)的可变区中的框架区。 Al. [0008] The present invention provides a humanized antibody specific for vWF binding fragment thereof, comprising: (a) the heavy chain and light chain complementarity determining regions (⑶R), which corresponds to the presence in the NMC-4 murine antibody heavy chain and light chain variable regions (SEQ ID N0: 1 and 2) CDR; (b) a heavy chain framework region and / or light chain framework regions, the former corresponding to the presence in the VH 4-59 human-derived antibody (for example, Antibody AAC18165 1 (SEQ ID NO: 4)) of the variable region framework region, which corresponds to the presence of human antibodies AAK94808 (VL 018) (SEQ ID NO: 6) of the variable region framework regions.

[0009] 本发明提供具有vWF特异性的人源化抗体或其结合片段,包含下列重链CDR其中一种或多种:HCDR1 :GFSLTDYGVD (SEQ ID NO :7) ,HCDR2 =MIffGDGSTDYNSALKS (SEQ ID NO :8), 和/ 或HCDR3 :DPADYGNYDYALDY(SEQID NO :9)。 Al. [0009] The present invention provides a humanized antibody specific for vWF binding fragment thereof, comprising the heavy chain CDR wherein one or more of: HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2 = MIffGDGSTDYNSALKS (SEQ ID NO : 8), and / or HCDR3: DPADYGNYDYALDY (SEQID NO: 9).

[0010] 本发明还提供具有vWF特异性的人源化抗体或其结合片段,包含HCDRl : GFSLTDYGVD(SEQ ID NO :7), HCDR2 =MIffGDGSTDYNSALKS(SEQ ID NO :8), HCDR3 :DPADYGNYDYALDY(SEQ ID NO :9)。 [0010] The present invention further provides human vWF having specificity for the humanized antibody or binding fragment thereof, comprising HCDRl: GFSLTDYGVD (SEQ ID NO: 7), HCDR2 = MIffGDGSTDYNSALKS (SEQ ID NO: 8), HCDR3: DPADYGNYDYALDY (SEQ ID NO: 9). 在一些实施方案中,该人源化抗体或其结合片段还可包含来自人类抗体AAC18165. 1(SEQ ID NO :4)的可变区的重链框架区。 In some embodiments, the humanized antibody or binding fragment thereof may further comprise AAC18165 1 from a human antibody. (SEQ ID NO: 4) regions of the heavy chain variable region framework.

[0011] 本发明还提供具有vWF特异性的人源化抗体或其结合片段,包含下列轻链CDR其中一种或多种:LCDRl :SASQDINKYLN(SEQ ID N0:10)、LCDR2 :YTSSLHS(SEQ ID 而:11)、和/ 或LCDR3 =QQYEKLPffT(SEQ ID NO :12)。 [0011] The present invention further provides human vWF having specificity for the humanized antibody or binding fragment thereof, comprising the following light chain CDR wherein one or more of: LCDRl: SASQDINKYLN (SEQ ID N0: 10), LCDR2: YTSSLHS (SEQ ID and: 11), and / or LCDR3 = QQYEKLPffT (SEQ ID NO: 12).

[0012] 本发明还提供具有vWF特异性的人源化抗体或其结合片段,包含下列轻链CDR :LCDR1 :SASQDINKYLN(SEQ ID NO :10)、LCDR2 :YTSSLHS (SEQID N0:11)、及LCDR 3 : QQYEKLPffT (SEQ ID N0:12)。 [0012] The present invention further provides human vWF having specificity for the humanized antibody or binding fragment thereof, comprising the following light chain CDR: LCDR1: SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQID N0: 11), and LCDR 3: QQYEKLPffT (SEQ ID N0: 12). 在某些实施方案中,人源化抗体或其结合片段还可包含:来自人类抗体AAK94808 (SEQ ID NO :6)的可变区的轻链框架区。 In certain embodiments, a humanized antibody or binding fragment thereof may further comprise: a light chain framework region: (6 SEQ ID NO) of the variable region derived from a human antibody AAK94808.

[0013] 本发明还提供具有vWF特异性的人源化抗体或其结合片段,包含:重链⑶R, HCDRl :GFSLTDYGVD(SEQ ID NO :7)、HCDR2 =MIffGDGSTDYNSALKS (SEQ ID NO :8)、及HCDR3 : DPADYGNYDYALDY (SEQ ID NO :9);及轻链CDR,LCDRl :SASQDINKYLN(SEQ ID NO :10)、LCDR2 : YTSSLHS(SEQ ID N0:11)、&LCDR3:QQYEKLPWT(SEQ ID N0:12)。 [0013] The present invention further provides human vWF having specificity for the humanized antibody or binding fragment thereof, comprising: a heavy chain ⑶R, HCDRl: GFSLTDYGVD (SEQ ID NO: 7), HCDR2 = MIffGDGSTDYNSALKS (SEQ ID NO: 8), and HCDR3: DPADYGNYDYALDY (SEQ ID NO: 9); and the light chain CDR, LCDRl: SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQ ID N0: 11), & LCDR3: QQYEKLPWT (SEQ ID N0: 12). 在某些实施方案中,人源化抗体或其结合片段还可包含来自人类抗体MK94808(SEQ ID NO :6)的可变区的轻链框架区、和/或来自人类抗体AAC18165. 1(SEQ ID NO :4)的可变区的重链框架区。 In certain embodiments, a humanized antibody or binding fragment thereof may further comprise a human antibody derived from MK94808: light chain framework region (SEQ ID NO 6) of the variable region, and / or from a human antibody AAC18165 1 (SEQ. ID NO: 4) heavy chain variable region framework region.

[0014] 本发明提供具有vWF特异性的人源化抗体或其结合片段,包含下列重链可变区其中一种或多种:H2 (SEQ ID NO :13)、H4(SEQ ID NO : 14)、H5 (SEQ ID NO : 15)、H6 (SEQ ID NO :16)、H7(SEQ ID NO : 17)、H8 (SEQID NO : 18)、H9 (SEQ ID NO : 19)、H12 (SEQ ID NO :20)、 H13(SEQ ID NO :21)、H14(SEQ ID NO :22)、H15 (SEQ ID N0:145)、或H 16 (SEQ ID N0:146)。 [0014] The present invention provides human vWF having specificity for the humanized antibody or binding fragment thereof, comprising the heavy chain variable region wherein one or more of: H2 (SEQ ID NO: 13), H4 (SEQ ID NO: 14 ), H5 (SEQ ID NO: 15), H6 (SEQ ID NO: 16), H7 (SEQ ID NO: 17), H8 (SEQID NO: 18), H9 (SEQ ID NO: 19), H12 (SEQ ID NO: 20), H13 (SEQ ID NO: 21), H14 (SEQ ID NO: 22), H15 (SEQ ID N0: 145), or H 16 (SEQ ID N0: 146).

[0015] 本发明提供具有vWF特异性的人源化抗体或其结合片段,包含下列轻链可变区其中一种或多种:L5(SEQ ID NO :23)、L4(SEQ ID NO :24)、L6(SEQ ID NO :25)、L7(SEQ ID NO : 26)、L8(SEQ ID NO :27)、L9 (SEQID NO:28)、LlO(SEQ ID NO :29)或Lll(SEQ ID NO :30)。 [0015] The present invention provides human vWF having specificity for the humanized antibody or binding fragment thereof, comprising the light chain variable region wherein one or more of: L5 (SEQ ID NO: 23), L4 (SEQ ID NO: 24 ), L6 (SEQ ID NO: 25), L7 (SEQ ID NO: 26), L8 (SEQ ID NO: 27), L9 (SEQID NO: 28), LlO (SEQ ID NO: 29) or Lll (SEQ ID NO: 30).

[0016] 本发明提供具有vWF特异性的人源化抗体或其结合片段,包含(1)下列重链可变区其中一种或多种:H2(SEQ ID NO :13)、H4(SEQ ID NO : 14)、H5(SEQ ID NO : 15)、H6(SEQ ID NO :16)、H7(SEQ ID NO : 17)、H8 (SEQID NO : 18)、H9 (SEQ ID NO : 19)、H12 (SEQ ID NO :20)、 H13(SEQ ID NO :21)、H14(SEQ ID NO :22)、H15 (SEQ ID NO : 145)、或H16 (SEQ ID NO: 146); 及(2)下列轻链可变区其中一种或多种:L5(SEQ ID NO :23)、L4 (SEQID NO :24)、L6 (SEQ ID NO :25)、L7(SEQ ID NO :26)、L8 (SEQ ID NO :27)、L9 (SEQ ID NO :28)、LlO (SEQ ID NO :29) 或Lll (SEQ ID NO :30)。 [0016] The present invention provides human vWF having specificity for the humanized antibody or binding fragment thereof, comprising (1) of the following heavy chain variable region wherein one or more of: H2 (SEQ ID NO: 13), H4 (SEQ ID NO: 14), H5 (SEQ ID NO: 15), H6 (SEQ ID NO: 16), H7 (SEQ ID NO: 17), H8 (SEQID NO: 18), H9 (SEQ ID NO: 19), H12 (SEQ ID NO: 20), H13 (SEQ ID NO: 21), H14 (SEQ ID NO: 22), H15 (SEQ ID NO: 145), or H16 (SEQ ID NO: 146); and (2) the following light chain variable region wherein one or more of: L5 (SEQ ID NO: 23), L4 (SEQID NO: 24), L6 (SEQ ID NO: 25), L7 (SEQ ID NO: 26), L8 (SEQ ID NO: 27), L9 (SEQ ID NO: 28), LlO (SEQ ID NO: 29) or Lll (SEQ ID NO: 30).

[0017] 举例而言,人源化抗体或其结合片段可包含:L5(SEQ ID NO :23)和H2(SEQ ID NO : 13) ;L5(SEQ ID NO :23)禾口H4(SEQ ID NO :14) ;L5 (SEQID NO :23)禾口H5 (SEQ ID N0:15); L5 (SEQ ID NO :23)和H6(SEQ ID NO :16) ;L5(SEQ ID NO : 23)禾口H7 (SEQ ID NO :17) ; L5(SEQ ID NO :23)和H8(SEQ ID NO :18) ; L4(SEQ ID NO :24)禾口H2 (SEQ ID NO :13) ; L6(SEQ ID NO :25)禾口H2(SEQ ID NO :13) ;Lll (SEQ ID NO :30)和H2 (SEQID NO :13) ; L7(SEQ ID NO: 26)禾口H2(SEQ ID NO :13) ;L9 (SEQ ID NO :28)和H9 (SEQ ID NO :19) ; L8(SEQ ID NO :27) 和H9(SEQ ID NO :19) ;L7(SEQ ID NO :26)和H9(SEQ ID NO :19) ;L6(SEQ ID NO :25)和H9 (SEQID NO :19) ;L4(SEQ ID NO :24)和H9 (SEQ ID NO :19) ;L5(SEQ ID NO :23)禾口H9 (SEQ ID NO: 19) ;LlO (SEQ ID NO :29)和H9 (SEQ ID NO :19) ; L9(SEQ ID NO :28)和H9 (SEQ ID NO :19) ; L9(SEQ ID NO :28)和H12(SEQID NO :20) ; L9(SEQ ID NO :28) ^P Hl 3 (SEQ ID NO: [0017] For example, a humanized antibody or binding fragment thereof may comprise: L5 (SEQ ID NO: 23) and H2 (SEQ ID NO: 13); L5 (SEQ ID NO: 23) Wo port H4 (SEQ ID NO: 14); L5 (SEQID NO: 23) Wo port H5 (SEQ ID N0: 15); L5 (SEQ ID NO: 23) and H6 (SEQ ID NO: 16); L5 (SEQ ID NO: 23) Wo mouth H7 (SEQ ID NO: 17); L5 (SEQ ID NO: 23) and H8 (SEQ ID NO: 18); L4 (SEQ ID NO: 24) Wo port H2 (SEQ ID NO: 13); L6 (SEQ ID NO: 25) Wo port H2 (SEQ ID NO: 13); Lll (SEQ ID NO: 30) and H2 (SEQID NO: 13); L7 (SEQ ID NO: 26) Wo port H2 (SEQ ID NO: 13 ); L9 (SEQ ID NO: 28) and H9 (SEQ ID NO: 19); L8 (SEQ ID NO: 27) and H9 (SEQ ID NO: 19); L7 (SEQ ID NO: 26) and H9 (SEQ ID NO: 19); L6 (SEQ ID NO: 25) and H9 (SEQID NO: 19); L4 (SEQ ID NO: 24) and H9 (SEQ ID NO: 19); L5 (SEQ ID NO: 23) Wo mouth H9 (SEQ ID NO: 19); LlO (SEQ ID NO: 29) and H9 (SEQ ID NO: 19); L9 (SEQ ID NO: 28) and H9 (SEQ ID NO: 19); L9 (SEQ ID NO: 28) and H12 (SEQID NO: 20); L9 (SEQ ID NO: 28) ^ P Hl 3 (SEQ ID NO:

1021) ; L9(SEQ ID NO :28)和H14(SEQ ID NO :22) ;Lll (SEQ ID NO :30)和H 9 (SEQ ID NO: 19);或Lll (SEQ ID NO :30)和H14(SEQ ID NO :22)。 1021); L9 (SEQ ID NO: 28) and H14 (SEQ ID NO: 22); Lll (SEQ ID NO: 30) and H 9 (SEQ ID NO: 19); or Lll (SEQ ID NO: 30) and H14 (SEQ ID NO: 22).

[0018] 本发明还提供具有vWF特异性的人源化抗体或其结合片段,包含(1)下列重链CDR 其中一种或多种:HCDR1 :GFSLTDYGVD(SEQ ID NO :7), HCDR2 =MIffGDGSTDYNSALKS (SEQ ID NO :8),禾口/ 或HCDR 3 =DPADYGNYDYALDY (SEQID NO :9);及(2)下列轻链CDR 其中一禾中或多种=LCDRl =SASQDI NKYLN (SEQID NO :10),LCDR2 :YTSSLHS (SEQ ID NO :11),禾Π / 或IXDR3 :QQYEKLPWT(SEQ ID N0:12)。 [0018] The present invention further provides human vWF having specificity for the humanized antibody or binding fragment thereof, comprising (1) a heavy chain CDR of the following one or more of: HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2 = MIffGDGSTDYNSALKS (SEQ ID NO: 8), Hekou / or HCDR 3 = DPADYGNYDYALDY (SEQID NO: 9); and (2) The following light chain CDR Wo one or more = LCDRl = SASQDI NKYLN (SEQID NO: 10), LCDR2: YTSSLHS (SEQ ID NO: 11), Wo Π / or IXDR3: QQYEKLPWT (SEQ ID N0: 12). 在某些实施方案中,人源化抗体或其结合片段还可包含来自人类抗体AAK94808 (SEQ ID NO :6)的可变区的轻链框架区、和/或来自人类抗体AAC18165. KSEQ ID NO :4)的可变区的重链框架区。 In certain embodiments, a humanized antibody or binding fragment thereof may further comprise a human antibody derived from AAK94808: light chain framework region (SEQ ID NO 6) of the variable region, and / or from a human antibody AAC18165 KSEQ ID NO. : 4) a heavy chain variable region framework region.

[0019] 本发明提供如此处所述的结合至vWF的人源化抗体或结合片段,其对vWF具有IOnM或更小的亲和力(Kd),优选为5nM或更小,更优选InM或更小,最优选为至少约0. 2nM 至约0. 4nM。 [0019] The present invention provides a combination as described herein to vWF humanized antibody or binding fragment of vWF having IOnM or less affinity (Kd), preferably 5nM or less, more preferably InM or less , most preferably at least about 0. 2nM to about 0. 4nM. 本发明还提供如此处所述的可竞争结合至vWF的人源化抗体或结合片段,其具有IOOnM或更小的亲和力(Ki),优选为50nM或更小,更优选IOnM或更小,最优选为至少约0. 2nM 至约5. OnM。 The present invention also provides as described herein may compete for binding to vWF humanized antibody or binding fragment thereof having IOOnM or less affinity (Ki), preferably 50nM or less, more preferably IOnM or less, and most preferably at least about 0. 2nM to about 5. OnM.

[0020] 本发明还提供结合至vWF的Al结构域的人源化抗体或结合片段,其具有IOnM或更小的亲和力(Kd),优选为5nM或更小,更优选InM或更小,最优选为至少约0. 2nM至约0. 4nM。 [0020] The present invention further provides a binding to the vWF Al domain of a humanized antibody or binding fragment thereof having IOnM or less affinity (Kd), preferably 5nM or less, more preferably InM or less, most preferably at least about 0. 2nM to about 0. 4nM. 本发明还提供可竞争结合至vWF的Al结构域的人源化抗体或其结合片段,其对vWF 具有IOOnM或更小的亲和力(Ki),优选为50nM或更小,更优选IOnM或更小,最优选为至少约0. 2nM 至约5. OnM。 The present invention also provides a competitive binding to vWF Al domain of a humanized antibody or binding fragment thereof for vWF having IOOnM or less affinity (Ki), preferably 50nM or less, more preferably IOnM or less , most preferably at least about 0. 2nM to about 5. OnM.

[0021] 本发明还提供Fab片段热稳定温度大于65C的人源化抗体或结合片段。 [0021] The present invention also provides a thermally stable Fab fragments temperature above 65 C humanized antibody or binding fragment thereof.

[0022] 本发明提供具有vWF特异性的人源化抗体或其结合片段,包含: HCDRl (GFSLTDYGVD ;SEQ ID NO :7), HCDR2(MIWGDGSTDYNSALKS ;SEQ ID NO :8), HCDR3 (DPADYGNYDYALDY ;SEQ ID NO :9);及轻链CDRl、轻链CDR2 及LCDR3 (QQYEKLPWT ;SEQ ID NO :12),附带条件为LCDl和/或LCD2至少其中一者分别不为SASQDINKYLN(SEQ ID NO: 10)或YTSSLHS (SEQ ID NO: 11)。 Al. [0022] The present invention provides a humanized antibody specific for vWF binding fragment thereof, comprising: HCDRl (GFSLTDYGVD; SEQ ID NO: 7), HCDR2 (MIWGDGSTDYNSALKS; SEQ ID NO: 8), HCDR3 (DPADYGNYDYALDY; SEQ ID NO: 9); and a light chain CDRl, CDR2 light chain and LCDR3 (QQYEKLPWT; SEQ ID NO: 12), with the proviso that LCDl and / or at least one of them LCD2 were not SASQDINKYLN (SEQ ID NO: 10) or YTSSLHS (SEQ ID NO: 11).

[0023] 本发明还提供具有vWF特异性的人源化抗体或其结合片段,包含: HCDRl (GFSLTDYGVD ;SEQ ID NO :7) , HCDR2 (MIWGDGSTDYNSALKS ;SEQ IDNO :8), HCDR3(DPADYGNYDYALDY ;SEQ ID NO :9), LCDRl(SASQDINKYLN ;SEQ ID NO :10), LCDR2 (YTSSLHS ; SEQ ID NO : 11)及LCDR 3 (QQYEKLPWT ; SEQ ID NO :12);对应于人类抗体种系家族VH4的框架区1、2和3的重链框架区1、2和3 ;以及对应于人类抗体种系家族VKl的框架区1、2和3的轻链框架区1、2和3。 Al. [0023] The present invention also provides a vWF having specificity for the humanized antibody or binding fragment thereof, comprising: HCDRl (GFSLTDYGVD; SEQ ID NO: 7), HCDR2 (MIWGDGSTDYNSALKS; SEQ IDNO: 8), HCDR3 (DPADYGNYDYALDY; SEQ ID NO: 9), LCDRl (SASQDINKYLN; SEQ ID NO: 10), LCDR2 (YTSSLHS; SEQ ID NO: 11) and LCDR 3 (QQYEKLPWT; SEQ ID NO: 12); corresponding to human germline family VH4 antibody framework region heavy chain framework region 1, 2 and 3, 1, 2 and 3; and the corresponding human antibody germline family VKl framework regions of the light chain framework region 1, 2 and 3, 1, 2 and 3.

[0024] 本发明总体还涉及分离核酸,其编码本发明所公开的具有人类vWF特异性的人源化抗体。 [0024] The present invention generally relates to an isolated nucleic acid, people with human vWF specific encoding the disclosed humanized antibodies. 在某些实施方案中,载体可包含本发明所公开的核酸;在另一实施方案中,宿主细胞可包含所公开的核酸。 In certain embodiments, the vector may comprise a nucleic acid of the present invention are disclosed; In another embodiment, the host cell may comprise a nucleic acid disclosed.

[0025] 本发明总体还涉及具有vWF特异性的人源化抗体的制造方法,包含:培养本发明的宿主细胞,使得核酸被表达并产生抗体。 Overall [0025] The present invention further relates to human vWF having a specific method for producing a humanized antibody, comprising: culturing a host cell of the present invention, so that the nucleic acid is expressed and the antibody produced. 在某些实施方案中,该方法还包含自宿主细胞培养物回收抗体;在某些实施方案中,抗体从宿主细胞培养基(medium)中回收;在某些实施方案中,于培养之前,用包含编码重链可变区的核酸的载体及包含编码轻链可变区的核酸的载体共同转染宿主细胞。 In certain embodiments, the method further comprises recovering from the host cell culture antibody; In certain embodiments, the antibody recovered from the host cell culture medium (medium); and in some embodiments, prior to incubation, vector comprising a nucleic acid encoding the heavy chain variable region and a vector comprising a nucleic acid encoding the light chain variable region of co-transfected host cells. [0026] 本发明基本涉及包含具有vWF特异性的人源化抗体及可药用载体的组合物。 [0026] The present invention relates to compositions comprising people with basic vWF-specific humanized antibody and a pharmaceutically acceptable carrier composition.

[0027] 本发明还提供这样的组合物,其包含如此处所述的第一人源化抗体或其结合片段、及结合至vWF的Al结构域的第二抗体。 [0027] The present invention also provides a composition as described herein comprising a first one of the humanized antibody or binding fragment, and binding to the vWF Al domain of a second antibody. 在某些实施方案中,第二抗体为AJW200。 In certain embodiments, the second antibody is AJW200.

[0028] 本发明总体还涉及对受试者(例如病患)的vWF介导的疾病或紊乱(例如血栓病或紊乱)的治疗方法,其通过给予该受试者有效治疗量的具有vWF特异性的人源化抗体或其片段进行。 [0028] The present invention generally relates to a subject (eg patient) of vWF mediated disease or disorder (such as thrombotic disease or disorder) treatment, which has a specific vWF by administering to the subject a therapeutically effective amount of of the humanized antibody or fragment. 在某些实施方案中,受试者为人类;在某些实施方案中,治疗有效量足以抑制血小板聚集但并不足以引发明显的临床出血迹象。 In certain embodiments, the subject is a human; In certain embodiments, the therapeutically effective amount sufficient to inhibit platelet aggregation but not enough to lead to significant clinical signs of bleeding.

[0029] 本发明还提供如此处所述的人源化抗体或其结合片段作为药物的用途;本发明还提供如此处所述的人源化抗体或其结合片段在制备治疗vWF介导的疾病或紊乱的药物中的用途。 [0029] The present invention also provides a person as described herein humanized antibody or binding fragment thereof as a medicament; The present invention also provides a person as described herein humanized antibody or binding fragment thereof in the manufacture of therapeutic vWF-mediated diseases or disorder medicament. 在某些实施方案中,治疗有效量足以抑制血小板聚集但并不足以引发明显的临床出血迹象。 In certain embodiments, the therapeutically effective amount sufficient to inhibit platelet aggregation but not enough to lead to significant clinical signs of bleeding.

[0030] 在某些实施方案中,vWF介导的疾病或紊乱为血栓性疾病或紊乱;在某些实施方案中,血栓性异常为心血管疾病或例如局部缺血性中风(ischemic stroke)的脑血管疾病; 在某些实施方案中,心血管疾病为动脉粥状硬化症、再狭窄、心绞痛(angina)、急性心肌梗塞、急性冠状动脉综合症(acute coronary syndrome)、或与糖尿病相关联的心血管异常; 在某些实施方案中,血栓性疾病或紊乱为血管发炎、静脉血栓形成、镰刀形细胞病、异种移植排斥、外周血管病、血栓性血小板减少性紫癜症、囊性纤维化、血管性痴呆、雷诺病、类风湿性关节炎、或糖尿病;在某些实施方案中,脑血管疾病为血管性痴呆、缺血性卒中、或预防再发性中风。 [0030] In some embodiments, vWF-mediated thrombotic disease or disorder is a disease or disorder; in some embodiments, the thrombotic cardiovascular diseases or abnormalities such as ischemic stroke (ischemic stroke) of cerebrovascular disease; in some embodiments, the cardiovascular disease is atherosclerotic sclerosis, restenosis, angina (angina), acute myocardial infarction, acute coronary syndrome (acute coronary syndrome), or associated with diabetes cardiovascular abnormalities; in some embodiments, the thrombotic disease or disorder is inflammation of blood vessels, venous thrombosis, sickle cell disease, xenograft rejection, peripheral vascular disease, thrombotic thrombocytopenic purpura disease, cystic fibrosis, vascular dementia, Raynaud's disease, rheumatoid arthritis, or diabetes; in some embodiments, cerebrovascular disease, vascular dementia, ischemic stroke, or preventing recurrent stroke.

[0031] 在某些实施方案中,具有vWF特异性的人源化抗体或其结合片段缺乏效应子功能;在某些实施方案中,人源化抗体包含源自于I gG4的Fc区。 [0031] In certain embodiments, the human has a specific vWF humanized antibody or binding fragment lacking effector function; In certain embodiments, the humanized antibody derived from the I gG4 comprises an Fc region.

[0032] 在某些实施方案中,具有vWF特异性的人源化抗体或其结合片段结合至冯维勒布兰德氏因子的Al结构域。 [0032] In certain embodiments, the human has a specific vWF humanized antibody or binding fragment thereof binds to the Al domain of von Willebrand Factor.

[0033] 在某些实施方案中,具有vWF特异性的抗体结合片段为Fab,Fab,,Fab,-SH, Fv, scFv,F(ab,)2 或双链抗体(diabody)。 [0033] In certain embodiments, an antibody having specificity for vWF binding fragments Fab, Fab ,, Fab, -SH, Fv, scFv, F (ab,) 2, or double-stranded antibody (diabody).

[0034] 在某些实施方案中,具有vWF特异性的抗体结合片段不为Fab。 [0034] In certain embodiments, an antibody having specific binding vWF fragments not Fab.

[0035] 在某些实施方案中,具有vWF特异性的人源化抗体为全长抗体。 [0035] In certain embodiments, the human has a vWF-specific antibody is full length humanized antibodies.

[0036] 在某些实施方案中,人源化抗体可在HCDRl中包含一个或多个取代,例如F27G, L29I,T30S和/或V34W取代;在某些实施方案中,人源化抗体可在HCDR2中包含一个或多个取代,例如S61P和/或A62S取代;在某些实施方案中,人源化抗体可在IXDRl中包含一个或多个取代,例如S24Q,N30S和/或K31N取代;在某些实施方案中,人源化抗体可在IXDR2 中包含一个或多个取代,例如Y50D,T51A,S53N, H55E和/或S56T取代。 [0036] In some embodiments, the humanized antibodies may comprise one or more substituents HCDRl such F27G, L29I, T30S and / or V34W substituted; in some embodiments, the humanized antibody may HCDR2 contains one or more substituents, such as S61P and / or A62S substitution; In certain embodiments, the humanized antibody may comprise one or more substituents in IXDRl such S24Q, N30S and / or K31N substituted; in some embodiments, the humanized antibodies may comprise one or more substituents IXDR2 such Y50D, T51A, S53N, H55E and / or S56T substitution. 在某些实施方案中,人源化抗体可包含:(1)在HCDRl中的一个或多个的取代,例如? In certain embodiments, the humanized antibody may comprise: a substituted or plurality of (1) an HCDRl in, e.g.,? 276丄2913305和/或V34W取代;(2)在HCDR2中的一个或多个的取代,例如S61P和/或A62S取代;(3)在LCDRl 中的一个或多个的取代,例如S24Q,N30S和/或K31N取代;及(4)在IXDR2中的一个或多个的取代,例如Y50D, T51A, S53N, H55E和/或S56T取代。 276 Shang 2,913,305 and / or V34W groups; substituted or more (2) an HCDR2 in such S61P and / or A62S substitution; replace one or more of (3) in one LCDRl, such S24Q, N30S and / or K31N substituted; and substituted with one or more of (4) in an IXDR2, such Y50D, T51A, S53N, H55E and / or S56T substitution.

[0037] 附图简述 [0037] Brief Description

[0038] 图1显示NMC-4嵌合抗体在瑞斯特霉素诱导的vWF介导的血小板凝集测定中的抑制活性,相较于原始NMC-4单株抗体及另一抗vWF抗体AJW200。 [0038] FIG. 1 shows the NMC-4 chimeric antibody measurement of platelet aggregation induced by doxorubicin in Rist vWF-mediated inhibitory activity of the original NMC-4 monoclonal antibody and another anti-vWF antibody AJW200 compared to. [0039] 图2A-B显示以单独及组合使用无标记NMC-4嵌合抗体(同源竞争)及AJW200,对以Eu标记的NMC-4结合的竞争(图2A)。 [0039] Figures 2A-B alone and in combination are shown in unlabeled NMC-4 chimeric antibody (homologous competition) and AJW200, NMC-4 competition binding (Fig. 2A) of the labeled with Eu. 图2A为以无标记的NMC-4单克隆抗体、同种型对照IgG、AJW200及具有称为H9,L9的可变区的NMC-4抗体的人源化衍生物对以Eu标记的NMC-4嵌合抗体的竞争;图2B为在20nM AJW200存在或不存在时的NMC-4竞争的希尔图(Hill plot)。 2A is a person in a non-labeled NMC-4 monoclonal antibodies, isotype control IgG, AJW200 and NMC-4 antibody having known H9, L9 variable region of the humanized derivatives labeled with Eu NMC- Chimeric antibody competition 4; FIG. 2B is NMC-4 in the presence or absence of competition at 20nM AJW200 FIG Hill (Hill plot).

[0040] 图3A-E显示NMC-4在剪切流情形下阻断血小板附着至内皮vWF的能力,包括附着至HUVEC细胞的血小板的显微照片。 [0040] Figure 3A-E display NMC-4 blockade in the case of platelet adhesion under shear flow to the ability of endothelial vWF, including platelet adhesion to HUVEC cells photomicrographs. 以PBS (图A)或25 μ M组胺(图BE)、加上10 μ g/ml 抗vWF 抗体、NMC-4 (图C)、18 μ g/ml 抗GPIb- α 抗体、ΑΚ2 (图D)、或18 μ g/ml 小鼠IgG (图E)处理HUVEC细胞单层。 With PBS (panel A) or 25 μ M histamine (Fig BE), plus 10 μ g / ml anti-vWF antibody, NMC-4 (Fig. C), 18 μ g / ml anti GPIb- α antibody, ΑΚ2 (Fig. D), or 18 μ g / ml mouse IgG (Figure E) treated HUVEC cell monolayers. 在灌注单层各处之前,还立即将不同抗体包含于对应的血小板悬浮液中。 Before perfusion throughout the monolayer, also immediately different antibodies contained in the corresponding platelet suspension.

[0041 ] 图4A-C显示相较于AJW200 (图4C),在动脉血栓形成的大鼠氯化铁模型中的NMC-4嵌合体(图4A)及具有名为H14,L10的可变区的人源化抗体(图4B)的活性。 [0041] FIG. 4A-C show compared to AJW200 (Fig. 4C), ferric chloride rat model of arterial thrombosis in the NMC-4 chimera (Fig. 4A) and has called H14, L10 of the variable region humanized antibody (Fig. 4B) activity. 将此三抗体在剂量应答研究中比较。 This triabodies compare dose response study.

[0042] 图5显示递增剂量(0. 03-10mg/kg)的GBR 600在狒狒中的周期性血流减少(CFR) 上的效应。 [0042] Figure 5 shows an increasing dose (0. 03-10mg / kg) of GBR 600 baboons periodic blood flow reduction effect (CFR) on.

[0043] 图6显示递增剂量(0. 01-10mg/kg)的GBR 600在狒狒中的CFR中的效应。 [0043] Figure 6 shows an increasing dose (0. 01-10mg / kg) of GBR 600 baboons in effect in the CFR.

[0044] 图7显示累积剂量(0. 005-0. 07mg/kg)的GBR 600在狒狒中的CFR中的效应。 [0044] Figure 7 shows the cumulative dose (0. 005-0. 07mg / kg) of GBR 600 baboons in effect in the CFR.

[0045] 图8显示累积剂量的GBR 600在狒狒中的CFR中的剂量应答曲线。 [0045] Figure 8 shows the cumulative dose of GBR 600 doses in baboon in the CFR in the response curve.

[0046] 图9显示递增剂量(l-10mg/kg)的氯吡格雷(clopidogrel)在狒狒中的效应。 [0046] Figure 9 shows an increasing dose (l-10mg / kg) of clopidogrel (clopidogrel) effect of baboons.

[0047] 图10显示递增剂量的GBR 600及氯吡格雷输注在切口出血测试上的效应比较。 [0047] Figure 10 shows increasing doses of GBR 600 and clopidogrel infusion test the effect of the cut bleeding compared. 已将剂量表成有效剂量的倍数(例如CFR降至零时的累积剂量)。 It has been effective dose dosage form into multiple (eg CFR dropped to zero the cumulative dose).

[0048] 图11显示由示差扫描量热法所测量的人源化NMC-4变体的热稳定性。 [0048] Figure 11 shows the differential scanning calorimetry measured thermal stability of the humanized NMC-4 variants.

[0049] 发明详述 [0049] DETAILED DESCRIPTION

[0050] 本发明提供具有人类冯维勒布兰德氏因子(vWF)特异性的人源化抗体或其结合片段,包括具有对应于存在于鼠类抗体NMC-4中的一个或多个CDR或部分CDR的CDR区; NMC-4抗体结合vWF的Al结构域上的GPlb- α结合部位(见例如Fujimura等,Blood,77 : 113-20,1991 ;Shima 等,J NaraMed Assoc. ,36 :662,1985)。 [0050] The present invention provides people with human von Willebrand factor (vWF) specific humanized antibody or binding fragment thereof, comprises a corresponding murine antibodies present in the NMC-4 to one or more CDR GPlb- α NMC-4 antibody binds to the Al domain of vWF binding site (see, e.g., Fujimura et al, Blood, 77; the CDR regions or CDR portions: 113-20,1991; Shima et, J NaraMed Assoc, 36:. 662,1985). 本发明的人源化抗体还可包含修饰或未修饰人类框架区,例如对应于人类抗体AAC18165. 1(SEQ ID NO :4)的可变区中的框架区的重链框架区,及对应于人类抗体AAK94808(SEQ ID NO :6)的可变区中的框架区的轻链框架区。 The present invention may further comprise humanized antibody modified or unmodified human framework region, e.g., corresponding to a human antibody AAC18165 1.: Heavy chain framework region (SEQ ID NO 4) of the variable region framework regions, and corresponding to human antibodies AAK94808 (SEQ ID NO: 6) of the variable region of the light chain framework region framework region. 有极多种人类框架区被视为NMC-4⑶R的潜在受体分子,包含对鼠类NMC-4重链及轻链区所属的亚科(subfamily)展现高度同源性的那些。 There are very many human framework regions are considered as potential receptor molecules NMC-4⑶R, including subfamily (subfamily) of murine NMC-4 heavy and light chain region belongs to those who show a high degree of homology. 最令人意外地,在无额外改变的情况下,将NMC-4CDR移植至所选择的重链可变区人类架构其中一者及轻链可变区人类架构其中一者上,就足以维持人源化抗体在阻断vWF介导的血小板应答上的能力。 Most surprisingly, in the absence of additional changes, the NMC-4CDR transplanted to the selected heavy chain variable region and human architecture in which one human light chain variable region framework in which one person is enough to sustain people Humanized antibodies on the ability to block vWF mediated platelet responses.

[0051] “嵌合抗体”一词包含可变区序列源自于一个物种而恒定区序列源自于另一物种的抗体,例如可变区序列源自于小鼠抗体而恒定区序列源自于人类抗体的抗体。 [0051] "Chimeric antibody" word comprises a variable region sequence derived from one species and the constant region sequences are derived from an antibody of another species, such as the variable region sequences derived from a mouse antibody and the constant region sequences are derived from antibodies to human antibodies.

[0052] “人源化抗体”一词包含其中已经将源自于另一哺乳动物物种(例如小鼠)的种系的CDR序列移植至人类构架序列上的抗体。 [0052] "humanized antibody" word contained therein has been derived from another mammalian species (such as a mouse) CDR sequences transplanted to an antibody germline human framework sequences. 在人类构架序列及源自于另一哺乳动物物种的种系的CDR序列内可对框架区进行额外修饰。 Framework regions may be additional modifications in the human framework sequences and CDR sequences derived from the germline of another mammalian species.

[0053] “人类抗体” 一词包含具有其中框架区及CDR区两者均源自于人类种系免疫球蛋 [0053] The "human antibody" wherein the term immunoglobulin comprises a framework region and CDR regions derived from human germline both

13白序列的可变区的抗体;此外,若抗体包含恒定区,则恒定区也源自于人类种系免疫球蛋白序列。 13 White antibody variable region sequence; In addition, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. 本发明的人类抗体可包含非由人类种系免疫球蛋白序列所编码的氨基酸残基(例如由体外随机或定点诱变、或由体内体细胞突变所引入的突变);然而,如此处所使用的术语“人类抗体”并不包含已将源自于另一哺乳动物物种(例如小鼠)的种系的CDR序列移植至人类构架序列上的抗体。 Human antibodies of the present invention may contain non-immunoglobulin sequences from the human germline encoded amino acid residues (for example, a random or site-directed mutagenesis in vitro or in vivo somatic mutation by the introduced mutations); however, as used herein The term "human antibody" does not include CDR sequences derived from another mammalian species have (e.g., mouse) antibody germline transplanted human framework sequences.

[0054] 如此处所使用,若抗体的可变区得自于利用人类种系免疫球蛋白基因的系统,则人类抗体包含“源自于”特定种系序列的重链或轻链可变区。 [0054] As used herein, if the variable regions of antibodies derived from the use of the system of human germline immunoglobulin genes, the human antibody with "derived from" heavy or light chain variable region specific germline sequences. 此种系统包含利用目的抗原使带有人类免疫球蛋白基因的转基因小鼠免疫,或者利用目的抗原来筛选展示于噬菌体上的人类免疫球蛋白基因库。 Such systems include the use of an antigen transgenic mice immunized with human immunoglobulin genes, or the use of an antigen to screen display on the phage human immunoglobulin gene libraries. 通过比较人类抗体的氨基酸序列与人类种系免疫球蛋白的氨基酸序列,并选择在序列上最接近(最大同一性% )人类抗体序列的人类种系免疫球蛋白序列, 可如此鉴定出“源自于”人类种系免疫球蛋白序列的人类抗体。 By amino acid sequence of human germline immunoglobulin compare human antibodies, and select the sequence closest (maximum identity%) immunoglobulin sequences of human germline human antibody sequences can be so identified "from in "human germline immunoglobulin sequences of a human antibody. 例如,由于自然发生的体细胞突变或有意的引入定点突变,“源自于”特定人类种系免疫球蛋白序列的人类抗体可能含有相较于种系序列的氨基酸差异;然而,所选择的人类抗体或其片段一般而言至少有80% 与由人类种系免疫球蛋白序列所编码的氨基酸序列相同,且包含在与其它物种的种系免疫球蛋白氨基酸序列(例如鼠类种系序列)相较时将人类抗体识别为人类的氨基酸残基。 For example, since the naturally occurring somatic mutations or intentional introduction of site-directed mutagenesis, "derived from" a particular human antibody human germline immunoglobulin sequence may contain amino acid differences as compared to the germline sequence; however, the choice of the human Generally antibody or fragment thereof of at least 80% by the human germline immunoglobulin sequences encoded by the same amino acid sequence, and contained in the germline immunoglobulin amino acid sequences of other species (e.g., murine germline sequences) Phase When compared with human antibody recognizes human amino acid residues. 在某些情况下,人类抗体的氨基酸序列可以有至少90 %、或甚至至少95 %,96 %,97 %,98 %, 或99 %的与由种系免疫球蛋白基因所编码的氨基酸序列的同一性,例如包含80 %,81 %, 82 %, 83 %, 84 %, 85 %, 86 %, 87 %, 88 %, 89 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97%,98%,99%,R IOOV0o In some cases, the amino acid sequence of a human antibody can have at least 90%, or even at least 95%, 96%, 97%, 98%, or by the germline immunoglobulin gene encoding the amino acid sequence of the 99% identity, for example, comprise 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98%, 99%, R IOOV0o

[0055] 本发明提供具有vWF特异性的人源化抗体或其结合片段(例如Fab,Fab', Fab,-SH,Fv,scFv,F(ab,)2,双链抗体或单链抗体),包含如SEQ IDNO :19中所示的重链可变区序列及如SEQ ID NO :28中所示的轻链可变区序列;本发明还提供具有vWF特异性的人源化抗体或其结合片段,包含如SEQ ID NO :237中所示的重链序列及如SEQ ID NO :238中所示的轻链序列。 [0055] The present invention provides human vWF having a specific humanized antibody or binding fragment (e.g. Fab, Fab ', Fab, -SH, Fv, scFv, F (ab,) 2, diabody, or a single chain antibody) comprising as SEQ IDNO: a heavy chain variable region sequence shown in Figure 19 and as SEQ ID NO: light chain variable region sequence shown in 28; The present invention also provides people with a specific humanized vWF antibody or binding fragments comprising e.g. SEQ ID NO: heavy chain sequence and 237 as shown in SEQ ID NO: 238 in the light chain sequence shown.

[0056] 本发明还提供具有vWF特异性的人源化抗体或其结合片段,包含(1)下列重链⑶R 其中一种或多种:HCDRl :GFSLTDYGVD(SEQ ID NO :7),HCDR2 :MIWGDGSTDYNSALKS(SEQ ID NO :8),禾口/ 或HCDR 3 :DPADYGNYDYALDY (SEQID NO :9);及(2)下列轻链CDR 其中一种或多种=LCDRl :SASQDINKYLN(SEQID NO :10),LCDR2 :YTSSLHS (SEQ ID NO :11),禾Π / 或LCDR3 : QQYEKLPffT(SEQ ID NO :12)。 [0056] The present invention further provides human vWF having specificity for the humanized antibody or binding fragment thereof, comprising (1) a heavy chain ⑶R following one or more of: HCDRl: GFSLTDYGVD (SEQ ID NO: 7), HCDR2: MIWGDGSTDYNSALKS (SEQ ID NO: 8), Hekou / or HCDR 3: DPADYGNYDYALDY (SEQID NO: 9); and (2) The following light chain CDR wherein the one or more = LCDRl: SASQDINKYLN (SEQID NO: 10), LCDR2: YTSSLHS (SEQ ID NO: 11), Wo Π / or LCDR3: QQYEKLPffT (SEQ ID NO: 12).

[0057] 本发明还提供具有vWF特异性的人源化抗体或其结合片段,包含(1)下列重链⑶R 其中一种或多种:HCDRl :GFSLTDYGVD(SEQ ID NO :7),HCDR2 :MIWGDGSTDYNSALKS(SEQ ID NO :8),和/ 或HCDR3 =DPADYGNYDYALDY(SEQID NO :9);或(2)下列轻链CDR 其中一种或多种=LCDRl :SASQDINKYLN(SEQID NO :10),LCDR2 :YTSSLHS (SEQ ID NO :11),禾Π / 或LCDR3 : QQYEKLPffT(SEQ ID NO :12)。 [0057] The present invention further provides human vWF having specificity for the humanized antibody or binding fragment thereof, comprising (1) a heavy chain ⑶R following one or more of: HCDRl: GFSLTDYGVD (SEQ ID NO: 7), HCDR2: MIWGDGSTDYNSALKS (SEQ ID NO: 8), and / or a HCDR3 = DPADYGNYDYALDY (SEQID NO: 9); or (2) The following light chain CDR wherein the one or more = LCDRl: SASQDINKYLN (SEQID NO: 10), LCDR2: YTSSLHS ( SEQ ID NO: 11), Wo Π / or LCDR3: QQYEKLPffT (SEQ ID NO: 12).

[0058] 本发明还提供具有vWF特异性的人源化抗体或其结合片段,包含(1)重链⑶R, HCDRl :GFSLTDYGVD(SEQ ID NO :7),HCDR2 =MIffGDGSTDYNSALKS(SEQ ID NO :8),及HCDR3 : DPADYGNYDYALDY (SEQ ID NO :9);及(2)轻链CDR,LCDRl :SASQDINKYLN(SEQ ID NO :10), LCDR2 :YTSSLHS (SEQ IDNO :11),及LCDR3 =QQYEKLPffT (SEQ ID NO :12)。 [0058] The present invention further provides human vWF having specificity for the humanized antibody or binding fragment thereof, comprising (1) a heavy chain ⑶R, HCDRl: GFSLTDYGVD (SEQ ID NO: 7), HCDR2 = MIffGDGSTDYNSALKS (SEQ ID NO: 8) and HCDR3: DPADYGNYDYALDY (SEQ ID NO: 9); and (2) a light chain CDR, LCDRl: SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQ IDNO: 11), and LCDR3 = QQYEKLPffT (SEQ ID NO : 12).

[0059] 本发明还提供具有vWF特异性的人源化抗体或其结合片段,包含(1)重链⑶R,HCDRl :GFSLTDYGVD(SEQ ID NO :7),HCDR2 =MIffGDGSTDYNSALKS(SEQ ID NO :8),及HCDR3 : DPADYGNYDYALDY (SEQ ID NO :9);或(2)轻链CDR,LCDRl :SASQDINKYLN(SEQ ID NO :10), LCDR2 :YTSSLHS (SEQ IDNO :11),及LCDR3 =QQYEKLPffT (SEQ ID NO :12)。 [0059] The present invention also provides people with vWF-specific humanized antibody or binding fragment thereof, comprising (1) a heavy chain ⑶R, HCDRl: GFSLTDYGVD (SEQ ID NO: 7), HCDR2 = MIffGDGSTDYNSALKS (SEQ ID NO: 8) and HCDR3: DPADYGNYDYALDY (SEQ ID NO: 9); or (2) a light chain CDR, LCDRl: SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQ IDNO: 11), and LCDR3 = QQYEKLPffT (SEQ ID NO : 12).

[0060] 在某些实施方案中,人源化抗体或其结合片段可包含重链可变区框架区,其中该框架区包含存在于来自人类VH4家族的抗体中的一个或多个(例如1,2,3和/或4)重链构架序列(例如构架1 (Fffl)、构架2 (FW2)、构架3 (FW3)、和/或构架4 (FW4))。 [0060] In certain embodiments, a humanized antibody or binding fragment can comprise a heavy chain variable region framework region, wherein the region comprises a framework derived from human antibodies present in the VH4 family of one or more (e.g. 1 2, 3 and / or 4) a heavy chain framework sequences (e.g., the frame 1 (Fffl), frame 2 (FW2), framework 3 (FW3), and / or framework 4 (FW4)).

[0061] 在某些实施方案中,人源化抗体或其结合片段可包含轻链可变区框架区,其中该框架区包含存在于来自人类VKl家族的抗体中的一个或多个(例如1,2,3和/或4)轻链框架区序列(例如构架1 (Fffl)、构架2 (FW2)、构架3 (FW3)、和/或构架4 (FW4))。 [0061] In certain embodiments, a humanized antibody or binding fragment can comprise a light chain variable region framework region, wherein the framework region comprises the human VKl family exist in antibodies derived from one or more (e.g. 1 2,3 / or 4) and a light chain framework region sequence (e.g., framework 1 (Fffl), frame 2 (FW2), framework 3 (FW3), and / or framework 4 (FW4)).

[0062] 在某些实施方案中,人源化抗体或其结合片段可包含一个或多个(例如一、二、 三、和/或四)存在于来自人类VH4家族的抗体中的重链框架区序列(例如构架1(FW1)、构架2 (FW2)、构架3 (FW3)、和/或构架4(FW4))、以及一个或多个(例如一、二、三、和/或四) 存在于来自人类VK 1家族的抗体中的轻链框架区序列(例如构架I(FWl)、构架2 (FW2)、构架3 (FW3)、和/ 或构架4 (FW4))。 [0062] In some embodiments, the humanized antibody or binding fragment heavy chain framework may comprise one or more (such as one, two, three, and / or d) the presence of antibodies derived from human VH4 family of region sequences (such as frame 1 (FW1), the frame 2 (FW2), frame 3 (FW3), and / or frame 4 (FW4)), and one or more (such as one, two, three, and / or d) present in the light chain framework region sequences from human VK 1 family antibodies (e.g., frame I (FWl), frame 2 (FW2), framework 3 (FW3), and / or framework 4 (FW4)).

[0063] VH4家族的成员及其各自重链框架区1、2和3包含:4_04(分别为SEQID NO -.Ul, 148 及149),4-28 (分别为SEQ ID NO : 150,151 及152),4-30. 1 (分别为SEQ ID NO: 153,154 及155),4-30. 2 (分别为SEQ ID NO : 156,157 及158),4-30. 4 (分别为SEQ ID NO: 159,160 及161),4-31 (分别为SEQ ID NO : 162,163 及164),4-34 (分别为SEQ ID NO: 165,166 及167),4-39 (分别为SEQ ID NO : 168,169 及170),4-59 (分别为SEQ IDNO : 171,172 及173), 4-61 (分别为SEQ ID NO: 174,175 及176)及4-b(分别为SEQ ID NO : 177,178 及179)。 [0063] and their respective family members VH4 heavy chain framework region 1, 2 and 3 contain: 4_04 (respectively SEQID NO -.Ul, 148 and 149), 4-28 (respectively SEQ ID NO: 150,151 and . 152), 4-30 1 (respectively SEQ ID NO: 153,154 and 155), 2 4-30 (respectively SEQ ID NO:.. 156,157 and 158), 4 4-30 (SEQ ID NO: 159,160 and 161), 4-31 (respectively SEQ ID NO: 162,163 and 164), 4-34 (respectively SEQ ID NO: 165,166 and 167), 4-39 (respectively SEQ ID NO: 168,169 and 170), 4-59 (respectively SEQ IDNO: 171,172 and 173), 4-61 (respectively SEQ ID NO: 174,175, and 176) and 4-b (respectively SEQ ID NO: 177,178, and 179).

[0064] VKl家族的成员及其各自轻链框架区1、2和3包含:012(分别为SEQID NO: 180, 181 及182),02 (分别为SEQ ID NO : 183,184 及185),018 (分别为SEQ ID N0:186,187 及188),08 (分别为SEQ ID NO : 189,190 及191),A20 (分别为SEQ ID NO : 192,193 及194), A30(分别为SEQ IDNO : 195,196 及197),L14(分别为SEQ ID NO : 198,199 及200),Ll (分别为SEQ ID NO : 201,202 及203),L15(分别为SEQ ID NO : 204,205 及206),L4 (分别为SEQ ID NO :207,208 及209),L18(分别为SEQ IDNO :210,211 及212),L5 (分别为SEQ ID NO :213,214 及215),L19(分别为SEQ ID NO :216,217 及218),L8 (分别为SEQ ID NO :219, 220 R 221), L23 (分别为SEQ ID NO :222,223 及224),L9 (分别为SEQ ID NO :225,226 及227),L24(分别为SEQ ID NO :228,229 及230),Lll (分别为SEQ ID NO :231,232 及233) 及L12 (分别为SEQ ID NO :234,235 及236)。 [0064] VKl family members and their respective light chain framework region 1, 2 and 3 comprising: 012 (respectively SEQID NO: 180, 181 and 182), 02 (respectively SEQ ID NO: 183,184 and 185), 018 (respectively SEQ ID N0: 186,187 and 188), 08 (respectively SEQ ID NO: 189,190 and 191), A20 (respectively SEQ ID NO: 192,193 and 194), A30 (SEQ respectively IDNO: 195,196 and 197), L14 (respectively SEQ ID NO: 198,199 and 200), Ll (respectively SEQ ID NO: 201,202 and 203), L15 (respectively SEQ ID NO: 204,205 and 206), L4 (respectively SEQ ID NO: 207,208 and 209), L18 (respectively SEQ IDNO: 210,211 and 212), L5 (respectively SEQ ID NO: 213,214 and 215), L19 ( respectively SEQ ID NO: 216,217 and 218), L8 (respectively SEQ ID NO: 219, 220 R 221), L23 (respectively SEQ ID NO: 222,223 and 224), L9 (SEQ ID NO respectively : 225, 226 and 227), L24 (respectively SEQ ID NO: 228,229 and 230), Lll (respectively SEQ ID NO: 231,232, and 233) and L12 (respectively SEQ ID NO: 234,235 and 236).

[0065] 本发明提供具有vWF特异性的人源化抗体或其结合片段,包含下列重链CDR其中一种或多种:HCDR1 :GFSLTDYGVD(SEQ ID NO :7),HCDR2 =MIffGDGSTDYNSALKS (SEQ ID NO :8), 和/ 或HCDR3 :DPADYGNYDYALDY(SEQID NO :9)。 Al. [0065] The present invention provides a humanized antibody specific for vWF binding fragment thereof, comprising the heavy chain CDR wherein one or more of: HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2 = MIffGDGSTDYNSALKS (SEQ ID NO : 8), and / or HCDR3: DPADYGNYDYALDY (SEQID NO: 9).

[0066] 本发明提供具有vWF特异性的人源化抗体或其结合片段,包含下列重链CDR其中一种或多种:HCDR1 :GFSLTDYGVD(SEQ ID NO :7),HCDR2 =MIffGDGSTDYNSALKS (SEQ ID NO :8), 和/ 或HCDR3 :DPADYGNYDYALDY(SEQID NO :9)以及来自人类抗体AAC18165. 1 (SEQ ID NO: 4)的重链框架区。 Al. [0066] The present invention provides a humanized antibody specific for vWF binding fragment thereof, comprising the heavy chain CDR wherein one or more of: HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2 = MIffGDGSTDYNSALKS (SEQ ID NO : 8), and / or HCDR3: DPADYGNYDYALDY (SEQID NO: 9) and AAC18165 1 (SEQ ID NO from a human antibody: 4) of the heavy chain framework region.

[0067] 本发明提供具有vWF特异性的人源化抗体或其结合片段,包含下列重链CDR其中一种或多种:HCDR1 :GFSLTDYGVD(SEQ ID NO :7),HCDR2 =MIffGDGSTDYNSALKS (SEQ ID NO :8),和/ 或HCDR3 :DPADYGNYDYALDY(SEQID NO :9)以及来自人类抗体AAC18165. 1 (SEQ ID NO: 4)的重链框架区,其中该重链框架区不包含一个或多个鼠类残基。 Al. [0067] The present invention provides a humanized antibody specific for vWF binding fragment thereof, comprising the heavy chain CDR wherein one or more of: HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2 = MIffGDGSTDYNSALKS (SEQ ID NO : 8), and / or HCDR3: DPADYGNYDYALDY (SEQID NO: 9) and AAC18165 1 from a human antibody (SEQ ID NO:. 4) of the heavy chain framework region, wherein the heavy chain framework region does not contain one or more of the murine residues.

[0068] 本发明提供具有vWF特异性的人源化抗体或其结合片段,包含下列重链CDR其中一种或多种:HCDR1 :GFSLTDYGVD(SEQ ID NO :7),HCDR2 =MIffGDGSTDYNSALKS (SEQ ID NO :8), 和/ 或HCDR 3 :DPADYGNYDYALDY(SEQID NO :9)以及来自人类抗体AAC18165. 1 (SEQ ID NO: 4)的重链框架区,其中该重链框架区还包含一个或多个的鼠类残基。 Al. [0068] The present invention provides a humanized antibody specific for vWF binding fragment thereof, comprising the heavy chain CDR wherein one or more of: HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2 = MIffGDGSTDYNSALKS (SEQ ID NO : 8), and / or HCDR 3: DPADYGNYDYALDY (SEQID NO: 9) and from a human antibody AAC18165 1 (SEQ ID NO: 4 with one or more) of the heavy chain framework region, wherein the heavy chain framework region is also included. murine residues.

[0069] 本发明还提供具有vWF特异性的人源化抗体或其结合片段,包含:HCDR1 : GFSLTDYGVD(SEQ ID NO :7), HCDR2 =MIffGDGSTDYNSALKS(SEQ IDNO :8),及HCDR 3 : DPADYGNYDYALDY(SEQ ID NO :9)。 [0069] The present invention further provides human vWF having specificity for the humanized antibody or binding fragment thereof, comprising: HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2 = MIffGDGSTDYNSALKS (SEQ IDNO: 8), and HCDR 3: DPADYGNYDYALDY ( SEQ ID NO: 9).

[0070] 本发明还提供具有vWF特异性的人源化抗体或其结合片段,包含:HCDR1 : GFSLTDYGVD(SEQ ID NO :7), HCDR2 =MIffGDGSTDYNSALKS(SEQ IDNO :8),及HCDR3 : DPADYGNYDYALDY (SEQ ID NO :9)以及来自人类抗体AAC18165. 1 (SEQ ID NO :4)的重链框架区。 [0070] The present invention further provides human vWF having specificity for the humanized antibody or binding fragment thereof, comprising: HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2 = MIffGDGSTDYNSALKS (SEQ IDNO: 8), and HCDR3: DPADYGNYDYALDY (SEQ ID NO: 9) and AAC18165 1 (SEQ ID NO from a human antibody: 4) of the heavy chain framework region.

[0071] 本发明还提供具有vWF特异性的人源化抗体或其结合片段,包含:HCDR1 : GFSLTDYGVD(SEQ ID NO :7), HCDR2 =MIffGDGSTDYNSALKS(SEQ IDNO :8),及HCDR 3 : DPADYGNYDYALDY (SEQ ID NO :9)以及来自人类抗体AAC18165. 1 (SEQ ID NO :4)的重链框架区,其中该重链框架区不包含一个或多个鼠类残基。 [0071] The present invention further provides human vWF having specificity for the humanized antibody or binding fragment thereof, comprising: HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2 = MIffGDGSTDYNSALKS (SEQ IDNO: 8), and HCDR 3: DPADYGNYDYALDY ( SEQ ID NO:. 9) and AAC18165 1 (SEQ ID NO from a human antibody: 4) a heavy chain framework region, wherein the heavy chain framework region does not contain one or more murine residues.

[0072] 本发明还提供具有vWF特异性的人源化抗体或其结合片段,包含:HCDR1 : GFSLTDYGVD(SEQ ID NO :7), HCDR2 =MIffGDGSTDYNSALKS(SEQ IDNO :8),及HCDR3 : DPADYGNYDYALDY (SEQ ID NO :9)以及来自人类抗体AAC18165. 1 (SEQ ID NO :4)的重链框架区,其中该重链框架区还包含一个或多个鼠类残基。 [0072] The present invention further provides human vWF having specificity for the humanized antibody or binding fragment thereof, comprising: HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2 = MIffGDGSTDYNSALKS (SEQ IDNO: 8), and HCDR3: DPADYGNYDYALDY (SEQ ID NO: 9) and AAC18165 1 (SEQ ID NO from a human antibody: 4) of the heavy chain framework region, wherein the heavy chain framework region further comprises one or more murine residues.

[0073] 本发明还提供具有vWF特异性的人源化抗体或其结合片段,包含下列轻链CDR其中一种或多种:LCDRl :SASQDINKYLN(SEQ ID N0:10),LCDR2 :YTSSLHS(SEQ ID 而:11),和/ 或LCDR3 =QQYEKLPffT(SEQ ID NO :12)。 [0073] The present invention further provides human vWF having specificity for the humanized antibody or binding fragment thereof, comprising the following light chain CDR wherein one or more of: LCDRl: SASQDINKYLN (SEQ ID N0: 10), LCDR2: YTSSLHS (SEQ ID and: 11), and / or LCDR3 = QQYEKLPffT (SEQ ID NO: 12).

[0074] 本发明还提供具有vWF特异性的人源化抗体或其结合片段,包含下列轻链CDR其中一种或多种:LCDR1 :SASQDINKYLN(SEQ ID N0:10),LCDR2 :YTSSLHS(SEQ ID 而:11),和/ 或LCDR3 :QQYEKLPWT(SEQ ID NO : 12)、以及来自人类抗体AAK94808 (SEQ ID NO :6)的轻链框架区。 [0074] The present invention further provides human vWF having specificity for the humanized antibody or binding fragment thereof, comprising the following light chain CDR wherein one or more of: LCDR1: SASQDINKYLN (SEQ ID N0: 10), LCDR2: YTSSLHS (SEQ ID and: 11), and / or LCDR3: QQYEKLPWT (SEQ ID NO: 12), as well as from a human antibody AAK94808 (SEQ ID NO: 6) a light chain framework region.

[0075] 本发明还提供具有vWF特异性的人源化抗体或其结合片段,包含下列轻链CDR其中一种或多种:LCDRl :SASQDINKYLN(SEQ ID N0:10),LCDR2 :YTSSLHS(SEQ ID 而:11),和/ 或LCDR3 :QQYEKLPWT(SEQ ID NO :12)以及来自人类抗体AAK94808 (SEQ ID NO :6)的轻链框架区,其中该轻链框架区不包含一个或多个鼠类残基。 [0075] The present invention further provides human vWF having specificity for the humanized antibody or binding fragment thereof, comprising the following light chain CDR wherein one or more of: LCDRl: SASQDINKYLN (SEQ ID N0: 10), LCDR2: YTSSLHS (SEQ ID and: 11), and / or LCDR3: QQYEKLPWT (SEQ ID NO: 12) as well as from a human antibody AAK94808 (SEQ ID NO: 6 light chain framework region), wherein the light chain framework region does not contain one or more of the murine residues.

