U.S. patent application number 14/361013 was filed with the patent office on 2015-02-26 for drug containing carrier into cell for forming immune complex.
This patent application is currently assigned to CHUGAI SEIYAKU KABUSHIKI KAISHA. The applicant listed for this patent is Chugai Seiyaku Kabushiki Kaisha. Invention is credited to Naoka Hironiwa, Tomoyuki Igawa.
Application Number | 20150056182 14/361013 |
Document ID | / |
Family ID | 48535590 |
Filed Date | 2015-02-26 |
United States Patent
Application |
20150056182 |
Kind Code |
A1 |
Igawa; Tomoyuki ; et
al. |
February 26, 2015 |
DRUG CONTAINING CARRIER INTO CELL FOR FORMING IMMUNE COMPLEX
Abstract
The present inventors discovered that by forming a large immune
complex comprising antigens containing two or more antigenic
binding units (epitopes) and two or more antigen-binding molecules
(for example, antibodies), elimination from the plasma of the
antigens containing two or more antigenic binding units can be
accelerated. Moreover, they found that by using this characteristic
and by further using antigen-binding molecules having an
ion-dependent antigen-binding activity, elimination of the antigens
can further be accelerated and the above problem can be solved.
Inventors: |
Igawa; Tomoyuki; (Shizuoka,
JP) ; Hironiwa; Naoka; (Shizuoka, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Chugai Seiyaku Kabushiki Kaisha |
Tokyo |
|
JP |
|
|
Assignee: |
CHUGAI SEIYAKU KABUSHIKI
KAISHA
Tokyo
JP
|
Family ID: |
48535590 |
Appl. No.: |
14/361013 |
Filed: |
November 30, 2012 |
PCT Filed: |
November 30, 2012 |
PCT NO: |
PCT/JP2012/081185 |
371 Date: |
May 28, 2014 |
Current U.S.
Class: |
424/131.1 ;
435/69.6; 435/7.92; 436/501 |
Current CPC
Class: |
C07K 16/248 20130101;
C07K 2317/31 20130101; C07K 16/4291 20130101; A61P 37/02 20180101;
C07K 16/303 20130101; C07K 2317/92 20130101; G01N 33/686 20130101;
C07K 2317/94 20130101; G01N 2500/04 20130101; C07K 2317/52
20130101; C07K 16/4283 20130101; A61K 2039/505 20130101 |
Class at
Publication: |
424/131.1 ;
436/501; 435/69.6; 435/7.92 |
International
Class: |
C07K 16/42 20060101
C07K016/42; G01N 33/68 20060101 G01N033/68 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 30, 2011 |
JP |
PCT/JP2011/077619 |
May 30, 2012 |
JP |
2012-123773 |
Claims
1-29. (canceled)
30. A method of reducing the amount of an antigen in the plasma of
a subject, the method comprising identifying a subject as being in
need of a reduction in the amount of the antigen in the subject's
plasma; and administering to the subject's plasma an
antigen-binding molecule comprising (i) an Fc region, and (ii) two
or more antigen-binding domains, including a first antigen-binding
domain that binds to a first epitope on the antigen and a second
antigen-binding domain that binds to a second epitope on the
antigen, wherein (a) the first and second epitopes may be identical
or different, (b) the antigen-binding activity of at least one of
the antigen-binding domains varies depending on pH or on calcium
ion concentration, and (c) the antigen-binding molecule forms an
immune complex comprising two or more copies of the antigen-binding
molecule and two or more copies of the antigen.
31. The method of claim 30, wherein the antigen-binding activity of
at least one of the antigen-binding domains varies depending on
calcium ion concentration.
32. The method of claim 31, wherein the antigen-binding activity of
at least one of the antigen-binding domains is lower at a calcium
ion concentration between 0.5 .mu.M and 10 .mu.M than at a calcium
ion concentration between 200 .mu.M and 5 mM.
33. The method of claim 30, wherein the antigen-binding activity of
at least one of the antigen-binding domains varies with pH.
34. The method of claim 33, wherein the antigen-binding activity of
at least one of the antigen-binding domains is lower at a pH in the
range of 4.0 to 6.5 than at a pH in the range of 6.7 to 10.0.
35. The method of claim 30, wherein the antigen is a multimer
comprising two or more subunits that may be the same or
different.
36. The method of claim 35, wherein each of at least two of the
subunits comprises at least one of the epitopes.
37. The method of claim 35, wherein the antigen is any one of GDF,
GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13,
CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15
(MIC-1), TNF, TNF-alphabeta, TNF-beta2, TNFSF10 (TRAIL, Apo-2
ligand, TL2), TNFSF11 (TRANCE/RANK ligand, ODF, OPG ligand),
TNFSF12 (TWEAK, Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL, TALL2),
TNFSF13B (BAFF, BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT, HVEM
ligand, LTg), TNFSF15 (TL1A/VEGI), TNFSF18 (GITR ligand, AITR
ligand, TL6), TNFSF1A (TNF-a, Cachectin, DIF, TNFSF2), TNFSF1B
(TNF-b, LTa, TNFSF1), TNFSF3 (LTb, TNFC, p33), TNFSF4 (OX40 ligand,
gp34, TXGP1), TNFSF5 (CD40 ligand, CD154, gp39, HIGM1, IMD3, TRAP),
TNFSF6 (Fas ligand, Apo-1 ligand, APT1 ligand), TNFSF7 (CD27
ligand, CD70), TNFSF8 (CD30 ligand, CD153), TNFSF9 (4-1BB ligand,
CD137 ligand), VEGF, IgE, IgA, IgG, IgM, RANKL, TGF-alpha,
TGF-beta, TGF-beta Pan Specific, or IL-8.
38. The method of claim 30, wherein the antigen is a monomer
comprising the first and second epitopes.
39. The method of claim 30, wherein the antigen-binding molecule is
a multispecific or multiparatopic antigen-binding molecule.
40. The method of claim 30, wherein the Fc region of (i) comprises
the sequence of any one of SEQ ID NOs: 13, 14, 15, and 16.
41. The method of claim 30, wherein the Fc region of (i) has, at a
pH between 4 and 6.5, an FcRn-binding activity greater than that of
a second Fc region at the same pH, wherein the second Fc region is
represented by any one of SEQ ID NOs: 13, 14, 15, and 16.
42. The method of claim 41, wherein the Fc region of (i) comprises
the amino acid sequence of any one of SEQ ID NOs: 13, 14, 15, and
16, except with an amino acid substitution at one or more positions
selected from positions 238, 244, 245, 249, 250, 251, 252, 253,
254, 255, 256, 257, 258, 260, 262, 265, 270, 272, 279, 283, 285,
286, 288, 293, 303, 305, 307, 308, 309, 311, 312, 314, 316, 317,
318, 332, 339, 340, 341, 343, 356, 360, 362, 375, 376, 377, 378,
380, 382, 385, 386, 387, 388, 389, 400, 413, 415, 423, 424, 427,
428, 430, 431, 433, 434, 435, 436, 438, 439, 440, 442, and 447 (EU
numbering).
43. The method of claim 42, wherein the Fc region of (i) comprises,
at one or more of the following positions, the indicated amino acid
(all positions by EU numbering): Leu at position 238; Leu at
position 244; Arg at position 245; Pro at position 249; Gln or Glu
at position 250; Arg, Asp, Glu, or Leu at position 251; Phe, Ser,
Thr, or Tyr at position 252; Ser or Thr at position 254; Arg, Gly,
Ile, or Leu at position 255; Ala, Arg, Asn, Asp, Gln, Glu, Pro, or
Thr at position 256; Ala, Ile, Met, Asn, Ser, or Val at position
257; Asp at position 258; Ser at position 260; Leu at position 262;
Lys at position 270; Leu or Arg at position 272; Ala, Asp, Gly,
His, Met, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr at position 279;
Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser,
Thr, Trp, or Tyr at position 283; Asn at position 285; Phe at
position 286; Asn or Pro at position 288; Val at position 293; Ala,
Glu, Gln, or Met at position 307; Ile, Pro, or Thr at position 308;
Ala, Glu, Ile, Lys, Leu, Met, Ser, Val, or Trp at position 311; Pro
at position 309; Ala, Asp, or Pro at position 312; Ala or Leu at
position 314; Lys at position 316; Pro at position 317; Asn or Thr
at position 318; Phe, His, Lys, Leu, Met, Arg, Ser, or Trp at
position 332; Asn, Thr, or Trp at position 339; Pro at position
341; Glu, His, Lys, Gln, Arg, Thr, or Tyr at position 343; Arg at
position 375; Gly, Ile, Met, Pro, Thr, or Val at position 376; Lys
at position 377; Asp, Asn, or Val at position 378; Ala, Asn, Ser,
or Thr at position 380; Phe, His, Ile, Lys, Leu, Met, Asn, Gln,
Arg, Ser, Thr, Val, Trp, or Tyr at position 382; Ala, Arg, Asp,
Gly, His, Lys, Ser, or Thr at position 385; Arg, Asp, Ile, Lys,
Met, Pro, Ser, or Thr at position 386; Ala, Arg, His, Pro, Ser, or
Thr at position 387; Asn, Pro, or Ser at position 389; Asn at
position 423; Asn at position 427; Leu, Met, Phe, Ser, or Thr at
position 428; Ala, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln,
Arg, Ser, Thr, Val, or Tyr at position 430; His or Asn at position
431; Arg, Gln, His, Ile, Lys, Pro, or Ser at position 433; Ala,
Gly, His, Phe, Ser, Trp, or Tyr at position 434; Arg, Asn, His,
Ile, Leu, Lys, Met, or Thr at position 436; Lys, Leu, Thr, or Trp
at position 438; Lys at position 440; and Lys at position 442.
44. The method of claim 30, wherein the Fc region of (i) has, at a
pH between 6.7 and 10.0, an FcRn-binding activity greater than that
of a second Fc region at the same pH, wherein the second Fc region
is represented by any one of SEQ ID NOs: 13, 14, 15, and 16.
45. The method of claim 44, wherein the Fc region of (i) comprises
the amino acid sequence of any one of SEQ ID NOs: 13, 14, 15, and
16, except with an amino acid substitution at one or more positions
selected from positions 237, 248, 250, 252, 254, 255, 256, 257,
258, 265, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312,
314, 315, 317, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387,
389, 424, 428, 433, 434, and 436 (EU numbering).
46. The method of claim 45, wherein the Fc region of (i) comprises,
at one or more of the following positions, the indicated amino acid
(all positions by EU numbering): Met at position 237; Ile at
position 248; Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr at
position 250; Phe, Trp, or Tyr at position 252; Thr at position
254; Glu at position 255; Asp, Asn, Glu, or Gln at position 256;
Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val at position 257; His
at position 258; Ala at position 265; Ala or Glu at position 286;
His at position 289; Ala at position 297; Ala at position 303; Ala
at position 305; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,
Pro, Gln, Arg, Ser, Val, Trp, or Tyr at position 307; Ala, Phe,
Ile, Leu, Met, Pro, Gln, or Thr at position 308; Ala, Asp, Glu,
Pro, or Arg at position 309; Ala, His, or Ile at position 311; Ala
or His at position 312; Lys or Arg at position 314; Ala, Asp, or
His at position 315; Ala at position 317; Val at position 332; Leu
at position 334; His at position 360; Ala at position 376; Ala at
position 380; Ala at position 382; Ala at position 384; Asp or His
at position 385; Pro at position 386; Glu at position 387; Ala or
Ser at position 389; Ala at position 424; Ala, Asp, Phe, Gly, His,
Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr at
position 428; Lys at position 433; Ala, Phe, His, Ser, Trp, or Tyr
at position 434; His, Ile, Leu, Phe, Thr, or Val at position
436.
47. The method of claim 30, wherein the Fc region of (i) has a
higher Fc.gamma. receptor-binding activity than does the Fc region
of a native human IgG.
48. The method of claim 47, wherein the amino acid sequence of the
Fc region of (i) differs from the amino acid sequence of the Fc
region of the native human IgG at one or more positions, including
at least one of the following positions (EU numbering): 221, 222,
223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237,
238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 254,
255, 256, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270,
271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284,
285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299,
300, 301, 302, 303, 304, 305, 311, 313, 315, 317, 318, 320, 322,
323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335,
336, 337, 339, 376, 377, 378, 379, 380, 382, 385, 392, 396, 421,
427, 428, 429, 434, 436, 440.
49. The method of claim 48, wherein the Fc region of (i) comprises,
at one or more of the following positions, the indicated amino acid
(all positions by EU numbering): Lys or Tyr at position 221; Phe,
Trp, Glu, or Tyr at position 222; Phe, Trp, Glu, or Lys at position
223; Phe, Trp, Glu, or Tyr at position 224; Glu, Lys, or Trp at
position 225; Glu, Gly, Lys, or Tyr at position 227; Glu, Gly, Lys,
or Tyr at position 228; Ala, Glu, Gly, or Tyr at position 230; Glu,
Gly, Lys, Pro, or Tyr at position 231; Glu, Gly, Lys, or Tyr at
position 232; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,
Gln, Arg, Ser, Thr, Val, Trp, or Tyr at position 233; Ala, Asp,
Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr,
Val, Tip, or Tyr at position 234; Ala, Asp, Glu, Phe, Gly, His,
Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Tip, or Tyr at
position 235; Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn,
Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr at position 236; Asp,
Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr,
Val, Trp, or Tyr at position 237; Asp, Glu, Phe, Gly, His, Ile,
Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr at
position 238; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,
Pro, Gln, Arg, Thr, Val, Trp, or Tyr at position 239; Ala, Ile,
Met, or Thr at position 240; Asp, Glu, Leu, Arg, Trp, or Tyr at
position 241; Leu, Glu, Leu, Gln, Arg, Trp, or Tyr at position 243;
His at position 244; Ala at position 245; Asp, Glu, His, or Tyr at
position 246; Ala, Phe, Gly, His, Ile, Leu, Met, Thr, Val, or Tyr
at position 247; Glu, His, Gln, or Tyr at position 249; Glu or Gln
at position 250; Phe at position 251; Phe, Met, or Tyr at position
254; Glu, Leu, or Tyr at position 255; Ala, Met, or Pro at position
256; Asp, Glu, His, Ser, or Tyr at position 258; Asp, Glu, His, or
Tyr at position 260; Ala, Glu, Phe, Ile, or Thr at position 262;
Ala, Ile, Met, or Thr at position 263; Asp, Glu, Phe, Gly, His,
Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr at
position 264; Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,
Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr at position 265; Ala,
Ile, Met, or Thr at position 266; Asp, Glu, Phe, His, Ile, Lys,
Leu, Met, Asn, Pro, Gln, Arg, Thr, Val, Trp, or Tyr at position
267; Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr,
Val, or Trp at position 268; Phe, Gly, His, Ile, Lys, Leu, Met,
Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr at position 269; Glu,
Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr
at position 270; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met,
Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr at position 271; Asp,
Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or
Tyr at position 272; Phe or Ile at position 273; Asp, Glu, Phe,
Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr
at position 274; Leu or Trp at position 275; Asp, Glu, Phe, Gly,
His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr at
position 276; Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Pro,
Gln, Arg, Ser, Thr, Val, or Trp at position 278; Ala at position
279; Ala, Gly, His, Lys, Leu, Pro, Gln, Trp, or Tyr at position
280; Asp, Lys, Pro, or Tyr at position 281; Glu, Gly, Lys, Pro, or
Tyr at position 282; Ala, Gly, His, Ile, Lys, Leu, Met, Pro, Arg,
or Tyr at position 283; Asp, Glu, Leu, Asn, Thr, or Tyr at position
284; Asp, Glu, Lys, Gln, Trp, or Tyr at position 285; Glu, Gly,
Pro, or Tyr at position 286; Asn, Asp, Glu, or Tyr at position 288;
Asp, Gly, His, Leu, Asn, Ser, Thr, Trp, or Tyr at position 290;
Asp, Glu, Gly, His, Ile, Gln, or Thr at position 291; Ala, Asp,
Glu, Pro, Thr, or Tyr at position 292; Phe, Gly, His, Ile, Leu,
Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr at position 293;
Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val,
Trp, or Tyr at position 294; Asp, Glu, Phe, Gly, His, Ile, Lys,
Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr at position 295;
Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser,
Thr, or Val at position 296; Asp, Glu, Phe, Gly, His, Ile, Lys,
Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr at position
297; Ala, Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr,
Val, Trp, or Tyr at position 298; Ala, Asp, Glu, Phe, Gly, His,
Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr at
position 299; Ala, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Asn,
Pro, Gln, Arg, Ser, Thr, Val, or Trp at position 300; Asp, Glu,
His, or Tyr at position 301; Ile at position 302; Asp, Gly, or Tyr
at position 303; Asp, His, Leu, Asn, or Thr at position 304; Glu,
Ile, Thr, or Tyr at position 305; Ala, Asp, Asn, Thr, Val, or Tyr
at position 311; Phe at position 313; Leu at position 315; Glu or
Gln at position 317; His, Leu, Asn, Pro, Gln, Arg, Thr, Val, or Tyr
at position 318; Asp, Phe, Gly, His, Ile, Leu, Asn, Pro, Ser, Thr,
Val, Trp, or Tyr at position 320; Ala, Asp, Phe, Gly, His, Ile,
Pro, Ser, Thr, Val, Trp, or Tyr at position 322; Ile at position
323; Asp, Phe, Gly, His, Ile, Leu, Met, Pro, Arg, Thr, Val, Trp, or
Tyr at position 324; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu,
Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr at position 325;
Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val,
Trp, or Tyr at position 326; Ala, Asp, Glu, Phe, Gly, His, Ile,
Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, or Tyr at position
327; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln,
Arg, Ser, Thr, Val, Trp, or Tyr at position 328; Asp, Glu, Phe,
Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or
Tyr at position 329; Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met,
Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr at position 330; Asp,
Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, or Tyr at
position 331; Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn,
Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr at position 332; Ala,
Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, or Tyr
at position 333; Ala, Glu, Phe, Ile, Leu, Pro, or Thr at position
334; Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val,
Trp, or Tyr at position 335; Glu, Lys, or Tyr at position 336; Glu,
His, or Asn at position 337; Asp, Phe, Gly, Ile, Lys, Met, Asn,
Gln, Arg, Ser, or Thr at position 339; Ala or Val at position 376;
Gly or Lys at position 377; Asp at position 378; Asn at position
379; Ala, Asn, or Ser at position 380; Ala or Ile at position 382;
Glu at position 385; Thr at position 392; Leu at position 396; Lys
at position 421; Asn at position 427; Phe or Leu at position 428;
Met at position 429; Trp at position 434; Ile at position 436; Gly,
His, Ile, Leu, or Tyr at position 440.
50. The method of claim 30, wherein the Fc region has a higher
binding activity toward an inhibitory Fc.gamma. receptor than
toward an activating Fc.gamma. receptor.
51. The method of claim 50, wherein the inhibitory Fc.gamma.
receptor is human Fc.gamma.RIIb, and the activating Fc.gamma.
receptor is human Fc.gamma.RIa, human Fc.gamma.RIIa (R), human
Fc.gamma.RIIa (H), human Fc.gamma.RIIIa (V), or human
Fc.gamma.RIIIa (F).
52. The method of claim 50, wherein the amino acid at position 238
or 328 (EU numbering) in the Fc region of (i) is different from the
amino acid in the corresponding position of a native human IgG Fc
region.
53. The method of claim 52, wherein the amino acid at position 238
of the Fc region of (i) is Asp, or the amino acid at position 328
of the Fc region of (i) is Glu (all positions by EU numbering).
54. The method of claim 53, wherein the Fc region of (i) comprises,
at one or more of the following positions, the indicated amino acid
(all positions by EU numbering): Asp at position 233; Tip or Tyr at
position 234; Ala, Asp, Glu, Leu, Met, Phe, Trp, or Tyr at position
237; Asp at position 239; Ala, Gln, or Val at position 267; Asn,
Asp, or Glu at position 268; Gly at position 271; Ala, Asn, Asp,
Gln, Glu, Leu, Met, Ser, or Thr at position 326; Arg, Lys, or Met
at position 330; Ile, Leu, or Met at position 323; Asp at position
296.
55. A method of reducing the amount of an antigen in the plasma of
a subject, the method comprising identifying a subject as being in
need of a reduction in the amount of the antigen in the subject's
plasma; and administering to the subject at least two different
antigen-binding molecules including (i) a first antigen-binding
molecule that comprises a first Fc region and a first
antigen-binding domain that binds to a first epitope on the
antigen, and (ii) a second antigen-binding molecule that comprises
a second Fc region and a second antigen binding domain that binds
to a second epitope on the antigen, wherein (a) the first and
second epitopes may be identical or different, (b) the
antigen-binding activity of at least one of the antigen-binding
domains varies depending on pH or on calcium ion concentration, and
(c) when contacted with molecules of the antigen, the first and
second antigen-binding molecules form an immune complex comprising
the first and second antigen-binding molecules and two or more
copies of the antigen.
56. The method of claim 55, wherein the at least two different
antigen-binding molecules are administered in admixture.
57. A pharmaceutical composition comprising at least two different
antigen-binding molecules including (i) a first antigen-binding
molecule that comprises a first Fc region and a first
antigen-binding domain that binds to a first epitope on the
antigen, and (ii) a second antigen-binding molecule that comprises
a second Fc region and a second antigen binding domain that binds
to a second epitope on the antigen, wherein (a) the first and
second epitopes may be identical or different, (b) the
antigen-binding activity of at least one of the antigen-binding
domains varies depending on pH or on calcium ion concentration, and
(c) when contacted with molecules of the antigen, the first and
second antigen-binding molecules form an immune complex comprising
the first and second antigen-binding molecules and two or more
copies of the antigen.
58. A method of screening for an antigen-binding molecule able to
remove an antigen from plasma, the method comprising: (a) providing
a composition comprising multiple copies of an antigen-binding
molecule that comprises an Fc region and two or more
antigen-binding domains, including a first antigen-binding domain
that binds to a first epitope on the antigen and a second
antigen-binding domain that binds to a second epitope on the
antigen, wherein (i) the first and second epitopes may be identical
or different, and (ii) the antigen-binding activity of at least one
of the antigen-binding domains varies depending on pH or on calcium
ion concentration; (b) contacting the composition with an antigen
composition comprising multiple copies of the antigen, thereby
forming immune complexes comprising the antigen and the
antigen-binding molecule; (c) determining that the immune complexes
formed in (b) include immune complexes containing multiple copies
of the antigen-binding molecule and multiple copies of the antigen
in a single immune complex; and (d) based on the determination of
(c), identifying the antigen-binding molecule as potentially being
able to remove the antigen from plasma.
59. The method of claim 58, further comprising (e) testing whether
the antigen-binding molecule removes the antigen from a subject's
plasma in vivo.
60. A method of manufacturing an antigen-binding molecule able to
remove an antigen from plasma, the method comprising: (a) providing
a cell comprising nucleic acid encoding an antigen-binding molecule
comprising an Fc region and two or more antigen-binding domains,
including a first antigen-binding domain that binds to a first
epitope on the antigen and a second antigen-binding domain that
binds to a second epitope on the antigen, wherein (i) the first and
second epitopes may be identical or different, (ii) the
antigen-binding activity of at least one of the antigen-binding
domains varies depending on pH or on calcium ion concentration, and
(iii) the antigen-binding molecule forms an immune complex
comprising two or more molecules of the antigen-binding molecule
and two or more molecules of the antigen; (b) culturing the cell,
thereby producing the antigen-binding molecule by expression of the
nucleic acid; and (c) obtaining the antigen-binding molecule from
the cell culture.
61. The method of claim 60, further comprising confirming that the
antigen-binding molecule obtained in (c) is able to remove the
antigen from a subject's plasma in vivo.
62. A method of screening for an antigen-binding molecule able to
remove an antigen from plasma, the method comprising (a) providing
a plurality of different antigen-binding molecules that bind to the
antigen, each comprising an Fc region and two or more
antigen-binding domains, wherein at least one of the
antigen-binding domains of each molecule of the plurality has an
antigen-binding activity that varies depending on pH or on calcium
ion concentration; (b) contacting each of the plurality of
different antigen-binding molecules with molecules of the antigen,
thereby forming immune complexes comprising antigen-binding
molecules and molecules of the antigen; and (c) selecting an
antigen-binding molecule that forms an immune complex comprising at
least two copies of an antigen-binding molecule and at least two
molecules of the antigen.
63. The method of claim 62, comprising (d) culturing a cell
containing nucleic acid encoding the antigen-binding molecule
selected in (c), thereby producing the selected antigen-binding
molecule.
64. The method of claim 63, comprising (e) administering the
antigen-binding molecule produced in (d) to a subject having plasma
that comprises the antigen; and (f) confirming that the level of
antigen in the plasma of the subject decreases after the
antigen-binding molecule is administered.
Description
TECHNICAL FIELD
[0001] The present invention provides uses of antigen-binding
molecules for eliminating antigens from plasma; methods for
eliminating antigens from plasma, which comprise administering
antigen-binding molecules; pharmaceutical compositions comprising
antigen-binding molecules that are capable of eliminating antigens
from plasma; methods of screening for antigen-binding molecules for
eliminating antigens from plasma; and methods for producing
antigen-binding molecules for eliminating antigens from plasma.
BACKGROUND ART
[0002] Antibodies are drawing attention as pharmaceuticals as they
have a high stability in plasma and have few side effects. At
present, a number of IgG-type antibody pharmaceuticals are
available on the market and many antibody pharmaceuticals are
currently under development (Non-patent Documents 1 and 2).
Meanwhile, various technologies applicable to second-generation
antibody pharmaceuticals have been reported, including those that
enhance effector function, antigen-binding ability,
pharmacokinetics, and stability, and those that reduce the risk of
immunogenicity (Non-patent Document 3). In general, the requisite
dose of an antibody pharmaceutical is very high. This in turn has
led to problems such as high production cost, as well as the
difficulty in producing subcutaneous formulations. In theory, the
dose of an antibody pharmaceutical may be reduced by improving
antibody pharmacokinetics or improving the affinity between
antibodies and antigens.
[0003] The literature has reported methods for improving antibody
pharmacokinetics using artificial substitution of amino acids in
constant regions (Non-patent Documents 4 and 5). Similarly,
affinity maturation has been reported as a technology for enhancing
antigen-binding ability or antigen-neutralizing activity
(Non-patent Document 6). This technology enables enhancement of
antigen-binding activity by introducing amino acid mutations into
the CDR region of a variable region or such. The enhancement of
antigen-binding ability enables improvement of in vitro biological
activity or reduction of dosage, and further enables improvement of
in vivo (in the body) efficacy (Non-patent Document 7).
[0004] Meanwhile, the antigen-neutralizing capacity of a single
antibody molecule depends on its affinity. By increasing the
affinity, an antigen can be neutralized by a smaller amount of an
antibody. Various methods can be used to enhance antibody affinity
(Non-patent Document 6). Furthermore, if the affinity could be made
infinite by covalently binding the antibody to the antigen, a
single antibody molecule could neutralize one antigen molecule (a
divalent antibody can neutralize two antigen molecules). However,
conventional methods have a limitation in which a single antibody
molecule binds to one single antigen molecule (two antigens when it
is bivalent). However, it has been recently reported that a single
antigen-binding molecule can bind to a plurality of antigen
molecules by using an antigen-binding molecule that binds to
antigen in a pH-dependent manner (Patent Document 1 and Non-Patent
Document 8). A pH-dependent antigen-binding molecule binds strongly
to an antigen under the neutral condition in plasma, and releases
the antigen under the acidic condition in the endosome.
Furthermore, after release of the antigen, the antigen-binding
molecule is released into the plasma via FcRn and binds to an
antigen again; therefore, a single pH-dependent antigen-binding
molecule can repeatedly bind to a number of antigens.
[0005] Furthermore, since pH-dependent antigen-binding molecules
which have been modified to enhance FcRn binding under a neutral
condition (pH7.4) have the advantage of being able to bind
repeatedly to antigens and the effect of eliminating antigens from
plasma, administration of such antigen-binding molecules has been
reported to enable antigen elimination from plasma (Patent Document
2). Conventional pH-dependent antigen-binding molecules comprising
an IgG antibody Fc region hardly show FcRn binding under neutral
conditions. Therefore, uptake of complexes formed between an
antigen-binding molecule and an antigen into cells may be mainly
through non-specific uptake. According to this report, pH-dependent
antigen-binding molecules that have been modified to enhance FcRn
binding under a neutral condition (pH7.4) can accelerate antigen
elimination more than conventional pH-dependent antigen-binding
molecules comprising an IgG antibody Fc region (Patent Document
2).
[0006] Since antigens have very short retentivity in plasma
compared to antibodies having an FcRn-mediated recycling mechanism,
binding of an antigen in plasma to an antibody having a recycling
mechanism (wherein the binding is not pH-dependent) lengthens the
normal retentivity in plasma and increases the plasma antigen
concentration. For example, when a plasma antigen has multiple
types of physiological functions, even if one type of physiological
activity is blocked by antibody binding, the plasma concentration
of the antigen may exacerbate symptoms caused by other
physiological functions due to antibody binding. From such
viewpoint, eliminating plasma antigens is sometimes favorable, and
methods similar to those described above for making modifications
to the Fc region to enhance FcRn binding with the objective of
accelerating antigen elimination have been reported, but other
methods for accelerating antigen elimination have not been reported
so far.
[0007] Prior art documents of the present invention are shown
below.
PRIOR ART DOCUMENTS
Patent Documents
[0008] [Patent Document 1] International Publication WO2009/125825
[0009] [Patent Document 2] International Publication
WO2011/122011
Non-Patent Documents
[0009] [0010] [Non-patent Document 1] Monoclonal antibody successes
in the clinic, Janice M Reichert, Clark J Rosensweig, Laura B Faden
& Matthew C Dewitz, Nat. Biotechnol. (2005) 23, 1073-1078
[0011] [Non-patent Document 2] Pavlou A K, Belsey M J. The
therapeutic antibodies market to 2008. Eur. J. Pharm. Biopharm.
(2005) 59 (3), 389-396 [0012] [Non-patent Document 3] Kim S J, Park
Y, Hong H J. Antibody engineering for the development of
therapeutic antibodies, Mol. Cells. (2005) 20 (1), 17-29 [0013]
[Non-patent Document 4] Hinton P R, Xiong J M, Johlfs M G, Tang M
T, Keller S, Tsurushita N, J. Immunol. (2006) 176 (1), 346-356
[0014] [Non-patent Document 5] Ghetie V, Popov S, Borvak J, Radu C,
Matesoi D, Medesan C, Ober R J, Ward E S. Nat. Biotechnol. (1997)
15 (7), 637-640 [0015] [Non-patent Document 6] Rajpal A, Beyaz N,
Haber L, Cappuccilli G, Yee H, Bhatt R R, Takeuchi T, Lerner R A,
Crea R. Proc. Natl. Acad. Sci. USA. (2005) 102 (24), 8466-8471
[0016] [Non-patent Document 7] Wu H, Pfarr D S, Johnson S, Brewah Y
A, Woods R M, Patel N K, White W I, Young J F, Kiener P A. J. Mol.
Biol. (2007) 368, 652-665 [0017] [Non-patent Document 8] Igawa T,
et al., Nat. Biotechnol. (2010) 28, 1203-1207
SUMMARY OF THE INVENTION
Problems to be Solved by the Invention
[0018] The present invention was achieved in view of the above
circumstances. An objective of the present invention is to provide
uses of antigen-binding molecules for eliminating antigens from
plasma; methods for eliminating antigens from plasma, which
comprise administering antigen-binding molecules; pharmaceutical
compositions comprising antigen-binding molecules that are capable
of eliminating antigens from plasma; methods of screening for
antigen-binding molecules for eliminating antigens from plasma; and
methods for producing antigen-binding molecules for eliminating
antigens from plasma.
Means for Solving the Problems
[0019] The present inventors conducted dedicated studies to achieve
the above-described objectives. As a result, the present inventors
produced antigen-binding molecules comprising (i) an Fc region and
(ii) two or more antigen-binding domains where at least one of the
domains is an antigen-binding domain whose antigen binding activity
varies depending on the ion concentration condition, which are
antigen-binding molecules that can form an immune complex
comprising (a) two or more of the antigen-binding molecules and (b)
two or more antigen molecules comprising two or more antigenic
binding units. Furthermore, the present inventors discovered that
the antigen-binding molecules can be used to eliminate antigens
from plasma. Furthermore, the present inventors discovered that the
antigen-binding molecules are useful as pharmaceutical
compositions, and they produced methods for eliminating antigens
from plasma, which comprise administering the antigen-binding
molecules. Furthermore, the present inventors discovered methods of
screening for antigen-binding molecules having the aforementioned
properties, and created methods for producing the molecules,
thereby completing the present invention.
[0020] More specifically, the present invention provides the
following:
[1] use of an antigen-binding molecule comprising (i) an Fc region
and (ii) two or more antigen-binding domains, wherein at least one
of the domains is an antigen-binding domain of which
antigen-binding activity varies depending on an ion concentration
condition, and wherein the antigen-binding molecule can form an
immune complex which comprises (a) two or more of the
antigen-binding molecules and (b) two or more antigens, wherein the
antigens comprise two or more antigenic binding units, for
eliminating the antigens from plasma; [2] the use of [1], wherein
the ion concentration condition is a calcium ion concentration
condition; [3] the use of [2], wherein the antigen-binding domain
has an antigen-binding activity under a low calcium-ion
concentration condition that is lower than the antigen-binding
activity under a high calcium-ion concentration condition; [4] the
use of any one of [1] to [3], wherein the ion concentration
condition is a pH condition; [5] the use of [4], wherein the
antigen-binding domain has an antigen binding activity in an acidic
pH range that is lower than the antigen-binding activity in a
neutral pH range condition; [6] the use of any one of [1] to [5],
wherein the antigens comprising two or more antigenic binding units
are multimers; [7] the use of [6], wherein the antigen is any one
of GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6
(BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9,
GDF-15 (MIC-1), TNF, TNF-alpha, TNF-alphabeta, TNF-beta2, TNFSF10
(TRAIL Apo-2 ligand, TL2), TNFSF11 (TRANCE/RANK ligand ODF, OPG
ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL
TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT
HVEM ligand, LTg), TNFSF15 (TL1A/VEGI), TNFSF18 (GITR ligand AITR
ligand, TL6), TNFSF1A (TNF-.alpha. Conectin, DIF, TNFSF2), TNFSF1B
(TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand
gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP),
TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand
CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137
ligand), VEGF, IgE, IgA, IgG, IgM, RANKL, TGF-alpha, TGF-beta,
TGF-beta Pan Specific, and IL-8; [8] the use of any one of [1] to
[5], wherein the antigens comprising two or more antigenic binding
units are monomers; [9] the use of any one of [1] to [8], wherein
the antigen-binding molecules are a multispecific or multiparatopic
antigen-binding molecule, or a cocktail of antigen-binding
molecules; [10] the use of any one of [1] to [9], wherein the Fc
region is represented by any one of SEQ ID NOs: 13, 14, 15, and 16;
[11] the use of any one of [1] to [9], wherein the Fc region is an
Fc region with an enhanced FcRn-binding activity under an acidic pH
range condition compared to that of the Fc region represented by
any one of SEQ ID NOs: 13, 14, 15, and 16; [12] the use of [11],
wherein the Fc region is an Fc region with a substitution of at
least one or more amino acids selected from the group consisting of
amino acids at positions 238, 244, 245, 249, 250, 251, 252, 253,
254, 255, 256, 257, 258, 260, 262, 265, 270, 272, 279, 283, 285,
286, 288, 293, 303, 305, 307, 308, 309, 311, 312, 314, 316, 317,
318, 332, 339, 340, 341, 343, 356, 360, 362, 375, 376, 377, 378,
380, 382, 385, 386, 387, 388, 389, 400, 413, 415, 423, 424, 427,
428, 430, 431, 433, 434, 435, 436, 438, 439, 440, 442, and 447 (EU
numbering) in the amino acid sequence of the Fc region represented
by any one of SEQ ID NOs: 13, 14, 15, and 16; [13] the use of [12],
wherein the Fc region comprises at least one or more amino acids
selected from the group consisting of: Leu for the amino acid of
position 238; Leu for the amino acid of position 244; Arg for the
amino acid of position 245; Pro for the amino acid of position 249;
Gln or Glu for the amino acid of position 250; Arg, Asp, Glu, or
Leu for the amino acid of position 251; Phe, Ser, Thr, or Tyr for
the amino acid of position 252; Ser or Thr for the amino acid of
position 254; Arg, Gly, Ile, or Leu for the amino acid of position
255; Ala, Arg, Asn, Asp, Gln, Glu, Pro, or Thr for the amino acid
of position 256; Ala, Ile, Met, Asn, Ser, or Val for the amino acid
of position 257; Asp for the amino acid of position 258; Ser for
the amino acid of position 260; Leu for the amino acid of position
262; Lys for the amino acid of position 270; Leu or Arg for the
amino acid of position 272; Ala, Asp, Gly, His, Met, Asn, Gln, Arg,
Ser, Thr, Trp, or Tyr for the amino acid of position 279; Ala, Asp,
Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or
Tyr for the amino acid of position 283; Asn for the amino acid of
position 285; Phe for the amino acid of position 286; Asn or Pro
for the amino acid of position 288; Val for the amino acid of
position 293; Ala, Glu, Gln, or Met for the amino acid of position
307; Ala, Glu, Ile, Lys, Leu, Met, Ser, Val, or Trp for the amino
acid of position 311; Pro for the amino acid of position 309; Ala,
Asp, or Pro for the amino acid of position 312; Ala or Leu for the
amino acid of position 314; Lys for the amino acid of position 316;
Pro for the amino acid of position 317; Asn or Thr for the amino
acid of position 318; Phe, His, Lys, Leu, Met, Arg, Ser, or Trp for
the amino acid of position 332; Asn, Thr, or Trp for the amino acid
of position 339; Pro for the amino acid of position 341; Glu, His,
Lys, Gln, Arg, Thr, or Tyr for the amino acid of position 343; Arg
for the amino acid of position 375; Gly, Ile, Met, Pro, Thr, or Val
for the amino acid of position 376; Lys for the amino acid of
position 377; Asp, Asn, or Val for the amino acid of position 378;
Ala, Asn, Ser, or Thr for the amino acid of position 380; Phe, His,
Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for
the amino acid of position 382; Ala, Arg, Asp, Gly, His, Lys, Ser,
or Thr for the amino acid of position 385; Arg, Asp, Ile, Lys, Met,
Pro, Ser, or Thr for the amino acid of position 386; Ala, Arg, His,
Pro, Ser, or Thr for the amino acid of position 387; Asn, Pro, or
Ser for the amino acid of position 389; Asn for the amino acid of
position 423; Asn for the amino acid of position 427; Leu, Met,
Phe, Ser, or Thr for the amino acid of position 428; Ala, Phe, Gly,
His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, or Tyr for
the amino acid of position 430; His or Asn for the amino acid of
position 431; Arg, Gln, His, Ile, Lys, Pro, or Ser for the amino
acid of position 433; Ala, Gly, His, Phe, Ser, Trp, or Tyr for the
amino acid of position 434; Arg, Asn, His, Ile, Leu, Lys, Met, or
Thr for the amino acid of position 436; Lys, Leu, Thr, or Trp for
the amino acid of position 438; Lys for the amino acid of position
440; and Lys for the amino acid of position 442; Ile, Pro, or Thr
for the amino acid of position 308; as indicated by EU numbering in
the amino acid sequence of the Fc region represented by any one of
SEQ ID NOs: 13, 14, 15, and 16; [14] the use of any one of [1] to
[9], wherein the Fc region is an Fc region with an enhanced
FcRn-binding activity under a neutral pH range condition compared
to that of the Fc region represented by any one of SEQ ID NOs: 13,
14, 15, and 16; [15] the use of [14], wherein the Fc region is an
Fc region with a substitution of at least one or more amino acids
selected from the group consisting of positions 237, 248, 250, 252,
254, 255, 256, 257, 258, 265, 286, 289, 297, 298, 303, 305, 307,
308, 309, 311, 312, 314, 315, 317, 332, 334, 360, 376, 380, 382,
384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 (EU numbering)
in the amino acid sequence of the Fc region represented by any one
of SEQ ID NOs: 13, 14, 15, and 16; [16] the use of [15], wherein
the Fc region comprises at least one or more amino acids selected
from the group consisting of: Met for the amino acid of position
237; Ile for the amino acid of position 248; Ala, Phe, Ile, Met,
Gln, Ser, Val, Trp, or Tyr for the amino acid of position 250; Phe,
Trp, or Tyr for the amino acid of position 252; Thr for the amino
acid of position 254; Glu for the amino acid of position 255; Asp,
Asn, Glu, or Gln for the amino acid of position 256; Ala, Gly, Ile,
Leu, Met, Asn, Ser, Thr, or Val for the amino acid of position 257;
His for the amino acid of position 258; Ala for the amino acid of
position 265; Ala or Glu for the amino acid of position 286; His
for the amino acid of position 289; Ala for the amino acid of
position 297; Ala for the amino acid of position 303; Ala for the
amino acid of position 305; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu,
Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr for the amino acid
of position 307; Ala, Phe, Ile, Leu, Met, Pro, Gln, or Thr for the
amino acid of position 308; Ala, Asp, Glu, Pro, or Arg for the
amino acid of position 309; Ala, His, or Ile for the amino acid of
position 311; Ala or His for the amino acid of position 312; Lys or
Arg for the amino acid of position 314; Ala, Asp, or His for the
amino acid of position 315; Ala for the amino acid of position 317;
Val for the amino acid of position 332; Leu for the amino acid of
position 334; His for the amino acid of position 360; Ala for the
amino acid of position 376; Ala for the amino acid of position 380;
Ala for the amino acid of position 382; Ala for the amino acid of
position 384; Asp or His for the amino acid of position 385; Pro
for the amino acid of position 386; Glu for the amino acid of
position 387; Ala or Ser for the amino acid of position 389; Ala
for the amino acid of position 424; Ala, Asp, Phe, Gly, His, Ile,
Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr for the amino
acid of position 428; Lys for the amino acid of position 433; Ala,
Phe, His, Ser, Trp, or Tyr for the amino acid of position 434; and
His, Ile, Leu, Phe, Thr, or Val for the amino acid of position 436;
as indicated by EU numbering in the amino acid sequence of the Fc
region represented by any one of SEQ ID NOs: 13, 14, 15, and 16;
[17] the use of any one of [1] to [13], wherein the Fc region
includes an Fc region that has a higher Fc.gamma. receptor-binding
activity than that of the Fc region of a native human IgG; [18] the
use of [17], wherein the Fc region comprises in its amino acid
sequence at least one or more amino acids that are different from
amino acids of the native human IgG Fc region selected from the
group consisting of positions 221, 222, 223, 224, 225, 227, 228,
230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243,
244, 245, 246, 247, 249, 250, 251, 254, 255, 256, 258, 260, 262,
263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275,
276, 278, 279, 280, 281, 282, 283, 284, 285, 286, 288, 290, 291,
292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304,
305, 311, 313, 315, 317, 318, 320, 322, 323, 324, 325, 326, 327,
328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 339, 376, 377,
378, 379, 380, 382, 385, 392, 396, 421, 427, 428, 429, 434, 436,
and 440 (EU numbering); [19] the use of [18], wherein the Fc region
comprises in its amino acid sequence at least one or more amino
acids selected from the group consisting of: Lys or Tyr for the
amino acid of position 221; Phe, Trp, Glu, or Tyr for the amino
acid of position 222; Phe, Trp, Glu, or Lys for the amino acid of
position 223; Phe, Trp, Glu, or Tyr for the amino acid of position
224; Glu, Lys, or Trp for the amino acid of position 225; Glu, Gly,
Lys, or Tyr for the amino acid of position 227; Glu, Gly, Lys, or
Tyr for the amino acid of position 228; Ala, Glu, Gly, or Tyr for
the amino acid of position 230; Glu, Gly, Lys, Pro, or Tyr for the
amino acid of position 231; Glu, Gly, Lys, or Tyr for the amino
acid of position 232; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met,
Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of
position 233; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn,
Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of
position 234; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn,
Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of
position 235; Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn,
Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of
position 236; Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro,
Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of position
237; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,
Ser, Thr, Val, Trp, or Tyr for the amino acid of position 238; Asp,
Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr,
Val, Trp, or Tyr for the amino acid of position 239; Ala, Ile, Met,
or Thr for the amino acid of position 240; Asp, Glu, Leu, Arg, Trp,
or Tyr for the amino acid of position 241; Leu, Glu, Leu, Gln, Arg,
Trp, or Tyr for the amino acid of position 243; His for the amino
acid of position 244; Ala for the amino acid of position 245; Asp,
Glu, His, or Tyr for the amino acid of position 246; Ala, Phe, Gly,
His, Ile, Leu, Met, Thr, Val, or Tyr for the amino acid of position
247; Glu, His, Gln, or Tyr for the amino acid of position 249; Glu
or Gln for the amino acid of position 250; Phe for the amino acid
of position 251; Phe, Met, or Tyr for the amino acid of position
254; Glu, Leu, or Tyr for the amino acid of position 255; Ala, Met,
or Pro for the amino acid of position 256; Asp, Glu, His, Ser, or
Tyr for the amino acid of position 258; Asp, Glu, His, or Tyr for
the amino acid of position 260; Ala, Glu, Phe, Ile, or Thr for the
amino acid of position 262; Ala, Ile, Met, or Thr for the amino
acid of position 263; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met,
Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr for the amino acid of
position 264; Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,
Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of
position 265; Ala, Ile, Met, or Thr for the amino acid of position
266; Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,
Thr, Val, Trp, or Tyr for the amino acid of position 267; Asp, Glu,
Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, or Trp for
the amino acid of position 268; Phe, Gly, His, Ile, Lys, Leu, Met,
Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of
position 269; Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg,
Ser, Thr, Trp, or Tyr for the amino acid of position 270; Ala, Asp,
Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr,
Val, Trp, or Tyr for the amino acid of position 271; Asp, Phe, Gly,
His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for
the amino acid of position 272; Phe or Ile for the amino acid of
position 273; Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro,
Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of position 274;
Leu or Trp for the amino acid of position 275; Asp, Glu, Phe, Gly,
His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the
amino acid of position 276; Asp, Glu, Gly, His, Ile, Lys, Leu, Met,
Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp for the amino acid of
position 278; Ala for the amino acid of position 279; Ala, Gly,
His, Lys, Leu, Pro, Gln, Trp, or Tyr for the amino acid of position
280; Asp, Lys, Pro, or Tyr for the amino acid of position 281; Glu,
Gly, Lys, Pro, or Tyr for the amino acid of position 282; Ala, Gly,
His, Ile, Lys, Leu, Met, Pro, Arg, or Tyr for the amino acid of
position 283; Asp, Glu, Leu, Asn, Thr, or Tyr for the amino acid of
position 284; Asp, Glu, Lys, Gln, Trp, or Tyr for the amino acid of
position 285; Glu, Gly, Pro, or Tyr for the amino acid of position
286; Asn, Asp, Glu, or Tyr for the amino acid of position 288; Asp,
Gly, His, Leu, Asn, Ser, Thr, Trp, or Tyr for the amino acid of
position 290; Asp, Glu, Gly, His, Ile, Gln, or Thr for the amino
acid of position 291; Ala, Asp, Glu, Pro, Thr, or Tyr for the amino
acid of position 292; Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg,
Ser, Thr, Val, Trp, or Tyr for the amino acid of position 293; Phe,
Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or
Tyr for the amino acid of position 294; Asp, Glu, Phe, Gly, His,
Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the
amino acid of position 295; Ala, Asp, Glu, Gly, His, Ile, Lys, Leu,
Met, Asn, Gln, Arg, Ser, Thr, or Val for the amino acid of position
296; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg,
Ser, Thr, Val, Trp, or Tyr for the amino acid of position 297; Ala,
Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, or
Tyr for the amino acid of position 298; Ala, Asp, Glu, Phe, Gly,
His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr
for the amino acid of position 299; Ala, Asp, Glu, Gly, His, Ile,
Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp for the
amino acid of position 300; Asp, Glu, His, or Tyr for the amino
acid of position 301; Ile for the amino acid of position 302; Asp,
Gly, or Tyr for the amino acid of position 303; Asp, His, Leu, Asn,
or Thr for the amino acid of position 304; Glu, Ile, Thr, or Tyr
for the amino acid of position 305; Ala, Asp, Asn, Thr, Val, or Tyr
for the amino acid of position 311; Phe for the amino acid of
position 313; Leu for the amino acid of position 315; Glu or Gln
for the amino acid of position 317; His, Leu, Asn, Pro, Gln, Arg,
Thr, Val, or Tyr for the amino acid of position 318; Asp, Phe, Gly,
His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, or Tyr for the amino
acid of position 320; Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr,
Val, Trp, or Tyr for the amino acid of position 322; Ile for the
amino acid of position 323; Asp, Phe, Gly, His, Ile, Leu, Met, Pro,
Arg, Thr, Val, Trp, or Tyr for the amino acid of position 324; Ala,
Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser,
Thr, Val, Trp, or Tyr for the amino acid of position 325; Ala, Asp,
Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr
for the amino acid of position 326; Ala, Asp, Glu, Phe, Gly, His,
Ile, Lys, Leu, Met, Asn, Pro,
Arg, Thr, Val, Trp, or Tyr for the amino acid of position 327; Ala,
Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser,
Thr, Val, Trp, or Tyr for the amino acid of position 328; Asp, Glu,
Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val,
Trp, or Tyr for the amino acid of position 329; Cys, Glu, Phe, Gly,
His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr
for the amino acid of position 330; Asp, Phe, His, Ile, Leu, Met,
Gln, Arg, Thr, Val, Trp, or Tyr for the amino acid of position 331;
Ala, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg,
Ser, Thr, Val, Trp, or Tyr for the amino acid of position 332; Ala,
Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, or Tyr
for the amino acid of position 333; Ala, Glu, Phe, Ile, Leu, Pro,
or Thr for the amino acid of position 334; Asp, Phe, Gly, His, Ile,
Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, or Tyr for the amino acid
of position 335; Glu, Lys, or Tyr for the amino acid of position
336; Glu, His, or Asn for the amino acid of position 337; Asp, Phe,
Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, or Thr for the amino acid
of position 339; Ala or Val for the amino acid of position 376; Gly
or Lys for the amino acid of position 377; Asp for the amino acid
of position 378; Asn for the amino acid of position 379; Ala, Asn,
or Ser for the amino acid of position 380; Ala or Ile for the amino
acid of position 382; Glu for the amino acid of position 385; Thr
for the amino acid of position 392; Leu for the amino acid of
position 396; Lys for the amino acid of position 421; Asn for the
amino acid of position 427; Phe or Leu for the amino acid of
position 428; Met for the amino acid of position 429; Trp for the
amino acid of position 434; Ile for the amino acid of position 436;
and Gly, His, Ile, Leu, or Tyr for the amino acid of position 440;
as indicated by EU numbering; [20] the use of any one of [1] to
[16], wherein the Fc region has a higher binding activity toward an
inhibitory Fc.gamma. receptor than toward an activating Fc.gamma.
receptor; [21] the use of [20], wherein the inhibitory Fc.gamma.
receptor is human Fc.gamma.RIIb; [22] the use of [20] or [21],
wherein the activating Fc.gamma. receptor is human Fc.gamma.RIa,
human Fc.gamma.RIIa (R), human Fc.gamma.RIIa (H), human
Fc.gamma.RIIIa (V), or human Fc.gamma.RIIIa (F); [23] the use of
any one of [20] to [22], wherein the amino acid at position 238 or
328 (EU numbering) in the Fc region is different from the amino
acid in the native human IgG Fc region; [24] the use of [23],
wherein the amino acid at position 238 of the Fc region is Asp or
the amino acid of position 328 of the Fc region is Glu as indicated
by EU numbering; [25] the use of [23] or [24], wherein the amino
acid sequence of the Fc region comprises at least one or more amino
acids selected from the group consisting of: Asp for the amino acid
of position 233; Trp or Tyr for the amino acid of position 234;
Ala, Asp, Glu, Leu, Met, Phe, Trp, or Tyr for the amino acid of
position 237; Asp for the amino acid of position 239; Ala, Gln, or
Val for the amino acid of position 267; Asn, Asp, or Glu for the
amino acid of position 268; Gly for the amino acid of position 271;
Ala, Asn, Asp, Gln, Glu, Leu, Met, Ser, or Thr for the amino acid
of position 326; Arg, Lys, or Met for the amino acid of position
330; Ile, Leu, or Met for the amino acid of position 323; and Asp
for the amino acid of position 296; as indicated by EU numbering;
[26] a method of screening for an antigen-binding molecule having a
function of eliminating an antigen from the plasma, wherein the
method comprises: (a) obtaining an antigen-binding domain of which
antigen-binding activity varies depending on an ion concentration
condition; (b) obtaining a gene encoding the antigen-binding domain
selected in (a) above; (c) operably linking the gene obtained in
(b) above with a gene encoding an Fc region; (d) culturing a host
cell comprising the genes operably linked in (c) above; (e)
isolating an antigen-binding molecule from a culture solution
obtained in (d) above; (f) contacting the antigen-binding molecule
obtained in (e) above with an antigen; and (g) evaluating the
formation of an immune complex comprising the antigen-binding
molecule and the antigen; [27] a method for producing an
antigen-binding molecule having a function of eliminating an
antigen from the plasma, wherein the method comprises: (a)
contacting an antigen with an antigen-binding molecule comprising
an Fc region and two or more antigen-binding domains, wherein at
least one of the antigen-binding domains has an antigen-binding
activity that varies depending on an ion concentration condition;
(b) evaluating formation of an immune complex comprising the
antigen-binding molecule and the antigen; (c) culturing a host cell
comprising a vector that carries a gene encoding an antigen-binding
molecule which is confirmed to form an immune complex in (b) above;
and (d) isolating the antigen-binding molecule from a culture
solution obtained in (c) above; [28] a method for producing an
antigen-binding molecule having a function of eliminating an
antigen from the plasma, wherein the method comprises: (a)
obtaining an antigen-binding domain of which antigen-binding
activity varies depending on an ion concentration condition; (b)
obtaining a gene encoding the antigen-binding domain selected in
(a) above; (c) operably linking the gene obtained in (b) above with
a gene encoding an Fc region; (d) culturing a host cell comprising
the genes operably linked in (c) above; (e) isolating an
antigen-binding molecule from a culture solution obtained in (d)
above; (f) contacting the antigen-binding molecule obtained in (e)
above with an antigen; (g) evaluating formation of an immune
complex comprising the antigen-binding molecule and the antigen;
(h) culturing a host cell comprising a vector that carries a gene
encoding an antigen-binding molecule which is confirmed to form an
immune complex in (g) above; and (i) isolating the antigen-binding
molecule from a culture solution obtained in (h) above; and [29] a
method for producing an antigen-binding molecule having a function
of eliminating an antigen from the plasma, wherein the method
comprises: (a) obtaining an antigen-binding domain of which
antigen-binding activity varies depending on an ion concentration
condition; (b) obtaining a gene encoding the antigen-binding domain
selected in (a) above; (c) operably linking the gene obtained in
(b) above with a gene encoding an Fc region; (d) culturing a host
cell comprising the genes operably linked in (c) above; and (e)
isolating an antigen-binding molecule from a culture solution
obtained in (d) above; and wherein the method further comprises
contacting the antigen-binding molecule obtained by the production
method with an antigen, and evaluating the formation of an immune
complex comprising the antigen-binding molecule and the
antigen.
[0021] The above-mentioned [1] to [25] may be restated as
follows:
[1'] a pharmaceutical composition for eliminating an antigen from
plasma, wherein the pharmaceutical composition comprises an
antigen-binding molecule comprising (i) an Fc region and (ii) two
or more antigen-binding domains, wherein at least one of the
domains is an antigen-binding domain of which antigen-binding
activity varies depending on an ion concentration condition, and
wherein the antigen-binding molecule can form an immune complex
which comprises (a) two or more of the antigen-binding molecules
and (b) two or more antigens, wherein the antigens comprise two or
more antigenic binding units; [2'] the pharmaceutical composition
of [l'], wherein the ion concentration condition is a calcium ion
concentration condition; [3'] the pharmaceutical composition of
[2'], wherein the antigen-binding domain has an antigen-binding
activity under a low calcium-ion concentration condition that is
lower than the antigen-binding activity under a high calcium-ion
concentration condition; [4'] the pharmaceutical composition of any
one of [1'] to [3'], wherein the ion concentration condition is a
pH condition; [5'] the pharmaceutical composition of [4'], wherein
the antigen-binding domain has an antigen binding activity in an
acidic pH range that is lower than the antigen-binding activity in
a neutral pH range condition; [6'] the pharmaceutical composition
of any one of [1'] to [5'], wherein the antigens comprising two or
more antigenic binding units are multimers; [7'] the pharmaceutical
composition of [6'], wherein the antigen is any one of GDF, GDF-1,
GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2),
GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1),
TNF, TNF-alpha, TNF-alphabeta, TNF-beta2, TNFSF10 (TRAIL Apo-2
ligand, TL2), TNFSF11 (TRANCE/RANK ligand ODF, OPG ligand), TNFSF12
(TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B
(BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand,
LTg), TNFSF15 (TL1A/VEGI), TNFSF18 (GITR ligand AITR ligand, TL6),
TNFSF1A (TNF-.alpha. Conectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa,
TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1),
TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas
ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70),
TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand),
VEGF, IgE, IgA, IgG, IgM, RANKL, TGF-alpha, TGF-beta, TGF-beta Pan
Specific, and IL-8; [8'] the pharmaceutical composition of any one
of [1'] to [5'], wherein the antigens comprising two or more
antigenic binding units are monomers; [9'] the pharmaceutical
composition of any one of [1'] to [8'], wherein the antigen-binding
molecules are a multispecific or multiparatopic antigen-binding
molecule, or a cocktail of antigen-binding molecules; [10'] the
pharmaceutical composition of any one of [1'] to [9'], wherein the
Fc region is represented by any one of SEQ ID NOs: 13, 14, 15, and
16; [11'] the pharmaceutical composition of any one of [1'] to
[9'], wherein the Fc region is an Fc region with an enhanced
FcRn-binding activity under an acidic pH range condition compared
to that of the Fc region represented by any one of SEQ ID NOs: 13,
14, 15, and 16; [12'] the pharmaceutical composition of [11'],
wherein the Fc region is an Fc region with an amino acid
substitution of at least one or more amino acids selected from the
group consisting of positions 238, 244, 245, 249, 250, 251, 252,
253, 254, 255, 256, 257, 258, 260, 262, 265, 270, 272, 279, 283,
285, 286, 288, 293, 303, 305, 307, 308, 309, 311, 312, 314, 316,
317, 318, 332, 339, 340, 341, 343, 356, 360, 362, 375, 376, 377,
378, 380, 382, 385, 386, 387, 388, 389, 400, 413, 415, 423, 424,
427, 428, 430, 431, 433, 434, 435, 436, 438, 439, 440, 442, and 447
(EU numbering) in the amino acid sequence of the Fc region
represented by any one of SEQ ID NOs: 13, 14, 15, and 16; [13'] the
pharmaceutical composition of [12'], wherein the Fc region
comprises at least one or more amino acids selected from the group
consisting of: Leu for the amino acid of position 238; Leu for the
amino acid of position 244; Arg for the amino acid of position 245;
Pro for the amino acid of position 249; Gln or Glu for the amino
acid of position 250; Arg, Asp, Glu, or Leu for the amino acid of
position 251; Phe, Ser, Thr, or Tyr for the amino acid of position
252; Ser or Thr for the amino acid of position 254; Arg, Gly, Ile,
or Leu for the amino acid of position 255; Ala, Arg, Asn, Asp, Gln,
Glu, Pro, or Thr for the amino acid of position 256; Ala, Ile, Met,
Asn, Ser, or Val for the amino acid of position 257; Asp for the
amino acid of position 258; Ser for the amino acid of position 260;
Leu for the amino acid of position 262; Lys for the amino acid of
position 270; Leu or Arg for the amino acid of position 272; Ala,
Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr for the
amino acid of position 279; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu,
Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr for the amino acid of
position 283; Asn for the amino acid of position 285; Phe for the
amino acid of position 286; Asn or Pro for the amino acid of
position 288; Val for the amino acid of position 293; Ala, Glu,
Gln, or Met for the amino acid of position 307; Ala, Glu, Ile, Lys,
Leu, Met, Ser, Val, or Trp for the amino acid of position 311; Pro
for the amino acid of position 309; Ala, Asp, or Pro for the amino
acid of position 312; Ala or Leu for the amino acid of position
314; Lys for the amino acid of position 316; Pro for the amino acid
of position 317; Asn or Thr for the amino acid of position 318;
Phe, His, Lys, Leu, Met, Arg, Ser, or Trp for the amino acid of
position 332; Asn, Thr, or Trp for the amino acid of position 339;
Pro for the amino acid of position 341; Glu, His, Lys, Gln, Arg,
Thr, or Tyr for the amino acid of position 343; Arg for the amino
acid of position 375; Gly, Ile, Met, Pro, Thr, or Val for the amino
acid of position 376; Lys for the amino acid of position 377; Asp,
Asn, or Val for the amino acid of position 378; Ala, Asn, Ser, or
Thr for the amino acid of position 380; Phe, His, Ile, Lys, Leu,
Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid
of position 382; Ala, Arg, Asp, Gly, His, Lys, Ser, or Thr for the
amino acid of position 385; Arg, Asp, Ile, Lys, Met, Pro, Ser, or
Thr for the amino acid of position 386; Ala, Arg, His, Pro, Ser, or
Thr for the amino acid of position 387; Asn, Pro, or Ser for the
amino acid of position 389; Asn for the amino acid of position 423;
Asn for the amino acid of position 427; Leu, Met, Phe, Ser, or Thr
for the amino acid of position 428; Ala, Phe, Gly, His, Ile, Lys,
Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, or Tyr for the amino acid
of position 430; His or Asn for the amino acid of position 431;
Arg, Gln, His, Ile, Lys, Pro, or Ser for the amino acid of position
433; Ala, Gly, His, Phe, Ser, Trp, or Tyr for the amino acid of
position 434; Arg, Asn, His, Ile, Leu, Lys, Met, or Thr for the
amino acid of position 436; Lys, Leu, Thr, or Trp for the amino
acid of position 438; Lys for the amino acid of position 440; and
Lys for the amino acid of position 442; Ile, Pro, or Thr for the
amino acid of position 308; as indicated by EU numbering in the
amino acid sequence of the Fc region represented by any one of SEQ
ID NOs: 13, 14, 15, and 16; [14'] the pharmaceutical composition of
any one of [1'] to [9'], wherein the Fc region is an Fc region with
an enhanced FcRn-binding activity under a neutral pH range
condition compared to that of the Fc region represented by any one
of SEQ ID NOs: 13, 14, 15, and 16; [15'] the pharmaceutical
composition of [14'], wherein the Fc region is an Fc region with a
substitution of at least one or more amino acids selected from the
group consisting of positions 237, 248, 250, 252, 254, 255, 256,
257, 258, 265, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311,
312, 314, 315, 317, 332, 334, 360, 376, 380, 382, 384, 385, 386,
387, 389, 424, 428, 433, 434, and 436 (EU numbering) in the amino
acid sequence of the Fc region represented by any one of SEQ ID
NOs: 13, 14, 15, and 16; [16'] the pharmaceutical composition of
[15'], wherein the Fc region comprises at least one or more amino
acids selected from the group consisting of: Met for the amino acid
of position 237; Ile for the amino acid of position 248; Ala, Phe,
Ile, Met, Gln, Ser, Val, Trp, or Tyr for the amino acid of position
250; Phe, Trp, or Tyr for the amino acid of position 252; Thr for
the amino acid of position 254; Glu for the amino acid of position
255; Asp, Asn, Glu, or Gln for the amino acid of position 256; Ala,
Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val for the amino acid of
position 257; His for the amino acid of position 258; Ala for the
amino acid of position 265; Ala or Glu for the amino acid of
position 286; His for the amino acid of position 289; Ala for the
amino acid of position 297; Ala for the amino acid of position 303;
Ala for the amino acid of position 305; Ala, Asp, Phe, Gly, His,
Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr for
the amino acid of position 307; Ala, Phe, Ile, Leu, Met, Pro, Gln,
or Thr for the amino acid of position 308; Ala, Asp, Glu, Pro, or
Arg for the amino acid of position 309; Ala, His, or Ile for the
amino acid of position 311; Ala or His for the amino acid of
position 312; Lys or Arg for the amino acid of position 314; Ala,
Asp, or His for the amino acid of position 315; Ala for the amino
acid of position 317; Val for the amino acid of position 332; Leu
for the amino acid of position 334; His for the amino acid of
position 360; Ala for the amino acid of position 376; Ala for the
amino acid of position 380; Ala for the amino acid of position 382;
Ala for the amino acid of position 384; Asp or His for the amino
acid of position 385; Pro for the amino acid of position 386; Glu
for the amino acid of position 387; Ala or Ser for the amino acid
of position 389; Ala for the amino acid of position 424; Ala, Asp,
Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or
Tyr for the amino acid of position 428; Lys for the amino acid of
position 433; Ala, Phe, His, Ser, Trp, or Tyr for the amino acid of
position 434; and His, Ile, Leu, Phe, Thr, or Val for the amino
acid of position 436; as indicated by EU numbering in the amino
acid sequence of the Fc region represented by any one of SEQ ID
NOs: 13, 14, 15, and 16; [17'] the pharmaceutical composition of
any one of [1'] to [13'], wherein the Fc region includes an Fc
region that has a higher Fc.gamma. receptor-binding activity than
that of the Fc region of a native human IgG; [18'] the
pharmaceutical composition of [17'], wherein the Fc region
comprises in its amino acid sequence at least one or more amino
acids that are different from amino acids of the native human IgG
Fc region selected from the group consisting of positions 221, 222,
223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237,
238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 254,
255, 256, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270,
271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284,
285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299,
300, 301, 302, 303, 304, 305, 311, 313, 315, 317, 318, 320, 322,
323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335,
336, 337, 339, 376, 377, 378, 379, 380, 382, 385, 392, 396, 421,
427, 428, 429, 434, 436, and 440 (EU numbering); [19'] the
pharmaceutical composition of [18'], wherein the Fc region
comprises in its amino acid sequence at least one or more amino
acids selected from the group consisting of: Lys or Tyr for the
amino acid of position 221; Phe, Trp, Glu, or Tyr for the amino
acid of position 222; Phe, Trp, Glu, or Lys for the amino acid of
position 223; Phe, Trp, Glu, or Tyr for the amino acid of position
224; Glu, Lys, or Trp for the amino acid of position 225; Glu, Gly,
Lys, or Tyr for the amino acid of position 227; Glu, Gly, Lys, or
Tyr for the amino acid of position 228; Ala, Glu, Gly, or Tyr for
the amino acid of position 230; Glu, Gly, Lys, Pro, or Tyr for the
amino acid of position 231; Glu, Gly, Lys, or Tyr for the amino
acid of position 232; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met,
Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of
position 233; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn,
Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of
position 234; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Met, Asn,
Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of
position 235; Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn,
Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of
position 236; Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro,
Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of position
237; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,
Ser, Thr, Val, Trp, or Tyr for the amino acid of position 238; Asp,
Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr,
Val, Trp, or Tyr for the amino acid of position 239; Ala, Ile, Met,
or Thr for the amino acid of position 240; Asp, Glu, Leu, Arg, Trp,
or Tyr for the amino acid of position 241; Leu, Glu, Leu, Gln, Arg,
Trp, or Tyr for the amino acid of position 243; His for the amino
acid of position 244; Ala for the amino acid of position 245; Asp,
Glu, His, or Tyr for the amino acid of position 246; Ala, Phe, Gly,
His, Ile, Leu, Met, Thr, Val, or Tyr for the amino acid of position
247; Glu, His, Gln, or Tyr for the amino acid of position 249; Glu
or Gln for the amino acid of position 250; Phe for the amino acid
of position 251; Phe, Met, or Tyr for the amino acid of position
254; Glu, Leu, or Tyr for the amino acid of position 255; Ala, Met,
or Pro for the amino acid of position 256; Asp, Glu, His, Ser, or
Tyr for the amino acid of position 258; Asp, Glu, His, or Tyr for
the amino acid of position 260; Ala, Glu, Phe, Ile, or Thr for the
amino acid of position 262; Ala, Ile, Met, or Thr for the amino
acid of position 263; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met,
Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr for the amino acid of
position 264; Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,
Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of
position 265; Ala, Ile, Met, or Thr for the amino acid of position
266; Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,
Thr, Val, Trp, or Tyr for the amino acid of position 267; Asp, Glu,
Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, or Trp for
the amino acid of position 268; Phe, Gly, His, Ile, Lys, Leu, Met,
Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of
position 269; Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg,
Ser, Thr, Trp, or Tyr for the amino acid of position 270; Ala, Asp,
Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr,
Val, Trp, or Tyr for the amino acid of position 271; Asp, Phe, Gly,
His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for
the amino acid of position 272; Phe or Ile for the amino acid of
position 273; Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro,
Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of position 274;
Leu or Trp for the amino acid of position 275; Asp, Glu, Phe, Gly,
His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the
amino acid of position 276; Asp, Glu, Gly, His, Ile, Lys, Leu, Met,
Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp for the amino acid of
position 278; Ala for the amino acid of position 279; Ala, Gly,
His, Lys, Leu, Pro, Gln, Trp, or Tyr for the amino acid of position
280; Asp, Lys, Pro, or Tyr for the amino acid of position 281; Glu,
Gly, Lys, Pro, or Tyr for the amino acid of position 282; Ala, Gly,
His, Ile, Lys, Leu, Met, Pro, Arg, or Tyr for the amino acid of
position 283; Asp, Glu, Leu, Asn, Thr, or Tyr for the amino acid of
position 284; Asp, Glu, Lys, Gln, Trp, or Tyr for the amino acid of
position 285; Glu, Gly, Pro, or Tyr for the amino acid of position
286; Asn, Asp, Glu, or Tyr for the amino acid of position 288; Asp,
Gly, His, Leu, Asn, Ser, Thr, Trp, or Tyr for the amino acid of
position 290; Asp, Glu, Gly, His, Ile, Gln, or Thr for the amino
acid of position 291; Ala, Asp, Glu, Pro, Thr, or Tyr for the amino
acid of position 292; Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg,
Ser, Thr, Val, Trp, or Tyr for the amino acid of position 293; Phe,
Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or
Tyr for the amino acid of position 294; Asp, Glu, Phe, Gly, His,
Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the
amino acid of position 295; Ala, Asp, Glu, Gly, His, Ile, Lys, Leu,
Met, Asn, Gln, Arg, Ser, Thr, or Val for the amino acid of position
296; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg,
Ser, Thr, Val, Trp, or Tyr for the amino acid of position 297; Ala,
Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, or
Tyr for the amino acid of position 298; Ala, Asp, Glu, Phe, Gly,
His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr
for the amino acid of position 299; Ala, Asp, Glu, Gly, His, Ile,
Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp for the
amino acid of position 300; Asp, Glu, His, or Tyr for the amino
acid of position 301; Ile for the amino acid of position 302; Asp,
Gly, or Tyr for the amino acid of position 303; Asp, His, Leu, Asn,
or Thr for the amino acid of position 304; Glu, Ile, Thr, or Tyr
for the amino acid of position 305; Ala, Asp, Asn, Thr, Val, or Tyr
for the amino acid of position 311; Phe for the amino acid of
position 313; Leu for the amino acid of position 315; Glu or Gln
for the amino acid of position 317; His, Leu, Asn, Pro, Gln, Arg,
Thr, Val, or Tyr for the amino acid of position 318; Asp, Phe, Gly,
His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, or Tyr
for the amino acid of position 320; Ala, Asp, Phe, Gly, His, Ile,
Pro, Ser, Thr, Val, Trp, or Tyr for the amino acid of position 322;
Ile for the amino acid of position 323; Asp, Phe, Gly, His, Ile,
Leu, Met, Pro, Arg, Thr, Val, Trp, or Tyr for the amino acid of
position 324; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met,
Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of
position 325; Ala, Asp, Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln,
Ser, Thr, Val, Trp, or Tyr for the amino acid of position 326; Ala,
Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr,
Val, Trp, or Tyr for the amino acid of position 327; Ala, Asp, Glu,
Phe, Gly, His, Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val,
Trp, or Tyr for the amino acid of position 328; Asp, Glu, Phe, Gly,
His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr
for the amino acid of position 329; Cys, Glu, Phe, Gly, His, Ile,
Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the
amino acid of position 330; Asp, Phe, His, Ile, Leu, Met, Gln, Arg,
Thr, Val, Trp, or Tyr for the amino acid of position 331; Ala, Asp,
Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr,
Val, Trp, or Tyr for the amino acid of position 332; Ala, Asp, Glu,
Phe, Gly, His, Ile, Leu, Met, Pro, Ser, Thr, Val, or Tyr for the
amino acid of position 333;
[0022] Ala, Glu, Phe, Ile, Leu, Pro, or Thr for the amino acid of
position 334;
Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg, Ser, Val, Trp, or
Tyr for the amino acid of position 335; Glu, Lys, or Tyr for the
amino acid of position 336; Glu, His, or Asn for the amino acid of
position 337; Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln, Arg, Ser, or
Thr for the amino acid of position 339; Ala or Val for the amino
acid of position 376; Gly or Lys for the amino acid of position
377; Asp for the amino acid of position 378; Asn for the amino acid
of position 379; Ala, Asn, or Ser for the amino acid of position
380; Ala or Ile for the amino acid of position 382; Glu for the
amino acid of position 385; Thr for the amino acid of position 392;
Leu for the amino acid of position 396; Lys for the amino acid of
position 421; Asn for the amino acid of position 427; Phe or Leu
for the amino acid of position 428; Met for the amino acid of
position 429; Trp for the amino acid of position 434; Ile for the
amino acid of position 436; and Gly, His, Ile, Leu, or Tyr for the
amino acid of position 440; as indicated by EU numbering; [20'] the
pharmaceutical composition of any one of [1'] to [16'], wherein the
Fc region has a higher binding activity toward an inhibitory
Fc.gamma. receptor than toward an activating Fc.gamma. receptor;
[21'] the pharmaceutical composition of [20'], wherein the
inhibitory Fc.gamma. receptor is human Fc.gamma.RIIb; [22'] the
pharmaceutical composition of [20'] or [21'], wherein the
activating Fc.gamma. receptor is human Fc.gamma.RIa, human
Fc.gamma.RIIa (R), human Fc.gamma.RIIa (H), human Fc.gamma.RIIIa
(V), or human Fc.gamma.RIIIa (F); [23'] the pharmaceutical
composition of any one of [20'] to [22'], wherein the amino acid at
position 238 or 328 (EU numbering) in the Fc region is different
from the amino acid in the native human IgG Fc region; [24'] the
pharmaceutical composition of [23'], wherein the amino acid at
position 238 of the Fc region is Asp or the amino acid of position
328 of the Fc region is Glu as indicated by EU numbering; and [25']
the pharmaceutical composition of [23'] or [24'], wherein the amino
acid sequence of the Fc region comprises at least one or more amino
acids selected from the group consisting of: Asp for the amino acid
of position 233; Trp or Tyr for the amino acid of position 234;
Ala, Asp, Glu, Leu, Met, Phe, Trp, or Tyr for the amino acid of
position 237; Asp for the amino acid of position 239; Ala, Gln, or
Val for the amino acid of position 267; Asn, Asp, or Glu for the
amino acid of position 268; Gly for the amino acid of position 271;
Ala, Asn, Asp, Gln, Glu, Leu, Met, Ser, or Thr for the amino acid
of position 326; Arg, Lys, or Met for the amino acid of position
330; Ile, Leu, or Met for the amino acid of position 323; and Asp
for the amino acid of position 296; as indicated by EU
numbering;
[0023] Furthermore, the above-mentioned [1] to [25] may also be
restated as follows:
[1''] a method for eliminating an antigen from plasma of a subject,
wherein the method comprises administering to the subject an
antigen-binding molecule comprising (i) an Fc region and (ii) two
or more antigen-binding domains, wherein at least one of the
domains is an antigen-binding domain of which antigen-binding
activity varies depending on an ion concentration condition, and
wherein the antigen-binding molecule can form an immune complex
which comprises (a) two or more of the antigen-binding molecules
and (b) two or more antigens, wherein the antigens comprise two or
more antigenic binding units; [2''] the method of [1''], wherein
the ion concentration condition is a calcium ion concentration
condition; [3''] the method of [2''], wherein the antigen-binding
domain has an antigen-binding activity under a low calcium-ion
concentration condition that is lower than the antigen-binding
activity under a high calcium-ion concentration condition; [4'']
the method of any one of [1''] to [3''], wherein the ion
concentration condition is a pH condition; [5''] the method of
[4''], wherein the antigen-binding domain has an antigen binding
activity in an acidic pH range that is lower than the
antigen-binding activity in a neutral pH range condition; [6''] the
method of any one of [1''] to [5''], wherein the antigens
comprising two or more antigenic binding units are multimers; [7'']
the method of [6''], wherein the antigen is any one of GDF, GDF-1,
GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2),
GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1),
TNF, TNF-alpha, TNF-alphabeta, TNF-beta2, TNFSF10 (TRAIL Apo-2
ligand, TL2), TNFSF11 (TRANCE/RANK ligand ODF, OPG ligand), TNFSF12
(TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B
(BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand,
LTg), TNFSF15 (TL1A/VEGI), TNFSF18 (GITR ligand AITR ligand, TL6),
TNFSF1A (TNF-.alpha. Conectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa,
TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1),
TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas
ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70),
TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand),
VEGF, IgE, IgA, IgG, IgM, RANKL, TGF-alpha, TGF-beta, TGF-beta Pan
Specific, and IL-8; [8''] the method of any one of [1''] to [5''],
wherein the antigens comprising two or more antigenic binding units
are monomers; [9''] the method of any one of [1''] to [9''],
wherein the antigen-binding molecules are a multispecific or
multiparatopic antigen-binding molecule, or a cocktail of
antigen-binding molecules; [10''] the method of any one of [1''] to
[9''], wherein the Fc region is represented by any one of SEQ ID
NOs: 13, 14, 15, and 16; [11''] the method of any one of [1''] to
[9''], wherein the Fc region is an Fc region with an enhanced
FcRn-binding activity under an acidic pH range condition compared
to that of the Fc region represented by any one of SEQ ID NOs: 13,
14, 15, and 16; [12''] the method of [11''], wherein the Fc region
is an Fc region with a substitution of at least one or more amino
acids selected from the group consisting of positions 238, 244,
245, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 260, 262,
265, 270, 272, 279, 283, 285, 286, 288, 293, 303, 305, 307, 308,
309, 311, 312, 314, 316, 317, 318, 332, 339, 340, 341, 343, 356,
360, 362, 375, 376, 377, 378, 380, 382, 385, 386, 387, 388, 389,
400, 413, 415, 423, 424, 427, 428, 430, 431, 433, 434, 435, 436,
438, 439, 440, 442, and 447 (EU numbering) in the amino acid
sequence of the Fc region represented by any one of SEQ ID NOs: 13,
14, 15, and 16; [13''] the method of [12''], wherein the Fc region
comprises at least one or more amino acids selected from the group
consisting of: Leu for the amino acid of position 238; Leu for the
amino acid of position 244; Arg for the amino acid of position 245;
Pro for the amino acid of position 249; Gln or Glu for the amino
acid of position 250; Arg, Asp, Glu, or Leu for the amino acid of
position 251; Phe, Ser, Thr, or Tyr for the amino acid of position
252; Ser or Thr for the amino acid of position 254; Arg, Gly, Ile,
or Leu for the amino acid of position 255; Ala, Arg, Asn, Asp, Gln,
Glu, Pro, or Thr for the amino acid of position 256; Ala, Ile, Met,
Asn, Ser, or Val for the amino acid of position 257; Asp for the
amino acid of position 258; Ser for the amino acid of position 260;
Leu for the amino acid of position 262; Lys for the amino acid of
position 270; Leu or Arg for the amino acid of position 272; Ala,
Asp, Gly, His, Met, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr for the
amino acid of position 279; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu,
Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr for the amino acid of
position 283; Asn for the amino acid of position 285; Phe for the
amino acid of position 286; Asn or Pro for the amino acid of
position 288; Val for the amino acid of position 293; Ala, Glu,
Gln, or Met for the amino acid of position 307; Ala, Glu, Ile, Lys,
Leu, Met, Ser, Val, or Trp for the amino acid of position 311; Pro
for the amino acid of position 309; Ala, Asp, or Pro for the amino
acid of position 312; Ala or Leu for the amino acid of position
314; Lys for the amino acid of position 316; Pro for the amino acid
of position 317; Asn or Thr for the amino acid of position 318;
Phe, His, Lys, Leu, Met, Arg, Ser, or Trp for the amino acid of
position 332; Asn, Thr, or Trp for the amino acid of position 339;
Pro for the amino acid of position 341; Glu, His, Lys, Gln, Arg,
Thr, or Tyr for the amino acid of position 343; Arg for the amino
acid of position 375; Gly, Ile, Met, Pro, Thr, or Val for the amino
acid of position 376; Lys for the amino acid of position 377; Asp,
Asn, or Val for the amino acid of position 378; Ala, Asn, Ser, or
Thr for the amino acid of position 380; Phe, His, Ile, Lys, Leu,
Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid
of position 382; Ala, Arg, Asp, Gly, His, Lys, Ser, or Thr for the
amino acid of position 385; Arg, Asp, Ile, Lys, Met, Pro, Ser, or
Thr for the amino acid of position 386; Ala, Arg, His, Pro, Ser, or
Thr for the amino acid of position 387; Asn, Pro, or Ser for the
amino acid of position 389; Asn for the amino acid of position 423;
Asn for the amino acid of position 427; Leu, Met, Phe, Ser, or Thr
for the amino acid of position 428; Ala, Phe, Gly, His, Ile, Lys,
Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, or Tyr for the amino acid
of position 430; His or Asn for the amino acid of position 431;
Arg, Gln, His, Ile, Lys, Pro, or Ser for the amino acid of position
433; Ala, Gly, His, Phe, Ser, Trp, or Tyr for the amino acid of
position 434; Arg, Asn, His, Ile, Leu, Lys, Met, or Thr for the
amino acid of position 436; Lys, Leu, Thr, or Trp for the amino
acid of position 438; Lys for the amino acid of position 440; and
Lys for the amino acid of position 442; Ile, Pro, or Thr for the
amino acid of position 308; as indicated by EU numbering in the
amino acid sequence of the Fc region represented by any one of SEQ
ID NOs: 13, 14, 15, and 16; [14''] the method of any one of [1'']
to [9''], wherein the Fc region is an Fc region with an enhanced
FcRn-binding activity under a neutral pH range condition compared
to that of the Fc region represented by any one of SEQ ID NOs: 13,
14, 15, and 16; [15''] the method of [14''], wherein the Fc region
is an Fc region with a substitution of at least one or more amino
acids selected from the group consisting of positions 237, 248,
250, 252, 254, 255, 256, 257, 258, 265, 286, 289, 297, 298, 303,
305, 307, 308, 309, 311, 312, 314, 315, 317, 332, 334, 360, 376,
380, 382, 384, 385, 386, 387, 389, 424, 428, 433, 434, and 436 (EU
numbering) in the amino acid sequence of the Fc region represented
by any one of SEQ ID NOs: 13, 14, 15, and 16; [16''] the method of
[15''], wherein the Fc region comprises at least one or more amino
acids selected from the group consisting of: Met for the amino acid
of position 237; Ile for the amino acid of position 248; Ala, Phe,
Ile, Met, Gln, Ser, Val, Trp, or Tyr for the amino acid of position
250; Phe, Trp, or Tyr for the amino acid of position 252; Thr for
the amino acid of position 254; Glu for the amino acid of position
255; Asp, Asn, Glu, or Gln for the amino acid of position 256; Ala,
Gly, Ile, Leu, Met, Asn, Ser, Thr, or Val for the amino acid of
position 257; His for the amino acid of position 258; Ala for the
amino acid of position 265; Ala or Glu for the amino acid of
position 286; His for the amino acid of position 289; Ala for the
amino acid of position 297; Ala for the amino acid of position 303;
Ala for the amino acid of position 305; Ala, Asp, Phe, Gly, His,
Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr for
the amino acid of position 307; Ala, Phe, Ile, Leu, Met, Pro, Gln,
or Thr for the amino acid of position 308; Ala, Asp, Glu, Pro, or
Arg for the amino acid of position 309; Ala, His, or Ile for the
amino acid of position 311; Ala or His for the amino acid of
position 312; Lys or Arg for the amino acid of position 314; Ala,
Asp, or His for the amino acid of position 315; Ala for the amino
acid of position 317; Val for the amino acid of position 332; Leu
for the amino acid of position 334; His for the amino acid of
position 360; Ala for the amino acid of position 376; Ala for the
amino acid of position 380; Ala for the amino acid of position 382;
Ala for the amino acid of position 384; Asp or His for the amino
acid of position 385; Pro for the amino acid of position 386; Glu
for the amino acid of position 387; Ala or Ser for the amino acid
of position 389; Ala for the amino acid of position 424; Ala, Asp,
Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Gln, Ser, Thr, Val, Trp, or
Tyr for the amino acid of position 428; Lys for the amino acid of
position 433; Ala, Phe, His, Ser, Trp, or Tyr for the amino acid of
position 434; and His, Ile, Leu, Phe, Thr, or Val for the amino
acid of position 436;
[0024] as indicated by EU numbering in the amino acid sequence of
the Fc region represented by any one of SEQ ID NOs: 13, 14, 15, and
16;
[17''] the method of any one of [1''] to [13''], wherein the Fc
region includes an Fc region that has a higher Fc.gamma.
receptor-binding activity than that of the Fc region of a native
human IgG; [18''] the method of [17''], wherein the Fc region
comprises in its amino acid sequence at least one or more amino
acids that are different from amino acids of the native human IgG
Fc region selected from the group consisting of positions 221, 222,
223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235, 236, 237,
238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250, 251, 254,
255, 256, 258, 260, 262, 263, 264, 265, 266, 267, 268, 269, 270,
271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282, 283, 284,
285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299,
300, 301, 302, 303, 304, 305, 311, 313, 315, 317, 318, 320, 322,
323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335,
336, 337, 339, 376, 377, 378, 379, 380, 382, 385, 392, 396, 421,
427, 428, 429, 434, 436, and 440 (EU numbering); [19''] the method
of [18''], wherein the Fc region comprises in its amino acid
sequence at least one or more amino acids selected from the group
consisting of: Lys or Tyr for the amino acid of position 221; Phe,
Trp, Glu, or Tyr for the amino acid of position 222; Phe, Trp, Glu,
or Lys for the amino acid of position 223; Phe, Trp, Glu, or Tyr
for the amino acid of position 224; Glu, Lys, or Trp for the amino
acid of position 225; Glu, Gly, Lys, or Tyr for the amino acid of
position 227; Glu, Gly, Lys, or Tyr for the amino acid of position
228; Ala, Glu, Gly, or Tyr for the amino acid of position 230; Glu,
Gly, Lys, Pro, or Tyr for the amino acid of position 231; Glu, Gly,
Lys, or Tyr for the amino acid of position 232; Ala, Asp, Phe, Gly,
His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr
for the amino acid of position 233; Ala, Asp, Glu, Phe, Gly, His,
Ile, Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for
the amino acid of position 234; Ala, Asp, Glu, Phe, Gly, His, Ile,
Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the
amino acid of position 235; Ala, Asp, Glu, Phe, His, Ile, Lys, Leu,
Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino
acid of position 236; Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn,
Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of
position 237; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,
Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of position
238; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln,
Arg, Thr, Val, Trp, or Tyr for the amino acid of position 239; Ala,
Ile, Met, or Thr for the amino acid of position 240; Asp, Glu, Leu,
Arg, Trp, or Tyr for the amino acid of position 241; Leu, Glu, Leu,
Gln, Arg, Trp, or Tyr for the amino acid of position 243; His for
the amino acid of position 244; Ala for the amino acid of position
245; Asp, Glu, His, or Tyr for the amino acid of position 246; Ala,
Phe, Gly, His, Ile, Leu, Met, Thr, Val, or Tyr for the amino acid
of position 247; Glu, His, Gln, or Tyr for the amino acid of
position 249; Glu or Gln for the amino acid of position 250; Phe
for the amino acid of position 251; Phe, Met, or Tyr for the amino
acid of position 254; Glu, Leu, or Tyr for the amino acid of
position 255; Ala, Met, or Pro for the amino acid of position 256;
Asp, Glu, His, Ser, or Tyr for the amino acid of position 258; Asp,
Glu, His, or Tyr for the amino acid of position 260; Ala, Glu, Phe,
Ile, or Thr for the amino acid of position 262; Ala, Ile, Met, or
Thr for the amino acid of position 263; Asp, Glu, Phe, Gly, His,
Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr for
the amino acid of position 264; Ala, Leu, Phe, Gly, His, Ile, Lys,
Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the
amino acid of position 265; Ala, Ile, Met, or Thr for the amino
acid of position 266; Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn,
Pro, Gln, Arg, Thr, Val, Trp, or Tyr for the amino acid of position
267; Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr,
Val, or Trp for the amino acid of position 268; Phe, Gly, His, Ile,
Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the
amino acid of position 269; Glu, Phe, Gly, His, Ile, Leu, Met, Pro,
Gln, Arg, Ser, Thr, Trp, or Tyr for the amino acid of position 270;
Ala, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,
Ser, Thr, Val, Trp, or Tyr for the amino acid of position 271; Asp,
Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or
Tyr for the amino acid of position 272; Phe or Ile for the amino
acid of position 273; Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn,
Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of position
274; Leu or Trp for the amino acid of position 275; Asp, Glu, Phe,
Gly, His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for
the amino acid of position 276; Asp, Glu, Gly, His, Ile, Lys, Leu,
Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp for the amino acid
of position 278; Ala for the amino acid of position 279; Ala, Gly,
His, Lys, Leu, Pro, Gln, Trp, or Tyr for the amino acid of position
280; Asp, Lys, Pro, or Tyr for the amino acid of position 281; Glu,
Gly, Lys, Pro, or Tyr for the amino acid of position 282; Ala, Gly,
His, Ile, Lys, Leu, Met, Pro, Arg, or Tyr for the amino acid of
position 283; Asp, Glu, Leu, Asn, Thr, or Tyr for the amino acid of
position 284; Asp, Glu, Lys, Gln, Trp, or Tyr for the amino acid of
position 285; Glu, Gly, Pro, or Tyr for the amino acid of position
286; Asn, Asp, Glu, or Tyr for the amino acid of position 288; Asp,
Gly, His, Leu, Asn, Ser, Thr, Trp, or Tyr for the amino acid of
position 290; Asp, Glu, Gly, His, Ile, Gln, or Thr for the amino
acid of position 291; Ala, Asp, Glu, Pro, Thr, or Tyr for the amino
acid of position 292; Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg,
Ser, Thr, Val, Trp, or Tyr for the amino acid of position 293; Phe,
Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or
Tyr for the amino acid of position 294; Asp, Glu, Phe, Gly, His,
Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the
amino acid of position 295; Ala, Asp, Glu, Gly, His, Ile, Lys, Leu,
Met, Asn, Gln, Arg, Ser, Thr, or Val for the amino acid of position
296; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg,
Ser, Thr, Val, Trp, or Tyr for the amino acid of position 297; Ala,
Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, or
Tyr for the amino acid of position 298; Ala, Asp, Glu, Phe, Gly,
His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr
for the amino acid of position 299; Ala, Asp, Glu, Gly, His, Ile,
Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp for the
amino acid of position 300; Asp, Glu, His, or Tyr for the amino
acid of position 301; Ile for the amino acid of position 302; Asp,
Gly, or Tyr for the amino acid of position 303; Asp, His, Leu, Asn,
or Thr for the amino acid of position 304; Glu, Ile, Thr, or Tyr
for the amino acid of position 305; Ala, Asp, Asn, Thr, Val, or Tyr
for the amino acid of position 311; Phe for the amino acid of
position 313; Leu for the amino acid of position 315; Glu or Gln
for the amino acid of position 317; His, Leu, Asn, Pro, Gln, Arg,
Thr, Val, or Tyr for the amino acid of position 318; Asp, Phe, Gly,
His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, or Tyr for the amino
acid of position 320; Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr,
Val, Trp, or Tyr for the amino acid of position 322; Ile for the
amino acid of position 323; Asp, Phe, Gly, His, Ile, Leu, Met, Pro,
Arg, Thr, Val, Trp, or Tyr for the amino acid of position 324; Ala,
Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser,
Thr, Val, Trp, or Tyr for the amino acid of position 325; Ala, Asp,
Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr
for the amino acid of position 326; Ala, Asp, Glu, Phe, Gly, His,
Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, or Tyr for the
amino acid of position 327; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys,
Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino
acid of position 328; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met,
Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of
position 329; Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,
Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of position
330; Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, or Tyr
for the amino acid of position 331; Ala, Asp, Glu, Phe, Gly, His,
Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for
the amino acid of position 332; Ala, Asp, Glu, Phe, Gly, His, Ile,
Leu, Met, Pro, Ser, Thr, Val, or Tyr for the amino acid of position
333; Ala, Glu, Phe, Ile, Leu, Pro, or Thr for the amino acid of
position 334; Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg,
Ser, Val, Trp, or Tyr for the amino acid of position 335; Glu, Lys,
or Tyr for the amino acid of position 336; Glu, His, or Asn for the
amino acid of position 337; Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln,
Arg, Ser, or Thr for the amino acid of position 339; Ala or Val for
the amino acid of position 376; Gly or Lys for the amino acid of
position 377; Asp for the amino acid of position 378; Asn for the
amino acid of position 379; Ala, Asn, or Ser for the amino acid of
position 380; Ala or Ile for the amino acid of position 382; Glu
for the amino acid of position 385; Thr for the amino acid of
position 392; Leu for the amino acid of position 396; Lys for the
amino acid of position 421; Asn for the amino acid of position 427;
Phe or Leu for the amino acid of position 428; Met for the amino
acid of position 429; Trp for the amino acid of position 434; Ile
for the amino acid of position 436; and Gly, His, Ile, Leu, or Tyr
for the amino acid of position 440; as indicated by EU numbering;
[20''] the method of any one of [1''] to [16''], wherein the Fc
region has a higher binding activity toward an inhibitory Fc.gamma.
receptor than toward an activating Fc.gamma. receptor; [21''] the
method of [20''], wherein the inhibitory Fc.gamma. receptor is
human Fc.gamma.RIIb; [22''] the method of [20''] or [21''], wherein
the activating Fc.gamma. receptor is human Fc.gamma.RIa, human
Fc.gamma.RIIa (R), human Fc.gamma.RIIa (H), human Fc.gamma.RIIIa
(V), or human Fc.gamma.RIIIa (F); [23''] the method of any one of
[20''] to [22''], wherein the amino acid at position 238 or 328 (EU
numbering) in the Fc region is different from the amino acid in the
native human IgG Fc region; [24''] the method of [23''], wherein
the amino acid at position 238 of the Fc region is Asp or the amino
acid of position 328 of the Fc region is Glu as indicated by EU
numbering; and [25''] the method of [23''] or [24''], wherein the
amino acid sequence of the Fc region comprises at least one or more
amino acids selected from the group consisting of: Asp for the
amino acid of position 233; Trp or Tyr for the amino acid of
position 234; Ala, Asp, Glu, Leu, Met, Phe, Trp, or Tyr for the
amino acid of position 237; Asp for the amino acid of position 239;
Ala, Gln, or Val for the amino acid of position 267; Asn, Asp, or
Glu for the amino acid of position 268; Gly for the amino acid of
position 271; Ala, Asn, Asp, Gln, Glu, Leu, Met, Ser, or Thr for
the amino acid of position 326; Arg, Lys, or Met for the amino acid
of position 330; Ile, Leu, or Met for the amino acid of position
323; and Asp for the amino acid of position 296; as indicated by EU
numbering;
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1 is a diagram showing that an antibody with
pH-dependent binding repeatedly binds to soluble antigens. (i) An
antibody binds to soluble antigens; (ii) the antibody is
non-specifically internalized into a cell by pinocytosis; (iii) the
antibody binds to FcRn within the endosome, and the soluble
antigens dissociate from the antibody; (iv) the soluble antigens
are transferred to the lysosome and degraded; (v) after
dissociation from the soluble antigens, the antibody is recycled to
the plasma via FcRn; (vi) the recycled antibody can bind to soluble
antigens again.
[0026] FIG. 2 is a diagram showing that enhancing FcRn binding
under neutral conditions results in improving the effect of an
antibody with pH-dependent binding to repeatedly bind to antigens:
(i) an antibody binds to soluble antigens; (ii) the antibody is
internalized into a cell by pinocytosis via FcRn; (iii) the soluble
antigens dissociate from the antibody in the endosome; (iv) the
soluble antigens are transferred into the lysosome and degraded;
(v) after dissociation of the soluble antigens, the antibody is
recycled to the plasma via FcRn; and (vi) the recycled antibody can
bind to soluble antigens again.
[0027] FIG. 3 presents Biacore sensorgrams showing the interaction
of anti-human IgA antibodies with human IgA under the conditions of
1.2 mM Ca.sup.2+ and 3 .mu.M Ca.sup.2+.
[0028] FIG. 4 is a graph showing the time-course changes in plasma
antibody concentration in normal mice for the human IgA+GA1-IgG1
antibody-administered group, the human IgA+GA2-IgG1
antibody-administered group, the human IgA+GA2-Fc.gamma.R(-) or
GA2-N434W antibody-administered groups.
[0029] FIG. 5 is a graph showing the time-course changes in plasma
concentration of human IgA in normal mice for the group
administered with human IgA alone, the human IgA+GA1-IgG1
antibody-administered group, the human IgA+GA2-IgG1
antibody-administered group, the human IgA+GA2-Fc.gamma.R(-)
antibody-administered group, and the human IgA+GA2-N434W
antibody-administered group.
[0030] FIG. 6 is a graph showing the time-course changes in plasma
concentration of unbound human IgA in normal mice for the human
IgA+GA1-IgG1 antibody-administered group, the human IgA+GA2-IgG1
antibody-administered group, the human IgA+GA2-Fc.gamma.R(-)
antibody-administered group, and the human IgA+GA2-N434W
antibody-administered group.
[0031] FIG. 7 is an illustrative diagram showing the efficiency of
antigen elimination per antibody molecule for a pH/Ca-dependent
antibody having the constant region of natural IgG1 which forms a
large immune complex with a multimeric antigen.
[0032] FIG. 8 is an illustrative diagram showing the efficiency of
antigen elimination per antibody molecule for a pH/Ca-dependent
multispecific antibody that recognizes two or more epitopes in a
monomeric antigen and is suitable for formation of a large immune
complex.
[0033] FIG. 9 shows the results of gel filtration chromatographic
analyses which confirmed that human IgE and clone 278, which is a
pH-dependent anti-IgE antibody, form large immune complexes in a
pH-dependent manner.
[0034] FIG. 10 is a graph showing the time-course changes in plasma
antibody concentration in normal mice for the human IgE+clone
278-administered group and the human IgE+Xolair
antibody-administered group.
[0035] FIG. 11 is a graph showing the time-course changes in plasma
concentration of human IgE in normal mice for the group
administered with human IgE alone, the human IgE+clone 278
antibody-administered group, and the human IgE+clone 278
antibody-administered group.
[0036] FIG. 12 is a graph showing the time-course changes in plasma
concentrations of GA2-IgG1 and GA2-F1087 antibodies in normal
mice.
[0037] FIG. 13 is a graph showing the time-course changes in plasma
concentration of hIgA in normal mice administered with GA2-IgG1 or
GA2-F1087.
[0038] FIG. 14 is a graph showing the time-course changes in plasma
concentrations of 278-IgG1 and 278-F1087 antibodies in C57BL/6J
mice.
[0039] FIG. 15 is a graph showing the time-course changes in plasma
concentration of hIgE(Asp6) in C57BL/6J mice administered with
278-IgG1 or 278-F1087.
[0040] FIG. 16 is a graph showing the time-course changes in plasma
concentration of GA2-F760 or GA2-F1331 in human FcRn transgenic
mice administered with GA2-F760 or GA2-F1331.
[0041] FIG. 17 is a graph showing the time-course changes in plasma
concentration of human IgA in human FcRn transgenic mice
administered with GA2-F760 or GA2-F1331.
[0042] FIG. 18 is a graph showing the time-course changes in plasma
concentration of 278-F760 or 278-F1331 in human FcRn transgenic
mice administered with 278-F760 or 278-F1331.
[0043] FIG. 19 is a graph showing the time-course changes in plasma
concentration of human IgE in human FcRn transgenic mice
administered with 278-F760 or 278-F1331.
[0044] FIG. 20 presents sensorgrams showing the interaction of
PHX-IgG1 with hsIL-6R at pH7.4 and pH6.0.
[0045] FIG. 21 shows the result of evaluating simultaneous binding
of Fv4-IgG1 and PHX-F29 with IL6R by the electrochemiluminescence
(ECL) method.
[0046] FIG. 22 is a graph showing the time-course changes in
anti-IL6R antibody concentration in plasma of normal mice for the
hsIL6R+Fv4-IgG1-administered group, and the
hsIL6R+Fv4-IgG1+PHX-IgG1-administered groups.
[0047] FIG. 23 is a graph showing the time-course changes in human
IL6R concentration in plasma of normal mice for the
hsIL6R+Fv4-IgG1-administered group, and the
hsIL6R+Fv4-IgG1+PHX-IgG1-administered groups.
[0048] FIG. 24 presents Biacore sensorgrams obtained when a
solution of antibody alone or a mixed antibody-antigen solution was
applied to Fc.gamma.R. The dashed line shows the sensorgram
obtained when the solution of antibody alone was applied, and the
solid line shows the sensorgram obtained when the mixed
antibody-antigen solution was applied.
[0049] FIG. 25 presents Biacore sensorgrams obtained when a
solution of antibody alone or a mixed antibody-antigen solution was
applied to human FcRn. The solid line shows the sensorgram obtained
when the solution of antibody alone was applied, and the dashed
line shows the sensorgram obtained when the mixed antibody-antigen
solution was applied.
[0050] FIG. 26 presents Biacore sensorgrams obtained when a
solution of antibody alone or a mixed antibody-antigen solution was
applied to mouse FcRn. The dashed line shows the sensorgram
obtained when the solution of antibody alone was applied, and the
solid line shows the sensorgram obtained when the mixed
antibody-antigen solution was applied.
[0051] FIG. 27 presents Biacore sensorgrams obtained when a
solution of antibody alone or a mixed antibody-antigen solution was
applied to mouse FcRn. The dashed line shows the sensorgram
obtained when the solution of antibody alone was applied, and the
solid line shows the sensorgram obtained when the mixed
antibody-antigen solution was applied.
MODE FOR CARRYING OUT THE INVENTION
[0052] The definitions and detailed description below are provided
to help the understanding of the present invention illustrated
herein.
Amino Acids
[0053] Herein, amino acids are described in one- or three-letter
codes or both, for example, Ala/A, Leu/L, Arg/R, Lys/K, Asn/N,
Met/M, Asp/D, Phe/F, Cys/C, Pro/P, Gln/Q, Ser/S, Glu/E, Thr/T,
Gly/G, Trp/W, His/H, Tyr/Y, Ile/I, or Val/V.
Modification of Amino Acids
[0054] For amino acid modification in the amino acid sequence of an
antigen-binding molecule, known methods such as site-directed
mutagenesis methods (Kunkel et al. (Proc. Natl. Acad. Sci. USA
(1985) 82, 488-492)) and overlap extension PCR may be appropriately
adopted. Furthermore, several known methods may also be adopted as
amino acid alteration methods for substitution to non-natural amino
acids (Annu Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; and
Proc. Natl. Acad. Sci. U.S.A. (2003) 100 (11), 6353-6357). For
example, it is suitable to use a cell-free translation system
(Clover Direct (Protein Express)) containing a tRNA that has a
non-natural amino acid bound to a complementary amber suppressor
tRNA of the UAG codon (amber codon) which is one of the stop
codons.
[0055] In the present specification, the meaning of the term
"and/or" when describing the site of amino acid alteration includes
every combination where "and" and "or" are suitably combined.
Specifically, for example, "the amino acids at positions 33, 55,
and/or 96 are substituted" includes the following variation of
amino acid modifications:
amino acid(s) at (a) position 33, (b) position 55, (c) position 96,
(d) positions 33 and 55, (e) positions 33 and 96, (f) positions 55
and 96, and (g) positions 33, 55, and 96.
Antigenic Binding Units
[0056] Herein, "antigenic binding units" refers to "the number of
epitopes present in a single antigen molecule in a form normally
present in plasma in the absence of an antigen-binding molecule,
wherein the antigen molecule comprises an epitope that binds to a
monovalent binding unit of an antigen-binding domain contained in
an antigen-binding molecule of the present invention". Examples of
an antigen that has two antigenic binding units are antigens
including multimers generally present in plasma in the form of
homodimers such as GDF, PDGF, or VEGF. For example, in
homodimerized GDF molecules, there are two units of epitopes each
bound by a monovalent binding unit of a variable region contained
in an anti-GDF antibody molecule that comprises two variable
regions having the same sequence (i.e., not the bispecific antibody
to be described later). Immunoglobulin molecules such as IgE are
also included as examples of antigens with two antigenic binding
units. IgE normally exists in plasma as a tetramer comprising a
heavy-chain dimer and a light-chain dimer; and in the tetramer,
there are two units of epitopes each bound by a single valence of a
variable region contained in an anti-IgE antibody molecule that
comprises two variable regions having the same sequence (i.e., not
the later-described bispecific antibody). IgA normally exists in
the plasma in two forms: a tetramer comprising a heavy-chain dimer
and a light-chain dimer, and an octamer produced when the tetramers
further form a complex via a J chain; and the tetramer and octamer
respectively have two units and four units of epitopes each bound
by a single valence of a variable region contained in an anti-IgA
antibody molecule that comprises two variable regions having the
same sequence (i.e., not the later-described bispecific antibody).
Examples of antigens with three antigenic binding units are
antigens including multimers generally present in plasma in the
form of homotrimers such as TNF.alpha., RANKL, or CD154 of the TNF
superfamily.
[0057] Examples of antigen molecules with one antigenic binding
unit are molecules generally present in plasma in the form of
monomers such as soluble IL-6 receptor (hereinafter also referred
to as sIL-6R), IL-6, HMGB-1, and CTGF. Heterodimers such as IL-12
comprising IL-12p40 and IL-12p35, IL-23 comprising IL-12p40 and
IL-23p19 (also referred to as IL-30B), IL-23 comprising EBI3 and
IL27p28, and IL-35 comprising IL-12p35 and EBI3 contain two
structurally different subunits molecules. In an anti-subunit
antibody molecule comprising two variable regions that bind to one
of these subunits (i.e., not the later-described bispecific
antibody), the epitope bound by a single valence of the variable
regions is one unit; therefore, the antigenic binding unit of these
heterodimers is one. The antigenic binding unit of heterotrimers
such as the multisubunit complex TNF.alpha.-TNF.beta.-hCG is
similarly one unit.
[0058] Since "antigenic binding unit" means the number of epitopes
bound by a monovalent binding unit of an antigen-binding domain,
when there is one type of paratope or multiple types of paratopes
present in an antigen-binding molecule, the antigenic binding unit
of the antigen will be different even if the antigen bound by these
antigen-binding molecules is the same antigen. For example, in the
case of the above-mentioned heterodimers, when the monovalent
binding unit in the antigen-binding domain contained in the
antigen-binding molecule binds to one type of subunit in the
heterodimer, the antigenic binding unit of this antigen is one,
whereas when the antigen-binding molecule is a biparatopic or a
bispecific antigen-binding molecule comprising two monovalent
binding units that bind to each of the subunits forming the
heterodimer, the antigenic binding unit of this antigen is two.
Multimers
[0059] Herein, when the phrase "two or more multimers" is simply
recited, the meaning of the term "multimer" includes both
homomultimer and heteromultimer. The modes of binding between
subunits contained in a multimer are covalent bonds such as peptide
bonds or disulfide bonds, and stable non-covalent bonds such as
ionic bonds, van der Waals bonds, and hydrogen bonds, but are not
limited thereto. Homomultimers include a plurality of identical
subunits, whereas heteromultimers contain a plurality of different
subunits. For example, the meaning of the term "dimer" includes
both homodimer and heterodimer; and while a homodimer contains two
identical subunits, a heterodimer contains two different subunits.
Furthermore, in the case of a polypeptide, the term "monomer" which
expresses each of the units forming a multimer refers to each of
the continuous structural units linked by peptide bonds.
Antigens
[0060] Herein, "antigens" are not particularly limited in their
structure, as long as they comprise epitopes to which
antigen-binding domains bind. In other words, antigens can be
inorganic or organic substances. Other antigens include, for
example, the molecules below: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a,
8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2,
activin, activin A, activin AB, activin B, activin C, activin RIA,
activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB,
ADAM, ADAM 10, ADAM 12, ADAM 15, ADAM17/TACE, ADAMS, ADAM9, ADAMTS,
ADAMTS4, ADAMTS5, addressin, aFGF, ALCAM, ALK, ALK-1, ALK-7,
alpha-1-antitrypsin, alpha-V/beta-1 antagonist, ANG, Ang, APAF-1,
APE, APJ, APP, APRIL, AR, ARC, ART, artemin, anti-Id, ASPARTIC,
atrial natriuretic peptide, av/b3 integrin, Axl, b2M, B7-1, B7-2,
B7-H, B-lymphocyte stimulating factor (BlyS), BACE, BACE-1, Bad,
BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF,
b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a,
BMP-3 Osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1),
BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6),
BRK-2, RPK-1, BMPR-II (BRK-3), BMP, b-NGF, BOK, bombesin,
bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement
factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin,
cAMP, carcinoembryonic antigen (CEA), cancer associated antigen,
cathepsin A, cathepsin B, cathepsin C/DPPI, cathepsin D, cathepsin
E, cathepsin H, cathepsin L, cathepsin 0, cathepsin S, cathepsin V,
cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12, CCL13,
CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21,
CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5,
CCL6, CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3,
CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5,
CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16,
CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30,
CD30L, CD32, CD33 (p67 protein), CD34, CD38, CD40, CD40L, CD44,
CD45, CD46, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74,
CD80 (B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147,
CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, Botulinum toxin,
Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMV UL, CNTF,
CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1,
CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7,
CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15,
CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin
tumor associated antigen, DAN, DCC, DcR3, DC-SIGN, complement
regulatory factor (Decay accelerating factor), des (1-3)-IGF-I
(brain IGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV/CD26, Dtk,
EGAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN,
ENA, endothelin receptor, enkephalinase, eNOS, Eot, eotaxin 1,
EpCAM, ephrin B2/EphB4, EPO, ERCC, E-selectin, ET-1, factor IIa,
factor VII, factor VIIIc, factor IX, fibroblast activation protein
(FAP), Fas, FcR1, FEN-1, ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8,
FGFR, FGFR-3, fibrin, FL, FLIP, Flt-3, Flt-4, follicle stimulating
hormone, fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7,
FZD8, FZD9, FZD10, G250, Gas6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1,
GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2),
GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15 (MIC-1),
GDNF, GDNF, GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR,
glucagon, Glut4, glycoprotein IIb/IIIa (GPIIb/IIIa), GM-CSF, gp130,
gp72, GRO, growth hormone releasing hormone, hapten (NP-cap or
NIP-cap), HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV gH
envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF),
Hep B gp120, heparanase, Her2, Her2/neu (ErbB-2), Her3 (ErbB-3),
Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD
glycoprotein, HGFA, high molecular weight melanoma-associated
antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3 loop, HLA, HLA-DR,
HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human
cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309,
IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE,
IGF, IGF binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1,
IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8,
IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon
(INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain,
insulin B chain, insulin-like growth factor1, integrin alpha2,
integrin alpha3, integrin alpha4, integrin alpha4/beta1, integrin
alpha4/beta7, integrin alpha5 (alpha V), integrin alpha5/beta1,
integrin alpha5/beta3, integrin alpha6, integrin beta1, integrin
beta2, interferon gamma, IP-10, I-TAC, JE, kallikrein 2, kallikrein
5, kallikrein 6, kallikrein 11, kallikrein 12, kallikrein 14,
kallikrein 15, kallikrein L1, kallikrein L2, kallikrein L3,
kallikrein L4, KC, KDR, keratinocyte growth factor (KGF), laminin
5, LAMP, LAP, LAP (TGF-1), latent TGF-1, latent TGF-1 bpl, LBP,
LDGF, LECT2, lefty, Lewis-Y antigen, Lewis-Y associated antigen,
LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn,
L-selectin, LT-a, LT-b, LTB4, LTBP-1, lung surface, luteinizing
hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC,
MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES, MGDF
receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK,
MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15,
MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP,
mucin (Mucl), MUC18, Mullerian-inhibiting substance, Mug, MuSK,
NAIP, NAP, NCAD, N-C adherin, NCA 90, NCAM, NCAM, neprilysin,
neurotrophin-3, -4, or -6, neurturin, nerve growth factor (NGF),
NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG,
OPN, OSM, OX40L, OX40R, p150, p95, PADPr, parathyroid hormone,
PARC, PARP, PBR, PBSF, PCAD, P-cadherin, PCNA, PDGF, PDGF, PDK-1,
PECAM, PEM, PF4, PGE, PGF, PGI2, PGD2, PIN, PLA2, placental
alkaline phosphatase (PLAP), PlGF, PLP, PP14, proinsulin,
prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane
antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES,
RANTES, relaxin A chain, relaxin B chain, renin, respiratory
syncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid factor, RLIP76,
RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, serum albumin, sFRP-3, Shh,
SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat,
STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated
glycoprotein-72), TARC, TCA-3, T-cell receptor (for example, T-cell
receptor alpha/beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT,
testis PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha,
TGF-beta, TGF-beta Pan Specific, TGF-betaRl (ALK-5), TGF-betaRII,
TGF-betaRIIb, TGF-betaRIII, TGF-beta1, TGF-beta2, TGF-beta3,
TGF-beta4, TGF-beta5, thrombin, thymus Ck-1, thyroid-stimulating
hormone, Tie, TIMP, TIQ, tissue factor, TMEFF2, Tmpo, TMPRSS2, TNF,
TNF-alpha, TNF-alphabeta, TNF-beta2, TNFc, TNF-RI, TNF-RII,
TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER,
TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D
(TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R),
TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B
(TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R,
TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR
AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF
RI CD120a, p55-60), TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26
(TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (OX40 ACT35,
TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95),
TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9
(4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2),
TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3,
TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 ligand, TL2), TNFSF11
(TRANCE/RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand,
DR3 ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1,
THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15
(TL1A/VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-a
Conectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb
TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand
CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand,
APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand
CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL,
TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferrin receptor, TRF,
Trk, TROP-2, TSG, TSLP, tumor associated antigen CA125, tumor
associated antigen expressing Lewis-Y associated carbohydrates,
TWEAK, TXB2, Ung, uPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD,
VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3
(flt-4), VEGI, VIM, virus antigen, VLA, VLA-1, VLA-4, VNR integrin,
von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A,
WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A,
WNT9B, WNT10A, WNT 10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1,
XEDAR, XIAP, XPD, HMGB1, IgA, AP, CD81, CD97, CD98, DDR1, DKK1,
EREG, Hsp90, IL-17/IL-17R, IL-20/IL-20R, oxidized LDL, PCSK9,
prekallikrein, RON, TMEM16F, SOD1, Chromogranin A, Chromogranin B,
tau, VAP1, high molecular weight kininogen, IL-31, IL-31R, Nav1.1,
Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8, Nav1.9,
EPCR, C1, C1q, C1r, C1s, C2, C2a, C2b, C3, C3a, C3b, C4, C4a, C4b,
C5, C5a, C5b, C6, C7, C8, C9, factor B, factor D, factor H,
properdin, sclerostin, fibrinogen, fibrin, prothrombin, thrombin,
tissue factor, factor V, factor Va, factor VII, factor VIIa, factor
VIII, factor VIIIa, factor IX, factor IXa, factor X, factor Xa,
factor XI, factor XIa, factor XII, factor XIIa, factor XIII, factor
XIIIa, TFPI, antithrombin III, EPCR, thrombomodulin, TAPI, tPA,
plasminogen, plasmin, PAI-1, PAI-2, GPC3, Syndecan-1, Syndecan-2,
Syndecan-3, Syndecan-4, LPA, and SIP; and receptors for hormone and
growth factors.
[0061] As described later, when an antigen-binding molecule binds
to a plurality of epitopes in an antigen molecule as with a
bispecific antibody and such, the antigen that can form a complex
with the antigen-binding molecule may be any of the antigens
exemplified above, or combinations thereof, i.e., monomers or
heteromultimers. Non-limiting examples of heteromultimers include
heterodimers such as IL-12 comprising IL-12p40 and IL-12p35, IL-23
comprising IL-12p40 and IL-23p19 (also referred to as IL-30B),
IL-23 comprising EBI-3 and IL27p28, and IL-35 comprising IL-12p35
and EBI-3.
[0062] While receptors are recited as examples of the
above-mentioned antigens, when these receptors exist in soluble
forms in biological fluids such as plasma, they can form complexes
with the antigen-binding molecules of the present invention.
Therefore, as long as the above-mentioned receptors exist in their
soluble forms in biological fluids such as plasma, they may be used
as antigens that may form complexes of the present invention by
binding to an antigen-binding molecule of the present invention. An
example of a non-limiting embodiment of such a soluble receptor is
soluble IL-6R, which is a protein consisting of the amino acids at
positions 1 to 357 in the IL-6R polypeptide sequence of SEQ ID NO:
1 as described in Mullberg et al. (J. Immunol. (1994) 152 (10),
4958-4968).
[0063] Soluble antigens are recited as examples of the
above-mentioned antigens, and the solutions in which the antigens
exist are not limited. Soluble antigens may exist in biological
fluids, or more specifically in all fluids filling the space
between tissues and cells or vessels in organisms. In a
non-limiting embodiment, the antigens to which antigen-binding
molecules of the present invention bind may be present in
extracellular fluids. In vertebrates, extracellular fluid is a
general term for plasma, interstitial fluid, lymph, compact
connective tissue, cerebrospinal fluid, spinal fluid, puncture
fluid, synovial fluid, or such components in the bone and
cartilage, alveolar fluid (bronchoalveolar lavage fluid),
peritoneal fluid, pleural fluid, pericardial fluid, cyst fluid,
aqueous humor (hydatoid), or such transcellular fluids (various
fluids in the glandular cavities and fluids in the digestive tract
cavity and other body cavity fluids produced as a result of active
transport/secretory activities of cells).
[0064] When the epitope to which the antigen-binding domain
contained in an antigen-binding molecule binds is a single epitope,
a non-limiting embodiment of an antigen that may form a complex of
the present invention upon binding with the antigen-binding
molecule includes the molecules exemplified below, which include a
homodimer, homotrimer, or such, as an antigenic binding unit: GDF,
GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13,
CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (myostatin), GDF-9, GDF-15
(MIC-1), TNF, TNF-alpha, TNF-alphabeta, TNF-beta2, TNFSF10 (TRAIL
Apo-2 ligand, TL2), TNFSF11 (TRANCE/RANK ligand ODF, OPG ligand),
TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRIL TALL2),
TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM
ligand, LTg), TNFSF15 (TL1A/VEGI), TNFSF18 (GITR ligand AITR
ligand, TL6), TNFSF1A (TNF-.alpha. Conectin, DIF, TNFSF2), TNFSF1B
(TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand
gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP),
TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand
CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137
ligand), VEGF, IgE, IgA, IgG, IgM, RANKL, TGF-alpha, TGF-beta,
TGF-beta Pan Specific, and IL-8.
Epitopes
[0065] "Epitope" means an antigenic determinant in an antigen, and
refers to an antigen site to which the antigen-binding domain of an
antigen-binding molecule disclosed herein binds. Thus, for example,
the epitope can be defined according to its structure.
Alternatively, the epitope may be defined according to the
antigen-binding activity of an antigen-binding molecule that
recognizes the epitope. When the antigen is a peptide or
polypeptide, the epitope can be specified by the amino acid
residues forming the epitope. Alternatively, when the epitope is a
sugar chain, the epitope can be specified by its specific sugar
chain structure.
[0066] A linear epitope is an epitope that contains an epitope
whose primary amino acid sequence has been recognized. Such a
linear epitope typically contains at least three and most commonly
at least five, for example, about 8 to 10 or 6 to 20 amino acids in
a specific sequence.
[0067] In contrast to the linear epitope, a "conformational
epitope" is an epitope in which the primary amino acid sequence
containing the epitope is not the only determinant of the
recognized epitope (for example, the primary amino acid sequence of
a conformational epitope is not necessarily recognized by an
epitope-defining antibody). Conformational epitopes may contain a
greater number of amino acids compared to linear epitopes. A
conformational epitope-recognizing antibody recognizes the
three-dimensional structure of a peptide or protein. For example,
when a protein molecule folds and forms a three-dimensional
structure, amino acids and/or polypeptide main chains that form a
conformational epitope become aligned, and the epitope is made
recognizable by the antibody. Methods for determining epitope
conformations include, for example, X ray crystallography,
two-dimensional nuclear magnetic resonance, site-specific spin
labeling, and electron paramagnetic resonance, but are not limited
thereto. See, for example, Epitope Mapping Protocols in Methods in
Molecular Biology (1996), Vol. 66, Morris (ed.).
[0068] The structure of the antigen-binding domain which binds to
an epitope is called a paratope. An epitope and a paratope bind
with stability through the action of hydrogen bonds, electrostatic
force, van der Waals force, hydrophobic bonds, and such between the
epitope and the paratope. This strength of binding between the
epitope and paratope is called affinity. The total sum of binding
strength when a plurality of epitopes and a plurality of paratopes
bind is referred to as avidity. When an antibody comprising a
plurality of paratopes (i.e., multivalent antibody) or such binds
to a plurality of epitopes, the affinity acts additively or
synergistically, and therefore avidity becomes higher than
affinity.
Binding Activity
[0069] Examples of a method for assessing epitope binding by a test
antigen-binding molecule containing an antigen-binding domain
directed to IgA are described below. According to the examples
below, methods for assessing epitope binding by a test
antigen-binding molecule containing an antigen-binding domain for
an antigen other than IgA can also be appropriately conducted.
[0070] For example, whether a test antigen-binding molecule
containing an antigen-binding domain against IgA recognizes a
linear epitope in the IgA molecule can be confirmed for example as
mentioned below. For example, a linear peptide comprising an amino
acid sequence forming an IgA constant region is synthesized for the
above purpose. The peptide can be synthesized chemically, or
obtained by genetic engineering techniques using a region encoding
the amino acid sequence corresponding to the constant region in an
IgA cDNA. Then, a test antigen-binding molecule containing an
antigen-binding domain toward IgA is assessed for its binding
activity towards a linear peptide comprising the amino acid
sequence forming the constant region. For example, an ELISA using
an immobilized linear peptide as an antigen can be performed to
evaluate the binding activity of the antigen-binding molecule
towards the peptide. Alternatively, the binding activity towards a
linear peptide can be assessed based on the level of inhibition by
the linear peptide of the binding of the antigen-binding molecule
toward IgA cells. These tests can demonstrate the binding activity
of the antigen-binding molecule towards the linear peptide.
[0071] Recognition of a conformational epitope by a test
antigen-binding molecule comprising an antigen-binding domain
targeting the IgA protein may be confirmed as stated below. For the
above-mentioned objective, as described herein, a general genetic
recombination technique is used to transfer an IgA-encoding
recombinant gene into host cells (for example, animal cells, insect
cells, or yeast cells) that enable formation of the native
conformational epitope in the IgA protein. IgA containing the
conformational epitope is prepared from the culture of recombinant
cells produced in this manner. Recognition of a conformational
epitope by a test antigen-binding molecule comprising an
antigen-binding domain targeting IgA is, for example, when the test
antigen-binding molecule binds strongly to the IgA molecule when it
is contacted with immobilized IgA containing the conformational
epitope, while the antigen-binding molecule does not substantively
bind to a linear peptide comprising an amino acid sequence
constituting the amino acid sequence of the immobilized IgA.
Alternatively, it is also possible to use, instead of the
above-mentioned linear peptide, the test IgA-targeting
antigen-binding molecule that has been denatured by a reducing
agent that cleaves disulfide bonds, such as dithiothreitol,
dithioerythritol, .beta.-mercaptoethanol, phosphines, and sodium
borohydride, and/or chaotropic agents such as surfactants including
guanidine hydrochloride, urea, and sodium lauryl sulfate. Here, the
phrase "does not substantively bind" refers to a binding activity
not greater than 80%, normally not greater than 50%, preferably not
greater than 30%, or particularly preferably not greater than 15%
of the human-IgA-binding activity.
[0072] Methods for assaying the binding activity toward IgA of a
test antigen-binding molecule containing an antigen-binding domain
against IgA include, for example, the methods described in
Antibodies: A Laboratory Manual (Ed Harlow, David Lane, Cold Spring
Harbor Laboratory (1988) 359-420). Specifically, the assessment can
be performed based on the principle of ELISA or EIA using IgA as
antigen.
[0073] In the ELISA format, the binding activity of a test
antigen-binding molecule containing an IgA antigen-binding domain
towards IgA can be assessed quantitatively by comparing the levels
of signal generated by enzymatic reaction. Specifically, a test
antigen-binding molecule is added to an ELISA plate onto which IgA
has been immobilized. Then, the test antigen-binding molecule that
bound to IgA immobilized on the plate is detected using an
enzyme-labeled antibody that recognizes the test antigen-binding
molecule. In the ELISA, a serial dilution of the test
antigen-binding molecule can be prepared and the antibody binding
titer toward IgA be determined to compare the binding activity of
the test antigen-binding molecule towards IgA.
[0074] The binding of a test antigen-binding molecule towards an
antigen expressed on the surface of cells suspended in buffer or
the like can be detected using a flow cytometer. Known flow
cytometers include, for example, the following devices:
FACSCanto.TM. II
FACSAria.TM.
FACSArray.TM.
FACSVantage.TM. SE
[0075] FACSCalibur.TM. (all are trade names of BD Biosciences)
EPICS ALTRA HyPerSort
Cytomics FC 500
EPICS XL-MCL ADC EPICS XL ADC
[0076] Cell Lab Quanta/Cell Lab Quanta SC (all are trade names of
Beckman Coulter).
[0077] Preferable methods for assaying the binding activity towards
an antigen of a test antigen-binding molecule containing an
antigen-binding domain against IgE include, for example, the
following method. First, IgE-expressing cells are reacted with a
test antigen-binding molecule, and then this is stained with an
FITC-labeled secondary antibody that recognizes the antigen-binding
molecule. The test antigen-binding molecule is appropriately
diluted with a suitable buffer to prepare the molecule at a desired
concentration. For example, the molecule can be used at a
concentration within the range of 10 .mu.g/ml to 10 ng/ml. Then,
the fluorescence intensity and cell count are determined using
FACSCalibur (BD). The fluorescence intensity obtained by analysis
using the CELL QUEST Software (BD), i.e., the Geometric Mean value,
reflects the quantity of antibody bound to cells. That is, the
binding activity of a test antigen-binding molecule, which is
represented by the quantity of the test antigen-binding molecule
bound, can be determined by measuring the Geometric Mean value.
[0078] Whether a test antigen-binding molecule containing an IgA
antigen-binding domain shares a common epitope with another
antigen-binding molecule can be assessed based on the competition
between the two molecules for the same epitope. The competition
between antigen-binding molecules can be detected by cross-blocking
assay or the like. For example, the competitive ELISA assay is a
preferred cross-blocking assay.
[0079] Specifically, in cross-blocking assay, the IgA protein
immobilized to the wells of a microtiter plate is pre-incubated in
the presence or absence of a candidate competitor antigen-binding
molecule, and then a test antigen-binding molecule is added
thereto. The quantity of test antigen-binding molecule bound to the
IgA protein in the wells is indirectly correlated with the binding
ability of a candidate competitor antigen-binding molecule that
competes for the binding to the same epitope. That is, the greater
the affinity of the competitor antigen-binding molecule for the
same epitope, the lower the binding activity of the test
antigen-binding molecule towards the IgA protein-coated wells.
[0080] The quantity of the test antigen-binding molecule bound to
the wells via the IgA protein can be readily determined by labeling
the antigen-binding molecule in advance. For example, a
biotin-labeled antigen-binding molecule is measured using an
avidin/peroxidase conjugate and appropriate substrate. In
particular, cross-blocking assay that uses enzyme labels such as
peroxidase is called "competitive ELISA assay". The antigen-binding
molecule can also be labeled with other labeling substances that
enable detection or measurement. Specifically, radiolabels,
fluorescent labels, and such are known.
[0081] When the candidate competitor antigen-binding molecule can
block the binding by a test antigen-binding molecule containing an
IgA antigen-binding domain by at least 20%, preferably at least 20
to 50%, and more preferably at least 50% compared to the binding
activity in a control experiment conducted in the absence of the
competitor antigen-binding molecule, the test antigen-binding
molecule is determined to substantially bind to the same epitope
bound by the competitor antigen-binding molecule, or compete for
the binding to the same epitope.
[0082] When the structure of an epitope bound by a test
antigen-binding molecule containing an IgA antigen-binding domain
has already been identified, whether the test and control
antigen-binding molecules share a common epitope can be assessed by
comparing the binding activities of the two antigen-binding
molecules towards a peptide prepared by introducing amino acid
mutations into the peptide forming the epitope.
[0083] To measure the above binding activities, for example, the
binding activities of test and control antigen-binding molecules
towards a linear peptide into which a mutation is introduced are
compared in the above ELISA format. Besides the ELISA methods, the
binding activity towards the mutant peptide bound to a column can
be determined by flowing test and control antigen-binding molecules
in the column, and then quantifying the antigen-binding molecule
eluted in the elution solution. Methods for adsorbing a mutant
peptide to a column, for example, in the form of a GST fusion
peptide, are known.
[0084] Alternatively, when the identified epitope in an antigen
expressed on a cell is a conformational epitope, whether test and
control antigen-binding molecules share a common epitope can be
assessed, for example, by the following method. Explanation is
provided below, using the case of IgE as the antigen as an example.
First, IgE-expressing cells and cells expressing IgE with a
mutation introduced into the epitope are prepared. The test and
control antigen-binding molecules are added to a cell suspension
prepared by suspending these cells in an appropriate buffer such as
PBS. Then, the cell suspensions are appropriately washed with a
buffer, and an FITC-labeled antibody that recognizes the test and
control antigen-binding molecules is added thereto. The
fluorescence intensity and number of cells stained with the labeled
antibody are determined using FACSCalibur (BD). The test and
control antigen-binding molecules are appropriately diluted using a
suitable buffer, and used at desired concentrations. For example,
they may be used at a concentration within the range of 10 .mu.g/ml
to 10 ng/ml. The fluorescence intensity determined by analysis
using the CELL QUEST Software (BD), i.e., the Geometric Mean value,
reflects the quantity of labeled antibody bound to cells. That is,
the binding activities of the test and control antigen-binding
molecules, which are represented by the quantity of labeled
antibody bound, can be determined by measuring the Geometric Mean
value.
[0085] In the above method, whether an antigen-binding molecule
does "not substantially bind to cells expressing mutant IgE" can be
assessed, for example, by the following method. First, the test and
control antigen-binding molecules bound to cells expressing mutant
IgE are stained with a labeled antibody. Then, the fluorescence
intensity of the cells is determined. When FACSCalibur is used for
fluorescence detection by flow cytometry, the determined
fluorescence intensity can be analyzed using the CELL QUEST
Software. From the Geometric Mean values in the presence and
absence of the polypeptide complex, the comparison value
(.DELTA.Geo-Mean) can be calculated according to the following
formula to determine the ratio of increase in fluorescence
intensity as a result of the binding by the antigen-binding
molecule. .DELTA.Geo-Mean=Geo-Mean (in the presence of the
polypeptide complex)/Geo-Mean (in the absence of the polypeptide
complex)
[0086] The Geometric Mean comparison value (.DELTA.Geo-Mean value
for the mutant IgE molecule) determined by the above analysis,
which reflects the quantity of a test antigen-binding molecule
bound to cells expressing mutant IgE, is compared to the
.DELTA.Geo-Mean comparison value that reflects the quantity of the
test antigen-binding molecule bound to IgE-expressing cells. In
this case, the concentrations of the test antigen-binding molecule
used to determine the .DELTA.Geo-Mean comparison values for
IgE-expressing cells and cells expressing mutant IgE are
particularly preferably adjusted to be equal or substantially
equal. An antigen-binding molecule that has been confirmed to
recognize an epitope in IgE is used as a control antigen-binding
molecule.
[0087] If the .DELTA.Geo-Mean comparison value of a test
antigen-binding molecule for cells expressing mutant IgE is smaller
than the .DELTA.Geo-Mean comparison value of the test
antigen-binding molecule for IgE-expressing cells by at least 80%,
preferably 50%, more preferably 30%, and particularly preferably
15%, then the test antigen-binding molecule "does not substantially
bind to cells expressing mutant IgE". The formula for determining
the Geo-Mean (Geometric Mean) value is described in the CELL QUEST
Software User's Guide (BD biosciences). When the comparison shows
that the comparison values are substantially equivalent, the
epitope for the test and control antigen-binding molecules can be
determined to be the same.
Antigen-Binding Domain
[0088] Herein, an "antigen-binding domain" may be of any structure
as long as it binds to an antigen of interest. Such domains
preferably include, for example:
antibody heavy-chain and light-chain variable regions; a module of
about 35 amino acids called A domain which is contained in the in
vivo cell membrane protein Avimer (International Publication No. WO
2004/044011, International Publication No. WO 2005/040229);
Adnectin containing the 10Fn3 domain which binds to the protein
moiety of fibronectin, a glycoprotein expressed on cell membrane
(International Publication No. WO 2002/032925); Affibody which is
composed of a 58-amino acid three-helix bundle based on the
scaffold of the IgG-binding domain of Protein A (International
Publication No. WO 1995/001937); Designed Ankyrin Repeat proteins
(DARPins) which are a region exposed on the molecular surface of
ankyrin repeats (AR) having a structure in which a subunit
consisting of a turn comprising 33 amino acid residues, two
antiparallel helices, and a loop is repeatedly stacked
(International Publication No. WO 2002/020565); Anticalins and
such, which are domains consisting of four loops that support one
side of a barrel structure composed of eight circularly arranged
antiparallel strands that are highly conserved among lipocalin
molecules such as neutrophil gelatinase-associated lipocalin (NGAL)
(International Publication No. WO 2003/029462); and the concave
region formed by the parallel-sheet structure inside the
horseshoe-shaped structure constituted by stacked repeats of the
leucine-rich-repeat (LRR) module of the variable lymphocyte
receptor (VLR) which does not have the immunoglobulin structure and
is used in the system of acquired immunity in jawless vertebrate
such as lampery and hagfish (International Publication No. WO
2008/016854). Preferred antigen-binding domains of the present
invention include, for example, those having antibody heavy-chain
and light-chain variable regions. Preferred examples of
antigen-binding domains include "single chain Fv (scFv)", "single
chain antibody", "Fv", "single chain Fv 2 (scFv2)", "Fab", and
"F(ab')2".
[0089] The antigen-binding domains of antigen-binding molecules of
the present invention can bind to an identical epitope. Such
identical epitope can be present, for example, in a protein
comprising the amino acid sequence of SEQ ID NO: 2. Alternatively,
each of the antigen-binding domains of antigen-binding molecules of
the present invention can bind to a different epitope. Herein, the
different epitope can be present in, for example, a protein
comprising the amino acid sequence of SEQ ID NO: 2.
Immune Complex
[0090] Immune complex refers to a relatively stable structure
produced when at least one antigen and at least one antigen-binding
molecule bind with each other to form a larger molecular-weight
complex. A non-limiting embodiment of the immune complex is, for
example, an antigen-antibody aggregate. A method for evaluating the
formation of immune complexes comprising two or more
antigen-binding molecules and two or more antigenic binding units
will be described later in this specification.
Specificity
[0091] "Specific" means that one of the molecules that specifically
bind does not show any significant binding to molecules other than
the single or plurality of partner molecules it binds to.
Furthermore, "specific" is also used when an antigen-binding domain
is specific to a particular epitope among multiple epitopes in an
antigen. When an epitope bound by an antigen-binding domain is
contained in multiple different antigens, antigen-binding molecules
containing the antigen-binding domain can bind to various antigens
that have the epitope. Here, "does not show any significant
binding" means showing no more than 50%, generally no more than
30%, preferably no more than 15%, particularly preferably no more
than 10%, and even more preferably no more than 5% of the binding
activity to a partner molecule towards molecules other than the
partner molecule.
Antibody
[0092] Herein, "antibody" refers to a natural immunoglobulin or an
immunoglobulin produced by partial or complete synthesis.
Antibodies can be isolated from natural sources such as
naturally-occurring plasma and serum, or culture supernatants of
antibody-producing hybridomas. Alternatively, antibodies can be
partially or completely synthesized using techniques such as
genetic recombination. Preferred antibodies include, for example,
antibodies of an immunoglobulin isotype or subclass belonging
thereto. Known human immunoglobulins include antibodies of the
following nine classes (isotypes): IgG1, IgG2, IgG3, IgG4, IgA1,
IgA2, IgD, IgE, and IgM. Of these isotypes, antibodies of the
present invention include IgG1, IgG2, IgG3, and IgG4. A number of
allotype sequences of human IgG1, human IgG2, human IgG3, and human
IgG4 constant regions due to gene polymorphisms are described in
"Sequences of proteins of immunological interest", NIH Publication
No. 91-3242. Any of such sequences may be used in the present
invention. In particular, for the human IgG1 sequence, the amino
acid sequence at positions 356 to 358 as indicated by EU numbering
may be DEL or EEM. Several allotype sequences due to genetic
polymorphisms have been described in "Sequences of proteins of
immunological interest", NIH Publication No. 91-3242 for the human
Ig.kappa. (Kappa) constant region and human Ig.lamda. (Lambda)
constant region, and any of the sequences may be used in the
present invention.
[0093] Methods for producing an antibody with desired binding
activity are known to those skilled in the art. Below is an example
that describes a method for producing an antibody that binds to IgA
(anti-IgA antibody). Antibodies that bind to an antigen other than
IgA can also be produced according to the example described
below.
[0094] Anti-IgA antibodies can be obtained as polyclonal or
monoclonal antibodies using known methods. The anti-IgA antibodies
preferably produced are monoclonal antibodies derived from mammals.
Such mammal-derived monoclonal antibodies include antibodies
produced by hybridomas or host cells transformed with an expression
vector carrying an antibody gene by genetic engineering techniques.
Meanwhile, "humanized antibodies" or "chimeric antibodies" are
included in the monoclonal antibodies of the present invention.
[0095] Monoclonal antibody-producing hybridomas can be produced
using known techniques, for example, as described below.
Specifically, mammals are immunized by conventional immunization
methods using an IgA protein as a sensitizing antigen. Resulting
immune cells are fused with known parental cells by conventional
cell fusion methods. Then, hybridomas producing an anti-IgA
antibody can be selected by screening for monoclonal
antibody-producing cells using conventional screening methods.
[0096] Specifically, monoclonal antibody production is performed,
for example, as shown below. For example, a purified native IgA
protein may be used as a sensitizing antigen. Furthermore, a
recombinant protein comprising an IgA polypeptide sequence of SEQ
ID NO: 2, which is registered as L00022|IGHE*02 in IMGT/GENE-DB, is
purified to obtain soluble IgA from the culture supernatant. To
express the recombinant protein, first, an IgA heavy chain constant
region gene of which nucleotide sequence is disclosed in SEQ ID NO:
3 may be expressed to obtain the IgA protein shown in SEQ ID NO: 2,
which is used as a sensitizing antigen for obtaining antibodies.
That is, suitable host cells are transformed by inserting an
IgA-encoding gene sequence into a known expression vector. The
desired IgA protein is purified by known methods from the host
cells or from their culture supernatant. For expression of the IgA
heavy chain constant region gene, the polynucleotide sequence of
SEQ ID NO: 3 may be operably linked to the 3' end of the signal
sequence. In another non-limiting embodiment, the polynucleotide
sequence of SEQ ID NO: 3 may be operably linked to the 3' end of a
polynucleotide sequence encoding a heavy chain variable region
comprising a signal sequence at the 5' end. Furthermore, to purify
the recombinant protein, a tag peptide for purification may be
added appropriately to the amino terminus or carboxy terminus of
SEQ ID NO: 2. GLNDIFEAQKIEWHE (SEQ ID NO: 4) which is an Avi tag
linked via the GGGGS (SEQ ID NO: 31) linker is an example of a
non-limiting embodiment of such. A non-limiting example of the
production of a recombinant protein similar to that mentioned above
is a method such as that described in Example 1, where a
recombinant IgA protein which has been expressed in combination
with a light chain is collected from the recombinant host cell
culture, and then purified.
[0097] The purified IgA protein may be used as a sensitizing
antigen for immunization of mammals. A partial IgA peptide may also
be used as a sensitizing antigen. In this case, a partial peptide
can be prepared by chemical synthesis based on the amino acid
sequence of human IgA, or by inserting a partial IgA gene into an
expression vector for expression. Alternatively, a partial peptide
can be produced by degrading an IgA protein with a protease. The
length and region of the partial IgA peptide are not limited to
particular embodiments. A preferred region can be arbitrarily
selected from the amino acid sequence of SEQ ID NO: 2. The number
of amino acids forming a peptide to be used as a sensitizing
antigen is preferably at least five or more, six or more, or seven
or more. More specifically, a peptide of 8 to 50 residues, more
preferably 10 to 30 residues can be used as a sensitizing
antigen.
[0098] For sensitizing antigen, alternatively it is possible to use
a fusion protein prepared by fusing a desired partial polypeptide
or peptide of the IgA protein with a different polypeptide. For
example, antibody Fc fragments and peptide tags are preferably used
to produce fusion proteins to be used as sensitizing antigens.
Vectors for expression of such fusion proteins can be constructed
by fusing in frame genes encoding two or more desired polypeptide
fragments and inserting the fusion gene into an expression vector
as described above. Methods for producing fusion proteins are
described in Molecular Cloning 2nd ed. (Sambrook, J et al.,
Molecular Cloning 2nd ed., 9.47-9.58 (1989) Cold Spring Harbor Lab.
Press). Methods for preparing IgA to be used as a sensitizing
antigen, and immunization methods using IgA are specifically
described in PCT/JP2011/077619 and such.
[0099] There is no particular limitation on the mammals to be
immunized with the sensitizing antigen. However, it is preferable
to select the mammals by considering their compatibility with the
parent cells to be used for cell fusion. In general, rodents such
as mice, rats, and hamsters, rabbits, and monkeys are preferably
used.
[0100] The above animals are immunized with a sensitizing antigen
by known methods. Generally performed immunization methods include,
for example, intraperitoneal or subcutaneous injection of a
sensitizing antigen into mammals. Specifically, a sensitizing
antigen is appropriately diluted with PBS (Phosphate-Buffered
Saline), physiological saline, or the like. If desired, a
conventional adjuvant such as Freund's complete adjuvant is mixed
with the antigen, and the mixture is emulsified. Then, the
sensitizing antigen is administered to a mammal several times at 4-
to 21-day intervals. Appropriate carriers may be used in
immunization with the sensitizing antigen. In particular, when a
low-molecular-weight partial peptide is used as the sensitizing
antigen, it is sometimes desirable to couple the sensitizing
antigen peptide to a carrier protein such as albumin or keyhole
limpet hemocyanin for immunization.
[0101] Alternatively, hybridomas producing a desired antibody can
be prepared using DNA immunization as mentioned below. DNA
immunization is an immunization method that confers
immunostimulation by expressing a sensitizing antigen in an animal
immunized as a result of administering a vector DNA constructed to
allow expression of an antigen protein-encoding gene in the animal.
As compared to conventional immunization methods in which a protein
antigen is administered to animals to be immunized, DNA
immunization is expected to be superior in that:
[0102] in the case the antigen is a membrane protein,
immunostimulation can be provided while retaining the structure of
the membrane protein; and
[0103] there is no need to purify the antigen for immunization.
[0104] In order to prepare a monoclonal antibody of the present
invention using DNA immunization, first, a DNA expressing an IgA
protein is administered to an animal to be immunized. The
IgA-encoding DNA can be synthesized by known methods such as PCR.
The obtained DNA is inserted into an appropriate expression vector,
and then this is administered to an animal to be immunized.
Preferably used expression vectors include, for example,
commercially-available expression vectors such as pcDNA3.1. Vectors
can be administered to an organism using conventional methods. For
example, DNA immunization is performed by using a gene gun to
introduce expression vector-coated gold particles into cells in the
body of an animal to be immunized. Antibodies that recognize IgA
can also be produced by the methods described in
PCT/JP2011/077619.
[0105] After immunizing a mammal as described above, an increase in
the titer of an IgA-binding antibody is confirmed in the serum.
Then, immune cells are collected from the mammal, and then
subjected to cell fusion. In particular, splenocytes are preferably
used as immune cells.
[0106] A mammalian myeloma cell is used as a cell to be fused with
the above-mentioned immune cells. The myeloma cells preferably
comprise a suitable selection marker for screening. A selection
marker confers characteristics to cells for their survival (or
death) under a specific culture condition. Hypoxanthine-guanine
phosphoribosyltransferase deficiency (hereinafter abbreviated as
HGPRT deficiency) and thymidine kinase deficiency (hereinafter
abbreviated as TK deficiency) are known as selection markers. Cells
with HGPRT or TK deficiency have hypoxanthine-aminopterin-thymidine
sensitivity (hereinafter abbreviated as HAT sensitivity).
HAT-sensitive cells cannot synthesize DNA in a HAT selection
medium, and are thus killed. However, when the cells are fused with
normal cells, they can continue DNA synthesis using the salvage
pathway of the normal cells, and therefore they can grow even in
the HAT selection medium.
[0107] HGPRT-deficient and TK-deficient cells can be selected in a
medium containing 6-thioguanine, 8-azaguanine (hereinafter
abbreviated as 8AG), or 5'-bromodeoxyuridine, respectively. Normal
cells are killed because they incorporate these pyrimidine analogs
into their DNA. Meanwhile, cells that are deficient in these
enzymes can survive in the selection medium, since they cannot
incorporate these pyrimidine analogs. In addition, a selection
marker referred to as G418 resistance provided by the
neomycin-resistant gene confers resistance to 2-deoxystreptamine
antibiotics (gentamycin analogs). Various types of myeloma cells
that are suitable for cell fusion are known.
[0108] For example, myeloma cells including the following cells can
be preferably used:
P3(P3x63Ag8.653) (J. Immunol. (1979) 123 (4), 1548-1550);
P3x63Ag8U.1 (Current Topics in Microbiology and Immunology (1978)
81, 1-7); NS-1 (C. Eur. J. Immunol. (1976)6 (7), 511-519);
MPC-11 (Cell (1976) 8 (3), 405-415);
SP2/0 (Nature (1978) 276 (5685), 269-270);
[0109] FO (J. Immunol. Methods (1980) 35 (1-2), 1-21);
S194/5.XXO.BU.1 (J. Exp. Med. (1978) 148 (1), 313-323);
R210 (Nature (1979) 277 (5692), 131-133), etc.
[0110] Cell fusions between the immunocytes and myeloma cells are
essentially carried out using known methods, for example, a method
by Kohler and Milstein et al. (Methods Enzymol. (1981) 73:
3-46).
[0111] More specifically, cell fusion can be carried out, for
example, in a conventional culture medium in the presence of a cell
fusion-promoting agent. The fusion-promoting agents include, for
example, polyethylene glycol (PEG) and Sendai virus (HVJ). If
required, an auxiliary substance such as dimethyl sulfoxide is also
added to improve fusion efficiency.
[0112] The ratio of immune cells to myeloma cells may be determined
at one's own discretion, preferably, for example, one myeloma cell
for every one to ten immunocytes. Culture media to be used for cell
fusions include, for example, media that are suitable for the
growth of myeloma cell lines, such as RPMI1640 medium and MEM
medium, and other conventional culture medium used for this type of
cell culture. In addition, serum supplements such as fetal calf
serum (FCS) may be preferably added to the culture medium.
[0113] For cell fusion, predetermined amounts of the above immune
cells and myeloma cells are mixed well in the above culture medium.
Then, a PEG solution (for example, the average molecular weight is
about 1,000 to 6,000) prewarmed to about 37.degree. C. is added
thereto at a concentration of generally 30% to 60% (w/v). This is
gently mixed to produce desired fusion cells (hybridomas). Then, an
appropriate culture medium mentioned above is gradually added to
the cells, and this is repeatedly centrifuged to remove the
supernatant. Thus, cell fusion agents and such which are
unfavorable to hybridoma growth can be removed.
[0114] The hybridomas thus obtained can be selected by culture
using a conventional selective medium, for example, HAT medium (a
culture medium containing hypoxanthine, aminopterin, and
thymidine). Cells other than the desired hybridomas (non-fused
cells) can be killed by continuing culture in the above HAT medium
for a sufficient period of time. Typically, the period is several
days to several weeks. Then, hybridomas producing the desired
antibody are screened and singly cloned by conventional limiting
dilution methods.
[0115] The hybridomas thus obtained can be selected using a
selection medium based on the selection marker possessed by the
myeloma used for cell fusion. For example, HGPRT- or TK-deficient
cells can be selected by culture using the HAT medium (a culture
medium containing hypoxanthine, aminopterin, and thymidine).
Specifically, when HAT-sensitive myeloma cells are used for cell
fusion, cells successfully fused with normal cells can selectively
proliferate in the HAT medium. Cells other than the desired
hybridomas (non-fused cells) can be killed by continuing culture in
the above HAT medium for a sufficient period of time. Specifically,
desired hybridomas can be selected by culture for generally several
days to several weeks. Then, hybridomas producing the desired
antibody are screened and singly cloned by conventional limiting
dilution methods.
[0116] Desired antibodies can be preferably selected and singly
cloned by screening methods based on known antigen/antibody
reaction. For example, the activity of an antibody to bind to
immobilized IgA can be assessed based on the principle of ELISA.
For example, IgA is immobilized to the wells of an ELISA plate.
Culture supernatants of hybridomas are contacted with the IgA in
the wells, and antibodies that bind to the IgA are detected. When
the monoclonal antibodies are derived from mouse, antibodies bound
to the cells can be detected using an anti-mouse immunoglobulin
antibody. Hybridomas producing a desired antibody having the
antigen-binding ability are selected by the above screening, and
they can be cloned by a limiting dilution method or the like.
[0117] Monoclonal antibody-producing hybridomas thus prepared can
be passaged in a conventional culture medium, and stored in liquid
nitrogen for a long period.
[0118] The above hybridomas are cultured by a conventional method,
and desired monoclonal antibodies can be prepared from the culture
supernatants. Alternatively, the hybridomas are administered to and
grown in compatible mammals, and monoclonal antibodies are prepared
from the ascites. The former method is suitable for preparing
antibodies with high purity.
[0119] Antibodies encoded by antibody genes that are cloned from
antibody-producing cells such as the above hybridomas can also be
preferably used. A cloned antibody gene is inserted into an
appropriate vector, and this is introduced into a host to express
the antibody encoded by the gene. Methods for isolating antibody
genes, inserting the genes into vectors, and transforming host
cells have already been established, for example, by Vandamme et
al. (Eur. J. Biochem. (1990) 192(3), 767-775). Methods for
producing recombinant antibodies are also known as described
below.
[0120] For example, a cDNA encoding the variable region (V region)
of an anti-IgA antibody is prepared from hybridoma cells producing
the anti-IgA antibody. For this purpose, total RNA is first
extracted from hybridomas. Methods used for extracting mRNAs from
cells include, for example: [0121] the guanidine
ultracentrifugation method (Biochemistry (1979) 18(24), 5294-5299),
and [0122] the AGPC method (Anal. Biochem. (1987) 162(1),
156-159)
[0123] Extracted mRNAs can be purified using the mRNA Purification
Kit (GE Healthcare Bioscience) or such. Alternatively, kits for
extracting total mRNA directly from cells, such as the QuickPrep
mRNA Purification Kit (GE Healthcare Bioscience), are also
commercially available. mRNAs can be prepared from hybridomas using
such kits. cDNAs encoding the antibody V region can be synthesized
from the prepared mRNAs using a reverse transcriptase. cDNAs can be
synthesized using the AMV Reverse Transcriptase First-strand cDNA
Synthesis Kit (Seikagaku Co.) or such. Furthermore, the SMART RACE
cDNA amplification kit (Clontech) and the PCR-based 5'-RACE method
(Proc. Natl. Acad. Sci. USA (1988) 85(23), 8998-9002; Nucleic Acids
Res. (1989) 17(8), 2919-2932) can be appropriately used to
synthesize and amplify cDNAs. In such a cDNA synthesis process,
appropriate restriction enzyme sites described below may be
introduced into both ends of a cDNA.
[0124] The cDNA fragment of interest is purified from the resulting
PCR product, and then this is ligated to a vector DNA. A
recombinant vector is thus constructed, and introduced into E. coli
or such. After colony selection, the desired recombinant vector can
be prepared from the colony-forming E. coli. Then, whether the
recombinant vector has the cDNA nucleotide sequence of interest is
tested by a known method such as the dideoxy nucleotide chain
termination method.
[0125] The 5'-RACE method which uses primers to amplify the
variable region gene is conveniently used for isolating the gene
encoding the variable region. First, a 5'-RACE cDNA library is
constructed by cDNA synthesis using RNAs extracted from hybridoma
cells as a template. A commercially available kit such as the SMART
RACE cDNA amplification kit is appropriately used to synthesize the
5'-RACE cDNA library.
[0126] The antibody gene is amplified by PCR using the prepared
5'-RACE cDNA library as a template. Primers for amplifying the
mouse antibody gene can be designed based on known antibody gene
sequences. The nucleotide sequences of the primers vary depending
on the immunoglobulin subclass. Therefore, it is preferable that
the subclass is determined in advance using a commercially
available kit such as the Iso Strip mouse monoclonal antibody
isotyping kit (Roche Diagnostics).
[0127] Specifically, for example, primers that allow amplification
of genes encoding .gamma.1, .gamma.2a, .gamma.2b, and .gamma.3
heavy chains and .kappa. and .lamda. light chains are used to
isolate mouse IgG-encoding genes. In general, a primer that anneals
to a constant region site close to the variable region is used as a
3'-side primer to amplify an IgG variable region gene. Meanwhile, a
primer attached to a 5' RACE cDNA library construction kit is used
as a 5'-side primer.
[0128] PCR products thus amplified are used to reshape
immunoglobulins composed of a combination of heavy and light
chains. A desired antibody can be selected using the IgA-binding
activity of a reshaped immunoglobulin as an indicator. For example,
when the objective is to isolate an antibody against IgA, it is
more preferred that the binding of the antibody to IgA is specific.
An IgA-binding antibody can be screened, for example, by the
following steps:
[0129] (1) contacting an IgA with an antibody comprising the V
region encoded by a cDNA isolated from a hybridoma;
[0130] (2) detecting the binding of the antibody to the IgA;
and
[0131] (3) selecting an antibody that binds to the IgA-expressing
cell.
[0132] Methods for detecting the binding of an antibody to IgA are
known. Specifically, the binding of an antibody to IL-6R-expressing
cells can be detected by the above-described techniques such as
ELISA.
[0133] Preferred antibody screening methods that use the binding
activity as an indicator also include panning methods using phage
vectors. Screening methods using phage vectors are advantageous
when the antibody genes are isolated from heavy-chain and
light-chain subclass libraries from a polyclonal
antibody-expressing cell population. Genes encoding the heavy-chain
and light-chain variable regions can be linked by an appropriate
linker sequence to form a single-chain Fv (scFv). Phages presenting
scFv on their surface can be produced by inserting a gene encoding
scFv into a phage vector. The phages are contacted with an antigen
of interest. Then, a DNA encoding scFv having the binding activity
of interest can be isolated by collecting phages bound to the
antigen. This process can be repeated as necessary to enrich scFv
having the binding activity of interest.
[0134] After isolation of the cDNA encoding the V region of the
anti-IgA antibody of interest, the cDNA is digested with
restriction enzymes that recognize the restriction sites introduced
into both ends of the cDNA. Preferred restriction enzymes recognize
and cleave a nucleotide sequence that occurs in the nucleotide
sequence of the antibody gene at a low frequency. Furthermore, a
restriction site for an enzyme that produces a sticky end is
preferably introduced into a vector to insert a single-copy
digested fragment in the correct orientation. The cDNA encoding the
V region of the anti-IgA antibody is digested as described above,
and this is inserted into an appropriate expression vector to
construct an antibody expression vector. In this case, if a gene
encoding the antibody constant region (C region) and a gene
encoding the above V region are fused in-frame, a chimeric antibody
is obtained. Herein, "chimeric antibody" means that the origin of
the constant region is different from that of the variable region.
Thus, in addition to mouse/human heterochimeric antibodies,
human/human allochimeric antibodies are included in the chimeric
antibodies of the present invention. A chimeric antibody expression
vector can be constructed by inserting the above V region gene into
an expression vector that already has the constant region.
Specifically, for example, a recognition sequence for a restriction
enzyme that excises the above V region gene can be appropriately
placed on the 5' side of an expression vector carrying a DNA
encoding a desired antibody constant region (C region). A chimeric
antibody expression vector is constructed by fusing in frame the
two genes digested with the same combination of restriction
enzymes.
[0135] To produce an anti-IgA monoclonal antibody, antibody genes
are inserted into an expression vector so that the genes are
expressed under the control of an expression regulatory region. The
expression regulatory region for antibody expression includes, for
example, enhancers and promoters. Furthermore, an appropriate
signal sequence may be attached to the amino terminus so that the
expressed antibody is secreted to the outside of cells. In the
Examples described later, a peptide having the amino acid sequence
MGWSCIILFLVATATGVHS (SEQ ID NO: 5) are used as a signal sequence.
Meanwhile, other appropriate signal sequences may be attached. The
expressed polypeptide is cleaved at the carboxyl terminus of the
above sequence, and the resulting polypeptide is secreted to the
outside of cells as a mature polypeptide. Then, appropriate host
cells are transformed with the expression vector, and recombinant
cells expressing the anti-IgA antibody-encoding DNA are
obtained.
[0136] DNAs encoding the antibody heavy chain (H chain) and light
chain (L chain) are separately inserted into different expression
vectors to express the antibody gene. An antibody molecule having
the H and L chains can be expressed by co-transfecting the same
host cell with vectors into which the H-chain and L-chain genes are
respectively inserted. Alternatively, host cells can be transformed
with a single expression vector into which DNAs encoding the H and
L chains are inserted (see International Publication No. WO
1994/011523).
[0137] There are various known host cell/expression vector
combinations for antibody preparation by introducing isolated
antibody genes into appropriate hosts. All of these expression
systems are applicable to isolation of the antigen-binding
molecules of the present invention. Appropriate eukaryotic cells
used as host cells include animal cells, plant cells, and fungal
cells. Specifically, the animal cells include, for example, the
following cells.
(1) mammalian cells: CHO (Chinese hamster ovary cell line), COS
(Monkey kidney cell line), myeloma (Sp2/O, NSO, etc), BHK (baby
hamster kidney cell line), HEK293 (human embryonic kidney cell line
with sheared adenovirus (Ad)5 DNA), PER.C6 cell (human embryonic
retinal cell line transformed with the Adenovirus Type 5 (Ad5) ElA
and E1B genes), HeLa, Vero, HEK293 (human embryonic kidney cell
line with sheared adenovirus (Ad)5 DNA), or such (Current Protocols
in Protein Science (May, 2001, Unit 5.9, Table 5.9.1)); (2)
amphibian cells: Xenopus oocytes, or such; and (3) insect cells:
sf9, sf21, Tn5, or such.
[0138] In addition, as a plant cell, an antibody gene expression
system using cells derived from the Nicotiana genus such as
Nicotiana tabacum is known. Callus cultured cells can be
appropriately used to transform plant cells.
[0139] Furthermore, the following cells can be used as fungal
cells: [0140] yeasts: the Saccharomyces genus such as Saccharomyces
serevisiae, and the Pichia genus such as Pichia pastoris; and
[0141] filamentous fungi: the Aspergillus genus such as Aspergillus
niger.
[0142] Furthermore, antibody gene expression systems that utilize
prokaryotic cells are also known. For example, when using bacterial
cells, E. coli cells, Bacillus subtilis cells, and such can
suitably be utilized in the present invention. Expression vectors
carrying the antibody genes of interest are introduced into these
cells by transfection. The transfected cells are cultured in vitro,
and the desired antibody can be prepared from the culture of
transformed cells.
[0143] In addition to the above-described host cells, transgenic
animals can also be used to produce a recombinant antibody. That
is, the antibody can be obtained from an animal into which the gene
encoding the antibody of interest is introduced. For example, the
antibody gene can be constructed as a fusion gene by inserting in
frame into a gene that encodes a protein produced specifically in
milk. Goat .beta.-casein or such can be used, for example, as the
protein secreted in milk. DNA fragments containing the fused gene
inserted with the antibody gene is injected into a goat embryo, and
then this embryo is introduced into a female goat. Desired
antibodies can be obtained as a protein fused with the milk protein
from milk produced by the transgenic goat born from the
embryo-recipient goat (or progeny thereof). In addition, to
increase the volume of milk containing the desired antibody
produced by the transgenic goat, hormones can be administered to
the transgenic goat as necessary (Ebert, K. M. et al.,
Bio/Technology (1994) 12 (7), 699-702).
[0144] When an antigen-binding molecule described herein is
administered to human, an antigen-binding domain derived from a
genetically recombinant antibody that has been artificially
modified to reduce the heterologous antigenicity against human and
such, can be appropriately used as the antigen-binding domain of
the antigen-binding molecule. Such genetically recombinant
antibodies include, for example, humanized antibodies. These
modified antibodies are appropriately produced by known
methods.
[0145] An antibody variable region used to produce the
antigen-binding domain of an antigen-binding molecule described
herein is generally formed by three complementarity-determining
regions (CDRs) that are separated by four framework regions (FR5).
CDR is a region that substantially determines the binding
specificity of an antibody. The amino acid sequences of CDRs are
highly diverse. On the other hand, the FR-forming amino acid
sequences often have high identity even among antibodies with
different binding specificities. Therefore, generally, the binding
specificity of a certain antibody can be introduced to another
antibody by CDR grafting.
[0146] A humanized antibody is also called a reshaped human
antibody. Specifically, humanized antibodies prepared by applying
the CDR grafting technology, which grafts the CDRs of a non-human
animal antibody such as a mouse antibody to a human antibody, and
such are known. Common genetic engineering techniques for obtaining
humanized antibodies are also known. Specifically, for example,
overlap extension PCR is known as a method for grafting a mouse
antibody CDR to a human FR. In overlap extension PCR, a nucleotide
sequence encoding a mouse antibody CDR to be grafted is added to
primers for synthesizing a human antibody FR. Primers are prepared
for each of the four FRs. It is generally considered that when
grafting a mouse CDR to a human FR, selecting a human FR that has
high identity to a mouse FR is advantageous for maintaining the CDR
function. That is, it is generally preferable to use a human FR
comprising an amino acid sequence which has high identity to the
amino acid sequence of the FR adjacent to the mouse CDR to be
grafted.
[0147] Nucleotide sequences to be ligated are designed so that they
will be connected to each other in frame. Human FRs are
individually synthesized using the respective primers. As a result,
products in which the mouse CDR-encoding DNA is attached to the
individual FR-encoding DNAs are obtained. Nucleotide sequences
encoding the mouse CDR of each product are designed so that they
overlap with each other. Then, complementary strand synthesis
reaction is conducted to anneal the overlapping CDR regions of the
products synthesized using a human antibody gene as template. Human
FRs are ligated via the mouse CDR sequences by this reaction.
[0148] The full length V region gene, in which three CDRs and four
FRs are ultimately ligated, is amplified using primers that anneal
to its 5'- or 3'-end, which are added with suitable restriction
enzyme recognition sequences. An expression vector for humanized
antibody can be produced by inserting the DNA obtained as described
above and a DNA that encodes a human antibody C region into an
expression vector so that they will ligate in frame. After the
recombinant vector is transfected into a host to establish
recombinant cells, the recombinant cells are cultured, and the DNA
encoding the humanized antibody is expressed to produce the
humanized antibody in the cell culture (see, European Patent
Publication No. EP 239400 and International Patent Publication No.
WO 1996/002576).
[0149] By qualitatively or quantitatively measuring and evaluating
the antigen-binding activity of the humanized antibody produced as
described above, one can suitably select human antibody FRs that
allow CDRs to form a favorable antigen-binding site when ligated
through the CDRs. Amino acid residues in FRs may be substituted as
necessary, so that the CDRs of a reshaped human antibody form an
appropriate antigen-binding site. For example, amino acid sequence
mutations can be introduced into FRs by applying the PCR method
used for grafting a mouse CDR into a human FR. More specifically,
partial nucleotide sequence mutations can be introduced into
primers that anneal to the FR. Nucleotide sequence mutations are
introduced into the FRs synthesized by using such primers. Mutant
FR sequences having the desired characteristics can be selected by
measuring and evaluating the activity of the amino acid-substituted
mutant antibody to bind to the antigen by the above-mentioned
method (Cancer Res. (1993) 53: 851-856).
[0150] Alternatively, desired human antibodies can be obtained by
immunizing transgenic animals having the entire repertoire of human
antibody genes (see International Publication Nos. WO 1993/012227;
WO 1992/003918; WO 1994/002602; WO 1994/025585; WO 1996/034096; WO
1996/033735) by DNA immunization.
[0151] Furthermore, techniques for preparing human antibodies by
panning using human antibody libraries are also known. For example,
the V region of a human antibody is expressed as a single-chain
antibody (scFv) on phage surface by the phage display method.
Phages expressing an scFv that binds to the antigen can be
selected. The DNA sequence encoding the human antibody V region
that binds to the antigen can be determined by analyzing the genes
of selected phages. The DNA sequence of the scFv that binds to the
antigen is determined. An expression vector is prepared by fusing
the V region sequence in frame with the C region sequence of a
desired human antibody, and inserting this into an appropriate
expression vector. The expression vector is introduced into cells
appropriate for expression such as those described above. The human
antibody can be produced by expressing the human antibody-encoding
gene in the cells. These methods are already known (see
International Publication Nos. WO 1992/001047; WO 1992/020791; WO
1993/006213; WO 1993/011236; WO 1993/019172; WO 1995/001438; WO
1995/015388).
[0152] In addition to the techniques described above, techniques of
B cell cloning (identification of each antibody-encoding sequence,
cloning and its isolation; use in constructing expression vector in
order to prepare each antibody (IgG1, IgG2, IgG3, or IgG4 in
particular); and such) such as described in Bernasconi et al.
(Science (2002) 298: 2199-2202) or in International Publication No.
WO 2008/081008 can be appropriately used to isolate antibody
genes.
EU Numbering and Kabat Numbering
[0153] According to the methods used in the present invention,
amino acid positions assigned to antibody CDR and FR are specified
according to Kabat's numbering (Sequences of Proteins of
Immunological Interest (National Institute of Health, Bethesda,
Md., 1987 and 1991)). Herein, when an antigen-binding molecule is
an antibody or antigen-binding fragment, variable region amino
acids are indicated according to Kabat's numbering system (Kabat
numbering), while constant region amino acids are indicated
according to EU numbering system based on Kabat's amino acid
positions.
Conditions of Ion Concentration
Conditions of Metal Ion Concentration
[0154] In one embodiment of the present invention, the ion
concentration refers to a metal ion concentration. "Metal ions"
refer to ions of group I elements except hydrogen such as alkaline
metals and copper group elements, group II elements such as
alkaline earth metals and zinc group elements, group III elements
except boron, group IV elements except carbon and silicon, group
VIII elements such as iron group and platinum group elements,
elements belonging to subgroup A of groups V, VI, and VII, and
metal elements such as antimony, bismuth, and polonium. Metal atoms
have the property of releasing valence electrons to become cations.
This is referred to as ionization tendency. Metals with strong
ionization tendency are deemed to be chemically active.
[0155] In the present invention, preferred metal ions include, for
example, calcium ion. Calcium ion is involved in modulation of many
biological phenomena, including contraction of muscles such as
skeletal, smooth, and cardiac muscles; activation of movement,
phagocytosis, and the like of leukocytes; activation of shape
change, secretion, and the like of platelets; activation of
lymphocytes; activation of mast cells including secretion of
histamine; cell responses mediated by catecholamine a receptor or
acetylcholine receptor; exocytosis; release of transmitter
substances from neuron terminals; and axoplasmic flow in neurons.
Known intracellular calcium ion receptors include troponin C,
calmodulin, parvalbumin, and myosin light chain, which have several
calcium ion-binding sites and are believed to be derived from a
common origin in terms of molecular evolution. There are also many
known calcium-binding motifs. Such well-known motifs include, for
example, cadherin domains, EF-hand of calmodulin, C2 domain of
Protein kinase C, Gla domain of blood coagulation protein Factor
IX, C-type lectins of acyaroglycoprotein receptor and
mannose-binding receptor, A domains of LDL receptors, annexin,
thrombospondin type 3 domain, and EGF-like domains.
[0156] In the present invention, when the metal ion is calcium ion,
the conditions of calcium ion concentration include low calcium ion
concentrations and high calcium ion concentrations. "The binding
activity varies depending on calcium ion concentrations" means that
the antigen-binding activity of an antigen-binding molecule varies
due to the difference in the conditions between low and high
calcium ion concentrations. For example, the antigen-binding
activity of an antigen-binding molecule may be higher at a high
calcium ion concentration than at a low calcium ion concentration.
Alternatively, the antigen-binding activity of an antigen-binding
molecule may be higher at a low calcium ion concentration than at a
high calcium ion concentration.
[0157] Herein, the high calcium ion concentration is not
particularly limited to a specific value; however, the
concentration may preferably be selected between 100 .mu.M and 10
mM. In another embodiment, the concentration may be selected
between 200 .mu.M and 5 mM. In an alternative embodiment, the
concentration may be selected between 400 .mu.M and 3 mM. In still
another embodiment, the concentration may be selected between 200
.mu.M and 2 mM. Furthermore, the concentration may be selected
between 400 .mu.M and 1 mM. In particular, a concentration selected
between 500 .mu.M and 2.5 mM, which is close to the plasma (blood)
concentration of calcium ion in vivo, is preferred.
[0158] Herein, the low calcium ion concentration is not
particularly limited to a specific value; however, the
concentration may preferably be selected between 0.1 .mu.M and 30
In another embodiment, the concentration may be selected between
0.2 .mu.M and 20 In still another embodiment, the concentration may
be selected between 0.5 .mu.M and 10 In an alternative embodiment,
the concentration may be selected between 1 .mu.M and 5
Furthermore, the concentration may be selected between 2 .mu.M and
4 In particular, a concentration selected between 1 .mu.M and 5
.mu.M, which is close to the concentration of ionized calcium in
early endosomes in vivo, is preferred.
[0159] Herein, "the antigen-binding activity is lower at a low
calcium ion concentration than at a high calcium ion concentration"
means that the antigen-binding activity of an antigen-binding
molecule is weaker at a calcium ion concentration selected between
0.1 .mu.M and 30 .mu.M than at a calcium ion concentration selected
between 100 .mu.M and 10 mM. Preferably, it means that the
antigen-binding activity of an antigen-binding molecule is weaker
at a calcium ion concentration selected between 0.5 .mu.M and 10
.mu.M than at a calcium ion concentration selected between 200
.mu.M and 5 mM. It particularly preferably means that the
antigen-binding activity at the calcium ion concentration in the
early endosome in vivo is weaker than that at the in vivo plasma
calcium ion concentration; and specifically, it means that the
antigen-binding activity of an antigen-binding molecule is weaker
at a calcium ion concentration selected between 1 .mu.M and 5 .mu.M
than at a calcium ion concentration selected between 500 .mu.M and
2.5 mM.
[0160] Whether the antigen-binding activity of an antigen-binding
molecule is changed depending on metal ion concentrations can be
determined, for example, by the use of known measurement methods
such as those described in the section "Binding Activity" above.
For example, in order to confirm that the antigen-binding activity
of an antigen-binding molecule becomes higher at a high calcium ion
concentration than at a low calcium ion concentration, the
antigen-binding activity of the antigen-binding molecule at low and
high calcium ion concentrations is compared.
[0161] In the present invention, the expression "the
antigen-binding activity is lower at a low calcium ion
concentration than at a high calcium ion concentration" can also be
expressed as "the antigen-binding activity of an antigen-binding
molecule is higher at a high calcium ion concentration than at a
low calcium ion concentration". In the present invention, "the
antigen-binding activity is lower at a low calcium ion
concentration than at a high calcium ion concentration" is
sometimes written as "the antigen-binding ability is weaker at a
low calcium ion concentration than at a high calcium ion
concentration". Also, "the antigen-binding activity at a low
calcium ion concentration is reduced to be lower than that at a
high calcium ion concentration" may be written as "the
antigen-binding ability at a low calcium ion concentration is made
weaker than that at a high calcium ion concentration".
[0162] When determining the antigen-binding activity, the
conditions other than calcium ion concentration can be
appropriately selected by those skilled in the art, and are not
particularly limited. For example, the activity can be determined
at 37.degree. C. in HEPES buffer. For example, Biacore (GE
Healthcare) or such can be used for the determination. When the
antigen is a soluble antigen, the antigen-binding activity of an
antigen-binding molecule can be assessed by flowing the antigen as
an analyte over a chip onto which the antigen-binding molecule is
immobilized. When the antigen is a membrane antigen, the binding
activity of an antigen-binding molecule to the membrane antigen can
be assessed by flowing the antigen-binding molecule as an analyte
over a chip onto which the antigen is immobilized.
[0163] As long as the antigen-binding activity of an
antigen-binding molecule of the present invention is weaker at a
low calcium ion concentration than at a high calcium ion
concentration, the ratio of the antigen-binding activity between
low and high calcium ion concentrations is not particularly
limited. However, the ratio of the KD (dissociation constant) of
the antigen-binding molecule for an antigen at a low calcium ion
concentration with respect to the KD at a high calcium ion
concentration, i.e. the value of KD (3 .mu.M Ca)/KD (2 mM Ca), is
preferably 2 or more, more preferably 10 or more, and still more
preferably 40 or more. The upper limit of the KD (3 .mu.M Ca)/KD (2
mM Ca) value is not particularly limited, and may be any value such
as 400, 1000, or 10000 as long as the molecule can be produced by
techniques known to those skilled in the art.
[0164] When the antigen is a soluble antigen, KD (dissociation
constant) can be used to represent the antigen-binding activity.
Meanwhile, when the antigen is a membrane antigen, apparent KD
(apparent dissociation constant) can be used to represent the
activity. KD (dissociation constant) and apparent KD (apparent
dissociation constant) can be determined by methods known to those
skilled in the art, for example, using Biacore (GE healthcare),
Scatchard plot, or flow cytometer.
[0165] Alternatively, for example, the dissociation rate constant
(kd) can also be preferably used as an index to represent the ratio
of the antigen-binding activity of an antigen-binding molecule of
the present invention between low and high calcium concentrations.
When the dissociation rate constant (kd) is used instead of the
dissociation constant (KD) as an index to represent the binding
activity ratio, the ratio of the dissociation rate constant (kd)
between low and high calcium concentrations, i.e. the value of kd
(low calcium concentration)/kd (high calcium concentration), is
preferably 2 or more, more preferably 5 or more, still more
preferably 10 or more, and yet more preferably 30 or more. The
upper limit of the Kd (low calcium concentration)/kd (high calcium
concentration) value is not particularly limited, and can be any
value such as 50, 100, or 200 as long as the molecule can be
produced by techniques known to those skilled in the art.
[0166] When the antigen is a soluble antigen, kd (dissociation rate
constant) can be used to represent the antigen-binding activity.
Meanwhile, when the antigen is a membrane antigen, apparent kd
(apparent dissociation rate constant) can be used to represent the
antigen-binding activity. The kd (dissociation rate constant) and
apparent kd (apparent dissociation rate constant) can be determined
by methods known to those skilled in the art, for example, using
Biacore (GE healthcare) or flow cytometer. In the present
invention, when the antigen-binding activity of an antigen-binding
molecule is determined at different calcium ion concentrations, it
is preferable to use the same conditions except for the calcium
concentrations.
[0167] For example, an antigen-binding domain or antigen-binding
molecule whose antigen-binding activity is lower at a low calcium
ion concentration than at a high calcium ion concentration, which
is one embodiment of the present invention, can be obtained via
screening of antigen-binding domains or antibodies including the
steps of:
(a) determining the antigen-binding activity of an antigen-binding
domain or antigen-binding molecule at a low calcium concentration;
(b) determining the antigen-binding activity of an antigen-binding
domain or antigen-binding molecule at a high calcium concentration;
and (c) selecting an antigen-binding domain or antigen-binding
molecule whose antigen-binding activity is lower at a low calcium
concentration than at a high calcium concentration.
[0168] Moreover, an antigen-binding domain or antigen-binding
molecule whose antigen-binding activity is lower at a low calcium
ion concentration than at a high calcium ion concentration, which
is one embodiment of the present invention, can be obtained via
screening of antigen-binding domains or antigen-binding molecules,
or a library thereof, including the steps of:
(a) contacting an antigen with an antigen-binding domain or
antigen-binding molecule, or a library thereof at a high calcium
concentration; (b) incubating at a low calcium concentration an
antigen-binding domain or antigen-binding molecule that has bound
to the antigen in step (a); and (c) isolating an antigen-binding
domain or antigen-binding molecule dissociated in step (b).
[0169] Furthermore, an antigen-binding domain or antigen-binding
molecule whose antigen-binding activity is lower at a low calcium
ion concentration than at a high calcium ion concentration, which
is one embodiment of the present invention, can be obtained via
screening of antigen-binding domains or antigen-binding molecules,
or a library thereof, including the steps of:
(a) contacting an antigen with a library of antigen-binding domains
or antigen-binding molecules at a low calcium concentration; (b)
selecting an antigen-binding domain or antigen-binding molecule
which does not bind to the antigen in step (a); (c) allowing the
antigen-binding domain or antigen-binding molecule selected in step
(c) to bind to the antigen at a high calcium concentration; and (d)
isolating an antigen-binding domain or antigen-binding molecule
that has bound to the antigen in step (c).
[0170] In addition, an antigen-binding domain or antigen-binding
molecule whose antigen-binding activity is lower at a low calcium
ion concentration than at a high calcium ion concentration, which
is one embodiment of the present invention, can be obtained by a
screening method comprising the steps of:
(a) contacting at a high calcium concentration a library of
antigen-binding domains or antigen-binding molecules with a column
onto which an antigen is immobilized; (b) eluting an
antigen-binding domain or antigen-binding molecule that has bound
to the column in step (a) from the column at a low calcium
concentration; and (c) isolating the antigen-binding domain or
antigen-binding molecule eluted in step (b).
[0171] Furthermore, an antigen-binding domain or antigen-binding
molecule whose antigen-binding activity is lower at a low calcium
ion concentration than at a high calcium ion concentration, which
is one embodiment of the present invention, can be obtained by a
screening method comprising the steps of:
(a) allowing at a low calcium concentration a library of
antigen-binding domains or antigen-binding molecules to pass
through a column onto which an antigen is immobilized; (b)
collecting an antigen-binding domain or antigen-binding molecule
that has been eluted without binding to the column in step (a); (c)
allowing the antigen-binding domain or antigen-binding molecule
collected in step (b) to bind to the antigen at a high calcium
concentration; and (d) isolating an antigen-binding domain or
antigen-binding molecule that has bound to the antigen in step
(c).
[0172] Moreover, an antigen-binding domain or antigen-binding
molecule whose antigen-binding activity is lower at a low calcium
ion concentration than at a high calcium ion concentration, which
is one embodiment of the present invention, can be obtained by a
screening method comprising the steps of:
(a) contacting an antigen with a library of antigen-binding domains
or antigen-binding molecules at a high calcium concentration; (b)
obtaining an antigen-binding domain or antigen-binding molecule
that has bound to the antigen in step (a); (c) incubating at a low
calcium concentration the antigen-binding domain or antigen-binding
molecule obtained in step (b); and (d) isolating an antigen-binding
domain or antigen-binding molecule whose antigen-binding activity
in step (c) is weaker than the criterion for the selection of step
(b).
[0173] The above-described steps may be repeated twice or more
times. Thus, the present invention provides antigen-binding domains
or antigen-binding molecules whose antigen-binding activity is
lower at a low calcium ion concentration than at a high calcium ion
concentration, which are obtained by screening methods that further
comprises the step of repeating twice or more times steps (a) to
(c) or (a) to (d) in the above-described screening methods. The
number of cycles of steps (a) to (c) or (a) to (d) is not
particularly limited, but generally is 10 or less.
[0174] In the screening methods of the present invention, the
antigen-binding activity of an antigen-binding domain or
antigen-binding molecule at a low calcium concentration is not
particularly limited as long as it is antigen-binding activity at
an ionized calcium concentration of between 0.1 .mu.M and 30 .mu.M,
but preferably is antigen-binding activity at an ionized calcium
concentration of between 0.5 .mu.M and 10 .mu.M. More preferably,
it is antigen-binding activity at the ionized calcium concentration
in the early endosome in vivo, specifically, between 1 .mu.M and 5
.mu.M. Meanwhile, the antigen-binding activity of an
antigen-binding domain or antigen-binding molecule at a high
calcium concentration is not particularly limited, as long as it is
antigen-binding activity at an ionized calcium concentration of
between 100 .mu.M and 10 mM, but preferably is antigen-binding
activity at an ionized calcium concentration of between 200 .mu.M
and 5 mM. More preferably, it is antigen-binding activity at the
ionized calcium concentration in plasma in vivo, specifically,
between 0.5 mM and 2.5 mM.
[0175] The antigen-binding activity of an antigen-binding domain or
antigen-binding molecule can be measured by methods known to those
skilled in the art. Conditions other than the ionized calcium
concentration can be determined by those skilled in the art. The
antigen-binding activity of an antigen-binding domain or
antigen-binding molecule can be evaluated as a dissociation
constant (KD), apparent dissociation constant (apparent KD),
dissociation rate constant (kd), apparent dissociation constant
(apparent kd), and such. These can be determined by methods known
to those skilled in the art, for example, using Biacore (GE
healthcare), Scatchard plot, or FACS.
[0176] In the present invention, the step of selecting an
antigen-binding domain or antigen-binding molecule whose
antigen-binding activity is higher at a high calcium concentration
than at a low calcium concentration is synonymous with the step of
selecting an antigen-binding domain or antigen-binding molecule
whose antigen-binding activity is lower at a low calcium
concentration than at a high calcium concentration.
[0177] As long as the antigen-binding activity is higher at a high
calcium concentration than at a low calcium concentration, the
difference in the antigen-binding activity between high and low
calcium concentrations is not particularly limited; however, the
antigen-binding activity at a high calcium concentration is
preferably twice or more, more preferably 10 times or more, and
still more preferably 40 times or more than that at a low calcium
concentration.
[0178] Antigen-binding domains or antigen-binding molecules of the
present invention to be screened by the screening methods described
above may be any antigen-binding domains and antigen-binding
molecules. For example, it is possible to screen the
above-described antigen-binding domains or antigen-binding
molecules. For example, antigen-binding domains or antigen-binding
molecules having natural sequences or substituted amino acid
sequences may be screened.
Libraries
[0179] In an embodiment, an antigen-binding domain or
antigen-binding molecule of the present invention can be obtained
from a library that is mainly composed of a plurality of
antigen-binding molecules whose sequences are different from one
another and whose antigen-binding domains have at least one amino
acid residue that alters the antigen-binding activity of the
antigen-binding molecules depending on ion concentrations. The ion
concentrations preferably include, for example, metal ion
concentration and proton concentration.
[0180] Herein, a "library" refers to a plurality of antigen-binding
molecules or a plurality of fusion polypeptides containing
antigen-binding molecules, or nucleic acids or polynucleotides
encoding their sequences. The sequences of a plurality of
antigen-binding molecules or a plurality of fusion polypeptides
containing antigen-binding molecules in a library are not
identical, but are different from one another.
[0181] Herein, the phrase "sequences are different from one
another" in the expression "a plurality of antigen-binding
molecules whose sequences are different from one another" means
that the sequences of antigen-binding molecules in a library are
different from one another. Specifically, in a library, the number
of sequences different from one another reflects the number of
independent clones with different sequences, and may also be
referred to as "library size". The library size of a conventional
phage display library ranges from 10.sup.6 to 10.sup.12. The
library size can be increased up to 10.sup.14 by the use of known
techniques such as ribosome display. However, the actual number of
phage particles used in panning selection of a phage library is in
general 10-10000 times greater than the library size. This excess
multiplicity is also referred to as "the number of library
equivalents", and means that there are 10 to 10,000 individual
clones that have the same amino acid sequence. Thus, in the present
invention, the phrase "sequences are different from one another"
means that the sequences of independent antigen-binding molecules
in a library, excluding library equivalents, are different from one
another. More specifically, the above means that there are 10.sup.6
to 10.sup.14 antigen-binding molecules whose sequences are
different from one another, preferably 10.sup.7 to 10.sup.12
molecules, more preferably 10.sup.8 to 10.sup.11 molecules, and
particularly preferably 10.sup.8 to 10.sup.10 molecules whose
sequences are different from one another.
[0182] Herein, the phrase "a plurality of" in the expression "a
library mainly composed of a plurality of antigen-binding
molecules" generally refers to, in the case of, for example,
antigen-binding molecules, fusion polypeptides, polynucleotide
molecules, vectors, or viruses of the present invention, a group of
two or more types of the substance. For example, when two or more
substances are different from one another in a particular
characteristic, this means that there are two or more types of the
substance. Such examples may include, for example, mutant amino
acids observed at specific amino acid positions in an amino acid
sequence. For example, when there are two or more antigen-binding
molecules of the present invention whose sequences are
substantially the same or preferably the same except for flexible
residues or except for particular mutant amino acids at
hypervariable positions exposed on the surface, there are a
plurality of antigen-binding molecules of the present invention. In
another example, when there are two or more polynucleotide
molecules whose sequences are substantially the same or preferably
the same except for nucleotides encoding flexible residues or
nucleotides encoding mutant amino acids of hypervariable positions
exposed on the surface, there are a plurality of polynucleotide
molecules of the present invention.
[0183] In addition, herein, the phrase "mainly composed of" in the
expression "a library mainly composed of a plurality of
antigen-binding molecules" reflects the number of antigen-binding
molecules whose antigen-binding activity varies depending on ion
concentrations, among independent clones with different sequences
in a library. Specifically, it is preferable that there are at
least 10.sup.4 antigen-binding molecules having such binding
activity in a library. More preferably, antigen-binding domains of
the present invention can be obtained from a library containing at
least 10.sup.5 antigen-binding molecules having such binding
activity. Still more preferably, antigen-binding domains of the
present invention can be obtained from a library containing at
least 10.sup.6 antigen-binding molecules having such binding
activity. Particularly preferably, antigen-binding domains of the
present invention can be obtained from a library containing at
least 10.sup.7 antigen-binding molecules having such binding
activity. Yet more preferably, antigen-binding domains of the
present invention can be obtained from a library containing at
least 10.sup.8 antigen-binding molecules having such binding
activity. Alternatively, this may also be preferably expressed as
the ratio of the number of antigen-binding molecules whose
antigen-binding activity varies depending on ion concentrations
with respect to the number of independent clones having different
sequences in a library. Specifically, antigen-binding domains of
the present invention can be obtained from a library in which
antigen-binding molecules having such binding activity account for
0.1% to 80%, preferably 0.5% to 60%, more preferably 1% to 40%,
still more preferably 2% to 20%, and particularly preferably 4% to
10% of independent clones with different sequences in the library.
In the case of fusion polypeptides, polynucleotide molecules, or
vectors, similar expressions may be possible using the number of
molecules or the ratio to the total number of molecules. In the
case of viruses, similar expressions may also be possible using the
number of virions or the ratio to total number of virions.