[0076] 本发明还提供具有vWF特异性的人源化抗体或其结合片段,包含下列轻链CDR其中一种或多种:LCDR1 :SASQDINKYLN(SEQ ID NO :10) ,LCDR2 :YTSSLHS(SEQ ID 而:11),和/ 或LCDR3 :QQYEKLPWT(SEQ ID NO: 12)以及来自人类抗体AAK94808 (SEQ ID NO :6)的轻链框架区,其中该轻链框架区还包含一个或多个鼠类残基。 [0076] The present invention further provides human vWF having specificity for the humanized antibody or binding fragment thereof, comprising the following light chain CDR wherein one or more of: LCDR1: SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQ ID and: 11), and / or LCDR3: QQYEKLPWT (SEQ ID NO: 12) as well as from a human antibody AAK94808 (SEQ ID NO: 6 light chain framework region), wherein the light chain framework region further comprises one or more of the murine residues.

[0077] 本发明提供具有vWF特异性的人源化抗体或其结合片段,包含轻链CDRLCDR1 : SASQDINKYLN(SEQ ID NO : 10),LCDR2 :YTSSLHS(SEQ ID NO :11),及LCDR3 :QQYEKLPWT(SEQ ID NO :12)。 [0077] The present invention provides human vWF having specificity for the humanized antibody or binding fragment thereof, comprising a light chain CDRLCDR1: SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQ ID NO: 11), and LCDR3: QQYEKLPWT ( SEQ ID NO: 12). [0078] 本发明提供具有vWF特异性的人源化抗体或其结合片段,包含轻链CDRLCDR1 : SASQDINKYLN(SEQ ID NO : 10),LCDR2 :YTSSLHS(SEQ ID NO :11),及LCDR3 :QQYEKLPWT(SEQ ID NO :12)以及来自人类抗体AAK94808(SEQID NO :6)的轻链框架区。 [0078] The present invention provides human vWF having specificity for the humanized antibody or binding fragment thereof, comprising a light chain CDRLCDR1: SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQ ID NO: 11), and LCDR3: QQYEKLPWT ( SEQ ID NO: 12) as well as from a human antibody AAK94808 (SEQID NO: 6) a light chain framework region.

[0079] 本发明提供具有vWF特异性的人源化抗体或其结合片段,包含轻链CDRLCDR1 : SASQDINKYLN(SEQ ID NO : 10),LCDR2 :YTSSLHS(SEQ ID NO :11),及LCDR3 :QQYEKLPWT(SEQ ID NO :12)以及来自人类抗体AAK94808(SEQID NO :6)的轻链框架区,其中该轻链框架区不包含一个或多个鼠类残基。 [0079] The present invention provides human vWF having specificity for the humanized antibody or binding fragment thereof, comprising a light chain CDRLCDR1: SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQ ID NO: 11), and LCDR3: QQYEKLPWT ( SEQ ID NO: 12) as well as from a human antibody AAK94808 (SEQID NO: 6) of the light chain framework region, wherein the light chain framework region does not contain one or more murine residues.

[0080] 本发明提供具有vWF特异性的人源化抗体或其结合片段,包含轻链CDRLCDR1 : SASQDINKYLN(SEQ ID NO : 10),LCDR2 :YTSSLHS(SEQ ID NO :11),及LCDR3 :QQYEKLPWT(SEQ ID NO :12)以及来自人类抗体AAK94808(SEQID NO :6)的轻链框架区,其中该轻链框架区还 [0080] The present invention provides human vWF having specificity for the humanized antibody or binding fragment thereof, comprising a light chain CDRLCDR1: SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQ ID NO: 11), and LCDR3: QQYEKLPWT ( SEQ ID NO: 12) as well as from a human antibody AAK94808 (SEQID NO: 6 light chain framework region), wherein the light chain framework region further

包含一个或多个鼠类残基。 It contains one or more murine residues.

[0081] 本发明提供具有vWF特异性的人源化抗体或其结合片段,包含:重链⑶R HCDRl :GFSLTDYGVD(SEQ ID NO :7),HCDR2 =MIffGDGSTDYNSALKS (SEQID NO :8),及HCDR 3 : DPADYGNYDYALDY (SEQ ID NO :9);轻链CDR LCDRl :SASQDINKYLN(SEQ ID NO :10),LCDR2 : YTSSLHS (SEQ ID NO :11),及LCDR3 :QQYEKLPWT(SEQ ID NO :12);以及任选地包含来自人类抗体AAK94808(SEQ ID NO :6)的可变区的轻链框架区和/或来自人类抗体AAC18165. 1 (SEQ ID NO :4)的可变区的重链框架区。 Al. [0081] The present invention provides a humanized antibody specific for vWF binding fragment thereof, comprising: a heavy chain ⑶R HCDRl: GFSLTDYGVD (SEQ ID NO: 7), HCDR2 = MIffGDGSTDYNSALKS (SEQID NO: 8), and HCDR 3: DPADYGNYDYALDY (SEQ ID NO: 9); light chain CDR LCDRl: SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQ ID NO: 11), and LCDR3: QQYEKLPWT (SEQ ID NO: 12); and optionally light chain variable region framework region: (6 SEQ ID NO) of and / or AAC18165 1 (SEQ ID NO: 4) containing a human antibody derived from human antibody AAK94808. The heavy chain variable region framework region.

[0082] 本发明还提供具有vWF特异性的人源化抗体或其结合片段的氨基酸序列变体。 [0082] The present invention also provides people with vWF-specific humanized antibody or binding fragment of amino acid sequence variants. 通常具有vWF特异性的人源化抗体或其结合片段的氨基酸序列变体将具有重链和/或轻链框架区的氨基酸序列,其分别有至少80% (具有至少80%氨基酸序列同一性)与具有重链或轻链(例如具有SEQ ID N0:19或SEQ ID NO :28中的重链及轻链可变区序列)的原始人源化抗体的重链和/或轻链框架区的氨基酸序列相同。 People generally have a specific vWF humanized antibody or binding fragment of amino acid sequence variants having an amino acid sequence of the heavy and / or light chain framework regions, which are at least 80% (at least 80% amino acid sequence identity) and having a heavy or light chain (for example, a SEQ ID N0: 19 or SEQ ID NO: 28 in the heavy and light chain variable region sequences) of the heavy chain of the humanized antibody primitive and / or light chain framework regions identical amino acid sequence. 优选为重链和/或轻链框架区序列的氨基酸序列同一性为至少85%,更优选至少90%,最优选至少95%,尤其96%、更尤其97%、再更特别98%,最特别为99%,包含例如80%,81%,82%,83%,84%,85%,86%, 87 %,88 %,89 %,90 %,91 %,92 %,93 %,94 %,95 %,96 %,97 %,98 %,99 %,及100 %。 Preferably a heavy and / or light chain amino acid sequence identity to the sequence of framework region is at least 85%, more preferably at least 90%, most preferably at least 95%, in particular 96%, more in particular 97%, still more in particular 98%, most especially 99%, comprising e.g. 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99% and 100%. 此处将关于此序列的同一性及同源性定义为:在比对序列及若有必要引进空位(gap)以得到最大百分比序列同一性之后,候选序列中与具有vWF特异性的人源化抗体或其结合片段相同的氨基酸残基的百分比。 The identity of this sequence and homology is defined here as follows: after aligning the sequences and introducing gaps, if necessary (gap) in order to obtain the maximum percent sequence identity, the candidate sequences and people with specific humanized vWF antibody or binding fragment the same percentage of amino acid residues. 如此,可通过一般用以比较两多肽的氨基酸的位置相似性的标准方法来决定序列同一性。 Thus, the position can be used to compare the two general amino acid polypeptide similarity standard methods to determine the sequence identity. 利用计算机程序例如BLAST或FASTA,针对其各自氨基酸的最佳匹配(沿着一或两序列的全长,或者沿着一或两序列的预定部分)而排列两多肽。 Using a computer program such as BLAST or FASTA, the best match for their respective amino acids (along the entire length of one or both sequences, or along a predetermined portion of one or both sequences) are arranged two polypeptides. 该程序提供默认开口罚分(opening penalty)及默认空位罚分(gap penalty),且可结合计算机程序而使用例如PAM250(标准得分矩阵;见Dayhoff等,Atlas of Protein Sequence andStructure,卷5,supp. 3(1978))的得分矩阵。 The program provides a default opening penalty (opening penalty) and a default gap penalty (gap penalty), and can be combined with the use of computer programs such as PAM250 (standard scoring matrix; see Dayhoff, etc., Atlas of Protein Sequence andStructure, Volume 5, supp. 3 (1978)) of the scoring matrix. 例如可以下列方式计算同一性百分比:将相同匹配的总数乘以100后,再除以匹配跨距(span)内的较长序列的长度加上为比对两序列而引进至较长序列中的空位的数目的总和。 For example, the percentage of identity can be calculated in the following manner: The total number of identical matches multiplied by 100 and divided by the length of the longer sequence match span (span) than on the inside, plus the introduction of the two sequences to a longer sequence the sum of the number of vacancies.

[0083] 因此,本发明提供具有vWF特异性的人源化抗体或其结合片段,其中该人源化抗体或其结合片段包含重链可变区序列,该重链可变区序列包含至少80%与SEQ ID NO :19 的框架区相同的框架区、和/或具有至少80%与SEQ ID NO :28的框架区相同的框架区的轻链可变区序列。 [0083] Accordingly, the present invention provides human vWF having specificity for the humanized antibody or binding fragment thereof, wherein the humanized antibody or binding fragment thereof comprises a heavy chain variable region sequences, the heavy chain variable region sequence comprises at least 80 % with SEQ ID NO: same framework region framework regions 19, and / or having at least 80% with SEQ ID NO: light chain variable region framework region sequences of the framework 28 of the same area. 本发明还提供具有vWF特异性的人源化抗体或其结合片段,其中该人源 The present invention further provides human vWF having specificity for the humanized antibody or binding fragment thereof, wherein the humanized

17化抗体或其结合片段包含重链可变区序列,该重链可变区序列包含至少80%与SEQ ID NO: 237的框架区相同的框架区、和/或具有至少80%与SEQ ID NO :238的框架区相同的框架区的轻链可变区序列。 17 antibody or binding fragment thereof comprises a heavy chain variable region sequences, the heavy chain variable region sequence comprises at least 80% with SEQ ID NO: framework region of the same frame 237, and / or having at least 80% with SEQ ID NO: light chain variable region framework region sequences of framework region 238 of the same.

[0084] 人源化抗体可任选地包含一个或多个的取代,例如在HCDRl中的F27G,L29I,T30S 和/或V34W取代;在某些实施方案中,人源化抗体可包含一个或多个的取代,例如在HCDR2 中的S61P,和/或A62S取代;在某些实施方案中,人源化抗体可包含一个或多个的取代,例如在IXDRl中的S24Q,N30S和/或K31N取代;在某些实施方案中,人源化抗体可包含一个或多个的取代,例如在LCDR2中的Y50D,T51A,S53N,H55E和/或S56T取代。 [0084] The humanized antibodies may optionally contain one or more substituents, such F27G in HCDRl in, L29I, T30S / or substituted and V34W; In certain embodiments, the humanized antibody may comprise one or a plurality of substituents, such as S61P in HCDR2 in, and / or A62S substitution; In certain embodiments, the humanized antibody may contain one or more substituents, such as S24Q in IXDRl in, N30S and / or K31N replace; in some embodiments, the humanized antibody may contain one or more substituents, such as Y50D in LCDR2 in, T51A, S53N, H55E and / or S56T substitution. 在某些实施方案中,人源化抗体可包含:(1) 一个或多个的取代,例如在HCDRl中的F27G,L29I,T30S和/ 或V34W取代;(2) 一个或多个的取代,包含在HCDR2中的S61P和/或A62S取代;(3) —个或多个的取代,包含在IXDRl中的S24Q,N30S和/或K31N取代;(4) 一个或多个的取代,例如在LCDR2 中的Y50D, T51A, S53N, H55E 和/ 或S56T 取代。 In certain embodiments, the humanized antibody may comprise: replace (1) one or more of, for example, in HCDRl in F27G, L29I, T30S and / or V34W substituted; (2) one or more substituents, HCDR2 included in the S61P and / or A62S substitution; (3) - substituted one or more, including S24Q in IXDRl in, N30S and / or K31N substituted; (4) one or more substituents, such as in LCDR2 The Y50D, T51A, S53N, H55E and / or S56T substitution.

[0085] 本发明提供具有vWF特异性的人源化抗体或其结合片段,包含下列重链可变区其中之一:H2(SEQ ID NO :13)、H4(SEQ ID NO : 14)、H5 (SEQID NO :15)、H6 (SEQ ID NO: 16)、 H7 (SEQ ID NO :17)、H8(SEQ ID NO :18)、H9 (SEQ ID NO :19)、H12 (SEQ ID NO :20)、H13 (SEQ ID NO :21)、H14(SEQ ID NO :22)、H15 (SEQ ID NO : 145)、或H16 (SEQ ID NO : 146)(编码上述重链可变区的多核苷酸分别由SEQ ID NO :128,129,130,131,132,133,134,135,136,137, 138及139加以提供)。 Al. [0085] The present invention provides a humanized antibody specific for vWF binding fragment thereof, comprising a heavy chain variable region where one of the following: H2 (SEQ ID NO: 13), H4 (SEQ ID NO: 14), H5 (SEQID NO: 15), H6 (SEQ ID NO: 16), H7 (SEQ ID NO: 17), H8 (SEQ ID NO: 18), H9 (SEQ ID NO: 19), H12 (SEQ ID NO: 20 polynucleotides 146) (encoding said heavy chain variable region:), H13 (SEQ ID NO: 21), H14 (SEQ ID NO: 22), H15 (SEQ ID NO: 145), or H16 (SEQ ID NO respectively by the SEQ ID NO: 128,129,130,131,132,133,134,135,136,137, 138 and 139 to be provided).

[0086] 本发明提供具有vWF特异性的人源化抗体或其结合片段,包含下列轻链可变区其中之一:L5(SEQ ID NO :23)、L4(SEQ ID NO :24)、L6 (SEQID NO :25)、L7 (SEQ ID NO :26)、 L8 (SEQ ID NO :27)、L9(SEQ ID NO :28)、LlO (SEQ ID NO :29)或Lll (SEQ ID NO :30)(编码上述轻链可变区的多核苷酸分别由SEQ ID NO :120,121,122,123,124,125,126,及127加以提供)。 [0086] The present invention provides human vWF having specificity for the humanized antibody or binding fragment thereof, comprising one of the following light chain variable region: L5 (SEQ ID NO: 23), L4 (SEQ ID NO: 24), L6 (SEQID NO: 25), L7 (SEQ ID NO: 26), L8 (SEQ ID NO: 27), L9 (SEQ ID NO: 28), LlO (SEQ ID NO: 29) or Lll (SEQ ID NO: 30 ) (polynucleotide encoding said light chain variable region, respectively, by SEQ ID NO: 120,121,122,123,124,125,126, and 127 to be provided).

[0087] 本发明还提供具有vWF特异性的人源化抗体或其结合片段,包含:(1)下列重链可变区其中之一:H2 (SEQ ID NO :13)、H4(SEQ ID NO : 14)、H5 (SEQ ID NO : 15)、H6 (SEQ ID NO :16)、H7(SEQ ID NO : 17)、H8 (SEQID NO : 18)、H9 (SEQ ID NO :19)、H12 (SEQ ID NO :20)、 H13(SEQ ID NO :21)、H14(SEQ ID NO :22)、H15 (SEQ ID NO : 145)、或H16 (SEQ ID NO : 146); (2)下列轻链可变区其中之一:L5(SEQ ID NO :23)、L4(SEQ ID NO :24)、L6(SEQ ID NO :25)、 L7 (SEQ ID NO :26)、L8(SEQ ID NO :27)、L9 (SEQ ID NO :28)、L10(SEQ ID NO :29)或Lll(SEQ ID NO :30)。 [0087] The present invention further provides human vWF having specificity for the humanized antibody or binding fragment thereof, comprising: one of the following heavy chain variable region wherein (1): H2 (SEQ ID NO: 13), H4 (SEQ ID NO : 14), H5 (SEQ ID NO: 15), H6 (SEQ ID NO: 16), H7 (SEQ ID NO: 17), H8 (SEQID NO: 18), H9 (SEQ ID NO: 19), H12 ( SEQ ID NO: 20), H13 (SEQ ID NO: 21), H14 (SEQ ID NO: 22), H15 (SEQ ID NO: 145), or H16 (SEQ ID NO: 146); (2) the following light chain One variable region: L5 (SEQ ID NO: 23), L4 (SEQ ID NO: 24), L6 (SEQ ID NO: 25), L7 (SEQ ID NO: 26), L8 (SEQ ID NO: 27 ), L9 (SEQ ID NO: 28), L10 (SEQ ID NO: 29) or Lll (SEQ ID NO: 30).

[0088] 本发明提供具有vWF特异性的人源化抗体或其结合片段,包含:L5 (SEQID NO :23) 和H2(SEQ ID NO: 13) ;L5 (SEQ ID NO :23)禾口H4(SEQ ID NO: 14) ; L5(SEQ ID NO :23)和H5 (SEQ ID NO :15) ;L5 (SEQ ID NO :23)和H6 (SEQ ID NO :16) ;L5(SEQ ID NO :23)禾口H7 (SEQ ID NO :17) ;L5(SEQ ID NO :23)和H8(SEQ ID NO :18) ;L4(SEQ ID NO :24)禾口H2(SEQID NO: 13) ;L6 (SEQ ID NO :25)和H2 (SEQ ID NO: 13) ;Lll (SEQ ID NO :30)和H2 (SEQ ID NO: 13) ;L7(SEQ ID NO :26)禾口H2 (SEQ ID NO :13) ;L9(SEQ ID NO : 28)禾口H 9 (SEQ ID NO :19); L8 (SEQ ID NO :27)和H9(SEQID NO :19) ;L7 (SEQ ID NO :26)和H9 (SEQ ID NO :19) ;L6 (SEQ ID NO :25)和H9(SEQ ID NO: 19) ;L4(SEQ ID NO :24)和H9 (SEQ ID NO: 19) ; L5(SEQ ID NO :23)和H9(SEQ ID NO :19) ;LlO (SEQ ID NO :29)和H9 (SEQID NO :19) ;L9 (SEQ ID NO:28)和H9(SEQ ID NO: 19) ; L9(SEQ ID NO :28)和H12(SEQ ID NO :20) ; L9(SEQ ID NO :28) 和H13(SEQ ID NO :21) ;L9(SEQ ID NO :28)和H14(SEQ ID NO :22) ;L11(SEQ ID NO :30)和H9 (SEQ ID NO: 19);或Lll (SEQ ID NO :30)和H14(SEQ ID NO :22)。 Al. [0088] The present invention provides a humanized antibody specific for vWF binding fragment thereof, comprising: L5 (SEQID NO: 23) and H2 (SEQ ID NO: 13); L5 (SEQ ID NO: 23) H4 Hekou (SEQ ID NO: 14); L5 (SEQ ID NO: 23) and H5 (SEQ ID NO: 15); L5 (SEQ ID NO: 23) and H6 (SEQ ID NO: 16); L5 (SEQ ID NO: 23) Hekou H7 (SEQ ID NO: 17); L5 (SEQ ID NO: 23) and H8 (SEQ ID NO: 18); L4 (SEQ ID NO: 24) Hekou H2 (SEQID NO: 13); L6 (SEQ ID NO: 25) and H2 (SEQ ID NO: 13); Lll (SEQ ID NO: 30) and H2 (SEQ ID NO: 13); L7 (SEQ ID NO: 26) Wo port H2 (SEQ ID NO : 13); L9 (SEQ ID NO: 28) Hekou H 9 (SEQ ID NO: 19); L8 (SEQ ID NO: 27) and H9 (SEQID NO: 19); L7 (SEQ ID NO: 26) and H9 (SEQ ID NO: 19); L6 (SEQ ID NO: 25) and H9 (SEQ ID NO: 19); L4 (SEQ ID NO: 24) and H9 (SEQ ID NO: 19); L5 (SEQ ID NO : 23) and H9 (SEQ ID NO: 19); LlO (SEQ ID NO: 29) and H9 (SEQID NO: 19); L9 (SEQ ID NO: 28) and H9 (SEQ ID NO: 19); L9 ( SEQ ID NO: 28) and H12 (SEQ ID NO: 20); L9 (SEQ ID NO: 28) and H13 (SEQ ID NO: 21); L9 (SEQ ID NO: 28) and H14 (SEQ ID NO: 22 ); L11 (SEQ ID NO: 30) and H9 (SEQ ID NO: 19); or Lll (SEQ ID NO: 30) and H14 (SEQ ID NO: 22).

[0089] 本发明提供如此处所述的结合至vWF的人源化抗体或结合片段,其对vWF具有IOnM或更小的亲和力(Kd),优选为5nM或更小,更优选InM或更小,最优选为至少约0. 2nM 至约0. 4nM(例如自约0. 21,0. 28或0. 34至约0. 25,0. 32或0. 38nM)。 [0089] The present invention provides a combination as described herein to vWF humanized antibody or binding fragment of vWF having IOnM or less affinity (Kd), preferably 5nM or less, more preferably InM or less , most preferably at least about 0. 2nM to about 0. 4nM (for example, from about 0.5 21,0 28 or 0.34 to about 0.1 25,0 32 or 0. 38nM). 本发明还提供如此处所述的竞争结合至vWF的人源化抗体或结合片段,其对vWF具有IOOnM或更小的亲和力(Ki),优选为50nM或更小,更优选IOnM或更小,最优选为至少约0. 2nM至约5. OnM (例如自约0. 22,0. 28 或0. 34 至约2. 3,3. 5 或4. 7nM)。 The present invention also provides a competitive binding as described herein to vWF humanized antibody or binding fragment of vWF having IOOnM or less affinity (Ki), preferably 50nM or less, more preferably IOnM or less, most preferably at least about 0. 2nM to about 5. OnM (for example, from about 0.1 22,0 28 or 0.34 to about 2 3,3 5 or 4. 7nM).

[0090] 本发明还提供此处所述结合至vWF的Al结构域的人源化抗体或结合片段,其对vWF的Al结构域具有IOnM或更小的亲和力(Kd),优选为5nM或更小,更优选InM或更小,最优选为至少约0. 2nM至约0. 4nM(例如自约0. 21,0. 28或0. 34至约0. 25,0. 32或0. 38nM)。 [0090] The present invention also provides herein binds to vWF Al domain of the humanized antibody or binding fragment of Al domain of vWF having IOnM or less affinity (Kd), or more preferably 5nM less, more preferably InM or less, and most preferably at least about 0. 2nM to about 0. 4nM (for example, from about 0.5 21,0 28 or 0.34 to about 0.1 25,0 32 or 0. 38nM ). 本发明还提供竞争结合至vWF的Al结构域的人源化抗体或其结合片段,其对vWF 具有IOOnM或更小的亲和力(Ki),优选为50nM或更小,更优选IOnM或更小,最优选为至少约0. 2nM 至约5. OnM (例如自约0. 22,0. 28 或0. 34 至约2. 3,3. 5 或4. 7nM)。 The present invention also provides a competitive binding to the vWF Al domain of a humanized antibody or binding fragment thereof against vWF having IOOnM or less affinity (Ki), preferably 50nM or less, more preferably IOnM or less, most preferably at least about 0. 2nM to about 5. OnM (for example, from about 0.1 22,0 28 or 0.34 to about 2 3,3 5 or 4. 7nM).

[0091] 本发明还提供Fab片段的热稳定性温度大于65C的人源化抗体或结合片段,优选为大于70C,更优选大于75C,最优选大于80C。 [0091] The present invention also provides a Fab fragment thermal stability greater than 65 C temperature humanized antibody or binding fragment thereof, preferably greater than 70 C, more preferably greater than 75 C, most preferably greater than 80 C. 分析Fab片段热稳定性使用示差扫描量热法,而识别出在全长IgG的情境中的Fab片段的中点熔解温度。 Thermal Stability Analysis Fab fragments using a differential scanning calorimetry, the melting temperature of the midpoint and identified in the context of the full-length IgG Fab fragments. 此类量热法为技术人员所已知,且可根据例如Garber和Demarest (2007),BBRC 355 :751_7加以实施。 Such calorimetry known to the skilled person, and may be based on, for example Garber and Demarest (2007), BBRC 355: 751_7 be implemented. 令人意外地, 已发现本发明的人源化抗体具有大于亲本非人源化抗体的Fab片段热稳定性温度,该亲本非人源化抗体通常为鼠类抗体,尤其是鼠类抗体NMC-4。 Surprisingly, it has been found that the present invention is a humanized antibody has greater than the parental non-humanized antibody Fab fragments temperature thermal stability, the parent non-humanized antibody is typically murine antibody, particularly murine antibody NMC- 4. 本发明还提供Fab片段热稳定性温度大于亲本非人源化抗体的具有vWF特异性的人源化抗体或其结合片段。 The present invention also provides a Fab fragment thermal stability temperature is greater than the parental non-humanized antibodies specific for people with vWF humanized antibody or binding fragment thereof.

[0092] 本发明提供具有vWF特异性的人源化抗体或其结合片段,包含下列高变区氨基酸序列=HCDRl (GFSLTDYGVD ;SEQ ID NO :7),HCDR2 (MIWGDGSTDYNSALKS ;SEQ ID NO :8), HCDR3(DPADYGNYDYALDY ;SEQ ID NO :9);及LCDR3 (QQYEKLPWT ;SEQ ID N0:12),附带条件为LCDl 和/ 或LCD2 至少其中之一分别不为SASQDINKYLN(SEQ ID NO :10)或YTSSLHS (SEQ IDNO : 11)。 Al. [0092] The present invention provides a humanized antibody specific for vWF binding fragment thereof, comprising the following hypervariable region amino acid sequence = HCDRl (GFSLTDYGVD; SEQ ID NO: 7), HCDR2 (MIWGDGSTDYNSALKS; SEQ ID NO: 8), HCDR3 (DPADYGNYDYALDY; SEQ ID NO: 9); and LCDR3 (QQYEKLPWT; SEQ ID N0: 12), with the proviso that LCDl and / or at least one of LCD2 were not SASQDINKYLN (SEQ ID NO: 10) or YTSSLHS (SEQ IDNO: 11). 令人意外地,缺乏NMC-4IXDR1和/或IXDR2的人源化匪C-4抗体保有对于vWF 的纳摩尔结合亲和力。 Surprisingly, the lack of NMC-4IXDR1 and / or IXDR2 humanized antibodies retain bandit C-4 nanomolar binding affinity for vWF.

[0093] 在某些实施方案中,人源化抗体还可包含人类抗体重链框架区。 [0093] In certain embodiments, the humanized antibody may further comprise a human antibody heavy chain framework region. 在某些实施方案中,重链框架区对应于存在于4-59衍生人类抗体中的重链框架区。 In certain embodiments, the heavy chain framework region corresponding to the presence in the 4-59-derived human antibody heavy chain framework region. 在某些实施方案中,存在于4-59衍生人类抗体中的重链框架区还包含一个或多个鼠类残基;在某些实施方案中, 存在于4-59衍生人类抗体中的重链框架区不包含一个或多个鼠类残基。 In certain embodiments, the present in 4-59-derived human antibody heavy chain framework region further comprises one or more of the murine residue; In certain embodiments, the present in 4-59 weight derivative of human antibodies chain frame does not contain one or more murine residues.

[0094] 在某些实施方案中,人源化抗体还可包含人类抗体轻链框架区;在某些实施方案中,轻链框架区对应于存在于018衍生人类抗体中的轻链框架区;在某些实施方案中,存在于018衍生人类抗体中的轻链框架区还包含一个或多个鼠类残基;在某些实施方案中,存在于018衍生人类抗体中的轻链框架区不包含一个或多个鼠类残基。 [0094] In certain embodiments, the humanized antibody may further comprise a human antibody light chain framework region; In certain embodiments, the light chain framework region corresponding to the 018 present in the human antibody-derived light chain framework regions; In certain embodiments, human antibodies present in the 018-derived light chain framework region further comprises one or more of the murine residue; In certain embodiments, human antibodies present in the 018-derived light chain framework region is not It contains one or more murine residues.

[0095] IXDRl和/或IXDR2可由人类来源获得。 [0095] IXDRl and / or IXDR2 by human sources. 在某些实施方案中,IXDRl和/或IXDR2 可由相同抗体(例如一个人类抗体)而获得;在其它实施方案中,LCDRl和/或LCDR2可由不同抗体(例如两个人类抗体)而获得。 In certain embodiments, IXDRl and / or IXDR2 by the same antibody (e.g. a human antibody) is obtained; In other embodiments, LCDRl and / or LCDR2 by different antibodies (e.g., two human antibody) is obtained. 若LCDR2得自人类来源,其优选为DASNLET(SEQID NO :118)。 If LCDR2 obtained from human origin, which is preferably DASNLET (SEQID NO: 118).

[0096] 本发明还提供具有vWF特异性的人源化抗体或其结合片段,包含: HCDRl (GFSLTDYGVD ;SEQ ID NO :7) , HCDR2 (MIWGDGSTDYNSALKS ;SEQ IDNO :8), HCDR3(DPADYGNYDYALDY ;SEQ ID NO :9), LCDRl(SASQDINKYLN ;SEQ ID NO :10), LCDR2(YTSSLHS ;SEQ ID NO :11)及LCDR3(QQYEKLPWT ; SEQ ID NO :12);对应于人类抗体重链种系序列4-59中的框架区的重链框架区1、2和3,其中重链框架区1为QVQLQESGPGLVKPSETLSLTCTVS (SEQ IDNO : 171),重链框架区2 为WI RQPPGKGLEffIG (SEQ ID NO : 172),且重链框架区3 为RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR(SEQ ID NO :173);以及对应于存在于人类抗体轻链种系序列018中的框架区的轻链框架区1、2和3,其中轻链框架区1 为DIQMTQSPSSLSASV⑶RVTITC(SEQ ID NO :186),轻链框架区2 为WYQQKPGKAPKLLIY(SEQ ID NO :187),且轻链框架区3 为GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC(SEQ ID NO: 188)。 Al. [0096] The present invention also provides a vWF having specificity for the humanized antibody or binding fragment thereof, comprising: HCDRl (GFSLTDYGVD; SEQ ID NO: 7), HCDR2 (MIWGDGSTDYNSALKS; SEQ IDNO: 8), HCDR3 (DPADYGNYDYALDY; SEQ ID NO: 9), LCDRl (SASQDINKYLN; SEQ ID NO: 10), LCDR2 (YTSSLHS; SEQ ID NO: 11) and LCDR3 (QQYEKLPWT; SEQ ID NO: 12); corresponding to the human germline antibody heavy chain sequences 4-59 heavy chain framework region framework regions 1, 2 and 3, wherein the heavy chain framework region 1 QVQLQESGPGLVKPSETLSLTCTVS (SEQ IDNO: 171), the heavy chain framework region 2 WI RQPPGKGLEffIG (SEQ ID NO: 172), and a heavy chain framework Zone 3 is RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: 173); and the light chain corresponding to the presence of human antibody light chain framework region germline sequence 018 in the framework regions 2 and 3, wherein the light chain framework region 1 DIQMTQSPSSLSASV⑶RVTITC (SEQ ID NO: 186), the light chain framework region 2 WYQQKPGKAPKLLIY (SEQ ID NO: 187), and the light chain framework region 3 GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC (SEQ ID NO: 188).

[0097] 本发明提供具有vWF特异性的人源化抗体或其结合片段,包含: HCDRl (GFSLTDYGVD ;SEQ ID NO :7), HCDR2(MIWGDGSTDYNSALKS ;SEQ ID NO :8), HCDR3(DPADYGNYDYALDY ;SEQ ID NO :9), LCDRl(SASQDINKYLN ;SEQ ID NO :10), LCDR2 (YTSSLHS ; SEQ ID NO :11)及LCDR3 (QQYEKLPWT ; SEQ ID NO : 12);对应于人类抗体重链种系序列4-34中的框架区的重链框架区1、2和3,其中重链框架区1为QVQLQQffGAGLLKPSETLSLTCAVY (SEQ ID NO : 165),重链框架区2 为WIRQPPGKGLEWIG (SEQ ID NO : 166),且重链框架区3 为RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR(SEQ ID NO :167);以及对应于存在于人类抗体轻链种系序列018中的框架区的轻链框架区1、2和3,其中轻链框架区1 为DIQMTQSPSSLSASV⑶RVTITC(SEQ ID NO : 186),轻链框架区2 为WYQQKPGKAPKLLIY(SEQ ID NO :187),且轻链框架区3 为GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC(SEQ ID NO: 188)。 Al. [0097] The present invention provides a humanized antibody specific for vWF binding fragment thereof, comprising: HCDRl (GFSLTDYGVD; SEQ ID NO: 7), HCDR2 (MIWGDGSTDYNSALKS; SEQ ID NO: 8), HCDR3 (DPADYGNYDYALDY; SEQ ID NO: 9), LCDRl (SASQDINKYLN; SEQ ID NO: 10), LCDR2 (YTSSLHS; SEQ ID NO: 11) and LCDR3 (QQYEKLPWT; SEQ ID NO: 12); corresponding to the human germline antibody heavy chain sequences 4-34 heavy chain framework region framework regions 1, 2 and 3, wherein the heavy chain framework region 1 QVQLQQffGAGLLKPSETLSLTCAVY (SEQ ID NO: 165), the heavy chain framework region 2 is WIRQPPGKGLEWIG (SEQ ID NO: 166), and a heavy chain framework Zone 3 is RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: 167); and the light chain corresponding to the presence of human antibody light chain framework region germline sequence 018 in the framework regions 2 and 3, wherein the light chain framework region 1 DIQMTQSPSSLSASV⑶RVTITC (SEQ ID NO: 186), the light chain framework region 2 WYQQKPGKAPKLLIY (SEQ ID NO: 187), and the light chain framework region 3 GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC (SEQ ID NO: 188).

[0098] 本发明还提供对vWF的Al结构域具有特异性的人源化抗体或其结合片段, 包含:HCDRl (GFSLTDYGVD ; SEQ ID NO :7) , HCDR2 (MIWGDGSTDYNSALKS ; SEQ ID NO: 8), HCDR3(DPADYGNYDYALDY ; SEQ ID NO :9), LCDRl(SASQDINKYLN;SEQ ID NO: 10), LCDR2 (YTSSLHS ; SEQ ID NO :11)及LCDR3 (QQYEKLPWT ; SEQ ID NO :12);对应于存在于来自人类抗体种系家族VH4的抗体中的框架区1、2和3的重链框架区1、2和3 ;以及对应于存在于来自人类抗体种系家族VKl的抗体中的框架区1、2和3的轻链框架区1、2和3。 [0098] The present invention also provides for the Al domain of vWF having a specific humanized antibody or binding fragment thereof, comprising: HCDRl (GFSLTDYGVD; SEQ ID NO: 7), HCDR2 (MIWGDGSTDYNSALKS; SEQ ID NO: 8), HCDR3 (DPADYGNYDYALDY; SEQ ID NO: 9), LCDRl (SASQDINKYLN; SEQ ID NO: 10), LCDR2 (YTSSLHS; SEQ ID NO: 11) and LCDR3 (QQYEKLPWT; SEQ ID NO: 12); corresponding to the presence in the human from antibody germline family VH4 framework regions of antibody heavy chain framework region 1, 2 and 3, 1, 2 and 3; and corresponds to the presence of antibodies derived from human antibody germline family VKl in the framework regions 1, 2 and 3 The light chain framework region 1, 2 and 3.

[0099] 人源化抗体或其结合片段可包含对应于存在于重链可变种系序列4-04中的框架区1、2 和3 的框架区1、2 禾口3(例如Fffl :QVQLQESGPGLVKPSGTLSLTCAVS (SEQ ID NO :147), FW2 =WVRQPPGKGLEffIG(SEQ ID NO :148)及FW3 :RVTISVDKSKNQFSLKLSSVTAADTAVYYCAR(SEQ ID NO :149))。 [0099] The humanized antibody or binding fragment thereof may be included to correspond to the presence of heavy chain germline sequence variants in 4-04 frame framework region 1, 2 and 3, 3, 2 Hekou (eg Fffl: QVQLQESGPGLVKPSGTLSLTCAVS ( SEQ ID NO: 147), FW2 = WVRQPPGKGLEffIG (SEQ ID NO: 148) and FW3: RVTISVDKSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: 149)).

[0100] 人源化抗体或其结合片段可包含对应于存在于重链可变种系序列4-28中的框架区1、2 和3 的框架区1、2 和3(例如Fffl :QVQLQESGPGLVKPSDTLSLTCAVS (SEQ ID NO :150), FW2 =WIRQPPGKGLEffIG(SEQ ID NO: 151)及FW3 :RVTMSVDTSKNQFSLKLSSVTAVDTAVYYCAR(SEQ ID NO :152))。 [0100] The humanized antibody or binding fragment thereof may comprise corresponding to the presence in the heavy chain of 4-28 germline sequence variants framework regions 1, 2 and 3 of the framework regions 1, 2 and 3 (for example Fffl: QVQLQESGPGLVKPSDTLSLTCAVS (SEQ ID NO: 150), FW2 = WIRQPPGKGLEffIG (SEQ ID NO: 151) and FW3: RVTMSVDTSKNQFSLKLSSVTAVDTAVYYCAR (SEQ ID NO: 152)).

[0101] 人源化抗体或其结合片段可包含对应于存在于重链可变种系序列4-30. 1中的框架区1、2 和3 的框架区1、2 和3(例如FWl :QVQLQESGPGLVKPSQTLSLTCTVS (SEQ ID NO: 153), FW2 =WIRQHPGKGLEffIG(SEQ ID NO :154)及FW3 :RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR(SEQ IDNO :155))。 [0101] The humanized antibody or binding fragment thereof may be included to correspond to the presence of heavy chain germline sequence variants 4-30 1 framework regions 1, 2 and 3 of the framework regions 1, 2 and 3 (for example FWl:. QVQLQESGPGLVKPSQTLSLTCTVS (SEQ ID NO: 153), FW2 = WIRQHPGKGLEffIG (SEQ ID NO: 154) and FW3: RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ IDNO: 155)).

20[0102] 人源化抗体或其结合片段可包含对应于存在于重链可变种系序列4-30. 2中的框架区1、2 和3 的框架区1、2 和3(例如FWl :QLQLQESGSGLVKPSQTLSLTCAVS (SEQ ID NO: 156), FW2 =WIRQPPGKGLEffIG(SEQ ID NO :157)及FW3 :RVTISVDRSKNQFSLKLSSVTAADTAVYYCAR(SEQ ID NO :158))。 20 [0102] The humanized antibody or binding fragment thereof can comprise a heavy chain present in the corresponding germline sequences may be variants in the framework region 4-30 2 1, 2 and 3 of the framework regions 1, 2 and 3 (e.g. FWl.: QLQLQESGSGLVKPSQTLSLTCAVS (SEQ ID NO: 156), FW2 = WIRQPPGKGLEffIG (SEQ ID NO: 157) and FW3: RVTISVDRSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: 158)).

[0103] 人源化抗体或其结合片段可包含对应于存在于重链可变种系序列4-30. 4中的框架区1、2 和3 的框架区1、2 和3(例如FWl :QVQLQESGPGLVKPSQTLSLTCTVS (SEQ ID NO: 159), FW2 =WIRQPPGKGLEffIG(SEQ ID NO :160)及FW3 :RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR(SEQ ID NO :161)。 [0103] The humanized antibody or binding fragment thereof may be included to correspond to the presence of heavy chain germline sequence variants 4-30 4 framework regions 1, 2 and 3 of the framework regions 1, 2 and 3 (for example FWl:. QVQLQESGPGLVKPSQTLSLTCTVS (SEQ ID NO: 159), FW2 = WIRQPPGKGLEffIG (SEQ ID NO: 160) and FW3: RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: 161).

[0104] 人源化抗体或其结合片段可包含对应于存在于重链可变种系序列4-31中的框架区1、2 和3 的框架区1、2 禾口3(例如Fffl :QVQLQESGPGLVKPSQTLSLTCTVS (SEQ ID NO :162), FW2 =WIRQHPGKGLEffIG(SEQ ID NO: 163)及FW3 : RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR(SEQ ID NO :164))。 [0104] The humanized antibody or binding fragment thereof may be included to correspond to the presence of heavy chain germline sequence variants in 4-31 frame framework region 1, 2 and 3, 3, 2 Hekou (eg Fffl: QVQLQESGPGLVKPSQTLSLTCTVS ( SEQ ID NO: 162), FW2 = WIRQHPGKGLEffIG (SEQ ID NO: 163) and FW3: RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: 164)).

[0105] 人源化抗体或其结合片段可包含对应于存在于重链可变种系序列4-34中的框架区1、2 和3 的框架区1、2 和3 (例如Fffl =QVQLQQffGAGLLKPSETLSLTCAVY (SEQ ID NO :165), FW2 =WIRQPPGKGLEffIG(SEQ ID N0:166)及FW3 :RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR(SEQ ID NO :167))。 [0105] The humanized antibody or binding fragment thereof may comprise corresponding to the presence in the heavy chain of 4-34 germline sequence variants framework regions 1, 2 and 3 of the framework regions 1, 2 and 3 (for example Fffl = QVQLQQffGAGLLKPSETLSLTCAVY (SEQ ID NO: 165), FW2 = WIRQPPGKGLEffIG (SEQ ID N0: 166) and FW3: RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: 167)).

[0106] 人源化抗体或其结合片段可包含对应于存在于重链可变种系序列4-39中的框架区1、2 和3 的框架区1、2 禾口3(例如Fffl :QLQLQESGPGLVKPSETLSLTCTVS (SEQ ID NO :168), FW2 =WIRQPPGKGLEffIG(SEQ ID NO: 169)及FW3 : RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR(SEQ ID NO :170))。 [0106] The humanized antibody or binding fragment thereof may be included to correspond to the presence of heavy chain germline sequence variants in 4-39 frame framework region 1, 2 and 3, 3, 2 Hekou (eg Fffl: QLQLQESGPGLVKPSETLSLTCTVS ( SEQ ID NO: 168), FW2 = WIRQPPGKGLEffIG (SEQ ID NO: 169) and FW3: RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: 170)).

[0107] 人源化抗体或其结合片段可包含对应于存在于重链可变种系序列4-59中的框架区1、2 和3 的框架区1、2 和3(例如Fffl :QVQLQESGPGLVKPSETLSLTCTVS (SEQ ID NO :171), FW2 =WIRQPPGKGLEffIG(SEQ ID NO: 172)及FW3 : RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR(SEQ ID NO :173))。 [0107] The humanized antibody or binding fragment thereof may be included to correspond to the presence of heavy chain germline sequence variants 4-59 in the framework regions 1, 2 and 3 of the framework regions 1, 2 and 3 (for example Fffl: QVQLQESGPGLVKPSETLSLTCTVS (SEQ ID NO: 171), FW2 = WIRQPPGKGLEffIG (SEQ ID NO: 172) and FW3: RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: 173)).

[0108] 人源化抗体或其结合片段可包含对应于存在于重链可变种系序列4-61中的框架区1、2 和3 的框架区1、2 禾口3(例如Fffl :QLQLQESGPGLVKPSETLSLTCTVS (SEQ ID NO :174), FW2 :ffl RQPPGKGLEffIG(SEQ ID NO :175)及FW3 :RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR(SEQ ID NO :176))。 [0108] The humanized antibody or binding fragment thereof may be included to correspond to the presence of heavy chain germline sequence variants in 4-61 frame framework region 1, 2 and 3, 3, 2 Hekou (eg Fffl: QLQLQESGPGLVKPSETLSLTCTVS ( SEQ ID NO: 174), FW2: ffl RQPPGKGLEffIG (SEQ ID NO: 175) and FW3: RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: 176)).

[0109] 人源化抗体或其结合片段可包含对应于存在于重链可变种系序列4-b中的框架区1、2 和3 的框架区1、2 禾口3(例如Fffl :QVQLQESGPGLVKPSETLSLTCAVS(SEQ ID NO :177), FW2 =WIRQPPGKGLEffIG(SEQ ID NO :178)及FW3 :RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR(SEQ ID NO :179))。 [0109] The humanized antibody or binding fragment thereof may be included to correspond to the presence of heavy chain germline sequence variants 4-b in the framework framework region 1, 2 and 3, 3, 2 Hekou (eg Fffl: QVQLQESGPGLVKPSETLSLTCAVS ( SEQ ID NO: 177), FW2 = WIRQPPGKGLEffIG (SEQ ID NO: 178) and FW3: RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR (SEQ ID NO: 179)).

[0110] 人源化抗体或其结合片段可包含对应于存在于κ链可变种系序列012中的框架区1、2 和3 的框架区1、2 和3(例如FWl :DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO : 180) ,Fff 2 : WYQQKPGKAPKLLIY(SEQID NO: 181)及FW3 :GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO :182))。 [0110] The humanized antibody or binding fragment thereof may comprise corresponding to the presence in the κ chain germline sequences may be variants of the frame area 012 in the frame region 3, 2 and 1, 2 and 3 (for example FWl: DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO : 180), Fff 2: WYQQKPGKAPKLLIY (SEQID NO: 181) and FW3: GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 182)).

[0111] 人源化抗体或其结合片段可包含对应于存在于K链可变种系序列02中的框架区1、2 和3 的框架区1、2 禾口3(例如Fffl :DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO :183),FW2 : WYQQKPGKAPKLLIY(SEQID NO : 184)及FW3 :GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ IDNO :185))。 [0111] The humanized antibody or binding fragment thereof may comprise corresponding to the presence in the K chain germline sequences may be variants of the frame 02 framework region 1, 2 and 3, 3, 2 Hekou (eg Fffl: DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 183), FW2: WYQQKPGKAPKLLIY (SEQID NO: 184) and FW3: GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ IDNO: 185)).

[0112] 人源化抗体或其结合片段可包含对应于存在于κ链可变种系序列018中的框架区1、2和3 的框架区1、2和3(例如FWl :DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO :186) ,FW2 : WYQQKPGKAPKLLIY(SEQID NO : 187)及FW3 :GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC(SEQ ID NO :188))。 [0112] The humanized antibody or binding fragment thereof may comprise corresponding to the presence in the κ chain germline sequences may be variants of the frame area 018 in the frame region 3, 2 and 1, 2 and 3 (for example FWl: DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO : 186), FW2: WYQQKPGKAPKLLIY (SEQID NO: 187) and FW3: GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC (SEQ ID NO: 188)).

[0113] 人源化抗体或其结合片段可包含对应于存在于κ链可变种系序列08中的框架区1、2 和3 的框架区1、2 禾口3(例如Fffl :DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO :189),FW2 : WYQQKPGKAPKLLIY(SEQID NO : 190)及FW3 :GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC(SEQ ID NO :191))。 [0113] The humanized antibody or binding fragment thereof may comprise corresponding κ chain can be present in the germline sequence variant framework region 08 of the framework regions 1, 2 and 3 of port 3 1,2 Wo (e.g. Fffl: DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 189), FW2: WYQQKPGKAPKLLIY (SEQID NO: 190) and FW3: GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC (SEQ ID NO: 191)).

[0114] 人源化抗体或其结合片段可包含对应于存在于κ链可变种系序列A20中的框架区1、2和3 的框架区1、2和3(例如FWl :DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO :192) ,FW2 : WYQQKPGKVPKLLIY(SEQID NO : 193)及FW3 :GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC(SEQ ID NO :194))。 [0114] The humanized antibody or binding fragment thereof may comprise corresponding to the presence in the κ chain germline sequences may be variants A20 in the framework regions 1, 2 and 3 of the framework regions 1, 2 and 3 (for example FWl: DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO : 192), FW2: WYQQKPGKVPKLLIY (SEQID NO: 193) and FW3: GVPSRFSGSGSGTDFTLTISSLQPEDVATYYC (SEQ ID NO: 194)).

[0115] 人源化抗体或其结合片段可包含对应于存在于κ链可变种系序列A 30中的框架区1、2 和3 的框架区1、2 禾口3(例如Fffl :DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO :195), FW2 :WYQQKPGKAPKRLIY(SEQID NO :196)及FW3 :GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC(SEQ ID NO :197))。 [0115] The humanized antibody or binding fragment thereof may comprise corresponding κ chain can be present in the germline sequence variants A 30 in the framework region the framework regions 1, 2 and 3 of port 3 1,2 Wo (e.g. Fffl: DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 195), FW2: WYQQKPGKAPKRLIY (SEQID NO: 196) and FW3: GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC (SEQ ID NO: 197)).

[0116] 人源化抗体或其结合片段可包含对应于存在于κ链可变种系序列L14中的框架区1、2和3 的框架区1、2和3(例如FWl :NIQMTQSPSAMSASVGDRVTITC(SEQ ID NO :198) ,FW2 : WFQQKPGKVPKHLIY(SEQID NO : 199)及FW3 :GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC(SEQ ID NO :200))。 [0116] The humanized antibody or binding fragment thereof may comprise corresponding to the presence in the κ chain germline sequences may be variants of L14 framework regions 1, 2 and 3 of the framework regions 1, 2 and 3 (for example FWl: NIQMTQSPSAMSASVGDRVTITC (SEQ ID NO : 198), FW2: WFQQKPGKVPKHLIY (SEQID NO: 199) and FW3: GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC (SEQ ID NO: 200)).

[0117] 人源化抗体或其结合片段可包含对应于存在于κ链可变种系序列Ll中的框架区1、2 和3 的框架区1、2 和3(例如Fffl :DIQMTQSPSSLSASVGDRVTITC(SEQ ID NO :201),FW2 : WFQQKPGKAPKSLIY (SEQID NO : 202)及FW3 :GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO :203))。 [0117] The humanized antibody or binding fragment thereof may comprise corresponding to the presence in the κ chain germline sequences may be variants of Ll framework regions 1, 2 and 3 of the framework regions 1, 2 and 3 (for example Fffl: DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO : 201), FW2: WFQQKPGKAPKSLIY (SEQID NO: 202) and FW3: GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 203)).

[0118] 人源化抗体或其结合片段可包含对应于存在于κ链可变种系序列L15中的框架区1、2 和3 的框架区1、2 和3(例如FWl :DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO : 204) ,Fff 2 : WYQQKPEKAPKSLIY(SEQID NO :205)及FW3 :GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO :206))。 [01] The humanized antibody or binding fragment thereof may comprise corresponding to the presence in the κ chain can be a variant of L15 germline sequence framework regions 1, 2 and 3 of the framework regions 1, 2 and 3 (for example FWl: DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO : 204), Fff 2: WYQQKPEKAPKSLIY (SEQID NO: 205) and FW3: GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 206)).

[0119] 人源化抗体或其结合片段可包含对应于存在于κ链可变种系序列L4中的框架区1、2 和3 的框架区1、2 和3(例如Fffl :AIQLTQSPSSLSASVGDRVTITC(SEQ ID NO :207),FW2 : WYQQKPGKAPKLLIY(SEQID NO : 208)及FW3 :GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO :209))。 [0119] The humanized antibody or binding fragment thereof may comprise corresponding to the presence in the κ chain germline sequences may be variants L4 region in the framework and the framework regions 1, 2, 3, 1, 2 and 3 (for example Fffl: AIQLTQSPSSLSASVGDRVTITC (SEQ ID NO : 207), FW2: WYQQKPGKAPKLLIY (SEQID NO: 208) and FW3: GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 209)).

[0120] 人源化抗体或其结合片段可包含对应于存在于κ链可变种系序列L18中的框架区1、2 和3 的框架区1、2 和3(例如FWl :AIQLTQSPSSLSASVGDRVTITC (SEQ ID NO :210) ,FW2 : WYQQKPGKAPKLLIY(SEQID NO :211)及FW3 :GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO :212))。 [0120] The humanized antibody or binding fragment thereof may comprise corresponding to the presence in the κ chain germline sequences may be variants of L18 framework regions 1, 2 and 3 of the framework regions 1, 2 and 3 (for example FWl: AIQLTQSPSSLSASVGDRVTITC (SEQ ID NO : 210), FW2: WYQQKPGKAPKLLIY (SEQID NO: 211) and FW3: GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 212)).

[0121] 人源化抗体或其结合片段可包含对应于存在于κ链可变种系序列L5中的框架区1、2 和3 的框架区1、2 和3(例如Fffl :DIQMTQSPSSVSASVGDRVTITC(SEQ ID NO :213),FW2 :WYQQKPGKAPKLLIY(SEQID NO : 214)及FW3 :GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO :215))。 [0121] The humanized antibody or binding fragment thereof may comprise corresponding to the presence in the κ chain germline sequences may be variants of L5 in the framework regions 1, 2 and 3 of the framework regions 1, 2 and 3 (for example Fffl: DIQMTQSPSSVSASVGDRVTITC (SEQ ID NO : 213), FW2: WYQQKPGKAPKLLIY (SEQID NO: 214) and FW3: GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 215)).

[0122] 人源化抗体或其结合片段可包含对应于存在于κ链可变种系序列L19中的框架区1、2和3 的框架区1、2和3(例如FWl :DIQMTQSPSSVSASV⑶RVTITC(SEQ ID NO :216) ,FW2 : WYQQKPGKAPKLLIY(SEQID NO :217)及FW3 :GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO :218))。 [0122] The humanized antibody or binding fragment thereof may comprise corresponding to the presence in the κ chain germline sequences may be variants of L19 framework regions 1, 2 and 3 of the framework regions 1, 2 and 3 (for example FWl: DIQMTQSPSSVSASV⑶RVTITC (SEQ ID NO : 216), FW2: WYQQKPGKAPKLLIY (SEQID NO: 217) and FW3: GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 218)).

[0123] 人源化抗体或其结合片段可包含对应于存在于κ链可变种系序列L8中的框架区1、2 和3 的框架区1、2 和3(例如FWl :DIQLTQSPSFLSASVGDRVTITC(SEQ ID NO :219),FW2 : WYQQKPGKAPKLLIY(SEQID NO : 220)及FW3 :GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC(SEQ ID NO :221))。 [0123] The humanized antibody or binding fragment thereof may comprise corresponding to the presence in the κ chain germline sequences may be variants of L8 in the framework regions 1, 2 and 3 of the framework regions 1, 2 and 3 (for example FWl: DIQLTQSPSFLSASVGDRVTITC (SEQ ID NO : 219), FW2: WYQQKPGKAPKLLIY (SEQID NO: 220) and FW3: GVPSRFSGSGSGTEFTLTISSLQPEDFATYYC (SEQ ID NO: 221)).

[0124] 人源化抗体或其结合片段可包含对应于存在于κ链可变种系序列L23中的框架区1、2和3 的框架区1、2和3(例如FWl :AIRMTQSPFSLSASVGDRVTITC(SEQ ID NO :222) ,FW2 : WYQQKPAKAPKLFIY(SEQID NO :223)及FW3 :GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC(SEQ ID NO :224))。 [0124] The humanized antibody or binding fragment thereof may comprise corresponding to the presence in the κ chain germline sequences may be variants of L23 framework regions 1, 2 and 3 of the framework regions 1, 2 and 3 (for example FWl: AIRMTQSPFSLSASVGDRVTITC (SEQ ID NO : 222), FW2: WYQQKPAKAPKLFIY (SEQID NO: 223) and FW3: GVPSRFSGSGSGTDYTLTISSLQPEDFATYYC (SEQ ID NO: 224)).

[0125] 人源化抗体或其结合片段可包含对应于存在于κ链可变种系序列L9中的框架区1、2 和3 的框架区1、2 和3(例如Fffl :AIRMTQSPSSFSASTGDRVTITC(SEQ ID NO :225),FW2 : WYQQKPGKAPKLLIY(SEQID NO :226)及FW3 :GVPSRFSGSGSGTDFTLTISCLQSEDFATYYC(SEQ ID NO :227))。 [0125] The humanized antibody or binding fragment thereof may comprise corresponding to the presence in the κ chain germline sequences may be variants of L9 in the framework regions 1, 2 and 3 of the framework regions 1, 2 and 3 (for example Fffl: AIRMTQSPSSFSASTGDRVTITC (SEQ ID NO : 225), FW2: WYQQKPGKAPKLLIY (SEQID NO: 226) and FW3: GVPSRFSGSGSGTDFTLTISCLQSEDFATYYC (SEQ ID NO: 227)).

[0126] 人源化抗体或其结合片段可包含对应于存在于κ链可变种系序列L24中的框架区1、2和3 的框架区1、2和3(例如FWl :VIWMTQSPSLLSASTGDRVTISC(SEQ ID NO :228) ,FW2 : WYQQKPGKAPELLIY (SEQID NO :229)及FW3 :GVPSRFSGSGSGTDFTLTISCLQSEDFATYYC(SEQ ID NO :230))。 [0126] The humanized antibody or binding fragment thereof may comprise corresponding to the presence in the κ chain germline sequences may be variants of L24 framework regions 1, 2 and 3 of the framework regions 1, 2 and 3 (for example FWl: VIWMTQSPSLLSASTGDRVTISC (SEQ ID NO : 228), FW2: WYQQKPGKAPELLIY (SEQID NO: 229) and FW3: GVPSRFSGSGSGTDFTLTISCLQSEDFATYYC (SEQ ID NO: 230)).

[0127] 人源化抗体或其结合片段可包含对应于存在于κ链可变种系序列Lll中的框架区1、2和3 的框架区1、2和3(例如FWl :AIQMTQSPSSLSASVGDRVTITC(SEQ ID NO :231) ,FW2 : WYQQKPGKAPKLLIY(SEQID NO :232)及FW3 :GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO :233))。 [0127] The humanized antibody or binding fragment thereof may comprise corresponding to the presence in the κ chain germline sequences may be variants of Lll framework regions 1, 2 and 3 of the framework regions 1, 2 and 3 (for example FWl: AIQMTQSPSSLSASVGDRVTITC (SEQ ID NO : 231), FW2: WYQQKPGKAPKLLIY (SEQID NO: 232) and FW3: GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 233)).

[0128] 人源化抗体或其结合片段可包含对应于存在于κ链可变种系序列L12中的框架区1、2和3 的框架区1、2和3(例如FWl :DIQMTQSPSTLSASV⑶RVTITC(SEQ ID NO :234) ,FW2 : WYQQKPGKAPKLLIY(SEQID NO :235)及FW3 :GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC(SEQ ID NO :236))。 [0128] The humanized antibody or binding fragment thereof may comprise corresponding to the presence in the κ chain germline sequences may be variants of L12 framework regions 1, 2 and 3 of the framework regions 1, 2 and 3 (for example FWl: DIQMTQSPSTLSASV⑶RVTITC (SEQ ID NO : 234), FW2: WYQQKPGKAPKLLIY (SEQID NO: 235) and FW3: GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC (SEQ ID NO: 236)).

[0129] 本发明还提供对vWF的Al结构域具有特异性的人源化抗体或其结合片段,包含: HCDRl :GFSLTDYGVD(SEQ ID NO :7),HCDR2 :MIWGDGSTDYNSALKS (SEQ ID NO :8),及HCDR3 : DPADYGNYDYALDY(SEQ ID NO :9),LCDRI :SASQDINKYLN(SEQ ID N0:10),LCDR2 :YTSSLHS (SEQ ID N0:11),&LCDR3:QQYEKLPWT(SEQ ID NO :12);对应于存在下列人类抗体中的框架区1、2和3的重链框架区1、2和3 :4-04(分别为SEQ ID NO :147,148及149),4_28 (分别为SEQ ID N0:150,151 及152),4-30. 1(分别为SEQ ID NO : 153,154 及155),4-30. 2 (分别为SEQ ID NO :156,157 及158),4-30. 4(分别为SEQ ID NO :159,160 及161),4-31 (分别为SEQ IDNO : 162,163 及164),4-34 (分别为SEQ ID NO : 165,166 及167),4-39 (分别为SEQ ID NO : 168,169 及170),4-59 (分别为SEQ ID NO : 171,172 及173),4-61 (分别为SEQ IDNO : 174,175及176)或4_b(分别为SEQ IDNO : 177,178及179);及对应于存在下列人类抗体中的框架区1、2和3的轻链框架区1、2和3 :012(分别为SEQ ID NO : 180,181及182), 02 (分别为SEQ ID NO : 183,184 及185),018 (分别为SEQ ID NO : 186,187 及188),08 (分别为SEQ ID NO :189,190 R 191), A20(分别为SEQ IDNO :192,193 及194),A30(分别为SEQ ID NO : 195,196 及197),L14(分别为SEQ ID NO : 198,199 及200),Ll (分别为SEQ ID NO: 201,202 及203),L15(分别为SEQ ID NO :204,205 及206),L4 (分别为SEQ ID NO :207, 208 及209),L18(分别为SEQ ID NO :210,211 及212),L5(分别为SEQ ID NO :213,214 及215),L19 (分别为SEQ ID NO :216,217 及218),L8 (分别为SEQ ID NO :219,220 R 221), L23(分别为SEQ ID NO :222,223 及224),L9 (分别为SEQ ID NO :225,226 及227),L24 (分别为SEQ ID NO :228,229 及230),Lll (分别为SEQ ID NO :231,232 及233)及L12 (分别为SEQ ID NO :234,235 及236)。 [0129] The present invention also provides for the Al domain of vWF having a specific humanized antibody or binding fragment thereof, comprising: HCDRl: GFSLTDYGVD (SEQ ID NO: 7), HCDR2: MIWGDGSTDYNSALKS (SEQ ID NO: 8), and HCDR3: DPADYGNYDYALDY (SEQ ID NO: 9), LCDRI: SASQDINKYLN (SEQ ID N0: 10), LCDR2: YTSSLHS (SEQ ID N0: 11), & LCDR3: QQYEKLPWT (SEQ ID NO: 12); corresponding to the presence of the following human antibody framework regions 1, 2 and 3 of the heavy chain framework region 1, 2 and 3: 4-04 (respectively SEQ ID NO: 147,148 and 149), 4_28 (respectively SEQ ID N0: 150,151 and . 152), 4-30 1 (respectively SEQ ID NO: 153,154 and 155), 2 4-30 (respectively SEQ ID NO:.. 156,157 and 158), 4 4-30 (SEQ ID NO: 159,160 and 161), 4-31 (respectively SEQ IDNO: 162,163 and 164), 4-34 (respectively SEQ ID NO: 165,166 and 167), 4-39 (SEQ ID NO: 168,169 and 170), 4-59 (respectively SEQ ID NO: 171,172 and 173), 4-61 (respectively SEQ IDNO: 174,175 and 176) or 4_b (respectively SEQ IDNO: 177, 178 and 179); and corresponds to the presence of these antibodies in the human framework regions 1, 2 and 3 of the light chain framework region 1, 2 and 3: 012 (respectively SEQ ID NO: 180,181 and 182), 02 (respectively SEQ ID NO: 183,184 and 185), 018 (respectively SEQ ID NO: 186,187 and 188), 08 (respectively SEQ ID NO: 189,190 R 191), A20 (respectively SEQ IDNO : 192 and 193 and 194), A30 (respectively SEQ ID NO: 195,196 and 197), L14 (respectively SEQ ID NO: 198,199 and 200), Ll (respectively SEQ ID NO: 201,202 and 203), L15 (respectively SEQ ID NO: 204,205 and 206), L4 (respectively SEQ ID NO: 207, 208 and 209), L18 (respectively SEQ ID NO: 210,211 and 212), L5 (respectively SEQ ID NO: 213,214 and 215), L19 (respectively SEQ ID NO: 216,217 and 218), L8 (respectively SEQ ID NO: 219,220 R 221), L23 (respectively SEQ ID NO: 222 , 223 and 224), L9 (respectively SEQ ID NO: 225,226 and 227), L24 (respectively SEQ ID NO: 228,229 and 230), Lll (respectively SEQ ID NO: 231,232 and 233) and L12 (respectively SEQ ID NO: 234,235, and 236).