Amino Acids that Alter the Antigen-Binding Activity of
Antigen-Binding Domains Depending on Calcium Ion Concentrations
[0184] Antigen-binding domains or antigen-binding molecules of the
present invention to be screened by the above-described screening
methods may be prepared in any manner. For example, when the metal
ion is calcium ion, it is possible to use preexisting
antigen-binding molecules, preexisting libraries (phage library,
etc.), antibodies or libraries prepared from hybridomas obtained by
immunizing animals or from B cells of immunized animals, antibodies
or libraries obtained by introducing amino acids capable of
chelating calcium (for example, aspartic acid and glutamic acid) or
unnatural amino acid mutations into the above-described antibodies
or libraries (calcium-cheletable amino acids (such as aspartic acid
and glutamic acid), libraries with increased content of unnatural
amino acids, libraries prepared by introducing calcium-chelatable
amino acids (such as aspartic acid and glutamic acid) or unnatural
amino acid mutations at particular positions, or the like.
[0185] Examples of the amino acids that alter the antigen-binding
activity of antigen-binding molecules depending on ion
concentrations as described above may be any types of amino acids
as long as the amino acids form a calcium-binding motif.
Calcium-binding motifs are well known to those skilled in the art
and have been described in details (for example, Springer et al.
(Cell (2000) 102, 275-277); Kawasaki and Kretsinger (Protein Prof.
(1995) 2, 305-490); Moncrief et al. (J. Mol. Evol. (1990) 30,
522-562); Chauvaux et al. (Biochem. J. (1990) 265, 261-265);
Bairoch and Cox (FEBS Lett. (1990) 269, 454-456); Davis (New Biol.
(1990) 2, 410-419); Schaefer et al. (Genomics (1995) 25, 638-643);
Economou et al. (EMBO J. (1990) 9, 349-354); Wurzburg et al.
(Structure. (2006) 14, 6, 1049-1058)). Specifically, any known
calcium-binding motifs, including type C lectins such as ASGPR,
CD23, MBR, and DC-SIGN, can be included in antigen-binding
molecules of the present invention. Preferred examples of such
preferred calcium-binding motifs also include, in addition to those
described above, for example, the calcium-binding motif in the
antigen-binding domain of SEQ ID NO: 6.
[0186] Furthermore, as amino acids that alter the antigen-binding
activity of antigen-binding molecules depending on calcium ion
concentrations, for example, amino acids having metal-chelating
activity may also be preferably used. Examples of such
metal-chelating amino acids include, for example, serine (Ser(S)),
threonine (Thr(T)), asparagine (Asn(N)), glutamine (Gln(Q)),
aspartic acid (Asp(D)), and glutamic acid (Glu(E)).
[0187] Positions in the antigen-binding domains at which the
above-described amino acids are contained are not particularly
limited to particular positions, and may be any positions within
the heavy chain variable region or light chain variable region that
forms an antigen-binding domain, as long as they alter the
antigen-binding activity of antigen-binding molecules depending on
calcium ion concentrations. Specifically, antigen-binding domains
of the present invention can be obtained from a library mainly
composed of antigen-binding molecules whose sequences are different
from one another and whose heavy chain antigen-binding domains
contain amino acids that alter the antigen-binding activity of the
antigen-binding molecules depending on calcium ion concentrations.
In another embodiment, antigen-binding domains of the present
invention can be obtained from a library mainly composed of
antigen-binding molecules whose sequences are different from one
another and whose heavy chain CDR3 domains contain the
above-mentioned amino acids. In still another embodiment,
antigen-binding domains of the present invention can be obtained
from a library mainly composed of antigen-binding molecules whose
sequences are different from one another and whose heavy chain CDR3
domains contain the above-mentioned amino acids at positions 95,
96, 100a, and/or 101 as indicated according to the Kabat numbering
system.
[0188] Meanwhile, in an embodiment of the present invention,
antigen-binding domains of the present invention can be obtained
from a library mainly composed of antigen-binding molecules whose
sequences are different from one another and whose light chain
antigen-binding domains contain amino acids that alter the
antigen-binding activity of antigen-binding molecules depending on
calcium ion concentrations. In another embodiment, antigen-binding
domains of the present invention can be obtained from a library
mainly composed of antigen-binding molecules whose sequences are
different from one another and whose light chain CDR1 domains
contain the above-mentioned amino acids. In still another
embodiment, antigen-binding domains of the present invention can be
obtained from a library mainly composed of antigen-binding
molecules whose sequences are different from one another and whose
light chain CDR1 domains contain the above-mentioned amino acids at
positions 30, 31, and/or 32 as indicated according to the Kabat
numbering system.
[0189] In another embodiment, antigen-binding domains of the
present invention can be obtained from a library mainly composed of
antigen-binding molecules whose sequences are different from one
another and whose light chain CDR2 domains contain the
above-mentioned amino acid residues. In yet another embodiment, the
present invention provides libraries mainly composed of
antigen-binding molecules whose sequences are different from one
another and whose light chain CDR2 domains contain the
above-mentioned amino acid residues at position 50 as indicated
according to the Kabat numbering system.
[0190] In still another embodiment of the present invention,
antigen-binding domains of the present invention can be obtained
from a library mainly composed of antigen-binding molecules whose
sequences are different from one another and whose light chain CDR3
domains contain the above-mentioned amino acid residues. In an
alternative embodiment, antigen-binding domains of the present
invention can be obtained from a library mainly composed of
antigen-binding molecules whose sequences are different from one
another and whose light chain CDR3 domains contain the
above-mentioned amino acid residues at position 92 as indicated
according to the Kabat numbering system.
[0191] Furthermore, in a different embodiment of the present
invention, antigen-binding domains of the present invention can be
obtained from a library mainly composed of antigen-binding
molecules whose sequences are different from one another and in
which two or three CDRs selected from the above-described light
chain CDR1, CDR2, and CDR3 contain the aforementioned amino acid
residues. Moreover, antigen-binding domains of the present
invention can be obtained from a library mainly composed of
antigen-binding molecules whose sequences are different from one
another and whose light chains contain the aforementioned amino
acid residues at any one or more of positions 30, 31, 32, 50,
and/or 92 as indicated according to the Kabat numbering system.
[0192] In a particularly preferred embodiment, the framework
sequences of the light chain and/or heavy chain variable region of
an antigen-binding molecule preferably contain human germ line
framework sequences. Thus, in an embodiment of the present
invention, when the framework sequences are completely human
sequences, it is expected that when such an antigen-binding
molecule of the present invention is administered to humans (for
example, to treat diseases), it induces little or no immunogenic
response. In the above sense, the phrase "containing a germ line
sequence" in the present invention means that a part of the
framework sequences of the present invention is identical to a part
of any human germ line framework sequences. For example, when the
heavy chain FR2 sequence of an antigen-binding molecule of the
present invention is a combination of heavy chain FR2 sequences of
different human germ line framework sequences, such a molecule is
also an antigen-binding molecule of the present invention
"containing a germ line sequence".
[0193] Preferred examples of the frameworks include, for example,
fully human framework region sequences currently known, which are
included in the website of V-Base (http://vbase.mrc-cpe.cam.ac.uk/)
or others. Those framework region sequences can be appropriately
used as a germ line sequence contained in an antigen-binding
molecule of the present invention. The germ line sequences may be
categorized according to their similarity (Tomlinson et al. (J.
Mol. Biol. (1992) 227, 776-798); Williams and Winter (Eur. J.
Immunol. (1993) 23, 1456-1461); Cox et al. (Nat. Genetics (1994) 7,
162-168)). Appropriate germ line sequences can be selected from VK,
which is grouped into seven subgroups; V.lamda., which is grouped
into ten subgroups; and VH, which is grouped into seven
subgroups.
[0194] Fully human VH sequences preferably include, but are not
limited to, for example, VH sequences of:
subgroup VH1 (for example, VH1-2, VH1-3, VH1-8, VH1-18, VH1-24,
VH1-45, VH1-46, VH1-58, and VH1-69); subgroup VH2 (for example,
VH2-5, VH2-26, and VH2-70); subgroup VH3 (VH3-7, VH3-9, VH3-11,
VH3-13, VH3-15, VH3-16, VH3-20, VH3-21, VH3-23, VH3-30, VH3-33,
VH3-35, VH3-38, VH3-43, VH3-48, VH3-49, VH3-53, VH3-64, VH3-66,
VH3-72, VH3-73, and VH3-74); subgroup VH4 (VH4-4, VH4-28, VH4-31,
VH4-34, VH4-39, VH4-59, and VH4-61); subgroup VH5 (VH5-51);
subgroup VH6 (VH6-1); and subgroup VH7 (VH7-4 and VH7-81). These
are also described in known documents (Matsuda et al. (J. Exp. Med.
(1998) 188, 1973-1975)) and such, and thus persons skilled in the
art can appropriately design antigen-binding molecules of the
present invention based on the information of these sequences. It
is also preferable to use other fully human frameworks or framework
sub-regions.
[0195] Fully human V.kappa. sequences preferably include, but are
not limited to, for example:
A20, A30, L1, L4, L5, L8, L9, L11, L12, L14, L15, L18, L19, L22,
L23, L24, O2, O4, O8, O12, O14, and O18 grouped into subgroup Vk1;
A1, A2, A3, A5, A7, A17, A18, A19, A23, O1, and O11, grouped into
subgroup Vk2; A11, A27, L2, L6, L10, L16, L20, and L25, grouped
into subgroup Vk3; B3, grouped into subgroup Vk4; B2 (herein also
referred to as Vk5-2), grouped into subgroup Vk5; and A10, A14, and
A26, grouped into subgroup Vk6 (Kawasaki et al. (Eur. J. Immunol.
(2001) 31, 1017-1028); Schable and Zachau (Biol. Chem.
[0196] Hoppe Seyler (1993) 374, 1001-1022); Brensing-Kuppers et al.
(Gene (1997) 191, 173-181)).
[0197] Fully human V.lamda., sequences preferably include, but are
not limited to, for example:
V1-2, V1-3, V1-4, V1-5, V1-7, V1-9, V1-11, V1-13, V1-16, V1-17,
V1-18, V1-19, V1-20, and V1-22, grouped into subgroup VL1; V2-1,
V2-6, V2-7, V2-8, V2-11, V2-13, V2-14, V2-15, V2-17, and V2-19,
grouped into subgroup VL1; V3-2, V3-3, and V3-4, grouped into
subgroup VL3; V4-1, V4-2, V4-3, V4-4, and V4-6, grouped into
subgroup VL4; and V5-1, V5-2, V5-4, and V5-6, grouped into subgroup
VL5 (Kawasaki et al. (Genome Res. (1997) 7, 250-261)).
[0198] Normally, these framework sequences are different from one
another at one or more amino acid residues. These framework
sequences can be used in combination with "at least one amino acid
residue that alters the antigen-binding activity of an
antigen-binding molecule depending on ion concentrations" of the
present invention. Other examples of the fully human frameworks
used in combination with "at least one amino acid residue that
alters the antigen-binding activity of an antigen-binding molecule
depending on ion concentrations" of the present invention include,
but are not limited to, for example, KOL, NEWM, REI, EU, TUR, TEI,
LAY, and POM (for example, Kabat et al. (1991) supra; Wu et al. (J.
Exp. Med. (1970) 132, 211-250)).
[0199] Without being bound by a particular theory, one reason for
the expectation that the use of germ line sequences precludes
adverse immune responses in most individuals is believed to be as
follows. As a result of the process of affinity maturation during
normal immune responses, somatic mutation occurs frequently in the
variable regions of immunoglobulin. Such mutations mostly occur
around CDRs whose sequences are hypervariable, but also affect
residues of framework regions. Such framework mutations do not
exist on the germ line genes, and also they are less likely to be
immunogenic in patients. On the other hand, the normal human
population is exposed to most of the framework sequences expressed
from the germ line genes. As a result of immunotolerance, these
germ line frameworks are expected to have low or no immunogenicity
in patients. To maximize the possibility of immunotolerance,
variable region-encoding genes may be selected from a group of
commonly occurring functional germ line genes.
[0200] Known methods such as site-directed mutagenesis (Kunkel et
al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and overlap
extension PCR can be appropriately employed to produce the
antigen-binding molecules of the present invention in which the
above-described variable region sequences, heavy or light chain
variable region sequences, CDR sequences, or framework sequences
contain amino acids that alter the antigen-binding activity of the
antigen-binding molecules depending on calcium ion
concentrations.
[0201] For example, a library which contains a plurality of
antigen-binding molecules of the present invention whose sequences
are different from one another can be constructed by combining
heavy chain variable regions prepared as a randomized variable
region sequence library with a light chain variable region selected
as a framework sequence originally containing at least one amino
acid residue that alters the antigen-binding activity of the
antigen-binding molecule depending on calcium ion concentrations.
As a non-limiting example, when the ion concentration is calcium
ion concentration, such preferred libraries include, for example,
those constructed by combining the light chain variable region
sequence of SEQ ID NO: 6 (Vk5-2) and the heavy chain variable
region produced as a randomized variable region sequence
library.
[0202] Alternatively, a light chain variable region sequence
selected as a framework region originally containing at least one
amino acid residue that alters the antigen-binding activity of an
antigen-binding molecule as mentioned above can be design to
contain various amino acid residues other than the above amino acid
residues. Herein, such residues are referred to as flexible
residues. The number and position of flexible residues are not
particularly limited as long as the antigen-binding activity of the
antigen-binding molecule of the present invention varies depending
on ion concentrations. Specifically, the CDR sequences and/or FR
sequences of the heavy chain and/or light chain may contain one or
more flexible residues. For example, when the ion concentration is
calcium ion concentration, non-limiting examples of flexible
residues to be introduced into the light chain variable region
sequence of SEQ ID NO: 6 (Vk5-2) include the amino acid residues
listed in Tables 1 or 2.
TABLE-US-00001 TABLE 1 CDR Position 70% of the total CDR1 28 S;
100% 29 I; 100% 30 E; 72% H; 14% S; 14% 31 D; 100% 32 D; 100% 33 L;
100% 34 A; 70% N; 30% CDR2 50 E; 100% 51 A; 100% 52 S; 100% 53 H;
5% N; 25% S; 45% T; 25% 54 L; 100% 55 Q; 100% 56 S; 100% CDR3 90 Q;
100% 91 H; 25% S; 15% R; 15% Y; 45% 92 D; 80% N; 10% S; 10% 93 D;
5% G; 10% N; 25% S; 50% R; 10% 94 S; 50% Y; 50% 95 P; 100% 96 L;
50% Y; 50% (Position indicates Kabat numbering)
TABLE-US-00002 TABLE 2 Kabat CDR numbering 30% amino acids of the
total CDR1 28 S: 100% 29 I: 100% 30 E: 83% S: 17% 31 D: 100% 32 D:
100% 33 L: 100% 34 A: 70% N: 30% CDR2 50 H: 100% 51 A: 100% 52 S:
100% 53 H: 5% N: 25% S: 45% T: 25% 54 L: 100% 55 Q: 100% 56 S: 100%
CDR3 90 Q: 100% 91 H: 25% S: 15% R: 15% Y: 45% 92 D: 80% N: 10% S:
10% 93 D: 5% G: 10% N: 25% S: 50% R: 10% 94 S: 50% Y: 50% 95 P:
100% 96 L: 50% Y: 50% (Position indicates Kabat numbering)
[0203] Herein, flexible residues refer to amino acid residue
variations present at hypervariable positions at which several
different amino acids are present on the light chain and heavy
chain variable regions when the amino acid sequences of known
and/or native antibodies or antigen-binding domains are compared.
Hypervariable positions are generally located in the CDR regions.
In an embodiment, the data provided by Kabat, Sequences of Proteins
of Immunological Interest (National Institute of Health Bethesda
Md.) (1987 and 1991) is useful to determine hypervariable positions
in known and/or native antibodies. Furthermore, databases on the
Internet (http://vbase.mrc-cpe.cam.ac.uk/,
http://www.bioinf.org.uk/abs/index.html) provide the collected
sequences of many human light chains and heavy chains and their
locations. The information on the sequences and locations is useful
to determine hypervariable positions in the present invention.
According to the present invention, when a certain amino acid
position has preferably about 2 to about 20 possible amino acid
residue variations, preferably about 3 to about 19, preferably
about 4 to about 18, preferably 5 to 17, preferably 6 to 16,
preferably 7 to 15, preferably 8 to 14, preferably 9 to 13, and
preferably 10 to 12 possible amino acid residue variations, the
position is hypervariable. In some embodiments, a certain amino
acid position may have preferably at least about 2, preferably at
least about 4, preferably at least about 6, preferably at least
about 8, preferably about 10, and preferably about 12 amino acid
residue variations.
[0204] Alternatively, a library containing a plurality of
antigen-binding molecules of the present invention whose sequences
are different from one another can be constructed by combining
heavy chain variable regions produced as a randomized variable
region sequence library with light chain variable regions into
which at least one amino acid residue that alters the
antigen-binding activity of antigen-binding molecules depending on
ion concentrations as mentioned above is introduced. When the ion
concentration is calcium ion concentration, non-limiting examples
of such libraries preferably include, for example, libraries in
which heavy chain variable regions produced as a randomized
variable region sequence library are combined with light chain
variable region sequences in which a particular residue(s) in a
germ line sequence such as SEQ ID NO: 7 (Vk1), SEQ ID NO: 8 (Vk2),
SEQ ID NO: 9 (Vk3), or SEQ ID NO: 10 (Vk4) has been substituted
with at least one amino acid residue that alters the
antigen-binding activity of an antigen-binding molecule depending
on calcium ion concentrations. Non-limiting examples of such amino
acid residues include amino acid residues in light chain CDR1.
Furthermore, non-limiting examples of such amino acid residues
include amino acid residues in light chain CDR2. In addition,
non-limiting examples of such amino acid residues also include
amino acid residues in light chain CDR3.
[0205] Non-limiting examples of such amino acid residues contained
in light chain CDR1 include those at positions 30, 31, and/or 32 in
the CDR1 of light chain variable region as indicated by EU
numbering. Furthermore, non-limiting examples of such amino acid
residues contained in light chain CDR2 include an amino acid
residue at position 50 in the CDR2 of light chain variable region
as indicated by Kabat numbering. Moreover, non-limiting examples of
such amino acid residues contained in light chain CDR3 include an
amino acid residue at position 92 in the CDR3 of light chain
variable region as indicated by Kabat numbering. These amino acid
residues can be contained alone or in combination as long as they
form a calcium-binding motif and/or as long as the antigen-binding
activity of an antigen-binding molecule varies depending on calcium
ion concentrations. Meanwhile, as troponin C, calmodulin,
parvalbumin, and myosin light chain, which have several calcium
ion-binding sites and are believed to be derived from a common
origin in terms of molecular evolution, are known, the light chain
CDR1, CDR2, and/or CDR3 can be designed to have their binding
motifs. For example, it is possible to use cadherin domains, EF
hand of calmodulin, C2 domain of Protein kinase C, Gla domain of
blood coagulation protein FactorlX, C type lectins of
acyaroglycoprotein receptor and mannose-binding receptor, A domains
of LDL receptors, annexin, thrombospondin type 3 domain, and
EGF-like domains in an appropriate manner for the above
purposes.
[0206] When heavy chain variable regions produced as a randomized
variable region sequence library and light chain variable regions
into which at least one amino acid residue that alters the
antigen-binding activity of an antigen-binding molecule depending
on ion concentrations has been introduced are combined as described
above, the sequences of the light chain variable regions can be
designed to contain flexible residues in the same manner as
described above. The number and position of such flexible residues
are not particularly limited to particular embodiments as long as
the antigen-binding activity of antigen-binding molecules of the
present invention varies depending on ion concentrations.
Specifically, the CDR sequences and/or FR sequences of heavy chain
and/or light chain can contain one or more flexible residues. When
the ion concentration is calcium ion concentration, non-limiting
examples of flexible residues to be introduced into the sequence of
light chain variable region include the amino acid residues listed
in Tables 1 and 2.
[0207] The preferred heavy chain variable regions to be combined
include, for example, randomized variable region libraries. Known
methods are combined as appropriate to produce a randomized
variable region library. In a non-limiting embodiment of the
present invention, an immune library constructed based on antibody
genes derived from lymphocytes of animals immunized with a specific
antigen, patients with infections, persons with an elevated
antibody titer in blood as a result of vaccination, cancer
patients, or auto immune disease patients, may be preferably used
as a randomized variable region library.
[0208] In another non-limiting embodiment of the present invention,
a synthetic library produced by replacing the CDR sequences of V
genes in genomic DNA or functional reshaped V genes with a set of
synthetic oligonucleotides containing sequences encoding codon sets
of an appropriate length can also be preferably used as a
randomized variable region library. In this case, since sequence
diversity is observed in the heavy chain CDR3 sequence, it is also
possible to replace the CDR3 sequence only. A criterion of giving
rise to diversity in amino acids in the variable region of an
antigen-binding molecule is that diversity is given to amino acid
residues at surface-exposed positions in the antigen-binding
molecule. The surface-exposed position refers to a position that is
considered to be able to be exposed on the surface and/or contacted
with an antigen, based on structure, ensemble of structures, and/or
modeled structure of an antigen-binding molecule. In general, such
positions are CDRs. Preferably, surface-exposed positions are
determined using coordinates from a three-dimensional model of an
antigen-binding molecule using a computer program such as the
InsightII program (Accelrys). Surface-exposed positions can be
determined using algorithms known in the art (for example, Lee and
Richards (J. Mol. Biol. (1971) 55, 379-400); Connolly (J. Appl.
Cryst. (1983) 16, 548-558)). Determination of surface-exposed
positions can be performed using software suitable for protein
modeling and three-dimensional structural information obtained from
an antibody. Software that can be used for these purposes
preferably includes SYBYL Biopolymer Module software (Tripos
Associates). Generally or preferably, when an algorithm requires a
user input size parameter, the "size" of a probe which is used in
the calculation is set at about 1.4 Angstrom or smaller in radius.
Furthermore, methods for determining surface-exposed regions and
areas using software for personal computers are described by Pacios
(Comput. Chem. (1994) 18 (4), 377-386; J. Mol. Model. (1995) 1,
46-53).
[0209] In another non-limiting embodiment of the present invention,
a naive library, which is constructed from antibody genes derived
from lymphocytes of healthy persons and whose repertoire consists
of naive sequences, which are antibody sequences with no bias, can
also be particularly preferably used as a randomized variable
region library (Gejima et al. (Human Antibodies (2002) 11,
121-129); Cardoso et al. (Scand. J. Immunol. (2000) 51, 337-344)).
Herein, an amino acid sequence comprising a naive sequence refers
to an amino acid sequence obtained from such a naive library.
[0210] In one embodiment of the present invention, an
antigen-binding domain of the present invention can be obtained
from a library containing a plurality of antigen-binding molecules
of the present invention whose sequences are different from one
another, prepared by combining light chain variable regions
constructed as a randomized variable region sequence library with a
heavy chain variable region selected as a framework sequence that
originally contains "at least one amino acid residue that alters
the antigen-binding activity of an antigen-binding molecule
depending on ion concentrations". When the ion concentration is
calcium ion concentration, non-limiting examples of such libraries
preferably include those constructed by combining light chain
variable regions constructed as a randomized variable region
sequence library with the sequence of heavy chain variable region
of SEQ ID NO: 11 (6RL#9-IgG1) or SEQ ID NO: 12 (6KC4-1#85-IgG1).
Alternatively, such a library can be constructed by selecting
appropriate light chain variable regions from those having germ
line sequences, instead of light chain variable regions constructed
as a randomized variable region sequence library. Such preferred
libraries include, for example, those in which the sequence of
heavy chain variable region of SEQ ID NO: 11 (6RL#9-IgG1) or SEQ ID
NO: 12 (6KC4-1#85-IgG1) is combined with light chain variable
regions having germ line sequences.
[0211] Alternatively, the sequence of a heavy chain variable region
selected as a framework sequence that originally contains "at least
one amino acid residue that alters the antigen-binding activity of
an antigen-binding molecule" as mentioned above can be designed to
contain flexible residues. The number and position of the flexible
residues are not particularly limited as long as the
antigen-binding activity of an antigen-binding molecule of the
present invention varies depending on ion concentrations.
Specifically, the CDR and/or FR sequences of heavy chain and/or
light chain can contain one or more flexible residues. When the ion
concentration is calcium ion concentration, non-limiting examples
of flexible residues to be introduced into the sequence of heavy
chain variable region of SEQ ID NO: 11 (6RL#9-IgG1) include all
amino acid residues of heavy chain CDR1 and CDR2 and the amino acid
residues of the heavy chain CDR3 except those at positions 95, 96,
and/or 100a. Alternatively, non-limiting examples of flexible
residues to be introduced into the sequence of heavy chain variable
region of SEQ ID NO: 12 (6KC4-1#85-IgG1) include all amino acid
residues of heavy chain CDR1 and CDR2 and the amino acid residues
of the heavy chain CDR3 except those at amino acid positions 95
and/or 101.
[0212] Alternatively, a library containing a plurality of
antigen-binding molecules whose sequences are different from one
another can be constructed by combining light chain variable
regions constructed as a randomized variable region sequence
library or light chain variable regions having germ line sequences
with heavy chain variable regions into which "at least one amino
acid residue responsible for the ion concentration-dependent change
in the antigen-binding activity of an antigen-binding molecule" has
been introduced as mentioned above. When the ion concentration is
calcium ion concentration, non-limiting examples of such libraries
preferably include those in which light chain variable regions
constructed as a randomized variable region sequence library or
light chain variable regions having germ line sequences are
combined with the sequence of a heavy chain variable region in
which a particular residue(s) has been substituted with at least
one amino acid residue that alters the antigen-binding activity of
an antigen-binding molecule depending on calcium ion
concentrations. Non-limiting examples of such amino acid residues
include amino acid residues of the heavy chain CDR1. Further
non-limiting examples of such amino acid residues include amino
acid residues of the heavy chain CDR2. In addition, non-limiting
examples of such amino acid residues also include amino acid
residues of the heavy chain CDR3. Non-limiting examples of such
amino acid residues of heavy chain CDR3 include the amino acids of
positions 95, 96, 100a, and/or 101 in the CDR3 of heavy chain
variable region as indicated by the Kabat numbering. Furthermore,
these amino acid residues can be contained alone or in combination
as long as they form a calcium-binding motif and/or the
antigen-binding activity of an antigen-binding molecule varies
depending on calcium ion concentrations.
[0213] When light chain variable regions constructed as a
randomized variable region sequence library or light chain variable
regions having germ line sequence are combined with a heavy chain
variable region into which at least one amino acid residue that
alter the antigen-binding activity of an antigen-binding molecule
depending on ion concentrations as mentioned above has been
introduced, the sequence of the heavy chain variable region can
also be designed to contain flexible residues in the same manner as
described above. The number and position of flexible residues are
not particularly limited as long as the antigen-binding activity of
an antigen-binding molecule of the present invention varies
depending on ion concentrations. Specifically, the heavy chain CDR
and/or FR sequences may contain one or more flexible residues.
Furthermore, randomized variable region libraries can be preferably
used as amino acid sequences of CDR1, CDR2, and/or CDR3 of the
heavy chain variable region other than the amino acid residues that
alter the antigen-binding activity of an antigen-binding molecule.
When germ line sequences are used as light chain variable regions,
non-limiting examples of such sequences include those of SEQ ID NO:
7 (Vk1), SEQ ID NO: 8 (Vk2), SEQ ID NO: 9 (Vk3), and SEQ ID NO: 10
(Vk4).
[0214] Any of the above-described amino acids that alter the
antigen-binding activity of an antigen-binding molecule depending
on calcium ion concentrations can be preferably used, as long as
they form a calcium-binding motif. Specifically, such amino acids
include electron-donating amino acids. Preferred examples of such
electron-donating amino acids include serine, threonine,
asparagine, glutamic acid, aspartic acid, and glutamic acid.
Condition of Proton Concentrations
[0215] In an embodiment of the present invention, the condition of
ion concentrations refers to the condition of proton concentrations
or pH condition. In the present invention, the concentration of
proton, i.e., the nucleus of hydrogen atom, is treated as
synonymous with hydrogen index (pH). When the activity of proton in
an aqueous solution is represented as aH+, pH is defined as -log
10aH+. When the ionic strength of the aqueous solution is low (for
example, lower than 10.sup.-3), aH+ is nearly equal to the proton
strength. For example, the ionic product of water at 25.degree. C.
and 1 atmosphere is Kw=aH+aOH=10.sup.-14, and therefore in pure
water, aH+=aOH=10.sup.-7. In this case, pH=7 is neutral; an aqueous
solution whose pH is lower than 7 is acidic or whose pH is greater
than 7 is alkaline.
[0216] In the present invention, when pH condition is used as the
ion concentration condition, pH conditions include high proton
concentrations or low pHs, i.e., an acidic pH range, and low proton
concentrations or high pHs, i.e., a neutral pH range. "The binding
activity varies depending on pH condition" means that the
antigen-binding activity of an antigen-binding molecule varies due
to the difference in conditions of a high proton concentration or
low pH (an acidic pH range) and a low proton concentration or high
pH (a neutral pH range). This includes, for example, the case where
the antigen-binding activity of an antigen-binding molecule is
higher in a neutral pH range than in an acidic pH range and the
case where the antigen-binding activity of an antigen-binding
molecule is higher in an acidic pH range than in a neutral pH
range.
[0217] In the present specification, neutral pH range is not
limited to a specific value and is preferably selected from between
pH6.7 and pH10.0. In another embodiment, the pH can be selected
from between pH6.7 and pH 9.5. In still another embodiment, the pH
can be selected from between pH7.0 and pH9.0. In yet another
embodiment, the pH can be selected from between pH7.0 and pH8.0. In
particular, the preferred pH includes pH 7.4, which is close to the
pH of plasma (blood) in vivo.
[0218] In the present specification, an acidic pH range is not
limited to a specific value and is preferably selected from between
pH4.0 and pH6.5. In another embodiment, the pH can be selected from
between pH4.5 and pH6.5. In still another embodiment, the pH can be
selected from between pH5.0 and pH6.5. In yet another embodiment,
the pH can be selected from between pH5.5 and pH6.5. In particular,
the preferred pH includes pH 5.8, which is close to the ionized
calcium concentration in the early endosome in vivo.
[0219] In the present invention, "the antigen-binding activity of
an antigen-binding molecule at a high proton concentration or low
pH (an acidic pH range) is lower than that at a low proton
concentration or high pH (a neutral pH range)" means that the
antigen-binding activity of an antigen-binding molecule at a pH
selected from between pH4.0 and pH6.5 is weaker than that at a pH
selected from between pH6.7 and pH10.0; preferably means that the
antigen-binding activity of an antigen-binding molecule at a pH
selected from between pH4.5 and pH6.5 is weaker than that at a pH
selected from between pH6.7 and pH9.5; more preferably, means that
the antigen-binding activity of an antigen-binding molecule at a pH
selected from between pH5.0 and pH6.5 is weaker than that at a pH
selected from between pH7.0 and pH9.0; still more preferably means
that the antigen-binding activity of an antigen-binding molecule at
a pH selected from between pH5.5 and pH6.5 is weaker than that at a
pH selected from between pH7.0 and pH8.0; particularly preferably
means that the antigen-binding activity at the pH in the early
endosome in vivo is weaker than the antigen-binding activity at the
pH of plasma in vivo; and specifically means that the
antigen-binding activity of an antigen-binding molecule at pH5.8 is
weaker than the antigen-binding activity at pH 7.4.
[0220] Whether the antigen-binding activity of an antigen-binding
molecule has changed by the pH condition can be determined, for
example, by the use of known measurement methods such as those
described in the section "Binding Activity" above. Specifically,
the binding activity is measured under different pH conditions
using the measurement methods described above. For example, the
antigen-binding activity of an antigen-binding molecule is compared
under the conditions of acidic pH range and neutral pH range to
confirm that the antigen-binding activity of the antigen-binding
molecule changes to be higher under the condition of neutral pH
range than that under the condition of acidic pH range.
[0221] Furthermore, in the present invention, the expression "the
antigen-binding activity at a high proton concentration or low pH,
i.e., in an acidic pH range, is lower than that at a low proton
concentration or high pH, i.e., in a neutral pH range" can also be
expressed as "the antigen-binding activity of an antigen-binding
molecule at a low proton concentration or high pH, i.e., in a
neutral pH range, is higher than that at a high proton
concentration or low pH, i.e., in an acidic pH range". In the
present invention, "the antigen-binding activity at a high proton
concentration or low pH, i.e., in an acidic pH range, is lower than
that at a low proton concentration or high pH, i.e., in a neutral
pH range" may be described as "the antigen-binding activity at a
high proton concentration or low pH, i.e., in an acidic pH range,
is weaker than the antigen-binding ability at a low proton
concentration or high pH, i.e., in a neutral pH range".
Alternatively, "the antigen-binding activity at a high proton
concentration or low pH, i.e., in an acidic pH range, is reduced to
be lower than that at a low proton concentration or high pH, i.e.,
in a neutral pH range" may be described as "the antigen-binding
activity at a high proton concentration or low pH, i.e., in an
acidic pH range, is reduced to be weaker than the antigen-binding
ability at a low proton concentration or high pH, i.e., in a
neutral pH range".
[0222] The conditions other than proton concentration or pH for
measuring the antigen-binding activity may be suitably selected by
those skilled in the art and are not particularly limited.
Measurements can be carried out, for example, at 37.degree. C.
using HEPES buffer. Measurements can be carried out, for example,
using Biacore (GE Healthcare). When the antigen is a soluble
antigen, the antigen-binding activity of an antigen-binding
molecule can be determined by assessing the binding activity to the
soluble antigen by pouring the antigen as an analyte into a chip
immobilized with the antigen-binding molecule. When the antigen is
a membrane antigen, the binding activity to the membrane antigen
can be assessed by pouring the antigen-binding molecule as an
analyte into a chip immobilized with the antigen.
[0223] As long as the antigen-binding activity of an
antigen-binding molecule of the present invention at a high proton
concentration or low pH, i.e., in an acidic pH range is weaker than
that at a low proton concentration or high pH, i.e., in a neutral
pH range, the ratio of the antigen-binding activity between that at
a high proton concentration or low pH, i.e., an acidic pH range,
and at a low proton concentration or high pH, i.e., a neutral pH
range is not particularly limited, and the value of KD (pH5.8)/KD
(pH7.4), which is the ratio of the dissociation constant (KD) for
an antigen at a high proton concentration or low pH, i.e., in an
acidic pH range to the KD at a low proton concentration or high pH,
i.e., in a neutral pH range, is preferably 2 or more; more
preferably the value of KD (pH5.8)/KD (pH7.4) is 10 or more; and
still more preferably the value of KD (pH5.8)/KD (pH7.4) is 40 or
more. The upper limit of KD (pH5.8)/KD (pH7.4) value is not
particularly limited, and may be any value such as 400, 1000, or
10000, as long as the molecule can be produced by the techniques of
those skilled in the art.
[0224] When the antigen is a soluble antigen, the dissociation
constant (KD) can be used as the value for antigen-binding
activity. Meanwhile, when the antigen is a membrane antigen, the
apparent dissociation constant (KD) can be used. The dissociation
constant (KD) and apparent dissociation constant (KD) can be
measured by methods known to those skilled in the art, and Biacore
(GE healthcare), Scatchard plot, flow cytometer, and such can be
used.
[0225] Alternatively, for example, the dissociation rate constant
(kd) can be suitably used as an index for indicating the ratio of
the antigen-binding activity of an antigen-binding molecule of the
present invention between that at a high proton concentration or
low pH, i.e., an acidic pH range and a low proton concentration or
high pH, i.e., a neutral pH range. When kd (dissociation rate
constant) is used as an index for indicating the binding activity
ratio instead of KD (dissociation constant), the value of kd (in an
acidic pH range)/kd (in a neutral pH range), which is the ratio of
kd (dissociation rate constant) for the antigen at a high proton
concentration or low pH, i.e., in an acidic pH range to kd
(dissociation rate constant) at a low proton concentration or high
pH, i.e., in a neutral pH range, is preferably 2 or more, more
preferably 5 or more, still more preferably 10 or more, and yet
more preferably 30 or more. The upper limit of kd (in an acidic pH
range)/kd (in a neutral pH range) value is not particularly
limited, and may be any value such as 50, 100, or 200, as long as
the molecule can be produced by the techniques of those skilled in
the art.
[0226] When the antigen is a soluble antigen, the dissociation rate
constant (kd) can be used as the value for antigen-binding activity
and when the antigen is a membrane antigen, the apparent
dissociation rate constant (kd) can be used. The dissociation rate
constant (kd) and apparent dissociation rate constant (kd) can be
determined by methods known to those skilled in the art, and
Biacore (GE healthcare), flow cytometer, and such may be used. In
the present invention, when the antigen-binding activity of an
antigen-binding molecule is measured at different proton
concentrations, i.e., pHs, conditions other than the proton
concentration, i.e., pH, are preferably the same.
[0227] For example, an antigen-binding domain or antigen-binding
molecule whose antigen-binding activity at a high proton
concentration or low pH, i.e., in an acidic pH range is lower than
that at a low proton concentration or high pH, i.e., in a neutral
pH range, which is one embodiment provided by the present
invention, can be obtained via screening of antigen-binding domains
or antigen-binding molecules, comprising the following steps (a) to
(c):
(a) obtaining the antigen-binding activity of an antigen-binding
domain or antigen-binding molecule in an acidic pH range; (b)
obtaining the antigen-binding activity of an antigen-binding domain
or antigen-binding molecule in a neutral pH range; and (c)
selecting an antigen-binding domain or antigen-binding molecule
whose antigen-binding activity in the acidic pH range is lower than
that in the neutral pH range.
[0228] Alternatively, an antigen-binding domain or antigen-binding
molecule whose antigen-binding activity at a high proton
concentration or low pH, i.e., in an acidic pH range, is lower than
that at a low proton concentration or high pH, i.e., in a neutral
pH range, which is one embodiment provided by the present
invention, can be obtained via screening of antigen-binding domains
or antigen-binding molecules, or a library thereof, comprising the
following steps (a) to (c):
(a) contacting an antigen-binding domain or antigen-binding
molecule, or a library thereof, in a neutral pH range with an
antigen; (b) placing in an acidic pH range the antigen-binding
domain or antigen-binding molecule bound to the antigen in step
(a); and (c) isolating the antigen-binding domain or
antigen-binding molecule dissociated in step (b).
[0229] An antigen-binding domain or antigen-binding molecule whose
antigen-binding activity at a high proton concentration or low pH,
i.e., in an acidic pH range is lower than that at a low proton
concentration or high pH, i.e., in a neutral pH range, which is
another embodiment provided by the present invention, can be
obtained via screening of antigen-binding domains or
antigen-binding molecules, or a library thereof, comprising the
following steps (a) to (d):
(a) contacting in an acidic pH range an antigen with a library of
antigen-binding domains or antigen-binding molecules; (b) selecting
the antigen-binding domain or antigen-binding molecule which does
not bind to the antigen in step (a); (c) allowing the
antigen-binding domain or antigen-binding molecule selected in step
(b) to bind with the antigen in a neutral pH range; and (d)
isolating the antigen-binding domain or antigen-binding molecule
bound to the antigen in step (c).
[0230] An antigen-binding domain or antigen-binding molecule whose
antigen-binding activity at a high proton concentration or low pH,
i.e., in an acidic pH range, is lower than that at a low proton
concentration or high pH, i.e., in a neutral pH range, which is
even another embodiment provided by the present invention, can be
obtained by a screening method comprising the following steps (a)
to (c):
(a) contacting in a neutral pH range a library of antigen-binding
domains or antigen-binding molecules with a column immobilized with
an antigen; (b) eluting in an acidic pH range from the column the
antigen-binding domain or antigen-binding molecule bound to the
column in step (a); and (c) isolating the antigen-binding domain or
antigen-binding molecule eluted in step (b).
[0231] An antigen-binding domain or antigen-binding molecule whose
antigen-binding activity at a high proton concentration or low pH,
i.e., in an acidic pH, range is lower than that at a low proton
concentration or high pH, i.e., in a neutral pH range, which is
still another embodiment provided by the present invention, can be
obtained by a screening method comprising the following steps (a)
to (d):
(a) allowing, in an acidic pH range, a library of antigen-binding
domains or antigen-binding molecules to pass a column immobilized
with an antigen; (b) collecting the antigen-binding domain or
antigen-binding molecule eluted without binding to the column in
step (a); (c) allowing the antigen-binding domain or
antigen-binding molecule collected in step (b) to bind with the
antigen in a neutral pH range; and (d) isolating the
antigen-binding domain or antigen-binding molecule bound to the
antigen in step (c).
[0232] An antigen-binding domain or antigen-binding molecule whose
antigen-binding activity at a high proton concentration or low pH,
i.e., in an acidic pH range, is lower than that at a low proton
concentration or high pH, i.e., in a neutral pH range, which is yet
another embodiment provided by the present invention, can be
obtained by a screening method comprising the following steps (a)
to (d):
(a) contacting an antigen with a library of antigen-binding domains
or antigen-binding molecules in a neutral pH range; (b) obtaining
the antigen-binding domain or antigen-binding molecule bound to the
antigen in step (a); (c) placing in an acidic pH range the
antigen-binding domain or antigen-binding molecule obtained in step
(b); and (d) isolating the antigen-binding domain or
antigen-binding molecule whose antigen-binding activity in step (c)
is weaker than the standard selected in step (b).
[0233] The above-described steps may be repeated twice or more
times. Thus, the present invention provides antigen-binding domains
and antigen-binding molecules whose antigen-binding activity in an
acidic pH range is lower than that in a neutral pH range, which are
obtained by a screening method that further comprises the steps of
repeating twice or more times steps (a) to (c) or (a) to (d) in the
above-described screening methods. The number of times that steps
(a) to (c) or (a) to (d) is repeated is not particularly limited;
however, the number is 10 or less in general.
[0234] In the screening methods of the present invention, the
antigen-binding activity of an antigen-binding domain or
antigen-binding molecule at a high proton concentration or low pH,
i.e., in an acidic pH range, is not particularly limited, as long
as it is the antigen-binding activity at a pH of between 4.0 and
6.5, and includes the antigen-binding activity at a pH of between
4.5 and 6.6 as the preferred pH. The antigen-binding activity also
includes that at a pH of between 5.0 and 6.5, and that at a pH of
between 5.5 and 6.5 as another preferred pH. The antigen-binding
activity also includes that at the pH in the early endosome in vivo
as the more preferred pH, and specifically, that at pH5.8.
Meanwhile, the antigen-binding activity of an antigen-binding
domain or antigen-binding molecule at a low proton concentration or
high pH, i.e., in a neutral pH range, is not particularly limited,
as long as it is the antigen-binding activity at a pH of between
6.7 and 10, and includes the antigen-binding activity at a pH of
between 6.7 and 9.5 as the preferred pH. The antigen-binding
activity also includes that at a pH of between 7.0 and 9.5 and that
at a pH of between 7.0 and 8.0 as another preferred pH. The
antigen-binding activity also includes that at the pH of plasma in
vivo as the more preferred pH, and specifically, that at pH7.4.
[0235] The antigen-binding activity of an antigen-binding domain or
antigen-binding molecule can be measured by methods known to those
skilled in the art. Those skilled in the art can suitably determine
conditions other than ionized calcium concentration. The
antigen-binding activity of an antigen-binding domain or
antigen-binding molecule can be assessed based on the dissociation
constant (KD), apparent dissociation constant (KD), dissociation
rate constant (kd), apparent dissociation rate constant (kd), and
such. These can be determined by methods known to those skilled in
the art, for example, using Biacore (GE healthcare), Scatchard
plot, or FACS.
[0236] Herein, the step of selecting an antigen-binding domain or
antigen-binding molecule whose antigen-binding activity at a low
proton concentration or high pH, i.e., in a neutral pH range, is
higher than that at a high proton concentration or low pH, i.e., in
an acidic pH range, is synonymous with the step of selecting an
antigen-binding domain or antigen-binding molecule whose
antigen-binding activity at a high proton concentration or low pH,
i.e., in an acidic pH range, is lower than that at a low proton
concentration or high pH, i.e., in a neutral pH range.
[0237] As long as the antigen-binding activity at a low proton
concentration or high pH, i.e., in a neutral pH range, is higher
than that at a high proton concentration or low pH, i.e., in an
acidic pH range, the difference between the antigen-binding
activity at a low proton concentration or high pH, i.e., a neutral
pH range, and that at a high proton concentration or low pH, i.e.,
an acidic pH range, is not particularly limited; however, the
antigen-binding activity at a low proton concentration or high pH,
i.e., in a neutral pH range, is preferably twice or more, more
preferably 10 times or more, and still more preferably 40 times or
more than that at a high proton concentration or low pH, i.e., in
an acidic pH range.
[0238] The antigen binding domain or antigen-binding molecule of
the present invention that is screened by the screening methods
described above may be any antigen-binding domain or
antigen-binding molecule, and for example, an above-mentioned
antigen-binding domain or antigen-binding molecule may be screened.
For example, antigen-binding domains or antigen-binding molecules
having a native sequence may be screened, and antigen-binding
domains or antigen-binding molecules in which their amino acid
sequences have been substituted may also be screened.
[0239] The antigen-binding domain or antigen-binding molecule of
the present invention to be screened by the above-described
screening methods may be prepared in any manner. For example,
preexisting antigen-binding molecules, preexisting libraries (phage
library, etc.), antibodies or libraries prepared from B cells of
immunized animals or from hybridomas obtained by immunizing
animals, antibodies or libraries (libraries with increased content
of amino acids with a side chain pKa of 4.0-8.0 (for example,
histidine and glutamic acid) or unnatural amino acids, libraries
introduced with amino acids with a side chain pKa of 4.0-8.0 (for
example, histidine and glutamic acid) or unnatural amino acid
mutations at specific positions, etc.) obtained by introducing
amino acids with a side chain pKa of 4.0-8.0 (for example,
histidine and glutamic acid) or unnatural amino acid mutations into
the above-described antibodies or libraries may be used.
[0240] Methods for obtaining an antigen-binding domain or
antigen-binding molecule whose antigen-binding activity at a low
proton concentration or high pH, i.e., in a neutral pH range, is
higher than that at a high proton concentration or low pH, i.e., in
an acidic pH range, from an antigen-binding domains or antibodies
prepared from hybridomas obtained by immunizing animals or from B
cells of immunized animals preferably include, for example, the
antigen-binding molecule or antigen-binding molecule in which at
least one of the amino acids of the antigen-binding domain or
antigen-binding molecule is substituted with an amino acid with a
side chain pKa of 4.0-8.0 (for example, histidine and glutamic
acid) or an unnatural amino acid mutation, or the antigen-binding
domain or antigen-binding molecule inserted with an amino acid with
a side chain pKa of 4.0-8.0 (for example, histidine and glutamic
acid) or unnatural amino acid, such as those described in
International Publication No. WO 2009/125825.
[0241] The sites of introducing mutations of amino acids with a
side chain pKa of 4.0-8.0 (for example, histidine and glutamic
acid) or unnatural amino acids are not particularly limited, and
may be any position as long as the antigen-binding activity in an
acidic pH range becomes weaker than that in a neutral pH range (the
value of KD (in an acidic pH range)/KD (in a neutral pH range) or
kd (in an acidic pH range)/kd (in a neutral pH range) is increased)
as compared to before substitution or insertion. For example, when
the antigen-binding molecule is an antibody, antibody variable
region and CDRs are suitable. Those skilled in the art can
appropriately determine the number of amino acids to be substituted
with or the number of amino acids with a side chain pKa of 4.0-8.0
(for example, histidine and glutamic acid) or unnatural amino acids
to be inserted. It is possible to substitute with a single amino
acid having a side chain pKa of 4.0-8.0 (for example, histidine and
glutamic acid) or a single unnatural amino acid; it is possible to
insert a single amino acid having a side chain pKa of 4.0-8.0 (for
example, histidine and glutamic acid) or a single unnatural amino
acid; it is possible to substitute with two or more amino acids
having a side chain pKa of 4.0-8.0 (for example, histidine and
glutamic acid) or two or more unnatural amino acids; and it is
possible to insert two or more amino acids having a side chain pKa
of 4.0-8.0 (for example, histidine and glutamic acid) or two or
more unnatural amino acids. Alternatively, other amino acids can be
deleted, added, inserted, and/or substituted concomitantly, aside
from the substitution into amino acids having a side chain pKa of
4.0-8.0 (for example, histidine and glutamic acid) or unnatural
amino acids, or the insertion of amino acids having a side chain
pKa of 4.0-8.0 (for example, histidine and glutamic acid) or
unnatural amino acids. Substitution into or insertion of amino
acids with a side chain pKa of 4.0-8.0 (for example, histidine and
glutamic acid) or unnatural amino acids can performed randomly by
methods such as histidine scanning, in which the alanine of alanine
scanning known to those skilled in the art is replaced with
histidine. Antigen-binding molecules exhibiting a greater value of
KD (in an acidic pH range)/KD (in a neutral pH range) or kd (in an
acidic pH range)/kd (in a neutral pH range) as compared to before
the mutation can be selected from antigen-binding domains or
antibodies introduced with random insertions or substitution
mutations of amino acids with a side chain pKa of 4.0-8.0 (for
example, histidine and glutamic acid) or unnatural amino acids.
[0242] Preferred examples of antigen-binding molecules containing
the mutation into amino acids with a side chain pKa of 4.0-8.0 (for
example, histidine and glutamic acid) or unnatural amino acids as
described above and whose antigen-binding activity in an acidic pH
range is lower than that in a neutral pH range include,
antigen-binding molecules whose antigen-binding activity in the
neutral pH range after the mutation into amino acids with a side
chain pKa of 4.0-8.0 (for example, histidine and glutamic acid) or
unnatural amino acids is comparable to that before the mutation
into amino acids with a side chain pKa of 4.0-8.0 (for example,
histidine and glutamic acid) or unnatural amino acids. Herein, "an
antigen-binding molecule after the mutation with amino acids having
a side chain pKa of 4.0-8.0 (for example, histidine and glutamic
acid) or unnatural amino acids has an antigen-binding activity
comparable to that before the mutation with amino acids having a
side chain pKa of 4.0-8.0 (for example, histidine and glutamic
acid) or unnatural amino acids" means that, when taking the
antigen-binding activity of an antigen-binding molecule before the
mutation with amino acids having a side chain pKa of 4.0-8.0 (for
example, histidine and glutamic acid) or unnatural amino acids as
100%, the antigen-binding activity of an antigen-binding molecule
after the mutation with amino acids having a side chain pKa of
4.0-8.0 (for example, histidine and glutamic acid) or unnatural
amino acids is at least 10% or more, preferably 50% or more, more
preferably 80% or more, and still more preferably 90% or more. The
antigen-binding activity after the mutation of amino acids with a
side chain pKa of 4.0-8.0 (for example, histidine and glutamic
acid) or unnatural amino acids at pH 7.4 may be higher than that
before the mutation of amino acids with a side chain pKa of 4.0-8.0
(for example, histidine and glutamic acid) or unnatural amino acids
at pH 7.4. If the antigen-binding activity of an antigen-binding
molecule is decreased due to insertion of or substitution into
amino acids with a side chain pKa of 4.0-8.0 (for example,
histidine and glutamic acid) or unnatural amino acids, the
antigen-binding activity can be made to be comparable to that
before the insertion of or substitution into amino acids with a
side chain pKa of 4.0-8.0 (for example, histidine and glutamic
acid) or unnatural amino acids, by introducing a substitution,
deletion, addition, and/or insertion of one or more amino acids of
the antigen-binding molecule. The present invention also includes
antigen-binding molecules whose binding activity has been adjusted
to be comparable by substitution, deletion, addition, and/or
insertion of one or more amino acids after substitution or
insertion of amino acids with a side chain pKa of 4.0-8.0 (for
example, histidine and glutamic acid) or unnatural amino acids.
Amino Acids that Alter the Antigen-Binding Activity of
Antigen-Binding Domain Depending on the Proton Concentration
Conditions
[0243] Antigen-binding domains or antigen-binding molecules of the
present invention to be screened by the above-described screening
methods may be prepared in any manner. For example, when ion
concentration condition is proton concentration condition or pH
condition, preexisting antigen-binding molecules, preexisting
libraries (phage library, etc.), antibodies or libraries prepared
from B cells of immunized animals or from hybridomas obtained by
immunizing animals, antibodies or libraries (libraries with
increased content of amino acids with a side chain pKa of 4.0-8.0
(for example, histidine and glutamic acid) or unnatural amino
acids, libraries introduced with mutations of amino acids with a
side chain pKa of 4.0-8.0 (for example, histidine and glutamic
acid) or unnatural amino acids at specific positions, etc.)
obtained by introducing mutations of amino acids with a side chain
pKa of 4.0-8.0 (for example, histidine and glutamic acid) or
unnatural amino acids into the above-described antibodies or
libraries may be used.
[0244] In one embodiment of the present invention, a library
containing multiple antigen-binding molecules of the present
invention whose sequences are different from one another can also
be constructed by combining heavy chain variable regions, produced
as a randomized variable region sequence library, with light chain
variable regions introduced with "at least one amino acid residue
that changes the antigen-binding activity of an antigen-binding
molecule depending on the proton concentration condition".
[0245] Such amino acid residues include, but are not limited to,
for example, amino acid residues contained in the light chain CDR1.
The amino acid residues also include, but are not limited to, for
example, amino acid residues contained in the light chain CDR2. The
amino acid residues also include, but are not limited to, for
example, amino acid residues contained in the light chain CDR3.
[0246] The above-described amino acid residues contained in the
light chain CDR1 include, but are not limited to, for example,
amino acid residues of positions 24, 27, 28, 31, 32, and/or 34
according to Kabat numbering in the CDR1 of light chain variable
region. Meanwhile, the amino acid residues contained in the light
chain CDR2 include, but are not limited to, for example, amino acid
residues of positions 50, 51, 52, 53, 54, 55, and/or 56 according
to Kabat numbering in the CDR2 of light chain variable region.
Furthermore, the amino acid residues in the light chain CDR3
include, but are not limited to, for example, amino acid residues
of positions 89, 90, 91, 92, 93, 94, and/or 95A according to Kabat
numbering in the CDR3 of light chain variable region. Moreover, the
amino acid residues can be contained alone or can be contained in
combination of two or more amino acids as long as they allow the
change in the antigen-binding activity of an antigen-binding
molecule depending on the proton concentration.
[0247] Even when the heavy chain variable region produced as a
randomized variable region sequence library is combined with the
above-described light chain variable region introduced with "at
least one amino acid residue that changes the antigen-binding
activity of an antigen-binding molecule depending on the proton
concentration condition", it is possible to design so that the
flexible residues are contained in the sequence of the light chain
variable region in the same manner as described above. The number
and position of the flexible residues are not particularly limited
to a specific embodiment, as long as the antigen-binding activity
of an antigen-binding molecule of the present invention changes
depending on the proton concentration condition. Specifically, the
CDR and/or FR sequences of heavy chain and/or light chain can
contain one or more flexible residues. For example, flexible
residues to be introduced into the sequences of the light chain
variable regions include, but are not limited to, for example, the
amino acid residues listed in Tables 3 and 4. Meanwhile, amino acid
sequences of light chain variable regions other than the flexible
residues and amino acid residues that change the antigen-binding
activity of an antigen-binding molecule depending on the proton
concentration condition suitably include, but are not limited to,
germ line sequences such as Vk1 (SEQ ID NO: 7), Vk2 (SEQ ID NO: 8),
Vk3 (SEQ ID NO: 9), and Vk4 (SEQ ID NO: 10).
TABLE-US-00003 TABLE 3 Position Amino acid CDR1 28 S: 100% 29 I:
100% 30 N: 25% S: 25% R: 25% H: 25% 31 S: 100% 32 H: 100% 33 L:
100% 34 A: 50% N: 50% CDR2 50 H: 100% or A: 25% D: 25% G: 25% K:
25% 51 A: 100% A: 100% 52 S: 100% S: 100% 53 K: 33.3% N: 33.3% S:
33.3% H: 100% 54 L: 100% L: 100% 55 Q: 100% Q: 100% 56 S: 100% S:
100% CDR3 90 Q: 100% or Q: 100% 91 H: 100% S: 33.3% R: 33.3% Y:
33.3% 92 G: 25% N: 25% S: 25% Y: 25% H: 100% 93 H: 33.3% N: 33.3%
S: 33.3% H: 33.3% N: 33.3% S: 33.3% 94 S: 50% Y: 50% S: 50% Y: 50%
95 P: 100% P: 100% 96 L: 50% Y: 50% L: 50% Y: 50% (Position
indicates Kabat numbering)
TABLE-US-00004 TABLE 4 CDR Position Amino acid CDR1 28 S: 100% 29
I: 100% 30 H: 30% N: 10% S: 50% R: 10% 31 N: 35% S: 65% 32 H: 40%
N: 20% Y: 40% 33 L: 100% 34 A: 70% N: 30% CDR2 50 A: 25% D: 15% G:
25% H: 30% K: 5% 51 A: 100% 52 S: 100% 53 H: 30% K: 10% N: 15% S:
45% 54 L: 100% 55 Q: 100% 56 S: 100% CDR3 90 Q: 100% 91 H: 30% S:
15% R: 10% Y: 45% 92 G: 20% H: 30% N: 20% S: 15% Y: 15% 93 H: 30%
N: 25% S: 45% 94 S: 50% Y: 50% 95 P: 100% 96 L: 50% Y: 50%
(Position indicates Kabat numbering)
[0248] Any amino acid residue may be suitably used as the
above-described amino acid residues that change the antigen-binding
activity of an antigen-binding molecule depending on the proton
concentration condition. Specifically, such amino acid residues
include amino acids with a side chain pKa of 4.0-8.0. Such
electron-releasing amino acids preferably include, for example,
naturally occurring amino acids such as histidine and glutamic
acid, as well as unnatural amino acids such as histidine analogs
(US2009/0035836), m-NO2-Tyr (pKa 7.45), 3,5-Br2-Tyr (pKa 7.21), and
3,5-12-Tyr (pKa 7.38) (Bioorg. Med. Chem. (2003) 11 (17),
3761-3768). Particularly preferred amino acid residues include, for
example, amino acids with a side chain pKa of 6.0-7.0. Such
electron-releasing amino acid residues preferably include, for
example, histidine.
[0249] Known methods such as site-directed mutagenesis (Kunkel et
al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and Overlap
extension PCR can be appropriately employed to modify the amino
acids of antigen-binding domains. Furthermore, various known
methods can also be used as an amino acid modification method for
substituting amino acids by those other than natural amino acids
(Annu Rev. Biophys. Biomol. Struct. (2006) 35, 225-249; Proc. Natl.
Acad. Sci. U.S.A. (2003) 100 (11), 6353-6357). For example, a
cell-free translation system (Clover Direct (Protein Express))
containing tRNAs in which amber suppressor tRNA, which is
complementary to UAG codon (amber codon) that is a stop codon, is
linked with an unnatural amino acid may be suitably used.
[0250] The preferred heavy chain variable region that is used in
combination includes, for example, randomized variable region
libraries. Known methods are appropriately combined as a method for
producing a randomized variable region library. In a non-limiting
embodiment of the present invention, an immune library constructed
based on antibody genes derived from animals immunized with
specific antigens, patients with infection or persons with an
elevated antibody titer in blood as a result of vaccination, cancer
patients, or lymphocytes of auto immune diseases may be suitably
used as a randomized variable region library.
[0251] In another non-limiting embodiment of the present invention,
in the same manner as described above, a synthetic library in which
the CDR sequences of V genes from genomic DNA or functional
reconstructed V genes are replaced with a set of synthetic
oligonucleotides containing the sequences encoding codon sets of an
appropriate length can also be suitably used as a randomized
variable region library. In this case, the CDR3 sequence alone may
be replaced because variety in the gene sequence of heavy chain
CDR3 is observed. The basis for giving rise to amino acid
variations in the variable region of an antigen-binding molecule is
to generate variations of amino acid residues of surface-exposed
positions of the antigen-binding molecule. The surface-exposed
position refers to a position where an amino acid is exposed on the
surface and/or contacted with an antigen based on the conformation,
structural ensemble, and/or modeled structure of an antigen-binding
molecule, and in general, such positions are the CDRs. The
surface-exposed positions are preferably determined using the
coordinates derived from a three-dimensional model of the
antigen-binding molecule using computer programs such as Insightll
program (Accelrys). The surface-exposed positions can be determined
using algorithms known in the art (for example, Lee and Richards
(J. Mol. Biol. (1971) 55, 379-400); Connolly (J. Appl. Cryst.
(1983) 16, 548-558)). The surface-exposed positions can be
determined based on the information on the three dimensional
structure of antibodies using software suitable for protein
modeling. Software which is suitably used for this purpose includes
the SYBYL biopolymer module software (Tripos Associates). When the
algorithm requires the input size parameter from the user, the
"size" of probe for use in computation is generally or preferably
set at about 1.4 angstrom or less in radius. Furthermore, a method
for determining surface-exposed region and area using PC software
is described by Pacios (Comput. Chem. (1994) 18 (4), 377-386; and
J. Mol. Model. (1995) 1, 46-53).
[0252] In still another non-limiting embodiment of the present
invention, a naive library constructed from antibody genes derived
from lymphocytes of healthy persons and consisting of naive
sequences, which are unbiased repertoire of antibody sequences, can
also be particularly suitably used as a randomized variable region
library (Gejima et al. (Human Antibodies (2002) 11, 121-129); and
Cardoso et al. (Scand. J. Immunol. (2000) 51, 337-344)).
Fc Region
[0253] An Fc region contains the amino acid sequence derived from
the heavy chain constant region of an antibody. An Fc region is a
portion of the heavy chain constant region of an antibody, starting
from the N terminal end of the hinge region, which corresponds to
the papain cleavage site at an amino acid around position 216
according to the EU numbering system, and contains the hinge, CH2,
and CH3 domains. While the Fc region may be obtained from human
IgG1, it is not limited to a particular subclass of IgG. As
described later, a favorable example of the Fc region is an Fc
region that has an FcRn-binding activity in an acidic pH range.
Furthermore, a favorable example of the Fc region is an Fc region
that has an Fc.gamma. receptor-binding activity as described later.
A non-limiting embodiment of such an Fc region is, for example, the
Fc region of human IgG1 (SEQ ID NO: 13), IgG2 (SEQ ID NO: 14), IgG3
(SEQ ID NO: 15), or IgG4 (SEQ ID NO: 16).
FcRn
[0254] Unlike Fc.gamma. receptor belonging to the immunoglobulin
superfamily, human FcRn is structurally similar to polypeptides of
major histocompatibility complex (MHC) class I, exhibiting 22% to
29% sequence identity to class I MHC molecules (Ghetie el al.,
Immunol. Today (1997) 18 (12): 592-598). FcRn is expressed as a
heterodimer consisting of soluble r3 or light chain (.beta.2
microglobulin) complexed with transmembrane a or heavy chain. Like
MHC, FcRn .alpha. chain comprises three extracellular domains
(.alpha.1, .alpha.2, and .alpha.3) and its short cytoplasmic domain
anchors the protein onto the cell surface. .alpha.1 and .alpha.2
domains interact with the FcRn-binding domain of the antibody Fc
region (Raghavan et al., Immunity (1994) 1: 303-315).
[0255] FcRn is expressed in maternal placenta and york sac of
mammals, and is involved in mother-to-fetus IgG transfer. In
addition, in neonatal small intestine of rodents, where FcRn is
expressed, FcRn is involved in transfer of maternal IgG across
brush border epithelium from ingested colostrum or milk. FcRn is
expressed in a variety of other tissues and endothelial cell
systems of various species. FcRn is also expressed in adult human
endothelia, muscular blood vessels, and hepatic sinusoidal
capillaries. FcRn is believed to play a role in maintaining the
plasma IgG concentration by mediating recycling of IgG to serum
upon binding to IgG. Typically, binding of FcRn to IgG molecules is
strictly pH dependent. The optimal binding is observed in an acidic
pH range below 7.0.
[0256] Human FcRn whose precursor is a polypeptide having the
signal sequence of SEQ ID NO: 17 (the polypeptide with the signal
sequence is shown in SEQ ID NO: 18) forms a complex with human
.beta.2-microglobulin in vivo. Soluble human FcRn complexed with
.beta.2-microglobulin is produced by using conventional recombinant
expression techniques. FcRn regions of the present invention can be
assessed for their binding activity to such a soluble human FcRn
complexed with .beta.2-microglobulin. Herein, unless otherwise
specified, human FcRn refers to a form capable of binding to an
FcRn region of the present invention. Examples include a complex
between human FcRn and human .beta.2-microglobulin. A non-limiting
embodiment is exemplified by a complex formed between a mouse FcRn
(SEQ ID NO: 73) and a mouse .beta.2-microglobulin (SEQ ID NO:
74).
Binding Activity of the Fc Region to FcRn, in Particular, Human
FcRn
[0257] The binding activity of an Fc region of the present
invention to FcRn, human FcRn in particular, can be measured by
methods known to those skilled in the art, as described in the
section "Binding Activity" above. Those skilled in the art can
appropriately determine the conditions other than pH. The
antigen-binding activity and human FcRn-binding activity of an
antigen-binding molecule can be assessed based on the dissociation
constant (KD), apparent dissociation constant (KD), dissociation
rate (kd), apparent dissociation rate (kd), and such. These can be
measured by methods known to those skilled in the art. For example,
Biacore (GE healthcare), Scatchard plot, or flow cytometer may be
used.
[0258] When the human FcRn-binding activity of an Fc region of the
present invention is measured, conditions other than the pH are not
particularly limited, and can be appropriately selected by those
skilled in the art. Measurements can be carried out, for example,
at 37.degree. C. using MES buffer, as described in International
Publication No. WO 2009125825. Alternatively, the human
FcRn-binding activity of an Fc region of the present invention can
be measured by methods known to those skilled in the art, and may
be measured by using, for example, Biacore (GE Healthcare) or such.
The binding activity of an Fc region of the present invention to
human FcRn can be assessed by pouring, as an analyte, human FcRn,
an Fc region, or an antigen-binding molecule of the present
invention containing the Fc region into a chip immobilized with an
Fc region, an antigen-binding molecule of the present invention
containing the Fc region, or human FcRn.
[0259] A neutral pH range as the condition where the Fc region
contained in an antigen-binding molecule of the present invention
has the FcRn-binding activity means pH6.7 to pH10.0 in general.
Preferably, the neutral pH range is a range indicated with
arbitrary pH values between pH7.0 and pH8.0, and is preferably
selected from pH7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9,
and 8.0, and is particularly preferably pH7.4 that is close to the
pH of plasma (blood) in vivo. When the binding affinity between the
human FcRn-binding domain and human FcRn at pH7.4 is too low to
assess, pH7.0 may be used instead of pH7.4. Herein, an acidic pH
range as the condition where the Fc region contained in an
antigen-binding molecule of the present invention has the
FcRn-binding activity means pH4.0 to pH6.5 in general. Preferably,
the acidic pH range means pH5.5 to pH6.5, particularly preferably
pH5.8 to pH6.0 which is close to the pH in the early endosome in
vivo. Regarding the temperature used as the measurement condition,
the binding affinity between the human FcRn-binding domain and
human FcRn may be assessed at any temperature between 10.degree. C.
and 50.degree. C. Preferably, the binding affinity between the
human FcRn-binding domain and human FcRn can be determined at
15.degree. C. to 40.degree. C. More preferably, the binding
affinity between the human FcRn-binding domain and human FcRn can
be determined in the same manner at an arbitrary temperature
between 20.degree. C. and 35.degree. C., such as any one
temperature of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,
33, 34, and 35.degree. C. In an embodiment of the present
invention, the temperature includes, but is not limited to, for
example, 25.degree. C.
[0260] According to the Journal of Immunology (2009) 182,
7663-7671, the human FcRn-binding activity of native human IgG1 in
an acidic pH range (pH 6.0) is 1.7 .mu.M (KD), and the activity is
almost undetectable in a neutral pH range. Thus, in a preferred
embodiment, antigen-binding molecules comprising an Fc region of
which human FcRn-binding activity in an acidic pH range is 20 .mu.M
(KD) or stronger may be screened. In a more preferred embodiment,
the antigen-binding molecules comprising an Fc region of which
human FcRn-binding activity in an acidic pH range is 2.0 .mu.M (KD)
or stronger may be screened. In a still more preferred embodiment,
the antigen-binding molecules comprising an Fc region of which
human FcRn-binding activity in an acidic pH range is 0.5 .mu.M (KD)
or stronger may be screened. The above-mentioned KD values are
determined by the method described in the Journal of Immunology
(2009) 182: 7663-7671 (by immobilizing the antigen-binding molecule
onto a chip and loading human FcRn as an analyte).
[0261] In the present invention, preferred Fc regions have an
FcRn-binding activity in an acidic pH range condition. When an Fc
region originally has an FcRn-binding activity under an acidic pH
range condition, the domain can be used as it is. When the domain
has a weak or no FcRn-binding activity under an acidic pH range
condition, an Fc region having a desired FcRn-binding activity can
be obtained by modifying amino acids of an antigen-binding
molecule. Fc regions having a desired or enhanced FcRn-binding
activity under an acidic pH range condition can also be suitably
obtained by modifying the amino acids of an Fc region. Amino acid
modifications of an Fc region that result in such a desired binding
activity can be found by comparing the FcRn-binding activity under
an acidic pH range condition before and after amino acid
modification. Those skilled in the art can appropriately modify the
amino acids using known techniques similar to the aforementioned
techniques used to modify the Fc.gamma.-receptor-binding
activity.
[0262] Fc regions comprised in the antigen-binding molecules of the
present invention, which have an FcRn-binding activity under an
acidic pH range condition, can be obtained by any method.
Specifically, FcRn-binding domains having an FcRn-binding activity
or an enhanced FcRn-binding activity under an acidic pH range
condition can be obtained by modifying the amino acids of an
IgG-type human immunoglobulin used as a starting Fc region.
Preferred Fc regions of an IgG-type immunoglobulin for modification
include, for example, those of human IgGs (IgG1, IgG2, IgG3, and
IgG4, and variants thereof). As long as the Fc region has an
FcRn-binding activity under an acidic pH range condition or can
increase the human FcRn-binding activity under an acidic pH range
condition, amino acids at any position may be modified into other
amino acids. When the antigen-binding molecule contains the Fc
region of human IgG1 as the Fc region, it is preferable that the
resulting Fc region contains a modification that results in the
effect of enhancing FcRn binding under an acidic pH range condition
as compared to the binding activity of the starting human IgG1 Fc
region. Amino acids that allow such modification include, for
example, amino acids of positions 252, 254, 256, 309, 311, 315,
433, and/or 434 according to EU numbering, and their combination
amino acids at positions 253, 310, 435, and/or 426 as described in
WO 1997/034631. Favorable examples include amino acids of positions
238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307,
309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 386,
388, 400, 413, 415, 424, 433, 434, 435, 436, 439, and/or 447 as
indicated by EU numbering as described in WO 2000/042072.
Similarly, favorable examples of amino acids that allow such
modification include, amino acids of positions 251, 252, 254, 255,
256, 308, 309, 311, 312, 385, 386, 387, 389, 428, 433, 434, and/or
436 according to EU numbering as described in WO 2002/060919.
Furthermore, amino acids that allow such modification include, for
example, amino acids of positions 250, 314, and 428 according to EU
numbering as described in WO2004/092219. In addition, favorable
examples of amino acids that allow such modification include amino
acids of positions 238, 244, 245, 249, 252, 256, 257, 258, 260,
262, 270, 272, 279, 283, 285, 286, 288, 293, 307, 311, 312, 316,
317, 318, 332, 339, 341, 343, 375, 376, 377, 378, 380, 382, 423,
427, 430, 431, 434, 436, 438, 440, and/or 442 as described in WO
2006/020114. Furthermore, favorable examples of amino acids that
allow such modification include amino acids of positions 251, 252,
307, 308, 378, 428, 430, 434, and/or 436 according to EU numbering
as described in WO 2010/045193. Modification of these amino acids
enhances FcRn binding of the Fc region of an IgG-type
immunoglobulin under an acidic pH range condition.
[0263] When the Fc region of human IgG1 is comprised as the Fc
region, a non-limiting embodiment of the modification that results
in the effect of enhancing FcRn binding under an acidic pH range
condition as compared to the binding activity of the starting Fc
region of human IgG1 includes at least one or more amino acid
modifications selected from the group consisting of:
Arg or Leu for the amino acid of position 251; Phe, Ser, Thr, or
Tyr for the amino acid of position 252; Ser or Thr for the amino
acid of position 254; Arg, Gly, Ile, or Leu for the amino acid of
position 255; Ala, Arg, Asn, Asp, Gln, Glu, or Thr for the amino
acid of position 256; Ile or Thr for the amino acid of position
308; Pro for the amino acid of position 309; Glu, Leu, or Ser for
the amino acid of position 311; Ala or Asp for the amino acid of
position 312; Ala or Leu for the amino acid of position 314; Ala,
Arg, Asp, Gly, His, Lys, Ser, or Thr for the amino acid of position
385; Arg, Asp, Ile, Lys, Met, Pro, Ser, or Thr for the amino acid
of position 386; Ala, Arg, His, Pro, Ser, or Thr for the amino acid
of position 387; Asn, Pro, or Ser for the amino acid of position
389; Leu, Met, Phe, Ser, or Thr for the amino acid of position 428;
Arg, Gln, His, Ile, Lys, Pro, or Ser for the amino acid of position
433; His, Phe, or Tyr for the amino acid of position 434; and Arg,
Asn, His, Lys, Met, or Thr for the amino acid of position 436, as
indicated by EU numbering. Meanwhile, the number of amino acids to
be modified is not particularly limited; and amino acid may be
modified at only one site or amino acids may be modified at two or
more sites.
[0264] When the Fc region of human IgG1 is comprised as the Fc
region, a non-limiting embodiment of the modification that results
in the effect of enhancing FcRn binding in an acidic pH range
condition as compared to the binding activity of the starting Fc
region of human IgG1 may be modifications including Ile for the
amino acid of position 308, Pro for the amino acid of position 309,
and/or Glu for the amino acid of position 311 according to EU
numbering. Another non-limiting embodiment of this modification may
include Thr for the amino acid of position 308, Pro for the amino
acid of position 309, Leu for the amino acid of position 311, Ala
for the amino acid of position 312, and/or Ala for the amino acid
of position 314. Furthermore, another non-limiting embodiment of
this modification may include Ile or Thr for the amino acid of
position 308, Pro for the amino acid of position 309, Glu, Leu, or
Ser for the amino acid of position 311, Ala for the amino acid of
position 312, and/or Ala or Leu for the amino acid of position 314.
Another non-limiting embodiment of this modification may include
Thr for the amino acid of position 308, Pro for the amino acid of
position 309, Ser for the amino acid of position 311, Asp for the
amino acid of position 312, and/or Leu for the amino acid of
position 314.
[0265] When the Fc region of human IgG1 is comprised as the Fc
region, a non-limiting embodiment of the modification that results
in the effect of enhancing FcRn binding under an acidic pH range
condition as compared to the binding activity of the starting Fc
region of human IgG1 may be modifications including Leu for the
amino acid of position 251, Tyr for the amino acid of position 252,
Ser or Thr for the amino acid of position 254, Arg for the amino
acid of position 255, and/or Glu for the amino acid of position 256
according to EU numbering.
[0266] When the Fc region of human IgG1 is comprised as the Fc
region, a non-limiting embodiment of the modification that results
in the effect of enhancing FcRn binding under an acidic pH range
condition as compared to the binding activity of the starting Fc
region of human IgG1 may be modifications including Leu, Met, Phe,
Ser, or Thr for the amino acid of position 428, Arg, Gln, His, Ile,
Lys, Pro, or Ser for the amino acid of position 433, His, Phe, or
Tyr for the amino acid of position 434, and/or Arg, Asn, His, Lys,
Met, or Thr for the amino acid of position 436 according to EU
numbering. Another non-limiting embodiment of this modification may
include His or Met for the amino acid of position 428, and/or His
or Met for the amino acid of position 434.
[0267] When the Fc region of human IgG1 is comprised as the Fc
region, a non-limiting embodiment of the modification that results
in the effect of enhancing FcRn binding under an acidic pH range
condition as compared to the binding activity of the starting Fc
region of human IgG1 may be modifications including Arg for the
amino acid of position 385, Thr for the amino acid of position 386,
Arg for the amino acid of position 387, and/or Pro for the amino
acid of position 389 according to EU numbering. Another
non-limiting embodiment of this modification may include Asp for
the amino acid of position 385, Pro for the amino acid of position
386, and/or Ser for the amino acid of position 389.
[0268] Furthermore, when the Fc region of human IgG1 is comprised
as the Fc region, a non-limiting embodiment of the modification
that results in the effect of enhancing FcRn binding under an
acidic pH range condition as compared to the binding activity of
the starting Fc region of human IgG1 include at least one or more
amino acid modifications selected from the group consisting of:
Gln or Glu for the amino acid of position 250; and Leu or Phe for
the amino acid of position 428 according to EU numbering. The
number of amino acids to be modified is not particularly limited;
and amino acid may be modified at only one site or amino acids may
be modified at two sites.
[0269] When the Fc region of human IgG1 is comprised as the Fc
region, a non-limiting embodiment of the modification that results
in the effect of enhancing FcRn binding under an acidic pH range
condition as compared to the binding activity of the starting Fc
region of human IgG1 may be modifications including Gln for the
amino acid of position 250, and/or Leu or Phe for the amino acid of
position 428 according to EU numbering. Another non-limiting
embodiment of this modification may include Glu for the amino acid
of position 250, and/or Leu or Phe for the amino acid of position
428.
[0270] When the Fc region of human IgG1 is comprised as the Fc
region, a non-limiting embodiment of the modification that results
in the effect of enhancing FcRn binding under an acidic pH range
condition as compared to the binding activity of the starting Fc
region of human IgG1 include at least two or more amino acid
modifications selected from the group consisting of:
Asp or Glu for the amino acid of position 251; Tyr for the amino
acid of position 252; Gln for the amino acid of position 307; Pro
for the amino acid of position 308; Val for the amino acid of
position 378; Ala for the amino acid of position 380; Leu for the
amino acid of position 428; Ala or Lys for the amino acid of
position 430; Ala, His, Ser, or Tyr for the amino acid of position
434; and Ile for the amino acid of position 436, as indicated by EU
numbering. The number of amino acids to be modified is not
particularly limited; and amino acid may be modified at only two
sites or amino acids may be modified at three or more sites.
[0271] When the Fc region of human IgG1 is comprised as the Fc
region, a non-limiting embodiment of the modification that results
in the effect of enhancing FcRn binding under an acidic pH range
condition as compared to the binding activity of the starting Fc
region of human IgG1 may be modifications including Gln for the
amino acid of position 307, and Ala or Ser for the amino acid of
position 434 according to EU numbering. Another non-limiting
embodiment of this modification may include Pro for the amino acid
of position 308, and Ala for the amino acid of position 434.
Furthermore, another non-limiting embodiment of this modification
may include Tyr for the amino acid of position 252, and Ala for the
amino acid of position 434. A different non-limiting embodiment of
this modification may include Val for the amino acid of position
378, and Ala for the amino acid of position 434. Another different
non-limiting embodiment of this modification may include Leu for
the amino acid of position 428, and Ala for the amino acid of
position 434. Another different non-limiting embodiment of this
modification may include Ala for the amino acid of position 434,
and Ile for the amino acid of position 436. Furthermore, another
non-limiting embodiment of this modification may include Pro for
the amino acid of position 308, and Tyr for the amino acid of
position 434. In addition, another non-limiting embodiment of this
modification may include Gln for the amino acid of position 307,
and Ile for the amino acid of position 436.
[0272] When the Fc region of human IgG1 is comprised as the Fc
region, a non-limiting embodiment of the modification that results
in the effect of enhancing FcRn binding under an acidic pH range
condition as compared to the binding activity of the starting Fc
region of human IgG1 may be modifications including any one of Gln
for the amino acid of position 307, Ala for the amino acid of
position 380, and Ser for the amino acid of position 434 according
to EU numbering. Another non-limiting embodiment of this
modification may include Gln for the amino acid of position 307,
Ala for the amino acid of position 380, and Ala for the amino acid
of position 434. Furthermore, another non-limiting embodiment of
this modification may include Tyr for the amino acid of position
252, Pro for the amino acid of position 308, and Tyr for the amino
acid of position 434. A different non-limiting embodiment of this
modification may include Asp for the amino acid of position 251,
Gln for the amino acid of position 307, and His for the amino acid
of position 434.
[0273] When the Fc region of human IgG1 is comprised as the Fc
region, a non-limiting embodiment of the modification that results
in the effect of enhancing FcRn binding under an acidic pH range
condition as compared to the binding activity of the starting Fc
region of human IgG1 include modification of at least two or more
amino acids selected from the group consisting of:
Leu for the amino acid of position 238; Leu for the amino acid of
position 244; Arg for the amino acid of position 245; Pro for the
amino acid of position 249; Tyr for the amino acid of position 252;
Pro for the amino acid of position 256; Ala, Ile, Met, Asn, Ser, or
Val for the amino acid of position 257; Asp for the amino acid of
position 258; Ser for the amino acid of position 260; Leu for the
amino acid of position 262; Lys for the amino acid of position 270;
Leu or Arg for the amino acid of position 272; Ala, Asp, Gly, His,
Met, Asn, Gln, Arg, Ser, Thr, Trp, or Tyr for the amino acid of
position 279; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro,
Gln, Arg, Ser, Thr, Trp, or Tyr for the amino acid of position 283;
Asn for the amino acid of position 285; Phe for the amino acid of
position 286; Asn or Pro for the amino acid of position 288; Val
for the amino acid of position 293; Ala, Glu, or Met for the amino
acid of position 307; Ala, Ile, Lys, Leu, Met, Val, or Trp for the
amino acid of position 311; Pro for the amino acid of position 312;
Lys for the amino acid of position 316; Pro for the amino acid of
position 317; Asn or Thr for the amino acid of position 318; Phe,
His, Lys, Leu, Met, Arg, Ser, or Trp for the amino acid of position
332; Asn, Thr, or Trp for the amino acid of position 339; Pro for
the amino acid of position 341; Glu, His, Lys, Gln, Arg, Thr, or
Tyr for the amino acid of position 343; Arg for the amino acid of
position 375; Gly, Ile, Met, Pro, Thr, or Val for the amino acid of
position 376; Lys for the amino acid of position 377; Asp or Asn
for the amino acid of position 378; Asn, Ser, or Thr for the amino
acid of position 380; Phe, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,
Ser, Thr, Val, Trp, or Tyr for the amino acid of position 382; Asn
for the amino acid of position 423; Asn for the amino acid of
position 427; Ala, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln,
Arg, Ser, Thr, Val, or Tyr for the amino acid of position 430; His
or Asn for the amino acid of position 431; Phe, Gly, His, Trp, or
Tyr for the amino acid of position 434; Ile, Leu, or Thr for the
amino acid of position 436; Lys, Leu, Thr, or Trp for the amino
acid of position 438; Lys for the amino acid of position 440; and
Lys for the amino acid of position 442 according to EU numbering.
The number of amino acids to be modified is not particularly
limited and amino acid at only two sites may be modified and amino
acids at three or more sites may be modified.
[0274] When the Fc region of human IgG1 is comprised as the Fc
region, a non-limiting embodiment of the modification that results
in the effect of enhancing FcRn binding under an acidic pH range
condition as compared to the binding activity of the starting Fc
region of human IgG1 may be modifications including Ile for the
amino acid of position 257, and Ile for the amino acid of position
311 according to EU numbering. Another non-limiting embodiment of
this modification may include Ile for the amino acid of position
257, and His for the amino acid of position 434. Another
non-limiting embodiment of this modification may include Val for
the amino acid of position 376, and His for the amino acid of
position 434.
[0275] Furthermore, in another non-limiting embodiment, one may
screen for antigen-binding molecules comprising an Fc region with
the characteristic of having a human FcRn-binding activity in the
neutral pH range instead of the above-described characteristic of
having a human FcRn-binding activity in the acidic pH range. In a
preferred embodiment, one may screen for antigen-binding molecules
comprising an Fc region whose human FcRn-binding activity in the
neutral pH range is 40 .mu.M (KD) or stronger. In a more preferred
embodiment, one may screen for antigen-binding molecules comprising
an Fc region whose human FcRn-binding activity in the neutral pH
range is 15 .mu.M (KD) or stronger.
[0276] Furthermore, in another non-limiting embodiment, one may
screen for antigen-binding molecules comprising an Fc region with
the characteristic of having a human FcRn-binding activity in the
neutral pH range in addition to the above-described characteristic
of having a human FcRn-binding activity in the acidic pH range. In
a preferred embodiment, one may screen for antigen-binding
molecules comprising an Fc region whose human FcRn-binding activity
in the neutral pH range is 40 .mu.M (KD) or stronger. In a more
preferred embodiment, one may screen for antigen-binding molecules
comprising an Fc region whose human FcRn-binding activity in the
neutral pH range is 15 .mu.M (KD) or stronger.
[0277] In the present invention, preferred Fc regions have a human
FcRn-binding activity in the acidic pH range and/or neutral pH
range. When an Fc region originally has a human FcRn-binding
activity in the acidic pH range and/or neutral pH range, it can be
used as it is. When an Fc region has a weak or no human
FcRn-binding activity in the acidic pH range and/or neutral pH
range, antigen-binding molecules comprising an Fc region having a
desired human FcRn-binding activity can be obtained by modifying
amino acids of the Fc region comprised in the antigen-binding
molecules. Fc regions having a desired human FcRn-binding activity
in the acidic pH range and/or neutral pH range can also be suitably
obtained by modifying amino acids of a human Fc region.
Alternatively, antigen-binding molecules comprising an Fc region
having a desired human FcRn-binding activity can be obtained by
modifying amino acids of an Fc region that originally has a human
FcRn-binding activity in the acidic pH range and/or neutral pH
range. Amino acid modifications of a human Fc region that result in
such a desired binding activity can be found by comparing the human
FcRn-binding activity in the acidic pH range and/or neutral pH
range before and after amino acid modification. Those skilled in
the art can appropriately modify amino acids using known
methods.
[0278] Furthermore, an altered Fc region modified from a starting
Fc region which has been already modified can also be used
preferably as an altered Fc region of the present invention. The
"starting Fc region" can refer to the polypeptide itself, a
composition comprising the starting Fc region, or an amino acid
sequence encoding the starting Fc region. Starting Fc regions can
comprise a known IgG antibody Fc region produced via recombination
described briefly in section "Antibodies". The origin of starting
Fc regions is not limited, and they may be obtained from human or
any nonhuman organisms. Such organisms preferably include mice,
rats, guinea pigs, hamsters, gerbils, cats, rabbits, dogs, goats,
sheep, bovines, horses, camels and organisms selected from nonhuman
primates. In another embodiment, starting Fc regions can also be
obtained from cynomolgus monkeys, marmosets, rhesus monkeys,
chimpanzees, or humans. Starting Fc regions can be obtained
preferably from human IgG1; however, they are not limited to any
particular IgG subclass. This means that an Fc region represented
by human IgG1 (SEQ ID NO: 13), IgG2 (SEQ ID NO: 14), IgG3 (SEQ ID
NO: 15), or IgG4 (SEQ ID NO: 16) can be used appropriately as a
starting Fc region, and herein also means that an Fc region of an
arbitrary IgG class or subclass derived from any organisms
described above can be preferably used as a starting Fc region.
Examples of naturally-occurring IgG variants or modified forms are
described in published documents (Curr. Opin. Biotechnol. (2009) 20
(6): 685-91; Curr. Opin. Immunol. (2008) 20 (4), 460-470; Protein
Eng. Des. Sel. (2010) 23 (4): 195-202; International Publication
Nos. WO 2009/086320, WO 2008/092117, WO 2007/041635, and WO
2006/105338); however, they are not limited to the examples.
[0279] Examples of alterations include those with one or more
mutations, for example, mutations by substitution of different
amino acid residues for amino acids of starting Fc regions, by
insertion of one or more amino acid residues into starting Fc
regions, or by deletion of one or more amino acids from starting Fc
region. Preferably, the amino acid sequences of altered Fc regions
comprise at least a part of the amino acid sequence of a non-native
Fc region. Such variants necessarily have sequence identity or
similarity less than 100% to their starting Fc region. In a
preferred embodiment, the variants have amino acid sequence
identity or similarity about 75% to less than 100%, more preferably
about 80% to less than 100%, even more preferably about 85% to less
than 100%, still more preferably about 90% to less than 100%, and
yet more preferably about 95% to less than 100% to the amino acid
sequence of their starting Fc region. In a non-limiting embodiment
of the present invention, at least one amino acid is different
between a modified Fc region of the present invention and its
starting Fc region. Amino acid difference between a modified Fc
region of the present invention and its starting Fc region can also
be preferably specified based on amino acid differences at
above-described particular amino acid positions according to EU
numbering system.
[0280] Known methods such as site-directed mutagenesis (Kunkel et
al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and Overlap
extension PCR can be appropriately employed to modify the amino
acids of Fc regions. Furthermore, various known methods can also be
used as an amino acid modification method for substituting amino
acids by those other than natural amino acids (Annu Rev. Biophys.
Biomol. Struct. (2006) 35, 225-249; Proc. Natl. Acad. Sci. U.S.A.
(2003) 100 (11), 6353-6357). For example, a cell-free translation
system (Clover Direct (Protein Express)) containing tRNAs in which
amber suppressor tRNA, which is complementary to UAG codon (amber
codon) that is a stop codon, is linked with an unnatural amino acid
may be suitably used.
[0281] Fc regions comprised in the antigen-binding molecules of the
present invention that have a human FcRn-binding activity in the
acidic pH range can be obtained by any method. Specifically, one
can screen for antigen-binding molecules comprising an Fc region of
which human FcRn-binding activity in the acidic pH range is 20
.mu.M (KD) or stronger; in a more favorable embodiment, an Fc
region of which human FcRn-binding activity in the acidic pH range
is 2.0 .mu.M (KD) or stronger; and in an even more favorable
embodiment, an Fc region of which human FcRn-binding activity in
the acidic pH range is 0.5 .mu.M (KD) or stronger as a result of
modifying amino acids of an IgG-type human immunoglobulin used as a
starting Fc region. Preferred Fc regions of IgG-type
immunoglobulins for modification include, for example, those of
human IgGs such as IgG1, IgG2, IgG3, and IgG4 shown in SEQ ID NOs:
13, 14, 15, and 15, respectively, and variants thereof.
[0282] When an antigen-binding molecule comprises the Fc region of
human IgG1 as the Fc region, suitable examples of amino acids that
may be modified to achieve the above-mentioned desired effects on
FcRn binding under an acidic pH range condition by modifying amino
acids of an IgG-type human immunoglobulin as a starting Fc region,
include amino acids of positions 238, 252, 253, 254, 255, 256, 265,
272, 286, 288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360,
362, 376, 378, 380, 382, 386, 388, 400, 413, 415, 424, 433, 434,
435, 436, 439, and/or 447 according to EU numbering as described in
WO 2000/042072. Similarly, favorable examples of amino acids that
allow such modification include amino acids of positions 251, 252,
254, 255, 256, 308, 309, 311, 312, 385, 386, 387, 389, 428, 433,
434, and/or 436 according to EU numbering as described in WO
2002/060919. Furthermore, amino acids that allow such modification
include, for example, amino acids of positions 250, 314, and 428
according to EU numbering as described in WO2004/092219.
Furthermore, favorable examples of amino acids that allow such
modification include amino acids of positions 251, 252, 307, 308,
378, 428, 430, 434, and/or 436 according to EU numbering as
described in WO 2010/045193. Modification of these amino acids
enhances FcRn binding of the Fc region of an IgG-type
immunoglobulin under an acidic pH range condition.
[0283] Fc regions having human FcRn-binding activity in the neutral
pH range can also be obtained by modifying amino acids of human
immunoglobulin of IgG type used as the starting Fc region. The Fc
regions of IgG type immunoglobulins adequate for modification
include, for example, those of human IgGs such as IgG1, IgG2, IgG3,
and IgG4 respectively represented by SEQ ID NOs: 13, 14, 15, and
16, and modified forms thereof. Amino acids of any positions may be
modified into other amino acids, as long as the Fc regions have the
human FcRn-binding activity in the neutral pH range or can increase
the human FcRn-binding activity in the neutral range. When the
antigen-binding molecule contains the Fc region of human IgG1 as
the human Fc region, it is preferable that the resulting Fc region
contains a modification that results in the effect of enhancing the
human FcRn binding in the neutral pH range as compared to the
binding activity of the starting Fc region of human IgG1. Amino
acids that allow such modification include, for example, one ore
more amino acids selected from the group of positions 221 to 225,
227, 228, 230, 232, 233 to 241, 243 to 252, 254 to 260, 262 to 272,
274, 276, 278 to 289, 291 to 312, 315 to 320, 324, 325, 327 to 339,
341, 343, 345, 360, 362, 370, 375 to 378, 380, 382, 385 to 387,
389, 396, 414, 416, 423, 424, 426 to 438, 440, and 442 according to
EU numbering. Modification of these amino acids augments the human
FcRn binding of the Fc region of IgG-type immunoglobulin in the
neutral pH range.
[0284] From those described above, modifications that augment the
human FcRn binding in the neutral pH range are appropriately
selected for use in the present invention. Particularly preferred
amino acids of the modified Fc regions include, for example, amino
acids of positions 237, 248, 250, 252, 254, 255, 256, 257, 258,
265, 286, 289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314,
315, 317, 332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389,
424, 428, 433, 434, and 436 according to the EU numbering system.
The human FcRn-binding activity in the neutral pH range of the Fc
region contained in an antigen-binding molecule can be increased by
substituting at least one amino acid selected from the above amino
acids into a different amino acid.
[0285] Particularly preferred modifications include, for
example:
Met for the amino acid of position 237; Ile for the amino acid of
position 248; Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr for
the amino acid of position 250; Phe, Trp, or Tyr for the amino acid
of position 252; Thr for the amino acid of position 254; Glu for
the amino acid of position 255; Asp, Asn, Glu, or Gln for the amino
acid of position 256; Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or
Val for the amino acid of position 257; His for the amino acid of
position 258: Ala for the amino acid of position 265; Ala or Glu
for the amino acid of position 286; His for the amino acid of
position 289; Ala for the amino acid of position 297; Ala for the
amino acid of position 303; Ala for the amino acid of position 305;
Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,
Ser, Val, Trp, or Tyr for the amino acid of position 307; Ala, Phe,
Ile, Leu, Met, Pro, Gln, or Thr for the amino acid of position 308;
Ala, Asp, Glu, Pro, or Arg for the amino acid of position 309; Ala,
His, or Ile for the amino acid of position 311; Ala or His for the
amino acid of position 312; Lys or Arg for the amino acid of
position 314; Ala, Asp, or His for the amino acid of position 315;
Ala for the amino acid of position 317; Val for the amino acid of
position 332; Leu for the amino acid of position 334; His for the
amino acid of position 360; Ala for the amino acid of position 376;
Ala for the amino acid of position 380; Ala for the amino acid of
position 382; Ala for the amino acid of position 384; Asp or His
for the amino acid of position 385; Pro for the amino acid of
position 386; Glu for the amino acid of position 387; Ala or Ser
for the amino acid of position 389; Ala for the amino acid of
position 424; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro,
Gln, Ser, Thr, Val, Trp, or Tyr for the amino acid of position 428;
Lys for the amino acid of position 433; Ala, Phe, His, Ser, Trp, or
Tyr for the amino acid of position 434; and His, Ile, Leu, Phe,
Thr, or Val for the amino acid of position 436 of the Fc region
according to EU numbering. Meanwhile, the number of amino acids to
be modified is not particularly limited and amino acid at only one
site may be modified and amino acids at two or more sites may be
modified. Combinations of these amino acid modifications include,
for example, the amino acid modifications shown in Tables 5-1 to
5-33.
TABLE-US-00005 TABLE 5-1 Variant KD (M) Site of amino acid
modification F1 8.10E-07 N434W F2 3.20E-06 M252Y/S254T/T256E F3
2.50E-06 N434Y F4 5.80E-06 N434S F5 6.80E-06 N434A F7 5.60E-06
M252Y F8 4.20E-06 M252W F9 1.40E-07 M252Y/S254T/T256E/N434Y F10
6.90E-08 M252Y/S254T/T256E/K434W F11 3.10E-01 M252Y/N434Y F12
1.70E-07 M252Y/N434W F13 3.20E-07 M252W/N434Y F14 1.80E-07
M252W/N434W F19 4.60E-07 P257L/N434Y F20 4.60E-07 V308F/N434Y F21
3.00E-08 M252Y/V308P/N434Y F22 2.00E-06 M428L/N434S F25 9.20E-09
M252Y/S254T/T256E/V308P/N434W F26 1.00E-06 I332V F27 7.40E-06 G237M
F29 1.40E-06 I332V/N434Y F31 2.80E-06 G237M/V308F F32 8.00E-07
S254T/N434W F33 2.30E-06 S254T/N434Y F34 2.80E-07 T256E/N434W F35
8.40E-07 T256E/N434Y F36 3.60E-07 S254T/T256E/N434W F37 1.10E-06
S254T/T256E/N434Y F38 1.00E-07 M252Y/S254T/N434W F39 3.00E-07
M252Y/S254T/N434Y F40 8.20E-08 M252Y/T256E/N434W F41 1.50E-07
M252Y/T25GE/N434Y
Table 5-2 is a continuation of Table 5-1.
TABLE-US-00006 TABLE 5-2 F42 1.00E-06 M252Y/S254T/T256E/N434A F43
1.70E-06 M252Y/N434A F44 1.10E-06 M252W/N434A F47 2.40E-07
M252Y/T256Q/K434W F48 3.20E-07 M252Y/T256Q/N434Y F49 5.10E-07
M253F/T256D/N434W F50 1.20E-06 M252F/T256D/N434Y F51 8.10E-06
N434F/Y436H F52 3.10E-06 H433K/N434F/Y436H F53 1.00E-06 I332V/N434W
F54 8.40E-08 V308P/N434W F56 9.40E-07 I332V/M428L/N434Y F57
1.10E-05 G385D/Q386P/N389S F58 7.70E-07 G385D/Q386P/N389S/N434W F59
2.40E-06 G385D/Q386P/N389S/N434Y F60 1.10E-05 G385H F61 9.70E-07
G385H/N434W F62 1.90E-06 G385H/N434Y F63 2.50E-06 N434F F64
5.30E-06 N434H F65 2.90E-07 M252Y/S254T/T256E/N434F F66 4.30E-07
M252Y/S254T/T256E/N434H F67 6.30E-07 M252Y/N434F F68 9.30E-07
M252Y/N434H F69 5.10E-07 M428L/N434W F70 1.50E-06 M428L/N434Y F71
8.30E-08 M252Y/S254T/T256E/M428L/N434W F72 2.00E-07
M252Y/S254T/T256E/M428L/N434Y F73 1.70E-07 M252Y/M428L/N434W F74
4.60E-07 M252Y/M428L/N434Y F75 1.40E-06 M252Y/M428L/N434A F76
1.00E-06 M252Y/S254T/T256E/M428L/N434A F77 9.90E-07
T256E/M428L/N434Y F78 7.80E-07 S254T/M428L/N434W
Table 5-3 is a continuation of Table 5-2.
TABLE-US-00007 TABLE 5-3 F79 5.90E-06 S254T/256E/N434A F80 2.70E-06
M252Y/T256Q/N434A F81 1.60E-06 M252Y/T256E/N434A F82 1.10E-06
T256Q/N434W F83 2.60E-06 T256Q/N434Y F84 2.80E-07 M252W/T256Q/N434W
F85 5.50E-07 M252W/T256Q/N434Y F86 1.50E-06 S254T/T256Q/N434W F87
4.30E-06 S254T/T256Q/N434Y F88 1.90E-07 M252Y/S254T/T256Q/N434W F89
3.60E-07 M252Y/S254T/T256Q/N434Y F90 1.90E-08
M252Y/T256E/V308P/N434W F91 4.80E-08 M252Y/V308P/M428L/N434Y F92
1.10E-08 M252Y/S254T/T256E/V308P/M428L/N434W F93 7.40E-07
M252W/M428L/N434W F94 3.70E-07 P257L/M428L/N434Y F95 2.60E-07
M252Y/S254T/T256E/M428L/N434F F99 6.20E-07 M252Y/M256E/N434H F101
1.10E-07 M252W/T256Q/P257L/N434Y F103 4.40E-08
P238A/M252Y/V308P/N434Y F104 3.70E-08 M252Y/D265A/V308P/N434Y F105
7.50E-08 M252Y/T307A/V308P/N434Y F106 3.70E-08
M252Y/V303A/V308P/N434Y F107 3.40E-08 M252Y/V308P/D376A/N434Y F108
4.10E-08 M252Y/V305A/V308P/N434Y F109 3.20E-08
M252Y/V308P/Q311A/N434Y F111 3.20E-08 M252Y/V308P/K317A/N434Y F112
6.40E-08 M252Y/V308P/E380A/N434Y F113 3.20E-08
M252Y/V308P/E382A/N434Y F114 3.80E-08 M252Y/V308P/S424A/N434Y F115
6.60E-06 T307A/N434A F116 8.70E-06 E380A/N434A F118 1.40E-05 M428L
F119 5.40E-06 T250Q/M428L
Table 5-4 is a continuation of Table 5-3.
TABLE-US-00008 TABLE 5-4 F120 6.30E-08 P257L/V308P/M428L/N434Y F121
1.50E-08 M252Y/T256E/V308P/M428L/N434W F122 1.20E-07
M252Y/T256E/M428L/N434W F123 3.00E-08 M252Y/T256E/V308P/N434Y F124
2.90E-07 M252V/T256E/M428L/N434Y F125 2.40E-08
M252Y/S254T/T256E/V308P/M428L/N434Y F128 1.70E-07 P257L/M428L/N434W
F129 2.20E-07 P257A/M428L/N434Y P131 3.00E-06 P257G/M428L/N434Y
F132 2.10E-07 P257I/M428L/N434Y F133 4.10E-07 P257M/M428L/N434Y
F134 2.70E-07 P257N/M428L/N434V F135 7.50E-07 P257S/M428L/N434Y
F136 3.80E-07 F257T/M428L/N434Y F137 4.60E-07 P257V/M428L/N434Y
F139 1.50E-08 M252W/V308P/N434W F140 3.60E-08
S239K/M252Y/V308P/N434Y F141 3.50E-08 M252Y/S298G/V308P/N434Y F142
3.70E-08 M252Y/D270F/V308P/N434Y F143 2.00E-07 M252Y/V308A/N434Y
F145 5.30E-08 M252Y/V308F/N434Y F147 2.40E-07 M252Y/V308I/N434Y
F149 1.90E-07 M252Y/V308L/N434Y F150 2.00E-07 M252Y/V308M/N434Y
F152 2.70E-07 M252Y/V308Q/N434Y F154 1.80E-07 M252Y/V308T/N434Y
F157 1.50E-07 P257A/V308P/M428L/N434Y F158 5.90E-08
P257T/V308P/M428L/N434Y F159 4.40E-08 P257V/V308P/M428L/N434Y F160
8.50E-07 M252W/M428I/N434Y F162 1.60E-07 M252W/M428Y/N434Y F163
4.20E-07 M252W/M428F/N434Y F164 3.70E-07 P238A/M252W/N434Y F165
2.90E-07 M252W/D265A/N434Y
Table 5-5 is a continuation of Table 5-4.
TABLE-US-00009 TABLE 5-5 F166 1.50E-07 M252W/T307Q/N434Y F167
2.90E-07 M252W/V303A/N434Y F168 3.20E-07 M252W/D376A/N434Y F169
2.90E-07 M252W/V305A/N434Y F170 1.70E-07 M252W/Q311A/N434Y F171
1.90E-07 M352W/D312A/N434Y F172 2.20E-07 M252W/K317A/N434Y F173
7.70E-07 M252W/E380A/N434Y F174 3.40E-07 M252W/E382A/N434Y F175
2.70E-07 M252W/S424A/N434Y F176 2.90E-07 S239K/M252W/N434Y F177
2.80E-07 M252W/S298G/N434Y F178 2.70E-07 M252W/D270F/N434Y F179
3.10E-07 M252W/N325G/N434Y F182 6.60E-08 P257A/M428L/N434W F183
2.20E-07 P257T/M428L/N434W F184 2.70E-07 P257V/M428L/N434W F185
2.60E-07 M252W/I332V/N434Y F188 3.00E-06 P257I/Q311I F189 1.90E-07
M252Y/T307A/N434Y F190 1.10E-07 M252Y/T307Q/N434Y F191 1.60E-07
P257L/T307A/M428L/N434Y F192 1.10E-07 P257A/T307A/M428L/N434Y F193
8.50E-08 P257T/T307A/M428L/N434Y F194 1.20E-07
P257V/T307A/M428L/N434Y F195 5.60E-08 P257L/T307Q/M428L/N434Y F196
3.50E-08 P257A/T307Q/M428L/N434Y F197 3.30E-08
P257T/T307Q/M428L/N434Y F198 4.80E-08 P257V/T307Q/M428L/N434Y F201
2.10E-07 M252Y/T307D/N434Y F203 2.40E-07 M252Y/T307F/N434Y F204
2.10E-07 M252Y/T307G/N434Y F205 2.00E-07 M252Y/T307H/N434Y F206
2.30E-07 M253Y/T307I/N434Y
Table 5-6 is a continuation of Table 5-5.
TABLE-US-00010 TABLE 5-6 F207 9.40E-07 M252Y/T307K/N434Y F208
3.90E-07 M252Y/T307L/N434Y F209 1.30E-07 M252Y/T307M/N434Y F210
2.90E-07 M252Y/T307N/N434Y F211 2.40E-07 M252Y/T307P/N434Y F212
6.80E-07 M252Y/T307R/N434Y F213 2.30E-07 M252Y/T307S/N434Y F214
1.70E-07 M252Y/T307V/N434Y F215 9.60E-08 M252Y/T307W/N434Y F216
2.30E-07 M252Y/T307Y/N434Y F217 2.30E-07 M252Y/K334L/N434Y F218
2.60E-07 M252V/G385H/N434Y F219 2.50E-07 M252Y/T289H/N434Y F220
2.50E-07 M252Y/Q311H/N434Y F221 3.10E-07 M252Y/D312H/N434Y F222
3.40E-07 M252Y/M315H/N434Y F223 2.70E-07 M252Y/K360H/N434Y F225
1.50E-06 M252Y/L314R/N434Y F226 5.40E-07 M252Y/L314K/N434Y F227
1.20E-07 M252V/N286E/N434Y F228 2.30E-07 M252Y/L309E/N434Y F229
5.10E-07 M252Y/R255E/N434Y F230 2.50E-07 M252Y/P387E/N434Y F236
8.90E-07 K248I/M428L/N434Y F237 2.30E-07 M252Y/M428A/N434Y F238
7.40E-07 M252Y/M428D/N434Y F240 7.20E-07 M252Y/M428F/N434Y F241
1.50E-06 M252Y/M428G/N434Y F242 8.50E-07 M252Y/M428H/N434Y F243
1.80E-07 M252Y/M428I/N434Y F244 1.30E-06 M252Y/M428K/N434Y F245
4.70E-07 M252Y/M428N/N434Y F246 1.10E-06 M252Y/M428P/N434Y F247
4.40E-07 M252Y/M428Q/N434Y
Table 5-7 is a continuation of Table 5-6.
TABLE-US-00011 TABLE 5-7 F249 6.40E-07 M252Y/M428S/N434Y F250
2.90E-07 M252Y/M428T/N434Y F251 1.90E-07 M252Y/M428V/N434Y F252
1.00E-06 M252Y/M428W/K434Y F253 7.10E-07 M252Y/M428Y/N434Y F254
7.50E-08 M252W/T307Q/M428Y/N434Y F255 1.10E-07
M252W/Q311A/M428Y/N434Y F256 5.40E-08 M252W/T307Q/Q311A/M428Y/N434Y
F257 5.00E-07 M252Y/T307A/M428Y/N434Y F258 3.20E-07
M252Y/T307Q/M428Y/N434Y F259 2.80E-01 M252Y/D270F/N434Y F260
1.30E-07 M252Y/T307A/Q311A/N434Y F261 8.40E-08
M252Y/T307Q/Q311A/N434Y F262 1.90E-07 M252Y/T307A/Q311H/N434Y F263
1.10E-07 M252Y/T307Q/Q311H/N434Y F264 2.80E-07 M252Y/E382A/N434Y
F265 6.80E-07 M252Y/E382A/M428Y/N434Y F266 4.70E-07
M252Y/T307A/E382A/M428Y/N434Y F267 3.20E-07
M252Y/T307Q/E382A/M428Y/N434Y F268 6.30E-07 P238A/M252Y/M428F/N434Y
F269 5.20E-07 M252Y/V305A/M428F//N434Y F270 6.60E-07
M252Y/N325G/M428/N434Y F271 6.90E-07 M252Y/D376A/M428F/N434Y F272
6.80E-07 M252Y/E380A/M428F/N434Y F273 6.50E-07
M252Y/E382A/M428F/N434Y F274 7.60E-07 M252Y/E380A/E382A/M428F/N434Y
F275 4.20E-08 S239K/M252Y/V308P/E382A/N434Y F276 4.10E-08
M252Y/D270F/V308P/E382A/N434Y F277 1.30E-07
S239K/M252Y/V308P/M428Y/N434Y F278 3.00E-08
M252Y/T307Q/V308P/E382A/N434Y F279 6.10E-08
M252Y/V308P/Q311H/E382A/N434Y F280 4.10E-08
S239K/M252Y/D270F/V308P/N434Y F281 9.20E-08
M252Y/V308P/E382A/M428F/N434Y F282 2.90E-08
M252Y/V308P/E382A/M428L/N434Y
Table 5-8 is a continuation of Table 5-7.
TABLE-US-00012 TABLE 5-8 F283 1.00E-07
M252Y/V308P/E382A/M428Y/N434Y F284 1.00E-07 M252Y/V308P/M428Y/N434Y
F285 9.90E-08 M252Y/V308P/M428F/N434Y F286 1.20E-07
S239K/M252Y/V308P/E382A/M428Y/N434Y F287 1.00E-07
M252Y/V308P/E380A/E382A/M428F/N434Y F288 1.90E-07
M252Y/T256E/E382A/N434Y F289 4.80E-07 M252Y/T256E/M428Y/N434Y F290
4.60E-07 M252Y/T256E/E382A/M428Y/N434Y F292 2.30E-08
S239K/M252Y/V308P/E382A/M428I/N434Y F293 5.30E-08
M252Y/V308P/E380A/E382A/M428I/N434Y F294 1.10E-07
S239K/M252Y/V308P/M428F/N434Y F295 6.80E-07
S239K/M252Y/E380A/E382A/M428F/N434Y F296 4.90E-07
M252Y/Q311A/M428Y/N434Y F297 5.10E-07 M252Y/D312A/M428Y/N434Y F298
4.80E-07 M252Y/Q311A/D312A/M428Y/N434Y F299 9.40E-08
S239K/M252Y/V308P/Q311A/M428Y/N434Y F300 8.30E-08
S239K/M252Y/V308P/D312A/M428Y/N434Y F301 7.20E-08
S239K/M252Y/V308P/Q311A/D312A/M428Y/N434Y F302 1.90E-07
M252Y/T256E/T307P/N434Y F303 6.70E-07 M252Y/T307P/M428Y/N434Y F304
1.60E-08 M252W/V308P/M428Y/N434Y F305 2.70E-08
M252Y/T256E/V308P/E382A/N434Y F306 3.60E-08 M252W/V308P/E382A/N434Y
F307 3.60E-08 S239K/M252W/V308P/E382A/N434Y F308 1.90E-08
S239K/M252W/V308P/E382A/M428Y/N434Y F310 9.40E-08
S239K/M252W/V308P/E382A/M428I/N434Y F311 2.80E-08
S239K/M252W/V308P/M428F/N434Y F312 4.50E-07
S239K/M252W/E380A/E382A/M428F/N434Y F313 6.50E-07
S239K/M252Y/T307P/M428Y/N434Y F314 3.20E-07
M252Y/T256E/Q311A/D312A/M428Y/N434Y F315 6.80E-07
S239K/M252Y/M428Y/N434Y F316 7.00E-07 S239K/M252Y/D270F/M428Y/N434Y
F317 1.10E-07 S239K/M252Y/D270F/V308P/M428Y/N434Y F318 1.80E-08
S239K/M252Y/V308P/M428I/N434Y
Table 5-9 is a continuation of Table 5-8.
TABLE-US-00013 TABLE 5-9 F320 2.00E-08
S239K/M252Y/V308P/N325G/E382A/M428I/N434Y F321 3.20E-08
S239K/M252Y/D270F/V308P/N325G/N434Y F322 9.20E-08
S239K/M252Y/D270F/T307P/V308P/N434Y F323 2.70E-08
S239K/M252Y/T256E/D270F/V308P/N434Y F324 2.80E-08
S239K/M252Y/D270F/T307Q/V308P/N434Y F325 2.10E-08
S239K/M252Y/D270F/T307Q/V308P/Q311A/N434Y F326 7.50E-08
S239K/M252Y/D270F/T307Q/Q311A/N434Y F327 6.50E-08
S239K/M252Y/T256E/D270F/T307Q/Q311A/N434Y F328 1.90E-08
S239K/M252Y/D270F/V308P/M428I/N434Y F329 1.20E-08
S239K/M252Y/D270F/N286E/V308P/N434Y F330 3.60E-08
S239K/M252Y/D270F/V308P/L309E/N434Y F331 3.00E-08
S239K/M252Y/D270F/V308P/P387E/N434Y F333 7.40E-08
S239K/M252Y/D270F/T307Q/L309E/Q311A/N434Y F334 1.90E-08
S239K/M252Y/D270F/V308P/N325G/M428I/N434Y F335 1.50E-08
S239K/M252Y/T256E/D270F/V308P/M428I/N434Y F336 1.40E-08
S239K/M252Y/D270F/T307Q/V308P/Q311A/M428I/ N434Y F337 5.60E-08
S239K/M252Y/D270F/T307Q/Q311A/M428I/N434Y F338 7.70E-09
S239K/M252Y/D270F/N286E/V308P/M428I/N434Y F339 1.90E-08
S299K/M252Y/D270F/V308P/L309E/M428I/N434Y F343 3.20E-08
S239K/M252Y/D270F/V308P/M428L/N434Y F344 3.00E-08
S239K/M252Y/V308P/M428L/N434Y F349 1.50E-07
S239K/M252Y/V308P/L309P/M428L/N434Y F350 1.70E-07
S239K/M252Y/V308P/L309R/M428L/N434Y F352 6.00E-07
S239K/M252Y/L309P/M428L/N434Y F353 1.10E-06
S239K/M252Y/L309R/M428L/N434Y F354 2.80E-08
S239K/M252Y/T307Q/V308P/M428L/N434Y F356 3.40E-08
S239K/M252Y/D270F/V308P/L309E/P387E/N434Y F357 1.60E-08
S239K/M252Y/T256E/D270F/V308P/N325G/M428I/ N434Y F358 1.00E-07
S239K/M252Y/T307Q/N434Y F359 4.20E-07 P257V/T307Q/M428I/N434Y F360
1.30E-06 P257V/T307Q/M428V/N434Y F362 5.40E-08
P257V/T307Q/N325G/M428L/N434Y F363 4.10E-08
P257V/T307Q/Q311A/M428L/N434Y F364 3.50E-08
P257V/T307Q/Q311A/N325G/M428L/N434Y
Table 5-10 is a continuation of Table 5-9.
TABLE-US-00014 TABLE 5-10 F365 5.10E-08
P257V/V305A/T307Q/M438L/N434Y F367 1.50E-08
S239K/M252Y/E258H/D270F/T307Q/V308P/ Q311A/N434Y F368 2.00E-08
S239K/M252Y/D270F/V308P/N325G/E382A/ M428I/N434Y F369 7.50E-08
M252Y/P257V/T307Q/M428I/N434Y F372 1.30E-08
S239K/M252W/V308P/M428Y/N434Y F373 1.10E-08
S239K/M252W/V308P/Q311A/M428Y/N434Y F374 1.20E-08
S239K/M252W/T256E/V308P/M428Y/N434Y F375 5.50E-09
S239K/M252W/N286E/V308P/M428Y/N434Y F376 9.60E-09
S239K/M252Y/T256E/D270F/N286E/V308P/N434Y F377 1.30E-07
S239K/M252W/T307P/M428Y/N434Y F379 9.00E-09
S239K/M252W/T256E/V308P/Q311A/M428Y/N434Y F380 5.60E-09
S239K/M252W/T256E/N286E/V308P/M428Y/N434Y F381 1.10E-07
P257V/T307A/Q311A/M428L/N434Y F382 8.70E-08
P257V/V305A/T307A/M428L/N434Y F386 3.20E-08 M252Y/V308P/L309E/N434Y
F387 1.50E-07 M252Y/V308P/L309D/N434Y F388 7.00E-08
M252Y/V308P/L309A/N434Y F389 1.70E-08 M252W/V308P/L309E/M428Y/N434Y
F390 6.80E-08 M253W/V308P/L309D/M428Y/N434Y F391 3.60E-08
M252W/V308P/L309A/M428Y/N434Y F392 6.90E-09
S239K/M252Y/N286E/V308P/M428I/N434Y F393 1.20E-08
S239K/M252Y/N286E/V308P/N434Y F394 5.30E-08
S239K/M252Y/T307Q/Q311A/M428I/N434Y F395 2.40E-08
S239K/M252Y/T256E/V308P/N434Y F396 2.00E-08
S239K/M252Y/D270F/N286E/T307Q/Q311A/M428I/ N434Y F397 4.50E-08
S239K/M252Y/D270F/T307Q/Q311A/P387E/M428I/ N434Y F398 4.40E-09
S239K/M252Y/D270F/N286E/T307Q/V308P/Q311A/ M428I/N434Y F399
6.50E-09 S239K/M252Y/D270F/N286E/T307Q/V308P/M428I/ N434Y F400
6.10E-09 S239K/M252Y/D270F/N286E/V308P/Q311A/M428I/ N434Y F401
6.90E-09 S239K/M252Y/D270F/N286E/V308P/P387E/M428I/ N434Y F402
2.30E-08 P257V/T307Q/M428L/N434W F403 5.10E-08
P257V/T307A/M428L/N434W F404 9.40E-08 P257A/T307Q/L309P/M428L/N434Y
F405 1.70E-07 P257V/T307Q/L309P/M428L/N434Y
Table 5-11 is a continuation of Table 5-10.
TABLE-US-00015 TABLE 5-11 F406 1.50E-07
P257A/T307Q/L309R/M428L/N434Y F407 1.60E-07
P257V/T307Q/L309R/M428L/N434Y F408 2.50E-07 P257V/M286E/M428L/N434Y
F409 2.00E-07 P257V/P387E/M428L/N434Y F410 2.20E-07
P257V/T307H/M428L/N434Y F411 1.30E-07 P257V/T307N/M428L/N434Y F412
8.80E-08 P257V/T307G/M428L/N434Y F413 1.20E-07
P257V/T307P/M428L/N434Y F414 1.10E-07 P257V/T307S/M428L/N434Y F415
5.60E-08 P257V/N286E/T307A/M428L/N434Y F416 9.40E-08
P257V/T307A/P387E/M428L/N434Y F418 6.20E-07
S239K/M252Y/T307P/N325G/M428Y/N434Y F419 1.60E-07
M252Y/T307A/Q311H/K360K/N434Y F420 1.50E-07
M252Y/T307A/Q311H/P387E/N434Y F421 1.30E-07
M252Y/T307A/Q311H/M428A/N434Y F422 1.80E-07
M252Y/T307A/Q311H/E382A-/N434Y F423 8.40E-08
M252Y/T307W/Q311H/N434Y F424 9.40E-08 S239K/P257A/V308P/M428L/N434Y
F425 8.00E-08 P257A/V308P/L309E/M428L/N434Y F426 8.40E-08
P257V/T307Q/N434Y F427 1.10E-07 M252Y/P257V/T307Q/M428V/N434Y F428
8.00E-08 M252Y/P257V/T307Q/M428L/N434Y F429 3.70E-08
M252Y/P257V/T307Q/N434Y F430 8.10E-08 M252Y/P257V/T307Q/M428Y/N434Y
F431 6.50E-08 M252Y/P257V/T307Q/M428F/N434Y F432 9.20E-07
P257V/T307Q/Q311A/N325G/M428V/N434Y F433 6.00E-08
P257V/T307Q/Q311A/N325G/M434Y F434 2.00E-08
P257V/T307Q/Q311A/N325G/M428Y/N434Y F435 2.50E-08
P257V/T307Q/Q311A/N325G/M428F/N434Y F436 2.50E-07
P257A/T307Q/M428V/N434Y F437 5.70E-08 P257A/T307Q/N434Y F438
3.60E-08 P257A/T307Q/M428Y/N434Y F439 4.00E-08
P257A/T307Q/M428F/N434Y F440 1.50E-08
P257V/N286E/T307Q/Q311A/N325G/M438L/N434Y
Table 5-12 is a continuation of Table 5-11.
TABLE-US-00016 TABLE 5-12 F441 1.80E-07 P257A/Q311A/M428L/N434Y
F442 2.00E-07 P257A/Q311H/M428L/N434Y F443 5.50E-08
P257A/T307Q/Q311A/M428L/N434Y F444 1.40E-07
P257A/T307A/Q311A/M428L/N434Y F445 6.20E-08
P257A/T307Q/Q311H/M428L/N434Y F446 1.10E-07
P257A/T307A/Q311H/M428L/N434Y F447 1.40E-08
P257A/N286E/T307Q/M428/N434Y F448 5.30E-08
P257A/N286E/T307A/M428L/N434Y F449 5.70E-07
S239K/M252Y/D270F/T307P/N325G/M428Y/N434Y F450 5.20E-07
S239K/M252Y/T307P/L309E/N325G/M428Y/N434Y F451 1.00E-07
P257S/T307A/M428L/N434Y F452 1.40E-07 P257M/T307A/M428L/N434Y F453
7.80E-08 P257N/T307A/M428L/N434Y F454 9.60E-08
P257I/T307A/M428L/N434Y F455 2.70E-08 P257V/T307Q/M428Y/N434Y F456
3.40E-08 P257V/T307Q/M428F/N434Y F457 4.00E-08
S239K/P257V/V308P/M428L/N434Y F458 1.50E-08
P257V/T307Q/V308P/N325G/M428L/N434Y F459 1.30E-08
P257V/T307Q/V308P/Q311A/N325G/M428L/N434Y F460 4.70E-08
P257V/T307A/V308P/N325G/M428L/N434Y F462 8.50E-08
P257A/V308P/N325G/M428L/N434Y F463 1.30E-07
P257A/T307A/V308P/M428L/N434Y F464 5.50E-08
P257A/T307Q/V308P/M428L/N434Y F465 2.10E-08
P257V/N286E/T307Q/N325G/M428L/N434Y F466 3.50E-07 T256E/P257V/N434Y
F467 5.70E-07 T256E/P257T/N434Y F468 5.70E-08
S239K/P257T/V308P/M428L/N434Y F469 5.60E-08
P257T/V308P/N325G/M428L/N434Y F470 5.40E-08
T256E/P257T/V308P/N325G/M428L/N434Y F471 6.60E-08
P257T/V308P/N325G/E382A/M428L/N434Y F472 5.40E-08
P257T/V308P/N325C/P387E/M428L/N434Y F473 4.50E-07
P257T/V308P/L309P/N325G/M428L/N434Y F474 3.50E-07
P257T/V308P/L309R/N325G/M428L/N434Y F475 4.30E-08
T256E/P257V/T307Q/M428L/N434Y
Table 5-13 is a continuation of Table 5-12.
TABLE-US-00017 TABLE 5-13 F476 5.50E-08
P257V/T307Q/E382A/M428L/N434Y F477 4.30E-08
P257V/T307Q/P387E/M428L/N434Y F480 3.90E-08 P257L/V308P/N434Y F481
5.60E-08 P257T/T307Q/N434Y F482 7.00E-08 P257V/T307Q/N325G/N434Y
F483 5.70E-08 P257V/T307Q/Q311A/N434Y F484 6.20E-08
P257V/V305A/T307Q/N434Y F485 9.70E-08 P257V/N286E/T307A/N434Y F486
3.40E-07 P257V/T307Q/L309R/Q311H/M428L/N434Y F488 3.50E-08
P257V/V308P/N325G/M428L/N434Y F490 7.50E-08
S239K/P257V/V308P/Q311H/M428L/N434Y F492 9.80E-08
P257V/V305A/T307A/N325G/M428L/N434Y F493 4.90E-07
S239K/D270F/T307P/N325G/M428Y/N434Y F497 3.10E-06
P257T/T307A/M428V/N434Y F498 1.30E-06 P257A/M428V/N434Y F499
5.20E-07 P257A/T307A/M428V/N434Y F500 4.30E-08
P257S/T307Q/M428L/N434Y F506 1.90E-07 P257V/N297A/T307Q/M428L/N434Y
F507 5.10E-08 P257V/N286A/T307Q/M428L/N434Y F508 1.10E-07
P257V/T307Q/N315A/M428L/N434Y F509 5.80E-08
P257V/T307Q/N384A/M428L/N434Y F510 5.30E-08
P257V/T307Q/N389A/M428L/N434Y F511 4.20E-07 P257V/N434Y F512
5.80E-07 P257T/N434Y F517 3.10E-07 P257V/N286E/N434Y F518 4.20E-07
P257T/N286E/N434Y F519 2.60E-08 P257V/N286E/T307Q/N434Y F521
1.10E-08 P257V/N286E/T307Q/M428Y/N434Y F523 2.60E-08
P257V/V305A/T307Q/M428Y/N434Y F526 1.90E-08 P257T/T307Q/M428Y/N434Y
F527 9.40E-09 P257V/T307Q/V308P/N325G/M428Y/N434Y F529 2.50E-08
P257T/T307Q/M428F/N434Y F533 1.20E-08 P257A/N286E/T307Q/M428F/N434Y
F534 1.20E-08 P257A/N286E/T307Q/M428Y/N434Y
Table 5-14 is a continuation of Table 5-13.
TABLE-US-00018 TABLE 5-14 F535 3.90E-08
T250A/P257V/T307Q/M428L/N434Y F538 9.90E-08
T250F/P257V/T307Q/M428L/N434Y F541 6.00E-08
T250I/P257V/T307Q/M428L/N434Y F544 3.10E-08
T250M/P257V/T307Q/M428L/N434Y F549 5.40E-08
T250S/P257V/T307Q/M428L/N434Y F550 5.90E-08
T250V/P257V/T307Q/M428L/N434Y F551 1.20E-07
T250W/P257V/T307Q/M428L/N434Y F552 1.10E-07
T250Y/P257V/T307Q/M428L/N437Y F553 1.70E-07 M252Y/Q311A/N434Y F554
2.80E-08 S239K/M252Y/S254T/V308P/N434Y F556 1.50E-06
M252Y/T307Q/Q311A F559 8.00E-08 M252Y/S254T/N286E/N434Y F560
2.80E-08 M252Y/S254T/V308P/N434Y F561 1.40E-07
M252Y/S254T/T307A/N434Y F562 8.30E-08 M252Y/S254T/T307Q/N434Y F563
1.30E-07 M252Y/S254T/Q311A/N434Y F564 1.90E-07
M252Y/S254T/Q311H/N434Y F565 9.20E-08 M252Y/S254T/T307A/Q311A/N434Y
F566 6.10E-08 M252Y/S254T/T307Q/Q311A/N434Y F567 2.20E-07
M252Y/S254T/M428I/N434Y F568 1.10E-07 M252Y/T256E/T307A/Q311H/N434Y
F569 2.00E-07 M252Y/T256Q/T307A/Q311H/N434Y F570 1.30E-07
M252Y/S254T/T307A/Q311H/N434Y F571 8.10E-08
M252Y/N286E/T307A/Q311H/N434Y F572 1.00E-07
M252Y/T307A/Q311H/M428I/N434Y F576 1.06E-06 M252Y/T256E/T307Q/Q311H
F577 1.30E-06 M252Y/N286E/T307A/Q311A F578 5.70E-07
M252Y/M286E/T307Q/Q311A F580 8.60E-07 M252Y/N286E/T307Q/Q311H F581
7.20E-08 M252Y/T256E/N286E/N434Y F582 7.50E-07 S239K/M252Y/V308P
F583 7.80E-07 S239K/M252Y/V308P/E382A F584 6.30E-07
S239K/M252Y/T256E/V306P F585 2.90E-07 S239K/M252Y/N286E/V308P
Table 5-15 is a continuation of Table 5-14.
TABLE-US-00019 TABLE 5-15 F586 1.40E-07
S239K/M252Y/N286E/V308P/M428I F587 1.90E-07 M252Y/N286E/M428L/N434Y
F592 2.00E-07 M252Y/S254T/E382A/N434Y F593 3.10E-08
S239K/M252Y/S254T/V308P/M428I/N434Y F594 1.60E-08
S239K/M252Y/T256E/V308P/M428I/N434Y F595 1.80E-07
S239K/M252Y/M428I/N434Y F596 4.00E-07 M252Y/D312A/E382A/M428Y/N434Y
F597 2.20E-07 M252Y/E382A/P387E/N434Y F598 1.40E-07
M252Y/D312A/P387E/N434Y F599 5.20E-07 M252Y/P387E/M428Y/N434Y F600
2.80E-07 M252Y/T356Q/E382A/N434Y F601 9.60E-09
M252Y/N286E/V308P/N434Y F608 G236A/S239D/I332E F611 2.80E-07
M252Y/V305T/T307P/V308I/L309A/N434Y F612 3.60E-07
M252Y/T307P/V308I/L309A/N434Y F613 S239D/A330L/I332E F616
S239D/K326D/L328Y F617 7.40E-07 S239K/N434W F618 6.40E-07
S239K/V308F/N434Y F619 3.10E-07 5239K/M252Y/N434Y F620 2.10E-07
S239K/M252Y/S254T/N434Y F621 1.50E-07 S239K/M252Y/T307A/Q311H/N434Y
F622 3.50E-07 S239K/M252Y/T256Q/N434Y F623 1.80E-07
S239K/M252W/N434W F624 1.40E-08 S239K/P257A/N286E/T307Q/M428L/N434Y
F625 7.60E-08 S239K/P257A/T307Q/M428L/N434Y F626 1.30E-06 V308P
F629 3.90E-08 M252Y/V279L/V308P/N434Y F630 3.70E-08
S239K/M252Y/V279L/V308P/N434Y F633 2.40E-08 M252Y/V282D/V308P/N434Y
F634 3.20E-06 S239K/M252Y/V282D/V308P/N434Y F635 4.50E-08
M252Y/V284K/V308P/N434Y F636 4.80E-08 S239K/M252Y/V284K/V308P/N434Y
F637 1.50E-07 M252Y/K288S/V308P/N434Y
Table 5-16 is a continuation of Table 5-15.
TABLE-US-00020 TABLE 5-16 F638 1.40E-07
S239K/M252Y/K288S/V308P/N434Y F639 2.70E-08 M252Y/V308P/G385R/N434Y
F640 3.60E-08 S239K/M252Y/V308P/G385R/N434Y F641 3.00E-08
M252Y/V308P/Q386K/N434Y F642 3.00E-08 S239K/M252Y/V308P/Q386K/N434Y
F643 3.20E-08 L235G/G236R/S239K/M252Y/V308P/N434Y F644 3.00E-08
G236R/S239K/M252Y/V308P/N434Y F645 3.30E-08
S239K/M252Y/V308P/L328R/N434Y F646 3.80E-08
S239K/M252Y/N297A/V308P/N434Y F647 2.90E-08 P238D/M252Y/V308P/N434Y
F648 P238D F649 1.20E-07 S239K/M252Y/N286E/N434Y F650 1.70E-07
S239K/M252Y/T256E/N434Y F651 1.80E-07 S239K/M252Y/Q311A/N434Y F652
2.40E-07 P238D/M252Y/N434Y F654 3.20E-08
L235K/S239K/M252Y/V308P/N434Y F655 3.40E-08
L235R/S239K/M252Y/V308P/S434Y F656 3.30E-08
G237K/S239K/M252Y/V308P/N434Y F657 3.20E-08
G237R/S239K/M252Y/V308P/N434Y F658 3.20E-08
P238K/S239K/M252Y/V308P/N434Y F659 3.00E-08
P238R/S239K/M252Y/V308P/N434Y F660 3.10E-08
S239K/M252Y/V308P/P329K/N434Y F661 3.40E-08
S239K/M252Y/V308P/P329R/N434Y F663 6.40E-09
S239K/M252Y/N286E/T307Q/V308P/Q311A/N434Y F664 3.90E-08
M252Y/N286A/V308P/N434Y F665 2.00E-08 M252Y/N286D/V308P/N434Y F666
2.10E-08 M252Y/N286F/V308P/N434Y F667 3.00E-08
M252Y/N286G/V308P/N434Y F668 4.00E-08 M252Y/N286H/V308P/N434Y F669
3.50E-08 M252Y/N286I/V308P/N434Y F670 2.10E-07
M252Y/N286K/V308P/N434Y F671 2.20E-08 M252Y/N286L/V308P/N434Y F672
2.40E-08 M252Y/N286M/V308P/N434Y F673 2.30E-08
M252Y/N286P/V308P/N434Y
Table 5-17 is a continuation of Table 5-16.
TABLE-US-00021 TABLE 5-17 F674 3.20E-08 M252Y/N286Q/V308P/N434Y
F675 5.10E-08 M252Y/N286R/V308P/N434Y F676 3.20E-08
M252Y/N286S/V308P/N434Y F677 4.70E-08 M252Y/N286T/V308P/N434Y F678
3.30E-08 M252Y/N286V/V308P/N434Y F679 1.70E-08
M252Y/N286W/V308P/N434Y F680 1.50E-08 M252Y/N286Y/V308P/N434Y F681
4.90E-08 M252Y/K288A/V308P/N434Y F682 8.20E-08
M252Y/K288D/V308P/N434Y F683 5.00E-08 M252Y/K288E/V308P/N434Y F684
5.10E-08 M252Y/K288F/V308P/N434Y F685 5.30E-08
M252Y/K288G/V308P/N434Y F686 4.60E-08 M252Y/K288H/V308P/N434Y F687
4.90E-08 M252Y/K288I/V308/N434Y F688 2.80E-08
M252Y/K288L/V308P/N434Y F689 4.10E-08 M252Y/K288M/V308P/N434Y F690
1.00E-07 M252Y/K288N/V308P/N434Y F691 3.20E-07
M252Y/K288P/V308P/N434Y F692 3.90E-08 M252Y/K288Q/V308P/N434Y F693
3.60E-08 M252Y/K288R/V308P/N434Y F694 4.70E-08
M252Y/K288V/V308P/N434Y F695 4.00E-08 M252Y/K288W/V308P/N434Y F696
4.40E-08 M252Y/K288V/V308P/N434Y F697 3.10E-08
S239K/M252Y/V308P/N325G/N434Y F698 2.20E-08
M252Y/N286E/T307Q/Q311A/N434Y F699 2.30E-08
S239K/M252Y/N286E/T307Q/Q311A/N434Y F700 5.20E-08
M252Y/V308P/L328E/N434Y F705 7.10E-09 M252Y/N286E/V308P/M428I/N434Y
F706 1.80E-08 M252Y/N286E/T307Q/Q311A/M428I/N434Y F707 5.90E-09
M252Y/N286E/T307Q/V308P/Q311A/N434Y F708 4.10E-09
M252Y/N286E/T307Q/V308P/Q311A/M428I/N434Y F709 2.00E-08
S239K/M252Y/N286E/T307Q/Q311A/M428I/N434Y F710 1.50E-08
P238D/M252Y/N286E/T307Q/Q311A/M428I/N434Y F711 6.50E-08
S239K/M252Y/T307Q/Q311A/N434Y
Table 5-18 is a continuation of Table 5-17.
TABLE-US-00022 TABLE 5-18 F712 6.00E-08
P238D/M252Y/T301Q/Q311A/N434Y F713 2.00E-08
P238D/M252Y/N286E/T307Q/Q311A/N434Y F714 2.30E-07
P238D/M252Y/N325S/N434Y F715 2.30E-07 P238D/M252Y/N325M/N434Y F716
2.70E-07 P238D/M252Y/N325L/N434Y F717 2.60E-07
P238D/M252Y/N325I/N434Y F718 2.80E-07 P238D/M252Y/Q295M/N434Y F119
7.40E-08 P238D/M252Y/N325G/N434Y F720 2.40E-08
M252Y/T307Q/V308P/Q311A/N434Y F721 1.50E-08
M252Y/T307Q/V308P/Q311A/M428I/N434Y F722 2.70E-07
P238D/M252Y/A327G/N434Y F723 2.80E-07 P238D/M252Y/L328D/N434Y F724
2.50E-07 P238D/M252Y/L32BE/N434Y F725 4.20E-08
L235K/G237R/S239K/M252Y/V308P/N434Y F726 3.70E-08
L235K/P238K/S239K/M252Y/V308P/N434Y F729 9.20E-07 T307A/Q311A/N434Y
F730 6.00E-07 T307Q/Q311A/N434Y F731 8.50E-07 T307A/Q311H/N434Y
F732 6.80E-07 T307Q/Q311H/N434Y F733 3.20E-07 M252Y/L328E/N434Y
F734 3.10E-07 G236D/M252Y/L328E/N434Y F736 3.10E-07
M252Y/S267M/L328E/N434Y F737 3.10E-07 M252Y/S267L/L328E/N434Y F738
3.50E-07 P238D/M252Y/T307P/N434Y F739 2.20E-07
M252Y/T307P/Q311A/N434Y F740 2.90E-07 M252Y/T307P/Q311H/N434Y F741
3.10E-07 P238D/T250A/M252Y/N434Y F744 9.90E-07
P238D/T250F/M252Y/N434Y F745 6.60E-07 P238D/T250G/M252Y/N434Y F746
6.00E-07 P238D/T250H/M252Y/N434Y F747 2.80E-07
P238D/T250I/M252Y/N434Y F749 5.10E-07 R238D/T250L/M252Y/N434Y F750
3.00E-07 P238D/T250M/M252Y/N434Y F751 5.30E-07
P238D/T250N/M252Y/N434Y
Table 5-19 is a continuation of Table 5-18.
TABLE-US-00023 TABLE 5-19 F753 1.80E-07 P238D/T250Q/M252Y/N434Y
F755 3.50E-07 F238D/T250S/M252Y/N434Y F756 3.70E-07
P235D/T250V/M252Y/N434Y F757 1.20E-06 P238D/T250W/M252Y/N434Y F758
1.40E-06 P238D/T250Y/M252Y/N434Y F759 L235K/S239K F760 L235R/S239K
F761 1.10E-06 P238D/N434Y F762 3.60E-08
L235K/S239K/M252Y/N286E/T307Q/Q311A/N434Y F763 3.50E-08
L235R/S239K/M252Y/N286E/T307Q/Q311A/N434Y F764 6.30E-07
P238D/T307Q/Q311A/N434Y F765 8.50E-08
P238D/M252V/T307Q/L309E/Q311A/N434Y F766 6.00E-07
T307A/L309E/Q311A/N434Y F767 4.30E-07 T307Q/L309E/Q311A/N434Y F768
6.40E-07 T307A/L309E/Q311H/N434Y F769 4.60E-07
T307Q/L309E/Q311H/N434Y F770 3.00E-07 M252Y/T256A/N434Y F771
4.00E-07 M252Y/E272A/N434Y F772 3.80E-07 M252Y/K274A/N434Y F773
3.90E-07 M252Y/V282A/N434Y F774 4.00E-07 M252Y/N286A/N434Y F775
6.20E-07 M252Y/K338A/N434Y F776 3.90E-07 M252Y/K340A/N434Y F777
3.90E-07 M252Y/E345A/N434Y F779 3.90E-07 M252Y/N361A/N434Y F780
3.90E-07 M252Y/Q362A/N434Y F781 3.70E-07 M252Y/S375A/N434Y F782
3.50E-07 M252Y/Y391A/N434Y F783 4.00E-07 M252Y/D413A/N434Y F784
5.00E-07 M252Y/L309A/N434Y F785 7.40E-07 M252Y/L309H/N434Y F786
2.80E-08 M252Y/S254T/N286E/T307Q/Q311A/N434Y F787 8.80E-08
M252Y/S254T/T307Q/L309E/Q311A/N434Y F788 4.10E-07
M252Y/N315A/M434Y
Table 5-20 is a continuation of Table 5-19.
TABLE-US-00024 TABLE 5-20 F789 1.50E-07 M252Y/N315D/N434Y F790
2.70E-07 M252Y/N315E/N434Y F791 4.40E-07 M252Y/N315F/N434Y F792
4.40E-07 M252Y/N315G/N434Y F793 3.30E-07 M252Y/N315I/N434Y F794
4.10E-07 M252Y/N315K/N434Y F795 3.10E-07 M252Y/N315L/N434Y F796
3.40E-07 M252Y/N315M/N434Y F798 3.50E-07 M252Y/N315Q/N434Y F799
4.10E-07 M252Y/N315R/N434Y F800 3.80E-07 M252Y/N315S/N434Y F801
4.40E-07 M252Y/N315T/N434Y F802 3.30E-07 M252Y/N315V/N434Y F803
3.60E-07 M252Y/N315W/N434Y F804 4.00E-07 M252Y/N315Y/N434Y F805
3.00E-07 M252Y/N325A/N434Y F806 3.10E-07 M252Y/N384A/N434Y F807
3.20E-07 M252Y/N389A/N434Y F808 3.20E-07 M352Y/N389A/N390A/N434Y
F809 2.20E-07 M522Y/S254T/T256S/N434Y F810 2.20E-07
M252Y/A378V/N434Y F811 4.90E-07 M252Y/E380S/N434Y F812 2.70E-07
M252Y/E382V/N434Y F813 2.80E-07 M252Y/S424E/N434Y F814 1.20E-07
M252Y/N434Y/Y436I F815 5.50E-07 M252Y/N434Y/T437R F816 3.60E-07
P238D/T250V/M252Y/T307P/N434Y F817 9.80E-08
P238D/T250V/M252Y/T307Q/Q311A/N434Y F819 1.40E-07
P938D/M252Y/N286E/N434Y F820 3.40E-07 L235K/S239K/M252Y/N434Y F821
3.10E-07 L235R/S239K/M252Y/N434Y F822 1.10E-06
P238D/T250Y/M252Y/W313Y/N434Y F823 1.10E-06
P238D/T250Y/M252Y/W313F/N434Y F828 2.50E-06
P238D/T250V/M253Y/I253V/N434Y
Table 5-21 is a continuation of Table 5-20.
TABLE-US-00025 TABLE 5-21 F831 1.60E-06
P238D/T250V/M252Y/R255A/N434Y F832 2.60E-06
P238D/T250V/M252Y/R255D/N434Y F833 8.00E-07
P238D/T250V/M252Y/R255E/N434Y F834 8.10E-07
P238D/T250V/M252Y/R255F/N434Y F836 5.00E-07
P238D/T250V/M252Y/R255H/N434Y F837 5.60E-07
P238D/T250V/M252Y/R255I/N434Y F838 4.30E-07
P238D/T250V/M252Y/2255K/N434Y F839 3.40E-07
P238D/T250V/M252Y/R255L/N434Y F840 4.20E-07
P238D/T250V/M253Y/R255M/N434Y F841 1.10E-06
P238D/T250V/M252Y/R255N/N434Y F843 6.60E-07
P238D/T250V/M252Y/R255Q/N434Y F844 1.30E-06
P238D/T250V/M252Y/R255S/N434Y F847 3.40E-07
P238D/T250V/M252Y/R255W/N434Y F848 8.30E-07
P238D/T250V/M252Y/R255Y/N434Y F849 3.30E-07 M252Y/D280A/N434Y F850
2.90E-07 M252Y/D280E/N434Y F852 3.30E-07 M252Y/D280G/N434Y F853
3.20E-07 M252Y/D280H/N434Y F855 3.20E-07 M252Y/D280K/N434Y F858
3.20E-07 M252Y/D280N/N434Y F860 3.30E-07 M252Y/D280Q/N434Y F861
3.20E-07 M252Y/D280R/N434Y F862 3.00E-07 M252Y/D280S/N434Y F863
2.70E-07 M252Y/D280T/N434Y F867 2.80E-07 M252Y/N384A/N389A/N434Y
F868 2.00E-08 G236A/S239D/M252Y/N286E/T307Q/Q311A/N434Y F869
G236A/S239D F870 7.30E-08 L235K/S239K/M252Y/T307Q/Q311A/N434Y F871
7.10E-08 L235R/S239K/M252Y/T307Q/Q311A/N434Y F872 1.30E-07
L235K/S239K/M252Y/N286E/N434Y F873 1.20E-07
L235R/S239K/M252Y/N286E/N434Y F875 4.80E-07 M252Y/N434Y/Y436A F877
8.30E-07 M252Y/N434Y/Y436E F878 1.90E-07 M252Y/N434Y/Y436F
Table 5-22 is a continuation of Table 5-21.
TABLE-US-00026 TABLE 5-22 F879 9.20E-07 M252Y/N434Y/Y436G F880
3.90E-07 M252Y/N434Y/Y436H F881 3.10E-07 M252Y/N434Y/Y436K F882
1.30E-07 M252Y/N434Y/Y436L F883 2.10E-07 M252Y/N434Y/Y436M F884
4.00E-07 M252Y/N434Y/Y436N F888 4.80E-07 M252Y/N434Y/Y436S F889
2.20E-07 M252Y/N434Y/Y436T F890 1.10E-07 M252Y/N434Y/Y436V F891
1.70E-07 M252Y/N434Y/Y436W F892 7.10E-08 M252/S254T/N434Y/Y436I
F893 9.80E-08 L235K/S239K/M252Y/N434Y/Y436I F894 9.20E-08
L235R/S239K/M252Y/N434Y/Y436I F895 2.10E-08
L23SK/S239K/M252Y/N286E/T307Q/Q311A/N315E/ N434Y F896 2.00E-08
L235R/S239K/M252Y/N286E/T307Q/Q311A/N315E/ N434Y F897 9.70E-08
M252Y/N315D/N384A/N389A/N434Y F898 1.70E-07
M252Y/N315E/N384A/N389A/N434Y F899 1.10E-07
M252Y/N315D/G316A/N4344Y F900 1.70E-07 M252Y/N315D/G316D/N434Y F901
1.30E-07 M252Y/N315D/G316E/N434Y F902 2.20E-07
M252Y/N315D/G316F/N434Y F903 2.30E-07 M252Y/N315D/G316H/N434Y F904
1.00E-07 M252Y/N315D/G316I/N434Y F905 1.30E-07
M252Y/N315D/G316K/N434Y F906 1.50E-07 M252Y/N315D/G316L/N434Y F907
1.30E-07 M252Y/N315D/G316M/N434Y F908 1.50E-07
M252Y/N315D/G316N/N434Y F909 1.30E-07 M252Y/K315D/G316P/N434Y F910
1.40E-07 M252Y/N315D/G316Q/N434Y F911 1.30E-07
M252Y/N315D/G316R/N434Y F912 1.20E-07 M252Y/N315D/G316S/N434Y F913
1.10E-07 M252Y/N315D/G316T/N434Y F914 1.50E-07
M252Y/H315D/G316V/N434Y F915 2.30E-07 M252Y/N315D/G316W/N434Y
Table 5-23 is a continuation of Table 5-22.
TABLE-US-00027 TABLE 5-23 F917 2.50E-07 M252Y/N286S/N434Y F918
2.80E-07 M252Y/D280E/N384A/N389A/N434Y F919 3.30E-07
M252Y/D280G/N384A/N389A/N434Y F920 2.50E-07
M252Y/N286S/N384A/N389A/N434Y F921 1.20E-07
M252Y/N386E/N384A/N389A/N434Y F922 5.90E-08
L235K/S239K/M252Y/M86E/K434/Y436I F923 6.00E-08
L235R/S239K/M252Y/N286E/N434Y/Y436I F924 3.40E-08
L235K/S239K/M252Y/T307Q/Q311A/N434Y/Y436I F925 3.20E-08
L235R/S239K//M252Y/T307Q/Q311A/N434Y/Y436I F926 1.10E-07
L235K/S239K/M252Y/S254T/N434Y/Y456I F927 1.00E-07
L235R/S239K/M252Y/S254T/N434Y/Y436I F928 2.90E-08
M252Y/T307Q/Q311A/N434Y/Y436I F929 2.90E-08
M252Y/S254T/T307Q/Q311A/N434Y/Y436I F930 1.40E-07
P238D/T250V/M252Y/N286E/N434Y F931 1.20E-07
P238D/T250V/M252Y/N434Y/Y436I F932 3.20E-07 T250V/M252Y/N434Y F933
3.00E-07 L234R/P238D/T250V/M252Y/N434Y F934 3.10E-07
G236K/P238D/T250V/M252Y/N434Y F935 3.20E-07
G237K/P238D/T250V/M252Y/N434Y F936 3.20E-07
G237R/P238D/T250V/M252Y/N434Y F937 3.10E-07
P238D/S239K/T250V/M252Y/N434Y F938 1.60E-07
L235K/S239K/M252Y/N434Y/Y436V F939 1.50E-07
L235R/S239K/M253Y/N434Y/Y436V F940 1.50E-07
P238D/T250V/M252Y/N434Y/Y436V F941 1.20E-08
M252Y/N286E/T307Q/Q311A/N434Y/Y436V F942 4.20E-08
L235K/S239K/M252Y/T307Q/Q311A/N434Y/Y436V F943 4.00E-08
L235R/S239K/M352Y/T307Q/Q311A/N434V/Y436V F944 1.70E-07
T250V/M252Y/N434Y/Y436V F945 1.70E-08 T250V/M252Y/V308P/N434Y/Y436V
F946 4.30E-08 T250V/M252Y/T301Q/Q311A/N434Y/Y436V F947 1.10E-08
T250V/M252Y/T307Q/V308P/Q311A/N434Y/Y436V F954 5.30E-07
M252Y/N434Y/H435K/Y436V F957 7.70E-07 M252Y/N434Y/H435N/436V F960
8.00E-07 M252Y/N434Y/H435R/Y436V
Table 5-24 is a continuation of Table 5-23.
TABLE-US-00028 TABLE 5-24 F966 3.10E-07 M252Y/S254A/N434Y F970
2.50E-06 M252Y/S254G/N434Y F971 2.60E-06 M252Y/S254H/N434Y F972
2.60E-07 M252Y/S254I/N434Y F978 1.30E-06 M252Y/S254Q/N434Y F980
1.80E-07 M252Y/S254V/N434Y F987 4.00E-08
P238D/T250V/M252Y/T307Q/Q311A/N434Y/ Y436V F988 6.90E-08
P238D/T250V/M252Y/N286E/N434Y/Y436V F989 1.40E-08
L235R/S239K/M252Y/V308P/N434Y/Y436V F990 9.40E-09
L235R/S39K/M252Y/T307Q/V308P/Q311A/ N434Y/Y436V F991 1.30E-08
L235R/S239K/M252Y/N286E/T307Q/Q311A/ N434Y/Y436V F992 5.10E-08
L235R/S239K/M252Y/T307Q/Q311A/M428I/ N434/Y436V P993 3.80E-08
M252Y/T307Q/Q311A/N434Y/Y436V F994 2.80E-07 M262Y/N325G/N434Y F995
2.90E-07 L235R/P238D/S239K/M252Y/N434Y F996 1.30E-07
L235R/P238D/S239K/M252Y/N434Y/Y436V F997 3.80E-07
K248I/T250V/M252Y/N434Y/Y436V F998 8.50E-07
K248Y/T250V/M252Y/N434Y/Y436V F999 2.10E-07
T250V/M252Y/E258H/N434Y/Y436V F1005 N325G F1008 1.70E-07
L235R/S239K/T250V/M252Y/N434Y/Y436V F1009 1.20E-08
L235R/S239K/T250V/M252Y/T307Q/V308P/ Q311A/N434Y/Y436V F1010
1.90E-07 L235R/S239K/M252Y/T307A/Q311H/N434Y F1011 4.50E-08
T250V/M252Y/V308P/N434Y F1012 4.70E-08
L235R/S239K/T250V/M252Y/V308P/N434Y F1013 3.00E-08
T250V/M252Y/T307Q/V308P/Q311A/N434Y F1014 3.20E-08
L235R/S239K/T250V/M252Y/T307Q/V308P/ Q311A/N434Y F1015 2.20E-08
L235R/S239K/M252Y/T307Q/V308P/Q311A/ N434Y F1016 3.80E-09
T250V/M252Y/N286E/T307Q/V308P/Q311A/ N434Y/Y436V F1017 4.20E-09
L235R/S239K/T250V/M252Y/N286E/T307Q/ V308P/Q311A/N431Y/Y436V F1018
3.20E-09 L235R/S239K/M252Y/N286E/T307Q/V308P/ Q311A/N434Y/Y436V
F1019 3.40E-07 P238D/T250V/M252Y/N325G/N434Y F1020 8.50E-08
P238D/T250V/M252Y/T307Q/Q311A/N325G/ N434Y
Table 5-25 is a continuation of Table 5-24.
TABLE-US-00029 TABLE 5-25 F1021 3.30E-07
P238D/T250V/M252Y/N325A/N434Y F1022 K326D/L328Y F1023 4.40E-08
S239D/T250V/M252Y/T307Q/Q311A/N434Y/ Y436V F1024 4.00E-08
T250V/M252Y/T307Q/Q311A/K326D/L328Y/ N434Y/Y436V F1025 3.60E-08
S239D/T250V/M252Y/T307Q/Q311A/K326D/ L328Y/N434Y/Y436V F1026
8.40E-08 M252Y/T307A/Q311H/N434Y/Y436V F1027 8.60E-08
L235R/S239K/M252Y/T307A/Q311H/N434Y/ Y436V F1028 4.60E-08
G236A/S239D/T250V/M252Y/T307Q/Q311A/ N434Y/Y436V F1029 5.10E-08
T250V/M252Y/T307Q/Q311A/1332E/N434Y/ Y436V F1030 I332E F1031
5.30E-08 G236A/S239D/T250V/M252Y/T307Q/Q311A/ I332E/N434Y/Y436V
F1032 4.30E-08 P238D/T250V/M252Y/T307Q/311A/N325G/ Y436V F1033
1.00E-06 P238D/N434W F1034 1.50E-08
L235K/S239K/M252Y/V308P/N434Y/Y436V F1035 1.00E-08
L235K/S239K/M252Y/T307Q/V30P/Q311A/ N434Y/Y436V F1036 1.40E-08
L235K/S239K/M252Y/N286E/T307Q/Q311A/ N434Y/Y436V F1037 6.10E-08
L235K/S239K/M252Y/T307Q/Q311A/M428I/ N434Y/Y436V F1038 2.80E-07
L235K/P238D/S239K/M252Y/N434Y F1039 1.30E-07
L235K/P238D/S239K/M252Y/N434Y/Y436V F1040 2.00E-07
L235K/S239K/T250V/M252Y/N434Y/Y436V F1041 1.40E-08
L235K/S239K/T250V/M252Y/T307Q/V308P/ Q311A/N434Y/Y436V F1042
3.00E-07 L235K/S239K/M252Y/T307A/Q311H/N434Y F1043 5.20E-08
L233K/S239K/T250V/M252Y/V308P/N434Y F1044 3.50E-08
L235K/S239K/T250V/M252Y/T307Q/V308P/ Q311A/N434Y F1045 2.50E-08
L235K/S239K/M252Y/T307Q/V308P/Q311A/ N434Y F1046 4.50E-09
L235K/S339K/T250V/M352Y/N286E/T307Q/ V308P/Q311A/N434Y/Y436V F1047
3.40E-09 L235K/S239K/M252Y/N286E/T307Q/V308P/ Q311A/N434Y/Y436V
F1048 9.90E-08 L235K/S239K/M252Y/T307A/Q311H/N434Y/ Y436V F1050
3.50E-09 T250V/M252Y/N286E/T307Q/V308P/Q311A/ M428I/N434Y/Y436V
F1051 3.90E-09 L235R/S239K/T250V/M252Y/N286E/T307Q/
V308P/Q311A/M428I/N434Y/Y436V F1052 3.20E-09
L235R/S239K/M252Y/N386E/T307Q/V308P/ Q311A/M428I/N434Y/Y436V
Table 5-26 is a continuation of Table 5-25.
TABLE-US-00030 TABLE 5-26 F1053 4.23E-08
L235R/S239K/T250V/M252Y/T307Q/ Q311A/N434Y/Y436V F1058 1.31E-07
M252V/Q386E/N434Y/Y436V F1059 1.39E-07 M252Y/Q386R/N434Y/Y436V
F1060 1.43E-07 M252Y/Q386S/N434Y/Y436V F1061 1.19E-07
M252Y/P387E/N434Y/Y436V F1062 1.2E-07 M252Y/F387R/N434Y/Y436V F1063
1.43E-07 M252Y/P387S/N434Y/436V F1064 1.32E-07
M252Y/V422E/N434Y/Y436V F1065 1.38E-07 M252Y/V422R/N434Y/Y436V
F1066 1.45E-07 M252Y/V422S/N434Y/Y436V F1067 1.26E-07
M252Y/S424E/N434Y/Y436V F1068 1.69E-07 M252Y/S424R/N434Y/Y436V
F1069 1.39E-07 M252Y/N434Y/Y436V/Q438E F1070 1.73E-07
M252Y/N434Y/Y436V/Q438R F1071 1.24E-07 M252Y/N434Y/Y436V/Q438S
F1072 1.35E-07 M252Y/N434Y/Y436V/S440E F1073 1.34E-07
M252Y/N434Y/Y436V/S440R F1074 1.32E-07 S239D/M252Y/N434Y/Y436V
F1075 1.4E-07 M252Y/K326D/L328Y/N434Y/Y436V F1076 1.27E-07
S239D/M252Y/K326D/L328Y/N434Y/ Y436V F1077 2.03E-06
K248N/M252Y/N434Y F1078 4.7E-07 M252Y/E380N/E382S/N434Y F1079
3.44E-07 M252Y/E382N/N384S/N434Y F1080 3.19E-07 M252Y/S424N/N434Y
F1081 6.2E-07 M252Y/N434Y/Y436N/Q438T F1082 2.76E-07
M252Y/N434Y/Q438N F1083 3.45E-07 M252Y/N434Y/S440N F1094 2.6E-07
M252Y/N434Y/S442N F1095 2.86E-07 M252Y/S383N/G385S/N434Y F1096
2.72E-07 M352Y/Q386T/N434Y F1097 2.82E-07 M252Y/0385N/P387S/N434Y
F1098 2.58E-07 S239D/M252Y/N434Y F1099 2.57E-07
M352Y/K326D/L328Y/N434Y F1100 2.41E-07
S239D/M252Y/K326D/L328Y/N434Y F1101 6.59E-08
S239D/M252Y/T307Q/0311A/N434Y F1102 6.46E-08
M252Y/T307Q/Q311A/K326D/L328V/ N434Y F1103 6.11E-08
S239D/M252Y/T307Q/Q311A/K326D/ L328Y/N434Y F1104 1.77E-07
M252Y/V422E/S424R/N434Y/Y436V F1105 1.54E-07
N252Y/V422S/S424R/N434Y/Y436V F1106 1.42E-07
M252Y/N434Y/Y436V/Q438R/S440E F1107 1.23E-07
M252Y/V422D/N434Y/Y436V
Table 5-27 is a continuation of Table 5-26.
TABLE-US-00031 TABLE 5-27 F1108 1.26E-07 M252Y/V422K/N434Y/Y436V
F1109 1.27E-07 M252Y/V422T/N434Y/Y436V F1110 1.33E-07
M252Y/V422Q/N434Y/Y436V F1111 1.65E-07 M252Y/S424K/N434Y/Y436V
F1112 1.23E-07 M252Y/N434Y/Y436V/Q438K F1113 1.18E-07
M252Y/N434Y/Y436V/S440D F1114 1.31E-07 M252Y/N434Y/Y436V/S440Q
F1115 1.35E-07 M252Y/S424N/N434Y/Y436V F1116 7.44E-08
M252Y/T307Q/Q311A/S424N/N434Y F1117 4.87E-08
T250V/M252Y/T307Q/Q311A/S424N/N434Y/ Y436V F1118 1.32E-08
T250V/M252Y/T307Q/V308P/Q311A/S424N/ N434Y/Y436V F1119 1.03E-08
T250V/M252Y/T307Q/V308P/Q311A/V422F/ N434Y/Y436V F1120 1.04E-08
T250V/M252Y/T307Q/V308P/Q311A/S424R/ N434Y/Y436V F1121 1.04E-08
T250V/M252Y/T307Q/V308P/Q311A/V422E/ S424R/N434Y/Y436V F1122
1.37E-08 T250V/M252Y/T307Q/V308P/Q311A/N434Y/ Y436V/Q436R F1123
9.55E-09 T250V/MS52Y/T307Q/V308P/Q311A/N434Y/ Y436V/S440E F1124
1.22E-08 T250V/M252Y/T307Q/V308P/Q311A/N434Y/ Y436V/Q438R/S440E
F1125 5.18E-08 M252Y/T307Q/N434Y/Y436V F1126 8.95E-08
M252Y/T307A/N434Y/Y436V F1127 7.94E-08 M252Y/Q311A/N434Y/Y436V
F1128 1.17E-07 M252Y/Q311H/N434Y/Y436V F1129 4.48E-08
M252Y/T307Q/Q311H/N434Y/Y436V F1130 5.54E-08
M252Y/T307A/Q311A/N434Y/Y436V F1131 1.29E-07
L235R/S239K/M252Y/V422E/N434Y/ Y436V F1132 1.4E-07
L235R/S239K/M252Y/V422S/N434Y/ Y436V F1133 1.58E-07
L235R/S239K/M253Y/S424R/N434Y/ Y436V F1134 1.66E-07
L235R/S239K/M252Y/N434Y/Y436V/ Q438R F1135 1.26E-07
L23SR/S239K/M252Y/N434Y/Y436V/ S440E F1136 1.63E-07
L235R/S239K/M252Y/V422E/S424R/ N434Y/Y436V F1137 1.58E-07
L235R/S239K/M252Y/V422S/S242R/N434Y/ Y436V F1138 1.65E-07
L235R/S239K/M252Y/N434Y/Y436V/ Q438R/S440E F1139 1.52E-07
L235R/S239K/M252Y/S424N/N434Y/Y436V F1140 1.62E-07
M252Y/V422E/S424R/N434Y/Y436V/ Q438R/S440E F1141 1.77E-07
M252Y/V422S/S424R/N434Y/Y436V/ Q438R/S440E F1142 1.87E-07
L235R/S239K/M252Y/V422E/S424R/ N434Y/Y436V/Q43SR/S440E F1143
1.98E-07 L235R/S239K/M252Y/V422S/S424R/ N434Y/Y436V/Q438R/S440E
F1144 1.44E-08 L235R/S239K/T250V/M252Y/T307Q/
V308P/Q311A/N434Y/Y436V/Q438R/S440E F1145 5.23E-08
T250V/M252Y/T307Q/Q311A/N434Y/ Y436V/Q438R/S440E F1146 6.24E-08
L235R/S239K/T250V/M252Y/T307Q/ Q311A/N434Y/Y436V/Q438R/S440E F1147
7.19E-08 M252Y/T307Q/Q311A/N434Y/Q438R/S440E
Table 5-28 is a continuation of Table 5-27.
TABLE-US-00032 TABLE 5-28 F1148 7.63E-08
L235R/S239K/M252Y/T307Q/Q311A/N434Y/ Q438R/S440E F1151 2.51E-07
L235R/S239K/M252Y/S424N/N434Y F1152 7.38E-08
L235R/S239K/M252Y/T307Q/Q311A/S424N/ N434Y F1153 4.85E-08
L235R/S239K/T250V/M252Y/T307Q/Q311A/ S424N/N434Y/Y436V F1154
1.34E-08 L235R/S239K/T250V/M252Y/T307Q/V308P/
Q311A/S424N/N434Y/Y436V F1157 2.09E-07 M252Y/N434Y/Q438R/S440E
F1158 2.44E-07 L235R/S239K/M252Y/N434Y/Q438R/S440E F1159 4.79E-07
S424N/N434W F1160 2.88E-07 V308F/S424N/N434Y F1161 1.07E-06
I332V/S424N/N434Y F1162 3.43E-07 P238D/T250Y/M252Y/N434Y/Y436V
F1163 1.54E-07 P238D/T250Y/M252Y/T307Q/Q311A/N434Y F1164 6.96E-08
P238D/T250Y/M252Y/T307Q/Q311A/N434Y/ Y436V F1165 1.63E-08
P238D/T250Y/M252Y/T307Q/V308P/Q311A/ N434Y/Y436V F1174 4.9E-07
P257I/N434H F1176 1.98E-06 V308F F1178 8.72E-07 V259I/V308F/M428L
F1183 1.28E-06 E380A/M428L/N434S F1184 1E-06 T307A/M428L/N434S
F1185 9.17E-07 T307A/E380A/M428L/N434S F1188 1.72E-06
T307A/E380A/N434H F1189 1.57E-07
M252Y/H433D/N434Y/Y436V/Q438R/S440E F1190 2.4E-07
M252Y/H433E/N434Y/Y436V/Q438R/S440E F1191 2.11E-07
M252Y/N434Y/Y436V/T437A/Q438R/S440E F1192 1.27E-07
M252Y/N434Y/Y436V/T437G/Q438R/S440E F1194 1.55E-07
M252Y/N434Y/Y436V/Q438R/K439D/S440E F1195 1.76E-07
M252Y/N434Y/Y436V/Q438R/S440E/L441A F1196 1.51E-07
M252Y/N434Y/Y436V/Q438R/S440E/L441E F1197 9.46E-08
M252Y/S254T/N434Y/Y436V/Q438R/S440E F1198 7.83E-08
M252Y/T256E/N434Y/Y436V/Q438R/S440E F1199 6.25E-08
M252Y/S254T/T256E/N434Y/Y436V/Q438R/ S440E F1200 1.26E-07
T250V/M252Y/S254T/N434Y/Y436V/Q438R/ S440E F1201 1.07E-07
T250V/M252Y/T256E/N434Y/Y436V/Q438R/ S440E F1202 8.81E-08
T250V/M252Y/S254T/T256E/N434Y/Y436V/ Q438R/S440E F1203 1.52E-07
M252Y/T256Q/N434Y/Y436V/Q438R/S440E F1204 1.18E-07
M252Y/S254T/T256Q/N434Y/Y436V/Q438R/ S440E F1205 1.98E-07
T250V/M252Y/T256Q/N434Y/Y436V/Q438R/ S440E F1206 1.69E-07
T250V/M252Y/S254T/T256Q/N434Y/Y436V/ Q438R/S440E F1207 1.11E-06
I332E/M428L/N434S F1208 5.71E-07 L251A/M252Y/N434Y/Y436V F1211
1.23E-06 L251H/M252Y/N434Y/Y436V
Table 5-29 is a continuation of Table 5-28.
TABLE-US-00033 TABLE 5-29 F1213 6.33E-07 L251N/M252Y/N434Y/Y436V
F1216 1.16E-06 L251S/M252Y/N434Y/Y436V F1217 1.14E-06
L251T/M252Y/N4S4Y/Y436V F1218 2.51E-07 L251V/M252Y/N434Y/Y436V
F1229 2.81E-06 M252Y/I253V/N434Y/Y436V F1230 1.12E-07
M252Y/N434Y/Y436V/Q438R/S440D F1231 9.73E-08
M252Y/N434Y/Y436V/Q4S8K/S440E F1232 9.79E-08
M252Y/N434Y/Y436V/Q438K/S440D F1243 1.25E-07
L235R/S239K/M252Y/S254T/N434Y/Y436V/ Q438R/S440E F1244 1.02E-07
L235R/S239K/M252Y/T256E/N434Y/Y436V/ Q438R/S440E F1245 8.2E-08
L235R/S239K/M252Y/S254T/T256E/N434Y/ Y436V/Q438R/S440E F1246
1.73E-07 L23SR/S239K/T250V/M252Y/S254T/N434Y/ Y436V/Q438R/S440E
F1247 1.45E-07 L235R/S239K/T250V/M252Y/T256E/N434Y/
Y436V/Q438R/S440E F1248 1.2E-07
L23SR/S239K/T250V/M252Y/S254T/T256E/ N434Y/Y436V/Q438R/S440E F1249
2.06E-07 L235R/S239K/M252Y/T256Q/N434Y/Y436V/ Q438R/S440E F1250
1.66E-07 L235R/S239K/M252Y/S254T/T256Q/N434Y/ Y436V/Q438R/S440E
F1251 2.77E-07 L235R/S239K/T250V/M252Y/T256Q/N434Y/
Y436V/Q438R/S440E F1252 2.33E-07
L235R/S239K/T250V/M252Y/S254T/T256Q/ N434Y/Y436V/Q438R/S440E F1253
1.12E-07 L235R/S239K/M252Y/T307A/N434Y/Y436V/ Q438R/S440E F1254
6.42E-08 L235R/S239K/M252Y/T307Q/N434Y/Y436V/ Q438R/S440E F1255
1.11E-07 L235R/S239K/M252Y/Q311A/N434Y/Y436V/ Q438R/S440E F1256
1.56E-07 L235R/S239K/M252Y/Q311H/N434Y/Y436V/ Q438R/S440E F1257
7.81E-08 L235R/S239K/M252Y/T307A/Q311A/N434Y/ Y436V/Q438R/S440E
F1258 1.05E-07 L235R/S239K/M252Y/T307A/Q311H/N434Y/
Y436V/Q438R/S440E F1259 4.46E-08
L235R/S239K/M252Y/T307Q/Q311A/N434Y/ Y436V/Q438R/S440E F1260
6.53E-08 L23SR/S239K/M252Y/T307Q/Q311H/N434Y/ Y436V/Q438R/S440E
F1261 1.35E-07 L335R/S239K/M252Y/N434Y/Y436V/Q438R/ S440E F1262
1.26E-07 L235R/S239K/M252Y/N434Y/Y436V/Q438K/ S440E F1263 1.24E-07
L235R/S239K/M252Y/N434Y/Y436V/Q438R/ S440E F1264 1.27E-07
L235R/S239K/M252Y/T256A/N434Y/Y436V/ Q438R/S440E F1265 1.57E-07
L235R/S239K/M252Y/T256G/N434Y/Y436V/ Q438R/S440E F1266 9.99E-08
L235R/S239K/M252Y/T256N/N434Y/Y436V/ Q458R/S440E F1267 1.5E-07
L235R/S239K/M252Y/S254A/N434Y/Y436V/ Q438R/S440E F1268 2E-07
L235R/S239K/M252Y/H433D/N434Y/Y436V/ Q438R/S440E F1269 1.69E-07
L235R/S239K/M252Y/H433D/N434Y/Y436V/ Q438K/S440D F1270 1.18E-07
L235R/S239K/M252Y/S254A/N434Y/Y436V/ Q438K/S440D F1271 2.05E-07
L235R/S239K/M252Y/S254A/H433D/N434Y/ Y436V/Q438R/S440E F1272
1.71E-07 L235R/S239K/M252Y/S254A/H433D/N434Y/ Y436V/Q438K/S440D
F1273 1.53E-07 L235R/S239K/M252Y/T256Q/N434Y/Y436V/ Q438K/S440D
F1274 2.48E-07 L235R/S239K/M252Y/T256Q/H433D/N434Y/
Y436V/Q438R/S440E F1275 2.09E-07
L235R/S239K/M252Y/T256Q/H433D/N434Y/ Y436V/Q438K/S440D
Table 5-30 is a continuation of Table 5-29.
TABLE-US-00034 TABLE 5-30 F1276 1.02E-07
L235R/S239K/M252Y/T256A/N434Y/Y436V/ Q438K/S440D F1277 1.69E-07
L235R/S239K/M252Y/T256A/H433D/N434Y/ Y436V/Q438K/S440E F1278
1.4E-07 L235R/S239K/M252Y/T256A/H433D/N434Y/ Y436V/Q438K/S440D
F1279 1.23E-07 L235R/S239K/M252Y/T256G/N434Y/Y436V/ Q438K/S440D
F1280 2.09E-07 L235R/S239K/M252Y/T256G/H433D/N434Y/
Y436V/Q438R/S440E F1281 1.74E-07
L235R/S239K/M252Y/T256G/H433D/N434Y/ Y436V/Q438K/S440E F1282
7.69E-08 L235R/S239K/M252Y/T256N/N434V/Y436V/ Q438K/S440D F1283
1.34E-07 L235R/S239K/M252Y/T256N/H433D/N434Y/ Y436V/Q438R/S440E
F1284 1.12E-07 L235R/S239K/M252Y/T256N/H433D/N434Y/
Y436V/Q438K/S440D F1285 9.36E-08
L235K/S239K/M252Y/S254T/N434Y/Y436V/ Q438K/S440D F1286 1.57E-07
L235R/S239K/M252Y/S254T/H433D/N434Y/ Y436V/Q438R/S440E F1287
1.5E-07 L235R/S239K/M252Y/S254T/H433D/N434Y/ Y436V/Q438K/S440D
F1288 7.95E-08 L235R/S239K/M252Y/T256E/N434Y/Y436V/ Q438K/S440D
F1289 1.33E-07 L235R/S239K/M252Y/T256E/H433D/N434Y/
Y436V/Q438R/S440E F1290 1.11E-07
L235R/S239K/M252Y/T256E/H433D/N434Y/ Y436V/Q438K/S440D F1291
1.51E-07 L235R/S239K/M252Y/H433D/N434Y/Y436V F1292 4.24E-07
L235R/S239K/H433D/N434W/Y436V/Q438R/ S440E F1293 1.61E-07
L235R/S239K/M252Y/T256E/N434Y/Q438R/ S440E F1294 2E-07
L235R/S239K/M252Y/T256E/N434Y/Y436T/ Q438R/S440E F1295 9.84E-08
L235R/S239K/M252Y/T256E/N434Y/Y436F/ Q438R/S440E F1296 2.27E-07
L235R/S239K/M252Y/T256E/H433D/N434Y/ Q438R/S440E F1297 2.5E-07
L235R/S239K/M252Y/T256E/H433D/N434Y/ Y436T/Q438R/S440E F1298
1.47E-07 L235R/S239K/M252Y/T256E/H433D/N434Y/ Y436F/Q438R/S440E
F1299 1.5E-07 L235R/S239K/M252Y/T256E/N434Y/Q438K/ S440D F1300
1.63E-07 L235R/S239K/M252Y/T256E/N434Y/Y436T/ Q438K/S440D F1301
8.3E-08 L235R/S239K/M252Y/T256E/N434Y/Y436F/ Q438K/S440D F1302
2.15E-07 L235R/S239K/M252Y/T256E/H433D/N434Y/ Q438K/S440D F1303
2.1E-07 L235R/S239K/M252Y/T256E/H433D/N434Y/ Y436T/Q438K/S440D
F1304 1.24E-07 L235R/S239K/M252Y/T256E/H433D/N434Y/
Y436F/Q438K/S440D F1305 2.05E-07
L235R/S239K/M252Y/H433D/M434Y/Y436V/ Q438R/S440D F1306 1.92E-07
L235R/S239K/M252Y/H433D/N434Y/Y436V/ Q438K/S440E F1307 1.44E-07
L235R/S239K/M252Y/V422A/S424A/N434Y/ Y436V F1308 2.06E-07
L235R/S239K/M252Y/V422L/S424L/N434Y/ Y436V F1309 1.26E-07
L235R/S239K/M252Y/N434Y/Y436V/Q438A/ S440A F1310 2.28E-07
L235R/S239K/M252Y/N434Y/Y436V/Q438L/ S440L F1311 1.69E-07
L235R/S239K/M252Y/V422A/S424A/H433D/ N434Y/Y436V F1312 1.79E-07
L235R/S239K/M252Y/V422L/S424L/H433D/ N434Y/Y436V F1313 1.77E-07
L235R/S239K/M252Y/H433D/N434Y/Y436V/ Q438A/S440A F1314 2.27E-07
L235R/S239K/M252Y/H433D/N434Y/Y436V/ Q438L/S440L F1315 1.52E-07
G237K/S239K/M252Y/N434Y/Y436V F1316 1.49E-07
G237R/S239K/M252Y/N434Y/Y436V
Table 5-31 is a continuation of Table 5-30.
TABLE-US-00035 TABLE 5-31 F1317 1.38E-07
S239K/M252Y/P329K/H434Y/Y436V F1318 1.43E-07
S239K/M252Y/P329R/N434Y/Y436V F1319 2.67E-07 M252Y/L328Y/N434Y
F1320 1.22E-07 L235R/S239K/M252Y/S254T/N434Y/Y436V/ Q438R/S440D
F1321 1.03E-07 L23SR/S239K/M252Y/S254T/N434Y/Y436V/ Q438K/S440E
F1322 1.6E-07 L235R/S239K/M252Y/S254T/H433D/N434Y/
Y436V/Q438R/S440D F1323 1.49E-07
L235R/S239K/M252Y/S254T/H433D/N434Y/ Y436V/Q438K/S440E F1324
1.32E-07 L234A/L235A/M252Y/N434Y/Y436V F1325 2.13E-07
L234A/L235A/M252Y/N297A/N434Y/Y436V F1326 1.09E-08
L234A/L235A/T250V/M252Y/T307Q/V308P/ Q311A/N434Y/Y436V F1327
1.41E-08 L234A/L235A/T250V/M252Y/N297A/T307Q/
V308P/Q311A/N434Y/Y436V F1328 1.52E-07
L235R/G236R/S239K/M252Y/N434Y/Y436V/ Q438R/S440E F1329 1.29E-07
L235R/G236R/S239K/M252Y/S254T/N434Y/ Y436V/Q438R/S440E F1330
1.03E-07 L235R/G236R/S239K/M252Y/T256E/N434Y/ T436V/Q438R/S440E
F1331 7.75E-08 L235R/G236R/S239K/M252Y/S254T/T256E/
N434Y/Y436V/Q438R/S440E F1333 1.23E-07
L235R/G236R/S239K/M252Y/N434Y/Y436V F1334 1.04E-07
L235R/G236R/S239K/M252Y/N434Y/Y436V/ Q438K/S440D F1335 8.78E-08
L235R/G236R/S239K/M252Y/S254T/N434Y/ Y436V/Q438K/S440D F1336
7.18E-08 L235R/G236R/S239K/M252Y/T256E/N434Y/ Y436V/Q438K/S440D
F1337 7.41E-08 L235R/S239K/M252Y/T256E/N434Y/Y436V/ Q438K/S440E
F1338 1.04E-07 L235R/S239K/M252Y/T256E/H433D/N434Y/
Y436V/Q438K/S440E F1339 2.51E-07
L235R/S239K/M252Y/S254T/T256E/H433D/ N434Y/Y436T/0438K/S440E F1340
5.58E-08 L235R/S239K/M252Y/S254T/T2S6E/N434Y/ Y436V/Q438K/S440E
F1341 3.32E-07 L235R/S239K/M252Y/S254T/N434Y/Y436T/ Q438K/S440E
F1342 2.51E-07 L235R/S239K/M252Y/T256E/N434Y/Y436T/ Q438K/S440E
F1343 2.01E-07 L235R/S239K/M252Y/S254T/T256E/N434Y/
Y436T/Q438K/S440E F1344 3.96E-07
L235R/S239K/M252Y/N434Y/Y436T/Q438K/ S440E F1345 1.05E-07
L235R/G236R/S239K/M252Y/N434Y/Y436V/ Q438K/S440E F1346 8.59E-08
L235R/G236R/S239K/M252Y/S254T/N434Y/ Y436V/Q438K/S440E F1347
7.14E-08 L235R/G236R/S239K/M252Y/T256E/N434Y/ Y436V/Q438K/S440E
F1348 5.52E-08 L235R/G236R/S239K/M252Y/S254T/T256E/
N434Y/Y436V/Q438K/S440E F1349 3.36E-07
L235R/S239K/M252Y/N434Y/Y436T/Q438R/ S440E F1350 1.18E-07
L335R/S239K/M252Y/N434Y/Y436F/Q438K/ S440E F1351 1.62E-07
L235R/S239K/M252Y/N434Y/Y436F/Q438R/ S440E F1352 3.93E-07
L235R/S239K/M252Y/H433D/N434Y/Y436T/ Q438K/S440E F1353 4.33E-07
L235R/S239K/M252Y/K433D/N434Y/Y436T/ Q438R/S440E F1354 2.29E-07
L235R/S239K/M252Y/H433D/N434Y/Y436F/ Q438K/S440E F1355 2.47E-07
L235R/S239K/M252V/H433D/N434V/Y436F/ Q438R/S440E F1356 1.58E-07
G236R/M252Y/L328R/N434Y/Y436V F1357 2.81E-07
L235R/S239K/M252Y/S254T/N434Y/Y436T/ Q438R/S440E F1358 9.07E-08
L235R/S239K/M252Y/S254T/N434Y/Y436F/ Q438K/S440E
Table 5-32 is a continuation of Table 5-31.
TABLE-US-00036 TABLE 5-32 F1359 1.28E-07
L235R/S239K/M252Y/S254T/N434Y/Y436F/ Q438R/S440E F1360 3.12E-07
L235R/S239K/M252Y/S254T/H433D/N434Y/ Y436T/Q438K/S440E F1361
3.52E-07 L235R/S239K/M252Y/S254T/H433D/N434Y/ Y436T/Q438R/S440E
F1362 1.41E-07 L235R/S239K/M252Y/8254T/H433D/N434Y/
Y436F/Q438K/S440E F1363 1.9E-07
L235R/S239K/M252Y/S254T/H433D/N434Y/ Y436F/Q438R/S440E F1364
7.49E-08 L235R/S239K/M252Y/T256E/N434Y/Y436F/ Q438K/S440E F1365
3.14E-07 L235R/S239K/M252Y/T256E/H433D/N434Y/ Y436T/Q438K/S440E
F1366 1.17E-07 L235R/S239K/M252Y/T256E/H433D/N434Y/
Y436F/Q438K/S440E F1367 1.79E-07
L235R/S239K/M252Y/S254T/T25GE/N434Y/ Y436T/Q438R/S440E F1368
5.49E-08 L235R/S239K/M252Y/S254T/T256E/N434Y/ Y436F/Q438K/S440E
F1369 7.6E-08 L235R/S239K/M252Y/S254T/T256E/N434Y/
Y436F/Q438R/S440E F1370 9.14E-08
L235R/S239K/M252Y/S254T/T256E/H433D/ N434Y/Y436V/Q438K/S440E F1371
1.09E-07 L235R/S239K/M252Y/S254T/T256E/H433D/
N434Y/Y436V/Q438R/S440E F1372 2.28E-07
L235R/S239K/M252Y/S254T/T256E/H433D/ N434Y/Y436T/Q438R/S440E F1373
8.67E-08 L235R/S239K/M252Y/S254T/T256E/H433D/
N434Y/Y436F/Q438K/S440E F1374 1.2E-07
L235R/S239K/M252Y/S254T/T256E/H433D/ N434Y/Y436F/Q438R/S440E F1375
1.03E-07 L235R/S239K/M252Y/S254T/N434Y/Y436V F1376 9.09E-08
L235R/S239K/M2G2Y/S254T/T256E/N434Y/ Y436V F1377 8.27E-08
L235R/S239K/M252Y/T256E/N434Y/Y436V F1378 3.61E-07
L235R/S239K/M252Y/N434Y/Y436T F1379 2.85E-07
L235R/S239K/M252Y/N434Y/Y436F F1410 1.90E-06 V308P/I332V F1411
1.70E-07 V308P/I332V/M428L/N434S F1413 3.70E-08
L235R/S239K/M252Y/S254T/T256E/T307Q/
Q311A/H433D/N434Y/Y436V/Q438K/S440E F1414 5.60E-08
L235R/S239/M252Y/S254T/T256E/T307Q/ H433D/N434Y/Y436V/Q438K/S440E
F1415 5.90E-08 L235R/S239K/M252Y/S254T/T256E/Q311A/
H433D/N434Y/Y436V/Q438K/S440E F1416 1.30E-08
L235R/S239K/M252Y/S254T/T256E/V308P/ H433D/N434Y/Y436V/Q438K/S440E
F1417 5.90E-08 L235R/S239K/M252Y/S254T/T256E/H433D/
N434W/Y436V/Q438K/S440E F1418 7.50E-08
L235R/S239K/M252Y/S254T/T256E/H433D/ N434W/Y436V/Q438R/S440E F1419
1.50E-07 L235R/S239K/M252Y/H433D/N434W/Y436V/ Q438R/S440E F1420
1.30E-07 L235R/S239K/M252Y/H433D/N434W/Y436V/ Q438K/S440E F1421
3.20E-08 V308P/M428L/N434W F1422 1.90E-08
L235R/S239K/M252Y/T256B/V308P/H433D/ N434Y/Y436V/Q438R/S440E F1423
1.60E-08 L235R/S239K/M252Y/T256E/V302D/V308P/
H433D/N434Y/Y436V/Q438R/S440E F1424 1.60E-08
L235R/S239K/M252Y/T256E/V302E/V308P/
H433D/N434Y/Y436V/Q438R/S440E
Table 5-33 is a continuation of Table 5-32.
TABLE-US-00037 TABLE 5-33 F1425 1.90E-08
L235R/S239K/M252Y/T256E/V303D/V308P/ H433D/N434Y/Y436V/Q438R/S440E
F1426 1.80E-08 L235R/S239K/M252Y/T256E/V303E/V308P/
H433D/N434Y/Y436V/Q438R/S440E F1428 1.50E-08
L235R/S239K/M352Y/T256E/S304E/V308P/ H433D/N434Y/Y436V/Q438R/S440E
F1430 3.10E-08 L235R/S239K/M252Y/T256E/V305E/V308P/
H433D/N434Y/Y436V/Q438R/S440E F1433 4.50E-08
L23SR/S239K/M252Y/T256E/T307D/V308P/ H433D/N434Y/Y436V/Q438R/S440E
F1434 3.60E-08 L235R/S239K/M252Y/256E/T307E/V308P/
H433D/N434Y/Y436V/Q438R/S440E
Fc.gamma. Receptor
[0286] Fc.gamma. receptor (also described as Fc.gamma.R) refers to
a receptor capable of binding to the Fc region of monoclonal IgG1,
IgG2, IgG3, or IgG4 antibodies, and includes all members belonging
to the family of proteins substantially encoded by an Fc.gamma.
receptor gene. In human, the family includes Fc.gamma.RI (CD64)
including isoforms Fc.gamma.RIa, Fc.gamma.RIb and Fc.gamma.RIc;
Fc.gamma.RII (CD32) including isoforms Fc.gamma.RIIa (including
allotypes H131 and R131, i.e., Fc.gamma.RIIa(H) and
Fc.gamma.RIIa(R)), Fc.gamma.RIIb (including Fc.gamma.RIIb-1 and
Fc.gamma.RIIb-2), and Fc.gamma.RIIc; and Fc.gamma.RIII (CD16)
including isoforms Fc.gamma.RIIIa (including allotypes V158 and
F158, i.e., Fc.gamma.RIIIa(V) and Fc.gamma.RIIIa(F)) and
Fc.gamma.RIIIb (including allotypes Fc.gamma.RIIIb-NA1 and
Fc.gamma.RIIIb-NA2); as well as all unidentified human Fc.gamma.Rs,
Fc.gamma.R isoforms, and allotypes thereof. However, Fc.gamma.
receptor is not limited to these examples. Without being limited
thereto, Fc.gamma.R includes those derived from humans, mice, rats,
rabbits, and monkeys. Fc.gamma.R may be derived from any organisms.