[0130] 本发明还提供如此处所述的人源化抗体或其结合片段,其保留与亲本非人源化抗体或包含来自亲本非人源化抗体及人类Fc区的嵌合抗体相同的活性。 [0130] The present invention also provides a person as described herein humanized antibody or binding fragment thereof which retains parental non-humanized antibody or a chimeric antibody derived from a parental non-humanized antibodies and human Fc region of the same activity . 亲本非人源化抗体通常为鼠类抗体,尤其为鼠类抗体NMC-4 ;包含来自亲本非人源化抗体的嵌合抗体通常为包含来自鼠类抗体(尤其来自鼠类抗体NMC-4)的可变区及人类Fc区的抗体。 Parental non-humanized antibody is typically murine antibodies, in particular murine antibody NMC-4; contains from parental non-humanized antibody chimeric antibody typically contains from murine antibody (in particular from the murine antibody NMC-4) antibody variable region and human Fc region. 优选以本发明所述的人类Fc区作为人类Fc区。 Preferably human Fc regions of the present invention as a human Fc region.

[0131] 亲本非人源化抗体及嵌合抗体的如此处所述的人源化抗体或其结合片段的活性, 可通过例如实施例1中所述判定EC5tl活性而以瑞斯特霉素诱导血小板凝集活性加以测量。 [0131] parental non-humanized antibodies and chimeric antibodies as described herein humanized antibody or binding fragment of activity, may for example, by the determination EC5tl active in Example 1 but with Rist Induced platelet aggregation activity is measured. 当如此处所述的人源化抗体或其结合片段的EC5tl活性与EC5tl活性相同,或有高达50%、优选为高达30%、优选为高达20%不同于(例如更高或更低)亲本非人源化抗体或嵌合抗体的EC5tl活性时,如此处所述的人源化抗体或其结合片段可被视为保留着与亲本非人源化抗体或嵌合抗体相同的活性。 When as described herein humanized antibody or binding fragment of EC5tl activity EC5tl same activity, or up to 50%, preferably up to 30%, preferably up to 20% different from (such as higher or lower) Parents When the non-humanized antibody or chimeric antibody EC5tl activity, so people at the humanized antibody or binding fragment thereof may be regarded as a reservation of the parent non-humanized antibody or chimeric antibody same activity.

[0132] 在本发明的优选实施方案中,如此处所述的人源化抗体或其结合片段还包含来自人类抗体的重链框架区,其中人类重链框架区不包含一个或多个鼠类残基。 [0132] In a preferred embodiment of the present invention, as described herein humanized antibody or binding fragment further comprises a heavy chain framework region from a human antibody, wherein the human heavy chain framework region does not contain one or more of the murine residues.

[0133] 在本发明的其他优选实施方案中,如此处所述的人源化抗体或其结合片段还包含来自人类抗体的轻链框架区,其中人类轻链框架区不包含一个或多个鼠类残基。 [0133] In a further preferred embodiment of the present invention, as described herein humanized antibody or binding fragment further comprises a light chain framework region from a human antibody, wherein the human light chain framework region does not contain one or more mouse Class residues.

[0134] “不包含一个或多个鼠类残基的人类重链框架区”或“不包含一个或多个鼠类残基的人类轻链框架区”指不包含一个或多个仅存在于鼠类中的鼠类残基的人类重链或轻链框架区,例如不包含回复突变(backmutation)至仅存在于鼠类且不存在于人类中的残基。 [0134] "does not contain one or more murine residues of human heavy chain framework regions" or "does not contain one or more murine residues of human light chain framework regions" that does not contain one or more exist only human heavy or light chain framework region rodent rodents residues, for example does not include reverse mutation (backmutation) to exist only in the presence of rodents and not in humans residues. 由此定义,并不排除包含也存在于鼠类中的人类残基的人类重链或轻链框架区;并且,由此定义,不排除残基已突变至一般人类(例如大多数人类框架区共同的残基)、但还存在于鼠类中的人类重链或轻链框架区。 Thus the definition does not exclude also exists in rodents contains human heavy or light chain human framework region residues; and, thus defined, does not exclude the residue has been mutated to general human (such as most human framework region Common residues), but also exists in rodents human heavy or light chain framework regions.

[0135] 在本发明来自人类抗体的重链或轻链框架区还包含一个或多个鼠类残基的实施方案情况中,其通常包含10个或更少的鼠类残基,优选为9个或更少,更优选为8个或更少,又更优选7个或更少,最优选为6个或更少,特别5个或更少,更特别4个或更少,又更特别3个或更少,最特别2个或更少,最特别优选1个。 Embodiments [0135] In the present invention, the heavy or light chain framework region from a human antibody further comprises one or more murine residues case, which usually contains 10 or fewer murine residues, preferably 9 or less, more preferably 8 or less, and more preferably 7 or less, and most preferably 6 or fewer, especially 5 or less, more particularly 4 or less, and more particularly 3 or less, and most especially 2 or less, most particularly preferably one.

[0136] 本发明提供编码具有vWF特异性的人源化抗体或其结合片段的分离核酸,其包含如SEQ ID NO :19中所述的重链可变区序列及如SEQ ID NO :28中所述的轻链可变区序列。 [0136] The present invention provides specificity for vWF encoding a humanized antibody or binding fragment isolated nucleic acid comprising e.g. SEQ ID NO: heavy chain variable region sequence as described in 19 and SEQ ID NO: 28 in The light chain variable region sequence.

[0137] 本发明提供编码具有vWF特异性的人源化抗体或其结合片段的分离核酸,其包含如SEQ ID NO :237中所述的重链序列及如SEQ ID NO :238中所述的轻链序列。 [0137] The present invention provides specificity for vWF encoding a humanized antibody or binding fragment of an isolated nucleic acid, which comprises as SEQ ID NO: heavy chain sequence and 237 as described in SEQ ID NO: 238 as described in light chain sequence.

[0138] 本发明还提供编码具有对人vWF的特异性的人源化抗体或其结合片段的分离核酸,其包含:对应于存在于鼠类抗体NMC-4中的CDR的CDR区;对应于存在于人类抗体AAC18165. KSEQ ID NO :4)的可变区中的框架区的重链框架区;及对应于存在于人类抗体AAK94808 (SEQ ID NO :6)的可变区中的框架区的轻链框架区。 [0138] The present invention further provides encoding having specificity to human vWF humanized antibody or binding fragment isolated nucleic acid, comprising: corresponding to the presence in the murine antibody NMC-4 in the CDR CDR regions; corresponding to present in the human antibody AAC18165 KSEQ ID NO:. 4) of the variable region framework region heavy chain framework regions; and corresponds to the presence of human antibodies AAK94808 (SEQ ID NO: 6) of the variable region framework regions light chain framework region.

[0139] 本发明还提供编码具有对人vWF的特异性的人源化抗体或其片段的分离核酸,其包含=HCDRl :GFSLTDYGVD (SEQ ID NO :7)、HCDR2 =MIffGDGSTDYNSALKS (SEQ ID NO :8)、HCDR3 : DPADYGNYDYALDY (SEQ ID NO :9)、以及来自人类抗体AAC18165. 1 (SEQ ID NO :4)的可变区的重链框架区。 [0139] The present invention also provides a human vWF encodes a specific humanized antibodies or fragments thereof isolated nucleic acid comprising = HCDRl: GFSLTDYGVD (SEQ ID NO: 7), HCDR2 = MIffGDGSTDYNSALKS (SEQ ID NO: 8 ), HCDR3: DPADYGNYDYALDY (SEQ ID NO: 9), and AAC18165 1 (SEQ ID NO from a human antibody: 4) heavy chain variable region framework region. 例示性人类重链框架区的核苷酸序列公开于SEQ ID N0:116中。 Exemplary nucleotide sequences of human heavy chain framework region is disclosed in SEQ ID N0: 116 in.

[0140] 本发明还提供编码具有人vWF特异性的人源化抗体或其结合片段的分离核酸,其包含下列轻链CDR :LCDR1 :SASQDINKYLN(SEQ ID N0:10)、LCDR2 :YTSSLHS(SEQ ID N0:11)、 及LCDR3 :QQYEKLPWT(SEQ ID NO : 12)、及来自人类抗体AAK94808 (SEQ ID NO :6)的可变区的轻链框架区。 [0140] The present invention further provides human vWF encodes a specific binding humanized antibody or fragment of an isolated nucleic acid, which comprises the following light chain CDR: LCDR1: SASQDINKYLN (SEQ ID N0: 10), LCDR2: YTSSLHS (SEQ ID N0: 11), and LCDR3: QQYEKLPWT (SEQ ID NO: 12), and from a human antibody AAK94808 (SEQ ID NO: 6 variable region) of the light chain framework regions. 例示性人类轻链框架区的核苷酸序列公开于SEQ ID N0:117中。 Exemplary nucleotide sequences of human light chain framework region is disclosed in SEQ ID N0: 117 in.

[0141] 本发明还提供编码具有人vWF特异性的人源化抗体或其结合片段的分离核酸,其包含下列重链可变区其中之一:H2 (SEQ ID NO :13)、H4(SEQID NO :14)、H5(SEQ ID NO :15)、 H6 (SEQ ID NO :16)、H7(SEQ ID NO : 17)、H8 (SEQ ID NO :18)、H9 (SEQ ID NO : 19)、H12 (SEQ ID NO :20)、H13(SEQ ID NO :21)、H14 (SEQ ID NO :22)、H15 (SEQ ID NO : 145)、或H16 (SEQ ID NO :146)。 [0141] The present invention further provides human vWF encodes a specific binding humanized antibody or fragment of an isolated nucleic acid, comprising one of the following heavy chain variable region: H2 (SEQ ID NO: 13), H4 (SEQID NO: 14), H5 (SEQ ID NO: 15), H6 (SEQ ID NO: 16), H7 (SEQ ID NO: 17), H8 (SEQ ID NO: 18), H9 (SEQ ID NO: 19), H12 (SEQ ID NO: 20), H13 (SEQ ID NO: 21), H14 (SEQ ID NO: 22), H15 (SEQ ID NO: 145), or H16 (SEQ ID NO: 146).

[0142] 本发明还提供编码具有人vWF特异性的人源化抗体或其结合片段的分离核酸,其包含下列轻链可变区其中之一:L5(SEQ ID NO :23)、L4(SEQID NO :24)、L6(SEQ ID NO :25)、 L7 (SEQ ID NO :26)、L8(SEQ ID NO :27)、L9(SEQ ID NO :28)、L10(SEQ ID NO :29)或Lll(SEQ ID NO :30)。 [0142] The present invention further provides human vWF encodes a specific binding humanized antibody or fragment of an isolated nucleic acid, comprising one of the following light chain variable region: L5 (SEQ ID NO: 23), L4 (SEQID NO: 24), L6 (SEQ ID NO: 25), L7 (SEQ ID NO: 26), L8 (SEQ ID NO: 27), L9 (SEQ ID NO: 28), L10 (SEQ ID NO: 29) or Lll (SEQ ID NO: 30).

[0143] 本发明还提供编码具有人vWF特异性的人源化抗体或其结合片段的分离核酸, 其包含(1)下列重链可变区其中之一:H2 (SEQ ID NO :13)、H4(SEQID NO : 14)、H5 (SEQ ID NO :15)、H6(SEQ ID NO : 16)、H7 (SEQ ID NO : 17)、H8 (SEQ ID NO : 18)、H9 (SEQ ID NO: 19)、 H12(SEQ ID NO :20)、H13(SEQ ID NO :21)、H14 (SEQ ID NO :22)、H15 (SEQ ID N0:145)、或H16(SEQ ID NO :146);及(2)下列轻链可变区其中之一:L5 (SEQ ID NO :23)、L4 (SEQ ID NO :24)、L6 (SEQ ID NO :25)、L7 (SEQ ID NO :26)、L8 (SEQ ID NO :27)、L9 (SEQ ID NO :28)、 LlO (SEQ ID NO :29)或Lll(SEQ ID NO :30)。 [0143] The present invention further provides human vWF encodes a specific binding humanized antibody or fragment of an isolated nucleic acid, comprising (1) a heavy chain variable region where one of the following: H2 (SEQ ID NO: 13), H4 (SEQID NO: 14), H5 (SEQ ID NO: 15), H6 (SEQ ID NO: 16), H7 (SEQ ID NO: 17), H8 (SEQ ID NO: 18), H9 (SEQ ID NO: 19), H12 (SEQ ID NO: 20), H13 (SEQ ID NO: 21), H14 (SEQ ID NO: 22), H15 (SEQ ID N0: 145), or H16 (SEQ ID NO: 146); and one of the following light chain variable region wherein (2): L5 (SEQ ID NO: 23), L4 (SEQ ID NO: 24), L6 (SEQ ID NO: 25), L7 (SEQ ID NO: 26), L8 (SEQ ID NO: 27), L9 (SEQ ID NO: 28), LlO (SEQ ID NO: 29) or Lll (SEQ ID NO: 30).

[0144] 本发明还提供包含载体GS264的编码轻链的核酸序列的分离核酸,载体GS264已在2008年1月23日在DSMZ保藏于微生物中,其登记号为DSM21059。 The isolated nucleic acid [0144] The present invention also provides a nucleic acid sequence encoding the light chain vector GS264 contained carrier GS264 has in January 23, 2008 at DSMZ deposited in microorganisms, which registration number DSM21059.

[0145] 本发明还提供包含载体GS 265的编码重链的核酸序列的分离核酸,载体GS265已在2008年1月23日在DSMZ保藏于微生物中,其登记号为DSM21060。 [0145] The present invention also provides an isolated nucleic acid comprising a nucleic acid sequence encoding the heavy carrier chain GS 265, the carrier GS265 has in January 23, 2008 at DSMZ deposited in microorganisms, which registration number DSM21060.

[0146] 本发明还提供具有vWF特异性的人源化抗体或其结合片段,其由载体GS264的编码轻链的核酸序列、及载体GS265的编码重链的核酸序列所编码。 [0146] The present invention also provides people with vWF-specific humanized antibody or binding fragment thereof, encoded by the vector nucleic acid sequence encoding the light chain of GS264, and nucleic acid sequences encoding the heavy chain of the carrier GS265.

[0147] 本发明提供包含分离核酸的载体,该分离核酸编码具有vWF特异性的人源化抗体或其结合片段,该人源化抗体或其结合片段包含如SEQ ID N0:19中所述的重链可变区序列及如SEQ ID NO :28中所述的轻链可变区序列。 [0147] The present invention provides a vector comprising an isolated nucleic acid, which isolated nucleic acid encoding a vWF-specific humanized antibody or binding fragment thereof, the humanized antibody or binding fragment thereof comprising as SEQ ID N0: 19 in the a heavy chain variable region sequence and as SEQ ID NO: 28 in the light chain variable region sequence.

[0148] 本发明提供包含分离核酸的载体,该分离核酸编码具有vWF特异性的人源化抗体或其结合片段,该人源化抗体或其结合片段包含如SEQ ID NO :237中所述的重链序列及如SEQ ID NO :238中所述的轻链序列。 [0148] The present invention provides a vector comprising an isolated nucleic acid, which isolated nucleic acid encoding a vWF-specific humanized antibody or binding fragment thereof, the humanized antibody or binding fragment thereof comprising as SEQ ID NO: 237 in the heavy chain sequence and as SEQ ID NO: 238 light chain sequence described.

[0149] 本发明提供包含核酸的载体,该核酸编码具有人vWF特异性的人源化抗体或其结合片段,该人源化抗体或其结合片段包含:对应于存在于鼠类抗体NMC-4中的CDR的CDR 区;对应于人类抗体AAC18165. 1(SEQ ID NO :4)的可变区中的框架区的重链框架区;及对应于人类抗体AAK94808 (SEQID NO :6)的可变区中的框架区的轻链框架区。 [0149] The present invention provides a vector comprising a nucleic acid, which nucleic acid encodes a human vWF-specific humanized antibody or binding fragment thereof, the humanized antibody or binding fragment thereof comprising: present in the corresponding murine antibody NMC-4 The CDR variable CDR regions;::; corresponding human antibody AAC18165 1 (SEQ ID NO 4). and the corresponding human antibody AAK94808 (6 SEQID NO) variable region framework region heavy chain framework regions region framework regions of the light chain framework region.

[0150] 本发明还提供包含核酸的载体,该核酸编码具有人vWF特异性的人源化抗体或其结合片段,而该人源化抗体或其结合片段包含HCDRl :GFSLTDYGVD(SEQ ID NO :7),HCDR2 : MIffGDGSTDYNSALKS (SEQ ID NO :8),HCDR3 :DPADYGNYDYALDY (SEQ ID NO :9),以及来自人类抗体AAC18165. 1(SEQ ID NO :4)的可变区的重链框架区。 [0150] The present invention further provides a vector comprising a nucleic acid, which nucleic acid encodes a human vWF-specific humanized antibody or binding fragment thereof, which humanized antibody or binding fragment thereof comprising HCDRl: GFSLTDYGVD (SEQ ID NO: 7 ), HCDR2: MIffGDGSTDYNSALKS (SEQ ID NO: 8), HCDR3: DPADYGNYDYALDY (SEQ ID NO: 9), and AAC18165 1 (SEQ ID NO from a human antibody: 4) heavy chain variable region framework region.

[0151] 本发明还提供包含核酸的载体,该核酸编码具有人vWF特异性的人源化抗体或其结合片段,而该人源化抗体或其结合片段包含轻链⑶R =IXDRl :SASQDINKYLN(SEQ ID NO: 10)、LCDR2 :YTSSLHS (SEQ ID NO :11)、及LCDR3 =QQYEKLPffT (SEQ ID NO :12)、及来自人类抗体AAK94808 (SEQ IDNO :6)的可变区的轻链框架区。 [0151] The present invention further provides a vector comprising a nucleic acid, which nucleic acid encodes a human vWF-specific humanized antibody or binding fragment thereof, which humanized antibody or binding fragment thereof comprising a light chain ⑶R = IXDRl: SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQ ID NO: 11), and LCDR3 = QQYEKLPffT (SEQ ID NO: 12), and from a human antibody AAK94808 (SEQ IDNO: 6 light chain framework regions) of the variable region.

[0152] 本发明还提供包含核酸的载体,该核酸编码具有人vWF特异性的人源化抗体或其结合片段,而该人源化抗体或其结合片段包含下列重链可变区其中之一:H2 (SEQ ID NO: 13)、H4(SEQ ID NO : 14)、H5 (SEQ ID NO : 15)、H6(SEQ ID NO : 16)、H7 (SEQ ID NO :17)、H8 (SEQ ID NO :18)、H9(SEQID NO :19)、H12 (SEQ ID NO :20)、H13 (SEQ ID NO :21)、H14 (SEQ IDNO : [0152] The present invention further provides a vector comprising a nucleic acid, which nucleic acid encodes a human vWF-specific humanized antibody or binding fragment thereof, which humanized antibody or binding fragment thereof comprising a heavy chain variable region wherein one of the following : H2 (SEQ ID NO: 13), H4 (SEQ ID NO: 14), H5 (SEQ ID NO: 15), H6 (SEQ ID NO: 16), H7 (SEQ ID NO: 17), H8 (SEQ ID NO: 18), H9 (SEQID NO: 19), H12 (SEQ ID NO: 20), H13 (SEQ ID NO: 21), H14 (SEQ IDNO:

22)、H15(SEQ ID NO : 145)、或H16 (SEQ ID NO: 146)。 22), H15 (SEQ ID NO: 145), or H16 (SEQ ID NO: 146).

[0153] 本发明还提供包含核酸的载体,该核酸编码具有人vWF特异性的人源化抗体或其结合片段,而该人源化抗体或其结合片段包含下列轻链可变区其中之一:L5 (SEQ ID N0: [0153] The present invention further provides a vector comprising a nucleic acid, which nucleic acid encodes a human vWF-specific humanized antibody or binding fragment thereof, which humanized antibody or binding fragment thereof comprising a light chain variable region wherein one of the following : L5 (SEQ ID N0:

23)、L4(SEQ ID NO :24)、L6(SEQ ID NO :25)、L7(SEQ ID NO :26)、L8(SEQ ID NO :27)、L9(SEQ ID NO :28)、LlO(SEQID NO :29)或Lll(SEQ ID NO :30)。 23), L4 (SEQ ID NO: 24), L6 (SEQ ID NO: 25), L7 (SEQ ID NO: 26), L8 (SEQ ID NO: 27), L9 (SEQ ID NO: 28), LlO ( SEQID NO: 29) or Lll (SEQ ID NO: 30).

[0154] 本发明还提供包含核酸的载体,该核酸编码具有人vWF特异性的人源化抗体或其结合片段,而该人源化抗体或其结合片段包含(1)下列重链可变区其中之一:H2 (SEQ ID NO :13)、H4(SEQ ID NO : 14)、H5 (SEQ ID NO : 15)、H6 (SEQ ID NO : 16)、H7 (SEQ ID NO: 17)、 H8 (SEQ ID NO :18)、H9(SEQ ID NO : 19)、H12(SEQ ID NO :20)、H13 (SEQ ID NO :21)、H14(SEQ ID NO :22)、H15(SEQ ID NO : 145)、或H16 (SEQ ID NO : 146);及(2)下列轻链可变区其中之一:L5(SEQ ID NO :23)、L4(SEQ ID NO :24)、L6 (SEQ ID NO :25)、L7 (SEQ ID NO :26)、L8 (SEQ ID NO :27)、L9(SEQID NO :28)、LlO (SEQ ID NO :29)或Lll (SEQ ID NO :30)。 [0154] The present invention further provides a vector comprising a nucleic acid, which nucleic acid encodes a human vWF-specific humanized antibody or binding fragment thereof, which humanized antibody or binding fragment thereof comprising (1) a heavy chain variable region below One: H2 (SEQ ID NO: 13), H4 (SEQ ID NO: 14), H5 (SEQ ID NO: 15), H6 (SEQ ID NO: 16), H7 (SEQ ID NO: 17), H8 (SEQ ID NO: 18), H9 (SEQ ID NO: 19), H12 (SEQ ID NO: 20), H13 (SEQ ID NO: 21), H14 (SEQ ID NO: 22), H15 (SEQ ID NO: 145), or H16 (SEQ ID NO: 146); and (2) one of the following light chain variable region wherein: L5 (SEQ ID NO: 23), L4 (SEQ ID NO: 24), L6 (SEQ ID NO : 25), L7 (SEQ ID NO: 26), L8 (SEQ ID NO: 27), L9 (SEQID NO: 28), LlO (SEQ ID NO: 29) or Lll (SEQ ID NO: 30).

[0155] 本发明还提供包含分离核酸的载体,该分离核酸包含载体GS 264的编码轻链的核酸序列,载体GS264已在2008年1月23日在DSMZ保藏于微生物中,其登记号为DSM 21059。 [0155] The present invention further provides a vector comprising an isolated nucleic acid, the vector comprising an isolated nucleic acid encoding GS 264 light chain nucleic acid sequences, vectors GS264 has January 23, 2008 at the DSMZ deposited microorganisms, which registration number DSM 21059.

[0156] 本发明还提供包含分离核酸的载体,该分离核酸包含载体GS265的编码重链的核酸序列,载体GS265已在2008年1月23日在DSMZ保藏于微生物中,其登记号为DSM 21060。 [0156] The present invention also provides a vector comprising an isolated nucleic acid, the isolated nucleic acid comprising a nucleic acid sequence of the vector encoding the heavy chain of the GS265 and GS265 carrier already in January 23, 2008 at DSMZ deposited in microorganisms, which registration number DSM 21060 .

[0157] 本发明提供包含核酸的宿主细胞,该核酸编码具有vWF特异性的人源化抗体或其结合片段,而该人源化抗体或其结合片段包含如SEQ ID NO :19中所述的重链可变区序列及如SEQ ID NO :28中所述的轻链可变区序列。 [0157] The present invention provides a host cell comprising a nucleic acid, which nucleic acid encoding a humanized vWF-specific antibody or binding fragment thereof, which humanized antibody or binding fragment thereof comprising as SEQ ID NO: 19 as described in a heavy chain variable region sequence and as SEQ ID NO: 28 in the light chain variable region sequence.

[0158] 本发明提供包含核酸的宿主细胞,该核酸编码具有vWF特异性的人源化抗体或其结合片段,而该人源化抗体或其结合片段包含如SEQ ID NO :237中所述的重链序列及如SEQ ID NO :238中所述的轻链序列。 [0158] The present invention provides a host cell comprising a nucleic acid, which nucleic acid encoding a humanized vWF-specific antibody or binding fragment thereof, which humanized antibody or binding fragment thereof comprising as SEQ ID NO: 237 in the heavy chain sequence and as SEQ ID NO: 238 light chain sequence described.

[0159] 本发明还提供包含分离核酸的宿主细胞,该分离核酸编码具有人vWF特异性的人源化抗体或其结合片段,而该人源化抗体或其结合片段包含:对应于存在于鼠类抗体NMC-4内的⑶R的⑶R区;对应于存在于人类抗体AAC18165. 1 (SEQ ID NO :4)的可变区中的框架区的重链框架区;及对应于存在于人类抗体AAK94808 (SEQ ID NO :6)的可变区中的框架区的轻链框架区。 [0159] The present invention further provides a host cell comprising an isolated nucleic acid, which isolated nucleic acid encoding a human vWF-specific humanized antibody or binding fragment thereof, which humanized antibody or binding fragment comprises: corresponding to the presence in the rat ⑶R of ⑶R area within class antibody NMC-4; corresponds to the presence of human antibody AAC18165 1 (SEQ ID NO: 4). The variable region framework regions of the heavy chain framework regions; and corresponds to the presence of human antibodies AAK94808 (SEQ ID NO: 6) of the variable region framework regions of the light chain framework region.

[0160] 本发明还提供包含分离核酸的宿主细胞,该分离核酸编码具有人vWF特异性的人源化抗体或其结合片段,而该人源化抗体或其结合片段包含:HCDR1 :GFSLTDYGVD(SEQ ID NO :7)、HCDR2 =MIffGDGSTDYNSALKS (SEQ IDNO :8)、HCDR3 :DPADYGNYDYALDY(SEQ ID NO :9)、 以及来自人类抗体AAC18165. 1(SEQ ID NO :4)的可变区的重链框架区。 [0160] The present invention further provides a host cell comprising an isolated nucleic acid, the isolated nucleic acid encoding a human vWF-specific humanized antibody or binding fragment thereof, which humanized antibody or binding fragment thereof comprising: HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2 = MIffGDGSTDYNSALKS (SEQ IDNO: 8), HCDR3: DPADYGNYDYALDY (SEQ ID NO:. 9), as well as from a human antibody AAC18165 1 (SEQ ID NO: 4) heavy chain variable region framework region .

[0161] 本发明还提供包含分离核酸的宿主细胞,该分离核酸编码具有人vWF特异性的人源化抗体或其结合片段,而该人源化抗体或其结合片段包含下列轻链CDR =LCDRl : SASQDINKYLN (SEQ ID NO :10) ,LCDR2 :YTSSLHS (SEQID NO :11)、及LCDR3 :QQYEKLPWT(SEQ ID NO :12)、及来自人类抗体AAK94808(SEQ ID NO :6)的可变区的轻链框架区。 [0161] The present invention further provides a host cell comprising an isolated nucleic acid, the isolated nucleic acid encoding a human vWF-specific humanized antibody or binding fragment thereof, which humanized antibody or binding fragment thereof comprising the light chain CDR = LCDRl : SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQID NO: 11), and LCDR3: QQYEKLPWT (SEQ ID NO: 12), and from a human antibody AAK94808: Light (SEQ ID NO 6) of the variable region chain frame.

[0162] 本发明还提供包含分离核酸的宿主细胞,该分离核酸编码具有人vWF特异性的人源化抗体或其结合片段,而该人源化抗体或其结合片段包含下列重链可变区其中之一: H2 (SEQ ID NO :13)、H4(SEQ ID NO :14)、H5 (SEQID NO :15)、H6 (SEQ ID NO :16)、H7 (SEQ ID NO :17)、H8(SEQ ID NO :18)、H9(SEQ ID NO :19)、H12(SEQ ID NO :20)、H13(SEQ ID NO :21)、 H14(SEQ ID NO :22)、H15(SEQ ID NO : 145)、或H16 (SEQ ID NO: 146)。 [0162] The present invention further provides a host cell comprising an isolated nucleic acid, the isolated nucleic acid encoding a human vWF-specific humanized antibody or binding fragment thereof, which humanized antibody or binding fragment thereof comprising the heavy chain variable region One: H2 (SEQ ID NO: 13), H4 (SEQ ID NO: 14), H5 (SEQID NO: 15), H6 (SEQ ID NO: 16), H7 (SEQ ID NO: 17), H8 ( SEQ ID NO: 18), H9 (SEQ ID NO: 19), H12 (SEQ ID NO: 20), H13 (SEQ ID NO: 21), H14 (SEQ ID NO: 22), H15 (SEQ ID NO: 145 ), or H16 (SEQ ID NO: 146).

[0163] 本发明还提供包含分离核酸的宿主细胞,该分离核酸编码具有人vWF特异性的人源化抗体或其结合片段,而该人源化抗体或其结合片段包含下列轻链可变区其中之一: L5 (SEQ ID NO :23)、L4(SEQ ID NO :24)、L6(SEQID NO :25)、L7 (SEQ ID NO :26)、L8(SEQ ID NO :27)、L9(SEQ ID NO :28)、LlO (SEQ ID NO :29)或Lll(SEQ ID NO :30)。 [0163] The present invention further provides a host cell comprising an isolated nucleic acid, the isolated nucleic acid encoding a human vWF-specific humanized antibody or binding fragment thereof, which humanized antibody or binding fragment thereof comprising the light chain variable region One: L5 (SEQ ID NO: 23), L4 (SEQ ID NO: 24), L6 (SEQID NO: 25), L7 (SEQ ID NO: 26), L8 (SEQ ID NO: 27), L9 ( SEQ ID NO: 28), LlO (SEQ ID NO: 29) or Lll (SEQ ID NO: 30).

[0164] 本发明还提供包含分离核酸的宿主细胞,该分离核酸编码具有人vWF特异性的人源化抗体或其结合片段,而该人源化抗体或其结合片段包含下列重链可变区其中之一: H2 (SEQ ID NO :13)、H4(SEQ ID NO :14)、H5 (SEQID NO :15)、H6 (SEQ ID NO :16)、H7 (SEQ ID NO :17)、H8(SEQ ID NO :18)、H9 (SEQ ID NO :19)、H12 (SEQ ID NO :20)、H13 (SEQ ID N0:21)、 H14(SEQ ID NO :22)、H15(SEQ ID NO : 145)、或H16 (SEQ ID NO: 146);及(2)下列轻链可变区其中之一:L5 (SEQ ID NO :23)、L4 (SEQ ID NO :24)、L6 (SEQ ID NO :25)、L7 (SEQ ID NO : 26)、L8(SEQ ID NO :27)、L9(SEQID NO :28)、LlO (SEQ ID NO :29)或Lll(SEQ ID NO :30)。 [0164] The present invention further provides a host cell comprising an isolated nucleic acid, the isolated nucleic acid encoding a human vWF-specific humanized antibody or binding fragment thereof, which humanized antibody or binding fragment thereof comprising the heavy chain variable region One: H2 (SEQ ID NO: 13), H4 (SEQ ID NO: 14), H5 (SEQID NO: 15), H6 (SEQ ID NO: 16), H7 (SEQ ID NO: 17), H8 ( SEQ ID NO: 18), H9 (SEQ ID NO: 19), H12 (SEQ ID NO: 20), H13 (SEQ ID N0: 21), H14 (SEQ ID NO: 22), H15 (SEQ ID NO: 145 ), or H16 (SEQ ID NO: 146); and (2) one of the following light chain variable region wherein: L5 (SEQ ID NO: 23), L4 (SEQ ID NO: 24), L6 (SEQ ID NO: 25), L7 (SEQ ID NO: 26), L8 (SEQ ID NO: 27), L9 (SEQID NO: 28), LlO (SEQ ID NO: 29) or Lll (SEQ ID NO: 30).

[0165] 本发明还提供包含分离核酸的宿主细胞,该分离核酸包含载体GS264的编码轻链的核酸序列,载体GS264已在2008年1月23日在DSMZ保藏于微生物中,其登记号为DSM [0165] The present invention also provides a host cell comprising an isolated nucleic acid, the isolated nucleic acid vector comprising a nucleic acid sequence encoding the light chain of GS264, GS264 carrier already in January 23, 2008 at DSMZ deposited in microorganisms, which registration number DSM

21059。 21059.

[0166] 本发明还提供包含分离核酸的宿主细胞,该分离核酸包含载体GS265的编码重链的核酸序列,载体GS265已在2008年1月23日在DSMZ保藏于微生物中,其登记号为DSM [0166] The present invention further provides a host cell comprising an isolated nucleic acid, which isolated nucleic acid comprising a nucleic acid sequence encoding the heavy chain vector GS265, GS265 has a carrier 23 January 2008 at the DSMZ deposited microorganisms, which registration number DSM

21060。 21060.

[0167] 本发明还提供具有vWF特异性的人源化抗体或其结合片段的生产方法,包含培养本发明的宿主细胞,以表达核酸及制造抗体;本发明的人源化vWF抗体或其结合片段的生 [0167] The present invention also provides people with vWF-specific humanized antibody or binding fragment production methods, including the culture of the present invention, a host cell to express the nucleic acid and produce antibodies; the present invention is a humanized antibody or a binding vWF Health Fragment

27产方法还可包含自宿主细胞培养物回收抗体。 Production method 27 may further comprise recovered from host cell culture antibody. 在某些实施方案中,抗体可自宿主细胞培养基加以回收;在某些实施方案中,于培养之前,可用包含编码重链可变区的核酸的载体及包含编码轻链可变区的核酸的载体共同感染宿主细胞。 In certain embodiments, the antibody can be recovered from the host cell culture; In certain embodiments, prior to culture, can be used comprising a heavy chain variable region encoding nucleic acids and vectors comprising a nucleic acid encoding the light chain variable region vector co-infected host cells.

[0168] 本发明提供包含具有vWF特异性的人源化抗体或其结合片段的组合物,该人源化抗体或其结合片段包含如SEQ ID NO : 19中所述的重链可变区序列及如SEQ ID N0:28中所述的轻链可变区序列。 [0168] The present invention provides a human comprising vWF having a specific humanized antibody or binding fragment compositions, the humanized antibody or binding fragment thereof comprising as SEQ ID NO: heavy chain variable region sequence in the 19 and as SEQ ID N0: 28 the light chain variable region sequence described.

[0169] 本发明提供包含具有vWF特异性的人源化抗体或其结合片段的组合物,该人源化抗体或其结合片段包含如SEQ ID NO :19中所述的重链可变区序列、如SEQ ID NO :28中所述的轻链可变区序列、及可药用的载体(carrier)。 [0169] The present invention provides a human comprising vWF having a specific humanized antibody or binding fragment compositions, the humanized antibody or binding fragment thereof comprising as SEQ ID NO: heavy chain variable region sequence in the 19 as SEQ ID NO: light chain variable region sequence of the 28, and a pharmaceutically acceptable carrier (carrier).

[0170] 本发明提供包含具有vWF特异性的人源化抗体或其结合片段的组合物,该人源化抗体或其结合片段包含如SEQ ID NO :237中所述的重链序列及如SEQ ID N0:238中所述的轻链序列。 [0170] The present invention provides a human comprising vWF having a specific humanized antibody or binding fragment compositions, the humanized antibody or binding fragment thereof comprising as SEQ ID NO: heavy chain sequence as described in SEQ 237 and ID N0: 238 light chain sequence described.

[0171] 本发明提供包含具有vWF特异性的人源化抗体或其结合片段的组合物,该人源化抗体或其结合片段包含如SEQ ID NO :237中所述的重链序列、如SEQ ID NO :238中所述的轻链序列、及可药用的载体。 [0171] The present invention provides a human comprising vWF having a specific humanized antibody or binding fragment compositions, the humanized antibody or binding fragment thereof comprising as SEQ ID NO: heavy chain sequence described in 237, SEQ e.g. ID NO: light chain sequence in the 238, and a pharmaceutically acceptable carrier.

[0172] 本发明提供包含具有人类冯维勒布兰德氏因子(vWF)特异性的人源化抗体或其结合片段的组合物,该人源化抗体或其结合片段包含:对应于存在于鼠类抗体NMC-4中的⑶R的⑶R区;对应于人类抗体AAC18165. 1 (SEQID NO :4)的可变区中的框架区的重链框架区;对应于人类抗体AAK94808(SEQ ID NO :6)的可变区中的框架区的轻链框架区;及可药用的载体。 [0172] The present invention provides comprising people with human von Willebrand factor (vWF) specific humanized antibody or binding fragment compositions, the humanized antibody or binding fragment thereof comprising: corresponding to the presence in murine antibody NMC-4 in the ⑶R the ⑶R area; corresponds to the human antibody AAC18165 1 (SEQID NO: 4) heavy chain variable region framework region framework regions; corresponds to the human antibody AAK94808 (SEQ ID NO.: and a pharmaceutically acceptable carrier; light chain framework region 6) of the variable region framework regions.

[0173] 本发明还提供包含具有vWF特异性的人源化抗体或其结合片段的组合物,该人源化抗体或其结合片段包含HCDRl :GFSLTDYGVD (SEQ ID NO :7)、HCDR2 : MIffGDGSTDYNSALKS (SEQ ID NO :8)、HCDR3 :DPADYGNYDYALDY(SEQID NO :9)、来自人类抗体AAC18165. KSEQ ID NO :4)的可变区的重链框架区、以及可药用的载体。 [0173] The present invention also provides a human comprising vWF having a specific humanized antibody or binding fragment compositions, the humanized antibody or binding fragment thereof comprising HCDRl: GFSLTDYGVD (SEQ ID NO: 7), HCDR2: MIffGDGSTDYNSALKS ( SEQ ID NO: 8), HCDR3: DPADYGNYDYALDY (SEQID NO: 9), KSEQ ID NO derived from a human antibody AAC18165: heavy chain framework region 4) of the variable region, and a pharmaceutically acceptable carrier.

[0174] 本发明还提供包含具有vWF特异性的人源化抗体或其结合片段的组合物,该人源化抗体或其结合片段包含下列轻链CDR =LCDRl :SASQDI NKYLN (SEQ ID N0:10)、 LCDR2 :YTSSLHS (SEQ ID NO :11)、及LCDR3 :QQYEKLPWT(SEQ ID NO : 12)、及来自人类抗体AAK94808 (SEQ ID NO :6)的可变区的轻链框架区、以及可药用的载体。 [0174] The present invention also provides a human comprising vWF having a specific humanized antibody or binding fragment compositions, the humanized antibody or binding fragment thereof comprising the light chain CDR = LCDRl: SASQDI NKYLN (SEQ ID N0: 10 ), LCDR2: YTSSLHS (SEQ ID NO: 11), and LCDR3: QQYEKLPWT (SEQ ID NO: 12), and from a human antibody AAK94808 (SEQ ID NO: 6 variable region) of the light chain framework regions, and a drug with the carrier.

[0175] 本发明还提供包含具有vWF特异性的人源化抗体或其结合片段的组合物,该人源化抗体或其结合片段包含:重链CDR,HCDRl :GFSLTDYGVD (SEQID NO :7)、HCDR2 : MIffGDGSTDYNSALKS (SEQ ID NO :8)、及HCDR3 :DPADYGNYDYALDY (SEQ ID NO :9);及轻链CDR, LCDRl :SASQDINKYLN(SEQID NO :10)、LCDR2 :YTSSLHS(SEQ ID NO : 11)、及LCDR3 : QQYEKLPffT (SEQID NO: 12);任选地,来自人类抗体AAK94808 (SEQ ID NO :6)的可变区的轻链框架区和/或来自人类抗体AAC18165. 1(SEQ ID NO :4)的可变区的重链框架区;以及可药用的载体。 [0175] The present invention also provides a human comprising vWF having a specific humanized antibody or binding fragment compositions, the humanized antibody or binding fragment comprises: a heavy chain CDR, HCDRl: GFSLTDYGVD (SEQID NO: 7), HCDR2: MIffGDGSTDYNSALKS (SEQ ID NO: 8), and HCDR3: DPADYGNYDYALDY (SEQ ID NO: 9); and the light chain CDR, LCDRl: SASQDINKYLN (SEQID NO: 10), LCDR2: YTSSLHS (SEQ ID NO: 11), and LCDR3: QQYEKLPffT (SEQID NO: 12);. Optionally, from a human antibody AAK94808: light chain framework region (SEQ ID NO 6) of the variable region and / or AAC18165 1 from a human antibody (SEQ ID NO: 4) and a pharmaceutically acceptable carrier; a heavy chain variable region framework region.

[0176] 本发明还提供包含具有vWF特异性的人源化抗体或其结合片段的组合物,该人源化抗体或其结合片段包含下列重链可变区之一:H2 (SEQ ID N0:13)、H4(SEQ ID N0:14)、 H5 (SEQ ID NO :15)、H6(SEQ ID NO : 16)、H7 (SEQ ID NO : 17)、H8 (SEQ ID NO : 18)、H9 (SEQ ID NO :19)、H12(SEQID NO :20)、H13 (SEQ ID NO :21)、H14 (SEQ ID NO :22)、H15 (SEQ IDNO :145)、或H16(SEQ ID NO : 146)以及可药用的载体。 [0176] The present invention also provides a human comprising vWF having a specific humanized antibody or binding fragment compositions, the humanized antibody or binding fragment thereof comprising a heavy chain variable region of one of the following: H2 (SEQ ID N0: 13), H4 (SEQ ID N0: 14), H5 (SEQ ID NO: 15), H6 (SEQ ID NO: 16), H7 (SEQ ID NO: 17), H8 (SEQ ID NO: 18), H9 ( SEQ ID NO: 19), H12 (SEQID NO: 20), H13 (SEQ ID NO: 21), H14 (SEQ ID NO: 22), H15 (SEQ IDNO: 145), or H16 (SEQ ID NO: 146) and a pharmaceutically acceptable carrier.

[0177] 本发明还提供包含具有vWF特异性的人源化抗体或其结合片段的组合物,该人源化抗体或其结合片段包含下列轻链可变区之一:L5 (SEQ ID N0:23)、L4(SEQ ID NO :24)、 L6 (SEQ ID NO :25)、L7(SEQ ID NO :26)、L8 (SEQ ID NO :27)、L9 (SEQ ID NO :28)、LlO (SEQ ID NO :29)或Lll (SEQ ID NO :30)以及可药用的载体。 [0177] The present invention also provides a human comprising vWF having a specific humanized antibody or binding fragment compositions, the humanized antibody or binding fragment thereof comprising a light chain variable region of one of the following: L5 (SEQ ID N0: 23), L4 (SEQ ID NO: 24), L6 (SEQ ID NO: 25), L7 (SEQ ID NO: 26), L8 (SEQ ID NO: 27), L9 (SEQ ID NO: 28), LlO ( SEQ ID NO: 29) or Lll (SEQ ID NO: 30), and a pharmaceutically acceptable carrier.

[0178] 本发明还提供包含具有vWF特异性的人源化抗体或其结合片段的组合物,该人源化抗体或其结合片段包含:下列重链可变区其中之一:H2(SEQID NO :13)、H4(SEQ ID NO: 14)、H5(SEQ ID NO :15)、H6(SEQ ID NO :16)、H7 (SEQ ID NO : 17)、H8 (SEQ ID NO :18)、H9(SEQ ID NO :19)、H12(SEQ ID NO :20)、H13 (SEQ ID NO :21)、H14 (SEQ ID NO :22)、H15 (SEQ ID NO :145)、或H16(SEQ ID NO : 146);下列轻链可变区其中之一_L5 (SEQ ID NO :23)、L4 (SEQ ID NO :24)、L6(SEQ ID NO :25)、L7 (SEQID NO :26)、L8 (SEQ ID NO :27)、L9 (SEQ ID N0:28)、 LlO (SEQ ID NO :29)或Lll (SEQ ID NO :30);以及可药用的载体。 [0178] The present invention also provides a human comprising vWF having a specific humanized antibody or binding fragment compositions, the humanized antibody or binding fragment comprises: a heavy chain variable region where one of the following: H2 (SEQID NO : 13), H4 (SEQ ID NO: 14), H5 (SEQ ID NO: 15), H6 (SEQ ID NO: 16), H7 (SEQ ID NO: 17), H8 (SEQ ID NO: 18), H9 (SEQ ID NO: 19), H12 (SEQ ID NO: 20), H13 (SEQ ID NO: 21), H14 (SEQ ID NO: 22), H15 (SEQ ID NO: 145), or H16 (SEQ ID NO : 146); following light chain variable region wherein one _L5 (SEQ ID NO: 23), L4 (SEQ ID NO: 24), L6 (SEQ ID NO: 25), L7 (SEQID NO: 26), L8 (SEQ ID NO: 27), L9 (SEQ ID N0: 28), LlO (SEQ ID NO: 29) or Lll (SEQ ID NO: 30); and a pharmaceutically acceptable carrier.

[0179] 本发明还提供包含如此处所述的第一人源化抗体或其结合片段及结合至vWF的Al结构域的第二抗体的组合物。 The first one [0179] The present invention also provides as described herein comprising a humanized antibody or binding fragment binds to vWF and the Al composition of the second antibody domain.

[0180] 在一些实施方案中,第二抗体是AJW-200。 [0180] In some embodiments, the second antibody is AJW-200.

[0181] 本发明还提供在一受试者(例如人类)中的vWF介导的疾病或紊乱(例如血栓形成疾病或紊乱)的治疗方法,该方法包含给予该受试者治疗有效量的具有人vWF特异性的人源化抗体或其结合片段,其包含如SEQ ID N0:19中所示的重链可变区序列及如SEQ ID NO :28中所示的轻链可变区序列。 [0181] The present invention also provides a subject (e.g., a human) in vWF mediated disease or disorder (e.g., thrombotic disease or disorder) treatment, the method comprising administering to the subject a therapeutically effective amount of a human vWF-specific humanized antibody or binding fragment thereof, which comprises as SEQ ID N0: 19 the heavy chain variable region sequence as shown in and SEQ ID NO: light chain variable region sequence shown in 28.

[0182] 本发明还提供在一受试者(例如人类)中的vWF介导的疾病或紊乱(例如血栓形成疾病或紊乱)的治疗方法,该方法包含给予该受试者治疗有效量的具有人vWF特异性的人源化抗体或其结合片段,其包含如SEQ ID NO :237中所示的重链序列及如SEQ ID NO :238 中所示的轻链序列。 [0182] The present invention also provides a subject (e.g., a human) in vWF mediated disease or disorder (e.g., thrombotic disease or disorder) treatment, the method comprising administering to the subject a therapeutically effective amount of a human vWF-specific humanized antibody or binding fragment thereof, which comprises as SEQ ID NO: heavy chain sequence and 237 as shown in SEQ ID NO: 238 in the light chain sequence shown.

[0183] 本发明还提供在一受试者(例如人类)中的vWF介导的疾病或紊乱(例如血栓形成疾病或紊乱)的治疗方法,该方法包含给予该受试者治疗有效量的具有人vWF特异性的人源化抗体或其结合片段,其包含:对应于存在于鼠类抗体NMC-4中的CDR的CDR区;对应于人类抗体AAC18165. 1(SEQ IDNO :4)的可变区中的框架区的重链框架区;对应于人类抗体AAK94808 (SEQID NO :6)的可变区中的框架区的轻链框架区。 [0183] The present invention also provides a subject (e.g., a human) in vWF mediated disease or disorder (e.g., thrombotic disease or disorder) treatment, the method comprising administering to the subject a therapeutically effective amount of a human vWF-specific humanized antibody or binding fragment thereof, comprising: corresponding to the presence in the murine antibody NMC-4 in the CDR CDR regions; corresponding human antibody AAC18165 1. (SEQ IDNO: 4) Variable heavy chain framework region region framework regions; corresponding to human antibody AAK94808 (SEQID NO: 6) of the variable region framework regions of the light chain framework region.

[0184] 本发明还提供在一受试者(例如人类)中的vWF介导的疾病或紊乱(例如血栓形成疾病或紊乱)的治疗方法,该方法包含给予该受试者治疗有效量的具有vWF特异性的人源化抗体或其结合片段,其包含:HCDR1 :GFSLTDYGVD(SEQ ID NO :7)、HCDR2 : MIffGDGSTDYNSALKS (SEQ ID NO :8)、HCDR3 :DPADYGNYDYALDY (SEQ ID NO :9)、及来自人类抗体AAC18165. 1(SEQ ID NO :4)的可变区的重链框架区。 [0184] The present invention also provides a subject (e.g., a human) in vWF mediated disease or disorder (e.g., thrombotic disease or disorder) treatment, the method comprising administering to the subject a therapeutically effective amount of a vWF-specific humanized antibody or binding fragment thereof, comprising: HCDR1: GFSLTDYGVD (SEQ ID NO: 7), HCDR2: MIffGDGSTDYNSALKS (SEQ ID NO: 8), HCDR3: DPADYGNYDYALDY (SEQ ID NO: 9), and from a human antibody AAC18165 1 (SEQ ID NO: 4). heavy chain variable region framework region.

[0185] 血栓形成疾病或紊乱可为心血管疾病或例如缺血性卒中的脑血管疾病。 [0185] thrombotic disease or disorder may be cardiovascular diseases such as ischemic stroke or cerebrovascular disease. 在某些实施方案中,心血管疾病为动脉粥状硬化症(atherosclerosis)、再狭窄(restenosis)、心绞痛(angina)、急性心肌梗塞(acute myocardialinfarction)、急性冠状动脉综合症(acute coronary syndrome)、或与糖尿病(diabetes)相关联的心血管异常;在某些实施方案中, 血栓性疾病或紊乱为血管发炎、静脉血栓(venous thrombosis)、镰刀形细胞病、异种移植排斥(xenograft rejection)、夕卜周血管病(peripheral vasculardisease)、血栓性血小板 In certain embodiments, the cardiovascular disease is atherosclerotic sclerosis (atherosclerosis), restenosis (restenosis), angina (angina), acute myocardial infarction (acute myocardialinfarction), acute coronary syndrome (acute coronary syndrome), or with diabetes mellitus (diabetes) associated with cardiovascular abnormalities; in some embodiments, the thrombotic disease or disorder is inflammation of blood vessels, venous thromboembolism (venous thrombosis), sickle cell disease, xenograft rejection (xenograft rejection), evening Bu peripheral vascular disease (peripheral vasculardisease), thrombotic thrombocytopenic

29减少性紫癜症(thrombotic thrombocytopenicpurpura)、囊性纤维化(cystic fibrosis)、 血管性痴呆(vasculardementia)、雷诺病(Raynaud,s disease)、类风湿性关节炎、或糖尿病;在某些实施方案中,脑血管异常可包含由于大脑动脉梗塞(cerebralartery infarct) 以及小腔隙梗塞(small lacunar infarct)的缺血性卒中及血管性痴呆(vascular dementia) 0人源化vWF抗体还可用于预防再发性中风、或由脑血管发炎所引起的初期中风。 29 Purpura syndrome (thrombotic thrombocytopenicpurpura), cystic fibrosis (cystic fibrosis), vascular dementia (vasculardementia), Raynaud's disease (Raynaud, s disease), rheumatoid arthritis, or diabetes; in some embodiments, , cerebral vascular abnormalities may include cerebral artery occlusion due (cerebralartery infarct) as well as small lacunar infarct (small lacunar infarct) of ischemic stroke and vascular dementia (vascular dementia) 0 humanized vWF antibodies may also be used to prevent recurrent stroke, or cerebral vascular inflammation caused by the initial stroke.

[0186] 在某些实施方案中,血栓形成疾病或紊乱可包含癌症。 [0186] In certain embodiments, the thrombotic disease or disorder may contain cancer.

[0187] 本发明还提供在一受试者(例如人类)中的vWF介导的疾病或紊乱(例如血栓形成疾病或紊乱)的治疗方法,该方法包含给予该受试者治疗有效量的具有vWF特异性的人源化抗体或其结合片段,其包含:下列轻链CDR =LCDRl :SASQDINKYLN(SEQ ID NO :10)、 LCDR2 :YTSSLHS (SEQ ID NO : 11)、及LCDR3 =QQYEKLPffT (SEQ ID NO : 12)、及来自人类抗体AAK94808 (SEQ IDNO :6)的可变区的轻链框架区。 [0187] The present invention also provides a subject (e.g., a human) in vWF mediated disease or disorder (e.g., thrombotic disease or disorder) treatment, the method comprising administering to the subject a therapeutically effective amount of a vWF-specific humanized antibody or binding fragment thereof, comprising: the following light chain CDR = LCDRl: SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQ ID NO: 11), and LCDR3 = QQYEKLPffT (SEQ ID NO: 12), and from a human antibody AAK94808 (SEQ IDNO: 6 light chain framework regions) of the variable region.

[0188] 本发明还提供在一受试者(例如人类)中的vWF介导的疾病或紊乱(例如血栓形成疾病或紊乱)的治疗方法,该方法包含给予该受试者治疗有效量的具有vWF特异性的人源化抗体或其片段,其包含:下列重链可变区其中之一:H2 (SEQ ID N0:13)、H4(SEQ ID NO :14)、H5(SEQ ID NO : 15)、H6 (SEQ ID NO : 16)、H7 (SEQ ID NO : 17)、H8 (SEQ ID NO: 18)、 H9 (SEQID NO :19)、H12(SEQ ID NO :20)、H13 (SEQ ID NO :21)、H14 (SEQ IDNO :22)、H15 (SEQ ID NO :145)、或H16(SEQ ID NO: 146)。 [0188] The present invention also provides a subject (e.g., a human) in vWF mediated disease or disorder (e.g., thrombotic disease or disorder) treatment, the method comprising administering to the subject a therapeutically effective amount of a vWF-specific humanized antibody or fragment thereof, which comprises: a heavy chain variable region where one of the following: H2 (SEQ ID N0: 13), H4 (SEQ ID NO: 14), H5 (SEQ ID NO: 15 ), H6 (SEQ ID NO: 16), H7 (SEQ ID NO: 17), H8 (SEQ ID NO: 18), H9 (SEQID NO: 19), H12 (SEQ ID NO: 20), H13 (SEQ ID NO: 21), H14 (SEQ IDNO: 22), H15 (SEQ ID NO: 145), or H16 (SEQ ID NO: 146).

[0189] 本发明还提供在一受试者(例如人类)中的vWF介导的疾病或紊乱(例如血栓形成疾病或紊乱)的治疗方法,该方法包含给予该受试者治疗有效量的具有vWF特异性的人源化抗体或其片段,其包含:下列轻链可变区其中之一:L5 (SEQ ID N0:23)、L4(SEQ ID NO :24)、L6(SEQ ID NO :25)、L7 (SEQ ID NO :26)、L8 (SEQ ID NO :27)、L9 (SEQ ID NO :28)、 LlO (SEQID NO :29)或Lll (SEQ ID NO :30)。 [0189] The present invention also provides a subject (e.g., a human) in vWF mediated disease or disorder (e.g., thrombotic disease or disorder) treatment, the method comprising administering to the subject a therapeutically effective amount of a vWF-specific humanized antibody or fragment thereof, comprising: a light chain variable region wherein one of the following: L5 (SEQ ID N0: 23), L4 (SEQ ID NO: 24), L6 (SEQ ID NO: 25 ), L7 (SEQ ID NO: 26), L8 (SEQ ID NO: 27), L9 (SEQ ID NO: 28), LlO (SEQID NO: 29) or Lll (SEQ ID NO: 30).

[0190] 本发明还提供在一受试者(例如人类)中的vWF介导的疾病或紊乱(例如血栓形成疾病或紊乱)的治疗方法,该方法包含给予该受试者治疗有效量的具有vWF特异性的人源化抗体或其片段,其包含:下列重链可变区其中之一:H2 (SEQ ID N0:13)、H4(SEQ ID NO :14)、H5(SEQ ID NO : 15)、H6 (SEQ ID NO : 16)、H7 (SEQ ID NO : 17)、H8 (SEQ ID NO: 18)、 H9 (SEQID NO :19)、H12(SEQ ID NO :20)、H13 (SEQ ID NO :21)、H14 (SEQ IDNO :22)、H15 (SEQ ID NO :145)、或H16(SEQ ID NO : 146);下列轻链可变区其中之一:L5 (SEQ ID NO :23)、 L4 (SEQ ID NO :24)、L6(SEQ IDNO :25)、L7 (SEQ ID NO :26)、L8 (SEQ ID NO :27)、L9 (SEQ ID NO :28)、L10(SEQ ID NO :29)或Lll (SEQ ID NO :30)。 [0190] The present invention also provides a subject (e.g., a human) in vWF mediated disease or disorder (e.g., thrombotic disease or disorder) treatment, the method comprising administering to the subject a therapeutically effective amount of a vWF-specific humanized antibody or fragment thereof, which comprises: a heavy chain variable region where one of the following: H2 (SEQ ID N0: 13), H4 (SEQ ID NO: 14), H5 (SEQ ID NO: 15 ), H6 (SEQ ID NO: 16), H7 (SEQ ID NO: 17), H8 (SEQ ID NO: 18), H9 (SEQID NO: 19), H12 (SEQ ID NO: 20), H13 (SEQ ID NO: 21), H14 (SEQ IDNO: 22), H15 (SEQ ID NO: 145), or H16 (SEQ ID NO: 146); one of the following light chain variable region wherein: L5 (SEQ ID NO: 23) , L4 (SEQ ID NO: 24), L6 (SEQ IDNO: 25), L7 (SEQ ID NO: 26), L8 (SEQ ID NO: 27), L9 (SEQ ID NO: 28), L10 (SEQ ID NO : 29) or Lll (SEQ ID NO: 30).

[0191] 在某些实施方案中,具有vWF特异性的人源化抗体或其结合片段缺乏效应子(effector)功能;在某些实施方案中,人源化抗体包含源自于IgG4的Fc区。 Al. [0191] In certain embodiments, having specificity for vWF humanized antibody or binding fragment lacking effector (effector) function; In certain embodiments, the humanized antibody comprises an Fc region derived from IgG4 .

[0192] 本发明提供具有冯维勒布兰德氏因子(vWF)特异性的人类抗体或其结合片段,其可以自EDiqq的约1至约250倍的治疗有效量来提供,而不致引发明显的临床出血迹象。 [0192] The present invention provides a von Willebrand factor (vWF) specific for human antibody or binding fragment thereof, which may be from about 1 to about 250 times the therapeutically effective amount EDiqq to offer, and does not cause any significant The clinical signs of bleeding. 人类抗体或其结合片段优选地为具有人类vWF的Al结构域特异性,具有vWF特异性的人类抗体或其结合片段更优选地为具有vWF特异性的人源化抗体或其结合片段。 Human antibody or binding fragment thereof preferably having a specific human vWF Al domain of vWF having a specific human antibody or binding fragment thereof more preferably one having specificity for vWF humanized antibody or binding fragment thereof.

[0193] 本发明还提供vWF介导的疾病或紊乱的治疗方法,其通过给予一受试者(优选人类)治疗有效量的如此处所述的人源化抗体或其结合片段。 [0193] The present invention also provides a method of treating a disease or disorder mediated by vWF, which humanized antibody or a binding fragment thereof by administering to a subject (preferably a human) as described herein in the treatment of humans an effective amount. 该治疗有效量从约0.001至约100mg/kg,优选为自约0. 002至约20mg/kg,更优选为自约0. 002至约10mg/kg,又更优选为自约0. 002至约0. 4mg/kg,再更优选为自约0. 005至约0. 2mg/kg,最优选为自约0. 01至约0.lmg/kg0 The therapeutically effective amount of from about 0.001 to about 100mg / kg, preferably from about 0.002 to about 20mg / kg, more preferably from about 0.002 to about 10mg / kg, and more preferably from about 0.002 to approximately 0. 4mg / kg, even more preferably from about 0.005 to about 0. 2mg / kg, and most preferably from about 0.01 to about 0.lmg / kg0

[0194] 本发明还提供一种vWF介导的疾病或紊乱的治疗方法,其通过给予一需要的受试者治疗有效量的如此处所述的人源化抗体或其结合片段来达成。 [0194] The present invention also provides a method of treating a disease or a disorder of vWF-mediated, who said so at their treatment by administering to the subject an effective amount of a required humanized antibody or binding fragment thereof to achieve. 该治疗有效量从EDiciq的约1至约250倍,优选为自EDltltl的约1至约200倍,更优选自EDltltl的约1至约100倍。 The therapeutically effective amount of from about 1 to about 250 times EDiciq, and preferably from about 1 to about EDltltl 200 times, more preferably from about 1 to about EDltltl 100 times.

[0195] 本发明还提供一种vWF介导的疾病或紊乱的治疗方法,其通过将单一或多次小剂量(sub-dose)的治疗有效量的如此处所述的人源化抗体或其结合片段施予需要此类处理的受试者来达成。 [0195] The present invention also provides a method of treating a disease or a disorder of vWF-mediated, which by the person so at the single or multiple small doses (sub-dose) of a therapeutically effective amount of a humanized antibody or binding fragment administered to a subject in need of such treatment to achieve.

[0196] 本发明还提供一种vWF介导的疾病或紊乱的治疗方法,其通过将治疗有效量的如此处所述的人源化抗体或其结合片段以皮下方式施予需要此类处理的受试者来达成。 [0196] The present invention also provides a method of treating a disease or disorder mediated by vWF, by which a therapeutically effective amount as described herein of the humanized antibody or binding fragment thereof administered subcutaneously in need of such treatment Subjects to achieve.

[0197] 本发明还提供一种vWF介导的疾病或紊乱的治疗方法,其通过将治疗有效量的如此处所述的人源化抗体或其结合片段透过静脉方式施予需要此类处理的受试者来达成。 [0197] The present invention also provides a method of treating a disease or a disorder mediated by vWF, by which a therapeutically effective amount as described herein of the humanized antibody or binding fragment thereof in need of such treatment by administering intravenously subject to achieve.

[0198] 本发明还提供一种vWF介导的疾病或紊乱的治疗方法,其通过连续地或结合放射性治疗法(例如照射或引入放射性物质,如UICC(编),Klinische Onkologie, Springer-Verlag (1982)),将治疗有效量的如此处所述的人源化抗体或其结合片段通过静脉方式施予需要此类处理的受试者来达成。 [0198] The present invention also provides a method of treating a disease or a disorder mediated by vWF, which by means of a continuous radiation therapy or in combination (e.g., irradiation or introduction of radioactive substances, such as UICC (series), Klinische Onkologie, Springer-Verlag ( 1982)), as described herein in a human a therapeutically effective amount of a humanized antibody or binding fragment by intravenously administered to a subject in need of such treatment to achieve.

[0199] 在实施或测试本发明时,虽然类似或等同于此处所述的任何方法及材料均可使用,但以下仍说明优选方法及材料。 [0199] In the practice or testing of the present invention, although any similar or equivalent methods and materials described herein can be used, but the following description of the preferred methods and materials still.

[0200] 人源化冯维勒布兰德氏因子抗体的生产 [0200] The humanized antibody Von Willebrand factor production

[0201] 此处提供人源化冯维勒布兰德氏因子(vWF)抗体(例如鼠类NMC-4)或其结合片段的生产方法。 [0201] provided herein humanized von Willebrand factor (vWF) antibody (such as murine NMC-4) or a binding fragment production methods. 具有vWF特异性的人源化抗体或其结合片段,可通过自非人类动物(例如小鼠)的VH和/或VL区转移一个或多个CDR或其部分至人类VH和/或VL区的一个或多个框架区。 Having vWF-specific humanized antibody or binding fragment thereof, can be from a non-human animal (such as a mouse) VH and / or VL region, or CDR transfer one or more portions to human VH and / or VL region one or more framework regions. 任选地,当需要或期望维持结合亲和力时,如此存在于VH和/或VL区中的人类构架残基,可用对应的非人类(例如小鼠)残基加以替代。 Optionally, when necessary or desirable to maintain binding affinity, so present in the VH and / or VL region of human framework residues (such as mice) can be radical corresponding non-human residues to replace. 任选地,存在于CDR中的非人类氨基酸残基可以人类残基加以替代。 Optionally, present in the CDR of non-human amino acid residues may be to replace human residues radical.

[0202] 如此处所述的构架及⑶R的分类(除了HFRl及⑶Rl以外)基于卡巴记数制(Kabat numbering system)。 [0202] so at the architecture and ⑶R free (except HFRl and ⑶Rl) Kaba based number system (Kabat numbering system). 在此定义中,重链的CDR 包含残基31-35 (HCDRl), 50-65 (HCDR2),及95-102 (HCDR3);轻链的CDR 被定义成包含残基24-34 (LCDRl), 50-56 (LCDR2),及89-97 (LCDR3)。 In this definition, the heavy chain CDR comprising residues 31-35 (HCDRl), 50-65 (HCDR2), and 95-102 (HCDR3); light chain CDR is defined as comprising residues 24-34 (LCDRl) , 50-56 (LCDR2), and 89-97 (LCDR3). 将VH(例如重链框架区1 (HFRl)、重链框架区2 (HFR)、重链框架区3(HFR3)和/或重链框架区4(HFR4))中的框架区定义成包含残基1_30 (HFRl), 36-49 (HFR2),66-94 (HFR3),及103-113 (HFR4),而VL 中的框架区包含残基1-23 (LFRl), 35-49(LFR2),57-88(LFR 3)及98-107 (LFR4) (Wu和Kabat,1970 J. Exp. Med. 132 :211)。 The definition VH ((HFR4) such as the heavy chain framework region 1 (HFRl), heavy chain framework region 2 (HFR), heavy chain framework region 3 (HFR3) and / or heavy chain framework region 4) framework region to contain residues group 1_30 (HFRl), 36-49 (HFR2), 66-94 (HFR3), and 103-113 (HFR4), while the VL framework region comprising residues 1-23 (LFRl), 35-49 (LFR2) , 57-88 (LFR 3) and 98-107 (LFR4) (. Wu and Kabat, 1970 J. Exp Med 132:. 211). 然而,基于CDR的结构,Chothia将CDRl-H定义成包含残基26-32 (Chothia等,1992 J. Mol. Biol. 227 :799),AbM(抗体模拟)定义为CDR1-H 包含残基26-35 的Oxford Molecular,s AbM抗体模拟软件所使用的两者之间的折衷。 However, CDR-based structure, Chothia the CDRl-H is defined as comprising residues 26-32 (Chothia et, 1992 J. Mol Biol 227:.. 799), AbM (antibody mimics) as CDR1-H comprising residues 26 -35 Oxford Molecular, compromise both s AbM antibody modeling software used between. 此为此处所述的利用NMC-4的人源化方法所用的定义。 This is where the use of NMC-4, humanized methods used to define said.

[0203] 具有冯维勒布兰德氏因子(vWF)特异性的人源化抗体或其结合片段,可通过下列方式加以生产:将来自NMC-4的重链互补决定区(OTR)转移至对应于人类抗体AAC18165. KSEQ ID NO :4)的可变区中的框架区的重链框架区;以及将来自匪C-4的轻链⑶R转移至对应于人类抗体AAK94808 (SEQID NO :6)的可变区中的框架区的轻链框架区。 [0203] have von Willebrand factor (vWF) specific humanized antibody or binding fragment thereof, can be produced by the following ways: from NMC-4, heavy chain complementarity determining region (OTR) was transferred to corresponds to the human antibody AAC18165 KSEQ ID NO:. 4) of the variable region framework region heavy chain framework region; and light chain from bandit ⑶R C-4 is transferred to the corresponding human antibody AAK94808 (SEQID NO: 6) the variable region of the light chain framework region framework region.

[0204] 具有vWF特异性的人源化抗体或其结合片段还可通过下列方式加以生产:将来自鼠类匪C-4 的重链CDR(例如HCDRl :GFSLTDYGVD (SEQ ID NO :7), HCDR2 : MIffGDGSTDYNSALKS (SEQ ID NO :8),及HCDR 3 =DPADYGNYDYALDY (SEQ ID NO :9))其中一禾中或多种转移至人类框架区(例如来自AAC18165. 1 (SEQ ID NO :4)的可变区)。 [0204] people with vWF-specific humanized antibody or binding fragment thereof may also be produced by the following ways: from the bandit C-4 murine heavy chain CDR (for example HCDRl: GFSLTDYGVD (SEQ ID NO: 7), HCDR2 : MIffGDGSTDYNSALKS (SEQ ID NO: 8), and HCDR 3 = DPADYGNYDYALDY (SEQ ID NO: 9)) wherein one or more of Wo is transferred to the human framework region (e.g., from AAC18165 1 (SEQ ID NO:. 4) may be variable region).

[0205] 具有vWF特异性的人源化抗体或其结合片段还可通过下列方式加以生产:将轻链CDR (例如LCDRl :SASQDINKYLN (SEQ ID N0:10),LCDR2 :YTSSLHS (SEQ ID NO :11),及LCDR3 : QQYEKLPffT (SEQ ID NO :12))其中一种或多种转移至人类框架区(例如来自AAK94808 (SEQ ID NO :6)的可变区)。 [0205] vWF people with specific binding of the humanized antibody or fragment may also be produced by the following methods: light chain CDR (e.g., LCDRl: SASQDINKYLN (SEQ ID N0: 10), LCDR2: YTSSLHS (SEQ ID NO: 11 ), and LCDR3: QQYEKLPffT (SEQ ID NO: 12)) was transferred to one or more of the human framework region (e.g., from AAK94808 (SEQ ID NO: 6) of the variable region).

[0206] 具有vWF特异性的人源化抗体可通过下列方式加以生产:将来自鼠类NMC-4的重链CDR(例如HCDRl :GFSLTDYGVD (SEQ ID NO :7) ,HCDR2 =MIffGDGSTDYNSALKS (SEQ ID NO :8), 及HCDR3 :DPADYGNYDYALDY(SEQ IDNO :9))其中一种或多种转移至人类框架区(例如来自AAC18165. 1(SEQ IDNO :4)的可变区);及将轻链CDR (例如LCDRl : SASQDINKYLN (SEQ ID NO: 10),LCDR2 :YTSSLHS (SEQ ID NO :11),及LCDR 3 =QQYEKLPffT (SEQ ID NO :12))其中一种或多种转移至人类框架区(例如来自AAK94808 (SEQ ID NO :6)的可变区)。 [0206] people with vWF-specific humanized antibodies can be produced by the following ways: from the murine heavy chain CDR NMC-4 (for example HCDRl: GFSLTDYGVD (SEQ ID NO: 7), HCDR2 = MIffGDGSTDYNSALKS (SEQ ID NO : 8) and HCDR3: DPADYGNYDYALDY (SEQ IDNO: 9)) was transferred to one or more human framework regions (e.g., from AAC18165 1 (SEQ IDNO:. 4) of the variable region); and the light chain CDR ( For example LCDRl: SASQDINKYLN (SEQ ID NO: 10), LCDR2: YTSSLHS (SEQ ID NO: 11), and LCDR 3 = QQYEKLPffT (SEQ ID NO: 12)) was transferred to one or more of the human framework region (e.g., from AAK94808 (SEQ ID NO: 6) of the variable region).

[0207] 具有vWF特异性的人源化抗体可通过下列方式加以生产:将包含存在于NMC-4及人类框架区中的CDR的修饰重链可变区(例如H2 (SEQ ID NO : 13)、H4 (SEQ ID NO :14), H5 (SEQ ID NO :15)、H6(SEQ ID NO : 16)、H7 (SEQ ID NO : 17)、H8 (SEQ ID NO :18)、H9 (SEQ ID NO :19)、H12(SEQID NO :20)、H13 (SEQ ID NO :21)、H14 (SEQ ID NO :22)、H15 (SEQ IDNO : 145)、或H16(SEQ ID NO: 146))转移至人类恒定区。 [0207] vWF having a specific humanized antibodies can be produced by the following methods: containing NMC-4 in the presence of human framework and CDR regions of a modified heavy chain variable region (e.g., H2 (SEQ ID NO: 13) , H4 (SEQ ID NO: 14), H5 (SEQ ID NO: 15), H6 (SEQ ID NO: 16), H7 (SEQ ID NO: 17), H8 (SEQ ID NO: 18), H9 (SEQ ID NO: 19), H12 (SEQID NO: 20), H13 (SEQ ID NO: 21), H14 (SEQ ID NO: 22), H15 (SEQ IDNO: 145), or H16 (SEQ ID NO: 146)) Transfer to a human constant region.

[0208] 具有vWF特异性的人源化抗体还可通过下列方式加以生产:将包含存在于NMC-4 及人类框架区中的CDR的修饰轻链可变区(例如L5 (SEQ ID NO :23)、L4 (SEQ ID NO :24)、 L6 (SEQ ID NO :25)、L7(SEQ ID NO :26)、L8 (SEQ ID NO :27)、L9 (SEQ ID NO :28)、LlO (SEQ ID NO :29)或Lll (SEQ ID NO :30))转移至人类恒定区。 [0208] people with vWF-specific humanized antibodies may also be produced by the following methods: will present within NMC-4 and human framework region CDR modified light chain variable region (such as L5 (SEQ ID NO: 23 ), L4 (SEQ ID NO: 24), L6 (SEQ ID NO: 25), L7 (SEQ ID NO: 26), L8 (SEQ ID NO: 27), L9 (SEQ ID NO: 28), LlO (SEQ ID NO: 29) or Lll (SEQ ID NO: 30)) were transferred to human constant regions.

[0209] 具有vWF特异性的人源化抗体可通过下列方式加以生产:将包含存在于NMC-4及人类框架区中的CDR的修饰重链可变区(例如H2 (SEQ ID NO : 13)、H4 (SEQ ID NO :14), H5 (SEQ ID NO :15)、H6(SEQ ID NO : 16)、H7 (SEQ ID NO : 17)、H8 (SEQ ID NO :18)、H9 (SEQ ID NO :19)、H12(SEQID NO :20)、H13 (SEQ ID NO :21)、H14 (SEQ ID NO :22)、H15 (SEQ IDNO : 145)、或H16(SEQ ID NO : 146))转移至人类恒定区;以及将包含存在于NMC-4及人类框架区中的CDR 的修饰轻链可变区(例如L5(SEQ ID NO :23)、L4 (SEQ ID NO :24)、L6 (SEQ ID NO :25)、L7(SEQ ID NO :26)、L8 (SEQ ID NO :27)、L9 (SEQ ID NO :28)、LlO (SEQ ID NO :29) 或Lll (SEQ ID NO :30))转移至人类恒定区。 [0209] vWF having a specific humanized antibodies can be produced by the following methods: containing NMC-4 in the presence of human framework and CDR regions of a modified heavy chain variable region (e.g., H2 (SEQ ID NO: 13) , H4 (SEQ ID NO: 14), H5 (SEQ ID NO: 15), H6 (SEQ ID NO: 16), H7 (SEQ ID NO: 17), H8 (SEQ ID NO: 18), H9 (SEQ ID NO: 19), H12 (SEQID NO: 20), H13 (SEQ ID NO: 21), H14 (SEQ ID NO: 22), H15 (SEQ IDNO: 145), or H16 (SEQ ID NO: 146)) Transfer to human constant regions; and containing NMC-4 in the presence of human framework and CDR regions of the modified light chain variable region (e.g., L5 (SEQ ID NO: 23), L4 (SEQ ID NO: 24), L6 (SEQ ID NO: 25), L7 (SEQ ID NO: 26), L8 (SEQ ID NO: 27), L9 (SEQ ID NO: 28), LlO (SEQ ID NO: 29) or Lll (SEQ ID NO: 30) ) was transferred to a human constant region.

[0210] 为试图进一步降低人源化抗体的抗原性,可将CDR中的残基(例如鼠类残基)变为(例如取代为)人类氨基酸残基。 [0210] In an attempt to further reduce the humanized antibody antigen, can be CDR residues (such as murine residues) become (for example, substitution of) human amino acid residues. 举例而言,人源化抗体可包含HCDRl中的F27G,L29I, T30S和/或V34W取代其中一种或多种。 For example, humanized antibodies may comprise HCDRl the F27G, L29I, T30S and / or V34W substituted with one or more. 在某些实施方案中,人源化抗体可包含HCDR2中的S61P和/或A62S取代其中一种或多种;在某些实施方案中,人源化抗体可包含IXDRl中的S24Q,N30S和/或K31N取代其中一种或多种;在某些实施方案中,人源化抗体可包含一个或多个的取代,例如LCDR2中的Y50D,T51A,S53N, H55E和/或S56T取代。 In certain embodiments, the humanized antibody may comprise HCDR2 the S61P and / or A62S substituted one or more; in some embodiments, the humanized antibody may comprise IXDRl the S24Q, N30S and / or K31N substituted one or more; in some embodiments, the humanized antibody may contain one or more substituents, such as LCDR2 the Y50D, T51A, S53N, H55E and / or S56T substitution. 在某些实施方案中,人源化抗体可包含:HCDR1中的F27G,L29I,T30S和/或V34W取代其中一种或多种; HCDR2中的S61P和/或A62S取代其中一种或多种;LCDRl中的S24Q,N30S和/或K31N取代其中一种或多种;及LCDR2中的Y50D,T51A,S53N, H55E和/或S56T取代其中一种或多种。 In certain embodiments, the humanized antibody may comprise: HCDR1 the F27G, L29I, T30S and / or V34W replace one or more of; HCDR2 the S61P and / or A62S substituted one or more; LCDRl the S24Q, N30S and / or substituted with one or more of K31N; and LCDR2 the Y50D, T51A, S53N, H55E and / or substituted with one or more of S56T.

[0211] 考虑了各种形式的人源化抗体。 [0211] consider various forms of humanized antibodies. 举例而言,人源化抗体可为例如Fab的抗体片段, 其任选地与一个或多个的细胞毒性物质缀合以产生免疫缀合物(immunoconjugate)。 For example, the humanized antibody may be an antibody fragment such as Fab, optionally with one or more cytotoxic substance is conjugated to immune conjugates (immunoconjugate). 可选地,人源化抗体或亲和力成熟抗体可为完整(intact)抗体,例如完整IgGl抗体。 Alternatively, the humanized antibody or affinity matured antibody may be a complete (intact) antibody, e.g., the complete IgGl antibody.

[0212] 已开发出生产人源化抗体的抗体片段的各种技术。 [0212] have been developed to produce human antibodies of the humanized antibody fragments Various techniques. 传统上,这些片段系通过完整抗体的蛋白水解消化而获得(见例如Morimoto等,Journal ofBiochemical和Biophysical Methods,24 :107-117 (1992);及Brennan 等,Science,229 :81 (1985));然而,如今这些片段可通过重组宿主细胞来直接产生,例如抗体片段可由以上所讨论的抗体噬菌体文库分离出来;可选地,Fab' -SH片段可直接由大肠杆菌回收并将其化学交联以形成F(ab')2片段(Carter 等,Bio/Technology,10 : 163-167 (1992))。 Traditionally, these fragments were obtained by the Department of proteolytic digestion of intact antibodies (see, for example, Morimoto et al, Journal ofBiochemical and Biophysical Methods, 24: 107-117 (1992); and Brennan et al., Science, 229: 81 (1985)); Today, however, these fragments can be directly by recombinant host cells to produce, for example, an antibody fragment antibody phage libraries discussed above can be isolated; alternatively, Fab '-SH fragments can be directly recovered from E. coli and chemically cross-linked to formation of F (ab ') 2 fragments (Carter et, Bio / Technology, 10: 163-167 (1992)). 根据另一方法,F(ab,)2 片段可直接由重组宿主细胞培养物中分离出来,而其它生产抗体片段的方法对技术人员将为显而易见。 According to another approach, F (ab,) 2 fragments can be isolated directly from recombinant host cell culture, and other methods for the production of antibody fragments will be apparent to the art. 在其它实施方案中,选定的抗体为单链Fv片段(scFv),见WO 1993/16185 ;美国专利第5,571,894号;及美国专利第5,587,458号。 In other embodiments, the selected single-chain Fv antibody fragment (scFv), see WO 1993/16185; US Patent No. 5,571,894; and US Patent No. 5,587,458. 抗体片段还可为“线性抗体”,例如在美国专利第5,641,870中所述。 Antibody fragment may also be a "linear antibody", e.g., described in U.S. Patent No. 5,641,870 in.

[0213] 根据不同方法,可将具有期望的结合特异性(抗体_抗原结合位点)的抗体可变结构域融合至免疫球蛋白恒定结构域序列。 Antibody variable domains [0213] Depending on the method, with the desired binding specificities (antibody _ antigen binding site) fused to immunoglobulin constant domain sequences. 该融合优选地为利用免疫球蛋白重链恒定结构域,其包含至少部分铰链区、CH2区及CH3区。 The fusion preferably utilizing an immunoglobulin heavy chain constant domain, comprising at least part of the hinge region, CH2 region, and CH3 regions. 优选使包含轻链结合所必须的位点的第一重链恒定区(CHl)存在于至少一融合物中。 Preferably the first heavy chain constant region comprises necessary for light chain binding site (CHl) is present in at least one of the fusions. 将编码免疫球蛋白重链融合物及(若有需要)免疫球蛋白轻链的DNA插入于分别的表达载体内,并共同转染至一适当宿主生物。 Encoding the immunoglobulin heavy chain fusions and (if necessary) an immunoglobulin light chain DNA inserted into separate expression vectors and co-transfected into a suitable host organism. 当建构时所使用的不等比率的三多肽链提供最优选化产率时,此在调整三多肽片段的相互比例的实施方案中可提供灵活性;然而,当等比例的至少两多肽链的表达导致高产率或是当比率不具特殊意义时,可在一表达载体中插入该两或三多肽链的编码序列。 When the three polypeptide chains ranging from construction of ratios when used with the most preferred of yield, this in adjusting the mutual proportions of the three polypeptide fragments in embodiments provide flexibility; however, when equal proportions of at least two polypeptide Expression chain resulted in high yields or when the ratio does not have special significance, can be inserted into the coding sequence of the two or three polypeptide chains in one expression vector.

[0214] 本发明还涉及包含抗体的免疫缀合物,该抗体缀合至细胞毒性物质(例如化疗剂、毒素(例如细菌、真菌、植物或动物来源的小分子毒素或酶活性毒素,包含其片段和/或变体))或放射性同位素(即放射性缀合物)。 [0214] The present invention further relates to an immunoconjugate comprising an antibody, the antibody is conjugated to a cytotoxic substances (e.g., a chemotherapeutic agent, toxin (e.g. bacterial, fungal, plant or animal origin, small molecule toxins or enzymatically active toxins, which comprises fragments and / or variants thereof)), or a radioactive isotope (i.e., radioactive conjugate).

[0215] 本发明进一步考虑形成于抗体与具有核溶解(nucleolytic)活性的化合物(例如核糖核酸酶或例如脱氧核糖核酸酶的DNA核酸内切酶(DNase))之间的免疫球蛋白。 [0215] The present invention further contemplates the formation of an antibody and (nucleolytic) having a core dissolving the active compound (e.g., a ribonuclease or deoxyribonuclease e.g. a DNA endonuclease (DNase)) between an immunoglobulin.

[0216] 有多种放射性同位素可用于生产辐射缀合(radioconjugated)的人源化vWF抗体,例子包含At211,I131, I125, Y90, Re186, Re188, Sm153,Bi212,P32 及Lu 的放射性同位素。 [0216] There are a variety of radioactive isotopes are available for the production of radiation conjugation (radioconjugated) humanized vWF antibody, examples contain At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32 and radioactive isotopes of Lu.

[0217] 抗体及细胞毒性物质的缀合物可利用多种双功能蛋白质交联剂加以生产,例如3-(2-吡啶二硫代)丙酸-N-琥珀酰亚胺酯(N-succinimidyl-3-(2-pyridyldithio 1)propionate (SPDP))、4_(N-马来酰亚胺基甲基)环己烷羧酸琥珀酰亚胺酯(sue cinimidyl-4- (N-maleimidomethyl) cyclohexane-l-carboxylate)、亚氨基硫烧盐酸盐(iminothiolanedT))、亚氨酸酯(imidoesters)的双官能基衍生物(例如二亚胺代己二酸二甲酯盐酸盐(dimethyladipimidate HCl))、活性酯类(例如双琥珀酰亚胺辛二酸酯(disuccinimidyl suberate))、酸类(例如戊二酸(glutaraldehyde))、叠氮(bis-azido)化合物(例如双(对叠氮苯甲酰基)己二胺(bis(p-azidobenzoyl)hexanediamine))、双重氮(bis-diazonium)衍生物(例如双_(对重氮苯酰基)_乙二胺(bis-(p-diazoniumbe nzoyl)-ethylenediamine))、二异氰酸酯类(diisocyanates)(例如2,6_ 二异氰酸甲苯酯(toluene2,6-diisocyanate))、及双活性氟(bis-active fluorine)化合物(例如1,5_ 二氟-2,4- 二硝基苯(1, 5-dif luoro-2,4-dinitrobenzene)) „ 举例而言,可如Vitetta 等, Science, 238 =1098(1987)中所述般制备蓖麻毒蛋白(ricin)免疫毒素。碳14标记的1-异氰硫苯基-3-甲基二乙烯三胺五乙酸(l-isothiocyanatobenzyl-3-methyldiethylenetri aminepentaacetic acid(MX-DTPA))为例示的放射性核苷酸(radionucleotide)缀合至抗体的螯合剂,见WO 1994/11026。接头可为在细胞中辅助细胞毒性物质药物的释放的“可切割接头”(cleavablelinker),例如可使用酸敏感接头、肽酶敏感性接头、二甲基接头、或含二硫化物接头(Chari 等,Cancer Research,52 : 127-131 (1992))。 [0217] conjugate antibody and cytotoxic substance may utilize a variety of bifunctional protein crosslinking agents be produced, for example, 3- (2-pyridyldithio) propionate succinimide ester -N- (N-succinimidyl -3- (2-pyridyldithio 1) propionate (SPDP)), 4_ (N- maleimido-methyl) cyclohexanecarboxylic acid succinimidyl ester (sue cinimidyl-4- (N-maleimidomethyl) cyclohexane -l-carboxylate), imino sulfur burning hydrochloride (iminothiolanedT)), imidate (imidoesters) bifunctional derivatives (e.g., dimethyl adipate, carbodiimide hydrochloride substituting (dimethyladipimidate HCl) ), active esters (e.g., bis-succinimidyl suberate (disuccinimidyl suberate)), acids (e.g., glutaric acid (glutaraldehyde)), azide (bis-azido) compounds (e.g., bis (p-azido benzene formyl) hexamethylene diamine (bis (p-azidobenzoyl) hexanediamine)), dual nitrogen (bis-diazonium) derivatives (such as bis _ (diazo benzoyl) _ ethylene diamine (bis- (p-diazoniumbe nzoyl) -ethylenediamine)), diisocyanate (diisocyanates) (for example 2,6_ two toluene diisocyanate (toluene2,6-diisocyanate)), and bis-active fluorine (bis-active fluorine) compounds (such 1,5_ difluoro - 2,4-dinitrophenyl (1, 5-dif luoro-2,4-dinitrobenzene)) "for example, and so can be e.g. Vitetta, Science, 238 = 1098 (1987) was prepared as described ricin (ricin) immunotoxin carbon 14-labeled l-phenyl-3-methyl isocyanate sulfur diethylenetriamine pentaacetic acid (l-isothiocyanatobenzyl-3-methyldiethylenetri aminepentaacetic acid (MX-DTPA)) illustrating radioactive nucleoside acid (radionucleotide) antibody conjugated to a chelating agent, see WO 1994/11026. connector can assist in cells release cytotoxic drug substance of "cleavable linker" (cleavablelinker), for example, an acid-sensitive linker, peptidase sensitive linker, dimethyl linker or disulfide-containing linker (Chari et, Cancer Research, 52: 127-131 (1992)).

[0218] 可选地,可例如通过重组技术或肽合成来生产包含人源化vWF抗体及细胞毒性物质的融合蛋白。 [0218] Alternatively, for example, by recombinant techniques or peptide synthesis to produce comprising the humanized vWF antibody and cytotoxic substances fusion protein.

[0219] 在又一实施方案中,可将人源化vWF抗体缀合至“受体”(例如抗生蛋白链菌素), 以用于肿瘤预定位,其中将抗体-受体缀合物给予病人,接着利用清除剂而由循环中移除未结合的缀合物,且接着施用缀合至细胞毒性物质(例如放射性核苷酸)的“配体”(例如抗生物素蛋白)。 [0219] In yet another embodiment, it can be humanized vWF antibody conjugated to a "receptor" (such as anti-streptavidin) for use in tumor pre-positioning, in which the antibody - receptor conjugate is administered patient, followed by the use of scavengers is removed from the circulation of unbound conjugate, and then administered conjugated to cytotoxic substances (e.g. avidin) a "ligand" (e.g. avidin).

[0220] 还可通过将人源化vWF抗体缀合至转化前药(例如肽基化疗剂,见W01981/01145) 为活性抗癌药(见例如WO 198807378及美国专利第4,975,278号)的前药活化酶上,而将本发明的抗体使用于ADEPT中。 [0220] can also be humanized vWF antibody conjugated to a conversion of the prodrug (eg a peptidyl chemotherapeutic agent, see W01981 / 01145) to an active anti-cancer drug (see for example WO 198807378 and U.S. Patent No. 4,975,278 first ) on the prodrug activating enzyme, and the antibodies of the invention in use in ADEPT.

[0221] 可用于ADEPT的免疫缀合物的酶成分包含能够以将其转换成更具活性和细胞毒性的方式作用于前药的任何酶。 Enzyme component [0221] can be used in ADEPT immunoconjugate comprises can be converted into the more active cytotoxic manner and acting on a prodrug of any enzyme.

[0222] 可使用的酶包含但不限于:有助于将含磷酸盐的前药转化成游离药(free drug) 的碱性磷酸酶(phosphatase);有助于将含硫酸盐的前药转化成游离药的芳基硫酸酯酶(arylsulfatase);有助于将无毒5_氟胞嘧啶(5_fluorocytosine)转化成抗癌药5_氟尿嘧啶(5-f luorouracil)的胞嘧啶脱氨酶(cytosine deaminase);有助于将含肽前药转化成游离药的蛋白酶(protease),例如沙雷菌属(serratia)蛋白酶、嗜热菌蛋白酶(thermolysin)、枯草杆菌蛋白酶(subtilisin)、羧肽酶(carboxypeptidase)及组织蛋白酶(cath印sin)(例如组织蛋白酶B及L);可用以转化包含β-氨基酸取代基的前药的D-丙氨酰羧肽酶(D-alanylcarboxyp印tidase);有助于将糖基化(glycosylated) 前药转化成游离药的糖类切割酶(carbohydrate-cleaving enzyme),例如β-半乳糖苷酶(β -galactosidase)及神经氨酸酶(neuraminidase);有助于将以β _内酰胺(β-lactam)衍生的药物转化成游离药的β _内酰胺酶(β-lactamase);及有助于分别将以苯氧乙酰基或苯乙酰基在其胺氮上加以衍生的药物转化成游离药的青霉素酰胺酶,例如为青霉素V酰胺酶及青霉素G酰胺酶。 [0222] Enzymes that may be used include, but are not limited to: contribute to the phosphate-containing prodrugs into free drugs (free drug) alkaline phosphatase (phosphatase); facilitates the conversion of sulfate-containing prodrugs into free drugs aryl sulfatase (arylsulfatase); contribute to toxic 5_ fluorocytosin (5_fluorocytosine) 5_ converted into the anti-cancer drug fluorouracil (5-f luorouracil) cytosine deaminase (cytosine deaminase ); contribute to the peptide-containing prodrugs into free drugs protease (protease), such as Serratia (serratia) protease, thermolysin (thermolysin), subtilisin (subtilisin), carboxypeptidase (carboxypeptidase ) and cathepsin (cath printing sin) (e.g., cathepsins B and L); β- amino acid can be used to convert the substituent containing prodrugs D- alanyl carboxypeptidase (D-alanylcarboxyp printed tidase); help The glycosylation (glycosylated) converted to a prodrug cleaving enzyme drug free sugars (carbohydrate-cleaving enzyme), such as β- galactosidase (β -galactosidase) and neuraminidase (neuraminidase); help to inner to inner β _ lactam (β-lactam) derived drugs into free drugs β _ lactamase (β-lactamase); and help each will phenoxyacetyl or phenylacetyl be on its amine nitrogen into free drugs derivatized drug penicillin amidase, such as penicillin V amidase of penicillin G amide enzymes. 可选地,可利用具有酶活性的抗体(在此领域中还称为“催化性抗体”(abzyme)),将本发明的前药转化成游离活性药(见例如Massey,Nature, 328 :457-458 (1987))。 Alternatively, use of antibodies with enzymatic activity (also referred to in this field "catalytic antibodies" (abzyme)), the prodrugs of the invention into free active drugs (see, e.g., Massey, Nature, 328: 457 -458 (1987)). 可如此处所述来制备抗体-催化性抗体缀合物,以将催化性抗体递送至肿瘤细胞族群。 It can be prepared as described herein antibodies - catalytic antibody conjugate to the catalytic antibodies delivered to the tumor cell population.

[0223] 通过本领域中所熟知的技术,例如上述使用异双功能(heterobifunctional)交 [0223] are well known in the art techniques, such as the above using different bifunctional (heterobifunctional) cross

34联剂,可使酶共价结合至人源化vWF抗体。 34 linking agent, which makes the enzyme is covalently bound to human humanized vWF antibody. 可选地,可利用本领域中所熟知的重组DNA技术, 建构至少包含本发明的抗体的抗原结合区的融合蛋白,该抗原结合区连结至一适当酶的至少功能活性部分(见例如Neuberger 等,Nature, 312 :604-608 (1984))。 Alternatively, utilizing well known in the art of recombinant DNA technology, the construction of fusion proteins comprising at least an antibody of the invention antigen-binding region of the antigen-binding region linked to at least one suitable enzyme functionally active portion (e.g., see Neuberger et , Nature, 312: 604-608 (1984)).

[0224] 此处考虑抗体的其它修饰。 [0224] Other modifications of the antibody are considered here. 举例而言,可将抗体连结至各种非蛋白质类聚合物其中之一,例如聚乙二醇(polyethylene glycol)、聚丙二醇(polypropylene glycol)、 聚氧化烯烃类(polyoxyalkylene)、或聚乙二醇及聚丙二醇的共聚合物;举例而言,还可使抗体埋入于通过例如凝聚(coacervation)技术或界面聚合(例如分别为羟甲基纤维素(hydroxymethyIcellulose)或明胶-微胶囊(gelatin—microcapsule)及聚甲基丙烯酸甲酯(poly-Onethylmethacylate))微胶囊)所制备的微胶囊内,埋入胶体药物递送系统(例如脂质体、白蛋白微球体、微乳液、纳米颗粒及纳米胶囊)或巨乳液中。 For example, the antibody may be linked to one of a variety of non-proteinaceous polymers wherein, for example, polyethylene glycol (polyethylene glycol), polypropylene glycol (polypropylene glycol), polyalkylene oxide (polyoxyalkylene), or polyethylene glycol and polypropylene glycol copolymer; for example, the antibody can be embedded in the example, by agglomeration (coacervation) techniques or by interfacial polymerization (for example, hydroxymethyl cellulose, respectively (hydroxymethyIcellulose) or gelatin - microcapsules (gelatin-microcapsule ) and microcapsules polymethyl methacrylate (poly-Onethylmethacylate)) microcapsules) prepared, embedded in colloidal drug delivery systems (e.g. liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or giant emulsion. 此类技术公开于Remington' sPharmaceutical Sciences (第16 版,由Oslo, Α所编(1980))中。 Such techniques are disclosed in Remington 'sPharmaceutical Sciences (16th edition, the Oslo, Α ed. (1980)) in.

[0225] 还可将此处所公开的人源化vWF抗体配制成免疫脂质体。 [0225] disclosed herein may also be humanized vWF antibody formulated as immunoliposomes. 通过本领域中已知的方法,制备包含抗体的脂质体,例如:Epstein 等,Proc. Natl. Acad. Sci. USA, 82 :3688 (1985); Hwang 等,Proc. Natl Acad. Sci. USA, 77 :4030 (1980);美国专利第4,485,045 及4,544,545 号;及1997年10月23日公开的WO 1997/38731中所述。 By methods known in the art, liposomes comprising an antibody preparation, for example: Epstein et, Proc Natl Acad Sci USA, 82: 3688 (1985); Hwang et, Proc Natl Acad Sci USA....... 77: 4030 (1980); US Patent No. 4,485,045 and 4,544,545; October 23, 1997 as described in WO 1997/38731 published and. 具有增加的循环时间的脂质体则公开于美国专利第5,013,556号中。 Liposomes have an increased circulation time are disclosed in U.S. Patent No. 5,013,556.

[0226] 特别有用的脂质体可利用包含卵磷脂(phosphatidylcholine)、胆固醇、及PEG衍生磷脂酰乙醇胺(PEG-derived phosphatidylethanolamine,PEG-PE)的脂质组合物并通过反相蒸发法加以产生。 [0226] Particularly useful liposomes can be used comprise lecithin (phosphatidylcholine), cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-derived phosphatidylethanolamine, PEG-PE) and lipid composition to be generated by the reverse phase evaporation method. 透过确定孔径的过滤器来挤出脂质体,以产生具有期望直径的脂质体。 Through filters to determine the pore size of extruded liposomes to produce liposomes with the desired diameter. 可透过二硫化物互换反应,将本发明的抗体的Fab'片段缀合至如Martin等,J.Biol. Chem.,257 :286-288(1982)中所述的脂质体。 Via a disulfide interchange reaction may be the Fab 'fragment of an antibody of the present invention as described in Martin et conjugated to, J.Biol Chem, 257:.. 286-288 (1982) described liposomes. 可任选地将化疗剂包含于脂质体内。 Chemotherapeutic agents may optionally be included in the liposomes. 见Gabizon 等,J. National Cancer Inst. ,81(19) : 1484 (1989)。 See Gabizon et, J National Cancer Inst, 81 (19):. 1484 (1989).

[0227] 载体、宿主细胞及重组方法 [0227] vectors, host cells and recombinant methods

[0228] 本发明提供编码具有vWF特异性的人源化抗体及其结合片段的分离核酸、载体、 及包含该核酸的宿主细胞、及生产抗体或其结合片段的重组技术。 [0228] The present invention provides encoding a vWF-specific humanized antibodies and binding fragments isolated nucleic acids, vectors, and host cells comprising the nucleic acid, and the production of antibodies or binding fragments recombinant techniques.

[0229] 就抗体的重组生产而言,可将编码该抗体的核酸分离出来或将其插入可复制载体中,以更进一步进行克隆(扩增DNA)或用于表达。 [0229] For recombinant production of the antibody, the nucleic acid encoding the antibody may be separated out or inserted into a replicable vector for further cloning (amplification of DNA) or for expression. 可利用传统方法(例如利用能够特定地结合至编码抗体的重链及轻链的基因的寡核苷酸探针)分离并测序编码单株抗体的DNA。 Conventional methods can be used (e.g., use can specifically bind to the gene encoding the antibody heavy chain and light chain oligonucleotide probes) isolating and sequencing of monoclonal antibody encoding DNA. 可利用许多载体。 Many vectors are available. 载体组成通常包含但不限于下列其中一种或多种:信号序列、复制起点、 一个或多个的标记基因、增强子元件(enhanced element)、启动子(promoter)、及转录终止序歹lj (transcription-termination sequence)。 Carrier composition typically contains, but is not limited to one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element (enhanced element), a promoter (promoter), and a transcription termination sequence bad lj ( transcription-termination sequence).

[0230] (i)信号序列组成 [0230] (i) the signal sequence of

[0231] 如此处所述的人源化vWF抗体不仅可直接以重组方式生产,还可为具有异源(heterologous)多肽的融合多肽形式,其优选为在成熟蛋白质或多肽的N端(N-terminus) 上具有特定断裂位点。 [0231] As described herein humanized vWF antibody recombinantly not only directly produce, but also for having a heterologous (heterologous) polypeptides form a fusion polypeptide, which is preferably at the N-terminus of the mature protein or polypeptide (N- terminus having a specific cleavage site) on. 异源讯号序列优选地可为宿主细胞所识别及加工(即由信号肽酶加以断裂)。 Heterologous signal sequence may be preferably a host cell recognition and processing (ie be a signal peptidase break). 就未能识别及处理天然人源化vWF抗体信号序列的原核生物宿主细胞而言,可利用由例如碱性磷酸酶、青霉素酶、lpp、或耐热性肠毒素II前导区(enterotoxin IIleader)中所选择出的原核生物信号序列来取代信号序列;就酵母菌分泌而言,天然信号序列可通过例如酵母菌转化酶前导区、α-因子前导区(包含酵母菌属(Saccharomyces)及克鲁维酵母菌属(Kluyveromyces)的α -因子前导区)、酸性磷酸酶前导区、白色念珠菌(C. albicans)葡糖淀粉酶前导区、或WO 1990/13646中所述的信号加以取代。 It fails to recognize and deal with natural humanized vWF antibody signal sequence, prokaryotic host cells, can be used by, for example alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leader (enterotoxin IIleader) in The prokaryotic signal sequence selected to replace the signal sequence; for yeast secretion, the native signal sequence can be, for example yeast invertase leader, α- factor leader (including Saccharomyces (Saccharomyces) and Kluyveromyces Saccharomyces (Kluyveromyces) of α - factor signal 1990/13646 in the leader), acid phosphatase leader, Candida albicans (C. albicans) glucoamylase leader, or WO be substituted. 在哺乳类细胞表达中,可使用哺乳类细胞信号序列以及病毒分泌前导区(viral secretoryleader),例如单纯疱疹病毒(herpes simplex) gD信号。 In mammalian cell expression, you can use mammalian cells as well as viral secretory signal sequence leader (viral secretoryleader), such as herpes simplex virus (herpes simplex) gD signal.

[0232] 此类前体区所用的DNA可在读码框中被连接至编码人源化vWF抗体的DNA。 [0232] Such precursor region of DNA can be used to connect to encoding a humanized vWF antibody DNA in reading frame.

[0233] (ii)复制元件的起点 Start [0233] (ii) Copy elements

[0234] 表达及克隆载体两者皆包含能够使载体在一个或多个的选定宿主细胞中复制的核酸序列。 [0234] Both cloning and expression vectors are capable of containing the nucleic acid sequence of the vector to replicate in one or more selected host cells. 一般而言,在克隆载体中,此序列可使载体独立于宿主染色体DNA而复制,且包含复制的起点或自发性复制序列(autonomously!印licating sequences)。 Generally, in cloning vectors, this sequence allows the vector to replicate independently of the host chromosomal DNA replication, and contains spontaneity origin of replication or replicating sequences (autonomously! India licating sequences). 已熟知许多细菌、酵母、及病毒的此类序列。 Many well known bacteria, yeast, and viruses such sequences. 来自质粒PBR322的复制起点适用于大多数革兰氏阴性菌, 2 μ质粒起点适用于酵母,且各种的病毒起点(SV40、多瘤病毒、腺病毒、VSV或BPV)可用于在哺乳动物细胞的克隆载体。 The origin of replication from the plasmid PBR322 suitable for most Gram-negative bacteria, 2 μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) may be used in mammalian cells The cloning vector. 一般而言,哺乳动物表达载体(一般可使用SV40起源,仅因为其含有早期启动子)并不需要复制元件的起点。 In general, the mammalian expression vectors (the SV40 origin may typically only because it contains the early promoter) origin of replication component is not required.

[0235] (iii)筛选基因元件 [0235] (iii) screening of genetic elements

[0236] 表达及克隆载体可包含筛选基因,还称为筛选标记(selectablemarker)。 [0236] Expression and cloning vectors may contain a selection gene, also known as selection markers (selectablemarker). 代表性筛选基因编码下列蛋白质:(a)赋予对抗生素或其它毒素(例如氨苄青霉素、新霉素(neomycin)、甲氨蝶呤、或四环霉素)的抗性、(b)补充营养缺陷(auxotrophic deficiency)、或(c)供应无法由复合培养基中得到的必须或期望营养,例如编码杆菌(Bacilli)的D-丙胺酸(D-alanine)消旋酶(racemase)的基因。 Representative screening of genes encoding the following proteins: (a) confer resistance to antibiotics or other toxins (such as ampicillin, neomycin (neomycin), methotrexate, or tetracycline) resistance, (b) supplementary nutrition deficiencies (auxotrophic deficiency), or (c) can not be supplied by the complex nutrient medium necessary or desired, e.g., encoding Bacillus (Bacilli) of D- alanine (D-alanine) racemase (racemase) gene.

[0237] 筛选方案的实例为利用药物以阻止宿主细胞的生长。 Examples [0237] Drug screening program for use to inhibit growth of the host cell. 这些成功地以异源基因加以转化(transformed)的细胞生产提供抗药性的蛋白质,因此在选择方案中存活下来。 These succeeded in heterologous gene to be converted (transformed) cell production to provide resistance protein, thus survive the selection scheme. 此种显性选择的实例使用新霉素、霉酚酸(mycophenolic acid)、及潮霉素(hygromycin)等药物。 Examples of such dominant selection use neomycin, mycophenolic acid (mycophenolic acid), and hygromycin (hygromycin) and other drugs.

[0238] 哺乳动物的适当筛选标记的另一例为可识别能够占有人源化vWF抗体编码核酸的细胞者,例如DHFR、胸腺激酶(thymidine kinase)、重金属蛋白质-I及II (metallothionein-I&II)(优选为灵长类重金属蛋白质基因)、腺核苷脱胺酶(adenosine deaminase)、鸟月安■去_ (ornithinedecarboxylase)等。 [0238] Examples of suitable selective marker another mammal to be able to identify the person in possession of a nucleic acid encoding a humanized vWF antibody cells are, for example, DHFR, thymidine kinase (thymidine kinase), heavy metals protein -I and II (metallothionein-I & II) ( preferably a primate heavy metal protein gene), gland nucleoside deaminase (adenosine deaminase), birds Ann month ■ go _ (ornithinedecarboxylase) and so on.

[0239] 举例而言,通过在含有灭杀除癌(methotrexate,Mtx)培养基中培养转化体(transformant),首先识别出以DHFR筛选基因加以转化的细胞。 [0239] For example, by containing in addition to killing cancer (methotrexate, Mtx) medium culturing the transformant (transformant), first to identify the DHFR selection gene to transformed cells. 当使用野生型DHFR时的适当宿主细胞为缺乏DHFR活性的中国仓鼠卵巢细胞株(CH0 cell line)。 When appropriate host cell when wild-type DHFR is lacking DHFR activity Chinese hamster ovary cell line (CH0 cell line).

[0240] 可选地,可通过在含有筛选标记的选择剂(像是胺基糖苷类抗体,例如康霉素0^皿!^(^11)、新霉素、或6418,见美国专利第4,965,199号)的培养基中的细胞生长,选择以编码人源化vWF抗体、野生型DHFR蛋白质、及另一筛选标记(例如胺基糖苷3' -磷酸转移酶(aminoglycoside 3' -phosphotransferase,ΑΡΗ))加以转化或共转化的DNA 序列宿主细胞(尤其是包含内生DHFR的野生型宿主)。 [0240] Alternatively, by U.S. Patent No. containing a selectable marker selection agent (such as aminoglycoside class of antibodies, such as Kang dish neomycin 0 ^! ^ (^ 11), neomycin, or 6418, see No. 4,965,199) in the cell growth medium, chosen to encode the humanized vWF antibody, wild-type DHFR protein, and another selectable marker (e.g., aminoglycoside 3 '- phosphotransferase (aminoglycoside 3' - phosphotransferase, ΑΡΗ)) to be transformed or co-transformed host cell DNA sequences (particularly containing endogenous wild-type host DHFR).

[0241] 用于酵母菌中的适当筛选基因可为存在于酵母菌质粒YRp7中的trpl基因(Stinchcomb 等,Nature, 282 :39 (1979)),trpl 基因为在色胺酸(tryptophen)中缺乏生长能力的酵母菌突变株(例如ATCC 44,076 或PEP4-1. Jones,Genetics,85 : 12 (1997))提供筛选标记。 [0241] The appropriate screening for yeast genes may be present in the yeast plasmid YRp7 the trpl gene (Stinchcomb et, Nature, 282: 39 (1979)), trpl gene is lacking in tryptophan (tryptophen) in ability to grow yeast mutant strain (e.g., ATCC 44,076 or PEP4-1 Jones, Genetics, 85:. 12 (1997)) provides a selection marker. 酵母菌宿主细胞染色体组(genome)中trpl损伤(lesion)的存在接着为通过 The presence of yeast host cell genome (genome) in trpl damage (lesion) is followed by

36在无色胺酸下的生长而检测转化(transformation)提供了一有效环境;同理,可利用带有Leu2基因的已知质粒,补充缺乏Leu2(Leu2-deficeient)的酵母菌株(ATCC 20,622或38, 626)。 36 as a colorless growth under histidine detecting transformation (transformation) provides an effective environment; Similarly, using known plasmids bearing the Leu2 gene supplement the lack Leu2 (Leu2-deficeient) yeast strains (ATCC 20, 622 or 38, 626).

[0242] 此外,可利用衍生自1.6μπι圆形质粒pKDl的载体,进行克鲁维酵母菌的转化;可选地,K. lactis. Van den Berg, Bio/Technology, 8 :135(1990)中有描述大规模生产重组小牛凝乳酶的表达系统。 [0242] In addition, derived from 1.6μπι available circular plasmid pKDl vectors, transformed yeast Kluyveromyces; alternatively, K lactis Van den Berg, Bio / Technology, 8: 135 (1990) in. described large-scale production of recombinant calf chymosin expression systems. 吾人已公开用于以克鲁维酵母菌属的工业菌株来分泌成熟重组人类血清蛋白的稳定多复制数(multi-copy)表达载体,见Fleer等,Bio/Technolog,9 : 968-975(1991)。 It had been publicly used to belong to an industrial yeast strain Kluyveromyces secrete mature recombinant human serum proteins more stable copy number (multi-copy) expression vectors, see Fleer, etc., Bio / Technolog, 9: 968-975 (1991 ).

[0243] (iv)启动子组成 [0243] (iv) promoter component

[0244] 表达及克隆载体通常包含可由宿主生物加以识别的启动子,且可被操作以连结至vWF抗体编码核酸。 [0244] Expression and cloning vectors usually contain a host organism to be identified by a promoter, and can be operated to link to vWF antibody-encoding nucleic acid. 原核生物宿主适用的启动子包含PhoA启动子、β-内酰胺酶及乳糖启动子系统、碱性磷酸酶、色胺酸(trp)启动子系统、及例如tac启动子的嵌合(hybrid)启动子;然而,其它已知的细菌启动子还为合适。 Suitable prokaryotic host PhoA promoter comprises a promoter, β- lactam enzymes lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter system and the tac promoter such as chimeric (hybrid) Start promoter; however, other known bacterial promoters suitable also. 用于细菌系统的启动子还可包含可被操作连结至编码人源化vWF抗体DNA的Shine-Dalgarno (SD)序列。 Promoters for use in bacterial systems also contain operable linked to the humanized vWF antibody encoding DNA of Shine-Dalgarno (SD) sequence.

[0245] 已知真核生物的启动子序列。 [0245] known eukaryotic promoter sequence. 真核生物基因具有位于转录起始的位点上游约25 至30个碱基(bases)的富AT(AT_rich)区,在许多基因的转录起始点上游70至80个碱基所发现的另一序列为CNCAAT(SEQ ID NO :31)区,其中N可为任何核苷酸。 Eukaryotic genes located upstream from the site of transcription initiation is about 25-30 bases (bases) Rich AT (AT_rich) area, another upstream of the transcription initiation in many genes 70-80 bases found sequence CNCAAT (SEQ ID NO: 31) region where N may be any nucleotide. 在大多数真核生物的3'端为AATAAA(SEQ IDNO :32)序列,此序列可能为附加多A尾部(poly A tail)至编码序列的3'端的信号。 In most eukaryotes 3 'end of AATAAA (SEQ IDNO: 32) sequence, this sequence may be additional multi A tail (poly A tail) to the coding sequence of the 3' end of the signal. 可将这些序列适当地插入真核生物的表达载体内,用于酵母菌的适当启动序列(promoting sequences)包含:用于3_磷酸甘油酸激酶(3-phosphoglycerate kinase)或其它糖解酶(例如烯醇酶(enolase)、甘油醛_3_磷酸去S1 BS (glyceraldehyde-3-phosphate dehydrogenase)、己H 激Sl (hexokinase)、 丙Sl 酸去幾Bl (pyruvate decarboxylase)、ΐ舞酸果H ■ Bl (phosphofructokinase)、 葡萄糖-6-磷酸异构酶(glucose-6-phosphate isomerase)、3-磷酸甘油酸变位酶(3-phosphoglycerate mutase) > M # Bl (pyruvate kinase) > HH ΐ^ ^ I^J g| (triosephosphate isomerase)、憐酸葡萄糖异构酶(phosphoglucose isomerase)、及葡萄糖激酶(glucokinase))的启动子。 These sequences can be properly inserted into the eukaryotic expression vector for yeast appropriate start-up sequence (promoting sequences) comprising: means for 3_ phosphoglycerate kinase (3-phosphoglycerate kinase) or other glycolytic enzymes (for example, enolase (enolase), glyceraldehyde phosphate _3_ to S1 BS (glyceraldehyde-3-phosphate dehydrogenase), has stimulated H Sl (hexokinase), propionic acid to several Sl Bl (pyruvate decarboxylase), ΐ dance cranberries H ■ Bl (phosphofructokinase), glucose-6-phosphate isomerase (glucose-6-phosphate isomerase), 3- phosphoglycerate mutase (3-phosphoglycerate mutase)> M # Bl (pyruvate kinase)> HH ΐ ^ ^ I ^ J g | (triosephosphate isomerase), pity acid glucose isomerase (phosphoglucose isomerase), and glucokinase (glucokinase)) promoter.

[0246] 其它为具有由生长条件加以控制的额外转录优势的可诱导启动子的酵母菌启动子可为下列蛋白质的启动子区:乙醇去氢酶2、异细胞色素C、酸性磷酸酶、与氮代谢相关联的降解酶、重金属蛋白质、甘油醛-3-磷酸去氢酶、及负责麦芽糖及半乳糖利用的酶。 [0246] Other forms of yeast growth conditions have to be controlled by the additional advantage of transcription inducible promoter promoter may be the promoter region of these proteins: alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, and degradative enzymes associated with nitrogen metabolism, heavy metal protein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization enzyme. 更进一步说明用于酵母菌表达中的适当载体及启动子,见例如欧洲专利第73,657号。 Further instructions for the appropriate yeast expression vector and promoter, see for example, European Patent No. 73,657. 酵母菌增强子(enhancer)与酵母菌启动子一同使用还相当有利。 Yeast enhancers (enhancer) used in conjunction with yeast promoters still quite favorable.

[0247] 由哺乳类宿主细胞中的载体转录人源化vWF抗体,可例如通过由多瘤(polyoma) 病毒、禽痘(fowlpox)病毒、腺病毒(如腺病毒2)、牛乳突状瘤病毒、禽类肉瘤病毒、细胞巨大病毒、反转录病毒、B型肝炎病毒且最优选为猿猴病毒40(SV40)的染色体组所得到的启动子、异源哺乳类启动子(例如肌动蛋白启动子或免疫球蛋白启动子)、热休克启动子加以控制,条件是此类启动子可兼容于宿主细胞系统。 [0247] by the mammalian host cell transcription vector humanized vWF antibody, for example, by the polyomavirus (polyoma) virus, avian pox (fowlpox), adenovirus (such as Adenovirus 2), bovine papilloma virus , avian sarcoma virus, cytomegalovirus promoter, a retrovirus, B hepatitis virus and most preferably Simian Virus Genome 40 (SV40) of the resulting heterologous mammalian promoters (eg actin promoter or immunoglobulin promoters), heat shock promoter to control the condition that such promoters are compatible with the host cell systems.

[0248] 吾人可便利地取得SV40病毒的早期及晚期启动子,以作为还包含复制的SV40病毒起源的SV40限制片段;吾人可便利地取得人类细胞巨大病毒的立即早期(immediateearly)启动子,以作为HindIII E限制片段。 [0248] 吾人 can conveniently be made early and late promoters of the SV40 virus, as further contains the SV40 viral origin of replication of SV40 restriction fragment; 吾人 may conveniently be made of human cytomegalovirus immediate early (immediateearly) promoter, in order to As a HindIII E restriction fragment. 已揭示利用牛乳突状瘤病毒作为载体而表达哺乳类宿主中的DNA的系统,见例如美国专利第4,419,446号,此系统的修饰系说明于例如美国专利第4,601,978号中。 Have revealed that the use of bovine papilloma virus as a vector to express the DNA of mammalian host systems, see for example, U.S. Patent No. 4,419,446, a modified system of this system is described for example in U.S. Patent No. 4,601,978 in. Reyes等,Nature,297 :598-601 (1982)描述在控制来自单纯疱疹病毒的胸腺激酶(thymidine kinase)启动子的下,将人类β _干扰素cDNA表达于鼠类细胞中。 Reyes et, Nature, 297: 598-601 (1982) describe under control from the herpes simplex virus thymidine kinase (thymidine kinase) promoter, human β _ interferon cDNA expression in murine cells.

[0249] (ν)增强子元素组成 [0249] (ν) enhancer elements

[0250] 通过较高等真核生物而转录编码本发明的人源化vWF抗体的DNA,可利用将增强子序列插入于载体中来提升。 [0250] by higher eukaryotes and transcription of the coding of the present invention is a humanized vWF antibody DNA, can take advantage of the enhancer sequence was inserted in the carrier to ascend. 今已知来自哺乳动物基因(血球蛋白、弹性蛋白酶、蛋白素、 甲型胎儿蛋白及胰岛素)的有用增强子序列;然而,一般来说,吾人可使用来自真核生物细胞病毒的增强子,例子包括:在复制起点的晚期侧上的SV40增强子(bp 100-270)、细胞巨大病毒早期启动子的增强子、在复制起点的晚期侧上的多瘤增强子、及腺病毒增强子。 Now known from mammalian genes (globin, elastase, a protein hormone, alpha-fetoprotein and insulin) useful enhancer sequences; however, in general, 吾人 can use an enhancer from a eukaryotic cell virus, Examples include: SV40 replication origin on the late side of the enhancer (bp 100-270), cytomegalovirus early promoter enhancer on the late side of the replication origin of polyoma enhancer, and adenovirus enhancers. 还说明用于活化真核生物增强子的增强元素。 Also shows enhanced strengthening elements for activation of eukaryotic promoter. 可在人源化vWF抗体编码序列的5'或3'位置将增强子切成载体,但优选为在由启动子起算的5'位点。 It can be humanized vWF antibody coding sequence 5 'or 3' position will cut enhancer carrier, but preferably at the 5 'site of the promoter starting.

[0251] (vi)转录终止组成 [0251] (vi) transcription termination composition

[0252] 用于真核生物宿主细胞(例如酵母菌、真菌、昆虫、植物、动物、人类、或来自其它多细胞生物体的有核细胞)中的表达载体可包含终止转录或稳定mRNA所必须的序列,此类序列一般可得自于真核生物或病毒DNAs或cDNAs的未转译区的5'端(有时为3'端),这些未转译区包含在编码人源化vWF抗体的mRNA的未转译部分中经转录成为多聚腺苷酸化(polyadenylated)片段的核苷酸节段。 [0252] For eukaryotic host cells (e.g., yeast, fungi, insect, plant, animal, human, or from other multicellular organisms nucleated cells) in the expression vector may comprise the termination of transcription or mRNA stability must sequence, the 5 'end (sometimes 3' Such sequences may generally be derived from eukaryotic or viral DNAs or cDNAs untranslated region of the end), which is included in the untranslated region encoding the humanized antibody of vWF mRNA of untranslated transcribed portion becomes polyadenylation (polyadenylated) nucleotide fragment segments. 一有用的转录终止组成为牛生长荷尔蒙多聚腺苷酸化(polyadenylation)区(见例如WO 1994/11026及其中所公开的表达载体)。 One useful transcription termination consisting of bovine growth hormone polyadenylation (polyadenylation) area (see for example WO 1994/11026 and the expression vector disclosed).

[0253] (vii)宿主细胞的筛选及转化 [0253] (vii) Selection and transformation of host cells

[0254] 用于克隆或表达载体中的DNA的适当宿主细胞包含各种的原核生物、酵母菌、 或较高等的原核生物细胞,用于此目的的当适原核生物包含真细菌类(eubacteria)(例如革兰氏阴性或革兰氏阳性生物体,像是肠杆菌科(Enterobacteriaceae)如大肠菌属(Escherichia)(例如大肠杆菌))、肠杆菌属(Enterobacter)、伊文氏杆菌属(Erwinia)、 克留氏菌属(Klebsiella)、变形杆菌数(Proteus)、沙门杆菌属(Salmonella)(例如Salmonella typhimurium)、锯杆菌属(Serratia)(例如Serratiamarcescans)、志贺杆菌属(Shigella)、以及杆菌(Bacilli)如B. subtilis 及B. licheniformis、假单孢菌属(Pseudomonas)如P. aeruginosa及链霉菌(Sti^ptomyces)0大肠杆菌克隆宿主包含大肠杆菌294 (ATCC31,446)、大肠杆菌B、大肠杆菌Xl776 (ATCC 31,537)及大肠杆菌W3110 (ATCC 27,325)。 [0254] suitable host cells for cloning or expression vector DNA containing a variety of prokaryotes, yeast, or equal to the higher of prokaryotes, for this purpose when appropriate prokaryotes contain true bacteria (eubacteria) (such as Gram-negative or Gram-positive organisms, such as Enterobacteriaceae (Enterobacteriaceae) as the genus Escherichia coli (Escherichia) (such as E. coli)), Enterobacter (Enterobacter), Evans genus (Erwinia) , g stay genus (Klebsiella), the number of Proteus (Proteus), Salmonella spp (Salmonella) (such as Salmonella typhimurium), saw the genus (Serratia) (for example Serratiamarcescans), Shigella spp (Shigella), and tuberculosis (Bacilli) such as B. subtilis and B. licheniformis, Pseudomonas (Pseudomonas), such as P. aeruginosa and Streptomyces (Sti ^ ptomyces) 0 contain E. coli cloning host 294 (ATCC31,446), E. coli B E. coli Xl776 (ATCC 31,537) and E. coli W3110 (ATCC 27,325).