Mouse Fc.gamma.R includes, without being limited to, Fc.gamma.RI
(CD64), Fc.gamma.RII (CD32), Fc.gamma.RIII (CD16), and
Fc.gamma.RIII-2 (Fc.gamma.RIV, CD16-2), as well as all unidentified
mouse Fc.gamma.Rs, Fc.gamma.R isoforms, and allotypes thereof. Such
preferred Fc.gamma. receptors include, for example, human
Fc.gamma.RI (CD64), Fc.gamma.RIIa (CD32), Fc.gamma.RIIb (CD32),
Fc.gamma.RIIIa (CD16), and/or Fc.gamma.RIIIb (CD16). The
polynucleotide sequence and amino acid sequence of Fc.gamma.RI are
shown in SEQ ID NOs: 19 (NM.sub.--000566.3) and 20
(NP.sub.--000557.1), respectively; the polynucleotide sequence and
amino acid sequence of Fc.gamma.RIIa (allotype H131) are shown in
SEQ ID NOs: 21 (BC020823.1) and 22 (AAH20823.1) (allotype R131 is a
sequence in which amino acid at position 166 of SEQ ID NO: 22 is
substituted with Arg), respectively; the polynucleotide sequence
and amino acid sequence of Fc.gamma.IIB are shown in SEQ ID NOs: 23
(BC146678.1) and 24 (AAI46679.1), respectively; the polynucleotide
sequence and amino acid sequence of Fc.gamma.RIIIa are shown in SEQ
ID NOs: 25 (BC033678.1) and 26 (AAH33678.1), respectively; and the
polynucleotide sequence and amino acid sequence of Fc.gamma.RIIIb
are shown in SEQ ID NOs: 27 (BC128562.1) and 28 (AAI28563.1),
respectively (RefSeq accession number is shown in each
parentheses). For example, as described as Fc.gamma.RIIIaV when
allotype V158 is used, unless otherwise specified, allotype F158 is
used; however, the allotype of isoform Fc.gamma.RIIIa described
herein should not be interpreted as being particularly limited.
[0287] In Fc.gamma.RI (CD64) including Fc.gamma.RIa, Fc.gamma.RIb,
and Fc.gamma.RIc, and Fc.gamma.RIII (CD16) including isoforms
Fc.gamma.RIIIa (including allotypes V158 and F158) and
Fc.gamma.RIIIb (including allotypes Fc.gamma.RIIIb-NA1 and
Fc.gamma.RIIIb-NA2), a chain that binds to the Fc portion of IgG is
associated with common .gamma. chain having ITAM responsible for
transduction of intracellular activation signal. Meanwhile, the
cytoplasmic domain of Fc.gamma.RII (CD32) including isoforms
Fc.gamma.RIIa (including allotypes H131 and R131) and Fc.gamma.RIIc
contains ITAM. These receptors are expressed on many immune cells
such as macrophages, mast cells, and antigen-presenting cells. The
activation signal transduced upon binding of these receptors to the
Fc portion of IgG results in enhancement of the phagocytic activity
of macrophages, inflammatory cytokine production, mast cell
degranulation, and the enhanced function of antigen-presenting
cells. Fc.gamma. receptors having the ability to transduce the
activation signal as described above are also referred to as
activating Fc.gamma. receptors.
[0288] Meanwhile, the intracytoplasmic domain of Fc.gamma.RIIb
(including Fc.gamma.RIIb-1 and Fc.gamma.RIIb-2) contains ITIM
responsible for transduction of inhibitory signals. The
crosslinking between Fc.gamma.RIIb and B cell receptor (BCR) on B
cells suppresses the activation signal from BCR, which results in
suppression of antibody production via BCR. The crosslinking of
Fc.gamma.RIII and Fc.gamma.RIIb on macrophages suppresses the
phagocytic activity and inflammatory cytokine production. Fc.gamma.
receptors having the ability to transduce the inhibitory signal as
described above are also referred to as inhibitory Fc.gamma.
receptor.
Fc.gamma.R-Binding Activity of Fc Region
[0289] As mentioned above, Fc regions having an Fc.gamma.
receptor-binding activity are examples of Fc regions comprised in
the antigen-binding molecules of the present invention. A
non-limiting embodiment of such an Fc region includes the Fc region
of human IgG1 (SEQ ID NO: 13), IgG2 (SEQ ID NO: 14), IgG3 (SEQ ID
NO: 15), or IgG4 (SEQ ID NO: 16). Whether an Fc.gamma. receptor has
binding activity to the Fc region of a monoclonal IgG1, IgG2, IgG3,
or IgG4 antibody can be assessed by ALPHA screen (Amplified
Luminescent Proximity Homogeneous Assay), surface plasmon resonance
(SPR)-based BIACORE method, and others (Proc. Natl. Acad. Sci. USA
(2006) 103(11), 4005-4010), in addition to the above-described FACS
and ELISA formats.
[0290] ALPHA screen is performed by the ALPHA technology based on
the principle described below using two types of beads: donor and
acceptor beads. A luminescent signal is detected only when
molecules linked to the donor beads interact biologically with
molecules linked to the acceptor beads and when the two beads are
located in close proximity. Excited by laser beam, the
photosensitizer in a donor bead converts oxygen around the bead
into excited singlet oxygen. When the singlet oxygen diffuses
around the donor beads and reaches the acceptor beads located in
close proximity, a chemiluminescent reaction within the acceptor
beads is induced. This reaction ultimately results in light
emission. If molecules linked to the donor beads do not interact
with molecules linked to the acceptor beads, the singlet oxygen
produced by donor beads do not reach the acceptor beads and
chemiluminescent reaction does not occur.
[0291] For example, a biotin-labeled antigen-binding molecule
comprising Fc region is immobilized to the donor beads and
glutathione S-transferase (GST)-tagged Fc.gamma. receptor is
immobilized to the acceptor beads. In the absence of an
antigen-binding molecule comprising a competitive Fc region
variant, Fc.gamma. receptor interacts with a polypeptide complex
comprising a wild-type Fc region, inducing a signal of 520 to 620
nm as a result. The antigen-binding molecule having a non-tagged Fc
region variant competes with the antigen-binding molecule
comprising a native Fc region for the interaction with Fc.gamma.
receptor. The relative binding affinity can be determined by
quantifying the reduction of fluorescence as a result of
competition. Methods for biotinylating the antigen-binding
molecules such as antibodies using Sulfo-NHS-biotin or the like are
known. Appropriate methods for adding the GST tag to an Fc.gamma.
receptor include methods that involve fusing polypeptides encoding
Fc.gamma. and GST in-frame, expressing the fused gene using cells
introduced with a vector to which the gene is operablye linked, and
then purifying using a glutathione column. The induced signal can
be preferably analyzed, for example, by fitting to a one-site
competition model based on nonlinear regression analysis using
software such as GRAPHPAD PRISM (GraphPad; San Diego).
[0292] One of the substances for observing their interaction is
immobilized as a ligand onto the gold thin layer of a sensor chip.
When light is shed on the rear surface of the sensor chip so that
total reflection occurs at the interface between the gold thin
layer and glass, the intensity of reflected light is partially
reduced at a certain site (SPR signal). The other substance for
observing their interaction is injected as an analyte onto the
surface of the sensor chip. The mass of immobilized ligand molecule
increases when the analyte binds to the ligand. This alters the
refraction index of solvent on the surface of the sensor chip. The
change in refraction index causes a positional shift of SPR signal
(conversely, the dissociation shifts the signal back to the
original position). In the Biacore system, the amount of shift
described above (i.e., the change of mass on the sensor chip
surface) is plotted on the vertical axis, and thus the change of
mass over time is shown as measured data (sensorgram). Kinetic
parameters (association rate constant (ka) and dissociation rate
constant (kd)) are determined from the curve of sensorgram, and
affinity (KD) is determined from the ratio between these two
constants. Inhibition assay is preferably used in the BIACORE
methods. Examples of such inhibition assay are described in Proc.
Natl. Acad. Sci. USA (2006) 103(11), 4005-4010.
[0293] In addition to the Fc region of human IgG1 (SEQ ID NO: 13),
IgG2 (SEQ ID NO: 14), IgG3 (SEQ ID NO: 15), or IgG4 (SEQ ID NO:
16), an Fc region with modified Fc.gamma.R binding, which has a
higher Fc.gamma. receptor-binding activity than an Fc region of a
native human IgG may be appropriately used as an Fc region included
in the present invention. Herein, "Fc region of a native human IgG"
refers to an Fc region in which the sugar chain bonded to position
297 (EU numbering) of the Fc region of human IgG1, IgG2, IgG3, or
IgG4 shown in SEQ ID NOs: 13, 14, 15, or 16 is a fucose-containing
sugar chain. Such Fc regions with modified Fc.gamma.R binding may
be produced by modifying amino acids of the Fc region of a native
human IgG. Whether the Fc.gamma.R-binding activity of an Fc region
with modified Fc.gamma.R binding is higher than that of an Fc
region of a native human IgG can be determined appropriately using
methods described in the abovementioned section on binding
activity.
[0294] In the present invention, "modification of amino acids" or
"amino acid modification" of an Fc region includes modification
into an amino acid sequence which is different from that of the
starting Fc region. The starting Fc region may be any Fc region, as
long as a variant modified from the starting Fc region can bind to
human Fc.gamma. receptor in a neutral pH range. Furthermore, an Fc
region modified from a starting Fc region which had been already
modified can also be used preferably as an Fc region of the present
invention. The "starting Fc region" can refer to the polypeptide
itself, a composition comprising the starting Fc region, or an
amino acid sequence encoding the starting Fc region. Starting Fc
regions can comprise known Fc regions produced via recombination
described briefly in the section "Antibodies". The origin of
starting Fc regions is not limited, and they may be obtained from
human or any nonhuman organisms. Such organisms preferably include
mice, rats, guinea pigs, hamsters, gerbils, cats, rabbits, dogs,
goats, sheep, bovines, horses, camels and organisms selected from
nonhuman primates. In another embodiment, starting Fc regions can
also be obtained from cynomolgus monkeys, marmosets, rhesus
monkeys, chimpanzees, or humans. Starting Fc regions can be
obtained preferably from human IgG1; however, they are not limited
to any particular IgG class. This means that an Fc region of human
IgG1, IgG2, IgG3, or IgG4 can be used appropriately as a starting
Fc region, and herein also means that an Fc region of an arbitrary
IgG class or subclass derived from any organisms described above
can be preferably used as a starting Fc region. Examples of
naturally-occurring IgG variants or modified forms are described in
published documents (Curr. Opin. Biotechnol. (2009) 20 (6): 685-91;
Curr. Opin. Immunol. (2008) 20 (4), 460-470; Protein Eng. Des. Sel.
(2010) 23 (4): 195-202; International Publication Nos. WO
2009/086320, WO 2008/092117, WO 2007/041635, and WO 2006/105338);
however, they are not limited to the examples.
[0295] Examples of alterations include those with one or more
mutations, for example, mutations by substitution of different
amino acid residues for amino acids of starting Fc regions, by
insertion of one or more amino acid residues into starting Fc
regions, or by deletion of one or more amino acids from starting Fc
region. Preferably, the amino acid sequences of altered Fc regions
comprise at least a part of the amino acid sequence of a non-native
Fc region. Such variants necessarily have sequence identity or
similarity less than 100% to their starting Fc region. In a
preferred embodiment, the variants have amino acid sequence
identity or similarity about 75% to less than 100%, more preferably
about 80% to less than 100%, even more preferably about 85% to less
than 100%, still more preferably about 90% to less than 100%, and
yet more preferably about 95% to less than 100% to the amino acid
sequence of their starting Fc region. In a non-limiting embodiment
of the present invention, at least one amino acid is different
between an Fc.gamma.R-binding modified Fc region of the present
invention and its starting Fc region. Amino acid difference between
an Fc.gamma.R-binding modified Fc region of the present invention
and its starting Fc region can also be preferably specified based
on the specific amino acid differences at the above-described
specific amino acid positions according to EU numbering system.
[0296] Known methods such as site-directed mutagenesis (Kunkel et
al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and Overlap
extension PCR can be appropriately employed to modify the amino
acids of Fc regions. Furthermore, various known methods can also be
used as an amino acid modification method for substituting amino
acids by those other than natural amino acids (Annu Rev. Biophys.
Biomol. Struct. (2006) 35, 225-249; Proc. Natl. Acad. Sci. U.S.A.
(2003) 100 (11), 6353-6357). For example, a cell-free translation
system (Clover Direct (Protein Express)) containing tRNAs in which
amber suppressor tRNA, which is complementary to UAG codon (amber
codon) that is a stop codon, is linked with an unnatural amino acid
may be suitably used.
[0297] Included in the antigen-binding molecules of the present
invention, an Fc region with modified Fc.gamma.R binding, which has
a higher Fc.gamma. receptor-binding activity than that of an Fc
region of a native human IgG, (an Fc.gamma.R binding-modified Fc
region) may be obtained by any method. Specifically, the Fc region
with modified Fc.gamma.R binding may be obtained by modifying amino
acids of an IgG-type human immunoglobulin used as a starting Fc
region. Preferred Fc regions of the IgG-type immunoglobulins for
modification include, for example, those of human IgGs shown in SEQ
ID NOs: 13, 14, 15, or 16 (IgG1, IgG2, IgG3, or IgG4, respectively,
and variants thereof).
[0298] Amino acids of any positions may be modified into other
amino acids, as long as the binding activity toward the Fc.gamma.
receptor is higher than that of the Fc region of a native human
IgG. When the antigen-binding molecule contains a human IgG1 Fc
region as the human Fc region, it preferably contains a
modification that yields the effect of a higher Fc.gamma.
receptor-binding activity than that of the Fc region of a native
human IgG, in which the sugar chain bound at position 297 (EU
numbering) is a fucose-containing sugar chain. Such amino acid
modifications have been reported, for example, in international
publications such as WO2007/024249, WO2007/021841, WO2006/031370,
WO2000/042072, WO2004/029207, WO2004/099249, WO2006/105338,
WO2007/041635, WO2008/092117, WO2005/070963, WO2006/020114,
WO2006/116260, and WO2006/023403.
[0299] Examples of such amino acids that may be modified include at
least one or more amino acids selected from the group consisting of
positions 221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233,
234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247,
249, 250, 251, 254, 255, 256, 258, 260, 262, 263, 264, 265, 266,
267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280,
281, 282, 283, 284, 285, 286, 288, 290, 291, 292, 293, 294, 295,
296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 311, 313, 315,
317, 318, 320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331,
332, 333, 334, 335, 336, 337, 339, 376, 377, 378, 379, 380, 382,
385, 392, 396, 421, 427, 428, 429, 434, 436, and 440 (EU
numbering). An Fc region (Fc region with modified Fc.gamma.R
binding) having a higher Fc.gamma. receptor-binding activity than
that of an Fc region of a native human IgG can be obtained by
modifying these amino acids.
[0300] Examples of particularly preferable modifications for use in
the present invention include at least one or more amino acid
modifications selected from the group consisting of:
Lys or Tyr for the amino acid of position 221; Phe, Trp, Glu, or
Tyr for the amino acid of position 222; Phe, Trp, Glu, or Lys for
the amino acid of position 223; Phe, Trp, Glu, or Tyr for the amino
acid of position 224; Glu, Lys, or Trp for the amino acid of
position 225; Glu, Gly, Lys, or Tyr for the amino acid of position
227; Glu, Gly, Lys, or Tyr for the amino acid of position 228; Ala,
Glu, Gly, or Tyr for the amino acid of position 230; Glu, Gly, Lys,
Pro, or Tyr for the amino acid of position 231; Glu, Gly, Lys, or
Tyr for the amino acid of position 232; Ala, Asp, Phe, Gly, His,
Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for
the amino acid of position 233; Ala, Asp, Glu, Phe, Gly, His, Ile,
Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the
amino acid of position 234; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys,
Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino
acid of position 235; Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met,
Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid
of position 236; Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro,
Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of position
237; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,
Ser, Thr, Val, Trp, or Tyr for the amino acid of position 238; Asp,
Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr,
Val, Trp, or Tyr for the amino acid of position 239; Ala, Ile, Met,
or Thr for the amino acid of position 240; Asp, Glu, Leu, Arg, Trp,
or Tyr for the amino acid of position 241; Leu, Glu, Leu, Gln, Arg,
Trp, or Tyr for the amino acid of position 243; His for the amino
acid of position 244; Ala for the amino acid of position 245; Asp,
Glu, His, or Tyr for the amino acid of position 246; Ala, Phe, Gly,
His, Ile, Leu, Met, Thr, Val, or Tyr for the amino acid of position
247; Glu, His, Gln, or Tyr for the amino acid of position 249; Glu
or Gln for the amino acid of position 250; Phe for the amino acid
of position 251; Phe, Met, or Tyr for the amino acid of position
254; Glu, Leu, or Tyr for the amino acid of position 255; Ala, Met,
or Pro for the amino acid of position 256; Asp, Glu, His, Ser, or
Tyr for the amino acid of position 258; Asp, Glu, His, or Tyr for
the amino acid of position 260; Ala, Glu, Phe, Ile, or Thr for the
amino acid of position 262; Ala, Ile, Met, or Thr for the amino
acid of position 263; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met,
Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr for the amino acid of
position 264; Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,
Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of
position 265; Ala, Ile, Met, or Thr for the amino acid of position
266; Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,
Thr, Val, Trp, or Tyr for the amino acid of position 267; Asp, Glu,
Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, or Trp for
the amino acid of position 268; Phe, Gly, His, Ile, Lys, Leu, Met,
Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of
position 269; Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg,
Ser, Thr, Trp, or Tyr for the amino acid of position 270; Ala, Asp,
Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr,
Val, Trp, or Tyr for the amino acid of position 271; Asp, Phe, Gly,
His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for
the amino acid of position 272; Phe or Ile for the amino acid of
position 273; Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro,
Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of position 274;
Leu or Trp for the amino acid of position 275; Asp, Glu, Phe, Gly,
His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the
amino acid of position 276; Asp, Glu, Gly, His, Ile, Lys, Leu, Met,
Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp for the amino acid of
position 278; Ala for the amino acid of position 279; Ala, Gly,
His, Lys, Leu, Pro, Gln, Trp, or Tyr for the amino acid of position
280; Asp, Lys, Pro, or Tyr for the amino acid of position 281; Glu,
Gly, Lys, Pro, or Tyr for the amino acid of position 282; Ala, Gly,
His, Ile, Lys, Leu, Met, Pro, Arg, or Tyr for the amino acid of
position 283; Asp, Glu, Leu, Asn, Thr, or Tyr for the amino acid of
position 284; Asp, Glu, Lys, Gln, Trp, or Tyr for the amino acid of
position 285; Glu, Gly, Pro, or Tyr for the amino acid of position
286; Asn, Asp, Glu, or Tyr for the amino acid of position 288; Asp,
Gly, His, Leu, Asn, Ser, Thr, Trp, or Tyr for the amino acid of
position 290; Asp, Glu, Gly, His, Ile, Gln, or Thr for the amino
acid of position 291; Ala, Asp, Glu, Pro, Thr, or Tyr for the amino
acid of position 292; Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg,
Ser, Thr, Val, Trp, or Tyr for the amino acid of position 293; Phe,
Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or
Tyr for the amino acid of position 294; Asp, Glu, Phe, Gly, His,
Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the
amino acid of position 295; Ala, Asp, Glu, Gly, His, Ile, Lys, Leu,
Met, Asn, Gln, Arg, Ser, Thr, or Val for the amino acid of position
296; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg,
Ser, Thr, Val, Trp, or Tyr for the amino acid of position 297; Ala,
Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, or
Tyr for the amino acid of position 298; Ala, Asp, Glu, Phe, Gly,
His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr
for the amino acid of position 299; Ala, Asp, Glu, Gly, His, Ile,
Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp for the
amino acid of position 300; Asp, Glu, His, or Tyr for the amino
acid of position 301; Ile for the amino acid of position 302; Asp,
Gly, or Tyr for the amino acid of position 303; Asp, His, Leu, Asn,
or Thr for the amino acid of position 304; Glu, Ile, Thr, or Tyr
for the amino acid of position 305; Ala, Asp, Asn, Thr, Val, or Tyr
for the amino acid of position 311; Phe for the amino acid of
position 313; Leu for the amino acid of position 315; Glu or Gln
for the amino acid of position 317; His, Leu, Asn, Pro, Gln, Arg,
Thr, Val, or Tyr for the amino acid of position 318; Asp, Phe, Gly,
His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, or Tyr for the amino
acid of position 320; Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr,
Val, Trp, or Tyr for the amino acid of position 322; Ile for the
amino acid of position 323; Asp, Phe, Gly, His, Ile, Leu, Met, Pro,
Arg, Thr, Val, Trp, or Tyr for the amino acid of position 324; Ala,
Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser,
Thr, Val, Trp, or Tyr for the amino acid of position 325; Ala, Asp,
Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr
for the amino acid of position 326; Ala, Asp, Glu, Phe, Gly, His,
Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, or Tyr for the
amino acid of position 327; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys,
Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino
acid of position 328; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met,
Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of
position 329; Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,
Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of position
330; Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, or Tyr
for the amino acid of position 331; Ala, Asp, Glu, Phe, Gly, His,
Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for
the amino acid of position 332; Ala, Asp, Glu, Phe, Gly, His, Ile,
Leu, Met, Pro, Ser, Thr, Val, or Tyr for the amino acid of position
333; Ala, Glu, Phe, Ile, Leu, Pro, or Thr for the amino acid of
position 334; Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg,
Ser, Val, Trp, or Tyr for the amino acid of position 335; Glu, Lys,
or Tyr for the amino acid of position 336; Glu, His, or Asn for the
amino acid of position 337; Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln,
Arg, Ser, or Thr for the amino acid of position 339; Ala or Val for
the amino acid of position 376; Gly or Lys for the amino acid of
position 377; Asp for the amino acid of position 378; Asn for the
amino acid of position 379; Ala, Asn, or Ser for the amino acid of
position 380; Ala or Ile for the amino acid of position 382; Glu
for the amino acid of position 385; Thr for the amino acid of
position 392; Leu for the amino acid of position 396; Lys for the
amino acid of position 421; Asn for the amino acid of position 427;
Phe or Leu for the amino acid of position 428; Met for the amino
acid of position 429; Trp for the amino acid of position 434; Ile
for the amino acid of position 436; and Gly, His, Ile, Leu, or Tyr
for the amino acid of position 440; as indicated by EU numbering.
The number of amino acids to be modified is not particularly
limited; and amino acid may be modified at only one site or amino
acids may be modified at two or more sites. Examples of
combinations for amino acid modifications at two or more sites
include those described in Table 6 (Tables 6-1 to 6-3).
TABLE-US-00038 TABLE 6-1 Combination of Combination of amino acids
amino acids K370E/P396L/D270E S239Q/I332Q Q419H/P396L/D270E
S267D/I332E V240A/P3996L/D270E S267E/I332E R255L/P396L/D270E
S267L/A327S R255L/P396L/D270E S267Q/A327S R255L/P396L/D270E/R292G
S298A/I332E R255L/P396L/D270E S304T/I332E R255L/P396L/D270E/Y300L
S324G/I332D P243L/D270E/K392N/P396L S324G/I332E
F243L/R255L/D270E/P396L S324I/I332D F243L/R292P/Y300L/V305I/P396L
S324I/I332E F243L/R292P/Y300L/P396L T260H/I332E F243L/R292P/Y300L
T335D/I332E F243L/R292P/P396L V240I/V266I F243L/R292P/V305I
V264I/I332E F243L/R292P D265F/M297E/I332E S298A/E333A/K334A
D265Y/N297D/I332E E380A/T307A F243L/V262I/V264W K326M/E333S
N297D/A330Y/I332E K326A/E333A N297D/T299E/I332E S317A/K353A
N297D/T299F/I332E A327D/I332E N297D/T299H/I332E A330L/I332E
N297D/T299I/I332E A330Y/I332E N297D/T299L/I332E E258H/I332E
N297D/T299V/I332E E272H/I332E P230A/E233D/I332E K272I/N276D
P244H/P245A/P247V E272R/I332E S239D/A330L/I332E E283H/I332E
S239D/A330V/I332E E293R/I332E S239D/H268E/A330Y F241L/V262I
S239D/I332E/A327A F241W/F243W S239D/I332E/A330I
Table 6-2 is a continuation of Table 6-1.
TABLE-US-00039 TABLE 6-2 F243L/V264I S239D/N297D/I332E H268D/A330Y
S239D/S298A/I332E H268E/A330Y S239D/V264I/I332E K246H/I332E
S239E/N297D/I332E L234D/I332E S239E/V264J/I332E L234E/I332E
S239N/A330L/I332E L234G/I332E S239N/A330Y/I332E L234I/I332E
S239N/S298A/I332E L234I/L235D S239Q/V264I/I332E L234Y/I332E
V264E/N297D/I332E L235D/I332E V264I/A330L/I332E L235E/I332E
V264I/A330Y/I332E L235I/I332E V264I/S298A/I332E L235S/I332E
Y296D/N297D/I332E L328A/I332D Y296E/N297D/I332E L328D/I332D
Y296H/N297D/I332E L328D/I332E Y296N/N297D/I332E L328E/I332D
Y296Q/N297D/I332E L328E/I332E Y296T/N297D/I332E L328F/I332D
D265Y/N297D/T299L/I332E L328F/I332E F241E/F243Q/V262T/V264E
L328H/I332E F241E/F243R/V262E/V264R L328I/I332D
F241E/F243Y/V262T/V264R L328I/I332E P241L/F243L/V262I/V264I
L328M/I332D F241R/F243Q/V262T/V264R L328M/I332E
F2418/F243H/V262T/V264T L328N/I332D F241W/F243W/V262A/V264A
L328N/I332E F241Y/F243Y/V262Y/V264T L328Q/I332D
I332E/A330Y/H268E/A327A L328Q/I332E N297D/I332E/S2390/A330L
L328T/I332D N297D/S298A/A330Y/I332E L328T/I332E
S239D/A330Y/I332E/K326E L328V/I332D S239D/A330Y/I332E/K326T
L328V/I332E S239D/A330Y/I332E/L234I L328Y/I332D
S239D/A330Y/I332E/L235D
Table 6-3 is a continuation of Table 6-2.
TABLE-US-00040 TABLE 6-3 L328Y/I332E S239D/A330Y/I332E/V240I
N297D/I332E S239D/A330Y/I332E/V264T N297E/I332E
S239D/A330Y/I332E/V266I N397S/I322E S239D/D268F/N297D/I332E
P227G/I332E S239D/D265H/N297D/I332E P230A/E233D
S239D/D265I/N297D/I332E Q295E/I332E S239D/D265L/N29ID/I332E
R255Y/I332E S239D/D265T/N297D/I332E S239D/I332D
S239D/D265V/N297D/I332E S239D/I332E S239D/D265Y/N297D/I332E
S239D/I332N S239D/I332E/A330Y/A327A S239D/I332Q
S239D/I332E/H268E/A327A S239E/D265G S239D/I332E/H268E/A330Y
S239E/D265N S239D/N297D/I332E/A330Y S239E/D265Q
S239D/N297D/I332E/K326E S239E/I332D S239D/N297D/I332E/L235D
S239E/I332E S239D/V264I/A330L/I332E S239E/I332N
S239D/V264I/S298A/I332E S239E/I332Q S239E/Y264I/A330Y/I332E
S239N/I332D F241E/F243Q/V262T/V264E/I332E S239N/I332E
F241E/F243R/V262E/V264R/I332E S239N/I332N
F241E/F243Y/V262T/V264R/I332E S239N/I332Q
F241R/F243Q/V262T/V264R/I332E S239Q/I332D
S239D/I332E/H268E/A330Y/A327A S239Q/I332E
S239E/V264I/S298A/A330V/I332E S239Q/I332N
F241Y/F243Y/V262T/V264T/N297D/I332E S267E/L328F G236D/S267E
S239D/S267E
[0301] For the pH conditions to measure the binding activity of the
Fc region and the Fc.gamma. receptor contained in the
antigen-binding molecule of the present invention, conditions in an
acidic pH range or in a neutral pH range may be suitably used. The
neutral pH range, as a condition to measure the binding activity of
the Fc region and the Fc.gamma. receptor contained in the
antigen-binding molecule of the present invention, generally
indicates pH 6.7 to pH10.0. Preferably, it is a range indicated
with arbitrary pH values between pH 7.0 and pH8.0; and preferably,
it is selected from pH 7.0, pH7.1, pH7.2, pH7.3, pH7.4, pH7.5,
pH7.6, pH7.7, pH7.8, pH7.9, and pH 8.0; and particularly
preferably, it is pH 7.4, which is close to the pH of plasma
(blood) in vivo. Herein, the acidic pH range, as a condition for
having a binding activity of the Fc region and the Fc.gamma.
receptor contained in the antigen-binding molecule of the present
invention, generally indicates pH 4.0 to pH6.5. Preferably, it
indicates pH 5.5 to pH6.5, and particularly preferably, it
indicates pH5.8 to pH6.0, which is close to the pH in the early
endosome in vivo. With regard to the temperature used as a
measurement condition, the binding affinity between an Fc region
and an Fc.gamma. receptor can be evaluated at any temperature
between 10.degree. C. and 50.degree. C. Preferably, a temperature
between 15.degree. C. and 40.degree. C. is used to determine the
binding affinity between an Fc region and an Fc.gamma. receptor.
More preferably, any temperature between 20.degree. C. and
35.degree. C., such as any single temperature from 20.degree. C.,
21.degree. C., 22.degree. C., 23.degree. C., 24.degree. C.,
25.degree. C., 26.degree. C., 27.degree. C., 28.degree. C.,
29.degree. C., 30.degree. C., 31.degree. C., 32.degree. C.,
33.degree. C., 34.degree. C., and 35.degree. C., can be similarly
used to determine the binding affinity between an Fc region and an
Fc.gamma. receptor. A temperature of 25.degree. C. is a
non-limiting example in an embodiment of the present invention.
[0302] Herein, "Fc region with modified Fc.gamma.R binding has a
higher Fc.gamma. receptor-binding activity than the native Fc
region" means that the human Fc.gamma. receptor-binding activity of
the Fc region with modified Fc.gamma.R binding toward any of the
human Fc.gamma. receptors of Fc.gamma.RI, Fc.gamma.RIIa,
Fc.gamma.RIIb, Fc.gamma.RIIIa, and/or Fc.gamma.RIIIb is higher than
the binding activity of the native Fc region toward these human
Fc.gamma. receptors. For example, it means that based on an
above-described analytical method, in comparison to the binding
activity of an antigen-binding molecule containing a native human
IgG Fc region as a control, the binding activity of the
antigen-binding molecule comprising an Fc region with modified
Fc.gamma.R binding is 105% or more, preferably 110% or more, 115%
or more, 120% or more, 125% or more, particularly preferably 130%
or more, 135% or more, 140% or more, 145% or more, 150% or more,
155% or more, 160% or more, 165% or more, 170% or more, 175% or
more, 180% or more, 185% or more, 190% or more, 195% or more,
2-fold or more, 2.5-fold or more, 3-fold or more, 3.5-fold or more,
4-fold or more, 4.5-fold or more, 5-fold or more, 7.5-fold or more,
10-fold or more, 20-fold or more, 30-fold or more, 40-fold or more,
50-fold or more, 60-fold or more, 70-fold or more, 80-fold or more,
90-fold or more, or 100-fold or more. The starting Fc region may be
used as a native Fc region, and native Fc regions of antibodies of
the same subclass may also be used.
[0303] In the present invention, an Fc region of a native human IgG
in which the sugar chain bonded to the amino acid at position 297
(EU numbering) is a fucose-containing sugar chain, is suitably used
as a native Fc region of human IgG to be used as a control. Whether
or not the sugar chain bonded to the amino acid at position 297 (EU
numbering) is a fucose-containing sugar chain can be determined
using the technique described in Non-Patent Document 6. For
example, it is possible to determine whether or not the sugar chain
bonded to the native human IgG Fc region is a fucose-containing
sugar chain by a method such as the one below. Sugar chain is
dissociated from a native human IgG to be tested, by reacting the
test native human IgG with N-Glycosidase F (Roche diagnostics)
(Weitzhandler et al. (J. Pharma. Sciences (1994) 83, 12,
1670-1675)). Next, a dried concentrate of a reaction solution from
which protein has been removed by reaction with ethanol (Schenk et
al. (J. Clin. Investigation (2001) 108 (11) 1687-1695)) is
fluorescently labeled with 2-aminopyridine or 2-aminobenzamide
(Bigge et al. (Anal. Biochem. (1995) 230 (2) 229-238)). Reagents
are removed by solid extraction using a cellulose cartridge, and
the fluorescently labeled 2-AP or 2-AB-modified sugar chain is
analyzed by normal-phase chromatography. It is possible to
determine whether or not the sugar chain bonded to the native Fc
region of a human IgG is a fucose-containing sugar chain by
observing the detected chromatogram peaks.
[0304] As an antigen-binding molecule containing an Fc region of a
native antibody of the same subclass, which is to be used as a
control, an antigen-binding molecule having an Fc region of a
monoclonal IgG antibody may be suitably used. The structures of the
Fc regions are described in SEQ ID NO: 13 (A is added to the N
terminus of RefSeq Accession No. AAC82527.1), SEQ ID NO: 14 (A is
added to the N terminus of RefSeq Accession No. AAB59393.1), SEQ ID
NO: 15 (RefSeq Accession No. CAA27268.1), and SEQ ID NO: 16 (A is
added to the N terminus of RefSeq Accession No. AAB59394.1).
Further, when an antigen-binding molecule containing an Fc region
of a particular antibody isotype is used as the test substance, the
effect of the antigen-binding molecule containing the test Fc
region on Fc.gamma. receptor-binding activity is tested by using as
a control an antigen-binding molecule having an Fc region of a
monoclonal IgG antibody of that particular isotype. In this way,
antigen-binding molecules containing an Fc region of which
Fc.gamma. receptor-binding activity is demonstrated to be high are
suitably selected.
Fc Regions Having a Selective Binding Activity to an Fc.gamma.
Receptor
[0305] Examples of Fc regions suitable for use in the present
invention include Fc regions having a higher binding activity to a
particular Fc.gamma. receptor than to other Fc.gamma. receptors (Fc
regions having a selective binding activity to an Fc.gamma.
receptor). When an antibody is used as the antigen-binding
molecule, a single antibody molecule can only bind to a single
Fc.gamma. receptor molecule. Therefore, a single antigen-binding
molecule cannot bind to other activating Fc.gamma.Rs in an
inhibitory Fc.gamma. receptor-bound state, and cannot bind to other
activating Fc.gamma. receptors or inhibitory Fc.gamma. receptors in
an activating Fc.gamma. receptor-bound state.
[0306] As described above, suitable examples of activating
Fc.gamma. receptors include Fc.gamma.RI (CD64) which includes
Fc.gamma.RIa, Fc.gamma.RIb, and Fc.gamma.RIc; Fc.gamma.RIII (CD16)
which includes isoforms Fc.gamma.RIIIa (including allotypes V158 or
F158) and Fc.gamma.RIIIb (including allotypes Fc.gamma.RIIIb-NA1
and Fc.gamma.RIIIb-NA2); and Fc.gamma.RIIa (including allotypes
H131 or R131). Meanwhile, suitable examples of inhibitory Fc.gamma.
receptors include Fc.gamma.RIIb (including Fc.gamma.RIIb-1 or
Fc.gamma.RIIb-2).
[0307] The Fc region comprised in an antigen-binding molecule of
the present invention that contains a selective Fc.gamma.R-binding
domain, and an antigen-binding molecule comprising this Fc region
may have a maintained or reduced binding activity to activating
Fc.gamma.R (Fc.gamma.RIa, Fc.gamma.RIb, Fc.gamma.RIc,
Fc.gamma.RIIIa including allotype V158, Fc.gamma.RIIIa including
allotype F158, Fc.gamma.RIIIb including allotype
Fc.gamma.RIIIb-NA1, Fc.gamma.RIIIb including allotype
Fc.gamma.RIIIb-NA2, Fc.gamma.RIIa including allotype H131,
Fc.gamma.RIIa including allotype R131 and/or Fc.gamma.RIIc), when
compared to the Fc region of human IgG1 (SEQ ID NO: 13), IgG2 (SEQ
ID NO: 14), IgG3 (SEQ ID NO: 15), or IgG4 (SEQ ID NO: 16)
(hereinafter referred to as wild-type Fc region) and an
antigen-binding molecule comprising this wild-type Fc region.
[0308] In comparison to a wild-type Fc region and an
antigen-binding molecule comprising a wild-type Fc region, an Fc
region comprised in an antigen-binding molecule of the present
invention that contains a selective Fc.gamma.R-binding domain, and
an antigen-binding molecule comprising this Fc region are reduced
in their binding activity to the aforementioned activating
Fc.gamma.R at a level of, for example, 99% or less, 98% or less,
97% or less, 96% or less, 95% or less, 94% or less, 93% or less,
92% or less, 91% or less, 90% or less, 88% or less, 86% or less,
84% or less, 82% or less, 80% or less, 78% or less, 76% or less,
74% or less, 72% or less, 70% or less, 68% or less, 66% or less,
64% or less, 62% or less, 60% or less, 58% or less, 56% or less,
54% or less, 52% or less, 50% or less, 45% or less, 40% or less,
35% or less, 30% or less, 25% or less, 20% or less, 15% or less,
10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or
less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, 0.1%
or less, 0.05% or less, 0.01% or less, or 0.005% or less.
[0309] The Fc region comprised in an antigen-binding molecule of
the present invention that contains a selective Fc.gamma.R-binding
domain, and an antigen-binding molecule comprising this Fc region
may have an enhanced binding activity to inhibitory Fc.gamma.R
(Fc.gamma.RIIb-1 and/or Fc.gamma.RIIb-2) when compared to the Fc
region of human IgG1 (SEQ ID NO: 13), IgG2 (SEQ ID NO: 14), IgG3
(SEQ ID NO: 15), or IgG4 (SEQ ID NO: 16) (hereinafter referred to
as wild-type Fc region), and an antigen-binding molecule comprising
the wild-type Fc region.
[0310] In comparison to a wild-type Fc region and an
antigen-binding molecule comprising a wild-type Fc region, the Fc
region comprised in an antigen-binding molecule of the present
invention that contains a selective Fc.gamma.R-binding domain, and
an antigen-binding molecule comprising this Fc region, are enhanced
in their binding activity to the aforementioned inhibitory
Fc.gamma.R at a level of, for example, 101% or greater, 102% or
greater, 103% or greater, 104% or greater, 105% or greater, 106% or
greater, 107% or greater, 108% or greater, 109% or greater, 110% or
greater, 112% or greater, 114% or greater, 116% or greater, 118% or
greater, 120% or greater, 122% or greater, 124% or greater, 126% or
greater, 128% or greater, 130% or greater, 132% or greater, 134% or
greater, 136% or greater, 138% or greater, 140% or greater, 142% or
greater, 144% or greater, 146% or greater, 148% or greater, 150% or
greater, 155% or greater, 160% or greater, 165% or greater, 170% or
greater, 175% or greater, 180% or greater, 185% or greater, 190% or
greater, 195% or greater, 2-fold or greater, 3-fold or greater,
4-fold or greater, 5-fold or greater, 6-fold or greater, 7-fold or
greater, 8-fold or greater, 9-fold or greater, 10-fold or greater,
20-fold or greater, 30-fold or greater, 40-fold or greater, 50-fold
or greater, 60-fold or greater, 70-fold or greater, 80-fold or
greater, 90-fold or greater, 100-fold or greater, 200-fold or
greater, 300-fold or greater, 400-fold or greater, 500-fold or
greater, 600-fold or greater, 700-fold or greater, 800-fold or
greater, 900-fold or greater, 1000-fold or greater, 10000-fold or
greater, or 100000-fold or greater.
[0311] The Fc region comprised in an antigen-binding molecule of
the present invention that contains a selective Fc.gamma.R-binding
domain, and an antigen-binding molecule comprising this Fc region
may have
a maintained or reduced binding activity to activating Fc.gamma.R
(Fc.gamma.RIa, Fc.gamma.RIb, Fc.gamma.RIc, Fc.gamma.RIIIa including
allotype V158, Fc.gamma.RIIIa including allotype F158,
Fc.gamma.RIIIb including allotype Fc.gamma.RIIIb-NA1,
Fc.gamma.RIIIb including allotype Fc.gamma.RIIIb-NA2, Fc.gamma.RIIa
including allotype H131, Fc.gamma.RIIa including allotype R131,
and/or Fc.gamma.RIIc), when compared to the Fc region of human IgG1
(SEQ ID NO: 13), IgG2 (SEQ ID NO: 14), IgG3 (SEQ ID NO: 15), or
IgG4 (SEQ ID NO: 16) (hereinafter referred to as wild-type Fc
region), and an antigen-binding molecule comprising the wild-type
Fc region; and an enhanced binding activity to inhibitory
Fc.gamma.R (Fc.gamma.RIIb-1 and/or Fc.gamma.RIIb-2) when compared
to the Fc region of human IgG1 (SEQ ID NO: 13), IgG2 (SEQ ID NO:
14), IgG3 (SEQ ID NO: 15), or IgG4 (SEQ ID NO: 16) (hereinafter
referred to as wild-type Fc region), and an antigen-binding
molecule comprising the wild-type Fc region.
[0312] In comparison to the Fc region of human IgG1 (SEQ ID NO:
13), IgG2 (SEQ ID NO: 14), IgG3 (SEQ ID NO: 15), or IgG4 (SEQ ID
NO: 16) (hereinafter referred to as wild-type Fc region) and an
antigen-binding molecule comprising the wild-type Fc region, the Fc
region comprised in an antigen-binding molecule of the present
invention that contains a selective Fc.gamma.R-binding domain, and
an antigen-binding molecule comprising this Fc region, have a
higher level of enhancement in their binding activity to inhibitory
Fc.gamma.R (Fc.gamma.RIIb-1 and/or Fc.gamma.RIIb-2) than the level
of enhancement in their binding activity to activating
Fc.gamma.receptor (Fc.gamma.RIa, Fc.gamma.RIb, Fc.gamma.RIc,
Fc.gamma.RIIIa including allotype V158, Fc.gamma.RIIIa including
allotype F158, Fc.gamma.RIIIb including allotype
Fc.gamma.RIIIb-NA1, Fc.gamma.RIIIb including allotype
Fc.gamma.RIIIb-NA2, Fc.gamma.RIIa including allotype H131, and
Fc.gamma.RIIa including allotype R131).
[0313] In the present invention, it is possible to add at least
another modification to the Fc region whose amino acid at position
238 (EU numbering) is Asp or to the Fc region whose amino acid at
position 328 (EU numbering) is Glu by the embodiments and such
described in the aforementioned section on amino acid
modifications. In addition to these modifications, additional
modifications may also be included. Additional modifications can be
selected, for example, from any of amino acid substitutions,
deletions, and modifications, and combinations thereof. For
example, modifications that enhance the Fc.gamma.RIIb-binding
activity while maintaining or decreasing the binding activity to
Fc.gamma.RIIa (H type) and Fc.gamma.RIIa (R type) may be added.
Addition of such modifications improves binding selectivity to
Fc.gamma.RIIb over Fc.gamma.RIIa.
[0314] Furthermore, whether or not the binding activities of the
polypeptides of the present invention towards various Fc.gamma.Rs
were maintained, enhanced, or decreased can be determined from the
increase or decrease in the amount of binding of the various
Fc.gamma.Rs to the polypeptides of the present invention, which
were determined according to the examples described above. Here,
the amount of binding of the various Fc.gamma.Rs to the
polypeptides refers to values obtained by determining the
difference in the RU values of sensorgrams that changed before and
after interaction of various Fc.gamma.Rs as the analyte with each
polypeptide, and dividing them by differences in the RU values of
sensorgrams that changed before and after capturing polypeptides to
the sensor chips.
[0315] It is possible to determine whether the Fc.gamma.RIIa (R
type and H type)-binding activity of the polypeptides of the
present invention is maintained or decreased, and whether the
peptides are peptides with enhanced Fc.gamma.RIIb-binding activity
can be determined from the binding amount of the polypeptides to
Fc.gamma.RIIa and the binding amount of the polypeptides to
Fc.gamma.RIIb by following the above-mentioned examples.
[0316] An exemplary case is when the amount of a polypeptide of the
present invention bound to Fc.gamma.RIIb is increased compared to
the Fc.gamma.RIIb-binding amount of the parent polypeptide, and the
amount of the polypeptide of the present invention bound to
Fc.gamma.RIIa (R type and H type) is equivalent to (maintained) or
preferably decreased relative to the binding amount of the parent
polypeptide to Fc.gamma.RIIa (R type and H type). It is also
possible to determine by appropriately combining the
Fc.gamma.RIa-binding amount and the Fc.gamma.RIIIa-binding amount
of the polypeptide determined by following the above-mentioned
examples.
[0317] In a non-limiting embodiment of the present invention, an
example of an Fc region having a higher inhibitory Fc.gamma.
receptor-binding activity than an activating Fc.gamma.
recptor-binding activity (having a selective binding activity
toward an inhibitory Fc.gamma. receptor) is the Fc region presented
in US2009/0136485 or WO 2012/115241; and another suitable example
is an Fc region in which the amino acid at position 238 or 328 as
indicated by EU numbering in the aforementioned Fc region has been
modified to an amino acid different from that of the native Fc
region.
[0318] In a non-limiting embodiment of the present invention, a
suitable example of an Fc region that has a higher binding activity
toward an inhibitory Fc.gamma.receptor than toward an activating
Fc.gamma.receptor (i.e., having a selective binding activity toward
an inhibitory Fc.gamma.receptor) is an Fc region with one or more
of the following modifications in the amino acids (indicated by EU
numbering) of the aforementioned Fc region: the amino acid at
position 238 is modified to Asp and the amino acid at position 328
is modified to Glu. The Fc regions and modifications described in
US2009/0136485 or WO 2012/115241 may be selected appropriately as
the Fc region having a selective binding activity to an inhibitory
Fc.gamma.receptor.
[0319] In a non-limiting embodiment of the present invention, a
suitable example is an Fc region in which one or more of the amino
acids indicated by EU numbering at positions 238 and 328 according
to EU numbering are respectively modified to Asp or Glu in the
aforementioned Fc region.
[0320] Furthermore, in a non-limiting embodiment of the present
invention, suitable examples of the Fc regions are those with
substitution of Asp for Pro at position 238 (EU numbering), and one
or more modifications selected from among Trp for the amino acid of
position 237, Phe for the amino acid of position 237, Val for the
amino acid of position 267, Gln for the amino acid of position 267,
Asn for the amino acid of position 268, Gly for the amino acid of
position 271, Leu for the amino acid of position 326, Gln for the
amino acid of position 326, Glu for the amino acid of position 326,
Met for the amino acid of position 326, Asp for the amino acid of
position 239, Ala for the amino acid of position 267, Trp for the
amino acid of position 234, Tyr for the amino acid of position 234,
Ala for the amino acid of position 237, Asp for the amino acid of
position 237, Glu for the amino acid of position 237, Leu for the
amino acid of position 237, Met for the amino acid of position 237,
Tyr for the amino acid of position 237, Lys for the amino acid of
position 330, Arg for the amino acid of position 330, Asp for the
amino acid of position 233, Asp for the amino acid of position 268,
Glu for the amino acid of position 268, Asp for the amino acid of
position 326, Ser for the amino acid of position 326, Thr for the
amino acid of position 326, Ile for the amino acid of position 323,
Leu for the amino acid of position 323, Met for the amino acid of
position 323, Asp for the amino acid of position 296, Ala for the
amino acid of position 326, Asn for the amino acid of position 326,
and Met for the amino acid of position 330, according to EU
numbering.
Antigen-Binding Molecule
[0321] In the present invention, an "antigen-binding molecule" is
used in the broadest sense to refer to a molecule containing an
antigen-binding domain and an Fc region. Specifically, the
antigen-binding molecules include various types of molecules as
long as they exhibit the antigen-binding activity. Molecules in
which an antigen-binding domain is linked to an Fc region include,
for example, antibodies. Antibodies may include single monoclonal
antibodies (including agonistic antibodies and antagonistic
antibodies), human antibodies, humanized antibodies, chimeric
antibodies, and such. Alternatively, when used as antibody
fragments, they preferably include antigen-binding domains and
antigen-binding fragments (for example, Fab, F(ab')2, scFv, and
Fv). Scaffold molecules where three dimensional structures, such as
already-known stable .alpha./.beta. barrel protein structure, are
used as a scaffold (base) and only some portions of the structures
are made into libraries to construct antigen-binding domains are
also included in antigen-binding molecules of the present
invention.
[0322] An antigen-binding molecule of the present invention may
contain at least some portions of an Fc region that mediates the
binding to FcRn and Fc.gamma. receptor. In a non-limiting
embodiment, the antigen-binding molecule includes, for example,
antibodies and Fc fusion proteins. A fusion protein refers to a
chimeric polypeptide comprising a polypeptide having a first amino
acid sequence that is linked to a polypeptide having a second amino
acid sequence that would not naturally link in nature. For example,
a fusion protein may comprise the amino acid sequence of at least a
portion of an Fc region (for example, a portion of an Fc region
responsible for the binding to FcRn or a portion of an Fc region
responsible for the binding to Fc.gamma. receptor) and a
non-immunoglobulin polypeptide containing, for example, the amino
acid sequence of the ligand-binding domain of a receptor or a
receptor-binding domain of a ligand. The amino acid sequences may
be present in separate proteins that are transported together to a
fusion protein, or generally may be present in a single protein;
however, they are included in a new rearrangement in a fusion
polypeptide. Fusion proteins can be produced, for example, by
chemical synthesis, or by genetic recombination techniques to
express a polynucleotide encoding peptide regions in a desired
arrangement.
[0323] Each of the domains of the antigen-binding domain, Rc
region, and such of the present invention can be linked together
via linkers or directly via polypeptide binding. The linkers
comprise arbitrary peptide linkers that can be introduced by
genetic engineering, synthetic linkers, and linkers disclosed in,
for example, Protein Engineering (1996) 9(3), 299-305. However,
peptide linkers are preferred in the present invention. The length
of the peptide linkers is not particularly limited, and can be
suitably selected by those skilled in the art according to the
purpose. The length is preferably five amino acids or more (without
particular limitation, the upper limit is generally 30 amino acids
or less, preferably 20 amino acids or less), and particularly
preferably 15 amino acids.
[0324] For example, such peptide linkers preferably include:
Ser
Gly.cndot.Ser
Gly.cndot.Gly.cndot.Ser
Ser.cndot.Gly.cndot.Gly
Gly.cndot.Gly.cndot.Gly.cndot.Ser (SEQ ID NO: 29)
Ser.cndot.Gly.cndot.Gly.cndot.Gly (SEQ ID NO: 30)
Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Ser (SEQ ID NO: 31)
Ser.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly (SEQ ID NO: 32)
Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Ser (SEQ ID NO:
33)
Ser.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly (SEQ ID NO:
34)
Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Ser
(SEQ ID NO: 35)
Ser.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly
(SEQ ID NO: 36)
(Gly.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Ser (SEQ ID NO: 31))n
(Ser.cndot.Gly.cndot.Gly.cndot.Gly.cndot.Gly (SEQ ID NO: 32))n
[0325] where n is an integer of 1 or larger. The length or
sequences of peptide linkers can be selected accordingly by those
skilled in the art depending on the purpose.
[0326] Synthetic linkers (chemical crosslinking agents) is
routinely used to crosslink peptides, and for example: [0327]
N-hydroxy succinimide (NHS), [0328] disuccinimidyl suberate (DSS),
[0329] bis(sulfosuccinimidyl) suberate (BS.sup.3), [0330]
dithiobis(succinimidyl propionate) (DSP), [0331]
dithiobis(sulfosuccinimidyl propionate) (DTSSP), [0332] ethylene
glycol bis(succinimidyl succinate) (EGS), [0333] ethylene glycol
bis(sulfosuccinimidyl succinate) (sulfo-EGS), [0334] disuccinimidyl
tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), [0335]
bis[2-(succinimidoxycarbonyloxy)ethyl]sulfone (BSOCOES), [0336] and
bis[2-(sulfosuccinimidoxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES).
These crosslinking agents are commercially available.
[0337] When multiple linkers for linking the respective domains are
used, they may all be of the same type, or may be of different
types.
[0338] In addition to the linkers exemplified above, linkers with
peptide tags such as His tag, HA tag, myc tag, and FLAG tag may
also be suitably used. Furthermore, hydrogen bonding, disulfide
bonding, covalent bonding, ionic interaction, and properties of
binding with each other as a result of combination thereof may be
suitably used. For example, the affinity between CH1 and CL of
antibody may be used, and Fc regions originating from the
above-described bispecific antibodies may also be used for hetero
Fc region association. Moreover, disulfide bonds formed between
domains may also be suitably used.
[0339] In order to link the respective domains via peptide linkage,
polynucleotides encoding the domains are linked in frame. Known
methods for linking polynucleotides in frame include techniques
such as ligation of restriction fragments, fusion PCR, and
overlapping PCR. Such methods can be appropriately used alone or in
combination to produce the antigen-binding molecules of the present
invention. In the present invention, the terms "linked" and
"fused", or "linkage" and "fusion" are used interchangeably. These
terms mean that two or more elements or components such as
polypeptides are linked together to form a single structure by any
means including the above-described chemical linking means and
recombination techniques. When two or more domains, elements, or
components are polypeptides, linking in frame means linking two or
more units of reading frames to form a longer continuous reading
frame while maintaining the correct reading frames of the
polypeptides. When two molecules of Fab are used as the
antigen-binding domain, an antibody which is an antigen-binding
molecule of the present invention in which the antigen-binding
domain is linked in frame to an Fc region by a peptide bond and not
via a linker can be used as a suitable antigen-binding molecule of
the present application.
Complex Containing Two or More Antigen-Binding Molecules and Two or
More Antigenic Binding Units
[0340] As described in Example 1 (as shown in WO 2011/122011),
Fv4-IgG1, of which sIL-6R-binding activity changes depending on the
pH condition more greatly than that of H54/L28-IgG1, accelerates
elimination of sIL-6R but does not accelerate the elimination more
than when sIL-6R is used alone. To accelerate the elimination more
than when sIL-6R is used alone, antigen-binding molecules
comprising an Fc region with enhanced FcRn binding in the neutral
pH range (for example, Fv4-IgG1-v2 and such of Example 1) must be
used.
[0341] Meanwhile, surprisingly, GA2-IgG1, of which human
IgA-binding activity varies depending on the Ca ion concentration,
was revealed to accelerate the elimination of human IgA more than
when human IgA is used alone, even though it contains an Fc region
derived from native IgG1, which FcRn binding in the neutral pH
range is not enhanced. Similarly, clone 278 of which human
IgE-binding activity varies depending on the pH condition, was
revealed to accelerate the elimination of human IgE more than when
human IgE is used alone, even though it contains an Fc region
derived from native IgG1, which does not have enhanced FcRn binding
in the neutral pH range. Without being restricted to a particular
theory, the following exemplary mechanism is thought to account for
what happened with GA2-IgG1 and clone 278.
[0342] When the antigenic binding unit is one unit (i.e., a
homomonomer), such as in sIL-6R, two molecules ((i.e., two
antigenic binding units) of antigens bind to a single antibody
molecule containing a divalent antigen-binding domain, and a
complex of one anti-sIL-6R antibody molecule and two antigen
molecules containing two antigenic binding units is formed.
Therefore, this type of antigen-antibody complex has only one Fc
region (native IgG1 Fc region) as shown in FIG. 1. Since this
complex binds to two molecules of FcRn or one molecule of
Fc.gamma.R via a single Fc region, the affinity toward these
receptors is the same as that of a general IgG antibody, and uptake
into cells is thought to occur mostly non-specifically.
[0343] On the other hand, when the antigenic binding unit is two
units as in human IgA, which is a dimer of a hetero-complex of
heavy chains and light chains, there are two units of epitopes to
which the antigen-binding domains will bind in the antigenic
binding unit. However, when a bivalent anti-IgA antibody (i.e., the
antigen-binding domains contained in one anti-IgA antibody molecule
bind to a same epitope) binds to its antigen, IgA, it is thought
that binding of each of the bivalent antigen-binding domains
contained in the single anti-IgA antibody molecule to each of the
two epitope units present on a single IgA molecule is difficult in
view of the position of the epitopes. As a result, it is thought
that separate anti-IgA antibody molecules bind to the two antigenic
binding units present in the two molecules of IgA that bind to the
bivalent antigen-binding domains present in a single anti-IgA
antibody molecule, and consequently, antigen-antibody complexes
(immune complexes) containing at least four molecules (that is, two
IgA molecules which are antigen molecule, and two anti-IgA antibody
molecules which are antigen-binding molecules) are formed.
[0344] When an antigen-binding molecule such as an antibody binding
to an antigen molecule that contains two or more antigenic binding
units, forms a large immune complex which is at least a tetramer,
the immune complex can bind strongly with avidity through at least
two or more multivalent Fc regions to Fc.gamma.R, FcRn, complement
receptors, and such. Therefore, as shown in FIG. 7, the complex is
taken up into cells expressing these receptors with a higher
efficiency than native IgG1. On the other hand, since the Fc
region-mediated affinity toward these receptors of immune complexes
formed of antigen molecules and antigen-binding molecules that for
example bind to (monomeric) antigen molecules containing one
antigenic binding unit is insufficient as mentioned above, the
immune complexes are taken up mostly nonspecifically (less
efficiently as compared to uptake mediated by avidity binding) into
cells expressing these receptors, as shown in FIG. 1. Thus, the
uptake is more inefficient than uptake mediated by avidity
binding.
[0345] When the antigen-binding molecule such as an antibody that
binds to an antigen molecule containing two or more antigenic
binding units is an antibody that contains antigen-binding domains
of which antigen-binding varies depending on an ion concentration
condition such as pH- or Ca-dependent binding and which forms an
antigen-antibody complex (immune complex) containing at least four
molecules (two antigen molecules and two antibody molecules) in the
plasma, once the immune complex is taken up into cells, the
antigens dissociate from the antibodies in the endosomes where the
ion concentration conditions are different from those in the
plasma. Therefore, the immune complex formation is dissolved in the
endosomes of cells that have taken up the immune complexes. Since
the dissociated antigens cannot bind to FcRn in the endosomes, they
are degraded after translocating to the lysosomes. On the other
hand, the antigen-dissociated antibodies are thought to be recycled
to the plasma after binding to FcRn in the endosomes (FIG. 7).
[0346] As described above, if an antibody that contains a native
IgG1-type constant region against a multimeric antigen containing
two or more antigenic binding units and shows pH- or Ca-dependent
binding can form a large immune complex and bind to Fc.gamma.R,
FcRn, complement receptors, and such with avidity, it is thought
that antigen elimination alone can be selectively and greatly
accelerated. It is thought that when GA2-IgG1 which binds to human
IgA is administered, such large immune complexes are formed.
Indeed, as shown in Example 3, GA2-IgG1-Fc.gamma.R(-) formed by
introducing into GA2-IgG1 modifications that impair binding to
mouse Fc.gamma.R could not substantially accelerate elimination of
human IgA like GA2-IgG 1 when compared to human IgA alone, and
showed an equivalent level of elimination as human IgA alone.
Therefore, the reason that GA2-IgG1 could accelerate elimination of
human IgA is because the immune complex containing GA2-IgG1 and
human IgA, which is a multimeric antigen containing two or more
antigenic binding units, binds with avidity to Fc.gamma.R and is
quickly taken up into cells expressing Fc.gamma.R. The IgA that
dissociates from the immune complex in the endosomes of cells that
have taken up the immune complex is degraded in the lysosomes. At
the same time, the IgA-dissociated antibody, which was bound to
FcRn in the endosomes, is subsequently recycled to the plasma and
can bind again to IgA in the plasma. Elimination of human IgA in
the plasma is thought to be greatly accelerated in this manner. A
method using an amino-acid-variant of the Fc region which binds to
FcRn in the pH neutral range is described in WO2011/122011 as a
method for accelerating elimination of antigens from the plasma.
The present invention is useful as a method for accelerating the
elimination from the plasma of multimeric antigens containing two
or more antigenic binding units without using the above-mentioned
variants, and as shown using GA2-N434W, can further accelerate the
elimination of the multimeric antigens containing two or more
antigenic binding units from the plasma through combination with
the above-mentioned variants.
Method for Evaluating the Formation of Immune Complexes Comprising
Two or More Antigen-Binding Molecules and Two or More Antigenic
Binding Units
[0347] In order to exhibit the effect of further accelerating the
elimination of multimeric antigens comprising two or more antigenic
binding units from the plasma as described above, it is considered
preferable that a large immune complex of antigen-binding molecules
and antigens are formed, and the Fc regions comprised in the
antigen-binding molecules strongly bind to Fc.gamma.R and/or FcRn
with avidity. When the antigen contains two or more antigenic
binding units, a large immune complex comprising two or more
antigen-binding molecules and two or more antigenic binding units
may be formed. Therefore, by screening for antigen-binding
molecules of which binding activity to multimeric antigens
containing two or more antigenic binding units changes according to
an ion concentration and which bind with avidity to the
above-mentioned receptors, antigen-binding molecules that further
accelerate the elimination of multimeric antigens containing two or
more antigenic binding units from plasma may be obtained.
Method for Evaluating Complex Formation
[0348] Examples of methods for evaluating the formation of immune
complexes comprising antigen-binding molecules and antigens include
techniques in analytical chemistry, including methods that make use
of the property that immune complexes become larger molecules than
an antigen-binding molecule alone or an antigen molecule alone such
as size exclusion (gel filtration) chromatography,
ultracentrifugation analysis method, light-scattering method,
electron microscopy, and mass spectrometry (Molecular Immunology
(2002), 39, 77-84; Molecular Immunology (2009), 47, 357-364). For
example, when size exclusion (gel filtration) chromatography is
used as shown in FIG. 9, whether an immune complex is formed is
evaluated by observing whether there are molecular species that are
larger than those in analyses of the antigen molecule alone or the
antigen-binding molecule alone.
[0349] Furthermore, when the antigen-binding molecule or antigen
has an immunoglobulin constant region, examples include
immunochemical methods including methods that use the property of
the immune complex to bind more strongly to an Fc receptor or a
complement component than the antigen-binding molecule alone or the
antigen alone, such as ELISA, FACS, or SPR methods (for example,
methods using Biacore) (The Journal of Biological Chemistry (2001)
276 (9), 6591-6604; Journal of Immunological Methods (1982) 50,
109-114; Journal of Immunology (2010) 184 (4), 1968-1976; mAbs
(2009) 1 (5) 491-504). For example, when ELISA is performed by
immobilizing an Fc receptor, formation of an immune complex is
evaluated by observing whether the detected signal is increased as
compared to when an antigen alone or an antigen-binding molecule
alone is evaluated.
Method for Promoting the Elimination of Monomeric Antigens from
Plasma Using a Cocktail of Antigen-Binding Molecules
[0350] As described above, when the antigen is a multimeric antigen
(a non-limiting example is an immunoglobulin such as IgA or IgE, or
a member of the TNF superfamily such as TNF or CD154), a large
immune complex comprising two or more antigen-binding molecules and
two or more antigenic binding units may be formed. On the other
hand, even when the antigen is monomeric, a mixture of two or more
appropriate antigen-binding molecules that each binds to a separate
epitope present on the monomeric antigen, where binding to the
epitopes varies depending on an ion concentration condition (such
as pH or Ca concentration) could also form a large immune complex
containing two or more antigen-binding molecules and two or more
antigenic binding units (monomeric antigens). Herein, the mixture
of two or more appropriate antigen-binding molecules that each
binds to a separate epitope present on a monomeric antigen, where
binding to the epitopes varies depending on an ion concentration
condition (such as pH or Ca concentration) is called an
antigen-binding molecule cocktail. Among these antigen-binding
molecules, at least one of (the antigen-binding domains comprised
in) the antigen-binding molecules forming the immune complex needs
to be an antigen-binding domain of which antigen-binding activity
varies depending on an ion concentration condition.
Method for Promoting Elimination of Monomeric Antigens from Plasma
Using Multispecific or Multiparatopic Antigen-Binding Molecules
[0351] Even in the case of a monomeric antigen, an antigen-binding
molecule containing antigen-binding domains that each binds to a
separate epitope present on the monomeric antigen, where binding of
the antigen-binding domains to the respective epitopes varies
depending on an ion concentration condition (such as pH or Ca
concentration) could also form a large immune complex containing
two or more antigen-binding molecules and two or more antigenic
binding units (monomeric antigens). Examples of a non-limiting
embodiment of such an antigen-binding molecule are multiparatopic
antibodies or multispecific antibodies containing appropriate
variable regions that bind to epitopes present on the monomeric
antigen that differ from each other. As a non-limiting embodiment
of such multispecific antibodies or multiparatopic antibodies,
antibodies whose variable regions show pH- or Ca-dependent binding
(bispecific antibodies or biparatopic antibodies containing a
right-arm variable region that recognizes epitope A and a left-arm
variable region that recognizes epitope B, as shown in FIG. 8)
could also form a large immune complex containing two or more
antibodies and two or more antigenic binding units (monomeric
antigens).
[0352] Antigen-binding molecules that further accelerate the
elimination of monomeric antigens from the plasma can be obtained
by screening for combinations of antigen-binding domains that
target different epitopes of a monomeric antigen, where the binding
activity to each of the epitopes varies depending on an ion
concentration condition, and which can bind with avidity to the
abovementioned receptors. The ion concentration condition that
changes the binding activity of a multispecific or multiparatopic
antigen-binding domain toward each of the epitopes may be the same
ion concentration condition or a different ion concentration
condition. An antigen-binding molecule containing bispecific or
biparatopic antigen-binding domains, where the epitope-binding
activity of one of the bispecific or biparatopic antigen-binding
domains varies depending on the pH condition or a metal ion
concentration condition such as the Ca ion concentration, may be
presented as an example of a non-limiting embodiment of the
antigen-binding molecules of the present invention. Furthermore, an
antigen-binding molecule containing bispecific or biparatopic
antigen-binding domains, where the epitope-binding activity of one
of the bispecific or biparatopic antigen-binding domains varies
depending on the pH condition and the epitope-binding activity of
the other antigen-binding domain varies depending on a metal ion
concentration condition such as the Ca ion concentration, may also
be presented as an example of a non-limiting embodiment of the
antigen-binding molecules of the present invention. An
antigen-binding molecule containing bispecific or biparatopic
antigen-binding domains, where the epitope-binding activity of one
of the bispecific or biparatopic antigen-binding domains varies
depending on the pH condition and the epitope-binding activity of
the other antigen-binding domain also varies depending on the pH
condition, may also be presented as an example of a non-limiting
embodiment of the antigen-binding molecules of the present
invention. An antigen-binding molecule containing bispecific or
biparatopic antigen-binding domains, where the epitope-binding
activity of one of the bispecific or biparatopic antigen-binding
domains varies depending on a metal ion concentration condition
such as the Ca ion concentration and the epitope-binding activity
of the other antigen-binding domain also varies depending on a
metal ion concentration condition such as the Ca ion concentration,
may also be presented as an example of a non-limiting embodiment of
the antigen-binding molecules of the present invention.
Multispecific Antigen-Binding Molecules and Multiparatopic
Antigen-Binding Molecules
[0353] An antigen-binding molecule containing at least two
antigen-binding domains, wherein at least one of the
antigen-binding domains binds to a first epitope in an antigen
molecule and at least another one of the antigen-binding domains
binds to a second epitope in the antigen molecule, is called a
multispecific antigen-binding molecule from the standpoint of its
reaction specificity. When a single antigen-binding molecule binds
to two different epitopes through two types of antigen-binding
domains contained in the antigen-binding molecule, this
antigen-binding molecule is called a bispecific antigen-binding
molecule. When a single antigen-binding molecule binds to three
different epitopes through three types of antigen-binding domains
contained in the antigen-binding molecule, this antigen-binding
molecule is called a trispecific antigen-binding molecule.
[0354] The paratope in an antigen-binding domain that binds to a
first epitope in the antigen molecule has a different structure
from that of the paratope in an antigen-binding domain that binds
to a second epitope that is structurally different from the first
epitope. Therefore, an antigen-binding molecule containing at least
two antigen-binding domains, wherein at least one of the
antigen-binding domains binds to a first epitope in an antigen
molecule and at least another one of the antigen-binding domains
binds to a second epitope in the antigen molecule, is called a
multiparatopic antigen-binding molecule from the standpoint of its
structure and specificity. When a single antigen-binding molecule
binds to two different epitopes through two types of
antigen-binding domains contained in the antigen-binding molecule,
this antigen-binding molecule is called a biparatopic
antigen-binding molecule. When a single antigen-binding molecule
binds to three different epitopes through three types of
antigen-binding domains contained in the antigen-binding molecule,
this antigen-binding molecule is called a triparatopic
antigen-binding molecule.
[0355] Multivalent multispecific or multiparatopic antigen-binding
molecules comprising one or several antigen-binding domains and
methods for preparing them have been described in Non-Patent
Documents such as Conrath et al. (J. Biol. Chem. (2001) 276 (10)
7346-7350), Muyldermans (Rev. Mol. Biotech. (2001) 74, 277-302),
and Kontermann R. E. (2011) Bispecific Antibodies (Springer-Verlag)
and in Patent Documents such as WO 1996/034103 and WO 1999/023221.
Antigen-binding molecules of the present invention can be produced
using the multispecific or multiparatopic antigen-binding molecules
and methods for preparing them described in these documents.
Bispecific Antibodies and Methods for Production Thereof
[0356] Bispecific antibodies and methods for production thereof are
presented below as examples of an embodiment of the aforementioned
multispecific or multiparatopic antigen-binding molecules and
methods for production thereof. Bispecific antibodies are
antibodies comprising two types of variable regions that bind
specifically to different epitopes. IgG-type bispecific antibodies
can be secreted from a hybrid hybridoma (quadroma) produced by
fusing two types of hybridomas that produce IgG antibodies
(Milstein et al., Nature (1983) 305, 537-540).
[0357] When a bispecific antibody is produced by using
recombination techniques such as those described in the
above-mentioned section on antibodies, one may adopt a method that
introduces genes encoding heavy chains containing the two types of
variable regions of interest into cells to co-express them.
However, even when only the heavy-chain combination is considered,
such a co-expression method will produce a mixture of (i) a
combination of a pair of heavy chains in which one of the heavy
chains contains a variable region that binds to a first epitope and
the other heavy chain contains a variable region that binds to a
second epitope, (ii) a combination of a pair of heavy chains which
include only heavy chains containing a variable region that binds
to the first epitope, and (iii) a combination of a pair of heavy
chains which include only heavy chains containing a variable region
that binds to the second epitope, which are present at a molecular
ratio of 2:1:1. It is difficult to purify antigen-binding molecules
containing the desired combination of heavy chains from the mixture
of three types of heavy chain combinations.
[0358] When producing bispecific antibodies using such
recombination techniques, bispecific antibodies containing a
heteromeric combination of heavy chains can be preferentially
secreted by adding appropriate amino acid substitutions in the CH3
domains constituting the heavy chains. Specifically, this method is
conducted by substituting an amino acid having a larger side chain
(knob (which means "bulge")) for an amino acid in the CH3 domain of
one of the heavy chains, and substituting an amino acid having a
smaller side chain (hole (which means "void")) for an amino acid in
the CH3 domain of the other heavy chain so that the knob is placed
in the hole. This promotes heteromeric heavy chain formation and
simultaneously inhibits homomeric heavy chain formation
(International Publication No. WO 1996027011; Ridgway et al.,
Protein Engineering (1996) 9, 617-621; Merchant et al., Nature
Biotechnology (1998) 16, 677-681).
[0359] Furthermore, there are also known techniques for producing a
bispecific antibody by applying methods for controlling polypeptide
association, or association of polypeptide-formed heteromeric
multimers to the association between heavy chains. Specifically,
methods for controlling heavy chain formation may be employed to
produce a bispecific antibody (International Publication No. WO
2006/106905), in which amino acid residues forming the interface
between the heavy chains are altered to inhibit the association
between the heavy chains having the same sequence and to allow the
formation of heavy chains of different sequences. Such methods can
be used for generating bispecific antibodies.
[0360] In a non-limiting embodiment of the present invention, two
polypeptides forming an Fc region derived from a bispecific
antibody described above can be suitably used as the Fc region
contained in an antigen-binding molecule. More specifically, two
polypeptides forming an Fc region may be suitably used, in which,
of the amino acid sequence of one of the polypeptides, the amino
acid at position 349 as indicated by EU numbering is Cys and the
amino acid at position 366 is Trp, and of the amino acid sequence
of the other of the polypeptides, the amino acid at position 356 as
indicated by EU numbering is Cys, the amino acid at position 366 is
Ser, the amino acid at position 368 is Ala, and the amino acid at
position 407 is Val.
[0361] In another non-limiting embodiment of the present invention,
two polypeptides forming an Fc region, in which, of the amino acid
sequence of one of the polypeptides, the amino acid at position 409
according to EU numbering is Asp, and of the amino acid sequence of
the other of the polypeptides, the amino acid at position 399
according to EU numbering is Lys, may be suitably used as the Fc
region. In the above embodiment, the amino acid at position 409 may
be Glu instead of Asp, and the amino acid at position 399 may be
Arg instead of Lys. Moreover, in addition to the amino acid Lys at
position 399, Asp may suitably be added as amino acid at position
360 or Asp may suitably be added as amino acid at position 392.
[0362] In still another non-limiting embodiment of the present
invention, two polypeptides forming an Fc region, in which, of the
amino acid sequence of one of the polypeptides, the amino acid at
position 370 according to EU numbering is Glu, and of the amino
acid sequence of the other of the polypeptides, the amino acid at
position 357 according to EU numbering is Lys, may be suitably used
as the Fc region.
[0363] In yet another non-limiting embodiment of the present
invention, two polypeptides forming an Fc region, in which, of the
amino acid sequence of one of the polypeptides, the amino acid at
position 439 according to EU numbering is Glu, and of the amino
acid sequence of the other of the polypeptides, the amino acid at
position 356 according to EU numbering is Lys, may be suitably used
as the Fc region.
[0364] In still yet another non-limiting embodiment of the present
invention, any of the embodiments indicated below, in which the
above have been combined, may be suitably used as the Fc
region:
[0365] (i) two polypeptides forming an Fc region, in which, of the
amino acid sequence of one of the polypeptides, the amino acid at
position 409 according to EU numbering is Asp and the amino acid at
position 370 is Glu, and of the amino acid sequence of the other of
the polypeptides, the amino acid at position 399 according to EU
numbering is Lys and the amino acid at position 357 is Lys (in this
embodiment, the amino acid at position 370 according to EU
numbering may be Asp instead of Glu, and the amino acid Asp at
position 392 according to EU numbering may be used instead of the
amino acid Glu at position 370 according to EU numbering);
[0366] (ii) two polypeptides forming an Fc region, in which, of the
amino acid sequence of one of the polypeptides, the amino acid at
position 409 according to EU numbering is Asp and the amino acid at
position 439 is Glu, and of the amino acid sequence of the other of
the polypeptides, the amino acid at position 399 according to EU
numbering is Lys and the amino acid at position 356 is Lys (in this
embodiment, the amino acid Asp at position 360 according to EU
numbering, the amino acid Asp at position 392 according to EU
numbering, or the amino acid Asp at position 439 according to EU
numbering may be used instead of the amino acid Glu at position 439
according to EU numbering);
[0367] (iii) two polypeptides forming an Fc region, in which, of
the amino acid sequence of one of the polypeptides, the amino acid
at position 370 according to EU numbering is Glu and the amino acid
at position 439 is Glu, and of the amino acid sequence of the other
of the polypeptides, the amino acid at position 357 according to EU
numbering is Lys and the amino acid at position 356 is Lys; and
[0368] two polypeptides forming an Fc region, in which, of the
amino acid sequence of one of the polypeptides, the amino acid at
position 409 according to EU numbering is Asp, the amino acid at
position 370 is Glu, and the amino acid at position 439 is Glu, and
of the amino acid sequence of the other of the polypeptides, the
amino acid at position 399 according to EU numbering is Lys, the
amino acid at position 357 is Lys, and the amino acid at position
356 is Lys (in this embodiment, the amino acid at position 370
according to EU numbering may not be substituted to Glu, and
furthermore, when the amino acid at position 370 is not substituted
to Glu, the amino acid at position 439 may be Asp instead of Glu,
or the amino acid Asp at position 392 may be used instead of the
amino acid Glu at position 439).
[0369] Further, in another non-limiting embodiment of the present
invention, two polypeptides forming an Fc region, in which, of the
amino acid sequence of one of the polypeptides, the amino acid at
position 356 according to EU numbering is Lys, and of the amino
acid sequence of the other of the polypeptides, the amino acid at
position 435 according to EU numbering is Arg and the amino acid at
position 439 is Glu, may also be suitably used.
[0370] In still another non-limiting embodiment of the present
invention, two polypeptides forming an Fc region, in which, of the
amino acid sequence of one of the polypeptides, the amino acid at
position 356 according to EU numbering is Lys and the amino acid at
position 357 is Lys, and of the amino acid sequence of the other of
the polypeptides, the amino acid at position 370 according to EU
numbering is Glu, the amino acid at position 435 is Arg, and the
amino acid at position 439 is Glu, may also be suitably used.
[0371] Furthermore, in addition to the above-mentioned technique of
associating heterologous heavy chains, the CrossMab technology
which is known as a technology for associating heterologous light
chains, in which a light chain forming a variable region that binds
to a first epitope and a light chain forming a variable region that
binds to a second epitope are respectively associated with a heavy
chain forming a variable region that binds to the first epitope and
a heavy chain forming a variable region that binds to the second
epitope (Scaefer et al. (Proc. Natl. Acad. Sci. U.S.A. (2011) 108,
11187-11192)), may also be used to produce the multispecific or
multiparatopic antigen-binding molecules provided by the present
invention.
Use of an Antigen-Binding Molecule to Eliminate Antigens Containing
Two or More Antigenic Binding Units from the Plasma
[0372] The present invention provides use of an antigen-binding
molecule comprising (i) an Fc region and (ii) an antigen-binding
domain of which antigen-binding activity varies depending on an ion
concentration condition;
wherein the antigen-binding molecule can form an immune complex
comprising (a) two or more of the antigen-binding molecules and (b)
two or more antigen molecules provided that the antigens comprise
two or more antigenic binding units, for eliminating the antigens
from the plasma.
[0373] In the present invention, embodiments of the use of an
antigen-binding molecule are not particularly limited as long as
antigens can be eliminated from the plasma. Examples of a
non-limiting embodiment of such use are pharmaceutical compositions
containing an antigen-binding molecule provided by the present
invention, and methods comprising administering to a subject an
antigen-binding molecule provided by the present invention. An
example of another non-limiting embodiment is use of the
antigen-binding molecule in an ex vivo method for eliminating
antigens from plasma, which includes contacting an immune complex
formed through contact of an antigen-binding molecule of the
present invention with plasma isolated from a subject, and
containing two or more of the antigen-binding molecules and two or
more antigens (provided that the antigens include two or more
antigenic binding units) with FcRn- or Fc.gamma.
receptor-expressing cells.
Improvement of Pharmacokinetics
[0374] In the present invention, the "ability to eliminate antigens
in plasma" refers to the ability to eliminate from the plasma
antigens that are present in the plasma when the antigen-binding
molecules are administered in vivo or when the antigen-binding
molecules are secreted in vivo. Thus, in the present invention, the
phrase "the ability of an antigen-binding molecule to eliminate an
antigen in the plasma is increased" can be used when the rate of
antigen elimination from the plasma is accelerated when the
antigen-binding molecule is administered, as compared to
administration of antigen-binding molecules that cannot form the
immune complexes disclosed in the present invention,
antigen-binding molecules containing antigen-binding domains of
which antigen-binding activity is independent of the ion
concentration, or antigen-binding molecules containing Fc regions
with compromised binding activity toward Fc.gamma.R or FcRn.
Whether or not the ability of an antigen-binding molecule to
eliminate an antigen in the plasma is increased can be assessed,
for example, by administering a soluble antigen and the
antigen-binding molecule in vivo and measuring the plasma
concentration of the soluble antigen after administration. The
ability of an antigen-binding molecule to eliminate an antigen in
the plasma is judged to be increased if the concentration of a
soluble antigen in the plasma is decreased after administration of
the soluble antigen, and the antigen-binding molecule which is an
antigen-binding molecule capable of forming an immune complex
containing two or more antigen-binding molecules and antigens with
two or more antigenic binding units, and of which antigen-binding
activity changes depending on ion concentrations such as reduction
of the antigen-binding activity in the acidic pH range compared to
in the neutral pH range (or reduction of the antigen-binding
activity at a low calcium ion concentration compared to at a high
calcium ion concentration). The soluble antigen may be an antigen
that is bound to an antigen-binding molecule or an antigen that is
not bound to an antigen-binding molecule in the plasma, and its
concentration can be determined as a "plasma concentration of the
antigen bound to the antigen-binding molecule" or as a "plasma
concentration of the antigen not bound to the antigen-binding
molecule", respectively (the latter is synonymous with "free
antigen concentration in plasma"). The "total antigen concentration
in plasma" means the sum of concentrations of the antigen-binding
molecule-bound antigen and the antigen not bound by an
antigen-binding molecule, or the "free antigen concentration in
plasma" which is the concentration of the antigen not bound by an
antigen-binding molecule. Thus, the soluble antigen concentration
can be determined as the "total antigen concentration in plasma".
Various methods for measuring the "total antigen concentration in
plasma" or the "free antigen concentration in plasma" are well
known in the art as described hereinafter.
[0375] Herein, "enhancement of pharmacokinetics", "improvement of
pharmacokinetics", and "superior pharmacokinetics" can be restated
as "enhancement of plasma (blood) retention", "improvement of
plasma (blood) retention", "superior plasma (blood) retention", and
"prolonged plasma (blood) retention". These terms are
synonymous.
[0376] Herein, "improvement of pharmacokinetics" means not only
prolongation of the period until elimination from the plasma (for
example, until the antigen-binding molecule is degraded
intracellularly or the like and cannot return to the plasma) after
administration of the antigen-binding molecule to humans, or
non-human animals such as mice, rats, monkeys, rabbits, and dogs,
but also prolongation of the plasma retention of the
antigen-binding molecule in a form that allows antigen binding (for
example, in an antigen-free form of the antigen-binding molecule)
during the period of administration to elimination due to
degradation. Human IgG having wild-type Fc region can bind to FcRn
from non-human animals. For example, mouse can be preferably used
to be administered in order to confirm the property of the
antigen-binding molecule of the invention since human IgG having
wild-type Fc region can bind to mouse FcRn stronger than to human
FcRn (Int Immunol. (2001) 13(12): 1551-1559). As another example,
mouse in which its native FcRn genes are disrupted and a transgene
for human FcRn gene is harbored to be expressed (Methods Mol Biol.
2010; 602: 93-104) can also be preferably used to be administered
in order to confirm the property of the antigen-binding molecule of
the invention described hereinafter. Specifically, "improvement of
pharmacokinetics" also includes prolongation of the period until
elimination due to degradation of the antigen-binding molecule not
bound to antigens (the antigen-free form of antigen-binding
molecule). The antigen-binding molecule in plasma cannot bind to a
new antigen if the antigen-binding molecule has already bound to an
antigen. Thus, the longer the period that the antigen-binding
molecule is not bound to an antigen, the longer the period that it
can bind to a new antigen (the higher the chance of binding to
another antigen). This enables reduction of the time period that an
antigen is free of the antigen-binding molecule in vivo and
prolongation of the period that an antigen is bound to the
antigen-binding molecule. The plasma concentration of the
antigen-free form of antigen-binding molecule can be increased and
the period that the antigen is bound to the antigen-binding
molecule can be prolonged by accelerating the antigen elimination
from the plasma by administration of the antigen-binding molecule.
Specifically, herein "improvement of the pharmacokinetics of
antigen-binding molecule" includes the improvement of a
pharmacokinetic parameter of the antigen-free form of the
antigen-binding molecule (any of prolongation of the half-life in
plasma, prolongation of mean retention time in plasma, and
impairment of plasma clearance), prolongation of the period that
the antigen is bound to the antigen-binding molecule after
administration of the antigen-binding molecule, and acceleration of
antigen-binding molecule-mediated antigen elimination from the
plasma. The improvement of pharmacokinetics of antigen-binding
molecule can be assessed by determining any one of the parameters,
half-life in plasma, mean plasma retention time, and plasma
clearance for the antigen-binding molecule or the antigen-free form
thereof ("Pharmacokinetics: Enshu-niyoru Rikai (Understanding
through practice)" Nanzando). For example, the plasma concentration
of the antigen-binding molecule or antigen-free form thereof is
determined after administration of the antigen-binding molecule to
mice, rats, monkeys, rabbits, dogs, or humans. Then, each parameter
is determined. When the plasma half-life or mean plasma retention
time is prolonged, the pharmacokinetics of the antigen-binding
molecule can be judged to be improved. The parameters can be
determined by methods known to those skilled in the art. The
parameters can be appropriately assessed, for example, by
noncompartmental analysis using the pharmacokinetics analysis
software WinNonlin (Pharsight) according to the appended
instruction manual. The plasma concentration of antigen-free
antigen-binding molecule can be determined by methods known to
those skilled in the art, for example, using the assay method
described in Clin Pharmacol. 2008 April; 48(4): 406-417.
[0377] Herein, "improvement of pharmacokinetics" also includes
prolongation of the period that an antigen is bound to an
antigen-binding molecule after administration of the
antigen-binding molecule. Whether the period that an antigen is
bound to the antigen-binding molecule after administration of the
antigen-binding molecule is prolonged can be assessed by
determining the plasma concentration of free antigen. The
prolongation can be judged based on the determined plasma
concentration of free antigen or the time period required for an
increase in the ratio of free antigen concentration to the total
antigen concentration.
[0378] The plasma concentration of free antigen not bound to the
antigen-binding molecule or the ratio of free antigen concentration
to the total antigen concentration can be determined by methods
known to those skilled in the art, for example, by the method used
in Pharm Res. 2006 January; 23 (1): 95-103. Alternatively, when an
antigen exhibits a particular function in vivo, whether the antigen
is bound to an antigen-binding molecule that neutralizes the
antigen function (antagonistic molecule) can be assessed by testing
whether the antigen function is neutralized. Whether the antigen
function is neutralized can be assessed by assaying an in vivo
marker that reflects the antigen function. Whether the antigen is
bound to an antigen-binding molecule that activates the antigen
function (agonistic molecule) can be assessed by assaying an in
vivo marker that reflects the antigen function.
[0379] Determination of the plasma concentration of free antigen
and ratio of the amount of free antigen in plasma to the amount of
total antigen in plasma, in vivo marker assay, and such
measurements are not particularly limited; however, the assays are
preferably carried out after a certain period of time has passed
after administration of the antigen-binding molecule. In the
present invention, the period after administration of the
antigen-binding molecule is not particularly limited; those skilled
in the art can determine the appropriate period depending on the
properties and the like of the administered antigen-binding
molecule. Such periods include, for example, one day after
administration of the antigen-binding molecule, three days after
administration of the antigen-binding molecule, seven days after
administration of the antigen-binding molecule, 14 days after
administration of the antigen-binding molecule, and 28 days after
administration of the antigen-binding molecule. Herein, the concept
"plasma antigen concentration" comprises both "total antigen
concentration in plasma" which is the sum of antigen-binding
molecule bound antigen and non-bound antigen concentration or "free
antigen concentration in plasma" which is antigen-binding molecule
non-bound antigen concentration.
[0380] The total antigen concentration in the plasma can be lowered
by administration, as antigen-binding molecule, of the
antigen-binding molecule of the present invention by 2-fold,
5-fold, 10-fold, 20-fold, 50-fold, 100-fold, 200-fold, 500-fold,
1,000-fold, or even higher as compared to administration of an
antigen-binding molecule containing an antigen-binding domain whose
antigen-binding activity is ion concentration-independent or an
antigen-binding molecule containing an Fc region with an impaired
Fc.gamma.R-binding activity, or compared to when the
antigen-binding domain molecule of the present invention is not
administered.
[0381] Molar antigen/antigen-binding molecule ratio can be
calculated as shown below:
value A: Molar antigen concentration at each time point value B:
Molar antigen-binding molecule concentration at each time point
value C: Molar antigen concentration per molar antigen-binding
molecule concentration (molar antigen/antigen-binding molecule
ratio) at each time point
C=A/B.
[0382] Smaller value C indicates higher efficiency of antigen
elimination per antigen-binding molecule whereas higher value C
indicates lower efficiency of antigen elimination per
antigen-binding molecule.
[0383] Molar antigen/antigen-binding molecule ratio can be
calculated as described above.
[0384] Administering an antigen-binding molecule of the present
invention can lower the molar antigen/antigen-binding molecule
ratio by 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold,
200-fold, 500-fold, 1000-fold or more as compared to administration
of antigen-binding molecules that cannot form the immune complexes
disclosed in the present invention, antigen-binding molecules
containing antigen-binding domains of which antigen-binding
activity is independent of ion concentrations, or antigen-binding
molecules containing Fc regions with compromised binding activity
toward Fc.gamma.R or FcRn.
[0385] In the present invention, as reference for comparison with
the antigen-binding molecules of the present invention,
antigen-binding molecules that cannot form the immune complexes
disclosed in the present invention, antigen-binding molecules
containing antigen-binding domains of which antigen-binding
activity is independent of ion concentrations, or antigen-binding
molecules containing Fc regions with compromised binding activity
toward Fc.gamma.R or FcRn.
[0386] When an FcRn-mediated pathway is used in the incorporation
of antigen-binding molecules of the present invention from the
plasma into cells, reduction in the total antigen concentration in
plasma or the molar antigen/antibody ratio can also be assessed
using human FcRn transgenic mouse line 32 or line 276 (Jackson
Laboratories, Methods Mol Biol. 2010; 602: 93-104) by either
antigen-antibody co-injection model or steady-state antigen
infusion model when the antigen-binding molecule do not cross-react
to the mouse counterpart antigen. When the antigen-binding molecule
cross-react with the mouse counterpart, they can also be assessed
by simply injecting the antigen-binding molecule to human FcRn
transgenic mouse line 32 or line 276 (Jackson Laboratories). In the
co-injection model, mixture of the antigen-binding molecule and
antigen is administered to mice. In the steady-state antigen
infusion model, infusion pump filled with an antigen solution is
embedded into mice to achieve a constant plasma antigen
concentration, and then the antigen-binding molecule is injected to
the mice. Test antigen-binding molecules are administered at the
same dosage. The total antigen concentration in plasma, free
antigen concentration in plasma, and antigen-binding molecule
concentration in plasma are measured at appropriate time points
using method known to those skilled in the art.
[0387] When an Fc.gamma.R-mediated pathway is used in the
incorporation of antigen-binding molecules of the present invention
from the plasma into cells, reduction in the total antigen
concentration in plasma or the molar antigen/antibody ratio can be
assessed by either the antigen-antibody co-injection model or the
steady-state antigen infusion model using the conventionally used
C57BL/6J mice (Charles River Japan) when the antigen-binding
molecule does not cross-react with the mouse counterpart antigen.
If the antigen-binding molecule cross-reacts with the mouse
counterpart, assessment can be carried out simply by injecting the
antigen-binding molecule into the conventionally used C57BL/6J mice
(Charles River Japan).
[0388] In the co-injection model, a mixture of the antigen-binding
molecule and antigen is administered to mice. In the steady-state
antigen infusion model, an infusion pump filled with an antigen
solution is embedded into mice to achieve a constant plasma antigen
concentration, and then the antigen-binding molecule is injected
into the mice. Test antigen-binding molecules are administered at
the same dose. The total antigen concentration in plasma, free
antigen concentration in plasma, and antigen-binding molecule
concentration in plasma are measured at appropriate time points
using methods known to those skilled in the art.
[0389] Total or free antigen concentration in plasma and molar
antigen/antigen-binding molecule ratio can be measured at 2, 4, 7,
14, 28, 56, or 84 days after administration to evaluate the
long-term effect of the present invention. In other words, a long
term plasma antigen concentration is determined by measuring total
or free antigen concentration in plasma and molar
antigen/antigen-binding molecule ratio at 2, 4, 7, 14, 28, 56, or
84 days after administration of an antigen-binding molecule in
order to evaluate the property of the antigen-binding molecule of
the present invention. Whether the reduction of plasma antigen
concentration or molar antigen/antigen-binding molecule ratio is
achieved by antigen-binding molecule described in the present
invention can be determined by the evaluation of the reduction at
any one or more of the time points described above.
[0390] Total or free antigen concentration in plasma and molar
antigen/antigen-binding molecule ratio can be measured at 15 min,
1, 2, 4, 8, 12, or 24 hours after administration to evaluate the
short-term effect of the present invention. In other words, a short
term plasma antigen concentration is determined by measuring total
or free antigen concentration in plasma and molar
antigen/antigen-binding molecule ratio at 15 min, 1, 2, 4, 8, 12,
or 24 hours after administration of an antigen-binding molecule in
order to evaluate the property of the antigen-binding molecule of
the present invention.
[0391] The route of administration of an antigen-binding molecule
of the present invention can be selected from intradermal,
intravenous, intravitreal, subcutaneous, intraperitoneal,
parenteral and intramuscular injection.
[0392] In the present invention, it is preferable that the
pharmacokinetics of the antigen-binding molecule in human is
improved. When the plasma retention in human is difficult to
determine, it may be predicted based on the plasma retention in
mice (for example, normal mice, human antigen-expressing transgenic
mice, human FcRn-expressing transgenic mice) or monkeys (for
example, cynomolgus monkeys).
[0393] Herein, "the improvement of the pharmacokinetics and
prolonged plasma retention of an antigen-binding molecule" means
improvement of any pharmacokinetic parameter (any of prolongation
of the half-life in plasma, prolongation of mean retention time in
plasma, reduction of plasma clearance, and bioavailability) after
in vivo administration of the antigen-binding molecule, or an
increase in the concentration of the antigen-binding molecule in
the plasma in an appropriate time after administration. It may be
determined by measuring any parameter such as half-life in plasma,
mean retention time in plasma, plasma clearance, and
bioavailability of the antigen-binding molecule (Pharmacokinetics:
Enshu-niyoru Rikai (Understanding through practice), (Nanzando)).
For example, when an antigen-binding molecule is administered to
mice (normal mice and human FcRn transgenic mice), rats, monkeys,
rabbits, dogs, humans, and so on, and the concentration of the
antigen-binding molecule in the plasma is determined and each of
the parameters is calculated, the pharmacokinetics of the
antigen-binding molecule can be judged to be improved when the
plasma half-life or mean retention time in the plasma is prolonged.
These parameters can be determined by methods known to those
skilled in the art. For example, the parameters can be
appropriately assessed by non-compartmental analysis using
pharmacokinetics analysis software WinNonlin (Pharsight) according
to the attached instruction manual.
[0394] Without being restricted to a particular theory, the
following mechanism is an example of a mechanism that allows
elimination of antigenic binding units from the plasma by an
antigen-binding molecule of the present invention which comprises
(i) an Fc region and (ii) an antigen-binding domain of which
antigen-binding activity varies depending on an ion concentration
condition, wherein the antigen-binding molecule can form an immune
complex containing two or more of the antigen-binding molecules and
antigens comprising two or more antigenic binding units. When the
antigenic binding unit is one unit (i.e., a monomer), such as in
sIL-6R, two molecules ((i.e., two antigenic binding units) of
antigens bind to a single antibody molecule containing a divalent
antigen-binding domain, and a complex of one anti-sIL-6R antibody
molecule and two antigen molecules containing two antigenic binding
units is formed. Therefore, this type of antigen-antibody complex
has only one Fc region (native IgG1 Fc region) as shown in FIG. 1.
Since this complex binds to two molecules of FcRn or one molecule
of Fc.gamma.R via a single Fc region, the affinity toward these
receptors is the same as that of a general IgG antibody, and uptake
into cells is thought to occur mostly non-specifically.
[0395] On the other hand, when the antigenic binding unit is two
units as in human IgA, which is a dimer of a hetero-complex of
heavy chains and light chains, there are two units of epitopes to
which the antigen-binding domains will bind in the antigenic
binding unit. However, when a bivalent anti-IgA antibody (i.e., the
antigen-binding domains contained in one anti-IgA antibody molecule
bind to a same epitope) binds to its antigen, IgA, it is thought
that binding of each of the bivalent antigen-binding domains
contained in the single anti-IgA antibody molecule to each of the
two epitope units present on a single IgA molecule is difficult in
view of the position of the epitopes. As a result, it is thought
that separate anti-IgA antibody molecules bind to the two antigenic
binding units present in the two molecules of IgA that bind to the
bivalent antigen-binding domains present in a single anti-IgA
antibody molecule, and consequently, antigen-antibody complexes
(immune complexes) containing at least four molecules (that is, two
IgA molecules which are antigen molecule, and two anti-IgA antibody
molecules which are antigen-binding molecules) are formed.
[0396] When an antigen-binding molecule such as an antibody binding
to an antigen molecule that contains two or more antigenic binding
units, forms a large immune complex which is at least a tetramer,
the immune complex can bind strongly with avidity through at least
two or more multivalent Fc regions to Fc.gamma.R, FcRn, complement
receptors, and such. Therefore, as shown in FIG. 7, the complex is
taken up efficiently into cells expressing these receptors. On the
other hand, since the Fc region-mediated affinity toward these
receptors of immune complexes formed of antigen molecules and
antigen-binding molecules that for example bind to (monomeric)
antigen molecules containing one antigenic binding unit is
insufficient as mentioned above, the immune complexes are taken up
mostly nonspecifically (less efficiently as compared to uptake
mediated by avidity binding) into cells expressing these receptors,
as shown in FIG. 1. Thus, the uptake is more inefficient than
uptake mediated by avidity binding.
[0397] When the antigen-binding molecule such as an antibody that
binds to an antigen molecule containing two or more antigenic
binding units is an antibody that contains antigen-binding domains
of which antigen-binding varies depending on an ion concentration
condition such as pH- or Ca-dependent binding and which forms an
antigen-antibody complex (immune complex) containing at least four
molecules (two antigen molecules and two antibody molecules) in the
plasma, once the immune complex is taken up into cells, the
antigens dissociate from the antibodies in the endosomes where the
ion concentration conditions are different from those in the
plasma. Therefore, the immune complex formation is dissolved in the
endosomes of cells that have taken up the immune complexes. Since
the dissociated antigens cannot bind to FcRn in the endosomes, they
are degraded after translocating to the lysosomes. On the other
hand, the antigen-dissociated antibodies are thought to be recycled
to the plasma after binding to FcRn in the endosomes (FIG. 7).
[0398] As described above, if an antibody that contains a native
IgG1-type constant region against a multimeric antigen containing
two or more antigenic binding units and shows pH- or Ca-dependent
binding can form a large immune complex and bind to Fc.gamma.R,
FcRn, complement receptors, and such with avidity, it is thought
that antigen elimination alone can be selectively and greatly
accelerated. It is thought that when GA2-IgG1 which binds to human
IgA is administered, such large immune complexes are formed.
Indeed, as shown in Example 3, GA2-IgG1-Fc.gamma.R(-) formed by
introducing into GA2-IgG1 modifications that impair binding to
mouse Fc.gamma.R could not substantially accelerate elimination of
human IgA like GA2-IgG 1 when compared to human IgA alone, and
showed an equivalent level of elimination as human IgA alone.
Therefore, the reason that GA2-IgG1 could accelerate elimination of
human IgA is because the immune complex containing GA2-IgG1 and
human IgA, which is a multimeric antigen containing two or more
antigenic binding units, binds with avidity to Fc.gamma.R and is
quickly taken up into cells expressing Fc.gamma.R. The IgA that
dissociates from the immune complex in the endosomes of cells that
have taken up the immune complex is degraded in the lysosomes. At
the same time, the IgA-dissociated antibody, which was bound to
FcRn in the endosomes, is subsequently recycled to the plasma and
can bind again to IgA in the plasma. Elimination of human IgA in
the plasma is thought to be greatly accelerated in this manner. A
method using an amino-acid-variant of the Fc region which binds to
FcRn in the pH neutral range is described in WO2011/122011 as a
method for accelerating elimination of antigens from the plasma.
The present invention is useful as a method for accelerating the
elimination from the plasma of multimeric antigens containing two
or more antigenic binding units without using the above-mentioned
variants, and can further accelerate the elimination of the
multimeric antigens containing two or more antigenic binding units
from the plasma through combination with the above-mentioned
variants.
Ex Vivo Method for Eliminating Antigens from the Plasma
[0399] An example of a non-limiting embodiment of the use of an
antigen-binding molecule in the method provided by the present
invention for eliminating antigens from the plasma includes use of
the antigen-binding molecule in a so-called ex vivo method for
eliminating antigens from the plasma, which includes forming an
immune complex containing two or more of the antigen-binding
molecules and two or more antigen molecules (provided that the
antigens contain two or more antigenic binding units) by allowing
the antigen-binding molecules of the present invention to contact
plasma isolated from a subject, and allowing the immune complex to
contact cells expressing FcRn and/or Fc.gamma. receptors. The speed
of antigen elimination from the plasma can also be promoted by
substituting or combining a method for administering
antigen-binding molecules in vivo with a so-called ex vivo method,
in which the plasma containing antigen-binding molecules and
antigens that bind to the antigen-binding molecules is temporarily
taken out of the body and then contacted with cells expressing FcRn
and/or Fc.gamma. receptors for a certain period of time, and the
plasma containing extracellularly recycled (or re-secreted or
re-circulated) antigen-binding molecules that are not bound to
antigen is returned to the body.
[0400] Furthermore, an example of a non-limiting embodiment of the
use of an antigen-binding molecule in the method provided by the
present invention for eliminating antigens from plasma includes use
of the antigen-binding molecule in a so-called ex vivo method for
eliminating antigens from the plasma, which includes contacting an
immune complex containing two or more of the antigen-binding
molecules and two or more antigen molecules (provided that the
antigens comprise two or more antigenic binding units) present in
the plasma isolated from a subject to whom the antigen-binding
molecules of the present invention are administered with cells
expressing FcRn and/or Fc.gamma. receptors.
[0401] Whether or not an antigen is eliminated from the plasma can
be confirmed, for example, by evaluating whether or not the speed
of antigen elimination from the aforementioned plasma is promoted
by using as a control, antigen-binding molecules that cannot form
the immune complexes disclosed in the present invention,
antigen-binding molecules containing antigen-binding domains with
ion-concentration-independent antigen-binding activity, or
antigen-binding molecules containing an Fc region with impaired
binding activity toward Fc.gamma.R or FcRn, instead of the
antigen-binding molecules of the present invention.
Method of Screening for Antigen-Binding Molecules Containing an Fc
Region and an Antigen-Binding Domain Whose Antigen-Binding Activity
is Ion Concentration-Dependent
[0402] The present invention provides a method of screening for an
antigen-binding molecule having a function of eliminating antigens
from the plasma, the method comprising:
(a) obtaining an antigen-binding domain of which antigen-binding
activity varies depending on an ion concentration condition; (b)
obtaining a gene encoding the antigen-binding domain selected in
(a) above; (c) operably linking the gene obtained in (b) above with
a gene encoding an Fc region; (d) culturing a host cell comprising
the genes operably linked in (c) above; (e) isolating an
antigen-binding molecule from a culture solution obtained in (d)
above; (f) contacting the antigen-binding molecule obtained in (e)
above with an antigen; and (g) evaluating the formation of an
immune complex comprising the antigen-binding molecule and the
antigen.
[0403] In a non-limiting embodiment of the present invention, after
isolating a polynucleotide encoding an antigen-binding domain whose
binding activity changes depending on the condition selected as
described above, the polynucleotide is inserted into an appropriate
expression vector. For example, when the antigen-binding domain is
an antibody variable region, once a cDNA encoding the variable
region is obtained, the cDNA is digested with restriction enzymes
that recognize the restriction sites inserted at the two ends of
the cDNA. Preferably, the restriction enzymes recognize and digest
a nucleotide sequence that appears at a low frequency in the
nucleotide sequence composing the gene of the antigen-binding
molecule. Furthermore, restriction enzymes that provide cohesive
ends are preferably inserted to insert a single copy of a digested
fragment into the vector in the correct orientation. The cDNA
encoding a variable region of an antigen-binding molecule digested
as described above is inserted into an appropriate expression
vector to obtain an expression vector for the antigen-binding
molecule of the present invention. At this time, a gene encoding an
antibody constant region (C region) may be fused in frame with the
gene encoding the variable region.
[0404] To produce an antigen-binding molecule of interest, a
polynucleotide encoding the antigen-binding molecule is inserted in
a manner operably linked to a regulatory sequence into an
expression vector. Regulatory sequences include, for example,
enhancers and promoters. Furthermore, an appropriate signal
sequence may be linked to the N terminus so that the expressed
antigen-binding molecule is secreted to the outside of the cells.
As signal sequence, for example, a peptide having the amino acid
sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 5) is used; however, it is
also possible to link other appropriate signal sequences. The
expressed polypeptide is cleaved at the carboxyl terminus of the
above-described sequence, and the cleaved polypeptide is secreted
as a mature polypeptide to the outside of cells. Then, appropriate
host cells are transformed with this expression vector so that
recombinant cells expressing the polynucleotide encoding the
antigen-binding molecule of interest can be obtained. The
antigen-binding molecules of the present invention can be produced
from the recombinant cells by following the methods described above
in the section on antibodies.
[0405] For a nucleic acid, "operably linked" means that the nucleic
acid has a functional relationship with another nucleic acid
sequence. For example, a DNA encoding a presequence or a secretory
leader is operably linked to a DNA encoding a certain polypeptide
if it is to be expressed as a precursor protein involved in the
secretion of the polypeptide. A promoter or enhancer is operably
linked to a coding sequence if it affects the transcription of the
coding sequence. A ribosome binding site is operably linked to a
coding sequence if it is in a position that facilitates
translation. Generally, "operably linked" means that the linked DNA
sequences are contiguous, and in the case of a secretory leader, it
means that the linked DNA sequences are contiguous and in a reading
frame. However, enhancers do not have to be contiguous. Linking is
accomplished by ligation at suitable restriction sites. If such
sites do not exist, synthetic oligonucleotide adaptors or linkers
are used in accordance with conventional practice. Furthermore,
linked nucleic acids may be produced by the above-mentioned overlap
extension PCR technique.
[0406] In a non-limiting embodiment of the present invention, after
isolating a polynucleotide encoding the above-described
antigen-binding molecule whose binding activity varies depending on
a selected condition, a variant of the polynucleotide is inserted
into an appropriate expression vector. Such variants preferably
include those prepared via humanization based on the polynucleotide
sequence encoding an antigen-binding molecule of the present
invention obtained by screening as a randomized variable region
library a synthetic library or an immune library constructed
originating from nonhuman animals. The same methods as described
above for producing above-described humanized antibodies can be
used as a method for producing humanized antigen-binding molecule
variants.
[0407] In another embodiment, such variants preferably include
those obtained by introducing an alteration that increases the
antigen affinity (affinity maturation) of an antigen-binding
molecule of the present invention into an isolated polynucleotide
sequence for the molecule obtained by screening using a synthetic
library or a naive library as a randomized variable region library.
Such variants can be obtained by various known procedures for
affinity maturation, including CDR mutagenesis (Yang et al. (J.
Mol. Biol. (1995) 254, 392-403)), chain shuffling (Marks et al.
(Bio/Technology (1992) 10, 779-783)), use of E. coli mutant strains
(Low et al. (J. Mol. Biol. (1996) 250, 359-368)), DNA shuffling
(Patten et al. (Curr. Opin. Biotechnol. (1997) 8, 724-733)), phage
display (Thompson et al. (J. Mol. Biol. (1996) 256, 77-88)), and
sexual PCR (Clameri et al. (Nature (1998) 391, 288-291)).
[0408] As described above, antigen-binding molecules that are
produced by the production methods of the present invention include
antigen-binding molecules having an Fc region. Various variants can
be used as Fc regions. In an embodiment, variants of the present
invention preferably include polynucleotides encoding
antigen-binding molecules having a heavy chain in which a
polynucleotide encoding an Fc region variant as described above is
linked in frame to a polynucleotide encoding the above-described
antigen-binding domain whose binding activity varies depending on a
selected condition.
[0409] In a non-limiting embodiment of the present invention, Fc
regions preferably include, for example, Fc constant regions of
antibodies such as IgG1 of SEQ ID NO: 13 (Ala is added to the N
terminus of AAC82527.1), IgG2 of SEQ ID NO: 14 (Ala is added to the
N terminus of AAB59393.1), IgG3 of SEQ ID NO: 15 (CAA27268.1), and
IgG4 of SEQ ID NO: 16 (Ala is added to the N terminus of
AAB59394.1). The plasma retention of IgG molecules is relatively
long (the elimination from plasma is slow) since FcRn, particularly
human FcRn, functions as a salvage receptor for IgG molecules. IgG
molecules incorporated into endosomes by pinocytosis bind under the
endosomal acidic condition to FcRn, particularly human FcRn,
expressed in endosomes. IgG molecules that cannot bind to FcRn,
particularly human FcRn, are transferred to lysosomes, and degraded
there. Meanwhile, IgG molecules bound to FcRn, particularly human
FcRn, are transferred to cell surface, and then return to plasma as
a result of dissociation from FcRn, particularly human FcRn, under
the neutral condition in plasma.
[0410] Since antibodies comprising a typical Fc region do not have
a binding activity to FcRn, particularly to human FcRn, under the
plasma neutral pH range condition, typical antibodies and
antibody-antigen complexes are incorporated into cells by
non-specific endocytosis and transferred to cell surface by binding
to FcRn, particularly human FcRn, in the endosomal acidic pH range
condition. FcRn, particularly human FcRn, transports antibodies
from the endosome to the cell surface. Thus, some of FcRn,
particularly human FcRn, is thought to be also present on the cell
surface. However, antibodies are recycled to plasma, since they
dissociated from FcRn, particularly human FcRn, in the neutral pH
range condition on cell surface.
[0411] Fc regions having human FcRn-binding activity in the neutral
pH range, which can be included in the antigen-binding molecules of
the present invention, can be obtained by any method. Specifically,
Fc regions having human FcRn-binding activity in the neutral pH
range can be obtained by altering amino acids of human IgG-type
immunoglobulin as a starting Fc region. Preferred Fc regions of
human IgG-type immunoglobulin for alteration include, for example,
those of human IgGs (IgG1, IgG2, IgG3, and IgG4, and variants
thereof). Amino acids at any positions may be altered to other
amino acids as long as the resulting regions have the human
FcRn-binding activity in the neutral pH range or increased human
FcRn-binding activity in the neutral range. When an antigen-binding
molecule comprises the Fc region of human IgG1 as human Fc region,
it is preferable that the resulting region comprises an alteration
that results in the effect to enhance the human FcRn binding in the
neutral pH range as compared to the binding activity of the
starting Fc region of human IgG1. Amino acids that allow such
alterations include, for example, one or more amino acids selected
from the group of positions 221 to 225, 227, 228, 230, 232, 233 to
241, 243 to 252, 254 to 260, 262 to 272, 274, 276, 278 to 289, 291
to 312, 315 to 320, 324, 325, 327 to 339, 341, 343, 345, 360, 362,
370, 375 to 378, 380, 382, 385 to 387, 389, 396, 414, 416, 423,
424, 426 to 438, 440, and 442 (indicated by EU numbering). More
specifically, such amino acid alterations include those listed in
Table 5. Alteration of these amino acids enhances the human FcRn
binding of the Fc region of IgG-type immunoglobulin in the neutral
pH range.
[0412] Among those described above, appropriate alterations that
enhance the human FcRn binding in the neutral pH range are selected
for use in the present invention. Particularly preferred amino
acids for such Fc region variants include, for example, amino acids
at positions 237, 248, 250, 252, 254, 255, 256, 257, 258, 265, 286,
289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317,
332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428,
433, 434, and 436 (indicated by EU numbering). The human
FcRn-binding activity of the Fc region included in an
antigen-binding molecule can be increased in the neutral pH range
by substituting at least one amino acid with a different amino
acid.
[0413] Particularly preferred alterations in the Fc region include,
for example, at least one or more amino acid alterations selected
from the group of:
Met for the amino acid at position 237; Ile for the amino acid at
position 248; Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr for
the amino acid at position 250; Phe, Trp, or Tyr for the amino acid
at position 252; Thr for the amino acid at position 254; Glu for
the amino acid at position 255; Asp, Asn, Glu, or Gln for the amino
acid at position 256; Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or
Val for the amino acid at position 257; His for the amino acid at
position 258; Ala for the amino acid at position 265; Ala or Glu
for the amino acid at position 286; His for the amino acid at
position 289; Ala for the amino acid at position 297; Ala for the
amino acid at position 303; Ala for the amino acid at position 305;
Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,
Ser, Val, Trp, or Tyr for the amino acid at position 307; Ala, Phe,
Ile, Leu, Met, Pro, Gln, or Thr for the amino acid at position 308;
Ala, Asp, Glu, Pro, or Arg for the amino acid at position 309; Ala,
His, or Ile for the amino acid at position 311; Ala or His for the
amino acid at position 312; Lys or Arg for the amino acid at
position 314; Ala, Asp, or His for the amino acid at position 315;
Ala for the amino acid at position 317; Val for the amino acid at
position 332; Leu for the amino acid at position 334; His for the
amino acid at position 360; Ala for the amino acid at position 376;
Ala for the amino acid at position 380; Ala for the amino acid at
position 382; Ala for the amino acid at position 384; Asp or His
for the amino acid at position 385; Pro for the amino acid at
position 386; Glu for the amino acid at position 387; Ala or Ser
for the amino acid at position 389; Ala for the amino acid at
position 424; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro,
Gln, Ser, Thr, Val, Trp, or Tyr for the amino acid at position 428;
Lys for the amino acid at position 433; Ala, Phe, His, Ser, Trp, or
Tyr for the amino acid at position 434; and His, Ile, Leu, Phe,
Thr, or Val for the amino acid at position 436 in the EU numbering
system. Meanwhile, the number of altered amino acids is not
particularly limited; such amino acid alterations include single
amino acid alteration and alteration of amino acids at two or more
sites. Combinations of amino acid alterations at two or more sites
include, for example, those described in Tables 5-1 to 5-32.
[0414] In addition to the Fc region of human IgG1 (SEQ ID NO: 13),
IgG2 (SEQ ID NO: 14), IgG3 (SEQ ID NO: 15), or IgG4 (SEQ ID NO:
16), as Fc regions included in the present invention, Fc regions
with modified Fc.gamma.R binding, which have a higher Fc.gamma.
receptor-binding activity than the Fc region of a native human IgG
in which the sugar chain bound at position 297 (EU numbering) is a
fucose-containing sugar chain, may be suitably used. Such Fc
regions with modified Fc.gamma.R binding may be produced by
modifying amino acids in the Fc region of a native human IgG.
Whether the Fc.gamma.R-binding activity of an Fc region is higher
than that of the Fc region of a native human IgG, in which the
sugar chain bound at position 297 (EU numbering) is a
fucose-containing sugar chain, can be appropriately determined
using methods such as those described above.
[0415] In the present invention, "modification of amino acids" or
"amino acid modification" of an Fc region includes modification
into an amino acid sequence which is different from that of the
starting Fc region. The starting Fc region may be any Fc region, as
long as a variant modified from the starting Fc region can bind to
human Fc.gamma. receptor in a neutral pH range. Furthermore, an Fc
region modified from a starting Fc region which had been already
modified can also be used preferably as an Fc region of the present
invention. The "starting Fc region" can refer to the polypeptide
itself, a composition comprising the starting Fc region, or an
amino acid sequence encoding the starting Fc region. Starting Fc
regions can comprise a known Fc region produced via recombination
described briefly in the section "Antibodies". The origin of
starting Fc regions is not limited, and they may be obtained from
human or any nonhuman organisms. Such organisms preferably include
mice, rats, guinea pigs, hamsters, gerbils, cats, rabbits, dogs,
goats, sheep, bovines, horses, camels and organisms selected from
nonhuman primates. In another embodiment, starting Fc regions can
also be obtained from cynomolgus monkeys, marmosets, rhesus
monkeys, chimpanzees, or humans. Starting Fc regions can be
obtained preferably from human IgG1; however, they are not limited
to any particular IgG class. This means that an Fc region of human
IgG1, IgG2, IgG3, or IgG4 can be used appropriately as a starting
Fc region, and herein also means that an Fc region of an arbitrary
IgG class or subclass derived from any organisms described above
can be preferably used as a starting Fc region. Examples of
naturally-occurring IgG variants or modified forms are described in
published documents (Curr. Opin. Biotechnol. (2009) 20 (6): 685-91;
Curr. Opin. Immunol. (2008) 20 (4), 460-470; Protein Eng. Des. Sel.
(2010) 23 (4): 195-202; International Publication Nos. WO
2009/086320, WO 2008/092117, WO 2007/041635, and WO 2006/105338);
however, they are not limited to the examples.
[0416] Examples of alterations include those with one or more
mutations, for example, mutations by substitution of different
amino acid residues for amino acids of starting Fc regions, by
insertion of one or more amino acid residues into starting Fc
regions, or by deletion of one or more amino acids from starting Fc
region. Preferably, the amino acid sequences of altered Fc regions
comprise at least a part of the amino acid sequence of a non-native
Fc region. Such variants necessarily have sequence identity or
similarity less than 100% to their starting Fc region. In a
preferred embodiment, the variants have amino acid sequence
identity or similarity about 75% to less than 100%, more preferably
about 80% to less than 100%, even more preferably about 85% to less
than 100%, still more preferably about 90% to less than 100%, and
yet more preferably about 95% to less than 100% to the amino acid
sequence of their starting Fc region. In a non-limiting embodiment
of the present invention, at least one amino acid is different
between an Fc.gamma.R-binding modified Fc region of the present
invention and its starting Fc region. The amino acid differences
between an Fc.gamma.R-binding modified Fc region of the present
invention and its starting Fc region can also be suitably specified
based on the amino acid differences at the above-described
particular amino acid positions specified by the EU numbering
system.
[0417] The Fc region with modified Fc.gamma.R binding, which has a
higher Fc.gamma. receptor-binding activity than that of the Fc
region of a native human IgG in which the sugar chain bound at
position 297 (EU numbering) is a fucose-containing sugar chain,
contained in the antigen-binding molecules of the present invention
may be obtained by any method. Specifically, the Fc region with
modified Fc.gamma.R binding can be obtained by modifying amino
acids in a human IgG-type immunoglobulin, and is used as the
starting Fc region. Preferred Fc regions of IgG-type
immunoglobulins for modification include, for example, Fc regions
of human IgGs (IgG1, IgG2, IgG3, IgG4, and variants thereof).
[0418] Amino acids of any positions may be modified into other
amino acids, as long as the binding activity toward the Fc.gamma.
receptor is higher than that of the Fc region of a native human
IgG, in which the sugar chain bound at position 297 (EU numbering)
is a fucose-containing sugar chain. When the antigen-binding
molecule contains a human IgG1 Fc region as the human Fc region, it
preferably contains a modification that yields the effect of a
higher Fc.gamma. receptor-binding activity than that of the Fc
region of a native human IgG, in which the sugar chain bound at
position 297 (EU numbering) is a fucose-containing sugar chain.
Such amino acid modifications have been reported, for example, in
international publications such as WO2007/024249, WO2007/021841,
WO2006/031370, WO2000/042072, WO2004/029207, WO2004/099249,
WO2006/105338, WO2007/041635, WO2008/092117, WO2005/070963,
WO2006/020114, WO2006/116260, and WO2006/023403.
[0419] Examples of such amino acids that can be modified include at
least one or more amino acids selected from the group of positions
221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235,
236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250,
251, 254, 255, 256, 258, 260, 262, 263, 264, 265, 266, 267, 268,
269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282,
283, 284, 285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297,
298, 299, 300, 301, 302, 303, 304, 305, 311, 313, 315, 317, 318,
320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333,
334, 335, 336, 337, 339, 376, 377, 378, 379, 380, 382, 385, 392,
396, 421, 427, 428, 429, 434, 436, and 440 (EU numbering).
Modification of these amino acids can yield Fc regions (Fc regions
with modified Fc.gamma.R binding) having a higher Fc.gamma.
receptor-binding activity than the Fc.gamma. receptor-binding
activity of an Fc region of a native human IgG, in which the sugar
chain bound at position 297 (EU numbering) is a fucose-containing
sugar chain.
[0420] Examples of particularly preferred modifications for use in
the present invention include at least one or more amino acid
modifications in the Fc region selected from the group of:
Lys or Tyr for the amino acid of position 221; Phe, Trp, Glu, or
Tyr for the amino acid of position 222; Phe, Trp, Glu, or Lys for
the amino acid of position 223; Phe, Trp, Glu, or Tyr for the amino
acid of position 224; Glu, Lys, or Trp for the amino acid of
position 225; Glu, Gly, Lys, or Tyr for the amino acid of position
227; Glu, Gly, Lys, or Tyr for the amino acid of position 228; Ala,
Glu, Gly, or Tyr for the amino acid of position 230; Glu, Gly, Lys,
Pro, or Tyr for the amino acid of position 231; Glu, Gly, Lys, or
Tyr for the amino acid of position 232; Ala, Asp, Phe, Gly, His,
Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for
the amino acid of position 233; Ala, Asp, Glu, Phe, Gly, His, Ile,
Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the
amino acid of position 234; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys,
Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino
acid of position 235; Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met,
Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid
of position 236; Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro,
Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of position
237; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,
Ser, Thr, Val, Trp, or Tyr for the amino acid of position 238; Asp,
Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr,
Val, Trp, or Tyr for the amino acid of position 239; Ala, Ile, Met,
or Thr for the amino acid of position 240; Asp, Glu, Leu, Arg, Trp,
or Tyr for the amino acid of position 241; Leu, Glu, Leu, Gln, Arg,
Trp, or Tyr for the amino acid of position 243; His for the amino
acid of position 244; Ala for the amino acid of position 245; Asp,
Glu, His, or Tyr for the amino acid of position 246; Ala, Phe, Gly,
His, Ile, Leu, Met, Thr, Val, or Tyr for the amino acid of position
247; Glu, His, Gln, or Tyr for the amino acid of position 249; Glu
or Gln for the amino acid of position 250; Phe for the amino acid
of position 251; Phe, Met, or Tyr for the amino acid of position
254; Glu, Leu, or Tyr for the amino acid of position 255; Ala, Met,
or Pro for the amino acid of position 256; Asp, Glu, His, Ser, or
Tyr for the amino acid of position 258; Asp, Glu, His, or Tyr for
the amino acid of position 260; Ala, Glu, Phe, Ile, or Thr for the
amino acid of position 262; Ala, Ile, Met, or Thr for the amino
acid of position 263; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met,
Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr for the amino acid of
position 264; Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,
Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of
position 265; Ala, Ile, Met, or Thr for the amino acid of position
266; Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,
Thr, Val, Trp, or Tyr for the amino acid of position 267; Asp, Glu,
Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, or Trp for
the amino acid of position 268; Phe, Gly, His, Ile, Lys, Leu, Met,
Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of
position 269; Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg,
Ser, Thr, Trp, or Tyr for the amino acid of position 270; Ala, Asp,
Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr,
Val, Trp, or Tyr for the amino acid of position 271; Asp, Phe, Gly,
His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for
the amino acid of position 272; Phe or Ile for the amino acid of
position 273; Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro,
Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of position 274;
Leu or Trp for the amino acid of position 275; Asp, Glu, Phe, Gly,
His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the
amino acid of position 276; Asp, Glu, Gly, His, Ile, Lys, Leu, Met,
Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp for the amino acid of
position 278; Ala for the amino acid of position 279; Ala, Gly,
His, Lys, Leu, Pro, Gln, Trp, or Tyr for the amino acid of position
280; Asp, Lys, Pro, or Tyr for the amino acid of position 281; Glu,
Gly, Lys, Pro, or Tyr for the amino acid of position 282; Ala, Gly,
His, Ile, Lys, Leu, Met, Pro, Arg, or Tyr for the amino acid of
position 283; Asp, Glu, Leu, Asn, Thr, or Tyr for the amino acid of
position 284; Asp, Glu, Lys, Gln, Trp, or Tyr for the amino acid of
position 285; Glu, Gly, Pro, or Tyr for the amino acid of position
286; Asn, Asp, Glu, or Tyr for the amino acid of position 288; Asp,
Gly, His, Leu, Asn, Ser, Thr, Trp, or Tyr for the amino acid of
position 290; Asp, Glu, Gly, His, Ile, Gln, or Thr for the amino
acid of position 291; Ala, Asp, Glu, Pro, Thr, or Tyr for the amino
acid of position 292; Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg,
Ser, Thr, Val, Trp, or Tyr for the amino acid of position 293; Phe,
Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or
Tyr for the amino acid of position 294; Asp, Glu, Phe, Gly, His,
Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the
amino acid of position 295; Ala, Asp, Glu, Gly, His, Ile, Lys, Leu,
Met, Asn, Gln, Arg, Ser, Thr, or Val for the amino acid of position
296; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg,
Ser, Thr, Val, Trp, or Tyr for the amino acid of position 297; Ala,
Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, or
Tyr for the amino acid of position 298; Ala, Asp, Glu, Phe, Gly,
His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr
for the amino acid of position 299; Ala, Asp, Glu, Gly, His, Ile,
Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp for the
amino acid of position 300; Asp, Glu, His, or Tyr for the amino
acid of position 301; Ile for the amino acid of position 302; Asp,
Gly, or Tyr for the amino acid of position 303; Asp, His, Leu, Asn,
or Thr for the amino acid of position 304; Glu, Ile, Thr, or Tyr
for the amino acid of position 305; Ala, Asp, Asn, Thr, Val, or Tyr
for the amino acid of position 311; Phe for the amino acid of
position 313; Leu for the amino acid of position 315; Glu or Gln
for the amino acid of position 317; His, Leu, Asn, Pro, Gln, Arg,
Thr, Val, or Tyr for the amino acid of position 318; Asp, Phe, Gly,
His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, or Tyr for the amino
acid of position 320; Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr,
Val, Trp, or Tyr for the amino acid of position 322; Ile for the
amino acid of position 323; Asp, Phe, Gly, His, Ile, Leu, Met, Pro,
Arg, Thr, Val, Trp, or Tyr for the amino acid of position 324; Ala,
Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser,
Thr, Val, Trp, or Tyr for the amino acid of position 325; Ala, Asp,
Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr
for the amino acid of position 326; Ala, Asp, Glu, Phe, Gly, His,
Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, or Tyr for the
amino acid of position 327; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys,
Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino
acid of position 328; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met,
Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of
position 329; Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,
Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of position
330; Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, or Tyr
for the amino acid of position 331; Ala, Asp, Glu, Phe, Gly, His,
Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for
the amino acid of position 332; Ala, Asp, Glu, Phe, Gly, His, Ile,
Leu, Met, Pro, Ser, Thr, Val, or Tyr for the amino acid of position
333; Ala, Glu, Phe, Ile, Leu, Pro, or Thr for the amino acid of
position 334; Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg,
Ser, Val, Trp, or Tyr for the amino acid of position 335; Glu, Lys,
or Tyr for the amino acid of position 336; Glu, His, or Asn for the
amino acid of position 337; Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln,
Arg, Ser, or Thr for the amino acid of position 339; Ala or Val for
the amino acid of position 376; Gly or Lys for the amino acid of
position 377; Asp for the amino acid of position 378; Asn for the
amino acid of position 379; Ala, Asn, or Ser for the amino acid of
position 380; Ala or Ile for the amino acid of position 382; Glu
for the amino acid of position 385; Thr for the amino acid of
position 392; Leu for the amino acid of position 396; Lys for the
amino acid of position 421; Asn for the amino acid of position 427;
Phe or Leu for the amino acid of position 428; Met for the amino
acid of position 429; Trp for the amino acid of position 434; Ile
for the amino acid of position 436; and Gly, His, Ile, Leu, or Tyr
for the amino acid of position 440; as indicated by EU numbering.
Meanwhile, the number of amino acids to be modified is not
particularly limited, and an amino acid at only one site may be
modified or amino acids at two or more sites may be modified.
Examples of combinations for the amino acid modifications at two or
more sites include those described in Table 6 (Tables 6-1 to
6-3).
[0421] Among the Fc regions suitable for use in the present
invention, a suitable example of an Fc region that has a higher
binding activity toward an inhibitory Fc.gamma. receptor than
toward an activating Fc.gamma. receptor (i.e., having a selective
binding activity toward an inhibitory Fc.gamma. receptor), which is
used as a non-limiting embodiment of an Fc region with the property
of having a higher binding activity toward a specific Fc.gamma.
receptor than toward other Fc.gamma. receptors (i.e., an Fc region
having a selective Fc.gamma. receptor-binding activity), is an Fc
region with one or more of the following modifications in the amino
acids (indicated by EU numbering) of the aforementioned Fc region:
the amino acid at position 238 is modified to Asp and the amino
acid at position 328 is modified to Glu. The Fc regions and
modifications described in US2009/0136485 may be selected
appropriately as the Fc region having a selective binding activity
to an inhibitory Fc.gamma. receptor.
[0422] In a non-limiting embodiment of the present invention, a
suitable example is an Fc region in which one or more of the amino
acids indicated by EU numbering at positions 238 and 328 according
to EU numbering are respectively modified to Asp or Glu in the
aforementioned Fc region.
[0423] Furthermore, in a non-limiting embodiment of the present
invention, suitable examples of the Fc regions are those with
substitution of Asp for Pro at position 238 (EU numbering), and one
or more modifications selected from among Trp for the amino acid of
position 237, Phe for the amino acid of position 237, Val for the
amino acid of position 267, Gln for the amino acid of position 267,
Asn for the amino acid of position 268, Gly for the amino acid of
position 271, Leu for the amino acid of position 326, Gln for the
amino acid of position 326, Glu for the amino acid of position 326,
Met for the amino acid of position 326, Asp for the amino acid of
position 239, Ala for the amino acid of position 267, Trp for the
amino acid of position 234, Tyr for the amino acid of position 234,
Ala for the amino acid of position 237, Asp for the amino acid of
position 237, Glu for the amino acid of position 237, Leu for the
amino acid of position 237, Met for the amino acid of position 237,
Tyr for the amino acid of position 237, Lys for the amino acid of
position 330, Arg for the amino acid of position 330, Asp for the
amino acid of position 233, Asp for the amino acid of position 268,
Glu for the amino acid of position 268, Asp for the amino acid of
position 326, Ser for the amino acid of position 326, Thr for the
amino acid of position 326, Ile for the amino acid of position 323,
Leu for the amino acid of position 323, Met for the amino acid of
position 323, Asp for the amino acid of position 296, Ala for the
amino acid of position 326, Asn for the amino acid of position 326,
and Met for the amino acid of position 330, according to EU
numbering.
[0424] Known methods such as site-directed mutagenesis (Kunkel et
al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and Overlap
extension PCR can be appropriately employed to modify the amino
acids of Fc regions. Furthermore, various known methods can also be
used as an amino acid modification method for substituting amino
acids by those other than natural amino acids (Annu Rev. Biophys.
Biomol. Struct. (2006) 35, 225-249; Proc. Natl. Acad. Sci. U.S.A.
(2003) 100 (11), 6353-6357). For example, a cell-free translation
system (Clover Direct (Protein Express)) containing tRNAs in which
amber suppressor tRNA, which is complementary to UAG codon (amber
codon) that is a stop codon, is linked with an unnatural amino acid
may be suitably used.
[0425] As described above, the antigen-binding molecules isolated
from the culture solution of host cells containing operably linked
genes encoding the antigen-binding molecules are contacted with
antigens in the screening method of the present invention. In a
non-limiting embodiment of the contacting step, the conditions
described in, e.g., the aforementioned section on antigen binding
antigens may be appropriately employed.
[0426] Next, whether immune complexes containing the test
antigen-binding molecule and the test antigen are formed is
evaluated. Examples of methods for evaluating the formation of
immune complexes containing the antigen-binding molecules and
antigens include methods that make use of the property that the
immune complexes become larger molecules than the antigen-binding
molecule alone or the antigen molecule alone, such as size
exclusion (gel filtration) chromatography, ultracentrifugal
analysis method, light-scattering method, electron microscopy, and
mass spectrometry (Molecular Immunology (2002), 39, 77-84;
Molecular Immunology (2009), 47, 357-364). For example, when size
exclusion (gel filtration) chromatography is used as shown in FIG.
9, whether immune complexes are formed is evaluated based on
whether larger molecular species are observed as compared to when
the antigen molecule alone or the antigen-binding molecule alone is
analyzed.
[0427] Furthermore, when the antigen-binding molecule or antigen
has an immunoglobulin constant region, examples also include ELISA
and FACS which use the property of the immune complex to bind more
strongly to an Fc receptor or a complement component than the
antigen-binding molecule alone or antigen alone (The Journal of
Biological Chemistry (2001) 276 (9), 6591-6604; Journal of
Immunological Methods (1982) 50, 109-114). For example, when ELISA
is performed by immobilizing an Fc receptor, formation of an immune
complex is evaluated by observing whether the detected signal is
increased compared to when an antigen alone or an antigen-binding
molecule alone is evaluated.
Methods for Producing an Antigen-Binding Molecule Containing an Fc
Region and an Antigen-Binding Domain Showing
Ion-Concentration-Dependent Antigen-Binding Activity
[0428] The present invention provides a method for producing
antigen-binding molecules having a function of eliminating antigens
from plasma, the method comprising:
(a) contacting an antigen with an antigen-binding molecule
comprising an Fc region and two or more antigen-binding domains,
wherein at least one of the antigen-binding domains has an
antigen-binding activity that varies depending on an ion
concentration condition; (b) evaluating formation of an immune
complex comprising the antigen-binding molecule and the antigen;
(c) culturing a host cell comprising a vector that carries a gene
encoding an antigen-binding domain which is confirmed to form an
immune complex in (b) above; and (d) isolating the antigen-binding
molecule from a culture solution obtained in (c) above.
[0429] The present invention provides a method for producing an
antigen-binding molecule having a function of eliminating antigens
from the plasma, the method comprising:
(a) obtaining an antigen-binding domain of which antigen-binding
activity varies depending on an ion concentration condition; (b)
obtaining a gene encoding the antigen-binding domain selected in
(a) above; (c) operably linking the gene obtained in (b) above with
a gene encoding an Fc region; (d) culturing a host cell comprising
the genes operably linked in (c) above; (e) isolating an
antigen-binding molecule from a culture solution obtained in (d)
above; (f) contacting the antigen-binding molecule obtained in (e)
above with an antigen; (g) evaluating formation of an immune
complex comprising the antigen-binding molecule and the antigen;
(h) culturing a host cell comprising a vector that carries a gene
encoding an antigen-binding domain which is confirmed to form an
immune complex in (g) above; and (i) isolating the antigen-binding
molecule from a culture solution obtained in (h) above.
[0430] The present invention further provides a method for
producing an antigen-binding molecule having a function of
eliminating antigens from the plasma, the method comprising:
(a) obtaining an antigen-binding domain of which antigen-binding
activity varies depending on an ion concentration condition; (b)
obtaining a gene encoding the antigen-binding domain selected in
(a) above; (c) operably linking the gene obtained in (b) above with
a gene encoding an Fc region; (d) culturing a host cell comprising
the genes operably linked in (c) above; and (e) isolating an
antigen-binding molecule from a culture solution obtained in (d)
above; and wherein the method further comprises contacting the
antigen-binding molecule obtained by the production method with an
antigen, and evaluating the formation of an immune complex
comprising the antigen-binding molecule and the antigen.
[0431] In a non-limiting embodiment of the present invention, after
isolating a polynucleotide encoding an antigen-binding domain whose
binding activity changes depending on the condition selected as
described above, the polynucleotide is inserted into an appropriate
expression vector. For example, when the antigen-binding domain is
an antibody variable region, once a cDNA encoding the variable
region is obtained, the cDNA is digested with restriction enzymes
that recognize the restriction sites inserted at the two ends of
the cDNA. Preferably, the restriction enzymes recognize and digest
a nucleotide sequence that appears at a low frequency in the
nucleotide sequence composing the gene of the antigen-binding
molecule. Furthermore, restriction enzymes that provide cohesive
ends are preferably inserted to insert a single copy of a digested
fragment into the vector in the correct orientation. The cDNA
encoding a variable region of an antigen-binding molecule digested
as described above is inserted into an appropriate expression
vector to obtain an expression vector for the antigen-binding
molecule of the present invention. At this time, a gene encoding an
antibody constant region (C region) may be fused in frame with the
gene encoding the variable region.
[0432] To produce an antigen-binding molecule of interest, a
polynucleotide encoding the antigen-binding molecule is inserted in
a manner operably linked to a regulatory sequence into an
expression vector. Regulatory sequences include, for example,
enhancers and promoters. Furthermore, an appropriate signal
sequence may be linked to the N terminus so that the expressed
antigen-binding molecule is secreted to the outside of the cells.
As signal sequence, for example, a peptide having the amino acid
sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 5) is used; however, it is
also possible to link other appropriate signal sequences. The
expressed polypeptide is cleaved at the carboxyl terminus of the
above-described sequence, and the cleaved polypeptide is secreted
as a mature polypeptide to the outside of cells. Then, appropriate
host cells are transformed with this expression vector so that
recombinant cells expressing the polynucleotide encoding the
antigen-binding molecule of interest can be obtained. The
antigen-binding molecules of the present invention can be produced
from the recombinant cells by following the methods described above
in the section on antibodies.
[0433] For a nucleic acid, "operably linked" means that the nucleic
acid has a functional relationship with another nucleic acid
sequence. For example, a DNA encoding a presequence or a secretory
leader is operably linked to a DNA encoding a certain polypeptide
if it is to be expressed as a precursor protein involved in the
secretion of the polypeptide. A promoter or enhancer is operably
linked to a coding sequence if it affects the transcription of the
coding sequence. A ribosome binding site is operably linked to a
coding sequence if it is in a position that facilitates
translation. Generally, "operably linked" means that the linked DNA
sequences are contiguous, and in the case of a secretory leader, it
means that the linked DNA sequences are contiguous and in a reading
frame. However, enhancers do not have to be contiguous. Linking is
accomplished by ligation at suitable restriction sites. If such
sites do not exist, synthetic oligonucleotide adaptors or linkers
are used in accordance with conventional practice. Furthermore,
linked nucleic acids may be produced by the above-mentioned overlap
extension PCR technique.
[0434] In a non-limiting embodiment of the present invention, after
isolating a polynucleotide encoding the above-described
antigen-binding molecule whose binding activity varies depending on
a selected condition, a variant of the polynucleotide is inserted
into an appropriate expression vector. Such variants preferably
include those prepared via humanization based on the polynucleotide
sequence encoding an antigen-binding molecule of the present
invention obtained by screening as a randomized variable region
library a synthetic library or an immune library constructed
originating from nonhuman animals. The same methods as described
above for producing above-described humanized antibodies can be
used as a method for producing humanized antigen-binding molecule
variants.
[0435] In another embodiment, such variants preferably include
those obtained by introducing an alteration that increases the
antigen affinity (affinity maturation) of an antigen-binding
molecule of the present invention into an isolated polynucleotide
sequence for the molecule obtained by screening using a synthetic
library or a naive library as a randomized variable region library.
Such variants can be obtained by various known procedures for
affinity maturation, including CDR mutagenesis (Yang et al. (J.
Mol. Biol. (1995) 254, 392-403)), chain shuffling (Marks et al.
(Bio/Technology (1992) 10, 779-783)), use of E. coli mutant strains
(Low et al. (J. Mol. Biol. (1996) 250, 359-368)), DNA shuffling
(Patten et al. (Curr. Opin. Biotechnol. (1997) 8, 724-733)), phage
display (Thompson et al. (J. Mol. Biol. (1996) 256, 77-88)), and
sexual PCR (Clameri et al. (Nature (1998) 391, 288-291)).
[0436] As described above, antigen-binding molecules that are
produced by the production methods of the present invention include
antigen-binding molecules having an Fc region. Various variants can
be used as Fc regions. In an embodiment, variants of the present
invention preferably include polynucleotides encoding
antigen-binding molecules having a heavy chain in which a
polynucleotide encoding an Fc region variant as described above is
linked in frame to a polynucleotide encoding the above-described
antigen-binding molecule whose binding activity varies depending on
a selected condition.
[0437] In a non-limiting embodiment of the present invention, Fc
regions preferably include, for example, Fc constant regions of
antibodies such as IgG1 of SEQ ID NO: 13 (Ala is added to the N
terminus of AAC82527.1), IgG2 of SEQ ID NO: 14 (Ala is added to the
N terminus of AAB59393.1), IgG3 of SEQ ID NO: 15 (CAA27268.1), and
IgG4 of SEQ ID NO: 16 (Ala is added to the N terminus of
AAB59394.1). The plasma retention of IgG molecules is relatively
long (the elimination from plasma is slow) since FcRn, particularly
human FcRn, functions as a salvage receptor for IgG molecules. IgG
molecules incorporated into endosomes by pinocytosis bind under the
endosomal acidic condition to FcRn, particularly human FcRn,
expressed in endosomes. IgG molecules that cannot bind to FcRn,
particularly human FcRn, are transferred to lysosomes, and degraded
there. Meanwhile, IgG molecules bound to FcRn, particularly human
FcRn, are transferred to cell surface, and then return to plasma as
a result of dissociation from FcRn, particularly human FcRn, under
the neutral condition in plasma.
[0438] Since antibodies comprising a typical Fc region do not have
a binding activity to FcRn, particularly to human FcRn, under the
plasma neutral pH range condition, typical antibodies and
antibody-antigen complexes are incorporated into cells by
non-specific endocytosis and transferred to cell surface by binding
to FcRn, particularly human FcRn, in the endosomal acidic pH range
condition. FcRn, particularly human FcRn, transports antibodies
from the endosome to the cell surface. Thus, some of FcRn,
particularly human FcRn, is thought to be also present on the cell
surface. However, antibodies are recycled to plasma, since they
dissociated from FcRn, particularly human FcRn, in the neutral pH
range condition on cell surface.
[0439] Fc regions having human FcRn-binding activity in the neutral
pH range, which can be included in the antigen-binding molecules of
the present invention, can be obtained by any method. Specifically,
Fc regions having human FcRn-binding activity in the neutral pH
range can be obtained by altering amino acids of human IgG-type
immunoglobulin as a starting Fc region. Preferred Fc regions of
human IgG-type immunoglobulin for alteration include, for example,
those of human IgGs (IgG1, IgG2, IgG3, and IgG4, and variants
thereof). Amino acids at any positions may be altered to other
amino acids as long as the resulting regions have the human
FcRn-binding activity in the neutral pH range or increased human
FcRn-binding activity in the neutral range. When an antigen-binding
molecule comprises the Fc region of human IgG1 as human Fc region,
it is preferable that the resulting region comprises an alteration
that results in the effect to enhance the human FcRn binding in the
neutral pH range as compared to the binding activity of the
starting Fc region of human IgG1. Amino acids that allow such
alterations include, for example, at least one amino acid selected
from the group of positions 221 to 225, 227, 228, 230, 232, 233 to
241, 243 to 252, 254 to 260, 262 to 272, 274, 276, 278 to 289, 291
to 312, 315 to 320, 324, 325, 327 to 339, 341, 343, 345, 360, 362,
370, 375 to 378, 380, 382, 385 to 387, 389, 396, 414, 416, 423,
424, 426 to 438, 440, and 442 (indicated by EU numbering). More
specifically, such amino acid alterations include those listed in
Table 5. Alteration of these amino acids enhances the human FcRn
binding of the Fc region of IgG-type immunoglobulin in the neutral
pH range.
[0440] Among those described above, appropriate alterations that
enhance the human FcRn binding in the neutral pH range are selected
for use in the present invention. Particularly preferred amino
acids for such Fc region variants include, for example, amino acids
at positions 237, 248, 250, 252, 254, 255, 256, 257, 258, 265, 286,
289, 297, 298, 303, 305, 307, 308, 309, 311, 312, 314, 315, 317,
332, 334, 360, 376, 380, 382, 384, 385, 386, 387, 389, 424, 428,
433, 434, and 436 (indicated by EU numbering). The human
FcRn-binding activity of the Fc region included in an
antigen-binding molecule can be increased in the neutral pH range
by substituting at least one amino acid with a different amino
acid.
[0441] Particularly preferred alterations in the Fc region include,
for example, at least one or more amino acid alterations selected
from the group of:
Met for the amino acid at position 237; Ile for the amino acid at
position 248; Ala, Phe, Ile, Met, Gln, Ser, Val, Trp, or Tyr for
the amino acid at position 250; Phe, Trp, or Tyr for the amino acid
at position 252; Thr for the amino acid at position 254; Glu for
the amino acid at position 255; Asp, Asn, Glu, or Gln for the amino
acid at position 256; Ala, Gly, Ile, Leu, Met, Asn, Ser, Thr, or
Val for the amino acid at position 257; His for the amino acid at
position 258; Ala for the amino acid at position 265; Ala or Glu
for the amino acid at position 286; His for the amino acid at
position 289; Ala for the amino acid at position 297; Ala for the
amino acid at position 303; Ala for the amino acid at position 305;
Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,
Ser, Val, Trp, or Tyr for the amino acid at position 307; Ala, Phe,
Ile, Leu, Met, Pro, Gln, or Thr for the amino acid at position 308;
Ala, Asp, Glu, Pro, or Arg for the amino acid at position 309; Ala,
His, or Ile for the amino acid at position 311; Ala or His for the
amino acid at position 312; Lys or Arg for the amino acid at
position 314; Ala, Asp, or His for the amino acid at position 315;
Ala for the amino acid at position 317; Val for the amino acid at
position 332; Leu for the amino acid at position 334; His for the
amino acid at position 360; Ala for the amino acid at position 376;
Ala for the amino acid at position 380; Ala for the amino acid at
position 382; Ala for the amino acid at position 384; Asp or His
for the amino acid at position 385; Pro for the amino acid at
position 386; Glu for the amino acid at position 387; Ala or Ser
for the amino acid at position 389; Ala for the amino acid at
position 424; Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro,
Gln, Ser, Thr, Val, Trp, or Tyr for the amino acid at position 428;
Lys for the amino acid at position 433; Ala, Phe, His, Ser, Trp, or
Tyr for the amino acid at position 434; and His, Ile, Leu, Phe,
Thr, or Val for the amino acid at position 436 in the EU numbering
system. Meanwhile, the number of altered amino acids is not
particularly limited; such amino acid alterations include single
amino acid alteration and alteration of amino acids at two or more
sites. Combinations of amino acid alterations at two or more sites
include, for example, those described in Tables 5-1 to 5-33.
[0442] In addition to the Fc region of human IgG1 (SEQ ID NO: 13),
IgG2 (SEQ ID NO: 14), IgG3 (SEQ ID NO: 15), or IgG4 (SEQ ID NO:
16), as Fc regions included in the present invention, Fc regions
with modified Fc.gamma.R binding, which have a higher Fc.gamma.
receptor-binding activity than the Fc region of a native human IgG
in which the sugar chain bound at position 297 (EU numbering) is a
fucose-containing sugar chain, may be suitably used. Such Fc
regions with modified Fc.gamma.R binding may be produced by
modifying amino acids in the Fc region of a native human IgG.
Whether the Fc.gamma.R-binding activity of an Fc region is higher
than that of the Fc region of a native human IgG, in which the
sugar chain bound at position 297 (EU numbering) is a
fucose-containing sugar chain, can be appropriately determined
using methods such as those described above.
[0443] In the present invention, "modification of amino acids" or
"amino acid modification" of an Fc region includes modification
into an amino acid sequence which is different from that of the
starting Fc region. The starting Fc region may be any Fc region, as
long as a variant modified from the starting Fc region can bind to
human Fc.gamma. receptor in a neutral pH range. Furthermore, an Fc
region modified from a starting Fc region which had been already
modified can also be used preferably as an Fc region of the present
invention. The "starting Fc region" can refer to the polypeptide
itself, a composition comprising the starting Fc region, or an
amino acid sequence encoding the starting Fc region. Starting Fc
regions can comprise known Fc regions produced via recombination
described briefly in the section "Antibodies". The origin of
starting Fc regions is not limited, and they may be obtained from
human or any nonhuman organisms. Such organisms preferably include
mice, rats, guinea pigs, hamsters, gerbils, cats, rabbits, dogs,
goats, sheep, bovines, horses, camels and organisms selected from
nonhuman primates. In another embodiment, starting Fc regions can
also be obtained from cynomolgus monkeys, marmosets, rhesus
monkeys, chimpanzees, or humans. Starting Fc regions can be
obtained preferably from human IgG1; however, they are not limited
to any particular IgG class. This means that an Fc region of human
IgG1, IgG2, IgG3, or IgG4 can be used appropriately as a starting
Fc region, and herein also means that an Fc region of an arbitrary
IgG class or subclass derived from any organisms described above
can be preferably used as a starting Fc region. Examples of
naturally-occurring IgG variants or modified forms are described in
published documents (Curr. Opin. Biotechnol. (2009) 20 (6): 685-91;
Curr. Opin. Immunol. (2008) 20 (4), 460-470; Protein Eng. Des. Sel.
(2010) 23 (4): 195-202; International Publication Nos. WO
2009/086320, WO 2008/092117, WO 2007/041635, and WO 2006/105338);
however, they are not limited to the examples.
[0444] Examples of alterations include those with one or more
mutations, for example, mutations by substitution of different
amino acid residues for amino acids of starting Fc regions, by
insertion of one or more amino acid residues into starting Fc
regions, or by deletion of one or more amino acids from starting Fc
region. Preferably, the amino acid sequences of altered Fc regions
comprise at least a part of the amino acid sequence of a non-native
Fc region. Such variants necessarily have sequence identity or
similarity less than 100% to their starting Fc region. In a
preferred embodiment, the variants have amino acid sequence
identity or similarity about 75% to less than 100%, more preferably
about 80% to less than 100%, even more preferably about 85% to less
than 100%, still more preferably about 90% to less than 100%, and
yet more preferably about 95% to less than 100% to the amino acid
sequence of their starting Fc region. In a non-limiting embodiment
of the present invention, at least one amino acid is different
between an Fc.gamma.R-binding modified Fc region of the present
invention and its starting Fc region. Amino acid difference between
an Fc.gamma.R-binding modified Fc region of the present invention
and its starting Fc region can also be preferably specified based
on the specific amino acid differences at the above-described
specific amino acid positions according to EU numbering system.
[0445] The Fc region with modified Fc.gamma.R binding, which has a
higher Fc.gamma. receptor-binding activity than that of the Fc
region of a native human IgG in which the sugar chain bound at
position 297 (EU numbering) is a fucose-containing sugar chain,
contained in the antigen-binding molecules of the present invention
may be obtained by any method. Specifically, the Fc region with
modified Fc.gamma.R binding can be obtained by modifying amino
acids in a human IgG-type immunoglobulin, and is used as the
starting Fc region. Preferred Fc regions of IgG-type
immunoglobulins for modification include, for example, Fc regions
of human IgGs (IgG1, IgG2, IgG3, IgG4, and variants thereof).
[0446] Amino acids of any positions may be modified into other
amino acids, as long as the binding activity toward the Fc.gamma.
receptor is higher than that of the Fc region of a native human
IgG, in which the sugar chain bound at position 297 (EU numbering)
is a fucose-containing sugar chain. When the antigen-binding
molecule contains a human IgG1 Fc region as the human Fc region, it
preferably contains a modification that yields the effect of a
higher Fc.gamma. receptor-binding activity than that of the Fc
region of a native human IgG, in which the sugar chain bound at
position 297 (EU numbering) is a fucose-containing sugar chain.
Such amino acid modifications have been reported, for example, in
international publications such as WO2007/024249, WO2007/021841,
WO2006/031370, WO2000/042072, WO2004/029207, WO2004/099249,
WO2006/105338, WO2007/041635, WO2008/092117, WO2005/070963,
WO2006/020114, WO2006/116260, and WO2006/023403.
[0447] Examples of such amino acids that can be modified include at
least one or more amino acids selected from the group of positions
221, 222, 223, 224, 225, 227, 228, 230, 231, 232, 233, 234, 235,
236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247, 249, 250,
251, 254, 255, 256, 258, 260, 262, 263, 264, 265, 266, 267, 268,
269, 270, 271, 272, 273, 274, 275, 276, 278, 279, 280, 281, 282,
283, 284, 285, 286, 288, 290, 291, 292, 293, 294, 295, 296, 297,
298, 299, 300, 301, 302, 303, 304, 305, 311, 313, 315, 317, 318,
320, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333,
334, 335, 336, 337, 339, 376, 377, 378, 379, 380, 382, 385, 392,
396, 421, 427, 428, 429, 434, 436, and 440 (EU numbering). By
modifying these amino acids, one can obtain Fc regions (Fc regions
with modified Fc.gamma.R binding) with a higher binding activity
toward the Fc.gamma. receptor than the binding activity toward the
Fc.gamma. receptor of the Fc region of a native human IgG, in which
the sugar chain bound at position 297 (EU numbering) is a
fucose-containing sugar chain.
[0448] Examples of particularly preferred modifications for use in
the present invention include at least one or more amino acid
modifications in the Fc region selected from the group of:
Lys or Tyr for the amino acid of position 221; Phe, Trp, Glu, or
Tyr for the amino acid of position 222; Phe, Trp, Glu, or Lys for
the amino acid of position 223; Phe, Trp, Glu, or Tyr for the amino
acid of position 224; Glu, Lys, or Trp for the amino acid of
position 225; Glu, Gly, Lys, or Tyr for the amino acid of position
227; Glu, Gly, Lys, or Tyr for the amino acid of position 228; Ala,
Glu, Gly, or Tyr for the amino acid of position 230; Glu, Gly, Lys,
Pro, or Tyr for the amino acid of position 231; Glu, Gly, Lys, or
Tyr for the amino acid of position 232; Ala, Asp, Phe, Gly, His,
Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for
the amino acid of position 233; Ala, Asp, Glu, Phe, Gly, His, Ile,
Lys, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the
amino acid of position 234; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys,
Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino
acid of position 235; Ala, Asp, Glu, Phe, His, Ile, Lys, Leu, Met,
Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid
of position 236; Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro,
Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of position
237; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg,
Ser, Thr, Val, Trp, or Tyr for the amino acid of position 238; Asp,
Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr,
Val, Trp, or Tyr for the amino acid of position 239; Ala, Ile, Met,
or Thr for the amino acid of position 240; Asp, Glu, Leu, Arg, Trp,
or Tyr for the amino acid of position 241; Leu, Glu, Leu, Gln, Arg,
Trp, or Tyr for the amino acid of position 243; His for the amino
acid of position 244; Ala for the amino acid of position 245; Asp,
Glu, His, or Tyr for the amino acid of position 246; Ala, Phe, Gly,
His, Ile, Leu, Met, Thr, Val, or Tyr for the amino acid of position
247; Glu, His, Gln, or Tyr for the amino acid of position 249; Glu
or Gln for the amino acid of position 250; Phe for the amino acid
of position 251; Phe, Met, or Tyr for the amino acid of position
254; Glu, Leu, or Tyr for the amino acid of position 255; Ala, Met,
or Pro for the amino acid of position 256; Asp, Glu, His, Ser, or
Tyr for the amino acid of position 258; Asp, Glu, His, or Tyr for
the amino acid of position 260; Ala, Glu, Phe, Ile, or Thr for the
amino acid of position 262; Ala, Ile, Met, or Thr for the amino
acid of position 263; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met,
Asn, Pro, Gln, Arg, Ser, Thr, Trp, or Tyr for the amino acid of
position 264; Ala, Leu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,
Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of
position 265; Ala, Ile, Met, or Thr for the amino acid of position
266; Asp, Glu, Phe, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg,
Thr, Val, Trp, or Tyr for the amino acid of position 267; Asp, Glu,
Phe, Gly, Ile, Lys, Leu, Met, Pro, Gln, Arg, Thr, Val, or Trp for
the amino acid of position 268; Phe, Gly, His, Ile, Lys, Leu, Met,
Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of
position 269; Glu, Phe, Gly, His, Ile, Leu, Met, Pro, Gln, Arg,
Ser, Thr, Trp, or Tyr for the amino acid of position 270; Ala, Asp,
Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr,
Val, Trp, or Tyr for the amino acid of position 271; Asp, Phe, Gly,
His, Ile, Lys, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for
the amino acid of position 272; Phe or Ile for the amino acid of
position 273; Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Pro,
Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of position 274;
Leu or Trp for the amino acid of position 275; Asp, Glu, Phe, Gly,
His, Ile, Leu, Met, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the
amino acid of position 276; Asp, Glu, Gly, His, Ile, Lys, Leu, Met,
Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp for the amino acid of
position 278; Ala for the amino acid of position 279; Ala, Gly,
His, Lys, Leu, Pro, Gln, Trp, or Tyr for the amino acid of position
280; Asp, Lys, Pro, or Tyr for the amino acid of position 281; Glu,
Gly, Lys, Pro, or Tyr for the amino acid of position 282; Ala, Gly,
His, Ile, Lys, Leu, Met, Pro, Arg, or Tyr for the amino acid of
position 283; Asp, Glu, Leu, Asn, Thr, or Tyr for the amino acid of
position 284; Asp, Glu, Lys, Gln, Trp, or Tyr for the amino acid of
position 285; Glu, Gly, Pro, or Tyr for the amino acid of position
286; Asn, Asp, Glu, or Tyr for the amino acid of position 288; Asp,
Gly, His, Leu, Asn, Ser, Thr, Trp, or Tyr for the amino acid of
position 290; Asp, Glu, Gly, His, Ile, Gln, or Thr for the amino
acid of position 291; Ala, Asp, Glu, Pro, Thr, or Tyr for the amino
acid of position 292; Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg,
Ser, Thr, Val, Trp, or Tyr for the amino acid of position 293; Phe,
Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or
Tyr for the amino acid of position 294; Asp, Glu, Phe, Gly, His,
Ile, Lys, Met, Asn, Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the
amino acid of position 295; Ala, Asp, Glu, Gly, His, Ile, Lys, Leu,
Met, Asn, Gln, Arg, Ser, Thr, or Val for the amino acid of position
296; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg,
Ser, Thr, Val, Trp, or Tyr for the amino acid of position 297; Ala,
Asp, Glu, Phe, His, Ile, Lys, Met, Asn, Gln, Arg, Thr, Val, Trp, or
Tyr for the amino acid of position 298; Ala, Asp, Glu, Phe, Gly,
His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Val, Trp, or Tyr
for the amino acid of position 299; Ala, Asp, Glu, Gly, His, Ile,
Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, or Trp for the
amino acid of position 300; Asp, Glu, His, or Tyr for the amino
acid of position 301; Ile for the amino acid of position 302; Asp,
Gly, or Tyr for the amino acid of position 303; Asp, His, Leu, Asn,
or Thr for the amino acid of position 304; Glu, Ile, Thr, or Tyr
for the amino acid of position 305; Ala, Asp, Asn, Thr, Val, or Tyr
for the amino acid of position 311; Phe for the amino acid of
position 313; Leu for the amino acid of position 315; Glu or Gln
for the amino acid of position 317; His, Leu, Asn, Pro, Gln, Arg,
Thr, Val, or Tyr for the amino acid of position 318; Asp, Phe, Gly,
His, Ile, Leu, Asn, Pro, Ser, Thr, Val, Trp, or Tyr for the amino
acid of position 320; Ala, Asp, Phe, Gly, His, Ile, Pro, Ser, Thr,
Val, Trp, or Tyr for the amino acid of position 322; Ile for the
amino acid of position 323; Asp, Phe, Gly, His, Ile, Leu, Met, Pro,
Arg, Thr, Val, Trp, or Tyr for the amino acid of position 324; Ala,
Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser,
Thr, Val, Trp, or Tyr for the amino acid of position 325; Ala, Asp,
Glu, Gly, Ile, Leu, Met, Asn, Pro, Gln, Ser, Thr, Val, Trp, or Tyr
for the amino acid of position 326; Ala, Asp, Glu, Phe, Gly, His,
Ile, Lys, Leu, Met, Asn, Pro, Arg, Thr, Val, Trp, or Tyr for the
amino acid of position 327; Ala, Asp, Glu, Phe, Gly, His, Ile, Lys,
Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino
acid of position 328; Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Met,
Asn, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of
position 329; Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn,
Pro, Arg, Ser, Thr, Val, Trp, or Tyr for the amino acid of position
330; Asp, Phe, His, Ile, Leu, Met, Gln, Arg, Thr, Val, Trp, or Tyr
for the amino acid of position 331; Ala, Asp, Glu, Phe, Gly, His,
Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr, Val, Trp, or Tyr for
the amino acid of position 332; Ala, Asp, Glu, Phe, Gly, His, Ile,
Leu, Met, Pro, Ser, Thr, Val, or Tyr for the amino acid of position
333; Ala, Glu, Phe, Ile, Leu, Pro, or Thr for the amino acid of
position 334; Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Pro, Arg,
Ser, Val, Trp, or Tyr for the amino acid of position 335; Glu, Lys,
or Tyr for the amino acid of position 336; Glu, His, or Asn for the
amino acid of position 337; Asp, Phe, Gly, Ile, Lys, Met, Asn, Gln,
Arg, Ser, or Thr for the amino acid of position 339; Ala or Val for
the amino acid of position 376; Gly or Lys for the amino acid of
position 377; Asp for the amino acid of position 378; Asn for the
amino acid of position 379; Ala, Asn, or Ser for the amino acid of
position 380; Ala or Ile for the amino acid of position 382; Glu
for the amino acid of position 385; Thr for the amino acid of
position 392; Leu for the amino acid of position 396; Lys for the
amino acid of position 421; Asn for the amino acid of position 427;
Phe or Leu for the amino acid of position 428; Met for the amino
acid of position 429; Trp for the amino acid of position 434; Ile
for the amino acid of position 436; and Gly, His, Ile, Leu, or Tyr
for the amino acid of position 440; as indicated by EU numbering.
Furthermore, the number of amino acids to be modified is not
particularly limited, and amino acid at only one site may be
modified or amino acids at two or more sites may be modified.
Examples of combinations for the amino acid modifications at two or
more sites include those described in Table 6 (Tables 6-1 to
6-3).
[0449] Among the Fc regions suitable for use in the present
invention, a suitable example of an Fc region that has a higher
binding activity toward an inhibitory Fc.gamma. receptor than
toward an activating Fc.gamma. receptor (i.e., having a selective
binding activity toward an inhibitory Fc.gamma. receptor), which is
used as a non-limiting embodiment of an Fc region with the property
of having a higher binding activity toward a specific Fc.gamma.
receptor than toward other Fc.gamma. receptors (i.e., an Fc region
having a selective Fc.gamma. receptor-binding activity), is an Fc
region with one or more of the following modifications in the amino
acids (indicated by EU numbering) of the aforementioned Fc region:
the amino acid at position 238 is modified to Asp and the amino
acid at position 328 is modified to Glu. The Fc regions and
modifications described in US2009/0136485 or WO 2012/115241 may be
selected appropriately as the Fc region having a selective binding
activity to an inhibitory Fc.gamma. receptor.
[0450] In a non-limiting embodiment of the present invention, a
suitable example is an Fc region in which one or more of the amino
acids indicated by EU numbering at positions 238 and 328 according
to EU numbering are respectively modified to Asp or Glu in the
aforementioned Fc region.
[0451] Furthermore, in a non-limiting embodiment of the present
invention, suitable examples of the Fc regions are those with
substitution of Asp for Pro at position 238 (EU numbering), and one
or more modifications selected from among Trp for the amino acid of
position 237, Phe for the amino acid of position 237, Val for the
amino acid of position 267, Gln for the amino acid of position 267,
Asn for the amino acid of position 268, Gly for the amino acid of
position 271, Leu for the amino acid of position 326, Gln for the
amino acid of position 326, Glu for the amino acid of position 326,
Met for the amino acid of position 326, Asp for the amino acid of
position 239, Ala for the amino acid of position 267, Trp for the
amino acid of position 234, Tyr for the amino acid of position 234,
Ala for the amino acid of position 237, Asp for the amino acid of
position 237, Glu for the amino acid of position 237, Leu for the
amino acid of position 237, Met for the amino acid of position 237,
Tyr for the amino acid of position 237, Lys for the amino acid of
position 330, Arg for the amino acid of position 330, Asp for the
amino acid of position 233, Asp for the amino acid of position 268,
Glu for the amino acid of position 268, Asp for the amino acid of
position 326, Ser for the amino acid of position 326, Thr for the
amino acid of position 326, Ile for the amino acid of position 323,
Leu for the amino acid of position 323, Met for the amino acid of
position 323, Asp for the amino acid of position 296, Ala for the
amino acid of position 326, Asn for the amino acid of position 326,
and Met for the amino acid of position 330, according to EU
numbering.
[0452] Appropriate known methods such as site-directed mutagenesis
(Kunkel et al. (Proc. Natl. Acad. Sci. USA (1985) 82, 488-492)) and
overlap extension PCR can be applied to alter the amino acids of Fc
regions. Furthermore, various known methods can also be used as an
amino acid alteration method for substituting amino acids with
those other than natural amino acids (Annu. Rev. Biophys. Biomol.
Struct. (2006) 35, 225-249; Proc. Natl. Acad. Sci. U.S.A. (2003)
100 (11), 6353-6357). For example, it is also preferable to use a
cell-free translation system (Clover Direct (Protein Express))
comprising tRNAs in which an unnatural amino acid is linked to an
amber suppressor tRNA, which is complementary to UAG stop codon
(amber codon).
[0453] In an embodiment of variants of the present invention,
polynucleotides encoding antigen-binding molecules which have a
heavy chain where a polynucleotide encoding an Fc region modified
to have an amino acid mutation as described above is linked in
frame to a polynucleotide encoding the above-described
antigen-binding domain whose binding activity varies depending on a
selected condition.
[0454] The present invention provides methods for producing
antigen-binding molecules, comprising collecting the
antigen-binding molecules from culture media of cells introduced
with vectors in which a polynucleotide encoding an Fc region is
operably linked in frame to a polynucleotide encoding an
antigen-binding domain whose binding activity varies depending on
ion concentration condition. Furthermore, the present invention
also provides methods for producing antigen-binding molecules,
comprising collecting the antigen-binding molecules from culture
media of cells introduced with vectors constructed by operably
linking a polynucleotide encoding an antigen-binding domain whose
binding activity varies depending on ion concentration condition to
a polynucleotide encoding an Fc region which is in advance operably
linked to a vector.
[0455] In the production methods of the present invention, one can
add suitable modifications to the domain after obtaining an
"antigen-binding domain confirmed to form an immune complex", as
long as formation of the immune complex is enabled. Furthermore, a
method for producing an antigen-binding molecule is also provided,
which includes collecting the antigen-binding molecule from a
culture solution of cells containing a vector that carries a
polynucleotide encoding an antigen-binding molecule that has a
heavy chain in which a polynucleotide encoding an antigen-binding
domain capable of forming an immune complex and modified this way
is linked in frame to a polynucleotide encoding an Fc region
variant in which the above-mentioned amino acid mutations have been
added.
Pharmaceutical Compositions
[0456] When a conventional neutralizing antibody against a soluble
antigen is administered, the plasma retention of the antigen is
expected to be prolonged by binding to the antibody. In general,
antibodies have a long half-life (one week to three weeks) while
the half-life of antigen is generally short (one day or less).
Meanwhile, antibody-bound antigens have a significantly longer
half-life in plasma as compared to when the antigens are present
alone. For this reason, administration of existing neutralizing
antibody results in an increased antigen concentration in plasma.
Such cases have been reported with various neutralizing antibodies
that target soluble antigens including, for example, IL-6 (J.
Immunotoxicol. (2005) 3, 131-139), amyloid beta (mAbs (2010) 2 (5),
1-13), MCP-1 (ARTHRITIS & RHEUMATISM (2006) 54, 2387-2392),
hepcidin (AAPS J. (2010) 4, 646-657), and sIL-6 receptor (Blood
(2008) 112 (10), 3959-64). Administration of existing neutralizing
antibodies has been reported to increase the total plasma antigen
concentration to about 10 to 1,000 times (the level of increase
varies depending on antigen) the base line. Herein, the total
plasma antigen concentration refers to a concentration as a total
amount of antigen in plasma, i.e., the sum of concentrations of
antibody-bound and antibody-unbound antigens. An increase in the
total plasma antigen concentration is undesirable for such antibody
pharmaceuticals that target a soluble antigen. The reason is that
the antibody concentration has to be higher than at least the total
plasma antigen concentration to neutralize the soluble antigen.
Specifically, "the total plasma antigen concentration is increased
to 10 to 1,000 times" means that, in order to neutralize the
antigen, the plasma antibody concentration (i.e., antibody dose)
has to be 10 to 1,000 times higher as compared to when increase in
the total plasma antigen concentration does not occur. Conversely,
if the total plasma antigen concentration can be reduced by 10 to
1,000 times as compared to the existing neutralizing antibody, the
antibody dose can also be reduced to similar extent. Thus,
antibodies capable of decreasing the total plasma antigen
concentration by eliminating the soluble antigen from plasma are
highly useful as compared to existing neutralizing antibodies.
[0457] Without being restricted to a particular theory, the
following mechanism is an example of a mechanism that can cause
elimination of the antigenic binding units from the plasma by an
antigen-binding molecule of the present invention, which contains
(i) an Fc region and (ii) an antigen-binding domain of which
antigen-binding activity varies depending on an ion concentration
condition, and where the antigen-binding molecule can form an
immune complex containing two or more of the antigen-binding
molecules and antigens having two or more antigenic binding units.
When the antigenic binding unit is one unit (i.e., a homomonomer),
such as in sIL-6R, two molecules ((i.e., two antigenic binding
units) of antigens bind to a single antibody molecule containing a
divalent antibody-binding domain, and a complex of one anti-sIL-6R
antibody molecule and two antigen molecules containing two
antigenic binding units is formed. Therefore, this type of
antigen-antibody complex has only one Fc region (native IgG1 Fc
region) as shown in FIG. 1. Since this complex binds to two
molecules of FcRn or one molecule of Fc.gamma.R via a single Fc
region, the affinity toward these receptors is the same as that of
a general IgG antibody, and uptake into cells is thought to occur
mostly non-specifically.
[0458] On the other hand, when the antigenic binding unit is two
units as in human IgA, which is a dimer of a hetero-complex of
heavy chains and light chains, there are two units of epitopes to
which the antigen-binding domains will bind in the antigenic
binding unit. However, when a bivalent anti-IgA antibody (i.e., the
antigen-binding domains contained in one anti-IgA antibody molecule
bind to a same epitope) binds to its antigen, IgA, it is thought
that binding of each of the bivalent antigen-binding domains
contained in the single anti-IgA antibody molecule to each of the
two epitope units present on a single IgA molecule is difficult in
view of the position of the epitopes. As a result, it is thought
that separate anti-IgA antibody molecules bind to the two antigenic
binding units present in the two molecules of IgA that bind to the
bivalent antigen-binding domains present in a single anti-IgA
antibody molecule, and consequently, antigen-antibody complexes
(immune complexes) containing at least four molecules (that is, two
IgA molecules which are antigen molecule, and two anti-IgA antibody
molecules which are antigen-binding molecules) are formed.
[0459] When an antigen-binding molecule such as an antibody binding
to an antigen molecule that contains two or more antigenic binding
units, forms a large immune complex which is at least a tetramer,
the immune complex can bind strongly with avidity through at least
two or more multivalent Fc regions to Fc.gamma.R, FcRn, complement
receptors, and such. Therefore, as shown in FIG. 7, the complex is
taken up efficiently into cells expressing these receptors. On the
other hand, since the Fc region-mediated affinity toward these
receptors of immune complexes formed of antigen molecules and
antigen-binding molecules that for example bind to (monomeric)
antigen molecules containing one antigenic binding unit is
insufficient as mentioned above, the immune complexes are taken up
mostly nonspecifically (less efficiently as compared to uptake
mediated by avidity binding) into cells expressing these receptors,
as shown in FIG. 1. Thus, the uptake is more inefficient than
uptake mediated by avidity binding.
[0460] When the antigen-binding molecule such as an antibody that
binds to an antigen molecule containing two or more antigenic
binding units is an antibody that contains antigen-binding domains
of which antigen-binding varies depending on an ion concentration
condition such as pH- or Ca-dependent binding and which forms an
antigen-antibody complex (immune complex) containing at least four
molecules (two antigen molecules and two antibody molecules) in the
plasma, once the immune complex is taken up into cells, the
antigens dissociate from the antibodies in the endosomes where the
ion concentration conditions are different from those in the
plasma. Therefore, the immune complex formation is dissolved in the
endosomes of cells that have taken up the immune complexes. Since
the dissociated antigens cannot bind to FcRn in the endosomes, they
are degraded after translocating to the lysosomes. On the other
hand, the antigen-dissociated antibodies are thought to be recycled
to the plasma after binding to FcRn in the endosomes (FIG. 7).
[0461] As described above, if an antibody that contains a native
IgG1-type constant region against a multimeric antigen containing
two or more antigenic binding units and shows pH- or Ca-dependent
binding can form a large immune complex and bind to Fc.gamma.R,
FcRn, complement receptors, and such with avidity, it is thought
that antigen elimination alone can be selectively and greatly
accelerated. It is thought that when GA2-IgG1 which binds to human
IgA is administered, such large immune complexes are formed.
Indeed, as shown in Example 3, GA2-IgG1-Fc.gamma.R(-) formed by
introducing into GA2-IgG1 modifications that impair binding to
mouse Fc.gamma.R could not substantially accelerate elimination of
human IgA like GA2-IgG 1 when compared to human IgA alone, and
showed an equivalent level of elimination as human IgA alone.
Therefore, the reason that GA2-IgG1 could accelerate elimination of
human IgA is because the immune complex containing GA2-IgG1 and
human IgA, which is a multimeric antigen containing two or more
antigenic binding units, binds with avidity to Fc.gamma.R and is
quickly taken up into cells expressing Fc.gamma.R. The IgA that
dissociates from the immune complex in the endosomes of cells that
have taken up the immune complex is degraded in the lysosomes. At
the same time, the IgA-dissociated antibody, which was bound to
FcRn in the endosomes, is subsequently recycled to the plasma and
can bind again to IgA in the plasma. Elimination of human IgA in
the plasma is thought to be greatly accelerated in this manner. A
method using an amino-acid-variant of the Fc region which binds to
FcRn in the pH neutral range is described in WO2011/122011 as a
method for accelerating elimination of antigens from the plasma.
The present invention is useful as a method for accelerating the
elimination from the plasma of multimeric antigens containing two
or more antigenic binding units without using the above-mentioned
variants, and as shown in GA2-N434W, can further accelerate the
elimination of the multimeric antigens containing two or more
antigenic binding units from the plasma through combination with
the above-mentioned variants. Moreover, multimeric antigens
containing two or more antigenic binding units may be eliminated,
other than from the plasma, from the interstitial fluid, synovial
fluid, peritoneal fluid, pleural fluid, and pericardial fluid, as
long as the cells contacting interstitial fluid, synovial fluid,
peritoneal fluid, pleural fluid, or pericardial fluid express
Fc.gamma.R or FcRn. A non-limiting embodiment of such cells
includes immune cells and such present in the interstitial fluid,
synovial fluid, peritoneal fluid, pleural fluid, and pericardial
fluid.
[0462] Specifically, the present invention also relates to
pharmaceutical compositions comprising antigen-binding molecules of
the present invention, antigen-binding molecules produced by
alteration methods of the present invention, or antigen-binding
molecules produced by production methods of the present invention.
Antigen-binding molecules of the present invention or
antigen-binding molecules produced by production methods of the
present invention are useful as pharmaceutical compositions since
they, when administered, have the strong effect to reduce the
plasma antigen concentration as compared to typical antigen-binding
molecules, and exhibit the improved in vivo immune response,
pharmacokinetics, and others in animals administered with the
molecules. The pharmaceutical compositions of the present invention
may comprise pharmaceutically acceptable carriers.
[0463] In the present invention, pharmaceutical compositions
generally refer to agents for treating or preventing, or testing
and diagnosing diseases.
[0464] The pharmaceutical compositions of the present invention can
be formulated by methods known to those skilled in the art. For
example, they can be used parenterally, in the form of injections
of sterile solutions or suspensions including water or other
pharmaceutically acceptable liquid. For example, such compositions
can be formulated by mixing in the form of unit dose required in
the generally approved medicine manufacturing practice, by
appropriately combining with pharmacologically acceptable carriers
or media, specifically with sterile water, physiological saline,
vegetable oil, emulsifier, suspension, surfactant, stabilizer,
flavoring agent, excipient, vehicle, preservative, binder, or such.
In such formulations, the amount of active ingredient is adjusted
to obtain an appropriate amount in a pre-determined range.
[0465] Sterile compositions for injection can be formulated using
vehicles such as distilled water for injection, according to
standard formulation practice. Aqueous solutions for injection
include, for example, physiological saline and isotonic solutions
containing dextrose or other adjuvants (for example, D-sorbitol,
D-mannose, D-mannitol, and sodium chloride). It is also possible to
use in combination appropriate solubilizers, for example, alcohols
(ethanol and such), polyalcohols (propylene glycol, polyethylene
glycol, and such), non-ionic surfactants (polysorbate 80.TM.,
HCO-50, and such).
[0466] Oils include sesame oil and soybean oils. Benzyl benzoate
and/or benzyl alcohol can be used in combination as solubilizers.
It is also possible to combine buffers (for example, phosphate
buffer and sodium acetate buffer), soothing agents (for example,
procaine hydrochloride), stabilizers (for example, benzyl alcohol
and phenol), and/or antioxidants. Appropriate ampules are filled
with the prepared injections.
[0467] The pharmaceutical compositions of the present invention are
preferably administered parenterally. For example, the compositions
in the dosage form for injections, transnasal administration,
transpulmonary administration, or transdermal administration are
administered. For example, they can be administered systemically or
locally by intravenous injection, intramuscular injection,
intraperitoneal injection, subcutaneous injection, or such.
[0468] Administration methods can be appropriately selected in
consideration of the patient's age and symptoms. The dose of a
pharmaceutical composition containing an antigen-binding molecule
can be, for example, from 0.0001 to 1,000 mg/kg for each
administration. Alternatively, the dose can be, for example, from
0.001 to 100,000 mg per patient. However, the present invention is
not limited by the numeric values described above. The doses and
administration methods vary depending on the patient's weight, age,
symptoms, and such. Those skilled in the art can set appropriate
doses and administration methods in consideration of the factors
described above.
[0469] Furthermore, the present invention provides kits for use in
the methods of the present invention, which comprise at least an
antigen-binding molecule of the present invention. In addition to
the above, pharmaceutically acceptable carriers, media, instruction
manuals describing the using method, and such may be packaged into
the kits.
[0470] Furthermore, the present invention relates to pharmaceutical
agents for eliminating, from the plasma, complexes containing two
or more antigenic binding units and two or more antigen-binding
molecules present in the plasma, which contain as an active
ingredient the antigen-binding molecules of the present invention
or the antigen-binding molecules produced by the production methods
of the present invention.
[0471] The present invention relates to methods for treating a
disease, which includes administering to subjects (patients, human
subjects, etc.) the antigen-binding molecules of the present
invention or the antigen-binding molecules produced by the
production methods of the present invention. A non-limiting example
of the disease includes cancer and inflammatory diseases.
[0472] The present invention also relates to use of the
antigen-binding molecules of the present invention or the
antigen-binding molecules produced by the production methods of the
present invention in the manufacture of a pharmaceutical agent for
eliminating from the plasma complexes containing two or more
antigenic binding units and two or more antigen-binding molecules
present in the plasma.
[0473] The present invention further relates to use of the
antigen-binding molecules of the present invention or the
antigen-binding molecules produced by the production methods of the
present invention for eliminating, from the plasma, complexes
containing two or more antigenic binding units and two or more
antigen-binding molecules present in the plasma.
[0474] In addition, the present invention relates to
antigen-binding molecules of the present invention and
antigen-binding molecules produced by the production methods of
present invention for use in the methods of the present
invention.
[0475] Amino acids contained in the amino acid sequences of the
present invention may be post-translationally modified (for
example, the modification of an N-terminal glutamine into a
pyroglutamic acid by pyroglutamylation is well-known to those
skilled in the art). Naturally, such post-translationally modified
amino acids are included in the amino acid sequences in the present
invention.
[0476] All prior art documents cited in this specification are
incorporated herein by reference.
EXAMPLES
[0477] Herein below, the present invention will be specifically
described with reference to examples, however, it is not to be
construed as being limited thereto.
Example 1
Preparation of Antibodies that Bind to Human IgA in a
Calcium-Dependent Manner
[0478] 1-1. Preparation of Human IgA (hIgA)
[0479] Human IgA (hereinafter also abbreviated as "hIgA") was
prepared as an antigen by using the following recombinant
techniques. hIgA was expressed by culturing host cells carrying
recombinant vectors inserted with H (WT)-IgA1 (SEQ ID NO: 49) and L
(WT) (SEQ ID NO: 50) and purified by a method known to those
skilled in the art using ion-exchange chromatography and gel
filtration chromatography.
1-2. Antibodies with Calcium-Dependent Binding
[0480] H54/L28-IgG1 described in International Publication No. WO
2009/125825 is a humanized anti-IL-6 receptor antibody. Fv4-IgG1 is
a humanized anti-IL-6 receptor antibody that results from
conferring H54/L28-IgG1 with the property of binding to soluble
human IL-6 receptor in a pH-dependent manner (i.e., of binding
under neutral condition and dissociating under acidic condition).
The in vivo test described in International Publication No. WO
2009/125825 using mice demonstrated that elimination of soluble
human IL-6 receptor is greatly accelerated in a group administered
with a mixture of Fv4-IgG1 and soluble human IL-6 receptor as
antigen as compared to a group administered with a mixture of
H54/L28-IgG1 and soluble human IL-6 receptor as antigen.
[0481] Soluble human IL-6 receptor bound to a general antibody that
binds to soluble human IL-6 receptor is recycled to the plasma
along with the antibody via FcRn. Meanwhile, an antibody that binds
to soluble human IL-6 receptor in a pH-dependent manner dissociates
under the acidic conditions in the endosome from the soluble human
IL-6 receptor that was bound to the antibody. The dissociated
soluble human IL-6 receptor is degraded in the lysosome. Thus, this
can greatly accelerate the elimination of soluble human IL-6
receptor from the plasma. Moreover, the antibody that binds to
soluble human IL-6 receptor in a pH-dependent manner is recycled to
the plasma via FcRn after it has dissociated from the soluble human
IL-6 receptor, so that the recycled antibody can bind to a soluble
human IL-6 receptor again. By repeating this cycle (Antibody that
bound to antigens is taken up into cells>the antigens are
dissociated from the antibody>the antigens are degraded and the
antibody is recycled back to plasma), a single antibody molecule
can repeatedly bind to soluble human IL-6 receptors multiple times
(FIG. 1).
[0482] Meanwhile, as described in International Publication No. WO
2009/125825, H54/L28-IgG1 is a humanized anti-IL-6 receptor
antibody and Fv4-IgG1 is a humanized anti-IL-6 receptor antibody
that results from conferring H54/L28-IgG1 with the property of
binding to soluble human IL-6 receptor in a pH-dependent manner
(i.e., binding under neutral condition and dissociating under
acidic condition). Fv4-IgG1-v2 is a humanized anti-IL-6 receptor
antibody in which FcRn binding is increased over Fv4-IgG1 under
neutral conditions. The in vivo test described in International
Publication No. WO 2011/122011 using mice demonstrated that
elimination of soluble human IL-6 receptor is greatly accelerated
in a group administered with a mixture of Fv4-IgG1-v2 and soluble
human IL-6 receptor as antigen as compared to a group administered
with a mixture of Fv4-IgG1 and soluble human IL-6 receptor as
antigen. Thus, it was reported that, by enhancing the binding
toward FcRn under neutral condition (pH7.4) of an antibody that
binds to antigens in a pH-dependent manner, the effect of the
enhanced modified antibody to repeatedly bind to antigens and the
effect of promoting elimination of antigens from the plasma can be
further improved, and antigens can be eliminated from the plasma
through administration of the antibody FIG. 2.
[0483] In the actions of the antibodies that bind to antigens in a
pH-dependent manner shown in FIGS. 1 and 2, the property of the
antibodies to strongly bind to antigens in the plasma and to
dissociate from the antigens in the endosomes based on the
environmental difference between the plasma and endosomes, i.e., pH
difference (pH 7.4 in the plasma; pH 6.0 in the endosomes), is
used. The properties of environmental factors in the plasma and
endosomes as well as the degree of their difference are important
for using such differences to the antigen-binding ability of the
antibody that binds in a pH-dependent manner in the plasma and
endosomes. A pH difference corresponds to a difference in proton
concentration. Specifically, the proton concentration in the plasma
(pH 7.4) is about 40 nM, while the proton concentration in the
endosome (pH 6.0) is about 1,000 nM; thus, the proton concentration
which is regarded as an environmental factor in the plasma and
endosome differs by about 25 times.
[0484] Moreover, the present inventors conceived that, in order to
achieve the actions illustrated in FIGS. 1 and 2 by different
embodiments or to achieve these embodiments in combination, it
would be beneficial to use an antibody that binds to antigens
depending on an environmental factor with a large difference in the
plasma and endosome, other than the difference in proton
concentration. Thus, the inventors searched for an environmental
factor whose concentration is considerably different between the
plasma and the endosome, and as a result, found calcium. The
ionized calcium concentration is about 1.1 mM to 1.3 mM in the
plasma and about 3 .mu.M in the endosome; thus, the difference in
the concentration of calcium ion, which is regarded as an
environmental factor in the plasma and endosome, is about 400 times
and was found to be greater than the difference in proton
concentration (25 times). Thus, it was considered that, by using an
antibody that binds to an antigen under a high calcium
concentration condition (1.1 mM to 1.3 mM) and dissociates from the
antigen under a low calcium concentration condition (3 .mu.M), the
antibody could dissociate from the antigen in the endosome at an
equivalent or higher level as compared to an antibody that binds to
the antigen in a pH-dependent manner.
1-3. Expression and Purification of Antibodies that Bind to
hIgA
[0485] GA1-IgG1 (heavy chain SEQ ID NO: 37; light chain SEQ ID NO:
38) and GA2-IgG1 (heavy chain SEQ ID NO: 39; light chain SEQ ID NO:
40) are antibodies that bind to hIgA. The DNA sequences encoding
GA1-IgG1 (heavy chain SEQ ID NO: 37; light chain SEQ ID NO: 38) and
GA2-IgG1 (heavy chain SEQ ID NO: 39; light chain SEQ ID NO: 40)
were inserted into animal cell expression plasmids by a method
known to those skilled in the art. The antibodies were expressed by
the method described below. Cells of human fetal kidney
cell-derived line FreeStyle 293-F (Invitrogen) were suspended in
FreeStyle 293 Expression Medium (Invitrogen). The cell suspension
was seeded into a 6-well plate (3 mL/well) at a cell density of
1.33.times.10.sup.6 cells/ml. Then, the constructed plasmids were
introduced into the cells by a lipofection method. The cells were
cultured for four days in a CO.sub.2 incubator (37.degree. C., 8%
CO.sub.2, 90 rpm). The antibodies were purified from the isolated
culture supernatants by a method known to those skilled in the art
using rProtein A Sepharose.TM. Fast Flow (Amersham Biosciences).
The absorbance (wavelength: 280 nm) of the purified antibody
solutions was measured using a spectrophotometer. The antibody
concentrations were determined from the measured values using the
absorption coefficient calculated by the PACE method (Protein
Science (1995) 4, 2411-2423).
1-4. Assessment of Obtained Antibodies for Calcium-Dependent
hIgA-Binding Activity
[0486] The antibodies isolated as described in 1-3 were assessed
for their hIgA-binding activity (dissociation constant K.sub.D (M))
using Biacore T200 (GE Healthcare). Running buffers used in the
measurement were:
0.05% tween20/20 mmol/L ACES/150 mmol/L NaCl (pH 7.4 or 5.8)
containing 3 .mu.M or 1.2 mM CaCl.sub.2; and 0.05% tween20/20
mmol/L ACES/150 mmol/L NaCl (pH 8.0) containing 0.1 .mu.M or 10 mM
CaCl.sub.2.