[0255] 除了原核生物以外,真核微生物(例如丝状真菌或酵母菌)为适合的人源化vWF抗体编码载体的克隆或表达宿主,可将Saccharomycescerevisiae或常见的面包酵母菌用于表达。 [0255] In addition to prokaryotes, eukaryotic microbes (such as filamentous fungi or yeast) suitable humanized vWF antibody-encoding vector of cloning or expression hosts may be Saccharomycescerevisiae or common bread yeast used for expression. 此外,其它若干属、种、及菌株还为一般可得到且可使用者,例如; Schizosaccharomyces pombe ;克鲁维酵母菌属(Kluyveromyces)宿主,例如K. Iactis, K. fragilis(ATCC12,424) , K. bulgaricus(ATCC 16,045), K. wickeramii(ATCC 24, 178), K. waltii(ATCC 56,500), K. drosophilarum(ATCC 36,906), K. thermotolerans, R K. marxianus ;yarrowia(EP 402,226) ;Pichiapastoris(EP 183,070);念珠菌属(Candida) ;Trichoderma reesia(EP244, 234);粗厚神经孢子菌(Neurospora crassa);Schwanniomyces 例如Schwanniomyces occidentalis ;及丝状真菌,例如红霄菌属(Neurospora)、青霉菌属(Penicillium)、Tolypocladium、及曲菌属(Aspergillus)宿主如A. nidulans及A. niger。 In addition, several other genera, species, and strains are also available for the general user and may, for example; Schizosaccharomyces pombe; Kluyveromyces Saccharomyces (Kluyveromyces) hosts, such as K. Iactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24, 178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, R K. marxianus; yarrowia (EP 402,226); Pichiapastoris (EP 183,070); Candida species (Candida); Trichoderma reesia (EP244, 234); thick nerve spores (Neurospora crassa); Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi, for example, red Xiao genus (Neurospora), green Streptomyces (Penicillium), Tolypocladium, and the song of the genus (Aspergillus) hosts such as A. nidulans and A. niger.

[0256] 用于表达糖化(glycosylated)的人源化vWF抗体的适当宿主细胞可得自于多细胞生物体,真核生物细胞的例子包含植物、昆虫及脊椎动物细胞。 Al. [0256] for the expression of glycated (glycosylated) vWF humanized antibody suitable host cells derived from multicellular organisms, eukaryotic cells contain examples of plants, insects and vertebrate cells. 吾人已识别出许多昆虫病毒(baculoviral)株及变体、以及来自例如Spodoptera frugiperda(毛虫)、Aedes aegypti (岐子)、Aedes albopictus (岐子)>Drosophila melanogaster (果妮)、及Bombyx mori等宿主的对应可容许的昆虫宿主细胞。吾人 has identified a number of insect viruses (baculoviral) strains and variants, as well as from such as Spodoptera frugiperda (caterpillar), Aedes aegypti (Qi son), Aedes albopictus (TOKI child)> corresponds Drosophila melanogaster (fruit Ni), and Bombyx mori and other hosts allowable insect host cells. 有许多种转染(transfection)用的病毒株可资公众利用,例如Autographa californica NPV R Bombyx mori NPV 的Bm_5 株,且可利用此类病毒作为此处根据本发明的病毒,尤其用于Spodoptera frugiperda细胞的转染上。 There are many strains transfection (transfection) can be funded with public use, such as Autographa californica NPV R Bombyx mori NPV of Bm_5 strains of these viruses and can be used as the virus herein according to the present invention, in particular for the Spodoptera frugiperda cell It turns infected.

[0257] 还可利用棉花、玉米、马铃薯、黄豆、牵牛花、蕃茄、及烟草的植物细胞培养作为宿主。 [0257] also use cotton, corn, potato, soybean, petunia, tomato, and tobacco plant cell culture as host.

[0258] 然而,最大的兴趣已经在脊椎动物细胞,且在培养(组织培养)中的脊椎细胞的繁殖已成为例行程序。 [0258] However, interest has been greatest in vertebrate cells, and in culture (tissue culture) reproductive cells spine has become routine. 可使用的哺乳类宿主细胞株的范例包含:ChK2细胞(Chromos Molecular Systems Inc.);由SV40 (COS-7,ATCC CRL1651)加以转化的猴子肾脏CVl 细胞株;人类胚胎肾脏细胞株(293或用以在悬浮培养基中生长而加以次克隆(subcloned)的293 细胞,Graham 等,J. Gen Virol. ,36 :59(1977));幼仓鼠肾脏细胞(BHK,ATCC CCL 10); 人类胚胎肾脏(HEK) 293 细胞(Simmons, 1990 Exp Physiol. 75 :309) ;SP2 脾脏-骨髓瘤融合细胞(Haas 和Wabl,1984,PNAS 81 :7185);中国仓鼠卵巢细胞/_DHFR(CH0,Urlaub 等,Proc. Natl. Acad. Sci. USA, 77 :4216(1980),包含DG44(Urlaub 等,Som. Cell 和Mol. Gen. , 12 :555_566 (1986))及DP12 细胞株);小鼠赛特利(Sertoli)细胞(TM4, Mather, Biol.Reprod. ,23 =243-251(1980));猴子肾脏细胞(CVl ATCC CCL 70);非洲绿猴肾脏细胞(VERO-76, ATCC CRL-1587);人类子宫颈癌细胞(HELA,ATCC CCL 2);犬肾脏细胞(MDCK,ATCC CCL 70);水牛鼠肝脏细胞(BRL3A, ATCC CRL 1442);人类肺脏细胞(W138, ATCC CCL 75);人类肝脏细胞(Hep G2,HB 8065);小鼠乳房瘤细胞(MMT 060562,ATCCCCL 51) ;TRI 细胞(Mather 等,Annals NY Acad. Sci.,383 :44_68 (1982)) ;MRC 5 细胞;FS4 细胞;及人类肝肿瘤细胞株(Hep G2)。 Examples can be used mammalian host cell lines contain: ChK2 cells (Chromos Molecular Systems Inc.); CVl be transformed monkey kidney cell line from the SV40 (COS-7, ATCC CRL1651); human embryonic kidney cell line (293 or . to grow in suspension culture and to be sub-cloned (subcloned) 293 cells, Graham et, J Gen Virol, 36: 59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); human embryonic kidney. (HEK) 293 cells (Simmons, 1990 Exp Physiol 75: 309.); SP2 spleen - fused myeloma cells (Haas and Wabl, 1984, PNAS 81: 7185); Chinese hamster ovary cells / _DHFR (CH0, Urlaub et al, Proc .... Natl Acad Sci USA, 77: 4216 (1980), comprising DG44 (Urlaub et, Som Cell and Mol Gen., 12: 555_566 (1986)..) and DP12 cell lines); mouse Saite Li ( Sertoli) cells (TM4, Mather, Biol.Reprod, 23 = 243-251 (1980.)); monkey kidney cells (CVl ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 70); buffalo rat liver cells (BRL3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells ( Hep G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCCCCL 51); TRI cells (Mather et, Annals NY Acad Sci, 383:.. 44_68 (1982)); MRC 5 cells; FS4 cells; and human liver tumor cell lines (Hep G2). 利用上述用于生产人源化vWF抗体的表达或克隆载体而将宿主细胞转化,并于视需要而调整的传统培养基中培养,以诱发启动子、筛选转化体(transformant)、或放大编码期望序列的基因。 VWF antibodies using the above expression or cloning vectors and transformed host cells used to produce the humanized, conventional medium and adjusted as necessary to the culture to induce the promoter, screening transformant (transformant), or encoding a desired amplification gene sequence.

[0259] 还可使用表达高水平抗体的稳定细胞株,以生产本发明的人源化抗体。 [0259] also express high levels of antibodies using stable cell lines for the production of the present invention is a humanized antibody. 例如,可利用突变的“整合酶(integrase)(例如ACE整合酶)结合穿梭载体(shuttle vector), 以异源基因序列载入基于鼠类人工染色体表达(ACE)平台的高产量哺乳类蛋白质表达系统中,该ACE平台已被设计成含有多个具位点特异性的重组受体位点(recombination acceptor sites) (Lindenbaum 等,(Nucl. Acid Res. 32(21) :el72(2004);美国专利申请案第2003/0119104 Al号及第2006/0246586 Al号)。可利用此系统产生用于表达所筛选的人源化变体及NMC-4嵌合体的稳定细胞株。 For example, you can use mutation "Integration enzyme (integrase) (such as ACE integrase) combined with shuttle vector (shuttle vector), loaded with the gene sequence which is heterologous protein expression based high yield mammalian artificial chromosome expression of murine (ACE) Platform system, the ACE platform has been designed to contain more than one site recombinant receptor points with site-specific (recombination acceptor sites) (Lindenbaum, etc., (Nucl Acid Res 32 (21):.. el72 (2004); United States Patent Application No. 2003/0119104 Al and the second number 2006/0246586 Al). You can use this system to produce screened for expression of humanized variants and NMC-4 chimera of stable cell lines.

[0260] (viii)培养宿主细胞 [0260] (viii) culturing the host cell

[0261] 可将可用于生产人源化vWF抗体的宿主细胞培养于多种培养基中。 [0261] can be used to produce humanized vWF antibody host cells were cultured in a variety of media. 商品化培养基例如Ham' s FlO(Sigma)、最低必需培养基(Minimal EssentialMedium, MEM(Sigma)),RPMI-1640 (Sigma)、及Dulbecco ' s ModifiedEagle ' s Medium ((DMEM),Sigma)均适合用来培养宿主细胞;此外,在例如:Ham等,Meth. Enz. 58 :44(1979) ;Barnes等,Anal. Biochem.,102 :255 (1980);美国专利第4,767,704 号、第4,657,866 号、第4,927,762 号、第4,560,655 号、或第5,122,469 号;WO 1990/03430 ;WO 1987/00195 ;或美国专利第Re. 30,985号中所述的任何培养基,皆可用作宿主细胞的培养基。 Commercially available media such as Ham 's FlO (Sigma), Minimal Essential Medium (Minimal EssentialMedium, MEM (Sigma)), RPMI-1640 (Sigma), and Dulbecco' s ModifiedEagle 's Medium ((DMEM), Sigma) are suitable for culturing the host cells; in addition, for example: Ham et, Meth Enz 58:... 44 (1979); Barnes et, Anal Biochem, 102:. 255 (1980); U.S. Patent No. 4,767,704 WO 1987/00195;;., or U.S. Patent No. Re 30,985, No. 4,657,866, No. 4,927,762, No. 4,560,655, or No. 5,122,469; WO 1990/03430 Any medium according to, can be used as host cells in the culture medium. 可视需要以荷尔蒙和/或其它生长因子(例如胰岛素、运铁蛋白(transferrin)或表皮生长因子)、盐类(例如氯化钠、钙、镁、及磷酸盐)、缓冲液(例如HEPES)、核苷酸(例如腺核苷酸、胸腺嘧啶核苷酸)、抗生素(例如GENTAMYCIN 药物)、微量元素(定义成通常以微莫耳范围的最终浓度存在的无机化合物)、及葡萄糖或等量能源补充至任何这些培养基;还可包含技术人员已知的适当浓度的任何其它必需补充料。 Optionally with hormones and / or other growth factors (such as insulin, transferrin (transferrin) or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES) , nucleotides (such as adenovirus nucleotides, thymine nucleotides), antibiotics (such as GENTAMYCIN drug), trace elements (defined as generally micromolar final concentration of inorganic compounds present in the range), and glucose or an equivalent amount energy supplement to any of these media; also contains an appropriate concentration techniques known any other necessary supplementary material. 可对所筛选的表达用宿主细胞使用各种培养条件,例如温度、pH。 It may be screened for expression of the host cell culture using a variety of conditions, such as temperature, pH.

[0262] (ix)人源化vWF抗体的纯化 Purification of vWF antibody [0262] (ix) humanized

[0263] 当使用重组技术时,抗体可产生于胞内或间膜区域(periplasmicspace)中、或者直接分泌至培养基内。 [0263] When using recombinant techniques, the antibody can be produced in the intracellular membrane area or room (periplasmicspace), or directly secreted into the culture medium. 若抗体产生于胞内,第一步可通过例如离心或超过滤而移除宿主细胞或细胞溶解片段的微粒碎片。 Where the antibody produced in the intracellular, the first step by, for example centrifugation or ultra-filtration and removal of host cell or cell lysate particulate debris fragments. Carter等,Bio/Technology,10 : 163-167 (1992)描述分泌至大肠杆菌间膜区域的抗体的分离程序;要言的,于醋酸钠(PH 3.5)、EDTA、及苯甲磺酰基氟(phenylmethylsulfonylf luoride (PMSF))存在下将细胞糊(cell paste)解冻约30 分钟,可通过离心而移除细胞碎片。 Carter et al., Bio / Technology, 10: 163-167 (1992) described the secretion of the antibody to E. coli membrane region between the separation procedures; to words, and sodium acetate (PH 3.5), EDTA, and phenylmethylsulfonyl fluoride ( phenylmethylsulfonylf luoride (PMSF)) in the presence of cell paste (cell paste) for about 30 minutes to thaw, cell debris can be removed by centrifugation. 在抗体分泌至培养基内的情况下,通常首先浓缩来自此类系统的上澄液,包含利用商品化蛋白质浓缩过滤器,例如AMICON或MILLIP0RE PELLIC0N超过滤单元。 In the antibody secreted into the culture medium, the usual solution is first concentrated on Cheng from such systems, including the use of a commercially available protein concentrate filter, for example AMICON or MILLIP0RE PELLIC0N ultrafiltration unit. 可将如苯甲磺酰基氟(phenylmethylsulfonylf luoride (PMSF))的蛋白酶抑制剂包含于前述步骤任一者中以抑制蛋白质分解(proteolysis),且可包含抗生素以防止偶发性污染扩大。 Sulfonyl groups such as benzyl may be fluoro (phenylmethylsulfonylf luoride (PMSF)) protease inhibitor containing any one of the foregoing steps to inhibit protein degradation (proteolysis), and may contain an antibiotic to prevent accidental contamination expand.

[0264] 可利用例如氢氧磷灰石层析法(hydroxylapatite chromatography)、凝胶电泳法、透析法、及亲和层析法而纯化出由细胞制备而来的抗体成分,其中亲和层析法为优选的层析技术。 [0264] can be used, for example hydroxyapatite chromatography (hydroxylapatite chromatography), gel electrophoresis, dialysis, and affinity chromatography and purified from the cells prepared from the antibody component, wherein the affinity chromatography France is the preferred chromatographic techniques. 蛋白质A作为亲和力配位体(Iigand)的适合性系取决于存在于抗体中的任何免疫球蛋白Fc结构域的物种及同种型(isotype)。 Protein A as an affinity ligand (Iigand) system suitability depends on the presence in the antibody of any immunoglobulin Fc domain species and isotypes (isotype). 可利用蛋白质A来纯化基于人类Y 1> Y 2、或Y 4 重链的抗体(Lindmark 等,J. Immunol. Meth, 62 : 1-13 (1983));可利用蛋白质G作为小鼠抗体同种型及人类γ 3(Guss等,EMBO J.,5 : 15671575 (1986))。 A purified protein can be used based on human Y 1> Y 2, or Y 4 heavy chains (Lindmark et, J Immunol Meth, 62:.. 1-13 (1983)); G protein can be used as a mouse antibody with isoform and human γ 3 (Guss et, EMBO J., 5: 15671575 (1986)). 亲和力配位体黏附的基质(matrix)可为洋菜糖(agarose),但还可使用其它基质。 Adhesion affinity ligand matrix (matrix) may be a sugar agar (agarose), but also other matrices. 机械上稳定的基质,例如可控孔径玻璃(controlled-pore glass)或聚(苯乙烯-二乙烯基苯) (polyktyrenedivinyl-benzene)),容许比使用洋菜糖所能达到者更快的流速及更短的处理时间。 Mechanically stable matrices such as controlled pore glass (controlled-pore glass) or poly (styrene - divinylbenzene) (polyktyrenedivinyl-benzene)), to allow the use of agar sugar than can be achieved by faster flow rates and shorter processing time. 在抗体包含CH3结构域的情形中,可将BAKERB0ND ABX 树脂(JT Baker, Phillipsburg, NJ)用于纯化;还可根据待回收的抗体而使用其它的蛋白质纯化技术, 例如离子交换管柱分馏、乙醇沉淀、逆相HPLC、硅土层析(chromatography on silica)、 肝素(heparin) SEPHAR0SE层析、阳离子或阴离子交换树脂层析(例如聚天门冬胺酸(polyasparticacid)管柱)、色层焦集(chromatofocusing)、SDS-PAGE、及硫酸铵沉淀法。 In the case of the antibody comprises a CH3 domain may be BAKERB0ND ABX resin (JT Baker, Phillipsburg, NJ) for purification; also based on antibody to be recovered using other protein purification techniques such as ion-exchange column fractionation, ethanol precipitation, reverse phase HPLC, silica chromatography (chromatography on silica), heparin (heparin) SEPHAR0SE chromatography, cation or anion exchange resin chromatography (e.g., poly aspartate (polyasparticacid) column), the focus set colored layer ( chromatofocusing), SDS-PAGE, and ammonium sulfate precipitation.

[0265] 药物制剂 [0265] The pharmaceutical formulation

[0266] 兹提供包含具有vWF特异性的人源化抗体的药物配方。 [0266] vWF is hereby provided having specificity for human comprising the humanized antibody pharmaceutical formulations. 可通过混合具有期望纯度的抗体与非必须的药物上可接受的载体、赋形剂、或安定剂(Remington' s 40Pharmaceutical Sciences 16th edition,Osol,A. Ed. (1980)),而制备具有冷冻干燥配方或水溶液形式的人源化vWF抗体的储存用配方。 Antibodies with non-drug may be having the desired purity by mixing acceptable carriers, excipients, or stabilizers (Remington 's 40Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), and preparing a freeze- dried formulation or in the form of an aqueous solution of a humanized vWF antibody formulations stored. 可接受的载体、赋形剂、或安定剂为在所使用的剂量及浓度下对接受者不具毒性者,且其含有:缓冲剂,例如磷酸盐、柠檬酸盐、及其它有机酸;抗氧化剂,包含抗坏血酸、甲硫胺酸(methionine);防腐剂,例如十八烧基二甲基节基氯化铵(octadecyldimethylbenzyl ammoniumchloride)、氯化六轻季铵(hexamethonium chloride)、轻基氯苯胺(benzalkonium chloride)、氯化苯胺松宁(benzethonium chloride)、酷(phenol)、丁酉享(butyl alcohol)或节酉享(benzyl alcohol)、 如对羟基苯甲酸甲酯(methyl paraben)或对羟基苯甲酸丙酯(propyl paraben)的烷基对羟基苯甲酸酯(alkyl parabens)、儿茶酚(catechol)、间苯二酚(resorcinol)、环己醇(cyclohexanol)、3-戊醇(3-pentanol)、及间甲苯酚(m-cresol);低分量(小于约10 个残基)多肽;蛋白质,例如血清蛋白、白明胶或免疫球蛋白;亲水性聚合物,例如聚乙烯四氢吡咯酮(polyvinylpyrrolidone);氨基酸,例如甘胺酸(glycine)、麸酰胺(glutamine)、天门冬酰胺酸(asparagine)、组胺酸(histidine)、精胺酸(arginine)或离胺酸(lysine); 单糖、双糖及其它包含葡萄糖、甘露糖(marmose)、或糊精(dextrins)的碳水化合物;螯合剂,例如EDTA ;糖类,例如蔗糖、甘露醇(marmitol)、海藻糖(trehalose)或山梨醇(sorbitol);盐类形成相对离子(coimter-ion),例如钠;金属错合物(例如锌-蛋白质错合物);和/或非离子性界面活性剂,例如TWEEN、PLUR0NICS、或聚乙二醇(polyethylene glycol,PEG) 0优选的冷冻干燥人源化vWF配方说明于WO 1997/04801中,其以参考数据并入于此。 Acceptable carrier, excipient, or stabilizer is at the dosages and concentrations used were not toxic to the recipient, and which comprises: a buffer, such as phosphate, citrate, and other organic acids; antioxidants containing ascorbic acid, methionine (methionine); preservatives, for example eighteen burn dimethyl ammonium chloride Festival (octadecyldimethylbenzyl ammoniumchloride), six light quaternary ammonium chloride (hexamethonium chloride), light base chloroaniline (benzalkonium chloride), chlorinated aniline Songning (benzethonium chloride), Cool (phenol), Ding enjoy (butyl alcohol) or Section unitary share (benzyl alcohol), such as methylparaben (methyl paraben) or hydroxy propyl p ester (propyl paraben) alkyl parabens (alkyl parabens), catechol (catechol), resorcinol (resorcinol), cyclohexanol (cyclohexanol), 3- pentanol (3-pentanol) , and m-cresol (m-cresol); low weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone tetrahydro- ( polyvinylpyrrolidone); amino acids such as glycine (glycine), bran amide (glutamine), asparagine acid (asparagine), histidine (histidine), arginine (arginine) or lysine (lysine); monosaccharide , disaccharides, and other comprising glucose, mannose (marmose), or dextrin (dextrins) carbohydrates; chelating agents such as EDTA; sugars such as sucrose, mannitol (marmitol), trehalose (trehalose) or sorbitol (sorbitol); salts formed relative ion (coimter-ion), such as sodium; metal complexes (eg zinc - protein complexes); and / or non-ionic surfactants, such as TWEEN, PLUR0NICS, or polyethylene glycols (polyethylene glycol, PEG) 0 A preferred lyophilized formulation of humanized vWF described in WO 1997/04801, which is incorporated herein by reference data.

[0267] 此处的配方还可包含一种以上特定的治疗症状所必须的活性化合物,优选为具有不会彼此产生不利影响的互补活性者;可选地,或此外,组成还可包含化疗剂、细胞毒性剂、细胞介素(cytokine)、生长抑制剂、抗激素剂、人源化vWF药物、抗血管新生剂(anti-angiogenic agent)、和/或保心药(cardioprotectant),此类分子适合以能够针对需要目的发挥效用的量的组合存在。 [0267] The formulation herein may also contain one or more symptoms of a particular treatment necessary active compounds, each preferably having no adverse effects are complementary activities; alternatively, or in addition, the composition may further comprise a chemotherapeutic agent , cytotoxic agents, cell interleukin (cytokine), growth inhibitors, anti-hormonal agents, humanized vWF drugs, anti-angiogenic agents (anti-angiogenic agent), and / or heart-protecting drugs (cardioprotectant), such molecules adapted to be able to be effective needs to aim for a combined amount of presence.

[0268] 还可将有效成分网罗在通过例如凝块(coacervation)技术或接口聚合(例如分别为羟甲基纤维素(hydroxymethylcellulose)或明胶-微胶囊(gelatin-microcapsule) 及聚甲基丙烯酸甲酯(poly-Onethylmethacylate))微胶囊)而在胶体药物传递系统(例如脂质粒、卵蛋白微球体、微乳液、纳米粒子及纳米胶囊)或巨乳液中加以制备的微胶囊内。 [0268] The active ingredient may also be recruited by e.g. clot (coacervation) or interface polymerization techniques (e.g., hydroxymethyl cellulose, respectively (hydroxymethylcellulose) or gelatin - microcapsules (gelatin-microcapsule) and polymethyl methacrylate (poly-Onethylmethacylate)) microcapsules) in colloidal drug delivery systems (e.g. liposomes grain, egg albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions microcapsules to be prepared. 此类技术公开于Remington' s Pharmaceutical Sciences (第16 版,由Oslo,Α.所编着(1980))的专书中。 Such techniques are disclosed in Remington 's Pharmaceutical Sciences (16th edition, the Oslo, Α. The ed (1980)) the special book.

[0269] 可制备持续释放(sustained-release)制剂。 [0269] may be prepared sustained release (sustained-release) formulations. 持续释放制剂的例子包括含有抗体的固态疏水性聚合物的半透性基质,其中基质用成型物品的形式存在,例如薄膜或微胶囊。 Examples of sustained-release preparations include antibody containing solid hydrophobic semipermeable polymer matrices, wherein a matrix form of shaped articles, e.g., films, or microcapsules. 持续释放基质的例子包含聚酯、水凝胶(例如聚(甲基丙烯酸2-羟乙酯)或聚乙烯醇)、聚乳酸交酯(polylactide)(美国专利第3,773,919号)、L-麸胺酸及-乙基-L-麸胺酸的共聚物、非可降解的乙烯-乙酸乙烯酯共聚物、可降解的乳酸__甘醇酸(lacticacid-glycolic acid)共聚物(例如LUPRON DEPO(由乳酸__甘醇酸共聚物及柳菩林(leuprolide acetate)所组成的可注射微球体)及聚_D_(-) _3_羟丁酸)。 Examples of sustained-release matrices comprise polyesters, hydrogels (e.g., poly (methacrylate 2-hydroxyethyl methacrylate) or polyvinyl alcohol), poly lactide (polylactide) (U.S. Patent No. 3,773,919), L- glutamate and - ethyl -L- glutamic acid copolymers, non-degradable ethylene - vinyl acetate copolymer, degradable lactic acid glycolic __ (lacticacid-glycolic acid) copolymers (e.g. LUPRON DEPO (copolymer of lactic acid and glycolic __ Liu Pu Lin (leuprolide acetate) consisting of injectable microspheres), and poly _D _ (-) _3_ hydroxybutyrate).

[0270] 待用于活体内(in vivo)服用的配方必须为无菌,此系透过经由无菌过滤膜的过滤加以完成。 [0270] to be used in vivo (in vivo) administered formulations must be sterile, this system through filtration through sterile filtration membranes to be completed. [0271] 利用人源化vWF抗体的治疗 [0271] The use of a humanized vWF antibody therapy

[0272] 可利用人源化vWF抗体或其结合片段来治疗各种的vWF相关疾病或紊乱,例示的疾病或病症包含血栓性疾病或紊乱。 [0272] can utilize humanized vWF antibody or binding fragment thereof to treat a variety of vWF-related disease or disorder, disease or condition illustrated contain thrombotic disease or disorder. 血栓性疾病或紊乱可包含心血管疾病或例如缺血性卒中的脑血管疾病。 Thrombotic diseases or disorders may include cardiovascular diseases such as ischemic stroke, or cerebrovascular disease. 在某些实施方案中,心血管疾病可为动脉粥状硬化症、再狭窄、心绞痛、急性心肌梗塞、急性冠状动脉综合症、或与糖尿病相关联的心血管异常;在某些实施方案中, 血栓性疾病或紊乱为血管发炎、静脉血栓、镰刀形细胞病、异种移植排斥、外周血管病、血栓性血小板减少性紫癜症、囊性纤维化、血管性痴呆、雷诺病、类风湿性关节炎、或糖尿病;在某些实施方案中,脑血管疾病包含由大脑动脉梗塞及小腔隙梗塞两者所引起的缺血性卒中以及血管性痴呆。 In certain embodiments, the cardiovascular disease may be atherosclerotic sclerosis, restenosis, cardiovascular angina, acute myocardial infarction, acute coronary syndrome, or are associated with abnormal diabetes; In certain embodiments, thrombotic disease or disorder is inflammation of blood vessels, venous thrombosis, sickle cell disease, xenograft rejection, peripheral vascular disease, thrombotic thrombocytopenic purpura disease, cystic fibrosis, vascular dementia, Raynaud's disease, rheumatoid arthritis , or diabetes; in some embodiments, cerebrovascular diseases comprising ischemic stroke and vascular dementia by the cerebral artery infarction lacunar infarct both small and caused. 还可利用人源化vWF抗体来预防再发性中风或由脑血管发炎所引发的中风起始。 Also use humanized vWF antibody to prevent recurrent stroke or stroke starting from the brain caused by inflammation of blood vessels. 在急性冠状动脉综合症的情况中,人源化vWF抗体或其结合片段尤其适合用来治疗ST段(ST-segment)诊断的受试者;还可将人源化vWF抗体或其结合片段用于术后治疗, 以预防血块形成。 In the case of acute coronary syndrome, a humanized vWF antibody or binding fragment thereof is particularly suitable for the treatment of ST segment (ST-segment) diagnosis of a subject; also humanized vWF antibody or binding fragment postoperative treatment to prevent clot formation.

[0273] 再者,可利用体内诊断测定法来评估vWF的过表达或扩增,例如通过施用结合待测分子且以可检测的标记(例如放射性同位素)加以标记的分子(例如抗体),并在外部扫描病患以定位该标签。 [0273] Further, in vivo diagnostic assays can be used to assess the vWF overexpression or amplification, e.g., with a detectable label and (e.g. a radioisotope) to a molecule (e.g. an antibody) labeled analyte binding molecule by administering, and externally scanning the patient to locate the label.

[0274] 在某些实施方案中,可将包含与细胞毒性物质缀合的人源化vWF抗体的免疫缀合物给予病患;优选免疫缀合物和/或其结合的人源化vWF抗体被细胞内化,从而提升了免疫缀合物在杀死其所结合的癌细胞上的治疗效用。 [0274] In certain embodiments, it may comprise a cytotoxic substance-conjugated humanized vWF antibody immunoconjugate given patient; and preferably immunoconjugate or binding human / humanized vWF antibody It is internalized by the cell, thereby increasing the immunoconjugate in killing the cancer cells to which it binds therapeutic utility. 在一优选实施方案中,细胞毒性物质(包含例如美登醇(maytansinoid)、卡奇霉素、核糖核酸酶及DNA内切酶)靶定或干扰癌细胞内的核酸;在另一实施方案中,细胞毒性物质(例如紫杉烷(taxane) 或埃博霉素(印othilone))可靶定或干扰癌细胞内的微管及依赖微管的有丝分裂(microtubule-dependent mitosis)0 In a preferred embodiment, the cytotoxic substance (containing, for example maytansinol (maytansinoid), calicheamicin, inside DNA endonuclease enzyme RNA) targeting cancer cells or interfering nucleic acid; and in another embodiment, , cytotoxic substances (such as taxanes (taxane) or epothilones (India othilone)) may be targeted or microtubule-dependent interference and microtubules in cancer cells in mitosis (microtubule-dependent mitosis) 0

[0275] 可根据已知的方法,例如以团注方式或通过一段时间内的连续式输注的静脉内施用,通过肌肉内、腹膜内、脑脊髓内、皮下、关节内、滑膜内、鞘内、口服、局部、或吸入等途径, 将人源化vWF抗体或免疫缀合物施用于病患。 [0275] According to known methods, for example by bolus injection or by continuous intravenous infusion administered over time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, articular, intrasynovial, intrathecal, oral, topical, or inhalation route, humanized vWF antibody or immunoconjugate is administered to patients. 抗体的静脉内、腹膜内、或皮下给药为优选; 特别优选腹膜内或皮下途径。 The antibody intravenously, intraperitoneally, subcutaneously, or preferably; particularly preferably intraperitoneal or subcutaneous routes. 优选的给药计划可为针对急性疾病单一剂量,而针对慢性疾病则约每三至四周一次,取决于接受治疗的特定哺乳动物、抗体类型、及行医者熟知的其它因素;然而,此处可采用其它的给药计划。 The preferred dosing schedule for a single dose for acute diseases, chronic diseases and for the once every about three to four weeks, depending on the particular mammal being treated, the type of antibody, and other factors known to medical practitioners; however, here Other uses dosing schedule.

[0276] 可将其它治疗方针结合人源化vWF抗体的施用方式。 [0276] can be combined with other therapeutic approach mode of administration of humanized vWF antibody. 结合的给药方式包含同时用药(co-administration)、利用各自制剂或单一药物制剂、及以任一次序连续给药,其中优选两种(或全部)活性剂同时展现其生物活性的时段。 The mode of administration comprises simultaneous administration of the combination (co-administration), use of their formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably both (or all) active agents simultaneously exhibit its biological activity period.

[0277] 在一实施方案中,治疗方式可包括人源化抗vWF抗体与纤维蛋白溶解剂(fibrinolytic agent)和/或抗血小板剂的结合施用,纤维蛋白溶解剂的例子如阿替普酶(alt印Iase)、去氨普酶(desmot印Iase)或微纤微蛋白溶酶(microplasmin),抗血小板剂的例子如用于治疗由心肌梗塞或脑梗塞或其它脑血管异常所诱发的局部缺血的阿司匹林、 双嘧达莫(dipyridamol)或氯吡格雷(clopidogrel)。 [0277] In one embodiment, the treatment may include a humanized anti-vWF antibody and fibrinolytic agents (fibrinolytic agent), and / or antiplatelet agents administered in combination, examples of fibrinolytic agents such as alteplase ( alt India Iase), Desmoteplase (desmot India Iase) or a micro-fiber micro-plasmin (microplasmin), antiplatelet agents such as for example the treatment of myocardial infarction or cerebral infarction or other cerebrovascular abnormalities induced by partial lack blood aspirin, dipyridamole (dipyridamol) or clopidogrel (clopidogrel).

[0278] 还可期望结合人源化vWF抗体的施用及针对另一肿瘤相关抗原的抗体的施用。 [0278] may also be combined with a desired humanized antibodies against vWF administration and administration of other tumor-associated antigen antibody.

[0279] 在一实施方案中,本发明的治疗方式涉及人源化vWF抗体、哺乳动物中的免疫功能的一个或多个调节剂(例如细胞因子)、以及化疗剂或生长抑制剂的组合给药,包括不同 [0279] In one embodiment, the treatment of the present invention relates to a humanized vWF antibody, a mammal's immune function or more modifiers (such as cytokines), and combinations of chemotherapeutic agents or growth inhibitors to drugs, including different

42化疗剂的鸡尾酒疗法的组合用药;优选的化疗剂包含紫杉烷(例如帕尼特西(paclitaxel) 及多西紫杉醇(docetaxel))和/或蒽环抗生素。 42 chemotherapeutic agents in combination HAART medication; preferred chemotherapeutic agent comprises a taxane (such as Trapani Teixido (paclitaxel) and docetaxel (docetaxel)) and / or anthracycline antibiotics. 可根据生产厂的用法说明或由熟悉的行医者依经验判定,而使用此类化疗剂的制剂及剂量计划;此类化疗剂的制剂及剂量计划还说明于Chemotherapy Service,编,Μ C. Perry,Williams & ffilkins,Baltimore,Md. (1992)。 Can explain or practice under the terms of the familiar experience of judgment, and the use of such chemotherapeutic agents and dose formulations according to the manufacturer's usage plan; preparation of such chemotherapeutic agents and dose plan also illustrated in Chemotherapy Service, edited, Μ C. Perry, Williams & ffilkins, Baltimore, Md. (1992).

[0280] 可以已知剂量的抗激素化合物,将人源化vWF抗体与此类分子相结合。 [0280] anti-hormonal compounds known dose of humanized vWF antibody molecule with such a combination. 抗激素化合物的例子包含:例如他莫昔芬(tamoxifen)的抗雌激素化合物或例如阿那曲唑(anastrozole)的芳香族酶抑制剂;例如奥那司酮(onapristone)的抗黄体酮(anti-progesterone)(见EP 616 812);或例如氟他胺(flutamide)的抗雄性素(anti-androgen)。 Examples of anti-hormonal compound comprising: for example, tamoxifen (tamoxifen) or an anti-estrogen compounds such as anastrozole (anastrozole) aromatic inhibitor; such as onapristone (onapristone) anti-progesterone (anti- progesterone) (see EP 616 812); or such as flutamide (flutamide) anti-androgen (anti-androgen). 若欲治疗的癌症为非激素依赖性的,病患先前可能已接受过抗激素疗法,且在癌症变成非激素依赖性之后,可对病患施用人源化vWF抗体(及如此处所述的其它非必须剂方)。 Ruoyu non-hormone-dependent cancer therapy, the patient may have previously received anti-hormonal therapy and, after the cancer becomes hormone-independent, can be administered to patients with a humanized vWF antibody (and as described herein Other non-essential agent side).

[0281] 就疾病的预防或治疗而言,抗体的适当剂量将取决于如上定义的待治疗疾病的类型、疾病的严重性及病程(不论施用抗体系为预防或治疗的目的)、先前的治疗、病患的临床病史与对抗体的反应、及主治医师的判断。 [0281] For the prevention or treatment of disease, the appropriate dosage of antibody will depend on the severity and course of the disease to be treated, as defined above, the type of disease (whether for the purpose of administering an anti-system prevention or treatment), previous treatments , the patient's clinical history and response to the antibody, and the discretion of the attending physician. 可将抗体一次或在一系列治疗中适当地对病患施用。 Antibodies can be at once or over a series of treatments for patients administered properly. 根据疾病的类型与严重性以及抗体的清除速率,对病患施用的起始候选剂量为约1 μ g/kg〜15mg/kg (例如0. l_20mg/kg)的抗体,不论例如通过一次或多次的分别给药或通过连续输注。 Depending on the type and severity of the disease and the rate of clearance of the antibody, the starting dose in patients administered the candidate is about 1 μ g / kg~15mg / kg (for example 0. l_20mg / kg) of antibody, regardless example, by one or more times, respectively, or administered by continuous infusion. 一般的日剂量范围可自约1 μ g/kg〜100mg/kg或以上,依据上述的因子而定。 A typical daily dosage may range from about 1 μ g / kg~100mg / kg or more, according to the above factors may be. 根据状况,为了若干天或更久的重复给药,可持续治疗至产生疾病症状的期望抑制效果为止。 Depending on the situation, in order to repeat the administration several days or longer, sustainable treatment to produce the desired effect of suppressing disease symptoms so far.

[0282] 人源化vWF抗体的优选剂量可在自约0. 05mg/kg〜约10mg/kg的范围内,因此,可对病患施用约0. 3mg/kg、0. 5mg/kg、2. 0mg/kg、4. 0mg/kg、或lOmg/kg(或其组合)的一个或多个的剂量。 [0282] The humanized vWF antibody preferred dosage may be from the range of about 0. 05mg / kg~ about 10mg / kg range, therefore, it can be administered to patients about 0. 3mg / kg, 0. 5mg / kg, 2 . 0mg / kg, 4. 0mg / kg, or lOmg / kg (or combinations thereof) one or more doses. 可间歇性地施用此剂量,例如每周、每两周、每三周或每四周(例如使病患接受自约2至约20 (如6)剂量的人源化vWF抗体);可在开始时施用较高起始剂量(loading dose),接着施用一次或多次较低剂量。 May be intermittently administered this dose, such as weekly, biweekly, every three weeks or every four weeks (for example to make patients received from about 2 to about 20 (eg 6) doses of humanized vWF antibody); in the beginning When high initial dose is administered (loading dose), followed by one or more lower doses administered. 例示性的剂量方案包含施用人源化vWF抗体或其结合片段约4mg/kg的起始剂量,接着维持每周约2mg/kg的剂量;然而,还可使用其它剂量方案。 Exemplary dosage regimen comprising administering a humanized vWF antibody or binding fragment of about 4mg / kg initial dose, followed by weekly to maintain about 2mg / kg dose; however, other dosage regimens may also be used. 此种疗法的进行可通过传统技术及方法加以监测。 Such therapy can be monitored by conventional techniques and methods.

[0283] 在评估如此处所述的人源化vWF抗体或其结合片段在狒狒上的有效性及安全性的研究中,已出人意表地发现此种抗体可以极低的剂量(例如在低μ g/kg的范围内)有效地预防(例如降低、减轻、或改善)体内的血小板凝集,此为治疗vWF介导的疾病或紊乱的未预期且前所未有的结果。 [0283] In assessing this at the humanized vWF antibody or binding studies of the efficacy and safety clips on baboons, it has been unexpectedly found that such antibodies may be very low doses (for example, low within μ g / kg range) effectively prevent (e.g. decrease, reduce, or ameliorate) in vivo platelet aggregation, for the treatment of this vWF mediated disease or disorder and an unprecedented result not expected. 在这些浓度下,皆未观察到出血的临床症状,除了小伤口的出血增加以外(例如在切口出血测试中所测量到的模板出血时间延长和/或出血量);甚至更令人惊讶的是,在自EDiqq的约1至250倍的剂量下,还未观察到明显的临床出血迹象,尽管有观察到小伤口的出血增加。 At these concentrations, garnered observed clinical symptoms of bleeding, except for a small increase in bleeding wounds (such as bleeding in the notch in the measured test template prolonged bleeding and / or bleeding); even more surprising is that in from about 1-250 times the dose EDiqq and we have not observed significant clinical signs of bleeding, although there was observed a small increase in bleeding wounds. 因此,如此处所述的人源化vWF抗体或其结合片段似乎可有效地治疗人类的vWF介导的疾病或紊乱。 Therefore, people as described herein humanized vWF antibody or binding fragment appears to be effective in the treatment of human vWF mediated disease or disorder.

[0284] 因此,可以范围自约0.001至约100mg/kg的治疗有效量,将如此处所述的人源化vWF抗体或其结合片段施用于一受试者(优选为人类);优选的治疗有效量范围为自约0. 002至约20mg/kg,更优选自约0. 002至约10mg/kg,特别是自约0. 002至约0. 4mg/kg、更特别自约0. 005至约0. 2mg/kg,最优选是施用受试者自约0. 01至约0. lmg/kg的治疗有效量,优选的受试者为人类。 [0284] Therefore, it can range from about 0.001 to about 100mg / kg of a therapeutically effective amount, thus at the humanized vWF antibody or binding fragment thereof is administered to a subject (preferably a human); the preferred treatment effective amount in the range of from about 0.002 to about 20mg / kg, more preferably from about 0.002 to about 10mg / kg, in particular from about 0.002 to about 0. 4mg / kg, more particularly from about 0.005 to about 0. 2mg / kg, and most preferably is administered to the subject from about 0.01 to about 0. lmg / kg therapeutically effective amount, preferably the subject is human. 可以一或多个有效治疗剂量,将治疗有效量人源化vWF抗体或其结合片段施用于一受试者,所施用的有效治疗剂量通常不足以引发明显的临床出血迹象, 但却足以抑制血小板凝集;换言之,可施用有效治疗剂量而不会引发明显的临床出血迹象(例如不会产生出血的临床症状,除了小伤口的出血增加以外)。 May be one or more of the therapeutically effective dose, a therapeutically effective amount of a humanized vWF antibody or binding fragment is administered to a subject, a therapeutically effective amount administered is usually enough to trigger significant clinical signs of bleeding, but it is sufficient to inhibit platelet agglutination; in other words, can be administered a therapeutically effective amount without causing significant clinical signs of bleeding (e.g. no bleeding of clinical symptoms, in addition to a small increase in bleeding wounds).

[0285] 可以范围自约0. 002至约0. 4mg/kg或特别为自约0. 005至约0. 2mg/kg、更特别为自约0. 01至约0. lmg/kg的治疗有效量,将如此处所述的人源化VWF抗体或其结合片段施用于一受试者(优选为人类),以在受试者上产生治疗效果(例如减少血栓形成)。 [0285] may range from about 0.002 to about 0. 4mg / kg or especially, from about 0.005 to about 0. 2mg / kg, more particularly from about 0.01 to about 0. lmg / kg treatment from effective amount, thus at the VWF humanized antibody or binding fragment is administered to a subject (preferably a human), to produce a therapeutic effect on the subject (e.g., reduce thrombosis).

[0286] 可以范围自EDiqq的约1至250倍、优选为自EDltltl的约1至200倍、更优选自EDltltl 的约1至100倍的治疗有效量,施用如此处所述的人源化VWF抗体或其结合片段,而不致引发明显的临床出血迹象(例如不会产生出血的临床症状,除了小伤口的出血增加以外)。 [0286] may range from about 1-250 times EDiqq, and preferably from EDltltl about 1-200-fold, about 1-100 times the therapeutically effective amount is more preferably from EDltltl of administration as described herein humanized VWF an antibody or binding fragment thereof, without significant clinical signs of bleeding caused (e.g. no bleeding of clinical symptoms, in addition to a small increase in bleeding wounds outside). 可以单一或多个亚剂量(sub-dose),将如此处所述的治疗有效量人源化vWF抗体或其结合片段施用至受试者。 Sub-doses may be single or multiple (sub-dose), as described herein the therapeutically effective amount of a humanized vWF antibody or binding fragment thereof is administered to a subject. 优选使用静脉注射方式施用上述有效治疗剂量。 Intravenous administration is preferred to use the above-described manner a therapeutically effective amount. 在透过皮下途径的给药中,优选的给药总量可在经由静脉注射的给药量的约1至3倍的范围内,优选为约2倍。 In administration via the subcutaneous route, the total amount may be administered preferably in the range of about 1 to 3 times the intravenous dose range via, preferably about 2 times.

[0287] 出血的临床症状可参考由Serebruany及Atar所述(American Journalof Cardiology, 2007,卷99,册2,15,01,2007,页288-290)并加以应用至动物模型的BleedScore分级,已将BleedScore具体地开发成可就出血的类型计分,出血的类型系抗血小板疗法的特征。 [0287] The clinical symptoms of bleeding and can be referred to by the Serebruany Atar said (American Journalof Cardiology, 2007, volume 99, book 2,15,01,2007, page 288-290) and apply to BleedScore grade animal models have been BleedScore will be specifically developed into the type of bleeding may score, feature type of antiplatelet therapy system bleeding. BleedScore基于根据出血的严重性来分派点数至临床发现(finding) 上,通过加总所有发现的点数,便可获得所产生的分数。 BleedScore depending on the severity of bleeding to assign points to clinical findings (finding) -based, found by summing all the points you can score points arising. 出血症状依渐增的严重性被分成三类:1)表浅(supreficial)出血(每事件1得分点数);2)内部(internal)出血(每事件3 得分点数);3)警示性(alarming)出血或以上的组合(每事件6得分点数),此方法在判定并记述与现代抗血小板及抗血栓疗法相关联的缓和至中等的出血事件时尤其有用,同时还说明了最严重的出血并发症。 Increasing according to the severity of bleeding are divided into three categories: 1) superficial (supreficial) hemorrhage (1 per event score points); 2) Internal (internal) bleeding (3 score points for each event); 3) warning (alarming ) bleeding or a combination thereof (6 score points per event), this method is particularly useful in the determination and description of modern antiplatelet and antithrombotic therapy is associated with ease to moderate bleeding episodes, and also shows the most serious hemorrhage disease. 表浅出血包含下列准则:容易瘀血(bruising)、小伤口的出血(例如延长的模板出血时间)、出血点(petechia)、瘀斑(ecchymosis);内部出血包含下列准则:血肿(hematoma)、流鼻血(印istaxis)、来自口腔或阴道的失血、黑粪症(melena)、 眼睛出血、血尿症(hematuria)、吐血(hematemesis);警示性出血包含下列准则:需要输血(transfusion)、卢页内出il (intracranial) Superficial bleeding include the following criteria: easy bleeding (bruising), minor wound bleeding (such as extending the template bleeding time), bleeding (petechia), bruising (ecchymosis); internal bleeding include the following criteria: hematoma (hematoma), epistaxis (India istaxis), bleeding from the mouth or the vagina, melena (melena), eye bleeding, hematuria (hematuria), vomiting blood (hematemesis); precautionary bleeding contains the following guidelines: need for blood transfusion (transfusion), Lu Page within the il (intracranial)

[0288] 在通过监测血液流经动脉所测量的动脉创伤的动物模型中,血小板凝集的抑制(抑制血小板凝集或足以抑制血小板凝集)可表示所施用的治疗有效量足以抑制闭塞性血栓形成于人为损伤的动脉中。 [0288] In animal models of arterial trauma by monitoring blood flow through arteries measured in platelet aggregation inhibition (inhibition of platelet aggregation or sufficient to inhibit platelet aggregation) may be represented by administering a therapeutically effective amount sufficient to inhibit the formation of occlusive thrombus on human arterial injury in. 一种以定量方式决定体内血小板凝集抑制的方法为透过在动脉创伤模型中的循环流减缩量(cyclic flow reductions,CFR)的测量,如此,例如,血小板凝集的抑制可表示所施用的治疗有效量足以减少动物中的CFR的数目。 Determine a quantitative manner vivo inhibition of platelet aggregation method in the arterial circulation through a flow model of trauma reduction shrinkage (cyclic flow reductions, CFR) measurements, so, for example, inhibition of platelet aggregation may be indicative of the treatment administered an effective amount sufficient to reduce the number of animals in the CFR.

[0289] 治疗有效量或有效量可指可有效地改善或预防症状、或延长所治疗受试者的存活时间的剂量,而判定治疗有效量在技术人员的能力范围内,特别是根据此处所提供的详细公开内容。 [0289] A therapeutically effective amount or effective amount means effective to ameliorate or prevent the symptoms, or prolong the survival time of the subject therapeutic dose, and determining the therapeutically effective amount is within the ability of the skilled person, in particular according to herein It provides detailed disclosure. 如此处所述的治疗有效量包含可有效地治疗受试者的vWF介导的疾病或紊乱的vWF抗体量;vWF抗体的治疗有效量包含治疗或抑制血小板凝集(例如在主动脉、末梢动脉、 小动脉、静脉的血栓形成过程中)所需要的用量。 As described herein the therapeutically effective amount may comprise an amount effective to treat the subject of vWF vWF antibody mediated disease or disorder; vWF antibody comprises a therapeutically effective amount to treat or inhibit platelet aggregation (e.g. aortic, peripheral artery, arterioles, the process amount) of the desired venous thrombosis.

[0290] 有效治疗剂量或有效剂量可指可有效地改善或预防症状、或延长所治疗受试者的存活时间的剂量。 [0290] therapeutically effective dose or the effective dose may refer effective to ameliorate or prevent the symptoms, or prolong the survival of the subject being treated, the dose time. 如此处所述的有效治疗剂量包含可有效地治疗受试者的vWF介导的疾病或紊乱的vWF抗体量;如此处所述的有效治疗剂量包含治疗或抑制血小板凝集(例如在主动脉、末梢动脉、小动脉、静脉的血栓形成过程中)的剂量。 Therapeutically effective amount as described herein comprises an amount effective to treat the subject of vWF vWF antibody mediated disease or disorder; therapeutically effective amount as described herein comprises the treatment or inhibition of platelet aggregation (e.g. aortic, peripheral arteries, arterioles, veins during thrombus) formation of a dose. 有效治疗剂量包含ED,其为足 Comprising a therapeutically effective dose ED , which is sufficient

44以将如同由血管中的血流减少量所测量的血栓形成降低约100%的有效剂量;如此处所述的EDiqq包含足以在30分钟的时间内将由血管中的血流减少量降至零的vWF抗体量。 As the vessel 44 to reduce the amount of blood flow measured thrombosis reduced to about 100% of the effective dose; as described herein EDiqq contained within 30 minutes is sufficient time to zero by reducing the amount of blood vessel The amount of vWF antibody. 有效治疗剂量包含能够减少如同由血管中的血流减少量、或由测量血小板凝集减少量的适当离体测试加以测量的血栓形成的剂量,例如至少足以减少约15%,优选为减少至少30%,更优选为减少至少50%,最优选为减少至少80%,特别是减少至少100%的血栓形成。 Comprising a therapeutically effective amount can be reduced as the reduction of blood flow in the vessel, or by the appropriate dose measuring platelet aggregation in vitro test to measure the amount of reduction of thrombus formation, for example at least sufficient to reduce about 15%, preferably at least 30% reduction , more preferably at least 50%, most preferably by at least 80%, especially by at least 100% of thrombosis.

[0291] 可选地,可将人源化vWF抗体连续地或结合放射性治疗(例如照射或引进放射性物质,像是参照UICC(编),Klinische Onkologie, Springer-Verlag(1982)中所述而适当地施用。 [0291] Alternatively, humanized vWF antibody or in combination with radiation therapy continuously (such as irradiation or introduction of radioactive substances, such as reference to the UICC (series), Klinische Onkologie, (1982) in the Springer-Verlag and appropriate administered.

[0292] 如此,本发明更提供具有冯维勒布兰德氏因子(vWF)特异性的人类抗体或其结合片段,其可以范围自EDltltl的约1至约250倍的治疗有效量来施用,而不致引发明显的临床出血迹象。 [0292] Thus, the present invention further provide a von Willebrand factor (vWF) specific for human antibody or binding fragment thereof, which may range from about 1 to about 250 times the therapeutically effective amount EDltltl be administered, without significant clinical signs of bleeding caused. 优选人类抗体或其结合片段具有人类vWF的Al结构域的特异性,具有vWF特异性的人类抗体或其结合片段更优选地为具有vWF特异性的人源化抗体或其结合片段。 Preferably a human antibody or binding fragment thereof specific for human vWF Al domain of vWF having a specific human antibody or binding fragment thereof more preferably one having specificity for vWF humanized antibody or binding fragment thereof.

[0293] 制剂 [0293] Formulation

[0294] 在本发明的另一实施方案中,提供了包含可用于治疗上述疾病的材料的制剂(例如人源化vWF抗体)。 [0294] In another embodiment of the present invention, there is provided comprising materials useful for the treatment of diseases of the formulations described above (e.g., humanized vWF antibody). 制剂可包含容器及在容器上或与容器相关联的标签(label)或包装说明书;适当的容器包含例如瓶子、小玻璃瓶(vial)、或注射器。 Formulations may contain container and on the container label or associated with the container (label) or package insert; suitable containers include, for example, bottles, glass bottles (vial), or syringe. 容器可由多种材料形成, 例如玻璃或塑料;容器盛装了可有效地治疗疾病的组合物,且可具有无菌的接取口(例如容器可为点滴液袋(intravenous solution bag)或者具有可被皮下注射针刺穿的瓶塞的小玻璃瓶)。 Container can be formed a variety of materials, such as glass or plastic; container containing an effective in the treatment of diseases of the composition, and may have a sterile access port taken (e.g., the container may be a liquid drip bags (intravenous solution bag) or may be having hypodermic needle through the stopper vials). 在该组合物中的至少一活性剂可为此处所述的人源化vWF抗体,标签或包装说明书可指出可使用该组合物来治疗特定的疾病,例如癌症。 In the composition of at least one active agent as described herein may be humanized vWF antibody, label or package insert may indicate that the composition can be used to treat specific diseases, such as cancer. 在一实施方案中,标签或包装说明书可指出可使用包含人源化vWF抗体的该组合物来治疗vWF相关的疾病。 In one embodiment, the label or package insert may indicate that the use of includes humanized vWF antibody composition to treat the vWF related diseases.

[0295] 再者,制剂可包含:(a)其中含有组合物的第一容器,其中该组合物包含此处的人源化抗体;及(b)其中含有组合物的第二容器,其中该组合物包含除了人源化抗体以外的治疗剂。 [0295] Further, the formulations may comprise: (a) a first container which contains a composition, wherein the composition comprises one humanized antibody herein; and (b) wherein the second container containing the composition, wherein the In addition to the composition comprising the humanized antibody therapeutic agent. 在本发明的此实施方案中的制剂还可包含指出可组合使用第一及第二组合物以治疗vWF相关疾病或紊乱的包装说明书,此治疗剂可为前面段落中所述的任何附加疗法(例如血栓溶解剂、抗血小板剂、化疗剂、抗血管生成剂、抗激素化合物、保心药、和/或哺乳动物中的免疫功能调节剂,包含细胞因子)。 In this embodiment of the present invention may be noted that the formulations may comprise a combination of first and second compositions used to treat a disease or disorder associated vWF package insert, the additional therapeutic agent can be any therapy described in the preceding paragraph ( such as thrombolytic agents, antiplatelet agents, chemotherapeutic agents, anti-angiogenic agents, anti-hormonal compound, Baoxin drugs, and / or mammalian immune function modulator comprising cytokines). 可选地,或此外,制剂还可包含第二(或第三) 容器,其具有可药用的缓冲剂,例如注射用的抑菌水(BWFI)、磷酸盐缓冲的盐水、林格溶液(Ringer' s solution)及葡萄糖液;其还可包含其它由商业及使用者立场看来为合宜的材料,包含其它缓冲液、稀释液、过滤器、注射针、及注射器。 Alternatively, or in addition, the formulation may further comprise a second (or third) container having a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution ( Ringer 's solution) and glucose; it may also contain other by commercial and user standpoint it seems as suitable material, including other buffers, diluents, filters, needles, and syringes.

[0296] 人源化vWF抗体的非治疗性用途 [0296] The humanized vWF antibody non-therapeutic use

[0297] 人源化vWF抗体及其结合片段具有其他的非治疗性应用。 [0297] The humanized vWF antibodies and binding fragments with other non-therapeutic applications. 举例而言,可将抗体作为亲合力纯化剂;在此程序中,可利用本领域中已知的方法,将抗体固定化在固相上,例如SEPHADEX™树脂或滤纸。 For example, antibodies can be used as affinity purification agents; in this program, you can use a method known in the art, the antibody is immobilized on a solid phase, for example, SEPHADEX ™ resin or filter paper. 可使固定化抗体与含有待纯化的人源化vWF蛋白质(或其片段)相接触,之后,可以实质上将移除样本中除了人源化vWF蛋白质(其可结合至固定化抗体)以外的所有物质的适当溶剂来洗涤支持物(support)。 Immobilized antibody can be purified with people humanized vWF containing proteins (or fragments thereof) in contact, after which the sample may be substantially removed in addition to the humanized vWF protein (which can bind to the immobilized antibody) except All materials suitable solvent to wash the support (support). 最后,可以另一适当溶剂(例如pH5.0 的甘氨酸缓冲液)来洗涤支持物,此溶剂将人源化vWF蛋白质与抗体脱离。 Finally, another suitable solvent (e.g., glycine buffer, pH5.0) to wash the support, the solvent and the humanized antibodies from the vWF protein.

[0298] 人源化vWF抗体还可用于人类vWF蛋白质的诊断测定上,例如检测特殊细胞、组织、或血清内的表达。 [0298] The humanized vWF antibody diagnostic assays can also be used on human vWF protein, such as detection of specific cells, tissues, or serum expression within. 就诊断应用而言,可以可检测部分来标记抗体。 On diagnostic applications, it can be labeled with a detectable moiety antibody. 有一般可归类成下列种类的多种标签可使用: There are generally categorized into the following types of multiple tags can be used:

[0299] (a)放射性同位素,例如35S、14C、125I、3H、及1311。 [0299] (a) radioisotopes, such as 35S, 14C, 125I, 3H, and 1311. 举例而言,可利用Current Protocols in Immunology,卷1&2, Coligen 等,编WiIey-Interscience, New York, NY,Pubs. (1991)中所述的技术用放射性同位素来标记抗体,例如,可利用闪烁计数法(scintillationcounting)来测量放射性。 For example, you can take advantage of Current Protocols in Immunology, Volume 1 & 2, Coligen etc., compiled WiIey-Interscience, New York, NY, Pubs. (1991), wherein the antibody labeled with a radioisotope techniques, for example, using a scintillation counter France (scintillationcounting) to measure radioactivity.

[0300] (b)可使用荧光标签例如稀土螯合剂(铕螯合剂)或荧光素(fluorescein)及其衍生物、罗丹明(rhodamine)及其衍生物、丹磺酰基(dansyl)、丽丝胺(Lissamine)、藻红蛋白(phycoerthrin)及德克萨其Jf红。 [0300] (b) Fluorescent labels such as rare earths can be used chelating agent (europium chelator) or fluorescein (fluorescein) and its derivatives, rhodamine (rhodamine) and its derivatives, dansyl (dansyl), lissamine (Lissamine), phycoerythrin (phycoerthrin) and as large as Texas its Jf red. 可禾1J用例如Current Protocols in Immunology,同上中所公开的技术,将荧光标记缀合至抗体;可利用荧光计来定量荧光标记。 He can, for example 1J Current Protocols in Immunology, supra disclosed techniques, the antibody conjugated to a fluorescent label; fluorometer can be used to quantify a fluorescent label.

[0301] (c)可使用各种的酶-底物标记(例如见美国专利第4,275,149号),这些酶通常催化可利用各种技术加以测量的显色底物的化学变化作用。 [0301] (c) using a variety of enzyme - substrate labels (see e.g., U.S. Patent No. 4,275,149), the enzyme generally catalyzes a variety of techniques can be used to measure the effect of chemical changes chromogenic substrate . 例如,酶可催化底物中的颜色变化,其可以分光光度测定方式加以测量;可选地,酶可以改变荧光性或化学发光(chemiluminescence),定量荧光变化的技术如上所述。 For example, the enzyme may catalyze a color change in a substrate, which can be measured spectrophotometrically measuring method; alternatively, the enzyme may alter the fluorescence or chemiluminescence (chemiluminescence), quantitative change in fluorescence technique described above. 化学发光底物可通过化学反应电子激发,且其接着可发出可被测量的光(例如用化学发光光度计)或供给能量至荧光受体。 Chemiluminescent substrate may be electronically excited by a chemical reaction, and it may then emit light that can be measured (e.g., using chemiluminescence photometer) or supplied energy to a fluorescent acceptor. 酶标记的例子包含荧光素酶(luciferases,例如萤火虫荧光素酶及细菌荧光素酶;美国专利第4,737,456 号)、荧光素(Iuciferin)、邻苯二甲腈(2, 3-dihydrophthalazinediones)、 苹果酸脱S酶(malatedehydrogenase)、尿素酶(urease)、过氧化酶(peroxidase,例如辣根过氧化物酶(horseradish peroxidase) (HRPO))、碱性磷酸酶(alkalin印hosphatase)、 β -半乳糖苷酶(β -galactosidase)、葡糖淀粉酶(glucoamylase)、溶菌酶(lysozyme)、糖类氧化酶(saccharide oxidases)(例如葡萄糖氧化酶、半乳糖氧化酶、及葡萄糖_6_磷酸脱氢酶)、杂环氧化酶(例如尿酸酶(uricase)及黄嘌呤氧化酶(xanthine oxidase))、乳过氧化酶(Iactoperoxidase)、及微过氧化酶(microperoxidase)。 Examples of enzyme labels containing luciferase (luciferases, such as firefly luciferase enzyme and bacterial luciferase; U.S. Patent No. 4,737,456), luciferin (Iuciferin), phthalonitrile (2, 3-dihydrophthalazinediones ), malic enzyme de S (malatedehydrogenase), urease (urease), peroxidase (peroxidase, such as horseradish peroxidase (horseradish peroxidase) (HRPO)), alkaline phosphatase (alkalin printed hosphatase), β - galactosidase (β -galactosidase), glucoamylase (glucoamylase), lysozyme (lysozyme), saccharide oxidase (saccharide oxidases) (e.g., glucose oxidase, galactose oxidase, and glucose phosphate _6_ dehydrogenase), heterocyclic oxidases (such as uricase (uricase) and xanthine oxidase (xanthine oxidase)), lactoperoxidase (Iactoperoxidase), and micro-peroxidase (microperoxidase). 将酶缀合至抗体的技术说明于O' SulIivan"Methods for thePreparation of Enzyme-Antibody Conjugates for use in Enzymelmmunoassay, "Methods in Enzym.(编,J. Langone & H. Van Vunaki s), Academic Press, New York,73 :147-166(1981)。 The enzyme conjugated to antibodies are described in O 'SulIivan "Methods for thePreparation of Enzyme-Antibody Conjugates for use in Enzymelmmunoassay," Methods in Enzym. (Edited, J. Langone & H. Van Vunaki s), Academic Press, New York, 73: 147-166 (1981).

[0302] 酶-底物组合的例子包含: [0302] enzyme - substrate combinations example contains:

[0303] (i)以过氧化氢酶作为底物的辣根过氧化物酶(HRPO),其中过氧化氢酶氧化染料前体(例如邻苯二胺(orthophenylene diamine)或3,3,,5,5,-四甲基联苯胺盐酸盐(3, 3,,5, 5,-tetramethyl benzidine hydrochloride (TMB))); [0303] (i) with hydrogen peroxide as a substrate for the enzyme horseradish peroxidase (HRPO), wherein the hydrogen peroxidase oxidation dye precursor (e.g., ortho-phenylenediamine (orthophenylene diamine) or 3,3 ,, 5,5, - tetramethyl benzidine hydrochloride (3, 3,, 5, 5, -tetramethyl benzidine hydrochloride (TMB)));

[0304] (ii)以对硝苯磷酸盐(para-nitrophenyl phosphate)为显色底物的碱性磷酸盐(AP) •'及 [0304] (ii) in order to nifedipine phosphate (para-nitrophenyl phosphate) is a chromogenic substrate of alkaline phosphatase (AP) • 'and

[0305] (iii)具有显色底物(例如对硝苯-β-D-半乳糖酶)或荧光底物4-甲基伞形酮-β-D-半乳糖苷酶(4-methylumbelliferyl-^-D-galactosidase)的β _D_ 半乳糖苷酶(β -D-Gal)。 [0305] (iii) with a chromogenic substrate (e.g., nifedipine -β-D- galactosidase) or fluorogenic substrate 4-methylumbelliferyl -β-D- galactosidase (4-methylumbelliferyl- ^ -D-galactosidase) of β _D_ galactosidase (β -D-Gal).

[0306] 技术人员可使用多种其它酶-底物组合(见例如美国专利第4,275,149号及第4,318,980 号)。 [0306] the art can use a variety of other enzyme - substrate combinations (see e.g., U.S. Patent No. 4,275,149 and in the No. 4,318,980).

[0307] 有时可使标记间接地缀合至抗体,技术人员应知达成此目的的各种技术。 [0307] can sometimes labeled indirectly conjugated to an antibody, technical personnel should be aware that a variety of techniques to achieve this purpose. 举例而言,可使抗体与生物素(biotin)缀合,且可将上述三大范围的任一标记与抗生物素蛋白(avidin)缀合,反之亦然。 For example, the antibody can biotin (biotin) conjugated, and may be any one of these three ranges with labeled avidin (avidin) conjugated to, or vice versa. 生物素选择性地与抗生物素蛋白结合,故可以此间接方式将标记与抗体相缀合;可选地,为达到标记与抗体的间接缀合,可将抗体与小型半抗原(hapten) (例如地高辛(digoxin))缀合,且可将上述不同种类标记其中之一与抗半抗原抗体(例如抗地高辛抗体)相缀合。 Biotin and avidin selectively binding, it is possible to indirectly labeled with this antibody conjugated; alternatively, to achieve indirect conjugation of antibodies labeled with the antibodies may be to small hapten (hapten) ( such as digoxin (digoxin)) conjugation, and it may be one of the above-mentioned different types of tags with an anti-hapten antibody (eg anti-digoxin antibody) conjugated. 如此,可完成标记与抗体的间接缀合。 So, to be completed indirectly labeled with an antibody conjugated.

[0308] 在本发明的另一实施方案中,人源化vWF抗体不需要被加上标记,且其存在可利用结合至人源化vWF抗体的标记抗体加以检测。 [0308] In another embodiment of the present invention, a humanized vWF antibody need not be marked, and its presence can be used in combination to human vWF antibody labeled humanized antibody to be detected.

[0309] 可将本发明的抗体使用于任何已知的测定方法中,例如竞争性结合测定、直接及间接夹心式测定(sandwich assays)、及免疫沉淀(immunoprecipitation)测定,见Zola, Monoclonal Antibodies :A Manualof Techniques,页147-158 (CRC Press, Inc.1987)。 [0309] Antibodies of the invention can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assay (sandwich assays), and immunoprecipitation (immunoprecipitation) determinations, see Zola, Monoclonal Antibodies: A Manualof Techniques, pages 147-158 (CRC Press, Inc.1987).

[0310] 就免疫组织化学而言,肿瘤样本可为新鲜或冷冻或经埋置于石蜡中并以例如福尔马林的防腐剂加以固定。 [0310] For immunohistochemistry, the tumor sample may be fresh or frozen or been embedded in paraffin and formalin preservative, for example, be fixed.

[0311] 还可将抗体用于体内的诊断测定上。 [0311] The antibody can also be used for in vivo diagnostic assays. 一般而言,可以放射性核素(radionuclide) (例如mIn,99Tc,14C,mI,125I,3H,32P或35S)将抗体加上标记,使例如可利用免疫闪烁成像(immunoscintigraphy)而将月中瘤定位。 In general, you can radionuclides (radionuclide) (for example mIn, 99Tc, 14C, mI, 125I, 3H, 32P or 35S) antibody marked, for example, can be used to make the immune scintigraphy (immunoscintigraphy) and the mid-tumor Location.

[0312] 为方便,本发明的抗体可以试剂盒的形式来提供(例如预定量的试剂与用以施行诊断测定的使用说明书的套装式组合)。 [0312] For convenience, the antibodies of the present invention may be provided in kit form (such as combo kit for a predetermined amount of reagent and diagnostic assays for the purposes of the operating instructions). 若为抗体用酶来标记的情况,试剂盒可包含酶所需要的底物及辅因子(cofactor),例如提供可检测的发色团或荧光团的底物前体;此外,可包含其它添加剂,例如稳定剂、缓冲液(例如阻断缓冲液(block buffer)或细胞溶解缓冲液(Iysisbuffer))等。 If the case is an antibody labeled with an enzyme, the kit may contain enzymes and substrates required cofactor (cofactor), such as providing a detectable chromophore or fluorophore precursor substrates; in addition, may include other additives , such as stabilizers, buffers (e.g. blocking buffer (block buffer) or cell lysis buffer (Iysisbuffer)) and the like. 可大范围地改变各种试剂的相对量,以提供溶液中实质上将测定的灵敏度最优选化的试剂浓度;尤其,试剂可以干粉末(通常为冷冻干燥)的形式加以提供, 包括在溶解时将提供具有适当浓度的试剂溶液的赋形剂。 Can be a wide range of varying the relative amounts of the various reagents, the solution to provide substantially the most preferred assay of the sensitivity of the reagent concentration; in particular, the reagent can be a dry powder (usually lyophilized) form to be provided, including at the time of dissolution excipients will provide a reagent solution having the appropriate concentration.

[0313] 人源化vWF抗体还可用于体内成像,其中可将标记抗体施用于宿主(优选为血流),并测定宿主中标记抗体的存在及位置;此成像技术可适用于血管栓塞的定位(localization)或肿瘤的分级及治疗上。 Location This imaging technique can be applied to blood clots; [0313] Humanized vWF antibodies may also be used for in vivo imaging, which can be labeled antibody is administered to the host (preferably blood), and determining the presence of antibodies labeled host and location (localization) or tumor grade and the treatment. 抗体可以用在宿主中可检测到的任何部分适当地标记,包括例如可由如核磁共振或其它本领域中已知的装置加以检测的非放射性指示齐ϋ。 Antibodies can be used in any part of the host can be detected suitably labeled, e.g., by including the indication to be non-radioactive detection such as magnetic resonance, or other means known in the art Qi ϋ. 然而,优选标记可为放射性标记,包括碘(例如125I及131D、硒、双功能螯合剂、铜(例如67Cu)、锝(例如99mTc)、及铼(例如186Re及188Re)等。可通过任何方法将放射性同位素缀合至蛋白质,包括例如金属螯合化合物或乳过氧化酶、或碘化(iodination)用的iodogen技术。 Preferably, however, it can be labeled as a radioactive label, including the iodine (e.g., 125I and 131D, selenium, bifunctional chelator, copper (e.g., 67Cu), technetium (e.g., 99mTc), and rhenium (e.g., 186Re and 188Re), etc. by any method radioisotopes conjugated to proteins, including for example a metal chelate compound iodogen technology or lactoperoxidase, or iodide (iodination) used.

[0314] 材料的保藏: [0314] Materials deposited:

[0315] 下列材料已经保藏于德国不伦瑞克(Braunschweig)的国家菌种保存中心(Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(DSMZ)): [0315] The following materials have been deposited in Braunschweig (Braunschweig) National Culture Collection (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ)):

[0316] (1)2008年1月23日保藏于DSMZ、登记号为DSM 21059的微生物(大肠杆菌), 其包含载体GS264,而GS264包含具有编码人源化NMC-4变体H9L9IgG4轻链的核酸序列的分离核酸;(2) 2008年1月23日保藏于DSMZ、登记号为DSM 21060的微生物(大肠杆菌),其包含载体GS265,而GS265包含具有编码人源化NMC-4变体H9L9IgG4重链的核酸序列的分离核酸。 [0316] (1) January 23, 2008 deposited at DSMZ, registration number DSM 21059 microorganism (E. coli), which comprises a carrier GS264, and GS264 containing NMC-4 variant H9L9IgG4 light chain having the encoding humanized isolated nucleic acid nucleic acid sequence; (2) January 23, 2008 deposited at DSMZ, registration number DSM 21060 microorganism (E. coli), which comprises a carrier GS265, and GS265 contains an encoding humanized NMC-4 variant H9L9IgG4 isolated nucleic acid sequences of the heavy chain nucleic acid. 此等保藏皆基于微生物有机体保藏专利程序的国际认可布达佩斯协议(International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure)及其(布达佩斯协议)下的规定而为。 These microorganisms are deposited on organisms preserved Patent Procedure Budapest Treaty on the international recognition (International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure) and its regulations (the Budapest Protocol) and for the under.

[0317] 在无进一步说明之下,人们相信:在此领域中普通技术人员使用前述说明及下列例示性实施例,可生产并利用本发明的试剂且实施权利要求的方法。 [0317] Under no further explanation, it is believed that: In this field of ordinary skill in the use of the foregoing description and the following exemplary embodiments of the present invention can be produced by using agents and the implementation of a method claim. 提供下列工作实施例以辅助本发明的实施,且其不应被解释为限制性。 The following working examples are provided to assist the implementation of the present invention, and it should not be construed as limiting.

实施例 Example

[0318] 实施例1 :嵌合抗体的建构 [0318] Example 1: Construction of the chimeric antibody

[0319] 产生NMC-4-人类Fc的嵌合体:包含来自鼠类抗体NMC-4的可变区及人类Fc区的嵌合抗体用下述方式加以建构。 [0319] NMC-4- produce human Fc chimera: chimeric antibodies comprising human Fc region and a variable region from a murine antibody NMC-4, to construct in the following manner. 在一例示方法中,是利用抗vWF抗体NMC-4(例如具有已被公开的可变区氨基酸序列的IgGl κ )作为模板来产生NMC-4的VH及VL区的合成基因序列(Celikel 等,1997,BloodCells,Molecules 和Diseases 23 :123_134)。 In one exemplary method, using anti-vWF antibody NMC-4 (e.g., IgGl variable region having the amino acid sequence has been disclosed in κ) generating a synthetic gene sequences of VH and VL regions of NMC-4 (Celikel the like as a template, 1997, BloodCells, Molecules and Diseases 23: 123_134). 举例而言,NMC-4 的VH及VL所用的合成基因序列通过采用Celikel等中所述的氨基酸序列及利用VECTOR NTI 软件产生对应的核苷酸序列而产生。 For example, the synthetic gene sequence NMC-4, used for VH and VL amino acid sequences by using the Celikel like and use VECTOR NTI software generates a corresponding nucleotide sequence is generated.

[0320] 表1用来产生NMC-4嵌合表达质粒的引物(primers) [0320] Table 1 for generating NMC-4 expression plasmid of chimeric primers (primers)

[0321] [0321]

48 48

[0322] 在一例示方法中,是利用Accuprime PFX DNA聚合酶试剂盒(Invitrogen)来进行PCR反应;例如,组合包含下列的50 μ 1反应混合物(mix) =IXPFX缓冲液、0. 2 μ M dNTP混合物、1单位的PFX聚合酶、ΙμΜ正向引物、IyM反向引物及IOOng的DNA模板。 [0322] In one exemplary method, using Accuprime PFX DNA Polymerase kit (Invitrogen) to perform a PCR reaction; e.g., a combination of 50 μ 1 comprising the following reaction mixture (mix) = IXPFX buffer, 0 2 μ M. dNTP mix, 1 unit of PFX polymerase, ΙμΜ forward primer, IyM IOOng reverse primer and a DNA template. 标准的PCR 程序包含:在94C下开始进行变性(denaturation)持续1分钟,接着以每一循环为94C持续30秒、55C持续30秒、68C持续1分钟进行30个循环,以及在68C下持续10分钟的最后延长(extension)步骤。 Standard PCR procedure comprising: at 94 C under denaturing starts (denaturation) for 1 min, followed by 94 C each cycle of 30 seconds, 55 C for 30 seconds, 68 C for 1 min 30 circulation, and finally extended (extension) step down at 68 C for 10 minutes. 以在0. 8% TAE胶上的琼脂糖凝胶电泳纯化PCR产物,切除一条或多条的期望大小的条带,并以Qiagen凝胶提取试剂盒纯化。 In the 0. 8% TAE agarose gel purified PCR product gel electrophoresis, excision of one or more strips of a desired size, and purified by Qiagen Gel Extraction Kit. 室温下,在包含1 XT4DNA连接酶(Iigase)缓冲液(NEB)、0. 5 μ 1 Τ4 DNA 连接酶(NEB)及各DNA IOOng 的10 μ 1 体积中,连接(Iigate)DNA片段持续30分钟;接着,利用1 μ 1的连接反应物来转化JM109大肠杆菌细胞,且通过利用贝克曼(Beckman)CEQ 8000DNA分析仪加以测序,以验证PCR所产生的插入物(inserts)。 At room temperature, containing 1 XT4DNA ligase (Iigase) buffer (NEB), 0. 5 μ 1 Τ4 DNA ligase (NEB) and each DNA IOOng volume of 10 μ 1, the connection (Iigate) DNA fragment for 30 minutes ; Next, the ligation reaction was used to transform 1 μ 1 of JM109 E. coli cells, and to be sequenced by using a Beckman (Beckman) CEQ 8000DNA analyzer to verify the generated PCR inserts (inserts).

[0323] 此等包含来自鼠类抗体NMC-4的VH和/或VL以及人类Fc的嵌合抗体,是根据本领域中已知的标准规则(protocol)利用PCR加以合成。 [0323] These include information from murine antibody VH NMC-4 and / or VL and human Fc chimeric antibody is synthesized by PCR to be known in the art according to standard rules (protocol). 在一例示方法中,通过以NMC-4专用的引物及人类Fc专用的引物,将来自NMC-4的VH和/或VL融合至人类Fe。 In one exemplary method, by NMC-4-specific primers and human Fc-specific primers from NMC-4, VH and / or VL fused to human Fe.

[0324] 任选地,将氨基酸取代(例如突变)引入IgGlFc区,以破坏Fc及补体结合位点(complement binding sites)而消除假设由野生型Y 1 Fc恒定区介导的细胞毒性(见例如SEQ ID NO : 143)。 [0324] Optionally, the amino acid substitutions (such as mutations) into IgGlFc areas to destroy Fc and complement binding sites (complement binding sites) and eliminate the assumption by the cytotoxicity of wild type Y 1 Fc constant region-mediated (see, for example SEQ ID NO: 143). 例如,衍生自IMAGE cDNA 克隆#4764579 (ATCC) (SEQ ID NO : 33)人类IgGl 恒定区(如Fe)是利用引物hIgG-F(SEQ ID NO :36) ^P HIgG-R (SEQ ID NO: 37)(表1)加以扩增。 E.g., derived from IMAGE cDNA clone # 4764579 (ATCC) (SEQ ID NO: 33) human IgGl constant region (e.g. Fe) using primers hIgG-F (SEQ ID NO: 36) ^ P HIgG-R (SEQ ID NO: 37) (Table 1) to be amplified. 通过进行例如定点突变(site directed mutagenesis) (Duncan& Winter ;Nature. 332(6166) :738_40 (1988)),将氨基酸取代(例如L235E (FcR 结合区)及E318A,K320A,K332A(在Clq补体结合位点))引入IgGl Fc区。 By performing such as site-directed mutagenesis (site directed mutagenesis) (Duncan & Winter; Nature 332 (6166):. 738_40 (1988)), the amino acid substitutions (e.g., L235E (FcR binding region) and E318A, K320A, K332A (in Clq complement binding Point)) introduced IgGl Fc region. 举例而言,利用引物对hFc-L235E-F(SEQ ID NO :39)及hFc_L235E_R(SEQ ID NO :40)将L235E 突变引入恒定区; 利用引物对CH2-Clq(-)_F(SEQ ID NO :41)及CH2_Clq(-)-R) (SEQ ID NO :42)(表1)引入补体位点突变(complement site mutations)。 For example, using primers of hFc-L235E-F (SEQ ID NO: 39) and hFc_L235E_R (SEQ ID NO: 40) The L235E mutations into a constant region; the use of primers CH2-Clq (-) _ F (SEQ ID NO: 41) and CH2_Clq (-) - R) (SEQ ID NO: 42) (Table 1) into the fill position of point mutations (complement site mutations). 利用两外部引物hlgG-F (SEQ ID NO :36) 及IgGl-BamHI-R(SEQ ID NO :38)来连结包含四种突变的合成的PCR产物,以产生编码修饰IgGl Fc 区(称为IgGl (dm))的PCR 产物。 The use of two external primers hlgG-F (SEQ ID NO: 36) and IgGl-BamHI-R (SEQ ID NO: 38) for coupling includes four mutant PCR product synthesized, to produce a modified IgGl Fc coding region (referred IgGl (dm)) PCR product.

[0325] 在一例示方法中,通过本领域中已知的重组技术而将NMC-4的重链可变区融合至修饰IgGl Fc区(例如IgGl (dm))。 [0325] In one exemplary method, known in the art of recombinant technology and the heavy chain variable region NMC-4 fused to a modified IgGl Fc region (e.g., IgGl (dm)). 例如,利用在NMC-VH的N端上游引入EcoRI克隆位点的引物匪C-VH-EcoRI-F (SEQ ID NO :34)及匪C-VH-IgGl-R(SEQ ID N0:35),扩增编码NMC-4的重链可变区(SEQ IDNO=I)的核苷酸序列;简言之,通过两步骤的重组PCR,可使PCR产物与重链恒定区(例如IgGl (dm))连接,首先利用引物对NMC-VH-EcoRI-F(SEQID NO :34)和HIgG-R (SEQ ID NO :37),接着利用引物对匪C-VH-EcoRI_F(SEQ ID NO :34)及IgGl-BamHI-R(SEQ ID NO :38)进行PCR 反应。 For example, the use of the N-terminus upstream NMC-VH introduced EcoRI cloning site primers bandit C-VH-EcoRI-F (SEQ ID NO: 34) and bandit C-VH-IgGl-R (SEQ ID N0: 35), encoding the heavy chain variable region was amplified NMC-4, (SEQ IDNO = I) a nucleotide sequence; Briefly, two-step by recombinant PCR, the PCR product allows the heavy chain constant region (e.g., IgGl (dm) ) is connected, firstly primers NMC-VH-EcoRI-F (SEQID NO: 34) and HIgG-R (SEQ ID NO: 37), followed by the use of primers bandit C-VH-EcoRI_F (SEQ ID NO: 34) and IgGl-BamHI-R (SEQ ID NO: 38) PCR reactions were performed. 以EcoRI 及BamHtI 来消化(digest)最终的PCR 产物,并将PCR 产物克隆至pIRES2-EGFP-IgK 载体(Clontech,Palo Alto, CA)的EcoRI及BamHI位点中,该载体经过修饰成包含被克隆至XhoI及EcoRI位点的Ig κ先导序歹Ij (METDTLLLWVLLLWVPGSTGD) (SEQ ID NO :107))(由SEQ ID NO :140 的多核苷酸加以编码)。 Digested with EcoRI and BamHtI (digest) the final PCR product, and the PCR product was cloned into pIRES2-EGFP-IgK vector (Clontech, Palo Alto, CA) the EcoRI and BamHI sites of the vector has been modified to contain cloned and Ig EcoRI to XhoI site κ pilot sequence bad Ij (METDTLLLWVLLLWVPGSTGD) (SEQ ID NO: 107)) (the SEQ ID NO: 140 polynucleotides are coded).

[0326] 在一例示方法中,通过如下所述的重组技术而将NMC-4的轻链可变区融合至修饰IgGl Fc区(例如IgGl (dm))。 [0326] In one exemplary method, by recombinant techniques as described below and the light chain variable region NMC-4 fused to a modified IgGl Fc region (e.g., IgGl (dm)). 例如,利用在IgK轻链恒定区的3'端更进一步引入BamHI 限制位点的引物κ -F(SEQ ID NO :47)及κ -BamHI-R(SEQ ID N0:48),扩增来自由I. Μ. AG E 克隆#4704496 (ATCC) (SEQ ID NO : 108)所制成的DNA 中的Ig κ 轻链恒定区(例如κ Cl (SEQ ID NO :141));同理,利用在5,端引入EcoRI 位点的引物NMC-VL-EcoRI-F (SEQ IDNO :45)及NMC-VL-κ-R (SEQ ID NO :46)(表1),扩增来自合成VL基因的轻链可变区;接着,利用引物匪C-VL-EcoRI-F (SEQ ID NO :45)及κ -BamHI-R (SEQ ID NO :48),以重组PCR 来连结NMC-4可变区及κ Cl片段。 For example, the use of the IgK light chain constant region of the 3 'end of the BamHI restriction site introduced further primers κ -F (SEQ ID NO: 47) and κ -BamHI-R (SEQ ID N0: 48), amplification free . I. Μ AG E clone # 4704496 (ATCC) (SEQ ID NO: 108) DNA made in the Ig κ light chain constant region (e.g., κ Cl (SEQ ID NO: 141)); Similarly, the use of the 5, the end of the EcoRI site introduced primer NMC-VL-EcoRI-F (SEQ IDNO: 45), and NMC-VL-κ-R (SEQ ID NO: 46) (Table 1), synthesized from the amplified light VL gene chain variable region; then, using primers bandit C-VL-EcoRI-F (SEQ ID NO: 45) and κ -BamHI-R (SEQ ID NO: 48), recombinant PCR was used to link the variable regions and NMC-4 κ Cl fragments. 以EcoRI及BamHI来分解最终的PCR产物,并将其克隆至pIRES2-DsRed2-Ig κ载体中的相同位点。 With EcoRI and BamHI to break down the final PCR product, and cloned into pIRES2-DsRed2-Ig κ vector in the same sites. [0327] 相较于表达轻链小鼠-人类嵌合体的pIRES-DsRed质粒,在pIRES2_EGFP载体中的重链的表达水平可能较低,尽管对于轻链及重链两者皆使用相同的IgK先导序列。 [0327] Compared to the expression of the light chain of mouse - human chimeric plasmid of pIRES-DsRed expression levels of the heavy chain in pIRES2_EGFP carrier may be low, although both use for both the heavy chain and light chain the same IgK pilot sequence. 因此,为改善重链的表达水平,利用引物对4-59前导-HindIII-NMC-4(SEQ ID NO :43)和IgGl-BamHI-R(SEQ ID NO :38)并以pIRES2-EGFP-WC4_IgG(mut)载体作为模板(表1),以来自人类种系4-59VH的前导序列(MKHLWFFLLLVAAPRWVLS) (SEQ ID NO :109)取代Ig κ前导序列;片段则以HindIII及PmeI加以消化,并亚克隆至pcDNA6-cMyc_A载体(Invitrogen, Carlsbad, CA) Hindi 11 及PmeI 的位点。 Therefore, in order to improve the level of expression of the heavy chain, the use of primers 4-59 preamble -HindIII-NMC-4 (SEQ ID NO: 43) and IgGl-BamHI-R (SEQ ID NO: 38) and pIRES2-EGFP-WC4_IgG (mut) vector as a template (Table 1), in order to come from the human germline 4-59VH leader sequence (MKHLWFFLLLVAAPRWVLS) (SEQ ID NO: 109) replace the Ig κ leader sequence; fragment HindIII and PmeI places to be digested, and subcloned to pcDNA6-cMyc_A vector (Invitrogen, Carlsbad, CA) Hindi 11 and PmeI sites.

[0328] AJW200参考抗体的建构:AJW200为针对vWF的Al结构域的另一抗体,其具有阻断vWF Al结构域与GPIb α之间的相互作用的能力。 [0328] AJW200 reference antibody Construction: AJW200 as another antibody against the Al domain of vWF, which has the ability to block the interaction of vWF Al domain and GPIb α between. AJW200参考抗体通过改造美国专利第6,228,360中所述的VH及VL序列,以包含例如改善表达用的额外功能性Kozak序列而产生。 AJW200 reference antibody through the transformation of US Patent 6,228,360 VH and VL sequences described in the first to include the expression such as improved functionality with additional Kozak sequence is generated. 举例而言,合成的AJW200VH基因是利用引物Hind III-Ko-AJff-F (SEQ ID NO :49)和HuFab-HR(SEQ IDNO :50)(表2)加以扩增,并将其克隆至包含人类IgGl (dm)重链恒定区的以HindIII-ApaI消化的pcDNA6-cMyc_A载体的HindIII及ApaI位点中(以取代匪C-4 的VH);而AJW200VL 是利用引物对XhoI-Ko-AJW-F (SEQ ID NO :51)及κ -BamHI-R(SEQ ID NO :48)加以扩增,并将其亚克隆至带有NMC-4轻链嵌合体的pIRES-DsRed载体的XhoI及BamHI位点中(藉以取代NMC-4的VL)。 For example, the synthetic gene using primers AJW200VH Hind III-Ko-AJff-F (SEQ ID NO: 49) and HuFab-HR (SEQ IDNO: 50) (Table 2) to be amplified, and cloned into comprising Human IgGl (dm) heavy chain constant region to HindIII-ApaI digested vector pcDNA6-cMyc_A HindIII and ApaI sites (to replace the bandit VH C-4); and AJW200VL using primers XhoI-Ko-AJW- F (SEQ ID NO: 51) and κ -BamHI-R (SEQ ID NO: 48) to be amplified and subcloned into the vector pIRES-DsRed with NMC-4 light chain chimera XhoI and BamHI sites point (thereby replace VL NMC-4 in).

[0329] 表2用以产生AJW200表达质粒的引物 [0329] Table 2 expression plasmid for generating AJW200 primer

[0330] [0330]

[0331] 抗体生产:嵌合抗体可通过本领域中已知的任何方法加以产生。 [0331] Antibody Production: chimeric antibodies can be any method known in the art to produce. 在一例示方法中, HEK239F 细胞在120rpm,37C,8% CO2 的摇瓶中以Freestyle293 表达培养基(Invitrogen) 来培养,细胞在IOOxg下离心沉淀,悬浮于30ml的Freestyle 293表达培养基中,并施以涡旋(vortexed)达20秒,以得到单细胞悬浮液。 In one exemplary method, HEK239F cells 120rpm, 37 C, 8% CO2 in shake flasks to Freestyle293 expression medium (Invitrogen) to culture, cell pellet was centrifuged at IOOxg, suspended in 30ml of Freestyle 293 expression medium and subjected to vortex (vortexed) for 20 seconds to obtain a single cell suspension. 计算细胞数目,且以3. 3 X IO8细胞接种于含有总体积330ml的Freestyle 293表达培养基的2L摇瓶中。 Calculate the number of cells and to 3. 3 X IO8 cells were seeded in a total volume of 330ml containing Freestyle 293 expression medium 2L shake flasks. 转染混合物是由等量的DNA/ OptiMEM (例如165 μ g的HC表达质粒、165 μ g的LC表达质粒、及室温OptiMEM (Invitrogen) 至总体积Ilml)及293fectin/0ptiMEM(例如433 μ 1 的293fectin (Invitrogen)及室温OptiMEM(Invitrogen)至总体积11ml)所组成。 The transfection mixture is the same amount of DNA / OptiMEM (for example 165 μ g of HC expression plasmids, 165 μ g of LC expression plasmid, and room OptiMEM (Invitrogen) to a total volume Ilml) and 293fectin / 0ptiMEM (for example, 433 μ 1 of 293fectin (Invitrogen) and room temperature OptiMEM (Invitrogen) to a total volume of 11ml) of the composition. 将DNA混合物添加至293fectin混合物中, 接着加以混合并在室温下培养20分钟,且将其添加至含有293F细胞的现存培养基中;以37C,8% CO2,120rpm的振荡速率培养细胞。 293fectin the DNA mixture was added to the mixture, then mixed and incubated for 20 min at room temperature, and add it to an existing medium containing 293F cells; to 37 C, the oscillation rate of 8% CO2,120rpm cultured cells. 于转染后72小时时,在IOOxg下离心悬浮液5 分钟,以使细胞沉淀;利用0. 2 μ m滤膜过滤含Mab (单克隆抗体)上清液,且利用蛋白质-A 亲和柱纯化的。 At 72 hours after transfection, the suspension was centrifuged IOOxg 5 minutes to precipitate the cells; the use of 0. 2 μ m membrane filter containing Mab (monoclonal antibody) supernatant, and use of protein affinity column -A Purified.

[0332] 将来自瞬时转染HEK-293F细胞的少量条件培养基(CM)施用于已以PBS来平衡的0. 3ml 蛋白质-A SEPHAR0SE 滴注柱(drip column),以IOml PBS 清洗柱并以0. 1M, pH 2. 7的甘氨酸洗脱蛋白质。 [0332] The small amount of conditioned medium from transiently transfected HEK-293F cell culture medium (CM) has applied to be balanced with PBS 0. 3ml protein -A SEPHAR0SE column infusion (drip column), the column was washed with IOml PBS and 0. 1M, pH 2.7 glycine eluted proteins. 将Iml的级分收集至0. Iml的1M,pH 8. 0 Tris-HCl,而大部分抗体会在前两个洗脱级分中洗脱;利用例如Vivaspin 0. 5ml离心装置,合并浓缩此两级分至最终体积(例如0. 2-0. 3ml)。 The fractions were collected in Iml 0. Iml of 1M, pH 8. 0 Tris-HCl, and most of the antibody is eluted in the first two fractions eluted; using, for example Vivaspin 0. 5ml centrifuge device, combined concentrated two minutes to the final volume (for example 0. 2-0. 3ml). 在此浓缩步骤期间,利用PBS进行中间稀释,以将缓冲液由Tris-甘氨酸换成PBS。 During this concentration step, intermediate diluted with PBS to the buffer Tris- glycine replaced by the PBS. 利用透过例如0. 2 μ m注射器式滤器的过滤而使最终浓缩液无菌化,并利用劳里(Lowry)蛋白质测定法(BioRad DC蛋白质测定法)来决定含抗体样本的蛋白质浓度。 For example through the use of 0. 2 μ m syringe filter leaving the final concentrate was filtered sterile, and to determine the protein concentration of antibody-containing samples using Lauri (Lowry) protein assay (BioRad DC protein assay).

[0333] 就大规模纯化而言,以超过滤在中空纤维柱(例如AmershamBiosciences 30,000NMWC/290cm2的中空纤维柱UFP-30-C-3X2MA)上浓缩来自瞬时转染粘附细胞(例如HEK-293T)的2L条件培养基,直至体积降至〜200ml为止。 [0333] For the purposes of large-scale purification, ultra filtration hollow fiber column (for example AmershamBiosciences 30,000NMWC / 290cm2 hollow fiber column UFP-30-C-3X2MA) concentrate from transient transfection adherent cells (for example on HEK- 293T) of 2L conditioned media, until the volume was reduced ~200ml up. 将此浓缩物质(或用于较小型转染,则为纯CM)抽取通过已利用0. ΙΜ,ρΗ 8.0的Tris-HCl加以平衡的12m 1蛋白质A的SEPHAR0SE柱,再以0. 1M,pH 8. 0的Tris-HCl清洗柱,直至UV280的读数确立基线为止。 This concentrate materials (or for smaller transfection, compared with pure CM) has been used to extract by 0. ΙΜ, ρΗ 8.0 of Tris-HCl to balance the 12m 1 protein A SEPHAR0SE column, then 0. 1M, pH 8.0 of Tris-HCl washing the column until UV280 establish a baseline reading so far. 以0. ΙΜ,ρΗ 2. 7的甘氨酸洗脱抗体并收集3ml的级分,通过加入0. ΙΜ,ρΗ 8. 0的Tris-HCl至0. IM的最终浓度来调整含峰蛋白质(peakprotein)的级分的pH值;集合峰级分,通过超过滤(例如在Amicon Ultral5ml离心装置上)将其浓缩至小于7ml的体积,接着利用在PD-10 柱上(Amersham Biosciences/GE-Healthcare)的两独立进程进行去盐化至PBS中。 To 0. ΙΜ, ρΗ glycine 2.7 eluted antibody and collecting 3ml fractions, by adding 0. ΙΜ, ρΗ Tris-HCl at a final concentration of 8.0 to 0. IM adjusted peak containing protein (peakprotein) fractions of pH values; set peak fractions by ultrafiltration (for example, on Amicon Ultral5ml centrifuge device) it is concentrated to a volume of less than 7ml, and then use the PD-10 column (Amersham Biosciences / GE-Healthcare) of two independent processes to Salt to PBS.

[0334] 通过本领域中已知的方法(例如劳里(Lowry)蛋白质测定法(BioRadDC蛋白质测定法)),定量并分析得自于培养上清液的蛋白质。 [0334] by methods known in the art (for example Lowry (Lowry) protein assay (BioRadDC protein assay)), and quantitative analysis derived from the culture supernatant proteins. 在一例示方法中,用SDS-PAGE分析培养上清液;简言之,将蛋白质转移至硝酸纤维素膜,在室温下以5%牛奶/PBS阻断持续1小时,并与缀合有HRP的小鼠抗人类IgG(例如γ -链特异性(Y -chain specific), 1 : 10,000)及小鼠抗人类κ (例如κ -链特异性,1 : 1,000) (Southern Biotech, Cat#9042-05 和#9220-05,Birmingham, AL) 一起培养,以ECL 试剂盒检测讯号。 In one exemplary method, by SDS-PAGE analysis of culture supernatants; Briefly, proteins were transferred to nitrocellulose membranes, at room temperature with 5% milk / PBS blocking for 1 hour with HRP conjugated mouse anti-human IgG (such as γ - chain-specific (Y -chain specific), 1: 10,000) and mouse anti-human κ (such as κ - chain specific, 1: 1,000) (Southern Biotech, Cat # 9042-05 and # 9220-05, Birmingham, AL) incubated in ECL kit signal.

[0335] 在瑞斯特霉素诱导血小板凝集测定中的体内抑制活性:在一例示方法中,测试嵌合NMC-4人类Fc抗体的活性,包括例如对于vWF的结合特异性。 [0335] In Rist adriamycin-induced platelet aggregation inhibitory activity in vivo assays: In one exemplary method, the test NMC-4 human Fc chimeric antibody activity, including for example, vWF binding specificity. 举例而言,血小板凝集测定用标准血小板凝集测定仪(aggregometer) (Bio/Data, model PAP-4)、利用冷冻干燥的人类血小板(Bio/Data,Horsham PA)来进行。 For example, platelet aggregation was measured using a standard platelet aggregation meter (aggregometer) (Bio / Data, model PAP-4), the use of freeze-dried human platelets (Bio / Data, Horsham PA) to carry out. 简言之,将50 μ 1的瑞斯特霉素(例如原液(stock)浓度=15mg/mL) (Bio/Data)及48. 5 μ 1的TBS或测试抗体添加至在400 μ 1体积中含有1 X IO8个冷冻干燥血小板的试管内,在将1. 5 μ 1的纯化vWF (例如终浓度1. 5 μ g/ mL)加入至试管以启动凝集反应前10秒,记录基线读数。 Briefly, 50 μ 1 of Rist adding neomycin TBS or test antibody (e.g. stock solution (stock) concentration = 15mg / mL) (Bio / Data) and 48. 5 μ 1 to 400 μ 1 in volume the tubes containing 1 X IO8 a freeze-dried platelets, the purified vWF (e.g., final concentration 1. 5 μ g / mL) 1. 5 μ 1 is added to the tubes to initiate agglutination 10 seconds before, the recording baseline readings. 估算测试抗体的EC5tl值,其为抑制50%血小板凝集的浓度;与亲本单克隆抗体相较,嵌合体表达出等同于亲本鼠类NMC-4 抗体的效力。 EC5tl estimated value antibody test, which is a 50% inhibition of platelet aggregation concentration; compared with the parent monoclonal antibody, a chimeric expression equivalent to the parent murine NMC-4 antibody potency.

[0336] 再者,为更精确地判定EC5tl值,利用例如改自微孔板(microplate)法的读板器法来测定嵌合抗体。 [0336] Furthermore, in order to more accurately determine EC5tl value, using, for example adapted from microplates (microplate) method was used for measurement plate reader chimeric antibody. 在一例示方法中,利用96孔COSTAR 3603板添加每孔150ml TBS (pH 7. 5)中包含4. 5X IO7个经低聚甲醛固定的血小板,并添加纯化人类vWF(Calbi0Chem,San Diego, CA)至每孔1.5 μ g/mL的最终浓度。 In one exemplary method, using a 96-well plate per well COSTAR 3603 150ml TBS (pH 7. 5) contains a through 4. 5X IO7 paraformaldehyde fixed platelets, and adding purified human vWF (Calbi0Chem, San Diego, CA ) to each well of 1.5 μ g / mL final concentration. 添加一连串浓度的测试抗体后,加入瑞斯特霉素至每孔1. 5mg/mL的最终浓度,以启动凝集反应,并利用设定于37C下持续6分钟、而读取循环之间有20秒机载振荡(on-board shaking)的SPECTRAMAXPLUS读板器(Molecular Devices)监测浊度(例如在405nm下的吸收度)。 After adding a series of concentrations of test antibodies, the final concentration was added to each well Rist ADM 1. 5mg / mL to start agglutination, and the use of the set in 6 minutes at 37 C, and read between cycles There are 20 seconds airborne oscillation (on-board shaking) of SPECTRAMAXPLUS plate reader (Molecular Devices) monitoring turbidity (such as absorbance at 405nm under). 加入(例如20 μ 1/孔)抑制剂化合物或参考MAb (例如AVW-3),培养且监测该混合物达2分钟。 Added (e.g. 20 μ 1 / well) inhibitor compound or the reference MAb (e.g., AVW-3), and the monitoring of the mixture was cultured for 2 minutes. 最后,加入瑞斯特霉素或美洲予 Finally, neomycin or the Americas to join Rist

52头蝮毒蛋白(botrocetin)(例如20 μ 1/孔)培养且监测该混合物达40分钟。 52 venomous snake toxin protein (botrocetin) (for example, 20 μ 1 / well) Training and monitoring the mixture for 40 minutes. 监测吸收度减少的程度(例如吸收度)作为凝集讯号。 Monitoring the absorbance of the reduced level (eg absorbance) as an aggregating signals.

[0337] 比较NMC-4-人类Fc嵌合体与原始NMC-4单克隆抗体、以及所克隆的AJW200抗体。 [0337] To compare the NMC-4- human Fc chimera with the original NMC-4 monoclonal antibodies, and antibodies cloned AJW200. 如图1所示,在瑞斯特霉素诱导血小板凝集测定中,NMC-4嵌合体在活性方面类似于原始匪C-4MAb,且略优于AJW200,其EC5tl值分别为0. 1,0. 17,及0. 27nM。 1, at Wrest adriamycin-induced platelet aggregation assay, NMC-4 chimera similar to the original bandit C-4MAb in the active side, and slightly better than AJW200, its EC5tl values were 0.5 1,0 17, and 0. 27nM.

[0338] 实施例2 :人源化抗体的构建 [0338] Example 2: Construction of humanized antibody

[0339] 人类受体构架的筛选:可利用数据库(例如人类种系数据库、V区数据库(V base)、或卡巴(Kabat)数据库)或公开发表物(例如Kabat等,Sequences of Proteins of Immunological Interest, 1992)来识别鼠类重链及轻链V区所属的亚科(subfamily), 并判定最适的人类种系构架,以用作受体分子。 [0339] Screening of human acceptor framework: You can use a database (such as the human germline database, V region database (V base), or carbachol (Kabat) databases) or published (for example, Kabat et al, Sequences of Proteins of Immunological Interest , 1992) to identify subfamily (subfamily) murine heavy and light chain V region belongs, and determines the optimum human germline framework to be used as acceptor molecules. 其中可利用此等亚科内的VH及VL序列作为受体序列的筛选,可基于序列同源性和/或CDRl及CDR2区的规范结构的匹配性,以帮助保持六个CDR在移植后的适当相对呈递。 Wherein the VH and VL sequences can be used such as a screening receptor subfamily within the sequence may be based on sequence homology matching and / or regulatory structure of CDRl and CDR2 region to help maintain the six CDR after transplantation Relative presenting appropriate.

[0340] 举例而言,假定识别出NMC-4VL构架与κ亚科1 (VKl)的成员之间具有良好同源性,则使用人类V区数据库表示NMC-4的κ轻链属于κ 1亚科。 [0340] For example, assume that the identified frame and NMC-4VL κ good homology between subfamily 1 (VKl) member, using a database of human V regions showing NMC-4 belongs to the κ 1 κ light chain alkylene Section. 观察到:种系序列018 (SEQ ID NO :5)具有⑶R环的规范结构的最高同源性及最佳保存性,就上至⑶R3的全部序列而言,其具有78%的序列同一性(sequence identy);就框架区而言,其具有84%的序列同一性。 Observed: germline sequence 018 (SEQ ID NO: 5) having the highest homology and the best preservation of the canonical structure of ⑶R ring, it is up to the entire sequence ⑶R3 terms, which has 78% sequence identity ( sequence identy); to framework regions concerned, which has 84% sequence identity. NMC-4轻链、人类轻链018(SEQ ID NO :5)、及AAK94808 (得自于018的成熟抗体,用来提供IXDR3及构架4序列,以与此区中的NMC-4相比较)(SEQ IDNO :6)的比对(alignment)显示于表3中,NMC-4与人类抗体之间的差异用粗体字表示(编号是基于卡巴记数制(Kabat, 1978))。 NMC-4 light chain, human light chain 018 (SEQ ID NO: 5), and AAK94808 (obtained from mature antibody 018, used to provide the framework sequences IXDR3 and 4, in this region compared to NMC-4) (SEQ IDNO: 6) alignment (alignment) shown in Table 3, and NMC-4 difference between human antibodies are shown in bold (ID number system based on Kaba (Kabat, 1978)).

[0341 ] 同样地,使用人类V区数据库指出直至构架3的VH序列落在VH亚科IV中。 [0341] Similarly, the use of human V region framework VH sequence database noted until 3 in the fall VH subfamily IV. 在人类VH的IV亚科内,NMC-4VH显示与4_59种系序列(SEQ ID NO :3)具有最高序列同源性, 就直至⑶R3的整个VH而言,其展现对鼠类VH 56%的序列同一性;单就框架区而言,则展现67%的同一性(表4)。 IV within the human VH subfamilies, NMC-4VH display 4_59 germline sequence (SEQ ID NO: 3) having the highest sequence homology to until ⑶R3 entire VH, its show on VH 56% of rodents 47; framework regions alone, it is to show 67% identity (Table 4). 在不受限于本发明的理论下,选择AAC18165. 1(SEQ ID NO :4)以提供HCDR3及构架4比较序列(comparator sequence),因为其在构架1至3与HCDRl及HCDR2中具有与人类种系4-59VH相同的氨基酸序列。 Under the present invention is not limited by theory, selection AAC18165 1. (SEQ ID NO: 4) to provide framework 4 and Comparative HCDR3 sequence (comparator sequence), because of its architecture and 1-3 and HCDRl HCDR2 having the human germline same amino acid sequence 4-59VH. 已知HCDR3为高度分歧的,且FW4为取自于独立基因产物(J)的不同结构域,于是HCDR3及构架4并未被包含在人类V区数据库的VH种系序列中。 HCDR3 known as highly divisive and FW4 is taken from the independent gene product (J) of different domains, so HCDR3 and framework 4 has not been included in the VH germline sequence of the human V region database. 在表4中,将NMC-4VH与AAC18165. 1 (SEQ ID NO :4)序列之间的氨基酸差异以粗体字、其位置加星号以强调。 In Table 4, the NMC-4VH and AAC18165 1 (SEQ ID NO: 4). Amino acid differences between the sequences in bold letters, the position of an asterisk to emphasize.

[0342] 表3NMC-4VL与人类抗体AAK94808的比对,后者具有与人类种系VL,018相同的氨基酸构架、⑶Rl及⑶R2序列 [0342] Table 3NMC-4VL human antibody AAK94808 alignment, which has human germline VL, 018 identical amino acid framework, ⑶Rl and ⑶R2 sequence

[0343] [0343]

53名称 FWl CDRl FW2 53 Name FWl CDRl FW2

—. — — —------ i —---——申~ — — — 2 — * ι ■ — — — * ^― — — — 1 1 “— — - * * * — ^― * -. - - ------- I ------ Application ~ - - - 2 - * ι ■ - - - * ^ - - - - 1 1 "- - - * * * - ^ - *

卡巴号: 1 2345678901234567890123 45678901234 56789012 3456789 NMC-4 VL Kabbah number: 1 2345678901234567890123 45678901234 56789012 3456789 NMC-4 VL

(SEQ ID NO: 2) DIOMTOSPSSLSASLGDRVTISC SASQDlNKYLN WYQQKPDGAVKLLIF (SEQ ID NO: 2) DIOMTOSPSSLSASLGDRVTISC SASQDlNKYLN WYQQKPDGAVKLLIF

018 (ΛΛΚ948ΰ8) 018 (ΛΛΚ948ΰ8)

iSEO ID NO: 6) DIQMTQSPSSLSASVGDRVTITC QASQDISNYLN WYQQKPGKAPKLLIY 名称FW 3 CDR 3 FW4 iSEO ID NO: 6) DIQMTQSPSSLSASVGDRVTITC QASQDISNYLN WYQQKPGKAPKLLIY name FW 3 CDR 3 FW4

卡巴号: 78901 234567890123456789012345678 901234567 8901 234567 NMC-4 VL GVPSRFSC.SGSGTDYSLTISNLEPEDIATYYC QQYEKLPWT FGGGTKLEVK 018(ΑΛΚ94808) GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQYDNLPLT FGGGTKVEIK Kabbah number: 78901 234567890123456789012345678 901234567 8901 234567 NMC-4 VL GVPSRFSC.SGSGTDYSLTISNLEPEDIATYYC QQYEKLPWT FGGGTKLEVK 018 (ΑΛΚ94808) GVPSRFSGSGSGTDFTFTISSLQPEDIATYYC QQYDNLPLT FGGGTKVEIK

[0344] 表4NMC-4VH与人类抗体AAC18165. 1的比对,后者具有与人类种系4_59相同的构架、⑶Rl及⑶R2氨基酸序列 [0344] Table 4NMC-4VH and human antibodies than AAC18165. 1, which has the same 4_59 human germline framework, ⑶Rl and ⑶R2 amino acid sequence

[0345] [0345]

名称 FWl CDRl FW2 CDR 2 Name FWl CDRl FW2 CDR 2

----本------1—水—Jj4 Jjs—;J4------* 一ψ 3 样一4: ^c 一^c—4-------中一5 _ * 氺尔----木一5 * *--- ---- ------ 1- The water -Jj4 Jjs-; J4 ------ * a ψ 3 like a 4: ^ c ^ c-4 ------- one in a _ * Shui Seoul 5 ---- wood a 5 * * ---

卡巴号: 1234567890123456789012345 6789012 345 67890123456789 0123456789012 345 Kabbah number: 6789012345 1234567890123456789012345 67890123456789 0,123,456,789,012,345

NMC-4 VH NMC-4 VH

(SEQ ID NO: 1) QVQLKESGPGLVAPSQSLSITCTVSGFSLTDYGVDWVRQPPGKGLEWLGMIWGDGSTDYNSALKS (SEQ ID NO: 1) QVQLKESGPGLVAPSQSLSITCTVSGFSLTDYGVDWVRQPPGKGLEWLGMIWGDGSTDYNSALKS

4-59(ACC18165. 1) 4-59 (ACC18165. 1)

(SEQ ID NO: 4) QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKS 名称 FW 3 CDR 3 FW4 (SEQ ID NO: 4) QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWIGYIYYSGSTNYNPSLKS Name FW 3 CDR 3 FW4

—*本一7*—*—***一—♦ 9—泽—*一— ———一一11 —— - * This a 7 * - * - *** a - ♦ 9- Ze - * a * - --- eleven 11--

卡巴号: 6789012 3456789012abc3456789 01234 567890abcdef 12 34567890123 NMC-4 VH RLSITKDNSKSQVFLKMNSLQTDDTARYYCVR DPADYGNYDYALDY WGQGTSVTVSS 4-59(ΑΛ€18165. 1) RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR GYRPGVAAHSPFDY WGQGTLVTVSS Kabbah number: 6789012 3456789012abc3456789 01234 567890abcdef 12 34567890123 NMC-4 VH RLSITKDNSKSQVFLKMNSLQTDDTARYYCVR DPADYGNYDYALDY WGQGTSVTVSS 4-59 (. ΑΛ € 18165 1) RVTISVDTSKNQFSLKLSSVTAADTAVYYCAR GYRPGVAAHSPFDY WGQGTLVTVSS

[0346] 由于假设来自小鼠抗体的⑶R直接移植至人类抗体构架造成了抗原结合亲和力的损失(Foote 和Winter J. Mol. Biol. 224 :487-499 (1992) ;Xiang 等,J Mol Biol. 253 : 385-90(1995) ;Homes 等,J. Immunol. 167 :296_301 (2001)),故可能期望使构架中的某些残基突变回到在该等位置上的小鼠残基,此程序称为回复突变(back-mutation)。 [0346] Since it is assumed ⑶R transplanted directly from a mouse antibody to a human antibody framework resulting in a loss of antigen binding affinity (Foote and Winter J. Mol Biol 224: 487-499 (1992); Xiang et, J Mol Biol... 253: 385-90 (1995); Homes, etc., J Immunol 167:.. 296_301 (2001)), it may be desirable to make certain residues framework mutations Back in those positions residue mice, this program called reverse mutation (back-mutation). 例如,表5 显示可能影响CDR构象且可能为回复突变至鼠类残基的潜力候选者的残基(例如以斜粗体字来强调NMC-4与人类构架之间在这些位置上的氨基酸差异)。 For example, Table 5 shows that may affect CDR conformation and possibly back mutation to the murine residue potential candidate residues (such as amino acid differences in these positions between NMC-4 and human framework that emphasizes bold oblique ).

[0347] 表5影响⑶R的构象的构架残基;比较NMC-4及人类受体可变区 [0347] Table 5 ⑶R influence the conformation of framework residues; Comparative NMC-4 receptor and the human variable region

[0348] [0348]

CDR 2 CDR 2

$氺一* 一冰* $ Shui Yibing * a *

0123456 YTSSLHS DASNLET 0123456 YTSSLHS DASNLET

[0349] [0349]

[0350] 已识别出可能涉及重链及轻链的配对以协调⑶R呈递的氨基酸残基(Holmes等, J Immunol. 167 :296-301 (2001)),且其可能为回复突变的候选者。 (. Holmes et, J Immunol 167: 296-301 (2001)) [0350] has identified the pairing may involve heavy and light chains to coordinate ⑶R presentation of amino acid residues, and it may reverse mutation candidates. 在不受限于本发明的理论下,VL区内的残基44,96及98被认为具有重要性,尚有来自残基34,36,38,46,87,89及91的额外贡献。 Under the present invention is not limited by theory, residues VL region 44,96 and 98 are considered important, there are residues from additional contributions 34,36,38,46,87,89 and 91. 在这些残基中,关于VL区的NMC-4与受体018之间唯一有显著不同为残基44,其在NMC-4VL中为缬氨酸(valine),而在人类018构架中为脯胺酸(proline)。 In these residues, on VL region NMC-4 receptor the only significant difference between the residue of 44 018, the NMC-4VL in valine (valine), and 018 in the human framework is preserved tryptophan (proline). 在不受限于本发明的理论下,就VH而言,残基45及103具有重要性,残基35,37,39,47,91,93及95还对VH与VL的界面堆积(interface packing)有贡献。 Under the present invention is not limited to theory, it VH, the residues 45 and 103 importance, residues 35,37,39,47,91,93 and 95 also piled VH and VL interface (interface packing) contribute. 在NMC-4及受体4-59构架的这些界面残基中,唯一差异仅在残基93,其中存在NMC-4中的缬氨酸相较于4-59中的丙氨酸的保守差异(表5)。 In NMC-4 receptor and these interface residues 4-59 frame, the only difference in the residue 93, where there is only a conservative difference NMC-4 in Val compared to 4-59 in alanine (Table 5).

[0351] 比较鼠类及人类种系4-59VH序列的构架序列可得知:在假设对⑶R呈递及界面堆积重要的残基中,许多差异群集在构架3中(残基67,71,73,78,93),额外的差异则在构架2中的残基37及48 ;这些残基在原型(prototype)人源化序列中为回复突变的推定候选者。 [0351] framework sequence comparison of murine and human germline sequences 4-59VH can know: the assumption that the accumulation of ⑶R presentation and interface residues important, many of the differences in the framework of the Cluster 3 (residues 67,71,73 , 78,93), differences in the framework of the additional 2 residues 37 and 48; these residues in the prototype (prototype) humanized sequences for reply putative candidate mutations. 然而,两构架2残基(37V vs 371及48L vs 481)的差异具保守性,因此,第一原型VH 序列可集中于构架3区中的差异,且载有下列由人类至小鼠的回复突变:671^,711(373队F78V及A93V (表6)。合成基因是由Retrogen (加州圣地亚哥)定制合成且被表示为H2,并被克隆入细菌载体pRSFDuet (Novagen,威斯康星州麦迪逊)的NheI及XhoI位点,且被用作以4-59-hu匪CF (SEQID NO : 54)和Hu-VH-R (SEQ ID NO : 55)引物来扩增插入物的模板(表7及9)。以ApaI来消化所合成的片段,以HindIII及ApaI来消化包含在实施例1中所产生的NMC-4嵌合重链的质粒pcDNA6-NMC-HC,且在NTP存在下以克列诺夫片段(Klenow fragment)对含有pcDNA-IgGldm片段的DNA片段进行钝端处理。将含VH的PCR产物缀合至经钝端处理的pcDNA-IgGldm片段,接着利用质粒DNA来转化大肠杆菌宿主菌株JM 109。 利用贝克曼(Beckman) CEQ 8000DNA测序仪来测序各自克隆(clones),以识别出以正确位向表达插入物且具有正确序列的克隆(克隆pcDNA-huVH-IgGldm)。利用此载体作为H4,H5,H6,H7及H8变体的模板,此等变体被用来评估单独地将各残基回复成人类残基的效果(总结于表6中)。利用表7中所述的引物对(对应序列显示于表9)来构建这些变体。 Two frame 2 residues (37V vs 371 and 48L vs 481) difference, however, a conservative, and therefore, the first prototype VH sequences can be concentrated in three districts in the framework of different, and contained the following reply from human to mouse mutation:. 671 ^, 711 (373 teams F78V and A93V (Table 6) synthetic gene is Retrogen (San Diego, CA) custom synthesis and is expressed as H2, and cloned into the bacterial vector pRSFDuet (Novagen, Madison, Wisconsin ) The NheI and XhoI sites, and it is used in 4-59-hu bandit CF (SEQID NO: 54) and Hu-VH-R (SEQ ID NO: 55) primers to amplify insert template (Table 7 and 9). In ApaI digested synthesized fragment was digested with HindIII and ApaI containing NMC-4 to the chimeric heavy chain plasmid pcDNA6-NMC-HC in Example 1 was produced, and in the presence of NTP in grams Lie Nuofu fragment (Klenow fragment) of DNA fragment containing pcDNA-IgGldm fragment was blunt-ended process. The VH PCR product containing conjugated to pcDNA-IgGldm fragment was blunt end treatment, followed by the use of plasmid DNA was used to transform E. coli host strain JM 109. utilization Beckman (Beckman) CEQ 8000DNA sequencer for sequencing the respective clones (clones), to identify the correct orientation and having the correct expression of the insert sequence of clone (clone pcDNA-huVH-IgGldm). Using this vector as a template H4, H5, H6, H7 and H8 variants, such variants are used to evaluate separately the effect of each residue reply residues adult class (summarized in Table 6). Use Table 7 in the The primer pairs (corresponding sequences are shown in Table 9) to build these variants.

[0352] 表6对不同VL及VH变体,并入种系4_59及018种系受体序列的构架残基突变 [0352] Table 6 different VL and VH variants, incorporated into the germline 4_59 and 018 kinds of lines acceptor framework residues mutated sequence

[0353] [0353]

[0354] 表7用来构建人源化重链变体的模板及引物总结 [0354] Table 7 template and primers used to construct humanized heavy chain variants summary

[0355] [0355]

[0356] [0356]

[0357] 比较假设可影响规范结构及界面堆积的残基可知:在NMC-4VL与018人类VL受体构架之间有三处差异(例如残基44,49,及71),因此,可设计出带有三个至鼠类残基的回复突变(例如P44V,Y49F,及F71Y)的原型人源化变体L5 ;此外,此变体被设计成具有由苯丙氨酸改变成亮氨酸(leucine)的残基73,因为在人类抗体谱中,亮氨酸为在此位置上更常见的残基。 [0357] To compare the assumptions can affect the structure and interface specifications accumulate residues known: there are three different (for example, residues 44, 49, and 71) between the NMC-4VL and 018 human VL acceptor framework, therefore, can be designed prototype with three human residues to murine back mutations (e.g. P44V, Y49F, and F71Y) a humanized variant L5; In addition, this variation is designed to have changed by a phenylalanine-leucine (leucine ) residues 73, as in the human antibody repertoire, leucine in this position more common residues. 为产生此变体,便定制合成称为VL4、带有构架变化(例如Y49F,F71Y,及F73V)的变体(Regrogen),并利用表8所列的引物对将其克隆入pETDuet载体内。 To generate this variant, it is custom synthesis called VL4, the variant with the frame change (eg Y49F, F71Y, and F73V) of (Regrogen), and using the primers listed in Table 8 pETDuet cloned into the vector. 以所得的L4-pETDuet载体作为模板,利用表8所列的引物对引进L5的P44V突变;接着将L5变体亚克隆入pIRES DsRed2载体的EcoRI及BamHI位点,以取代鼠类NMC-4VL ;接着利用L5_pIRES DsRed载体作为模板,利用表8所列的引物对来产生L6及L7载体。 In the resulting vector as a template L4-pETDuet using the primers listed in Table 8 for the introduction of mutation P44V of L5; L5 then subcloned into pIRES DsRed2 variant vector the EcoRI and BamHI sites, to replace the murine NMC-4VL; Then use L5_pIRES DsRed vector as a template, primers listed in Table 8 to generate L6 and L7 carrier.