[0487] The antibody was allowed to bind to Sensor chip CM5 (GE
Healthcare) immobilized with a suitable amount of recombinant
Protein A/G (Thermo Scientific) by the amino coupling method. Then,
an appropriate concentration of hIgA (described in 1-1) was
injected as an analyte to allow interaction with the antibody on
the sensor chip. The measurement was carried out at 37.degree. C.
After the measurement, 10 mmol/L glycine-HCl (pH 1.5) was injected
to regenerate the sensor chip. The dissociation constant K.sub.D
(M) was calculated from the measurement result by curve fitting
analysis and equilibrium parameter analysis using Biacore T200
Evaluation Software (GE Healthcare). The result and obtained
sensorgrams are shown in Table 7 and FIG. 3, respectively. It was
revealed that GA2-IgG1 bound strongly to hIgA at a Ca.sup.2+
concentration of 1.2 mM whereas the antibody bound weakly to hIgA
at a Ca.sup.2+ concentration of 3 Furthermore, at a Ca.sup.2+
concentration of 1.2 mM, GA2-IgG1 was shown to bind to human IgA
strongly at pH 7.4 but weakly at pH 5.8. More specifically,
GA2-IgG1 was revealed to bind to human IgA in a pH- and
calcium-dependent manner.
TABLE-US-00041 TABLE 7 Antibody name Conditions Fit ka kd KD[M]
GA1-IgG1 pH 8.0, 10 mM Ca 1:1binding model 1.2E+06 1.2E-01 1.0E-07
pH 8.0, 0.1 .mu.M Ca 1:1binding model 1.1E+06 2.4E-01 2.2E-07 pH
7.4, 1.2 mM Ca 1:1binding model 5.7E+05 8.4E-02 1.5E-07 pH 7.4, 3
.mu.M Ca 1:1binding model 6.4E+05 1.2E-01 1.9E-07 pH 5.8, 1.2 mM Ca
1:1binding model 6.8E+05 9.9E-02 1.4E-07 pH 5.8, 3 .mu.M Ca
1:1binding model 7.1E+05 1.1E-01 1.5E-07 GA2-IgG1 pH 7.4, 1.2 mM Ca
1:1binding model 4.0E+05 1.6E-02 3.9E-08 pH 7.4, 3 .mu.M Ca Steady
State Affinity -- -- 6.7E-06 pH 5.8, 1.2 mM Ca Steady State
Affinity -- -- 4.0E-06 pH 5.8, 3 .mu.M Ca Steady State Affinity --
-- 5.0E-06
Example 2
Preparation of Modified Antibodies that Bind to hIgA in a
Calcium-Dependent Manner
[0488] Next, to further enhance antigen (hIgA) elimination from
plasma, GA2-N434W (heavy chain SEQ ID NO: 41; light chain SEQ ID
NO: 40) was constructed by introducing amino acid substitution
N434W into GA2-IgG1, which binds to hIgA in a calcium-dependent
manner, to potentiate the binding to mouse FcRn at pH 7.4.
Furthermore, GA2-Fc.gamma.R(-) (heavy chain SEQ ID NO: 42; light
chain SEQ ID NO: 40) was constructed by introducing amino acid
substitutions L235R and S239K into GA2-IgG1 to eliminate the
Fc.gamma.R-binding affinity. The modified antibodies were expressed
by the method described above using animal expression plasmids
inserted with DNA sequences encoding GA2-N434W (heavy chain SEQ ID
NO: 41; light chain SEQ ID NO: 40) and GA2-Fc.gamma.R(-) (heavy
chain SEQ ID NO: 42; light chain SEQ ID NO: 40) by a method known
to those skilled in the art. The antibody concentrations were
determined after purification. GA2-Fc.gamma.R(-) was assessed for
its binding to various mouse Fc.gamma.R (mFc.gamma.RI,
mFc.gamma.RII, mFc.gamma.RIII, and mFc.gamma.RIV). The result
showed that GA2-Fc.gamma.R(-) did not bind to any of the
receptors.
Example 3
Assessment of the Effect of Ca-Dependent hIgA-Binding Antibodies on
Plasma Retention of an Antigen Using Normal Mice
3-1. In Vivo Test Using Normal Mice
[0489] In vivo kinetics of hIgA and anti-hIgA antibody was assessed
after administration of hIgA (human IgA; prepared as described in
Example 1) alone or in combination with an anti-hIgA antibody to
normal mice (C57BL/6J mice; Charles River Japan). An hIgA solution
(80 .mu.g/mL) or a mixture of hIgA and anti-hIgA antibody was
administered once at a dose of 10 mL/kg via the caudal vein.
Anti-hIgA antibodies used were GA1-IgG1, GA2-IgG1, GA2-N434W, and
GA2-Fc.gamma.R(-) described above.
[0490] In every mixture, the hIgA concentration was 80 .mu.g/mL.
Meanwhile, the anti-hIgA antibody concentration varied depending on
the antibody affinity for hIgA. GA1-IgG1 was prepared at 10 mg/mL;
GA2-IgG1 at 2.69 mg/mL; GA2-N434W at 1 mg/mL; and GA2-Fc.gamma.R(-)
at 2.69 mg/mL. Under the conditions described above, the majority
of hIgA is predicted to bind to the antibody since the anti-hIgA
antibody is present sufficiently in excess over hIgA. Blood was
collected from the mice five minutes, seven hours, one day, two
days, three days, and seven days after administration. The
collected blood was immediately centrifuged at 12,000 rpm and
4.degree. C. for 15 minutes to obtain plasma. The separated plasma
was stored in a freezer at -20.degree. C. or below until
measurement.
3-2. Determination of Plasma Anti-hIgA Antibody Concentration in
Normal Mice by ELISA
[0491] Anti-hIgA antibody concentrations in mouse plasma were
determined by ELISA. First, Anti-Human IgG-immobilized plates were
prepared by aliquoting Anti-Human IgG (.gamma.-chain specific)
F(ab')2 Fragment of Antibody (SIGMA) to each well of Nunc-Immuno
Plate, MaxiSorp (Nalge nunc International) and allowing the plates
to stand at 4.degree. C. overnight. Anti-hIgA antibody standard
curve samples prepared as standard solutions at plasma
concentrations of 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.01563, and
0.07813 .mu.g/mL and assay samples prepared by diluting mouse
plasma samples 100-fold or more were aliquoted into the Anti-Human
IgG-immobilized plates, and then the plates were incubated at
25.degree. C. for one hour. Next, Goat Anti-Human IgG (.gamma.
chain specific) Biotin (BIOT) Conjugate (Southern Biotechnology
Associates Inc.) was aliquoted into each well of the plates, and
then the plates were incubated at 25.degree. C. for one hour. Then,
Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was
added to each well of the plates, after which the plates were
incubated at 25.degree. C. for one hour. The chromogenic reaction
using as a substrate TMB One Component HRP Microwell Substrate
(BioFX Laboratories) was terminated with 1N sulfuric acid (Showa
Chemical), and then the reaction mixture in each well was measured
using a microplate reader to measure the absorbance at 450 nm. The
anti-hIgA antibody concentration in mouse plasma was calculated
from the absorbance of the standard curve using analysis software
SOFTmax PRO (Molecular Devices). The time course of plasma antibody
concentrations of GA1-IgG1, GA2-IgG1, GA2-N434W, and
GA2-Fc.gamma.R(-) in normal mice, which were determined by the
method described above, is shown in FIG. 4.
3-3. Determination of hIgA Concentration in Plasma by ELISA
[0492] hIgA concentrations in mouse plasma were measured by ELISA.
First, Anti-Human
[0493] IgA-immobilized plates were prepared by aliquoting Goat
anti-Human IgA Antibody (BETHYL) into each well of Nunc-Immuno
Plate, MaxiSorp (Nalge nunc International) and allowing the plates
to stand at 4.degree. C. overnight. hIgA standard curve samples
prepared as standard solutions at plasma concentrations of 0.4,
0.2, 0.1, 0.05, 0.025, 0.0125, and 0.00625 .mu.g/mL and assay
samples prepared by diluting mouse plasma samples 100-fold or more,
were aliquoted at 100 .mu.L/well into the Anti-Human
IgA-immobilized plates, after which 500 ng/mL hsIL-6R was added at
200 .mu.L/well. The resulting plates were allowed to stand at room
temperature for one hour. Next, after adding Biotinylated
Anti-human IL-6R Antibody (R&D) into each well of the plates,
the plates were incubated at room temperature for one hour. Then,
after aliquoting Streptavidin-PolyHRP80 (Stereospecific Detection
Technologies) into each well of the plates, the plates were
incubated at room temperature for one hour. The chromogenic
reaction using as a substrate TMB One Component HRP Microwell
Substrate (BioFX Laboratories) was terminated with 1N sulfuric acid
(Showa Chemical), and then the reaction mixture in each well was
measured using a microplate reader to measure the absorbance at 450
nm. The concentration in mouse plasma was calculated from the
absorbance of the standard curve using analysis software SOFTmax
PRO (Molecular Devices). The time course of plasma hIgA
concentrations in normal mice after intravenous administration, as
determined by the method described above, is shown in FIG. 5.
[0494] The result showed that the elimination of hIgA was retarded
when hIgA was administered in combination with GA1-IgG1, an
antibody that exhibits weak Ca-dependent binding (the degree of
dependency is low), as compared to when hIgA was administered
alone. In contrast, when hIgA was administered in combination with
GA2-IgG1 whose Ca-dependent binding activity is 100 or more times
greater, the elimination of hIgA was considerably accelerated, as
compared to when hIgA was administered alone. The free hIgA
concentration in plasma was determined from the plasma antibody
concentration shown in FIG. 4, the plasma hIgA concentration shown
in FIG. 5, and the KD value for each antibody shown in Table 7. The
result is shown in FIG. 6. As shown in FIG. 6, the concentration of
free antigen (hIgA) in the group administered with GA2-IgG1 which
binds to hIgA in a calcium-dependent manner was lower than that in
the group administered with GA1-IgG1. This demonstrates that the
antibody-free antigen (hIgA) (antibody unbound form) can be reduced
by accelerating antigen elimination by using antibodies that bind
in a calcium-dependent manner. Furthermore, GA2-N434W with enhanced
FcRn binding at pH 7.4 accelerated the antigen elimination more
than GA2-IgG1 did, and seven hours after administration, the plasma
hIgA concentration was below the detection limit.
Example 4
Preparation of pH-Dependent Anti-IgE Antibody
4-1. Preparation of Anti-Human IgE Antibody
[0495] To prepare pH-dependent anti-human IgE antibodies, human IgE
(heavy chain SEQ ID NO: 43; light chain SEQ ID NO: 44) (the
variable region is derived from an anti-human glypican3 antibody)
as an antigen was expressed using FreeStyle293 (Life Technologies).
Human IgE was prepared by purifying the expressed human IgE using a
conventional chromatographic method known to those skilled in the
art.
[0496] An antibody that binds to human IgE in a pH-dependent manner
and forms a large immune complex consisting of two or more
molecules of anti-IgE antibody and two or more molecules of IgE
were selected from a number of obtained antibodies. The selected
anti-human IgE antibody was expressed using human IgG1 heavy chain
constant region and human light chain constant region, and then
purified. The produced antibody was named clone 278 (heavy chain
SEQ ID NO: 45; light chain SEQ ID NO: 46).
4-2. Assessment of Anti-Human IgE Antibodies for their Binding
Activity and pH-Dependent Binding Activity
[0497] Antibodies capable of dissociating from antigens within the
endosome can be created not only by designing them so as to bind to
antigens in a pH-dependent manner, but also by designing them so as
to bind to antigens in a Ca-dependent manner. Thus, clone 278 and
the control Xolair (omalizumab; Novartis) whose IgE-binding
activity does not depend on pH/Ca were assessed for their pH
dependency and pH/Ca dependency of the human IgE (hIgE)-binding
activity.
[0498] More specifically, the hIgE-binding activities (dissociation
constant K.sub.D (M)) of clone 278 and Xolair were assessed using
Biacore T200 (GE Healthcare). Running buffers used in the assay
were:
1.2 mmol/l CaCl.sub.2/0.05% tween20, 20 mmol/l ACES, 150 mmol/l
NaCl, pH 7.4; 1.2 mmol/l CaCl.sub.2/0.05% tween20, 20 mmol/l ACES,
150 mmol/l NaCl, pH 5.8; and 3 .mu.mol/l CaCl.sub.2/0.05% tween20,
20 mmol/l ACES, 150 mmol/l NaCl, pH 5.8.
[0499] A chemically-synthetized peptide having a human glypican 3
protein-derived sequence (SEQ ID NO: 47) whose C-terminal Lys is
biotinylated (hereinafter abbreviated as "biotinylated GPC3
peptide") was added in an appropriate amount and immobilized onto
Sensor chip SA (GE Healthcare) based on the affinity between biotin
and streptavidin. Human IgE was immobilized onto the chip by
injecting it at an appropriate concentration so as to be trapped by
the biotinylated GPC3 peptide. As an analyte, clone 278 was
injected at an appropriate concentration and allowed to interact
with the human IgE on the sensor chip. Then, 10 mmol/L glycine-HCl
(pH 1.5) was injected to regenerate the sensor chip. The
interaction was always measured at 37.degree. C. The measurement
result was analyzed by curve fitting using Biacore T200 Evaluation
Software (GE Healthcare) to calculate the association rate constant
ka (1/Ms) and dissociation rate constant kd (1/s). The dissociation
constant K.sub.D (M) was calculated from the above-described
constants. Furthermore, the KD ratios in each antibody under the
conditions of [pH 5.8, 1.2 mM Ca] to [pH 7.4, 1.2 mM Ca] were
calculated to assess the pH-dependent binding, while the KD ratios
in each antibody under the conditions of [pH 5.8, 3 .mu.M Ca] to
[pH 7.4, 1.2 mM Ca] were calculated to assess the pH/Ca-dependent
binding. The result is shown in Table 8.
TABLE-US-00042 TABLE 8 pH dependency pH/Ca dependency Antibody name
KD(pH 5.8, 1.2 mM Ca)/ KD(pH 5.8, 3 .mu.M Ca)/ (abbreviation)
Buffer conditions ka (1/Ms) kd (1/s) KD (M) KD(pH 7.4, 1.2 mM Ca)
KD(pH 7.4, 1.2 mM Ca) Clone 278 pH 7.4, 1.2 mM Ca 1.5E+06 3.6E-03
2.4E-09 842.5 1636.5 pH 5.8, 1.2 mM Ca 1.2E+05 2.3E-01 2.0E-06 pH
5.8, 3 .mu.M Ca 6.2E+04 2.4E-01 3.9E-06 Xolair pH 7.4, 1.2 mM Ca
2.5E+06 1.1E-02 4.4E-09 2.3 2.9 pH 5.8, 1.2 mM Ca 2.4E+06 2.4E-02
9.9E-09 pH 5.8, 3 .mu.M Ca 1.4E+06 1.7E-02 1.3E-08
4-3. Evaluation of the Formation of Immune Complexes of Clone
278
[0500] Whether clone 278 forms large immune complexes of 2:2 or
more with human IgE under neutral condition (pH7.4) and whether the
immune complexes dissociate under acidic condition (pH5.8) were
evaluated using gel filtration chromatography. Clone 278 which was
dialyzed against 100 mM NaCl was diluted using a 20 mM Tris-HCl,
150 mM NaCl, 1.2 mM CaCl.sub.2, pH 7.4 buffer for samples under
neutral condition and using a 20 mM Bis-tris-HCl, 150 mM NaCl, 3
.mu.M CaCl.sub.2, pH 5.8 buffer for samples under acidic condition.
Mixtures in which 100 .mu.g/mL (0.60 .mu.M) hIgE(Asp6) which is a
human IgE (prepared in Example 5) and clone 278 were mixed at a
molar ratio of 1:1 or 1:6 were left for two hours or longer at room
temperature or in an autosampler at 25.degree. C., and were then
analyzed by gel filtration chromatography. A mobile phase of 20 mM
Tris-HCl, 300 mM NaCl, 1.2 mM CaCl.sub.2, pH 7.4 was used under
neutral condition, and a mobile phase of 20 mM Bis-tris-HCl, 300 mM
NaCl, 3 .mu.M CaCl.sub.2, pH 5.8 was used under acidic condition.
Analyses were carried out using a G4000SWx1 (TOSOH) column and
under conditions of a flow rate of 0.5 mL/min and 25.degree. C. The
results are shown in FIG. 9. As shown in FIG. 9, it was confirmed
that clone 278 and human IgE formed large immune complexes
consisting of tetramers (when assuming that one antibody molecule
is a monomer) or larger multimers with an apparent molecular weight
of about 670 kDa under neutral condition. Moreover, such immune
complexes were not observed under acidic condition. Thus, these
immune complexes were confirmed to dissociate in a pH-dependent
manner, similarly as in the above-described evaluation on binding
using Biacore.
[0501] From these results, it was considered that clone 278 was
able to accelerate elimination of human IgE, similarly to the
aforementioned anti-IgA antibody GA2-IgG1.
Example 5
In Vivo Assessment of Clone 278 and Xolair
[0502] 5-1. Preparation of Human IgE (hIgE(Asp6)) for In Vivo
Assessment
[0503] hIgE(Asp6) (the variable region is derived from an
anti-human glypican3 antibody), which is a human IgE for in vivo
assessment consisting of a heavy chain (SEQ ID NO: 48) and a light
chain (SEQ ID NO: 44), was produced by the same method as described
in Example 1. hIgE(Asp6) is a modified molecule resulting from
asparagine-to-aspartic acid alteration at the six N-linked
glycosylation sites in human IgE so that the heterogeneity in the
N-linked sugar chain of human IgE is not affected by time-dependent
changes in the plasma concentration of human IgE as an antigen.
5-2. Assessment of Clone 278 and Xolair for the Effect of
Accelerating Human IgE Elimination Using Normal Mice
[0504] In vivo kinetics of hIgE(Asp6) and anti-human IgE antibody
was assessed after administration of hIgE(Asp6) alone or in
combination with an anti-hIgE antibody (clone 278 and Xolair) to
C57BL/6J mice (Charles river Japan). An hIgE(Asp6) solution (20
.mu.g/mL) or a mixture of hIgE(Asp6) and anti-human IgE antibody
(the concentrations are shown in Table 9) was administered once at
a dose of 10 mL/kg via the caudal vein. Under the conditions
described above, hIgE(Asp6) is predicted to bind almost completely
to the antibody since each antibody is present sufficiently in
excess over hIgE(Asp6). Blood was collected from the mice five
minutes, two hours, seven hours, one day, two days, four or five
days, seven days, 14 days, 21 days, and 28 days after
administration. The collected blood was immediately centrifuged at
15,000 rpm and 4.degree. C. for 5 minutes to obtain plasma. The
separated plasma was stored in a freezer at -20.degree. C. or below
until measurement.
TABLE-US-00043 TABLE 9 Concentration of Concentration of hIgE(Asp6)
in the anti-hIgE antibody in the Anti-hIgE administered solution
administered solution antibody (.mu.g/mL) (.mu.g/mL) Clone 278 20
100 Xolair 20 308
5-3. Determination of Plasma hIgE(Asp6) Concentration in Normal
Mice
[0505] hIgE(Asp6) concentrations in mouse plasma were determined by
ELISA. Standard curve samples were prepared at plasma
concentrations of 192, 96, 48, 24, 12, 6, and 3 ng/mL. Xolair
(Novartis) was added at 10 .mu.g/mL to the standard curve samples
and mouse plasma assay samples to equalize the immune complex of
hIgE(Asp6) and anti-hIgE antibody. After 30 minutes of incubation
at room temperature, the standard curve samples and mouse plasma
assay samples were aliquoted into immunoplates (MABTECH)
immobilized with anti-human IgE antibody or immunoplates (Nunc F96
MicroWell Plate (Nalge nunc International)) immobilized with
anti-human IgE antibody (clone 107; MABTECH). The plates were
allowed to stand at room temperature for two hours or at 4.degree.
C. overnight. Then, human GPC3 core protein (SEQ ID NO: 51),
anti-GPC3 antibody biotinylated with NHS-PEG4-Biotin (Thermo Fisher
Scientific) (prepared in Chugai pharmaceutical Co., Ltd.), and
Sterptavidin-PolyHRP80 (Stereospecific Detection Technologies) were
reacted sequentially for one hour each. The chromogenic reaction
using as a substrate TMB One Component HRP Microwell Substrate
(BioFX Laboratories) was terminated with 1N sulfuric acid (Showa
Chemical), and then the concentration in mouse plasma was
determined by a method in which the color development is assessed
by measuring the absorbance at 450 nm using a microplate reader or
a method in which a luminescent reaction is carried out using
SuperSignal(r) ELISA Pico Chemiluminescent Substrate (Thermo Fisher
Scientific) as a substrate and the luminescence intensity is
measured with a microplate reader. The concentration in mouse
plasma was calculated from the absorbance or luminescence intensity
of the standard curve using analysis software SOFTmax PRO
(Molecular Devices). The time course of plasma hIgE(Asp6)
concentration after intravenous administration, which was
determined by the method described above, is shown in FIG. 11.
5-4. Determination of Plasma Anti-Human IgE Antibody Concentration
in Normal Mice
[0506] Anti-hIgE antibody concentrations in mouse plasma were
determined by ELISA. Standard curve samples were prepared at plasma
concentrations of 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, and 0.00625
.mu.g/mL. hIgE(Asp6) was added at 1 .mu.g/mL to the standard curve
samples and mouse plasma assay samples to equalize the immune
complex of hIgE(Asp6) and anti-hIgE antibody. After 30 minutes of
incubation at room temperature, the standard curve samples and
mouse plasma assay samples were aliquoted into immunoplates
(Nunc-Immuno Plate, MaxiSorp (Nalge nunc International))
immobilized with Anti-Human Kappa Light Chain Antibody (Bethyl
Laboratories). The plates were allowed to stand at room temperature
for two hours or at 4.degree. C. overnight. Then, Rabbit anti-Human
IgG (Fc) Secondary antibody, Biotin conjugate (Pierce
Biotechnology) and Streptavidin-Poly HRP80 (Stereospecific
Detection Technologies) were reacted sequentially for one hour
each. The chromogenic reaction using as a substrate TMB One
Component HRP Microwell Substrate (BioFX Laboratories) was
terminated with 1N sulfuric acid (Showa Chemical), and then the
concentration in mouse plasma was determined by a method in which
the color development is assessed by measuring the absorbance at
450 nm with a microplate reader. The concentration in mouse plasma
was calculated from the absorbance of the standard curve using
analysis software SOFTmax PRO (Molecular Devices). A time course of
the plasma antibody concentration after intravenous administration,
which was determined by the method described above, is shown in
FIG. 10.
[0507] The result showed that the elimination of human IgE was
retarded when human IgE was administered in combination with
Xolair, a control anti-IgE antibody, as compared to when human IgE
was administered alone. Meanwhile, the elimination of human IgE was
markedly accelerated when administered in combination with clone
278, which has a strong pH-dependent human IgE-binding activity, as
compared to when human IgE was administered alone. Specifically, it
was demonstrated that not only in the case of IgA, but also in the
case of IgE, antigen elimination was accelerated by administering
an antibody that forms a large immune complex as compared to when
the antigen is administered alone.
Example 6
Preparation of Antibody Variants that Show Calcium-Dependent hIgA
Binding
[0508] Next, with the objective of further augmenting antigen
(hIgA) elimination from plasma, Leu at position 328 (EU numbering)
in GA2-IgG1, which shows calcium-dependent hIgA binding, was
substituted with Tyr to enhance its binding to mouse Fc.gamma.R,
producing GA2-F1087 (heavy chain SEQ ID NO: 52). A DNA sequence
encoding GA2-F1087 (heavy chain SEQ ID NO: 52, and light chain SEQ
ID NO: 40) was inserted into an animal expression plasmid by a
method known to those skilled in the art. These antibody variants
were expressed using the plasmid according to the abovementioned
method, and their concentrations were determined after
purification. Antibodies containing this modification showed
greatly enhanced binding to mouse Fc.gamma.R as shown in Reference
Example 5.
Example 7
Assessment of the Effect on Plasma Retention of Antigen in Normal
Mice Administered with Ca-Dependent hIgA-Binding Antibodies
7-1. In Vivo Test Using Normal Mice
[0509] Normal mice (C57BL/6J mouse; Charles River Japan) were
administered with hIgA (human IgA: produced in Example (1-1)) alone
or co-administered with hIgA and an anti-hIgA antibody, and then
assessed for the in vivo dynamics of hIgA and the anti-hIgA
antibody. An hIgA solution (80 .mu.g/mL), or a mixed solution of
hIgA and an anti-hIgA antibody was administered once at a dose of
10 mL/kg into the tail vein. The anti-hIgA antibodies used were
GA2-IgG1 and GA2-F1087 described above.
[0510] The hIgA concentration was 80 .mu.g/mL in all cases, and the
anti-hIgA antibody concentration was 2.69 mg/mL in the mixed
solutions. The anti-hIgA antibody was sufficiently present in
excess with respect to hIgA, and therefore almost all hIgA was
considered to be bound by the antibody. From the
GA-IgG1-administered group, blood was collected from the mice 5
minutes, 7 hours, 1 day, 2 days, 3 days, and 7 days after
administration. From the GA-F1087-administered group, blood was
collected from the mice 5 minutes, 30 minutes, 1 hour, 2 hours, 1
day, 3 days, and 7 days after administration. The collected blood
samples were immediately centrifuged at 4.degree. C. and 12,000 rpm
for 15 minutes to obtain the plasma samples. The separated plasma
samples were stored in a freezer at -20.degree. C. or below until
measurement.
7-2. Measurement of the Anti-hIgA Antibody Concentration in Normal
Mouse Plasma by ELISA
[0511] The anti-hIgA antibody concentration in mouse plasma was
determined by ELISA. First, Anti-Human IgG (.gamma.-chain specific)
F(ab')2 Fragment of Antibody (SIGMA) was dispensed into each well
of Nunc-Immuno Plates, MaxiSorp (Nalge nunc International), and
allowed to stand overnight at 4.degree. C. to prepare Anti-Human
IgG-immobilized plates. Standard samples of anti-hIgA antibodies to
be used as calibration curve samples were prepared at plasma
concentrations of 0.5, 0.25, 0.125, 0.0625, 0.03125, 0.01563, and
0.007813 .mu.g/mL; and mouse plasma assay samples diluted 100-fold
or more were prepared and aliquoted into the above-mentioned
Anti-Human IgG-immobilized plates, and then the plates were
incubated at 25.degree. C. for one hour. Then, Goat Anti-Human IgG
(.gamma. chain specific) Biotin (BIOT) Conjugate (Southern
Biotechnology Associats Inc.) was dispensed into each well of the
aforementioned plates, and then the plates were incubated to allow
reaction to take place at 25.degree. C. for one hour. Then,
Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was
dispensed into each well of the aforementioned plates, and then the
plates were incubated to allow reaction to take place at 25.degree.
C. for one hour. A color development reaction was carried out using
TMB One Component HRP Microwell Substrate (BioFX Laboratories) as a
substrate. After stopping the reaction with 1N Sulfuric acid (Showa
Chemical), absorbance of the reaction solution in each well at 450
nm was measured on a microplate reader. The mouse plasma
concentration of the anti-hIgA antibody was calculated based on
absorbance from the calibration curve using the analytical software
SOFTmax PRO (Molecular Devices). Time-course changes in the plasma
concentrations of the GA2-IgG1 and GA2-F1087 antibodies in normal
mice determined by this method after intravenous administration are
shown in FIG. 12. The results confirmed that the plasma antibody
concentration of clone GA2-IgG1, which has a strong pH- and
Ca-dependent hIgA-binding activity, does not decrease greatly even
when Fc.gamma.R binding is enhanced.
7-3. Determination of the Plasma hIgA Concentration by ELISA
[0512] Concentration of hIgA in mouse plasma was determined by
ELISA. First, Goat anti-Human IgA Antibody (BETHYL) was aliquoted
into each well of a Nunc-Immuno Plate, MaxiSorp (Nalge nunc
International). The plate was left to stand at 4.degree. C.
overnight to prepare an anti-human IgA-immobilized plate. Standard
samples of hIgA to be used as calibration curve samples were
prepared at plasma concentrations of 0.4, 0.2, 0.1, 0.05, 0.025,
0.0125, and 0.00625 .mu.g/mL. Mouse plasma assay samples were
prepared at 100-fold or greater dilution. 200 .mu.l of 500 ng/ml
hsIL-6R was added to 100 .mu.L of each of the calibration curve
samples and plasma samples. The resulting mixtures were allowed to
stand at room temperature for one hour, and then the mixed
solutions were aliquoted at 100 .mu.l into the above-mentioned
anti-human IgA-immobilized plate; and this was left to stand at
room temperature for one hour. Next, a biotinylated Anti-human
IL-6R antibody (R&D) was aliquoted into each well of the
aforementioned plate, and then the plate was incubated at room
temperature for one hour to allow reaction to take place. In
addition, Streptavidin-PolyHRP80 (Stereospecific Detection
Technologies) was aliquoted into each well of the aforementioned
plate, and the plate was incubated at room temperature for one hour
to allow reaction to take place. A color development reaction was
carried out using the TMB One Component HRP Microwell Substrate
(BioFX Laboratories) as a substrate. After the reaction was
terminated with 1 N sulfuric acid (Showa Chemical), absorbance of
the reaction solutions in each well at 450 nm was measured using a
microplate reader. The concentrations in mouse plasma were
calculated based on absorbance from the calibration curve using the
analytical software SOFTmax PRO (Molecular Devices). Time-course
changes in the plasma concentration of hIgA determined by this
method in normal mice subjected to intravenous administration are
shown in FIG. 13.
[0513] The results showed that when hIgA was co-administered to
mice with GA2-IgG1, which exhibits a 100-fold or more
Ca-dependent-binding activity, the elimination of hIgA was
accelerated as compared to when hIgA was administered alone.
Furthermore, when hIgA and GA2-F1087, which has enhanced binding to
Fc.gamma.R, were administered to mice, the plasma hIgA
concentration decreased below the measurement range (0.006 .mu.g/mL
or more) one day after administration, and the elimination of
plasma hIgA was significantly accelerated compared to when GA-IgG1
was administered to mice. The above indicates that in mice
administered with an anti-hIgA antibody and hIgA forming an immune
complex, the effect of an antibody with enhanced Fc.gamma.R binding
in removing antigen (hIgA) from plasma is enhanced compared to the
effect of antigen (hIgA) removal by an antibody serving as the
source for the antibody with enhanced Fc.gamma.R binding.
Example 8
Preparation of Antibody Variants that Show pH-Dependent Human IgE
Binding
[0514] Next, with the objective of further augmenting antigen
(human IgE) elimination from plasma, Leu at position 328 (EU
numbering) in 278-IgG1, which shows pH-dependent human IgE binding,
was substituted with Tyr to enhance its binding to mouse
Fc.gamma.R, producing 278-F1087 (heavy chain SEQ ID NO: 53 and
light chain SEQ ID NO: 46). A DNA sequence encoding 278-F1087 was
inserted into an animal expression plasmid by a method known to
those skilled in the art. Antibody variants were expressed
according to the abovementioned method using animal cells
introduced with the plasmid, and their concentrations were
determined after purification.
Example 9
In Vivo Assessment of 278-IgG1
9-1. Preparation of Human IgE(hIgE(Asp6)) for In Vivo
Assessment
[0515] A method similar to the method described in Example 5-1 was
used to prepare hIgE (Asp6) (in which the variable region is that
of an anti-human Glypican 3 antibody), which is a human IgE for use
in in vivo assessment. Human IgE (Asp6) is a molecule in which
asparagine has been modified to aspartic acid at six
N-glycosylation sites of human IgE, so that the heterogeneity of
the N-glycosides of human IgE will not be affected by change in the
plasma concentration of the antigen human IgE.
9-2. Verification of the Effect of Accelerating Removal of Human
hIgE from Plasma of Normal Mouse Administered with Clone 278
[0516] It was demonstrated in Example 7 that the plasma antigen
concentration was significantly reduced in mice administered with a
molecule with enhanced binding to mouse Fc.gamma.R through
pH-dependent binding to the antigen human IgA. When mouse
Fc.gamma.R binding is enhanced, to further verify if the
elimination effect on soluble antigen in plasma can be similarly
observed in an organism administered with an antibody that shows
enhanced binding to mouse Fc.gamma.R and binds in a pH-dependent
manner to antigens other than human IgA, tests using antibodies
against the antigen human IgE were also carried out.
[0517] C57BL/6J mice (Charles River Japan) were administered with
hIgE (Asp6) alone or co-administered with hIgE (Asp6) and an
anti-hIgE antibody (278-IgG1 or 278-F1087), and then assessed for
the in vivo dynamics of hIgE (Asp6) and the anti-human IgE
antibody. An hIgE (Asp6) solution (20 .mu.g/ml) or a mixed solution
of hIgE (Asp6) and an anti-human IgE antibody (all antibody
concentrations were adjusted to be the same concentrations as shown
in Table 10) was administered once at 10 mL/kg into the tail vein.
In this case, since each antibody was sufficiently present in
excess with respect to hIgE (Asp6), nearly all hIgE (Asp6) was
considered to be bound by the antibody. Blood was collected from
the mice 5 minutes, 2 hours, 7 hours, 1 day, 2 days, 4 days, 5
days, 7 days, 14 days, and 21 days after administration in the
clone 278 (278-IgG1)-administered group. Blood was collected from
the mice 5 minutes, 30 minutes, 1 hour, 2 hours, 1 day, 3 days, 7
days, 14 days, and 21 days after administration in the
278-F1087-administered group. The collected blood samples were
immediately centrifuged at 4.degree. C. and 15,000 rpm for 5
minutes to obtain plasma samples. The separated plasma samples were
stored in a freezer set to -20.degree. C. or below until the assay
was performed.
TABLE-US-00044 TABLE 10 Concentration of Concentration of
hIgE(Asp6) in the anti-hIgE antibody in the Anti-hIgE administered
solution administered solution antibody (.mu.g/mL) (.mu.g/mL)
278-IgG1 20 100 278-F1087 20 100
9-3. Determination of the Anti-Human IgE Antibody Concentration in
Normal Mouse Plasma
[0518] The anti-hIgE antibody concentration in mouse plasma was
determined by ELISA. Calibration curve samples were prepared at
plasma concentrations of 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, and
0.00625 .mu.g/mL. To make the immune complex formed between
hIgE(Asp6) and the anti-hIgE antibody homogeneous, hIgE(Asp6) was
added at 1 .mu.g/mL to the calibration curve samples and mouse
plasma assay samples; and the 278-hIgG1-administered group and the
corresponding calibration curve samples were left to stand at room
temperature for 30 minutes. Furthermore, the 278-F1087-administered
group and the corresponding calibration curve samples were stirred
overnight at 37.degree. C. The calibration curve samples and mouse
plasma assay samples which were left to stand or stirred were
aliquoted into the Anti-Human Kappa Light Chain Antibody (Bethyl
Laboratories)-immobilized immuno plate (Nunc-Immuno Plate, MaxiSorp
(Nalge nunc International)), and were left to stand/stirred at room
temperature for two hours (samples of the 278-F1087-administered
group and calibration curve samples of 278-F1087) or left to stand
overnight at 4.degree. C. (samples of the 278-hIgG1-administered
group and the calibration curve samples of 278-hIgG1). Then, Rabbit
anti-Human IgG (Fc) Secondary antibody, Biotin conjugate (Pierce
Biotechnology), and Streptavidin-PolyHRP80 (Stereospecific
Detection Technologies) were each reacted sequentially for one
hour. A color development reaction was carried out using TMB One
Component HRP Microwell Substrate (BioFX Laboratories) as a
substrate. After the reaction was stopped with 1N Sulfuric acid
(Showa Chemical), the mouse plasma concentration was determined by
color development by measuring absorbance at 450 nm on a microplate
reader. The concentrations in mouse plasma were calculated based on
absorbance from the calibration curve using the analytical software
SOFTmax PRO (Molecular Devices). Time-course changes in the plasma
concentrations of antibodies after intravenous administration
determined by this method are shown in FIG. 14. The results
confirmed that in mice administered with a variant produced by
enhancing Fc.gamma.R binding of 278-IgG1, which has a strong
pH-dependent binding activity to human IgE, antibody concentrations
in the serum of these mice are not greatly decreased even when they
are compared to those of 278-IgG1.
9-4. Determination of the hIgE(Asp6) Concentration in Normal Mouse
Plasma
[0519] The hIgE(Asp6) concentration in mouse plasma was determined
by ELISA. Calibration curve samples were prepared at plasma
concentrations of 192, 96, 48, 24, 12, 6, and 3 ng/mL. To make the
immune complex formed between hIgE(Asp6) and the anti-hIgE antibody
homogeneous, Xolair (Novartis) was added at 10 .mu.g/mL to the
calibration curve samples and the mouse plasma assay samples for
the 278-hIgG1-administered group, and the samples were left to
stand at room temperature for 30 minutes. In the
278-F1087-administered group, 278-F1022 (heavy chain SEQ ID NO: 54
and light chain SEQ ID NO: 46, prepared in a similar manner to
Example 8) or 278-F760 (heavy chain SEQ ID NO: 55 and light chain
SEQ ID NO: 46, prepared in a similar manner to Example 8) was added
at 20 .mu.g/mL, and then stirred at 37.degree. C. for 60 hours. The
mouse plasma assay samples were aliquoted into the anti-human
IgE-immobilized immuno plate (MABTECH) or anti-human IgE (clone
107, MABTECH)-immobilized immuno plate (Nunc F96 MicroWell Plate
(Nalge nunc International)); and the samples were left to stand or
stirred at room temperature for two hours, or they were left to
stand overnight at 4.degree. C. Then, the human GPC3 core protein
(SEQ ID NO: 51), an anti-GPC3 antibody (prepared in-house)
biotinylated with NHS-PEG4-Biotin (Thermo Fisher Scientific), and
Sterptavidin-PolyHRP80 (Stereospecific Detection Technologies) were
each reacted sequentially for one hour. The concentration in mouse
plasma was determined by the method of carrying out a color
development reaction using TMB One Component HRP Microwell
Substrate (BioFX Laboratories) as a substrate, stopping the
reaction with 1N Sulfuric acid (Showa Chemical), and then measuring
color development by the absorbance at 450 nm measured on a
microplate reader; or the method of carrying out a color
development reaction using the SuperSignal(r) ELISA Pico
Chemiluminescent Substrate (Thermo Fisher Scientific) as a
substrate, and then measuring luminescence intensity on a
microplate reader. The concentrations in mouse plasma were
calculated from the luminescence intensity, or based on absorbance
from the calibration curve using the analytical software SOFTmax
PRO (Molecular Devices). Time-course changes in plasma
concentration of hIgE(Asp6) after intravenous administration
determined by this method are shown in FIG. 15.
[0520] As a result, as opposed to elimination in the case of human
IgE alone, elimination of human IgE was accelerated in mice
co-administered with human IgE and 278-IgG1, which has a strong
pH-dependent binding activity, as compared to human IgE alone.
Furthermore, human IgE elimination was significantly accelerated in
mice administered with human IgE and 278-F1087, which is produced
by enhancing Fc.gamma.R binding of 278-IgG1, compared to mice
administered with human IgE alone and mice co-administered with
human IgE and 278-IgG1. That is, antigen elimination was shown to
be accelerated not only in mice administered with anti-IgA
antibodies with enhanced Fc.gamma.R binding discussed so far, but
also in mice administered with anti-IgE antibodies with enhanced
Fc.gamma.R binding. The above-mentioned results showed that antigen
elimination can be further accelerated by enhancing Fc.gamma.R
binding in each of the immune complex-forming hIgA-anti-hIgA
antibody pair and hIgE-anti-hIgE antibody pair.
Example 10
Preparation of Antibody Variants that Show Calcium-Dependent hIgA
Binding
[0521] Next, with the objective of augmenting antigen (hIgA)
elimination from plasma, variants with enhanced binding to mouse
FcRn were produced from GA2-IgG1, which shows calcium-dependent
hIgA binding. First, with the objective of decreasing Fc.gamma.R
binding by the Fc region, GA2-F760 (heavy chain SEQ ID NO: 57) was
produced by substituting Arg for Leu at position 235 and Lys for
Ser at position 239 as indicated by EU numbering in GA2-IgG1.
Furthermore, GA2-F1331, a variant showing stronger FcRn binding at
pH 7.4 than GA2-F760, was produced by introducing the following
substitutions into GA2-F760: Arg for Gly at position 236, Tyr for
Met at position 252, Thr for Ser at position 254, Glu for Thr at
position 256, Tyr for Asn at position 434, Val for Tyr at position
436, Arg for Gln at position 438, and Glu for Ser at position 440
as indicated by EU numbering. DNA sequences encoding GA2-F760
(heavy chain SEQ ID NO: 57, and light chain SEQ ID NO: 40) and
GA2-F1331 (heavy chain SEQ ID NO: 56, and light chain SEQ ID NO:
40) were inserted into animal expression plasmids by a method known
to those skilled in the art. These plasmids were used for
expression by the method described above, and the concentrations of
these antibody variants were determined after purification. Binding
activities of GA2-F760 to mouse Fc.gamma.Rs (mFc.gamma.RI,
mFc.gamma.RII, mFc.gamma.RIII, and mFc.gamma.RIV) were determined.
As a result, GA2-F760 did not show significant binding to mouse
Fc.gamma.Rs.
Example 11
Assessment of the Effect on Plasma Retention of Antigen in Human
FcRn Transgenic Mice Administered with Ca-Dependent hIgA-Binding
Antibodies
11-1. In Vivo Testing Using Human FcRn Transgenic Mice
[0522] Human FcRn transgenic mice (B6.mFcRn-/-.hFcRn Tg line
32+/+mouse, Jackson Laboratories; Methods Mol Biol. (2010) 602,
93-104) were administered with hIgA (human IgA: produced in Example
(1-1)) alone or co-administered with hIgA and an anti-hIgA
antibody; and then the in vivo dynamics of hIgA and the anti-hIgA
antibody were assessed. An hIgA solution (80 .mu.g/mL) or a mixed
solution of hIgA and the anti-hIgA antibody was administered once
at a dose of 10 mL/kg into the tail vein. Any one of the
above-mentioned GA2-IgG1, GA2-F760, and GA2-F1331 was used as an
anti-hIgA antibody for administration.
[0523] Concentration of hIgA in the mixed solution was 80 .mu.g/mL
in all cases, and the anti-hIgA antibody concentration was 2.69
mg/mL. Since the anti-hIgA antibody was sufficiently present in
excess with respect to hIgA, most of hIgA was considered to be
bound by the antibody. Blood was collected from the mice 15
minutes, 1 hour, 2 hours, 7 hours, 1 day, 3 days, 7 days, and 14
days after antibody administration. The collected blood samples
were immediately centrifuged at 4.degree. C. and 12,000 rpm for 15
minutes to obtain the plasma samples. The separated plasma samples
were stored in a freezer at -20.degree. C. or below until
measurement.
11-2. ELISA Determination of the Anti-hIgA Antibody Concentration
in Human FcRn Transgenic Mouse Plasma
[0524] The anti-hIgA antibody concentration in mouse plasma was
determined by ELISA. First, Anti-Human IgG (.gamma.-chain specific)
F(ab')2 Fragment of Antibody (SIGMA) was dispensed into each well
of Nunc-Immuno Plates, MaxiSorp (Nalge nunc International), and
this was left to stand overnight at 4.degree. C. to prepare
Anti-Human IgG-immobilized plates. Standard samples of the
anti-hIgA antibodies to be used as calibration curve samples were
prepared at plasma concentrations of 0.5, 0.25, 0.125, 0.0625,
0.03125, 0.01563, and 0.007813 .mu.g/mL; and mouse plasma assay
samples diluted 100-fold or more were prepared and aliquoted into
the Anti-Human IgG-immobilized plates, and then the plates were
incubated at 25.degree. C. for one hour. Then, Goat Anti-Human IgG
(.gamma. chain specific) Biotin (BIOT) Conjugate (Southern
Biotechnology Associates Inc.) was dispensed into each well of the
aforementioned plates, and then the plates were incubated at
25.degree. C. for one hour to allow reaction to take place. Then,
Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was
dispensed into each well of the aforementioned plates, and then the
plates were incubated at 25.degree. C. for one hour to allow
reaction to take place. A color development reaction was carried
out using TMB One Component HRP Microwell Substrate (BioFX
Laboratories) as a substrate. After stopping the reaction with 1N
Sulfuric acid (Showa Chemical), absorbance of the reaction solution
in each well at 450 nm was measured on a microplate reader. The
anti-hIgA antibody concentration in mouse plasma was calculated
based on absorbance from the calibration curve using the analytical
software SOFTmax PRO (Molecular Devices). Time-course changes in
plasma concentrations of the GA2-F1331 and GA2-F760 antibodies in
human FcRn transgenic mice after intravenous administration
determined by this method are shown in FIG. 16.
11-3. Determination of the Plasma hIgA Concentration by ELISA
[0525] Concentration of hIgA in mouse plasma was determined by
ELISA. First, Goat anti-Human IgA antibody (BETHYL) was aliquoted
into each well of a Nunc-Immuno Plate, MaxiSorp (Nalge nunc
International). The plate was left to stand overnight at 4.degree.
C. to prepare an anti-human IgA-immobilized plate. Standard samples
of hIgA to be used as calibration curve samples were prepared at
plasma concentrations of 0.4, 0.2, 0.1, 0.05, 0.025, 0.0125, and
0.00625 .mu.g/mL. Mouse plasma assay samples were prepared at a
100-fold or greater dilution. 200 .mu.l of 500 ng/mL hsIL6R was
added to 100 .mu.L of each of the calibration curve samples and
plasma samples. The resulting mixtures were left to stand at room
temperature for one hour, and then the mixed solutions were
aliquoted at 100 .mu.l into the above-mentioned anti-human
IgA-immobilized plate; and the plate was left to stand at room
temperature for one hour. Next, Biotinylated Anti-human IL-6R
Antibody (R&D) was aliquoted into each well of the
aforementioned plate, and then the plate was incubated at RT for
one hour to allow reaction to take place. In addition,
Streptavidin-PolyHRP80 (Stereospecific Detection Technologies) was
aliquoted into each well of the aforementioned plate, and then the
plate was incubated at room temperature for one hour to allow
reaction to take place. A color development reaction was carried
out using TMB One Component HRP Microwell Substrate (BioFX
Laboratories) as a substrate. After the reaction was terminated
with 1 N sulfuric acid (Showa Chemical), absorbance of the reaction
solution in each well at 450 nm was measured using a microplate
reader. The concentrations in mouse plasma were calculated based on
absorbance from the calibration curve using the analytical software
SOFTmax PRO (Molecular Devices). Time-course changes in plasma
concentration of hIgA in human FcRn transgenic mice determined by
this method after intravenous administration are shown in FIG.
17.
[0526] The results showed that the elimination of hIgA from plasma
was markedly accelerated in mice co-administered with hIgA and
GA2-F1331, which has enhanced human FcRn binding, in comparison to
the plasma hIgA elimination in mice co-administered with hIgA and
GA2-F760, which has a low human FcRn-binding activity.
Example 12
Preparation of Antibody Variants that Show pH-Dependent Human IgE
Binding
[0527] Next, with the objective of augmenting antigen (human IgE)
elimination from plasma, variants with enhanced binding to mouse
FcRn were produced from 278-IgG1, which shows pH-dependent human
IgE binding. First, with the objective of decreasing mouse
Fc.gamma.R binding, 278-F760 (SEQ ID NO: 55) was produced by
substituting Arg for Leu at position 235 and Lys for Ser at
position 239 as indicated by EU numbering in 278-IgG1. Furthermore,
278-F1331, a variant showing stronger FcRn binding at pH 7.4 than
278-F760, was produced by introducing the following substitutions
into 278-F760: Arg for Gly at position 236, Tyr for Met at position
252, Thr for Ser at position 254, Glu for Thr at position 256, Tyr
for Asn at position 434, Val for Tyr at position 436, Arg for Gln
at position 438, and Glu for Ser at position 440 as indicated by EU
numbering. A DNA sequence encoding 278-F1331 (heavy chain SEQ ID
NO: 58, and light chain SEQ ID NO: 46) or 278-F760 (heavy chain SEQ
ID NO: 55, and light chain SEQ ID NO: 46) was inserted into an
animal expression plasmid by a method known to those skilled in the
art. These antibody variants were expressed by the above-mentioned
method using animal cells introduced with the plasmid, and their
concentrations were determined after purification.
Example 13
Assessment of the Effect on Plasma Retention of Antigen in Human
FcRn Transgenic Mice Administered with a pH-Dependent hIgE-Binding
Antibody
13-1. In Vivo Test Using Human FcRn Transgenic Mice
[0528] Human FcRn transgenic mice (B6.mFcRn-/-.hFcRn Tg line
32+/+mouse, Jackson Laboratories; Methods Mol Biol. (2010) 602,
93-104) were co-administered with hIgE(Asp6) (human IgE(Asp6):
produced in Example (5-1)), an anti-hIgE antibody (278-F760 or
278-F1331), and Sanglopor (human normal immunoglobulin, CSL
Behring); and then the in vivo dynamics of hIgE(Asp6) and the
anti-hIgE antibody were assessed. A mixed solution of hIgE(Asp6),
an anti-hIgE antibody, and Sanglopor (concentrations are shown in
Table 11) was administered once at a dose of 10 mL/kg into the tail
vein. The above-mentioned 278-F760 or 278-F1331 was used as the
anti-hIgE antibody for administration.
[0529] Since the anti-hIgE antibody was sufficiently present in
excess with respect to hIgE(Asp6), most of the hIgE(Asp6) was
considered to be bound by the antibody. Blood was collected from
the mice 5 minutes, 2 hours, 7 hours, 1 day, 2 days, 4 days, 5
days, 7 days, 14 days, 21 days, and 28 days after antibody
administration. The collected blood samples were immediately
centrifuged at 4.degree. C. and 12,000 rpm for 15 minutes to obtain
plasma samples. The separated plasma samples were stored in a
freezer at -20.degree. C. or below until measurement.
TABLE-US-00045 TABLE 11 Concentration of Concentration of
Concentration of hIgE(Asp6) in hIgE antibody in Sanglopor in the
administered the administered the administered Anti-hIgE solution
solution solution antibody (.mu.g/mL) (.mu.g/mL) (mg/mL) 278-F760
20 100 100 278-F1331 20 100 100
13-2. Determination of the Anti-hIgE Antibody Concentration in
Human FcRn Transgenic Mouse Plasma by ELISA
[0530] The anti-hIgE antibody concentration in mouse plasma was
determined by electrochemiluminescence (ECL) assay. Standard
samples of the anti-hIgE antibodies to be used as calibration curve
samples were prepared at plasma concentrations of 32, 16, 8, 4, 2,
1, 0.5, and 0.25 .mu.g/mL. Each of the calibration curve samples
and mouse plasma samples was aliquoted into each well of the
hIgE(Asp6)-immobilized ECL plate, and then the plate was incubated
at 4.degree. C. for one hour/overnight to allow reaction to take
place. Then, Goat Anti-Human IgG (.gamma. chain specific) Biotin
(BIOT) Conjugate (Southern Biotechnology Associates Inc.) was
dispensed into each well of the aforementioned plate, and then the
plate was incubated at 25.degree. C. for one hour to allow reaction
to take place. Furthermore, Streptavidin-PolyHRP80 (Stereospecific
Detection Technologies) was dispensed into each well of the
aforementioned plate, and then the plate was incubated at
25.degree. C. for one hour to allow reaction to take place. Next,
each reaction solution in the plate was made to react with a
SULFO-tagged goat anti-rabbit antibody (Meso Scale Discovery) at
room temperature for one hour. Finally, Read Buffer T (.times.4)
(Meso Scale Discovery) was dispensed into each reaction solution,
and this was immediately followed by luminescence measurement of
the reaction solution using the Sector Imager 2400 Reader (Meso
Scale Discovery). The anti-hIgE antibody concentration in mouse
plasma was calculated based on response from the calibration curve
using the analytical software SOFTmax PRO (Molecular Devices).
Time-course changes in the plasma concentrations of the 278-F1331
and 278-F760 antibodies in human FcRn transgenic mice determined by
this method after intravenous administration are shown in FIG.
18.
13-3. Determination of the Plasma hIgE(Asp6) Concentration in Human
FcRn Transgenic Mice
[0531] Concentration of hIgE(Asp6) in mouse plasma was determined
by ELISA. Calibration curve samples were prepared at plasma
concentrations of 192, 96, 48, 24, 12, 6, and 3 ng/mL. To make the
immune complex formed between hIgE(Asp6) and an anti-hIgE antibody
homogeneous, calibration curve samples and mouse plasma assay
samples were prepared by adding Xolair (Novartis) at 10 .mu.g/mL
for the 278-hIgG1-administered group, and the samples were left to
stand at room temperature for 30 minutes. The mouse plasma assay
samples were aliquoted into the anti-human IgE-immobilized immuno
plate (MABTECH) or anti-human IgE (clone 107, MABTECH)-immobilized
immuno plate (Nunc F96 MicroWell Plate (Nalge nunc International)),
and the samples were left to stand at room temperature for two
hours, or at 4.degree. C. overnight. Then, the human GPC3 core
protein (SEQ ID NO: 51), an anti-GPC3 antibody (prepared in-house)
biotinylated with NHS-PEG4-Biotin (Thermo Fisher Scientific), and
Sterptavidin-PolyHRP80 (Stereospecific Detection Technologies) were
each reacted sequentially for one hour. The concentrations in mouse
plasma were determined by the method of carrying out a color
development reaction using TMB One Component HRP Microwell
Substrate (BioFX Laboratories) as a substrate, stopping the
reaction with 1N Sulfuric acid (Showa Chemical), and then measuring
color development by the absorbance at 450 nm measured on a
microplate reader; or the method of carrying out a luminescence
reaction using SuperSignal(r) ELISA Pico Chemiluminescent Substrate
(Thermo Fisher Scientific) as a substrate, and then measuring
luminescence intensity on a microplate reader. The concentrations
in mouse plasma were calculated from luminescence intensity, or
based on absorbance from the calibration curve using the analytical
software SOFTmax PRO (Molecular Devices). Time-course changes in
the plasma concentration of hIgE(Asp6) determined by this method
after intravenous administration are shown in FIG. 19.
[0532] The results showed that mouse plasma hIgE elimination was
markedly accelerated in mice co-administered with hIgE and
278-F1331, which has enhanced human FcRn binding, in comparison to
the plasma hIgE elimination in mice co-administered with hIgE and
278-F760, which has a low human FcRn-binding activity. That is,
antigen elimination was shown to be accelerated not only in mice
administered with anti-IgE antibodies having enhanced FcRn binding
discussed so far, but also in mice administered with anti-IgE
antibodies having enhanced FcRn binding. The above-mentioned
results showed that antigen elimination can be further accelerated
by enhancing FcRn binding in each of the immune complex-forming
hIgA-anti-hIgA antibody pair and hIgE-anti-hIgE antibody pair.
Example 14
Assessment of the Effect on Plasma Retention of Antigen in Normal
Mice Co-Administered with Two Types of Anti-Human IL6 Receptor
Antibodies
14-1. Preparation of Two Types of Anti-IL6R Antibodies
[0533] Fv4-IgG1 (heavy chain SEQ ID NO: 59, and light chain SEQ ID
NO: 60) is an anti-human IL6 receptor antibody having the property
of binding to human IL6R in a pH-dependent manner (binds under a
neutral condition and dissociates under an acidic condition) as
described in WO 2011/122011. PHX-IgG1 (heavy chain SEQ ID NO: 61,
and light chain SEQ ID NO: 62) is a human IL6R-binding antibody. A
DNA sequence encoding Fv4-IgG1 (heavy chain SEQ ID NO: 59, and
light chain SEQ ID NO: 60), PHX-IgG1 (heavy chain SEQ ID NO: 61,
and light chain SEQ ID NO: 62), or PHX-F29 (heavy chain SEQ ID NO:
63, and light chain SEQ ID NO: 62) produced by making amino acid
modifications to the heavy chain constant region of PHX-IgG1 was
inserted into an animal expression plasmid by a method known to
those skilled in the art. These antibody variants were expressed by
the aforementioned method (described in Example 1) using the
plasmids, and their concentrations were determined after
purification.
14-2. Assessment of Human IL6 Receptor-Binding of PHX-IgG1
[0534] Interaction between IL-6R and PHX-IgG1 prepared in 14-1 was
analyzed using Biacore T200 (GE Healthcare) to calculate the
dissociation constant (KD). A 10 mM ACES, 150 mM NaCl, and 0.05%
Tween20, pH 7.4 buffer was used as the running buffer to analyze
interactions at 37.degree. C. Protein A/G (Thermo Scientific) was
immobilized onto Series S Sensor chip CM4 (GE Healthcare) by an
amine coupling method, and the chip was allowed to capture an
antibody of interest. Then, the running buffer and IL-6R which was
diluted using the running buffer to 800, 400, 200, 100, 50, 25, and
12.5 nM were made to flow over the antibody-captured chip at a flow
rate of 2 .mu.L/min to allow interaction to take place for 15
minutes. Antibodies captured onto the chips were washed by reaction
with 10 mM glycine-HCl at pH 1.5; and the washed chips were
regenerated, and used repeatedly for interaction analyses.
[0535] The dissociation constant KD (mol/L) of PHX-IgG1 for IL-6R
was calculated by performing a steady-state affinity analysis on
the sensorgrams obtained as the Biacore measurement results using
the Biacore Evaluation Software. The dissociation constant (KD) of
PHX-IgG1 for IL-6R at pH 7.4 calculated by this method was 1.4 E-7
(M).
[0536] Next, pH dependency of PHX-IgG1 in hIL-6R binding was
evaluated using Biacore T100. A 10 mM ACES, 150 mM NaCl, 0.05%
Tween20, pH 7.4 buffer and a 10 mM ACES, 150 mM NaCl, 0.05%
Tween20, pH 6.0 buffer were used as running buffers to determine
the binding of PHX-IgG1 to hIL-6R at 37.degree. C. under conditions
of pH 7.4 and pH6.0, respectively. Protein A/G (Thermo Scientific)
was immobilized onto Series S Sensor chip CM4 (GE Healthcare) by an
amine coupling method, and the chip was allowed to capture an
antibody of interest. Then, the running buffer and IL-6R which was
diluted using the running buffer to 1000, 250, and 62.5 nM were
allowed to interact with the antibody-captured chip.
[0537] The sensorgrams obtained through measurements made by this
method at pH 7.4 and pH 6.0 are shown in FIG. 20. FIG. 20 shows the
hIL-6R-bound phase and the hIL-6R-dissociated phase of PHX-IgG1
when the amount of antibodies captured is normalized to 100 RU.
Comparison of the results shown in FIG. 20 indicates that binding
of PHX-IgG1 to hIl-6R is decreased at pH 6.0 when compared to the
case at pH 7.4.
14-3. Assessment of the Property of Two Types of Antibodies to
Simultaneously Bind the Same Antigen by an Electrochemiluminescence
Method
[0538] Whether or not two types of antibodies can bind to a single
antigen simultaneously was evaluated by an electrochemiluminescence
method. First, Fv4-IgG1 (1 .mu.g/mL, 100 .mu.L) biotinylated using
EZ-Link Sulfo-NHS-Biotin (Thermo SCIENTIFIC) was added to a
MULTI-ARRAY(r) 96-well Streptavidin Gold Plate, and this was
incubated at room temperature for one hour to allow reaction to
take place. After washing, 100 .mu.L of the hsIL-6R solutions
prepared at 0, 0.2, 1, 5, and 25 .mu.g/mL was added, and they were
allowed to react at room temperature for one hour. After another
wash, PHX-F29 subjected to ruthenium labeling using SULFO-TAG NHS
Ester (Meso Scale Discovery) (0, 0.2, 1, 5, 25, and 125 .mu.g/mL;
100 .mu.L) or Fv4-IgG1 subjected to ruthenium labeling by the same
method (5 .mu.g/mL; 100 .mu.L) was added and allowed to react at
room temperature for one hour. After washing, 150 .mu.L of Read
Buffer T (.times.4) (Meso Scale Discovery) was dispensed into each
well, and this was immediately followed by chemiluminescence
measurement using the SECTOR IMAGER 2400 Reader (Meso Scale
Discovery).
[0539] The results are shown in FIG. 21. When ruthenium-labeled
Fv4-IgG1 was allowed to react, reaction was not observed regardless
of antigen concentration. On the other hand, when ruthenium-labeled
PHX-F29 was allowed to react, antigen (IL6R)
concentration-dependent reaction was observed. These results show
that PHX-F29 and Fv4-IgG1 bind simultaneously to IL6R. Therefore,
this indicates that the epitope of IL6R recognized by PHX-F29 and
the epitope recognized by Fv4-IgG1 are different; and by using
antibodies carrying the PHX-F29 variable region and the Fv4-IgG1
variable region, respectively, two antigen-binding molecules can
bind to a single IL6R molecule.
14-4. In Vivo Tests Using Normal Mice
[0540] Normal mice (C57BL/6J mouse; Charles River Japan) were
administered with hsIL-6R (soluble human IL-6 receptor: prepared in
Reference Example 1) alone or co-administered with hsIL-6R and
anti-human IL-6 receptor antibodies, and then assessed for the in
vivo dynamics of hsIL-6R and the anti-human IL-6 receptor
antibodies. An hsIL-6R solution (5 .mu.g/ml) or a mixed solution of
hsIL-6R and an anti-human IL-6 receptor antibody was administered
once at 10 mL/kg into the tail vein. The anti-human IL-6 receptor
antibodies used were Fv4-IgG1 and PHX-IgG1.
[0541] Concentration of hsIL-6R in the mixed solution was 5
.mu.g/mL in every case, and the anti-human IL-6 receptor antibody
concentrations were different for each administration group as
shown in Table 12. Since the anti-human IL-6 receptor antibody was
sufficiently present in excess with respect to hsIL-6R, most of the
hsIL-6R was considered to be bound by the antibody. Blood was
collected from the mice 5 minutes, 7 hours, 1 day, 2 days, 3 days,
7 days, 14 days, and 21 days after administration. Blood was
collected from the Fv4-IgG1 (1 mg/kg)-administered group (#1) 5
minutes, 7 hours, 1 day, 2 days, 3 days, 7 days, 15 days, and 22
days after antibody administration. The collected blood samples
were immediately centrifuged at 4.degree. C. and 12,000 rpm for 15
minutes to obtain the plasma samples. The separated plasma samples
were stored in a freezer at -20.degree. C. or below until
measurement.
TABLE-US-00046 TABLE 12 Amount of Amount of Administration
administered Fv4-IgG1 administered PHX-IgG1 group (mg/kg) (mg/kg)
#1 1 0 #2 1 1 #3 1 5 #4 1 25
14-5. Determination of the Anti-Human IL-6 Receptor Antibody
Concentration in Normal Mouse Plasma by an Electrochemiluminescence
Method
[0542] The anti-human IL-6 receptor antibody concentration in mouse
plasma was determined by an electrochemiluminescence method. First,
an anti-human kappa capture antibody (Antibody Solutions) was
dispensed onto Multi-ARRAY 96-Well plates (Meso Scale Discovery),
and stirred at room temperature for one hour, and then a PBS-Tween
solution containing 5% BSA (w/v) was used for blocking at room
temperature for two hours to prepare anti-human IgG-immobilized
plates. Calibration curve samples were prepared at plasma
concentrations of 40.0, 13.3, 4.44, 1.48, 0.494, 0.165, and 0.0549
.mu.g/mL; and mouse plasma assay samples prepared at 500-fold or
more dilution were aliquoted into each well of the anti-human
IgG-immobilized plates, and then the plates were stirred for one
hour at room temperature. Then, an anti-human kappa capture
antibody Biotin conjugate (Antibody Solutions) was dispensed into
each well of the aforementioned plates, and then the plates were
stirred at room temperature for one hour to allow reaction to take
place. Furthermore, SULFO-TAG Labeled streptavidin (Meso Scale
Discovery) was aliquoted into each well of the plates, and then the
plates were stirred at room temperature for one hour to allow
reaction to take place. After washing each well, Read Buffer T
(.times.1) (Meso Scale Discovery) was dispensed into the wells, and
chemiluminescence of the reaction solutions was measured
immediately using the Sector Imager 2400 Reader (Meso Scale
Discovery). The concentrations in mouse plasma were calculated
based on the response from the calibration curve using the
analytical software SOFTmax PRO (Molecular Devices). Time-course
changes in the anti-human IL-6R antibody concentration are shown in
FIG. 22.
14-6. Determination of the Plasma hsIL-6R Concentration by an
Electrochemiluminescence Method
[0543] The hsIL-6R concentration in mouse plasma was determined by
an electrochemiluminescence method. Calibration curve samples of
hsIL-6R were prepared at plasma concentrations of 12.5, 6.25, 3.13,
1.56, 0.781, 0.391, and 0.195 ng/mL. Mouse plasma assay samples
were prepared at 50-fold or greater dilution. A mixed solution of
the hsIL-6R sample or mouse plasma assay sample, a monoclonal
anti-human IL-6R antibody (R&D) which has been
ruthenium-labeled using SULFO-TAG NHS Ester (Meso Scale Discovery),
a biotinylated anti-human IL-6 R antibody (R&D), and
tocilizumab (heavy chain SEQ ID NO: 64, light chain SEQ ID NO: 65)
were allowed to react overnight at 37.degree. C. Then, a PBS-Tween
solution containing 0.5% BSA (w/v) was used for blocking a
Streptavidin Gold Multi-ARRAY Plate (Meso Scale Discovery) by
incubation at 5.degree. C. overnight, and the mixed solution was
aliquoted into each well of that plate. After another two-hour
incubation of this plate at room temperature to allow reaction to
take place, each well of the plate was washed, and then Read Buffer
T (.times.2) (Meso Scale Discovery) was dispensed into each well;
and chemiluminescence of the reaction solutions was measured
immediately using the SECTOR Imager 2400 Reader (Meso Scale
Discovery). The hsIL-6R concentrations were calculated based on
response from the calibration curve using the analytical software
SOFTmax PRO (Molecular Devices).
[0544] The calculated time-course changes in the concentration of
human IL6R (which is also described as hsIL6R or hsIL-6R, and
refers to the same protein) are shown in FIG. 23. Elimination of
plasma IL6R was accelerated in mice administered with PHX-IgG1
together with Fv4-IgG1 compared to mice administered with Fv4-IgG1
alone.
[0545] Without being restricted to a particular theory, the
following explanation may be given for the above-mentioned
acceleration of plasma antigen elimination. Even when administered
alone, Fv4-IgG1 was present in sufficient amount with respect to
hsIL6R, and thus most of the hsIL6R was considered to be bound by
Fv4-IgG1 in plasma. When PHX-IgG1, which has a different epitope
from that of Fv4-IgG1 and can bind to hsIL6R with Fv4-IgG1, is
administered, an immune complex comprising Fv4-IgG1 and PHX-IgG1
for a single hsIL6R molecule is formed. As a result, uptake into
cells was promoted by enhanced binding to an Fc.gamma. receptor
and/or FcRn, and this may have resulted in acceleration of hsIL6R
elimination from plasma. Specifically, formation of large immune
complexes comprising two or more antibodies and two or more
antigenic binding units (monomeric antigens) by multispecific
antibodies comprising appropriate variable regions that bind to
epitopes which are different from each other and are present on a
monomeric antigen, or multiparatopic antibodies in a non-limiting
embodiment, which are antibodies whose variable regions show pH- or
Ca-dependent binding (bispecific antibodies or biparatopic
antibodies comprising a right-arm variable region that recognizes
epitope A and a left-arm variable region that recognizes epitope B,
as shown in FIG. 8) enables acceleration of antigen
elimination.
Example 15
Assessment of Binding of an Immune Complex with Fc.gamma.R Using
Biacore
[0546] 15-1. Regarding Binding of an Immune Complex with
Fc.gamma.R
[0547] As methods for evaluating formation of immune complexes
comprising an antigen-binding molecule and an antigen, gel
filtration (size exclusion) chromatography was used in Example 4,
and an electrochemiluminescence (ECL) method was used in Example
14. When the antigen or antigen-binding molecule comprised in an
immune complex contains an Fc.gamma.R-binding domain such as an
immunoglobulin constant region, the immune complex binds to
Fc.gamma.R with avidity. Therefore, formation of an immune complex
comprising an Fc.gamma.R-binding domain may be confirmed by a
method that uses the property of stronger Fc.gamma.R binding (in
particular, the property of slower dissociation) than the
antigen-binding molecule alone or the antigen alone (The Journal of
Biological Chemistry (2001) 276(9), 6591-6604, mAbs (2009) 1 (5),
491-504).
15-2. Preparation of Antibodies and Antigens for Evaluation, and
Histidine-Tagged Human Fc.gamma.R IIIaV
[0548] Xolair (Novartis), clone 278-IgG1 (prepared in Example 4),
GA2-IgG1 (prepared in Example 1), Fv4-IgG1 (prepared in Example
14), and PHX-IgG1 (prepared in Example 14) to be used for
assessment of immune complex formation were prepared by the
aforementioned methods.
[0549] The hIgE (hIgE(Asp6); prepared in Example 5) and IL6R
(Reference Example 1) to be used for assessment were prepared by
the aforementioned methods, respectively. A recombinant of human
IgA, hIgA-v2 (GC-hIgA), was prepared by the method below. A gene
fragment encoding GC-hIgA-MYC (heavy chain SEQ ID NO: 66 and light
chain SEQ ID NO: 67) was inserted into an animal cell expression
vector. The constructed plasmid vector was introduced into
FreeStyle 293 (Invitrogen) using 293Fectin (Invitrogen) along with
an EBNA1-expressing gene. Then, the transfected cells were cultured
at 37.degree. C. under 8% CO.sub.2 for six days, and the GC-hIgA
protein was secreted into the culture supernatant. The cell culture
containing GC-hIgA-MYC was filtered through a 0.22-.mu.m bottle top
filter to obtain the culture supernatant. Purified GC-hIgA-MYC was
obtained using ion exchange chromatography and gel filtration
chromatography according to methods known to those skilled in the
art.
[0550] Histidine tagged human Fc.gamma.R IIIaV was prepared by the
method of Reference Example 2.
15-3. Assessment of Binding of an Immune Complex with
Fc.gamma.R
[0551] Binding of an antigen-antibody immune complex with
Fc.gamma.R was assessed using Biacore T200 (GE Healthcare).
[0552] A suitable amount of the Penta-His Antibody (QIAGEN) was
immobilized onto Sensor chip CM5 (GE Healthcare) by an amine
coupling method; and a suitable concentration of his-tagged human
Fc.gamma.R IIIaV was injected. Human Fc.gamma.R IIIaV was
immobilized onto the chip by allowing the penta-His antibody
immobilized on the chip to capture human Fc.gamma.R IIIaV. A
solution containing 200 nM antibody or a mixed solution containing
200 nM antibody and 200 nM antigen was injected as the analyte; and
it was allowed to interact with human Fc.gamma.R IIIaV immobilized
on the sensor chip. Subsequently, a 10 mmol/L Glycine-HCl, pH2.5
solution was injected to regenerate the sensorchip. A 1.2 mmol/L
CaCl.sub.2/0.05% tween20, 20 mmol/L ACES, 150 mmol/L NaCl, pH 7.4
buffer was used as the running buffer, and binding between the
antigen-antibody immune complex and Fc.gamma.R was measured at
25.degree. C.
[0553] The results of binding analyses of reacting an
antibody-containing solution or a mixed antibody-antigen solution
with human Fc.gamma.R IIIaV are shown in FIG. 24. To focus on the
dissociation, FIG. 24 shows sensorgrams in which the binding levels
have been normalized to 100. Although Fv4-IgG1 binds to IL6R, it
does not form an immune complex. Therefore, no differences were
observed between dissociation in the reaction of an
antibody-containing solution and Fc.gamma.R, and dissociation when
an antigen-antibody mixture was used. Meanwhile, when a mixed
solution of hIgE and Xolair (J. Pharmacol. Exp. Ther. (1996) 279
(2) 1000-1008), which were reported to form an immune complex, was
allowed to bind to Fc.gamma.R, dissociation from Fc.gamma.R was
observed to be slower than when Xolair alone was allowed to bind to
Fc.gamma.R. Furthermore, regarding clone 278-IgG1, which has been
confirmed to form an immune complex using size exclusion (gel
filtration) chromatography in Example 4, when a mixed solution of
clone 278-IgG1 and an antigen (hIgE) was allowed to bind to
Fc.gamma.R, dissociation from Fc.gamma.R became slower than when
clone 278-IgG1 alone was allowed to bind to Fc.gamma.R.
Furthermore, when a mixed solution of GA2-IgG1 and hIgA was allowed
to bind to Fc.gamma.R, dissociation from Fc.gamma.R also became
slower than when GA2-IgG1 alone was allowed to bind to Fc.gamma.R.
In addition, when a mixed solution of Fv4-IgG1, PHX-IgG1, and
antigen (IL6R) was allowed to bind with Fc.gamma.R, dissociation
from Fc.gamma.R also became slower than when Fv4-IgG1 or PHX-IgG1
was allowed to bind to Fc.gamma.R.
[0554] Accordingly, when the antigen or antigen-binding molecule
comprised in an immune complex contains an Fc.gamma.R-binding
domain such as an immunoglobulin constant region, the immune
complex binds to Fc.gamma.R with avidity. Therefore, a method for
assessing delay in dissociation from Fc.gamma.R when a mixed
solution of an antigen-binding molecule containing an
Fc.gamma.R-binding domain or such and its antigen is allowed to
bind to Fc.gamma.R, or when a mixed solution of an antigen
containing an Fc.gamma.R-binding domain and an antigen-binding
molecule that binds to this antigen is allowed to bind to
Fc.gamma.R, as compared to when a molecule containing an
Fc.gamma.R-binding domain such as an immunoglobulin constant region
is allowed to bind by itself to Fc.gamma.R, was found to be an
effective method for assessing formation of immune complexes
comprising an antigen-binding molecule and an antigen. The
above-mentioned assessment of immune complexes using dissociation
from Fc.gamma.R as the criteria was shown to be effective in the
case of immune complexes formed by an antibody against an antigen
bound by a plurality of monomers, and also in the case of immune
complexes formed by a monomeric antigen and a plurality of
antibodies binding to different epitopes.
Example 16
Assessment of Binding of an Immune Complex with FcRn Using
Biacore
[0555] 16-1. Regarding Binding of an Immune Complex with FcRn
[0556] In a method for evaluating formation of immune complexes
comprising an antigen-binding molecule and an antigen, when the
antigen or antigen-binding molecule comprised in an immune complex
contains an FcRn-binding domain such as an immunoglobulin constant
region, the immune complex binds to FcRn with avidity. Therefore,
formation of an immune complex comprising an FcRn-binding domain
can be confirmed by a method that uses the property of stronger
FcRn binding (in particular, the property of slower dissociation)
than the antigen-binding molecule alone or the antigen alone
(Journal of Immunology (2010) 184 (4) 1968-1976). However, in the
methods reported so far, binding between FcRn and the FcRn-binding
domain has been assessed under the condition of pH 6.0. This
condition is not only different from the condition in vivo, but is
also inappropriate for assessing samples of which avidity changes
with the change in pH. Such condition may have been used because
binding at pH 7.4 could not be assessed due to the very low avidity
between human FcRn and human IgG1. Thus, whether assessment of
immune complexes under a pH7.4 condition, which is closer to the in
vivo condition, is possible was examined.
16-2. Preparation of Antibodies and Antigens for Assessment and
FcRn
[0557] Fv4-F22 used in the assessment is a heavy-chain constant
region variant of Fv4-IgG1 (described in Example 14). Xolair-F22 is
a heavy-chain constant region variant of Xolair (Novartis). A DNA
sequence encoding Fv4-F22 (heavy chain SEQ ID NO: 68, and light
chain SEQ ID NO: 60) and a DNA sequence encoding Xolair-F22 (heavy
chain SEQ ID NO: 69, and light chain SEQ ID NO: 70) were inserted
into an animal cell expression plasmid by a method known to those
skilled in the art. Antibody variants were expressed according to
the above-described method (described in Example 1) using animal
cells introduced with the plasmid, and their concentrations were
determined after purification. Clone 278-IgG1 (prepared in Example
4), Fv4-IgG1 (prepared in Example 14), and PHX-IgG1 (prepared in
Example 14) were prepared by the aforementioned methods.
[0558] The hIgE (hIgE(Asp6), prepared in Example 5) and IL6R
(prepared in Reference Example 1) used for the assessment were
prepared by the respective aforementioned methods.
[0559] Human FcRn and mouse FcRn were prepared by methods described
in Reference Examples 3 and 4, respectively.
16-3. Assessment of Binding of an Immune Complex with Human
FcRn
[0560] Binding of an antigen-antibody immune complex with human
FcRn was assessed using Biacore T100 (GE Healthcare).
[0561] A solution containing an antibody alone or a mixed
antibody-antigen solution shown in Table 13 was injected as an
analyte onto Sensor chip CM4 (GE Healthcare) onto which a suitable
amount of human FcRn (hFcRn) had been immobilized by an amine
coupling method, and interaction was allowed to take place with
hFcRn on the sensor chip. Subsequently, a 20 mM Tris-HCl, 150 mM
NaCl, pH 9.0 or pH 9.5 solution was injected to regenerate the
sensor chip. A 50 mmol/L Na-Phosphate (phosphoric acid), 150 mmol/L
NaCl, 0.05% Tween20, pH 7.4 buffer was used as the running buffer,
and binding between the antigen-antibody immune complex and FcRn
was measured at 25.degree. C.
TABLE-US-00047 TABLE 13 Sample name Antibody Antigen Fv4-F22
Fv4-F22 (10 .mu.g/mL) -- Fv4-F22 + IL6R Fv4-F22 (10 .mu.g/mL)
hsIL6R (2.5 .mu.g/mL) Xolair-F22 Xolair-F22(10 .mu.g/mL) --
Xolair-F22 + IgE Xolair-F22(10 .mu.g/mL) hIgE(Asp6) (11.2
.mu.g/mL)
[0562] The results of binding analyses, when an antibody-containing
solution or a mixed antibody-antigen solution was allowed to react
with hFcRn, are shown in FIG. 25. To focus on the dissociation,
FIG. 25 shows sensorgrams in which the binding levels have been
normalized to 100. While Fv4-F22 binds with IL6R, it does not form
an immune complex containing a plurality of molecules that carry
immunoglobulin constant regions; and thus no differences were
observed between dissociation in the reaction of an
antibody-containing solution and FcRn, and dissociation when a
mixed antigen-antibody solution was used. Meanwhile, when a mixed
solution of hIgE and Xolair-F22 carrying the variable region of
Xolair which has been reported to form an immune complex with hIgE
(J. Pharmacol. Exp. Ther. (1996) 279 (2) 1000-1008) was allowed to
bind to FcRn, dissociation from FcRn was observed to be slower than
the dissociation from FcRn when Xolair-F22 alone was bound to FcRn.
The above showed that even under the condition of pH 7.4, hFcRn
binding can be detected by using antibodies with enhanced binding
to hFcRn. When the antigen-binding molecule or antigen comprised in
an immune complex contains an FcRn-binding domain such as an
immunoglobulin constant region, the immune complex binds more
strongly to FcRn. Therefore, a method for assessing delay in
dissociation from FcRn when a mixed solution of an antigen-binding
molecule containing an FcRn-binding domain or such and its antigen
is allowed to bind to FcRn, or when a mixed solution of an antigen
containing an FcRn-binding domain and an antigen-binding molecule
that binds to this antigen is allowed to bind to FcRn, as compared
to when a molecule containing an FcRn-binding domain such as an
immunoglobulin constant region or such is allowed to bind by itself
to FcRn, was found to be an effective method for assessing
formation of immune complexes comprising an antigen-binding
molecule and an antigen.
16-4. Assessment of Binding of an Immune Complex with FcRn Using
Biacore
[0563] In 16-3, FcRn binding of immune complexes containing
variants of immunoglobulin constant regions with enhanced hFcRn
binding was evaluated. Since native human IgG can bind more
strongly to human FcRn than to mouse FcRn (Int. Immunol. (2001) 13
(12), 1551-1559), binding of an antibody containing a native human
IgG constant region to mouse FcRn was assessed to examine if
assessment of an immune complex formation is possible without
modifying the immunoglobulin constant region.
[0564] Binding of an antigen-antibody immune complex with FcRn was
assessed using Biacore T100 (GE Healthcare). A solution containing
only antibodies or a mixed antibody-antigen solution as shown in
Table 14 was injected as an analyte onto Sensor chip CM4 (GE
Healthcare) that has been immobilized with a suitable amount of
mouse FcRn (mFcRn) by an amine coupling method, and interaction was
allowed to take place with mFcRn on the sensor chip. Subsequently,
a 20 mM Tris-HCl, 150 mM NaCl, pH 9.0 or pH 9.5 solution was
injected to regenerate the sensor chip. A 50 mmol/L Na-Phosphate
(phsophoric acid), 150 mmol/L NaCl, 0.05% Tween20, pH 7.4 buffer
was used as the running buffer, and binding between the
antigen-antibody immune complex and FcRn was measured at 25.degree.
C.
TABLE-US-00048 TABLE 14 Sample name Antibody Antigen Xolair
Xolair-IgG1(50 .mu.g/mL) -- Xolair + hIgE(Asp6) Xolair-IgG1(50
.mu.g/mL) hIgE(Asp6) (56 .mu.g/mL) Clone 278 278 Clone 278-IgG1 --
gG1 (50 .mu.g/mL) Clone 278 278 + Clone 278-IgG1- hIgE(Asp6)
hIgE(Asp6) F22 (50 .mu.g/mL) (56 .mu.g/mL) Fv4-IgG1 Fv4-IgG1 (50
.mu.g/mL) -- Fv4-IgG1 + IL6R Fv4-IgG1 (50 .mu.g/mL) hsIL6R (12.5
.mu.g/mL) PHX-IgG1 PHX-IgG1 (50 .mu.g/mL) -- PHX-IgG1 + IL6R
PHX-IgG1 (50 .mu.g/mL) hsIL6R (12.5 .mu.g/mL) Pv4-IgG1 + PHX-IgG1
(25 .mu.g/mL) + -- PHX-IgG1 Fv4-IgG1 (25 .mu.g/mL) Fv4-1 gG1 +
PHX-IgG1 (25 .mu.g/mL) + hsIL6R PHX-IgG1 + IL6R Fv4-IgG1 (25
.mu.g/mL) (12.5 .mu.g/mL
[0565] The results of binding analyses, when a solution containing
only antibodies or a mixed antibody-antigen solution was allowed to
react with mFcRn, are shown in FIGS. 26 and 27. To focus on the
dissociation, FIGS. 26 and 27 show sensorgrams in which the binding
levels have been normalized to 100. When a mixed solution of an
antigen (hIgE) and clone 278-IgG1, which has been confirmed to form
an immune complex by size exclusion (gel filtration) chromatography
in Example 4, was allowed to bind with mFcRn, dissociation from
mFcRn also became slower than when clone 278-IgG1 alone was allowed
to bind to mFcRn. Furthermore, when a mixed solution of hIgE and
Xolair, which has been reported to form an immune complex (J.
Pharmacol. Exp. Ther. (1996) 279 (2) 1000-1008), was allowed to
bind with mFcRn, dissociation from mFcRn was observed to become
slower than when Xolair alone was allowed to bind to mFcRn (FIG.
26). On the other hand, difference was not observed between
dissociation from mFcRn when a solution containing Fv4-IgG1 was
allowed to bind with mFcRn, and dissociation from mFcRn when a
mixed solution of Fv4-IgG1 and IL6R was allowed to bind with mFcRn.
Furthermore, difference was not observed between dissociation from
mFcRn when a solution containing PHX-IgG1 was allowed to bind with
mFcRn, and dissociation from mFcRn when a mixed solution of
PHX-IgG1 and IL6R was allowed to bind with mFcRn (FIG. 27). This
may be because the antigenic binding unit is one in IL6R when
binding to a variable region contained in Fv4-IgG1; and when
Fv4-IgG1 alone is used as the antigen-binding molecule for IL6R, an
immune complex comprising two or more Fv4-IgG1 molecules for one
molecule of IL6R is not formed. Since the antigenic binding unit is
also one in IL6R when binding to a variable region in PHX-IgG1,
there is only one epitope in IL6R; and therefore it is thought that
when PHX-IgG1 alone is used as an antigen-binding molecule
targeting IL6R, an immune complex comprising two or more PHX-IgG1
molecules for one molecule of IL6R is not formed. Herein, when a
mixed solution of IL6R, and Fv4-IgG1 and PHX-IgG1, which are found
in Example 14 to have epitopes that are different from each other,
was allowed to bind to mFcRn, dissociation from mFcRn became slower
than when a solution containing the Fv4-IgG1 antibody and the
PHX-IgG1 antibody (only antibodies) was allowed to bind with mFcRn.
Such phenomenon of slower dissociation is similar to the phenomenon
observed with clone 278. This indicates that when a single type of
antigen-binding molecule is used for the antigen-binding molecule
that binds to a target antigen, even if the antigenic binding unit
is one, the antigenic binding unit of the antigen can be changed to
two or more by using a combination of antibody-binding molecules
that have epitopes different from each other as antigen-binding
molecules targeting the antigen; and as a result, an immune complex
containing two or more antigen-binding molecules can be formed.
[0566] The above showed that by using mouse FcRn, even a native
human IgG1 molecule without modifications that would affect
interaction with FcRn can be assessed for FcRn binding under the
condition of pH 7.4 which is the same as the in vivo condition.
Furthermore, when an antigen-binding molecule or an antigen
contains an FcRn-binding domain such as an immunoglobulin constant
region, the immune complex binds strongly with FcRn; therefore,
immune complex formation could be confirmed by evaluating delay in
dissociation from FcRn of an immune complex comprising a plurality
of molecules containing an FcRn-binding domain such as an
immunoglobulin constant region as compared to dissociation from
FcRn of a molecule containing an FcRn-binding domain of an
immunoglobulin constant region. It was confirmed that the
above-mentioned assessment of immune complex formation using FcRn
can be used to verify the formation of both an immune complex
formed from an antigen comprising a plurality of bound monomers and
an antibody that binds to this antigen, and an immune complex
formed from a monomeric antigen and a plurality of antibodies
having different binding epitopes from each other that bind to this
antigen.
Reference Example 1
Preparation of Soluble Human IL-6 Receptor (hsIL-6R)
[0567] A recombinant form of human IL-6 receptor, which is an
antigen, was prepared as follows. A CHO line that constantly
expresses a soluble human IL-6 receptor (hereinafter referred to as
hsIL-6R) composed of the amino acid sequence from the first to
357th amino acid from the N terminus as reported in J. Immunol.
(1994) 152, 4958-4968 was constructed using a method known to those
skilled in the art. hsIL-6R was expressed by culturing this CHO
line. hsIL-6R was purified from the culture supernatant of the
resulting CHO line by a two-step process involving the Blue
Sepharose 6 FF column chromatography and gel filtration column
chromatography. The fraction that eluted as the main peak in the
final step was used as the final purified product.
Reference Example 21
Preparation of Histidine-Tagged Human Fc.gamma.RIIIaV
[0568] Histidine-tagged human Fc.gamma.RIIIaV was prepared by the
following method. First, a gene of the extracellular domain of
Fc.gamma.R was synthesized by a method well known to those skilled
in the art. At that time, the sequence of each Fc.gamma.R was
produced based on the information registered at NCBI. Specifically,
Fc.gamma.RIIIa was produced based on the sequence of NCBI Accession
No. NM.sub.--001127593.1, and a His tag was attached to the C
terminus. Moreover, polymorphism is known for Fc.gamma.RIIIa;
however, production was carried out by referring to J. Clin.
Invest., 1997, 100 (5): 1059-1070 for Fc.gamma.RIIIa.
[0569] The obtained gene fragments were inserted into an animal
cell expression vector, and expression vectors were produced. The
produced expression vectors were introduced transiently into human
embryonic kidney cancer cell line-derived FreeStyle293 cells
(Invitrogen) to express the proteins of interest. Cells were
cultured, and after collection of the obtained culture supernatant,
this was passed through a 0.22 .mu.m filter to obtain the culture
supernatant. In principle, the obtained culture supernatants were
purified in the following four steps. The steps carried out were,
cation exchange column chromatography (SP Sepharose FF) in step 1,
affinity column chromatography (HisTrap HP) for His tag in step 2,
gel filtration column chromatography (Superdex200) in step 3, and
aseptic chromatography in step 4. The purified proteins were
subjected to absorbance measurements at 280 nm using a
spectrophotometer; and from the obtained values, the concentrations
of the purified proteins were calculated using the absorption
coefficient calculated using methods such as PACE (Protein Science
1995; 4: 2411-2423).
Reference Example 3
Preparation of Soluble Human FcRn (hFcRn)
[0570] Human FcRn forms a complex with .beta.2-microglobulin.
Oligo-DNA primers were designed based on the reported human FcRn
gene sequence (J Exp Med. 1994 Dec. 1; 180(6): 2377-81). A cDNA was
amplified by PCR from a human cDNA (Human Placenta Marathon-Ready
cDNA, Clontech) using the designed primers. The amplified cDNA
encoding the extracellular domain containing the signal sequence
(Met1-Leu290) was inserted into an animal cell expression vector.
Similarly, primers were designed for .beta.2-microglobulin based on
the reported human .beta.2-microglobulin sequence, and the cDNA was
amplified by PCR. The cDNA fragment of Met1-Met119 containing a
signal sequence was amplified and inserted into an animal cell
expression vector.
[0571] Soluble human FcRn (called hFcRn) was prepared according to
the procedure below. The vector for expressing human FcRn (SEQ ID
NO: 71) and the vector for expressing .beta.2-microglobulin (SEQ ID
NO: 72) were transduced into HEK293H cells (Invitrogen) by the
lipofection method using PEI (Polyscience). The transduced cells
were cultured, and the culture solution was collected. Human FcRn
was purified from the collected culture by each of the
chromatographic methods, IgG Sepharose 6 Fast Flow (Amersham
Biosciences) and HiTrap Q HP (GE Healthcare) (J Immunol. 2002 Nov.
1; 169(9): 5171-80).
Reference Example 4
Preparation of Soluble Mouse FcRn (mFcRn)
[0572] Soluble mouse FcRn was prepared in a similar manner to
soluble human FcRn. First, a gene encoding the extracellular domain
of mouse FcRn and a gene encoding mouse .beta.2-microglobulin were
synthesized by methods known to those skilled in the art. For this
operation, mouse FcRn and mouse .beta.2-microglobulin sequences
were produced based on the information registered at NCBI.
Specifically, for mouse FcRn, a gene fragment encoding the amino
acids of positions 1 to 290 containing the signal sequence was
produced as the extracellular domain based on the sequence of NCBI
accession # NP.sub.--034319.2 (SEQ ID NO: 73). Furthermore, for
.beta.2-microglobulin, a gene fragment was produced based on the
sequence of NCBI accession # NP.sub.--033865 (SEQ ID NO: 74). An
expression vector was produced by inserting the obtained gene
fragments into an animal cell expression vector. The produced
expression vector was introduced transiently into human embryonic
kidney carcinoma cell-derived FreeStyle 293 cells (Invitrogen) to
express the protein of interest. The transduced cells were
cultured, and the culture solution was collected. Mouse FcRn was
purified from the collected culture by each of the chromatographic
methods, IgG Sepharose 6 Fast Flow (Amersham Biosciences) and
Superdex 200 (GE Healthcare).
Reference Example 5
Production of an Antigen-Binding Molecule with Higher
Fc.gamma.R-Binding Activity than that of the Native Human IgG Fc
Region, and Enhanced Human FcRn-Binding Activity Under an Acidic pH
Condition
[0573] (5-1) Production of an Anti-Human IL-6 Receptor Antibody
with Enhanced Binding to Mouse Fc.gamma.R
[0574] VH3-IgG1-F1087 (SEQ ID NO: 75), which is formed by
substituting Asp for Lys at position 326 (EU numbering) in
VH3-IgG1, and VH3-IgG1-F1182 (SEQ ID NO: 76), which is formed by
substituting Asp for Ser at position 239, and Glu for Ile at
position 332 (EU numbering) in VH3-IgG1, were produced as
antigen-binding molecules with enhanced binding to mouse
Fc.gamma.R. Fv4-IgG1-F1087 containing VH3-IgG1-F1087 as the heavy
chain and VL3-CK (SEQ ID NO: 77) as the light chain, and
Fv4-IgG1-F1182 containing VH3-IgG1-F1182 as the heavy chain and
VL3-CK as the light chain were produced using the method of
Reference Example 2.
(5-2) Confirmation of Mouse Fc.gamma.R-Binding Activity
[0575] VH3/L (WT)-IgG1-F1087 and VH3/L (WT)-IgG1-F1182, which
contain VH3-IgG1-F1087 and VH3-IgG1-F1182 respectively as the heavy
chain, and L (WT)-CK (SEQ ID NO: 78) as the light chain were
produced. The mouse Fc.gamma.R-binding activities of these
antibodies and VH3/L (WT)-IgG1-F1022 were assessed, and the results
are shown in Table 15. The number of folds enhanced in the mouse
Fc.gamma.R-binding activity of each of the variants compared to
that of IgG1 before modification is shown in Table 16.