[0358] 表8用来构建人源化轻链变体的模板及引物总结 [0358] Table 8 template and primers used to construct humanized light chain variant summary

[0359] [0359]

[0360] [0360]

[0361] 表9用来构建各种的可变区的引物序列 Primer sequences [0361] Table 9 is used to construct a variety of variable regions

[0362] [0362]

[0364] 平行于克隆这些第一组变体,利用在BLAST搜索中经识别为具有上至HCDR3的所有序列的最高序列同一性(88% )及框架区的89%序列同一性的lDNO.pdb结构(例如2. 3埃分辨率),建立计算机产生的4-59受体种系序列的三维模型。 [0364] parallel to the first set of variants of these clones using the BLAST searches identified as having up to HCDR3 of all sequences of the highest sequence identity of 89% sequence (88%) and framework regions of identity lDNO.pdb structure (for example, 2.3 resolution), the establishment of computer-generated three-dimensional model 4-59 acceptor germline sequence. 选择结构1A0K.Pdb作为具有81%序列同一性的人源化VH序列的模板。 Select structure 1A0K.Pdb as people with 81% sequence identity template VH humanized sequences. 可利用鼠类NMC-4FV的两结晶结构:lA0K.pdb (例如2. 2人分辨率),其已结合抗原(Celikel等,Nat. Struct. Biol. 5 : 189-194 (1998))且还被选为在原型人源化变体的VH结构域中的最佳适配;及1FNS. Pdb (2. OA分辨率),已结合至突变抗原。 Two available crystal structure of murine NMC-4FV: lA0K.pdb (e.g., 2.2 people resolution), which has been combined with antigen (.. Celikel etc., Nat Struct Biol 5:. 189-194 (1998)) and also He was selected as the prototype of variant humanized VH domain of best fit;. and 1FNS Pdb (2. OA resolution), bound to the mutant antigen. 1A0K. pdb及1FNS. pdb两者具有实质上相同的VH/VL界面角(interface angle),故将1A0K. pdb用于重迭人类受体的模型及人源化VH及VL序列的模型。 1A0K. And 1FNS. Pdb pdb both have substantially the same VH / VL interface angle (interface angle), it will 1A0K. Pdb human receptors for overlapping models and humanized VH and VL series model.

[0365] 当重迭NMC-4VH及4-59的骨架结构时,可观察到三区差异。 [0365] When the overlapping skeletal structure of NMC-4VH and 4-59, three-zone differences can be observed. 第一,差异存在于残基H27至H33,其包含HCDRl的一部分。 First, the differences in residues H27 to H33, which includes a portion HCDRl of. 不受限于本发明的理论,这些残基接触共同形成抗原结合位点的一部分的HCDR2及HCDR3。 The present invention is not limited to theory, these residues together form the contact portion of the antigen binding site of HCDR2 and HCDR3. 预测残基34(在鼠类NMC-4VH中为Val,在4_59 序列中为Trp)可能为H27-33环的构象改变的原因,且因此为回复突变的候选者。 Prediction residue 34 (in murine NMC-4VH for Val, in 4_59 sequence of Trp) may H27-33 ring conformational changes of reasons, and thus to reverse mutation candidates. 第二,差异存在于残基H52至H55,其形成⑶R2环的一部分。 Second, differences in residues H52 to H55, which forms part of ⑶R2 ring. 认为构架残基71 (小鼠中的Lys,4_59 中的Val)可能与此差异有关,且因此为回复突变的候选者。 That framework residues 71 (mice Lys, in 4_59 Val) may be related to this difference, and therefore the revertant candidate. 在4-59结构中的三额外残基被识别出可能阻碍抗原结合:H37,相较于小鼠中的Val,其为IIe的保守性变化;H73(相较于小鼠中的Asp,其代表4-59中的和缓变化Thr);及H78,相较于小鼠中的Val,其代表4_59 中的较大变化Phe。 Three additional residues 4-59 of the identified structure may hinder antigen binding: H37, compared to mice Val, which is conserved changes IIe; H73 (compared to mice Asp, which The moderate changes on behalf 4-59 Thr); and H78, compared to mice Val, which represents the major changes 4_59 Phe. 第三,差异存在于CDR3中,可预期其中的多样性。 Third, differences in CDR3 in which diversity can be expected.

[0366] 对于种系018及原型人源化抗体VL结构域的模拟,结构1IGM. pdb (例如2. 3人分辨率)与018具有优异的序列同一性(例如95% ),有四处差异在残基L34,L45,L47, 及L92。 [0366] For the prototype germline and humanized 018 antibody VL domain simulation, the structure 1IGM. Pdb (e.g., 2.3 people resolution) and 018 excellent in sequence identity (e.g. 95%), there are four differences residues L34, L45, L47, and L92. 选择OlBJl.pdb (例如2. 4 A分辨率)作为原型人源化VL的模板,因为其可匹配97/107残基(例如91%序列同一性)。 Select OlBJl.pdb (for example 2. 4 A resolution) as a prototype humanized VL templates, because it can match 97/107 residues (for example 91% sequence identity).

[0367] NMC-4的VL骨架及018VL结构的良好适配性符合序列同一性,唯一明显的结构差异在由残基L39-L45所组成的环中,其接触VH且因此影响堆积接着影响结合口袋(binding pocket)的形状。 Good adaptation of VL skeleton and 018VL structure [0367] NMC-4 in line sequence identity, the only significant structural differences in the ring by the residues L39-L45 composed in contact and thus influence the accumulation of VH then affect binding shape pocket (binding pocket) of. 不受限于本发明的理论,残基44(例如小鼠中的Val、4-59中的Pro)可能为此差异的原因,因此基于计算机模拟而可能为回复突变的候选者。 The present invention is not limited by theory, the reasons residues 44 (e.g., mice Val, in 4-59 Pro) possible for this difference, and therefore may be based on computer simulation and revertant candidate.

[0368] 在瑞斯特霉素诱导血小板凝集测定中的体外(in vitro)抑制活性:为测试计算机模拟预测是否可准确地预测在活性上的效应,将VH2原型变体与VL变体(例如L4,5,6 及7)配对,且将原型L5与VH变体(例如H2,H4,H5,H6,H7及H8)组合。 [0368] In Rist adriamycin-induced platelet aggregation in vitro assays (in vitro) inhibitory activity: In order to test whether a computer simulation to predict accurately predict the effect on the activity, the prototype will VH2 variant VL variants (such as L4,5,6 and 7) pair, and the prototype L5 and VH variants (such as H2, H4, H5, H6, H7 and H8) combination. 通过HEK293T细胞的瞬时转染来产生抗体变体,并如同针对实施方案1中的NMC-4嵌合抗体所述,将其由经过滤的培养上清液中纯化出来。 HEK293T cells by transient transfection to generate antibody variants, and chimeric antibodies as described for the embodiment 1 NMC-4, which was purified by filtration of the culture supernatant were purified by the.

[0369] 瑞斯特霉素诱导血小板凝集测定是利用如实施例1中所述的冷冻干燥血小板加以施行。 [0369] Wrest adriamycin-induced platelet aggregation was measured using as described in Example 1 to implement the purposes of freeze-dried platelets. 针对抗体(一式两份)的一连串稀释液,利用Spectromax读板器(Molecular Devices)测量吸收度的变化、并利用Prism软件来分析数据,以判定各种的纯化抗体变体的EC50值(见例如表9)。 For antibody (in duplicate) of a series of dilutions, use Spectromax plate reader (Molecular Devices) measuring the change in absorbance, and use Prism software to analyze data, to determine EC50 values for various purified antibody variants (see, for example Table 9).

[0370] 人源化抗体的第一型(由H2及L5所组成)展现与亲本嵌合体(例如EC5。为0. 18nM)相同的活性(例如EC5tl为0. 13)(表10),接着测试具有回复至人类构架残基的连续点突变的重链变体(例如变体H2-H6)。 [0370] The first humanized antibody type (composed by the H2 and L5) to show the parental chimera (eg EC5. To 0. 18nM) the same activity (for example EC5tl to 0.13) (Table 10), followed by Testing has returned to continuous point mutations of human framework residues of the heavy chain variants (e.g., variant H2-H6). 变体展现极相似的活性,除了显示出低1. 5倍的EC5_L5-H8变体以外(表9)。 Variants show a very similar activity, in addition to showing the low 1.5-fold beyond EC5_L5-H8 variants (Table 9). 有趣的是,此变体的产率不高(例如其它变体的1/10),此显示人源化重链与轻链之间的相互作用应该已受到此突变组合的负面影响,因为抗体的稳定性取决于重链及轻链的正确组装。 Interestingly, the yield of this variant is not high (e.g., other variants of 1/10), this humanized interaction between the heavy chain and the light chain should have been adversely affected by a combination of mutation, because antibodies Stability depends on the correct assembly of heavy and light chains. 同理,生产并测试L5变体抗体,同样地,回复至人类的变化似乎不影响活性,此出人意表地显示着:不需要改变构架。 Similarly, the production and testing L5 variants of the antibody, in the same manner, changes in response to human activity does not seem to affect this unexpectedly showing: no need to change the frame. 这些结果显示:本预期会产生问题的受体构架与NMC-4可变区构架之间的差异,包括被认为引起由计算机模拟所观察到的结构差异的最显著差异、包括被视为诱导最大的构象差异的差异(例如VH的V71K及T73N与VL的P44V),在效力上完全无影响。 These results show that: a difference of the expected problems with the acceptor framework NMC-4 between the variable region framework, including structural difference is believed to cause by a computer simulation of the observed significant differences, including the maximum induction is considered difference (for example, VH and VL of V71K and T73N the P44V) conformational differences, completely without influence on the effectiveness. 以这些结果作为前提,构建带有完全人类构架的重链变体(例如H9),其代表简单的⑶R-移植VH。 Based on these results as a prerequisite to build heavy chain variants with a fully human framework (eg H9), which represents a simple ⑶R- transplant VH. 测试此变体、以及组合轻链变体、代表将移植至完全人类构架上的简单⑶R的额外变体L9。 Test this variant, and combinations of light chain variants, on behalf of the graft to the simple ⑶R fully human framework on additional variants L9. 出人意表地,直接(straight)⑶R-移植的抗体保留了完整的活性(EC5tl为0. OSnM ;表10)。 Unexpectedly, direct (straight) ⑶R- grafted antibody retains full activity (EC5tl is 0. OSnM; Table 10). 出乎意料之外,这些资料说明:直接由鼠类抗体将CDR移植至所选择的人类构架上,保留了原始鼠类抗体的完整活性,即使所公开的数据显示多个CDR对抗原结合起作用(Celikel等,Blood Cells Mol Dis. 23:123; Celikel等,Nat. Struct Biol 5 :189),因此,预期在6个CDR的相对呈递中的任何微扰皆会影响亲和力。 Surprisingly, these data illustrate: directly from the CDR murine antibody grafted onto selected human framework retains intact the original murine antibody activity, even if the disclosed data show more CDR works for antigen binding (Celikel etc., Blood Cells Mol Dis 23:.. 123; Celikel etc., Nat Struct Biol 5: 189), therefore, contemplated that any perturbation in the opposite presenting six CDR can affect both the affinity.

[0371 ] 表10在瑞斯特霉素诱导血小板凝集测定中,亲本NMC-4嵌合抗体相较于人源化变体的EC5tl值比较 [0371] Table 10 Rist rapamycin-induced platelet aggregation assay, the parent NMC-4 chimeric humanized antibody variants EC5tl value compared to compare

[0372] [0372]

[0373] 人源化⑶R区:分子模拟显示:在NMC-4IXDR1 (例如残基24,30及31)及NMC-4HCDR1 (例如残基27,29,30及34)中,可容忍经改造成人源化轻链及重链的⑶Rl的额外变化;此外,还认为在HCDR2序列中的两残基(例如61及62)代表可容忍的变化(例如残基61的Ser至Pro及残基62的Ala至Ser)。 [0373] The humanized ⑶R Area: Molecular modeling show: NMC-4IXDR1 (for example, residues 24, 30 and 31) and NMC-4HCDR1 (such as residues 27,29,30 and 34), the tolerable engineered adult Additional changes ⑶Rl humanized light chain and heavy chain; moreover, also believes in HCDR2 sequence two residues (such as 61 and 62) on behalf of tolerable variations (such as residues Ser 61 to Pro and residues 62 Ala to Ser). 因此,一连串所谓的“超级人源化”变体(Tan等,2002J Immunol. 169 :1119_1125)便由如表12中所列的模板及引物对(例如代表具有完全人类IXDRl的变体的LlO ;部分代表人源化HCDRl区的H12及H13 (表11))构建出来。 Thus, a series of so-called "super-humanized" variant (Tan et, 2002J Immunol 169: 1119_1125.) Will be the template and the primers listed in Table 12 on (such as fully human IXDRl LlO represents a variant; section representative humanized H12 and H13 HCDRl region (Table 11)) to build it.

[0374] 表11改变鼠类⑶R残基至其4-59对应物的突变[0375] [0374] Table 11 changes to the mutant rodents ⑶R its residues 4-59 counterparts [0375]

超^lA源化 超MA源化VH (鼠类至又类) VL (鼠类至人类) Super ^ lA humanized super MA humanized VH (murine to another category) VL (rodents to humans)

[0376] [0376]

[0377] 在一例示方法中,通过施行一个或多个的测定来测试计算机模拟预测,以判定人源化抗体的活性。 [0377] In one exemplary method, through the implementation of one or more of the assay to test the computer simulation projections to determine the activity of the humanized antibody. 举例而言,H9与Lll变体共转染,且在瑞斯特霉素诱导血小板凝集测定中测试抗体。 For example, H9 and Lll variants were co-transfected, and in Rist rapamycin-induced platelet aggregation assay test antibody. 如表14所示,可容忍引入三处变异以将IXDRl转化成完全为018的IXDRl,且不致引发效力上的明显损失。 As shown in Table 14, it can tolerate the introduction of three mutation to IXDRl transformed into completely 018 IXDRl, and does not cause any significant loss of potency on. 当H12,13,及14变体与L9组合时,H12-L9变体抗体在效力上显示出略有损失;但额外V34W突变恢复了完全的效力,且加入S61P及A62S突变在效力上几无影响,此表示这些残基可在不影响活性的情况下被转化成4-59序列。 When H12,13, and 14 variants L9 combination, H12-L9 variants of the antibody showed a slight loss in potency; but additional mutation V34W resumed in full force, and added S61P and A62S mutation in almost no effect effect, this indicates that these residues can affect the activity without being converted into 4-59 sequence. 其次,IXDR2在维持vWF阻断活性上的重要性,通过构建LlO链的变体加以评价,在该LlO链的变体中,整个LCDR2 (YTSSLHS) (SEQID NO :11)皆利用编码VL变体LlO的质粒作为模板及表11及12 中所列的引物对,而由人类种系018 IXDR2 (DASNLET) (SEQ ID NO : 118)加以取代。 Secondly, IXDR2 importance in maintaining blocking activity on vWF, be evaluated by constructing variants LlO chain, in a variant of the LlO chain, the entire LCDR2 (YTSSLHS) (SEQID NO: 11) encoding VL variants are the use of LlO plasmid as a template and primers in Table 11 and 12 are listed in the right, and from human germline 018 IXDR2 (DASNLET) (SEQ ID NO: 118) to be replaced. 接着将此新构建的变体Lll与H14配对,以产生抗体变体L11-H14。 Then construct this new variant Lll paired with H14, to generate antibody variants L11-H14. 虽然此变体仍展现nM级效力, 但相较于嵌合体,血小板凝集测试中,其活性却降低10倍(EC5tl为1. 63ηΜ),此说明LCDR2可能为人源化抗体达到最佳活性所不可或缺。 Although this variant still show nM potency level, but compared to the chimera, platelet aggregation test, the activity was reduced by 10-fold (EC5tl is 1. 63ηΜ), this description may LCDR2 humanized antibody is not optimal activity or missing.

[0378] 表12如何构建“超级人源化”变体的总结 Summary [0378] Table 12 how to build a "super-humanized" variants

[0379] [0379]

[0382] 接着,剩余鼠类残基(例如H31,H33,及H35)在变体H14的人源化HCDRl中的重要性,通过将这些残基改变至其在VH种系4-59序列中的人类对应部分(例如D31S,G33Y,及D35S)检测。 [0382] Next, the remaining murine residues (such as H31, H33, and H35) in variant H14 humanized HCDRl importance, by changing these residues to their germline VH 4-59 sequence in Human corresponding portion (eg D31S, G33Y, and D35S) testing. 所得的构建体,H15相较于H14中的部分人源化序列GGSISDYGWD(SEQ ID NO: 111),具有HCDRl 的序列GGSISSYYWS(SEQ ID NO :110);最后,将H15 的整个HCDR2转化成VH 4-59 中的人类对应部分(由MIWGDGSTDYNSALKS (SEQ ID NO :8)至YIYYSGSTNYNPSLKS (SEQ ID NO :119),总计7个残基有差异),以建立另一具有完全的人类HCDRl及HCDR2的变体H16。 Finally, the entire HCDR2 converted H15 VH 4;:, HCDRl sequence having GGSISSYYWS (110 SEQ ID NO): The resulting construct, H15 compared to H14 in partially humanized sequence GGSISDYGWD (111 SEQ ID NO) in -59 in the corresponding portion of the human (the MIWGDGSTDYNSALKS (SEQ ID NO: 8) to YIYYSGSTNYNPSLKS (SEQ ID NO: 119), a total of seven residues are different) in order to establish another and having a fully human HCDRl HCDR2 variants H16. 变体H15及H16各自与轻链变体LlO配对,以分别产生抗体变体H15-L10及H16-L10。 H15 and H16 each variant light chain variant LlO paired to generate antibody variants H15-L10 and H16-L10.

[0383] 以血小板凝集测定法评价这些变体,以判定其活性。 [0383] In the platelet aggregation assay such variants evaluated to determine its activity. 表14中所示的数据显示:以人类序列取代整个HCDRl破坏了抗vWF活性。 Data shown in Table 14 show: replace the entire sequence of human anti-vWF activity HCDRl destroyed. 此显示着HCDRl中剩余的三残基(例如在位置H31上的D、在位置H33上的G、及在位置H35上的D)对于保留活性而言具有重要性,即使这些残基可能不会直接接触抗原,如同由Celikel,等(Nat. Struct Biol 5 :189)提出的晶体结构所指示。 This displays the HCDRl remaining three residues (e.g., at position H31 D, G, at position H33 and H35 and D on the position) in terms of importance for the retention of activity, even if these residues may not direct contact with the antigen, as the Celikel, etc. (Nat Struct Biol 5:. 189) crystal structure of the proposed indication.

[0384] 表14 “超级人源化”变体的EC5tl值 [0384] Table 14 "Super humanized" EC5tl value variants

[0385] [0385]

[0386] 实施例3 :抗体的同种型的重构(reformat) [0386] Example 3: reconstruction isotype antibody (reformat)

[0387] 在一例示方法中,由于IgGl为关于补体(complement)活化及引发效应子应答的活性同种型,可能期望将VH变体由突变的IgGl形式重构成IgG4形式,尽管IgG4相对而言较不具活性。 [0387] In one exemplary method, since the IgGl is on complement (complement) activation and priming activity of isotype effector response, it may be desirable to form VH variants by the mutant IgGl IgG4 reconstructed form, although relatively IgG4 with less activity. 例如,将候选VH变体由突变的IgGl形式转化成IgG4形式(见例如SEQ ID NO :144),以产生研发用的候选者;此外,重新改造轻链及重链开放阅读框架(open readingframes),以并入5,端(例如XhoI及HindIII位点)及3,端(例如BamHI及NotI 位点)上的限制核酸内切酶位点,以辅助将其亚克隆至表达载体PST0518的多重克隆位点, 而用于下游细胞系发展及大规模抗体生产(表15)。 For example, the candidate variant VH mutated form of the IgGl converted into IgG4 form (see for example SEQ ID NO: 144), to produce candidates for R & D; in addition, to reinvent the light chain and heavy chain open reading frame (open readingframes) to incorporate the 5 enzyme sites within the limits of nucleic acid end (for example XhoI and HindIII sites) and 3, the end (for example BamHI and NotI site) on to assist subcloned into the expression vector PST0518 multiple cloning site, while the downstream cell line developed for large scale production of antibodies and (Table 15).

[0388] 举例而言,通过以I gG4恒定区取代IgGl恒定区,并在重链表达盒(cassette) 的5,端上引入XhoI及BamHI位点、而在3,端上引入HindIII及NotI位点,将两人源化VH变体H9及H14转化成IgG4形式。 [0388] For example, by I gG4 constant region IgGl constant region substitution and heavy chain expression cassette (cassette) of 5, XhoI and BamHI sites of the introduction of the end, and in the 3, end introduced HindIII and NotI site point, the two variants of the humanized VH H9 and H14 converted IgG4 form. IgGl及IgG4两者在靠近可变区及恒定区的接合处皆包含自然生成的ApaI位点,此位点被用来在IgG4(取代在IgGl)中克隆。 Both IgGl and IgG4 in the variable region and constant region near the junction of all contain naturally occurring ApaI site, this site is used IgG4 (substituted IgGl in) clone. 将BamHI及NotI限制位点置放于序列的3'端上,以辅助稍后至PST0518载体内的亚克隆。 The BamHI and NotI restriction sites placed on the 3 'end of the sequence to aid later to subcloning PST0518 within the vector. 利用Blue HeronBiotechnology (Bothell,WA),定制化合成具有加入BamHI及NotI位点、由ApaI位点至终止密码子(termination codon)的内含子缺失(intron-deleted)的IgG4恒定区序列。 Use Blue HeronBiotechnology (Bothell, WA), IgG4 constant region sequences BamHI and NotI site added by ApaI site to the termination codon (termination codon) deletion of an intron (intron-deleted) customized synthesis. 再者,通过以被改造成在重链可变区的5'端包含HindIII及XhoI且插入IgK信号序列的引物进行重叠PCR(overlapping PCR),将pST0518载体中的信号序列变成IgK前导序列。 Moreover, by being transformed into a heavy chain variable region 5 'end contains HindIII and XhoI and inserted IgK signal sequence primers overlap PCR (overlapping PCR), the signal sequence pST0518 carrier into IgK leader sequence.

[0389] H9及H14重链序列有相同的引物区,因此两者均使用相同的引物及克隆策略。 [0389] H9 and H14 heavy chain sequence with the same primer region, therefore both using the same primers and cloning strategy. 产生两独立的PCR产物,各包含在huNMC4-H9 (及huNMC4_Hl 4)重链可变区中所必须的变化其中之一。 Generates two independent PCR products each containing one change which huNMC4-H9 (and huNMC4_Hl 4) heavy chain variable region as necessary. 利用PlRESdsRed-HULlO作为模板以及引物IgKLF(SEQ ID N0:100)及IgKHnmcR(SEQ ID NO :101)(见表15a及15b),进行在正向引物上包含XhoI及HindIII限制位点且扩增IgK信号肽的PCR反应,接着进行利用ρ⑶NA6-H9(或ρ⑶NA6-H14)作为模板以及引物14VHF(SEQ ID NO : 102)及14VHR(SEQ ID NO : 103)、并与第一PCR 反应重叠30 个核苷酸的第二反应步骤。 PlRESdsRed-HULlO use as a template and the primers IgKLF (SEQ ID N0: 100) and IgKHnmcR (SEQ ID NO: 101) (see table 15a and 15b), carried on the forward primer and contains the XhoI and HindIII restriction site was amplified IgK PCR reaction signal peptide, followed by the use of ρ⑶NA6-H9 (or ρ⑶NA6-H14) as template and the primers 14VHF (SEQ ID NO: 102) and 14VHR (SEQ ID NO: 103), and a first overlap PCR reaction with 30 nuclear nucleotide of the second reaction step. PCR产物包含H9(或H14)可变区以及IgGl恒定区经过ApaI 位点的前五个氨基酸;恒定区的前五个氨基酸在IgGl与IgG4之间并无不同。 PCR product contains H9 (or H14) variable region and a constant region IgGl After the first five amino acids ApaI sites; the first five amino acids of the constant region did not differ between the IgGl and IgG4. 该反应在ApaI位点之后加入NotI位点,以辅助在插入IgG4恒定区之前的可变区的克隆。 After the reaction was added ApaI sites NotI site to aid in the insertion IgG4 constant region before the variable region clone. 通过第三PCR步骤(利用前两步骤的反应产物作为模板、第一反应之正向引物(IgKLF)、及第二反应的反向引物(14VHR)),在上游加入κ前导区;以XhoI/NotI消化来自此反应的产物, 并将其插入以相似方式消化的质粒骨架PCIneo中,此连接(ligation)产生了克隆中间体pCI-WC4-VH9var (或pCI-WC4_VH14var),其含有Ig κ 前导区及NMC4-H9 (或Η14)的可变区。 By a third PCR step (using the first two steps of the reaction product as a template, the forward primer of the first reaction (IgKLF), and the second reaction reverse primer (14VHR)), was added upstream of κ leader; with XhoI / NotI digestion products from the reaction, in a similar manner and inserted into digested plasmid backbone PCIneo in this connection (ligation) clones generated intermediate pCI-WC4-VH9var (or pCI-WC4_VH14var), containing Ig κ leader and NMC4-H9 (or Η14) variable region. 以ApaI及NotI消化来自于Blue Heron Biotechnology且含有重新(denovo)合成的IgG4恒定区的质粒,以凝胶纯化(gel-purified) Ikb的IgG4恒定区片段并连接入ApaI/ NotI 消化的pCI-NMC4-VH9var (或pCI-WC4_VH14var)),如此便产生了pCI_NMC4_VH9 及PCI-NMC4-VH14。 Was digested with ApaI and NotI from Blue Heron Biotechnology and re plasmid containing (denovo) synthesized IgG4 constant region, gel purified (gel-purified) Ikb the IgG4 constant region fragment and ligated into ApaI / NotI digested pCI-NMC4 -VH9var (or pCI-WC4_VH14var)), so they had a pCI_NMC4_VH9 and PCI-NMC4-VH14. 在转化成DH5 α细胞后,对来自各自克隆的质粒插入物进行测序以确认其为正确。 In transformed into DH5 α cells, from each of the plasmid clone inserts were sequenced to confirm that it is correct.

[0390] 表15a用于重链PCR反应中的引物 [0390] Table 15a for the heavy chain PCR reaction primers

[0391] [0391]

[0392] 表15b用于重链构建的PCR反应 PCR reaction [0392] Table 15b built for heavy chain

[0393] [0393]

66 66

[0394] L9及LlO轻链中的变化通过PCR来完成。 [0394] L9 and LlO light chain changes by PCR to complete. 由于L9及LlO轻链有相同的引物区,因此两者均使用相同的引物及克隆策略。 Since L9 and LlO light chains have the same primer region, therefore both using the same primers and cloning strategy. 每一轻链产生两独立的PCR模板。 Each light chain to generate two separate PCR template. 第一PCR步骤并入在5'端上的XhoI及HindIII限制位点;第二反应步骤与第一反应步骤重叠30个核苷酸,且在片段的3'端并入BamHI及NotI位点,此两独立的重叠PCR产物在第三PCR反应中被用作模板,以通过利用来自第一PCR步骤的正向引物及来自第二步骤的反向引物将其扩增,而产生包含这些变化的最终重叠PCR产物;来自第三PCR反应的产物以XhoI/NotI加以消化, 并将其插入于以类似方式消化的质粒pCI-neo (Invitrogen)中,产生质粒pCI-NMC4_VL9及PCI-NMC4-VL10 (用于轻链构建的例示引物及策略见表16a及16b)。 The first PCR step is incorporated at the 5 'end of the XhoI and HindIII restriction sites; the second reaction step of the first reaction step with overlapping 30 nucleotides, and fragments of the 3' end of the BamHI and NotI site incorporated, This two separate overlapping PCR products in a third PCR reaction was used as a template for PCR from the first step by using the forward primer and the reverse primer from the second step will be amplified, generated comprising these changes final overlap PCR product; the product from the third PCR reaction with XhoI / NotI digested them, and insert it in a similar manner digested plasmid pCI-neo (Invitrogen), resulting in the plasmid pCI-NMC4_VL9 and PCI-NMC4-VL10 ( for light chain constructs illustrative primers and strategies Table 16a and 16b).

[0395] 表16a用于轻链PCR反应中的引物 [0395] Table 16a for the light chain PCR reaction primers

[0396] [0396]

[0397] 表16b用于轻链构建的PCR反应 PCR reaction [0397] Table 16b for light chain constructs of

[0398] [0398]

[0399] H9-L9及H14-L10的IgG4同种型是由以上在实施例1中所述的HEK293T细胞而产生,且利用蛋白质A亲和层析法加以纯化;接着以vWF介导的血小板凝集测定法来测试纯化抗体,以判定相对效力。 [0399] H9-L9 and H14-L10 of IgG4 isotype is in HEK293T cells described above in Example 1 to produce, and be purified by Protein A affinity chromatography; followed by vWF-mediated platelet agglutination assays to test the purified antibodies to determine relative potency. 如表17所示,转化至IgG4同种型对效力不生影响。 As shown in Table 17, converted to IgG4 isotype can not have influence on the effectiveness.

[0400] 表17前导抗vWF IgGl及IgG4变体的瑞斯特霉素诱导血小板凝集活性的比较 [0400] Table 17 leading anti-vWF IgGl and IgG4 variants Rist compare rapamycin-induced platelet aggregation activity

[0401] [0401]

[0402] 实施例4 :抗体至vWF或Al结构域的结合 4 [0402] Example: antibody to vWF Al domain binding or

[0403] 克隆His标记(His-tagged)的Al结构域抗原:不受限于本发明的理论,假设NMC-4结合至vWF的Al结构域,此一般仅在vWF被活化时(例如在高剪切情况下)方可达到。 [0403] Cloning of His-tagged (His-tagged) an Al domain antigen: the present invention is not limited by theory, it is assumed NMC-4 to vWF binding domain of Al, this is generally only when activated vWF (e.g. high shear case) in order to achieve. 另一方法,可能期望表达vWF的分离Al结构域,其被揭示可以等于完全活化vWF者的效力结合GPIb-α (Celikel等,1997),因此,克隆Al结构域以作为微孔结合研究的底物。 Another method of separation may be desired expression of vWF Al domain, which is completely disclosed may be equal to those of the effectiveness of activated vWF binding GPIb-α (Celikel et al., 1997), therefore, clone Al domain as a microporous substrate binding studies thereof. 包含全长人类vWF cDNA的质粒克隆是购自于ATCC (目录编号67122),vWF Al结构域(例如残基499-729)是利用引物vWF-Al-For(5,-CCCAGGAATTCCTCGGAACCGCGTTGCAC-3,) (SEQ ID NO: 112)及vWF-Al-Rev(5,-CCGATGCGGCCGCTCACCTCTTGGGCCC CAG-3,)(SEQ ID NO: 113)而自此克隆加以扩增。 Contains full-length human vWF cDNA plasmid clones were purchased from ATCC (Cat. No. 67122), vWF Al domain (e.g., residues 499-729) using primers vWF-Al-For (5, -CCCAGGAATTCCTCGGAACCGCGTTGCAC-3,) ( SEQ ID NO: 112) and vWF-Al-Rev (5, -CCGATGCGGCCGCTCACCTCTTGGGCCC CAG-3,) (SEQ ID NO: 113) to be amplified and cloned since. 将PCR产物以凝胶纯化、再以EcoRI及NotI消化,并将其克隆入pETDuet-Ι载体内。 The PCR product was gel-purified, and then digested with EcoRI and NotI, and cloned into the vector pETDuet-Ι. 将所连接成的产物转化成DH5 α感受态细胞(competent cell),以产生Al结构域的氧化形式。 The product was converted into the connected into DH5 α competent cells (competent cell), to produce an oxidized form of the Al domain.

[0404] 为构建表达大鼠Al结构域的质粒,遵循产品生产商的说明(MolecularResearch Center, Inc.,Cat#DN127, Cincinnati, Ohio),利用DNAzol试剂由大鼠肝脏分离出大鼠染色体DNA ;接着,利用引物Rat-vWF-Al-F(5,-AGCGAATTCCCCCGAACCCCCCCTGCAC AACTTC-3,) (SEQ ID NO: 114)及Rat_vWF_Al-R(5,-AGTGCGGCCGCTTATCACCTTTTGGGTCCTGGTGATGAAACC-3,)(SEQ ID NO :115),将染色体DNA用于PCR反应。 [0404] To construct the expression plasmid Al domain of rat, to follow the product manufacturer's instructions (MolecularResearch Center, Inc., Cat # DN127, Cincinnati, Ohio), use DNAzol agent rat chromosomal DNA was isolated from the rat liver; Subsequently, using primers Rat-vWF-Al-F (5, -AGCGAATTCCCCCGAACCCCCCCTGCAC AACTTC-3,) (SEQ ID NO: 114) and Rat_vWF_Al-R (5, -AGTGCGGCCGCTTATCACCTTTTGGGTCCTGGTGATGAAACC-3,) (SEQ ID NO: 115), the Chromosomal DNA used for the PCR reactions. 将PCR产物以EcoRI及NotI加以消化,并克隆入pETDuct-Ι载体的相同位点中;将所连接成的产物转化入DH5 α感受态细胞。 The PCR product was digested with EcoRI and NotI to, and cloned into the same vector pETDuct-Ι sites; connected to the product transformed into DH5 α competent cells.

[0405] 以25ml的过夜细菌培养物(例如带有质粒p35 [pET-Duet-Rat-A或p36 [pET-Duet-human-A 的Origami B 菌株),接种含有抗生素羧苄西林(Carbencillin)、 卡那霉素、四环素的1公升细菌培养基(LB或2XYT),使培养液在37C下的摇瓶中生长至OD600为0. 6-0. 8。 [0405] In 25ml overnight bacterial culture (eg with plasmids p35 [pET-Duet-Rat-A or p36 [pET-Duet-human-A of Origami B strains), inoculated antibiotics carbenicillin (Carbencillin), kanamycin, tetracycline 1 liter bacterial culture (LB or 2XYT), the culture fluid in shake flasks grown at 37 C to OD600 0. 6-0. 8. 通过加入IPTG至ImM的最终浓度来诱导重组蛋白质的表达;接着,在37C 下继续培养培养液持续另外4-5小时;之后在JA-IO转子(贝克曼)中以6000rpm离心, 收集细菌。 By adding IPTG to a final concentration of ImM to induce expression of the recombinant protein; and then, at 37 C for medium to continue to foster sustained additional 4-5 hours; after JA-IO rotor (Beckman) and centrifuged with 6000rpm collect bacteria . 将细胞沉淀物(cell pellet)于-80C下冷冻,或立即通过使沉淀物在含有两片溶解的完全蛋白酶抑制剂(Roche)的20ml PBS中重新悬浮加以处理,对所得的细胞悬浮液在冰块上施行两次2分钟的细胞破裂循环(例如使用在1-2的固定负载循环设定及输出控制设定下、配备着微量滴管尖头(micro-tip)的Branson Sonifier 250)。 The cell pellets (cell pellet) freezing, or immediately by the precipitate resuspended be treated at -80 C under full dissolution containing two protease inhibitors (Roche) in 20ml PBS, and the resulting cell suspension implementation of two 2-minute cell rupture cycle on ice (for example at a fixed duty cycle setting and output control settings 1-2, is equipped with micro-dropper tip (micro-tip) the Branson Sonifier 250) . 在4C下,以16000rpm于JA-20转子(贝克曼)中离心这些细胞溶解产物(cell lysate)持续30分钟;以0. 45 μ m的针头过滤器过滤上清液,之后利用已在结合缓冲液(5mM的咪唑,0. 3M NaCl, 50mM Tris-HCl, pH 8.0)中平衡过且填充着His-Select HF镍亲和性凝胶(Sigma)的柱, 以将流速调整于lml/min的注射泵来施行层析法。 In at 4 C, the cells were centrifuged at 16000rpm to JA-20 rotor (Beckman) in the lysate (cell lysate) for 30 minutes; to 0. 45 μ m syringe filter of the supernatant was filtered, after which it has binding buffer (5mM imidazole, 0. 3M NaCl, 50mM Tris-HCl, pH 8.0) and equilibrated His-Select HF filled with a nickel affinity gel (Sigma) column, in order to adjust the flow rate of lml / min injection pump to the implementation of chromatography. 在以20ml的结合缓冲液冲洗柱后,以250mM的咪唑,0. 3M NaCl, 50mM Tris-HCl,pH 8.0洗脱蛋白质,并收集Iml的级分;大多数蛋白质在前4个洗脱馏分内便被洗脱。 After 20ml binding buffer to wash the column, to 250mM imidazole, 0 3M NaCl, 50mM Tris-HCl, pH 8.0 proteins were eluted and fractions collected Iml; most proteins within the first four elution fractions then eluted. 在经考马斯(Coomassie)蓝染色的SDS-聚丙烯酰胺凝胶(polyacrylamide gel)上监测蛋白质的大小(〜28kD)及完整性。 Monitoring the size of the protein (~28kD) and integrity in Coomassie (Coomassie) blue stained SDS- polyacrylamide gel (polyacrylamide gel) on. 集合峰级分,若有必要则将其浓缩至2. 5ml,接着利用PD-10柱(Amersham/GE-Healthcare)去盐化成PBS。 Peak fractions collection, if necessary then it is concentrated to 2. 5ml, followed by the use of PD-10 column (Amersham / GE-Healthcare) to salified into PBS. 利用劳里(Lowry)蛋白质测定法(BioRad DC蛋白质测定法)来决定蛋白质浓度。 Protein concentration is determined utilizing Lauri (Lowry) protein assay (BioRad DC protein assay).

[0406] 结合动力学:施行敏感性测定以评估K。 [0406] The binding kinetics: Determination of sensitivity to assess the implementation K. n、K。 n, K. ff及&数值,从而合成并纯化铕(m 螯合剂)抗体缀合物。 Numerical ff and thus synthesized and purified europium (m chelator) antibody conjugates. 利用解离增强型镧荧光免疫测定法(DELFIA),测量这些以铕标记的NMC-4嵌合体及同种型对照抗体至固定化Al抗原的结合。 Utilizing dissociation enhanced lanthanide fluorescence immunoassay (DELFIA), a combination of these to measure Europium-labeled NMC-4 chimeras and isotype control antibody to the antigen immobilized Al.

[0407] 举例而言,以铕标记对照抗体(例如同种型对照组M0PC-21,来自人类骨髓瘤的IgGl/κ ;Sigma-Aldrich, St Louis M0)及NMC-4嵌合体;简言之,将抗体加入经无菌过滤处理的磷酸钠缓冲液(96mM,pH 7. 4),并广泛地透析成磷酸盐缓冲液(PBS,1. 47mM KH2PO4, 8. ImM Na2HPO4,pH 7. 4,138mM NaCl 及2. 67mM KCl),以移除低分子量一级胺。 [0407] For example, europium labeled control antibody (e.g., isotype control group M0PC-21, IgGl from human myeloma / κ; Sigma-Aldrich, St Louis M0) and NMC-4 chimera; short , the antibody was added by sterile-filtered sodium phosphate buffer treatment (96mM, pH 7. 4), and extensively dialyzed into phosphate buffered saline (PBS, 1. 47mM KH2PO4, 8. ImM Na2HPO4, pH 7. 4, 138mM NaCl and 2. 67mM KCl), to remove low molecular weight primary amine. 在4C下,以9500rpm (7000 X g)的JA-20转子在洗过的MicroS印浓缩器中浓缩经透析的抗体持续20分钟,以含有最终浓度为IOOmM NaHCO3, pH 9. 3的PBS调整抗体至4. Omg/ml。 In at 4 C to 9500rpm (7000 X g) of the JA-20 rotor concentrate dialyzed antibody continued for 20 minutes washed MicroS India concentrator to contain a final concentration of IOOmM NaHCO3, pH PBS 9. 3 of adjust antibodies to 4. Omg / ml. 通过以温和地上下吸液,将mAb/碳酸氢盐(bicarbonate)混合物(0. 250ml)混合至含有以Eu3+(Eu-N1_ITC ; Perkin Elmer LifeSciences, Waltham MA)螯合的0. 2mg Nl-(对异硫氰苄基)-二亚乙基三胺-N1,N2,N3,N3-四乙酸(Ni-(p-isothiocyanatobenzyl)-diethylenetriamine-N1, N2, N3, N3-tetra acetic acid)中,使抗体及胺反应性螯合剂的混合物在4C、不搅拌的情况下反应过夜。 By gentle pipetting up and down, the mAb / bicarbonate (bicarbonate) mixture (0. 250ml) was blended to contain with Eu3 + (Eu-N1_ITC; Perkin Elmer LifeSciences, Waltham MA) chelated 0. 2mg Nl- (for benzyl isothiocyanate) - diethylene triamine -N1, N2, N3, N3- acid (Ni- (p-isothiocyanatobenzyl) -diethylenetriamine-N1, N2, N3, N3-tetra acetic acid), allowing a mixture of amine-reactive antibodies and the chelating agent in the 4 C, without stirring overnight.

[0408] 将经标记的抗体混合物上样至以PBS预平衡的独立NAP-10柱(Amersham Biosciences, Piscataway, NJ),利用作为柱缓冲液的PBS收集级分(例如0. 5ml); 利用SpectraMax 384吸收度读板器测定样本的总蛋白质(例如布莱德福试剂(Bradford reagent) ;Bio-Rad Laboratories, Inc., Hercules, CA);于DELFIA 增强溶液(Perkin-Elmer)中以1 : 10,000稀释之后,通过利用Victor2多标记读板器(Perkin-Elmer)的时间分辨荧光法(time-resolved fluorescence, TRF)测定铕。 [0408] The mixture was loaded onto an antibody labeled with PBS pre-equilibrated NAP-10 column independently (Amersham Biosciences, Piscataway, NJ), used as the column buffer PBS fractions were collected (e.g., 0. 5ml); use SpectraMax 384 absorbance plate reader measuring total protein samples (such as Bradford reagent (Bradford reagent); Bio-Rad Laboratories, Inc., Hercules, CA); in DELFIA Enhancement Solution (Perkin-Elmer) to 1:10 After 000 dilution, measured by using a europium Victor2 multi-label plate reader (Perkin-Elmer) time-resolved fluorescence (time-resolved fluorescence, TRF). 集合对蛋白质及铕两者皆为阳性反应的级分,并将其上样至以电泳缓冲液(Running Buffer) (50mM Tris,pH 7. 4和138mM NaCl)预平衡的新NAP-10柱;集合来自这些柱的对蛋白质及以铕标记两者皆为阳性反应的级分,并将其上样至以电泳缓冲液预平衡的PD-10柱,集合对蛋白质及以铕标记两者皆为阳性反应的级分,并通过铕标准液(Perkin-Elmer)加以校正的TRF来测定总蛋白质及铕含量。 Class set of protein and europium both of which are positive points, and loaded onto electrophoretic buffer (Running Buffer) (50mM Tris, pH 7. 4 and 138mM NaCl) pre-equilibrated with the new NAP-10 column; protein and are all set to mark both positive Eu fractions from these columns, and loaded onto a pre-equilibrated with running buffer of PD-10 column, set both the protein and labeled with europium are all positive fractions to determine the total protein content and to TRF and europium europium calibration standard solution (Perkin-Elmer). 接着计算荧光:蛋白质比例。 Then calculate fluorescence: protein ratio.

[0409] 以来自人类(例如在100μ 1/孔中,30mM Tris,pH 7 4及300mMNaCl或不含二价阳离子的PBS中的25ng)或大鼠(例如50ng/孔)的vffF的His-Al结构域包被Immulon-4 板的孔,4C下隔夜培养。 [0409] In (e.g. 100μ 1 / hole, 30mM Tris, pH 7 4 and 300mMNaCl or PBS without divalent cations of 25ng) or rat (e.g., 50ng / well) from the human vffF the His- Al domain coat Immulon-4 hole plate, cultured overnight at 4 C. 在室温下,用清洗缓冲液(Wash Buffer,例如50mM HEPES,pH 7.4, 150mM NaCl,0. 5% Tween-20)清洗反应盘三次,利用以300 μ 1/孔阻断缓冲液(例如含有3. 0mg/ml的无IgG的BSA及0. 叠氮化钠的清洗缓冲液)阻断1小时,且在使用前以清洗缓冲液清洗5次。 At room temperature with wash buffer (Wash Buffer, e.g. 50mM HEPES, pH 7.4, 150mM NaCl, 0. 5% Tween-20) three times washing the reaction disk, with the use of 300 μ 1 / hole blocking buffer (e.g., comprising 3 No IgG in BSA 0mg / ml of sodium azide and 0.5 wash buffer) to block for one hour prior to use and to wash buffer 5 times.

69[0410] 如下施行平衡结合测定。 69 [0410] A binding assay purposes of balance. 将铕_抗体预先稀释入结合缓冲液(例如含有100 μ g/ ml的无IgG的BSA及0. 叠氮化钠的清洗缓冲液)中并上样至96孔板的各孔(例如10 μ 1/孔),以SEALPLATE膜密封住反应盘。 _ Europium antibody diluted into the binding buffer in advance (e.g., containing no IgG in BSA 100 μ g / ml of sodium azide and 0.5 wash buffer) and loaded onto each well of a 96 well plate (e.g. 10 μ 1 / well) in order to seal the reaction disk SEALPLATE film. 振荡反应盘(例如在室温下彡15秒或者60 秒,将Titer Plate Shaker速率设定在4),将其置入含有湿纸巾的Nalgene盒内,于37C 下在封闭盒中培养2小时。 Oscillating reaction disk (for example 彡 15 seconds or 60 seconds at room temperature, the Titer Plate Shaker rate was set at 4), which was placed in Nalgene box containing wet tissue, cultured at 37 C for two hours in a closed box . 就游离标记的测量而言,将上清液样本(4.0μ1)由含有结合混合物的各孔转移至含有DELFIA增强溶液(ΙΟΟμΙ/孔)之一平行孔组中。 Free label on the measurements, the supernatant samples (4.0μ1) is transferred from each well containing a combined mixture to contain DELFIA Enhancement Solution (ΙΟΟμΙ / well), one parallel hole group. 为评估被结合的抗体,以清洗缓冲液清洗具有剩余结合混合物的Al包被的孔五次,在纸巾上轻拍干,且将DELFIA增强溶液(100 μ 1/孔)加入至空孔以测量被结合的抗体。 To assess antibody bound to wash buffer having Al mixture was combined with the remaining packets of five holes, pat dry on paper towels, and the DELFIA enhancement solution (100 μ 1 / well) was added to the void to measure bound antibody. 就测定校正而言,将DELFIA增强溶液(ΙΟΟμΙ/孔)加入未用孔并加入铕标准液(Ι.ΟμΙ/孔)。 Determination of the correction terms on the DELFIA enhancement solution (ΙΟΟμΙ / well) was added unused holes and adding europium standard solution (Ι.ΟμΙ / well). 振荡反应盘(例如在室温下将TiterPlate Shaker速率设定在5持续10分钟),利用Victor2多标记读板器(Perkin-Elmer ffallac, Boston, MA)读取时间分辨荧光(TRF)强度,通过各自抗体的荧光:蛋白质比例(F : P)而将结合情形就铕螯合物含量加以归一化。 Oscillating reaction disk (for example, at room temperature TiterPlate Shaker rate was set at 5 for 10 minutes), use Victor2 multi-label plate reader (Perkin-Elmer ffallac, Boston, MA) to read the time-resolved fluorescence (TRF) strength, through their Fluorescent antibody: protein ratio (F: P) and will be combined with the situation normalized content on europium chelates. 通过将总结合(例如Eu-NMC-4的结合)扣除非特定结合(例如以Eu标记的同种型对照组的平均结合)计算特定结合。 By specific binding total binding (e.g. Eu-NMC-4 binding) deduction of non-specific binding (e.g., labeled with Eu average binding isotype control group) calculations. 以Scatchard (1949)法计算结合位点的数目及Kd值,通过将1 Og (「/ (/?~;7)) 对游离Eu-NMC-4浓度的对数作图以绘制出希尔图来估算结合状况,其中η为每孔的高亲和性结合位点的数目,「为每孔的特异结合Eu-NMC-4mAb的平均数,且游离Eu-NMC-4嵌合体则由溶液相中所测量的TRF读数加以计算。 In Scatchard (1949) method to calculate the number of binding sites and the Kd value by 1 Og ("/ (/ ~;? 7)) of the number of free Eu-NMC-4 concentration plotted to draw a diagram Hill to estimate the binding condition in which η is the high-affinity binding sites for each of the number of holes, "for the specific binding of each hole Eu-NMC-4mAb average, and a free Eu-NMC-4 chimera by the solution phase readings measured in TRF be calculated.

[0411] 此分析揭示出两类结合位点:Kd为0. 37nm的高亲和性位点及Kd为5nM的低亲和性位点;同理,对来自抗His mAb所捕获的大鼠vWF的His_rAl的结合还展现出具有0. 19 及3 4nM的Kd值(表17)的两类结合位点。 [0411] This analysis revealed two classes of binding sites: Kd is 0. 37nm high affinity site and a low affinity Kd of 5nM sites; Similarly, the rats from anti-His mAb captured binding of vWF His_rAl also show two classes of binding sites with a Kd value of 0.19 and 3 4nM (Table 17).

[0412] 利用相同规则决定结合动力学,除了在不同时点以指示浓度(100μ 1/孔)的以铕标记的抗体代替清洗缓冲液以外。 [0412] using the same rules to determine the binding kinetics, in addition to different points in the indicated concentrations (100μ 1 / well) with europium-labeled antibody instead of washing buffer outside. 立即封住反应盘、振荡(例如在室温下将Titer Plate Shaker速率设定在4持续15秒)、且在37C下培养持续一段指定时间;以清洗缓冲液清洗含有结合混合物的反应盘五次,在纸巾上轻拍干,记录每一反应盘的清洗时间。 Immediately sealed reaction plate, oscillation (for example, at room temperature Titer Plate Shaker rate was set at 4 for 15 seconds), and incubated for a specified period at 37 C for; to wash buffer reaction mixture containing binding plate five times, pat dry on paper towels, cleaning disc recording each reaction time. 如上所述,将DELFIA增强溶液(ΙΟΟμΙ/孔)添加空孔中以测量结合标记。 As described above, the DELFIA enhancement solution (ΙΟΟμΙ / well) added an empty hole to measure binding tag. 通过利用Prism软件(GraphPad Software Inc.,San Diego, CA)以下列方程式来对时间拟合特异结合,测量每一抗体浓度的表观结合速率(apparent on-rate)k。 By using Prism software (GraphPad Software Inc., San Diego, CA) in the following equation to fit the specific binding of time, measuring the apparent concentration of each antibody binding rate (apparent on-rate) k. n,app。 n, app.

[0413] B = Bmm-(l_e-k。一.') [0413] B = Bmm- (l_e-k. A. ')

[0414] 将表观结合速率(k。n,app)对Eu-mAb浓度作图,数据用线性方程式拟合: [0414] The apparent binding rate (k.n, app) plotted against the concentration of Eu-mAb, the data fit a linear equation:

[0415] k。 [0415] k. n,app = k。 n, app = k. n[mAb]+koff n [mAb] + koff

[0416] 其中结合速率常数k。 [0416] wherein the binding rate constant k. n为拟合出的斜率,[mAb]为Eu-NMC-4的浓度,而解离速率k。 n is the slope fitted, [mAb] is the concentration of Eu-NMC-4, and the dissociation rate k. ff 则为拟合出的截距。 ff the intercept was fitted.

[0417] 所计算出的与来自人类或大鼠vWF的His-Al结合的NMC-4嵌合体的解离半衰期通过下列方程式加以计算: [0417] The calculated solution NMC-4 chimera with His-Al or rat derived human vWF binding to be calculated from the half-life by the following equation:

_ In(2) _ In (2)

[0418] '\'2.dis.wc =T [0418] '\' 2.dis.wc = T

Κΰ Κ ΰ

[0419] Eu-NMC-4至人类或大鼠vWF的His-Al的特异结合符合单指数结合方程式,由此式可获得关于每一标记抗体的浓度的表观结合速率k。 [0419] Eu-NMC-4 to His-Al specific human or rat vWF binding accord binding single exponential equation, whereby the formula can be obtained on the apparent concentration of each labeled antibody binding rate k. n, app(例如来自指数结合曲线拟合的常数k)。 n, app (such as binding constants k from the exponential curve fitting). 如k。 As k. n,app对Eu-NMC-4浓度的图式中所示:对两抗原的表观结合速率皆为剂量依赖型的。 n, app for Eu-NMC-4 concentrations shown in the drawings: The two antigen binding rates are all apparent dose-dependent. 结果整理于表17中,且揭示出NMC-4嵌合体对于人类Al结构域具有0. 32士0. 07nM 的Kd值,而对于大鼠Al结构域则具有0. 28士0. OlnM的Kd值。 Collate the results in Table 17, and reveals the NMC-4 chimera humans Al domain has 0.32 persons Kd value 0. 07nM, and for rats Al domain is having 0.28 Guests 0. OlnM of Kd value. 在两情况中,这些结果与由平衡结合所测定的Kd值极为一致。 In both cases, these results are very consistent with the Kd determined by equilibrium binding value. 这些结果还显示:两Al物种的抗原-抗体复合物皆可长久存在,人类及大鼠抗原的体外解离半衰期则分别为44及69分钟。 These results also show: two Al species antigen - antibody complexes Jieke long existence, human and rat antigens in vitro dissociation half-life of respectively 44 and 69 minutes. [0420] 表18 Eu-NMC-4嵌合体与来自人类及大鼠vWF的His-Al结构域的动力及平衡结合(37C下) [0420] Table 18 Eu-NMC-4 chimera with the power and balance in humans and rats from the His-Al vWF binding domain (37 C below)

[0423] tk。 [0423] tk. n app_t剂量依赖的截距 n app_t dose-dependent intercept

[0424] t tEU-NMC-4与直接包被的抗原(人vs.大鼠)的结合的Kd,对T检测的显著性为不具统计学显著性(P = ο. 6892)。 [0424] t bound Kd tEU-NMC-4 directly coated antigen (human vs. rat), for the detection of significant T is not statistically significant (P = ο. 6892).

[0425] 误差以SEM表示。 [0425] In the error SEM.

[0426] 可施行竞争结合研究以判定Ki值(例如抗原的相对亲合力的量度)。 [0426] Competitive binding studies can be implemented to determine Ki values (such as antigen affinity relative measure). 除了在此案例中以外,如上所述施行结合动力的测定,施用80μ 1/孔的结合缓冲液(例如含有100μ g/ ml的无IgG的BSA及0. 1 %叠氮化钠的清洗缓冲液),接着施用10 μ 1/孔Eu-NMC-4或无铕标记的竞争剂及10 μ 1/孔的无标记竞争抗体,范围自10_12Μ至10_7Μ的连续稀释一式两份; 以铕标记的抗体的最终浓度为为ΙΟΟρΜ。 In addition to this case, the implementation of dynamic binding assay as described above, administration of binding buffer 80μ 1 / well (e.g., containing 100μ g / IgG-free BSA ml and 0.1% sodium azide wash buffer ), followed by administration of 10 μ 1 / hole Eu-NMC-4 or no competition europium labeled agent 10 μ 1 / hole competition unmarked antibodies range from duplicate serial dilutions 10_12Μ to 10_7Μ; and with europium-labeled antibody The final concentration of ΙΟΟρΜ. 在螯合剂DTPA(1 μ Μ)存在下,非特异背景结合的水平大幅降低,故此也包含在竞争结合测定中;此外,通过已利用碘乙酰基(iodoacetyl) 凝胶以从蛋白质制备液中移除任何还原的Al而加以纯化的His-Al令各孔的包被最优化。 In the chelator DTPA (1 μ Μ) the presence of non-specific background binding levels greatly reduced, therefore also included in the competition binding assay; In addition, by utilizing already iodoacetyl (iodoacetyl) to move from the gel preparation liquid protein In addition to any reduction of Al and be purified His-Al makes each hole package is optimized. 培养混合物达3. 75小时,以使反应达到完全平衡,清洗并如上述以TRF判定结合的标记抗体。 Incubation mixture of 3.75 hours to complete the reaction to reach equilibrium, as described above in order to clean and TRF determining bound labeled antibody. 利用Prism软件(GraphPad,Inc.),以“一位点竞争”模型拟合出抑制曲线以获得IC5tl 值;并利用由平衡结合实验的Scatchard分析所测量的Kd值,以Cheng & Prusoff (1973 Biochem Pharm. 22 :3099)的方程式计算&。 Using Prism software (GraphPad, Inc.), A "one site competition" model fitted inhibition curves for IC5tl value; and using Kd values from the Scatchard analysis of equilibrium binding experiments measured to Cheng & Prusoff (1973 Biochem Pharm 22:. equation 3099) calculations &.

[0427] 通过比较由无标记的NMC-4嵌合体的同源竞争所获得的Ki值,与Eu-NMC-4结合至抗原所测量到的亲和力(Kd),来说明竞争结合测定的标准化。 [0427] Ki values by comparing the unmarked homologous competition NMC-4 chimera obtained with Eu-NMC-4 binding to antigen measured affinity (Kd), to illustrate the standardized competition binding assay. 就对人类His-Al的同源竞争而言,NMC-4嵌合体为Eu-NMC-4结合的强力抑制剂,Ki值为0. 28士0. 06nM(表19),与所观察到的Ki值0. 32士0. 07nM—致;同理,就对大鼠His-Al的同源竞争而言,NMC-4嵌合体具有0.297士0. 128nM的&值(表18),与0. 276士0. OllnM的Kd值一致。 Homologous to compete in terms of human His-Al, a potent inhibitor of NMC-4 chimera of Eu-NMC-4 binding, Ki value of 0.28 persons 0. 06nM (Table 19), and the observed Ki values of 0.32 persons 0. 07nM- cause; Similarly, the homologous rat His-Al competitive terms, NMC-4 chimera has 0.297 disabilities 0. 128nM of & values (Table 18), and 0 consistent with Kd values. 276 persons of 0. OllnM. 相较之下,无标 In contrast, non-standard

71记的同种型对照(来自人类骨髓瘤血浆的IgGl/ κ )在Eu-NMC-4结合至Al抗原上并无抑制效应。 71 remember isotype control (plasma from human myeloma IgGl / κ) bound Eu-NMC-4 to no inhibitory effect on Al antigen.

[0428] 表19所选择的人源化NMC变体的结合活性(竞争测定的Ki)比较 The selected binding activity [0428] Table 19 NMC humanized variants (competitive assay Ki) Comparison

[0429] [0429]

[0430] 接着利用竞争结合测定来测试人源化NMC-4变体H9L9IgGl及IgG4的效力。 [0430] A competition binding assays to test the use of humanized NMC-4 variant H9L9IgGl and IgG4 effectiveness. 此两同种型的⑶R-移植H9L9变体显现出相同nM活性,尽管效力上小于同源NMC-4嵌合体(表19)。 This two isoform ⑶R- transplant H9L9 variants showing the same activity nM, although the effect is less than the homologous NMC-4 chimera (Table 19).

[0431] 以竞争结合测定法来测试AJW200抗体。 [0431] In competitive binding assay to test AJW200 antibodies. 相较于与Eu-NMC-4结合至His-Al竞争的人源化NMC-4变体,Eu-NMC-4结合至His-Al被AJW200强化,其EC50为2IOpM(图2)。 Compared with the Eu-NMC-4 competition binding to His-Al humanized NMC-4 variants, Eu-NMC-4 binding to His-Al is strengthened AJW200, EC50 of 2IOpM (FIG. 2). Eu-NMC-4结合至His-Al的希尔图显示出接近统一的希尔斜率(nH = 0. 984及0. 957),与在无协同性(cooperativity)下与单一类型的结合位点的结合一致。 Eu-NMC-4 binding to His-Al Hill diagram showing nearly uniform Hill slope (nH = 0. 984 and 0.957), and in the absence of cooperativity (cooperativity) under a single type of binding site binding agreement. 相较之下,在1.8nM 或20nM AJW200存在时(分别见图2C及2D)的Eu-NMC-4结合至His-Al的希尔图显现大于1的希尔斜率(nH = 1. 548及1. 201),此表示正协同性(cooperativity)。 In contrast, when the presence of 1.8nM or 20nM AJW200 (see Figures 2C and 2D) of Eu-NMC-4 binding to His-Al Hill pattern exhibits greater than 1 Hill slope (nH = 1. 548 and 1.201), this represents a positive synergistic (cooperativity). 由AJW200介导的正协同性在两不同日所进行的两独立实验中被观察到,这些结果不仅证实了NMC-4结合至与AJW200不同的vWF的Al结构域上的结合位点,还指出至少就分离的Al片段而言, AJW200使匪C-4能够结合至GPIb- α结合位点。 AJW200 mediated by the positive synergy in two independent experiments conducted by two different days was observed. These results not only confirmed the binding sites NMC-4 binding to and AJW200 different from the Al domain of vWF, but also pointed out Al fragment at least in terms of the separation, AJW200 make bandit C-4 is capable of binding to GPIb- α binding sites.

[0432] 实施例5 :抗体阻断血小板附着的能力 [0432] Example 5: Antibodies ability to block platelet adhesion

[0433] 抗体封闭在vWF上的GPIb-α受体结合位点的证实是其在天然流动条件下对抗vWF-GPIb相互作用的能力。 [0433] vWF antibody blocking on the GPIb-α receptor binding sites proved its ability to fight vWF-GPIb interaction under natural flow conditions. 已由Moake及其同僚(1986, J ClinInvest. 78 : 1456-61)发展出的一方法发现以下事实:当以组胺活化内皮细胞时,其分泌出超大型vWF(ULvWF),其中Al结构域是处于开放(例如活性)构象中。 By Moake and colleagues (1986, J ClinInvest 78:. 1456-61) developed a method discovered the following facts: When activated endothelial cells when histamine, which secrete large vWF (ULvWF), in which the Al domain It is in the open (for example, active) conformation. 因ADAMS-13介导的ULvWF的切割,ULvWF在引入血菜时迅速瓦解(Dong等,2002 Blood 100:4033-9)。 ADAMS-13 because ULvWF mediated cleavage, ULvWF dish when introducing blood rapid disintegration (Dong et, 2002 Blood 100: 4033-9).

[0434] 在一例示方法中,分出第一代(passage) (Pl)HUVEC,并以IX IO5细胞/培养皿的密度接种至35mm的培养皿上,培养7天,且在第7天(在其100%汇合后2_3天)时使用。 [0434] In one exemplary method, the separation of the first generation (passage) (Pl) HUVEC, and IX IO5 cells / dish seeded at a density on 35mm petri dishes, cultured for 7 days and on day 7 ( Use 2_3 days) after its 100% confluence. 在1. 2mL/min的流速下,以CFSE标记的人类血小板轻易地附着至HUVEC (图3A);当在室温下以25 μ M的组胺预处理细胞达10分钟时,更多血小板附着至HUVEC (图3Β);当血小板在含有浓度为10μ g/ml的NMC-4的缓冲液中灌注上单层时,此种血小板的附着被完全抑制(图3C);而当血小板在浓度为18 μ g/ml的抗GPIb-α抗体(例如ΑΚ2)存在下被灌注时, 则血小板的附着受到部分阻断(图3D)。 At a flow rate of 1. 2mL / min to CFSE labeled human platelet easily attached to HUVEC (Fig. 3A); when at room temperature at 25 μ M of histamine pretreatment of cells with up to 10 minutes, more platelet adhesion to HUVEC (Fig 3Β); when the concentration of platelets containing 10μ g / ml of buffer perfusion NMC-4, on the single, this was completely inhibited platelet adhesion (Fig. 3C); and when the platelet concentration of 18 μ g / ml of anti-GPIb-α antibody (e.g. ΑΚ2) is the presence of perfusion, the platelets is partially blocked by the adhesion (FIG. 3D). 相较之下,浓度为18μ g/ml的小鼠对照IgG并未阻止血小板附着至vWF聚合物上(图3E),此显示血小板附着至HUVEC确实是由内皮衍生的vWF与血小板GPIb- α之间的相互作用介导。 In contrast, the concentration of 18μ g / ml of the control mouse IgG did not prevent platelet adhesion to vWF polymer (Figure 3E), this is indeed a platelet adhesion to HUVEC by the endothelium-derived vWF and platelet GPIb- α of mediated by the interaction between. 当从每回合所截取的20个图像测量被血小板所覆盖的面积并利用Compix软件加以定量时,相较于对照抗体所造成的可忽略效应, NMC-4将血小板附着性减少> 95%。 When measuring size from 20 images taken by each turn of the platelet covered and use Compix software to quantify the effect is negligible compared to the control antibody caused, NMC-4 platelet adhesion reduction> 95%.

[0435] 实施例6 :抗体预防血管阻塞的能力 6 [0435] Example: ability to prevent blood vessels blocking antibodies

[0436] 利用动脉血栓的氯化铁模型,以评估NMC-4嵌合体及人源化衍生物(例如Η14, L10)相较于AJW200的抗血栓活性。 [0436] The use of arterial thrombosis ferric chloride model to assess NMC-4 chimeric and humanized derivatives (for example Η14, L10) compared to AJW200 antithrombotic activity. 对侧颈动脉是透过唾腺及随附脂肪组织重新定位至切口的头颅侧而分离。 Contralateral carotid artery is through salivary glands and the accompanying fat tissue repositioned to the head side of the incision and separation. 使颈动脉外露,并将其置于折叠成可作为摆放颈动脉的支架且提供氯化铁(7. 5% )溶液的表面的一片滤纸(例如4mmX5mm)上。 So that the exposed carotid artery, and folded so as to be placed as a stent placed in the carotid artery and provides ferric chloride (7.5%) solution of a surface of the filter paper (e.g. 4mmX5mm) on. 在施加FeCl3溶液4分钟后, 置放流量探测器于颈动脉周围,并利用Transonic Systems Inc.流动系统(Ithaca,NY)测量流量,直至阻塞时(在对照大鼠中一般为10分钟)或45分钟为止。 After 4 minutes FeCl3 solution applied, flow detector placed around the carotid artery, and the use of a flow system Transonic Systems Inc. (Ithaca, NY) measuring the flow rate till the congestion (in control rats typically 10 minutes) or 45 minutes so far. 在1 μ 1/g大鼠体重的体积下,4只大鼠的各组(盐水的η = 6)皆投以范围例如由5至0. 01mg/kg的NMC-4嵌合体、V14、L10、AJW200或对照IgG的4种剂量。 In volume 1 μ 1 / g body weight of rats, four rats in each group (saline η = 6) are cast in the range, for example from 5 to 0. 01mg / kg of NMC-4 chimera, V14, L10 , AJW200 or control IgG four doses. 无菌过滤抗体制备液,并在使用作动物研究之前,在遵照生产商的规则下利用LIMULUS AM0EB0CYTE ASSAY试剂盒(BioWhittaker) 加以测试,以确保低内毒素含量;以HPLC分析估计单分散性(mono-dispersity)。 Antibody preparation was sterile filtered and before use as animal studies, in accordance with the rules under the manufacturer's use LIMULUS AM0EB0CYTE ASSAY kit (BioWhittaker) to be tested to ensure low endotoxin content; HPLC analysis to estimate monodisperse (mono -dispersity).

[0437] 如图4所示,NMC-4及AJW200两者大幅地抑制了血管阻塞。 [0437] As shown in FIG, NMC-4 and significantly inhibited both AJW200 4 vessel occlusion. 匪C-4在剂量为0. 03 及0. lmg/kg下显现出类似于AJW200的ED5tl,人源化衍生物H14,LlO还显现出类似的活性, ED50介于0. 03及0. lmg/kg剂量之间。 Bandit C-4 at a dose of 0.03 and 0. lmg / kg showing similar AJW200 of ED5tl, humanized derivative H14, LlO also showing similar activity, ED50 of between 0.03 and 0. lmg / kg dose between.

[0438] 实施例7 :抗体对出血时间及失血的影响 7 [0438] Example: antibodies on bleeding time and blood loss

[0439] 失血有时为与抗血小板剂(例如抗vWF抗体)相关的有害副作用,因此,可能必须评估人源化NMC-4抗体促成出血并发症的可能性。 [0439] hemorrhagic adverse side effects sometimes associated with the anti-platelet agent (e.g., anti-vWF antibodies), therefore, it may be necessary to assess the humanized antibody NMC-4 to promote the possibility of bleeding complications. 为此,实施标准出血时间测定,在施行尾部横切之前先施用30分钟的抗体对照、NMC-4嵌合体、或AJW200。 To this end, the implementation of standards bleeding time measurement, in the implementation of the rear cross 30 minutes before the first administration of antibody control, NMC-4 chimera, or AJW200. 就尾部横切而言,切下尾部的末端(例如0. 5mm)并将尾部置入已知体积的温盐水中,测量停止出血所需要的时间;于估算出血时间过程中,还通过估算在盐水中所收集的血球的血红蛋白含量来测量失血量。 Cross on the tail, the cut end of the tail (for example 0. 5mm) and tail into a known volume of water temperature and salinity, measure needed to stop the bleeding time; in bleeding time estimation process, but also by estimating the brine hemoglobin blood cells collected to measure the amount of blood loss. 为此,以低速离心使红血球成粒状,将其悬浮于含有TritonXlOO的盐水中,调整最终体积为5ml,通过测定在420nm下的吸收度而测量溶液的血红蛋白浓度。 For this reason, in order to make the red blood cells into a granular low-speed centrifugation, suspended in saline containing TritonXlOO, the final volume adjusted to 5ml, by measuring the absorbance at 420nm measured under the hemoglobin concentration of the solution.

[0440] NMC-4嵌合体显现出对于显著增加的出血时间,与人源化衍生物H14-L10相同的ED5tl剂量0. 09mg/kg ;在与此两抗体在动脉血栓的FeCl3模型中的效力相关联的剂量0. 03mg/kg,并未有出血时间延长或失血量增加的情形。 [0440] NMC-4 chimera showing the significant increase in bleeding time, humanized derivatives of H14-L10 ED5tl same dose of 0. 09mg / kg; in the other two antibody efficacy in arterial thrombosis FeCl3 model dose associated 0. 03mg / kg, did not appear to have prolonged bleeding or increased blood loss situation. NMC-4嵌合体及其人源化衍生物显现出比AJW200 (其在大鼠中出血时间增加时的ED5tl更接近其抗血栓活性时的ED5tl剂量)略高的ED5tl剂量应答(dose response),此显示NMC-4相较于AJW200提供了更加改良的治疗窗口。 NMC-4 chimera and humanized derivatives show than AJW200 (ED5tl increase bleeding time in the rat when it is closer to ED5tl dose when its antithrombotic activity) ED5tl slightly higher dose response (dose response), this AJW200 NMC-4 compared to provide a more improved therapeutic window.

[0441] 表20 NMC-4嵌合体及其人源化衍生物H14,LlO相较于AJW200在大鼠中的出血时间及失血量上的效应 [0441] Table 20 NMC-4 chimera and humanized derivatives of H14, LlO compared to the effect on bleeding time and blood loss in rats of AJW200 in

[0442] [0442]

[0444] 这些抗体的有害副作用在止血上的另一参数为失血量,其是在测定出血时间时针对所收集的血液加以测定。 [0444] harmful side effects of these antibodies on the other parameters of hemostasis is blood loss, which is measured in bleeding time be measured against the collected blood. 又,在剂量高于0. lmg/kg时,这些抗体诱发出明显的失血量; 然而,在剂量为0. 3及3mg/kg时,相同剂量下AJW200引发比NMC-4嵌合体高出更多的失血量,尽管这些差异仅在3. Omg/kg组(其中n = 4而非η = 3)接近在统计上的显著性(表20)。 And when, at doses higher than 0. lmg / kg, these antibodies induce a significant blood loss; however, when at a dose of 0.3 and 3mg / kg, the same dose higher than AJW200 initiator NMC-4 chimera more more blood loss, although these differences only in 3. Omg / kg group (where n = 4 instead of η = 3) close in statistically significant (Table 20). Η14,LlO抗体变体对亲本NMC-4嵌合体并未表达出明显的差异。 Η14, LlO antibody variants of the parent NMC-4 chimera did not express significant differences.

[0445] 实施例8 :抗体在血小板及白血球循环数量上的效应 8 [0445] Example: antibodies and white blood cells circulating in the number of platelet effects

[0446] 假设某些抗血小板剂可抑制血栓形成,而同时可能引发血小板减少症(thrombocytopenia)(Hansen 等,J Pharmacol Exp Ther. 298 : 165-71 (2001))。 [0446] Certain assumptions antiplatelet agents inhibit thrombus formation, while may cause thrombocytopenia (thrombocytopenia) (Hansen et, J Pharmacol Exp Ther 298:. 165-71 (2001)). 为测定NMC-4在被治疗动物中的白血细胞(WBC)及血小板循环数目上的效应,便以lmg/ml 的NMC-4注射(例如以静脉方式)至重量230-260g的5只大鼠的一组,而以载体对照(vehicle control)(例如外科手术级PBS)注射至3只大鼠的对照组。 NMC-4 as measured in animals treated white blood cells (WBC) and platelet effect on the number of cycles, then to lmg / ml for NMC-4 injection (e.g. intravenously) to five rats of 230-260g weight a group, and to vehicle control (vehicle control) (e.g., surgical grade PBS) was injected into 3 rats in the control group. 在注射之前,以例如HEMAVETHEMATOLOGY ANALYZER™(Drew Scientific),利用尾部出血测量基线血细胞数量。 Prior to injection, for example HEMAVETHEMATOLOGY ANALYZER ™ (Drew Scientific), the use of the number of baseline tail bleeding blood cell measurements. 注射入抗体或盐水后,在通过利用毛细吸管从未麻醉大鼠眼后抽取,在上至48小时(例如30分钟、2,4,24,及48小时)的预定时间点时收集血液样本,接着将约40 μ L的血液转移至含有L酸化柠檬酸葡萄糖抗凝液(ACD)的管中,并立即在HEMAVET细胞计数器中取样, 以测定血小板及白血细胞的数量。 After injection into the antibody or saline, blood samples were collected at the time by using a capillary pipette never anesthetized rat eyes after extraction, in up to 48 hours (for example 30 minutes, 2,4,24, and 48 hours) of a predetermined point in time, then about 40 μ L of blood was transferred to contain L acidified citrate dextrose anticoagulant liquid (ACD) tubes, and immediately HEMAVET cell counter sampling, to determine the number of platelets and white blood cells. 就各次抽血而言,两眼交替进行取样。 On blood each time, the two alternately sampled.

[0447] 在所分析的任何时间点,NMC-4对血小板数量的影响甚微;在注射后30分钟时,观察到白血细胞短暂减少了37. 5% (ρ = 0. 016),但在注射入PBS药物时,也观察到类似的减少现象。 [0447] At any point in time analysis, NMC-4 has little effect on platelet count; 30 minutes after the injection, the white blood cells was observed transient reduction 37. 5% (ρ = 0. 016), but in When injected into PBS drugs, also observed a similar phenomenon is reduced. 在NMC-4及对照载体处理组两者中,白血细胞水平皆在注射后2-4小时之间回到基线。 In both treatment groups and the control vector NMC-4, the white blood cell levels are between 2-4 hours after injection returned to baseline.

[0448] 实施例9 :抗体表达用的细胞系的建立 9 [0448] Example: To establish antibody-expressing cell lines used

[0449] 可开发出基于鼠类人工染色体表达(ACE)平台的高产量哺乳类蛋白质表达系统, 该ACE平台已被改造成含有可利用突变的λ-整合酶(integrase)(例如ACE整合酶)结合 [0449] to develop a high yield mammalian protein expression system (ACE) platform based on murine artificial chromosome expression, the ACE platform has been transformed to contain mutations λ- available integrase (integrase) (such as ACE integrase) combine

0. 094 + 0. 0350. 630 土 0. 2941. 883 土 0. 312靶向穿梭载体(targeting shuttlevector)而载入异源基因序列的多个具位点特异性的重组受体位点(recombination acceptor sites) (Lindenbaum 等,(Nucl. Acid Res. 32 (21): el72(2004);美国专利申请第2003/0119104 Al号及第2006/0246586 Al号)。可利用此系统产生用于表达所选的人源化变体及NMC-4嵌合体的稳定细胞系。 0.094 + 0.5 0.5 2941.883 0350.630 Soil Soil .312 targeted shuttle vector (targeting shuttlevector) while loading a plurality of heterologous gene sequence with site-specific recombination acceptor sites (recombination acceptor sites) (Lindenbaum, etc., (Nucl Acid Res 32 (21):.. el72 (2004); US Patent Application No. 2003/0119104 Al and the second number 2006/0246586 Al) can use this system for the expression of the produce. selected humanized variants and NMC-4 chimera stable cell lines.

[0450] 以NotI加上HindiII (轻链载体)或XhoI及BamHI (重链载体)来消化质粒PCI-WC4-VL10及pCI-WC4-VH14的插入物,随后将其克隆入pST0518载体的MCS 1 (轻链) 及MCS 2(重链)中。 [0450] In NotI plus HindiII (light chain vector) or XhoI and BamHI (heavy chain vector) digested plasmid PCI-WC4-VL10 and insert pCI-WC4-VH14 subsequently cloned into vector MCS 1 pST0518 (light chain) and MCS 2 (heavy chain) in. 带有前后串联的重链及轻链插入物的PST0518载体作为用以递送至具有不同抗性基因(resistance genes)的ACE靶向载体(ATV)内的穿梭载体,此是得自于由Lindenbaum等人所说明的靶向载体(Nucl. Acid Res. 32(21) :el72(2004);美国专利申请: 2003/0119104A1 & 2006/0246586A1)。 PST0518 carrier with around a series of heavy and light chains as inserts for delivery to a different resistance genes (resistance genes) of ACE shuttle vector targeting vector (ATV) inside, this is derived from the Lindenbaum, etc. People described the targeting vector (Nucl Acid Res 32 (21): el72 (2004); US Patent Application:.. 2003 / 0119104A1 & 2006 / 0246586A1). 为了将含有抗体重链及轻链两者的盒(cassette) 递送至ATV 内,以I-Ceul 及PI-Scel 寻靴内切酶(homing endonuclease) (New England Biolabs,MA)来消化pST0518-VH14,VLlO载体;凝胶纯化VH14加VLlO片段,并将其克隆入以相同的I-Ceul及PI-Scel内切酶预先消化的pZeo及pHygro-ATV的相同位点中。 In order to contain the antibody heavy and light chains of both cassette (cassette) delivered into the ATV, to I-Ceul and PI-Scel to find Endo boots enzyme (homing endonuclease) (New England Biolabs, MA) to digest pST0518-VH14 , VLlO carrier; VH14 plus VLlO fragment gel purified and cloned into with the same amount of I-Ceul and PI-Scel endonuclease predigested pZeo and pHygro-ATV same sites. 此即产生质粒pNHT605-H14L10-IgG4 (hygE 基因)及pNHT607-H14L10_IgG4 (ρ zeoE 基因)。 Namely generating plasmid pNHT605-H14L10-IgG4 (hygE gene) and pNHT607-H14L10_IgG4 (ρ zeoE genes).

[0451] 同理,构建带有NMC_4IgG4嵌合体的pST0518靶向载体,并将前后串联插入物亚克隆入pZeo及pHygro-ATV载体内,如此产生质粒pNHT623 (人类IgG4嵌合体加hygK基因) 及pNHT624 (人类IgG4嵌合体加zeoR基因)。 [0451] Similarly, with the targeting vector constructed pST0518 NMC_4IgG4 chimera and tandem around the insert and subcloned into the pZeo pHygro-ATV vector, thus generating plasmid pNHT623 (plus hygK human IgG4 chimeric gene) and pNHT624 (plus zeoR human IgG4 chimeric gene).

[0452] 为靶向整合至平台ACE,以6孔培养皿的每孔0. 4X IO5个细胞的密度接种宿主ChK2 ACE平台细胞并隔夜培养。 [0452] The targeted integration platform to ACE, to each well of 6-well culture dishes 0. 4X IO5 cells were seeded at a density ChK2 ACE internet host cells and cultured overnight. 在转染前3小时,以无血清培养基更换培养基;3小时后,根据生产商的使用说明,Wlyg的载体及1 μ g ACE整合酶表达载体用LipofectAMINE PLUS 试剂(Invitrogen)加以复合进行转染。 In the three hours before transfection, the medium was changed to serum-free medium; three hours later, according to the manufacturer's instructions, Wlyg carriers and 1 μ g ACE integrase expression vector to be complex with LipofectAMINE PLUS reagent (Invitrogen) were transfected infection. 24小时之后,将细胞扩充至15cm的培养皿上;第二天,将3.0yg/ml的zeomycin或潮霉素(取决于所用的载体)加入培养基中;在14天的筛选后,利用克隆环分离出抗药性菌落,扩增各自克隆以用于抗体生产的分析。 After 24 hours, the cells were expanded to a 15cm dish; the next day, the 3.0yg / ml of zeomycin or hygromycin (depending on the carrier) was added to the culture medium; after 14 days of screening, using cloning ring resistant colonies were isolated, amplified each clone analysis for antibody production.

[0453] 实施例10 :NMC-4抗体的体内效力及安全性 10 [0453] Example: NMC-4 antibody in vivo efficacy and safety

[0454] 以一体内动物(例如狒狒)模型来测试人源化NMC-4抗体的效力及安全性。 [0454] In an in vivo animal (such as baboon) model to test the humanized NMC-4 antibody efficacy and safety.

[0455] 在一例示方法中,以盐酸氯胺酮(ketamine hydrochloride)(购自Premier制药公司的Anaket-V™)麻醉狒狒(10mg/kg頂/30分钟或有必要时维持全身麻醉),并以加热桌将其体温维持于37C。 [0455] In one exemplary method to ketamine hydrochloride (ketamine hydrochloride) (available from Premier pharmaceutical companies Anaket-V ™) anesthetized baboon (when necessary to maintain or 10mg / kg top / 30 minutes of general anesthesia), and to heat Tables will maintain their body temperature at 37 C. 其次,缓慢地解剖开一节4-5cm、周围无组织的股血管,连接所有在股动脉及股静脉中的附近分支。 Secondly, slowly dissected section of 4-5cm, shares perivascular unorganized, connecting all branches of the femoral artery and nearby femoral vein. 接着,在股动脉及股静脉中切出一小切口,插入血管尖端并以外科手术线固定,接将硅氧烷管贴附至血管尖端,以使动脉血液分流入股静脉;绕过毛细管而由动脉直接至静脉循环的分流增加血液流量至约150-300mL/分钟。 Then, in the femoral artery and femoral vein cut a small incision into the blood vessel tip and surgical cable fixed, then the silicone tube attached to the vessel tip to the arterial venous shunting shares; bypassing the capillaries and the artery bypass directly to the venous circulation increases blood flow to about 150-300mL / min. 将管型超因波流量探针(Transonic Systems Inc, Maastricht, the Netherlands))贴附至硅氧烷管,容许血流稳定约20分钟;整个实验中连续地测量平均及位向(phasic)血液流量,将分流用于给药以及血液取样。 Due to the super-wave tube flow probe (Transonic Systems Inc, Maastricht, the Netherlands)) attached to the silicone tube to allow blood flow to stabilize for about 20 minutes; continuously measured throughout the experiment and the average orientation (phasic) Blood traffic will be diverted for administration and blood sampling.

[0456]其次,利用马丁针持针器(Hegar-Baumgartner TC Gold 14cm,产品码20. 634. 14),通过以最大凹陷程度重压内皮层持续10秒钟,在极靠近血管尖端处损伤股动脉的内皮层。 [0456] Secondly, Martin needle needle holder (Hegar-Baumgartner TC Gold 14cm, 20. The product code 634.14), by the weight of the cortex to the maximum level of depression for 10 seconds, at the tip of the pole near the vascular injury Shares cortical arteries. 生产出两重叠创伤,并将一可调式塑料缩窄器置于创伤位点上方,以将血液流量减少至基线值的10% -20%。 Produce two overlapping trauma, and an adjustable plastic constrictor is placed above the wound site, to reduce blood flow to 10% -20% of the baseline value. 观察到因血栓形成而使血液流量逐渐减少,当血液流量减少至彡5mL/min时,打开缩窄器以取出富含血小板的血栓;其次,再度使外部变狭窄并重新开始血栓形成的程序。 Thrombosis was observed due to decreased blood flow leaving, while reducing the blood flow to 彡 5mL / min, the open constrictors to remove the platelet-rich thrombus; secondly, once again narrowing of the external and restart procedures thrombosis. 此种在机械复位之后的血液流量减少的重复模式被称为循环流减缩(CFR)。 Such mechanical reset in blood flow reduction after repeated pattern is called cyclic flow reduction (CFR). 测量为时间的函数的CFR数目,用三十分钟记录基线循环流减缩量;注射盐水后并监测CFR持续另一个三十分钟。 CFR was measured as a function of the number of times, in 30 minutes were recorded at baseline circulating flow shrinkage reduction; and to monitor continued after saline CFR another thirty minutes. 使用如实施例3中所述的人源化NMC-4变体H9L9 IgG4 (此处还称为GBR 600)。 Use as described in Example 3 people humanized NMC-4 variant H9L9 IgG4 (herein also referred GBR 600).

[0457] 可利用两方法来评估给药时的出血状况。 [0457] Two methods can be used to assess the condition of bleeding when administered. 在第一方法中,于前臂表面处测定皮肤模板出血时间。 In the first method, the template bleeding time was measured in the forearm skin surface. 在手臂附近使用压力袖并在40mmHg下膨胀,接着以Surgicut装置(ITC, Edison, NJ)诱导伤口。 Use a pressure cuff around the arm and inflated under 40mmHg, followed by Surgicut means (ITC, Edison, NJ) induced wounds. 将表皮出血时间定义为伤口的诱发与视觉观察出血中止之间的时间。 The skin bleeding time was defined as a wound-induced bleeding time and visual observation between the suspension. 每15秒钟以滤纸小心地轻擦血液,而勿碰触伤口。 Every 15 seconds to paper carefully graze blood, Erwu touch the wound. 当表皮出血时间超过900秒(例如15分钟)时停止测量,并将出血时间视为900秒。 Stop measurement when the skin bleeding time over 900 seconds (for example 15 minutes), and bleeding time regarded as 900 seconds.

[0458] 在第二方法中,通过重组armexin V而以兔颈动脉创伤模型来估算切口的失血量(见例如P. Thiagarajan 等,(1997)Circulation96(7) :2339-47)。 [0458] In the second method, by recombinant armexin V and rabbit carotid artery incision wound model to estimate blood loss (see for example P. Thiagarajan, etc., (1997) Circulation96 (7): 2339-47). 在腹股沟上切出2cmX0. 8cm 切口,并埋入预先秤重的纱布消毒棉块,在每三十分钟剂量注入周期结束或其充满血液时更换纱布消毒棉块;在研究结束时将所有纱布秤重,以得到失血量。 On groin cut 2cmX0 8cm incision, and embedded in a pre-weighed sterile cotton gauze block, replace sterile cotton gauze block every 30 minutes during dose injection cycle is completed or filled with blood; at the end of the study all the gauze scales weight, to give blood loss. 以盐水对照状态纱布的比例来表示每一剂量的数值。 In saline gauze proportion of state to represent the value of each dose. 在整个研究期间,以10分钟为间隔连续地监测心跳速率及血压。 Throughout the study period, 10-minute intervals to continuously monitor the heart rate and blood pressure.

[0459] 在每一剂量周期结束时,抽取Iml的EDTA血液及IOml的含柠檬酸血液,并判定FBC血小板数量、凝血酶原(prothrombin)时间、部分促凝血酶原时间(activated partial thromboplastin time)、因子VIII及vWF。 [0459] At the end of each dose cycle, taking blood and IOml Iml of EDTA blood containing citric acid, and determination FBC platelet count, prothrombin (prothrombin) time, partial thromboplastin time (activated partial thromboplastin time) Factor VIII and vWF. 若有必要,于_80C下冷冻两管300 μ 1的小剂量,以递送至研究者处供额外体外实验室研究用。 If necessary, at _80 C frozen two tubes of small doses of 300 μ 1, for delivery to the researchers at the laboratory for additional in vitro research use only. 同理,在流量研究结束后0. 5,1,2,8,24 及48小时进行抽血,而在最终剂量结束时,以测试及对照样本施行血小板凝集测试。 Similarly, 5,1,2,8,24 and 48 hours at 0. After completion of blood flow studies, and at the end of the final dose, to test and control samples of blood platelet aggregation test purposes.

[0460] 在累积剂量(例如当观察到CFR的完全抑制时)之后,以2. 2 μ g/kg/min的剂量注入肾上腺素(Intramed)持续20分钟并再度测量CFR。 [0460] In the accumulated dose (for example, when complete inhibition was observed when CFR) Thereafter, 2. 2 μ g / kg / min dose injection of epinephrine (Intramed) for 20 min and re-measured CFR. 仅有肾上腺素不会在狒狒中引发血小板凝集,但可通过强化其它血小板凝集因子而恢复已遭破坏的循环流量变化(见例如G.Anfossi 等,(1996)Eur J ClinInvest. 26 :353_370)。 Epinephrine will not only lead to platelet aggregation in baboons, but other platelet aggregation by enhancing factor has been damaged and restored circulation flow change (see, for example G.Anfossi etc., (1996) Eur J ClinInvest 26:. 353_370).

[0461] GBR 600的效力及安全性研究:进行下述的研究1至4以判定GBR 600的效力及 [0461] GBR 600 of efficacy and safety studies: Studies conducted following 1-4 to determine the effectiveness of GBR 600 and

安全性。 Security.

[0462] 研究1 :以η = 1动物进行先导研究,建立含量递增的GBR 600的剂量应答(dose response)曲线,并识别出观察到最大CFR抑制时的有效剂量。 [0462] Study 1: η = 1 pilot study animals, increasing the content of GBR establishing dose response 600 (dose response) curve, and identify the maximum effective dose observed suppression of the CFR. 针对所有的测试剂量判定模板出血及切口出血;在高达48小时内取出血液样本,以建立最高剂量下的抗体药物动力学。 Judgment template bleeding and wound bleeding for all doses tested; blood samples taken up to 48 hours in order to establish the highest doses of antibody drug kinetics.

[0463] 间隔30分钟注射下列递增剂量的GBR 600,并在研究的持续时间内记录流量:剂量1,0. 03mg/kg ;剂量2,0. lmg/kg ;剂量3,0. 3mg/kg ;剂量4,lmg/kg ;及剂量5,0. 03mg/kg。 [0463] 30 minute intervals following injection of increasing doses of GBR 600, and record the flow rate for the duration of the study: Dose 1,0 03mg / kg; dose 2,0 lmg / kg; dose 3,0 3mg / kg. ; dose 4, lmg / kg;. and dose 5,0 03mg / kg. 接着在每一剂量的注射之后10分钟时施行出血测试。 Then test purposes bleeding 10 minutes after each dose injection.

[0464] 图5及表21说明递增剂量的GBR 600对CFR的效应。 Effect [0464] FIG. 5 and Table 21 illustrates the increasing doses of GBR 600 对 CFR's. 若CFR并未稳定,在接近三十分钟基线阶段结束时再度损伤动脉。 If the CFR has not stabilize, near the end of the stage again thirty minutes baseline arterial damage. 相较于盐水阶段的8/30分钟,0. 03mg/kg的GBR 600将CFR的数目降低至5/30分钟;注入额外的0. lmg/kg可完全地抑制CFR,此现象是由动脉再度受损伤并未使得CFR回复的事实加以确认。 Compared to saline stage 8/30 minutes, 0 03mg / CFR reduce the number of GBR 600 kg to 5/30 minutes;. Inject additional 0. lmg / kg completely inhibited CFR, this phenomenon is caused by arterial again CFR injured did not make a reply to confirm the facts. 就下列递增剂量而言,有观察到抑制现象。 On the following increasing doses, the inhibition was observed. 在注入最高剂量的GBR 600(10mg/kg)后,在2. 2 μ g/kg/min的速率下注入肾上腺素, 以建立究竟达到血小板沉积的强力或微弱抑制。 In the highest dose injection GBR 600 (10mg / kg) after, at a rate of 2. 2 μ g / kg / min of adrenaline injection to establish whether platelet deposition to achieve strong or weak inhibition. 由于肾上腺素在血压上的效应,注入肾上腺素导致血液流量的短暂增加,但并未逆转CFR的抑制。 Due to the effects of adrenaline on blood pressure, injection of adrenaline cause a brief increase in blood flow, but not reversed CFR suppressed.

[0465] 表21递增剂量的GBR 600在CFR中的效应(0. 03_10mg/kg) [0465] Table 21 increasing doses of GBR 600 in the CFR in effect (0. 03_10mg / kg)

[0466] [0466]

[0467] [0467]

[0468] 研究2 :研究2用类似于研究1的方式进行,除了在开始逐步升高剂量时(0.03mg/ kg剂量之前)使用0. 01mg/kg的剂量以外,因为在研究1中已观察到在剂量0. 03mg/kg下存在CFR的部分抑制。 [0468] Study 2: Study conducted in a manner similar research methods 1, except at the beginning and gradually increase the dose (0.03mg / kg dose) before using 0. 01mg / kg dose outside, because it has been observed in Study 1 partial inhibition CFR presence at a dose of 0. 03mg / kg.

[0469] 在研究2中(见例如图6及表22),观察到0. 01mg/kg的GBR 600在CFR中的效应(7CFR/30分钟,相较于盐水的9CFR/30分钟);然而,注入额外的0. 03mg/kg(累积剂量=0. 04mg/ kg)造成CFR的完全抑制,GBR 600的EDltltl因此为0. 04mg/kg。 [0469] In Study 2 (see e.g., FIG. 6 and Table 22), was observed to 0. 01mg / kg the effect of GBR 600 (7CFR / 30 min compared to saline 9CFR / 30 min) in the CFR; however, injecting extra 0. 03mg / kg (cumulative dose = 0. 04mg / kg) caused complete inhibition of CFR, GBR 600 of EDltltl therefore is 0. 04mg / kg. 动脉的再度损伤并未逆转CFR的抑制,表示此为真实抑制。 Once again did not reverse the damage artery CFR of rejection, this is the real suppression. 抑制效应维持于较高剂量下,上至最大剂量10mg/kg。 Maintaining the inhibitory effect at higher doses, up to a maximum dose of 10mg / kg. 由于肾上腺素在血压上的效应,注入肾上腺素导致血液流量的短暂增加,但并未逆转CFR的抑制。 Due to the effects of adrenaline on blood pressure, injection of adrenaline cause a brief increase in blood flow, but not reversed CFR suppressed.

[0470] 表22递增剂量的GBR 600在CFR中的效应(0. Ol-lOmg/kg) [0470] Table 22 Effect of increasing doses of GBR 600 in the CFR (0. Ol-lOmg / kg)

[0471] [0471]

[0472] 研究3 :研究3用类似于研究1的方式进行,除了施用0. 005mg/kg的起始剂量、接着施用另一次0. 005mg/kg剂量(累积剂量=0. 01mg/kg)、且接着以增量0. Olmg/kg的方式增加6次以外。 [0472] Study 3: Study 3 Study analogous manner using 1, except that administration of 0. 005mg / kg initial dose, followed by administration of another 0. 005mg / kg dose (cumulative dose = 0 01mg / kg.), and then in increments of 0. Olmg / kg of ways to increase six times outside.

[0473] 在研究3中(见例如图7),观察到注入0. 005mg/kg的GBR 600在CFR中的效应(8CFR/30分钟降至7CFR/30分钟)。 [0473] In Study 3 (see e.g., FIG. 7), the injection was observed 0. 005mg / kg the effect of GBR 600 (8CFR / 30 分钟 dropped 7CFR / 30 min) in the CFR. CFR随着GBR 600的剂量增加而呈线性方式减少,每剂量周期的CFR数目显示于表23与图8中;图8说明与递增剂量的GBR 600相关联的CFR 数目的线性减少。 With the GBR 600 CFR dose increase tended to decrease in a linear manner, the number of cycles per dose CFR are shown in Table 23 and Figure 8; CFR number of linear Figure 8 illustrates the increasing doses of GBR 600 associated reduction. CFR数目与GBR 600剂量之间的关系是由下列方程式表示的,数据拟合此方程式时的R2 = 0. 9901。 Relationship between the number of doses between CFR and GBR 600 is represented by the following equation, the data fit this equation when R2 = 0. 9901.

[0474] CFR的数目/剂量周期=-109 XGBR 600的剂量+7. 4517 [0474] The number of CFR / dose cycle = -109 XGBR 600 doses +7. 4517

[0475] 相较于研究2中因累积剂量.04mg/kg所引起的完全抑制,研究3中的EDltltl为0. 07mg/kg。 [0475] Compared to the Study due to the cumulative dose .04mg / kg caused complete inhibition study 3 EDltltl of 0. 07mg / kg. 在研究3中,递增剂量之间的时间为30分钟,而所观察到的此EDltltl上的差异可能由血液中因药物的起始清除结果导致的GBR 600浓度下降所引发。 In Study 3, increasing the time between doses was 30 minutes, while the differences observed GBR EDltltl on this may be provided by the blood clearance results depending on the starting drug concentration decreased resulting 600 triggered. 注入肾上腺素逆转了CFR的抑制,此可能与在0. 07mg/kg累积剂量时的CFR曲线的形状有关。 CFR injected adrenaline reversal of inhibition, this may shape at 0. 07mg / kg cumulative dose of CFR curve related. 在0. 07mg/kg 累积剂量下,抑制情形被缓慢地逆转,其显示血栓正在发展中。 At 0. 07mg / kg cumulative dose, inhibition situation is slowly reversed, showing thrombus under development. 在这些特殊条件下,肾上腺素似乎能够逆转CFR。 In these special conditions, adrenaline seems to be able to reverse the CFR.

[0476]表 23 累积剂量的GBR 600 在CFR 中的效应(0. 005-0. 07mg/kg) [0476] Table 23 cumulative doses of GBR 600 in the CFR in effect (0. 005-0. 07mg / kg)

[0477] [0477]

[0478] 研究4:研究4用类似于研究1的方式进行,除了在三只狒狒中以氯吡格雷(clopidogrel)作为阳性(positive)对照外,目的是为比较GBR600在1,1. 5,2. 5,5及10mg/kg剂量下对抗氯吡格雷的效力及出血倾向。 [0478] Study: Study conducted in a manner similar research methods 1, except in three baboons to clopidogrel (clopidogrel) as positive (positive) control, the aim is to compare GBR600 at 1,1 5. against clopidogrel efficacy and bleeding tendency under 2. 5,5 and 10mg / kg dose.

[0479] 在研究4中,氯吡格雷于狒狒1中在10mg/kg累积剂量下、于狒狒2&3中在5mg/kg 累积剂量下完全地抑制了CFR,如表24及图9(显示表24中的狒狒3的结果)所示。 [0479] In Study 4 clopidogrel in baboons 1 at 10mg / kg cumulative dose, in baboons & 3 at 5mg / kg cumulative dose completely inhibited CFR 2, as shown in Table 24 and 9 (display table 24 The baboons 3 result) below. 注入肾上腺素逆转了CFR的抑制。 CFR injected adrenaline reversal of inhibition.

[0480] 表24氯吡格雷在狒狒中的递增剂量(l-10mg/kg)的效应 [0480] Table 24 clopidogrel in increasing doses in baboon (l-10mg / kg) in effect

[0482] 模板出血时间:在研究1及2中,所有高于0. 04mg/kg剂量下的模板出血时间皆长于15 分钟;在包含氯吡格雷(Bristol-Myers Squibb/Sanof!Pharmaceuticals)的阳性(positive)对照研究中,模板出血时间延长至与大于2. 5mg/kg累积剂量时相同的程度;在研究3中,模板出血时间从未延长至超过15分钟。 [0482] Template bleeding time: In Study 1 and 2, all higher than 0. 04mg / kg dose template bleeding time under both longer than 15 minutes; positive containing clopidogrel (Bristol-Myers Squibb / Sanof Pharmaceuticals!) Of (positive) control studies, when the template bleeding time was extended to more than 2. 5mg / kg cumulative dose of the same extent; study 3, the template bleeding time has never been extended to over 15 minutes. 由于模板出血时间呈现高基线变化性(见例如氯吡格雷中狒狒1,2,3的基线值),故其并非极精准的出血倾向量度,模板出血时间本身不被视为可极准确地预测临床上相关出血,例如在手术前调整中(见例如Lind等, Platelets,第二版,p485-493,Michelson AD 编,Academic Press)。 Since the template bleeding time exhibit high baseline variability (see e.g. clopidogrel in baboons 1,2,3 baseline value), so it is not a very precise measure of the bleeding tendency, template bleeding time itself can not be considered as a very accurate prediction Clinically relevant bleeding, such as adjustment before surgery (see for example Lind, etc., Platelets, Second Edition, p485-493, Michelson AD series, Academic Press). 切口出血测试显示较少变化性,因其是定量经由切口的实际失血量且具有较大动态范围。 Incision bleeding tests showed less variability, because it is the quantitative blood loss via real cuts and has a large dynamic range. 因此,除了模板出血测试以外尚执行切口出血测试。 Therefore, in addition to the template bleeding test still perform incision bleeding test. 这些数据整理于表25及26中。 The data processing in Tables 25 and 26.

[0483] 表25GBR 600的模板失血时间[分钟] Template bleeding time [0483] Table 25GBR 600 of [minutes]

[0484] [0484]

[0485] [0485]

[0486] 表26氯吡格雷的模板失血时间[分钟] [0486] Table 26 clopidogrel template bleeding time [min]

[0487] [0487]

[0488] 表27及28显示以氯吡格雷及GBR 600的切口出血测试所获得的结果。 [0488] Table 27 and 28 shows the results of clopidogrel and GBR incision bleeding test 600 gained. 由纱布所吸收的血液量起初随剂量增加,而在高剂量时呈现自限性。 The amount of blood absorbed by the gauze initially increased with dose, and showed self-limiting at high doses. 在所有研究中,所观察到的最高失血量为在第四剂量时,其后由纱布所吸收的血液量便减少,且似乎发生伤口的愈合;在研究1及2中,最大出血量类似于氯吡格雷者,尽管氯吡格雷用2-4倍的EDltltl、而GBR 600用高达250的倍数加以测试;在研究3中,观察到全部的注射剂量下的出血情况微不足道。 The maximum amount of blood loss in all studies was observed at the fourth dose, followed by the amount of blood absorbed by the gauze will be decreased, and wound healing seems to happen; in Study 1 and 2, the maximum amount of bleeding similar clopidogrel, clopidogrel although 2-4 fold EDltltl, and GBR 600 to be used in multiples of up to 250 tests; 3 in the study, all bleeding was observed at the dose negligible.

[0489] 表27GBR 600的切口失血测试[盐水值的倍数单位] [0489] Table 27GBR 600 incision bleeding test [saline value multiple units]

[0490] [0490]

[0491] 表28氯吡格雷的切口失血测试[盐水值的倍数单位] [0491] Table 28 clopidogrel incision bleeding test [saline value multiple units]

[0492] [0492]

[0493] [0493]

[0494] GBR 600 的治疗窗口(therapeutic window)及出血得分(bleedscore):在图10 中,将来自研究1及2及三项氯吡格雷研究的切口测试结果对GBR600及氯吡格雷的剂量作图(将剂量表示成其EDltltl的倍数并在对数标度上作图)。 [0494] GBR 600 therapeutic window (therapeutic window) and bleeding score (bleedscore): In FIG. 10, the test results from the study incision 1 and 2, and three studies of clopidogrel and clopidogrel dose for GBR600 Figure (dose expressed as a multiple of its EDltltl and plotted on a logarithmic scale in degrees).

[0495] 即使在大于其EDltltl的100倍的剂量下,GBR 600仍导致氯吡格雷中在仅高至其EDltltl的4倍下可见的出血程度。 [0495] Even at 100 times greater than the dose EDltltl, GBR 600 degree still cause bleeding with clopidogrel in only up to four times its EDltltl under visible. 令人意外地,GBR 600关于出血风险是具有前所未见的安全性的治疗窗口。 Surprisingly, GBR 600 on bleeding risk is an unprecedented security window treatment.

[0496] 此项研究中所观察到出血的唯一临床上相关的增加为来自表皮伤口的自限性出血的增加,如同由模板出血及切口出血法所判定。 [0496] This study observed that the only clinically relevant increase in bleeding increased bleeding from self-limited skin wounds, as the cut is determined by the template bleeding and bleeding method. 在外科手术后密切地观察动物达48小时,并未检测到额外的表皮出血信号,例如容易瘀血、出血点、瘀斑等;更重要的是,未检测到内部出血的信号,例如血肿(hematoma)、流鼻血(印istaxis)、来自口腔或阴道的失血、 黑粪症(melena)、眼睛出血、血尿症(hematuria)、或吐血(hematemesis)等,这些手术伤口不会出血且可正常地愈合。 After surgery the animals were observed closely for 48 hours, did not detect the extra skin bleeding signals, such as easy bleeding, bleeding, bruising and so on; and more importantly, internal bleeding signals are detected, such as hematoma ( hematoma), epistaxis (India istaxis), bleeding from the mouth or the vagina, melena (melena), eye bleeding, hematuria (hematuria), or vomiting blood (hematemesis), these surgical wounds do not bleed and can normally healing.

[0497] 如表29所示,氯吡格雷及GBR 600两者在BleedScore计分方案中的得分皆为1。 [0497] As shown in Table 29, both clopidogrel and GBR 600 in BleedScore score scoring scheme are all 1. 除了表皮伤口上的出血增加外,在实验期间或得出研究的结论后48小时的观察期期间,并未在动物上检测到其它症状。 During the 48 hours after the addition to the increased bleeding wound on the outer skin, during the experiment or study concluded that the observation period, the animals have not detected other symptoms.

[0498] 表29氯吡格雷及GBR 600的BleedScore判定 [0498] Table 29 Clopidogrel and GBR 600 of BleedScore determination

[0499] [0499]

[0501] 效力及安全性研究的发现:表30-32显示在研究1-3中的GBR 600在下列方面的效应:vWF水平、因子VIII水平、白血细胞数量、血红蛋白浓度(Hb)、血小板数量(Pit)、凝血酶原时间(PT)、激活部分促凝血酶原激酶时间(aPTT)。 [0501] found that efficacy and safety studies: GBR 600 Table 30-32 show effects in the following areas of study 1-3: vWF levels, factor VIII levels, the number of white blood cells, hemoglobin (Hb), platelet count (Pit), prothrombin time (PT), activated partial thromboplastin time (aPTT).

[0502] 关于在研究1-3中所获得的冯维勒布兰德氏水平,于研究1中并未观察到任何模式;但于研究2&3中清楚地观察到冯维勒布兰德氏水平降低。 [0502] For the study gained 1-3 von Willebrand levels, in Study 1, did not observe any mode; but in Study 2 & 3 clearly observed in the level of von Willebrand lower. 在使用更高剂量的研究2中, 效应比使用相对低剂量的研究3更加显著;在使用未与vWF结合的对照人源化单克隆IgG4 抗体的研究中,未观察到对于冯维勒布兰德氏水平的效应,在未来研究中应仔细监测其此类效应及涵义。 Research studies using higher doses of 2, effect than the use of relatively low doses of 3 is more significant; not binding and vWF using a control study humanized monoclonal IgG4 antibodies, was not observed for Von Willebrandt Deshi effect level in the next study such effects should be carefully monitored and their meanings. 在所有研究中,GBR 600对于因子VIII水平均无显著效应。 In all studies, GBR 600 for factor VIII levels had no significant effect.

[0503] 虽然观察到WBC增加,但此为侵入性程序的已知效应,且至目前为止,此结果与针对未结合vWF的对照人源化单克隆IgG4抗体、及在此模型中所测试的一切其它药物所观察到的结果具有良好关联性;未能观察到因注入GBR 600所引发的对于血红蛋白浓度的显著效应;注入GBR 600对于血小板数量有一效应,由于血小板沉积为CFR期间动脉阻塞的原因,故血小板在这些程序期间被消耗掉,因此,CFR的有效抑制将减少血小板的消耗量。 [0503] Although the observed increase in WBC, but this is a known effect of invasive procedures, and so far, the results are not binding for the control of vWF humanized monoclonal IgG4 antibody, and in this model being tested All other drugs observed showed good correlation; 600 for the number of platelets have a effect due to injection GBR Since platelet deposition for the duration CFR arterial obstruction; not observed for hemoglobin concentrations significantly effect due to injection GBR triggered 600 , so that during these procedures platelets are consumed, and therefore, inhibit the CFR will reduce the consumption of platelets. 此解释了在未结合vWF的对照人源化单克隆IgG4抗体中所观察到的较大血小板消耗量,其中并未观察到CFR的抑制。 This explains the non-bound vWF controls humanized IgG4 monoclonal antibody that observed large platelet consumption, which was not observed CFR suppressed.

[0504] GBR 600对于指示凝血蛋白的完整性的PT及aPTT似乎无显著效应,在未结合vWF 的对照人源化单克隆I gG4抗体中还观察到类似结果。 [0504] GBR 600 seems to be no significant effect on blood coagulation protein for indicating the integrity of PT and aPTT, in unbound control vWF I gG4 humanized monoclonal antibodies were also observed similar results.

[0505]表 30.研究1 [0505] Table 30. Study 1

[0511] [0511]

表31.研究2 Table 31. Study

此抑制反应,对氯吡格雷也是。 This inhibitory response, clopidogrel is. 使用GBR 600时未发现严重的有害出血,即使当药物以目前看来像是高达有效剂量的250倍的剂量被输注时也是。 When using GBR 600 No serious adverse bleeding, even when the drugs it seems like an effective dose of up to 250 times the dose by infusion, too. 在这些剂量下,通过切口出血模型所测量的出血量,产生与以有效剂量4-8倍加以输注的氯吡格雷类似的结果。 At these doses, bleeding through incision bleeding measurement model, resulting in an effective dose and infusion of clopidogrel 4-8 doubly to similar results. GBR 600对于凝血蛋白不具影响力,此由PT及aPTT结果加以显示;然而,存在冯维勒布兰德氏因子水平降低的情形,但在因子VIII水平上却未观察到清楚的效应。 GBR 600 for coagulation proteins does not have influence, this to be displayed by the PT and aPTT results; however, the presence of von Willebrand factor in reducing the case, but the factor VIII level not observed a clear effect. 由于杀鼠灵(warfarin)通过降低机能性维生素K依赖的凝血蛋白的循环水平来抑制凝血系统,故此方面并非问题。 Since Warfarin (warfarin) to inhibit the function of the coagulation system by reducing circulating levels of vitamin K-dependent coagulation proteins, and therefore is not a problem aspects. 在此研究中,对于全血计量参数并未观察到意料之外的效应。 In this study, the measurement of blood parameters not observed unexpected effects.

[0512] 实施例11 :人源化NMC-4变体的热稳定性 [0512] Example 11: humanized NMC-4 variant thermal stability

[0513] 利用量热法比较鼠类NMC-4的Fab片段的人源化NMC-4变体及NMC—4-IgGl嵌合体的热稳定性。 [0513] To compare the use of calorimetry rodent Fab fragment NMC-4, humanized NMC-4 thermal stability of variants and NMC-4-IgGl chimera. 单克隆抗体的熔融谱为其同种型的特征(Garber和Demarest (2007), BBRC 355 :751-7);然而,即使在全长IgG的情况中,仍可轻易地识别出Fab片段的中点熔融温度。 The melting profile for monoclonal antibody isotype characterized (Garber and Demarest (2007), BBRC 355: 751-7); however, even in the case of full-length IgG can still easily identify the Fab fragment of point melting temperature. 利用此种Fab片段的中点熔融温度来监测人源化候选者的单克隆抗体稳定性。 Utilizing midpoint melting temperature of such Fab fragments monitoring candidate humanized monoclonal antibody stability.

[0514] 在VP-DSC示差高灵敏度扫描热量计(differential scanningmicrocalorimeter, MicroCal, Northampton, UK)上进行量热法。 [0514] In the VP-DSC differential scanning calorimeter High sensitivity (differential scanningmicrocalorimeter, MicroCal, Northampton, UK) on the calorimetric method. 槽(cell)体禾只为0. 128ml ;加热速率为1C /min ;且将超压(excess pressure)维持于64psi ;所有蛋白质片段皆于PBS(pH 7.4)中l-0.5mg/mL(74yM)浓度下使用。 Slot (cell) Wo body only 0. 128ml; heating rate of 1 C / min; pressure and ultra (excess pressure) is maintained at 64psi; all protein fragments are in PBS (pH 7.4) in l-0.5mg / mL (74yM) concentrations under. 通过与含有相同缓冲液的一式两份样本(蛋白质以从中去除)相比较,估计每一蛋白质的摩尔热容量;利用标准程序分析部分摩尔热容量及熔融曲线。 By the same buffer containing a duplicate samples (protein is removed therefrom) compared to the estimated molar heat capacity of each protein; using standard procedures analysis section molar heat capacity and melting curve. 在进一步于软件Origin v7. 0中利用非两状态模型(Non-Two State model)分析温度记录图之前,以基线校正的并将浓度归一化。 Before further use of non two-state model (Non-Two State model) software Origin v7. 0 in the analysis of temperature recording chart to baseline-corrected and normalized concentration. 针对如实施例3中提及的H14L10-IgG4所获得的数据的例子显示于图11。 As an example of the data for Example 3 mentioned H14L10-IgG4 obtained are shown in FIG. 11. 鼠类NMC-4的Fab片段在74. 7C时显现单一转化,而H14L10-IgG4的Fab片段转化则在81. 1C时显现,其对应于稳定性上的明显差异(6. 4C)。 Fab fragments murine NMC-4, appeared in 74. 7 C when a single conversion, and the conversion H14L10-IgG4 Fab fragment of the show at 81. 1 C, the stability of which corresponds to the significant differences (6.4 C). 为了探知人类Fab稳定结构域的影响,制备由经移植至人类IgGl(最稳定的人类同种型;Garber和Demarest (2007),BBRC 355 :751_7)上的鼠类NMC-4可变结构域所组成的嵌合体。 In order to ascertain the impact of human Fab stability domains were prepared by a transplanted human IgGl (the most stable human isoforms; Garber and Demarest (2007), BBRC 355: 751_7) murine NMC-4 on the variable domain chimeras formed. 就HH-LlOFab而言,H14L10-IgG4及NMC-4-IgGl嵌合体的表观(apparent) Fab Tm值仍显示出稳定性上的明显增加(ΔΤπι> 1C ) On HH-LlOFab, the apparent H14L10-IgG4 and NMC-4-IgGl chimera (apparent) Fab Tm value still shows a significant increase in stability on the (ΔΤπι> 1 C)

[0515] 实施例12 :编码GBR 600重链(VH9)及轻链(VL9)的基因的克隆 12 [0515] Example: GBR 600 clones encoding the heavy chain (VH9) and light chain (VL9) gene

[0516] 用于克隆编码GBR 600的基因的材料及方法列出如下: [0516] clones encoding GBR 600 materials and methods for gene are listed below:

[0517] PfuUltra(Stratagene,Cat.-No. :600380) [0517] PfuUltra (Stratagene, Cat.-No:. 600380)

[0518] SpeI (NEB, Cat. -No. :R0133) [0518] SpeI (NEB, Cat -No:.. R0133)

[0519] HindIII (NEB, Cat. -No. :R0104) [0519] HindIII (NEB, Cat -No:.. R0104)

[0520] CIP (NEB, Cat.-No. :M0290) [0520] CIP (NEB, Cat.-No.: M0290)

[0521] pCR-钝端(Invitrogen,Cat.-No. :44_0302) [0521] pCR- blunt end (Invitrogen, Cat.-No:. 44_0302)

[0522] 弓丨物:Operon, Cologne, Germany [0522] bow Shu thing: Operon, Cologne, Germany

[0523] GLNPR107 :TAACTAGTCGTGAGGCTCCGGTGCCCGTC [0523] GLNPR107: TAACTAGTCGTGAGGCTCCGGTGCCCGTC

[0524] GLNPR108 :AAGCTTACGGCTAGCTCACGACACCTGAAATGGAAG [0524] GLNPR108: AAGCTTACGGCTAGCTCACGACACCTGAAATGGAAG

[0525] GLNPRl39 :CCTCAGACAGTGGTTCAAAG [0525] GLNPRl39: CCTCAGACAGTGGTTCAAAG

[0526] GLNPRl76 :GCTAGCGCCACCATGGAGACAGACACAC [0526] GLNPRl76: GCTAGCGCCACCATGGAGACAGACACAC

[0527] GLNPRl77 :TAAGCTTCTATCATTTACCCAGAGACAGGG [0527] GLNPRl77: TAAGCTTCTATCATTTACCCAGAGACAGGG

[0528] GLNPRl78 :TAAGCTTCTATCAACACTCTCCCCTGTTG[0529] BGHREV :由Fasteris 所提供 [0528] GLNPRl78: TAAGCTTCTATCAACACTCTCCCCTGTTG [0529] BGHREV: is provided by Fasteris

[0530] TMC 载体pCI-WC4-VL9 (pl56)及pCI_WC4_VH9 (pl58)(由Chromos 所提供) [0530] TMC vector pCI-WC4-VL9 (pl56) and pCI_WC4_VH9 (pl58) (provided by the Chromos)

[0531] Qiaquick 凝胶提取试剂盒(Qiagen,Cat.-No. :28706) [0531] Qiaquick Gel Extraction Kit (Qiagen, Cat.-No:. 28706)

[0532] lkb+ladder (Fermentas, Cat. -No. :R0491) [0532] lkb + ladder (Fermentas, Cat -No:.. R0491)

[0533] pcDNA3. 1(-)(Invitrogen, Cat.-No. :V795_20) . [0533] pcDNA3 1 (-) (Invitrogen, Cat.-No.: V795_20)

[0534] pEF-Dest51[CDl(RZPD, Cat. -No. :RZPDo839G0167-pEF_DEST51) [0534] pEF-Dest51 [CDl (RZPD, Cat -No:.. RZPDo839G0167-pEF_DEST51)

[0535] IlJj^ :Fasteris SA(Geneva, Switzerland) [0535] IlJj ^: Fasteris SA (Geneva, Switzerland)

[0536] Gigapr印试剂盒(Macherey-Nagel, Cat. -No. :Nucleobond PC10000) [0536] Gigapr printing kit (Macherey-Nagel, Cat -No:.. Nucleobond PC10000)

[0537] 表达载体pEFcDNA3. 1的克隆 [0537] The expression vector pEFcDNA3. Clone 1

[0538] 表达载体pEFcDNA通过以来自pEF_DEST51的EFl - α启动子取代来自pcDNA3. 1 (-) (Invitrogen)的CMV启动子而加以构建。 [0538] By the expression vector pEFcDNA pEF_DEST51 from the EFl - α promoter substituents from pcDNA3 1 - CMV promoter (Invitrogen) and be constructed in. (). 为此目的,故利用引物GLNPR107,108 及PfuUltra (Stratagene,退火温度55C,30个循环)来扩增EFl-α启动子;引物扩增全部的EFl-a启动子,并在所扩增片段的5'端贴附SpeI侧而在3'端贴附HindIII侧。 For this purpose, it is using primers GLNPR107,108 and PfuUltra (Stratagene, annealing temperature 55 C, 30 cycles) to amplify EFl-α promoter; primer all EFl-a promoter, and the amplified 5 fragment 'end attached SpeI side and 3' end is attached HindIII side. 将PCR扩增子(amplicon)克隆入pCR-钝端(Invitrogen),并通过Spel/Hindlll消化来分析克隆。 The PCR amplicons (amplicon) was cloned into pCR- blunt end (Invitrogen), and through Spel / Hindlll digestion Cloning. 切下来自克隆#4的Spel/Hindlll片段,并将其克隆入利用相同酶组合及CIPed加以消化的pcDNA3. 1 (-)骨架中。 Cut Spel / Hindlll fragment from clone # 4 and Kelongruli combination with the same enzymes and CIPed be digested pcDNA3 1. (-) Backbone. 利用SpeI及HindIII分析克隆,克隆#2似乎呈阳性;以骨架及插入物进行第二次消化更证实了启动子片段的正确大小。 Use of SpeI and HindIII Cloning, seems positive clones # 2; and to insert the skeleton was subjected to a second digestion confirmed more promoter fragment of the correct size.

[0539]将 GBR 600 克隆至pEFcDNA [0539] The GBR 600 cloned pEFcDNA

[0540] 利用PfuUltra (标准条件,退火温度55C,30个循环)及引物GLNPR176及177来扩增GBR 600VH9,模板为TMC载体pl56 ;以如同对于重链所述,利用引物GLNPR176及178 来扩增GBR 600VL9,所使用的模板为TMC载体ρ 158。 [0540] The use of PfuUltra (standard conditions, an annealing temperature of 55 C, 30 cycles) and primer GLNPR176 and 177 to amplify GBR 600VH9, TMC template vector pl56; the same manner as for the heavy chain, using primers and 178 to GLNPR176 Amplification GBR 600VL9, templates used for TMC carrier ρ 158. 引物将NheI限制位点5,及HindIII 限制位点3'附加至各自扩增子,将所获得的PCR片段克隆入pCR-钝端,并利用NheI及HindIII通过限制消化加以分析。 Primers NheI restriction sites 5, and HindIII restriction site 3 'appended to each amplicon, the PCR fragment was cloned into pCR- blunt end, and using NheI and HindIII be analyzed by restriction digest. 切下轻链的克隆#1及重链的克隆#3,并将其克隆入利用酶NheI及HindIII及CIPed打开的pEFcDNA中。 Cut light chain and heavy chain clone # 1 clone # 3, and Kelongruli enzymes NheI and HindIII and CIPed open pEFcDNA in. 该限制消化显示:轻链的克隆#6及重链的克隆#1含有正确大小的片段,将此两克隆送至用作测序对照的Fasteris,以作为样本GS256及GS257。 The restriction digest display: light chain and heavy chain clone # 6 clone # 1 contains fragments of the correct size, this is used to the two clones sequenced control Fasteris, as a sample GS256 and GS257. 测序结果比对参考序列。 The sequencing results compared to the reference sequence. 由于少量制备(minipr印)DNA的质量不佳,故重链序列GS257无法确认至100%程度。 Because a small amount of poor quality preparation (minipr India) DNA, so the heavy chain sequence GS257 can not confirm the extent of 100%. 利用编码GBR 600重链VH9 (GS257)及GBR 600轻链VL9 (GS256)以制备多量制备(Gigapr印s)。 The use of a heavy chain coding GBR 600 VH9 (GS257) and GBR 600 light chain VL9 (GS256) to prepare a lot of preparation (Gigapr India s). 将质粒制备再度送至Fasteris以进行序列确认,此次的样本名为GS265(GBR 600重链VH9)及GSS264 (GBR 600重链VL9)。 The plasmid preparation Fasteris again sent for sequence confirmation, the samples called GS265 (GBR 600 heavy chain VH9) and GSS264 (GBR 600 heavy chain VL9). 由于其DNA 质量较好,故可确认重链及轻链对于参考序列的序列同一性。 Because of its good quality DNA, it can be confirmed that the heavy and light chain sequence identity to the reference sequence.

[0541] 虽然本发明于此已通过参照各种特定材料、程序、及实施例加以说明,但应了解技术人员在阅读以上说明书及研究图式时将可实现各种的修改、增加、变更及其均等物。 [0541] While the invention has been herein by reference to various specific materials, procedures, and examples will be described, it is to be understood in the art upon reading the above description and study of the drawings will realize various modifications, additions, changes and their equivalents. 因此,本发明实施例应被视为例示性而非限制性,其真实范围及精神是由下列权利要求书所表示。 Accordingly, embodiments of the invention should be considered as illustrative and not restrictive, the true scope and spirit of the following claims represents. 本案中所参照的有参考数据、专利、专利申请通过参考文献方式将其整体并入于此。 There are case referenced reference data, patents, patent applications by way of reference incorporated herein in its entirety.

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