TABLE-US-00049 TABLE 15 Name of KD (M) variant mFc .gamma. RI mFc
.gamma. RIIb mFc .gamma. RIII mFc .gamma. RIV IgG1 5.3E-08 9.8E-07
2.4E-06 8.6E-08 F1022 7.6E-09 1.0E-08 5.5E-09 1.4E-07 F1087 2.9E-08
5.6E-08 5.2E-08 3.3E-07 F1182 2.4E-09 1.1E-07 4.8E-07 5.3E-10
TABLE-US-00050 TABLE 16 Name of IgG1 binding ratio variant mFc
.gamma. RI mFc .gamma. RIIb mFc .gamma. RIII mFc .gamma. RIV IgG1
1.0 1.0 1.0 1.0 F1022 7.0 93.6 440.5 0.6 F1087 1.8 17.5 46.2 0.3
F1182 22.1 9.1 5.0 162.3
INDUSTRIAL APPLICABILITY
[0576] The antigen-binding molecules of the present invention
enable acceleration of elimination of antigens with two or more
antigenic binding units from the plasma, by being equipped with the
feature of forming large immune complexes comprising antigens with
two or more antigenic binding units (epitopes) and two or more
antigen-binding molecules (for example, antibodies).
Antigen-binding molecules of the present invention also enable
further acceleration of elimination of the antigens by being
equipped with an ion-dependent antigen-binding activity in addition
to the above-mentioned features. By being equipped with such
technical features, antigen-binding molecules of the present
invention become very useful as pharmaceuticals for treating
diseases or symptoms (such as cancer and inflammatory disease).
Sequence CWU 1
1
781468PRTHomo sapiens 1Met Leu Ala Val Gly Cys Ala Leu Leu Ala Ala
Leu Leu Ala Ala Pro 1 5 10 15 Gly Ala Ala Leu Ala Pro Arg Arg Cys
Pro Ala Gln Glu Val Ala Arg 20 25 30 Gly Val Leu Thr Ser Leu Pro
Gly Asp Ser Val Thr Leu Thr Cys Pro 35 40 45 Gly Val Glu Pro Glu
Asp Asn Ala Thr Val His Trp Val Leu Arg Lys 50 55 60 Pro Ala Ala
Gly Ser His Pro Ser Arg Trp Ala Gly Met Gly Arg Arg 65 70 75 80 Leu
Leu Leu Arg Ser Val Gln Leu His Asp Ser Gly Asn Tyr Ser Cys 85 90
95 Tyr Arg Ala Gly Arg Pro Ala Gly Thr Val His Leu Leu Val Asp Val
100 105 110 Pro Pro Glu Glu Pro Gln Leu Ser Cys Phe Arg Lys Ser Pro
Leu Ser 115 120 125 Asn Val Val Cys Glu Trp Gly Pro Arg Ser Thr Pro
Ser Leu Thr Thr 130 135 140 Lys Ala Val Leu Leu Val Arg Lys Phe Gln
Asn Ser Pro Ala Glu Asp 145 150 155 160 Phe Gln Glu Pro Cys Gln Tyr
Ser Gln Glu Ser Gln Lys Phe Ser Cys 165 170 175 Gln Leu Ala Val Pro
Glu Gly Asp Ser Ser Phe Tyr Ile Val Ser Met 180 185 190 Cys Val Ala
Ser Ser Val Gly Ser Lys Phe Ser Lys Thr Gln Thr Phe 195 200 205 Gln
Gly Cys Gly Ile Leu Gln Pro Asp Pro Pro Ala Asn Ile Thr Val 210 215
220 Thr Ala Val Ala Arg Asn Pro Arg Trp Leu Ser Val Thr Trp Gln Asp
225 230 235 240 Pro His Ser Trp Asn Ser Ser Phe Tyr Arg Leu Arg Phe
Glu Leu Arg 245 250 255 Tyr Arg Ala Glu Arg Ser Lys Thr Phe Thr Thr
Trp Met Val Lys Asp 260 265 270 Leu Gln His His Cys Val Ile His Asp
Ala Trp Ser Gly Leu Arg His 275 280 285 Val Val Gln Leu Arg Ala Gln
Glu Glu Phe Gly Gln Gly Glu Trp Ser 290 295 300 Glu Trp Ser Pro Glu
Ala Met Gly Thr Pro Trp Thr Glu Ser Arg Ser 305 310 315 320 Pro Pro
Ala Glu Asn Glu Val Ser Thr Pro Met Gln Ala Leu Thr Thr 325 330 335
Asn Lys Asp Asp Asp Asn Ile Leu Phe Arg Asp Ser Ala Asn Ala Thr 340
345 350 Ser Leu Pro Val Gln Asp Ser Ser Ser Val Pro Leu Pro Thr Phe
Leu 355 360 365 Val Ala Gly Gly Ser Leu Ala Phe Gly Thr Leu Leu Cys
Ile Ala Ile 370 375 380 Val Leu Arg Phe Lys Lys Thr Trp Lys Leu Arg
Ala Leu Lys Glu Gly 385 390 395 400 Lys Thr Ser Met His Pro Pro Tyr
Ser Leu Gly Gln Leu Val Pro Glu 405 410 415 Arg Pro Arg Pro Thr Pro
Val Leu Val Pro Leu Ile Ser Pro Pro Val 420 425 430 Ser Pro Ser Ser
Leu Gly Ser Asp Asn Thr Ser Ser His Asn Arg Pro 435 440 445 Asp Ala
Arg Asp Pro Arg Ser Pro Tyr Asp Ile Ser Asn Thr Asp Tyr 450 455 460
Phe Phe Pro Arg 465 2428PRTHomo sapiens 2Ala Ser Thr Gln Ser Pro
Ser Val Phe Pro Leu Thr Arg Cys Cys Lys 1 5 10 15 Asn Ile Pro Ser
Asn Ala Thr Ser Val Thr Leu Gly Cys Leu Ala Thr 20 25 30 Gly Tyr
Phe Pro Glu Pro Val Met Val Thr Trp Asp Thr Gly Ser Leu 35 40 45
Asn Gly Thr Thr Met Thr Leu Pro Ala Thr Thr Leu Thr Leu Ser Gly 50
55 60 His Tyr Ala Thr Ile Ser Leu Leu Thr Val Ser Gly Ala Trp Ala
Lys 65 70 75 80 Gln Met Phe Thr Cys Arg Val Ala His Thr Pro Ser Ser
Thr Asp Trp 85 90 95 Val Asp Asn Lys Thr Phe Ser Val Cys Ser Arg
Asp Phe Thr Pro Pro 100 105 110 Thr Val Lys Ile Leu Gln Ser Ser Cys
Asp Gly Gly Gly His Phe Pro 115 120 125 Pro Thr Ile Gln Leu Leu Cys
Leu Val Ser Gly Tyr Thr Pro Gly Thr 130 135 140 Ile Asn Ile Thr Trp
Leu Glu Asp Gly Gln Val Met Asp Val Asp Leu 145 150 155 160 Ser Thr
Ala Ser Thr Thr Gln Glu Gly Glu Leu Ala Ser Thr Gln Ser 165 170 175
Glu Leu Thr Leu Ser Gln Lys His Trp Leu Ser Asp Arg Thr Tyr Thr 180
185 190 Cys Gln Val Thr Tyr Gln Gly His Thr Phe Glu Asp Ser Thr Lys
Lys 195 200 205 Cys Ala Asp Ser Asn Pro Arg Gly Val Ser Ala Tyr Leu
Ser Arg Pro 210 215 220 Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser Pro
Thr Ile Thr Cys Leu 225 230 235 240 Val Val Asp Leu Ala Pro Ser Lys
Gly Thr Val Asn Leu Thr Trp Ser 245 250 255 Arg Ala Ser Gly Lys Pro
Val Asn His Ser Thr Arg Lys Glu Glu Lys 260 265 270 Gln Arg Asn Gly
Thr Leu Thr Val Thr Ser Thr Leu Pro Val Gly Thr 275 280 285 Arg Asp
Trp Ile Glu Gly Glu Thr Tyr Gln Cys Arg Val Thr His Pro 290 295 300
His Leu Pro Arg Ala Leu Met Arg Ser Thr Thr Lys Thr Ser Gly Pro 305
310 315 320 Arg Ala Ala Pro Glu Val Tyr Ala Phe Ala Thr Pro Glu Trp
Pro Gly 325 330 335 Ser Arg Asp Lys Arg Thr Leu Ala Cys Leu Ile Gln
Asn Phe Met Pro 340 345 350 Glu Asp Ile Ser Val Gln Trp Leu His Asn
Glu Val Gln Leu Pro Asp 355 360 365 Ala Arg His Ser Thr Thr Gln Pro
Arg Lys Thr Lys Gly Ser Gly Phe 370 375 380 Phe Val Phe Ser Arg Leu
Glu Val Thr Arg Ala Glu Trp Glu Gln Lys 385 390 395 400 Asp Glu Phe
Ile Cys Arg Ala Val His Glu Ala Ala Ser Pro Ser Gln 405 410 415 Thr
Val Gln Arg Ala Val Ser Val Asn Pro Gly Lys 420 425 3 1482DNAHomo
sapiens 3gcctccacac agagcccatc cgtcttcccc ttgacccgct gctgcaaaaa
cattccctcc 60aatgccacct ccgtgactct gggctgcctg gccacgggct acttcccgga
gccggtgatg 120gtgacctgcg acacaggctc cctcaacggg acaactatga
ccttaccagc caccaccctc 180acgctctctg gtcactatgc caccatcagc
ttgctgaccg tctcgggtgc gtgggccaag 240cagatgttca cctgccgtgt
ggcacacact ccatcgtcca cagactgggt cgacaacaaa 300accttcagcg
tctgctccag ggacttcacc ccgcccaccg tgaagatctt acagtcgtcc
360tgcgacggcg gcgggcactt ccccccgacc atccagctcc tgtgcctcgt
ctctgggtac 420accccaggga ctatcaacat cacctggctg gaggacgggc
aggtcatgga cgtggacttg 480tccaccgcct ctaccacgca ggagggtgag
ctggcctcca cacaaagcga gctcaccctc 540agccagaagc actggctgtc
agaccgcacc tacacctgcc aggtcaccta tcaaggtcac 600acctttgagg
acagcaccaa gaagtgtgca gattccaacc cgagaggggt gagcgcctac
660ctaagccggc ccagcccgtt cgacctgttc atccgcaagt cgcccacgat
cacctgtctg 720gtggtggacc tggcacccag caaggggacc gtgaacctga
cctggtcccg ggccagtggg 780aagcctgtga accactccac cagaaaggag
gagaagcagc gcaatggcac gttaaccgtc 840acgtccaccc tgccggtggg
cacccgagac tggatcgagg gggagaccta ccagtgcagg 900gtgacccacc
cccacctgcc cagggccctc atgcggtcca cgaccaagac cagcggcccg
960cgtgctgccc cggaagtcta tgcgtttgcg acgccggagt ggccggggag
ccgggacaag 1020cgcaccctcg cctgcctgat ccagaacttc atgcctgagg
acatctcggt gcagtggctg 1080cacaacgagg tgcagctccc ggacgcccgg
cacagcacga cgcagccccg caagaccaag 1140ggctccggct tcttcgtctt
cagccgcctg gaggtgacca gggccgaatg ggagcagaaa 1200gatgagttca
tctgccgtgc agtccatgag gcagcgagcc cctcacagac cgtccagcga
1260gcggtgtctg taaatcccga gctggacgtg tgcgtggagg aggccgaggg
cgaggcgccg 1320tggacgtgga ccggcctctg catcttcgcc gcactcttcc
tgctcagcgt gagctacagc 1380gccgccctca cgctcctcat ggtgcagcgg
ttcctctcag ccacgcggca ggggaggccc 1440cagacctccc tcgactacac
caacgtcctc cagccccacg cc 1482415PRTArtificial Sequencean
artificially synthesized sequence 4Gly Leu Asn Asp Ile Phe Glu Ala
Gln Lys Ile Glu Trp His Glu 1 5 10 15 519PRTArtificial Sequencean
artificially synthesized sequence 5Met Gly Trp Ser Cys Ile Ile Leu
Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser
6107PRTArtificial Sequencean artificially synthesized sequence 6Glu
Thr Thr Leu Thr Gln Ser Pro Ala Phe Met Ser Ala Thr Pro Gly 1 5 10
15 Asp Lys Val Asn Ile Ser Cys Lys Ala Ser Gln Asp Ile Asp Asp Asp
20 25 30 Met Asn Trp Tyr Gln Gln Lys Pro Gly Glu Ala Ala Ile Phe
Ile Ile 35 40 45 Gln Glu Ala Thr Thr Leu Val Pro Gly Ile Ser Pro
Arg Phe Ser Gly 50 55 60 Ser Gly Tyr Gly Thr Asp Phe Thr Leu Thr
Ile Asn Asn Ile Glu Ser 65 70 75 80 Glu Asp Ala Ala Tyr Tyr Phe Cys
Leu Gln His Asp Asn Phe Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Leu Glu Ile Lys 100 105 7107PRTArtificial Sequencean
artificially synthesized sequence 7Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Ser Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala
Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr
Pro Phe 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys 100
105 8112PRTArtificial Sequencean artificially synthesized sequence
8Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly 1
5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His
Ser 20 25 30 Asn Gly Asp Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro
Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg
Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly
Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp
Val Gly Val Tyr Tyr Cys Met Gln Val 85 90 95 Leu Arg Asn Pro Leu
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Gln 100 105 110
9107PRTArtificial Sequencean artificially synthesized sequence 9Glu
Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10
15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu
Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala
Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Arg Ser Asn Trp Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr
Lys Val Glu Ile Lys 100 105 10112PRTArtificial Sequencean
artificially synthesized sequence 10Asp Ile Val Met Thr Gln Ser Pro
Glu Ser Leu Val Leu Ser Leu Gly 1 5 10 15 Gly Thr Ala Thr Ile Asn
Cys Arg Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Asn Asn Lys
Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro
Thr Leu Leu Phe Ser Trp Ala Ser Ile Arg Asp Ser Gly Val 50 55 60
Pro Asp Arg Phe Ser Ala Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 65
70 75 80 Ile Ser Asp Leu Gln Ala Glu Asp Ala Ala Val Tyr Tyr Cys
Gln Gln 85 90 95 Tyr Tyr Arg Ala Pro Ser Phe Gly Gln Gly Thr Lys
Leu Gln Ile Lys 100 105 110 11121PRTArtificial Sequencean
artificially synthesized sequence 11Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Met His Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile
Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65
70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Asp Asp Pro Gly Gly Gly Glu Tyr Tyr Phe
Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115
120 12126PRTArtificial Sequencean artificially synthesized sequence
12Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Glu Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ala Pro
Tyr Tyr Tyr Asp Ser Ser Gly Tyr Thr Asp Ala 100 105 110 Phe Asp Ile
Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125
13330PRTArtificial Sequencean artificially synthesized sequence
13Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1
5 10 15 Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser
Ser Ser Leu Gly Thr Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His
Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135
140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245
250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu
Ser Pro Gly Lys 325 330 14326PRTArtificial Sequencean artificially
synthesized sequence 14Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser
Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr
Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90
95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro
100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp 115 120 125 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe
Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val
Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190 Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala
Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215
220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu
Ser Pro Gly Lys 325 15377PRTHomo sapiens 15Ala Ser Thr Lys Gly Pro
Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50
55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr 65 70 75 80 Tyr Thr Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys 85 90 95 Arg Val Glu Leu Lys Thr Pro Leu Gly Asp Thr
Thr His Thr Cys Pro 100 105 110 Arg Cys Pro Glu Pro Lys Ser Cys Asp
Thr Pro Pro Pro Cys Pro Arg 115 120 125 Cys Pro Glu Pro Lys Ser Cys
Asp Thr Pro Pro Pro Cys Pro Arg Cys 130 135 140 Pro Glu Pro Lys Ser
Cys Asp Thr Pro Pro Pro Cys Pro Arg Cys Pro 145 150 155 160 Ala Pro
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 165 170 175
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 180
185 190 Val Val Asp Val Ser His Glu Asp Pro Glu Val Gln Phe Lys Trp
Tyr 195 200 205 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu 210 215 220 Gln Tyr Asn Ser Thr Phe Arg Val Val Ser Val
Leu Thr Val Leu His 225 230 235 240 Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys 245 250 255 Ala Leu Pro Ala Pro Ile
Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln 260 265 270 Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 275 280 285 Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 290 295 300
Ser Asp Ile Ala Val Glu Trp Glu Ser Ser Gly Gln Pro Glu Asn Asn 305
310 315 320 Tyr Asn Thr Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe
Phe Leu 325 330 335 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Ile 340 345 350 Phe Ser Cys Ser Val Met His Glu Ala Leu
His Asn Arg Phe Thr Gln 355 360 365 Lys Ser Leu Ser Leu Ser Pro Gly
Lys 370 375 16327PRTArtificial Sequencean artificially synthesized
sequence 16Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys
Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val
Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr
Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn
Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115
120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp
Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235
240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu
Gly Lys 325 17365PRTHomo sapiens 17Met Gly Val Pro Arg Pro Gln Pro
Trp Ala Leu Gly Leu Leu Leu Phe 1 5 10 15 Leu Leu Pro Gly Ser Leu
Gly Ala Glu Ser His Leu Ser Leu Leu Tyr 20 25 30 His Leu Thr Ala
Val Ser Ser Pro Ala Pro Gly Thr Pro Ala Phe Trp 35 40 45 Val Ser
Gly Trp Leu Gly Pro Gln Gln Tyr Leu Ser Tyr Asn Ser Leu 50 55 60
Arg Gly Glu Ala Glu Pro Cys Gly Ala Trp Val Trp Glu Asn Gln Val 65
70 75 80 Ser Trp Tyr Trp Glu Lys Glu Thr Thr Asp Leu Arg Ile Lys
Glu Lys 85 90 95 Leu Phe Leu Glu Ala Phe Lys Ala Leu Gly Gly Lys
Gly Pro Tyr Thr 100 105 110 Leu Gln Gly Leu Leu Gly Cys Glu Leu Gly
Pro Asp Asn Thr Ser Val 115 120 125 Pro Thr Ala Lys Phe Ala Leu Asn
Gly Glu Glu Phe Met Asn Phe Asp 130 135 140 Leu Lys Gln Gly Thr Trp
Gly Gly Asp Trp Pro Glu Ala Leu Ala Ile 145 150 155 160 Ser Gln Arg
Trp Gln Gln Gln Asp Lys Ala Ala Asn Lys Glu Leu Thr 165 170 175 Phe
Leu Leu Phe Ser Cys Pro His Arg Leu Arg Glu His Leu Glu Arg 180 185
190 Gly Arg Gly Asn Leu Glu Trp Lys Glu Pro Pro Ser Met Arg Leu Lys
195 200 205 Ala Arg Pro Ser Ser Pro Gly Phe Ser Val Leu Thr Cys Ser
Ala Phe 210 215 220 Ser Phe Tyr Pro Pro Glu Leu Gln Leu Arg Phe Leu
Arg Asn Gly Leu 225 230 235 240 Ala Ala Gly Thr Gly Gln Gly Asp Phe
Gly Pro Asn Ser Asp Gly Ser 245 250 255 Phe His Ala Ser Ser Ser Leu
Thr Val Lys Ser Gly Asp Glu His His 260 265 270 Tyr Cys Cys Ile Val
Gln His Ala Gly Leu Ala Gln Pro Leu Arg Val 275 280 285 Glu Leu Glu
Ser Pro Ala Lys Ser Ser Val Leu Val Val Gly Ile Val 290 295 300 Ile
Gly Val Leu Leu Leu Thr Ala Ala Ala Val Gly Gly Ala Leu Leu 305 310
315 320 Trp Arg Arg Met Arg Ser Gly Leu Pro Ala Pro Trp Ile Ser Leu
Arg 325 330 335 Gly Asp Asp Thr Gly Val Leu Leu Pro Thr Pro Gly Glu
Ala Gln Asp 340 345 350 Ala Asp Leu Lys Asp Val Asn Val Ile Pro Ala
Thr Ala 355 360 365 18119PRTHomo sapiens 18Met Ser Arg Ser Val Ala
Leu Ala Val Leu Ala Leu Leu Ser Leu Ser 1 5 10 15 Gly Leu Glu Ala
Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg 20 25 30 His Pro
Ala Glu Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser 35 40 45
Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu 50
55 60 Arg Ile Glu Lys Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp
Trp 65 70 75 80 Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr
Glu Lys Asp 85 90 95 Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu
Ser Gln Pro Lys Ile 100 105 110 Val Lys Trp Asp Arg Asp Met 115
191125DNAHomo sapiensCDS(1)..(1125) 19atg tgg ttc ttg aca act ctg
ctc ctt tgg gtt cca gtt gat ggg caa 48Met Trp Phe Leu Thr Thr Leu
Leu Leu Trp Val Pro Val Asp Gly Gln 1 5 10 15 gtg gac acc aca aag
gca gtg atc act ttg cag cct cca tgg gtc agc 96Val Asp Thr Thr Lys
Ala Val Ile Thr Leu Gln Pro Pro Trp Val Ser 20 25 30 gtg ttc caa
gag gaa acc gta acc ttg cac tgt gag gtg ctc cat ctg 144Val Phe Gln
Glu Glu Thr Val Thr Leu His Cys Glu Val Leu His Leu 35 40 45 cct
ggg agc agc tct aca cag tgg ttt ctc aat ggc aca gcc act cag 192Pro
Gly Ser Ser Ser Thr Gln Trp Phe Leu Asn Gly Thr Ala Thr Gln 50 55
60 acc tcg acc ccc agc tac aga atc acc tct gcc agt gtc aat gac agt
240Thr Ser Thr Pro Ser Tyr Arg Ile Thr Ser Ala Ser Val Asn Asp Ser
65 70 75 80 ggt gaa tac agg tgc cag aga ggt ctc tca ggg cga agt gac
ccc ata 288Gly Glu Tyr Arg Cys Gln Arg Gly Leu Ser Gly Arg Ser Asp
Pro Ile 85 90 95 cag ctg gaa atc cac aga ggc tgg cta cta ctg cag
gtc tcc agc aga 336Gln Leu Glu Ile His Arg Gly Trp Leu Leu Leu Gln
Val Ser Ser Arg 100 105 110 gtc ttc acg gaa gga gaa cct ctg gcc ttg
agg tgt cat gcg tgg aag 384Val Phe Thr Glu Gly Glu Pro Leu Ala Leu
Arg Cys His Ala Trp Lys 115 120 125 gat aag ctg gtg tac aat gtg ctt
tac tat cga aat ggc aaa gcc ttt 432Asp Lys Leu Val Tyr Asn Val Leu
Tyr Tyr Arg Asn Gly Lys Ala Phe 130 135 140 aag ttt ttc cac tgg aat
tct aac ctc acc att ctg aaa acc aac ata 480Lys Phe Phe His Trp Asn
Ser Asn Leu Thr Ile Leu Lys Thr Asn Ile 145 150 155 160 agt cac aat
ggc acc tac cat tgc tca ggc atg gga aag cat cgc tac 528Ser His Asn
Gly Thr Tyr His Cys Ser Gly Met Gly Lys His Arg Tyr 165 170 175 aca
tca gca gga ata tct gtc act gtg aaa gag cta ttt cca gct cca 576Thr
Ser Ala Gly Ile Ser Val Thr Val Lys Glu Leu Phe Pro Ala Pro 180 185
190 gtg ctg aat gca tct gtg aca tcc cca ctc ctg gag ggg aat ctg gtc
624Val Leu Asn Ala Ser Val Thr Ser Pro Leu Leu Glu Gly Asn Leu Val
195 200 205 acc ctg agc tgt gaa aca aag ttg ctc ttg cag agg cct ggt
ttg cag 672Thr Leu Ser Cys Glu Thr Lys Leu Leu Leu Gln Arg Pro Gly
Leu Gln 210 215 220 ctt tac ttc tcc ttc tac atg ggc agc aag acc ctg
cga ggc agg aac 720Leu Tyr Phe Ser Phe Tyr Met Gly Ser Lys Thr Leu
Arg Gly Arg Asn 225 230 235 240 aca tcc tct gaa tac caa ata cta act
gct aga aga gaa gac tct ggg 768Thr Ser Ser Glu Tyr Gln Ile Leu Thr
Ala Arg Arg Glu Asp Ser Gly 245 250 255 tta tac tgg tgc gag gct gcc
aca gag gat gga aat gtc ctt aag cgc 816Leu Tyr Trp Cys Glu Ala Ala
Thr Glu Asp Gly Asn Val Leu Lys Arg 260 265 270 agc cct gag ttg gag
ctt caa gtg ctt ggc ctc cag tta cca act cct 864Ser Pro Glu Leu Glu
Leu Gln Val Leu Gly Leu Gln Leu Pro Thr Pro 275 280 285 gtc tgg ttt
cat gtc ctt ttc tat ctg gca gtg gga ata atg ttt tta 912Val Trp Phe
His Val Leu Phe Tyr Leu Ala Val Gly Ile Met Phe Leu 290 295 300 gtg
aac act gtt ctc tgg gtg aca ata cgt aaa gaa ctg aaa aga aag 960Val
Asn Thr Val Leu Trp Val Thr Ile Arg Lys Glu Leu Lys Arg Lys 305 310
315 320 aaa aag tgg gat tta gaa atc tct ttg gat tct ggt cat gag aag
aag 1008Lys Lys Trp Asp Leu Glu Ile Ser Leu Asp Ser Gly His Glu Lys
Lys 325 330 335 gta att tcc agc ctt caa gaa gac aga cat tta gaa gaa
gag ctg aaa 1056Val Ile Ser Ser Leu Gln Glu Asp Arg His Leu Glu Glu
Glu Leu Lys 340 345 350 tgt cag gaa caa aaa gaa gaa cag ctg cag gaa
ggg gtg cac cgg aag 1104Cys Gln Glu Gln Lys Glu Glu Gln Leu Gln Glu
Gly Val His Arg Lys 355 360 365 gag ccc cag ggg gcc acg tag 1125Glu
Pro Gln Gly Ala Thr 370 20374PRTHomo sapiens 20Met Trp Phe Leu Thr
Thr Leu Leu Leu Trp Val Pro Val Asp Gly Gln 1 5 10 15 Val Asp Thr
Thr Lys Ala Val Ile Thr Leu Gln Pro Pro Trp Val Ser
20 25 30 Val Phe Gln Glu Glu Thr Val Thr Leu His Cys Glu Val Leu
His Leu 35 40 45 Pro Gly Ser Ser Ser Thr Gln Trp Phe Leu Asn Gly
Thr Ala Thr Gln 50 55 60 Thr Ser Thr Pro Ser Tyr Arg Ile Thr Ser
Ala Ser Val Asn Asp Ser 65 70 75 80 Gly Glu Tyr Arg Cys Gln Arg Gly
Leu Ser Gly Arg Ser Asp Pro Ile 85 90 95 Gln Leu Glu Ile His Arg
Gly Trp Leu Leu Leu Gln Val Ser Ser Arg 100 105 110 Val Phe Thr Glu
Gly Glu Pro Leu Ala Leu Arg Cys His Ala Trp Lys 115 120 125 Asp Lys
Leu Val Tyr Asn Val Leu Tyr Tyr Arg Asn Gly Lys Ala Phe 130 135 140
Lys Phe Phe His Trp Asn Ser Asn Leu Thr Ile Leu Lys Thr Asn Ile 145
150 155 160 Ser His Asn Gly Thr Tyr His Cys Ser Gly Met Gly Lys His
Arg Tyr 165 170 175 Thr Ser Ala Gly Ile Ser Val Thr Val Lys Glu Leu
Phe Pro Ala Pro 180 185 190 Val Leu Asn Ala Ser Val Thr Ser Pro Leu
Leu Glu Gly Asn Leu Val 195 200 205 Thr Leu Ser Cys Glu Thr Lys Leu
Leu Leu Gln Arg Pro Gly Leu Gln 210 215 220 Leu Tyr Phe Ser Phe Tyr
Met Gly Ser Lys Thr Leu Arg Gly Arg Asn 225 230 235 240 Thr Ser Ser
Glu Tyr Gln Ile Leu Thr Ala Arg Arg Glu Asp Ser Gly 245 250 255 Leu
Tyr Trp Cys Glu Ala Ala Thr Glu Asp Gly Asn Val Leu Lys Arg 260 265
270 Ser Pro Glu Leu Glu Leu Gln Val Leu Gly Leu Gln Leu Pro Thr Pro
275 280 285 Val Trp Phe His Val Leu Phe Tyr Leu Ala Val Gly Ile Met
Phe Leu 290 295 300 Val Asn Thr Val Leu Trp Val Thr Ile Arg Lys Glu
Leu Lys Arg Lys 305 310 315 320 Lys Lys Trp Asp Leu Glu Ile Ser Leu
Asp Ser Gly His Glu Lys Lys 325 330 335 Val Ile Ser Ser Leu Gln Glu
Asp Arg His Leu Glu Glu Glu Leu Lys 340 345 350 Cys Gln Glu Gln Lys
Glu Glu Gln Leu Gln Glu Gly Val His Arg Lys 355 360 365 Glu Pro Gln
Gly Ala Thr 370 21951DNAHomo sapiensCDS(1)..(951) 21atg act atg gag
acc caa atg tct cag aat gta tgt ccc aga aac ctg 48Met Thr Met Glu
Thr Gln Met Ser Gln Asn Val Cys Pro Arg Asn Leu 1 5 10 15 tgg ctg
ctt caa cca ttg aca gtt ttg ctg ctg ctg gct tct gca gac 96Trp Leu
Leu Gln Pro Leu Thr Val Leu Leu Leu Leu Ala Ser Ala Asp 20 25 30
agt caa gct gct ccc cca aag gct gtg ctg aaa ctt gag ccc ccg tgg
144Ser Gln Ala Ala Pro Pro Lys Ala Val Leu Lys Leu Glu Pro Pro Trp
35 40 45 atc aac gtg ctc cag gag gac tct gtg act ctg aca tgc cag
ggg gct 192Ile Asn Val Leu Gln Glu Asp Ser Val Thr Leu Thr Cys Gln
Gly Ala 50 55 60 cgc agc cct gag agc gac tcc att cag tgg ttc cac
aat ggg aat ctc 240Arg Ser Pro Glu Ser Asp Ser Ile Gln Trp Phe His
Asn Gly Asn Leu 65 70 75 80 att ccc acc cac acg cag ccc agc tac agg
ttc aag gcc aac aac aat 288Ile Pro Thr His Thr Gln Pro Ser Tyr Arg
Phe Lys Ala Asn Asn Asn 85 90 95 gac agc ggg gag tac acg tgc cag
act ggc cag acc agc ctc agc gac 336Asp Ser Gly Glu Tyr Thr Cys Gln
Thr Gly Gln Thr Ser Leu Ser Asp 100 105 110 cct gtg cat ctg act gtg
ctt tcc gaa tgg ctg gtg ctc cag acc cct 384Pro Val His Leu Thr Val
Leu Ser Glu Trp Leu Val Leu Gln Thr Pro 115 120 125 cac ctg gag ttc
cag gag gga gaa acc atc atg ctg agg tgc cac agc 432His Leu Glu Phe
Gln Glu Gly Glu Thr Ile Met Leu Arg Cys His Ser 130 135 140 tgg aag
gac aag cct ctg gtc aag gtc aca ttc ttc cag aat gga aaa 480Trp Lys
Asp Lys Pro Leu Val Lys Val Thr Phe Phe Gln Asn Gly Lys 145 150 155
160 tcc cag aaa ttc tcc cat ttg gat ccc acc ttc tcc atc cca caa gca
528Ser Gln Lys Phe Ser His Leu Asp Pro Thr Phe Ser Ile Pro Gln Ala
165 170 175 aac cac agt cac agt ggt gat tac cac tgc aca gga aac ata
ggc tac 576Asn His Ser His Ser Gly Asp Tyr His Cys Thr Gly Asn Ile
Gly Tyr 180 185 190 acg ctg ttc tca tcc aag cct gtg acc atc act gtc
caa gtg ccc agc 624Thr Leu Phe Ser Ser Lys Pro Val Thr Ile Thr Val
Gln Val Pro Ser 195 200 205 atg ggc agc tct tca cca atg ggg gtc att
gtg gct gtg gtc att gcg 672Met Gly Ser Ser Ser Pro Met Gly Val Ile
Val Ala Val Val Ile Ala 210 215 220 act gct gta gca gcc att gtt gct
gct gta gtg gcc ttg atc tac tgc 720Thr Ala Val Ala Ala Ile Val Ala
Ala Val Val Ala Leu Ile Tyr Cys 225 230 235 240 agg aaa aag cgg att
tca gcc aat tcc act gat cct gtg aag gct gcc 768Arg Lys Lys Arg Ile
Ser Ala Asn Ser Thr Asp Pro Val Lys Ala Ala 245 250 255 caa ttt gag
cca cct gga cgt caa atg att gcc atc aga aag aga caa 816Gln Phe Glu
Pro Pro Gly Arg Gln Met Ile Ala Ile Arg Lys Arg Gln 260 265 270 ctt
gaa gaa acc aac aat gac tat gaa aca gct gac ggc ggc tac atg 864Leu
Glu Glu Thr Asn Asn Asp Tyr Glu Thr Ala Asp Gly Gly Tyr Met 275 280
285 act ctg aac ccc agg gca cct act gac gat gat aaa aac atc tac ctg
912Thr Leu Asn Pro Arg Ala Pro Thr Asp Asp Asp Lys Asn Ile Tyr Leu
290 295 300 act ctt cct ccc aac gac cat gtc aac agt aat aac taa
951Thr Leu Pro Pro Asn Asp His Val Asn Ser Asn Asn 305 310 315
22316PRTHomo sapiens 22Met Thr Met Glu Thr Gln Met Ser Gln Asn Val
Cys Pro Arg Asn Leu 1 5 10 15 Trp Leu Leu Gln Pro Leu Thr Val Leu
Leu Leu Leu Ala Ser Ala Asp 20 25 30 Ser Gln Ala Ala Pro Pro Lys
Ala Val Leu Lys Leu Glu Pro Pro Trp 35 40 45 Ile Asn Val Leu Gln
Glu Asp Ser Val Thr Leu Thr Cys Gln Gly Ala 50 55 60 Arg Ser Pro
Glu Ser Asp Ser Ile Gln Trp Phe His Asn Gly Asn Leu 65 70 75 80 Ile
Pro Thr His Thr Gln Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn 85 90
95 Asp Ser Gly Glu Tyr Thr Cys Gln Thr Gly Gln Thr Ser Leu Ser Asp
100 105 110 Pro Val His Leu Thr Val Leu Ser Glu Trp Leu Val Leu Gln
Thr Pro 115 120 125 His Leu Glu Phe Gln Glu Gly Glu Thr Ile Met Leu
Arg Cys His Ser 130 135 140 Trp Lys Asp Lys Pro Leu Val Lys Val Thr
Phe Phe Gln Asn Gly Lys 145 150 155 160 Ser Gln Lys Phe Ser His Leu
Asp Pro Thr Phe Ser Ile Pro Gln Ala 165 170 175 Asn His Ser His Ser
Gly Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr 180 185 190 Thr Leu Phe
Ser Ser Lys Pro Val Thr Ile Thr Val Gln Val Pro Ser 195 200 205 Met
Gly Ser Ser Ser Pro Met Gly Val Ile Val Ala Val Val Ile Ala 210 215
220 Thr Ala Val Ala Ala Ile Val Ala Ala Val Val Ala Leu Ile Tyr Cys
225 230 235 240 Arg Lys Lys Arg Ile Ser Ala Asn Ser Thr Asp Pro Val
Lys Ala Ala 245 250 255 Gln Phe Glu Pro Pro Gly Arg Gln Met Ile Ala
Ile Arg Lys Arg Gln 260 265 270 Leu Glu Glu Thr Asn Asn Asp Tyr Glu
Thr Ala Asp Gly Gly Tyr Met 275 280 285 Thr Leu Asn Pro Arg Ala Pro
Thr Asp Asp Asp Lys Asn Ile Tyr Leu 290 295 300 Thr Leu Pro Pro Asn
Asp His Val Asn Ser Asn Asn 305 310 315 23876DNAHomo
sapiensCDS(1)..(876) 23atg gga atc ctg tca ttc tta cct gtc ctt gcc
act gag agt gac tgg 48Met Gly Ile Leu Ser Phe Leu Pro Val Leu Ala
Thr Glu Ser Asp Trp 1 5 10 15 gct gac tgc aag tcc ccc cag cct tgg
ggt cat atg ctt ctg tgg aca 96Ala Asp Cys Lys Ser Pro Gln Pro Trp
Gly His Met Leu Leu Trp Thr 20 25 30 gct gtg cta ttc ctg gct cct
gtt gct ggg aca cct gca gct ccc cca 144Ala Val Leu Phe Leu Ala Pro
Val Ala Gly Thr Pro Ala Ala Pro Pro 35 40 45 aag gct gtg ctg aaa
ctc gag ccc cag tgg atc aac gtg ctc cag gag 192Lys Ala Val Leu Lys
Leu Glu Pro Gln Trp Ile Asn Val Leu Gln Glu 50 55 60 gac tct gtg
act ctg aca tgc cgg ggg act cac agc cct gag agc gac 240Asp Ser Val
Thr Leu Thr Cys Arg Gly Thr His Ser Pro Glu Ser Asp 65 70 75 80 tcc
att cag tgg ttc cac aat ggg aat ctc att ccc acc cac acg cag 288Ser
Ile Gln Trp Phe His Asn Gly Asn Leu Ile Pro Thr His Thr Gln 85 90
95 ccc agc tac agg ttc aag gcc aac aac aat gac agc ggg gag tac acg
336Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn Asp Ser Gly Glu Tyr Thr
100 105 110 tgc cag act ggc cag acc agc ctc agc gac cct gtg cat ctg
act gtg 384Cys Gln Thr Gly Gln Thr Ser Leu Ser Asp Pro Val His Leu
Thr Val 115 120 125 ctt tct gag tgg ctg gtg ctc cag acc cct cac ctg
gag ttc cag gag 432Leu Ser Glu Trp Leu Val Leu Gln Thr Pro His Leu
Glu Phe Gln Glu 130 135 140 gga gaa acc atc gtg ctg agg tgc cac agc
tgg aag gac aag cct ctg 480Gly Glu Thr Ile Val Leu Arg Cys His Ser
Trp Lys Asp Lys Pro Leu 145 150 155 160 gtc aag gtc aca ttc ttc cag
aat gga aaa tcc aag aaa ttt tcc cgt 528Val Lys Val Thr Phe Phe Gln
Asn Gly Lys Ser Lys Lys Phe Ser Arg 165 170 175 tcg gat ccc aac ttc
tcc atc cca caa gca aac cac agt cac agt ggt 576Ser Asp Pro Asn Phe
Ser Ile Pro Gln Ala Asn His Ser His Ser Gly 180 185 190 gat tac cac
tgc aca gga aac ata ggc tac acg ctg tac tca tcc aag 624Asp Tyr His
Cys Thr Gly Asn Ile Gly Tyr Thr Leu Tyr Ser Ser Lys 195 200 205 cct
gtg acc atc act gtc caa gct ccc agc tct tca ccg atg ggg atc 672Pro
Val Thr Ile Thr Val Gln Ala Pro Ser Ser Ser Pro Met Gly Ile 210 215
220 att gtg gct gtg gtc act ggg att gct gta gcg gcc att gtt gct gct
720Ile Val Ala Val Val Thr Gly Ile Ala Val Ala Ala Ile Val Ala Ala
225 230 235 240 gta gtg gcc ttg atc tac tgc agg aaa aag cgg att tca
gcc aat ccc 768Val Val Ala Leu Ile Tyr Cys Arg Lys Lys Arg Ile Ser
Ala Asn Pro 245 250 255 act aat cct gat gag gct gac aaa gtt ggg gct
gag aac aca atc acc 816Thr Asn Pro Asp Glu Ala Asp Lys Val Gly Ala
Glu Asn Thr Ile Thr 260 265 270 tat tca ctt ctc atg cac ccg gat gct
ctg gaa gag cct gat gac cag 864Tyr Ser Leu Leu Met His Pro Asp Ala
Leu Glu Glu Pro Asp Asp Gln 275 280 285 aac cgt att tag 876Asn Arg
Ile 290 24291PRTHomo sapiens 24Met Gly Ile Leu Ser Phe Leu Pro Val
Leu Ala Thr Glu Ser Asp Trp 1 5 10 15 Ala Asp Cys Lys Ser Pro Gln
Pro Trp Gly His Met Leu Leu Trp Thr 20 25 30 Ala Val Leu Phe Leu
Ala Pro Val Ala Gly Thr Pro Ala Ala Pro Pro 35 40 45 Lys Ala Val
Leu Lys Leu Glu Pro Gln Trp Ile Asn Val Leu Gln Glu 50 55 60 Asp
Ser Val Thr Leu Thr Cys Arg Gly Thr His Ser Pro Glu Ser Asp 65 70
75 80 Ser Ile Gln Trp Phe His Asn Gly Asn Leu Ile Pro Thr His Thr
Gln 85 90 95 Pro Ser Tyr Arg Phe Lys Ala Asn Asn Asn Asp Ser Gly
Glu Tyr Thr 100 105 110 Cys Gln Thr Gly Gln Thr Ser Leu Ser Asp Pro
Val His Leu Thr Val 115 120 125 Leu Ser Glu Trp Leu Val Leu Gln Thr
Pro His Leu Glu Phe Gln Glu 130 135 140 Gly Glu Thr Ile Val Leu Arg
Cys His Ser Trp Lys Asp Lys Pro Leu 145 150 155 160 Val Lys Val Thr
Phe Phe Gln Asn Gly Lys Ser Lys Lys Phe Ser Arg 165 170 175 Ser Asp
Pro Asn Phe Ser Ile Pro Gln Ala Asn His Ser His Ser Gly 180 185 190
Asp Tyr His Cys Thr Gly Asn Ile Gly Tyr Thr Leu Tyr Ser Ser Lys 195
200 205 Pro Val Thr Ile Thr Val Gln Ala Pro Ser Ser Ser Pro Met Gly
Ile 210 215 220 Ile Val Ala Val Val Thr Gly Ile Ala Val Ala Ala Ile
Val Ala Ala 225 230 235 240 Val Val Ala Leu Ile Tyr Cys Arg Lys Lys
Arg Ile Ser Ala Asn Pro 245 250 255 Thr Asn Pro Asp Glu Ala Asp Lys
Val Gly Ala Glu Asn Thr Ile Thr 260 265 270 Tyr Ser Leu Leu Met His
Pro Asp Ala Leu Glu Glu Pro Asp Asp Gln 275 280 285 Asn Arg Ile 290
25765DNAHomo sapiensCDS(1)..(765) 25atg tgg cag ctg ctc ctc cca act
gct ctg cta ctt cta gtt tca gct 48Met Trp Gln Leu Leu Leu Pro Thr
Ala Leu Leu Leu Leu Val Ser Ala 1 5 10 15 ggc atg cgg act gaa gat
ctc cca aag gct gtg gtg ttc ctg gag cct 96Gly Met Arg Thr Glu Asp
Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30 caa tgg tac agg
gtg ctc gag aag gac agt gtg act ctg aag tgc cag 144Gln Trp Tyr Arg
Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45 gga gcc
tac tcc cct gag gac aat tcc aca cag tgg ttt cac aat gag 192Gly Ala
Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60
agc ctc atc tca agc cag gcc tcg agc tac ttc att gac gct gcc aca
240Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr
65 70 75 80 gtt gac gac agt gga gag tac agg tgc cag aca aac ctc tcc
acc ctc 288Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser
Thr Leu 85 90 95 agt gac ccg gtg cag cta gaa gtc cat atc ggc tgg
ctg ttg ctc cag 336Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp
Leu Leu Leu Gln 100 105 110 gcc cct cgg tgg gtg ttc aag gag gaa gac
cct att cac ctg agg tgt 384Ala Pro Arg Trp Val Phe Lys Glu Glu Asp
Pro Ile His Leu Arg Cys 115 120 125 cac agc tgg aag aac act gct ctg
cat aag gtc aca tat tta cag aat 432His Ser Trp Lys Asn Thr Ala Leu
His Lys Val Thr Tyr Leu Gln Asn 130 135 140 ggc aaa ggc agg aag tat
ttt cat cat aat tct gac ttc tac att cca 480Gly Lys Gly Arg Lys Tyr
Phe His His Asn Ser Asp Phe Tyr Ile
Pro 145 150 155 160 aaa gcc aca ctc aaa gac agc ggc tcc tac ttc tgc
agg ggg ctt gtt 528Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys
Arg Gly Leu Val 165 170 175 ggg agt aaa aat gtg tct tca gag act gtg
aac atc acc atc act caa 576Gly Ser Lys Asn Val Ser Ser Glu Thr Val
Asn Ile Thr Ile Thr Gln 180 185 190 ggt ttg tca gtg tca acc atc tca
tca ttc ttt cca cct ggg tac caa 624Gly Leu Ser Val Ser Thr Ile Ser
Ser Phe Phe Pro Pro Gly Tyr Gln 195 200 205 gtc tct ttc tgc ttg gtg
atg gta ctc ctt ttt gca gtg gac aca gga 672Val Ser Phe Cys Leu Val
Met Val Leu Leu Phe Ala Val Asp Thr Gly 210 215 220 cta tat ttc tct
gtg aag aca aac att cga agc tca aca aga gac tgg 720Leu Tyr Phe Ser
Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp 225 230 235 240 aag
gac cat aaa ttt aaa tgg aga aag gac cct caa gac aaa tga 765Lys Asp
His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp Lys 245 250
26254PRTHomo sapiens 26Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu
Leu Leu Val Ser Ala 1 5 10 15 Gly Met Arg Thr Glu Asp Leu Pro Lys
Ala Val Val Phe Leu Glu Pro 20 25 30 Gln Trp Tyr Arg Val Leu Glu
Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40 45 Gly Ala Tyr Ser Pro
Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu 50 55 60 Ser Leu Ile
Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala Ala Thr 65 70 75 80 Val
Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90
95 Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln
100 105 110 Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu
Arg Cys 115 120 125 His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr
Tyr Leu Gln Asn 130 135 140 Gly Lys Gly Arg Lys Tyr Phe His His Asn
Ser Asp Phe Tyr Ile Pro 145 150 155 160 Lys Ala Thr Leu Lys Asp Ser
Gly Ser Tyr Phe Cys Arg Gly Leu Val 165 170 175 Gly Ser Lys Asn Val
Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln 180 185 190 Gly Leu Ser
Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln 195 200 205 Val
Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala Val Asp Thr Gly 210 215
220 Leu Tyr Phe Ser Val Lys Thr Asn Ile Arg Ser Ser Thr Arg Asp Trp
225 230 235 240 Lys Asp His Lys Phe Lys Trp Arg Lys Asp Pro Gln Asp
Lys 245 250 27702DNAHomo sapiensCDS(1)..(702) 27atg tgg cag ctg ctc
ctc cca act gct ctg cta ctt cta gtt tca gct 48Met Trp Gln Leu Leu
Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala 1 5 10 15 ggc atg cgg
act gaa gat ctc cca aag gct gtg gtg ttc ctg gag cct 96Gly Met Arg
Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu Pro 20 25 30 caa
tgg tac agc gtg ctt gag aag gac agt gtg act ctg aag tgc cag 144Gln
Trp Tyr Ser Val Leu Glu Lys Asp Ser Val Thr Leu Lys Cys Gln 35 40
45 gga gcc tac tcc cct gag gac aat tcc aca cag tgg ttt cac aat gag
192Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp Phe His Asn Glu
50 55 60 agc ctc atc tca agc cag gcc tcg agc tac ttc att gac gct
gcc aca 240Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile Asp Ala
Ala Thr 65 70 75 80 gtc aac gac agt gga gag tac agg tgc cag aca aac
ctc tcc acc ctc 288Val Asn Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn
Leu Ser Thr Leu 85 90 95 agt gac ccg gtg cag cta gaa gtc cat atc
ggc tgg ctg ttg ctc cag 336Ser Asp Pro Val Gln Leu Glu Val His Ile
Gly Trp Leu Leu Leu Gln 100 105 110 gcc cct cgg tgg gtg ttc aag gag
gaa gac cct att cac ctg agg tgt 384Ala Pro Arg Trp Val Phe Lys Glu
Glu Asp Pro Ile His Leu Arg Cys 115 120 125 cac agc tgg aag aac act
gct ctg cat aag gtc aca tat tta cag aat 432His Ser Trp Lys Asn Thr
Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135 140 ggc aaa gac agg
aag tat ttt cat cat aat tct gac ttc cac att cca 480Gly Lys Asp Arg
Lys Tyr Phe His His Asn Ser Asp Phe His Ile Pro 145 150 155 160 aaa
gcc aca ctc aaa gat agc ggc tcc tac ttc tgc agg ggg ctt gtt 528Lys
Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg Gly Leu Val 165 170
175 ggg agt aaa aat gtg tct tca gag act gtg aac atc acc atc act caa
576Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile Thr Ile Thr Gln
180 185 190 ggt ttg gca gtg tca acc atc tca tca ttc tct cca cct ggg
tac caa 624Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Ser Pro Pro Gly
Tyr Gln 195 200 205 gtc tct ttc tgc ttg gtg atg gta ctc ctt ttt gca
gtg gac aca gga 672Val Ser Phe Cys Leu Val Met Val Leu Leu Phe Ala
Val Asp Thr Gly 210 215 220 cta tat ttc tct gtg aag aca aac att tga
702Leu Tyr Phe Ser Val Lys Thr Asn Ile 225 230 28233PRTHomo sapiens
28Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu Leu Val Ser Ala 1
5 10 15 Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val Phe Leu Glu
Pro 20 25 30 Gln Trp Tyr Ser Val Leu Glu Lys Asp Ser Val Thr Leu
Lys Cys Gln 35 40 45 Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln
Trp Phe His Asn Glu 50 55 60 Ser Leu Ile Ser Ser Gln Ala Ser Ser
Tyr Phe Ile Asp Ala Ala Thr 65 70 75 80 Val Asn Asp Ser Gly Glu Tyr
Arg Cys Gln Thr Asn Leu Ser Thr Leu 85 90 95 Ser Asp Pro Val Gln
Leu Glu Val His Ile Gly Trp Leu Leu Leu Gln 100 105 110 Ala Pro Arg
Trp Val Phe Lys Glu Glu Asp Pro Ile His Leu Arg Cys 115 120 125 His
Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr Tyr Leu Gln Asn 130 135
140 Gly Lys Asp Arg Lys Tyr Phe His His Asn Ser Asp Phe His Ile Pro
145 150 155 160 Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys Arg
Gly Leu Val 165 170 175 Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn
Ile Thr Ile Thr Gln 180 185 190 Gly Leu Ala Val Ser Thr Ile Ser Ser
Phe Ser Pro Pro Gly Tyr Gln 195 200 205 Val Ser Phe Cys Leu Val Met
Val Leu Leu Phe Ala Val Asp Thr Gly 210 215 220 Leu Tyr Phe Ser Val
Lys Thr Asn Ile 225 230 294PRTArtificial Sequencean artificially
synthesized sequence 29Gly Gly Gly Ser 1 304PRTArtificial
Sequencean artificially synthesized sequence 30Ser Gly Gly Gly 1
315PRTArtificial Sequencean artificially synthesized sequence 31Gly
Gly Gly Gly Ser 1 5 325PRTArtificial Sequencean artificially
synthesized sequence 32Ser Gly Gly Gly Gly 1 5 336PRTArtificial
Sequencean artificially synthesized sequence 33Gly Gly Gly Gly Gly
Ser 1 5 346PRTArtificial Sequencean artificially synthesized
sequence 34Ser Gly Gly Gly Gly Gly 1 5 357PRTArtificial Sequencean
artificially synthesized sequence 35Gly Gly Gly Gly Gly Gly Ser 1 5
367PRTArtificial Sequencean artificially synthesized sequence 36Ser
Gly Gly Gly Gly Gly Gly 1 5 37450PRTArtificial Sequencean
artificially synthesized sequence 37Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly
Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65
70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Val Ala Pro Gly Asn Trp Gly Ser Pro Tyr
Phe Asp Tyr Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro 115 120 125 Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr 130 135 140 Ala Ala Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr 145 150 155 160 Val Ser Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175 Ala
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185
190 Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
195 200 205 His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser 210 215 220 Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu 225 230 235 240 Gly Gly Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu 245 250 255 Met Ile Ser Arg Thr Pro Glu
Val Thr Cys Val Val Val Asp Val Ser 260 265 270 His Glu Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu 275 280 285 Val His Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr 290 295 300 Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn 305 310
315 320 Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
Pro 325 330 335 Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu Pro Gln 340 345 350 Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
Thr Lys Asn Gln Val 355 360 365 Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr Pro Ser Asp Ile Ala Val 370 375 380 Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr Lys Thr Thr Pro 385 390 395 400 Pro Val Leu Asp
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr 405 410 415 Val Asp
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val 420 425 430
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu 435
440 445 Ser Pro 450 38214PRTArtificial Sequencean artificially
synthesized sequence 38Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Glu Asp Asp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Ser
Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Arg Asp Ser Ser Pro Leu 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe
Asn Arg Gly Glu Cys 210 39451PRTArtificial Sequencean artificially
synthesized sequence 39Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro
Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg
Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Pro Arg Trp Glu Thr Ala Ile Ser Ser Asp Ala Phe Asp Ile
100 105 110 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr
Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215
220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340
345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg
Trp Gln Gln
Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His
Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro 450
40214PRTArtificial Sequencean artificially synthesized sequence
40Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asp
Asp 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Glu Ala Ser Asn Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln His Ser Ser Ser Pro Leu 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
41451PRTArtificial Sequencean artificially synthesized sequence
41Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn
Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp
Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Pro Arg Trp
Glu Thr Ala Ile Ser Ser Asp Ala Phe Asp Ile 100 105 110 Trp Gly Gln
Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135
140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Leu Gly
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260
265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385
390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Trp His Tyr
Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro 450
42451PRTArtificial Sequencean artificially synthesized sequence
42Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser
Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn
Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp
Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Pro Arg Trp
Glu Thr Ala Ile Ser Ser Asp Ala Phe Asp Ile 100 105 110 Trp Gly Gln
Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115 120 125 Pro
Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 130 135
140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val
His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr
Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr
Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Arg Gly
Gly Pro Lys Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260
265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys
Val Ser Asn Lys Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385
390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro 450
43543PRTArtificial Sequencean artificially synthesized sequence
43Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala
Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp
Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Tyr Ser
Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser
Ala Ser Thr Gln Ser Pro Ser Val Phe Pro Leu Thr Arg 115 120 125 Cys
Cys Lys Asn Ile Pro Ser Asn Ala Thr Ser Val Thr Leu Gly Cys 130 135
140 Leu Ala Thr Gly Tyr Phe Pro Glu Pro Val Met Val Thr Trp Asp Thr
145 150 155 160 Gly Ser Leu Asn Gly Thr Thr Met Thr Leu Pro Ala Thr
Thr Leu Thr 165 170 175 Leu Ser Gly His Tyr Ala Thr Ile Ser Leu Leu
Thr Val Ser Gly Ala 180 185 190 Trp Ala Lys Gln Met Phe Thr Cys Arg
Val Ala His Thr Pro Ser Ser 195 200 205 Thr Asp Trp Val Asp Asn Lys
Thr Phe Ser Val Cys Ser Arg Asp Phe 210 215 220 Thr Pro Pro Thr Val
Lys Ile Leu Gln Ser Ser Cys Asp Gly Gly Gly 225 230 235 240 His Phe
Pro Pro Thr Ile Gln Leu Leu Cys Leu Val Ser Gly Tyr Thr 245 250 255
Pro Gly Thr Ile Asn Ile Thr Trp Leu Glu Asp Gly Gln Val Met Asp 260
265 270 Val Asp Leu Ser Thr Ala Ser Thr Thr Gln Glu Gly Glu Leu Ala
Ser 275 280 285 Thr Gln Ser Glu Leu Thr Leu Ser Gln Lys His Trp Leu
Ser Asp Arg 290 295 300 Thr Tyr Thr Cys Gln Val Thr Tyr Gln Gly His
Thr Phe Glu Asp Ser 305 310 315 320 Thr Lys Lys Cys Ala Asp Ser Asn
Pro Arg Gly Val Ser Ala Tyr Leu 325 330 335 Ser Arg Pro Ser Pro Phe
Asp Leu Phe Ile Arg Lys Ser Pro Thr Ile 340 345 350 Thr Cys Leu Val
Val Asp Leu Ala Pro Ser Lys Gly Thr Val Asn Leu 355 360 365 Thr Trp
Ser Arg Ala Ser Gly Lys Pro Val Asn His Ser Thr Arg Lys 370 375 380
Glu Glu Lys Gln Arg Asn Gly Thr Leu Thr Val Thr Ser Thr Leu Pro 385
390 395 400 Val Gly Thr Arg Asp Trp Ile Glu Gly Glu Thr Tyr Gln Cys
Arg Val 405 410 415 Thr His Pro His Leu Pro Arg Ala Leu Met Arg Ser
Thr Thr Lys Thr 420 425 430 Ser Gly Pro Arg Ala Ala Pro Glu Val Tyr
Ala Phe Ala Thr Pro Glu 435 440 445 Trp Pro Gly Ser Arg Asp Lys Arg
Thr Leu Ala Cys Leu Ile Gln Asn 450 455 460 Phe Met Pro Glu Asp Ile
Ser Val Gln Trp Leu His Asn Glu Val Gln 465 470 475 480 Leu Pro Asp
Ala Arg His Ser Thr Thr Gln Pro Arg Lys Thr Lys Gly 485 490 495 Ser
Gly Phe Phe Val Phe Ser Arg Leu Glu Val Thr Arg Ala Glu Trp 500 505
510 Glu Gln Lys Asp Glu Phe Ile Cys Arg Ala Val His Glu Ala Ala Ser
515 520 525 Pro Ser Gln Thr Val Gln Arg Ala Val Ser Val Asn Pro Gly
Lys 530 535 540 44219PRTArtificial Sequencean artificially
synthesized sequence 44Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu
Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser Cys Arg Ser
Ser Gln Ser Leu Val His Ser 20 25 30 Asn Arg Asn Thr Tyr Leu His
Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile
Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser
Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn 85 90
95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 45443PRTArtificial
Sequencean artificially synthesized sequence 45Gln Ser Leu Glu Glu
Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu
Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Ser Tyr His 20 25 30 Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40
45 Val Ile Asn Ser Ala Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly
50 55 60 Arg Phe Thr Val Ser Lys Thr Ser Thr Thr Val Asp Leu Asn
Leu Thr 65 70 75 80 Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys
Ala Arg Tyr Val 85 90 95 Phe Ser Ser Gly Ser His Asp Ile Trp Gly
Pro Gly Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170
175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly
180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn
Thr Lys 195 200 205 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys
Thr His Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val
Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295
300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys
305 310 315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr
Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro Pro Ser
340 345 350 Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro 435 440 4621PRTArtificial
Sequencean artificially synthesized sequence 46Ala Tyr Asp Met Thr
Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly 1 5 10 15 Gly Thr Val
Thr Ile Lys Cys Gln Ala Ser Gln Ser Ile Gly Ser Trp 20 25 30 Leu
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Glu Leu Ile 35 40
45 Tyr Gly Thr Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ile Gly
50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Gly Val
Glu Cys 65 70 75 80 Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Tyr
Ser Glu Asp Asn 85 90 95 Ile Asp Asn Ala Phe Gly Gly Gly Thr Glu
Val Val Val Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170
175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
4732PRTArtificial Sequencean artificially synthesized sequence
47Val Asp Asp Ala Pro Gly Asn Ser Gln Gln Ala Thr Pro Lys Asp Asn 1
5 10 15 Glu Ile Ser Thr Phe His Asn Leu Gly Asn Val His Ser Pro Leu
Lys 20 25 30 48543PRTArtificial Sequencean artificially synthesized
sequence 48Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr
Phe Thr Asp Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45 Gly Ala Leu Asp Pro Lys Thr Gly
Asp Thr Ala Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu
Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser
Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg
Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110
Val Ser Ser Ala Ser Thr Gln Ser Pro Ser Val Phe Pro Leu Thr Arg 115
120 125 Cys Cys Lys Asn Ile Pro Ser Asp Ala Thr Ser Val Thr Leu Gly
Cys 130 135 140 Leu Ala Thr Gly Tyr Phe Pro Glu Pro Val Met Val Thr
Trp Asp Thr 145 150 155 160 Gly Ser Leu Asp Gly Thr Thr Met Thr Leu
Pro Ala Thr Thr Leu Thr 165 170 175 Leu Ser Gly His Tyr Ala Thr Ile
Ser Leu Leu Thr Val Ser Gly Ala 180 185 190 Trp Ala Lys Gln Met Phe
Thr Cys Arg Val Ala His Thr Pro Ser Ser 195 200 205 Thr Asp Trp Val
Asp Asp Lys Thr Phe Ser Val Cys Ser Arg Asp Phe 210 215 220 Thr Pro
Pro Thr Val Lys Ile Leu Gln Ser Ser Cys Asp Gly Gly Gly 225 230 235
240 His Phe Pro Pro Thr Ile Gln Leu Leu Cys Leu Val Ser Gly Tyr Thr
245 250 255 Pro Gly Thr Ile Asp Ile Thr Trp Leu Glu Asp Gly Gln Val
Met Asp 260 265 270 Val Asp Leu Ser Thr Ala Ser Thr Thr Gln Glu Gly
Glu Leu Ala Ser 275 280 285 Thr Gln Ser Glu Leu Thr Leu Ser Gln Lys
His Trp Leu Ser Asp Arg 290 295 300 Thr Tyr Thr Cys Gln Val Thr Tyr
Gln Gly His Thr Phe Glu Asp Ser 305 310 315 320 Thr Lys Lys Cys Ala
Asp Ser Asn Pro Arg Gly Val Ser Ala Tyr Leu 325 330 335 Ser Arg Pro
Ser Pro Phe Asp Leu Phe Ile Arg Lys Ser Pro Thr Ile 340 345 350 Thr
Cys Leu Val Val Asp Leu Ala Pro Ser Lys Gly Thr Val Asp Leu 355 360
365 Thr Trp Ser Arg Ala Ser Gly Lys Pro Val Asp His Ser Thr Arg Lys
370 375 380 Glu Glu Lys Gln Arg Asn Gly Thr Leu Thr Val Thr Ser Thr
Leu Pro 385 390 395 400 Val Gly Thr Arg Asp Trp Ile Glu Gly Glu Thr
Tyr Gln Cys Arg Val 405 410 415 Thr His Pro His Leu Pro Arg Ala Leu
Met Arg Ser Thr Thr Lys Thr 420 425 430 Ser Gly Pro Arg Ala Ala Pro
Glu Val Tyr Ala Phe Ala Thr Pro Glu 435 440 445 Trp Pro Gly Ser Arg
Asp Lys Arg Thr Leu Ala Cys Leu Ile Gln Asn 450 455 460 Phe Met Pro
Glu Asp Ile Ser Val Gln Trp Leu His Asn Glu Val Gln 465 470 475 480
Leu Pro Asp Ala Arg His Ser Thr Thr Gln Pro Arg Lys Thr Lys Gly 485
490 495 Ser Gly Phe Phe Val Phe Ser Arg Leu Glu Val Thr Arg Ala Glu
Trp 500 505 510 Glu Gln Lys Asp Glu Phe Ile Cys Arg Ala Val His Glu
Ala Ala Ser 515 520 525 Pro Ser Gln Thr Val Gln Arg Ala Val Ser Val
Asn Pro Gly Lys 530 535 540 49464PRTArtificial Sequencean
artificially synthesized sequence 49Gln Val Gln Leu Gln Glu Ser Gly
Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys
Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser
Trp Val Arg Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly
Tyr Ile Ser Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60
Lys Ser Arg Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65
70 75 80 Leu Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr
Trp Gly Gln Gly 100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Pro
Thr Ser Pro Lys Val Phe 115 120 125 Pro Leu Ser Leu Cys Ser Thr Gln
Pro Asp Gly Asn Val Val Ile Ala 130 135 140 Cys Leu Val Gln Gly Phe
Phe Pro Gln Glu Pro Leu Ser Val Thr Trp 145 150 155 160 Ser Glu Ser
Gly Gln Gly Val Thr Ala Arg Asn Phe Pro Pro Ser Gln 165 170 175 Asp
Ala Ser Gly Asp Leu Tyr Thr Thr Ser Ser Gln Leu Thr Leu Pro 180 185
190 Ala Thr Gln Cys Leu Ala Gly Lys Ser Val Thr Cys His Val Lys His
195 200 205 Tyr Thr Asn Pro Ser Gln Asp Val Thr Val Pro Cys Pro Val
Pro Ser 210 215 220 Thr Pro Pro Thr Pro Ser Pro Ser Thr Pro Pro Thr
Pro Ser Pro Ser 225 230 235 240 Cys Cys His Pro Arg Leu Ser Leu His
Arg Pro Ala Leu Glu Asp Leu 245 250 255 Leu Leu Gly Ser Glu Ala Asn
Leu Thr Cys Thr Leu Thr Gly Leu Arg 260 265 270 Asp Ala Ser Gly Val
Thr Phe Thr Trp Thr Pro Ser Ser Gly Lys Ser 275 280 285 Ala Val Gln
Gly Pro Pro Glu Arg Asp Leu Cys Gly Cys Tyr Ser Val 290 295 300 Ser
Ser Val Leu Pro Gly Cys Ala Glu Pro Trp Asn His Gly Lys Thr 305 310
315 320 Phe Thr Cys Thr Ala Ala Tyr Pro Glu Ser Lys Thr Pro Leu Thr
Ala 325 330 335 Thr Leu Ser Lys Ser Gly Asn Thr Phe Arg Pro Glu Val
His Leu Leu 340 345 350 Pro Pro Pro Ser Glu Glu Leu Ala Leu Asn Glu
Leu Val Thr Leu Thr 355 360 365 Cys Leu Ala Arg Gly Phe Ser Pro Lys
Asp Val Leu Val Arg Trp Leu 370 375 380 Gln Gly Ser Gln Glu Leu Pro
Arg Glu Lys Tyr Leu Thr Trp Ala Ser 385 390 395 400 Arg Gln Glu Pro
Ser Gln Gly Thr Thr Thr Phe Ala Val Thr Ser Ile 405 410 415 Leu Arg
Val Ala Ala Glu Asp Trp Lys Lys Gly Asp Thr Phe Ser Cys 420 425 430
Met Val Gly His Glu Ala Leu Pro Leu Ala Phe Thr Gln Lys Thr Ile 435
440 445 Asp Arg Leu Ala Gly Lys Glu Gln Lys Leu Ile Ser Glu Glu Asp
Leu 450 455 460 50214PRTArtificial Sequencean artificially
synthesized sequence 50Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys
Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg
Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu
Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90
95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp
Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe
Asn Arg Gly Glu Cys 210 51545PRTHomo sapiens 51Gln Pro Pro Pro Pro
Pro Pro Asp Ala Thr Cys His Gln Val Arg Ser 1 5 10 15 Phe Phe Gln
Arg Leu Gln Pro Gly Leu Lys Trp Val Pro Glu Thr Pro 20 25 30 Val
Pro Gly Ser Asp Leu Gln Val Cys Leu Pro Lys Gly Pro Thr Cys 35 40
45 Cys Ser Arg Lys Met Glu Glu Lys Tyr Gln Leu Thr Ala Arg Leu Asn
50 55 60 Met Glu Gln Leu Leu Gln Ser Ala Ser Met Glu Leu Lys Phe
Leu Ile 65 70 75 80 Ile Gln Asn Ala Ala Val Phe Gln Glu Ala Phe Glu
Ile Val Val Arg 85 90 95 His Ala Lys Asn Tyr Thr Asn Ala Met Phe
Lys Asn Asn Tyr Pro Ser 100 105 110 Leu Thr Pro Gln Ala Phe Glu Phe
Val Gly Glu Phe Phe Thr Asp Val 115 120 125 Ser Leu Tyr Ile Leu Gly
Ser Asp Ile Asn Val Asp Asp Met Val Asn 130 135 140 Glu Leu Phe Asp
Ser Leu Phe Pro Val Ile Tyr Thr Gln Leu Met Asn 145 150 155 160 Pro
Gly Leu Pro Asp Ser Ala Leu Asp Ile Asn Glu Cys Leu Arg Gly 165 170
175 Ala Arg Arg Asp Leu Lys Val Phe Gly Asn Phe Pro Lys Leu Ile Met
180 185 190 Thr Gln Val Ser Lys Ser Leu Gln Val Thr Arg Ile Phe Leu
Gln Ala 195 200 205 Leu Asn Leu Gly Ile Glu Val Ile Asn Thr Thr Asp
His Leu Lys Phe 210 215 220 Ser Lys Asp Cys Gly Arg Met Leu Thr Arg
Met Trp Tyr Cys Ser Tyr 225 230 235 240 Cys Gln Gly Leu Met Met Val
Lys Pro Cys Gly Gly Tyr Cys Asn Val 245 250 255 Val Met Gln Gly Cys
Met Ala Gly Val Val Glu Ile Asp Lys Tyr Trp 260 265 270 Arg Glu Tyr
Ile Leu Ser Leu Glu Glu Leu Val Asn Gly Met Tyr Arg 275 280 285 Ile
Tyr Asp Met Glu Asn Val Leu Leu Gly Leu Phe Ser Thr Ile His 290 295
300 Asp Ser Ile Gln Tyr Val Gln Lys Asn Ala Gly Lys Leu Thr Thr Thr
305 310 315 320 Ile Gly Lys Leu Cys Ala His Ser Gln Gln Arg Gln Tyr
Arg Ser Ala 325 330 335 Tyr Tyr Pro Glu Asp Leu Phe Ile Asp Lys Lys
Val Leu Lys Val Ala 340 345 350 His Val Glu His Glu Glu Thr Leu Ser
Ser Arg Arg Arg Glu Leu Ile 355 360 365 Gln Lys Leu Lys Ser Phe Ile
Ser Phe Tyr Ser Ala Leu Pro Gly Tyr 370 375 380 Ile Cys Ser His Ser
Pro Val Ala Glu Asn Asp Thr Leu Cys Trp Asn 385 390 395 400 Gly Gln
Glu Leu Val Glu Arg Tyr Ser Gln Lys Ala Ala Arg Asn Gly 405 410 415
Met Lys Asn Gln Phe Asn Leu His Glu Leu Lys Met Lys Gly Pro Glu 420
425 430 Pro Val Val Ser Gln Ile Ile Asp Lys Leu Lys His Ile Asn Gln
Leu 435 440 445 Leu Arg Thr Met Ser Met Pro Lys Gly Arg Val Leu Asp
Lys Asn Leu 450 455 460 Asp Glu Glu Gly Phe Glu Ala Gly Asp Cys Gly
Asp Asp Glu Asp Glu 465 470 475 480 Cys Ile Gly Gly Ala Gly Asp Gly
Met Ile Lys Val Lys Asn Gln Leu 485 490 495 Arg Phe Leu Ala Glu Leu
Ala Tyr Asp Leu Asp Val Asp Asp Ala Pro 500 505 510 Gly Asn Ser Gln
Gln Ala Thr Pro Lys Asp Asn Glu Ile Ser Thr Phe 515 520 525 His Asn
Leu Gly Asn Val His Ser Pro Leu Lys His His His His His 530 535 540
His 545 52451PRTArtificial Sequencean artificially synthesized
sequence 52Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr
Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly
Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Ile Phe Gly
Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile
Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Pro Arg Trp Glu Thr Ala Ile Ser Ser Asp Ala Phe Asp Ile 100 105 110
Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly 115
120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215 220 Ser
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 225 230
235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Tyr Pro Ala 325 330 335 Pro Ile
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340 345 350
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 355
360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser Pro 450
53443PRTArtificial Sequencean artificially synthesized sequence
53Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Thr Pro 1
5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu Ser Ser Tyr
His 20 25 30 Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Ile Gly 35 40 45 Val Ile Asn Ser Ala Gly Asn Thr Tyr Tyr Ala
Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Val Ser Lys Thr Ser Thr
Thr Val Asp Leu Asn Leu Thr 65 70 75 80 Ser Leu Thr Thr Glu Asp Thr
Ala Thr Tyr Phe Cys Ala Arg Tyr Val 85 90 95 Phe Ser Ser Gly Ser
His Asp Ile Trp Gly Pro Gly Thr Leu Val Thr 100 105 110 Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115 120 125 Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 130 135
140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala
145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln
Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220 Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255
Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 260
265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Ala Tyr Pro Ala
Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg Asp Glu Leu
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385
390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Pro 435 440 5444PRTArtificial Sequencean artificially synthesized
sequence 54Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly
Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser Leu
Ser Ser Tyr His 20 25 30 Met Ser Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Ile Gly 35 40 45 Val Ile Asn Ser Ala Gly Asn Thr
Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr Val Ser Lys
Thr Ser Thr Thr Val Asp Leu Asn Leu Thr 65 70 75 80 Ser Leu Thr Thr
Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Tyr Val 85 90 95 Phe Ser
Ser Gly Ser His Asp Ile Trp Gly Pro Gly Thr Leu Val Thr 100 105 110
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 115
120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu
Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn
Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205 Val Asp Lys Lys
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215 220 Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu 225 230 235
240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu
245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu
Val Lys 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Asp Ala
Tyr Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360
365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro 435 440 5544PRTArtificial Sequencean artificially
synthesized sequence 55Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val
Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr Ala Ser Gly
Phe Ser Leu Ser Ser Tyr His 20 25 30 Met Ser Trp Val Arg Gln Ala
Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Val Ile Asn Ser Ala
Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60 Arg Phe Thr
Val Ser Lys Thr Ser Thr Thr Val Asp Leu Asn Leu Thr 65 70 75 80 Ser
Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg Tyr Val 85 90
95 Phe Ser Ser Gly Ser His Asp Ile Trp Gly Pro Gly Thr Leu Val Thr
100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu
Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu
Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185 190 Thr Gln Thr
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 195 200 205 Val
Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys 210 215
220 Pro Pro Cys Pro Ala Pro Glu Leu Arg Gly Gly Pro Lys Val Phe Leu
225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser His Glu
Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 325 330 335
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340
345 350 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val
Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro 435 440 5645PRTArtificial Sequencean
artificially synthesized sequence 56Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys
Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp
Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly
Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65
70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Pro Arg Trp Glu Thr Ala Ile Ser Ser Asp
Ala Phe Asp Ile 100 105 110 Trp Gly Gln Gly Thr Met Val Thr Val Ser
Ser Ala Ser Thr Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185
190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu Leu 225 230 235 240 Arg Arg Gly Pro Lys Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Tyr Ile Thr Arg Glu Pro
Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310
315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430
Val Met His Glu Ala Leu His Tyr His Val Thr Arg Lys Glu Leu Ser 435
440 445 Leu Ser Pro 450 57451PRTArtificial Sequencean artificially
synthesized sequence 57Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val
Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser
Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln
Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro
Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg
Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Pro Arg Trp Glu Thr Ala Ile Ser Ser Asp Ala Phe Asp Ile
100 105 110 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr
Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe 165
170 175 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val 180 185 190 Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val 195 200 205 Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys 210 215 220 Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu 225 230 235 240 Arg Gly Gly Pro Lys Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 245 250 255 Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 260 265 270 Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val 275 280 285
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290
295 300 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu 305 310 315 320 Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala 325 330 335 Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro 340 345 350 Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln 355 360 365 Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 370 375 380 Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 385 390 395 400 Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu 405 410
415 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
420 425 430 Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser 435 440 445 Leu Ser Pro 450 58443PRTArtificial Sequencean
artificially synthesized sequence 58Gln Ser Leu Glu Glu Ser Gly Gly
Arg Leu Val Thr Pro Gly Thr Pro 1 5 10 15 Leu Thr Leu Thr Cys Thr
Ala Ser Gly Phe Ser Leu Ser Ser Tyr His 20 25 30 Met Ser Trp Val
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile Gly 35 40 45 Val Ile
Asn Ser Ala Gly Asn Thr Tyr Tyr Ala Ser Trp Ala Lys Gly 50 55 60
Arg Phe Thr Val Ser Lys Thr Ser Thr Thr Val Asp Leu Asn Leu Thr 65
70 75 80 Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg
Tyr Val 85 90 95 Phe Ser Ser Gly Ser His Asp Ile Trp Gly Pro Gly
Thr Leu Val Thr 100 105 110 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe Pro Leu Ala Pro 115 120 125 Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu Gly Cys Leu Val 130 135 140 Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala 145 150 155 160 Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 165 170 175 Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 180 185
190 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
195 200 205 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His
Thr Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Leu Arg Arg Gly Pro
Lys Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu
Tyr Ile Thr Arg Glu Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp Pro Glu Val Lys 260 265 270 Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310
315 320 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser
Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
Pro Pro Ser 340 345 350 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Tyr 420 425 430
His Val Thr Arg Lys Glu Leu Ser Leu Ser Pro 435 440
59447PRTArtificial Sequencean artificially synthesized sequence
59Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1
5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His
Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly
Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala
Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260
265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385
390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro 435 440 445 60214PRTArtificial Sequencean
artificially synthesized sequence 60Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Ser Val Thr Ile Thr
Cys Gln Ala Ser Thr Asp Ile Ser Ser His 20 25 30 Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile 35 40 45 Tyr Tyr
Gly Ser His Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala 65
70 75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu
Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu Arg
Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185
190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205 Phe Asn Arg Gly Glu Cys 210 61452PRTArtificial
Sequencean artificially synthesized sequence 61Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala
Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe
50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Pro Val Pro Gly Val Tyr
Tyr Tyr Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Met Val
Thr Val Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170
175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
180 185 190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn 195 200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu 225 230 235 240 Leu Leu Gly Gly Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met Ile Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser His
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295
300 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
305 310 315 320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro 325 330 335 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400 Thr Pro
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415
Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420
425 430 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu 435 440 445 Ser Leu Ser Pro 450 62215PRTArtificial Sequencean
artificially synthesized sequence 62Glu Ile Val Leu Thr Gln Ser Pro
Ala Thr Leu Ser Val Ser Pro Gly 1 5 10 15 Glu Arg Val Thr Leu Ser
Cys Arg Ala Ser Gln Ser Val Ala Asn His 20 25 30 Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Gln Pro Pro Ser Leu Leu Ile 35 40 45 Tyr Asp
Ala Ser Thr Arg Ala Thr Asp Val Pro Ala Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Ser Ile Ser Gly Leu Gln Ser 65
70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Lys Asp Trp
Pro Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Phe Lys
Arg Thr Val Ala 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser 115 120 125 Gly Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 Ala Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 Gln Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu 165 170 175 Ser
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185
190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
195 200 205 Ser Phe Asn Arg Gly Glu Cys 210 215 63452PRTArtificial
Sequencean artificially synthesized sequence 63Gln Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys
Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala
Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40
45 Gly Gly Ile Ile Pro Ile Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe
50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr
Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ser Pro Val Pro Gly Val Tyr
Tyr Tyr Tyr Gly Met Asp 100 105 110 Val Trp Gly Gln Gly Thr Met Val
Thr Val Ser Ser Ala Ser Thr Lys 115 120 125 Gly Pro Ser Val Phe Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 130 135 140 Gly Thr Ala Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 145 150 155 160 Val
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 165 170
175 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
180 185 190 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn 195
200 205 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
Pro 210 215 220 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
Ala Pro Glu 225 230 235 240 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp 245 250 255 Thr Leu Met Ile Ser Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp 260 265 270 Val Ser His Glu Asp Pro
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 275 280 285 Val Glu Val His
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn 290 295 300 Ser Thr
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 305 310 315
320 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
325 330 335 Ala Pro Val Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu 340 345 350 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn 355 360 365 Gln Val Ser Leu Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile 370 375 380 Ala Val Glu Trp Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr 385 390 395 400 Thr Pro Pro Val Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 405 410 415 Leu Thr Val
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 420 425 430 Ser
Val Met His Glu Ala Leu His Tyr His Tyr Thr Gln Lys Ser Leu 435 440
445 Ser Leu Ser Pro 450 64449PRTArtificial Sequencean artificially
synthesized sequence 64Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu
Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser
Gly Tyr Ser Ile Thr Ser Asp 20 25 30 His Ala Trp Ser Trp Val Arg
Gln Pro Pro Gly Arg Gly Leu Glu Trp 35 40 45 Ile Gly Tyr Ile Ser
Tyr Ser Gly Ile Thr Thr Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg
Val Thr Met Leu Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu
Arg Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Gln Gly
100 105 110 Ser Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser
Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu
Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu
Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser
Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215
220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro
225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp
Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu
Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys
Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340
345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu
Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly
Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys
65214PRTArtificial Sequencean artificially synthesized sequence
65Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser
Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr
Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135
140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu
Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210
66460PRTArtificial Sequencean artificially synthesized sequence
66Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
Tyr 20 25 30 Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu
Glu Trp Met 35 40 45 Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala
Tyr Ser Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Ala Asp
Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Thr
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Phe Tyr Ser
Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser
Ala Ser Pro Thr Ser Pro Lys Val Phe Pro Leu Ser Leu 115 120 125 Cys
Ser Thr Gln Pro Asp Gly Asn Val Val Ile Ala Cys Leu Val Gln 130 135
140 Gly Phe Phe Pro Gln Glu Pro Leu Ser Val Thr Trp Ser Glu Ser Gly
145 150 155 160 Gln Gly Val Thr Ala Arg Asn Phe Pro Pro Ser Gln Asp
Ala Ser Gly 165 170 175 Asp Leu Tyr Thr Thr Ser Ser Gln Leu Thr Leu
Pro Ala Thr Gln Cys 180 185 190 Leu Ala Gly Lys Ser Val Thr Cys His
Val Lys His Tyr Thr Asn Pro 195 200 205 Ser Gln Asp Val Thr Val Pro
Cys Pro Val Pro Ser Thr Pro Pro Thr 210 215 220 Pro Ser Pro Ser Thr
Pro Pro Thr Pro Ser Pro Ser Cys Cys His Pro 225 230 235 240 Arg Leu
Ser Leu His Arg Pro Ala Leu Glu Asp Leu Leu Leu Gly Ser 245 250 255
Glu Ala Asn Leu Thr Cys Thr Leu Thr Gly Leu Arg Asp Ala Ser Gly 260
265 270 Val Thr Phe Thr Trp Thr Pro Ser Ser Gly Lys Ser Ala Val Gln
Gly 275 280 285 Pro Pro Glu Arg Asp Leu Cys Gly Cys Tyr Ser Val Ser
Ser Val Leu 290 295 300 Pro Gly Cys Ala Glu Pro Trp Asn His Gly Lys
Thr Phe Thr Cys Thr 305 310 315 320 Ala Ala Tyr Pro Glu Ser Lys Thr
Pro Leu Thr Ala Thr Leu Ser Lys 325 330 335 Ser Gly Asn Thr Phe Arg
Pro Glu Val His Leu Leu Pro Pro Pro Ser 340 345 350 Glu Glu Leu Ala
Leu Asn Glu Leu Val Thr Leu Thr Cys Leu Ala Arg 355 360 365 Gly Phe
Ser Pro Lys Asp Val Leu Val Arg Trp Leu Gln Gly Ser Gln 370 375 380
Glu Leu Pro Arg Glu Lys Tyr Leu Thr Trp Ala Ser Arg Gln Glu Pro 385
390 395 400 Ser Gln Gly Thr Thr Thr Phe Ala Val Thr Ser Ile Leu Arg
Val Ala 405 410 415 Ala Glu Asp Trp Lys Lys Gly Asp Thr Phe Ser Cys
Met Val Gly His 420 425 430 Glu Ala Leu Pro Leu Ala Phe Thr Gln Lys
Thr Ile Asp Arg Leu Ala 435 440 445 Gly Lys Glu Gln Lys Leu Ile Ser
Glu Glu Asp Leu 450 455 460 67219PRTArtificial Sequencean
artificially synthesized sequence 67Asp Val Val Met Thr Gln Ser Pro
Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 Glu Pro Ala Ser Ile Ser
Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Arg Asn Thr
Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln
Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65
70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser
Gln Asn 85 90 95 Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys
Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile
Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser
Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys
Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185
190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215
68447PRTArtificial Sequencean artificially synthesized sequence
68Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1
5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His
Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly
Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn
Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp
Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala
Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro
Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135
140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp
145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile
Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys
Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260
265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385
390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Leu His Glu 420 425 430 Ala Leu His Ser His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro 435 440 445 69449PRTArtificial Sequencean
artificially synthesized sequence 69Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys
Ala Val Ser Gly Tyr Ser Ile Thr Ser Gly 20 25 30 Tyr Ser Trp Asn
Trp Ile Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp 35 40 45 Val Ala
Ser Ile Thr Tyr Asp Gly Ser Thr Asn Tyr Asn Pro Ser Val 50 55 60
Lys Gly Arg Ile Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr Phe Tyr 65
70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
Tyr Cys 85 90 95 Ala Arg Gly Ser His Tyr Phe Gly His Trp His Phe
Ala Val Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185
190 Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205 Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys
Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310
315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu 420 425 430
His Glu Ala Leu His Ser His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435
440 445 Pro 70218PRTArtificial Sequencean artificially synthesized
sequence 70Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser
Val Asp Tyr Asp 20 25 30 Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro 35 40 45 Lys Leu Leu Ile Tyr Ala Ala Ser
Tyr Leu Glu Ser Gly Val Pro Ser 50 55 60 Arg Phe Ser Gly Ser Gly
Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Pro
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser His 85 90 95 Glu Asp
Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115
120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 His Lys Val Tyr Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 Val Thr Lys Ser
Phe Asn Arg Gly Glu Cys 210 215 71365PRTHomo sapiens 71Met Gly Val
Pro Arg Pro Gln Pro Trp Ala Leu Gly Leu Leu Leu Phe 1 5 10 15 Leu
Leu Pro Gly Ser Leu Gly Ala Glu Ser His Leu Ser Leu Leu Tyr 20 25
30 His Leu Thr Ala Val Ser Ser Pro Ala Pro Gly Thr Pro Ala Phe Trp
35 40 45 Val Ser Gly Trp Leu Gly Pro Gln Gln Tyr Leu Ser Tyr Asn
Ser Leu 50 55 60 Arg Gly Glu Ala Glu Pro Cys Gly Ala Trp Val Trp
Glu Asn Gln Val 65 70 75 80 Ser Trp Tyr Trp Glu Lys Glu Thr Thr Asp
Leu Arg Ile Lys Glu Lys 85 90 95 Leu Phe Leu Glu Ala Phe Lys Ala
Leu Gly Gly Lys Gly Pro Tyr Thr 100 105 110 Leu Gln Gly Leu Leu Gly
Cys Glu Leu Gly Pro Asp Asn Thr Ser Val 115 120 125 Pro Thr Ala Lys
Phe Ala Leu Asn Gly Glu Glu Phe Met Asn Phe Asp 130 135 140 Leu Lys
Gln Gly Thr Trp Gly Gly Asp Trp Pro Glu Ala Leu Ala Ile 145 150 155
160 Ser Gln Arg Trp Gln Gln Gln Asp Lys Ala Ala Asn Lys Glu Leu Thr
165 170 175 Phe Leu Leu Phe Ser Cys Pro His Arg Leu Arg Glu His Leu
Glu Arg 180 185 190 Gly Arg Gly Asn Leu Glu Trp Lys Glu Pro Pro Ser
Met Arg Leu Lys 195 200 205 Ala Arg Pro Ser Ser Pro Gly Phe Ser Val
Leu Thr Cys Ser Ala Phe 210 215 220 Ser Phe Tyr Pro Pro Glu Leu Gln
Leu Arg Phe Leu Arg Asn Gly Leu 225 230 235 240 Ala Ala Gly Thr Gly
Gln Gly Asp Phe Gly Pro Asn Ser Asp Gly Ser 245 250 255 Phe His Ala
Ser Ser Ser Leu Thr Val Lys Ser Gly Asp Glu His His 260 265 270 Tyr
Cys Cys Ile Val Gln His Ala Gly Leu Ala Gln Pro Leu Arg Val 275 280
285 Glu Leu Glu Ser Pro Ala Lys Ser Ser Val Leu Val Val Gly Ile Val
290 295 300 Ile Gly Val Leu Leu Leu Thr Ala Ala Ala Val Gly Gly Ala
Leu Leu 305 310 315 320 Trp Arg Arg Met Arg Ser Gly Leu Pro Ala Pro
Trp Ile Ser Leu Arg 325 330 335 Gly Asp Asp Thr Gly Val Leu Leu Pro
Thr Pro Gly Glu Ala Gln Asp 340 345 350 Ala Asp Leu Lys Asp Val Asn
Val Ile Pro Ala Thr Ala 355 360 365 72119PRTHomo sapiens 72Met Ser
Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser 1 5 10 15
Gly Leu Glu Ala Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg 20
25 30 His Pro Ala Glu Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val
Ser 35 40 45 Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu Leu Lys
Asn Gly Glu 50 55 60 Arg Ile Glu Lys Val Glu His Ser Asp Leu Ser
Phe Ser Lys Asp Trp 65 70 75 80 Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu
Phe Thr Pro Thr Glu Lys Asp 85 90 95 Glu Tyr Ala Cys Arg Val Asn
His Val Thr Leu Ser Gln Pro Lys Ile 100 105 110 Val Lys Trp Asp Arg
Asp Met 115 73290PRTMus musculus 73Met Gly Met Pro Leu Pro Trp Ala
Leu Ser Leu Leu Leu Val Leu Leu 1 5 10 15 Pro Gln Thr Trp Gly Ser
Glu Thr Arg Pro Pro Leu Met Tyr His Leu 20 25 30 Thr Ala Val Ser
Asn Pro Ser Thr Gly Leu Pro Ser Phe Trp Ala Thr 35 40 45 Gly Trp
Leu Gly Pro Gln Gln Tyr Leu Thr Tyr Asn Ser Leu Arg Gln 50 55 60
Glu Ala Asp Pro Cys Gly Ala Trp Met Trp Glu Asn Gln Val Ser Trp 65
70 75 80 Tyr Trp Glu Lys Glu Thr Thr Asp Leu Lys Ser Lys Glu Gln
Leu Phe 85 90 95 Leu Glu Ala Leu Lys Thr Leu Glu Lys Ile Leu Asn
Gly Thr Tyr Thr 100 105 110 Leu Gln Gly Leu Leu Gly Cys Glu Leu Ala
Ser Asp Asn Ser Ser Val 115 120 125 Pro Thr Ala Val Phe Ala Leu Asn
Gly Glu Glu Phe Met Lys Phe Asn 130 135 140 Pro Arg Ile Gly Asn Trp
Thr Gly Glu Trp Pro Glu Thr Glu Ile Val 145 150 155 160 Ala Asn Leu
Trp Met Lys Gln Pro Asp Ala Ala Arg Lys Glu Ser Glu 165 170 175 Phe
Leu Leu Asn Ser Cys Pro Glu Arg Leu Leu Gly His Leu Glu Arg 180 185
190 Gly Arg Arg Asn Leu Glu Trp Lys Glu Pro Pro Ser Met Arg Leu Lys
195 200 205 Ala Arg Pro Gly Asn Ser Gly Ser Ser Val Leu Thr Cys Ala
Ala Phe 210 215 220 Ser Phe Tyr Pro Pro Glu Leu Lys Phe Arg Phe Leu
Arg Asn Gly Leu 225 230 235 240 Ala Ser Gly Ser Gly Asn Cys Ser Thr
Gly Pro Asn Gly Asp Gly Ser 245 250 255 Phe His Ala Trp Ser Leu Leu
Glu Val Lys Arg Gly Asp Glu His His 260 265 270 Tyr Gln Cys Gln Val
Glu His Glu Gly Leu Ala Gln Pro Leu Thr Val 275 280 285 Asp Leu 290
74119PRTMus musculus 74Met Ala Arg Ser Val Thr Leu Val Phe Leu Val
Leu Val Ser Leu Thr 1 5 10 15 Gly Leu Tyr Ala Ile Gln Lys Thr Pro
Gln Ile Gln Val Tyr Ser Arg 20 25 30 His Pro Pro Glu Asn Gly Lys
Pro Asn Ile Leu Asn Cys Tyr Val Thr 35 40 45 Gln Phe His Pro Pro
His Ile Glu Ile Gln Met Leu Lys Asn Gly Lys 50 55 60 Lys Ile Pro
Lys Val Glu Met Ser Asp Met Ser Phe Ser Lys Asp Trp 65 70 75 80 Ser
Phe Tyr Ile Leu Ala His Thr Glu Phe Thr Pro Thr Glu Thr Asp 85 90
95 Thr Tyr Ala Cys Arg Val Lys His Ala Ser Met Ala Glu Pro Lys Thr
100 105 110 Val Tyr Trp Asp Arg Asp Met 115 75447PRTArtificial
Sequencean artificially synthesized sequence 75Gln Val Gln Leu Gln
Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser
Leu Thr Cys Ala Val Ser Gly His Ser Ile Ser His Asp 20 25 30 His
Ala Trp Ser Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp 35 40
45 Ile Gly Phe Ile Ser Tyr Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu
50 55 60 Gln Gly Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Leu Ala Arg Thr Thr Ala Met
Asp Tyr Trp Gly Glu Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser
Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170
175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser
180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val
Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295
300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Tyr Pro Ala Pro
Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr
Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420
425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445 76447PRTArtificial Sequencean artificially synthesized
sequence 76Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Ala Val Ser Gly His Ser
Ile Ser His Asp 20 25 30 His Ala Trp Ser Trp Val Arg Gln Pro Pro
Gly Glu Gly Leu Glu Trp 35 40 45 Ile Gly Phe Ile Ser Tyr Ser Gly
Ile Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Gln Gly Arg Val Thr Ile
Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn
Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg
Ser Leu Ala Arg Thr Thr Ala Met Asp Tyr Trp Gly Glu Gly 100 105 110
Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115
120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln
Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys
Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His
Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235
240 Asp Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
Asn Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His
Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala Pro Glu Glu Lys 325 330 335 Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu
Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360
365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 77214PRTArtificial
Sequencean artificially synthesized sequence 77Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Ser Val
Thr Ile Thr Cys
Gln Ala Ser Thr Asp Ile Ser Ser His 20 25 30 Leu Asn Trp Tyr Gln
Gln Lys Pro Gly Lys Ala Pro Glu Leu Leu Ile 35 40 45 Tyr Tyr Gly
Ser His Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser
Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Glu Ala 65 70
75 80 Glu Asp Ala Ala Thr Tyr Tyr Cys Gly Gln Gly Asn Arg Leu Pro
Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Glu Arg Thr
Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn
Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr
Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195
200 205 Phe Asn Arg Gly Glu Cys 210 78214PRTArtificial Sequencean
artificially synthesized sequence 78Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Asp Ile Ser Ser Tyr 20 25 30 Leu Asn Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Tyr
Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu
Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185
190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205 Phe Asn Arg Gly Glu Cys 210
* * * * *
References